Design, synthesis and biological evaluation of D-ring opened galantamine analogs as multifunctional anti-Alzheimer agents

Article abstract of DOI:10.1016/j.ejmech.2014.02.035

Facing the multifactorial nature of Alzheimer's disease, twelve dibenzofuran/carbazole derivatives, which can be considered as the D-ring opened analogs of galantamine, have been designed and synthesized as multifunctional anti-Alzheimer agents. In vitro tests revealed that compounds 3 and 5, which bear a nitrate moiety in the molecule, showed a potent inhibition activity towards AChE and compound 3 showed a good Aβ42 aggregation inhibitory activity. Moreover, 3 and 5 could also release a relative low concentration of NO in vitro and they did not show toxicity to neuronal cells, while exerted a neuroprotective effect against the Aβ-induced toxicity. More importantly, compound 3 showed a significant spatial memory improving effect in vivo, and a good safety in the ex vivo toxicity study.

Full text of DOI:10.1016/j.ejmech.2014.02.035

European Journal of Medicinal Chemistry 76 (2014) 376e386
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of D-ring opened
galantamine analogs as multifunctional anti-Alzheimer agents
,
,
c
c
a
Lei Fang a b, Xubin Fang a, Shaohua Gou a , Amelie Lupp , Isabell Lenhardt , Yanyan Sun ,
*
Zhangjian Huang b, Yao Chen d, Yihua Zhang b, Christian Fleck c
,
*
a Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, Pharmaceutical Research Center, School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, PR China
b State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
c Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Jena, Germany
d Institute of Pharmacy, Friedrich Schiller University Jena, Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Facing the multifactorial nature of Alzheimer’s disease, twelve dibenzofuran/carbazole derivatives, which
can be considered as the D-ring opened analogs of galantamine, have been designed and synthesized as
multifunctional anti-Alzheimer agents. In vitro tests revealed that compounds 3 and 5, which bear a
nitrate moiety in the molecule, showed a potent inhibition activity towards AChE and compound 3
Received 13 October 2013
Received in revised form
12 February 2014
Accepted 13 February 2014
Available online 14 February 2014
showed a good A
concentration of NO in vitro and they did not show toxicity to neuronal cells, while exerted a neuro-
protective effect against the A -induced toxicity. More importantly, compound 3 showed a significant
spatial memory improving effect in vivo, and a good safety in the ex vivo toxicity study.
Ó 2014 Elsevier Masson SAS. All rights reserved.
b42 aggregation inhibitory activity. Moreover, 3 and 5 could also release a relative low
b
Keywords:
Anti-Alzheimer
AChE inhibitors
Neuroprotective agents
Ab aggregation inhibitors
Galantamine analogs
1. Introduction
donepezil and galantamine; the oldest AChEI tacrine was with-
drawn from the market due to its serious hepatotoxicity) and one
N-methyl-D-aspartic acid (NMDA) receptor antagonist (mem-
antine). The rationale of AChEIs, which were developed based on
the cholinergic hypothesis [3,4], is mainly aiming to enhance the
levels of synaptic ACh and consequently improve the cholinergic
function in the central nervous system. AChEIs can slow the pro-
gression of AD and postpone the cognitive deterioration of the
patients, acting as the mainstay for the symptomatic treatment of
AD. Nevertheless, as mentioned above, AD is a multifactorial dis-
ease, and traditional AChEIs like tacrine and donepezil can only hit
AChE one target without influence on other pathogenic factors of
Alzheimer’s disease (AD) is the most prominent form of de-
mentia affecting the aged with high incidence. Despite AD has been
found for more than 100 years, it is still incurable up to date. The
pathophysiology of AD is very complicated. Many studies revealed
that AD is a multifactorial syndrome derived from a complex array
of neurochemical factors, including the deficiency of synaptic
acetylcholine (ACh) and other related neurotransmitters, the for-
mation of neurotoxic b-amyloid peptide (Ab), oxidative stress, the
inflammation of neurons, and so on [1,2]. Thanks to the elucidation
of the AD mechanism during the past 2 decades, a variety of eti-
ology hypotheses (e.g. cholinergic hypothesis, amyloid hypothesis,
oxidative hypothesis, etc.) have been brought out, leading to
various therapeutic strategies. However, only four drugs are clini-
cally available for the treatment of AD so far; they are three
acetylcholinesterase inhibitors (AChEIs, including rivastigmine,
AD, especially Ab and its aggregates which play a key role in trig-
gering a neurotoxic cascade and have been considered as a primary
target for developing novel anti-AD candidates (e.g. carbazole-
based fluorophores [5] and bifunctional metal chelators [6]).
Thereby, AChEIs can offer only a palliative effect, but not halt the
progression of the disease. Facing the multifactorial nature of AD,
researchers have now turned to develop the so called multi-target
directed ligands which are able to simultaneously modulate
different targets involved in the AD cascade [7]. As the cholinergic
* Corresponding authors.
E-mail addresses: sgou@seu.edu.cn (S. Gou), Fleck-Christian@t-online.de
(C. Fleck).
and Ab-related pathways play key roles in the progression of the
http://dx.doi.org/10.1016/j.ejmech.2014.02.035
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
377
disease, they have been considered as the main targets of the
multifunctional ligands.
galantamine is also made broken to generate a flexible aliphatic
side chain, on which an additional pharmacophore, such as a nitric
oxide (NO) donor (e.g. organic nitrate), can be introduced, leading
to novel dibenzofuran/carbazole-NO donor hybrid compounds
(Fig. 1). Our design strategy is upon the following factors that i) the
designed compounds, which maintain the aromatic moiety and the
nitrogen atom, will probably retain or even improve the AChE
inhibitory activity of galantamine; ii) the simplified structure of the
target molecules makes them possible to be prepared via common
synthetic reactions; iii) more importantly, the introduction of the
NO-donor moiety, which is able to release NO in vivo, will benefit
the anti-AD activity. NO is a key signaling molecule involved in the
regulation of many physiological processes. It is also reported that
NO plays an important role in the nervous system [17]. Our previ-
ous studies indicated that tacrine-NO donor hybrid compounds can
enhance the anti-AD activity both in vitro and in vivo [18,19].
Therefore, a synergic action of the dibenzofuran/carbazole scaffold
and the NO donor can be expected. In order to predict whether the
target compounds retain the AChE inhibitory activity of galant-
amine we preliminarily performed a simulant study using MOE
software. The overlap study of galatamine and the D-ring opened
analog showed that the benzofuran ring almost total covers the B
and C ring of galantamin; a clear difference between these two
molecules is that the tricyclic aromatic system of the
analog extends about 7.9 Ang while the partially saturated galant-
amine tricyclic system is bent over, thus it is 2.5 Ang shorter (Fig. S3,
supporting information). Docking compound 2a to AChE (Protein
Data Bank code 3I6M) also revealed that the ligand could effectively
Galantamine (1, Fig. 1) is a natural AChEI approved for the
treatment of AD in many countries. It has a unique dual mechanism
of action, combining allosteric modulation of nicotinic receptors
with reversible, competitive inhibition of AChE [8]. Besides, it also
exhibits less toxicity than other AChEIs like tacrine. Due to such
unique properties, galantamine has been presented as an ideal
leading structure for developing multi-target directed ligands. It is
noted that much effort has been made in the past decade. The
structural modifications of galantamine by previous researches
were mostly carried out at the 6-position hydroxyl group and the
11-position nitrogen atom to which a second pharmacophore was
incorporated to enhance the anti-AD activity [9e13]. However, such
structural modifications usually took galantamine as a starting
material, resulting in larger and more complex molecules than
galantamine itself, which may possibly cause pharmacokinetic
problems. There are also some reports concerning the galantamine
analogs whose structural backbone is different from that of gal-
antamine. [14,15] However, the focus of these studies was on the
synthetic chemistry, while not on the medicinal chemistry.
Our drug design strategy is to develop novel facile-prepared
galantamine analogs by simplifying the backbone of galantamine.
From the chemical point of view, galantamine contains two struc-
tural units of a universal tricyclic benzofuran core with a sterically
congested quaternary carbon and a complex seven-member aze-
pine ring. Notably, the sterical C2 allylic alcohol moiety and the
quaternary carbon are very difficult to be constructed via common
synthetic procedures. However, according to the classic structuree
activity relationship theory of AChEIs, these two fragments are not
essential for the activity of galantamine, while the aromatic moiety
(i.e. the benzene ring) and the nitrogen atom are. X-ray crystal
structure of galantamine bound in the active site of AChE also
revealed that the AChE inhibitory activity of galantamine is due to
enter the enzyme pocket and block the catalytic site of AChE via
p-
p
conjugation and H-bond. These results preliminarily proved our
drug design rationale that the simplified molecules could retain the
AChE inhibitory activity of galantamine (Fig. S4, supporting
information).
the interaction of the benzene ring with Trp84 via
pe
p
stacking
2. Results and discussion
action and the interaction of the nitrogen atom with Asp72 via H-
bond action [16]. Therefore, we can hypothesize that the benzo-
furan fragment and the nitrogen atom are exact pharmacophores
responsible for the activity, while the other parts of the molecular
structure, including the complex congested quaternary carbon and
the seven-membered azepine ring, can be simplified to generate
novel facile-prepared galantamine analogs. With this regard in
mind, we attempt to use dibenzofuran/carbazole as the basic
skeleton instead of the complex tetracyclic structure of galant-
amine. Furthermore, the seven-membered azepine ring of
2.1. Synthesis of the target compounds
For the synthesis of the dibenzofuran derivatives, the previous
reported protocol was employed with minor modifications [20].
Generally, 3-bromo-4-nitrophenyl methyl ether reacted with m-
cresol in the presence of potassium hydroxide to give the nitro-
diaryl ether intermediate (6), which was further reduced catalyti-
cally by hydrogen, forming the amine product (7). The cyclization to
dibenzofuran was carried out via two steps: firstly treating 7 with
NaNO2 under acid condition yielded the diazotized o-(ary1oxy)
aniline intermediate (8); and secondly a mixture of the dibenzo-
furan derivatives (9a, 9b) was obtained via a free-radical cycliza-
tion. The mixture (as judged from 1H NMR spectra and HPLC result
the ratio of 9a:9b is about 3:2, see Supporting Information, Fig. S1)
can hardly be separated, but after bromination by N-bromosucci-
nimide (NBS) and the treatment of the corresponding amines the
products 2ae2e can be successfully obtained (Scheme 1). Com-
pound 2a was hydrolyzed by treating with 48% HBr aqueous solu-
tion, giving the phenol product 2f (Scheme 3). To introduce the
organic nitrate moiety, compound 2a was acylated by 3-
chloropropanoyl chloride to give the chloride intermediate, which
was further treated with silver nitrate to give product 3 (Scheme 3).
