Experimental and Density Functional Theory Study of a New Dimer with Tetrasubstituted Cyclobutane Ring System Isolated from Psoralea plicata Seeds

A new cyclobutane ring system related to plicatin B has been isolated from the methanol soluble fraction of Psoralea plicata seeds and named as -diplicatin B. In addition, the anti-repellent (-)-loliolide and phloretic acid ethyl ester [3-(4-hydroxyphenyl) propanoic acid ethyl ester) have been isolated from the same extract, their structures being determined on the basis of 1D (HNMR and CNMR) and 2D (DQF-COSY, HMQC and HMBC) NMR and mass spectra. Stereoisomers of -diplactin B have been optimized by means of density functional theory using the hybrid functional B3LYP.


Introduction
Many molecular properties of organic compounds, such as chemical reactivity, catalytic, biological, and pharmacological activities, are critically affected not only by their functional groups but also by their spatial position.Thus, the disclosure of the relative configuration has a great impact on the full understanding of their chemical behaviors.Different approaches to determine the exact structure and/or configuration of organic products have been devised (Seco et al., 2004;Nicolaou et al., 2005;Bifulco et al., 2007).Stereochemical information on organic compounds is usually derived from vicinal H, H coupling constants.The values of cyclobutane system vary widely with overlapping ranges from cis to trans couplings (Fleming & Williams, 1967).Psoralea plicata Del. (Syn = Cullen plicatum Del.) is an Egyptian wild perennial plant and it is known in Arabic as Marmid (Tӓckholm, 1974).Genus Psoralea is one of the most popular traditional Chinese medicines and is officially listed in the Chinese Pharmacopoeia.It used for the treatment of enuresis, pollakiuria, weak kidney, and pain and cold in the waist and knees (Qiao et al., 2006a(Qiao et al., , 2006b)).The wide use of the plant seeds as anthelmintic, diuretic, diaphoretic, tonic or an aphrodisiac against impotence and menstruation disorders; or externally the treatment of leucodermia, psoriases and leprosy in Indian folkloric remedy prompted worldwide investigation Psoralea spp. to isolate its active components (Perry & Metzger, 1980).
In the previous publications, we have isolated many structurally different compounds from the aerial parts including (Perry & Metzger, 1980;Hamed et al., 1997) and from seed extract; we were able to isolate a group of benzofuran glycosides (Hamed et al., 1999).By thorough chromatographic separation and purification, we obtained three more compounds 2, 3 and 4.
B3LYP hybrid functional has been used in describing potential energy surfaces (PES) (Becke, 1993;Lee et al., 1988).The geometries of the five stereoisomers were fully optimized using analytic gradients.The harmonic vibrational frequencies of the stationary points of the PES have been calculated at the same level of theory in order to identify the local minima as well as to estimate the corresponding zero point vibrational energy (ZPE).A 6-31G (d,p) Basis Set was employed for each atom (Cimino et al., 2004).

Plant Material
The seeds of Psoralea plicata were collected from Aswan Governorate, South of Egypt in March 2010.The plant was identified by Dr. Hamed A. I. according to Täckholm (1974).The voucher specimen was deposited at the herbarium of the Botany Department, Faculty of Science, Aswan, Egypt.

Results and Discussions
Compound 2, showed a molecular ion peak in the MS spectrum [M] + , m/z 492, suggesting the molecular formula C 30 H 36 O 6 .Its IR spectrum displayed absorption bands at 3420 and 1715 cm-1, indicated the presence of hydroxyl and carbonyl functions respectively.The 13 CNMR spectrum indicated the presence of two molecules of plicatin B with the absence of the olefinic carbon signals (Perry & Metzger 1980), which were replaced by four signals at  43.53, 44.38, 47.72, and 48.92 characters of a cyclobutane structure.The cyclobutane structure confirmed by 1 H-1 H and 1 H-13 C COSY (Hodges & Porte 1964).The remaining carbon signals disclosed the presence of four methyl groups at  17. 76, 17.83, 25.71 (two methyl); two O-methyl groups ( 52.04 and 52.12); eight methines at  115.11, 115.72, 121.66, 125.77, 126.50, 129.63 and 130.59.Also, it showed signals of two carboxylic carbons at  173.28 and eight quaternary carbons at  126.35, 128.31, 128.42, 133.09, 134.29, 134.67, 152.61, and 153.67.It has been stated that, from the magnitude of the vicinal coupling in cyclobutane ring, it might be possible to determine the stereochemistry of the substituents on the cyclobutyl ring by deriving the values of the cis-and trans-vicinal coupling from the analysis of the spectra.However, vicinal coupling in cyclobutane system has been found to be sensitive to substitutions and strain effects.Furthermore, they vary over a sufficient range, so that some overlap between the values occurs.Due to the presence of phenyl ring appear shifted the resonance signals of cis-vicinal protons to higher field than those of trans-vicinal (Fleming & Williams, 1967;Chi et al., 1997).This is the first report to isolate such dimer from natural sources and we have given the name -diplicatin B (2) (Figure 1).
The configuration analyses of the -diplicatin-B prove the probability presence of five isomers (Figure 2).The relative stability of these sterioisomers was evaluated using DFT approach.All stereoisomer's were optimized using DFT/B3LYP/6-31G (d, p) and localized as a minimum in the PES.For each stereoisomer, harmonic vibrational frequencies were calculated to confirm that all configurations are stable.The relative energies as well as corrections of zero point energy (ZPE) and free energy of the five stereoisomers are gathered at table 2. The structure of configurations 2a, 2b, 2c, 2d and 2e was illustrated in figures 2and 3. DFT calculations give energies in the order 2a<2b<2c<2d<2e at the same order of steric effects.The theoretical results to which we have succeeded, shows that the stereoisomer 2a is the most stable one.The calculated free energies indicated that compound 2a to be the most populated gas-phase stereoisomers (99.94% at 298 K).These interpretations are in good agreements with our experimental choice.
Compound 3, showed optical rotation, UV and IR spectra identical with those reported for (-)-loloilide (Hodges & Porte, 1964;Tanaka & Matsunga, 1989).Its mass spectrum can be used conveniently for its identification.A side from the molecular ion peak, characteristic fragment ion peak at m/z 181 [M- and 124 base peak could be traced in the spectrum.The compound is easily acetylated in the usual manner (pyridine/acetic anhydride, overnight), giving its acetate derivative as seen from the 1 HNMR spectrum of the acetate.The 1 HNMR spectrum of compound 3 is very informative, showing three quaternary methyl signals at  1.26, 1.46 and 1.80 and one methine proton broad singlet at  5.68.The remaining signals could be resolved through decoupling experiment.The 13 CNMR spectrum of compound 3, allowed for simple assignment of its carbon skeleton and DEPT experiment displayed resonances at  182.34 (s), 171.15 (s), 112.93 (d), 86.61 (s), 66.83 (d), 47.31 (t), 45.63 (t), 30.63 (q), and 27.00 (q) and 26.49(q), confirming the structure of loliolide, a monoterpene lactone compound which has not been reported before from Psoralea plicata.116.70 (d), 130.81 (d), 128.70 (s), 174.41 (s), 42.31 (t), 34.11 (t), 29.50 (t), and 25.40 (q).From the given data, compound 4 is supposed to be the ethyl ester of phloretic acid or 3(4-hydroxyphenyl)-propanoic acid, an acid isolated from the Heartwood of Haplomosia monophylla (Devon and Scott 1975), but this is the first report of isolation such ester from Psoralea plicata.
a Assignments were confirmed by DQF-COSY, HMQC and DEPT experiments.