Stanozolol Case Study - American Chemical Society

Article pubs.acs.org/crystal

How Cocrystallization Affects Solid-State Tautomerism: Stanozolol Case Study Anael̈ le Tilborg,† Géraldine Springuel,‡ Bernadette Norberg,† Johan Wouters,† and Tom Leyssens*,‡ †

Unité de Chimie Physique Théorique et Structurale, Department of Chemistry, University of Namur, 61, rue de Bruxelles, B-5000 Namur, Belgium ‡ IMCN, MOST, UCL, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium S Supporting Information *

ABSTRACT: Three original cocrystals of stanozolol with monoacidic and diacidic coformers are presented and fully characterized in this study. Powder X-ray diffraction (PXRD) permits cocrystal formation to be highlighted, with the help of liquid-assisted grinding (LAG) from the two starting coformers. Single-crystal X-ray diffraction (SCXRD) gives a detailed structural characterization, which allows comparison with existing structures coming from the Cambridge Structural Database (essentially stanozolol solvates) and determination of structural similarities between the new cocrystal structures and the existing ones. As stanozolol can exist under two tautomeric forms (on its pyrazole moiety), statistical and theoretical studies have been performed in order to better apprehend the potential appearance of one of its tautomers at the cost of the other in crystal structures, and the eventuality of “freezing” the molecule in one of these forms by cocrystallization.

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INTRODUCTION Stanozolol (17β-hydroxy-17α-methylandrostano[3,2-c]pyrazole, IUPAC systematic name: (1S,3aS,3bR,5aS,10aS,10bS, 12aS)-1,10a,12a-trimethyl 1,2,3,3a,3b,4,5,5a,6,7,10,10a,10b,11, 12,12a-hexadecahydrocyclopenta[5,6]naphtho[1,2-f ]indazol-1ol, CAS number: 10418-03-8, MW: 328.49 g/mol) is a therapeutic anabolic steroid used for treatment of anemia, hereditary angioedema, and osteoporosis.1,2 Stanozolol is a direct testosterone derivative and has been abused by several high profile professional athletes.3,4 It has been produced and marketed since 1962 by Winthrop Laboratories, under the trade name Winstrol or Stromba. Two polymorphs and 10 solvates of this molecule have already been reported.5−7 Stanozolol exhibits tautomerism on the unsubstituted pyrazole moiety (Scheme 1). Not only can both tautomers be encountered at the solid state, stanozolol is

furthermore prone to solvation with up to 10 solvates identified by single-crystal X-ray diffraction. So far, whereas the two known polymorphs contain tautomer 2, the solvates predominantly orient stanozolol toward tautomer 2 (nine encounters vs three).6 Considering these observations, we investigated the possibility of steering the tautomeric form of stanozolol using cocrystallization principles.8−10 By addition of a second cocrystallization agent (coformer), we hoped to be able to tune the tautomeric form of stanozolol. Furthermore, newly identified cocrystals of stanozolol could help to improve the poor water-solubility of this compound substantially. Even if cocrystals of steroid molecules have already been studied,11−13 we recently identified the first cocrystal including stanozolol, and to our knowledge no other cocrystals of this compound have been reported so far.

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EXPERIMENTAL SECTION

Materials. Stanozolol was sourced from TCI Europe (>99% chemical purity) and used as received. Coformers used (malonic acid, L-phenyllactic acid (D-form) and 6-hydroxy-2-naphthoic acid, 99% chemical purity) came from Sigma-Aldrich (Steinheim, germany) (Scheme 2). Solvents used (essentially acetonitrile and ethanol from Acros Organic, Geel, Belgium) are commercially available and were used without further purification. Grinding Experiments. Liquid-assisted grinding (LAG)14,15 was performed with a Retsch MM 400 mixer mill, equipped with two grinding jars in which five 2 mL Eppendorf tubes can be installed (with five stainless steel grinding balls of 1 mm diameter).

Scheme 1. Stanozolol, Tautomer 1 (Left), and Tautomer 2 (Right)

Received: March 14, 2014 Revised: May 20, 2014 Published: May 30, 2014 © 2014 American Chemical Society

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dx.doi.org/10.1021/cg500358h | Cryst. Growth Des. 2014, 14, 3408−3422

Crystal Growth & Design

Article

for hydrogen bonds are presented in Table 2. For the three cocrystals studied here, CIF files are provided as Supporting Information. Calculated Morphology. Simulated morphologies have been determined from structural data with the geometric method from Bravais, Friedel, Donnay and Harker (BFDH) included in Mercury v. 3.0 software,17 a comprehensive range of tools for structure visualization and exploration of crystal packing. Simulation of PXRD Diffractograms. Simulated powder patterns and representation of the experimental crystal network were carried out with Mercury v. 3.0 software.17 Quantum Mechanics of Crystal Hydrogen-Bonding Stability and Intramolecular Transition State. To quantify relative energies of the new cocrystalline structures formed, comparison between calculated energies of the cocrystal hydrogen-bonding patterns (for each different hydrogen bond existing in each cocrystal stucture) and isolated hydrogen bond partners were estimated using ab initio DFT calculations (GAUSSIAN program,18 pbepbe/6-311++G(d,p) level of theory). The intramolecular transition-state conformation between stanozolol tautomers has been identified using Gaussian QST3 (Synchronous Transit-Guided Quasi-Newton or STQN) method with a first geometrical guess19,20 on pbepbe/6-311++G(d,p)).

