Synthesis, biological evaluation and molecular modeling of novel triazole-containing berberine derivatives as acetylcholinesterase and β-amyloid aggregation inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.02.025

A series of novel triazole-containing berberine derivatives were synthesized via the azide-alkyne cycloaddition reaction. Their biological activity as inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were evaluated. Among t

Full text of DOI:10.1016/j.bmc.2011.02.025

Bioorganic & Medicinal Chemistry 19 (2011) 2298–2305
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular modeling of novel
triazole-containing berberine derivatives as acetylcholinesterase
and b-amyloid aggregation inhibitors
⇑
⇑
Anding Shi, Ling Huang, Chuanjun Lu, Feng He , 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:
A series of novel triazole-containing berberine derivatives were synthesized via the azide–alkyne
cycloaddition reaction. Their biological activity as inhibitors of both acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) were evaluated. Among them, compound 16d, which featured a diisopro-
pylamino substitution at the 4-position of triazole ring, was found to be a potent inhibitor of AChE, with
Received 2 December 2010
Revised 14 February 2011
Accepted 15 February 2011
Available online 18 February 2011
IC50 value of 0.044
the highest potency of b-amyloid aggregation inhibition (77.9% at 20
indicated that the triazole moiety of berberine derivatives displayed a face-to-face
l
M. Compound 18d, which beares a butyl at the 4-position of the triazole ring, showed
M). Molecular modeling studies
stacking interac-
l
Keywords:
p–p
Acetylcholinesterase inhibitor
b-Amyloid aggregation inhibitors
Click chemistry
tion in a ‘sandwich’ form with the Trp84 (4.09 Å) and Phe330 (4.33 Å) in catalytic sites of AChE.
Ó 2011 Elsevier Ltd. All rights reserved.
Berberine derivatives
Triazole-containing compounds
1. Introduction
triazole-containing compounds for pharmacodynamic and phar-
macological studies.
Click chemistry, which provides near-perfect properties of
green chemistry including simple and benign procedures, high
yields, few byproducts and mild reaction conditions, has found
applications in many research fields.1,2 In pharmaceutical chemis-
try, with the choice of appropriate building blocks, it has been used
In recent years, numerous studies have indicated that besides
cholinergic hypothesis, AChE is also involved with the etiology of
Alzheimer0s disease (AD) through PAS-induced b-amyloid (Ab)
aggregation, which is an early event in the neurodegenerative cas-
cade of AD.13 In consideration of these effects, different kinds of
dual binding site AChEIs have been reported to possess inhibitory
activities of both AChE and AChE-induced Ab aggregation.14–16
Usually, these AChEIs are made up of two pharmacophores linked
by a appropriate chain, so that they can simultaneously bind to
for synthesis of the peroxisome proliferator-activated receptor
c
(PPAR-
c
) agonists for the treatment of type II diabetes,3 multiva-
lent triazole-linked neoglycoconjugates 1,3-dipolar cycloaddi-
tions,4,5 preparation of an enzyme-bound inhibitor with
acetylcholinesterase as a reaction vessel,6 identification of HIV-1-
PR inhibitors,7 synthesis of drugs with anti-HIV activity and anti-
microbial activity against Gram-positive bacteria,8,9 the synthesis
of a divalent single-chain fragment (di-scFv) of a monoclonal anti-
body (mAb),10 and so on. Among them, the application of the
azide–alkyne cycloaddition (AAC) reaction to acetylcholinesterase
inhibitors (AChEIs) screening,11,12 reported by Sharpless et al.
made significant contributions to this field. The X-ray crystallo-
graphic studies of complexes of chosen AChEIs and mouse acetyl-
cholinesterase (AChE)11 suggested that the newly AAC-formed
triazole moiety was employed as a valuable pharmacophore for
its van der waals contract with the Phe-297 and Tyr-341 side
chains. Therefore, from both fundamental and practical stand-
points, it is still desirable to design, synthesize and evaluate new
both the peripheral and catalytic sites (CAS) of AChE, which are
0
separated by about 14 ÅA, located at the mouth and the bottom of
the gorge of AChE, respectively. In our previous work, we designed
and synthesized a series of novel molecules using berberine as the
lead structure and found that they were capable of binding both
the CAS and PAS of AChE.17 Following this study, several series of
novel berberine derivatives have been constructed and evaluated
as potential dual binding site AChEIs. This paper discloses our work
of designing, synthesizing, and evaluating a new series of triazole-
containing berberine derivatives as multi-valent AchEIs and Ab
aggregation inhibitors.
2. Results and discussion
2.1. Design and synthesis of targeting compounds
Inspired from the excellent works of Sharpless, we designed a
series of triazole-containing berberine derivatives and expected
⇑
Corresponding authors. Tel./fax: +86 20 3994305 (X.L.).
E-mail address: lixsh@mail.sysu.edu.cn (X. Li).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.025

A. Shi et al. / Bioorg. Med. Chem. 19 (2011) 2298–2305
2299
Scheme 1. 9-Substituted berberine derivatives 11–18c, 11–18d. Reagents and conditions:(i) RH, Cs2CO3, acetone; (ii) 190 °C, 20–30 mmHg, 15 min; (iii)Br(CH2)nBr, DMF,
80 °C, 1–4 h; (iv) NaN3, TBAI, DMF, 60 °C; (v) intermediates 5–10, phenyl-allylene or 1-hexyne, CuSO4, sodium ascorbate, DMF, 50 °C.
them to behave the follwing three discrete loci for effective inhib-
itory activity to AChE: (1) the berberine moiety binds to the PAS of
AChE; (2) the triazole moeity binds to the intervening site; (3) the
amino substituent at the 4-position of triazole ring binds to the
CAS. The synthetic route of triazole-containing berberine deriva-
tives is shown in Scheme 1. To begin with, berberrubine 2 was ob-
tained in 66% yield through selective demethylation of berberine 1
at 190 °C under vacuum,18 and then alkylated with 1,3-dibromo-
propane or 1,4-dibromobutane in DMF19 to afford 3c and 3d in
66% and 75% yields, respectively. Compound 3c or 3d was
equipped with an azide group by reacting with NaN3 in the pres-
ence of tetrabutylammonium iodide (TBAI). On the other hand,
intermediates 5–10 containing the propargyl substituted tertiary
amine could be easily synthesized by displacement reaction of
the corresponding secondary amine with propargyl bromide.20 Fi-
nally, the generated azides 4c or 4d were reacted with the corre-
sponding alkynes through Cu-catalyzed AAC to give the final
products.
