Synthesis and biological evaluation of novel aromatic-heterocyclic biphenyls as potent anti-leukemia agents

Article abstract of DOI:10.1016/j.ejmech.2015.07.015

As a continuation to our previous research, twenty-eight aromatic-heterocyclic biphenyls were designed and synthesized as novel Bcr-Abl inhibitors. The title compounds were investigated for their antiproliferative activities against wild K562 cells and Imatinib-resistant K562 cells (K562R). The results indicated that most of them exhibited potent Bcr-Abl inhibition and moderate antiproliferative potency against K562 cells. Furthermore, three compounds 3, 7 and 21 displayed moderate antiproliferative activities against K562R cells. Molecular docking indicated that 3 bound more tightly with Bcr-Abl^T315I compared to Bcr-Abl^WT. The higher affinity was consistent with its relatively promising K562R cell growth inhibition. These aromatic-heterocyclic biphenyls could be considered as novel lead compound for optimized as Bcr-Abl^T315I inhibitors. They provide a good starting point for the further development of novel anti-leukemia agents capable of dealing with clinical acquired resistance against Imatinib.

Full text of DOI:10.1016/j.ejmech.2015.07.015

Accepted Manuscript
Synthesis and biological evaluation of novel aromatic-heterocyclic biphenyls as potent
anti-leukemia agents
Jinyun Dong, Xiaoyan Pan, Jinfeng Wang, Ping Su, Lin Zhang, Fen Wei, Jie Zhang
PII:
S0223-5234(15)30147-1
10.1016/j.ejmech.2015.07.015
EJMECH 7996
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 February 2015
Revised Date: 7 July 2015
Accepted Date: 7 July 2015
Please cite this article as: J. Dong, X. Pan, J. Wang, P. Su, L. Zhang, F. Wei, J. Zhang, Synthesis and
biological evaluation of novel aromatic-heterocyclic biphenyls as potent anti-leukemia agents, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.07.015.
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ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel aromatic heterocyclic biphenyls as
potent anti-leukemia agents
Jinyun Dong, Xiaoyan Pan, Jinfeng Wang, Ping Su, Lin Zhang, Fen Wei, Jie Zhang*
School of Pharmacy, Health Science Center, Xi'an Jiaotong University, No. 76, Yanta West Road,
Xi’an, Shaanxi Province, 710061, P.R. China
T315I
wt
As a continuation to our previous research, a series of aromatic-heterocyclic biphenyls were
developed as novel Bcr-Abl inhibitors.

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis and biological evaluation of novel aromatic-heterocyclic biphenyls as potent anti-leukemia
agents
∗
Jinyun Dong, Xiaoyan Pan, Jinfeng Wang, Ping Su, Lin Zhang, Fen Wei, Jie Zhang
School of Pharmacy, Health Science Center, Xi'an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi
Province, 710061, P.R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
As a continuation to our previous research, twenty-eight aromatic-
heterocyclic biphenyls were designed and synthesized as novel Bcr-Abl
inhibitors. The title compounds were investigated for their antiproliferative
activities against wild K562 cells and Imatinib-resistant K562 cells (K562R).
The results indicated that most of them exhibited potent Bcr-Abl inhibition
and moderate antiproliferative potency against K562 cells. Furthermore,
three compounds 3, 7 and 21 displayed moderate antiproliferative activities
against K562R cells. Molecular docking indicated that 3 bound more tightly
Available online
Keywords:
CML
Bcr-Abl inhibitors
biphenyls
T315I
WT
with Bcr-Abl
compared to Bcr-Abl . The higher affinity was consistent
with its relatively promising K562R cell growth inhibition. These aromatic-
heterocyclic biphenyls could be considered as novel lead compound for
T315I
Resistant
optimized as Bcr-Abl
inhibitors. They provide a good starting point for
the further development of novel anti-leukemia agents capable of dealing
with clinical acquired resistance against Imatinib.
2
009 Elsevier Ltd. All rights reserved.
∗
Corresponding author. Tel/Fax: +86-29-82655451; E-mail: zhj8623@mail.xjtu.edu.cn (Jie Zhang).
1

ACCEPTED MANUSCRIPT
1
. Introduction
Chronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl
1]. Bcr-Abl plays a critical role in cellular activities, including cell growth, proliferation, survival,
[
adhesion and migration [2]. Development of potent Bcr-Abl inhibitors has become a well
validated approach for the treatment of CML. Imatinib (STI571, Gleevec), the first Bcr-Abl
inhibitors, has achieved tremendous clinical success in early-stage disease. However, a significant
proportion of patients eventually develop resistance to Imatinib. The primary mechanism of
T315I
Imatinib resistance is point mutations in the Bcr-Abl kinase domain. Bcr-Abl
is the notorious
point mutation that causes resistance to imatinib [3]. Several classes of second generation Bcr-Abl
inhibitors (Figure 1), such as Nilotinib (AMN107), Dasatinib (BMS-354825), Bosutinib (SKI-
6
06) and Bafetinib (NS-187, INNO-406), have been approved for the treatment of patients in
whom resistance to imatinib develops. However, resistance to Bcr-Abl inhibitors is still the major
reason for the failure of chemotherapy [4]. Most recently, Ponatinib (AP24534) has been approved
as third generation anti-leukaemia drugs. Despite of its potent inhibitory activity against refractory
T315I mutant, the serious side effects appeared in approximately 12% of the patient population
[5].
<Insert Figure 1 here>
Many efforts have been made to identify novel inhibitors which have efficacy against various
point mutations in Bcr-Abl. Fortunately, dozens of novel compounds possessing new scaffolds
exhibit potent Bcr-Abl inhibitory activity as well as mutants (Figure 2). The allosteric inhibitor
GNF-2 could bind to myristate binding pocket instead of ATP binding site to stabilize Bcr-Abl
into inactive state [6]. The pyrrolo-pyridine PPY-A and PPY-B could effectively inhibit the T315I
mutation [7]. PD166326, the most potent of pyrido[2,3-d]pyrimidine derivatives, provided a
possible mechanism for maintaining activity against fifty-seven Imatinib-resistant mutations [8,9].
These Bcr-Abl inhibitors shared the same aromatic-heterocyclic biphenyl scaffold. Preclinical
study had further heightened interest in identifying small molecule Bcr-Abl inhibitors to fight
against clinical acquired resistance.
<Insert Figure 2 here>
We previously developed pyrimidine biphenyl (a) possessing an aniline unit on pyrimidine ring as
Bcr-Abl inhibitor (Figure 3). It displayed potent Bcr-Abl inhibitory activity with an IC50 value of
1
7 nM. However, it only modestly suppressed Bcr-Abl induced proliferation of K562 cells with an
IC50 value of 17.8 ꢀM. It might be due to its poor solubility. In light of diverse pharmacological
activities of aromatic-heterocyclic including protein kinase inhibition, structural modification of
the pyrimidine-aniline moiety (via fragment fusion) was carried out to improve solubility and
binding affinity. Various pyrimidine bioisosteres were employed as potential favorable hinge-
binding scaffolds. Firstly, piperazine was introduced to biphenyl core as a solubilizing group.
Additionally, the flexibility and conformational diversity of piperazine might contribute to span
certain bulky amino acid side chains presented in Bcr-Abl mutations. Inspired by the hypothesis
that amide moiety would provide additional hydrogen bonds to improve binding affinity, the two
nitrogen atoms of piperazine group were acylated. This optimization is significantly distinguished
from previously reported Bcr-Abl inhibitors. A ring was expected to occupy the hydrophobic
pocket of Bcr-Abl to make favorable hydrophobic interactions. We further investigated the
potential influence of A ring by introducing different substituents. Herein we described the
synthesis and biological evaluation of aromatic-heterocyclic biphenyls as novel Bcr-Abl
2