Scheme 2. Malonic Acid (Left, MA), D-Phenyllactic Acid (Middle, PA) and 6-Hydroxy-2-Naphthoic Acid (Right, HNA) with Their Systematic IUPAC Name

Powder X-ray Diffraction (PXRD). Powder X-ray diffraction data were collected on a PANalytical Bragg−Brentano−geometry diffractometer, using Ni-filtered Cu Kα radiation (λ = 1.54179 Å) at 40 kV and 40 mA with a X′Celerator detector. Each sample was analyzed between 4 and 50° in 2θ with a step size of ca. 0.0167° and a total scan time of 3 min 48 s. Single-Crystal X-ray Diffraction (SCXRD). Single crystal X-ray diffraction was performed on a Gemini Ultra R system (4-circle kappa platform, Ruby CCD detector) using Mo Kα (λ = 0.71073 Å) radiation. Selected crystals were mounted on a quartz needle using commercial glue (cyanoacrylate). Cell parameters were estimated from a preexperiment run, and full data sets were collected at room temperature (293 K). Structures were solved by direct methods with the SHELXS-97 program and then refined on F2 using SHELXL-97 software.16 Nonhydrogen atoms were anisotropically refined, and the hydrogen atoms (not present in H-bonds) were fixed in the riding mode with isotropic temperature factors fixed at 1.2 times U(eq) of the parent atoms (1.5 times for methyl groups). Hydrogen atoms implicated in hydrogen bonds were localized by Fourier difference maps (ΔF), except for cocrystal structure stanozolol/PA/CAN where hydrogen atom positions have been calculated. CCDC 988950, 988951, and 989733 entries contain the supplementary crystallographic data for this paper and can be obtained free of charge via www.ccdc.cam.ac.uk/conts/ retrieving.html (or from the Cambridge Crystallographic Data Center, 12, Union Road, Cambridge CB21EZ, UK; fax: +44−1223−336033; or [email protected]. Details of data collection and structure refinement are listed in Table 1, and selected geometrical parameters

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RESULTS AND DISCUSSION

Preparation of Solid Phases. Cocrystal of Stanozolol and Malonic acid (MA) (1:1). A powder sample of the stanozolol/ malonic acid cocrystal was produced by LAG (49.5 mg of stanozolol (0.15 mmol) and 15.84 mg of malonic acid (0.15 mmol), 10 μL of EtOH) with an optimized grinding time of 90′, followed by PXRD analysis. Figure 1 presents powder patterns of stanozolol and malonic acid before the experiment, the pattern of the ground sample (cocrystal), and the simulated pattern coming from the SCXRD data (see below). Essentially pure cocrystalline product is formed through grinding, without visible traces of the starting coformers. Although other techniques such as TeraHertz spectroscopy21,22 can provide further information about the cocrystal formation process during grinding, PXRD remains

Table 1. Crystallographic Data, Data Collection and Structure Refinement Details

crystal data

collection data

refinement

empirical formula fw crystal system space group a, b, c (Å)

α, β, γ (deg) V (Å3) Z ρcalcd (g/cm3) Mu (Mo Kα) (/mm) F(000) crystal size (mm) T (K) radiation (Å) θ min and max (deg) tot., uniq., R (int) obs. data R[I > 2σ(I)] wR2 [all] GOF residual density

1:1 Stan - MA

2:1:1 Stan - PA - ACN

1:1 Stan - HNA

C21H32N2O, C3H4O4 432.55 monoclinic P21 (No. 4) 10.6644(7) 7.2335(5) 15.094(1) 90, 106.016(7), 90 1119.2(1) 2 1.283 0.089 468 0.10/0.22/0.32 293(2) Mo Kα: 0.710 73 3.50−29.40 6073, 4372, 0.019 3899 0.0457 0.1118 1.02 −0.22, 0.24

2(C21H32N2O), C9H10O3, C2H3N 864.20 monoclinic C2 (No. 5) 58.610(1) 7.5624(2) 10.7139(4) 90, 97.989(2), 90 4702.6(2) 4 1.221 0.612 1880 0.10/0.15/0.45 293(2) Cu Kα: 1.541 84 3.00−66.60 18654, 6280, 0.097 5357 0.0838 0.2264 1.04 −0.34, 0.35

C21H32N2O, C11H8O3 516.66 triclinic P1 (No. 1) 7.9530(6) 8.6403(7) 10.4582(7) 83.337(6), 86.973(6), 75.431(7) 690.6(9) 1 1.242 0.081 278 0.08/0.18/0.53 293(2) Mo Kα: 0.710 73 3.30−25.50 8869, 4457, 0.032 3966 0.0465 0.1073 1.03 −0.18, 0.22