The synthesis of the carbazole derivatives is different from that
of dibenzofurans since the carbazole scaffold can not be con-
structed via the cyclization reaction due to the NH group of
carbazole. Therefore, Suzuki reaction and the modified Cadogan
reaction [21] were employed to prepare the carbazole scaffold. In
detail, 1-bromo-4-methoxy-2-nitrobenzene was treated with 2-
methylphenylboronic acid in the presence of catalytic amount of
Fig. 1. The molecular structures of galantamine (1), dibenzofuran derivatives (2ae2f,
3) and carbazole derivatives (4ae4d, 5).

378
L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
CH3
CH3
NO2
Br
NO2
NH2
a
b
H3CO
H3CO
+ H2CO
f
O
6
H3CO
O
7
c
H3C
O
BF4
N2
CH3
d
H3CO
CH3
H3CO
O
8
O
9b
9a
R1
N
e
Br
R2
H3CO
H3CO
O
10
O
2a, R1=CH(CH3)2, R2=H;
2b, R1=CH2CH3, R2=H;
2c, R1=CH3, R2=CH2CH3;
Scheme 3. Synthetic route of NO donor hybrid compounds. Reagents and condition: a)
pyridine, CH2Cl2, 3-chloropropanoyl chloride, ꢁ5 ꢀC, 1 h; b) AgNO3, anhydrous CH3CN,
reflux, 24 h; c) 48% HBr aqueous solution, reflux, 3 h.
2d, R1=CH2CH3, R2=CH2CH3;
2e, R1=CH2Ph, R2=H
Scheme 1. Synthetic route of dibenzofuran derivatives. Reagents and condition: a)
KOH, m-cresol, 130 ꢀC / 170 ꢀC, 2 h; b) H2 (5 Mpa), 10% Pd/C, 50 ꢀC, 5 h; c) 18% HCl,
NaNO2, NaBF4, ꢁ5 ꢀC, 20 min; d) 1 M H2SO4, Cu2O, 70 ꢀC, 3 h; e) NBS, AIBN, CCl4, reflux,
3 h; f) alkylamine, KI, anhydrous K2CO3, CH2Cl2, RT, overnight.
amines, compound 15ae15c was obtained, respectively, and then
transformed into compound 4ae4c by removing the protecting
group (Scheme 2). The synthesis procedure of compound 4d and 5
was as same as that of compound 2f and 3, respectively (Scheme 3).
Pd(PPh3)4 to give the nitrobiphenyl intermediate (11). Subse-
quently, the cyclization of the nitrobiphenyl was achieved via
reductive deoxygenation of the nitro group using a slight excess of
triphenylphosphine in o-dichlorobenzene, resulting in the carba-
zole compound (12). Thereafter, we tried to directly treat 12 with
NBS, as we did in the dibenzofuran case, to obtain the benzylic
bromination intermediate. However, only the electrophilic aro-
matic bromination products were obtained after this reaction,
which is probably because of the electron donating ability of the NH
group of compound 12. Thus, compound 12 was firstly reacted with
benzenesulfonyl chloride to yield the sulfonylated compound 13,
which was then treated with NBS to offer the desired benzylic
bromination product (14). After treating 14 with corresponding
2.2. Cholinesterase inhibition assay in vitro
The AChE inhibitory activity of the analogs was screened by
Ellman’s assay in vitro (Table 1). Most of the synthesized com-
pounds showed moderate to potent inhibitory effect on the cho-
linesterases, except for 2c, 2e and 4c towards AChE. The AChE
inhibitory activity of compounds 2a and 5 is in magnitude at the
same order of galantamine, with IC50 values in the single to double
digit micromole range, while the activity of compound 3 (IC50
0.18 mM) is 60-fold higher than galantamine (IC50 10.53 mM). As far
as butyrylcholinesterase (BChE, another cholinesterase isomer
responsible for the hydrolysis of ACh) is concerned, all analogs
showed higher inhibitory activity than galantamine (Table 1),
indicating the analogs can be considered as dual inhibitors towards
both AChE and BChE. Analysis of the structureeactivity relationship
of the synthesized compounds revealed that the activity of diben-
zofuran derivatives is generally better than that of carbozole de-
rivatives. The substituents of the nitrogen atom seem have
influence on the activity. The alkyl substituents are superior to the
aralkyl substituents since 2e and 4c only showed a very weak ac-
tivity. Comparing the activity of 2a and 4a with 2e and 4d,
respectively, we could also found that converting the methyl ether
group to phenol group would slightly decrease the inhibition ac-
tivity. Interestingly, compounds 3 and 5, which are hybrid com-
pounds derived from compounds 2a and 4a connected to a NO
donor moiety, showed a significantly improved inhibitory activity.
This finding is consistent with our previous result [18,19]. The
improved activity is probably due to the introduction of the nitrate
group, as our former study indicated that it could interact on the
residue Trp279 of the peripheral anionic site of AChE via a hydro-
phobic action. [19]
H3C
OH
Br
B
b
a
HO
H3C
+
H3CO
H3CO
NO2
H3CO
NO2
11
Br
H3C
N
H3C
c
d
H3CO
H3CO
N
S
Ph
N
H
O
S
O
O
O
Ph
13
12
R1
14
R1
N
R2
N
R2
e
f
H3CO
H3CO
N
N
H
O
S
O
Ph
15a, R1= CH(CH3)2, R2= H;
4a, R1= CH(CH3)2, R2= H;
4b, R1=CH2CH3, R2=CH2CH3;
4c, R1=CH2Ph, R2=H
2.3. In vitro nitric oxide release assay
15b, R1=CH2CH3, R2=CH2CH3;
15c, R1=CH2Ph, R2=H
Furthermore, in order to investigate the NO-releasing ability of
the target compounds, compounds 3 and 5 were selected to
perform Griess assay in vitro. It is well known that a nitrate group is
able to release NO via either enzymatic or nonenzymatic pathways
[22]. Thus, the in vitro nitric oxide release was tested under two
Scheme 2. Synthetic route of carbazole derivatives. Reagents and condition: a)
Pd(PPh3)4, Na2CO3, DME, reflux, 20 h; b) PPh3, DCB, reflux, 3 h; c) PhSO2Cl, NaH, THF,
0
ꢀC / RT, overnight; d) NBS, AIBN, CCl4, reflux, 3 h; e) alkylamine, KI, K2CO3, anhy-
drous acetone, RT, overnight; f) 2 N NaOH, ethanol, reflux, overnight.

L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
379
Table 1
increased to 29% and 25%, respectively, which is indicative of
dosage-dependent manner of the inhibition. As for galantamine, no
significant inhibition was observed no matter low or high con-
centration of galantamine was used. Clearly, in comparison with
Inhibitory effect of 2ae2f, 3, 4ae4d, 5 and galantamine on AChE and BChE (IC50
values).
Compound
IC50
(
m
M) ꢂ S.E.M.a
Selectivity ratioc
AChEb
BChEb
galantamine compound 3 showed additional anti-A
b
42 aggrega-
tion activity, which may alleviate the A
eventually benefit the treatment of AD.
b-induced toxicity and
Galantamine
10.53 ꢂ 3.11
11.60 ꢂ 3.41
18.92 ꢂ 4.08
>>50
39.03 ꢂ 12.11
13.5 ꢂ 3.1
0.27
0.86
0.85
e
2a
2b
2c
2d
2e
2f
3
4a
4b
4c
4d
5
22.18 ꢂ 7.12
Not tested
2.5. Atomic force microscopy
39.11 ꢂ 8.80
27.39 ꢂ 7.76
18.1 ꢂ 9.4
1.43
e
>>50
22.6 ꢂ 7.4
0.18 ꢂ 0.04
50.83 ꢂ 12.13
37.91 ꢂ 8.44
>>50
18.36 ꢂ 3.1
14.3 ꢂ 3.2
1.23
0.13
5.03
2.54
e
It is well known that a fluorescence intensity-based assay is
prone to produce false positives because of fluorescence quenching
due to absorbance from colored compounds [24]. Therefore, the
10.08 ꢂ 7.7
14.88 ꢂ 2.08
29.9 ꢂ 1.79
17.27 ꢂ 1.16
2.50 ꢂ 0.90
formation of Ab aggregates was visualized using Atomic Force Mi-
croscopy (AFM) to confirm the inhibition activity of the target
compound. As compared with the blank sample (see Supporting
Information Fig. S2, a), the model sample (see Supporting
Information Fig. S2, b) without any additive contained high den-
43.49 ꢂ 5.02
3.54
0.88
2.21 ꢂ 0.51
a
Data are the means of at least three determinations.
AChE from Electric Eel and BChE from equine serum were used.
Selectivity ratio¼ (IC50 of AChE)/(IC50 of BChE).
b
c
sity of typical A
with compound 3 (50
b
amyloid fibrils. In contrast, the sample incubated
M) contained only a number of aggregates,
m
different conditions: i) incubation of the test compound with only
phosphate buffer solution (PBS) at pH 7.4 and 37 ꢀC for 24 h, ii)
incubation of the test compound in PBS at pH 7.4 and 37 ꢀC to which
NO release assay
**
10
8
90 mL rat serum had been added. The results (Fig. 2) showed that
Blank
the %NO released from the nitrate of compounds 3 and 5 in PBS at
pH 7.4 varied over a 4.8e6.1% range, which is indicative of a mod-
erate release of NO. The NO release ability of 3 and 5 was slightly
improved when the measurement was performed in PBS contain-
ing rat serum. This is probably due to that the thiols or enzymes
contained in the serum are able to promote the decomposition of
the nitrate. Low concentration of NO is essential for the growth of
neurons. It was reported that some NO donors or NO mimetic
molecules had shown a positive effect on the treatment of AD [17].
Our previous studies also indicated that NO-releasing tacrine de-
rivatives can enhance the anti-AD activity both in vitro and in vivo,
probably due to the vasorelaxant effect of NO [18]. In this context, it
may be anticipated that the NO release ability of 3 and 5 may
present some additional anti-AD properties, though further works
need be done to confirm it.
