Synthesis and structure-activity relationship of 2-aminobenzophenone derivatives as antimitotic agents

Article abstract of DOI:10.1021/jm010365+

A new type of inhibitor of tubulin polymerization was discovered on the basis of the combretastatin molecular skeleton. The lead compounds in this series, compounds 6 and 7, strongly inhibited tubulin polymerization in vitro and significantly arrested cel

Full text of DOI:10.1021/jm010365+

2556
J . Med. Chem. 2002, 45, 2556-2562
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip of 2-Am in oben zop h en on e
Der iva tives a s An tim itotic Agen ts
J ing-Ping Liou,† Chun-Wei Chang,† J eng-Shin Song,‡ Yung-Ning Yang,‡ Ching-Fang Yeh,† Huan-Yi Tseng,‡
Yu-Kang Lo,‡ Yi-Ling Chang,‡ Chung-Ming Chang,‡ and Hsing-Pang Hsieh*,†
Medicinal Synthetic Laboratory and Molecular Biology Laboratory, Division of Biotechnology and Pharmaceutical Research,
National Health Research Institutes, Taipei, Taiwan, Republic of China
Received August 3, 2001
A new type of inhibitor of tubulin polymerization was discovered on the basis of the
combretastatin molecular skeleton. The lead compounds in this series, compounds 6 and 7,
strongly inhibited tubulin polymerization in vitro and significantly arrested cells at the G2/M
phase. Compounds 6 and 7 yielded 50- to 100-fold lower IC50 values than did combretastatin
A-4 against Colo 205, NUGC3, and HA22T human cancer cell lines as well as similar or greater
growth inhibitory activities than did combretastain A-4 against DLD-1, HR, MCF-7, DU145,
HONE-1, and MES-SA/DX5 human cancer cell lines. Structure-activity relationship informa-
tion revealed that introduction of an amino group at the ortho position of the benzophenone
ring plays an integral role for increased growth inhibition.
In tr od u ction
tion. The trans-forms of these compounds display dra-
matically reduced inhibition of cancer cell growth and
tubulin polymerization.8c Pettit’s group recently syn-
thesized two new benzophenones, phenstatin and hy-
droxyphenstatin, which display potent anticancer and
antimitotic activities comparable to CA-4.9 On the basis
of geometric comparisons, it was suggested that the sp2-
hybridized carbonyl group present in phenstatin and
hydroxyphenstatin constrains the two aryl rings in a
quasi “cis” orientation that appears to be necessary for
significant biological activity.9b
Benzophenone-type CA-4 analogues are attractive
targets for anti-tubulin agents as the benzophenone
backbone not only provides ease of synthesis without
the need to control the geometric selectivity (Z and E
geometry) but also increases the pharmacological po-
tential through increased drug stability and water
solubility.7f,g In the present study, introduction of an
amino group at the ortho position of the B-ring was
expected to maintain the quasi cis conformation to
obtain more potent anti-tubulin agents and also increase
water solubility by potential salt formation. Further-
more, we envisioned that variation of the substituents
on the B-ring would provide significant SAR information
about antitumor activity and the colchicine binding site
on â-tubulin.
The microtubule system of eukaryotic cells is an
important target for the development of anticancer
agents. Consequently, perturbation of normal tubulin
polymerization/depolymerization is a popular target for
new chemotherapeutic agents.1 A variety of clinically
used compounds such as vinca alkaloids, colchicine,
podophyllotoxin, paclitaxel, and epothilone act on mi-
crotubules or tubulin to disrupt the cellular microtubule
structure and cause mitotic arrest (Chart 1).2 Combre-
tastatin A-4 (CA-4), isolated by Pettit and co-workers
in 1982 from the stem wood of the South African tree
Combretum caffrum, is one of the most potent antimi-
totic agents.3 It exhibits strong growth inhibition against
a wide variety of human cancer cells, including multi-
drug resistant cancer cells.4,5 Moreover, CA-4 is also
structurally similar to colchicine and possesses a higher
affinity for the colchicine binding site on tubulin than
does colchicine itself.4b However, the low water solubility
of CA-4 limits its efficacy in vivo. A water-soluble
sodium phosphate prodrug (CA-4P, OXIGENE, Boston,
MA) is currently in phase I/II clinical trials.6
Because CA-4 is currently the simplest structure for
a natural product that binds to tubulin, its remarkable
anticancer activity has attracted the attention of many
medicinal chemists for the rational design of antitubulin
agents.7 A number of structure-activity relationships
(SARs) have been reported for the combretastatins.8
These studies indicate that the cis orientation of the two
aromatic rings (Z geometry) is the most important factor
for the inhibition of cancer cell growth. Z-Combretasta-
tin analogues, however, are prone to isomerization
(trans-forms/E-isomer) during storage and administra-
Ch em istr y
The general synthetic strategy shown in Scheme 1
was employed for the preparation of the new 2-ami-
nobenzophenone analogues. Grignard reaction of (3,4,5-
trimethoxyphenyl)magnesium bromide10 with several
commercially available or synthesized substituted 2-ni-
trobenzaldehydes yielded benzhydrol derivatives A in
80% yield. The substituted 2-nitrobenzophenone deriva-
tives B were obtained by pyridinium dichromate (PDC)
oxidation of the substituted benzhydrol derivatives. In
most cases, the benzhydrol derivatives were used with-
out purification. The desired 2-nitrobenzophenones were
obtained in 65% overall yield (two steps). Further
* Corresponding author. Phone: 886-2-2695-4246, ext. 305. Fax:
886-2-2695-3759. E-mail: hphsieh@nhri.org.tw. Corresponding ad-
dress: Division of Biotechnology and Pharmaceutical Research,
National Health Research Institutes, 128 Yen-Chiu-Yuan Road, Sec
II, Taipei 115, Taiwan, Republic of China.
