Identification of N-Acetyldopamine Dimers from the Dung Beetle Catharsius molossus and Their COX-1 and COX-2 Inhibitory Activities

Recent studies focusing on identifying the biological agents of Catharsius molossus have led to the identification of three new N-acetyldopamine dimers molossusamide A–C (1−3) and two known compounds 4 and 5. The structures of the new compounds were identified by comprehensive spectroscopic evidences. Compound 4 was found to have inhibitory effects towards COX-1 and COX-2.


Introduction
Catharsius molossus (Linnaeus), one of the most widespread coprophagous species on the Earth, plays a vital role in ecosystems because it uses faeces as a source of food and nesting material. However, this insect is also important for its medicinal values. It was recorded in Shen-Nong-Ben-Cao-Jing, a classical ancient Chinese medical book, and has been used for centuries in China due to its multiple OPEN ACCESS functions such as arresting convulsion, removing blood stasis, relaxing the bowels, and counteracting toxins [1]. Previous chemical investigations revealed that C. molossus contains melanin [2] and imidazole compounds [3]. The extract of C. molossus was found to have antitumor [4], anti-benign prostatic hyperplasia [5,6], and cardiovascular activities [7]. We have found that C. molossus extract possesses significant anxiolytic effects in mice [8]. Nonpeptide small molecules present in the insects often have intriguing chemical structures and biological activities, and some such compounds have been characterized from diverse insects [9], however, much still needs to be explored. As part of our broader efforts aimed at searching for and characterizing bioactive compounds extracted from insects, C. molossus was investigated. Three new N-acetyldopamine dimers 1-3 and two known compounds 4-5 were isolated ( Figure 1). The biological activities of all the compounds were evaluated using cytotoxicity, MDCK cell based anti-influenza, EV71 inhibition and cyclooxygenase inhibitory assays. Below, we describe the isolation, structure identification, and biological evaluation of these compounds.

Structural Identification
Compound 1 was obtained as a solid white substance. Its molecular formula was assigned as C20H21NO7, with eleven degrees of unsaturation, based on its HRESIMS data (m/z 410.1212 [M + Na] + , calcd for C20H21NO7Na, 410.1210). The 1 H-NMR spectrum (Table 1) (Table 1) contain resonances for 20 carbons, including two methyl, two aliphatic methylene (one oxygenated), eight methine (two oxygenated aliphatic, six olefinic), and eight quaternary carbons (two carbonyls, six olefinic including four oxygenated). The 1 H-1 H COSY spectrum ( Figure 2 HMBC correlation between H-6 (dd, J = 8.5, 2.3 Hz)/C-4a suggested the side chain attached to C-7. The coupling constant for H-2 is 7.2 Hz, suggesting a trans-H-2/H-3 relationship. Compound 1 was isolated as a racemic mixture indicated by its optical rotation. Further chiral separation was not carried out in this study. Consequently, the structure of 1 was determined as shown in Figure 1 and named molossusamide A. Compound 2 has a molecular formula C19H19NO7 (11 degrees of unsaturation) as derived from the HRESIMS data (m/z 396.1056 [M + Na] + , calcd for C19H19NO7Na, 396.1054). The 1 H-NMR spectrum (Table 1)  , suggesting the presence of two 1,3,4-trisubstituted benzene rings. The 13 C-NMR and DEPT spectra contain resonances for 19 carbons including two methyl, one aliphatic methylene, eight methine (two oxygenated, six olefinic), and eight quaternary carbons (two carbonyls, six olefinic including four oxygenated). The above NMR data of 2 was quite similar to those of 1. The main difference was a side chain at C-7. HMBC correlations (Figure 2) of H-1′′/C-2′′, C-6 and H-3′′/C-2′′ indicated the presence of C-1′′-C-2′′-O-C-3′′, which was positioned at C-7 by the observation of HMBC correlation of H-6 (dd, J = 8.3, 1.8 Hz)/C-4a. The trans-form of H-2/H-3 was determined by the large coupling constant of H-2 (7.3 Hz). Of note, 2 was also isolated as a racemate, successive chiral separation was not carried out. As a result, the structure of 2 was determined as shown in Figure 1 and named molossusamide B.
Compound 3 possesses a molecular formula C18H17NO7 from its HRESIMS (m/z 360.1076 [M + H] + , calcd for C18H18NO7, 360.1078). The NMR data of 3 are extremely similar to those of 2, differing in that an aliphatic methylene was missing in 3, which was supported by HMBC correlations (Figure 2) of H-6, H-8, H-2′′/C-1′′. Similarly, the trans-configuration of H-2/H-3 was evident from the J value of H-2 (7.3 Hz). The racemic nature of 3 was indicated by its null optical rotation. Thus, the structure of 3 was determined as shown in Figure 1 and named molossusamide C.
Considering the traditional uses of C. molossus for the treatment of furunculosis, diarrhea [12] and cancer [13], the biological activities of racemic compounds 1-5 were evaluated using several assays including cytotoxicity in cancer cells, anti-virus (influenza and EV71) and anti-inflammation (COX-1/2). The results showed that only compound 4 exhibited inhibitory effects towards COX-1 and COX-2 with respective IC50 values of 78.85 μM and 6.43 μM (Figure 3 and Table S2) (celecoxib, with IC50 values of 54.55 μM and 0.015 μM for COX-1 and COX-2, respectively, was used as a positive control). All the compounds were inactive against cancer cells, influenza and EV71 (Tables S3-S5). Because pyogenic infections are related to inflammation processes, the observations made in this study suggested that compound 4 might be responsible for the therapeutic applications of C. molossus.   column (MeOH/H2O, 20%-50%), followed by semi-preparative HPLC (MeOH/H2O, 35%) to give 5 (10 mg, Rt = 11.7 min).