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inhibitors.
<Insert Figure 3 here>
2
. Chemistry
Based on our previously findings, a convenient and feasible synthetic route of target compounds is
depicted in Scheme 1 [11].
<Insert Scheme 1 here>
The synthesis of title compounds mainly involved the construction of aromatic-heterocyclic
biphenyl core and the preparation of mono-benzoylation piperazine intermediates. 4-
Carboxyphenyl-boronic acid was prepared from commercial available 4-bromotoluene 1a [12,
1
3]. Compound 1a was converted to corresponding Grignard reagent 2a by Grignard reaction,
followed by esterification in tri-methylborate and acidic hydrolysis with dilute hydrochloric acid.
After recrystallization from water, the purified 4-methylphenylboronic acid 3a was oxidized with
KMnO in the presence of NaOH and TBAB (tetra-butylammonium bromide). After acidification
4
with hydrochloric acid, 4-carboxyphenyl boronic acid 4a was afforded and reacted with various
brominated aromatic heterocycles to yield key intermediates 5a-5f by palladium-catalyzed Suzuki
cross coupling reaction [14]. Mono-benzoylation piperazines 8a-8g were prepared from various
substituted benzoic acid and piperazine using CDI (N,N'-Carbonyldiimidazole) as condensing
agent [15]. Finally, the synthesis of title compounds 1-28 was accomplished via condensation
reaction using the mixed anhydride method (iso-butylchloroformate) [16].
3
. Results and discussion
Preliminary biological evaluation was performed for all the title compounds both on enzymatic
and cellular level with Imatinib as positive control. Both Bcr-Abl kinase inhibition and
antiproliferative activity against K562 cells were evaluated. Based on these results data, we
selected eight promising compounds to further test their effects on the proliferation against
Imatinib-resistant K562R cells. Concentration-dependent activity was observed and the results
obtained are summarized in Table 1.
<Insert Table 1 here>
As elucidated in Table 1, most compounds exhibited potent inhibitory activity against Bcr-Abl. In
particular, nine compounds (3, 21, 25, 7, 10, 12, 17, 22 and 26) displayed potent Bcr-Abl
inhibition with IC50 values ranging from 16 to 940 nM. The most potent compounds 25, 21 and 3
were comparable with Imatinib, with IC values of 16 nM, 66 nM and 73 nM, respectively. It was
5
0
indicated that the structures of heterocyclic in biphenyl core have no significant influence on the
inhibitory activity against Bcr-Abl. The results suggested that all of these aromatic-heterocyclics
could fit well with hinge region of Bcr-Abl. Moreover, compounds bearing different substituents
on A ring had an influence on inhibitory activity. Methoxyl substituent frequently showed better
inhibitory activity than other substituents. We speculated that the methoxyl group might form
hydrogen bond with amino acid in hydrophobic pocket of Bcr-Abl. The interaction of hydrogen
bond was benefit for the binding affinity and the activity.
In parallel, antiproliferative activities of these compounds were assessed against K562 cells. The
results revealed that antiproliferative effects were generally consistent with the enzymatic
inhibitory activity. Some compounds showed acceptable antiproliferative activity, but most of
them could moderately inhibit the growth of K562 cells. Compounds 3 and 19 exhibited relatively
higher antiproliferative activities with IC50 values of 12.5 ꢀM and 13.5 ꢀM, respectively.
Interestingly, incorporation of methyl group on pyridine ring and replacement of pyridine with
3

ACCEPTED MANUSCRIPT
thiophene were benefit for antiproliferative activity. Moreover, replacement pyridine ring with
thiophene could enhance the antiproliferative potency (IC values of compounds 19-23 ranging
5
0
from 13.5 to 152.4 ꢀM). However, replacement of pyridine with thiazole resulted in dramatic
potency loss (IC values of 24-28 ranging from 117.3 to more than 500 ꢀM ).
5
0
In order to discovery novel anti-leukemia agents, we focused on promising compounds which
were active against imatinib-resistant K562R cells. Eight compounds were evaluated for their
antiproliferative activity against K562R cells. As shown in Table 1, these compounds displayed
moderate inhibition on the growth of K562R cells. The IC50 values ranged from 57.3 ꢀM to even
more than 1000 ꢀM. Interestingly, three compounds 3, 7 and 21 displayed more sensitive to
Imatinib-resistant K562R cells than the others. From the IC50 values of 3, 7 and 21 we found that
they exhibited equivalent antiproliferative activity against Imatinib-resistant K562R cells
compared with wild K562 cells.
To rationalize the molecular basis for the inhibitory activity of these aromatic-heterocyclic
T315I
WT
biphenyls against Bcr-Abl
and Bcr-Abl , a molecular docking study was performed using
Sybyl-X 2.0. The most promising 3 showed a good balance of biological activities against Bcr-Abl
kinase and K562 cells. It was selected to carry out molecular modeling study. The crystal
WT
T315I
structures of Bcr-Abl -Imatinib complex (PDB code: 1IEP) and Bcr-Abl
-PPY-A complex
(
PDB code: 2Z60) were obtained from Protein Data Bank [17]. The binding modes of 3 with Bcr-
T315I
WT
Abl
(a) and Bcr-Abl (b) were illustrated in Figure 4. In agreement with biological results,
T315I
compound 3 showed a favorable binding affinity with Bcr-Abl
which is superior to the Bcr-
WT
Abl . It exhibited a similar binding pose to the corresponding ligand PPY-A with two hydrogen
bonds with hinge region. It can span the bulky iso-leucine side chain caused by “gatekeeper”
T315I
T315I mutation and penetrate through the central region of Bcr-Abl
. The nitrogen atom of
pyridine ring formed two hydrogen bonds with Asn 322 with distances of 2.46 Å and 2.48 Å,
respectively. Furthermore, carbonyl oxygen atom of amide formed an essential hydrogen bond
with the NH of Met 318 in the hinge region with distance of 1.91 Å. In addition, 3-methoxyphenyl
group also bound deeply into the DFG-out hydrophobic pocket. However, different from Bcr-
T315I
WT
Abl
were mediated by van de Waals interactions.
Insert Figure 4 here>
, no hydrogen bond was formed and the majority of contacts between 3 and Bcr-Abl
<
4
. Conclusion
In conclusion, a series of aromatic-heterocyclic biphenyls have been synthesized and evaluated as
novel Bcr-Abl inhibitors. The majority of these compounds displayed potent inhibitory activity
against Bcr-Abl. Fortunately, some of them exhibited acceptable antiproliferative activity against
both Bcr-Abl positive K562 cells and Imatinib-resistant K562R cells. Compounds 25, 21 and 3
displayed superior kinase inhibitory activities and compounds 3, 7 and 21 showed moderate
antiproliferative activities against Imatinib-resistant cells (K562R). In particular, the most
promising compound 3 with pyridine biphenyl incorporated with 3-methoxy substituent on A ring
exhibited meaningful antiproliferation against Imatinib-resistant K562R cells. Structure activity
relationship studies afforded a preliminary conclusion that the substituents on A ring played an
important role in Bcr-Abl inhibition, while modification of aromatic-heterocyclic had obvious
influence on cell proliferation inhibition. We will continue our efforts to optimize the present hit
compounds to overcome clinical acquired resistance against Imatinib.
5
. Experiment section
4

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5
5
.1. Chemistry
.1.1. General procedure
Chemicals and solvents were used as purchased from commercial suppliers. All anhydrous
solvents were dried according to standard methods and partly of them were freshly distilled prior
to use. NMR spectra were measured on a Bruker Advance 400 with 400 MHz proton frequency.
1
H-NMR spectra in DMSO-d were referenced to tetra-methyl silane (TMS 0.0 ppm). The solvent
6
peak and H O peak were set to 2.50 ppm and 3.33 ppm, respectively. All coupling constants are
2
given in Hz. Melting points were measured on an X-4 micromelting apparatus. Mass spectra was
partly measured using gas chromatography mass spectrometry (GC-MS) with electron impact
ionisation (EI), the other was measured using liquid chromatography mass spectrometry (LC-MS)
with electrospray ionization (ESI). Thin layer chromatography (TLC) analyses: TLC plates
covered silica gel GF254. All reaction progress was monitored by TLC and visualized with UV
light (254 nm). All compounds were purified by flash chromatography with silica gel.
5
.1.2. (4-methylphenyl)boronic acid (3a)
Dry magnesium turnings (1.8 g, 75 mmol) and 3 trace of iodine crystal were added to a 250 ml,
two-necked, round-bottomed flask is equipped with one rubber septum and a reflux condenser.
The reaction system is heated and stirred under nitrogen, and then the solution of 4-bromotoluene
(8.55 g, 50 mmol) in 40 mL anhydrous THF was added very slowly into the flask through a
syringe. After reaction has commenced, the remainder of the solution of 4-bromotoluene was
added slowly at a rate sufficient to maintain reflux. The reaction mixture was kept in refluxing
with stirring. Five hours later, the heating equipment is removed and the mixture is cooled to -30
°C. Then, the solution of trimethylborate (7.0 g, 75 mmol) in 60 mL anhydrous THF was added
slowly through a syringe. After addition finishing, the resulting mixture is allowed to stir at room
temperature for overnight and then treated with HCl (2 mol/L, 50 ml), stirred for an additional 1
hour at room temperature. The organic layer THF was separated under reduced pressure by rotary
evaporation and the aqueous layer was extracted with ethyl acetate (30 mL×3). The combined
organic layer was dried over Na SO for overnight, filtered, and concentrated by rotary
2
4
evaporation to give the crude product. The crude product was purified by recrystallization from
H O and finally dried to yield a white flocculent solid (4.2 g, 62%) of pure (4-methyl-
2
phenyl)boronic acid.
5
.1.3. 4-Carboxyphenylboronic acid (4a)
A solution of (4-methylphenyl)boronic acid (1) (3.4 g, 25 mmol) in 75 ml of 1mol/L aqueous
NaOH was added in one portion to a mixture of KMnO4 (11.8 g, 75 mmol) and
tetrabutylammoniumbromide (0.5 g) in 250 ml water. The mixture was stirred at room
temperature for overnight. 50 ml ethanol was added and stirred for an additional 1 hour. The dark
residue was filtered off and the clear filter liquor was acidified with conc.HCl (6 mol/L). The
resulting precipitate was filtered out and dried under vacuum to obtain a white solid (3.4 g, 82%).
5
.1.4. 4-pyridin-2-ylbenzoic acid (5a)
Pd(PPh ) (0.6 g, 0.5 mmol) was added to a degassed solution of 4-Carboxyphenyl-boronic acid
3
4
(
2a) (2.0 g,12 mmol), 2-bromopyridine (1. 58 g, 10 mmol) and Cs CO (13.0 g, 30 mmol) in 50
2 3
ml acetonitrile and 50 ml water. The reaction mixture was heated at 90 °C in an oil bath and
stirred under nitrogen for 48 h .The hot suspension was filtered and the filtrate distilled by rotary
evaporation to remove acetonitrile. Water was added and the mixture was exacted with ethyl
acetate (3×30 ml). The aqueous layer was acidified with conc.HCl and exacted with ethyl acetate
5