† Medicinal Synthetic Laboratory.
‡ Molecular Biology Laboratory.
10.1021/jm010365+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/11/2002

2-Aminobenzophenones as Antimitotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2557
Ch a r t 1
Sch em e 1a
panel of human cancer cell lines using the MTS assay.11
The IC50 values represent the drug concentrations
producing a 50% decrease in cell growth after 3 days of
incubation.
We first evaluated the effect of a hydroxyl substitution
at the C-2 position for growth inhibitory activity. A
hydroxyl group at the ortho position of the B-ring
resulted in less potent activity as compared to phen-
statin regardless of whether the methoxy substituent
was located at the C-4 or C-5 position (1 and 2). Removal
of the hydroxyl group at the C-2 position (4 and 5)
resulted in slightly reduced growth inhibition as com-
pared to phenstatin. Lack of a methoxy group (com-
pound 3) resulted in significantly decreased growth
inhibitory activity. A surprising observation was that
placement of the methoxy group at the C-4 position
on the B-ring is less important than previously
thought,7f,8d,12b since analogues 2 and 5 with the meth-
oxy group shifted to the C-5 position have growth
inhibitory properties similar to analogues 1 and 4 with
the methoxy group at the C-4 position.
a
(a) THF, 0-25 °C; (b) PDC, CH2Cl2, 25 °C; (c) Fe, AcOH, EtOH,
reflux.
Sch em e 2a
Introduction of an amino group at the ortho position
produced compounds 6 and 7, which showed signifi-
cantly increased growth inhibition against many of the
cancer cell lines compared to phenstatin. It is interesting
that a methoxy group at the C-3 position (8) resulted
in complete loss or significant reduction of growth
inhibition against the 10 cancer cell lines. The inactivity
of the 4,5-dimethoxy analogue 12 and 5,6-dimethoxy
analogue 13 indicates that bulky substituents on the
B-ring are detrimental to activity. The moderate potency
of the 4,5-methylenedioxy bridge analogue 14 suggests
that a methylenedioxy substitution on the B-ring may
mimic natural anticancer products such as podophyl-
lotoxin and steganacin.1 Replacement of a 5-methoxy
group (7) with a bulky N,N-dimethylamino group (9)
decreased growth inhibition by 2 orders of magnitude.
An order of magnitude increase in potency of the C-2
amino analogue 10 and the C-3 amino analogue 11 as
compared to the C-2 hydroxyl analogue 3 indicates that
introduction of an amino group at the appropriate
position positively contributes to growth inhibition.
a
(a) THF, 0-25 °C; (b) PDC, CH2Cl2, 25 °C; (c) TBAF, THF, 25
°C.
reduction of the nitro group of benzophenone B with
Fe/AcOH yielded the corresponding substituted 2-
aminobenzophenones C in yields over 95%.
The preparation of 2-hydroxybenzophenone analogues
is shown in Scheme 2. The nucleophilic reaction was
incomplete and complicated when 2 equiv of (3,4,5-
trimethoxyphenyl)magnesium bromide was reacted with
1 equiv of substituted salicylaldehydes. The method was
improved by first protecting the hydroxyl group of
substituted salicylaldehydes as silyl ethers by tert-
butyldimethylsilyl chloride (TBSCl) to obtain 2-(tert-
butyldimethylsilanyloxy)benzhydrol derivatives. Fol-
lowing the approach outlined above, preparation of the
2-hydroxybenzophenone analogues D was accomplished
in 70-90% yield by Grignard addition, PDC oxidation,
and tetrabutylammonium fluoride (TBAF) deprotection.
Biologica l Resu lts a n d Discu ssion
To summarize SAR information (1-14) about growth
inhibition, first, the growth inhibitory tendency at the
C-2 position was NH2 > H > OH, on the basis of the
IC50 values of 1 vs 6, 2 vs 7, 3 vs 10, 4 vs 6, and 5 vs 7.
(A) In Vitr o Cell Gr ow th In h ibition Assa y. A
comparison of IC50 values for benzophenone analogues
(1-14) is provided in Table 1. All 14 compounds were
evaluated for in vitro cell growth inhibition against a

2558 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Liou et al.

2-Aminobenzophenones as Antimitotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2559
Ta ble 2. Inhibition of Colchicine Binding to Tubulin for
Compounds 2, 6-8, and Phenstatin at 20 µM
% Inhibition ( SDa
2
6
7
8
R1 ) OH, R2 ) R3 ) R5 ) H, R4 ) OCH3
R1 ) NH2, R2 ) R4 ) R5 ) H, R3 ) OCH3
R1 ) NH2, R2 ) R3 ) R5 ) H, R4 ) OCH3
R1 ) NH2, R3 ) R4 ) R5 ) H, R1 ) OCH3
phenstatin
50 ( 2
82 ( 4
85 ( 2
8 ( 1
93 ( 3
a
All experiments were independently performed three times.
