Synthesis and biological evaluation of berberine analogues as novel up-regulators for both low-density-lipoprotein receptor and insulin receptor

Article abstract of DOI:10.1016/j.bmcl.2009.09.059

Berberine (BBR) is a natural compound with up-regulating activity on both low-density-lipoprotein receptor (LDLR) and insulin receptor (InsR). This one-drug-multiple-target characteristic might be suitable for the treatment of metabolic syndrome. In searching for up-regulators effective for both LDLR and InsR expression, the structure-activity relationship (SAR) analysis for BBR analogues was done. Fourteen BBR analogues were designed, synthesized and biologically evaluated. SAR analysis revealed that appropriate modifications on the phenyl ring A or D of BBR might retain the up-regulatory activities on the expression of both LDLR and InsR. Among these compounds, compound 13a bearing 9-methoxy and 10-hydroxyl on the ring D showed promising activities on either LDLR or InsR gene expression. The 10-hydroxyl of 13a could be an arm to connect proper chemical groups for optimizing drug-bioavailability in vivo. Thus, 13a could be considered to be a parent compound to make pro-drugs for either blood lipids or glucose.

Full text of DOI:10.1016/j.bmcl.2009.09.059

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6004–6008
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of berberine analogues as novel
up-regulators for both low-density-lipoprotein receptor and insulin receptor
Yan-Xiang Wang , Yu-Ping Wang , Hao Zhang, Wei-Jia Kong, Ying-Hong Li, Fei Liu, Rong-Mei Gao, Ting Liu,
*
*
Jian-Dong Jiang , Dan-Qing Song
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2009
Revised 13 September 2009
Accepted 16 September 2009
Available online 19 September 2009
Berberine (BBR) is a natural compound with up-regulating activity on both low-density-lipoprotein
receptor (LDLR) and insulin receptor (InsR). This one-drug-multiple-target characteristic might be suit-
able for the treatment of metabolic syndrome. In searching for up-regulators effective for both LDLR
and InsR expression, the structure–activity relationship (SAR) analysis for BBR analogues was done. Four-
teen BBR analogues were designed, synthesized and biologically evaluated. SAR analysis revealed that
appropriate modifications on the phenyl ring A or D of BBR might retain the up-regulatory activities
on the expression of both LDLR and InsR. Among these compounds, compound 13a bearing 9-methoxy
and 10-hydroxyl on the ring D showed promising activities on either LDLR or InsR gene expression.
The 10-hydroxyl of 13a could be an arm to connect proper chemical groups for optimizing drug-bioavail-
ability in vivo. Thus, 13a could be considered to be a parent compound to make pro-drugs for either blood
lipids or glucose.
Keywords:
Berberine
Low-density-lipoprotein receptor
Insulin receptor
Structure–activity relationship
Metabolic syndrome
Ó 2009 Elsevier Ltd. All rights reserved.
Berberine (BBR, 1, Fig. 1) is an isoquinoline natural product ex-
tracted from Coptis chinensis, and has been extensively used as a
nonprescription drug to treat diarrhea in China for decades with
a confirmed safety. Because of its poor solubility and bioavailabil-
ity in oral administration, most of the BBR remains in intestine.1,2
Our previous work has identified BBR as a new drug for hyperlip-
idemia causing reduction of cholesterol and triglyceride in hyper-
lipidemic patients.3,4 The results were confirmed by other
groups.5–7 BBR increases low-density-lipoprotein receptor (LDLR)
expression by stabilization of LDLR mRNA through activation of
extracellular signal-regulated kinase (ERK) pathway.4,8,9 In addi-
tion, we have recently discovered insulin receptor (InsR) to be an-
other key target of BBR with significant benefit of antagonizing
insulin resistance in type 2 diabetes.10 It activated the promoter
of InsR gene through a protein kinase C (PKC) pathway, increased
the expression of InsR in liver and muscle cells, and therefore en-
hanced glucose consumption.10 Clinical studies have shown a sig-
nificant hypoglycemic effect of BBR in patients with type 2
diabetes.11,12 Our findings suggest that BBR is an one-drug-multi-
ple-target compound with great potential for the treatment of
metabolism syndrome, which represents a group of metabolic dis-
orders including insulin resistance, obesity, dyslipidaemia, hyper-
tension and low level of high-density-lipoprotein (HDL). There
currently are no approved medicines for this complicated
disease.13
The structure–activity relationship (SAR) analysis of BBR for the
up-regulation of LDLR mRNA expression was elucidated in our lab-
oratory.14,15 Among these analogues, pseudoberberine (2) bearing
10,11-dimethoxy and compound 3 with 10-hydroxy on the ring
D are the representative compounds (Fig. 1), and exhibited potent
activity on LDLR mRNA expression (Table 1).14 As the lipid and su-
gar metabolism are dynamically associated, we then turned our
attention of chemical modifications to agents effective in lowering
both blood lipid and glucose. Therefore, SAR studies for the up-reg-
ulatory activities on both LDLR and InsR mRNA expression were
conducted by concentrating the modifications on the phenyl ring
A mainly through de novo synthesis.
In the present study, we retained the active fragment of the ring
D, that is, 9,10-dimethoxy of 1, or 10,11-dimethoxy of 2, or 10-hy-
droxy of 3, respectively (Fig. 1), and replaced the methylenedioxy
moiety at the 2,3-position of the ring A with various side chain
substituents including hydrogen, methoxy, hydroxyl and benzyl-
oxy groups. On the basis of this strategy, twelve new BBR ana-
logues (13b–m) were designed and de novo synthesized. The
non-active trimethoxy substitution was also employed in the ring
D, from which compound 13n was created. In addition, compound
13a (thalifendine) with 9-methoxy and 10-hydroxyl on the ring D
was also synthesized according to the pervious methods.14,15
* Corresponding authors. Tel.: +86 10 6318 8423; fax: +86 10 6301 7302 (J.D.J.);
tel./fax: +86 10 6316 5268 (D.Q.S.).
E-mail addresses: jiang.jdong@163.com (J.-D. Jiang), songdanqingsdq@hotmail.
com (D.-Q. Song).
These authors made equal contributions to the work.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.059