inhibitory activity decreased even severer when the 4-substitution
of triazole ring was replaced by a proline ester (11c–d, 12c–d). This
loss of potency could be caused by both the relatively rigid five-
membered ring and the ester group. Furthermore, the inhibition
activity was also affected by the linkage between the triazole ring
and berberine unit. Usually, a methylene tether length of three pro-
vided a better inhibitor than that of four, except for compounds
16c–d, which revealed the highest inhibitory affinities. It is also
worth noting that 17d, with a phenyl group at the 4-position of
the triazole ring, showed the most dramatic activity loss relative
to its 3-methylene tethered homologue 17c (1.32
lM vs
0.310 M), suggesting that with the aromatic ring to occupy the
l
CAS, a longer chain was not favorable. On the other hand, all these
berberine triazole derivatives were uniformly more potent than
berberine against butyrylcholinesterase (BuChE), which is consis-
tent with the known theory that berberine primarily binds to the
PAS of AChE, since there is no such domain within BuChE.
2.3. Kinetic study of AChE
2.2. In vitro inhibition studies of AChE and BuChE
To study the inhibitory mechanism for this class of AChEIs, one
of the most potent inhibitors, compound 16d, was chosen for fur-
ther kinetic studies with AChE. The graphical presentation of stea-
dy-state inhibition data of compound 16d for AchE is shown in
Figure 1. The results revealed that there was an increasing slope
and an increasing intercept at higher inhibitor concentrations,
indicating a mixed type inhibitory behavior for compound 16d
with binding at both the CAS and PAS of AChE, which was similar
with the model of berberine-derived AChEIs we previously
reported.
The AChE inhibitory effects of all triazole-containing berberine
derivatives were examined by the method of Ellman et al.21 on
AChE from electric eel using commercial galanthamine as the ref-
erence standard. The IC50 values for AChE and BuChE inhibition
are shown in Table 1. Most of the triazole-containing berberine
derivatives were potent AChEIs with IC50 values ranging from
micromolar to sub-micromolar. The optimal AChE inhibition po-
tency (IC50 = 0.044 lM) was provided by compound 16d featuring
a diisopropylanmino substitution at the 4-position of the triazole
ring. Mutation of this moiety from diisopropylanmino to straight-
chain secondary amino (14c–d, 15c–d) or cyclic secondary amino
substitutes (13c–d) lowered the inhibitory activity by an order of
magnitude. This result implied that a flexible bulk of branch-chain
secondary amines was favored toward the inhibitory activity. The
2.4. Molecular modeling studies
To obtain useful information about the binding mode between
triazole-containing berberine derivatives and AChE, a molecular

2300
A. Shi et al. / Bioorg. Med. Chem. 19 (2011) 2298–2305
Table 1
In vitro inhibition IC50 (lM) and selectivity of compounds 1, 11–18c, 11–18d, for AChE and BuChE
O
O
Br
N
N
N
N
R
O
O
n
Compound
R
n
IC50
(
lM)
Selectivity for AChEc
48.6
AChE/ACha
BuChE/BuChb
18.2 0.683
1
Berberine
—
0.374 0.024
11c
11d
3
4
0.785 0.023
0.903 0.047
5.30 0.106
5.27 0.378
6.75
5.84
O
N
O
12c
12d
3
4
0.601 0.147
0.606 0.052
2.40 0.258
2.32 0.304
3.99
3.83
O
N
O
13c
13d
3
4
0.108 0.001
0.274 0.011
4.24 0.071
5.73 0.09
39.3
20.9
N
N
14c
14d
3
4
0.243 0.006
0.469 0.040
2.06 0.106
5.46 0.106
8.48
11.6
17c
15d
3
4
0.140 0.011
0.270 0.018
1.58 0.099
4.77 0.148
11.3
17.7
N
N
16c
16d
3
4
0.067 0.003
0.044 0.001
2.86 0.396
6.21 0.127
42.7
141
17c
17d
3
4
0.310 0.029
1.32 0.196
2.21 0.138
1.50 0.131
7.13
1.14
18c
18d
3
4
0.165 0.015
0.201 0.017
4.06 0.250
2.02 0.500
24.6
10.0
Galanthamine
—
—
0.623 0.099
15.7 0.787
25.3
a
50% inhibitory concentration (means SEM of three experiments) of AChE from electric eel.
50% inhibitory concentration (means SEM of three experiments) of BuChE from equine serum.
Selectivity for AChE = IC50 (BuChE)/IC50 (AChE).
b
c
docking simulation was performed based on a well-known com-
plex between AChE from Torpedo californica (TcAChE) and
bis(5)-tacrine (PDB code 2CMF). Triazole-containing compound
16d, the most potent AChE inhibitor, was chosen to explore the li-
gand-protein interactions. The position of compound 16d with re-
spect to the key residues in the binding site is shown in Figure 2.
Similar to the berberine derivatives we previously reported,17 com-
pound 16d could simultaneously interact with the CAS and PAS of
AChE. The berberine moiety of 16d adopted an appropriate orien-
tation that allows its B ring to form a p–p stacking with the indole
ring Trp279 (4.29 Å) in PAS. A hydrogen bond between the 9-oxy-
gen atom of the berberine moiety and the backbone OH group of
Tyr121 (2.77 Å) existed, which was consistent with the results of
our previous reports. However, the triazole moiety, which was de-
signed to occupy the intervening site, was found to positioned at
the CAS, displaying an unexpected face-to-face p–p stacking inter-
action in a ‘sandwich’ form with Trp84 (4.09 Å) and Phe330
(4.33 Å). While the diisopropylamino moiety formed hydrophobic
interactions with Ser81, Ser122 and Try121, instead of occupying
the CAS as predicted. This difference from the sharpless report
might be due to the relatively long and narrow structure of berber-
Figure 1. Steady state inhibition by 16d of AChE hydrolysis of ATCh; the plots show
mixed-type inhibition for 16d on AchE.

