Optimization of 4-(N-cycloamino)phenylquinazolines as a novel class of tubulin-polymerization inhibitors targeting the colchicine site

Article abstract of DOI:10.1021/jm4016526

The 6-methoxy-1,2,3,4-tetrahydroquinoline moiety in prior leads 2-chloro- and 2-methyl-4-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)quinazoline (1a and 1b) was modified to produce 4-(N-cycloamino)quinazolines (4a-c and 5a-m). The new compounds were evaluated in cytotoxicity and tubulin inhibition assays, resulting in the discovery of new tubulin-polymerization inhibitors. 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (5f), the most potent compound, exhibited high in vitro cytotoxic activity (GI ₅₀ 1.9-3.2 nM), significant potency against tubulin assembly (IC ₅₀ 0.77 μM), and substantial inhibition of colchicine binding (99% at 5 μM). In mechanism studies, 5f caused cell arrest in G2/M phase, disrupted microtubule formation, and competed mostly at the colchicine site on tubulin. Compound 5f and N-methylated analogue 5g were evaluated in nude mouse MCF7 xenograft models to validate their antitumor activity. Compound 5g displayed significant in vivo activity (tumor inhibitory rate 51%) at a dose of 4 mg/kg without obvious toxicity, whereas 5f unexpectedly resulted in toxicity and death at the same dose.

Full text of DOI:10.1021/jm4016526

Article
pubs.acs.org/jmc
Open Access on 02/06/2015
Optimization of 4‑(N‑Cycloamino)phenylquinazolines as a Novel
Class of Tubulin-Polymerization Inhibitors Targeting the Colchicine
Site
Xiao-Feng Wang,†,⊥ Fang Guan,† Emika Ohkoshi,‡ Wanjun Guo,§ Lili Wang,† Dong-Qing Zhu,†
Sheng-Biao Wang,† Li-Ting Wang,‡ Ernest Hamel,∥ Dexuan Yang,§ Linna Li,§ Keduo Qian,‡
Susan L. Morris-Natschke,‡ Shoujun Yuan,§ Kuo-Hsiung Lee,*,‡,# and Lan Xie*,†
†Beijing Institute of Pharmacology and Toxicology, 27 Tai-Ping Road, Beijing 100850, China
‡Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, North
Carolina 27599, United States
§Beijing Institute of Radiation Medicine, 27 Tai-Ping Road, Beijing 100850, China
∥Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National
Cancer Institute, Frederick National Laboratory for Cancer Research, National Institutes of Health, Frederick, Maryland 21702,
United States
⊥Pharmacy Department, Urumqi General Hospital, Lanzhou Military Region, Urumqi 830000, China
#Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 40402, Taiwan
S
* Supporting Information
ABSTRACT: The 6-methoxy-1,2,3,4-tetrahydroquinoline
moiety in prior leads 2-chloro- and 2-methyl-4-(6-methoxy-
3,4-dihydroquinolin-1(2H)-yl)quinazoline (1a and 1b) was
modified to produce 4-(N-cycloamino)quinazolines (4a−c and
5a−m). The new compounds were evaluated in cytotoxicity
and tubulin inhibition assays, resulting in the discovery of new
tubulin-polymerization inhibitors. 7-Methoxy-4-(2-methylqui-
nazolin-4-yl)-3,4-dihydroquinoxalin- 2(1H)-one (5f), the most
potent compound, exhibited high in vitro cytotoxic activity
(GI50 1.9−3.2 nM), significant potency against tubulin
assembly (IC50 0.77 μM), and substantial inhibition of
colchicine binding (99% at 5 μM). In mechanism studies, 5f caused cell arrest in G2/M phase, disrupted microtubule
formation, and competed mostly at the colchicine site on tubulin. Compound 5f and N-methylated analogue 5g were evaluated
in nude mouse MCF7 xenograft models to validate their antitumor activity. Compound 5g displayed significant in vivo activity
(tumor inhibitory rate 51%) at a dose of 4 mg/kg without obvious toxicity, whereas 5f unexpectedly resulted in toxicity and death
at the same dose.
cause a significant shutdown in blood flow to solid tumors by
selectively targeting established tumor vasculature, leading to
cancer cell death via extensive necrosis and apoptosis while the
blood flow in normal tissues remains relatively intact.3
Therefore, this type of tubulin inhibitor might provide new
therapeutic approaches to treat cancers and overcome
limitations of existing tubulin-inhibiting drugs. Currently, a
dozen drug candidates targeted at the colchicine site are in
clinical development as anticancer VDAs4,5 (e.g., combretasta-
tin A-4 (CA4), its phosphate derivative CA4P,6 and verubulin
(MPC6827)7), as shown in Figure 1.