Y.-X. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6004–6008
6005
O
O
O
-
-
O
N+
Cl
-
Cl
A
B
N+
C
Cl
O
N+
O
OCH3
D
OCH3
OCH3
OH
OCH3
1
3
2
Figure 1. Structures of the compounds 1–3.
Table 1
Structures and up-regulation of LDLR and InsR expression by BBR analogues
R3
3
Cl-
N+
R2
2
R1
R4
R5
9
R7
12
11
R6
Compound
R1
R2
R3
R4
R5
R6
R7
LDLR
InsR
mRNAa
mRNAa
1
2
3
H
H
H
H
H
H
H
OMe
H
H
OMe
H
H
H
H
H
H
OCH2O
OCH2O
OCH2O
OCH2O
OMe
H
H
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
OMe
OMe
OH
H
OMe
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.9 0.1
3.1 0.8
1.9 0.2
1.6 0.2
0.8 0.1
1.3 0.1
1.5 0.2
1.1 0.1
0.9 0.1
1.0 0.1
1.1 0.1
1.1 0.1
1.1 0.1
1.0 0.1
1.0 0.1
1.4 0.2
1.0 0.1
2.3 0.4
1.2 0.1
1.4 0.1
1.7 0.2
1.2 0.1
1.3 0.1
1.7 0.1
1.2 0.1
0.7 0.1
0.8 0.1
0.8 0.1
0.8 0.1
1.1 0.1
0.6 0.1
0.9 0.1
1.2 0.1
1.0 0.1
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
OH
H
H
OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OH
OCH2Ph
OMe
OMe
OH
OCH2Ph
OMe
OMe
OMe
OH
OCH2Ph
OMe
H
H
OMe
OH
OMe
OMe
OMe
OMe
OMe
H
H
H
H
OMe
OH
OH
OH
OMe
H
OMe
OMe
a
Human liver HepG2 cells were cultured in the MEM medium, and incubated with BBR or its analogues (10 lg/mL) for 8 h. Up-regulation of LDLR or InsR mRNA expression
was determined with real-time RT-PCR method. The level of LDLR or InsR mRNA in the untreated cells was defined as 1, and the levels of LDLR or InsR mRNA from the cells
treated with study analogues were defined as a fold of the untreated control. The data shown were mean SD of three separate experiments.
The fourteen BBR analogues (13a–n) were synthesized as de-
scribed in Scheme 1. It includes three synthetic processes employ-
ing different starting materials.16,17 Compound 13a was obtained
directly by using homopiperonylamine (8) and 2-methoxy-3-
hydroxybenzaldehyde (9) as starting materials, using the method
reported previously.14
The desired products 13b, 13f, 13i, 13k and 13m–n were syn-
thesized through an eight-step process, using commercially avail-
able derivatives of hydroxyl benzaldehyde (4) as starting
materials.14 The hydroxyl was protected with a benzyl group using
benzyl bromide and K2CO3 as the base. Then, the intermediate 5
underwent condensation with nitromethane in the presence of
methylamine. Another key intermediate 8 was obtained through
a two-step reduction (58–65% yield) in which the double bond
was reduced by NaBH4 and the nitro group was reduced by Zinc
powder respectively. Using zinc powder or Pd/C hydrogen (10%)
alone as the reducing agent, resulted in lower yields of 8 due to
anol or ethanol. The final products were obtained through dehy-
dration, reduction and intramolecular cyclization from
substituted phenylethylamines (8) and benzaldehydes (9).14 Dur-
ing the course of the intramolecular cyclization of 11 under an acid
condition, the de-protection of benzyl group occurred simulta-
neously and the desired products were then obtained directly.
The desired compounds 13c–e, 13g–h, 13j and 13l were synthe-
sized with derivatives of benzaldehyde (5) as starting materials
using a seven-step sequence including condensation, reduction,
dehydration, reduction and cyclization.14 The final products in
the 13 series were purified with flash column chromatography
using methanol–dichloromethane as the eluent.
Next, the fourteen study analogues were examined in human li-
ver HepG2 cells for their up-regulatory activities on both LDLR and
InsR mRNA expression. The up-regulatory effect was quantitatively
measured with specific real-time RT-PCR assay.4 Structures of the
analogues and their LDLR and InsR up-regulatory effect are shown
in Table 1.
the poor solubility of a,b-unsaturated nitro compound 6 in meth-