ACCEPTED MANUSCRIPT
three times. The combined organic layer was washed with brine and dried over Na SO , filtered,
2
4
and concentrated by rotary evaporation to give the crude product, which was isolated by flash
chromatography on silica gel to obtain the targeted compound (1.5 g, 75% ), mp.: 238-240 °C.
The intermediates 4-pyridin-3-ylbenzoic acid (5b), 4-(4- methylpyridin-2-yl)benzoic acid (5c), 4-
(6-methylpyridin-3 -yl)benzoic acid (5d), 4-(2-thienyl)benzoic acid (5e) and 4-(1,3-thiazol-2-
yl)benzoic acid (5f) were prepared by commercially available materials 3-bromo-pyridine, 2-
bromo-4-methylpyridine, 5-bromo-2-methylpyridine, 2-bromothiophene and 2-bromo-1,3-thiazole
respectively using the general procedure described above.
5
.1.5. 1-[3-(trifluoromethyl)benzoyl]piperazine (8a)
A solution of 3-(trifluoromethyl)benzoic acid (9.5 g, 0.05 mol) and CDI (8.90 g, 0.055 mol) was
stirred in 30 ml anhydrous THF at room temperature for 30min. In a separate round bottom flask
added piperazine (10.76 g, 0.125 mol) and piperazine dihydrochloride (20 g, 0.125 mol ) in 60 ml
of water. The reaction mixture was stirred for 5 min and added 14 g NaCl. Then add the brine
solution to the round bottom flask containing acyl imidazole and stir the reaction mixture for 5
hour. The mixture was filtered and the filtrate distilled by rotary evaporation to remove THF. The
aqueous layer was washed with ethyl acetate (3×10 ml) to remove diacylated product. The pH of
the aqueous layer was adjusted to about 9 using saturated solution of NaOH and washed with ethyl
acetate (4×30 ml).The organic layer was washed with water (4×25 ml), dried over anhydrous
Na SO for overnight, concentrated by rotary evaporation and purified by flash chromatography to
2
4
afford 1-[3-(trifluoromethyl) benzoyl] piperazine as colourless solid (6.5 g, 50%).
The intermediate compounds 1-(3-fluorobenzoyl)piperazi-ne
methoxybenzoyl)piperazine (8c), 1-(4-tert-butylbenzoyl)piperazine (8d), N,N-dimethyl-3-
piperazin-1-ylcarbonyl)aniline (8e), 1-(4-bromobenzoyl) piperazine (8f) and 1-(2,4-
(8b),
1-(3-
(
dichlorobenzoyl) piperazine (8g) were prepared by commercially available materials 3-
fluorobenzoic acid, 3-methoxybenzoic acid, 4-tert-butylbenzoic acid, 3-(dimethylamino) benzoic
acid, 4-bromobenzoic acid and 2,4-dichlorobenzoic acid respectively using the similar procedure
of 1-[3-(trifluoromethyl)benzoyl]piperazine (8a) described above.
5
.1.6. 1-(4-pyridin-2-ylbenzoyl)-4-[3-(trifluoromethyl)-benzoyl]piperazine (1)
Isobutyl chloroformate (1.5 ml, 10 mmol) and anhydrous triethylamine 4 ml were dissolved in 20
ml of anhydrous THF and cooled to 0 °C, with vigorous stirring for 10 min. A solution of 4-
pyridin-2-ylbenzoic acid (5a) (1.0 g, 5 mmol) and 2.5 ml anhydrous triethylamine in 50 ml of
THF was added slowly, maintaining the temperature at room temperature and stirred for
overnight. A solution of 1-[3-(trifluoromethyl) benzoyl]piperazine (8a) (1.93 g, 7.5 mmol) in 10
ml anhydrous THF was added and stirred for 24 hours. The mixture was filtered and the filtrate
was concentrated under reduced pressure to remove THF. The residue was poured into water and
extracted with ethyl acetate three times. The combined organic layer was dried over anhydrous
Na SO for overnight. Evaporation of the ethyl acetate under reduced pressure to provide the
2
4
crude product, which was purified by flash chromatography on silica gel (EtOAc-petroleum =
1
2
8
3
1
5
:3). A single targeted compound (0.43 g) with write solid was obtained in 20% yield; mp.:
+
1
08~210 °C ; EI-MS (m/z): 439.3 (M ); H NMR (400 MHz, DMSO) δ 8.70-8.73 (m, 2H), 8.21-
.23 (m, 2H), 8.07 (s, 2H), 8.05 (d, J = 2.1 Hz, 2H), 7.90-7.98 (m, 2H), 7.36-7.46 (m, 2H), 3.32-
1
3
.79 (m, 8H). C NMR (101 MHz, DMSO) δ 169.37, 168.19, 149.36, 148.30, 138.73, 136.19,
35.63, 135.21, 134.73, 132.49, 131.78, 128.47, 126.73, 124.46, 124.26, 122.74.
.1.7. 1-(3-fluorobenzoyl)-4-(4-pyridin-2-ylbenzoyl)piperazine (2)
6

