Phenylimino-2H-chromen-3-carboxamide derivatives as novel small molecule inhibitors of β-secretase (BACE1)

Article abstract of DOI:10.1016/j.bmc.2013.01.064

The inhibition of β secretase (BACE1) is potentially important approach to treatment of Alzheimer disease (AD). A novel series of 4-bromophenyl piperazine derivatives coupled to the phenylimino-2H-chromen-3-carboxamide scaffold were investigated as BACE1 inhibitors in this study. Docking study suggested that the phenyl-imino group of the scaffold establishes favorable π-π stacking interaction with side chain of Phe108 of flap pocket. Some of the docking proposed derivatives were synthesized and evaluated for BACE1 inhibitory activity using a FRET-based assay. High BACE1 inhibitory activities were observed from derivatives containing fused heteroaromtic groups attached through the aliphatic linkage to the N4-piperazine moiety, which may be attributed to the engagement of effective interactions with S1-S′1 sub-pocket residues. Of the most potent compounds, 9e displayed an IC ₅₀ value for BACE1 of 98 nM. Some of these derivatives demonstrated good inhibitory activity on Aβ production in N2a-APPswe cells at 5 and 10 μM. These compounds might be considered as promising BACE1 inhibitory agents that could lower Aβ production in AD.

Full text of DOI:10.1016/j.bmc.2013.01.064

Bioorganic & Medicinal Chemistry 21 (2013) 2396–2412
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Phenylimino-2H-chromen-3-carboxamide derivatives as novel small
molecule inhibitors of b-secretase (BACE1)
Najmeh Edraki a,b, Omidreza Firuzi b, Alireza Foroumadi a,c, Ramin Miri b, Armin Madadkar-Sobhani d,
Mehdi Khoshneviszadeh a,b, Abbas Shafiee a,
⇑
a Department of Medicinal Chemistry, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
b Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
c Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran
d Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibition of b secretase (BACE1) is potentially important approach to treatment of Alzheimer disease
(AD). A novel series of 4-bromophenyl piperazine derivatives coupled to the phenylimino-2H-chromen-
3-carboxamide scaffold were investigated as BACE1 inhibitors in this study. Docking study suggested that
Received 17 November 2012
Revised 19 January 2013
Accepted 22 January 2013
Available online 8 February 2013
the phenyl-imino group of the scaffold establishes favorable p–p stacking interaction with side chain of
Phe108 of flap pocket. Some of the docking proposed derivatives were synthesized and evaluated for
BACE1 inhibitory activity using a FRET-based assay. High BACE1 inhibitory activities were observed from
derivatives containing fused heteroaromtic groups attached through the aliphatic linkage to the N4-
piperazine moiety, which may be attributed to the engagement of effective interactions with S1–S01
sub-pocket residues. Of the most potent compounds, 9e displayed an IC50 value for BACE1 of 98 nM. Some
of these derivatives demonstrated good inhibitory activity on Ab production in N2a-APPswe cells at 5 and
Keywords:
Alzheimer disease
BACE1 inhibitor
Phenylimino-2H-chromen-3-carboxamide
10
lM. These compounds might be considered as promising BACE1 inhibitory agents that could lower Ab
production in AD.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
mice have little or no endogenous Ab in their brains, demonstrat-
ing that there are no redundant molecular mechanisms for b-secre-
tase cleavage in mice. Interestingly, these mice were in good health
and did not show any side effects of BACE1 deficiency.8,9
Alzheimer’s disease (AD) characterized by progressive loss of
brain function and known as the main cause of irreversible demen-
tia in the elderly, is reported as one of the most costly and life
threatening disease worldwide.1–3 Extracellular deposition of amy-
loid-b peptide (Ab) in the brain is one of the major histopatholo-
gical hallmarks of AD.4 Ab is a short peptide (the most prevalent
forms consisting of 40 or 42 amino acids) generated by sequential
cleavage of transmembrane protein amyloid precursor protein
Development of BACE1 inhibitors was initiated from the sub-
strate-based design of transition-state compounds based on
replacing the cleavable amide bond of APP with a stable mimetic
of the putative transition-state structure.10,11 Many peptidomimet-
ic inhibitors such as hydroxyethylen and statine-based agents with
in vitro subnanomolar potencies have been designed. However, the
peptidic inhibitors coming out of this approach suffer from poor
pharmacological properties, which are attributed to their unfavor-
able physicochemical characteristics, and also poor brain penetra-
tion.12–16 Therefore, development of potent non-peptidic
small-molecule inhibitors of BACE1 has been the focus of many
pharmaceutical and academic researchers in the past decades.11,17
In late 2002, Vertex Pharmaceuticals Inc., disclosed several classes
of heterocyclic BACE1 inhibitors. Among these diverse proposed
scaffolds, halogen-substituted biaryl naphthalenes 1 were identi-
fied as one of the most potent reported classes.18 Garino et al.
published enzymatic and cell-based assay data on naphthyl and
coumarin derivatives of biarylpiperazines amides.19 Investigation
of different substitutes on the piperazine ring revealed that
employment of specific substitutes on the N4-position of this ring
(APP) by enzymes b-secretase (BACE1) and c
-secretase.5,6 Inhibi-
tors of these two proteases provide attractive candidates as poten-
tially disease-modifying, rather than palliative, treatments for AD.3
c-Secretase is involved in important physiological functions;
therefore its inhibitors have encountered many mechanism-based
toxicity drawbacks due to a deficiency in selectivity.3,7
BACE1 (b-site APP cleaving enzyme) or memapsin 2 (membrane
aspartic protease of the pepsin family) is an attractive drug target
for AD treatment, since it is well established that this enzyme
catalyses the first and rate limiting step of amyloidogenic path-
way.6 Furthermore, it has been demonstrated that BACE1 knockout
⇑
Corresponding author. Tel.: +98 2166406757; fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.064

N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
2397
significantly affects the potency, while, replacement of biphenyl
piperazine with the 4-bromophenyl piperazine derivatives re-
sulted in enhanced cellular potency of compounds.19 Furthermore,
replacement of naphtyl ring of 1 with other heterocyclic substi-
tutes resulted in enhanced BACE1 inhibitory activity of the
phenyl-piperazine derivatives in some cases (Fig. 1).20
imentally derived structure of this inhibitor bound to BACE1. The
best-docked and actual conformation of inhibitor correlate quite
well with an RMSD of 0.86 Å and binding free energies of
ꢀ11.45 kcal/mol (RMSD <2 Å is the commonly acceptable reported
limit).27
Although Vertex reported the first 3D pharmacophore map of
BACE1 inhibitors,18 no crystallographic data is available to clarify
the exact binding interaction of aryl piperazine derivatives with
the BACE1 enzyme.
2.1.2. AutoDock binding affinities and binding pose of naphtyl
and coumarin derivatives of arylpiperazines amides
The binding affinity was evaluated by the binding free energies
(DGb), inhibition constant (Ki) and hydrogen bonding. All of these
Based on the above findings and in an attempt to understand
the binding mode and interaction of arylpiperazine amide series
with catalytic site of BACE1 and in continuation of our study on de-
sign of novel small-molecule scaffolds against AD,21,22 herein, we
carried out an in silico molecular docking study to design a new
series of (4-bromophenyl)-piperazine derivatives coupled to phe-
nylimino-2H-chromen-3-carboxamide moiety as BACE1 inhibitors.
Selected and synthesized compounds were tested with a FRET
based enzyme assay to determine their BACE1 inhibitory activity.
The most potent compounds were further investigated in vitro
for their inhibitory effects on Ab production in neuroblastoma 2a
cells stably expressing human Swedish mutant APP (N2a-APPswe
cells).
data are summarized in Table 1.
BACE1 is a membrane bound aspartyl protease with a pair of ac-
tive-site aspartyl residues (Asp32 and Asp228) located in sub-
strate-binding cleft between N- and C-terminal lobes.12,28 BACE 1
functions optimally at acidic pH and is believed to be located in
acidic intracellular compartments (pH 5) and at such pH amino
and amidino groups are protonated.29,30 To this end, we assumed
that N4 atom of piperazine is protonated in such pH to form hydro-
gen bond interaction with catalytic aspartates. The binding pose of
top ranked conformers of compounds A5 and A3 are depicted in
Figure 2.
According to the reported pharmacophore by Vertex,18 for the
maximum inhibitory potency of small molecule BACE1 inhibitors,
the following interactions occur between BACE1 catalytic site res-
idues and inhibitor ligand:
2. Results and discussion
– HB-1: Hydrogen bond 1 between catalytic aspartates (Asp32 and
Asp228) and inhibitor ligand that is necessary for primary inhi-
bition of enzyme.
– HPB-1: Hydrophobic interaction 1 between inhibitor and S2
pocket of active site composed of Thr232, Asn233, Arg235 and
Gln73.
– HPB2: This additional hydrophobic interaction occurs due to
hydrophobic interaction between ligand and flap loop residues
including Trp76, Phe108, Phe109, Trp115 and Ile102.
– HPB-3: Hydrophobic interaction 3 arises between S02 site of
BACE1 active site and inhibitor. This pocket mainly covered by
amino acids such as Asn37, Ala39, Val69, Trp76, Ile118 and
Arg128.
– HPB-4: This interaction mainly occurred through the stacking
interaction of inhibitor with Tyr71, Phe 108 or Trp 76 of active
site.
2.1. Molecular docking study
Molecular docking is an effective means in structure-based ra-
tional design of potent biological scaffolds and accurate prediction
of the nature of interactions and binding energies of protein–ligand
complex in molecular design.23 Toward the optimization of afore-
mentioned BACE1 inhibitors, we first investigated the binding free
energy and binding map of some of the most potent naphtyl and
coumarin derivatives of arylpiperazine amide series A1–A519
(Table 1) into the crystal structure of BACE1 using docking program
Autodock 4.2.24 Since no crystallographic data is available for bind-
ing interactions of arylpyperazine amide derivatives, we defined
the active site of BACE1 enzyme, based on the center and radius
of non-peptidic binding substrate ((4S)-4-(2-amino-6-phenoxyqui-
nazolin-3(4H)-yl)-N,4-dicyclohexyl-N-methylbutanamide) (entry
1, Table 1) in an X-ray crystal structure complexed with BACE1
(PDB ID: 2Q15) with the resolution of 2.40 Å.25
– The additional hydrogen bond interactions with Gly34 (HB-2)
and/or Gly230 (HB-3) favor the ligand-enzyme binding. How-
ever, these two last ones are not necessary for the primary inter-
action of ligand with receptor.
2.1.1. Validation of the accuracy and performance of docking
Docking optimization and validation was performed according to
the cited method based on the RMSD(root mean square deviation) of
the best-docked conformation of cognate ligand from experimental
one (internal validation).26 Accordingly, docking performance
examined by re-docking the co-crystallized conformation of a native
ligand into the BACE1 active site. The top-docking score pose of cog-
nate ligand was superimposed over the X-ray coordinates of exper-
According to the above pharmacophore and according to the
docking results (Fig. 2 and Table 1), the binding interaction of aryl-
pyperazine amide derivatives into BACE1 active site could be sim-
ply explained as follows:
– Aryl group (naphtyl or coumarin) mainly directed toward the flap
and S02 pocket of enzyme and involves in hydrophobic interaction
with the corresponding amino acids. In addition, this part of
Figure 1. Chemical structure of biaryl naphthalenes 1.

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N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
Table 1
The docking results of naphtyl and coumarin derivatives of arylpyperazine amidesa A1–A5 and cognate ligand 1, docked into BACE1 enzyme (PDB ID: 2Q15)
b
Compound
D
Gb (kcal/mol)
Kic (nM)
Hydrogen bonds between atoms of compounds and amino acids
Atom of the ligand
Amino acid
Distance (Å)
ꢀ11.45
ꢀ10.53
4.03
(Quinazolin)NH
(Amine)NH
(Amine)NH
C@O of Asp32
C@O of Asp32
C@O of Asp228
2.51
1.88
2.32, 1.83
O
N
N
O
N
NH2
Br
1
19.13
(Piperazine)N4
(Amide)NH
(Coumarine)O
C@O of Asp228
C@O of Asp32
N–H of Trp76
1.82
2.11
1.85
O
N
H
N
N
O
O
A1
Br
ꢀ9.21
77.79
(Piperazine)N4
OH
C@O of Asp32
C@O of Asp228
1.86
1.94
O
O
(Amide)C=O
OH of Tyr 198
2.94
N
H
N
N
O
A2
OH
ꢀ11.13
6.97
(Piperazine)N4
(Coumarine)O
C@O of Asp228
N–H of Trp76
1.69
3.12
O
N
H
N
N
O
O
A3
ꢀ11.08
7.57
(Piperazine)N4
(Amide)NH
C@O of Asp32
C@O of Gly34
1.84
2.36
O
N
H
N
N
OH
A4
Br
ꢀ10.48
20.59
(Piperazine)N4
OH
C@O of Asp32
C@O of Thr231
2.29
1.79
O
OH
C@O of Asp32
3.10
N
H
N
N
A5
OH
a
b
c
The N4 piperazine group adopted as protonated state.
Binding free energy.
Inhibition constant.
inhibitor is oriented toward stacking pocket covered by
Tyr71 and Phe108. Moreover, in coumarin series, aromatic part
is involved in hydrogen bond interaction with Trp76 of flap
pocket.
– Amide bond could be involved in the hydrogen bond interaction
with residues such as Asp228 or Gly34.
– The substituted group on piperazine ring (X) is oriented toward
the S2 pocket of the enzyme. Therefore, introduction of suitable
groups favors hydrophobic interaction (HPB-1) with the amino
acid residues of this pocket. In addition, this part of inhibitor
engages in additional hydrogen bond network with Gly230 or
Thr231 of the enzyme.
– Piperazine ring is mainly responsible for key hydrogen bond
interaction with Asp32 or Asp228.
– The R group on phenyl ring is involved in hydrophobic interac-
tion with various parts of active site that differs between cases.

