Cladieunicellins M–Q, New Eunicellins from Cladiella sp.

Five new 7α-hydroxyeunicellin-based diterpenoids, designated as cladieunicellins M–Q (1–5), were isolated from a Formosan octocoral Cladiella sp. The structures of 1–5 were elucidated on the basis of spectroscopic methods and by comparison of the data with those of the related metabolites. Cytotoxicity of metabolites 1–5 against the human leukemia Molt 4 and HL 60 is also described. Among them, compounds 1, 3 and 5 exhibited moderate cytotoxicity toward Molt 4 cells with IC50 values 16.43, 14.17 and 15.55 μM, respectively. Preliminary SAR (structure activity relationship) information was obtained from these compounds and their analogues.


Introduction
During the course of our search for novel metabolites from marine invertebrates of Taiwanese waters, a series of eunicellin-type diterpenoids including cladieunicellins A-J, have been isolated from a soft coral identified as Cladiella sp. (family Alcyoniidae) collected in Taiwan waters [1][2][3]. Because of our interest in the chemistry of new natural products, the continuing investigation on the chemical constituents of the soft coral Cladiella sp. was carried out and resulted in the isolation of five new eunicellin-based diterpenoids, cladieunicellins M-Q (1-5) (Chart 1). This paper deals with the isolation, structure elucidation and cytotoxicity of compounds 1-5. Chart 1. The structures of cladieunicellins M-Q (1)(2)(3)(4)(5), krempfielins C and L (6 and 7) and cladieunicellin L (8).

