3 Fused Tetracyclic

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Klyflaccilides A and B, Diterpenoids with 6/5/8/3 Fused Tetracyclic Carbon Skeleton from the Hainan Soft Coral Klyxum flaccidum Geng Li,†,‡ Heng Li,‡,§ Wei Tang,§ Yue-Wei Guo,*,†,‡ and Xu-Wen Li*,†,‡ †

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State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China ‡ University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China § Laboratory of Immunopharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China S Supporting Information *

ABSTRACT: Two novel polyoxygenated diterpenoids, klyflaccilides A (1) and B (2), featuring an uncommon 6/5/8/3 tetracyclic ring system, were isolated from the Hainan soft coral Klyxum flaccidum. Their structures were established by extensive spectroscopic analysis, X-ray diffraction analysis, and/or a quantum chemical computation method. A plausible biosynthetic relationship of 1 and 2 with their potential precursor 3 was further proposed. Compounds 1 and 3 exhibited moderate anti-inflammatory activity by the inhibition of LPS-induced TNF-α protein release.

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oft corals of the genus Klyxum (order Alcyonacea, family Alcyoniidae), being commonly called cauliflower colt coral, were widely distributed over the tropical Indo-Pacific, including the South China Sea.1 Unlike the other genera such as Sinularia and Sarcophyton, only 11 species of soft corals in the genus Klyxum were discovered and identified and of which only three species have been chemically investigated, including Klyxum simplex, Klyxum mole, and Klyxum flaccidum.1,2 Until now, only approximately 70 secondary metabolites were discovered from Klyxum soft corals, and structurally complex eunicellin-type diterpenoids, featuring by a C-2, C-9 ether bridge on a tetradecahydrobenzo[10]annulene skeleton, were found to be the characteristic compounds, displaying biological activities such as cytotoxic, antibacterial, and anti-inflammatory effects.1,2 Among all the species of Klyxum, K. flaccidum was chemically investigated by Kashman’s group and Sheu’s group from the animals in the Kenya Sea and the Taiwan Sea, respectively, of which Kashman has only obtained two eunicellin-type diterpenoids,1 while Sheu has mainly discovered steroids and cembranoids.2a−c,e In the course of our continuous efforts in the search of novel and bioactive marine natural products from Chinese soft corals,3 and in order to find more diverse and complex eunicellin-type diterpenoids from Klyxum, the title animal was encountered off the coast of Ximao Island, Hainan Province, China, and chemically investigated for the first time, resulting in the isolation and characterization of two structurally unprecedented diterpenoids, namely klyflaccilides A (1) and B (2), and a related new eunicellin-type diterpene, klyflaccilin A (3) (Figure 1). Herein, we report the isolation, structure determination, bioactivity evaluation, as well as plausible biosynthetic pathway of the new compounds. © XXXX American Chemical Society

Figure 1. Structures of compounds 1−4.

The usual workup3 of the Et2O-soluble portion of the acetone extract of K. flaccidum yielded the pure compounds 1 (6.4 mg), 2 (1.0 mg), and 3 (8.5 mg), respectively. Klyflaccilide A (1) was obtained as a colorless crystal. Its molecular formula, C22H34O4, was determined by the HREIMS from the molecular ion peak at m/z 362.2450 ([M]+, calcd 362.2457), indicating six degrees of unsaturation. The 13C NMR, DEPT, and HSQC spectra of 1 revealed 22 carbon signals, including five methyls, four sp3 methylenes, eight sp3 methines (two oxygenated ones at δC 91.9 and 83.1), two oxygenated sp3 quaternary carbons (δC 83.7 and 72.4), one sp2 methylene, and one ester carbonyl (δC 169.7). The diagnostic 1 H and 13C NMR resonances, as well as coupling constants of the connected protons (Table 1), suggested the presence of one disubstituted terminal double bond (δH 4.76 and 4.63, δC 110.7, CH2; δC 147.3, qC). The above functionalities account for two of the six degrees of unsaturation, suggesting the presence of a tetracyclic ring system in 1. Received: June 11, 2019

A

DOI: 10.1021/acs.orglett.9b01998 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters Table 1. 1H NMR (δH) and 13C NMR (δC) Data for 1−3 in CDCl3 1 no. 1 2 3 4 5a 5b 6 7 8a 8b 9 10 11 12a 12b 13a 13b 14 15 16 17a 17b 18 19 20 1’ 2’ a