INTRODUCTION
■
Tubulin, a known target of anticancer drugs, has multiple drug-
binding sites, the most extensively studied being the taxoid,
vinca, and colchicine sites. Colchicine and its analogues (Figure
1) act at a unique site located between the α- and β-tubulin
monomers within an αβ heterodimer.1 These compounds
exhibit significant cytotoxicity by inhibiting tubulin polymer-
ization into microtubules, but they are also highly toxic, greatly
limiting their clinical use. However, other diverse small
molecules that also act at the colchicine site on tubulin have
recently come under intensive investigation. These compounds
not only potently inhibit the growth of a wide variety of human
cancer cell lines but also they show vascular-disrupting effects
on tumor endothelial cells required for the growth of the cancer
and thus they represent a new class of potential antitumor
drugs termed vascular-disrupting agents (VDAs).2 VDAs can
In our prior studies,8−10 we evaluated a series of N-aryl-
1,2,3,4-tetrahydroquinoline derivatives and found that lead
Received: October 25, 2013
Published: February 6, 2014
© 2014 American Chemical Society
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a
Scheme 1
Figure 1. Colchicine, DAMA-colchicine, drug candidates CA4, CA4P,
and verubulin, and the modification strategy from leads 1a and 1b to
new target compounds in this study.
a
(i) i-PrOH/HCl, reflux or rt; (ii) EtOH/NaHCO3, reflux, 1−4 h for
4a and 5d; (iii) NaBH4/MeOH, 0 °C to rt.
compounds 1a and 1b (Figure 1) exhibited low nanomolar GI50
values (1.5−18 nM) against a human tumor cell line (HTCL)
panel. Subsequent biological evaluations revealed that, like
CA4, 1a and 1b inhibited tubulin assembly and colchicine
binding to tubulin. These promising results with these new
chemotype inhibitors prompted us to elucidate structure−
activity relationships (SARs) and structure−property relation-
ships (SPRs) as well as to develop potential new drug
candidates. Our modification strategy first focused on the
tetrahydroquinoline moiety, as shown in Figure 1. We
maintained the quinazolinemoiety because it is a common
structural core in various antitumor agents with different
targets. These compounds include the tyrosine kinase inhibitor
gefitinib,11 β-catenin/Tcf4 inhibitors,12 and G9a inhibitors.13
According to the strategy shown in Figure 1, we first changed
the R group on the phenyl ring (A-ring) from methoxy to
methyl or bromo and determined the effect of these changes on
cytotoxic activity. Then, we focused on modifications of the
piperidine ring (B-ring) by introducing additional heteroa-
tom(s) or functional groups at the β (X) or γ (Y) position
while maintaining the α-methylene group on the B-ring. Next,
we changed the B-ring size by reduction to a five-membered
ring or expansion to a seven-membered ring. Meanwhile, the 2-
substituent on the quinazoline ring was kept as either chloro or
methyl, as in leads 1a and 1b, respectively. The newly
synthesized 4-cycloaminoquinazoline derivatives (4 and 5)
were evaluated in cellular cytotoxicity and tubulin assays.