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Table 2. Geometrical Parameters (Distance and Angle) of Hydrogen Bonds in the Cocrystals Structures20 H-bonds

D···A distance (Å)

N1--H1N···O2 O1--H1O···O5 O3−H3O···O1 O5−H5O···N2

2.795(3) 2.967(3) 2.631(3) 2.692(3)

O1--H1O···O2 N2--H2···O4 O2--H2O···N97 N3−H3···O3 N3−H3···O5 O3−H3O···N1 O4−H4O···N4

2.758(5) 2.917(5) 2.808(9) 2.900(4) 3.226(5) 2.786(5) 2.603(5)

O1--H1O···O2 N2--H1N···O1 O2−H2O···O4 O3−H3O···N1

2.937(4) 2.859(3) 2.712(3) 2.641(3)

H···A distance (Å) 1:1 Stan/MA 1.94(2) 2.28(3) 1.75(4) 1.76(3) 2:1:1 Stan/PA/ACN 1.98 2.11 2.19 2.10 2.54 2.05 1.77 1:1 Stan/HNA 2.10(4) 2.03(3) 1.89(4) 1.84(1)

D−H···A angle (deg) 173(3) 150(3) 175(3) 177(3) 154 157 129 155 137 158 163 167(4) 158(3) 172(3) 165(3)

symmetry code 1−x, −3/2+y, 1−z −x, −1/2+y, −z −x, 3/2+y, −z 1−x, 1/2+y, 1−z 1/2−x, −1/2+y, 1−z −x, −1/2+y, 1−z

−x, y, 1−z x, 1+y, z x, −1+y, 1+z 1+x, y, −1+z x, y, −1+z −1+x, y, 1+z

Figure 1. PXRD patterns of malonic acid (blue), the 90 min LAG-ground sample (pink dotted), the SCXRD powder pattern simulation (green dotted), and stanozolol (red dotted).

phenyllactic acid (0.19 mmol), 10 μL of EtOH) with a grinding time of 90′, followed by PXRD (Figure S1, Supporting Information). Crystallization trials were built up by slow evaporation of acetonitrile saturated solutions of the ground product after grinding. Needle-like single crystals suitable for single-crystal X-ray analysis were obtained after several days. Cocrystal of Stanozolol and 6-Hydroxy-2-Naphthoic Acid (HNA) (1:1). Powder samples of stanozolol/6-hydroxy-2naphthoic acid were produced by LAG (49.8 mg of stanozolol (0.15 mmol) and 28.7 mg of 6-hydroxy-2-naphthoic acid (0.15

the fastest and the most-employed method for cocrystal detection. Crystallization trials were built up by slow evaporation of acetonitrile saturated solutions of the ground product after grinding. Needle-like single crystals suitable for single-crystal Xray analysis were obtained after 3 days. Solvated Cocrystal (Acetonitrile) of Stanozolol and DPhenyllactic Acid (PA) (1:2:1). Powder samples of the stanozolol/D-phenyllactic acid cocrystal were synthesized by LAG (63.75 mg of stanozolol (0.19 mmol) and 32.13 mg of D3410



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Figure 2. Crystal conformation (ORTEP diagram) drawn with 50% probability ellipsoids of the cocrystals of (a) stanozolol/malonic acid [MA], (b) stanozolol/D-phenyllactic acid [PA], and (c) stanozolol/6-hydroxy-2-naphthoic acid [HNA]. Numbers in bold in (a), (b), and (c) are references for C− O bond lengths: 1 = 1.203(3) Å; 2 = 1.307(3) Å; 3 = 1.193(4) Å; 4 = 1.306(3) Å; 5 = 1.320(5) Å; 6 = 1.205(5) Å; 7 = 1.202(4) Å; 8 = 1.309(4) Å.

mmol), 10 μL of EtOH) with 90′ grinding time, and the ground product was analyzed through PXRD (Figure S2, Supporting Information). Crystallization trials were put up by slow evaporation of saturated acetonitrile solutions after LAG experiment. Block-like single crystals suitable for single-crystal X-ray analysis were obtained after 5 days. Structure Determination. Single-crystal XRD analysis was implemented on single crystals obtained by recrystallization from acetonitrile saturated solutions of ground samples from stanozolol/malonic acid, stanozolol/D-phenyllactic acid, and stanozolol/6-hydroxy-2-naphthoic acid cocrystals. Principal crystallographic data are provided in Table 1. The structural study reveals that cocrystals (not salts) have actually been formed because the structures contain protonated malonic acid (MA) and 6-hydroxy-2-naphthoic acid (HNA) in a 1:1 stoichiometry ratio and protonated D-phenyllactic acid (PA) in a 2:1 stoichiometry ratio. Protonation states within the malonic acid, the naphthoic acid, and the D-phenyllactic acid have been unambiguously determined by detailed investigation of Fourier difference maps and on the basis of geometrical features (C−O bond lengths, Figure 2). In the structure of the stanozolol/MA cocrystal, the hydrogenbonding network is composed of four types of hydrogen bonds, all between molecules of stanozolol and malonic acid (no hydrogen bonds between identical molecules are observed). Table 2 presents the geometrical parameters associated with these hydrogen bonds, and Figure 3 shows the cocrystalline network, highlighting malonic acid molecules in red. This figure

