Synthesis and in vitro evaluation of 12-(substituted aminomethyl) berberrubine derivatives as anti-diabetics

Article abstract of DOI:10.1016/j.bmcl.2014.02.032

By introducing various amino methyl groups into 12-position of berberrubine, a series of 12-(substituted aminomethyl) berberrubine derivatives were synthesized and evaluated for their anti-diabetic activity against type 2 diabetes mellitus. The results indicated that most of the prepared compounds exhibited moderate to good anti-diabetic activity, which were comparable to or even better than the berberine, the positive control rosiglitazone and insulin. Especially, compound 3b with an N-methyl piperazine-4-methyl group at C-12, exerted the most powerful anti-diabetic activity.

Full text of DOI:10.1016/j.bmcl.2014.02.032

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1762–1765
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of 12-(substituted aminomethyl)
berberrubine derivatives as anti-diabetics
⇑
Renjun Li, Jianbo Wu, Yun He, Li Hai, Yong Wu
Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, Department of Medicinal Chemistry, West China School of Pharmacy,
Sichuan University, Chengdu 610041, China
a r t i c l e i n f o
a b s t r a c t
Article history:
By introducing various amino methyl groups into 12-position of berberrubine, a series of 12-(substituted
aminomethyl) berberrubine derivatives were synthesized and evaluated for their anti-diabetic activity
against type 2 diabetes mellitus. The results indicated that most of the prepared compounds exhibited
moderate to good anti-diabetic activity, which were comparable to or even better than the berberine,
the positive control rosiglitazone and insulin. Especially, compound 3b with an N-methyl piperazine-
4-methyl group at C-12, exerted the most powerful anti-diabetic activity.
Received 18 December 2013
Revised 8 February 2014
Accepted 12 February 2014
Available online 21 February 2014
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
Anti-diabetic activity
Type 2 diabetes mellitus
12-(Substituted aminomethyl) berberrubine
derivatives
Synthesis
Type 2 diabetes mellitus (T2DM), formerly known as non-
insulin-dependent diabetes, accounts for most cases of diabetes
worldwide and is a well-recognized public health problem today.
It is a leading cause of death in developed countries and there is
substantial evidence that it has been shown to be an epidemic of
significant proportions in many developing and newly industrial-
ized countries. According to the International Diabetes Federation,
an estimated 366 million people worldwide have diabetes in 2011
and this number is projected to rise to 552 million by 2030.1
T2DM is a complex heterogeneous group of metabolic disorders
characterized by increased levels of blood glucose due to impaired
insulin action and/or insulin secretion.2 The partial failure of the
insulin producing b cells of the pancreas may lead to various com-
plications, including hyperglycemia, excessive urine production,
compensatory thirst, increased fluid intake, blurred vision, unex-
plained weight loss, lethargy, and changes in energy metabolism.3
The enormous and escalating economic and social costs of T2DM
present a major challenge for attempts to reduce the risk of devel-
oping the condition as well as for energetic management of the
established disease.
sensitizer biguanides caused gastrointestinal complaints and the
major side effects of another sensitizer thiazolidinediones (TZDs)
included weight gain and peripheral edema. Secretagogue agents
sulfonylureas and glinedes are always associated with weight
gain and hypoglycemia. Alpha glucosidase inhibitors also reduce
gastrointestinal complaints, such as bloating, abdominal cramps,
flatulence et al. Another method for treatment of T2DM is incretin
based therapies, including exenatide, liraglutide, dipeptidyl pepti-
dase 4 (DPP 4) inhibitors, pramlintide, bromocriptine and insulin.
However, these types of drugs also lead a series of side effects, such
as common gastrointestinal complaints, body weight’s change,
headache and hypotension.