A. Shi et al. / Bioorg. Med. Chem. 19 (2011) 2298–2305
2301
T-based fluorimetric assay with curcumin (Cur) as standard com-
pound. The results are summarized in Figure 3. It is interesting that
all berberine derivatives showed higher potencies compared to
that of the curcumin (52.1%) and the parent compound, berberine
(36.3%). Among them, compound 18d, with
a butyl at the
4-position of the triazole ring, showed the highest potency of Ab
aggregation inhibition (77.9%). On the other hand, compound
16d, which was the most potent AChE inhibitor, gave 52.8% of
the inhibition potency. Compound 14d, with a N,N-dibutyl amino
at the end of the 4-substitute of the triazole ring, gave 71.7% inhi-
bition potency, while its homologous 15d, with a N,N-dipropyl
amino at the same possition, provided 62.5% of that. The results
implied that straight chain structure with enough length at the
end of the 4-substitute of the triazole ring would be favourable
for Ab1–42 peptide aggregation inhibition. However, branched chain
(16d, a N,N-di-isobutyl amino group, 52.8%), ring structures (13d, a
cyclic secondary amino group, 63.7%, and 17d, a phenyl group,
60.6%) at the end of the 4-substitute of the triazole ring were found
less favourable.
3. Conclusion
In summary, this study revealed the construction of a new ser-
ies of triazole-containing berberine derivatives using click chem-
istry and their evaluation as potential multi-valent inhibitors of
AChE, BuChE and Ab aggregation. Among the triazole-containing
compounds tested, compound 16d, featuring a diisopropylanmino
substitute at the 4-position of the triazole ring, displayed the
highest inhibitory activity with an IC50 value of 0.044 lM against
AChE. Replacement of the diisopropylanmino substitution at
4-position of the triazole ring with straight-chain secondary
amino, cyclic secondary amino or nonamino weakened the
inhibition activity drastically. Meanwhile, compound 18d, which
beares a butyl at the 4-position of the triazole ring and showed
good inhibitory activity against AChE (with an IC50 value of
0.201 lM), gave the highest potency of Ab aggregation inhibition
(77.9%). Molecular modeling simulations of the AChE inhibitor
complex showed that the triazole moieties in berberine deriva-
tives actually contributed to the inhibitory activities through
interacting with the CAS of AChE. In addition, kinetic studies indi-
cated that these compounds exhibited a mixed type inhibition for
both the catalytic active site and the peripheral anionic site. Fur-
ther investigations of these compounds as AD candidates are in
progress.
4. Experimental section
4.1. Chemistry
Figure 2. Docking models of the compound-enzyme complex. (a) Stereoviews
looking down the gorge of TcAChE binding with 16d (colored in purple) and the
original ligand of the X-ray structure bis(5)-tacrine (colored green). (b) Superpo-
sition of modeled structure of compound 16d (colored in purple) with X-ray crystal
structure of TcAChE binding with bis(5)-tacrine (colored in green, PDB entry:
2CMF). (c) Representation of compound 16d docked into the binding site of AChE
highlighting the protein residues that form the main interactions with the inhibitor.
Hydrogen-bonding interaction between ligand and residues Tyr121 is shown with
the green line.
The 1H NMR spectra were recorded with TMS as the internal
standard on a Varian 400 MHz spectrometer. Coupling constants
were given in Hz. MS spectra were recorded on a Agilent LC–MS
6120 instrument with an ESI mass selective detector. High-resolu-
tion mass spectra were obtained by Shimadazu LCMS-IT-TOF mass
spectrometere. Flash column chromatography was performed with
silica gel (200–300 mesh) purchased from Qingdao Haiyang Chem-
ical Co. Ltd or alumina from Sinopharm Chemical Reagent Co. Ltd.
All the reactions were monitored by thin layer chromatography on
silica gel. Berberine chloride was isolated from Chinese herbal
medicine Cryptocarya chinensis Franch and recrystallized from
hot water. Compound 2 was prepared according to the reported
procedure.20 Intermediates 3c and 3d were also synthesized based
on previous report.21 Intermediates 5–10 were synthesized as the
following procedures.
ine, which uses B ring to bind to the PAS, C and D rings stretch into
the gorge. Thus, the triazloe moiety could reach the CAS of AChE.
2.5. Effects on the Ab1–42 peptide aggregation
The ability of several triazole-containing berberine derivatives
to reduce Ab1–42 self-aggregation was studied through a thioflavin

2302
A. Shi et al. / Bioorg. Med. Chem. 19 (2011) 2298–2305
Figure 3. Effects on the Ab1–42 peptide aggregation inhibition at 20
lM of some triazole-containing berberine derivatives.
4.2. General procedures for the preparation of 5–10
1.99–1.89 (m, 2H), 1.85–1.75 (m, 2H); LC/MS (ESI) m/z: [MꢀBr]+
419.3.
Propargyl bromide (0.5 mmol) was dropwise added to a mix-
ture of Cs2CO3 (0.5 mmol), acetone (30 mL) and the corresponding
secondary amine (0.5 mmol) at 0 °C. The mixture was stirred over-
night at room temperature and then filtered. The filtrates was
evaporated to dryness to afford the target productes as an or-
ange-brown oil. which was directly used for the next step without
purification.
4.4. General procedures for the preparation of 11–18c, 11–18d
CuSO4 (0.32 mmol), sodium ascorbate (1.3 mmol) and com-
pound 4c or 4d (0.65 mmol) were stirred in DMF, corresponding
terminal alkyne (dissoluted in DMF) was dropwise added as the
temperature rising to 50°C. The mixture was stirred at 50°C for
5 h, and then evaporated under vaccum, the crude product was
chromatographed on an Al2O3 column, eluted with CHCl3/MeOH
(100:1–50:1) to afford the final product.
4.3. General procedures for the preparation of 4c–d
To a solution of compounds 3c or 3d (2 mmol) in DMF (15 mL),
NaN3 (20 mmol) and TBAI (12 mmol) were added at the room tem-
perature. After stirred at 60 °C for 6 h, the mixture was cooled to
room temperature, filtered, washed with water and dried under
vaccum, giving the desired product as bright yellow solid.