Cytotoxicity Assay
Cell lines, K562, MCF-7, A549, Huh-7, Hela, DU145, H1975, and A431 were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Cells were routinely grown and maintained in mediums RPMI or DMEM with 10% FBS and with 1% penicillin/streptomycin. All cell lines were incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator (Greenbelt Maryland, MD, USA) with 5% CO2 in air at 37 °C. Cell viability was determined by the CCK8 (Dojindo, Kyushu, Japan) assay. Cells were seeded at a density of 400-800 cells/well in 384 well plates and treated with various concentrations of compounds or solvent control. After 72 h incubation, CCK8 reagent was added, and absorbance was measured at 450 nm using Envision 2104 multi-label Reader (Perkin Elmer, Waltham, MA, USA).

MDCK Cell-Based Anti-Influenza Assay
Cell-based anti-influenza virus inhibitor screening was based on the principle of cytopathic effect (CPE) protection assay. Madin-Darby canine kidney (MDCK) cells cultured to approximately 90% confluence were detached with 0.25% Trypsin-EDTA (Invitrogen, Shanghai, China), washed and re-suspended in complete EMEM, 2.5 × 10 4 MDCK cells were plated in triplicate in a 96-well plate and incubated overnight at 37 °C in a humidified 5% CO2 incubator. The confluent MDCK monolayers cells were rinsed twice with Hanks' solution devoid of serum, and then the cells were treated with 50 μL medium with 1 mg/mL TPCK and 0.3% BSA and infected by different influenza virus strains at a multiplicity of infection (MOI) of 0.01 PFU/cell. After 2.0 h incubation, serially diluted compounds were added. After 3 day incubation, the medium was removed and 50 μL medium containing 5 μL CCK8 reagent was added into each well followed by additional 2 h incubation, the absorbance was measured at 450 nm using an UV-star-Microplates Synergy HT plate reader (BioTek, Winnooski, VT, USA).

EV71 Inhibition CPE Assay
EV71 (GZ-08-02 strain, GenBank Accession No. FJ360545) was originally isolated and identified by the Guangzhou Children's Hospital, Guangzhou, China [14]. The CPE induced by EV71 infection was measured in Vero cells with CCK8 assay. Confluent VERO cells were plated into 384-well plate and inoculated in triplicate with the mixture of 100 times of the 50% tissue culture infectious dose (TCID50) of EV71, and serially diluted compounds in DMEM supplemented with 2% FCS. After that, the cells were incubated at 37 °C for additional 72 h. Then fresh medium containing CCK8 were added, and incubated in 37 °C for 2 h. the A450 was measured with an Envision Plate Reader (PerkinElmer).

Cyclooxygenase (COX) Inhibitory Assay
Compounds were evaluated for COX inhibitory activity in vitro by using Cayman's COX Fluorescent Inhibitor Screening Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) as previously described methods [15].

Conclusions
Three new and two known N-acetyldopamine dimers were isolated from dung beetle, and their structures were characterized by spectroscopic methods. One known compound was found to have inhibitory activities against COX-1 and COX-2. This contribution adds new facets to the chemistry and biological activity of insect-derived nonpeptide small molecules.