2.1. Structure Elucidation
Compound 1 was obtained as white, needle-like crystals. The molecular formula of 1 was determined to be C22H34O6 on the basis of positive HR-ESI-MS at m/z 417.22367 [M + Na]+ (calcd for C22H34O6Na+, 417.22467). Its IR spectrum displayed the absorption bands of hydroxyl group (3422 cm−1) and free carbonyl group (1731 cm−1). The NMR spectra revealed three methyls (δC 9.2, 33.3, 21.5(each q); δH 1.12 (3H, d, 7.1 Hz), 0.91 (3H, s), 0.85 (3H, s)), one acetoxyl group (δC 170.8(s), 21.5(q); δH 2.13(3H, s)), one ketone carbonyl group (δC 222.8), three oxy-methines (δC 81.6, 76.1, 75.4) and one oxy-methylene (δC 63.7). Considering the diterpenoids previously isolated from the plant, 1 was tentatively presumed to be a 7,20-non-epoxy-ent-kaurane skeleton, substituted with three hydroxyl groups and one acetoxyl group.
The
1H- and
13C-NMR data of
1 were nearly identical with that of a known diterpene henryin (
4) (
Table 1) [
17], and their only difference was in the moiety at C-16. For
1, the methyl signal at δ
H 1.12 (3H, d,
J = 7.2 Hz) has correlations with C-13 (δ
C 42.4) and C-15 (δ
C 222.8) in the HMBC spectrum (
Figure 2), revealing that the exo-methylene at C-16 in
4 had been replaced by a methyl at C-16 in
2.
Table 1.
1H- and 13C-NMR data of compounds 1–3 (500 and 125 MHz δ in ppm).
Table 1.
1H- and 13C-NMR data of compounds 1–3 (500 and 125 MHz δ in ppm).
No. | 1 (In CDCl3) | 2 (In DMSO-d6 and D2O) | 3 (In CDCl3) |
---|
δH (J in Hz) | δC | δH (J in Hz) | δC | δH (J in Hz) | δC |
---|
1 | β 3.31, dd, (11.0, 4.6) | 81.6 | β 3.09, dd, (10.8, 4.5) | 80.5 | β 4.57, dd, (11.2, 5.3) | 76.6 |
2 | α 1.82, m | 30.5 | α 1.63, overlapped | 29.2 | α 1.69, overlapped | 25.1 |
β 1.65, overlapped | β 1.53, overlapped | β 1.47, m |
3 | α 1.47, dt, (13.8, 3.7) | 39.4 | α 1.53, overlapped | 32.9 | α 1.44, m | 37.9 |
β 1.29, dt, (13.8, 4.4) | | β 1.05, m | | β 1.24, m | |
4 | – | 32.9 | – | 36.6 | – | 33.6 |
5 | β 0.98, dd, (11.6, 3.5) | 52.5 | β 1.21, d, (12.2) | 42.0 | β 1.33, ddd, (11.8, 7.4, 1.5) | 47.1 |
6 | 1.93, m | 28.6 | β 1.80, d, (12.2) | 28.6 | β 2.85, ddd, (14.0, 11.8, 1.9) | 25.1 |
α 1.63, overlapped | α 1.84, overlapped |
7 | β 4.20, dd (11.4, 6.0) | 75.4 | β 3.77, dd, (11.8, 4.3) | 73.8 | β 3.95, dd, (3.8, 1.9) | 64.5 |
8 | – | 61.0 | – | 59.8 | – | 58.7 |
9 | β 1.55, d, (8.8) | 55.5 | β 1.37, d, (8.6) | 55.7 | β 1.69, overlapped | 50.8 |
10 | – | 46.0 | – | 44.1 | – | 39.2 |
11 | α 2.77, q, (6.1) | 19.9 | α 2.82, dd, (15.9, 5.6) | 19.3 | α 1.84, overlapped | 17.8 |
