Synthesis and biological relationships of 3′,6-substituted 2-phenyl-4-quinolone-3-carboxylic acid derivatives as antimitotic agents

Article abstract of DOI:10.1016/j.bmc.2004.09.041

As part of a continuing search for potential anticancer drug candidates in the 2-phenyl-4-quinolone series, 3′,6-substituted 2-phenyl-4-quinolone-3- carboxylic acid derivatives and their salts were synthesized and evaluated. Preliminary screening showed t

Full text of DOI:10.1016/j.bmc.2004.09.041

Bioorganic & Medicinal Chemistry 13 (2005) 265–275
Synthesis and biological relationships of 30,6-substituted
2-phenyl-4-quinolone-3-carboxylic acid derivatives as
antimitotic agents
Ya-Yun Lai,a Li-Jiau Huang,a Kuo-Hsiung Lee,b Zhiyan Xiao,b Kenneth F. Bastow,b
Takao Yamoric and Sheng-Chu Kuoa,*
aGraduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
bNatural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
cDivision of Experimental Chemotherapy, Cancer Chemotherapy Cancer, Japanese Foundation for Cancer Research,
Tokyo 170-8455, Japan
Received 14 February 2004; accepted 21 September 2004
Available online 12 October 2004
Abstract—As part of a continuing search for potential anticancer drug candidates in the 2-phenyl-4-quinolone series, 30,6-substi-
tuted 2-phenyl-4-quinolone-3-carboxylic acid derivatives and their salts were synthesized and evaluated. Preliminary screening
showed that carboxylic acid analogs containing a m-fluoro substituted 2-phenyl group displayed the highest in vitro anticancer activ-
ity. Activity decreased significantly if a chlorine or methoxy group replaced the fluorine atom. 30-Fluoro-6-methoxy-2-phenyl-4-
quinolone-3-carboxylic acid (68) had the highest in vitro cytotoxic activity among all tested carboxylic acid derivatives and their
salts. The mechanism of action may be similar, but not identical, to that of tubulin binding drugs, such as navelbine and taxol. Com-
pound 68 merits further investigation as a novel hydrophilic antimitotic agent.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
has emerged as the pharmacological target for this class
of quinolone compounds.6–14 In our prior studies, sub-
stituted 2-phenyl-4-quinolones (A) were identified as
novel antimitotic agents,15,16 and structure–activity
relationships (SAR) were established with many related
Quinolone derivatives were initially discovered as bacte-
rial DNA gyrase inhibitors, and thus, developed as anti-
bacterial agents.1–5 Recently, DNA topoisomerase II
O
O
O
NH
R
R
R
N
H
N
N
H
N
R'
R'
R'
(A)
(B)
(C)
O
O
R6
H
R
R3'
N
H
N
H
R'
(A-1)
(D)
R6, R3' = F, Cl, OCH3
*
Corresponding author. Fax: +886 4 2203 0760; e-mail: sckuo@mail.cmu.edu.tw
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.09.041

266
Y.-Y. Lai et al. / Bioorg. Med. Chem. 13 (2005) 265–275
R1
synthetic analogs, including 2-phenylnaphthyridine-4-
ones (B),17 2-phenyl-4-quinazolones (C),18,19 and tetra-
hydro-2-phenyl-4-quinolones (D).20 Among these
analogs, many compounds, including 30,6-disubstituted
2-phenyl-4-quinolones (A-1),21 possessed potent cyto-
toxic activity against human tumor cell lines. However,
most of these compounds were quite lipophilic, and
therefore, not optimal for in vivo and clinical studies.
Introducing a carboxylic acid group into the 2-phenyl-
4-quinolone skeletone should increase polarity and
might improve the pharmacokinetic properties of this
cytotoxic compound class.
O
C
O
R. T.
toluene
+
R1
Cl
NH2
R2
N
H
8-18
R2
1-3
4-7
1. R1=F
2. R1=Cl
3. R1=OCH3
8. R1=F, R2=Cl
9. R1=F, R2=F
10. R1=F, R2=OCH3
11. R1=F, R2=H
12. R1=Cl, R2=F
13. R1=Cl, R2=Cl
4. R2=Cl
5. R2=F
6. R2=OCH3
7. R2=H
14. R1=Cl, R2=OCH3
15. R1=OCH3, R2=F
16. R1=OCH3, R2=Cl
17. R1=OCH3, R2=OCH3
18. R1=OCH3, R2=H
COOC2H5
R1
NaCH
Cl
COOC2H5
30
PCl5
110-140 o
R2
N
toluene, 110 oC reflux
C
19-29
The design of target compounds was based on compar-
ative Molecular Force Field (CoMFA) models derived
from 104 known colchicine binding site antimitotic
agents, including thiocolchicines, quinolones, and naph-
thyridinones,22 and catalyst common-feature pharmaco-
phore models, which were generated from 10
19. R1=F, R2=Cl
20. R1=F, R2=F
21. R1=F, R2=OCH3
22. R1=F, R2=H
23. R1=Cl, R2=F
24. R1=Cl, R2=Cl
25. R1=Cl, R2=OCH3
26. R1=OCH3, R2=F
27. R1=OCH3, R2=Cl
28. R1=OCH3, R2=OCH3
29. R1=OCH3, R2=H
H5C2OOC
COO
C2H5
C H
COO
R1
R1
H
2
5
structurally diverse, colchicine binding site agents.23
A
R2
R2
+
closer examination of these pharmacophore models
indicated that substitution at the 3-position did not
interfere with any molecular area that is critical for anti-
mitotic activity. Therefore, the antimitotic activities of
3-substituted and unsubstituted 2-phenyl-4-quinolones
should be similar, and the effects of different phenyl sub-
stituents should also be comparable (30,6-electron-rich
substitution is favored in the parent series). Based on
this hypothesis, 30,6-substituted 2-phenyl-4-quinolone-
3-carboxylic acids (61–71) and their salts (72–82) were
designed as potential hydrophilic antimitotic agents. In
this paper, the synthesis and in vitro cytotoxic activities
of novel derivatives of 2-phenyl-4-quinolone with im-
proved hydrophilicity are described.
N
H
N
H
31-41
37
42-45
42. R1=F, R2=Cl
43. R1=F, R2=F
44. R1=F, R2=OCH3
45
. R1=Cl, R2=Cl
31. R1=F, R2=Cl
32. R1=F, R2=F
. R1=Cl, R2=OCH3
. R1=OCH3, R2=F
. R1=OCH3, R2=Cl
40
. R1=OCH3, R2=OCH3
38
33. R =F, R2=OCH3 39
1
34. R1=F, R2=H
35. R1=Cl, R2=F
36. R1=Cl, R2=Cl
41
. R1=OCH3, R2=H
170 oC,
170 oC,
O
O
C H
COO
R1
R1
2
5
N
H
N
H
R2
R2
2. Results and discussion
2.1. Chemistry
46-56
57-60
46. R1=F, R2=Cl
47. R1=F, R2=F
48. R1=F, R2=OCH3
49. R1=F, R2=H
50. R1=Cl, R2=F
51. R1=Cl, R2=Cl
52. R1=Cl, R2=OCH3
53. R1=OCH3, R2=F
54. R1=OCH3, R2=Cl
55. R1=OCH3, R2=OCH3
56. R1=OCH3, R2=H
57. R1=F, R2=Cl
58. R1=F, R2=F
59. R1=F, R2=OCH3
60. R1=Cl, R2=Cl
The synthesis of the key intermediates, ethyl 30,6-disub-
stituted 2-phenyl-4-quinolone-3-carboxylates (46–56), is
illustrated in Scheme 1.24 Reaction of p-substituted ani-
lines (1–3) with m-substituted benzoyl chlorides (4–7)
yielded the corresponding N-(p-substituted phenyl)-3-
substituted benzamides (8–18). Subsequent chlorination
of compounds 8–18 with PCl5 afforded carboximidoyl
chlorides 19–29, which, without further purification, were
then treated with sodium diethylmalonate (30) to give the
corresponding N-[1-(3-substituted phenyl)-2-diethoxy-
carbonylvinyl]-N-(4-substituted phenyl) amines (31–41).
Scheme 1.
The synthetic route from key intermediates 46–56 to the
target compounds 61–82 is illustrated in Scheme 2. As
shown, compounds 46–56 were first hydrolyzed to the
carboxylic acids (61–71), which were subsequently trea-
ted with tromethamine to afford the corresponding salts
(72–82).
2.2. In vitro cytotoxic activity
These intermediates (31–41) were thermally cyclized to
the corresponding ethyl 30,6-disubstituted 2-phenyl-4-
quinolone-3-carboxylates (46–56). During the purifica-
tion of products 46, 47, 48, and 51, the 30,6-disubstituted
2-phenyl-4-quinolones (57–60) were also obtained.
Therefore, mono-ethoxycarbonyl vinyl derivatives (42–
45) might have also been produced during the prepara-
tion of intermediates 31, 32, 33, and 36. Indeed, the
mono-ethoxycarbonyl vinyl derivative (42) could be iso-
lated and thermally cyclized to 30-chloro-6-fluoro-2-phen-
yl-4-quinolone (57).
Initial screening was performed using the human lung
cancer (A549) cell line. As shown in Table 1, the tested
ethyl esters (50, 52–55) were inactive. Among the tested
3-carboxylic acid derivatives (61–71), only compounds
62, 65, and 68, which contain a fluorine atom at the
m-position of the 2-phenyl group, showed significant
activity.
30-Fluoro-6-methoxy-2-phenyl-4-quinolone-3-carboxy-
lic acid (68) was the most potent compound with an

Y.-Y. Lai et al. / Bioorg. Med. Chem. 13 (2005) 265–275
267
O
O
C H
COO
R1
2
5
R1
COOH
10% NaOH
N
H
N
H
R2
R2
61-71
46-56
61. R1=F, R2=Cl
62. R1=F, R2=F
63. R1=F, R2=OCH3
64. R1=F, R2=H
65. R1=Cl, R2=F
66. R1=Cl, R2=Cl
52. R1=Cl, R2=OCH3
53. R1=OCH3, R2=F
54. R1=OCH3, R2=Cl
55. R1=OCH3, R2=OCH3
56. R1=OCH3, R2=H
46. R1=F, R2=Cl
47. R1=F, R2=F
48. R1=F, R2=OCH3
49. R1=F, R2=H
50. R1=Cl, R2=F
51. R1=Cl, R2=Cl
67. R1=Cl, R2=OCH3
68. R1=OCH3, R2=F
69. R1=OCH3, R2=Cl
70. R1=OCH3, R2=OCH3
71. R1=OCH3, R2=H
O
.
H2NC(CH2OH)3
COOH
R1
N
H
Tromethamine
R2
72-82
72. R1=F, R2=Cl
78. R1=Cl, R2=OCH3
79. R1=OCH3, R2=F
80. R1=OCH3, R2=Cl
81. R1=OCH3, R2=OCH3
82. R1=OCH3, R2=H
73. R1=F, R2=F
74. R1=F, R2=OCH3
75. R1=F, R2=H
76. R1=Cl, R2=F
77. R1=Cl, R2=Cl
Scheme 2.
Table 1. Cytotoxicity data for compounds 50, 52–55, 61–63, and 65–71
against A549 lung cancer cells
Table 2. Cytotoxicity data for compounds 72–74 and 76–82 against
A549 lung cancer cells
O
O
O
R1
R1
COOR
OH
H2N(CH2OH)3
N
H
N
H
R2
R2
Compd
R
R1
R2
ED50 (lg/mL)a
Compd
R1
R2
ED50 (lg/mL)a
50
52
53
54
55
61
62
63
65
66
67
68
69
70
71
CH2CH3
CH2CH3
Cl
Cl
F
>20
>20
NA
>20
>20
20
72
73
74
76
77
78
79
80
81
82
F
Cl
F
17.6
4.3
OCH3
F
Cl
F
CH2CH3
CH2CH3
CH2CH3
OCH3
OCH3
OCH3
F
F
Cl
OCH3
F
Cl
NA
10.9
>20
>20
2.4
OCH3
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
OCH3
F
Cl
F
F
0.5
OCH3
OCH3
OCH3
OCH3
F
Cl
OCH3
F
Cl
>20
1.80
>20
>20
0.19
20
17.6
NA
>20
OCH3
H
Cl
Cl
OCH3
F
Cl
a Cytotoxicity expressed as ED50 against A549 (the concentration of
compounds that causes a 50% reduction in absorbance at 562nm
relative to untreated cells using the SRB assay).
OCH3
OCH3
OCH3
OCH3
OCH3
H
>20
>20
a Cytotoxicity expressed as ED50 against A549 (the concentration of
compounds that causes a 50% reduction in absorbance at 562nm
relative to untreated cells using the SRB assay).22
general order: 30-fluoro-derivatives (73, 76, and
79) ꢀ 30-chloro-derivatives (72, 77, and 80) > 30-meth-
oxy derivatives (74, 78, and 81). Similarly with the carb-
oxylic acids, the salt (79) of compound 68 showed the
best in vitro cytotoxic activity with an ED50 of 2.4lg/
mL.
ED50 of 0.19lg/mL. Activity decreased (ED50 P 20lg/
mL) when the m-position of the 2-phenyl group was sub-
stituted with OCH3 (63, 67, and 70), Cl (61, 66, and 69),
or H (71) rather than F.
The in vitro cytotoxic activity of compounds 68 and 79
was further tested in eight additional cancer cell lines
(CAKI, HOS, KB, KB-VIN, SK-MEL-2, U87-MG,
HCT-8, and 1A9). As shown in Table 3, these two com-
pounds demonstrated marked inhibition against most of
The cytotoxic activity data for the tromethamine salts
are presented in Table 2. The potencies followed the

268
Y.-Y. Lai et al. / Bioorg. Med. Chem. 13 (2005) 265–275
Table 3. Activity of compounds 68 and 79 against HTCL replication
Compd
ED50 (lg/mL)a
SK-MEL-2b
CAKIb
HOSb
KBb
KB-VINb
U87-MGb
HCT-8b
1A9b
68
79
10.0
NT
0.14
5.0
0.13
NT
0.13
NT
0.16
2.0
1.50
5.0
0.14
1.20
0.03
0.88
a Cell line/mean ED50 in microgram per milliliter (duplicates varied no more than 5%). Note: if inhibition is less than 50% at 10lg/mL then inhibition
values observed are shown in brackets. NT = not tested.
b Renal cancer (CAKI), bone (HOS), epidermoid carcinoma of the nasopharynx (KB), vincristine-resistant epidermoid carcinoma of the
nasopharynx (KB-VIN), melanoma (SK-MEL-2), glioblastoma (C187-MG), ileocecal carcinoma (HCT-8), human ovarian cancer (1A9) cell
lines.
Table 4. Results of COMPARE analysis of compound 68
the eight cancer cell lines, and most notably, were quite
active against human ovarian cancer cells (1A9), which
are known to have a high level of drug resistant P-glyco-
protein. Compound 68 showed an ED50 of 0.03lg/mL
against 1A9 cells, and also was equally active against
vincristine-sensitive and -resistant KB cells. Thus, this
compound merits further anticancer development,
particularly against drug resistant ovarian cancer and
vincristine-resistant epidermal carcinoma of the naso-
pharynx.
Rank
Compd
ra
Molecular targets/drug type
1
2
3
Navelbine
Vindesine
Taxol
0.512
0.461
0.452
Tubulin
Tubulin
Tubulin
a r: correlation coefficient.
which suggests that 68 shares a similar action mecha-
nism with these drugs. A possible mechanism is tubulin
binding and subsequent inhibition of microtubule
organization. However, to prove this possibility, the
tubulin binding activity of 68 must be examined. Com-
pound 68 may have a different mode of action compared
to the above tubulin binders, as the correlation coeffi-
cients were low. Further investigation is required to clar-
ify the action mechanism of this novel compound.
Based on the excellent preliminary results, compound 68
was further evaluated against the JCI human cancer cell
line panel,25 which includes five breast cancer, six CNS,
five colon, seven lung, one melanoma, five ovarian, two
renal, six stomach, and two prostate cancer cell lines.
Dose response curves at five different concentrations
between 10ꢁ4 and 10ꢁ8 M, which were obtained from
computer analysis. GI50 (50% growth inhibition), TGI
(total growth inhibition), and LC50 (50% lethal concen-
tration) were calculated and the corresponding mean
graphs (fingerprints) obtained.
3. Conclusions
In order to improve the pharmacokinetic properties of
antimitotic 2-phenyl-4-quinolone derivatives, molecular
modeling was used to design a series of 3-carboxylic acid
analogs. Initial cytotoxicity screening in the A549 cancer
cell line identified 30-fluoro-6-methoxy-2-phenyl-4-
quinolone-3-carboxylic acid (68) as the most active
compound. In further in vitro cytotoxic evaluation, this
compound showed impressive potency against OVCAR-
4 cancer cells. Analysis by the COMPARE program
suggested a similar, but not identical, mechanism of
action to that of navelbine and taxol. Compound 68 is
an attractive candidate for development as a novel anti-
cancer agent.
Compound 68 was active against most cancer cell lines,
with a MG-MID (mean growth midpoint) for logGI50
of ꢁ6.22. The highest potency was found against
HGC2998 cells (logGI50 ꢁ6.86), followed by OVCAR-
4 (logGI50 ꢁ6.76) cells. When evaluated for TGI (Table
2B), compound 68 had a mean log value of ꢁ4.61, with
marked inhibition against BSY-1 breast, SF-539 CNS,
HGC2998 colon, NCI-H552 lung, OVCAR-4 and
SK-OV-3 ovarian, RXF-631L renal, and DU-145 pros-
tate cancer cell lines. The mean log value for the LC50
values was ꢁ4.05. Compound 68 showed impressive
selective toxicity against the OVCAR-4 cell line with
logLC50 value of ꢁ6.02, which is 100-fold different in
comparison to the mean value. Therefore, compound
68 is an excellent lead compound and worthy of further
development.
4. Experimental
Melting points were determined on a Yanaco MP-500D
melting point apparatus and are uncorrected. IR spectra
were recorded on Shimadzu IR-440 and Nicolet Impact
400 FT-IR spectrophotometers as KBr pellets. NMR
spectra were obtained on a Bruker Advance DPX-200
FT-NMR spectrometer in DMSO-d6 The following
abbreviations are used: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; and br, broad. MS spectra were
measured with an HP 5995 GC–MS instrument. The
UV spectra were recorded on a Shimadzu UV-160A
UV–vis recording spectrophotometer as methanolic
solutions. Elemental analyses (C, H, N) were performed
Most drugs with the same mechanism of action will
show similar fingerprints against a cancer cell line data-
base. Therefore, the fingerprint of compound 68 was
submitted for data analysis by a computer pattern rec-
ognition (COMPARE) program, which has a database
covering fingerprints of over 100 known anticancer
agents with various action mechanisms. The three high-
est correlation coefficient (r) values that were statistically
significant (p < 0.05) are presented in Table 4. Com-
pound 68 was closest to navelbine (r = 0.521), followed
by vindesine (r = 0.461), and then taxol (r = 0.452),

Y.-Y. Lai et al. / Bioorg. Med. Chem. 13 (2005) 265–275
269
at China Medical University, Taiwan, and the results
were within 0.4% of the calculated values.
d 7.38–7.43 (2H, d, J = 8.8Hz, H-30, H50), 7.51–7.59
(1H, t, J = 7.6Hz, H-5), 7.63–7.68 (1H, d, J = 8.3Hz,
H-4), 7.78–7.82 (2H, d, J = 8.8Hz, H-20, H-60), 7.87–
7.92 (1H, dt, J = 1.5, 7.6Hz, H-6), 7.98-8.00 (1H, t,
J = 1.6Hz, H-2), 10.46 (1H, br, NH); IR (KBr) m 3353
(NH), 1652 (C@O) cmꢁ1; MS (M+) m/z 266.1; Anal.
(C13H9Cl2NO) C, H, N.
4.1. 3-Chlorophenyl-N-(4-fluorophenyl)benzamide (8)
p-Fluoroaniline (1) (11.11g, 0.1mol) in 100mL of tolu-
ene at 20 2ꢁC was added dropwise to m-chlorobenzoyl
chloride (4) (8.81g, 0.05mol). After being stirred for 3h,
the resulting precipitate was collected and recrystallized
from EtOH to give compound 9 as colorless needles
4.7. 3-Methoxyphenyl-N-(4-chlorophenyl)benzamide (14)
Obtained from compounds 2 and 6; colorless needles
1
(9.59g, 77%); mp 134–136ꢁC; H NMR (DMSO-d6): d
7.18 (2H, dd, J = 8.9Hz, H-30, H-50), 7.36–7.46 (1H,
m, H-4), 7.51–7.62 (1H, m, H-5), 7.74–7.84 (4H, m, H-
2, H-6, H-20, H-60), 10.41 (1H, br, NH); IR (KBr) m
3330 (NH), 1650 (C@O) cmꢁ1; MS (M+) m/z 249.7;
Anal. (C13H9ClFNO) C, H, N. Compounds 9–18 were
prepared in an analogous manner.
1
(9.92g, 76%); mp 123–124ꢁC; H NMR (DMSO-d6): d
3.99 (3H, s, OCH3), 7.13–7.16 (1H, dd, J = 2.4, 8.3Hz,
H-4), 7.38–7.41 (2H, d, J = 8.8Hz, H-20, H-60), 7.43–
7.44 (1H, t, H-5), 7.44–7.45 (1H, s, H-2), 7.50–7.52
(1H, d, J = 7.8Hz, H-6), 7.74–7.77 (2H, d, J = 11.7Hz,
H-6); IR (KBr) m 3300 (NH), 1649 (C@O) cmꢁ1; MS
(M+) m/z 261.7; Anal. (C14H12ClNO2) C, H, N.
4.2. 3-Fluorophenyl-N-(4-fluorophenyl)benzamide (9)
Obtained from compounds 1 and 5; colorless needles
4.8. 3-Fluorophenyl-N-(4-methoxyphenyl)benzamide (15)
Obtained from compounds 3 and 5; colorless needles
1
(9.32g, 80%); mp 147–148ꢁC; H NMR (DMSO-d6): d
7.16 (2H, dd, J = 8.8Hz, H-30, H-50), 7.34–7.43 (1H,
m, H-4), 7.49–7.60 (1H, m, H-5), 7.75–7.86 (4H, m, H-
2, H-6, H-20, H-60), 10.37 (1H, br, NH); IR (KBr) m
3333 (NH), 1651 (C@O) cmꢁ1; MS (M+) m/z 233.2;
Anal. (C13H9F2NO) C, H, N.
1
(10.78g, 88%); mp 161–163ꢁC; H NMR (DMSO-d6):
d 3.64 (3H, s, OCH3), 6.83 (2H, d, J = 9.1Hz, H-30, H-
50), 7.26–7.36 (1H, m, H-4), 7.48 (1H, m, H-5), 7.56
(2H, d, J = 9.1Hz, H-20, H-60), 7.62–7.72 (2H, m, H-2,
H-6), 10.10 (1H, br, NH); IR (KBr) m 3320 (NH),
1650 (C@O) cmꢁ1
;
MS (M+) m/z 245.2; Anal.
(C14H12FNO2) C, H, N.
4.3. 3-Methoxyphenyl-N-(4-fluorophenyl)benzamide (10)
Obtained from compounds 1 and 6; colorless needles
1
4.9. 3-Chlorophenyl-N-(4-methoxyphenyl)benzamide (16)
Obtained from compounds 3 and 4; colorless needles
(7.19g, 75%); mp 104–105ꢁC; H NMR (DMSO-d6): d
3.83 (3H, s, OCH3), 7.11–7.23 (3H, m, H-4, H-30, H-
50), 7.43 (1H, t, J = 7.7Hz, H-5), 7.47–7.56 (2H, m, H-
2, H-6), 7.79 (2H, dd, J = 9.1, 5.1Hz, H-20, H-60),
10.27 (1H, br, NH); IR (KBr) m 3305 (NH), 1650
(C@O) cmꢁ1; MS (M+) m/z 245.2; Anal. (C14H12FNO2)
C, H, N.
1
(10.57g, 81%); mp 146–148ꢁC; H NMR (DMSO-d6):
d 3.73 (3H, s, OCH3), 6.93 (2H, d, J = 9.0Hz, H-30, H-
50), 7.47–7.51 (1H, m, H-5), 7.58–7.62 (1H, m, H-4),
7.71 (2H, d, J = 9.0Hz, H-20, H-60), 7.93 (1H, d,
J = 7.4Hz, H-6), 8.02 (1H, m, H-2), 10.25 (1H, br,
NH); IR (KBr) m 3345 (NH), 1650 (C@O) cmꢁ1; MS
(M+) m/z 261.7; Anal. (C14H12ClNO2) C, H, N.
4.4. N-(4-Fluorophenyl)benzamide (11)
Obtained from compounds 1 and 7; colorless needles
1
(8.28g, 77%); mp 167–168ꢁC; H NMR (DMSO-d6): d
4.10. 3-Methoxyphenyl-N-(4-methoxyphenyl)benzamide
(17)
7.18 (2H, dd, J = 9.0, 9.0Hz, H-30, H-50), 7.47–7.58
(3H, m, H-4, H-20, H-60), 7.80 (2H, m, H-3, H-5), 7.95
(2H, m, H-2, H-6), 10.31(1H, br, NH); IR (KBr) m
3344 (NH), 1655 (C@O) cmꢁ1; MS (M+) m/z 215.2;
Anal. (C13H10FNO) C, H, N.
Obtained from compounds 3 and 6; colorless needles
1
(10.02g, 78%); mp 107–109ꢁC; H NMR (DMSO-d6):
d 3.74 (3H, s, 40-OCH3), 3.83 (3H, s, 3-OCH3), 6.93
(2H, d, J = 9.1Hz, H-30, H-50f), 7.50 (1H, m, H-2),
7.13 (1H, d, J = 9.1Hz, H-4), 7.42 (1H, d, J = 7.7Hz,
H-5), 7.55 (1H, d, J = 7.7Hz, H-6), 7.69 (2H, d,
J = 9.0Hz, H-20, H-60), 10.11 (1H, br, NH); IR (KBr)
m 3304 (NH), 1644 (C@O) cmꢁ1; MS (M+) m/z 257.3;
Anal. (C15H15NO3) C, H, N.
4.5. 3-Fluorophenyl-N-(4-chlorophenyl)benzamide (12)
Obtained from compounds 2 and 5; colorless needles
1
(10.21g, 82%); mp 156–158ꢁC; H NMR (DMSO-d6):
d 7.40 (2H, d, J = 8.9Hz, H-30, H50), 7.43–7.48 (1H,
m, H-5), 7.52–7.60 (1H, m, H-4), 7.71–7.82 (4H, m, H-
2, H-6, H-20, H-60), 10.42 (1H, br, NH); IR (KBr) m
3320 (NH), 1656 (C@O) cmꢁ1; MS (M+) m/z 249.7;
Anal. (C13H9ClFNO) C, H, N.
4.11. N-(4-Methoxyphenyl)benzamide (18)
Obtained from compounds 3 and 7; colorless needles
1
(9.65g, 85%); mp 153–155ꢁC; H NMR (DMSO-d6): d
4.6. 3-Chloro-N-(4-chlorophenyl)benzamide (13)
3.74 (3H, s, OCH3), 6.93 (2H, d, J = 9.0Hz, H-30, H-
50), 7.50–7.57 (3H, m, H-3, H-4, H-5), 7.71 (2H, d,
J = 9.0Hz, H-20, H-60), 7.97 (2H, dd, J = 7.4, 1.6Hz,
H-2, H-6), 10.16 (1H, br, NH); IR (KBr) m 3332 (NH),
Obtained from compounds 2 and 4; colorless needles
(10.64g, 80%); mp 124–126ꢁC; H NMR (DMSO-d6):
1

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Y.-Y. Lai et al. / Bioorg. Med. Chem. 13 (2005) 265–275
1649 (C@O) cmꢁ1
(C14H13NO2) C, H, N.
;
MS (M+) m/z 227.3; Anal.
12.56 (1H, br, NH); IR (KBr) m 1720 (C@O) cmꢁ1
;
MS (M+) m/z 319.8; Anal. (C17H15ClFNO2) C, H, N.
4.12. N-[1-(3-Chlorophenyl)-2,2-diethoxycarbonylvinyl]-
N-(4-fluorophenyl)amine (31), N-[1-(3-chlorophenyl)-2-eth-
oxy-carbonylvinyl]-N-(4-fluorophenyl)amine (42), ethyl
30-chloro-6-fluoro-phenyl-4-quinolone-3-carboxylate (46)
and 30-chloro-6-fluoro-2- phenyl-4-quinolone (57)
4.15. Compound 46
1
Colorless needles; mp 215–217ꢁC; H NMR (DMSO-
d6): d 0.92 (3H, t, J = 7.2Hz, CH3), 3.96 (2H, q,
J = 7.2Hz, CH2), 7.44–7.61 (3H, m, aromatic), 7.71–
7.92 (4H, m, aromatic), 12.30 (1H, br, NH); IR (KBr)
m 1723, 1615 (C@O) cmꢁ1; MS (M+) m/z 345.8; Anal.
(C18H13ClFNO3) C, H, N.
PCl5 (2.24g, 0.01mol) was added to compound 8 (2.50g,
0.01mol) and the mixture was heated to 110ꢁC, stirred
for 1h, and then evaporated under vacuum to yield car-
boximidoyl chloride (19) as viscous liquid. Meanwhile,
diethyl malonate (4.8g, 0.03mol) was introduced drop-
wise into the solution of sodium (0.72g, 0.03mol) in
absolute ethanol (50mL), at 20 2ꢁC. The mixture
was stirred for 1h at 50 2ꢁC. Subsequent removal of
ethanol under vacuum afforded sodium diethyl malo-
nate (30) as a gel. Then compound 19 was dissolved in
toluene (20mL) and added into a suspension of 30 in
toluene (20mL). The resulting mixture was allowed to
react 4h at 110ꢁC. The reaction mixture was filtered
and the filtrate was concentrated and extracted with
diethyl ether, washed with water, dried over MgSO4,
and concentrated under vacuum to yield 31 and 42 as
yellowish, viscous liquid. Without further purification,
the viscous liquid was heated for 4h at 170ꢁC to give
a yellowish solid that was purified further by column
chromatography (silica gel, 100:1 CHCl3–EtOH) to pro-
duce compounds 46 (1.21g, 35%) and 57 (0.82g, 30%).
4.16. Compound 57
Amorphous colorless; mp 271–289ꢁC (dec); 1H
NMR (DMSO-d6): d 6.38 (1H, s, H-3), 7.57–7.81 (6H,
m, H-5, H-7, H-8, H-40, H-50, H-60), 7.92 (1H, s, H-
20), 11.90 (1H, br, NH); IR (KBr) m 1630 (C@O)
cmꢁ1; MS (M+) m/z 273.7; Anal. (C15H9ClFNO) C,
H, N.
Compounds 47–60 were prepared in an analogous man-
ner without the intermediate purification step.
4.17. Ethyl 30,6-difluoro-2-phenyl-4-quinolone-3-carboxyl-
ate (47) and 30,6-difluoro-2-phenyl-4-quinolone (58)
Obtained from compound 9 (2.33g, 0.01mol).
4.18. Compound 47
After the initial reaction, compounds 31 and 42 could
also be separated by chromatography on silica gel using
CHCl3 as eluant to give pure.
Colorless needles (1.21g, 38%); mp 209–220ꢁC (dec); 1H
NMR (DMSO-d6): d 0.93 (3H, t, J = 7.2Hz, CH3), 3.98
(2H, q, J = 7.2Hz, CH2), 7.38–7.49 (3H, m, aromatic),
7.59–7.78 (4H, m, aromatic), 12.28 (1H, br, NH); IR
(KBr) m 3228 (NH), 1722, 1612 (C@O) cmꢁ1; MS
(M+) m/z 329.3; Anal. (C18H13F2NO3) C, H, N.
N-[1-(3-Chlorophenyl)-2,2-diethoxycarbonylvinyl]-N-(4-
fluorophenyl)amine (31) (1.84g, 47%) and N-[1-(3-
chlorophenyl)-2-ethoxycarbonylvinyl]-N-(4-fluorophenyl)-
amine (42) (1.09g, 34%). Compounds 31 (1.17g,
3mmol) and 42 (0.96g, 3mmol) were then heated sepa-
rately for 4h at 170ꢁC to give yellowish solids, which
were purified by individual column chromatography
(silica gel, CHCl3–EtOH) to produce compounds 46
(0.84g, 81%) and 57 (0.60g, 73%).
4.19. Compound 58
Amorphous colorless (0.54g, 21%); mp 252–278ꢁC
1
(dec); H NMR (DMSO-d6): d 6.43 (1H, s, H-3), 7.40
(1H, dd, J = 8.2, 7.4Hz, H-7), 7.57–7.85 (6H, m, H-5,
H-8, H-20, H-40, H-50, H-60), 11.87 (1H, br, NH); IR
(KBr) m 1635 (C@O) cmꢁ1; MS (M+) m/z 257.2; Anal.
(C15H9F2NO) C, H, N.
4.13. Compound 31
Mp 70–72ꢁC; 1H NMR (DMSO-d6): d 0.92 (3H, t,
J = 7.2Hz, CH3), 1.30 (3H, t, J = 7.2Hz, CH3), 3.90
(2H, q, J = 7.2Hz, CH2), 4.28 (2H, q, J = 7.2Hz,
CH2), 6.67–6.78 (2H, m, H-3, H-5), 6.81–6.83 (2H, m,
H-2, H-6), 7.20–7.28 (2H, m, H-40, H-50), 7.77–7.90
(2H, m, H-20, H-60), 11.30 (1H, br, NH); IR (KBr) m
1720, 1725 (C@O) cmꢁ1; MS (M+) m/z 391.8; Anal.
(C20H19ClFNO4) C, H, N.
4.20. Ethyl 30-methoxy-6-fluoro-2-phenyl-4-quinolone-3-
carboxylate (48) and 30-methoxy-6-fluoro-2-phenyl-4-
quinolone (59)
Obtained from compound 10.
4.21. Compound 48
Colorless needles (1.09g, 32%); mp 214–216ꢁC; 1H
NMR (DMSO-d6): d 0.95 (3H, t, J = 7.2Hz, CH3),
3.86 (3H, s, OCH3), 4.00 (2H, q, J = 7.2Hz, CH2),
7.12–7.15 (3H, m, H-20, H-40, H-60), 7.47 (1H, t,
J = 4.1Hz, H-50), 7.63 (1H, ddd, J = 8.4, 2.9Hz, H-7),
7.75–7.79 (2H, m, H-5, H-8), 12.19 (1H, br, NH); IR
(KBr) m 1711 (C@O) cmꢁ1; MS (M+) m/z 341.3; Anal.
(C19H16FNO4) C, H, N.
4.14. Compound 42
Mp 52–54ꢁC; 1H NMR (DMSO-d6): d 1.31 (3H, t,
J = 7.2Hz, CH3), 4.20 (2H, q, J = 7.2Hz, CH2), 4.99
(1H, s, CH2), 6.64–6.67 (2H, m, H-3, H-5), 6.78–6.83
(2H, m, H-2, H-6), 7.16–7.18 (2H, m, H-40, H-50),
7.27–7.36 (1H, m, H-60), 7.79–7.80 (1H, m, H-20),

Y.-Y. Lai et al. / Bioorg. Med. Chem. 13 (2005) 265–275
271
4.22. Compound 59
d, J = 2.3Hz, H-5); IR (KBr) m 1712 (–C@O) cmꢁ1
;
MS (M+) m/z 357.8; Anal. (C19H16ClNO4) C, H, N.
1
Amorphous colorless (0.67g, 25%); mp 236ꢁC (dec); H
NMR (DMSO-d6): d 3.86 (3H, s, OCH3), 6.43 (1H, s, H-
3), 7.53–7.63 (3H, m, H-7, H-40, H-50), 7.68–7.91 (4H,
m, H-5, H-8, H-20, H-60), 11.91 (1H, br, NH); IR
(KBr) m 1631 (C@O) cmꢁ1; MS (M+) m/z 269.3; Anal.
(C16H12FNO2) C, H, N.
4.29. Ethyl 30-fluoro-6-methoxy-2-phenyl-4-quinolone-3-
carboxylate (53)
Obtained from compound 15; colorless needles (6.44g,
63%); mp 211–214ꢁC; 1H NMR (DMSO-d6): d 0.93
(3H, t, J = 7.2Hz, CH3), 3.85 (3H, s, OCH3), 3.98
(2H, q, J = 7.1Hz, CH2), 7.34–7.48 (4H, m, H-20, H-
40, H-50, H-60), 7.51 (1H, d, J = 2.9Hz, H-5), 7.54–7.57
(1H, m, H-7), 7.64 (1H, d, J = 9.1Hz, H-8), 12.13 (1H,
br s, NH); IR (KBr) m 1709, 1632 (C@O) cmꢁ1; MS
(M+) m/z 341.3; Anal. (C19H16FNO4) C, H, N.
4.23. Ethyl 30,6-dichloro-phenyl-4-quinolone-3-carboxyl-
ate (51) and 30,6-dichloro-2-phenyl-4-quinolone (60)
Obtained from compound 13.
4.24. Compound 51
4.30. Ethyl 30-chloro-6-methoxy-2-phenyl-4-quinolone-3-
carboxylate (54)
Colorless needles (4.89g, 45%); mp 210–228ꢁC (dec); 1H
NMR (DMSO-d6): d 0.93 (3H, t, J = 7.1Hz, CH3), 3.99
(2H, q, J = 7.1Hz, CH2), 7.50–7.77 (6H, m, H-7, H-8,
H-20, H-40, H50, H-60), 8.04 (1H, s, H-5), 12.31 (1H,
br, NH); IR (KBr) m 1720, 1631 (C@O) cmꢁ1; MS
(M+) m/z 362.2; Anal. (C18H13Cl2NO3) C, H, N.
Obtained from compound 16; colorless needles (6.65g,
62%); mp 193–210ꢁC (dec); 1H NMR (DMSO-d6): d
0.90 (3H, t, J = 7.1Hz, CH3), 3.82 (3H, s, OCH3), 3.96
(2H, q, J = 7.1Hz, CH2), 7.37–7.52 (4H, m, H-20, H-
40, H-50, H-60), 7.48 (1H, d, J = 2.9Hz, H-5), 7.36 (1H,
dd, J = 9.0, 2.9Hz, H-7), 7.57 (1H, d, J = 9.1Hz, H-8),
4.25. Compound 60
12.12 (1H, br s, NH); IR (KBr) m 1712 (C@O) cmꢁ1
MS (M+) m/z 357.8; Anal. (C19H16ClNO4) C, H, N.
;
Amorphous colorless (0.87g, 30%); mp 275–286ꢁC
1
(dec); H NMR (DMSO-d6): d 6.47 (1H, s, H-3), 7.59–
7.80 (5H, m, H-20, H-40, H-50, H-60, H-8), 7.93 (1H,
m, H-7), 7.80 (1H, m, H-5); IR (KBr) m 1636 (C@O)
cmꢁ1; MS (M+) m/z 290.1; Anal. (C15H9Cl2NO) C, H,
N.
4.31. Ethyl 30,6-dimethoxy-2-phenyl-4-quinolone-3-carb-
oxylate (55)
Obtained from compound 17; colorless needles (6.25g,
59%); mp 242–243ꢁC (dec); 1H NMR (DMSO-d6): d
0.93 (3H, t, J = 7.2Hz, CH3), 3.79 (3H, s, OCH3), 3.83
(3H, s, OCH3), 3.97 (2H, q, J = 7.1Hz, CH2), 7.08–
7.13 (3H, m, H-20, H-40, H-60), 7.35 (1H, dd, J = 9.1,
2.9Hz, H-7), 7.44 (1H, t, J = 7.9Hz, H-50), 7.51 (1H,
d, J = 2.8Hz, H-5), 7.65 (1H, d, J = 9.2Hz, H-8),
12.10 (1H, s, NH); IR (KBr) m 1731 (C@O) cmꢁ1; MS
(M+) m/z 353.4; Anal. (C20H19NO5) C, H, N.
4.26. Ethyl 6-fluoro-2-phenyl-4-quinolone-3-carboxylate
(49)26
Obtained from compound 11; colorless needles (4.29g,
46%); mp 277–279ꢁC; 1H NMR (DMSO-d6): d 0.89
(3H, t, J = 7.0Hz, CH3), 3.96 (2H, q, J = 7.0Hz, CH2),
7.56–7.67 (5H, m, H-7, H-8, H-30, H-40, H-50), 7.73–
7.79 (3H, m, H-5, H-20, H-60), 12.25 (1H, br, NH); IR
(KBr) m 3232 (NH), 1720 (C@O) cmꢁ1; MS (M+) m/z
311.3; Anal. (C18H14FNO3) C, H, N.
4.32. Ethyl 6-methoxy-2-phenyl-4-quinolone-3-carboxyl-
ate (56)24
4.27. Ethyl 30-fluoro-6-chloro-2-phenyl-4-quinolone-3-
carboxylate (50)
Obtained from compound 18; colorless needles (5.72g,
59%); mp 225–227ꢁC; 1H NMR (DMSO-d6): d 0.89
(3H, t, J = 7.1Hz, CH3), 3.84 (3H, s, OCH3), 3.94
(2H, q, J = 7.1Hz, CH2), 7.36 (1H, dd, J = 9.0, 2.9Hz,
H-7), 7.51 (1H, d, J = 2.9Hz, H-5), 7.54 (5H, m, H-20,
H-30, H-40, H-50, H-60), 7.65 (1H, d, J = 9.0Hz, H-8),
Obtained from compound 12; colorless needles (5.91g,
57%); mp 237–238ꢁC; 1H NMR (DMSO-d6): d 0.92
(3H, t, J = 7.1Hz, CH3), 3.98 (2H, q, J = 7.1Hz, CH2),
7.37–7.67 (4H, m, H-20, H-40, H-50, H-60), 7.72–7.76
(2H, m, H-7, H-8), 8.05 (1H, d, J = 2.2Hz, H-5); IR
(KBr) m 1715, 1630 (C@O) cmꢁ1; MS (M+) m/z 345.8;
Anal. (C18H13ClFNO3) C, H, N.
12.08 (1H, br, NH); IR (KBr) m 1716 (C@O) cmꢁ1
MS (M+) m/z 323.3; Anal. (C19H17NO4) C, H, N.
;
4.33. 30-Chloro-6-fluoro-2-phenyl-4-quinolone-3-carb-
oxylic acid (61)
4.28. Ethyl 30-methoxy-6-chloro-2-phenyl-4-quinolone-3-
carboxylate (52)
Compound 46 (1.04g, 3mmol) was stirred in 10%
NaOH (100mL) until completely dissolved (ca. 1h).
The solution was cooled to 5 2ꢁC and acidified with
dil HCl. The resulting precipitate was collected by filtra-
tion, washed with water, and recrystallized from EtOH
to afford the desired carboxylic acid; amorphous color-
Obtained from compound 14; colorless needles (5.80g,
54%); mp 211–236ꢁC (dec); 1H NMR (DMSO-d6): d
0.80 (3H, t, J = 7.2Hz, CH3), 3.60 (3H, s, OCH3), 3.86
(2H, q, J = 7.2Hz, CH2), 6.66 (1H, m, H-60), 6.95–7.10
(3H, m, H-20, H-40, H-50), 7.49 (1H, dd, J = 8.9,
2.3Hz, H-7), 7.78 (1H, d, J = 8.9Hz, H-8), 7.87 (1H,
1
less (0.86g, 90%); mp 263ꢁC; H NMR (DMSO-d6): d
7.46 (1H, d, J = 7.4Hz, H-60), 7.52 (1H, d, J = 7.5Hz,

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Y.-Y. Lai et al. / Bioorg. Med. Chem. 13 (2005) 265–275
H-50), 7.58–7.61 (2H, m, H-20, H-40), 7.76 (1H, ddd,
J = 2.8, 8.8Hz, H-7), 7.85 (1H, dd, J = 9.0, 4.6Hz, H-
8), 7.90 (1H, dd, J = 9.3, 2.8Hz, H-5); IR (KBr) m
3454 (NH), 1631 (C@O) cmꢁ1; MS (M+) m/z 317.7;
Anal. (C16H9ClFNO3) C, H, N.
4.39. 30-Methoxy-6-chloro-2-phenyl-4-quinolone-3-carb-
oxylic acid (67)
Obtained from compound 52; amorphous colorless
1
(1.46g, 89%); mp 243ꢁC (dec); H NMR (DMSO-d6):
d 7.05 (3H, m, H-20, H-40, H-60), 7.40 (1H, dd,
J = 8.3Hz, H-50), 7.80 (1H, d, J = 8.9Hz, H-8), 7.91
(1H, dd, J = 8.8, 2.4Hz, H-7), 8.19 (1H, d, J = 2.4Hz,
H-5), 13.01 (1H, br s, NH), 15.37 (1H, br s, OH); IR
(KBr) m 3450 (NH), 1675 (C@O) cmꢁ1; MS (M+) m/z
329.7; Anal. (C17H12ClNO4) C, H, N.
Compounds 62–71 were prepared in an analogous
manner.
4.34. 30,6-Difluoro-2-phenyl-4-quinolone-3-carboxylic
acid (62)
Obtained from compound 47; amorphous colorless
4.40. 30-Fluoro-6-methoxy-2-phenyl-4-quinolone-3-carb-
oxylic acid (68)
1
(0.78g, 86%); mp 272ꢁC (dec); H NMR (DMSO-d6): d
7.27–7.33 (3H, m, H-20, H-40, H-60), 7.46–7.51 (1H, m,
H-50), 7.70 (1H, ddd, J = 3.0, 8.8, 8.7Hz, H-7), 7.84
(1H, dd, J = 9.0, 4.7Hz, H-8), 7.91 (1H, dd, J = 9.3,
3.0Hz, H-5); IR (KBr) m 1677, 1620 (C@O) cmꢁ1; MS
(M+) m/z 301.2; Anal. (C16H9F2NO3) C, H, N.
Obtained from compound 53; amorphous colorless
1
(1.46g, 93%); mp 268ꢁC (dec); H NMR (DMSO-d6):
d 3.76 (3H, s, OCH3), 7.07–7.12 (2H, m, H-20, H-40),
7.17 (1H, d, J = 7.9Hz, H-60), 7.28 (1H, dd, J = 2.9,
9.2Hz, H-7), 7.31–7.42 (1H, m, H-50), 7.56 (1H, d,
J = 2.9Hz, H-5), 7.65 (1H, d, J = 9.0Hz, H-8); IR
(KBr) m 1680, 1625 (C@O) cmꢁ1; MS (M+) m/z 313.3;
Anal. (C17H12FNO4) C, H, N.
4.35. 6-Fluoro-30-methoxy-2-phenyl-4-quinolone-3-carb-
oxylic acid (63)
Obtained from compound 48; amorphous colorless
1
(0.84g, 89%); mp 231ꢁC; H NMR (DMSO-d6): d 3.97
4.41. 30-Chloro-6-methoxy-2-phenyl-4-quinolone-3-carb-
oxylic acid (69)
(3H, s, OCH3), 7.06–7.11 (3H, m, H-20, H-40, H-60),
7.42 (1H, m, H-50), 7.77 (1H, dd, J = 2.9, 8.6Hz, H-7),
7.86–7.93 (2H, m, H-5, H-8); IR (KBr) m 3450 (NH),
1679, 1617 (C@O) cmꢁ1; MS (M+) m/z 313.3; Anal.
(C17H12FNO4) C, H, N.
Obtained from compound 54; amorphous colorless
1
(1.48g, 90%); mp 223ꢁC (dec); H NMR (DMSO-d6):
d 3.91 (3H, s, OCH3), 7.43–7.66 (6H, m, H-5, H-7, H-
20, H-40, H-50, H-60), 7.77 (1H, d, J = 9.1Hz, H-8),
13.15 (1H, br s, NH); IR (KBr) m 3444 (NH), 1679,
4.36. 6-Fluoro-2-phenyl-4-quinolone-3-carboxylic acid
(64)
1624 (C@O) cmꢁ1
;
MS (M+) m/z 329.7; Anal.
(C17H12ClNO4) C, H, N.
Obtained from compound 49; amorphous colorless
1
(0.76g, 89%); mp 297ꢁC; H NMR (DMSO-d6): d 7.52
4.42. 30,6-Dimethoxy-2-phenyl-4-quinolone-3-carboxylic
acid (70)
(5H, m, H-7, H-8, H-30, H-40, H-50), 7.74–7.95 (3H, m,
H-5, H-20, H-60), 13.08 (1H, br, NH), 15.50 (1H, br,
OH); IR (KBr) m 1674 (C@O) cmꢁ1; MS (M+) m/z
283.3; Anal. (C16H10FNO3) C, H, N.
Obtained from compound 55; amorphous colorless;
mp > 300ꢁC; 1H NMR (DMSO-d6): d 3.90 (3H, s,
OCH3), 7.03–7.08 (3H, m, H-40, H-50, H-60), 7.37–7.49
(2H, m, H-7, H-20), 7.59 (1H, m, H-5), 7.75 (1H, d,
J = 9.0Hz, H-8), 13.03 (1H, br, NH), 16.02 (1H, br,
4.37. 30-Fluoro-6-chloro-2-phenyl-4-quinolone-3-carb-
oxylic acid (65)
OH); IR (KBr) m 3455 (NH), 1677, 1624 (C@O) cmꢁ1
MS (M+) m/z 325.3; Anal. (C18H15NO5) C, H, N.
;
Obtained from compound 50; amorphous colorless
(1.40g, 88%); mp > 300ꢁC; 1H NMR (DMSO-d6): d
7.37–7.58 (4H, m, H-20, H-40, H-50, H-60), 7.72–7.76
(2H, m, H-7, H-8), 8.05 (1H, d, J = 2.2Hz, H-5), 12.38
(1H, br, NH); IR (KBr) m 3430 (NH), 1687, 1635
4.43. 6-Methoxy-2-phenyl-4-quinolone-3-carboxylic acid
(71)
(C@O) cmꢁ1
;
(C16H9ClFNO3) C, H, N.
MS (M+) m/z 317.7; Anal.
Obtained from compound 56; amorphous colorless;
mp > 300ꢁC; 1H NMR (DMSO-d6): d 3.91 (3H, s,
OCH3), 7.49–7.50 (5H, m, H-20, H-30, H-40, H-50, H-
60), 7.54 (1H, d, J = 2.9Hz, H-7), 7.65 (1H, d,
J = 2.8Hz, H-5), 7.79 (1H, d, J = 9.1Hz, H-8); IR
(KBr) m 3450 (NH), 1672, 1619 (C@O) cmꢁ1; MS
(M+) m/z 295.3; Anal. C17H13NO4) C, H, N.
4.38. 30,6-Dichloro-2-phenyl-4-quinolone-3-carboxylic
acid (66)
Obtained from compound 51; amorphous colorless
1
(1.52g, 91%); mp 260ꢁC (dec); H NMR (DMSO-d6):
d 7.47 (1H, d, J = 7.58Hz, H-40), 7.53 (1H, dd, J = 7.9,
7.9Hz, H-50), 7.59–7.63 (2H, m, H-20, H-60), 7.80 (1H,
d, J = 8.9Hz, H-8), 7.89 (1H, dd, J = 8.9, 2.6Hz, H-7),
8.20 (1H, d, J = 2.4Hz, H-5); IR (KBr) m 3452 (NH),
1680, 1635 (C@O) cmꢁ1; MS (M+) m/z 334.2; Anal.
(C16H9Cl2NO3) C, H, N.
4.44. 30-Chloro-6-fluoro-2-phenyl-4-quinolone-3-carb-
oxylic acid tromethamine salt (72)
A solution of compound 61 (0.32g, 1mmol) in butyl
chloride (20mL) was treated with a methanolic solution
(10mL) of tromethamine (0.12g, 1mmol) and an addi-

Y.-Y. Lai et al. / Bioorg. Med. Chem. 13 (2005) 265–275
273
tional 20mL of butyl chloride to yield 0.41g (93%) of a
white solid precipitate, which was dried overnight at
50ꢁC under reduced pressure; mp 241ꢁC (dec); 1H
NMR (DMSO-d6): d 3.46 (CH2OH), 5.16 (3H, br,
OH), 7.26–7.33 (4H, m, H-20, H-40, H-50, H-60), 7.49
(1H, ddd, J = 8.7, 3.0Hz, H-7), 7.74 (1H, dd, J = 9.2,
5.2Hz, H-8), 7.79 (1H, dd, J = 8.8, 3.1Hz, H-5); IR
(KBr) m 3432, 3228cmꢁ1 (NH); MS (M+) m/z 438.8;
Anal. (C20H20ClFN2O6) C, H, N.
d, J = 2.4Hz, H-5); IR (KBr) m 3228cmꢁ1 (NH); MS
(M+) m/z 455.3; Anal. (C20H20Cl2N2O6) C, H, N.
4.50. 30-Methoxy-6-chloro-2-phenyl-4-quinolone-3-carb-
oxylic acid tromethamine salt (78)
Obtained from compound 67; amorphous colorless (4.1g,
92%); mp 261ꢁC (dec); 1H NMR (DMSO-d6): d 3.75 (3H,
s, OCH3), 5.10 (3H, br, OH), 6.82–6.90 (3H, m, H-20, H-
40, H-60), 7.16–7.20 (1H, m, H-50), 7.57 (1H, dd, J = 8.8,
2.4Hz, H-7), 7.67 (1H, d, J = 8.8Hz, H-8), 8.11 (1H, d,
J = 2.3Hz, H-5); IR (KBr) m 3379 (NH) cmꢁ1; MS
(M+) m/z 450.9; Anal. (C21H23ClN2O7) C, H, N.
Salts 73–82 were obtained in a similar manner.
4.45. 30,6-Difluoro-2-phenyl-4-quinolone-3-carboxylic
acid tromethamine salt (73)
4.51. 30-Fluoro-6-methoxy-2-phenyl-4-quinolone-3-carb-
oxylic acid tromethamine salt (79)
Obtained from compound 62; amorphous colorless (0.40g,
94%); mp > 300ꢁC; 1H NMR (DMSO-d6): d 3.49
(CH2OH), 5.08 (br, OH), 7.04–7.15 (3H, m, H-20, H-40,
H-60), 7.31 (1H, m, H-50), 7.48 (1H, ddd, J = 9.0, 3.0Hz,
H-7), 7.69–7.82 (2H, m, H-5, H-8); IR (KBr) m 3391cmꢁ1
(NH); MS (M+) m/z 422.4; Anal. (C20H20F2N2O6) C, H, N.
Obtained from compound 68; amorphous colorless
1
(0.41g, 95%); mp 268-269ꢁC; H NMR (DMSO-d6): d
3.81 (3 · CH2OH), 3.86 (3H, s, OCH3), 4.78 (3H, br,
OH), 7.08–7.34 (5H, m, H-20, H-30, H-40, H-50), 7.54
(1H, d, J = 2.8Hz, H-5), 7.63 (1H, d, J = 9.8Hz, H-8);
IR (KBr) m 3256 (br, NH) cmꢁ1; MS (M+) m/z 434.4;
Anal. (C21H23FN2O7) C, H, N.
4.46. 30-Methoxy-6-fluoro-2-phenyl-4-quinolone-3-carb-
oxylic acid tromethamine salt (74)
Obtained from compound 63; amorphous colorless
(0.40g, 93%); mp 222ꢁC; H NMR (DMSO-d6): d 3.50
4.52. 30-Chloro-6-methoxy-2-phenyl-4-quinolone-3-carb-
oxylic acid tromethamine salt (80)
1
(CH2OH), 3.75 (3H, s, OCH3), 5.11 (3H, br, OH),
6.84–6.89 (3H, m, H-20, H-40, H-60), 7.20 (1H, m, H-
50), 7.48 (1H, ddd, J = 9.2, 2.9Hz, H-7), 7.67–7.80
(2H, m, H-5, H-8); IR (KBr) m 3384cmꢁ1 (NH); MS
(M+) m/z 434.4; Anal. (C21H23FN2O7) C, H, N.
Obtained from compound 69; amorphous colorless
(0.42g, 94%); mp 144ꢁC; H NMR (DMSO-d6): d 3.85
(3 · CH2OH), 3.87 (3H, s, OCH3), 4.81 (3H, br, OH),
7.27–7.37 (5H, m, H-20, H-40, H-50, H-60, H-7), 7.56
(1H, d, J = 2.6Hz, H-5), 7.66 (1H, d, J = 9.1Hz, H-8);
IR (KBr) m 3341cmꢁ1 (NH), 1621cmꢁ1 (–C@O); MS
(M+) m/z 450.9; Anal. (C21H23ClN2O7) C, H, N.
1
4.47. 6-Fluoro-2-phenyl-4-quinolone-3-carboxylic acid
tromethamine salt (75)
Obtained from compound 64; amorphous colorless
(0.36g, 89%); mp > 300ꢁC; 1H NMR (DMSO-d6): d
3.71 (3 · CH2OH), 5.12 (3H, br, OH), 7.26–7.36 (5H,
m, H-7, H-8, H-30, H-40, H-50), 7.5ꢁ81–7.91 (3H, m, H-5,
4.53. 30,6-Dimethoxy-2-phenyl-4-quinolone-3-carboxylic
acid tromethamine salt (81)
Obtained from compound 70; amorphous colorless
1
H-20, H-60); IR (KBr) m 3378cm
(NH); MS (M+)
(0.42g, 93%); mp 225ꢁC; H NMR (DMSO-d6): d 3.74
m/z 404.4; Anal. (C20H21FN2O6) C, H, N.
(3 · CH2OH), 4.85 (3H, br, OH), 6.83–6.91 (3H, m,
H-40, H-50, H-60), 7.17–7.29 (2H, m, H-7, H-20), 7.52
(1H, m, H-5), 7.63 (1H, d, J = 9.0Hz, H-8); IR (KBr)
4.48. 30-Fluoro-6-chloro-2-phenyl-4-quinolone-3-carb-
oxylic acid tromethamine salt (76)
m
(C22H26N2O8) C, H, N.
3252cmꢁ1 (NH); MS (M+) m/z 446.5; Anal.
Obtained from compound 65; amorphous colorless
1
(0.41g, 93%); mp 262ꢁC (dec); H NMR (DMSO-d6):
4.54. 6-Methoxy-2-phenyl-4-quinolone-3-carboxylic acid
tromethamine salt (82)
d 3.60 (3 · CH2OH), 5.15 (3H, br, OH), 7.06–7.15
(3H, m, H-40, H-50, H-60), 7.31 (1H, dd, J = 7.3Hz, H-
20), 7.59 (1H, dd, J = 9.0, 2.4Hz, H-7), 7.68 (1H, d,
J = 8.9Hz, H-8), 8.11 (1H, d, J = 2.3Hz, H-5); IR
(KBr) m 3375cmꢁ1 (NH); MS (M+) m/z 438.8; Anal.
(C20H20ClFN2O6) C, H, N.
Obtained from compound 71; amorphous colorless
(0.39g, 93%); mp > 300ꢁC; 1H NMR (DMSO-d6): d
3.37 (3 · CH2OH), 3.85 (3H, s, OCH3), 4.88 (3H, br,
OH), 7.22–7.33 (6H, m, H-20, H-30, H-40, H-50, H-60,
H-7), 7.52 (1H, d, J = 2.9Hz, H-5), 7.61 (1H, d,
J = 9.0Hz, H-8); IR (KBr) m 3382cmꢁ1 (NH); MS
(M+) m/z 416.4; Anal. (C21H24N2O7) C, H, N.
4.49. 30,6-Dichloro-2-phenyl-4-quinolone-3-carboxylic
acid tromethamine salt (77)
Obtained from compound 66; amorphous colorless
1
(0.43g, 94%); mp 272ꢁC (dec); H NMR (DMSO-d6):
5. Preliminary cytotoxicity assay
d 3.60 (3 · CH2OH), 5.15 (3H, br, OH), 7.25–7.33
(4H, m, H-20, H-40, H-50, H-60), 7.58 (1H, dd, J = 8.8,
2.4Hz, H-7), 7.69 (1H, d, J = 8.8Hz, H-8), 8.12 (1H,
Compounds were assayed for in vitro cytotoxicity in a
panel of human tumor cell lines at the School of

274
Y.-Y. Lai et al. / Bioorg. Med. Chem. 13 (2005) 265–275
Pharmacy, University of North Carolina at Chapel Hill,
according to procedures described previously.19,25,27,28
The cell lines included human ovarian cancer (1A9),
renal cancer (CAKI), ileocecal carcinoma (HCT-8), lung
carcinoma (A549), glioblastoma (U-87-MG), bone
(HOS), epidermoid carcinoma of the nasopharynx
(KB), P-gp-expressing epidermoid carcinoma of the
nasopharynx (KB-VIN), and melanoma (SKMEL-2)
cell line. The cytotoxic effects of each compound were
obtained as EC50 values, which represent the drug con-
centrations required to cause 50% inhibition.
Absorbance for the control well (C) and the tests well
(T) were measured at 525nm. Moreover, at time 0 (addi-
tion of drugs), absorbance for the test well (T0) was also
measured. Using these measurements, cell growth inhi-
bition (percentage of growth) by each concentration of
drug was calculated as: % growth = 100 · [(T ꢁ T0)/
(C ꢁ T0)], when T > T0 and 50% growth inhibition
parameter (GI50) was determined. The GI50 was calcu-
lated as 100 · [(T ꢁ T0)/(C ꢁ T0)] = 50. The mean
graph, which shows the differential growth inhibition
of the drug in the cell line panel, was drawn based on
a calculation using a set of GI50.27,32 To analyze the cor-
relation between the mean graphs of drug A and drug B,
the COMPARE computer algorithm was developed
according to the method described by Paull et al.32
6. Evaluation against human cancer cell line panel
6.1. Cell lines
Peason correlation coefficients were calculated using
the following formula: c = ( (Xi ꢁ Xm)(Yi ꢁ Ym))/
P
P
P
Compound 68 was evaluated against the human cancer
cell line panel at JCI, according to procedures described
previously.25 Human breast cancer MDA-MB-231 was
purchased from American type culture collection (Rock-
ville, MD) and the following human cancer cell lines29
were generously distributed by the National Cancer
Institute (Frederick, MD): lung cancer, NCI-H23,
NCI-H226, NCI-H522, NCI-H460, A549, DMS273,
and DMS114; colon cancer, HGC-2998, KM-12, HT-
29, HCT-15, and HCT-116; ovarian cancer, OVCAR-3,
OVCAR-4, OVCAR-5, OVCAR-8, and SKOV-3; breast
cancer, MCF-7; renal cancer, RXF-631L, and ACHN;
melanoma, LOX-IMVI; brain tumor, U251, SF-295,
SF-539, SF-268, SNB-75, and SNB-78, and prostate can-
cer, DU-145 and PC-3. Human stomach cancer, MKN-
1, MKN-7, MKN-28, MKN-45, MKN-74, and St-4, and
human breast cancer BSY-1, HBC-4, and HBC-5 were
described elsewhere.30,31 The cells were cultured in
RPM1 1640 supplemented with 5% fetal bovine serum,
penicillin (100units/mL), and streptomycin (100mg/
mL) at 37ꢁC in humidified air containing 5% CO2.
(
(Xi ꢁ Xm)2 (Yi ꢁ Ym)2)1/2, where Xi and Yi are log-
GI50 of drug A and drug B, respectively, against each
cell line and Xm and Ym are the mean values of Xi and
Yi, respectively.
Computer processing of the GI50s produced the mean
graph. The logGI50 for each cell line is indicated. One
scale represents one logarithm difference. Other calcu-
lated values are MG-MID, the mean of logGI50 values
for 39 cell lines; Delta, the logarithm of difference be-
tween the MG-MID and the logGI50 of the most sensi-
tive cell line; and range, the logarithm of difference
between the logGI50 of the most resistant cell line and
the logGI50 of the most sensitive one.
Acknowledgements
The investigation was supported by research grants
from the National Science Council of the Republic of
China (NSC90-2320-B039-039) and China Medical Uni-
versity awarded to S.-C.K. and in part by NIH grant
CA17625 awarded to K.-H.L.
6.2. Human cancer cell line panel and the database
To evaluate drugs for the cell growth inhibition profile,
a human cell line panel was combined with a database.
The system as a whole was developed according to the
method of the National Cancer Institute,27,32 with mod-
ification. The cell line panel consisted of 38 human can-
cer cell lines, described above. With this system, the
antiproliferative effect of more than 200 standard com-
pounds, including various anticancer drugs, was exam-
ined and a new database established, as described below.
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