Ta ble 3. Effectsa of Compounds 2, 6-8, and Phenstatin on
Cell Cycle Progression
compd
G0/G1 (%)
S-phase (%)
G2/M (%)
control (DMSO)
2 (1 µM)
67.7 ( 3.1
59.7 ( 2.8
6.3 ( 1.8
5.2 ( 1.1
67.6 ( 3.0
64.5 ( 1.6
13.6 ( 1.8
13.1 ( 3.1
3.8 ( 0.7
3.2 ( 1.1
12.5 ( 0.5
15.7 ( 0.4
19.0 ( 1.7
27.5 ( 3.3
90.1 ( 2.2
91.6 ( 3.2
20.2 ( 3.2
20.1 ( 1.6
6 (1µM)
7 (1 µM)
8 (1 µM)
phenstatin (1 µM)
F igu r e 1. Comparison of inhibition of tubulin polymerization
by compounds 2, 6, 7, 8, phenstatin, and control (DMSO). (All
experiments were performed at least twice.)
a
All experiments were independently performed three times.
Second, an amino group at the C-2 or C-3 position (10
vs 11) did not greatly affect growth inhibition. Third, a
methoxy substituent at the C-4 or C-5 position is
important for growth inhibition (1 or 2 vs 3, 6 or 7 vs
10 and 8, 12 or 13 vs 6 or 7). Finally, a methoxy group
located at either the C-4 or C-5 position resulted in
compounds with similar potencies (1 vs 2, 4 vs 5, and 6
vs 7). Thus, introduction of an amino group at the C-2-
position with a methoxy group at either the C-4 or C-5
position produced the greatest growth inhibitory activity
as seen for compounds 6 and 7.
Lead compounds 6 and 7 displayed similar or greater
growth inhibitory activities than phenstatin against all
10 human cell lines. Compounds 6 and 7 also showed
significantly increased growth inhibition against certain
cancer cell lines as compared to CA-4. For instance,
compounds 6 and 7 were a 50- to 100-fold more potent
than CA-4 to Colo 205, NUGC3, and HA22T cancer cells.
It is interesting that both phenstatin and CA-4 were
relatively inactive against Colo 205, NUGC3, and
HA22T cells, whereas compounds 6 and 7 displayed
potent growth inhibition against these cells. None of the
16 compounds, including phenstatin and CA-4, exhibited
selective resistance to multi-drug resistant MES-SA/
DX5 cells, emphasizing the potential use of benzo-
phenone-type CA-4 analogues for multi-drug resistant
cancer treatment.5a
(B) In Vitr o Tu bu lin Bin d in g Assa ys. To investi-
gate whether the activities of these compounds were
related to interactions with the microtubule system,
their in vitro polymerization inhibitory activities were
measured (Figure 1). The results demonstrated that
drug growth inhibition correlated with inhibition of
tubulin polymerization. For instance, lead compounds
6 and 7 displayed similar inhibition of GTP-induced
polymerization of Map-rich tubulin and their growth
inhibitory activities were strikingly similar. Compound
8, which was 100-fold less potent than compounds 6 and
7 and 10-fold less potent than compound 2, also poorly
inhibited tubulin polymerization. The order of tubulin
polymerization activity was phenstatin > 6, 7 > 2 . 8.
All five of the selected compounds competed with
colchicine for binding to tubulin (Table 2). The results
were consistent with their tubulin polymerization in-
hibition and growth inhibitory activities; i.e., the two
new potent analogues 6 and 7 displayed very strong
inhibition of tubulin polymerization, which helps ex-
plain their potent growth inhibitory activities.
(C) Cell Cycle An a lysis. The effects of these com-
pounds on the cell cycle were measured by flow cytom-
etry against NUGC3 human cancer cells after 24 h. The
results in Table 3 show that lead compounds 6 and 7
caused significant arrest of the cells at the G2/M phase
relative to the untreated control, consistent with their
high antiproliferative potency against NUGC3 cells. On
the contrary, the less potent compounds 2, 8, and
phenstatin produced minimal perturbation of the cell
cycle, consistent with the low growth inhibitory activi-
ties of these compounds against NUGC3 cells. Thus, the
effect of the CA-4 type analogues on cell cycle progres-
sion correlated well with their growth inhibitory and
antitubulin activities. In summary, lead compounds 6
and 7 have been identified as candidate antineoplastic
and antimitotic agents.
Con clu sion s
We have synthesized a series of benzophenone-type
analogues of combretastatin A-4 and have found two of
the fourteen compounds to be potent antiproliferative
agents, inhibitors of tubulin polymerization, and inhibi-
tors of colchicine binding to tubulin. In addition, those
compounds caused G2/M phase arrest of cells and are
considered to be potential new antimitotic agents for
clinical use. The two lead compounds 6 and 7 most likely
interact with tubulin at the colchicine site and display
potent growth inhibitory activity against human cancer
cells, including multi-drug resistant cancer cells. Most
importantly, compounds 6 and 7 showed a 10- to 100-
fold increase in growth inhibition compared to both
phenstatin and combretastatin A-4 against several
human cancer cell lines. Examination of the SAR in this
series of benzophenone-type analogues revealed that
introduction of an amino group at the ortho position was
important for increased growth inhibition. With an
amino group at the C-2 position, a methoxy group at
the C-4 or C-5 position produced maximal growth
inhibitory activity.
