Abstract
A new regular diterpene possessing an unusual 1,6-anti-3-methylcyclohex-2-en-1-ol ring system, dactyloditerpenol acetate (1), has been extracted from the tropical sea hare Aplysia dactylomela and its stereostructure elucidated by spectroscopic methods. The absolute configuration of 1 was determined as 1S, 6S, 7R, 10S, and 11R by application of Kishi’s method for the assignment of absolute configuration of alcohols. The new diterpene potently inhibited in vitro thromboxane B2 (TXB2) (IC50 0.4 μM) and superoxide anion (O2−) (IC50 1 μM) generation from E. coli lipopolysaccharide (LPS)-activated rat neonatal microglia, with concomitant low short-term toxicity.
Keywords: Aplysia dactylomela, diterpene, prenylbisabolane, anti-neuroinflammatory activity, tuberculosis
Marine organisms, comprising more than half of the total global diversity, offer an enormous and biodiverse source of novel and pharmacologically active compounds.1 Mollusks belonging to the order Anaspidea have provided a wealth of structurally diverse natural products with potential application as therapeutic agents.2 The sea hare Aplysia dactylomela is found worldwide in tropical to warm temperature waters. Previous chemical studies on this species from our group had led to the discovery of aplysqualenols A and B, two squalene-derived polyethers with interesting biological activity.3
As part of our ongoing search for bioactive natural products, we herein report the isolation and structural elucidation of a new prenylbisabolane-type diterpene named dactyloditerpenol acetate (1), along with seven known metabolites. Prenylbisabolane-based diterpenoids are very rare in Nature, and to the best of our knowledge, only nine compounds of this type have been thus far described from both marine and terrestrial sources.4 Arguably, the most prominent example of this intriguing family of metabolites is laurenditerpenol (2), an irregular diterpene from the red alga Laurencia intricata, which was found to inhibit HIF-1 by blocking the induction of the oxygen-regulated HIF-1α protein.5 The absolute stereostructure of diterpene 2, which inhibited HIF-1 activation in T47D breast tumor cells by hypoxia with IC50 value of 0.4 μM, was settled by a highly convergent asymmetric total synthesis that also clarified the stereochemistry of its conspicuous 1,6-syn-3-methylcyclohex-2-en-1-ol ring system.6,7
Spurred by a keen interest for the discovery of new secondary metabolites with biological activity of relevance, compound 1 was evaluated for in vitro cytotoxicity against A2058 (melanoma) and DU-145 (prostate) cancer cell lines, antibacterial activity against Mycobacterium tuberculosis (H37Rv), and anti-neuroinflammatory activity in Escherichia coli lipopolysaccharide (LPS)-activated rat brain microglia, specifically for thromboxane B2 (TXB2) and superoxide anion (O2−) generation inhibition. Enhanced generation of O2− and TXB2 by LPS-activated microglia, a nervous system mononuclear phagocyte involved in neuroinflammation, has been reported both in vitro8 and in vivo.9 Thus, modulation of O2− and TXB2 release by LPS-activated microglia has been hypothesized as a therapeutic approach to ameliorate neuroinflammatory disorders.10
About forty small specimens of the tropical sea hare Aplysia dactylomela (Rang, 1828), collected by hand at a depth of 1–2 ft off Mona Island, Puerto Rico, were freeze-dried, homogenized with a 1:1 mixture of MeOH–CHCl3, and filtered in vacuo.11 Preliminary in vitro evaluation of cytotoxicity of the n-hexane, CHCl3, and EtOAc extracts was not possible due to a spurious contamination during the assays.12 Notwithstanding, a portion of the n-hexane extract was subjected successively to bioassay-guided fractionation using normal-phase silica gel column chromatography with a mixture of n-hexane–EtOAc as eluent to afford pure dactyloditerpenol acetate (1, 19 mg).13 An array of known compounds co-isolated during this investigation, namely, brominated indol,14 allolaurinterol,15 elatol,16 trans-laurediol,17 parguerol,18 parguerol 16-acetate,18 and deoxyparguerol,18 was confidently identified from comparisons of their spectroscopic data with those previously described in the literature.
