6-alkylamino- and 2,3-dihydro-3′-methoxy-2-phenyl-4-quinazolinones and related compounds: Their synthesis, cytotoxicity, and inhibition of tubulin polymerization

Article abstract of DOI:10.1021/jm000151c

As part of our continuing search for potential anticancer candidates among 2-phenyl-4-quinolones and 2-phenyl-4-quinazolinones, two series of 6,7,2′,3′,4′,5′-substituted 2-phenyl-4-quinazolinones and 6,2′,3′,4′,5′-substituted 2,3-dihydro-2-phenyl-4-quinazolinones were synthesized and evaluated for cytotoxicity and as inhibitors of tubulin polymerization. In general, a good correlation was found between the two activities. Five of the 6-substituted heterocyclic 2-phenyl-4-quinozolinones (37-51) showed significant cytotoxicity against a panel of human tumor cell lines with EC₅₀ values in the low micromolar to nanomolar concentration ranges. Compound 38 was the most potent of these compounds, as well as the most potent inhibitor of tubulin polymerization in this series. The activity of 38 was in the same range as those of the antimitotic natural products, colchicine, podophyllotoxin, and combretastatin A-4. Substituted 2-phenyl-4-quinazolinones and 2,3-dihydro-2-phenyl-4-quinazolinones also displayed highly selective cytotoxicity against the ovarian cancer 1A9 and P-gp resistant KB-VIN cell lines.

Full text of DOI:10.1021/jm000151c

J . Med. Chem. 2000, 43, 4479-4487
4479
6-Alk yla m in o- a n d 2,3-Dih yd r o-3′-m eth oxy-2-p h en yl-4-qu in a zolin on es a n d
Rela ted Com p ou n d s: Th eir Syn th esis, Cytotoxicity, a n d In h ibition of Tu bu lin
P olym er iza tion
Mann-J en Hour,† Li-J iau Huang,† Sheng-Chu Kuo,*,† Yi Xia,‡ Kenneth Bastow,§ Yuka Nakanishi,§
Ernest Hamel,| and Kuo-Hsiung Lee*,‡
Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan, Natural Products Laboratory and
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North
Carolina 27599, and Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and
Diagnosis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702
Received J une 14, 2000
As part of our continuing search for potential anticancer candidates among 2-phenyl-4-
quinolones and 2-phenyl-4-quinazolinones, two series of 6,7,2′,3′,4′,5′-substituted 2-phenyl-4-
quinazolinones and 6,2′,3′,4′,5′-substituted 2,3-dihydro-2-phenyl-4-quinazolinones were syn-
thesized and evaluated for cytotoxicity and as inhibitors of tubulin polymerization. In general,
a good correlation was found between the two activities. Five of the 6-substituted heterocyclic
2-phenyl-4-quinozolinones (37-51) showed significant cytotoxicity against a panel of human
tumor cell lines with EC50 values in the low micromolar to nanomolar concentration ranges.
Compound 38 was the most potent of these compounds, as well as the most potent inhibitor of
tubulin polymerization in this series. The activity of 38 was in the same range as those of the
antimitotic natural products, colchicine, podophyllotoxin, and combretastatin A-4. Substituted
2-phenyl-4-quinazolinones and 2,3-dihydro-2-phenyl-4-quinazolinones also displayed highly
selective cytotoxicity against the ovarian cancer 1A9 and P-gp resistant KB-VIN cell lines.
In tr od u ction
Cl, and F (PQ1-7), were placed at the 6-position of the
A-ring and the 3′-position of the C-ring, activity was
very potent (Chart 1). These two functional groups are
about 10-11 Å apart, and these two groups possibly
may interact with the tubulin binding domain by acting
as receptors in hydrogen bonding. Thus, they might
contribute significantly to the potency of PQ compounds.
In the DHPQ system8, the 6- and 3′-substituents
(DHPQ1) also play a decisive role in activity. However,
in the PN system,9,10 when the 3′-substituent is fixed
(e.g. OCH3), the identity of the 6-substituent, e.g. H (PN-
1), CH3 (PN-2), or C1 (PN-3), does not noticeably affect
activity. This finding is unique to the PN system and
differentiates it from the PQ and DHPQ systems.
Microtubules provide an important framework sup-
porting cellular morphology in interphase, and they are
essential in cell division as the key component of the
mitotic spindle. Consequently, the microtubule has
become an important target for the design of new
antimitotic anticancer agents. The antimitotic agents
currently in clinical use include vinca alkaloids,1 which
inhibit microtubule polymerization, and taxoids,2 which
promote microtubule assembly. Colchicine3,4 is another
well-known antimitotic agent. However, it is too toxic
to be used as an anticancer agent, although it has
clinical utility in gout and other inflammatory diseases.
In recent years, we have designed and synthesized
three types of heterocyclic ketones, 2-phenyl-4-quino-
lones (PQ),4-7 2,3-dihydro-2-phenyl-4-quinolones (DH-
PQ),8 and 2-phenyl-1,8-naphthyridin-4-ones (PN)8-10
(Chart 1) as novel antimitotic agents. Among these three
types of heterocyclic ketones, the common structural
feature is a biaryl system composed of A- and C-rings
that are linked by an interposed B-ring. However, some
minor structural differences also exist. Preliminary
structure-activity relationships (SARs) for these three
series are described below.
Phenylquinazolines (PQZ) and dihydrophenylquinazo-
lines (DHPQZ) are additional related compound classes
(Chart 1). The antitumor activities of 2,3-dihydro-2-aryl-
4-quinazolinones (DHPQZ) were reported around
1970.11,12 A more recent reevaluation of this type of
compound by NCI against human tumor cell lines
reconfirmed that, like colchicine, they are effective
inhibitors of tubulin polymerization.13 Somewhat ear-
lier, 2-styrylquinazolin-4-ones (SQZ)14,15 were also iden-
tified as potent inhibitors of tubulin polymerization, but
in this series the most active compounds have a hydro-
carbon bridge between the B- and C-rings. More re-
cently, we prepared a series of 6,7-methoxy-2-aryl
quinazolin-4-one derivatives (PQZ) for evaluation of
their cytotoxicity and inhibitory effects on tubulin
polymerization.16 Although a few 6-methoxy derivatives
demonstrated moderate activity, most of the compounds
were essentially inactive.
In the PQ system, when functional groups with
nonbonding electrons, e.g. -OCH3, -OCH2O-, -NRR′,
* To whom correspondence should be addressed. For K.-H. Lee: tel,
919-962-0066; fax, 919-966-3893; e-mail, khlee@unc.edu. For S.C.
Kuo: tel, 886-4-205-7153; fax, 886-4-205-5105.
† China Medical College.
‡ Natural Products Laboratory, University of North Carolina.
§ Division of Medicinal Chemistry and Natural Products, University
of North Carolina.
| National Cancer Institute.
10.1021/jm000151c CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/28/2000

4480 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Ch a r t 1. Antimitotic Heterocyclic Ketones
Hour et al.
Sch em e 1
The PQ, PN, and PQZ series are related as bioisos-
teres; the latter two series contain a second nitrogen in
the A- and B-rings, respectively, rather than a carbon
as found in the PQ series. Thus, their structures,
physicochemical properties, and pharmacokinetics as
well as SAR results likely are similar but not identical.
Definitive SAR must be first established for each class
before formulating hypotheses or pharmacophores
through QSAR molecular modeling. Accordingly, we
chose the relatively less investigated 2-phenylquinazo-
lin-4-one (PQZ) and 2,3-dihydro-2-phenylquinazolin-4-
one (DHPQZ) as lead compounds and prepared addi-
tional derivatives, including 3′-methoxy-6-alkylamino
analogues, as our targets. These PQZ and DHPQZ
derivatives were evaluated for cytotoxicity and inhibi-
tion of tubulin polymerization in order to add to our
understanding of microtubule binding and extend our
SAR knowledge. The goal of our continuing studies is
to identify novel compounds for in vivo testing and,
ultimately, to aid in new antimitotic drug design.