Further studies on the mechanism of action were performed
with the most active compound, 5f, to identify the biological
target of this class of new anticancer agents. Subsequently,
several new potent compounds were assessed for essential
druglike properties, such as water solubility, log P, and in vitro
metabolic stability. Finally, antitumor activities of selected
compounds with a good balance between potency and
physicochemical properties were validated in vivo.
prepared cyclic amines (3a−h). As reported previously, the
coupling of 2a with 6-methyl-1,2,3,4-tetrahydroquinoline (3a)
selectively produced 4-substituted 2-chloroquinazoline 4a
because of the lower reactivity of the C2 as compared with
the C4 position.14 Similarly, 2b reacted with 6-methyl-1,2,3,4-
tetrahydroquinoline (3a) or 6-bromo-1,2,3,4-tetrahydroquino-
line (3b) in the presence of HCl to yield 4b and 4c,
respectively, in 75−80% yields. 6-Methoxy-2,3-dihydroquino-
lin-4(1H)-one (3c)15,16 and 7-methoxy-3,4-dihydro-2H-benzo-
[b][1,4]oxazine (3d)17,18 were prepared according to literature
methods and then coupled individually with 2a and 2b to afford
5a,b and 5d,e, respectively. Next, the ketone in 5b was
converted to the hydroxyl in 5c by reduction with NaBH4.
In contrast with these simple syntheses, lactam compounds
5f and 5g were prepared via multiple steps, as shown in Scheme
2. Commercially available 2-nitro-4-methoxyaniline (3e) and
2b were coupled to provide intermediate diarylamine 6.
Compound 5f with a new six-membered lactam ring was
prepared by a three-step sequence: reduction of the nitro group
in 6 by hydrogenation with Pd/C, acylation with 2-chloroacetyl
chloride, and subsequent ring closure. Treatment of 5f with
methyl iodide in the presence of NaH afforded N-methylated
5g.
The syntheses of compounds 5h−i with a five-membered B-
ring and 5j−m with two different seven-membered B-rings are
shown in Scheme 3. 4-Chloroquinazolines 2a and 2b were
coupled individually with 5-methoxyindoline (3f)19 under
alkaline conditions to afford 5h and 5i, respectively. In a
Schmidt reaction,20 commercially available 6-methoxy-3,4-
dihydronaphthalen-1(2H)-one (9) was treated with NaN3 in
the presence of CH3SO3H to generate a pair of lactam isomers
in a ratio of 1:4, which were separated by flash chromatography
to give 10a and 10b. Compounds 10a and 10b were separately
reduced with LiAlH4 to afford cyclic amines 3g and 3h,
respectively, which were coupled with 2a or 2b to produce
corresponding target compounds 5j−m, respectively. All newly
CHEMISTRY
■
The syntheses of new target series 4 and 5 are outlined in
Schemes 1−3. Basically, commercially available 2,4-dichlor-
oquinazoline (2a) and 4-chloro-2-methylquinazoline (2b)
underwent nucleophilic substitution with various available or
1
synthesized compounds were identified by H NMR and MS
spectra, and their purities were determined by HPLC.
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a
Scheme 2
a
(i) i-PrOH/HCl, reflux, 5 h, 75%; (ii) H2/Pd−C, in EtOAc, 2 h; (iii) ClCH2COCl, K2CO3, acetone, 0 °C, 1 h, 69% for two steps; (iv) K2CO3/
DMF, 100 °C, 2 h, 69%; (v) MeI/NaH, DMF, 0 °C, 1 h, 84%.
a
Scheme 3
a
(i) NaHCO3, EtOH, rt, 1 h for 5h−i, or reflux 2 h for 5j and 5l−m; (ii) CH3SO3H, NaN3, rt, 24 h; (iii) LiAlH4, THF, reflux, 16 h; (iv) i-PrOH/
HCl, reflux, 2 h, 64% for 5k.
resulted in hydroxyl compound 5c, which exhibited GI50 values
of 17−19 nM, similar to those of 5b. Subsequently, the γ-
methylene of the tetrahydropyridine B-ring in 1a and 1b was
replaced with an oxygen atom. However, the corresponding2H-
benzo[b][1,4]oxazine analogues 5d and 5e displayed reduced
cytotoxic activity (GI50 values of 0.153−0.211 μM). Interest-
ingly, when the tetrahydropyridine B-ring in 1b was converted
to a six-membered lactam ring (3,4-dihydropyrazin-2(1H)-
one), the resulting 3,4-dihydroquinoxalin-2(1H)-one com-
pound (5f) showed extremely high cytotoxic activity with low
nanomolar GI50 values (1.9−3.2 nM), more potent than the
positive-control drug paclitaxel against the HTCL panel. The
N-methylated lactam (5g) also displayed high potency with
GI50 values of 17−25 nM, although 5g was less potent than 5f.
