Acanthophoraine A, a new pyrrolidine alkaloid from the red alga Acanthophora spicifera

Abstract A new pyrrolidine alkaloid, acanthophoraine A (1), along with six known alkaloids (2–7), had been isolated from the red alga Acanthophora spicifera. The structures of these compounds were identified by spectroscopic analyses. The absolute configuration of 1 was established by ECD calculation. Compound 1 represents the first example of N-isobutyl pyrrolidone with an urea arm. The antimicrobial activity of 1 was also evaluated.


Introduction
The red alga Acanthophora spicifera, belonging to the family Rhodomalaceae (Xia 2011;Martins et al. 2018), was widely distributed in tropical and subtropical areas, which grew abundantly in sandy rocky areas in the upper intertidal zone where the plant was exposed to air during low tides. It was an important seaweed in folklore remedies as well as a food supplement in the Gulf of Mannar, coastal region of Tamil Nadu (India) (Davis and Vasanthi 2011). It possessed antibacterial, antitumor, antioxidant and anti-inflammatory activities due to the presence of bioactive components such as steroids (Han et al. 2009;Lang et al. 2007;Prakash et al. 1989;Shi et al. 2011;Wahidulla et al. 1987;Wang et al. 2003), flavonoids (El Shoubaky et al. 2016;Martins et al. 2018;Wang et al. 2003;Zeng et al. 2010), alkaloids (Wahidulla et al. 1991), terpenoids (Shi et al. 2007) and tannins (Yamamoto and Maruyama 1985).
In our ongoing systematic chemical investigations of marine organisms (Lai et al. 2018;Wang et al. 2017;Zhou et al. 2018;Zou et al. 2017), a new pyrrolidine alkaloid, acanthophoraine A (1), together with six known alkaloids (2-7), had been isolated from the red alga A. spicifera (Figure 1). Compound 1 represents the first example of N-isobutyl pyrrolidone with an urea arm. Herein, we describe the isolation, structural elucidation and antimicrobial activity of 1. With the aid of 1 H-1 H COSY, HSQC and HMBC experiments, the 1 H and 13 C NMR signals of 1 were assigned as shown in Table S2.

Results and discussion
The 1 H-1 H COSY data of 1 revealed the presence of two spin coupling systems in bold as shown in Figure S2. The HMBC correlations between H-3/H-4/H-5 and C-2, between H-6 and C-7 as well as between H-1 0 and C-2/C-5 verified the planar skeleton of 1. Additionally, according to the molecular formula information and the obvious downfield shifts at C-7, the remaining amino group should be attached to C-7. Thus, the planar structure of 1 was deduced.
The absolute configuration of 1 was determined by the quantum chemical CD calculation method. The overall predicted CD spectra of (5R)-1 and (5S)-1 were subsequently compared with the experimental one ( Figure S1). The calculated CD curve of (5R)-1 revealed a good agreement with the measured one. Herein, the absolute structure of 1 was determined as 5R.

Plant material
The red alga was collected from Xuwen County, Zhanjiang City, Guangdong Province in May 2017 and identified by the researcher Liu Cui as A. spicifera. The specimen has been deposited in the Department of Chemistry, College of Chemistry and Materials Science, Jinan University.

Bacterial strains and chemicals
The antimicrobial activity of 1 was evaluated against six bacterial strains of Staphylococcus aureus ATCC 25923, MRSA ATCC 43300, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Klebsiella Pneumoniae ATCC 700603, which were obtained from the American Type Culture Collection (ATCC). Vancomycin hydrochloride and Ciprofloxacin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cation adjusted Mueller-Hinton broth (CAMHB) was purchased from Hope Bio-Technology Co. Ltd (Qingdao, China).

Broth microdilution assay
The minimum inhibitory concentration (MIC) of 1 and control antibiotics (Vancomycin and Ciprofloxacin) was evaluated in accordance with the guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI) (Wayne 2012). Approximately 5 Â 10 5 CFU/mL of bacteria in CAMHB was incubated with two-fold serial dilutions of drugs at 37 C without agitation for 16-20 hours. The MIC represents the lowest concentration that prevented the growth of the bacteria by visual inspection. All MIC determinations were performed in triplicate.

Conclusions
A new pyrrolidine alkaloid, acanthophoraine A (1), with six known ones (2-7), had been isolated from the red alga A. spicifera. Compound 1 represents the first example of N-isobutyl pyrrolidone with an urea arm. Compounds 2-5 and 7 were isolated from the genus Acanthophora for the first time. However, compound 1 showed no antimicrobial activity against six bacterial strains.