Design, synthesis and biological evaluation of novel 6H-benzo[c]chromen-6-one, and 7,8,9,10-tetrahydro-benzo[c]chromen-6-one derivatives as potential cholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.08.016

Hydroxylated 6H-benzo[c]chromen-6-one derivatives (i.e., urolithins) are the main bioavailable metabolites, and biomarkers of ellagitannins present in various nutrition. Although these dietaries, the sources of urolithins, are employed in folk medicine as cognitive enhancer in the treatment of Alzheimer's Disease, urolithins have negligible potential to inhibit acetylcholinesterase and butyrylcholinesterase enzymes, the validated targets of Alzheimer's Disease. Therefore, within this research, a series of 6H-benzo[c]chromen-6-one, and 7,8,9,10-tetrahydro-benzo[c]chromen-6-one derivatives has been designed, synthesized, and their biological activities were evaluated as potential acetylcholinesterase and butyrylcholinesterase inhibitors. The compounds synthesized exerted comparable activity in comparison to rivastigmine, galantamine, and donepezil both in in vitro and in vivo studies.

Full text of DOI:10.1016/j.bmc.2014.08.016

Bioorganic & Medicinal Chemistry 22 (2014) 5141–5154
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel 6H-benzo[c]
chromen-6-one, and 7,8,9,10-tetrahydro-benzo[c]
chromen-6-one derivatives as potential cholinesterase inhibitors
a
a
a
a
a
Hayrettin Ozan Gulcan a,b, , Serdar Unlu , Ilker Esiringu , Tugba Ercetin , Yasemin Sahin , Demet Oz ,
⇑
_
Mustafa Fethi Sahin a,b
a Nobel Drug Pharmaceutical R&D Center (Nobel-Ilac Ar-Ge Merkezi), Sancaklar Koyu Mevkii, 81100 Duzce, Turkey
b Eastern Mediterranean University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Famagusta via Mersin 10, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydroxylated 6H-benzo[c]chromen-6-one derivatives (i.e., urolithins) are the main bioavailable metabo-
lites, and biomarkers of ellagitannins present in various nutrition. Although these dietaries, the sources of
urolithins, are employed in folk medicine as cognitive enhancer in the treatment of Alzheimer’s Disease,
urolithins have negligible potential to inhibit acetylcholinesterase and butyrylcholinesterase enzymes,
the validated targets of Alzheimer’s Disease. Therefore, within this research, a series of 6H-benzo[c]chro-
men-6-one, and 7,8,9,10-tetrahydro-benzo[c]chromen-6-one derivatives has been designed, synthesized,
and their biological activities were evaluated as potential acetylcholinesterase and butyrylcholinesterase
inhibitors. The compounds synthesized exerted comparable activity in comparison to rivastigmine, gal-
antamine, and donepezil both in in vitro and in vivo studies.
Received 16 June 2014
Revised 5 August 2014
Accepted 13 August 2014
Available online 22 August 2014
Keywords:
6H-Benzo[c]chromen-6-ones
Acetylcholinesterase
Butyrylcholinesterase
Alzheimer’s Disease
Passive avoidance test
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
in clinical trials. The agents targeting the cholinergic neurotrans-
mission (i.e., namely, acetylcholinesterase (AChE) and butyrylcho-
Alzheimer’s Disease, characterized by progressive deterioration
of memory and higher cortical functions that ultimately result in
total degradation of intellectual and mental activities, is a com-
plex neurodegenerative disease.1–3 The symptomatic develop-
ment of the disease starts with mild cognitive disorders, and
they are followed by difficulties in the interaction with the envi-
ronment, personal disorders, and finally end-stage character loss.4
Therefore, it also has devastating effects socioeconomically to
patient care-givers, and totally national health economics regard-
ing that the incidence of the disease reaches up to 50% at ages
above 85.5
Unfortunately, the complete etiology of the disease still remains
unknown. Various physiological targets, and pathways, including
but not limited to, beta-amyloid biosynthesis and degradation
pathways (i.e., beta-, and gamma-secretase inhibitors, antibeta-
amyloid immunization),6–8 tau-protein hyperphosphorylating
kinases (i.e., kinase inhibitors),9 oxidative stress (i.e., antioxi-
dants),10 brain iron homeostasis (e.g., chelating agents)11 have all
been tried, however, none of them so far found validated targets
linesterase (BuChE) inhibitors) are the only clinically approved
drugs, besides the NMDA receptor antagonist memantine, since
acetylcholine deficiency is characteristics in the development of
dementia and cognitive disorders in Alzheimer’s Disease.12 How-
ever, there are only several clinically approved drugs currently on
the market as cholinesterase inhibitors (i.e., rivastigmine, galanta-
mine, donepezil, and tacrine) and their clinical effectiveness is only
available for mild to moderate level cognitive disorders, generated
in the first couple years of the progressive development of Alzhei-
mer’s Disease.12,13 Furthermore, tacrine is currently not used due
to its serious hepatotoxic side effects.14 Although the employment
of cholinergic hypothesis in Alzheimer’s Disease has been known
for more than three decades, the number of AChE and BuChE inhib-
itor drugs on the market is questionable regarding the number of
drugs in other pharmacological groups. Therefore, more drug candi-
dates are expected not only to reach to market to be used, at least, in
the symptomatic treatment of the disease but also to provide alter-
native treatment, in particular, expected to act in a longer period,
and high levels of cognitive disability symptoms. That is the basic
reason of observing most developed drug companies still designing
AChE and BuChE inhibitor drugs and trying their effect in clinical
trials.15–17
⇑
Corresponding author. Tel.: +90 392 6302401; fax: +90 392 6302819.
E-mail address: ozan.gulcan@emu.edu.tr (H.O. Gulcan).
http://dx.doi.org/10.1016/j.bmc.2014.08.016
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

5142
H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
Based on these, our interest on this research was to design novel
successively performed in the presence of polyphosphoric acid,
since other traditional methods yielded too low yields for the syn-
thesis of the intermediates 3a–b, and 8. The intramolecular aryl-
coupling was optimized employing PdCl2(PPh3)2, NaOAc, and
DMA. Other palladium sources, bases, and organic solvents utilized
conditions, yielded no product formation or too poor yields. The
synthesis of the title compounds 9 and 10 were achieved in the
same reaction, although the reaction dominantly yielded the com-
pound 9 as the major product. The reasonable Rf difference in the
TLC monitoring of the reaction wherein acetone was used as the
mobile phase led us to quantitatively separate these compounds
(i.e., 9 and 10) employing column chromatography. HBr catalyzed
O-demethylation of 9, and 10 yielded the title compounds 11, and
12, respectively. Finally, O-carbamylations of 11 and 12 were per-
formed to obtain the title compounds 13, and 14.
AChE and BuChE inhibitor molecules. Our starting point was on the
common compounds present in some dietaries (i.e., pomegranate,
nuts, and some berries) since there is scientific data available on
the positive effects of these nutrients in the treatment of cognitive
disorders in Alzheimer’s Disease.18–20 Our findings have directed
us that ellagitannins are the common, and major compounds that
human are exposed to following the digestion of these dietar-
ies.18–20 However, ellagitannins are macromolecules and they have
no bioavailability.21 In fact, they are almost fully converted to uro-
lithins (i.e., hydroxylated 6H-benzo[c]chromen-6-one derivatives)
in the human gastrointestinal flora and these compounds are even
accepted as the biomarkers of ellagitannin metabolism.21,22 Our
initial trials with one of these metabolites of ellagitannin metabo-
lism (i.e., 3-hydroxy-6H-benzo[c]chromen-6-one, also referred to
as Urolithin B) have not provided potential AChE and BuChE inhib-
The second group compounds, 3-aminoalkyloxy-substituted-
6H-benzo[c]chromen-6-one (i.e., 19 series) and 3-aminoalkyloxy-
substituted-7,8,9,10-tetrahydro-benzo[c]chromen-6-one (i.e., 20
series) derivatives, were synthesized following the strategy
wherein, first, the 3-hydroxy-7,8,9,10-tetrahydro-benzo[c]chro-
men-6-one (i.e., 15) and 3-hydroxy-6H-benzo[c]chromen-6-one
(i.e., 16) were synthesized and the consequent alkylation and ami-
nation reactions were conducted (Scheme 4). Accordingly, the pro-
cedures described in the literature for the synthesis of 15 and 16
were modified to increase the yields. Literature suggests the reac-
tion of resorcinol and 2-oxo-cyclohexancarboxylate ethyl ester to
obtain 15 in the presence of a Lewis acid.25 Our trails gave the best
results in the presence of ZrCl4 without an organic solvent and
heating up the reaction to 80 °C. The synthesis of 16 from resor-
cinol and 2-bromobenzoic acid under CuSO4 catalyzed aqueous
base conditions is also described well in the literature.26 However,
employing 2-iodobenzoic acid in our studies resulted in very good
yields to synthesize 16. The following reactions on the compounds
15 and 16 were all in parallel. First, the corresponding alkyl halides
were prepared (i.e., 17a–c, and 18a–c). The synthesis of 17a and
18a was absolutely achieved with concomitant elimination reac-
tions, therefore, the procedure utilizing 1,2-dichloroethane was
improved to obtain around 50% yield, ideal for these synthesis
intermediates. The conventional methods for the amination of
17a–c, and 18a–c derivatives (e.g., the refluxing of corresponding
amine with an alkyl halide in the presence of base and NaI in an
appropriate organic solvent) under various temperatures yielded
out too low yields (e.g., mostly less than 50% conversion after
1 day reflux).27 This was overcome employing microwave assisted
amination reactions simply using the same reaction conditions of
conventional methods described above. Therefore, synthesis of
the title compounds 19a1–19c6 and 20a1–20c6 (i.e., totally 36
compounds) were efficiently conducted employing a single mode
microwave instrument and the average yields were around 70–
80% in a 32 min run time. All the title compounds were then con-
verted to their corresponding HCl salts.
itor molecule (i.e., IC50s were found higher than 50 lM for both
cholinesterase enzymes). Another disadvantage that literature sug-
gests on urolithins is that these compounds do not have the poten-
tial to penetrate to the central nervous system.23 Therefore, we
have designated mainly two group of compounds (i.e., rivastig-
mine-like and donepezil-like analogues) employing 6H-
benzo[c]chromen-6-one moiety, present in urolithins. Further-
more, since the central effect is required in the treatment of the
disease, the 7,8,9,10-tetrahydro-benzo[c]chromen-6-one deriva-
tive compounds (i.e., relatively more saturated urolithin deriva-
tives) were also designated regarding the potential of
tetrahydrocannabinol (i.e., a well-known cannabinoid receptor
partial agonist that also possesses tetrahydro-6H-benzo[c]chro-
men moiety) to penetrate the central nervous system.24 Therefore,
this study, for the first time, shows the generations of original 6H-
benzo[c]chromen-6-one, and 7,8,9,10-tetrahydro-benzo[c]chro-
men6-one moiety bearing compounds that possess promising
AChE and BuChE inhibitory potential. The title compounds and
their generation are illustrated in Scheme 1. As seen, the rivastig-
mine-like title compounds possess a meta-disubstitution (i.e., a
1-(dimethylamino)ethyl moiety and a –OR substituent) on a rigid
planar system as it appears on rivastigmine. On the other hand,
the structural organization in donepezil (i.e., a rigid planar system
connected to a tertiary amine with an alkylene spacer) is utilized in
donepezil like title compounds employing 6H-benzo[c]chromen-6-
one, 7,8,9,10-tetrahydro-benzo[c]chromen6-one moieties.
2. Results and discussion
2.1. Synthetic pathways
The synthetic strategy for the synthesis of the title compounds
within the first group (i.e., 4a, and 9–14) was designated to obtain
their racemic mixture except the compound 4b (i.e., the S enantio-
mer of the racemate 4a), which was obtained through directly
employing the commercially available 2b as the starting material
(i.e., Scheme 2 for the title compounds 4a–b and Scheme 3 for
the title compounds from 9 to 14). The synthetic strategy follows
the reductive amination of acetophenone derivatives, the esterifi-
cation, and finally the aryl-coupling reaction steps. Following the
reactions all the title compounds were converted to their corre-
sponding HCl salts.
The mono-O-methylation of 5 was quite critical not only to
make another substitution on the 1-(dimethylamino)ethyl substi-
tuted ring but also to protect one of the phenolic hydroxyls for
the esterification reaction in the total synthesis of the compounds
9–14. The reductive amination reactions (i.e., also a step pursued in
the commercial synthesis of rivastigmine) employing NaCNBH3
2.2. In vitro analysis of compounds: Cholinesterase assays
All the original compounds synthesized were tested for their
potential to inhibit AChE and BuChE enzymes. The IC50s obtained
for each test compound including the reference compounds riv-
astigmine, donepezil, and galantamine as well as their selectivity
are displayed in Table 1.
The first critical observation within the first group of com-
pounds (i.e., 4a–b, and 9–14) was on the effect of stereochemistry
regarding the comparison of the activities of 4a and 4b. The
employment of the S-enantiomer (4b) did not result in significant
difference for the inhibition of AChE and BuChE, since both the
racemate (4a) and the S enantiomer (4b) appeared to possess
similar activity. The compounds 9, 11, and 13 are meta-substituted
yielded the intermediates
2 and 7 in moderate yields. The
esterification of 2, and 7 employing 2-bromobenzoic acid was only

