Microbial transformations of buagarofuran, an anti-anxietic agent

Article abstract of DOI:10.1016/j.tet.2014.04.011

19 structurally diverse metabolites (2-20) were isolated from the transformation of buagarofuran (1), an anxiety agent, by three filamentous fungal strains, Cunninghamella echinulata AS 3.3402, Cunninghamella elegans AS 3.3400, and Absidia coerulea AS 3.3538. Their structures and identified on the basis of extensive spectroscopic data (HRMS, NMR, IR, and CD) and chemical methods. All of the metabolites were oxidized products of 1, and metabolite 20 was also one of mammalian metabolites. Metabolites 8, 10-12, and 20 significantly inhibited [³H]-NE re-uptake, and 8 and 9 strongly inhibited [³H]-5-HT re-uptake in the rat brain synaptosomes at 10 μM in vitro. The structural diversity of the microbial transformed metabolites of buagarofuran in the present report would give a favorable opportunity to probe its mammalian metabolism and better shed light on its pharmacokinetics and pharmacodynamics in vivo.

Full text of DOI:10.1016/j.tet.2014.04.011

Tetrahedron 70 (2014) 3560e3569
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Microbial transformations of buagarofuran, an anti-anxietic agent
,
Lirui Qiao a, Chengxue Ji a, Dewu Zhang a, Lin Yang b, Ridao Chen a, Jianjun Zhang a
*
,
,
Jungui Dai a
*
a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking Union Medical College & Chinese
Academy of Medical Sciences, Beijing 100050, China
b College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, China
a r t i c l e i n f o
a b s t r a c t
Article history:
19 structurally diverse metabolites (2e20) were isolated from the transformation of buagarofuran (1), an
anxiety agent, by three filamentous fungal strains, Cunninghamella echinulata AS 3.3402, Cunninghamella
elegans AS 3.3400, and Absidia coerulea AS 3.3538. Their structures and identified on the basis of ex-
tensive spectroscopic data (HRMS, NMR, IR, and CD) and chemical methods. All of the metabolites were
oxidized products of 1, and metabolite 20 was also one of mammalian metabolites. Metabolites 8, 10e12,
and 20 significantly inhibited [3H]-NE re-uptake, and 8 and 9 strongly inhibited [3H]-5-HT re-uptake in
Received 26 January 2014
Received in revised form 25 March 2014
Accepted 3 April 2014
Available online 12 April 2014
Keywords:
Microbial transformation
Buagarofuran
Anti-anxiety activity
Hydroxylation
the rat brain synaptosomes at 10
mM in vitro. The structural diversity of the microbial transformed
metabolites of buagarofuran in the present report would give a favorable opportunity to probe its
mammalian metabolism and better shed light on its pharmacokinetics and pharmacodynamics in vivo.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
analytical standards, and so on. The advantages of using microor-
ganisms for the production of metabolites are that the cultures are
Buagarofuran
named AF-5, 1, Fig. 1), a derivative of
((þ)-(5R,7R,10S)-4-butyl-
a
-agarofuran,
also
relatively easy to maintain and grow, and that scale-up to produce
milligram or gram amounts is readily accomplished. In addition,
a variety of metabolites could be obtained by microbial trans-
formations, from which more active metabolites might be found.8
In the above context, the microbial transformations of buagar-
ofuran were systematically investigated in our group. Totally 31
strains were used for screening tests, three strains of which were
selected for further preparative transformations to lead to the
yields of 19 metabolites. Herein, we first report the microbial
transformations of buagarofuran, the structural elucidation of the
metabolites, and their inhibitory effects in vitro on 5-
hydroxytryptamine (5-HT) and [3H]-NE re-uptaking in rat brain
synaptosomes.
a-agarofuran isolated from the
Chinese traditional medicine Gharu-wood (Aquillaria agallocha
Roxb.), is a novel drug candidate targeting anxiety disorder under
phase II clinical trial in China. Buagarofuran was proved to possess
significant anti-anxiety activity with a higher potency and a lower
toxicity compared with diazepam and buspirone.1,2 The anti-
anxiety mechanism of buagarofuran was related to the modula-
tion of central monoamine neurotransmitters, and it could greatly
reduce the level of dopamine in homogenates of striatum, mid-
brain, and cortex to alleviate anxiety.3 The metabolism study of
buagarofuran revealed that various metabolites were detected
when incubated with rat and human liver microsomal fractions.
However, only two metabolites, 2-hydroxy- and 2-oxo-buagar-
ofuran, were identified because of limited scale, ethnics, etc.4 As
reported, microbial transformations could be used for the prepa-
ration of mammalian drug metabolites.5e7 Once identities of the
microbial and mammalian drug metabolites have been established,
the microbial metabolites can be prepared in sufficient quantity to
be used in biological activity and toxicity tests, preparation of
2. Results and discussion
31 microbial strains (Table S1) belonging to prokaryotic organ-
isms (including bacteria, actinomycetes) and eukaryotic organisms
(including yeast, filamentous fungi), were used as biocatalysts for
the biotransformations of buagarofuran (1). On the basis of TLC,
HPLC, and LCeMS analyses, three strains of filamentous fungi
designated to Cunninghamella echinulata AS 3.3402, Cunning-
hamella elegans AS 3.3400, and Absidia coerulea AS 3.3538, which
showing good ability to convert buagarofuran, were selected for the
further preparative biotransformations.
* Corresponding authors. Tel.: þ86 10 63165195; fax: þ86 10 63017757 (J.D.); tel.:
þ86 10 63182392; fax: þ86 10 63017757 (J.Z.); e-mail addresses: jjzhang@imm.ac.
cn (J. Zhang), ledailedai@126.com, jgdai@imm.ac.cn (J. Dai).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.04.011

L. Qiao et al. / Tetrahedron 70 (2014) 3560e3569
3561
11
1
9
HO
OH
HO
O
HO
OH
5
7
3
12
13
O
15
14
O
O
O
1'
3'
2'
4 (2.7%)
2 (22.8%)
3 (2.2%)
Buagarofuran (1, AF-5)
O
OH
HO
HO
O
HO
O
O
O
O
HO
OH
6 (0.9%)
OH
8 (0.7%)
5 (2.3%)
7 (1.2%)
OH
OH
OH
O
O
O
O
HO
HO
HO
OH
11 (0.5%)
12 (0.5%)
10 (0.7%)
9 (0.9%)
Fig. 1. The chemical structures of buagarofuran (1) and its metabolites (2e12) with C. echinulata AS 3.3402 (yields in parentheses).
2.1. Transformation of 1 by C. echinulata AS 3.3402
and 13C NMR spectroscopic data (Tables 1 and 2), suggesting the
introduction of two additional OH groups as compared with 1. It
was further confirmed by the IR absorption at 3260 cmꢀ1. The 13C
NMR (Table 2) spectrum of 2 was similar to that of 1 except that the
signals corresponding to C-2 (dC 23.1) and C-8 (dC 24.7) in 1 dis-
appeared, while the oxygen-bearing methine signals C-2 (dC 64.8)
and C-8 (dC 70.5) were observed. The loci of OH groups at C-2 and C-
8 were further supported by the signals of H-2 and H-8 in 2 (Table
1), which significantly shifted downfield to dH 4.13 (t, J¼4.8 Hz) and
dH 4.06 (m) when compared with 1. Furthermore, the HMBC cor-
relations (Fig. 2) of H-2/C-1 and C-3, and H-8/C-9 and C-7 strongly
supported the above deduction. The enhancement of integration
value of H3-11 was not observed when H-2 irradiated, and the
Following a standard two-stage fermentation protocol,9 11 new
metabolites (2e12, Fig. 1) were obtained after incubation of 1 with
cell cultures of C. echinulata for 4 days. These metabolites were
purified by silica gel chromatography and semi-preparative HPLC.
Their structures were identified on the basis of the extensive
spectroscopic data (HRESIMS, IR, 1D NMR, 2D NMR, etc.) analysis
and chemical methods. As shown, the major reactions were hy-
droxylations at various positions (C-2, C-8, C-15, and C-10) and
oxidations of alcohol to ketone (C-2 and C-8).
