Development of tacrine-bifendate conjugates with improved cholinesterase inhibitory and pro-cognitive efficacy and reduced hepatotoxicity

Article abstract of DOI:10.1016/j.ejmech.2017.12.005

A novel series of tacrine-bifendate (THA-DDB) conjugates (7a-e) were synthesized and evaluated as potential anti-Alzheimer's agents. These compounds showed potent cholinesterase and self-induced β-amyloid (Aβ) aggregation inhibitory activities. A Lineweaver–Burk plot and molecular modeling study showed that these compounds can target both catalytic active site (CAS) and peripheral anionic site (PAS) of acetylcholinesterase (AChE). The cytotoxicity of the conjugate 7d against PC12 and HepG2 cells and hepatotoxicity against human hepatocyte cell line (HL-7702) were found to be considerably less compared to THA. Moreover, treatment with 7d did not exhibit significant hepatotoxicity in mice. Finally, in vivo studies confirmed that 7d significantly ameliorates the cognitive performances of scopolamine-treated ICR mice. Therefore, 7d has high potential for the treatment of Alzheimer's disease and warrants further investigation.

Full text of DOI:10.1016/j.ejmech.2017.12.005

Accepted Manuscript
Development of tacrine-bifendate conjugates with improved cholinesterase inhibitory
and pro-cognitive efficacy and reduced hepatotoxicity
Juan Cen, Huiyan Guo, Chen Hong, Jianwu Lv, Yacheng Yang, Ting Wang, Dong
Fang, Wen Luo, Chaojie Wang
PII:
S0223-5234(17)31011-5
10.1016/j.ejmech.2017.12.005
EJMECH 9983
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 October 2017
Revised Date: 29 November 2017
Accepted Date: 2 December 2017
Please cite this article as: J. Cen, H. Guo, C. Hong, J. Lv, Y. Yang, T. Wang, D. Fang, W. Luo, C. Wang,
Development of tacrine-bifendate conjugates with improved cholinesterase inhibitory and pro-cognitive
efficacy and reduced hepatotoxicity, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.12.005.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Development of Tacrine-Bifendate Conjugates with Improved
Cholinesterase Inhibitory and Pro-Cognitive Efficacy and
Reduced Hepatotoxicity
Juan Cen, a Huiyan Guo, a Chen Hong,b Jianwu Lv, a Yacheng Yang, a Ting Wang, a
Dong Fang, c Wen Luo, a,* Chaojie Wang a,*
a
Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University,
Kaifeng 475004, China
b Huaihe Hospital, Henan University, Kaifeng 475000, China
c Pharmceutical College, Henan University, Kaifeng 475004, China
*
Wen Luo: Email luowen83@henu.edu.cn, Tel 086 0371-22864665, Fax 086
0371–22864665
*
Chao-Jie Wang: Email wcjsxq@163.com, Tel 086 0371-22864665, Fax 086
0371–22864665
KEYWORDS
Tacrine
Bifendate
Alzheimer’s disease
Cholinesterase
Hepatoprotective

ACCEPTED MANUSCRIPT
ABSTRACT
A novel series of tacrine-bifendate (THA-DDB) conjugates (7a-e) were synthesized
and evaluated as potential anti-Alzheimer’s agents. These compounds showed potent
cholinesterase and self-induced β-amyloid (Aβ) aggregation inhibitory activities. A
Lineweaver–Burk plot and molecular modeling study showed that these compounds
can target both catalytic active site (CAS) and peripheral anionic site (PAS) of
acetylcholinesterase (AChE). The cytotoxicity of the conjugate 7d against PC12 and
HepG2 cells and hepatotoxicity against human hepatocyte cell line (HL-7702) were
found to be considerably less compared to THA. Moreover, treatment with 7d did not
exhibit significant hepatotoxicity in mice. Finally, in vivo studies confirmed that 7d
significantly ameliorates the cognitive performances of scopolamine-treated ICR mice.
Therefore, 7d has high potential for the treatment of Alzheimer’s disease and warrants
further investigation.

ACCEPTED MANUSCRIPT
1. Introduction
Alzheimer’s disease (AD), a chronic neurodegenerative disease is primarily
associated with dementia, and often characterized by cognitive impairment, and
memory loss. Elderly people are more susceptible to AD, although approximately 5%
of AD patients are aged below 65 [1]. In 2010, approximately, 36 million people were
suffering from dementia worldwide and that number could increase up to 115 million
by 2050 [2]. Despite of numerous researches and efforts, any substantial curative
therapy for AD could not be developed till date.
Limited numbers of U.S. Food and Drug Administration (FDA) approved drugs are
currently available for AD therapy, which includes three acetylcholinesterase (AChE)
inhibitors (donepezil, rivastigmine and galantamine) and one NMDA antagonist
(memantine). The most potent AChE inhibitor, tacrine (THA, Figure 1) was
withdrawn from the market for its severe reversible hepatotoxicity caused by
increased transaminase levels in 30-50% of patients treated with the drug [3].
THA, a well-known multitarget direct ligand (MTDL) with a relatively simple
chemical structure, can inhibit both AChE and butyrylcholinesterase (BuChE) in
nanomolar concentration [4]. THA molecule can be easily modified by linking with
other pharmacologically active moieties to produce different multifunctional
cholinesterase (ChE) inhibitors, such as THA-caffeic acid hybrids, THA-silibinin
codrug (Figure 1, D), THA-quinone hybrids, and THA-benzofuran hybrids [5-8].