H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
5143
Ellagitannins
Human microbial flora
Urolithins (Polyhydroxylated 3H-benzo(c)chromen-6-one derivatives)
HO
O
O
HO
O
O
7,8,9,10-Tetrahydrourolithin B
(IC50s 35 µM, and > 50 µM for AChE and BuChE, respectively)
Urolithin B, an Ellagitannin metabolite
(IC50s >50 µM for AChE and BuChE)
Title Compounds
Donepezil-like analogues
Rivastigmine-like analogues
R1
O
O
N
O
O
O
n
R2
R1 and R2: -H, OH, OCH3, -OCON(CH3)2, -CH(N(CH3)2)CH3
N
O
O
O
n
N
O
N
O
n: 2,3, and 4
(R,S)-Rivastigmine
N
R
N
N
N
R
R
R: -H, -OCH3
O
N
O
O
Donepezil
Scheme 1. The origin of development of title compounds.
O
N
N
OH
OH
a
b
O
O
H3C
H3C
Br
2a: Racemic mixture
1
3a: Racemic mixture
2b*: The S configuration
*: Commercially obtained
3b: The S configuration
c
N
O
O
H3C
. HCl
4a: Racemic mixture
4b: The S configuration
Scheme 2. Synthesis of the title compounds 4a–b. Reagents and conditions: (a) NaCNBH3, dimethylamine HCl, methanol, reflux, 17 h; (b) 2-bromobenzoic acid, PPA, 80 °C,
15 min; (c) PdCl2(PPh3)2, NaOAc, DMA, 130 °C, 16 h, HCl, acetone.
analogues of compound 4a. Very interestingly, among those meta-
substitutions, only the methoxy substituent present in 9 appeared
to increase the potential to inhibit AChE, since hydroxyl (i.e., in 11),
and O-carbamyl (i.e., in 13) substitutions resulted in lower poten-
tial to inhibit AChE. However, the compounds 11 and 13 displayed
lower IC50 values for the inhibition of BuChE. These results suggest
that particularly the compounds 11 and 13 display similarity in
activity in comparison to rivastigmine. Therefore, meta-substitu-
tion was found the key position to obtain either AChE selective
or BuChE selective analogues regarding the AChE selectivity of
compounds 4a and 9 and BuChE selectivity of the compounds 11
and 13. The comparison of the activities of the compounds 10,

5144
H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
O
O
N
O
OH
d
O
e
H3C
OH
OH
OH
6
7
5
f
. HCl
N
O
O
O
N
O
O
. HCl
H3C
O
g
H3C
+
O
Br
N
CH3
10
O
O
9
8
h
h
N
HO
O
O
O
O
H3C
. HCl
. HCl
N
CH3
OH
12
11
i
i
N
N
O
O
N
O
O
O
H3C
O
. HCl
. HCl
O
N
CH3
14
O
13
Scheme 3. Synthesis of the title compounds 9–14. Reagents and conditions: (d) NaOH, Dimethylsulfate, 50 °C, 2 h; (e) NaCNBH3, dimethylamine HCl, methanol, reflux, 17 h;
(f) 2-bromobenzoic acid, PPA, 80 °C, 15 min; (g) PdCl2(PPh3)2, NaOAc, DMA, 130 °C, 16 h; (h) 48% aqueous HBr, 130 °C, 24 h; (i) N-Ethyl-N-methylcarbamyl chloride, pyridine,
rt, 20 h.
12, and 14 with their corresponding regioisomers (i.e., 9, 11, and
13, respectively) has revealed out that the position of aminoalkyl
moiety in the compounds 9, 11 and 13 is better to obtain lower
IC50s for the inhibition of AChE. This observation could not be
extrapolated to BuChE inhibition, since compound 14 exhibited a
stronger potential to inhibit BuChE. Overall, the activities observed
for the first group of compounds were found in the at least compa-
rable to better range according to the activities obtained for riv-
astigmine. In addition, our research on this first group
compounds also pointed out significant SAR to convert selectivity
towards either of the two cholinesterase enzymes.
The second group of compounds (i.e., the 19 and 20 series) have
3 basic differences; the saturation level of the benzo[c]chromen-6-
one moiety, the carbon chain bridging the benzo[c]chromen-6-one
moiety to amine group, and the various amine substitutions. The
results obtained for this group absolutely have indicated that this
type of structural organization reveals out AChE-selective mole-
cules. Indeed, all members of this category displayed selectivity
for AChE. Therefore, the activity potential of these compounds
might be resembled to the activities of donepezil and galantamine,
since both of them are significant AChE-selective agents. However,
the average selectivity ratios of the test compounds were found far
behind the ones for those two drugs. Another important SAR for
the second group compounds revealed out that the 19 series com-
pounds, in general, have better potential and selectivity to inhibit
AChE. This is, in fact, in parallel with our initial observation that
compound 15 is more potent inhibitor of AChE in comparison to
the potential of compound 16 to inhibit AChE. Therefore, the 19
series developed from 15 kept their property in terms of more
potent inhibition of AChE.
It is critical to state that the compounds 19b5 and 20b5 were
found the most potent AChE and BuChE inhibitors within this
group. Although they appear to be more active than rivastigmine
and have comparable activity in comparison to galantamine in
terms of AChE inhibition, they definitely exert a better potential
to inhibit both cholinesterase enzymes that is absent in the current
3 reference drugs (i.e., rivastigmine, galantamine, donepezil), used
in the treatment of Alzheimer’s Disease, since these drugs have
higher IC50 values for BuChE.
An important observation was on the meta-methoxy substitu-
tion of the title compounds. All unsubstituted derivatives (i.e., no
meta-methoxy substitution as seen in a1–3–5, b1–3–5, and c1–
3–5 series) were found to have better potential to inhibit AChE
in comparison to the potential of their corresponding meta-meth-
oxy substituted analogues (i.e., a2–4–6, b2–4–6, and c2–4–6
series).
Among the amine derivatives utilized N-methylbenzylamine,
and, in particular, N-benzylpiperazine substituted derivatives
appeared to be the more active compounds in comparison to
4-benzylpiperidine containing derivatives. The N-benzylalkyl

H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
HO
5145
O
O
HO
O
O
HO
OH
b
a
16
15
c
c
X
O
O
O
X
O
O
O
n
n
X: -Cl, or -Br
17a: n=2
X: -Cl, or -Br
18a: n=2
17b: n=3
18b: n=3
17c: n=4
18c: n=4
d
d
R
O
O
O
R
O
O
O
n
n
19 series
19a 1- 19a 6: n=2
20 series
19b 1- 19b 6: n=3
19c 1- 19c 6: n=4
20a 1- 20a 6: n=2
20b 1- 20b 6: n=3
20c 1- 20c 6: n=4
n: 2, 3, and 4
R groups: 1 to 6
-N
O
-N
-N
1
2
3
-N
-N
-N
N
N
O
O
6
4
5
Scheme 4. Synthesis of the title compounds 19 and 20 series. Reagents and conditions: (a) ethyl 2-oxocyclohexanecarboxylate, ZrCl4, 70 °C, 1 h; (b) 2-iodobenzoic acid,
NaOH, CuSO4, H2O, reflux, 40 min; (c) alkyl halide (i.e., 1,2-dichloroethane, 1-bromo-3-chloropropane, or 1-bromo-4-chlorobutane), base (i.e., NaOEt for n = 2, NaH, for n = 3,
or 4); (d) K2CO3, NaI, Acetonitrile, amine derivative (i.e., 1–6), 32 min microwave assisted reaction, 105 °C.
Table 1
Choliesterase activities and selectivities of compounds (i.e., test compounds 4a–b, 9–14, 19a1–19c6, and 20a1–20c6)
Compounds
IC50
(
lM)
Selectivity*
Compounds
IC50
(lM)
Selectivity
AChE
BuChE
AChE
BuChE
4a
4b
9
10
11
12
13
14
19a1
19a2
19a3
19a4
19a5
19a6
19b1
19b2
19b3
19b4
19b5
19b6
19c1
19c2
19c3
10.9 0.5
7.9 0.9
2.8 0.2
15.1 0.5
18.9 1.0
33.1 0.6
39.7 0.4
>40
2.8 0.2
6.0 0.7
8.1 0.1
37.2 0.8
1.3 0.1
7.3 0.1
2.1 0.3
2.7 0.2
4.3 0.3
4.9 0.5
0.9 0.1
6.2 0.2
1.0 0.2
1.7 0.1
9.2 0.1
17.9 1.2
16.2 2.5
21.1 1.3
24.7 0.4
4.2 0.4
>40
12.7 1.2
8.5 0.2
>40
>40
>40
>40
32.0 0.5
>40
ꢀ1.6
ꢀ2.1
ꢀ7.5
ꢀ1.6
ꢀ4.5
>1
19c4
19c5
19c6
20a1
20a2
20a3
20a4
20a5
20a6
20b1
20b2
20b3
20b4
20b5
20b6
20c1
13.7 1.0
6.4 0.2
8.7 0.3
6.7 0.5
18.4 1.0
20.0 0.2
29.3 0.2
0.9 0.1
11.4 0.2
8.5 0.5
20.9 0.8
4.1 0.2
18.5 0.2
0.8 0.5
18.0 0.5
1.1 0.1
8.3 0.3
14.2 1.0
27.4 0.5
8.2 0.8
21.3 0.2
35.2 0.1
27.8 0.4
14.6 0.6
23.4 1.5
14.1 0.1
33.2 0.3
>40
>40
12.1 0.4
>40
14.9 0.5
29.2 0.2
21.3 0.2
>40
3.7 0.4
>40
6.5 0.1
17.6 1.1
>40
ꢀ2.0
ꢀ2.3
ꢀ2.7
ꢀ2.1
ꢀ1.8
>2
ꢀ3.5
>1
>4
ꢀ13.4
>14
>6
>5
>3
ꢀ1.7
ꢀ1.4
ꢀ5.2
>2
>1
ꢀ24.6
>5
ꢀ4.6
15.9 0.6
>40
>40
ꢀ7.6
>14
>9
>2
ꢀ5.9
ꢀ2.1
>2
>1
>4
20c2
20c3
20c4
20c5
>40
>8
3.1 0.1
18.4 0.6
18.9 0.5
>40
ꢀ3.4
ꢀ2.9
ꢀ18.9
>23
ꢀ2.2
>40
>40
>40
11.0 1.0
7.1 0.1
21.9 0.2
20c6
>1
Rivastigmine
Donepezil
Galantamine
ꢀ0.3
ꢀ888
ꢀ31
20.1 0.3
0.008
0
0.7 0.1
*
Selectivity either for AchE or BuChE.

5146
H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
structure is, in fact, also present in donepezil. Therefore, the activ-
ity loss found in derivatives formed through the employment of 4-
benzylpiperidine moiety (i.e., a3–4, b3–4, and c3–4 series) was
attributed to the absence of the N-benzylalkyl moiety in the
structure.
(i.e., 4a, 4b, 9, and 13) were all found reasonable. The difference
regarding stereochemistry seen for the compounds 4a and 4b
was found, again, negligible in in vivo studies. 19b5, the most
potent inhibitor of the second group series, also exerted a good
activity in in vivo experiments. 19c1, and 20c1, relatively weaker
cholinesterase inhibitors in comparison to 19b5 also exerted
promising results in in vivo studies. The most unexpected result
in in vivo studies was obtained with the compound 20b5, since it
was found as one of the most potent inhibitors of cholinesterase
enzymes. However, the effect obtained with 20b5 is still compara-
ble with rivastigmine.
In the evaluation of the number of carbon atoms that acts as the
spacer between the benzo[c]chromen-6-one moiety and amine
group within the 2nd group of compounds, the 2 or 3 carbon atom
distance were found ideal since the 19c and 20c series were found,
in general, to possess less potential to inhibit the cholinesterase
enzymes. However, this does not totally reflect the required dis-
tance from the Oxygen atom bound to 3rd position of the
benzo[c]chromen-6-one moiety and the Nitrogen atom, since N-
methylbenzylamine moiety containing structures have a shorter
distance (i.e., in a1, b1, and c1 series) in comparison to other amine
substitutions. Therefore, it is reasonable to observe that almost all
a1, b1, and c1 series compounds displayed significant potential to
inhibit AChE. This also means that, considering the 19c5 and 20c5
as the least active N-benzylpiperazine containing structures, the 4
carbon atom space is at the board to obtain AChE inhibitory poten-
tial. In fact, this evaluation is also completely straightforward for
BuChE inhibition, since the arrangement of inhibitory potential
was found as b5 > a5 > c5 in 19 and 20 series compounds. This is
also a reflection of significance of the 3 carbon chain spacer, ideal
for cholinesterase inhibition within this group of compounds.
Based on these results, 8 compounds (i.e., 4a, 4b, 9, 11, 19c1,
19b5, 20b5, and 20c1) of totally 44 new original molecules were
selected to test them in in vivo experiments.
3. Conclusion
Benzo[c]chromen-6-one moiety is present in various plant-
derived nutrition, however, it has negligible inhibitory potential
to inhibit cholinesterase enzymes. Therefore, this research, for
the first time, displayed the generation of benzo[c]chromen-6-
one derived compounds for the inhibition of cholinesterase
enzymes. The molecules designed were subcategorized to rivastig-
mine-like and donepezil-like analogues. Indeed, the synthesized
molecules tested within the two groups exerted similarity in terms
of potential to inhibit AChE and BuChE, regarding the activity
potentials of donepezil and rivastigmine. Moreover, the activities
of the compounds were further shown in the in vivo experiments.
The results definitely have shown that most of the original com-
pounds designed and synthesized are promising molecules to be
tested for further drug development stages. The at least compara-
ble activity of test compounds in comparison to donepezil and riv-
astigmine within the in vivo experiments is another parameter to
consider the oral bioavailability and activity of the molecules.
However, more information (e.g., the correct salt selection, basic
ADME properties, and the screening of activity in other in vivo
models) is required to reach to the hit molecule for the continua-
tion of drug-development stages. The compounds particularly 4a
and 11 from the first group, and the compounds 19b5, 19c1, and
20c1 appears to be the ideal molecules that might be chosen as
lead compounds for further generation of benzo[c]chromen-6-
one derivative hit molecules, since these compounds have reflected
well the activity expected according to the SARs driven throughout
the research.
2.3. In vivo testing of compounds: scopolamine induced passive
avoidance test
Although there is no ideal test in animal models for measuring
the cognition, the scopolamine induce passive avoidance test is one
of the most abundantly employed methods to measure the effects
of drug candidates on cognition in animal models. As expressed
previously, 8 of the 44 original compounds were selected and
tested their activity in the scopolamine induce passive avoidance
test. Following the administration of scopolamine and the test sub-
stances the time for the rats visiting the dark box (latency) was
measured for 5 min. The time difference between the no scopol-
amine administered group and the only scopolamine administered
group was taken as 100% and the effect of each compound was cal-
culated accordingly in terms of the percentage antagonism. Each
test compound was assessed employing ten animals per dose.
The results are described in Table 2. Accordingly, all test com-
pounds exhibited at least comparable activity with donepezil,
and rivastigmine, since the percent antagonism was found in 50–
85% range for all of them. Very interestingly, among the first series
of the title compounds, the most active one in in vivo experiments
appeared to be the compound 11, a BuChE selective inhibitor.
However, the activities of the other first group series compounds
4. Experimental
4.1. Synthesis
All chemicals, reagents, and solvents were analytical grade, pur-
chased from commercial suppliers (i.e., Sigma–Aldrich, Acros, and
Rare Chemicals), and used without purification unless otherwise
specified. The reactions were monitored employing thin-layer
chromatography (TLC), performed on Merck 60 F254 plates and
visualized under UV light. Melting points were determined on a
Q100 Model TA InstrumentsÒ apparatus and they are uncorrected.
The purity of the test substances were analyzed through UPLC
studies using UV detection both at 220 nm and 254 nm and the
purity of all the test compounds were greater than 99%. In UPLC
studies, a Waters Acquity Ultra Performance LC System with a
PDA detector was utilized and the analytical separations were
Table 2
In vivo activity results of selected compounds
Compound
Antagonism (%)
4a
4b
9
11
70
71
71
49
65
58
62
85
58
85
achieved using an Acquity UPLC BEH C18 column (1.7 lm
2.1 cm  50 mm) and a mobile phase with a linear gradient formed
between 25% of acetonitrile to 55% Acetonitrile both in pH 7 phos-
phate buffer. The column temperature was set to 25 °C and the
flow rate was 0.3 mL/min. The exact mass analyses were conducted
employing a Waters LCT Premier XE KE309 (TOF-MS) instrument.
The analyses were performed in positive mode and the capillary,
19c1
19b5
20c1
20b5
Donepezil
Rivastigmine

