Berberine derivatives, with substituted amino groups linked at the 9-position, as inhibitors of acetylcholinesterase/butyrylcholinesterase

Article abstract of DOI:10.1016/j.bmcl.2010.09.013

Berberine derivatives with substituted amino groups linked at the 9-position using different carbon spacers were designed, synthesized, and biologically evaluated as inhibitors of acetylcholinesterase. Compound 10b, with a cyclohexylamino group linked to berberine by a three carbon spacer, gave the most potent inhibitor activity with an IC₅₀ of 0.020 μM for AChE. Kinetic studies revealed mixed inhibition of AChE, and molecular modeling simulations of the AChE-inhibitor complex confirmed that compounds bound to both the catalytic active site and the peripheral anionic site.

Full text of DOI:10.1016/j.bmcl.2010.09.013

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6649–6652
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Berberine derivatives, with substituted amino groups linked at the 9-position,
as inhibitors of acetylcholinesterase/butyrylcholinesterase
⇑
Ling Huang, Zonghua Luo, Feng He, Anding Shi, Fangfei Qin, Xingshu Li
Institute of Drug Synthesis and Pharmaceutical Processing, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 June 2010
Revised 20 August 2010
Accepted 3 September 2010
Available online 15 September 2010
Berberine derivatives with substituted amino groups linked at the 9-position using different carbon spac-
ers were designed, synthesized, and biologically evaluated as inhibitors of acetylcholinesterase. Com-
pound 10b, with a cyclohexylamino group linked to berberine by a three carbon spacer, gave the most
potent inhibitor activity with an IC50 of 0.020 lM for AChE. Kinetic studies revealed mixed inhibition
of AChE, and molecular modeling simulations of the AChE–inhibitor complex confirmed that compounds
bound to both the catalytic active site and the peripheral anionic site.
Ó 2010 Elsevier Ltd. All rights reserved.
Dedicated to Professor Chan on the occasion
of his 60th birthday
Keywords:
Berberine derivatives
Dual inhibitors
Acetylcholinesterase
Butyrylcholinesterase
Molecular modeling
Alzheimer’s disease (AD) is the most prevalent form of dementia
affecting approximately 24 million people worldwide.1 One possi-
ble approach to treating this disease is to restore the level of acetyl-
choline (ACh) by inhibiting acetylcholinesterase (AChE) with
reversible inhibitors. The aim of AChE inhibitors is to improve the
endogenous levels of ACh in the brain of AD patients, thereby
increasing cholinergic neurotransmission.2 By contrast, numerous
studies in recent years found that butyrylcholinesterase (BuChE)
levels are unchanged or even rise in advanced AD while AChE activ-
ity in certain brain regions decreased.3–6 Therefore, these studies
indicate that a balance of inhibition of both AChE and BuChE may
be beneficial in treating the cognitive deficits seen in AD. Further-
more, it may not be an advantage to only have a selective inhibitor
of AChE.7–9 In addition, it has been reported that some peripheral
anionic site (PAS) ligands, for example, propidium,10 could inhibit
the aggregation of beta-amyloid peptide induced by AChE, but is
not involved in the catalytic active site (CAS) of AChE.11,12 Given this
observation, bis(7)-tacrine (A in Fig. 1),13 and a series of hybrid
compounds (e.g., B and C in Fig. 114,15) were studied for their inhib-
itory activity of AChE and AChE induced Ab aggregation.
berberine was linked with phenol by 4-carbon spacers (D in
Fig. 1),16 and the berberine linked with 3-methylpyridinium by a
2-carbon spacer (E in Fig. 1),17 exhibited potent AChE inhibition
with an IC50 of 0.097 lM and 0.048 lM, respectively. Herein, we re-
port the design, synthesis and biological evaluation of another ser-
ies of novel berberine derivatives which have an amino group
linked at the 9-position using different carbon spacers.
The synthetic pathway of 9-substituted berberine derivatives
4–11 are shown in Scheme 1. Partial demethylation of berberine
1 at 190 °C under vacuum for 15 min gave berberrubine 2, with a
68% yield.18 Alkylations of berberrubine 2 with 1,2-dibromoethane
and 1,3-dibromopropane in DMF afforded 3a and 3b with a 69%
and 77% yield, respectively. Final products 4–10 were prepared
by reactions 3a–b with commercially available secondary amines
(dimethylamine, pyrrolidine, etc.), which gave a 31–69% yield. All
the compounds were characterized using analytical and NMR spec-
troscopic data (see Supplementary data).
The AChE and BuChE inhibitory effects of the berberine deriv-
atives were determined using the spectroscopic method described
by Ellman et al.19 Results are summarized in Table 1. AChE (E.C.
3.1.1.7) was obtained from electric eel and BuChE (E.C. 3.1.1.8)
was obtained from equine serum. It is interesting that eight of
the twelve berberine derivatives where the amino group was
linked with berberine using different carbon spacers exhibited
better inhibitory activity for AChE than berberine. The carbon
spacers seem to be very important for the inhibitory activities
of these compounds. However, compounds 4a and 4b, which have
In our earlier studies, we have designed and synthesized a series
of novel molecules using berberine as the lead structure. Biological
evaluation indicated that these compounds could be used as inhib-
itors of both AChE and BuChE. Among them, the compounds where
⇑
Corresponding author.
E-mail address: lixsh@mail.sysu.edu.cn (X. Li).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.013

