Enigmazole C: A Cytotoxic Macrocyclic Lactone and Its Ring-Opened Derivatives from a New Species of Homophymia Sponge

A new macrolide, enigmazole C (1), and two additional analogues, enigmazoles E (2) and D (3), were obtained from a new species of the Homophymia genus as part of an ongoing discovery program at PharmaMar to study cytotoxic substances from marine sources. The structures were fully characterized by cumulative analyses of NMR, IR, and MS spectra, along with density functional theory computational calculations. All three of the new compounds feature an unusual 2,3-dihydro-4H-pyran-4-one moiety, but only enigmazoles C (1) and D (3) showed cytotoxic activity in the micromolar range against A-549 (lung), HT-29 (colon), MDA-MB-231 (breast), and PSN-1 (pancreas) tumor cells.

M arine sponges represent a prolific source of structurally unique macrolides possessing promising biological activities, including cytotoxic, anticancer, and neuroprotective properties, thus suggesting their potential value for the development of leads in drug discovery. 1 Particularly, sponges from the Neopeltidae family have not been extensively chemically investigated, with relatively few structures being described in the literature from each of the three genera Homophymia, Callipelta, and Daedalopelta. By way of illustration, the high molecular weight peptides homophymines A−E/A1−E1, 2,3 homophymamide A, 4 pipecolidepsins A− C, 5−7 and callipeltins A and B, 8−10 have all been reported to have significant cytotoxic and anti-HIV activities. Others metabolites isolated from Neopeltidae sponges that were assigned in 2013 to the suborder Astrophorina 11,12 are the bioactive tetramic acid glycoside aurantoside C 13 and the macrocycles callipeltosides A−C. 14,15 Furthermore, only two structures belonging to the Daedalopelta genus have been described, the cytotoxic cyclodepsipeptide daedophamide 16 and the 14-membered macrolide neopeltolide, 17 which has been extensively studied and synthesized because it is a potent cytochrome bc1 complex inhibitor, as well as a cytotoxic compound with activity against A-549 human lung adenocarcinoma, NCI/ADR-Res ovarian sarcoma, and P388 murine leukemia cell lines. 18−21 During continuing efforts at PharmaMar to discover new cytotoxic compounds from marine natural sources, we have evaluated a new sponge species of the Homophymia genus (Vacelet & Vasseur, 1971) collected off the coast of Gorontalo, Indonesia. In this paper, we describe the isolation of a new macrocyclic compound as well as two open-chain analogues, all isolated from a specimen of this sponge collected in Indonesia. Despite the fact that the new macrocycle shows structural similarities to neopeltolide, a clear resemblance to the macrolide enigmazole A isolated by Oku et al. in 2010 from the marine sponge Cynachyrella enigmatica, 22 has led us to designate the three new compounds as enigmazoles C (1), E (2), and D (3). The configurations of these enigmazoles have been solved using a combination of microscale chemical conversions and the use of an elegant J-based configurational analysis based on capillary NMR measurements. 23 A methanolic extract of the Homophymia sponge specimen showed cytotoxic activity against A-549 (lung), HT-29 (colon), MDA-MB-231 (breast), and PSN-1 (pancreas) tumor cells and was hence selected for a more detailed bioassay-guided chemical investigation.
The interconnection between the determined fragments A− E was achieved by using an HMBC experiment as follows: (a) The methine at δ H 5.20 (H-23) showed long-range correlation with three methyl groups at δ C 19.5 (C-24), δ C 17.7 (C-25), and δ C 56.6 (C-29), along with the sp 2 methine carbon at δ C 113.6 (C-21), implying the position of the methoxy group on C-23 and the connection between fragments A and B.  allowed us to connect fragments C and D, therefore assembling the cyclic macrolactone planar structure for 1 as is drawn in Figure 1. At this point, the resemblance of 1 to the known compound enigmazole A (4) was clear, as some structural features are common in both compounds: a disubstituted oxazole ring, a pyran ring, the presence of an exomethylene double bond, and a similar macrolactone size. Once the planar structure of 1 was established, the relative and absolute configuration were   To deduce the relative configuration of the entire spin system D, we were nicely able to relate the two stereogenic centers at C-2 and C-5 located three carbon−carbon bonds away through a J-based configurational analysis (JBCA). 24 To make this possible, we had to change the NMR solvent to acetone-d 6 , because it gave us a 1 H NMR spectrum where the two diastereotopic pairs at C-3 (H-3a and H-3b) and C-4 (H-4a and H-4b) are well separated and fully resolved.