4.4.1. 9-O-{3-{4-[(2-Methoxycarbonyl-pyrrolidin-1-yl)methyl]-
1H-1,2,3-triazol-1-yl}propyl}berberine bromide (11c)
Intermediate 4c was treated with CuSO4, sodium ascorbate and
methyl 1-(prop-2-ynyl)pyrrolidine-2-carboxylate (5) according to
general procedure to give the target product 11c as yellow semi-
solid. Yield 38.1%;
½
a 2D0
ꢁ
= ꢀ12.9 (0.1, CHCl3); 1H 1H NMR
4.3.1. 9-O-(3-Azido-propyl) berberine bromide (4c)
(400 MHz,) d 9.84 (s, 1H), 8.95 (s, 1H), 8.19 (d, J = 9.0 Hz, 1H),
8.07 (s, 1H), 8.00 (d, J = 8.9 Hz, 1H), 7.80 (s, 1H), 7.10 (s, 1H), 6.18
(s, 2H), 4.98 (t, J = 5.7 Hz, 2H), 4.67 (t, J = 6.3 Hz, 2H), 4.26 (t,
J = 5.8 Hz, 2H), 4.03 (s, 3H), 3.88 (d, J = 13.9 Hz, 1H), 3.70 (d,
J = 13.9 Hz, 1H), 3.57 (s, 3H), 3.23–3.20 (m, 2H), 2.92–2.85 (m,
1H), 2.43–2.32 (m, 2H), 2.04–1.86 (m, 3H), 1.81–1.72 (m, 1H),
1.70–1.62 (m, 2H); LC/MS (ESI) m/z: [MꢀBr]+ 572.3; HRMS m/z
[MꢀBr]+ Calcd for C31H33N5O6 572.2509, Found 572.2522.
Intermediate 3c was treated with NaN3 and TBAI according to
general procedure to give the target product 4c as bright yellow so-
lid. Yield 75%. 1H NMR (400 MHz,) d 9.78 (s, 1H), 8.93 (s, 1H), 8.20
(d, J = 9.2 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.79 (s, 1H), 7.09 (s, 1H),
6.17 (s, 2H), 4.94 (t, J = 6.0 Hz, 2H), 4.36 (t, J = 6.2 Hz, 2H), 4.06 (s,
3H), 3.66 (t, J = 6.7 Hz, 2H), 3.26–3.16 (m, 2H), 2.14 (p, J = 6.5 Hz,
2H). LC/MS (ESI) m/z: [MꢀBr]+ 405.4.
4.3.2. 9-O-(3-Azido-butyl) berberine bromide (4d)
4.4.2. 9-O-{3-{4-[(2-Methoxycarbonyl-pyrrolidin-1-yl)methyl]-
1H-1,2,3-triazol-1-yl}butyl}berberine bromide (11d)
Intermediate 3d was treated with NaN3 and TBAI according to
general procedure to give the target product 4d as bright yellow
solid. Yield 72%; 1H 1H NMR (400 MHz,) d 9.77 (s, 1H), 8.94 (s,
1H), 8.20 (d, J = 9.0 Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.80 (s, 1H),
7.09 (s, 1H), 6.18 (s, 2H), 4.95 (t, J = 5.8 Hz, 2H), 4.31 (t, J = 6.1 Hz,
2H), 4.06 (s, 3H), 3.47 (t, J = 6.6 Hz, 2H), 3.21 (t, J = 5.7 Hz, 2H),
Intermediate 4d was treated with CuSO4, sodium ascorbate and
methyl 1-(prop-2-ynyl)pyrrolidine-2-carboxylate (5) according to
general procedure to give the target product 11d as yellow solid.
Yield 33.8%; mp 172.4–173.1; ½a D20
ꢁ
= ꢀ12.5 (0.4, CHCl3); 1H NMR
(400 MHz,) d 9.75 (s, 1H), 8.93 (s, 1H), 8.19 (d, J = 9.0 Hz, 1H),

A. Shi et al. / Bioorg. Med. Chem. 19 (2011) 2298–2305
2303
7.99 (d, J = 9.3 Hz, 2H), 7.80 (s, 1H), 7.10 (s, 1H), 6.17 (s, 2H), 4.95 (t,
5.6Hz, 2H), 4.45 (t, J = 6.6 Hz, 2H), 4.28 (t, J = 5.9 Hz, 2H), 4.04 (s,
3H), 3.85 (d, J = 13.8 Hz, 1H), 3.69 (d, J = 13.9 Hz, 1H), 3.58 (s,
3H), 3.22 (t, J = 6.6 Hz 2H), 2.92–2.82 (m, 1H), 2.42–2.36 (m, 2H),
2.12–2.04 (m, 2H), 2.00–1.92 (m, 1H), 1.85–1.72 (m, 3H), 1.72–
1.62 (m, 2H); LC/MS (ESI) m/z: [MꢀBr]+ 586.3; HRMS m/z [MꢀBr]+
Calcd for C32H35N5O6 586.2666, Found 586.2643.
4.4.7. 9-O-{3-{4-[(N,N-Dibutyl-amino)methyl]-1H-1,2,3-triazol-
1-yl}propyl}berberine bromide (14c)
Intermediate 4c was treated with CuSO4, sodium ascorbate and
N-butyl-N-(prop-2-ynyl)butan-1-amine (8) according to general
procedure to give the target product 14c as yellow semisolid. Yield
34.2%; 1H NMR (400 MHz,) d 9.85 (s, 1H), 8.95 (s, 1H), 8.20 (d,
J = 9.0 Hz, 1H), 8.01 (t, J = 9.9 Hz, 2H), 7.80 (s, 1H), 7.10 (s, 1H),
6.18 (s, 2H), 4.98 (t, J = 5.2 Hz, 2H), 4.68 (t, J = 6.4 Hz, 2H), 4.26 (t,
J = 5.5 Hz, 2H), 4.03 (s, 3H), 3.64 (s, 2H), 3.24 (t, J = 6.3 Hz, 2H),
2.45–2.41 (m, 2H), 2.31 (m, 4H), 1.38 (m, 4H), 1.22 (m, 4H), 0.81
(t, J = 7.2 Hz, 6H); LC/MS (ESI) m/z: [MꢀBr]+ 572.3; HRMS m/z
[MꢀBr]+ Calcd for C33H41N5O4 572.3237, Found 572.3221.
