2-Amino-3,4,5-Trimethoxybenzophenones as Potent Tubulin Polymerization Inhibitors

Article abstract of DOI:10.1002/cmdc.201000479

A series of novel 2-amino-3,4,5-trimethoxybenzophenone analogues exhibited excellent activity as tubulin polymerization inhibitors by targeting the colchicine binding site of microtubules. The lead compound 17 exhibited an IC₅₀ value of 1.6μM, similar to that of combretastatin A-4 (IC₅₀=1.9μM). It also displayed remarkable anti-proliferative activity, with IC₅₀ values ranging from 7-16nM against a variety of human cancer cell lines and one MDR(+) cancer cell line. SAR information indicated that the introduction of an amino group at the C2 position of benzophenone ringA and the C3' position of benzophenone ringB play important roles in maximizing activity.

Full text of DOI:10.1002/cmdc.201000479

MED
DOI: 10.1002/cmdc.201000479
2-Amino-3,4,5-Trimethoxybenzophenones as Potent
Tubulin Polymerization Inhibitors
Hsun-Yueh Chuang,[a] Jang-Yang Chang,[b, c] Mei-Jung Lai,[a] Ching-Chuan Kuo,[b] Hsueh-
Yun Lee,[a] Hsing-Pang Hsieh,[d] Ying-Jen Chen,[a] Li-Tzong Chen,[b] Wen-Yu Pan,[b] and Jing-
Ping Liou*[a]
A series of novel 2-amino-3,4,5-trimethoxybenzophenone ana-
logues exhibited excellent activity as tubulin polymerization in-
hibitors by targeting the colchicine binding site of microtu-
bules. The lead compound 17 exhibited an IC50 value of
1.6 mm, similar to that of combretastatin A-4 (IC50 =1.9 mm). It
also displayed remarkable anti-proliferative activity, with IC50
values ranging from 7–16 nm against a variety of human
cancer cell lines and one MDR(+) cancer cell line. SAR informa-
tion indicated that the introduction of an amino group at the
C2 position of benzophenone ring A and the C3’ position of
benzophenone ring B play important roles in maximizing activ-
ity.
Introduction
A growing tumor requires the development of vasculature to
gather oxygen and nutrients. This suggests that if tumor vas-
culature formation is disrupted, tumor growth could be inhibit-
ed. In eukaryotic cells, microtubules are involved in a variety of
cellular processes. Suppression of microtubule polymerization
blocks cell division at mitosis, leading to cell death and disrup-
tion of tumor vasculature formation. Therefore, microtubules
represent a proven target for the development of anticancer
agents.[1] A variety of antimitotic agents, such as vincristine,
vinblastine, and paclitaxel, are currently in clinical use as che-
motherapies. Combretastatins, which are natural tubulin bind-
ing agents isolated from the bark of the South African Cape
Bushwillow (Combretum caffrum), have been shown to have
potential as anticancer agents.[2] Combretastatin A-4 (CA-4, 1a;
Figure 1) exhibits the most potent activity by binding to b-tu-
bulin at the colchicine binding site. Drugs binding to the col-
chicine domain are undergoing intensive investigation as vas-
cular-disrupting agents for cancer therapy. For example, clinical
candidates combretastatin A-4P (1b; fosbretabulin disodium)
and AVE-8062 (1c; ombrabulin) act as vascular-disrupting
agents (VDA) by rapidly depolymerizing microtubules of newly
formed vasculatures, subsequently shutting down blood
supply to tumors.[3]
Benzophenone-type analogues of combretastatins such as
compound 2,[4] phenstatin (3)[5] and hydroxyphenstatin (4),[6]
reported by the Cushman and Pettit groups, exhibited sub-
stantial anticancer and antimitotic activity, comparable to the
Z-stilbene derivatives. The benzophenone (diarylketone) or aryl
heteroaryl ketone compounds were easier to synthesize than
the combretastatins, which require control of geometric selec-
tivity, and have greater pharmacological potential due to im-
[a] H.-Y. Chuang,+ Dr. M.-J. Lai, Dr. H.-Y. Lee, Y.-J. Chen, Dr. J.-P. Liou
School of Pharmacy, College of Pharmacy, Taipei Medical University
No.250 Wuxing Street, Taipei 11031 (Taiwan, Republic of China)
Fax: (+886)2-2736-9558
E-mail: jpl@tmu.edu.tw
[b] Dr. J.-Y. Chang,+ Dr. C.-C. Kuo, Dr. L.-T. Chen, W.-Y. Pan
National Institute of Cancer Research
National Health Research Institutes, 2F
No.367, Sheng Li Road, Tainan 704 (Taiwan, Republic of China)
[c] Dr. J.-Y. Chang+
Division of Hematology/Oncology, Department of Internal Medicine
National Cheng Kung University Hospital
No.138, Sheng Li Road, Tainan 704 (Taiwan, Republic of China)
[d] Dr. H.-P. Hsieh
Institute of Biotechnology and Pharmaceutical Research
National Health Research Institutes
35 Keyan Road, Zhunan Town, Maioli County 350
(Taiwan, Republic of China)
[+] These authors contributed equally to this work.
Figure 1. Microtubule inhibitors targeting the colchicine binding domain of
tubulin.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000479.
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Tubulin Polymerization Inhibitors
proved metabolic stability.[7,8] Recently, we reported that the in-
troduction of an amino group at the C2’ and C3’ positions of
benzophenone ring B (5 and 6),[9,10] and at the C2 position on
ring A of Z-stilbene (8),[11] increased the polarity of the corre-
sponding structure without compromising bioactivity. On the
basis of this observation, we report herein the design and
structure–activity relationships (SAR) of the 2-amino-3,4,5-tri-
methoxybenzophenones as novel and potent antimitotic
agents.