Exp er im en ta l Section
(A) Gen er a l Meth od s. Melting points were determined on
a Yanaco (MP-500D) melting point apparatus and are uncor-

2560 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Liou et al.
rected. EI-MS spectra were recorded using a Hewlett-Packard
(1100MSD) spectrometer. High-resolution mass spectra (HRMS)
steps following procedures A, B, and D; mp 106-108 °C. 1H
NMR (300 MHz, CDCl3): δ 3.88 (s, 3H, OCH3), 3.90 (s, 6H,
2 × OCH3), 3.93 (s, 3H, OCH3), 6.44 (dd, J ) 9.0 Hz, 2.4 Hz,
1H, Ar-H5), 6.53 (d, J ) 2.4 Hz, 1H, Ar-H3), 6.88 (s, 2H, Ar-
H2′, H6′), 7.58 (d, J ) 9.0 Hz, 1H, Ar-H6), 12.56 (s, 1H, OH).
13C NMR (CDCl3): δ 55.62, 56.30, 60.94, 101.13, 106.58,
107.31, 113.00, 133.42, 134.96, 152.97, 166.19, 166.25, 199.05.
MS (EI) m/z: 318 (M+). HRMS (EI) for C17H18O6 (M+): calcd,
318.1103; found, 318.1108. Anal. Calcd for C17H18O6: C, 64.14;
H, 5.70. Found: C, 63.90; H, 5.90.
(c) (2-H yd r oxy-5-m et h oxyp h en yl)(3,4,5-t r im et h oxy-
p h en yl)m eth a n on e (2). The title compound was obtained in
65% overall yield from 3,4,5-trimethoxybromobenzene and
2-(tert-butyldimethylsilanyloxy)-5-methoxybenzaldehyde in three
steps following procedures A, B, and D; mp 125-127 °C. IR
(cm-1): 2988, 2936, 2831, 1578, 1485, 1411, 1350, 1282, 1218,
1174, 1134. 1H NMR (300 MHz, CDCl3): δ 3.73 (s, 3H, OCH3),
3.91 (s, 6H, 2 × OCH3), 3.95 (s, 3H, OCH3), 6.96 (s, 2H, Ar-
H2′, H6′), 7.01-7.05 (m, 1H), 7.13-7.18 (m, 2H), 11.45 (s, 1H,
OH). 13C NMR (CDCl3): δ 55.92, 56.33, 60.99, 106.90, 115.79,
118.61, 119.33, 124.13, 132.88, 151.41, 152.97, 157.41, 199.89.
MS (EI) m/z: 318 (M+). HRMS (EI) for C17H18O6 (M+): calcd,
318.1103; found, 318.1106. Anal. Calcd for C17H18O6: C, 64.14;
H, 5.70. Found: C, 64.15; H, 5.81.
(d ) (2-Hyd r oxyp h en yl)(3,4,5-tr im eth oxyp h en yl)m eth a -
n on e (3). The title compound was obtained in 68% overall
yield from 3,4,5-trimethoxybromobenzene and 2-(tert-butyldi-
methylsilanyloxy)benzaldehyde in three steps following pro-
cedures A, B, and D. 1H NMR (300 MHz, CDCl3): δ 3.90 (s,
6H, 2 × OCH3), 3.95 (s, 3H, OCH3), 6.90 (t, J ) 7.5 Hz, 1H,
Ar-H5), 6.94 (s, 2H, Ar-H2′, H6′), 7.30 (dt, J ) 8.4 Hz, 0.6 Hz,
1H, Ar-H3), 7.50 (m, 1H, Ar-H4), 7.67 (dd, J ) 8.4 Hz, 1.8
Hz, 1H, Ar-H6). 13C NMR (CDCl3): δ 56.21, 60.85, 102.55,
106.86, 118.32, 118.49, 118.99, 132.82, 133.16, 136.09, 141.44,
152.85, 162.97, 200.35. MS (ESI): 289 (M + 1).
(e) (4-Meth oxyp h en yl)(3,4,5-tr im eth oxyp h en yl)m eth a -
n on e (4). The title compound12a was obtained in 70% overall
yield from 3,4,5-trimethoxybromobenzene and 4-methoxybenz-
aldehyde in two steps following procedures A and B; mp 46-
48 °C. 1H NMR (300 MHz, CDCl3): δ 3.88 (s, 3H, OCH3), 3.90
(s, 6H, 2 × OCH3), 3.94 (s, 3H, OCH3), 6.98 (dd, J ) 6.8 Hz,
2.1 Hz, 2H, Ar-H2, H6), 7.03 (s, 2H, Ar-H2′, H6′), 7.83 (dd, J
) 6.8 Hz, 2.1 Hz, 2H, Ar-H3, H5). 13C NMR (CDCl3): δ 55.43,
56.26, 60.90, 107.45, 113.51, 130.26, 132.33, 133.31, 141.60,
152.80, 163.10, 194.59. MS (EI) m/z: 302 (M+). HRMS (EI)
for C17H18O5 (M+): calcd, 302.1154; found, 302.1157.
were recorded on
a Finnigan (MAT-95XL) spectrometer.
Elemental analyses were performed on a Heraeus CHN-O
Rapid microanalyzer. 1H NMR and 13C NMR spectra were
obtained with a Varian Mercury-300 spectrometer operating
at 300 MHz and at 75 MHz, respectively; all values are
reported in parts per million (δ) downfield from (CH3)4Si.