4.4.3. 9-O-{3-{4-[(2-Ethoxycarbonyl-pyrrolidin-1-yl)methyl]-
1H-1,2,3-triazol-1-yl}propyl}berberine bromide (12c)
Intermediate 4c was treated with CuSO4, sodium ascorbate and
ethyl 1-(prop-2-ynyl)pyrrolidine-2-carboxylate (6) according to
general procedure to give the target product 12c as yellow semi-
solid. Yield 42.3%; ½a D20
ꢁ
= ꢀ13.5 (0.2, CHCl3); 1H NMR (400 MHz,)
4.4.8. 9-O-{3-{4-[(N,N-Dibutyl-amino)methyl]-1H-1,2,3-triazol-
1-yl}butyl}berberine bromide (14d)
d 9.84 (s, 1H), 8.94 (s, 1H), 8.19 (d, J = 9.2 Hz, 1H), 8.06 (s, 1H),
8.00 (d, J = 9.1 Hz, 1H), 7.80 (s, 1H), 7.10 (s, 1H), 6.18 (s, 2H), 4.98
(t, J = 5.8Hz, 2H), 4.67 (t, J = 6.8 Hz, 2H), 4.26 (t, J = 5.9 Hz, 2H),
4.08–3.97 (m, 5H), 3.88 (d, J = 13.9 Hz, 1H), 3.70 (d, J = 13.8 Hz,
1H), 3.23 (t, J = 6.8 Hz, 4H), 2.91–2.84 (m, 1H), 2.45–2.41 (m, 2H),
2.03–1.88 (m, 1H), 1.81–1.72 (m, 1H), 1.72–1.59 (m, 2H), 1.15 (t,
J = 7.1 Hz, 3H); LC/MS (ESI) m/z: [MꢀBr]+ 586.3; HRMS m/z
[MꢀBr]+ Calcd for C32H35N5O6 586.2666, Found 586.2637.
Intermediate 4d was treated with CuSO4, sodium ascorbate and
N-butyl-N-(prop-2-ynyl)butan-1-amine (8) according to general
procedure to give the target product 14d as yellow semisolid. Yield
30.7%; mp 190.5–191.6; 1H NMR (400 MHz,) d 9.74 (s, 1H), 8.93 (s,
1H), 8.19 (d, J = 9.2 Hz, 1H), 8.01–7.94 (m, 2H), 7.80 (s, 1H), 7.10 (s,
1H), 6.17 (s, 2H), 4.96 (t, J = 6.1 Hz, 2H), 4.46 (t, J = 6.8 Hz, 2H), 4.28
(t, J = 6.3 Hz, 2H), 4.04 (s, 3H), 3.60 (s, 2H), 3.22 (t, J = 6.6 Hz, 2H),
2.27 (t, J = 7.0 Hz, 4H), 2.14–2.04 (m, 2H), 1.84–1.76 (m, 2H),
1.42–1.31 (m, 4H), 1.26–1.16 (m, 4H), 0.82 (t, J = 7.3 Hz, 6H); LC/
MS (ESI) m/z: [MꢀBr]+ 586.3; HRMS m/z [MꢀBr]+ Calcd for
4.4.4. 9-O-{3-{4-[(2-Ethoxycarbonyl-pyrrolidin-1-yl)methyl]-
1H-1,2,3-triazol-1-yl}butyl}berberine bromide (12d)
Intermediate 4d was treated with CuSO4, sodium ascorbate and
ethyl 1-(prop-2-ynyl)pyrrolidine-2-carboxylate (6) according to
general procedure to give the target product 12d as yellow semi-
C34H43N5O4 586.3393, Found 586.3384.
4.4.9. 9-O-{3-{4-[(N,N-Dipropyl-amino)methyl]1H-1,2,3-triazol-
1-yl}propyl}berberine bromide (15c)
solid. Yield 37.1%; ½a D20
ꢁ
= ꢀ14.0 (0.1, CHCl3); 1H NMR (400 MHz,)
d 9.76 (s, 1H), 8.94 (s, 1H), 8.19 (d, J = 9.2 Hz, 1H), 7.99 (d,
J = 10.1 Hz, 2H), 7.80 (s, 1H), 7.10 (s, 1H), 6.17 (s, 2H), 4.96 (t,
J = 6.4Hz, 2H), 4.46 (t, J = 6.8 Hz, 2H), 4.29 (t, J = 6.2 Hz, 2H), 4.08–
4.01 (m, 5H), 3.86 (d, J = 13.8 Hz, 1H), 3.69 (d, J = 13.8 Hz, 1H),
3.22 (t, J = 6.8Hz, 2H), 2.92–2.83 (m, 1H), 2.45–2.37 (m, 2H),
2.14–2.04 (m, 2H), 2.02–1.93 (m, 1H), 1.86–1.74 (m, 3H), 1.71–
1.62 (m, 2H), 1.16 (t, J = 7.1 Hz, 3H); LC/MS (ESI) m/z: [MꢀBr]+
600.3; HRMS m/z [MꢀBr]+ Calcd for C33H37N5O6 600.2822, Found
600.2818.
Intermediate 4c was treated with CuSO4, sodium ascorbate and
N,N-dipropylprop-2-yn-1-amine (9) according to general proce-
dure to give the target product 15c as yellow semisolid. Yield
39.3%; 1H NMR (400 MHz,) d 9.83 (s, 1H), 8.94 (s, 1H), 8.19 (d,
J = 9.2 Hz, 1H), 8.01 (t, J = J = 8.9 Hz, 2H), 7.80 (s, 1H), 7.10 (s, 1H),
6.18 (s, 2H), 4.97 (t, 5.9 Hz, 2H), 4.68 (t, J = 6.7 Hz, 2H), 4.26 (t,
J = 5.8 Hz, 2H), 4.03 (s, 3H), 3.65 (s, 2H), 3.23 (t, J = 6.7 Hz, 2H),
2.46–2.38 (m, 2H), 2.32–2.23 (m, 4H), 1.41 (m, 4H), 0.79 (t,
J = 7.2 Hz, 6H); LC/MS (ESI) m/z: [MꢀBr]+ 544.3; HRMS m/z
[MꢀBr]+ Calcd for C31H37N5O4 544.2924, Found 544.2937.
