Holophyllin A, a rearranged abietane-type diterpenoid from the trunk of Abies holophylla

Article abstract of DOI:10.1016/j.tetlet.2014.10.030

Holophyllin A (1), a novel rearranged abietane-type diterpenoid was isolated, together with a new diterpene glycoside, holophyllin B (2), from the trunk of Abies holophylla. The structures of 1 and 2 were established by extensive spectroscopic analyses and their absolute configurations were determined by ECD calculation. All the isolates were tested for their inhibitory effects on NO production in LPS-activated murine microglial cells.

Full text of DOI:10.1016/j.tetlet.2014.10.030

Tetrahedron Letters 55 (2014) 6504–6507
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Tetrahedron Letters
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Holophyllin A, a rearranged abietane-type diterpenoid
from the trunk of Abies holophylla
Chung Sub Kim a, Bora Shin b, Oh Wook Kwon c,d, Sun Yeou Kim d,e, Sang Un Choi f, Dong-Chan Oh b,
Ki Hyun Kim a, Kang Ro Lee a,
⇑
a Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Jangan-ku, Suwon 440-746, Republic of Korea
b Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
c Graduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Republic of Korea
d Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 406-799, Republic of Korea
e College of Pharmacy, Gachon University, Incheon 406-799, Republic of Korea
f Korea Research Institute of Chemical Technology, Daejeon 305-343, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Holophyllin A (1), a novel rearranged abietane-type diterpenoid was isolated, together with a new diter-
pene glycoside, holophyllin B (2), from the trunk of Abies holophylla. The structures of 1 and 2 were estab-
lished by extensive spectroscopic analyses and their absolute configurations were determined by ECD
calculation. All the isolates were tested for their inhibitory effects on NO production in LPS-activated
murine microglial cells.
Received 28 July 2014
Revised 2 October 2014
Accepted 7 October 2014
Available online 14 October 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Abies holophylla
Pinaceae
Abietane diterpene
Spiro compound
Abietane-type diterpenoids are rich in the genus Abies (Pinaceae)
specifically1 and associated with a variety of pharmacological
effects that include anti-inflammatory, antiproliferative, antiviral,
antiplasmodial, and antidepressant antivities.2–6 Several species in
this genus have been used in Korean traditional medicine for the
treatment of stomach ache, rheumatic diseases, and vascular dis-
eases.7 Abies holophylla MAXIM (Pinaceae) is a evergreen tree that
is widely distributed in Korea, China, and Russia. Our earlier phyto-
chemical investigation on A. holophylla resulted in the isolation of
anti-inflammatory lignans.8 In our continuing search for bioactive
constituents from Korean medicinal plants, we further isolated a
novel rearranged abietane-type diterpene with spiro[4.5]decane
structure, holophyllin A (1), together with a new diterpene glyco-
side, holophyllin B (2) (Fig. 1) from the n-hexane and EtOAc-soluble
layers of the MeOH extract of A. holophylla, respectively. Shimagaki
et al. reported two diterpenes as synthetic intermediates having the
same skeleton as 2.9,10 However, this Letter described the first isola-
tion of a diterpene possessing the 6/6/7 ring system from nature.
The trunk of A. holophylla (5.0 kg) was extracted with 80% aq
MeOH (3 ꢀ 5 L) under reflux and filtered. The filtrate was evapo-
rated under reduced pressure to obtain a MeOH extract (280 g),
17
HO
16
12
12
O
20
11
16
15
13
11
1
20
14 O
15
13
C
1
C
14
OH
9
9
A
5 B 8
O
8
H
A
B
3
17
7
3
7
H
6
H
COOH
19
COOR
19
18
18
1
2: R = β-
D-Glc
2a: R = H
Figure 1. Structures of compounds 1, 2, and 2a.
which was suspended in distilled H2O and successively partitioned
with n-hexane, CHCl3, EtOAc, and n-butanol. The n-hexane-soluble
fraction and EtOAc-soluble fraction were subjected to repeated
column chromatography on silica gel, Sephadex LH-20, and semi-
preparative HPLC to give compounds 1 (3.2 mg) and 2 (17.2 mg),
respectively.
