Antitumor agents. 181. Synthesis and biological evaluation of 6,7,2',3',4'-substituted-1,2,3,4-tetrahydro-2-phenyl-4-quinolones as a new class of antimitotic antitumor agents

Article abstract of DOI:10.1021/jm9707479

A novel series of 6,7,2',3',4'-substituted-1,2,3,4-tetrahydro-2-phenyl- 4-quinolones were synthesized and evaluated for interactions with tubulin and for cytotoxic activity against a panel of human tumor cell lines, including ileocecal carcinoma (HCT-8), breast cancer (MCF-7), lung carcinoma (A-549), epidermoid carcinoma of the nasopharynx (KB), renal cancer (CAKI-1), and melanoma cancer (SKMEL-2). Most compounds (18, 20, 22-27) showed potent cytotoxic and antitubulin effects. The most active compounds (23, 26, 27) demonstrated strong cytotoxic effects with ED₅₀ values in the nanomolar or subnanomolar range in almost all tumor cell lines. Three active racemates (20, 22, 25) were separated into the enantiomers, and generally, the optically pure (-)-isomers (20a, 22a, 25a) exhibited greater biological activity than the racemates or (+)-isomers. Cytotoxicity and antitubulin activity were closely correlated, with the most active compounds (23, 26, 27) having effects comparable to those of colchicine, podophyllotoxin, and combretastatin A-4.

Full text of DOI:10.1021/jm9707479

1155
J . Med. Chem. 1998, 41, 1155-1162
An titu m or Agen ts. 181.† Syn th esis a n d Biologica l Eva lu a tion of
6,7,2′,3′,4′-Su bstitu ted -1,2,3,4-tetr a h yd r o-2-p h en yl-4-qu in olon es a s a New Cla ss of
An tim itotic An titu m or Agen ts
Yi Xia,§ Zheng-Yu Yang,§ Peng Xia,§ Kenneth F. Bastow,§ Yoko Tachibana,§ Sheng-Chu Kuo,| Ernest Hamel,‡
Torben Hackl,‡ 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, China Medical College, Taichung, Taiwan, and Laboratory of
Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment and
Diagnosis, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702
Received November 3, 1997
A novel series of 6,7,2′,3′,4′-substituted-1,2,3,4-tetrahydro-2-phenyl-4-quinolones were synthe-
sized and evaluated for interactions with tubulin and for cytotoxic activity against a panel of
human tumor cell lines, including ileocecal carcinoma (HCT-8), breast cancer (MCF-7), lung
carcinoma (A-549), epidermoid carcinoma of the nasopharynx (KB), renal cancer (CAKI-1), and
melanoma cancer (SKMEL-2). Most compounds (18, 20, 22-27) showed potent cytotoxic and
antitubulin effects. The most active compounds (23, 26, 27) demonstrated strong cytotoxic
effects with ED50 values in the nanomolar or subnanomolar range in almost all tumor cell
lines. Three active racemates (20, 22, 25) were separated into the enantiomers, and generally,
the optically pure (-)-isomers (20a , 22a , 25a ) exhibited greater biological activity than the
racemates or (+)-isomers. Cytotoxicity and antitubulin activity were closely correlated, with
the most active compounds (23, 26, 27) having effects comparable to those of colchicine,
podophyllotoxin, and combretastatin A-4.
Ch a r t 1. Antimitotic Natural Products
In tr od u ction
Microtubules are an important target for development
of compounds potentially useful as anticancer chemo-
therapeutics. Examples of such drugs are the vinca
alkaloids,2 which inhibit microtubule polymerization,
and taxoids,3 which promote microtubule assembly.
Colchicine4,5 (Chart 1) is another well-known agent that
inhibits microtubule assembly. Although too toxic to
be useful for cancer therapy, colchicine has been an
important tool in studies of microtubule structure and
function. The vinca alkaloids, taxoids, and colchicine
each interact with tubulin by a unique mechanism,
probably involving distinct binding sites on the protein.
A large number of compounds act as antimitotic
agents through interactions at the colchicine binding
site on tubulin, including the natural products podo-
phyllotoxin,6 cornigerine,7 steganacin,8 and combretast-
atins A-2 and A-49,10 (Chart 1). In addition, a variety
of heterocyclic ketones (Chart 2) are potent antimitotic
agents that inhibit the tubulin-colchicine interaction
and presumably bind in the same site on the protein.
Over 2 decades ago, 2,3-dihydro-2-aryl-4(1H)-quinazoli-
none (DHQZ) derivatives were reported to display
antitumor activities11,12 and thus were reevaluated in
the National Cancer Institute cancer cell line screen.
Significant inhibition of tubulin assembly and of the
binding of radiolabeled colchicine to tubulin13 was
demonstrated. 2-Styrylquinazolin-4(3H)-one (SQZ) de-
rivatives and flavonols have also been found to inhibit
tubulin polymerization, colchicine binding, and the
growth of L1210 murine leukemia cells.14-18
In our continuing study aimed at the discovery and
development of potential anticancer drug candidates, we
synthesized two series of substituted heterocyclic ke-
† For part 180, see ref 1.
* To whom correspondence should be addressed.
§ University of North Carolina at Chapel Hill.
| China Medical College.
‡ NCI.
S0022-2623(97)00747-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/06/1998

1156 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Ch a r t 2. Antimitotic Heterocyclic Ketones
Xia et al.
tones, 2-phenyl-4-quinolones (PQ)19-21 and 2-phenyl-1,8-
naphthyridin-4-ones (PN),22,23 and identified potent
cytotoxic antimitotic agents in each series. Structure-
activity relationship (SAR) studies of the quinolone class
led to the discovery of the particularly potent compound
PQ1, which totally inhibited the growth of about half
of the NCI tumor cell lines at subnanomolar concentra-
tions (log TGI < -9.0). The activity of PQ1 as an
inhibitor of tubulin polymerization and colchicine bind-
ing was comparable to those of colchicine, podophyllo-
toxin, and combretastatin A-4 in these assays.
