"mirror-image" manipulation of curdione stereoisomer scaffolds by chemical and biological approaches: Development of a sesquiterpenoid library

Article abstract of DOI:10.1021/np500864e

The sesquiterpenoid curdione is one of the main bioactive components in the essential oil of Rhizoma Curcumae (Curcuma wenyujin, Curcuma phaeocaulis, and Curcuma kwangsiensis), which has been clinically used for the treatment of cancer in mainland China. Recently it was reported that natural curdione could be hydroxylated by Aspergillus niger and transferred to its corresponding curcumalactones under acidic conditions. Based on this study, the development of a sesquiterpenoid library through the "mirror-image" manipulation of bioactive (non)natural curdione scaffolds by chemical and biological approaches is presented herein. A. niger induced the hydroxylation of two pairs of curdione enantiomers, yielding the corresponding mirror-image hydroxylated curdiones. Simultaneously, the acid-mediated intramolecular "ene" rearrangements of these curdiones and hydroxylated curdione enantiomers yielded the corresponding mirror-image curcumalactones and hydroxylated curcumalactones. Among the 16 pairs of enantiomers obtained in this study, 23 compounds are new sesquiterpenoids. These curdione and curcumalactone derivatives are of particular interest, as they have the potential to be used as lead compounds and scaffolds in drug discovery.

Full text of DOI:10.1021/np500864e

Article
pubs.acs.org/jnp
“Mirror-Image” Manipulation of Curdione Stereoisomer Scaffolds by
Chemical and Biological Approaches: Development of a
Sesquiterpenoid Library
Bin Qin,† Yuxin Li,† Lingxin Meng,‡ Jingping Ouyang,‡ Danni Jin,† Lei Wu,† Xin Zhang,† Xian Jia,*,‡
and Song You*,†
‡
†School of Life Science and Biopharmaceutical Sciences and School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang 110016, People’s Republic of China
S
* Supporting Information
ABSTRACT: The sesquiterpenoid curdione is one of the main bioactive
components in the essential oil of Rhizoma Curcumae (Curcuma wenyujin,
Curcuma phaeocaulis, and Curcuma kwangsiensis), which has been clinically
used for the treatment of cancer in mainland China. Recently it was
reported that natural curdione could be hydroxylated by Aspergillus niger
and transferred to its corresponding curcumalactones under acidic
conditions. Based on this study, the development of a sesquiterpenoid
library through the “mirror-image” manipulation of bioactive (non)natural
curdione scaffolds by chemical and biological approaches is presented
herein. A. niger induced the hydroxylation of two pairs of curdione
enantiomers, yielding the corresponding mirror-image hydroxylated
curdiones. Simultaneously, the acid-mediated intramolecular “ene”
rearrangements of these curdiones and hydroxylated curdione enantiomers
yielded the corresponding mirror-image curcumalactones and hydroxylated curcumalactones. Among the 16 pairs of enantiomers
obtained in this study, 23 compounds are new sesquiterpenoids. These curdione and curcumalactone derivatives are of particular
interest, as they have the potential to be used as lead compounds and scaffolds in drug discovery.
atural products and their derivatives historically have
been a rich source of bioactive compounds for drug
isomers of natural products as substrates for biotransformation
have been reported.11,12
N
discovery and development.1 Of the 1130 new drugs approved
by the FDA from 1981 to 2010, approximately 50% are natural
products or their derivatives.2 However, in the past two
decades, natural product-based drug discovery has declined,
partly due to the difficulty in isolating or synthesizing natural
products and their analogues.3 Therefore, to retain their
usefulness in the future, it is important to construct structurally
diverse compounds from natural products.
Curdione [(4S,7S)-Cd, 1a, Figure 1] is one of the major
sesquiterpenoids in the essential oil of Rhizoma Curcumae
(Curcuma wenyujin Y.H. Chen et C. Ling, Curcuma phaeocaulis
Valeton, and Curcuma kwangsiensis S.G. Lee et C.F. Liang) and
has been used clinically as a remedy for the treatment of
cervical cancer in mainland China for a long time.13 This
compound with a germacrane skeleton possesses multiple
bioactive properties, such as anti-inflammatory and anticancer
activities.14 However, the lack of viable synthesis routes for the
derivatization of curdione represents a fundamental obstacle to
the continued discovery of new lead compounds. The
curdiones possess two stereogenic centers and thus have four
possible stereoisomers. Neocurdione [(4S,7R)-Cd, 1c], a minor
sesquiterpenoid in the essential oil of Rhizoma Curcumae, is the
7-epimer of curdione. The 4,7- (1b) and 4-epimers (1d) of
curdione, non-natural sesquiterpenoids, are enantiomers of
naturally occurring curdione and neocurdione, respectively.
Owing to the potential biological activities of curdione, both
chemical15 and biological procedures16 have been developed for
the preparation of curdione derivatives. For example, several
Biotransformation is considered an efficient method of
constructing complex and diverse compounds from natural
products.4,5 The biotransformation of bioactive natural
products has the potential to produce regio- and stereoselective
lead compounds for drug discovery under mild conditions.6,7
For example, many commercial steroidal drugs are produced
using biotransformations of steroid precursors by microbial
whole cells.8 It is not uncommon for natural products to have
more than one stereogenic center with a significant influence
on biological activity.9 The biotransformation of optically active
natural products may generate compounds with increased
complexity and additional stereogenic centers. Furthermore,
these compounds with particular molecular attributes might
contribute to their success throughout the drug discovery
process, including increased solubility and greater selectivity.10
However, only a few studies with complete sets of stereo-
Received: November 1, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np500864e
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Structures of natural curdione (1a) and its stereoisomers. Curdione [(4S,7S)-curdione, 1a] and neocurdione [(4S,7R)-curdione, 1c] are
natural products, while their enantiomers (1b and 1d) are synthetic non-natural compounds.
