**Mao-Sheng Zhang 1, Yan Deng 1, Shao-Bin Fu 1, Da-Le Guo <sup>2</sup> and Shi-Ji Xiao 1,\***


Received: 17 May 2018; Accepted: 22 June 2018; Published: 26 June 2018

**Abstract:** A novel isoquinoline alkaloid, mahimbrine A, possessing a rare benzotropolone framing scaffold, was isolated from the endemic plant of *Mahonia imbricata*. Its structure was established on the basis of extensive spectroscopic analysis. A plausible biosynthetic route of mahimbrine A was proposed. Mahimbrine A showed no antimicrobial activity at the concentration of 1 mg/mL.

**Keywords:** *Mahonia imbricata*; Berberidaceae; isoquinoline alkaloid; mahimbrine A

#### **1. Introduction**

*Mahonia imbricata* Ying *et* Boufford, as one of the endemics of seed plants in China, is a perennial shrub of the family Berberidaceae, distributed only in Guizhou and Yunan provinces of Southwest China [1]. Plants of the genus *Mahonia* have long been used as a traditional medicine to treat tuberculosis, periodontitis, dysentery, pharyngolaryngitis, eczema, and wounds [2]. Previous chemical investigations on this genus have involved a series of chemical constituents. Among these constituents, alkaloids are principal constituents of the genus *Mahonia* [3–5]. As part of an ongoing research program to isolate and determine structures of secondary metabolites from medicinal endemic plants of southwestern China [6,7], we performed a phytochemical study on *M. imbricate.* As a result, a novel isoquinoline alkaloid, possessing a rare benzotropolone substituent, was isolated (Figure 1). Its structure was established by means of spectroscopic analysis including one- and two-dimensional NMR spectroscopy. Moreover, the hypothetical biosynthetic route was proposed. Mahimbrine A was tested against four gram-positive bacterial strains and four gram-negative bacteria.

**Figure 1.** Structure of mahimbrine A.

#### **2. Results and Discussion**

#### *2.1. Structure Elucidation of Mahimbrine A*

Mahimbrine A was obtained as a brown gum. Its molecular formula was established as C24H23NO5 on the basis of the HR-ESI-MS ([M + H]<sup>+</sup> at *m*/*z* 406.1632, calcd. 406.1649 and [2M + H]+ at *m*/*z* 811.3226, calcd. 811.3225), which requires 14 degrees of unsaturation. The IR absorptions at 3434 and 1633 cm−<sup>1</sup> suggested the presence of hydroxyl and carbonyl groups in the molecule. UV absorptions at λmax 230 and 288 nm deduced the presence of an α, β-unsaturated carbonyl moiety [8]. The 1H, 13C NMR and HSQC spectra of mahimbrine A (Table 1) showed 24 carbon resonances due to a methine at δ<sup>H</sup> 2.91 (2H, m), δ<sup>C</sup> 25.8; four methoxyl groups at δ<sup>H</sup> 3.60 (3H, s), 3.93 (6H, s), 3.94 (3H, s), δ<sup>C</sup> 56.2, 56.2, 56.2, 61.8; a methine at δ<sup>H</sup> 3.90 (1H, m), 4.03 (1H, m), δ<sup>C</sup> 47.9; a 1,2,3,4-tetrasubstituted phenyl ring moiety at δ<sup>H</sup> 7.04 (1H, d, *J* = 9.1 Hz), 7.28 (1H, d, *J* = 9.1 Hz), δ<sup>C</sup> 153.7, 148.4, 1131.5, 129.2, 128.8, 114.5; a 1,2,4,5-tetrasubstituted phenyl ring moiety at δ<sup>H</sup> 6.57 (1H, s), 6.78 (1H, s), δ<sup>C</sup> 151.8, 147.9, 131.4, 121.9, 110.6, 110.6; a carbonyl at δ<sup>C</sup> 188.4; and an unsaturated system at δ<sup>H</sup> 8.20 (1H, d, *J* = 13.0 Hz), 6.87 (1H, dd, *J* = 13.0, 2.7 Hz), 6.74 (1H, d, *J* = 2.7 Hz), δ<sup>C</sup> 168.4, 148.8, 134.9, 133.6, 133.5.


