Synthesis and biological evaluation of N-alkyl-N-(4-methoxyphenyl)pyridin- 2-amines as a new class of tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.11.047

Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivatives were designed, synthesized and evaluated for cytotoxic activity against A549, KB, KB_VIN, and DU145 human tumor cell lines (HTCL). Subsequently, three new leads (6a, 7g, and 8c) with submicromolar GI₅₀ values of 0.19-0.41 μM in the cellular assays were discovered, and these compounds also significantly inhibited tubulin assembly (IC₅₀ 1.4-1.7 μM) and competitively inhibited colchicine binding to tubulin with effects similar to those of the clinical candidate CA-4 in the same assays. These promising results indicate that these tertiary diarylamine derivatives represent a novel class of tubulin polymerization inhibitors targeting the colchicine binding site and showing significant anti-proliferative activity.

Full text of DOI:10.1016/j.bmc.2012.11.047

Bioorganic & Medicinal Chemistry xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of N-alkyl-N-(4-methoxyphenyl)
pyridin-2-amines as a new class of tubulin polymerization inhibitors
Xiao-Feng Wang a, Emika Ohkoshi b, Sheng-Biao Wang a, Ernest Hamel c, Kenneth F. Bastow d,
Susan L. Morris-Natschke b, Kuo-Hsiung Lee b,e, Lan Xie a,
a Beijing Institute of Pharmacology and Toxicology, 27 Tai-Ping Road, Beijing 100850, PR China
⇑
b Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC 27599, USA
c Screening Technologies Branch, Development Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institute of Health, Frederick, MD 21702, USA
d Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC 25799, USA
e Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivatives
were designed, synthesized and evaluated for cytotoxic activity against A549, KB, KBVIN, and DU145
human tumor cell lines (HTCL). Subsequently, three new leads (6a, 7g, and 8c) with submicromolar
Received 18 September 2012
Revised 21 November 2012
Accepted 29 November 2012
Available online xxxx
GI50 values of 0.19–0.41
l
M in the cellular assays were discovered, and these compounds also signifi-
M) and competitively inhibited colchicine binding to
cantly inhibited tubulin assembly (IC50 1.4–1.7
l
tubulin with effects similar to those of the clinical candidate CA-4 in the same assays. These promising
results indicate that these tertiary diarylamine derivatives represent a novel class of tubulin polymeriza-
tion inhibitors targeting the colchicine binding site and showing significant anti-proliferative activity.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
N-Alkyl-N-phenylpyridin-2-amines
Cytotoxicity
Tubulin polymerization inhibitors
Colchicine binding site
1. Introduction
encourage further efforts to design and discover novel small mole-
cules that function as tubulin polymerization inhibitors targeted at
Microtubules are formed by polymerization of heterodimers of
the colchicine binding site.
a
- and b-tubulin, and play important roles in cellular activities,
In our prior studies, we discovered two synthetic hits
6-chloro-N-(4-methoxyphenyl)- 3-nitropyridin-2-amine (1a) and
6-chloro-2-(4-methoxyphenoxy)-3-nitro pyridine (1b) by using
cell assay screening. They showed promising cytotoxic activity
including cell structure maintenance, intracellular transport, mito-
sis, and cell division. Taxoids and vincristine alkaloids are two
well-known classes of anticancer drugs that act as tubulin inhibi-
tors by targeting two distinct binding sites (i.e., taxol and vinca
(GI50 values 2.40–13.5 lM and 1.18–2.03 lM, respectively) against
sites) on the
a
,b-tubulin heterodimer1,2 and disrupting the dynam-
a panel of human tumor cell lines (HTCL), including A549, KB,
KBVIN, and DU145.3 While the only structural difference between
1a and 1b is the linker (NH or O) between the two aryl rings (A
and B rings), 1b was two- to seven-fold more potent than 1a.
Therefore, we postulated that the linker might be associated with
the molecular antitumor potency. By performing a conformation
analysis using a molecular mechanics (MM2) method with energy
minimization and dynamics calculation,4 we found that the phenyl
and pyridine rings of compounds 1a and 1b have quite different
spatial orientations as shown in Figure 2. Compound 1a has a con-
jugated resonance system and a planar conformation, in which an
intramolecular hydrogen bond between the hydrogen of the NH
linker and an oxygen atom of the ortho-nitrogen group orients
the phenyl and pyridine rings (A and B rings) in the same plane.
The presence of the intramolecular H-bond was validated by a
down-field signal (d 10.17 ppm) for the NH proton in the 1H
ics of microtubule assembly and disassembly. They are effective in
the clinical treatment of cancers, but do have certain deficiencies,
including narrow therapeutic indexes and emergence of drug resis-
tance. In efforts to overcome these drawbacks, many new tubulin
inhibitors, which are targeted at the colchicine site, a third distinct
binding site on tubulin, have been discovered recently and are the
subjects of intense investigation. Figure 1 shows examples of such
inhibitors, including plant natural products and derivatives, as well
as synthetic small molecules. Because of their synthetic accessibil-
ity and structural diversity, certain small molecule drug candi-
dates. such as CA-4, ABT-751, and MPC-6827, are currently
undergoing clinical trials for treating cancers. These results greatly
⇑
Corresponding author. Tel./fax: +86 10 6931690.
E-mail address: lanxieshi@yahoo.com (L. Xie).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.047
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

2
X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
OH
O
MeO
MeO
MeO
MeO
MeO
MeO
O
O
O
O
O
MeO
MeO
N
H
O
NHAc
R
MeO
OAc
MeO
OMe
O
O
OPO3H2
MeO
O
OMe
R = H, Colchicine
ZD6126 (phase I)
OH
Steganacin
Podophyllotoxin
R = SH, DMDA-colchicine
MeO
OMe
MeO
MeO
O
N
HN
N
N
N
H
H
N
O
S
N
O
N
H
O
OMe
MeO
OR
R =
R =
H,
ABT-751 (phase II)
MPC-6827 (phase II)
TN16
CA4
PO
3Na2
, C
A4
-P
(ph
as
e II
/III)
Figure 1. Plant natural products, derivatives, and synthetic compounds as tubulin inhibitors targeted at the colchicine binding site. Some of them are in clinical trials.
O
N
O
N
O
N
A
R3
N
O
H
O
O
A
N
R2
R4
SAR
Cl
N
N
Cl
N
N
Cl
N
O
B
new
compounds
R1
1a
1b
3a
OMe
OMe
OMe
B
Figure 2. (A) Hits, design, and a general formula of new tertiary diarylamines.(B) Preferred conformations of 1a and 1b. The torsional angle C1–N2–C3–C4 is defined as
positive if, when viewed along the N2–C3 bond, atom C1 must be rotated clockwise to eclipse atom C4.
NMR spectrum of 1a. In contrast, the conformation of 1b shows a
torsional angle of 54.14° [C1–N2–C3–C4] between the two aryl
rings, caused by electronic repulsion between lone-pair electrons
of the linker oxygen and the negatively charged ortho-nitro group.
Therefore, we hypothesize that a non-planar molecular conforma-
tion might be favorable for enhancing antitumor potency. Even
though 1a is less active than 1b, the NH linker of 1a is modifiable
and steric hindrance can be introduced to promote a non-planar
molecular conformation. Consequently, 6-chloro-N-(4-methoxy-
phenyl)-N-methyl-3-nitropyridin-2-amine (3a), a tertiary amine
derivative of 1a, was designed and synthesized. In another positive
design direction, 3a contains a 4-methoxy-N-methylanilino moiety
as also found in MPC-6827. Compound 3a exhibited significant
cytotoxic activity against a panel of human tumor cell lines with
and possible binding site. Our new results on the tertiary diarylam-
ines, including chemical synthesis, cytotoxicity against human tu-
mor cells, SAR results, and identification of biological target, are
presented herein.
