Design, synthesis and biological evaluation of 7-(aryl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline derivatives as potential Hsp90 inhibitors and anticancer agents

Article abstract of DOI:10.1016/j.bmc.2016.12.050

A new series of quinoline analogues was designed and synthesized as Hsp90 inhibitors. The cytotoxic activity of the synthesized compounds was evaluated against three human cancer cell lines including MCF-7 (human breast cancer cells), DU145 (human prostate cancer cell lines), and A549 (adenocarcinomic human alveolar basal epithelial cells). Some of our compounds (13a-13f) showed significant cytotoxic activity on MCF-7 cells. The most potent anti-proliferative compounds were also tested against Her2, a client protein of Hsp90. Compound 13d that demonstrated the highest antiproliferative activity in the series, was found the most potent one for both Her2 protein degradation and Hsp70 protein induction as well. Molecular modeling studies displayed possible mode of interaction between this compound and N-terminal ATP-binding site of Hsp90.

Full text of DOI:10.1016/j.bmc.2016.12.050

Accepted Manuscript
Design, synthesis and biological evaluation of 7-(aryl)-2,3-dihydro-[1,4]dioxi-
no[2,3-g]quinoline derivatives as potential Hsp90 inhibitors and anticancer
agents
Sina Omid Malayeri, Khalil Abnous, Atefeh Arab, Maryam Akaberi, Soghra
Mehri, Afshin Zarghi, Razieh Ghodsi
PII:
S0968-0896(16)31529-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.050
BMC 13477
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 October 2016
18 December 2016
29 December 2016
Please cite this article as: Omid Malayeri, S., Abnous, K., Arab, A., Akaberi, M., Mehri, S., Zarghi, A., Ghodsi, R.,
Design, synthesis and biological evaluation of 7-(aryl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline derivatives as
potential Hsp90 inhibitors and anticancer agents, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.12.050
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Design, synthesis and biological evaluation of 7-(aryl)-2,3-dihydro-[1,4]dioxino[2,3-
g]quinoline derivatives as potential Hsp90 inhibitors and anticancer agents
Sina Omid Malayeriab, Khalil Abnousbc, Atefeh Arabd, Maryam Akaberie, Soghra Mehric,
Afshin Zarghif, and Razieh Ghodsiab*
aBiotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
bDepartment of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran
cPharmaceutical Research Center, Department of Pharmacodynamics and Toxicology, School of
Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
dDepartment of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran
eDepartment of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran
fDepartment of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
*Corresponding author: Tel: +98 51 38823255; Fax: +98 51 38823251; E-mail:
ghodsir@mums.ac.ir
Abstract
A new series of quinoline analogues was designed and synthesized as Hsp90 inhibitors. The
cytotoxic activity of the synthesized compounds was evaluated against three human cancer cell
lines including MCF-7 (human breast cancer cells), DU145 (human prostate cancer cell lines),
and A549 (adenocarcinomic human alveolar basal epithelial cells). Some of our compounds
(13a-13f) showed significant cytotoxic activity on MCF-7 cells. The most potent anti-
proliferative compounds were also tested against Her2, a client protein of Hsp90. Compound 13d
that demonstrated the highest antiproliferative activity in the series, was found the most potent
one for both Her2 protein degradation and Hsp70 protein induction as well. Molecular modeling
studies displayed possible mode of interaction between this compound and N-terminal ATP-
binding site of Hsp90.

Keywords: Quinolines, Heat Shock Protein 90, Anti-cancer activity, Her2, Molecular docking,
Hsp90 inhibitor
Introduction
The molecular chaperone heat shock protein 90 kDa (Hsp90) is one of the most attractive targets
for drug design and development, because it interacts with a diversity of client proteins.1-3 To
date, there are many Hsp90s clients that have been reported to be involved in cell divisions and
signal transduction, including a number of proteins that are involved in tumorgenesis. The
dependence of multiple oncogenes on Hsp90 renders cancer cells hypersensitive to Hsp90s
inhibition; therefore, Hsp90 inhibitors play a major role in cancer treatment.4
The discovery of Hsp90 as the target of anticancer activity of geldanamycin (1) (Fig. 1) devoted
much attention in the inhibition of Hsp90 as a policy for the treatment of cancer. This interest has
led to huge efforts to develop clinically practical small molecule Hsp90 inhibitors.5, 6
Hsp90 has been demonstrated to contain two nucleotide binding site, one at each terminus (N-
terminal and C-terminal domains).7 An ATPase cycle is fundamental to the chaperoning activity
of Hsp90. Geldanamycin 1 and its derivative, 7-allylaminogeldanamycin (17-AAG, 2) and
radicicol target N-terminal domain, resulting in reduced ATPase activity and avoiding
detachment of client proteins from the Hsp90 complex.8 Small-molecule inhibitors of HSP90
have huge structural diversity such as purine-based analogues 3 (PU3), pyrazole based structure
4 (CCT018159), β-lactam 5 and indazole 6 as the first stated Hsp90 and tubulin inhibitor (Fig.
1).8-11
12
Recently, Ganesh et al reported a new series of quinoline based derivatives as new class of
Hsp90 inhibitors by high-throughput screening. They identified amino quinoline 7 (SID: 850375)
as inhibitor of Hsp90 with moderate activity. In addition, benzodioxan was found as one of the
most common ring system which used in development of some potent Hsp90 inhibitors such as 4
(CCT018159) and 5. In the present study we decided to design new 7-(aryl)-2,3-dihydro-
[1,4]dioxino[2,3-g]quinolines as Hsp90 inhibitors (Fig. 1). The rational design of these
compounds was based on choosing quinoline scaffold for Hsp90 inhibitory potential and fused it
with a benzodioxan system that is a part of some potent Hsp90 inhibitors. The synthesized
compounds were evaluated for their cytotoxic activity against three different cancer cell lines
including MCF-7 (human breast cancer cells), DU145 (human prostate cancer cell lines), and
A549 (adenocarcinomic human alveolar basal epithelial cells). We also synthesized

corresponding imines which they possess the same functional group moieties of quinolines, to
check the role of quinoline ring in their cytotoxicity activity. The effects of the most anti-
proliferative compounds were also tested on Her2 (a client protein of Hsp90) degradation, and
Hsp70 protein induction. In addition, docking studies were carried out to predict the binding
mode of quinoline compounds and to rationalize the observed structure-activity relationships.
Figure 1. Chemical structures of known Hsp90 inhibitors and one of our designed compounds