A mixture of 2-aminobenzamide (14) and 3-methoxy-
benzaldehyde (17) in DMAC was heated to 80 °C for 1
h. Subsequent purification with column chromatography
afforded 43 (mp 148-150 °C) and 25 (mp 177-179 °C)
in yields of 75% and 15%, respectively. The chemical
structure of 25 was confirmed by IR, NMR, and MS
spectral analysis as 3′-methoxy-2-phenyl-4-quinazoli-
none. For 43, the molecular formula was determined by
elemental and MS analysis (m/z 254, M+) as C15H14N2O2,
which matches the expected 2,3-dihydro-3′-methoxy-2-
phenyl-4-quinazolinone. The 1H NMR of 43 showed
three additional proton signals not seen in the spectrum
of 25: 5.73 (H-2), 7.15 (N1-H), and 8.34 (N3-H). The 2D
HMBC spectrum of 43 showed the expected long-range
coupling between H-2 and C-4 (δ 163.77), C-8a (δ
147.98), C-2′ (δ 119.11), and C-6′ (δ 112.75). All spectral
data confirmed our assignment of 43 as 2,3-dihydro-3′-
methoxy-2-phenyl-4-quinazolinone.
To increase the yield of 43, reaction conditions were
adjusted to minimize the unwanted dehydrogenation of
43 and conversion to 25. First, we lowered the reaction
temperature to 20 ( 2 °C and extended the reaction
time to 4 h. However, these conditions only resulted in
a lower conversion as indicated by the considerable
amount of starting materials (14, 17) that was detected
by TLC in the reaction mixture. Next, we again carried
out the reaction, at 20 ( 2 °C in DMAC, but also
Ch em istr y
Starting 4,5-substituted 2-aminobenzamides (5-13)
were prepared using standard methodology8 from sub-
stituted 2-nitrobenzoic acids by reaction with SOCl2,
NH3, HNRR′′ (for 9-13), and H2 as shown in the general
reaction sequence in Scheme 1. The starting benzamides
(5-15) were reacted with methoxybenzaldehydes (16-
23) in N,N-dimethylacetamide (DMAC) in the presence
of NaHSO3 at 150 °C (Scheme 2). Thermal cyclodehy-
dration/dehydrogenation gave the target substituted
2-phenyl-4-quinazolinones (24-41) in high yields. How-
ever, to obtain the 2,3-dihydro-2-phenyl-4-quinazolino-
nes (42-50), 14 and 15 were reacted with 16-23 using
modified reaction conditions as shown in Scheme 3 and
described below for 2,3-dihydro-3′-methoxy-2-phenyl-4-
quinazolinone (43).

Quinazolinones and Related Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4481
Sch em e 2
Sch em e 3
When various benzamides (6-15) with electron-
donating substituents (e.g. -OCH3, -OCH2O-, -NRR′)
were used as starting materials under these conditions,
TLC check analysis of the crude products did show
materials resembling the expected 2,3-dihydro deriva-
tives. However, they were readily converted to their
corresponding dehydrogenated derivatives (34-41) via
spontaneous dehydrogenation during the workup pro-
cedures, such as column chromatography or recrystal-
lization. Also, although F and Cl belong to the same
class of electron-withdrawing group, the fluorinated 2,3-
dihydro derivative was much less stable than the Cl-
containing counterpart (50) and readily underwent
spontaneous dehydrogenation to give 33 during the final
workup. Thus, 2,3-dihydro-2-phenyl-4-quinazolones con-
taining electron-donating groups or fluorine at the 6-
or 7-position were not prepared for cytotoxic evaluation,
as they would likely undergo spontaneous dehydroge-
nation and would not be stable in the solvent required
for biological testing.
All 2,3-dihydro products (42-50) were prepared as
racemic mixtures. Resolution of these mixtures has not
been attempted in this work but will be pursued if
unusual biological activity is found in our forthcoming
studies.
incorporated a catalytic amount of p-toluenesulfonic
acid. The reaction was completed in 30 min, and the
subsequent workup resulted in a high yield (89.0%) of
43 and a minor yield (4.0%) of 25. Consequently, the
same reaction condition was adopted in the subsequent
preparation of 42 and 44-50 and resulted in high yields
of all desired products.
Resu lts a n d Discu ssion
a . Eva lu a tion of Cytotoxicity of P QZ a n d DH-
P QZ Der ivatives. The 6,7,2′,3′,4′,5′-substituted 2-phen-
yl-4-quinazolinones (24-32) and 6,2′,3′,4′,5′-substituted
2,3-dihydro-2-phenyl-4-quinazolinones (42-50) were as-

4482 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Hour et al.
sayed for cytotoxicity in vitro against 10 human tumor
cell lines, including ovarian cancer (1A9), ileocecal
carcinoma (HCT-8), lung carcinoma (A-549), glioblas-
toma (U-87-MG), bone (HOS), epidermoid carcinoma of
the nasopharynx (KB), P-gp-expressing epidermoid
carcinoma of the nasopharynx (KB-VIN), prostate can-
cer (PC3), breast cancer (MCF-7), and melanoma (SK-
MEL-2) cell lines. As shown in Table 2, the 6-alkylamino-
substituted 37-40 showed strong cytotoxic effects toward
a variety of tumor cell lines, with EC50 values < 1.0 µg/
mL, but 37 was inactive in the U-87-MG or HOS cell
lines. Notably, 37-40 showed highly selective effects
on the ovarian cancer line (1A9). For example, the EC50
value of 37 in the 1A9 cell line was 0.49 µM, and growth
from this more sensitive cell line was arrested at a
concentration approximately 20-30 times lower than
those in less sensitive cell lines (e.g. A-549, U087-MG,
PC3, and SKMEL-2). Similarly, high antiovarian cell
selectivity was also found with 38-40. In addition, 37-
40 displayed increased activity against the P-gp-
expressing cell line KB-VIN relative to the parental cell
line (KB), with 37 and 40 showing about 25 and 15
times greater activity against the drug-resistant cell
line. Although the structural difference between 37 and
38 is very small, 38 was significantly more cytotoxic.
However, 38 showed reduced selectivity against KB-
VIN.
(43) were about 15 or 20 times more active than
2′-methoxy (42) or 4′-methoxy (44) derivatives. In
addition, 43 was also more active than multimethoxy
compounds (45-49). This result is consistent with
observations in other heterocyclic ketones, such as the
PQ, DHPQ, and PN derivatives. 6-Cl-3-methoxy DHPQZ
(50) showed significantly increased cytotoxicity as com-
pared to 43. The EC50 of 50 was about 5-fold lower than
that of 43. Compound 50 was also substantially more
active than its cognate PQZ derivative (32).
As summarized in Table 2, the cytotoxic PQZ and
DHPQZ compounds showed strong sensitivity in the
ovarian cancer cell line and were selective against the
drug-resistant KB cell line. These interesting results
make PQZ and DHPQZ unique among structurally
similar heterocyclic ketones, which were generally ac-
tive against most human tumor cell lines in virtually
all cases. Further mechanistic studies may reveal
reasons for these differences.
b. In ter a ction of P QZ a n d DHP QZ Der iva tives
w ith Tu bu lin . Previously, PQ, DHPQ, and PN deriva-
tives were found to inhibit both tubulin polymerization
and the binding of radiolabeled colchicine to tubulin.5-10
The most active compound prepared so far, both in its
cytotoxicity and in the strength of its interaction with
tubulin, has been PQ7.7 In most cell lines examined IC50
values for cell growth inhibition by PQ7 were below 1
nM, about 100-500-fold lower than the IC50 values
obtained with compound 38. In the previous study,7 and
in a reevaluation performed in conjunction with the
current work (Table 3), PQ7 strongly inhibited tubulin
polymerization and the binding of [3H]colchicine to
tubulin. Under the reaction conditions used, 0.32 µM
PQ7 inhibited the assembly of 12 µM tubulin by 50%,
and 5 µM PQ7 nearly completely inhibited the binding
of 5 µM colchicine to 1 µM tubulin (Table 3). For
comparison, previously we obtained IC50 values of 0.80,
0.46, and 0.53 µM for colchicine, podophyllotoxin, and
combretastatin A-4 for inhibition of tubulin assembly,
and PQ7, podophyllotoxin,17 and combretastatin A-4
were all nearly identical as inhibitors of colchicine
binding.
