Synthesis and biological evaluation of 2,4-diaminoquinazoline derivatives as novel heat shock protein 90 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.01.117

Novel 2,4-diaminoquinazoline derivatives originating from a virtual screening approach were designed, synthesized and their biological activities as heat shock protein 90 (Hsp90) inhibitors were evaluated. The prepared compounds exhibited significant anti-proliferative activities against DU-145, HT-29, HCT-116, A375P and MCF-7 cancer cell lines. The selected compounds were tested against Her2, a client protein of Hsp90, and showed significant reduction in Her2 protein expression. Compound 6b was found the most potent, reduced Her2 protein expression levels and induced Hsp70 protein expression levels significantly.

Full text of DOI:10.1016/j.bmcl.2011.01.117

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1593–1597
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 2,4-diaminoquinazoline derivatives
as novel heat shock protein 90 inhibitors
Dhanaji Achyutrao Thorat a,c, Munikumar Reddy Doddareddy a,c, Seon Hee Seo a, Tae-Joon Hong b,
Yong Seo Cho a, Ji-Sook Hahn b, Ae Nim Pae a,
⇑
a Neuro-Medicine Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
b School of Chemical and Biological Engineering, Seoul National University, Sillim-dong, Gwanak-gu, Seoul 151-744, Republic of Korea
c School of Science, Korea University of Science and Technology, 52 Eoeun dong,Yuseong-gu, Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 2,4-diaminoquinazoline derivatives originating from a virtual screening approach were designed,
synthesized and their biological activities as heat shock protein 90 (Hsp90) inhibitors were evaluated.
The prepared compounds exhibited significant anti-proliferative activities against DU-145, HT-29,
HCT-116, A375P and MCF-7 cancer cell lines. The selected compounds were tested against Her2, a client
protein of Hsp90, and showed significant reduction in Her2 protein expression. Compound 6b was found
the most potent, reduced Her2 protein expression levels and induced Hsp70 protein expression levels
significantly.
Received 13 October 2010
Revised 19 January 2011
Accepted 27 January 2011
Available online 2 February 2011
Keywords:
Hsp90 inhibitors
Cancer
Ó 2011 Elsevier Ltd. All rights reserved.
2,4-Diaminoquinazolines
Hsp90 proteins are important molecular chaperones that are
responsible for the conformational maturation of numerous onco-
genic proteins.1 The Hsp90 family generally represent 1–2% of total
cellular proteins in unstressed cells and up to 4–6% of total cellular
proteins in cells under stress.2 Currently, four Hsp90 chaperones
the degradation of multiple key proteins that depend on the inter-
action with Hsp90 for maintaining their bioactive conformation.
Targeting multiple oncogenic proteins provides an advantage for
cancer therapy due to the potential to increase the efficacy of the
therapeutic and to overcome the drug resistance that occurs in
many cancers including prostate7 and breast cancer.8 Most pub-
lished work show Hsp90 expression in breast cancer cell lines.9
In 1994, geldanamycin (GDC) was identified as the first natural
product that inhibited of Hsp90.10 It was shown to bind to Hsp90
and interfere with the Hsp90-v-src heterocomplex formation.11
GDC alters chaperone function and drives the degradation of many
Hsp90 client proteins by stimulating Hsp90-mediated presentation
to the ubiquitin-proteosome machinery.12 Radicicol (RDC), the
most potent natural product inhibitor in vitro, is shown to be inac-
tive in vivo.13 A 17-carbon position derivative, 17-(allylamino)-
17-(demethoxygeldanamycin (17-AAG) is currently being tested
in ongoing phase 1 and phase 2 clinical trials. Although antitumor
activity has been observed in early clinical trials of 17-AAG, this
agent is poorly soluble and has limited oral bioavailability. In vivo,
RDC is rapidly converted into inactive metabolites that have little
or no affinity for Hsp90.