Results and Discussion
Chemistry
Figure 2. 2-aminobenzophenones and 2’,3’-diaminobenzophenones as anti-
mitotics.
The general syntheses of the 2-aminobenzophenones are de-
picted in Scheme 1. The 2-amino-3,4,5-trimethoxybenzophe-
nones (9–16, 18; Figure 2) were prepared in three steps, start-
nitrobenzaldehyde (26)[12] in
three steps: a Grignard reaction
with 4-methoxyphenylmagnesi-
um bromide and 3,4,5-trime-
thoxyphenylmagnesium
bro-
mide, respectively, followed by
PDC-mediated oxidation, then
Fe/NH4Cl-mediated reduction, as
shown
in
Scheme 2
and
Scheme 3.
Biological evaluation
In vitro cellular growth inhibito-
ry activity
Scheme 1. Syntheses of 2-amino-3,4,5-trimethoxybenzophenones 9–18 and 2-N,N-dimethylamino-3,4,5-trimethox-
ybenzophenone 19: a) 1. various substituted phenyl Grignard reagents, THF, 08C!RT, 2. PDC, CH2Cl2, molecular
sieves, RT; b) Fe, NH4Cl, iPrOH, H2O, reflux; c) CH3I, K2CO3, DMF, RT; d) HNO3, 08C.
Synthesized
nones 9–21 were evaluated for
anti-proliferative activities
aminobenzophe-
ing from 3,4,5-trimethoxy-2-nitrobenzaldehyde (22) and com-
mercially available substituted phenylmagnesium bromides
through Grignard reaction to obtain the corresponding secon-
dary benzyl alcohols. These alcohols were oxidized by pyridini-
um dichromate (PDC) to obtain the respective 2-nitrobenzo-
phenones, followed by reduction of the nitro group in the
presence of Fe/NH4Cl in isopropanol to prepare the desired 2-
aminobenzophenones (9–16, 18; Figure 2) in 15–79% yield
(three steps). The treatment of compound 9 with methyl
iodide in DMF, using potassium carbonate as base, afforded
the N,N-dimethylaminobenzophenone (19) in 26% yield. Com-
pound 17, with an additional amino group at the 3’-position
of ring B as compared with 9, was synthesized from key inter-
mediate 24. Compound 23 was subject to HNO3-mediated ni-
tration to synthesize the 3’-nitro-4’-methoxybenzophenone
(24) which, upon reduction of the dinitro moieties, afforded
the desired (2-amino-3,4,5-trimethoxyphenyl)(3’-amino-4’-me-
thoxyphenyl)methanone (17) in 34% yield. The 2-amino-3,5-di-
methoxybenzophenone (20) and 2’,3’-diaminobenzophenone
(21) were obtained in 36% and 43% yield, respectively, from
3,5-dimethoxy-2-nitrobenzaldehyde (25) and 4-methoxy-2,3-di-
Scheme 2. Synthesis of 2-amino-3,5-dimethoxybenzophenone 20: a) 1. 4-me-
thoxyphenylmagnesium bromide, THF, 08C!RT, 2. PDC, CH2Cl2, molecular
sieves, RT; b) Fe, NH4Cl, iPrOH, H2O, reflux.
Scheme 3. Synthesis of 2’,3’-diaminobenzophenone 21: a) 1. 3,4,5-trimethox-
yphenylmagnesium bromide, THF, 08C!RT, 2. PDC, CH2Cl2, molecular sieves,
RT; b) Fe, NH4Cl, iPrOH, H2O, reflux.
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against oral epidermoid carcinoma KB cells, colorectal carcino-
ma HT29 cells, non small cell lung carcinoma H460 cells, stom-
ach carcinoma MKN45 cells, and MDR(+), KBvin10 cells, which
overexpress P-gp 170/MDR (Table 1). First, we evaluated the
in 3’,4’-difluorobenzophenone (18), resulted in drastically de-
creased activity. This indicates that the methoxy group located
at the C4’ site of 2-amino-3,4,5-trimethoxybenzophenones
plays an important role for activity.
In an effort to understand the
role of the C2 amino group of
Table 1. In vitro cell growth inhibition by compounds 9–21 and reference compound 7.
IC50 ꢀSD [nm][a]
aminobenzophenones, we re-
placed the primary amino group
at the C2 position of ring A of 9
with a tertiary amine moiety,
yielding 2-N,N-dimethylamino-
benzophenone (19) which has
dramatically diminished cell
growth inhibitory activity. This
indicates that a free C2 amino
substitution on ring A is requi-
site for activity. Modifying the 2-
amino-3,4,5-trimethoxyphenyl
Compd
KB
H460
HT29
MKN45
KBvin10
9
46ꢀ6
1943ꢀ128
621ꢀ113
34ꢀ2
48ꢀ40
116ꢀ33
2828ꢀ662
ND
25ꢀ2
783ꢀ54
ND
35ꢀ4
1673ꢀ165
ND
10
11
12
13
14
15
16
17
18
19
20
21
5[9,10]
6[10]
7
2078ꢀ100
734ꢀ84
35ꢀ2
33ꢀ5
19ꢀ1
24ꢀ4
>10000
>10000
>10000
>10000
36ꢀ2
14ꢀ4
>10000
>10000
>10000
>10000
>10000
>10000
>10000
30ꢀ6
14ꢀ3
>10000
>10000
>10000
>10000
33ꢀ5
16ꢀ4
>10000
>10000
9817ꢀ1340
>10000
>10000
>10000
27ꢀ4
7.7ꢀ0.9
>10000
>10000
18ꢀ1
8ꢀ0.5
>10000
>10000
moiety of 9 with a 2-amino-3,5-
dimethoxyphenyl moiety (20) re-
sulted in a two- to threefold de-
crease in potency relative to the
parent compound. The 2-amino-
3,4,5-trimethoxybenophenones
(9, 12, 16, and 17) all displayed
strong anti-proliferative activity,
160ꢀ19
170ꢀ6
165ꢀ11
72 ꢀ4
104ꢀ20
17ꢀ4
32ꢀ17
32ꢀ2
13ꢀ1
21ꢀ7
ND
19ꢀ2
42ꢀ7
33ꢀ9
15ꢀ2
12ꢀ0.3
ND
19ꢀ1.0
24ꢀ8
ND
19ꢀ2
ND
11ꢀ4
31ꢀ6
130ꢀ9
[a] All experiments were independently performed at least three times.