Figure 2. Mirror-image chemical and biological transformations of curdione enantiomers (1a and 1b).
Figure 3. Synthesis of (4R,7R)-curdione (1b) using (4S,7R)-curdione (1c) as a substrate.
strains of filamentous fungi were used to convert curdione (1a)
into more polar metabolites.17,18 In previous papers, Asakawa
and co-workers19 and our group20 have reported site-specific
hydroxylation of curdione (1a) by the fungus Aspergillus niger,
which yielded 3α-hydroxycurdione [3α-OH-(4S,7S)-Cd, 2a] as
the major metabolite (Figure 2, Figure S1, Supporting
Information) and 2β-hydroxycurdione [2β-OH-(4S,7S)-Cd,
3a] as the minor metabolite. Moreover, curdione (1a), 3α-
hydroxycurdione (2a), and 2β-hydroxycurdione (3a) could be
transformed into curcumalactone [(1S,4S,5R,7S)-Cl, 4a], 3α-
hydroxycurcumalactone [3α-OH-(1S,4S,5R,7S)-Cl, 5a], and
2β-hydroxycurcumalactone [2β-OH-(1S,4S,5R,7S)-Cl, 6a] pos-
sessing a spirolactone skeleton formed via transannular
cyclization, upon acid treatment (Figure 2). Additionally,
Kuroyanagi and co-workers also reported that the transannular
cyclization of germacrene-type compounds might yield
eudesmane-type sesquiterpenoids.21 Although the spirolactone
structure and potential bioactivity of curcumalactone (4a) have
attracted significant interest, its challenging synthesis has been a
major obstacle in exploring its promising chemistry and
bioactivity. The first and only enantioselective total synthesis
of curcumalactone was reported in 2012.22
In an attempt to obtain new pharmacologically active
derivatives of curdione (1a) and curcumalactone (4a), herein
the development of a sesquiterpenoid library by mirror-image
B
DOI: 10.1021/np500864e
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Table 1. 13C NMR Data (δC) for Curdiones and Hydroxycurdiones (CDCl3, 150 MHz)
Article
a
position
1a
1c
2a
2c
2e
3a
3c
3e
1
131.5
26.3
131.2
25.5
32.7
45.7
210.3
42.0
52.5
212.6
55.2
129.1
30.9
20.2
21.0
18.2
18.2
127.0
36.4
73.6
53.3
210.9
44.0
53.1
211.6
55.3
133.0
29.8
21.0
19.8
15.1
16.9
126.6
34.6
74.4
50.9
210.3
43.0
52.5
212.8
55.8
131.7
30.5
20.0
21.0
17.8
17.8
126.8
34.0
74.7
48.7
209.7
43.0
53.4
213.5
55.1
131.9
30.7
21.0
20.3
14.1
17.7
133.8
68.1
42.4
44.2
215.1
44.4
54.1
210.5
56.2
130.8
29.9
19.7
21.1
18.5
17.0
134.6
67.1
41.0
40.0
208.6
43.6
52.5
212.3
57.5
128.7
31.6
20.9
20.3
18.9
17.3
133.7
67.6
41.6
44.1
211.9
42.1
52.7
209.4
53.8
131.4
30.9
21.0
20.4
18.7
20.0
2
3
34.0
4
46.7
5
211.1
44.2
6
7
53.6
8
214.3
55.8
9
10
11
12
13
14
15
129.8
29.9
19.8
21.1
18.5
16.50.
a
The 13C NMR data of 1b, 1d, 2b, 2d, 2f, 3b, 3d, and 3f are identical to those of 1a, 1c, 2a, 2c, 2e, 3a, 3c, and 3e, respectively.
a
Table 2. 13C NMR Data (δC) for Curcumalactones and Hydroxycurcumalactones (CDCl3, 150 MHz)
position
4a
4c
5a
5c
5e
6a
6c
6e
1
53.5
23.7
26.8
41.7
92.4
22.4
46.4
178.4
113.3
143.3
28.2
18.0
20.5
13.7
24.0
56.2
27.3
31.3
43.4
94.6
28.0
46.7
178.1
114.3
145.7
27.8
20.6
18.0
12.8
21.6
51.3
34.9
73.9
51.1
91.2
22.8
46.0
177.7
113.5
142.1
28.0
17.7
20.2
11.3
23.7
53.9
36.7
76.4
50.8
92.7
28.6
46.2
177.9
115.2
144.7
27.8
18.0
20.5
10.3
21.6
55.0
38.7
74.8
47.6
94.7
28.0
46.0
176.9
115.2
144.7
27.7
20.7
18.0
7.4
58.9
69.8
40.0
39.8
93.1
24.6
46.6
178.2
116.4
141.8
28.3
20.6
18.1
14.1
27.2
61.5
71.9
40.9
40.4
94.3
28.9
46.5
177.6
117.1
141.8
27.7
20.6
18.1
13.0
24.8
65.5
74.6
41.4
41.8
93.0
29.0
46.5
177.4
115.4
142.9
27.8
20.6
18.1
12.8
22.9
2
3
4
5
6
7
8
9
10
11
12
13
14
15
21.1
a
The 13C NMR data of 4b, 4d, 5b, 5d, 5f, 6b, 6d, and 6f are identical to those of 4a, 4c, 5a, 5c, 5e, 6a, 6c, and 6e, respectively.
chemical and biological transformations of curdione and its
three stereoisomers is described.