N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
2399
employment of different fused heteroaromatic moieties compris-
ing various hydrogen bond donor and acceptor groups in different
positions of the ring. We then further analyzed substitution of phe-
nyl moiety as simple aromatic group in order to establish supple-
mentary stacking and hydrophobic interactions with surrounding
residues of S02, flap or stacking site (the benzyl moiety adopted
as piperazine-attached group (X) in all cases). The binding free
energies (DGb), inhibition constant (Ki) and hydrogen bonding
interactions of some of these derivatives with BACE1 active site
are reported in Table 2. As illustrated, 2H-chromen-2-imine (2-imi-
nochromene) 6a and especially its phenylated derivative 7a (phe-
nylimino-2H-chromen derivative), demonstrated promising
binding interaction with the catalytic site. The corresponding bind-
ing free energy of compound 7a is ꢀ12.05 kcal/mol. Therefore, it
seems that introduction of phenyl group into 2-position of hetero-
aromatic-3-carboxamides is more favorable than the other posi-
tions. As depicted in Figure 3, the phenyl moiety of
phenyliminochromene derivative is well oriented toward the
stacking and S02 site that favors the probable stacking interaction.
2.1.4. Optimization of piperazine-pendant moiety (X)
As demonstrated in Figure 3, the benzyl substitute attached to
piperazine ring is mainly oriented toward the S2 hydrophobic
pocket of active site and is adjacent to Gly230 and Asp228 resi-
dues. This feature is more outstanding in phenylimino-2H-chro-
men derivative 7a. Therefore; introduction of suitable
hydrophobic and especially extended aromatic moieties containing
the appropriate hydrogen bond establishing groups to the pipera-
zine ring, that facilitates both hydrophobic and hydrogen-bond
interaction with the surrounded amino acids, would favor the
inhibitor–enzyme binding interaction. We performed docking
study in order to examine this assumption. Accordingly, the N4-
piperazine attached benzyl substitute replaced with different
hydrophobic moieties bearing heteroatoms with hydrogen-bond
interacting potential. The docking results together with relevant
chemical structures are illustrated in Table 3.
The docking results confirmed that attachment of heteroatom
comprising moieties into piperazine ring resulted in pivotal H bond
interaction with the Gly34, Gly230, Thr231 or Thr232. Moreover
the effective hydrophobic interaction of hydrophobic pendants
with the S2 pocket, affected the enzyme inhibitory potential that
is more predominant in fused heteroaromatic moieties than ali-
phatic ones. The binding pose of phenyliminochromene carboxam-
ide derivatives containing ethyl-2H-indole pendant attached to the
N4-piperazine ring 9h is depicted in Figure 4. The following inter-
actions were observed for the best-ranked pose: (i) The NH of
piperazine ring interact via H-bond with the side chain of catalytic
Asp32. (ii) The NH of indole ring establishes two hydrogen bonds
with CO side chain of Asp228 and CO amide linkage of Gly230.
(iii) The phenyl-imino group mounted on C2 atom of 2H-chromen
Figure 2. Binding orientation of compound A5 (A) and A3 (B) (ball and stick) within
BACE1 active site.
moiety, establishes favorable
chain of Phe108 of flap pocket. (iv) The choromene ring might
interact via or cation– interaction with Tyr71.
p–p stacking interaction with side
Based on these findings, primary optimization of phenylpiper-
azine amide scaffold is possible with respect to three different
parts including aromatic ring (Ar), substituted group on the phenyl
ring (R) and piperazine-pendant group (X). Since 4-bromophenyl
piperazine derivatives have demonstrated better cellular potency
than biphenylpiperazines,19 we performed optimizations on the
4-bromophenyl piperazine derivatives (R = Br).
p
–p
p
2.2. Chemistry
The target compounds 7a–7c and 9a–n were prepared accord-
ing to sequential procedures depicted in Schemes 1 and 2. The
compound 1a–b was synthesized by condensation of commercially
available piperazine derivatives (N-benzyl or N-tert-butoxycar-
bonyl piperazine) with 2,5-dibromonitro-benzene according to
literature procedure.19 Reduction of nitro group of compound
1a–b was performed using aqueous solution of sodium carbonate
and sodium dithionate at 70 °C in very good yield (95–97%).
Although we assessed different reductive procedures such as use
of Zn powder in K2HPO4 in refluxed THF for reduction of nitro
2.1.3. Optimization of the heteroaromatic ring
As we mentioned earlier, ‘Ar’ part of the scaffold is oriented to-
ward the S02 and flap pocket and surrounded by residues such as
Phe108 and Tyr76 involved in stacking interaction with suitable li-
gand. Apart from this, introduction of heteroatom into this ring
might generate additional hydrogen bonding with amino acid
residues such as Trp76. As part of our study, we first investigated

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N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
Table 2
Investigation of different heteraromatic moieties employment into 4-bromophenyl-benzylpiperazine derivativesa
c
Het. Ar
D
Gb (kcal/mol)
Ki (nM)
Hydrogen bonds between atoms of compounds and amino acids
Atom of the ligand
Amino acid
Distance (Å)
ꢀ10.71
ꢀ10.42
ꢀ10.16
14.04
22.9
(2-Oxo-Quinoline)NH
(Piperazine)N4
ILeu 126
Asp32
2.21
3.20
B
C
N
H
O
(Amino-quinoline)NH2
(Piperazine)N4
Tyr 198
Asp228
3.01
2.33
N
NH2
OH
35.93
(2-Oxo-Quinoline)NH
(Piperazine)N4
ILeu126
Asp32
3.02
3.1
D
E
F
N
H
O
ꢀ10.57
ꢀ11.03
17.72
8.21
(Piperazine)N4
Asp228
2.73
HO
N
O
H
(Iminochromene)NH
(Piperazine)N4
Trp76
Asp32
2.57
3.22
O
O
O
NH
NH
ꢀ11.26
5.57
(Benzyloxy)O
(Piperazine)N4
Trp76
Asp228
3.13
3.22
G
O
ꢀ11.11
ꢀ12.05
7.14
1.46
(Iminochromene)NH
(Piperazine)N4
Lys107
Asp32
1.86
2.24, 2.46
6a
O
O
NH
(Iminochromene)NH
(Piperazine)N4
Trp76
Asp228
3.37
1.81
N
7a
Results of docking study into BACE1 enzyme.
a
The N4 piperazine atom adopted as protonated state.
binding free energy
Inhibition constant.
b
c
group,31 the latter method led to low yield (50–55%) of desirable
compound after chromatographic purification. As mentioned ear-
lier, the use of sodium carbonate and sodium dithionate was more
favorable; since sodium hydrosulfate is quite effective in cleanly
reducing the nitro functionality.32 Moreover; under this condition,
the reductive reaction is complete and the pure amine products
2a–b was obtained after the work up procedure without further
purification.
The 2-cyanoacetyl-3,5-dimethyl pyrazole intermediate 4 was
synthesized following the known strategies (Scheme 1).33,34 Ried
and Schliemer provided this intermediate as a cheap cyanoacetyla-
tion reagent first synthesized in the late 1950th.33 in this regard, the
prepared 2-cyanoacetyl-3,5-dimethyl pyrazole 4 was further used
for cyanoacetylation of synthesized amine derivatives 2a–b in
refluxing toluene to synthesize the corresponding N-(2-(4-
benzylpiperazin-1-yl)-5-bromophenyl)-2-cyanoacetamide 5a and
N-(2-(4-tert-butoxycarbonyl-piperazin-1-yl)-5-bromophenyl)-
2-cyanoacetamide 5b in very good yield (75–80%). The resulted key
intermediates, methylene active nitriles of type 5a–b, were reacted
with the salicylaldehyde derivatives in presence of catalytic amount
of piperidine in methanol at room temperature (Knoevenagel con-
densation)35,36 to afford 2-iminochromene-3-carboxamide deriva-
tives 6a–c.
The 2-iminochromene-3-carboxamide derivatives 6a–c were
classified into two groups according to the substituted moiety on
the piperazine ring: benzyl piperazine (6a and 6c) and N-Boc-
piperazine (6b) derivatives. In our approach to synthesis of 2-phe-
nylimino-2H-chromen-3-carboxamide derivatives, we used two
different routes in benzyl piperazine (6a and 6c) and N-Boc-piper-
azine (6b) derivatives of 2-iminochromene-3-carboxamide. In gen-
eral, the synthesis of 2-(N-arylimino)chromenes is based on
aminolysis of cyclic imido esters of 2-iminochromen involving
nucleophilic attack on a C@N carbon and subsequent decomposi-
tion of the tetrahedral intermediate and is similar to the reaction
of simple imidates with amines.36,37 According to our literature re-
view, we found that a method based on using glacial acetic acid is
more common for preparation of 2-(N-arylimino)-2H-chromen
derivatives.38 The glacial acetic acid is an ideal solvent that