ACCEPTED MANUSCRIPT
NH2
O
O
O
O
HN
N
n-2
H
O
O
OH
O
N
A
N
MeOOC
Bifendate
COOMe
Tacrine
N
O
O
O
HN
N
N
O
O
O
n-2
HN
N
H
N
n-2
H
OMe
C
B
O
H
O
O
N
O
NH
N
HO
O
O
OH
OH
OCH3
OH
O
Tacrine-Silibinin Co-drug
D
Figure 1. Structures of tacrine (THA), bifendate (DDB), our previous THA hybrids (A, B and C)
and THA-silibinin codrug (D).
We have undertaken a novel approach to reduce or abate the THA-induced toxicity.
THA-vanillin conjugates and THA-flavonoid derivatives (Figure 1, A, B), synthesized
and reported in our earlier researches, have shown higher potency as AChE inhibitors
or antioxidants [9, 10], but lower cell toxicity in vitro (such as PC12 cell) than THA,
which encouraged us to explore Bifendate (DDB) conjugates (an analog of
schizandrin C, Figure 1) to specifically reduce the hepatotoxicity of THA.
DDB has been widely used for the treatment of chronic viral hepatitis B since the
last three decades in China [11]. DDB can reduce the CCl4-induced liver injury and
transaminase levels [12, 13], and was reported to protect the liver mitochondria in
mice from THA-induced injury [14, 15].
Compounds containing electron-rich aromatic groups like methoxy- and
methylenedioxybenzene (Figure 1, C) can bind to the peripheral anionic site (PAS) of

ACCEPTED MANUSCRIPT
AChE [16, 17]. Diamine moieties are present in various pharmaceutical compounds to
improve solubility, to enhance bioavailability and to increase metabolic stability in
vivo [18]. In this study, we conjugated THA and DDB moieties via a diamine linkage
to develop compounds with better cholinesterase (ChE) inhibitory activity and lower
hepatoxicity than the parent molecule.
The synthesized conjugates were evaluated for several biological and
pharmacological properties like, in vitro AChE and BuChE inhibitory activity,
cytotoxicity towards human hepatoma cell line HepG2 and rat pheochromocytoma
cell line PC12, and toxicity toward the human hepatocyte cell line HL-7702. A
conjugate 7d was assayed in vivo in mice to assess hepatotoxicity by observing the
histomorphological changes in the liver. The in vivo pro-cognitive effects of 7d were
evaluated through the application of scopolamine in mice and exposing to Morris
water maze (MWM) and Y-maze, as per the behavioral pharmacology protocol to
ascertain the antidementive potential of the compound 7d [19, 20].
2. Results and discussion
2.1. Chemistry
The THA conjugates 7a–e were prepared following a sequence of reactions as
shown in the Scheme 1. Intermediate 3 was synthesized from anthranilic acid with
90% overall yield following a previously reported procedure (Supporting Information)
[21]. Compound 3 and alkylamine were refluxed for 10 h in the presence of KI in
1-pentanol to obtain 9-alkylamino-tetra-hydroacridines (4a–e) with good yields.
Intermediate 6 was synthesized by following a three-stepped reaction process

ACCEPTED MANUSCRIPT
(Supporting Information) [22]. In the final step, the target compounds 7a–e were
obtained by the reaction of intermediates 4a–e with the intermediate 6 in the presence
of Et3N in CHCl3.
O
NH2
O
O
HOOC
MeOOC
MeOOC
O
O
1
2
a
O
5
O
Cl
N
c
O
3
O
b
MeOOC
ClOC
O
O
HN
N
n-2
NH2
O
4a-e
n = 2,4,6,8,10
6
O
d
O
O
MeOOC
O
O
H
H
N
N
n-2
N
O
O
O
7a-e
n = 2,4,6,8,10
Scheme 1. Synthesis of THA-DDB hybrids 7a-e. Reagents and conditions: (a) i. toluene, reflux
(Dean-Stark Trap), 2 h; ii. POCl3, 120 °C, 2 h; (b) diamine, KI, 1-pentanol, 160 °C, 10 h. (c) i.
5%KOH, reflux, 6 h; ii. (Ac)2O, reflux 12 h; iii. MeOH, reflux, 9 h; iv. SOCl2, 1,4-dioxane, DMF,
115 °C, 2 h; (d) CHCl3, Et3N, rt, 24 h.
2.2. Inhibition studies on AChE and BuChE
The inhibitory activities of the compounds were evaluated against AChE and
BuChE using THA as the positive control using the Ellman assay (Table 1). The IC50

ACCEPTED MANUSCRIPT
values and selectivity indexes (SI) for the inhibition of AChE and BuChE are
summarized in Table 1.
As expected, DDB (5) did not inhibit either AChE or BuChE even at a
concentration of 50 µM. Generally, the synthesized conjugates (7a-e) showed potent
inhibitory activities at the nanomolar concentrations, and good selectivity for BuChE
over AChE was observed. Among these compounds, the conjugate 7d with a
diaminooctane (n=8) linker between its two units was found to be the most potent
AChE inhibitor, with an IC50 of 27.32 nM. The compound 7b with a butanediamine
(n=4) linker showed the strongest inhibition towards BuChE, with an IC50 value of
4.02 nM. Recently, BuChE has been considered as a potential drug-target because of
its ability to regulate acetylcholine (ACh) levels [23]. Greig et al. showed that
cognitive performances of aged rats were improved and the amount of Aβ was
lowered through selective BuChE inhibition [24]. Therefore, concurrent inhibition of
AChE and BuChE may provide additional benefits for the treatment of AD.
Variation in the chain length of the inhibitors (7a-e) had a greater effect on AChE
inhibition than BuChE, which may be due to the conformational differences between
these enzymes. As the active site of BuChE is wider than that of AChE, BuChE can
accommodate the inhibitors with variable linker lengths.
The inhibitory potency of the compounds 7a-e was also assessed on recombinant
human AChE (hAChE) and BuChE (hBuChE) (Supporting Information, Table S1).
As the hAChE and hBuChE were highly expensive, we evaluated the compounds only
at the concentration of 100 nM. The compound 7d also showed higher inhibition than

ACCEPTED MANUSCRIPT
THA on both enzymes.
In summary, these conjugates were potent AChE and BuChE inhibitors in the
nanomolar range. The compound 7d exhibited better inhibitory activity towards both
AChE and BuChE than THA.
Table 1. Inhibition of AChE and BuChE, selectivity index (SI), and inhibition of
Aβ1-42 self-aggregation.
IC50a for AChE IC50b for BuChE
A
β
inhibition
(%) d
SI c
12.2
153.1
145.3
3.2
Compds.
n
(nM）
(nM)
7a
7b
7c
7d
7e
2
4
922.76±35.44
75.46±3.02
65.9±8.8
67.3±6.1
90.1±7.9
82.5±10.4
85.6±3.0
615.51±49.63
944.65±33.21
27.32±1.61
4.02±0.35
6.50±0.31
8.63±1.02
35.31±0.87
>50000
6
8
10
52.13±5.13
1.5
DDB
THA
-
-
-
>50000
143.5±4.15
-
-
5.9
-
-
24.27±1.32
5.6±1.6
53.1±3.3
Curcumin
-
a
b
c
AChE from electric eel; BuChE from equine serum; Selectivity Index = IC50 (AChE) / IC50
(BuChE) d The thioflavin-T (ThT) fluorescence method was used and the compounds were used at
a concentration of 20 µM. Data are represented as mean ± SD.
2.3 Kinetic characterization of AChE inhibition

