Scopariusic acid, a new meroditerpenoid with a unique cyclobutane ring isolated from Isodon scoparius

Article abstract of DOI:10.1021/ol401991u

Scopariusic acid (1), a new ent-clerodane-based meroditerpenoid with a unique cyclobutane ring and an unusual 1-octen-3-ol substituent, together with its biosynthetic related compound 2, were isolated from the aerial parts of Isodon scoparius. The structures of 1 and 2, including their absolute configurations, were determined by spectroscopic methods, single-crystal X-ray diffraction analysis, and chemical methods. Compound 1 showed weak cytotoxicity and moderate immunosuppressive activity.

Full text of DOI:10.1021/ol401991u

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Scopariusic Acid, a New Meroditerpenoid
with a Unique Cyclobutane Ring Isolated
from Isodon scoparius
Min Zhou,†,‡ Hai-Bo Zhang,† Wei-Guang Wang,† Ning-Bo Gong,§ Rui Zhan,† Xiao-Nian Li,†
Xue Du,† Li-Mei Li,^ Yan Li,† Yang Lu,§ Jian-Xin Pu,*,† and Han-Dong Sun*,†
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming
Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, P. R. China,
University of Chinese Academy of Sciences, Beijing 100039, P. R. China, Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, P. R. China, and Research Center, Chengdu Medical College, Chengdu, P. R. China
pujianxin@mail.kib.ac.cn; hdsun@mail.kib.ac.cn
Received July 16, 2013
ABSTRACT
Scopariusic acid (1), a new ent-clerodane-based meroditerpenoid with a unique cyclobutane ring and an unusual 1-octen-3-ol substituent,
together with its biosynthetic related compound 2, were isolated from the aerial parts of Isodon scoparius. The structures of 1 and 2, including
their absolute configurations, were determined by spectroscopic methods, single-crystal X-ray diffraction analysis, and chemical methods.
Compound 1 showed weak cytotoxicity and moderate immunosuppressive activity.
In 1908, the first photochemical [2 þ 2] cycloaddition
was reported by Ciamician and involved the intramolecu-
lar cyclobutanation of carvone on exposure to intense
sunlight for one year.1 After confirmation of this finding
in the late 1950s, the [2 þ 2] photocycloaddition reactions
involving R,β-unsaturated cyclic enones, cycloalkenyl esters,
and lactones have been established to be one of the most
useful and stereoselective methods in the synthesis of cyclo-
butane derivatives.2
ꢀ ~
(2) (a) Garcıa-Exposito, E.; Bearpark, M. J.; Ortuno, R. M.; Robb,
´
M. A.; Branchadell, V. J. Org. Chem. 2002, 67, 6070–6077. (b) Araki, T.;
Ozawa, T.; Yokoe, H.; Kanematsu, M.; Yoshida, M.; Shishido, K. Org.
Lett. 2013, 15, 200–203. (c) Deng, J.; Hsung, R. P.; Ko, C. H. Org. Lett.
2012, 14, 5562–5565. (d) Rasik, C. M.; Brown, M. K. J. Am. Chem. Soc.
2013, 135, 1673–1676. (e) Albrecht, L.; Dickmeiss, G.; Acosta, F. C.;
Rodriguez-Escrich, C .; Davis, R. L.; Jorgensen, K. A. J. Am. Chem. Soc.
2012, 134, 2543–2546. (f) Navarro, R.; Reisman, S. E. Org. Lett. 2012,
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† Kunming Institute of Botany
‡ University of Chinese Academy of Sciences
§ Chinese Academy of Medical Sciences and Peking union Medical College
^ Chengdu Medical College
(1) Ciamician, G.; Silber, P. Chem. Ber. 1908, 41, 1928–1935.
