Synthesis and antitumor activities of novel 1,4-disubstituted phthalazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.05.016

In an attempt to develop potent and selective antitumor agents,a series of novel 1,4-disubstituted phthalazine derivatives was designed and synthesized. All the prepared compounds were screened for their cytotoxic activities against A549,HT-29 and MDA-MB-231 cell lines in vitro. Among them,seven compounds (7a7e,7j and 7i) displayed excellent selectivity for MDA-MB-231 cells with IC50 values in the nM range,a desirable range for pharmacological testing. The most promising compound,7a (IC50 = 3.79 μ M,2.32 μ M,0.84 nM),was 5.6-,10.8-and 6.9 × 10⁴-times more active than PTK-787 (IC50 = 21.16 μ M,22.11 μ M,57.72 μ M),respectively.

Full text of DOI:10.1016/j.ejmech.2010.05.016

European Journal of Medicinal Chemistry 45 (2010) 3504e3510
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antitumor activities of novel 1,4-disubstituted
phthalazine derivatives
Shulan Zhang, Yanfang Zhao, Yajing Liu, Dong Chen, Weihuan Lan, Qiaoling Zhao,
*
Chengcheng Dong, Lin Xia, Ping Gong
Key Laboratory of Original New Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang,
Liaoning 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In an attempt to develop potent and selective antitumor agents, a series of novel 1,4-disubstituted
phthalazine derivatives was designed and synthesized. All the prepared compounds were screened for
their cytotoxic activities against A549, HT-29 and MDA-MB-231 cell lines in vitro. Among them, seven
compounds (7ae7e, 7j and 7i) displayed excellent selectivity for MDA-MB-231 cells with IC50 values in
the nM range, a desirable range for pharmacological testing. The most promising compound, 7a
Received 26 January 2010
Received in revised form
2 May 2010
Accepted 7 May 2010
Available online 12 May 2010
(IC50 ¼ 3.79
m
m
M, 2.32
m
M, 0.84 nM), was 5.6-, 10.8- and 6.9 ꢀ 104- times more active than PTK-787
(IC50 ¼ 21.16
M, 22.11 mM, 57.72 mM), respectively.
Keywords:
1,4-Disubstituted phthalazine
Synthesis
Ó 2010 Elsevier Masson SAS. All rights reserved.
Cytotoxic activities
1. Introduction
antitumor drugs [9e13]. From the viewpoint of molecular design,
the combination of two active molecules or pharmacophores with
a linker is a well-known approach for the build-up of drug-like
molecules, which allows us to find more potent agents. To provide
further insight into the cytotoxic activities of 1,4-disubstituted
phthalazine derivatives, we combined the inherent antitumor
agent PTK-787 and the piperazinyl group into one structure, and
introduced an acetyl-flexible linker into the structure to develop
more active agents. Therefore, we designed and synthesized
a series of novel 1,4-disubstituted phthalazine derivatives con-
taining piperazinyl groups. In addition, we screened their cytotoxic
activities against different cancer cell lines in vitro to find potent
and selective agents. Some of these compounds showed promising
cytotoxicity against cancer cell lines (Fig. 1).
Cancer is the major leading causes of death in the world.
Therefore, the design of new antitumor agents is one of the most
urgent research areas in medicinal chemistry. In the past few years,
a large number of phthalazine derivatives have been prepared and
studied for their antitumor activities [1e5]. Among them, Vatalanib
(PTK-787), a 1,4-disubstituted phthalazine VEGFR (vascular endo-
thelial growth factor receptor) inhibitor, is currently in Phase III
clinical trials for metastatic colorectal cancer [6]. Based on the
structure of PTK-787, it has been suggested that the structure
activity relationships (SARs) for 1,4-disubstituted phthalazines and
their analogues include the following: (i) a [6,6]-fused (or related)
aromatic system, (ii) a 4- or 3,4-disubstituted aniline function in the
position 1 of phthalazine, and (iii) a hydrogen-bond acceptor
(Lewis’ base: lone pair(s) of a nitrogen- or oxygen atom(s)) attached
to the position 4 via an appropriate linker (aryl or fused aryl group)
[7,8]. In this paper, our research interests have focused on the
synthesis and evaluation of the 1,4-disubstituted phthalazine
derivatives containing a piperazinyl group.
2. Chemistry
The synthetic routes of the target compounds 7ae7m and
12ae12m are illustrated in Scheme 1. Isobenzofuran-1(3H)-one (1)
was obtained by reduction of the phthalic anhydride starting
material in the presence of NaBH4 in dry THF. Next, compound 1
was treated with 4-pyridine carboxaldehyde using CH3ONa in
methanol and ethyl propionate to give the corresponding
3-hydroxy-2-(pyridin-4-yl)-1H-inden-1-one (2), which was then
reacted with 80% hydrazine hydrate to give 4-(pyridin-4-ylmethyl)
The piperazinyl group, a small and rigid heterocyclic backbone,
has been an attractive pharmacological scaffold present in several
* Corresponding author. Tel./fax: þ86 24 2398 6429.
E-mail address: gongpinggp@126.com (P. Gong).
phthalazin-1(2H)-one (3) as
a
white solid. Furthermore,
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.016

S. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 3504e3510
3505
as IC50 were summarized in Table 1. The IC50 values are the average
of at least two independent experiments.
Cl
HN
As shown in Table 1, most of the prepared compounds showed
moderate to excellent cytotoxic activities against different cancer
cell lines. Indeed, the cytotoxic activities of compounds 7ae7m
were more active than the reference drug PTK-787 against all three
human cancer cell lines, and most of compounds 12ae12m
exhibited better activities against MDA-MB-231 cells than PTK-787.
Interestingly, the pharmacological results indicated that the cyto-
toxicities of 7ae70m and 12ae12m against MDA-MB-231 cells was
higher than against A549 and HT-29 cells, which reflects excellent
selectivity for a particular human breast cancer cell type.
H
N
N
N
N
R
N
O
X
N
N
N
7a-7m: X=N, 12a-12m: X=C
Vatalanib (PTK-787)
Fig. 1. The structures of PTK-787 and the target compounds.
In most cases, the antitumor activities of 7ae7m were much
more potent than those of the corresponding compounds
12ae12m against all three human cancer cell lines. As shown in
Table 1, compounds 7ae7m displayed excellent cytotoxic activities
against A549 and HT-29 cell lines with IC50 values in the single-
chlorination of compound 3 with a solution of phosphoryl chloride
and acetonitrile formed 1-chloro-4-(pyridin-4-ylmethyl)phthala-
zine (4) [7].
Thereafter, the key important intermediate (compound 5) was
synthesized by the reaction of compound 4 with an excess of
piperazine in ethanol at 60 ꢁC. Another important intermediate
(compound 11) was obtained according to the same method as
described for compound 5 except that 4-pyridine carboxaldehyde
was replaced by benzaldehyde. The side chains 6ae6m were
synthesized via a series of substituted anilines with 2-chloroacetyl
chloride in the presence of Et3N in acetone at room temperature.
Finally, the target compounds 7ae7m and 12ae12m were
successfully obtained via the reaction of intermediate 5 and 11 with
6ae6m in the presence of K2CO3 in refluxing acetone, respectively.
