Discovery of phenylpiperazine derivatives as IGF-1R inhibitor with potent antiproliferative properties in vitro

Article abstract of DOI:10.1016/j.bmcl.2015.01.011

A series of phenylpiperazine derivatives (3a-3q) were designed and synthesized. In vitro assays indicated that several phenylpiperazine derivatives had excellent antiproliferative properties against four cancer cell lines including multidrug-resistant can

Full text of DOI:10.1016/j.bmcl.2015.01.011

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of phenylpiperazine derivatives as IGF-1R inhibitor with
potent antiproliferative properties in vitro
⇑
Feng-Jiao Guo , Juan Sun , Li-Li Gao, Xin-Yi Wang, Yang Zhang, Shao-Song Qian, Hai-Liang Zhu
School of Life Sciences, Shandong University of Technology, Zibo 255049, Shandong Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phenylpiperazine derivatives (3a–3q) were designed and synthesized. In vitro assays indicated
that several phenylpiperazine derivatives had excellent antiproliferative properties against four cancer
cell lines including multidrug-resistant cancer cell lines, with IC50 values in the low micromolar range.
Received 4 November 2014
Revised 18 December 2014
Accepted 6 January 2015
Available online xxxx
The average IC50 of the most active compound 3b is 0.024 lM to the MCF-7 cell line. In addition, the
mechanism of action of these new analogues was investigated by molecular docking studies, insulin-like
growth factor 1-receptor (IGF-1R) kinase assay and apoptosis induced assay. These studies confirmed that
these new phenylpiperazine derivatives maintain their mechanisms of action by disrupting IGF-1R
kinase.
Keywords:
Phenylpiperazine
IGF-1R inhibitors
Apoptosis
Ó 2015 Elsevier Ltd. All rights reserved.
Computer simulation
The insulin-like growth factor 1 receptor (IGF-1R), as part of the
large class of receptor tyrosine kinases (RTKs), is now considered a
potential cellular oncogene that plays a key role in various cellular
processes, such as proliferation, survival, transformation, differen-
tiation as well as cell-cell and cell-substrate interactions.1,2 Signal-
ing through IGF-1R is initiated upon the binding of the IGF-I ligand
to the receptor leading to receptor dimerization, autophosphoryla-
tion, and subsequent activation of the downstream substrates Shc,
IRS-1, and IRS-2.3
It has been demonstrated that IGF-1R expression is a funda-
mental prerequisite for cellular transformation because enhanced
IGF-1R levels and IGF-I signaling are considered key factors for
the cell to adopt the proliferative and oncogenic pathways.4 From
a clinical perspective, epidemiological studies have correlated ele-
vated IGF-I levels with increased risk of developing colon, breast,
prostate, and lung tumors, highlighting the importance of IGF-1R
signaling.5–10 In breast neoplastic cell lines, expression of IGF-1R
is a fundamental prerequisite for a malignant phenotype, poten-
tially facilitating cell survival and metastasis.11–14 For these rea-
sons, the IGF-1R has emerged as a therapeutic target for the
treatment of human cancer.
parthi et al.15 designed and synthesized a series of new compounds
that piperazine replacements of morpholine in BMS-536924
(Fig. 1B). With the participated of the piperazine ring, some of
the IGF-1R inhibitory activity were much better than BMS-
536924. Girnita et al.16 used molecular modeling showed that a
molecule consisting of two benzene rings separated by only one
carbon atom could mimic the suggested three-dimensional struc-
ture of the two tyrosines of IGF-1R, and thereby possibly inhibits
their phosphorylation. We therefore attempted to synthesis a ser-
ies of phenylpiperazine derivatives as novel IGF-1R inhibitors with
1-bromophenyl ethanol as the skeleton which could be beneficial
to patients suffering from various cancers.
Subsequently, in order to validate whether these designed com-
pounds can work on target protein IGF-1R, the molecular docking
was performed by fitting these designed compounds and reference
compound (BMS-536924) into the ATP binding site of IGF-1R. (PDB
code: 2OJ9).17 Then, the obtained results have been plotted as a
line-scatter graph and presented in Figure 2, which mainly displays
the corresponding CDOCKER_INTERACTION_ENERGY of the molec-
ular docking studies.18 Compared with the positive drug BMS-
536924, it was clearly seen that compounds 3b and 3a showed
lower interaction energy than positive that reached up to
À53.41 kcal/mol and À52.17 kcal/mol, respectively. Besides,
almost all the designed molecules possessed low interaction
energy, demonstrating that they are likely to exhibit potent inhib-
itory activity against IGF-1R tyrosine kinase. Therefore, this preli-
minary analysis served as a modest stimulant to induce us to
synthesize these 1-(4-bromophenyl)-2-(4-phenylpiperazin-1-
yl)ethanol compounds.