Holophyllin A (1)11 was obtained as a colorless gum and had a
molecular formula of C20H30O5 by the positive ion HRFABMS (m/z
351.2171 [M+H]+, calcd for C20H31O5 351.2171), requiring six
degrees of unsaturation. The IR spectrum of 1 displayed absorption
⇑
Corresponding author. Tel.: +82 31 290 7710; fax: +82 31 290 7730.
E-mail address: krlee@skku.ac.kr (K.R. Lee).
http://dx.doi.org/10.1016/j.tetlet.2014.10.030
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

C. S. Kim et al. / Tetrahedron Letters 55 (2014) 6504–6507
6505
Table 1
8
1H (700 MHz) and 13C (175 MHz) NMR data for 1 in CDCl3
Calculated ECD of 1
Calculated ECD of ent-1
Experimental CD of 1
6
4
Position
dH, mult (J in Hz)
dC
1ax
1eq
2
3
4
1.88, br d (13.3)
1.50, overlap
1.72, overlap
1.74, overlap
33.5 (CH2)
17.4 (CH2)
36.7 (CH2)
47.1 (C)
2
5
2.65, m
39.0 (CH)
29.7 (CH2)
0
6ax
6eq
7
8
9
2.24, br t (14.0)
1.72, overlap
3.96, br s
70.2 (CH)
61.1 (C)
210.6 (C)
47.8 (C)
-2
-4
-6
10
11a
11b
12a
12b
13
14
15
16
17
18
19
20
2.30, dt (13.3, 9.1)
1.82, overlap
1.95, dd (13.3, 9.1)
1.69, overlap
23.1 (CH2)
250
300
350
400
23.0 (CH2)
wavelength (nm)
72.5 (C)
Figure 3. Experimental CD spectra of 1 (black), calculated ECD spectra of 1 (red),
and ent-1 (blue).
3.35, s
66.4 (CH)
30.2 (CH)
19.0 (CH3)
18.6 (CH3)
181.4 (C)
16.6 (CH3)
18.6 (CH3)
1.81, overlap
1.02, d (7.0)
1.02, d (7.0)
deduced from the HMBC correlations of H3-16 and H3-17 with C-
13. The fact that the 13C NMR signals of C-13 (dC 72.5) and C-14
(dC 66.4) were shifted upfield in comparison with those of normal
oxyquaternary and oxymethine carbon groups,12,13 respectively,
implied an epoxide ring was to be located at C-13/C-14.
1.35, br s
1.31, s
16
A b orientation of H3-19 and H3-20 was confirmed from the NOE
correlations of H-6ax with H3-19 and H3-20. The distinct NOE
cross-peaks of H-14 with H-6ax, H-7, H3-16, and H3-20 indicated
O
20
a b orientation of H-7 and an
a orientation of H-14 and the isopro-
pyl group. Therefore, the relative configuration of 1 was assigned
as 4R⁄,5R⁄,7R⁄,8R⁄,10S⁄,13S⁄, and 14S⁄. The absolute configuration
of 1 was determined by comparing the CD spectrum of 1 and the
ECD spectra of the two possible enantiomers of 1. These spectra
were calculated using time-dependent density-functional theory
(TD-DFT) at the B3LYP/def2-TZVPP//B3LYP/def-SV(P) level for all
19
14
6ax
O
OH
COOH
7
1H-1H COSY
HMBC
Table 2
NOESY
1H (500 MHz) and 13C (125 MHz) NMR data for 2 in CD3OD
Figure 2. 1H–1H COSY, HMBC, and NOESY correlations of 1.