are known, including Hsp90a and Hsp90b, which are found in
cytoplasm, Grp94, which is found in the endoplasmic reticulum,
and Trap-1, which is found in the mitochondria.3 The cytoplasmic
forms of these chaperones exist predominantly as dimers within
the cell, and each subunit is composed of three domains: a 24–
28-kDa N-terminal ATP-binding domain, a 38–44 kDa middle do-
main and
a 11–15 kDa C-terminal domain. Conformational
changes that occur upon binding and hydrolysis of ATP regulate
the ability of the chaperone to bind to its client proteins.4 Its client
proteins include kinases, steroid hormone receptors, transcription
factors that are directly involved in malignancy and mutated onco-
genic proteins that are required for the transformed phenotype
(i.e., Her2, raf-1, Akt, Cdk4, Polo-1-kinase, cMet, mutant B-raf, mu-
tant P53, Ar, ER, Bct-Abl, Hif-1, alpha, and hTERT.5)
Hsp90 has attracted growing interest over the past few years
because of its role in the evolution, development and disease
pathology of cancer.6 Two natural products, radicicol (RDC) (1
Fig. 1) and geldanamycin (GDC) (2 Fig. 1), are known to be potent
inhibitors of Hsp90 chaperone activity. Inhibition of Hsp90 leads to
To overcome the limitations of natural product inhibitors, syn-
thetic Hsp90 inhibitors have also been reported (3–5, Fig. 1).
Gopalsamy et al. reported the benzisoxazole-based compound 3
with IC50 = 30 nM, in comparison to 20 nM for GDC.14 Hahn and
coworkers disclosed the quinazoline-4-one based compound 4
⇑
Corresponding author. Tel.: +82 2 958 5185; fax: +82 2 958 5189.
E-mail address: anpae@kist.re.kr (A.N. Pae).
with IC50 = 20 l
M in an ATPase assay.15 Chiosis and coworkers
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.117

1594
D. A. Thorat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1593–1597
O
R
O
OH
Cl
O
N
H
O
O
O
MeO
HO
H
OH
H
MeO
O
OCONH2
1. Radicicol
2. R=OMe; Geldanamycin
R=NHAll; 17-AAG
(Monorden)
NH2
N
OH
Cl
N
N
OCH3
N
O
N
N
Cl
F
HO
OH
OMe
OMe
H
OCH3
N
N
N
O
MeO
5
O
3
4
Figure 1. Structures of natural and synthetic inhibitors of Hsp90.
reported a purine-based inhibitor, compound 5 (PU24FCl), with
low micromolar in vitro potency.16 In the present study, we have
identified the 2,4-diaminoquinazolines, a new class of Hsp90
inhibitors based on the structure of the hit compound 6a (Fig. 2)
obtained by a virtual screening approach.17 The compound 6a
showed growth inhibition of human cancer cells with IC50 values
MCF-7. Synthesis and SAR studies lead to several 2,4-diaminoqui-
nazoline analogs with low micromolar inhibition against various
cancer cell lines. Based on the structure of hit compound 6a, the
synthesis and SAR of the 2,4-diaminoquinazoline derivatives are
described in detail below.
The 2,4-diaminoquinazoline derivatives (Table 1) were synthe-
sized as shown in Scheme 1. The quinazoline-2,4-(1H,3H)-
of 35.01
l
M for DU-145, 12.19
lM for HT-29, and 7.25 lM for
O
O
HN
HN
N
OMe
N
R1
R2
N
N
H
N
N
H
6a
6b-p
7a-p
8a-p
R1= H, 6F, 8F
R2=
Cl
Cl
N
N
N
N
N
N
O
a
b
c
d
Cl
Cl
N
N
O
N
O
N
e
N
f
h
g
i
O
N
N
N
l
N
N
k
m
n
o
j
Cl
Cl
N
N
p
Figure 2. Designed Hsp90 inhibitors.