Not many approaches of this kind have been applied to a natural compound with two sp 3 methylenes surrounded by two stereogenic centers, even though this approximation is a very good tool when the diastereotopic protons can be unequivocally assigned with their corresponding chemical shifts and their sets of proton−proton and carbon−proton coupling constants. Therefore, 13 C− 1 H-HSQC-TOCSY-HECADE and J-HMBC experiments were needed to measure key small coupling constants for 3 J C26−H3a and 3 J C26−H3b to place H 3 -26 in a gauche disposition to both H-3 diastereotopic protons at the C-2/C-3 single bond ( Figure 3a). These two experiments along with selective 1 H NMR irradiation spectra were satisfactory to deduce relationships from diastereotopic protons H-3a/H-3b and H-4a/H-4b to C-2 and C-5 as is drawn Figure 3b) for the C-3/C-4 bond. The hydroxylated carbon at C-5 helped us to deduce the relative dispositions for H-4a and H-4b to the OH group with regard to the C-4/C-5 bond (Figure 3c), as well as H-6a and H-6b to the mentioned OH relative to the C-5/C-6 bond ( Figure 3d).
The absolute configuration at position C-5 was elucidated by the application of the modified Mosher's method (MMM). 25,26 The derivatization of the secondary alcohol with R-and S-MTPA-Cl was completed directly in pyridine-d 5 in an NMR tube to give compounds 1-S and 1-R, respectively. Comparison of the Δδ values (δ S − δ R ) obtained from the MTPA esters ( Figure 4) indicated that the absolute configuration of C-5 was R, which implies a C-2S configuration.  Thus, the 1 H NMR spectrum of 2 displays a singlet signal at δ H 3.61 (s, 3H) that was assigned to a methoxy group, which shows an HMBC correlation to an carbonyl carbon at δ C 176.6 assigned to the new carbonyl ester at C-1. Moreover, the proton chemical shift at δ H 5.92, assigned to H-17 in 1, was clearly shifted to δ H 4.72 in 2 as expected due to the lack of the lactone moiety. The presence of a hydroxy group at C-17 in 2 due to the macrolactone ring opening of 1 could be used to determine the absolute configuration at this position by the application of the MMM using the α-methoxy-α-trifluoromethylphenylacetic (MTPA) esters.
In this case, the comparison between the proton chemical shifts of both MTPA esters at C-17 (δ 2-S − δ 2-R) established the absolute configuration of C-17 as S (see Figure 5). In addition, the esterification of 2 with MTPA-Cl generated also the MTPA esters at C-5; the comparison of proton chemical shift values confirmed again the configuration of this stereogenic center as R.
In the same way, a large proton−proton coupling constant of 11.0 Hz between H-13 and H-12a fixed these protons in an antiperiplanar disposition, making necessary the use of ROESY correlations between the pairs H-12a/H-27 and H-11/H-14 and the long-range 13 C− 1 H coupling constants 3 J C11−H13 0.5 Hz, 3 J C27−H12a 3.0 Hz, and 2 J C27−H12a 3.0 Hz to determine the presence of the rotamer drawn in Figure 6b. Once H-12a and H-12b are interrelated to conformers 6a and 6b, we were able to determine the relative configuration of the two stereogenic centers C-11 and C-13 as R* and R*.
