Design, synthesis, and mechanism of action of 2-(3-hydroxy-5-methoxyphenyl) -6-pyrrolidinylquinolin-4-one as a potent anticancer lead

Article abstract of DOI:10.1016/j.bmcl.2013.06.083

New 6- (or 6,7-) substituted 2-(hydroxyl substituted phenyl)quinolin-4-one derivatives were synthesized and screened for antiproliferative effects against cancer cell lines. Structure-activity relationship correlations were established and the most promis

Full text of DOI:10.1016/j.bmcl.2013.06.083

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5223–5227
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and mechanism of action
of 2-(3-hydroxy-5-methoxyphenyl)-6-pyrrolidinylquinolin-4-one
as a potent anticancer lead
Yung-Yi Cheng a, Chin-Yu Liu a, Meng-Tung Tsai a, Hui-Yi Lin a, Jai-Sing Yang b, Tian-Shung Wu c,
Sheng-Chu Kuo a,d, Li-Jiau Huang a, , Kuo-Hsiung Lee d,e,
⇑
⇑
a Graduate Institute of Pharmaceutical Chemistry, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan, ROC
b Department of Pharmacology, China Medical University, No. 91 Hsueh-Shih Road, Taichung 40402, Taiwan, ROC
c Department of Chemistry, National Cheng Kung University, No. 1 Dasyue Road, Tainan, Taiwan, ROC
d Chinese Medicine Research and Development Center, China Medical University Hospital, 2 Yuh-Der Road, Taichung 40447, Taiwan, ROC
e Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
New 6- (or 6,7-) substituted 2-(hydroxyl substituted phenyl)quinolin-4-one derivatives were synthesized
and screened for antiproliferative effects against cancer cell lines. Structure–activity relationship corre-
lations were established and the most promising compound 2-(3-hydroxy-5-methoxyphenyl)-6-pyrroli-
din-1-ylquinolin-4-one (6h) exhibited strong inhibitory activity against various human cancer cell lines,
particularly non-small cell lung cancer NCI-H522. Additional studies suggested a mechanism of action
resembling that of the antimitotic drug vincristine. The presence of a C-ring OH group in 6h will allow
this compound to be converted readily to a water soluble and physicochemically stable hydrophilic pro-
drug. Compound 6h is proposed as a new anticancer lead compound.
Received 3 May 2013
Revised 24 June 2013
Accepted 27 June 2013
Available online 4 July 2013
Keywords:
2-(Hydroxyphenyl)quinolin-4-one
derivatives
Antiproliferative activity
Anticancer lead development
Prodrug
Ó 2013 Elsevier Ltd. All rights reserved.
O
In prior studies, we synthesized 2-phenylquinolin-4-one deriv-
atives (2PQs)1–17 and identified various compounds, for example,
2PQ-1, 2PQ-2, 2PQ-3, and 2PQ-4(Chart 1), with potent anticancer
activity.3 These potential antitumor agents, however, suffer from
high toxicity and poor hydrophilicity, which has limited their fur-
ther development. To overcome such drawbacks of 2PQs, we intro-
duced a hydroxy group into the 2PQ skeleton to improve water
solubility. Moreover, this type of 2PQ could be further converted
to a water soluble prodrug. Accordingly, we synthesized a series
of 2PQs with a hydroxy group on the A-ring (2PQ-5ꢀ10, Chart 2),
and among them, 5-hydroxy-6-methoxy derivatives (2PQ-5, and
2PQ-6) exhibited good anticancer activity with IC50 values ranging
R6
R7
A
B
OCH3
N
C
H
: R , R7 = -OCH2O-
6
2PQ-1
2PQ-2
: R6 = pyrrolidinyl, R7 = H
2PQ-3: R6 = morpholino, R7 = H
: R6 = dimethylamino, R7 = H
2PQ-4
Chart 1. Structures of compounds 2PQ-1ꢀ4.