2. Chemistry
As shown in Scheme 1, the designed target compounds 3–7
could be synthesized from substituted 2-chloropyridines (2) and
various commercially available anilines. Most of the compounds
were prepared by a coupling reaction between 2 and a substituted
aniline in t-BuOH in the presence of potassium carbonate either at
room temperature for 12–24 h5 (method A for 3a, 3e, 3f) or under
microwave irradiation at 120–160 °C for 10–30 min (method B for
3c, 3d).6,7 As an exception, the coupling of 2,6-dichloro-3-nitropyr-
idine (2a) with N-methyl 4-cyanoaniline was carried out by
direct heating under solvent-free conditions to provide 3b in 37%
yield. Consistent with our previous results,8 the nucleophilic
N-substituted aniline preferred to attack the 2,6-dichloropyridine
(2)at the 2-chloro position, which is ortho to an electron-
withdrawing substituent R3 (such as NO2, COOMe, and COOEt in
2a, 2c, and 2e, respectively) to produce corresponding series 3–7
low micromolar GI50 values of 1.55–2.20 lM. Accordingly, addi-
tional tertiary diarylamine derivatives 3–8 were designed, synthe-
sized, and evaluated for antitumor activity. The new target
compounds are shown in the general formula in Figure 2. The im-
pact of substituents [R1 on the B-ring, R2 on the linker (Y), R3 and R4
on the A-ring] on the cytotoxic activity was investigated succes-
sively. Furthermore, new active leads with high potency were
tested in anti-tubulin assays to identify a potential biological target
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
3
R3
Cl
R3 = NO2, R4 = Cl
R2
HN
2a
2b
R1
R
3 = NO2, R4 = Me,
2c R3 = CO2Me, R4 = Cl
R4
a
N
2d R3 = CN,
R4 = Cl
2
2e R3 = CO2Et, R4 = Cl
R3
N
R3
OMe
NO2
OMe
R1
R4
N
Cl
N
N
Cl
N
N
R2
5 6
,
7
3 4
,
R2 = Me
R3 = NO2
7a R3 = CN
1 = 4-OMe
1 = 4-CN
3a
3b
R
R
R4 = Me
7b R3 = COOEt
5a
5b
5c
c
e
4 = OMe
4 = NHMe
R
R
R
3 = COOH
R
R
3c R1 = 4-OCF3
f
i
7c
7d
7e
3a
d
3 = COOi-Pr
3d R1 = 4-COOMe
3e R1 = 3,4-diOMe
6a
3 = CONHMe
R3 = COOMe
g
6a
6b
6c
7f R3 = CH2OH
7g R3 = CH2OMe
3f R1 = 3,4,5-triOMe
R
R
R
4 = Cl
h
c
4 = OMe
4 = NHMe
d
R
1 = 4-OMe
R2 = Allyl
4a
4b
4c
R
R
2 = cyclo-pentyl
2 = Ac
b
1a R2 = H
Scheme 1. Synthesis of 3–7 series. (a) neat, 140 °C, N2, 4 h for 3b; K2CO3/t-BuOH, rt, 12–24 h or microwave irradiation. 120–160 °C, 10–30 min for others; (b) Ac2O/H2SO4,
90 °C, 18 h; (c) NaOMe/MeOH, reflux, 2 h; (d) MeNH2/MeOH, reflux, 12 h; (e) (i) aq NaOH (3 N), THF/MeOH, rt, 36 h; (ii) HCl (2 N); (f) i-PrI, K2CO3, acetone, reflux, 4 h; (g)
LiBH4, THF, MeOH, rt, 2 h; (h) CH3I, NaH (60% oil suspension), THF, rt, 2 h; (i) (i) HOBt, EDCI, CH2Cl2, rt, 30 min; (ii) NH2Me (30% in MeOH), 1.5 h, rt.
compounds. However, when 3-cyano-2,6-dichloropyridine (2d)
was treated with N-methyl-4-methoxyaniline, 2- (7a) and 6-anili-
no products were formed in a 1:2 ratio, indicating that the pres-
ence of the cyano (CN), a weak electron-withdrawing group, led
to less selectivity between the two chlorinated ortho- and para-
positions on the pyridine ring. By using the same aromatic nucleo-
philic substitution reaction, compounds 4a, 4b, 5a, 6a, 7a, and 7b
were prepared from the appropriate chloropyridines 2a-e and N-
substituted 4-methoxyanilines. Acetylation of hit 1a with acetic
anhydride in the presence of a catalytic amount of concentrated
sulfuric acid provided compound 4c. The 6-chloro in 3a and 6a
was converted to a methoxy or methylamine group by treatment
with sodium methoxide or methylamine in dry MeOH to produce
corresponding compounds 5b, 6b or 5c, 6c, respectively. Hydroly-
sis of 6a under basic conditions gave the carboxylic acid compound
7c, which was esterified with 2-iodopropane in acetone to produce
7d. The treatment of 7c with 1-hydroxybenzotriazole (HOBt) in the
presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDCI) hydrochloride salt, followed by the reaction with methyl-
amine (30% in MeOH) gave 7e, with 3-CONHMe (R3) on the
pyridine ring, in moderate yield.9 Additionally, the ester group in
6a was reduced by reaction with lithium borohydride (LiBH4) to
give 3-hydroxymethyl compound 7f, which was converted to
3-methoxymethyl compound 7g by treatment with iodomethane
in the presence of sodium hydride in anhydrous DMF.
8f, respectively. Similarly to the preparation of 7g, methylation of
8h afforded methoxymethyl compound 8i. A tetrazolyl ring (R3)
was formed11 from the cyano group in 8j by treatment with so-
dium azide and triethylamine hydrochloride in refluxing toluene
to afford 8k. However, this reaction did not go to completion,
and ca. one-third of the starting 8j was recovered, even after
36 h. Furthermore, methylation of 8k with dimethyl sulfate pro-
duced the corresponding 8l. All target compounds were identified
by 1H NMR and MS spectroscopic data, and purity was determined
by HPLC.
3. Results and discussion
The 32 newly synthesized tertiary diarylamines (series 3–8)
were first evaluated for cytotoxic activity using a HTCL panel,
including A549 (lung carcinoma), DU145 (prostate cancer), KB
(epidermoid carcinoma of the nasopharynx), and KBVIN (vincris-
tine-resistant KB), with paclitaxel as a reference compound. The
in vitro anticancer activity (GI50) was determined using the estab-
lished sulforhodamine B (SRB) method.12 The cytotoxicity data of
all new tertiary amines in HTCL assays are listed in Tables 1 and
2. Some of the designed tertiary diarylamine compounds were
more potent than hit 1a (GI50 values 2.40–13.5) in the HTCL assays.
As shown in Table 1, compounds 3a with
a para-methoxy
(R1 = OMe) and 3d with a para-ester (R1 = COOMe) on the phenyl
Scheme 2 shows the synthesis of 6-trifluoromethylpyridine
compounds 8c–l from 3-substituted N-(4-methoxyphenyl)-6-tri-
fluoromethylpyridin-2-amine 8a (R3 = COOMe) or 8b (R3 = CN),
which were prepared by literature methods.10 Methylation of 8a
and 8b with methyl iodide in the presence of sodium hydride in
DMF at low temperature (0 °C) produced tertiary amines 8c and
8j, respectively. Hydrolysis, aminolysis, or reduction of the ester
group in 8c produced corresponding benzoic acid (8d), N-methyl
carboxamide (8g), and hydroxymethyl (8h) compounds, respec-
tively. Further esterification of 8d with iodoethane or 2-iodopro-
pane in refluxing acetone gave ethyl ester 8e and isopropyl ester
ring displayed low micromolar GI50 values ranging from 1.55 to
3.00
(21–27
l
M, and thus, were more potent than 3b with a para-cyano
M) and 3e with 3,4-dimethoxy (15–18 M) against the
l
l
HTCL panel. However, corresponding compounds 3c and 3f with
4-trifluoromethoxy or 3,4,5-trimethoxy substitution, respectively,
on the phenyl ring completely lost cytotoxic activity. These results
indicated that a para-substituent on the phenyl ring could enhance
the antiproliferative activity and a methoxy group was preferred to
other substituents or multi-substituents on the phenyl ring. Conse-
quently, in the series 4 compounds, the 4-methoxyphenyl moiety
was retained, while the R2 group on the linker was varied. In
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

4
X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
R3
N
COOMe
N
8d R3 = COOH
8e R3 = COOEt
8f R3 = COOi-Pr
8g R3 = CONHMe
8h R3 = CH2OH
8i R3 = CH2OMe
c
b for 8d
d for 8g
e for 8h
c
F3C
N
F3C
N
f
R3
OMe
OMe
8d-i
8c
F3C
N
NH
a
N
N
HN
N
N
N
N
CN
N
N
N
F3C
N
g
h
F3C
N
N
F3C
N
OMe
8a R3 = COOMe
8b R3 = CN
OMe
8j
OMe
OMe
8k
8l
Scheme 2. Synthesis of 8 series. (a) CH3I, NaH (60% oil suspension), DMF, 0 °C, 1 h; (b) (i) aq NaOH (3 N), THF/MeOH, rt, 36 h; (ii) HCl (2 N); (c) EtI or i-PrI, K2CO3, acetone,
reflux, 4 h; (d) NH2Me, MeOH, rt, 4 days; (e) LiBH4, THF, MeOH, rt, 2 h; (f) CH3I, NaH (60% oil suspension), THF, rt, 2 h; (g) NaN3, Et3NꢀHCl, toluene, reflux, 36 h; (h) Me2SO4,
K2CO3, acetone, reflux, 5 h.
Table 1
Inhibitory activity of 3–4 against HTCL panel
NO2
A
N
R1
B
Cl
N
R2
3, 4
R1
R2
GI50 SDa
(lM)
A549
KB
KBVIN
DU145
3a
3b
3c
4-OMe
4-CN
4-OCF3
Me
Me
Me
2.20 0.20
24.0 2.79
NAb
1.78 0.11
22.1 2.52
NA
1.86 0.30
27.0 4.22
NA
1.55 0.24
21.4 3.89
NA
3d
3e
3f
4a
4-COOMe
3,4-diOMe
3,4,5-triOMe
4-OMe
4-OMe
4-OMe
Me
Me
Me
Allyl
c-Pentyl
Ac
3.00 0.35
18.4 1.31
NA
3.33 0.41
NA
11.8 2.36
13.5
8.18 1.01 nM
2.89 0.24
16.0 0.62
NA
2.61 0.16
NA
14.3 2.05
7.19
7.77 0.84 nM
2.87 0.55
16.5 0.88
NA
3.15 0.57
NA
13.9 1.25
2.40
1803 302 nM
2.22 0.22
15.0 0.95
NA
2.69 0.35
NA
11.2 2.03
3.83
6.50 0.43 nM
4b
4c
1a3
4-OMe
H
Paclitaxelc
a
b
c
Concentration of compound that inhibits 50% human tumor cell growth, presented as mean standard deviation (SD), performed at least in triplicate.
