New synthetic method of 8-oxocoptisine starting from natural quaternary coptisine as anti-ulcerative colitis agent

Article abstract of DOI:10.1080/10286020.2014.932778

Quaternary coptisine (1), a natural bioactive quaternary protoberberine alkaloid (QPA), was treated with potassium ferricyanide in aqueous solution of 5 N sodium hydroxide leading to the acquisition of 8-oxocoptisine (2) with much higher yield than reported in the literature. This is the first report of the oxidation of a natural QPA by applying potassium ferricyanide as an oxidant. 8-Oxocoptisine showed significant anti-ulcerative colitis efficacy in vitro with EC₅₀ value being 8.12 × 10^-8 M.

Full text of DOI:10.1080/10286020.2014.932778

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New synthetic method of 8-
oxocoptisine starting from natural
quaternary coptisine as anti-ulcerative
colitis agent
Zhi-Hui Zhanga, Lian-Qiu Wub, An-Jun Denga, Jin-Qian Yua, Zhi-
Hong Lia, Hai-Jing Zhangb, Wen-Jie Wangb & Hai-Lin Qina
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences and Peking Union Medical College, Beijing
100050, China
b Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, China
Published online: 16 Jul 2014.
To cite this article: Zhi-Hui Zhang, Lian-Qiu Wu, An-Jun Deng, Jin-Qian Yu, Zhi-Hong Li, Hai-Jing
Zhang, Wen-Jie Wang & Hai-Lin Qin (2014) New synthetic method of 8-oxocoptisine starting from
natural quaternary coptisine as anti-ulcerative colitis agent, Journal of Asian Natural Products
Research, 16:8, 841-846, DOI: 10.1080/10286020.2014.932778
To link to this article: http://dx.doi.org/10.1080/10286020.2014.932778
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 8, 841–846, http://dx.doi.org/10.1080/10286020.2014.932778
New synthetic method of 8-oxocoptisine starting from natural
quaternary coptisine as anti-ulcerative colitis agent
Zhi-Hui Zhanga, Lian-Qiu Wub, An-Jun Denga, Jin-Qian Yua, Zhi-Hong Lia,
Hai-Jing Zhangb, Wen-Jie Wangb and Hai-Lin Qina*
aState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, China; bInstitute of Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing 100050, China
(Received 26 March 2014; final version received 5 June 2014)
Quaternary coptisine (1), a natural bioactive quaternary protoberberine alkaloid (QPA),
was treated with potassium ferricyanide in aqueous solution of 5 N sodium hydroxide
leading to the acquisition of 8-oxocoptisine (2) with much higher yield than reported in
the literature. This is the first report of the oxidation of a natural QPA by applying
potassium ferricyanide as an oxidant. 8-Oxocoptisine showed significant anti-
ulcerative colitis efficacy in vitro with EC50 value being 8.12 £ 1028 M.
Keywords: 8-oxocoptisine; quaternary coptisine; potassium ferricyanide; anti-
ulcerative colitis
1. Introduction
part, closely related to the abnormality of
key downstream transcription factor X-
box-binding protein 1 (xbp1) that associ-
ates with noncontrollable endoplasmic
reticulum stress response within intestinal
epithelial cells [3,4]. In other words, the
variation of the coding region and the
expressive abnormalities of xbp1 are often
characteristic of UC patients. Therefore, it
is speculated that xbp1 may be the
potential new drug targets of treating UC.
However, there has been no claim as yet
about the development of anti-UC drugs
targeting at xbp1 during recent research at
home and abroad in the literature. This
paper involves an investigation on anti-UC
activity of 8-oxocoptisine (2) based on
xbp1 pharmacological model in vitro,
which was obtained for the first time in
considerable yield from natural quaternary
coptisine (1) as the starting material by
treatment with potassium ferricyanide as
an oxidant and exhibited significantly
Ulcerative colitis (UC) is a kind of chronic
nonspecific inflammatory bowel disease
and has been identified as a nasty disease
by World Health Organization [1]. It has
been reported that the clinical incidence of
UC is 1.2–20.2 cases in 100,000 people
each year in China, with much higher rate
occurred in North Europe and North
America, and the etiology of UC has not
been clear [2]. Up to now, only several
drugs are used to treat UC clinically, such
as preparations from aminosalicylic acid,
glucocorticoids, and immunosuppressive
agents [1]. Realistically, many problems
abound nowadays, such as limited effi-
cacy, serious side effect, and relapse after
treatment, among others, leading to no
complete cure to UC. Therefore, signifi-
cantly effective anti-UC drugs are urgently
needed. Recent investigations have shown
that the pathogenesis of UC is, in large
*Corresponding author. Email: qinhailin@imm.ac.cn
q 2014 Taylor & Francis