Berberine (BBR, 1, Fig. 1), an isoquinoline alkaloid, is the major
pharmacological component of the Chinese herb Coptis chinensis
(Huang-Lian, a common herb in traditional Chinese medicine). As
a botanic drug, berberine or berberine-containing herbs have been
used to treat intestinal infections, particularly bacterial diarrhea,
for thousands of years in China.5 Recently, many research groups
have showed a strong impact of BBR on glucose homeostasis and
marked anti-diabetic effects on both human beings and rodent
models, especially, with type 2 diabetes mellitus.6–8 The action
mechanism of anti-diabetic effect of BBR has been widely investi-
gated.9 The up-to-date reports argued that the anti-diabetic
activity of BBR is related to activation of AMP-activated protein
kinase (AMPK), acute initiation of glucose transport activity of
glucose transporter GLUT1, and improvement of insulin signal
transduction in insulin-resistant myotubes in cultured cells.10,11
Current treatment options for T2DM include insulin, insulin
sensitizers, secretagogues, alpha glucosidase inhibitors, incretins,
pramlintide, and bromocriptine. However, current medicines for
T2DM usually cause various side effects.4 For instance, insulin
⇑
Corresponding author. Tel./fax: +86 28 85503666.
E-mail address: wyong@scu.edu.cn (Y. Wu).
http://dx.doi.org/10.1016/j.bmcl.2014.02.032
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

R. Li et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1762–1765
1763
4
1
5
control respectively. In 3T3-L1 adipocytes, berberine stimulated
glucose uptake to a similar magnitude as did rosiglitazone, but
the mechanism was not fully elucidated,17 thus we take the rosig-
litazone as positive control. The L6 myotubes has been widely used
to investigate the mechanism of insulin- and exercise-stimulated
glucose transport,18 and Cheng et al. found that berberine
stimulated glucose transport in myotubes.19 And then in the cell
studies, the 3T3-L1 adipocytes is selected to investigate the
insulin-resistant reversal activity while the L6 myotubes is
selected to investigate the glucose transport activity. The relative
sensitization rates of the compounds to 3T3-L1 adipocytes and
the sensitization rates of the compounds to L6 myotubes are
summarized in Table 1.
Comparing the 12-(substituted aminomethyl) berberrubine
derivatives and berberine at different concentrations in 3T3-L1 adi-
pocytes and L6 myotubes, the insulin-resistant reversal activity
and glucose transport activity of the berberrubine derivatives are
mostly comparable to or even better than the berberine. And com-
paring berberrubine derivatives and the positive control rosiglitaz-
O
O
6
O
O
N
Cl
8
Cl
N
O
OH
O
13
9
12
10 O
11
2
1
Figure 1. Structures of berberine (1) and berberrubine (2).
As berberine shows marked anti-diabetic effect, we set up to
study the synthesis and anti-diabetic activity of the berberine
derivatives using berberine as the lead compound in order to find
active hypoglycemic agents. In our earlier studies, we have studied
a new shortcut synthesis of 8-oxocoptisine, a natural protoberber-
ine alkaloid starting from readily available and inexpensive
berberine hydrochloride.12 Furthermore, in our research to the
anti-diabetic activity of berberine derivatives, we designed and
synthesized a series of berberrubine derivatives and accidentally
found that most of the 12-(substituted aminomethyl) berberrubine
derivatives (Fig. 2) owing good anti-diabetic activities, which are
better than that of berberine in the cell studies in vitro.
In this study, berberrubine derivatives were synthesized by
means of the introduction of various aminomethyl groups into
12-position of berberrubine to improve the anti-diabetic activity.
