Chemical and microbial semi-synthesis of tetrahydroprotoberberines as inhibitors on tissue factor procoagulant activity

Article abstract of DOI:10.1016/j.bmc.2012.11.002

To discover new inhibitors on tissue factor procoagulant activity, 21 tetrahydroprotoberberines were screened on the model of human THP-1 cells stimulated by lipopolysaccharide. Among these tetrahydroprotoberberines, several unique compounds were synthesized through microbial transformation: compound 6 (l-corydalmine) was obtained through regio-selective demethylation by Streptomyces griseus ATCC 13273, whereas compounds 4a, 4b, 5h, and 5i were microbial glycosylation products by Gliocladium deliquescens NRRL1086. The bioassay results showed that compounds 3 (tetrahydroberberine), 10 (tetrahydroberberrubine), and 5f (cinnamyl ester of 5) and 5i (glycosidic product of 5), exhibited the most potential effects, with IC₅₀ values of 8.35, 6.75, 3.75, and 8.79 nM, respectively. The preliminary structure and activity relationship analysis revealed that the 2,3-methylenedioxy group of the A ring was essential for the strong inhibitory effects, and the R configuration of the chiral center C-14 showed higher activity than S-form products. The formation of fatty acid or aromatic acid esters of compound 5, except the cinnamyl esters, would weaken its effects. It is also interesting to note that the glycosylation of tetrahydroprotoberberines will maintain and even enhance the inhibitory effects. Because of the importance of glycochemistry in new drug discovery and development, this deserves further exploration and may provide some guide on the semi-synthesis of tetrahydroprotoberberines as tissue factor pathway inhibitors. Our findings also provide some potential leading compounds for tissue factor-related diseases, such as cancer and cardiovascular diseases.

Full text of DOI:10.1016/j.bmc.2012.11.002

Bioorganic & Medicinal Chemistry 21 (2013) 62–69
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemical and microbial semi-synthesis of tetrahydroprotoberberines
as inhibitors on tissue factor procoagulant activity
Hai-Xia Ge a,c, , Jian Zhang a, , Ling Chen b, Jun-Ping Kou b, , Bo-Yang Yu a,
⇑
⇑
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24# Tongjia Xiang Street, Nanjing 210009, China
b Department of Complex Prescription of Traditional Chinese Medicine, China Pharmaceutical University, 639# Longmian Avenue, Nanjing 211198, China
c Department of Pharmacy, Huzhou Teachers College, Huzhou 313000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
To discover new inhibitors on tissue factor procoagulant activity, 21 tetrahydroprotoberberines were
screened on the model of human THP-1 cells stimulated by lipopolysaccharide. Among these tetrahydro-
protoberberines, several unique compounds were synthesized through microbial transformation: com-
pound 6 (l-corydalmine) was obtained through regio-selective demethylation by Streptomyces griseus
ATCC 13273, whereas compounds4a, 4b, 5h, and 5i were microbial glycosylation products by Gliocladium
deliquescens NRRL1086. The bioassay results showed that compounds 3 (tetrahydroberberine), 10 (tetr-
ahydroberberrubine), and 5f (cinnamyl ester of 5) and 5i (glycosidic product of 5), exhibited the most
potential effects, with IC50 values of 8.35, 6.75, 3.75, and 8.79 nM, respectively. The preliminary structure
and activity relationship analysis revealed that the 2,3-methylenedioxy group of the A ring was essential
for the strong inhibitory effects, and the R configuration of the chiral center C-14 showed higher activity
than S-form products. The formation of fatty acid or aromatic acid esters of compound 5, except the cin-
namyl esters, would weaken its effects. It is also interesting to note that the glycosylation of tetrahydro-
protoberberines will maintain and even enhance the inhibitory effects. Because of the importance of
glycochemistry in new drug discovery and development, this deserves further exploration and may pro-
vide some guide on the semi-synthesis of tetrahydroprotoberberines as tissue factor pathway inhibitors.
Our findings also provide some potential leading compounds for tissue factor-related diseases, such as
cancer and cardiovascular diseases.
Received 21 August 2012
Revised 31 October 2012
Accepted 2 November 2012
Available online 10 November 2012
Keywords:
Tissue factor
Tetrahydroprotoberberines
Biotransformation
Structure and activity relationship (SAR)
Cardiovascular diseases
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
a hypercoagulable state, and TF expression by cancer cells has re-
ceived widespread attention because of its significant contribution
Tissue factor (TF), a 47-kD membrane-bound glycoprotein and a
member of the cytokine-receptor superfamily, is the primary initi-
ator of the coagulation cascade in both hemostasis and thrombo-
sis.1 Elevated levels of TF are observed in patients with
cardiovascular risk factors, such as hypertension, diabetes, dyslipi-
demia, and smoking, as well as in those with acute coronary syn-
dromes.2 TF may indeed be involved in the pathogenesis of
atherosclerosis by promoting thrombus formation. Thus, inhibition
of TF procoagulant activity or induction appears to be an attractive
target for the treatment of cardiovascular diseases.3 Furthermore,
TF upregulation resulting from its enhanced exposure to clotting
factor FVII/FVIIa often manifests not only a hypercoagulable state
but also an inflammatory one.4 Advanced cancer is associated with
to the pathogenesis of cancer progression and metastasis.5 There-
fore, pharmaceutical intervention in the TF pathway could possibly
provide novel treatment strategies for cardiovascular disease, sep-
ticemia, and tumor. Screening for new TF pathway inhibitors is
thus becoming one of the hot points in the field of drug develop-
ment and correlative basic research.6 To explore new TF pathway
inhibitors, we optimized one convenient and high-throughput
chromogenic assay method by using a commercial human pro-
thrombin complex instead of purified coagulation factors.7 This
method was tested on simvastatin and curcumin, which showed
IC50 values of 0.1–1 lM, which fits well with the reported results
on previous models.8–10
Tetrahydroprotoberberines (THPB), a class of abundant, natu-
rally occurring tetracyclic alkaloids that contain an isoquinoline
skeleton with substituent methoxyl or hydroxyl groups on the
two benzene rings and a stereogenic center at C-14, have been dem-
onstrated to possess various biological activities. These include
analgesic activity; anticonvulsant, anti-anxiety, antidepressant,
and antipsychotic activity in the central nervous system; anti-
⇑
Corresponding author. Tel.: +86 25 83271321; fax: +86 25 83313080 (B.-Y.Y.);
tel.: +86 25 86185157; fax: +86 25 86185158 (J.-P.K.).
E-mail addresses: junpingkou@163.com (J.-P. Kou), boyangyu59@163.com
(B.-Y. Yu).
These authors contributed equally to the work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.002

H.-X. Ge et al. / Bioorg. Med. Chem. 21 (2013) 62–69
63
arrhythmic, antihemorrhagic, and hypotensive activities in the car-
2.1.2. Inhibitory effects of THPB analogues 1–10 on TF
procoagulant activity
diovascular system; and other activities.11 In the screening test, we
found that some protoberberines showed an inhibitory effect on TF
procoagulant activity in human monocytes stimulated by lipopoly-
saccharide (LPS).12 In this study, we prepared a series of THPB deriv-
atives by chemical and microbial semi-synthesis to further evaluate
their applications as TF pathway inhibitors. Because biocatalysis
has become one of the promising methods for the structural modi-
fication of natural products, we demonstrate several unique micro-
bial transformations. Compound 6 (l-corydalmine) was synthesized
through regio-selective demethylation by Streptomyces griseus
ATCC 13273, and compounds 4a, 4b, 5h, and 5i were microbial gly-
cosylation products by Gliocladium deliquescens NRRL1086. Their
inhibitory effects on TF procoagulant activity in human monocytes
stimulated by LPS were assayed, and the preliminary structure–
activity relationship was also investigated. This study should pro-
vide a better understanding of TF pathway inhibitor discovery
and the multi-activity of THPBs for further clinical application.
