Synthesis, growth, spectral, thermal and crystallographic studies of 5α,6α-epoxycholestane single crystals

Journal of Crystal Growth 384 (2013) 135–143

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Synthesis, growth, spectral, thermal and crystallographic studies of 5α,6α-epoxycholestane single crystals Shamsuzzaman a,n, Hena Khanam a, Ashraf Mashrai a, Musheer Ahmad b, Yahia Nasser Mabkhot c, Wolfgang Frey d, Nazish Siddiqui e a

Steroid Research Laboratory, Department of Chemistry, Aligarh Muslim University, Aligarh 202002, UP, India Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India c Department of Chemistry, Faculty of Science, King Saud University, Riyadh 11451, Saudi Arabia d Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring 55, Stuttgart 70569, Germany e AKT College, Aligarh Muslim University, Aligarh 202002, UP, India b

art ic l e i nf o Article history: Received 9 July 2013 Received in revised form 20 August 2013 Accepted 28 August 2013 Communicated by M. Fleck Available online 24 September 2013 Keywords: A1. Thermal stability A1. X-ray diffraction A1. Solubility A2. Growth from solution A2. Single crystal growth B1. Epoxycholestane

a b s t r a c t

α-Selective

epoxidation of 3β-chlorocholest-5-ene and 3β-acetoxycholest-5-ene has been performed with m-CPBA to synthesize 3β-chloro-5α,6α-epoxycholestane (1) and 3β-acetoxy-5α,6α-epoxycholestane (2). We provided an analysis of these compounds by means of FT-IR, FT-Raman, 1H NMR, 13C NMR, 2D cosy, NOESY, UV–visible and X-ray crystallography. The compound 1 crystallizes in the orthorhombic space group P212121 while compound 2 crystallizes in the monoclinic space group P21. We compared the conformations of both compounds in solid state and in solution by calculation of dihedral angles and coupling constant values. The powder X-ray diffraction (PXRD) of the compound was recorded to ascertain the purity of the grown crystals. Thermogravimetric analysis showed stability of the compounds up to 250 1C. Moreover, the ICH rule has been applied to test the stability of two crystals, which showed significant stability. & 2013 Elsevier B.V. All rights reserved.

1. Introduction Cholesterol is a tetracyclic lipid of biological importance since its discovery by François Poulletier de la Salle in 1758. Since the last century cholesterol is known to be subject to oxidation leading to the formation of mono- or poly-oxygenation products called oxysterols. Oxysterols attract much attention in cell biology and pathophysiology because of the wide range of biological phenomena in which they are involved. Oxysterols can be endogenously produced from cholesterol (Chola) by enzymatic and nonenzymatic oxidative processes or absorbed from diet sources [1]. They participate in the biosynthesis of bile acids and steroid hormones, acting also as signaling lipids that regulate cholesterol biosynthesis, cellular cholesterol efflux, lipoprotein uptake, intracellular cholesterol trafficking [2] and have been related to neurodegenerative diseases, such as Alzheimer, Parkinson, and Multiple sclerosis [1]. Most common oxysterols are known to interfere with the cell membrane structure and cellular receptors [3] to inhibit

n

Corresponding author. Mobile: þ 91 9411003465. E-mail addresses: [email protected], [email protected] (Shamsuzzaman). 0022-0248/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcrysgro.2013.08.037

cholesterol [2,4] and DNA biosynthesis [5], and to induce cell death in different cell lines by apoptosis or necrosis [1]. Among oxysterols, 5,6-epoxycholestanes have stimulated the interest of researchers some years after the photo-oxidation products of cholesterol were suspected to be involved in photocarcinogenesis [6]. Because of the presence of an oxirane group, it was supposed that 5,6-epoxycholestane could be electrophilic and behave like alkylating agents with direct carcinogenic properties. Recent data from literature ruled out that 5,6-epoxycholestanes could be direct alkylating substances [7] and provides evidence that 5,6-epoxycholestanes may be involved in physiological processes that result in metabolites with tumor promoter properties as well as to the production of steroidal alkaloids which are antioncogenic. Ring B oxysterols were reported to stimulate cholesterol ester formation in cultured fibroblasts [8] and 5α,6α-epoxycholestane was shown to be the most potent allosteric activator for ACAT-1 (acyl-coA: cholesterol acyl transferase) whereas 5β,6βepoxycholestane was found to be inefficient [9]. It was found that 5α,6α-epoxycholestane has tighter interactions with phospholipids than 5β,6β-epoxycholestane and would be considered a better raft-stabilizing sterol [10]. 5α,6α-Epoxycholestane was reported to inhibit Topoisomerase II [11]. Furthermore, ring