6006
Y.-X. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6004–6008
O
O
NO2
a
b
H
R2O
H
R2O
HO
R1
R1
R1
6
4
5
c
CHO
R2O
NH2
d
NO2
N
R2O
R3
R2O
e
R1
+
R1
R1
R3
7
8
9
10
f
-
Cl-
COOH
HO
HO
N+
N+
R1
R1
h
i
R3
R3
R2O
HCl
12
13(b,f,i,k,m-n)
HN
g
R1
R3
-
R2O
Cl-
R2O
11
COOH
N+
N+
h
R1
i
R1
R3
R3
13(a,c-e,g-h,j,l)
13
Scheme 1. Synthesis of the study compounds (13a–n). Reagents and conditions: (a) BnBr, K2CO3, DMF, 4 h; (b) CH3NO2, CH3NH2, methanol, 4 h; (c) NaBH4, 1,4-dioxane,
reflux, 2 h; (d) Zn, HCl; (e) 100 °C, 8 h; (f) NaBH4, methanol, reflux, 5 h; (g) glyoxal, formic acid, CuSO4, HCl, 100 °C, 5 h; (h) methanol/H2O, CaO, rt, 2 h; (i) ethanol/HCl, rt, 0.5 h.
First, the SAR analysis was done for the activity on LDLR mRNA
expression. Replacing methoxy at the 10-position of the ring D of
BBR with hydroxyl, compound 13a afforded a promising activity
on LDLR expression. Introducing different groups at the 2- or/and
3-position of BBR, compounds 13b–e were obtained. Replacement
of the methylenedioxy moiety of the ring A in BBR with a hydroxyl
at the 3-position, the obtained compound (13b) showed no activ-
ity. Replacing hydroxyl with benzyloxy at the 3-position, the com-
pound (13c) showed a weak activity. The compound 13d bearing a
benzyloxy at the 2-position and a methoxy at the 3-position exhib-
ited a good activity on LDLR expression. Three methoxy groups at
the 1, 2, and 3 positions of ring A (13e) afforded no activity on LDLR
mRNA expression.
At the 2- or/and 3-position, replacing the methylenedioxy of
compound 2 with various substituents, such as hydroxyl, methoxy
and benzyloxy groups, all of the analogs (13f–k) lost the up-regu-
latory effect on LDLR expression. Similarly, the methylenedioxy of
compound 3 was replaced with hydroxyl or methoxy at positions 2
and 3. The compound 13m, possessing a 2-methoxy and 3-hydro-
xyl, showed an activity lower than its parent compound 3 did.
Compound 13n bearing methoxy groups at the 9, 10 and 11 posi-
tions of ring D lost the activity completely.
up-regulatory effect of the study compounds (13a–n) on InsR
mRNA expression was basically consistent with that for LDLR
expression. Compounds 13a and 13d showed up-regulatory activ-
ities for both LDLR and InsR mRNA expression; especially, com-
pound 13a exhibited good activities for both receptors.
The up-regulation of LDLR and InsR mRNA expression by 13a
were further validated with flow cytometric analysis for the protein
profile on the surface of human HepG2 liver cells (Fig. 2A and B). The
results showed that either LDLR or InsR protein expression was in-
creased on the surface of the 13a-treated hepatocytes. Although
the up-regulatory activities of 13a on LDLR and InsR expression
was not as high as that of BBR, the hydroxyl at the 10-position pro-
vides an opportunity to prepare pro-drugs by connecting 13a with
acids or alcohols. Therefore, 13a could be an ideal form of BBR to im-
prove the bioavailability and therapy efficacy in vivo.18
In conclusion, we have synthesized fourteen BBR analogues with
substituents on the ring A or D and studied the biological effect of
their activities on LDLR and InsR up-regulation. The results sug-
gested that introducing suitable substituents at the ring A or D might
retain the up-regulatory activities for both LDLR and InsR. As 13a re-
tains good up-regulatory activities in both receptors and possesses a
free arm for chemical modification, it might be selected as a parent
structure to make pro-drug candidates for their potential in the
treatment of both hyperlipidemia and hyperglycemia.
Second, SAR analysis for the activity on InsR mRNA expression
was carried out as well (Table 1). SAR results showed that the