The THPB analogues (1–10) were screened for their inhibitory
effect on TF procoagulant activity in human THP-1 cells stimulated
by LPS. Table 1 shows the results. Most of the compounds exhib-
ited higher activity than the positive control curcumin. Compounds
3, 5, and 10 even showed IC50 values of 8.35, 12.17, and 6.75 nM,
respectively, indicating that THPBs were one type of effective TF
pathway inhibitors. After a careful analysis of the chemical struc-
tures of the related compounds, we found that the substitute
groups of the A ring may play an essential role in the TF inhibitory
effects. The compounds bearing the methylenedioxy group at C-2
and C-3 showed the highest activities. The substitution group of
two free hydroxyl groups or the methyloxy may decrease the ef-
fects; in particular, the compound with the substitution of ortho-
dihydroxyl groups in ring A exhibited the lowest activity with an
IC50 value of 968.90 nM, which was almost a hundredfold higher
compared with compounds 3 or 10. In our experiment, the substi-
tution groups on the D ring seemed to have less impact on THPB
analogues’ inhibitory effect.
2. Results and discussion
Another interesting aspect of the results was that the stereo-
chemistry of the chiral center of C-14 may also influence the
effects. In our tested compounds, there was one pair of isomers:
compound 9, bearing the S-form configuration of C-14, and com-
pound 10, which was the R-form. Compound 10 showed higher
activity than compound 9, with the IC50 values of 6.75 and
36.41 nM, respectively, indicating a higher activity of R-form com-
pounds compared with the corresponding S-form compounds.
Such results could be also deduced from the results of compounds
1 and 2. Compound 1 was the S-form, which exhibited an IC50 va-
lue of 22.78 nM, while compound 2, as the racemic mixtures, had a
much lower inhibitory effect. In the structure and activity research
on THPBs on some other bioassay models, the S-form compounds
usually had stronger activity than the R-form.18 We may hypothe-
size from this that the inner interactions between the small
molecular and the amino acid moieties of the target protein may
be much different from the previous research, which deserves fur-
ther exploration.
2.1. Synthesis of THPB analogues 1–10 and their inhibitory
effects on TF procoagulant activity
2.1.1. Synthesis of THPB analogues 1–10
Compounds 1 (l-tetrahydropalmatine) and 8 (l-stepholidine,
l-SPD) were obtained commercially, and compound 2 was the race-
mic mixture of compound 1. Compounds 2 and 3 were obtained by
palmatine and berberine hydrochloride reduction reaction with
NaBH4, respectively,13 and both has a rotation value of 0.
Compounds 4 and 5 were synthesized as in Scheme 1, in which
the chloride salt of palmatine and berberine undergoes pyrolysis
monodemethylation14 to yield a red compound, palmatrubine,
and berberrubine, reducing them to 4 and 5. Compound 7 was syn-
thesized from berberine hydrochloride reacted with phloroglucinol
in H2SO4 (60%) at 90–95 °C and reduction reaction (Scheme 2).13,15
Compound 6 (l-corydalmine, l-CDL) was prepared by bioconversion
of Streptomyces griseus ATCC 13273 from l-tetrahydropalmatine
(Scheme 3). Compounds 9 and 10, a pair of enantiomers of com-
pound 5, were obtained through the classical chemical resolution
Among all the tested compounds, compound 5 was the most
attractive, not only because of its strong activity but also because
of the free hydroxyl group substituted on C-9, which can provide
an active site for further esterification or glycosylation. Thus, we
synthesized a series of derivatives of compound 5 through chemi-
cal and microbial methods.
by using (+)-di-p-toluoyl-L-tartaric acid (DTTA) in 95% ethanol
(Scheme 4),16 and their absolute configuration was confirmed
through the chiral column HPLC and CD spectrum according to
the literature.17
R2O
R2O
R2O
R1O
Cl-
N
Cl-
N
R1O
(1) 195-205°c
R1O
N
NaBH4
MeOH
OH
OH
OCH3
(2) HCl
OCH3
OCH3
OCH3
R1,R2=CH3, palmatrubine
R1,R2=-CH2-,berberrubine
R1,R2= CH3, 4
R1,R2=-CH2-,
R1,R2=CH3, palmatine
R1,R2=-CH2-,berberine
5
Scheme 1. Synthetic routes of compounds 4 and 5.
HO
HO
HO
O
O
Cl-
N
Cl-
N
N
HO
60%H2SO4
NaBH4
MeOH
OCH3
OCH3
OCH3
OCH3
OCH3 Phloroglucinol
7
OCH3
Scheme 2. Synthetic routes of substrate 7.

64
H.-X. Ge et al. / Bioorg. Med. Chem. 21 (2013) 62–69
H3CO
H3CO
H3CO
N
N
Streptomyces griseus ATCC 13273
H3CO
H
H
OCH3
OH
OCH3
1
OCH3
6
Scheme 3. Microbial semi-synthesis of compound 6 by Streptomyces griseus ATCC13273
O
N
O
H
OH
NH3 H2O
(-)-1 (+)-DTTA
O
O
9: S-(-)-5
OCH3
N
(+)-DTTA
95% Ethanol
OH
O
O
NH3 H2O
(+)-1 (+)-DTTA
N
OCH3
5
H
OH
OCH3
10: R-(+)-5
Scheme 4. The resolution routes of compound 5.
2.2. Synthesis of derivatives of compound 5 and their effects on
TF procoagulant activity
was one of the most used strain in our research; it contained aston-
ishing glycosylation ability on natural products.21,22 Thus, com-
pounds 4–10 were employed in the screening test, and
compound 4 could also be transformed to 4a and 4b (Scheme 6).
The ESI-MS of compounds 4a and 4b showed the same quasi-
molecular ion at m/z 504.3, indicating a 162 amu mass increase
from that of 4. It was suggested that a hexose moiety was intro-
duced, as their 13C NMR spectra all showed the presence of six
new signals at d 103, 74, 76, 69, 77, and 61 ppm, which are
2.2.1. Synthesis of ester derivatives of compound 5
Compound 5a was synthesized according to the general acety-
lation reaction procedure by acetic anhydride and compound 5.
Compounds 5b–5g were synthesized by acylate reaction of the cor-
responding acyl chloride and compound 5 (Scheme 5). The struc-
tures of compounds 5a–5g were confirmed with MS, 13C NMR,
and 1H NMR.
characteristic of b-
10 indicated that the stereochemistry of the glycosidic linkage at
C-10 of
-glucose is b. This deduction was supported by the long-
D-glucose. A coupling constant of 7.5 Hz for H-
2.2.2. Inhibitory effects of the ester derivatives 5a–5g on TF
procoagulant activity
D
range coupling between H-10 and C-9 in their HMBC experiment.
All the 13C NMR and 1H NMR data of compounds 4a and 4b were
carefully and unambiguously assigned by extensive 2D-NMR tech-
niques (DEPT, HMBC, HSQC). The data revealed that the two com-
pounds were the glycosidic products of 4, and there was only one
hydroxyl group in the skeleton of compound 4. Further CD spectra
of compound 4a exhibited negative Cotton effects, whereas com-
pound 4b showed positive Cotton effects of around 210 nM. When
the CD spectra of substrate 4 were compared, the S-form substrate
of 4 showed negative Cotton effects, whereas the R-type isomer
showed positive Cotton effects of around 210 nM.17 It can be de-
duced that the chiral carbon of the aglycone part of compound
4a is of S configuration, whereas compound 4b is of R configura-
tion. This conclusion was further confirmed by their optical rota-
In the ester derivatives 5a–5g, different acyloxy substituents on
C-9 affected the TF inhibitory activity greatly. The straight chain
fatty acid esters of compounds 5 and 5a–5c showed less activity,
and their IC50 values declined considerably from 63.06 to
1501 nM along with the chain extension from C2–C4. Moreover,
compounds 5d and 5e, which bear aromatic acyloxy substituents
on C-9, also displayed much weaker activity than compound 5. It
was amazing that although compound 5f, which was the cinnam-
oyl ester of 5, showed the highest inhibitory activity with the an
IC50 value of 3.75, compound 5g, which was the acetylferuloyl ester
of 5, exhibited lower activity with an the IC50 value of 201.93 nM.