Flash-column chromatography was performed on silica gel
(Merck Kieselgel 60, No. 9385, 230-400 mesh ASTM). Thin
layer chromatography (TLC) was carried out on silica gel
plates (E. Merck 60 F254); zones were detected visually by
ultraviolet irradiation (254 nm) or by spraying with phospho-
molybdic acid reagent (Aldrich Chemical Co., Milwaukee, WI)
followed by heating at 100 °C. All reagents were used as
purchased unless otherwise stated. All solvents were dried
according to standard procedures. All reactions were carried
out under an atmosphere of dry nitrogen.
(1) Ch em istr y. (a ) Gen er a l P r oced u r e for th e P r ep a r a -
tion of Su bstitu ted Ben zop h en on e An a logu es (1-14).
P r oced u r e A. A Grignard reagent was prepared in an oven-
dried three-necked flask outfitted with a reflux condenser,
dropping funnel, and magnetic stirrer. Approximately 1/4 of
a 10 mmol aliquot of 3,4,5-trimethoxybromobenzene10 in 5 mL
of anhydrous tetrahydrofuran (THF) was added to a mixture
of magnesium turnings (10 mmol) in 2.5 mL of anhydrous THF
with a small piece of iodine. As soon as the solution became
colorless (heating sometimes necessary), the remaining 3,4,5-
trimethoxylbromobenzene solution was added dropwise to the
solution under mild reflux. Stirring was then continued for 1
h
at room temperature. A (trimethoxyphenyl)magnesium
bromide solution (10 mmol) was then slowly added to the given
substituted 2-nitrobenzoaldehyde (8.35 mmol) in 2.5 mL of
anhydrous THF solution at 0 °C. After complete addition, the
solution was allowed to stir at room temperature for another
20 min. A saturated NH4Cl solution (10 mL) was slowly added
to hydrolyze the adduct at 0 °C, and the mixture was stirred
for 10 min. The phases were separated, and the aqueous layer
was extracted with Et2O (10 mL × 3). The combined organic
layers were washed with brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo, and the residue was
purified by flash chromatography to furnish each benzhydrol.
P r oced u r e B. Pyridinium dichromate (PDC, 7.5 mmol) was
added to a stirred solution of benzhydrol (5 mmol) and powered
4 Å molecular sieves (0.75 g) in 50 mL of anhydrous CH2Cl2
at 0 °C. After complete addition, the mixture was stirred at
room temperature for 8 h. The mixture was diluted with
anhydrous ether (50 mL) and filtered through a pad of Celite.
The filtrate was concentrated in vacuo, and the residue was
purified by flash chromatography to yield each substituted
2-nitrobenzophenone.
P r oced u r e C. A mixture of substituted 2-nitrobenzophe-
none (1 mmol) and iron powder (431 mg) was added to a
mixture of ethanol (3.5 mL), acetic acid (3.5 mL), water (1.7
mL), and 35% HCl (1 drop). The suspension was refluxed with
vigorous stirring for 40 min, cooled, and filtered through Celite.
The filtrate was diluted with water (25 mL) and extracted with
CHCl3 (3 × 10 mL). The organic layers were combined,
sequentially washed with 9% aqueous sodium NaHCO3 (20
mL) and water (2 × 20 mL), dried over MgSO4, and evapo-
rated. The residue was further purified by flash chromatog-
raphy to yield each substituted 2-aminobenzophenone.
(f) (3-Meth oxyp h en yl)(3,4,5-tr im eth oxyp h en yl)m eth a -
n on e (5). The title compound was obtained in 71% overall
yield from 3,4,5-trimethoxybromobenzene and 3-methoxybenz-
aldehyde in two steps following procedures A and B; mp 70-
71 °C. 1H NMR (300 MHz, CDCl3): δ 3.87 (s, 3H, OCH3), 3.88
(s, 6H, 2 × OCH3), 3.95 (s, 3H, OCH3), 7.08 (s, 2H, Ar-H2′,
H6′), 7.12-7.16 (m, 1H), 7.33-7.42 (m, 3H). 13C NMR
(CDCl3): δ 55.45, 56.30, 60.94, 107.80, 114.26, 118.67, 122.52,
129.14, 132.59, 139.14, 142.15, 152.85, 159.58. MS (EI) m/z:
302 (M+). HRMS (EI) for C17H18O5 (M+): calcd, 302.1154;
found, 302.1153.
(g)
(2-Am in o-4-m et h oxyp h en yl)(3,4,5-t r im et h oxy-
p h en yl)m eth a n on e (6). The title compound was obtained in
67% overall yield from 3,4,5-trimethoxybromobenzene and
4-methoxy-2-nitrobenzaldehyde in three steps following pro-
1
cedures A-C; mp 88-90 °C. H NMR (300 MHz, CD3OD): δ
P r oced u r e D. To the substituted 2-(tert-butyldimethyl-
silanyloxy)benzophenone (1 mmol) in anhydrous THF (10 mL)
was added Bu4NF (1.0 M in THF; 3 mmol, 3 mL). After 1 h,
the mixture was poured into H2O (10 mL) and extracted with
Et2O (3 × 10 mL). The combined organic layers were washed
with brine (50 mL), dried (MgSO4), and evaporated. The
residue was chromatographed on silica gel to afford each
substituted 2-hydroxybenzophenone.