H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
5147
and the sample cone voltages were set to 2500 V, and 100 V,
respectively. The desolvation temperature was at 150 °C and the
source temperature was set to 100 °C. 1H NMR spectra were
recorded on a Varian-300 NMR spectrometer using TMS as an
internal standard in either DMSO-d6 or CDCl3 and the chemical
shifts are reported in d (ppm).
ppm): d 1.67 (d, 3H, ACHACH3), 2.52 (s, 3H, CH3ANACH3), 2.72
(s, 3H, CH3ANACH3), 4.56 (q, 1H, ANACHACH3), 7.60–7.73 (m,
3H, Ar-H), 7.94 (dt, 1H, Ar-H), 8.22 (dd, 1H, Ar-H), 8.39–8.49 (m,
2H, Ar-H), 11.40 (br s, 1H, N-H).
4.1.4. (S)-3-(1-(Dimethylamino)ethyl)phenyl 2-bromobenzoate
(3b)
4.1.1. ( )-3-(1-(Dimethylamino)ethyl)phenol (2)
The desired ester derivative was synthesized employing (S)-3-
(1-(dimethylamino)ethyl)phenol (i.e., obtained through hydrolysis
of the commercially available rivastigmine) as the starting com-
pound according to procedure given for the compound 3a. Yield
obtained: 78.0%. Mass spectrometry: peak at m/z 348 (M+1)+. 1H
NMR (300 MHz, CDCl3, ppm): d 1.36 (d, 3H, ACHACH3), 2.17 (s,
6H, CH3ANACH3), 3.26 (q, 1H, ANACHACH3), 7.04–7.21 (m, 3H,
Ar-H), 7.26–7.41 (m, 3H, Ar-H), 7.63–7.70 (m, 1H, Ar-H), 7.91–
7.99 (m, 1H, Ar-H).
A mixture of 27.23 g 3-hydroxyacetophenone (0.2 mol) (1),
3.60 g dimethylamine hydrochloride (0.4 mol), 10 g activated
molecular sieve (3 Å) and 10 g dry MgSO4 was refluxed in dry
methanol for 1–2 h. Then, 18.85 g NaCNBH3 was added to reaction
mixture and the mixture was refluxed for additional 18–20 h. After
hot filtration of reaction mixture, methanol was removed under
reduced pressure and the residue thus obtained was dissolved in
water (250 mL). The content was acidified with concd HCl to pH
1 and extracted with ethyl acetate (3 Â 100 mL). The aqueous solu-
tion was basified to pH 8–9 using NaHCO3(s) (i.e., solid NaHCO3)
and extracted with ethyl acetate (5 Â 100 mL). Combined ethyl
acetate extracts were washed with water dried over MgSO4 and
concentrated under reduced pressure to give thick oil. This oil
was dissolved in isopropyl ether (120 mL) under heating and
allowed to cool to 0 °C. The crystalline solid obtained was filtered
and dried to obtain a light yellow crystalline solid. Yield obtained:
56.0%. Purity by HPLC: 99%. Mass spectrometry: peak at m/z 166
(M+1)+. 1H NMR data in CD3OD: d 1.34 (d, 3H, ACHACH3), 2.17
(s, 6H, CH3ANACH3), 3.22 (q, 1H, ANACHACH3), 6.63–6.68 (m,
1H, Ar-H), 6.69–6.79 (m, 2H, Ar-H), 7.10 (t, 1H, Ar-H).
4.1.5. (S)-3-(1-(Dimethylamino)ethyl)-6H-benzo[c]chromen-6-
one hydrochloride (4b)
The procedure for synthesis and HCl salt preparation was the
same as the procedure given for the compound 4a. (i.e., yield
obtained 66.0%, 83.0%, respectively, for synthesis and salt prepara-
tion). HPLC purity: 99.95%, HRMS (ESI) C17H18NO2 calcd 268.1338
(M+1)+, found 268.1341, mp 254.1 °C, 1H NMR (300 MHz, DMSO-
d6, ppm): d 1.66 (d, 3H, ACHACH3), 2.51 (d, 3H, CH3ANACH3),
2.72 (d, 3H, CH3ANACH3), 4.50–4.62 (m, 1H, ANACHACH3),
7.59–7.74 (m, 3H, Ar-H), 7.92 (dt, 1H, Ar-H), 8.22 (dd, 1H, Ar-H),
8.37–8.49 (m, 2H, Ar-H), 11.36 (br s, 1H, N-H).
4.1.2. ( )-3-(1-(Dimethylamino)ethyl)phenyl 2-bromobenzoate
(3a)
4.1.6. 1-(3-Hydroxy-5-methoxyphenyl)ethanone (6)
Procedure A: 6.2 g NaOH (0.150 mol) was dissolved in 125 mL
water. 25 g 3,5-dihydroxyacetophenone (0.160 mol) (5) was
weighted in 250 mL round bottom flask and previously prepared
NaOH solution was added to the flask. After complete dissolution
of acetophenone derivative in basic aqueous media, the reaction
mixture was cooled to 0–10 °C. Then, 19.5 g DMS (0.156 mol)
was added to reaction flask dropwise. The content was mixed at
ambient temperature for one hour, and then it was heated at
50 °C for two hours. To separate 3,5-dimethoxyacetophenone
impurity, pH of medium was adjusted to 14 with NaOH pellet
and this solution was extracted with EtOAc (3 Â 50 mL). The alkali
solution was acidified with conc HCl to pH 1 and extracted with
ethyl acetate (3 Â 75 mL). Combined ethyl acetate extracts were
dried over MgSO4 and concentrated under reduced pressure to give
a mixture of 3,5-dihydroxyacetophenone and 1-(3-hydroxy-5-
methoxyphenyl)ethanone. To the mixture, 50 mL acetone and
25 g NaOH(s) was added. When, 8–10 mL cold water was put into
the reaction flask slowly, sodium salt of 3,5-dihydroxyacetophe-
none is precipitated. The liquid part of the content was decanted
to another round bottom flask. The solid part was washed with
acetone (3 Â 15 mL). Combined acetone parts were concentrated
under reduced pressure to give 1-(3-hydroxy-5-methoxy-
phenyl)ethanone as off white solid. Yield obtained: 56.2%. Mass
spectrometry: peak at m/z 166 (M+1). 1H NMR (300 MHz, CDCl3,
ppm): d 2.56 (s, 3H, CH3AC@O), 3.79 (s, 3H, AOACH3), 6.62–6.67
(m, 2H, Ar-H), 7.01–7.05 (m, 1H, Ar-H), 7.07–7.11 (m, 1H, Ar-H).
Procedure B: 11.36 mL NEt3 (82.14 mmol) was added to 137 mL
water. 25 g 3,5-dihydroxyacetophenone (0.160 mol) was weighted
in 250 mL round bottom flask and previously prepared NEt3 solu-
tion was added to the flask. The reaction mixture was cooled to
0–10 °C. Then, 7.78 mL DMS (82.14 mol) was added to reaction
flask dropwise. The content was mixed at ambient temperature
for 4 h. After the reaction mixture was cooled 0–5 °C, the content
was filtrated to remove undissolved 3,5-dihydroxyacetophenone.
pH of filtrate was adjusted to 1 with concd HCl and extracted with
EtOAc (3 Â 50 mL). Combined ethyl acetate extracts were dried
A
mixture of 1.65 g ( )-3-(1-(dimethylamino)ethyl)phenol
(10.0 mmol) (2) and 2.2 g 2-bromobenzoic acid (11.0 mmol) was
heated in polyphosphoric acid at 80 °C for 10–15 min. After com-
plete disappearance of aminophenol derivative, checked by TLC,
the reaction vessel was cooled. Then, 100 mL ethylacetate and
100 mL water were added to reaction mixture, respectively, and
pH of the content was adjusted to 8–9 using NaHCO3(s). Organic
phase was separated and concentrated under reduced pressure to
give oily ester product. Yield obtained: 78.0%. Mass spectrometry:
peak at m/z 348 (M+1)+. 1H NMR (300 MHz, CDCl3, ppm): d 1.36 (d,
3H, ACHACH3), 2.17 (s, 6H, CH3ANACH3), 3.26 (q, 1H,
ANACHACH3), 7.04–7.21 (m, 3H, Ar-H), 7.26–7.41 (m, 3H, Ar-H),
7.63–7.70 (m, 1H, Ar-H), 7.91–7.99 (m, 1H, Ar-H).
4.1.3. ( )-3-(1-(Dimethylamino)ethyl)-6H-benzo[c]chromen-6-
one hydrochloride (4a)
To a solution of ( )-3-(1-(dimethylamino)ethyl)phenyl 2-bro-
mobenzoate (1.006 g, 4.05 mmol) (3a) in 35 mL of dry DMA, NaOAc
(664 mg, 8.1 mmol) and PdCl2(PPh3)2 (284 mg, 0.405 mmol) were
added at 80 °C. After stirring the reaction mixture at 130 °C for
15–16 h, the solvent was evaporated under reduced pressure.
The content was acidified with 1 N HCl (200 mL) and extracted
with ethyl acetate (3 Â 75 mL). The aqueous solution was basified
to pH 8–9 using NaHCO3(s) and extracted with ethyl acetate
(5 Â 100 mL). Combined ethyl acetate extracts were dried over
MgSO4 and concentrated under reduced pressure to give yellow
thick oil. Column chromatography on silica gel employing acetone
gives desired benzo[c]chromen-6-one derivative (526 mg, 68.0%).
( )-3-(1-(Dimethylamino)ethyl)-6H-benzo[c]chromen-6-one (free
base) dissolved in dry acetone (10 mL) was filtrated from
0.45 lm filter. HCl gas was passed through the obtained acetone
solution until salt precipitation took place. The content was mixed
for additional 1 h and then filtrated. Yield obtained: 81.0%, HPLC
purity: 99.90%, HRMS (ESI) C17H18NO2 calcd 268.1338 (M+1)+,
found 268.1330, mp: 235.4 °C, 1H NMR (300 MHz, DMSO-d6,