However, their electronic circular dichroism (ECD) spectra
(Figure S4, Supporting Information) were opposite of those of
1a and 1c, indicating that the synthetic compounds (4R,7R)-
curdione (1b) and (4R,7S)-curdione (1d) are the enantiomers
of (4S,7S)-curdione (1a) and (4S,7R)-curdione (1c), respec-
tively.
RESULTS AND DISCUSSION
■
(4S,7S)-Curdione (1a) and (4S,7R)-curdione (1c) can be
isolated from the essential oil of Curcuma wenyujin. However,
the non-natural (4R,7R)-curdione (1b) and (4R,7S)-curdione
(1d) need to be synthesized. The synthesis of 1d was
performed following the reported approach by isomerization
at C-4 of natural 1a via a three-step reaction.23 The reported
method for the synthesis of 1b is a multistep reaction (12
steps) and gave a low overall yield.24 Inspired by the synthesis
of 1d from 1a, we have developed a new synthesis method for
non-natural (4R,7R)-curdione (1b) through isomerization at C-
4 of the naturally occurring (4S,7R)-curdione (neocurdione,
1c) via a similar three-step reaction, as described in Figure 3.
Thus, (4S,7R)-curdione (1c) was reduced by treatment with
NaBH4 in MeOH, followed by t-BuOK-mediated isomerization
at C-4 and subsequent oxidation, to yield (4R,7R)-curdione
(1b). The 1D 1H and 13C NMR data of (4R,7R)-curdione (1b)
and (4R,7S)-curdione (1d) matched those of natural (4S,7S)-
curdione (1a) and (4S,7R)-curdione (1c) well, respectively
(Tables 1 and 3, Figures S2 and S3, Supporting Information).
With curdione and its three stereoisomers in hand, the
compounds were subjected to microbial transformation. The
synthetic (4R,7R)-curdione (1b), the enantiomer of 1a, was
selected as the substrate for biotransformation, where we
anticipated it to behave similarly to 1a in the presence of A.
niger (As 3.739). The incubation of 1b with A. niger for 3 days
led to compounds 2b and 3b as major and minor metabolites,
respectively (Figure 2 and Figure S1, Supporting Information).
Compounds 2b and 3b were characterized from their HRMS,
chiral HPLC analysis, and 1D NMR spectra. The 13C NMR and
HRMS data of 2b and 3b were in accordance with the
molecular formula C15H24O3. The 1D 1H and 13C NMR
spectroscopic data of compound 2b were identical to those of
3α-OH-(4S,7S)-curdione (2a) (Tables 1 and 3, Figures S5 and
S6, Supporting Information), while the NMR data of 3b were
identical to those of 3a (Tables 1 and 3, Figures S8 and S9,
Supporting Information). Furthermore, compounds 2a and 2b,
C
DOI: 10.1021/np500864e
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
a
1
Table 3. H NMR Data (δH) for Curdiones and Hydroxycurdiones (CDCl3, 600 MHz, J in Hz)
position
1a
5.15, brs
1c
5.13, brs
2a
2c
5.27, brs
2e
5.29, brs
3a
3c
3e
1
2
5.10, brs
5.10, d (10.2)
5.56, d (7.2)
4.62, m
5.00, d (10.0)
α, 2.12, m
α, 2.16, m
α, 2.09, m
α, 2.26, m
α, 2.32, dt (14.8, 4.46, td (10.3,
8.1)
4.36, td (10.0,
4.7)
5.8)
β, 2.12, m
α,1.52, m
β, 2.07, m
α,1.93, m
β, 2.50, m
β, 2.51, m
β, 2.50, d (12.3)
4.22, m
3
4.00, m
3.97, m
α, 2.21, m
β, 1.86, m
α, 2.56, ddd (15.4,
α, 2.07, dt (13.3,
10.2, 5.7)
10.6)
β, 2.06, m
β, 1.72, m
β, 1.63, dt (14.6, 2.7) β, 1.91, m
4
6
2.30, m
2.48, m
2.37, m
2.60, m
2.61, m
2.32, dd (11.6,
6.8)
2.97, ddd (9.8, 7.1,
2.3)
2.43, m
α, 2.67, m
α, 2.69, dd (15.0, α, 2.66, m
10.0)
α, 2.70, dd (16.1, α, 2.42, m
9.2)
α, 2.44, m
α, 2.41, dd (12.9,
11.0)
α, 2.75, m
β, 2.38, dd (16.7, β, 2.38, dd (15.0, β, 2.41, m
β, 2.37, d (16.0) β, 2.79, m
β, 2.44, m