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opening of 5R,6R-epoxysteroids gives access to contraceptives, anti-inflammatory agents, and enzymatic inhibitors [12–14]. Several physical and spectroscopic characterizations are important for pharmaceutical compounds and crystals for determining their thermal stability, shelf-life, reactivity and their production point of view. Thermogravimetry is used for pharmaceutical compounds as well as crystals for assessing thermal stability and content of water either in moisture form or in crystallization form. Using DSC a polymorph of compound can be predicted. Properties of the compound in bulk can be studied through SEM and powder X-ray diffraction (PXRD) analysis. Encouraged by the above mentioned pioneering applications of steroidal epoxides and in continuation of our work towards synthesis of new steroidal derivatives [15] and single crystal X-ray analysis [16] in the present study we have synthesized 3β-chloro and 3βacetoxy-5α,6α-epoxycholestanes (1, 2) and attempted to grow their single crystals. As many properties are governed by the crystalline nature, the grown crystals were characterized by spectral techniques (FT-IR, FT-Raman, NMR, UV–vis), XRD, SEM, EDX and TG–DTA–DSC.

2. Experimental 2.1. General procedure for the synthesis of 5α,6α-epoxycholestanes 2.1.1. 3β-Chloro-5α,6α-epoxycholestane (1) 3β-Chlorocholest-5-ene (11 g), in chloroform (100 mL) was treated with a solution of m-chloroperbenzoic acid (1.1 mol equivalent) in chloroform and left for 20 h at  8 1C. The reaction mixture was then washed successively with ice-cooled aqueous solution of sodium bicarbonate (5%), water, sodium thiosulphate (5%) and again with water and dried over anhydrous sodium sulfate. Evaporation of solvents gave oil which was chromatographed over silica gel column. Elution with light petroleum ether/ ether (10:1) gave a solid compound which after repeated and selective recrystallization from methanol provided compound 1 as colorless needles, (8.1 g), m.p. 87–89 1C (reported m.p. 89.5– 90.5 1C) [17] (Fig. 1a). Anal. Calcd for C27H45ClO: C, 77.01; H, 10.77. Found: C, 77.11; H, 10.67; IR (KBr, cm  1): 2944, 2869 (C–H, stretching), 1464, 1385 (C–H, bending), 1166 (C–O), 706 (C–Cl); 1H NMR (CDCl3, 400 MHz): δ ppm 4.12 (1H, m, C3α–H), 2.91 (1H, d, C6–βH, J ¼4.36 Hz), 1.10 (3H, s, 10-CH3) 0.67 (3H, s, 13-CH3), 0.90 & 0.85 (other methyl protons); 13C NMR (CDCl3, 100 MHz): δ ppm 65.8 (C-5), 60.3, 59.5, 56.9, 56.7, 55.8, 50.0 (C-3), 43.3, 42.4, 41.4, 39.6, 39.2, 36.3, 36.1, 35.7, 34.7, 33.3, 32.7, 31.8, 29.7, 28.7, 28.0, 27.9, 24.2, 18.6, 15.8, 11.8. 2.1.2. 3β-Acetoxy-5α,6α-epoxycholestane (2) 3β-Acetoxycholest-5-ene (10 g), in chloroform (100 mL) was treated with a solution of m-chloroperbenzoic acid (1.1 mol