Bristol–Myers Squibb (BMS) company discovered 1H-ben-
zoimidazol-2-yl)-1H-pyridin-2-one (BMS-536924) (Fig. 1A) as a
novel ATP-competitive inhibitor of IGF-1R.3 We can clearly know
that the compound contains the structure of phenyl ethanol. Vela-
⇑
Corresponding author.
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.01.011
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Guo, F.-J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.011

2
F.-J. Guo et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
R1
O
N
O
O
H
N
H
N
N
NH
N
NH
N
N
HN
HN
CH3
HO
HO
Cl
Cl
A
B
Figure 1. The structure of compounds A and B.
pathway outlined in Scheme 1. They are prepared in two steps.
Firstly, the intermediates obtained by condensation reaction
between the 2-bromo-1-(4-bromophenyl)ethanone and the substi-
tuted phenylpiperazine, adding 1.5 times of K2CO3 in acetonitrile.
Secondly, reduction of intermediates with NaBH4 in ethanol leads
to the formation of the final phenylpiperazine derivatives.
All of the synthetic compounds gave satisfactory analytical and
spectroscopic data, which were in full accordance with their
depicted structures. Additionally, the structure of compound 3p
was further confirmed by X-ray diffraction. The CCDC deposition
number of 3p is 1039756. Its crystal data and hydrogen bond date
are presented in Tables 1 and 2, respectively, and Figure 3 gives a
perspective view of this compound together with the atomic label-
ing system.
To test the anticancer activities of the synthesized compounds,
the target compounds were evaluated in vitro antiproliferation
assays against four human cancer cell lines MCF-7, LS-741T,
SMMC-7721 and SGC-7901 cell lines. The results were summarized
in Table 3. With few exception, the active analogs showed a
remarkable potential antitumor activity, suggesting that the new
compounds could significantly enhance anticancer potency. For
the given compounds, it was observed that compound 3b showed
Figure 2. The CDOCKER_INTERACTION_ENERGY (kcal/mol) obtained from the
docking study of all synthesized compounds by the CDOCKER protocol (Discovery
Studio 3.1, Accelrys, Co. Ltd).
the most potent biological activity (IC50 = 0.024
l
M for MCF-7,
To continue our study of anticancer drugs,19 seventeen phenyl-
piperazine derivatives were synthesized for the first time. The syn-
thetic route of compounds 3a–3q was followed the general
IC50 = 0.068
IC50 = 0.116
l
l
M for LS-741T, IC50 = 0.150
M for SGC-7901).
lM for SMMC-7721 and
Cl
3j R=
3f R=
3g R=
3n R=
3a R=
3b R=
H3C
CH3
F
3k R=
O2N
3o R=
3p R=
3q R=
F
F
OCH3
OCH3
F
3c R=
3d R=
3e R=
CF3
Cl
Cl
3l R=
3h R=
3i R=
N
Cl
Cl
3m R=
H3CO
Cl
Scheme 1. General synthesis of compounds 3a–3q. Reagents and conditions: (i) 2-bromo-1-(4-bromophenyl)ethanone, 1.5 times of K2CO3, CH3CN, reflux; (ii) 1.5 times of
NaBH4, CH3CH2OH, rt.
Please cite this article in press as: Guo, F.-J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.011

F.-J. Guo et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
According to the data presented in Table 3, we could arrive at the
conclusion that the activity of the tested compounds may be corre-
lated to the variation and modifications of structure. Structure–
activity relationships in these new compounds demonstrated that
when the compounds (3a and 3b) were substituted by two phenyl
ring, they showed the most potent antiproliferative activity to four
Crystallographic data, details of data collection and structure refinement parameters
Compound
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
3p
C19H23BrN2O2
391.29
Monoclinic
P21/n
9.8057(6)
20.2577(12)
10.0973(6)
90
113.182(2)
90
1843.79(19)
4
1.410
2.0–25.1
808
17426/3268
2029/0/219
2.243
tumor cell lines, with IC50 concentration range of 0.024–0.253 lM.