R5
O
from 0.03 to 0.11 lM against HL-60, HCT116, Hep3B and NCI-H460
R6
cell lines.5 Subsequently, 2PQ-6 was converted to a water soluble
disodium monophosphate prodrug (2PQ-6P), which exhibited sig-
nificant tumor growth suppression, without appreciably affecting
normal biological function.5 Based on this finding, we have now
R2´
R3´
R4´
N
H
: R5 = OH, R6 = OCH3, R2´= F, R3´= R4´= H
2PQ-5
2PQ-6: R5 = OH, R6 = OCH3, R3´= F, R2´= R4´= H
2PQ-7: R5 = OH, R6 = OCH3, R4´= F, R2´= R3´= H
: R5 = R6 = OH, R2´= F, R3´= R4´= H
: R5 = R6 = OH, R3´= F, R2´= R4´= H
2PQ-8
2PQ-9
⇑
Corresponding authors. Tel./fax: +886 4 2203 0760 (L.-J.H.), tel.: +1 919 962
0066; fax: +1 919 966 3893 (K.-H. L.).
2PQ-10: R5 = R6 = OH, R4´= F, R2´= R3´= H
E-mail addresses: ljhuang@mail.cmu.edu.tw (L.-J. Huang), khlee@unc.edu
(K.-H. Lee).
2PQ-6P: R5 = OPO3Na2, R6=OCH3, R3´= F, R2´= R4´= H
Chart 2. Structures of compounds 2PQ-5ꢀ10 and 2PQ-6P.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.083

5224
Y.-Y. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5223–5227
designed a new series of 2PQs with a hydroxy group on the C-ring
(6a–k). Again, this hydroxy moiety should provide a ‘synthetic
handle’ for potential useful prodrug design. The benefits of this de-
sign are to decrease toxicity, increase hydrophility, and facilitate
O
O
R6
R7
6
R6
R , R7 = -OCH2O-
3a
3b
R
6 = pyrrolidinyl
R1
R4
O
R1
R4
O
R7
NH
R1
NH2
3c R6 = morpholinyl
R2
R3
R2
R3
3a-d
R4
R3
6 = dimethylamino
Et3N/THF
OH
Cl
3d
R
SOCl2
O
THF/ DMF
6
: R , R7 = -OCH2O-, R1 = OBn, R2 = R3 = R4 = H
R2
4a, 5a
4b, 5b: R6, R7 = -OCH2O-, R2 = OBn, R1 = R3 = R4 = H
4a-k
1a-f
2a-f
: R1 = OBn, R2 = R3 = R4 = H
6
: R , R7 = -OCH2O-, R3 = OBn, R1 = R2 = R4 = H
4c, 5c
4d, 5d
1a, 2a
6
: R , R7 = -OCH2O-, R2 = R4 = OBn, R1 = R3 = H
1b, 2b: R2 = OBn, R1 = R3 = R4 = H
4e, 5e: R6, R7 = -OCH2O-, R2 = OBn, R4 = OMe, R1 = R3 = H
: R3 = OBn, R1 = R2 = R4 = H
: R2 = R4 = OBn, R1 = R3 = H
1c, 2c
1d, 2d
NaOH
1,4-Dioxane
: R , R7 = -OCH2O-, R2 = OBn, R4 = OEt, R1 = R3 = H
6
4f, 5f
: R6 = pyrrolidinyl; R2 = R4 = OBn, R7 = H
4g, 5g
1e, 2e: R2 = OBn, R4 = OMe, R1 = R3 = H
4h, 5h: R6 = pyrrolidinyl; R2 = OBn, R4 = OMe, R7 = H
4i, 5i
: R2 = OBn, R4 = OEt, R1 = R3 = H
1f, 2f
: R6 = pyrrolidinyl; R2 = OBn, R4 = OEt, R7 = H
4j, 5j: R6 = morpholinyl; R2 = OBn, R4 = OMe, R7 = H
: R6 = dimethylamino; R2 = OBn, R4 = OMe, R7 = H
4k, 5k
O
O
R6
R7
R6
R7
R1
R4
Pd/C
THF, CH2Cl2 or MeOH
R1
R4
R2
R3
R2
R3
N
H
N
H
6a-k
5a-k
6a: R6, R7 = -OCH2O-, R1 = OH, R2 = R3 = R4 = H
6
: R , R7 = -OCH2O-, R2 = OH, R1 = R3 = R4 = H
6b
6c: R6, R7 = -OCH2O-, R3 = OH, R1 = R2 = R4 = H
6
: R , R7 = -OCH2O-, R2 = R4 = OH, R1 = R3 = H
6d
6e
6
: R , R7 = -OCH2O-, R2 = OH, R4 = OMe, R1 = R3 = H
6f: R6, R7 = -OCH2O-, R2 = OH, R4 = OEt, R1 = R3 = H
: R6 = pyrrolidinyl; R2 = R4 = OH, R7 = H
6g
6h: R6 = pyrrolidinyl; R2 = OH, R4 = OMe, R7 = H
6i
: R6 = pyrrolidinyl; R2 = OH, R4 = OEt, R7 = H
6j: R6 = morpholinyl; R2 = OH, R4 = OMe, R7 = H
: R6 = dimethylamino; R2 = OH, R4 = OMe, R7 = H
6k
Scheme 1. Synthesis of compounds 6a–k.
