Design, synthesis and evaluation of tacrine-flurbiprofen-nitrate trihybrids as novel anti-Alzheimer's disease agents

Article abstract of DOI:10.1016/j.bmc.2013.03.005

To search for multifunctional anti-Alzheimer's disease (AD) agents with good safety, the previously synthesized tacrine-flurbiprofen hybrids 1a and 1b were modified into tacrine-flurbiprofen-nitrate trihybrids 3a-h. These compounds displayed comparable or higher cholinesterase inhibitory activity relative to the bivalent hybrids. Compound 3a was the most potent, which released moderate NO, exerted blood vessel relaxative activity, and showed significant Aβ inhibitory effects whereas tacrine and flurbiprofen did not exhibit any Aβ inhibitory activity at the same dose. In addition, 3a was active in improving memory impairment in vivo. More importantly, the hepatotoxicity study showed that 3a was much safer than tacrine, suggesting it might be a promising anti-AD agent for further investigation.

Full text of DOI:10.1016/j.bmc.2013.03.005

Bioorganic & Medicinal Chemistry 21 (2013) 2462–2470
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of tacrine–flurbiprofen–nitrate
trihybrids as novel anti-Alzheimer’s disease agents
Yao Chen a,b,c, Jianfei Sun d, Zhangjian Huang a,b, Hong Liao d, , Sixun Peng a,b, Jochen Lehmann c,e,
⇑
⇑
,
Yihua Zhang a,b,
⇑
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
b Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
c Lehrstuhl für Pharmazeutische/Medizinische Chemie, Institut für Pharmazie, Friedrich-Schiller-Universität Jena, Philosophenweg 14, D-07743 Jena, Germany
d Neurobiology Lab, New Drug Screening Center, China Pharmaceutical University, Nanjing 210009, PR China
e College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
To search for multifunctional anti-Alzheimer’s disease (AD) agents with good safety, the previously syn-
thesized tacrine–flurbiprofen hybrids 1a and 1b were modified into tacrine–flurbiprofen–nitrate trihy-
brids 3a–h. These compounds displayed comparable or higher cholinesterase inhibitory activity
relative to the bivalent hybrids. Compound 3a was the most potent, which released moderate NO, exerted
blood vessel relaxative activity, and showed significant Ab inhibitory effects whereas tacrine and flurbi-
profen did not exhibit any Ab inhibitory activity at the same dose. In addition, 3a was active in improving
memory impairment in vivo. More importantly, the hepatotoxicity study showed that 3a was much safer
than tacrine, suggesting it might be a promising anti-AD agent for further investigation.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 18 January 2013
Revised 4 March 2013
Accepted 6 March 2013
Available online 13 March 2013
Keywords:
Tacrine
Flurbiprofen
Nitrate
Cholinesterase
Amyloid-b
1. Introduction
is to connect two distinct pharmacological classes of compounds
in one molecule via a proper spacer.15 Since the cooperation of
Alzheimer’s disease (AD) is a progressive neurological disease
leading to impairment in memory, language skills, judgment and
orientation.1 This disease is multifaceted and many factors such
as cholinergic dysfunction, deposits of amyloid-b (Ab) and
both b- and
c-secretase can cleave amyloid precursor protein
(APP) leading to the accumulation of b-amyloid (Ab) peptides,
the inhibitors of b- and c-secretase are therefore believed to pre-
vent or slow AD development.16–22 We previously synthesized
s-protein and poor blood supply in brain are believed to play
and evaluated hybrids 1a and 1b (Fig. 1) from tacrine and racemic
important roles in the development of AD.2 To our knowledge,
most currently prescribed anti-AD drugs are cholinesterase inhib-
itors (ChEI), for example, tactine and rivastigmine, which act by
increasing the acetylcholine (ACh) level in the brain via inhibiting
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).3,4
Unfortunately, ChEIs have been proved to be therapeutically lim-
ited because ChEIs are not able to delay or prevent the progression
of AD.5,6 Given that the pathogenesis of AD is complicated and cho-
linesterase may not be the only focused factor,7–11 an innovative
therapeutic approach based on the ‘multi-target-directed ligands’
(MTDLs) paradigm12 has recently been applied in the research of
ChEIs.13,14 The most common strategy to rationally design MTDLs
flurbiprofen, a c-secretase inhibitor, as highly potent anti-AD
agents.23 Kinetic studies revealed that they can block both the
catalytic active site (CAS)24,25 and the peripheral anionic site
(PAS),26–28 which are closely associated with hydrolysis of ACh.
Moreover, 1a inhibited the formation of Ab in vitro, probably due
to the ability of flurbiprofen moiety to lower Ab40/42 peptide pro-
duction by an allosteric modulation of c
-secretase.29
In continuation of our ongoing studies on novel multifunctional
ChEIs, we attempted to extend the MTDL strategy by conjugating a
third pharmacophore to the bivalent hybrids.30 Recently, increas-
ing evidences suggest that nitric oxide (NO), a free radical gas,
may be beneficial for the treatment of AD by increasing blood sup-
ply31 and regulating the cerebral circulation.32 Additionally, the ta-
crine–ferulic acid–nitrate trihybrids 2a–e (Fig. 1) from our group
exhibited lower hepatotoxicity compared to the dual-acting ta-
crine–ferulic acid hybrid, which could be beneficial for the treat-
ment of AD.33 Based on these investigations, we introduced a NO
⇑
Corresponding authors. Tel.: +86 25 83271043; fax: +86 25 83271142 (H.L.);
tel.: +49 3641949803; fax: +49 3641949802 (J.L.); tel./fax:+86 25 83271015 (Y.Z.).
E-mail addresses: hliao@cpu.edu.cn (H. Liao), j.lehmann@uni-jena.de (J. Leh-
mann), zyhtgd@163.com (Y. Zhang).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.005

Y. Chen et al. / Bioorg. Med. Chem. 21 (2013) 2462–2470
2463
F
O
NH2
COOH
HN
N
N
H
m
F
N
1a-b
,
m=6, 8
tacrine
flurbiprofen
O
ONO2
O
F
n
HN m N
H
O
O
n ONO2
HN m N
H
O
N
2a-e, m=2-4, 6, 8; n=2-4,6
3a-h, m=6, 8; n=2-4,6
N
Figure 1. Structures of tacrine, flurbiprofen, tacrine–flurbiprofen hybrids 1a and 1b, tacrine–ferulic acid–nitrate trihybrids 2a–e and tacrine–flurbiprofen–nitrate trihybrids 3a–h.
donor moiety (nitrate group) into the bivalent hybrids 1a and 1b to
HN m NH2
Cl
O
generate a group of novel tacrine–flurbiprofen–nitrate trihybrids
3a–h (Scheme 1). Herein, synthesis and biological evaluations
including inhibition of AChE and BuChE, kinetics of enzyme inhibi-
tion, NO releasing, vascular relaxation, cell-based Ab inhibition as
well as in vivo behavioral and hepatotoxicity studies are described.
COOH
NH2
a
b
N
N
5a, m=6; 5b, m=8
4
Scheme 1. The synthetic route of 5a and 5b. Reagents: (a) POCl3, reflux, 3 h; (b)
pentanol, NH2(CH2)mNH2, reflux, 18 h.
2. Results and discussion
2.1. Chemistry
3a and 3e, which were characterized by IC50 values of 9.1 and
12.5 nM on AChE and IC50 values of 2.5 and 1.0 nM against BuChE.
Interestingly, 3g and 3h showed much higher inhibitory activity
against BuChE (IC50 = 1.3 and 1.5 nM) relative to AChE
(IC50 = 70.9 and 255.6 nM), indicating that 3g and 3h were selec-
tive BuChE inhibitors with selectivity indexes of 54.5 and 150.4,
respectively.