The molecular formula C18H30O3 of 2 was determined by com-
bined analyses of its HRESIMS (m/z [MþH]þ 295.2257), 1H NMR,
Table 1
1H NMR spectroscopic data of 2e10a,b
Position 2a
3b
4b
5b
6a
7b
8a
9a
10a
1
2.38 (dd, 13.2, 5.2) 2.32 m
1.59 (dd, 13.2, 6.8) 1.60 m
1.80
2.75 (d, 15.6) 1.88 m,
2.80 (d, 15.6)
2.02 (d, 15.6)
1.88 (dd, 14.4, 6.0) 1.48 s
1.37 (d, 8.8)
1.44 m
(dd, 15.0, 4.8) 1.96 (d, 15.6) 1.36 m
1.54 (d, 15.0)
2
3
6
4.13 (t, 4.8)
5.61 (d, 3.6)
1.97 (dd, 12.4, 5.6) 2.66 m
4.34 (t, 4.5) 4.22 (t, 4.8)
5.78 (d, 3.3) 5.70 (d, 3.9) 5.90 s
2.38 (d, 11.1) 2.53
4.14 (t, 5.2)
5.91 (t, 4.4)
2.44 m
4.11 (t, 5.1)
5.75 (d, 4.2)
1.93 m
5.96 (t, 3.6)
1.93 m
5.73 br s
2.21 m
6.06 s
2.66 (dd, 12.6, 4.8) 2.20 (dd, 12.0, 3.6) 1.85 (d, 12.9)
1.73 (d, 12.4)
2.17 m
2.66 m
2.32 m
2.11 m
(dd, 2.4, 13.8) 1.84 m
1.98 (d, 12.6)
1.08 (d, 12.4)
1.97 m
1.79 (d, 11.7)
2.26 m
2.26 m
4.2 br s
7
8
9
2.04 m
4.06 m
2.07 m
2.20 m
4.15 br s
2.00 m,
1.45 m
1.26 s
1.61 s
1.27 s
1.50 m
1.94 m
1.73 m
1.84 m
1.75 m
1.57 m
1.41 m
1.05 s
1.97 m
1.72 m
1.58 m,
1.27 (dd, 9.6, 6.0)
1.04 s
1.97 m
1.56 m
1.97 m
1.55 m
2.01 (d, 7.8), 1.57 m
1.61 (d, 7.8) 1.24 m
3.80 (dd, 11.4, 6.0) 3.80
(dd, 11.6, 6.0)
0.81 s
1.30 s
1.18 s
11
12
13
15
1.03 s
1.19 s
1.43 s
2.02 m
1.12 s
1.30 s
1.33 s
2.17 m
1.21 s
1.37 s
1.33 s
2.26 m
1.03 s
1.30 s
1.18 s
0.82 s
1.30 s
1.18 s
1.43 s
1.30 s
1.30 s
1.18 s
4.07 (dd, 8.0, 4.8) 4.35 (dd, 6.9, 5.1) 2.27 m
2.01 (d, 9.2)
3.52 m
4.02 (dd, 5.4, 5.1) 2.16 m, 1.94 m
10
20
30
1.38e1.32 m
1.38e1.32 m
0.91 (t, 6.8)
1.52 m
1.45 m
0.96 (t, 6.6) 0.95 (t, 7.2)
1.45 m
1.37 m
1.52 m
1.57 m
2.09 m
1.48 m, 1.30 m
0.97 (t, 7.5)
1.60 m
1.30 m
0.91 (t, 7.2)
3.42 m
1.47 m
0.94 (t, 7.5)
1.44 m
0.96 (t, 6.6)
1.48 m, 1.30 m
0.91 (t, 7.6)
1.48 m
0.95 (t, 7.6)
a
Data were measured in CD3OD and recorded at 400 for MHz; Coupling constants (J in Hz) are given in parentheses. Assignments were based on gCOSY, HSQC, and HMBC
experiments.
b
Recorded at 300 MHz in CDCl3.

3562
L. Qiao et al. / Tetrahedron 70 (2014) 3560e3569
Table 2
13C NMR spectroscopic data of 2e10a,b
Position
2a
3b
4b
5b
6a
7b
8a
9a
10a
1
2
3
4
42.0, CH2
64.8, CH
128.3, CH
139.6, C
85.9, C
40.3, CH2
64.2, CH
127.0, CH
138.2, C
84.3, C
42.1, CH2
64.3, CH
126.2, CH
139.4, C
85.0, C
49.3, CH2
199.2, C
127.3, CH
159.4, C
85.0, C
42.4, CH2
64.4, CH
128.4, CH
141.9, C
86.6, C
49.7, CH2
200.1, C
126.7, CH
158.9, C
84.6, C
42.5, CH2
64.6, CH
131.1, CH
137.1, C
86.8, C
30.0, CH2
23.0, CH2
128.9, CH
139.5, C
87.3, C
31.6, CH2
23.2, CH2
131.5, CH
133.8, C
87.6, C
5
6
7
8
9
10
11
12
13
14
15
10
20
30
33.3, CH2
51.8, CH
70.5, CH
43.5, CH2
38.5, C
25.9, CH3
24.6, CH3
31.9, CH3
83.2, C
32.3, CH2
31.9, CH2
23.7, CH2
14.4, CH3
32.2, CH2
61.2, CH
210.0, C
52.0, CH2
39.6, C
26.3, CH3
23.7, CH3
29.3, CH3
80.7, C
31.1, CH2
30.4, CH2
22.7, CH2
14.0, CH3
27.3, CH2
51.3, CH
68.4, CH
41.1, CH2
36.3, C
26.2, CH3
22.8, CH3
30.5, CH3
80.0, C
30.9, CH2
27.3, CH2
22.8, CH2
14.0, CH3
27.3, CH2
51.3, CH
67.8, CH
41.9, CH2
40.6, C
24.9, CH3
22.7, CH3
30.4, CH3
81.1, C
31.3, CH2
29.5, CH2
22.6, CH2
13.9, CH3
34.6, CH2
45.3, CH
24.7, CH2
35.7, CH2
37.4, C
25.5, CH3
30.7, CH3
23.1, CH3
83.2, C
69.3, CH
39.7, CH2
20.8, CH2
14.3, CH3
33.9, CH2
44.0, CH
24.0, CH2
33.9, CH2
41.1, C
23.8, CH3
30.6, CH3
22.9, CH3
83.0, C
70.4, CH
38.2, CH2
19.4, CH2
13.8, CH3
34.3, CH2
45.5, CH
24.7, CH2
35.7, CH2
37.4, C
25.5, CH3
23.0, CH3
30.9, CH3
83.2, C
40.4, CH2
72.8, CH
31.4, CH2
10.6, CH3
33.5, CH2
44.1, CH
34.0, CH2
74.5, CH
42.9, C
16.9, CH3
23.2, CH3
30.7, CH3
82.9, C
69.1, CH
39.7, CH2
20.9, CH2
14.3, CH3
33.3, CH2
44.2, CH
34.0, CH2
74.4, CH
42.9, C
16.8, CH3
23.1, CH3
30.1, CH3
83.0, C
40.0, CH2
73.5, CH
30.1, CH2
10.6, CH3
a
Data were measured in CD3OD and recorded at 100 MHz; Assignments were based on gCOSY, HSQC, and HMBC experiments.
Data were measured in CDCl3 and recorded at 100 MHz.
b
H
H
H
H
OH
H
H
HO
OH
HO
HO
HO
OH
H
O
O
O
O
O
HO
H
OH
H
OH
2
6
8
9
19
Fig. 2. Key HMBC correlations (H/C) of compounds 2, 6, 8, 9, and 19.
irradiation of H-8 resulted in enhancement of H3-11 in the NOE
difference spectral experiments, suggesting the -orientation of 2-
OH and -orientation of 8-OH. In addition, the assignment for po-
sitions 12 and 13 was determined according to NOE different
spectrum experiments, mainly according to the NOE of H-7 /H3-12.
Thus, the structure of 2 was determined as 2 ,8 -dihydroxy-bua-
garofuran. Metabolite 4 (C18H30O3 established by HRESIMS) had
NMR spectroscopic features similar to those of 2, except that the 1H
NMR resonance of the H-8 (dH 4.06, m) in 2 instead of H-8 (dH 4.17,
br s). In addition, the enhancement of H3-11 was not observed
when H-2 and H-8 were irradiated, which indicated that 2- and 8-
a
,
b
-unsaturated carbonyl moiety. Therefore, 5 was 8
a
-hydroxy-2-
a
oxo-buagarofuran.
b
The molecular formula C18H30O3 of 6 was established by HRESI-
positive mass spectrum at m/z [MþNa]þ 317.2074, suggesting the
a
substitution of two additional OH groups as compared with 1. 1
H
a
b
NMR (Table 1) showed that two methylene signals responsible for
H-2 (dH 1.97, m) and H-15 (dH 1.36, m) in 1 disappeared, while a pair
of oxygenated methine signals at H-2 (dH 4.14, t, J¼5.2 Hz) and H-15
(
dH 4.07, dd, J¼8, 4.8 Hz) were observed. The HMBC correlations
(Fig. 2) of H-2/C-1, C-3, C-4, and C-10, and H-15/C-3, C-4, C-5, C-10,
and C-20 further supported this deduction. When H-2 was irradi-
ated, the enhancement of H3-11 was not observed, suggesting 2-OH
OH groups were both
,8 -dihydroxy-buagarofuran.