β 1.20, m | β 0.98, m | β 1.15, q, (6.5) |
12 | α 1.88, m | 24.2 | α 1.63, overlapped | 24.3 | α 2.42, dt, (14.1, 9.0) | 30.9 |
β 1.65, overlapped | | β 1.53, overlapped | | β 1.54, m | |
13 | α 2.42, m | 42.4 | α 2.24, m | 42.5 | α 2.99, br d, (9.9) | 42.0 |
14 | α 4.82, d, (1.1) | 76.1 | α 4.72, br. s | 75.2 | α 4.61, br s | 71.5 |
15 | – | 222.8 | – | 221.2 | – | 204.5 |
16 | 2.88, m | 43.2 | 2.67, m | 44.6 | – | 151.1 |
17 | 1.12, d, (7.1) | 9.2 | 1.00, d, (7.1) | 9.1 | 6.00, br s; 5.38, br.s | 117.5 |
18 | 0.91, s | 33.3 | 3.19, d, (10.6) | 69.5 | 0.88, s | 31.4 |
2.84, d, (10.6) |
19 | 0.85, s | 21.5 | 0.62, s | 17.3 | 1.08, s | 20.3 |
20 | α 4.74, d, (13.2) | 63.7 | α 4.35, d, (13.3) | 64.1 | α 4.09, dd, (10.3, 1.5) | 61.0 |
β 4.37, d, (13.2) | β 4.28, d, (13.3) | β 4.03, dd, (10.3, 1.6) |
OAc | – | 170.8 | – | 170.2 | – | 170.1 |
OAc | 2.13, s | 21.5 | 2.07, s | 21.2 | 1.95, s | 21.5 |
In the HMBC spectrum, correlations between H-1 (δ 3.31) with C-5 (δ 52.5), C-9 (δ 55.5) and C-20 (δ 63.7), H-7 (δ 4.20) with C-14 (δ
C 76.1), H-14 (δ 4.82) with C-12 (δ
C 24.2), and C-15 (δ
C 222.8), indicated that the hydroxy groups were at C-1, C-7, and C-14, respectively. Moreover, the acetoxyl group was assigned at C-20 based on the correlation of H-20 (δ
H 4.74 and 4.37) with the carbonyl at δ
C 170.8 (–OCOCH
3) in the HMBC spectrum (
Figure 2).
The relative configuration of the substituents was revealed by the ROESY spectrum, in which the correlations of H-1 with H-5 and H-9, Me-18 with H-5, H-7 with H-5 and H-9, and H-13 with H-14 and H-16, indicated that they were positioned on the same side, and that H-14, H-13, and H-16 were on the other side (
Figure 3).
Figure 2.
Key HMBC and 1H-1HCOSY correlations for compounds 1–3.
Figure 2.
Key HMBC and 1H-1HCOSY correlations for compounds 1–3.
Figure 3.
Key ROESY correlations for compounds 1–3.
Figure 3.
Key ROESY correlations for compounds 1–3.
The absolute configuration of
1 was confirmed using the CD spectrum. According to the octant rule for saturated cyclopentanone [
9], the negative Cotton effect at 305 nm (Δε-0.196), based on the n-π* transition of the saturated cyclopentanone moiety, indicated that the D ring was in a β-orientation (
Figure 4). Finally, the structure of compound
1 was elucidated as 1α,7α,14β-trihydroxy-20-acetoxy-
ent-
kaur-15-one.
Figure 4.
Experimental CD spectra of compounds 1 and 2.
Figure 4.
Experimental CD spectra of compounds 1 and 2.
