Antitumor agents. 178. Synthesis and biological evaluation of substituted 2-aryl-1,8-naphthyridin-4(1H)-ones as antitumor agents that inhibit tubulin polymerization

Article abstract of DOI:10.1021/jm970146h

As part of our continuing search for potential anticancer drug candidates in the 2-aryl-1,8-naphthyridin-4(1H)-one series, we have synthesized two series of 3'-substituted 2-phenyl-1,8-naphthyridin-4(1H)- ones and 2-naphthyl-1,8-naphthyridin-4(1H)-ones. A

Full text of DOI:10.1021/jm970146h

J . Med. Chem. 1997, 40, 3049
3049
An titu m or Agen ts. 178.† Syn th esis a n d Biologica l Eva lu a tion of Su bstitu ted
2-Ar yl-1,8-n a p h th yr id in -4(1H)-on es a s An titu m or Agen ts Th a t In h ibit Tu bu lin
P olym er iza tion
Ke Chen,‡ Sheng-Chu Kuo,§ Ming-Chieh Hsieh,§ Anthony Mauger,| Chii M. Lin, Ernest Hamel, and
Kuo-Hsiung Lee*,‡
Natural Products Laboratory, Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, Graduate Institute of Pharmaceutical Chemistry, China Medical
College, Taichung 400, Taiwan, Republic of China, Drug Synthesis and Chemistry Branch, Developmental Therapeutics
Program, Division of Cancer Treatment, Diagnosis and Centers, and Laboratory of Molecular Pharmacology, Division of Basic
Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Received March 10, 1997X
As part of our continuing search for potential anticancer drug candidates in the 2-aryl-1,8-
naphthyridin-4(1H)-one series, we have synthesized two series of 3′-substituted 2-phenyl-1,8-
naphthyridin-4(1H)-ones and 2-naphthyl-1,8-naphthyridin-4(1H)-ones. All compounds showed
significant cytotoxic effects (log GI50 < -4.0; log molar drug concentration required to cause
50% growth inhibition) against a variety of human tumor cell lines of the National Cancer
Institute’s in vitro screen, including cells derived from solid tumors such as non-small cell
lung, colon, central nervous system, melanoma, ovarian, prostate, and breast cancers. All 3′-
substituted compounds demonstrated strong cytotoxic effects in almost all tumor cell lines.
Introduction of an aromatic ring at the 2′- and 3′-positions also generated compounds with
potent antitumor activity. Incorporation of an aromatic ring at the 3′- and 4′-positions produced
compounds with reduced activity. Interestingly, introduction of a halogen at the 3′-position
yielded compounds with different selectivity for the tumor cell lines tested. All 3′-halogenated
compounds (29-36) and compounds 38 and 42-44 were potent inhibitors of tubulin polym-
erization with activities nearly comparable to those of the potent antimitotic natural products
colchicine, podophyllotoxin, and combretastatin A-4. Active agents also inhibited the binding
of [3H]colchicine to tubulin.
In tr od u ction
In our continuing search for antitumor agents, 2-phen-
yl-1,8-naphthyridin-4(1H)-ones were identified as anti-
tumor agents interacting with tubulin at the colchicine
site.2 We have continued our synthesis and evaluation
of related compounds, and in this paper, we describe
new active agents, a series of 3′-substituted 2-phenyl-
1,8-naphthyridin-4(1H)-ones (29-41) and 2-naphthyl-
F igu r e 1.
1,8-naphthyridin-4(1H)-ones (42-52). These compounds
displayed strong inhibitory effects against a variety of
retained significant, but reduced, antitubulin activity.
The effects on activity of substitutions in ring A de-
pended on the substitution in ring C, and this was most
readily evaluated by comparing inhibitory effects on
tubulin assembly. The work presented here describes
the synthesis and evaluation of new 3′-substituted
2-phenyl-1,8-naphthyridin-4(1H)-ones and of 2-naph-
thyl-1,8-naphthyridin-4(1H)-ones in order to investigate
further the steric and electronic effects of these positions
for compound activity.
human tumor cell lines, derived from solid tumors, when
evaluated in the National Cancer Institute’s (NCI) in
vitro screen.3,4 Like the initial members of the series,2
the most potent 2-aryl-1,8-naphthyridin-4(1H)-ones (29-
31, 33-36, 43-46) inhibited tubulin polymerization and
colchicine binding to tubulin. These activities were
comparable to those of the antimitotic natural products
colchicine (Figure 1),5,6 podophyllotoxin,7,8 and combre-
tastatin A-4.9,10
Our initial investigation of the structure-activity
relationships of the 2-phenyl-1,8-naphthyridin-4(1H)-
ones2 indicated that a methoxy group at the 3′-position
was required for potent cytotoxicity against most tumor
cell lines. Substitution at the 2′- and 4′-positions yield
compounds with little cytotoxic activity, although sev-
eral agents with a 4′-methyl or 4′-chloro substituent
Ch em istr y
The 3′-substituted 2-phenyl-1,8-naphthyridin-4(1H)-
ones and 2-naphthyl-1,8-naphthyridin-4(1H)-ones were
synthesized according to the previously reported meth-
ods.2 In brief, condensation of substituted 2-aminopy-
ridines (1) with substituted ethyl benzoylacetates (2) in
the presence of polyphosphoric acid (PPA) formed the
corresponding pyridopyrimidinones (5-28). Thermal
rearrangement in mineral oil at 350 °C yielded the
target compounds (29-52) (Scheme 1).11 The starting
substituted ethyl benzoylacetates (2) were prepared
† For part 177, see ref. 1.
* To whom correspondence should be addressed.
‡ University of North Carolina.
§ China Medical College.
| Drug Synthesis and Chemistry Branch, NCI.
Laboratory of Molecular Pharmacology, NCI.
X Abstract published in Advance ACS Abstracts, August 15, 1997.
S0022-2623(97)00146-5 CCC: $14.00 © 1997 American Chemical Society

3050 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Chen et al.
Sch em e 1. Synthetic Route to
2-Aryl-1,8-naphthyridin-4(1H)-ones
nervous system, renal, ovarian, prostate, and breast
cancers, plus a few leukemia cell lines. The cytotoxic
effects of each compound are presented as the molar
drug concentrations required to cause 50% growth
inhibition (GI50) and total growth inhibition (TGI),
respectively. The results are expressed as log GI50
values (Table 2) for each cell line and average log GI50
values (Table 1) and as log TGI values and average log
TGI values (Table 3).
All compounds had cytotoxic activity (log GI50
<
-4.00) against all cell lines tested. Among the three
subseries of 2-phenyl-1,8-naphthyridin-4(1H)-ones, only
compound 41 had limited activity, for compounds 29-
40 were inhibitory toward most cell lines in the submi-
cromolar (log GI50’s of -7 to -6) to nanomolar (log GI50’s
of -8 to -7) range. Ignoring compound 41, and con-
sidering only the average log GI50 values, which takes
into account all the cell lines tested (not just those
shown in Table 2), the agents with 3′-fluoride and
methyl groups were slightly more cytotoxic than those
with a 3′-chloride group. The 3′-fluoride compounds, in
particular, have cytotoxicity comparable to that reported
earlier for agents bearing a 3′-methoxy substituent.2
according to a literature method.12 Condensation of
substituted acetophenones (4) with diethyl carbonate (3)
in the presence of sodium hydride formed the required
ethyl benzoylacetates. The structures and chemical
features of newly synthesized compounds are sum-
marized in Table 1.
Resu lts a n d Discu ssion
(a ) Eva lu a tion of th e Cytotoxicity of 2-Ar yl-
n a p h th yr id in -4(1H)-on es. The 2-aryl-1,8-naphthyri-
din-4(1H)-ones were submitted to the NCI for testing
in its in vitro disease-oriented antitumor screen.3,4 This
assay involves determinations of a test agent’s effect on
growth parameters against a panel of approximately 60
human tumor cell lines, derived largely from solid
tumors, including non-small cell lung, colon, central
A fairly dramatic difference was observed between the
2-(R-naphthyl) (compounds 42-46) and the 2-(â-naph-
thyl) (compounds 47-52) derivatives. In the former
group, except for compound 46, low nanomolar GI50
values (log < -8) were observed with many cell lines.