In terms of SAR information, PQZ derivatives without
C-6 substitutions (24-31) were inactive. In comparing
the effects of electron-donating or electron-withdrawing
groups at the 6-position, the compound with an electron-
donating methoxy (34) was more active than the electron-
withdrawing 6-C1 (32) or 6-F (33) PQZ derivatives. The
(methylenendioxy)benzene moiety occurs commonly in
many antimitotic agents, such as podophyllotoxin, ste-
ganacin, and combretastatin A-2. However, the 6,7-
(methylenedioxy)-substituted compound (36) did not
show significantly increased activity compared with
either an unsubstituted (35), 6-methoxy (34), or 6,7-
dimethoxy (35) compound. Single substitution at the
6-position seemed to be beneficial for increased antitu-
mor activity. The 6-methoxy compound (34) was about
5-fold more active than its corresponding 6,7-dimethoxy
analogue (35) in virtually all cell lines. Compounds with
a heterocyclic ring or N(CH3)2 at the 6-position (37-
41) displayed the greatest potency, except for 41 with a
morpholine substituent. Compound 38 with a 6-pyrro-
lidinyl ring was the most potent compound, with ED50
values in the nanomolar concentration range. In our
previous studies, maximum activity has often been
observed with a 6-pyrrolidinyl substituent in other
heterocyclic ketone series.
The chief structural difference between the PQ agents
and the new PQZ series, as well as between DHPQ and
the new DHPQZ series, is the additional nitrogen atom
in the B-ring. The only structural difference between
the two newly synthesized PQZ and DHPQZ compounds
is the oxidation status of the bond between C-2 and N-3
in the B-ring. This modification results in configura-
tional and conformational changes in the relative posi-
tions of the aromatic rings A and C. Many studies have
suggested that the interaction between colchicine and
tubulin is stereoselective and is highly dependent on the
conformation and configuration of the biaryl system
formed by the trimethoxyphenyl A-ring and tropolonic
C-ring. Thus, evaluation of the new agents for interac-
tions with tubulin should provide additional insight into
the mechanism of ligand binding at the colchicine site.
A fairly dramatic difference was observed between the
PQZ (24-32) and DHPQZ (41-50) series. In the former
group, little activity was observed across all cell lines
examined. In contrast, in the latter group, highly
selective activities were obtained against the 1A9 and
KB-Vin cell lines. Generally, DHPQZ derivatives showed
greater activity than PQZ compounds with the same
substitution. However, as the DHPQZ compounds tested
in this study were all racemates, it is reasonable to
anticipate higher activity for one of the enantiomer pair.
In terms of cytotoxicity in the more sensitive cell lines,
DHPQZ compounds with a methoxy at the 3′-position
In the studies reported in the previous paper,16
selected molecules in the PQZ compounds were shown
to inhibit tubulin polymerization. This finding led us
to prepare additional PQZ and DHPQZ derivatives to
further delineate SARs. Table 3 summarizes our find-
ings when these compounds were evaluated as inhibi-

Quinazolinones and Related Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4483
Ta ble 1. Physical and Spectral Data of 6-Alkylamino-3′-methoxy-2-phenyl-4-quinazolinones,
2,3-Dihydro-3′-methoxy-2-phenyl-4-quinazolinones, and Related Compounds
UV, λmax
yield
(%)
mp
(MeOH)
IR, νCdO MS (M+)
compd
(°C)
(log C)
(cm-1
1678
)
m/z
1H NMR (DMSO-d6) δ
24
89
95
96
86
198-202
177-179
215-219
179-181
211 (4.50)
218 (4.41)
207 (4.38)
223 (4.52)
252
3.86 (3 H, s, OCH3), 7.05-7.21 (2 H, m, H-3′, H-5′), 7.49-7.58 (2 H, m, H-4,
H-6), 7.69-7.73 (2 H, m, H-6′, H-8), 7.83 (1 H, ddd, J ) 1.2, 8.0, 8.0 Hz,
H-7), 8.15 (1 H, dd, J ) 1.2, 8.0 Hz, H-5), 12.13 (1 H, br s, NH)
3.86 (3 H, s, OCH3), 7.14 (1 H, dd, J ) 2.2, 8.0 Hz, H-4′), 7.41-7.56 (2 H, m,
H-5′, H-6), 7.74-7.88 (4 H, m, H-2′, H-6′, H-7, H-8), 8.15 (1 H, dd, J ) 1.2,
8.0 Hz, H-5), 12.55 (1 H, br s, NH)
3.83 (3 H, s, OCH3), 7.08 (2 H, d, J ) 8.0 Hz, H-3′, H-5′), 7.47 (1 H, ddd, J
) 1.2, 8.0, 8.0 Hz, H-6), 7.67-7.85 (2 H, m, H-7, H-8), 8.10-8.20 (3 H, m,
H-2′, H-6′, H-5), 12.42 (1 H, br s, NH)
3.76 (3 H, s, 2′-OCH3 or 3′-OCH3), 3.87 (3 H, s, 2′-OCH3 or 3′-OCH3), 7.18-
7.28 (3 H, m, H-4′, H-5′, H-6′), 7.54 (1 H, ddd, J ) 1.2, 8.0, 8.0 Hz, H-6),
7.70 (1 H, dd, J ) 1.2, 8.0 Hz, H-8), 7.84 (1 H, ddd, J ) 1.2, 8.0, 8.0 Hz, H-7),
8.16 (1 H, dd, J ) 1.2, 8.0 Hz, H-5), 12.23 (1 H, br s, NH)
25
26
27
1672
1678
1678
252
252
282
28
29
30
31
84
88
89
84
184-186
143-144
240-242
200-203
207 (4.27)
220 (4.70)
226 (4.44)
221 (4.57)
1678
1698
1671
1674
282
282
282
282
3.85 (3 H, s, 2′-OCH3 or 4′-OCH3), 3.89 (3 H, s, 2′-OCH3 or 4′-OCH3), 6.65-
6.72 (2 H, m, H-3′, H-5′), 7.49 (1 H, ddd, J ) 1.2, 8.0, 8.0 Hz, H-6), 7.67