842
Z.-H. Zhang et al.
O
O
O
O
O
Cl
N
N
O
N
O
a
+
O
O
O
O
O
O
1
2
3
Scheme 1. Synthesis of 8-oxocoptisine. Reagents and conditions: (a) K3Fe(CN)6, 5 N NaOH in
water, reflux.
´
being observed for six steps [19]. Dostal
potent efficacy with EC50 value reaching
the level of 1028 M.
et al. have converted quaternary coptisine
to compound 2 during their NMR inves-
tigation on related compounds with
DMSO-d6 as a solvent, but the conversion
needed a very long duration of reaction
and underwent the preparation of 8-
hydroxy-7,8-dihydrocoptisine as an inter-
mediate product [20]. Recently, Cui et al.
have reported the synthesis of 2 by
reacting 1 with 50% KOH solution, and
also with poor yield being obtained [21].
Moreover, this method resulted in a
significant portion of dihydrocoptisine (3)
when repeated by us, which is very
difficult to separate from 2. Based on the
reality mentioned above, this paper
focused on searching for efficient synthetic
route of 2 (Scheme 1) starting from natural
quaternary coptisine as complementary
contents for the above-mentioned anti-UC
agent.
Quaternary coptisine (1) is a natural
quaternary protoberberine alkaloid (QPA)
isolated previously from some Coptis and
Corydalis species belonging to the plant
families of Ranunculaceae and Papaver-
aceae [5,6], respectively. It has been
demonstrated that quaternary coptisine
exhibits some kinds of biological and
pharmacological activities, including the
anti-fungal and cardioprotective proper-
ties, inhibition of monoamine oxidase of
type A, and so on [7–12]. 8-Oxocoptisine
(2) was initially isolated from Fumaria
indica as a natural protoberberine alkaloid
with amide moiety being one of its
structural features [13,14]. Wu et al. have
reported that the multidrug resistance
(MDR) inhibition activity of 2 is similar
to verapamil, which is an active inhibitor
of the p-glycol protein-induced MDR
[15,16]. However, too low natural yield
(0.004%) makes the obtaining of 2 from
natural resources very arduous [5]. There-
fore, ever more increasing interest has
been focused on developing convenient
synthetic route to get the artificial 8-
oxocoptisine and its derivatives. The
preparation of 2 through total synthesis
has been reported [17]. However, the
approach was uneconomical and tedious
due to the reality of multistep and low
yield (only 3% for six steps). A compli-
cated synthetic method has also been
reported [18]. Lamentedly, the raw
materials were noncommercially avail-
able. Reimann et al. have used isoquino-
line derivative as the starting material to
synthesize 2 with a total yield of only 10%
2. Results and discussion
It has been confirmed that quaternary
coptisine (1) yields one oxidative product
(8-oxocoptisine (2)) and one reductive
product (dihydrocoptisine (3)) as a result
of reaction with unknown mechanism
when reacted only with 50% KOH
solution. An idea was thus proposed
based on the common sense of organic
chemistry that whether an oxidant can be
added to make the reactant predominantly
or completely transformed to the oxidative
product (2). In the process of searching for
such an oxidizing agent, we found that
potassium ferricyanide is very suitable.
Potassium ferricyanide has ideal oxidizing
properties under alkaline conditions.