First, the commercially available berberine hydrochloride was
readily converted into the berberrubine (2)13 in a vacuum at
180 °C, and then the appropriate aliphatic amino or aryl amine
with formaldehyde were added, stirred at 80 °C or room tempera-
ture to obtain the 12-C amino methylation berberrubine deriva-
tives. Finally, salification of them with hydrochloric acid in
ethanol gave the products 3a–j14 (Scheme 1).
one at the concentration of 10 lmol/ml in 3T3-L1 adipocytes, all of
the experimental compounds have a certain degree of insulin-
resistant reversal activity, Therein the insulin-resistant reversal
activities of compounds 3a–e are better than the positive control
rosiglitazone, and they are concentration-dependent. Among them
the compound 3b shows the best activity and the sensitization
reaches 1.26 fold of rosiglitazone. Even the concentration is low-
ered to 1 lmol/ml, it still shows similar activity as the positive con-
trol. In the L6 myotubes, most of the compounds show middle to
high activity to increase the transport of glucose compared to insu-
lin. Especially, the compounds 3g and 3h are superior to positive
control at the concentration of 10
compounds 3b and 3g are similar to or even better than the posi-
tive control at the concentration of 1 mol/ml.
lmol/ml. And the activities of
l
All of the synthetic 12-(substituted aminomethyl) berberrubine
derivatives and berberine were evaluated for their anti-diabetic
activities against type 2 diabetes mellitus in 3T3-L1 adipocytes15
and L6 myotubes16 using rosiglitazone and insulin as positive
The results indicate that the introducing various aminomethyl
groups into 12-position of berberrubine can remarkably improve
the insulin-resistant reversal activity and stimulate glucose trans-
port activity against type 2 diabetes mellitus. This modification is
likely to enhance their ability binding to the target of drug action
mainly through hydrophobic effect, conjugation effect and hydro-
gen bond on 9-hydroxyl. Since compounds 3a–d differ structurally
from compounds 3e–j by the presence of a nitric heterocyclic
six-membered ring group, the nitric heterocyclic six-membered
ring group is perceived to be important for the enhanced insulin-
resistant reversal activity. And in the L6 myotubes, the difference
that 3e–g show in the presence of the insulin-resistant reversal
activity or glucose transport activity indicates that the single
straight chain hydrocarbon substituted aminomethyl is likely
important for the enhanced insulin sensitization as well. Among
them, the compound 3g shows the best glucose transport activity.
And with the chain hydrocarbon substituted aminomethyl gets
shorter from 3e to 3g, their insulin-resistant reversal activity is
decreasing, while their glucose transport activity is increasing.
If substituent is changed to benzyl and phenyl substituted
aminomethyl, the compounds 3h and 3i show good glucose
transport activity for the conjugation system might bind to the
3a: R =
3b: R =
3f: R =
3g: R =
N
O
O
N
N
Cl
N
N
N
N
OH
O
Boc
Bn
N
3c: R =
N
N
3h: R =
3i: R =
Bn
Ph
R
O
Ph
N
3d: R =
3e: R =
3j: R =
N
N
Figure 2. Structures of 12-(substituted aminomethyl) berberrubine derivatives.
O
O
O
O
O
N
Cl
N
Cl
N
Cl
O
a
b
OH
O
OH
O
O
O
R
1
2
3a-j
Scheme 1. Synthesis of 12-(substituted aminomethyl) berberrubine derivatives. Reagents and conditions: (a) 180 °C in a vacuum (98% yield); (b) aliphatic amino or aryl
amine, HCHO, HCl, C2H5OH (65–85% yield).