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This compound was prepared from compounds (5a) and (8b) using the same procedure as
described for the preparation of targeted compound (1): Yield 18%; mp.: 197~199 °C; EI-MS
+
1
(
m/z): 389.3 (M ); H NMR (400 MHz, DMSO) δ 8.70 (d, J = 4.3 Hz, 1H), 8.17 (d, J = 7.8 Hz,
2
1
1
1
5
H), 8.03 (d, J = 7.9 Hz, 1H), 7.92 (d, J = 1.4 Hz, 1H), 7.56 (d, J = 7.9 Hz, 3H), 7.44–7.37 (m,
1
3
H), 7.27-7.32 (m, 3H), 3.87–3.37 (m, 8H). C NMR (101 MHz, DMSO) δ 169.44, 168.25,
63.51, 161.08, 155.57, 150.15, 140.25, 138.42, 137.83, 136.41, 131.26, 128.08, 127.00, 123.57,
20.99, 117.16, 114.64, 47.35.
.1.8. 1-(3-methoxybenzoyl)-4-(4-pyridin-2-ylbenzoyl)piperazine (3)
This compound was prepared from compounds (5a) and (8c) using the same procedure as
described for the preparation of targeted compound (1): Yield 21%; mp.: 187~188 °C; EI-MS
1
(
m/z): 401 (M+); H NMR (400 MHz, DMSO) δ 8.70 (d, J = 4.1 Hz, 1H), 8.17 (d, J = 7.5 Hz,
2
H), 8.03 (d, J = 7.8 Hz, 1H), 7.92 (t, J = 7.7 Hz, 1H), 7.55 (d, J = 7.8 Hz, 2H), 7.44–7.32 (m,
1
3
2
H), 7.00 (dd, J = 19.1, 7.8 Hz, 3H), 3.79 (s, 3H), 3.75–3.37 (m, 8H). C NMR (101 MHz,
DMSO) δ 169.42, 169.39, 159.58, 155.56, 150.16, 140.22, 137.86, 137.48, 136.44, 130.17,
1
28.08, 126.99, 123.52, 121.00, 119.42, 115.80, 112.78, 55.69.
5
.1.9. 1-(4-tert-butylbenzoyl)-4-(4-pyridin-2-ylbenzoyl)piperazine (4)
This compound was prepared from compounds (5a) and (8d) using the same procedure as
described for the preparation of targeted compound (1): Yield 15%; mp.: 80~82 °C; EI-MS (m/z):
+
1
4
7
3
1
4
5
27 (M ); H NMR (400 MHz, DMSO) δ 8.70 (d, J = 3.8 Hz, 1H), 8.17 (d, J = 7.5 Hz, 2H), 8.09–
.99 (m, 1H), 7.95–7.87 (m, 1H), 7.56 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 7.6 Hz, 2H), 7.43–7.32 (m,
13
H), 3.78–3.56 (m, 8H), 1.30 (s, 9H). C NMR (101 MHz, DMSO-d ) δ 169.79, 169.84, 154.94,
6
52.79, 152.40, 140.30, 138.75, 136.68, 130.15, 129.60, 127.68, 126.58, 125.61, 114.68, 111.69,
7.52, 34.99, 31.45.
.1.10. N,N-dimethyl-3-{[4-(4-pyridin-2-ylbenzoyl)piperazin-1-yl]carbonyl}aniline (5)
This compound was prepared from compounds (5a) and (8e) using the same procedure as
described for the preparation of targeted compound (1):Yield 20%; mp.: 199~200 °C; ESI-MS
+
1
(
m/z): 415.10 (M+H) ; H NMR (400 MHz, DMSO) δ 8.70 (d, J = 4.3 Hz, 1H), 8.17 (d, J = 7.8
Hz, 2H), 8.02-8.04 (m, 1H), 7.90-7.94(m, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.39-7.42 (m, 1H), 7.22-
1
3
7
.24 (m, 1H), 6.79 (d, J = 7.9 Hz, 1H), 6.71–6.60 (m, 2H), 3.45-3.67 (m, 8H), 2.92 (s, 6H).
C
NMR (101 MHz, DMSO) δ 170.38, 169.42, 155.58, 150.65, 150.15, 140.21, 137.84, 136.84,
1
36.47, 129.43, 128.08, 126.99, 123.50, 120.99, 114.60, 113.69, 110.84, 40.45.
5
.1.11. 1-(4-pyridin-3-ylbenzoyl)-4-[3-(trifluoromethyl)benzoyl]piperazine (6)
This compound was prepared from compounds (5b) and (8a) using the same procedure as
described for the preparation of targeted compound (1): Yield 19%; mp.: 147~148 °C; ESI-MS
+
1
(
m/z): 440 (M+H) ; H NMR (400 MHz, DMSO) δ 8.94 (s, 1H), 8.61 (d, J = 3.5 Hz, 1H), 8.13 (d,
1
3
J = 7.7 Hz, 1H), 7.92–7.79 (m, 4H), 7.78–7.68 (m, 2H), 7.62–7.49 (m, 3H), 3.72 (m, 8H).
C
NMR (101 MHz, DMSO) δ 169.37, 168.19, 149.39, 148.20, 138.78, 137.19, 135.65, 135.21,
1
34.73, 131.49, 130.18, 128.41, 127.42, 126.78, 124.42, 124.26, 122.14.
5
.1.12. 1-(3-fluorobenzoyl)-4-(4-pyridin-3-ylbenzoyl)piperazine (7)
This compound was prepared from compounds (5b) and (8b) using the same procedure as
described for the preparation of targeted compound (1): Yield 22%; mp.: 199~201 °C; EI-MS
+
1
(
m/z): 389 (M ); H NMR (400 MHz, DMSO) δ 8.95 (s, 1H), 8.61 (dd, J = 4.7, 1.3 Hz, 1H), 8.13
(
d, J = 7.9 Hz, 1H), 7.83 (d, J = 7.7 Hz, 2H), 7.55-7.62(d, J = 7.8 Hz, 2H), 7.46-7.54 (m, 2H),
1
3
7
.24-7.36 (m, 3H), 3.39-3.81 (m, 8H). C NMR (101 MHz, DMSO) δ 169.34, 168.22, 163.50,
7

ACCEPTED MANUSCRIPT
1
1
5
61.07, 149.40, 148.21, 138.78, 135.65, 135.21, 134.74, 131.28, 128.41, 127.43, 124.42, 123.55,
17.15, 114.63.
.1.13. 1-(3-methoxybenzoyl)-4-(4-pyridin-3-ylbenzoyl)piperazine (8)
This compound was prepared from compounds (5b) and (8c) using the same procedure as
described for the preparation of targeted compound (1): Yield 24%; mp.: 179~180 °C; ESI-MS
+
1
(
m/z): 402.05 (M+H) ; H NMR (400 MHz, DMSO) δ 8.94 (s, 1H), 8.61 (d, J = 3.9 Hz, 1H), 8.13
d, J = 7.5 Hz, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 7.8 Hz, 2H), 7.54–7.48 (m,1H), 7.43–
(
1
3
7
1
1
5
.32 (m, 1H), 7.00–6.93 (m, 2H), 3.79 (s, 3H), 3.73–3.40 (m, 8H). C NMR (101 MHz, DMSO) δ
69.41, 159.59, 149.39, 148.20, 138.76, 137.47, 135.67, 135.22, 134.72, 130.17, 128.42, 127.41,
24.41, 122.17, 119.42, 115.80, 112.80, 55.68, 47.38, 42.27.
.1.14. N,N-dimethyl-3-{[4-(4-pyridin-3-ylbenzoyl)piperazin-1-yl]Carbonyl}aniline (9)
This compound was prepared from compounds (5b) and (8e) using the same procedure as
described for the preparation of targeted compound (1): Yield 20%; mp.: 151~153 °C; ESI-MS
+
1
(
m/z): 415.10 (M+H) ; H NMR (400 MHz, DMSO) δ 8.94 (s, 1H), 8.61 (d, J = 3.7 Hz, 1H), 8.13
d, J = 7.7 Hz, 1H), 7.83 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 7.9 Hz, 2H), 7.53–7.46 (m, 1H), 7.30–
(
7
6
1
5
.19 (m, J = 6.7 Hz, 1H), 6.79 (d, J = 7.5 Hz, 1H), 6.71–6.60 (m, 2H), 3.71–3.39 (m, 8H), 2.92 (s,
13
H). C NMR (101 MHz, DMSO) δ 170.42, 169.36, 150.65, 149.38, 148.20, 138.75, 136.82,
35.64, 134.73, 129.44, 128.41, 127.41, 124.41, 122.17, 114.59, 113.70, 110.83, 40.43.
.1.15. 1-[4-(4-methylpyridin-2-yl)benzoyl]-4-[3-(trifluoromethyl)benzoyl]piperazine (10)
This compound was prepared from compounds (5c) and (8a) using the same procedure as
described for the preparation of targeted compound (1): Yield 19%; mp.: 205~206 °C; ESI-MS
+
1
(
m/z): 454.05 (M+H) ; H NMR (400 MHz, DMSO) δ 8.55 (d, J = 4.9 Hz,1H), 8.21 (d, J = 8.5
Hz, 1H), 8.16 (d, J = 7.0 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.92 (s, 1H), 7.87 (s, 1H), 7.80 (s, 1H),
7
2
1
1
5
.78–7.57 (m, 2H), 7.54 (d, J = 7.5 Hz, 1H), 7.24 (dd, J = 10.6, 4.8 Hz, 1H), 3.84–3.36 (m, 8H),
13
.41 (s, J = 2.8 Hz, 3H). C NMR (101 MHz, DMSO-d ) δ 169.95, 169.45, 168.05, 153.25,
6
49.34, 138.30, 136.70, 135.84, 131.48, 130.41, 130.28, 126.75, 125.09, 124.38, 124.27, 122.93,
13.60, 24.49.
.1.16. 1-(3-fluorobenzoyl)-4-[4-(4-methylpyridin-2-yl)benzoyl]piperazine (11)
This compound was prepared from compounds (5c) and (8b) using the same procedure as
described for the preparation of targeted compound (1): Yield 18%; mp.: 129~130 °C; ESI-MS
+
1
(
m/z): 404.05 (M+H) ; H NMR (400 MHz, DMSO) δ 8.54 (d, J = 4.9 Hz, 1H), 8.16 (d, J = 7.8
Hz, 2H), 7.87 (s, 1H), 7.54 (d, J = 8.0 Hz, 3H), 7.37–7.26 (m, 3H), 7.23 (d, J = 4.8 Hz, 1H), 3.83–
1
3
3
1
1
5
.37 (m, 8H), 2.41 (s, 3H). C NMR (101 MHz, DMSO) δ 169.48, 168.25, 163.52, 161.08,
55.45, 149.83, 148.46, 140.38, 138.42, 136.27, 131.25, 128.02, 126.98, 124.29, 123.57, 121.75,
17.12, 114.64, 47.31, 42.14, 21.09.
.1.17. 1-(3-methoxybenzoyl)-4-[4-(4-methylpyridin-2-yl)benzoyl]piperazine (12)
This compound was prepared from compounds (5c) and (8c) using the same procedure as
described for the preparation of targeted compound (1): Yield 19%; mp.: 158~159 °C; ESI-MS
+
1
(
m/z): 416.10 (M+H) ; H NMR (400 MHz, DMSO) δ 8.54 (d, J = 4.7 Hz, 1H), 8.16 (d, J = 7.4
Hz, 2H), 7.87 (s, 1H), 7.54 (d, J = 7.6 Hz, 2H), 7.43–7.31 (m, 1H), 7.23 (d, J = 4.5 Hz, 1H), 7.03
1
3
(
d, J = 6.7 Hz, 1H), 7.00–6.88 (m, 2H), 3.79 (s, 3H), 3.75–3.37 (m, 8H), 2.41 (s, 3H). C NMR
101 MHz, DMSO) δ 169.45, 169.39, 159.58, 155.45, 149.85, 148.49, 140.35, 137.48, 136.31,
(
1
30.17, 128.03, 126.98, 124.31, 121.77, 119.42, 115.80, 112.78, 55.69, 21.12.
5
.1.18. 1-(4-tert-butylbenzoyl)-4-[4-(4-methylpyridin-2-yl)benzoyl]piperazine (13)
8