In succeeding experiments we investigated the activity of cinnamic
acid and ferulic acid. Both of them showed no significant inhibitory
effects (IC50 values higher than 10
lM). The structure differences
tion values, in which compounds 4a and 4b showed ½a D24
ꢁ221.3
ꢀ
between 5f and 5g were the acyl moieties, so the subunit of the
substituents on the benzene ring may have a great influence on
TF inhibitory effect.
(c 0.501, pyridine) and ½a D24
ꢀ
235.1 (c 0.520, pyridine), respectively.
Compounds 5h and 5i were the glycosidic products of the race-
mic substrate 5. Similar to compounds 4a and 4b, they were also a
pair of disasteoisomers, and their structures were elucidated
unambiguously based on the MS, 13C NMR, 1H NMR, DEPT, HMBC,
HSQC, and CD.21
2.3. Synthesis of glycosylation products of compounds 4 and 5
and their effects on TF procoagulant activity
2.3.1. Synthesis of compounds 4a, 4b, 5h, and 5i by
biotransformation pathway
2.3.2. Inhibitory effects of the compounds 4a, 4b, 5h, and 5i on
TF procoagulant activity
Because of the importance of glycochemistry in drug discov-
ery,19,20 we synthesized the glycosidic products 5h and 5i based
on our previous discovery.21 Gliocladium deliquescens NRRL1086
The IC50 values of the TF inhibitory activity of compounds 4a,
4b, 5h, and 5i were 16.80, 12.61, 38.59, and 8.79 nM, respectively.
These four compounds are two pairs of enantiomerical isomers.

H.-X. Ge et al. / Bioorg. Med. Chem. 21 (2013) 62–69
65
Table 1
Structures of compounds and their IC50 values on TF procoagulant activity
5
4
A
1
4a
3
2
R2O
R1O
6
B
14
N
C
8
14a
8a
H
9
OR3
OR4
13
12a
D
10
12
11
No.
C-14
R1
R2
R3
R4
IC50 (nM)
1
2
3
4
5
6
7
8
ꢁ
CH3
CH3
–CH2–
CH3
–CH2–
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
H
22.78 3.73
14.98 2.18
8.35 0.63
187.26 52.93
12.17 1.24
152.43 17.86
968.90 161.65
104.58 15.62
36.41 0.83
6.75 0.38
CH3
H
ꢁ
CH3
H
CH3
CH3
CH3
CH3
H
H
H
CH3
H
ꢁ
ꢁ
+
9
–CH2–
–CH2–
–CH2–
–CH2–
–CH2–
CH3
CH3
CH3
CH3
CH3
10
5a
5b
5c
H
COCH3
COCH2CH3
CO(CH2)4CH3
63.06 3.79
118.80 31.97
1501.43 98.06
O
C
5d
–CH2–
CH3
376.52 29.69
OCOCH3
O
C
5e
–CH2–
CH3
555.37 63.21
O
5f
–CH2–
–CH2–
CH3
CH3
3.75 0.34
O
OCH3
5g
201.93 14.73
OCOCH3
OH
OH
OH
OH
HO
OH
OH
5h
5i
ꢁ
+
–CH2–
–CH2–
CH3
CH3
CH3
CH3
CH3
38.59 6.56
8.79 0.72
16.80 1.97
O
HO
HO
HO
OH
OH
O
O
O
OH
OH
4a
ꢁ
+
CH3
CH3
OH
OH
4b
CH3
12.61 1.41
Curcumin
199.63 16.52
The R-form products also showed higher activity than the S-form.
Compared with the IC50 value of compound 4, the glycosidic prod-
ucts showed much enhanced activity. On the contrary, where com-
3. Conclusions
In summary, we prepared a series of THPB analogues 1–10 and
derivatives (5a–5i, 4a, 4b) through a combination of chemical and
microbial means, and examined their inhibitory effects on TF
procoagulant activity in human THP-1 monocytic cells stimulated
by LPS. Among them, several unique compounds were first synthe-
sized through microbial transformation. Compound 5 (l-corydal-
mine) was obtained through regio-selective demethylation by
Streptomyces griseus ATCC 13273. Compounds 4a, 4b, 5h, and 5i
were microbial glycosylation products that are enantio-selective
by Gliocladium deliquescens NRRL1086 and exhibited enhanced or
pound
5 was concerned, the activity of glycosidic products
exhibited no significant improvement. Glycosylated secondary
metabolites continue to serve as an important source for drug dis-
covery. A significant number of glycosylated small molecules, such
as heparin, amikacin, and cytarabine, have been shown to be clin-
ically useful for the treatment of a variety of diseases, including
bacterial and fungal infections, cancer, and other human dis-
eases.20–23 As the first synthesized glycosidic THPBs, their in vivo
bioassays are now under investigation.

66
H.-X. Ge et al. / Bioorg. Med. Chem. 21 (2013) 62–69
RCOOH
SOCl2
O
O
O
N
N
RCOCl
O
OH
OR
DMAP, triethylamine, CHCl3
OCH3
OCH3
5
5a-5g
Scheme 5. Synthetic routes of esters derivatives of compound 5.
R2O
N
R1O
OH
O
HO
O
H
OH
OH
R2O
R1O
OCH3
4a R1=R2= CH3
5h
H
R1,R2= -CH2
Gliocladium deliquescens
OH
NRRL 1086
+
OCH3
4 R1=R2= CH3
R2O
R1O
5
R1,R2=-CH2-
N
H
OH
HO
O
O
OH
OH
OCH3
4b R1=R2= CH3
5i
R1,R2=-CH2
Scheme 6. Biotransformation of compounds 4 and 5 by Gliocladium deliquescens NRRL 1086.
similar TF inhibitory activities with their aglycone. We found that
the configuration of C-14 would play an important role in their
inhibitory activity and that R-form compounds showed stronger
effects than the S-form. Compound 5f, with cinnamoyl group in
C-9, showed the most attractive activity among all the compounds.
Due to the multifunctional role of TF in many physiopathologic
processes, these potent THPB compounds would be promising lead
compounds in the discovery of new cardiovascular and anticarci-
nogenic drugs, and this information will provide a better under-
standing of the chemical and biological research of THPBs.
of glucose, 3 g of KH2PO4, and 1.5 g of MgSO4ꢂ7H2O were added
into the filtrate and diluted with distilled water to 1 L before being
autoclaved at 121 °C for 15 min before use.
4.2. Preparation and spectral properties of some compounds
4.2.1. ( )-2,3,10-Trimethoxy-9-hydroxy-5,6,13,14-tetrahydro-
8H-dibenzo[a,g]quinolizine (4)
Palmatine hydrochloride (5 g, 0.013 mol) was heated at 190 °C
in a dry over under vacuum (20–30 mmHg) for 15 min. The crude
product was recrystallized to provide 2.70 g (62%) of palmatrubine;
A mixture of palmatrubine (2.70 g, 0.0078 mol) and 65 mL EtOH/
HCl (95:5) was stirred at room temperature for 1 h, and then the so-
lid were filtered and washed with EtOH two times to supply palma-
trubine chloride 3.18 g (94%); A mixture of palmatrubine chloride
(2.74 g, 0.0074 mol) and MeOH (70 mL) was refluxed for 20 min
to dissolve, then NaBH4 (1.40 g, 0.037 mol) was added in small por-
tions with stirring at room temperature. Two hours later, the result-
ing solids were filtered, washed with MeOH to crude which
recrystallized from EtOAc, to provide 4 0.94 g (32%).
4. Experiments
4.1. General experimental methods
Thin-layer chromatography (TLC) was performed on silica gel
GF254 plates of 0.5 mm of thickness for analysis. Layers were air
dried and activated at 110 °C for 0.5 h before use. NMR spectra were
recorded on a Bruker Avance 500 spectrometer with tetramethylsil-
ane as the internal standard, and chemical shifts were expressed in
d (parts per million). ESIMS experiments were performed on an Agi-
lent 1100 Series MSD Trap mass spectrometer. CD spectra were re-
corded on a JASCO J-810 spectropolarimeter. Optical rotations were
obtained using a JASCO P-1020 digital polarimeter. Streptomyces
griseus ATCC 13273 and Gliocladium deliquescens NRRL 1086 was
obtained from a courtesy of Professor J.P.N. Rosazza of University
of Iowa, USA.