2. Results and discussion
2.1. Synthesis
A one-step Doebner reaction was used to prepare the target 7-(aryl)-2,3-dihydro-
[1,4]dioxino[2,3-g]quinoline derivatives (12a-12j). As shown in Scheme 1, substituted
benzaldehyde 8, pyruvic acid 9 and 1,4-benzodioxan-6-amine 10 were refluxed in ethanol to
obtain quinolines 12a-12j.13,14 In this situation, two cyclization paths were probable; angular (8-
aryl-2,3-dihydro-[1,4]dioxino[2,3-f]quinolines, 11) or linear (7-(aryl)-2,3-dihydro-[1,4]dioxino
1
[2,3-g]quinolines, 12). The H-NMR data showed that the only product of this reaction is
compound 12, whereas the angular analogue 11 was not obtained, this result can be attributed to
1
13
steric factors.15 The structure of 12 was confirmed by H and C NMR. Appearance of two
aromatic singlets (attributed to H-5 and H-10) confirmed the structure of 12. The Doebner-type
synthesis of quinoline did not produce the expected (12d) in ethanol solvent; the desired
quinoline derivative was prepared in acetic acid. Reduction of carboxyl substituent to an
alcoholic substituent was carried out using LiAlH4 in dry THF.16 Imines (14a-14j) were prepared
8
by condensation of the appropriate benzaldehydes and 1,4-benzodioxan-6-amine 10 in ethanol
(Scheme 2). All compounds were pure and stable. The synthesized quinolines were characterized
13
by nuclear magnetic resonance (1H-NMR, C-NMR), infrared, mass spectrometry and CHN
analysis.

2
1
2
3
1
1
1
1
1
1
1
2
3
2
2
3
2
3
2
3
2
3
3
3
3
3
2
3
3
1
3
2
3
1
2
3
1
3
2
2
1
3
2
3
1
3
2
3
1
2
3
1
2
3
2
2
3
1
1
1
1
2
3
3
3
3
3
2
3
3
1
3
2
3
Scheme 1. Reagents and conditions: (a) Ethanol, reflux, (b) LiAlH4, dry THF

Scheme 2. Reagents and conditions: (a) Ethanol, reflux
2.2. Biological evaluation
2.2.1. In vitro anticancer activity
The cytotoxic activity of the synthesized compounds was evaluated against MCF-7, DU145 and
A549 cancer cells employing the MTT assay. As illustrated in Table 1, all the alcoholic
derivatives exhibited cytotoxic activity in all three cancer cell lines. However carboxylic
quinoline derivatives did not show any significant cytotoxic activity at concentrations below 100
µM. This may be due to their poor ability of carboxylic quinoline derivatives to penetrate the cell
membrane because of their more polarity. The cytotoxic activity of some of imines (14c, 14d,
14f and 14h) was also evaluated against MCF-7, DU145 and A549 cancer cells, they did not
show cytotoxic activity at concentrations below 100 µM (data not shown).In general the
alcoholic quinoline derivatives (13a-13f) showed more cytotoxicity effects in MCF-7 cell line in
comparison to other two cell lines. DU145 cells were more sensitive to all the alcoholic
derivatives except 13d compared to A549 cancer cells, indicating that our compounds exerted
their cytotoxic activity with different mechanisms in different tumor cells. Compound 13c
possessing dimethoxy phenyl group at position 2 of quinoline ring, demonstrated the most
cytotoxicity stronger the other quinolines against the DU145cancer cell lines and compound 13d
possessing trimethoxy phenyl group at position 2 of quinoline ring demonstrated the most
cytotoxic activity against the MCF-7 and A549 cell lines and 13f showed more cytotoxic activity
than its isomer 13e. Comparison the cytotoxicity of the alcoholic quinolines derivatives with

their corresponding imines possessing the same functional groups revealed that, the quinoline
ring has a key role in their cytotoxicity activity.
Table 1. The in vitro antiproliferative activities of quinoline derivatives and 17-AAG against
human cancer cell lines.
IC50(µM)
X
R1
R2
R3
MCF-7
A549
DU145
13a
13b
13c
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
H
H
H
OCH3
OCH3
OCH3
OCH3
OH
H
H
22.66 1.98
23.98 1.55
6.7 1.38
4.53 1.87
39.99 6.35
25.70 4.67
>100
>100
>100
86.13 3.79
21.42 2.9
11.54 1.2
64.15 4.07
89.65 2.20
86.79 6.69
>100
OCH3
OCH3
OH
OCH3
H
H
82.13 9.23
46.88 3.8
94.22 7.32
87.82 5.43
>100
13d
13e
OCH3
H
13f
H
12a
12b
12c
H
H
H
OCH3
OCH3
OCH3
OCH3
OH
H
>100
>100
>100
OCH3
OCH3
OH
OCH3
OH
H
H
>100
>100
>100
12d
12e
OCH3
H
>100
>100
>100
>100
>100
>100
12f
H
>100
>100
>100
12g
12h
12i
H
H
>100
>100
>100
OH
H
>100
>100
>100
H
NHCOCH3
CH3
H
>100
>100
>100
12j
H
H
>100
>100
>100
17-AAG
0.192 0.08
0.286 0.1
0.282 0.09
IC50: compound concentration required to inhibit tumor cell proliferation by 50%. Data are
presented as the mean SD from the dose−response curves of three independent experiments