ACCEPTED MANUSCRIPT
This compound was prepared from compounds (5c) and (8d) using the same procedure as
described for the preparation of targeted compound (1): Yield 13%; mp.: 168~169 °C; EI-MS
+
1
(
m/z): 441 (M ); H NMR (400 MHz, DMSO) δ 8.55 (d, J = 4.8 Hz, 1H), 8.16 (d, J = 7.5 Hz, 2H),
7
.88 (s, 1H), 7.54 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 7.6 Hz, 2H), 7.38 (d, J = 7.8 Hz, 2H), 7.23 (d, J
1
3
=
4.4 Hz, 1H), 3.82–3.41 (m, 8H), 2.41 (s, 3H), 1.30 (s, 9H). C NMR (101 MHz, DMSO-d ) δ
6
1
1
5
69.47, 167.35, 153.96, 152.45, 152.31, 139.95, 139.73, 133.74, 131.82, 130.70, 127.58, 125.65,
16.57, 113.30, 31.97, 30.41, 24.15.
.1.19. N,N-dimethyl-3-({4-[4-(4-methylpyridin-2-yl)benzoyl]piperazin-1-yl}carbonyl)aniline
(14)
This compound was prepared from compounds (5c) and (8e) using the same procedure as
described for the preparation of targeted compound (1): Yield 20%; mp.: 126~127 °C; EI-MS
+
1
(
m/z): 428 (M ); H NMR (400 MHz, DMSO) δ 8.54 (d, J = 4.6 Hz, 1H), 8.16 (d, J = 7.2 Hz, 2H),
7
.87 (s, 1H), 7.54 (d, J = 7.6 Hz, 2H), 7.23 (d, J = 4.4 Hz, 2H), 6.79 (d, J = 6.5 Hz, 1H), 6.73–6.58
1
3
(m, 2H), 3.85–3.42 (m, 8H), 2.92 (s, 6H), 2.41 (s, 3H). C NMR (101 MHz, DMSO) δ 170.38,
1
1
4
69.45, 155.45, 150.64, 149.85, 148.48, 140.34, 136.84, 136.34, 129.43, 128.03, 126.97, 124.30,
21.76, 114.60, 113.69, 110.84, 47.46, 42.09, 40.45, 21.12.
.1.20. 1-[4-(6-methylpyridin-3-yl)benzoyl]-4-[3-(trifleoromethyl)benzoyl]piperazine (15)
This compound was prepared from compounds (5d) and (8a) using the same procedure as
described for the preparation of targeted compound (1):Yield 19%; mp.: 177~178 °C; ESI-MS
+
1
(
m/z): 454.10 (M+H) ; H NMR (400 MHz, DMSO) δ 8.80 (s, 1H), 8.11–7.96 (m, 1H), 7.9–7.65
1
3
(
m, 6H), 7.55 (d, J = 7.6 Hz, 2H), 7.37 (d, J = 8.1 Hz, 1H), 3.87–3.39 (m, 8H), 2.51 (s, 3H).
C
NMR (101 MHz, DMSO-d ) δ 169.41, 168.13, 152.15, 145.98, 137.32, 137.20, 135.70, 129.98
6
1
28.78, 126.25, 125.39, 124.38, 124.30, 122.93, 112.70, 42.34.
5
.1.21. 1-(3-fluorobenzoyl)-4-[4-(6-methylpyridin-3-yl)benzoyl]piperazine (16)
This compound was prepared from compounds (5d) and (8b) using the same procedure as
described for the preparation of targeted compound (1): Yield 20%; m.p = 200~201 °C; EI-MS
+
1
(
m/z): 403 (M ); H NMR (400 MHz, DMSO) δ 8.80 (s, 1H), 8.02 (d, J = 6.3 Hz, 1H), 7.80 (d, J =
7
8
1
2
5
.8 Hz, 2H), 7.60 – 7.47 (m, 3H), 7.37 (d, J = 8.1 Hz, 1H), 7.35 – 7.22 (m, 3H), 3.81 – 3.38 (m,
1
3
H), 2.52 (s, 3H). C NMR (101 MHz, DMSO) δ 169.39, 168.23, 163.50, 161.06, 157.89, 147.41,
38.89, 138.35, 135.31, 134.93, 132.33, 131.20, 128.39, 127.10, 123.71, 123.58, 117.14, 114.63,
4.18.
.1.22. 1-(3-methoxybenzoyl)-4-[4-(6-methylpyridin-3-yl)benzoyl]piperazine (17)
This compound was prepared from compounds (5d) and (8c) using the same procedure as
described for the preparation of targeted compound (1): Yield 21%; mp.: 177~178 °C; ESI-MS
+
1
(
m/z): 416.10 (M+H) ; H NMR (400 MHz, DMSO) δ 8.80 (s, 1H), 8.02 (d, J = 6.3 Hz, 1H), 7.80
(
d, J = 7.8 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.03 (d, J = 7.9 Hz, 1H),
1
3
7
1
1
4
.00–6.92 (m, 2H), 3.79 (s, 3H), 3.75–3.37 (m, 8H), 2.53 (s, 3H). C NMR (101 MHz, DMSO) δ
69.38, 159.58, 157.89, 147.41, 138.87, 137.47, 135.33, 134.93, 132.33, 130.17, 128.40, 127.10,
23.71, 119.42, 115.80, 112.78, 55.69, 24.19.
.1.23. N,N-dimethyl-3-({4-[4-(6-methylpyridin-3-yl) benzoyl]piperazin-1-yl}carbonyl)aniline
(18)
This compound was prepared from compounds (5d) and (8e) using the same procedure as
described for the preparation of targeted compound (1): Yield 16%; mp.: 187~188 °C; ESI-MS
+
1
(
m/z): 429.10 (M+H) ; H NMR (400 MHz, DMSO) δ 8.80 (s, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.79
9