6650
L. Huang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6649–6652
Figure 1. Structure of reported bifunctional AChE inhibitors.
Scheme 1. 9-Substituted berberine derivatives 4–11. Reagents and conditions: (i) 190 °C, 20–30 mm Hg, 15 min; (ii) Br(CH2)nBr, DMF, 1–4 h; (iii) amine, DMF, 80 °C.
a N,N-dimethyl amino group linked with berberine by two or
three carbon spacers, were less potent inhibitors when compared
compounds 7b and 8b, pyrrolidine and piperidine linked with
berberine by three carbon spacers, gave IC50 values of 0.093
and 0.052 M, respectively. Compounds 9a and 9b, morpholine
linked with berberine by two and three carbon spacers, showed
poor activity compared with the lead compound (IC50 = 1.80
for 9a, and IC50 = 1.456 M for 9b). The most potent inhibitor
was 10b (IC50 value = 0.020 M), which contained a cyclohexan-
amine linked with berberine by three carbon spacers and was
18-fold more potent than berberine. It was surprising that com-
pound 11b, which possessed a hydroxyl group at the end of the
side chain, exhibited much weaker anti-AChE activity than that
lM
with berberine (berberine, IC50 = 0.374
carbon spacer, IC50 = 1.74 M; compound 4b, three carbon spacer,
IC50 = 0.20 M). When considering the optimal carbon spacer, the
lM; compound 4a, two
l
l
l
lM
volume of the substituted amino groups is also important for
inhibitory activity. Among the three aliphatic substituted amino
groups, the N,N-diethyl amino group gave the best results (N,N-
l
l
diethyl amino group, IC50 = 0.105
lM; N,N-dimethyl amino group,
IC50 = 0.20 M; N,N-dipropyl amino group, IC50 = 0.310
l
lM). Inter-
estingly, cyclic substituted amino groups appeared to have better
activity than the chain substituted amino groups. For example,
of the lead compound berberine (IC50 = 2.37 lM). This result