Compound 3
Compound 5
**
**
6
**
4
2
0
PBS onlyb
PBS + Serumc
Fig. 2. NO release ability: a) Percent of nitric oxide released based on a theoretical
maximum release of 1 mol of NO/mol of the nitrate test compound. The result is the
mean value of 3 measurements (n ¼ 3) where variation from the mean % value was
ꢃ0.2%. b) A solution of the test compound (2.4 mL of a 100
mM) or solvent vehicle
(2.4 mL) in phosphate buffer at pH 7.4 was incubated at 37 ꢀC for 24 h. c) A solution of
the test compound (2.4 mL of a 100 M) or solvent vehicle (2.4 mL) in phosphate buffer
at pH 7.4, to which 90
L rat serum had been added, was incubated at 37 ꢀC for 24 h.
m
2.4. ThT fluorescence assay
m
Statistical analysis was performed by the one-way ANOVA followed by Tukey’s mul-
tiple comparison test. **p < 0.01 vs. blank.
A
b
and its aggregates have been believed to be key etiological
factors triggering a neurotoxic cascade and finally causing neuro-
degeneration in AD brains. In vivo monomeric A 42 peptides come
from the amyloid precursor protein (APP) cleaved by - and
secretase. The monomeric A 42 peptides, spontaneously or bio-
b
1.5
1.0
0.5
0.0
Curcumin
Galantamine
Aβ alone
3
b
g-
b
activated by various factors, prone to aggregate and subsequently
form toxic oligomeric intermediates and plaque-associated amyloid
fibrils. Thus, inhibition of the Ab aggregation has been presented as
a promising approach for the development of anti-AD agents. It was
reported previously that carbazole and benzofuran scaffolds had
ability to inhibit A
inhibitory effect of the synthesized analogs on A
compound 3 (5, 50, 100 M), which showed good activity in all
former tests, and galantamine (5, 50, 100 M) were selected to
*
**
**
**
b
aggregation [5,23]. In order to investigate the
b
aggregation,
m
m
perform Thioflavin T (ThT) assay. Curcumin was used as positive
control. The results are presented in Fig. 3. At a concentration of
5
m
M, neither curcumin nor the test compound showed significant
inhibitory effect on the aggregation of A 42. As the concentration
increased, the inhibition rate of curcumin and 3 was enhanced. At a
concentration of 50 M, the inhibition rate of curcumin and 3
reached 24% and 19%, respectively. When the concentration was
raised to 100 M, the inhibition rate of curcumin and 3 was also
5 µM
50 µM
100 µM
b
Fig. 3. Inhibition of amyloid formation by compound 3, curcumin and galantamine
monitored by ThT fluorescence. The result is the mean value of 3 measurements
(n ¼ 3) ꢂ SEM. Statistical analysis was performed by the one-way ANOVA followed by
m
m
Tukey’s multiple comparison test. *p < 0.05 vs. Ab alone, **p < 0.01 vs. Ab alone.

380
L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
but without fibrils (see Supporting Information Fig. S2, c). Ac-
cording to AFM histogram profile analysis, the sample with com-
pound 3 had the aggregates with the average height of 2.15 nm,
while aggregates contained in the control samples had the average
height of 8.68 nm. The findings, which are in accordance with the
ThT data, apparently indicate a moderate inhibition effect of com-
pound 3.
a)
8
6
4
2
0
Scop
tacrine
Compound 3
Compound 5
*
Galantamine
**
**
**
*
2.6. Cell viability analysis
**
*
**
**
Clinically, most AD patients are diagnosed at the middle to late
phase of the disease; that means a great number of Ab oligomers/
0 min
20 min
60 min
120 min
fibrils already exist in the patients’ brain when the treatment starts.
In this context, the agents capable of protecting the neurons from
Scop
the toxicity of A
aggregation inhibitors. Thus, it will be interesting to investigate
whether compound 3, which already showed A aggregation
inhibitory activity in vitro, also possesses neuron protective effect
against A -inducing toxicity. By using a previously reported pro-
b aggregates seem more attractive than the Ab
b)
400
tacrine
**
Compound 3
Compound 5
b
300
200
100
0
Galantamine
b
cedure [25] compound 3 was screened for such activity. When PC12
cells were incubated with different concentrations of compound 3
**
*
(0.08, 0.4, 2, 10
compared with the vehicle group (control), even at the highest
concentration tested (10 M). In contrast, A 42 (5 M) induced a
mM) for 24 h, no cytotoxicity was observed as
**
**
**
m
b
m
significant, albeit moderate, toxicity to PC12 cells upon exposure for
24 h as the viability of the cells dropped from 1 to 0.82. Interest-
0 min
20 min
60 min
120 min
ingly, when PC12 cells were co-treated with A
b42 (5
mM) and
different concentrations of compound 3 (0.08, 0.4, 2, 10
mM), the
Fig. 5. Influence of tacrine, galantamine, compounds 3 and 5 (each 1 mmol/100 g b.
toxicity was diminished in a dose-dependent manner, indicating a
neuroprotective effect of compound 3. As for galantamine, the
protective effect was not observed (Fig. 4). The neuroprotective
effect of 3 is probably due to the NO donor moiety. It was reported
that NO released from NO donor like nitrate or furoxans could
effectively prevent neurons from the treatment with neurotoxic
agents [26,27].
wt.) on scopolamine (0.05 mg/100 g b. wt.) induced impairment of working memory in
adult rats measured in an eight-arm radial maze. The result is the mean value of 14
measurements (n ¼ 14) ꢂ SEM. a) Errors made plus baits not found after 10 min. b)
Total searching time. Statistical analysis was performed by the one-way ANOVA fol-
lowed by Tukey’s multiple comparison test. *p < 0.01 vs. scop group, **p < 0.001 vs.
scop group.
cognition impaired adult rats (after scopolamine [scop] adminis-
tration) as animal model to measure the cognition improving ef-
fects of compounds 3 and 5. Galantamine and tacrine were chosen
as positive control, respectively. The measurement was performed
in an eight-arm radial maze, which is an accepted tool in behavioral
pharmacology [28]. After the rats were adapted to the maze and
trained to be able to quickly find the bait at the end of each arm,
they were treated with scopolamine (0.05 mg/100 g b. wt., i.p.).
Scopolamine distinctly blocks muscarinic cholinergic receptors,
and thus caused reversible impairment of the animals’ cognition.
2.7. Behavioral studies in vivo
In vivo study is essential for the pharmacological evaluation of
drug candidates, especially for anti-AD substances since many of
them met failure previously due to the poor penetration of the
blood-brain-barrier and low bioavailability in vivo. In order to
determine the in vivo activity of the target compounds, we used
Then tacrine (1 mmol/100 g b. wt.) as well as compounds 3, 5, and
Comp.3 + Aβ (5 µM)
Comp.3
galantamine (equimolar dose), which are supposed to more or less
compensate for the cholinergic dysfunction in the central nervous
system by inhibiting AChE, were administered to the animals,
respectively. Two behavioral parameters, i.e. the total searching
time for all baits and the sum of the number of errors made during
the exploration plus the number of the baits not found after 10 min
(errors plus baits not found), were recorded 0, 20, 60, and 120 min
after scopolamine administration to determine the cognition
improving activity of the selected compounds. Given the
scopolamine-induced cognition impairment usually fades within
2 h, the data obtained at 20 min are more valuable for discussion.
The results are shown in Fig. 5. At 20 min, the amount of errors
plus baits not found of the model group (i.e. scop group) increased
from 0 at 0 min to 6.2, and the total exploration time also increased
from 28 to 242 s, indicating an obvious impairment of working
memory and animals’ behavior caused by scopolamine. When the
animals were co-treated with tacrine, compounds 3 or 5, the
amount of errors plus baits not found significantly decreased to 2.1,
Galantamine
Galantamine + Aβ (5 µM)
1.5
1.0
0.5
0.0
**
**
*
*
#
Fig. 4. Neuroprotective effects of tested compounds against Ab42-induced cytotoxicity
towards PC12 cells. The result is the mean value of 3 measurements (n ¼ 3). Data were
subjected to one-way ANOVA followed by Dunnett’s multiple comparison post-test.
*p < 0.01 vs. Ab group, **p < 0.001 vs. Ab group, #p < 0.05 vs. control group.

L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
381
2.4, 2.5, respectively (Fig. 5, a). Besides, the total exploration time of
the compound 3 and 5 group also decreased from 242 s to 175 and
107 s, respectively, while the searching time of the tacrine group
(237 s) was not shortened (Fig. 5, b). For both errors plus baits not
found and the searching time parameters, statistically significant
differences can be observed between the scop group and the
compounds 3 and 5 groups, respectively. Apparently, both com-
pounds 3 and 5 effectively alleviated the cognitive deficits induced
by scopolamine. The activity of galantamine was not as good as the
other test compounds in the assay. This is probably because of its
relative low AChE inhibitory activity.
In order to more comprehensively evaluate the activity of the
test compounds, we developed a scoring system, which considers
both the exploration time and errors plus baits not found, to
quantify the performance of the rats in the assay. The calculation of
the score is based on the following equation: Score ¼ errors plus
baits not found þ total exploration time (seconds)/100. The lower
the score is, the better the performance of the rats is. Table 2 shows
the scores of the tacrine, galantamine, compound 3 and 5 groups,
respectively. The lowest scores of compound 3 and 5 groups sug-
gest the best performance of the corresponding rats in the assay.
The tacrine and galantamine groups got higher scores, which
attribute to the long searching time needed to find all food baits.
Notably, it was observed that the treatment with tacrine, no matter
with or without scopolamine, likely caused a significant slow-down
of the rats’ movement in the maze, which may result from the
tacrine-related cholinergic events, whereas the treatment with
compounds 3 and 5 had no influence on the rats’ movement at all.
These findings indicate that, in addition to the better spatial
memory improving effect in vivo, the target compounds 3 and 5
also possess advantages in terms of safety in comparison to tacrine.
was observed between the compound 3 group and the control
group, hinting no hepatotoxicity was caused by compound 3 at all.
As for brain and kidney tissues, both tacrine and compound 3
induced an increased ratio of GSH/GSSG and a lower level of lip-
idperoxidation products, suggesting no oxidative damage caused
by them.
