New Bicyclic Cembranoids from the South China Sea Soft Coral Sarcophyton trocheliophorum

Abstract

Nine new bicyclic cembranoids, sarcophytrols M–U(1–9), were isolated from the South China Sea soft coral Sarcophyton trocheliophorum as minor components, along with one known related cembranoid 10. Their structures were elucidated by detailed spectroscopic analysis and chemical conversion. The chemical structures of these metabolites are characterized by the different patterns of the additional cyclization within the 14-member skeleton, which leading to the formation of furan, pyran, oxepane, and peroxyl rings, respectively. Among them, sarcophytrols R and S(6 and 7) share a rare decaryiol skeleton with an unusual C12/C15 cyclization. In addition, the absolute configurations of sarcophytrols M and T(1 and 8) were determined by the modified Mosher’s method. The research of these new secondary metabolites provided a further understanding of the diversity of cyclized cembranoids from the title species.

Cyclization is an extraordinary artistry that nature turn the simple cembranoids to a prodigious variety of structurally novel compounds, and it often links to a network of oxygenation process¹, which lead to the formation of epoxyl²,³,⁴,⁵, furan⁴,⁶,⁷,⁸, pyran⁵,⁷,⁸, and oxepane⁵,⁹,¹⁰. Among them, the decaryiol-type cembranoids, characterized by a 6:14-fused ring system, are one of the most amazing examples. They are biogenetically derived from cembranoids by an uncommon transannular etherfication between C-12 and C-15 position and rarely discovered in nature. Actually, there are only four decaryiol-type cembranoids, decaryiols A–D, that have been reported from soft coral Sarcophyton decaryi¹¹ and Lobophytum sp¹². before.

It is widely recognized that the South China Sea soft coral S. trocheliophorum(phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea, family Alcyoniidae) contains unusual cembranoids with a diversity of cyclizations¹³. Interestingly, these cembranoids are regarded as chemical defense compounds against predators such as other corals and fishes as well as against settlements of microorganisms¹⁴,¹⁵. In our ongoing studies of the chemistry and biology of the Hainan soft corals, we have considered the soft coral S. trocheliophorum as an important issue. A previous study we conducted on a collection of the title animal from Hainan had resulted in the isolation of a series of new cembranoids and cembranoid derivatives³,⁴,⁹,¹⁶. Many of these new secondary metabolites with different patterns of cyclizations showed significant inhibitory activity against human protein tyrosine phosphatase 1B(PTP1B) enzyme³,⁴,¹⁶, a promising drug target for the treatment of type 2 diabetes and obesity¹⁷. To accumulate these compounds for further biological study, we made a different collection of the same species from the same location(Yalong Bay, Hainan Province). Surprisingly, our chemical investigation on the crude acetone extract of the title animal showed the absence of aforementioned compounds existed in the former collection, while resulting to the discovery of three new capnosane diterpenoids¹⁸. We have now focused our attentions on the cembrane-type metabolites with diverse kinds of cyclizations from the latter collection to find more chemically interesting and biologically active compounds. This continuous investigation has now resulted in the isolation of nine new cembranoids(1–9), together with one known related cembranoid 10(Fig. 1). Among them, the characteristic chemical features of them are the diverse types of cyclized rings: furan rings possessed by sarcophytrols M–P(1–4), pyran rings formed at different positions in sarcophytrols Q–S(5–7), while oxepane and peroxyl rings appeared in sarcophytrols T(8) and U(9), respectively. In addition, sarcophytrols R and S(6 and 7) share a rare decaryiol skeleton with an unusual C12/C15 cyclization. We herein report the isolation and structure elucidation of these new cembranoids.

Figure 1. Structures of compounds 1–13.

Results and Discussion

Samples of S. trocheliophorum(dry weight 400 g) were extracted exhaustively with acetone, and the extract was partitioned between water and Et₂O. The Et₂O solvable fraction was subjected to repeated chromatography as usual work³,⁴,⁹,¹⁶, to afford ten pure metabolites, compounds 1–10(Fig. 1). A preliminary NMR analysis revealed that all the new molecules shared the same cembrane skeleton. Among them, the known compound was readily identified as sarglaucol(10)¹⁹ by comparison of its spectral data and [α]_D values with those reported in the literatures.