The products obtained were purified by column chromatography
on silica gel. The chemical structures of these novel compounds
were confirmed by 1H NMR, 13C NMR, IR and MS spectra.
digit
mM range. In particular, seven of the compounds 7ae7m
(7ae7e, 7j and 7i) showed IC50 values in the nM range against
MDA-MB-231 cells, which were 102e103-fold more active than
those of the corresponding compounds 12ae12m (12ae12e, 12j
and 12i). It was noteworthy that the most promising compound, 7a
(IC50 ¼ 3.79
m
M, 2.32
m
M, 0.84 nM), was 5.6-, 10.8- and 6.9 ꢀ 104-
times more active than PTK-787 (IC50 ¼ 21.16
mM, 22.11 mM,
57.72
mM), respectively. The preliminary SAR showed that the
pyridinyl group at position 4 of the phthalazine scaffold plays an
important role in enhancing their antitumor activities of these
compounds.
We also studied these compounds and compared different
substituted groups of benzene ring were studied. Among
compounds 7ae7m, the cytotoxicity of compounds 7fe7i and
7m were less active than those of the other compounds against
all three different cancer cell lines. It can be concluded that the
introduction of halogen groups into the target compounds was
beneficial for enhancing their antitumor activities. Indeed,
halogen groups at the meta and para positions of the benzene
ring facilitated the increase of their cytotoxic activities of these
compounds.
3. Biological evaluation
All the compounds 7ae7m and 12ae12m were evaluated for
their cytotoxic activities on VEGFR overexpressing non-small-cell
lung cancer cell line (A549) [14], breast cancer cell line (MDA-MB-
231) [15] and human colorectal cancer cell line (HT-29) [16] by the
MTT assay, using PTK-787 as a standard. And the results expressed
OHC
O
O
OH
O
Cl
X
NH
a
c
e
d
O
X
O
N
N
N
b
O
O
1
2, X=N
8, X=C
X
H
N
X
3, X=N
9, X=C
4, X=N
10, X=C
N
H
N
R
g
N
N
6a: R= 3,4-difluoro
N
O
N
X
6b: R= 3-chloro-4-fluoro
6c: R= 3-trifluoromethyl-4-fluoro
6d: R= 2-methyl-5-fluoro
N
N
6e: R= 2-chloro-5-trifluoromethyl
6f: R= 2-chloro-6-methyl
6g: R= 2,6-difluoro
6h: R= 2,4-dimethyl
6i: R= 3,4-dimethoxy
6j: R= 3-fluoro
X
5, X=N
7a-7m, X=N
11, X=C
12a-12m, X=C
H
N
NH2
O
f
Cl
R
6k: R= 3-bromo
R
Cl
O
Cl
6l: R= 4-bromo
6m: R= 2-methyl
6a-6m
Scheme 1. Reagents and conditions: a) NaBH4/MeOH/THF, r.t. 3 h; b) CH3ONa/MeOH/ethyl propionate, r.f. 1 h; c) 80% NH2NH2$H2O, 100 ꢁC, 5 h; d) POCl3, 90 ꢁC, 3 h; e) piperazine/
EtOH, 60 ꢁC, 5 h; f) Et3N/acetone, r.t. 4e8 h; g) K2CO3/acetone, r.f. 7e12 h.

3506
S. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 3504e3510
Table 1
Cytotoxicity of the tested compounds against A549, HT-29 and MDA-MB-231 cell lines in vitro.
Compd.
X
R
IC50
A549b
3.79 ꢂ 0.01
(m
M)a
HT-29b
2.32 ꢂ 0.38
MDA-MB-231b
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
PTK-78d
N
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
C
C
C
3,4-difluoro
0.00084 ꢂ 0.00004
0.0040 ꢂ 0.0010
0.0019 ꢂ 0.0080
0.0064 ꢂ 0.0002
0.0037 ꢂ 0.0012
2.67 ꢂ 0.23
3-chloro-4-fluoro
3-trifluoromethyl-4-fluoro
2-methyl-5-fluoro
2-chloro-5-trifluoromethyl
2-chloro-6-methyl
2,6-difluoro
2,4-dimethyl
3,4-dimethoxy
3-fluoro
3-bromo
4-bromo
2-methyl
3,4-difluoro
3-chloro-4-fluoro
3-trifluoromethyl-4-fluoro
2-methyl-5-fluoro
2-chloro-5-trifluoromethyl
2-chloro-6-methyl
2,6-difluoro
2,4-dimethyl
3,4-dimethoxy
3-fluoro
12.28 ꢂ 0.27
16.01 ꢂ 0.31
38.89 ꢂ 5.00
1.85 ꢂ 0.52
7.19 ꢂ 1.02
54.37 ꢂ 2.24
45.01 ꢂ 1.31
26.48 ꢂ 1.32
4.60 ꢂ 0.41
1.53 ꢂ 0.21
2.71 ꢂ 1.16
43.09 ꢂ 1.35
10.14 ꢂ 0.62
0.88 ꢂ 0.15
1.11 ꢂ 0.11
0.47 ꢂ 0.14
4.81 ꢂ 0.22
22.59 ꢂ 2.53
28.69 ꢂ 0.78
1.93 ꢂ 0.23
40.11 ꢂ 1.44
6.13 ꢂ 0.72
1.35 ꢂ 0.17
1.99 ꢂ 0.34
15.03 ꢂ 3.1
9.51 ꢂ 0.34
13.29 ꢂ 0.24
4.59 ꢂ 0.31
170.50 ꢂ 21.30
66.77 ꢂ 5.40
53.59 ꢂ 1.78
19.65 ꢂ 2.43
NAc
1.90 ꢂ 0.31
0.24 ꢂ 0.11
0.10 ꢂ 0.02
0.0044 ꢂ 0.0003
0.0017 ꢂ 0.0002
0.0190 ꢂ 0.0030
0.20 ꢂ 0.012
2.75 ꢂ 0.26
NAc
2.48 ꢂ 0.12
NAc
7.36 ꢂ 1.25
0.76 ꢂ 0.17
19.18 ꢂ 2.54
19.47 ꢂ 1.35
3.92 ꢂ 0.41
89.03 ꢂ 3.55
7.42 ꢂ 0.52
84.53 ꢂ 4.45
5.49 ꢂ 0.71
NAc
180.50 ꢂ 15.80
32.18 ꢂ 0.79
3.73 ꢂ 0.36
NAc
144.80 ꢂ 35.90
94.96 ꢂ 4.45
13.39 ꢂ 0.56
60.18 ꢂ 7.10
113.0 ꢂ 23.80
22.11 ꢂ 0.15
C
C
C
C
129.60 ꢂ 7.12
3-bromo
4-bromo
2-methyl
NAc
3.88 ꢂ 0.21
2.00 ꢂ 0.25
NAc
21.16 ꢂ 0.89
12.42 ꢂ 0.47
17.29 ꢂ 0.88
57.72 ꢂ 6.18
a
IC50: concentration of the compound (mM) producing 50% cell growth inhibition after 72 h of drug exposure, as determined by the MTT assay. Each experiment was run at
least two times, and the results are presented as average values ꢂ standard deviation.
b
A549, non-small-cell lung adenocarcinoma cell line; HT-29, human colon cancer cell line; MDA-MB-231, human breast cancer cell line.
NA: Compounds having IC50 value > 200 mM.
Used as a positive control.
c
d
4. Conlusion
USA) with TMS as an internal standard. IR spectra (KBr disks) were
recorded with a Bruker IFS 55 instrument (Bruker). Unless other-
wise noted, all the materials were obtained from commercially
available sources and were used without further purification.