L. Huang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6649–6652
6651
Table 1
In vitro inhibition IC50 (lM) and selectivity of compounds 1, 4–13 on AChE and BuChE
O
O
Br
N
(CH2)n
R
O
O
Selectivity for AChEc
48.6
IC50 (l
M) BuChE/AChd
a
a
Compound
R
n
IC50
(
lM)
AChE/AChb
BuChE/BuChc
18.2 0.683
1
Berberine
CH3
0.374 0.024
n.t.e
4a
4b
2
3
1.74 0.135
0.201 0.022
1.65 0.106
10.4 0.578
0.95
51.7
0.936 0.044
14.2 0.297
N
CH3
CH3
N
N
5b
3
0.105 0.003
2.64 0.258
25.1
3.46 0.111
CH3
6b
7b
3
3
0.310 0.007
0.093 0.024
2.88 0.155
3.00 0.116
9.3
1.48 0.0.085
3.42 0.0.134
32.3
N
N
N
8a
8b
2
3
0.287 0.010
0.052 0.005
0.242 0.005
2.59 0.180
0.84
48.8
0.060 0.001
2.97 0.0.122
9a
9b
2
3
1.80 0.190
0.456 0.059
>30
>30
>16.7
>43.8
n.t.
n.t.
O
H
N
10b
3
3
0.020 0.002
4.17 0.391
208
1.35 0.0.07
11b
Tacrine
–OH
2.37 0.098
0.311 0.009
3.23 0.197
0.041 0.003
1.36
0.13
2.10 0.0.035
n.t.
a
Mean SD of at least three independent measurements.
Acetylcholine substrate for evaluation of antiacetylcholinesterase activity.
Butyrylcholine substrate for evaluation of antibutyrylcholinesterase activity.
Acetylcholine substrate for evaluation of antibutyrylcholinesterase activity.
n.t. = not tested.
b
c
d
e
indicated that the group at the end of the molecule influenced
inhibitory activity.
the most potent inhibitor for BuChE with an IC50 value of
0.242 M. Because BuChE preferentially acts on BuCh, but also
l
In vitro BuChE inhibition was also determined using the same
method. Most of these compounds demonstrated higher inhibitory
potency against BuChE than the lead compound berberine. Com-
hydrolyzes ACh, BuChE inhibition was also evaluated in the pres-
ence of ACh as the enzyme substrate. The IC50 values showed sim-
ilar trends as those when BuCh was employed as the enzyme
substrate.
pound 8a, which gave an IC50 value of 0.287 lM for AChE, was
The mechanism of inhibition of AChE was investigated using the
derivative 10b, one of the most potent AChE inhibitors. Steady-
state inhibition data of compound 10b for AChE is shown in Figure
2. Lineweaver–Burk reciprocal plots revealed that there was an
increasing slope and an increasing intercept at higher inhibitor
concentrations, indicating mixed inhibition. The similar binding
mode of compounds 10b, E and D16,17 was not surprising because
all of these compounds had similar structures with minor differ-
ences only in the terminal group at the 9-position.
To investigate the possible mode of interaction of the com-
pounds with T. californica enzyme (TcAChE), some of the test com-
pounds were docked to the AChE active site gorge using the
CDOCKER program in Discovery studio 2.1 software, based on the
structure of the complex of TcAChE (PDB entry 2CMF). The most
probable conformations of the ligands were chosen based on the
docked energy value. As an example, the position of compound
10b in the binding site with respect to the key residues is shown
in Figure 3. Even though it behaved differently in the binding mod-
el compared with the original ligand of the X-ray structure, bis(5)-
tacrine, compound 10b could interact with both CAS and PAS
which was consistent with the results from kinetic studies (see
Figure 2. Steady-state inhibition by 10b of AChE hydrolysis of ACh. Reciprocal plots
of initial velocity and substrate concentration showing mixed-type inhibition for
10b on AChE.