effect of a 2-amino-3,4,5-trimethoxyphenyl group in the benzo-
phenone system. Compound 9, with a methoxy group at the
C4’ position of aminobenzophenone ring B, displayed substan-
tial cellular growth inhibitory activity, with mean IC50 values of
54 nm against five human cancer cell lines. Replacement of the
methoxy group at the C4’ position of 9 with a methyl group
(10) or a methylthio group (11), respectively, resulted in a de-
crease in potency to micromolar range relative to parent 9. In-
terestingly, changing the C4’ methoxy group of 9 to a N,N-di-
methylamino group (12) resulted in slightly elevated cellular
growth inhibition, with a mean IC50 of 29.1 nm against five
human cancer cell lines. In contrast, replacing the C4’ methoxy
group with a halogen group such as fluoro (13) or a chloro
(14) resulted in a dramatic loss of activity. In order to under-
stand the positional effect of a methoxy group on ring B of the
2-aminobenzophenones, we shifted the methoxy group from
the C4’ position to the C3’ position, as in 15, resulting in a sub-
stantial loss of activity.
Reports[13] that introduction of a C3’ halogen or an amino
group on ring B causes an increase of cytotoxicity in the Z-stil-
bene (combretastatin) system inspired us to investigate similar
effects in the 2-amino-3,4,5-trimethoxybenzophenone series.
Compound 16, with a 3’-fluoro-4’-methoxyphenyl group, and
compound 17, with an 3’-amino-4’-methoxyphenyl group, dis-
played two- to fivefold improvements in potency as compared
with the parent 9, indicating that the addition of a fluoro or
amino group at the C3’ position of ring B in this series was
beneficial for bioactivity. Notably, compounds 16 and 17 exhib-
ited anti-proliferative activity against five cancer cell lines with
mean IC50 values of 28.7 and 11.7 nm, respectively. Replace-
ment of the C4’ methoxy group in 16 with a fluoro group, as
indicating that the polar amino group located at the C2 posi-
tion of ring A was both acceptable and beneficial for activity.
In summary of the benzophenone SAR, introduction of an
amino group at the C2 position of benzophenone ring A or
the C2’ or C3’ positions of ring B resulted in analogues with im-
proved anti-proliferative activity over those without an amino
group (for example, 5, 6, and 9 versus compound 2).[4] Com-
pound 17, which has amino groups at both the C2 (ring A)
and the C3’ (ring B) positions exhibited slightly elevated activi-
ty relative to compound 9, with only one amino group. This
sparked us to investigate the effects of a diamino group at the
C2’ and C3’ position of benzophenone ring B. A study of 2’,3’-
diaminobenzophenones showed that compound 21, with an
additional amino group as compared with 2’-aminobenzophe-
none (5) and 3’-aminobenzophenone (6), displayed slightly in-
creased or similar growth inhibition in cell lines. Overall, the in-
troduction of either one or two amino moieties in the C2, C2’,
or C3’ positions of benzophenones appear to be beneficial for
potency.
Inhibition of tubulin polymerization and colchicine binding ac-
tivity
To investigate whether the activities of these aminobenzophe-
nones were related to interactions with the microtubule
system, compounds 9, 12, 16, 17, 21, and reference com-
pounds 1a and 7, were tested for inhibition of tubulin poly-
merization and the ability to compete for the colchicine bind-
ing site. (Table 2) For in vitro microtubule assembly, we incu-
bated purified, unpolymerized, microtubule-associated protein
(MAP)-rich tubulin with various concentrations of aminobenzo-
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Tubulin Polymerization Inhibitors
aminobenzophenone (6), 2’,3’-diaminobenzophenone (21), and
2,3’-diaminobenzophenone (17) moieties, all exhibit substantial
anti-proliferative and microtubule destabilizing activities, dem-
onstrating that amino group(s) at the C2, C2’, and C3’ positions
of benzophenones can increase activity. Overall, the 2-amino-
3,4,5-trimethoxybenzophenone (17) exhibited strong inhibition
of tubulin polymerization and cancer cell growth and has po-
tential for further investigation as an anticancer agent.
Table 2. Inhibition of tubulin polymerization and colchicine binding by
compounds 9, 12, 16, 17, 21, and reference compounds 1a and 7.