N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
2401
potential. Comparison of compounds 6a and 7a demonstrated that
the introduction of phenyl moiety into the imino group of benzyl
piperazine derivative of imino-2H-chromene-3-carboxamide scaf-
fold 6a (IC50 = 1.216
of compound in phenylimino-2H-chromen-3-carboxamide deriva-
tive 7a (IC50 = 0.503 M). Similar results were also obtained for the
lM), resulted in enhanced inhibitory potential
l
tert-butoxy carbonyl piperazine derivative of imino-2H-chromene-
3-carboxamide type; compound 6b was almost inactive against
BACE1 enzyme (IC50 >10 lM), while replacement of the hydrogen
atom of imino moiety (of imino-2H-chromene ring) with phenyl
group, ameliorated the inhibitory potential of the phenylimino-
chromene compound 7b (IC50 = 9.61 lM). This finding is in accor-
dance with the results of the docking study. It could be deduced
that the introduced phenyl moiety might be engaged in the net-
work of hydrophobic and
p–p stacking interaction with the
Tyr71 or Phe108 of active site (S1–S01) that favors enzyme–ligand
interaction.
Figure 3. The comparative binding affinities of benzylpiperazine derivative imino-
2H-chromene-3-carboxamide 6a; ball and stick colored by element
Another important structural feature of phenyliminochromene
carboxamide based 4-bromophenyl piperazine derivatives that
should be considered in the design of potent BACE1 inhibitors of
this type is the substituted moiety on the N4-piperazine ring. As
the results of Table 4 indicate, the most potent compounds of this
series bear an extended heterocyclic moiety on the N4-piperazine
group. The 2-phthalimidoethyl piperazine derivative of phenylimi-
nochromene carboxamide type 9e with an IC50 value of 98 nM is
the most potent synthesized inhibitor of this type. This significant
inhibitory potential may be due to an additional hydrophobic
interaction with hydrophobic residue of the S2 sub-pocket of active
site and the network of hydrogen bonding interactions established
between the heteroatoms of phthalimide moiety and correspond-
ing residue of the active site. Elongation of methylene linker be-
tween the phthalimide group and piperazine ring partially
(
D
Gb = ꢀ11.11 kcal/mol), and its Phenylimino-2H-chromen-3-carboxamide ana-
logue 7a, yellow stick (
Asp are colored in red and Gly34 and 230 are colored in green (CPK representation).
D
Gb = ꢀ12.05 kcal/mol), into BACE1 active site. (Catalytic
increases the solubility of reagents and functions as protonating
agent. Moreover, it plays the important role as reactant, which
binds to the ammonia produced. According to this method the ben-
zyl piperazine derivatives of 2-iminochromene-3-carboxamides
(6a and 6c) and aniline were reacted in refluxed glacial acetic acid
to afford the corresponding 2-N-(2-(4-benzylpiperazin-1-yl)-5-
bromophenyl)-2-(phenylimino)-2H-chromene-3-carboxamide
derivatives (7a and 7c) in good yield (>70%).
In order to synthesize the N-Boc-piperazine derivatives of 2-
phenylimino-2H-chromen-3-carboxamide we employed another
acid catalyzed procedure using hydrochloride salt of aniline.39
The principle feature of this method is reaction of equimolar
amount of N-tert-butoxycarbonyl-piperazine derivative 6b and
aniline hydrochloride in boiling-dry methanol to afford the
2-N-(2-(4-tert-butoxycarbonylpiperazin-1-yl)-5-bromophenyl)-
2-(phenylimino)-2H-chromene-3-carboxamide 7b in a high yield
(90%). The N-Boc protected derivative 7b was then treated with tri-
fluoroacetic acid in dry CH2Cl2 at 0 °C in order to isolate the corre-
sponding free amino derivative 8 after work up procedure with sat.
K2CO3. The prepared key intermediate 8 was further functionalized
with various alkyl or aroyl halide derivatives in refluxing-dry tolu-
ene to give different 2-phenylimino-2H-chromen-3-carboxamide
derivatives 9a–n. The general structure of target compounds are
demonstrated in Table 4.
reduced the inhibitory potential of compound 9f (IC50 = 0.326
lM)
compared to 9g (IC50 = 0.854 M), which might be due to hin-
l
drance of the effective hydrogen bonding interactions at this site.
The binding interactions of these derivatives with the catalytic site
of BACE1 are reported in Table 3. Introduction of 1H-indol ring
through the ethyl linker into N4-piperazine of the scaffold is also
a favorable modification; compound 9h is a promising BACE1
inhibitor of this type (IC50 = 0.928 lM). The binding interaction of
this compound into BACE1 active site is depicted in Figure 4.
As it could be clearly understood from Table 4, attachment of
aryl moieties through a carbonyl linker into N4-piperazine ring sig-
nificantly reduces the BACE1 inhibitory activity of compounds in
most cases; For example, compound 9d bearing the p-methoxy
benzyl substitute attached to the N4-piperazine demonstrated
strong inhibitory activity (IC50 = 0.578
benzoyl derivative 9j is almost inactive against the BACE1 enzyme
(IC50 >10 M). This observation might be due to the reduced pro-
lM), while the p-methoxy
2.3. Biological data and structure–activity relationship
l
tonation capability of N4-piperazine, which is important for the
most important hydrogen bonding interactions with catalytic
aspartates and is necessary for the primary binding of ligand with
catalytic site of enzyme.
2.3.1. BACE1 enzyme inhibitory activity
The focus of this study was to optimize 4-bromophenyl pipera-
zine derivatives with the aim of discovery of more potent BACE1
inhibitors and finding the most important structural prerequisites
for the optimal activity of this scaffold. To this end and according to
the docking results, the BACE1 inhibitory activity of synthesized
phenylimino-2H-chromen-3-carboxamide derivatives was evalu-
ated using a fluorescence resonance energy transfer (FRET) based
BACE1 kit from Invitrogen (former Pan-Vera). This kit uses the
purified baculovirus expressed BACE1 enzyme and specific APP-
based peptide substrate (Rh-EVNLDAEFK-quencher). Different con-
centrations of each compound were tested and IC50 values were
calculated. Experiments were repeated three to four times for each
derivative. The results are summarized in Table 4.
Another important structural feature understood from the re-
sults of Tables 3 and 4, is the importance of key hydrogen binding
interaction between the studied ligand and catalytic aspartates;
According to the obtained binding model (Table 3) for compound
9b (bearing ‘cyanopropyl’ moiety attached to the piperazine ring),
the N4-piperazine atom of this compound was not involved in the
key hydrogen binding interaction with Asp32 or Asp228. In fact,
the mentioned key interaction was not seen between 9b and the
catalytic aspartates. As it is described earlier, the hydrogen binding
interaction of ligand with the catalytic aspartates (HB-1), is the
essential step in inhibiting the BACE1 catalytic activity. Therefore,
According to the obtained data, some of the phenyliminochrom-
ene derivatives demonstrated significant BACE1 inhibitory
the low inhibitory potential of compound 9b (IC50 >10
lM) might
be attributed to its inability for inhibiting the catalytic aspartates.

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N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
Table 3
Investigation of different piperazine–pendant moieties attachments (X) into Phenylimino-2H-chromen carboxamide derivativesa
b
Compound
X
D
Gb (kcal/mol)
Kic (nM)
Hydrogen bonds between atoms of compounds and amino acids
Atom of the ligand
(Piperazine)N4
Amino acid
Distance (Å)
8
H
ꢀ12.04
1.49
Asp32
Asp228
2.46, 1.87
2.03, 1.97
CH2CH2OH
(CH2)3CN
(Piperazine)N4
OH
(Piperazine)N4
CN
Asp228
Thr232
Gly230
Gly230
Trp76
1.62
2.09
1.75
3.20
3.15
9a
9b
ꢀ11.18
ꢀ11.31
6.37
5.11
(Chromene)O
(Piperazine)N4
CN
Asp228
Arg128
1.62
3.13
9c
ꢀ12.28
ꢀ12.25
0.99
1.05
CN
(Piperazine)N4
(Methoxy)O
(Amide)CO
Asp228
Gly34
Phe108
1.87, 2.45
3.02
3.11
9d
OCH3
O
(Piperazine)N4
(Phthalimide)CO
(Phthalimide)CO
(Amide)CO
Asp228, Thr231
Arg235, Thr231
Gly230
2.19, 2.25
3.31, 1.20
3.04
N
9e
9f
(CH2)2
(CH2)3
(CH2)4
ꢀ13.6
0.108
0.259
Ser35
2.79
O
O
(Piperazine)N4
(Phthalimide)CO
Asp32
Gly34
2.3, 1.92
2.75
N
ꢀ13.08
O
O
N
O
9g
9h
9i
ꢀ12.9
0.348
0.086
1.97
(Piperazine)N4
Asp32
1.74, 3.77
H
N
(Piperazine)N4
(Indole)NH
Asp32
Asp228, Gly230
2.24, 2.28
2.22, 2.30
ꢀ13.73
ꢀ11.77
O
—
—
—
Cl
O
9j
ꢀ11.55
ꢀ11.91
3.40
1.86
OMe
Gly 34
3.13
OCH3
O
(Piperazine)N4
C@O
Thr231
Gly230
1.70
2.70
OMe
9k
O
(NO2)O
C@O
(Amide)C@O
Arg235
Tyr71
Asp32
2.73
3.02
2.21
9l
ꢀ12.54
ꢀ11.61
0.63
3.08
NO2
O
(Piperazine)N4
C@O
Asp228, Asp32
Gly34
1.70, 3.18
3.18
9m
O
C@O
Asp228
2.89

N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
2403
Table 3 (continued)
b
Compound
X
D
Gb (kcal/mol)
Kic (nM)
Hydrogen bonds between atoms of compounds and amino acids
Atom of the ligand
Amino acid
Distance (Å)
O
(Piperazine)N4
C@O
Asp228
Gly34
1.95
2.42
9n
ꢀ11.30
5.22
S
Results of docking study into BACE1 enzyme.
a
The N4 piperazine atom adopted as protonated state.
Binding free energy.
Inhibition constant.
b
c
Figure 4. The binding mode of compound 9h at the catalytic domain of BACE-1 (PDB ID: 2Q15) with the key H bond interactions shown by red points. The
p–p stacking
demonstrated by red dash-line.
On the other hand, the resulted key hydrogen bond interaction be-
tween Asp228 and N4-piperazine moiety of compound 9a (con-
taining hydroxyl ethyl substitute attached to the piperazine ring),
(10 and 5 lM) were tested on N2a-APPswe cells. The values are ex-
pressed as the percent inhibition of Ab production (Table 5). The
data of ClogP (range from 4.6 to 7.09) as an index of compounds’
lipophilicity are also reported in Table 5. All compounds seem to
be sufficiently lipophilic to cross the cell membrane. Potential
made this compound more potent (IC50 = 0.849 lM) than 9b. This
observation firmly confirms the importance of HB-1 between the
designed ligand and BACE1 active site. This finding emphasizes
on the importance of the substituted moiety on the piperazine ring
which might considerably affects the BACE1 inhibitory potential of
phenylimino-2H-chromen-3-carboxamides and should be consid-
ered as the most important structural feature of these derivatives.
Correlation plot of AutoDock4.2 inhibition constant value (Ki)
versus experimental IC50s (FRET assay) obtained for the most po-
tent synthesized phenylimino-2H-chromen-3-carboxamide deriv-
atives is depicted in Figure 5.
compound toxicity at examined concentrations (5 and 10 lM)
was assessed by MTT assay. Values are reported as percent of cell
viability in Table 5 (the viability percent of more than 90% was con-
sidered as acceptable viability).
Based on the data of Table 5, novel phenyliminochromene
derivatives demonstrated considerable inhibitory potency for Ab
production especially at the concentration of 10 lM (the percent-
age of inhibition ranged from 22.9 to 65.8 at this concentration).
However, this inhibitory potential was not directly correlated with
the BACE1 enzyme inhibitory activity of compounds. As an exam-
ple, compound 9e (phenyliminochromene derivative bearing
phthalimidoethyl moiety attached to the N4-piperazine) was the
most potent compound in the enzymatic assay (IC50 = 98 nM),
however, this derivative was weakly effective on inhibition of Ab
2.3.2. Inhibition of amyloid-b production determined in
cultured cells
Ab lowering effects of synthesized compounds mediated by
BACE1 inhibition were evaluated in N2a-APPswe cells by measur-
ing Ab secreted in the culture media. An enzyme immunoassay
(ELISA) was used for determination of Ab(1–40).
We considered the BACE1 enzymatic assay as the first primary
screening for selection of more potent BACE1 derivatives. The ef-
fect of two different concentrations of the most potent derivatives
production in the cell-based assay (% inhibitions at 5 and 10 lM
were 13.6 and 39.4, respectively). This observation might be attrib-
uted to the poor physicochemical properties (such as low solubility
in the culture media) of this compound that compromise its
efficiency in the cell model. On the other hand, compound 9h

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N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
Scheme 1. Synthesis protocol of the compounds 6a–c. Reagents and conditions: (a) TEA, iPrOH, 70 °C, 20 h; (b) Na2S2O6, Na2CO3, MeOH/water, 70 °C, 3–4 h; (c) neat, rt; (d)
acetyl acetone, water, 2 h, rt; (e) Toluene, reflux (10 min), rt (overnight); (f) salicylalidehyde or 3-methoxysalisylaldehyde, piperidine, MeOH, 50 °C, 7 h.
(phenyliminochromene derivative containing 2-ethyl-1H-indole
moiety attached to the N4-piperazine) with much higher ClogP va-
lue (ClogP = 7.09) than the corresponding value of 9e
(ClogP=6.34), demonstrated moderately potent inhibitory poten-
Since this compound was toxic on the cells (viability less than
70% at both examined concentrations, Table 5), we could not reli-
ably measure its effect on Ab production.
tial in cell-based assay (%inhibitions at 5 and 10 lM were 37.8
3. Conclusion
and 57.6, respectively). The much lower lipophilic phenylimino-
chromene derivative 9m (bearing furanoyl moiety at N4-pipera-
zine position, ClogP = 4.07) shows potent inhibitory potential on
Ab production. Based on these finding and according to the re-
ported data (Table 5), although the lipophilicity of compounds is
not the only determinant parameter for inhibitory potential of
these derivatives, this factor along with other physicochemical
parameters, might affect the potency of phenyliminochromene
derivatives especially in cell-based assay.
In summary, a series of small molecule phenylimino-2H-chro-
men-3-carboxamide based BACE1 inhibitors containing different
4-bromophenyl piperazine derivatives were designed using in sil-
ico docking study. According to the results of computational study,
phenyl-imino group mounted on C2 atom of 2H-chromen moiety,
establishes favorable p–p stacking interaction with the side chain
of Phe108 of flap pocket. In addition, the docking results suggested
that the optimal BACE1 inhibitory activity would be achieved via
the attachment of heteroatom comprising moieties into piperazine
ring through the pivotal H bond interaction with the Gly34,
Gly230, Thr231 or Thr232. Moreover the effective hydrophobic
interaction of hydrophobic pendants with the S2 pocket, affected
the enzyme inhibitory potential that is more predominant in fused
heteroaromatic moieties than aliphatic ones. In this regard, some
of the best designed derivatives were synthesized and further eval-
uated for the BACE1 inhibitory activity using FRET-based BACE1 as-
say. Some of the synthesized derivatives bearing suitable
heterocyclic moieties at N4-piperazine position demonstrated high
potency in BACE1 enzymatic evaluation. The result of SAR study
was in agreement with the indicated binding interactions of dock-
ing evaluation. The results suggested that the presence of fused
The most potent phenyliminochromene derivatives for inhibit-
ing Ab production, compounds 7a, 9h and 9m, demonstrated the
inhibitory percent equal to 64.68, 57.61 and 65.67 at 10
respectively. These derivatives also exhibited considerable inhibi-
tory potential at 5 M (% inhibitions of 60.91, 37.76 and 53.16,
lM,
l
respectively). Moreover, according to the data of enzymatic assay,
introduction of extra phenyl ring into iminochromene derivative
6a, enhances its inhibitory potential in compound 7a, which is
more predominant at concentration equal to 5
inhibition(Ab) for 6a and 7a at is 26.4 and 60.9,
respectively).
We also examined the effect of OM-99 at the same concentra-
tions (5 and 10 M) on N2a-APPswe cells as a positive control.
lM (the reported
%
5 lM
l