In summary, we have designed and synthesized twenty-six 1,4-
disubstituted phthalazine derivatives, and evaluated their cytotoxic
activities against all three cancer cell lines (A549, HT-29 and MDA-
MB-231). Most of the prepared compounds displayed moderate to
excellent cytotoxic activity against all three different cancer cell
lines. In particular, seven compounds (7ae7e, 7j and 7i) showed
excellent selectivity for MDA-MB-231 cells with IC50 values in the
nM range, a desirable range for pharmacological testing. From the
structure activity relationships (SARs) we may conclude that
the introduction of the pyridinyl group at position 4 of the phtha-
lazine scaffold plays an important role in enhancing their antitumor
activities of these compounds. Further studies are in progress in our
laboratories and will be reported upon in the future.
5.2. Isobenzofuran-1(3H)-one (1)
NaBH4 (126.2 g, 3.32 mol) was added slowly to a stirred solution
of phthalic anhydride (123 g, 0.83 mol) in dry THF (750 mL) at
0e10 ꢁC. Anhydrous methanol was added to the reaction mixture
drop-wise at 0e10 ꢁC, and the reaction mixture was stirred at room
temperature for 3 h. The mixture was concentrated under reduced
pressure. The residue was poured into a 10% hydrochloric acid
aqueous solution (1000 mL), stirred intensively for 3 h and sepa-
rated by filtration. The solid was alkalized with a 10% Na2CO3
aqueous solution (1000 mL), stirred for 1 h and separated by
filtration to give compound 1 as a white crystal (78 g, 70%). m.p.:
72e73 ꢁC. MS[MHþ](m/z): 135.1(M þ 1).$1H NMR$(300 MHz,
5. Experimental protocols
DMSO)
d
: 7.85 (d, J ¼ 7.6 Hz, 1H), 7.78 (t, J ¼ 7.4 Hz, 1H), 7.68 (d,
5.1. Chemistry
J ¼ 7.7 Hz, 1H), 7.59 (t, J ¼ 7.4 Hz, 1H), 5.42 (s, 2H). C8H6O2 (134.13).
All melting points were obtained on a Büchi Melting Point B-540
apparatus (Büchi Labortechnik, Flawil, Switzerland) and were
uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent
1100 LC-MS (Agilent, Palo Alto, CA, USA). Proton (1H) nuclear
magnetic resonance spectroscopy were performed using Bruker
ARX-300, 300 MHz spectrometers (Bruker Bioscience, Billerica, MA,
5.3. 3-Hydroxy-2-(pyridin-4-yl)-1H-inden-1-one (2)
A solution of 1 50 g (0.37 mol) and 4-pyridine carboxaldehyde
42 g (0.39 mol) in ethyl propionate (200 mL) was portion-wise
added to a stirred solution of CH3ONa (82 g, 1.49 mol) in MeOH

S. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 3504e3510
3507
(800 mL) at 0e10 ꢁC. The reaction mixture was stirred at room
temperature for 1 h, heated to reflux for 1 h, and then concentrated
under reduced pressure. The residue was poured into ice water
(1000 mL), acidified with glacial acetic acid to pH 2e3 and sepa-
rated by filtration to give compound 2 as an orange solid (43.8 g,
52.6%). m.p.: >300 ꢁC. MS[MHþ](m/z): 224.3 (M þ 1). 1H NMR
reduced pressure, and purified by chromatography on silica gel
using MeOH/CH2Cl2 to obtain 7ae7m as light yellow crystals.
5.8.1. N-(3,4-Difluorophenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (7a)
Yield: 36%. m.p.: 207e208 ꢁC. MS[MHþ](m/z): 475.1 (M þ 1). IR
(KBr) cmꢃ1: 3263.3, 3056.8, 2932.0, 2846.7, 1676.1, 1604.5, 1517.5,
1411.9, 1368.3, 1264.0, 1204.7, 1150.4, 1134.7, 1015.6, 777.9. 1H NMR
(300 MHz, DMSO)
d: 13.23 (s, 1H), 8.72 (d, J ¼ 7.0 Hz, 2H), 8.18
(d, J ¼ 7.0 Hz, 2H), 7.51 (m, 4H). C14H9NO2 (223.23).
(300 MHz, DMSO)
d
: 10.12 (s, 1H), 8.45 (d, J ¼ 5.8 Hz, 2H), 8.22e8.14
(m, 1H), 8.14e8.07 (m, 1H), 8.01e7.82 (m, 3H), 7.51e7.36 (m, 2H),
7.31 (d, J ¼ 5.7 Hz, 2H), 4.62 (s, 2H), 3.47 (s, 4H), 3.29 (s, 2H), 2.83 (s,
5.4. 4-(Pyridin-4-ylmethyl)phthalazin-1(2H)-one (3)
4H). 13C NMR (150.9 MHz, DMSO)
d: 168.77, 159.43, 149.77, 148.01,
A solution of 80% hydrazine hydrate (NH2NH2$H2O) (306.6 mL,
4.9 mol) and compound 2 (43.8 g, 0.20 mol) was stirred at 100 ꢁC
for 5 h. The mixture was cooled, separated by filtration and washed
with EtOH to give compound 3 þas a light yellow crystal (34.9 g,
75%). m.p.: 210e211 ꢁC. MS[MH ](m/z): 238.3 (M þ 1). 1H NMR
135.88, 132.27, 132.01, 126.85, 125.26, 124.88, 124.29, 120.95, 117.55,
117.44, 115.97, 108.70, 61.79, 52.75, 50.85, 37.66. C26H24F2N6O
(474.51).
5.8.2. N-(3-Chloro-4-fluorophenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (þ7b)
(300 MHz, DMSO)
d
: 12.64 (s, 1H), 8.46 (d, J ¼ 4.3 Hz, 2H), 8.26
(d, J ¼ 7.4 Hz, 1H), 8.00e7.77 (m, 3H), 7.32 (d, J ¼ 5.2 Hz, 2H), 4.34
Yield: 33%. m.p.:169e170 ꢁC. MS[MH ](m/z): 491.1 (Cl ¼ 35),
493.1 (Cl ¼ 37). IR (KBr) cmꢃ1: 3421.8, 2923.4, 2851.8,1681.0,1601.3,
1524.2, 1499.6, 1411.6, 1386.3, 1264.8, 1013.2. 1H NMR (300 MHz,
(s, 2H). C14H11N3O (237.26).
5.5. 1-Chloro-4-(pyridin-4-ylmethyl)phthalazine (4)
DMSO)
d
: 10.11 (s, 1H), 8.45 (d, J ¼ 6.0 Hz, 2H), 8.14 (m, 2H), 8.02
(m,1H), 7.96e7.88 (m, 2H), 7.70e7.59 (m,1H), 7.38 (t, J ¼ 9.1 Hz,1H),
Compound 3 (17.5 g, 0.07 mol) was added to a stirred solution of
POCl3 (170 mL) and CH3CN (80 mL) at room temperature, and then
3 drops of DMF were added to the mixture. The reaction mixture
was heated to 90 ꢁC for 3 h. The mixture was concentrated under
reduced pressure. The residue was poured into ice water (500 mL),
alkalized with a 10% Na2CO3 aqueous solution to pH 7e8 and
separated by filtration to give compound 4 as a red solid (13.5 g,
71.6%). m.p.: 180e181 ꢁC. MS[MHþ](m/z): 255.8 (Cl ¼ 35), 257.8
7.31 (d, J ¼ 5.9 Hz, 2H), 4.62 (s, 2H), 3.47 (s, 4H), 3.29 (s, 2H), 2.83
(s, 4H). 13C NMR (75.5 MHz, DMSO)
d: 168.78, 159.41, 154.13, 149.75,
148.00, 136.14, 132.24, 131.98, 126.83, 125.23, 124.86,124.27, 121.07,
119.91, 117.08, 61.71, 52.71, 50.83, 37.64. C26H24ClFN6O (490.96).