D. A. Thorat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1593–1597
1595
Table 1
Cytotoxity (% inhibition) and IC50
(l
M) values of 2,4-diaminoquinazoline derivatives against various cancer cell lines at 10 lM concentration
O
HN
N
R1
R2
N
N
H
Compds
R1
R2
DU145
HT-29
HCT-116
A375P
MCF-7
%In.(IC50
)
%In.(IC50
)
%In.(IC50
)
%In.(IC50
)
%In.(IC50)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
7a
7b
7c
7e
7g
7h
7i
7l
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
6F
6F
6F
6F
6F
6F
6F
6F
6F
6F
6F
6F
8F
a
b
c
d
e
f
g
h
i
nd(35.01)
93.63(1.07)
91.52(0.93)
93.16(3.71)
94.32(3.87)
20.04(nd)
nd(nd)
nd(12.19)
nd(nd)
nd(nd)
nd(3.90)
nd(3.04)
Nd(3.36)
Nd(3.75)
nd(nd)
nd(nd)
nd(nd)
nd(nd)
nd(nd)
64.55(7.25)
95.78(3.14)
95.86(3.31)
95.85(2.98)
95.82(2.97)
nd(nd)
95.75(2.12)
nd(nd)
nd(nd)
nd(nd)
nd(nd)
nd(nd)
nd(nd)
66.70(nd)
nd(nd)
nd(nd)
97.65(3.13)
nd(nd)
nd(nd)
nd(nd)
94.42(nd)
nd(nd)
nd(nd)
nd(nd)
94.21(0.86)
93.99(0.84)
94.10(3.37)
94.75(3.37)
21.69(nd)
nd(nd)
nd(6.73)
nd(23.93)
nd(4.45)
87.48(6.01)
75.11(11.26)
80.02(10.29)
92.27(2.94)
68.68(1.34)
86.19(4.34)
96.12(1.83)
96.69(1.05)
96.30(0.39)
9.68(nd)
94.31(0.84)
93.21(0.77)
94.45(3.36)
94.73(3.2)
nd(nd)
nd(nd)
nd(nd)
nd(nd)
nd(nd)
67.50(16.12)
62.58(26.14)
50.25(25.91)
94.78(5.47)
79.79(2.10)
73.98(6.15)
97.39(0.82)
97.34(1.14)
97.09(0.52)
12.76(nd)
94.17(4.1)
52.09(25.28)
27.44(nd)
26.39(nd)
87.77(4.31)
81.98(2.14)
nd(1.01)
nd(11.53)
nd(79.91)
nd(51.03)
40.06(35.27)
30.25(>100)
28.48(>100)
89.90(4.99)
52.26(6.24)
55.38(11.63)
93.40(3.32)
95.93(2.66)
95.48(0.67)
27.76(nd)
83.59(7.24)
26.72(>100)
32.36(nd)
26.59(nd)
75.37(5.91)
57.57(6.46)
nd(nd)
j
k
l
nd(19.39)
nd(>100)
nd(34.88)
nd(9.2)
m
n
o
a
b
c
e
g
h
i
59.19(5.23)
nd(7.66)
97.55(3.28)
97.59(2.83)
97.35(2.65)
28.55(nd)
nd(7.34)
nd(22.51)
15.84(nd)
7.67(nd)
nd(8.48)
62.00(4.44)
nd(2.42)
nd(2.63)
nd(nd)
nd(4.03)
83.07(9.32)
34.83(nd)
27.52(nd)
88.09(3.04)
76.78(1.36)
nd(nd)
l
7m
7n
7o
7p
8n
Doxorubicin
m
n
o
p
n
nd(nd)
nd(nd)
95.48(nd)
86.98(nd)
nd(nd)
nd(nd)
nd(nd)
nd(nd)
nd(0.82)
nd(1.33)
nd(nd)
Du-145: Human prostate cancer cell line, HT-29: Human colon cancer, HCT-116: Human colon carcinoma cancer, A375P: Human melanoma cancer, MCF-7: Breast cancer cell
line; nd: not determined.
diones18(12–14) were synthesized from 2-amino benzoic acids or
methyl-2-amino (9–11) benzoates which were treated with
phosphoryl chloride in the presence of N,N-diethylaniline or N,N-
diisopropylethyleneamine to obtain 2,4-dichloroquinazolines19
(15–17). Further treatment of the 2,4-dichloroquinazolines with
furfurylamine in the presence of ethanol yielded the 2-chloro-N-
(furan-2-ylmethyl)quinazoline-4-amines20 (18–20).
Finally, the 2-chloro-N-(furan-2-ylmethyl)quinazoline-4-amines
(18–20) were treated with the appropriate amines in the presence of
ethanol and catalytic amounts of concentrated hydrochloric acid to
yield the target 2,4-diaminoquinazoline derivatives (6b–q, 7a–q,
8a–q).21 The amines that are not commercially available were syn-
thesized as per Scheme 1.22
The inhibitory activities of the 2,4-diaminoquinazoline deriva-
tives were measured against various cancer cell lines including
DU-145 (prostate), HT-29 (colon), HCT-116 (colon carcinoma),
A375P (melanoma). The IC50 values were also measured.23 As the
Human Epidermal growth Factor (Her2) is mainly found in breast
cancer cells, which are client proteins of Hsp90, the selected
compounds were tested for their anti-cancer activities against
the MCF-7 breast cancer cell line.