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H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
over MgSO4 and concentrated under reduced pressure to give mix-
ture of 3,5-dihydroxyacetophenone and 1-(3-hydroxy-5-methoxy-
phenyl)ethanone. To the mixture, 50 mL acetone and 25 g NaOH(s)
was added. When, 8–10 mL cold water is put into the reaction flask
slowly, sodium salt of 3,5-dihydroxyacetophenone was precipi-
tated. The liquid part of the content was decanted to another round
bottom flask. The solid part was washed with acetone (3 Â 15 mL).
Combined acetone parts were concentrated under reduced pres-
sure to give 1-(3-hydroxy-5-methoxyphenyl)ethanone as off white
solid. Yield obtained: 44.7%.
HBr (190 mL) at room temperature. The mixture was heated at
130 °C for 20–24 h. After cooling, the aqueous solution was basified
to pH 8–9 using NaHCO3(s) and extracted with CH2Cl2 (4 Â 50 mL).
Combined CH2Cl2 extracts were dried over MgSO4 and concen-
trated under reduced pressure to give yellow-brown oil. Column
chromatography on silica gel employing acetone gave desired
benzo[c]chromen-6-one derivative (1.37 g, 31.0%). In order to pre-
pare its HCl salt, the free base was dissolved in 30 mL dry metha-
nol/acetone (1:3) and filtrated from 0.45 lm filter. HCl gas was
passed through the obtained methanol–acetone solution. Liquid
part was concentrated under reduced pressure to give yellow oil.
When 10 mL dry acetone was added to resulted oil, HCl salt form
of 3-(1-(dimethylamino)ethyl)-1-hydroxy-6H-benzo[c]chromen-
6-one was precipitated. The content was mixed for 1 h and then fil-
trated. The residue was dried at 40 °C under vacuum. Yield
obtained: 79.3%. HPLC purity: 99.82%, HRMS (ESI) C17H18NO3 calcd
284.1287 (M+1)+, found 284.1293, mp: 253.5 °C 1H NMR
(300 MHz, DMSO-d6, ppm): d 1.62 (d, 3H, ACHACH3), 2.54 (s, 3H,
CH3ANACH3), 2.75 (s, 3H, CH3ANACH3), 4.43 (q, 1H,
ANACHACH3), 7.06 (s, 1H, Ar-H), 7.23 (s, 1H, Ar-H), 7.62 (t, 1H,
Ar-H), 7.90 (dt, 1H, Ar-H), 8.24 (d, 1H, Ar-H), 9.09 (d, 1H, Ar-H),
11.07 (br s, 1H, ANAH), 11.55 (s, 1H, Ar-H).
4.1.7. ( )-3-(1-(Dimethylamino)ethyl)-5-methoxyphenol (7)
The desired aminophenol derivative was synthesized according
to procedure given for the compound 2. Yield obtained 60.6%. Mass
spectrometry, peak at m/z 196 (M+1)+. 1H NMR (300 MHz, CDCl3,
ppm): d 1.29 (d, 3H, ACHACH3), 2.13 (s, 6H, CH3ANACH3), 3.15
(q, 1H, ANACHACH3), 3.61 (s, 3H, OACH3), 6.21 (t, 1H, Ar-H),
6.24–6.27 (m, 1H, Ar-H), 6.29–6.31 (m, 1H, Ar-H), 8.32 (br s, 1H,
AOAH).
4.1.8. ( )-3-(1-(Dimethylamino)ethyl)-5-methoxyphenyl 2-
bromobenzoate (8)
The compound was synthesized according to procedure given
for the compound 3a. Yield obtained: 70.2%. Mass spectrometry:
peak at m/z 378 (M+1)+. 1H NMR (300 MHz, CDCl3, ppm): d 1.33
(d, 3H, ACHACH3), 2.19 (s, 6H, CH3ANACH3), 3.23 (q, 1H,
ANACHACH3), 3.75 (s, 3H, OACH3), 6.64 (t, 1H, Ar-H), 6.75 (d,
2H, Ar-H), 7.32–7.39 (m, 2H, Ar-H), 7.64–7.69 (m, 1H, Ar-H),
7.92–7.97 (m, 1H, Ar-H).
4.1.12. ( )-1-(1-(Dimethylamino)ethyl)-3-hydroxy-6H-
benzo[c]chromen-6-one hydrochloride (12)
To
( )-1-(1-(dimethylamino)ethyl)-3-methoxy-6H-benzo[c]
chromen-6-one (1.49 g, 5.0 mmol) was added 48% aqueous HBr
(62 mL) at room temperature. The mixture was heated at 120 °C
for 4 h. After cooling, the aqueous solution was basified to pH 8–
9 using NaHCO3(s) and extracted with CH2Cl2 (4 Â 40 mL). Com-
bined CH2Cl2 extracts were dried over MgSO4 and concentrated
under reduced pressure to give yellow solid (1.20 g, 84.5%). Its
HCl salt was prepared according to procedure given for the com-
pound 4a. Yield obtained, 86.0%. HPLC purity: 99.07%, HRMS (ESI)
4.1.9. ( )-3-(1-(Dimethylamino)ethyl)-1-methoxy-6H-
benzo[c]chromen-6-one hydrochloride (9)
The compound was synthesized according to the procedure
given for the compound 4a. (i.e., yield obtained 46.2%, 81.3%,
respectively, for synthesis and salt preparation). HPLC purity:
99.40%, HRMS (ESI) C18H20NO3 calcd 298.1443 (M+1)+, found
298.1433, mp: 247.81 °C, 1H NMR (300 MHz, DMSO-d6, ppm): d
1.68 (d, 3H, ACHACH3), 2.56 (d, 3H, CH3ANACH3), 2.76 (d, 3H, CH3-
ANACH3), 4.07 (s, 3H, OACH3), 4.51 (q, 1H, ANACHACH3), 7.27 (d,
1H, Ar-H), 7.47 (s, 1H, Ar-H), 7.65 (dt, 1H, Ar-H), 7.90 (dt, 1H, Ar-H),
8.25 (dd, 1H, Ar-H), 8.93 (d, 1H, Ar-H), 11.41 (br s, 1H, ANAH). It is
important to note that the ring formation to obtain the title com-
pound from ( )-3-(1-(dimethylamino)ethyl)-5-methoxyphenyl2-
bromobenzoate (8) also yields ( )-1-(1-(dimethylamino)ethyl)-3-
methoxy-6H-benzo[c]chromen-6-one (10). Their separation was
achieved through silica-gel flash chromatography employing ace-
tone as the mobile phase (i.e., the Rf values were 0.6 and 0.2 on
TLC, respectively, for the compounds 9, and 10).
C
17H18NO3 calcd 284.1287 (M+1)+, found 284.1289, mp: 217.0 °C,
1H NMR (300 MHz, CD3OD, ppm): d 1.50 (d, 3H, ACHACH3), 2.17
(s, 6H, CH3ANACH3), 4.13 (q, 1H, ANACHACH3), 6.62 (d, 1H, Ar-
H), 7.09 (s, 1H, Ar-H), 7.48 (t, 1H, Ar-H), 7.78 (dt, 1H, Ar-H), 8.15
(br s, 1H, ANAH), 8.25 (dd, 1H, Ar-H).
4.1.13. ( )-3-(1-(Dimethylamino)ethyl)-6-oxo-6H-
benzo[c]chromen-1-yl ethyl(methyl)carbamate (13)
960 mg ( )-3-(1-(dimethylamino)ethyl)-1-hydroxy-6H-benzo
[c]chromen-6-one (11) (3.00 mmol) and 547.40 mg carbamoyl
chloride (4.30 mmol) was mixed in pyridine (18 mL) at ambient
temperature for 18–20 h. Pyridine was concentrated under
reduced pressure to give yellow-brown oil. Then pH was adjusted
to 8–9 using saturated NaHCO3 and extracted with CH2Cl2
(4 Â 50 mL). Combined CH2Cl2 extracts were dried over MgSO4
and concentrated under reduced pressure to give yellow thick oil.
Column chromatography on silica gel employing acetone gave
desired 13 (1.04 g, 95.0%). In order to prepare its HCl salt the free
base was dissolved in 30 mL dry acetone/heptane (1:3) and it
4.1.10. ( )-1-(1-(Dimethylamino)ethyl)-3-methoxy-6H-
benzo[c]chromen-6-one hydrochloride (10)
The compound was synthesized according to the procedure
described for the title compound 9 (i.e., yield obtained 14.2%,
83.7%, respectively, for synthesis and salt preparation) HPLC pur-
ity: 99.90%, HRMS (ESI) C18H20NO3 calcd 298.1443 (M+1)+, found
298.1431, mp: 110.4 °C,1H NMR (300 MHz, CD3OD, ppm): d 1.54
(d, 3H, ACHACH3), 2.18 (s, 6H, CH3ANACH3), 3.86 (s, 3H, OACH3),
4.15 (q, 1H, ANACHACH3), 6.81 (d, 1H, Ar-H), 7.24 (d, 1H, Ar-H),
7.53 (dt, 1H, Ar-H), 7.82 (dt, 1H, Ar-H), 8.20 (br s, 1H, ANAH),
8.28 (dd, 1H, Ar-H).
was filtrated from 0.45 lm filter. HCl gas was passed through the
obtained acetone-heptane solution, until salt precipitation took
place. The content was mixed for 2 h and then filtrated. Yield
obtained: 96.3%. HPLC purity: 99.16%, HRMS (ESI) C21H25N2O4
calcd 369.1814 (M+1)+, found 369.1825, mp: 256.3 °C, 1H NMR
(300 MHz, DMSO-d6, ppm): d 1.07–1.35 (m, 3H, CH3ACH2ANA),
1.67 (d, 3H, ACHACH3), 2.54 (d, 3H, CH3ANACH3), 2.71 (d, 3H, CH3-
ANACH3), 3.08 (d, 3H, CH3ANACH2ACH3), 3.28–3.68 (m, 2H, CH3-
ANACH2ACH3), 4.57 (q, 1H, ANACH-CH3), 7.42 (s, 1H, Ar-H), 7.65–
7.77 (m, 2H, Ar-H), 7.91–8.02 (m, 1H, Ar-H), 8.27–8.42 (m, 2H, Ar-
H), 11.37 (br s, 1H, ANAH).
4.1.11. ( )-3-(1-(Dimethylamino)ethyl)-1-hydroxy-6H-
benzo[c]chromen-6-one hydrochloride (11)
To
( )-3-(1-(dimethylamino)ethyl)-1-methoxy-6H-benzo[c]
chromen-6-one (9) (4.60 g, 15.47 mmol) was added 48% aqueous