ACCEPTED MANUSCRIPT
35
non inhibitor
[7d]=25 nM
[7d]=50 nM
30
[7d]=100 nM
25
20
15
10
5
0
-10
-5
0
5
10
-1
15
20
-1
[S] mM
Figure 2. Lineweaver-Burk plot for the inhibition of AChE by compound 7d.
We cultured the crystal of 7d with Torpedo californica AChE to elucidate the
interaction patterns of 7d with AChE; though, no positive results were obtained. The
interaction patterns of 7d with AChE were further studied through the graphical
analysis of steady state inhibition data, as shown in the Figure 2. Enzyme kinetic
analyses indicated a mixed-type inhibition, with an increasing slope (decreased Vmax
)
and an increasing intercept (higher Km) for higher inhibitor concentrations. The result
suggested that 7d could bind to both the catalytic active site (CAS) and the PAS of
AChE.
Figure 3. Docking models of the compound−enzyme complex. (A) Molecular structure of
conjugate 7d. (B) Representation of compound 7d docked into the binding site of AChE
highlighting the protein residues that form the main interactions with the inhibitor.

ACCEPTED MANUSCRIPT
2.4 Molecular modeling
Molecular docking is an important tool in recent years for elucidating the
interactions between ligands with receptors. We performed molecular docking using
docking program AUTODOCK 4.2 package to understand the binding interactions
between the hybrid 7d and AChE, and the results are shown in the Figure 3. The
results of the docking studies indicate that conjugate 7d was bonded with AChE via
multiple binding modes. In the TcAChE-7d complex, 7d occupied the entire
enzymatic CAS, mid-gorge and PAS. The THA moiety was bound to CAS by a
classic π-π stacking interaction between Trp84 and Phe330. The protonated nitrogen
atom of the quinoline ring was bound to the carbonyl group of His440 via a hydrogen
bond (3.4 Å) interaction. In the PAS, the DDB moiety was bound to the PAS by
hydrophobic interaction. Multiple interactions between 7d and AChE may be
responsible for more potent inhibitory effect of the molecule than THA. The docking
studies confirmed a mixed type of inhibition, which was consistent with our kinetic
analysis result.
2.5. Inhibition of self-mediated Aβ1-42 aggregation by Thioflavin-T (ThT)
Aβ1-42 and Aβ1-40 are the main forms of Aβ peptides found in amyloid plaques,
Aβ1-42 is more fibrillogenic than Aβ1-40 [25]. Numerous previous studies have
indicated that schizadrin derivatives and the compounds containing
a
methylenedioxybenzene moiety may possess significant inhibitory activity towards
Aβ self-induced aggregation [17, 26], which inspired us to select Aβ1-42 for evaluating
the inhibitory activities of the synthesized conjugates using a ThT fluorescence

ACCEPTED MANUSCRIPT
method. The compounds 7a–e prevented the self-mediated Aβ1-42 aggregation
(percentages of inhibition: 66% - 90%) in a significantly better way than the reference
compound curcumin (percentages of inhibition: 53%) (Table 1). However, THA was
quite inefficient to inhibit Aβ1-42 aggregation (percentages of inhibition: 5.6%).
Figure 4. TEM images of Aβ1-42 self-induced aggregation in the presence and absence of test
compounds. (A) 50 µM Aβ1–42 without inhibitors at 0 h, (B) 50 µM Aβ1–42 without inhibitors at 48
h, 37 °C, (C) 50 µM Aβ1–42 and 20 µM 7d, (D) 50 µM Aβ1–42 and 20 µM curcumin.
Aβ1-42 aggregation potential of 7d was also evaluated by Transmission Electron
Microscopic (TEM) study [27]. Fig 4A represents the Aβ1-42 aggregation at the start of
the incubation at 37 °C without any inhibitor. The Aβ1-42 after 48 h of incubation
formed a firm, dense and bulky aggregates (Figure 4B). The inhibitory effect was
clearly visible when treated with the compound 7d at 48 h (20 µM) and lesser
numbers of Aβ aggregates were visible (Figure 4, C) compared to the treatment with
standard curcumin (Figure 4, D). The results of TEM are consistent with the ThT
assay where compound 7d potentially inhibit Aβ self-induced aggregation.
2.6. In vitro cytotoxicity

ACCEPTED MANUSCRIPT
Figure 5. The effects of compounds on the HL-7702 cell viability by MTT assay. Data represents
the mean ± SD, (n=3; *P<0.05, **P<0.01 vs control group).
MTT assay was performed using the human hepatoma cell line HepG2 and the rat
pheochromocytoma cell line PC12 to evaluate the cytotoxicity of the compounds
(7a-7e). Most of the compounds showed less toxicity on cell viability than THA at the
concentrations of 10 µM (Supporting Information, Table S2), and 7d exhibited low
toxicity towards both the cell lines. The hepatotoxicity of the compound 7d was
investigated by treating the compound with the human normal hepatocyte cell line
HL-7702 for cell viability assessment (Figure 5). The compound 7d and DDB also
exhibited low hepatotoxicity compared to THA.
Consistent with the results of MTT assay, the apoptosis rates in 10 µM THA-treated
and 10 µM 7d-treated HL-7702 cells were 19.8±3.7%, and 10.8±2.1%, respectively,
while LY294002 enhanced 7d-induced apoptosis to 14.1±3.3% (Supporting
Information, Figure S2). Since LY294002 is a PI3K inhibitor, the results suggest that
phosphatidylinositol 3-kinase (PI3K) signal pathway may mediate its potential
hepatotoxicity/hepatoprotection. To reveal the potential mechanism, further
investigation is needed.
In conclusion, in vitro studies indicate that 7d possess less hepatotoxicity than