(b ) (2-H yd r oxy-4-m et h oxyp h en yl)(3,4,5-t r im et h oxy-
p h en yl)m eth a n on e (1). The title compound was obtained in
68% overall yield from 3,4,5-trimethoxybromobenzene and
2-(tert-butyldimethylsilanyloxy)-4-methoxybenzaldehyde in three
3.79 (s, 3H, OCH3), 3.82 (s, 6H, 2 × OCH3), 3.84 (s, 3H, OCH3),
6.13 (dd, J ) 8.7 Hz, 2.4 Hz, 1H, Ar-H5), 6.30 (d, J ) 2.4 Hz,
1H, Ar-H3), 6.82 (s, 2H, Ar-H2′, H6′), 7.33 (d, J ) 8.7 Hz, 1H,
Ar-H6). 13C NMR (CD3OD): δ 55.87, 56.87, 61.33, 100.00,
105.14, 107.64, 112.75, 137.82, 141.51, 154.30, 156.35, 166.50,
198.57. MS (EI) m/z: 317 (M+). HRMS (EI) for C17H19NO5
(M+): calcd, 317.1263; found, 317.1266. Anal. Calcd for C17H19
-
NO5: C, 64.34; H, 6.03; N, 4.41. Found: C, 64.20; H, 6.22; N,
4.46.
(h )
(2-Am in o-5-m et h oxyp h en yl)(3,4,5-t r im et h oxy-
p h en yl)m eth a n on e (7). The title compound was obtained in
65% overall yield from 3,4,5-trimethoxybromobenzene and

2-Aminobenzophenones as Antimitotic Agents
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2561
5-methoxy-2-nitrobenzaldehyde in three steps following pro-
cedures A-C; mp 127-129 °C. IR (cm-1): 3420, 3307, 3005,
2944, 2832, 1640, 1583, 1411, 1333, 1235, 1212, 1133. 1H NMR
(300 MHz, CDCl3): δ 3.68 (s, 3H, OCH3), 3.88 (s, 6H, 2 ×
OCH3), 3.95 (s, 3H, OCH3), 5.62 (br, 2H, NH2), 6.73 (dd, J )
8.0 Hz, 1.5 Hz, 1H, Ar-H4), 6.95 (s, 2H, Ar-H2′, H6′), 6.98 (d,
J ) 8.0 Hz, 1H, Ar-H3), 7.01 (d, J ) 1.5 Hz, 1H, Ar-H6). 13C
NMR (CDCl3): δ 55.98, 56.27, 60.94, 106.92, 116.67, 118.49,
122.73, 134.82, 141.02, 145.15, 149.87, 152.80, 197.40. MS (EI)
m/z: 317 (M+). HRMS (EI) for C17H19NO5 (M+): calcd, 317.1263;
found, 317.1271. Anal. Calcd for C17H19NO5: C, 64.34; H, 6.03;
N, 4.41. Found: C, 64.32; H, 6.20; N, 4.18.
CDCl3): δ 3.59 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.80 (s, 6H,
2 × OCH3), 3.88 (s, 3H, OCH3), 6.42 (d, J ) 8.7 Hz, 1H, Ar-
H3), 6.84 (d, J ) 8.7 Hz, 1H, Ar-H4), 7.07 (s, 2H, Ar-H2′, H6′).
13C NMR (CDCl3): δ 56.07, 56.90, 60.74, 61.23, 106.64, 111.36,
117.10, 120.03, 133.54, 139.55, 142.62, 144.50, 147.75, 152.84,
196.03. MS (EI) m/z: 347 (M+). HRMS (EI) for C18H21NO6
(M+): calcd, 347.1369; found, 347.1374.
(o) (6-Am in ob en zo[1,3]d ioxol-5-yl)(3,4,5-t r im et h oxy-
p h en yl)m eth a n on e (14). The title compound was obtained
in 65% overall yield from 3,4,5-trimethoxybromobenzene and
6-nitrobenzo[1,3]dioxole-5-carbaldehyde in three steps follow-
ing procedures A-C. 1H NMR (300 MHz, CDCl3): δ 3.88 (s,
6H, 2 × OCH3), 3.93 (s, 3H, OCH3), 5.91 (s, 2H, O-CH2-O),
6.23 (s, 1H, Ar-H3), 6.35 (br, 2H, NH2), 6.92 (s, 2H, Ar-H2′,
H6′), 7.00 (s, 1H, Ar-H6). 13C NMR (CDCl3): δ 56.27, 60.91,
96.81, 101.31, 106.17, 106.78, 110.28, 111.40, 135.98, 138.41,
150.28, 152.85, 153.18, 196.21. MS (EI) m/z: 331 (M+). HRMS
(EI) for C17H17NO6 (M+): calcd, 331.1056; found, 331.1044.
(2) Biology. (a ) Ma ter ia ls. DMEM medium, nonessential
amino acids and fetal bovine serum were purchased from
GIBCO BRL (Life Technologies, Grand Island, NY). MTS assay
kits were obtained from Promega (Madison, WI). Propidium
iodide, PIPES, and GTP were purchased from Sigma. [3H]-
Colchicine and Sephadex G50 columns were obtained from
Amersham Pharmacia (Piscataway, NJ ). MAP-rich tubulin
was purchased from Cytoskeleton Inc. (Denver, CO).