3. Conclusion
Due to the multifactorial nature of AD the multi-target directed
agents hold considerable promise as a therapeutic strategy to
combat this incurable disease. Hereby we have designed and syn-
thesized novel dibenzofuran/carbazole derivatives as multifunc-
tional anti-AD agents by means of simplifying the skeleton of the
galantamine molecule. In comparison with galantamine, the ana-
logs, especially compound 3, showed an improved anti-AD activity,
combining the cholinesterase inhibition and anti-Ab aggregation
ability. Compounds 3 and 5, which bear a nitrate moiety in the
molecule, could also release a relative low concentration of NO
in vitro. Furthermore, in vitro assay revealed that the analogs did
not show toxicity to neuronal cells, and could exert a neuro-
protective effect against the A
b oligomer-induced toxicity. More
importantly, compound 3 showed a significant spatial memory
improving effect in vivo, and the ex vivo toxicity study indicated
that no obvious toxicity was caused by compound 3, suggesting a
good safety. Altogether, the results suggest that the dibenzofuran/
carbazole derivatives, in particular compound 3, can be considered
as potential therapeutic agents for AD. The work also presents new
leading structures for developing novel anti-AD drugs.
4. Experimental protocols
4.1. Chemistry
2.8. Biochemical investigations ex vivo
4.1.1. Materials and instruments
For more precise information about the safety of the target
compounds, we further applied an ex vivo study to investigate the
toxicity of compound 3 towards liver, kidney and brain tissues,
using tacrine as a reference control. After treatment with tacrine (1
All chemicals and reagents were of analytical reagent grade and
were used without further purification. All the solvents were dried
by the standard methods wherever needed. IR spectra were
measured on KBr pellets with a Nicolet IR200 FTIR spectrometer in
the range from 4000 to 400 cmꢁ1 1H NMR and 13C NMR were
.
or 6 mmol/100 g b. wt.) or equimolar doses of the novel compound 3
for 24 h, respectively, the rats were killed and the corresponding
tissues were separated. Five parameters, including levels of reduced
glutathione (GSH), oxidized glutathione (GSSG), total glutathione,
lipidperoxidation products (LPO), and the weight of the organ, were
measured and recorded. For a more comprehensive study of hep-
atotoxicity, which is one of the serious side effects of tacrine,
additional data of protein content, activity of ethylmorphine N-
demethylation (END), ethoxycoumarin O-deethylation (EcOD) and
ethoxyresorufin O-deethylation (ErOD) were also collected (see
Supporting Information, Table S1).
recorded using a Bruker 300/500 MHz spectrometer and refer-
enced to the residue CHCl3 7.26 ppm or DMSO-d6 2.5 ppm. Low
resolution and High resolution mass spectrum measurements were
carried out by using Angilent technologies LC/MS TOF. The Purity of
the tested compounds was >95% based on the HPLC analysis
(Angilent 1260 consisting of G1312C Bin Pump and 1315D DAD UV
detector and the mobile phase is water:methanl ¼ 60:40, and the
detection wavelength was set at 210 nm). Retention times (tR) are
given in minutes.
It was found that the treatment with the highest tolerated dose
(6 mmol/100 g b. wt.) of tacrine led to severe toxicity towards liver
4.1.2. Synthesis of 4-methoxy-2-(3-methylphenoxy)-1-
nitrobenzene (6)
tissue as indicated by the significantly decreased liver weight,
reduced GSH and total glutathione content, and diminished END
and EcOD activity. In contrast, no statistically significant difference
A mixture of m-cresol (1.62 g, 15 mmol) and potassium hy-
droxide (0.84 g, 15 mmol) was heated to 120 ꢀC and stirred for 0.5 h,
and then 3-bromo-4-nitrophenyl methyl ether (2.31 g, 10.0 mmol)
and copper powder (0.1 g) were added, respectively. The resulting
mixture was stirred at 170 ꢀC for 2 h. After cooling down to room
temperature, ethyl acetate (50 mL) was added to the reaction
mixture. The deposit was filtrated off, and the filtrate was dried
with anhydrous Na2SO4, and the solvent was removed under vac-
uum to give the crude product which was furtehr purified by col-
umn chromatography (petro ether/EtOAc ¼ 15:1) to give the
desired product 6 (1.86 g) in 72% yield as a yellow oil. IR (KBr,
cmꢁ1): 3033, 1580, 1513, 1442, 1342, 1290. 1H NMR (300 MHz,
Table 2
Scores of tacrine, galantamine, compound 3 and 5 groups.
Time after scop treatment (min)
Scorea
Scop
Tacrine
3
5
1
20
60
120
8.6
4.3
2.4
4.5
2.3
1.2
3.7
3.5
1.0
3.1
1.9
1.8
7.4
4.3
1.3
a
Score ¼ errors plus baits not found þ total searching time (seconds)/100.
DMSO-d6):
d
2.31 (s, 3H), 3.81 (s, 3H), 6.59 (d, J ¼ 2.5 Hz, 1H), 6.86

382
L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
(d, J ¼ 8.0 Hz, 1H), 6.89 (s, 1H), 6.96 (dd, J ¼ 9.0 Hz, 2.5 Hz, 1H), 7.02
(d, J ¼ 8.0 Hz, 1H), 7.32 (t, J ¼ 8.0 Hz, 1H), 8.15 (d, J ¼ 9.0 Hz, 1H).
MS(ESI) m/z Calcd for C14H14NO4 [M þ H]þ 260.1 found 260.0.
(d, J ¼ 8.0 Hz, 6H, CH(CH3)2), 3.02e3.04 (m, 1H, CH(CH3)2), 3.90 (s,
3H, OCH3), 4.24 (s, 2H, CH2NCH), 6.97 (dd, J ¼ 8.0 Hz, 2.5 Hz, 1H,
arom), 7.11 (d, J ¼ 2.5 Hz, 1H, arom), 7.25 (d, J ¼ 7.5 Hz, 1H, arom),
7.34 (t, J ¼ 8.0 Hz, 7.5 Hz, 1H, arom), 7.44 (d, J ¼ 8.0 Hz, 1H, arom),
7.90 (d, J ¼ 8.0 Hz, 1H, arom). HPLC purity: tR ¼ 7.56, 98.67%. HRMS
(ESI) m/z Calcd for C17H20NO2 [M þ H]þ 270.1494 found 270.1488.
Anal. Calcd. for C17H19NO2: C, 75.81; H, 7.11; N, 5.20. Found C, 75.76;
H, 7.05; N, 5.35.
4.1.3. Synthesis of 4-methoxy-2-(3-methylphenoxy)phenylamine
(7)
Compound 6 (2.6 g, 10.0 mmol) was dissolved in 500 mL of
anhydrous ethanol; 5% palladium on charcoal (0.05 g) was added to
the solution and the reduction was carried out with hydrogen at an
initial pressure of 3 Mpa. After 3 h no more hydrogen was
consumed; the catalyst was filtered off and the solvent was
removed under vacuum. The obtained crude product was purified
by column chromatography (petro ether/EtOAc ¼ 5:1) to give the
desired product 7 (2.0 g) in 87% yield as a yellow oil. IR (KBr, cmꢁ1):
3449, 3365, 1583, 1511, 1444, 1156, 1037. 1H NMR (300 MHz, DMSO-
Analytical data for N-((7-methoxydibenzo[b,d]furan-1-yl)
methyl)ethanamine (2b): yellow oil. Yield 44%. IR (KBr, cmꢁ1):
3190, 1627, 1511, 1445, 1270. 1H NMR (300 MHz, CDCl3):
d 1.07 (t,
J ¼ 7.5 Hz, 3H), 2.59 (q, J ¼ 7.5 Hz, 2H), 3.91 (s, 3H), 4.39 (s, 2H), 6.97
(dd, J ¼ 7.5 Hz, 3.0 Hz, 1H), 7.11 (d, J ¼ 3.0 Hz, 1H), 7.27e7.26 (d,
J ¼ 7.5 Hz, 1H), 7.34 (t, J ¼ 7.5 Hz, 1H), 7.45 (d, J ¼ 7.5 Hz, 1H), 7.89 (d,
J ¼ 7.5 Hz,1H). HPLC purity: tR ¼ 7.69, 99.09%. HRMS (ESI) m/z Calcd
for C16H18NO2 [M þ H]þ 256.1337 found 256.1342.
d6):
d
2.26 (s, 3H), 3.60 (s, 3H), 4.41 (s, 2H), 6.41 (d, J ¼ 2.5 Hz, 1H),
6.60 (dd, J ¼ 9.0 Hz, 2.5 Hz, 1H), 6.66e6.76 (m, 3H), 6.87 (d,
J ¼ 7.5 Hz, 1H), 7.22 (t, J ¼ 9.0 Hz, 7.5 Hz, 1H). MS(ESI) m/z Calcd for
Analytical data for N-((7-methoxydibenzo[b,d]furan-1-yl)
methyl)-N-methylethanamine (2c): yellow oil. Yield 41%. IR (KBr,
cmꢁ1): 3132, 1631, 1510, 1456, 1270. 1H NMR (500 MHz, CDCl3):
C
14H16NO2 [M þ H]þ 230.1 found 230.1.
d
1.13 (t, J ¼ 8.00 Hz, 3H), 2.25 (s, 3H), 2.56e2.58 (m, 2H), 3.89 (s,
4.1.4. Synthesis of 7-methoxy-1-methyldibenzo[b,d]furan (9a) and
3-methoxy-7-methyldibenzo [b,d]furan (9b)
3H), 3.99 (s, 2H), 6.94 (dd, J ¼ 7.5 Hz, 1.5 Hz, 1H), 7.07 (d, J ¼ 1.5 Hz,
1H), 7.23 (d, J ¼ 7.5 Hz, 1H), 7.29 (t, J ¼ 7.5 Hz, 1H), 7.42 (d, J ¼ 7.5 Hz,
1H), 8.00 (d, J ¼ 7.5 Hz, 1H). HPLC purity: tR ¼ 8.69, 100%. HRMS
(ESI) m/z Calcd for C17H20NO2 [M þ H]þ 270.1494 found 270.1489.
Analytical data for N-ethyl-N-((7-methoxydibenzo[b,d]furan-
1-yl)methyl)ethanamine (2d): yellow oil. Yield 67%. IR (KBr,
cmꢁ1): 3089, 1632, 1583, 1437, 1280. 1H NMR (300 MHz, CDCl3):
To a solution of compound 7 (15.7 g, 68.5 mmol) in 50 mL of
6 mol/L HCl aqueous solution which was cooled to 0 ꢀC, a solution of
NaNO2 (5.7 g, 82.3 mmol) in 10 mL of water was added dropwise,
keeping the temperature of the reaction mixture not beyond 5 ꢀC.
Then the reaction mixture was stirred at 0 ꢀC for another 30 min.