Scheme 1 depicts that the condensation reaction of anthranilic
acid with cyclohexanone furnished 9-chloro-1,2,3,4-tetrahydroac-
ridine 4,34 which was subsequently treated with different alkylen-
ediamines to produce the intermediates 9-aminoalkylamino-
1,2,3,4-tetrahydroacridines 5a and 5b.
To synthesize the other key intermediates NO-donating flurbi-
profen derivatives 13a–d, the carboxyl group of racemic flurbipro-
fen was protected to form a methyl ester 6. Friedel–Crafts reaction
of 6 yielded acylated compound 7, which underwent the Baeyer–
Villiger oxidation rearrangement to furnish acetyl ester 8, followed
by hydrolysis to give 40-OH flubiprofen 9. Compound 9 was ester-
ified to give methyl ester 10, which was treated with the corre-
sponding dibromoalkanes to give the halogenated intermediates
11a–d. Subsequent treatment of 11a–d with AgNO3 in dry CH3CN
offered nitrates 12a–d. The nitrates were then de-protected under
basic conditions to generate acids 13a–d, which were coupled with
5a and 5b, respectively, in the presence of DCC/DMAP to provide
the target compounds 3a–h (Scheme 2). These target compounds
were purified by column chromatography and characterized by
IR, ESI-MS, 1H NMR, 13C NMR, and elementary analysis, and indi-
vidual compounds with chemical purity of >95% for subsequent
experiments.
2.3. Kinetic study of ChE inhibition
To get insight into the mechanism of action of this new family
of compounds on both AChE and BuChE, a kinetic study was con-
ducted for the most potent compound 3a. Graphical analysis of
the Lineweaver–Burk reciprocal plot of AChE inhibition (Fig. 2)
showed both increased slopes (decreased Vmax) and intercepts
(higher Km) with increased concentration of 3a. This pattern sug-
gested a mixed-type inhibition and accordingly supported the dual
site (CAS and PAS) binding of these compounds. A Ki value of
16.1 nM was derived from the plots of the slop versus the concen-
tration of 3a. As for BuChE, a different plot was observed, with dif-
ferent Km and constant Vmax in varied inhibitor concentrations
(Fig. 3), suggesting a competitive inhibition, in which 3a competes
with the substrate acetylcholine for the same binding site (CAS).
Replots of the slope versus concentration of 3a give a competitive
inhibition constant, Ki of 1.7 nM.
2.2. Pharmacology: AChE and BuChE inhibition
2.4. Detection of nitrite (Griess reaction)
Target compounds 3a–h were tested in vitro for inhibition of
AChE from Electrophorus electricus (eeAChE) and of BuChE from
equine serum, using the Ellman assay35 (Table 1). The inhibitory
activities of the hybrids were compared to those of tacrine, parent
compounds 1a and 1b. All target compounds (except 3h) exhibit
similar or even higher inhibitory activities compared to tacrine,
1a and 1b. The results revealed that the length of the alkyl, which
was connected to nitrate moiety of the target compounds, could
significantly influence the AChE inhibitory activity. When the
length increased, the activity decreased. The optimal spacer length
was two carbon atoms. It was observed that 3a–h were more active
against BuChE (IC50s = 0.6–2.5 nM) than tacrine (IC50 = 10.6 nM),
1a and 1b (IC50s = 2.1–3.7 nM). The most potent compounds were
The NO-releasing ability of compounds 3a–h was measured
using the Griess reaction36 by quantifying the nitrite produced
from the oxidative reaction of NO, oxygen and water (Table 1).
The results showed that all the target compounds released moder-
ate NO (0.283–0.565 lg/mL), more than tacrine, and comparable to
isosorbide mononitrate (ISMN, 0.412 lg/mL), a known NO donor.
2.5. Vascular relaxation study
Previous study have suggested that NO might have a positive
correlation with the vasorelaxation effect.33 Therefore, NO-donat-
ing compounds 3a and 3e were tested in an ex vivo organs bath

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Y. Chen et al. / Bioorg. Med. Chem. 21 (2013) 2462–2470
COOCH3
c
COOCH3
COOH
a
b
F
F
F
6
7
O
COOCH3
COOCH3
d
COOH
e
O
F
F
F
HO
O
HO
8
10
9
COOCH3
g
COOCH3
h
f
F
F
Br
O
n
O2NO
O
11a
11b
n=3
11c n=4; 11d n=6
n=2;
n
12a n=2; 12b n=3
12c 12d
n=4;
n=6
O
ONO2
F
n
O
COOH
HN
N
m NH
i
F
O2NO
O
n
13a n=2; 13b n=3
13c 13d
3a-h
n=4;
n=6
3a, m=6, n=2; 3b, m=6, n=3; 3c, m=6, n=4
3d, m=6, n=6; 3e, m=8, n=2; 3f, m=8, n=3
3g, m=8, n=4; 3h, m=8, n=6;
Scheme 2. The synthetic route of 13a–d and 3a–h. Reagents: (a) CH3OH, H2SO4, reflux, 3 h; (b) CH3COCl, AlCl3, anhydrous CH2Cl2, reflux, 12 h; (c) mCPBA, CH2Cl2, room temp,
48 h; (d) NaOH, THF/CH3OH/H2O, room temp, 6 h; (e) CH3OH, H2SO4, reflux, 3 h; (f) Br(CH2)nBr, DMF, K2CO3, 65 °C, 6 h; (g) AgNO3, CH3CN, 60 °C, 6 h; (h) LiOH, THF/CH3OH/
H2O, room temp, 24 h; (i) DCC, DMAP, anhydrous CH2Cl2, room temp, 24 h.
Table 1
Inhibitory IC50 values against AChE and BuChE and selectivity index for compounds
1a, 1b, 3a–h and tacrine; and release of nitrite from the organic nitrates measured by
Griess reaction
Compd
IC50 (nM) SEMa
Nitritec
(lg/mL)
AChE
BChE
Selectivity Indexb
1a
1b
3a
3b
3c
3d
3e
3f
3g
3h
Tacrine
ISMN
19.3 3.4
34.5 3.3
9.1 0.6
3.7 0.4
2.1 0.3
2.5 0.2
2.4 0.2
1.3 0.1
0.6 0.0
1.0 0.1
1.6 0.1
1.3 0.1
1.5 0.2
10.6 1.1
/
5.2
16.4
3.6
/
/
0.348 0.035
0.353
18.7 1.4
27.4 8.9
31.1 8.5
12.5 0.6
30.6 9.6
70.9 16.1
225.6 26.9
69.8 11.1
/
7.8
0.032
21.1
51.8
12.5
19.1
54.5
150.4
6.6
0.360 0.033
0.283 0.026
0.565 0.049
0.353 0.045
0.360 0.026
0.340 0.039
0.011 0.004
0.412 0.013
/
a
b
c
Data is the mean of three determinations.
Selectivity index = (IC50 of AChE)/(IC50 of BuChE).
All values are the mean SEM. Determined by Griess reaction.
Figure 2. Linewear–Burk plots resulting from sub-velocity curves of AChE activity
with varied substrate concentrations (25–450 lM) in the indicated concentrations
of 3a.
(coronary arteries from rat) by vascular relaxation assay, in which
tacrine was employed as a negative control. As can be seen from
Table 2, both 3a (28.6%) and 3e (28.8%) exhibited moderate blood
vessel relaxative activity and no vessel relaxation was observed
with tacrine. These results suggest that NO generated from the ni-
trate group of 3a and 3e might be crucial for the vascular relaxa-
tion potency.
that, 3a displayed significant Ab formation inhibitory effects with
17% reduction in levels of Ab40, whereas flurbiprofen and tacrine
did not exhibit any Ab formation inhibitory activity at the same
dose (0.5 lM).