The molecular formula of 3 was established to be C18H28O3 by
a-oriented. Accordingly, 4 was determined as
2
a
a
was
using Mosher’s method.10e13 The (S)- and (R)-MTPA [
(trifluoromethyl) phenylacetyl] esters of 6 (6a and 6b) were pre-
pared using the corresponding (R)-(ꢀ)- and (S)-(þ)- -methoxy-
(trifluoromethyl) phenylacetyl chloride (Fig. 3), respectively. The
determination of the chemical shift differences Dd dS dR) for the
a
-oriented. The absolute configuration of C-15 was determined
a
-methoxy-
a-
combined analyses of its HRESIMS, 1H NMR, and 13C NMR spec-
troscopic data (Tables 1 and 2), with 2 amu less than that of 2. The
absence of H-8 signal (dH 4.06, m) and the presence of one carbonyl
signal at dC 210.0 indicated that 3 was a C-8 ketonized product of 2.
It was further supported by the significant downshift of C-7 and C-9
at dC 61.2 and dC 52.0, respectively. The 2-OH group was determined
a
a-
(
ꢀ
protons neighboring C-15 functionality led to the assignment of S or
R configurations for C-15 of 6. Although 6 has two hydroxy groups
to be
a-configuration by NOE difference spectrum, in which the
enhancement of H3-11 was not observed when H-2 was irradiated.
Thus, the structure of 3 was determined as 2a-hydroxy-8-oxo-
buagarofuran.
-0.12
HO
HO
HO
-0.05
EDCI,DMAP/CH2Cl2
The HRESIMS, 1H and 13C NMR spectroscopic data of 5 were in
accordance with the molecular formula C18H28O3, with 2 amu less
than the corresponding value of 4. The 1H NMR spectrum of 5
showed the absence of oxygen-bearing methine signal of H-2 (dH
4.22, t, J¼4.8 Hz) in 4, whereas its 13C NMR displayed the presence
of one carbonyl signal at dC 199.2. Furthermore, the resonance for C-
4 of 5 was deshielded significantly as compared with that of 4. This
implied that 2-OH in 4 was oxidized to a carbonyl moiety in 5,
which was confirmed by the IR absorption at 1667 cmꢀ1 assigned to
-0.11
-0.18
-0.21
O
O
(S)-or (R)-MTPA
RO
+0.06
+0.18
+0.11
+0.06
6
6a R=(S)-MTPA
6b R=(R)-MTPA
Fig. 3. Dd
esters 6a and 6b.
(
dS
dR) values obtained from the 1H NMR spectra near C-15 of the MTPA
ꢀ

L. Qiao et al. / Tetrahedron 70 (2014) 3560e3569
3563
(2-OH and 15-OH), the steric hindrance of 2-OH from H3-11 and H-
3 resulted in selective esterification of 15-OH. The esterification of 6
with (S)-MTPA afforded a mixture of the (S)-MTPA monoester 6a
and the diester 6c in a ratio of 3:1. While, in the esterification of 6
with (R)-MTPA, only monoester (2-ester) was observed and ob-
tained. On the basis of these results, 15R configuration of 6 was
The molecular formula C18H30O3 of metabolite 9 was established
by HRESIMS at m/z 317.2085 [MþNa]þ. The 1H NMR data of 9 (Table
1) were similar to those of 6 except that the signal corresponding to
H-2 (dH 4.14, t, J¼5.2 Hz) in 6 disappeared, while the oxygen-
bearing methine signal at dH 3.80 (dd, J¼6.0, 5.7 Hz, H-9) was ob-
served. These indicated the introduction of OH group at C-9 posi-
tion instead of C-2 position, which was further supported by that
the signal of H3-11 in 9 significantly shifted upfield to dH 0.81 (s)
from dH 1.03 (s) in 6. Additionally, the HMBC correlations (Fig. 2) of
H-9/C-8, C-10, and C-11 solidly suggested the presence of 9-OH
group. The enhancement of integration value of H3-11 was not
observed when irradiation of H-9 in the NOE difference spectral
determined. Thus, 6 was identified as (15R)-2a,15-dihydroxy-
buagarofuran.
The HRESIMS, 1H NMR, and 13C NMR spectroscopic data (Tables
1 and 2) of 7 were in accordance with the molecular formula
C
18H28O3, with 2 amu less than the corresponding value of 6. The
NMR spectroscopic data were similar to those of 6, except the ab-
sence of oxygen-bearing methine signal corresponding to H-2 (dH
4.14, t, J¼5.2 Hz) in 6, and the presence of one carbonyl signal at dC
200.1. On the basis of the bulkiness rule for secondary alcohols,
a negative Cotton effect at w350 nm (the E band) in the Rh2(O-
COCF3)4-induced CD14 spectrum (Fig. 4) supported the 15R con-
figuration. Therefore, 7 was determined as (15R)-15-hydroxy-2-
oxo-buagarofuran.
experiments, revealing that 9-OH was
a-oriented. The absolute
configuration of C-15 was determined using Mosher’s method
(Fig. 6).11,12 The (S)- and (R)-MTPA esters of 9 (9a and 9b) were
prepared using the corresponding (R)-(ꢀ)-and (S)-(þ)-MPTA, re-
spectively. The determination of the chemical shift differences Dd
(
dS
ꢀ
dR) for the protons neighboring C-15 functionality led to the
assignment of S or R configurations for C-15 of 9. On the basis of
Fig. 4. Comparative CD spectra of 7. a, CD spectrum of 7 recorded in MeOH; b, Rh2(OCOCF3)4-induced CD spectrum of 7.
Metabolite 8 showed
a
quasi-molecular ion peak at m/z
these results, 15R configuration was determined. Thus, 9 was de-
295.2241 [MþH]þ in the HRESIMS spectrum, in agreement with the
molecular formula C18H30O3. The NMR spectroscopic (Tables 1 and
2) features of 8 were very similar to those of 6 except that the signal
corresponding to H-15 (dH 4.07, dd, J¼8.0, 4.8 Hz) in 6 disappeared,
while another oxygen-bearing methine signal (dH 3.52, m) was
observed. The HMBC correlations (Fig. 2) of the signals of dH 3.52/C-
15, C-20, and C-30 indicated the presence of 10-OH rather than 15-
OH. Selective oxidation of the allylic 2-OH to carbonyl by active
termined as (15R)-9a,15-dihydroxy-buagarofuran.
The HRESIMS, 1H and 13C NMR spectroscopic data of 10 were in
accordance with the molecular formula C18H30O3. The NMR spec-
OH
OH
-0.02
-0.08
-0.3
-0.1
-0.08
-0.07
-0.07
-0.10
15
-0.0O9
+0.10
MnO2 was undertaken to yield the derivative 8a (Fig. 5). On the
O
-0.15
-0.09
RO
RO
-0.07
-0.01
basis of the bulkiness rule for secondary alcohols, a positive Cotton
effect at w350 nm (the E band) in the Rh2(OCOCF3)4-induced
CD spectrum of 8a (Fig. 5) supported the 10S configuration. Thus,
-0.02
+0.04
+0.04
+0.08
the structure of 8 was deduced to be (10S)-2 ,10-dihydroxy-
a
9a R=(S)-MTPA
9b R=(R)-MTPA
14a R=(S)-MTPA
14b R=(R)-MTPA
buagarofuran.
Fig. 6. Dd
and 14.
(
dS
dR) values obtained from the 1H NMR spectra of the MTPA esters of 9
ꢀ
troscopic data (Tables 1 and 2) were analogous to those of 8 and 9.
Specifically, the spinespin splitting pattern and coupling constants
of H-9 (dH 3.80, dd, J¼11.6, 6.0 Hz) were similar to those of 9, while
those of H-10 in 10 was identical with those in 8. Therefore, 10 was
deduced as (10S)-9 ,10-dihydroxy-buagarofuran.
a
(þ)-HRESI mass spectrum of 11 exhibited the quasi-molecular
ion peaks at m/z [MþH]þ 295.2246, suggesting the molecular for-
mula of C18H30O3 and the introduction of two OH groups as com-
pared with 1. The 1H NMR (Table 3) data of 11 were similar to those
of 6 except that the signal corresponding to H-2 (dH 4.14, t, J¼5.2 Hz)
in 6 disappeared, while another oxygen-bearing methine signal at
dH 4.09 (m) was observed. The introduction of OH group at C-8
position was supported by comparing the signal of H-8 in 11 with
Fig. 5. Rh2(OCOCF3)4-induced CD spectrum of 8a.

