Two new diprenylated flavanones from Derris laxiflora Benth

Abstract Phytochemical reinvestigation on the whole plants of Derris laxiflora Benth. afforded two new diprenylated flavanones, derriflavanones B and C (1–2), together with thirty-two known compounds, including sixteen flavonoids (3–18), eleven aromatic compounds (19–29), and five chlorophylls (30–34). All known compounds were first isolated from this plant. The structures of these compounds were determined by analysis of the NMR spectroscopy, mass data, IR spectra, UV spectra, optical rotation and by comparison with literature data. Graphical Abstract


Results and discussion
Compound 1, a yellow solid, gave a molecular ion radical at m/z 426.2040 in a high resolution electron impact mass spectrum (HREIMS) corresponding to the molecular formula C 25 H 30 O 6 (cal. 426.2034). The infrared (IR) spectrum exhibited absorption bands at 3354 (OH) and 1619 cm À1 (chelated C ¼ O group). The UV spectrum of this compound had maxima bands at 225, 296, and 341 nm, indicating the presence of flavanone nucleus (Roussis et al. 1987), which was corroborated with a set of ABX signals in the 1 H NMR spectrum (Table 1S, Supplementary Material) at d 5.39, 3.11, and 2.73 for H-2 and H 2 -3 of the flavanone C-ring. The 1 H NMR spectrum showed a downfield resonance at d 12.47 attributing to a chelated phenolic proton (5-OH), while two doublets with 2H each in the aromatic region (at d 7.39 and 6.89, J ¼ 8.5 Hz) suggested the presence of a para-substituted aromatic B ring. The substituent at C-4 0 was proposed a hydroxyl group due to the ortho phenyl proton at high field-shift d 6.89 (2H, d, J ¼ 8.5 Hz, H-3 0 , 5 0 ). The 1 H signals of an isoprenyl group was at d 1.58 (6H, s), 3.22 (2H, br d, J ¼ 7.1 Hz, H-1 00 ), and 5.16 (1H, br t, J ¼ 7.1 Hz, H-2 00 ) and a 3-hydroxy-3-methylbutanyl group at d 1.25 (6H, s), 1.70 (2H, t, J ¼ 7.0 Hz, H-2 000 ), and 2.69 (2H, t, J ¼ 7. 0 Hz, H-1 000 ). The presence of an isoprenyl group and a 3-hydroxy-3-methylbutanyl group and only a pair of aromatic doubles (each 2H) at d 7.39 and 6.89, indicating that the A-ring is fully substituted flavanone as similar as 6,8-diprenylnaringenin (lonchocarpol A) (Meragelman et al. 2001). The 13 C NMR and DEPT spectra, along with the results of HMQC, show that the structure contains 25 carbon resonances including four methyls, four methylenes, six methines (one oxygenated aliphatic and four aromatics) and eleven guaternary carbons (one carbonyl, one oxygenated carbon, and nine olefinic carbons including four oxygenated). All the NMR spectra of this compound had many similar features to those of 6,8-diprenylnaringenin, beside an isoprenyl group instead of a 3-hydroxy-3-methylbutanyl group. The HMBC spectrum also confirmed the structures of the isoprenyl and 3-hydroxy-3-methylbutanyl moieties ( Figure 1S). The HMBC experiment showed correlations of 5-OH/C-5, -6, -10; H-1 000 /C-5, -6, -7; H-1 00 /C-7, -8, -9; confirming the isoprenyl and a 3-hydroxy-3-methylbutanyl groups are attached to C-8 and C-6, respectively. Assignment of the 1 H and 13 C NMR spectra of 1 was based on the HSQC, HMBC, COSY, and NOESY. Compound 1 consists of the sole chiral centre and has the specific rotation ½a 25 D À15.6 (Meragelman et al. 2001). The data showed the S configuration at C-2 was determined by the optical rotation and the coupling constant of axial-axial (J ¼ 12.7 Hz) between H-2 and H 2 -3 in the 1 H NMR spectrum (Decharchoochart et al. 2014). Based on the above deduction, 1 was elucidated as a new compound named derriflavanone B.
Compounds 1-6, 8, and 9 were evaluated for the cytotoxic activities against human tumor cell lines MCF-7, NCI-H460, and SF-268, but none of them showed significant activity.

General experimental procedures
Optical rotations were measured with a JASCO DIP-180 digital polarimeter, UV spectra with a Hitachi S-3200 spectrometer, and IR spectra with a Perkin-Elmer 983 G spectrophotometer. Melting points were determined with a Yanagimoto micromelting point apparatus and are uncorrected. 1 H, 13 C, DEPT, and two-dimensional NMR spectra were performed using Bruker AVANCE III-500 MHz spectrometer or Varian Unity Plus 400 spectrometers. EIMS was determined using a JEOL JMS-HX 300. Silica gel (Merck 70-230 mesh, 230-400 mesh, ASTM) were used for column chromatography. HPLC was performed on a LDC Analytical-III system, using a semi-preparative normal-phase HPLC column (250 x 10 mm, 7 lm, LiChrosorb Si 60).

Plant material
The whole plant of D. laxiflora was collected in Taitong County, Taiwan, in December 2001 and identified by Prof. Shang-Tzen Chang from School of Forestry and Resource Conservation, National Taiwan University. A voucher specimen (No. NTU200112DL) was deposited in the herbarium of School of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan.

Cytotoxic assay
Compounds 1-6, 8, and 9 were tested against MCF-7 (breast), NCI H460 (lung), and SF-268 (Central Nervous System, CNS) cell lines by using the MTT colorimetric method based on the described procedures (Liaw et al. 2015). Mytomycin c (purity > 97%, Sigma-Aldrich) was used as a positive control. The cells were cultured in RPMI-1640 medium supplemented with 10% FBS at 37 C under 5% CO 2 in a humidified incubator. After seeding 8000 cells/well in a 96-well microplates for 4 h, 20 lL of samples were placed in each well and incubated at 37 C for 48 h, and then 20 lL MTT was added for 3 h. After removing the medium, DMSO (200 lL/well) was added to each well and the microplate were shaken for 10 min to dissolve the formazan crystals formed. The absorbance was measured on a microtiter plate reader (Dynatech, MR 7000) at a wavelength of 570 nm, and the results were used to calculate the half maximal inhibitory concentration.

Disclosure statement
No potential conflict of interest was reported by the authors.