b (Å)
It was concluded that compounds 3b and 3a may be have broad-
spectrum antitumor activity against the mentioned four cancer cell
lines. The compounds that introduction of two substituent on the
phenyl ring (3n, 3m, 3l) has much better antiproliferative activity
than the compounds of single-substituent on the phenyl ring, while
cell-based activity is maintained relative to the compound that phe-
nyl ring substituent containing-CF3 (3h). Generally speaking, when
the compounds were single-substituent on the substituted phenyl
ring, the potency order was –NO2 > –OCH3 > –Cl > –F, and the anti-
proliferative activities that having –NO2 phenyl ring substituent
(3k) are much better than the other derivatives, even considerable
withthe positive control (BMS-536924). When the compoundswere
benzyl, pyridyl and phenyl-substituent derivatives (3c, 3d, 3e), their
antiproliferative activities were not remarkable compared with the
single-substituent phenyl ring derivatives, and they have one or two
orders of magnitude difference with compounds 3a and 3b.
c (Å)
a
(o)
b (o)
(o)
c
V (Å)
Z
D calc/g cmÀ3
h range (°)
F(000)
Reflections collected/unique
Data/restraints/parameters
Absorption coefficient (mmÀ1
R1/WR2 [I >2
R1/WR2 (all date)
GOOF
)
r
(I)]
0.0475/0.0960
0.0929/0.1107
1.013
Table 2
Hydrogen bond lengths (Å) and bond angles (°) of compound 3p
In order to verify whether it has damage to normal cell, we
tested the cytotoxicity of some new compounds to human hepato-
cytes QSG7701. The result showed that CC50 of all the tested com-
H-bond type
D–H. . .A
d(D–H)
d(H. . .A)
d(D. . .A)
\DHA
intra
O3–H3. . .N1
C2–H2. . .O3
0.82
0.93
0.96
0.96
2.18
2.44
2.58
2.59
2.678(4)
2.763(5)
3.524(6)
3.477(5)
119
101
168
153
pounds were larger than 200 lM, it means that when the low
concentrations new compounds act on human hepatocytes
QSG7701 in vitro, the normal cells almost no destruction. The data
of cytotoxicity assay and CC50 were presented in Table 4.
inter
C20–H20A. . .O3
C20–H20B. . .O1
Figure 3. Molecular structure of compound 3p with atomic numbering scheme.
Table 3
In vitro anticancer activities (IC50
,
l
M) against four human tumor cell lines and IGF-1R inhibitory activities (IC50
, lM)
Compounds
IGF-1R ( M) SD IC50 (lM) SD
l
MCF-7
LS-741T
SMMC-7721
SGC-7901
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
0.034 0.009
0.030 0.009
0.568 0.034
1.186 0.037
1.308 0.035
2.081 0.034
2.471 0.038
0.235 0.039
1.107 0.040
1.641 0.032
0.098 0.036
0.265 0.033
0.207 0.041
0.129 0.040
0.270 0.047
0.395 0.038
0.374 0.033
0.153 0.047
0.037 0.011
0.024 0.013
2.877 0.024
3.410 0.040
5.708 0.019
11.48 0.035
16.36 0.029
0.860 0.040
2.461 0.027
10.38 0.042
1.308 0.031
1.065 0.034
0.777 0.033
0.863 0.036
1.216 0.049
1.444 0.034
1.339 0.059
1.748 0.029
0.111 0.009
0.068 0.015
2.687 0.020
6.612 0.049
8.991 0.008
4.613 0.060
5.343 0.046
0.963 0.079
2.538 0.022
16.90 0.031
2.181 0.018
1.922 0.022
1.889 0.025
1.824 0.024
2.566 0.019
2.062 0.024
2.598 0.007
2.050 0.034
0.135 0.026
0.150 0.029
3.535 0.030
6.117 0.027
7.640 0.025
8.157 0.029
9.633 0.026
1.198 0.033
3.918 0.029
7.832 0.027
1.526 0.029
2.035 0.031
1.678 0.027
1.476 0.029
2.277 0.029
2.906 0.032
2.824 0.044
2.555 0.031
0.253 0.043
0.116 0.034
3.756 0.037
5.696 0.042
9.277 0.038
10.33 0.031
15.95 0.042
2.079 0.040
4.617 0.041
22.29 0.037
1.837 0.038
2.470 0.038
2.141 0.035
1.385 0.044
4.150 0.041
3.285 0.042
3.464 0.039
2.893 0.042
3q
BMS-536924
Please cite this article in press as: Guo, F.-J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.011

4
F.-J. Guo et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 4
In vitro cytotoxicity activities (CC50, lM) against human hepatocytes QSG7701
Compounds
CC50 M)
3a
3e
3h
3k
3m
BMS-536924
(
l
2.0 Â 103
2.5 Â 102
6.6 Â 103
1.6 Â 103
5.6 Â 105
3.2 Â 104
Figure 4. (A) 2D molecular docking model of compound 3b with 2OJ9. (B) 3D interaction map between compound 3b and 2OJ9 binding site. (C) 2D diagram of docking
structure of compound BMS-536924 with 2OJ9. (D) 3D Model of the interaction between compound BMS-536924 and 2OJ9 binding site.