β, 2.62, dd (12.9, 3.1) β, 2.40, m
2.3)
2.8)
7
9
2.81, m
2.87, m
2.76, m
2.90, m
2.79, m
2.86, dd (11.4,
7.7)
3.12, ddd (11.3, 8.7, 2.74, m
3.0)
α, 3.05, d (10.9) α, 2.86, m
α, 2.96, m
α, 3.06, m
β, 3.06, m
α, 2.94, d (11.8) α, 2.86, d (11.4) α, 2.75, d (10.0)
α, 2.74, m
β, 2.92, d (11.2) β, 3.02, d (12.7) β, 2.96, m
β, 3.06, d (11.9) β, 3.18, d (10.7) β, 3.17, d (10.0)
β, 3.20, d (13.7)
7.7)
11
12
13
14
15
1.85, m
1.84, m
1.78, m
1.85, m
1.86, m
1.86 (m, H)
0.95, d (6.6)
0.87, d (6.5)
1.01, d (7.0)
1.77, s
1.85, ddt (13.1, 8.9,
6.5)
1.86, dt (13.7,
7.3)
0.94, d (7.3)
0.87, d (6.7)
0.97, d (6.9)
1.64, s
0.95, d (6.7)
0.90, d (6.6)
1.03, d (7.2)
1.64, s
0.81, d
(6.5)
0.89, d (6.6)
0.95, d (6.6)
1.16, d (7.1)
1.66, s
0.90, d (6.6)
0.97, d (6.6)
1.25, d (4.5)
1.66, s
0.92, d (6.7)
0.98, d (6.7)
1.02, d (7.2)
1.47, s
0.95, d (6.8)
0.90, d (6.7)
1.07, d (7.2)
1.80, s
0.88, d
(6.6)
1.03, d
(7.1)
1.63, s
a
1
The H NMR data of 1b, 1d, 2b, 2d, 2f, 3b, 3d, and 3f are identical to those of 1a, 1c, 2a, 2c, 2e, 3a, 3c, and 3e, respectively.
a
1
Table 4. H NMR Data (δH) for Curcumalactones and Hydroxycurcumalactones (CDCl3, 600 MHz, J in Hz)
position
1
4a
4c
5a
5c
5e
6a
6c
6e
2.75, dd (11.7,
8.7)
2.78, dd (8.8,
6.5)
3.05, m
2.77, t (8.4)
3.09−3.03, m
2.60, d (6.5)
2.85, d (6.8)
2.70, d (5.7)
2
3
α, 1.72, m
β, 1.72, m
α, 1.20, m
α, 1.99, m
β, 1.60, m
α, 1.49, m
α, 1.74, m
β, 2.13, m
3.78, m
α, 1.60, m
β, 2.35, m
4.00, q (8.0)
α, 2.10, m
β, 2.05, m
4.17, t (5.6)
4.32, td (7.0, 4.3) 4.56, td (7.4, 5.0) 4.15, m
α, 2.27, m
β, 1.29, m
α, 1.94, m
β, 1.94, m
α, 2.34, dt (13.8,
7.3)
β, 1.93, m
β, 1.88, m
β, 1.57, m
4
6
2.41, ddq (11.0,
9.5, 6.8)
1.88, m
2.37, m
1.75 (m, 1H)
1.94, m
2.49, ddd (14.1,
9.1, 7.5)
2.35, ddt (16.7,
9.7, 6.9)
1.93, dq (11.5,
7.0)
α, 1.83, dd (10.6, α, 1.80, dd (12.9, α, 1.85, m
3.9) 12.0)
α, 2.10, dd (13.1, α, 2.05, m
9.2)
α, 2.02, dd (13.5, α, 2.21, dd (13.2, α, 2.08, dd (13.1,
11.2)
9.1)
9.2)
β, 1.83, dd (10.6, β, 2.04, m
3.9)
β, 1.85, m
β, 1.83, t (12.5) β, 1.88, t (12.7) β, 2.37, dd (13.5, β, 1.87, m
β, 1.79, m
9.8)
7
9
2.56, td (10.5,
4.8)
2.57, ddd (12.0,
2.56, m
2.56, ddd (11.9,
9.2, 4.9)
2.60, ddd (13.3,
9.1, 4.9)
2.75, ddd (11.2,
9.7, 5.0)
2.55, ddd (12.1,
9.1, 5.0)
2.57, ddd (11.9,
9.2, 4.9)
9.2, 5.0)
a, 4.99, m
b, 4.91, m
a, 4.88, p (1.6)
a, 5.02, s
b, 4.90, s
a, 4.94, s
b, 4.88, s
a, 4.93, s
b, 4.83, s
a, 5.20, s
b, 5.15, s
a, 5.14, s
b, 4.89, s
a, 5.03, s
b, 4.84, s
b, 4.79, dd (2.0,
1.0)
11
2.20, m
2.16, pd (6.8,
5.0)
2.20, m
2.17, pd (6.8,
4.7)
2.19, dt (13.3,
6.5)
2.16, pd (6.9,
4.9)
2.17, m
2.18, pd (6.9,
4.9)
12
13
14
15
0.90, d (6.8)
0.99, d (6.8)
0.94, d (6.9)
1.80, s
0.99, d (6.9)
0.90, d (6.8)
0.98, d (6.4)
1.71, s
0.90, d (6.8) 0.90, d (6.8)
0.98, d (7.1) 1.00, d (6.9)
1.06, d (6.9) 1.05, d (6.8)
1.02, d (6.8)
0.92, d (6.9)
1.06, d (7.0)
1.70, s
1.00, d (6.9)
0.91, d (6.8)
1.03, d (6.9)
1.90, s
1.01, d (7.2),
0.91, d (6.8)
1.00, d (7.2)
1.87, s
1.01, d (6.9)
0.91, d (6.8)
1.05, d (6.8)
1.79, s
1.81, s
1.77, s
a
1
The H NMR data of 4b, 4d, 5b, 5d, 5f, 6b, 6d, and 6f are identical to those of 4a, 4c, 5a, 5c, 5e, 6a, 6c, and 6e, respectively.
as well as compounds 3a and 3b, were eluted at different
retention times by chiral column chromatography, while each
of the pairs exhibited mirror-image-like ECD spectra (Figures
S7 and S10, Supporting Information). These results indicated
that compounds 2b and 2a as well as 3b and 3a are pairs of
enantiomers.