However, 5-methoxyindoline compounds 5h and 5i with a five-
membered 2,3-dihydro-1H-pyrrole B-ring showed decreased
potency (GI50 values of 0.17−0.32 μM) compared with 5a−5g
or leads 1a and 1b with various six-membered B-rings.
Therefore, the conformational changes and restricted torsional
angles of the dihydropyrrole may affect molecular antitumor
activity greatly. When the N-heterocyclic B-ring of 1b was
expanded to a seven-membered ring, resulting 5k with a
benzo[b]azepine moiety exhibited high potency (GI50 values of
RESULTS AND DISCUSSION
■
Antiproliferative Activity in Cellular Assays and SAR
Analysis. Newly synthesized 4-cycloaminoquinazolines (series
4 and 5) were initially evaluated for antitumor activity against
an HTCL panel, including A549 (lung carcinoma), KB
(epidermoid carcinoma of the mouth), KBvin, a P-gp-
expressing multidrug-resistant cell line (vincristine-resistant
KB),21,22 and DU145 (prostate cancer), in parallel with
paclitaxel as a positive reference (Table 1). The in vitro
anticancer activity (GI50) was determined using the established
sulforhodamine B (SRB) method.23
With a 6-methyl or 6-bromo rather than a 6-methoxy (R)
group on the A-ring, compounds 4a−c showed significant
cytotoxic activity (GI50 0.043−0.057 μM for 4a and 0.169−
0.211 μM for 4b and 4c) but were at least 10-fold less potent
than leads 1a (GI50 1.5−1.7 nM) and 1b (GI50 0.013−0.018
μM). Thus, the 6-methoxy (R) group is more favorable than
methyl or bromo groups.
Next, we focused on modifications of the N-heterocycle (B-
ring) in series 5 derivatives. Compounds 5a and 5b, with a 4-
oxo group added on the tetrahydropyridine B-ring relative to 1a
and 1b, exhibited high potency with GI50 values of 27−33 and
19−24 nM, respectively. Reduction of the carbonyl in 5b
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Table 1. Antiproliferative Activities of Series 4 and 5 against Human Tumor Cell Lines
a
GI50 (μM SD)
compound
R1
R
A549
KB
KBvin
DU145
4a
Cl
Me
0.043 0.011
0.211 0.038
0.211 0.034
0.048 0.004
0.204 0.035
0.178 0.028
0.046 0.002
0.176 0.031
0.169 0.011
0.057 0.011
0.187 0.007
0.198 0.034
4b
Me
Me
R1
Me
Br
4c
compound
X
Y
5a
5b
5c
5d
5e
5f
Cl
CH2
CH2
CH2
CH2
CH2
CO
CO
0.027 0.005
0.024 0.001
0.019 0.003
0.199 0.004
0.189 0.016
0.0032 0.0007
0.019 0.002
0.233 0.013
0.268 0.021
0.191 0.029
0.021 0.002
7.57 0.55
0.028 0.005
0.019 0.003
0.018 0.002
0.153 0.028
0.211 0.036
0.0023 0.0005
0.017 0.003
0.227 0.026
0.320 0.056
0.227 0.015
0.019 0.001
11.30 0.58
0.033 0.005
0.021 0.001
0.017 0.001
0.195 0.031
0.169 0.033
0.0022 0.0004
0.025 0.002
0.197 0.024
0.216 0.025
0.197 0.024
0.020 0.001
5.45 0.90
0.031 0.006
0.021 0.004
0.018 0.004
0.169 0.021
0.198 0.033
0.0019 0.0004
0.022 0.003
0.165 0.035
0.243 0.039
0.165 0.035
0.018 0.004
6.81 1.34
Me
Me
Cl
CO
CHOH
O
Me
Me
Me
Cl
O
NH
NMe
5g
5h
5i
CO
CH2
CH2
(CH2)2
(CH2)2
Me
Cl
5j
CH2
CH2
5k
5l
Me
Cl
5m
Me
16.71 2.90
19.59 3.33
14.76 2.32
1.21 0.19
14.63 1.33
0.006 0.001
b
paclitaxel
0.0076 0.0017
0.0064 0.0014
a
Concentration of compound that inhibits 50% human tumor cell growth, presented as the mean standard deviation (SD) and performed at least
b
in triplicate. Positive control.
a
18−21 nM), similar to the values obtained with 1b, 5b, and 5g.