Table 1
In vitro cytotoxic activity of 6- (or 6,7-) substituted 2-(hydroxyl substituted phenyl)quinolin-4-one derivatives (6a–k)
O
R6
R2'
R3'
R7
N
H
R4'
5'
Compounds 6Ra-k
a,b
Compds
IC50
(lM)
0
0
0
0
R6
R7
R2
R3
R4
R5
HL-60
Hep3B
NCI-H460
Detroit 551
2PQ-1
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
Pyrrolidinyl
Pyrrolidinyl
Pyrrolidinyl
Morpholinyl
Dimethylamino
H
OMe
H
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
OH
OMe
OEt
OH
OMe
OEt
OMe
OMe
0.08
7.62
0.25
3.26
8.71
0.50
0.50
0.34
0.10
0.02
3.90
0.22
0.08
8.04
1.05
22.33
>25
0.20
19.13
17.00
>100
>25
4.77
48.85
21.80
40.98
NDc
OH
H
H
H
H
H
H
H
H
H
H
>50
25
>50
>25
>50
NDc
H
H
H
H
H
1.58
0.50
0.07
6.03
6.15
13.89
2.12
0.59
>100
>50
>25
>50
13.23
>100
>50
a
b
c
Data are presented as IC50 (lM) for each cell line; the concentration of compound that caused a 50% proliferation-inhibitory effect after 48 h incubation.
Cell lines include human promyelocytic leukemia (HL-60), human hepatoma (Hep3B), human lung cancer (H460), and embryonic skin fibroblast (Detroit551) cell lines.
ND = not determined.

Y.-Y. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5223–5227
5225
Table 2
moderate cytotoxicity compared with 2PQ-1. Compound 6d with
an additional hydroxy group at the C-ring 5-position exhibited
only weak activity. Based on the structures of active compounds
2PQ-1ꢀ4, we also synthesized compounds with a 3-hydroxy-5-
alkoxyphenyl C-ring (6e–f) and changed the A-ring substitution
from 6,7-methylenedioxy to 6-pyrrolidinyl (6g–i), 6-morpholinyl
(6j), and 6-dimethylamino (6k). These seven compounds (6e–k)
showed the highest cytotoxicity against the HL-60 cancer cell line.
With hydroxy and methoxy groups on the C-ring, the rank order of
HL-60 inhibitory activity was 6-pyrrolidinyl (6h) > 6-dimethyl-
Inhibition of in vitro tumor cell growth by compound 6ha
b
d
Cell linee
logGI50
logTGIc
logLC50
SR
<ꢁ8.00
<ꢁ8.00
ꢁ7.27
ꢁ6.96
ꢁ7.52
ꢁ8.00
ꢁ7.61
ꢁ7.63
ꢁ7.21
ꢁ7.63
ꢁ7.39
ꢁ7.51
ꢁ6.58
>ꢁ4.00
<ꢁ8.00
ꢁ6.66
ꢁ4.95
ꢁ4.80
ꢁ7.53
ꢁ7.18
ꢁ7.05
ꢁ6.29
ꢁ4.60
ꢁ4.62
ꢁ6.59
ꢁ5.70
>ꢁ4.00
ꢁ5.41
NCI-H522
Colo205
SF-295
f
—
>ꢁ4.00
M14
>ꢁ4.00
f
MDA-MB-435
SK-MEL-5
OVCAR-3
NCI/ADR-RES
SK-OV-3
RXF 393
DU-145
—
ꢁ6.31
ꢁ5.19
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
amino (6k) > 6,7-methylenedioxy (6e) > 6-morpholinyl (6j).
A
change from 50-methoxy (6h) to 50-ethoxy (6i) significantly
increased anti-proliferative activity, but replacement with 50-hy-
droxy (6g) decreased activity significantly, as also seen with 6e
versus 6d. While 6i was more potent than 6h, unfortunately, 6i
also exhibited unsatisfactory toxicity against the Detroit 551 cell
line. Therefore, among compounds 6a–k, we selected6h as the lead
compound for further investigation, and submitted it to NCI for
anticancer evaluation.
MDA-MB-468
a
Data obtained from NCI0s in vitro disease-oriented human tumor cells screen.
Log concentrations that reduced cell growth to 50% of level at start of
b
experiment.
c
Log concentrations that caused total growth inhibition.
d
Log concentrations that
population.
a given compound required to kill 50% of a test
The resulting activity profile fingerprint (Table 2 and Supple-
mentary data) of 6h against the NCI-60 human cancer cell line
panel indicated significant inhibitory activity against a variety of
cancer cell lines. Compound 6h was particularly active against SR
leukemia (logGI50 <ꢁ8.00), MDA-MB-435 melanoma (logGI50
<ꢁ8.00), and NCI-H522 non-small cell lung cancer (logTGI
<ꢁ8.00) cell lines. We also evaluated the potency of 6h in an
anti-proliferation assay with NCI-H522 cells. MTT assay results
showed that 6h effectively inhibited proliferation of NCI-H522
cells with an IC50 value of 42.3 1.2 nM at 48 h-incubation. The
activity profile fingerprint of 6h was further analyzed by COMPARE
correlation at the GI50 level. As shown in Table 3, the fingerprint of
6h correlated most closely with that of antimitotic Vinca alkaloids,
such as vincristine and vinblastine. To support this supposition, we
examined whether 6h has an effect on tubulin assembly. In an
in vitro tubulin polymerization assay, 6h inhibited tubulin poly-
merization in a concentration-dependent manner, which was sim-
ilar to the effects caused by vincristine and vinblastine (Fig. 1A).24
Results from an in vivo tubulin assembly assay showed that
e
SR, leukemia; NCI-H522, non-small cell lung cancer; Colo205, colon cancer; SF-
295, CNS cancer; M14, MDA-MB-435, SK-MEL-5, melanoma, M14; OVCAR-3, NCI/
ADR-RES, SK-OV-3, ovarian cancer; RXF 393, renal cancer; DU-145, prostate cancer;
MDA-MB-468, breast cancer cell lines.
f
‘—’ no data.
possible hydrophilic prodrug derivatization. This report describes
the chemical synthesis, antitumor activity screening in vitro, and
mechanism of action of the new series of 6- (or 6,7-) substituted
2-(hydroxyl substituted phenyl)quinolin-4-ones (6a–k).
The synthesis of target compounds 6a–k is illustrated in
Scheme 1. As shown, variously substituted benzoic acids (1a–f)
were chlorinated with thionyl chloride to afford compounds 2a–
f.18 Without purification, 2a–f were reacted with o-aminoacetoph-
enones (3a–d)2,3 to give the desired amides (4a–k),19 which were
then cyclized in the presence of basic dioxane solution to yield
5a–k.20 Catalytic hydrogenolysis of 5a–k with palladium on active
charcoal gave the final compounds, 6- (or 6,7-) substituted
2-(hydroxyphenyl)quinolin-4-ones (6a–k).21 The spectroscopic
data (1H, 13C NMr and HR-ESIMS) were consistent with the pro-
posed structures.
The newly synthesized compounds were screened for antipro-
liferative activity against HL-60 leukemia, Hep3B hepatoma, NCI-
H460 non-small cell lung cancer, and Detroit 551 human skin
fibroblast cells.22,23 The results are given in Table 1. Among the
three mono-hydroxy derivatives (6a–c), 6b with meta-hydroxy
substitution was the most potent (Table1), but showed only
6h inhibited
a-tubulin and b-tubulin accumulation concentra-
tion-dependently in the cytoskeletal fraction, the same effect as
vincristine and vinblastine, whereas paclitaxel caused tubulin
polymerization (Fig. 1B).25 Thus, the preliminary results indicated
that the mechanism of action of 6h resembles that of vincristine
and vinblastine (Fig. 2).
In summary, 6- (or 6,7-) substituted 2-(hydroxyl substituted
phenyl)quinolin-4-one derivatives were designed, synthesized,
and evaluated for in vitro antitumor activity. Preliminary SAR
correlations of the new analogs were established. The most prom-
ising compound 6h demonstrated low toxicity against a normal
cell line and significant inhibition against several cancer cell lines.
In addition, the results of a COMPARE analysis suggested that 6h
might function as an antimitotic agent, such as Vinca alkaloids.
Therefore, we believe that 6h is a promising lead compound that
deserves further optimization and derivatization as a hydrophilic
prodrug.
Table 3
Results of COMPARE correlations at GI50 level for compound 6h
Rank
Compound (NCI number)
ra
1
2
3
4
5
6
7
8
9
10
Vincristine sulfate (NSC 67574)
Maytansine (NSC 153858)
Vinblastine sulfate (NSC 49842)
Rhizoxin (NSC 332598)
DON (NSC 7365)
Didemnin B (NSC 325319)
AT-125 (NSC 163501)
S-Trityl-L-cysteine (NSC 83265)
0.538
0.497
0.489
0.476
0.452
0.450
0.442
0.421
0.402
0.401
Acknowledgments
The investigation was supported by research grants from the
National Science Council of the Republic of China awarded to
S.C.K. (NSC101-2320-B-039-008). Thanks are also due to support
by Grant CA177584from the National Cancer Institute, NIH
awarded to K.H.L.
Tiazofurin (NSC 286193)
Trimetrexate (NSC 352122)
a
r = correlation coefficient.

5226
Y.-Y. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5223–5227
Figure 1. Action mechanism of 6h for anti-proliferation of NCI-H522 non-small cell lung cancer cells. (A) Compound 6h, vincristine, vinblastine, and paclitaxel were
incubated with pure tubulin proteins in GPEM buffer. Alternation of tubulin assembly was recorded at absorbance 340 nm. (B) Cells were treated with vehicle (DMSO, as
control), 6h (10, 30, and 100 nM), 100 nM vincristine, and 100 nM paclitaxel for 24 h. Cytosolic (S, soluble) and cytoskeletal (P, polymerized tubulin) fractions were separated
and followed by Western blot analysis for detection of a-tubulin, b-tubulin, and b-actin protein expression.
Figure 2. Immunostaining of non-small cell lung cancer NCI-H522 with
vinblastine at 100 nM; (d–f) 24 h treatment with 6h at 50, 100, 200 nM.
a-tubulin-FITC. (a) Control; (b) 24 h treatment with paclitaxel at 100 nM; (c) 24 h treatment with
2. Li, L.; Wang, H.-K.; Kuo, S.-C.; Wu, T.-S.; Lednicer, D.; Lin, C. M.; Hamel, E.; Lee,
K.-H. J. Med. Chem. 1994, 37, 1126.
Supplementary data
3. Li, L.; Wang, H.-K.; Kuo, S.-C.; Wu, T.-S.; Mauger, A.; Lin, C. M.; Hamel, E.; Lee,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.06.083.
K.-H. J. Med. Chem. 1994, 37, 3400.
4. Zhang, S.-X.; Feng, J.; Kuo, S.-C.; Brossi, A.; Hamel, E.; Tropsha, A.; Lee, K.-H. J.
Med. Chem. 2000, 43, 167.
5. Chou, L.-C.; Tsai, M.-T.; Hsu, M.-H.; Lin, H.-Y.; Huang, L.-J.; Kuo, S.-C.; Wang, S.-
H.; Way, T.-D.; Huang, C.-H.; Qian, K.; Dong, Y.; Lee, K.-H. J. Med. Chem. 2010, 53,
8047.