ACCEPTED MANUSCRIPT
THA.
2.7. In vivo studies on hepatotoxiciy/hepatoprotection in mice
Figure 6. Histomorphological appearance of the liver of a male control mice (A) 48 h after
treatment with THA (B), compound 7d (C) DDB plus THA (D) or DDB (E), respectively. H&E;
original magnification: ×200. V, central vein; P, portal field.
The H&E-stained sections of the mice liver from the control group, THA and 7d
treated group can represent the in vivo hepatotoxic nature of the compounds. Figure 6
showed that the control liver had normal architecture, characterized by complete and
clear liver tissue structure, close-connected liver cells, and normal hepatic lobules. In
contrast, the THA-treated liver tissue demonstrated cellular edema, vacuolar
degeneration in the perivascular tissue, focal necrosis, and a large number of
inflammatory cells infiltrates (Figure 6B). The treatment with 7d, THA plus DDB or
DDB alone at the same dosage level exhibited less adverse histomorphological
changes compared to the treatment with THA alone (Figure 6C, D and E).
The severe hepatotoxicity of THA is the primary limitation for the clinical use of
the compound. THA can cause a reversible hepatotoxicity in 30-50% of patients, as

ACCEPTED MANUSCRIPT
indicated by the increase in transaminase levels during clinical use [3]. Compound 7d,
DDB and THA were assayed at equimolar concentrations in adult mice, and the
toxicity profiles of the compounds were compared. Heparinized serum was collected
at 48 h after the administration of DDB, THA and 7d, and the levels of alanine
aminotransferase (ALT) and aspartate aminotransferase (ASAT) were evaluated
(Table 2). The serum levels of ALT and ASAT are directly proportional to the
severity of the liver damage. Significantly elevated ALT and ASAT activities in the
THA-treated mice indicate acute liver damage. Specifically, after 48 h of THA
administration, statistically significant increases in both ALT (1.5-fold, p ≤ 0.01) and
ASAT (1.3-fold, p ≤ 0.05) were observed; whereas, negligible increase in ALT
(1.1-fold, not statistically significant) and a small decrease in ASAT (1.1- fold, not
statistically significant) was noticed after 7d administration. As expected, DDB had
little influence on ALT and ASAT levels (not statistically significant) at the tested
concentration [28]. THA plus DDB also did not increase the levels of ALT and ASAT.
Finally, the results show that 7d possess a significantly lower hepatotoxicity than
THA when tested at equimolar doses.
Table 2. ALT and ASAT activity after 48 h of the administration of 7d, DDB, THA
and THA plus DDB a
Compds.
Control
7d
ALT(U/L)
ASAT(U/L)
42.7±6.3
45.2±14.8
41.9±10.2
101.5±22.3
92.3±19.4
110.0±28.5
DDB+THA

ACCEPTED MANUSCRIPT
DDB
THA
39.3±6.5
105.0±8.1
136.1±38.7*
62.6±19.6**
a Values are expressed as the mean ± SD (n = 8; *p ≤ 0.05, **p ≤ 0.01 vs control group).
2.8. Behavioral studies
Figure 7. Effect of 7d treatment on spatial memory performance. (A) Escape latencies during
acquisition training trials measured for 90 s for each trial; (B) The path to find the platform on the
4th day of acquisition training trials; (C) The number to cross the platform in probe trial without
platform; (D) The percentage of path length in the target quadrant measured for 90 s in probe trial
without platform; (E) The swimming time in the target quadrant measured for 90 s in probe trial
without platform. Data are presented as mean ± S.D. (n=8; *p<0.05, **p<0.01 vs control group,
and #p<0.05, ##p<0.01 vs model group).
The in vitro cholinesterase inhibitory activities of anti-AD agents must translate
into behavioral effects and improve the cognition in the experimental animal. The
ameliorating potential of 7d against scopolamine-induced cognition impairment in
ICR mice was investigated in MWM and Y-maze [29, 30]. The results show that the
treatment with 7d significantly ameliorated the cognition impairment in mice

ACCEPTED MANUSCRIPT
compared to THA in equimolar doses of 20 µmol/kg body weight (Figure 7). In
MWM test, mice in control group exhibited a reduction of mean escape latency from
62.2 to 42.3s (p < 0.01) over the course of 4 training days. Compared to the model
group, the 7d-treated and THA-treated group showed significantly shortened escape
latency in the 4th day of training trial sessions (p < 0.05); the compound 7d reduced
the mean escape latency (MEL) from 60.2 to 46.4 s while THA decreased the MEL
from 59.8 to 48.3 s (p < 0.05) (Figure 7 A). Furthermore, in the probe trial on day
15, the administration of 7d significantly improved the overall target quadrant
preference (the number to cross the platform, the percentage of path length in the
target quadrant, and the swimming time in the target quadrant) compared to the model
group (p < 0.05) (Figure 7, C, D, E), while no significant differences were observed
between the two groups in the total distance traveled and swimming speed
(Supporting Information, Figure S4). We also measured the protective effect of 7d
against memory deficit in scopolamine-treated mice using the Y-maze task (Figure 8).
Treatment with 7d significantly reversed the lowered spontaneous alteration induced
by scopolamine when compared to the control group (p < 0.01), and exhibited higher
potency than that of THA (p < 0.05 vs. control). These results indicate that 7d
significantly ameliorated the cognition impairment in the scopolamine treated mice
probably by penetrating the blood−brain barrier to affect the central nervous system.
Figure 8. Effects of 7d on scopolamine-induced memory deficit in the Y-maze tasks. (A) Total
number of arm entries. (B) The percentage of spontaneous alteration. Data are presented as mean
± S.D. (n=8; *p<0.05, **p<0.01 vs control group, and #p<0.05, ##p<0.01 vs model group).
3. Conclusion
A series of novel conjugates of THA-DDB (7a–e) were designed, synthesized and