2

δH, mult (J, Hz)a 2.46 dd (11.6, 8.1) 4.00 s 0.76 0.85 0.73 1.36

m m m m

2.30 1.99 4.20 3.20

dd (15.2, 4.2) m ddd (10.1, 4.1, 2.4) t (9.2)

2.26 2.07 1.75 1.01 1.28 1.29 1.17 4.76 4.63 1.90 0.98 0.78

dt (13.7, 3.1) t (13.7) dd (12.9, 3.4) dd (13.1, 2.9) m s s s s m d (7.0) d (6.9)

1.99 s b

3

δCc

δH, mult (J, Hz)a

δC c

44.4 91.9 83.7 19.7 5.1

2.70 t (7.7) 3.89 s

39.9 92.8 84.0 20.0 5.2

26.4 72.4 45.5 83.1 46.9 147.3 31.3 25.0

0.92 0.82 0.75 1.34

m m m s

2.40 2.04 4.09 3.10

dd (15.3, 3.7) m m dd (10.5, 7.5)

26.4 72.6 47.6

5.49 dd (10.2, 2.3)

80.8 44.5 79.3 126.0

5.99 dd (10.2, 3.2)

135.6

44.3 17.8 24.9 110.7

1.94 1.28 1.19 1.34

m s s s

45.3 18.2 24.1 23.2

28.5 21.9 15.4 169.7 22.3

1.89 m 1.05 d (6.8) 0.85 d (6.8)

30.7 21.2 17.4 169.9 22.4

2.02 s

δH, mult (J, Hz)b 2.57 m 4.04 d (8.9) 5.15 dd (10.1, 7.1) 2.96, m 2.03, m 5.48, t (9.1) 2.53, 1.99, 4.16, 2.39,

dd (13.4, 5.8) d (13.3) dd (5.9, 2.3) d (7.6)

5.40, m 2.17, 1.94, 1.56, 1.45, 1.91, 1.67,

m m m s s s

1.55, m 0.99, d (6.1) 0.85, d (6.2) 2.11 s

δCc 39.7 89.3 77.1 75.0 29.1 123.6 131.6 44.4 80.8 47.1 132.5 121.7 22.9 38.4 22.7 19.3 22.0 29.2 21.5 21.3 170.8 21.5

c

Recorded at 600 MHz. Recorded at 500 MHz. Recorded at 125 MHz. Assignments were deduced by analysis of 1D and 2D NMR spectra.

(δH 0.73), H-4/H-6 (δH 1.36), and H2-5/H-6, and another structural fragment II by the correlation of H2-8/H-9/H-10/ H-1, H-1/H-14/H2-13/H2-12, H-14/H-18/H3-19 and H-18/ H3-20. The connection of the two structure moieties with other quaternary carbons were further determined by the detailed interpretation of the well resolved HMBC spectrum (Figure 2). The HMBC correlations from H2-17 to C-10/C11/C-12 and from H-10 to C-11/C-12/C-14 revealed the presence of a cyclohexane ring (ring A) with a terminal double bond at C-11 and an isopropyl group at C-14. The cross peaks from H3-15 to C-2/C-3/C-4, from H-4 to C-3/C-6/C-7, from H2-5 to C-3, and from H3-16 to C-6/C-7/C-8, suggested a cyclononane ring fused with rings A and D at C-1/C-10 and C4/C-6, respectively. Furthermore, the clear key HMBC correlation from H-2 to C-9, bearing in mind of one remaining degree of unsaturation, suggested an ether bridge between C-2 and C-9, which divided the cyclononane ring into a tetrahydrofuran ring B and an oxocane ring C. Finally, the presence of an acetoxy group at C-3, rather than at C-7, was determined by characteristic carbon resonances of the acetoxy carbon at C-3 (δC 83.7) and hydroxy carbon at C-7 (δC 72.4). Therefore, the planar structure of 1 was determined as shown in Figure 2, featuring by a novel 6/5/8/3 fused tetracyclic ring system. The relative configuration of 1 was established by analysis of its NOESY spectrum (Figure 2). The clear NOE correlations of H-1/H-10 suggested the cis-fused rings A and B, of which H-1 was arbitrarily assigned as β-configuration. Then the cross peaks of H-10/H3-16 and H-10/H-4 indicated the βorientation of the Me-16 and the H-4. Furthermore, the

The planar structure of 1 was further determined by the H−1H COSY and HMBC experiments (Figure 2). Analysis of the COSY spectrum of 1 rapidly identified a cyclopropane ring (part I, ring D) by the clear correlations of H-4 (δH 0.76)/H2-5