r
10.1021/ol401991u
XXXX American Chemical Society

Meanwhile, cyclobutane-containing natural products
were also found in plants or microorganisms as a small
yet diverse family with a variety of biological activities.3
Interestingly, structureꢀactivity relationship studies per-
formed on several monoterpene alkaloids suggested that
the cyclobutyl motifs play a crucial role in the expression
of their antinociceptive activity in a formalin-induced pain
model in mice.4 Moreover, some of them have been
synthesized by using a photochemical [2 þ 2] approach
as the key step, such as incarvillateine,3b,5 an antinocicep-
tive monoterpene alkaloid characterized by a tetrasubsti-
tuted cyclobutyl moiety, biyouyanagin A,3c,6 a unique
hydrophobic compound exhibiting significant activity
against HIV and inhibiting cytokine production, and
littoralisone,3d,7 a neuritogenic iridolactone having a novel
heptacyclic skeleton. Therefore, such naturally occurring
cyclobutanesoftenprovoke interesting questionsanddraw
widespread attention of the scientific community.3a,8
In our further study of the chemical constituents of
Isodon scoparius,9 scopariusic acid (1), a new ent-clerodane-
based meroditerpenoid with a unique cyclobutane
ring, together with its biosynthetic related compound
(2), have been isolated from the aerial parts of this plant.
Interestingly, compound 1 might be biosynthetically
formed via an intermolecular [2 þ 2] cycloaddition be-
tween the acyclic side chains of two absolutely different
units, one moiety possessing an ent-clerodane diterpenoid
nucleus (part A) and the other derived from 10-octen-30-
yl-4-hydroxycinnamate, an unusual ester of trans-4-
hydroxycinnamic acid (part B) and (3R)-1-octen-3-ol
(part C). To the best of our knowledge, compound 1
represents a new class of meroditerpenoid with a unique
cyclobutane ring and a novel 1-octen-3-ol substituent.
Herein, we report the isolation and structure determina-
tion of compounds 1 and 2 as well as their cytotoxic and
immunosuppressive activities.
Isoscoparin P (2) was obtained as colorless crystals. Its
molecular formula C20H32O3 was determined on the basis
of the HREIMS at m/z 343.2244 [M þ Na]þ (calcd
343.2249), corresponding to 5 degrees of unsaturation.
1
The H NMR spectrum of compound 2 exhibited three
olefinic protons (δH 4.66, 4.66, and 6.32), an oxymethine
(δH 3.97), two tertiary methyl groups (δH 1.11 and 1.13),
one secondary methyl group (δH 0.85), and one olefinic
methyl group (δH 2.50) (Table S1, Supporting Information).
The HSQC spectrum resolved the 20 carbon signals as four
methyls, seven methylenes (including one sp2 methylene),
four methine (of which one was oxygenated), and five
quaternary carbons (including two sp2 carbons and one
carbonyl) (Table 1), which was consistent with a skeleton
of a clerodane diterpenoid.9 One oxymethine proton at δH
1
3.97 (1H, br d, J = 10.0 Hz) observed in the H NMR
spectrum, was located at C-11, which was proven by the
HMBC correlations from H-11 to C-8, C-10, and C-13
(Figure 1). These features indicated the gross structure
of 2 as 11-hydroxy-clerodane-4(18),13-dien-15-oic acid.
However, the relative configuration at C-11 in 2 could
not be determined from spectroscopic data alone. Finally,
its absolute configuration was established by single-crystal
X-ray diffraction analysis (Figure 3). Thus, compound 2 was
identified as (11S,13E)-11-hydroxy-ent-clerodane-4(18),13-
dien-15-oic acid and named isoscoparin P.
Scopariusic acid (1) was isolated as colorless needles. Its
molecular formula C37H54O6 was determined on the basis
of the HREIMS at m/z 594.3907 [M]þ (calcd 594.3920),
corresponding to 11 degrees of unsaturation. The 1H NMR
spectrum of compound 1 exhibited nine olefinic protons
(δH 4.46, 4.64, 5.40, 5.50, 6.01, 7.08, 7.08, 7.39, and 7.39),
two oxymethines (δH 4.22 and 5.59), three tertiary methyl
groups (δH 1.12, 1.17, and 1.80), and two secondary methyl
groups (δH 0.80 and 0.88) (Table S1, Supporting
Information). The 13C NMR and DEPT spectra resolved
the 37 carbon signals (Table 1) as 5 methyls, 12 methylenes
(including two sp2 methylenes), 12 methines (of which
five were sp2 methines and two were oxygenated), and
8 quaternary carbons (including three sp2 carbons and
two carbonyls). Among them, two carbonyl carbons and
10 olefinic carbons occupied seven degrees of unsaturation.