2.7. Behavioral studies
2.6. Cell-based Ab inhibition assay
Scopolamine-induced impairment in passive avoidance test was
commonly employed for screening test to predict the potential of a
ChE inhibitor as a cognitive enhancer drug. To evaluate the thera-
peutic potential of the target compounds, we used the passive
avoidance test38 for behavioral studies. Compound 3a was tested
in comparison with tacrine and 1a, using scopolamine-induced im-
paired adult mice as the animal model. Figure 5 described the
To further explore the influence of the nitrate group on the Ab
formation inhibitory activity, compound 3a was assessed by an en-
zyme-linked immune sandwich assay (ELISA).37 DMSO and a
known Ab formation inhibitory agent L-685458 were used as neg-
ative and positive controls, respectively. The results (Fig. 4) showed

Y. Chen et al. / Bioorg. Med. Chem. 21 (2013) 2462–2470
2465
Figure 4. Ab40 reduction of flurbiprofen, tacrine, 3a (0.5
lM) in the cell-based
Figure 3. Linewear–Burk plots resulting from sub-velocity curves of BuChE activity
with varied substrate concentrations (25–450 lM) in the indicated concentrations
assay. (⁄⁄p 60.01, ⁄⁄⁄p 60.001 compared to the control group).
of 3a.
transfer lantency time (TLT in seconds) of the tested compounds
and the TLT in second trial was taken as the parameter to deter-
mine the cognitive improving potency of the compounds. The re-
sults showed that, the TLT in second trial for each group was
much higher than the TLT in first trial, indicating the rationality
and validity. A significant increase in the 1a and 3a group was ob-
served compared to the scopolamine group, indicating that both
two compounds were active in improving memory impairment.
Compound 3a showed no significant difference in comparison with
tacrine and 1a (p >0.05), respectively, suggesting that the nitrate
group might not be crucial for the in vivo cognition improving
potency.
2.8. Hepatotoxicity studies
Given that our previous study has proved that moderate levels
of NO are beneficial for decreasing hepatotoxicity of the anti-AD
agents,39–42 3a and 1a were tested to examine whether the ta-
crine–flurbiprofen–nitrate trihybrids show lower hepatotoxicity
in adult mice, compared to the non-nitrate parent compound. After
mice were treated with tacrine, 1a and 3a at equimolar doses, the
heparinized serum of the mice was obtained at various time inter-
val and the levels of aspartate aminotransferase (ASAT) and alanine
aminotransferase (ALT) were determined (Fig. 6). The result
showed that tacrine caused significant increase of ASAT and ALT
activity, indicating its strong hepatotoxicity compared to the con-
trol. Compounds 1a and 3a displayed higher safety than tacrine,
as evidenced by the lower values of ASAT and ALT, and 3a showed
the lowest hepatotoxicity among all the compounds.
Figure 5. Effects of 1a, 3a and tacrine (tac) on transfer latency time (TLT) in the PA
test. Values are expressed as mean SEM; (n = 8–9, t test, ⁄⁄⁄p 60.001 compared to
the control group, #p 60.05 compared to the scop group).
zones were observed in the tacrine treated section (Fig. 7B). In con-
trast, there were only minor morphological changes after the treat-
ment with 1a and 3a (Fig. 7C and D). All these findings strongly
support that coupling nitrate to tacrine–flurbiprofen hybrids could
improve the safety of the parent compounds.
Compounds 1a, 3a and tacrine were further evaluated in histo-
logical studies, in which liver tissues taken from the animals were
stained with hematoxylin and eosin (HE). Paraffin sections were
observed 30 h after administration of the tested compounds.
Complete pericentral necrosis and distinct fatty degeneration of
the hepatocytes of the surrounding intermediate and periportal
3. Conclusions
A series of tacrine–flurbiprofen–nitrate trihybrid compounds
have been designed and synthesized as novel anti-AD agents. All
of the compounds except 3h showed comparable or stronger ChEs
inhibitory activity compared to the parent compounds 1a and 1b,
and compounds 3a and 3e were the most potent ChEIs. All the
compounds could moderately release NO in vitro, and the activity
of 3a and 3e was comparable to ISMN in vascular relaxation assay.
In addition, 3a displayed significant Ab formation inhibitory effects
whereas flurbiprofen and tacrine did not exhibit any Ab formation
inhibitory activity at the same dose. Furthermore, 3a showed com-
parable cognition improving activity in vivo relative to 1a. Signifi-
cantly, the hepatotoxicity of 3a was much lower than that of
tacrine and 1a, and the higher safety of the trihybrid qualified it
Table 2
Relaxative activity (%) of 3a and 3e at the concentration 100 lM
Compounds
Relaxative activitya (%)
Tacrine
ISMN
3a
3.4 3.7
77.1 10.7
28.6 9.3
28.8 7.9
3e
a
All values are the mean SEM.

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Y. Chen et al. / Bioorg. Med. Chem. 21 (2013) 2462–2470
(250 MHz). ESI-MS and HRMS data were recorded using Hewlett–
Packard 1100 LC/MSD spectrometer and Agilent technologies LC/
MSD TOF, respectively. TLC was performed on silica gel F254 plates
(Merck). Column chromatography was carried out with silica gel
60, 63–200 lm (Baker). Purities of the compounds were deter-
mined by elemental analysis, performed on a Heraeus Vario EL III
apparatus (Firma Elementar Analysensysteme GmbH, Germany).
All values for C, H, and N were found to be within 0.4% of the the-
oretical values. All compounds showed >95% purity.
4.1.1. 9-Aminoalkylamino-1,2,3,4-tetrahydroacridines 5a and
5b: General procedure I
To a solution of 4 in 10 mL of pentanol 3 molar equiv of diami-
nolkane were added. After refluxing for 18 h under a nitrogen, the
solution was cooled to 0 °C and then acidified to pH 2 with HCl/
ether solution. The precipitate was collected and dissolved in
10 mL of water, then the solution was basified to pH 10 with a sat-
urated aqueous solution of Na2CO3 and extracted with CH2Cl2
(3 ꢀ 10 mL). The combined organic layers were washed with brine,
dried over anhydrous Na2SO4 and evaporated in vacuo. The residue
was purified through column chromatography (CH2Cl2/MeOH = 7/
3, v/v, plus 5 mL triethylamine per 1000 mL).
ALT (U/L)
ASAT (U/L)
4.1.2. Methyl 2-[40-(bromoalkoxy)-2-fluorobiphenyl-4-yl]
propanoate 11a–d: General procedure II
Groups
8h
24h
8h
24h
control
tacrine
1a
25.1 2.6
28.1 2.8
22.7 3.7
23.3 3.2
32.0 4.1
45.3 4.6
35.5 4.5
30.9 2.3
39.1 5.1
46.0 7.3
36.4 2.4
36.1 4.0
43.5 6.2
77.4 14.5
55.6 8.0
49.2 9.1
A mixture of given amount of 10 and 3 molar equiv of dibro-
moalkane in DMF (50 mL) was treated with 0.75 molar of K2CO3.
After being stirred at 60 °C for 6 h, the reaction mixture was cooled
down to ambient temperature, poured into water and extracted
with ethyl acetate (3 ꢀ 15 mL). The combined organic layers were
washed with brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure to give the crude product. The purification
was performed by column chromatography (ethyl acetate/petrol
ether = 1/6, v/v).