(1 H, dd, J ) 1.2, 8.0 Hz, H-8), 7.76-7.85 (2 H, m, H-6′, H-7), 8.12 (1 H, dd,
J ) 1.2, 8.0 Hz, H-5), 11.82 (1 H, br s, NH)
3.77 (3 H, s, 2′-OCH3 or 5′-OCH3), 3.81 (3 H, s, 2′-OCH3 or 5′-OCH3), 7.10-
7.12 (2 H, m, H-3′, H-4′), 7.29-7.31 (1 H, m, H-6′), 7.53 (1 H, ddd, J ) 1.2,
8.0, 8.0 Hz, H-6), 7.71 (1 H, dd, J ) 1.2, 8.0 Hz, H-8), 7.83 (1 H, ddd, J )
1.2, 8.0, 8.0 Hz, H-7), 8.14 (1 H, dd, J ) 1.2, 8.0 Hz, H-5), 12.10 (1 H, br s, NH)
3.84 (3 H, s, 3′-OCH3 or 4′-OCH3), 3.88 (3 H, s, 3′-OCH3 or 4′-OCH3), 7.10
(1 H, d, J ) 8.0 Hz, H-5′), 7.48 (1 H, ddd, J ) 1.2, 8.0, 8.0 Hz, H-6), 7.69-
7.89 (4 H, m, H-2′, H-6′, H-7, H-8), 8.13 (1 H, dd, J ) 1.2, 8.0 Hz, H-5), 12.44
(1 H, br s, NH)
3.83 (6 H, s, 3′-OCH3, 5′-OCH3), 6.69 (1 H, dd, J ) 2.2, 2.2 Hz, H-4′), 7. 38
(2 H, d, J ) 2.2 Hz, H-2′, H-6′), 7.52 (1 H, ddd, J ) 1.2, 8.0, 8.0 Hz, H-6), 7.74
(1 H, dd, J ) 1.2, 8.0 Hz, H-8), 7.83 (1 H, ddd, J ) 1.2, 8.0, 8.0 Hz, H-7), 8.15
(1 H, dd, J ) 1.2, 8.0 Hz, H-5), 12.52 (1 H, br s, NH)
3.86 (3 H, s, 3′-OCH3), 7.16 (1 H, dd, J ) 2.5, 8.0 Hz, H-4′), 7.46 (1 H, dd,
J ) 8.0, 8.0 Hz, H-5′), 7.73-7.80 (3 H, m, H-2′, H-6′, H-8), 7.87 (1 H, dd, J )
2.5, 8.0 Hz, H-7), 8.09 (1 H, d, J ) 2.5 Hz, H-5), 12.70 (1 H, br s, NH)
3.84 (3 H, s, 3′-OCH3), 7.13 (1 H, dd, J ) 2.5, 8.0 Hz, H-4′), 7.44 (1 H, dd,
J ) 8.0, 8.0 Hz, H-5′), 7.68-7.84 (5 H, m, H-2′, H-6′, H-5, H-7, H-8), 12.61
(1 H, br s, NH)
3.86 (3 H, s, 3′-OCH3 or 6-OCH3), 3.96 (3 H, s, 3′-OCH3 or 6-OCH3), 7.11
(1 H, dd, J ) 2.5, 8.0 Hz, H-4′), 7.29-7.49 (2 H, m, H-5′, H-7), 7.53 (1 H,
d, J ) 2.5 Hz, H-5), 7.67-7.82 (3 H, m, H-2′, H-6′, H-8), 12.48 (1 H, br s, NH)
3.86 (3 H, s, 3′-OCH3 or 6-OCH3 or 7-OCH3), 3.89 (3 H, s, 3′-OCH3 or 6-OCH3
or 7-OCH3), 3.94 (3 H, s, 3′-OCH3 or 6-OCH3 or 7-OCH3), 7.11 (1 H, dd, J )
1.2, 8.0 Hz, H-4′), 7.21 (1 H, s, H-8), 7.43 (1 H, dd, J ) 8.0, 8.0 Hz, H-5′),
7.48 (1 H, s, H-5), 7.73-7.78 (2 H, m, H-2′, H-6′), 12.40 (1 H, br s, NH)
3.85 (3 H, s, 3′-OCH3), 6.20 (2 H, s, OCH2O), 7.11 (1 H, dd, J ) 2.2, 8.0 Hz,
H-4′), 7.18 (1 H, s, H-8), 7.43 (1 H, dd, J ) 8.0, 8.0 Hz, H-5′), 7.44 (1 H, s,
H-5), 7.68-7.75 (2 H, m, H-2′, H-6′), 12.46 (1 H, br s, NH)
32
33
34
35
94
245-248
246-247
216-218
266-267
218 (4.60)
217 (4.70)
218 (4.69)
253 (4.55)
1678
1680
1659
1658
286.5
270
46.8
42
282
30
312
36
37
8.8
51
269-270
239-241
249 (4.48)
224 (4.79)
1665
1659
296
295
3.01 (s, 6 H, N(CH3)2), 3.85 (3 H, s, 3′-OCH3), 7.07 (1 H, dd, J ) 2.5, 8.0 Hz,
H-4′), 7. 21 (1 H, d, J ) 2.5 Hz, H-5), 7.33 (1 H, dd, J ) 2.5, 8.0 Hz, H-7),
7.41 (1 H, dd, J ) 8.0, 8.0 Hz, H-5′), 7.61 (1 H, d, J ) 8.0 Hz, H-8), 7.70-
7.76 (2 H, m, H-2′, H-6′), 12.26 (1 H, br s, NH)
38
39
40
40
55
57
261-263
222-225
212-215
227 (4.54)
223 (4.71)
224 (4.61)
1653
1668
1663
321
335
349
1.96-2.03 (4 H, m, CH2CH2NCH2CH2), 3.30-3.34 (4 H, m, CH2NCH2),
3.85 (3 H, s, 3′-OCH3), 7.03-7.17 (3 H, m, H-4′, H-5, H-7), 7.41 (1 H, dd,
J ) 8.0, 8.0 Hz, H-5′), 7.60 (1 H, d, J ) 8.0 Hz, H-8), 7.68-7.75 (2 H, m, H-2′,
H-6′), 12.22 (1 H, br s, NH)
1.55-1.60 (6 H, m, (CH2)2CH2NCH2CH2), 3.25-3.30 (4 H, m, CH2NCH2),
3.85 (3 H, s, 3′-OCH3), 7.09 (1 H, dd, J ) 22, 8.0 Hz, H-4′), 7.38-7.46 (2 H,
m, H-5′, H-5), 7.53 (1 H, dd, J ) 2.2, 8.0 Hz, H-7), 7.60 (1 H, d, J ) 8.0 Hz,
H-8), 7.70-7.76 (2 H, m H-2′,H-6′), 12.33 (1 H, br s, NH)
0.93 (3 H, d, J ) 6.2 Hz, CH3CH(CH2)2N(CH2)2), 1.20-1.26, 1.68-1.74 (2 H
each, both m, (NCH2CH2) × 2), 1.48-1.58 (1 H, m, CH3CH(CH2)2N(CH2)2),
2.71-2.82, 3.75-3.83 (2 H each, both m, (NCH2) × 2), 3.85 (3 H, s, 3′-OCH3),
7.09 (1 H, dd, J ) 2.2, 8.0 Hz, H-4′), 7.38-7.46 (2 H, m, H-5′, H-5), 7.54
(1 H, dd, J ) 2.2, 8.0 Hz, H-7), 7.61 (1 H, d, J ) 8.0 Hz, H-8), 7.71-7.77
(2 H, m, H-2′, H-6′), 12.34 (1 H, br s, NH)
41
48
254-257
225 (4.66)
1673
337
3.23 (4 H, t, J ) 4.7 Hz, CH2NCH2), 3.75 (4 H, t, J ) 4.7 Hz, CH2OCH2),
3.85 (3 H, s, 3′-OCH3), 7.10 (1 H, dd, J ) 2.5, 8.0 Hz, H-4′), 7.43 (1 H, dd,
J ) 8.0, 8.0 Hz, H-5′), 7.45 (1 H, d, J ) 2.5 Hz, H-5), 7.57 (1 H, dd, J ) 2.5,
8.0 Hz, H-7), 7.65 (1 H, d, J ) 8.0 Hz,H-8), 7.71-7.77 (2 H, m, H-2′, H-6′),
12.39 (1 H, br s, NH)
42
43
90
89
163-165
148-105
222 (4.54)
222 (4.50)
1668
1647
254
254
3.83 (3 H, s, OCH3), 6.02 (1 H, s, H-2), 6.66 (1 H, dd, J ) 7.5, 7.5 Hz, H-6),
6.74-6.80 (2 H, m, H-8, N1H), 6.94 (1 H, dd, J ) 7.5, 7.5 Hz, H-5′), 7.04
(1 H, d, J ) 8.0 Hz, H-3′), 7.18-7.41 (3 H, m, H-4′, H-6′, H-7), 7.62 (1 H, dd,
J ) 1.0, 1.0 Hz, H-5), 8.02 (1 H, br s, N3H)
3.73 (3 H, s, OCH3), 5.73 (1 H, s, H-2), 6.68 (1 H, dd, J ) 7.5, 7.5 Hz, H-6),
6.77 (1 H, d, J ) 8.0 Hz, H-8), 6.90 (1 H, dd, J ) 8.0, 2.5 Hz, H-4′), 7.04-
7.07 (2 H, m, H-2′, H-6′), 7.15 (1 H, br s, N1H), 7.21-7.33 (2 H, m, H-5′,
H-7), 8.15 (1 H, dd, J ) 1.0, 7.5 Hz, H-5), 8.34 (1 H, br s, N3H)

4484 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Hour et al.