The HRESIMS of sarcophytrol M(1) established the molecular formula C₂₀H₃₄O₄. ¹H and ¹³C NMR spectra of 1(Tables 1 and 2) were reminiscent of a known cembranoid,(2E,7E)-4,11-dihydroxy-1,12-oxidocembra-2,7-diene(11), previously isolated from Sinularia ovispiculata²⁰. The distinct difference between them was the presence of a hydroxyl at C-15(δ_C 72.4) in 1, which was further confirmed by the observation of HMBC correlations from H₃-16(δ_H 1.12)/H₃-17(δ_H 1.06) to C-15(Fig. 2). Similar to 11, a trans-disubstituted olefin at C-2/C-3 was recognized by the doublet coupling constant(15.4 Hz) of H-2(δ_H 5.62) and H-3(δ_H 5.92), while the chemical shift of Me-19(δ_C < 20 ppm) indicated the 7E assignment in 1²⁰,²¹. The relative configurations of the stereogenic centers were determined by NOE relationships(Fig. 2), which exhibited similar key cross-peaks to those in the cembranoids sinulariols D and F⁷. The NOE correlation between H-3 and H-11(δ_H 3.54) implied that the trans-disubstituted olefin and the hydroxyl group at C-11 were hind the same face toward the five-membered ring. Thus, Me-20 was oriented to the same face with the isopropyl group, and tentatively assigned to be α-oriented. In addition, the significant NOE correlations of H-2/H₃-16 and H-2/H-14b(δ_H 1.67) suggested that H-2 and the isopropyl group to be on the same side of 1. The trans-disubstituted olefin group favoring ‘up’ or ‘down’-orientation toward the 14-membered ring, and thus H-3 was oriented to ‘up’ face. The configuration of H-11 was assigned as β mainly on the basis of the aforementioned strong NOE correlation between H-11 and H-3 and the very weak NOE correlation between H-11 and H₃-20(δ_H 1.11). According to the NOE correlation from H-3 to H₃-18(δ_H 1.27) and the lack of correlation from H-2 to H₃-18, we tentatively assigned the configuration of H₃-18 to β face. To obtain its absolute configuration, two aliquots of compound 1 were treated with(R)- and(S)-α-methoxy-α-trifluoromethylphenyl acetyl(MTPA) chlorides to obtain the(S)- and(R)-esters, respectively. Analysis of Δδ^SR values(δ_S - δ_R) observed for the signals of the protons close to 11-OH(Fig. 3) indicated the S configuration at this carbon. Consequently, the absolute configuration of compound 1 was determined as(1S, 4S, 11S, 12R).

Table 1. ¹H NMR spectroscopic data for compounds 1–9^a.

No.	1	2	3	4	5	6	7	8	9
1
2	5.62 d(15.4)	5.68 d(15.6)	5.64 d(15.4)	5.55 d(15.7)	5.27 d(16.0)	5.46 dd(7.3, 1.4)	5.36 d(7.0)	6.09 d(10.9)	6.33 d(16.6)
3	5.92 d(15.4)	5.88 d(15.6)	6.00 d(15.4)	6.07 d(15.7)	5.98 d(16.0)	4.84 d(7.3)	4.19 d(7.0)	5.66 d(10.9)	5.97 d(16.6)
4
5	1.82 m	1.83 ddd(13.4 11.5, 1.9)	1.82 m	1.82 m	1.90 m	2.35 m	1.92 m	2.22 m	1.99 ddd(13.6, 4.8, 2.5)
	1.57 m	1.59 ddd(13.4, 13.2, 8.1)	1.58 m	1.57 m	1.60 m	2.30 m	1.69 m	1.98 m	1.87 dd(13.6, 4.8)
6	2.26 t(11.4)	2.27 m	2.28 t(11.6)	2.27 t(11.3)	2.20 m	2.36 m	2.17 m	2.25 m	2.15 m
	2.15 m	2.16 m	2.49 m	2.47 m		2.21 m		2.03 m	1.54 m
7	5.19 dd(10.9, 3.8)	5.20 dd(11.3, 4.5)	5.44 dd(10.9, 4.0)	5.21 dd(9.9, 4.5)	5.21 t(7.3)	5.32 t(6.6)	5.30 t(5.9)	5.05 m	4.71 dd(12.0, 2.9)
8
9	2.09 m	2.10 dd(12.6, 2.4)	2.09 m	2.10 m	2.19 m	2.11 dd(13.2, 3.6)	2.12 m	2.40 m	2.42 dd(13.4, 6.1)
		2.07 m			2.01 m	2.11 dt(13.2, 4.0)		2.16 m	2.13 m
10	1.89 dt(13.3, 2.5)	2.14 m	1.88 dt(13.2, 2.4)	1.88 m	1.90 m	1.74 ddt(14.0, 3.6, 1.6)	1.85 m	4.26, dd(20.1, 3.0)	2.00 m
	1.35 m	1.83 ddd(13.2, 8.1, 1.9)			1.39 m	1.20 m		1.25 m	1.41 m
11	3.54 d(9.5)	3.53 d(9.6)	5.09 d(9.6)	3.54 d(9.4)	3.34 t(4.06)	3.31 d(10.0)	3.57 d(10.5)	5.07 m	3.44 dd(10.2, 2.2)
12
13	1.77 m	1.74 m	1.79 m	1.79 m	1.70 m	2.12 m	2.17 m	2.79 dd(19.6, 11.2)	1.62 m
		1.72 m			1.51 m	1.87 m	1.91 m	2.13 m
14	2.41 td(12.1, 7.7)	2.39 m	2.45 td(12.1, 7.6)	2.41 td(12.0, 7.7)	2.19 m	2.49 ddd(17.5, 11.7, 10.0)	2.85 dd(17.8, 9.8)	2.60 ddd(13.6, 10.2, 8.2)	2.49 m
	1.67 m	1.72 m	1.66 m	1.67 m	1.53 m	2.25 m	2.40 dd(17.8, 9.0)	2.35 t(12.1)	1.93 m
15
16	1.12 s	1.15 s	1.15 s	1.13 s	1.12 s	1.34 s	1.38 s	1.38 s	1.77 s
17	1.06 s	1.06 s	1.07 s	1.10 s	1.07 s	1.28 s	1.31 s	1.35 s	1.73 s
18	1.27 s	1.30 s	1.27 s	1.33 s	1.36 s	5.13 s	1.27 s	1.75 s	1.28 s
						4.93 s
19	1.68 s	1.68 s	1.67 s	1.72 s	1.65 s	1.60 s	1.61 s	1.38 s	5.11 d(1.8)
									5.09 brs
20	1.11 s	1.09 s	1.12 s	1.12 s	1.16 s	1.04 s	1.06 s	4.25 d(16.4)	1.28 s
								4.07 dd(16.4, 1.5)
OMe		3.23 s
OAc			2.04 s