Dactyloditerpenol acetate (1) was obtained as a colorless oil whose molecular formula was established as C22H38O4 by HRESI-MS (m/z 389.2664 [M+Na]+, calcd 389.2668), indicating four degrees of unsaturation. The presence of hydroxyl groups was implied from the broad stretch at 3418 cm−1 in the IR spectrum. The 13C NMR and DEPT data (Table 1) showed resonances for four quaternary, six methine, six methylene, and six methyl carbons. Inspection of the 1H–1H COSY, HSQC and HMBC spectra indicated the presence of an acetate group [δC 171.2, C-21; 21.1, C-22; δH 2.10, (3H, s, H3-22)], two trisubstituted double bonds [δC 137.3, C-3; 125.6, C-2; δH 5.37 (1H, br s, H-2); δC 132.1, C-15; 124.1, C-14; δH 5.09 (1H, br t, J = 7.1 Hz, H-14)], two oxymethines [δC 69.0, C-1; δH 4.00 (1H, br s, H-1) and δC 79.2, C-10; δH 4.85 (1H, dd, J = 9.7, 3.5 Hz, H-10)], a secondary methyl at δH 0.79 (3H, d, J = 6.9 Hz, H3-19), and four singlet-methyl protons (δH 1.68, 1.66, 1.61, and 1.15). Two isolated 1H–1H spin systems were quickly established from interpretation of the 1H–1H COSY spectrum (Fig. 1). These segments were in turn interconnected through the long-range 1H–13C couplings detected in the HMBC spectrum and summarized in Table 1.
Table 1.
13C (125 MHz), 1H NMR (500 MHz), and long-range correlation data for dactyloditerpenol acetate (1)a
| N° | δC, typeb | δH (mult., J in Hz)c | HMBCd |
|---|---|---|---|
| 1 | 69.0, CH | 4.00 (br s) | 2, 3, 6, 7 |
| 2 | 125.6, CH | 5.37 (br s) | 1, 4, 6, 20 |
| 3 | 137.3, C | ||
| 4 | 30.3, CH2 | 1.98–1.85 (br m) | 2, 3, 6 |
| 5 | 20.9, CH2 | 1.57 (m) 1.26 (m) |
1, 6 |
| 6 | 46.8, CH | 1.26 (m) | 1, 4, 19 |
| 7 | 31.1, CH | 1.89 (m) | 1, 5, 6, 8, 9, 19 |
| 8 | 31.7, CH2 | 1.26–1.24 (br m) | 7, 10, 19 |
| 9 | 27.1, CH2 | 1.64–1.60 (br m) | 10 |
| 10 | 79.2, CH | 4.85 (dd, 9.7, 3.5) | 8, 9, 11, 12, 18, 21 |
| 11 | 74.1, C | ||
| 12 | 37.6, CH2 | 1.54 (ddd, 16.4, 11.1, 5.5) 1.43 (ddd, 16.8, 11.1, 5.8) |
10, 11, 13, 14 |
| 13 | 22.0, CH2 | 2.12–2.02 (br m) | 12, 14, 15 |
| 14 | 124.1, CH | 5.09 (br t, 7.1) | 12, 13, 16, 17 |
| 15 | 132.1, C | ||
| 16 | 17.6, CH3 | 1.61 (br s) | 14, 15, 17 |
| 17 | 25.7, CH3 | 1.68 (br s) | 14, 15, 16 |
| 18 | 23.5, CH3 | 1.15 (s) | 10, 11, 12 |
| 19 | 14.2, CH3 | 0.79 (d, 6.9) | 6, 7, 8 |
| 20 | 23.1, CH3 | 1.66 (br s) | 2, 3, 4 |
| 21 | 171.2, C | ||
| 22 | 21.1, CH3 | 2.10 (s) | 21 |
Spectra recorded in CDCl3 at 25 °C.
Chemical shifts refer to CDCl3 (δC = 77.0). Carbon types were determined from DEPT NMR experiments.
Chemical shifts refer to CDCl3 (δH = 7.26).
HMBC experiment recorded with a delay of 50 ms.
Figure 1.
Spin systems deduced through the COSY (bold lines) and key 2,3J H→C correlations exhibited by the HMBC spectrum of compound 1.
Further analysis of the 2D-NMR data revealed that the two terminal vinyl methyls at δH 1.61 and 1.68 (H3-16 and H3-17) showed HMBC correlations to δC 124.1 (C-14) and 132.1 (C-15). The sp2 methine at δH 5.09 (H-14) showed a COSY correlation to the aliphatic methylene protons at δH 2.12–2.02 (H2-13), which in turn showed COSY correlations to a pair of diastereotopic protons at δH 1.43/1.54 (H2-12). These NMR data established a terminal isoprene unit, which was expanded to two adjacent asymmetric centers by the observation of HMBC correlations from the methyl singlet at δH 1.15 (H3-18) to δC 79.2 (C-10), 74.1 (C-11) and 37.6 (C-12), and from cross peaks between the oxyacetyl methine proton at δH 4.85 (H-10) and δC 31.7 (C-8), 27.1 (C-9), 74.1 (C-11), 37.6 (C-12), 23.5 (C-18), and 171.2 (C-21). These correlations also established unambiguously the locus of the acetate group at C-10. A series of COSY correlations between H-10 and two contiguous aliphatic methylenes at δH 1.64–1.60 (2H, br m, H2-9) and 1.26–1.24 (2H, br m, H2-8) enabled us to further elongate the aliphatic chain. Key HMBC correlations from H3-19 to C-6 and C-8 helped us place the secondary methyl at C-7. Moreover, HMBC correlations from the oxymethine proton at δH 4.00 (1H, br s, H-1) to δC 137.3 (C-3), 125.6 (C-2), and 31.1 (C-7); from the sp2 methine proton at δH 5.37 (1H, br s, H-2) to δC 46.8 (C-6) and 23.1 (C-20), and from the methyl singlet at δH 1.66 (3H, s, H3-20) to δC 125.6 (C-2) and 30.3 (C-4), revealed the presence in 1 of a 3-methylcyclohex-2-en-1-ol ring moiety similar to that present in laurenditerpenol (2). On the basis of the analyses outlined above, the planar structure of dactyloditerpenol acetate was elucidated as depicted in 1.