27

21

2

Cl

1

19

10 5 3 4 O

11 9

12

18

17 20

H14 13

H 8 H 15 6 7 H

23

25

equivalent) in chloroform and left for 20 h at  8 1C. The reaction mixture was then washed successively with ice-cooled aqueous solution of sodium bicarbonate (5%), water, sodium thiosulphate (5%) and again with water and dried over anhydrous sodium sulfate. Evaporation of solvents gave a solid which after repeated and selective recrystallization from methanol provided compound 2 as colorless blocks, (8.5 g), m.p. 98 1C (reported m.p. 97 1C) [18]. (Fig. 1b) Anal. Calcd for C29H48O3: C, 78.33; H, 10.88. Found: C, 78.20; H, 11.01; IR (KBr, cm  1): 1736 (C ¼O), 1242 (C–O); 1H NMR (CDCl3, 400 MHz): δ ppm 4.94 (1H, m, C3α-H), 2.88 (1H, d, C6–βH, J¼ 4.36 Hz), 2.01 (3H, s, OCOCH3), 1.10 (3H, s, 10-CH3) 0.63 (3H, s, 13-CH3), 0.89 & 0.85 (other methyl protons). 13C NMR (100 Mz, CDCl3): δ ppm 170.2 (CQO), 71.3 (C–O), 65.2 (C-5), 63.6, 59.1, 56.7, 55.8, 50.9, 42.4, 39.7, 39.3, 38.0, 36.6, 35.7, 35.0, 32.4, 29.8, 28.7, 28.0, 27.2, 24.1, 23.9, 23.8, 22.5, 21.9, 20.5, 18.6,15.8, 11.8. 2.2. Solubility and crystal growth studies Selection of suitable solvent is very definitive for the growth of good quality single crystals. Equilibrium solubility and its temperature dependence are essential for solution growth. The data from the solubility curve will suffice to start growing fair quality single crystals. The solubility of compounds in methanol was assessed as a function of temperature in the temperature range 30–45 1C. There were slight difference in the solubility of compounds 1 and 2. The amount of epoxycholestane required to make the saturated solution at different temperatures was estimated gravimetrically and the obtained solubility curve is shown in Fig. 2. From the solubility curve, it is clear that the compound exhibits a positive solubility-temperature gradient which is very important to grow good quality crystals from solution. The quality of single crystal depends on the purity of the used material. Hence, the synthesized materials were recrystallized several times to obtain highly purified compounds. The saturated solution (about 50 ml) was prepared in methanol according to the solubility curve (Fig. 2). Then, the prepared solution was passed through anhydrous sodium sulfate and covered with aluminum foil. Finally, it was kept at room temperature. Good quality colorless crystals of epoxycholestanes were harvested after a span of about 8–10 days. 3. Material characterization Three dimensional intensity data for the compound 1 were collected at 100 K on Bruker KAPPA APEXII DUO diffractometer using Mo Kα radiation (λ ¼0.71073 Ǻ). The structure was solved by direct methods using SHELXS-97 software (SHELDRICK, 1990). Isotropic refinement of the structure by least-squares methods was carried out by using SHELXL-97 (SHELDRICK, 1997) followed

27

26

24

21

22

16

1

2

O O

19

10 5 3

4 O

11 9

12

18

23

17 20

H14 13

25

26

24

22

16

H 8 H 15 6 7 H

Fig. 1. Molecular structure of (a) 3β-chloro-5α,6α-epoxycholestane and (b) 3β-acetoxy-5α,6α-epoxycholestane.

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performed at ambient temperature. Thin layer chromatography (TLC) plates were coated with silica gel G and exposed to iodine vapors to check the homogeneity as well as the progress of reaction. Sodium sulfate (anhydrous) was used as a drying agent.

4. Results and discussion

Fig. 2. Solubility curve of 3β-chloro-5α,6α-epoxycholestane (1) in methanol.