In the latter group, in contrast, submicromolar GI50
values were rarely obtained with any agent except
Ta ble 1. Physical Properties and Antimicrotubule Effects of Substituted 2-Aryl-1,8-naphthyridin-4(1H)-ones
ITPa
ICBb
averagec
compd R5
R6
R7
R2′
R3′
R4′
IC50 (µM) ( SD (% inhibition) log GI50
formulad
mp, °C
>300
yield, %e
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
H
H
CH3
H
CH3
H
H
CH3
H
CH3
H
H
CH3
H
CH3
H
H
CH3
H
CH3
H
H
CH3
H
CH3
H
H
H
H
CH3
H
H
H
H
CH3
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
F
F
Cl
Cl
Cl
Cl
Cl
CH3
CH3
CH3
CH3
CH3
H
0.63 ( 0.20
0.53 ( 0.08
0.74 ( 0.06
1.50 ( 0.10
1.00 ( 0.03
0.72 ( 0.08
0.89 ( 0.09
0.77 ( 0.20
3.30 ( 0.60
1.80 ( 0.50
1.50 ( 0.30
1.90 ( 0.50
2.30 ( 0.20
1.10 ( 0.30
0.93 ( 0.20
0.55 ( 0.05
0.66 ( 0.10
0.78 ( 0.20
14.00 ( 2.00
1.80 ( 0.06
2.10 ( 0.50
5.10 ( 0.90
43 ( 1
41 ( 2
29 ( 1
-7.30
-7.37
-7.07
-6.64
-6.80
-6.57
-6.77
-6.46
-7.02
-7.24
-6.19
-7.01
-4.42
-7.45
-7.45
-7.72
-7.18
-5.98
-5.09
-5.92
-6.13
-5.34
-4.87
-5.24
-7.24
-7.54
-8.18
C15H11FN2O
C15H11FN2O
C16H13FN2O
C14H9ClN2O
C15H11ClN2O
C15H11ClN2O
C15H11ClN2O
C16H13ClN2O
C15H12N2O
C16H14N2O
C16H14N2O‚0.25H2O
C16H14N2O
C17H16N2O
C18H12N2O
C19H14N2O
C19H14N2O
C19H14N2O‚0.5H2O
C20H16N2O
C18H12N2O
C19H14N2O
C19H14N2O
C19H14N2O
C20H16N2O‚2H2O
C18H11ClN2O
57
48
57
26
21
49
17
49
33
32
36
52
55
27
27
25
34
37
38
29
34
45
59
51
CH3
CH3
H
H
H
CH3
CH3
H
H
H
CH3
CH3
H
H
H
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
270-272f
236-238
295-297f
260-262
290-292f
255-257f
232-234
205-207
175-177
223-225
193-195
197-198
282-283f
228-230f
236-238
240-242
276-278f
259-261
217-219
255-257
244-246
149-151
32 ( 1
33 ( 2
38 ( 1
22 ( 2
CHdCHsCHdCH
CHdCHsCHdCH
CHdCHsCHdCH
CHdCHsCHdCH
CHdCHsCHdCH
37 ( 4
46 ( 3
40 ( 4
15 ( 10
H
H
H
H
H
H
CHdCHsCHdCH
CHdCHsCHdCH
CHdCHsCHdCH
CHdCHsCHdCH
CHdCHsCHdCH >40
CHdCHsCHdCH
CH3
H
H
CH3
CH3
H
Cl
2.50 ( 0.50
>300
colchicine
podophyllotoxin
combretastatin A-4
0.80 ( 0.07g
0.46 ( 0.02g
0.53 ( 0.05g
76 ( 5
91 ( 2
a
b
ITB ) inhibition of tubulin polymerization. ICB ) inhibition of colchicine binding and evaluated only when polymerization IC50
e
1 µM. c Data obtained from NCI’s 60 human tumor cell line in vitro screen and calculated from all cell lines tested. All compounds were
d
analyzed for C, H, and N, and results agreed to (0.4% of the theoretical values. e All yields were calculated from aminopyridines.
f Decomposed. Previously obtained data, see ref 16.
g

Antitumor Agents 178
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3051
Ta ble 2. Inhibition of in Vitro Tumor Cell Growth by Substituted 2-Aryl-1,8-naphthyridin-4(1H)-onesa
cytotoxicity log GI50 (M)b
compd HL-60 (TB)c NCI-H460 HCT-116 SF-295
U-251 SK-MEL-5 OVCAR-3 CAKI-1
PC-3
MDA-MB-435 MDA-N
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
-7.69
-7.65
-7.43
-7.98
< -8.00
-7.51
-7.54
-7.13
-7.72
-7.74
-6.76
-7.57
-4.89
< -8.00
< -8.00
< -8.00
< -8.00
-6.61
-5.74
-6.69
-6.85
-5.75
-5.46
-5.75
-7.41
-7.48
-7.27
-7.06
-7.41
-7.30
-7.38
-6.65
-7.59
-7.44
-6.41
-7.75
-4.35
-7.41
< -8.00
< -8.00
-7.76
-6.20
-5.42
-5.98
-6.40
-5.42
-4.88
-5.39
-7.62
-7.55
< -8.00
-7.54
-7.80
-7.52
-7.59
-6.77
-7.65
-7.33
-6.39
-6.92
-4.75
-7.56
-7.54
-7.48
-7.20
-7.39
-7.60
-7.64
-7.32
-7.43
-7.52
-6.38
-7.22
-4.19
-7.39
-7.42
-7.19
-6.84
-7.28
-7.16
-7.19
-6.53
-7.35
-7.27
-6.77
-6.70
-4.27
-7.99
-7.55
-7.65
-7.43
-7.28
-7.38
-7.15
-7.50
-6.93
-7.59
-7.47
-6.54
-6.90
-4.46
< -8.00
< -8.00
< -8.00
-7.86
-6.33
-5.46
-6.36
-6.44
-5.48
-5.66
-5.53
-7.87
-7.71
-7.55
-7.34
-7.45
-7.25
-7.48
-7.46
-7.46
-7.21
-7.65
-7.81
< -8.00
-7.91
-7.93
-7.84
-7.74
< -8.00
< -8.00
< -8.00
< -8.00
< -8.00
-7.88
-7.89
-7.34
-7.76
-7.93
-7.50
-7.65
-7.37 > -4.00
-7.38 > -4.00
> -4.00
> -4.00
-7.72
< -8.00
-6.77
-6.69
-7.43
-7.27
-6.36
-6.37
-4.25
-7.82
-7.92
-7.56
-7.37
-7.48
-6.51
-6.86
-4.50
-7.75
-7.70
-7.38
<- 8.00
< -8.00
<- 8.00
-7.49
< -8.00
-7.96
-7.65
-6.60
< -8.00
-7.50
-6.31
-4.52
-4.70
-4.68
< -8.00 < -8.00
< -8.00
< -8.00 < -8.00 < -8.00
< -8.00
< -8.00
< -8.00
< -8.00
< -8.00
< -8.00
-6.72
< -8.00
< -8.00
< -8.00
< -8.00
-6.63
-7.97 < -8.00
< -8.00 < -8.00 < -8.00
< -8.00
-6.42
-5.42
-6.37
-6.33
-5.40
-4.90
-5.40
-7.62
-6.23
-5.04
-5.83
-6.26
-5.42
-4.83
-5.26
-7.81
-6.43
-5.36
-6.19
-6.33
-5.34
-4.80
-5.32
< -8.00
-6.46
-5.08
-5.89
-6.45
-5.44
-4.70
-5.58
-5.22
-5.02
-5.25
-5.62
-6.21
-5.27
-4.29
-5.12
-7.52
-6.12
-4.98
-5.86
-6.39
-5.58
-5.26
-5.51
-5.75
-5.78
-6.69
-6.69
-6.98
-6.88
-5.80
-5.71
-5.53
-5.49
-6.07
-5.81
a
b
Data obtained from NCI’s in vitro disease-oriented human tumor cells screen (see refs 3 and 4 for details). Log concentrations which
reduced cell growth to 50% of level at start of experiment. c HL-60 (TB), leukemia cell line; NCI-H266, non-small cell lung cancer cell
line; HCT-116, colon cancer cell line; SF-295, U251, CNS cancer cell lines; SK-MEL-5, melanoma cell line; OVCAR-3, ovarian cancer cell
line; 786-0, renal cancer cell line; PC-3, prostate cancer cell line; MDA-MB-435, MDA-N, breast cancer cell lines.
Ta ble 3. Total Inhibition of in Vitro Tumor Cell Growth by 2-Phenyl-1,8-naphthyridin-4(1H)-ones (29-36)a
Cytotoxicity log TGI (M)b
cancer
average
leukemia
non-small cell lung
colon
CNS
melanoma
ovarian
29
30
31
32
33
34
35
36
-5.29
-5.57
-4.79
-6.49
-5.51
-4.49
-4.57
-4.26
-6.16
-6.27
-5.33
-5.56
-5.24
-6.26
-5.65
-4.62
-4.99
-4.19
-5.80
-6.24
-5.25
-5.61
-5.60
-5.93
-5.01
-4.86
-5.26
-4.31
-4.31
-6.00
-4.62
-4.41
-4.07
-4.79
-4.78
-4.01
-4.50
-4.31
-5.58
-5.93
-4.59
> -4.00
> -4.00
-4.92
-4.51
-4.14
-4.35
-5.02
-5.72
-4.32
-4.80
-4.06
> -4.00
-4.89
-4.67
> -4.00
-4.61
-4.58
-4.09
> -4.00
-4.54
-5.30
-4.14
-4.52
-4.23
-5.51
-5.91
-5.51
-4.74
-5.71
-4.15
-4.16
-4.56
-4.89
renal
prostate
breast
-4.16
> -4.00
> -4.00
-5.42
-5.63
-6.09
a
b
Data obtained from NCI’s in vitro disease-oriented human tumor cells screen (see refs 3 and 4 for details). log molar concentrations
required to cause total growth inhibition.
compound 49. Note, moreover, that 2-(â-naphthyl)
derivative compound 49 was at least 50-fold less active
than its 2-(R-naphthyl) congener compound 44.
As in the earlier study,2 substituents in the C ring
altered substituent effects in the A ring. With a
3′-halogen, cytotoxicity was apparently unaffected by A
ring substituents, within the limited range of available
compounds. With a 3′-methyl group and with both
2-naphthyl series, cytotoxicity was unfavorably affected
by methyl groups at both positions 5 and position 7.