3564
L. Qiao et al. / Tetrahedron 70 (2014) 3560e3569
Table 3
1H NMR spectroscopic data of 11e19a,b
Position 11a
12b
13b
14a
15b
16a
17b
18b
19a
1
1.08 m
1.07 m
1.30 m
1.53 m
2.03 m, 1.42 m 1.47 m, 1.89 m
2.85 (d, 15.6)
2.07 (d, 15.6)
2.75 (d, 15.6) 1.89 (dd, 14.4, 6.4)
1.96 (d, 15.6) 1.35 m
4.09 (t, 5.2)
2
3
6
2.00 m
5.88 br s
2.49
(dd, 13.2, 4.8) 1.85 m
1.76 (d, 13.2)
2.06 m
4.09 m
2.00 m
2.05 m
5.92 (t, 3.3)
2.17 m
2.03 m
5.64 br s
2.26 (d, 12.0)
1.75 (d, 12.0)
1.93 m
5.58 br s
2.20 (d, 12.3)
1.72 (d, 12.3)
4.22 (t, 4.2)
5.73 (d, 3.3)
2.33 m
4.04 m
5.62 br s
2.21 (d, 12.6)
5.91 s
5.90 s
5.62 (d, 4.0)
2.52 (dd, 12.6, 5.1) 2.66 m
1.82 (d, 12.6)
2.29 (dd,12.0, 3.3)
1.79 (d, 12.0)
1.76 (d, 15.0) 1.77 (d, 12.6)
2.17 m
2.66 m
2.32 m
7
8
9
2.03 m
4.15 br s
1.85
2.03 m
1.56 m
1.94 m
1.53 m, 1.94 m
1.98 m
1.69 m
1.97 m
1.58 m
2.03 m
4.18 m
2.10 m
1.73 m
1.59 m, 1.26 m
3.96 (dd, 11.1, 6.3) 3.76 (dd, 11.0, 5.7) 1.69 m, 1.32 m 3.82 (dd, 11.4, 6.6) 2.27 m
(dd, 15.3, 5.1)
1.66 m
1.11 s
1.32 s
1.30 s
11
12
13
0.91 s
1.20 s
1.44 s
0.85 s
1.36 s
1.24 s
0.75 s
1.26 s
1.16 s
1.07 s
1.35 m
1.24 s
0.81 s
1.30 s
1.18 s
1.04 s
1.59 s
1.33 s
1.12 s
1.30 s
1.33 s
1.02 s
1.22 s
3.78 (d, 10.8)
3.54 (d, 10.8)
2.02 m, 1.34 m
2.02 m, 1.34 m
1.34 m
15
10
4.04 m
2.06 m
1.57 m
0.91 (t, 7.5)
4.21 m
2.08 m
1.66 m
0.96 (t, 6.9)
2.11 m
1.52 m
1.48 m
0.94 (t, 7.5)
2.10 m, 1.94 m
1.53 m, 1.45 m
3.65 m
2.14 m, 2.23 m 1.38 m
1.65 m
3.83 br s
1.22 (d, 6.0)
1.75 m
2.17 m
1.52 m
1.45 m
1.91 m
1.30 m
0.91 (t, 6.6)
1.38e1.51 m
1.32e1.51 m
0.96(t, 6.9)
20
30
1.09(d, 6.6)
0.96 (t, 6.6) 0.92 (t, 6.8)
a
Data were measured in CD3OD and recorded at 400 MHz for compounds 19, others for 300 MHz; Coupling constants (J in Hz) are given in parentheses. Assignments were
based on gCOSY, HSQC, and HMBC experiments.
b
Data were measured in CDCl3 and recorded at 300 MHz.
the methine proton signal of H-8 in 2. 8-OH was determined to be
-oriented by NOE difference spectrum experiments, in which H3-
11 enhancement was observed as H-8 irradiated. Therefore, 11 was
deduced to be (15R)-8 ,15-dihydroxy-buagarofuran. Metabolite 12
had the same molecular formula of C18H30O3 as 11, and the spec-
troscopic data of 12 were very similar to those of 11. Comparison of
the spinespin splitting pattern and coupling constants of H-8 in 12
OH was
hydroxy-buagarofuran.
a-oriented. Thus, the structure of 13 was determined as 9a-
b
The MS and NMR spectroscopic data of 14 demonstrated that it
was the 20-OH derivative of 13. This was verified by 2D NMR data
analysis of 14, which supported the 1D NMR data (Tables 3 and 4)
assignments. It was further confirmed by the doublet of H3-30. The
absolute configuration of C-20 was determined by Mosher’s method
as before, and the results (Fig. 6) suggested that R configuration of
b
and 11 indicated that
a-orientation of H-8 in 12. Thus, 12 was
assigned as (15R)-8 ,15-dihydroxy-buagarofuran.
a
C-20. Therefore, 14 was determined to be (20R)-9 ,20-dihydroxy-
a
buagarofuran.
2.2. Transformation of 1 by A. coerulea AS 3.3538
2.3. Transformation of 1 by C. elegans AS 3.3400
Three metabolites were obtained from the conversion of 1 by A.
coerulea AS 3.3538, their structures were identified to be (15R)-
After incubation of 1 with cell cultures of C. elegans for 4 days,
seven metabolites (2, 15e20, Fig. 8) were obtained by a combina-
tion of open silica gel chromatography and semi-preparative HPLC
purification. Their structures were elucidated based on the exten-
sive spectroscopic data analyses. The occurred reactions included
hydroxylation (C-8, C-9, etc.) and ketonization (C-2 and C-8 alco-
hols). Though the two strains of C. echinulata AS 3.3402 and C.
elegans AS 3.3400 belong to the same genus, the generated me-
tabolites of 1 were substantially different both in reaction position
and in yield. It suggests the different enzymes (probably P450s)
existence in the two different strains.
9
a
,15-dihydroxy-buagarofuran (9), 9
a-hydroxy-buagarofuran (13),
and (20R)-9 ,20-dihydroxy-buagarofuran (14) (Fig. 7). The pre-
a
dominant reaction was 9a-hydroxylation by this strain.
OH
OH
OH
O
O
O
HO
9 (2.1%)
14 (3.9%)
13 (17.0%)
The NMR spectroscopic data (Tables 3 and 4) of 15 were similar
to those of 14, except the signal H-9 was disappeared, while H-2 at
dH 4.22 (t, J¼4.2 Hz) was observed, implying the presence of an OH
HO
Fig. 7. The chemical structures of metabolites (9, 13, 14) of buagarofuran (1) with A.
coerulea AS 3.3538 (yields in parentheses).
group at C-2 rather than at C-9. The
a-orientation of 2-OH was
established according to the NOE difference spectrum, in which the
enhancement of H3-11 was not observed when irradiated the H-2.
The molecular formula of C18H30O2 of 13 was determined by
HRESIMS with the quasi-molecular ion peak at m/z [MþH]þ
279.2150, suggesting the substitution of an additional OH group as
compared with 1. The 1H NMR (Table 3) data of 13 were similar to
those of 1 except that the signal corresponding to H-9 (dH 1.17, m) in
1 disappeared, while an oxygen-bearing methine signal (dH 3.96,
dd, J¼11.0, 6.3 Hz) was observed. These indicated the introduction
of OH group at C-9 position, which was further supported by the
significantly shifting upfield of signal of H3-11 to dH 0.85 (s) in
comparison with 1. The enhancement of H3-11 was not observed
when H-9 irradiated in the NOE difference spectra, revealing that 9-
Thus, 15 was deduced as (20R)-2 ,20-dihydroxy-buagarofuran.
a
The HRESIMS combined with the NMR data (Tables 3 and 4)
indicated that metabolite 16 had the molecular formula C18H30O3.
The NMR data of 16 were very similar to those of 13 except that one
additional oxy-bearing methine was observed, and the position was
assigned to C-2 by HMBC correlations of H-2/C-1, and C-3. The
enhancement of H3-11 when H-2 irradiated suggested the
b
-ori-
entation of 2-OH. Therefore, 16 was identified as 2b,9a-dihydroxy-
buagarofuran.