The deposit was filtrated off, and the filtrate was treated with so-
dium tetrafluoroborate (9.0 g, 82.2 mmol) to form white deposit
which was collected and added to a mixture of cuprous oxide
(11.8 g, 82.2 mmol) in 200 mL of 1 mol/L H2SO4 aqueous solution.
The resulting mixture was stirred at 60 ꢀC for 2 h. After cooling the
reaction mixture to room temperature, the deposit was filtrated off
and the filtrate was extracted with AcOEt (100 mL ꢄ 3). The organic
phase was dried with anhydrous Na2SO4, and the solvent was
removed under vacuum to give the crude product which was fur-
tehr purified by column chromatography (petro ether/
EtOAc ¼ 30:1) to give the mixture of 9a and 9b (9a:9b ¼ 3:2). For 9a,
d
0.97 (t, J ¼ 8.00 Hz, 6H), 2.66 (q, J ¼ 7.5 Hz, 4H), 3.91 (s, 3H), 4.04 (s,
2H), 6.94 (dd, J ¼ 7.5 Hz, 3.0 Hz, 1H), 7.09 (d, J ¼ 3.0 Hz, 1H), 7.28 (d,
J ¼ 7.5 Hz, 1H), 7.34 (t, J ¼ 7.5 Hz, 1H), 7.91 (d, J ¼ 3.0 Hz, 1H), 8.04 (d,
J ¼ 7.5 Hz, 1H). HPLC purity: tR ¼ 11.60, 98.91%. HRMS (ESI) m/z
Calcd for C18H22NO2 [M þ H]þ 284.1651 found 284.1656.
Analytical data for N-benzyl-1-(7-methoxydibenzo[b,d]furan-
1-yl)methanamine (2e): yellow oil. Yield 66%. IR (KBr, cmꢁ1):
3026, 1632, 1584, 1503, 1437, 1279. 1H NMR (500 MHz, CDCl3):
d
3.89 (s, 3H), 3.93 (s, 2H), 4.23 (s, 2H), 6.88 (dd, J ¼ 7.5 Hz, 2.5 Hz,
1H), 7.08 (d, J ¼ 2.5 Hz, 1H), 7.28e7.26 (m, 2H), 7.34e7.31 (m, 3H),
7.37e7.35 (m, 2H), 7.43e7.42 (m, 1H), 7.71 (d, J ¼ 7.5 Hz, 1H). HPLC
purity: tR ¼ 9.80, 100%. HRMS (ESI) m/z Calcd for C21H20NO2
[M þ H]þ 318.1494 found 318.1498.
1H NMR (500 MHz, DMSO-d6):
d 2.72 (s, 3H), 3.87 (s, 3H), 7.03 (dd,
J ¼ 8.5 Hz, 2.5 Hz, 1H), 7.15 (d, J ¼ 7.5 Hz, 1H), 7.31 (d, J ¼ 2.5 Hz, 1H),
7.32 (t, J ¼ 7.5 Hz, 7.5 Hz, 1H), 7.46 (d, J ¼ 7.5 Hz, 1H), 7.97 (d,
J ¼ 8.5 Hz, 1H). MS(ESI) m/z Calcd for C14H13O2 [M þ H]þ 212.1
4.1.6. Synthesis of 4-methoxy-20-methyl-2-nitro-1,10-biphenyl (11)
To a solution of 1-bromo-4-methoxy-2-nitrobenzene (1.2 g,
5.0 mmol) in 15 mL of glycol dimethyl ether was added a solution of
palladium-tetrakis(triphenylphosphine) (0.3 g, 0.25 mmol) in
15 mL of glycol dimethyl ether. The resulting mixture was stirred
for 15 min at room temperature in a nitrogen atmosphere. Then 2-
methylphenylboronic acid (0.95 g, 7.0 mmol) and 20 mL of 2 N
Na2CO3 aqueous solution were added, respectively. The reaction
mixture was stirred and refluxed for 20 h. After cooling the reaction
mixture to room temperature, the organic phase was separated and
concentrated and finally purified by column chromatography
(petro ether/EtOAc ¼ 20:1) to give the desired product 11 (1.20 g) in
98% yield as a yellow solid, m.p. 56e58 ꢀC. IR (KBr, cmꢁ1): 3100,
3010, 2977, 1621, 1536, 1480, 1455, 1356, 1294, 1228, 1119, 1083, 874,
found 212.2. For 9b, 1H NMR (500 MHz, DMSO-d6):
d 2.46 (s, 3H),
3.86 (s, 3H), 6.98 (dd, J ¼ 8.5 Hz, 2.5 Hz, 1H), 7.18 (d, J ¼ 8.0 Hz, 1H),
7.27 (d, J ¼ 2.5 Hz, 1H), 7.45 (s, 1H), 7.89 (d, J ¼ 8.0 Hz, 1H), 7.94 (d,
J ¼ 8.5 Hz, 1H). MS(ESI) m/z Calcd for C14H13O2 [M þ H]þ 212.1
found 212.2.
4.1.5. General procedure for the synthesis of 2ae2e
To the mixture of 9a and 9b (0.5 g, 2.4 mmol) dissovled in 30 mL
of carbon tetrachloride were added N-bromosuccinimide (0.45 g,
2.5 mmol) and azobisisobutyronitrile (0.25 g, 1.5 mmol). The
resulting mixture was stirred and refluxed for 3 h. After cooling to
room temperature, the deposit was filtrated off and the filtrate was
concentrated to give a yellow oil which was then dissolved in 20 mL
of CH2Cl2. To the obtained solution corresponding amines
(20 mmol), potassium carbonate (0.70 g, 5 mmol) and catalytic
amount of potassium iodide were added and the reaction mixture
was stirred at room temperature for 3 h. The deposit was filtered off
and the solvent was removed under vacuum. The obtained crude
product was purified by column chromatography (petro ether/
EtOAc ¼ 5:1) to give the desired product.
812, 793, 768, 734, 568. 1H NMR (300 MHz, CDCl3):
d 2.09 (s, 3H),
3.92 (s, 3H), 7.08 (dd, J ¼ 7.5 Hz, 1.5 Hz, 1H), 7.18 (dd, J ¼ 8.5 Hz,
2.5 Hz, 1H), 7.23e7.29 (m, 2H), 7.18e7.22 (m, 2H), 7.51 (d, J ¼ 2.5 Hz,
1H). MS(FAB) m/z Calcd for C14H13NO3 [M]þ 243.1 found 243.2.
4.1.7. Synthesis of 2-methoxy-5-methyl-9H-carbazole (12)
The solution of compound 11 (2.4 g, 10.0 mmol) and triphenyl-
phosphine (6.6 g, 25.0 mmol) in 50 mL of 1,2-dichlorobenzene was
stirred at 180 ꢀC in a nitrogen atmosphere for 3 h. Then the reaction
solution was cooled to 35 ꢀC and the solvent was removed under
Analytical data for N-((7-methoxydibenzo[b,d]furan-1-yl)
methyl)propan-2-amine (2a): yellow oil. Yield 56%. IR (KBr, cmꢁ1):
2958, 1632, 1584, 1437, 1282. 1H NMR (300 MHz, DMSO-d6):
d 1.18

L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
383
reduced pressure. The obtained crude product was purified by
column chromatography (petro ether/EtOAc ¼ 3:1) to give the
desired product 12 (1.80 g) in 85.3% yield as a yellow crystal. m.p.
120e122 ꢀC. IR (KBr, cmꢁ1): 3401, 3053, 2951, 1624, 1607, 1572,
1496, 1306, 1228, 1197, 1119, 819, 754, 725. 1H NMR (300 MHz,
1H), 7.92e7.94 (m, 1H), 8.10 (d, J ¼ 8.5 Hz, 1H), 8.20e8.22 (m, 1H).
MS(ESI) m/z Calcd for C23H24N2O3SNa [M þ Na]þ 431.1 found 431.2.
Analytical
data
for
N-ethyl-N-((7-methoxy-9-(phenyl-
sulfonyl)-9H-carbazol-4- yl)methyl)ethanamine (15b): yellow
solid, m.p. 90e92 ꢀC. Yield 61%. IR (KBr, cmꢁ1): 3018, 2931, 1625,
1580, 1415, 1280, 1117, 855, 756, 679. 1H NMR (300 MHz, CDCl3):
DMSO-d6):
d
2.74 (s, 3H), 3.85 (s, 3H), 6.81 (dd, J ¼ 8.0 Hz, 2.1 Hz,
1H), 6.91 (d, J ¼ 7.2 Hz, 1H), 6.99 (d, J ¼ 2.1 Hz, 1H), 7.21 (t, J ¼ 7.2 Hz,
1H), 7.28 (d, J ¼ 7.2 Hz, 1H), 7.97 (d, J ¼ 8.0 Hz, 1H), 11.14 (s, 1H).
MS(ESI) m/z Calcd for C14H14NO [M þ H]þ 212.1 found 213.2.
d
1.05 (t, J ¼ 7.0 Hz, 6H), 2.58 (d, J ¼ 7.0 Hz, 4H), 3.97 (s, 3H), 4.21 (s,
2H), 7.10 (dd, J ¼ 8.5 Hz, 2.5 Hz, 1H), 7.31 (d, J ¼ 8.0 Hz, 2H), 7.55 (t,
J ¼ 8.0 Hz, 7.5 Hz, 2H), 7.69 (d, J ¼ 7.5 Hz, 1H), 7.81 (d, J ¼ 2.5 Hz, 1H),
7.90 (d, J ¼ 1.5 Hz, 1H), 7.92e7.94 (m, 1H), 8.04 (d, J ¼ 8.5 Hz, 1H),
8.18e8.20 (m, 1H). MS(ESI) m/z Calcd for C24H27N2O3S [M þ H]þ
423.2 found 423.2.
4.1.8. Synthesis of 2-methoxy-5-methyl-9-(phenylsulfonyl)-9H-
carbazole (13)
Analytical data for N-benzyl-1-(7-methoxy-9-(phenyl-
sulfonyl)-9H-carbazol-4-yl)methanamine (15c): yellow solid,
m.p. 121e122 ꢀC. Yield 65%. IR (KBr, cmꢁ1): 3412, 2981, 1610, 1481,
1321, 1219, 1190, 947, 775, 760, 730, 696. 1H NMR (300 MHz, CDCl3):
To a solution of sodium hydride (1.2 g, 50.0 mmol) in 50 mL of
anhydrous THF which was cooled to 5 ꢀC was added dropwise a
solution of compound 12 (2.11 g, 10.0 mmol) in 10 mL of anhydrous
THF. The resulting solution was stirred at 5 ꢀC for 1 h. Benzene
sulfochloride (2.1 g, 12.0 mmol) was then added dropwise to the
reaction solution, controlling the reaction temperature not beyond
d
3.85 (s, 3H), 3.88 (s, 2H), 4.18 (s, 2H), 6.86e6.92 (m, 4H), 7.27e7.29
(m, 3H), 7.41 (d, J ¼ 7.2 Hz, 2H), 7.49e7.55 (m, 3H), 7.75e7.77 (m,
2H), 8.25e8.30 (m, 2H). MS(ESI) m/z Calcd for C27H25N2O3S
[M þ H]þ 457.2 found 457.2.