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R. Li et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1762–1765
Table 1
Relative sensitization rates of 12-(substituted aminomethyl) berberrubine derivatives to 3T3-L1 adipocytes and sensitization rates of 12-(substituted aminomethyl) berberrubine
derivatives to L6 myotubes
a
b
Compound
R =
Concentration (lmol/ml)
Relative sensitization rate (%)
Sensitization rate (%)
10
1.0
102.93
41.02
43.53
15.78
3a
N
N
0.10
8.33
10
1.0
0.10
126.29
90.23
À1.09
107.30
3.14
À43.33
115.14
68.72
48.55
41.90
N
3b
3c
3d
N
Boc
O
10
1.0
0.10
52.51
23.32
N
10
1.0
0.10
57.22
26.16
N
28.22
10
1.0
112.25
28.02
46.08
17.97
N
N
3e
0.10
À64.34
10
1.0
0.10
75.63
23.48
2.03
48.83
5.81
3f
10
1.0
0.10
51.59
78.50
51.30
À17.72
3g
3h
3i
N
À16.79
Bn
N
10
1.0
0.10
65.58
70.02
28.39
À9.51
Bn
Ph
À17.34
Ph
N
10
1.0
96.66
62.08
53.05
12.43
0.10
33.38
10
1.0
0.10
10
1.0
73.15
40.73
7.95
À12.10
3j
N
À22.09
74.23
13.27
À2.93
Berberine
43.28
21.30
0.10
1.0
Insulin
48.21
a
The relative sensitization rates of the compounds to 3T3-L1 adipocytes in vitro. Sensitization rate = [1 À (sample’s OD – blank’s OD)/(negative’s OD – blank’s OD)] Â 100%,
relative sensitization rate = the sensitization rate of sample/the sensitization rate of rosiglitazone  100%. Each sample is diluted into three concentrations in the screening.
b
The sensitization rates of the compounds to L6 myotubes in vitro. Sensitization rate = [(negative’s OD À sample’s OD)/(negative’s OD – blank’s OD)] Â 100%. Each sample
is diluted into two concentrations in the screening.
target through conjugation effect. However, it remains to be
further investigated that whether their target is AMPK as berberine
and the structural change of the group is effective to generate more
potent anti-diabetic agents.
In conclusion, the 12-(substituted aminomethyl) berberrubine
derivatives have a certain degree of stimulating glucose uptake
for the insulin-resistant 3T3-L1 adipocytes and L6 myotubes. It
indicates that these compounds can be used in the treatment
caused by non-insulin dependent (type 2 diabetes mellitus) DM,
obesity, fatty liver disease and their complications. Structural
features as mentioned above can provide useful information for
the modification of berberrubine derivatives with an enhanced
activity in the future.
References and notes
1. Seng, C. K.; Willett, W. C.; Hu, F.; Dam, R. M.; Cheung, L. L.; Mohan, V.; Naldoo,
N.; Rebello, S. A.; Prasad, R. O. Health in Asia (NIHA) Forum White Paper 2011;
Saw Swee Hock School of Public Health, National University of Singapore and
Harvard School of Public Health, 2011.
2. Das, S. K.; Elbein, S. C. Cell Sci. 2006, 2, 100.
3. Lin, Y.; Sun, Z. J. J. Endocrinol. 2010, 204, 1.
4. Skugor, M. Presented at the 5th Annual Diabetes and the Heart: Prevention and
Management of Diabetes and its Cardiovascular Complications, Cleveland,
Ohio, October 21–22, 2012; paper 3.
5. Tang, J.; Feng, Y. B.; Tsao, S. W.; Wang, N.; Curtain, R.; Wang, Y. W. J.
Ethnopharmacol. 2009, 126, 5.
6. Wang, T.; Wang, N.; Song, H.; Xi, X. N.; Wang, J. A.; Hao, A. J.; Li, T. F. Eur. J.
Pharm. Sci. 2011, 44, 127.
7. Yin, J.; Xing, H.; Ye, J. Metabolism 2008, 57, 712.
8. Wang, Y.; Campbell, T.; Perry, B.; Beaurepaire, C.; Qin, L. Metabolism 2011, 60,
298.
Acknowledgments
9. Yin, J.; Zhang, H.; Ye, J. Endocr. Metab. Immune Disord. Drug Targets 2008, 99.
10. Cok, A.; Plaisier, C.; Salie, M. J.; Oram, D. S.; Chenge, J.; Louters, L. L. Biochimie
2011, 93, 1187.
11. Liu, L. Z.; Cheung, S. C.; Lan, L. L.; Ho, S. K.; Xu, H. X.; Chan, J. C.; Tong, P. C. Mol.
Endocrinol. Cell. 2010, 317, 148.