clearly proves the absence of interaction between stanozolol or malonic acid molecules themselves. The network can be seen as a two-dimensional one: layers of stanozolol and malonic acid are piled up, but no hydrogen bonding interactions occur between layers. The crystal structure of the stanozolol/HNA cocrystal is organized through a three-dimensional lattice, with four types of hydrogen bonding interaction between naphthoic acid and stanozolol moieties but also between stanozolol molecules, in contrast to the stanozolol/MA cocrystal (Table 2). In Figure 3, it can be observed that the cocrystal lattice consists essentially of rows of stanozolol and naphthoic acid linked by hydrogen bonds in the three directions of space. For the cocrystal structure of stanozolol/PA with acetonitrile, seven different hydrogen bonding patterns are present in the crystalline network. Once more, no interactions occur between molecules of similar nature (no hydrogen bonds between the same molecules, as for stanozolol/MA cocrystal) (Table 2). In this cocrystal, a 3D hydrogen bonding network is observed: hydrogen-bonding interactions between different layers of stanozolol and D-phenyllactic acid or acetonitrile molecules are present, and CH2−π interactions between D-phenyllactic acid entities of different layers can also be observed (Figure 3). First-level graph sets for the stanozolol/MA (Figure 4a) and stanozolol/PA (Figure 4b) cocrystals are of the type D11(2) finite elements. For stanozolol/HNA acid cocrystal, C11(10) and C11(12) chain elements are also present (Figure 4c). Secondlevel graph-sets consist principally of infinite C22(18) or C22(8) 3411



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Figure 3. (a, b) Illustration of the hydrogen bonding network within the single crystal structure of (a) stanozolol/MA cocrystal and (b) stanozolol/PA cocrystal. Two representations are given for each molecular packing: (a) On the left, the packing organization with hydrogen bonds highlighted and on the right, malonic acid molecules shown in space-filling models and (b) On top, the packing organization with hydrogen bonds highlighted and on the bottom, D-phenyllactic acid molecules (red) and acetonitrile molecules (blue) shown in space-filling models. (c) Illustration of the hydrogen bonding network within the single crystal structure of stanozolol/HNA cocrystal. Two representations are given for each molecular packing: On top, the packing organization with hydrogen bonds highlighted and on the bottom, 6-hydroxy-naphthoic acid molecules (red) shown in space-filling model.

chains for the stanozolol/MA cocrystal and stanozolol/PA cocrystal, whereas D33(13, 16, or 17) finite elements are present for stanozolol/HNA cocrystal. In comparison with existing structures including stanozolol (essentially solvates), this type of structural organization is quite common.6 Particular to cocrystals is the fact that hydrogen bonds only occur between different kinds of molecules. Furthermore, each stanozolol molecule sees

all potential hydrogen-bond donor and acceptor moieties used, in contrast to the polymorph I structure.6 This is in concordance with the first rule of Etter’s hydrogen-bonding statements: All the good proton donors and acceptors are used in hydrogen bonds.23,24,25 Structural Comparison with Reported Structures involving Stanozolol. Each structure involving stanozolol 3412



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Figure 4. Selected first-level and second-level graph-set patterns for the (a) stanozolol/MA cocrystal, (b) stanozolol/PA cocrystal, (c) stanozolol/HNA cocrystal.

(already available in CSD and described in this work, Table 3) seems to present a common cell parameter (close to 10 Å), with two exceptions: solvates with isopropanol and 2-butanol. A

careful inspection of the network organization of each structure does not indicate a particular common symmetry element. The two solvates (with isopropanol and 2-butanol) are monoclinic, 3413



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Table 3. Main Characteristics and Differences between Solvates Structures from CSD and ref Cocrystals from ref 7 and from the work Discussed Here structure name (CSD refcode)

formula

space group

cocrystal from the work

C21H32N2O, C3H4O4

P21


2(C21H32N2O), C9H10O3, C2H3N

C2

lattice parameters (Å)

lattice parameters (deg)

a 10.6644(7) b 7.2335(5) c 15.095(1) a 58.610(1)

α 90.00 β 106.016(7) γ 90.00 α 90.00 β 97.989(2) γ 90.00 α 83.337(6) β 86.973(6) γ 75.431(7) α 90.00


C21H32N2O, C11H8O3

P1

cocrystal from ref 7, methylsuccinic acid (1:1)

C21H32N2O, C5H8O4

P21

b 7.5624(2) c 10.7139(4) a 7.9530(6) b 8.6403(7) c 10.4582(7) a 6.8807(1)

P212121

b 11.0656(2) c 16.4085(3) a 7.26590(10)