Five out of the 22 carbons of dactyloditerpenol acetate are stereogenic, and the location of three of them along a C10 acyclic tail made the configurational assignment somewhat difficult. The 2D NOESY experiment allowed us to reduce the number of possible stereoisomers about the 3-methylcyclohex-2-en-1-ol and monoacetylated 1,2-glycol systems within the structure of 1 (Fig. 2). Indeed, the NOESY cross-peaks between the allylic hydroxyl proton at δH 4.00 (H-1) and the signals at δH 1.89 (H-7) and 0.79 (H3-19), combined with the fact that no cross-peaks were detected between H-1 and H-6 (δH 1.26), clearly pointed to a trans-diaxial orientation of the H-1 and H-6 protons. The coupling constant for H-1 and H-6 (J = 8.7 Hz) measured in Bz-d6 (see Table 3 in Supplementary data) is in full agreement with their trans-diaxial orientation. Based on this characterization and comparison of the C-1/H-1 and C-2/H-2 NMR chemical shift values of natural product 1 with reported spectroscopic data of similar systems,19 it became clear that dactyloditerpenol acetate must possess a 1,6-anti-3-methylcyclohex-2-en-1-ol functionality within its structure. Interestingly, while the cross-peaks of H-6 with H-7 and H3-19 observed in the NOESY spectrum could not be used to unambiguously assign the relative configuration at C-7 due in part to signal overlap near the 1.26 ppm region of the 1H NMR spectrum, the spatial coupling of H-7 with H-10 indicated the 7R* relative arrangement (vide infra).20 Analogously, the anti relationship between the tertiary hydroxy and acetate groups in 1 was deduced from the additional spatial couplings of H-10 at δH 4.85 and the signals ascribed to H3-16 (δH 1.61), H3-18 (δH 1.15), and H2-12 (δH 1.54 and 1.43) (Fig. 2). Moreover, deacetylation of 1 with KCN in MeOH at 25 °C afforded glycol 3 in 26% isolated yield (Scheme 1),21,22 and when comparisons were made of the 13C NMR spectroscopic data of natural product 1 and derivative 3 with reported spectroscopic data of similar systems, we concluded that a 1,2-anti-glycol terpenoid structural unit [δC 79.2 (C-10) and 74.1 (C-11)] in 1 and 78.9 (C-10) and 74.8 (C-11) in 3], would exhibit the characteristic 13C NMR signal difference in chemical shift values.23 Additionally, by comparison of the carbon shifts of C-12 (δC 37.6) and C-18 (δC 23.5) of 1 with those of environmentally similar carbons of previously reported squalene-derived metabolites,23d it was suggested that 1 should possess a 10,11-erythreo relative configuration. On the basis of the above observations, the relative configuration at C-10 and C-11 in 1 was assigned as S* and R*, respectively.
Figure 2.
Key NOESY correlations for the energy-minimized structure of dactyloditerpenol acetate (1).
Scheme 1.
Synthesis of derivative 3.
At this stage, reduction of the number of stereoisomers was accomplished by application of the modified Kishi method to assess the absolute configuration at the two stereogenic centers bearing hydroxyl groups in 1, namely, C-1 and C-11.24 Upon analysis of the 13C NMR chemical shifts of the carbons adjacent to the secondary and tertiary alcoholic centers in the presence of chiral lanthanide shift reagents, the absolute configuration of C-1 and C-11 were established as S and R, respectively (Table 2 and Fig. 3). These data, coupled with our proposed assignments of relative configuration on the basis of proton-proton coupling constant and NOESY data (Fig. 2), established the absolute configuration of all the stereogenic carbons in dactyloditerpenol acetate (1) as 1S, 6S, 7R, 10S, and 11R. Finally, when a comparison was made between the NMR spectroscopic data of dactyloditerpenol acetate (1) with those reported for 4, an unnatural stereoisomer of laurenditerpenol (2) prepared by diastereoselective synthesis, the two sets of chemical shift values for comparable centers were almost identical.6
Table 2.