H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
5149
4.1.14. ( )-1-(1-(Dimethylamino)ethyl)-6-oxo-6H-
benzo[c]chromen-3-yl ethyl(methyl)carbamate hydrochloride
(14)
and concentrated under reduced pressure to give the white solid
product. Yield obtained: 49.6%. (300 MHz, CDCl3, ppm): d 1.69–
1.82 (m, 4H, ACH2ACH2ACH2ACH2A), 2.41–2.54 (m, 2H, ACH2-
ACH2AC@CA), 2.62–2.73 (m, 2H, ACH2ACH2ACACOOA), 3.79 (t,
2H, ClACH2A), 4.21 (t, 2H, AOACH2ACH2A), 6.72 (d, 1H, Ar-H),
6.80 (dd, 1H, Ar-H), 7.40 (d, 1H, Ar-H).
660 mg ( )-1-(1-(dimethylamino)ethyl)-3-hydroxy-6H-benzo
[c]chromen-6-one (12) (2.33 mmol) and 424.76 mg carbamoyl
chloride (3.49 mmol) was mixed in pyridine (16 mL) at ambient
temperature for 17–19 h. Pyridine was concentrated under
reduced pressure to give yellow-brown oil. Then pH was adjusted
to 8–9 using saturated NaHCO3 and extracted with CH2Cl2
(4 Â 50 mL). Combined CH2Cl2 extracts were dried over MgSO4
and concentrated under reduced pressure to give yellow thick oil.
Column chromatography on silica gel (acetone/dichloromethane;
2:1) gave desired benzo[c]chromen-6-one derivative (511 mg,
60.0%). Then, the free base dissolved in 30 mL dry ether/acetone
4.1.18. 3-(3-Chloropropoxy)-7,8,9,10-tetrahydro-6H-
benzo[c]chromen-6-one (17b)
3-Hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one (10 g,
46.2 mmol) and NaH (2.76 g, 69 mmol) were mixed in 40 mL DMF.
1-Bromo-3-chloropropane (18.17 g, 115.5 mmol) was added to this
mixture and the reaction was mixed at ambient temperature for
6 h. After completion of the reaction, the reaction mixture was
poured onto 200 mL cold 15% NaOH solution. The mixture was
mixed for 5 min and then 50 mL of hexane was added. The solid
product precipitated was filtered and washed with cold water.
Yield obtained: 81.2% (300 MHz, CDCl3, ppm): d 1.68–1.82 (m,
4H, ACH2ACH2ACH2ACH2A), 2.20 (p, 2H, ClACH2ACH2ACH2AOA),
2.47–2.51 (m, 2H, ACH2ACH2AC@CA), 2.65–2.70 (m, 2H,
ACH2ACH2ACACOOA), 3.70 (t, 2H, ClACH2A), 4.09 (t, 2H,
AOACH2ACH2A), 6.73–6.79 (m, 2H, Ar-H), 7.39 (d, 1H, Ar-H).
(1:3) was filtrated from 0.45 lm filter. HCl gas was passed through
the obtained ether–acetone solution, until salt precipitation took
place. The content was mixed for 2 h and then filtrated. Yield
obtained: 71.7%. HPLC purity: 99.62%, HRMS (ESI) C21H25N2O4
calcd 369.1814 (M+1)+, found 369.1808, mp: 127.9 °C, 1H NMR
(300 MHz, CD3OD, ppm): d 1.13–1.31 (m, 3H, CH3ACH2ANA),
1.67 (d, 3H, ACHACH3), 2.33 (s, 6H, CH3ANACH3), 3.04 (d, 3H,
CH3ANACH2ACH3), 3.34–3.57 (m, 2H, CH3ANACH2ACH3), 4.48
(br s, 1H, ANACHACH3), 7.13 (s, 1H, Ar-H), 7.43 (d, 1H, Ar-H),
7.63 (dt, 1H, Ar-H), 7.89 (dt, 1H, Ar-H), 8.16 (br s, 1H, ANAH),
8.33 (dd, 1H, Ar-H).
4.1.19. 3-(4-Chlorobutoxy)-7,8,9,10-tetrahydro-6H-
benzo[c]chromen-6-one (17c)
3-(4-Chlorobutoxy)-7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-
one was synthesized according to procedure given for 17b, except
the employment of 1-bromo-4-chlorobutane (19.81 g, 115.5
mmol) instead of 1-bromo-3-chloropropane. Yield obtained:
82.5%. 1H NMR (300 MHz, CDCl3, ppm): d 1.58–1.72 (m, 4H,
ACH2ACH2ACH2ACH2A), 1.73–2.06 (m, 4H, ClACH2ACH2
ACH2ACH2AOA), 2.37–2.56 (m, 2H, ACH2ACH2AC@CA), 2.57–
2.76 (m, 2H, ACH2ACH2ACACOOA), 3.56 (t, 2H, ClACH2A), 3.96
(t, 2H, AOACH2ACH2A), 6.61–6.79 (m, 2H, Ar-H), 7.37 (d, 1H,
Ar-H).
4.1.15. 3-Hydroxy-7,8,9,10-tetrahydro-benzo[c]chromen-6-one
(15)
A mixture of resorcinol (5.0 g, 45.45 mmol) and ethyl 2-oxocy-
clohexanecarboxylate (7.73 g, 45.45 mmol) was heated at 70 °C
in the presence of zirconium(IV) chloride (1.06 g, 10 mol %) for
1 h. After completion of the reaction, the reaction mixture was
cooled to room temperature and poured onto crushed ice (200 g).
The solid product obtained was filtered off, and washed with ice-
cold water. Yield obtained: 91%, 1H NMR (DMSO-d6) d 1.68–1.73
(m, 4H, ACH2ACH2ACH2ACH2A), 2.27–2.40 (m, 2H, ACH2A
CH2AC@CA), 2.62–2.76 (m, 2H, ACH2ACH2ACACOOA), 6.65 (d,
1H, Ar-H), 6.74 (dd, 1H, Ar-H), 7.47 (d, 1H, Ar-H), 10.35 (s, 1H,
AOAH).
4.1.20. 3-(2-Chloroethoxy)-6H-benzo[c]chromen-6-one (18a)
3-(2-Chloroethoxy)-6H-benzo[c]chromen-6-one was synthe-
sized according to procedure given for the compound 17a
(14.7 g, 69.4 mmol), except the employment of 3-hydroxy-6H-
benzo[c]chromen-6-one instead of 3-hydroxy-7,8,9,10-tetrahy-
dro-6H-benzo[c]chromen-6-one. Yield obtained: 46.8%. 1H NMR
(300 MHz, CDCl3, ppm): d 3.80 (t, 2H, ClACH2A), 4.23 (t, 2H,
AOACH2ACH2ACl), 6.79 (d, 1H, Ar-H), 6.87 (dd, 1H, Ar-H), 7.45
(dt, 1H, Ar-H), 7.72 (dt, 1H, Ar-H), 7.87–7.95 (m, 2H, Ar-H), 8.28
(dd, 1H, Ar-H).
4.1.16. 3-Hydroxy-6H-benzo[c]chromen-6-one (16)
A mixture of 2-iodobenzoic acid (30 g, 0.12 mol), resorcinol
(40 g, 0.36 mol), and NaOH (17.4 g, 0.44 mol) in water (150 mL)
was heated under reflux for 30 min. After the addition of aqueous
CuSO4 (28%, 25 mL) the mixture was refluxed for additional
10 min, during which time the product (0.62 g) precipitated as a
white powder. The precipitate was filtered and washed with cold
water. Yield obtained: 80.1%, 1H NMR (DMSO-d6) d 6.71 (d, 1H),
6.80 (dd, 1H, Ar-H), 7.51 (t, 1H, Ar-H), 7.83 (t, 1H, Ar-H), 8.08–
8.22 (m, 3H, Ar-H), 10.32 (s, 1H, AOAH).
4.1.21. 3-(3-Chloropropoxy)-6H-benzo[c]chromen-6-one (18b)
3-(3-Chloropropoxy)-6H-benzo[c]chromen-6-one was synthe-
sized according to procedure given for the compound 17b, except
the employment of 3-hydroxy-6H-benzo[c]chromen-6-one (9.8 g,
46.2 mmol) instead of 3-hydroxy-7,8,9,10-tetrahydro-6H-benzo[c]
chromen-6-one. The reaction was completed in 40 min. Yield
obtained: 79.3%. 1H NMR (300 MHz, CDCl3, ppm): d 2.22 (p, 2H,
ClACH2ACH2ACH2AOA), 3.71 (t, 2H, ClACH2A), 4.12 (t, 2H,
AOACH2ACH2A), 6.79 (d, 1H, Ar-H), 6.84 (dd, 1H, Ar-H), 7.44 (t,
1H, Ar-H), 7.72 (tt, 1H, Ar-H), 7.86–7.94 (m, 2H, Ar-H) 8.28 (dd,
1H, Ar-H).
4.1.17. 3-(2-Chloroethoxy)-7,8,9,10-tetrahydro-6H-
benzo[c]chromen-6-one (17a)
Na(s) (1.9 g, 82.6 mmol) was dissolved in 50 mL dry ethanol to
give NaOEt solution. To 3-hydroxy-7,8,9,10-tetrahydro-6H-
benzo[c]chromen-6-one (15 g, 69.4 mmol) solution in 150 mL Eth-
anol, previously prepared NaOEt solution was added at ambient
temperature. Ethanol was distilled out under reduced vacuum
and then 100 mL dry DMF was added onto the residue. 100 mL
1,2-dichloroethane was put into the reaction mixture and the con-
tent was refluxed for 7 h. The reaction mixture was cooled to room
temperature and it was poured onto 250 mL cold 0.8 N NaOH solu-
tion. 100 mL 1,2-dichloroethane was added to the medium and
organic phase was separated. Then, the aqueous phase was
extracted with additional 1,2-dichloroethane (2 Â 50 mL). Com-
bined organic extracts were washed with water, dried over MgSO4
4.1.22. 3-(4-Chlorobutoxy)-6H-benzo[c]chromen-6-one (18c)
3-(4-Chlorobutoxy)-6H-benzo[c]chromen-6-one was synthe-
sized according to procedure given for the compound 17b, except
the employment of 3-hydroxy-6H-benzo[c]chromen-6-one (9.8 g,
46.2 mmol) instead of 3-hydroxy-7,8,9,10-tetrahydro-6H-benzo
[c]chromen-6-one. The reaction was completed in 40 min. Yield
obtained: 83.6%. 1H NMR (300 MHz, CDCl3, ppm): d 1.89–1.99

5150
H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
(m, 4H, ClACH2ACH2ACH2ACH2AOA), 3.56–3.60 (m, 2H, ClACH2-
A), 3.98–4.01 (m, 2H, AOACH2ACH2A), 6.77 (d, 1H, Ar-H), 6.83
(dd, 1H, Ar-H), 7.43 (dt, 1H, Ar-H), 7.71 (dt, 1H, Ar-H), 7.86 (d,
1H, Ar-H), 7.93 (d, 1H, Ar-H), 8.28 (dd, 1H, Ar-H).
HPLC purity: 99.85%, HRMS (ESI) C27H32NO3 calcd 418.2382
[M+H]+, found 418.2392, mp: 153.1 °C.
4.1.27. 3-(2-(4-(3-Methoxybenzyl)piperidin-1-yl)ethoxy)-
7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one hydrochloride
(19a4)
4.1.23. General procedure for the synthesis of 19, and 20-series
title compounds
Yield obtained 76.1%, 88.3%, respectively, for the reaction and
HCl salt preparation.1H NMR (300 MHz, CDCl3, ppm): d1.28–1.36
(m, 2H, AOACH2ACH2ANACH2ACH2(ax)ACHA), 1.44–1.52 (m, 1H,
AOACH2ACH2ANACH2ACH2ACHACH2ACH2A), 1.60 (d, 2H,
AOACH2ACH2ANACH2ACH2(eq)ACHA), 1.71–1.79 (m, 4H, ACH2-
ACH2ACH2ACH2A), 2.02 (t, 2H, AOACH2ACH2ANACH2(ax)A),
2.44–2.50 (m, 4H, ACH2ACH2AC@CAand AOACH2ACH2ANA
CH2(eq)A), 2.62–2.71 (m, 2H, ACH2ACH2ACACOOA), 2.76 (t, 2H,
ANACH2ACH2AOA), 2.94 (d, 2H, ACHACH2APh), 3.73 (s, 3H,
AOACH3), 4.08 (t, 2H, AOACH2ACH2A), 6.62–6.78 (m, 5H, Ar-H),
7.12 (t, 1H, Ar-H), 7.37 (d, 1H, Ar-H). HPLC purity: 99.66%, HRMS
(ESI) C28H34NO4 calcd 448.2488 [M+H]+, found 448.2496, mp:
101.9 °C.
A mixture of 2.73 mmol appropriate alkyl halide (i.e., 17a–c, or
18a–c), 0.6 g K2CO3 (4.34 mmol), and 0.6 g NaI (4.00 mmol) in ACN
(12 mL) was mixed for 5 min in a 30 mL microwave reaction vessel.
Then, 8.2 mmol appropriate amine derivative (i.e., 1–6, as shown in
Scheme 3) was added and the resulting content was mixed for
additional 2 min. The reaction vessel was capped, and inserted
inside the microwave instrument (i.e., A CEM Model Single Mode
Microwave Instrument) and heated for 32 min at 105 °C at the
dynamic mode of the instrument automatically calibrating the
radiation and temperature balance with respect to the change in
pressure. Then, acetonitrile was distilled out under vacuum and
the residue thus obtained was mixed with 20 mL of K2CO3 solution
(5.0%) and this mixture was heated at 60 °C for 1 h. Then the mix-
ture was cooled down to room temperature and the aqueous phase
was extracted 3 times with 20 mL of ethylacetate. Combined
organic extracts were dried over MgSO4 and concentrated under
reduced pressure to give the product (i.e., free base). The free base
was dissolved in 10 mL acetone and HCl gas was continuously
passed until HCl salt precipitation took place.
4.1.28. 3-(2-(4-Benzylpiperazin-1-yl)ethoxy)-7,8,9,10-
tetrahydro-6H-benzo[c]chromen-6-one dihydrochloride (19a5)
Yield obtained 70.3%, 88.8%, respectively, for the reaction and
HCl salt preparation.1H NMR (300 MHz, CDCl3, ppm): d 1.69–1.81
(m, 4H, ACH2ACH2ACH2ACH2A), 2.35–2.62 (m, 10H, ACH2ACH2-
AC@CA and piperazine protons), 2.63–2.70 (m, 2H, ACH2ACH2-
ACACOOA), 2.78 (t, 2H, ANACH2ACH2AOA), 3.46 (s, 2H,
NACH2APh), 4.07 (t, 2H, AOACH2ACH2A), 6.71 (d, 1H, Ar-H),
6.76 (dd, 1H, Ar-H), 7.15–7.27 (m, 5H, Ar-H), 7.36 (d, 1H, Ar-H).
HPLC purity: 99.70%, HRMS (ESI) C26H31N2O3 calcd 419.2335
[M+H]+, found 419.2333, mp: 129.6 °C.
4.1.24. 3-(2-(Benzyl(methyl)amino)ethoxy)-7,8,9,10-tetrahydro-
6H-benzo[c]chromen-6-one hydrochloride (19a1)
Yield obtained 75.1%, 83.3%, respectively, for the reaction
and HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm):
d
1.71–1.80 (m, 4H, ACH2ACH2ACH2ACH2A), 2.31 (s, 3H,
ANACH3), 2.43–2.54 (m, 2H, ACH2ACH2AC@CA), 2.62–2.73 (m,
2H, ACH2ACH2ACACOOA), 2.80 (t, 2H, ANACH2ACH2AOA), 3.58
(s, 2H, ANACH2APh), 4.06 (t, 2H, AOACH2ACH2A), 6.71 (d, 1H,
Ar-H), 6.76 (dd, 1H, Ar-H), 7.15–7.31 (m, 5H, Ar-H), 7.37 (d, 1H,
Ar-H). HPLC purity: 99.67%, HRMS (EI) C23H26NO3 calcd 364.1913
(M+), found 364.1909, mp: 196.0 °C.
4.1.29. 3-(2-(4-(3-Methoxybenzyl)piperazin-1-yl)ethoxy)-
7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one
dihydrochloride (19a6)
Yield obtained 66.0%, 80.6%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.71–
1.79 (m, 4H, ACH2ACH2ACH2ACH2A), 2.38–2.63 (m, 10H, m,
10H, ACH2ACH2AC@CA and piperazine protons), 2.64–2.73 (m,
2H, ACH2ACH2ACACOOA), 2.78 (t, 2H, ANACH2ACH2AOA), 3.44
(s, 2H, NACH2APh), 3.74 (s, 3H, AOACH3), 4.07 (t, 2H, AO
ACH2ACH2A), 6.69–6.88 (m, 5H, Ar-H), 7.16 (t, 1H, Ar-H), 7.37
(d, 1H, Ar-H). HPLC purity: 100.0%, HRMS (ESI) C27H33N2O4 calcd
449.2440 [M+H]+, found 449.2453, mp: 125.6 °C.
4.1.25. 3-(2-((3-Methoxybenzyl)(methyl)amino)ethoxy)-
7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one hydrochloride
(19a2)
Yield obtained 68.1%, 90.1%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.71–
1.80 (m, 4H, ACH2ACH2ACH2ACH2A), 2.35 (s, 3H, ANACH3),
2.42–2.55 (m, 2H, ACH2ACH2AC@CA), 2.63–2.72 (m, 2H, ACH2-
ACH2ACACOOA), 2.84 (br s, 2H, ANACH2ACH2AOA), 3.60 (s, 2H,
ANACH2APh), 3.75 (s, 3H, AOACH3), 4.10 (t, 2H, AOACH2ACH2A),
6.71–6.79 (m, 3H, Ar-H), 6.83–6.91 (m, 2H, Ar-H), 7.18 (t, 1H, Ar-
H), 7.38 (d, 1H, Ar-H). 90.1%, HPLC purity: 99.98%, HRMS (ESI)
4.1.30. 3-(3-(Benzyl(methyl)amino)propoxy)-7,8,9,10-
tetrahydro-6H-benzo[c]chromen-6-one hydrochloride (19b1)
Yield obtained 69.8%, 88.2%, respectively, for the reaction and
HCl salt preparation.1H NMR (300 MHz, CDCl3, ppm): d 1.66–1.89
(m, 4H, ACH2ACH2ACH2ACH2A), 1.90–2.08 (m, 2H, AOACH2A
CH2ACH2ANA), 2.22 (s, 3H, ANACH3), 2.40–2.62 (m, 4H,
ACH2ACH2AC@CA and ANACH2ACH2A), 2.63–2.79 (m, 2H,
ACH2ACH2ACACOOA), 3.50 (s, 2H, ANACH2APh), 4.02 (t, 2H,
AOACH2ACH2A), 6.67–6.82 (m, 2H, Ar-H), 7.12–7.33 (m, 5H, Ar-
H), 7.34–7.46 (m, 1H, Ar-H). HPLC purity: 99.00%, HRMS (ESI)
C
24H28NO4 calcd 394.2018 [M+H]+, found 394.2000, mp: 176.5 °C.
4.1.26. 3-(2-(4-Benzylpiperidin-1-yl)ethoxy)-7,8,9,10-
tetrahydro-6H-benzo[c]chromen-6-one hydrochloride (19a3)
Yield obtained 66.3%, 87.4%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.32–
1.45 (m, 2H, AOACH2ACH2ANACH2ACH2(ax)ACHA), 1.46–1.53
(m, 1H, AOACH2ACH2ANACH2ACH2ACHACH2ACH2A), 1.57 (d,
2H, AOACH2ACH2ANACH2ACH2(eq)ACHA), 1.63–1.78 (m, 4H,
ACH2ACH2ACH2ACH2A), 2.05 (t, 2H, AOACH2ACH2ANACH2(ax)A),
2.39–2.54 (m, 4H, ACH2ACH2AC@CA and AOACH2ACH2-
ANACH2(eqA), 2.62–2.73 (m, 2H, ACH2ACH2ACACOOA), 2.81 (t,
2H, ANACH2ACH2AOA), 2.99 (d, 2H, ACHACH2APh), 4.13 (t, 2H,
AOACH2ACH2A), 6.72 (d, 1H, Ar-H), 6.77 (dd, 1H, Ar-H), 7.06–
7.15 (m, 3H, Ar-H), 7.18–7.24 (m, 2H, Ar-H), 7.37 (d, 1H, Ar-H).
C
24H28NO3 calcd 378.2069 [M+H]+, found 378.2070, mp: 84.8 °C.
4.1.31. 3-(3-((3-Methoxybenzyl)(methyl)amino)propoxy)-
7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one hydrochloride
(19b2)
Yield obtained 60.2%, 86.4%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d1.68–1.84
(m, 4H, ACH2ACH2ACH2ACH2A), 1.89–2.01 (m, 2H, AOACH2ACH2-
ACH2ANA), 2.19 (s, 3H, ANACH3), 2.45–2.54 (m, 4H, ACH2ACH2-
AC@CA and ANACH2ACH2A), 2.64–2.73 (m, 2H, ACH2ACH2