However, the related 2-chloroquinazoline 5j was much less
potent than 5k. In addition, 5l and 5m with the isomericbenzo-
[c]azepine moiety were 300- to 1000-fold less active (GI50
5.45−19.59 μM) than 5k. Thus, the position of the N atom in
the B-ring is important for optimal antitumor activity.
From these results, seven new active 4-aminoquinazoline
compounds (4a, 5a−c, 5f−g, and 5k) displayed low GI50 values
ranging from 1.9 to 57 nM (Table 1). Structure−activity
relationships conclude that (1) a six-membered or seven-
membered B-ring is desirable but the N atom should connect
directly to the phenyl ring (A-ring), (2) the six-membered
lactam of 5f and 5g led to a new favorable chemical scaffold
with enhanced cytotoxic activity, and (3) the para-methoxy
group (R) on the phenyl ring (A-ring) is more favorable than a
methyl or bromo group.
Inhibition of Tubulin Polymerization. Five of the most
cytotoxic compounds (5b-c, 5f−g, and 5k, GI50 1.9−33 nM)
were evaluated in tubulin assembly and colchicine binding
assays in parallel with CA4, a clinical trial drug candidate, as
reference. CA4 is a well-described, highly potent competitive
inhibitor of the binding of colchicine to tubulin.24 The results in
these assays are shown in Table 2. Compound 5f, which
displayed the highest cytotoxic potency, also exhibited greater
inhibition of tubulin assembly with an IC50 value of 0.77 μM
and greater potency (99% at 5 μM and 93% at 1 μM) against
colchicine binding to tubulin than CA4 (0.96 μM) and leads 1a
and 1b in the same assays. The other four compounds also
showed significant activity in both assays, inhibiting tubulin
assembly with IC50 values of 0.87−1.3 μM and colchicine
binding by 87−96% at 5 μM, similar to CA4 in the same assays.
Table 2. Inhibition of Tubulin Polymerization and
Colchicine Binding to Tubulin
b
inhibition of colchicine
binding
(%) inhibition SD
inhibition of tubulin assembly
compound
IC50 (μM) SD
5 μM
1 μM
5b
5c
5f
0.94 0.03
0.97 0.1
0.77 0.07
1.3 0.03
0.87 0.1
0.96 0.07
87
1
56
4
94 0.7
99 0.02
75 0.6
93 0.8
82 0.2
46 0.6
90 0.2
5g
5k
96
89
2
1
c
CA4
98 0.6
a
The tubulin assembly assay measured the extent of assembly of 10
b
μM tubulin after 20 min at 30 °C. Tubulin, 1 μM; [3H]colchicine, 5
μM; and inhibitor, 5 or 1 μM. Incubation was performed for 10 min at
c
37 °C. The reference compound is a drug candidate in phase II/III
clinical trials.
Therefore, these 4-(N-cyclo)aminoquinazolines have been
identified as a new class of tubulin inhibitors, comparable to
our previously discovered N-aryl-1,2,3,4-tetrahydroquinolines.
To validate the biological target further, we investigated the
effects of the most active compound, 5f, on the cell cycle. A549
cells were treated with 5f at 3 nM for 24 h in parallel with
colchicine at 300 nM. After staining with propidium iodide, the
cells were analyzed by flow cytometry. As shown in Figure 2,
cells treated with either colchicine or 5f were arrested at the
G2/M phase, whereas control cells were mainly in the G0/G1
phase. The effects of both compounds on cell cycle distribution
patterns were dose-dependent, as shown in Figure 2D.