5.8.3. N-(4-Fluoro-3-(trifluoromethyl)phenyl)-2-(4-(4-(pyridin-4-
ylmethyl)phthalazin-1-yl)piperazin-1-yl) acetamide (7c)
Yield: 22%. m.p.: 121e122 ꢁC. MS[MHþ](m/z): 525.4 (M þ 1). IR
(KBr) cmꢃ1: 3412.3, 2922.1, 2849.2, 1692.2, 1601.1, 1506.9, 1450.2,
1411.6, 1370.7, 1217.6, 1194.4, 1164.1, 1131.0, 1012.6. 1H NMR
(Cl ¼ 37). 1H NMR (300 MHz, DMSO)
: 8.46 (d, J ¼ 4.3 Hz, 2H), 8.35
d
(m, 2H), 8.13 (m, 2H), 7.33 (d, J ¼ 5.2 Hz, 2H), 4.76 (s, 2H).
C14H10ClN3 (255.70).
(300 MHz, DMSO) d: 10.22 (s, 1H), 8.45 (s, 2H), 8.18 (m, 3H), 7.93
(m, 3H), 7.49 (m, 1H), 7.31 (s, 2H), 4.62 (s, 2H), 3.47 (s, 4H),
3.31e3.27 (m, 2H), 2.83 (s, 4H). C27H24F4N6O (524.51).
5.6. 1-(Piperazin-1-yl)-4-(pyridin-4-ylmethyl)phthalazine (5)
Compound 4 (13.5 g, 0.05 mol) was added portion-wise to
a stirred solution of piperazine (30.4 g, 0.4 mol) in anhydrous
ethanol at room temperature. The mixture was heated to 60 ꢁC for
5 h, and then concentrated under reduced pressure. The residue
was poured into ice water (200 mL), stirred intensively for 1 h and
separated by filtration to give compound 5 as a light yellow solid
(11.45 g, 71%). m.p.: 209e210 ꢁC. MS[MHþ](m/z): 306.4 (M þ 1).
C18H19N5 (305.38).
5.8.4. N-(5-Fluoro-2-methylphenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (7d)
Yield: 21%. m.p.: 192e193 ꢁC. MS[MHþ](m/z): 471.4 (M þ 1). IR
(KBr) cmꢃ1: 3311.6, 2919.0, 2838.9, 1685.4, 1600.8, 1523.2, 1450.2,
1413.0, 1394.8, 1149.4, 1131.2, 1270.1, 1013.2, 778.8. 1H NMR
(300 MHz, DMSO)
d
: 9.61 (s, 1H), 8.45 (d, J ¼ 5.5 Hz, 2H), 8.21e8.16
(m,1H), 8.13 (m, 1H), 7.93 (m, 2H), 7.82 (dd, J ¼ 11.3, 2.6 Hz,1H), 7.32
(d, J ¼ 5.7 Hz, 2H), 7.30e7.23 (m, 1H), 6.88 (t, J ¼ 8.4 Hz, 1H), 4.63
(s, 2H), 3.48 (s, 4H), 3.31 (s, 2H), 2.89 (s, 4H), 2.26 (s, 3H). 13C NMR
5.7. General procedure for preparation of 2-chloro-N-(substituted-
phenyl)acetamide (6ae6m)
(75.5 MHz, DMSO) d: 168.36, 159.32, 154.21, 149.70, 147.92, 137.38,
132.23, 131.97, 131.49, 126.82, 125.20, 124.77, 124.25, 123.86, 120.91,
110.25, 107.71, 61.43, 52.79, 51.22, 37.62. C27H27FN6O (470.54).
Triethylamine (36.7 mL, 0.3 mol) was added to a solution of
substituted aniline (0.2 mol) in acetone (250 mL), and then
2-chloroacetyl chloride (21.4 mL, 0.26 mol) was added drop-wise to
the reaction mixture at 0e10 ꢁC. The reaction mixture was stirred at
room temperature for 4e8 h. The mixture was concentrated under
reduced pressure. The residue was poured into ice water (200 mL),
stirred at room temperature for 1 h and separated by filtration to
obtain 6ae6m.
5.8.5. N-(2-Chloro-5-(trifluoromethyl)phenyl)-2-(4-(4-(pyridin-4-
ylmethyl)phthalazin-1-yl)piperazin-1-yl) acetamide (7e)
Yield: 24%. m.p.: 189e190 ꢁC. MS[MHþ](m/z): 541.2 (Cl ¼ 35),
543.1 (Cl ¼ 37). IR (KBr) cmꢃ1: 3252.3, 2919.2, 2846.2, 1700.1,
1601.6, 1588.2, 1526.4, 1430.3, 1331.4, 1262.5, 1169.6, 1129.2, 1079.2,
1012.1, 778.8. 1H NMR (300 MHz, DMSO)
d: 10.23 (s, 1H), 8.69
(s, 1H), 8.45 (d, J ¼ 5.6 Hz, 2H), 8.23e8.16 (m, 1H), 8.13 (m, 1H),
7.99e7.86 (m, 2H), 7.82 (d, J ¼ 8.4 Hz, 1H), 7.53 (d, J ¼ 8.4 Hz, 1H),
7.32 (d, J ¼ 5.6 Hz, 2H), 4.63 (s, 2H), 3.50 (s, 4H), 3.38 (s, 2H), 2.91
(s, 4H). C27H24ClF3N6O (540.97).
5.8. General procedure for preparation of compound (7ae7m)
K2CO3 (0.23 g, 1.64 mmol) was added to a stirred solution of
compound 5 (0.2 g, 0.65 mmol) and 2-chloro-N-(substituted-
phenyl)acetamide (0.72 mmol) in acetone (2 mL). The reaction
mixture was heated to reflux for 7e12 h. The mixture was cooled,
separated by filtration. The filter liquor was concentrated under
5.8.6. N-(2-Chloro-6-methylphenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (7f)
Yield: 26%. m.p.: 85e86 ꢁC. MS[MHþ](m/z): 487.1 (Cl ¼ 35),
489.2 (Cl ¼ 37). IR (KBr) cmꢃ1: 3418.2, 2920.2, 2846.0, 1682.6,

3508
S. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 3504e3510
1598.9, 1496.1, 1411.9, 1266.0, 1178.0, 1150.8, 1132.1, 1012.5, 777.4. 1H
NMR (300 MHz, DMSO)
(m, 2H), 7.92 (m, 2H), 7.40e7.28 (m, 3H), 7.22 (m, 2H), 4.62 (s, 2H),
3.50 (s, 4H), 3.30 (s, 2H), 2.89 (s, 4H), 2.21 (s, 3H). C27H27ClN6O
(487.00).
5.8.12. N-(4-Bromophenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (7l)
d
: 9.54 (s, 1H), 8.45 (d, J ¼ 5.8 Hz, 2H), 8.15
Yield: 28%. m.p.: 78e79 ꢁC. MS[MHþ](m/z): 517.1 (Br ¼ 79), 519.1
(Br ¼ 81). IR (KBr) cmꢃ1: 3407.6, 2919.9, 2845.5, 1688.8, 1600.6,
1514.0, 1412.2, 1395.0, 1306.9, 1266.7, 1010.5, 826.7, 778.2. 1H NMR
(300 MHz, DMSO)
d
: 10.00 (s, 1H), 8.45 (d, J ¼ 5.8 Hz, 2H), 8.20e8.14
5.8.7. N-(2,6-Difluorophenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (7g)
(m, 1H), 8.12 (m, 1H), 8.03e7.82 (m, 2H), 7.67 (d, J ¼ 8.9 Hz, 2H),
7.59e7.38 (d, J ¼ 8.7 Hz, 2H), 7.31 (d, J ¼ 5.9 Hz, 2H), 4.62 (s, 2H),
3.46 (s, 4H), 3.28 (s, 2H), 2.83 (s, 4H). C26H25BrN6O (517.42).