2.2.2. Her2 degradation assay
Since our compounds showed more cytotoxicity effects in MCF-7 cell line in comparison to
other two cell lines and human epidermal growth factor receptor 2 (Her2) is mainly found in
breast cancer cells17 which is a client protein of Hsp90, we selected Her2 client protein to
evaluate the selected compounds for their effects on Her2 degradation in MCF-7 breast cancer
cell line. Since Her2 is a client protein of Hsp90, Hsp90 inhibitors can decrease the expression
levels of Her2. Hsp90 inactivates heat shock transcription factor (Hsf1), so most of the Hsp90
inhibitors such as geldanamycin trigger expression of Hsf1 target genes, such as Hsp70.17, 18 The
potent alcoholic derivatives (13a-13f) were further investigated for their effects on Her2
degradation in MCF-7 breast cancer cell line. In addition we evaluated them for the induction of
Hsp70 to evaluate their Hsp90 inhibition ability. We found that all the tested quinolines (13a-
13f) at concentration of 50 μM conferred more than 50% reduction of Her2 protein levels
compared to vehicle control. All the alcoholic derivatives induced Hsp70 expression. Whereas β-
tubulin levels remained unchanged which is not dependent on the Hsp90 chaperoning activity.
Compound 13d was identified as the most potent analogue in Her2 protein degradation and
Hsp70 protein induction as well. As shown in figure 2, the order of their ability to degrade Her2
was 13d> 13c> 13f> 13e> 13b> 13a. These data showed that the presence and the number of
methoxy and hydroxyl groups at the positions 3, 4 and 5 of phenyl ring are important for their
effects on Her2 degradation and Hsp90 inhibition, suggested that these functional groups may act
as hydrogen binding acceptors in the binding site of Hsp90. Both, Her2 client protein
degradation and heat shock protein70 induction indicates that 7-(aryl)-2,3-dihydro-
[1,4]dioxino[2,3-g]quinoline derivatives act as Hsp90 inhibitors, in a similar way to other Hsp90
inhibitors.19 Generally the cytotoxicity effects of our compounds on cell growth of MCF-7 were
consistent with their Her2 protein degradation effects, indicating that their cytotoxic activity is a
consequence of Hsp90 chaperone inhibition.

A
13c 13e 13a
13f
13b
13d 17-AAG CTR
Her-2
Hsp70
Β-Tubulin
B
Figure 2. (A) Effects of quinolines on Her2 degradation and Hsp70 induction. MCF-7 cells were
treated with 0.5 μM of 17-AAG and 50μM of quinoline compounds, DMSO (CTR) for control.
The protein level of Her2 and Hsp70 were detected by western blotting. B-Tubulin was used as a
loading control. (B) Quantification of Her2 degradation and Hsp70 induction.

2.3. Molecular modeling
In order to explore the mode of interactions between the human Hsp90 and 13d, molecular
modeling studies were performed with the N-terminal ATP-binding site of Hsp90.
Our studies showed that alcoholic quinoline derivative 13d, could bind in the binding site of
Hsp90 well (Fig. 3) and is expected to interact with the ATP-binding site of Hsp90 in a similar
9
way to PU3, the known small molecule Hsp90 inhibitor. Modeling studies revealed a great
degree of overlap between the purine ring of PU3 and 2,3-dihydro-[1,4]dioxino[2,3-g]quinoline
tricyclic ring of 13d (Figs 3, 4). The trimethoxy phenyl in the position 7 of 13d interacts with
Hsp90 similar to trimethoxy phenyl ring of PU3. It could be predictable since the same
substitution pattern on the phenyl rings of the two compounds.
The important interaction at the bottom of the binding site between an oxygen atom of 13d with
Asp93 is existing. Interactions with Asp93 are common to all Hsp90 ligands and can be reflected
9
to be essential for interaction with Hsp90. Interactions with Gly97 and Thr184 are also
observed for 13d, imitating key interactions of the adenine base of PU3 and ATP (the natural
ligand of HSP90). Oxygen atom in the position 1 of 13d interacts with an Asp93-Thr184-Gly 97-
water quadripartite in a similar manner to the C6-NH2 of PU3. Oxygen atom in the position 4
and hydroxyl group of 13d, both make hydrogen bonds with amino acid Asn51 mediated by a
water molecule, similar to purine-ring nitrogen’s of PU3. The three methoxy groups of phenyl
ring of 13d form hydrogen bonds with a network of several amino acid residues, like Tyr139,
Leu103, Val150 and two water molecules. The phenyl ring of 13d interacts with the aromatic
ring of Phe138 and side chain of Leu107, the quinoline ring can make hydrophobic interaction
with Met98. Molecular modeling studies revealed that quinoline tricyclic ring of 13d can
incorporate adenine-type interactions. (Compare the interactions of adenine ring of PU3 bound to
human HSP90, with those of quinoline tricyclic ring of 13d docked in human HSP90 (1uym.pdb)
(Figs 3, 4). Therefore, replacement of the adenine moiety with the 2,3-dihydro-[1,4]dioxino[2,3-
g]quinoline tricyclic ring one, can be considered as bioisosteric.

Figure 3. The 2D representation of the interaction between compound 13d in the crystal
structure of human Hsp90 (1uym.pdb) using LigX in MOE

Figure 4. PU3 bound to human HSP90 (1uym.pdb)
3. Conclusions
A new series of 7-(aryl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline analogues was designed,
synthesized and evaluated for their cytotoxic activity against three human cancer cell lines
including MCF-7, A549 and DU-145. All the alcoholic derivatives (13a-13f) showed significant
cytotoxic activity against MCF-7 cells. The biological data showed that the presence and the
number of methoxy and hydroxyl groups at the positions 3, 4 and 5 of phenyl rings are important
for their effects on Her2 degradation and Hsp90 inhibition and their cytotoxicity. Compound 13d