ACCEPTED MANUSCRIPT
evaluated both in vitro and in vivo. The conjugate 7d was found to be the most potent
AChE inhibitor with stronger inhibition than THA, and less hepatotoxic than THA
against HL-7702 cell line without any obvious cytotoxicity towards HepG2 and PC12
cell lines. Histopathology studies show that the necrosis was reduced in the 7d-treated
group to a lesser degree compared to the THA-treated group. In addition, no
significant increases in ALT and ASAT levels were observed after the administration
of 7d in mice. Both the in vitro and in vivo studies indicated that 7d possess lesser
hepatotoxicity than THA. Furthermore, administration of 7d considerably ameliorated
the cognition impairment in the scopolamine treated ICR mice in MWM and Y-maze
test. Finally, the THA−DDB conjugate 7d can be a promising lead compound for
further development of newer anti-AD drugs.
4. Experimental section
4.1 Chemistry
All chemicals (reagent grade) were purchased from J&K and Sigma-Aldrich
(China). Intermediate compounds 4a−e and 6 were synthesized following previously
reported protocols (Supporting Information) [22, 23]. Reactions were followed by
TLC on silica gel GF254 plates (Qingdao Haiyang Chemical Plant, Qingdao, China),
and the spots were visualized using a UV lamp (λ = 254 nm). Chromatographic
separations were performed using silica gel columns (200-300 mesh; Qingdao Marine
1
13
Chemical Inc., Qingdao, China) by flash chromatography. H and C NMR spectra
data were obtained from a Bruker AV-400 spectrometer (400 and 100 MHz,

ACCEPTED MANUSCRIPT
respectively). Chemical shifts are reported in parts per millions (ppm) relative to
tetramethylsilane (TMS), and spin multiplicities are given as s (singlet), brs (broad
singlet), d (doublet), t (triplet), q (quartet), or m (multiplet). High resolution mass
spectra (HRMS) were recorded on Shimadzu LCMS-IT-TOF. Elemental analyses
were performed on a Gmbe VarioEL Elemental Instrument.
4.2. General procedure for the preparation of target compounds 7a e.
−
The intermediate 6 (2.0 mmol, freshly prepared, cf. Supporting Information) was
dissolved in CHCl3 (10 mL), and mixed with Et3N (1.0 mL) and the intermediate 4
(2.0 mmol). The mixture was stirred at room temperature for 24 h, washed with water
and brine, dried over MgSO4, and concentrated under reduced pressure. The residue
was purified by column chromatography (CHCl3: MeOH: NH4OH = 30:1:0.5%) to
obtain the target compounds 7a-e.
4.2.1.
Methyl-7,7'-dimethoxy-5'-(2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethylcarbamoyl)-4,
4'-bibenzo[d][1,3]dioxole-5-carboxylate (7a)
Yellow oil, yield 63%. 1H NMR (400 MHz, CDCl3): δ ppm 7.91 (d, J = 8.4 Hz, 1H,
Ar-H), 7.88 (d, J = 8.4 Hz, 1H, Ar-H), 7.45 (t, J = 7.6 Hz, 1H, Ar-H), 7.30 (t, J = 6.0
Hz, 1H, Ar-H), 7.23 (t, J = 6.0 Hz, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 6.99 (s, 1H, Ar-H),
5.90 (s, 1H, -CONH-), 5.84 (s, 2H, -OCH2O-), 5.75 (d, J = 1.2 Hz, 1H, -OCHHO-),
5.68 (d, J = 1.2 Hz, 1H, -OCHHO-), 5.03 (brs, 1H, -NHAr), 3.84 (s, 3H, -OCH3), 3.70
(s, 3H, -OCH3), 3.63 (s, 3H, -OCH3), 3.60 – 3.53 (m, 1H, -CONHCHH-), 3.53 – 3.50
(m, 2H, -CH2NHAr-), 3.42 – 3.35 (m, 1H, -CONHCHH-), 2.96 (s, 2H, -CH2Ar), 2.50

ACCEPTED MANUSCRIPT
(s, 2H, -CH2Ar), 1.78 – 1.72 (m, 4H, 2× -CH2-). 13C NMR (100 MHz, CDCl3): δ ppm
170.19, 167.38, 155.67, 152.59, 147.78, 146.74, 144.06, 143.12, 142.66, 138.28,
136.56, 129.68, 129.59, 125.33, 124.23, 124.12, 123.32, 118.29, 113.90, 110.85,
110.63, 108.65, 108.17, 102.59, 102.14, 56.60, 56.56, 52.41, 50.79, 40.65, 31.86,
24.75, 22.70, 21.96. HRMS (ESI): calcd for [M+H]+ (C34H33N3O9) requires m/z
628.2295, found 628.2288. Elemental Anal. Calcd for C34H33N3O9: C, 65.06; H, 5.30;
N, 6.69. Found: C, 65.08; H, 5.23; N, 6.48.
4.2.2.
Methyl-7,7'-dimethoxy-5'-(4-(1,2,3,4-tetrahydroacridin-9-ylamino)butylcarbamoyl)
-4,4'-bibenzo[d][1,3]dioxole-5-carboxylate (7b)
1
Yellow oil, yield 61%，1H NMR (400 MHz, CDCl3): δ ppm H NMR (400 MHz,
CDCl3) δ 7.91 (d, J = 1.6 Hz, 1H, Ar-H), 7.90 (d, J = 1.6 Hz, 1H, Ar-H), 7.51 (t, J =
8.4 Hz, 1H, Ar-H), 7.31 (t, J = 8.0 Hz, 1H, Ar-H), 7.17 (s, 1H, Ar-H), 7.00 (s, 1H,
Ar-H), 6.27 (t, J = 6.0 Hz, 1H, -CONH-), 5.94 (dd, J = 14.8, 1.2 Hz, 2H, -OCH2O-),
5.89 (s, 2H, -OCH2O-), 4.33 (brs, 1H, -NHAr), 3.88 (s, 3H, -OCH3), 3.82 (s, 3H,
-OCH3), 3.72 (s, 3H, -OCH3), 3.37 (t, J = 7.2 Hz, 2H, -CH2NHAr), 3.34 – 3.27 (m,
1H, -CONHCHH-), 3.11 – 3.08 (m, 1H, -CONHCHH-), 3.02 (s, 2H, -CH2Ar), 2.63 (s,
2H, -CH2Ar), 1.87 – 1.83 (m, 4H, 2× -CH2CH2Ar), 1.46 – 1.38 (m, 2H, -CH2-), 1.34 –
13
1.26 (m, 2H, -CH2-). C NMR (100 MHz, CDCl3): δ ppm 168.13, 167.51, 157.47,
151.27, 147.87, 146.45, 146.12, 143.28, 142.83, 138.42, 136.24, 130.53, 128.84,
127.50, 124.44, 123.92, 122.90, 119.63, 115.51, 110.60, 108.19, 107.81, 102.68,
102.13, 56.68, 56.44, 52.53, 48.77, 39.17, 33.16, 28.60, 26.96, 24.65, 22.87, 22.44.