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Figure 2. Cell cycle analysis was performed using a FACSCalibur (BD Biosciences) after treatment of A549 cells with 5f and analysis by a standard
propidium iodide procedure as described in the Experimental Section. (A) DMSO-treated (0.1%) cells served as a control. (B, C) A549 cells were
harvested after treatment with 5f (3nM, B) or colchicine (300 nM, C). (D) Cell cycle contributions resulting from treatment with 5f (1 and 3 nM)
or colchicine (100 and 300 nM) for 24 h.
Next, we performed immunocytochemistry studies to
examine effects of 5f treatment on microtubule structure
using an α-tubulin antibody to stain cellular microtubules. As
shown in Figure 3, a hairlike microtubule network of slim,
fibrous microtubules (red) wrapped around the cell nucleus
(blue) was visualized upon immunofluorescence staining of
A549 cells. However, the microtubule network disappeared
when cells were treated with 5f or colchicine for 24 h (Figure
3B,D), whereas cells treated with paclitaxel retained a spindle-
shaped microtubule network around the nucleus.25 These
results indicated that 5f is unlike paclitaxel as a microtubule-
stabilizing agent. To define the actual binding site of 5f further,
competitive assays for the colchicine site or vinblastine site
were performed, respectively. We found that compound 5f
decreased the fluorescence intensity of colchicine−tubulin
complex in a concentration-dependent fashion with a low
IC50 value of 0.55 μM, as shown in Figure 4A. In contrast, 5f
did not significantly change the binding of BODIPY FL-
vinblastine to tubulin even at 30−100 μM. Vincristine, a
vinblastine analogue, did show inhibition in the same assay at
10 μM with inhibition rates of 50−60% (Figure 4B), which is
consistent with the literature.26 Therefore, these results
demonstrated that 5f binds at the colchicine site rather than
at the vinblastine site on tubulin.
Molecular Modeling. To elucidate the binding character-
istics of these new compounds with tubulin, we performed
docking studies with the most active compound, 5f, at the
colchicine binding pocket using the CDOCKER program in the
Discovery Studio 3.0 software and the tubulin crystal structure
(PDB code: 1SA0)27,28 in comparison with the original ligand,
N-deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-colchi-
cine, Figure 1). As described in our previous publications8,10
and shown in Figure 5, active compound 5f (orange stick
mode) displayed a binding torsional angle of 66.04° between
the two fused rings and a binding conformation with low
energy (−39.04 kcal/mol), which superimposed well with
DAMA-colchicine (cyan) in the binding site. Like most
colchicine-binding inhibitors, key amino acid Cys241 on β-
H7 tubulin at the colchicine site forms a hydrogen bond with
the methoxy group on the phenyl of 5f, similar to the 2′-OCH3
on the A-ring of DAMA-colchicine. It is noteworthy that the
carbonyl group in the lactam of 5f interacts with Leuβ252 and
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Figure 3. Effects of tested compounds on microtubules. A549 cells were treated with (A) 0.1% DMSO, (B) 5f (3 nM), (C) paclitaxel (100 nM), or
(D) colchicine (100 nM) for 24 h. Microtubules were visualized with an anti-α-tubulin antibody (red), and the cell nucleus was visualized with
Hoechst 33342 (blue). Images were acquired with an Incell Analyzer 1000 using a 20× objective.
Figure 4. (A) Compound 5f bound tubulin at the colchicine site. The inhibition curve of 5f competing with colchicine−tubulin was plotted as
inhibition rate vs concentration. The inhibition rates were expressed as the percentage (%) of decreased fluorescence of the tubulin−colchicine
complex. An IC50 value of 0.55 0.09 μM was determined using GraphPad Prism V5.01. (B) Compound 5f did not compete for the vinblastine site.
Compound 5f or vincristine at the indicated concentrations competed with BODIPY FL-vinblastine to tubulin. The reduction in the fluorescence
intensity of tubulin−BODIPY FL-vinblastine complex was measured and converted into inhibition rates. All results were expressed as the mean
SD of at least three independent experiments.