N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
2405
Scheme 2. Synthesis of 2-phenylimino-2H-chromen-3-carboxamide derivatives. Reagents and conditions: (i) aniline, glacial AcOH, reflux, overnight; (ii) aniline
hydrochloride, dry MeOH, under N2, reflux, overnight; (iii) TFA, dry CH2Cl2, under N2, 0 °C to rt, 12 h; (iv) different alkyl or aroyl halides, dry toluene, under N2, reflux,
overnight.
heteroaromatic groups such as Indole and phthalimide rings linked
through a suitable linker to the N4-piperazine position offers high
enzyme inhibitory activity. Some of these derivatives demon-
strated good activity at inhibition of Ab production in N2a
neuroblastoma cells stably expressing human Swedish APP (N2a-
APPswe cells). The results of BACE1 enzymatic and cell-based assay
confirmed that the introduction of extra phenyl ring into imino-
2H-chromene 3-carboxamide scaffold, resulted in enhanced inhib-
itory potential of phenylimino-2H-chromen derivatives. It should
be added that some physicochemical parameters are also to be
considered in design of more potent derivatives of this scaffold.
These compounds could be considered as promising BACE1 inhib-
itory agents that could be considered for Ab lowering therapy in
Alzheimer disease.
azine amides and design of phenylimino-2H-chromen-3-carbox-
amide derivatives
4.1.1. Preparation of the protein
The X-ray crystal structure of BACE1 in complex with (4S)-4-(2-
amino-6-phenoxyquinazolin-3(4H)-yl)-N,4-dicyclohexyl-N-meth-
ylbutanamid (PDB ID: 2Q15) was retrieved from the Protein Data
Bank (http://www.rcsb.org). Water molecules and cognate ligand
were removed from the receptor. All hydrogens were properly
added to the receptor PDB and non-polar hydrogens were merged
into related carbon atoms of the receptor using ADT and Kollman
charges were also assigned.
4.1.2. Preparation of ligands
The three-dimensional structures of the ligands were con-
structed using Chem3D Ultra 8.0 software to obtain standard 3D
structures. The constructed compounds were energetically mini-
mized (100 steepest descent steps using MM+ force field with a
gradient convergence value of 0.1 kcal/mol) using HyperChem soft-
ware. The Gastiger charges (empirical atomic partial charges) and
torsional degrees of freedom were assigned on the generated
PDB files by ADT 1.5.4.
4. Experimental protocols
4.1. Molecular docking
Autodock 4.2 and Auto Dock Tools 1.5.4 (ADT) were used for
docking study of naphtyl and coumarin derivatives of arylpiper-

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N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
Table 4
Table 4 (continued)
BACE1 inhibitory activity of synthesized compounds
R1
R2
X
BACE1 IC50
(lM)
a,b
Compound
O
O
OMe
9k
H
Phe
25.74( 1.35)
9l
H
H
Phe
Phe
26.43( 6.02)
0.741( 0.21)
NO2
O
R1
R2
X
BACE1 IC50
(lM)
a,b
Compound
9m
O
6a
6b
6c
H
H
H
H
1.216( 0.34)
>10
O
9n
H
—
Phe
—
3.55( 1.70)
S
COOtBut
H
OM99–2
—
0.003( 0.00)
a
OCH3
>10
BACE enzymatic inhibition was determined using BACE1-FRET assay kit, from
Pan Vera corporation (Madison WI).
b
Values are means of at least three independent experiments with standard
errors (SEM).
7a
7b
H
H
Phe
Phe
0.503( 0.10)
9.81( 0.98)
COOtBut
7c
8
OCH3
H
Phe
Phe
>10
H
9.267( 1.34)
CH2CH2OH
(CH2)3CN
9a
9b
H
H
Phe
Phe
0.849( 0.19)
>10
9c
H
H
Phe
Phe
0.119( 0.31)
0.578( 0.12)
CN
9d
Figure 5. Correlation plot of AutoDock4.2 inhibition constant value (Ki) versus
experimental IC50s (FRET assay) obtained for the most potent synthesized phenyli-
mino-2H-chromen-3-carboxamide derivatives.
OCH3
O
4.1.3. Calculation of Grid map
N
9e
9f
H
H
Phe
Phe
(CH2)2
(CH2)3
(CH2)4
0.098( 0.01)
0.326( 0.04)
The grid maps were calculated with AutoGrid (part of the Auto-
Dock package). The created three-dimensional grids were
60 ꢁ 60 ꢁ 60 (x,y,z) with a grid spacing of 0.375 Å and the cubic
grids were centered on the binding site of native ligand.
O
O
N
O
O
4.1.4. Preparation of docking parameters and running the
automated docking simulation
Automated docking studies were carried out using Lamarckian
Genetic Algorithm (LGA) to model the interaction between BACE1
and ligands. For LGA method; 25,000,000 energy evaluation, popu-
lation size of 150, a gene mutation rate of 0.02; a crossover rate of
0.8 and number of 250 independent docking runs were used for
each case. Cluster analysis was performed on the docked results
using a root mean square (RMS) tolerance of 2.0 Å. After validating
of our computational modeling approach in which X-ray structures
served as reference structures for the calculation of the root mean
square deviation (RMSD), the same modeling computational proto-
cols were applied to the target compounds.
N
O
9g
9h
9i
H
H
H
Phe
Phe
Phe
0.854( 0.19)
0.928( 0.09)
>10
H
N
O
Cl
O
9j
H
Phe
>10
4.2. Chemistry
OCH3
The starting materials, chemical reagents and solvents used in
this study were obtained from commercial sources. NMR-spectra

N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
2407
Table 5
Effect of synthesized BACE1 inhibitors on Ab1–40 production and cell viability in N2a-APPswe cells
d
b
Compound
CLogP
% Inhibition of Ab 1–40a
% Cell viability
10
l
M
5
lM
10
lM
5 lM
6a
7a
9a
9c
9d
9e
9f
9h
4.7
6.9
4.6
6.4
6.9
6.3
6.7
7.1
4.8
—
58.3 ( 2.6)
64.7 ( 11.1)
48.2 ( 6.0)
51.8( 0.01)
22.9 ( 4.2)
39.4 ( 6.3)
ND
57.6 ( 6.4)
65.8 ( 2.2)
NDe
26.4 ( 0.5)
60.9 ( 5.4)
41.3 ( 8.0)
NDb
98.9( 8.2)
95.0( 2.7)
101.4( 3.1)
88.7( 6.3)
98.3( 2.5)
96.5( 2.0)
NDc
111.4( 4.4)
95.4( 5.3)
<70
99.7( 8.90)
101.5( 5.7)
105.2( 2.7)
91.2( 11.8)
101.4( 4.4)
108.1( 7.4)
101.3( 8.3)
104.6( 3.7)
93.0( 0.3)
<70
ND
13.6 ( 5.5)
44.3 ( 5.1)
37.8 ( 4.8)
53.1 ( 2.0)
ND
9m
OM99–2
a
Percent inhibition of Ab1–40 production compared to control (non-treated cells). Ab1–40 levels were measured by an ELISA kit. Values represent the average of 2–3
different experiments ( SEM).
b
Percent cell viability determined by MTT assay compared to control (non-treated cells). Values represent the average of 3–4 experiments ( SEM).
Not determined.
The value of CLogP was calculated by ChemDraw Ultra software.
Effect on Ab production could not be measured due to toxicity.
c
d
e
were recorded on Brucker 500 spectrometer or Varian unity 400
spectrometer and chemical shifts (d) are reported in parts per mil-
lion (ppm) relative to tetramethylsilane (TMS) as an internal stan-
dard using CDCl3 or DMSO-d6 as solvent. The mass spectra were
run on an Agilent 6410. The IR spectra were obtained on a FT-IR
Perkin–Elmer Precisely system spectrophotometer (potassium bro-
mide disks). Melting points were determined on a Kofler hot stage
apparatus and are uncorrected. Elemental analyses were carried
out on a CHN-rapid elemental analyzer (Heraeus, Hanau, Germany)
for C, H and N, and the results are within 0.4% of the theoretical
values.
CH2 (piperazine)), 3.59 (s, 2H, NCH2phenyl), 4.02 (s, 2H, NH2),
6.82–6.87 (m, 3H, ArH), 7.27–7.36 ppm (m, 5H, ArH); Anal. Calcd
for C17H20BrN3: C 58.97, H 5.82, N 12.184. Found: C 58.44, H
5.64, N 13.01.
4.3.1.2. 4-(2-Amino-4-bromo-phenyl)-1-tert-butoxycarbonyl-
piperazine (2b).
Compound 2b was prepared as pale orange
foam (yield 97%): Rf = 0.42 (PE/EtOAc 4:1); 1H NMR (CDCl3,
400 MHz): dH = 1.48 (s, 9H, tBut) 2.80 (br s, 4H, CH2 (piperazine)),
3.55 (br s, 4H, CH2 (piperazine)), 4.04 (s, 2H, NH2), 6.82–
6.87 ppm (m, 3H, ArH); Anal. Calcd for C15H22BrN3O2: C 50.57, H
6.22, N 11.79. Found: C 50.22, H 6.15, N 11.81.
Merck silica gel 60 F254 plates were used for analytical TLC.
Preparative thin-layer chromatography was done with prepared
glass-backed plates (20 ꢁ 20 cm2, 500
l
) using silica gel (Merk Kie-
4.3.2. General procedure for synthesis of N-(2-(4-substituted
piperazine-1-yl)-5-bromophenyl)-2-cyanoacetamide (5a–b)
The 2-cyanoacetyl-3, 5-dimethyl pyrazole 4 (1.2 g, 7 mmol) was
added to the solution of 2-(4-substitutedpiperazin-1-yl)-5-bromo-
aniline 2a–b in toluene (10 ml). The mixture was refluxed for
10 min and then stirred at room temperature overnight.33,41 The
resulted precipitate was filtered off, washed with Et2O and further
purified by preparative column chromatography with increasing
proportion of petroleum ether/ethyl acetate (4:1 to 1:1) to afford
compound 5a–b as white crystalline solid.
selgel 60 HF254, Art. 7739). Preparative column chromatographies
were carried out on silica gel (230–400 mesh, G60 Merck). Methy-
lene dichloride (CH2Cl2) was distilled over CaH2 just prior to use.
Methanol was typically dried by distillation over iodine activated
magnesium with a magnesium loading of 5.0 g/L.40
4.3. General procedures
The 1-benzyl-4-(4-bromo-2-nitrophenyl) piperazine 1a and 4-
(4-bromo-2-nitro-phenyl)-1-tert-butoxycarbonyl-piperazine
1b
were prepared according to the previously described procedure.19
Cyanoacetic acid hydrazide 3 and 2-cyanoacetyl-3,5-dimethyl pyr-
azole 4 were also obtained following the previously published
protocols.33,34
4.3.2.1. N-(2-(4-Benzylpiperazin-1-yl)-5-bromophenyl)-2-cya-
noacetamide (5a).
(Yield 81%): Rf = 0.38 (PE/EtOAc 2:1);
mp = 196–198 °C; 1H NMR (CDCl3, 500 MHz): dH = 2.75 (br s, 4H,
CH2 (piperazine)), 2.90 (br s, 4H, CH2 (piperazine)), 3.59 (s, 4H,
CH2-phenyl and CH2CN), 7.13 (d, J = 8.4 Hz, 1H, ArH), 7.26–.29
(m, 3H, ArH), 7.33–7.36 (m, 3H, ArH), 8.55 (s, 1H, ArH), 9.59 ppm
4.3.1. Synthesis of 2-(4-substitutedpiperazin-1-yl)-5-
bromoaniline (2a–b)
(br s, 1H, NH(amide)); IR (KBr):
m = 3300, 3082, 2935, 2247, 1701,
The nitro compound 1a–b (6.65 mmol) was dissolved in meth-
anol (25 mL) and was added drop wise to the aqueous solution of
sodium carbonate (6.5 g, 61.3 mmol) and sodium dithionite (13 g,
74.4 mmol) in 100 ml water at 70 °C. When all of the nitro starting
material is consumed, the methanol was evaporated under re-
duced pressure and the resultant aqueous mixture was extracted
with ethyl acetate and washed with water, brine, and dried over
Na2SO4, filtered, and concentrated to give the amine product 2a–
b as pale yellow foam. The complete experimental data of com-
pounds 2a and 2b have been reported previously.19
1581 cmꢀ1; MS (EI) m/z (%): 414 ((M+2)+, 14), 412 (M+, 14), 346
(22), 295 (33), 146 (38), 120 (63), 91 (100); Anal. Calcd for
C20H21BrN4O: C 58.12, H 5.12, N 13.56. Found: C 57.98, H 5.38, N
13.41.
4.3.2.2. N-(2-(4-tert-butoxycarbonyl-piperazin-1-yl)-5-bromo-
phenyl)-2-cyanoacetamide (5b).
(Yield 75%): Rf = 0.33 (PE/
EtOAc 2:1); mp = 180–183 °C; 1H NMR (CDCl3, 500 MHz):
dH = 1.49 (s, 9H, t-but), 2.81 (br s, 4H, CH2 (piperazine)), 3.60 (s,
2H, CH2CN), 3.67 (br s, 4H, CH2 (piperazine)), 7.09 (d, J = 8.5 Hz,
1H, ArH), 7.27 (m, 1H, ArH), 8.57 (br s, 1H, ArH), 9.63 ppm (br s,
4.3.1.1. 2-(4-Benzylpiperazin-1-yl)-5-bromoaniline (2a).
(Yield
1H, NHCO); IR (KBr): m = 3285, 3116, 2980, 2262, 1682, 1579,
95%): Rf = 0.74 (petroleum ether (PE)/EtOAc 1:1); 1H NMR (CDCl3,
500 MHz): dH = 2.61 (br s, 4H, CH2 (piperazine)), 2.91 (br s, 4H,
1517 cmꢀ1; MS (EI) m/z (%): 424 ((M+2)+, 54), 422 (M+, 54), 366
(58), 351 (25), 292 (100), 266 (25), 213 (23), 199 (25), 57 (98),