ACCEPTED MANUSCRIPT
HRMS (ESI): calcd for [M+H]+ (C36H37N3O9) requires m/z 656.2608, found 656.2610.
Calcd for C36H37N3O9: C, 65.94; H, 5.69; N, 6.41. Found: C, 65.57; H, 5.49; N, 6.60.
4.2.3.
Methyl-7,7'-dimethoxy-5'-(6-(1,2,3,4-tetrahydroacridin-9-ylamino)hexylcarbamoyl
)-4,4'-bibenzo[d][1,3]dioxole-5-carboxylate (7c)
1
Yellow oil, yield 41%，H NMR (400 MHz, CDCl3): δ ppm 8.00 (d, J = 8.4 Hz, 1H,
Ar-H), 7.97 (d, J = 8.4 Hz, 2H, Ar-H), 7.55 (t, 1H, J = 8.0 Hz, Ar-H), 7.35 (t, 1H, J =
8.4 Hz, Ar-H), 7.23 (s, 1H, Ar-H), 7.05 (s, 1H, Ar-H), 6.21 (t, 1H, J = 5.6 Hz,
-CONH-), 5.97 (dd, J = 7.6, 1.2 Hz, 2H, -OCH2O-), 5.92 (s, 2H, -OCH2O-), 4.47 (brs,
1H, -NHAr), 3.92 (s, 3H, -OCH3), 3.90 (s, 3H, -OCH3), 3.76 (s, 3H, -OCH3), 3.51 (t,
2H, J = 6.8 Hz, -CH2NHAr), 3.32 (m, 1H, -CONHCHH-), 3.08 (s, 2H, -CH2Ar), 3.05
– 2.99 (m, 1H, -CONHCHH-), 2.70 (s, 2H, -CH2Ar), 1.90 (s, 4H, 2× -CH2CH2Ar),
1.65 – 1.55 (m, 2H, -CH2-), 1.32 – 1.17 (m, 4H, 2× -CH2-), 1.13 – 1.03 (m, 2H,
-CH2-). 13C NMR (100 MHz, CDCl3): δ ppm 167.95, 167.41, 157.18, 151.57, 147.90,
146.45, 145.93, 143.27, 142.82, 138.48, 136.20, 130.70, 128.93, 127.27, 124.45,
123.85, 123.08, 119.49, 115.13, 110.76, 110.69, 108.31, 107.89, 102.63, 102.08, 56.74,
56.45, 52.44, 49.09, 39.39, 33.04, 31.37, 29.35, 26.49, 26.35, 24.65, 22.87, 22.41.
HRMS (ESI): calcd for [M+H]+ (C38H41N3O9) requires m/z 684.2921, found 684.2924.
Calcd for C38H41N3O9: C, 66.75; H, 6.04; N, 6.15. Found: C, 66.42; H, 6.23; N, 6.24.
4.2.4.
Methyl-7,7'-dimethoxy-5'-(8-(1,2,3,4-tetrahydroacridin-9-ylamino)octylcarbamoyl)
-4,4'-bibenzo[d][1,3]dioxole-5-carboxylate (7d)

ACCEPTED MANUSCRIPT
1
Yellow oil, yield 66%，H NMR (400 MHz, CDCl3): δ ppm 7.98 (d, J = 8.0 Hz, 1H,
Ar-H), 7.93 (d, J = 8.4 Hz, 1H, Ar-H), 7.55 (t, J = 7.2 Hz, 1H, Ar-H), 7.34 (t, J = 7.6
Hz, 1H, Ar-H), 7.23 (s, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 6.11 (t, J = 5.6 Hz, 1H,
-CONH-), 5.98 (dd, J = 8.4, 1.2 Hz, 2H, -OCH2O-), 5.92 (s, 2H, -OCH2O-), 4.17 (brs,
1H, -NHAr), 3.93 (s, 3H, -OCH3), 3.92 (s, 3H, -OCH3), 3.76 (s, 3H, -OCH3), 3.50 (t, J
= 7.2 Hz, 2H, -CH2NHAr), 3.35 – 3.26 (m, 1H, -CONHCHH-), 3.07 (t, J = 6.0 Hz, 2H,
-CH2Ar), 3.04 – 2.97 (m, 1H, -CONHCHH-), 2.70 (s, 2H, -CH2Ar), 1.93 – 1.90 (m,
4H, 2× -CH2CH2Ar), 1.69 – 1.60 (m, 2H, -CH2-), 1.40 – 1.31 (m, 2H, -CH2-), 1.26 –
1.14 (m, 6H, 3× -CH2-), 1.07 – 0.97 (m, 2H, -CH2-). 13C NMR (100 MHz, CDCl3): δ
ppm 167.87, 167.42, 157.94, 151.11, 147.92, 146.87, 146.42, 143.31, 142.85, 138.51,
136.19, 130.78, 128.54, 128.18, 124.42, 123.69, 122.95, 119.94, 115.54, 110.75,
110.67, 108.32, 107.90, 102.65, 102.08, 56.71, 56.44, 52.43, 49.41, 39.61, 33.65,
31.67, 29.34, 29.16, 26.88, 26.60, 24.73, 23.00, 22.66. HRMS (ESI): calcd for
[M+H]+ (C40H45N3O9) requires m/z 712.3234, found 712.3247. Elemental Anal. Calcd
for C40H45N3O9: C, 67.50; H, 6.37; N, 5.90. Found: C, 67.33; H, 6.13; N, 5.66.
4.2.5.
Methyl-7,7'-dimethoxy-5'-(10-(1,2,3,4-tetrahydroacridin-9-ylamino)decylcarbamoy
l)-4,4'-bibenzo[d][1,3]dioxole-5-carboxylate (7e)
1
Yellow oil, yield 51%，H NMR (400 MHz, CDCl3): δ ppm 7.97 (t, J = 8.8 Hz, 2H,
2× Ar-H), 7.56 (t, J = 7.6 Hz, 1H, Ar-H), 7.35 (t, J = 7.6 Hz, 1H, Ar-H), 7.23 (s, 1H,
Ar-H), 7.06 (s, 1H, Ar-H), 6.08 (t, J = 5.6 Hz, 1H, -CONH-), 5.99 (dd, J = 8.0, 1.2 Hz,
2H, -OCH2O-), 5.93 (s, 2H, -OCH2O-), 4.15 (brs, 1H, -NHAr), 3.94 (s, 3H, -OCH3),