The molecular formula C18H28O3 of 17 was established by
HRESIMS, IR, and NMR spectroscopic data (Tables 3 and 4). The

L. Qiao et al. / Tetrahedron 70 (2014) 3560e3569
3565
Table 4
1
a,b
3C NMR spectroscopic data of 11e19
Position
11a
12b
13b
14a
15b
16a
17b
18b
19a
1
2
3
4
33.3, CH2
23.3, CH2
128.7, CH
139.3, C
86.1, C
32.2, CH2
22.1, CH2
128.3, CH
138.6, C
85.3, C
30.1, CH2
22.1, CH2
126.3, CH
135.9, C
85.5, C
30.2, CH2
23.1, CH2
128.1, CH
136.9, C
87.7, C
41.4, CH2
64.4, CH
126.7, CH
139.2, C
84.8, C
39.6, CH2
65.9, CH
131.5, CH
138.6, C
87.1, C
49.1, CH2
199.2, C
127.5, CH
159.0, C
84.0, C
48.4, CH2
197.8, C
127.7, CH
157.7, C
84.3, C
42.4, CH2
64.7, CH
128.1, CH
140.0, C
87.0, C
5
6
7
8
9
10
11
12
13
14
15
10
20
30
33.2, CH2
51.5, CH
69.4, CH
43.5, CH2
39.3, C
22.8, CH3
24.8, CH3
32.2, CH3
82.8, C
70.8, CH
39.5, CH2
20.8, CH2
14.3, CH3
27.1, CH2
51.3, CH
68.3, CH
42.3, CH2
36.9, C
22.9, CH3
23.2, CH3
30.8, CH3
80.0, C
71.8, CH
38.6, CH2
19.9, CH2
14.0, CH3
33.7, CH2
42.9, CH
32.1, CH2
74.1, CH
41.6, C
16.1, CH3
22.9, CH3
30.8, CH3
81.1, C
31.3, CH2
29.2, CH2
22.8, CH2
14.1, CH3
33.1, CH2
44.3, CH
34.1, CH2
74.5, CH
42.8, C
16.8, CH3
23.1, CH3
30.4, CH3
83.0, C
28.7, CH2
39.9, CH2
68.4, CH
23.7, CH3
33.2, CH2
44.1, CH
23.9, CH2
34.2, CH2
38.1, C
25.2, CH3
22.7, CH3
30.4, CH3
81.6, C
27.1, CH2
37.8, CH2
67.5, CH
24.0, CH3
32.8, CH2
44.0, CH
33.4, CH2
73.8, CH
44.2, C
17.6, CH3
23.0, CH3
30.3, CH3
82.8, C
31.5, CH2
32.1, CH2
23.7, CH2
14.3, CH3
32.7, CH2
50.6, CH
70.0, CH
42.5, CH2
41.6, C
24.1, CH3
24.2, CH3
31.5, CH3
82.7, C
31.6, CH2
29.5, CH2
22.5, CH2
13.9, CH3
32.2, CH2
61.1, CH
207.9, C
51.1, CH2
43.0, C
26.2, CH3
23.7, CH3
29.2, CH3
81.8, C
30.9, CH2
29.5, CH2
22.6, CH2
13.9, CH3
34.0, CH2
43.6, CH
24.2, CH2
35.9, CH2
37.3, C
25.7, CH3
25.7, CH3
65.8, CH2
85.3, C
32.1, CH2
32.1, CH2
23.8, CH2
14.4, CH3
a
Data were measured in CD3OD and recorded at 100 MHz for compounds 11e14, 17, and 18, and 150 MHz for 16; Assignments were based on gCOSY, HSQC, and HMBC
experiments.
b
Data were measured in CDCl3 and recorded at 100 MHz for compound 15, 19 and 125 MHz for others.
OH
HO
OH
HO
HO
HO
O
O
O
2 (0.5%)
15 (0.7%)
16 (1.1%)
HO
O
O
O
O
OH
O
O
O
O
OH
18 (4.1%)
20 (2.0%)
17 (1.4%)
19 (0.3%)
Fig. 8. The chemical structures of metabolites (2, 15e20) of buagarofuran with C. elegans AS 3.3402 (yields in parentheses).
NMR data were similar to those of 5. The different spinespin pat-
terns of H-8 in 17 and 5 indicated the different orientation of the
introduced 8-OH group. It was confirmed by the enhancement of
the HMBC correlations (Fig. 2) of H2-13/C-7, C-12, and C-14 strongly
confirmed the deduction. Accordingly, 19 was elucidated as 2a,13-
dihydroxy-buagarofuran.
H3-11 when H-8 irradiated. Accordingly, 17 was identified as 8
hydroxy-2-oxo-buagarofuran.
b
-
The HRESIMS combined with the NMR data (see Section 4.4.3)
indicated that 20 had the molecular formula C18H28O2, requiring 5
degrees of unsaturation. The NMR data of 20 resembled those of 17
except the disappearance of the resonance for 8-OH signal.
Therefore, 20 was determined to be 2-oxo-buagarofuran, which
was one of the metabolites of 1 incubated with rat and human liver
microsomes in vitro.
The HRESIMS combined with the NMR data (Tables 3 and 4)
indicated that 18 had the molecular formula C18H26O3. The NMR
data of 18 resembled those of 17, except the presence of one car-
bonyl moiety instead of one OH group. The resonances for C-7 and
C-9 were deshielded significantly to dC 61.1 and dC 51.1, respectively,
as compared with that of 17, further suggesting the carbonyl moiety
at C-8. Thus, 18 was determined to be 2,8-dioxo-buagarofuran.
HRESI-positive mass spectrum of 19 exhibited two quasi-mo-
lecular ion peaks at [MþH]þ 295.2243 and [MþNa]þ 317.2091,
consistent with the molecular formula of C18H30O3. It suggested the
substitution of two additional OH groups in comparison with 1. The
1H NMR spectrum (Table 3) of 19 was similar to that of 2 except that
the signal corresponding to H3-13 (dH 1.43, s) in 2 disappeared,
while the oxy-bearing methylene signals at dH 3.78 (d, J¼10.8 Hz)
and dH 3.54 (d, J¼10.8 Hz) were observed. These indicated the in-
troduction of OH group at C-13 position, which was further sup-
ported by that the signal of C-13 in 19 significantly shifted
downfield to dC 65.8 (t) compared with dC 31.9 (q) in 2. Additionally,
2.4. Pharmacological evaluation of 5-hydroxytryptamine (5-
HT) and [3H]-NE re-uptaking in rat brain synaptosomes in-
hibitory activity
Serotonin and norepinephrine re-uptake inhibitors may be
potential anti-anxiety. The in vitro anti-anxiety activity of the
above 19 (2e20) metabolites along with buagarofuran (1) were
preliminarily evaluated using rat brain synaptosomes by inhibit-
ing both [3H]-5-HT and [3H]-NE re-uptake16,17 using duloxetine as
a positive control (Table 5). The results showed that metabolites 8,
10e12, and 20 exhibited remarkable in vitro anti-anxiety activity
at 10
m
M, inhibiting [3H]-NE re-uptake in the rat brain

3566
L. Qiao et al. / Tetrahedron 70 (2014) 3560e3569
Table 5
a JASCO J-815 spectropolarimeter. The IR spectra were measured
using a Nicolet 5700 FT-IR microscope spectrometer (FT-IR Micro-
scope Transmission). NMR spectra were obtained on Mercury-300,
Mercury-plus-400, and Bruker ARX-500 spectrometers. Chemical
The inhibitory effects on 5-hydroxytryptamine (5-HT)-re-uptaking in rat brain
synaptosomes and re-uptake of [3H]-NE experiments
Compounds
Inhibitory rates on
5-HT re-uptaking (%)
Inhibitory rates on
[3H]-NE re-uptaking (%)
shifts (d) are given in parts per million, and coupling constants (J)
Duloxetine
1
2
3
4
5
6
7
89.5
37.0
33.7
38.6
39.1
38.9
37.9
40.3
57.0
50.0
45.1
40.5
35.9
44.9
34.2
40.2
35.9
33.4
41.6
36.8
44.1
81.4
56.3
42.2
31.4
21.3
43.4
31.7
44.2
64.9*
8.2
60.5*
65.3*
66.9*
19.2
35.1
33.2
37.1
39.2
32.5
21.8
71.2
are given in Hertz (Hz). ESIMS data and HRESIMS spectra were
carried out using an Agilent Technologies 6520 Accurate Mass Q-
TOF LC/MS spectrometer. Column chromatography was performed
with silica gel (200e300 mesh, Qingdao Marine Chemical Inc.
Qingdao, PR China). Semi-preparative HPLC was carried out on
a Shimadzu LC-6AD instrument equipped with a Shimadzu RID-
10A detector and a YMC-Pack ODS-A column (250 mmꢁ10 mm
8
9
i.d., 5
m
m) or a Grace Allsphere silica column (250 mmꢁ10 mm, i.d.,
10
11
12
13
14
15
16
17
18
19
20
5
mm). Analytical TLC was performed on pre-coated silica gel GF254
plates (Qingdao Marine Chemical Inc.), and visualized under UV
light or by spraying with 10% H2SO4 in EtOH followed by heating at
120 ꢂC. Unless otherwise stated, all chemicals were of analytic
grade and obtained from commercially available sources and used
without further purification.
4.2. Substrate, organisms, and cultivation conditions
Buagarofuran (1, 98% by HPLC) was gifted from Professor Dali
Yin of Institute of Materia Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College, and characterized by
comparison of the NMR data with the references.1 The sample was
dissolved in N,N-dimethylformamide (DMF) at the concentration of
50 mg/mL as stock solution before use. The strains including pro-
karyotic organisms and eukaryotic organisms listed in Table S1
were purchased from Chinese General Microorganism Culture
Collection, Chinese Academy of Sciences, and kept on slats. The
medium was used after autoclaving at 121 ꢂC for 25 min. The seed
cultures were prepared in 250 mL flasks with 70 mL liquid medium
by incubating 2 days. A volume of 5 mL of the seed cultures was
inoculated into each 1000 mL flask with 300 mL medium, and
shaken at 120 r/min and 25 ꢂC in the dark for the use of preparative
biotransformation.