Not active; test compound (20
Positive control.
lg/mL) did not reach 50% inhibition.
comparison with 3a (R2 = Me, GI50 1.55–2.20
l
M), compound 4a
(0.20–0.26
(GI50 1.55–9.63
showed ten-fold higher potency against KBVIN cell growth (GI50
0.92 M) compared with 5c (GI50 9.63 M). The results from series
l
M) and was ten-fold more potent than 3a and 5a-c
with a N-allyl group showed comparable potency, but a bulky N-
cyclopentyl group or an electron-withdrawing N-acetyl group re-
sulted in significantly reduced or no potency (see 4b and 4c). Thus,
as we expected, a small alkyl group on the linker might favor en-
hanced cytotoxic activity.
lM), 6b displayed similar potency to 5b, while 6c
l
l
6 indicated that the R3 on the pyridine ring can be modified to en-
hance potency and might have a greater impact on the potency than
R4. Furthermore, two additional compound series, 7 (a–g) and 8 (c–l)
with a chloro or trifluoromethyl R4 group, respectively, were synthe-
sized in parallel to investigate the impact of various R3 groups on the
antiproliferative activity. Among them, 3-methyl or -ethyl ester pyr-
idine compounds 7b, 8c, and 8e (R3 = COOMe or COOEt) showed
high potency with low to sub-micromolar GI50 values (0.19–
Next, we turned to modifications of the R3 and R4 substituents on
the pyridine ring (A ring) (see Table 2). The R4 on the pyridine ring
was changed to methyl, methoxy, or N-methylamine, rather than
the chlorine atom in 3a. This change resulted in active compounds
5a–5c with GI50 values of 2.70–9.63 lM. Then, because a nitro group
is generally considered to be a moiety associated with risk and, thus,
unacceptable in pharmaceutical agents,13 compounds 6a–6c with a
methoxy carbonyl (R3) rather than the nitro group in the series 5
compounds and various R4 groups (Cl, OMe, and NHMe, respec-
tively) on the A-ring were synthesized and evaluated against the
HTCL panel. Compound 6a exhibited sub-micromolar GI50 values
1.81 l
M) similar to series 6, regardless of whether R4 was chloro
or trifluoromethyl. In contrast, the presence of a 3-cyano (7a and
8j), 3-isopropyl ester (7d and 8f), 3-carboxylic acid (7c and 8d), or
3-methylamide (8g and 7e) on the pyridine ring resulted in substan-
tially reduced (GI50 >7.21 lM) or no cytotoxic activity. Therefore,
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
5
Table 2
Inhibitory activity of 5–8 against HTCL panel
R3
OMe
A
N
B
R4
N
5-8
R3
R4
GI50 SDa
(
lM)
A549
KB
KBVIN
DU145
3a
5a
5b
5c
6a
6b
6c
7a
7b
7c
7d
7e
7f
7g
8c
8d
8e
8f
8g
8h
8i
NO2
NO2
NO2
NO2
COOMe
COOMe
COOMe
CN
COOEt
COOH
COOPr-i
CONHMe
CH2OH
CH2OMe
COOMe
COOH
COOEt
COOPr-i
CONHMe
CH2OH
CH2OMe
CN
Cl
2.20 0.20
4.42 0.44
3.27 0.69
4.50 0.35
0.23 0.01
3.15 0.75
3.43 0.30
20.4 2.51
1.81 0.12
NAb
22.0 1.64
NA
3.25 0.27
0.30 0.03
0.35 0.05
NA
1.78 0.11
2.92 0.25
3.31 0.46
4.53 0.70
0.26 0.04
4.01 0.75
1.36 0.34
22.1 1.94
1.80 0.10
NA
14.5 0.40
NA
2.26 0.10
0.33 0.07
0.41 0.09
NA
1.86 0.30
4.19 0.97
2.83 0.71
9.63 1.14
0.20 0.03
4.02 0.57
0.92 0.09
18.1 3.73
1.43 0.18
NA
17.3 1.58
NA
2.12 0.16
0.20 0.01
0.19 0.01
NA
1.55 0.24
3.35 0.55
2.70 0.25
4.45 0.85
0.21 0.04
2.45 0.49
1.67 0.13
20.4 4.59
1.43 0.09
NA
17.8 0.34
NA
1.96 0.11
0.25 0.01
0.25 0.04
NA
Me
OMe
NHMe
Cl
OMe
NHMe
Cl
Cl
Cl
Cl
Cl
Cl
Cl
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
1.46 0.13
NA
NA
2.13 0.09
1.20 0.02
7.21 1.20
1.52 0.10
NA
NA
1.92 0.28
1.64 0.04
15.3 2.33
1.30 0.15
NA
NA
1.75 0.11
1.43 0.20
7.91 1.56
1.30 0.13
NA
NA
1.71 0.08
1.29 0.11
10.7 2.49
8j
N
N
N
8k
CF3
CF3
NA
NA
NA
NA
N
H
N
N
N
8l
1.82 0.50
1.47 0.34
1.26 0.09
1.16 0.05
N
Paclitaxelc
8.18 1.01 nM
7.77 0.84 nM
1803 302 nM
6.50 0.43 nM
a
b
c
Concentration of compound that inhibits 50% human tumor cell growth, presented as mean standard deviation (SD), performed at least in triplicate.
Not active; test compound (20
Positive control.
lg/mL) did not reach 50% inhibition.
while a methyl or ethyl ester R3 group was beneficial, a bulky (isopro-
pyl ester) or negatively charged (such as carboxylic acid at physiolog-
ical pH) R3 substituent impaired the cytotoxic activity. Interestingly,
we observed that 3-methoxymethylpyridines 7g and 8i (R3 = CH2OMe)
and 3-hydroxymethylpyridines 7f and 8h (R3 = CH2OH) also were po-
of P-glycoprotein and can overcome paclitaxel-resistance, provid-
ing a therapeutic advantage over paclitaxel.
In investigating possible biological target(s), the three most ac-
tive compounds 6a, 7g, and 8c (GI50 0.19–0.41 lM) were tested in
tubulin assays. As shown in Table 3, they showed high activity for
tent with GI50 values of 0.20–3.25 lM, generally comparable with 3-
inhibition of tubulin assembly, with IC50 values ranging from 1.4 to
methyl and -ethyl ester compounds. Notably, compounds 6a
1.7 lM, which are comparable to those with CA-4 (IC50 1.2 lM), a
(R3 = COOMe, R4 = Cl), 7g (R3 = CH2OMe, R4 = Cl), and 8c (R3 = COOMe,
tubulin inhibitor currently in phase II clinical trials, in the same as-
say. Furthermore, they inhibited the binding of colchicine to tubu-
lin (P80%). These results suggest that these active diarylamines
might exert their effects by inhibiting tubulin polymerization tar-
geted at the colchicine binding site.
To better understand how the diarylamine analogues interact
with tubulin, we investigated the binding mode of 6a at the
colchicine binding domain in the tubulin dimer by using CDOCKER
in the Discovery Studio 3.0 software. We attempted to dock 6a
into two different crystal structures, the tubulin/TN16 complex
(PDB ID code: 3HKD) and the tubulin/DMDA-colchicine complex
(PDB ID code: 1SA0). We selected the former crystal structure as
our modeling system, because 6a showed a lower binding energy
(ꢁ14.50 kcal/mol) in 3HKD than in 1SA0 (ꢁ9.74 kcal/mol) and
superposed very well with the native ligand TN16 in the 3HKD
R4 = CF3) exhibited GI50 values between 0.19 and 0.41
lM against all
four HTCL. Similarly to 7c and 8d, compound8k with a 3-tetrazole ring,
a bio-isostere of a carboxylic acid, on the pyridine ring was inactive.
However, with a methylated tetrazole that cannot ionize, compound
8l exhibited high potency with low micromolar GI50 values ranging
from 1.16 to 1.82 lM against the HTCL panel.
More interestingly, we found that most active compounds in
the series of tertiary diarylamine showed equal or slightly better
potency against both the KB and paclitaxel-resistant KBVIN cell
lines (see Tables 1 and 2). In contrast, paclitaxel showed nanomolar
activity against KB cells (GI50 8 nM), but significantly lower po-
tency (GI50 1800 nM) against the KBVIN cell line, which over-
expresses P-glycoprotein, a drug efflux transporter. These results
indicated that these types of active compounds are not substrates
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

6
X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
Table 3
cromolar GI50 values, significant potency against tubulin assembly,
Inhibition of tubulin assemblya and colchicine bindingb
and inhibitory activity for colchicine binding to tubulin protein.
Notably, the active tertiary diarylamine compounds showed high
potency against paclitaxel-resistant KBVIN cell lines, providing a po-
tential advantage over paclitaxel. In addition, molecular modeling
studies revealed that the active 6a and CA-4 might have different
binding modes. Therefore, our active tertiary diarylamines might
be a novel class of tubulin inhibitors targeting the colchicine
binding site. Current SAR studies on several series of N-methyl-N-
(4-methoxyphenyl)pyridin-2-amines revealed the following con-
clusions: (1) a tertiary amine linker is crucial for enhanced
antitumor activity, and a methyl group is better than others, (2) a
para-methoxyphenyl moiety is favorable for antitumor activity,
(3) the R3 group on the pyridine ring can be modified to improve po-
tency, and methyl ester and methoxymethyl groups are better than
other groups tested, (4) the R4 substituent on the pyridine ring also
contributes to the cytotoxic activity, but seems to have less impact
than R3. We will use these initial promising results to guide our fur-
ther optimization of new compounds as tubulin inhibitors targeting
the colchicine binding site. In addition, because various tubulin
inhibitors targeting the colchicine binding site have been reported
to selectively disrupt the cytoskeleton of proliferating endothelial
cells as vascular disrupting agents (VDAs),16,17 further studies on
our newly discovered active compounds will be necessary to
develop a new class of antitumor drug with a new mechanism of
action as a new approach for treating cancers and tumors.