by anisotropic refinement on F2of all the non-hydrogen atoms. Single crystal X-ray data of compound 2 was collected at 100 K on a Bruker SMART APEX CCD diffractometer using graphite monochromated MoKα radiation (λ ¼ 0.71073 Å). The linear absorption coefficients, scattering factors for the atoms, and the anomalous dispersion corrections were taken from the International Tables for X-ray Crystallography [19].The data integration and reduction were carried out with SAINT [20] software. Empirical absorption correction was applied to the collected reflections with SADABS [21] and the space group was determined using XPREP [22].The structure was solved by the direct methods using SHELXTL-97 [23] and refined on F2 by full-matrix least-squares using the SHELXL-97 [24] program package. All non-hydrogen atoms were refined anisotropically. Crystallographic data (excluding structure factors) for the structures reported in this article have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as deposition no. CCDC 896093 (for compound 1) and 917627 (for compound 2). All H-atom positions were calculated geometrically with Uiso (H)¼ 1.2–1.5 Ǻ Ueq (parent atom). A riding model was used in their refinement (C–H¼ 0.98–1.00 Ǻ). Melting points were determined on a Kofler apparatus and are uncorrected. The IR spectra were recorded on KBr pellets with Interspec 2020 FT-IR Spectrometer spectro Lab and values are given in cm  1. FT-Raman spectra were recorded on WITec alpha 300 scanning Near Field Optical Microscope (SNOM), Germany. 1H and 13 C NMR spectra were run in CDCl3 on a Bruker Avance II 400 NMR Spectrometer at 400 MHz and 100 MHz respectively. Chemical shifts (δ) are reported in ppm relative to the TMS (1H NMR, 400 MHz) and to the solvent signal (13C NMR spectra, 100 MHz). Elemental analyses of the compounds were recorded on Perkin Elmer 2400 CHN Elemental Analyzer. The thermal studies of the compounds were carried out using TGA/DTA- 60H and DSC-60 instrument (SHIMADZU) at a heating rate of 20 1C min  1 from ambient temperature to 800 1C (for DSC 500 1C ). The surface morphology of the compounds was monitored using JEOL JSM-6510LV scanning electron microscope (SEM), equipped with energy-dispersive X-ray spectroscopy (EDX) analyzer. UV–visible spectra were recorded on UV–vis spectrophotometer (Perkin Elmer Life and Analytical Sciences, CT, USA) in the wavelength range of A200–800 nm. The Stokes shift (δν, cm  1) and oscillator strength (f) were calculated by the following equations, (VA VF)¼(1/VA  1/VF)  107 and f¼4.32  10  9 δν1/2 εmax, (Where εmax is molar extinction coefficient and δν 1/2 is the width of the absorption band (cm  1) at (εmax)). X-ray diffraction (PXRD) pattern of powdered sample was recorded on MiniFlex™ II benchtop XRD system (Rigaku Corporation, Tokyo, Japan) operating at 40 kV and a current of 30 mA with Cu Kα radiation (λ ¼ 1.54 Ǻ). The diffracted intensities were recorded from 201 to 801 2θ angles with scan rate of 21/min and a step size of 0.021 and all XRD measurements were

The starting materials 3β-chlorocholest-5-ene/3β-acetoxycholest-5-ene for the present investigation were synthesized by literature method [25]. α-Selective epoxidation of 3β-chlorocholest-5-ene/3β-acetoxycholest-5-ene were performed with 3-chloroperoxybenzoic acid (m-CPBA) by reported methods [17,18]. The preferred α-diastereoselection in peroxyacid epoxidation of cholest-5-enes can be easily explained by the steric encumbrance imposed by the C-10 and C-13 angular methyl groups. The FT-IR, FT-Raman, 1H NMR, 13C NMR, 2D 1H–1H cosy and NOESY spectra of compounds 1 and 2 were found consistent with their crystal structure. 4.1. Single crystal X-ray crystallography The two crystals (1 and 2) belong to orthorhombic and monoclinic crystal system with Sohncke space groups P212121 (Z¼8) and P21 (Z¼4) respectively. The occurrence of these types of space groups can be explained by the presence of 10 chiral centers for both of them [26]. All rings of the steroid skeleton are trans fused. The cyclohexane rings A and C are identical in chair conformations, whereas ring B assumes a distorted chair conformation. The cyclopentane ring D is distorted envelop while the oxirane ring is α-oriented. The epoxycholestane side-chain is fully extended with a gauche–trans conformation of the terminal methyl groups. Due to the paucity of hydrogen-bonds, the epoxycholestane molecules interact through only van der Waals interactions, which are the major intermolecular forces in most pharmaceutical crystals. And because of the existence of these forces only, one can observes different molecular conformations of the independent molecules. Pertinent crystallographic data and refinement details for the structural analyses of the compounds 1 and 2 are summarized in Table 1. Single crystal X-ray structures are given in Figs. 3 and 4. The numbering scheme in the discussion of single crystal X-ray is according to CIF file. The asymmetric unit of the compound, C27H45ClO, (1) consists of two crystallographically independent molecules. The angle C1–C2–C3 is 120.21 and the angle C6–C7–C8 is 110.351. The distance between Cl and C18 is 3.684 Ǻ. The chlorine atom at position-4 is equatorial and antiperiplanar to the C2–C3 bond with a torsion angle 179.711 (16) and not involved in any hydrogen bond. The distance between the chlorine atom and the nearest hydrogen atom is 2.84 Å. The C4–Cl bond distance is 1.814 (2) Ǻ being slightly longer than the average value reported for Cl–C ¼ 1.805 (14) Ǻ [27]. There are 10 chiral centers in the molecule, the absolute configuration of these sites have been determined from the structure presented, these sites exhibit the following chiralities: C1 ¼S, C2¼ R, C4 ¼S, C7¼ R, C8¼ S, C11¼ R, C12¼ R, C15 ¼ S, C16¼ S and C20¼R. The asymmetric unit of the compound C29H48O (2) contains two crystallographically independent molecules. The angle C9–C5–C4 is 120.71 and the angle C7–C6–C12 is 111.11. The acetoxy group at position-3 is equatorial and antiperiplanar to the C4–C5 bond with a torsion angle 174.531 and also not involved in any hydrogen bond. The distance between acetoxy group and C13 is 3.649 Ǻ and the bond C–O (OCOCH3) is 1.455 Ǻ which is slightly longer than the average values reported [27]. There are 10 chiral centers in the molecule. The absolute configurations of these sites have been determined from the structure presented, these sites exhibit the following chiralities: C3 ¼R, C5 ¼S, C6 ¼S, C9¼ R, C11¼ R, C12¼ R,