3a
Figure 6. The values of ALT and ASAT measured after the administration of tacrine,
1a and 3a, which are expressed as mean SEM; (n = 8–9, t test, compared to control
of the same time after administration, ⁄p 60.05).
more beneficial for the treatment of AD. Therefore, 3a might be a
promising lead compound for further investigation.
4.1.3. Methyl 2-{2-fluoro-40-[(nitrooxy) alkoxy]biphenyl-4-yl}
propanoate 12a–d: General procedure III
4. Experimental section
4.1. Chemistry
A mixture of given amount compound 11a–d and 1.2 molar e-
quiv of AgNO3 in 50 mL of CH3CN was stirred at 60 °C overnight un-
der dark condition. Then the resulting mixture was cooled to room
temperature and filtrated to give a brown filtrate, which was con-
centrated under reduced pressure. The residue was purified by
recrystallization from CH3CN.
Melting points were measured in open capillary tubes using a
Gallenkamp melting point apparatus without correcting. IR spectra
were run on Shimadzu FTIR-8400s. 1H and 13C NMR spectral data
were obtained from
a Bruker Advance 250 spectrometer
4.1.4. 2-{2-Fluoro-40-[(nitrooxy) alkoxy]biphenyl-4-
yl}propanoic acid 13a–d: General procedure IV
Three molar equivalent of LiOHꢁH2O was added to a solution of
given amount of compound 12a–d in a 15/5/5 mL mixture of THF/
MeOH/H2O and the reaction was stirred at room temperature for
24 h. After removing the solvent under reduced pressure, the resi-
due was diluted with H2O (30 mL), acidified to pH 4 with 10% HCl,
and extracted with ethyl acetate (3 ꢀ 15 mL). The combined organ-
ic layers were washed with brine, dried over anhydrous Na2SO4
and concentrated in vacuo to give crude product, which was
recrystallized from ethyl acetate.
4.1.5. {20-Fluoro-40-[1-oxo-1-((1,2,3,4-tetrahydroacridin-9-
ylamino) alkylamino)propan-2-yl]biphenyl-4-yloxy}alkyl
nitrate 3a–h: General procedure V
Given amount of 13a–d and equiv molar of N,N0-dicyclohexyl-
carbodiimide (DCC) were dissolved in 20 mL of dry CH2Cl2 and
the mixture was stirred at room temperature for 0.5 h. Then a solu-
tion of equiv molar of 5a–b in 5 mL of dry CH2Cl2 was added into
the reaction, followed by a catalytic amount of 4-dimethylamino-
pyridine (DMAP). After being stirred for 24 h at room temperature,
the reaction was quenched by adding 10 mL of H2O. Then the
Figure 7. Histomorphological appearance of livers of male mice after treatment
with the solvent only (control) (A) or 30 h after administration of tacrine (B), 1a (C)
or 3a (D). HE, original magnification ꢀ 200.

Y. Chen et al. / Bioorg. Med. Chem. 21 (2013) 2462–2470
2467
resulting mixture was filtrated and the filtrate was extracted with
CH2Cl2 (3 ꢀ 15 mL). The combined organic phases were washed
with brine, dried over anhydrous Na2SO4 and concentrated in va-
cuo. The residue was purified through column chromatography
(CH2Cl2/MeOH = 10/1, v/v).
(0.31 g, 47%); mp 34–36 °C. IR (KBr): 669, 1279, 1401, 1560,
1627, 2361 cmꢂ1 1H NMR (CDCl3): d 7.95–7.88 (t, 2H, J = 7.4 Hz,
.
arom), 7.57–7.50 (t, 1H, J = 8.1 Hz, arom), 7.47–7.43 (m, 2H, arom),
7.38–7.29 (m, 2H, arom), 7.12–7.06 (m, 2H, arom), 6.94–6.91 (d,
2H, J = 8.7 Hz, arom), 5.65–5.61 (t, 1H, J = 5.6 Hz, NHCO), 4.56–
4.51 (t, 2H, J = 6.2 Hz, OCH2), 4.04–3.99 (t, 3H, CH2ONO2, NH),
3.57–3.41 (m, 3H, CHCH3, NHCH2), 3.24–3.17 (q, 2H, CH2NHCO),
3.05 (br, 2H, C4-H2), 2.67 (br, 2H, C1-H2), 1.94–1.88 (m, 8H,
OCH2CH2, C3-H2, C2-H2, NHCH2CH2), 1.64–1.26 (m, 11H, CH2CH2O-
NO2, NHCH2CH2CH2CH2CH2, CHCH3). 13C NMR (CDCl3): d 173.47
4.1.6. 2-{20-Fluoro-40-[1-oxo-1-(6-(1,2,3,4-tetrahydroacridin-9-
ylamino)hexylamino)propan-2-yl]biphenyl-4-yloxy}ethyl
nitrate (3a)
Following general procedure V, compound 5a (0.30 g, 1 mmol)
and 13a (0.35 g, 1 mmol) combined to offer 3a as a yellow solid
(0.28 g, 45%); mp 42–44 °C. IR (KBr): 758, 1280, 1401, 1492,
(NHCO), 161.69, 157.74 (arom, C3), 158.41 (arom), 158.28 (arom,
0
C4 ), 150.79 (arom), 147.25 (arom), 142.44, 142.33 (arom, C1),
0
0
1560, 1637, 2363 cmꢂ1
.
1H NMR (CDCl3):
d
7.95–7.89 (t,
130.67, 130.61 (arom, C5), 130.04 (arom, C6 ), 129.99 (arom, C2 ),
0
J = 7.4 Hz, 2H, arom), 7.56–7.43 (m, 3H, arom), 7.34–7.33 (m, 2H,
arom), 7.11–7.06 (m, 2H, arom), 6.95–6.91 (d, J = 10.0 Hz, 2H,
arom), 5.69 (br, 1H, NHCO), 4.81 (br, 2H, OCH2), 4.25 (br, 2H, CH2O-
NO2), 3.99 (br, 1H, NH), 3.57–3.42 (m, 3H, CHCH3, NHCH2), 3.22–
3.19 (m, 2H, CH2NHCO), 3.04 (br, 2H, C4-H2), 2.66 (br, 2H, C1-
H2), 1.89 (br, 4H, C3-H2, C2-H2), 1.60–1.39 (m, 11H,
128.50 (arom), 128.36 (arom), 127.86 (arom, C1 ), 127.46, 127.25
(arom, C4), 123.63 (arom), 123.56, 123.51 (arom, C6), 122.84
(arom), 120.12 (arom), 115.80 (arom), 115.39, 115.01 (arom, C2),
0
0
114.46 (arom, C3 , C5 ), 72.90 (OCH2), 66.98 (CH2ONO2), 49.23
(NHCH2), 46.59 (CHCH3), 39.45 (CH2NHCO), 33.88 (NHCH2CH2),
31.58 (CH2CH2NHCO), 29.44 (C4), 26.45 (NHCH2CH2CH2CH2),
25.53 (OCH2CH2), 24.75 (C1), 23.87 (CH2CH2ONO2), 23.01 (C3),
22.72 (C2), 18.55 (CHCH3). ESI-MS: m/z 657 [M+H]+. Anal. Calcd
for (C38H45FN4O5 ꢀ 1/2 H2O): found C, 68.55; H, 6.63; N, 8.06; re-
quires C, 68.55; H, 6.96; N, 8.42.