Ta ble 1 (Continued)
UV, λmax
yield
(%)
mp
(MeOH)
IR, νCdO MS (M+)
compd
(°C)
(log C)
(cm-1
1655
)
m/z
1H NMR (DMSO-d6) δ
44
93
78
86
75
80
188-190 225 (4.64)
232-233 224 (4.54)
182-183 225 (4.45)
163-165 223 (4.53)
210-213 223 (4.61)
254
3.73 (3 H, s, OCH3), 5.70 (1 H, s, H-2), 6.67-6.75 (2 H, m, H-6, H-8), 6.91-
6.95 (2 H, m, H-3′, H-5′), 7.00 (1 H, br s, NIH), 7.24 (1 H, ddd, J ) 1.5, 8.0,
8.0 Hz, H-7), 7.36-7.62 (2 H, m, H-2′, H-6′), 7.60 (1 H, dd, J ) 1.2, 7.5 Hz,
H-5), 8.19 (1 H, br s, N3H)
3.78 (3 H, s, 2′-OCH3 or 3′-OCH3), 3.81 (3 H, s, 2′-OCH3 or 3′-OCH3), 6.03
(1 H, s, H-2), 6.63-6.75 (2 H, m, H-6, H-8), 6.79 (1 H, br s, N1H), 7.04-7.10
(3 H, m, H-4′, H-5′, H-6′), 7.23 (1 H, ddd, J ) 1.0, 8.0, 8.0 Hz, H-7), 7.62
(1 H, dd, J ) 1.0, 8.0 Hz, H-5), 8.02 (1 H, br s, N3H)
3.75 (3 H, s, 2′-OCH3 or 4′-OCH3), 3.81 (3 H, s, 2′-OCH3 or 4′-OCH3), 5.94
(1 H, s, H-2), 6.49-6.77 (5 H, m, H-3′, H-5′, H-6, H-8, N1H), 7.21 (1 H, ddd,
J ) 1.0, 8.0, 8.0 Hz, H-7), 7.31 (1 H, d, J ) 8.0, H-6′), 7.61 (1 H, dd, J ) 1.0,
7.5 Hz, H-5), 7.94 (1 H, br s, N3H)
45
46
47
48
1651
1663
1653
1655
284
284
284
284
3.66 (3 H, s, 2′-OCH3 or 5′-OCH3), 3.78 (3 H, s, 2′-OCH3 or 5′-OCH3), 5.98
(1 H, s, H-2), 6.68 (1 H, dd, J ) 7.5, 7.5 Hz, H-6), 6.77 (1 H, d, J ) 8.0 Hz,
H-8), 6.84-7.00 (4 H, m, H-3′, H-4′, H-6′, N1H), 7.23 (1 H, ddd, J ) 1.0, 8.0,
8.0 Hz, H-7), 7.63 (1 H, dd, J ) 1.0, 7.5 Hz, H-5), 8.02 (1 H, br s, N3H)
3.73 (3 H, s, 3′-OCH3 or 4′-OCH3), 3.74 (3 H, s, 3′-OCH3 or 4′-OCH3), 5.70
(1 H, s, H-2), 6.66 (1 H, dd, J ) 7.5, 7.5 Hz, H-6), 6.75 (1 H, d, J ) 7.5 Hz,
H-8), 6.91-7.02 (3 H, m, H-2′, H-5′, H-6′), 7.12 (1 H, br s, N1H), 7.25 (1 H,
ddd, J ) 1.0, 7.5, 7.5 Hz, H-7), 7.61 (1 H, dd, J ) 1.0, 7.5 Hz, H-5), 8.19 (1 H,
br s, N3H)
3.72 (6 H, s, 3′-OCH3, 5′-OCH3), 5.67 (1 H, s, H-2), 6.46 (1 H, s, H-4′), 6.64-
6.77 (4 H, rn, H-2′, H-6′, H-6, H-8), 7.13 (1 H, br s, N1H), 7.24 (1 H, ddd, J
) 1.0, 7.5, 7.5 Hz, H-7), 7.60 (1 H, dd, J ) 1.0, 7.5 Hz, H-5), 8.30 (1 H, br s, N3H)
3.74 (3 H, s, OCH3), 5.75 (1 H, s, H-2), 6.79 (1 H, d, J ) 8.0 Hz, H-8), 6.92
(1 H, dd, J ) 2.5, 8.0 Hz, H-4′), 7.02-7.05 (2 H, m, H-2′, H-6′), 7.25-7.36
(3 H, m, H-5′, H-7, NH), 7.53 (1 H, d, J ) 2.5 Hz, H-5), 8.50 (1 H, br s, N3H)
49
50
93
95
89-92
223 (4.54)
1658
1658
284
193-195 223 (4.57)
288.5
Ta ble 2. In Vitro Cytotoxicity of 6,7,2′,3′,4′,5′-Substituted 2-Phenyl-4-quinazolinones (24-41) and 6,2′,3′,4′,5′-Substituted
2,3-Dihydro-2-phenyl-4-quinazolinones (42-50)
ED50 (g/mL)a
compd
IA9b
HCT-8b
A-549b
U-87-MGb
HOS
KBb
KB-VINb
PC3b
NA
MCF-7b
SKMEL-2b
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
>20 (8)c
>20 (27)
>20 (22)
NA
NA
>20 (15)
>20 (21)
>20 (42)
>20 (19)
>20 (31)
>20 (33)
>20 (29)
>20 (29)
3.2
16.5
12.5
>20 (49)
>20 (11)
14.0
0.50
2.8
6.0
10.9
>20 (44)
3.0
NA
NA
NA
NA
>20 (16)
NA
NA
NA
>20 (5)
>20 (6)
>20 (21)
NA
>20 (7)
>20 (20)
>20 (11)
NA
>20 (6)
NA
>20 (23)
NA
>20 (5)
>20 (10)
>20 (12)
NA
11.5
>20 (39)
17.5
>20 (45)
>20 (16)
10.0
0.23
10
10.8
8.2
>20 (32)
3.0
>20 (14)
>20 (41)
>20 (43)
>20 (40)
>20 (28)
4.7
>20 (9)
>20 (12)
>20 (40)
>20 (11)
>20 (8)
>20 (21)
>20 (22)
>20 (12)
6.5
18.0
3.5
19.8
>20 (19)
0.42
0.10
0.60
2.5
3.7
10.2
1.20
>20 (39)
8.6
7.6
8.4
>20 (23)
26
NA
>20 (8)
>20 (29)
NA
>20 (14)
>20 (21)
>20 (15)
NA
6.0
>20 (31)
16.5
>20 (37)
NA
1.05
0.06
4.3
4.0
10.4
>20 (21)
1.5
>20 (16)
>20 (32)
>20 (17)
>20 (16)
>20 (30)
>20 (26)
>20 (8)
4.5
20.0
>20 (37)
15.0
>20 (12)
15.0
15.0
10.0
12.4
>20 (12)
>20 (24)
NA
>20 (5)
>20 (9)
>20 (15)
>20 (5)
4.8
>20 (41)
>20 (49)
18.0
>20 (14)
19.0
0.09
4.5
7.5
4.8
<20 (52)d
>20 (49)
>20 (23)
<20 (53)
>20 (37)
>20 (44)
>20 (24)
4.4
>20 (42)
>20 (24)
>20 (15)
>20 (20)
>20 (48)
>20 (28)
17.8
>20 (40)
8.0
12.0
>20 (40)
0.85
0.22
8.5
10.0
4.5
>20 (48)
>20 (48)
>20 (44)
>20 (40)
>20 (44)
>20 (42)
20
NA
>20 (19)
>20 (34)
NA
>20 (29)
NA
3.4
16.5
>20 (26)
0.49.