Journal of Asian Natural Products Research
843
Table 1. Reaction conditions and results.
Potassium
along with 2 at different ratios. The
structures of the products were settled
based on their physical and spectroscopic
(1H and 13C NMR, ESI-MS, and IR)
properties, although the detailed reaction
mechanism was not entirely clear.
ferricyanide:1
(mol/mol)
Total yield
(%)
Entry
2:3
1
2
3
4
0:1
2:1
4:1
5:1
1:1
5:1
20:1
1:0
52.3
68.5
72.1
73.0
8-Oxocoptisine (2) was evaluated for
its selective activation effect on xbp1
in vitro with a blank group (nontransfec-
tion) as control 1 and a group transfected
with pGL3-pxbp1 plasmid (promoter
2330 to þ60) as control 2, and the result
is shown in Figure 1. No positive control
was used in this paper because 8-
oxocoptisine (2) was the first reported
xbp1 activator. The result indicated that 2
had certain transcription-activating effect
on xbp1. Further experiment confirmed
that 2 exhibited a significant anti-UC
efficacy in vitro with its EC50 value being
8.12 £ 1028 M (Figure 2).
In our work, the treatment of 1 with
potassium ferricyanide has been con-
ducted with several ratios of reactants
being investigated under alkaline con-
dition and the results are depicted in
Table 1. Potassium ferricyanide was
dissolved in the solution of 5 N NaOH in
water and 1 was added to give a
suspension. The reaction of the mixture
was carried out under reflux conditions
leading to products 2 and 3 being obtained
(Scheme 1) in most cases. It was
substantiated that the relative amount of
potassium ferricyanide had significant
effect on the resultant proportion of 2
and 3, as well as the total yield. As shown
in Table 1, a ratio of 5:1 of potassium
ferricyanide/1 (mol/mol) was found to be
optimal for the preparation of 2, with 1
completely being transformed to 2 under
this condition. When 1 was more than one-
fifth of potassium ferricyanide by mole in
amount added to the reaction system,
compound 3 would always be obtained
To sum up, due to the rich natural
resources of quaternary coptisine (1), the
method described here is convenient and
economic in practice for the preparation of
2, with considerable yield being obtained.
Moreover, this method can be expected to
expand for oxidation of other QPAs, such
as quaternary berberine. However, our
preliminary investigation showed that the
yield of oxidative product is not as good as
oxidizing quaternary coptisine in the case
of quaternary berberine under the same
condition (data not shown), and searching
160
140
120
100
80
60
40
20
0
con1
con2
50nM
100nM
500nM
1uM
5uM
concentration of 8-oxocoptisine 2
Figure 1. Activity of 2 as xbp1 activator.