NHCH2CH2CH2CH2CH2, CHCH3). 13C NMR (CDCl3): d 173.47 (NHCO),
0
161.66, 157.72 (arom, C3), 158.18 (arom), 157.60 (arom, C4 ),
150.87 (arom), 147.10 (arom), 142.65, 142.54 (arom, C1), 130.67,
0
0
130.61 (arom, C5), 130.13 (arom, C6 ), 130.09 (arom, C2 ), 128.68
0
(arom, C1 ), 128.42 (arom), 128.35 (arom), 127.23, 127.02 (arom,
C4), 123.65 (arom), 123.59, 123.54 (arom, C6), 122.86 (arom),
120.04 (arom), 115.71 (arom), 115.40, 115.03 (arom, C2), 114.59
4.1.9. 6-{20-Fluoro-40-[1-oxo-1-(6-(1,2,3,4-tetrahydroacridin-9-
ylamino)hexylamino)propan-2-yl]biphenyl-4-yloxy}hexyl
nitrate (3d)
0
0
(arom, C3 , C5 ), 70.92 (OCH2), 64.10 (CH2ONO2), 49.20 (NHCH2),
46.58 (CHCH3), 39.45 (CH2NHCO), 33.78 (NHCH2CH2), 31.56
(CH2CH2NHCO), 29.43 (C4), 26.44 (NHCH2CH2CH2CH2), 24.72
(C1), 22.97 (C3), 22.67 (C2), 18.55 (CHCH3). ESI-MS: m/z 629
[M+H]+. Anal. Calcd for (C36H41FN4O5 ꢀ 1/4 H2O): found C, 68.05;
H, 6.56; N, 8.80; requires C, 68.28; H, 6.61; N, 8.85.
Following general procedure V, compound 5a (0.30 g, 1 mmol)
and 13d (0.42 g, 1 mmol) combined to offer 3d as a yellow solid
(0.31 g, 45%); mp 30–32 °C. IR (KBr): 760, 1278, 1401, 1493,
1564, 1625, 2361 cmꢂ1 1H NMR (CDCl3): d 7.94–7.87 (t, 2H,
.
J = 8.5 Hz, arom), 7.55–7.49 (t, 1H, J = 7.0 Hz, arom), 7.46–7.42
(m, 2H, arom), 7.38–7.29 (m, 2H, arom), 7.11–7.05 (m, 2H, arom),
6.95–6.91 (d, 2H, J = 8.9 Hz, arom), 5.74–5.70 (t, 1H, J = 5.8 Hz,
NHCO), 4.47–4.42 (t, 2H, J = 6.6 Hz, OCH2), 4.00–3.95 (t, 3H, CH2O-
NO2, NH), 3.56–3.40 (m, 3H, CHCH3, NHCH2), 3.24–3.16 (q, 2H,
CH2NHCO), 3.04 (br, 2H, C4-H2), 2.66 (br, 2H, C1-H2), 1.90–1.70
(m, 8H, C3-H2, C2-H2, OCH2CH2, NHCH2CH2), 1.63–1.25 (m, 15H,
CH2CH2CH2CH2ONO2, NHCH2CH2CH2CH2CH2, CHCH3). 13C NMR
4.1.7. 3-{20-Fluoro-40-[1-oxo-1-(6-(1,2,3,4-tetrahydroacridin-9-
ylamino)hexylamino)propan-2-yl]biphenyl-4-yloxy}propyl
nitrate (3b)
Following general procedure V, compound 5a (0.30 g, 1 mmol)
and 13b (0.36 g, 1 mmol) combined to offer 3b as a yellow solid
(0.29 g, 45%); mp 38–40 °C. IR (KBr): 764, 1278, 1401, 1638 cmꢂ1
.
1H NMR (CDCl3): d 7.95–7.89 (t, J = 7.4 Hz, 2H, arom), 7.57–7.50
(t, J = 8.2 Hz, 1H, arom), 7.47–7.43 (m, 2H, arom), 7.38–7.29 (m,
2H, arom), 7.12–7.06 (m, 2H, arom), 6.95–6.91 (d, J = 7.4 Hz, 2H,
arom), 5.66–5.61 (t, J = 5.5 Hz, 1H, NHCO), 4.70–4.65 (t, J = 6.4 Hz,
2H, OCH2), 4.11–3.99 (m, 3H, CH2ONO2, NH), 3.57–3.42 (m, 3H,
CHCH3, NHCH2), 3.25–3.17 (q, 2H, CH2NHCO), 3.05 (br, 2H, C4-
H2), 2.67 (br, 2H, C1-H2), 2.27–2.17 (m, 2H, OCH2CH2), 1.91–1.88
(t, 4H, C3-H2, C2-H2), 1.67–1.26 (m, 11H, NHCH2CH2CH2CH2CH2,
(CDCl3): d 173.51 (NHCO), 161.68, 157.74 (arom, C3), 158.70
0
(arom), 158.32 (arom, C4 ), 150.77 (arom), 147.31 (arom), 142.39,
0
142.26 (arom, C1), 130.66, 130.59 (arom, C5), 129.98 (arom, C6 ),
0
129.93 (arom, C2 ), 128.52 (arom), 128.32 (arom), 127.60 (arom,
0
C1 ), 127.52, 127.30 (arom, C4), 123.61 (arom), 123.54, 123.49
(arom, C6), 122.85 (arom), 120.15 (arom), 115.82 (arom), 115.36,
0
0
114.99 (arom, C2), 114.48 (arom, C3 , C5 ), 73.24 (OCH2), 67.64
(CH2ONO2), 49.24 (NHCH2), 46.56 (CHCH3), 39.45 (CH2NHCO),
33.92 (NHCH2CH2), 31.59 (CH2CH2NHCO), 29.43 (C4), 29.00
(OCH2CH2), 26.70 (CH2CH2ONO2), 26.46 (NHCH2CH2CH2CH2),
25.67 (OCH2CH2CH2), 25.44 (CH2CH2CH2ONO2), 24.75 (C1), 23.01
(C3), 22.73 (C2), 18.56 (CHCH3). ESI-MS: m/z 685 [M+H]+. Anal.
Calcd for (C40H49FN4O5 ꢀ 1/2 H2O): found C, 69.15; H, 7.36; N,
7.92; requires C, 69.24; H, 7.26; N, 8.07.
CHCH3). 13C NMR (CDCl3): d 173.47 (NHCO), 161.69, 157.74 (arom,
0
C3), 158.25 (arom), 158.10 (arom, C4 ), 150.82 (arom), 147.19
(arom), 142.52, 142.40 (arom, C1), 130.68, 130.61 (arom, C5),
0
0
130.08 (arom, C6 ), 130.03 (arom, C2 ), 128.45 (arom), 128.38
0
(arom), 128.15 (arom, C1 ), 127.39, 127.17 (arom, C4), 123.63
(arom), 123.57, 123.52 (arom, C6), 122.84 (arom), 120.09 (arom),
0
0
115.77 (arom), 115.39, 115.02 (arom, C2), 114.47 (arom, C3 , C5 ),
69.92 (OCH2), 63.58 (CH2ONO2), 49.23 (NHCH2), 46.59 (CHCH3),
39.45 (CH2NHCO), 33.85 (NHCH2CH2), 31.58 (CH2CH2NHCO),
29.44 (C4), 26.98 (OCH2CH2), 26.45 (NHCH2CH2CH2CH2), 24.74
(C1), 23.00 (C3), 22.70 (C2), 18.56 (CHCH3). ESI-MS: m/z 643
[M+H]+. Anal. Calcd for (C37H43FN4O5 ꢀ 1/2 H2O): found C, 67.99;
H, 6.96; N, 8.54; requires C, 68.18; H, 6.80; N, 8.60.