Tubulin[a]
IC50 ꢀSD [mm]
Colchicine Binding ꢀSD [%][b]
Compd
[I]=1 mm
[I]=5 mm
9
3.7ꢀ0.7
1.7ꢀ0.3
2.0ꢀ0.2
1.6ꢀ0.2
2.2ꢀ0.3
4.1ꢀ0.5
1.9ꢀ0.2
44ꢀ6
71ꢀ4
80ꢀ1
76ꢀ2
71ꢀ2
78ꢀ3
88ꢀ2
88ꢀ3
93ꢀ1
85ꢀ2
12
16
17
21
7
1a
86ꢀ1
94ꢀ2
Experimental Section
[a] Inhibition of tubulin polymerization.[14] [b] Inhibition of [3H]colchicine
binding;[15] experiments used 1 mm tubulin or 5 mm [3H]colchicine.
Chemistry
Nuclear magnetic resonance (1H NMR) spectra were obtained with
a Bruker DRX-500 spectrometer (500 MHz), with chemical shifts in
parts per million (ppm, d) downfield from TMS as an internal stan-
dard. High-resolution mass spectra (HRMS) were measured with a
JEOL (JMS-700) electron impact (EI) mass spectrometer. Purity of
the final compounds were determined using an Agilent 1100 series
HPLC system using a C18 column (Agilent ZORBAX Eclipse XDB-
C18 5 mm ꢁ 4.6 mm ꢁ 150 mm) and were found to be ꢁ 95%.
Flash column chromatography was carried out using silica gel
(Merck Kieselgel 60, No. 9385, 230–400 mesh ASTM). All reactions
were performed under an atmosphere of dry nitrogen.
phenones in the presence of GTP and measured polymeri-
zation. Compounds 9, 12, 16, 17, and 21 were efficacious in in-
hibiting microtubule assembly, with IC50 values of 3.7, 1.7, 2.0,
1.6, and 2.2 mm, respectively, which positively correlated with
their anti-proliferative activity. The microtubule destabilizing
activities of compounds 12, 16, 17, and 21 were similar to that
of 1a. In the [3H]colchicine binding assay, results indicated that
2-amino-3,4,5-trimethoxybenzophenones (12, 16, and 17) and
2’,3’-diaminobenzophenone (21) bound strongly to the colchi-
cines binding site of tubulin.
General procedure for preparation of 2-amino-3,4,5-trime-
thoxybenzophenones
Aminobenzophenones 5, 6, 17, and 21 all substantially in-
hibited tubulin polymerization, revealing that the amino
group(s) located at benzophenone positions C2, C2’, or C3’
result in maximal activity. A similar phenomenon was also ob-
served in the case of hydroxybenzophenones 3 and 4, where
the introduction of a 3’-hydroxy group and a 2’,3’-dihydroxy
group, respectively, resulted in strong inhibitory effects toward
tubulin polymerization (3,[5] IC50 =1.0 mm; 4,[6] IC50 =0.82 mm)
similar to 1a.
(2-Amino-3,4,5-trimethoxyphenyl)(4-methoxyphenyl)methanone
(9): Grignard reagent 4-methoxyphenylmagnesium bromide
(6.22 mL, 3.11 mmol, 0.5 m) was added to a stirred solution of 22
(0.5 g, 2.07 mmol) and anhydrous THF (10 mL) under nitrogen at
08C, which was warmed to room temperature and stirred for 1 h.
The mixture was quenched with H2O and extracted with EtOAc
(3ꢁ10 mL). The combined organic extract was dried over anhy-
drous MgSO4 and evaporated to yield a crude residue, which was
dissolved in CH2Cl2 (20 mL). Molecular sieves (1.56 g, 4 ꢂ) and PDC
(1.56 g, 4.14 mmol) were added to the reaction mixture and stirred
at room temperature for 12 h. The reaction mixture was filtered
through a pad of Celite, and the filtrate was evaporated to yield a
residue that was purified by silica gel flash column chromatogra-
phy (EtOAc/n-hexane=1:2) to afford 2-nitrobenzophenone 23;
Conclusions
A series of concisely synthesized 2-amino-3,4,5-trimethoxyben-
zophenones were identified as a novel tubulin polymerization
inhibitors that act at the colchicine binding site of tubulin.
Lead compounds 12, 16, and 17 exhibited significant inhibi-
tion of tubulin polymerization with IC50 values of 1.7, 2.0, and
1.6 mm, respectively, comparable to the activity of compound
1a (IC50 =1.9 mm). They also demonstrated substantial anti-pro-
liferative activity, with IC50 values ranging from 7–36 nm in a
variety of human cancer cell lines, including one MDR(+) resist-
ant cell line (KBvin10). The SAR information indicated that
ring B of the 2-aminobenzophenone system, substituted with a
4’-N,N-dimethylamino group (12), 3’-fluoro-4’-methoxy group
(16), or 3’-amino-4’-methoxy group (17), exhibited maximal ac-
tivity. Replacement of the C2 amino group of ring A with a 2-
N,N-dimethylamino group (19) resulted in a drastic loss of ac-
tivity, revealing that the introduction of a free amino group at
the benzophenone C2 position plays a critical role for activity.
