**3. Discussion**

Marine organisms and microorganisms living in extreme marine environments were considered to be important sources of halogenated compounds [28]. Chlorinated metabolites, as a key part of the halogenated compounds, have demonstrated a wide range of significant activities. Due to the high concentration of chloride ions in mangrove ecosystem, many chlorinated compounds have been excavated from mangrove-derived microorganisms [9–11]. In this study, two new chlorinated metabolites, **1** and **3**, were isolated from mangrove sediments-derived fungus. Although 3,4-dichloro-1*H*-pyrrole-2,5-dione (**3**) has been previously reported as a synthetic product, it was discovered as a new metabolite of natural origin in our study. So, mangrove sediments-derived microbes have proved to be a promising source of novel and unique chlorine-containing bioactive secondary metabolites.

An important result of this study is the discovery of stemphone C (**4**) with significant anti-prostate cancer activity in vitro. Stemphone C (**4**) had been reported from an *Aspergillus* strain as a potentiator of imipenem activity against methicillin-resistant *Staphylococcus aureus*, with other stemphone derivatives [21,29]. Stemphone was found to inhibit over contraction of portal vein, induced by high glucose levels [30], and its derivatives were also revealed to inhibit lipid droplet accumulation in macrophages [31]. In our study, stemphone C (**4**) exhibited obvious antimicrobial activity against the bacteria *Erysipelothrix rhusiopathiae* WH13013, with the MIC value of 1.56 μg/mL. In addition, obvious antiproliferative activities of stemphone C (**4**) were also revealed against two prostate cancer cell lines (PC-3 and 22Rv1) and three other cancer cell lines (HepG2, WPMY-1, and MC3T3-E1), with IC50 values less than 10 μM. Furthermore, it showed reducing PC-3 cells colony formation, inducing apoptosis, and blocking the cell cycle at S-phase in a dose-dependent manner. Our study showed that stemphone C (**4**) could be considered as a potential antiproliferative agent, especially as a promising anti-prostate cancer lead compound. Importantly, we could able to determine the absolute configuration of **4**, the first time in this study, which is necessary for further development of this active compound.

#### **4. Materials and Methods**

## *4.1. General Experimental Procedures*

The UV and IR spectra were recorded on a Shimadzu UV-2600 PC spectrometer (Shimadzu, Beijing, China) and an IR Affinity-1 spectrometer (Shimadzu), respectively. Optical rotations were determined with an Anton Paar MPC 500 (Anton, Graz, Austria) polarimeter. High resolution electrospray ionization mass spectroscopy (HRESIMS) spectra were acquired on a Bruker maXis Q-TOF mass spectrometer (Bruker BioSpin International AG, Fällanden, Swizerland). The NMR spectra were recorded on a Bruker Avance spectrometer (Bruker) operating at 500 and 700 MHz for 1H NMR and 125 and 175 MHz for 13C NMR that used tetramethylsilane as an internal standard. Semipreparative high-performance liquid chromatography (HPLC) was performed on the Hitachi Primaide with a DAD detector, using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm). Column chromatography was performed over silica gel (200–300 mesh) (Qingdao Marine Chemical Factory, Qingdao, China). Spots were detected on TLC (Qingdao Marine Chemical Factory) under 254 nm UV light. All solvents employed were of analytical grade (Tianjin Fuyu Chemical and Industry Factory, Tianjin, China).