(b) Cell Gr ow th In h ibitor y Assa y. Carcinoma cells were
maintained in DMEM medium supplemented with 10% fetal
bovine serum. For in vitro treatment, 6 × 103 cells/well were
seeded in 96-well plates and incubated in a CO2 incubator at
37 °C for 24 h. The cells were treated with at least five different
concentrations of test compounds in a CO2 incubator for 72 h.
The number of viable cells was estimated using the tetrazolium
dye reduction assay (MTS assay),11 and the experiment was
performed as the manufacturer recommended (Promega,
Madison, WI). The absorbance was measured at 490 nm on a
Wallac 1420 VICTOR2 Multilabel counter (Perkin-Elmer,
Boston, MA). The results of these assays were used to obtain
the dose-response curves from which IC50 (nM) values were
determined. The IC50 value is calculated as follows: % of
(i)
(2-Am in o-3-m et h oxyp h en yl)(3,4,5-t r im et h oxy-
p h en yl)m eth a n on e (8). The title compound was obtained in
61% overall yield from 3,4,5-trimethoxybromobenzene and
3-methoxy-2-nitrobenzaldehyde in three steps following pro-
1
cedures A-C. H NMR (300 MHz, CDCl3): δ 3.87 (s, 6H, 2 ×
OCH3), 3.90 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 6.56 (t, J )
8.1 Hz, Ar-H5), 6.89 (dd, J ) 8.1 Hz, 0.9 Hz, 1H, Ar-H3), 6.98
(s, 2H, Ar-H2′, H6′), 7.14 (dd, J ) 8.1, 1.2 Hz, 1H, Ar-H6). 13
C
NMR (CDCl3): δ 55.63, 56.09, 60.76, 106.70, 112.75, 113.73,
117.39, 125.43, 140.57, 141.81, 147.17, 152.57, 197.77. MS (EI)
m/z: 317 (M+). HRMS (EI) for C17H19NO5 (M+): calcd, 317.1263;
found, 317.1266. Anal. Calcd for C17H19NO5: C, 64.34; H, 6.03;
N, 4.41. Found: C, 64.46; H, 6.17; N, 4.24.
(j) (2-Am in o-5-d im et h yla m in op h en yl)(3,4,5-t r im et h -
oxyp h en yl)m eth a n on e (9). The title compound was obtained
in 60% overall yield from 3,4,5-trimethoxybromobenzene and
3,5-(dimethylamino)-2-nitrobenzaldehyde in three steps fol-
1
lowing procedures A-C. H NMR (300 MHz, CDCl3): δ 2.75
(s, 6H, 2 × CH3), 3.87 (s, 6H, 2 × OCH3), 3.92 (s, 3H, OCH3),
6.73 (d, J ) 9.0 Hz, 1H, Ar-H3), 6.92-7.00 (m, 4H, Ar-H2′,
H6′, Ar-H4, H6). 13C NMR (CDCl3): δ 29.64, 42.18, 56.21, 60.90,
107.01, 118.32, 118.38, 119.02, 122.88, 134.96, 142.06, 143.26,
152.66, 197.71. MS (ESI): 331 (M + 1).
(k ) (2-Am in op h en yl)(3,4,5-tr im eth oxyp h en yl)m eth a -
n on e (10). The title compound was obtained in 65% overall
yield from 3,4,5-trimethoxybromobenzene and 2-nitrobenz-
aldehyde in three steps following procedures A-C. 1H NMR
(300 MHz, CDCl3): δ 3.89 (s, 6H, 2 × OCH3), 3.94 (s, 3H,
OCH3), 6.00 (br, 2H, NH2), 6.63 (t, J ) 7.2 Hz, 1H, Ar-H5),
6.66 (d, J ) 8.4 Hz, 1H, Ar-H3), 6.91 (s, 2H, Ar-H2′, H6′), 7.30
(td, J ) 7.2 Hz, 1.5 Hz, 1H, Ar-H4), 7.50 (dd, J ) 8.4 Hz, 1.5
Hz, 1H, Ar-H6). 13C NMR (CDCl3): δ 56.26, 60.91, 102.55,
106.86, 11.50, 117.00, 118.24, 134.10, 134.15, 135.13, 150.76,
152.77, 153.18, 198.01. MS (EI) m/z: 287 (M+). HRMS (EI)
for C16H17NO4 (M+): calcd, 287.1158; found, 287.1153.
(l) (3-Am in op h en yl)(3,4,5-t r im et h oxyp h en yl)m et h a -
n on e (11). The title compound was obtained in 64% overall
yield from 3,4,5-trimethoxybromobenzene and 3-nitrobenzal-
dehyde in three steps following procedures A-C. 1H NMR (300
MHz, CDCl3): δ 3.87 (s, 6H, 2 × OCH3), 3.94 (s, 3H, OCH3),
5.30 (br, 2H, NH2), 6.87-6.91 (m, 1H), 7.08 (s, 2H, Ar-H2′,
H6′), 7.09-7.12 (m, 2H), 7.24 (m, 1H). 13C NMR (CDCl3): δ
56.24, 60.87, 107.68, 115.63, 118.72, 120.21, 128.90, 132.71,
138.80, 141.92, 146.59, 152.72, 195.94. MS (ESI): 288 (M +
1).
control ) 100% × [(OD490compd - OD490blank)/(OD490DMSO
-
OD490blank)] from a 50% of control inhibitory concentration.
