(±)-(4bS,8aR,10aS)-10a-Ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahydrophenanthrene-2,6-dicarbonitrile

The anti-inflammatory and cytoprotective tricyclic title compound, C21H18N2O2, also known as TBE-31, crystallizes with two nearly superimposable molecules in the asymmetric unit. In both molecules, the three ring systems conform to an envelope–chair–planar arrangement. The central ring, in a cyclohexane chair conformation, contains an axial ethynyl group that bends slightly off from a nearby axial methyl group because of the 1,3-diaxial repulsion between the two groups. In the crystal, weak C—H⋯N and C—H⋯O interactions form chains along [001].

The anti-inflammatory and cytoprotective tricyclic title compound, C 21 H 18 N 2 O 2 , also known as TBE-31, crystallizes with two nearly superimposable molecules in the asymmetric unit. In both molecules, the three ring systems conform to an envelope-chair-planar arrangement. The central ring, in a cyclohexane chair conformation, contains an axial ethynyl group that bends slightly off from a nearby axial methyl group because of the 1,3-diaxial repulsion between the two groups. In the crystal, weak C-HÁ Á ÁN and C-HÁ Á ÁO interactions form chains along [001].

Experimental
Data collection: CrysAlis PRO (Oxford Diffraction, 2010); cell refinement: CrysAlis RED (Oxford Diffraction, 2010); data reduction: CrysAlis RED; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: WinGX (Farrugia, 1999); software used to prepare material for publication: Mercury (Macrae et al., 2006), PLATON (Spek, 2009) and publCIF (Westrip, 2010 8,4b,7,8,8a,9,10, with nonenolizable cyanoenones in rings A and C is a potential antiinflammatory, growth suppressive, and proapoptotic compound. TBE-31 inhibits nitric oxide (NO) production at low nanomolar concentrations in RAW 264.1 cells and mouse primary macrophages stimulated with IFN-γ (Honda et al., 2007;2011). TBE-31 induces cytoprotective enzymes HO-1 in RAW cells and in mice (Honda et al., 2007) and NQO1 in Hepa1c1c murine hepatoma cells (Honda et al., 2011). Incorporation of small quantities of TBE-31 in the diet robustly induces NQO1 and GST in the liver, skin, and stomach in mice (Dinkova-Kostova et al., 2010). TBE-31 is orally highly active against aflatoxin-induced liver cancer in rats (Liby et al., 2008). The nonenolizable cyanoenone in ring C of TBE-31 is a highly reactive Michael acceptor and the reactivity is higher than that of MCE-1 (Fig. 3a), which has the same structure as that of ring C (Dinkova-Kostova et al., 2010). Moreover, in this series of Michael acceptors, the reactivity is closely related to the biological potency in the bioassays for inhibition of inflammation and carcinogenesis (Zheng et al., 2012). It has been speculated that the reactivity of the cyanoenone in ring C of TBE-31 would be enhanced because the same structure as that of MCE-1 exists in an unusually unsaturated tricyclic ring system containing eight sp 2 carbons. Thus, we have synthesized TBE-31 from cyclohexanone in 14 steps (Honda et al., 2011) and determined the crystal structure of TBE-31. We herein report the crystal structure determination of the title compound, C 21 H 18 N 2 O 2 , TBE-31, and subsequently discuss its reactivity.
This reported crystal structure confirms previous assignments made by NMR spectroscopy (Honda et al., 2007;2011).
Aside from structural confirmation, the X-ray crystal structure of TBE-31 is interesting due to the presence of two independent molecules in the asymmetric unit (Z′ = 2) (Fig. 1). Steiner (2000) reported finding 73% of the organic crystal structures in the Cambridge Structural Database (CSD) with a Z′ = 1 while only 9% have Z′ = 2. Strong intermolecular interactions have been suspect in the phenomenon of Z′ > 1, but Steed concluded that it is not possible to use this information to predict the contents of the asymmetric unit (Steed, 2003). Further analysis of the organic crystal structures in the CSD by Gavezzotti showed that certain space groups show higher frequency of Z′ = 2 (Gavezzotti, 2008). In the space group P1, 