These antimitotic heterocyclic ketones share a com-
mon structural feature, a biaryl system composed of
rings A and C, which are linked by an interposed B ring
and sometimes a hydrocarbon bridge. Their biaryl
system may be analogues to similar biaryl systems that
occur in the natural products shown in Chart 1. The
DHQZ, SQZ, PQ, and PN derivatives are aza com-
pounds, in which one or more nitrogen atoms are
present at the 1-, 3-, or 8-position, while the flavones
have an oxygen atom at position 1. In particular, the
DHQZ derivatives differ from the other classes in the
oxidation status of the bond between C(2) and N(3). In
a direct comparison, oxidation of the C(2)-N(3) bond
converted the 2,3-dihydroquinazolinone ring (DHQZ1)
to the quinazolinone ring (QZ1) and resulted in loss of
activity in the tubulin-based biochemical assays.13 This
finding suggests that configurational and conforma-
tional changes in the biaryl system are important in the
drug-tubulin interaction, as has been demonstrated
with colchicinoids and allocolchicinoids.1,4,5,24 Moreover,
the DHQZ studies11-13 were performed with racemic
mixtures, and it was reasonable to anticipate dif-
ferential activities in diastereoisomeric pairs, similar to
that which occur in colchicinoids and allocolchicino-
ids.1,4,5,24 These considerations prompted us to design,
synthesize, and evaluate a series of 2,3-dihydro-2-
phenyl-4(1H)-quinolone (DHPQ) derivatives, represent-
ing analogues of PQ derivatives with a reduced B ring
double bond. We also resolve the racemates of several
active compounds into the corresponding optically pure
enantiomers. As expected, the two isomers differed in
their biological activity, with the (-)-agents more active
than the (+)-isomers.
Ch em istr y
DHPQ derivatives were prepared from substituted 2′-
aminoacetophenones (3-5). Scheme 1 shows the syn-
thesis of 2′-amino-5′-pyrrolinylacetophenone (4) follow-
ing the literature methods.21,25 Nitration of 3′-chloro-
acetophenone (1) gave 2′-nitro-5′-chloroacetophenone.
Nucleophilic displacement of the 5′-chloro group by
pyrroline followed by hydrogenation gave compound 4.
Condensation of 3-5 with the appropriate benzaldehyde
(7) followed by acid-or base-catalyzed cyclization gave
26-29
the final products 18-27
(Scheme 2).
The optically active quinolones were not obtained
successfully by chromatography of the racemic mixtures

Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1157
Sch em e 1. Synthesis of 2′-Amino-5′-pyrrolinylacetophenone (4)
Sch em e 2. General Synthetic Routes to DHPQ Derivatives
on a chiral column or by separation of amide diastereo-
isomers obtained with optically pure (1S)-(-)-camphanic
chloride.
(MCF-7), lung carcinoma (A-549), epidermoid carcinoma
(KB), renal cancer (CAKI-1), and melanoma cancer
(SKMEL-2). As shown in Table 1, compounds 18, 20,
and 22-27 displayed significant activity, with ED50
values < 1.0 µg/mL in virtually all cases. In terms of
SAR information, compounds substituted at the 4′-
position (i.e., 21, 24) or 2′-position (i.e., 19, 22) were
substantially less active than those substituted at the
3′-position (i.e., 20, 23, 25), while compound 21 with a
methoxy group at the 4′-position was the least active.
(Methylenedioxy)benzene is a common moiety in many
antimitotic agents, such as podophyllotoxin, steganacin,
and combretastatin A-2 (Chart 1). However, the 6,7-
(methylenedioxy)-substituted compounds (22-25) did
not show any significant increase in activity compared
to an unsubstituted compound (18). Compound 26, with
a heterocyclic ring at the 6-position, was the most potent
compound with ED50 values in the nanomolar concen-
tration range. The effects of substitutions at the 6- and/
or 7-positions in ring A depend on the substitution in
ring C.
Because quinolones are the aza analogues of fla-
vonoids which react smoothly with an oxo reagent to
give the expected hydrazone, azine, and oxime deriva-
tives,30 19, 22, and 25 were reacted with the optically
active oxo reagent (-)-5-(R-phenethyl)semioxamazide,
as shown in Scheme 3. Compounds 19, 22, and 25 were
treated in MeOH with (-)-5-(R-phenethyl)semioxam-
azide to give diastereoisomeric mixtures (28a -30a ,
28b-30b). Separation of diastereomeric oxamazones
was achieved by means of fractional crystallization,
yielding crystalline, strongly levorotatory products (28a-
30a ) and less levorotatory products (28b-30b). Both
diastereoisomeric oxamazones 28a -30a and 28b-30b
were crystallized from methanol. Decomposition of the
oxamazones with dilute sulfuric acid afforded the opti-
cally active enantiomers of 19, 22, and 25. The enan-
tiomers showed identical NMR spectra but opposite
optical rotations.