H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
5151
ACACOOA), 3.45 (s, 2H, ANACH2APh), 3.71 (s, 3H, AOACH3), 3.99
4.1.36. 3-(4-(Benzyl(methyl)amino)butoxy)-7,8,9,10-
(t, 2H, AOACH2ACH2A), 6.67–6.76 (m, 3H, Ar-H), 6.82 (dd, 2H, Ar-
H), 7.14 (t, 1H, Ar-H), 7.37 (dd, 1H, Ar-H). HPLC purity: 99.45%,
HRMS (ESI) C25H30NO4 calcd 408.2175 [M+H]+, found 408.2166,
mp: 215.6 °C.
tetrahydro-6H-benzo[c]chromen-6-one hydrochloride (19c1)
Yield obtained 61.6%, 86.2%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm):
d
1.62–1.88 (m, 8H, AOACH2ACH2ACH2ACH2ANA and CACH2A
CH2ACH2ACH2AC), 2.20 (s, 3H, ANACH3), 2.43 (t, 2H, ACH2A
CH2ACH2ANA), 2.49–2.58 (m, 2H, ACH2ACH2AC@CA), 2.68–2.76
(m, 2H, ACH2ACH2ACACOOA), 3.49 (s, 2H, ANACH2APh), 3.96 (t,
2H, AOACH2ACH2ACH2A), 6.74 (d, 1H, Ar-H), 6.79 (dd, 1H, Ar-
H), 7.18–7.32 (m, 5H, Ar-H), 7.42 (d, 1H, Ar-H). HPLC purity:
99.98%, HRMS (ESI) C25H30NO3 calcd 392.2226 [M+H]+, found
392.2227, mp: 150.7 °C.
4.1.32. 3-(3-(4-Benzylpiperidin-1-yl)propoxy)-7,8,9,10-
tetrahydro-6H-benzo[c]chromen-6-one hydrochloride (19b3)
Yield obtained 66.7%, 82.5%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.22–
1.42 (m, 2H, ANACH2ACH2(ax)ACHACH2ACH2A), 1.43–1.56 (m,
1H, ANACH2ACH2ACHACH2ACH2A), 1.57–1.69 (m, 2H, ANACH2-
ACH2(eq)ACHACH2A), 1.70–1.86 (m, 4H, ACH2ACH2ACH2ACH2A),
1.87–2.09 (m, 4H, ANACH2(ax)ACH2ACHACH2A and AOA
CH2ACH2ACH2ANA), 2.39–2.61 (m, 6H, ACH2ACH2AC@CA,
ANACH2(eq)ACH2ACHACH2A, and OACH2ACH2ACH2ANA), 2.63–
2.76 (m, 2H, ACH2ACH2ACACOOA), 2.95 (d, 2H, ACHACH2APh),
4.01 (t, 2H, AOACH2ACH2A), 6.70–6.83 (m, 2H, Ar-H), 7.03–7.28
(m, 5H, Ar-H), 7.39 (d, 1H, Ar-H). HPLC purity: 99.59%, HRMS
(ESI) C28H34NO3 calcd 432.2539 [M+H]+, found 432.2559, mp:
121.7 °C.
4.1.37. 3-(4-((3-Methoxybenzyl)(methyl)amino)butoxy)-
7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one hydrochloride
(19c2)
Yield obtained 59.4%, 89.2%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm):
d
1.59–1.84 (m, 8H, AOACH2ACH2ACH2ACH2ANA and CACH2A
CH2ACH2ACH2AC), 2.19 (s, 3H, ANACH3), 2.41 (t, 2H, ACH2
ACH2ACH2ANA), 2.46–2.53 (m, 2H, ACH2ACH2AC@CA), 2.63–
2.72 (m, 2H, ACH2ACH2ACACOOA), 3.46 (s, 2H, ANACH2APh),
3.73 (s, 3H, AOACH3), 3.92 (t, 2H, AOACH2ACH2ACH2A), 6.67–
6.77 (m, 3H, Ar-H), 6.81–6.88 (m, 2H, Ar-H), 7.16 (t, 1H, Ar-H),
7.37 (d, 1H, Ar-H). HPLC purity: 99.67%, HRMS (ESI) C26H32NO4
calcd 422.2331 [M+H]+, found 422.2345, mp: 184.8 °C.
4.1.33. 3-(3-(4-(3-Methoxybenzyl)piperidin-1-yl)propoxy)-
7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one hydrochloride
(19b4)
Yield obtained 65.6%, 81.1%, respectively, for the reaction and
HCl salt preparation.1H NMR (300 MHz, CDCl3, ppm): d 1.30–1.42
(m, 2H, ANACH2ACH2(ax)ACHACH2ACH2A), 1.46–1.57 (m, 1H,
ANACH2ACH2ACHACH2ACH2A), 1.58–1.71 (m, 2H, ANA
CH2ACH2(eq)ACHACH2A), 1.72–1.88 (m, 4H, ACH2ACH2ACH2-
ACH2A), 1.92–2.02 (m, 4H, ANACH2(ax)ACH2ACHACH2A and
–OACH2ACH2ACH2ANA), 2.42–2.61 (m, 6H, ACH2ACH2AC@CA,
ANACH2(eq)ACH2ACHACH2A, and OACH2ACH2ACH2ANA), 2.66–
2.78 (m, 2H, ACH2ACH2ACACOOA), 2.97 (d, 2H, ACHACH2APh),
3.77 (s, 3H, AOACH3), 4.03 (t, 2H, AOACH2ACH2A), 6.63–6.83
(m, 5H, Ar-H), 7.17 (t, 1H, Ar-H), 7.42 (d, 1H, Ar-H). HPLC purity:
99.00%, HRMS (ESI) C29H36NO4 calcd 462.2644 [M+H]+, found
462.2644, mp: 119.1 °C.
4.1.38. 3-(4-(4-Benzylpiperidin-1-yl)butoxy)-7,8,9,10-
tetrahydro-6H-benzo[c]chromen-6-one hydrochloride (19c3)
Yield obtained 70.4%, 80.3%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm):
d
1.19–1.38 (m, 2H, ANACH2ACH2(ax)ACHACH2ACH2A), 1.39–1.53
(m, 1H, ANACH2ACH2ACHACH2ACH2A), 1.53–1.94 (m,
12H, ANACH2ACH2(eq)ACHACH2ACH2A, ACACH2ACH2ACH2-
ACH2ACA, AOACH2ACH2ACH2ACH2ANA and ANACH2(ax)ACH2-
ACHACH2A), 2.34 (t, 2H, AOACH2ACH2ACH2ACH2ANA),
2.42–2.56 (m, 4H, ANACH2(eq)ACH2ACHACH2A and ACH2ACH2-
AC@CA), 2.62–2.73 (m, 2H, ACH2ACH2ACACOOA), 2.89 (d, 2H,
ACHACH2APh), 3.94 (t, 2H, AOACH2ACH2A), 6.70 (d, 1H, Ar-H),
6.74 (dd, 1H, Ar-H), 7.03–7.25 (m, 5H, Ar-H), 7.36 (d, 1H, Ar-H).
HPLC purity: 99.71%, HRMS (ESI)
C29H36NO3 calcd 446.2695
4.1.34. 3-(3-(4-Benzylpiperazin-1-yl)propoxy)-7,8,9,10-
[M+H]+, found 446.2695, mp: 94.6 °C.
tetrahydro-6H-benzo[c]chromen-6-one dihydrochloride (19b5)
Yield obtained 68.0%, 90.2%, respectively, for the reaction and
HCl salt preparation.1H NMR (300 MHz, DMSO-d6, ppm): d1.57–
1.78 (m, 4H, ACH2ACH2ACH2ACH2A), 1.83 (p, 2H, AOACH2A
CH2ACH2ANA), 2.14–2.52 (m, 12H, ACH2ACH2AC@CA, AOA
CH2ACH2ACH2ANA and piperazine protons), 2.64–2.78 (m, 2H,
ACH2ACH2ACACOOA), 3.40 (s, 2H, ANACH2APh), 4.04 (t, 2H,
AOACH2ACH2), 6.83–6.93 (m, 2H, Ar-H), 7.15–7.34 (m, 5H, Ar-
H), 7.55 (d, 1H, Ar-H). HPLC purity: 100.0%, HRMS (ESI)
4.1.39. 3-(4-(4-(3-Methoxybenzyl)piperidin-1-yl)butoxy)-
7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one hydrochloride
(19c4)
Yield obtained 59.5%, 81.9%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.20–1.41
(m, 2H, ANACH2ACH2(ax)ACHACH2ACH2A), 1.41–1.55 (m, 1H,
ANACH2ACH2ACHACH2ACH2A), 1.55–1.95 (m, 12H, ANACH2-
ACH2(eq)ACHACH2ACH2A, ACACH2ACH2ACH2ACH2ACA, AOA
CH2ACH2ACH2ACH2ANA and ANACH2(ax)ACH2ACHACH2A), 2.36
(t, 2H, AOACH2ACH2ACH2ACH2ANA), 2.45–2.57 (m, 4H,
ANACH2(eq)ACH2ACHACH2A and ACH2ACH2AC@CA), 2.62–2.76
(m, 2H, ACH2ACH2ACACOOA), 2.91 (d, 2H, ACHACH2APh), 3.75
(s, 3H, AOACH3), 3.96 (t, 2H, AOACH2ACH2A), 6.62–6.80 (m, 5H,
Ar-H), 7.14 (t, 1H, Ar-H), 7.39 (d, 1H, Ar-H). HPLC purity: 96.54%,
HRMS (ESI) C30H38NO4 calcd 476.2801 [M+H]+, found 476.2816,
mp: 151.9 °C.
C
27H33N2O3 calcd 433.2491 [M+H]+, found 433.2488, mp: 96.3 °C.
4.1.35. 3-(3-(4-(3-Methoxybenzyl)piperazin-1-yl)propoxy)-
7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one
dihydrochloride (19b6)
Yield obtained 64.6%, 80.3%, respectively, for the reaction and
HCl salt preparation.1H NMR (300 MHz, CDCl3, ppm): d 1.72–1.79
(m, 4H, ACH2ACH2ACH2ACH2A), 1.93–2.10 (m, 2H, AOACH2ACH2-
ACH2ANA), 2.39–2.73 (m, 14H, ACH2ACH2AC@CA, AOACH2-
ACH2ACH2ANA, ACH2ACH2ACACOOA and piperazine protons),
3.50 (s, 2H, NACH2APh), 3.75 (s, 3HAOACH3), 4.00 (t, 2H, O-CH2-
ACH2A), 6.71–6.91 (m, 5H, Ar-H), 7.17 (t, 1H, Ar-H), 7.38 (d, 1H,
4.1.40. 3-(4-(4-Benzylpiperazin-1-yl)butoxy)-7,8,9,10-
tetrahydro-6H-benzo[c]chromen-6-one dihydrochloride (19c5)
Yield obtained 64.3%, 84.7%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.58–
1.85 (m, 8H, ACACH2ACH2ACH2ACH2ACA and AOACH2ACH2-
Ar-H). HPLC purity: 98.72%, HRMS (ESI)
C28H35N2O4 calcd
463.2597 [M+H]+, found 463.2597, mp: 274.2 °C.

