3β-Hydroxylup-20(29)-en-28-yl 1H-imidazole-1-carboxylate

The title triterpene, C34H52N2O3, is a C-28 carbamate derivative of betulin prepared in a one-step reaction from the commercially available 1,1′-carbonyldiimidazole (CDI). All rings are fused trans. The X-ray study shows the retention of the configuration of C-28 with respect to the known chiral centres of the molecule. In the crystal, the molecules are O—H⋯O hydrogen bonded via the hydroxy group and the carbonyl group of the carbamate function into chains running along the c axis. A quantum-mechanical ab initio Roothaan Hartree–Fock calculation of the equilibrium geometry of the isolated molecule gives values for bond-lengths and valency angles close to the experimental values. The calculations also reproduce the molecular conformation well, with calculated puckering parameters that agree well with the observed values.

The title triterpene, C 34 H 52 N 2 O 3 , is a C-28 carbamate derivative of betulin prepared in a one-step reaction from the commercially available 1,1 0 -carbonyldiimidazole (CDI). All rings are fused trans. The X-ray study shows the retention of the configuration of C-28 with respect to the known chiral centres of the molecule. In the crystal, the molecules are O-HÁ Á ÁO hydrogen bonded via the hydroxy group and the carbonyl group of the carbamate function into chains running along the c axis. A quantum-mechanical ab initio Roothaan Hartree-Fock calculation of the equilibrium geometry of the isolated molecule gives values for bond-lengths and valency angles close to the experimental values. The calculations also reproduce the molecular conformation well, with calculated puckering parameters that agree well with the observed values.  (2007). For puckering and asymmetry parameters, see: Cremer & Pople (1975); Duax & Norton (1975). The quantum chemical calculations were performed with the computer program GAMESS (Schmidt et al., 1993 Table 1 Hydrogen-bond geometry (Å , ). 3 -Hydroxylup-20(29)-en-28-yl 1H-imidazole-1-carboxylate R. C. Santos, A. Matos Beja, J. A. R. Salvador and J. A. Paixão

Comment
Cancer is the second most important disease leading to death in both the developing and developed countries nowadays.
Numerous experimental and epidemiological studies have shown that several plant derived natural products may serve as effective anticancer drugs, among which are plant triterpenes (Kinghorn et al., 2004 andSetzer et al., 2003). Betulin and betulinic acid, two pentacyclic lupane triterpenes were reported to display several biological effects including anti-inflammatory, antiviral, antimalarial and in particular anticancer (Dzubak et al., 2006 andTolstikova et al., 2006). The therapeutic characteristics of betulinic acid regarding specificity and mode of action make it a promising anticancer agent presently under evaluation in phase I studies (Petronelli et al., 2009).
As part of our current interest in the synthesis of new triterpenoid derivatives with cytotoxic activity, we have recently reported the synthesis and evaluation of novel carbamates and N-acylheterocyclic derivatives of betulin and betulinic acid for potential use as chemotherapeutic agents (Santos et al., 2009).
The general procedure for the synthesis of the novel lupane derivatives involved dissolution of the corresponding lupanes and CDI, in THF at reflux, under N 2 . The reaction of CDI with alcohols and phenols has been reported to afford either N-alkylimidazoles (Tang et al., 2004 andTotleben et al., 1997) or imidazole carboxylic esters (carbamates) (Herbez et al., 2005;Moreira et al., 2008;Ramos Silva et al., 2007;Tang et al., 2004 andTotleben et al., 1997) depending both on alcohol type and on the reaction conditions used. In this case the reaction afforded the carbamate derivative 3β-hydroxy-lup-20 (29)en-28-yl-1H-imidazole-1-carboxylate in good yield. This compound had been found to induce a selective dose-dependent decrease in the viability of HepG2, HeLa and Jurkat cells after 72 h of treatment according to the determined IC 50 values (4.2 µ M, 7.6 µ M and 16.3µ M, respectively), which were 2-8 times lower than that obtained with betulinic acid.
Mindful of the biological and synthetic importance of such molecules, we report in this communication the molecular structure of the 3β-hydroxy-lup-20 (29)-en-28-yl-1H-imidazole-1-carboxylate determined by single-crystal X-ray diffraction, and compare it with that of the free molecule as given by a quantum mechanical ab initio calculation. The structure of this compound with the corresponding atomic numbering scheme is shown in Fig. 1. This triterpenoid compound is a lupane-type with an imidazole carbonyloxy at C-28. The retention of configuration of C-28 was unequivocally demonstrated by this X-ray crystallographic study. The molecules are hydrogen bonded involving the hydroxyl group at C3 and the carbonyl group of the carbamate moiety, forming infinite chains running along the c axis. In addition, two short distances between C16-H16A and C28-H28B and the O28A and O28B atoms, respectively may be due to weak intramolecular C-H···O interactions.
In order to gain some insight on how the crystal packing of (I) might affect the molecular geometry we have performed a quantum chemical calculation on the equilibrium geometry of the free molecule. These calculations were performed with the computer program GAMESS (Schmidt et al., 1993).
The ab initio calculations reproduce the observed experimental bond lengths and valency angles of the molecule well, with the exception of the bond C20-C30 for which the calculations gave a distance of 1.5103 Å instead of the observed value of 1.433 (6) Å. Also, the calculated conformations of the rings are very close to the experimental values, with the exception of ring E for which the calculations gave a conformation closer to envelope on C17, instead of the observed twisted conformation around C17-C18.

Experimental
All reagents were obtained from Sigma-Aldrich Co. THF was dried and purified before use according to standard procedures.
A solution of betulin (200 mg, 0.45 mmol) and CDI (219 mg, 1.35 mmol) was refluxed in anyhdrous THF (8 ml). After 7 h the reaction was complete (TLC control). Water (30 ml) was added to the mixture and the resulting precipitate was dissolved in ethyl ether (50 ml). The aqueous phase was extracted twice with diethyl ether (2 x 30 ml). The organic phase was then washed with water (30 ml), brine (30 ml), dried with anhydrous Na 2 SO 4 , filtered, and concentrated under reduced pressure to give a yellowish solid. This solid was submitted to f.c.c. with petroleum ether 40-60°C/ethyl acetate (3:2) and afforded the title compound (246 mg, 82%). Full analytical details for this compound (MS, IR, 1 H and 13 C NMR spectroscopy data) can be found in Santos et al., 2009. Recrystallization from acetone at room temperature gave colourless single crystals suitable for X-ray diffraction.
Ab initio calculations were based on a molecular orbital Roothaan Hartree-Fock method using an extended 6-31 G(d,p) basis set. Tight conditions for convergence of both the self-consistent field cycles and maximum density and energy gradient variations were imposed (10 -6 atomic units). The program was run on the Milipeia cluster of UC-LCA (using 16 Opteron cores at 2.2 GHz, runing Linux).

Refinement
All H atoms attached to C atoms were refined as riding on their parent atoms using SHELXL97 defaults. The H atom of the hydroxyl group was refined using an HFIX 147 instruction with U iso = 1.5 U eq of the O atom. The absolute configuration was not determined from the X-ray data, as the molecule lacks any strong anomalous scatterers at the Mo Kα wavelength, but was known from the synthetic route. Friedel pairs were merged for the refinement.   3β-Hydroxylup-20 (29)