An IC50 value represents the concentration (nM) of the test
compound which produces a 50% cell growth inhibition after
3 days of incubation. The values represent averages of three
independent experiments, each with duplicate samples.
(c) Tu bu lin P olym er iza tion In Vitr o Assa y. Turbidi-
metric assays of microtubules were performed as described by
Lopes et al.14 and the manual of Cytoskeleton Inc. (Denver,
CO) with some modifications. MAP-rich tubulin (2 mg/mL) was
preincubated in polymerization buffer (0.1 M PIPES, pH 6.9,
1 mM MgCl2) with drug at 4 °C for 2 min before the addition
of 1 mM GTP. The samples were then rapidly warmed to 37
°C in a 96-well plate thermostatically controlled spectropho-
tometer (BioCAD SPRINT, PE Biosystems), and the change
in absorbance at 350 nm was periodically measured.
(d ) Colch icin e Bin d in g Assa y. Binding of [3H]colchicine
was measured by column centrifugation as described by Singer
et al.15 with some modifications. Solutions of 2.5 µM pure
tubulin and 2.5 µM [3H]colchicine (0.1 Ci/mmol), with or
without drug, were incubated in polymerization buffer at room
temperature for 1 h before 50 µL samples were centrifuged
through Sephadex G-50 columns. The eluates were analyzed
for radioactivity by scintillation counting (Tri-Crab 2100TR,
Packard BioScience, Meriden, CT).
(e) Cell Cycle An a lysis. Flow cytometric evaluation of the
cell cycle status was performed according to Boquest et al.16
with some modifications.A 5 × 105 amount of untreated or
drug-treated NUGC3 cells were washed twice with PBS and
fixed in 60% methanol for 30 min on ice. The cells were washed
once with PBS and then incubated with propidium iodide (25
µg/mL)/RNase A (100 µg/mL) in PBS for 30 min at room
temperature in the dark. The samples were analyzed on a
FACSCalibur (Becton Dickinson, Heidelberg, Germany) flow
(m )
(2-Am in o-4,5-d im et h oxyp h en yl)(3,4,5-t r im et h -
oxyp h en yl)m eth a n on e (12). The title compound was ob-
tained in 61% overall yield from 3,4,5-trimethoxybromoben-
zene and 4,5-dimethoxy-2-nitrobenzaldehyde in three steps
following procedures A-C. 1H NMR (300 MHz, CDCl3): δ 3.70
(s, 3H, OCH3), 3.88 (s, 6H, 2 × OCH3), 3.91 (s, 3H, OCH3),
3.92 (s, 3H, OCH3), 6.17 (br, 2H, NH2), 6.22 (s, 1H, Ar-H3),
6.89 (s, 2H, Ar-H2′, H6′), 7.00 (s, 1H, Ar-H6). 13C NMR
(CDCl3): δ 55.84, 56.18, 56.56, 60.90, 99.30, 106.35, 109.79,
116.29, 135.77, 139.66, 140.24, 148.44, 152.76, 155.35, 196.09.
MS (EI) m/z: 347 (M+). HRMS (EI) for C18H21NO6 (M+): calcd,
347.1369; found, 347.1360.
(n )
(6-Am in o-2,3-d im et h oxyp h en yl)(3,4,5-t r im et h -
oxyp h en yl)m eth a n on e (13). The title compound was ob-
tained in 63% overall yield from 3,4,5-trimethoxybromoben-
zene and 2,3-dimethoxy-6-nitrobenzaldehyde in three steps
following procedures A-C; mp 89-91 °C. 1H NMR (300 MHz,

2562 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Liou et al.
B. S.; del Rey, B.; Lamamie de Clairac, R. P.; Caballero, E.;
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A-4. Bioorg. Med. Chem. Lett. 2000, 10, 2549-2551. (e) Shirai,
R.; Takayama, H.; Nishikawa, A.; Koiso, Y.; Hashimoto, Y.
Asymmetric Synthesis of Antimitotic Combretadioxolane with
Potent Antitumor Activity Against Multi-Drug Resistant Cells.
Bioorg. Med. Chem. Lett. 1998, 8, 1997-2000. (f) Ohsumi, K.;
Nakagawa, R.; Fukuda, Y.; Hatanaka, T.; Morinaga, Y.; Nihei,
Y.; Ohishi, K.; Suga, Y.; Akiyama, Y.; Tsuji, T. Novel Combre-
tastatin Analogues Effective Against Murine Solid Tumors:
Design and Structure-Activity Relationships. J . Med. Chem.
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R.; Fukuda, Y.; Morinaga, Y.; Suga, Y.; Nihei, Y.; Ohishi, K.;
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cytometer with excitation at 488 nm and emission measured
at 564-606 nm. The number of cells in G0/G1, S, and G2/M
phases was calculated using Cell Quest (version 3.1f) cell cycle
analysis software.
Ack n ow led gm en t. We thank Dr. Shu-J en Chen,
Ms. Ying-Ying Chang, Ms. Hsin-Hui Hung, and Mr.
Shih-Hau Liu for assistance in performing high-
throughput screening measurements and Drs. Steve
Roffler, Iwao Ojima, Kenneth J . O’Connor, and Uan-
Kang Tan for valuable suggestions concerning the
manuscript. The authors are also grateful for adminis-
trative support from Ms. Edith Hsiao-Wen Chu and Ms.
Shiau-Fen J ung and for financial support from the
National Health Research Institutes, Taiwan, Republic
of China.
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