Position
dH, mult (J in Hz)
dC
1ax
1eq
2
3ax
3eq
4
5
6
7ax
8
9
1.27, m
38.9 (CH2)
1.67, overlap
1.62, overlap
1.89, m
characteristic of hydroxy (3384 cmꢁ1
) and carbonyl groups
19.3 (CH2)
37.7 (CH2)
(1697 cmꢁ1). The 13C NMR spectrum revealed the presence of 20
carbon signals, which were assigned with the assistance of DEPT
spectrum as two sp2 quaternary carbonyl carbons [dC 210.6 and
181.4], four sp3 methyls [dC 19.0, 18.6 (ꢀ2), and 16.6], six sp3 meth-
ylenes [dC 36.7, 33.5, 29.7, 23.1, 23.0, and 17.4], four sp3 methines
[dC 70.2 (CAO), 66.4 (CAO), 39.0, and 30.2], and four sp3 quaternary
carbons [dC 72.5 (CAO), 61.1, 47.8, and 47.1]. From the 1H NMR
spectrum, the resonances of two oxymethine protons [dH 3.96, s;
3.35, s] and four methyls [dH 1.35, br s; 1.31, s; 1.02 (ꢀ2), d,
J = 7.0 Hz] were observed. These spectroscopic data (Table 1)
suggested an abietane-type origin diterpenoid for 1.
1.62, overlap
49.0 (C)
2.10, t (8.0)
1.54, m
2.30, overlap
50.2 (CH)
27.0 (CH2)
39.5 (CH2)
157.6 (C)
58.4 (CH)
40.8 (C)
2.36, dd (11.0, 5.0)
10
11a
11b
12
13
14
15
16
17
18
19
20
10
1.79, dd (14.3, 5.0)
1.64, overlap
3.75, d (8.8)
33.0 (CH2)
74.3 (CH)
55.9 (C)
Analysis of the 1H–1H COSY spectrum established four proton
sequences from H2-1 to H2-3, H-5 to H-7, H2-11 to H2-12, and
H3-16 to H3-17 (Fig. 2). HMBC correlations of H3-19 with C-3 and
C-5 provided the connection of C-3 and C-5 and formation of ring
A was confirmed by HMBC cross-peaks of H3-20 with C-1, C-5,
and C-10. Ring B, which was adjacent to ring A could be deduced
from the HMBC correlations of H-5, H-7 and H3-20 with C-9, and
H2-6 with C-8. The HMBC cross-peaks of H2-11 with C-7, C-8 and
C-9, and H-14 with C-7 and C-8 provided the connection from C-
11 to C-14 through the spiro carbon at C-8. The cyclopentane ring
C was confirmed by HMBC correlations of H2-11 with C-13 and
H2-12 with C-14, and the isopropyl group attached at C-13 was
211.0 (C)
125.8 (CH)
27.3 (CH3)
16.7 (CH3)
178.8 (C)
17.3 (CH3)
15.2 (CH3)
96.2 (CH)
74.1 (CH)
78.4 (CH)
71.2 (CH)
79.0 (CH)
62.5 (CH2)
5.81, br s
1.08, s
1.12, s
1.20, s
0.71, s
5.46, d (8.1)
3.35, overlap
3.41, overlap
3.37, overlap
3.37, overlap
3.83, d (11.5)
3.68, dd (11.5, 2.2)
20
30
40
50
60a
60b

6506
C. S. Kim et al. / Tetrahedron Letters 55 (2014) 6504–6507
HO
O
20
19
O
16
12
9
5
HO
HO
O
O
HO
OH
1H-1H COSY
HMBC
NOESY
Figure 4. 1H–1H COSY, HMBC, and NOESY correlations of 2.
B-
6
4
[O]
II
II
I
Calculated ECD of 2a
Calculated Ecd ofent-2a
Experimental CD of 2a
H
[O]
I
I
II
[O]
II
[O]
2
H
H
[O]
H
H
H+
II
0
COOH
COOH
Abietic acid
I
ii
-2
-4
-6
OH
250
300
wavelength (nm)
350
400
OH
O
O
H+
O
H
H+
Figure 5. Experimental CD spectra of 2a (black), calculated ECD spectra of 2a (red),
and ent-2a (blue).