^aSpectra measured at 500 MHz in CDCl₃.

Table 2. ¹³C NMR spectroscopic data for compounds 1–9^a, 11 and 12.

No.	11[12]	1	2	3	4	5	12[22]	6	7	8	9
1	88.2, s	91.3, s	90.8, s	91.8, s	91.0, s	81.0, s	77.9, s	141.5, s	146.3, s	140.2, s	130.3, s
2	129.0, d	128.1, d	129.4, d	127.9, d	130.4, d	122.1, d	125.0, d	123.5, d	121.5, d	123.5, d	129.0, d
3	137.7, d	138.4, d	137.3, d	138.6, d	137.0, d	142.3, d	140.9, d	75.2, d	74.0, d	123.4, d	132.8, d
4	74.4, s	74.5, s	74.5, s	74.5, s	72.5, s	74.3, s	74.2, s	147.9, s	76.8, s	137.2, s	79.4, s
5	42.9, t	43.9, t	43.6, t	43.6, t	41.8, t	44.5, t	44.5, t	28.3, t	36.9, t	40.0, t	33.1, t
6	28.4, t	24.4, t	24.4, t	24.4, t	24.4, t	23.8, t	23.6, t	22.8, t	21.5, t	26.0, t	24.2, t
7	128.4, d	129.3, d	129.0, d	130.1, d	129.4, d	129.8, d	129.1, d	124.8, d	126.7, d	125.5, d	85.3, d
8	133.6, s	133.5, s	133.5, s	131.9, s	133.3, s	132.8, s	133.1, s	134.6, s	135.5, s	133.5, s	145.9, s
9	34.6, t	35.5, t	35.3, t	35.4, t	35.3, t	38.6, t	37.5, t	34.9, t	34.4, t	50.2, t	31.1, t
10	29.6, t	29.4, t	29.5, t	30.7, t	29.5, t	26.2, t	25.8, t	26.5, t	26.7, t	64.8, d	31.0, t
11	76.2, d	76.4, d	76.4, d	77.3, d	76.2, d	75.0, d	74.8, d	68.8, d	69.5, d	124.8, d	76.9, d
12	84.6, s	85.4, s	85.4, s	84.2, s	85.5, s	70.6, s	70.6, s	74.8, s	75.3, s	145.9, s	74.4, s
13	36.6, t	36.6, t	36.2, t	34.8, t	36.6, t	37.0, t	37.2, t	28.7, t	28.9, t	32.0, t	37.8, t
14	35.2, t	31.0, t	31.9, t	30.7, t	30.9, t	28.1, t	30.3, t	18.9, t	20.4, t	24.2, t	25.1, t
15	39.2, d	72.4, s	78.4, s	72.4, s	72.2, s	74.6, s	39.5, d	74.3, s	74.4, s	78.9, s	123.0, s
16	18.4, q	24.4, q	20.2, q	24.3, q	24.4, q	23.7, q	17.1, q	31.2, q	30.5, q	24.0, q	20.7, q
17	17.6, q	26.0, q	21.9, q	25.9, q	25.9, q	24.4, q	17.0, q	29.5, q	29.2, q	27.9, q	20.5, q
18	29.3, q	28.4, q	28.7, q	28.0, q	28.7, q	27.7, q	29.0, q	111.4, t	22.9, q	16.5, q	25.5, q
19	16.7, q	16.3, q	16.4, q	16.0, q	16.4, q	14.8, q	14.8, q	15.6, q	16.8, q	16.1, q	119.1, t
20	20.0, q	19.4, q	19.3, q	20.6, q	19.4, q	19.1, q	19.5, q	22.6, q	22.6, q	62.4, t	25.9, q
OMe			50.6, q
OAc				21.3, q
				171.0, s

^aSpectra measured at 125 MHz in CDCl₃.