that identified the best antiproliferative activity in the series was found the most potent one for
Her2 protein degradation as well. Molecular modeling studies revealed that quinoline tricyclic
ring of 13d can incorporate adenine-type interactions with N-terminal ATP-binding site of
Hsp90, suggested that the 2,3-dihydro-[1,4]dioxino[2,3-g]quinoline scaffold affords fortunate
interactions with the N-terminal ATP binding site of Hsp90.
4. Experimental section
All chemicals, reagents and solvents used in this study were purchased from Merck AG and
Aldrich Chemical. Melting points were determined with a Thomas–Hoover capillary apparatus.
Infrared spectra were acquired using a Perkin Elmer Model 1420 spectrometer. Bruker FT-500
and 400 MHz instruments (Brucker Biosciences, USA) was used to acquire 1H-NMR spectra and
13
A Bruker FT-300 MHz instrument was used to acquire C-NMR spectra with TMS as internal
standard. Chloroform-D and DMSO-D6 were used as solvents. Coupling constant (J) values are
assessed in hertz (Hz) and spin multiples are given as s (singlet), d (double), t (triplet), q
(quartet), m (multiplet). The mass spectral measurements were performed on a 6410 Agilent
LCMS triple quadrupole mass spectrometer (LCMS) with an electrospray ionization (ESI)
interface.
4.1. Chemistry
4.1.1. General procedure for preparation of 7-aryl-2,3-dihydro-[1,4]dioxino[2,3-
g]quinoline-9-carboxylic acid (Doebner reaction)
A solution of appropriately substituted aldehydes (9.45 mmol) and pyruvic acid (1.26 g, 14.3
mmol) in ethanol (5 ml) was heated for 30 min, then 1,4-benzodioxan-6-amine (9.45 mmol) was
added to the solution and refluxed overnight. After cooling, the produced precipitate was filtered
and washed with ethanol and hexane and recrystallized from ethanol to obtain the pure
compounds.
7-phenyl-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid (12a)
Yield, 58%; mp= 349-351°C: IR (KBr) ν (cm-1):1695(C=O), 3428.5 (OH);1H NMR (400MHz-
DMSO-d6): δ (ppm) 4.34 (s, 4H, CH2-CH2), 7.5-7.574 (m, 4H, phenyl H3 & H4 & H5 & quinoline
H8), 8.15 (s, 1H, quinoline H3), 8.21-8.23 (d, 2H, phenyl H2 & H6, J = 8.8 Hz),8.31(s, 1H,
13
quinoline H5), 13.8 (s, 1H, COOH); C NMR (DMSO-d6, 75 MHz): δ 64.78, 64,78, 110.36,
114.37, 118.27, 120.03, 127.34, 127.34, 129.38, 129.38,130.01, 135.35, 138.61, 145.80, 145.88,

147.62, 154.41, 168.01.LC-MS (ESI): 308.1 (M+1). Anal. Calcd for C18H13NO4: C, 70.35; H,
4.26; N. 4.56. Found: C, 71.42; H, 4.31; N, 4.47.
7-(4-methoxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid (12b)
Yield, 42%; mp= 221-223°C: IR (KBr) ν (cm-1):1648(C=O), 3442.4 (OH);1H NMR (400MHz-
DMSO-d6): δ (ppm) 3.84 (s, 3H, OCH3), 4.42 (s, 4H, CH2-CH2), 7.08–7.1 (d, 2H, 4-methoxy
phenyl H3& H5, J = 8.8 Hz), 7.48 (s, 1H, quinoline H8), 8.12 (s, 1H, quinoline H5) 8.17–8.19 (d,
13
2H, phenyl H2& H6, J = 8.8 Hz), 8.26 (s, 1H, quinoline H3), 13.8 (s, 1H, COOH); C NMR
(DMSO-d6, 75 MHz): δ 55.75, 64.76, 64.76, 110.39, 114.18, 114.74, 114.74,117.84, 119.60,
128.77, 131.07, 131.07, 135.23, 145.39, 145.83, 147.51, 154.14, 161.02, 168.08; LC-MS (ESI):
338.1 (M+1). Anal. Calcd for C19H15NO5: C, 67.65; H, 4.48; N, 4.15. Found: C, 66.25; H, 4.35;
N, 3.95.
7-(3,4-dimethoxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid (12c)
Yield, 31%; mp= 235-237°C: IR (KBr) ν (cm-1):1695(C=O) , 3437(OH);1H NMR (400MHz-
DMSO-d6): δ (ppm) 3.84 (s, 3H, OCH3), 3.91(s, 3H, OCH3), 4.43(s, 4H, CH2-CH2), 7.09–7.11
(d, 1H, 3,4-dimethoxy phenyl H5, J = 8.8 Hz), 7.52 (s, 1H, quinoline H8), 7.76-7.78 (dd, 1H, 3,4-
methoxy phenyl H6 J=8.8 Hz , J= 2Hz) 7.82–7.82 (d, 1H, phenyl H2, J = 2 Hz), 8.09 (s, 1H,
quinoline H3), 8.28 (s, 1H, quinoline H5); 13C NMR (DMSO-d6, 75 MHz): δ 56.01, 56.01, 64.76,
64.76, 110.39, 110.39, 112.12, 114.19, 117.95, 119.60, 120.26, 131.29, 135.27, 145.36, 145.71,
147.47, 149.49, 150.76, 154.18, 168.15; LC-MS (ESI): 368.1 (M+1). Anal. Calcd for
C20H17NO6: C, 65.39; H, 4.66; N, 3.81. Found: C, 68.86; H, 4.79; N, 3.72.
7-(3,4,5-trimethoxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid (12d)
Yield, 48%; mp= 208-210°C: IR (KBr) ν (cm-1):1593(C=O) , 3423.8 (OH);1H NMR (500MHz-
DMSO-d6 ): δ (ppm) 3.69 (s, 3H, OCH3), 3.87(s, 6H, OCH3), 4.37(s, 4H, CH2-CH2), 7.44 (s, 2H,
3,4,5-trimethoxy phenyl H2 & H6), 7.47 (s, 1H, quinoline H8), 8.02(s, 1H, quinoline H3), 8.24 (s,
1H, quinoline H5); 13C NMR (DMSO-d6, 75 MHz): δ 56.51, 56.51, 60.60, 64.77, 64.77, 104.76,
104.76, 110.35, 114.26, 118.12, 119.82, 134.29, 135.93, 139.39, 145.56, 145.56, 147.55, 153.68,
153.68, 154.14, 168.29; LC-MS (ESI): 398.1 (M+1). Anal. Calcd for C21H19NO7: C, 63.47; H,
4.82; N, 3.52. Found: C, 65.43; H, 5.02; N, 3.41.