β 96.713(2) γ 90.00 α 90.00

b 10.3858(3) c 42.1865(10) a 7.3164(4) b 21.562(2) c 24.006(1) a 19.4540(7) b 18.7009(8) c 10.8348(4) a 7.1392(5) b 10.9541(8) c 26.8891(16) a 24.545(1) b 7.2894(3) c 15.2500(8) a 25.321(2) b 7.421(2) c 15.393(2) a 7.3934(2) b 27.5812(6) c 10.5964(3) a 10.5093(3) b 7.2906(2) c 15.5622(5) a 7.2098(5) b 10.4654(7) c 26.031(1) a 10.3775(8) b 7.4347(6) c 15.5801(8) a 7.5672(3) b 19.6475(9) c 26.3375(9) a 7.233(2) b 11.850(2) c 48.672(2) a 10.424(1) b 7.4432(5) c 27.018(2)

β 90.00 γ 90.00 α 90.00 β 90.00 γ 90.00 α 90.00 β 90.00 γ 90.00 α 90.00 β 90.00 γ 90.00 α 90.00 β 122.679(7) γ 90.00 α 90.00 β 123.40 γ 90.00 α 90.00 β 109.234(3) γ 90.00 α 90.00 β 94.812(3) γ 90.00 α 90.00 β 90.00 γ 90.00 α 90.00 β 91.895(6) γ 90.00 α 90.00 β 90.00 γ 90.00 α 90.00 β 90.00 γ 90.00 α 90.00 β 94.069(9) γ 90.00

cocrystal from ref 7, methylsuccinic acid (1:2)

C21H32N2O, 2(C5H8O4)

tautomer A6

C21H32N2O

P212121

tautomer B6

C21H32N2O

P21212

EtOH solvate (1:1)5,6

C21H32N2O, C2H6O

P212121

iPrOH solvate (1:1)6

C21H32N2O, C3H8O

C2

2-ButOH solvate (1:1)6

C21H32N2O, C4H10O

C2

EtOH, H2O solvate (2:1:1)6

2(C21H32N2O), C2H6O, H2O

P21

iPrOH, H2O solvate (1:1:2)6

C21H32N2O, C3H8O, 2(H2O)

P21

monohydrate (1:1)6

C21H32N2O, H2O

P212121

acetic acid solvate (1:2)6

C21H32N2O, 2(C2H4O2)

P21

formamide solvate (2:1)6

2(C21H32N2O), CH3NO

P212121

pyridine solvate (2:1)6

2(C21H32N2O), C5H5N

P212121

dimethylformamide, H2O solvate (2:1:1)6

2(C21H32N2O), C3H7NO, H2O

P21

belonging to the C2 space group. The solvated cocrystal of stanozolol and D-phenyllactic acid found in this work also belongs to this space group but does not present the same lattice arrangement.

cell volume

Z

R (%)

1119.2(1)

2

4.57

4702.6(2)

4

8.38

690.64(9)

1

4.65

1240.76

2

2.98

3183.49

4

3.23

3787.06

4

3.80

3941.78

8

4.87

2102.82

4

5.50

2296.61

4

3.18

2414.76

4

5.03

2040.19

4

3.27

1188.16

2

3.32

1964.15

4

3.72

1201.4

2

5.04

3915.77

8

4.76

4171.73

8

6.79

2091.05

4

4.93

In fact, several kinds of networks can appear in stanozolol cocrystals (Figure 5): they can either exhibit two-dimensional networks, as for stanozolol/MA and stanozolol/methylsuccinic acid (1:1) cocrystals,7 essentially consisting of simple 2D layers, 3414



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Figure 5. Network organization for (a) stanozolol/MA cocrystal, with 2D layer structure, (b) the stanozolol/methylsuccinic acid (1:1) cocrystal,7 also with 2D layer arrangement, (c) the stanozolol/methylsuccinic acid (1:2) cocrystal,7 with ≪ triple-ranks ≫ 2D layer structure, (d) the stanozolol/PA cocrystal, with two-dimensional layers linked between them through acetonitrile molecules, and (e) the stanozolol/HNA cocrystal, with a threedimensional lattice consisting of stanozolol and naphthoic acid rows interconnected.

molecules, these latter showing the same orientation in the same layer but with an inverted direction between each layer (Figure 5). A 3D structure can also appear in stanozolol cocrystals as illustrated by the stanozolol/PA cocrystal and the stanozolol/ HNA cocrystal. The stanozolol/PA cocrystal shows 2D layers parallel to the crystallographic ac-plane but presents hydrogen bonding interactions between layers, essentially between acetonitrile moieties. There are no hydrogen bonds between stanozolol molecules, but CH2−π interactions stabilize Dphenyllactic acid molecules from different layers (Figure 6)23. The stanozolol/HNA cocrystal presents a three-dimensional network with rows of stanozolol and rows of naphthoic acid connected by hydrogen bonds between each entity, parallel to the crystallographic a axis, and linked by hydrogen bond interactions between the two different kinds of molecules (Figure 5). In the case of the stanozolol/methylsuccinic acid (1:2) cocrystal,7 the structure exhibits layers with ≪ triple ≫ ranks parallel to the crystallographic ac-plane: one formed by methylsuccinic acid molecules, one composed of stanozolol molecules, and the last one consisting of methylsuccinic acid molecules. There is no interaction between these layers

Figure 6. Details of the stanozolol/PA cocrystal structure, highlighting the CH2−π interaction (length = 2.982 Å) between D-phenyllactic acid moieties.26

with a 45° tilting from the crystallographic bc-plane for the stanozolol/MA cocrystal, and double 2D-layers parallel to the crystallographic ac-plane for the stanozolol/methylsuccinic acid cocrystal. No hydrogen bonds are present between stanozolol 3415



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Figure 7. Network organization for (a) stanozolol monohydrate, (b) stanozolol isopropanol and H2O solvate, (c) stanozolol isopropanol solvate, and (d) stanozolol polymorph I.6

(principally sterical arrangement), and there are no hydrogen bonds between stanozolol molecules. Comparing other stanozolol involving CSD structures, essentially solvates and the two polymorphs of the title compound, we observe the same kind of arrangement. In the case of stanozolol monohydrate (Figure 7),6 2D layers are connected through hydrogen bonding interactions between water molecules, and hydrogen bonds are present between stanozolol molecules, these latter being in the same orientation in each layer but in an inverted direction between different layers. For the isopropanol and H2O solvate (Figure 7)6, double 2D layers are present, with hydrogen bonding interactions between water molecules in the same double layers, but no interactions between different double layers. Stanozolol molecules are in the

same direction in the upper part of the layer and in an inverted orientation in the lower part, without hydrogen bonds between stanozolol molecules. For the isopropanol solvate (Figure 7),6 the structure is essentially organized in two dimensions: layers of stanozolol and isopropanol moieties (45° tilted from the crystallographic bcplane) without hydrogen bonding interactions between them, and stanozolol moieties again in the same orientation in each layer but in an inverted configuration between differents layers. There are hydrogen bonds between stanozolol molecules in this case. For the stanozolol polymorph I structure (Figure 7),6 stanozolol moieties constitute a three-dimensional network, with staggered rows of molecules possessing hydrogen bond interactions between each row. 3416



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Table 4. Calculated Density for Stanozolol Cocrystal, Solvates/Polymorphic Forms6 structure name (CSD refcode) cocrystal from the work (MA) cocrystal from the work (PA) cocrystal from the work (HNA) cocrystal from ref 7 cocrystal from ref 7 Polymorph I6 Polymorph II6 EtOH solvate (1:1)5,6 IprOH solvate (1:1)6

formula

calculated density (g/cm3)

structure name (CSD refcode)

formula

calculated density (g/cm3)

C21H32N2O, C3H4O4

1.283

2-butOH solvate (1:1)6

C21H32N2O, C4H10O

1.107

2(C21H32N2O), C9H10O3, C2H3N C21H32N2O, C11H8O3

1.221

EtOH, H2O solvate (2:1:1)6

1.203

1.242

IprOH, H2O solvate (1:1:2)6

C21H32N2O, C5H8O4 C21H32N2O, 2(C5H8O4) C21H32N2O C21H32N2O C21H32N2O, C2H6O

1.233 1.237 1.152 1.107 1.183

monohydrate (1:1)6 acetic acid solvate (1:2)6 formamide solvate (2:1)6 pyridine solvate (2:1)6 dimethylformamide, H2O solvate (2:1:1)6

2(C21H32N2O), C2H6O, H2O C21H32N2O, C3H8O, 2(H2O) C21H32N2O, H2O C21H32N2O, 2(C2H4O2) 2(C21H32N2O), CH3NO 2(C21H32N2O), C5H5N 2(C21H32N2O), C3H7NO, H2O

C21H32N2O, C3H8O

1.124

1.187 1.172 1.240 1.186 1.172 1.188

Figure 8. Comparison between the calculated and the experimental morphologies for (a) stanozolol/MA, (b) stanozolol/PA, and (c) stanozolol/HNA cocrystals.

Calculated densities for each cocrystal structure, solvate, and polymorphic form are listed in Table 4. This table shows that cocrystals exhibit a slightly denser crystalline network compared to stanozolol solvates (and the two polymorphs). The hydrogenbonding network shaping the cocrystal structures and the greater potential of forming new interactions when a coformer is implicated in the network potentially explains this observation, but the hydrogen bond network cannot be considered as the

Table 5. Distribution of Tautomers for Structures Including Stanozolol in refs 6 and 7 and in This Worka tautomer

polymorphs

solvates

cocrystals

total

1 2

1 2

9 3

3 2

13 7

a

In total: 2 polymorphs, 10 solvates and 5 cocrystals with stanozolol were taken into account. 3417



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Figure 9. (a) Energy chart for the concerted bimolecular transition state for stanozolol, calculated with the STQN GAUSSIAN method and (b) theoretical conformation for the two tautomers and the transition state after geometrical optimization.