compound inhibition of tubulin assembly
IC50 M) SD
inhibition of colchicine
binding (%) SD
(l
6a
7g
1.40 0.01
1.50 0.20
1.70 0.20
1.20 0.20
80 2.0
88 0.5
82 1.0
98 0.6
8c
CA-4c
a
The tubulin assembly assay measured the extent of assembly of 10
after 20 min at 30 °C.
lM tubulin
b
Tubulin:
1 l 5 lM. Inhibitor: 5 lM. Incubation was per-
M. [3H]colchicine:
formed for 10 min at 37 °C.
c
Reference compound is a drug candidate in phase II clinical trial.
structure. Figure 3A illustrates the binding mode of 6a (orange)
with tubulin protein and its overlap with TN16 (cyan). The meth-
oxy group on the B-ring of 6a formed two hydrogen bonds with
the carboxyl group of Glu200 and the hydroxyl of Tyr202 in b-S6,
while TN-16 formed one H-bond also with Glu200 and another
H-bond with Val238 in b-H7. The pyridine moiety (A-ring) of 6a
bound in the lipophilic pocket formed by the side chains of amino
acids in b-S8 and b-H7, and the ester group on the A-ring showed
two hydrogen bonds with the side chain of Cys241, a key amino
acid in the colchicine binding site. For comparison, we superposed
6a, DMDA-colchicine (the native ligand of 1SA0), and CA-4 into the
same docking model, as shown in Figure 3B. While the TN16 bind-
ing site extended deeply into the b-subunit side and less into the
a
-subunit,14,15 DMDA-colchicine (green) and CA-4 (blue) bound
at the interface between the - and b-subunits of the tubulin di-
a
4. Experimental section
4.1. Chemistry
mer, and showed an overlap only with the pyridine ring of 6a.
The 3,4,5-trimethoxyphenyl moieties found in both DMDA-
colchicine and CA-4 overlapped well and also formed H-bonds with
the thiol group of Cys241, which plays a locating role for most tubu-
lin inhibitors binding at the colchicine binding site. The theoretical
model of 6a supports our bioassay results and can explain, at least
partially, the high potency of 6a and the preference for the ester
group on the pyridine ring. Meanwhile, it also reveals obvious dif-
ferences in the binding conformations and orientations between
our active compounds and CA-4 in the colchicine binding site.
In summary, three new leads 6a, 7g, and 8c exhibited promising
cytotoxic activity in human tumor cell line assays with submi-
The nuclear magnetic resonance (1H and 13C NMR) spectra were
measured on a JNM-ECA-400 (400 MHz and 100 MHz, respec-
tively) spectrometer using tetramethylsilane (TMS) as internal
standard. The solvent used was CDCl3 unless otherwise indicated.
Mass spectra (MS) were measured on API-150 mass spectrometer
with the electrospray ionization source from ABI, Inc. Melting
points were measured with a RY-1 melting apparatus without cor-
rection. The microwave reactions were performed on a microwave
reactor from Biotage, Inc. Medium-pressure column chromatogra-
Figure 3. (A) A predicted mode of compound 6a (orange stick model) binding with tubulin (PDB ID code: 3HKD), and overlapping with TN16 (cyan stick model, the native
ligand of 3HKD); (B) the superimposition of docked compound 6a with DMDA-colchicine (green stick model, the native ligand of 1SA0) and CA-4 (blue stick model).
Surrounding amino acid side chains are shown in gray stick format and labeled. The hydrogen bonds are shown by green dashed lines and the distance between the ligands
and protein is less than 3 Å
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
7
phy was performed using a CombiFlashÒ Companion system from
ISCO, Inc. Thin-layer chromatography (TLC) was performed on sil-
ica gel GF254 plates. Silica gel GF254 and H (200–300 mesh) from
Qingdao Haiyang Chemical Company were used for TLC, prepara-
tive TLC (PTLC), and column chromatography, respectively. All
commercial chemical reagents were purchased from Beijing Chem-
ical Works or Sigma–Aldrich, Inc. Purities of target compounds
were determined by using an Agilent HPLC-1200 with UV detector
7.16 (2H, d, J = 8.8 Hz, ArH-30,50), 7.96 (1H, d, J = 8.4 Hz , PyH-4);
MS m/z (%) 348 (M+1, 100), 350 (M+3, 25); HPLC purity 97.2%.
4.6. 6-Chloro-N-methyl-N-(4-methoxycarbonylphenyl)-3-
nitropyridin-2-amine (3d)
Method B. Starting with 193 mg of 2a and 248 mg of methyl N-
methyl-4-amino benzoate at 160 °C for 15 min to produce130 mg
of 3d, 41% yield, brown solid, mp 106–107 °C; 1H NMR d ppm
3.67 (1H, s, NCH3), 3.89 (1H, s, OCH3), 6.94 (1H, d, J = 8.4 Hz,
PyH-5), 7.07 (2H, d, J = 9.2 Hz, ArH-20,60), 7.98 (2H, d, J = 9.2 Hz,
ArH-30,50), 8.02 (1H, d, J = 8.4 Hz, PyH-4); MS m/z (%) 322 (M+1,
100), 324 (M+3, 28); HPLC purity 97.2%.
and an Agilent Eclipse XDB-C18 column (150 mm ꢂ 4.6 mm, 5
lm)
eluting with a mixture of solvents A and B (acetonitrile/water
80:20), flow rate 0.8 mL/min and UV detection at 254 nm.
4.2. 6-Chloro-N-(4-cyanophenyl)-N-methyl-3-nitropyridin-2-
amine (3b)
4.7. 6-Chloro-N-(3,4-dimethoxyphenyl)-N-methyl-3-
A mixture of 2,6-dichloro-3-nitropyridine (2a, 94 mg, 0.5 mmol)
and N-methyl 4-cyanoaniline (80 mg, 0.6 mmol) was heated neat
at 120 °C under N2 protection for 4 h with stirring. Then the mix-
ture was diluted with CH2Cl2 and purified by preparative TLC
(CH2Cl2/petroleum ether/MeOH = 1/1.5/0.04) to obtain 40 mg of
3b in 37% yield, together with recovery of 24 mg of 2a, yellow solid,
mp 98–100 °C; 1H NMR d ppm 3.66 (3H, s, NCH3), 7.02 (1H, d,
J = 8.4 Hz, PyH-5), 7.09 (2H, d, J = 8.8 Hz, ArH-20,60), 7.58 (2H, d,
J = 8.8 Hz, ArH-30,50), 8.07 (1H, d, J = 8.4 Hz, PyH-4); MS m/z (%)
289 (M+1, 100), 291 (M+3, 35); HPLC purity 97.7%.
nitropyridin-2-amine (3e)
Method A. Starting with 193 mg of 2a and 250 mg of N-methyl-
3,4-dimethoxyaniline to produce 262 mg of 3e, 81% yield, red solid,
mp 145–146 °C; 1H NMR d ppm 3.56 (s, 3H, NCH3), 3.83 (s, 3H,
OCH3), 3.85 (s, 3H, OCH3), 6.60 (2H, m, ArH-20 and 60), 6.78 (1H,
d, J = 8.0 Hz, PyH-5), 6.80 (1H, d, J = 9.2 Hz, ArH-40), 7.88 (1H, d,
J = 8.0 Hz, PyH-4); MS m/z (%), 324.3 (M+1, 18), 325.9 (M+3, 6),
290.1 (Mꢁ33, 100); HPLC purity 99.2%.
4.8. 6-Chloro-N-methyl-3-nitro-N-(3,4,5-
4.3. General procedure for coupling of substituted 2-
trimethoxyphenyl)pyridin-2-amine (3f)
chloropyridine and N-alkylaniline
Method A. Starting with 193 mg of 2a and 250 mg of N-methyl-
3,4,5-trimethoxyaniline to produce 268 mg of 3f, 76% yield, orange
solid, mp 138–140 °C; 1H NMR d ppm 3.60 (3H, s, NCH3), 3.79 (3H,
s, OCH3), 3.80 (3H, s, OCH3), 3.83 (3H, s, OCH3), 6.26 (2H, s, ArH-
20,60), 6.82 (1H, d, J = 8.4 Hz, PyH-5), 7.91 (1H, d, J = 8.4 Hz, PyH-
4); MS m/z (%) 324.3 (M+1, 18), 325.9 (M+3, 6), 290.1 (Mꢁ33,
100); HPLC purity 99.5%.
Method A (traditional): A mixture of a substituted 2-chloropyr-
idine (2, 1.0 mmol), N-methyl-4-methoxyaniline (1.5 mmol), and
anhydrous potassium carbonate (2 mmol) in t-BuOH was stirred at
rt for 12–24 h monitored by TLC until the reaction was complete.
The mixture was poured into ice-water, the pH adjusted to ca. 3.0
with aq HCl (2 N), and the resulting solution extracted with CH2Cl2
three times. The combined organic phases were washed with water
and brine, successively, until neutral and dried over anhydrous
Na2SO4 overnight. After removal of solvent in vacuo, the crude prod-
uct was purified by flash column chromatography (gradient elution:
EtOAc/petroleum ether, 0–50%) to obtain corresponding pure prod-
uct. Method B (microwave irradiation): A mixture of 2 (1.0 mmol),
substituted aniline (1.5 mmol), and anhydrous potassium carbonate
(414 mg, 3.0 mmol) in 3 mL of t-BuOH was heated at 120–160 °C
with microwave-assistance for 10–30 min with stirring. The reac-
tion mixture was then poured into ice-water, pH adjusted to ꢃ3.0
with aq HCl (2 N), and solid crude product was filtered. The product
was then purified by the same methods as in Method A above.