Yield: 46%. m.p.: 74e75 ꢁC. MS[MHþ](m/z): 475.3 (M þ 1). IR
(KBr) cmꢃ1: 3218.7, 2938.8, 2822.9, 1692.2, 1598.6, 1511.5, 1467.0,
1416.0, 1304.1, 1283.7, 1267.1, 1242.5, 1009.5, 780.1. 1H NMR
5.8.13. N-(2-Methylphenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (7m)
(300 MHz, DMSO)
d: 9.60 (s, 1H), 8.45 (d, J ¼ 5.7 Hz, 2H), 8.17
(m, 1H), 8.15e8.09 (m, 1H), 7.92 (m, 2H), 7.38 (m, 1H), 7.31
(d, J ¼ 5.8 Hz, 2H), 7.17 (t, J ¼ 8.1 Hz, 2H), 4.62 (s, 2H), 3.49 (s, 4H),
3.33 (s, 2H), 2.85 (s, 4H). C26H24F2N6O (474.51).
Yield: 36%. m.p.: 183e184 ꢁC. MS[MHþ](m/z): 453.3 (M þ 1). IR
(KBr) cmꢃ1: 3303.2, 2919.0, 2831.0, 1687.7, 1599.7, 1526.8, 1456.5,
1411.6, 1266.2, 1178.1, 1152.0, 1132.4, 1012.2, 755.0. 1H NMR
(300 MHz, DMSO)
d
: 9.48 (s, 1H), 8.45 (d, J ¼ 5.3 Hz, 2H), 8.18
5.8.8. N-(2,4-Dimethylphenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (7h)
(m, 1H), 8.13 (m, 1H), 7.99e7.88 (m, 2H), 7.82 (d, J ¼ 7.7 Hz, 1H), 7.32
(d, J ¼ 5.6 Hz, 2H), 7.21 (m, 2H), 7.05 (t, J ¼ 7.2 Hz, 1H), 4.63 (s, 2H),
3.49 (s, 4H), 3.29 (s, 2H), 2.89 (s, 4H), 2.27 (s, 3H). C27H28N6O
(452.55).
Yield: 33%. m.p.: 158e159 ꢁC. MS[MHþ](m/z): 467.1 (M þ 1). IR
(KBr) cmꢃ1: 3340.7, 2922.5, 2842.4, 1681.9, 1598.6, 1511.7, 1451.3,
1412.2, 1394.8, 1268.0, 1128.3, 1014.7. 1H NMR (300 MHz, DMSO)
d:
9.39 (s, 1H), 8.45 (d, J ¼ 5.9 Hz, 2H), 8.17 (m, 1H), 8.13 (m, 1H),
8.01e7.84 (m, 2H), 7.65 (d, J ¼ 8.1 Hz, 1H), 7.32 (d, J ¼ 5.7 Hz, 2H),
7.04 (s, 1H), 6.99 (d, J ¼ 8.1 Hz, 1H), 4.63 (s, 2H), 3.48 (s, 4H), 3.27
(s, 2H), 2.88 (s, 4H), 2.24 (s, 3H), 2.23 (s, 3H). C28H30N6O (466.58).
5.9. 3-Hydroxy-2-phenyl-1H-inden-1-one (8)
Prepared in a similar procedure as described for compound 2,
prepared from isobenzofuran-1(3H)-one 1 (30 g, 0.22 mol) and
benzaldehyde (23.7 mL, 0.23 mol) to give compound 8 as a light
yellow solid (34 g, 68.4%). m.p.: 147e148 ꢁC. MS[MHþ](m/z): 223.2
(M þ 1). C15H10O2 (222.24).
5.8.9. N-(3,4-Dimethoxyphenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (7i)
Yield: 32%. m.p.: 81e82 ꢁC. MS[MHþ](m/z): 499.4 (M þ 1). IR
(KBr) cmꢃ1: 3408.3, 2913.8, 2832.7, 1675.7, 1602.0, 1514.6, 1412.2,
1263.6,1234.0,1133.7,1025.0, 806.1, 780.0, 755.4.1H NMR (300 MHz,
5.10. 4-Benzylphthalazin-1-ol (9)
DMSO)
d
: 9.67 (s, 1H), 8.45 (d, J ¼ 5.5 Hz, 2H), 8.16 (m, 1H), 8.11 (m,
Prepared in a similar procedure as described for compound 3,
prepared from compound 8 (10 g, 0.045 mol) to give compound 9 as
a white crystal (7.8 g, 73.6%). m.p.: 200e201 ꢁC. MS[MHþ](m/z):
d: 12.60 (s, 1H), 8.25 (d,
J ¼ 7.7 Hz, 1H), 7.93 (d, J ¼ 7.6 Hz, 1H), 7.90e7.76 (m, 2H), 7.37e7.23
(m, 4H), 7.19 (t, J ¼ 6.5 Hz, 1H), 4.30 (s, 2H). C15H12N2O (236.27).
1H), 7.93 (m, 2H), 7.42 (d, J ¼ 4.0 Hz, 1H), 7.39 (s, 1H), 7.31 (d,
J ¼ 5.5 Hz, 2H), 7.05 (d, J ¼ 8.1 Hz,1H), 4.62 (s, 2H), 3.46 (s, 4H), 3.24 (s,
2H), 2.82 (s, 4H), 2.19 (s, 3H), 2.16 (s, 3H).13C NMR (75.5 MHz, DMSO)
237.1 (M þ 1). 1H NMR (300 MHz, DMSO)
d: 167.87,159.38,149.71,148.61,147.97,145.01,132.41,132.20,131.93,
126.81, 125.19, 124.83, 124.23, 120.91, 112.08, 111.44, 104.77, 61.83,
55.83, 55.51, 52.75, 50.87, 37.63. C28H30N6O3 (498.58).
5.11. 1-Chloro-4-benzylphthalazine (10)
5.8.10. N-(3-Fluorophenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamide (7j)
Prepared in a similar procedure as described for compound 4,
prepared from compound 9 (7.8 g, 33 mmol) to give compound 10
as a milk white solid (8.3 g, 99%). m.p.: 145e146 ꢁC. MS[MHþ](m/z):
Yield: 37%. m.p.: 74e75 ꢁC. MS[MHþ](m/z): 457.2 (M þ 1). IR
(KBr) cmꢃ1: 3280.9, 2920.2, 2826.8, 1687.8, 1600, 1528.6, 1493.4,
1443.0, 1414.7, 1265.6, 1134.2, 1013.4, 777.9. 1H NMR (300 MHz,
255.0 (Cl ¼ 35), 257.0 (Cl ¼ 37). 1H NMR (300 MHz, DMSO)
d: 8.38
DMSO)
d
: 10.09 (s, 1H), 8.45 (d, J ¼ 5.4 Hz, 2H), 8.17 (m, 1H), 8.12
(dd, J ¼ 6.2, 3.1 Hz, 1H), 8.31 (dd, J ¼ 6.3, 3.1 Hz, 1H), 8.17e8.06 (m,
2H), 7.37e7.24 (m, 4H), 7.19 (t, J ¼ 7.1 Hz, 1H), 4.72 (s, 2H).
C15H11ClN2 (254.71).