To improve the activity, a variety of different linkers were intro-
duced between the quinazoline and terminal amine moieties. In
general, compounds with phenylpipierazine moiety (6b–e, 7b, 7c,
7e, 7p) showed enhanced potency (Table 1) over the compounds
without phenylpiperazine moiety (6h–o, 7h– n). The compounds
which shown better potency against DU-145 (prostate), HT-29
(colon), HCT-116 (colon carcinoma), A375P (melanoma) were
chosen to tested against MCF-7 breast cancer cell line. As seen in
Table 1, all the selected compound (6b–e and 7b) having phenylpip-
erazine moiety exhibited more potent activity against MCF-7 breast
cancer cell line compared to lead compound 6a.
The compound which shown the better potency against MCF-7
breast cancer cell line were tested for their affects on Her2 degra-
dation in MCF-7 breast cancer cell line,24 in addition we tested for
the induction of Hsp70 to evaluate Hsp90 inhibitors. Since heat
shock transcription factor (Hsf1) is inactivated by Hsp90, most of
the Hsp90 inhibitors such as geldanamycin activate expression of
Hsp1 target genes including Hsp70.25 MCF-7 cells were treated
with selected compounds for 24 h and the expression levels of
Her2 and Hsp70 were detected by western blotting analysis. We
found compounds 6b, 6d and 6e conferred more than 50% reduc-
tion of Her2 protein levels compared to vehicle control where as
compounds 6c and 7b showed less than 50% reduction of Her2 pro-
tein levels. Compounds 6b–e, 7b induced Hsp70 expression and
compound 6b was found the most potent one for both Her2 protein
degradation and Hsp70 protein induction (Fig. 3), whereas b-actin
levels remained unchanged which is not dependent on the Hsp90
protein folding machinery. Both, Her2 client protein degradation
and heat shock protein70 induction indicates that 2,4-diaminoqui-
nazoline acts as Hsp90 inhibitors, which is consistent with other
Hsp90 inhibitors.

1596
D. A. Thorat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1593–1597
Cl
O
O
R
i
ii
N
NH
O
R1
R1
R1
N
Cl
Cl
N
H
O
NH2
15 R1=H
16 R1=6F
17 R1=8F
12 R1=H
9
R=H, R1=H
iii
13 R1=6F
14 R1=8F
10 R=Me, R1=5F
11 R=H, R1=3F
O
O
HN
HN
N
iv
N
N
R1
R1
( )n
N
R2
N
H
18 R1=H
19 R1=6F
20 R1=8F
6a-q
7a-q
8a-q
n=0,1,2,3,4
R2=1-(2,3-dimethylphenyl)piperazinyl,
1-(2,3-dichlorophenyl)piperazinyl,
n-pyrrolidinyl, dimethylamine, diethylamine,
morpholine, butyl, pentyl, 4-methoxyphenyl
O
O
R
v
vi
H2N
R
( )n
N
( )n
Br
N
( )n
O
O
21
22
23
n= 2,3,4
R= 1-(2,3-dimethylphenyl)piperazine,
1-(2,3-dichlorophenyl)piperazine,
Morpholine
Scheme 1. Reagents and conditions: (i) Urea, phenol, 150 °C; (ii) POCl3, N,N-diisopropylethylamine or N,N-diethylaniline; (iii) furfurylamine, ethanol, reflux; (iv) appropriate
amine, ethanol, concn HCl, reflux; (v) Appropriate amine, N-bromoethylphthalimide or N-bromopropylphthalimide or N-bromobutylphthalimide, acetomitrile, potassium
carbonate, reflux; (vi) hydrazine hydrate, ethanol, reflux.
found that the extension of the methylene linker chain enhances
the inhibitory activity. Furthermore, the replacement of the termi-
nal dialkylamine moiety with phenyl piperazine led to new deriv-
atives with 10- to 30-fold enhanced anti-proliferative activity
than the hit compound 6a. Western blot analysis supports that 2,4-
iaminoquinazoline derivatives acts as Hsp90 inhibitors by degrad-
ing client protein, Her2 and increasing heat shock protein70 level.
The data resulted from compounds 6b–e, 7b seems promising and
have prompted further evaluation of this 2,4-diaminoquinazoline
scaffold and additional work in improving the results is in progress.
Acknowledgement
This work was supported by the Korea Institute of Science and
Technology.
References and notes
1. (a) Zhang, H.; Burrows, F. J. Mol. Med. 2004, 82, 488; (b) Isaacs, J. S.; Xu, W.;
Neckers, L. Cancer Cell 2003, 213–217; (c) Mosser, D. D.; Morimotto, R. I.
Oncogene 2004, 23, 2907.
2. Chiosis, G. Expert Opin. Ther. Targets 2006, 10. 37.
3. (a) Yamakazi, M.; Akaogi, K.; Miwa, T.; Imai, T.; Soeda, E.; Yokoyama, K. Nucleic
Acid Res. 1989, 17, 7108; (b) Rebbe, N.; Ware, J.; Bertina, R. M.; Modric, P.;
Staford, D. W. Gene 1987, 53, 235.
4. Prodromou, C.; Pearl, L. H. Curr. Cancer Drug Targets 2003, 3, 301.
Figure 3. (A) Effects of 2,4-diaminoquinazolines on Her2 degradation and Hsp70
induction. MCF-7 cells were treated with 0.5 lM of 17AAG and 5 lM of all other
2,4-diaminoquinazoline compounds, dissolved in 0.4% DMSO, for 24 h, DMSO (C) 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.
5. Maloney, A.; Workman, P. Expert Opin. Emerg. Drugs 2005, 1, 137.
6. Maloney, A.; Workman, P. Expert Opin. Biol. Ther. 2002, 2, 3.
7. Solit, D. B.; Scher, H. I.; Rosen, N. Semin. Oncol. 2003, 30, 709.
8. Beliakoff, J.; Begatell, R., et al Clin. Cancer Res. 2003, 9, 4961.
9. Connel, P.; Ballinger, C. A.; Jiang, J., et al Nat. Cell Biol. 2001, 3, 93.
10. Chnur, R. C.; Corman, M. L. J. Org. Chem. 1994, 59, 2581.
11. Whitesell, L.; Mimnaugh, E. G.; De Costa, B.; Myers, C. E.; Neckers, L. M. Proc.
Natl. Acad. Sci. 1994, 91, 8324.
In conclusion, we have reported a novel series of 2,4-diamino-
quinazolines as Hsp90 inhibitors. Several compounds shown excel-
lent growth inhibition activity against cancer cell lines. It was
12. Mimnaugh, E. G.; Chavany, C.; Neckers, L. J. Biol. Chem. 1996, 271, 22796.
13. Roe, S. M.; Prodromou, C.; O’Brien, R.; Ladbury, J. E.; Piper, P. W.; Pearl, L. H. J.
Med. Chem. 1999, 42, 260.

D. A. Thorat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1593–1597
1597
14. Gopalsamy, A.; Shi, M.; Golas, J.; Vogan, E.; Jacob, J.; Johnson, M.; Lee, F.;
Nilakantan, R.; Peterson, R.; Svenson, K.; Boshcelli, F. J. Med. Chem. 2008, 51,
373.
15. Park, H.; Kim, Y. J.; Hahn, J. Biorg. Med. Chem. Lett. 2007 2007(17), 6345.
16. Vilenchik, M.; Solit, D.; Basso, A.; Huezo, H.; Lucas, B.; He, H.; Rosen, N.;
Spampinato, C.; Modrich, P.; Chiosis, G. Chem. Biol. 2004, 11, 787.
17. Doddareddy, M. R.; Thorat, D. A.; Seo, S. H.; Hong, T.-J.; Cho, Y. S.; Hahn, J.-S.;
Pae, A. N. Bioorg. Med. Chem. 2011, doi:10.1016/j.bmc.2011.01.023.
Human colon cancer (HT-29), Human colon carcinoma cancer (HCT-116),
Human melanoma cancer (375P) and Human breast cancer (MCF-7) were
supplied from the Korean Cell Line Bank at Seoul National University. All cell
lines were grown in RPMI 1640 medium (Gibco BRL) supplemented with 10%
(v/v) heat inactivated Fetal Bovine Serum (FBS) and maintained at 37 °C in a
humidified atmosphere with 5% CO2. The cells (3 Â 103 cells/well) were seeded
into 96-well plates. Various concentrations of samples were added to each well
in duplicates and then incubated at 37 °C with 5% CO2 for two days such that
time cells are in the exponential phase of growth at the time of drug addition.