7-(3-hydroxy-4-methoxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid
(12e)
1
Yield, 50%; mp= 306-308°C: IR (KBr) ν (cm-1):1644(C=O) , 3433(OH); H NMR (400MHz-
DMSO-d6): δ (ppm) 3.80 (s, 3H, OCH3), 4.37 (s, 4H, CH2-CH2), 6.99-7.01 (d, 1H, Phenyl H5,
J=6.8 Hz), 7.42 (s, 1H, quinoline H8), 7.56–7.58 (dd, 1H, phenyl H6, J = 6.8 Hz, J=2 Hz), 7.68-
7.69 (d, 1H, phenyl H2, J=2 Hz), 8.08 (s, 1H, quinoline H5), 8.16 (s, 1H, quinoline H3), 9.21 (s,
1H, OH); 13C NMR (DMSO-d6, 75 MHz): δ 56.05, 64.74, 64.74, 110.48, 112.56, 117.92, 118.74,
119.13,119.64, 131.41, 135.11, 145.31, 145.82, 147.07, 147.24, 147.43, 149.71, 154.26, 168.15;
LC-MS (ESI): 354.1 (M+1). Anal. Calcd for C19H15NO6: C, 64.59; H, 4.28; N, 3.96. Found: C,
66.11; H,4.35; N,3.86.
7-(4-hydroxy-3-methoxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid
(12f)
1
Yield, 59%; mp= 279-281°C: IR (KBr) ν (cm-1):1639(C=O) , 3428.5(OH); H NMR (500MHz-
DMSO-d6): δ (ppm) 3.86 (s, 3H, OCH3), 4.36 (s, 4H, CH2-CH2), 6.87-6.88 (d, 1H, Phenyl H5,
J=8 Hz), 7.43 (s, 1H, quinoline H8), 7.60–7.62 (dd, 1H, phenyl H6, J = 8 Hz, J=2 Hz), 7.754-
7.758 (d, 1H, phenyl H2, J=2 Hz), 8.04 (s, 1H, quinoline H5), 8.19 (s, 1H, quinoline H3), 9.38 (s,
1H, OH),13.7(s, 1H, COOH); 13C NMR (DMSO, 75 MHz): δ 56.23, 64.73, 64.73, 110.43,
110.98, 114.11, 116.12, 117.90, 119.48, 120.63, 130.08, 135.20, 145.18, 145.71, 147.38, 148.42,
148.84, 154.45, 168.23; LC-MS (ESI): 354.1 (M+1). Anal. Calcd for C19H15NO6: C, 64.59; H,
4.28; N, 3.96. Found: C, 65.66; H, 4.25; N, 3.83.
7-(3-hydroxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid (12g)
Yield, 16%; mp= 290-292°C: IR (KBr) ν (cm-1):1678.1(C=O) , 3397.5(OH); 1H NMR (400MHz-
DMSO-d6): δ (ppm) 4.43 (s, 4H, CH2-CH2), 6.88–6.90 (dd, 1H, 3-Hydroxy phenyl H4, J = 8 Hz,
J=2 Hz), 7.32-7.36 (t, 1H, 3-Hydroxy phenyl H5, J= 8),7.51( s, 1H, 3-Hydroxy phenyl H2), 7.60-
7.62 (d, 1H, 3-Hydroxy phenyl H6, J=8 Hz) 7.65 (s, 1H, quinoline H8), 8.16(s, 1H, quinoline H3),
8.23 (s, 1H, quinoline H5),9.62(s, 1H, OH), 13.8(s, 1H, COOH); 13C NMR (DMSO-d6, 75 MHz):
δ 64.76, 64.76, 110.41, 113.85, 114.30, 117.09, 118.15, 118.36, 120.09, 130.45, 135.11, 139.98,
145.73, 145.83, 147.56, 154.39, 158.26, 168.01; LC-MS (ESI): 324.1 (M+1). Anal. Calcd for
C18H13NO5: C, 66.87; H, 4.05; N, 4.33. Found: C, 67.56; H, 4.13; N, 4.22.
7-(4-hydroxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid (12h)

Yield, 19%; mp= 290-292°C: IR (KBr) ν (cm-1):1709(C=O) , 3414.5 (OH); 1H NMR (400MHz-
DMSO-d6): δ (ppm) 4.41 (s, 4H, CH2-CH2), 6.90–6.92 (d, 2H, 4-Hydroxy phenyl H3 & H5 , J =
8.4 Hz),7.47( s, 1H, quinolineH8), 8.068-8.09(d, 2H, 4-Hydroxy phenyl H2&H6 , J=8.4 Hz), 8.11
(s, 1H, quinoline H3), 8.22(s, 1H, quinoline H5), 9.87(s, 1H, OH), 13.8(s, 1H, COOH); 13C NMR
(DMSO-d6, 75 MHz): δ 64.73, 64.73, 110.08, 114.06, 116.07, 116.07, 119.09, 119.46, 128.84,
134.30, 135.07, 145.18, 154.33, 154.43, 159.38, 166.44, 168.12; LC-MS (ESI): 324.1 (M+1).
Anal. Calcd for C18H13NO5: C, 66.87; H, 4.05; N, 4.33. Found: C, 65.95; H, 4.18; N, 4.23.
7-(4-acetamidophenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid (12i)
Yield, 39%; mp= 296-298°C: IR (KBr) ν (cm-1): 1653.5, 1630.2(C=O), 3442.4 (OH), 3061.4
(NH); 1H NMR (400MHz-DMSO-d6): δ (ppm) 2.09(s, 3H, CH3) 4.42 (s, 4H, CH2-CH2), 7.50( s,
1H, quinoline H8)7.75–7.77 (d, 2H, 4-Acetamido phenyl H3 & H5 , J = 8.4 Hz),8.127 (s, 1H,
quinoline H3), 8.16-8.18(d, 2H, 4-Acetamido phenyl H2&H6 , J=8.4 Hz), 8.27 (s, 1H, quinoline
13
H5), 10.15(s, 1H, NH), 13.8(s, 1H, COOH); C NMR (DMSO-d6, 75 MHz): δ 64.77, 64.77,
110.41, 114.20, 117.92, 119.38, 119.38, 119.75, 127.85, 127.85, 133.08, 135.31, 141.02, 145.50,
145.85, 147.53, 154.03, 168.11, 168.96; LC-MS (ESI): 365.1 (M+1). Anal. Calcd for
C20H16N2O5: C, 65.93; H, 4.43; N, 7.69. Found: C, 66.71; H, 4.51; N, 7.62.
7-(p-tolyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline-9-carboxylic acid (12j)
1
Yield, 28%; mp= 276-278°C: IR (KBr) ν (cm-1):1704 (C=O), 2931.3(OH); H NMR (400MHz-
DMSO-d6): δ (ppm) 2.39 (s, 3H, CH3), 4.42 (s, 4H, CH2-CH2), 7.34-7.36 (d, 2H, 4-methyl
phenyl H3 & H5, J= 8 Hz), 7.51 (s, 1H, quinoline H8), 7.67 (s, 1H, quinoline H5) 8.11-8.13 (d,
2H, phenyl H2 & H6, J=8), 8.28(s, 1H, quinoline H3), 13.5 (s, 1H, COOH); 13C NMR (DMSO-d6,
75 MHz): δ 21.33, 64.74, 64.74, 110.39, 114.28, 118.08, 119.90, 127.19, 127.19, 129.93, 129.93,
135.16, 135.85, 139.60, 145.57, 145.84, 147.49, 154.32, 168.06; LC-MS (ESI): 322.1 (M+1).
Anal. Calcd for C19H15NO4: C, 71.02; H, 4.71; N, 4.36. Found: C, 70.84; H, 4.87; N, 4.29.
4.1.2. General procedure for preparation of 2-aryl- 5,6,7-trimethoxyquinoline-4-
yl)methanol
LiAlH4 (0.165 g, 4.32 mmol) was added to dry THF (20 ml) under a nitrogen atmosphere. Under
vigorous stirring a solution of appropriate acid (1 mmol) in dry THF was added drop wise to
keep the reaction mixture somewhat boiling. After stirring for 5 h at room temperature, the
resulted suspension was carefully hydrolyzed with NaOH solution (10%) till no more hydrogen