Figure 2. Selected key COSY, HMBC and ROESY correlations for compound 1.

Figure 3. The Δδ^SR [Δ(δ_S-δ_R)] data for the MTPA esters of compounds 1 and 8.

Sarcophytrol N(2) has a molecular formula of C₂₁H₃₆O₄, as established by HRESIMS and NMR data, 14 mass units more than that of 1. The ¹H NMR data of 2(Tables 1 and 2) were closely reminiscent to those of 1, except for the newly appeared signal at δ_H 3.23(3H, s), which suggested the presence of an additional methoxyl group in 2, consistent with one carbon resonance at δ_C 50.6(q) in its ¹³C NMR spectrum. The introduction of the methoxyl group in 2 resulted in the significant downfield shift of C-15 from δ_C 72.4(s) in 1 to 78.4(s) in 2 and the upfield shifts of C-16/C-17. Furthermore, the methoxyl group was secured at C-15 by a diagnostic HMBC correlation from methoxyl group to C-15. Furthermore, ROESY correlations of compound 2 were similar with those of 1. On the basis of above evidences, compound 2 was identified as 15-methoxyl derivative of 1.

Sarcophytrol P(3) possesses a molecular formula of C₂₂H₃₆O₅ as determined by HRESIMS data, 42 mass units more than that of 1. The NMR spectroscopic features of 3(Tables 1 and 2) mostly resembled those of 1. In fact, the only difference was at C-11 position, where the hydroxyl group(δ_H 3.54; δ_C 76.4) in 1 was replaced by an acetyl(δ_H 5.09, 2.04; δ_C 77.3, 21.3, 171.0) in 3. A detailed 2D NMR analysis further confirmed the planar structure of compound 3. As the patterns of ROESY correlations of 3 were similar with those of 1, accordingly the structure of 3 was established as the 11-acetyl derivative of 1. The chemical conversion of 1 into 3 by simple acetylation in Ac₂O/pyridine further indicated the absolute configuration of 3 is the same as 1.

Sarcophytrol P(4) was isolated as an optically active colorless oil with molecular formula C₂₀H₃₄O₄, the same as 1. The NMR spectra of 4(Tables 1 and 2) were strongly reminiscent of those of 1 and a careful 2D NMR analysis suggested that they share the same gross structure. In fact, the only difference was found in the segment from C-2 to C-5, where ¹³C NMR chemical shifts of C-3, C-4 and C-5 in 4 were all upfield shifted while that of γ-carbon C-2 was downfield shifted with respect of 1. This evidence clearly suggested that the relative configuration of the hydroxyl group at C-4 of 4 was different from that of 1. The NOE interactions between H-2(δ_H 5.55) and H₃-18(δ_H 1.33) in 1 rather than H-3(δ_H 6.07) and H₃-18 in 4 confirmed this assignment. Herein, 4 was 4-epimer of 1.

Sarcophytrol Q(5) possesses the same molecular formula C₂₂H₃₆O₅ as 4, established by HRESIMS, whereas its NMR data(Tables 1 and 2) were almost identical to those of sinulariol Z(12)²². The distinction was attributed to the NMR data of 5 presenting an extra hydroxyl group at C-15, which was further confirmed by the observation of HMBC correlations from H₃-16(δ_H 1.12)/H₃-17(δ_H 1.07) to C-15(δ_C 74.6). Due to the presence of the 15-OH, ¹³C NMR chemical shifts of C-15, C-16, C-17, and C-1 were all downfield shifted reasonably while those of γ-carbons 2 and 14 were upfield shifted with respect of those of 12. Furthermore, the significant NOE interactions between H-3(δ_H 5.98) and H-11(δ_H 3.34) and the lack of NOE correlations of H-11/H₃-16(δ_H 1.12), H-11/H₃-17(δ_H 1.07), H-11/H₃-20(δ_H 1.16) confirmed the isopropyl group and H₃-20 were in opposite face against H-11 in the hexatomic ring. Additional strong NOE interaction between H-3 and H₃-18 indicated H₃-18 at the same side as H-3 in the case of 5. Thus, 5 had the same relative configuration relationships as those of 12, which were further confirmed by the closely identical spectral data for the segment C-3–C-13.