ACCEPTED MANUSCRIPT
(
d, J = 7.5 Hz, 2H), 7.54 (d, J = 7.9 Hz, 2H), 7.37 (d, J = 8.1 Hz, 1H), 7.32–7.19 (m, 1H), 6.79 (d,
1
3
J = 7.4 Hz, 1H), 6.72–6.58 (m, 2H), 3.88–3.41 (m, 8H), 2.92 (s, 6H), 2.52 (s, 3H). C NMR (101
MHz, DMSO) δ 170.37, 169.38, 157.88, 150.64, 147.41, 138.85, 136.83, 135.36, 134.93, 132.34,
1
29.43, 128.40, 127.09, 123.71, 114.59, 113.69, 110.83, 40.46, 24.19.
5
.1.24. 1-(3-fluorobenzoyl)-4-[4-(2-thienyl)benzoyl]piperazine (19)
This compound was prepared from compounds (5e) and (8b) using the same procedure as
described for the preparation of targeted compound (1): Yield 17%; mp.: 193~194 °C; ESI-MS
+
1
(
m/z): 395.00 (M+H) ; H NMR (400 MHz, DMSO) δ 7.74 (d, J = 7.7 Hz, 2H), 7.65–7.59 (m,
1
3
2
H), 7.56–7.45 (m, 3H), 7.36–7.25 (m, 3H), 7.20–7.16 (m, 1H), 3.78–3.37 (m, 8H). C NMR
(101 MHz, DMSO) δ 169.30, 168.21, 161.07, 142.83, 138.43, 135.46, 134.78, 131.27, 129.17,
1
28.57, 127.02, 125.73, 125.06, 123.54, 117.13, 114.63.
5
.1.25. 1-(3-methoxybenzoyl)-4-[4-(2-thienyl)benzoyl]piperazine (20)
This compound was prepared from compounds (5e) and (8c) using the same procedure as
described for the preparation of targeted compound (1): Yield 22%; mp.: 180~181 °C; EI-MS
+
1
(
m/z): 406 (M ); H NMR (400 MHz, DMSO) δ 7.74 (d, J = 7.6 Hz, 2H), 7.66–7.58 (m, 2H), 7.48
(
d, J = 7.9 Hz, 2H), 7.41–7.32 (m, 1H), 7.21–7.15 (m, 1H), 7.07–6.93 (m, 3H), 3.79 (s, 3H), 3.75–
1
3
3
1
4
5
.37 (m, 8H). C NMR (101 MHz, DMSO) δ 169.39, 169.29, 159.59, 142.84, 137.49, 135.45,
34.81, 130.17, 129.17, 128.57, 127.01, 125.73, 125.05, 119.42, 115.80, 112.79, 55.70, 47.54,
2.09.
.1.26. 1-(4-bromobenzoyl)-4-[4-(2-thienyl)benzoyl]pipe -razine (21)
This compound was prepared from compounds (5e) and (8f) using the same procedure as
described for the preparation of targeted compound (1): Yield 18%; mp.: 222~223 °C; EI-MS
+
1
(
m/z): 456 (M ); H NMR (400 MHz, DMSO) δ 7.74 (d, J = 7.8 Hz, 2H), 7.67 (d, J = 7.7 Hz, 2H),
7
3
1
5
.64–7.60 (m, 2H), 7.48 (d, J = 7.9 Hz, 2H), 7.4 (d, J = 8.0 Hz, 2H), 7.21–7.15 (m, 1H), 3.83 –
1
3
.38 (m, 8H). C NMR (101 MHz, DMSO-d ) δ 169.95, 169.36, 168.75, 153.22, 146.24, 137.30,
6
36.77, 135.94, 131.38, 126.78, 125.60, 124.38, 124.21, 122.93, 113.56.
.1.27. N,N-dimethyl-3-({4-[4-(2-thienyl)benzoyl]piperazin-1-yl}carbonyl)aniline (22)
This compound was prepared from compounds (5e) and (8e) using the same procedure as
described for the preparation of targeted compound (1): Yield 23%; mp.: 207~209 °C; ESI-MS
+
1
(
m/z): 420 (M+H) ; H NMR (400 MHz, DMSO) δ 7.74 (d, J = 7.8 Hz, 2H), 7.64–7.59 (m, 2H),
7
8
1
5
.48 (d, J = 8.1 Hz, 2H), 7.28–7.20 (m,1H), 7.20–7.16 (m, 1H), 6.71–6.62 (m, 2H), 3.78–3.39 (m,
1
3
H), 2.92 (s, 6H). C NMR (101 MHz, DMSO) δ 170.36, 169.28, 150.64, 142.83, 136.83, 135.43,
34.84, 129.43, 129.17, 128.57, 127.02, 125.72, 125.05, 114.59, 113.69, 110.83, 40.46.
.1.28. 1-(2,4-dichlorobenzoyl)-4-[4-(2-thienyl)benzoyl]-piperazine (23)
This compound was prepared from compounds (5e) and (8g) using the same procedure as
described for the preparation of targeted compound (1): Yield 22%; mp.:140~141 °C; EI-MS
+
1
(
m/z): 444 (M ); H NMR (400 MHz, DMSO) δ 7.79–7.69 (m, 3H), 7.65–7.59 (m, 2H), 7.58–7.52
1
3
(
m, 1H), 7.51–7.43 (m, 3H), 7.21–7.14 (m, 1H), 3.85–3.35 (m, 8H). C NMR (101 MHz, DMSO-
d6) δ 169.35, 168.85, 167.37, 153.21, 146.55, 136.73, 135.69, 135.15, 133.06, 132.37, 130.51,
1
28.88, 126.57, 124.87, 121.06, 113.57.
5
.1.29. 1-(3-fluorobenzoyl)-4-[4-(1,3-thiazol-2-yl)benzoyl]piperazine (24)
This compound was prepared from compounds (5f) and (8b) using the same procedure as
described for the preparation of targeted compound (1): Yield 20%; mp.: 224~225 °C; EI-MS
+
1
(
m/z): 395 (M ); H NMR (400 MHz, DMSO) δ 8.08 – 8.01 (m, 2H), 7.99 (d, J = 3.1 Hz, 1H),
1
0

ACCEPTED MANUSCRIPT
1
3
7
.87 (d, J = 3.1 Hz, 1H), 7.61–7.48 (m, 3H), 7.36 –7.24 (m, 3H), 3.78–3.39 (m, 8H). C NMR
(101 MHz, DMSO) δ 169.01, 168.24, 166.66, 161.07, 144.60, 138.34, 137.35, 134.51, 131.27,
1
28.55, 126.68, 123.54, 121.65, 117.13, 114.63.
5
.1.30. 1-(3-methoxybenzoyl)-4-[4-(1,3-thiazol-2-yl)benzoyl]piperazine (25)
This compound was prepared from compounds (5f) and (8c) using the same procedure as
described for the preparation of targeted compound (1): Yield 19%; mp.: 204~205 °C; EI-MS
+
1
(
m/z): 407 (M ); H NMR (400 MHz, DMSO) δ 8.01-8.08(m, 2H), 7.98 (d, J = 3.2 Hz, 1H), 7.86
(
d, J = 3.2 Hz, 1H), 7.56 (d, J = 7.9 Hz, 2H), 7.42 –7.23 (m, 2H), 6.98–6.94 (m, 2H), 3.79 (s, 3H),
1
3
3
1
5
.75–3.45 (m, 8H). C NMR (101 MHz, DMSO) δ 169.41, 169.02, 166.68, 159.59, 144.59,
37.46, 135.63, 134.50, 130.16, 128.55, 126.68, 121.62, 119.41, 115.79, 112.79, 55.68.
.1.31. 1-(4-bromobenzoyl)-4-[4-(1,3-thiazol-2-yl)benzoyl]piperazine (26)
This compound was prepared from compounds (5f) and (8f) using the same procedure as
described for the preparation of targeted compound (1): Yield 20%; mp.: 245~246 °C; EI-MS
+
1
(
m/z): 457 (M ); H NMR (400 MHz, DMSO) δ 8.08–8.01 (m, 2H), 7.99 (d, J = 3.1 Hz, 1H), 7.87
(
d, J = 3.2 Hz, 1H), 7.67 (d, J = 7.4 Hz, 2H), 7.56 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H),
1
3
3
1
5
.84–3.39 (m, 8H). C NMR (101 MHz, DMSO) δ 168.99, 168.72, 166.66, 144.61, 137.36,
35.20, 134.50, 131.94, 129.74, 128.56, 126.68, 123.57, 121.67.
.1.32. N,N-dimethyl-3-({4-[4-(1,3-thiazol-2-yl)benzoyl] piperazin-1-yl}carbonyl)aniline (27)
This compound was prepared from compounds (5f) and (8e) using the same procedure as
described for the preparation of targeted compound (1): Yield 23%; mp.: 86~88 °C; EI-MS (m/z):
+
1
4
3
3
1
5
20 (M ); H NMR (400 MHz, DMSO) δ 8.01-8.08 (m, 2H), 7.98 (d, J = 3.2 Hz, 1H), 7.86 (d, J =
.2 Hz, 1H), 7.56 (d, J = 8.0 Hz, 2H), 7.28–7.19 (m, 1H), 6.83–6.75 (m, 1H), 6.72–6.58 (m, 2H),
1
3
.85–3.50 (m, 8H), 2.92 (s, 6H). C NMR (101 MHz, DMSO) δ 170.55, 169.12, 166.74, 150.66,
44.56, 137.37, 136.78, 135.73, 129.46, 128.51, 126.69, 122.20, 114.57, 113.71, 110.81, 40.35.
.1.33. 1-(2,4-dichlorobenzoyl)-4-[4-(1,3-thiazol-2-yl)benzoyl]piperazine (28)
This compound was prepared from compounds (5f) and (8g) using the same procedure as
described for the preparation of targeted compound (1): Yield 21%; mp.: 175~176 °C; EI-MS
+
1
(
m/z): 445 (M ); H NMR (400 MHz, DMSO) δ 8.09–8.00 (m, 2H), 7.98 (d, J = 2.8 Hz, 1H), 7.86
1
3
(
d, J = 2.9 Hz, 1H), 7.76 (s, 1H), 7.62–7.51 (m, 3H), 7.50–7.41 (m, 1H), 3.85–3.36 (m, 8H).
C
NMR (101 MHz, DMSO) δ 169.02, 166.65, 165.41, 144.60, 137.28, 134.85, 134.52, 130.85,
1
29.88, 129.57, 128.57, 128.42, 126.66, 121.64.
5
.2. Cell culture and proliferation assay [18]
The MTT assays were performed to evaluate the antiproliferative activity of the title compounds.
Human chronic myeloid leukemia K562 cell line was kindly provided by Dr. Tao Zhang (Xi'an
Jiaotong University, Xi'an, China). Leukemia cell lines were cultured in RPMI-1640 medium
supplemented with 10% foetal bovine serum (FBS), 100 units/ml penicillin G, and 100 units/ml
streptomycin at 37°C, 5% CO humidified incubator. Dimethylsulfoxide (DMSO) was used as a
2
vehicle with the final concentration less than 0.05% to avoid toxicity. The imatinib-resistant K562
cell line (K562R) was purchased from Institute of Hematology, Chinese Academy of Medical
Sciences and Peking Union Medical College, Tianjin, China. K562R was established with
1
5.2±3.0 fold resistant to imatinib (1 ꢀM) [19]. Leukemia cells were seeded into 96-well
microtiter plates (2000 cells in 0.18 mL of nutrient medium per well), and 20 ꢀL of investigated
compounds solution with four different concentrations were added 48 h after the cell seeding, and
various concentrations were set up in triplicate. After 48 h treatment, 20 ꢀL of MTT solution (5
1
1