*P<0.05.
synaptosomes by 64.9%, 60.5%, 65.3%, 66.9%, and 71.2%, re-
spectively. In addition, compounds 8 and 9 at 10 M showed
m
strongly in vitro inhibiting [3H]-5-HT re-uptake in the rat brain
synaptosomes by 57% and 50%, respectively. These metabolites,
especially for 20, one mammalian metabolite in vitro, displayed
much more potent inhibitory activity on [3H]-NE and (5-HT) re-
uptaking than 1. It suggested that metabolism of buagarofuran
might generated pharmacologically active metabolites (bio-
activation). These oxidized metabolites might be generated by
P450 enzymes, one kind of common phase I metabolism enzymes
in human and/or mammalian in vivo.
3. Conclusion
4.3. Screening biotransformations
In summary, we report the successful microbial transformations
of an anti-anxiety agent, buagarofuran, by three filamentous fungi,
which led to the yield of 19 metabolites. The occurred reactions
included mono-hydroxylation, di-hydroxylation, and oxidation of
alcohol to ketone. The absolute configurations of the metabolites
were determined by NOE difference spectral experiments, CD
spectral data, and chemical methods. In particular, the Rh2(O-
COCF3)4-induced CD spectroscopic data together with the Mosher’s
methods, and selective oxidation of the double bond allylic hy-
droxyl group to carbonyl moiety were undertaken to determine the
absolute configurations of several products. The structural diversity
of the microbial transformed metabolites of buagarofuran in the
present report would give a favorable opportunity to probe the
mammalian metabolism of buagarofuran and better shed light on
its pharmacokinetics and pharmacodynamics. Moreover, the more
active metabolites generated both microbes and mammalian liver
microsomes suggest that the anti-anxiety effects of buagarofuran
in vivo might be attributed to the combination of the parent mol-
ecule and its active metabolite(s).
For screening test, the microbial strains were incubated as
described above. 2-day-old cell cultures were employed for bio-
transformation. A volume of 140 mL of substrate solution with final
concentration of 100 mg/L was fed to one flask. The incubation
was allowed to proceed for 4 days, after which the cultures were
filtered and the filtrate was extracted with ethyl acetate
(3ꢁ100 mL). The extract was dried with anhydrous Na2SO4 and
concentrated under vacuum at 45 ꢂC to give residue, and analyzed
by TLC and HPLC.
4.4. Preparative biotransformations, extraction, isolation,
and identification of metabolites
The 2-day-old seed cultures of the fungal strains were added to
the 1000 mL Erlenmeyer flasks containing 300 mL of liquid me-
dium. After 2 days of incubation, 1 (500 mg in 10 mL DMF for each
strain) was evenly distributed into 5 L of cultures, and incubated at
25 ꢂC in a rotary shaker at 120 r/min for 4 days. After which the cell
cultures were filtrated. The filtrate was extracted with ethyl acetate
(3ꢁ5 L), dried over anhydrous Na2SO4, and evaporated under re-
duced pressure to afford the crude extract.
4. Experimental section
4.1. General experimental procedures
4.4.1. Metabolites from 1 by C. echinulata AS 3.3402. The crude
extract (1.8 g) was subjected to silica gel column (200 g,
200e300 mesh) chromatography eluting with a gradient of pe-
troleum ether (60e90 ꢂC) in ethyl acetate to afford 11 fractions. The
Optical rotations were measured on a PerkineElmer Model-343
digital polarimeter. The UV and CD spectra were recorded on

L. Qiao et al. / Tetrahedron 70 (2014) 3560e3569
3567
fraction 10 (180 mg) was submitted to semi-preparative HPLC and
eluted with n-hexane in ethyl acetate (5:1, v/v, 4.0 mL/min) to yield
2 (128 mg, 22.8%) and 8 (4 mg, 0.7%). Fraction 8 (170 mg) was
fractionated by Sephadex LH-20 (MeOH) then purified by normal
phase semi-preparative HPLC (n-hexane/ethyl acetate¼5:1, v/v,
4.0 mL/min) to give 4 (15 mg, 2.7%) and 6 (5 mg, 0.9%). Fraction 7
(310 mg) was applied to reverse phase semi-preparative HPLC (30%
ACN in H2O, v/v) to afford 5 (13.0 mg, 2.3%), 9 (5.0 mg, 0.9%), 11
(3 mg, 0.5%), and 12 (3 mg, 0.5%). Fraction 6 was underwent
chromatography through a Sephadex LH-20 column eluted with
30% CHCl3 in MeOH (v/v) and further purified by reverse phase
semi-preparative HPLC (35% ACN in H2O, v/v, 3.0 mL/min) to yield 3
(9.0 mg, 2.2%), and 10 (4 mg, 0.7%). Fraction 5 (95 mg) was purified
by reverse phase semi-preparative HPLC (35% ACN in H2O, v/v,
3.0 mL/min) to give 7 (7 mg, 1.2%).
J¼4.2 Hz, H-3), 2.38 (dd, J1¼12.0 Hz, J2¼4.2 Hz, H-6a), 1.81 (d,
J¼12.0 Hz, H-6b), 1.59 (m, H-10), 1.37 (m, H-20a), 1.25 (m, H-20b),
0.84(t, J¼7.8 Hz, H3-30).
20
4.4.1.7. (15R)-15-Hydroxy-2-oxo-buagarofuran (7). Pale oil, [a]
D
ꢀ11.8 (c 0.07, MeOH); IR nmax 3424, 2964, 2934, 2873, 1670, 1459,
1382, 1229, 1148, 1131, 1071, 1009, 960, 885 cmꢀ1; 1H (Table 1) and
13C NMR data (Table 2); ESIMS m/z 293 [MþH]þ; HRESIMS (posi-
tive) m/z 293.2090 [MþH]þ (calcd for C18H29O3, 293.2111).
4.4.1.8. (10S)-2 ,10-Dihydroxy-buagarofuran (8). White powder,
a
20
[a
]
ꢀ12.7 (c 0.06, MeOH); IR nmax 3370, 3253, 2949, 2926, 2858,
D
1651, 1459, 1291, 1007, 881 cmꢀ1 1H (Table 1) and 13C NMR data
;
(Table 2); ESIMS m/z 295 [MþH]þ; HRESIMS (positive) m/z
295.2241 [MþH]þ (calcd for C18H31O3, 295.2268).
20
4.4.1.1. 2
a
,8
b
-Dihydroxy-buagarofuran (2). White powder, [
a
]
4.4.1.9. Preparation of 8a. A solution of 2.0 mg of 8 in 0.5 mL of
ether and 5.0 mg of MnO2 was stirred at room temperature for 4 h,
after which the reaction was quenched with the addition of 10 mL
of ethyl acetate. After purified by semi-preparative HPLC, 1.5 mg of
8a was obtained. 1H NMR (600 MHz, CD3OCD) dH 2.05 (m, H-1), 5.91
(s, H-3), 2.66 (d, 15.6, H-6a), 2.20 (dd, 15.6, 9.6, H-6b), 1.92 (m, H-7),
1.75 (2H, m, H-8), 1.54 (m, H-9a), 1.27 (m, H-9b), 1.02 (3H, s, H-11),
1.37 (3H, s, H-12), 1.23 (3H, s, H-13), 2.42 (dd, 15.6, 3.6, H-15a), 2.32
(dd, 15.6, 5.4, H-15b), 3.56 (m, H-10), 1.53 (2H, m, H-20), 0.96 (3H, t,
7.8, H-30).
D
þ62.4 (c 0.50, MeOH); IR nmax 3260, 2966, 2938, 2879, 1663, 1460,
1381, 1360, 1326, 1056, 1010, 882 cmꢀ1 1H (Table 1) and 13C NMR
;
data (Table 2); ESIMS m/z 295 [MþH]þ; HRESIMS (positive) m/z
295.2257 [MþH]þ (calcd for C18H31O3, 295.2268).
20
4.4.1.2. 2
a
-Hydroxy-8-oxo-buagarofuran (3). Pale oil,
[a]
D
ꢀ1.33 (c 0.30, MeOH); IR nmax 3439, 2981, 2980, 2873, 1714, 1677,
1462, 1387, 1254, 1133, 1032, 903, 872 cmꢀ1 1H (Table 1) and 13C
;
NMR data (Table 2); ESIMS m/z 293 [MþH]þ; HRESIMS (positive) m/
z 293.2096 [MþH]þ (calcd for C18H29O3, 293.2111).