White crystals. ESI-MS m/z: 342.2 [M+H]+. 1H NMR (CDCl3,
500 MHz) d: 2.68 (m, 2H, 6-H, 13-H), 2.86 (m, 1H, 5-H), 3.22 (m,
3H, 13-H, 6-H, 5-H), 3.54 (d, J = 15.0 Hz, 1H, 8-H), 3.58 (m, 1H,
14-H), 3.87 (s, 6H, –OCH3), 3.89 (s, 3H, –OCH3), 4.25 (d,
J = 15.0 Hz, 1H, 8-H), 5.68 (s, 1H, –OH), 6.68 (d, J = 8.0 Hz, 1H, 12-
H), 6.74 (d, J = 8.0 Hz, 1H, 11-H), 6.62 (s, 1H, 4-H), 6.74 (s, 1H, 1-
H). 13C NMR (CDCl3, 125 MHz) d: 108.70 (C1), 147.54 (C2),
147.48 (C3), 109.00 (C4), 121.29 (C4a), 29.31 (C5), 51.46 (C6),
53.51 (C8), 126.88 (C8a), 141.56 (C9), 144.06 (C10), 111.42 (C11),
119.28 (C12), 128.08 (C12a), 36.41 (C13), 59.30 (C14), 129.81
(C14a), 56.17 (2-OCH3), 56.11 (3-OCH3), 55.87 (10-OCH3).
Cultures were grown by a two-stage procedure in 30 mL of po-
tato medium (PD) held in a 150 mL Erlenmeyer flask. The PD med-
ium was prepared as follows: 200 g of peeled potatoes were cut
into pieces, boiled in water for 20 min, and filtered. Twenty grams

H.-X. Ge et al. / Bioorg. Med. Chem. 21 (2013) 62–69
67
4.2.2. ( )-5,6,13,14-Tetrahydro-9-hydroxy-10-methoxy-2,3-
4.2.5. ( )-5,6,13,14-Tetrahydro-9-acetoxy-10-methoxy-2,3-
(methylenedioxy)-8H-dibenzo[a,g]quinolizine (5)
(methylenedioxy)-8H-dibenzo[a,g]quinolizine (5a)
Preparation and purification of 5 was carried out according to
the procedure of compound 4, starting from berberine hydrochlo-
ride (5 g, 0.0123 mol). Yield: 2.02 g (62%).
Compound 5 (2 g, 0.006 mol) was added to CHCl3 (30 mL) and
stirred for dissolving at room temperature, acetic anhydride
(2.8 mL, 0.03 mol), 2 mL triethylamine and slight 4-dimethylami-
opryidine were added to the solution. The mixture was stirred
for 3 h at room temperature, then washed three times by water,
saturated NaHCO3 solution and saturated NaCl solution, respec-
tively. The CHCl3 phase was dried over sodium sulfate and evapo-
rated to crude which recrystallized from EtOAC to obtain 5a 1.86 g
(82%).
White crystal. ESI-MS m/z: 326.2 [M+H]+. 1H NMR (CDCl3,
500 MHz) d: 2.63 (m, 2H, 13-H, 6-H), 2.82 (m, 1H, 5-H), 3.16 (m,
3H, 13-H, 6-H, 5-H), 3.51 (d, J = 16.0 Hz, 1H, 8-H), 3.52 (m, 1H,
14-H), 3.86 (s, 3H, –OCH3), 4.23 (d, J = 16.0 Hz, 1H, 8-H), 5.67 (s,
1H, 9-OH), 5.91 (d,d, J = 1.5 Hz, 2H, –OCH2O–), 6.59 (s, 1H, 4-H),
6.66 (d, J = 8.5 Hz, 1H, 12-H), 6.73 (d, J = 8.5 Hz, 1H, 11-H), 6.73
(s, 1H, 1-H). 13C NMR (CDCl3, 125 MHz) d: 105.59 (C1), 144.07
(C2), 145.93 (C3), 108.44 (C4), 121.30 (C4a), 29.67 (C5), 51.34
(C6), 53.47 (C8), 127.93 (C8a), 141.57 (C9), 146.15 (C10), 109.02
(C11), 119.32 (C12), 128.10 (C12a), 36.57 (C13), 59.62 (C14),
131.02 (C14a), 100.76 (–OCH2O–), 56.20 (10-OCH3).
Light yellow crystal. ESI-MS m/z: 368.2 [M+H]+. 1H NMR (CDCl3,
500 MHz) d: 2.33 (s, 3H, COCH3), 2.62 (m, 2H, 13-H, 6-H), 2.83 (m,
1H, 5-H), 3.11 (m, 2H, 6-H, 5-H), 3.20 (m, 1H, 13-H), 3.45 (d,
J = 16.0 Hz, 1H, 8-H), 3.56 (m, 1H, 14-H), 3.80 (s, 3H, –OCH3),
4.01 (d, J = 16.0 Hz, 1H, 8-H), 5.90, 5.91 (d, d, J = 1.5 Hz, 2H, –
OCH2O–), 6.58 (s, 1H, 4-H), 6.71 (s, 1H, 1-H), 6.82 (d, J = 8.5 Hz,
1H, 12-H), 6.99 (d, J = 8.5 Hz, 1H, 11-H). 13C NMR (CDCl3,
125 MHz) d: 105.56 (C1), 146.00 (C2), 146.19 (C3), 108.41 (C4),
127.71 (C4a), 29.56 (C5), 51.12 (C6), 53.44 (C8), 127.65 (C8a),
136.19 (C9), 149.01 (C10), 110.59 (C11), 126.44 (C12), 128.25
(C12a), 36.19 (C13), 59.33 (C14), 130.71 (C14a), 100.78
(–OCH2O–), 56.07 (10-OCH3), 168.58(–CO–), 20.43 (–CH3).
4.2.3. (ꢁ)-2,3,9-Trimethoxy-10-hydroxy-5,6,13,14-tetrahydro-
8H-dibenzo[a,g]quinolizine (6)24
Using 24-h-old stage II cultures of Streptomyces griseus ATCC
13273, a total of 500 mg of 1 was distributed evenly among fifty
150 mL Erlenmeyer flasks. Substrate-containing cultures were
incubated for 120 h, then filtrated and extracted with equal
amount of EtOAc. The organic solvent layer was removed, evapo-
rated to dryness. The extract was subjected to silica gel column
chromatography eluted with petroleum ether/acetone (9:1, v/v)
to afford the product 6 0.173 g (38%).
4.2.6. ( )-5,6,13,14-Tetrahydro-9-propionyloxy-10-methoxy-
2,3-(methylenedioxy)-8H-dibenzo[a,g]quinolizine (5b)
Preparation and purification of 5b was carried out according to
the general procedure, starting from compound 5 and propionyl
chloride. Yield: 87%.