0.09
0.90
0.80
3.8
14.7
1.0
20
11.4
12.5
10.0
17.0
NA
>20 (30)
>20 (11)
>20 (28)
NA
>20 (17)
13.8
9.0
15.5
20
NA
>20 (35)
NA
NA
NA
>20 (5)
NA
>20 (37)
>20 (37)
>20 (43)
>20 (27)
>20 (6)
>20 (46)
7.0
15.4
14.4
12.5
>20 (38)
>20 (42)
>20 (13)
>20 (34)
>20 (40)
>20 (40)
>20 (24)
>20 (41)
20.0
13.4
>20 (30)
>20 (43)
>20 (14)
>20 (20)
>20 (28)
>20 (22)
>20 (30) 20
>20 (44)
2.5
>20 (19)
>20 (39)
>20 (29)
>20 (32)
>20 (25)
3.0
>20 (34)
>20 (39)
>20 (43)
>20 (42)
>20 (41)
5.0
10.0
14.6
1.92
0.27
16.0
1.6
0.42
>20 (49)
16.0
0.48
0.6
0.95
<2.5 (56)
a
ED50 was the concentration of compound which afforded 50% reduction in cell number after 3-4 days of incubation. bHuman ovarian
cancer (1A9), ileocecal carcinoma (HCT-8), lung carcinoma (A-549), glioblastoma (U-87-MG), bone (HOS), epidermoid carcinoma of the
nasopharynx (KB), P-gp-expressing epidermoid carcinoma of the nasopharynx (KB-VIN), prostate cancer (PC3), breast cancer (MCF-7),
and melanoma (SKMEL-2) cell lines. cInhibition of <50% at highest test concentration (percent observed is given in parentheses). dPlateau
dose response apparent to maximum inhibition of 50% seen at low dose but plateaus over broad dose range (range indicated with inhibition
values given in parentheses).
tors of tubulin polymerization. In addition, the most
potent of the new compounds (38) was compared to PQ7
for its effects on the binding of [3H]colchicine to tubulin.
Considering first the PQZ derivatives (24-41), they
differ from the previously studied PQ, DHPQ, and PN
derivatives in that most of these compounds have only
moderate inhibitory effects on tubulin assembly and
their relative potencies varied only slightly. IC50 values
in a relatively narrow range (7-17 µM) were obtained
for compounds 24-36, 39, and 40. Only three com-
pounds with a m-methoxy group in the C-ring and
nitrogen-containing substituents at C-6 were more
potent inhibitors of the tubulin assembly reaction. These
compounds were 37, with a dimethylamino C-6 subsitu-
ent (IC50 3.5 µM), 41, with a morpholino group at C-6
(IC50 4.4 µM), and especially 38, with a pyrrolidinyl

Quinazolinones and Related Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4485
Ta ble 3. Antitubulin Effects of 6,7,2′,3′,4′,5′-Substituted 2-Phenyl-4-quinazolinones 24-41 and 6,2′,3′,4′,5′-Substituted
2,3-Dihydro-2-phenyl-4-quinazolinones 42-50
inhib of colchicine
binding (%) ( SD
compd
R6
R7
R2′
R3′
R4′
R5′
ITPa IC50 (µM) ( SD
24
25
26
27
28
29
30
31
32
33
34
35
36
37
H
H
H
H
H
H
H
H
Cl
F
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
OCH3
H
OCH3
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
17 ( 1
H
12 ( 1
H
OCH3
12 ( 2
OCH3
H
15 ( 0.07
10 ( 0.4
11 ( 0.9
15 ( 2
OCH3
OCH3
OCH3
H
OCH3
H
H
H
H
H
H
H
H
OCH3
H
OCH3
H
H
H
H
H
H
H
11 ( 2
12 ( 0.9
14 ( 2
6.7 ( 0.08
9.1 ( 0.9
9.6 ( 0.6
3.5 ( 0.3
H
H
H
H
H
H
OCH3
OCH3
OCH2O
OCH3
H
38
39
H
H
H
H
OCH3
OCH3
H
H
H
H
1.1 ( 0.02
9.3 ( 2.26
24 ( 10
40
41
H
H
H
H
OCH3
OCH3
H
H
H
H
12 ( 0.3
4.4 ( 0.1
42
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
OCH3
H
H
OCH3
H
OCH3
H
H
OCH3
OCH3
OCH3
H
H
>40
43
H
H
5.6 ( 1
>40
44
H
OCH3
H
OCH3
H
OCH3
H
H
H
45
OCH3
OCH3
OCH3
H
H
H
H
>40
>40
32 ( 4
>40
12 ( 3
1.5 ( 0.01
0.32 ( 0.06
46
H
47
OCH3
H
48
49
OCH3
H
50
PQ-7
97 ( 3
a
ITP ) inhibition of tubulin polymerization.
group at C-6 (IC50 1.1 µM). Thus, only a modest
correlation exists between the cytotoxicity data and
inhibitory effects on tubulin polymerization, with the
important exception that the most active agent in both
assays was compound 38. It should also be noted that
38 is structurally identical to PQ7, except for the
additional nitrogen atom in the B-ring. This modifica-
tion results in a major loss in cytotoxic activity for 38
relative to PQ7 (cf. data in Table 2 with the cytotoxicity
data in ref 7), a 3-4-fold loss in activity of 54 vs PQ7
as an inhibitor of tubulin polymerization, and a sub-
stantial reduction in activity of 54 vs PQ7 as an
inhibitor of colchicine binding (Table 3).
Turning to DHPQZ derivatives, the data obtained
show a wider range in activities depending on substitu-
ent pattern. Only two derivatives, compounds 43 and
50, showed significant inhibitory effects on tubulin
assembly, with IC50 values of 5.6 and 1.5 µM, respec-
tively. The third most active analogue, compound 49,
had an IC50 value of 12 µM. On comparing the data in
Tables 2 and 3 for the DHPQZ derivatives, good cor-
relation was found between inhibitory effects on tubulin
assembly and cytotoxicity. The most active analogue in
both assays was 50, followed in succession by 43 and
49. For this series, these derivatives indicate that
activity requires a m-methoxy substituent in the C-ring,
with substantial enhancement by a substituent at C-6.
This pattern repeats that previously observed with the
PZ, DHPQ, and PN series.
However, in comparing cognate PQZ and DHPQZ
derivatives, an interesting divergent pattern was ob-
served. In most cases, reduction of the B-ring resulted
in a loss of activity in inhibiting tubulin polymerization
(cf. 42 with 24, 44 with 26, 45 with 27, 46 with 28, 47
with 29, and 49 with 31). These compounds have either
no m-methoxy substituent in the C-ring or a second
C-ring methoxy group. The opposite was observed with
the two most active members of the DHPZQ series,
those with a single, m-methoxy substituent in the
C-ring. When 25 was reduced to 43, a 2-fold increase
occurred in the inhibitory effect on tubulin polymeri-
zation (if only one enantiomer of 43 is active, as is likely,
then a 4-fold increase in activity occurred). The en-
hancement of activity by reduction is even more dra-
matic with compounds 32 and 50: 8-fold for the racemic
mixture and, presumably, 16-fold for the active enan-
tiomer. These observations suggest some subtle interac-
tion, which has not been elucidated, exists between the
meta substituent in the C-ring and the heterocyclic
B-ring.

4486 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23
Hour et al.
2-(3′,4′-Dim eth oxyp h en yl)-4-qu in a zolin on e (30): 1.8 g
from 14 and 3,4-dimethoxybenzaldehyde (22) (1.2 g, 7.3 mmol),
pale yellow prism crystals.
2-(3′,5′-Dim eth oxyp h en yl)-4-qu in a zolin on e (31): 1.7 g
from 14 and 3,5-dimethoxybenzaldehyde (23) (1.2 g, 7.3 mmol),
pale yellow needles.