Following biotransformation, the synthetic (4R,7R)-curdione
(1b) was used as a substrate for chemical transformation under
acidic conditions. The observed results showed that the
sesquiterpenoid 1b could be readily converted to compound
4b in a 91% yield by treatment with hydrochloric acid.
Similarly, the isolated 4b was found to be identical in all aspects
to naturally occurring (1S,4S,5R,7S)-curcumalactone (4a)
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DOI: 10.1021/np500864e
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Figure 4. Mirror-image chemical and biological transformations of neocurdione enantiomers (1c and 1d).
(Tables 2 and 4, Figures S11 and S12, Supporting
Information), except that its ECD spectrum was opposite
that of 4a (Figure S13, Supporting Information). This evidence
confirmed that the absolute stereochemistry of synthetic 4b is
opposite that of (1S,4S,5R,7S)-Cl (4a). Moreover, chemical
transformation of 3β-OH-(4R,7R)-Cd (2b) and 2α-OH-
(4R,7R)-Cd (3b) yielded the corresponding 3β-OH-
(1R,4R,5S,7R)-Cl (5b) and 2α-OH-(1R,4R,5S,7R)-Cl (6b),
which are the enantiomers of 3α-OH-(1S,4S,5R,7S)-Cl (5a)
and 2β-OH-(1S,4S,5R,7S)-Cl (6a), respectively (Tables 2 and
4, Figures S14−19, Supporting Information). As mentioned
above, it is interesting to observe that the chemical and
biological transformations of one pair of curdione enantiomers
(1a and 1b) displayed mirror-image pathways (Figure 2). A.
niger induced the hydroxylation of curdione enantiomers (1a
and 1b), yielding the corresponding oxidized mirror-image
products (2a and 2b, 3a and 3b), while the acid-mediated
transannular cyclization of curdione enantiomers (1a and 1b)
and hydroxycurdiones (2a and 2b, 3a and 3b) afforded the
corresponding mirror-image products (4a and 4b, 5a and 5b,
6a and 6b).
first fed into the culture of A. niger. After 3 days, four products
were detected by TLC and HPLC analysis in the culture
medium (Figure S1, Supporting Information). Further
characterization by MS and NMR spectroscopic data (Tables
1 and 3) was used to identify the structures of the major
metabolites 2e and 3c as 3β-hydroxyneocurdione [3β-OH-
(4S,7R)-Cd, Figure 4] and 2α-hydroxyneocurdione [2α-OH-
(4S,7R)-Cd], respectively. The minor metabolites 2c and 3e
were identified as 3α-hydroxyneocurdione [3α-OH-(4S,7R)-
Cd] and 2β-hydroxyneocurdione [2β-OH-(4S,7R)-Cd], respec-
tively. Next, compounds 1c, 2c, 2e, 3c, and 3e were subjected
to chemical transformations under acidic conditions. The
corresponding compounds 4c, 5c, 5e, 6c, and 6e were
produced. After purification, further analysis by NMR spec-
troscopy (Tables 2 and 4) confirmed the structures of 4c and
5c/5e/6c/6e to be (1R,4S,5S,7R)-curcumalactone and 3α/3β/
2α/2β-hydroxy-(1R,4S,5S,7R)-curcumalactones (Figure 4), re-
spectively. Therefore, A. niger induced hydroxylation of
neocurdione (1c) to yield the corresponding hydroxylated
derivatives, while the acid-mediated cyclization of 1c and its
hydroxylated derivatives produced the corresponding spirolac-
tones (Figure 4), in a similar manner to the curdione
enantiomers (1a and 1b). Moreover, it is interesting to observe
that the configuration at C-5 of 4c (5c) was opposite that of 4a
(5a). These results indicated that the facile conversion of
curdione stereoisomers to curcumalactone stereoisomers seems
Inspired by the results of the mirror-image chemical and
biological transformations of curdione enantiomers (1a and
1b), we next focused on the corresponding transformations of
the naturally occurring neocurdione [(4S,7R)-Cd, 1c] and its
synthetic enantiomer [(4R,7S)-Cd, 1d]. Neocurdione (1c) was
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to involve different steric courses (Figure S20, Supporting
Information). Since the configuration of 1a and 1c at C-7 is
different, it can be determined that the isopropyl group might
play an important role in the regio- and stereospecific
spirolactone-forming reaction.
obtained, which could be used for further evaluation of
biological activities in vitro or in vivo.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The electronic circular
dichroism spectra were obtained by spectral scanning (220−420
nm) using a JASCO CD-2095 circular dichroism chiral detector. 1D
and 2D NMR spectra were recorded using a Bruker AVANCE-600 in
CDCl3 with TMS as an internal standard. High-resolution mass
spectra (HRMS) were recorded on a Bruker micrOTOF-Q
spectrometer. High-performance liquid chromatography (HPLC)
analysis was performed on a JASCO LC-2000 system using a Dikma
Diamonsil C18 column (5 μm, 200 × 4.6 mm) with a JASCO UV-2075
detector.