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Table 1 Crystal data and structure refinement for crystals 1 and 2.

Empirical formula Formula weight Wavelength (λ) Crystal system Space group Unit cell dimensions a, Ǻ b, Ǻ c, Ǻ α β γ Volume, Ǻ3 No. of molecules per unit cell (Z) Calculated density, Mg m  3 Absorption coefficient (μ, mm  1) F(000) Crystal size θ range for data collection Limiting indices Reflections collected Data/restraints/parameters Goodness-of-fit on F2 Final R indices [I 42s(I)] R indices (all data)

Compound 1

Compound 2

C27H45ClO 421.08 0.71073 Ǻ Orthorhombic P212121

C29H48O3 444.67 0.71073 Å Monoclinic P21

7.6571 (8) 22.056 (2) 28.418 (2) 90.001 90.001 90.001 4799.5 (8) 8 1.165 0.175 1856 0.83  0.16  0.13 mm3 1.70–26.431  9o ¼h o ¼ 9,  27o ¼k o ¼ 25,  22o ¼l o ¼35 33,568 9853/0/533 1.045 R1¼ 0.0528, wR2¼ 0.0726 R1¼ 0.0963, wR2¼ 0.0796

12.7820 (13) 10.1722 (10) 20.321 (2) 90.000 90.465 (2) 90.000 2642.1 (5) 4 1.118 0.070 984 0.21  0.19  0.16 mm3 2.00–25.001  15o ¼ h o ¼15,  12o ¼ k o ¼12,  24o ¼ lo ¼ 24 10,069 5186/1/577 1.027 R1¼ 0.0320, wR2¼0.0759 R1¼ 0.0364 wR2¼0.0784

R1 ¼Σ||Fo|–|Fc||/Σ|Fo| with Fo2 4 2s(Fo2). wR2 ¼[Σw(|Fo2|–|Fc2|)2/Σ|Fo2|2]1/2.

C16 ¼S, C17 ¼ R, C20 ¼S and C22¼ S. The two molecules (1 and 2) in the present study have different substituents at the same position of ring-A. Therefore, it is of interest to investigate bond distances and bond angles of the same ring. The substitution at ring-A of the steroid nucleus causes significant changes in the bond distance and bond angle. The bond distances and bond angles in compound 1 are found to be C3–C4¼1.514 Ǻ, C4–C5¼1.508 Ǻ and C3–C4–C5¼ 111.231 while for compound 2 these are recorded as C3–C4¼1.504 Ǻ, C3–C8¼1.519 Ǻ and C4–C3–C8¼111.481. The observed bond distances are shorter than the standard value of C (sp3)–C(sp3) bond. The bond angles in the two molecules show some deviation from the average value of 109.51 for sp3-type hybridization [28]. We examined the conformations of compounds 1 and 2 in solution (through NMR data) and solid state (through X-ray data). The calculation of the dihedral angles deduced from the measured coupling constants are given in Table 2. Interestingly, we scrutinized less differences in the conformations in solution and in the solid state, showing that the bending of rings at the A, B junction was also less in solution than in the solid state. 4.2. FT-IR/FT-Raman spectral analysis The infrared spectral analysis was carried out to understand the chemical bonding and it provides useful information regarding the molecular structure of the compound. In the infrared spectrum of the compound 1 characteristic peak observed at 706, was assigned to ν (C–Cl) stretching vibrations and 1166 cm  1 was attributed to C–O–C stretching of oxirane ring. The peaks at 1297 and 1333 cm  1, respectively, correspond to C–H out-of-plane and C–H in-plane bends, whereby the symmetric and asymmetric bends of CH group were detected at 1385 and 1464 cm  1, respectively. The characteristic bands in the range of 2800–2944 cm  1 were ascribed to C–H stretching vibrations. The IR spectra of compound 2 showed strong peaks at 1736 and 1242 cm  1 ascribed to CQO and C–O stretching vibrations respectively. The Raman spectra of compounds 1 and 2 in the 1250–4000 cm  1 spectral range are