Sarcophytrol R(6) was obtained as a colorless, optically active oil {[α − 13.3(c 0.06, MeOH)}. HRESIMS and ¹³C NMR spectra analysis established the molecular formula of 6 as C₂₀H₃₄O₄. Thus, four degrees of unsaturation were determined for 6. The NMR data(Tables 1 and 2) revealed the presence of two trisubstituted double bonds(δ_H 5.36, δ_C 146.33, 121.54; δ_H 5.30, δ_C 126.73, 135.48), which accounted for two degrees of unsaturation. The remaining two degrees of unsaturation strongly indicated that 6 has a bicyclic structure with a carbocyclic ring bridged by oxygen. The ¹³C NMR spectrum of 6 contained five oxygenated carbon signals at δ_C 74.04(CH), 76.80(C), 69.49(CH), 75.32(C) and 74.44(C), which were assigned by 2D NMR spectrum to C-3, C-4, C-11, C-12 and C-15, respectively. In order to define the carbon atoms linked to these functionalities, 2D NMR experiments(in DMSO-d₆) of 6 were measured. The formation of an ether bridge across C-12 and C-15 was deduced from the observation of OH-3(δ_H 4.31), OH-4(δ_H 3.95) and OH-11(δ_H 4.10). The location of these three –OH groups are determined by the ¹H-¹H COSY correlations of H-3(δ_H 3.90) with OH-3 and H-11(δ_H 3.40) with OH-11, and the HMBC correlations from OH-4 to C-3(δ_C 72.9), C-4(δ_C 75.1), C-5(δ_C 38.1).

The E geometry of the Δ¹ and Δ⁹ double bonds was deduced by the ROESY correlations of H-2(δ_H 5.36)/H₃-17(δ_H 1.31) and of H-7(δ_H 5.30)/H₂-9(δ_H 2.12)(Fig. 4). Moreover, H-14a(δ_H 2.85) correlated with H-3(δ_H 4.19) and H-11(δ_H 3.57), suggesting H-3 and H-11 to be on the same side of 6. When H-3 was assigned tentatively as β-orientation, H-11 was accordingly oriented in β-face. The presence of the cross-peak between H-2 and H₃-18(δ_H 1.27) in combination with the absence of the correlations of H-3/H-2 and H-3/H₃-18 in the ROESY spectrum, clarified H₃-18 and H-2 to be oriented in opposite to H-3. Finally, the α-oriented H₃-20 was tentatively deduced by the absence of correlations between H₃-20 and H-11.

Figure 4. Selected key ROESY correlations for compounds 6 and 7.

Sarcophytrol S(7) was isolated as an optically active, colorless oil {[α − 39.3(c 0.07, MeOH)}. Its molecular formula, C₂₀H₃₂O₃, was established by HRESIMS, 18 units less than that of 6. Careful comparison of NMR data of 7 and 6(Tables 1 and 2) revealed that the former differs from the latter only by the presence of a terminal methylene group(δ_H 5.13, 4.93; δ_C 111.37, 147.92) in 7 instead of a methyl(δ_H 1.27; δ_C 22.91) and a oxygenated quarternary carbon(δ_C 76.8) in 6, in agreement with 18 mass units difference between them, while the rest of the molecules was the same. Base on the HMBC correlations between H₂-18(δ_H 5.13, 4.93) and C-3(δ_C 75.24) and C-5(δ_C 28.32), the terminal methylene group was located at C-4. Moreover, ¹H-¹H COSY, HSQC, and HMBC experiments allowed the unambiguous definition of the structure of 7. Analogously to 6, the relative stereochemistry of three chiral centers at C-3, C-11 and C-12 was elucidated to be the same as those of 6 by the ROESY experiments(Fig. 4).

The HRESIMS of sarcophytrol T(8) established the molecular formula C₂₀H₃₀O₂, indicating six degrees of unsaturation. The presence of four double bonds(δ_H 6.09, δ_C 140.2, 123.5; δ_H 5.66, δ_C 137.2, 123.4; δ_H 5.05, δ_C 125.5, 133.5; δ_H 5.07, δ_C 124.8, 145.9) accounted for four degrees of unsaturation. The remaining two degrees of unsaturation strongly indicated that 8 has a bicyclic structure. A HMBC relationship between H₂-20(δ_H 4.25 and 4.07) and C-15(δ_C 78.9) ascertained that an ether bridge formed across C-20 and C-15. Key NOE correlations from H-3(δ_H 5.66) to H-5b(δ_H 1.98) and H-14a(δ_H 2.60) and from H-2(δ_H 6.09) to H₃-18(δ_H 1.75) and H₃-17(δ_H 1.35) suggested the olefinic geometries were assigned to 1E and 3E. The chemical shift of Me-19(δ_C < 20 ppm) indicated the 7E assignment²¹. The additional hydroxylation at C-10 was supported by the ¹H-¹H COSY correlations of H-10(δ_H 4.26)/H-11(δ_H 5.07) and H-10/H-9(δ_H 2.40 and 2.16). Since compound 8 contained a secondary alcohol at C-10, its absolute configuration of C-10 was determined to be S, by applying the modified Mosher’s method following the same protocol as used for 1(Fig. 3).