Extensive analyses of 1D and 2D NMR data prompted
us to consider that 1 was a meroterpenoid, which might
be assembled by three subunits: one moiety possessing a di-
terpenoid nucleus (part A), one phenol or phenylpropanoid
(3) (a) Dembitsky, V. M. J. Nat. Med. 2008, 62, 1–33. (b) Chi, Y. M.;
Yan, W. M.; Li, J. S. Phytochemistry 1990, 29, 2376–2378. (c) Tanaka,
N.; Okasaka, M.; Ishimaru, Y.; Takaishi, Y.; Sato, M.; Okamoto, M.;
Oshikawa, T.; Ahmed, S. U.; Consentino, L. M.; Lee, H. K.; Lee, H. K.
Org. Lett. 2005, 7, 2997–2999. (d) Li, Y. S.; Matsunaga, K.; Ishibashi,
M.; Ohizumi, Y. J. Org. Chem. 2001, 66, 2165–2167. (e) Martin, M. J.;
Fernandez, R.; Francesch, A.; Amade, P.; de Matos-Pita, S. S.; Reyes,
F.; Cuevas, C. Org. Lett. 2010, 12, 912–914. (f) Yang, C. S.; Wang, X. B.;
Wang, J. S.; Luo, J. G.; Luo, J.; Kong, L. Y. Org. Lett. 2011, 13, 3380–
3383. (g) Marrero, J.; Rodriguez, A. D.; Baran, P.; Raptis, R. G.;
Sanchez, J. A.; Ortega-Barria, E.; Capson, T. L. Org. Lett. 2004, 6,
1661–1664.
(4) Nakamura, M.; Chi, Y. M.; Yan, W. M.; Yonezawa, A.; Nakasugi,
Y.; Yoshizawa, T.; Hashimoto, F.; Kinjo, J.; Nohara, T.; Sakurada, S.
Planta Med. 2001, 67, 114–117.
(5) Ichikawa, M.; Takahashi, M.; Aoyagi, S.; Kibayashi, C. J. Am.
Chem. Soc. 2004, 126, 16553–16558.
(6) (a) Nicolaou, K. C.; Sarlah, D.; Shaw, D. M. Angew. Chem., Int.
Ed. 2007, 46, 4708–4711. (b) Nicolaou, K. C.; Wu, T. R.; Sarlah, D.;
Shaw, D. M.; Rowcliffe, E.; Burton, D. R. J. Am. Chem. Soc. 2008, 130,
11114–11121. (c) Nicolaou, K. C.; Sanchini, S.; Sarlah, D.; Lu, G.; Wu,
T. R.; Nomura, D. K.; Cravatt, B. F.; Cubitt, B.; de la Torre, J. C.;
Hessell, A. J.; Burton, D. R. Proc. Natl. Acad. Sci. U.S.A. 2011, 108,
6715–6720.
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Lett. 2013, 15, 314–317.
B
Org. Lett., Vol. XX, No. XX, XXXX

C-16, H-70 with C-13, C-14, C-15, and H-14 with C-80,
C-70, C-10 (HMBC). Thus, the direct connections between
C-80 and C-13, C-70 and C-14 formed a cyclobutane ring.
The remaining 1H and 13C NMR resonances were sugges-
tive of a C8 fatty alcohol (part C). This aliphatic side
Table 1. 13C NMR Spectroscopic Data (δ in ppm)a of
Compounds 1, 2, and 4 in Pyridine-d5
no.
1
2
4
no.