25% of all the organic crystal structures analyzed by Gavezzotti have Z′ = 2 (Gavezzotti, 2008). He also found that 64% of all the organic crystal structures with Z′ = 2 contain the ketone functional group. In addition, hydrogen bond accepting nitrogen groups were found in 58% of the Z′ = 2 crystal structures (Gavezzotti, 2008). TBE-31 solves in the P1 space group, possesses the ketone functional group, and displays weak C-H···N and C-H···O intermolecular interactions (Table 1)  The crystal structure of the title compound, TBE-31, is the latest addition to an important class of cyanoenone-based drugs. The only previously reported crystal structure of a cyanoenone-based drug, to our knowledge, is methyl 2cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (bardoxolone methyl, CDDO-Me, (Fig. 3(b)) (Bore et al., 2002), which is in phase 3 clinical trials for the treatment of chronic kidney disease in type 2 diabetic patients. It is essential to further study the role of ring strain in these drugs to understand their chemical reactivity as Michael acceptors because it is closely related to their biological potency (Zheng et al., 2012). Ring A (C6A-C11A or C6B-C11B) assumes a slightly distorted envelope conformation (Q = 0.4295 (2) or 0.433 (2), θ = 124.8 (3)° or 57.7 (3)°, φ = 120.7 (3)° or 304.7 (3)° for the 1 st and 2 nd molecule, respectively) (Cremer & Pople, 1975). A typical envelope conformation in this case would maintain a θ value of 54.7° (or 180° -54.7° = 125.3°) so the distortion is minor. Ring A maintains this conformation due to the rigid Csp 2 hybridization at C7A, C8A, and C9A or C7B, C8B and C9B, respectively. Atoms C6A and C10A or C6B and C10B deviate from the Cremer-Pople plane by 0.204 (2) Å and 0.200 (2) Å or 0.215 (2) Å and 0.185 (2) Å, for the 1 st or 2 nd molecule, respectively. This deviation is most likely influenced by ring B. Ring B (C5A/C6A/C11A-C14A or C5B/C6B/C11B-C14B) is found to be in a slightly distorted cyclohexane chair conformation: Q = 0.5901 (2) or 0.5914 (2), θ = 6.93 (2)° or 173.74 (2)°. A typical θ value for a chair conformtion is 0.00° or 180°. The rigid dienone functionality in ring C (C1A-C5A/C14A or C1B-C5B/C14B) leads to a fully planar geometry (r.m.s.d. = 0.0111 (9) Å or 0.0167 (2) Å for the 1 st and 2 nd molecule). Therefore, in summary, while both rings A and B are distorted from ideal conformations, Ring C, has a very slight deviation from planarity.
Cremer-Pople analysis, therefore, supports the assignments of the ring systems in TBE-31 as envelope-chair-planar. The distortions from the ideal parameters can be attributed to the result of the rigidity of ring C. The X-ray structure reveals that the methyl group at C6 and hydrogen at C11 is trans and that the methyl group at C6 and alkyne group at C14 is cis.
Consequently, two 1,3-diaxial interactions between the methyl group on C6 and ethynyl group on C14 and between the methyl groups on C6 and C10 are observed. In addition, the ethynyl group at C14 bends slightly off from the axial methyl group at C6 because of the 1,3-diaxial repulsion between both groups. These observations contribute to the higher strain seen in TBE-31 as compared to the all-trans perhydrophenanthrene with all chairs, (Marcos et al., 2005). Overall, the Xray structure of TBE-31 indicates that the unusually unsaturated tricyclic ring systems containing eight sp 2 carbons and two 1,3-diaxial interactions, which are closely affected with each other, impose rigid constraints on the conformation of TBE-31. This high strain would increase the reactivity of the nonenolizable cyanoenone in ring C in comparison with that of MCE-1, because MCE-1, which is monocyclic, does not have such strain.

Figure 1
The molecular structure of the title compound with the atom numbering scheme. Displacement ellipsoids are drawn at the 30% probability level. H atoms are presented as small spheres of arbitrary radius.  The packing arrangement of the title compound, C 21 H 18 N 2 O 2 , containing both entaniomers viewed along the a axis.

Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.