illustrated in Figs. S1 and S2. The spectra demonstrated the position and relative intensity of the Raman bands. The spectra of both the compounds displayed a strong band at 2750–3000 cm  1, attributed to the stretching vibrations of the C–H units. A sharp medium peak at 1750 cm  1 in the Raman spectrum of compound 2 was assigned to CQO stretching vibrations. 4.3. NMR analysis 1 H NMR spectra of compound 1 (Fig. 5) gives doublet at 2.91 ppm (J ¼4.36 Hz) corresponding to C6–βH and a broad multiplet at 4.12 ppm due to C3–αH. The 2D 1H–1H cosy showed momentous H–H interactions (Fig. 6). The signal at δ 2.91 in the 1 H NMR spectrum of the compound 1 was unambiguously assigned to the C6–H function. It showed cross peak at δ 1.9 (C7–H) in the 2D spectrum. The C3–αH proton coupled to C2–H and C4–H, thus showed cross peaks at δ 1.5, 1.9 (C2–αH, C2–βH) and at δ 2.1, 2.4 (C4–αH, C4–βH). We determined the stereochemistry of epoxycholestanes through NOESY NMR experiments and observed potential interactions between hydrogen atoms from ring-A and ring-B (Fig. 7). The hydrogen H6 carried by C6 of compound 1, gave NOESY signals showing the interactions (spatial H–H correlations) with C19–H, indicating that both are on the same face (β-face) of the steroid ring-A. The other 1,3 interaction between the hydrogen at C-6 and the β-hydrogen at C-8 confirmed this assignment. Moreover, C6–H does not show any interaction with C3–αH, thus strongly supporting its β-nature and αorientation of oxirane ring at 5,6 position. These observations highlighted a similar stereochemistry to that observed in the crystal structure. The 1H NMR spectra of compound 2 showed diagnostic signals (Supporting information Fig. S3). A doublet at 2.88 (J ¼4.36 Hz), was assigned to C6–βH while a broad multiplet at 4.94 ppm was attributed to C3–αH. The 2D 1H–1H cosy spectra (Supporting information Fig. S4) of compound 2 also showed distinct 1H–1H interactions. C6–βH showed cross peak at δ 1.9 ppm (C7–H). NOESY spectra also showed spatial H–H

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Fig. 3. Crystallographic structure of 3β-Chloro-5α,6α-epoxycholestane (1): (a) A perspective view ellipsoid (50% Probability) and (b) 2D-view.

Fig. 4. Crystallographic structure of 3β-Acetoxy-5α,6α-epoxycholestane (2): (a) A perspective view ellipsoid (50% Probability) and (b) 2D-view.

correlations (Supporting information Fig. S5). Fig. 8 shows significant C6–H interactions with C8–H and C19–H in compounds 1 and 2, supporting the α–nature of oxirane ring. 13C NMR spectra of

compounds (Supporting information Fig. S6 and S7) were also found to be in good agreement to the proposed structure. The signals in the 13C NMR of compound 1 at 65.8 and 50.0 ppm

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Table 2 Comparison of torsion angles of epoxycholestanes in the solid state and in solution. For solid state data, angles were measured from CIF files. 3JH–H were determined from 1H NMR analysis of the molecule in CDCl3, and angles were calculated using the on-line interactive data table. When 3JH–H corresponded to two or more possible angles, the closest one (between  90 and þ 901 from the solid state angle) was chosen. Torsion angle