1
4
1
chain was identified as 1-octen-3-ol by the 1Hꢀ H COSY
1
2
3
4
5
6
7
8
24.7 t
29.2 t
34.1 t
161.1 s
41.2 s
38.0 t
29.2 t
37.5 d
45.5 s
49.1 d
74.6 d
38.5 t
42.2 s
53.5 d
175.9 s
23.7 q
17.5 q
103.4 t
21.2 q
14.9 q
24.4 t
29.0 t
34.0 t
160.9 s
41.1 s
37.9 t
29.1 t
37.8 d
45.0 s
49.3 d
75.7 d
44.6 t
158.8 s
119.8 d
169.6 s
19.7 q
17.3 q
103.4 t
21.1 q
15.1 q
24.7 t
29.4 t
34.1 t
161.2 s
41.1 s
38.0 t
29.1 t
37.5 d
45.5 s
49.4 d
74.5 d
38.7 t
41.7 s
54.1 d
175.5 s
23.6 q
17.5 q
103.3 t
21.1 q
14.8 q
10
132.1 s
129.0 d
116.2 d
157.5 s
116.2 d
129.0 d
38.0 d
50.4 d
172.6 s
118.3 t
137.9 d
75.5 d
34.9 t
132.7 s
129.2 d
116.2 d
157.4 s
116.2 d
129.2 d
38.4 d
20
experiment which showed the presence of the fragment
CH2(100)ꢀCH(200)ꢀCH(300)ꢀCH2(400)ꢀCH2(500)ꢀCH2(600)ꢀ
CH2(700)ꢀCH3(800) and the HMBC correlations from H-300
to C-100, C-500, H-400 to C-200, C-600, and H-800 to C-500, C-600,
as shown in Figure 1. Furthermore, the connection
between part B and part C through C-90 was supported
by the HMBC correlation of H-300 with C-90. Therefore,
the planar structure of 1 was established to be a new ent-
clerodane-based meroditerpenoid with a unique cyclobu-
tane ring and a 1-octen-3-ol substituent at C-90. However,
the relative stereochemistry of C-11, C-300, and the cyclo-
butane ring could not be easily determined by the NOESY
spectrum. Fortunately, a single-crystal X-ray diffraction
experiment not only confirmed a unique cyclobutane
ring as elucidated above but also determined the relative
stereochemistry (the Flack parameter was too high to
determine its absolute configuration) (Figure 2).
30
40
50
60
70
800
50.5 d
176.0 s
9
900
100
200
3
10
11
12
13
14
15
16
17
18
19
20
400
500
600
700
800
25.5 t
32.1 t
23.1 t
14.5 q
a Data of compounds 1, 2, and 4 were recorded at 125 MHz.
1
Figure 1. Key 1Hꢀ H COSY and selected HMBC correlations
of 1 and 2.
Figure 2. X-ray crystal structures of compound 1.
derivative (part B), and a substituent group of a medium-
chain fatty acid or alcohol (part C).
In order to determine the absolute configuration, com-
pound 1 was hydrolyzed with concentrated aq NaOH in
MeOH to yield 1-octen-3-ol (3) and 4 (Scheme 1), which
were determined by spectroscopic methods. Moreover, the
absolute configuration of 3 was determined to be R (C-300)
by comparison of its optical rotation value with that of
an authentic sample. Thus, the absolute configuration
of compound 1 was established to be 11S,13S,14R,70S,-
80R,300R and named as scopariusic acid (1).
Compound 1 represents a new class of meroditerpenoid
with a unique cyclobutane ring and a novel 1-octen-3-ol
substituent. Although no “[2 þ 2]-ase” has been identified,
it is generally presumed that such cyclobutane derivatives
originate from the direct coupling of the two olefinic
carbon bonds in Nature.3a,10 Our postulated biosynthetic
The 13C NMR signals belong to the diterpenoid nucleus
(part A) were similar to those of 2, and the only difference
was that the C-13/C-14 double bond on the side chain was
replaced by a saturated bond (a methine carbon and a
quaternary carbon, respectively), which was proven by the
HMBC correlations from H-11 to C-13 and H-14 to C-12.