was formed. The solid was filtered off and washed thoroughly with chloroform. The filtrate was
extracted by chloroform, dried over Na2SO4 and the solvent was removed under reduced pressure
to yield a solid which was washed with hexane and cold diethyl ether to obtain the pure
compounds.
(7-phenyl-2,3-dihydro-[1,4]dioxino[2,3-g]quinolin-9-yl)methanol (13a)
1
Yield, 81%; mp= 137-139°C: IR (KBr) ν (cm-1): 3168 (OH); H NMR (300MHz-CDCl3): δ
(ppm) 3.35 (s, 1H, OH),4.37(s, 4H, CH2-CH2), 4.98( s, 2H, CH2), 7.2(s, 1H, quinoline H3), 7.41–
7.48 (m, 3H, phenyl H3 & H4 & H5 ), 7.6(s, 1H, quinoline H5) 7.66 (s, 1H, quinoline H8), 7.99-
8.01(d, 2H, phenyl H2 & H6, J= 6.6 Hz); 13C NMR (DMSO-d6, 75 MHz): δ 60.62, 64.72, 108.44,
114.19, 114.35, 120.90, 127.26, 127.26, 129.25, 129.25, 129.58, 139.62, 144.35, 144.49, 147.04,
147.50, 154.67; LC-MS (ESI): 294.1 (M+1). Anal. Calcd for C18H15NO3: C, 73.71; H, 5.15; N,
4.78. Found: C, 75.21; H, 5.36; N, 4.69.
(7-(4-methoxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinolin-9-yl)methanol (13b)
Yield, 21%; mp= 90-92°C: IR (KBr) ν (cm-1): 3400 (OH);1H NMR (300MHz-CDCL3): δ (ppm)
2.53(s, 1H, OH),3.97 (s, 3H, OCH3), 4.47 (s, 4H, CH2-CH2), 5.08( s, 2H, CH2), 7.05–7.08(d, 2H,
4-methoxy phenyl H3& H5, J = 8.1 Hz), 7.39 (s, 1H, quinoline H8), 7.67 (s, 1H, quinoline H5),
13
7.71 (s, 1H, quinoline H3), 8.05-8.08 (d, 2H, 4-methoxy phenyl H2 & H6 J=8.1 Hz); C NMR
(CDCl3, 75 MHz): δ 55.36, 62.04, 64.41, 64.41, 107.62, 114.07, 114.07, 114.26, 114.59, 120.32,
128.63, 128.63, 132.23, 143.73,144.54,145.08, 146.58, 155.36, 160.54; LC-MS (ESI): 324.1
(M+1). Anal. Calcd for C19H17NO4: C, 70.58; H, 5.30; N, 4.33. Found: C, 72.31; H, 5.54; N,
4.24.
(7-(3,4-dimethoxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinolin-9-yl)methanol (13c)
Yield, 74%; mp= 216-218°C: IR (KBr) ν (cm-1): 3270 (OH);1H NMR (300MHz-DMSO-d6): δ
(ppm) 3.84 (s, 3H, OCH3),4.45(s, 1H, OH), 4.48-4.49 (m, 4H, CH2-CH2), 5.11( s, 2H, CH2)7.23–
7.26 (d, 1H, 3,4-methoxy phenyl H5, J = 8.7 Hz), 7.66 (s, 1H, quinoline H8), 7.75-7.79 (dd, 1H,
3,4-methoxy phenyl H6, J=8.7 Hz , J=2 Hz) 7.88–7.89 (d, 1H, 3,4-methoxy phenyl H2, J = 2 Hz),
8.05-8.11(m, 2H, quinoline H3& H5); LC-MS (ESI): 354.1 (M+1). Anal. Calcd for C20H19NO5:
C, 67.98; H, 5.42; N, 3.96. Found: C, 69.16; H, 5.64; N, 3.84.
(7-(3,4,5-trimethoxyphenyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinolin-9-yl)methanol (13d)