12. Cui, X. J.; Lu, Y. Y.; Wu, Y. Chin. Chem. Lett. 2010, 21, 1281.
13. Berberine hydrochloride (1, 20 g, 62.11 mmol) was placed in the round bottom
flask and in a vacuum, stirred at 180 °C for 20 min to give the crude product,
which was purified using flash silica gel column chromatography (CH2Cl2/
MeOH = 10:1) to give 22 g of the red solid compound berberrubine (2, 98%
yield). 1H NMR (400 MHz, DMSO-d6): d 9.22 (s, 1H), 8.06 (s, 1H), 7.56 (d, 1H,
J = 8.0 Hz), 7.43 (s, 1H), 6.96 (s, 1H), 6.85 (d, 1H, J = 8.0 Hz), 6.10 (s, 2H), 4.70 (t,
2H, J = 6.0 Hz), 3.78 (s, 3H), 3.23 (t, 2H, J = 6.0 Hz). MS (ESI+) m/z: 322 [M+H]+.
14. General procedure for synthesis of the compounds 3a–j: Hydrochloride
berberrubine (322 mg, 1.0 mmol) was dissolved in 5.0 ml of the anhydrous
ethanol, aliphatic amino or aryl amine (5.0 mmol) and 0.4 ml formaldehyde
aqueous solution (37%, 5.0 mmol) were added, stirred at room temperature or
We thank the National Science Foundation of China (No.
81373259) and the Science & Technology Pillar Program of Sichuan
Province of China (No. 2011SZ0014) for financial support of this
study.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
02.032. These data include MOL files and InChiKeys of the most
important compounds described in this article.

R. Li et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1762–1765
1765
at 80 °C for 24 h. Concentrated under reduced pressure to give the crude
products, which were purified using flash silica gel column chromatography
(CH2Cl2/MeOH = 20:1) to give the dark red solid, salification of them with
hydrochloric acid in ethanol gave the products 3a–j.
DMEM supplemented with 10% FBS, PSP, Ins, Dex, IBMX when the confluence
degree reaches 90%. Then the cells are digested and inoculated in 24-well
plates, cultured in high glucose DMEM supplemented with 10% FBS and PSP for
two days. The cells are induced insulin resistance in the high glucose DMEM
supplemented with 10% FBS, PSP, low concentration of Ins, high concentration
of Dex for four days, and switch to the high glucose DMEM supplemented with
the sample but no PSP, meanwhile we set up the blank control (normal cells
after being induced differentiation) and the negative control (insulin-resistant
cells after being induced differentiation) containing no samples. The cells are
12-(Piperidine-1-ylmethyl)-berberrubine (3a): 1H NMR (400 MHz, CDCl3): d 9.18
(s, 1H), 8.16 (s, 2H), 7.26 (s, 1H), 7.22 (s, 1H), 6.74 (s, 1H), 6.06 (s, 2H), 4.38 (t,
2H, J = 6.0 Hz), 3.93 (s, 3H), 3.61 (s, 2H), 3.08 (t, 2H, J = 6.0 Hz), 2.41 (s, 4H), 1.56
(m, 6H). MS (ESI+) m/z: 420 [M+H]+.
12-(N-Methyl piperazine-4-methyl)-berberrubine (3b): 1H NMR (400 MHz,
CDCl3): d 9.20 (s, 1H), 8.09 (s, 2H), 7.24 (s, 1H), 7.22 (s, 1H), 6.75 (s, 1H),
6.07 (s, 2H), 4.39 (t, 2H, J = 6.0 Hz), 3.92 (s, 3H), 3.66 (s, 2H), 3.08 (t, 2H,
J = 6.0 Hz), 2.45 (s, 8H). MS (ESI+) m/z: 435 [M+H]+.
continued to be cultivated for 50 h. Then we take 10 ll supernatant to the
enzyme label plate, examine the glucose level via Kit assay (Chengdu Mike
Technology Company), and determine the OD (optical density) value in
490 nm. This experiment adopts the positive control rosiglitazone (purchased
from GSK).