White powders. ESI-MS m/z: 342.2[M+H]+. 1H NMR (CDCl3,
500 MHz) d:2.65 (m, 2H, 6-H, 13-H), 2.82 (m, 1H, 5-H), 3.16 (m,
1H, 5-H), 3.23 (m, 2H, 13-H, 6-H), 3.56 (m, 2H, 14-H, 8-H), 3.77,
3.85, 3.88 (s, 9H, 2,3,9-OCH3), 4.22 (d, J = 15.0 Hz, 1H, 8-H), 6.23
(brs, 1H, –OH), 6.61 (s, 1H, 4-H), 6.70 (d, J = 8.0 Hz, 1H, 12-H),
6.72 (s, 1H, 1-H), 6.75 (d, J = 8.0 Hz, 1H, 11-H). 13C NMR (CDCl3,
125 MHz) d: 108.63 (C1), 147.55 (C2), 147.48 (C3), 111.39 (C4),
126.55 (C4a), 28.86 (C5), 51.46 (C6), 53.77 (C8), 126.96 (C8a),
143.52 (C9), 146.54 (C10), 114.66 (C11), 124.59 (C12), 127.74
(C12a), 35.97 (C13), 60.37 (C14), 129.53 (C14a), 55.80 (2-OCH3),
Light yellow powders. ESI-MS m/z: 382.2 [M+H]+. 1H NMR
(CDCl3, 500 MHz) d: 1.34 (t, 3H, J = 7.5 Hz, –CH3), 2.66 (m, 4H, 6-
H, 13-H, –CH2–), 2.86 (m, 2H, 5-H), 3.15 (m, 2H, 5-H, 6-H), 3.26,
3.23 (dd, 1H, J = 16.0 Hz, 3.5 Hz, 13-H), 3.48 (d, 1H, J = 16.0 Hz, 8-
H), 3.61 (m, 1H, 14-H), 3.83 (s, 3H, –OCH3), 4.04 (d, 1H,
J = 16.0 Hz, 8-H), 5.94, 5.95 (d, d, 2H, J = 1.5 Hz, –OCH2O–), 6.61
(s, 1H, 4-H), 6.75 (s, 1H, 1-H), 6.86 (d, 1H, J = 8.0 Hz, 12-H), 7.03
(d, 1H, J = 8.5 Hz, 11-H). 13C NMR (CDCl3, 125 MHz) d: 105.59
(C1), 149.08 (C2), 146.19 (C3), 108.41 (C4), 127.66 (C4a), 29.59
(C5), 51.14 (C6), 53.45 (C8), 127.74 (C8a), 136.27 (C9), 146.0
(C10), 110.63 (C11), 126.31 (C12), 128.30 (C12a), 36.21 (C13),
59.34 (C14), 130.79 (C14a), 100.78 (–OCH2O–), 56.11 (10-OCH3),
172.05 (–CO–), 27.29 (–CH2–), 9.35 (–CH3).
56.05 (3-OCH3), 59.38 (9-OCH3). The optical rotations was ½a D20
ꢀ
ꢁ380 (c 0.1, CHCl3).
4.2.4. ( )-9,10-Dimethoxy-2,3-dihydroxy-5,6,13,14-tetrahydro-
8H-dibenzo[a,g]quinolizine (7)
Berberine hydrochloride (5 g, 0.013 mol) and phloroglucin (5 g,
0.04 mol) were mixed with 60% H2SO4 (40 mL) and stirred at 95 °C
in an oil bath for 20 min. The mixture was cooled, concentrated,
and resuspended in H2O/acetone (1:1), and then the intermediate
was purified by silica gel chromatography (CHCl3/MeOH, 15:1) to
4.2.7. ( )-5,6,13,14-Tetrahydro-9-hexanoyloxy-10-methoxy-2,3-
(methylenedioxy)-8H-dibenzo[a,g]quinolizine (5c)
Preparation and purification of 5c was carried out according to
the general procedure, starting from compound 5 and hexanoyl
chloride. Yield: 83%.
obtain 2.18 g (44%).
A mixture of the intermediate (2.18 g,
0.006 mol) and MeOH (45 mL) was refluxed for 20 min to dissolve,
then NaBH4 (1.13 g, 0.03 mol) was added in small portions with
stirring at room temperature. Two hours later, the resulting solids
were filtered, washed with MeOH and purified by silica gel chro-
matography (CHCl3/MeOH, 25:1) to obtain 7 1.05 g (53%).
White powders. ESI-MS m/z: 424.3[M+H]+. 1H NMR (CDCl3,
500 MHz) d: 0.94 (t, 3H, J = 7.0 Hz, 60-CH3), 1.42 (m, 4H, 50,40-
CH2CH2–), 1.79 (m, 2H, 30-CH2–), 2.60 (t, 2H, J = 7.5 Hz, 20-CH2–),
2.64 (m, 2H, 6-H, 13-H), 2.83 (m, 1H, 5-H), 3.10 (m, 2H, 5-H, 6-
H), 3.20, 3.23 (dd, 1H, J = 16.0 Hz, 3.5 Hz, 13-H), 3.45 (d, 1H,
J = 16.0 Hz, 8-H), 3.57 (m, 1H, 14-H), 3.79 (s, 3H, –OCH3), 4.0 (d,
1H, J = 16.0 Hz, 8-H), 5.90, 5.91 (d, d, 2H, J = 1.5 Hz, –OCH2O–),
6.58 (s, 1H, 4-H), 6.71 (s, 1H, 1-H), 6.82 (d, 1H, J = 8.5 Hz, 12-H),
6.99 (d, 1H, J = 8.5 Hz, 11-H),. 13C NMR (CDCl3, 125 MHz) d:
105.59 (C1), 149.08 (C2), 146.21 (C3), 108.42 (C4), 127.66 (C4a),
29.60 (C5), 51.14 (C6), 53.54 (C8), 127.74 (C8a), 136.29 (C9),
146.0 (C10), 110.63 (C11), 126.31 (C12), 128.30 (C12a), 36.22
(C13), 59.34 (C14), 130.80 (C14a), 100.79 (–OCH2O–), 56.06 (10-
OCH3), 171.37 (–CO–), 33.95 (20-CH2–), 31.32 (30-CH2–), 24.86
(40-CH2–), 22.36 (50-CH2–), 13.97 (60-CH3).
Pale yellow powders. ESI-MS m/z: 328.2 [M+H]+. 1H NMR
(DMSO-d6, 500 MHz) d: 2.79 (m, 1H, 6-H), 3.04 (m, 1H, 13-H),
3.23 (m, 1H, 5-H), 3.41 (m, 2H, 6-H, 5-H), 3.58 (m, 1H, 13-H), 3.77
(m, 1H, 8-H), 3.79 (s, 3H, –OCH3), 3.82 (s, 3H, –OCH3), 4.35 (m,
1H, 14-H), 4.59 (m, 1H, 8-H), 6.60 (s, 1H, 4-H), 6.79 (s, 1H, 1-H),
7.04 (d, J = 8.5 Hz, 1H, 12-H), 7.07 (d, J = 8.5 Hz, 1H, 11-H), 8.99 (s,
1H, –OH), 9.28 (s, 1H, –OH). 13C NMR (DMSO-d6, 125 MHz) d:
109.05 (C1), 144.39 (C2), 144.12 (C3), 109.36 (C4), 114.91 (C4a),
28.89 (C5), 51.06 (C6), 53.23 (C8), 123.65 (C8a), 139.72 (C9),
143.58 (C10), 111.29 (C11), 112.40 (C12), 124.56 (C12a), 35.84
(C13), 59.52 (C14), 127.57 (C14a), 58.56 (–OCH3), 55.71 (–OCH3).

68
H.-X. Ge et al. / Bioorg. Med. Chem. 21 (2013) 62–69
4.2.8. ( )-5,6,13,14-Tetrahydro-9-benzoyloxy-10-methoxy-2,3-
(methylenedioxy)-8H-dibenzo[a,g]quinolizine (5d)
Preparation of 5d was carried out according to the general pro-
cedure, starting from compound 5 and benzoyl chloride and puri-
fication by silica gel column chromatography. Yield: 76%.
4.2.11. ( )-5,6,13,14-Tetrahydro-9-[3-(3-methoxyl-4-
acetoxy)phenyl]acryloxy-10-methoxy-2,3-(methylenedioxy)-
8H-dibenzo[a,g]quinolizine (5g)
Preparation and purification of 5g was carried out according to
the general procedure, starting from compound 5 and acetylferu-
loyl chloride and purification by silica gel column chromatography.
Yield: 65%.
White powders. ESI-MS m/z: 430.2 [M+H]+. 1H NMR (CDCl3,
500 MHz) d: 2.58(m, 2H, 6-H, 13-H), 2.85(m, 1H, 5-H), 3.07(m,
2H, 6-H, 5-H), 3.27, 3.24 (dd, 1H, J = 16.0, 3.5 Hz, 13-H), 3.50 (d,
1H, J = 15.0 Hz, 8-H), 3.58 (m, 1H, 14-H), 3.79 (s, 3H, –OCH3),
4.07 (d, 1H, J = 15.0 Hz, 8-H), 5.91, 5.90 (d, d, 2H, J = 1.5 Hz, –
OCH2O–), 6.57(s, 1H, 4-H), 6.73 (s, 1H, 1-H), 6.87 (d, 1H,
J = 8.5 Hz, 12-H), 7.05 (d, 1H, J = 8.5 Hz, 11-H), 7.53 (m, 2H, 30-
ArH, 50-ArH), 7.65(m, 1H, 40-ArH), 8.24(m, 2H, 20-ArH, 60-ArH).