**Table 1.** 1H and 13C NMR data for mahimbrine A (400/100 MHz, in CDCl3).

The obvious HMBC correlations (Figure 2) from H-3 to C-1, C-4a, H-4 to C-5, C-4a, C-8a, H-5 to C-4, C-7, C-8a, H-8 to C-1, C-4a, C-6, OCH3 to C-6 and OCH3 to C-7 evidenced the presence of a dihydroisoquinoline unit with two methoxyl groups located at C-6 and C-7, respectively. The HMBC correlations of H-9 to C-7 (δ<sup>C</sup> 188.4), C-4 a, H-8 to C-6 , C-9 a (δ<sup>C</sup> 131.5), and H-6 to C-4 a (δ<sup>C</sup> 129.2), C-8 a, established the presence of a tropolone moiety, which was further verified by the vicinal coupling constants of H-8 /H-9 (*J* = 13.0 Hz) [9]. Moreover, the HMBC correlations of H-9 to C-1 , C-4 a, H-4 to C-2 , C-5 , C-9 a confirmed the tropolone moiety and 1,2,3,4-tetrasubstituted phenyl ring moiety connected via a bridged bond at C-4 a and C-9 a. Similarly, two methoxyl groups at C-1 and C-2 were also assigned. The HMBC correlations of H-6 to C-1, C-4 a and H-4 to C-5 indicated that the dihydroisoquinoline unit and benzotropolone unit were connected to each other by C-1 and C-5 . Thus, the final structure of this compound was determined and named mahimbrine A.

**Figure 2.** Key COSY, HMBC and NOESY correlations of mahimbrine A.

## *2.2. Plausible Biogenetic Pathway*

A plausible biogenetic pathway for mahimbrine A was postulated (Scheme 1). As a precursor, naphthylacetic acid and dopamine via condensation reaction to give an amide intermediate **1** [10], which subsequently undergoes a Bischler-Napieralski reaction to generate intermediate **2** [11]. Intermediate **2** is then oxidized to yield intermediate **3** [12]. Intermediate **3** can spontaneously convert to its enol form. This enol intermediate then undergoes ring expansion rearrangement to give intermediate **4** [13], which is finally methylated to get mahimbrine A.

**Scheme 1.** Proposed Biogenetic Pathway of mahimbrine A.

#### *2.3. Antimicrobial Activity*

The antimicrobial activity of mahimbrine A was evaluated against four gram-positive bacterial strains *Staphylococcus aureus* ATCC 6538, *Micrococcus luteus* CMCC 28001, *Staphylococcus epidermidis* ATCC 12228, and *Bacillus subtilis* ATCC 21332; and four gram-negative bacteria *Pseudomonas aeruginosa* ATCC 27853, *Escherichia coli* ATCC 25922, *Salmonella typhimurium* ATCC 14028, and *Enterobacter aerogenes* ATCC 13048, by a microdilution titre technique; neither was active [14]. Gentamicin and streptomycin were used as positive controls. Discs containing 10 μL DMSO solutions were used as a negative control. All tests were performed in triplicate.

#### **3. Experimental Section**

#### *3.1. General Procedures*

UV spectra were recorded on a Perkin-Elmer Lambda 35 UV-VIS spectrophotometer (Perkin-Elmer, Waltham, MA, USA). IR spectra were measured on a PerkinElmer one FT-IR spectrometer (KBr) (Perkin-Elmer, Waltham, MA, USA). 1D and 2D-NMR spectra were recorded on a Bruker-Ascend-400 or an Agilent DD2400-MR instrument using TMS as the internal reference. HR-ESI-MS spectra were measured on a LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, USA). Column chromatography was performed using silica gel (300–400 mesh, Qingdao marine Chemical Ltd., Qingdao, China). Semi-preparative HPLC was performed on LC3000 system (Beijing ChuangXingTongHeng Science And Technology Co., Ltd., Beijing, China) equipped with an ODS column (5 μm, i.d. 10 mm × 250 mm, YMC). Original raw data of 1D, 2D NMR spectra and HR-ESI-MS of mahimbrine A are available in the supplementary materials.