stanozolol tautomer developed structures). Tautomer 1 is the predominant one, but the two tautomers can appear in all solid forms. In fact, tautomer 1 is present in the structures of polymorph I, solvate with EtOH (1:1), iPrOH (1:1), 2-butanol (1:1), solvate with EtOH and H2O (2:1:1), iPrOH and H2O (1:1:2), acetic acid (1:2), formamide (2:1), pyridine (2:1), DMF and H2O (2:1:1), and also in the structures of cocrystals with methylsuccinic acid (1:2), methylsuccinic acid (1:1), and malonic acid (MA) (1:1). On the other hand, tautomer 2 is present in the structures of polymorph II but also in polymorph I, presenting the two different tautomers in the asymmetric unit. Tautomer 2 is also present in the structures of the mixed solvate with EtOH and H2O (2:1:1) (same case as for polymorph I),

unique driving force to a dense crystalline network: other effects like geometric fit and sterical hindrance should also be taken into account.27−29 Simulated crystal morphologies (BFDH model30) for the stanozolol/MA, stanozolol/HNA, and stanozolol/PA cocrystals are represented in Figure 8 and compared with morphologies of the experimental single crystals which have been analyzed. The prediction for the shape-based on BFDH model reproduces the morphologies of crystals of stanozolol/MA cocrystal, stanozolol/ HNA cocrystal, and stanozolol/PA cocrystals fairly well. Theoretical Study of Stanozolol Tautomerism. A statistical distribution of the two potential tautomer forms for stanozolol is presented in Table 5 (see Scheme 1 for the two 3418



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Figure 10. Intrinsic reaction coordinate path (IRC) calculated for intramolecular transition state of stanozolol.

H2O (1:1), DMF and H2O (2:1:1) (same case as for polymorph I) and in the structures of cocrystals with D-phenyllactic acid (PA) and 6-hydroxy-2-naphthoic acid (HNA). Table 5 summarizes the tautomer distribution. Theoretical energy calculations have been carried out in the gas phase for the two tautomers, using the GAUSSIAN09 program10 (pbepbe functional, 6-311++G(d,p) basis set), and the energy difference found between the two forms is around 1 kJ/mol in favor of tautomer 2, a value consistent with the one calculated in earlier work.6 We furthermore decided to quantify the barrier for tautomer interconversion. The synchronous transit-guided quasi-Newton (STQN) theoretical method19,20 has been used in order to approach the quadratic region near the transition state and to optimize the transition state conformation. A geometrical guess has to be provided for this calculation, and we have selected an intramolecular transition state conformation, in accordance with theoretical data for pyrazole derivatives.31 The geometrical guess has been provided by manually lengthening covalent bonds between hydrogen and nitrogen atoms and bending the valence bond angle between the hydrogen atom and the two nitrogen atoms. The resulting energy chart for this STQN calculation is provided in Figure 9, with a difference in energy for the

intramolecular transition state of 187.2 kJ/mol in comparison with the lowest energy value for tautomer 2. Distances between the hydrogen moiety and the two nitrogen atoms from the pyrazole moiety are of 1.246 and 1.251 Å, respectively, and the valence bond angle value between these three partners is 72.6°. One imaginary frequency has been calculated for this transition state (−1578 cm−1, corresponding to the single proton transfer), which confirms the validity of the starting geometrical guess considered for this calculation. The intrinsic reaction coordinate path (IRC) has also been determined for the intramolecular transition state considered here (Figure 10). In cocrystals, even if the two tautomers exist among the different structures, only one tautomer form is present in each structure, in contrast to the situation for solvates and polymorphs of stanozolol, which simultaneously possess the two tautomers in the crystalline lattice for several structures (see Table 5). Cocrystallization could be considered as a ≪ tautomer form blocker ≫ allowing fixing of the solid state in one conformation to the detriment of the other one. In fact, the presence of a cocrystal former in the crystal lattice, that is to say another molecule possessing several hydrogen bonding potentialities (more so than in the case of a solvent molecule), seems to block by hydrogen bond implication the pyrazole moiety of the 3419



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stanozolol molecule (see crystalline structures) and prevent a possible intramolecular tautomer transition. To investigate this further, future calculations of transition state taking into account several molecules (not only stanozolol, but also solvent molecules or cocrystal formers for example) are being performed. On a larger scale, comparison between cocrystal structures and well-claimed Z′ > 1 structures32 might be performed, in order to justify a potential tautomer transition at the solid-state. To the best of our knowledge, no analogy has been drawn between cocrystal structures and Z′ > 1 structures,32 presenting nonconventional crystallographic symmetry operations. This new observation could lead to a better understanding of cocrystal formation and reactivity at the solid-state for several cases of cocrystallization study. Theoretical Simulations of Hydrogen-Bonding Interactions for Stanozolol Cocrystals. A geometrical optimization and energy determination has been carried out on each pair of molecules linked by hydrogen bonds present in the three cocrystal structures including stanozolol in this work, in order to evaluate the potential importance of some interactions over others. The GAUSSIAN program18 with DFT method (pbepbe functional, 6-311++G(d,p) basis set) has been used, and stabilizing energies are summarized in Table 6. It can be noticed