12-(Morpholinomethyl)-berberrubine (3d): 1H NMR (400 MHz, CDCl3): d 9.22 (s,
1H), 8.10 (s, 2H), 7.27 (s, 1H), 7.21 (s, 1H), 6.75 (s, 1H), 6.07 (s, 2H), 4.40 (t, 2H,
J = 6.0 Hz), 3.93 (s, 3H), 3.70 (t, 4H, J = 4.4 Hz), 3.66 (s, 2H), 3.09 (t, 2H,
J = 6.0 Hz), 2.50 (t, 4H, J = 4.4 Hz). MS (ESI+) m/z: 421 [M+H]+.
16. L6 myotubes are cultured in 24-well plates with high glucose DMEM
containing 10% FBS, PSP for 24 h, and differentiated in the high glucose
DMEM supplemented with 2% FBS and PSP. The differentiation medium is
charged every 48 h. The cells are disposed overnight with high glucose DMEM
supplemented with no serum after being differentiated for 6–8 days, and then
disposed for 4–6 h in the DMEM supplemented with no serum, no glucose. At
12-(N,N-Diethyl amino-methyl)-berberrubine (3f): 1H NMR (400 MHz, DMSO-
d6): d 9.10 (s, 1H), 7.97 (s, 1H), 7.65 (s, 1H), 7.10 (s, 1H), 6.97 (s, 1H), 6.15 (s,
2H), 4.46 (t, 2H, J = 6.0 Hz), 3.73 (s, 3H), 3.54 (s, 2H), 2.57 (m, 4H), 1.05 (m, 6H).
MS (ESI+) m/z: 408 [M+H]+.
12-(Dimethylamino-methyl)-berberrubine (3g): 1H NMR (400 MHz, DMSO-d6): d
9.07 (s, 1H), 7.98 (s, 1H), 7.64 (s, 1H), 7.13 (s, 1H), 6.96 (s, 1H), 6.10 (s, 2H), 4.47
(t, 2H, J = 6.0 Hz), 3.72 (s, 3H), 3.56 (s, 2H), 3.03 (t, 2H, J = 6.0 Hz), 2.16 (s, 6H).
MS (ESI+) m/z: 379 [M+H]+.
last, the cells are disposed in the 180
ll serum-free DMEM containing 20 mmol
glucose for 16–24 h. Then 10 l supernatant is taken to the enzyme label plate.
l
We examine the glucose level via Kit assay (Chengdu Mike Technology
Company), and determine the OD value in 490 nm. This experiment adopts the
positive control insulin (purchased from the SIGMA).
12-(Pyrrolidin-1-ylmethyl)-berberrubine (3j): 1H NMR (400 MHz, CDCl3): d 9.17
(s, 1H), 8.01 (s, 2H), 7.26 (s, 2H), 6.74 (s, 1H), 6.05 (s, 2H), 4.36 (t, 2H, J = 6.0 Hz),
3.93 (s, 3H), 3.78 (s, 2H), 3.05 (t, 2H, J = 6.0 Hz), 2.55 (s, 4H), 1.68 (s, 4H). MS
(ESI+) m/z: 406 [M+H]+.
17. Wang, S. H.; Wang, W. J.; Wang, X. F.; Chen, W. Zhongguo Zhong Xi Yi Jie He Za
Zhi 2004, 24, 926.
18. Klip, A.; Paquet, M. R. Diabetes Care 1990, 13, 228.
19. Cheng, Z.; Pang, T.; Gu, M.; Gao, A. H.; Xie, C. M.; Li, J. Y.; Nan, F. J.; Li, J. Biochim.
Biophys. Acta 2006, 1760, 1682.
15. 3T3-L1 adipocytes are cultured in six-well plates with high glucose Dulbecco’s
modified Eagle medium (DMEM) containing 10% Fetal bovine serum (FBS) and
phenolsulfonphthalein (PSP), differentiated for four days in the high glucose