5
ꢀC. The reaction solution was stirred over night at room tem-
perature and then poured into 300 mL of cooled water, forming
white deposit which was collected via filtration and recrystallized
from ethanol to give the desired product 13 (3.23 g) in 92% yield as a
light yellow crystal, m.p. 177e179 ꢀC. IR (KBr, cmꢁ1): 3435, 2981,
1621, 1585, 1502, 1479, 1413, 1365, 1287, 1179, 1149, 986, 849, 780,
4.1.11. General procedure for the synthesis of 4ae4c
A solution of compound 15 (2.5 mmol), 10 mL of 2 N NaOH
aqueous solution and 40 mL of ethanol was refluxed over night, and
then ethanol was removed under reduced pressure. The formed
yellow deposit was collected and recrystallized from ethanol to give
the desired product.
Analytical data for N-((7-methoxy-9H-carbazol-4-yl)methyl)
propan-2-amine (4a): yellow solid, m.p. 71e72 ꢀC. Yield 85%. IR
(KBr, cmꢁ1): 3295, 2967,1626,1610,1582,1375,1236,1172, 874, 806,
756, 599. 1H NMR (500 MHz, DMSO-d6):
d 2.67 (s, 3H), 3.92 (s, 3H),
7.09 (dd, J ¼ 8.7 Hz, 2.4 Hz, 1H), 7.20 (d, J ¼ 7.5 Hz, 1H), 7.40 (t,
J ¼ 8.1 Hz, 7.8 Hz, 1H), 7.53 (t, J ¼ 8.1 Hz, 7.5 Hz, 2H), 7.66 (t,
J ¼ 7.8 Hz, 7.2 Hz, 1H), 7.83e7.86 (m, 3H), 8.00 (d, J ¼ 8.7 Hz, 1H),
8.10 (d, J ¼ 8.7 Hz, 1H). MS(ESI) m/z Calcd for C20H18NO3S [M þ H]þ
352.1 found 352.1.
750, 726. 1H NMR (300 MHz, DMSO-d6):
d
1.10 (d, J ¼ 6.5 Hz, 6H,
CH(CH3)), 2.92e2.94 (m, 1H, CH(CH3)2), 3.84 (s, 3H, OCH3), 4.19 (s,
2H, CH2NCH), 6.80 (dd, J ¼ 8.6 Hz, 2.5 Hz, 1H, arom), 6.97 (d,
J ¼ 2.5 Hz, 1H, arom), 7.11 (d, J ¼ 7.5 Hz, 1H, arom), 7.22e7.24 (m, 1H,
arom), 7.31 (d, J ¼ 7.5 Hz, 1H, arom), 7.98 (d, J ¼ 8.6 Hz, 1H, arom),
11.11 (s, 1H, NH). HPLC purity: tR ¼ 9.34, 100%. MS(ESI) m/z Calcd for
4.1.9. Synthesis of 5-(bromomethyl)-2-methoxy-9-
(phenylsulfonyl)-9H-carbazole (14)
A solution of 9b (3.5 g, 10 mmol), N-bromosuccinimide (1.98 g,
11.0 mmol), catalystic amount of azobisisobutyronitrile in 30 mL of
carbon tetrachloride were stirred and refluxed for 2 h. After cooling
to room temperature, the deposit was filtrated off and the filtrate
was concentrated to give a yellow oil which was further purified by
column chromatography (petro ether/EtOAc ¼ 5:1) to give the
desired product 14 (4.09 g) in 95.0% yield as a yellow solid. m.p.
116e118 ꢀC. IR (KBr, cmꢁ1): 3445, 2999,1619,1498,1444,1290,1178,
C
17H21N2O [M þ H]þ 269.1 found 269.1. Anal. Calcd. for C17H20N2O:
C, 76.09; H, 7.51; N, 10.44. Found C, 76.01; H, 7.55; N, 10.35.
Analytical data for N-ethyl-N-((7-methoxy-9H-carbazol-4-yl)
methyl)ethanamine (4b): yellow solid, m.p. 97e99 ꢀC. Yield 91%.
IR (KBr, cmꢁ1): 3383, 2967, 2934, 1626, 1577, 1497, 1314, 1289, 1114,
993, 819, 808, 757, 729. 1H NMR (300 MHz, DMSO-d6):
d 1.03 (t,
1152, 856, 759, 721, 683. 1H NMR (300 MHz, DMSO-d6):
d 3.95 (s,
J ¼ 7.0 Hz, 6H), 2.60 (q, J ¼ 7.0 Hz, 4H), 3.84 (s, 3H), 4.02 (s, 2H), 6.78
(dd, J ¼ 8.0 Hz, 2.4 Hz, 1H), 6.96 (d, J ¼ 2.4 Hz, 1H), 7.12 (d, J ¼ 7.5 Hz,
1H), 7.25 (t, J ¼ 7.5 Hz, 8.0 Hz, 1H), 7.34 (d, J ¼ 8.0 Hz, 1H), 8.08 (d,
J ¼ 8.0 Hz, 1H), 11.15 (s, 1H). HPLC purity: tR ¼ 9.09, 100%. HRMS
(ESI) m/z Calcd for C18H21N2O [MꢁH]- 281.1652 found 281.1653.
Analytical data for N-benzyl-1-(7-methoxy-9H-carbazol-4-yl)
methanamine (4c): yellow solid, m.p. 111e113 ꢀC. Yield 81%. IR
(KBr, cmꢁ1): 3399, 2823,1621,1494,1309,1226,1197,1166, 940, 775,
3H), 5.14 (s, 2H), 7.16 (dd, J ¼ 8.7 Hz, 2.4 Hz, 1H), 7.47 (d, J ¼ 8.1 Hz,
2H), 7.55 (t, J ¼ 8.1 Hz, 7.5 Hz, 2H), 7.65 (d, J ¼ 7.5 Hz, 1H), 7.85 (d,
J ¼ 2.4 Hz, 1H), 7.88 (d, J ¼ 1.5 Hz, 1H), 7.92e7.90 (m, 1H), 8.11 (d,
J ¼ 8.7 Hz, 1H), 8.24e8.26 (m, 1H). MS(ESI) m/z Calcd for
C
20H17BrNO3S [M þ H]þ 430.0 found 430.0.
4.1.10. General procedure for the synthesis of 15ae15c
To a mixture of corresponding amines (25.0 mmol), potassium
carbonate (0.70 g, 5.0 mmol) and catalytic amount of potassium io-
dide in 30 mL of anhydrous acetone was added dropwise a solution of
14 (1.07 g, 2.5 mmol) in 10 mL of anhydrous acetone. The resulting
mixture was stirred over night at room temperature. The deposit was
filtered off and the filtrate was concentrated under reduced pressure.
The obtained crude product was purified by column chromatography
(petro ether/EtOAc ¼ 5:1) to give the desired product.
760, 737, 698. 1H NMR (300 MHz, DMSO-d6):
d 3.83 (s, 3H), 3.85 (s,
2H), 4.18 (s, 2H), 6.75 (dd, J ¼ 8.0 Hz, 2.4 Hz, 1H), 6.97 (d, J ¼ 2.4 Hz,
1H), 7.14 (d, J ¼ 7.2 Hz, 1H), 7.27 (t, J ¼ 7.2 Hz, 8.0 Hz, 2H), 7.39e7.31
(m, 3H), 7.41 (d, J ¼ 6.9 Hz, 2H), 7.85 (d, J ¼ 8.0 Hz, 1H), 11.16 (s, 1H).
HPLC purity: tR ¼ 8.04, 100%. HRMS (ESI) m/z Calcd for C21H19N2O
[MꢁH]- 315.1496 found 315.1495.
4.1.12. General procedure for the synthesis of 3-chloro-N-isopropyl-
N- [(7-methoxydibenzo[b,d]furan-1-yl)methyl]propanamide (16a)
and 3-chloro-N-isopropyl-N- [(7-methoxy-9H-carbazol-4-yl)
methyl]propanamide (16b)
To an ice-cooled solution of compound 2a (0.67 g, 2.5 mmol) or
4a (0.67 g, 2.5 mmol), pyridine (0.24 g, 3.0 mmol) in 30 mL of
anhydrous CH2Cl2 was added chloroacetyl chloride (0.32 g,
2.8 mmol) dropwise. The resulting solution was stirred at room
Analytical data for N-((7-methoxy-9-(phenylsulfonyl)-9H-car-
bazol-4-yl)methyl)propan-2-amine (15a): yellow solid, m.p. 111e
112 ꢀC. Yield 55%. IR (KBr, cmꢁ1): 3440, 2950, 1630, 1581, 1437, 1282,
1117, 1098, 855, 759, 688. 1H NMR (300 MHz, CDCl3):
d
1.11 (d,
J ¼ 6.3 Hz, 6H), 2.90e2.93 (m, 1H), 3.94 (s, 3H), 4.19 (s, 2H), 7.06 (dd,
J ¼ 8.5 Hz, 2.5 Hz,1H), 7.37 (d, J ¼ 8.0 Hz, 2H), 7.58 (t, J ¼ 8.0 Hz, 7.5 Hz,
2H), 7.66 (d, J ¼ 7.5 Hz, 1H), 7.88 (d, J ¼ 2.5 Hz, 1H), 7.91 (d, J ¼ 1.5 Hz,

384
L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
temperature for 1 h, and then washed with saturated Na2CO3
aqueous solution (50 mL ꢄ 3) and saturated NaCl aqueous solution
(50 mL ꢄ 3), respectively. The organic phase was dried with
anhydrous Na2SO4 and concentrated to give crude product which
was further purified by column chromatography (petro ether/
EtOAc ¼ 5:1) to give the desired product.
3 h, and then basified by 10% NaOH aqueous solution to pH ¼ 8. The
resulted solution was extracted with AcOEt (100 mL ꢄ 3). The
organic phase was dried with anhydrous Na2SO4, and the solvent
was removed under vacuum to give the crude product which was
furtehr purified by column chromatography (petro ether/
EtOAc ¼ 10:1) to give the desired product.