(m, 1H), 8.00e7.84 (m, 2H), 7.68 (d, J ¼ 11.8 Hz, 1H), 7.44
(d, J ¼ 8.3 Hz, 1H), 7.40e7.33 (m, 1H), 7.31 (d, J ¼ 5.4 Hz, 2H), 6.89
(t, J ¼ 7.2 Hz, 1H), 4.62 (s, 2H), 3.47 (s, 4H), 3.30 (s, 2H), 2.83 (s, 4H).
13C NMR (150.9 MHz, DMSO)
d: 168.77, 162.96, 161.37, 159.35,
5.12. 1-(Piperazin-1-yl)-4-benzylphthalazine (11)
154.03, 149.68, 147.93, 140.59, 140.51, 132.15, 131.88, 130.34, 130.28,
126.79, 125.14, 124.78, 124.21, 120.89, 115.28, 109.91, 109.77, 106.37,
106.19, 61.71, 52.65, 50.82, 37.61. C26H25FN6O (456.51).
Prepared in a similar procedure as described for compound 5,
prepared from compound 10 (7.5 g, 29.4 mmol) to give compound
11 as a white solid (5.5 g, 61%). m.p.: 77e78 ꢁC. MS[MHþ](m/z):
305.3 (M þ 1). C19H20N4 (304.39).
5.8.11. N-(3-Bromophenyl)-2-(4-(4-(pyridin-4-ylmethyl)
phthalazin-1-yl)piperazin-1-yl)acetamiþde (7k)
Yield: 52%. m.p.: 74e75 ꢁC. MS[MH ](m/z): 517.0 (Br ¼ 79), 519.0
(Br ¼ 81). IR (KBr) cmꢃ1: 3418.0, 2919.0, 2846.6, 1686.2, 1593.0,
1516.8, 1477.7, 1415.3, 1307.3, 1266.0, 1012.7, 777.9. 1H NMR
5.13. General procedure for preparation of compound (12ae12m)
Prepared in a similar procedure as described for 7ae7m.
(300 MHz, DMSO)
d
: 10.05 (s, 1H), 8.45 (d, J ¼ 5.7 Hz, 2H), 8.17 (m,
1H), 8.12 (m, 1H), 8.04 (s, 1H), 7.99e7.86 (m, 2H), 7.64 (d, J ¼ 7.3 Hz,
5.13.1. N-(3,4-Difluorophenyl)-2-(4-(4-benzylphthalazin-1-yl)
piperazin-1-yl)acetamide (12a)
1H), 7.31 (d, J ¼ 5.6 Hz, 2H), 7.26 (m, 2H), 4.62 (s, 2H), 3.47 (s, 4H),
3.29 (s, 2H), 2.83 (s, 4H). 13C NMR (75.5 MHz, DMSO)
d: 168.84,
Yield: 44%. m.p.: 73e75 ꢁC. MS[MHþ](m/z): 474.4 (M þ 1). IR
(KBr) cmꢃ1: 3283.8, 3061.2, 2921.0, 2827.3, 1689.4, 1615.5, 1517.2,
1411.2, 1264.9, 1205.4, 1132.6, 1013.4, 763.1. 1H NMR (300 MHz,
159.37, 154.05, 149.70, 147.96, 140.45, 132.18. 131.92, 130.71, 126.81,
126.02, 125.17, 124.81, 124.24, 121.89, 121.54, 120.90, 118.34, 61.68,
52.65, 50.84, 37.63. C26H25BrN6O (517.42).
DMSO) d: 10.24 (s, 1H), 8.18 (m, 1H), 8.09 (m, 1H), 7.88 (m, 3H),

S. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 3504e3510
3509
7.51e7.36 (m, 2H), 7.36e7.21 (m, 4H), 7.16 (t, J ¼ 7.1 Hz, 1H), 4.58
(d, J ¼ 6.8 Hz, 1H), 7.97e7.82 (m, 2H), 7.38e7.30 (m, 3H), 7.25
(m, 4H), 7.17 (t, J ¼ 6.7 Hz, 1H), 4.58 (s, 2H), 3.49 (s, 4H), 3.30
(s, 2H), 3.47 (s, 4H), 3.37 (s, 2H), 2.88 (s, 4H). 13C NMR (75.5 MHz,
DMSO)
d: 168.72, 159.28, 155.17, 139.10, 131.98, 131.71, 128.63,
(s, 2H), 2.89 (s, 4H), 2.22 (s, 3H). 13C NMR (75.5 MHz, DMSO)
d:
128.55, 126.82, 126.35, 125.44, 124.70, 121.00, 117.50, 117.26, 115.93,
108.73, 108.45, 61.72, 52.71, 50.82, 38.48. C27H25F2N5O (473.20).
168.30, 159.26, 155.13, 139.09, 138.36, 133.80, 131.95, 131.77,
131.68, 129.01, 128.61, 128.53, 127.87, 126.90, 126.82, 126.32,
125.41, 124.71, 120.99, 61.29, 52.94, 50.82, 38.47. C28H28ClN5O
(485.20).
5.13.2. N-(3-Chloro-4-fluorophenyl)-2-(4-(4-benzylphthalazin-1-
yl)piperazin-1-yl)acetamide (12b)
Yield: 41%. m.p.: 77e79 ꢁC. MS[MHþ](m/z): 490.3 (Cl ¼ 35),
492.3 (Cl ¼ 37). IR (KBr) cmꢃ1: 3277.0, 3028.3, 2918.6, 2826.3,
1692.0, 1602.6, 1498.7, 1400.3, 1263.8, 1210.5, 1131.6, 1013.0, 819.9,
5.13.7. N-(2,6-Difluorophenyl)-2-(4-(4-benzylphthalazin-1-yl)
piperazin-1-yl)acetamide (12g)
Yield: 32%. m.p.: 177e178 ꢁC. MS[MHþ](m/z): 474.3 (M þ 1). IR
(KBr) cmꢃ1: 3417.9, 2920.9, 2849.4, 1701.5, 1598.8, 1512.4, 1466.7,
1412.9, 1266.9, 1132.4, 1178.7, 1152.3, 1132.4, 1010.2, 779.7. 1H NMR
762.5. 1H NMR (300 MHz, DMSO)
d: 10.12 (s, 1H), 8.18 (m, 1H), 8.10
(m, 1H), 8.03 (dd, J ¼ 6.9, 2.6 Hz, 1H), 7.90 (m, 2H), 7.64 (m, 1H), 7.40
(d, J ¼ 9.1 Hz, 1H), 7.30 (m, 4H), 7.17 (t, J ¼ 7.1 Hz, 1H), 4.58 (s, 2H),
(300 MHz, DMSO)
7.94e7.83 (m, 2H), 7.41e7.22 (m, 5H), 7.16 (m, 3H), 4.57 (s, 2H), 3.46
(s, 4H), 3.31 (s, 2H), 2.83 (s, 4H). 13C NMR (75.5 MHz, DMSO)
d:
d: 9.61 (s, 1H), 8.17 (m, 1H), 8.09 (m, 1H),
3.46 (s, 4H), 3.29 (s, 2H), 2.83 (s, 4H). 13C NMR (75.5 MHz, DMSO)
d:
169.42, 159.96, 155.86, 139.77, 132.67, 132.40, 129.30, 129.23, 127.47,
127.03, 126.13, 125.38, 121.69, 120.54, 117.70, 62.35, 53.37, 51.48,
39.14. C27H25ClFN5O (489.17).
168.87, 159.28, 155.17, 139.10, 131.99, 131.73, 128.63, 128.55, 126.82,
126.35, 125.44, 124.71, 121.01, 111.98, 111.68, 60.98, 52.72, 50.78,
38.49. C27H25F2N5O (473.20).
5.13.3. N-(4-Fluoro-3-(trifluoromethyl)phenyl)-2-(4-(4-
benzylphthalazin-1-yl)piperazin-1-yl)acetamide (12c)
5.13.8. N-(2,4-Dimethylphenyl)-2-(4-(4-benzylphthalazin-1-yl)
piperazin-1-yl)acetamide (12h)
Yield: 33%. m.p.: 77e79 ꢁC. MS[MHþ](m/z): 524.1 (M þ 1). IR
(KBr) cmꢃ1: 3284.6, 3063.4, 2931.8, 2827.8, 1695.6, 1606.0, 1506.4,
1424.3, 1327.3 1267.7, 1247.2, 1132.1, 1053.7, 1013.6, 899.3, 824.2. 1H
Yield: 31%. m.p.: 176e178 ꢁC. MS[MHþ](m/z): 466.2 (M þ 1). IR
(KBr) cmꢃ1: 3323.7, 3027.1, 2919.8, 2844.7, 1693.0, 1590.7, 1521.6,
1409.4, 1265.8, 1178.4, 1151.4, 1130.5, 1013.7, 824.0, 765.2. 1H NMR
NMR (300 MHz, DMSO)
d: 10.25 (s, 1H), 8.19 (m, 2H), 8.09
(d, J ¼ 6.8 Hz, 1H), 7.99 (m, 1H), 7.95e7.83 (m, 2H), 7.49 (t, J ¼ 9.8 Hz,
(300 MHz, DMSO)
d
: 9.40 (s, 1H), 8.19 (d, J ¼ 8.5 Hz, 1H), 8.10
1H), 7.39e7.21 (m, 4H), 7.18 (t, J ¼ 6.8 Hz, 1H), 4.58 (s, 2H), 3.46
(d, J ¼ 7.8 Hz, 1H), 7.97e7.85 (m, 2H), 7.65 (d, J ¼ 8.0 Hz, 1H),
7.37e7.23 (m, 4H), 7.18 (t, J ¼ 6.9 Hz, 1H), 7.04 (s, 1H), 6.99
(d, J ¼ 8.4 Hz, 1H), 4.58 (s, 2H), 3.47 (s, 4H), 3.26 (s, 2H), 2.87 (s, 4H),
2.23 (d, J ¼ 4.4 Hz, 6H). C29H31N5O (465.25).
(s, 4H), 3.31 (s, 2H), 2.83 (s, 4H). 13C NMR (150.9 MHz, DMSO)
d:
168.98, 159.28, 155.14, 139.09, 135.65, 131.94, 131.67, 128.61, 128.52,
126.81, 126.32, 125.67, 125.62, 125.40, 124.68, 121.00, 117.61, 117.46,
61.66, 52.69, 50.80, 38.47. C28H25F4N5O (523.20).
5.13.9. N-(3,4-Dimethoxyphenyl)-2-(4-(4-benzylphthalazin-1-yl)
piperazin-1-yl)acetamide (12i)
5.13.4. N-(5-Fluoro-2-methylphenyl)-2-(4-(4-benzylphthalazin-1-
yl)piperazin-1-yl)acetamide (12d)
Yield: 34%. m.p.: 97e99 ꢁC. MS[MHþ](m/z): 498.4 (M þ 1). IR
(KBr) cmꢃ1: 3461.5, 2935.2, 2834.4, 1686.1, 1610.1, 1516.7, 1453.6,
1409.1, 1262.9, 1236.0, 1133.1, 1028.0, 774.8. 1H NMR (300 MHz,
Yield: 33%. m.p.: 191e192 ꢁC. MS[MHþ](m/z): 470.1 (M þ 1). IR
(KBr) cmꢃ1: 3305.0, 2920.2, 2827.8, 1692.6, 1602.0, 1527.7, 1494.9,
1453.0, 1410.2, 1266.7, 1010.8, 728.6. 1H NMR (300 MHz, DMSO)
d:
DMSO)
d
: 9.73 (s, 1H), 8.18 (d, J ¼ 9.1 Hz, 1H), 8.09 (d, J ¼ 6.6 Hz, 1H),
9.61 (s, 1H), 8.18 (d, J ¼ 7.8 Hz, 1H), 8.10 (d, J ¼ 8.1 Hz, 1H), 7.99e7.87
(m, 2H), 7.83 (d, J ¼ 11.5 Hz, 1H), 7.44e7.22 (m, 5H), 7.18
(t, J ¼ 6.7 Hz, 1H), 6.89 (t, J ¼ 7.2 Hz, 1H), 4.59 (s, 2H), 3.47 (s, 4H),
7.88 (m, 2H), 7.38 (d, J ¼ 2.0 Hz, 1H), 7.36e7.22 (m, 5H), 7.21e7.14
(m,1H), 6.89 (d, J ¼ 8.8 Hz,1H), 4.58 (s, 2H), 3.73 (s, 3H), 3.71 (s, 3H),
3.46 (s, 4H), 3.26 (s, 2H), 2.84 (s, 4H). 13C NMR (75.5 MHz, DMSO)
d:
3.31 (s, 2H), 2.89 (s, 4H), 2.27 (s, 3H). 13C NMR (75.5 MHz, DMSO)
d:
167.89, 159.29, 155.15, 148.61, 144.99, 139.10, 132.47, 131.97, 131.70,
128.62, 128.54, 126.81, 126.34, 125.41, 124.69, 121.00, 112.10, 111.45,
104.82, 61.78, 55.84, 55.52, 52.75, 50.88, 38.48. C29H31N5O3
(497.24).
168.38, 159.25, 155.33,139.08, 132.06, 131.79, 131.39, 128.66, 128.56,
126.84, 126.37, 125.49, 124.69, 121.01, 110.26, 108.05, 61.45, 52.82,
51.25, 38.49. C28H28FN5O (469.23).
5.13.5. N-(2-Chloro-5-(trifluoromethyl)phenyl)-2-(4-(4-
5.13.10. N-(3-Fluorophenyl)-2-(4-(4-benzylphthalazin-1-yl)
piperazin-1-yl)acetamide (12j)
benzylphthalazin-1-yl)piperazin-1-yl)acetamide (12e)
Yield: 24%. m.p.: 168e169 ꢁC. MS[MHþ](m/z): 540.3 (Cl ¼ 35),
542.3 (Cl ¼ 37). IR (KBr) cmꢃ1: 3271.8, 3029.9, 2922.1, 2885.3,
2824.1, 1702.2, 1585.1, 1523.3, 1455.0, 1415.1, 1331.6, 1263.6, 1162.7,
1127.5, 1082.0, 1013.3, 903.2, 824.5, 732.3. 1H NMR (300 MHz,
Yield: 44%. m.p.: 77e78 ꢁC. MS[MHþ](m/z): 456.4 (M þ 1). IR
(KBr) cmꢃ1: 3282.2, 3062.3, 2920.1, 2826.8, 1687.4, 1612.4, 1524.4,
1493.4,1442.7,1415.2,1308.6,1265.4,1134.4,1013.5, 868.5, 778.1. 1H
NMR (300 MHz, DMSO)
d: 10.20 (s, 1H), 8.18 (d, J ¼ 6.2 Hz, 1H), 8.09
DMSO)
d
: 10.23 (s, 1H), 8.69 (s, 1H), 8.20 (d, J ¼ 7.8 Hz, 1H), 8.11
(d, J ¼ 6.0 Hz, 1H), 7.89 (d, J ¼ 3.7 Hz, 2H), 7.70 (d, J ¼ 11.7 Hz, 1H),
7.46 (d, J ¼ 7.9 Hz,1H), 7.39e7.23 (m, 5H), 7.17 (t, J ¼ 6.8 Hz,1H), 6.88
(t, J ¼ 7.2 Hz, 1H), 4.58 (s, 2H), 3.46 (s, 4H), 3.33 (s, 2H), 2.85 (s, 4H).