Sarcophytrol U(9), a colorless oil, had a molecular formula of C₂₀H₃₂O₄ established by HRESIMS(m/z 359.2199 [M + Na]⁺) and NMR data, indicating five degrees of unsaturation. The presence of a secondary and a tertiary hydroxyl group was clearly deduced from NMR signals at δ_C 76.9(d), 74.4(s) and δ_H 3.44(1H, dd, J = 10.2, 2.2 Hz, H-11). One additional terminal double bond was inferred by ¹³C NMR signals at δ_C 145.9(C) and 119.1(CH₂). The conjugated olefinic group was also evident by four sp² carbon signals in the ¹³C NMR spectrum at δ_C 130.3(s), 129.0(d), 132.8(d), and 123.0(s), and two olefinic doublets in the ¹H NMR spectrum at δ_H 6.33(1H, d, J = 16.6 Hz, H-2) and 5.97(1H, d, J = 16.6 Hz, H-3). Detailed analysis of the 2D spectra(Fig. 5), allowed assigning all the chemical shifts in the NMR spectra, which led to the cembrane skeleton of 9. An exo-CH₂, the conjugated olefinic groups and the 14-member ring of cembrane skeleton accounted for four degrees of unsaturation. As a consequence, there must be two oxygen atoms unassigned had to be ascribed to a peroxide bridge that linked at C-4 and C-7, respectively, to complete the required unsaturation degrees of five. In addition, the analysis of the NMR data of 9, in comparison with that of a known cembranoid,(1S,2E,4S,6R,7S,8R,11S)-8,11-epidioxy-2,12(20)-cembradiene-4,6,7-triol(13), previously reported from Greek tobacco²³, confirmed the partial structure from C-4-C-9. Thus, the gross planar structure of 9 was determined as a 4,7-epidioxy-15(1),2,8(19)-cembratriene-11,12-diol.

Figure 5. Selected key COSY, HMBC and ROESY correlations for compound 9.

The relative stereochemistry of the chiral centers at C-4, C-7, C-11 and C-12 was established by a ROESY experiment running on 9. In MM2 energy-minimized conformation(Fig. 5), H-7 was suggested to be axial position toward the hexatomic ring(β-orientation). The diagnostic correlations between H-7(δ_H 4.71) and H-5a(δ_H 1.87) as well as H₃-18(δ_H 1.28) and H-5a suggested H-5a and H₃-18 were determined as axial and equatorial position, respectively(β-orientation). The ROESY correlation of H-11(δ_H 3.44) with H₃-20(δ_H 1.28) suggested the H-11 and H₃-20 are in the same face, which was compatible with that of the co-exist cembranoid sarglaucol(10)¹⁹. However, the relationship of H-7 and H-11 cannot be determined from the ROESY spectrum.

In conclusion, nine new cembranoids, sarcophytrols M–U(1–9), were isolated from the South China Sea soft coral Sarcophyton trocheliophorum, along with one known related cembranoid 10. Among them, the characteristic chemical features of them are the diverse types of cyclized rings: furan rings possessed by sarcophytrols M-P(1–4), pyran rings formed in sarcophytrols Q-S(5–7), while oxepane and peroxyl rings appeared in sarcophytrols T(8) and U(9), respectively. In addition, sarcophytrols R and S(6 and 7) share a rare bicyclic skeleton of the decaryiol-type. These group of diterpenes were first reported from the same genus of soft coral S. decaryi¹¹, and described so far only from the species of soft corals Lobophytum sp.¹². The co-isolation of these diterpenes is an example of the productivity of the title animal.

In light of a wide range of biological activities and pharmacological properties of cembranoids¹³, we performed in vitro investigation of inhibitory activity against human protein tyrosine phosphatase 1B(PTP1B) enzyme, a promising drug target for the treatment of type 2 diabetes and obesity¹⁷, for compounds 1–10, since the previously reported cembranoids from the title animals displayed significant PTP1B inhibitory activity³,⁴,¹⁶. Unfortunately, the bioassay result showed that none of the tested compounds exhibited interesting PTP1B inhibitory activities. In addition, the antitumor and antibacterial activities were also tested for compounds 1–10. However, they exhibited neither cytotoxicities against the human tumor cell lines HL-60 and K-562, nor antibacterial activity against Pseudomonas aeruginosa.