1
Yield, 38%; mp= 201-203°C: IR (KBr) ν (cm-1): 3214 (OH); H NMR (300MHz-DMSO-d6): δ
(ppm) 3.90 (s, 3H, OCH3), 3.97 (s, 3H, 2OCH3), 4.40 (s, 4H, CH2-CH2), 5.10 ( s, 2H, CH2), 7.30
(s, 1H, quinoline H8), 7.32 (s, 2H, phenyl H2& H6), 7.63(s, 1H, quinoline H5) 7.70(s, 1H,
13
quinoline H3); C NMR (CDCl3, 75 MHz): δ 56.26, 56.2,60.97, 62.04, 64.40, 64.44, 104.50,
104.50, 107.57, 114.42, 114.79, 120.57, 135.46, 139.05, 144.05, 144.53, 145.10, 146.71, 153.39,
153.39, 155.37; LC-MS (ESI): 384.2 (M+1). Anal. Calcd for C21H21NO6: C, 65.79; H, 5.52; N,
3.65. Found: C, 64.86; H, 6.04; N, 3.41.
5-(9-(hydroxymethyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinolin-7-yl)-2-methoxyphenol (13e)
1
Yield, 18%; mp=205 °C (decomposed): IR (KBr) ν (cm-1): 3368 (OH); H NMR (300MHz-
DMSO-d6): δ (ppm)3.84 (s, 3H, OCH3), 4.39 (m, 5H, CH2-CH2 & OH), 4.93 ( s, 2H, CH2),7.04-
7.07 (d, 1H, Phenyl H5, J=8.4 Hz), 7.39-7.40 (m, 2H, quinoline H3 & H5), 7.58–7.61 (dd, 1H,
phenyl H6, J = 8.4 Hz, J=1.8 Hz), 7.72-7.73 (d, 1H, phenyl H2, J=1.8 Hz), 7.8 (s, 1H, quinoline
H8), 9.25 (s, 1H, OH); 13C NMR (DMSO, 75 MHz): δ 56.08, 60.52, 64.68, 64.74, 108.46,
112.56, 113.74, 113.95, 114.19, 114.30, 118.62, 120.50, 143.99, 146.96, 147.04, 147.18, 149.40,
149.46, 154.46; LC-MS (ESI): 340.1 (M+1). Anal. Calcd for C19H17NO5: C, 67.25; H, 5.05; N,
4.13. Found: C, 65.27; H, 5.15; N, 3.89.
4-(9-(hydroxymethyl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinolin-7-yl)-2-methoxyphenol (13f)
Yield, 62%; mp= 110-112°C: IR (KBr) ν (cm-1):3442 (OH);1H NMR (400MHz-DMSO-d6): δ
(ppm) 3.86 (s, 3H, OCH3), 4.37(m, 5H, CH2-CH2 & OH), 4.9 (s, 2H, CH2), 6.80-6.82(d, 1H, 4-
hydroxy,3-methoxy phenyl H5, J = 8.4 Hz), 7.35-7.36 (m, 2H, quinoline H5 & H8), 7.55-7.58
(dd, 1H, 4-hydroxy,3-methoxy phenyl H6, J=8.4 Hz , J=2 Hz) 7.72–7.73(d, 1H, 4-hydroxy,3-
13
methoxy phenyl H2, J = 2 Hz), 7.85(s, 1H, quinoline H3), 8.51 (s, 1H, OH); C NMR (DMSO-
d6, 75 MHz): δ 56.14, 60.66, 64.67, 64.73, 108.51, 111.00, 113.96, 114.07, 116.17, 120.47,
130.39, 143.77, 144.42, 146.81, 146.98, 148.48, 149.38, 154.85, 167.28; LC-MS (ESI): 340.1
(M+1). Anal. Calcd for C19H17NO5: C, 67.25; H, 5.05; N, 4.13. Found: C, 66.24; H, 5.21; N,
3.98.
4.1.3. General procedure for imine preparation

1,4-benzodioxan-6-amine (1 mmol) was heated at reflux with the appropriate aldehyde (1 mmol)
in ethanol (5 mL) for 3 h. The reaction mixture was cooled and then the solvent was removed
under reduced pressure. The obtaining solid product was recrystallized in ethanol.
N-benzylidene-2,3-dihydrobenzo[b][1,4]dioxin-6-amine (14a)
1
Yield, 88%; liquid; IR (KBr) ν (cm-1): 1630(C=N); H NMR (400MHz-DMSO-d6): δ (ppm),
4.29 (s, 4H, CH2-CH2), 6.82-6.83(d, 1H, benzodioxine H5, J=2.4 Hz), 6.85-6.87(dd,
1H,benzodioxine H7, J=8.4 Hz, J=2.4 Hz), 6.91-6.93(d, 1H, benzodioxine H8 , J= 8.4 Hz), 7.48-
7.49(m, 3H, phenyl H3& H4 & H5), 7.9-7.92(m, 2H, phenyl H2& H6), 8.48 (s, 1H, CH).
N-(4-methoxybenzylidene)-2,3-dihydrobenzo[b][1,4]dioxin-6-amine (14b)
1
Yield, 58%; mp= 68-70°C; IR (KBr): ν (cm-1) 1625 (C=N); H NMR (400MHz-DMSO-d6): δ
(ppm), 3.30(s, 3H, CH3O), 4.27(s, 4H, CH2-CH2), 6.78-6.79(d , 1H, benzodioxine H5, J=2.4 Hz),
6.80-6.83(dd, 1H,benzodioxine H7, J=8.4 Hz, J=2.4 Hz), 6.89-6.91(d, 1H, benzodioxine H8 , J=
8.4 Hz), 6.97-6.99(d, 2H, 4-methoxyphenyl H3 & H5, J=8.8 Hz), 7.831-7.853(d, 2H, 4-
methoxyphenyl H2& H6, J= 8.8 Hz), 8.39 (s, 1H, CH).
N-(3,4-dimethoxybenzylidene)-2,3-dihydrobenzo[b][1,4]dioxin-6-amine (14c)
1
Yield, 81%; mp= 65-67°C; IR (KBr): ν (cm-1) 1625.6 (C=N); H NMR (400MHz-DMSO-d6): δ
(ppm), 3.95(s, 3H, CH3O), 3.99 (s, 3H, CH3O), 4.28 (s, 4H, CH2-CH2), 6.80-6.94 (m , 4H, 3,4-
dimethoxy phenyl H5, benzodioxine H5, H7 & H8), 7.28(s, 1H, phenyl H6), 7.6(s, 1H, phenyl H2),
8.39 (s, 1H, CH).
N-(3,4,5-trimethoxybenzylidene)-2,3-dihydrobenzo[b][1,4]dioxin-6-amine (14d)
Yield, 71%; mp= 148-150°C; IR (KBr): ν (cm-1) 1625.6 (C=N); 1H NMR (400MHz-DMSO-d6):
δ (ppm), 3.92(s, 3H, OCH3), 3.95 (s, 6H, CH3O) 4.28(s, 4H, CH2-CH2), 6.78-6.90(M, 3H,
benzodioxine), 7.14 (s, 2H,trimethoxyphenyl H2& H6), 8.35 (s, 1H, CH).
5-(((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)imino)methyl)-2-methoxyphenol (14e)
Yield, 85%; mp= 180-182°C; IR (KBr): ν (cm-1) 1625.6 (C=N), 3066(OH); 1H NMR (400MHz-
CDCl3): δ (ppm), 3.98(s, 3H, OCH3), 4.29(s, 4H, CH2-CH2), 5.67(s, 1H, OH), 6.81-6.90(m , 3H,
benzodioxine) 6.92-6.95(d, 1H, phenyl H5 , J=8.4 Hz), 7.36-7.38(dd, 1H,phenyl H6, J=8.4 Hz,
J=2 Hz), 7.52-7.53 (d, 1H, phenyl H2, J=2 Hz), 8.35(s, 1H, CH).