With a 3′-methyl group, a methyl group at position 6
was less favorable than either no substituent or a
methyl group at positions 5 or 7. In the R-naphthyl
series, a methyl substituent at position 5, 6, or 7 was
equivalent, and cytotoxicity differed little from that of
the unsubstituted compound. In the â-naphthyl series,
greater activity was observed in the compounds with
methyl groups at positions 5 or 6 than in the unsubsti-
tuted agent or that bearing a methyl group at position
7.
from more sensitive panels was arrested at a concentra-
tion approximately 1-2 log concentrations lower than
was growth of less sensitive panels. As summarized in
Table 3, all 3′-halogenated compounds, except for com-
pound 34, exhibited a highly selective effect at the TGI
level on the breast cancer panel. In the 3′-fluoro
analogs, the compound with a methyl group at position
6 (29) showed high selectivity for the colon and breast
panels and the two prostate cancer cell lines; the analog
with a methyl group at position 7 (30) demonstrated
high selectivity for the colon and breast panels; and the
compound with methyl groups at positions 5 and 7 (31)
exhibited highly selective effects on the colon and breast
panels. In the 3′-chloro compounds, substitution of a
methyl group at position 5 (32) or no substitution (33)
in ring A produced high selectivity for the two prostate
cancer cell lines and breast panel; a methyl at position
6 (34) conferred high selectivity for the CNS panel and
moderate selectivity for the colon, ovarian, and breast
panels; a methyl substituent at position 7 (35) gave
highly selective effects on the colon, CNS, and breast
panels; and methyl groups at positions 5 and 7 (36)
produced high selectivity for the CNS and breast panels
Interestingly, all 3′-halogenated compounds showed
different selectivity in the tested tumor panels at the
total growth inhibition (TGI) levels. Growth of cells

3052 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Chen et al.
as well as the provided two prostate cancer cell lines.
The reasons for this selectivity of the 3′-halogenated
derivatives and its modulation by ring A substituents
are not clear.
positions 5 and 7 abolished activity. Finally, in the 2-(â-
naphthyl) series, we found no significant difference
between methyl and chloride substituents at position
6.
All compounds that inhibited tubulin assembly with
IC50 values of 1.0 µM or less were compared with
podophyllotoxin and combretastatin A-4 for inhibitory
effects on the binding of [3H]colchicine to tubulin (data
summarized in Table 1). In these experiments inhibitor
and colchicine were equimolar and in 5-fold molar excess
over tubulin. All agents, except possibly compound 46,
significantly inhibited colchicine binding to tubulin, as
had the previously studied active members of this
group,2 but none was as potent as either podophyllotoxin
or combretastatin A-4. We believe the quantitative
differences between the assembly assay and the
[3H]colchicine assay result from differences in relative
rates of drug binding and dissociation from tubulin and
from the stoichiometric character of the [3H]colchicine
assay versus the cooperative character of the assembly
assay. However, the [3H]colchicine binding assay does
detect reduced activity in the 5,7-dimethyl derivatives
that was not apparent in either the cytotoxicity or
polymerization assays (cf. results with 31 vs 29 and 30;
with 36 vs 33-35; and with 46 vs 43-45).
(c) Su m m a r y. We found that the 3′-halogenated
compounds were highly active in both antitumor and
antitubulin assays. Substitution in ring A at different
positions produced different selective cytotoxicity in the
tumor types. The 2-(R-naphthyl)-1,8-naphthyridin-
4(1H)-ones also exhibited high activities in both assays.
However, the 2-(â-naphthyl)-1,8-naphthyridin-4(1H)-
ones showed reduced and variable activity in the anti-
tubulin polymerization assay and relatively weak cyto-
toxicity. Further structure-activity studies with
additional compounds should provide new insights into
the optimal substitution pattern in this new class of
antineoplastic agents.
(b) In ter action s of 2-Ar yl-1,8-n aph th yr idin -4(1H)-
on es w ith Tu bu lin . In our previous study2 we dem-
onstrated that the most potently cytotoxic 2-phenyl-1,8-
naphthyridin-4(1H)-ones (those with a 3′-methoxy
substituent) strongly interacted with tubulin at the
colchicine binding site. They inhibited the polymeriza-
tion of 12 µM tubulin by 50% at concentrations below
1.0 µM, as did colchicine, podophyllotoxin, and combre-
tastatin A-4, and they were potent inhibitors of radio-
labeled colchicine binding to tubulin, showing 22-35%
inhibition when present in an equimolar concentration
with [3H]colchicine. Data presented in Table 1 demon-
strate that the new agents described here, both the new
2-phenyl-1,8-naphthyridin-4(1H)-ones and the 2-naph-
thyl derivatives, also interact with tubulin, with good
correlation between their cytotoxic properties and their
relative activity as inhibitors of tubulin assembly.
Among the compounds examined here, only one
compound was inactive (51; IC50 > 40 µM), one weakly
active (47; IC50 ) 14 µM), and one moderately active
(50; IC50 ) 5.1 µM). These three compounds were
among the least cytotoxic agents, in terms of the average
log GI50 values. At the opposite extreme, eleven com-
pounds (29-31, 33-36, and 43-46) were highly potent
inhibitors of tubulin polymerization, with IC50 values
of 1.0 µM or less. This is the group that has activity
comparable to that observed with the natural products
colchicine, podophyllotoxin, and combretastatin A-4. Six
of these agents had average log GI50 values below -7,
and the remainder fell between -7 and -6. The
remaining 10 compounds (32, 37-42, 48, 49, and 52)
inhibited tubulin assembly with IC50 values of 1.1-3.3
µM. Four of these compounds (37, 38, 40, and 42) were
among the most cytotoxic agents (log GI50 ) -7), three
(32, 39, and 49) were moderately cytotoxic with log GI50
values between -7 and -6, and three (41, 48, and 52)
had little cytotoxicity (log GI50 > -6).
Considering structure-activity aspects solely from
the point of view of inhibition of tubulin polymerization,
the 3′-fluoride and 3′-chloride substituents appeared to
be equivalent, differed little in their activity from the
previously described 3′-methoxy series, and were more
active than the analogous derivatives with the 3′-methyl
substituent. With the 3′-halogen series and the 3′-
methyl series, derivatives with different methyl sub-
stituents in the A ring all had nearly equivalent activity.
In the chloride and methyl series the data suggest that
the derivatives that are unsubstituted in the A ring
(compounds 32 and 37) were less active than those
bearing methyl substituent(s).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on a Fisher-J ohn melting point apparatus and
are uncorrected. Elemental analyses were performed on a
Carlo Erba EA 1108 elemental analyzer. 1H NMR spectra
were measured on a Bruker AC-300 spectrometer with TMS
as internal standard. Chemical shifts are reported in δ (ppm).
Mass spectra (MS) were obtained on a TRIO 1000 mass
spectrometer EI. Flash column chromatography was per-
formed on silica gel (mesh 25-150 µm) using a mixture of
CH2Cl2 and EtOAc as eluant. Precoated silica gel plates
(Kieselgel 60 F254 0.25 mm, Merck) were used for TLC analysis.
All substituted 2-aminopyridines were purchased from Aldrich
Chemical Company.
P r ep a r a tion of Su bstitu ted Eth yl Ben zoyla ceta tes.
The substituted ethyl benzoylacetates (2) were prepared
according to procedures described by Krapcho et al.12 To a
vigorously stirring suspension of NaH and CO(OEt)2 (3) in
toluene was added dropwise a solution of substituted ac-
etophenone (4) in toluene under reflux. The mixture was
allowed to reflux and was stirred for 20 min after the addition
was complete. When cooled to room temperature, the mixture
was acidified with glacial HOAc. After ice-cold water was
added, the mixture was extracted with toluene. The extract
was then dried over MgSO4. After the toluene was evaporated
at atmospheric pressure, the residue was distilled in vacuo to
give the corresponding substituted ethyl benzoylacetates.
P r oced u r es for P r ep a r a tion of 2-Ar ylp yr id o[1,2-a ]p y-
r im idin -4-on es (5-28) an d 2-Ar yl-1,8-n aph th yr idin -4(1H)-
on es (29-52).11 A mixture of substituted 2-aminopyridine (1),
The 2-(R-naphthyl) derivatives (compounds 42-46)
were comparable in activity to the 3′-halogen deriva-
tives. In this series, too, substituents in the A ring had
little effect on inhibitory activity. It was only in the less
active 2-(â-naphthyl) series (compounds 47-52) that A
ring substitution affected the apparent interaction with
tubulin. A methyl substituent at either position 5 (48)
or 6 (49) decreased the IC50 value 7-fold relative to the
value obtained with the unsubstituted compound 47,
while a methyl substituent at position 7 decreased the
IC50 value 3-fold. In contrast, two methyl groups at

Antitumor Agents 178
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3053
substituted ethyl benzoylacetate (2), and polyphosphoric acid
was heated at 125 °C with stirring. The reaction was
monitored by TLC. After the reaction was complete, the
mixture was cooled to room temperature and neutralized with
4 M NaOH. After extraction with CH2Cl2, the extract was
passed through a silica gel column to give the 2-arylpyrido[1,2-
a]pyrimidin-4-one. The 2-arylpyrido[1,2-a]pyrimidin-4-one
was added to liquid paraffin at 350 °C with stirring. The oil
was maintained at 350 °C for 2 h after the addition was
complete. The cooled mixture was subjected to silica gel
column chromatography, and elution with CH2Cl2-EtOAc
gave the corresponding 2-aryl-1,8-naphthyridin-4(1H)-one.
2-(3′-F lu or op h en yl)-7-m eth ylp yr id o[1,2-a ]p yr im id in -4-
on e (5) was obtained from ethyl (3′-fluorobenzoyl)acetate and
2-amino-5-picoline: amorphous, mp 163-165 °C; 1H NMR
(CDCl3) δ 8.90 (s, 1 H, H-6), 7.85 (d, J ) 8.5 Hz, 1 H, H-6′),
7.83 (s, 1 H, H-2′), 7.71 (d, J ) 9.0 Hz, 1 H, H-9), 7.65 (dd, J
) 9.0, 1.5 Hz, 1 H, H-8), 7.48 (m, 1 H, H-5′), 7.19 (dt, J ) 2.0,
8.5 Hz, 1 H, H-4′), 6.88 (s, 1 H, H-3), 2.46 (s, 3 H, CH3-7); MS
m/z 254 (M+). Anal. C, H, N.
2-(3′-F lu or op h en yl)-6-m eth ylp yr id o[1,2-a ]p yr im id in -4-
on e (6) was obtained from ethyl (3′-fluorobenzoyl)acetate and
2-amino-6-picoline: amorphous, mp 145-146 °C; 1H NMR
(CDCl3) δ 7.82 (d, J ) 7.5, Hz, 1 H, H-6′), 7.80 (d, J ) 1.5, Hz,
1 H, H-2′), 7.51 (d, J ) 7.5 Hz, 1 H, H-9), 7.48 (m, 1 H, H-5′),
7.45 (dd, J ) 7.5, 6.0 Hz, 1 H, H-8), 7.18 (dt, J ) 2.5, 8.0 Hz,
1 H, H-4′), 6.72 (s, 1 H, H-3), 6.69 (d, J ) 6.0 Hz, 1 H, H-7),
3.10 (s, 3 H, CH3-6); MS m/z 254 (M+). Anal. C, H, N.
2-(3′-F lu or op h en yl)-6,8-d im eth ylp yr id o[1,2-a ]p yr im i-
d in -4-on e (7) was obtained from ethyl (3′-fluorobenzoyl)-
acetate and 2-amino-4,6-dimethylpyridine: amorphous, mp
162-164 °C; 1H NMR (CDCl3) δ 7.80 (d, J ) 8.0 Hz, 1 H, H-6′),
7.79 (d, J ) 1.0 Hz, 1 H, H-2′), 7.45 (m, 1 H, H-5′), 7.30 (br s,
1 H, H-9), 7.17 (dt, J ) 2.5, 8.0 Hz, 1 H, H-4′), 6.64 (s, 1 H,
H-3), 6.53 (br s, 1 H, H-7), 3.07 (s, 3 H, CH3-6), 2.37 (s, 3 H,
CH3-8); MS m/z 268 (M+). Anal. C, H, N.
nopyridine: needles, mp 118-120 °C; 1H NMR (CDCl3) δ 9.09
(d, J ) 7.0 Hz, 1 H, H-6), 7.94 (s, 1 H, H-2′), 7.87 (d, J ) 7.5,
Hz, 1 H, H-6′), 7.76 (d, J ) 3.5 Hz, 2 H, H2-8, 9), 7.41 (t, J )
7.5 Hz, 1 H, H-5′), 7.32 (d, J ) 7.5 Hz, 1 H, H-4′), 7.15 (ddd,
J ) 7.0, 3.5, 1.5 Hz, 1 H, H-7), 6.92 (s, 1 H, H-3), 2.47 (s, 3 H,
CH3-3′); MS m/z 236 (M+). Anal. C, H, N.
2-(3′-Meth ylp h en yl)-8-m eth ylp yr id o[1,2-a ]p yr im id in -
4-on e (14) was obtained from ethyl (3′-methylbenzoyl)acetate
and 2-amino-4-picoline: amorphous, mp 146-148 °C; 1H NMR
(CDCl3) δ 8.97 (d, J ) 7.2 Hz, 1 H, H-6), 7.92 (s, 1 H, H-2′),
7.86 (d, J ) 7.5, 1 H, H-6′), 7.54 (s, 1 H, H-9), 7.40 (t, J ) 7.5
Hz, 1 H, H-5′), 7.31 (d, J ) 7.5, Hz, 1 H, H-4′), 6.98 (d, J ) 7.2
Hz, 1 H, H-7), 6.85 (s, 1H, H-3), 2.51 (s, 3 H, CH3-8), 2.45 (s,
3 H, CH3-3′); MS m/z 250 (M+). Anal. C, H, N.
2-(3′-Meth ylp h en yl)-7-m eth ylp yr id o[1,2-a ]p yr im id in -
4-on e (15) was obtained from ethyl (3′-methylbenzoyl)acetate
and 2-amino-5-picoline: prisms, mp 158-160 °C; 1H NMR
(CDCl3) δ 8.90 (s, 1 H, H-6), 7.92 (s, 1 H, H-2′), 7.86 (d, J )
7.7, 1 H, H-6′), 7.69 (d, J ) 9.0 Hz, 1 H, H-9), 7.62 (dd, J )
9.0, 2.0 Hz, 1 H, H-8), 7.40 (t, J ) 7.7, Hz, 1 H, H-5′), 7.31 (d,
J ) 7.7, Hz, 1 H, H-4′), 6.90 (s, 1 H, H-3), 2.47 (s, 3 H, CH3-7),
2.46 (s, 3 H, CH3-3′); MS m/z 250(M+). Anal. C, H, N.
2-(3′-Meth ylp h en yl)-6-m eth ylp yr id o[1,2-a ]p yr im id in -
4-on e (16) was obtained from ethyl (3′-methylbenzoyl)acetate
and 2-amino-6-picoline: prisms, mp 152-154 °C; 1H NMR
(CDCl3) δ 7.90 (s, 1 H, H-2′), 7.84 (d, J ) 7.7, 1 H, H-6′), 7.51
(d, J ) 8.5, Hz, 1 H, H-9), 7.46 (dd, J ) 8.5, 6.4 Hz, 1 H, H-8),
7.39 (t, J ) 7.5 Hz, 1 H, H-5′), 7.30 (d, J ) 7.7, Hz, 1 H, H-4′),
6.73 (s, 1 H, H-3), 6.66 (d, J ) 6.4 Hz, 1 H, H-7), 3.09 (s, 3 H,
CH3-6), 2.46 (s, 3 H, CH3-3′); MS m/z 250(M+). Anal. C, H,
N.
2-(3′-Meth ylp h en yl)-6,8-d im eth ylp yr id o[1,2-a ]p yr im i-
d in -4-on e (17) was obtained from ethyl (3′-methylbenzoyl)-
acetate and 2-amino-4,6-dimethylpyridine: prisms, mp 145-
1
147 °C; H NMR (CDCl3) δ 7.88 (br s, 1 H, H-2′), 7.82 (d, J )
7.5, Hz, 1 H, H-6′), 7.38 (t, J ) 7.5 Hz, 1 H, H-5′), 7.35 (br s,
1 H, H-9), 7.28 (d, J ) 7.5, Hz, 1 H, H-4′), 6.66 (s, 1 H, H-3),
6.50 (br s, 1 H, H-7), 3.07 (s, 3 H, CH3-6), 2.45 (s, 3 H, CH3-3′),
2.36 (s, 3 H, CH3-8); MS m/z 264 (M+). Anal. C, H, N.
2-(3′-Ch lor oph en yl)pyr ido[1,2-a ]pyr im idin -4-on e (8) was
obtained from ethyl (3′-chlorobenzoyl)acetate and 2-amino-
pyridine: amorphous, mp 160-161 °C; 1H NMR (CDCl3) δ 9.08
(d, J ) 7.2 Hz, 1 H, H-6), 8.15 (t, J ) 1.5 Hz, 1 H, H-2′), 7.94
(td, J ) 7.0, 1.5 Hz, 1 H, H-6′), 7.79 (m, 2 H, H2-8, 9), 7.46 (m,
2 H, H2-4′, 5′), 7.17 (dt, J ) 6.5, 1.5 Hz, 1 H, H-7), 6.90 (s, 1 H,
H-3); MS m/z 256 (M+). Anal. C, H, N.
2-(r-Na p h th yl)p yr id o[1,2-a ]p yr im id in -4-on e (18) was
obtained from ethyl (R-naphthylbenzoyl)acetate and 2-amino-
pyridine: amorphous, mp 169-170 °C; 1H NMR (CDCl3) δ 9.19
(d, J ) 7.0 Hz, 1 H, H-6), 8.24 (dd, J ) 6.5, 2.5 Hz, 1 H, H-8′),
7.97 (d, J ) 8.5 Hz, 1 H, H-2′), 7.95 (m, 1 H, H-5′), 7.81 (d, J
) 2.5 Hz, 2 H, H2-8, 9), 7.72 (d, J ) 6.5 Hz, 1 H, H-4′), 7.58
(dd, J ) 8.5, 6.5 Hz, 1 H, H-3′), 7.52 (m, 2 H, H2-6′, 7′), 7.24
(m, 1 H, H-7), 6.80 (s, 1 H, H-3); MS m/z 272 (M+). Anal. C,
H, N.
2-(r-Na p h t h yl)-8-m e t h ylp yr id o[1,2-a ]p yr im id in -4-
on e (19) was obtained from ethyl (R-naphthylbenzoyl)acetate
and 2-amino-4-picoline: needles, mp 146-148 °C; 1H NMR
(CDCl3) δ 9.08 (d, J ) 7.2 Hz, 1 H, H-6), 8.24 (dd, J ) 6.5, 2.5
Hz, 1 H, H-8′), 7.96 (d, J ) 8.7 Hz, 1 H, H-2′), 7.93 (m, 1 H,
H-5′), 7.71 (d, J ) 6.8 Hz, 1 H, H-4′), 7.62 (br s, 1 H, H-9),
7.58 (dd, J ) 8.7, 6.8 Hz, 1 H, H-3′), 7.53 (m, 2 H, H2-6′, 7′),
7.08 (dd, J ) 7.2, 1.5 Hz, 1 H, H-7), 6.72 (s, 1H, H-3), 2.54 (s,
3 H, CH3-8); MS m/z 286 (M+). Anal. C, H, N.
2-(r-Na p h t h yl)-7-m e t h ylp yr id o[1,2-a ]p yr im id in -4-
on e (20) was obtained from ethyl (R-naphthylbenzoyl)acetate
and 2-amino-5-picoline: amorphous, mp 138-140 °C; 1H NMR
(CDCl3) δ 8.99 (s, 1 H, H-6), 8.24 (dd, J ) 6.5, 2.5 Hz, 1 H,
H-8′), 7.96 (d, J ) 8.7 Hz, 1 H, H-2′), 7.93 (m, 1 H, H-5′), 7.72
(d, J ) 7.0 Hz, 1 H, H-4′), 7.70 (d, J ) 9.0 Hz, 1 H, H-9), 7.66
(dd, J ) 9.0, 1.5 Hz, 1 H, H-8), 7.56 (dd, J ) 8.7, 6.8 Hz, 1 H,
H-3′), 7.50 (m, 2 H, H2-6′, 7′), 6.77 (s, 1 H, H-3), 2.49 (s, 3 H,
CH3-7); MS m/z 286(M+). Anal. C, H, N.
2-(r-Na p h t h yl)-6-m e t h ylp yr id o[1,2-a ]p yr im id in -4-
on e (21) was obtained from ethyl (R-naphthylbenzoyl)acetate
and 2-amino-6-picoline: prisms, mp 149-150 °C; 1H NMR
(CDCl3) δ 8.28 (dd, J ) 5.0, 2.5 Hz, 1 H, H-8′), 7.95 (d, J ) 8.3
Hz, 1 H, H-2′), 7.92 (m, 1 H, H-5′), 7.71 (d, J ) 6.5 Hz, 1 H,
H-4′), 7.56 (dd, J ) 8.7, 6.8 Hz, 1 H, H-3′), 7.52 (m, 2 H, H2-8,
9), 7.50 (m, 2 H, H2-6′, 7′), 6.74 (d, J ) 5.5 Hz, 1 H, H-7), 6.61
(s, 1 H, H-3), 3.16 (s, 3 H, CH3-6); MS m/z 286 (M+). Anal. C,
H, N.
2-(3′-Ch lor op h en yl)-8-m et h ylp yr id o[1,2-a ]p yr im id in -
4-on e (9) was obtained from ethyl (3′-chlorobenzoyl)acetate
and 2-amino-4-picoline: prisms, mp 142-144 °C; 1H NMR
(CDCl3) δ 8.97 (d, J ) 7.2 Hz, 1 H, H-6), 8.13 (t, J ) 2.0, 1 H,
H-2′), 7.92 (td, J ) 7.5, 2.0 Hz, 1 H, H-6′), 7.54 (br s, 1 H,
H-9), 7.45 (m, 2 H, H2-4′, 5′), 7.00 (dd, J ) 7.2, 2.0 Hz, 1 H,
H-7), 2.52 (s, 3 H, CH3-8); MS m/z 270 (M+). Anal. C, H, N.
2-(3′-Ch lor op h en yl)-7-m et h ylp yr id o[1,2-a ]p yr im id in -
4-on e (10) was obtained from ethyl (3′-chlorobenzoyl)acetate
and 2-amino-5-picoline: prisms, mp 175-176 °C; 1H NMR
(CDCl3) δ 8.89 (s, 1 H, H-6), 8.13 (s, 1 H, H-2′), 7.92 (td, J )
7.0, 1.0 Hz, 1 H, H-6′), 7.69 (d, J ) 9.0 Hz, 1 H, H-9), 7.64 (dd,
J ) 9.0, 1.5 Hz, 1 H, H-8), 7.45 (m, 2 H, H2-4′, 5′), 6.86 (s, 1 H,
H-3), 2.46 (s, 3 H, CH3-7); MS m/z 270 (M+). Anal. C, H, N.
2-(3′-Ch lor op h en yl)-6-m et h ylp yr id o[1,2-a ]p yr im id in -
4-on e (11) was obtained from ethyl (3′-chlorobenzoyl)acetate
and 2-amino-6-picoline: needles, mp 150-152 °C; 1H NMR
(CDCl3) δ 8.10 (t, J ) 1.5 Hz, 1 H, H-2′), 7.91 (td, J ) 8.0, 1.5
Hz, 1 H, H-6′), 7.52 (d, J ) 7.0 Hz, 1 H, H-9), 7.48 (dd, J )
8.0, 7.0 Hz, 1 H, H-8), 7.45 (m, 2 H, H2-4′, 5′), 6.70 (s, 1 H,
H-3), 6.68 (d, J ) 8.0 Hz, 1 H, H-7), 3.10 (s, 3 H, CH3-6); MS
m/z 270 (M+). Anal. C, H, N.
2-(3′-Ch lor op h en yl)-6,8-d im eth ylp yr id o[1,2-a ]p yr im i-
d in -4-on e (12) was obtained from ethyl (3′-chlorobenzoyl)-
acetate and 2-amino-4,6-dimethylpyridine: prisms, mp 125-
126 °C; 1H NMR (CDCl3) δ 8.09 (t, J ) 2.0 Hz, 1 H, H-2′), 7.89
(dd, J ) 6.7, 2.0 Hz, 1 H, H-6′), 7.43 (m, 2 H, H2-4′, 5′), 7.31
(br s, 1 H, H-9), 6.63 (s, 1 H, H-3), 6.53 (br s, 1 H, H-7), 3.07
(s, 3 H, CH3-6), 2.37 (s, 3 H, CH3-8); MS m/z 284 (M+). Anal.
C, H, N.
2-(3′-Meth ylp h en yl)p yr id o[1,2-a ]p yr im id in -4-on e (13)
was obtained from ethyl (3′-methylbenzoyl)acetate and 2-ami-

3054 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Chen et al.
2-(r-Na p h th yl)-6,8-d im eth ylp yr id o[1,2-a ]p yr im id in -4-
on e (22) was obtained from ethyl (R-naphthylbenzoyl)acetate
and 2-amino-4,6-dimethylpyridine: prisms, mp 160-162 °C;
1H NMR (CDCl3) δ 8.27 (dd, J ) 5.0, 2.5 Hz, 1 H, H-8′), 7.94
(d, J ) 7.5 Hz, 1 H, H-2′), 7.92 (m, 1 H, H-5′), 7.70 (d, J ) 7.5
Hz, 1 H, H-4′), 7.54 (t, J ) 7.5 Hz, 1 H, H-3′), 7.51 (m, 2 H,
H2-6′, 7′), 7.35 (br s, 1 H, H-9), 6.60 (br s, 1 H, H-7), 6.53 (s, 1
H, H-3), 3.13 (s, 3 H, CH3-6), 2.39 (s, 3 H, CH3-8); MS m/z 300
(M+). Anal. C, H, N.
2-(â-Na p h t h yl)p yr id o[1,2-a ]p yr im id in -4-on e (23) was
obtained from ethyl (â-naphthylbenzoyl)acetate and 2-amino-
pyridine: amorphous, mp 201-202 °C; 1H NMR (CDCl3) δ 9.11
(d, J ) 7.0 Hz, 1 H, H-6), 8.68 (s, 1 H, H-1′), 8.18 (dd, J ) 8.5,
1.0 Hz, 1 H, H-3′), 7.99 (m, 1 H, H-8′), 7.98 (d, J ) 8.5 Hz, 1
H, H-4′), 7.91 (m, 1 H, H-5′), 7.81 (m, 2 H, H2-8, 9), 7.57 (m, 2
H, H2-6′, 7′), 7.17 (dt, J ) 7.0, 2.0 Hz, 1 H, H-7), 7.08 (s, 1 H,
H-3); MS m/z 272 (M+). Anal. C, H, N.
2-(â-Na p h t h yl)-8-m e t h ylp yr id o[1,2-a ]p yr im id in -4-
on e (24) was obtained from ethyl (â-naphthylbenzoyl)acetate
and 2-amino-4-picoline: amorphous, mp 168-169 °C; 1H NMR
(CDCl3) δ 9.00 (d, J ) 7.2 Hz, 1 H, H-6), 8.66 (s, 1 H, H-1′),
8.16 (d, J ) 8.5 Hz, 1 H, H-3′), 7.99 (s, 1 H, H-9), 7.98 (m, 1 H,
H-8′), 7.97 (d, J ) 8.5 Hz, 1 H, H-4′), 7.90 (br d, J ) 7.0 Hz, 1
H, H-5′), 7.57 (m, 2 H, H2-6′, 7′), 7.01 (s, 1H, H-3), 6.99 (d, J
) 7.2 Hz, 1 H, H-7), 2.54 (s, 3 H, CH3-8); MS m/z 286 (M+).
Anal. C, H, N.
2-(â-Na p h t h yl)-7-m e t h ylp yr id o[1,2-a ]p yr im id in -4-
on e (25) was obtained from ethyl (â-naphthylbenzoyl)acetate
and 2-amino-5-picoline: needles, mp 228-229 °C; 1H NMR
(CDCl3) δ 8.93 (s, 1 H, H-6), 8.66 (s, 1 H, H-1′), 8.17 (dd, J )
8.7, 1.0 Hz, 1 H, H-3′), 7.99 (m, 1 H, H-8′), 7.97 (d, J ) 8.7 Hz,
1 H, H-4′), 7.90 (m, 1 H, H-5′), 7.75 (d, J ) 9.0 Hz, 1 H, H-9),
7.65 (dd, J ) 9.0, 1.2 Hz, 1 H, H-8), 7.56 (m, 2 H, H2-6′, 7′),
7.06 (s, 1 H, H-3), 2.47 (s, 3 H, CH3-7); MS m/z 286(M+). Anal.
C, H, N.
2-(â-Na p h t h yl)-6-m e t h ylp yr id o[1,2-a ]p yr im id in -4-
on e (26) was obtained from ethyl (â-naphthylbenzoyl)acetate
and 2-amino-6-picoline: prisms, mp 160-162 °C; 1H NMR
(CDCl3) δ 8.64 (s, 1 H, H-1′), 8.14 (d, J ) 8.6 Hz, 1 H, H-3′),
7.99 (m, 1 H, H-8′), 7.96 (d, J ) 8.6 Hz, 1 H, H-4′), 7.90 (m, 1
H, H-5′), 7.55 (m, 2 H, H2-6′, 7′), 7.53 (d, J ) 8.8 Hz, 1 H, H-9),
7.49 (dd, J ) 8.8, 6.5 Hz, 1 H, H-8), 6.88 (s, 1 H, H-3), 6.68 (d,
J ) 6.5 Hz, 1 H, H-7), 3.12 (s, 3 H, CH3-6); MS m/z 286 (M+).
Anal. C, H, N.
2-(â-Na p h th yl)-6,8-d im eth ylp yr id o[1,2-a ]p yr im id in -4-
on e (27) was obtained from ethyl (â-naphthylbenzoyl)acetate
and 2-amino-4,6-dimethylpyridine: needles, mp 189-190 °C;
1H NMR (CDCl3) δ 8.62 (s, 1 H, H-1′), 8.12 (d, J ) 8.9 Hz, 1 H,
H-3′), 7.98 (m, 1 H, H-8′), 7.94 (d, J ) 8.9 Hz, 1 H, H-4′), 7.89
(m, 1 H, H-5′), 7.55 (m, 2 H, H2-6′, 7′), 7.36 (br s, 1 H, H-9),
6.81 (s, 1 H, H-3), 6.53 (br s, 1 H, H-7), 3.09 (s, 3 H, CH3-6),
2.38 (s, 3 H, CH3-8); MS m/z 300 (M+). Anal. C, H, N.
2-(â-Na p h t h yl)-7-ch lor o-p yr id o[1,2-a ]p yr im id in -4-
on e (28) was obtained from ethyl (â-naphthylbenzoyl)acetate
and 2-amino-5-chloro-pyridine: amorphous, mp 227-229 °C;
1H NMR (CDCl3) δ 9.12 (d, J ) 2.0 Hz, 1 H, H-6), 8.66 (s, 1 H,
H-1′), 8.15 (dd, J ) 8.7, 1.5 Hz, 1 H, H-3′), 8.00 (m, 1 H, H-8′),
7.97 (d, J ) 8.7 Hz, 1 H, H-4′), 7.90 (m, 1 H, H-5′), 7.75 (d, J
) 9.3 Hz, 1 H, H-9), 7.70 (dd, J ) 9.3, 2.0 Hz, 1 H, H-8), 7.57
(m, 2 H, H2-6′, 7′), 7.09 (s, 1 H, H-3); MS m/z 306(M+). Anal.
C, H, N.
5′), 7.39 (dd, J ) 9.3, 1.8 Hz, 1 H, H-6′), 7.24 (m, 1 H, H-4′),
6.92 (s, 1 H, H-6), 6.49 (s, 1 H, H-3), 2.93 (s, 3 H, CH3-5), 2.51
(s, 3 H, CH3-7); MS m/z 268 (M+). Anal. C, H, N.
2-(3′-Ch lor oph en yl)-1,8-n aph th yr idin -4(1H)-on e (32) was
1
obtained from compound 8: needles; H NMR (CDCl3) δ 8.66
(dd, J ) 4.5, 2.0 Hz, 1 H, H-5), 8.63 (dd, J ) 8.0, 2.0 Hz, 1 H,
H-7), 7.72 (t, J ) 1.5 Hz, 1 H, H-2′), 7.61 (td, J ) 7.0, 1.5 Hz,
1 H, H-6′), 7.48 (m, 2 H, H2-4′, 5′), 7.36 (dd, J ) 8.0, 4.5 Hz, 1
H, H-6), 6.58 (s, 1 H, H-3); MS m/z 256 (M+). Anal. C, H, N.
2-(3′-Ch lor op h en yl)-5-m eth yl-1,8-n a p h th yr id in -4(1H)-
1
on e (33) was obtained from compound 9: needles; H NMR
(CDCl3 + CD3OD) δ 8.37 (d, J ) 5.0 Hz, 1 H, H-7), 7.70 (t, J
) 2.0 Hz, 1 H, H-2′), 7.59 (td, J ) 7.2, 2.0 Hz, 1 H, H-6′), 7.46
(m, 2 H, H2-4′, 5′), 7.02 (d, J ) 5.0 Hz, 1 H, H-6), 6.49 (s, 1 H,
H-3), 2.91 (s, 3 H, CH3-5); MS m/z 270 (M+). Anal. C, H, N.
2-(3′-Ch lor op h en yl)-6-m eth yl-1,8-n a p h th yr id in -4(1H)-
on e (34) was obtained from compound 10: amorphous; 1H
NMR (CDCl3 + CD3OD) δ 8.49 (d, J ) 2.0 Hz, 1 H, H-5), 8.42
(d, J ) 2.0 Hz, 1 H, H-7), 7.72 (t, J ) 1.5 Hz, 1 H, H-2′), 7.61
(td, J ) 7.2, 1.5 Hz, 1 H, H-6′), 7.48 (m, 2 H, H2-4′, 5′), 6.57 (s,
1 H, H-3), 2.46 (s, 3 H, CH3-6); MS m/z 270 (M+). Anal. C, H,
N.
2-(3′-Ch lor op h en yl)-7-m eth yl-1,8-n a p h th yr id in -4(1H)-
on e (35) was obtained from compound 11: amorphous; 1H
NMR (CDCl3 + CD3OD) δ 8.54 (d, J ) 8.0 Hz, 1 H, H-5), 7.72
(t, J ) 2.0 Hz, 1 H, H-2′), 7.61 (td, J ) 6.8, 2.0 Hz, 1 H, H-6′),
7.50 (m, 2 H, H2-4′, 5′), 7.23 (d, J ) 8.0 Hz, 1 H, H-6), 2.64 (s,
3 H, CH3-7); MS m/z 270 (M+). Anal. C, H, N.
2-(3′-Ch lor op h e n yl)-5,7-d im e t h yl-1,8-n a p h t h yr id in -
4(1H)-on e (36) was obtained from compound 12: needles; 1H
NMR (CDCl3) δ 7.67 (t, J ) 1.5 Hz, 1 H, H-2′), 7.58 (td, J )
7.0, 1.5 Hz, 1 H, H-6′), 7.50 (m, 2 H, H2-4′, 5′), 6.93 (s, 1 H,
H-6), 6.49 (s, 1 H, H-3), 2.94 (s, 3 H, CH3-5), 2.53 (s, 3 H, CH3-
7); MS m/z 284 (M+). Anal. C, H, N.
2-(3′-Met h ylp h en yl)-1,8-n a p h t h yr id in -4(1H)-on e (37)
was obtained from compound 13: prisms; 1H NMR (CDCl3) δ
10.57, (br s, 1 H, NH-1), 8.70 (dd, J ) 8.0, 2.0 Hz, 1 H, H-5),
8.18 (dd, J ) 4.5, 2.0 Hz, 1 H, H-7), 7.54 (d, J ) 1.5 Hz, 1 H,
H-2′), 7.53 (d, J ) 7.5 Hz, 1 H, H-6′), 7.45 (t, J ) 7.5, Hz, 1 H,
H-5′), 7.40 (d, J ) 7.5 Hz, 1 H, H-4′), 7.27 (dd, J ) 8.0, 4.5 Hz,
1 H, H-6), 6.60 (s, 1 H, H-3), 2.44 (s, 3 H, CH3-3′); MS m/z 236
(M+). Anal. C, H, N.
2-(3′-Meth ylp h en yl)-5-m eth yl-1,8-n a p h th yr id in -4(1H)-
on e (38) was obtained from compound 14: needles; 1H NMR
(CDCl3) δ 9.77, (br s, 1 H, NH-1), 8.10 (d, J ) 4.8 Hz, 1 H,
H-7), 7.52 (s, 1 H, H-2′), 7.50 (br d, J ) 6.0 Hz, 1 H, H-6′),
7.43 (t, J ) 7.5, Hz, 1 H, H-5′), 7.37 (d, J ) 7.5 Hz, 1 H, H-4′),
6.98 (d, J ) 4.8 Hz, 1 H, H-6), 6.52 (s, 1 H, H-3), 2.98 (s, 3 H,
CH3-5), 2.45 (s, 3 H, CH3-3′); MS m/z 250 (M+). Anal. C, H,
N.