5152
H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
ACH2ACH2ANA), 2.30–2.62 (m, 12H, AOACH2ACH2ACH2ACH2-
ANA, piperazine protons and ACH2ACH2AC@CA), 2.63–2.74 (m,
2H, ACH2ACH2ACACOOA), 3.46 (s, 2H, NACH2APh), 3.95 (t, 2H,
AOACH2ACH2A), 6.68–6.78 (m, 2H, Ar-H), 7.14–7.29 (m, 5H, Ar-
H), 7.37 (d, 1H, Ar-H). HPLC purity: 99.36%, HRMS (ESI)
AOACH3), 4.15 (t, 2H, ANACH2ACH2AOA), 6.58–6.72 (m, 3H, Ar-
H), 6.78 (d, 1H, Ar-H), 6.86 (dd, 1H, Ar-H), 7.14 (t, 1H, Ar-H), 7.47
(d, 1H, Ar-H), 7.74 (dt, 1H, Ar-H), 7.86–7.98 (m, 2H, Ar-H), 8.30
(dd, 1H, Ar-H). HPLC purity: 99.49%, HRMS (ESI) C28H30NO4 calcd
444.2175 [M+H]+, found 444.2171, mp: 174.3 °C.
C
28H35N2O3 calcd 447.2648 [M+H]+, found 447.2637, mp: 270.7 °C.
4.1.46. 3-(2-(4-Benzylpiperazin-1-yl)ethoxy)-6H-benzo[c]chro-
men-6-one dihydrochloride (20a5)
4.1.41. 3-(4-(4-(3-Methoxybenzyl)piperazin-1-yl)butoxy)-
7,8,9,10-tetrahydro-6H-benzo[c]chromen-6-one
dihydrochloride (19c6)
Yield obtained 71.8%, 90.8%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 2.35–
2.71 (m, 8H, piperazine protons), 2.80 (t, 2H, ANACH2ACH2AOA),
3.47 (s, 2H, ANACH2APh), 4.10 (t, 2H, ANACH2ACH2AOA), 6.79 (d,
1H, Ar-H), 6.85 (dd, 1H, Ar-H), 7.16–7.29 (m, 5H, Ar-H), 7.44 (dt,
1H, Ar-H), 7.72 (dt, 1H, Ar-H), 7.87 (d, 1H, Ar-H), 7.93 (d, 1H, Ar-
H), 8.29 (dd, 1H, Ar-H). HPLC purity: 99.97%, HRMS (ESI)
Yield obtained 72.6%, 82.2%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.62–
1.88 (m, 8H, ACACH2ACH2ACH2ACH2ACA and AOACH2ACH2-
ACH2ACH2ANA), 2.35–2.66 (m, 12H, AOACH2ACH2ACH2ACH2-
ANA, piperazine protons and ACH2ACH2AC@CA), 2.67–2.78 (m,
2H, ACH2ACH2ACACOOA), 3.47 (s, 2H, N-CH2APh), 3.76 (s, 3H,
AOACH3), 3.97 (t, 2H, AOACH2ACH2A), 6.71–6.79 (m, 3H, Ar-H),
6.82–6.88 (m, 2H, Ar-H), 7.18 (t, 1H, Ar-H), 7.39 (d, 1H, Ar-H). HPLC
purity: 99.54%, HRMS (ESI) C29H37N2O4 calcd 477.2753 [M+H]+,
found 477.2762, mp: 243.4 °C.
C
26H27N2O3 calcd 415.2022 [M+H]+, found 415.2039, mp: 176.6 °C.
4.1.47. 3-(2-(4-(3-Methoxybenzyl)piperazin-1-yl)ethoxy)-6H-
benzo[c]chromen-6-one dihydrochloride (20a6)
Yield obtained 75.6%, 88.2%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 2.36–
2.73 (m, 8H, piperazine protons), 2.80 (t, 2H, ANACH2ACH2AOA),
3.44 (s, 2H, ANACH2APh), 3.74 (s, 3H, AOACH3), 4.10 (t, 2H,
ANACH2ACH2AOA), 6.69–6.89 (m, 5H, Ar-H), 7.16 (t, 1H, Ar-H),
7.44 (dt, 1H, Ar-H), 7.72 (dt, 1H, Ar-H), 7.87 (d, 1H, Ar-H), 7.93
(d, 1H, Ar-H), 8.29 (dd, 1H, Ar-H). HPLC purity: 99.62%, HRMS
(ESI) C27H29N2O4 calcd 445.2127 [M+H]+, found 445.2143, mp:
282.3 °C.
4.1.42. 3-(2-(Benzyl(methyl)amino)ethoxy)-6H-benzo[c]chrom-
en-6-one hydrochloride (20a1)
Yield obtained 79.2%, 83.2%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 2.35 (s,
3H, ANACH3), 2.84 (t, 2H, ANACH2ACH2AOA), 3.62 (s, 2H,
ANACH2APh), 4.12 (t, 2H, ANACH2ACH2AOA), 6.81 (d, 1H,
Ar-H), 6.85 (dd, 1H, Ar-H), 7.19–7.32 (m, 5H, Ar-H), 7.47 (t,
1H, Ar-H), 7.75 (dt, 1H, Ar-H), 7.84–7.98 (m, 2H, Ar-H), 8.32 (d,
1H, Ar-H). HPLC purity: 99.54%, HRMS (ESI) C23H22NO3 calcd
360.1600 [M+H]+, found 360.1613, mp: 221.7 °C.
4.1.48. 3-(3-(Benzyl(methyl)amino)propoxy)-6H-benzo[c]
chromen-6-one hydrochloride (20b1)
Yield obtained 75.2%, 80.8%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.97 (p,
2H, AOACH2ACH2ACH2ANA), 2.19 (s, 3H, ANACH3), 2.52 (t, 2H,
AOACH2ACH2ACH2ANA), 3.47 (s, 2H, ANACH2APh), 4.03 (t, 2H,
AOACH2ACH2ACH2ANA), 6.75–6.85 (m, 2H, Ar-H), 7.17–7.25 (m,
5H, Ar-H), 7.44 (dt, 1H, Ar-H), 7.73 (dt, 1H, Ar-H), 7.87 (d, 1H, Ar-
H), 7.95 (d, 1H, Ar-H), 8.30 (dd, 1H, Ar-H). HPLC purity: 99.92%,
HRMS (ESI) C24H24NO3 calcd 374.1756 [M+H]+, found 374.1750,
mp: 186.9 °C.
4.1.43. 3-(2-((3-Methoxybenzyl)(methyl)amino)ethoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20a2)
Yield obtained 74.4%, 85.1%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CD3OD, ppm): d 2.96 (s,
3H, ANACH3), 3.63 (br s, 2H, ANACH2ACH2AOA), 3.78 (s, 3H,
AOACH3), 4.34–4.54 (m, 4H, ANACH2APh and ANACH2ACH2-
AOA), 6.88–7.17 (m, 5H, Ar-H), 7.37 (t, 1H, Ar-H), 7.53 (t, 1H, Ar-
H) 7.81 (t, 1H, Ar-H), 7.99–8.14 (m, 2H, Ar-H), 8.21 (d, 1H, Ar-H).
HPLC purity: 99.40%, HRMS (ESI)
C24H24NO4 calcd 390.1705
4.1.49. 3-(3-((3-Methoxybenzyl)(methyl)amino)propoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20b2)
[M+H]+, found 390.1709, mp: 205.9 °C.
Yield obtained 75.8%, 80.3%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.96 (p,
2H, AOACH2ACH2ACH2ANA), 2.20 (s, 3H, ANACH3), 2.51 (t, 2H,
AOACH2ACH2ACH2ANA), 3.44 (s, 2H, ANACH2APh), 3.71 (s, 3H,
AOACH3), 4.02 (t, 2H, AOACH2ACH2ACH2ANA), 6.67–6.87 (m,
5H, Ar-H), 7.14 (t, 1H, Ar-H), 7.43 (dt, 1H, Ar-H), 7.72 (dt, 1H, Ar-
H), 7.85 (d, 1H, Ar-H), 7.94 (d, 1H, Ar-H), 8.29 (dd, 1H, Ar-H). HPLC
purity: 99.95%, HRMS (ESI) C25H26NO4 calcd 404.1862 [M+H]+,
found 404.1856, mp: 215.1 °C.
4.1.44. 3-(2-(4-Benzylpiperidin-1-yl)ethoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20a3)
Yield obtained 70.1%, 88.6%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.16–
1.40 (m, 2H, AOACH2ACH2ANACH2ACH2(ax)ACHA), 1.42–1.56
(m, 1H, AOACH2ACH2ANACH2ACH2ACHACH2ACH2A), 1.57–
1.70 (m, 2H, AOACH2ACH2ANACH2ACH2(eq)ACHA), 2.03 (t, 2H,
AOACH2ACH2ANACH2(ax)A), 2.50 (d, 2H, AOACH2ACH2ANA
CH2(eq)A), 2.78 (t, 2H, ANACH2ACH2AOA), 2.96 (d, 2H, ACHA
CH2APh), 4.12 (t, 2H, ANACH2ACH2AOA), 6.74–6.91 (m, 2H,
Ar-H), 7.03–7.28 (m, 5H, Ar-H), 7.46 (t, 1H, Ar-H), 7.73 (dt, 1H,
Ar-H), 7.82–7.99 (m, 2H, Ar-H), 8.31 (d, 1H, Ar-H). HPLC purity:
99.37%, HRMS (ESI) C27H28NO3 calcd 414.2069 [M+H]+, found
414.2075, mp: 199.6 °C.
4.1.50. 3-(3-(4-Benzylpiperidin-1-yl)propoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20b3)
Yield obtained 70.0%, 88.8%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm):
d
1.30–1.75 (m, 7H, AOACH2ACH2ACH2ANACH2ACH2ACHA,AN
ACH2ACH2ACHACH2ACH2A and AOACH2ACH2ACH2AN ACH2(ax)-
A), 2.10–2.23 (m, 2H, AOACH2ACH2ACH2ANA), 2.55 (d, 2H,
AOACH2ACH2ACH2ANACH2(eq)A), 2.75–3.06 (m, 2H, AOACH2-
ACH2ACH2ANA), 3.19–3.44 (m, 2H, ANACH2APh), 4.06 (t, 2H,
AOACH2ACH2ACH2ANA), 6.77 (d, 1H, Ar-H), 6.82 (dd, 1H, Ar-H),
7.12–7.26 (m, 5H, Ar-H), 7.46 (m, 1H, Ar-H), 7.73 (dt, 1H, Ar-H),
7.89 (d, 1H, Ar-H), 7.94 (d, 1H, Ar-H), 8.3 (dd, 1H, Ar-H). HPLC pur-
ity: 99.09%, HRMS (ESI) C28H30NO3 calcd 428.2226 [M+H]+, found
428.2212, mp: 232.4 °C.
4.1.45. 3-(2-(4-(3-Methoxybenzyl)piperidin-1-yl)ethoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20a4)
Yield obtained 70.1%, 88.6%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm):
d
1.29–1.95 (m, 7H, AOACH2ACH2ANACH2ACH2ACHA, ANA
CH2ACH2ACHACH2ACH2A and AOACH2ACH2ANACH2(ax)A), 2.53
(d, 2H, AOACH2ACH2ANACH2(eq)A), 3.13–3.33 (m, 2H,
- N-
CH2ACH2AOA), 3.38–3.56 (m, 2H, ACHACH2APh), 3.73 (s, 3H,