References and notes
6. Chen, H. Y.; Lu, H. F.; Yang, J. S.; Kuo, S. C.; Lo, C.; Yang, M. D.; Chiu, T. H.; Chueh,
F. S.; Ho, H. C.; Ko, Y. C. Anticancer Res. 2010, 30, 4187.
1. Kuo, S.-C.; Lee, H.-Z.; Juang, J.-P.; Lin, Y.-T.; Wu, T.-S.; Chang, J.-J.; Lednicer, D.;
Paull, K. D.; Lin, C. M.; Hamel, E.; Lee, K.-H. J. Med. Chem. 1993, 36, 1146.

Y.-Y. Cheng et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5223–5227
7. Chen, Y.-C.; Lu, P.-H.; Pan, S.-L.; Teng, C.-M.; Kuo, S.-C.; Lin, T.-P.; Ho, Y.-F.;
Huang, Y.-C.; Guh, J.-H. Biochem. Pharmacol. 2007, 74, 10.
8. Wang, J.-P.; Raung, S.-L.; Huang, L.-J.; Kuo, S.-C. Biochem. Pharmacol. 1998, 56,
1505.
9. Huang, L.-J.; Hsieh, M.-C.; Teng, C.-M.; Lee, K.-H.; Kuo, S.-C. Bioorg. Med. Chem.
1998, 6, 1657.
10. Lai, Y.-Y.; Huang, L.-J.; Lee, K.-H.; Xiao, Z.; Bastow, K. F.; Yamori, T.; Kuo, S.-C.
Bioorg. Med. Chem. 2005, 13, 265.
5227
21. Preparation of 2-(hydroxyl substituted phenyl)quinolin-4-ones (6a–k):
A
suspension of 5a–k and palladium (10 wt.% on activated carbon) in MeOH or
CH2Cl2 was stirred at room temperature under hydrogen gas atmosphere for
24 h. The precipitate was collected and dissolved in 10% NaOH solution and
then filtered. The filtrate was acidified with dil aq HCl and the resulting
precipitate was collected and washed with acetone and water to yield 2-
(hydroxyl substituted phenyl)quinolin-4-ones (6a–k).
22. Cell culture: Human leukemia HL-60 and non-small-cell-lung cancer H460 cells
were maintained in RPMI-1640 medium supplemented with 10% fetal bovine
11. Xia, Y.; Yang, Z.-Y.; Xia, P.; Bastow, K. F.; Tachibana, Y.; Kuo, S.-C.; Hamel, E.;
Hackl, T.; Lee, K.-H. J. Med. Chem. 1998, 41, 1155.
serum (GIBCO/BRL), penicillin (100 U/mL)/streptomycin (100
lg/mL) (GIBCO/
12. Hsu, S.-C.; Yang, J.-S.; Kuo, C.-L.; Lo, C.; Lin, J.-P.; Hsia, T.-C.; Lin, J.-J.; Lai, K.-C.;
Kuo, H.-M.; Huang, L.-J.; Kuo, S.-C.; Wood, W. G.; Chung, J.-G. J. Orthop. Res.
2009, 27, 1637.
BRL) and 1% -glutamine (GIBCO/BRL) at 37 °C in a humidified atmosphere
L
containing 5% CO2. Human hepatocellular carcinoma cell line Hep3B was
obtained from America Type Culture Collection (Manassas, VA, USA). Hep3B
cells were cultured in DMEM/F12 medium supplemented with 10% FBS and
13. Lai, T. Y.; Yang, J. S.; Wu, P. P.; Huang, W. W.; Kuo, S. C.; Ma, C. Y.; Wood, W. G.;
Chung, J. G. Leuk. Lymphoma 2010, 51, 2098.
penicillin (100 U/mL)/streptomycin (100
l
g/mL) and maintained in
a
14. Wang, S. W.; Pan, S. L.; Huang, Y. C.; Guh, J. H.; Chiang, P. C.; Huang, D. Y.; Kuo,
S. C.; Lee, K. H.; Teng, C. M. Mol. Cancer Ther. 2008, 7, 350.
15. Wen, C. C.; Juang, S. H.; Chou, L. C.; Chung, J. G.; Kuo, S. C.; Yang, N. S. Pharm.
Biol. 2012, 50, 570.
humidified incubator containing 5% CO2. Normal skin Detroit 551 cells were
maintained in DMEM medium supplemented with 10% fetal bovine serum
(GIBCO/BRL), penicillin (100 U/mL)/streptomycin (100
lg/mL) (GIBCO/BRL)
and 1% -glutamine (GIBCO/BRL) at 37 °C in humidified atmosphere
L
a
16. Chang, Y.-H.; Hsu, M.-H.; Wang, S.-H.; Huang, L.-J.; Qian, K.; Morris-Natschke,
S. L.; Hamel, E.; Kuo, S.-C.; Lee, K.-H. J. Med. Chem. 2009, 52, 4883.
17. Chou, L.-C.; Chen, C.-T.; Lee, J.-C.; Huang, S.-M.; Huang, L.-J.; Kuo, S.-C.;
Qian, K.; Morris-Natschke, S. L.; Lee, K.-H.; Way, T.-D.; Huang, C.-H.; Teng,
C.-M.; Yamori, T.; Wu, T.-S.; Sun, C.-M.; Chien, D.-S. J. Med. Chem. 2010, 53,
1616.
18. Preparation of arylcarbonyl chlorides (2a–f): Arylcarboxylic acids (1a–f) were
suspended in dry THF at room temperature. Thionyl chloride and DMF were
added dropwise. The reaction mixtures were stirred for 12 h at room
temperature and then evaporated to dryness. The residues were used
directly in the next step without purification.
19. Preparation of benzamides (4a–k): Into solutions of 1a–f in dry THF were added
triethylamine and o-aminoacetophenones (3a–d).2,3 The mixtures were stirred
at room temperature for 12 h and evaporated. The residue was purified by
column chromatography eluting with a mixture of n-hexane and EtOAc (1/1) to
afford pure carboxamides (4a–k).
containing 5% CO2. Logarithmically growing cancer cells were used for all
experiments.
23. Cell viability assay: The cell viability was detected by 3(4,5-dimethythiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were cultured in 96-
well plates at 37 °C and incubated with complete medium containing the
vehicle (DMSO) or compounds for indicated times and concentrations. After
treatment, cells were incubated with MTT solution (1 mg/mL in 1ꢂ PBS) at
37 °C for 2 h. The absorbance of the samples was read at 570 nm and corrected
for inference at 630 nm.
24. In vitro microtubule polymerization: In vitro assay of microtubule
polymerization was detected with the tubulin polymerization assay (Cat.#
BK011P, Cytoskeleton Inc, Denver, CO, USA) Briefly, pure porcine brain tubulin
proteins (300 lg) in 100 lL of GPEM buffer (80 mM PIPES, pH 6.9, 2 mM MgCl2,
0.5 mM EGTA, 1 mM GTP, 5% glycerol) were incubated with test compounds at
37 °C. The alternation of tubulin polymerization was measured at absorbance
340 nm every 30 s for 30 min (Synergy 2; BioTek, Winooski, VT, USA).
20. Preparation of 2-(benzyloxy substituted phenyl)quinolin-4-ones (5a–k):
A
25. In vivo tubulin assembly assay: Cells were lysed in hypotonic buffer (1 mM
MgCl2, 2 mM EGTA, 0.5% NP-40, 2 mM PMSF, 200 Units/mL aprotinin, 5 mM
amino caproic acid, 1 mM benzamidine, and 20 mM Tris–HCl, pH 6.8) for 5 min
mixture of carboxamide (4a–k) and NaOH was suspended in 1,4-dioxane.
The reaction mixture was refluxed for 4 h. After cooling to room temperature,
the mixture was evaporated and then the residue was added to 10% NH4Cl
solution. The precipitate was collected, washed with water and acetone, and
then purified by silica gel column chromatography to obtain 2-(benzyloxy
substituted phenyl)quinolin-4-ones (5a–k).
at 37 °C followed by centrifugation, 15,000
g for 10 min at 25 °C. The
supernatant contained cytosolic tubulin. The pellets of polymerized tubulin
were resuspended in hypotonic buffer and sonication. Both fractions were
subjected to Western blot analysis for detection of tubulin contents.