Results and Discussion
Cladieunicellin M (1) was obtained as colorless oil and its molecular formula of 1 was established as C 28 H 44 O 9 (7° of unsaturation) by the HRESIMS at m/z 547.28760 (calcd for C 28 H 44 O 9 Na, 547.28775). The IR absorptions at υ max 3462 (broad) and 1734 cm −1 revealed the presence of hydroxy and ester carbonyl functionalities. The 13 C NMR of 1 showed 28 carbon signals (Table 1), which were assigned with the assistance of the DEPT spectrum to six methyls, seven sp 3 methylenes (including an oxymethylene), an sp 2 methylene, eight sp 3 methines (including four oxymethines), two sp 3 oxygenated quaternary carbons and four sp 2 quaternary carbons (including three carbonyls). The 13 C resonances at δ C 172.3, 171.9 and 171.2 demonstrated the presence of three ester carbonyls. Two of these signals were identified as acetate carbonyls by the presence of two methyl resonances in the 1 H NMR spectrum at δ H 2.09 and 2.08 (each 3H × s) and the other one was identified as an n-butyrate carbonyl by the presence of seven contiguous protons at δ H 0.99 (3H, t, J = 7.2 Hz), 1.66 (2H, m) and 2.32 (2H, m). From the 13 C NMR data, an exocyclic carbon-carbon double bond was deduced from the signals at δ C 147.8 (C-11) and 111.1 (CH 2 -17), and confirmed by two olefin proton signals at δ H 4.91 (1H, br s, H-17) and 4.79 (1H,dd,J = 2.0,1.6 Hz, in the 1 H NMR spectrum. In addition, a suite of resonances of proton signals at δ H 3.84 (1H,dd,J = 8.8,6.8 Hz,, 3.57 (1H, s, H-2), 3.38 (1H,dd,J = 7.2,6.8 Hz,dd,J = 10.8,7.2 Hz, and carbon signals at δ C 92.7 (CH-2), 81.5 (CH-9), 53.5 (CH-10) and 45.1 (CH-1), indicated the presence of a tetrahydrofuran moiety. Comparison of the 13 C NMR and DEPT spectra with the molecular formula indicated that there must be two exchangeable protons, requiring the presence of two hydroxy groups. From the above data, compound 1 was proven to be a diterpenoid with three rings.  1 H-1 H couplings in the COSY spectrum of 1 enabled identification of the C-4/-5/-6, C-8/-9/-10/-1/ -14/-13/-12, C-14/-18/-19 and C-18/-20 units (Table 1 and Figure 1), which were assembled with the assistance of an HMBC experiment. The HMBC correlations between protons and quaternary carbons of 1 (Table 1 and Figure 1), such as H-1, H-2, H 2 -4, H 2 -5/C-3; H 2 -5, H-6/C-7; and H-9, H-10, H 2 -17/C-11, permitted the elucidation of the main carbon skeleton of 1. The exocyclic carbon-carbon double bond at C-11 was confirmed by the HMBC correlations between H-10/C-17 and H 2 -17/C-10, -11, -12. The ether bridge between C-2 and C-9 was supported by an HMBC correlation between H-9/C-2. The C-15 and C-16 tertiary methyls bonded to the C-3 and C-7 oxygenated quaternary carbons were established by the HMBC correlations between H 3 -15/C-2, -3, -4 and H 3 -16/C-6, -7, -8, respectively. The hydroxy proton signal at δ H 1.82 was revealed by its 1 H-1 H COSY and HMBC correlations to δ H 3.58 (H-8) and δ C 80.0 (CH-8), respectively, indicating its attachment to C-8. The location of a hydroxy group at C-7, an oxygenated quaternary carbon, was confirmed by the HMBC correlations between a hydroxy proton at δ H 2.36 and C-6, -7 and C-16. Furthermore, the acetoxy groups at C-6 and C-19 were confirmed by the HMBC correlations from oxymethine (δ H 5.72, H-6) and acetate methyl (δ H 2.08) to the ester carbonyl at δ C 171.9 (C); and oxymethylene (δ H 3.95, H 2 -19) and acetate methyl (δ H 2.09) to the ester carbonyl at δ C 171.2 (C), respectively. Thus, the remaining n-butyrate ester had to be positioned at C-3, an oxygen-bearing quaternary carbon resonating at δ C 86.0 ppm. Based on the above findings, the planar structure of 1 was established. Naturally occurring eunicellin analogues from soft corals belonging to the genus Cladiella have H-1 and H-10 in the β-orientation [4]. In the NOESY experiment ( Figure 1), observation of the correlations between H-10 with H-1 and H-8, suggested that H-1, H-8 and H-10 are β-oriented. Also, correlations of H-2 with H 3 -15 and H-14; H-9 with H-6 and OH-8; and H-8 with H 3 -16, suggested that H-2, H-6, H-9, H-14, Me-15 and both the hydroxy groups at C-7 and C-8 are α-oriented. The C-18 asymmetric center was assigned to be R*-configured on the basis of correlations between the β-oriented H-1 and H 3 -20 and between the α-oriented H-2 and H-18. Based on the above findings, the structure of 1 was elucidated and the chiral carbons for 1 were assigned as 1R*, 2R*, 3R*, 6S*, 7S*, 8S*, 9S*, 10R*, 14R* and 18R*. The NMR data of 1 was found to be similar to those of a known compound, krempfielin C (6)  between both compounds arises from the replacement of the C-19 methyl at C-18 in 6 by a acetoxymethyl group in 1.
The new metabolite cladieunicellin N (2) was found to have the molecular formula C 24 H 38 O 7 and six degrees of unsaturation, as indicated from the HRESIMS at m/z 461.25067 (calcd for C 24 H 38 O 7 Na, 461.25097). NMR data of 2 (Tables 2 and 3) showed the presence of two acetoxy group (δ H 2.08 and 2.06, each 3H × s; δ C 169. 5 and 22.4; 171.8 and 21.4). The 1 H and 13 C NMR data of 2 was found to be similar to those of a known compound, krempfielin L (7) (Chart 1) [6]. By comparison of the 1D and 2D NMR data of these two compounds revealed that the hydroxy group at C-6 in 7 was replaced by an acetoxy group in 2 (Tables 2 and 3; Figure 2). The stereochemistry of 2 was confirmed by comparison of the NMR data and NOESY correlations of eunicellins 7 and 2 (Tables 2 and 3; Figure 2).     Tables 2 and 3) are almost identical with those of 2 except for the presence of an additional acetoxy group in 3 (δ H 2.04, 3H, s; δ C 170.4 and 21.6) in 3. Furthermore, the placement of an acetoxy group at C-12 was established by the HMBC experiment which showed correlations from an oxymethine proton (δ H 5.48) and acetate methyl (δ H 2.04) to the ester carbonyl at δ C 170.4 (C) (Figure 3). The NOESY correlations of 3 ( Figure 3) also showed that the relative stereochemistry of this metabolite is similar with that of 2. Thus the structure of eunicellin 3 was elucidated.  Cladieunicellin P (4) had the same molecular formula as that of 1, C 28 H 44 O 9 , as determined by HRESIMS, with seven degrees of unsaturation. In the HMBC spectrum, the 13 C signal at δ C 172.3 correlated with the signal of the methylene protons at δ H 2.29 ( Figure 4) and was consequently assigned as the carbon atom of the n-butyrate carbonyl. The positions of the two acetoxy groups at C-6 and C-12, were confirmed by the correlations the two methine protons at δ H 5.64 (H-6) and 5.41 (H-12) and the ester carbonyls at δ C 171.8 (s) and 170.8 (s), respectively, in the HMBC spectrum of 4. Thus, the remaining n-butyrate group was at C-3, an oxygenated quaternary carbon which bonded to the C-15 tertiary methyl and was confirmed by the HMBC correlations between H 3 -15/C-2, -3, -4. The relative configuration of 4 was mostly confirmed to be the same as that of 1 by comparison of the chemical shifts of both compounds (Tables 1-3) and was further confirmed by NOESY correlations (Figure 4). The coupling constants between H-12 and C-13 methylene protons (J = 4.0, 2.8 Hz) indicated that H-12 was positioned on equatorial direction and possessed a β-orientation in the cyclohexane ring of 4.
Cladieunicellin Q (5) exhibited the molecular ion peak [M + Na] + at m/z 461.25110 in the HRESIMS and established a molecular formula of C 24 H 38 O 7 (calcd for C 24 H 38 O 7 Na, 461.25097), appropriate with six degrees of unsaturation. The IR absorptions at υ max 3462 and 1732 cm −1 revealed the presence of hydroxy and ester carbonyl functionalities. The 13 C NMR spectrum of 5 showed signals of 24 carbons (Table 3), which were characterized by the DEPT spectrum of six methyls (including two acetate methyls), five methylenes (including an sp 2 methylene), eight methines (including four oxymethines) and five quaternary carbons (including two ester carbonyls and an sp 2 quaternary carbon of an olefin). The 1 H and 13 C NMR spectral data of 5 (Tables 2 and 3) also showed the presence of two acetoxy groups (δ H 2.10 and 2.07, each 3H × s; δ C 22.4 and 21.4, acetate methyls; δ C 169.6 and 171.8, acetate carbonyls). The remaining three degrees of unsaturation identified 5 as a tricyclic diterpenoid. The molecular framework was established by 1 H-1 H COSY and HMBC correlations ( Figure 5). Comparison of the NMR data of 5 with those of the known compound, cladieunicellin L (8) [2] revealed that 5 is the 12-deacetoxy derivative of cladieunicellin L. The stereochemistry of compound 5 was determined by the NOESY spectrum as shown in Figure 5.  Cytotoxicity of compounds 1-5 toward Molt 4 (human acute lymphoblastic leukemia) and HL 60 (human promyelocytic leukemia) cells was studied, and the results are shown in Table 4. Eunicellins 1, 3 and 5 was found to exhibit moderate cytotoxicity against Molt 4 cells. Eunicellin 2 did not show cytotoxicity toward Molt 4 cells, implying that the presence of a hydroxy substituent at C-12 would weaken the activity comparison with the structure and cytotoxicity of 3. Eunicellin 4 was found to be inactive against Molt 4 cells, indicating that the bulky n-butyrate group at C-3 could reduce cytotoxicity in comparison with the structure and cytotoxicity of cladieunicellin L (8) [2]. In a previous study, we reported the isolation of a natural eunicellin, litophynin I diacetate (9) [1,7]. However, based on the spectral data analysis and by comparing the 13 C NMR chemical shifts of C-7 and C-16 with those of its analogues [8], the C-7 should be revised as to possess an S*-configuration as presented in eunicellin 10 (Chart 2). (9)