4.1.10. 2-{20-Fluoro-40-[1-oxo-1-(8-(1,2,3,4-tetrahydroacridin-9-
ylamino)octylamino)propan-2-yl]biphenyl-4-yloxy}ethyl
nitrate (3e)
Following general procedure V, compound 5b (0.31 g, 1 mmol)
and 13a (0.35 g, 1 mmol) combined to offer 3e as a yellow solid
(0.30 g, 46%); mp 40–42 °C. IR (KBr): 751, 1280, 1401, 1492, 1561,
1638 cmꢂ1. 1H NMR (CDCl3): d 7.98–7.94 (d, 2H, J = 10.0 Hz, arom),
7.58–7.51 (t, 1H, J = 8.1 Hz, arom), 7.47–7.43 (m, 2H, arom), 7.37–
7.31 (m, 2H, arom), 7.14–7.08 (m, 2H, arom), 6.95–6.91 (d, 2H,
J = 8.7 Hz, arom), 5.76–5.72 (t, 1H, J = 5.4 Hz, NHCO), 4.82–4.79 (t,
2H, J = 4.6 Hz, OCH2), 4.26–4.15 (m, 3H, CH2ONO2, NH), 3.60–3.46
(m, 3H, CHCH3, NHCH2), 3.23–3.15 (q, 2H, CH2NHCO), 3.07 (br,
4.1.8. 4-{20-Fluoro-40-[1-oxo-1-(6-(1,2,3,4-tetrahydroacridin-9-
ylamino)hexylamino)propan-2-yl]biphenyl-4-yloxy}butyl
nitrate (3c)
Following general procedure V, compound 5a (0.30 g, 1 mmol)
and 13c (0.39 g, 1 mmol) combined to offer 3c as a yellow solid

2468
Y. Chen et al. / Bioorg. Med. Chem. 21 (2013) 2462–2470
0
2H, C4-H2), 2.67 (br, 2H, C1-H2), 1.90 (br, 4H, C3-H2, C2-H2), 1.66–
1.57 (m, 2H, NHCH2CH2), 1.53–1.50 (d, 3H, J = 7.0 Hz, CHCH3),
1.44–1.24 (m, 10H, NHCH2CH2CH2CH2CH2CH2CH2). 13C NMR
C4 ), 151.14 (arom), 146.81 (arom), 142.51, 142.39 (arom, C1),
0
0
130.66, 130.59 (arom, C5), 130.05 (arom, C6 ), 130.00 (arom, C2 ),
0
128.56 (arom), 128.10 (arom), 127.93 (arom, C1 ), 127.42, 127.20
(CDCl3): d 173.41 (NHCO), 161.66, 157.65 (arom, C3), 157.71 (arom),
(arom, C4), 123.67 (arom), 123.58, 123.53 (arom, C6), 122.95
(arom), 119.86 (arom), 115.40 (arom), 115.40, 115.02 (arom, C2),
0
157.58 (arom, C4 ), 151.36 (arom), 146.40 (arom), 142.74, 142.62
0
0
0
(arom, C1), 130.63, 130.57 (arom, C5), 130.14 (arom, C6 ), 130.09
114.45 (arom, C3 , C5 ), 72.89 (OCH2), 66.97 (CH2ONO2), 49.34
(NHCH2), 46.59 (CHCH3), 39.61 (CH2NHCO), 33.60 (NHCH2CH2),
31.64 (CH2CH2NHCO), 29.43 (C4), 29.12 (NHCH2CH2CH2CH2),
0
0
(arom, C2 ), 128.76 (arom, arom), 127.70 (arom, C1 ), 127.17,
126.95 (arom, C4), 123.74 (arom), 123.61, 123.56 (arom, C6),
123.04 (arom), 119.65 (arom), 115.41 (arom), 115.17, 115.04 (arom,
29.00
(CH2CH2CH2CH2NHCO),
26.72
(OCH2CH2),
26.61
0
0
C2), 114.59 (arom, C3 , C5 ), 70.92 (OCH2), 64.10 (CH2ONO2), 49.28
(NHCH2), 46.56 (CHCH3), 39.61 (CH2NHCO), 33.32 (NHCH2CH2),
31.59 (CH2CH2NHCO), 29.42 (C4), 29.09 (NHCH2CH2CH2CH2),
28.98 (CH2CH2CH2CH2NHCO), 26.69 (NHCH2CH2CH2), 26.60
(CH2CH2CH2NHCO), 24.61 (C1), 22.89 (C3), 22.51 (C2), 18.57
(CHCH3). ESI-MS: m/z 657 [M+H]+. Anal. Calcd for
(C38H45FN4O5 ꢀ 1/2 H2O): found C, 68.79; H, 6.58; N, 8.19; requires
C, 68.55; H, 6.96; N, 8.41.
(NHCH2CH2CH2), 25.53 (CH2CH2CH2NHCO), 24.67 (C1), 23.86
(CH2CH2ONO2), 22.96 (C3), 22.62 (C2), 18.56 (CHCH3). ESI-MS:
m/z 685 [M+H]+. Anal. Calcd for (C40H49FN4O5 ꢀ 1/2 H2O): found
C, 69.22; H, 7.01; N, 7.91; requires C, 69.24; H, 7.26; N, 8.07.
4.1.13. 6-{20-Fluoro-40-[1-oxo-1-(8-(1,2,3,4-tetrahydroacridin-9-
ylamino)octylamino)propan-2-yl]biphenyl-4-yloxy}hexyl
nitrate (3h)
Following general procedure V, compound 5b (0.31 g, 1 mmol)
and 13d (0.42 g, 1 mmol) combined to offer 3h as a yellow solid
(0.30 g, 42%); mp 29–31 °C. IR (KBr): 759, 1278, 1401, 1493, 1561,
4.1.11. 3-{20-Fluoro-40-[1-oxo-1-(8-(1,2,3,4-tetrahydroacridin-9-
ylamino)octylamino)propan-2-yl]biphenyl-4-yloxy}propyl
nitrate (3f)
1624, 2932 cmꢂ1 1H NMR (CDCl3): d 7.97–7.92 (t, 2H, J = 6.0 Hz,
.
Following general procedure V, compound 5b (0.31 g, 1 mmol)
and 13b (0.36 g, 1 mmol) combined to offer 3f as a yellow solid
(0.31 g, 46%); mp 35–36 °C. IR (KBr): 759, 1279, 1401, 1493,
arom), 7.57–7.53 (t, 1H, J = 5.1 Hz,arom), 7.48–7.46 (d, 2H,
J = 5.4 Hz, arom), 7.39–7.32 (m, 2H, arom), 7.14–7.09 (m, 2H, arom),
6.96–6.94 (d, 2H, J = 5.4 Hz, arom), 5.55 (br, 1H, NHCO), 4.49–4.45 (t,
2H, J = 4.1 Hz, OCH2), 4.01–3.98 (t, 3H, CH2ONO2, NH), 3.58–3.52 (q,
1H, CHCH3), 3.49–3.46 (t, J = 4.4 Hz, 2H, NHCH2), 3.24–3.19 (q, 2H,
CH2NHCO), 3.08 (br, 2H, C4-H2), 2.70 (br, 2H, C1-H2), 1.92 (br, 4H,
C3-H2, C2-H2), 1.84–1.76 (m, 4H, OCH2CH2, NHCH2CH2), 1.67–1.60
(m, 2H, CH2CH2ONO), 1.55–1.50 (m, 7H, CH2CH2NHCO, CHCH3,
OCH2CH2CH2), 1.44–1.36 (m, 4H, CH2CH2CH2ONO, NHCH2CH2CH2),
1.27 (br, 6H, NHCH2CH2CH2CH2CH2CH2). 13C NMR (CDCl3): d
1561, 1631, 2929 cmꢂ1 1H NMR (CDCl3): d 7.96–7.89 (t, 2H,
.