2408
N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
41 (33); Anal. Calcd for C18H23BrN4O3: C 51.07, H 5.48, N 13.24.
Found: C 50.91, H 5.29, N 13.49.
tion, the reaction mixture was cooled to room temperature and
was quenched by crushed ice/water (30 ml). The precipitated prod-
uct was filtered, washed with cold water and dried under the vac-
uum and recrystallized from iPrOH to afford compound 7a or 7c as
yellow crystals.
4.3.2.3.
N-(2-(4-Benzylpiperazin-1-yl)-5-bromophenyl)-2-
An equimolar
imino-2H-chromene-3-carboxamide (6a).
mixture of salicylaldehyde (5 mmol, 0.625 g), N-(2-(4-benzylpi-
perazin-1-yl)-5-bromophenyl)-2-cyanoacetamide 5a (5 mmol,
2 g), methanol (10 ml) and catalytic amount of piperdine (0.1 ml)
was stirred at 50 °C for 6–7 h. After cooling, the yellow precipitate
was filtered, washed with Et2O and subsequently dried under re-
duced pressure (yield: 87%): Rf = 0.69 (CHCl3/MeOH 4:1); mp:
224–226 °C; 1H NMR (CDCl3, 400 MHz): dH = 2.67 (br s, 4H, CH2
(piperazine)), 2.91 (t, 4H, J = 4.8 Hz, CH2 (piperazine)), 3.63 (s, 2H,
NCH2phenyl), 7.03 (d, J = 8.4 Hz, 1H, ArH), 7.15–7.25 (m, 4H, ArH
and C@NH), 7.30–7.37 (m, 4H, ArH), 7.51–7.57 (m, 3H, ArH), 8.55
(s, 1H, CH(chromene)), 8.79 (d, J = 2.4 Hz, 1H, ArH), 12.65 ppm
4.3.3.1. N-(2-(4-Benzylpiperazin-1-yl)-5-bromophenyl)-2-(phe-
nylimino)-2H-chromene-3-carboxamide (7a).
(Yield 78%):
Rf = 0.81 (CH2Cl2/MeOH 5:1); mp: 202–205 °C; 1H NMR (CDCl3,
400 MHz): dH = 2.51 (br s, 4H, CH2 (piperazine)), 3.10 (m, 6H, CH2
(piperazine)) and NCH2phenyl), 7.05 (d, J = 8.4 Hz, 1H, ArH), 7.09–
7.11 (m, 1H, ArH), 7.21 (dd, J = 2.2 and 8.4 Hz, 1H. ArH), 7.29–
7.34 (m, 8H, ArH), 7.48–7.51 (m, 2H, ArH), 7.55–7.6 (m, 3H, ArH),
8.71 (s, 1H, CH(chromene)), 8.76 (d, J = 2.2 Hz, 1H, ArH),
12.52 ppm (br s, 1H, NHCO); 13C NMR (CDCl3, 125 MHz):
dC = 50.74, 52.10, 52.92, 61.60, 115.49, 116.63, 117.87, 118.96,
121.37, 121.68, 122.59, 123.35, 124.38, 124.61, 124.79, 125.44,
127.43, 127.88, 128.41, 129.20, 129.54, 129.83, 129.96, 129.98,
133.05, 134.41, 134.67, 142.79, 145.18, 149.18, 149.35, 153.44,
(br s, 1H, NHCO); IR (KBr):
m = 3441, 3293, 3110, 3001, 2907,
1673, 1603, 1574 cmꢀ1; MS (EI) m/z (%): 518 ((M+2)+, 75), 516
(M+, 75), 370 (23), 345 (46), 226 (21), 174 (100), 145 (54), 118
(38), 91 (96); Anal. Calcd for C27H25BrN4O2: C 62.67, H 4.87, N
10.83. Found: C 62.34, H 11.02, N 10.61.
160.18 ppm; IR (KBr):
m = 3060, 2941, 2824, 1682, 1592,
1567 cmꢀ1; MS (EI) m/z (%): 594 ((M+2)+, 34), 592 (M+, 34), 345
(37), 248 (100), 220 (69), 173 (37), 146 (21), 91 (100); Anal. Calcd
for C33H29BrN4O2: C 66.78, H 4.92, N 9.44. Found: C 66.84, H 5.01, N
9.31.
4.3.2.4. N-(2-(4-tert-Butoxycarbonyl-piperazin-1-yl)-5-bromo-
phenyl)-2-imino-2H-chromene-3-carboxamide (6b).
The
yellow solid product 6b was prepared from N-(2-(4-tert-butoxy-
carbonyl-piperazin-1-yl)-5-bromophenyl)-2-cyanoacetamide 5b
as described for compound 6a (yield 93%): Rf = 0.52 (PE/EtOAc
1:1); mp: 216–219 °C; 1H NMR (CDCl3, 400 MHz): dH = 1.51 (s,
9H, t-But), 2.85 (br s, 4H, CH2 (piperazine)), 3.65 (br s, 4H, CH2
(piperazine)), 6.99 (d, J = 8.5 Hz, 1H, ArH), 7.16–7.26 (m, 3H,
ArH), 7.49–7.56 (m, 2H, ArH), 7.73 (s, 1H, C=NH), 8.58 (s, 1H,
CH(chromene)), 8.81 (br s, 1H, ArH), 12.68 ppm (s, 1H, NHCO); IR
4.3.3.2. N-(2-(4-Benzylpiperazin-1-yl)-5-bromophenyl)-8-meth-
oxy-2-(phenylimino)-2H-chromene-3-carboxamide
(7c).
The titled compound 7c was prepared similar to 7a.
(yield 79%): Rf = 0.58 (PE/EtOAc (2:1)); mp: 184–186 °C; 1H NMR
(CDCl3, 500 MHz): dH = 3.04 (br s, 2H, CH2 (piperazine)), 3.38 (br
s, 2H, CH2 (piperazine)), 3.48 (br s, 2H, CH2 (piperazine)), 3.73 (br
s, 2H, CH2 (piperazine)), 3.82 (s, 2H, NCH2phenyl), 4.08 (s, 3H,
OCH3), 7.21–7.22 (m, 2H, ArH), 7.25–7.27 (m, 2H, ArH), 7.30 (dd,
J = 1.5 and 8.0 Hz, 1H, ArH), 7.36–7.42 (m, 3H, ArH), 7.48–7.52
(m, 4H, ArH), 7.83–7.84 (m, 3H, ArH), 8.84 (d, J = 2.1 Hz, 1H,
ArH), 9.04 (s, 1H, ArH), 1.67 ppm (br s, 1H, NHCO); IR (KBr):
(KBr):
m ;
= 3438, 3320, 3064, 3010, 2975, 1673, 1601, 1577 cmꢀ1
MS (EI) m/z (%): 528 ((M+2)+, 10), 526 (M+, 10), 426 (25), 253
(21), 174 (79), 145 (100), 118 (38), 57 (54); Anal. Calcd for
C25H27BrN4O4: C 56.93, H 5.16, N 10.62. Found: C 57.05, H 5.09,
N 10.54.
m
= 3200, 2963, 2839, 1709, 1666, 1606, 1575 cmꢀ1; MS (EI) m/z
(%): 624 ((M+2)+, 9), 622 (M+, 9), 547 (17), 401 (42), 344 (21),
278 (18), 250 (12), 203 (100), 146 (25), 91 (70); Anal. Calcd for
4.3.2.5.
N-(2-(4-Benzylpiperazin-1-yl)-5-bromophenyl)-2-
The
imino-8-methoxy-2H-chromene-3-carboxamide (6c).
C34H31BrN4O3: C 65.49, H 5.01, N 8.99. Found: C 65.66, H 4.97, N
titled compound 6c was synthesized by the similar procedure de-
scribed for compound 6a using 3-methoxysalicylaldehyde (yield
88%): Rf = 0.43 (PE/EtOAc 2:1); mp: 199–200 °C; 1H NMR (CDCl3,
500 MHz): dH = 2.67 (br s, 4H, CH2 (piperazine)), 2.93 (t,
J = 4.6 Hz, 4H, CH2 (piperazine)), 3.62 (s, 2H, NCH2phenyl), 4.02 (s,
3H, OCH3), 7.04 (d, J = 8.4 Hz, 1H, ArH), 7.10 (dd, J = 1.7 and
7.8 Hz, 1H, ArH), 7.13–7.18 (m, 2H, ArH), 7.21 (dd, J = 2.4 and
8.4 Hz, 1H, ArH), 7.30–7.32 (m, 1H, ArH), 7.37–7.38 (m, 4H, ArH),
7.79 (s, 1H, C@NH), 8.53 (d, J = 1.7 Hz, 1H, CH(chromene)), 8.79
(d, J = 2.4 Hz, 1H, ArH), 12.71 ppm (br s, 1H, NHCO); 13C NMR
(CDCl3, 125 MHz): dC = 52.01, 53.11, 56.30, 63.17, 114.78, 117.76,
119.58, 121.21, 121.77, 123.90, 124.01, 127.10, 127.22, 128.26,
129.35, 135.38, 138.3, 137.73, 142.25, 142.50, 143.28, 146.21,
8.73.
4.3.4. Synthesis of N-(2-(4-tert-butoxycarbonylpiperazin-1-yl)-
5-bromophenyl)-2-(phenylimino)-2H-chromene-3-carbox
amide (7b)
4.3.4.1. Synthesis of aniline hydrochloride.
A fast stream of
dry HCl gas was passes with vigorous stirring through a solution of
aniline (10 ml) in dry diethyl ether (50 ml) at room temperature.
The precipitated aniline hydrochloride was filtered off, washed
with dry ether and dried.
Equimolar amount of N-(2-(4-tert-butoxycarbonyl-piperazin-1-
yl)-5-bromophenyl)-2-imino-2H-chromene-3-carboxamide
(1 g, 2 mmol) and aniline hydrochloride (0.25 g, 2 mmol) in 20 ml
dry methanol were refluxed under nitrogen overnight. The reaction
mixture was concentrated under reduced pressure and then di-
luted by addition of 50 ml water. The resulted precipitate was fil-
tered off, washed with cold water, and dried under vacuum to
afford compound 7b as shiny yellow solid (yield 91%): Rf = 0.7
(PE/EtOAc 2:1): mp: 208–213 °C; 1H NMR (CDCl3, 500 MHz):
dH = 1.42 (s, 9H, tert-But), 2.75 (br s, 4H, CH2 (piperazine)), 3.18
(br s, 4H, CH2 (piperazine)), 6.95 (d, J = 8.4 Hz, 1H, ArH), 7.08 (d,
J = 8.2 Hz, 1H, ArH), 7.17–7.19 (m, 3H. ArH), 7.22–7.27 (m, 2H,
ArH), 7.40 (m, 2H, ArH), 7.46- 7.47 (m, 1H, ArH), 7.57–7.59 (m,
1H, ArH), 8.65 (s, 1H, ArH), 8.71 (s, 1H, ArH), 12.62 ppm (br s,
1H, NHCO). MS (EI) m/z (%):604 ((M+2)+, 11), 602 (M+, 11), 586
(21), 504 (39), 486 (26), 446 (17), 289 (32), 248 (100), 220 (98),
6b
149.10, 156.36, 160.30 ppm; IR (KBr):
m = 3445, 3304, 2939, 1675,
1607, 1574 cmꢀ1; MS (EI) m/z (%) 548 ((M+2)+, 83), 546 (M+, 83),
400 (28), 345 (46), 226 (21), 204 (100), 175 (50), 146 (26), 91
(88); Anal. Calcd for C28H27BrN4O3: C 61.43, H 4.97, N 10.23.
Found: C 61.56, H 4.67, N 10.35.
4.3.3. General method for synthesis of N-(2-(4-benzylpiperazin-
1-yl)-5-bromophenyl)-2-(phenylimino)-2H-chromene-3-
carboxamide derivatives (7a and 7c)
To a well-stirred warm (70–75 °C) solution of 6a or 6c (2 mmol)
in 10 ml glacial acetic acid was added equivalent amount of aniline
(0.2 g, 2 mmol). The reaction mixture was refluxed overnight. The
completion of the reaction was monitored by TLC. After comple-

