Davidones F and G, Two Novel Flavonoids from Sophora davidii (Franch.) Skeels

An unprecedented novel flavanone davidone F (1) with a seven-membered ring side chain, and a novel flavanonol davidone G (2), along with 11 known flavonoids, were isolated from the ethyl acetate fraction of Sophora davidii (Franch.) Skeels. Their planar structures were established by UV, IR, HRESIMS, 1D and 2D NMR data. The relative configurations of 1 and 2 were determined by calculation of NMR chemical shift values, the absolute configuration of 1 and 2 were assigned by comparing their experimental and calculated electronic circular dichroism (ECD) spectra. Moreover, compounds 1–13 were screened for the translocation activity of glucose transporter 4 (GLUT-4), and the fluorescence intensity was increased to the range of 1.56 and 2.79 folds. Compounds 1 and 2 showed moderate GLUT-4 translocation activity with 1.64 and 1.79 folds enhancement, respectively, at a concentration of 20 μg/mL.


Introduction
Sophora davidii (Franch.) Skeels are shrubs or dungarungas, which belong to the family Fabaceae, mainly distributed in Guizhou, Ningxia, Yunnan and Sichuan provinces of China [1]. As a folk herbal medicine, this plant is grown in hillsides, roadsides and bushes at an altitude of 1300-2500 m, and its roots, leaves, flowers and fruits are traditionally used to treat diarrhea, cystitis, stomachache, edema and sarcoptic mange [2]. The plant has proven to be a rich source of flavonoids, alkaloids, steroids, lignans and phenolic acids [3][4][5]. Flavonoids, as the main compounds of S. davidii, have a broad spectrum of biological activities. Notably, for the prenylated flavonoids, prenylation increased the lipophilicity of flavonoids, which resulted in an increased affinity to biological receptors [6]. To enhance our understanding of the chemical and biological diversity of the Sophora species, the chemical investigation of S. davidii led to the isolation of one new prenylated flavanone, davidone F (1), with a novel seven-membered oxygen ring, and one new prenylated flavanonol, davidone G (2), together with one known flavanonol (3), two known chalcones (5)(6), four known isoflavones (7)(8)(9)(10)(11)(12) and one known flavonol (13) (Figure 1). Additionally, we describe the isolation, structure elucidation and GLUT-4 translocation activities of these isolated compounds.

Results and Discussion
Compound 1 was isolated as a brown oil. The molecular formula was established as C 26  . The 13 C NMR spectrum of 1 displayed 26 carbon signals, including three methyls, one methoxy group, four methylenes, seven methines and 11 non-protonated carbons. The pendant aromatic B-ring suggested a 1,2,4-trisubstituted arrangement, due to the HMBC correlations of H-6 and H-3 with C-2 (δ C 75.5), C-3 (δ C 99.8), C-2 (δ C 159.3), C-4 (δ C 160.5) and C-5 (δ C 108.0), C-1 (δ C 119.2), respectively. Meanwhile, the pentasubstituted phenyl ring system assigned to the A-ring was determined by HMBC correlations between H-6 and C-10 (δ C 103.3), C-8 (δ C 108.2), C-9 (δ C 162.5), C-5 (δ C 163.3), C-7 (δ C 166.3) and C-4 (δ C 198.7). The methoxy group located on C-2 was established by HMBC correlation from H 3 -2 -OCH 3 to C-3 (δ C 99.8) and C-2 (δ C 159.3). Besides 15 skeletal carbon atoms of flavanone and the methoxy moiety, the remaining 10 carbons were connected to form a substituted seven-membered ring from the detailed analysis of the COSY and HMBC correlations. Through interpretation of the COSY correlations between H-1 /H-2 /H-3 /H-4 ( Figure 2), it was possible to establish the presence of a CH 2 -CH-CH 2 -CH=C spin system. Combining with the HMBC cross-peaks ( Figure 2) from H 3 -7 to C-6 (δ C 65.7), C-4 (δ C 126.6) and C-5 (δ C 136.8), from H 2 -6 to C-7 (δ C 20.9) and C-8 (δ C 80.3) and from H 3 -9 or H 3 -10 to C-2 (δ C 48.9) and C-8" (δ C 80.3), allowed the construction of a seven-membered ring. The HMBC correlation between H 2 -1 and C-8 (δ C 108.2), C-8 (δ C 162.5) and C-9 (δ C 166.5) suggested the seven-membered ring containing oxygen in the ring was connected to the A-ring. Thus, the planar structure of compound 1 was determined as shown. In order to further verify the relative configuration of 1, we use the DP4 analysis to solve the stereochemistry problems [8]. NMR chemical shift (GIAO) was calculated at the mPW1PW91/6-311G(d,p) level of theory as required for DP4 analysis [9]. Importantly, the calculated DP4 probability of isomer 2S*, 2 R*-1 was assigned to 100% ( Figure S21). Therefore, the relative configuration of 1 was defined as 2S*, 2 R* ( Figure 3). To ascertain the absolute configuration of 1, its ECD spectrum was determined in MeOH and then comparing the experimental and time-dependent density functional theory (TDDFT)-calculated electronic circular dichroism (ECD) spectra. The experimental ECD data of 1 showed positive CEs at 331 nm due to n → π* transitions of the carbonyl group ( Figure 4) [10,11]. Based on the above evidence, the absolute configuration of 1 was elucidated as 2S, 2 R, and named davidone F.  [12], a flavanone previously isolated from S. davidii, except for an oxygenated aliphatic carbon C-3 (δ C 72.5), where an additional hydroxy group moiety was attached. The location of the additional hydroxy group substituent was established by the COSY relationship between H-2 and H-3 as well as the HMBC correlations ( Figure 2) from H-3 to C-2 (δ C 79.8), C-1 (δ C 116.9) and C-4 (δ C 199.3), from H-2 to C-3 (δ C 72.5), C-1 (δ C 116.9), C-6 (δ C 130.9), C-9 (δ C 161.6) and C-4 (δ C 199.3). According to the J-based configuration analysis protocol, the large coupling constant (11.6 Hz) observed between H-2 and H-3 indicated that the two hydrogens tended to adopt an "anti" arrangement. The ECD spectrum of 2 contained the characteristic CEs induced by C-4 ketone carbonyl in its flavanonol framework, namely, a positive one at λ max 320 nm ( Figure 4). The absolute configuration of C-2 and C-3 was thus proposed to be 2R, 3R [11,13]. Subsequently, we performed computational predictions of NMR chemical shifts of both the possible isomers 2R, 3R, 2 R and 2R, 3R, 2 S (Figure 3) using the GIAO method at the mPW1PW91/6-311G(d,p) level with the conductor polarizable calculation model (CPCM) in MeOH [8]. As a result, DP4 probability analysis showed that 2R, 3R, 2 S was predicted to be correct with probabilities of 99.56% for the NMR data ( Figure S22). The calculated ECD spectrum of 2R, 3R, 2 S-2 at the B3LYP/6-311+G(d,p) level with the CPCM in MeOH fit well with the experimental spectrum of 2 ( Figure 4). Finally, the absolute configuration of 2 was determined as 2R, 3R, 2 S, and named davidone G.