2-(3′-Met h oxyp h en yl)-6-ch lor o-4-qu in a zolin on e (32):
1.6 g from 2-amino-5-chlorobenzamide (15) (1.0 g, 5.9 mmol)
and 17 (0.8 g, 5.9 mmol), pale yellow needles.
2-(3′-Meth oxyph en yl)-6-flu or o-4-qu in azolin on e (33): 0.8
g from 2-amino-5-fluorobenzamide (5) (1.0 g, 6.5 mmol) and
17 (0.9 g, 6.5 mmol), pale yellow needles.
Con clu sion
This work has established preliminary SAR of PQZ
compounds and provided important information for
future QSAR molecular modeling study. Although 38,
the most potent compound in this study, is not as potent
as the previously identified PQ7, 38 belongs to a related
but different compound class and is worthwhile for
future in vivo investigation.
Exp er im en ta l Section
Ch em istr y. 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 spec-
trometer 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 at
China Medical College, Taiwan, and the results were within
(0.4% of the calculated values.
Gen er a l P r oced u r e for Syn th esis of Sta r tin g 2-Am i-
n oben za m id es. (a) Thionyl chloride (1.5 g, 12.6 mmol) was
added dropwise to a suspension of a 5-chloro-2-nitrobenzoic
acid (1) (1.0 g, 6.3 mmol) in benzene (30 mL) under reflux.
The resulting mixture was stirred under reflux for 4 h and
dried under vacuum. (b) The residue was dissolved in 200 mL
of benzene and treated with anhydrous ammonia gas at room
temperature. (c) After removing solvent, the intermediate
5-chloro-2-nitrobenzamide was dissolved in DMF (10 mL),
treated with the appropriate amine at 110 °C for 3 h, and
poured into ice water (300 mL). (d) The precipitate was
collected, washed with water, dried in vacuo, dissolved in
MeOH and hydrogenated over 10% Pd/C for 6 h. The catalyst
was removed by filtration, and the solution was dried under
vacuum to afford the substituted 2-aminobenzamides 9-13.
Compound 5 was prepared from 2-aminofluorobenzamide
by steps a and b only. Compounds 6 and 7 were prepared from
5-methoxy- or 4,5-dimethoxy-2-nitrobenzoic acid using steps
a, b, and d. Compound 8 was prepared by oxidation of 4,5-
methylenedioxy-2-nitrobenzaldehyde to the corresponding ben-
zoic acid using Conforth reagent (CrO3-pyridine-water)
followed by steps a, b, and d. Compounds 14 and 15 were
commercially available.
2-(3′-Meth oxyp h en yl)-6-m eth oxy-4-qu in a zolin on e (34):
0.7 g from 2-amino-5-methoxybenzamide (6) (1.0 g, 6.0 mmol)
and 17 (0.8 g, 6.0 mmol), pale yellow needles.
2-(3′-Met h oxyp h en yl)-6,7-d im et h oxy-4-q u in a zolin o-
n e (35): 0.5 g from 2-amino-4-methoxybenzamide (7) (1.0 g,
5.1 mmol) and 17 (0.7 g, 5.1 mmol), pale yellow prism crystals.
2-(3′-Meth oxyp h en yl)-6,7-m eth ylen ed ioxy-4-qu in a zoli-
n on e (36): 0.1 g from 2-amino-4,5-methylenedioxybenzamide
(8) (1.0 g, 5.5 mmol) and 17 (0.7 g, 5.5 mmol), pale yellow
needles.
2-(3′-M e t h o x y p h e n y l)-6-(N ,N -d i m e t h y la m i n o )-4-
qu in a zolin on e (37): 0.8 g from 2-amino-5-(N,N-dimethylami-
no)benzamide (9) (1.0 g, 5.6 mmol) and 17 (0.8 g, 5.6 mmol),
pale yellow needles.
2-(3′-Met h oxyp h en yl)-6-p yr r olid in yl-4-q u in a zolin o-
n e (38): 0.6 g from 2-amino-5-pyrrolidinylbenzamide (10) (1.0
g, 4.9 mmol) and 17 (0.7 g, 4.9 mmol), pale yellow prism
crystals.
2-(3′-Met h oxyp h en yl)-6-p ip er id in yl-4-q u in a zolin on e
(39): 0.8 g from 2-amino-5-piperidinylbenzamide (11) (1.0 g,
4.6 mmol) and 17 (0.6 g, 4.6 mmol), pale yellow prism crystals.
2-(3′-Me t h o x y p h e n y l)-6-(4-m e t h y lp ip e r id in y l)-4-
qu in a zolin on e (40): 0.9 g from 2-amino-5-(4-methylpiperidi-
nyl)benzamide 12 (1.0 g, 4.3 mmol) and 17 (0.6 g, 4.3 mmol),
pale yellow prism crystals.
2-(3′-Met h oxyp h en yl)-6-m or p h olin yl-4-q u in a zolin o-
n e (41): 0.7 g from 2-amino-5-morpholinylbenzamide (13) (1.0
g, 4.5 mmol) and 17 (0.6 g, 4.5 mmol), pale yellow needles.
2,3-Dih ydr o-2-(3′-m eth oxyph en yl)-4-qu in azolin on e (43).
Meth od A: 2-Aminobenzamide (14) (1.0 g, 7.3 mmol) and 17
(1.0 g, 7.3 mmol) were heated with stirring in DMAC (20 mL)
at 80 °C for 2 h and then poured into ice water (200 mL). The
precipitate was collected, washed with water, dried, and
purified by column chromatography (silica gel; ethyl acetate/
n-hexane) to afford 25 (0.3 g, 15.0%) and 43 (1.4 g, 75.2%) as
pale yellow powders.
2-(2′-Meth oxyp h en yl)-4-qu in a zolin on e (24). Sodium hy-
drogen sulfite (0.8 g, 7.5 mmol) was added to a solution of
2-aminobenzamide (14) (1.0 g, 7.3 mmol) and 2-methoxyben-
zaldehyde (16) (1.0 g, 7.3 mmol) in N,N-dimethylacetamide
(DMAC) (20 mL). The mixture was heated with stirring at 150
°C for 2 h and poured into ice water (200 mL). The precipitate
was collected, washed with water, and dried in vacuo. After
recrystallization from EtOH, 24 was obtained (1.6 g) as pale
yellow needles. Yield, melting point, and spectral data of 24
and all subsequent compounds are summarized in Table 1.
The method used to prepare 24 was used with the indicated
substituted benzaldehyde and benzamide to afford 25-41.
2-(3′-Meth oxyp h en yl)-4-qu in a zolin on e (25): 1.7 g from
14 and 3-methoxybenzaldehyde (17) (1.0 g, 7.3 mmol), pale
yellow needles.
Meth od B: p-Toluenesulfonic acid monohydrate (0.1 g, 0.3
mmol) was added to a solution of 14 (1.0 g, 7.3 mmol) and 17
(1.0 g, 7.3 mmol) in DMAC (20 mL). The mixture was stirred
at room temperature for 2 h and poured into ice water (200
mL). The precipitate was collected, washed with water, dried,
and purified by column chromatography (silica gel; ethyl
acetate/n-hexane) to afford 25 (70 mg, 4.0%) and 43 (1.7 g,
89.0%) as pale yellow powders.
Method B used for the preparation of 43 was used with the
appropriate benzaldehyde and benzamide to afford 42 and 44-
50, together with 24 and 44-32.
2,3-Dih ydr o-2-(2′-m eth oxyph en yl)-4-qu in azolin on e (42):
pale yellow powder, 24 (90.0 mg) and 42 (1.7 g) from 14 (1.0
g, 7.3 mmol) and 16 (1.0 g, 7.3 mmol).
2,3-Dih ydr o-2-(4′-m eth oxyph en yl)-4-qu in azolin on e (44):
pale yellow powder, 26 (40.0 mg) and 44 (1.7 g) from 14 (1.0
g, 7.3 mmol) and 18 (1.0 g, 7.3 mmol).