C27H26FN5O (455.21).
(d, J ¼ 6.8 Hz, 1H), 8.00e7.87 (m, 2H), 7.82 (d, J ¼ 8.4 Hz, 1H), 7.52
(d, J ¼ 8.4 Hz, 1H), 7.37e7.22 (m, 4H), 7.18 (t, J ¼ 7.1 Hz, 1H), 4.59
(s, 2H), 3.49 (s, 4H), 3.38 (s, 2H), 2.91 (s, 4H). 13C NMR (75.5 MHz,
DMSO)
d: 169.21, 159.20, 155.35, 139.07, 135.36, 132.07, 131.80,
130.62, 128.66, 128.56, 126.85, 126.37, 125.49, 124.68, 121.40, 121.01,
117.28, 61.24, 52.76, 51.25, 38.49. C28H25ClF3N5O (539.17).
5.13.11. N-(3-Bromophenyl)-2-(4-(4-benzylphthalazin-1-yl)
piperazin-1-yl)acetamide (12k)
Yield: 44%. m.p.: 80e81 ꢁC. MS[MHþ](m/z): 516.3 (Br ¼ 79),
518.3 (Br ¼ 81). IR (KBr) cmꢃ1: 3282.1, 3060.9, 2919.8, 2825.3,
1687.4, 1591.4, 1516.0, 1415.4, 1306.7, 1265.6, 1177.2, 1131.8, 1012.7,
5.13.6. N-(2-Chloro-6-methylphenyl)-2-(4-(4-benzylphthalazin-1-
yl)piperazin-1-yl)acetamide (12f)
Yield: 42%. m.p.: 80e81 ꢁC. MS[MHþ](m/z): 486.4 (Cl ¼ 35),
488.4 (Cl ¼ 37). IR (KBr) cmꢃ1: 3300.8, 2921.0, 2827.7, 1698.0,
1494.3, 1410.1, 1266.0, 1177.2, 1132.1, 1012.7, 766.5. 1H NMR
778.0. 1H NMR (300 MHz, DMSO)
d: 10.11 (s, 1H), 8.25e8.14 (m, 1H),
8.14e8.02 (m, 2H), 7.96e7.82 (m, 2H), 7.65 (d, J ¼ 7.2 Hz, 1H), 7.28
(m, 6H), 7.16 (t, J ¼ 6.9 Hz, 1H), 4.58 (s, 2H), 3.45 (s, 4H), 3.30 (s, 2H),
2.83 (s, 4H). C27H26BrN5O (515.13).
(300 MHz, DMSO)
d
: 9.55 (s, 1H), 8.18 (d, J ¼ 8.7 Hz, 1H), 8.11

3510
S. Zhang et al. / European Journal of Medicinal Chemistry 45 (2010) 3504e3510
5.13.12. N-(4-Bromophenyl)-2-(4-(4-benzylphthalazin-1-yl)
piperazin-1-yl)acetamide (12l)
Approximately 4 ꢀ 103 cells, suspended in MEM medium, were
plated onto each well of a 96-well plate and incubated in 5% CO2 at
37 ꢁC for 24 h. The test compounds at indicated final concentrations
were added to the culture medium and the cell cultures were
continued for 72 h. Fresh MTT was added to each well at a terminal
Yield: 21%. m.p.: 192e193 ꢁC. MS[MHþ](m/z): 516.1 (Br ¼ 79),
518.1 (Br ¼ 81). IR (KBr) cmꢃ1: 3226.2, 3056.5, 2932.2, 2823.4,
1670.3, 1587.0, 1505.0, 1393.3, 1306.1, 1267.7, 1176.4, 1132.5,
1011.7821.0, 762.4, 711.9. 1H NMR (300 MHz, DMSO)
d: 10.03 (s, 1H),
concentration of 5
m
g/mL and incubated with cells at 37 ꢁC for 4 h.
L DMSO each well,
8.23e8.14 (m, 1H), 8.14e8.04 (m, 1H), 7.90 (m, 2H), 7.68
(d, J ¼ 8.8 Hz, 2H), 7.50 (d, J ¼ 8.7 Hz, 2H), 7.29 (m, 4H), 7.17
(t, J ¼ 7.0 Hz, 1H), 4.58 (s, 2H), 3.45 (s, 4H), 3.29 (s, 2H), 2.83 (s, 4H).
The formazan crystals were dissolved in 100
m
and the absorbency at 492 nm (for absorbance of MTT formazan)
and 630 nm (for the reference wavelength) was measured with the
ELISA reader. All of the compounds were tested twice in each of the
cell lines. The results expressed as IC50 (inhibitory concentration
50%) were the averages of two determinations and calculated by
using the Bacus Laboratories Incorporated Slide Scanner (Bliss)
software.
13C NMR (75.5 MHz, DMSO)
d: 168.62, 159.27, 155.15, 139.10, 138.26,
131.97, 131.70, 131.48, 128.62, 128.54, 126.81, 126.34, 125.43, 124.69,
121.51, 120.99, 114.98, 61.70, 52.68, 50.87, 38.49. C27H26BrN5O
(515.13).
5.13.13. N-(2-Methylphenyl)-2-(4-(4-benzylphthalazin-1-yl)
piperazin-1-yl)acetamide (12m)
Yield: 35%. m.p.: 170e171 ꢁC. MS[MHþ](m/z): 452.3 (M þ 1). IR
References
(KBr) cmꢃ1: 3296.9, 2919.2, 2824.5, 1688.4, 1585.5, 1526.7, 1454.8,
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3954e3968.
1412.6, 1386.8, 1262.9, 1012.9, 762.8. 1H NMR (300 MHz, DMSO)
d:
9.49 (s, 1H), 8.19 (d, J ¼ 8.1 Hz, 1H), 8.10 (d, J ¼ 8.1 Hz, 1H), 7.98e7.86
(m, 2H), 7.83 (d, J ¼ 7.8 Hz, 1H), 7.28 (m, 5H), 7.18 (t, J ¼ 7.5 Hz, 2H),
7.05 (t, J ¼ 7.3 Hz, 1H), 4.59 (s, 2H), 3.48 (s, 4H), 3.29 (s, 2H), 2.89
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3942e3945.
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1040e1044.
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585e592.
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(s, 4H), 2.28 (s, 3H). 13C NMR (150.9 MHz, DMSO)
d: 167.93, 159.21,
155.23, 139.05, 136.16, 131.99, 131.71, 130.31, 129.00, 128.51, 126.82,
126.32, 126.28, 125.42, 124.65, 124.40, 122.18, 120.99, 61.54, 52.84,
51.14, 38.46. C28H29N5O (451.24).
5.14. Cytotoxicity assay in vitro
The cytotoxic activities of compounds 7ae7m and 12ae12m
were evaluated with A549, HT-29 and MDA-MB-231 cell lines
(A549: non-small-cell lung cancer cell line, HT-29: human colon
cancer cell line, MDA-MB-231: human breast cancer cell line) by
the standard MTT assay [17] in vitro, with PTK-787 as the positive
control. The cancer cell lines were cultured in minimum essential
medium (MEM) supplement with 10% fetal bovine serum (FBS).