An efficient method for identifying natural common homoisoflavonoid by 1H-NMR

Abstract Homoisoflavone contains 16 carbon atoms in the skeleton. The homoisoflavonoid skeleton from natural products can be roughly divided into 13 kinds, among which 5 kinds of common skeletons contain a large amount of compounds and 8 kinds of abnormal skeletons comprise a small amount of compounds. In this article, the structure identification experience of homoisoflavonoids found in Caesalpinia mimosoides was used as references and an efficient 1H NMR spectroscopic method for identifying homoisoflavonoid structure has been established. Using the chemical shift differences of H-2, 3, 4 and 9, the common natural homoisoflavonoids can be quickly and conveniently determined. Graphical Abstract


Introduction
There are many kinds of active ingredients in traditional Chinese medicine, among which flavonoid has attracted more and more research and their pharmacological effects are becoming clearly. Flavonoid has a wide range of pharmacological effects, such as anti-tumor, antibacterial, anti-inflammatory and other activities, etc. As we all know, rutin, quercetin, hyperoside, silymarin, luteolin, etc, for example, have good therapeutic effects on human diseases.
As a special kind of flavone, homoisoflavone contains 16 carbon atoms in skeleton. In recent years, researchers gradually found an increasing number of homoisoflavonoids in natural medicinal plants, and its extensive pharmacological activities have attracted more and more scholars' interest. Jiang and Lin summarized the naturally occurring homoisoflavone and pharmacological activities in 2007 and 2014, respectively. Several homoisoflavonoid skeletons and hundreds of compounds of homoisoflavonoid were summarized, as well as their anti-inflammation, antidiabetic, antioxidant, phosphorylation inhibition, protein tyrosine kinase inhibition, antimutagenesis, antitumor, antifungal, antiangiogenesis, liver protection, estrogen-like, and immunomodulation activities. This article builds on their work [1,2].
This research group has been devoted to studying the chemical constituents of medicinal plants in Caesalpinia (Leguminosae), and found that the seeds of medicinal plants in this genus are rich in cassane diterpenes, but the roots and stems are rich in homoisoflavonoids [3][4][5][6][7]. In this article, the structural identification experience of homoisoflavonoid found in Caesalpinia mimosoides was used as references, and 1 H-NMR spectroscopy was used to quickly and effectively identify the common natural homoisoflavonoid.

Results and discussion
At present, the homoisoflavonoid skeleton originate from natural products can be roughly divided into 13 kinds, among which 5 kinds of common skeletons (Figure 1, C-I $ C-V) contain a large amount of compounds and 8 kinds of abnormal skeletons ( Figure 2, U-I $ U-VIII) comprise a small amount of compounds. The chemical shift characteristics of protons in A and B rings of homoisoflavonoid are relatively constant and easy to recognize, and common substitution patterns on aromatic rings include AB spin system (H-7, 8 were replaced), ABX spin system (H-7 was replaced, or H-3 0 ,4 0 were replaced), AA' BB 'spin system (H-4 0 was replaced). These homoisoflavonoid skeletons are distinguished by the protons in C ring (H-2, 3, 4) and the protons at C-9 (H-9). We will only discuss H-2, 3, 4, 9 to quickly and accurately identify the homoisoflavonoid using 1 H-NMR spectrum.
The biggest imparity between C-IV and C-V-type homoisoflavonoid is the protons H 2 -2 and H 2 -9 split into double double peaks (C-IV) and double peaks (C-V), respectively. In C-IV-type skeleton, the chemical shifts of H 2 -2 are d H 4.04.2 (dd,  The distinctions of chemical shift signals of five common natural homoisoflavonoid skeletons C-I, C-II, C-III, C-IV, and C-V are shown in Figure 5. Based on the 1 H NMR data of 21 homoisoflavonoid compounds, a relationship between the chemical shift differences (H-2, 3 and 9) and their corresponding skeleton types may be summarized as follows ( Figure 5): Compound 6 was isolated as white powder. Its molecular formula was established as C 18  .77 to C-4 0 (d C 159.9), indicated that two methoxy substituents are located at C-8 and C-4 0 respectively ( Table 1). The electronic circular dichroism (ECD) spectrum of compound 6 showed a positive Cotton effect at De 286 þ13.00, suggesting a 3S configuration by comparison with literature data (Fig. S18, Supplementary data) [19]. Thus, the structure of 8-methoxy-dihydrobonducellin (6) was identified as (3S)-7-hydroxy-8, 4 0 -dimethoxy-3,9-dihydrohomoisoflavonoid.
The U-V (Q) and U-VII (10) types have the protons on 2, 3, 4, and 9 positions, and two protons of H 2 -9 are magnetically equivalent without splitting. In addition, there is an olefinic proton signal on H-4 and an oxygen-methylene on H-2 of U-VII, while U-V has no protons. U-IV (P) and U-VI (R) types have the protons in 2, 3, 4, and 9 positions, and their chemical shifts are similar except that the H 2 -9 of U-IV  type have a slightly bigger chemical shift than U-VI because they were attached to aromatic ring. But the coupling splitting of U-VI is more complete than U-IV. The left four homoisoflavonoid skeletons U-I (M), U-II (N), U-III (O), and U-VIII (S) have around 45 protons. And the biggest feature of U-I type is that it has a hemiacetal proton. There are many variations for these eight unusual skeletons, and it is difficult to determine their type only by 1 H-NMR spectroscopy, sometimes with the help of 13 C-NMR and other two-dimensional spectra.

General experimental procedures
Optical rotations were measured with a polarimeter (Anton-Paar MCP 200; Austria) at room temperature. UV spectra were measured on a Shimadzu UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan). HRESIMS data was acquired on a micro-TOF-Q mass spectrometer (Bruker, Karlsruhe, Germany). NMR spectra were recorded by ARX-400 and AV-600 spectrometers (Bruker), and chemical shifts were given in d

Plant material
The stems of Caesalpinia mimosoides were collected in October 2016 from a mountain near Fapa Hot Spring in Dehong (Yunnan Province, China), and identified by Associated Prof. Jiuzhi Yuan, Shenyang Pharmaceutical University, Shenyang, China. A voucher specimen (No. SY-20161027-06A) was deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, China.

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