The oxamazone derivatives 28-30 had minimal ac-
tivity as inhibitors of cell growth (Table 1). Neverthe-
less, these compounds displayed the widest range in
cytotoxic activity and may show tissue selectivity. In
particular, compound 29a showed highly selective ef-
fects on the ileocecal carcinoma line (HCT-8). Growth
of HCT-8 cell was inhibited by a 10-fold lower concen-
Resu lts a n d Discu ssion
a . Eva lu a tion of Cytotoxicty of DHP Q Der iva -
tives. The 6,7,2′,3′,4′-substituted DHPQ derivatives
and related compounds (18-30) were assayed for their
cytotoxicity in vitro against six human tumor cell lines,
including ileocecal carcinoma (HCT-8), breast cancer

1158 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Xia et al.
Sch em e 3. Resolution of (()-DHPQ Derivatives 20, 22, and 25 with (-)-5-(R-Phenethyl)semioxamazide
tration than the concentration required with the less
sensitive cell lines.
to tubulin. Close structural analogues that had been
studied previously (PQ1, PQ2, PQ3, and PN1; structures
in Chart 2) were reevaluated. Table 2 also summarizes
previous data, obtained under identical reaction condi-
tions, for colchicine, podophyllotoxin, and combretasta-
tin A-4. The inhibitory effects on tubulin activities were
in excellent agreement with the cytotoxicity data. The
cytotoxic compounds (18, 20, 22-27) all were substo-
ichiometric inhibitors of tubulin polymerization, and the
highly cytotoxic compounds (23, 26, 27) were also the
most potent inhibitors of colchicine binding. These
compounds had effects virtually identical to those of the
three natural products included for comparison. Con-
versely, the least cytotoxic compounds (19, 21, 28a ,b-
30a ,b) had little or no inhibitory effect on tubulin
polymerization (IC50 > 40 µM).
b. In ter a ction of DHP Q Der iva tives w ith Tu bu -
lin . Previously, PQ derivatives were found to inhibit
both tubulin polymerization and the binding of radio-
labeled colchicine to tubulin.19-21 The chief structural
difference between the PQ agents and the new series
of DHPQ derivatives is the oxidation status of the bond
between C(2) and C(3) in the B ring. This modification
results in configurational and conformational changes
in the relative positions 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 configuration and conformation
of the biaryl system formed by the trimethoxyphenyl A
ring and tropolonic C ring.1,4,5,24,31 Thus, evaluation of
the new agents for interactions with tubulin should
provide additional insight into the mechanism of ligand
binding at the colchicine site.
As with the PQ21 and PN22 derivatives, DHPQ com-
pounds with 3′-substitution were more active than those
with the same substituent at the 2′- or 4′-position.
Compounds with an o-methoxy (19) or p-methoxy (21)
substituent were inactive. This total loss of activity
with the methoxy substituent is probably steric in
Table 2 summarizes the effects of the DHPQ deriva-
tives as inhibitors of tubulin polymerization and, for the
most active compounds, on the binding of [3H]colchicine

Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1159
Ta ble 1. In Vitro Cytotoxic Activities of 2′,3′,4′,6,7-Substituted DHPQ Derivatives
ED50 (µg/mL)a
compd
KBb
A-549b
HCT-8b
CAKI-1b
MCF-7b
SKMEL-2b
18
19
20
20a
20b
21
0.07
1.8
0.11
0.12
0.50
0.013
6
0.013
6
0.013
25
0.13
6.5
0.13
12.5
0.2
0.23
0.15
0.50
>25
0.08
0.18
0.6
0.12
0.09
0.50
>25
0.13
0.09
0.50
0.016
0.4
0.32
0.15
3.8
0.32
0.30
0.60
>25
0.06
0.1
NDc
ND
>25
NDc
1.0
ND
0.125
ND
ND
0.016
0.2
22
0.06
0.08
0.43
0.012
0.3
22a
22b
23
1.0
1.25
0.016
0.8
0.85
0.032
0.4
0.012
1
24
25
0.02
0.02
0.07
0.008
0.05
8
0.04
0.03
0.09
0.01
0.09
8
0.04
0.02
0.08
0.016
0.09
3.8
0.06
0.25
0.25
0.008
0.95
25
>25
>25
18
0.04
0.03
0.12
0.11
0.31
8
0.06
ND
ND
0.016
0.08
ND
ND
ND
ND
ND
25a
25b
26
27
28a
28b
29a
29b
30a
30b
colchicine
>25
18
10
1.8
12
0.002
>25
13
9
2.4
11.5
0.002
>25
>25
2.1
9
1.0
1.5
8.5
25
3.0
6.3
>0.4
12.5
>25
0.4
ND
0.008
0.016
a
Cytotoxicity as ED50 for each cell line, the concentration of compound that causes a 50% reduction in adsorbance at 562 nm relative
to untreated cells using the SRB assay.32 bHuman ileocecal carcinoma (HCT-8), human breast cancer (MCF-7), human lung carcinoma
(A-549), human epidermoid carcinoma of the nasopharynx (KB), human renal cancer (CAKI-1), and human melanoma cancer (SKMEL-
2). c ND, not determined.