General Experimental Procedures
Optical rotations were measured on a Jasco P-1010 digital polarimeter (Japan Spectroscopic Corporation, Tokyo, Japan). Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrometer (Varian Inc., Palo Alto, CA, USA) or a Jasco 4100 FT-IR spectrometer (Japan Spectroscopic Corporation, Tokyo, Japan); peaks are reported in cm −1 . NMR spectra were recorded on a Varian Mercury Plus 400 NMR spectrometer (Varian Inc., Palo Alto, CA, USA) using the residual CHCl 3 signal (δ H 7.26 ppm) as the internal standard for 1 H NMR and CDCl 3 (δ C 77.1 ppm) for 13 C NMR. Coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 Tesla solariX FTMS system (Bruker, Bremen, Germany). Column chromatography was performed on silica gel (230-400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F 254 (0.25 mm, Merck, Darmstadt, Germany); spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. The normal phase HPLC (NP-HPLC) was performed using a system comprised of a Hitachi L-7110 pump (Hitachi Ltd., Tokyo, Japan) and a Rheodyne 7725 injection port (Rheodyne LLC, Rohnert Park, CA, USA). Two normal phase columns (Supelco Ascentis ® Si Cat

MTT Antiproliferative Assay
HL 60 (human promyelocytic leukemia) and Molt 4 (Human acute lymphoblastic leukemia) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere of 5% CO 2 . Cells were seeded at 4 × 10 4 per well in 96-well culture plates before treatment with different concentrations of the tested compounds. The compounds were dissolved in dimethyl sulfoxide (less than 0.02%) and made immediately of 1.25, 2.5, 5, 10 and 20 μg/μL prior to experiments. After treatment for 72 h, the cytotoxicity of the tested compounds was determined using MTT cell proliferation assay (thiazolyl blue tetrazolium bromide, Sigma-M2128, St. Louis, MO, USA). The MTT is reduced by the mitochondrial dehydrogenases of viable cells to a purple formazan product. The MTT-formazan product dissolved in DMSO. Light absorbance values (OD = OD 570 − OD 620 ) were recorded at wavelengths of 570 and 620 nm using an ELISA reader (Anthos labtec Instrument, Salzburg, Austria) for calculating the concentration which caused 50% inhibition (IC 50 ), i.e., the cell concentration at which the light absorbance value of the experimental group is half that of the control group. These results were expressed as a percentage of the control ± SD established from n = 4 wells per one experiment from three separate experiments [10].

Conclusions
Five new 7α-hydroxyeunicellin-based diterpenoids, cladieunicellins M-Q (1)(2)(3)(4)(5), were isolated from the soft coral Cladiella sp. The eunicellins 1, 3 and 5 are found to show moderate cytotoxicity against the Molt 4 human acute lymphoblastic leukemia. The soft coral Cladiella sp. will be transplanted to culturing tanks located in the National Museum of Marine Biology and Aquarium, Taiwan, for extraction of additional natural products to establish a stable supply of bioactive material.