Analytical data for 16a: yellow oil. Yield 92%. IR (KBr, cmꢁ1): 2958,
Analytical data for 2f: yellow oil. Yield 65%. IR (KBr, cmꢁ1): 3384,
2958, 1630, 1585, 1415, 1279, 1177, 874, 750, 715. 1H NMR (300 MHz,
1632,1584,1437,1282.1H NMR(500 MHz,CDCl3):
d
1.18 (d, J¼ 7.50 Hz,
6H), 2.99e3.01 (m,1H), 3.90 (s, 3H), 4.24 (s, 2H), 4.87 (s, 2H), 6.96 (dd,
J ¼ 10.0 Hz, 2 Hz, 1H), 7.10 (d, J ¼ 2.0 Hz, 1H), 7.26 (d, J ¼ 10.0 Hz, 1H),
7.32 (t, J ¼ 10.0 Hz, 1H), 7.42 (d, J ¼ 10.0 Hz,1H), 7.89 (d, J ¼ 10.0 Hz,1H).
MS(ESI) m/z Calcd for C19H21ClNO3 [M þ H]þ 346.1 found 346.1.
Analytical data for 16b: pale yellow solid, m.p. 119e121 ꢀC. Yield
90%. IR (KBr, cmꢁ1): 3394, 3280, 2977, 1732, 1647, 1497, 1437, 1306,
1198, 1159, 1119, 1034, 824, 802, 758, 731. 1H NMR (300 MHz,
DMSO-d6):
d
1.16 (d, J ¼ 7.5 Hz, 6H), 3.02e3.04 (m, 1H), 4.24 (s, 2H),
6.89 (dd, J ¼ 7.5 Hz, 2.5 Hz, 1H), 7.08 (d, J ¼ 2.5 Hz, 1H), 7.24 (d,
J ¼ 7.5 Hz, 1H), 7.31 (t, J ¼ 8.0 Hz, 7.5 Hz, 1H), 7.45 (d, J ¼ 8.0 Hz, 1H),
7.88 (d, J ¼ 7.5 Hz, 1H). HPLC purity: tR ¼ 7.56, 98.67%. HRMS (ESI)
m/z Calcd for C16H18NO2 [M þ H]þ 256.1338 found 256.1336.
Analytical data for 4d: yellow solid. Yield 71%. m.p. 111e112 ꢀC.
IR (KBr, cmꢁ1): 3398, 3273, 2966, 1626, 1580, 1437, 1374, 1235, 1171,
DMSO-d6):
d
1.17 (d, J ¼ 6.9 Hz, 6H), 3.06e3.08 (m, 1H), 3.86 (s, 3H),
824, 803, 752, 727. 1H NMR (500 MHz, DMSO-d6):
d 1.12 (d,
4.11 (s, 2H), 4.96 (s, 2H), 6.80 (dd, J ¼ 8.65 Hz, 2.35 Hz, 1H), 7.02 (d,
J ¼ 2.25 Hz, 1H), 7.19 (d, J ¼ 7.15 Hz, 1H), 7.25 (t, J ¼ 7.35 Hz, 7.9 Hz,
1H), 7.37 (d, J ¼ 8.05 Hz, 1H), 8.03 (d, J ¼ 8.65 Hz, 1H), 11.32 (s, 1H).
MS(ESI) m/z Calcd for C19H22ClN2O2 [M þ H]þ 345.1 found 345.1.
J ¼ 6.3 Hz, 6H), 2.97e2.93 (m, 1H), 3.17 (s, 1H), 4.20 (s, 2H), 6.66 (dd,
J ¼ 8.4 Hz, 2.4 Hz,1H), 6.82 (d, J ¼ 2.4 Hz,1H), 7.09 (d, J ¼ 6.9 Hz,1H),
7.22 (t, J ¼ 6.9 Hz, 8.4 Hz, 1H), 7.28 (d, J ¼ 7.8 Hz, 1H), 7.86 (d,
J ¼ 7.8 Hz, 1H), 9.39 (s, 1H), 10.99 (s, 1H). HPLC purity: tR ¼ 7.69,
99.09%. HRMS (ESI) m/z Calcd for C16H17N2O [MꢁH]- 253.1379
found 373.1348.
4.1.13. General procedure for the synthesis of N-isopropyl-N-[(7-
methoxydibenzo[b,d]furan-1-yl)methyl]-3-nitropropanamide (3)
and N-isopropyl-N-[(7-methoxy-9H-carbazol-4-yl)methyl]- 3-
nitropropanamide (5)
4.2. Biological studies
A mixture of compound 16a (0.69 g, 2.0 mmol) or 16b (0.69 g,
2.0 mmol), silver nitrate (1.7 g, 10.0 mmol) in 50 mL of anhydrous
acetonitrile was refluxed in the dark for 24 h. After cooling to room
temperature, the deposit was filtrated off and the filtrate was
concentrated under reduced pressure to give crude product which
was further purified by column chromatography (petro ether/
EtOAc ¼ 5:1) to give the desired product.
4.2.1. Cholinesterase inhibition assay in vitro
The cholinesterase inhibitory activity of the target compounds
was measured using Ellman’s assay. AChE (E.C.3.1.1.7, Type VI-S,
from Electric Eel) and BChE (E.C.3.1.1.8, from equine serum) were
purchased from SigmaeAldrich (Steinheim, Germany). DTNB (Ell-
man’s reagent), ATC and BTC iodides were obtained from Fluka
(Buchs, Switzerland). The assay was performed as described in the
following procedure: stock solutions of the test compounds were
Analytical data for 3: yellow oil. Yield 54%. IR (KBr, cmꢁ1): 2988,
2847, 1648, 1579, 1484, 1503, 1282, 848. 1H NMR (500 MHz, DMSO-
prepared in ethanol, 100 mL of which gave a final concentration of
d6):
d
1.20 (d, J ¼ 8.0 Hz, 6H, CH(CH3)2), 2.82 (t, J ¼ 8 Hz, 2H,
10ꢁ3 M when diluted to the final volume of 3.32 mL. The highest
concentration of the test compounds applied in the assay was
10ꢁ4 M (10% EtOH in the stock solution did not influence enzyme
activity). In order to obtain an inhibition curve, at least five different
concentrations (normally 10ꢁ4e10ꢁ9 M) of the test compound were
measured at 25 ꢀC at 412 nm, each concentration in triplicate. For
buffer preparation, 1.36 g of potassium dihydrogen phosphate
(10 mmol) were dissolved in 100 mL of water and adjusted with
KOH to pH ¼ 8.0 ꢂ 0.1. Enzyme solutions were prepared to give 2.5
units mLꢁ1 in 1.4 mL aliquots. Furthermore, 0.01 M DTNB solution,
0.075 M ATC and BTC solutions, respectively, were used. A cuvette
COCH2CH2), 3.02e3.05 (m, 1H, CH(CH3)2), 3.74 (s, 3H, OCH3), 4.43
(s, 2H, CH2NCH), 4.98 (t, J ¼ 8 Hz, 2H, CH2ONO2), 6.55 (dd, J ¼ 8.0 Hz,
2.5 Hz, 1H, arom), 6.67 (d, J ¼ 2.5 Hz, 1H), 6.88 (d, J ¼ 8.0 Hz, 1H,
arom), 6.97 (d, J ¼ 2.5 Hz, 1H, arom), 7.22e7.24 (m, 1H, arom), 7.32
(d, J ¼ 8.0 Hz,1H, arom). 13C NMR (500 MHz, DMSO-d6):
d 21.5, 33.2,
44.9, 47.0, 53.0, 62.0, 100.1, 115.5, 120.0, 122.8, 123.0, 123.8, 124.2,
125.1, 130.4, 157.9, 159.6, 164.2, 186.4. HPLC purity: tR ¼ 3.69,
99.87%. HRMS (ESI) m/z Calcd for C20H23N2O6 [M þ H]þ 387.1556
found 387.1556. Anal. Calcd. for C20H22N2O6: C, 62.17; H, 5.74; N,
7.25. Found C, 62.01; H, 5.65; N, 7.35.
Analytical data for 5: yellow oil. Yield 43%. IR (KBr, cmꢁ1): 3317,
2921, 2850, 1641, 1610, 1498, 1454, 1307, 1282, 1198, 851, 805, 753,
containing 3.0 mL of phosphate buffer, 100
enzyme, and 100 L of the test compound solution was allowed to
stand for 5 min, then 100 L of DTNB were added, and the reaction
was started by addition of 20 L of the substrate solution (ATC/BTC).
mL of the respective
m
726. 1H NMR (500 MHz, DMSO-d6):
d
1.13 (d, J ¼ 6.5 Hz, 6H,
m
CH(CH3)2), 2.80 (t, J ¼ 8 Hz, 2H, COCH2CH2), 3.05e3.07 (m, 1H,
CH(CH3)2), 3.86 (s, 3H, OCH3), 4.22 (s, 2H, CH2NCH), 5.08 (t, J ¼ 8 Hz,
2H, CH2ONO2), 6.86 (dd, J ¼ 8.0 Hz, 2.5 Hz, 1H, arom), 6.99 (d,
J ¼ 2.5 Hz, 1H, arom), 7.15 (d, J ¼ 7.2 Hz,1H, arom), 7.22 (t, J ¼ 8.0 Hz,
7.2 Hz, 1H, arom), 7.33 (d, J ¼ 8.0 Hz, 1H, arom), 7.99 (d, J ¼ 8.0 Hz,
m
The solution was mixed immediately, and exactly 2 min after
substrate addition the absorption was measured. For the reference
value, 100
mL of water replaced the test compound solution. For
determining the blank value, additionally 100
mL of water replaced
1H, arom), 11.28 (s, 1H, NH). 13C NMR (500 MHz, DMSO-d6):
d
20.3,
the enzyme solution. The inhibition curve was obtained by plotting
the percentage enzyme activity (100% for the reference) vs. loga-
rithm of test compound concentration.