4-(((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)imino)methyl)-2-methoxyphenol (14f)
1
Yield, 84%;mp= 156-158°C; IR (KBr): ν (cm-1)1625.6 (C=N), 2926 (OH); H NMR (400MHz-
CDCl3): δ (ppm), 3.84(s, 3H, OCH3), 4.24(s, 4H, CH2-CH2), 6.87-7.49 (m, 5H, benzodioxine&
phenyl H5 & H6), 7.49(s, 1H, phenyl H2), 8.43(s, 1H, CH), 9.68(s, 1H, OH).
3-(((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)imino)methyl)phenol (14g)
Yield, 91%; mp= 130-132°C; IR (KBr): ν (cm-1) 1625.6 (C=N), 3382 (OH); 1H NMR (400MHz-
DMSO-d6): δ (ppm), 4.29 (s, 4H, CH2-CH2), 6.82-6.99 (m ,4H, benzodioxine & 3-hydroxy
phenyl H4), 7.28-7.42 (m, 4H,3-hydroxy phenyl H2& H5 & H6 & OH ), 8.39(s, 1H, CH).
4-(((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)imino)methyl)phenol (14h)
1
Yield, 95%; mp= 210-212°C; IR (KBr): ν (cm-1)1607(C=N), 2922 (OH); H NMR (400MHz-
DMSO-d6): δ (ppm), 4.24 (s, 4H, CH2-CH2), 6.77-6.87 (m, 5H, benzodioxine & 4-hydroxy
phenyl H3 & H5), 7.72-7.74 (d,2H,4-hydroxy phenyl H2& H6, J= 8.4),8.44 (s, 1H, CH), 10.06 (s,
1H, OH).
N-(4-methylbenzylidene)-2,3-dihydrobenzo[b][1,4]dioxin-6-amine (14i)
1
Yield, 85%; liquid; IR (KBr): ν (cm-1)1625.6 (C=N); H NMR (400MHz-DMSO-d6): δ (ppm),
3.35(s, 3H, CH3 ), 4.29(s, 4H, CH2-CH2), 6.81-6.92 (m , 3H, benzodioxine), 7.28-7.30(d, 2H, 4-
methyl phenyl H3& H5 , J= 8 Hz), 7.79-7.81 (d, 2H, 4-methyl phenyl H2& H6 , J= 8 Hz), 8.44 (s,
1H, CH).
4.2. Biology
4.2.1. Cytotoxicity assay
The MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) based assay was
carried out by seeding 5000 cancer cells per 180 µL RPMI complete culture medium in each
well of 96-well culture plates. The day after seeding, culture medium was replaced with medium
containing standard anti-tumor agent 17-AAG as well as different concentrations of newly
synthesized quinolines and RPMI control (no drug). Cells were then incubated at 37 ºC in 5%
CO2 incubator for 48 h. Then 25 µL of MTT solution (4mg Ml-1) were added to each well and
further incubated at 37 ºC for 3 h. At the end of incubation, formazan crystals were dissolved in
100 µL of DMSO and plates were read in a plate reader (Synergy H4, USA) at 540 nm. This
experiment was performed in triplicate determination each time. 14

4.2.2. Her2 degradation assay
MCF-7 breast cancer cells were seeded on to 25 cm2 flasks at a density of 106 cells per flask.
Cells were treated with all samples at the concentration of 50 µM for 24 h. The cells were then
lysed in a buffer containing 50 mM Tris-HCl with pH 7.4, 2 mM EGTA, 1 Mm phenyl methyl
sulfonyl fluoride, 10 mM β-Glycerophosphate, 10 mM β-mercaptoethanol, 1 mM sodium
orthovanadate, 0.1% deoxycholic acid sodium salt. Proteins (50 µg) were resolved by 10% SDS-
polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidene
difluoride (PVDF) membranes and then were blocked for 2 h in 5% skim milk in TBST (20 Mm
Tris-HCl pH 7.4, 100 mM NaCl, and 0.1% Tween 20) buffer at 4°C. After washing in TBST
buffer, they were then incubated for 2 h with primary antibodies: Rabbit Her-2 antibody
(1:3000), Rabbit Hsp-70 antibody (1:3000). After incubation with an anti-rabbit IgG (1:1000) as
secondary antibody the bands were detected using the ECL Prime Western Blotting Detection
System (Bio RaD, USA). Bands were scanned by GS-800, and band intensities were quantified
by measuring optical densities with Quantity One software (Bio-Rad). 17, 20
4.3. Molecular modeling
Mode of interaction between synthesized ligands and Hsp90 was investigated by docking. 2D
structure of chemicals was prepared in Chem Draw Ultera 8.0 software and 3D structures were
prepared by Hyperchem 7 software using molecular mechanic force filed pre-optimization
followed by AM1 semi empirical calculation. The X-ray crystal structure of Hsp90 (PDB ID:
1uym) was downloaded from the Protein Data Bank (www.rcsb.org). Further modification such
as polar hydrogen addition was done by MOE software. Synthesized chemicals were docked into
the binding site of Hsp90 by MOE software. All atoms within a 5 Å around the co-crystallized
ligand in crystal coordinates of Hsp90 was chosen as binding site. The docking simulations were
done employing triangle matcher placement algorithm in combination with London dG scoring
function and force field as refinement method. For each compound, the top-score docking poses
were chosen for final ligand–target interaction analysis employing LigX module in MOE
Software.16
Acknowledgment

We are grateful to Research deputy of Mashhad University of Medical Sciences for financial
support of this research as part of thesis of Sina Omid Malayeri.
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