ACCEPTED MANUSCRIPT
3.94 (s, 3H, -OCH3), 3.76 (s, 3H, -OCH3), 3.52 (t, J = 7.2 Hz, 2H, -CH2NHAr), 3.34 –
3.25 (m, 1H, -CONHCHH-), 3.08 (s, 2H, -CH2Ar), 3.05 – 2.99 (m, 1H,
-CONHCHH-), 2.70 (s, 2H, -CH2Ar), 1.94 – 1.88 (m, 4H, 2× -CH2CH2Ar), 1.72 –
1.63 (m, 2H, -CH2-), 1.40 – 1.36 (m, 2H, -CH2-), 1.30 – 1.12 (m, 10H, 5× -CH2-),
1.06 – 0.97 (m, 2H, -CH2-). 13C NMR (100 MHz, CDCl3): δ ppm 167.89, 167.46,
157.76, 151.30, 147.93, 146.41, 143.33, 142.88, 138.54, 136.19, 130.79, 128.71,
127.97, 124.40, 123.75, 123.00, 119.80, 115.35, 110.73, 110.66, 108.31, 107.90,
102.68, 102.10, 56.71, 56.44, 52.45, 49.47, 39.68, 33.47, 31.74, 29.48, 29.36, 29.34,
29.26, 26.93, 26.71, 24.68, 22.97, 22.60. HRMS (ESI): calcd for [M+H]+
(C42H49N3O9) requires m/z 740.3547, found 740.3573. Elemental Anal. Calcd for
C42H49N3O9: C, 68.18; H, 6.68; N, 5.68. Found: C, 67.92; H, 7.74; N, 5.40.
5. Biological activity
5.1. In vitro inhibition studies on AChE and BChE
AChE (E.C.3.1.1.7, Type VI−S, from electric eel) and BuChE (E.C.3.1.1.8, from
equine serum) were utilized for the inhibition studies, and the assays were performed
as previously described [31] (Supporting Information).
5.2. Kinetic characterization of AChE inhibition
Kinetic characterization of AChE was performed as per our previously reported
method [10] (Supporting Information).
5.3. Molecular modeling
The crystal structure of the torpedo AChE (code ID: 1ACJ) was obtained from the
Protein Data Bank after eliminating the inhibitor and water molecules. Docking

ACCEPTED MANUSCRIPT
studies were carried out using the AUTODOCK 4.2 program using ADT. Polar
hydrogen atoms were added and Gasteiger charges were assigned to the enzyme. The
resulting enzyme structure was used as an input for the AUTOGRID program.
AUTOGRID performed a pre-calculated atomic affinity grid map for each atom type
in the ligand, and plus an electrostatics map and a separate desolvation map present in
the substrate molecule. All maps were calculated with 0.375 Å spacing between grid
points. The center of the grid box was placed at the bottom of the active site gorge
(AChE [2.781 64.383 67.971]). The dimensions of the active site box were set at 50 ×
50 × 50 Å.
Flexible ligand docking was performed for the compounds. Docking calculations
were carried out using the Lamarckian genetic algorithm (LGA) and all parameters
were remained same for each docking. To ensure the reliability of the results, the
docking procedures were repeated 10 independent times for the compounds and the
obtained orientations were analyzed.
5.4. Inhibition of self-mediated Aβ(1-42) aggregation
Aβ1-42 peptide (Millipore) was dissolved in ammonium hydroxide (1% v/v), and
diluted with phosphate buffer (pH 7.40, 20 mM) to 200 µM before use. Compounds
were firstly dissolved in DMSO to obtain a 10 mM solution. The peptide was
incubated in phosphate buffer (pH 7.40, 20 mM) in 37 °C for 48 h (final Aβ
concentration of 50 µM) with or without the tested compounds at 20 µM. After
incubation, thioflavin-T (5 µM in 50 mM glycine-NaOH buffer, pH 8.00) was added.
Then a scan of fluorescence intensity was performed (λex = 450 nm; λem = 485 nm).

ACCEPTED MANUSCRIPT
The fluorescence intensities were recorded, and the percentage of inhibition on
aggregation was calculated by the following expression: (1-IFi/IFc)*100% in which IFi
and IFc were the fluorescence intensities obtained for absorbance in the presence and
absence of inhibitors, respectively, after subtracting the fluorescence of respective
blanks. Each assay was performed in triplicate.
5.5. Hepatotoxicity
All experiments were performed in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. The procedures were
approved by the Animal Care and Use Committee of the Henan University. Kunming
Mice, (male, 20 ± 2g) were purchased form Experimental Animal Center of
Zhengzhou University (Zhengzhou, China). The mice were housed in SPF grade
animal room, and kept in plastic cages in the Experimental Animal Center of Henan
University (Kaifeng, China). The animals were provided with free access to food and
water. THA was dissolved in sodium carboxymethyle cellulose (CMC-Na) solution,
35 mg/kg body weight, corresponding to 176.5 µmol/kg body weight, were given
intragastrically (ig). The tested compounds were dissolved in CMC-Na solution and
an equimolar dose (106 mg/kg body weight) corresponding to THA was administered
ig.
5.2.1. Histopathology
The liver tissue samples were collected after 48 hours of drug administration and
fixed with 4% paraformaldehyde, and then embedded by paraffin and cut. Paraffin
sections were stained with hematoxylin and eosin (H&E) for histopathology