Compound 1 C4–C3–C2–C1 C3–C2–C1–C17 C5–C6–C7–C8 C6–C7–C8–C16 H1–C1–C17–H17α H1–C1–C17–H17β H17α–C17–C16–H16 H17β–C17–C16–H16 Compound 2 C3–C4–C5–C9 C4–C5–C9–C10 C8–C7–C6–C12 C7–C6–C12–C11 H9–C9–C10–H10α H9–C9–C10–H10β H10α–C10–C11–H11 H10β– C10–C11–H11

Measured angle Solid state

3 JH–H (Hz)

Calculated angle Solution

 145.0 (2)  158.9 (2) 168.3 (2)  176.29 (19) 87.6  28.85  147.4  31.1

0 4.36 9.35 5.90

79.54  42.36  148  31.49

140.1 (2) 160.1 (3)  171.2 (2) 171.5 (2)  93.6 22.8 155.8 39.3

0 4.36 9.34 5.91

 99.88 42.36 148 31.42

corresponded to (C-5) and (C-3) respectively while compound 2 showed peaks at 170.2 (CQO), 71.3 (C–O) and 65.2 ppm (C-5). 4.4. Phase identification X-ray powder diffraction (PXRD) The powder forms of the grown crystals (1 and 2) were subjected to powder X-ray diffraction analysis. The appearances of sharp and strong peaks confirmed the good crystallinity of the grown crystals (Supporting information S8 and S9). The lattice parameters of crystals were calculated theoretically using the powder XRD data and were found in good agreement with the values obtained from single crystals. 4.5. Thermal analysis (TG–DTA–DSC) Thermogravimetric/Differential thermal analysis/Differential scanning calorimetry (TG/DTA/DSC) measurements were performed under nitrogen atmosphere to examine the thermal stabilities of the crystalline samples (1 and 2) and to define the conditions for the thermal treatment on it. Melting of the grown crystals had been estimated by differential thermal analysis (DSC). The thermograms observed from simultaneous TG/DTA and DSC are illustrated in Figs. 9 and 10. The TG curve of compound 1 revealed that it is stable up to 250 1C (no weight loss) and does not undergo any phase transition. A single stage weight loss (75.09%) occurred between 306 1C and 561 1C. The disintegration process continued with the confiscation of almost all fragments as gaseous products, leading to the bulk decomposition of the compound before 650 1C since the initial mass of the sample was 9.387 mg and at a temperature of about 650 1C, all the mass was lost and nothing was left as residue. The absence of any weight loss or phase transition around or before its melting point, confirmed the nonexistence of any lattice entrapped solvent or moisture on the grown material. The corresponding DTA curve showed three notable thermal events. The endothermic peak at 93 1C showed melting of the compound. The exothermic peaks at 263 1C and 566 1C depicted the crystallization of some of the phases of the decomposed material. From DSC (Differential scanning calorimetry) curve the melting point in the present investigation was found to be 86 1C. The TG curve of compound 2 revealed that it is stable

Fig. 5. 1H NMR spectra of 3β-chloro-5α,6α-epoxycholestane (1).

Fig. 6. 1H–1H cosy spectrum of 3β-chloro-5α,6α-epoxycholestane (1).

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Fig. 7. NOESY spectrum of 3β-chloro-5α,6α-epoxycholestane (1).

H19 H8

H6

Fig. 9. TG–DTA/DSC curve of 3β-chloro-5α,6α-epoxycholestane.

endotherm was indicative of a solid-state transition for relatively pure material. The physical stability of two crystals was tested against at various relative humidity (RH) and temperature conditions recommended by the ICH guidelines for pharmaceutical stability testing [29] (Supporting information S10). Our observations indicated the two crystals exhibited significant stability to varying temperature and humidity over a period of 13 weeks. Fig. 8. Spatial correlation of C6–H with H8 and H19 in (a) 3β-chloro-5α,6αepoxycholestane (1) and (b) 3β-acetoxy-5α,6α-epoxycholestane (2).