Furthermore, the chemical and biological transformations of
(4R,7S)-curdione (1d), the enantiomer of neocurdione (1c),
were carried out. In vivo biotransformation revealed that A.
niger accepted 1d as a substrate to generate four products
(Figure S1, Supporting Information). The metabolites 2d, 2f,
3d, and 3f were found to have 1D 1H and 13C NMR
spectroscopic data identical to those of 2c, 2e, 3c, and 3e
(Tables 1 and 3, Figures S5, 6 and S8, 9, Supporting
Information), while their ECD spectra were opposite those of
2c, 2e, 3c, and 3e, respectively (Figures S7 and S10, Supporting
Information). This evidence confirmed that compounds 2d, 2f,
3d, and 3f are the corresponding enantiomers of 2c, 2e, 3c, and
3e. The chemical transformations of 1d and 2d/2f/3d/3f
yielded compounds 4d and 5d/5f/6d/6f, respectively. After
NMR and ECD analysis (Tables 2 and 4, Figures S11−S19,
Supporting Information), the structures of 4d and 5d/5f/6d/6f
were confirmed as (1S,4R,5R,7S)-curcumalactone and 3β/3α/
2β/2α-hydroxy-(1S,4R,5R,7S)-curcumalactones, which are the
corresponding enantiomers of 4c and 5c/5e/6c/6e. Therefore,
the chemical and biological transformations of one pair of
neocurdione enantiomers (1c and 1d) also displayed mirror-
image pathways (Figure 4), which are the same as the mirror-
image chemical and microbial transformations of the curdione
enantiomers (1a and 1b).
In this report, the development of a sesquiterpenoid library
through the mirror-image chemical and biological trans-
formations of curdione stereoisomers is presented. A. niger
induced the hydroxylation of two pairs of curdione enantiomers
(1a:1b and 1c:1d) and produced the corresponding mirror-
image hydroxylated derivatives of curdione (2a−f and 3a−f) via
C−H activation. Through the biotransformation of three
curdione stereoisomers (1b−d), 10 new hydroxylated
curdiones (2b−f and 3b−f) were obtained. Furthermore, the
acid-mediated cyclization of these curdione enantiomers (1a:1b
and 1c:1d) and related hydroxylated curdione enantiomers
(2a−f and 3a−f) yielded the corresponding mirror-image
curcumalactones (4a:4b and 4c:4d) and hydroxylated
curcumalactones (5a−f and 6a−f) via transannular cyclization.
Through the transannular cyclization of three curdione
stereoisomers (1b−d) and 10 related hydroxylated curdiones
(2b−f and 3b−f), three new curcumalactones (4b−d) and 10
new hydroxylated curcumalactones (5b−f and 6b−f) were
obtained. Among the 32 compounds (16 pairs of enantiomers)
purified in the course of our study, 23 are new sesquiterpenoids
(enantiomers, 2b−f, 3b−f, 4a−d, 5b−f, and 6b−f).
Strain and Medium. Aspergillus niger AS 3.739, purchased from
the China General Microbiological Culture Collection Center (Beijing,
People’s Republic of China), was cultured in the medium (15.0 g
dextrose, 15.0 g sucrose, 5.0 g peptone, 1.0 g KH2PO4, 0.5 g MgSO4,
0.5 g KCl, 0.01 g FeSO4, and 1000 mL H2O, pH 7.0) at 28 °C and 210
rpm for 48 h.
Plant Material. The dried rhizomes of Curcuma wenyujin were
collected from Ruian, Zhejiang Province, China, in April 2010. The
plant was identified by Dr. Y. H. Chen, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical
College. A voucher specimen (No. 4587) of the rhizomes is available
from the plant collection in the Chinese Virtual Herbarium.
Preparation of Curdione (1a) and Neocurdione (1c). The
essential oil was obtained by steam distillation from the rhizomes of C.
wenyujin. Curdione (1a) and neocurdione (1c) were isolated from the
essential oil of C. wenyujin by column chromatography with petroleum
ether−EtOAc (50:1, v/v). The compounds were identified by NMR
1
and MS techniques, and their H and 13C NMR and MS data were
identical to those reported in the literature.27,28
Preparation of (4R,7R)-Curdione (1b) and (4R,7S)-Curdione
(1d). The synthesis of (4R,7S)-curdione (1d) was performed based on
the reported approach23 by isomerization at C-4 of natural (4S,7S)-
curdione (1a) via a three-step reaction. The synthesis of (4R,7R)-
curdione (1b) was carried out using a similar method by isomerization
at C-4 of natural (4S,7R)-curdione (neocurdione, 1c). Step 1:
Neocurdione (1c, 230 mg) was dissolved in 3 mL of methanol.