The introduction of an amino group or groups into the benzo-
phenone system, for example: 2’-aminobenzophenone (5), 3’-
1
mp: 111–1138C; H NMR (500 MHz, CDCl3): d=3.88 (s, 3H), 3.90 (s,
3H), 3.97 (s, 3H), 4.04 (s, 3H), 6.74 (s, 1H), 6.94 (d, J=8.8 Hz, 2H),
7.78 ppm (d, J=8.8 Hz, 2H). Compound 23 (0.57 g, 2.47 mmol),
iron powder (0.41 g, 7.40 mmol) and ammonium chloride (0.57 g,
4.94 mmol) were added to a mixture solution of isopropyl alcohol
(24.7 mL, 0.1m) and H2O (6 mL). The mixture was stirred at reflux
for 5 h, then cooled and filtered through a pad of Celite. The fil-
trate was concentrated and extracted with H2O (10 mL) and EtOAc
(3ꢁ10 mL). The combined organic extracts were dried over anhy-
drous MgSO4 and evaporated to give a residue that was purified
by flash chromatography (EtOAc/n-hexane=1:3) to yield the de-
sired compound as a yellow solid (0.49 g, 63% over three steps);
1
mp: 86–878C; H NMR (500 MHz, CDCl3): d=3.64 (s, 3H), 3.82 (s,
3H), 3.86 (s, 3H), 3.95 (s, 3H), 5.60–6.00 (b, 2H), 6.77 (s, 1H), 6.91
(d, J=8.7 Hz, 2H), 7.64 ppm (d, J=8.7 Hz, 2H); 13C NMR (125 MHz,
CDCl3): d=55.5, 56.9, 60.5, 61.0, 112.5, 113.4, 113.5, 131.6, 132.7,
140.5, 141.3, 142.8, 147.5, 162.2, 196.6 ppm; MS (EI) m/z (%):318
(16.4), 317 (100) [M+H]+; HRMS-EI m/z [M+H]+ calcd for
C17H19NO5: 317.1258, found: 317.1264.
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3H), 3.93 (s, 3H), 3.99 (s, 3H), 4.26 (s, 3H), 6.65 (s, 1H), 7.01 (dd, J=
1.9, 7.7, 8.0 Hz, 1H), 7.44 (d, J=1.9 Hz, 1H), 7.47 (d, J=8.0 Hz, 1H),
7.50 ppm (d, J=7.7 Hz, 1H); MS (EI) m/z (%): 317 (11.5), 257 (42.9),
300 (100) [M+H]+; HRMS-EI m/z [M+H]+ calcd for C17H19NO5:
317.1263, found: 317.1264.
(2-Amino-3,4,5-trimethoxyphenyl)(p-tolyl)methanone (10): com-
pound 10 was obtained as a yellow solid (0.88 g, 89%) from 3,4,5-
trimethoxy-2-nitrobenzaldehyde and p-tolymagnesium bromide in
a similar manner as described for the preparation of 9; mp: 73–
1
748C; H NMR (500 MHz, CDCl3): d=2.34 (s, 3H), 3.59 (s, 3H), 3.83
(s, 3H), 3.92 (s, 3H), 6.76 (s, 1H), 7.18 (d, J=8.0 Hz, 2H), 7.50 ppm
(d, J=8.0 Hz, 2H); 13C NMR (125 MHz, CDCl3): d=21.6, 56.8, 60.4,
61.0, 112.7, 113.0, 128.8, 129.3, 137.5, 140.4, 141.5, 141.6, 142.7,
147.7, 197.6 ppm; MS (EI) m/z (%): 300 (6.6), 302 (7.1), 301 (100)
[M+H]+; HRMS-EI m/z [M+H]+ calcd for C17H19NO4: 301.1309,
found: 301.1316.
(2-Amino-3,4,5-trimethoxyphenyl)(3-fluoro-4-methoxyphenyl)-
methanone (16): compound 16 was obtained as a yellow solid
(0.53 g, 77%) from 3,4,5-trimethoxy-2-nitrobenzaldehyde and 3-
fluoro-4-methoxyphenylmagnesium bromide in a similar manner as
described for the preparation of 9; mp: 117–1198C; 1H NMR
(500 MHz, CDCl3): d=3.69 (s, 3H), 3.90 (s, 3H), 3.97 (s, 3H), 3.99 (s,
3H), 6.03 (s, 2H), 6.77 (s, 1H), 7.01 (d, J=8.2 Hz, 1H) 7.46 (dd, J=
1.8, 8.2 Hz, 1H), 7.48 ppm (d, J=1.8 Hz, 1H); 13C NMR (125 MHz,
CDCl3): d=56.4, 56.9, 60.5, 61.0, 112.3, 112.7, 117.3, 126.4, 133.0,
140.5, 141.7, 142.9, 147.8, 150.4, 150.8, 152.8, 195.3 ppm; MS (EI)
m/z (%): 320 (29.4), 335 (100) [M+H]+; HRMS-EI m/z [M+H]+ calcd
for C17H18FNO5: 335.1164, found: 335.1166.
(2-Amino-3,4,5-trimethoxyphenyl)(4-(methylthio)phenyl)metha-
none (11): compound 11 was obtained as a yellow solid (1.22 g,
75%) from 3,4,5-trimethoxy-2-nitrobenzaldehyde and (4-(methyl-
thio)phenyl)magnesium bromide in a similar manner as described
for the preparation of 9; mp: 105–1068C; 1H NMR (500 MHz,
CDCl3): d=2.56 (s, 3H), 3.93 (s, 3H), 4.00 (s, 3H), 4.25 (s, 3H), 6.62
(s, 1H), 7.39 (d, J=8.4 Hz, 2H), 7.84 ppm (d, J=8.4 Hz, 2H); MS (EI)
m/z (%): 333 (19.2), 284 (56.5), 331 (100) [M+H]+; HRMS-EI m/z
[M+H]+ calcd for C17H19NO4S: 333.1035, found: 333.1035.