1

Figure 2. 1H−1H COSY, key HMBC, and NOESY correlations of compounds 1 and 2. B

DOI: 10.1021/acs.orglett.9b01998 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

especially the typical signals related to the ether bridge (δC/H 92.8/3.89 C-2, δC/H 80.8/4.09, C-9) and the cyclopropane ring (δC/H 20.0/0.92, C-4; δC/H 5.2/0.75 and 0.82, C-5; δC/H 26.4/ 1.34, C-6) in 2, suggesting the same skeleton of the two compounds with the same rings B−D, which was further confirmed by 1H−1H COSY and HMBC experiments (Figure 2). After careful NMR data analysis, the main differences between these two compounds were found to be the presence of a 1,2-disubstituted double bond (δC/H 126.0/5.49, δC/H 135.6/6.01) and an additional oxygenated tertiary carbon (δC 79.3) in 2, instead of the terminal double bond in ring A of 1. The double bond of 2 was deduced to be Δ12,13 by the clear COSY correlations of H-12/H-13/H-14/H-1/H-10, and the oxygenated tertiary carbon at δC 79.3 was revealed to be C-11 by the well resolved HMBC cross peaks from H-13 (δH 6.01) to C-11 (Figure 2). Finally, the presence of a hydroperoxyl group at C-11 was supported by its downfield shifted 13C NMR datum (δC 79.3) compared to the typical hydroxyl carbons and as well as the molecular formula of 2. The relative configuration in rings B−D of 2 was assigned to be the same as that of 1 by the clear NOE correlations of H-1/ H-10, H-10/H3-16, H3-16/H-5b, H3-16/H-4, H-10/H-4, H-2/ H3-15, H3-15/H-5a, H3-15/H-6, and H-17/H-9 (Figure 2) to be 1R*,2R*,3R*,4S*,6S*,7S*,9R*,10S*. The stereochemistry of C-11 and C-14 in ring A was determined as 11R*,14S* by the characteristic NOE cross peaks of H3-17/H-9 and H-14/ H-2. Because of the co-occurrence of 1 and 2 and the same structural features of their rings B−D, the absolute stereochemistry of 2 was tentatively determined to be 1R,2R,3R,4S,6S,7S,9R,10S,11R,14S on a biogenetic consideration. The molecular formula of klyflaccilin A (3) was deduced to be C22H34O4 by HREIMS (m/z 362.2452 [M]+, calcd 362.2452), indicating six degrees of unsaturation. The 1H and 13C NMR data of 3 showed high similarity to those of hirsutalin G (4), an eunicullin-type diterpenoid previously isolated from Cladiella hirsuta,4 except for the absence of a hydroxy at C-19 in 3. In fact, the chemical shifts in C-18 and C-19 were upfield shifted from δC 36.4 and 66.3 in 4 to δC 29.2 and 21.5 in 3, respectively, bearing in mind the 16 mass units less molecular weight of 3 than that of 4, indicating that 3 should be a 19-dehydrxyl derivative of 4. The planar structure and absolute configuration of 3 were further unambiguously determined as shown in Figure 1 by single crystal X-ray diffraction analysis with Ga Kα radiation [Flack parameter was −0.04 (7)] (Figure 5, CCDC 1921884). The scaffolds of klyflaccilides A and B (1 and 2) are unprecedented and formally different from the co-occurring eunicellin-based diterpene klyflaccilin A (3). While intriguingly, 1−3 share the common rings A and B, sparking our curiosity to explore the biogenetic origin of 1 and 2 as well as the possible biosynthetic relationship between 1−3. A plausible biosynthetic connection from 3 to 1 and 2 was thus proposed after detailed analysis. As outlined in Scheme 1, under acidic conditions, the electron would be delivered from the olefin Δ6,7 to the acetoxy group at C-4 on 3 to release an acetic acid and form a carbon cation at C-7 toward the intermediate 3a, which could rapidly catch a molecule of water to obtain compound 1a. Further, on the one hand, 1a would undergo a double bond migration from Δ11,12 to Δ11,17 and an acylation of 3-hydroxyl toward the new compound 1; on the other hand, 1a could also be oxidized to 2a, followed by a Δ12,13 and a hydroperoxyl formation5 as well as an acylation to

NOE correlations of H3-16/H-5b, H3-16/H-4, H-5a/H-6, H5a/H3-15, H3-15/H-2, H3-15/H-9, and H-2/H-14 were indicative of the α-orientation of H-2, H-6, H-9, H-14, and Me-15. With the relative configuration having been established, the remaining task was the absolute stereochemistry of 1, which was initially determined by time-dependent density functional theory−electronic circular dichroism (TDDFT-ECD) calculation because this method has been verified to be reliable and powerful for the determination of natural products.3 As shown in Figure 3, the Boltzmann-averaged ECD spectrum of

Figure 3. Experimental ECD spectrum of 1 (black) and the calculated ECD spectrum of (1R,2R,3R,4S,6S,7S,9R,10R,14R)-1 (red).