4.4.5. 9-O-{3-{4-[(Piperidin-1-yl)methyl]-1H-1,2,3-triazol-1-
yl}propyl}berberine bromide (13c)
4.4.10. 9-O-{3-{4-[(N,N-Dipropyl-amino)methyl]-1H-1,2,3-
triazol-1-yl}butyl}berberine bromide (15d)
Intermediate 4c was treated with CuSO4, sodium ascorbate and
1-(prop-2-ynyl)piperidine (7) according to general procedure to
give the target product 13c as yellow solid. Yield 30.7%; mp
140.5–143.6; 1H NMR (400 MHz,) d 9.84 (s, 1H), 8.95 (s, 1H), 8.20
(d, J = 9.2 Hz, 1H), 8.09 (s, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.80 (s,
1H), 7.10 (s, 1H), 6.18 (s, 2H), 4.99 (t, J = 6.0 Hz, 2H), 4.68 (t,
J = 6.9 Hz, 2H), 4.28 (t, J = 6.0 Hz, 2H), 4.03 (s, 3H), 3.54 (s, 2H),
3.23 (t, J = 6.8 Hz, 2H), 2.47–2.41 (m, 2H), 2.38–2.31 (m, 4H),
1.50–1.41 (m, 4H), 1.38–1.29 (m, 2H); LC/MS (ESI) m/z: [MꢀBr]+
528.2; HRMS m/z [MꢀBr]+ Calcd for C30H33N5O4 528.2611, Found
528.2598.
Intermediate 4d was treated with CuSO4, sodium ascorbate and
N,N-dipropylprop-2-yn-1-amine (9) according to general proce-
dure to give the target product 15d as yellow solid. Yield 38.0%;
mp 169.6–170.5; 1H NMR (400 MHz,) d 9.75 (s, 1H), 8.94 (s, 1H),
8.19 (d, J = 9.2 Hz, 1H), 7.99 (d, J = 9.1 Hz, 2H), 7.80 (s, 1H), 7.10
(s, 1H), 6.18 (s, 2H), 4.95 (t, J = 6.2 Hz, 2H), 4.47 (t, J = 6.8Hz, 2H),
4.29 (t, J = 6.3 Hz, 2H), 4.04 (s, 3H), 3.43 (s, 2H), 3.22 (t, J = 6.9 Hz,
2H), 2.43–2.17 (m, 4H), 2.14–2.05 (m, 2H), 1.87–1.76 (m, 2H),
1.59–1.28 (m, 4H), 0.81 (t, J = 7.2 Hz, 6H); LC/MS (ESI) m/z: [MꢀBr]+
558.2; HRMS m/z [MꢀBr]+ Calcd for C32H39N5O4 558.3080, Found
558.3072.
4.4.6. 9-O-{3-{4-[(Piperidin-1-yl)methyl]-1H-1,2,3-triazol-1-
yl}butyl}berberine bromide (13d)
4.4.11. 9-O-{3-{4-[(N,N-Di-isopropyl-amino)methyl]-1H-1,2,3-
triazol-1-yl}propyl}berberine bromide (16c)
Intermediate 4d was treated with CuSO4, sodium ascorbate and
1-(prop-2-ynyl)piperidine (7) according to general procedure to
give the target product 13d as yellow solid. Yield 33.0%; mp
191.7–192.5; 1H NMR (400 MHz,) d 9.75 (s, 1H), 8.94 (s, 1H), 8.19
(d, J = 9.2 Hz, 1H), 8.05–7.96 (m, 2H), 7.80 (s, 1H), 7.10 (s, 1H),
6.18 (s, 2H), 4.95 (t, J = 6.0 Hz, 2H), 4.46 (t, J = 6.9 Hz, 2H), 4.29 (t,
J = 6.3 Hz, 2H), 4.05 (s, 3H), 3.54 (s, 2H), 3.22 (t, J = 6.8 Hz, 2H),
2.46–2.24 (m, 4H), 2.15–2.05 (m, 2H), 1.88–1.78 (m, 2H), 1.52–
1.41 (m, 4H), 1.40–1.30 (m, 2H); LC/MS (ESI) m/z: [MꢀBr]+ 542.3;
HRMS m/z [MꢀBr]+ Calcd for C31H35N5O4 542.2767, Found
542.2750.
Intermediate 4c was treated with CuSO4, sodium ascorbate and
N,N-diisopropylprop-2-yn-1-amine (10) according to general pro-
cedure to give the target product 16c as yellow solid. Yield
40.5%; mp 182.3–183.1; 1H NMR (400 MHz,) d 9.83 (s, 1H), 8.93
(s, 1H), 8.19 (d, J = 9.2 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.91 (s,
1H), 7.79 (s, 1H), 7.10 (s, 1H), 6.17 (s, 2H), 4.97 (t, J = 6.0 Hz, 2H),
4.65 (t, J = 6.8 Hz, 2H), 4.26 (t, J = 6.0 Hz, 2H), 4.03 (s, 3H), 3.64 (s,
2H), 3.23 (t, J = 6.7 Hz, 2H), 3.02–2.92 (m, 2H), 2.45–2.40 (m, 2H),
0.95 (d, J = 6.6 Hz, 12H); HRMS m/z [MꢀBr]+ Calcd for C31H37N5O4
544.2924, Found 544.2929.

2304
A. Shi et al. / Bioorg. Med. Chem. 19 (2011) 2298–2305
4.4.12. 9-O-{3-{4-[(N,N-Di-isopropyl-amino)methyl]-1H-1,2,3-
triazol-1-yl}butyl}berberine bromide (16d)
4.5. Biological activity
Intermediate 4d was treated with CuSO4, sodium ascorbate and
N,N-diisopropylprop-2-yn-1-amine (10) according to general pro-
cedure to give the target product 16d as yellow solid. Yield
37.3%; mp 187.8–188.9; 1H NMR (400 MHz,) d 9.74 (s, 1H), 8.92
(s, 1H), 8.19 (d, J = 9.2 Hz, 1H), 7.99 (d, J = 9.1 Hz, 1H), 7.86 (s,
1H), 7.79 (s, 1H), 7.09 (s, 1H), 6.17 (s, 2H), 4.95 (t, J = 6.2 Hz, 2H),
4.44 (t, J = 6.9 Hz, 2H), 4.28 (t, J = 6.4 Hz, 2H), 4.04 (s, 3H), 3.62 (s,
2H), 3.22 (t, J = 6.1 Hz, 2H), 3.00–2.93 (m, 2H), 2.11–2.04 (m, 2H),
1.84–1.77 (m, 2H), 0.95 (d, J = 6.6 Hz, 12H); HRMS m/z [MꢀBr]+
Calcd for C32H39N5O4 558.3080, Found 558.3082.