(2-Amino-3,4,5-trimethoxyphenyl)(3-amino-4-methoxyphenyl)-
methanone (17): compound 23 (0.5 g, 1.44 mmol) was added to a
solution of nitric acid (13 mL) at 08C and was stirred for 2 h. The
mixture was quenched with H2O and extracted with H2O (10 mL)
and CH2Cl2 (3ꢁ10 mL). The combined organic extract was dried
over anhydrous MgSO4 and evaporated. The residue was purified
by flash chromatography (EtOAc/n-hexane=2:1) to yield dinitro-
(2-Amino-3,4,5-trimethoxyphenyl)(4-(dimethylamino)phenyl)me-
thanone (12): compound 12 was obtained as a yellow solid
(0.03 g, 15%) from 3,4,5-trimethoxy-2-nitrobenzaldehyde and 4-
(N,N-dimethyl)aniline magnesium bromide in a similar manner as
described for the preparation of 9; mp: 93–958C; 1H NMR
(500 MHz, CDCl3): d=3.04 (s, 6H), 3.70 (s, 3H), 3.87 (s, 3H), 3.96 (s,
3H), 5.67 (s, 2H), 6.66 (d, J=8.8 Hz, 2H), 6.85 (s, 1H), 7.69 ppm (d,
J=8.8 Hz, 2H); 13C NMR (125 MHz, CDCl3): d=40.2, 57.0, 60.4, 61.0,
110.7, 112.4, 114.8, 127.0, 132.1, 140.1, 140.7, 142.9, 146.8, 152.8,
196.2 ppm; MS (EI) m/z (%): 331 (14.9), 330 (100) [M+H]+; HRMS-EI
m/z [M+H]+ calcd for C18H22N2O4: 330.1574, found: 330.1568.
1
benzophenone 24; H NMR (500 MHz, CDCl3): d=3.92 (s, 3H), 3.98
(s, 3H), 4.00 (s, 3H), 4.04 (s, 3H), 6.73 (s, 1H), 7.17 (d, J=8.8 Hz,
1H), 8.02 (dd, J=2.0, 8.8 Hz, 1H), 8.24 ppm (d, J=2.0 Hz, 1H).
Compound 24 (0.56 g, 1.44 mmol), iron powder (0.48 g, 8.64 mmol)
and ammonium chloride (0.31 g, 5.75 mmol) were added to a solu-
tion of isopropyl alcohol (14.4 mL, 0.1m) and H2O (4 mL). The mix-
ture was stirred at reflux for 5 h, then cooled and filtered through
a pad of Celite. The filtrate was concentrated in vacuo, and the res-
idue was extracted with H2O (20 mL) and EtOAc (3ꢁ20 mL). The
combined organic extract was dried over anhydrous MgSO4 and
evaporated to give the residue, which was purified by flash chro-
matography (EtOAc/n-hexane=1:1) to afford the desired com-
pound as a yellow solid (0.16 g, 34%); mp: 109–1118C; 1H NMR
(500 MHz, CDCl3): d=3.68 (s, 3H), 3.89 (s, 3H), 3.91 (s, 3H), 3.97 (s,
3H), 5.91 (s, 2H), 6.79 (d, J=8.2 Hz, 1H), 6.86 (s, 1H), 7.06 (d, J=
2.1, 8.2 Hz, 1H), 7.09 ppm (d, J=2.1 Hz, 1H); 13C NMR (125 MHz,
CDCl3): d=55.7, 56.9, 60.4, 61.0, 109.1, 112.8, 113.7, 115.6, 121.2,
133.0, 136.1, 140.4, 141.0, 142.7, 147.4, 150.0, 197.1 ppm; MS (EI)
m/z (%): 333 (16.4), 332 (100) [M+H]+; HRMS-EI m/z [M+H]+ calcd
for C17H20N2O5: 332.1367, found: 332.1370.
(2-Amino-3,4,5-trimethoxyphenyl)(4-fluorophenyl)methanone
(13): compound 13 was obtained as a yellow solid (0.12 g, 41%)
from 3,4,5-trimethoxy-2-nitrobenzaldehyde and 4-fluorophenyl-
magnesium bromide in a similar manner as described for the prep-
1
aration of 9; mp: 85–868C; H NMR (500 MHz, CDCl3): d=3.65 (s,
3H), 3.90 (s, 3H), 3.98 (s, 3H), 6.71 (s, 1H), 7.12 (d, J=8.6 Hz, 2H),
7.66 ppm (d, J=8.6 Hz, 2H); 13C NMR (125 MHz, CDCl3): d=56.9,
60.5, 61.1, 112.4, 115.3, 131.5, 136.5, 140.4, 142.0, 142.9, 148.0,
163.5, 165.5, 196.3 ppm; MS (EI) m/z (%): 290 (59.3), 291 (9.6), 304
(11.1), 306 (17.3), 305 (100) [M+H]+; HRMS-EI m/z [M+H]+ calcd for
C16H16FNO4: 305.1058, found: 305.1069.
(2-Amino-3,4,5-trimethoxyphenyl)(4-chlorophenyl)methanone
(14): compound 14 was obtained as a yellow solid (0.38 g, 72%)
from 3,4,5-trimethoxy-2-nitrobenzaldehyde and 4-chlorphenylmag-
nesium bromide in a similar manner as described for the prepara-
(2-Amino-3,4,5-trimethoxyphenyl)(3,4-difluorophenyl)metha-
none (18): compound 18 was obtained as a yellow solid (0.24 g,
89%) from 3,4,5-trimethoxy-2-nitrobenzaldehyde and 3,4-difluoro-
phenylmagnesium bromide in a similar manner as described for
the preparation of 9; mp: 111–1128C; 1H NMR (500 MHz, CDCl3):
d=3.67 (s, 3H), 3.90 (s, 3H), 4.00 (s, 3H), 6.68 (s, 1H), 7.23 (d, J=
8.3 Hz, 1H), 7.41 (d, J=2.1 Hz, 1H), 7.51 ppm (dd, J=2.1, 8.3 Hz,
1H); 13C NMR (125 MHz, CDCl3): d=56.9, 60.5, 61.1, 111.8, 118.5,
125.7, 125.8, 137.2, 137.3, 140.4, 142.3, 143.0, 148.3, 194.7 ppm; MS
(EI) m/z (%): 322 (12.5), 324 (16.8), 323 (100) [M+H]+; HRMS-EI m/z
[M+H]+ calcd for C16H15F2NO4: 323.0964, found: 323.0968.