6652
L. Huang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6649–6652
which may contribute significantly to the inhibition activity of
compound 10b. Near the bottom of the gorge, the cyclohexylamine
moiety of compound 10b might interact with Phe330 and Trp84, as
protonated amino groups at physiological pH, via hydrophobic
interactions to form a cation–p interaction with these aromatic
residues.
In conclusion, a series of berberine derivatives, with various
amino groups linked at the 9-position of berberine with different
carbon spacers, were designed, synthesized, and biologically eval-
uated as inhibitors of AChE and BuChE. All these berberine deriva-
tives were potent inhibitors of AChE, with IC50 values ranging from
micromolar to sub-micromolar. Among them, compound 10b with
a cyclohexylamino group linked to berberine by a three carbon
spacer, gave the most potent inhibitor activity with an IC50 value
of 0.020 lM against AChE. In addition, all these compounds
showed moderate to potent activity towards BuChE. Kinetic stud-
ies indicated that these compounds exhibited a mixed type inhibi-
tion for both the CAS and the PAS, which were suggested by
molecular modeling simulations of the AChE–inhibitor complex.
Studies into the inhibitory activity of aggregation of beta-amyloid
peptide induced by AChE are in progress.
Acknowledgments
We thank the Natural Science Foundation of China (20972198)
and the Ministry of Science and Technology of China (No.
2009ZX09501-017) for financial support of this study.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.09.013.
References and notes
1. Ferri, C.; Prince, M.; Brayne, C.; Brodaty, H.; Fratiglioni, L.; Ganguli, M.; Hall, K.;
Hasegawa, K.; Hendrie, H.; Huang, Y.; Jorm, A.; Mathers, C.; Menezes, P.;
Rimmer, E.; Scazufca, M. Lancet 2005, 366, 2112.
2. Talesa, V. N. Mech. Ageing Dev. 2001, 122, 1961.
3. Darvesh, S.; Hopkins, D. A.; Geula, C. Nat. Rev. Neurosci. 2003, 4, 131.
4. Eskander, M. F.; Nagykery, N. G.; Leung, E. Y.; Khelghati, B.; Geula, C. Brain Res.
2005, 1060, 144.
5. Decker, M.; Krauth, F.; Lehmann, J. Bioorg. Med. Chem. 2006, 49, 1966.
6. Decker, M. J. Med. Chem. 2006, 49, 5411.
7. Giacobini, E. Pharmacol. Res. 2004, 50, 433.
8. Greig, N. H.; Utsuki, T.; Ingram, D. K.; Wang, Y.; Pepeu, G.; Scali, C.; Yu, Q. S.;
Mamczarz, J.; Holloway, H. W.; Giordano, T.; Chen, D.; Furukawa, K.;
Sambamurti, K.; Brossi, A.; Lahiri, D. K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
17213.
9. Greig, N. H.; Utsuki, T.; Yu, Q.; Zhu, X.; Holloway, H. W.; Perry, T.; Lee, B.;
Ingram, D. K.; Lahiri, D. K. Curr. Med. Res. Opin. 2001, 17, 159.
10. Bolognesi, M. L.; Andrisano, V.; Bartolini, M.; Cavalli, A.; Minarini, A.;
Recanatini, M.; Rosini, M.; Tumiatti, V.; Melchiorre, C. Farmaco 2005, 60, 465.
11. Campos, E. O.; Alvarez, A.; Inestrosa, N. C. Neurochem. Res. 1998, 23, 135.
12. Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.; Vicente, M.; Linker, C.;
Casanueva, O. I.; Soto, C.; Garrido, J. Neuron 1996, 16, 881.
13. Pang, Y. P.; Quiram, P.; Jelacic, T.; Hong, F.; Brimijoin, S. J. Biol. Chem. 1996, 271,
23646.
14. Camps, P.; Formosa, X.; Galdeano, C.; MunozTorrero, D.; Ramirez, L.; Gomez, E.;
Isambert, N.; Lavilla, R.; Badia, A.; Clos, M. V.; Bartolini, M.; Mancini, F.;
Andrisano, V.; Arce, M. P.; Rodriguez-Franco, M. I.; Huertas, O.; Dafni, T.; Luque,
F. J. J. Med. Chem. 2009, 52, 5365.
15. Xie, Q.; Wang, H.; Xia, Z.; Lu, M.; Zhang, W.; Wang, X.; Fu, W.; Tang, Y.; Sheng,
W.; Li, W.; Zhou, W.; Zhu, X.; Qiu, Z.; Chen, H. J. Med. Chem. 2008, 51, 2027.
16. Huang, L.; Shi, A. D.; He, F.; Li, X. S. Bioorg. Med. Chem. 2010, 18, 1244.
17. Huang, L.; Luo, Z. H.; He, F.; Lu, J.; Li, X. S. Bioorg. Med. Chem. 2010, 18, 4475.
18. Iwasa, K.; Kamigauchi, M.; Ueki, M.; Taniguchi, M. Eur. J. Med. Chem. 1996, 31,
469.
Figure 3. Docking models of the compound–enzyme complex. (a): stereoviews
looking down the gorge of TcAChE binding with 10b (colored purple) and the
original ligand of the X-ray structure bis(5)-tacrine (colored green). (b) Represen-
tation of compound 10b docked into the binding site of AChE highlighting the
protein residues that form the main interactions with the inhibitor. Compound 10b
is shown in purple. Hydrogen-bonding interaction between ligand and residues
Tyr121 is shown with the green line.
Fig. 3a). As shown in Figure 3b, the berberine moiety was firmly
bound to the peripheral sites of AChE by its B ring which formed
a tight connection with the electron-rich indole ring of Trp279
(average distance between rings of 4.2 Å). A classic parallel cat-
ion–p stacking of the quaternary nitrogen of berberine occurred
with the aromatic rings of Tyr121, Phe330 and Phe331 (average
distance of 5.0 Å, 5.1 Å and 5.8 Å, respectively), which snaked along
the gorge of the enzyme active site. It is interesting that the 9-oxy-
gen atom of berberine forms a direct hydrogen-bond contact with
the backbone OH group of Tyr121 (average OH distance of 2.4 Å),
19. Ellman, G. L.; Courtney, K. D.; Andres, B. J.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.