4.5.1. In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), buty-
lcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum), 5,50-dithi-
obis-(2-nitrobenzoic acid) (Ellman0s reagent, DTNB), acetyl-
thiocholine chloride (ATC) and butylthiocholine chloride (BTC)
were purchased from Sigma Aldrich. Berberine derivatives were
dissolved in DMSO and then diluted in 0.1 M KH2PO4/K2HPO4 buf-
fer (pH 8.0) to provide a final concentration range.
4.5.2. In vitro AChE assay
All the assays were carried out under 0.1 M KH2PO4/K2HPO4
buffer, pH 8.0, using a Shimadzu UV-2450 Spectrophotometer.
AChE and BuChE solutions were prepared to give 2.0 units/mL in
2 mL aliquots. The assay medium (1 mL) consisted of phosphate
4.4.13. 9-O-{3-[4-(Benzyl)-1H-1,2,3-triazol-1-yl]proyl}berberine
bromide (17c)
Intermediate 4c was treated with CuSO4, sodium ascorbate and
phenyl acetylene according to general procedure to give the target
product 17c as yellow semisolid. Yield 38.6%; 1H NMR (400 MHz,) d
9.85 (s, 1H), 8.92 (s, 1H), 8.67 (s, 1H), 8.19 (d, J = 9.2 Hz, 1H), 7.99
(d, J = 9.1 Hz, 1H), 7.83 (d, J = 7.1 Hz, 2H), 7.79 (s, 1H), 7.44 (dd,
J = 7.1, 7.5 Hz, 2H), 7.33 (t, J = 7.4 Hz, 1H), 7.10 (s, 1H), 6.18 (s,
2H), 4.97 (t, J = 6.1 Hz, 2H), 4.75 (t, J = 6.8 Hz, 2H), 4.33 (t,
J = 6.0 Hz, 2H), 4.02 (s, 3H), 3.23 (t, J = 6.9 Hz, 2H), 2.58–2.54 (m,
2H); LC/MS (ESI) m/z: [MꢀBr]+ 507.2; HRMS m/z [MꢀBr]+ Calcd
for C30H27N4O4 507.2032, Found 507.2035.
buffer (pH 8.0), 50 ll of 0.01 M DTNB, 10 ll of enzyme, and 50 ll
of 0.01 M substrate (ACh chloride solution). Test compounds were
added to the assay solution and preincubated at 37 °C with the en-
zyme for 15 min followed by the addition of substrate. The activity
was determined by measuring the increase in absorbance at
412 nm at 1 min intervals at 37 °C. Calculations were performed
according to the method of the equation in Ellman et al.21 Each
concentration was assayed in triplicate.
In vitro BuChE assay was similar with the method described
above.
4.4.14. 9-O-{3-[4-(Benzyl)-1H-1,2,3-triazol-1-yl]butyl}berberine
bromide (17d)
4.6. Determination of the inhibitory effect on the self-mediated
Ab(1-42) aggregation
Intermediate 4d was treated with CuSO4, sodium ascorbate and
phenyl acetylene according to general procedure to give the target
product 17d as yellow semisolid. Yield 37.9%; 1H NMR (400 MHz,)
d 9.75 (s, 1H), 8.90 (s, 1H), 8.62 (s, 1H), 8.18 (d, J = 9.2 Hz, 1H), 7.98
(d, J = 9.1 Hz, 1H), 7.82 (d, J = 7.1 Hz, 2H), 7.78 (s, 1H), 7.44 (dd,
J = 7.2, 7.4 Hz, 2H), 7.33 (t, J = 7.4 Hz, 1H), 7.09 (s, 1H), 6.17 (s,
2H), 4.93 (t, J = 5.9 Hz, 2H), 4.53 (t, J = 6.8 Hz, 2H), 4.32 (t,
J = 6.3 Hz, 2H), 4.04 (s, 3H), 3.20 (t, J = 6.8 Hz, 2H), 2.18–2.11 (m,
2H), 1.90–1.84 (m, 2H); LC/MS (ESI) m/z: [MꢀBr]+ 521.3; HRMS
m/z [MꢀBr]+ Calcd for C31H29N4O4 521.2243, Found 521.2250.
HFIP pretreated Ab1–42 samples (AnaSpec) were resolubilized
with a 50 mM phosphate buffer (pH 7.4) in order to have a stable
stock solution ([Ab] = 200
50 mM phosphate buffer (pH 7.4) at 37 °C for 48 h (final Ab con-
centration 50 M) with and without the tested compound at
20 M. After incubation, the samples were diluted to a final vol-
l
M).22 The peptide was incubated in
l
l
ume of 200 uL with 50 mM glycine–NaOH buffer (pH 8.0) contain-
ing thioflavin T. Then, a 300-seconds-time scan of fluorescence
intensity was performed (kexc = 450 nm; kem = 485 nm), and values
at plateau were averaged after subtracting the background fluores-
cence of thioflavin T solution.
4.4.15. 9-O-{3-[4-(Butyl)-1H-1,2,3-triazol-1-yl]propyl}berberine
bromide (18c)
Intermediate 4c was treated with CuSO4, sodium ascorbate and
1-hexyne according to general procedure to give the target product
18c as yellow semisolid. Yield 39.7%; 1H NMR (400 MHz,) d 9.84 (s,
1H), 8.92 (s, 1H), 8.17 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H),
7.94 (s, 1H), 7.77 (s, 1H), 7.08 (s, 1H), 6.16 (s, 2H), 4.98 (t,
J = 5.8 Hz, 2H), 4.64 (t, J = 6.6 Hz, 2H), 4.27 (t, J = 5.7 Hz, 2H), 4.02
(s, 3H), 3.23 (t, J = 6.8 Hz, 2H), 2.60 (t, J = 7.5 Hz, 2H), 2.45–2.37
(m, 2H), 1.62–1.49 (m, 2H), 1.37–1.24 (m, 2H), 0.86 (t, J = 7.3 Hz,
3H); LC/MS (ESI) m/z: [MꢀBr]+ 487.2; HRMS m/z [MꢀBr]+ Calcd
for C28H31N4O4 487.2304, Found 487.2308.