Assignment of absolute configuration of dactyloditerpenol acetate (1) at 15 mol% per OH of the (R)- and (S)-Eu(tfc)3a
| center | ΔδR = δCXR − δCYR
|
ΔδS = δCXS − δCYS
|
ΔΔδ = ΔδR − ΔδS | ||||
|---|---|---|---|---|---|---|---|
| δCXR | δCYR | ΔδR | ΔCXS | δCYS | ΔδS | ||
| C-1 | 47.31 | 125.64 | −78.33 | 47.28 | 125.77 | −78.49 | +0.16 |
| C-11 | 80.55 | 37.78 | 42.77 | 80.57 | 37.76 | 42.81 | −0.04 |
13C NMR data for 1 collected in CDCl3 solution.
Figure 3.
Empirical rules for the determination of absolute configuration of secondary and tertiary alcohols within dactyloditerpenol acetate (1).
Upon screening for cancer cell cytotoxicity against A2058 melanona and DU-145 prostate cancer cell lines, dactyloditerpenol acetate (1) displayed weak activity as the observed reduction for cell viability was only 67% and 77%, respectively. When tested for in vitro antituberculosis activity, compound 1 was found to inhibit growth of Mycobacterium tuberculosis H37Rv marginally (MIC 59.4 μg/mL). In contrast, as shown in Fig. 4, dactyloditerpenol acetate (1) potently inhibited both TXB2 generation (IC50 0.4 μM), and O2− release (IC50 1.0 μM), with minimal effect on short-term in vitro toxicity (LDH50 > 10 μM). Thus, in our experimental conditions, significant inhibition of LPS-activated rat brain neonatal microglia TXB2 and O2− release generation appeared to result from a pharmacologic rather than a toxic effect of compound 1 on microglia in vitro. Interestingly, compound 1, appears more potent than acetylsalicylic acid (aspirin) (IC50 3.12–10 μM),25 and of similar potency to flurbiprofen (apparent IC50 100 nM),25 two U.S. Food and Drug Administration approved nonsteroidal anti-inflammatory drugs used to treat pain, fever, and inflammation. Thus, our current data support further development of 1 as a novel anti-neuroinflammatory agent designed to modulate activated brain microglia release of TXB2 and O2−, both inflammatory mediators associated with neuroinflammation.10b
Figure 4.
Differential effects of compound 1 on PMA (1 μM)-stimulated O2−, TXB2 and LDH generation by E. coli LPS-activated rat neonatal microglia. The O2−, TXB2 and LDH assays, and the statistical analysis of the data, were performed as described in reference 27. Data corresponds to three independent experiments. ** P< 0.01, *** P<0.001.
Compound 1 appears to be biogenetically related to laurenditerpenol (2), this in spite of the fact that the latter metabolite is an irregular diterpene with the opposite 1,6-syn-3-methylcyclohex-2-en-1-ol ring system.5 As Aplysia dactylomela frequently grazes upon several Laurencia species, it is therefore envisioned that 1 is most likely of dietary origin and that it may actually be a biosynthetically-related Laurencia secondary metabolite.26 Thus, dactyloditerpenol acetate could be derived from “regular” head-to-tail arrangement of four isoprene units followed by bromine-assisted cyclization of a suitable diterpene precursor and subsequent oxidation.2b It is not unlikely that the sea hare further undertakes the acetylation of an ingested algal-derived alcohol.
Supplementary Material
Acknowledgments
We thank E. Avilés, J. J. La Clair, M. Burkart, J. Vicente, J. Asencio, and the crew of the R/V Sultana for providing logistic support during the collection of A. dactylomela. The assistance of G. Torres during molecular-modeling studies is gratefully acknowledged. The City of Hope Comprehensive Cancer Center (COH) and the Institute for Tuberculosis Research at the University of Illinois at Chicago provided in vitro cytotoxicity and antituberculosis activity data, respectively. A. M. S. M. thanks the Office of Research and Sponsored Programs at Midwestern University for generous funding, and Mary L. Hall for expert technical assistance with the O2−, TXB2 and LDH assays. Mass spectral determinations were provided by the Mass Spectrometry Laboratory of the University of Illinois at Urbana-Champaign. Financial support to C.J.-R. was provided by the IFARHU-SENACYT Program of the Republic of Panama. This work was supported by a grant from the NIH-SC1 Program (Grant 1SC1GM086271-01A1) awarded to A. D. R.
Footnotes
Underwater photograph of Aplysia dactylomela, full spectral data in CDCl3 for dactyloditerpenol acetate (1), 1H and 13C NMR spectra for 3, Tables 3 and 4, and Fig. 5. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/
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References and notes
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