Compound
2 was obtained as a white crystal. The molecular formula was established as C
22H
34O
7 by HR-ESI-MS at
m/
z 433.22012 [M + Na]
+ (calcd for C
22H
34O
7Na
+, 433.21967),which indicated that
2 had an additional oxygen atom compared to
1. The molecular formulas, NMR, and IR data suggested that
2 was an oxygenated analog of
1. Comparison of the NMR spectral data of
2 with those of
1 indicated that one angular methyl (δ
C 33.3, δ
H 0.91 (3H, s)) at C-4 in
1 had been replaced by one hydroxymethyl (δ
C 69.5, δ
H 3.19 (1H, d,
J = 10.6) and 2.82 (1H, d,
J = 10.6)) in
2. Furthermore, the downfield shift of C-4 and the upfield shift of C-3, C-5 and C-19 suggested the presence of one hydroxyl group at C-18 in
2. The planar structure of
2 was indicated by the HMBC data (
Figure 2). In the HMBC spectrum, correlations were observed for δ
H 3.19 (H-18) with δ
C 32.9 (C-3) and 17.3 (C-19) also confirmed that a hydroxymethyl group was linked to C-4.
The same relative stereo-structure for
1 and
2 was deduced from their similar ROESY correlations (
Figure 3) and almost identical
1H- and
13C-NMR data. In addition, compound
2 exhibited almost the same CD absorption as that of
1 (
Figure 4). Thus, the structure of
2 was determined to be 1α,7α,14β,18-tetrahydroxy-20-acetoxy-
ent-kaur-15-one(
Figure 1).
Compound
3 was obtained as a white crystalline powder. The molecular formula of
3 was deduced to be C
22H
30O
5 by positive HR-ESI-MS at
m/
z 397.1992 [M + Na]
+ (calcd for C
22H
30O
5Na
+, 397.19855). The UV spectrum of
3 showed an absorption maximum at 230 nm. The IR spectrum of
3 showed the presence of hydroxyl (3418 cm
−1), carbonyl (1732 cm
−1), and double bond (1647 cm
−1) groups. The
1H- and
13C-NMR spectra of
3, together with the results from a HSQC experiment showed the presence of one exocyclic double bond (δ
H 6.00, 5.38 (each 1H, brs); δ
C 117.5, 151.1), one acetoxyl group (δ
H 1.95 (3H, s); δ
C 170.1 (s), 21.5 (q)), one ketone carbonyl (δ
C 204.6), and two angular methyl groups (δ
H 0.88 and 1.08 (each 3H, s); δ
C 31.4 (q) and 21.3 (q)). In addition, the other carbon signals were assigned to six methenes (including one oxygenated signal), six methine carbons (including three oxygenated signals), and three quaternary carbons. These carbon signals were the characteristic signals of the structures of the diterpenoids isolated from the
Isodon genus. The
1H- and
13C-NMR spectra of
3 were very similar to those of kamebacetal A (
7) [
18], except for the absence of a dioxygenated methine (δ
H 5.51 (1H, d,
J = 1.1 Hz, H-20); δ
C 101.9) and a methoxyl group (δ
H 3.38 (3H, s); δ
C 54.9) as well as the presence of an acetoxyl group (δ
H 1.95 (s, 3H); δ
C170.1 (s), 21.5 (q)) and an oxygenated methylene (δ
H 4.09 (1H, dd,
J = 10.3, 1.5 Hz, H-20) and 4.03 (1H, dd,
J = 10.3, 1.6 Hz, H-20); δ
C 61.0) in
3. Meanwhile, the following cross-peaks were observed in the HMBC spectrum: δ
H 3.95 (H-7β) with δ
C 61.0 (C-20), δ
H 4.57 (H-1) with δ
C 170.1 (OAc), and δ
H 4.61 (H-14) with δ
C 30.9 (C-12), 204.5 (C-15) and 151.1 (C-16). Thus, the basic skeleton of
3 was assumed to be 1-acetoxy-14-hydroxy-7,20-epoxy-
ent-kaur-16-en-15-one.
The relative configuration of
3 was revealed by ROESY experiments (
Figure 3). In the ROESY spectrum, the correlations of H-1/H-5 and H-9, Me-18/H-5, H-7/H-5 and H-9, H-14/H-13 were observed. These results indicated that H-1, H-5, H-9, and Me-18 should be on the same side of the molecule, and H-14 and H-13 should be on the other side.