N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
2409
77 (28), 41 (25); Anal. Calcd for C31H31BrN4O4: C 61.69, H 5.18, N
9.28. Found: C 61.53, H 5.21, N 9.33.
1.95 (t, J = 6.9 Hz, 2H,NCH2–CH2–CH2–CN), 2.33 (t, J = 7.1 Hz, 4H,
CH2 (piperazine)), 2.87 (br t, 4H, CH2 (piperazine)), 2.96 (br t,
J = 6.9 Hz, 2H, NCH2–CH2–CH2–CN), 7.01 (d, J = 8.6 Hz, 1H, ArH),
7.08–7.11 (m, 1H, ArH), 7.22–7.26 (m, 5H, ArH), 7.44–7.50 (m,
3H, ArH), 7.57–7.59 (dd, J = 2.2 and 7.7 Hz, 1H, ArH), 8.70 (s, 1H,
ArH), 8.85 (d, J = 2.2 Hz, 1H, ArH), 12.51 ppm (br s, 1H, NHCO);
13C NMR (CDCl3, 125 MHz): dC = 14.15, 22.22, 29.73, 51.58, 55.31,
115.60, 116.70, 118.90, 121.13, 121.77, 122.99, 123.42, 124.53,
124.83, 125.12, 125.61, 127.58, 129.49, 129.66, 133.31, 134.63,
134.79, 135.12, 143.26, 145.29, 153.44, 154.58, 160.19 ppm; IR
4.3.5. General protocol for synthesis of N-(5-bromo-2-
(piperazin-1-yl)phenyl)-2-(phenylimino)-2H-chromene-3-
carboxamide (8)
To a stirred solution of the N-Boc derivative 7b (0.1 g, 0.2 mmol)
in dry CH2Cl2 (10 mL) was added trifluoroacetic acid (TFA) (100 lL,
1.2 mmol) dropwise under nitrogen at 0 °C. The reaction mixture
was stirred overnight at room temperature. The completion of
reaction was monitored by TLC. After completion, the solvent and
excess TFA were removed under reduced pressure. The saturated
solution of K2CO3 was added to the resulted brown oily residue
to afford the desirable product 8 as light yellow solid (yield 95%):
Rf = 0.38 (CH2Cl2/MeOH 5:1;.mp: 212–214 °C; 1H NMR (CDCl3,
500 MHz): dH = 2.1 (br s, 1H, NH(piperazine), 2.82 (br s, 4H, CH2
(piperazine)), 2.96 ((br s, 4H, CH2 (piperazine)), 7.01 (d, J = 8.4 Hz,
1H, ArH), 7.07 (m, 1H, ArH), 7.17 (m, 2H, ArH), 7.23–7.25 (m, 3H,
ArH), 7.41–7.49 (m, 3H, ArH), 7.56–7.58 (m, 1H, ArH), 8.67 (s, 1H,
ArH), 8.70 (d, J = 2.1 Hz, 1H, ArH).12.63 ppm (s, 1H, NHCO); MS
(EI) m/z (%): 504 ((M+2)+, 16), 502 (M+, 16), 486 (16), 289 (31),
248 (100), 220 (96), 77 (32).
(KBr):
m ;
= 3430, 3119, 2924, 2852, 2246, 1678, 1643, 1593 cmꢀ1
MS (EI) m/z (%): 571 ((M+2)+, 33), 569 (M+, 33), 553 (18), 446
(38), 320 (38), 248 (100), 220 (83), 77 (21); Anal. Calcd for
C30H28BrN5O2: C 63.16, H 4.95, N 12.28. Found: C 62.97, H 4.98,
N 11.31.
4.3.6.3.
N-(2-(4-(3-Cyanobenzyl)piperazin-1-yl)-5-bromophe-
nyl)-2-(phenylimino)-2H-chromene-3-carboxamide
(9c). The yellow solid product 9c was prepared in similar de-
scribed manner iv using 3-(bromomethyl)benzonitrile (20 mg,
0.09 mmol): (yield 57%), Rf = 0.48 (PE/EtOAc 1:1); mp: 189–
194 °C; 1H NMR (CDCl3, 500 MHz): dH = 2.22 (br s, 4H, CH2 (piper-
azine)), 2.83 (br s, 4H, CH2 (piperazine)), 2.90 (s, 2H, NCH2-(3-CN-
phenyl), 7.01 (d, J = 8.5 Hz, 1H, ArH), 7.10 (d, J = 8.3 Hz, 1H, ArH),
7.23–7.26 (m, 2H, ArH), 7.27–7.3 (m, 3H, ArH), 7.39–7.45 (m, 3H,
ArH), 7.49–7.54 (m, 4H, ArH), 7.58 (dd, 1H, J = 8.6 and 2.2 Hz,
ArH), 8.69 (s, 1H, ArH), 8.76 (d, J = 2.0 Hz, 1H, ArH), 12.49 ppm
(br s, 1H, NHCO); 13C NMR (CDCl3, 125 MHz): dC = 48.52, 51.81,
59.18, 112.98, 115.63, 117.81, 118.84, 119.51, 120.79, 122.05,
123.35, 124.57, 125.09, 125.30, 127.76, 129.62, 129.81, 129.91,
130.67, 133.64, 134.40, 134.69, 136.45, 139.66, 143, 97, 145.91,
4.3.6. General procedure for synthesis of 2-N-(5-bromo-2-(4-
substitutedpiperazin-1-yl)phenyl)-2 (phenylimino)-2H-
chromene-3-carboxamide derivatives 9a–n
Appropriate derivative of alkyl or aroyl halides (0.09 mmol) was
added to the vigorous stirred mixture of compound 8 (40 mg,
0.08 mmol) and anhydrous K2CO3 (18 mg, 0.13 mmol) in dry tolu-
ene (7–10 ml). The reaction mixture was refluxed for 10–12 h un-
der nitrogen. The completion of reaction was monitored by TLC.
After completion, the solvent was removed under reduced pres-
sure. The concentrated residue was dissolved in ethyl acetate or
CH2Cl2 (5–10 ml) and extracted with water (3 ꢁ 5 ml) and washed
with brine (2 ꢁ 5 ml). The resulted organic phase was dried over
Na2SO4 and then purified by preparative thin layer chromatogra-
phy on silica gel with appropriate eluent in each case to yield the
desirable products 9a–n.
150.69, 153.33, 160.02 ppm; IR (KBr):
m = 3445, 3080, 2933, 2843,
2225, 1675, 1640, 1591 cmꢀ1; MS (EI) m/z (%): 619 ((M+2)+, 30),
617 (M+, 30), 601 (17), 446 (30), 368 (25), 248 (100), 220 (75),
116 (28), 77 (18); Anal. Calcd for C34H28BrN5O2: C 66.02, H 4.56,
N 11.32. Found: C 61.96, H 4.54, N 11.44.
4.3.6.4. N-(2-(4-(4-Methoxybenzyl)piperazin-1-yl)-5-bromophe-
nyl)-2-(phenylimino)-2H-chromene-3-carboxamide
4.3.6.1. N-(5-Bromo-2-(4-(2-hydroxyethyl)piperazin-1-yl)phe-
nyl)-2-(phenylimino)-2H-chromene-3-carboxamide
(9d).
The compound 9d was synthesized as yellow solid using
4-methoxybenzylbromide (18.1 mg, 0.09 mmol) in similar manner
describe in procedure iv. (yield 43%): Rf = 0.45 (CH2Cl2/MeOH 5:1);
mp: 198–201 °C; 1H NMR (CDCl3, 500 MHz): dH = 1.70 (br s, 2H,
CH2 (piperazine)), 2.23 (br s, 2H, CH2 (piperazine)), 2.83 (br s, 4H,
CH2 (piperazine)), 2.86 (s, 2H, NCH2-(p-methoxy)phenyl), 3.79 (s,
3H, OCH3), 6.82 (d, J = 8.4 Hz, 1H, ArH), 6.99 (d, 1H, J = 8.9 Hz,
ArH), 7.07–7.11 (m, 4H, ArH), 7.22–7.29 (m, 5H, ArH), 7.47–7.53
(m, 3H, ArH), 7.57–7.59 (dd, J = 8.4 and 2 Hz, 1H, ArH), 8.69 (s,
1H, ArH), 8.74 (d, J = 2.0 Hz, 1H, ArH), 12.52 ppm (s, 1H, NHCO);
13C NMR (CDCl3, 125 MHz): dC = 29.73, 31.95, 46.33, 52.10, 55.28,
113.77, 113.97, 114.15, 115.54, 115.66, 119.01, 121.47, 122.34,
123.44, 124.49, 124.72, 124.77, 124.98, 127.49, 128.63, 129.25,
129.59, 129.88, 130.09, 133.10, 134.72, 134.93, 141.30, 142.88,
(9a).
The yellow solid compound 9a was prepared by the de-
scribed procedure iv using 14 mg (0.09 mmol) of 2-bromoethanol
(yield 78%): Rf = 0.6 (CH2Cl2/MeOH 5:1); mp: 203–207 °C; 1H
NMR (CDCl3, 500 MHz): dH = 2.1 (t, J = 4.9 Hz, 2H, NCH2CH2OH),
3.02 (br s, 4H, CH2 (piperazine)), 3.44 (br s, 4H, CH2 (piperazine)),
3.58 (t, J = 4.9 Hz, 2H, NCH2CH2OH), 7.04–7.09 (m, 2H, ArH),
7.21–7.23 (m, 3H, ArH), 7.25–7.26 (m, 2H, ArH), 7.44–7.46 (m,
2H, ArH), 7.47–7.51 (m, 1H, ArH), 7.60 (dd, J = 2.3 and 7.8 Hz, 1H,
ArH), 8.73 (s, 1H, ArH), 8.78 (d, J = 2.3 Hz, 1H. ArH), 12.47 ppm (s,
1H, NHCO); 13C NMR (CDCl3, 125 MHz): dC = 40.31, 51.24, 55.67,
58.34, 76.44, 114.71, 117.76, 119.05, 120.31, 120.82, 122.19,
122.37, 123.68, 123.94, 125.70, 126.12, 128.46, 128.82, 132.41,
133.93, 140.17, 142.38, 148.43, 148.93, 152.59, 159.36,
144.59, 145.20, 153.50, 160.25 ppm; IR (KBr):
m
= 3442, 3127,
159.46 ppm; IR (KBr):
m
= 3382, 3150, 2923, 2849, 1675, 1637,
3060, 2927, 1676, 1627, 1591 cmꢀ1
; MS (EI) m/z (%): 624
1590 cmꢀ1; MS (EI) m/z (%): 548 ((M+2)+, 38), 546 (M+, 38), 446
(21), 297 (46), 248 (100), 220 (71), 77 (44), 42 (29); Anal. Calcd
for C28H27BrN4O3: C 61.43, H 4.97, N 10.23. Found: C 61.54, H
4.84, N 10.12.
((M+2)+, 39), 622 (M+, 39), 448 (17), 373 (31), 248 (92), 220 (53),
121 (100), 77 (29); Anal. Calcd for C34H31BrN4O3: C 65.49, H 5.01,
N 8.99. Found: C 65.60, H 4.95, N 9.03.
4.3.6.5.
yl)phenyl)-2-(phenylimino)-2H
(9e). The titled compound 9e was obtained by a procedure
iv using N-(2-bromoethyl)phthalimide (23 mg, 0.09 mmol) as light
yellow solid (yield 52%): Rf = 0.61 (CH2Cl2/MeOH 5:1); mp: 220–
225 °C; 1H NMR (CDCl3, 500 MHz): dH = 2.11 (br t, 2H, NCH2CH2-
phthalimide), 2.32 (br s, 4H, CH2 (piperazine)), 2.77 (br s, 4H, CH2
N-(5-Bromo-2-(4-(2-phthalimidoethyl)piperazin-1-
4.3.6.2.
nyl)-2-(phenylimino)-2H-chromene-3-carboxamide
(9b). The titled compound was synthesized by the procedure
iv from 4-bromobutanenitrile (13 mg, 0.09 mmol) as yellow solid
(yield 84%): Rf = 0.57 (CH2Cl2/MeOH 5:1); mp: 193–196 °C; 1H
NMR (CDCl3, 500 MHz): dH = 1.64 (m, 2H, NCH2–CH2–CH2–CN),
N-(5-Bromo-2-(4-(3-cyanopropyl)piperazin-1-yl)phe-
chromene-3-carboxamide