4.6. UV–visible absorption spectra

up to 250 1C (no weight loss) and does not undergo any phase transition. The disintegration process started after 250 1C and continued till complete decomposition and at a temperature of about 450 1C, all the mass was lost and nothing was left as residue. The absence of any weight loss or phase transition around or before its melting point, confirmed the nonexistence of any lattice entrapped solvent or moisture on the grown material. The corresponding DTA curve showed two notable endothermic peaks at 122 1C and 368 1C. From DSC curve the melting point of compound 2 in the present investigation was found to be 99 1C. The sharpness of the endothermic peaks observed with DSC show the good degree of crystallinity of the material. No decomposition up to the melting point ensured the high stability of the steroidal epoxides. The sharp

The UV–vis absorption properties of the two compounds (1 and 2) in different solvents with the concentration of 2  10  5 M were shown in Fig. S11 and Table S. Absorption wavelength maxima values of crystals 1 and 2 in different solvents varied from 237 to 282 and 259 to 189 nm while molar extinction coefficient varied from 1.18 to 1.41 (106 M  1 cm  1) and 1.29–145(106 M  1 cm  1) respectively. The magnitude of this shift suggested that the ground state of the molecule was polar [30]. With increasing the polarity of solvents, the absorption peak was hypsochromic, which was because the ground state of compounds was more stabilized in a solvent cage of already partly oriented solvent molecules with stronger polarity. The oscillator strength of this transition was estimated to be varied from 6.54 to 2.29 (compound 1) and 6.67 to 2.41 (compound 2) in different solvents.

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Fig. 12. SEM micrograph of 3β-acetoxy-5α,6α-epoxycholestane (2).

Fig. 10. TG–DTA/DSC curve of 3β-acetoxy-5α,6α-epoxycholestane.

Fig. 13. EDX of (a) 3β-chloro-5α,6α-epoxycholestane and (b) 3β-acetoxy-5α,6αepoxycholestane.

surface followed by few micro crystals on it. The SEM image of compound 2 (Fig. 12) showed the presence of elongated crystals with rough surface. The composition of the grown crystals were determined by using EDX analysis (Fig. 13) which depicted the presence of C, O and Cl in compound 1 and C, O in compound 2. Fig. 11. SEM micrograph of 3β-chloro-5α,6α-epoxycholestane (1).

4.7. Microstructural studies (SEM/EDX) The surface morphology of the compounds 1 and 2 was investigated by scanning electron microscopy (SEM). Figs. 11 and 12 show SEM images recorded on the grown crystal surface. The SEM micrograph of compound 1 (Fig. 11) clearly showed the presence of brick shaped epoxide particles, extending over 135μm in length, with less pronounced edge sharpness and rough

5. Conclusion 3β-chloro and 3β-acetoxy-epoxycholestanes derived from corresponding cholest-5-enes have been characterized by spectral techniques. Their lattice parameters have been determined by single-crystal X-ray diffraction analysis, which confirmed the identity of the synthesized materials. The powder X-ray diffraction patterns (PXRD) of compounds are equivalent to those simulated from single-crystal X-ray data. The TG–DTA studies established that the compounds undergo no phase transition and were found

Shamsuzzaman et al. / Journal of Crystal Growth 384 (2013) 135–143

to be stable up to 250 1C. The sharpness of the endothermic peaks observed with DSC show the good degree of crystallinity of the material. No decomposition up to the melting point ensured the stability of the steroidal epoxides. In addition, the ICH rule has been applied to test the stability of two crystals at various relative humidity (RH) and temperature conditions, which showed significant stability. Acknowledgments The authors would like to thank chairman, Department of Chemistry Aligarh Muslim University, Aligarh for providing necessary research facilities. HK acknowledges UGC, New Delhi India for providing BSR fellowship (R.no. Acad/D-742/MR). YNM thanks the Deanship of Scientific Research at King Saud University for the support. We are also thankful to Dr. M. Poirot (Université de Toulouse, France) and Dr. H. Gornitzka (Université Paul Sabatier, France) for their valuable discussion. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jcrysgro.2013.08.037. References [1] A. Vejux, G. Lizard, Molecular Aspects of Medicine 30 (2009) 153–170. [2] G.J.J. Schroepfer, Physiological Reviews 80 (2000) 361–554. [3] V.M. Olkkonen, R. Hynynen, Molecular Aspects of Medicine 30 (2009) 123–133. [4] A. Radhakrishnan, Y. Ikeda, H.J. Kwon, M.S. Brown, J.L. Goldstein, Proceedings of the National Academy of Sciences of United States of America 104 (2007) 6511–6518. [5] C. Ishimaru, Y. Yonezawa, I. Kuriyama, M. Nishida, H. Yoshida, Y. Mizushina, Lipids 43 (2008) 373–382. [6] A.H. Roffo, American Journal of Cancer 17 (1933) 42–57.

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