The negative Cotton effect at 356 nm (Δε-0.02), based on the n-π* transition of the unsaturated cyclopentanone moiety, indicated that the D ring was in a β-orientation [
11]. According to the analysis described above, the structure of
3 was determined to be 1α-acetoxy-14β-hydroxy-7α,20-epoxy-
ent-kaur-16-en-15-one.The six known compounds isolated from
I.excisoides were identified as henryin (
4) [
17], kamebanin (
5) [
19], reniformin C (
6) [
20], kamebacetal A (
7) [
18], kamebacetal B (
8) [
21], and oridonin (
9) [
22], by comparison of their spectral data to the reported in the literature.
2.3. Analysis of Structure-Activity Relationships
We assessed the structure-activity relationships of the isolated compounds, based on the results of cytotoxic activity test. Compounds
1,
2,
4, and
5 were 7, 20-non-epoxy kaurane diterpenoids and were present in large amounts in
I. excisoides. Compounds
4 and
5 contained α,β-unsaturated pentone and exocyclic methylene. In addition, compound
4 also contained 20-OAc. No exocyclic double bond was found in compounds
1 or
2; however, 20-OAc was present in these compounds. Previous reports have suggested that α,β-unsaturated pentones and exocyclic methylene are essential structural requirements for the cytotoxic activity of diterpenoids [
24,
25,
26]. The results of the cytotoxic activity tests indicated that all 4 compounds had cytotoxic activity. Although the NCI-H1650 cell line was resistant to compounds
1 and
2, these compounds exhibited significant cytotoxic activities (IC
50: 2.94–6.47 μM) against the other four tumor cell lines, while compound
4 displayed the highest cytotoxic activity. This suggests that α,β-unsaturated pentone and exocyclic methylene are not the only moieties required for the cytotoxic activity of diterpenoids. Furthermore, 7,20-non-epoxy kaurane diterpenoid, and 20-OAc may also be responsible for the cytotoxic activity of diterpenoids. this possibility should be further investigated.
Compounds
3,
6,
7,
8, and
9 are 7,20-epoxy kaurane diterpenoids composed of α,β-unsaturated pentone and exocyclic methylene. A large number of studies have confirmed that compound
9 has definite cytotoxic activity [
25,
26,
27]. Pre-clinical studies are being conducted on the use of compound
9 as a potential new drug. Compounds
3 and
6–
9 exhibited expected levels of cytotoxic activity consistent with previous reports suggesting that α,β-unsaturated pentones and exocyclic methylene are responsible for the cytotoxic activity of diterpenoids.
Compounds 6–8 are variants of diterpenoids comprising an oxygen-containing substituent on the C-20 chiral carbon, but have different configurations (compounds 6 and 7 have an α-configuration, while compound 8 has a β-configuration). A desirable result is that compounds of different configurations display selective activity against NCI-H1650 and HepG2 cell lines. Compound 8 exhibited significant cytotoxic effects on both NCI-H1650 and HepG2 cells (IC50: 1.09–2.58 μM), whereas compounds 6 and 7 had an insignificant effect on the NCI-H1650 cell line (IC50 > 10 μM), suggesting that the relative configuration of the C-20 chiral carbon affected the cytotoxic effect of the tested diterpenoids on some of the tumor cell lines (NCI-H1650 and HepG2), whereas the β-configuration enhanced their cytotoxic activity.
Among the tested compounds, compound 4 exhibited the highest cytotoxic activity against the five tested tumor cell lines (IC50: 1.31–2.07 μM). The cytotoxic activity of compound 4 was almost 5-fold higher than that of compound 9. These results indicate that 7,20-non-epoxy, α,β-unsaturated pentones and exocyclic methylene, as well as the 20-OAc group, had a positive effect on the cytotoxic activity of diterpenoids. α,β-unsaturated pentones (compounds 1 and 2), exocyclic methylene, and configurations of C-20 chiral carbon (compounds 6–8) significantly affected the cytotoxic activity of the tested diterpenoids in some of the cell lines, such as NCI-H1650 and HepG2.