Methods

General experimental procedures

Optical rotations were measured on a Perkin-Elmer 341 polarimeter. IR spectra were recorded on a Perkin-Elmer 577 spectrometer with KBr disks. HRESIMS spectra were recorded on a Waters-Micromass Q-TOF Ultima Global electrospray mass spectrometer. NMR spectra were measured on a Bruker Avance III 500 and Varian INOVA 600 spectrometers with the residual CHCl₃(δ_H 7.26 ppm, δ_C 77.0 ppm) as internal standard. Chemical shifts are expressed in δ(ppm) and coupling constants(J) in Hz. ¹H and ¹³C NMR assignments were supported by ¹H-¹H COSY, HSQC, and HMBC experiments. Commercial Silica gel(Qing Dao Hai Yang Chemical Group Co., 200–300 and 400–600 mesh), C18 reversed-phase silica gel(150–200 mesh, Merck) and Sephadex LH-20(Amersham Biosciences) were used for column chromatography. Reversed phase HPLC(Agilent 1100 series liquid chromatography using a VWDG1314A detector at 210 nm and a semi-preparative ODS-HG-5 [5 μm, 10 mm(i.d.) × 25 cm] column was also employed. Pre-coated silica gel GF₂₅₄ plates(Qing Dao Hai Yang Chemical Group Co. Ltd. Qingdao, People’s Republic of China) were used for analytical thin-layer chromatography(TLC). All solvents used were of analytical grade(Shanghai Chemical Reagents Company, Ltd.).

Animal materials

The soft corals S. trocheliophorum was collected by scuba at Yalong Bay, Hainan Province, China, in February 26, 2006, at a depth of −15 to −20 m, and identified by Professor R.-L. Zhou of South China Sea Institute of Oceanology, Chinese Academy of Sciences. The voucher sample is deposited at the Shanghai Institute of Materia Medica, CAS, under registration No. YAL-4.

Extraction and isolation

The lyophilized bodies of S. trocheliophorum(400 g, dry weight) were minced into pieces and exhaustively extracted with Me₂CO at room temperature(3 × 1 L). The solvent-free Me₂CO extract was partitioned between Et₂O and H₂O. The organic phase was evaporated under reduced pressure to give a dark brown residue(16 g), which was subjected to Si gel column chromatography(CC) and eluted with petroleum ether(PE) in acetone(0–100%, gradient) to yield 14 fractions(A–M). These fractions were subjected to repeated chromatography as usual work³,⁴,⁹,¹⁶, to afford ten pure metabolites: 1(5.7 mg), 2(4.8 mg), 3(4.2 mg), 4(3.6 mg), 5(6.7 mg), 6(3.1 mg), 7(2.0 mg), 8(5.6 mg), 9(1.0 mg), 10(2.1 mg).

Chemical structure data

All investigated compounds were ≥95% pure(HPLC, wavelength = 210 nm).

The NMR spectra of the compounds are provided in the Supporting Information.

Sarcophytrol M(1): colorless oil;  − 60.8(c 0.17, MeOH). IR(KBr) ν_max 3438, 2936, 1645, 1098, 950, 754 cm⁻¹; for ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 361.2356 [M + Na]⁺(calcd for 361.2355, C₂₀H₃₄O₄Na).

Sarcophytrol N(2): colorless oil;  − 120.0(c 0.15, MeOH). IR(KBr) ν_max 3435, 2922, 1648, 1116, 954 cm⁻¹; for ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 375.2504 [M + Na]⁺(calcd for 375.2511, C₂₁H₃₆O₄Na).

Sarcophytrol O(3): colorless oil;  − 30.0(c 0.10, MeOH). IR(KBr) ν_max 3433, 2932, 1733, 1638, 1262, 1024 cm⁻¹; for ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 403.2458 [M + Na]⁺(calcd for 403.2455, C₂₂H₃₆O₅Na).

Sarcophytrol P(4): colorless oil;  + 24.4(c 0.10, MeOH). IR(KBr) ν_max 3441, 2942, 1648, 1262, 1098, 802 cm^-1; for ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 361.2345 [M + Na]⁺(calcd for 361.2355, C₂₀H₃₄O₄Na).

Sarcophytrol Q(5): colorless oil;  + 45.9(c 0.22, MeOH). IR(KBr) ν_max 3458, 2950, 1650, 1232, 1028, 756 cm⁻¹; for ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 361.2354 [M + Na]⁺(calcd for 361.2355, C₂₀H₃₄O₄Na).

Sarcophytrol R(6): colorless oil;  − 13.3(c 0.06, MeOH). IR(KBr) ν_max 3455, 2932, 1653, 1255, 768 cm⁻¹; ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 361.2346 [M + Na]⁺(calcd for 361.2355, C₂₀H₃₄O₄Na).