J = 8.9 Hz, arom), 7.57–7.51 (t, 1H, J = 7.0 Hz, arom), 7.48–7.44
(m, 2H, arom), 7.39–7.30 (m, 2H, arom), 7.13–7.07 (m, 2H, arom),
6.96–6.92 (d, 2H, J = 8.9 Hz, arom), 5.63–5.59 (t, 1H, J = 5.6 Hz,
NHCO), 4.70–4.65 (t, 2H, J = 6.2 Hz, OCH2), 4.10–3.98 (m, 3H, CH2O-
NO2, NH), 3.58–3.43 (m, 3H, CHCH3, NHCH2), 3.24–3.16 (q, 2H,
CH2NHCO), 3.05 (br, 2H, C4-H2), 2.69 (br, 2H, C1-H2), 2.26–2.17
(m, 2H, OCH2CH2), 1.92–1.89 (t, 4H, C3-H2, C2-H2), 1.68–1.59 (m,
2H, NHCH2CH2), 1.54–1.51 (d, 3H, J = 7.2 Hz, CHCH3), 1.44–1.24
173.38 (NHCO), 161.00, 158.19 (arom, C3), 158.75 (arom), 158.54
0
(arom, C4 ), 150.95 (arom), 147.18 (arom), 142.42, 142.35 (arom,
0
(m, 10H, NHCH2CH2CH2CH2CH2CH2CH2). 13C NMR (CDCl3):
d
C1), 130.67, 130.63 (arom, C5), 130.00 (arom, C6 ), 129.97 (arom,
0
0
173.39 (NHCO), 161.69, 157.74 (arom, C3), 158.20 (arom), 158.09
C2 ), 128.45 (arom), 128.39 (arom), 127.68 (arom, C1 ), 127.52,
127.39 (arom, C4), 123.62 (arom), 123.55, 123.51 (arom, C6),
122.86 (arom), 120.10 (arom), 115.69 (arom), 115.33, 115.09 (arom,
0
(arom, C4 ), 150.94 (arom), 147.18 (arom), 142.56, 142.44 (arom,
0
C1), 130.67, 130.61 (arom, C5), 130.09 (arom, C6 ), 130.04 (arom,
0
0
0
0
C2 ), 128.39 (arom, arom), 128.20 (arom, C1 ), 127.36, 127.15 (arom,
C4), 123.60 (arom), 123.60, 123.53 (arom, C6), 122.90 (arom),
120.06 (arom), 115.65 (arom), 115.41, 115.04 (arom, C2), 114.46
C2), 114.53 (arom, C3 , C5 ), 73.20 (OCH2), 67.69 (CH2ONO2), 49.37
(NHCH2), 46.67 (CHCH3), 39.64 (CH2NHCO), 33.83 (NHCH2CH2),
31.67 (CH2CH2NHCO), 29.45 (C4), 29.14 (NHCH2CH2CH2CH2),
29.02 (CH2CH2CH2CH2NHCO, OCH2CH2), 26.75 (CH2CH2ONO2,
NHCH2CH2CH2), 26.62 (CH2CH2CH2NHCO), 25.68 (OCH2CH2CH2),
25.45 (CH2CH2CH2ONO2), 24.74 (C1), 23.03 (C3), 22.72 (C2), 18.54
(CHCH3). ESI-MS: m/z 713 [M+H]+. Anal. Calcd for
(C42H53FN4O5 ꢀ 1/2 H2O): found C, 69.57; H, 7.19; N, 7.54; requires
C, 69.88; H, 7.54; N, 7.76.
0
0
(arom, C3 , C5 ), 69.92 (OCH2), 63.57 (CH2ONO2), 49.38 (NHCH2),
46.63 (CHCH3), 39.63 (CH2NHCO), 33.83 (NHCH2CH2), 31.68
(CH2CH2NHCO), 29.44 (C4), 29.13 (NHCH2CH2CH2CH2), 29.01
(CH2CH2CH2CH2NHCO), 26.98 (NHCH2CH2CH2), 26.74 (OCH2CH2),
26.61 (CH2CH2CH2NHCO), 24.72 (C1), 23.01 (C3), 22.71 (C2),
18.57 (CHCH3). ESI-MS: m/z 671 [M+H]+. Anal. Calcd for
(C39H47FN4O5 ꢀ 1/2 H2O): found C, 68.97; H, 7.02; N, 8.09; requires
C, 68.90; H, 7.12; N, 8.24.
4.2. Cholinesterase inhibition assay in vitro
4.1.12. 4-{20-Fluoro-40-[1-oxo-1-(8-(1,2,3,4-tetrahydroacridin-9-
ylamino)octylamino)propan-2-yl]biphenyl-4-yloxy}butyl
nitrate (3g)
The assay followed the method of Ellman et al., using a Shima-
dzu 160 Spectrophotometer. AChE (E.C.3.1.1.7, Type VI-S, from
Electric Eel) and BuChE (E.C.3.1.1.8, from equine serum), 5,50-dithi-
obis(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthiocho-
line (ATC), and butyrylthiocholine (BTC) iodides were purchased
from Sigma–Aldrich (Steinheim, Germany). AChE/BChE stock solu-
tion was prepared by adjusting 500 units of the enzyme and 1 mL
of gelatine solution (1% in water) to 100 mL with water. This en-
zyme solution was further diluted before use, to give 2.5 units/
mL in 1.4 mL aliquots. 0.075 M ATC/BTC iodide solution was pre-
pared in water. A 0.01 M DTNB solution was prepared in water
containing 0.15% (w/v) sodium bicarbonate. For buffer preparation,
potassium dihydrogen phosphate (1.36 g, 10 mmol) were dissolved
in 100 mL of water and adjusted with KOH to pH = 8.0 0.1. Stock
solutions of the test compounds were prepared in methanol,
Following general procedure V, compound 5b (0.31 g, 1 mmol)
and 13c (0.39 g, 1 mmol) combined to offer 3g as a yellow solid
(0.31 g, 45%); mp 31–33 °C. IR (KBr): 1401, 1637, 2361 cmꢂ1 1H
.
NMR (CDCl3): d 7.97–7.92 (m, 2H, arom), 7.57–7.52 (t, 1H,
J = 7.0 Hz, arom), 7.47–7.43 (m, 2H, arom), 7.39–7.31 (m, 2H,
arom), 7.14–7.07 (m, 2H, arom), 6.95–6.91 (d, 2H, J = 8.7 Hz,
arom), 5.67–5.62 (t, 1H, J = 5.6 Hz, NHCO), 4.56–4.51 (t, 2H,
J = 6.2 Hz, OCH2), 4.03–3.99 (m, 3H, CH2ONO2, NH), 3.59–3.45
(m, 3H, CHCH3, NHCH2), 3.24–3.16 (q, 2H, CH2NHCO), 3.07 (br,
2H, C4-H2), 2.68 (br, 2H, C1-H2), 1.94–1.91 (m, 8H, OCH2CH2,
C3-H2, C2-H2, NHCH2CH2), 1.68–1.57 (m, 2H, CH2CH2ONO), 1.54–
1.51 (d, 3H, J = 7.0 Hz, CHCH3), 1.44–1.24 (m, 10H,
NHCH2CH2CH2CH2CH2CH2CH2). 13C NMR (CDCl3):
(NHCO), 161.69, 157.74 (arom, C3), 158.40 (arom), 157.89 (arom,
d
173.41
100
to the final volume of 3.32 mL. For each compound, a dilution
l
L of which gave a final concentration of 10ꢂ4 M when diluted

Y. Chen et al. / Bioorg. Med. Chem. 21 (2013) 2462–2470
2469
series of at least five different concentrations (normally 10ꢂ4–10
ꢂ9 M) was prepared.
humidified atmosphere. Cells were incubated 24 h with test
compound at concentration 0.25 M. {1S-Benzyl-4R-[1-(1S-car-
bamoyl-2-phenylethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-
2R-hydrox-5-phenylpentyl}carbamic acid tert-butyl ester
(L-685458) was used as positive control. DMSO (0.3%) was used
as negative control. Following incubation with the test compounds
the supernatant was collected and the Ab40 levels were determined
using a sandwich Human/Rat b-Amyloid(40) ELISA Kit II, per-
formed in 96-well microtiter plates. Conditioned media was incu-
bated with capture antibody for 16 h at 4 °C, followed by washing.
