2-Aroylquinoline-5,8-diones as potent anticancer agents displaying tubulin and heat shock protein 90 (HSP90) inhibition

Article abstract of DOI:10.1039/c5ob02100f

This study reports the synthesis of a series of 2-aroylquinoline-5,8-diones (11-23) on the basis of scaffold hopping. The presence of a methoxy group at C6 assists the highly regioselective incorporation with various amines, and simplifies the structural

Full text of DOI:10.1039/c5ob02100f

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Biomolecular Chemistry
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2
-Aroylquinoline-5,8-diones as potent anticancer
agents displaying tubulin and heat shock protein
0 (HSP90) inhibition†
Cite this: DOI: 10.1039/c5ob02100f
9
a
a
a
a
Kunal Nepali,‡ Sunil Kumar,‡ Hsiang-Ling Huang, Fei-Chiao Kuo,
a
b
b
a
Cheng-Hsin Lee, Ching-Chuan Kuo, Teng-Kuang Yeh, Yu-Hsuan Li,
Jang-Yang Chang, Jing-Ping Liou and Hsueh-Yun Lee*
c
a
a
This study reports the synthesis of a series of 2-aroylquinoline-5,8-diones (11–23) on the basis of scaffold
hopping. The presence of a methoxy group at C6 assists the highly regioselective incorporation with
various amines, and simplifies the structural identification process. Among the synthetic compounds,
6
-dimethylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (12) and 7-pyrrolidin-1-yl-2-(3,4,5-
trimethoxybenzoyl)-quinoline-5,8-dione (23) exhibit remarkable anti-proliferative activity against the
cancer cell lines tested with mean IC50 values of 0.14 and 0.27 μM, respectively. Compound 23 showed
moderate inhibitory activity against tubulin polymerization with an IC50 value of 5.9 μM. In a western blot
analysis, 23 caused induction of HSP70 and degradation of Akt, revealing that it possesses HSP90 inhibi-
tory activity.
Received 12th October 2015,
Accepted 9th November 2015
DOI: 10.1039/c5ob02100f
www.rsc.org/obc
6
7
quinoline-5,8-diones 5 and 6 have also shown antiprolifera-
tive activity (Fig. 1).
Introduction
Natural sources have been widely exploited in medicinal chem-
Geldanamycin (GA, 7) is a benzoquinone ansamycin anti-
8
istry, and natural products provide diverse templates for biotic isolated from Streptomyces hygroscopicus. It was identi-
further structural modification. Streptonigrin (1) was isolated
1
from Streptomyces flocculus, and recognized in 1973 as a
2
potent antitumor and antiviral agent. Lavendamycin (2),
which has the same quinoline-5,8-dione core as streptonigrin,
and is derived from Streptomyces lavendulae has been found to
3
exhibit antimicrobial and antitumor properties. The quino-
line-5,8-dione skeleton in streptonigrin (1) and lavendamycin
(
2) has triggered much research centered on quinoline-5,8-
dione as component of potentially bioactive agents.
-Chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione
DA 3003-2, 3) for example, was identified as a potential inhibi-
tor of cdc25 phosphatases, which are crucial to the cell cycle
a
7
(
4
progression.
(
7-Chloro-6-piperidin-1-yl-quinoline-5,8-dione
5
PT-262, 4) was found as a new ROCK inhibitor and other
a
School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 11031,
Taiwan. E-mail: hyl@tmu.edu.tw; Tel: +886-2-27361661 ext 6134
b
Institute of Biotechnology and Pharmaceutical Research, National Health Research
Institutes, Zhunan Town, Miaoli County, Taiwan
c
National Institute of Cancer Research, National Health Research Institutes, Tainan,
Taiwan
1
13
†
Electronic supplementary information (ESI) available: H-NMR and C-NMR
spectra of compounds 11–23. See DOI: 10.1039/c5ob02100f
These authors contributed equally to this work.
‡
Fig. 1 Natural and synthetic quinonin-5,8-diones.
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fied as an inhibitor of the heat shock protein (HSP90) and
entered clinical trials as an anticancer agent in 1994, but the
Results and discussion
Chemistry
9
clinical evaluations were hampered by its serious hepato-
1
0
toxicity. Replacement of the methoxy group in geldanamycin The synthetic routes to 6-substituted 2-aroylquinoline-5,8-
with an allylamino group afforded 17-N-allylamino-17- diones (11–20) are shown in Scheme 1. The commercially avail-
demethoxygeldanamycin (17-AAG, 8) which also displayed able 1,2,4-trimethoxybenzene (24) underwent nitration to
1
1
12
2
HSP90 inhibitory activity and was less toxic. In 2003, Skibo produce 25 whose nitro group was reduced by Pd/C and H .
et al. modified the 2-aminobenzoquinone moiety of geldana- The resulting amine was reacted with crotonaldehyde in the
mycin, using the concept of ring expansion to afford a series presence of boiling concentrated HCl and then subjected to
1
3
of quinoline-5,8-diones and continued work demonstrated benzylic oxidation by selenium dioxide to give 26. The reaction
1
4
that compound 9 exhibited an HSP90 response. This result of 26 with 3,4,5-trimethoxyphenylmagnesium bromide fol-
attracted our attention due to the presence in 9 of a 3,4,5- lowed by the oxidation with pyridinium dichromate (PDC)
trimethoxybenzene moiety which had also been used in our yielded compound 27. This was subsequently oxidized by ceric
1
5
previous work on 2-aroylquinolines. The continuing studies (IV) ammonium nitrate (CAN) to furnish 6-methoxyquinoline-
on compound 10 demonstrated that the carbonyl linkage can 5,8-dione (11) which, when subjected to substitution reactions
1
7
be replaced with various groups such as sulfone or sulfide with various amines, yielded compounds 12–20. The pres-
without affecting the compound’s cellular activity and pharma- ence of a methoxy group at C6 allows amination specifically at
1
6
cological function. In an attempt to widen the biological the C6 position without advanced instrumental analysis to
window of this series of compounds, the present study confirm the structures of the products.
explores scaffold hopping in this case, changing the central
Scheme 2 describes the synthesis of 7-substituted quino-
quinoline to a quinoline-5,8-dione. A series of 2-aroylquino- line-5,8-diones (21–23). The starting material, 2,5-dimethoxy-
line-5,8-diones (11–23) were synthesized and the effect of sub- aniline (28) was reacted with crotonaldehyde and
stitution on biological activity is examined in this study. subsequently, selenium dioxide to afford the corresponding
Additionally, we provide an efficient synthetic methodology to aldehyde 29. This aldehyde was reacted with 3,4,5-trimethoxy-
overcome the regioselectivity of amination of target com- phenylmagnesium bromide and then oxidized with pyridi-
pounds, without a requirement for advanced instrumental nium dichromate (PDC) to furnish 30. Treatment of 30 with
analytical techniques such as COSY and HMBC (Fig. 2).
ceric(IV) ammonium nitrate (CAN) provided the quinoline-5,8-
dione (31) which upon amination, gave compounds 21–23.
Unlike the methodology illustrated in Scheme 1, the absence
of C6-OMe resulted in the generation of C6- and C7-substi-
tuted regio-isomers. The C7-substituted regio-isomers (21–23)
were readily purified by column chromatography after the
C6-substituted isomers (12, 18, and 19) were identified.
Scheme 1 Reagents and conditions: (a) HNO
i. Pd/C, H , MeOH, rt; ii. crotonaldehyde, 6 N HCl, toluene, 100–110 °C;
iii. SeO , p-xylene, 100–110 °C, 23.7%; (c) i. 3,4,5-trimethoxyphenylmag-
nesium bromide, THF, rt; ii. PDC, MS, DCM, rt, 75.9%; (d) CAN, CH CN,
O, 0 °C, 21.2%; (e) amines, DMF in air, 70–80 °C, 19–57%.
3
, AcOH, rt, 86.8%; (b)
2
2
Fig. 2 The diagram of the design of 2-aroylquinoline-5,8-diones
3
(
11–23).
H
2
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enhancement of inhibitory potency toward HCT116 cells with
an IC50 value of 0.03 μM, but a 6-fold decrease of inhibitory
activity of KB cells as compared with 17-AAG. Compound 15,
with a N-(N,N-dimethylaminoethyl)amino group at C6 exhibits
moderate inhibitory activity towards the cancer cells tested
with a mean IC50 value of 0.67 μM. The cyclic amino groups in
compounds 18–20 fail to produce significant improvement of
cytotoxicity; compound 19, with a 6-morpholino group displays
moderate inhibitory activity toward HONE-1 and HCT116 cells,
with IC50 values of 0.30 and 0.45 μM, respectively. The shift of
Scheme 2 Reagents and conditions: (a) i. crotonaldehyde, 6 N HCl, –NMe from C6 (12) to C7 (22) leads to a minor decrease in
2
toluene, 100–110 °C; ii. SeO
trimethoxyphenylmagnesium bromide, THF, rt; ii. PDC, MS, DCM, rt,
0.1%; (c) CAN, CH CN, H O, 0 °C, 46.5%; (d) amines, DMF in air,
0–80 °C, 23–87%.
2
, p-xylene, 100–110 °C, 21.2%; (b) i. 3,4,5-
activity and the 7-morpholino group in 21 causes a 1- to 6-fold
decrease of antiproliferative activity as compared with com-
pound 19. In contrast, the pyrrolidin-1-yl group in compound
8
7
3
2
23 causes a 6- to 10-fold increase in the inhibition of the
growth of HCT116 and KB cells with IC50 values of 0.07 and
0
.18 μM, respectively.
Inhibition of tubulin polymerization and colchicine binding
Biological evaluation
In vitro cell growth inhibitory activity. In an attempt to activity. To investigate the relationship of the synthetic pro-
evaluate the effect of various 2-aroylquinoline-5,8-diones on ducts in the current study with the microtubule system, com-
cancer cell inhibition, all the synthetic compounds (11–23) pounds 12, 15, 22, 23, and reference compounds colchicine
and the reference compound 17-AAG (8), were evaluated for and CA4 were evaluated for tubulin polymerization inhibitory
the antiproliferative activities of three human cancer cell lines: activity, producing the results shown in Table 2. Among the
nasopharyngeal carcinoma HONE-1 cells, colon carcinoma compounds tested, 23 shows the greatest activity in inhibiting
HCT116 cells, and cervical carcinoma KB cells. The results are tubulin polymerization (IC50 = 5.9 μM), but it is less potent
shown in Table 1. Compound 11 shows inhibitory activity com- than colchicine and CA4. As shown in Fig. 3, compound 23
parable to that of 17-AAG, with IC50 values of 0.30, 0.19, and inhibits polymerization of pure MAP-rich tubulins in a concen-
0
.36 μM against HONE-1, HCT116, and KB cell lines, respecti- tration-dependent manner and disrupts tubulin assembly with
vely. The replacement of –OMe by –NMe
2
(12) contributes to an IC50 value of 5.9 µM.
an improvement of the inhibitory activity in the HCT116 and
Effect on HSP90 client proteins. Inhibition of HSP90 activity
KB cells. Compound 12 exhibits 2- to 4-fold more cytotoxicity results in the induction of HSP70 and degradation of Akt.
1
9,20
than 17-AAG in inhibiting HONE-1, HCT116, and KB cells’ Six compounds (compounds 11, 12, 13, 15, 19, and 23) and
growth with IC50 values of 0.3, 0.06, and 0.05 μM, respectively. reference compound 8 were tested for their interaction with
The linear amines at C6 in 14 and 16 cause a dramatic loss of these two client proteins of HSP90 (Fig. 4). Fig. 4A shows that
cellular potency. The 6-NHEt of compound 13 leads to a 6-fold compound 23 causes dose-independent induction of HSP70
but the other compounds have no significant influence.
Cell treatment with compound 23 also leads to the degradation
of Akt in a concentration-dependent manner (Fig. 4B).
Thus compound 23 appears to be able to inhibit the function
Table 1 Antiproliferative activity of compounds 11–23
of HSP90.
a
IC50 ± SD (µM)
Compd
HONE-1
HCT116
KB
1
1
1
1
1
1
1
1
1
2
2
2
2
1
1
2
3
4
5
6
7
8
9
0
1
2
0.30 ± 0.10
0.30 ± 0.14
0.39 ± 0.18
1.04 ± 0.01
0.53 ± 0.09
7.27 ± 2.6
>50
12.2 ± 3.2
0.30 ± 0.00
37.3 ± 2.0
1.85 ± 0.01
1.42 ± 0.13
0.55 ± 0.07
0.76 ± 0.21
0.19 ± 0.02
0.06 ± 0.03
0.03 ± 0.02
2.13 ± 0.66
0.86 ± 0.08
1.91 ± 1.28
>50
12.4 ± 2.12
0.45 ± 0.23
33.5 ± 4.12
0.81 ± 0.14
0.05 ± 0.01
0.07 ± 0.03
0.20 ± 0.04
0.36 ± 0.04
0.05 ± 0.03
1.19 ± 1.6
1.31 ± 0.22
Table 2 Inhibition of tubulin polymerization by compounds 12, 15, 22,
0.62 ± 0.07 23, and CA4
1.89 ± 0.14
43.6 ± 8.93 Compd
19.2 ± 0.52
1.74 ± 0.01 12
35.7 ± 6.93 15
1.82 ± 0.05 22
0.18 ± 0.01 23
a
Tubulin IC50 ± SD (µM)
8.9
>10
6.2
5.9
4.2
b
3
0.18 ± 0.01 Colchicine
0.20 ± 0.16 CA4
c
7-AAG (8)
1.9
a
a
b
c
SD: standard deviation, all experiments were independently
Inhibition of tubulin polymerization. Data from ref. 18. Data from
ref. 16.
performed at least three times.
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17-AAG. Compounds 12 and 23 inhibit the growth of cancer
cells tested with mean IC50 values of 0.14 and 0.27 μM, respecti-
vely. Compound 23 inhibits tubulin polymerization with an
IC50 value of 5.9 μM. Compound 23 also induces the
expression of HSP70 and triggers the degradation of Akt in a
dose-dependent manner. These results indicate that these
2-aroylquinoline-5,8-diones are potential anticancer agents
with novel promising pharmacological mechanisms.
Experimental section
Chemistry
Fig. 3 Effect of compound 23 on in vitro tubulin polymerization. MAP-
rich tubulins were incubated at 37 °C in the absence [dimethyl sulfoxide
(
1
13
DMSO) control] or presence of drugs (colchicine or serial concen- Nuclear magnetic resonance ( H and C NMR) spectra were
trations of compound 23). The absorbance at 350 nm was measured
every 30 s for 30 min and is presented as the increased polymerized
microtubule.
obtained with a Bruker Fourier 300 and DRX-500 spectro-
meters, with chemical shifts in parts per million (ppm, δ)
downfield from TMS as an internal standard. High-resolution
mass spectra (HRMS) were recorded with a FINNIGAN MAT
95S mass spectrometer. Purity of the final compounds was
determined using a Hitachi 2000 series HPLC system using a
C-18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm, 4.6 mm
×
150 mm) with the solvent system (elution conditions: mobile
phase A consisting of MeCN; mobile phase B consisting of
O containing 0.1% formic acid + 10 mmol NH OAc) and
H
2
4
was found to be ≥95%. Flash column chromatography was
performed using silica gel (Merck Kieselgel 60, no. 9385,
2
30–400 mesh ASTM). All reactions were carried out under an
atmosphere of dry nitrogen.
-Methoxy-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione
11). A solution of 27 (0.2 g, 0.48 mmol) in ACN (15 mL) was
6
(
placed in a round-bottomed flask which was equipped with an
addition funnel. A solution of CAN (1.0 g, 1.8 mmol) in H O
2
(
10 mL) was added to the funnel. The solution was added
slowly into the round-bottomed flask at room temperature,
and then the resulting solution was stirred for 1 h. The reac-
tion was quenched with H
2
O (30 mL) and extracted with
CH Cl (3 × 30 mL). The organic layer was collected and puri-
2
2
fied by column chromatography (EtOAc/n-hexane = 2 : 1) to
afford 11 (0.1 g, 48.4%) as a red solid. mp = 174–175 °C;
H NMR (500 MHz, CDCl ) δ 3.96 (s, 6H), 3.97 (s, 3H), 3.98 (s,
3
Fig. 4 Investigation of the effect of tested compounds on the
expression level of HSP70 and AKT proteins in human nasopharyngeal
1
carcinoma HONE-1 cells. The HSP90 inhibitory activity of test com- 3H), 6.43 (s, 1H), 7.86 (s, 2H), 8.41 (d, J = 8.5 Hz, 1H), 8.65 (d,
1
3
pounds was assessed by HSP70 induction (A) and HSP90 client protein
AKT degradation (B) in HONE-1 cells. Cells were treated with various
concentrations of compounds 11, 12, 13, 15, 19, or 23 for 24 hours. Cell
lysates were prepared after the treatment and subjected to western blot
analysis for assessment of HSP70 and AKT protein levels. β-Actin was
J = 8.0 Hz, 1H); C NMR (75 MHz, CDCl
3
) δ 56.45, 56.95,
1.14, 109.60, 111.31, 128.10, 128.85, 130.17, 136.27, 143.58,
46.32, 152.99, 159.33, 160.55, 179.19, 182.25, 188.92. HRMS
6
1
+
(
7
ESI) for C20H17NNaO (M + Na ) calcd 406.0903, found
used as a loading control. Cells treated with 1 μM of compound 8 406.0906. HPLC purity of 100.0% (retention time = 32.31).
17-AAG) was used as a positive control in this study.
(
6-Dimethylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-
dione (12). A solution of 11 (0.04 g, 0.1 mmol), N,N-dimethyl-
amine (40% w/v, 0.015 mL, 0.13 mmol), and DMF (1 mL) was
stirred at 70 °C for 1 h. The reaction was quenched with H O
2
Conclusions
(
20 mL) and extracted with EtOAc (30 mL × 3). The organic
We have synthesized a series of 2-aroylquinoline-5,8-diones layer was collected and purified by column chromatography
11–23). The highly regioselective incorporation of compound (EtOAc/n-hexane = 2 : 1) to afford 12 (0.02 g, 48.4%) as a red
(
1
1
1 with various amines assists in the straightforward gen- solid. mp = 202–203 °C; H NMR (300 MHz, CDCl ) δ 2.98–3.00
3
eration of the designed compounds (12–20). In antiproliferation (m, 6H), 3.96 (s, 9H), 5.98 (s, 1H), 7.89 (s, 2H), 8.31 (d, J =
1
3
inhibition assays, compounds 12 and 23 are more potent than 8.1 Hz, 1H), 8.57 (d, J = 8.1 Hz, 1H); C NMR (75 MHz, CDCl3)
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δ 29.55, 29.93, 56.58, 61.21, 102.63, 109.75, 127.13, 128.38,
6-Phenylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-
1
1
4
30.46, 135.73, 143.55, 148.14, 148.90, 153.06, 159.48, 180.43, dione (17). The title compound was obtained in 25.9% overall
+
81.27, 189.25. HRMS (ESI) for C21
19.1219, found 419.1222. HPLC purity of 100.0% (retention for the preparation of 12: H NMR (500 MHz, CDCl
6H), 3.96 (s, 3H), 6.65 (s, 1H), 7.27–7.57 (m, 5H), 7.89 (s, 2H),
20 2 6
H N NaO (M + Na ) calcd yield from compound 11 in a manner similar to that described
1
3
) δ 3.96 (s,
time = 31.23).
-Ethylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione 8.37 (d, J = 8.0 Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H); C NMR
13). The title compound was obtained in 57.1% overall yield (75 MHz, CDCl ) δ 56.03, 56.61, 94.28, 106.79, 112.34, 116.74,
1
3
6
(
3
from compound 11 in a manner similar to that described 121.78, 123.96, 127.25, 129.67, 131.27, 136.05, 136.22, 138.34,
1
for the preparation of 12: mp = 180.9–181.7 °C; H NMR 141.38, 142.09, 153.28, 157.99, 171.03, 182.42, 190.30. HRMS
+
(500 MHz, CDCl
3
) δ 1.38 (t, J = 7.5 Hz, 3H), 3.27–3.33 (m, 2H), (ESI) for C25
H
20
N
2
NaO
6
(M + Na ) calcd 467.1219, found
3
.96 (s, 3H), 3.97 (s, 6H), 5.86–5.90 (m, 1H), 5.99 (s, 1H), 7.91 464.1216. HPLC purity of 97.91% (retention time = 33.77).
1
3
(s, 2H), 8.32 (d, J = 8.0 Hz, 1H), 8.55 (d, J = 8.0 Hz, 1H);
C
6-Pyrrolidin-1-yl-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-
NMR (75 MHz, CDCl
3
) δ 13.60, 37.77, 56.43, 61.13, 100.97, dione (18). The title compound was obtained in 36.3% overall
1
1
C
09.57, 129.24, 130.38, 131.94, 135.83, 143.37, 145.27, 148.58, yield from compound 11 in a manner similar to that described
1
52.92, 157.61, 179.64, 180.82, 189.09. HRMS (ESI) for for the preparation of 12: mp = 178.8–179.5 °C; H NMR
(M − H) calcd 395.1243, found 395.1253. HPLC (500 MHz, CDCl ) δ 1.50–1.57 (m, 4H), 2.00–2.10 (m, 4H), 3.97
purity of 99.33% (retention time = 29.83). (4, 9H), 6.02 (s, 1H), 7.91 (s, 2H), 8.31 (d, J = 8.0 Hz, 1H), 8.53
-Allylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-dione (d, J = 8.0 Hz, 1H); C NMR (75 MHz, CDCl
14). The title compound was obtained in 56.3% overall yield 61.26, 106.82, 109.83, 126.90, 129.43, 130.65, 136.00, 143.51,
from compound 11 in a manner similar to that described for 147.77, 148.74, 153.08, 159.10, 179.93, 182.50, 189.52. HRMS
21
H
19
N
2
O
6
3
1
3
6
3
) δ 29.62, 56.64,
(
1
+
the preparation of 12: mp = 167.5–168.2 °C; H NMR (ESI) for C23
300 MHz, CDCl ) δ 3.90 (t, J = 5.7 Hz, 2H), 3.96 (s, 9H), 445.1372. HPLC purity of 97.99% (retention time = 30.67).
.30–5.38 (m, 2H), 5.83–5.97 (m, 1H), 6.01 (s, 1H), 6.06 (t, J = 6-Morpholin-4-yl-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-
.7 Hz), 7.89 (s, 2H), 8.32 (d, J = 8.1 Hz, 1H), 8.56 (d, J = 8.1 Hz, dione (19). The title compound was obtained in 30.6% overall
22 2 6
H N NaO (M + Na ) calcd 445.1376, found
(
3
5
5
1
1
1
1
3
H);
03.21, 109.53, 118.65, 126.92, 128.19, 130.22, 131.23, 135.50, for the preparation of 12: mp = 172–173 °C; H NMR
43.29, 147.56, 147.74, 152.80, 159.18, 180.33, 181.04, 188.94. (500 MHz, CDCl ) δ 3.58–3.61 (m, 4H), 3.88–3.91 (m, 4H), 3.95
(M + Na ) calcd 431.1219, (s, 6H), 3.96 (s, 3H), 6.26 (s, 1H), 7.86 (s, 1H), 8.34 (d, J =
3
C NMR (75 MHz, CDCl ) δ 45.09, 56.35, 60.97, yield from compound 11 in a manner similar to that described
1
3
+
HRMS (ESI) for C22
found 431.1227. HPLC purity of 97.40% (retention time = 8.0 Hz, 1H), 8.53 (d, J = 8.0 Hz, 1H); C NMR (75 MHz, CDCl3)
0.48). δ 49.30, 56.45, 61.12, 66.50, 109.61, 112.65, 127.48, 130.29,
-(3-Dimethylamino-propylamino)-2-(3,4,5-trimethoxybenzoyl)- 136.27, 143.48, 146.55, 152.89, 152.96, 159.05, 181.21, 182.04,
20 2 6
H N NaO
1
3
3
6
+
quinoline-5,8-dione (15). The title compound was obtained in 189.20. HRMS (ESI) for C H N NaO (M + Na ) calcd
2
3
22
2
7
2
9.6% overall yield from compound 11 in a manner similar to 461.1325, found 461.1340. HPLC purity of 100.0% (retention
that described for the preparation of 12: mp = 151.2–151.5 °C; time = 31.29).
1
H NMR (500 MHz, CDCl ) δ 1.90 (t, J = 9.5 Hz, 2H), 2.55 (t, J =
6-(4-Methyl-piperazin-1-yl)-2-(3,4,5-trimethoxybenzoyl)-quino-
3
9
.5 Hz, 2H), 3.36 (q, J = 9.0 Hz, 2H), 3.99 (s, 3H), 4.00 (s, 6H), line-5,8-dione (20). The title compound was obtained in
5
1
.98 (s, 1H), 7.94 (s, 2H), 8.00–8.10 (m, 1H), 8.33 (d, J = 8.0 Hz, 21.2% overall yield from compound 11 in a manner similar to
1
3
1
H), 8.56 (d, J = 8.0 Hz, 1H); C NMR (75 MHz, CDCl3) that described for the preparation of 12: mp 171–172 °C; H
) δ 2.36 (s, 3H), 2.59–2.62 (m, 4H),
25.37, 130.86, 131.31, 133.70, 135.34, 142.53, 150.68, 151.50, 3.61–3.64 (m, 4H), 3.95 (s, 6H), 3.96 (s, 3H), 6.27 (s, 1H), 7.86
δ 56.16, 56.19, 57.12, 60.99, 61.61, 98.60, 109.81, 121.88, NMR (500 MHz, CDCl
1
1
3
+
13
52.62, 153.80, 190.24. HRMS (ESI) for C H N O (M + H ) (s, 2H), 8.33 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 8.5 Hz, 1H);
C
2
4
28 3 6
calcd 454.1978, found 454.1973. HPLC purity of 95.23% (reten- NMR (75 MHz, CDCl
tion time = 17.73). 109.61, 112.50, 127.36, 130.34, 136.22, 143.43, 146.67, 152.95,
-(2-Morpholin-4-yl-ethylamino)-2-(3,4,5-trimethoxybenzoyl)- 158.96, 181.12, 182.17, 189.27. HRMS (ESI) for C H N NaO
6
3
) δ 46.02, 48.97, 54.65, 56.45, 61.10,
6
2
4
25
3
+
quinoline-5,8-dione (16). The title compound was obtained in (M + Na ) calcd 474.1641, found 474.1644. HPLC purity of
9.9% overall yield from compound 11 in a manner similar to 96.03% (retention time = 18.93).
that described for the preparation of 12: mp = 167.5–168.2 °C; 7-Morpholin-4-yl-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8-
) δ 2.50–2.54 (m, 4H), 2.72 (t, J = dione (21). The title compound was obtained in 24.2% overall
1
1
H NMR (300 MHz, CDCl
3
6
3
8
.0 Hz, 2H), 3.27 (q, J = 6.0 Hz, 2H), 3.74–3.78 (m, 4H), yield from compound 31 in a manner similar to that described
1
.96–3.97 (m, 10H), 5.97 (s, 1H), 7.90 (s, 2H), 8.33 (d, J = for the preparation of 12: mp = 171–172 °C; H NMR
1
3
.1 Hz, 1H), 8.56 (d, J = 8.1 Hz, 1H); C NMR (75 MHz, CDCl
3
)
3
(300 MHz, CDCl ) δ 3.62–3.66 (m, 4H), 3.90–3.95 (m, 4H), 4.01
δ 30.01, 38.76, 53.49, 55.74, 56.67, 61.31, 67.19, 103.06, 109.86, (s, 9H), 6.17 (s, 1H), 7.84 (s, 2H), 8.42 (d, J = 8.1 Hz, 1H), 8.60
1
3
1
1
27.22, 128.55, 130.56, 135.81, 148.03, 153.17, 159.56, 161.08, (d, J = 8.1 Hz, 1H); C NMR (75 MHz, CDCl ) δ 47.94, 55.15,
3
+
28 3 7
80.59, 181.36, 189.31. HRMS (ESI) for C25H N O (M + H ) 59.79, 65.17, 108.20, 109.26, 127.42, 128.73, 128.94, 134.04,
calcd 482.1927, found 482.1924. HPLC purity of 95.42% (reten- 138.54, 142.09, 145.63, 151.63, 152.81, 179.30, 180.15, 188.05.
+
tion time = 17.80).
HRMS (ESI) for C H N NaO (M + Na ) calcd 461.1325,
23 22 2 7
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found 461.1313. HPLC purity of 100.0% (retention time = 10.5 mmol) in THF (60 mL) and the mixture was stirred for
1.29). 3 h. The mixture was then filtered and the filtrate was concen-
-Dimethylamino-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8- trated in vacuo to afford an oily adduct. PDC (5.0 g,
3
7
dione (22). The title compound was obtained in 35.7% overall 10.3 mmol), molecular sieves (5.0 g), and DCM (50 mL) were
yield from compound 31 in a manner similar to that described added to this residue and then the mixture was stirred at room
1
for the preparation of 12: mp = 212–213 °C; H NMR temperature for 1 h. The mixture was filtered and the filtrate
(
(
500 MHz, CDCl
s, 2H), 8.36 (d, J = 8.0 Hz, 1H), 8.56 (d, J = 8.0 Hz, 1H);
) δ 40.07, 53.64, 58.30, 103.63, 106.72, (300 MHz, CDCl
25.87, 127.59, 127.83, 132.38, 140.48, 143.88, 150.12, 150.38, 3H), 6.95 (s, 1H), 8.02 (s, 2H), 8.27 (d, J = 9.0 Hz, 1H), 8.59 (d,
54.70, 177.56, 178.30, 186.64. HRMS (ESI) for C H N NaO J = 8.7 Hz, 1H).
3
) δ 3.29 (s, 6H), 3.97 (s, 9H), 5.97 (s, 1H), 7.80 was purified by flash column chromatography over silica gel
1
3
1
C
(EtOAc/n-hexane = 1 : 1) to yield 27 (3.3 g, 75.9%). H NMR
) δ 3.95–4.05 (m, 12H), 4.10 (s, 3H), 4.12 (s,
NMR (75 MHz, CDCl
1
1
3
3
2
1
20
2
6
+
(M + Na ) calcd 419.1219, found 419.1224. HPLC purity of
5,8-Dimethoxy-quinoline-2-carbaldehyde (29). A mixture of
1
00.0% (retention time = 31.13).
2,5-dimethoxyaniline (3.0 g, 19.6 mmol), crotonaldehyde
7
-Pyrrolidin-1-yl-2-(3,4,5-trimethoxybenzoyl)-quinoline-5,8- (4.25 mL, 51.5 mmol), 6 N HCl (80 mL), and toluene (10 mL)
dione (23). The title compound was obtained in 33.5% overall was heated to reflux for 2 h. After cooling down, the resulting
yield from compound 31 in a manner similar to that described mixture was basified with concentrated NaOH solution fol-
1
for the preparation of 12: mp = 183.2–183.9 °C; H NMR lowed by the extraction of EtOAc (3 × 300 mL). The organic
3
(500 MHz, CDCl ) δ 1.58–1.59 (m, 4H), 2.03–2.07 (m, 4H), 3.96 layers were collected and purified by flash column chromato-
(
8
s, 6H), 3.97 (s, 3H), 5.88 (s, 1H), 7.81 (s, 2H), 8.37 (d, J = graphy to give a crude product. The residue was further
.0 Hz, 1H), 8.59 (d, J = 8.0 Hz, 1H); C NMR (75 MHz, CDCl3) reacted with SeO (3.0 g, 27.0 mmol) in the presence of reflux-
2
1
3
δ 29.82, 31.05, 56.43, 61.10, 104.92, 109.53, 128.78, 130.48, ing p-xylene (30 mL) for 6 h. The mixture was cooled down and
1
1
4
31.13, 135.24, 143.27, 145.98, 149.74, 152.92, 157.32, 180.01, filtered with a pad of celite. The filtrate was purified by flash
+
80.84, 189.54. HRMS (ESI) for C H N NaO (M + Na ) calcd column chromatography over silica gel (EtOAc/n-hexane = 1 : 1)
2
3
22
2
6
1
45.1376, found 445.1377. HPLC purity of 97.64% (retention to give 29 (0.9 g, 21.2%) as a yellow solid: mp = 188–191 °C; H
NMR (500 MHz, CDCl ): δ 3.99 (s, 3H), 4.11 (s, 3H), 6.91 (d, J =
,2,4-Trimethoxy-5-nitrobenzene (25). A mixture of 1,2,4-tri- 8.5 Hz, 1H), 7.04 (d, J = 8.5 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H),
methoxybenzene (5.0 g, 29.7 mmol), AcOH (150 mL), and 8.71 (d, J = 8.5 Hz, 1H), 10.31 (s, 1H).
HNO (2.0 mL, 47.9 mmol) was stirred for 12 h. The reaction (5,8-Dimethoxyquinolin-2-yl)-(3,4,5-trimethoxyphenyl)-
time = 30.70).
3
1
3
was quenched with H O (500 mL), and the resulting product methanone (30). The title compound was obtained in 80.1%
2
was collected by filtration and washed with a large amount of overall yield from compound 17 in a manner similar to that
1
1
H
2
O to afford 25 as a yellow solid (5.5 g, 86.8%); H NMR described for the preparation of 18: H NMR (300 MHz, CDCl
3
)
(
(
300 MHz, CDCl ) δ 3.89 (s, 3H), 3.97 (s, 3H), 3.98 (s, 3H), 6.56 δ 3.96 (s, 6H), 4.00 (s, 3H), 4.02 (s, 3H), 6.99 (d, J = 8.7 Hz, 1H),
3
s, 1H), 7.58 (s, 1H).
7.01 (d, J = 8.7 Hz, 1H), 7.99 (s, 2H), 8.21 (d, J = 8.7 Hz, 1H),
2-(3,4,5-Trimethoxybenzoyl)-quinoline-5,8-dione (31). The
5,6,8-Trimethoxy-quinoline-2-carbaldehyde (26). A mixture 8.73 (d, J = 8.7 Hz, 1H).
of compound 25 (4.0 g, 18.8 mmol), Pd/C (0.5 g) and MeOH
(100 mL) was stirred under hydrogen for 3 h. The Pd/C was fil- title compound was obtained in 46.5% overall yield from com-
tered off and the filtrate was concentrated in vacuo. The result- pound 17 in a manner similar to that described for the prepa-
1
ing residue was dissolved in 6 N HCl (60 mL) and heated to ration of 18: H NMR (300 MHz, CDCl ) δ 8.66 (d, J = 8.1 Hz,
3
reflux for 10 min. To the mixture was added a solution of cro- 1H), 8.49 (d, J = 8.1 Hz, 1H), 7.90 (s, 2H), 7.25 (s, 1H), 7.20
tonaldehyde (4.0 mL, 48.5 mmol) in toluene (10 mL) and then (s, 1H), 4.01 (s, 3H), 4.00 (s, 6H).
heated at 110 °C for an additional 6 h. After cooling, the
Biology
mixture was basified with concentrated NaOH solution in an
ice-bath followed by extraction with EtOAc (3 × 300 mL). The Reagents for cell culture were obtained from Gibco-BRL Life
organic layers were collected and purified by flash column Technologies (Gaitherburg, MD). Microtubule-associated
chromatography over silica gel (EtOAc/n-hexane = 1 : 1) to give protein (MAP)-rich tubulin was purchased from Cytoskeleton,
3
a crude product. To the product was added SeO
2
2
(2.8 g, Inc. (Denver, CO). [ H]Colchicine (specific activity, 60–87
5.2 mmol) and p-xylene (50 mL) and the mixture was then Ci mmol− ) was purchased from PerkinElmer Life Sciences
1
allowed to reflux for 2 h. The mixture was cooled and filtered (Boston, MA).
with a pad of celite. The filtrate was purified by flash column
Cell growth inhibitory assay. Human nasopharyngeal carci-
chromatography over silica gel (EtOAc/n-hexane = 1 : 1) to give noma HONE-1, cervical carcinoma KB, and colorectal carci-
1
2
6 as a yellow solid. (1.1 g, 23.7%); H NMR (500 MHz, CDCl
3
): noma HCT116 cells were grown in Dulbecco’s modified
δ 3.94 (s, 3H), 4.08 (s, 3H), 4.15 (s, 3H), 6.94 (s, 1H), 8.04 (d, J = Eagle’s medium, minimal essential medium, or RPMI
.0 Hz, 1H), 8.52 (d, J = 8.5 Hz, 1H), 10.25 (s, 1H). 1640 medium. All cell cultures were supplemented with 10%
3,4,5-Trimethoxy-phenyl)-(5,6,8-trimethoxy-quinolin-2-yl)- fetal bovine serum, 2 μM glutamine, 100 units per mL penicil-
9
(
methanone (27). 3,4,5-Trimethoxyphenylmagnesium bromide lin, and 100 μg mL− streptomycin and incubated under a
1
(
0.5 M in THF, 25 mmol) was added to a solution of 26 (2.6 g, humidified atmosphere (95% air and 5% CO ) at 37 °C. In vitro
2
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growth inhibition was assessed with the methylene blue antibody against β-actin protein was purchased from Santa
2
1
assay. Exponentially growing cells were seeded into 24-well Cruz Biotechnology (Santa Cruz, CA, USA).
culture plates at a density of 8000–20000 cells per mL per well
(
depending on the doubling time of the cell line) and allowed
Acknowledgements
to adhere overnight. The cells were incubated with various con-
centrations of drugs for 72 h. Then the A595 of the resulting
solution from 1% N-lauroylsarcosine extraction was measured.
The IC50 was calculated on the basis of the A595 of untreated
cells (taken as 100%). The values shown are the means and
standard errors of at least three independent experiments
performed in duplicate.
This research was supported by the Ministry Science Technol-
ogy of the Republic of China (grant no. MOST 103-2113-M-038-
001-MY3).
Notes and references
2
2,23
Tubulin polymerization in vitro assay.
Turbidimetric
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2
4
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1
1
mM GTP] to prepare a 4 mg mL
tubulin solution.
The tubulin solution (240 μg of MAP-rich tubulin per well)
was placed in a 96-well microtiter plate in the presence of test
compounds or 2% (v/v) DMSO as a vehicle control. The
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2
5–27
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0
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3
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