Table 6. Difference Energy Values for Hydrogen Bond Pairs in Stanozolol Cocrystals from This Work ΔE value (kJ/mol) H-bonds

(E(stan+coformer) − E(stan) − E(coformer)) 1:1 Stanozolol/MA −29.94 - 30.61 −26.11 −33.12

O1--H1O···O5 (a) N1--H1N···O2 O3−H3O···O1 O5−H5O···N2 2:1:1 Stanozolol/PA/ACN

−22.65 −22.75 −71.01 −10.56 −72.97 −50.23 −47.85

O1--H1O···O2 (a) N2--H2···O4 O2--H2O···N97 N3−H3···O3 N3−H3···O5 O3−H3O···N1 O4−H4O···N4 1:1 Stanozolol/HNA O1--H1O···O2 (d) N2---H1N···O1 O2−H2O···O4 O3−H3O···N1

−14.90 −17.22 −23.47 −25.87

Figure 11. (a) Starting geometry and optimized geometry for hydrogen bond (a) (numbering from graph-set, see Table 6) in stanozolol/MA cocrystal, (b) starting geometry and optimized geometry for hydrogen bond (a) (numbering from graph-set, see Table 6) in stanozolol/PA cocrystal, and (c) starting geometry and optimized geometry for hydrogen bond (b) (numbering from graph-set, see Table 6) in stanozolol/HNA cocrystal. 3420


Crystal Growth & Design that all values are of the same order of magnitude, with respect to the partners implicated in each bond. In Figure 11, examples from starting geometries (directly isolated from the crystal structure) and resulting geometry after optimization are represented. The DFT calculation method seems to correctly mimic the conformation of the partners in the highlighted hydrogen bonding interaction and reproduce adequately the length of a hydrogen bond between entities in the cocrystal structures.

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REFERENCES

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CONCLUSIONS Stanozolol, a therapeutic anabolic steroid, has been investigated in the context of cocrystallization with malonic acid, Dphenyllactic acid, and 6-hydroxy-2-naphthoic acid in order to study the eventuality of “freezing” stanozolol in one of its tautomeric forms. Single-crystal X-ray diffraction provides detailed insight in the crystal structure of cocrystals formed and is compared with existing solid-state data (polymorphs, solvates, and other cocrystals) to establish a first statistical tautomer distribution in the different solid forms. Stanozolol presents a lot of solvated structures, but up to now, no salt form has been identified. Cocrystal formation seems an interesting approach on this pharmaceutical molecule. The two tautomers appear in each possible solid-state (true polymorphs, solvates, or cocrystals) which is likely explained by the small stabilization energy difference between the two forms. Possibly this can be also linked to the potential belonging of cocrystals to a well-debated category of solid states compounds, Z′ > 1 structures, which possess interactions that do not conform to classical symmetry operations. In the case of stanozolol cocrystals, only one tautomer is present in each crystal structure, whereas the two tautomer forms can simultaneously appear in solvates or in the true polymorphs. Cocrystallization therefore seems to orient the tautomerism, although further examples are required to statistically confirm this tendency. Theoretical calculations give first clues of an understanding of the increased occurrence of tautomer 1 to the detriment of tautomer 2 in crystal structures and provide quantification for hydrogen bonding interactions present in each stanozolol cocrystal structure. ASSOCIATED CONTENT

S Supporting Information *

Additional Figures S1 and S2 and X-ray crystallographic information files (CIFs) are available for all original compounds in the study. This material is available free of charge via the Internet at http://pubs.acs.org.

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ABBREVIATIONS

SCXRD, single-crystal X-ray diffraction; PXRD, powder X-ray diffraction; DFT, density functional theory; MA, malonic acid; PA, D-phenyllactic acid; HNA, 6-hydroxy-2-naphthoic acid

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AUTHOR INFORMATION

Corresponding Author

*Tel: +32 (0)10 47 28 11. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.T. thanks the FRS-FNRS for its grant and support (PDR postdoctoral research grant). Financial support of the FNRS (Grant No. 2.4511.07) is also acknowledged. 3421



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(27) Temesvári, I.; G. Liptay, G.; Pungor, E. J. Therm. Anal. 1971, 3, 293−295. (28) Takata, N.; Shiraki, K.; Takano, R.; Hayashi, Y.; Terada, K. Cryst. Growth Des. 2008, 8 (8), 3032−3037. (29) Schultheiss, N.; Newman, A. Cryst. Growth Des. 2009, 9 (6), 2950−2967. (30) Donnay, J. D. H.; Harker, D. Am. Mineral. 1937, 22, 446−447. (31) Salimi Beni, A.; Jafari Chermahini, Z. Struct. Chem. 2013, 24, 1713−1723. (32) Steed, J. W. CrystEngComm 2003, 5 (32), 169−179.

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Stanozolol Case Study - American Chemical Society

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