20
4.4.1.10. (15R)-9a,15-Dihydroxy-buagarofuran (9). Pale oil, [a]
D
20
4.4.1.3. 2
a
,8
a
-Dihydroxy-buagarofuran (4). White powder, [
a
]
þ28.4 (c 0.07, MeOH); IR nmax 3384, 2999, 2933, 2867, 1456,
D
þ13.3 (c 0.06, MeOH); IR nmax 3376, 2996, 2968, 2925, 2862, 1714,
1382, 1148, 1065, 1019, 969, 883, 855 cmꢀ1 1H (Table 1) and 13C
;
1678, 1660, 1459, 1365, 1295, 1136, 1085, 992, 871, 850 cmꢀ1
;
1H
NMR data (Table 2); ESIMS m/z 295 [MþH]þ, 317 [MþNa]þ; HRE-
SIMS (positive) m/z 317.2085 [MþNa]þ (calcd for C18H30NaO3,
317.2087).
(Table 1) and 13C NMR data (Table 2); ESIMS m/z 295 [MþH]þ, 611
[2MþNa]þ; HRESIMS (positive) m/z 295.2242 [MþH]þ (calcd for
C
18H31O3, 295.2268).
4.4.1.4. 8
4.4.1.11. Preparation of (S)-(9a) and (R)-MTPA (9b) esters. A
sample of 9 (5.0 mg, 0.017 mmol), (S)-MTPA (15 mg, 0.064 mmol),
and anhydrous dichloromethane (0.5 mL), EDC 10 mg, and DMAP
10 mg were allowed to react in a 5 mL tube at ambient temperature
for 24 h. The mixture was evaporated to dryness and purified by
normal phase semi-preparative HPLC (n-hexane/ethyl acetate¼2:1,
v/v) to afford the 9a (1.0 mg): colorless oil; 1H NMR (CD3OD,
600 MHz) dH 1.68 (m, H-1), 1.90 (m, H-2), 5.77 (d, 3.6, H-3), 2.25 (dd,
9.6, 3.0, H-6a), 1.68 (d, 10.2, H-6b), 3.74 (dd, 10.8, 6.0, H2-9), 1.70 (m,
H-10), 1.28 (m, H2-2), 0.89 (t, 7.2, H3-30). Likewise, the reaction
mixture was processed as described above for 9a to afford 9b
(0.7 mg). Colorless oil; 1H NMR (CD3OD, 600 MHz) dH 1.70 (m, H-1),
1.98 (m, H-2), 6.07 (d, 3.6, H-3), 2.32 (dd,10.2, 3.0, H-6a),1.68 (d, 6.6,
H-6b), 3.77 (dd, 10.8, 6.6, H2-9), 1.60 (m, H-10), 1.24 (m, H2-2), 0.85
(t, J¼7.8 Hz, H3-30).
20
a
-Hydroxy-2-oxo-buagarofuran (5). Pale oil,
[a]
D
ꢀ16.0 (c 0.16, MeOH); IR nmax 3443, 2960, 2938, 2873, 1667, 1624,
1464, 1388, 1351, 1248, 1136, 1077, 994, 871 cmꢀ1; 1H (Table 1) and
13C NMR data (Table 2); ESIMS m/z 293 [MþH]þ; HRESIMS (posi-
tive) m/z 293.2105 [MþH]þ (calcd for C18H29O3, 293.2111).
4.4.1.5. (15R)-2
a
,15-Dihydroxy-buagarofuran (6). White pow-
20
der, [a]
D
þ43.2 (c 0.07, MeOH); IR nmax 3265, 2957, 2927, 2869,
1726, 1676, 1459, 1383, 1299, 1126, 1071, 1013, 958, 880 cmꢀ1 1H
;
(Table 1) and 13C NMR data (Table 2); ESIMS m/z 295 [MþH]þ, 611
[2MþNa]þ; HRESIMS (positive) m/z 317.2074 [MþNa]þ (calcd for
C
18H30NaO3, 317.2087).
4.4.1.6. Preparation of (S)-(6a) and (R)-MTPA (6b) esters. To
a solution of 6 (5.0 mg, 0.017 mmol) in anhydrous dichloromethane
(0.5 mL) were added (S)-MTPA chloride (15 mg, 0.064 mmol), EDC
[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] 10 mg, and
4.4.1.12. (10S)-9 ,10-Dihydroxy-buagarofuran (10). White pow-
a
der, [
a
]
20 ꢀ3.3 (c 0.03, MeOH); IR nmax 3400, 2966, 2928, 2877, 1724,
D
DMAP
[
a
-methoxy-
a
-(trifluoromethyl) phenylacetyl chloride]
1667, 1458, 1141, 1062, 1028, 983, 871 cmꢀ1 1H (Table 1) and 13C
;
10 mg, then the mixture was allowed to stir for 48 h at room
temperature. The reaction was quenched by addition of 1.0 mL of
H2O, and the mixture was subsequently extracted with ethyl ace-
tate (3ꢁ1.0 mL). The organic layers were combined, dried over
anhydrous NaSO4, and evaporated. The residue was subjected to
column chromatography over silica gel chromatography using n-
hexane/ethyl acetate (3:1) to yield the (S)-MTPA ester (6a, 1.1 mg).
The same procedure was used to prepare the (R)-MTPA ester (6b,
0.8 mg). 1H NMR (CD3OD, 600 MHz, 6a): dH 1.87 (m, H-1), 4.10 (t,
J¼5.4 Hz, H-2), 5.97 (d, J¼4.2 Hz, H-3), 2.20 (dd, J1¼12.0 Hz,
J2¼4.2 Hz, H-6a), 1.60 (d, J¼12.0 Hz, H-6b), 1.65 (m, H-10), 1.52 (m,
H-20a), 1.36 (m, H-20b), 0.90 (t, J¼7.8 Hz, H3-30). 1H NMR (CD3OD,
600 MHz, 6b): dH 1.98 (m, H-1), 4.15 (t, J¼5.4 Hz, H-2), 6.08 (d,
NMR data (Table 2); ESIMS m/z 295 [MþH]þ; HRESIMS (positive) m/
z 295.2246 [MþH]þ (calcd for C18H31O3, 295.2268).
4.4.1.13. (15R)-8b,15-Dihydroxy-4-buagarofuran (11). Pale oil,
20
[a
]
þ35.2 (c 0.13, MeOH); IR nmax 3383, 2961, 2929, 2872, 1657,
D
1461, 1379, 1245, 1137, 1053, 1012, 959, 876 cmꢀ1; 1H (Table 3) and
13C NMR data (Table 4); ESIMS m/z 295 [MþH]þ, 317 [MþNa]þ;
HRESIMS (positive) m/z 317.2104 [MþNa]þ (calcd for C18H30NaO3,
317.2087).
4.4.1.14. (15R)-8
a
,15-Dihydroxy-4-buagarofuran
(12). White
powder, [
a
]
20 ꢀ4.8 (c 0.12, MeOH); IR nmax 3409, 2960, 2931, 2873,
D
1708, 1669, 1462, 1384, 1136, 1023, 879 cmꢀ1
;
1H (Table 3) and 13C

3568
L. Qiao et al. / Tetrahedron 70 (2014) 3560e3569
20
NMR data (Table 4); ESIMS m/z 295 [MþH]þ; HRESIMS (positive) m/
z 317.2072 [MþNa]þ (calcd for C18H30NaO3, 317.2087).
4.4.3.3. 8
b-Hydroxy-2-oxo-buagarofuran (17). Pale oil, [a]
D
þ16.5 (c 0.12, MeOH); IR nmax 3330, 2961, 2935, 2874, 1715, 1676,
1462, 1387, 1254, 1132, 1032, 903, 873, 829 cmꢀ1 1H (Table 3) and
;
4.4.2. Metabolites from 1 by A. coerulea AS 3.3538. The giving crude
extract (0.9 g) was submitted to silica gel column (200 g,
200e300 mesh) chromatography eluting with a gradient of pe-
troleum ether (60e90 ꢂC) in acetone to afford 8 fractions. Com-
pound 13 (90 mg, 17.0%) was crystallized from Fr. 4 (154 mg). Fr. 5
(132 mg) was purified by reverse phase semi-preparative HPLC
(35% ACN in H2O, v/v, 3 mL/min) to give 9 (12 mg, 2.1%). Fr. 8
(75 mg) was subjected to reverse phase semi-preparative HPLC to
yield 14 (22 mg, 3.9%).
13C NMR data (Table 4); ESIMS m/z 293 [MþH]þ; HRESIMS (posi-
tive) m/z 293.2111 [MþH]þ (calcd for C18H29O3, 293.2111).
20
4.4.3.4. 2,8-Dioxo-buagarofuran (18). Pale oil, [
a]
ꢀ41.3 (c
D
0.21, MeOH); IR nmax 3444, 2957, 2932, 2872, 1710, 1670, 1462, 1384,
1254, 1134, 1012, 879 cmꢀ1 1H (Table 3) and 13C NMR data (Table
;
4); ESIMS m/z 291 [MþH]þ; HRESIMS (positive) m/z 291.1948
[MþH]þ (calcd for C18H27O3, 291.1955).