ACCEPTED MANUSCRIPT
examination.
5.2.2. ALT and ASAT Activity
Heparinized serum was collected at 48 h after dosing from the retrobulbar plexus to
determine the ASAT and ALT activity, using routine colorimetric methods [8] (ALT
& ASAT Activity Assay Kit, Jiancheng Institute of Biotechnology, Nanjing, China).
5.3. Behavioral Studies
Behavioral studies were performed with adult male ICR mice (8−10 weeks old,
weight 20−25 g) from Beijing Vital River Laboratory Animal Technology Co., Ltd.
(Beijing, China) by MWM and Y-maze. The mice were divided into four groups:
control (vehicle), model (scopolamine), THA (scopolamine plus THA), and 7d
(scopolamine plus compound 7d). THA and 7d at the dose of 20 µmol/kg body
weight per day were orally administered to the mice at 30 min before the
intraperitoneal (ip) administration of scopolamine (1 mg/kg). Memory impairment
was induced by administering scopolamine and the maze task was performed after 30
min of scopolamine administration. The schematic diagram (Figure S3) depicts the
experimental procedure.
MWM is a circular pool (100 cm in diameter and 50 cm in height) with visual cues.
The circular pool was filled at a depth of 30 cm with water (25 °C). A platform (6 cm
in diameter and 29 cm in height) was centered in one of four quadrants of the pool.
Mice were trained during trial sessions of four trials each day for four consecutive
days. During each trial, the mouse’s escape latency was recorded. Once the mouse
located the platform, it was permitted to remain on it for 10s. If the mouse did not

ACCEPTED MANUSCRIPT
locate the platform within 90 s, it was placed on the platform for 10s. On the fifth day
after training, mice were given a probe trial session in which the platform was moved
from the pool and allowing the mice to swim for 90s. The time and the distance taken
in the target quadrant and the number of times the animals crossed the platform
location were recorded.
Y-maze is a three-arm horizontal maze (45 cm long, 14 cm wide, 16 cm high) in
which the arms are symmetrically disposed at 120° angles from each other. A mouse
was placed at the end of one arm and allowed to move freely through the maze for 8
min, and the sequence and number of arm entries were recorded. A spontaneous
alternation was defined as entries into all three arms on consecutive choices. The
percentage of alternations was defined according to the following equation: [(number
of alternations)/ (total number of arm entries − 2)] × 100.
Acknowledgements
W.L. acknowledges the National Natural Science Foundation of China (21302041),
the Foundation for University Key Teacher by Educational Commission of Henan
Province (2016GGJS-030), and the Educational Commission of Henan Province
(14A350008).
Conflict of interest
We declare that we have no conflict of interest.

ACCEPTED MANUSCRIPT
References
[1] J.B. Shaik, B.K. Palaka, M. Penumala, K.V. Kotapati, S.R. Devineni, S. Eadlapalli,
M.M. Darla, D.R. Ampasala, R. Vadde, G.D. Amooru, Synthesis, pharmacological
assessment, molecular modeling and in silico studies of fused tricyclic coumarin
derivatives as a new family of multifunctional anti-Alzheimer agents, Eur. J. Med.
Chem. 107 (2016) 219-232.
[2] N. Khunnawutmanotham, N. Chimnoi, P. Saparpakorn, S. Techasakul, Synthesis
and anti-acetylcholinesterase activity of scopoletin derivatives, Bioorg. Chem. 65
(2016) 137-145.
[3] D. Lagadic-Gossmann, M. Rissel, M.A. Le Bot, A. Guillouzo, Toxic effects of
tacrine on primary hepatocytes and liver epithelial cells in culture, Cell Biol. Toxicol.
14 (1998) 361-373.
[4] M.L. Bolognesi, A. Cavalli, L. Valgimigli, M. Bartolini, M. Rosini, V. Andrisano,
M. Recanatini, C. Melchiorre, Multi-target-directed drug design strategy: from a dual
binding site acetylcholinesterase inhibitor to a trifunctional compound against
Alzheimer's disease, J. Med. Chem. 50 (2007) 6446-6449.
[5] X. Chao, X. He, Y. Yang, X. Zhou, M. Jin, S. Liu, Z. Cheng, P. Liu, Y. Wang, J.
Yu, Y. Tan, Y. Huang, J. Qin, S. Rapposelli, R. Pi, Design, synthesis and
pharmacological evaluation of novel tacrine-caffeic acid hybrids as multi-targeted
compounds against Alzheimer's disease, Bioorg. Med. Chem. Lett. 22 (2012)
6498-6502.
[6] X. Chen, K. Zenger, A. Lupp, B. Kling, J. Heilmann, C. Fleck, B. Kraus, M.

ACCEPTED MANUSCRIPT
Decker, Tacrine-silibinin codrug shows neuro- and hepatoprotective effects in vitro
and pro-cognitive and hepatoprotective effects in vivo, J. Med. Chem. 55 (2012)
5231-5242.
[7] E. Nepovimova, E. Uliassi, J. Korabecny, L.E. Pena-Altamira, S. Samez, A.
Pesaresi, G.E. Garcia, M. Bartolini, V. Andrisano, C. Bergamini, R. Fato, D. Lamba,
M. Roberti, K. Kuca, B. Monti, M.L. Bolognesi, Multitarget Drug Design Strategy:
Quinone-Tacrine Hybrids Designed To Block Amyloid-β Aggregation and To Exert
Anticholinesterase and Antioxidant Effects, J. Med. Chem. 57 (2014) 8576-8589.
[8] X. Zha, D. Lamba, L. Zhang, Y. Lou, C. Xu, D. Kang, L. Chen, Y. Xu, A. De
Simone, S. Samez, A. Pesaresi, J. Stojan, M.G. Lopez, J. Egea, V. Andrisano, M.
Bartolini, Novel Tacrine-Benzofuran Hybrids as Potent Multitarget-Directed Ligands
for the Treatment of Alzheimer's Disease: Design, Synthesis, Biological Evaluation,
and X-ray Crystallography, J. Med. Chem. 59 (2016) 114-131.
[9] W. Luo, Y.P. Li, Y. He, S.L. Huang, D. Li, L.Q. Gu, Z.S. Huang, Synthesis and
evaluation of heterobivalent tacrine derivatives as potential multi-functional
anti-Alzheimer agents, Eur. J. Med. Chem. 46 (2011) 2609-2616.
[10] W. Luo, T. Wang, C. Hong, Y.C. Yang, Y. Chen, J. Cen, S.Q. Xie, C.J. Wang,
Design, synthesis and evaluation of 4-dimethylamine flavonoid derivatives as
potential multifunctional anti-Alzheimer agents, Eur. J. Med. Chem. 122 (2016)
17-26.
[11] J.B. Chang, Q. Wang, Y.F. Li, Synthesis and biological activity of Wuweizisu C
and analogs, Curr. Top. Med. Chem. 9 (2009) 1660-1675.