Ta ble 2. Antitubulin Effects of 2′,3′,4′,6,7-Substituted DHPQ Derivatives
ICBb (% inhib ( SD)
ITPa IC50
(µM) ( SD
compd
R6
R7
R2′
R3′
R4′
5 µMc
1 µMc
18
19
20
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
H
H
H
H
F
H
2.7 ( 0.5
>40
3.3 ( 0.1
1.2 ( 0.2
7.2 ( 0.9
>40
OCH3
H
H
H
H
F
H
H
H
H
OCH3
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
20a d
20bd
21
22
OCH2O
1.1 ( 0.2
0.56 ( 0.03
22a d
22bd
23
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
OCH2O
F
F
51 ( 2
12 ( 0.07
H
H
H
H
H
H
H
H
H
F
0.75 ( 0.04
2.0 ( 0.3
75 ( 0.9
24
25
H
OCH3
OCH3
OCH3
OCH3
Cl
OCH3
OCH3
H
0.82 ( 0.07
0.69 ( 0.01
2.3 ( 0.1
60 ( 3
67 ( 0.5
25a d
25bd
26
pyrrolinyl
pyrrolinyl
H
H
H
H
H
H
0.66 ( 0.03
0.76 ( 0.09
91 ( 0.3
89 ( 2
63 ( 5
63 ( 4
27
28a
28b
29a
29b
30a
30b
PQ1e
PQ2e
PQ3e
PN1e
>40
>40
>40
>40
>40
>40
OCH2O
OCH2O
OCH2O
OCH2O
F
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
pyrrolinyl
H
OCH2O
H
H
H
0.49 ( 0.07
1.5 ( 0.2
0.74 ( 0.09
0.79 ( 0.03
0.80 ( 0.07f
0.46 ( 0.02f
0.53 ( 0.05f
91 ( 0.2
65 ( 8
33 ( 2
24 ( 1
H
colchicine
podophyllotoxin
combretastatin A-4
92 ( 3
88 ( 0.4
a
ITP, inhibition of tubulin polymerization. bICB, inhibition of colchicine binding; evaluated only when polymerization IC50 e 1.0 µM.
c In the colchicine binding experiments, these values refer to the inhibitor concentration used. The [3H]colchicine concentration was 5
µM, and the tubulin concentration was 1 µM. See Scheme 3 for details of chiral configuration at C(2). e See Chart 2 for structures.
d
f Data from ref 20.

1160 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Xia et al.
(4.05 g, 30 mmol) and benzaldehyde (3.18 g, 30 mmol) in EtOH
(50 mL) was added NaOH (9 mL, 15%). The solution was
stirred at room temperature for 24 h and then filtered. The
resulting yellow precipitate was crystallized once from MeOH
origin, for compounds with the small F atom at the 2′-
position (22) or 4′-position (24) were still effective
inhibitors of polymerization, although maximum activ-
ity was still observed with the F at the 3′-position (23).
Methoxy, chloride, and fluoride groups at the 3′-position
all appeared to be equivalent in activity (i.e., 23 with
25, 26 with 27), whether an electron-donating group
(OCH3) or an electron-withdrawing group (F, Cl). The
size of the substituents can be as small as hydrogen or
as large as OCH3 without greatly affecting activity (i.e.,
18, 20). Methylenedioxy substitution in the A ring (25)
significantly (4-fold) increased activity (i.e., 25, 20), as
with PQ21 derivatives. Compounds with a pyrrolinyl
ring at the 6-position (26, 27) were exceptionally active,
particularly as inhibitors of colchicine binding. The
activity of the compounds with the 6-pyrrolinyl sub-
stituent approaches that of combretastatin A-4 as an
inhibitor of colchicine binding. Compounds 26 and 27,
as well as PQ1, inhibited colchicine binding over 60%
(as compared to 88% inhibition by combretastatin A-4)
when present at 1 µM in a reaction mixture containing
5 µM [3H]colchicine and 1 µM tubulin.
1
to afford yellow needles of 8 (4.35 g, 65%): mp 71-72 °C; H
NMR (CDCl3) δ 6.20 (brs, 2 H, NH2), 6.57-6.91 (m, 2 H, 3′-H
and 5′-H), 7.20-8.08 (m, 9 H); MS m/z 223 (M+). Anal.
(C15H13NO) C, H, N.
2′-Am in o-4-m eth oxych a lcon e (11): obtained from 2′-
aminoacetophenone and p-anisaldehyde; yield 86.0%, needles;
mp 91-92 °C; 1H NMR (CDCl3) δ 3.76 (s, 3 H, OCH3), 6.29
(brs, 2 H, NH2), 6.67-6.72 (m, 2 H, H-3′ and H-5′), 6.93 (m, 2
H, H-3 and H-5), 7.29 (m, 1 H, H-4′), 7.50 (d, 1 H, H-R, J )
15.3 Hz), 7.57-7.61 (m, 2 H, H-2 and H-6), 7.72 (d, 1 H, H-â,
J ) 15.3 Hz), 7.75 (m, 1 H, H-6′); MS m/ z 253 (M+). Anal.
(C16H15NO2) C, H, N.
2′-Am in o-4′,5′-(m et h ylen ed ioxy)-3-m et h oxych a lcon e
(15): obtained from 6′-amino-3′,4′-(methylenedioxy)aceto-
phenone and m-anisaldehyde; yield 54.5%, needles; mp 92-
1
94 °C; H NMR (CDCl3) δ 3.86 (s, 3 H, OCH3), 5.94 (s, 2 H,
OCH2O), 6.20 (s, 1 H, H-3′), 6.60 (brs, 2 H, NH2), 6.93 (m, 1
H, H-4), 7.13 (s, 1 H, H-6′), 7.20-7.35 (m, 3 H, H-2, H-5 and
H-6), 7.46 (d, 1 H, H-R, J ) 15.4 Hz), 7.67 (d, 1 H, H-â, J )
15.4 Hz); MS m/z 297 (M+). Anal. (C17H15NO2) C, H, N.
2′-Am in o-5′-p yr r olin yl-3-ch lor och a lcon e (17): obtained
from 2′-amino-5′-pyrrolinylacetophenone (4) and 3′-chlorobenz-
aldehyde; yield 69.4%, needles; mp 139-141 °C; 1H NMR
(CDCl3) δ 2.03 (m, 4 H, CH2CH2NCH2CH2), 3.30 (m, 4 H, CH2-
NCH2), 5.70 (brs, 2 H, NH2), 6.70 (d, 1 H, H-3′, J ) 8.8 Hz),
6.81 (dd, 1 H, H-4′, J ) 8.8, 2.6 Hz), 6.92 (d, 1 H, H-6′, J ) 2.6
Hz), 7.34-7.37 (m, 2 H, H-4 and H-6), 7.48 (m, 1 H, H-5), 7.54
(d, 1 H, H-R, J ) 15.6 Hz), 7.60 (s, 1 H, H-2), 7.66 (d, 1 H,
H-â, J ) 15.6 Hz); MS m/z 326 (M+). Anal. (C19H19ClN2O) C,
H, N.