The detecting antibody incubation was for 2 h at 4 °C, again fol-
l
For measurement, a cuvette containing 3.0 mL of phosphate
buffer, 100
compound solution was allowed to stand for 5 min before 100
of DTNB were added. The reaction was started by addition of
20 L of the substrate solution (ATC/BTC). The solution was mixed
lL of the respective enzyme, and 100 lL of the test
lL
l
immediately, and exactly 2 min after substrate addition the
absorption was measured at 25 °C at 412 nm. For the reference va-
lue, 100
lL of water replaced the test compound solution. For
determining the blank value, additionally 100
l
L of water replaced
the enzyme solution. Each concentration was measured in tripli-
cate at 25 °C. The inhibition curve was obtained by plotting per-
centage enzyme activity (100% for the reference) versus
logarithm of test compound concentration. Calculation of the IC50
values was performed with Graph Pad Prism™ 4.
Kinetic measurements were performed in the same manner,
while the substrate (ATC/BTC) was used in concentrations of 25,
50, 90, 150, 226, 452 lM for each test compound concentration
and the reaction was extended to 4 min before measurement of
the absorption. Vmax and Km values of the Michaelis–Menten kinet-
ics were calculated by non-linear regression from substrate-veloc-
ity curves using Graph Pad Prism™ 4. Linear regression was used
for calculating the Lineweaver–Burk plots.
lowed by the wash steps. Then 100
added and incubated for 30 min at room temperature in the dark,
and the reaction was stopped by adding 100 L of 2 M H2SO4.
Absorbance at 450 nm was recorded with a Microplate Reader TE-
CAN Safire2 (Tecan Group Ltd, Maennedorf, Switzerland). Ab40 was
used to generate standard curves. The percent of control Ab40 as a
function of the concentration of the test compound was recorded.
Triplicate measurements from each drug concentration were
averaged.
The cytotoxicity potential of test compound was assessed in the
same cells of the Ab assay using the 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) assay. After adding
increasing concentrations of test compound (ranging from 0.01
lL of TMB ultrasubstrate was
l
to 100
of culture medium containing 20
incubated for another 4 h at 37 °C, 5% CO2, and saturated humidity.
After removal of the medium, an amount of 120 L of 100% DMSO
was added to each well. The amount of formed formazan was
determined by reading the samples at 570 nm.
l
M), cells were incubated for 24 h. Then cells with 100
lL
4.3. Detection of nitrite (Griess reaction)
l
L (0.5 mg mLꢂ1) of MTT were
A solution of the appropriate compound (80
added to 320 L of phosphate buffer (pH 7.4) containing 5 mM
cysteine. The final concentration of the compound was 10ꢂ4 M.
After incubation for 4 h at 37 °C, 120 L of the reaction mixture
was treated with 25 L of Griess reagent (2 g of sulfanilamide,
l
L) in DMSO was
l
l
L
-
l
l
4.6. Behavioural studies
0.1 g of N-naphthylethylenediamine dihydrochloride, 85% phos-
phoric acid [5 mL] in distilled water [final volume, 50 mL]). After
30 min in darkness at room temperature, the absorbance was mea-
sured at 540 nm using a Microplate Reader TECAN Safire2 (Tecan
Group Ltd, Maennedorf, Switzerland) each compound was mea-
sured in triplicate. Standard sodium nitrite solutions (0, 0.01,
0.03, 0.05, 0.07, 0.09, 0.2, 0.4 lg/mL) were used to construct the
calibration curve, from which the amount of nitric oxide release
(quantitated as nitrite ion) was calculated.
The experiments were performed with adult male ICR mice
(weighing 18–22 g), which were purchased from Comparative
Medicine Centre, Yangzhou University (Certificate No: 2023793).
Scopolamine hydrobromide was obtained from Aladdin Reagents,
Shanghai, China. Tacrine hydrochloride hydrate was purchased
from Bio Trend, Wangen, Switerland.
Scopolamine (0.2 mg/100 g b wt) were dissolved in saline and
administered intraperitoneally (ip) 5 min before the training trial.
Tacrine was dissolved in CMC-Na solution (0.5 g CMC-Na in
100 mL distilled water) and 0.5 mg/100 g b wt, corresponding to
4.4. Vascular relaxation study
1.978 lmol/100 g b wt, were given in intragastric (id) administra-
Aortic rings isolated from rat heart (3–5 mm long) were sus-
pended between two L-shaped platinum hooks and mounted un-
der a passive tension(1–1.1 g) in a 10 mL organ bath filled with
modified Krebs–Henseleit solution, which was kept at 37 °C. Prep-
aration was connected to isometric force transducer JZ100 (New
Channel Instruments, Gaobeidian, China). After an equilibration
period of 60 min, contractions were induced by Norepinephrine
(NE, final concentration 10ꢂ5 M) to detect the activity of the vessel.
Endothelial integrity was further assessed by the acetylcholine (10
ꢂ5 M)-induced relaxation of NE-precontracted vessels. The ring
segments were initially made to contract with KCl (60 mM) and
tion 1 h before the training trial. The new substances were solved
in CMC-Na solution and an equimolar dose corresponding to ta-
crine was administered id 1 h before the training trial. Mice were
divided into four groups. (i) Vehicle, (ii) scopolamine, (iii) com-
pound 1a plus scopolamine and (iv) compound 3a plus scopol-
amine were given to each group, respectively.
The mice were subjected to Passive Avoidance Test by using
Step-through Passive Avoidance Apparatus for Mice RD1105-RA-
M-1 (Mice doctor, Shanghai, China). The apparatus contained
two symmetrical compartments (dark and light chamber), which
were separated by a dark board that had a door near the floor.
The grid floors of the dark chamber consisted of copper bars. To
begin with, the dark compartment was electrified to generate
low intensity foot shocks (0.4 mA). A trial of 300 s started as soon
as one animal was gently put inside the light compartment, with
the head facing away from the door. After entering to the dark
compartment, the mouse would return to the light one. After-
wards, the mouse would repeat the actions. A transfer from light
chamber into the dark chamber was recorded as transfer latency
time (TLT in seconds). The first trial was for training and the
retention was tested by a second trial 24 h later, in which the
subsequently with L
-cysteine (10ꢂ3 M). The relaxation response
to addition of test compounds was studied after the contractions
became constant. Relaxation responses were expressed as a per-
centage of the KCl (60 mM)-induced contraction.
4.5. Cell-based Ab inhibition assay
Ab40 were measured in culture medium of primary cortical neu-
ron cells (pregnant Wistar rats). Cells were seeded onto 48-well
plates and allowed to grow for 3–5 days in 5% CO2/95% air in a

2470
Y. Chen et al. / Bioorg. Med. Chem. 21 (2013) 2462–2470
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The authors thank the financial support from National Science
Foundation of China (NSFC, No. 30973608) and the Natural Science
Foundation of Jiangsu Province of China (No. BK2009296).
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Supplementary data (syntheses and spectra data of intermedi-
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online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.005.
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