H
H
COOH
COOH
atoms.14 The CD spectrum of 1 displayed a positive n–p⁄ Cotton
i
iii
effect at 304 nm (De = 3.8), matching well with the calculated
ECD spectrum for 1 (Fig. 3). Thus, the absolute configuration of 1
pinacol-like
pinacol-like
rearrangement
rearrangement
was assigned as 4R,5R,7R,8R,10S,13S, and 14S.
Holophyllin B (2)15 was purified as a colorless gum. The molecu-
lar formula was determined to be C26H40O9 from the [M+Na]+ ion in
the positive ion HRFABMS (m/z 519.2569; calcd for C26H40O9Na
519.2570), indicating seven degrees of unsaturation. The presence
HO
H
O
O
O
OH
of a,b-unsaturated ketone was deduced from the UV maximum at
239 nm. The analysis of 13C NMR spectrum revealed that 2 pos-
sessed 26 carbons, of which six carbons were characteristic of a
glucopyronosyl unit [dC 96.2, 79.0, 78.4, 74.1, 71.2, and 62.5]. The
remaining 20 carbons were assigned as four sp2 carbons [dC 211.0
(C@O), 178.8 (C@O), 157.6 (C@C), and 125.8 (C@C)], four sp3 meth-
yls [dC 27.3, 17.3, 16.7, and 15.2], six sp3 methylenes [dC 39.5, 38.9,
37.7, 33.0, 27.0, and 19.3], three sp3 methines [dC 74.3 (CAO),
58.4, and 50.2], and three sp3 quaternary carbons [dC 55.9, 49.0,
and 40.8] with the assistance of DEPT spectrum. The 1H NMR spec-
trum showed one olefinic proton [dH 5.81, br s], one oxymethine
proton [dH 3.75, d, J = 8.8 Hz], and four tertiary methyls [dH 1.20,
1.12, 1.08, and 0.71 (each s)], as well as one anomeric proton [dH
5.46, d, J = 8.1 Hz]. The above spectral data (Table 2) suggested that
2 could be a glycoside of abietane-type origin diterpenoid.
H
COOH
H
COOH
1
2a
Scheme 1. Hypothetical biogenetic pathway of 1 and 2a.
C-9 to C-14 (Fig. 4). The seven-membered ring C was confirmed
by HMBC correlations of H-15 with C-7, C-9, and C-13. The large
J value of H-10 (8.1 Hz) suggested that the sugar was a b-glucopyr-
onosyl unit and the HMBC cross-peak of H-10 with C-18 unambig-
uously located this sugar on the aglycon.
The relative configuration was deduced from the distinct NOESY
correlations of H3-19 with H3-20 and H-9 with H-5 and H-12
(Fig. 4). The absolute configuration of 2 was determined through
the same method as 1. The CD spectrum of 2a,17 the product of acid
hydrolysis of 2, showed a negative n?p⁄ Cotton effect at 320 nm
The 1H and 13C NMR data assigned to ring A and B, with the aid
of 1H–1H COSY, HMQC, HMBC, and NOESY, were similar to those of
8(14)-podocarpen-7,13-dion-18-oic acid.16 The 1H–1H COSY corre-
lations of H2-11 with H-9 and H-12 and the HMBC cross-peaks of
H3-16 with C-12, C-13, and C-14 provided the connections from
(
D
e
e
= ꢁ1.6) and
a positive p?