Compounds 18-27 were all racemic mixtures. Be-
cause protein-ligand interactions are almost always
stereoselective, we obtained the pure enantiomers (-)-
20a , (+)-20b, (-)-22a , (+)-22b, (-)-25a , and (+)-25b.
Invariably, the racemic mixtures (20, 22, 25) were less
active than the (-)-enantiomers (20a , 22a , 25a ) and
more active than the (+)-enantiomers (20b, 22b, 25b).
The difference between optical isomers as inhibitors of
tubulin assembly ranged from 4-fold between 25a and
25b to 20-fold between 22a and 22b. The apparent
superiority of one enantiomer confirms the postulate
that the binding interaction with tubulin is stereo-
selective and suggests that, for all racemates, it is the
(-)-isomer that is primarily responsible for the inhibi-
tion of tubulin polymerization.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
18, 21, 25, a n d 27. A mixture of 2′-amino-4-methoxychalcone
(11) (760 mg, 3 mmol), HOAc (12.5 mL), and orthophosphoric
acid (12.5 mL) was warmed at 100 °C for 20 min. After cooling,
the mixture was poured into ice water. The product that
precipitated was purified by column chromatography on silica
gel using hexane-EtOAc (4:1) as eluant. Recrystallization
from hexanes-EtOAc afforded yellow needles of 21 (540 mg,
Our earlier study13 suggested that the DHQZ deriva-
tives were probably more active than the structurally
similar QZ analogues. The data presented in Table 2
lead to an analogous conclusion for DHPQ derivatives
versus their conjugate PQ derivatives. Thus, compound
20a appears to be about 20% more active than PQ2, as
an inhibitor of assembly, and 25a is more active than
PQ3, as judged from the colchicine inhibition data.
Moreover, 26 has activities comparable to those of PQ1,
suggesting that the (-)-enantiomer of 26 would be more
active. Finally, PN1 appears to be more active than
either 20a or PQ2, suggesting that reduction of the
C(2)-C(3) double bond in the PN derivatives would yield
compounds with still greater potency.
71.1%): mp 131-132 °C; [R]25 0° (c 0.40, CHCl3); 1H NMR
D
(CDCl3) δ 2.72 (q, 1 H, H-3, J ) -16.5, 3.9 Hz), 2.86 (q, 1 H,
H-3, J ) -16.5, 13.8 Hz), 3.82 (s, 3 H, OCH3), 4.44 (brs, 1 H,
NH), 4.70 (q, 1 H, H-2, J ) 3.9, 13.8 Hz), 6.69 (d, 1 H, H-8, J
) 8.2 Hz), 6.76-6.81 (m, 4 H, H-2′, H-3′, H-5′, and H-6′), 7.30-
7.40 (m, 2 H, H-6 and H-7), 7.87 (d, 1 H, H-5, J ) 7.8 Hz); MS
m/z 253 (M+). Anal. (C16H15NO2) C, H, N.
1,2,3,4-Tetr a h yd r o-2-p h en yl-4-qu in olon e (18): obtained
from 8; yield 67%, yellow needles; mp 149-150 °C; [R]25D 0° (c
0.30, CHCl3); 1H NMR (CDCl3) δ 2.72 (q, 1 H, H-3, J ) -16.4,
7.4 Hz), 2.90 (q, 1 H, H-3, J ) -16.4, 10.3 Hz), 4.75 (q, 1 H,
H-2, J ) 10.6, 7.4 Hz), 4.75 (brs, 1 H, NH), 6.70-7.07 (m, 2 H,
H-6 and H-8), 7.19-7.40 (m, 1 H, H-7), 7.45 (s, 5 H, C6H5),
7.93 (q, 1 H, H-5, J ) 9.0, 1.5 Hz); MS m/z 223 (M+). Anal.
(C15H13NO) C, H, N.
3′-Meth oxy-6,7-(m eth ylen ed ioxy)-1,2,3,4-tetr a h yd r o-2-
p h en yl-4-qu in olon e (25): obtained from 15; yield 75.2%,
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Fisher-
J ohns melting point apparatus without correction. Elemental
analyses were performed by Atlantic Microlabs, Atlanta, GA.
Optical rotations were determined with a DIP-1000 polarim-
eter. 1H NMR spectra were measured on a Bruker AC-300
spectrometer with TMS as internal reference and CDCl3 as
solvent. Mass spectral (MS) data were obtained on a TRIO
1000 mass spectrometer. Flash chromatography was per-
formed on silica gel (mesh 25-150 µm) using a mixture of
hexanes-EtOAc as eluant. Where not noted, compounds were
recrystallized once from the same solvent given in the sample
procedure.
yellow plates; mp 215-216 °C; [R]25 0°(c 0.32, CHCl3); 1H
D
NMR (CDCl3) δ 2.80 (q, 1 H, H-3, J ) -16.8, 4.5 Hz), 2.75 (q,
1 H, H-3, J ) -16.8, 13.4 Hz), 3.84 (s, 3 H, OCH3), 4.39 (brs,
1 H, NH), 4.69 (q, 1 H, H-2, J ) 4.5, 13.4 Hz), 5.95 (s, 2 H,
OCH2O), 6.19 (s, 1 H, H-8), 6.88 (m, 1 H, H-4′), 6.90 (s, 1 H,
H-2′), 7.00 (m, 1 H, H-5), 7.27-7.35 (m, 2 H, H-5′ and H-6′);
MS m/z 297 (M+). Anal. (C17H15NO4) C, H, N.
3′-Ch lor o-6-p yr r olin yl-1,2,3,4-t et r a h yd r o-2-p h en yl-4-
q u in olon e (27): obtained from 17; yield 71.4%, orange
needles; mp 197-198 °C; [R]25 0°(c 0.32, CHCl3); 1H NMR
D
(CDCl3) δ 2.00 (m, 4 H, CH2CH2NCH2CH2), 2.70-2.87 (m, 2
H, H-3), 3.27 (m, 4 H, CH2NCH2), 4.10 (brs, 1 H, NH), 4.67 (q,
1 H, H-2, J ) 12.9, 4.1 Hz), 6.70 (m, 1 H, H-8), 6.82 (m, 1 H,
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
8, 11, 15, a n d 17.27 To a solution of 2′-aminoacetophenone

Antitumor Agents
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7 1161
H-7), 7.03 (s, 1 H, H-5), 7.33 (m, 3 H, H-4′, H-5′, and H-6′),
7.51 (s, 1 H, H-2′); MS m/z 326 (M+). Anal. (C19H19ClN2O) C,
H, N.
afforded 28a (310 mg, 35.1%): needles; mp 167-168 °C; [R]25
D
1
-176° (c 0.41, CHCl3); H NMR (CDCl3) δ 1.58 (d, 3 H, CH3),
2.61 (1 H, B-part of ABX, H-3a, J 3e,3a ) 16.1 Hz), 3.03 (1 H,
A-part of ABX, H-3e), 3.83 (s, 3 H, OCH3), 4.25 (s, 1 H, NH-1),
4.43 (dd, 1 H, H-2, J 2,3e ) 4.0 Hz, J 2,3a ) 12.9 Hz), 5.06 (m, 1
H, CH-Ph), 6.65 (d, 1 H, H-8), 6.80-7.27 (m, 11 H, aromatic),
7.76 (d, 1 H, CNH), 8.20 (d, 1 H, H-5), 10.06 (s, 1 H, NNH);
MS m/z 442 (M+). Anal. (C26H26N4O3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
19, 20, 22-24, a n d 26. NaOH (0.8 mL, 15%) was added to a
stirred solution of 2′-aminoacetophenone (2.7 g, 20 mmol) and
m-anisaldehyde (4.08 g, 30 mmol) in EtOH (8 mL) at room
temperature. After refluxing for 30 min, the mixture was
evaporated to dryness. The residue was added with stirring
in one portion to diphenyl ether (10 mL) at 180 °C. After 30
min, the mixture was cooled to room temperature and diluted
with hexanes. The precipitate was collected and purified by
chromatography on a silica gel column. Elution with hex-
anes-EtOAc (4:1) and recrystallization from hexanes-EtOAc
Concentration of the mother liquors of 28a and recrystal-
lization from MeOH afforded 28b (270 mg, 30.5%): needles;
mp 180-182 °C; [R]25
-42.3° (c 0.40, CHCl3); 1H NMR
D
spectrum of 28b was essentially identical with the spectrum
of compound 28a ; MS m/z 442 (M+). Anal. (C26H26N4O3) C,
H, N.
afforded 20 (2.26 g, 44.7%): needles; mp 211-213 °C; [R]25
D
Compounds 29a ,b and 30a ,b were prepared from 22 and
25, respectively, using the same procedure described above for
28a ,b. 1H NMR and MS data of the enantiomers were
comparable.
1
0° (c 0.345, CHCl3); H NMR (CDCl3) δ 2.76 (q, 1 H, H-3, J )
-16.2, 4.3 Hz), 2.87 (q, 1 H, H-3, J ) -16.2, 13.3 Hz), 3.80 (s,
3 H, OCH3), 4.50 (brs, 1 H, NH), 4.75 (q, 1 H, H-2, J ) 4.3,
13.3 Hz), 6.73 (m, 1 H, H-8), 6.81 (m, 1 H, H-4′), 6.90-6.91
(m, 3 H, H-2′, H-5′, and H-6′), 7.30-7.38 (m, 2 H, H-6 and
Dia ster eoisom er ic (-)-5-(r-p h en eth yl)oxa m a zon es of
2′-flu or o-6,7-m eth ylen edioxy-2-ph en yl-1,2,3,4-tetr ah ydr o-
4-qu in olon e (29a , 29b): yield 20%, needles; mp 239-241 °C;
H-7), 7.87 (m, 1 H, H-5); MS m/z 253 (M+). Anal. (C16H15
-
NO2) C, H, N.
[R]25 -17.4° (c 0.31, CHCl3); 1H NMR (CDCl3) δ 1.58 (d, 3 H,
D
2′-Meth oxy-1,2,3,4-tetr ah ydr o-2-ph en yl-4-qu in olon e (19):
obtained from 2′-aminoacetophenone and o-anisaldehyde; yield
CH3), 2.59 (1 H, B-part of ABX, H-3a, J 3e,3a ) 15.9 Hz), 3.03 (1
H, A-part of ABX, H-3e), 4.06 (s, 1 H, NH-1), 4.82 (dd, 1 H,
H-2, J 2,3e ) 3.8 Hz, J 2,3a ) 12.0 Hz), 5.05 (m, 1 H, CH-Ph),
5.92 (s, 2 H, OCH2O), 6.18 (s, 1 H, H-8), 7.06-7.56 (m, 9 H,
aromatic), 7.65 (s, 1 H, H-5), 7.78 (d, 1 H, CNH), 10.00 (s, 1 H,
NNH); MS m/z 474 (M+). Anal. (C26H23FN4O4) C, H, N.