4.9. N-Allyl-6-chloro-N-(4-methoxyphenyl)-3-nitropyridin-2-
amine (4a)
Method
B. Starting with 193 mg of 2a and 244 mg of N-allyl-4-
methoxyaniline at 120 °C for 10 min to produce 168 mg of 4a,
53% yield, red liquid. 1H NMR d ppm 3.77 (3H, s, OCH3), 4.63 (2H,
d, J = 5.6 Hz, NCH2), 5.16 and 5.18 (each 1H, dd, J = 17.6 Hz and
1.2 Hz, CH2@), 5.99 (1H, m, CH), 6.76 (1H, d, J = 8.4 Hz, PyH-5),
6.81 (2H, d, J = 9.2 Hz, ArH-20,60), 6.98 (2H, d, J = 9.2 Hz, ArH-30,50),
7.87 (1H, d, J = 8.4 Hz, PyH-4); MS m/z (%) 320 (M+1, 100), 322
(M+3, 25); HPLC purity 99.4%.
4.4. 6-Chloro-N-(4-methoxyphenyl)-N-methyl-3-nitropyridine-
4.10. 6-Chloro-N-cyclopentyl-N-(4-methoxyphenyl)-3-
2-amine (3a)
nitropyridin-2-amine (4b)
Method A. Starting with 193 mg of 2a and 206 mg of N-methyl
4-methoxyanaline to produce 240 mg of 3a, 82% yield, orange solid
recrystallized from MeOH, mp 96–97 °C; 1H NMR d ppm 3.54 (3H,
s, NCH3), 3.78 (3H, s, OCH3), 6.76 (1H, d, J = 8.4 Hz, PyH-5), 6.83
(2H, d, J = 9.2 Hz, ArH-20,60), 7.00 (2H, d, J = 9.2 Hz, ArH-30,50), 7.88
(1H, d, J = 8.4 Hz, PyH-4); MS m/z (%) 294 (M+1, 100), 296 (M+3,
32); HPLC purity 99.5%.
Method B. Starting with 194 mg of 2a and 288 mg of N-cyclo-
pentyl-N-4-methoxyaniline at 160 °C for 20 min to produce
196 mg of 4b, 50% yield, yellow solid, mp 93–94 °C; 1H NMR d
ppm 1.49–1.61 (6H, m, CH2 ꢂ 3), 2.02 (2H, m, CH2), 3.79 (1H, s,
OCH3), 4.93 (1H, m, CH), 6.70 (1H, d, J = 8.0 Hz, PyH-5), 6.83 (2H,
d, J = 9.2 Hz, ArH-20,60), 6.93 (2H, d, J = 9.2 Hz, ArH-30,50), 7.69 (1H,
d, J = 8.0 Hz, PyH-4); MS m/z (%) 348 (M+1, 100), 350 (M+3, 32);
HPLC purity 98.7%.
4.5. 6-Chloro-N-methyl-3-nitro-N-(4-
trifluoromethoxyphenyl)pyridin-2-amine (3c)
4.11. N-(4-Methoxyphenyl)-N-methyl-6-methyl-3-nitropyridin-
2-amine (5a)
Method B. Starting with 193 mg of 2a and 288 mg of N-methyl-
4-trifluoro methoxyaniline at 120 °C for 30 min to produce 198 mg
of 3c, 57% yield, yellow liquid. 1H NMR d ppm 3.59 (1H, s, NCH3),
6.87 (1H, d, J = 8.4 Hz, PyH-5), 7.08 (2H, d, J = 8.8 Hz, ArH-20,60),
Method B. Starting with 174 mg of 2b and 206 mg of N-methyl-
4-methoxyaniline at 140 °C for 20 min to produce 154 mg of 5a,
56% yield, yellow solid, mp 97–98 °C; 1H NMR d ppm 2.53 (3H, s,
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

8
X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
CH3), 3.53 (3H, s, NCH3), 3.77 (3H, s, OCH3), 6.64 (1H, d, J = 8.0 Hz,
PyH-5), 6.82 (2H, d, J = 9.2 Hz, ArH-20,60), 6.99 (2H, d, J = 9.2 Hz,
ArH-30,50), 7.86 (1H, d, J = 8.0 Hz, PyH-4); MS m/z (%) 274 (M+1,
100); HPLC purity 98.5%.
OCH3), 6.19 (1H, d, J = 8.8 Hz, PyH-5), 6.83 (2H, d, J = 9.2 Hz, ArH-
20,60), 7.02 (2H, d, J = 9.2 Hz, ArH-30,50), 8.03 (1H, d, J = 8.8 Hz,
PyH-4); MS m/z (%) 290 (M+1, 100); HPLC purity 95.6%.
4.17. Methyl 6-methoxy-2-(N-(4-methoxyphenyl)-N-
4.12. Methyl 6-chloro-2-(N-(4-methoxyphenyl)-N-
methyl)aminonicotinate (6b)
methyl)aminonicotinate (6a)
Prepared in the same manner as for 5b. Starting with 123 mg of
6a to obtain119 mg of 6b in 98% yield, white solid, mp 78–80 °C;
1H NMR d ppm 3.28 (3H, s, NCH3), 3.48 (3H, s, OCH3), 3.78 (3H, s,
OCH3), 3.96 (3H, s, OCH3), 6.18 (1H, d, J = 8.4 Hz, PyH-5), 6.83
(2H, d, J = 9.2 Hz, ArH-20,60), 7.03 (2H, d, J = 9.2 Hz, ArH-30,50), 7.71
(1H, d, J = 8.4 Hz, PyH-4). MS m/z (%)303 (M+1, 24), 271 (Mꢁ31,
100); HPLC purity 97.9%.
Method B. Starting with 206 mg of methyl 2,6-dichloronicoti-
nate (2c) and 206 mg of N-methyl-4-methoxyaniline at 160 °C for
30 min to produce 186 mg of 6a, 60% yield, pale yellow solid, mp
79–81 °C; 1H NMR d ppm 3.28 (3H, s, NCH3), 3.47 (3H, s, OCH3),
3.78 (3H, s, OCH3), 6.74 (1H, d, J = 8.0 Hz, PyH-5), 6.84 (2H, d,
J = 9.2 Hz, ArH-20,60), 7.02 (2H, d, J = 9.2 Hz, ArH-30,50), 7.61 (1H, d,
J = 8.0 Hz, PyH-4); 13C NMR d ppm 41.23, 51.79, 55.57, 113.21,
114.07, 114.74, 115.45, 125.55, 128.34, 141.30, 141.72, 151.33,
156.81, 156.98, 167.35; MS m/z (%) 307 (M+1, 100), 309 (M+3,
29); HPLC purity 99.2%.
4.18. N2,N6-Dimethyl-N-(4-methoxyphenyl)-3-nitropyridin-2,6-
diamine (5c)
Compound 3a (117 mg, 0.2 mmol) in 3 mL of 30% methylamine-
MeOH solution was refluxed for 12 h with stirring. After the reac-
tion was completed, the mixture was poured into ice-water, and
the resulting red solid was collected to give 86 mg of pure 5c,
75% yield, mp 169–170 °C; 1H NMR d ppm 3.03 (3H, d, J = 5.2 Hz,
NCH3), 3.49 (3H, s, NCH3), 3.76 (3H, s, OCH3), 5.01 (1H, br s, NH),
5.87 (1H, d, J = 9.2 Hz, PyH-5), 6.81 (2H, d, J = 8.8 Hz, ArH-20,60),
7.02 (2H, d, J = 8.8 Hz, ArH-30,50), 8.00 (1H, d, J = 9.2 Hz, PyH-4);
MS m/z (%) 289 (M+1,27), 255 (Mꢁ33, 100); HPLC purity 100.0%.
4.13. 6-Chloro-3-cyano-N-(4-methoxyphenyl)-N-methylpyridin-
2-amine (7a)
Method B. Starting with 173 mg of 3-cyano-2,6-dichloropyridine
(2e) and 205 mg of N-methyl-4-methoxyaniline at 160 °C for
15 min to produce 32 mg of 7a, 12% yield, white solid, mp 137–
138 °C; 1HNMR d ppm 3.47 (3H, s, NCH3), 3.85 (3H, s, OCH3), 6.18
(1H, d, J = 8.4 Hz, PyH-5), 6.99 (2H, d, J = 8.8 Hz, ArH-20,60), 7.14
(2H, d, J = 8.8 Hz, ArH-30,50), 7.38 (1H, d, J = 8.4 Hz, PyH-4). MS m/
z (%): 274 (M+1, 100), 276 (M+3, 31); HPLC purity 99.0%.
4.19. Methyl 2-[N-(4-methoxyphenyl)-N-methyl)]amino-6-
methylaminonicotinate (6c)
4.14. Ethyl 6-chloro-2-(N-(4-methoxyphenyl)-N-
methyl)aminonicotinate (7b)
Prepared in the same manner as for 5c. Starting with 123 mg of
6a to obtain 96 mg of 6c, 80% yield, white solid, mp 88–90 °C; 1H
NMR d ppm 2.97 (3H, d, J = 5.2 Hz, NHCH3), 3.28 (3H, s, NCH3),
3.43 (3H, s, OCH3), 3.76 (3H, s, OCH3), 4.69 (1H, br s, NH), 5.88
(1H, d, J = 8.8 Hz, PyH-5), 6.80 (2H, d, J = 9.2 Hz, ArH-20,60), 7.01
(2H, d, J = 9.2 Hz, ArH-30,50), 7.67 (1H, d, J = 8.8 Hz, PyH-4); MS m/
z (%) 302 (M+1, 1), 227 (Mꢁ7, 100); HPLC purity 100.0 %.