(1R,2R,3R,4S,6S,7S,9R,10R,14R)-1 displayed an ECD curve which highly matched to the experimental curve of 1, allowing the assignment of the absolute stereochemistry of 1 (see Supporting Information for detailed calculation procedure). To further confirm the structure of 1, we attempted and fortunately obtained its suitable quality crystals from the recrystallization of 1 in methanol, which allowed a successful performance of X-ray crystallography study using Ga Kα radiation (λ = 1.34139 Å). Analysis of the X-ray data unambiguously confirmed the planar structure of 1 and its absolute configuration as 1R,2R,3R,4S,6S,7S,9R,10R,14R [Flack parameter was −0.12 (15)] (Figure 4, CCDC 1921888). Klyflaccilide B (2) was isolated as an optically active colorless oil with the chemical formula of C22H34O6 as revealed by the HRESIMS ion peak at m/z 393.2277, ([M − H]−, calcd 393.2283), indicating six degrees of unsaturation. The 1H and 13 C NMR data of 2 were reminiscent of those of 1 (Table 1),

Figure 4. Perspective ORTEP drawing of X-ray structure of 1 (displacement ellipsoids are drawn at the 50% probability level). C

DOI: 10.1021/acs.orglett.9b01998 Org. Lett. XXXX, XXX, XXX−XXX

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Letter

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01998. General information on the experiment, experimental procedures, characterization data, biological activity assays, and NMR spectra for all the new compounds (PDF) Accession Codes

CCDC 1921884 and 1921888 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

Figure 5. Perspective ORTEP drawing of X-ray structure of 3 (displacement ellipsoids are drawn at the 50% probability level).

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Scheme 1. Plausible Biosynthetic Connection from Compound 3 to the New Skeleton of 1 and 2

AUTHOR INFORMATION

Corresponding Authors

*X.-W. Li: E-mail, [email protected]. *Y.-W. Guo: E-mail, [email protected]. ORCID

Xu-Wen Li: 0000-0001-7919-9726 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research work was financially supported by the National Key Research and Development Program of China (2018YFC0310903), the Drug Innovation Major Project (2018ZX09711-001-001-009), the National Natural Science Foundation of China (NSFC) (81520108028, 21672230, 81603022, 41676073), the NSFC/CNRS joint project (81811530284), the NSFC-Shandong Joint Fund for Marine Science Research Centers (U1606403), and the SKLDR/ SIMM Project (SIMM1903ZZ-04). X.-W. Li is also thankful for the support of the “Youth Innovation Promotion Association” (2016258) from Chinese Academy of Sciences and SA-SIBS Scholarship Program. We thank Prof. X.-B. Li from Hainan University for the taxonomic identification of the soft coral material.

afford the new compound 2. Such a rational biosynthetic proposal further confirmed the absolute configuration of compound 2. In the anti-inflammatory assay, 1 and 3 displayed moderate inhibition of LPS-induced TNF-α protein release in RAW264.7 macrophages with IC50 values of 35.3 and 25.9 μM, respectively. Interestingly, both compounds showed no obvious cytotoxicity against RAW264.7 cells, with CC50 values over 50 μM, which is worth further pharmacological investigation. In summary, this is the first chemical investigation of K. flaccidum from Ximao island in South China Sea. Two novel diterpenoids, klyflaccilides A and B (1 and 2), with unprecedented 6/5/8/3 tetracyclic ring systems, and a new eunicellin-type diterpene (3), were isolated and fully characterized. The discovery of 1−3 has expanded the diversity and complexity of marine diterpenes, and the interesting antiinflammatory potential of 1 and 3 with low toxicity would impel the biomimetic total synthesis, inspired by our biosynthetic hypothesis, and structure modification for their further medicinal applications.

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REFERENCES

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DOI: 10.1021/acs.orglett.9b01998 Org. Lett. XXXX, XXX, XXX−XXX