While the absolute configurations of C-2, C-5, and C-17 are already known as S, R, and S, respectively, the relative configurations of C-11 and C-13 as R* and R* along with the possible configuration at C-23 of either R or S give us four possible enigmazole C diastereomers (1a−d) that can be discriminated by an NMR−density functional theory (DFT) approximation. Initially we submitted all diastereoisomers to a conformational search with the Macromodel program using the protocol of Daranas, Sarotti, et al. 27 Thus, 58 conformers f o r 1 a -( 2 S , 5 R , 1 1 R , 1 3 R , 1 7 S , 2 3 R ) , 6 4 f o r 1 b -(2S,5R,11S,13S,17S,23S), 60 for 1c-(2S,5R,11S,13S,17S,23R), and 62 for 1d-(2S,5R,11R,13R,17S,23S) were found within a 5.0 kcal/mol window, which were further classified by energy and frequencies using the B3LYP/6-31G(d) functional. Once the duplicates and conformers with imaginary frequencies were removed, a combination of MPW1PW91/6-31G(d,p) and the polarizable continuum model was used for proton and carbon chemical shift calculations using MeCN as solvent. The sets of 1 H and 13 C chemical shifts were compared by the statistical  was achieved when six 3 J HH constraints were introduced. 27 Clearly both DP4+ and iJ-DP4 were able to discriminate 11R,13R diastereoisomers 1a and 1d from both 11S,13S-1b and 11S,13S-1c. At this point just C-23 remains to be assigned, and since the fragment C-18/C-25 showed similar NMR values (see chemical shifts in CD 3 OD in the Supporting Information) to that for enigmazole A (4), 22 we propose the final structure for enigmazole C (1) as 2S,5R,11R,13R,17S,23R (diastereoisomer 1a). In comparison with enigmazole A, 1 presents the same configurations of the stereogenic centers at the C-2, C-5, C-11, C-13, C-17, and C-23 positions.
Along with 1, an analogue of 3, enigmazole D, was also found during the process of isolation and purification. Its HR-ESITOF-MS data showed the [M + H] + adduct at m/z 516.2932, which was in perfect agreement for a molecular formula of C 29 H 42 NO 7 . Although the formula of 3 matches that found for enigmazole C, its HPLC retention time and 1 H NMR spectrum were different. Three new 1 H NMR signals corresponding to two vinylic protons (δ H 6.58 and 6.00) and one oxygenated methine at δ H 4.74, in addition to the absence    -17) found in 1 to a lower value of δ H 4.74 in 3 along with the expected chemical shift of the new methine proton is in agreement with the opening of the lactone ring. With all these data on hand, we were able to assign the structure of enigmazole E to 3, a metabolite resulted from the opening of enigmazole C and a further dehydration at position C-5. It is important to notice that this new double bond has an E configuration, based on the 15.6 Hz coupling constant found between H-5 and H-6. Compounds 2 and 3 can be considered derivatives of enigmazole C, and therefore we assumed the absolute configurations at positions C-2, C-5 (just for compound 2), C-11, C-13, C-17, and C-23 were the same as for 1; thus (2S,5R,11R,13R,17S,23R) for enigmazole E (2)   Computational Calculations. Conformational searches were performed by using the corresponding module implemented in the Maestro Quantum mechanical software. The MMFF force field with acetonitrile as solvent was used, and torsional enhanced sampling with 10 000 steps was fixed using an energy window of 5 kcal/mol. DP4+. Molecular geometry optimizations were performed for 1a− d at the DFT theoretical level using the Gaussian 16W package first at the B3LYP/6-31G(d) level for energy and frequency calculations. After removing redundant conformers and those with imaginary frequencies, theoretical Boltzmann energy population-weighted 1 H and 13 C NMR were calculated by using the combination MPW1PW91/6-31G(d,p). Results were input in Sarotti's and Darana's Excel spreadsheet 27 to calculate the best fit.
Preparation of the (S)-MTPA Ester of Enigmazole C (1-S). R-(−)-MTPA chloride (10 μL) was added to a solution of 1 (1.0 mg) in 0.5 mL of pyridine-d 5 in an NMR tube. The resulting mixture was allowed to stand at room temperature (rt) for 8 h to yield the (R)-MTPA ester of 1, which was monitored by recording 1 H NMR spectra at 500 MHz. 1  Biological Assays. The cytotoxic activities of compounds 1, 2, and 3 were tested against A-549 human lung carcinoma cells, MDA-MB-231 human breast adenocarcinoma cells, HT-29 human colorectal carcinoma cells, and PSN-1 pancreatic adenocarcinoma cells. The concentration giving 50% inhibition of cell growth (GI 50 ) was calculated according to the procedure described in the literature. Cell survival was estimated using the National Cancer Institute (NCI) algorithm. Three dose−response parameters were calculated for 1, 2, and 3.