Method B. Starting with 220 mg of ethyl 2,6-dichloronicotinate
(2d) and 205 mg of N-methyl-4-methoxyaniline at 160 °C for
30 min to produce 173 mg of 7b, 54% yield, yellow solid, mp 79–
81 °C; 1H NMR d ppm 1.08 (3H, t, J = 7.6 Hz, CH3), 3.47 (3H, s,
NCH3), 3.70 (2H, q, J = 7.2 Hz, CH2), 3.78 (3H, s, OCH3), 6.73 (2H,
d, J = 8.0 Hz, PyH-5), 6.83 (2H, d, J = 9.2 Hz, ArH-20,60), 7.03 (2H, d,
J = 9.2 Hz, ArH-30,50), 7.61 (1H, d, J = 8.0 Hz, PyH-4); MS m/z (%)
321.3 (M+1, 58), 323.3 (M+3, 20), 275.1 (Mꢁ45, 100); HPLC purity
96.7%.
4.20. 6-Chloro-2-(N-(4-methoxyphenyl)-N-
methyl)aminonicotinic acid (7c)
To a solution of 6a (154 mg, 0.5 mmol) in THF/MeOH (1.5 mL/
1.5 mL) was added 3 N aq NaOH (3.0 mL) dropwise with stirring
at rt for 36 h. The mixture was poured into 20 mL of water. After
removal of insoluble substance, the water phase was acidified with
aq. HCl (2 N) to pH 2. The precipitated solid was collected, washed
with water, and dried to give 112 mg of pure 7c, 77% yield, yellow
solid, mp 138–140 °C; 1H NMR d ppm 3.43 (3H, s, OCH3), 3.72 (3H,
s, OCH3), 6.81 (2H, d, J = 8.8 Hz, ArH-20,60), 6.90 (1H, d, J = 8.0 Hz,
PyH-5), 7.00 (2H, d, J = 8.8 Hz, ArH-30,50), 7.90 (1H, d, J = 8.0 Hz,
PyH-4). MS m/z (%) 293.2 (M+1, 100), 295.3 (M+3, 45); HPLC purity
95.2%.
4.15. 6-Chloro-N-(4-methoxyphenyl)-N-methylcarbonyl-3-
nitropyridin-2-amine (4c)
To a solution of 2a (280 mg, 1.0 mmol) in Ac2O (8 mL) was
added a drop of concentrated H2SO4. The mixture was heated at
100 °C under N2 protection for 18 h with stirring. After the reaction
was completed, the mixture was poured into ice-water and neu-
tralized with 5% aq NaOH. The collected solid was further purified
by flash chromatography (gradient eluant/EtOAc/petroleum ether,
0–50%) to obtain 249 mg of pure 4c, 77% yield, yellow solid, mp
146–147 °C; 1H NMR d ppm 2.01 (3H, s, CH3), 3.86 (3H, s, OCH3),
6.99 (2H, d, J = 8.8 Hz, ArH-20,60), 7.31 (1H, d, J = 8.4 Hz, PyH-5),
7.49 (2H, d, J = 8.8 Hz, ArH-30, 50), 8.22 (1H, d, J = 8.4 Hz, PyH-4);
MS m/z (%) 322 (M+1, 100), 324 (M+3, 28); HPLC purity 97.8%.
4.21. Isopropyl 6-chloro-2-(N-(4-methoxyphenyl)-N-
methyl)aminonicotinate (7d)
A mixture of 7c (59 mg, 0.2 mmol), i-PrI (68 mg , 0.4 mmol), and
K2CO3 (55 mg, 0.4 mmol) in 5 mL of acetone was refluxed for 4 h.
K2CO3 was filtered out, and acetone was removed under reduced
pressure. The solid residue was purified by flash column chroma-
tography (gradient eluant/EtOAc/petroleum ether, 0–40%) to give
55 mg of pure 7d, 82% yield, pale yellow solid, mp 56–58 °C; 1H
NMR d ppm 1.02 (6H, d, J = 6.0 Hz, CH3ꢂ2), 3.46 (3H, s, NCH3),
3.76 (3H, s, OCH3), 4.60 (1H, m, CH), 6.73 (1H, d, J = 8.0 Hz, PyH-
5), 6.82 (2H, d, J = 9.2 Hz, ArH-20,60), 7.02 (2H, d, J = 9.2 Hz, ArH-
4.16. 6-Methoxy-N-(4-methoxyphenyl)-N-methyl-3-
nitropyridin-2-amine (5b)
A mixture of 3a (117 mg, 0.4 mmol) and NaOMe (ca. 1.0 mmol)
in 3 mL of MeOH was refluxed for 2 h with stirring. After the reac-
tion was finished, the mixture was poured into ice-water to obtain
108 mg of pure 5b as a yellow solid, 93% yield, mp 56–57 °C; 1H
NMR d ppm 3.54 (3H, s, NCH3), 3.78 (3H, s, OCH3), 3.99 (3H, s,
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
9
30,50), 7.59 (1H, d, J = 8.0 Hz, PyH-4); MS m/z (%) 335.4 (M+1, 32),
337.3 (M+3, 11), 275.1 (Mꢁ59+, 100); HPLC purity 95.5%.
MeI (0.07 mL, 1.13 mmol) in DMF (3–5 mL) was added NaH
(44 mg, 1.1 mmol, 60% oil suspension) at 0 °C with stirring for
1 h. After the reaction was completed, the mixture was poured
into ice-water and extracted with EtOAc three times. The com-
bined organic phase was washed with water and brine succes-
sively, and dried over anhydrous Na2SO4 overnight. After
removal of solvent in vacuo, the crude product was purified by
flash chromatography (gradient eluant/EtOAc/petroleum ether,
0–50%) to produce 116 mg of 8c, 92% yield, yellow solid, mp
99–101 °C; 1H NMR d ppm 3.30 (3H, s, NCH3), 3.50 (3H, s,
OCH3), 3.79 (3H, s, OCH3), 6.85 (2H, d, J = 9.2 Hz, ArH-20,60),
7.04 (2H, d, J = 9.2 Hz, ArH-30,50), 7.06 (1H, d, J = 8.0 Hz, PyH-5),
7.74 (1H, d, J = 8.0 Hz, PyH-4); 13C NMR d ppm 40.94, 51.83,
55.47, 109.46, 114.66 (2C), 118.36, 125.78 (2C), 140.10, 140.92,
147.63, 147.97, 156.32, 157.05, 167.19; MS m/z (%) 341 (M+1,
6), 309 (Mꢁ31, 100); HPLC purity 97.2%.
4.22. N-Methyl 6-chloro-2-(N-(4-methoxyphenyl)-N-
methyl)aminonicotinamide (7e)
A mixture of 7c (84 mg, 0.29 mmol), EDCI (86 mg, 0.45 mmol)
and HOBt (61 mg, 0.45 mmol) in CH2Cl2 (4 mL) was stirred at rt
for 30 min, and then 30% methyl amine (0.18 mL)-MeOH solution
was added dropwise at 0 °C with stirring over 15 min, followed
by warming to to rt for an additional 90 min. After adding water
(20 mL), the product was extracted with CH2Cl2 three times. The
combined organic phase was washed with water and brine, succes-
sively, and dried over anhydrous Na2SO4 overnight. After removal
of solvent in vacuo, crude product was purified by flash column
chromatography (gradient eluant/EtOAc/petroleum ether, 0–50%)
to give 59 mg of pure 7e, 66% yield, white solid, mp 150–152 °C;
1H NMR d ppm 2.52 (3H, d, J = 7.2 Hz, NHCH3), 3.39 (3H, s,
NCH3), 3.77 (3H, s, OCH3), 6.16 (1H, br, NH), 6.83 (2H, d,
J = 8.8 Hz, ArH-20,60), 6.90 (1H, d, J = 7.6 Hz, PyH-5), 6.95 (2H, d,
J = 8.8 Hz, ArH-30,50), 7.74 (1H, d, J = 7.6 Hz, PyH-4); MS m/z (%)
306 (M+1, 16), 308 (M+3, 6), 275.1 (M-30, 100); HPLC purity 98.3%.
4.26. 2-(N-(4-Methoxyphenyl)-N-methyl)amino-6-
trifluoromethylnicotinic acid (8d)
Prepared in a similar manner as for 7c. Starting with 170 mg of
8c to produce 118 mg of 8d, 72% yield, mp 146–148 °C; 1H NMR d
ppm 3.48 (3H, s, NCH3), 3.71 (3H, s, OCH3), 6.80 (2H, d, J = 8.8 Hz,
ArH-20,60), 7.02 (2H, d, J = 8.8 Hz, ArH-30,50), 7.16 (1H, d, J = 7.6 Hz,
PyH-5), 7.95 (1H, d, J = 7.6 Hz, PyH-4); MS m/z (%) 261 (Mꢁ65ꢁ,
100), 325 (Mꢁ1ꢁ, 46); HPLC purity 97.4%.
4.23. 6-Chloro-3-hydroxymethyl-N-(4-methoxyphenyl)-N-
methylpyridin-2-amine (7f)
To a solution of 6a (123 mg, 0.4 mmol) in 5 mL of anhydrous
THF, LiBH4 (88 mg, 4.0 mmol) was added at 0 °C with stirring over
15 min, and then the solution was warmed to rt for 30 min. MeOH
(0.5 mL) was then added dropwise to the mixture with stirring for
an additional 30 min. The mixture was poured into ice-water and
extracted with EtOAc three times. Combined organic phase was
washed successively with water and brine, and dried over anhy-
drous Na2SO4 overnight. After removal of solvent in vacuo, the
crude product was purified by flash column chromatography (gra-
dient eluant/EtOAc/petroleum ether, 0–50%) to to produce 91 mg
of pure 7f, 82% yield, yellow oil. 1H NMR d ppm 3.35 (3H, s,
NCH3), 3.79 (3H, s, OCH3), 3.98 (2H, s, CH2), 6.84 (2H, d,
J = 8.8 Hz, ArH-20,60), 6.92 (1H, d, J = 8.0 Hz, PyH-4), 6.95 (2H, d,
J = 8.8 Hz, ArH-30,50), 7.60 (1H, d, J = 8.0 Hz, PyH-5); MS m/z (%)
279.2 (M+1, 100), 281.2 (M+3, 34); HPLC purity 99.4%.