13C NMR (CDCl3, 125 MHz) d: 105.60 (C1), 149.29 (C2), 146.21
(C3), 108.43 (C4), 26.52 (C4a), 29.61 (C5), 51.19 (C6), 53.57 (C8),
127.77 (C8a), 136.32 (C9), 146.00 (C10), 110.77 (C11), 128.58
(C12), 127.81 (C12a), 36.32 (C13), 59.44 (C14), 130.37 (C14a),
100.79 (–OCH2O–), 56.17 (–OCH3), 164.19 (–CO–), 130.82 (Ar-C10),
133.51(Ar-C40), 129.36 (Ar-C60, Ar-C20), 128.59 (Ar-C50, Ar-C30).
White powders. ESI-MS m/z: 544.3 [M+H]+. 1H NMR (CDCl3,
500 MHz) d: 2.33 (s, 3H, –COCH3), 2.62 (m, 2H, 6-H, 13-H), 2.85
(m, 1H, 5-H), 3.11 (m, 2H, 5-H, 6-H), 3.24 (dd, 1H, J = 16.0 Hz,
3.5 Hz, 13-H), 3.48 (d, 1H, J = 16.0 Hz, 8-H), 3.58 (m, 1H, 14-H),
3.82 (s, 3H, –OCH3), 3.89 (s, 3H, –OCH3), 4.05 (d, 1H, J = 16.0 Hz,
8-H), 5.91, 5.92 (d,d, 2H, J = 1.5 Hz, –OCH2O–), 6.58 (s, 1H, 4-H),
6.65 (d, 1H, J = 16.0 Hz, 20-CH@), 6.73 (s, 1H, 1-H), 6.86 (d, 1H,
J = 8.5 Hz, 12-H), 7.03 (d, 1H, J = 8.5 Hz, 300-ArH), 7.09 (d, 1H,
J = 8.5 Hz, 11-H), 7.20 (d, 1H, J = 8.5 Hz, 200-ArH), 7.20 (s, 1H, 600-
ArH), 7.85 (d, 1H, J = 16.0 Hz, 30@CH-Ar). 13C NMR (CDCl3,
125 MHz) d: 105.59 (C1), 149.18 (C2), 146.21 (C3), 108.44 (C4),
127.76 (C4a), 29.61 (C5), 51.20 (C6), 53.57 (C8), 127.80 (C8a),
136.14 (C9), 146.01 (C10), 110.67 (C11), 126.53 (C12), 128.55
(C12a), 36.30 (C13), 59.42 (C14), 130.80 (C14a), 100.80
(–OCH2O–), 56.01 (–OCH3), 56.16 (–OCH3), 168.67 (–CO–), 164.36
(–CO–Me), 121.62 (–C@), 141.88 (@C-Ar), 133.22 (Ar-C100), 117.03
(Ar-C200), 123.39 (Ar-C300), 145.95 (Ar-C400), 151.57 (Ar-C500),
111.49 (Ar-C600), 20.65 (–CH3).
4.2.9. ( )-5,6,13,14-Tetrahydro-9-(2-acetoxy)benzoyloxy-10-
methoxy-2,3-(methylenedioxy)-8H-dibenzo[a,g]quinolizine
(5e)
Preparation of 5e was carried out according to the general pro-
cedure, starting from compound 5 and o-acetylsalicylryl chloride
and purification by silica gel column chromatography. Yield: 56%.
White powders. ESI-MS m/z: 488.2 [M+H]+. 1H NMR (CDCl3,
500 MHz) d: 2.29 (s, 3H, -OCOCH3), 2.63(m, 2H, 6-H, 13-H),
3.0(m, 3H, 5-CH2, 6-H), 3.29(m, 1H, 13-H), 3.52(m, 2H, 14-H, 8-
H), 3.79 (s, 3H, –OCH3), 4.03 (m, 1H, 8-H), 5.92(s, 2H, –OCH2O–),
6.58 (s, 1H, 4-H), 6.72 (s, 1H, 1-H), 6.88 (d, 1H, J = 8.5 Hz, 12-H),
7.05 (d, 1H, J = 8.5 Hz, 11-H), 7.18 (dd, 1H, J = 8.0 Hz, J = 1.5 Hz,
30-ArH), 7.40 (m, 1H, 40-ArH), 7.65 (m, 1H, 50-ArH), 8.27 (dd, 1H,
J = 8.0 Hz, J = 1.5 Hz, 60-ArH). 13C NMR (CDCl3, 125 MHz) d: 105.56
(C1), 149.17 (C2), 146.20 (C3), 108.44 (C4), 127.79 (C4a), 29.71
(C5), 51.17 (C6), 53.48 (C8), 127.86 (C8a), 136.06 (C9), 145.99
(C10), 110.78 (C11), 126.68 (C12), 128.68 (C12a), 36.33 (C13),
59.43 (C14), 130.79 (C14a), 100.79 (–OCH2O–), 56.15 (–OCH3),
132.40 (Ar-C10), 151.33 (Ar-C20), 122.59 (Ar-C30), 134.41 (Ar-C40),
124.05 (Ar-C50), 126.15(Ar-C60), 161.60 (–COCH3), 169.59 (–CO-
Ar), 21.07 (–CH3).
4.2.12. (ꢁ)-2,3,10-Trimethoxy-9-b-
D-glucosyl-5,6,13,14-
tetrahydro-8H-dibenzo[a,g]quinolizine (4a)23
Using 24-h-old stage II cultures of G. deliquescens NRRL1086, a
total of 500 mg of 4 was distributed evenly among fifty 150 mL
Erlenmeyer flasks. Substrate-containing cultures were incubated
for 120 h, then filtrated and extracted with equal amount of EtOAc.
The organic solvent layer was removed, evaporated to dryness. The
extract was subjected to silica gel column chromatography eluted
with chloroform/methanol (30:1, v/v) to afford the product 4a
311 mg (43%).
Yellow powders. HR-ESI-MS m/z: 504.2229 [M+H]+. 1H NMR
(DMSO-d6, 500 MHz) d: 2.45 (m, 1H, 6-H), 2.56 (m, 1H, 13-H),
2.61 (m, 1H, 5-H), 2.91 (m, 1H, 5-H), 3.07 (m, 1H, 6-H), 3.08 (m,
1H, 50-H), 3.20 (m, 1H, 40-H), 3.23 (m, 1H, 20-H), 3.24 (m, 1H, 30-
H), 3.34 (m, 1H, 13-H), 3.37 (d, 1H, J = 16.0 Hz, 8-H), 3.39 (m, 1H,
14-H), 3.46 (m, 1H, 60-H), 3.62 (m, 1H, 60-H), 3.73 (s, 3H, –OCH3),
3.74 (s, 3H, –OCH3), 3.76 (s, 3H, –OCH3), 4.33 (d, 1H, J = 16.0 Hz,
8-H), 4.35 (t, 1H, J = 5.5 Hz, 60-OH), 4.93 (d, 1H, J = 7.5 Hz, 10-H),
4.93 (s, 1H, 40-OH), 4.99 (s, 1H, 30-OH), 4.99 (s, 1H, 20-OH), 6.68
(s, 1H, 4-H), 6.86 (s, 1H, 1-H), 6.88 (d, 1H, J = 11.0 Hz, 12-H), 6.89
(d, 1H, J = 11.0 Hz, 11-H). 13C NMR (DMSO-d6, 125 MHz) d:
109.49 (C1), 149.06 (C2), 147.14 (C3), 111.74 (C4), 126.33 (C4a),
28.57 (C5), 50.51 (C6), 53.90 (C8), 127.97 (C8a), 141.48 (C9),
147.13 (C10), 111.93 (C11), 123.88 (C12), 129.29 (C12a), 35.57
(C13), 58.59 (C14), 129.79 (C14a), 102.92 (C10), 74.25 (C20), 76.48
(C30), 69.79 (C40),77.10 (C50), 60.82 (C60), 55.39 (10-OCH3), 55.71
(3-OCH3), 56.34 (2-OCH3).