Materials
The roots of S. davidii (Franch.) Skeels (age 12-15 years) were collected from Xiuwen county, Guizhou province, China (at altitudes of 1200 to 1300 m) in June 2014. The roots were dried at room temperature, macerated into a fine powder and stored at room temperature. The identification was performed by Professor Dingrong Wan of School of Pharmaceutical Sciences, South-Central University for Nationalities (SCUN), Wuhan, China. A voucher specimen (SC0801) is deposited at the School of Pharmaceutical Sciences, SCUN, Wuhan, China.

Computation Details
Conformational searches were carried out via random searching in Sybyl-X 1.1.1 using the MMFF94S forcefield [28]. The conformers obtained in an energy window of 10 kcal·mol −1 were further applied to geometrical optimization and minimization using the B3LYP/6-31G(d,p) level in the gas phase in Gaussian 09. Subsequently, NMR chemical shift calculations were conducted using gauge-independent atomic orbitals (GIAO) method at the mPW1PW91/6-311G(d,p)/CPCM level in MeOH (1 and 2) [9]. The shielding constants were converted into unscaled chemical shifts (δ u ) by referencing TMS (δ u = σ TMS − σ Cal ), where the σ TMS was the shielding constant of TMS calculated at the same level. The NMR chemical shifts of the isomers were obtained by Boltzmann averaging the 1 H and 13 C NMR chemical shifts of the stable conformers. The calculated NMR properties of optimized structures were averaged based upon their respective Boltzmann populations, and calculations of DP4+ probability analysis were facilitated by the Excel sheet (DP4+) provided by Grimblat et al. [8]. In the part of ECD calculation, the optimized conformers were calculated using the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-311+G(d,p) level in MeOH. The ECD curves were simulated on the basis of rotatory strengths using SpecDis v1.71 with a half-band of 0.3 eV and averaged according to the Boltzmann distribution [29].

GLUT-4 Translocation Assay
Construction of myc-GLUT4-mOrange plasmid and cell line were performed as described previously. Myc-GLUT4-mOrange-L6 cells were cultured on glass coverslips for 12 h, and then L6 myoblasts were differentiated to L6 myotubes. Cells were starved in a PSS solution for 2 h. After starvation, mOrange fluorescence was detected by laser-scanning confocal microscopy at an excitation wavelength of 555 nm. Images were taken after addition of tested samples (10 µg/mL), insulin (10 nM) or normal control (NC) (0.1% DMSO) for 30 min using 555 nm excitation laser. Zen 2010 Software (Carl Zeiss, Jena, Germany) was used to analyze the fluorescence intensity of mOrange [30].
Institutional Review Board Statement: Not applicable.