Sarcophytrol S(7): colorless oil;  − 39.3(c 0.07, MeOH). IR(KBr) ν_max 3437, 2947, 1633, 1198, 879 cm⁻¹; for ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 343.2252 [M + Na]⁺(calcd for 343.2249, C₂₀H₃₂O₃Na).

Sarcophytrol T(8): colorless oil;  − 35.4(c 0.08, MeOH); UV(MeOH) λ_max 242 nm. IR(KBr) ν_max 3418, 2962, 1568, 1262, 782 cm⁻¹; for ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 325.2141 [M + Na]⁺(calcd for 325.2144, C₂₀H₃₀O₂Na).

Sarcophytrol U(9): colorless oil;  + 29.0(c 0.10, MeOH); UV(MeOH) λ_max 238 nm. IR(KBr) ν_max 3425, 2955, 1753, 1662, 778 cm⁻¹; for ¹H and ¹³C NMR data, see Tables 1 and 2; HRESIMS m/z 359.2199 [M + Na]⁺(calcd for 359.2198, C₂₀H₃₂O₄Na).

Preparation of(S)- and(R)-MTPA Esters of 1. The 1S derivative was obtained by treating 1(2.0 mg) with(R)-MTPA-Cl in dry pyridine for ca. 16 h under stirring at room temperature. The reaction mixture was purified by CC(silica gel) to afford pure 1S(1.4 mg). In a similar manner, 1R(1.2 mg) was prepared from(S)-MTPA-Cl.

1S: Selected ¹H NMR(CDCl₃, 400 MHz) δ 5.997(1H, d, J = 15.4 Hz, H-3), 5.657(1H, d, J = 15.4 Hz, H-2), 5.504(1H, dd, J = 10.4, 4.3 Hz, H-7), 5.273(1H, d, J = 9.3 Hz, H-11), 1.673(3H, s, Me-19), 1.284(3H, s, Me-18), 1.122(3H, s, Me-16), 1.100(3H, s, Me-20), 1.072(3H, s, Me-17).

1R: Selected ¹H NMR(CDCl₃, 400 MHz) δ 6.000(1H, d, J = 15.4 Hz, H-3), 5.656(1H, d, J = 15.4 Hz, H-2), 5.515(1H, dd, J = 11.1, 4.3 Hz, H-7), 5.288(1H, d, J = 9.9 Hz, H-11), 1.684(3H, s, Me-19), 1.285(3H, s, Me-18), 1.117(3H, s, Me-16), 1.072(3H, s, Me-20), 1.072(3H, s, Me-17).

Preparation of(S)- and(R)-MTPA Esters of 8. The 8S derivative was obtained by treating 8(2.0 mg) with(R)-MTPA-Cl in dry pyridine for ca. 16 h under stirring at room temperature. The reaction mixture was purified by CC(silica gel) to afford pure 8S(1.4 mg). In a similar manner, 8R(1.2 mg) was prepared from(S)-MTPA-Cl.

8S: Selected ¹H NMR(CDCl₃, 400 MHz) δ 6.111(1H, d, J = 10.9 Hz, H-2), 5.642(1H, d, J = 10.9 Hz, H-3), 5.591(1H, m, H-10), 5.098(1H, dd, J = 9.9, 5.2 Hz, H-7), 4.969(1H, d, J = 9.4 Hz, H-11), 4.206(1H, dd, J = 16.8, 1.4 Hz, Ha-20), 4.008(1H, dd, J = 16.8, 2.2 Hz, Hb-20), 1.767(3H, s, Me-18), 1.458(3H, s, Me-19), 1.379(3H, s, Me-16), 1.357(3H, s, Me-17).

8R: Selected ¹H NMR(CDCl₃, 400 MHz) δ 6.128(1H, d, J = 10.9 Hz, H-2), 5.659(1H, d, J = 10.9 Hz, H-3), 5.626(1H, dt, J = 10.2, 3.8 Hz, H-10), 5.070(1H, dd, J = 9.9, 5.2Hz, H-7), 5.070(1H, d, J = 9.4 Hz, H-11), 4.255(1H, dd, J = 16.8, 1.1 Hz, Ha-20), 4.055(1H, dd, J = 16.8, 1.9 Hz, Hb-20), 1.769(3H, s, Me-18), 1.449(3H, s, Me-19), 1.386(3H, s, Me-16), 1.357(3H, s, Me-17).

Additional Information

How to cite this article: Liang, L.-F. et al. New Bicyclic Cembranoids from the South China Sea Soft Coral Sarcophyton trocheliophorum. Sci. Rep. 7, 46584; doi: 10.1038/srep46584(2017).

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