2-(3′-Meth ylp h en yl)-6-m eth yl-1,8-n a p h th yr id in -4(1H)-
1
on e (39) was obtained from compound 15: prisms; H NMR
(CDCl3) δ 10.78, (s, 1 H, NH-1), 8.50 (d, J ) 2.0 Hz, 1 H, H-5),
7.92 (d, J ) 2.0 Hz, 1 H, H-7), 7.52 (br s, 2 H, H2-2′, 6′), 7.45
(t, J ) 7.5, Hz, 1 H, H-5′), 7.40 (d, J ) 7.5 Hz, 1 H, H-4′), 6.57
(s, 1 H, H-3), 2.43 (s, 3 H, CH3-3′), 2.38 (s, 3 H, CH3-6); MS
m/z 250 (M+). Anal. C, H, N.
2-(3′-Meth ylp h en yl)-7-m eth yl-1,8-n a p h th yr id in -4(1H)-
on e (40) was obtained from compound 16: amorphous; 1H
NMR (CDCl3) δ 9.23, (br s, 1 H, NH-1), 8.57 (d, J ) 8.0 Hz, 1
H, H-5), 7.49 (br s, 2 H, H2-2′, 6′), 7.42 (t, J ) 7.2, Hz, 1 H,
H-5′), 7.36 (d, J ) 7.2 Hz, 1 H, H-4′), 7.20 (d, J ) 8.0 Hz, 1 H,
H-6), 6.60 (s, 1 H, H-3), 2.58 (s, 3 H, CH3-7), 2.43 (s, 3 H, CH3-
3′); MS m/z 250 (M+). Anal. C, H, N.
2-(3′-Met h ylp h en yl)-5,7-d im et h yl-1,8-n a p h t h yr id in -
4(1H)-on e (41) was obtained from compound 17: amorphous;
1H NMR (CDCl3) δ 9.14, (br s, 1 H, NH-1), 7.47 (br s, 2 H,
H2-2′, 6′), 7.40 (t, J ) 7.5, Hz, 1 H, H-5′), 7.34 (d, J ) 7.5 Hz,
1 H, H-4′), 6.89 (s, 1 H, H-6), 6.50 (s, 1 H, H-3), 2.94 (s, 3 H,
CH3-5), 2.42 (s, 3 H, CH3-7), 2.47 (s, 3 H, CH3-3′); MS m/z 264
(M+). Anal. C, H, N.
2-(r-Na p h th yl)-1,8-n a p h th yr id in -4(1H)-on e (42) was ob-
tained from compound 18: plates; 1H NMR (CDCl3) δ 8.67 (dd,
J ) 8.0, 1.5 Hz, 1 H, H-5), 8.08 (d, J ) 8.0 Hz, 1 H, H-2′), 7.99
(d, J ) 8.0 Hz, 1 H, H-8′), 7.97 (d, J ) 8.0 Hz, 1 H, H-5′),
2-(3′-F lu or op h en yl)-6-m eth yl-1,8-n a p h th yr id in -4(1H)-
1
on e (29) was obtained from compound 5: needles; H NMR
(CDCl3 + CD3OD) δ 8.48 (d, J ) 2.0 Hz, 1 H, H-5), 8.47 (d, J
) 2.0 Hz, 1 H, H-7), 7.52 (m, 2 H, H2-2′, 5′), 7.45 (dd, J ) 8.0,
2.0 Hz, 1 H, H-6′), 7.25 (m, 1 H, H-4′), 6.57 (s, 1 H, H-3), 2.47
(s, 3 H, CH3-6); MS m/z 254 (M+). Anal. C, H, N.
2-(3′-F lu or op h en yl)-7-m eth yl-1,8-n a p h th yr id in -4(1H)-
on e (30) was obtained from compound 6: amorphous; 1H NMR
(CDCl3) δ 8.90, (br s, 1 H, NH-1), 8.56 (d, J ) 8.0 Hz, 1 H,
H-5), 7.52 (m, 2 H, H2-2′, 5′), 7.41 (d, J ) 9.5 Hz, 1 H, H-6′),
7.29 (m, 1 H, H-4′), 7.23 (d, J ) 8.0 Hz, 1 H, H-6), 6.57 (s, 1 H,
H-3), 2.64 (s, 3 H, CH3-7); MS m/z 254 (M+). Anal. C, H, N.
2-(3′-F lu or op h e n yl)-5,7-d im e t h yl-1,8-n a p h t h yr id in -
4(1H)-on e (31) was obtained from compound 7: amorphous;
1H NMR (CDCl3) δ 8.86, (br s, 1 H, NH-1), 7.50 (m, 2 H, H2-2′,

Antitumor Agents 178
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19 3055
7.68 (d, J ) 8.0 Hz, 1 H, H-4′), 7.62 (t, J ) 8.0 Hz, 1 H, H-3′),
7.57 (d, J ) 8.0 Hz, 1 H, H-6′), 7.52 (d, J ) 8.0 Hz, 1 H, H-7′),
7.49 (d, J ) 4.5 Hz, 1 H, H-7), 7.11 (dd, J ) 8.0, 4.5 Hz, 1 H,
H-6), 6.56 (s, 1 H, H-3); MS m/z 272 (M+). Anal. C, H, N.
2-(r-N a p h t h y l)-5-m e t h y l-1,8-n a p h t h y r id in -4(1H )-
on e (43) was obtained from compound 19: amorphous; 1H
NMR (CDCl3) δ 11.37, (br s, 1 H, NH-1), 8.06 (d, J ) 7.8 Hz,
1 H, H-2′), 7.97 (d, J ) 8.5 Hz, 2 H, H2-5′, 8′), 7.65 (br d, J )
6.5 Hz, 1 H, H-4′), 7.62 (t, J ) 7.8 Hz, 1 H, H-3′), 7.56 (d, J )
7.5 Hz, 1 H, H-6′), 7.48 (d, J ) 7.5 Hz, 1 H, H-7′), 7.00 (d, J )
4.8 Hz, 1 H, H-7), 6.71 (d, J ) 4.8 Hz, 1 H, H-6), 6.46 (s, 1 H,
H-3), 2.93 (s, 3 H, CH3-5); MS m/z 286 (M+). Anal. C, H, N.
2-(r-N a p h t h y l)-6-m e t h y l-1,8-n a p h t h y r id in -4(1H )-
on e (44) was obtained from compound 20: amorphous; 1H
NMR (CDCl3) δ 8.39 (d, J ) 1.0 Hz, 1 H, H-5), 8.10 (dd, J )
7.5, 1.5 Hz, 1 H, H-2′), 7.99 (d, J ) 8.0 Hz, 1 H, H-8′), 7.88 (d,
J ) 8.0 Hz, 1 H, H-5′), 7.66 (br d, J ) 7.0 Hz, 1 H, H-4′), 7.65
(d, J ) 1.0 Hz, 1 H, H-7), 7.63 (t, J ) 7.5 Hz, 1 H, H-3′), 7.54
(t, J ) 7.5 Hz, 1 H, H-6′), 7.44 (t, J ) 7.5 Hz, 1 H, H-7′), 6.50
(s, 1 H, H-3), 2.12 (s, 3 H, CH3-6); MS m/z 286 (M+). Anal. C,
H, N.
2-(r-N a p h t h y l)-7-m e t h y l-1,8-n a p h t h y r id in -4(1H )-
on e (45) was obtained from compound 21: amorphous; 1H
NMR (CDCl3) δ 10.78, (br s, 1 H, NH-1), 8.48 (d, J ) 8.0 Hz,
1 H, H-5), 7.97(d, J ) 8.0 Hz, 1 H, H-2′), 7.92 (d, J ) 8.0 Hz,
1 H, H-8′), 7.85 (d, J ) 8.0 Hz, 1 H, H-5′), 7.58 (dd, J ) 7.0,
1.0 Hz, 1 H, H-4′), 7.51 (t, J ) 8.0 Hz, 1 H, H-3′), 7.43 (dt, J
) 7,0, 1.0 Hz, 1 H, H-6′), 7.32 (dt, J ) 7.0, 1.0 Hz, 1 H, H-7′),
6.93 (d, J ) 8.0 Hz, 1 H, H-6), 6.50 (s, 1 H, H-3), 2.06 (s, 3 H,
CH3-7); MS m/z 286 (M+). Anal. C, H, N.
2-(r-Na p h t h yl)-5,7-d im et h yl-1,8-n a p h t h yr id in -4(1H )-
on e (46) was obtained from compound 22: amorphous; 1H
NMR (CDCl3) δ 7.98 (d, J ) 7.5 Hz, 1 H, H-2′), 7.95 (d, J )
8.0 Hz, 1 H, H-8′), 7.88 (d, J ) 8.0 Hz, 1 H, H-5′), 7.60 (dd, J
) 6.5, 1.0 Hz, 1 H, H-4′), 7.53 (t, J ) 7.5 Hz, 1 H, H-3′), 7.49
(dt, J ) 6.0, 2.0 Hz, 1 H, H-6′), 7.38 (dt, J ) 7.0, 1.0 Hz, 1 H,
H-7′), 6.69 (s, 1 H, H-6), 6.45 (s, 1 H, H-3), 2.91 (s, 3 H, CH3-
5), 2.10 (s, 3 H, CH3-7); MS m/z 300 (M+). Anal. C, H, N.