1
tion of 9; mp: 97–988C; H NMR (500 MHz, CDCl3): d=3.66 (s, 3H),
3.90 (s, 3H), 3.99 (s, 3H), 6.70 (s, 1H), 7.43 (d, J=8.5 Hz, 2H),
7.59 ppm (d, J=8.5 Hz, 2H); 13C NMR (125 MHz, CDCl3): d=56.9,
60.5, 61.0, 112.1, 112.2, 128.5, 130.5, 137.2, 138.8, 140.4, 142.2,
142.9, 148.1, 196.3 ppm; MS (EI) m/z (%): 323 (38.4), 321 (100)
[M+H]+; HRMS-EI m/z [M+H]+ calcd for C16H16ClNO4: 321.0762,
found: 321.0772.
(2-Amino-3,4,5-trimethoxyphenyl)(3-methoxyphenyl)methanone
(15): compound 15 was obtained as a yellow solid (1.07 g, 81%)
from 3,4,5-trimethoxy-2-nitrobenzaldehyde and 3-methoxyphenyl-
magnesium bromide in a similar manner as described for the prep-
(2-(Dimethylamino)-3,4,5-trimethoxyphenyl)(4-methoxyphenyl)-
methanone (19): potassium carbonate (0.44 g, 3.16 mmol) and io-
domethane (0.29 mL, 4.73 mmol) were added to a solution of com-
pound 9 (0.5 g, 1.58 mmol) in DMF (5 mL) under nitrogen, and the
mixture was stirred for 1 h. The mixture was extracted with H2O
1
aration of 9; mp: 115–1178C; H NMR (500 MHz, CDCl3): d=3.91 (s,
454
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ChemMedChem 2011, 6, 450 – 456

Tubulin Polymerization Inhibitors
(10 mL) and EtOAc (3ꢁ10 mL). The combined organic extract was
dried over anhydrous MgSO4 and evaporated to give a residue
that was purified by flash chromatography (EtOAc/n-hexane=1:3)
to yield compound 19 as a yellow solid (0.06 g, 26%); mp: 89–
918C; 1H NMR (500 MHz, CDCl3): d=2.59 (s, 6H), 3.81 (s, 3H),
3.85(s, 3H), 3.90 (s, 3H), 3.92 (s, 3H), 6.56 (s, 1H), 6.91 (d, J=8.8 Hz,
2H), 7.76 ppm (d, J=8.8 Hz, 2H); 13C NMR (125 MHz, CDCl3): d=
44.0, 55.5, 56.2, 60.9, 61.0, 106.2, 113.4, 131.0, 132.0, 133.1, 138.3,
144.5, 150.3, 151.8, 163.4, 196.7 ppm; MS (EI) m/z (%): 346 (51.7),
345 (100) [M+H]+; HRMS-EI m/z [M+H]+ calcd for C19H23NO5:
345.1571, found: 345.1577.
(Denver, CO, USA). [3H]Colchicine (specific activity=60–
87 Cimmolꢂ1
was purchased from PerkinElmer Life Sciences
)
(Boston, MA, USA).
Cell growth inhibitory assay
Human oral epidermoid carcinoma KB cells, non small cell lung car-
cinoma H460 cells, colorectal carcinoma HT29 cells, and stomach
carcinoma MKN45 cells were maintained in RPMI 1640 medium
supplemented with 5% fetal bovine serum. KB-vin10 cells were
maintained in growth medium supplemented with 10 nm vincris-
tine, generated from vincristine-driven selection, and displayed
overexpression of P-gp170/MDR. Cells in logarithmic phase were
cultured at a density of 5000 cells per mL per well in a 24-well
plate, and were cultured in drug-free medium for three days prior
to use. The cells were exposed to various concentrations of the
test compounds for 72 h. The methylene blue dye assay was used
to evaluate the effect of the test compounds on cell growth as
previously described.[16] The IC50 value, resulting from 50% inhibi-
tion of cell growth, was calculated graphically as a comparison
with the control. Compounds were examined in at least three inde-
pendent experiments, and the values shown for these compounds
are the mean and standard deviation of these data.
(2-Amino-3,5-dimethoxyphenyl)(4-methoxyphenyl)methanone
(20): compound 20 was obtained as a yellow solid (0.1 g, 35%)
from 3,5-dimethoxy-2-nitrobenzaldehyde (25) and 4-methoxyphe-
nylmagnesium bromide in a similar manner as described for the
1
preparation of 9; mp: 133–1358C; H NMR (500 MHz, CDCl3): d=
3.67 (s, 3H), 3.88 (s, 3H), 3.89 (s, 3H), 6.56 (d, J=2.3 Hz, 1H), 6.60
(d, J=2.3 Hz, 1H), 6.95 (d, J=8.7 Hz, 2H), 7.72 ppm (d, J=8.7 Hz,
2H); 13C NMR (125 MHz, CDCl3): d=55.4, 55.8, 55.8, 103.7, 106.3,
113.3, 117.9, 131.7, 132.3, 136.0, 148.4, 149.1, 162.3, 197.1 ppm; MS
(EI) m/z (%): 286 (51.9), 286 (8.8), 287 (100) [M+H]+; HRMS-EI m/z
[M+H]+ calcd for C16H17NO4: 287.1152, found: 287.1153.
(2’,3’-diamino-4-methoxyphenyl)(3,4,5-trimethoxyphenyl)metha-
none (21): Grignard reagent 3,4,5-trimethoxyphenylmagnesium
bromide (5.0 mL, 5.0 mmol, 1.0 m) was added to a stirred solution
of 4-methoxy-2,3-dinitrobenzaldehyde (26) (0.75 g, 3.34 mmol) and
anhydrous THF (15 mL) under nitrogen at 08C, then was warmed
to room temperature for 1 h. After completion of this addition, the
mixture was quenched with H2O, then extracted with H2O and
EtOAc (3ꢁ10 mL). The combined organic extract was dried over
anhydrous MgSO4 and evaporated to yield crude benzyl alcohol.
This crude alcohol (1.31 g, 3.34 mmol) was stirred with powdered
4 ꢂ molecular sieves (1.88 g) in anhydrous CH2Cl2 (20 mL). PDC
(1.88 g, 4.14 mmol) was added to the stirred solution under nitro-
gen at room temperature overnight. After completion of the reac-
tion, the mixture was filtered through a Celite-silica gel-Celite pad.
The filtrate was concentrated in vacuo, and the residue was puri-
fied by flash chromatography to obtain the substituted 2’,3’-dini-
trobenzophenone (70%).
Tubulin polymerization in vitro assay[10,15]
Turbidimetric assays of microtubules were performed as described
by Bollag et al.[17] Briefly, MAP-rich tubulin (from bovine brain) was
dissolved in reaction buffer (100 mm PIPES (pH 6.9), 2 mm MgCl2,
1 mm GTP) to prepare a 4 mgmLꢂ1 tubulin solution. This solution
(240 mg MAP-rich tubulin per well) was placed in 96-well microtiter
plate in the presence of test compounds or 2% (v/v) DMSO as a
vehicle control. The increase in absorbance was measured at
350 nm in a PowerWave X Microplate Reader (BIO-TEK Instruments,
Winooski, VT) at 378C and was recorded every 30 s for 30 min. The
area under the curve (AUC) was used to determine the IC50 for tu-
bulin polymerization. The AUC of the untreated control and 10 mm
of colchicine was set to 100 and 0% polymerization, respectively,
and the IC50 was calculated by nonlinear regression in at least
three experiments.
The 2’,3’-dinitrobenzophenone (0.92 g, 2.34 mmol), iron powder
(0.78 g, 14.04 mmol) and ammonium chloride (9.36 mmol, 0.50 g)
were added to a solution of isopropyl alcohol (23.4 mL, 0.1 m) and
H2O (6 mL). The mixture was stirred at reflux for 5 h, then cooled
and filtered through a pad of Celite. The filtrate was concentrated
in vacuo, and the residue was extracted with H2O (10 mL) and
EtOAc (3ꢁ10 mL). The combined organic extracts were dried over
anhydrous MgSO4 and evaporated. The residue was purified by
flash chromatography (EtOAc/n-hexane=1:1) to yield compound
21 as a yellow solid (1.37 g, 62%); mp: 124–1268C; 1H NMR
(500 MHz, CDCl3): d=3.87 (s, 9H), 3.92 (s, 3H), 6.33 (d, J=8.9 Hz,
1H), 6.89 (s, 2H), 7.13 ppm (d, J=8.9 Hz, 1H); 13C NMR (125 MHz,
CDCl3): d=55.8, 56.4, 61.0, 99.9, 106.9, 114.3, 122.1, 127.3, 135.9,
140.6, 141.9, 151.7, 152.8, 197.7 ppm; MS (EI) m/z (%):301 (17), 332
(100) [M+H]+; HRMS-EI m/z [M+H]+ calcd for C17H19NO5: 332.1372,
found: 332.1371.
Tubulin competition binding scintillation proximity assay[18–20]
This assay was performed in a 96-well plate. Briefly, 0.08 mm of
[3H]colchicine was mixed with the test compound and 0.5 mg of
special long-chain biotin-labeled tubulin, then incubated in 100 mL
of reaction buffer (80 mm PIPES (pH 6.8), 1 mm EGTA, 10% glycerol,
1 mm MgCl2, and 1 mm GTP) for 2 h at 378C. Streptavidin-labeled
SPA beads (80 mg) were added to each reaction mixture, and radio-
active counts were directly measured by a scintillation counter.
Acknowledgements
This research was supported by the National Science Council of
the Republic of China (grant nos. NSC 99-2323-B-038-001,
NSC 98-2113M-038-002-MY2, NSC 99-2323-B-400-006, NSC 99-
2323-B-400-007, and NSC 99-2120-M-006-00) and the Depart-
ment of Health of the Republic of China (grant no. DOH99-TD-C-
111-004).
Biology
Materials: Regents for cell culture were obtained from Gibco-BRL
Life Technologies (Gaithersburg, MD, USA). Microtubule-associated
protein (MAP)-rich tubulin was purchased from Cytoskeleton, Inc.
ChemMedChem 2011, 6, 450 – 456
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
455

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Keywords: antimitotic agents · benzophenones · inhibitors ·
polymerization · tubulin
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Received: November 9, 2010
Published online on January 4, 2011
456
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ChemMedChem 2011, 6, 450 – 456