4.7. Kinetic characterization of AChE inhibition
Kinetic characterization of AChE was performed using a re-
ported method.23 Test compound was added into the assay solu-
tion and pre-incubated with the enzyme at 37 °C for 15 min,
followed by the addition of substrate. Kinetic characterization of
the hydrolysis of ATC catalyzed by AChE was carried out spectro-
metrically at 412 nm. A parallel control was made with the assay
solution of no inhibitor for each times. The plots were assessed
by a weighted least squareanalysis that assumed the variance of
V to be a constant percentage of V for the entire data set. Slopes
of these reciprocal plots were then plotted against the concentra-
tion of the inhibitors in a weighted analysis and Ki was determined
as the ratio of the replot intercept to the replot slope.
4.4.16. 9-O-{3-[4-(Butyl)-1H-1,2,3-triazol-1-yl]butyl}berberine
bromide (18d)
Intermediate 4d was treated with CuSO4, sodium ascorbate and
1-hexyne according to general procedure to give the target prod-
uct 18d as yellow ssemiolid. Yield 38.1%; 1H NMR (400 MHz,) d
9.77 (s, 1H), 8.95 (s, 1H), 8.18 (d, J = 9.0 Hz, 1H), 7.99 (d,
J = 9.2 Hz, 1H), 7.90 (s, 1H), 7.79 (s, 1H), 7.09 (s, 1H), 6.17 (s, 2H),
4.97 (t, J = 6.3 Hz, 2H), 4.43 (t, J = 6.8 Hz, 2H), 4.29 (t, J = 6.1 Hz,
2H), 4.04 (s, 3H), 3.22 (t, J = 6.7 Hz, 2H), 2.58 (t, J = 7.6 Hz, 2H),
2.13–2.02 (m, 2H), 1.88–1.76 (m, 2H), 1.59–1.48 (m, 2H), 1.37–
1.24 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H); LC/MS (ESI) m/z: [MꢀBr]+
501.3; HRMS m/z [MꢀBr]+ Calcd for C29H33N4O4 501.2532, Found
501.2529.
4.8. Molecular modeling
The simulation system was built based on the X-ray crystal
structure of the bis(7)tacrine-AChE complex which was obtained
from the Protein Data Bank (PDB entry 2CMF). The original ligand
was removed while water molecules present in the PDB file were
maintained in their position. 3D structures of the 9-substituted
berberine derivatives were generated and optimized by DISCOVERY
studio 2.1 package (Accelrys Inc., San Diego, CA). The CDOCKER

A. Shi et al. / Bioorg. Med. Chem. 19 (2011) 2298–2305
2305
10. Natarajan, A.; Du, W.; Xiong, C. Y.; DeNardo, G. L.; DeNardo, S. J.; Gervay-Hague,
J. Chem. Commun. 2007, 7, 695.
11. Bourne, Y.; Kolb, H. C.; Radic, Z.; Sharpless, K. B.; Taylor, P.; Marchot, P. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 1449.
program in DISCOVERY STUDIO 2.1 software was used to perform dock-
ing simulations, which allows full flexibility of the ligand.
12. Krasinski, A.; Radic, Z.; Manetsch, R.; Raushel, J.; Taylor, P.; Sharpless, K. B.;
Kolb, H. C. J. Am. Chem. 2005, 127, 6686.
Acknowledgments
13. Recanatini, M.; Valenti, P. Curr. Pharm. Des. 2004, 10, 3157.
14. Isabel, F. B.; Concepcion, P.; Nuria, E.; Juan, A. P.; Gema, C. G.; Paola, U.; Esther,
G. P.; Manuela, G. L.; Mercedes, V.; Antonio, G. G.; Ana, M.; Isabel, R. F. Chem.
Med. Chem. 2009, 4, 828.
15. Guillou, C.; Mary, A.; Renko, D. Z.; Gras, E.; Thal, C. Bioorg. Med. Chem. Lett.
2000, 10, 637.
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.
References and notes
16. 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.
17. Huang, L.; Shi, A. D.; He, F.; Li, X. S. Bioorg. Med. Chem. 2010, 18, 1244.
18. Iwasa, K.; Kamigauchi, M.; Ueki, M.; Taniguchi, M. Eur. J. Med. Chem. 1996, 31,
469.
19. Zhang, W. J.; Ou, T. M.; Lu, Y. J.; Huang, Y. Y.; Wu, W. B.; Huang, Z. S.; Zhou, J. L.;
Wong, K. Y.; Gu, L. Q. Bioorg. Med. Chem. 2007, 15, 5493.
20. Tsou, H. R.; Mamuya, N.; Johnson, B. D. J. Med. Chem. 2001, 17, 2719.
21. Ellman, G. L.; Courtney, K. D.; Andres, B. Jr.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
1. Bemis, G. W.; Murcko, M. A. . J. Med. Chem. 1996, 39, 2887.
2. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
3. Zhang, H.; Ryono, D. E.; Devasthale, P. Bioorg. Med. Chem. Lett. 2009, 19, 1451.
4. Calvo-Flores, F. G.; Isac-Garcia, J.; Hernandez-Mateo, F. Org. Lett. 2000, 2, 2499.
5. Perez-Balderas, F.; Ortega-Munoz, M.; Morales-Sanfrutos, J. Org. Lett. 2003, 5,
1951.
6. Lewis, W. G.; Green, L. G.; Grynszpan, R. Angew. Chem., Int. Ed. 2002, 41, 1053.
7. Whiting, M.; Muldoon, J.; Lin, Y. C. Angew. Chem., Int. Ed. 2006, 45, 1435.
8. Alvarez, R.; Velazquez, S.; San-Felix, A.; Balzarini, J.; Camarasa, M. J. J. Med.
Chem. 1994, 37, 4185.
22. Michela, R.; Elena, S.; Manuela, B. J. Med. Chem. 2008, 51, 4381.
23. Maria, L. B.; Vincenza, A.; Manuela, B.; Rita, B.; Carlo, M. J. Med. Chem. 2005, 48,
24.
9. Genin, M. J.; Allwine, D. A.; Anderson, D. J. J. Med. Chem. 2000, 43, 953.