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N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
(piperazine)), 3.61 (br t, 2H, NCH2CH2-phthalimide), 6.97 (d,
J = 8.4 Hz, 1H, ArH), 7.07–7.09 (m, 1H, ArH), 7.11–7.14 (m, 1H,
ArH), 7.20–7.25 (m, 4H, ArH), 7.38–7.41 (m, 2H, ArH), 7.46–7.49
(m, 1H, ArH), 7.57–7.58 (dd, J = 8.4 and 2.0 Hz 1H, ArH), 7.72–
7.74 (m, 2H, ArH), 7.85–7.87 (m, 2H, ArH), 8.68 (s, 1H, ArH), 8.73
(d, J = 2.0 Hz, 1H, ArH), 12.46 ppm (br s, 1H, NHCO); IR (KBr):
(CH2Cl2/MeOH 5:1); mp: 206–209 °C; 1H NMR (CDCl3, 500 MHz):
dH = 2.24 (t, J = 7.9 Hz, 2H, NCH2CH2-(2-(1H-indol-2-yl), 2.62 (br
s, 4H, CH2 (piperazine)), 2.76 (t, J = 7.9 Hz, 2H, NCH2CH2-(2-(1H-in-
dol-2-yl)), 2.80 (br s, 4H, CH2 (piperazine)), 6.09–7.12 (m, 4H, ArH),
7.21–7.25 (m, 6H, ArH), 7.35–7.40 (m, 3H, ArH), 7.45–7.47 (m, 1H,
ArH), 7.54–7.59 (m, 2H, ArH), 8.03 (br s, 1H, NH(1H-indole-2-yl)),
8.68 (s, 1H, ArH), 8.73 (br s, 1H, ArH), 12.52 ppm (br s, 1H, NHCO);
13C NMR (CDCl3, 125 MHz): dC = 22.58, 28.93, 53.29, 58.43, 111.33,
115.76, 117.23, 118.92, 119.23, 119.43, 121.61, 121.75, 122.27,
123.42, 123.64, 124.79, 124.91, 127.58, 127.71, 129.2, 129.74,
130.23, 133.21, 134.53, 134.84, 135.48, 142.64, 142.30, 145.09,
m
= 3400, 3220, 2942, 2852, 1771, 1709, 1671, 1635, 1589 cmꢀ1
;
MS (EI) m/z (%): 677 ((M+2)+, 47), 675 (M+, 47), 657 (10), 448
(33.3), 428 (44), 248 (100), 220 (71), 174 (18), 77 (25); Anal. Calcd
for C36H30BrN5O4: C 63.91, H 4.47, N 10.35. Found: C 64.00, H 4.43,
N 10. 41.
149.34, 149.40, 153.23, 154.83, 160.52 ppm; IR (KBr):
m = 3396,
4.3.6.6. N-(5-Bromo-2-(4-(3-phthalimidopropyl)piperazin-1-yl)
phenyl)-2-(phenylimino)-2H-chromene-3-carboxamide
3198, 3055, 2923, 2819, 1715, 1674, 1635, 1589 cmꢀ1; MS (EI)
m/z (%): 647 ((M+2)+, 21), 645 (M+, 21), 517 (100), 497 (25), 442
(38), 294 (13), 268 (21), 248 (83), 220 (46), 173 (14), 130 (29),
77 (18); Anal. Calcd for C36H32BrN5O2: C 66.87, H 4.99, N 10.83.
Found: C 66.94, H 4.93, N 10. 79.
(9f).
The yellow solid compound 9f was prepared by the de-
scribed method iv using N-(3-bromopropyl)phthalimide (24 mg,
0.09 mmol. (yield 60%): Rf = 0.48 (CH2Cl2/MeOH 5:1); mp: 208–
223 °C; 1H NMR (CDCl3, 500 MHz): dH = 1.69–1.75 (m, 4H,
NCH2CH2CH2-phthalimide and CH2 (piperazine)), 1.96 (br t,
NCH2CH2CH2-phthalimide), 2.19 (br s, 4H, CH2 (piperazine)), 2.77
(br s, 4H, CH2 (piperazine)), 3.68 (t, J = 6.7 Hz, 2H, NCH2CH2CH2–
phthalimide), 6.89 (m, 1H, ArH), 6.99–7.01 (m, 1H, ArH), 7.06 (d,
J = 8.2 Hz, 1H, ArH), 7.17–7.25 (m, 4H, ArH), 7.31–7.34 (m, 2H,
ArH), 7.56–7.49 (m, 1H, ArH), 7.57 (dd, J = 8.2 and 2.4 Hz, 1H,
ArH), 7.75–7.77 (m, 2H, ArH), 7.87–7.89 (m, 2H, ArH), 8.68 (s, 1H,
ArH), 8.73 (d, J = 2.4 Hz, 1H, ArH), 12.45 ppm (br s, 1H, NHCO);
13C NMR (CDCl3, 125 MHz): dC = 22.41, 35.47, 48.87, 51.70, 51.67,
115.70, 118.30, 119.02, 121.05, 122.35, 123.08, 123.70, 124.61,
124.84, 125.16, 127.87, 129.66, 129.97, 131.61, 132.13, 133.61,
134.50, 134.66, 134.94, 135.11, 139.96, 143.85, 145.71, 150.51,
4.3.6.9. N-(2-(4-(3-Chlorobenzoyl)piperazin-1-yl)-5-bromophe-
nyl)-2-(phenylimino)-2H-chromene-3-carboxamide
(9i).
The yellow solid compound 9i was synthesized as the
similar manner described in procedure iv using 3-chlorobenzoyl
chloride (15.8 mg, 0.09 mmol) (yield: 45%): Rf = 0.47 (PE/EtOAc
1:1); mp: 288–291 °C; 1H NMR (CDCl3, 500 MHz): dH = 2.71 (br s,
2H, CH2 (piperazine)), 2.88 (br s, 2H, CH2 (piperazine)), 3.09 (br s,
2H, CH2 (piperazine)), 3.63 (br s, 2H, CH2 (piperazine)), 6.96 (d,
1H, J = 8.4 Hz, ArH), 7.09–7.15 (m, 2H, ArH), 7.21–7.27 (m, 2H,
ArH), 7.28–7.33 (m, 2H, ArH), 7.37–7.38 (m, 1H, ArH), 7.3–7.51
(m, 3H, ArH), 7.57–761 (m, 2H, ArH), 7.99–8.01 (dd, J = 8.4 and
2.2 Hz, 1H, ArH), 8.08–8.1 (m, 1H, ArH), 8.71 (s, 1H, ArH), 8.78 (d,
J = 2.2 HZ, ArH), 12.66 ppm (br s, 1H, NHCO); 13C NMR (CDCl3,
125 MHZ): dC = 41.17, 46.66, 51.38, 51.64, 115.11, 117.95, 118.39,
120.75, 121.09, 122.08, 124.11, 124.17, 124.34, 124.56, 126.53,
126.94, 127.78, 128.73, 129.10, 129.29, 129.73, 132.67, 133.25,
133.92, 134.42, 136.76, 141.13, 142.41, 144.37, 148.87, 153.00,
153.52, 160.19, 168.22 ppm; IR (KBr):
m
= 3462, 3060, 2938, 2827,
1771, 1709, 1675, 1639, 1590 cmꢀ1
;
MS (EI) m/z (%): 691
((M+2)+, 46), 689 (M+, 46), 442 (63), 248 (100), 220 (75), 188
(15), 160 (25), 77 (19); Anal. Calcd for C37H32BrN5O4: C 64.35, H
4.67, N 10.14. Found: C 64.46, H 4.59, N 10. 17.
159.84, 168.01 ppm; IR (KBr):
m = 3400, 3061, 2919, 1680. 1626,
4.3.6.7.
yl)phenyl)-2-(phenylimino)-2H
(9g). The titled compound 9g was synthesized by similar pro-
N-(5-Bromo-2-(4-(4-phthalimidobutyl)piperazin-1-
1594 cmꢀ1; MS (EI) m/z (%): 644 ((M+4)+, 2), 642 ((M+2)+, 8), 640
(M+, 6), 624 (38), 393 (17), 248 (75), 220 (100), 139 (18), 77
(14); Anal. Calcd for C33H26BrClN4O3: C 61.74, H 4.08, N 8.73.
Found: C 61.81, H 4.11, N 8. 60.
chromene-3-carboxamide
tocol as mentioned in procedure iv using N-(4-bromobutyl)phthal-
imide (25 mg, 0.09 mmol) as yellow solid (yield: 48%): Rf = 0.3
(CH2Cl2/MeOH 5:1); mp: 200–204 °C; 1H NMR (CDCl3, 500 MHz):
dH = 1.26 (m, 2H, NCH2CH2CH2CH2–phthalimide), 1.47 (br s, 2H,
CH2 (piperazine)), 1.58 (m, 2H, NCH2CH2CH2CH2-phthalimide),
1.96 (br s, 2H, CH2 (piperazine)), 2.02 (br t, 2H, NCH2CH2CH2CH2–
phthalimide), 3.02 (br s, 4H, 4H, CH2 (piperazine)), 3.65 (t,
J = 6.9 Hz, 2H, NCH2CH2CH2CH2–phthalimide), 7.04–7.08 (m, 2H,
ArH), 7.21–7.27 (m, 4H, ArH), 7.43–7.50 (m, 4H, ArH), 7.58–7.60
(dd, J = 6.9 and 2.2 Hz, 1H, ArH), 7.73–7.74 (m, 2H, ArH), 7.86–
7.87 (m, 2H, ArH), 8.72 (s, 1H, ArH), 8.78 (d, J = 2.2 Hz, 1H, ArH),
12.45 ppm (br s, 1H, NHCO); 13C NMR (CDCl3, 125 MHz):
dC = 20.80, 25.71, 36.79, 48.51, 51.44, 56.04, 115.58, 118.81,
119.38, 119.59, 122.30, 122.95, 123.67, 124.59, 125.17, 128.80,
127.91, 129.52, 131.94, 132.78, 133.58, 134.12, 134.71, 135.18,
139.90, 143.73, 145.69, 151.02, 153.17, 160.20, 168.23; IR (KBr):
4.3.6.10. N-(2-(4-(4-Methoxybenzoyl)piperazin-1-yl)-5-bromo-
phenyl)-2-(phenylimino)-2H-chromene-3-carboxamide
(9j).
The compound 9j was prepared according to the proce-
dure iv using 4-methoxybenzoyl chloride (15.3 mg, 0.09 mmol)
(yield 38%): Rf = 0.62 (CH2Cl2/MeOH 5:1); mp: 258–260 °C; 1H
NMR (CDCl3, 500 MHz): dH = 2.72 (br s, 2H, CH2(piperazine)) 2.85
(br s, 2H, CH2 (piperazine)), 3.20 (br s, 2H, CH2 (piperazine)), 3.61
(br s, 2H, CH2 (piperazine)), 3.83 (s, 3H, OCH3), 6.87 (d, J = 8.6 Hz,
2H, ArH), 6.95–6.96 (m, 1H, ArH), 7.08–7.1 (m, 1H, ArH), 7.18–
7.26 (m, 7H, ArH), 7.44–7.51 (m, 3H, ArH), 7.58–7.59 (dd, J = 8.6
and 2.0 Hz, 1H, ArH), 8.69 (s, 1H, ArH), 8.76 (d, J = 2.0 Hz, 1H,
ArH), 12.65 ppm(br s, 1H, NHCO); 13C NMR (CDCl3, 500 MHz):
dC = 41.72, 47.39, 52.09, 55.32, 113.58, 115.58, 118.24, 118.30,
118.91, 121.34, 121.47, 122.58, 122.60, 124.62, 124.69, 127.42,
127.69, 128.98, 129.12, 129.55, 133.10, 134.85, 141.88, 142.73,
144.80, 149.24, 153.49, 160.29, 160.66, 169.96 ppm; IR (KBr):
m
= 3461, 3116, 2937, 2832, 1770, 1716, 1674, 1635, 1588 cmꢀ1
;
MS (EI) m/z (%): 705 ((M+2)+, 42), 703 (M+, 42), 456 (53), 294
(15), 248 (100), 220 (75), 160 (42), 118 (13), 77 (23); Anal. Calcd
for C38H34BrN5O4: C 64.77, H 4.86, N 9.94. Found: C 64.80, H
4.78, N 10. 01.
m
= 3445, 3080, 2927, 1676. 1627, 1604, 1591 cmꢀ1; MS (EI) m/z
(%): 638 ((M+2)+, 25), 636 (M+, 25), 620 (21), 389 (23), 248 (92),
220 (100), 135 (63), 77 (25); Anal. Calcd for C34H29BrN4O4: C
64.05, H 4.58, N 8.79. Found: C 63.99, H 4.61, N 8. 75.
4.3.6.8.
bromophenyl)-2-(phenylimino)-2H-chromene-3-carboxamide
(9h). The compound 9h was prepared in similar manner as
N-(2-(4-(2-(1H-Indol-2-yl)ethyl)piperazin-1-yl)-5-
4.3.6.11. N-(2-(4-(2-Methoxybenzoyl)piperazin-1-yl)-5-bromo-
phenyl)-2-(phenylimino)-2H-chromene-3-carboxamide
described in procedure iv using 2-(2-bromoethyl)-1H-indole
(20 mg, 0.09 mmol) as yellow solid (yield: 51%): Rf = 0.63
(9k).
The titled compound 9k was synthesized in similar
manner described in procedure iv from 2-methoxybenzoyl