4.4.3.5. 2a,13-Dihydroxy-buagarofuran (19). White powder,
20
4.4.2.1. 9
a
-Hydroxy-buagarofuran (13). White powder,
[a
]
[a
]
20 þ18 (c 0.1, MeOH); IR nmax 3319, 2953, 2927, 2869, 1663, 1461,
D
D
þ2.1 (c 0.15, MeOH); IR nmax 3405, 2958, 2933, 2871, 1710, 1666,
1381, 1034, 1005, 948, 880 cmꢀ1
;
1H (Table 3) and 13C NMR data
1459, 1384, 1137, 1064, 1023, 973, 880 cmꢀ1
;
1H (Table 3) and 13C
(Table 4); ESIMS m/z 317 [MþNa]þ; HRESIMS (positive) m/z
295.2243 [MþH]þ (calcd for C18H31O3, 295.2268); m/z 317.2091
[MþNa]þ (calcd for C18H30NaO3, 317.2087).
NMR data (Table 4); ESIMS m/z 277 [MþH]þ; HRESIMS (positive) m/
z 277.2150 [MþH]þ (calcd for C18H29O2, 277.2162).
20
4.4.2.2. (20R)-9
a
,20-Dihydroxy-buagarofuran (14). Pale oil, [
a
]
4.4.3.6. 2-Oxo-buagarofuran (20). Colorless oil, [
a
]
20 þ3 (c 0.05,
D
D
þ3.4 (c 0.26, MeOH); IR nmax 3443, 2985, 2964, 2925, 2872, 1459,
MeOH); IR nmax 3298, 2959, 2931, 2869, 1675, 1624, 1460, 1383,
1367,1310,1233,1137,1059, 967, 871, 852 cmꢀ1; 1H (Table 3) and 13
C
1258, 1145, 1013, 960, 889, 867, 819 cmꢀ1 1H NMR (300 MHz,
;
NMR data (Table 4); ESIMS m/z 295 [MþH]þ; HRESIMS (positive) m/
CD3OCD) dH 2.75 (d, 15.0, H-1a), 1.97 (d, 15.0, H-1b), 5.89 (s, H-3),
2.38 (dd,12.5, 3.0, H-6a),1.85 (d,12.5, H-6b), 2.07 (m, H-7), 1.78 (2H,
m, H-8), 1.48 (2H, m, H-9), 1.03 (3H, s, H-11), 1.42 (3H, s, H-12), 1.29
(3H, s, H-13), 2.21 (2H, m, H-15), 1.40 (4H, m, H-10 and H-20), 0.93
(3H, t, 7.5, H-30); 13C NMR (100 MHz, CD3OCD) dC 49.6 (C-1), 199.4
(C-2), 127.3 (C-3), 159.8 (C-4), 84.8 (C-5), 33.4 (C-6), 44.1 (C-7), 24.1
(C-8), 29.6 (C-9), 40.6 (C-10), 23.7 (C-11), 22.6 (C-12), 30.3 (C-13),
82.6 (C-14), 34.0 (C-15), 31.1 (C-10), 22.6 (C-20),13.9 (C-30); ESIMS m/
z 299 [MþNa]þ; m/z 276 [MþH]þ.
z 295.2251 [MþH]þ (calcd for C18H31O3, 295.2268).
4.4.2.3. Preparation of (S)-(14a) and (R)-MTPA (14b) esters. The
reaction mixture of 14 (5.0 mg, 0.017 mmol), (S)-MTPA (15 mg,
0.064 mmol), and anhydrous dichloromethane (0.5 mL), EDC
10 mg, and DMAP 10 mg were processed as described above for to
afford 14a and 14b. Compound 14a: colorless oil; 1H NMR (CD3OD,
600 MHz) dH 1.59 (m, H-1), 1.85 (m, H-2), 5.48 (s, H-3), 2.04 (d, 12.6,
H-6a), 1.67 (d, 12.0, H-6b), 1.97 (m, H2-7), 3.74 (dd, 11.4, 6.0, H2-9),
1.85 (m, H-15a), 1.91 (m, H-15b), 1.59 (m, H-10a), 1.52 (m, H-10b),
1.35 (d, 6.0, H3-30). Compound 14b: colorless oil; 1H NMR (CD3OD,
600 MHz) dH 1.58 (m, H-1), 1.90 (m, H-2), 5.58 (s, H-3), 2.14 (d, 14.4,
H-6a), 1.74 (d, 12.6, H-6b), 2.00 (m, H2-7), 3.76 (dd, 11.4, J2¼6.0 Hz,
H2-9), 2.0 (m, H2-15), 1.65 (m, H-10a), 1.53 (m, H-10b), 1.27 (d, 6.0,
H3-30).
4.5. Inhibition of (5-HT) and [3H]-NE-serotonin re-uptake
bioassay
One male SD rat was decapitated and the brain was rapidly re-
moved.16,17 The whole brain minus cerebellum was weighed and
homogenized in 19 volumes of ice-cold 0.32 mol/L sucrose solution
by a PottereElvejhem homogenizer. The homogenate was centri-
fuged at 1000ꢁg at 4 ꢂC for 10 min. The supernatant was decanted
4.4.3. Metabolites from 1 by C. elegans AS 3.3400. The residue
(1.7 g) was subjected onto a silica gel chromatography column by
eluting with petroleum/ether (60e90 ꢂC)/acetone to provide 10
fractions (Fr. 1eFr. 10). Fr. 7 (430 mg) was separated by Sephadex
LH-20 column chromatography eluting with chloroform/MeOH
(40:60, v/v) and further purified by semi-preparative reverse phase
HPLC (37% ACN in H2O, v/v, flow rate 3 mL/min) to afford 15 (4 mg,
0.7%), and 16 (6 mg, 1.1%). The Fr. 5 (430 mg) was separated by
reverse phase HPLC column chromatography (37% ACN in H2O, v/v,
flow rate 3 mL/min) to 2 (3 mg, 0.5%) and 18 (23 mg, 4.1%). Fraction
4 (170 mg) was applied to semi-preparative reverse phase HPLC
(30% ACN in H2O, v/v, flow rate 3 mL/min) to afford 17 (8 mg, 1.4%),
19 (2 mg, 0.3%), and 20 (11 mg, 2.0%).
and used for re-uptake experiments. 100
with 50
tubes and sample tested tubes were preincubated at 37 ꢂC for
mL suspension was mixed
mL drug/sample solution or krebs buffer. The total binding
20 min, then added 400 m
L 40.1 nmol/L [3H]-5-HT ([3H]-NE) solu-
tion in krebs buffer and incubated at 37 ꢂC for 20 min. The non-
specific binding tubes were preincubated in 0 ꢂC ice water for
20 min, then added 400 m
L 40.1 nmol/L [3H]-5-HT ([3H]-NE) solu-
tion in krebs buffer and incubated in 0 ꢂC ice water for 20 min. All
tubes were filtrated by a cell harvester immediately after in-
cubation. The fiberglass filter membrane was washed five times by
ice-cold saline, then put into scintillation vials, and counted in 4 mL
of liquid scintillation cocktail. The percent inhibition of sample on
[3H]-5-HT ([3H]-NE) re-uptake¼(cpm of total binding tubesꢀcpm
of sample tubes)/(cpm of total binding tubesꢀcpm of nonspecific
binding tubes)ꢁ100%. The percent inhibition at each sample con-
centration was the mean of three replicates. IC50 values were cal-
culated by log-probit analysis.
4.4.3.1. (20R)-2 ,20-Dihydroxy-buagarofuran (15). White pow-
a
der, [
a
]
20 ꢀ6.0 (c 0.1, MeOH); IR nmax 3398, 3069, 2927, 2861, 1721,
D
1620, 1546, 1448, 1395, 1227, 1063, 1016, 948, 819 cmꢀ1; 1H (Table
3) and 13C NMR data (Table 4); ESIMS m/z 317 [MþNa]þ; HRESIMS
(positive) m/z 317.2084 [MþNa]þ (calcd for C18H30NaO3,
317.2087).
Acknowledgements
4.4.3.2. 2
b
,9
a
-Dihydroxy-buagarofuran (16). White powder,
This work was financially supported by the Science & Technol-
ogy Project of Guangdong Province (2011A080403020), the Fun-
damental Research Funds for the Central Universities (Grant No.
2012N06), and the National Science & Technology Major Project
‘Key New Drug Creation and Manufacturing’, China (No.
2012ZX09301002-001-005).
20
[
a
]
ꢀ9.7 (c 0.07, MeOH); IR nmax 3375, 2930, 2867, 1725, 1664,
D
1460, 1383, 1124, 1063, 1026, 978, 948, 880, 856 cmꢀ1; 1H (Table 3)
and 13C NMR data (Table 4); ESIMS m/z 317 [MþNa]þ; HRESIMS
(positive) m/z 317.2084 [MþNa]þ (calcd for C18H30NaO3,
317.2087).

L. Qiao et al. / Tetrahedron 70 (2014) 3560e3569
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