Hemimycalins C–E; Cytotoxic and Antimicrobial Alkaloids with Hydantoin and 2-Iminoimidazolidin-4-one Backbones from the Red Sea Marine Sponge Hemimycale sp.

In the course of our continuing efforts to identify bioactive secondary metabolites from Red Sea marine sponges, we have investigated the sponge Hemimycale sp. The cytotoxic fraction of the organic extract of the sponge afforded three new compounds, hemimycalins C–E (1–3). Their structural assignments were obtained via analyses of their one- and two-dimensional NMR spectra and HRESI mass spectrometry. Hemimycalin C was found to differ from the reported hydantoin compounds in the configuration of the olefinic moiety at C-5–C-6, while hemimycalins D and E were found to contain an 2-iminoimidazolidin-4-one moiety instead of the hydantoin moiety in previously reported compounds from the sponge. Hemimycalins C–E showed significant antimicrobial activity against Escherichia coli and Candida albicans and cytotoxic effects against colorectal carcinoma (HCT 116) and the triple-negative breast cancer (MDA-MB-231) cells.


Introduction
The marine environment has played an essential role in the discovery of compelling secondary metabolites with fascinating antitumor, immunomodulatory, analgesic, antiinflammatory, anti-allergic, antimicrobial, and antiviral effects [1,2]. Since 1963, more than 30,000 new chemical entities have been identified from marine organisms, including macroand micro-organisms [3]. Secondary metabolites obtained from marine invertebrates have received great attention from pharmacologists and chemists due to their remarkable chemical diversity and biological activities [4][5][6]. The fact that 14 marine-derived approved drugs and another 23 drug leads in different phases (I-III) of clinical trials [7], mostly from marine invertebrates [7], clearly indicates the role of marine invertebrates as a vigorous source for the drug-discovery process [7]. Sponges belonging to the genus Hemimycale are excellent producers of alkaloids with both guanidine [8,9] and hydantoin backbones [10,11]. Ptilomycalin A, with its exceptional polycyclic guanidine backbone linked with a ωhydroxyhexadecanoyl-spermidine moiety via an ester linkage, has displayed notable antimicrobial and antiviral activities [8,9].

General Experimental Procedures
The IR spectra of 1-3 were recorded on a Shimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). One-and two-dimensional NMR spectra were acquired on Bruker Avance DRX 600 MHz (Bruker, Rheinstetten, Germany) spectrometer. Positive ion HRESIMS data were obtained with a Micromass Q-ToF equipped with leucine enkephalin lock spray, using m/z 556.2771 [M + H] + as a reference mass. Sephadex LH-20 (0.25-0.1 mm, Pharmacia) was used for column chromatography. Silica gel 60 F-254 plates (Merck) were used for TLC.

Biological Materials
The sponge ( Figure 5) was collected by hand using SCUBA at a depth of 13 m off Al-lith, Saudi Arabia. The dark blue encrusting sponge was found to be composed of a 1.5-2.0 cm thick soft mass. The skeleton of the sponge was plumose and composed of parallel loose bundles of thin spicules running from the substratum upwards through the sponge and fanning out at the surface. In between, there were many loose spicules. Bundles had a diameter of 30-50 µm and contained 12-20 spicules in cross-section. Siliceous spicules were straight and thin, either strongyles or styles but otherwise similar in shape and size, ranging from 215-255 × 2-4 µm. These details conformed with the description of the type specimen of the Red Sea sponge Hemimycale arabica, with which the current specimen was compared. A voucher specimen is kept in the Red Sea Invertebrates Collection at King Abdulaziz University under the code # DY21.
Mar. Drugs 2021, 19, x 7 of 10 ranging from 215-255 × 2-4 μm. These details conformed with the description of the type specimen of the Red Sea sponge Hemimycale arabica, with which the current specimen was compared. A voucher specimen is kept in the Red Sea Invertebrates Collection at King Abdulaziz University under the code # DY21.

Evaluation of Antiproliferative Activity
The evaluation of the antiproliferative effects of 1-3 was performed using an MTT, assay as reported earlier [26,27]. The cells were incubated at 37 °C overnight in 5% CO2/air. After that, the compounds were added to the top row of a 96-well microtiter plate, and

Evaluation of Antiproliferative Activity
The evaluation of the antiproliferative effects of 1-3 was performed using an MTT, assay as reported earlier [26,27]. The cells were incubated at 37 • C overnight in 5% CO 2 /air. After that, the compounds were added to the top row of a 96-well microtiter plate, and descendant serial dilutions (1:4) of the concentration were performed followed via the incubation of the cells with the compounds for 72 h. Using the CellTiter 96 AQueous non-radioactive cell proliferation protocol, the cells' viability was estimated at 490 nm on a Molecular Devices Emax microplate reader. The IC 50 values of the compounds (expressed in µM) were determined using the program SOFTmax PRO. 5-Flourouracil and DMSO were used as positive and negative controls, respectively. A concentration of 25 µM was set as a cutoff value in this assay.

Disk Diffusion Assay
The antimicrobial effects of 1-3 were evaluated using a disc diffusion assay at 50 µg/disc against E. coli (ATCC 25922), C. albicans (ATCC 14053), and S. aureus (ATCC 25923), as described previously [28][29][30]. Ciprofloxacin and ketoconazole served as positive controls in the antimicrobial assay, while DMSO was used as a negative control.

Evaluation of the MIC Values
The determination of the MIC values of 1-3 against C. albicans and E. coli was performed using a macro-dilution assay, as previously reported [31].

Conclusions
The bioassay-directed partition and purification of the cytotoxic fraction of the Red Sea sponge Hemimycale sp. provided three new alkaloids: hemimycalins C-E (1-3). The structures of the compounds were assigned via analyses of their spectral data. Interestingly, hemimycalin C (1) was found to possess an E configuration [25] at ∆ 5,6 instead of the previously reported Z configuration of ∆ 5,6 . In addition, hemimycalins D and E (2 and 3) were found to possess the 2-iminoimidazolidin-4-one [25] backbone instead of hydantoin (imidazolidine-2,4-dione) moiety in previously reported alkaloids from the genus Hemimycale. Furthermore, hemimycalin D (2) was found to share the E configuration at ∆ 5,6 with hemimycalin C (1). Consequently, the E-configured 1 and 2 were shown to possess higher chemical shift values for C-6 than the Z-configured compounds, while H-6 [23][24][25] in the E-configured compounds [23][24][25] was found to resonate at lower chemical shift values than in the Z-configured ones.
Hemimycalins C-E showed significant cytotoxic effects and selective antimicrobial effects against E. coli and C. albicans, making them potential scaffolds for the development of drug leads.
The current findings provide a deeper insight and understanding of the chemical diversity and biological activities of the secondary metabolites of the Red Sea sponge Hemimycale sp.