NaBH4 (50 mg, 1.2 equiv) was then added at 0 °C, and the mixture
was stirred at 0 °C. The reaction was monitored by TLC. After the
substrate had almost disappeared (5 h), the reaction solution was
evaporated under reduced pressure. Water was added to the residue,
and the solution was further acidified with several drops of HCl to
reach pH 7. The mixture was extracted with EtOAc four times. The
combined organic phase was evaporated under reduced pressure to
yield the product compound 7 (Figure 2). Step 2: The crude oil of 7
was dissolved in 5 mL of benzene. Potassium tert-butoxide (30 mg, 0.3
equiv) was added to the solution. A reflux condenser was attached, and
the reaction mixture was stirred at 83 °C for 7 h. After cooling to room
temperature, the reaction solution was evaporated under reduced
pressure to yield a crude oil. The crude oil was loaded on a silica gel
column and eluted with a mixture of petroleum ether and EtOAc
(30:1, v/v) to yield compound 8. Step 3: Compound 8 was dissolved
in 2 mL of pyridine. CrO3 (100 mg, 2.8 equiv) was added to the
solution. After stirring at room temperature for 5 h, the precipitate was
removed by filtration with silica gel. The filtrate was concentrated in
vacuo, and the residue was purified by chromatography [petroleum
ether and EtOAc (30:1, v/v)] to obtain (4R,7R)-curdione (1b, 70
mg).
The compounds reported in this paper, especially the
hydroxylated curdione and curcumalactones, which are rare
natural products with a unique spirolactone ring system25 and
documented bioactivity,26 were of particular interest. These
compounds can provide valuable lead compounds or building
blocks that are not accessible by currently available synthesis
methods for drug discovery. To the best of our knowledge, no
information regarding the biological activities of curcumalac-
tone derivatives is currently available. In this regard, the
potential anti-inflammatory and anticancer activities of
curdiones, curcumalactones, and their derivatives will be further
investigated. Moreover, by the target-oriented derivation of the
sesquiterpenoid library, more diversified compounds may be
Biotransformation of Curdione Stereoisomers (1a−d) Using
A. niger. The biotransformation of curdione stereoisomers was carried
out according to the method of Asakawa et al.19 Precultured mycelium
(dry weight 5 g) was collected, washed, and incubated with 1000 mL
of phosphate buffer (43.70 g Na2HPO4·12H2O, 12.17 g NaH2PO4·
2H2O, and 1000 mL H2O, pH 7.0). Concomitantly, the cells were
supplemented with compounds 1a−d (0.3 mg/mL) and incubated at
28 °C and 210 rpm. The biotransformation was monitored by TLC
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and HPLC. After 3 days, the reaction solution was centrifuged, and the
supernatant was extracted with EtOAc. Then the organic phase was
evaporated under reduced pressure. The crude product was passed
through a silica column with a petroleum ether−EtOAc elution (30:1,
v/v) to yield the related hydroxylated curdione stereoisomers (2a−f
and 3a−f).
Chemical Transformation of Curdione and Hydroxycur-
dione Stereoisomers (1a−d, 2a−f, and 3a−f). A total of 50 mg of
curdione or hydroxycurdione stereoisomers dissolved in MeOH (2
mL) was added to 0.2 mL of HCl (1 M). The mixtures were stirred at
28 °C for 4 h. Water was then added to the mixture, and the solution
was further neutralized with NaOH to reach pH 7. The mixture was
extracted with CH2Cl2 four times. The combined organic phase was
concentrated in vacuo, and the residue was purified by chromatog-
raphy [petroleum ether and EtOAc (30:1, v/v)] to obtain related
curcumalactones and hydroxycurcumalactones (4a−d, 5a−f, and 6a−
f) in yields between 80% and 90%.
HPLC Analysis of the 16 Pairs of Sesquiterpenoid
Enantiomers. High-performance liquid chromatography analysis
was carried out using a JASCO LC-2000 system with a CD-2095
circular dichroism chiral detector. The chromatographic conditions
were as follows: chromatographic column: Diamonsil C18 column (5
μm, 200 × 4.6 mm, Dikma Technologies Inc., Beijing, People’s
Republic of China), mobile phase: (A) CH3CN−H2O (60:40, v/v),
(B) CH3CN−H2O (40:60, v/v), or (C) CH3CN−H2O (20:80, 0 min;
80:20, 30 min; 20:80, 35 min; v/v), flowing at a rate of 1.0 mL/min.
Detection: 220 nm. For the curdione enantiomers, the elution times
were 11.2 min for 1a and 1b using mobile phase A and 10.2 min for 1c
and 1d using mobile phase A. For the 3-hydroxycurdione enantiomers,
the elution times were 6.5 min for 2a and 2b using mobile phase B,
14.8 min for 2c and 2d using mobile phase C, and 14.4 min for 2e and
2f using mobile phase C. For the 2-hydroxylcurdione enantiomers, the
elution times were 5.9 min for 3a and 3b using mobile phase B, 13.3
min for 3c and 3d using mobile phase C, and 12.8 min for 3e and 3f
using mobile phase C. For the curcumalactone enantiomers, the
elution times were 19.5 min for 4a and 4b using mobile phase A and
20.3 min for 4c and 4d using mobile phase A. For the 3-
hydroxycurcumalactone enantiomers, the elution times were 10.4
min for 5a and 5b using mobile phase B, 16.1 min for 6c and 6d using
mobile phase B, and 16.0 min for 6c and 6d using mobile phase B. For
the 2-hydroxycurcumalactone enantiomers, the elution times were
18.2 min for 6a and 6b using mobile phase B, 12.0 min for 3c and 3d
using mobile phase B, and 13.8 min for 3e and 3f using mobile phase
B.