H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
5153
4.1.51. 3-(3-(4-(3-Methoxybenzyl)piperidin-1-yl)propoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20b4)
4.1.56. 3-(4-(4-Benzylpiperidin-1-yl)butoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20c3)
Yield obtained 76.5%, 80.4%, respectively, for the reaction and
Yield obtained 68.9%, 90.8%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm):
1.21–1.39 (m, 2H, ANACH2ACH2(ax)ACHACH2(ax)ACH2A),
1.40–1.54 (m, 1H, ANACH2ACH2ACHACH2ACH2A), 1.55–1.86
(m, 8H, AOACH2ACH2ACH2ACH2ANA, ANACH2ACH2(eq)-
ACHACH2(eq)ACH2A and ANACH2(ax)ACH2ACHACH2), 2.35 (t,
2H, AOACH2ACH2ACH2ACH2ANA), 2.48 (d, 2H, ANACH2(eq)
ACH2ACHACH2), 2.90 (d, 2H, ANACH2APh), 3.98 (t, 2H, AO
ACH2ACH2ACH2ACH2ANA), 6.79 (d, 1H, Ar-H), 6.84 (dd, 1H,
Ar-H), 7.03–7.26 (m, 5H, Ar-H), 7.44 (dt, 1H, Ar-H), 7.73 (dt, 1H,
Ar-H), 7.88 (d, 1H, Ar-H), 7.94 (d, 1H, Ar-H), 8.30 (dd, 1H, Ar-H).
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm):
d
d
1.20–1.35 (m, 2H, AOACH2ACH2ACH2ANACH2ACH2(ax)ACHA),
1.41–1.53 (m, 1H, ANACH2ACH2ACHACH2ACH2A), 1.54–1.66
(m, 2H, AOACH2ACH2ACH2ANACH2ACH2(eq)ACHA), 1.85 (t, 2H,
AOACH2ACH2ACH2ANACH2(ax)A), 1.95 (p, 2H, AOACH2
ACH2ACH2ANA), 2.37–2.53 (m, 4H, AOACH2ACH2ACH2ANA
CH2(eq)A and AOACH2ACH2ACH2ANA), 2.88 (d, 2H, ANACH2APh),
3.74 (s, 3H, AOACH3), 4.01 (t, 2H, AOACH2ACH2ACH2ANA), 6.59–
6.73 (m, 3H, Ar-H), 6.77–6.88 (m, 2H, Ar-H), 7.14 (t, 1H, Ar-H), 7.44
(dt, 1H, Ar-H), 7.72 (dt, 1H, Ar-H), 7.87 (d, 1H, Ar-H), 7.94 (d, 1H,
Ar-H), 8.29 (dd, 1H, Ar-H). HPLC purity: 99.19%, HRMS (ESI)
HPLC purity: 99.26%, HRMS (ESI)
C29H32NO3 calcd 442.2382
C
29H32NO4 calcd 458.2331 [M+H]+, found 458.2317, mp: 225.8 °C.
[M+H]+, found 442.2403, mp: 159.2 °C.
4.1.57. 3-(4-(4-(3-Methoxybenzyl)piperidin-1-yl)butoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20c4)
4.1.52. 3-(3-(4-Benzylpiperazin-1-yl)propoxy)-6H-
benzo[c]chromen-6-one dihydrochloride (20b5)
Yield obtained 75.4%, 82.2%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.20–
1.38 (m, 2H, (m, 2H, ANACH2ACH2(ax)ACHACH2(ax)ACH2A),
1.39–1.54 (m, 1H, ANACH2ACH2ACHACH2ACH2A), 1.55–1.82
(m, 8H, AOACH2ACH2ACH2ACH2ANA, ANACH2ACH2(eq)ACH
ACH2(eq)ACH2A and ANACH2(ax)ACH2ACHACH2), 2.33 (t, 2H, AO
ACH2ACH2ACH2ACH2ANA), 2.45 (d, 2H, ANACH2(eq)ACH2-
ACHACH2), 2.88 (d, 2H, ANACH2APh), 3.73 (s, 3H, AOACH3),
3.98 (t, 2H, AOACH2ACH2ACH2ACH2ANA), 6.57–6.73 (m, 3H, Ar-
H), 6.75–6.88 (m, 2H, Ar-H), 7.07–7.17 (m, 1H, Ar-H), 7.45 (t,
1H, Ar-H), 7.72 (t, 1H, Ar-H), 7.82–7.99 (m, 2H, Ar-H), 8.29 (d,
1H, Ar-H). HPLC purity: 99.01%, HRMS (ESI) C30H34NO4 calcd
472.2488 [M+H]+, found 472.2490, mp: 177.8 °C.
Yield obtained 59.8%, 80.0%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.95 (p,
2H, AOACH2ACH2ACH2ANA), 2.43–2.68 (m, 10H, piperazine pro-
tons and AOACH2ACH2ACH2ANA), 3.46 (s, 2H, ANACH2APh),
4.01 (t, 2H, AOACH2ACH2ACH2ANA), 6.78 (d, 1H, Ar-H), 6.83
(dd, 1H, Ar-H), 7.14–7.29 (m, 5H, Ar-H), 7.43 (t, 1H, Ar-H), 7.71
(dt, 1H, Ar-H), 7.89 (d, 1H, Ar-H), 7.93 (d, 1H, Ar-H), 8.28 (dd, 1H,
Ar-H). HPLC purity: 99.74%, HRMS (ESI)
C27H29N2O3 calcd
429.2178 [M+H]+, found 429.2171, mp: 283.8 °C.
4.1.53. 3-(3-(4-(3-Methoxybenzyl)piperazin-1-yl)propoxy)-6H-
benzo[c]chromen-6-one dihydrochloride (20b6)
Yield obtained 76.8%, 81.6%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.88–
2.03 (m, 2H, AOACH2ACH2ACH2ANA), 2.22–2.71 (m, 10H, pipera-
zine protons and AOACH2ACH2ACH2ANA), 3.45 (s, 2H, ANACH2-
APh), 3.73 (s, 3H, AOACH3), 4.02 (t, 2H, AOACH2ACH2ACH2
ANA), 6.69–6.89 (m, 5H, Ar-H), 7.16 (d, 1H, Ar-H), 7.44 (dt, 1H,
Ar-H), 7.72 (dt, 1H, Ar-H), 7.87 (d, 1H, Ar-H), 7.94 (d, 1H, Ar-H),
8.29 (dd, 1H, Ar-H). HPLC purity: 99.52%, HRMS (ESI) C28H31N2O4
calcd 459.2284 [M+H]+, found 459.2277, mp: 265.0 °C.
4.1.58. 3-(4-(4-Benzylpiperazin-1-yl)butoxy)-6H-
benzo[c]chromen-6-one dihydrochloride (20c5)
Yield obtained 68.3%, 84.3%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d1.56–1.84
(m, 4H, AOACH2ACH2ACH2ACH2ANA), 2.24–2.67 (m, 10H, piper-
azine protons and AOACH2ACH2ACH2ACH2ANA), 3.46 (s, 2H,
ANACH2APh), 3.97 (t, 2H, AOACH2ACH2ACH2ACH2ANA), 6.78
(d, 1H, Ar-H), 6.83 (dd, 1H, Ar-H), 7.14–7.28 (m, 5H, Ar-H), 7.44
(dt, 1H, Ar-H), 7.72 (dt, 1H, Ar-H), 7.87 (d, 1H, Ar-H), 7.94 (d, 1H,
Ar-H), 8.29 (dd, 1H, Ar-H). HPLC purity: 99.77%, HRMS (ESI)
4.1.54. 3-(4-(Benzyl(methyl)amino)butoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20c1)
C
28H31N2O3 calcd 443.2335 [M+H]+, found 443.2350, mp: 163.6 °C.
Yield obtained 78.3%, 85.6%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.66 (p,
2H, AOACH2ACH2ACH2ACH2ANA), 1.80 (p, 2H, AOACH2
ACH2ACH2ACH2ANA), 2.16 (s, 3H, ANACH3), 2.39 (t, 2H, AOACH2-
ACH2ACH2ACH2ANA), 3.45 (s, 2H, ANACH2APh), 3.94 (t,
2H, AOACH2ACH2ACH2ACH2ANA), 6.76 (d, 1H, Ar-H), 6.82 (dd,
1H, Ar-H), 7.13–7.29 (m, 5H, Ar-H), 7.43 (dt, 1H, Ar-H), 7.71 (dt,
1H, Ar-H), 7.86 (d, 1H, Ar-H), 7.93 (d, 1H, Ar-H), 8.29 (dd, 1H, Ar-
H). HPLC purity: 99.92%, HRMS (ESI) C25H26NO3 calcd 388.1913
[M+H]+, found 388.1911, mp: 169.2 °C.
4.1.59. 3-(4-(4-(3-Methoxybenzyl)piperazin-1-yl)butoxy)-6H-
benzo[c]chromen-6-one dihydrochloride (20c6)
Yield obtained 75.2%, 78.4%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.63
(p, 2H, AOACH2ACH2ACH2ACH2ANA), 1.77 (p, 2H, AOACH2
ACH2ACH2ACH2ANA), 2.28–2.65 (m, 10H, piperazine protons
and AOACH2ACH2ACH2ACH2ANA), 3.43 (s, 2H, ANACH2APh),
3.74 (s, 3H, AOACH3), 3.97 (t, 2H, AOACH2ACH2ACH2ACH2ANA),
6.69–6.87 (m, 5H, Ar-H), 7.15 (t, 1H, Ar-H), 7.43 (dt, 1H, Ar-H), 7.72
(dt, 1H, Ar-H), 7.86 (d, 1H, Ar-H), 7.93 (d, 1H, Ar-H), 8.29 (dd, 1H,
4.1.55. 3-(4-((3-Methoxybenzyl)(methyl)amino)butoxy)-6H-
benzo[c]chromen-6-one hydrochloride (20c2)
Ar-H). HPLC purity: 98.48%, HRMS (ESI)
C29H33N2O4 calcd
473.2440 [M+H]+, found 473.2448, mp: 251.2 °C.
Yield obtained 80.3%, 86.1%, respectively, for the reaction and
HCl salt preparation. 1H NMR (300 MHz, CDCl3, ppm): d 1.64 (p,
2H, AOACH2ACH2ACH2ACH2ANA), 1.80 (p, 2H, AOACH2ACH2-
ACH2ACH2ANA), 2.16 (s, 3H, ANACH3), 2.38 (t, 2H, AOACH2-
ACH2ACH2ACH2ANAANA), 3.42 (s, 2H, ANACH2APh), 3.73 (s,
3H, AOACH3), 3.94 (t, 2H, AOACH2ACH2ACH2ACH2ANA), 6.67–
6.87 (m, 5H, Ar-H), 7.15 (t, 1H, Ar-H), 7.42 (dt, 1H, Ar-H), 7.71
(dt, 1H, Ar-H), 7.85 (d, 1H, Ar-H), 7.92 (d, 1H, Ar-H), 8.28 (dd, 1H,
4.2. Enzyme assays: inhibition of AChE and BuChE
AChE inhibitory activities of the test compounds were deter-
mined by the modified Ellmann’s method.28,29 Briefly, each reac-
tion mixture (final volume of 200
of Tris HCl buffer (pH 8.0), 10 L of 6.8 mM DTNB solution (i.e.,
0.34 mM final) containing 20 mM MgCl2 and 100 mM NaCl, 10
of ACHE solution (0.4 U/mL from Human recombinant AChE), and
L of each sample solution. The reactions were initiated by the
lL) contained 168 lL of 50 mM
l
lL
Ar-H). HPLC purity: 99.89%, HRMS (ESI)
C26H28NO4 calcd
418.2018 [M+H]+, found 418.2024, mp: 209.5 °C.
2
l

5154
H.O. Gulcan et al. / Bioorg. Med. Chem. 22 (2014) 5141–5154
addition of 10
l
L of 10 mM acetylthiocholine iodide (i.e., 0.5 mM
scopolamine administered group was taken as 100% and the effect
of each compound of the present invention was calculated accord-
ingly in terms of the percentage antagonism.
final). After incubation for 15 min at 27 °C, the enzyme catalyzed
formation of the yellow color was measured at 412 nm using Bio-
tech Synergy 2 Spectrophotometer. The non-enzymatic hydrolysis
of acetylthiocholine iodide was also measured in enzyme-free
assay systems, and the results were employed as blank (i.e., also
referred to as blank in the calculation below). In control experi-
ments, inhibitor-free assay systems were utilized to measure the
full activity (i.e., also referred to as the control in the calculation
below). In positive control experiments, rivastigmine, galantha-
mine, and donepezil were employed as the reference inhibitors.
Each experiment was performed in triplicate, and the mean
standard deviation was calculated. Absorbance values for each
measurement were utilized in the percentage inhibition calculated
through [(Acontrol À Ablank) À (Asample À Ablank)/(Acontrol À
Ablank)] Â 100. The concentration of test compounds and refer-
ence materials which determined 50% inhibition of the AChE or
BuChE activity (IC50) was calculated using a sigmoidal hill slope
model. BuChE inhibitory activities of the test compounds and the
reference drugs were measured following the same methodology
described for the AChE assays except employing 1.6 U/mL BuChE
from human recombinant BuChE solution as the enzyme source
and the butyrylthiocholine iodide (i.e., 1.5 mM final) as the sub-
Acknowledgments
The support provided by Scientific and Technological Research
Council of Turkey (i.e., Project No: 3100373) is gratefully acknowl-
edged. The authors also would like to thank Dr. Manfred Windisch,
the president of JSW Life Sciences (i.e., a pre-clinical and clinical
drug development organization, a member of QPS Clinical Research
in Austria), for his guidance in in vivo studies. The authors have
declared no conflict of interest.
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