abilities to inhibit the activity of IGF-1R relevant to cancer. As
expected, all compounds displayed the potent inhibitory activity
for IGF-1R and the results were showed in Table 3. Among them,
compound 3b showed the most potent inhibitory with IC50 of
0.025 lM, and sixteen-fold improvement in enzymatic potency
than the positive control BMS-536924. The results of IGF-1R inhib-
itory activity of the tested compounds were in agreement to the
structure relationships (SAR) of their antiproliferative activities.
This agreement suggested that antiproliferative activities of the
synthesized compounds would derive from the inhibition of IGF-
1R enzymatic activities.
To help understand the new compounds observed at the IGF-1R
and guide further phenylpiperazine studies, molecular docking of
the most potent inhibitor 3b into active binding site of IGF-1R
was performed on the binding model based on the IGF-1R complex
structure (PDB code: 2OJ9). The obtained results were presented in
Figure 4. Figure 4A and B showed the binding mode of compound
3b interacting with IGF-1R protein, the docking results revealed
that two amino acids Asp1056 and THR1127 located in the binding
pocket of protein played a vital roles in the conformation with
compound 3b, which were stabilized by two hydrogen bonds. Fig-
ure 4C and D displayed 2D and 3D diagram of docking structure of
compound BMS-536924 with 2OJ9 binding site. Insight into those
two pictures, we can see that amino acid residue Asp1056 located
in the binding pocket also seemed very important for the active
Figure 5. The result of apoptosis induced assay of compound 3b to MCF-7 cancer
cell line. The first strip named M means the DL 2000 DNA Marker. The second strip
named 1 is the DNA Ladder that caused by compound 3b in the concentration of
IC50. The third strip named 2 is the negative control which without any drug
treatment to the normal cell.
To validate whether the above anti-proliferative effect was pro-
duced by interaction of IGF-1R protein and the synthesized com-
pounds, the synthesized compounds were evaluated for their
Please cite this article in press as: Guo, F.-J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.011

F.-J. Guo et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
conformation of the positive control. These results could provide a
References and notes
molecular level foundation to illustrate compound 3b can bind
well at the active site of IGF-1R tyrosine kinase.
1. Werner, H.; Bruchim, I. Arch. Physiol. Biochem. 2009, 115, 58.
2. Surmacz, E. J. Mammary Gland Biol. Neoplasia 2000, 5, 95.
We evaluated compound 3b for its ability to induce apoptosis in
the MCF-7 cell line used cellular DNA extraction kit to extract
genomic DNA. The results were shown in Figure 5. As can be seen,
compound 3b is very effective in induction the apoptosis of MCF-7
cell. This is consistent with its nice binding affinity to IGF-1R TK
and its potent activity in inhibition of cell growth.
In summary, novel phenylpiperazine analogues were designed
and synthesized. Structure-activity relationships were investi-
gated by introducing different substituent into the piperazine
ring. Several compounds showed excellent antiproliferative activ-
ity which were comparable to existing IGF-1R-targeting agents,
such as BMS-536924. Compound 3b demonstrated the most
potent inhibitory activity that inhibited the activity of IGF-1R
3. Wittman, M.; Carboni, J.; Attar, R.; Balasubramanian, B.; Balimane, P.; Brassil,
P.; Beaulieu, F.; Chang, C.; Clarke, W.; Dell, J.; Eummer, J.; Frennesson, D.;
Gottardis, M.; Greer, A.; Hansel, S.; Hurlburt, W.; Jacobson, B.; Krishnananthan,
S.; Lee, F. Y.; Li, A. X.; Lin, T. A.; Liu, P. Y.; Ouellet, C.; Sang, X. P.; Saulnier, M. G.;
Stoffan, K.; Sun, Y.; Velaparthi, U.; Wong, H.; Yang, Z.; Zimmermann, K.;
Zoeckler, M.; Vyas, D. J. Med. Chem. 2005, 48, 5639.