REFERENCES
■
(1) Eder, J.; Sedrani, R.; Wiesmann, C. Nat. Rev. Drug Discovery 2014,
13, 577−587.
(2) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311−335.
(3) Li, J. W.; Vederas, J. C. Science 2009, 325, 161−165.
(4) Turner, N. J.; Truppo, M. D. Curr. Opin. Chem. Biol. 2013, 17,
212−214.
́ ́
(5) Munoz Solano, D.; Hoyos, P.; Hernaiz, M. J.; Alcantara, A. R.;
̃
́
Sanchez-Montero, J. M. Bioresour. Technol. 2012, 115, 196−207.
(6) Liu, J.; Yu, B. Curr. Org. Chem. 2010, 14, 1400−1406.
(7) Groussin, A. L.; Antoniotti, S. Bioresour. Technol. 2012, 115, 237−
243.
(8) Donova, M.; Egorova, O. Appl. Microbiol. Biotechnol. 2012, 94,
1423−1447.
(9) Clemons, P. A.; Bodycombe, N. E.; Carrinski, H. A.; Wilson, J. A.;
Shamji, A. F.; Wagner, B. K.; Koehler, A. N.; Schreiber, S. L. Proc. Natl.
Acad. Sci. U.S.A. 2010, 107, 18787−18792.
(10) Koehn, F. E.; Carter, G. T. Nat. Rev. Drug Discovery 2005, 4,
206−220.
(11) Daramwar, P. P.; Rincy, R.; Niloferjahan, S.; Krithika, R.; Gulati,
A.; Yadav, A.; Sharma, R.; Thulasiram, H. V. Bioresour. Technol. 2012,
115, 70−74.
́
(12) Arseniyadis, S.; Ouazzani, J.; Rodriguez, R.; Rumbero, A.;
Ourisson, G. Tetrahedron Lett. 1991, 32, 3573−3576.
(13) Pharmacopoeia Commission of People’s Republic of China.
Pharmacopoeia of the People’s Republic of China (Part 1); Chinese
Medical Science and Technology Press: Beijing, 2010; pp 257−258.
(14) Aggarwal, B. B.; Yuan, W.; Li, S.; Gupta, S. C. Mol. Nutr. Food
Res. 2013, 57, 1529−1542.
(15) Kikuchi, H.; Sakurai, K.; Oshima, Y. Org. Lett. 2014, 16, 1916−
1919.
(16) Ma, X.; Deng, S.; Wang, C.; Shu, X.; Zhang, B. Prog. Mod.
Biomed. 2010, 18, 3537−3540.
(17) Ma, X.; Ye, M.; Wu, L.; Guo, D. Enzyme Microb. Technol. 2006,
38, 367−371.
(18) Zhou, L.; Xu, W.; Chen, Y.; Zhao, J.; Yu, N.; Fu, B.; You, S.
Catal. Commun. 2012, 28, 191−195.
(19) Asakawa, Y.; Takahashi, H.; Toyota, M. Phytochemistry 1991, 30,
3993−3997.
(20) Chen, Y.; Zhang, L.; Qin, B.; Zhang, X.; Jia, X.; Wang, X.; Jin,
D.; You, S. Nat. Prod. Res. 2014, 28, 454−460.
(21) Kuroyanagi, M.; Shirota, O.; Sekita, S. Chem. Pharm. Bull. 2014,
62, 725−728.
(22) Leverett, C. A.; Purohit, V. C.; Johnson, A. G.; Davis, R. L.;
Tantillo, D. J.; Romo, D. J. Am. Chem. Soc. 2012, 134, 13348−13356.
(23) Ohkura, T.; Gao, J.-F.; Harimaya, K.; Hikichi, M.; Iitaka, Y.;
Kawamata, T.; Kuroyanagi, M.; Fukushima, S.; Inayama, S. Chem.
Pharm. Bull. 1986, 34, 4435−4438.
ASSOCIATED CONTENT
* Supporting Information
1D NMR, UV, and CD spectra for related compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
(24) Zhao, R.; Wu, Y. Acta Chim. Sin. 1989, 7, 86−87.
(25) Chen, Y.; Jia, X.; Ma, X.; Qin, B.; Wan, X.; Wu, Y.; Xu, W.; Yin,
Y.; You, S. WO Patent 2012013016, 2012.
AUTHOR INFORMATION
Corresponding Authors
*Tel: 86-24-23986456. Fax: 86-24-23986436. E-mail:
jiaxian206@163.com (X. Jia).
*Tel: 86-24-23986436. Fax: 86-24-23986436. E-mail:
yousong207@163.com (S. You).
(26) Saifudin, A.; Tanaka, K.; Kadota, S.; Tezuka, Y. J. Nat. Prod.
2013, 76, 223−229.
■
(27) Yan, J.; Chen, G.; Tong, S.; Feng, Y.; Sheng, L.; Lou, J. J.
Chromatogr. A 2005, 1070, 207−210.
(28) Kuroyanagi, M.; Ueno, A.; Ujiie, K.; Sato, S. Chem. Pharm. Bull.
1987, 35, 53−59.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Scientific
Major Program (2010ZX09301-012), the Shenyang Municipal
Scientific and Technology Research Fund (F11-243-1-00), and
the Science and Technology Research Projects from the
Ministry of Education of the People’s Republic of China
(213007A).
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DOI: 10.1021/np500864e
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