p⁄ Cotton effect at 244 nm
(D
= 4.3), corresponding well with the calculated ECD spectrum

C. S. Kim et al. / Tetrahedron Letters 55 (2014) 6504–6507
6507
for 2a (Fig. 5). Therefore, the absolute configuration of 2 was
References and notes
assigned as 4R,5R,9S,10R, and 12R. The D-configuration of sugar
was determined by GC/MS analysis.8
1. Yang, X.-W.; Li, S.-M.; Shen, Y.-H.; Zhang, W.-D. Chem. Biodivers. 2008, 5, 56–81.
2. Yang, J.-H.; Du, X.; He, F.; Zhang, H.-B.; Li, X.-L.; Su, J.; Li, Y.; Pu, J.-X.; Sun, H.-D.
J. Nat. Prod. 2013, 76, 256–264.
3. Yang, L.; Qiao, L.; Ji, C.; Xie, D.; Gong, N.-B.; Lu, Y.; Zhang, J.; Dai, J.; Guo, S. J. Nat.
Prod. 2013, 76, 216–222.
4. Burmistrova, O.; Simões, M. F.; Rijo, P.; Quintana, J.; Bermejo, J.; Estéves, F. J.
Nat. Prod. 2013, 76, 1413–1423.
5. Wang, Y.-D.; Zhang, G.-J.; Qu, J.; Li, Y.-H.; Jiang, J.-D.; Liu, Y.-B.; Ma, S.-G.; Li, Y.;
Lv, H.-N.; Yu, S.-S. J. Nat. Prod. 2013, 76, 1976–1983.
6. Clarkson, C.; Campbell, W. E.; Smith, P. Planta Med. 2003, 69, 720–724.
7. Lee, J.-H.; Hong, S.-K. J. Microbiol. Biotechnol. 2009, 19, 372–377.
8. Kim, C. S.; Kwon, O. W.; Kim, S. Y.; Lee, K. R. J. Nat. Prod. 2013, 76, 2131–2135.
9. Shimagaki, M.; Anazawa, A.; Oishi, T.; Tahara, A. Heterocycles 1977, 8, 237–242.
The biogenetic origin of 1 and 2a can plausibly be tracked back to
the most common compound, abietic acid (Scheme 1). Allylic oxida-
tion at C-9 and epoxidation at C-7/C-8 and C-13/C-14 of abietic acid
generates intermediate i. After opening of the epoxide ring at C-7/C-
8 and a pinacol-like rearrangement, compound 1 might be formed
having a spiro[4,5]decane structure. Compound 2a could be derived
through biosynthetic intermediates ii and iii. Abietic acid under-
went isomerization to yield ii with the formation of D8(14) and
D
13(15), and further C-12 and C-14/C-15 may be oxidized and epox-
10. Shimagaki, M.; Tahara, A. Tetrahedron Lett. 1976, 17, 1103–1106.
idized, respectively, to give intermediate iii. The 6/6/7 ring system of
2a could be formed through a pinacol-like rearrangement.
25
11. Holophyllin A (1): Colorless gum; [
a
]
D
ꢁ12.3 (c 0.07, MeOH); IR (KBr) mmax
3384, 2950, 2843, 1697, 1649, 1457, 1033 cmꢁ1; CD (MeOH) kmax
(De): 304
Antineuroinflammatory activities of the 1, 2, and 2a were
evaluated for their inhibitory effects on NO production in lipopoly-
saccharide (LPS)-activated murine microglial cells because the
n-hexane and EtOAc-soluble layers of the MeOH extract of A. holo-
phylla showed inhibitory activities on NO production (IC50 8.21 and
(3.8) nm; 1H and 13C NMR data, see Table 1; positive HRFABMS m/z 351.2171
[M+H]+ (calcd for C20H31O5 351.2171).
12. Tapia, R.; Bouanou, H.; Alvarez, E.; Alvarez-Manzaneda, R.; Chahnoun, R.;
Alvarez-Manzaneda, E. J. Org. Chem. 2014, 79, 4405–4413.