4. Werner, H.; Maor, S. Trends Endocrinol. Metab. 2006, 17, 236.
5. Ma, J.; Pollak, M. N.; Giovannucci, E.; Chan, J. M.; Tao, Y.; Hennekens, C. H.;
Stampfer, M. J. J. Natl. Cancer Inst. 1999, 91, 620.
6. Hankinson, S. E.; Willett, W. C.; Colditz, G. A.; Hunter, D. J.; Michaud, D. S.;
Deroo, B.; Rosner, B.; Speizer, F. E.; Pollak, M. Lancet 1998, 351, 1393.
7. Chan, J. M.; Stampfer, M. J.; Giovannucci, E.; Gann, P. H.; Ma, J.; Wilkinson, P.;
Hennekens, C. H.; Pollak, M. Science (Washington, D.C.) 1998, 279, 563.
8. Wolk, A.; Mantzoros, C. S.; Andersson, S. O.; Bergstrom, R.; Signorello, L. B.;
Lagiou, P.; Adami, H. O.; Trichopoulos, D. J. Natl. Cancer. Inst. 1998, 90, 911.
9. Yu, H.; Spitz, M. R.; Mistry, J.; Gu, J.; Hong, W. K.; Wu, X. J. Natl. Cancer. Inst.
1999, 91, 151.
with IC50 of 0.03 lM. Docking simulation was performed to posi-
10. Shi, L.; Wu, T. T.; Wang, Z.; Xue, J. Y.; Xu, Y. J. Bioorg. Med. Chem. 2014, 22, 4735.
11. Sachdev, D.; Zhang, X.; Matise, I.; Gaillard-Kelly, M.; Yee, D. Oncogene 2010, 29,
251.
12. Klinakis, A.; Szabolcs, M.; Chen, G.; Xuan, S.; Hibshoosh, H.; Efstratiadis, A. Proc.
Natl. Acad. Sci. U.S.A. 2009, 106, 2359.
tion compound 3b into the IGF-1R active site to determine the
probable binding conformation and the result indicated that com-
pound 3b was a potent inhibitor of IGF-1R. Apoptosis assay result
showed the compound 3b can induce the programmed cell death
of MCF-7 cell line. These results strongly suggest that novel phe-
nylpiperazine analogues can be further developed as a promising
antitumor agent for the more efficacious treatment of advanced
cancers.
13. Sachdev, D.; Hartell, J. S.; Lee, A. V.; Zhang, X.; Yee, D. J. Biol. Chem. 2004, 279,
5017.
14. Shi, L.; Wu, T. T.; Wang, Z.; Xue, J. Y.; Xu, Y. J. Eur. J. Med. Chem. 2014, 84, 698.
15. Velaparthi, U.; Saulnier, M. G.; Wittman, M. D.; Liu, P. Y.; Frennesson, D. B.;
Zimmermann, K.; Carboni, J. M.; Gottardis, M.; Aixin Li, A. X.; Greer, A.; Clarke,
W.; Yang, Z.; Menard, K.; Lee, F. Y.; Trainor, G.; Vyas, D. Bioorg. Med. Chem. Lett.
2010, 20, 3182.
16. Girnita, A.; Girnita, L.; del Prete, F.; Bartolazzi, A.; Larsson, O.; Axelson, M.
Cancer Res. 2004, 64, 236.
17. Discovery Studio 3.5, Accelrys Software Inc., San Diego, 2011.
18. Sun, J.; Li, D. D.; Li, J. R.; Fang, F.; Du, Q. R.; Qian, Y.; Zhu, H. L. Org. Biomol. Chem.
2013, 11, 7676.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.01.
011.
19. Guo, F. J.; Sun, J.; Zhou, W.; Zhu, H. L. Chin. J. Struct. Chem. 2014, 33, 79.
Please cite this article in press as: Guo, F.-J.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.01.011