N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
2411
chloride (15.3 mg, 0.09 mmol) as yellow solid (yield: 40%):
Rf = 0.63 (CH2Cl2/MeOH 5:1); mp: 245–249 °C; 1H NMR (CDCl3,
500 MHz): dH = 2.71 (m, 2H, CH2 (piperazine)), 2.89 (t, J = 5.0 Hz,
2H, CH2 (piperazine)), 2.98–3.06 (m, 2H, CH2 (piperazine)), 3.51
(br s, 2H, CH2 (piperazine)), 3.79 (s, 3H, OCH3), 6.87 (d, J = 8.4 Hz,
1H, ArH), 6.95–6.98 (m, 2H, ArH), 7.06–7.10 (m, 2H, ArH), 7.19–
7.25 (m, 5H, ArH), 7.31–7.34 (m, 1H, ArH), 7.42–7.50 (m, 3H,
ArH), 7.56–7.59 (dd, J = 7.6 and 0.86 Hz, 1H, ArH), 8.68 (s, 1H,
ArH), 8.70 (d, J = 2.2 Hz, 1H, ArH), 12.66 ppm (br s, 1H, NHCO);
13C NMR (CDCl3, 125 MHz): dC = 41.08, 46.51, 51.76, 52.34, 55.51,
110.88, 115.55, 117.97, 118.89, 120.81, 121.23, 121.24, 122.65,
124.61, 124.72, 124.78, 125.50, 127.44, 127.74, 129.02, 129.53,
130.33, 133.07, 134.59, 142.13, 142.63, 144.54, 149.16, 153.46,
0.09 mmol) as yellow solid (yield 56%): Rf = 0.59 (PE/EtOAc 1:1);
mp: 285–288 °C; 1H NMR (CDCl3, 500 MHz): dH = 2.83 (br s, 4H,
CH2 (piperazine)), 3.52 (br s, 4H, CH2 (piperazine)), 6.96 (d,
J = 8.4 Hz, 1H, ArH), 7.01–7.05 (m, 2H, ArH), 7.09 (m, 1H, ArH),
7.20–7.28 (m, 5H, ArH), 7.42–7.51 (m, 4H, ArH), 7.58–7.60 (dd,
J = 8.4 and 2.3 Hz, 1H, ArH), 8.71 (s, 1H, ArH), 8.77 (d, J = 2.3 Hz,
1H, ArH), 12.69 ppm (br s, 1H, NHCO); 13C NMR (CDCl3,
125 MHz): dC = 52.22, 57.57, 115.67, 116.39, 118.33, 118.51,
118.96, 121.18, 121.55, 122.59, 124.79, 124.98, 125.07, 126.33,
126.59, 127.63, 128.44, 128.61, 128.70, 129.22, 129.27, 129.70,
131.68, 133.26, 134.81, 136.89, 141.74, 142.93, 148.97, 153.48,
160.48 ppm; IR (KBr):
m
= 3444, 3078, 1680. 1620, 1592 cmꢀ1; MS
(EI) m/z (%): 614 ((M+2)+, 13), 612 (M+, 13), 596 (33), 365 (17),
155.14, 160.26, 167.41 ppm; IR (KBr):
m
= 3444, 3059, 2957, 1679.
248 (75), 220 (100), 111 (33), 77 (21); Anal. Calcd for
C31H25BrN4O3S: C 60.69, H 4.11, N 9.13. Found: C 60.63, H 4.20,
N 9. 18.
1623, 1593 cmꢀ1; MS (EI) m/z (%): 638 ((M+2)+, 19), 636 (M+, 19),
620 (29), 389 (28), 248 (88), 220 (100), 135 (67), 77 (29); Anal.
Calcd for C34H29BrN4O4: C 64.05, H 4.58, N 8.79. Found: C 64.11,
H 4.54, N 8. 70.
4.4. Biological assays
4.3.6.12. N-(2-(4-(3-Nitrobenzoyl)piperazin-1-yl)-5-bromophe-
nyl)-2-(phenylimino)-2H-chromene-3-carboxamide
4.4.1. BACE1 enzymatic assay
Enzyme inhibition assay was carried out using BACE1 (b-secre-
tase) FRET assay kit, from Invitrogen (former Pan Vera corporation,
Madison, WI) according to the manufacturer instructions42,43 and
using a multiwell spectrofluorometer instrument capable of 530–
545 nm excitation and 570–590 nm emission wavelengths (BMG
LABTECH, Polar star, Germany).
The assay procedure was conducted as follows: The provided
BACE1 enzyme (purified baculovirus-expressed BACE1) was di-
luted with BACE1 assay buffer (50 mM sodium acetate, pH 4.5) to
make a working solution of (1 Unit/ml) (3X). The peptide substrate
(Rh-EVNLDAEFK-Quencher) was also diluted with assay buffer to
provide the 3X stock solution (750 nM). The inhibitors stock solu-
tion in DMSO were diluted with assay buffer to provide 3X solution
of test compounds at different concentrations (12% DMSO). The 3X
(9l).
The yellow solid product 9l was prepared from 3-nitro-
benzoyl chloride (16.7 mg, 0.09 mmol) in similar manner described
in procedure iv (yield: 41%): Rf = 0.47 (PE/EtOAc 1:1); mp: 289–
290 °C; 1H NMR (CDCl3, 500 MHz): dH = 2.72 (br s, 2H, CH2 (piper-
azine)), 2.90 (br s, 2H, CH2 (piperazine)), 3.11 (br s, 2H, CH2 (piper-
azine)), 3.69 (br s, 2H, CH2 (piperazine)), 6.96 (d, J = 8.6 Hz, 1H,
ArH), 7.09–7.1 (m, 1H, ArH), 7.21–7.26 (m, 3H, ArH), 7.28–7.33
(m, 2H, ArH), 7.45–7.53 (m, 3H, ArH), 7.59–7.61 (m, 3H, ArH),
8.02 (m, 1H, ArH), 8.26–8.29 (m, 1H, ArH), 8.71 (s, 1H, ArH), 8.79
(d, J = 2.2 Hz, 1H, ArH), 12.69 ppm (br s, 1H, ArH); 13C NMR (CDCl3,
125 MHz): dC = 41.61, 47.08, 52.17, 52.20, 116.03, 116.60, 116.94,
118.85, 119.11, 119.59, 120.37, 121.81, 122.33, 122.57, 122.83,
122.99, 124.83, 124.93, 125.94, 126.50, 128.60, 129.56, 129.63,
129.75, 130.10, 130.37, 133.36, 134.85, 137.15, 148.17, 149.41,
solution of BACE1 enzyme (10
(10 l of corresponding solution) were introduced in the 96-well
plate and gently mixed. The substrate 3X solution (10 l) was then
added to the described mixture in each well to start the reaction at
the final reaction volume of 30 l (the final concentration of DMSO
ll) and each inhibitor samples
153.24, 157.68 ppm; IR (KBr):
m
= 3446, 3065, 2834, 1675. 1636,
l
1593, 1568, 1352 cmꢀ1; MS (EI) m/z (%): 653 ((M+2)+, 14), 651
(M+, 14), 635 (33), 404 (10), 248 (83), 220 (100), 150 (17), 104
(12), 77 (19); Anal. Calcd for C33H26BrN5O5: C 60.74, H 4.02, N
10.73. Found: C 60.88, H 3.96, N 10. 67.
l
l
in each sample and control wells was less than 4%). The reaction
mixtures were incubated at 25 °C for 90 min in the dark and then
the reaction was stopped with 2.5 mM sodium acetate. Fluores-
cence was monitored at 544 nm (excitation wavelength) and
4.3.6.13. N-(5-Bromo-2-(4-(furan-2-carbonyl)piperazin-1-yl)phe-
nyl)-2-(phenylimino)-2H-chromene-3-carboxamide (9m).
The
compound 9m was obtained according the described procedure iv
using furan-2-carbonyl chloride (12 mg, 9.09 mmol) as yellow solid
(yield 47%): Rf = 0.49 (CH2Cl2/MeOH 5:1); mp: 269–273 °C; 1H NMR
(CDCl3, 500 MHz): dH = 2.86 (br s, 4H, CH2 (piperazine)), 3.59 (br s,
4H, CH2 (piperazine)), 6.45–6.47 (m, 1H, ArH), 6.86 (m, 1H, ArH),
6.97 (d, J = 8.4 Hz, 1H, ArH), 7.09–7.1 (m, 1H, ArH), 7.16–7.21 (m, 3H,
ArH), 7.24–7.28 (m, 2H, ArH), 7.38–7.41 (m, 2H, ArH), 7.46–7.51 (m,
2H, ArH), 7.58–7.60 (dd, J = 8.4 and 2.4 Hz, 1H, ArH), z, 1H, ArH),
8.71 (s, 1H, ArH), 8.74 (d, J = 2.4 Hz, 1H, ArH), 12.71 ppm (br s, 1H,
NHCO); 13C NMR (CDCl3, 125 MHz): dC = 53.00, 57.56, 111.19,
111.25, 115.67, 116.23, 116.39, 118.21, 118.37, 118.98, 121.27,
121.39, 121.50, 122.63, 124.76, 124.96, 125.16, 126.22, 127.64,
129.16, 129.23, 129.69, 133.23, 134.73, 141.88, 142.81, 143.76,
590 nm (emission wavelength). OM99-2 (Glu-Val-Asn-Leu-W-
Ala-Ala-Glu-Phe, Calbiochem) was used as a reference inhibitor
compound.
The percentage of enzyme inhibition for each concentration of
test compound was calculated compared to maximum enzyme
activity wells (containing substrate plus enzyme) and baseline
wells (containing substrate). IC50 values (concentration of the com-
pound that induces 50% inhibition of enzymatic activity) were cal-
culated with CurveExpert software version 1.34 for Windows. Each
experiment was repeated 3–4 times. Data are presented as
mean SD.
4.4.2. Determination of BACE1 inhibition in cultured cells
RPMI, OPTI-MEM, fetal bovine serum (FBS), trypsin and phos-
phate buffered saline (PBS) were purchased from Biosera (Ringmer,
UK) and Dulbecco’s modified Eagle’s medium (DMEM) was pur-
chased from Gibco (GibcoBRL, Grand Island, NY, USA). Geneticine
(G418) and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) was obtained from Sigma–Aldrich (Saint Louis,
MO, USA) and penicillin/streptomycin was purchased from Invitro-
gen (San Diego, CA, USA). The production of amyloid-b (Ab) was
carried out by commercially available human Ab (1–40) (FL) assay
kit (L)-IBL (IBL Co. Ltd, Japan, Code No. 27718).
147.67, 153.50, 158.88, 160.47 ppm; IR (KBr):
m = 3444, 3128, 3107,
2922, 1681. 1625, 1593 cmꢀ1; MS (EI) m/z (%):598 ((M+2)+, 17), 596
(M+, 17), 580 (33), 349 (21), 248 (75), 220 (100), 95 (25), 77 (17); Anal.
Calcd for C31H25BrN4O4: C 62.32, H 4.22, N 9.38. Found: C 62.19, H
4.31, N 9. 40.
4.3.6.14. N-(5-Bromo-2-(4-(thiophen-2-carbonyl)piperazin-1-
yl)phenyl)-2-(phenylimino)-2H-chromene-3-carboxamide
(9n).
The titled compound 9n was synthesized according to
the procedure iv using thiophen-2-carbonyl chloride (13.1 g,

2412
N. Edraki et al. / Bioorg. Med. Chem. 21 (2013) 2396–2412
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Mouse Neuro2a (N2a) cells stably
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maintained in 50% Dulbecco’s modified Eagle’s medium (DMEM),
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mycin, 10% fetal bovine serum in the presence of 200
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G418. Cells were maintained at 37 °C in humidified air containing
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4.4.3. Measurement of Ab secretion by cells
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The authors wish to thank Professor Paul Greengard, Rockefeller
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