Y.-X. Wang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6004–6008
6007
Figure 2. Up-regulation of LDLR or InsR protein expression by 13a in the liver cells. HepG2 cells were untreated or treated with 10
lg/mL of BBR or 13a for 12 h. Cells were
analyzed by flow cytometry for cell surface LDLR (A) or InsR (B) protein expression using a rabbit-polyclonal antibody against LDLR or InsR. Normal rabbit IgG was used as
control for the background staining. Fluorescence intensities on cell surface were analyzed in a FACSCalibur system. The count on Y-axis is the absolute number of cells; and
FL1 in the X-axis represents the fluorescent intensity of the LDLR or InsR protein expressed on the HepG2 cell surface.
3. Zhao, W.; Xue, R.; Zhou, Z. X.; Kong, W. J.; Jiang, J. D. Biomed. Pharmacother.
Acknowledgment
2008, 62, 730.
4. Kong, W. J.; Abidi, P.; Lin, M.; Inaba, S.; Li, C.; Wang, Y.; Wang, Z.; Si, S.; Pan, H.;
This work was supported by the ‘Innovative Drug R&D’ (11th 5-
Year Plan) of the Ministry of Science and Technology, PR China.
Wang, S.; Wu, J.; Wang, Y.; Li, Z.; Liu, J.; Jiang, J. D. Nat. Med. 2004, 10, 1344.
5. Cicero, A. F.; Rovati, L. C.; Setnikar, I. Arzneimittelforschung 2007, 57, 26.
6. Zhang, Y.; Li, X.; Zou, D.; Liu, W.; Yang, J.; Zhu, N.; Huo, L.; Wang, M.; Hong, J.;
Wu, P.; Ren, G.; Ning, G. J. Clin. Endocrinol. Metab. 2008, 93, 2559.
7. Yin, J.; Xing, H.; Ye, J. Metabolism 2008, 57, 712.
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