45.7%, yellow needles; mp 126-128 °C; [R]25 0° (c 0.38,
D
1
CHCl3); H NMR (CDCl3) δ 2.85 (q, 1 H, H-3, J ) -16.2, 4.7
Hz), 2.95 (q, 1 H, H-3, J ) -16.2, 11.4 Hz), 3.87 (s, 3 H, OCH3),
4.62 (brs, 1 H, NH), 5.19 (q, 1 H, H-2, J ) 4.7, 11.4 Hz), 6.70-
6.80 (m, 2 H, H-8 and Η-3′), 7.30-7.90 (m, 2 H, H-6 and H-7),
6.90-7.00 (m, 3 H, H-4′, H-5′, and H-6′), 7.88 (m, 1 H, H-5′);
MS m/z 253 (M+). Anal. (C16H15NO2) C, H, N.
29b: yield 29.4%, needles; mp 257-259 °C; [R]25 +100.5°
D
(c 0.39, CHCl3).
Dia ster eoisom er ic (-)-5-(r-p h en eth yl)oxa m a zon es of
3′-m eth oxy-6,7-(m eth ylen edioxy)-2-ph en yl-1,2,3,4-tetr ah y-
d r o-4-qu in olon e (30a ,b): yield 26%, needles; mp 198-200
2′-F lu or o-6,7-(m et h ylen ed ioxy)-1,2,3,4-t et r a h yd r o-2-
p h en yl-4-qu in olon e (22): obtained from 6′-amino-3′,4′-(me-
thylenedioxy)acetophenone and 2-fluorobenzaldehyde; yield
°C; [R]25 -143.6° (c 0.33, CHCl3); 1H NMR (CDCl3) δ 1.57 (d,
D
44.3%, amorphous; mp 229-231 °C; [R]25 0° (c 0.32, CHCl3);
D
3 H, CH3), 2.56 (1 H, B-part of ABX, H-3a, J 3e,3a ) 15.7 Hz),
3.00 (1 H, A-part of ABX, H-3e), 3.83 (s, 3 H, OCH3), 4.12 (s,
1 H, NH-1), 4.38 (dd, 1 H, H-2, J 2,3e ) 7.15 Hz, J 2,3a ) 14.6
Hz), 5.06 (m, 1 H, CH-Ph), 5.92 (s, 2 H, OCH2O), 6.17 (s, 1 H,
H-8), 6.89-7.38 (m, 9 H, aromatic), 7.65 (s, 1 H, H-5), 7.78 (d,
1 H, CNH), 9.99 (s, 1 H, NNH); MS m/z 486 (M+). Anal.
(C27H26N4O5) C, H, N.
1H NMR (CDCl3) δ 2.84 (m, 2 H, H-3), 4.39 (brs, 1 H, NH),
5.10 (m, 1 H, H-2), 5.95 (s, 2 H, OCH2O), 6.21 (s, 1 H, H-8),
7.07-7.59 (m, 5 H, aromatic); MS m/z 285 (M+). Anal. (C16H12
FNO3) C, H, N.
-
3′-F lu or o-6,7-(m et h ylen ed ioxy)-1,2,3,4-t et r a h yd r o-2-
p h en yl-4-qu in olon e (23): obtained from 6′-amino-3′,4′-(me-
thylenedioxy)acetophenone and 3-fluorobenzaldehyde; yield
48.1%, orange plates; mp 231-233 °C; [R]25D 0° (c 0.32, CHCl3);
1H NMR (CDCl3) δ 2.75 (q, 1 H, H-3, J ) -16.5, 5.6 Hz), 2.80
(q, 1 H, H-3, J ) -16.5, 12.1 Hz), 4.40 (brs, 1 H, NH), 4.72 (q,
1 H, H-2, J ) 5.6, 12.1 Hz), 5.94 (s, 2 H, OCH2O), 6.21 (s, 1 H,
H-8), 7.02-7.42 (m, 5 H, aromatic); MS m/z 285 (M+). Anal.
(C16H12FNO3) C, H, N.
30b: yield 31.2%, needles; mp 218-220 °C; [R]25D -14.9° (c
0.35, CHCl3).
(-)-3 ′-M e t h o x y -2 -p h e n y l -1 ,2 ,3 ,4 -t e t r a h y d r o -4 -
qu in olon e (20a ). H2SO4 (25%) (8 mL) was added to a solution
of 28a (200 mg, 0.45 mmol) in EtOH (8 mL). After the mixture
refluxed for 1 h, the EtOH was evaporated, and the reaction
mixture was cooled, made neutral with NaHCO3, and extracted
with CHCl3. The CHCl3 phase was washed with water, dried
over Na2SO4, and evaporated to dryness. The residue was
purified by column chromatography using hexane-EtOAc (4:
1) as eluant to afford 20a (70 mg, 61.1%): needles; mp 129-
4′-F lu or o-6,7-(m et h ylen ed ioxy)-1,2,3,4-t et r a h yd r o-2-
p h en yl-4-qu in olon e (24): obtained from 6′-amino-3′,4′-(me-
thylenedioxy)acetophenone and 4-fluorobenzaldehyde; yield
44.8%, amorphous; mp 238-241 °C; [R]25 0° (c 0.35, CHCl3);
D
1H NMR (CDCl3) δ 2.66-2.88 (m, 2 H, H-3), 4.36 (brs, 1 H,
NH), 4.70 (m, 1 H, H-2), 5.95 (s, 2 H, OCH2O), 6.20 (s, 1 H,
H-8), 7.12 (s, 1 H, H-5), 7.30 (m, 2 H, H-2′ and H-6′), 7.45 (m,
2 H, H-3′ and H-5′); MS m/z 285 (M+). Anal. (C16H12FNO3)
C, H, N.