35.8, 43.5, 44.8, 55.1, 62.0, 98.7,108.0,111.4,119.0,120.5,121.5,123.9,
125.0, 125.1, 137.8, 150.5, 155.8, 178.9. HPLC purity: tR ¼ 11.93,
98.62%. HRMS (ESI) m/z Calcd for C20H24N3O5 [M þ H]þ 386.1716
found 386.1713. Anal. Calcd. for C20H23N3O5: C, 62.33; H, 6.01; N,
10.90. Found C, 62.31; H, 6.95; N, 10.77.
4.2.2. In vitro nitric oxide release assay
In vitro nitric oxide release, upon incubation of the test com-
pound (2.4 mL of 100
(PBS) at pH 7.4 and 37 ꢀC for 24 h, ii) PBS at pH 7.4 and 37 ꢀC to
which 90 L rat serum had been added, was determined by quan-
tification of nitrite produced by the reaction of nitric oxide with
oxygen and water using the Griess reaction. Nitric oxide release
data were acquired for test compounds using the following
mM) with either i) phosphate buffer solution
4.1.14. General procedure for the synthesis of 9-[(isopropylamino)
methyl]dibenzo[b,d]furan-3-ol (2f) and 5-[(isopropylamino)
methyl]-9H-carbazol-2-ol (4d)
A solution of 2a (0.27 g, 1 mmol) or 4a (0.27 g, 1 mmol) disolved
in 30 mL of 48% HBr aqueous solution was stirred and refluxed for
m

L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
385
procedure. Phosphate buffer solution (PBS) was prepared by dis-
4.2.5. Cell viability analysis
solving KH2PO4 (50 mM) and NaH2PO4 (50 mM) in distilled water.
This solution was titrated to physiological pH (7.4) using 0.01 N
sodium hydroxide. Griess reagent was prepared by dissolving sul-
fanilamide (4.0 g) and N-naphtylenediamine dihydrochloride
(0.2 g) in a mixture of 85% H3PO4 (10 mL) and distilled water
(approximately 90 mL). An aliquot of 2.4 mL of a 5% dimethyl
sulfoxide (DMSO) solution in PBS (10 mL of dimethyl sulfoxide and
190 mL of PBS) was used as blank control solution, which was
maintained at 37 ꢀC for 1 h with gentle shaking. Griess reagent
(0.8 mL) was then added, and the mixture was maintained at 37 ꢀC
for 30 min with gentle shaking. A standard nitrite concentration-
absorbance plot was prepared by dissolving NaNO2 (0.1 mM) in
blank solution, and an aliquot of this solution (2.4 mL) was main-
tained at 37 ꢀC for 1 h with gentle shaking. Griess reagent (0.8 mL)
was then added and the mixture was maintained for 30 min at
37 ꢀC with gentle shaking. After incubation, this solution was
further diluted by mixing different aliquots with variable volumes
of dilution solvent (32 mL of Griess reagent and 96 mL of blank
solution). These concentrations of NaNO2 were used to plot a cali-
bration curve from which the nitrite concentration for each test
compound was calculated (ꢂSEM, n ¼ 3). The percentage nitric
oxide released from the test compounds was determined by pre-
paring 0.2 mM solutions in DMSO. An aliquot (0.12 mL) was diluted
with PBS solution, and then rat serum (90 mL) was added. This
solution was maintained at 37 ꢀC for 1 h with gentle shaking, Griess
reagent (0.8 mL) was added, and the mixture was maintained for
30 min at 37 ꢀC with gentle shaking. The ultraviolet absorbance
values for the control blank solution, calibration curve, and the test
compounds were measured at 540 nm. The absorbance value for
each test compound was corrected by subtracting the averaged
blank control absorbance. The nitrite concentration was deter-
mined from the standard nitrite concentration-absorbance curve,
which in turn was used to calculate the percentage of nitric oxide
released from each test compound.
PC12 cells, obtained from the Cell Bank of the Chinese Academy
of Sciences (Shanghai, China), were cultured in a humidified, 5%
CO2 atmosphere at 37 ꢀC, and maintained in monolayer culture in F-
12 medium supplemented with 10% fetal bovine serum (FBS),
100 mg/mL of streptomycin and 100 mg/mL of penicillin. The cell
viability was determined by MTT assay (MTT: 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). For the
preparation of Ab42 solution, Ab42 (Chinapeptides Co., Ltd) was
initially dissolved to 1 mM in hexafluoroisopropanol (Sigma) and
separated into aliquots in sterile microcentrifuge tubes. Hexa-
fluoroisopropanol was removed under vacuum in a Speed Vac, and
the peptide film was stored dessicated at ꢁ20 ꢀC. F-12 culture
medium was added to bring the peptide to a final concentration of
100 m
M and incubated at 4 ꢀC for 24 h. The tested compounds were
dissolved in DMSO and diluted to the required concentration with
culture medium (DMSO final concentration < 0.5%). The suspen-
sion of 2000 cells/well was plated in 96-well culture plates with
culture medium and was incubated for 24 h at 37 ꢀC, in a 5% CO2
incubator. Then 24
trations ranging between 0.008
compound solution (final concentrations ranging between
0.008 M and 10 M) containing A 42 (final concentration 5 M)
mL of tested compound solution (final concen-
m
M and 10 M), or 24 L of tested
m
m
m
m
b
m
was added. Cells were incubated at 37 ꢀC for 24 h. respectively.
After that, the cells were treated with 10 mL MTT dye solution
(5 mg/mL) for 4 h cultivation. The media with MTT solution were
removed with 100 mL of DMSO solution. The absorbance of for-
mazane solution was measured at 540 nm with an automatic
microplate ELISA reader. Wells without cells were used as blanks
and were subtracted as background from each sample. Results were
expressed as a percentage of control. Three independent trials were
analyzed and the results were expressed as mean ꢂ SEM.
4.2.6. Behavioral studies
Investigations were performed on 60-day-old female Wistar
rats (Han:Wist) of the out-bred stock of the Institute of Pharma-
cology and Toxicology, Jena University Hospital, Germany. Rats
were housed in a room that was automatically maintained on a 12-
h light/dark cycle at 25 ꢀC and proper humidity. Rats were given
food (Altromin 1326Ò, Altromin, Lage, Germany) and tap water ad
libitum. The permission of the animal protection commission of the
State of Thuringia, Germany, was given. Scopolamine hydro-
bromide trihydrate was obtained from Sigma Chemicals, Munich,
Germany. 0.05 mg/100 g b. wt. were dissolved in saline and
administered intraperitoneally (i. p.) 5 min after tacrine or the test
compounds, respectively. Tacrine hydrochloride (BioTrend Wan-
gen/Zürich, Switzerland) was dissolved in phosphate buffer (pH
4.2.3. ThT fluorescence assay
A
200
b
m
42 (Chinapeptides Co., Ltd) was dissolved in DMSO to make a
M stock solution. The stock solution was centrifuged at the
speed of 12,000 rpm for10 min. The above supernatant was used for
experiments. The tested compounds were dissolved in DMSO at
concentrations of 0.2, 2.0, 4.0 mM. A screening assay for tested
compounds to inhibit A
ThT fluorescence emission. Each compound (in concentrations of
0.2e4.0 mM) (2 L) and 2 L of 200 M A 42 were added into 76
of phosphate-buffered saline (PBS at pH 7.4) in a 96-well plate.
After incubation for 1 h at room temperature, 80 L of 5 M ThT
b aggregation was performed by measuring
m
m
m
b
mL
m
m
7.4) and 0.24 mg/100 g b. wt., corresponding to 1.0 mmol/100 g b.
solution (in 50 mM glycine-NaOH at pH 8.5) was added to the re-
action solution. Fluorescence emission was measured at 488 nm
with an excitation wavelength of 430 nm on a multilevel HTS
counter.
wt., were administered i. p. The new substances were dissolved in
saline.
We examined whether the new compounds could improve
spatial memory impairment induced by scopolamine using RAM
performance. Each arm (44 cm L, 30 cm H, 14 cm W) radiated from
an octagonal platform that served as a starting point. A food cup
(3 cm diameter) was located at the end of each arm. The entire arms
and food cups were painted gray and placed in a dark and calm
room. Animal behavior was monitored by a video camera. Image
analysis and pattern recognition from the monitor were performed
by a VideoMot 2 program (video tracking, motion analysis, and
behavior recognition system) provided by TSE Systems (Bad
Homburg, Germany). The computerized recording systems were
located in the same room.
4.2.4. Atomic force microscopy
The tested compound (2.5
42 were added to 45 L of PBS buffer (20% TFE at pH 7.4). As a
control, 2.5 L of 200 M A 42 was added to 47.5 L of PBS buffer
(20% TFE at pH 7.4). Samples were incubated at 37 ꢀC for 6 days, and
a 3 L sample solution was deposited on freshly cleaved mica and
mL) (1.0 mM) and 2.5 mL of 200 mM
A
b
m
m
m
b
m
m
incubated for 5 min, then blown dry. Amyloid formation was
visualized with Molecular Imaging Picoplus II workstation (Mo-
lecular Imaging Corp.). AFM probe was NSC35-type from Molecular
Imaging Corp. with resonant frequency of 170 kHz. The spring
constant of the cantilever having the AFM tip was 45 N/m.
Two phases of pre-training were performed: After a short
handling period (phase 1) in which the rats were in close contact
with the laboratory staff, three rats experienced free movement

386
L. Fang et al. / European Journal of Medicinal Chemistry 76 (2014) 376e386
and feeding in the RAM once a day for 5 days to adapt to the maze
and bait (phase 2 ¼ adaptation). The baits were scattered in all
arms. After handling and adaptation of the rats to the maze, food
was restricted to reduce the rat’s body weight by 10%. Then the
training trial was started. Each rat was placed once daily in the
centre of the RAM to visit all eight arms and eat all reward food
baits (Dustless precision Rodent Pellets, Bilaney Consultants Ltd.,
Sevenoaks, Kent, U.K.) in each food cup. Each trial was performed
until the rat entered and ate all the pellets in the eight arms. Rats
were trained to a criterion of trials without error in less than 60 s at
three consecutive days. Then the experiments started. The rats that
did not reach this standard were eliminated from the study.
One day after the training trials the experiments were carried
out. Before the administration of a substance a control run was
performed. Thus, each animal was its own control. Thereafter
cognition impairment was induced by administration of scopol-
amine. Tacrine or the new compounds were given i. p. 5 min before
scopolamine. The following parameters were registered at different
times after scopolamine:
Acknowledgments
This work is supported by National Natural Science Foundation
of China (No. 81001361), the New Drug Creation Project of the
National Science and Technology Major Foundation of China
(Project 2013ZX09402102-001-006), Ph.D. Programs Foundation of
Ministry of Education of China (No. 20100092120046) and the
Open Project Program of State Key Laboratory of Natural Medicines,
China Pharmaceutical University. Dr. Fang is grateful to the support
of Alxander von Humboldt Foundation.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.035.
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