4.2.10. ( )-5,6,13,14-Tetrahydro-9-(3-phenyl)acryloxy)-10-
methoxy-2,3-(methylenedioxy)-8H-dibenzo[a,g]quinolizine
(5f)
Preparation and purification of 5f was carried out according to
the general procedure, starting from compound 5 and cinnamoyl
chloride and purification by silica gel column chromatography.
Yield: 62%.
Light yellow powders. ESI-MS m/z: 456.2 [M+H]+. 1H NMR
(CDCl3, 500 MHz) d: 2.62 (m, 2H, 6-H, 13-H), 2.86 (m, 1H, 5-H),
3.10 (br, 2H, 5-H, 6-H), 3.24 (dd, 1H, J = 16.0 Hz, 3.0 Hz, 13-H),
3.49 (d, 1H, J = 15.0 Hz, 8-H), 3.58(br, 1H, 14-H), 3.81 (s, 3H, –
OCH3), 4.06 (d, 1H, J = 15.0 Hz, 8-H), 5.91 (s, 2H, –OCH2O–), 6.57
(s, 1H, 4-H), 6.70 (d, 1H, J = 16.0 Hz, –CH@), 6.72 (s, 1H, 1-H),
6.86 (d, 1H, J = 8.0 Hz, 12-H), 7.03 (d, 1H, J = 8.0 Hz, 11-H), 7.42
(m, 3H, 300-ArH, 400-ArH, 500-ArH,), 7.60 (m, 2H, 200-ArH, 600-ArH),
7.90(d, 1H, J = 16.0 Hz, @CH-Ar). 13C NMR (CDCl3, 125 MHz) d:
105.59 (C1), 149.21 (C2), 146.70 (C3), 108.43 (C4), 127.73 (C4a),
29.61 (C5), 51.18 (C6), 53.57 (C8), 127.80 (C8a), 136.16 (C9),
146.20 (C10), 110.67 (C11), 126.48 (C12), 128.55 (C12a), 36.29
(C13), 59.41 (C14), 130.81 (C14a), 100.79 (–OCH2O–), 56.16
(–OCH3), 164.50 (–CO–), 116.82 (–C@), 146.0 (@C-Ar), 134.30
(Ar-C100), 130.68 (Ar-C400), 129.0 (Ar-C300, Ar-C500), 128.36 (Ar-C200,
Ar-C600).
4.2.13. (+)-2,3,10-Trimethoxy-9-b-D-glucosyl-5,6,13,14-
tetrahydro-8H-dibenzo[a,g]quinolizine (4b)
Preparation and purification of 4b was carried out according to
the procedure of compound 4a, Yield: 16%.
Yellow powders. HR-ESI-MS m/z: 504.2231 [M+H]+. 1H NMR
(DMSO-d6, 500 MHz) d: 2.45 (m, 1H, 6-H), 2.56 (m, 1H, 13-H),
2.60 (m, 1H, 5-H), 2.91 (m, 1H, 5-H), 3.04 (m, 1H, 6-H), 3.06 (m,
1H, 50-H), 3.15 (m, 1H, 40-H), 3.20 (m, 1H, 20-H), 3.22 (m, 1H, 30-
H), 3.34 (m, 1H, 13-H), 3.42 (m, 1H, 14-H), 3.45 (m, 1H, 60-H),
3.51(d, 1H, J = 16.0 Hz, 8-H), 3.63 (m, 1H,60-H), 3.73 (s, 3H, –
OCH3), 3.74 (s, 3H, –OCH3), 3.76 (s, 3H, –OCH3), 4.13 (d, 1H,
J = 16.0 Hz, 8-H), 4.30 (s, 1H, 60-OH), 4.78 (d, J = 7.5 Hz, 1H, 10-H),

H.-X. Ge et al. / Bioorg. Med. Chem. 21 (2013) 62–69
69
4.92 (s, 1H, 40-OH), 4.98 (s, 1H, 30-OH), 4.98 (s, 1H, 20-OH), 6.68 (s,
1H, 4-H), 6.86 (s, 1H, 1-H), 6.88 (d, 1H, J = 9.0 Hz, 12-H), 6.89 (d, 1H,
J = 9.0 Hz, 11-H). 13C NMR (DMSO-d6, 125 MHz) d: 109.44 (C1),
148.88 (C2), 147.14 (C3), 111.72 (C4), 126.35 (C4a), 28.56 (C5),
50.66 (C6), 53.97 (C8), 127.86 (C8a), 141.65 (C9), 147.11 (C10),
111.74 (C11), 123.90 (C12), 129.71 (C12a), 35.54 (C13), 58.59
(C14), 129.85 (C14a), 103.47 (C10), 74.17 (C20), 76.50 (C30), 69.95
(C40), 77.05 (C50), 61.01 (C60), 55.38 (10-OCH3), 55.71 (3-OCH3),
56.28 (2-OCH3).
of Sciences), and cultured in RPMI-1640 medium with 10% heat-
inactivated fetal calf serum at 37 °C in 5% CO2.
4.3.2. Induction of TF and drug treatment
According to our previous reports,7,25 THP-1 cells (1 ꢃ 106 cells/
mL) were initially incubated with control vehicle (DMSO, 0.1% V/V)
or various compounds at indicated concentrations (0.01–1 lM) for
1 h in a 96-well plate, and then stimulated with 500 ng/mL LPS for
5 h to induce TF activity. At the end of incubation, cells were sed-
imented by centrifugation and resuspended in RPMI-1640. The cell
suspension was frozen at ꢁ20 °C until TF activity measurement.
The cell lysates were frozen and thawed three times before they
4.2.14. (ꢁ)-5,6,13,14-Tetrahydro-9-b-
D-glucosyl-10-methoxy-
2,3-(methylenedioxy)-8H-dibenzo[a,g]quinolizine (5h)
Using 24-h-old stage II cultures of G. deliquescens NRRL1086, a
total of 500 mg of 5 was distributed evenly among fifty 150 mL
Erlenmeyer flasks. Substrate-containing cultures were incubated
for 120 h, then filtrated and extracted with equal amount of EtOAc.
The organic solvent layer was removed, evaporated to dryness. The
extract was subjected to silica gel column chromatography eluted
with chloroform/methanol (30:1, v/v) to afford the product 5h
323 mg (44%).
were used in the assay. Cell lysates (45
a reagent mixture (5 L, pH 7.3) containing 10 g/L prothrombin
complex and 100 mM CaCl2 in a 96-well plate at 37 °C for
15 min. Then, 50 L of factor Xa chromogenic substrate (0.5 mM)
containing 100 mM EDTA (pH 8.4) was added, and the absorbance
was measured at 405 nM with a microplate reader (Sunrise, TECAN
Austria GmbH, Grodig, Austria). The IC50 of each compound on TF
procoagulatant activity was calculated.
lL) were incubated with
l
l
White powders. HR-ESI-MS m/z: 488.1915 [M+H]+. 1H NMR
(DMSO-d6, 500 MHz) d: 2.46 (m, 1H, 6-H), 2.54 (m, 1H, 13-H),
2.60 (m, 1H, 5-H), 2.89 (m, 1H,5-H), 3.06 (m, 1H, 6-H), 3.07 (m,
1H, 50-H), 3.18 (m, 1H, 40-H), 3.22 (m, 1H, 20-H), 3.23 (m, 1H, 30-
H), 3.26 (m, 1H, 13-H), 3.36 (m, 1H, 14-H), 3.37 (d, 1H,
J = 16.0 Hz, 8-H), 3.45 (m, 1H, 60-H), 3.62 (m, 1H, 60-H), 3.76 (s,
3H, –OCH3), 4.32 (d, 1H, J = 16.0 Hz, 8-H), 4.33 (t, 1H, J = 5.5 Hz,
60-OH), 4.92 (d, 1H, J = 5.0 Hz, 40-OH), 4.92 (d, 1H, J = 7.5 Hz, 10-
H), 4.95 (d, 1H, J = 4.0 Hz, 30-OH), 4.99 (d, 1H, J = 4.0 Hz, 20-OH),
5.94, 5.93 (d, d, J = 1.0 Hz, 2H, –OCH2O–), 6.66 (s, 1H, 4-H), 6.85
(d, 1H, J = 8.0 Hz, 12-H), 6.89 (s, 1H, 1-H), 6.89 (d, 1H, J = 8.0 Hz,
11-H). 13C NMR (DMSO-d6, 125 MHz) d: 105.66 (C1), 145.35 (C2),
145.62 (C3), 108.00 (C4), 127.42 (C4a), 29.00 (C5), 50.29 (C6),
53.79 (C8), 127.83 (C8a), 141.49 (C9), 149.10 (C10), 111.96 (C11),
123.94 (C12), 129.19 (C12a), 35.61 (C13), 58.82 (C14), 130.97
(C14a), 102.95 (C10), 74.23 (C20), 76.48 (C30), 69.80 (C40), 77.07
(C50), 60.84 (C60), 100.44 (–OCH2O–), 56.36 (10-OCH3).