131 °C; [R]25 -19.5° (c 0.40, CHCl3); 1H NMR spectrum of
D
20a was identical with the spectrum of racemic 20.
Compounds 20b, 22a , 22b, 25a , and 25b were prepared in
the same manner from 28b, 29a , 29b, 30a , and 30b, respec-
tively. 1H NMR spectra were identical with those of the
racemic compounds.
3′-Meth oxy-6-p yr r olin yl-1,2,3,4-tetr a h yd r o-2-p h en yl-4-
qu in olon e (26): obtained from compound 4 and m-anisalde-
hyde; yield 42.2%, yellow needles; mp 194-196 °C; [R]25D 0° (c
0.32, CHCl3); 1H NMR (CDCl3) δ 2.03 (m, 4 H, CH2CH2-
NCH2CH2), 2.78 (q, 1 H, H-3, J ) -16.5, 4.3 Hz), 2.87 (q, 1 H,
H-3, J ) -16.5, 13.4 Hz), 3.28 (m, 4 H, CH2NCH2), 3.84 (s, 3
H, OCH3), 4.14 (brs, 1 H, NH), 4.67 (q, 1 H, H-2, J ) 4.3, 13.4
Hz), 6.70-7.34 (m, 7 H, aromatic); MS m/z 322 (M+). Anal.
(C20H22N2O2) C, H, N.
Resolu tion of (()-1,2,3,4-Tetr a h yd r o-2-p h en yl-4-qu i-
n olon es 20, 22, a n d 25 w ith (-)-5-(R-P h en eth yl)sem iox-
a m a zid e. Racemic 20 (506 mg, 2 mmol) was dissolved in
boiling MeOH (30 mL), and (-)-5-(R-phenethyl)semioxamazide
(520 mg, 2.5 mmol) was added gradually together with a piece
of crystalline iodine. The mixture was refluxed for 3.5 h. After
cooling to room temperature, the yellow crystalline product
was separated by filtration. Recrystallization from MeOH
(+)-3 ′-M e t h o x y -2 -p h e n y l -1 ,2 ,3 ,4 -t e t r a h y d r o -4 -
qu in olon e (20b): yield 72.1%, needles; mp 129-131 °C; [R]25
D
+19.5° (c 0.40, CHCl3).
(-)-2′-F lu or o-6,7-(m et h ylen ed ioxy)-2-p h en yl-1,2,3,4-
tetr a h yd r o-4-qu in olon e (22a ): yield 61.1%, needles; mp
214-215 °C; [R]25 -139° (c 0.31, CHCl3).
D
(+)-2′-F lu or o-6,7-(m et h ylen ed ioxy)-2-p h en yl-1,2,3,4-
tetr a h yd r o-4-qu in olon e (22b): yield 68.2%, needles; mp
214-216 °C; [R]25 +143° (c 0.34, CHCl3).
D
(-)-2′-Meth oxy-6,7-(m eth ylen ed ioxy)-2-p h en yl-1,2,3,4-
tetr a h yd r o-4-qu in olon e (25a ): yield 65.5%, needles; mp
230-232 °C; [R]25 -48° (c 0.30, CHCl3).
D
(+)-2′-Meth oxy-6,7-(m eth ylen ed ioxy)-2-p h en yl-1,2,3,4-
tetr a h yd r o-4-qu in olon e (25b): yield 81.8%, needles; mp
229-232 °C; [R]25 +50° (c 0.29, CHCl3).
D

1162 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Xia et al.
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Biologica l Assa ys. The in vitro cytotoxicity assay was
carried out according to procedures described in Rubinstein
et al.32 Drug stock solutions were prepared in DMSO, and the
final solvent concentration was e2% DMSO (v/v), a concentra-
tion without effect on cell replication. The human tumor cell
line panel constituted of epiderimoid carcinoma of the na-
sopharynx (KB), lung carcinoma (A-549), ileocecal carcinoma
(HCT-8), renal cancer (CAKI-1), breast cancer (MCF-7), and
melanoma cancer (SKMEL-2). Cells were cultured at 37 °C
in RPMI-1640 with 100 µg/mL kanamycin and 10% (v/v) fetal
bovine serum in a humidified atmosphere containing 5% CO2.
Initial seeding densities varied among the cell lines to ensure
a final absorbance reading in control cultures in the range
1-2.5 A562 units. Drug exposure was for 3 days, and the ED50
value, the drug concentration that reduced the absorbance by
50%, was interpolated from dose-response data. Each test
was performed in triplicate, and absorbance readings varied
no more than 5%. ED50 values determined in independent
tests varied no more than 30%.
The tubulin polymerization and [3H]colchicine binding as-
says were performed as described previously.21 For the
polymerization assay a 25% difference in IC50 values repre-
sents a reproducible difference in the relative activity of two
agents, while in the colchicine binding assay a 10% difference
is usually reproducible.
Ack n ow led gm en t . This investigation was sup-
ported by a grant from the National Cancer Institute
(CA-17625) awarded to K. H. Lee. We would like to
thank Dr. Susan Morris-Natschke for her critical read-
ing of the manuscript, many valuable suggestions, and
assistance.
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