4.27. Ethyl 2-(N-(4-methoxyphenyl)-N-methyl)amino-6-
trifluoromethylnicotinate (8e)
Prepared in a similar manner as for 7d. Starting with 8d
(163 mg, 0.5 mmol), EtI (153 mg , 1.0 mmol), and K2CO3 (138 mg,
1.0 mmol) to produce 156 mg of 8e, 88% yield, yellow solid, mp
105–106 °C; 1H NMR d ppm 1.09 (3H, t, J = 5.6 Hz, CH3), 3.50 (3H,
s, NCH3), 3.70 (3H, q, J = 7.2 Hz, OCH2), 3.79 (3H, s, OCH3), 4.01
(2H, d, J = 5.6 Hz, CH2), 6.84 (2H, d, J = 9.2 Hz, ArH-20,60), 7.04 (2H,
d, J = 9.2 Hz, ArH-30,50), 7.06 (1H, d, J = 7.6 Hz, PyH-5), 7.74 (1H, d,
J = 7.6 Hz, PyH-4). MS m/z (%)355.2 (M+1, 28), 309.3 (Mꢁ45,
100); HPLC purity 100.0%.
4.28. Isopropyl 2-(N-(4-methoxyphenyl)-N-methyl)amino-6-
4.24. 6-Chloro-3-methoxymethyl-N-(4-methoxyphenyl)-N-
trifluoromethylnicotinate (8f)
methylpyridin-2-amine (7g)
Prepared in a similar manner as for 7d. Starting with 8d (42 mg,
To a solution of 7f (51 mg, 0.18 mmol) and MeI (0.03 mL,
0.54 mmol) in 6 mL of anhydrous THF was added NaH (22 mg,
0.54 mmol, 60% oil suspension) at 0 °C and stirred for 30 min at
0 °C and for 2 h at rt. The mixture was poured into ice-water and
extracted with EtOAc three times. The combined organic phase
was washed with water and brine, successively, and dried over
anhydrous Na2SO4 overnight. After removal of solvent in vacuo,
the crude product was purified by flash column chromatography
(gradient eluant/EtOAc/petroleum ether, 0–30%) to give 41 mg of
pure 7g, 78% yield, yellow oil. 1H NMR d ppm 3.15 (3H, s, OCH3),
3.36 (3H, s, NCH3), 3.69 (2H, s, OCH2), 3.79 (3H, s, OCH3), 6.82
(2H, d, J = 8.8 Hz, ArH-20,60), 6.91 (3H, m, PyH-5 and ArH-30,50),
7.58 (1H, d, J = 8.0 Hz, PyH-4); 13C NMR d ppm 42.07, 55.45,
58.29, 70.16, 114.59, 115.12, 116.09, 122.63, 125.09, 128.22,
138.86, 142.20, 147.50, 156.27, 157.05; MS m/z (%) 261 (Mꢁ31+,
100), 293 (M+1, 52), 295.3 (M+3, 18); HPLC purity 99.3%.
0.13 mmol), i-PrI (66 mg
, 0.39 mmol), and K2CO3 (54 mg,
0.39 mmol) to produce 45 mg of 8f, 94% yield, pale yellow solid,
mp 108–109 °C; 1H NMR d ppm 1.04 (6H, d, J = 6.4 Hz, CH3ꢂ2),
3.49 (3H, s, NCH3), 3.77 (3H, s, OCH3), 4.59 (1H, m, CH), 6.84 (2H,
d, J = 8.8 Hz, ArH-20,60), 7.04 (2H, d, J = 8.8 Hz, ArH-30,50), 7.06 (1H,
d, J = 7.6 Hz, PyH-5), 7.72 (1H, d, J = 7.6 Hz, PyH-4). MS m/z (%)
369.3 (M+1, 8), 309.3 (M-59, 100); HPLC purity 96.5%.
4.29. N-Methyl 2-(N-(4-methoxyphenyl)-N-methyl)amino-6-
trifluoromethyl nicotinamide (8g)
A solution of 8c (49 mg, 0.14 mmol) in 3 mL of 30% methyl-
amine-MeOH solution was stirred at rt for 4 days. After removal
of solvent in vacuo, the crude product was purified with flash
column chromatography (gradient eluant: EtOAc/petroleum ether,
0–60%) to provide 35 mg of 8g, 74% yield, white solid, mp 151–
152 °C; 1H NMR d ppm 2.51 (3H, d, J = 4.8 Hz, NHCH3), 3.42 (3H,
s, NCH3), 3.78 (3H, s, OCH3), 6.00 (1H, br, CONH), 6.84 (2H, d,
J = 8.8 Hz, ArH-20,60), 6.98 (2H, d, J = 8.8 Hz, ArH-30,50), 7.20 (1H,
J = 7.6 Hz, PyH-5), 7.84 (1H, J = 7.6 Hz, PyH-4); MS m/z (%) 340
(M+1, 4), 309 (Mꢁ30, 100); HPLC purity 100.0%.
4.25. Methyl 2-(N-(4-methoxyphenyl)-N-methyl)amino-6-
trifluoromethylnicotinate (8c)
To a solution of methyl 6-chloro-2-[N-(4-methoxyphenyl) ami-
no]-6-trifluoro-methylnicotinate (8a, 120 mg, 0.37 mmol), and
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

10
X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
4.30. 3-Hydroxymethyl-N-(4-methoxyphenyl)-N-methyl-6-
trifluoromethylpyridin-2-amine (8h)
4.08 (3H, s, OCH3), 6.60 (2H, d, J = 9.2 Hz, ArH-20,60), 6.83 (2H, d,
J = 9.2 Hz, ArH-30,50), 7.21 (1H, d, J = 7.6 Hz, PyH-5), 7.83 (1H, d,
J = 7.6 Hz, PyH-4); MS m/z (%) 365 (M+1, 3), 279 (Mꢁ85, 100); HPLC
purity 100.0%.
Prepared in a similar manner as for 7f. Starting with 8c (136 mg,
0.4 mmol) to produce 102 mg of 8h, 82% yield, yellow oil. 1H NMR d
ppm 3.38 (3H, s, NCH3), 3.80 (3H, s, OCH3), 4.01 (2H, d, J = 5.6 Hz,
CH2), 6.84 (2H, d, J = 9.2 Hz, ArH-20,60), 6.96 (2H, d, J = 9.2 Hz,
ArH-30,50), 7.26 (1H, d, J = 7.6 Hz, PyH-5), 7.80 (1H, d, J = 7.6 Hz,
PyH-4); MS m/z (%) 312 (M+1, 100); HPLC purity 99.1%.
4.35. Antiproliferative activity assay
Target compounds were assayed by SRB method for cytotoxic
activity using the HTCL assay according to procedures described
previously.18–20 The panel of cell lines included human lung carci-
noma (A-549), epidermoid carcinoma of the nasopharynx (KB),
P-gp-expressing epidermoid carcinoma of the nasopharynx
(KBVIN), and prostate cancer (DU145). The cytotoxic effects of each
compound were expressed as GI50 values, which represent the
molar drug concentrations required to cause 50% tumor cell
growth inhibition.
4.31. 3-Methoxymethyl-N-(4-methoxyphenyl)-N-methyl-6-
trifluoromethylpyridine-2-amine (8i)
Prepared in a similar manner as for 7g. Starting with 8h
(110 mg, 0.35 mmol), MeI (0.06 mL, 1.0 mmol), and NaH (40 mg,
1.0 mmol, 60% oil suspension) to produce 94 mg of 8i, 82% yield,
yellow oil. 1H NMR d ppm 3.17 (3H, s, OCH3), 3.39 (3H, s, NCH3),
3.72 (2H, s, OCH2), 3.80 (3H, s, OCH3), 6.83 (2H, d, J = 8.8 Hz,
ArH-20,60), 6.94 (2H, d, J = 8.8 Hz, ArH-30,50), 7.23 (1H, d, J = 7.6 Hz,
PyH-5), 7.76 (1H, d, J = 7.6 Hz, PyH-4). MS m/z (%) 327 (M+1, 27),
295 (Mꢁ31, 100); HPLC purity 99.6%.
4.36. Tubulin assays
Tubulin assembly was measured by turbidimetry at 350 nm as
described previously.21 Assay mixtures contained 1.0 mg/mL
(10 lM) tubulin and varying compound concentrations were pre-
incubated for 15 min at 30 °C without guanosine 50-triphosphate
(GTP). The samples were placed on ice, and 0.4 mM GTP was added.
Reaction mixtures were transferred to 0 °C cuvettes, and turbidity
development was followed for 20 min at 30 °C following a rapid
temperature jump. Compound concentrations that inhibited in-
crease in turbidity by 50% relative to a control sample were
determined.