13. Miyaichi, Y.; Segawa, A.; Tomimori, T. Chem. Pharm. Bull. 2006, 54, 1370–1379.
14. Woo, J.-K.; Kim, C.-K.; Kim, S.-H.; Kim, H.; Oh, D.-C.; Oh, K.-B.; Shin, J. Org. Lett.
2014, 16, 2826–2829.
38.33
inhibited NO production with IC50 values of 18.59, 29.78, and
24.80 M, respectively, in LPS-stimulated BV-2 cells without cell
toxicity (L-NMMA, positive control, IC50 17.24 M). We also tested
lg/mL, respectively). Compounds 1, 2, and 2a moderately
15. Holophyllin B (2): Colorless gum; [a] mmax 3382,
25 +16.4 (c 0.08, MeOH); IR (KBr)
D
2949, 2843, 1649, 1455, 1033 cmꢁ1; UV (MeOH) kmax (log
e): 239 (9.2) nm; CD
(MeOH) kmax
(D
e
): 319 (ꢁ1.6), 244 (4.3) nm; 1H and 13C NMR data, see Table 2;
l
positive HRFABMS m/z 519.2569 [M+Na]+ (calcd for C26H40O9Na 519.2570).
l
16. Yang, X.-W.; Li, S.-M.; Feng, L.; Shen, Y.-H.; Tian, J.-M.; Liu, X.-H.; Zeng, H.-W.;
the cytotoxicity of the compounds 1, 2, and 2a against A549, SK-
OV-3, SK-MEL-2, and HCT-15 human tumor cell lines using the sul-
forhodamine B (SRB) assays in vitro, but they were inactive
Zhang, C.; Zhang, W.-D. Tetrahedron 2008, 64, 4354–4362.
25
17. Holophyllin C (2a): Colorless gum; [
a
]
+43.3 (c 0.02, MeOH); IR (KBr) mmax
D
3381, 2948, 2842, 1648, 1454, 1032 cmꢁ1
; UV (MeOH) kmax (loge): 240
(10.0) nm; CD (MeOH) kmax
(D
e
): 320 (ꢁ1.6), 244 (4.4) nm; 1H NMR (CD3OD,
(IC50 > 10.0 lM).
500 MHz) dH 5.81 (1H, br s, H-15), 3.75 (1H, d, J = 8.8 Hz), 2.35 (1H, overlap,
H-9), 2.30 (1H, overlap, H-7ax), 2.28 (1H, td, J = 14.2, 5.5 Hz, H-7eq), 2.10 (1H,
dd, J = 12.0, 2.4 Hz, H-5), 1.83 (1H, overlap, H-3ax), 1.79 (1H, dd, J = 14.2,
5.5 Hz, H-11a), 1.66 (1H, overlap, H-1eq), 1.64 (1H, overlap, H-11b), 1.59 (2H,
overlap, H-2), 1.58 (1H, overlap, H-3eq), 1.55 (2H, overlap, H-6), 1.25 (1H, td,
J = 12.3, 4.4 Hz, H-1ax), 1.13 (3H, s, H-19), 1.11 (3H, s, H-17), 1.08 (3H, s, H-16),
0.69 (3H, s, H-20); 13C NMR (CD3OD, 125 MHz) dC 211.1 (C-14), 183.9 (C-18),
158.0 (C-8), 125.7 (C-15), 74.3 (C-12), 58.7 (C-9), 55.9 (C-13), 50.6 (C-5), 48.9
(C-4), 40.9 (C-10), 39.7 (C-7), 39.3 (C-1), 38.5 (C-3), 33.0 (C-11), 27.4 (C-6), 27.3
(C-16), 19.6 (C-2), 17.8 (C-19), 16.7 (C-17), 15.1 (C-20); positive HRFABMS m/z
335.2222 [M+H]+ (calcd for C20H31O4 335.2222).
Acknowledgments
This research was supported by the Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(2013R1A1A2A10005315). We are thankful to the Korea Basic Sci-
ence Institute (KBSI) for the measurements of NMR and MS spectra.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.10.
030.