ACCEPTED MANUSCRIPT
mg/ml) were added to each well and incubated for further 4 h. Supernatant was removed and the
formazan precipitate was dissolved in 150 ꢀL DMSO (1%, v/v). The absorbance at 490 nm of
each well was measured by a microplate reader (Bio-Rad Instruments). All IC50 values were
reported as a mean of three experiments.
5
.3. Kinase inhibition assay [20]
Bcr-Abl inhibitory activity assay was performed using ADP-Glo™ Kinase assay kit from
Promega (Madison, WI). Briefly, ADP-Glo™ Kinase Assay is a luminescent kinase assay that
measures ADP formed from a kinase reaction. The Abl reaction utilizes ATP and generates ADP.
Then the ADP-Glo™ reagent is added to simultaneously terminate the kinase reaction and
consume the remaining ATP. Finally, ADP is converted into ATP after the Kinase Detection
Reagent is added and the newly synthesized ATP is converted into light by the luciferase reaction.
According to the established protocols of the manufacturer’s instructions, general procedures are
as the following: Abl was incubated with substrate/ATP mix and inhibitors in a final buffer of Tris
4
0 mM, MgCl 10 mM, BSA 0.1 mg/mL, DTT 1 mM in the wells of 384 low volume plate with
2
the total volume of 5 ꢀL. The assay plate was incubated at 30 °C for 60 minutes and stopped with
the addition of 5 ꢀL ADP-Glo™ Kinase Assay. Incubated at room temperature for 40 minutes,
and 10 ꢀL of Kinase Detection Reagent was added to convert ADP into ATP and introduced
luciferase and luciferin to detect ATP. The reaction system was incubated at room temperature for
3
0 minutes and then the luminescence was measured by VICTOR X multilabel plate reader. The
luminescence signal is proportional to the amount of ATP present and inversely correlated to
kinase activity.
5
.4. Molecular docking [21]
T315I
Further research of the binding mode of the title compounds with both Bcr-Abl
and Bcr-
WT
Abl , docking studies were performed by means of the software package Sybyl/Surflex-dock
within the ATP binding site based on crystal structures of Bcr-Abl. The inhibitors were docked
WT
into the active site using Ligand-Based mode. The crystal structures of Bcr-Abl -Imatinib
T315I
complex (PDB code: 1IEP) and Bcr-Abl
-PPY-A complex (PDB code: 2Z60) were used for
this study retrieved from RCSB Protein Data Bank. All crystal water molecules and ligands were
removed from the crystal structures. Hydrogen and missing atoms of the protein were added and
minimized using Tripos force field and Gasteiger-Hückel charges. Over the refined model we
defined the active site of Bcr-Abl as 5.0 Å around the crystallized ligands. The title compound 3
was constructed with Sybyl/Sketch module and optimized applying Powell’s method with Tripos
force field with convergence criterion set at 0.05 kcal/(Å mol), and assigned with Gasteiger-
Hückel method. The co-crystallized ligands served as templates to generate protomol with a
threshold of 0.50 as default settings. Other docking parameters were kept at default and al docking
calculations were carried out with Surflex-Dock.
Acknowledgment
This work was supported by the National Natural Science Foundation (NNSF) of China (Grant
No. 81302641 and 81302737), the Natural Science Basic Research Plan in Shaanxi Province of
China (Program No. 2015JQ8312), and the Fundamental Research Funds for the Central
Universities.
Reference
1
.
Z. Lu, Y. Jin, C. Chen, J. Li, Q. Cao, J. Pan, Mol Cancer. 9 (2010) 112.
1
2

ACCEPTED MANUSCRIPT
2
.
C. Zhang, C. Tan, X. Zu, X. Zhai, F. Liu, B. Chu, X. Ma, Y. Chen, P. Gong, Y. Jiang, Eur. J.
Med. Chem. 46 (2011) 1404.
3
.
.
E. Jabbour, H. Kantarjian, Am. J. Hematol. 87 (2012) 1037.
4
Y. Li, M. Shen, Z. Zhang, J. Luo, X. Pan, X. Lu, H. Long, D. Wen, F. Zhang, F. Leng, L. Li,
Z. Tu, X. Ren, K. Ding, J. Med. Chem. 55 (2012) 10033.
5
.
.
G.D. Miller, B.J. Bruno, C.S. Lim, 8 (2014) 243.
6
R. Schneider, C. Becker, J.R. Simard, M. Getlik, N. Bohlke, P. Janning, D. Rauh, J. Am.
Chem. Soc. 134 (2012) 9138.
7
.
.
.
T. Zhou, L. Parillon, F. Li, Y. Wang, J. Keats, S. Lamore, Q. Xu, W. Shakespeare, D.
Dalgarno, X. Zhu, Chem. Biol. Drug. Des. 70 (2007) 171.
8
B. Nagar, W.G. Bornmann, P. Pellicena, T. Schindler, D.R. Veach, W.T. Miller, B. Clarkson,
J. Kuriyan, J. Cancer Res. 62 (2002) 4236.
9
1
A. Quintas-Cardama, H. Kantarjian, J. Cortes, Nat. Rev. Drug .Discov. 6 (2007) 834.
0. M. Mojzych, V. Subertova, A. Bielawska, K. Bielawski, V. Bazgier, K. Berka, T. Gucky, E.
Fornal, V. Krystof, Eur. J. Med. Chem. 78 (2014) 217.
1
1. J. Dong, W. Lu, X. Pan, P. Su, Y. Shi, J. Wang, J. Zhang, Bioorg. Med. Chem. 22 (2014)
6
876.
1
2. B.M. Rosen, D.A. Wilson, C.J. Wilson, M. Peterca, B.C. Won, C. Huang, L.R. Lipski, X.
Zeng, G. Ungar, P.A. Heiney, V. Percec, J. Am. Chem. Soc. 131 (2009) 17500.
3. J.C. Namyslo, D.E. Kaufmann, Holzforschung. 63 (2009) 627.
1
1
1
1
1
4. Y. Gong, H.W. Pauls, Synlett. 6 (2000) 829.
5. S.K. Verma, R. Ghorpade, A. Pratap, M.P. Kaushik, Green Chem. 14 (2012) 326.
6. T.H. Applewhite, J.S. Binder, J. Am. Oil. Chem. Soc. 44 (1967) 423.
7. S.W. Cowan-Jacob, G. Fendrich, A. Floersheimer, P. Furet, J. Liebetanz, G. Rummel, P.
Rheinberger, M. Centeleghe, D. Fabbro, P.W. Manley, Acta Crystallogr D Biol Crystallogr.
6
3 (2007) 80.
8. F. Wang, W. Lu, T. Zhang, J. Dong, H. Gao, P. Li, S. Wang, J. Zhang, Bioorg. Med. Chem.
1 (2013) 6973.
1
2
1
2
2
9. J. Qi, H. Peng, Z.L. Gu, Z.Q. Liang, C.Z. Yang, Chin. J. Hematol. 25 (2004) 337.
0. W. Lu, B.L. Dai, W.N. Ma, Y. Zhang, Oncol. Lett. 4 (2012) 1109.
1. J. Zhang, Y. Shan, X. Pan, C. Wang, W. Xu, L. He, Chem. Biol. Drug. Des. 78 (2011) 709.
1
3