2-(4′-Meth oxyp h en yl)-4-qu in a zolin on e (26): 1.8 g from
14 and 4-methoxybenzaldehyde (18) (1.0 g, 7.3 mmol), pale
yellow needles.
2,3-Dih yd r o-2-(2′,3′-d im eth oxyp h en yl)-4-qu in a zolin o-
n e (45): pale yellow powder, 27 (80.1 mg) and 45 (1.6 g) from
14 (1.0 g, 7.3 mmol) and 19 (1.2 g, 7.3 mmol).
2-(2′,3′-Dim eth oxyp h en yl)-4-qu in a zolin on e (27): 1.8 g
from 14 and 2,3-dimethoxybenzaldehyde (19) (1.2 g, 7.3 mmol),
pale yellow needles.
2,3-Dih yd r o-2-(2′,4′-d im eth oxyp h en yl)-4-qu in a zolin o-
n e (46): pale yellow powder, 28 (99.9 mg) and 46 (1.8 g) from
14 (1.0 g, 7.3 mmol) and 20 (1.2 g, 7.3 mmol).
2-(2′,4′-Dim eth oxyp h en yl)-4-qu in a zolin on e (28): 1.7 g
from 14 and 2,4-dimethoxybenzaldehyde (20) (1.2 g, 7.3 mmol),
pale yellow needles.
2-(2′,5′-Dim eth oxyp h en yl)-4-qu in a zolin on e (29): 1.8 g
from 14 and 2,5-dimethoxybenzaldehyde (21) (1.2 g, 7.3 mmol),
pale yellow prism crystals.
2,3-Dih yd r o-2-(2′,5′-d im eth oxyp h en yl)-4-qu in a zolin o-
n e (47): pale yellow powder, 29 (80.1 mg) and 47 (1.7 g) from
14 (1.0 g, 7.3 mmol) and 21 (1.2 g, 7.3 mmol).

Quinazolinones and Related Compounds
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 23 4487
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M.; Hamel, E.; Lee, K. H. Synthesis and biological evaluation of
3′,6′,7-substituted 2-phenyl-4-quinolones as antimitotic antitu-
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S. C.; Hamel, E.; Hackl, T.; Lee, K. H. Synthesis and biological
evaluation of 6,7,2′,3′,4′-substituted-1,2,3,4-tetrahydro-2-phenyl-
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(9) Chen, K.; Kuo, S. C.; Hsih, M. C.; Mauger, A.; Lin, C. M.; Hamel,
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polymerization. J . Med. Chem. 1996, 40, 2266-2275.
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2-aryl-1,8-naphthyridin-4(1H)-ones as antimitotic antitumor
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2,3-Dih yd r o-2-(3′,4′-d im eth oxyp h en yl)-4-qu in a zolin o-
n e (48): pale yellow powder, 30 (60.2 mg) and 48 (1.6 g) from
14 (1.0 g, 7.3 mmol) and 22 (1.2 g, 7.3 mmol).
2,3-Dih yd r o-2-(3′,5′-d im eth oxyp h en yl)-4-qu in a zolin o-
n e (49): pale yellow powder, 31 (40.2 mg) and 49 (1.9 g) from
14 (1.0 g, 7.3 mmol) and 23 (1.2 g, 7.3 mmol).
2,3-Dih ydr o-2-(3′-m eth oxyph en yl)-6-ch lor o-4-qu in azoli-
n on e (50): colorless powder, 32 (20.0 g, 1.1%) and 50 (1.6 g,
95.0%) from 15 (1.0 g, 5.9 mmol) and 17 (0.8 g, 5.9 mmol).
Cytotoxicity Assa ys. Compounds 24-50 were assayed for
in vitro cytotoxicity in a panel of human tumor cell lines at
the School of Pharmacy, University of North Carolina at
Chapel Hill, according to procedures described previously.18-20
The cell lines included human ovarian cancer (1A9), ileocecal
carcinoma (HCT-8), lung carcinoma (A-549), glioblastoma (U-
87-MG), bone (HOS), epidermoid carcinoma of the nasophar-
ynx (KB), P-gp-expressing epidermoid carcinoma of the na-
sopharynx (KB-VIN), prostate cancer (PC3), breast cancer
(MCF-7), and melanoma (SKMEL-2) cell lines. The cytotoxic
effects of each compound were obtained as EC50 values, which
represent the molar drug concentrations required to cause 50%
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An tim icr otu bu le Assa y. Electrophoretically homogeneous
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The tubulin polymerization7 and colchicine binding assays22
were performed as described previously, except that Beckman
DU7400/7500 spectrophotometers equipped with “high-per-
formance” temperature controllers were used in the former
assays. Unlike the manual control possible with the previously
used Gilford spectrophotometers, the polymerization assays
required use of programs provided by MD Analytical Associ-
ates, South Plainfield, NJ , since the Beckman instruments are
microprocessor controlled. The Beckman instruments were
unable to maintain 0 °C, and the lower temperature in the
assays fluctuated between 2 and 5 °C. Temperature changes
were, however, more rapid than in the Gilford instruments
with the jump from the lower temperature to 26 °C taking
about 20 s and the reverse jump about 95 s.
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1996, 51, 53-59.
(14) J iang, J . B.; Hesson, D. P.; Dusak, B. A.; Dexter, D. L.; Kang,
G. J .; Hamel, E. Synthesis and biological evaluation of 2-styryl-
quinazolin-4(3H)-ones, a new class of antimitotic anticancer
agents which inhibit tubulin polymerization. J . Med. Chem.
1990, 33, 1721-1728.
(15) Lin, C. M.; Kang, G. J .; Roach, M. C.; J iang, J . B.; Hesson, D.
P.; Luduena, R. F.; Hamel, E. Investigation of the mechanism
of the interaction of tubulin with derivatives of 2-styrylquinazo-
lin-4(3H)-one. Mol. Pharmacol. 1991, 40, 827-832.
(16) Xia, Y.; Yang, Z. Y.; Hour, M. J .; Kuo, S. C.; Xia, P.; Bastow, K.
F.; Nakanishi, Y.; Nampoothiri, P.; Hackl, T.; Hamel, E.; Lee,
K. H. Synthesis and biological evaluation of substituted
2-arylquinazolinones. Bioorg. Med. Chem. Lett., submitted.
(17) Xia, Y.; Wang, Z. Y.; Morris-Natschke, S. L.; Lee, K. H. Recent
advances in the discovery and development of quinolones and
analogues as antitumor agents. Curr. Med. Chem. 1999, 6, 179-
194.
(18) Lee, K. H.; Lin, Y. M.; Wu, T. S.; Zhang, D. C.; Yamagishi, T.;
Hayashi, T.; Hall, I. H.; Chang, J . J .; Wu, R. Y.; Yang, T. H.
The cytotoxic principles of Prunella vulgaris, Psychotria serpens,
and Hyptis captitata: Ursolic acid and related derivatives.
Planta Med. 1988, 54, 308-312.
(19) Boyd, M. R. Status of the NCI preclinical antitumor drug
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Updates; De Vita, V. T., Hellman, S., Rosenberg, S. A., Eds; J .
B. Lippincoft: Philadelphia, 1989; pp 1-12.
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Vistica, D.; Hose, C.; Langley, J .; Cronise, P.; Vaigro-Woiff, A.;
Gray-Goodrich, M.; Campbell, H.; Mayo, J .; Boyd, M. Feasibility
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(21) Hamel, E.; Lin, C. M. Separation of active tubulin and micro-
tubule-associated proteins by ultracentrifugation and isolation
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Ack n ow led gm en t . This investigation was sup-
ported by research grants from the National Science
Council of the Republic of China (NSC88-2314-B-039-
010) awarded to S.-C. Kuo and from the National Cancer
Institute (CA 17625) awarded to K.-H. Lee.
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