4.32. 3-Cyano-N-(4-methoxyphenyl)-N-methyl-6-
trifluoromethylpyridin-2-amine (8j)
The preparation, work-up, and purity are the same as
for 8c. Starting with 3-cyano-N-(4-methoxyphenyl)-6-trifluoro-
methylpyridin-2-amine (8b) (586 mg, 2.0 mmol), MeI (0.25 mL,
4.0 mmol), and NaH (160 mg, 60% oil suspension) in 3 ml of DMF
at 0 °C with stirring for 1 hour to afford 565 mg of pure 8j, 92%
yield, white solid, mp 83–84 °C; 1H NMR d ppm 3.47 (3H, s,
NCH3), 3.84 (3H, s, OCH3), 6.96 (2H, d, J = 9.2 Hz, ArH-20,60), 6.98
(1H, d, J = 7.6 Hz, PyH-5), 7.20 (2H, d, J = 9.2 Hz, ArH-30,50), 7.77
(1H, d, J = 7.6 Hz, PyH-4); MS m/z (%) 308 (M+1, 100); HPLC purity
97.6%.
Inhibition of the binding of [3H]colchicine to tubulin was mea-
sured as described previously.22 Incubation of 1.0
5.0 l lM inhibitor was for 10 min at 37 °C,
l
M tubulin with
M [3H]colchicine and 5.0
when about 40–60% of maximum colchicine binding occurs in con-
trol samples.
5. Molecular modeling studies
4.33. N-(4-Methoxyphenyl)-N-methyl-3-(1H-tetrazol-5-yl)-6-
trifluoromethylpyridin-2-amine (8k)
All molecular modeling studies were performed with Discov-
ery Studio 3.0 (Accelrys, San Diego, USA). The crystal structures
of tubulin in complex with TN16 (PDB: 3HKD) and with
DMDA-colchicine (PDB: 1SA0) were downloaded from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb) for possible use in
the modeling study. We selected the structure 3HKD as our
modeling system, because of lower binding energy of 6a with
it and matched binding orientation between 6a and TN16. The
CDOCKER was used to evaluate and predict in silico binding free
energy of the inhibitors and automated docking. The protein pro-
tocol was prepared by several operations, including standardiza-
tion of atom names, insertion of missing atoms in residues and
removal of alternate conformations, insertion of missing loop re-
gions based on SEQRES data, optimization of short and medium
size loop regions with Looper Algorithm, minimization of
remaining loop regions, calculation of pK, and protonation of
the structure. The receptor model was typed with the CHARMm
forcefield. A binding sphere with radius of 9 Å was defined
through the native ligand as the binding site for the study. The
docking protocol employed total ligand flexibility and the final
ligand conformations were determined by the simulated anneal-
ing molecular dynamics search method set to a variable number
of trial runs. The docked ligands (6a, TN16, DMDA-colchicine,
and CA-4) were further refined using in situ ligand minimization
with the Smart Minimizer algorithm by the standard parameters.
The ligand and its surrounding residues within the above defined
sphere were allowed to move freely during the minimization,
while the outer atoms were frozen. The implicit solvent model
To a solution of 8j (417 mg, 1.3 mmol) in toluene (5 mL) was
added 4 drops of water, NaN3 (266 mg, 4.1 mmol), and Et3 NꢀHCl
(562 mg, 4.1 mmol) successively. The mixture was heated at reflux
temperature for 36 h, After cooling to rt, the mixture was added to
saturated aq. Na2CO3 (5 mL) and water (5 mL), and then extracted
with petroleum ether three times. The organic phase was washed
successively with water and brine, and dried over MgSO4. After re-
moval of petroleum ether in vacuo, 176 mg of unreacted 8c was
recovered from the organic phase. The water phase was acidified
with aq. HCl (2 N) to pH 2–3, filtered, and dried to give 262 mg
of 8k, 95% yield, yellow solid, mp 225 °C (decomposition); 1H
NMR (DMSO-d6) d ppm 3.36 (3H, s, NCH3), 3.62 (3H, s, OCH3),
6.63 (2H, d, J = 8.8 Hz, ArH-20,60), 6.82 (2H, d, J = 8.8 Hz, ArH-30,50),
7.47 (1H, d, J = 7.6 Hz, PyH-5), 7.96 (1H, d, J = 7.6 Hz, PyH-4). MS
m/z (%) 349 (Mꢁ1, 60), 278 (Mꢁ72, 100); HPLC purity 95.5%.
4.34. N-(4-Methoxyphenyl)-N-methyl-3-(1-methyl-tetrazol-5-
yl)-6-trifluoromethylpyridin-2-amine (8l)
A
mixture of 8k (101 mg, 0.29 mmol), Me2SO4 (0.05 mL,
0.58 mmol), and K2CO3 (80 mg, 0.58 mmol) in acetone (5 mL)
was refluxed for 5 h. After cooling to rt, additional acetone (ca.
10 mL) was added and solid K2CO3 was filtered out. After removal
of solvent in vacuo, the residue was purified with flash column
chromatography (gradient eluant/EtOAc/petroleum ether, 0–30%)
to afford 55 mg of 8l, 52% yield, pale yellow solid, mp 115–
116 °C; 1H NMR d ppm 3.51 (3H, s, NCH3), 3.69 (3H, s, NCH3),
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047

X.-F. Wang et al. / Bioorg. Med. Chem. xxx (2012) xxx–xxx
11
4. Using the ChemBioDraw Ultra 12.0 software.
5. Schmid, S.; Röttgen, M.; Thewalt, U.; Austel, V. Org. Biomol. Chem. 2005, 3, 3408.
6. Frlan, R.; Kikelj, D. Synthesis 2006, 14, 2271.
of Generalized Born with Molecular Volume (GBMV) was also
used to calculate the binding energies.
7. Li, F.; Wang, Q. R.; Ding, Z. B.; Tao, F. G. Org. Lett. 2003, 5, 2169.
8. Qin, B. J.; Jiang, X. K.; Lu, H.; Tian, X. T.; Barbault, F.; Huang, L.; Qian, K.; Chen, C.
H.; Huang, R.; Jiang, S.; Lee, K. H.; Xie, L. J. Med. Chem. 2010, 53, 4906.
9. Onnis, V.; Cocco, M. T.; Lilliu, V.; Congiu, C. Bioorg. Med. Chem. 2008, 16, 2367.
10. Cocco, M. T.; Congiu, C.; Onnis, V.; Morelli, M.; Felipo, V.; Cauli, O. Bioorg. Med.
Chem. 2004, 12, 4169.
Acknowledgements
This investigation was supported by Grants 81120108022 and
30930106 from the Natural Science Foundation of China (NSFC)
awarded to Lan Xie and NIH Grant CA17625-32 from the National
Cancer Institute awarded to K. H. Lee. This study was also sup-
ported in part by the Taiwan Department of Health, China Medical
University Hospital Cancer Research Center of Excellence
(DOH100-TD-C-111-005).
11. He, X. Y.; Zou, P.; Qiu, J.; Hou, L.; Jiang, S.; Liu, S.; Xie, L. Bioorg. Med. Chem. 2011,
19, 6726.
12. Rubinstein, L. V.; Shoemaker, R. H.; Paull, R. M.; Tosini, S.; Skehan, P.; Scudiero,
D. A.; Monks, A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
13. Walsh, J. S.; Miwa, G. T. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 145.
14. Dorleans, A.; Gigant, B.; Ravelli, R. B. G.; Mailliet, P.; Mikol, V.; Knossow, M.
PNAS 2009, 106, 13775.
15. Barbier, P.; Dorleans, A.; Devred, F.; Sanz, L.; Allegro, D.; Alfonso, C.; Knossow,
M.; Peyrot, V.; Andreu, J. M. J. Biol. Chem. 2010, 285, 31672.
16. Mason, R. P.; Zhao, D.; Liu, L.; Trawick, L.; Pinney, K. G. Integr. Biol. 2011, 3, 375.
17. Flynn, B. L.; Gill, G. S.; Grobelny, D. W.; Chaplin, J. H.; Paul, D.; Leske, A. F.;
Lavranos, T. C.; Chalmers, D. K.; Charman, S. A.; Kostewicz, E.; Shackleford, D.
M.; Morizzi, J.; Hamel, E.; Jung, M. K.; Kremmidiotis, G. J. Med. Chem. 2011, 54,
6014.
18. Boyd, M. R. Status of the NCI preclinical antitumor drug discovery screen. In
Cancer: Principles and Practice of Oncology Updates; J. B. Lippincott: Philadelphia,
1989; pp 1–12.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.047.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
19. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose,
C.; Langley, J.; Cronise, P.; Vaigro-Woiff, A.; Gray-Goodrich, M.; Campbell, H.;
Mayo, J.; Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757.
20. Houghton, P.; Fang, R.; Techatanawat, I.; Steventon, G.; Hylands, P. J.; Lee, C. C.
Methods 2007, 42, 377.
References and notes
1. Stanton, R. A.; Gernert, K. M.; Nettles, J. H.; Aneja, R. Med. Res. Rev. 2011, 31,
443.
2. Dumontet, C.; Jordan, M. A. Nat. Rev. Drug Disc. 2010, 9, 790.
3. Wang, X. F.; Tian, X. T.; Ohkoshi, E.; Qin, B. J.; Liu, Y. N.; Wu, P. C.; Hour, M. J.;
Hung, H. Y.; Huang, R.; Bastow, K. F.; Janzen, W. P.; Jin, J.; Morris-Natschke, S.;
Lee, K. H.; Xie, L. Bioorg. Med. Chem. Lett. 2012, 22, 6224.
21. Hamel, E. Cell Biochem. Biophys. 2003, 38, 1.
22. Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Biochemistry 1989, 28, 6984.
Please cite this article in press as: Wang, X.-F.; et al. Bioorg. Med. Chem. (2012), http://dx.doi.org/10.1016/j.bmc.2012.11.047