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Table 1. Structure and activities of aromatic-heterocyclic biphenyls (1-28)
Compounds
Ar
R1
Abl IC ( µM ) K562 IC ( µM ) K562R IC (µM)
50
50
50
1
2
3
4
5
6
7
8
9
3-CF3
3-F
>500
ND
>500
ND*
>500
ND
57.3 ± 8.4
ND
3-OCH3
4-tBu
0.066 ± 0.02
1.156 ± 0.47
45.6 ± 5.2
ND
12.5 ± 2.6
177.9 ± 21.3
29.7 ± 3.7
346.5 ± 46.9
63.4 ± 8.5
140.5 ± 20.1
>500
3-N(CH3)2
3-CF3
ND
ND
3-F
0.276 ± 0.09
60.67 ± 5.7
10.22 ± 2.1
0.112 ± 0.03
63.3 ± 7.0
0.94 ± 0.25
>500
81.6 ± 9.6
ND
3-OCH3
3-N(CH3)2
3-CF3
ND
1
0
1
41.6 ± 5.5
74.2 ± 7.1
76.1 ± 9.5
58.3 ± 6.8
31.4 ± 5.9
47.1 ± 4.7
64.5 ± 5.1
260.9 ± 24.0
109.5 ± 14.4
13.5 ± 0.94
152.4 ± 11.3
29.9 ± 2.7
96.7 ± 7.5
30.5 ± 5.9
>500
946.9 ± 105.3
>1000
494.4 ± 60.7
ND
1
3-F
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
3-OCH3
4-tBu
3-N(CH3)2
3-CF3
19.07 ± 4.2
ND
ND
ND
3-F
>500
ND
3-OCH3
3-N(CH3)2
3-F
0.143 ± 0.02
>500
303.9 ± 40.3
ND
5.05 ± 0.61
89.7 ± 10.5
0.073 ± 0.01
0.321 ± 0.11
ND
ND
3-OCH3
4-Br
ND
86.3 ± 10.6
ND
3-N(CH3)2
2,4-diCl
3-F
ND
444.18 ± 61.7
0.016 ± 0.006
0.421 ± 0.13
1.837 ± 0.39
>500
ND
3-OCH3
4-Br
271.1 ± 30.4
>500
492.4 ± 47.8
ND
S
N
3-N(CH3)2
2,4-di-Cl
---------
117.3 ± 15.7
>500
ND
ND
Imatinib
------
0.014 ± 0.005
1.16 ± 0.21
15.7 ± 2.8
*ND = Not Determined

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N
N
N
F
Cl
O
F
F
N
O
HN
S
N
N
HN
O
NH
N
N
N
H
N
N
N
N
H
N
N
N
H
OH
N
N
Nilotinib,AMN107
Dasatinib, BMS-345825
Imatinib,STI571
N
N
F
N
N
F
Cl
Cl
F
F
F
O
NH
F
N
O
O
HN
O
N
O
NH
N
N
N
N
N
N
N
N
H
N
N
Bosutinib, SKI-606
Ponatinib, AP24534
Bafetinib,INNO-406
Figure 1. Structures of approved Bcr-Abl inhibitors

ACCEPTED MANUSCRIPT
OH
OH
COOH
CH3
Br
COOH
CH3
B
a
b
c
d
O
B
Ar
MgBr
CH3
3a
OH
HO
N
1
a
2
a
4a
5a-5f
R
f
N
O
Ar
H
N
COOH
O
N
e
+
R
NH
1~28
R
N
H
7
a-7g
8a-8g
6
a
1
2
3
4
5
R=3-CF3
R=3-F
R=3-OCH3
R=4-tBu
10 R=3-CF3
11 R=3-F
12 R=3-OCH3
13 R=4-tBu
6
7
R=3-CF3
R=3-F
8 R=3-OCH3
R=3-N(CH3)2
Ar=
Ar=
Ar=
Ar=
N
N
N
N
9
R=3-N(CH3)2
14 R=3-N(CH3)2
1
9 R=3-F
20 R=3-OCH3
21 R=4-Br
22 R=3-N(CH3)2
3 R=2,4-diCl
24 R=3-F
25 R=3-OCH3
26 R=4-Br
27 R=3-N(CH3)2
28 R=2,4-diCl
1
5 R=3-CF3
6 R=3-F
7 R=3-OCH3
S
S
N
1
1
1
Ar=
Ar=
8 R=3-N(CH3)2
2
Scheme 1. Synthetic route of aromatic heterocyclic biphenyls (1~28).
Reagents and conditions: (a) Mg, I , THF, reflux, N (b) B(OMe) , THF, -20 ºC, HCl; (c) NaOH
2
2;
3
(
1M), KMnO , TBAB, H O; (d) brominated aromatic heterocycle, Pd(Pph ) , Cs CO ,
4
2
3 4
2
3
CH CN/H O (V:V=1:1), 90 ºC; (e) CDI, THF, piperazine/piperazine dihydrochloride (1:1), NaCl
3
2
(20%), rt; (f) ClCOO-iBu, THF, TEA, 0 ºC→rt.

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Figure 2. Several aromatic heterocyclic biphenyls potently inhibit Bcr-Abl mutations

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Figure 3. Rational design of aromatic heterocyclic biphenyls as novel Bcr-Abl inhibitors

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3
15I
WT
Figure 4. Docking of 3 with Bcr-AblT (a) and Bcr-Abl (b).
Hydrogen bonds are indicated by yellow hatched lines to key amino acids.

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Highlights
ꢀ
ꢀ
ꢀ
Discovery of aromatic-heterocyclic biphenyls as novel Bcr-Abl
inhibitors.
Two compounds showed promising antiproliferative activity against
imatinib-resistant K562 cells.
Molecular docking indicated that 3 fit well with active site of
T315I
Bcr-Abl
.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel aromatic heterocyclic
biphenyls as potential anti-leukemia agents
Jinyun Dong, Xiaoyan Pan, Jinfeng Wang, Ping Su, Lin Zhang, Fen Wei, Jie Zhang*
School of Pharmacy, Health Science Center, Xi'an Jiaotong University, No. 76, Yanta West
Road, Xi’an, Shaanxi Province, 710061, P.R. China
Table of Contents
1
13
The Name, structure, melting point, MS, H-NMR spectra, and C-NMR spectra
of Title Compounds (1-28)

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1
-(4-pyridin-2-ylbenzoyl)-4-[3-(trifluoromethyl)benzoyl]piperazine (1)
%
182.2
100.0
75.0
50.0
25.0
154.2
58.1
127.2
439.3
69.1
371.3
257.2
199.2
101.1
237.2
420.2
0.0
50
100
150
200
250
300
350
400
450
1
-(3-fluorobenzoyl)-4-(4-pyridin-2-ylbenzoyl)piperazine (2)

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%
123.2
100.0
182.1
7
5
2
5.0
0.0
5.0
1
54.2
9
5.1
3
21.2
3
89.3
6
9.1
207.2
211.2
5
6.0
77.1
252.2
250.0
2
37.2
266.2
275.0
1
47.2
323.2
325.0
361.2
350.0
0
.0
5
0.0
75.0
100.0
125.0
150.0
175.0
200.0
225.0
300.0
375.0
400.0
1
-(3-methoxybenzoyl)-4-(4-pyridin-2-ylbenzoyl)piperazine (3)
%
135
100.0
182
75.0
50.0
25.0
154
107
333
77
401
9
2
219
58
190
200
266
237
289
318
373 386
427 441
0.0
50
100
150
250
300
350
400
450

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1
-(4-tert-butylbenzoyl)-4-(4-pyridin-2-ylbenzoyl)piperazine (4)
%
161
100.0
182
75.0
50.0
25.0
359
118
58
91
245
427
77
196
200
225
266
373
441
280
303
300
344
350
399
400
458
0.0
50
100
150
250
450

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N,N-dimethyl-3-{[4-(4-pyridin-2-ylbenzoyl)piperazin-1-yl]carbonyl}aniline (5)

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1
-(4-pyridin-3-ylbenzoyl)-4-[3-(trifluoromethyl)benzoyl]piperazine (6)
1
-(3-fluorobenzoyl)-4-(4-pyridin-3-ylbenzoyl)piperazine (7)
%
123
100.0
7
5
2
5.0
0.0
5.0
1
82
58
95
1
69
321
154
389
207
250
250
01
191
200
235
277
303
300
333
371
442
450
0
.0
50
100
150
350
400

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1
-(3-methoxybenzoyl)-4-(4-pyridin-3-ylbenzoyl)piperazine (8)
Inten.(x1,000,000)
2.0
1.0
0.0
4
02.05
104.90
2.85
1
45.85
150
8
193.00
200
261.05 305.90
250 300
375.90
350 400
50
100

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N,N-dimethyl-3-{[4-(4-pyridin-3-ylbenzoyl)piperazin-1-yl]carbonyl}aniline (9)