Acknowledgments
This work was supported by 2011’ Program for Excellent Scien-
tific and Technological Innovation Team of Jiangsu Higher Educa-
tion. Thanks also give to the financial support from the State
Administration of Foreign Expert Affairs of China (No. 111-2-07),
the ‘111 Project’ from the Ministry of Education of China, and the
Fundamental Research Funds for the Central Universities
(JKZ2009010, JKZ2011017).
References and notes
1. Banner, D. W.; D’Arcy, A.; Chene, C.; Winkler, F. K.; Guha, A.; Konigsberg, W. H.
Nature 1996, 380, 41.
2. Semeraro, N.; Colucci, M. Thromb. Haemost. 1997, 78, 759.
3. Steffel, J.; Lüscher, T. F.; Tanner, F. C. Circulation 2006, 113, 722.
4. Chu, J. Arch. Biochem. Biophys. 2005, 440, 123.
5. Mattias, B.; Jasimuddin, A.; Wolfram, R. Arterioscler. Thromb. Vasc. Biol. 2005, 25,
1545.
6. Suleymanov, O. D.; Szalony, J. A.; Salyers, A. K.; LaChance, R. M.; Parlow, J. J.;
South, M. S.; Wood, R. S.; Nicholson, N. S. J. Pharmacol. Exp. Ther. 2003, 306,
1115.
4.2.15. (+)-5,6,13,14-Tetrahydro-9-b-D-glucosyl-10-methoxy-2,3-
(methylenedi oxy)-8H-dibenzo[a,g]quinolizine (5i)
Preparation and purification of 5i was carried out according to
the procedure of compound 5h. Yield: 5%.
7. Jiang, W. W.; Kou, J. P.; Yuan, S. T.; Sun, L.; Yu, B. Y. J. Biomol. Screen. 2011, 16,
295.
White powders. HR-ESI-MS m/z: 488.1917 [M+H]+. 1H NMR
(DMSO-d6, 500 MHz) d: 2.46 (m, 1H, 6-H), 2.53 (m, 1H, 13-H),
2.59 (m, 1H, 5-H), 2.89 (m, 1H, 5-H), 3.05 (m, 1H, 6-H), 3.06 (m,
1H, 50-H), 3.14 (m, 1H, 40-H), 3.20 (m, 1H, 20-H), 3.21 (m, 1H, 30-
H), 3.28 (m, 1H, 13-H), 3.40 (m, 1H, 14-H), 3.44 (m, 1H, 60-H),
3.50 (d, 1H, J = 16.0 Hz, 8-H), 3.62 (m, 1H, 60-H), 3.76 (s, 3H, –
OCH3), 4.12 (d, 1H, J = 16.0 Hz, 8-H), 4.29 (t, 1H, J = 5.5 Hz, 60-
OH), 4.78 (d, 1H, J = 7.5 Hz, 10-H), 4.87 (d, 1H, J = 5.0 Hz, 40-OH),
4.94 (d, 1H, J = 4.5 Hz, 30-OH), 4.96 (d, 1H, J = 4.0 Hz, 20-OH), 5.95,
5.94 (d, d, 2H, J = 1.0 Hz, –OCH2O–), 6.66 (s, 1H, 4-H), 6.85 (d, 1H,
J = 8.5 Hz, 12-H), 6.89 (s, 1H, 1-H), 6.89 (d, 1H, J = 8.5 Hz, 11-H).
13C NMR (DMSO-d6, 125 MHz) d: 105.59 (C1), 145.31 (C2), 145.60
(C3), 107.97 (C4), 127.42 (C4a), 28.98 (C5), 50.45 (C6), 53.86 (C8),
127.74 (C8a), 141.65 (C9), 148.89 (C10), 111.76 (C11), 123.94
(C12), 129.59 (C12a), 35.60 (C13), 58.84 (C14), 131.01 (C14a),
103.49 (C10), 74.16 (C20), 76.50 (C30), 69.94 (C40), 77.06 (C50),
60.98 (C60), 100.43 (–OCH2O–), 56.30 (10-OCH3).
8. Steiner, S.; Speidl, W. S.; Pleiner, J.; Seidinger, D.; Zom, G.; Kaun, C.; Wojta, J.;
Huber, K.; Minar, E.; Wolzt, M. Circulation 2005, 111, 1841.
9. Eto, M.; Kozai, T.; Cosentino, F.; Joch, H.; Luscher, T. F. Circulation 2002, 105,
1756.
10. Pendurthi, U. R.; Williams, J. T.; Rao, L. V. Arterioscler. Thromb. Vasc. Biol. 1997,
17, 3406.
11. Emidio, V. L. D.; Ivana, M. M. F.; Diego, N. G. The Alkaloids: Chemistry and
Biology; Academic Press: New York, 2005. p 62:1.
12. Yu, B. Y.; Jiang, W. W.; Kou, J. P.; Zhang, J.; Lu, D. C. CN Patent 10,022,092,
2008.
13. Li, Y. H.; Yang, P.; Kong, W. J.; Wang, Y. X.; Hu, C. Q.; Zuo, Z. Y.; Wang, Y. M.;
Gao, H.; Gao, L. M.; Feng, Y. C.; Du, N. N.; Liu, Y.; Song, D. Q.; Jiang, J. D. J. Med.
Chem. 2009, 52, 492.
14. Iwasa, K.; Kamigauchi, M.; Ueki, M.; Taniguchi, M. Eur. J. Med. Chem. 1996, 31,
469.
15. Feng, Z.; Norio, N.; Teruaki, A.; Masao, H. Drug Metab. Dispos. 2006, 34, 2064.
16. Zhang, H. F.; Xue, L. L.; Tong, J. Y.; Zhang, C. YaoXueJinZhan 2010, 34, 459.
17. Iwasa, K.; Cui, W.; Takahashi, T.; Nishiyama, Y.; Kamigauchi, M.; Koyama, J.;
Takeuchi, A.; Moriyasu, M.; Takeda, K. J. Nat. Prod. 2010, 73.
18. Mo, J.; Guo, Y.; Yang, Y. S.; Shen, J. S.; Jin, G. Z.; Zhen, X. C. Curr. Med. Chem.
2007, 14, 2996.
19. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461.
20. Weymouth-Wilson, A. C. Nat. Prod. Rep. 1997, 14, 99.
21. Ge, H. X.; Zhang, J.; Cui, K.; Liu, J. H.; Yu, B. Y. Appl. Microbiol. Biotechnol. 2012,
93, 2357.
4.3. The measurement of TF procoagulant activity
22. Chen, N. D.; Zhang, J.; Cui, K.; Liu, J. H.; Yu, B. Y. Appl. Microbiol. Biotechnol. 2010,
86, 491.
23. Martinkova, V. K. L. Curr. Med. Chem. 2001, 8, 1303.
24. Betts, R. E.; Walters, D. E.; Rosazza, J. P. N. J. Med. Chem. 1974, 17, 599.
25. Jiang, W. W.; Kou, J. P.; Zhang, Z.; Yu, B. Y. Thromb. Res. 2009, 124, 714.
4.3.1. Cell culture
The human monocytic cell line (THP-1) cells were obtained
from the cell bank of type culture collection of Chinese Academy
of Sciences (Shanghai Institute of Cell Biology, Chinese Academy