2-(â-Na p h th yl)-1,8-n a p h th yr id in -4(1H)-on e (47) was ob-
tained from compound 23: amorphous; 1H NMR (CDCl3) δ 9.98
(br s, 1H, NH-1), 8.71 (dd, J ) 8.0, 1.5 Hz, 1 H, H-5), 8.44 (d,
J ) 4.5 Hz, 1 H, H-7), 8.24 (d, J ) 1.0 Hz, 1 H, H-1′), 8.04 (d,
J ) 8.5 Hz, 1 H, H-4′), 7.94 (m, 2 H, H2-5′, 8′), 7.80 (dd, J )
8.5, 2.0 Hz, 1 H, H-3′), 7.63 (m, 2 H, H2-6′, 7′), 7.28 (dd, J )
8.0, 4.5 Hz, 1 H, H-6), 6.76 (s, 1 H, H-3); MS m/z 272 (M+).
Anal. C, H, N.
2-(â-N a p h t h y l)-5-m e t h y l-1,8-n a p h t h y r id in -4(1H )-
on e (48) was obtained from compound 24: needles; 1H NMR
(CDCl3) δ 10.09, (br s, 1 H, NH-1), 8.21 (d, J ) 1.0 Hz, 1 H,
H-1′), 8.08 (d, J ) 5.0 Hz, 1 H, H-7), 8.01 (d, J ) 8.5 Hz, 1 H,
H-4′), 7.93 (m, 2 H, H2-5′, 8′), 7.79 (dd, J ) 8.5, 2.0 Hz, 1 H,
H-3′), 7.61 (m, 2 H, H2-6′, 7′), 6.93 (d, J ) 5.0 Hz, 1 H, H-6),
6.67 (s, 1 H, H-3), 2.99 (s, 3 H, CH3-5); MS m/z 286 (M+). Anal.
C, H, N.
2-(â-N a p h t h y l)-6-m e t h y l-1,8-n a p h t h y r id in -4(1H )-
on e (49) was obtained from compound 25: amorphous; 1H
NMR (CDCl3) δ 8.49 (d, J ) 2.0 Hz, 1 H, H-5), 8.21 (d, J ) 1.0
Hz, 1 H, H-1′), 8.03 (d, J ) 8.5 Hz, 1 H, H-4′), 7.97 (m, 2 H,
H2-5′, 8′), 7.87 (d, J ) 1.0 Hz, 1 H, H-7), 7.79 (dd, J ) 8.5, 2.0
Hz, 1 H, H-3′), 7.62 (m, 2 H, H2-6′, 7′), 6.72 (s, 1 H, H-3), 2.24
(s, 3 H, CH3-6); MS m/z 286 (M+). Anal. C, H, N.
2-(â-N a p h t h y l)-7-m e t h y l-1,8-n a p h t h y r id in -4(1H )-
on e (50) was obtained from compound 26: needles; 1H NMR
(CDCl3) δ 9.32, (br s, 1 H, NH-1), 8.59 (d, J ) 8.0 Hz, 1 H,
H-5), 8.19 (d, J ) 2.0 Hz, 1 H, H-1′), 8.01 (d, J ) 8.5 Hz, 1 H,
H-4′), 7.92 (m, 2 H, H2-5′, 8′), 7.76 (dd, J ) 8.5, 2.0 Hz, 1 H,
H-3′), 7.61 (m, 2 H, H2-6′, 7′), 7.21 (d, J ) 8.0 Hz, 1 H, H-6),
6.74 (s, 1 H, H-3), 2.60 (s, 3 H, CH3-7); MS m/z 286 (M+). Anal.
C, H, N.
2-(â-Na p h t h yl)-5,7-d im et h yl-1,8-n a p h t h yr id in -4(1H )-
on e (51) was obtained from compound 27: amorphous; 1H
NMR (CDCl3) δ 9.20, (br s, 1 H, NH-1), 8.17 (d, J ) 1.5 Hz, 1
H, H-1′), 7.99 (d, J ) 8.5 Hz, 1 H, H-4′), 7.92 (m, 2 H, H2-5′,
8′), 7.75 (dd, J ) 8.5, 1.5 Hz, 1 H, H-3′), 7.60 (m, 2 H, H2-6′,
7′), 6.90 (s, 1 H, H-6), 6.65 (s, 1 H, H-3), 2.96 (s, 3 H, CH3-5),
2.50 (s, 3 H, CH3-7); MS m/z 300 (M+). Anal. C, H, N.
2-(â-Naph th yl)-6-ch lor o-1,8-n aph th yr idin -4(1H)-on e (49)
was obtained from compound 28: amorphous; 1H NMR (CDCl3)
δ 8.59 (d, J ) 2.5 Hz, 1 H, H-5), 8.56 (d, J ) 2.5 Hz, 1 H, H-7),
8.22 (d, J ) 1.0 Hz, 1 H, H-1′), 7.95 (d, J ) 8.5 Hz, 1 H, H-4′),
7.91 (m, 1 H, H-8′), 7.86 (m, 1 H, H-5′), 7.74 (dd, J ) 8.5, 2.0
Hz, 1 H, H-3′), 7.55 (m, 2 H, H2-6′, 7′), 6.68 (s, 1 H, H-3); MS
m/z 306 (M+). Anal. C, H, N.
Cytotoxicity Assa ys. Compounds 5-28 were assayed for
in vitro cytotoxicity in a panel of human and murine tumor
cell lines at the School of Medicine, University of North
Carolina at Chapel Hill, and according to procedures described
previously.13 The cell lines include human epidermoid carci-
noma of the nasopharynx (KB), lung carcinoma (A-549),
ileocecal carcinoma (HCT-8), melanoma (PRMI-7951), and
medulloblastoma (TE-671), as well as one murine leukemia
cell line (P-388). The results (data not shown) demonstrated
that essentially all 2-arylpyrido[1,2-a]pyrimidin-4-ones (5-28)
were inactive (EC50 > 4 µg/mL); only a few compounds showed
marginal activity. Compounds 29-52 were submitted to NCI
and assayed in the NCI’s in vitro disease-oriented antitumor
screen, which involves determination of a test compound’s
effects on the growth of approximately 60 human tumor cell
lines.3,4 These lines include leukemia, non-small cell lung,
colon, CNS, melanoma, ovarian, renal, prostate, and breast
cancers. The cytotoxic effects of each compound were obtained
as GI50 or TGI values, which represent the molar drug
concentrations required to cause 50% inhibition or total growth
inhibition, respectively.
An t im icr ot u b u le Assa y. Electrophoretically homoge-
neous bovine brain tubulin was purified as described previ-
ously.14 Combretastatin A-4 was a generous gift of Dr. G. R.
Pettit, Arizona State University.
The tubulin polymerization assay was performed as de-
scribed previously.15 In brief, tubulin at 1.2 mg/mL (12 µM)
was preincubated for 15 min at 26 °C in a 0.24-mL volume of
0.8 M monosodium glutamate (pH 6.6 with NaOH in a 2 M
stock solution) with varying drug concentrations. The drug
stock solutions were in DMSO, and the final solvent concen-
tration was 4% (v/v). All concentrations are in terms of the
final reaction volume (0.25 mL). The reaction mixtures were
chilled on ice, and 10 µL of 10 mM GTP was added to each
reaction mixture. Samples were transferred to cuvettes held
at 0 °C by an electronic temperature controller in Gilford
spectrophotometers. Baselines were established at 350 nm,
and polymerization was initiated by a temperature jump to
26 °C. The jump took about 50 s to complete. After 20 min,
turbidity readings were recorded, and the temperature con-
troller was set to 0 °C. When depolymerization was complete,
turbidity readings were again recorded. Generally, turbidity
readings were about 90% cold-reversible, and the cold-revers-
ible turbidity was taken to represent the extent of assembly
for each reaction mixture. IC50 values were obtained graphi-
cally from inhibition of polymerization by different drug
concentrations. Four spectrophotometers were used for each
experimental sequence, with two control reactions (no drug)
in each set. Generally, the control reactions were within 5%
of their average and IC50 values obtained with this polymer-
ization assay are usually highly reproducible. Generally,
standard deviations were within 20% of the mean values, but
in some cases, the standard deviations were 30-35% of the
mean. Therefore, we can conservatively estimate that a 50%
difference in IC50 values represents a difference in the relative
activity of two agents.
Colch icin e Bin d in g Assa y. The binding of radiolabeled
colchicine to tubulin was measured by the DEAE-cellulose
filter technique, as described previously.16 In brief, each 0.1-
mL reaction mixture contained 0.1 mg (1.0 µM) of tubulin, 1.0
M commercial monosodium glutamate (pH 6.6 with HCl), 1
mM MgCl2, 0.1 mM GTP, 5.0 µM [3H]colchicine, 5% (v/v)
DMSO, and 5.0 µM inhibitor. Incubation was for 20 min at
37 °C. Each reaction mixture was filtered under reduced
vacuum through a stack of two DEAE-cellulose paper filters
that were washed with water, and the radioactivity was
quantitated in a liquid scintillation counter.

3056 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Chen et al.
(8) Andreu, S. M.; Timasheff, S. Conformational states of tubulin
liganded to colchicine, tropolone methyl ether, and podophyllo-
toxin. Biochemistry 1982, 21, 6465-6476.
(9) Lin, C. M.; Ho, H, H.; Pettit, G. R.; Hamel, E. Antimitotic natural
products combretastatin A-4 and combretastatin A-2; studies on
the mechanism of their inhibition of binding of colchicine to
tubulin. Biochemistry 1989, 28, 6984-6991.
Ack n ow led gm en t . This investigation was sup-
ported by a grant from the National Cancer Institute
No. CA-17625 awarded to K.H.L.
Refer en ces
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