844
Z.-H. Zhang et al.
120
100
80
y = 11.911Ln(x) + 244.47
R2 = 0.956
60
40
20
0
1.00E-08
1.00E-07
1.00E-06
1.00E-05
Figure 2. Experiment of EC50 value of 2 (8.12 £ 1028 mol/l).
for optimal procedure is in progress in our
laboratory. Based on the above-mentioned
advantages of this procedure, exploitation
of 2 can be considered for its known, or
possibly unknown, biological properties.
chemical and spectroscopic evidences. All
reagents and solvents were reagent grade.
3.2 Procedure for the synthesis of
compound 2
Potassium ferricyanide (2.2 g, 6.68 mmol)
was dissolved in the solution of 5 N NaOH
in water (15 ml). To the suspension was
added slowly 500 mg of 1 (1.41 mmol).
When the addition was completed, the
mixture was refluxed for 10 h and then
allowed to cool down to room temperature.
The precipitate was filtered and washed
with water and dried in high vacuum to
give pure 8-oxocoptisine (2) (344 mg,
73.0%). 8-Oxocoptisine (2): 1H NMR
(300 MHz, DMSO-d6) d: 2.86 (t,
J ¼ 5.4 Hz, 2H, NCH2CH2), 4.09 (t,
J ¼ 5.4 Hz, 2H, NCH2CH2), 6.07 (s, 2H,
OCH2O), 6.19 (s, 2H, OCH2O), 6.92 (s,
1H, ArH), 7.11 (s, 1H, ArH), 7.15 (d,
J ¼ 8.1 Hz, 1H, ArH), 7.34 (d, J ¼ 8.1 Hz,
1H, ArH), 7.47 (s, 1H, ZCHv); 13C NMR
(100 MHz, DMSO-d6) d: 158.2, 148.0,
147.0, 146.1, 145.7, 134.9, 131.6, 129.9,
123.2, 119.4, 114.0, 109.8, 107.9, 104.8,
102.1, 101.7, 101.4, 38.9, 27.7; IR (cm21):
3. Experimental
3.1 General experimental procedures
IR spectrum was obtained on a Nicolet
5700 spectrometer (Thermo Electron,
Madison, WI, USA). 1H and 13C NMR
spectra were recorded on either a Varian
Mercury-300 NMR spectrometer or a
Varian Mercury-400 NMR spectrometer
(Varian, Inc., Palo Alto, CA, USA),
respectively, and reported with TMS as
the internal standard and DMSO-d6 and
pyridine-d5, respectively, as solvents.
Chemical shifts (d values) and coupling
constants (J values) are given in ppm and
Hz, respectively. ESI-MS was obtained
using an Agilent 1100 series LC/MSD
Trap SL mass spectrometer (Santa Clara,
CA, USA). TLC analyses were carried out
on glass plates with silica gel GF254 being
precoated (Qingdao Marine Chemical,
Inc., Qingdao, China). Starting material
was isolated and purified by our group
from natural resources of Coptis chinensis
and other plants with the purity of
quaternary coptisine being determined to
be over 99% by HPLC procedure (data not
shown). The structure of quaternary
coptisine was confirmed on the basis of
y
max: 3047, 2904, 2787, 1655, 1596, 1483,
1389, 1270, 1228, 1101, 1044, 941 [21].
On the case of other ratio of reactants
as shown in Table 1, both 8-oxocoptisine
(2) and dihydrocoptisine (3) were obtained
as a mixture after the same procedure as
described above. It is very difficult to
separate 3 from 2. The following spectro-

Journal of Asian Natural Products Research
845
scopic data of dihydrocoptisine (3) were
recorded with pure compound prepared by
reduction of starting material (quaternary
coptisine 1) with sodium borohydride
(NaBH4) and potassium carbonate
(K2CO3), which were used to determine
the existence of dihydrocoptisine (3) in the
resultant of the reaction. Dihydrocoptisine
(3): 1H NMR (300 MHz, DMSO-d6) d:
2.78 (t, J ¼ 5.7 Hz, 2H, NCH2CH2), 3.04
(t, J ¼ 5.7 Hz, 2H, NCH2CH2), 4.15 (s,
2H, NCH2), 5.96 (s, 2H, OCH2O), 5.99 (s,
2H, OCH2O), 6.08 (s, 1H, ArH), 6.46 (d,
J ¼ 7.8 Hz, 1H, ArH), 6.68 (d, J ¼ 7.8 Hz,
1H, ArH), 6.74 (s, 1H, ArH), 7.28 (s, 1H,
ArH). Considering the unsteadiness of
dihydroprotoberberine alkaloid in DMSO-
d6, the 1H and 13C NMR spectra of 3 were
further recorded with pyridine-d5 as
detected using dual luciferase report gene
detection reagent kit (Promega, Madison,
WI, USA) on experimental sample, and
the EC50 value was determined.
Acknowledgments
This work was financially supported by grants
from National Science and Technology Project
of China (No. 2011ZX09307-002-01) and
National Natural Science Foundation of China
(No. 81373269; 81202546).
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