´
18. Godde, F.; Toulme, J.-J.; Moreau, S. Biochemistry 1998, 37, 13765.
19. Kosuke, K.; Katsunori, O., et al Bioorg. Med. Chem. 2006, 14(10), 3307.
20. Sirisoma; Nilantha; Kasibhatla, S., et al J. Med. Chem. 2008, 51(15), 4771.
21. 2N-(3-(4-(2,3-dichlorophenyl) piperazine-1-yl)propyl)-8-fluoro-4N-(furan-2-
ylmethyl)quinazoline-2,4-diamine (8d):
Then 15
The plate was incubated at 37 C for up to 4 h in
atmosphere. After incubation, 100 L of the Solubilization Solution/Stop Mix
(Promrga, CellTiter96) was added to each well. The plates were allowed to
stand overnight in a sealed container with a humidified atmosphere at room
temperature to completely solubilize the formazan crystals. The optical density
was measured by using a microplate reader (Versamax, Molecular Devices)
with a wavelength of 570 nm and the anticancer concentration was expressed
l
L of the Dye Solution (Promega, CellTiter96) was added to each well.
a
humidified, 5% CO2
l
To a solution of 2-chloro-8-fluoro-N-(furan-2-ylmethyl)quinazolin-4-amine 20
(0.03 g, 0.1 mmol) and 3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propan-1-
amine 23d (0.057 g, 0.2 mmol) in ethanol (2 mL), a catalytic amount of concn
HCl was added and the reaction mixture was refluxed for 22 h. The ethanol was
then removed under reduced pressure and the residue was extracted with
dichloromethane, dried over magnesium sulfate, concentrated and purified by
column chromatography on silica gel to obtain 2N-(3-(4-(2,3-
dichlorophenyl)piperazine-1-yl)propyl)-8-fluoro-4N-(furan-2-yl
as an IC50
.
24. Her2 degradation assay: MCF-7 cells were seeded into 6-well plates at a density
of 105 cells/well and grown up to 60% confluency. Grown cells were treated
with the compounds, 17-AAG which was dissolved in DMSO, or DMSO alone as
a control. Cells were incubated for an additional 24 h and then harvested and
lysed in TNES buffer [50 mM Tris–HCl, pH 7.4, 1% NP-40, 2 mM EDTA, 100 mM
NaCl, 1 mM PMSF, 0.1% protease inhibitor cocktail solution]. The clarified cell
lysates were collected after centrifugation and the protein concentration of
each lysates were determined by Protein assay solution (Bio-rad). Each lysates
methyl)quinazoline-2,4-diamine 8d in 18% (0.01 g) yield.
1H NMR (400 MHz, CD3OD): d 8.15 (q, J = 8.5 Hz, 1H), 7.45 (s, 1H), 7.24 (m, 2H),
7.09 (m, 3H), 6.38 (s, 2H), 4.87 (s, 2H), 3.63 (t, J = 7.4 Hz, 2H), 3.14 (s, 4H), 2.93
(s, 4H), 2.82 (s, 2H), 2.01 (quin, J = 7.4 Hz, 2H).
13C NMR (400 MHz, CD3OD): 150.98, 150.54, 142.16, 133.56, 127.73, 127.03,
124.83, 118.84, 110.17, 107.48, 55.25, 52.65, 49, 92, 39.19, 37.78.
containing 50 lg of total protein was loaded, subjected to SDS–PAGE and
IR (KBr):3244, 2948, 2823, 1650, 1579 cmÀ1
.
transferred to nitrocellulose membrane. After being probed with an antibody
against Her2-neu (Santa Cruz Biotechnology), the membrane was stripped and
also probed with antibodies against b-tubulin (Santa Cruz Biotechnology) and
HSP70 (Stressgen Bioreagents).
HRMS: C26H27Cl2FN6O, calcd: 528.1607, found: 529.1688[M+H]+.
22. Jo, M. N.; Seo, H. J.; Kim, Y.; Seo, S. H.; Rhim, H.; Cho, Y. S.; Cha, J. H.; Koh, H. Y.;
Choo, H.; Pae, A. N. Bioorg. Med. Chem. 2007, 15, 365.
23. Cytotoxic activities of the anticancer drugs against human cancer cell lines
were investigated using the MTT assay. Human prostate cancer (DU145),
25. Zou, J.; Guo, Y.; Guettouche, T.; Smith, D. F.; Voellmy, R. Cell 1998, 94, 471.