This indicated that part A was linked with other moieties
(part B or C) through C-13 and C-14. The structure of
partial unit B was established to be a phenylpropanoid unit
1
by the 1Hꢀ H COSY correlation of H-80 with H-70 and the
HMBC correlations from H-80 to C-90, C-10 and from H-70
to C-90, C-20, C-60. The connections of part A and Part B
were deduced based on the following keycorrelations: H-70
1
with H-14 (1Hꢀ H COSY); H-80 with C-12, C-13, C-14,
Org. Lett., Vol. XX, No. XX, XXXX
C

Scheme 2. Hypothetical Biosynthetic Pathway of 1
Table 2. Cytotoxic Activity of Compounds 1 and 2
IC50 (μM)
compd
HL-60
SMMC-7721
A-549
16.55
MCF-7
SW480
Figure 3. X-ray crystal structures of compound 2.
Scheme 1. Alkali Hydrolysis of Compound 1
1
2
18.07
>40
17.28
>40
22.54
>40
26.62
>40
>40
DDP
Taxol
2.03
<0.008
13.54
<0.008
12.56
<0.008
18.65
<0.008
19.70
<0.008
the laboratory, and enzymatic control might be essential in
this case.12
Compounds 1 and 2 were screened for cytotoxic activities
against the A-549, HL-60, MCF-7, SMMC-7721, and SW-
480 human cell lines, using the MTT method, as reported
previously,13 with cisplatin and paclitaxel as the positive
controls. Compound 1exhibited weak cytotoxicity against all
the five cell lines with IC50 values in the range of 16.6ꢀ26.6 μM
(Table 2). In addtion, compounds 1 and 2 were screened for
immuno-suppressive activity mediated through inhibition
of mouse T cell proliferation in vitro and with BD750 as a
positive control (IC50 = 1.5 μM).14 The nontoxic concentra-
tions of compound 1exhibited moderate immunosuppressive
activity (IC50 = 2.6 μM), while compound 2 showed no
activity (Table S4 and Figure S57, Supporting Information).
pathway of compound 1 from the related ent-clerodane
diterpenoid (2), trans-4-hydroxycinnamic acid (5), and
(3R)-1-octen-3-ol (3) is shown in Scheme 2. The key step
was the formation of a cyclobutane ring by an intermole-
cular [2 þ 2] cycloaddition between (11S,13E)-11-hydroxy-
ent-clerodane-4(18),13-dien-15-oic acid and 10-octen-30-yl-
4-hydroxy-cinnamate (Scheme 2).
As far as we know, such a crossed intermolecular [2 þ 2]
photocycloaddition between acyclic side chains of two
different molecules is uncommon in the natural products
and is still a long-standing unsolved problem in synthetic
chemistry.11 Therefore, we suggested that the biomimetic
synthesis of compound 1 could not be easily carried out in
Acknowledgment. This project was supported financially
by the National Natural Science Foundation of China (No.
81172939), the Major State Basic Research Development
Program of China (No. 2009CB522300), the reservation-
talent project of Yunnan Province (2011CI043), the Science
and Technology Program of Yunnan Province (No.
2008IF010), the Major Direction Projection Foundation
of CAS Intellectual Innovation Project (No. KSCX2-EW-
J-24), and WestLight Foundation ofthe Chinese Academy
of Sciences (J.-X.P.).
(10) Gutekunst, W. R.; Baran, P. S. J. Am. Chem. Soc. 2011, 133,
19076–19079.
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(b) Garcıa-Exposito, E.; Gonzalez-Moreno, R.; Martın-Vila, M.;
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Muray, E.; Rife, J.; Bourdelande, J. L.; Branchadell, V.; Ortuno,
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Supporting Information Available. 1H NMR assign-
ments of compounds 1, 2, and 4 in Table S1, immunosup-
pressive activity of compounds 1and 2in Table S4 and Figure
S57, 1D, 2D NMR spectra, X-ray crystallographic data of 1
and 2, and detailed experimental procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
(12) Gravel, E.; Poupon, E. Eur. J. Org. Chem. 2008, 1, 27–42.
(13) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker,
R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589–601.
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S. X.; Zheng, J.; Luo, X. Y.; Lai, Y.; Yang, P.; Li, L. M.; Zou, Q. Br. J.
Pharmacol. 2013, 168, 632–634.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX