Synthesis and antiproliferative evaluation of certain pyrido[3,2-g]quinoline derivatives

Article abstract of DOI:10.1016/j.bmc.2006.07.030

The present report describes the synthesis and evaluation of tricyclic pyrido[3,2-g]quinoline derivatives in which an additional pyridine ring is linearly fused on the antibacterial quinoline-3-carboxylic acid. Among them, only diethyl 4,6-diamino-10-meth

Full text of DOI:10.1016/j.bmc.2006.07.030

Bioorganic & Medicinal Chemistry 14 (2006) 7370–7376
Synthesis and antiproliferative evaluation of certain
pyrido[3,2-g]quinoline derivatives
Shu-Yu Li,a,c Yeh-Long Chen,a Chihuei Wangb,* and Cherng-Chyi Tzenga,*
aFaculty of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
bFaculty of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
cGraduate Institute of Pharmaceutical Sciences, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City 807, Taiwan
Received 24 May 2006; revised 7 July 2006; accepted 11 July 2006
Available online 2 August 2006
Abstract—The present report describes the synthesis and evaluation of tricyclic pyrido[3,2-g]quinoline derivatives in which an addi-
tional pyridine ring is linearly fused on the antibacterial quinoline-3-carboxylic acid. Among them, only diethyl 4,6-diamino-10-
methylpyrido[3,2-g]quinoline-3,7-dicarboxylate (9a) and diethyl 4,6-bis-(3-dimethylaminopropylamino)-10-methylpyrido[3,2-
g]quinoline-3,7-dicarboxylate (9d) were able to completely inhibit cell proliferation of MCF7 (Breast), NCI-H460 (Lung), and
SF-268 (CNS) implying either amino or dimethylaminopropyl moiety at C-4 and C-6 positions is crucial for the antiproliferative
activity of pyrido[3,2-g]quinoline derivatives. Compounds 9a–9d were further evaluated for their activity against the growth of
MCF-7 and two prostate cancer cell lines, LNCaP and PC-3. Results indicated the antiproliferative activity decreased in an order
9d > 9a ꢀ 9b and 9c. Compound 9d was the most cytotoxic with an IC50 value of 5.63 and 3.96 lM, respectively, against LNCaP
and MCF-7. Flow cytometric analyses revealed that growth inhibition of LNCaP by 9d was due to cell cycle arrest in G1 phase, and
followed by apoptosis.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
activity from antibacterial to DNA-intercalating.7,8
Extensive SAR studies with DNA-intercalating chro-
mophores reveal a positive correlation between the
strength of reversible DNA binding and the cytotoxic
Tricyclic 4,6-dihydroxy-10-methylpyrido[3,2-g]quino-
line-3,7-dicarboxylic acid (3) and its diethyl ester 2 have
been discovered to be useful for the treatment of disease
or syndrome which is initiated by an antigen–antibody
reaction, for example, allergic asthma, hay fever, urti-
caria, or an auto-immune disease.1 The skeleton of these
compounds belongs to DNA-intercalating coplanar aro-
matic chromophores and therefore, cytotoxic evaluation
of pyrido[3,2-g]quinoline derivatives becomes crucial be-
cause highly cytotoxic agents will hardly be developed as
anti-allergic drug candidates. For the past few years, we
were interested in the synthesis and biological evaluation
of quinolone-3-carboxylic acids.2–5 These compounds
are potent antibacterial agents that target the bacterial
type II DNA topoisomerases (DNA gyrase and topoiso-
merase IV).6 Due to structural and functional similari-
ties between bacterial DNA gyrase and mammalian
topoisomerase II, a series of modified tricyclic benzoqu-
inolones have been synthesized in an attempt to shift the
potency.9–11 For example, benzoacronycine (IC50
=
1.9 lM), the acronycine analogue with an additional
aromatic ring linearly fused on its A-ring to enhance
DNA-intercalating capability, exhibits 10-fold more
potent than the parent acronycine (IC50 = 19.9 lM) in
inhibiting L1210 cell proliferation.12
Due to DNA-intercalating capability exhibited by
coplanar tricyclic benzoquinolones, the present report
describes the synthesis and antiproliferative evaluation
of potential anti-allergic pyrido[3,2-g]quinolin-4(1H)-
ones 2 and 3. Their substituted amino- and anilino-
derivatives have also been synthesized for evaluation
because a number of 4-anilinoquinoline derivatives were
found to possess potent antiproliferative activity.13–15
2. Chemistry
Keywords: Pyrido[3,2-g]quinoline; Antiproliferative.
*
Thermal condensation of 2,6-diaminotoluene with
diethyl ethoxymethylenemalonate (EMME) by the
Corresponding authors. Tel.: +886 7 3121101x6985; fax: +886 7
3125339; e-mail: tzengch@kmu.edu.tw
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.030

S.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 7370–7376
7371
Marcos procedure16 gave diethyl 4,6-dihydroxy-
10-methylpyrido[3,2-g]quinoline-3,7-dicarboxylate (2)
which was hydrolyzed with 2 N HCl to give dicarboxylic
acid 3 in a fairly good overall yield (Scheme 1).
ed anilines to afford 8a and its substituted derivatives 8b
and 8c. Accordingly, reaction of 7 with amine or substi-
tuted amines gave 9a and its substituted derivatives 9b–
9d (see Scheme 2).
Alkylation of 2 with methyl iodide or ethyl iodide gave
diethyl 4,6-dimethoxy-10-methylpyrido[3,2-g]quinoline-
3,7-dicarboxylate (4a) or its 4,6-diethoxy counterpart
4b. Hydrolysis of either 4a or 4b with 2 N HCl afforded
their respective dicarboxylic acids 5a or 5b as depicted in
Scheme 2. Treatment of 2 with ammonia afforded 4,6-di-
hydroxy-10-methylpyrido[3,2-g]quinoline-3,7-dicarbox-
amide (6a). Accordingly, 6b and 6c were synthesized
from 2 by reaction with methylamine and ethylamine,
respectively. Chlorination of 2 with POCl3 gave diethyl
4,6-dichloro-10-methylpyrido[3,2-g]quinoline-3,7-dicarb-
oxylate (7) which was treated with aniline or 4-substitut-
3. Pharmacological results and discussion
All the pyrido[3,2-g]quinoline derivatives 2–9 were eval-
uated in vitro against a 3-cell line panel consisting of
MCF7 (Breast), NCI-H460 (Lung), and SF-268 (CNS)
in US National Cancer Institute (NCI). In this protocol,
each cell line was inoculated and preincubated on a
microtiter plate. Test agents were then added at a single
concentration (100 lM) and the culture was incubated
for 48 h. End-point determinations were made with ala-
mar blue.17 Results for each test agent were reported as
OH
N
OH
N
CO2Et EtO2C
EtO2C
CO2Et
EtO2C
CO2Et
EMME
Ph2O
250oC
80%
EtOH, reflux
90%
H2N
NH2
N
N
H
H
Me
Me
Me
2
1
OH
OH
N
HOOC
COOH
2N HCl
67%
N
Me
3
Scheme 1.
OR
N
OR
N
OR
OR
HOOC
COOH
EtO2C
RI, K2CO3
CO2Et
2N HCl
67%
N
N
DMF
34-46%
Me
Me
4a R = Me
4b R = Et
5a R = Me
5b R = Et
OH
OH
RHNOC
CONHR
RNH2
2
N
N
58-64%
Me
6a R = H
6b R = Me
6c R = Et
X
X
Cl
Cl
NH
N
HN
EtO2C
CO2Et
EtO2C
CO2Et
NH2C6H4-p-X
POCl3
72%
K2CO3, DMF
50-86%
N
N
N
Me
Me
7
8a X = H
8b X = Cl
8c X = OMe
RNH2
NHR
N
NHR
CO2Et
EtO2C
N
Me
9a R = H
9b R = Me
9c R = CH2CH2NMe2
9d R = CH2CH2CH2NMe2
Scheme 2.

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S.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 7370–7376
90
the percent of growth of the treated cells in comparison
with the untreated control cells. Compounds which re-
duced the growth of any one of the cell lines to 32%
or less are considered active. The results indicated most
of them were inactive against the primary 3-cell line pan-
el except diethyl 4,6-diamino-10-methylpyrido[3,2-g]-
quinoline-3,7-dicarboxylate (9a) and diethyl 4,6-bis-(3-
dimethylaminopropylamino)-10-methylpyrido[3,2-g]quin-
oline-3,7-dicarboxylate (9d) that were able to completely
inhibit cell proliferation of all three cells (Table 1).
Either amino or dimethylaminopropyl moiety at C-4
and C-6 positions is crucial for the antiproliferative
activity of pyrido[3,2-g]quinoline derivatives. Com-
pounds 9a–9d were further evaluated for their activity
against the growth of two prostate cancer cells, LNCaP
and PC-3, and a breast cancer cell, MCF-7. For each
compound, dose–response curves for each cell line were
measured with 10 different drug concentrations, and the
concentration for 50% of cell growth inhibition (IC50)
was calculated by SigmaPlot. Results from Table 2
indicated the antiproliferative activity decreased in an
order 9d > 9a ꢀ 9b and 9c. Compound 9d was the most
cytotoxic with an IC50 value of 5.63 and 3.96 lM,
80
70
60
50
40
30
20
10
0
G1
S
G2/M
μM
C
10
LNCaP
20
40
C
10
20
40
MCF-7
Figure 1. Dose–response effects of compound 9d on cell cycle in
LNCaP and MCF-7 cancer cells for 24 h.
respectively, against LNCaP and MCF-7, and therefore
was selected for further evaluation of its effect on cell cy-
cle distribution of LNCaP cells. As shown in Figure 1,
the proportion of cells was slightly accumulated in G1
phase, however, was decreased in the S phase of the cell
cycle after 24 h treatment. After 48 h, the accumulation
of G1 DNA content significantly decreased, while the
hypodiploid (sub G1 phase) cells increased as shown
in Figure 2. Compound 9d inhibited proliferation of
LNCaP by the alteration of cell division, accumulation
of cells in G1 phase at early 24 h, and it was then de-
creased followed by the increase of apoptotic cells (sub
G1 phase) after 48 h treatment. This suggested that
compound 9d induces cell cycle arrest in G1 phase fol-
lowed by apoptosis.
Table 1. Antiproliferative activity of pyrido[3,2-g]quinoline
derivativesa
Compound
Growth percentages
NCI-H460
(Lung)
MCF7
(Breast)
SF-268
(CNS)
2
118
107
122
95
115
97
93
81
3
4a
4b
5a
5b
6a
6b
6c
8a
8b
8c
9a
9c
9d
104
105
108
106
85
99
4. Conclusion
119
97
111
120
96
A number of pyrido[3,2-g]quinoline derivatives were
synthesized and evaluated for their antiproliferative
activity. The results indicated most of them, with excep-
tion of 9a and 9d, are non-cytotoxic against MCF7,
NCI-H460, and SF-268 cells at a concentration of
100 lM although their skeleton belongs to DNA-inter-
calating coplanar aromatic chromophores. These results
are valuable because 9a and 9d are new leads for further
design and synthesis of potential anticancer agents,
while others (2–8 and 9c) can be developed as anti-aller-
gic drug candidates.
114
107
84
76
75
104
111
108
115
0
114
117
111
120
0
110
108
108
110
0
74
0
101
0
99
0
a Compounds were evaluated in vitro against a 3-cell line panel con-
sisting of MCF7 (Breast), NCI-H460 (Lung), and SF-268 (CNS). In
this protocol, each cell line is inoculated and preincubated on a
microtiter plate. Test agents are then added at a single concentration
(100 lM) and the culture is incubated for 48 h. End-point determi-
nations are made with alamar blue.17 Results for each test agent are
reported as the percent of growth of the treated cells when compared
to the untreated control cells. Compounds which reduced the growth
of any one of the cell lines to 32% or less are active.
5. Experimental
5.1. General
TLC: precoated (0.2 mm) silica gel 60 F254 plates
from EM Laboratories, Inc.; detection by UV light
(254 nm). All chromatographic separations were
performed using silica gel (Merck 60 230–400
mesh). Mp: Electrothermal IA9100 melting point
apparatus; uncorrected. 1H and 13C NMR spectra:
Table 2. IC50 (lM) values of selective pyrido[3,2-g]quinoline deriva-
tives against LNCaP, PC-3 (Prostate cancer), and MCF-7 (Breast
cancer) cells
Varian-Unity-400
spectrometer
at
400
and
Compound
LNCaP
PC-3
MCF-7
100 MHz or Varian-Gemini-200 spectrometer at
200 and 50 MHz chemical shifts d in ppm with
SiMe4 as an internal standard (=0 ppm), coupling
constants J in Hz. Mass spectra (HRMS) were
recorded on Finnigan/Thermo Quest MAT 95XL.
9a
9b
9c
9d
16.91
51.04
62.49
5.63
14.73
71.98
59.57
6.42
14.54
76.38
69.52
3.96

S.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 7370–7376
7373
A
(1) Sub G1 = 1.08%
(2) Sub G1 = 1.29%
(3) Sub G1 = 2.96%
(4) Sub G1 = 33.22%
B
(1) Sub G1 = 8.10%
(2) Sub G1 = 6.55%
(3) Sub G1 = 6.70%
(4) Sub G1 = 57.86%
DNA content
Figure 2. Apoptosis is induced by 9d. LNCaP cells were treated in (A) 24 h and (B) 48 h culture with: (1) 0.1% DMSO control, (2) 5 lM, (3) 10 lM,
or (4) 20 lM of 9d prior to PI staining and analysis of DNA content. Apoptosis is apparent by the large population of cells with increased DNA
content in sub G1.
Elemental analyses were carried out on a Heraeus
CHN-O-Rapid elemental analyzer, and results were
within 0.4% of calculated values.
5.1.3.
4,6-Dihydroxy-10-methylpyrido[3,2-g]quinoline-
3,7-dicarboxylic acid (3). To a suspension of 2 (0.37 g,
1 mmol) in EtOH (5 mL) was added a solution of 2 N
HCl (20 mL) and the mixture heated at reflux for 2 h.
The mixture was evaporated under reduced pressure
and the residual solid was crystallized from MeOH to
give 3 (0.21 g, 67%). Mp > 300 ꢁC [lit.1 mp 358–
5.1.1. Diethyl 2-{[3-(2,2-bisethoxycarbonylvinylamino)-2-
methylphenylamino]-methylene}malonate (1). A mixture
of 2,6-diaminotoluene (0.61 g, 5 mmol) and diethyl
ethoxymethylenemalonate (2.16 g, 10 mmol) in EtOH
(15 mL) was heated at reflux for 5 h. The mixture
was evaporated under reduced pressure and then
n-hexane (100 mL) was added. The resulting precipi-
tate was collected and crystallized from EtOH to af-
ford 1 (2.08 g, 90%). Mp 138–140 ꢁC. 1H NMR
(200 MHz, CDCl3): 1.30–1.43 (m, 12H, OCH2CH3),
2.34 (s, 3H, 2-CH3), 4.20–4.39 (m, 8H, OCH2CH3),
7.07 (d, J = 8.2 Hz, 2H–C(4,6)), 7.32 (t, J = 8.2 Hz,
1H–C(5)), 8.49 (d, J = 13.2 Hz, 2H, NHCH@), 11.20
(d, J = 13.2 Hz, 2H, NHCH@). 13C NMR (50 MHz,
CDCl3): 11.27 (2-CH3), 14.26 (2C), 14.36 (2C), 60.13
(2C), 60.51 (2C), 94.51 (2C), 112.96 (2C), 117.42,
128.04, 139.39 (2C), 152.68 (2C), 165.57 (2C), 169.19
(2C). Anal. Calcd for C23H30N2O8Æ0.2 EtOH: C,
59.57; H, 6.68; N, 5.94. Found: C, 59.25; H, 6.49;
N, 5.98.
1
360 ꢁC]. H NMR (200 MHz, TFA-d): 3.23 (s, 3H, 10-
CH3), 9.64 (s, 2H–C(2, 8)), 9.96 (s, 1H–C(5)). 13C
NMR (50 MHz, TFA-d): 11.75 (10-CH3), 107.84 (2C),
122.24 (2C), 123.10, 127.69, 142.18 (2C), 152.88 (2C),
171.80 (2C), 179.89 (2C). Anal. Calcd for
C15H10N2O6Æ0.8 H2O: C, 54.81; H, 3.56; N, 8.52. Found:
C, 54.78; H, 3.95; N, 8.19.
5.1.4. Diethyl 4,6-dimethoxy-10-methylpyrido[3,2-g]quin-
oline-3,7-dicarboxylate (4a). A mixture of 2 (0.37 g,
1 mmol), K2CO3 (1.10 g, 8 mmol), and CH3I (0.85 g,
6 mmol) in DMF (50 mL) was heated at 40–45 ꢁC for
22 h (TLC monitoring). The mixture was evaporated
under reduced pressure and then H2O (200 mL) was
added. The resulting precipitate was collected, purified
by flash column chromatography (FC, silica gel; n-hex-
ane/EtOAc = 1:1), and crystallized from EtOH to afford
4a (0.14 g, 34%). Mp > 300 ꢁC. 1H NMR (200 MHz,
TFA-d): 1.66 (t, J = 7.0 Hz, 6H, OCH2CH3), 3.34 (s,
3H, 10-CH3), 4.87 (m, 10H, 4,6-OCH3, OCH2CH3),
9.70 (s, 2H–C(2, 8)), 10.06 (s, 1H–C(5)). 13C NMR
(50 MHz, TFA-d): 12.69 (10-CH3), 14.42 (2C), 52.14
(2C), 67.83 (2C), 108.57 (2C), 123.56 (2C), 126.44,
127.89, 149.15 (2C), 160.38 (2C), 168.92 (2C), 176.85
(2C). Anal. Calcd for C21H22N2O6ÆH2O: C, 60.56; H,
5.82; N, 6.73. Found: C, 60.30; H, 5.80; N, 6.87.
5.1.2. Diethyl 4,6-dihydroxy-10-methylpyrido[3,2-g]quin-
oline-3,7-dicarboxylate (2). A solution of 1 (0.93 g,
2 mmol) in Ph2O (30 mL) was heated at 250–260 ꢁC
for 2 h (TLC monitoring). The reaction mixture was
cooled and then n-hexane (100 mL) was added. The
resulting precipitate was filtered, washed with n-hexane,
CH2Cl2, MeOH, and dried under vacuum to afford 2
(0.59 g, 80%). Mp > 300 ꢁC. 1H NMR (200 MHz,
TFA-d): 1.61 (t, J = 7.2 Hz, 6H, OCH2CH3), 3.31 (s,
3H, 10-CH3), 4.80 (q, J = 7.2 Hz, 4H, OCH2CH3),
9.68 (s, 2H–C(2, 8)), 10.09 (s, 1H–C(5)). 13C NMR
(50 MHz, TFA-d): 12.31 (10-CH3), 14.40 (2C), 67.86
(2C), 107.71 (2C), 122.04 (2C), 124.25, 127.37, 141.49
(2C), 153.26 (2C), 169.31 (2C), 178.41 (2C). Anal. Calcd
for C19H18N2O6: C, 61.62; H, 4.90; N, 7.56. Found: C,
61.63; H, 4.98; N, 7.69.
5.1.5. Diethyl 4,6-diethoxy-10-methylpyrido[3,2-g]quino-
line-3,7-dicarboxylate (4b). Prepared from 2 by the same
procedure as described for 4a. Compound 4b was puri-
fied by FC (n-hexane/EtOAc = 3:1) and crystallized
from EtOH in a 46% yield. Mp 156–157 ꢁC. H NMR
(200 MHz, CDCl3): 1.48 and 1.62 (two t, J = 7.0 Hz,
12H, OCH2CH3), 3.32 (s, 3H, 10-CH3), 4.48 (m, 8H,
1

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S.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 7370–7376
OCH2CH3), 9.25 (s, 1H–C(5)), 9.34 (s, 2H–C(2, 8)). 13C
NMR (50 MHz, CDCl3): 12.36 (10-CH3), 14.24 (2C),
15.79 (2C), 61.54 (2C), 72.54 (2C), 112.40 (2C), 118.15
(2C), 122.52, 136.06, 148.19 (2C), 153.00 (2C), 165.08
(2C), 165.22 (2C). Anal. Calcd for C23H26N2O6: C,
64.78; H, 6.15; N, 6.57. Found: C, 64.79; H, 6.17; N,
6.54.
5.1.10. 4,6-Dihydroxy-10-methylpyrido[3,2-g]quinoline-
3,7-dicarboxylic acid bis-ethylamide (6c). Prepared from
2 and 70% EtNH2 by the same procedure as described
for 6a. Compound 6c was crystallized from EtOH in a
1
58% yield. Mp > 300 ꢁC. H NMR (200 MHz, TFA-d):
1.47 (t, J = 7.4 Hz, 6H, NCH2CH3), 3.08 (s, 3H, 10-
CH3), 3.79 (q, J = 7.4 Hz, 4H, NCH2CH3), 9.55 (s,
2H–C(2, 8)), 9.77 (s, 1H–C(5)). 13C NMR (50 MHz,
TFA-d): 11.27 (2C), 14.23 (10-CH3), 38.66 (2C), 107.73
(2C), 120.32 (2C), 124.18, 127.61, 141.66 (2C), 149.56
(2C), 170.02 (2C), 181.06 (2C). Anal. Calcd for
C19H20N4O4Æ1.3 H2O: C, 58.23; H, 5.82; N, 14.30.
Found: C, 58.27; H, 5.85; N, 14.17.
5.1.6.
4,6-Dimethoxy-10-methylpyrido[3,2-g]quinoline-
3,7-dicarboxylic acid (5a). Prepared from 4a by the same
procedure as described for 3. Compound 5a was crystal-
lized from EtOH in a 67% yield. Mp > 300 ꢁC. 1H NMR
(200 MHz, TFA-d): 3.15 (s, 3H, 10-CH3), 4.64 (s, 6H,
OCH3), 9.51 (s, 2H–C(2, 8)), 9.81 (s, 1H–C(5)). 13C
NMR (50 MHz, TFA-d): 14.49 (10-CH3), 51.26 (2C),
108.35 (2C), 124.60 (2C), 124.77, 128.19, 149.96 (2C),
160.22 (2C), 171.49 (2C), 178.46 (2C). Anal. Calcd for
C17H14N2O6ÆH2O: C, 56.66; H, 4.48; N, 7.78. Found:
C, 56.47; H, 4.52; N, 7.52.
5.1.11. Diethyl 4,6-dichloro-10-methylpyrido[3,2-g]quino-
line-3,7-dicarboxylate (7). A mixture of 2 (0.37 g,
1 mmol) in POCl3 (10 mL) was heated at 90 ꢁC for 4 h
(TLC monitoring). After cooling, the mixture was
poured into ice-water (50 mL) and neutralized with sat-
urated Na2CO3until pH 7 resulted. This aqueous mix-
ture was extracted with CH2Cl2 (50 mL 3·) dried
(MgSO4), and concentrated to yield a brown solid.
The crude product was purified by FC (using n-hex-
ane/EtOAc = 9:1 as the eluent) to give a yellow solid,
which was recrystallized with MeOH to give 7 (0.29 g,
5.1.7. 4,6-Diethoxy-10-methylpyrido[3,2-g]quinoline-3,7-
dicarboxylic acid (5b). Prepared from 4b by the same
procedure as described for 3. Compound 5b was crystal-
lized from EtOH in a 77% yield. Mp > 300 ꢁC. 1H NMR
(200 MHz, TFA-d): 1.60 (t, J = 7.0 Hz, 6H, OCH2CH3),
3.30 (s, 3H, 10-CH3), 4.78 (q, J = 7.0 Hz, 4H,
OCH2CH3), 9.67 (s, 2H–C(2, 8)), 10.07 (s, 1H–C(5)).
13C NMR (50 MHz, TFA-d): 12.26 (10-CH3), 14.35
(2C), 67.79 (2C), 107.61 (2C), 122.93 (2C), 124.20,
127.32, 141.40 (2C), 153.21 (2C), 169.26 (2C), 178.35
(2C). Anal. Calcd for C19H18N2O6ÆH2O: C, 58.75; H,
5.20; N, 7.21. Found: C, 58.40; H, 5.30; N, 7.12.
1
72%). Mp 125–126 ꢁC. H NMR (200 MHz, CDCl3):
1.51 (t, J = 7.0 Hz, 6H, OCH2CH3), 3.33 (s, 3H, 10-
CH3), 4.55 (q, J = 7.0 Hz, 4H, OCH2CH3), 9.33 (s,
2H–C(2, 8)), 9.37 (s, 1H–C(5)). 13C NMR (50 MHz,
CDCl3): 12.85 (10-CH3), 14.23 (2C), 62.30 (2C), 122.46
(2C), 122.87 (2C), 124.92, 138.42, 144.87 (2C), 146.42
(2C), 150.94 (2C), 164.13 (2C). Anal. Calcd for
C19H16Cl2N2O4: C, 56.03; H, 3.97; N, 6.88. Found: C,
56.32; H, 3.97; N, 7.07.
5.1.8.
3,7-dicarboxamide (6a).
4,6-Dihydroxy-10-methylpyrido[3,2-g]quinoline-
mixture of (0.50 g,
A
2
1.35 mmol) and 30% NH4OH (50 mL) in a sealed ves-
sel was heated at 70 ꢁC for 8 h (TLC monitoring). The
resulting mixture was evaporated under reduced pres-
sure and then H2O (100 mL) was added. The precipi-
tate was collected, washed with H2O, and then
crystallized from EtOH to give 6a (0.25 g, 60%)as a
yellow powder. Mp > 300 ꢁC. 1H NMR (200 MHz,
TFA-d): 3.32 (s, 3H, 10-CH3), 9.77 (s, 2H–C(2, 8)),
10.00 (s, 1H–C(5)). 13C NMR (50 MHz, TFA-d):
11.35 (10-CH3), 107.57 (2C), 120.49 (2C), 124.34,
126.69, 142.00 (2C), 150.75 (2C), 175.33 (2C), 180.28
(2C). HRMS (ESI) Calcd for C15H13FN4O4(M+1):
313.0937, found: 313.0940.
5.1.12. Diethyl 10-methyl-4,6-bisphenylaminopyrido[3,2-
g]quinoline-3,7-dicarboxylate (8a).
A mixture of 7
(0.20 g, 0.5 mmol), aniline (0.28 g, 3 mmol), and dry
DMF (20 mL) was heated in 100 ꢁC for 6 h (TLC mon-
itoring). The mixture was then cooled and evaporated in
vacuo to yield a yellow residue, treated with H2O
(50 mL), and the resulting precipitate was filtered and
washed with H2O. The crude product was purified by
FC (using n-hexane/EtOAc = 3:1 as the eluent) to give
a yellow solid, which was recrystallized with EtOH to
give 8a (0.15 g, 56%). Mp 251–253 ꢁC. 1H NMR
(200 MHz, CDCl3): 1.43 (t, J = 7.2 Hz, 6H, OCH2CH3),
3.27 (s, 3H, 10-CH3), 4.40 (q, J = 7.2 Hz, 4H,
OCH2CH3), 6.59 (m, 4H, Ar–H), 7.72 (m, 6H, Ar–H),
8.38 (s, 1H–C(5)), 9.33 (s, 2H–C(2, 8)), 10.52 (br s,
2H, NH). 13C NMR (50 MHz, CDCl3): 12.62 (10-
CH3), 14.23 (2C), 61.36 (2C), 104.56 (2C), 115.30 (2C),
119.72, 122.52 (4C), 124.90 (2C), 125.30, 129.47 (4C),
141.26 (2C), 146.22 (2C), 151.96 (2C), 154.22 (2C),
168.15 (2C). Anal. Calcd for C31H28N4O4: C, 71.51;
H, 5.43; N, 10.76. Found: C, 71.31; H, 5.50; N, 10.76.
5.1.9.
4,6-Dihydroxy-10-methylpyrido[3,2-g]quinoline-
3,7-dicarboxylic acid bis-methylamide (6b). Prepared
from 2 and 40% CH3NH2 by the same procedure as
described for 6a. Compound 6b was crystallized from
EtOH in
a
64% yield. Mp > 300 ꢁC. 1H NMR
(200 MHz, TFA-d): 3.11 (s, 3H, 10-CH3), 3.32 (s, 6H,
NCH3), 9.57 (s, 2H–C(2, 8)), 9.78 (s, 1H–C(5)). 13C
NMR (50 MHz, TFA-d): 11.26 (10-CH3), 28.56 (2C),
108.08 (2C), 120.28 (2C), 124.14, 127.61, 141.69 (2C),
149.61 (2C), 170.69 (2C), 180.79 (2C). Anal. Calcd
for C17H16N4O4Æ2.0H2O: C, 54.24; H, 5.37; N, 14.89.
Found: C, 54.46; H, 5.46; N, 14.46. HRMS (ESI)
Calcd for C17H17N4O4(M+1): 341.1250, found:
341.1253.
5.1.13. Diethyl 4,6-bis-(4-chlorophenylamino)-10-methyl-
pyrido[3,2-g]quinoline-3,7-dicarboxylate (8b). Prepared
from 7 and p-chloroaniline by the same procedure as de-
scribed for 8a. Compound 8b was crystallized from
EtOH in a 68% yield. Mp 308–309 ꢁC. 1H NMR
(200 MHz, CDCl3): 1.44 (t, J = 7.2 Hz, 6H, OCH2CH3),

S.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 7370–7376
7375
3.29 (s, 3H, 10-CH3), 4.43 (q, J = 7.2 Hz, 4H,
OCH2CH3), 6.60 (m, 4H, Ar–H), 7.16 (m, 4H, Ar–H),
8.42 (s, 1H–C(5)), 9.38 (s, 2H–C(2, 8)), 10.71 (br s,
2H, NH). 13C NMR (50 MHz, CDCl3): 12.70 (10-
CH3), 14.24 (2C), 61.37 (2C), 105.10 (2C), 115.52 (2C),
120.74, 123.14 (4C), 124.11 (2C), 129.38 (4C), 129.61
(2C), 135.72, 140.23 (2C), 147.81, 152.06 (2C), 153.12
(2C), 168.37 (2C). Anal. Calcd for C31H26Cl2N4O4Æ0.5
H2O: C, 62.20; H, 4.56; N, 9.36. Found: C, 62.13; H,
4.47; N, 9.27.
7.2 Hz, 6H, OCH2CH3), 2.35 (s, 12H, N(CH3)2), 2.62
(t, J = 6.0 Hz, 4H, NCH2CH2N(CH3)2), 3.17 (s, 3H,
10-CH3), 3.89 (q, J = 6.0 Hz, 4H, NCH2CH2N(CH3)2),
4.40 (q, J = 7.2 Hz, 4H, OCH2CH3), 8.98 (s, 1H–C(5)),
9.21 (s, 2H–C(2, 8)), 9.59 (br s, 2H, NH). 13C NMR
(50 MHz, CDCl3): 12.62 (10-CH3), 14.29 (2C), 45.35
(4C), 47.02 (2C), 59.43 (2C), 60.51 (2C), 101.60 (2C),
116.28 (2C), 122.58 (2C), 133.78, 148.51, 152.46 (2C),
158.00 (2C), 168.37 (2C). Anal. Calcd for C27H38N6O4:
C, 63.49; H, 7.52; N, 16.46. Found: C, 63.10; H, 7.49;
N, 16.43.
5.1.14. Diethyl 4,6-bis-(4-methoxyphenylamino)-10-meth-
ylpyrido[3,2-g]quinoline-3,7-dicarboxylate (8c). Prepared
from 7 and p-anisidine by the same procedure as de-
scribed for 8a. Compound 8c was crystallized from
EtOH in a 50% yield. Mp 290–292 ꢁC. 1H NMR
(400 MHz, CDCl3): 1.43 (t, J = 7.2 Hz, 6H, OCH2CH3),
3.25 (s, 3H, 10-CH3), 3.84 (s, 6H, OCH3), 4.41 (q,
J = 7.2 Hz, 4H, OCH2CH3), 6.57 (m, 4H, Ar–H), 6.73
(m, 4H, Ar–H), 8.46 (s, 1H–C(5)), 9.34 (s, 2H–C(2,
8)), 10.71 (br s, 2H, NH). 13C NMR (100 MHz, CDCl3):
12.620 (10-CH3), 14.23 (2C), 55.62 (2C), 61.35 (2C),
103.79 (2C), 114.73 (4C), 115.03 (2C), 122.92, 123.99
(4C), 125.12 (2C), 134.12 (2C), 135.16, 151.90 (2C),
154.71 (2C), 157.13 (2C), 168.18 (2C). Anal. Calcd for
C33H32N4O6Æ0.2 H2O: C, 67.83; H, 5.60; N, 9.59. Found:
C, 67.94; H, 5.56; N, 9.58
5.1.18. Diethyl 4,6-bis-(3-dimethylaminopropylamino)-10-
methylpyrido[3,2-g]quinoline-3,7-dicarboxylate (9d). Pre-
pared from 7 and N,N-dimethylpropane-1,3-diamine
by the same procedure as described for 6a. Compound
9d was crystallized from EtOH in a 46% yield. Mp
184–186 ꢁC. 1H NMR (200 MHz, CDCl3): 1.43
(t, J = 7.2 Hz, 6H, OCH2CH3), 1.94 (q, J = 6.6 Hz,
4H, NCH2CH2CH2N(CH3)2), 2.22 (s, 12H, N(CH3)2),
2.44 (t, J = 6.6 Hz, 4H, NCH2CH2CH2N(CH3)2),
3.16 (s, 3H, 10-CH3), 3.91 (q, J = 6.6 Hz, 4H,
NCH2CH2CH2N(CH3)2), 4.39 (q, J = 7.2 Hz, 4H,
OCH2CH3), 9.02 (s, 1H–C(5)), 9.20 (s, 2H–C(2, 8)),
9.58 (br s, 2H, NH). 13C NMR (50 MHz, CDCl3):
12.66 (10-CH3), 14.29 (2C), 29.10 (2C), 45.44 (4C),
47.46 (2C), 56.64 (2C), 60.55 (2C), 100.97 (2C), 116.21
(2C), 123.38 (2C), 133.76, 148.68, 152.40 (2C), 158.34
(2C), 168.92 (2C). Anal. Calcd for C29H42N6O4Æ0.5
HCl: C, 62.53; H, 7.71; N, 15.09. Found: C, 62.46; H,
7.89; N, 15.02.
5.1.15. Diethyl 4,6-diamino-10-methylpyrido[3,2-g]quino-
line-3,7-dicarboxylate (9a). Prepared from 7 and 30%
NH4OH by the same procedure as described for 6a.
Compound 9a was crystallized from EtOH in a 56%
1
yield. Mp > 300 ꢁC. H NMR (200 MHz, TFA-d): 1.57
5.2. Flow cytometric analysis
(t, J = 7.0 Hz, 6H, OCH2CH3), 3.08 (s, 3H, 10-CH3),
4.66 (q, J = 7.0 Hz, 4H, OCH2CH3), 9.33 (s, 2H–C(2,
8)), 9.97 (s, 1H–C(5)). 13C NMR (50 MHz, TFA-d):
12.10 (10-CH3), 14.47 (2C), 66.36 (2C), 103.94 (2C),
118.28 (2C), 121.44, 124.72, 140.22 (2C), 150.97 (2C),
161.97 (2C), 167.72 (2C). Anal. Calcd for
C19H20N4O4Æ1.5 HCl: C, 53.92; H, 5.13; N, 13.24.
Found: C, 53.68; H, 5.28; N, 13.33.
5.2.1. Chemicals. RPMI 1640 medium, fetal bovine ser-
um (FBS), trypsin-EDTA, phosphate-buffered saline
(PBS), penicillin G, and streptomycin were obtained
from GIBCO BRL (Gaithersburg, MD). Trypan blue,
dimethylsulfoxide (DMSO), ribonuclease A (RNase
A), Triton X-100, and propidium iodide (PI) were pur-
chased from Sigma Chemical (St. Louis, MD).
5.1.16. Diethyl 4,6-bismethylamino-10-methylpyrido[3,2-
g]quinoline-3,7-dicarboxylate (9b). Prepared from 7 and
40% CH3NH2 by the same procedure as described for
6a. Compound 9b was crystallized from EtOH in a
5.2.2. Cell culture. LNCaP, MCF-7, and PC-3 cells were
obtained from the American Type Culture Collection
(ATCC, Manassas, VA). Cells were maintained in
RPMI 1640 medium supplemented with 10% fetal bo-
vine serum, 2 mM glutamine, and antibiotics (100 U/
ml penicillin and 100 lg/ml streptomycin) at 37 ꢁC in a
humidified atmosphere of 5% CO2.
1
41% yield. Mp 219–221 ꢁC. H NMR (200 MHz, TFA-
d): 1.54 (t, J = 7.2 Hz, 6H, OCH2CH3), 2.98 (s, 3H,
10-CH3), 3.94 (s, 6H, NCH3), 4.60 (q, J = 7.2 Hz, 4H,
OCH2CH3), 9.15 (s, 2H–C(2, 8)), 9.70 (s, 1H–C(5)).
13C NMR (50 MHz, TFA-d): 11.99 (10-CH3), 14.42
(2C), 37.37 (2C), 66.32 (2C), 104.93 (2C), 116.69 (2C),
119.39, 131.16, 141.62 (2C), 148.60 (2C), 162.65 (2C),
168.58 (2C). Anal. Calcd for C21H24N4O4Æ0.2 HCl: C,
62.46; H, 6.05; N, 13.87. Found: C, 62.74; H, 5.96; N,
13.53.
5.2.3. Cell viability and cytotoxicity. The viability and
cytotoxicity of cells were determined by the ATPLite as-
say system (Perkin-Elmer). Exponentially growing cells
(5 · 103 cells) were plated in 96-well plates and treated
with a series of concentrations of compounds dissolved
in RPMI 1640 medium. Incubation was carried out at
37 ꢁC for 48 h. ATPLite assay reagents (Perkin-Elmer)
were added and luminescence measured by TopCounter
(Perkin-Elmer). The concentration that killed 50% of
cells (IC50) was determined from the curve by calculat-
ing the concentration of agent that reduced the readout
of luciferase activity in treated cells, compared to con-
trol cells by SigmaPlot (SYSTAT).
5.1.17. Diethyl 4,6-bis-(2-dimethylaminoethylamino)-10-
methylpyrido[3,2-g]quinoline-3,7-dicarboxylate (9c). Pre-
pared from 7 and N,N-dimethylethylenediamine by the
same procedure as described for 6a. Compound 9c was
crystallized from EtOH in a 63% yield. Mp 186–
188 ꢁC. 1H NMR (200 MHz, CDCl3): 1.43 (t, J =

7376
S.-Y. Li et al. / Bioorg. Med. Chem. 14 (2006) 7370–7376
2. Fang, K. C.; Chen, Y. L.; Sheu, J. Y.; Wang, T. C.; Tzeng,
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5.2.4. Flow cytometric analysis. Cells were seeded onto
10-cm dishes and treated with or without compounds
for 24 and 48 h. Cells were harvested, washed twice with
ice-cold PBS, and collected by centrifugation at 2000g
for 5 min at 4 ꢁC. The harvested cells were fixed in
70% (v/v) ethanol at 4 ꢁC for 30 min. After fixation, cells
were centrifuged and the solution was discarded, and
then the fixed cells were incubated with 500 ll PBS con-
taining 5% of Triton X-100 and 2 lg/mL RNase A at
37 ꢁC for 1 h. Then, 50 ll of propidium iodide (500 lg/
mL propidium iodide in PBS) was added and shaked
in dark for 30 min at room temperature. Cells were
detected using a cytometer (Coulter EpicsXL, Beckman)
and analyzed by Win cycle and Win MDI 2.8 programs.
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Financial support to this work by the National Science
Council of the Republic of China is gratefully acknowl-
edged. We also thank National Cancer Institute (NCI)
of the United States for the anticancer screenings, and
the National Center for High-Performance Computing
for providing computer resources and chemical database
services.
15. Chen, Y. L.; Huang, C. J.; Huang, Z. Y.; Tseng, C. H.;
Chang, F. S.; Yang, S. H.; Lin, S. R.; Tzeng, C. C. Bioorg.
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