Leuβ255 of the β-H8 region of tubulin. The two additional H-
bonds could enhance the binding affinity of 5f with tubulin and
subsequently result in the observed higher inhibitory activity on
tubulin polymerization. Because the carbonyl group projected
deeper into the pocket, the lactam ring (B-ring) of 5f might
have additional hydrophobic interactions with surrounding
amino acids. However, the 2-methylquinazoline ring and the
phenyl ring of 5f superimposed well with the two aromatic
rings in DAMA-colchicine, suggesting that the two aromatic
rings might be important to anchor the ligand at the colchicine-
binding pocket and to maintain the required binding
conformation. Thus, this modeling investigation provided
insight and rationale for the high potency exhibited by 5f in
biological assays (Table 2).
Druglike Properties and Antitumor Activity in Vivo.
Prior lead 1b displayed better druglike properties than 1a
(Figure 1); thus, we chose five active 4-(N-cycloamino)phenyl-
2-methylquinazolines (5b−c, 5f−g, and 5k) for assessment of
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To validate further the antitumor activity in vivo, nude
mouse MCF7 xenograft models were established, and
compounds 5f, 5g, 1a, and 1b were administered by
intravenous (i.v.) injection at a dose of 4 mg/kg every 3
days. Data are shown in Table 4. Compound 5f exhibited
Table 4. In Vivo Antitumor Data of Compounds 1a, 1b, 5f,
and 5g
antitumor
activity
administration
toxicity
body
weight
change
(%)
dose
compound (mg/kg) schedule
inhibitory
a
route
i.v.
death rate (%)
vehicle
control
Q3D × 6
+0.10
0/7
1a
1b
5f
4
4
4
4
Q3D × 6
Q3D × 6
Q3D × 6
Q3D × 6
i.v.
i.v.
i.v.
i.v.
−7.9
−15.7
0/5
1/6
7/7
0/7
22.9
47.3
Figure 5. Predicted binding mode of 5f (orange stick) with tubulin
(PDB code: 1SA0) and overlapping with DAMA-colchicine (cyan, the
bound ligand of 1SA0). Surrounding amino acid side chains are shown
in gray stick format and are labeled. Hydrogen bonds are shown by
green dashed lines, and the distance between ligands and protein is less
than 3 Å.
5g
+2.28
51.0
a
Q3D, every 3 days.
unexpected toxicity, and all seven mice died during treatment.
No obvious signs of toxicity were observed with 5g at the same
dose and schedule. Mice treated with 5g even had increased
body weight after treatment. The higher in vivo toxicity of 5f
might be explained by its better aqueous solubility (Table 3),
which should allow 5f to reach a higher free concentration in
blood with less binding to plasma proteins than the more
liposoluble 5g when given by i.v. administration. The result is
also consistent with the high antiproliferative activity of 5f
(Table 1). Nevertheless, 5g significantly inhibited the growth of
MCF7 tumor xenografts. Statistically significant differences (p <
0.05) compared with the control tumor volumes were reached
from day 10 onward (Figure 6A). At the end of treatment, the
mice were sacrificed for autopsy, and tumors were recovered
and weighed. The average tumor weight of the 5g-treated group
was 0.703 0.323 g, which was much less than that of control
mice (1.436 0.531 g), and the tumor growth inhibitory rate
was 51.0%. Hence, compound 5g showed strong antitumor
activity on a well-tolerated dose schedule and was more
efficacious than 1a and 1b.
essential druglike properties aimed at reaching the critical
balance between potency and physicochemical properties
required for potential drug candidates. Aqueous solubility, log
P values, and metabolic stability in a human liver microsome
assay were measured according to methods described
previously,29 and data are summarized in Table 3. In
Table 3. Physicochemical Parameters of Selected
a
Compounds
pH 7.4
aqueous solubility
human liver
microsome t1/2 (min)
compound
(μg/mL)
log P
b
1a
0.45 0.06
7.67 0.40
1.21 0.08
8.72 0.13
8.28 0.23
4.20 0.12
2.94 0.11
4.13 0.05
3.65 0.03
2.91 0.05
2.55 0.06
2.97 0.01
2.99 0.03
4.16 0.06
10.59
25.2
b
1b
5b
5c
5f
20.89
42.44
54.81
22.00
24.85
40.82
21.14
5g
5k
c
propranolol
CONCLUSIONS
d
■
terfenadine
By structural modifications on the 6-methoxy-1,2,3,4-tetrahy-
droquinoline moiety in prior leads 1a and 1b, a series of 4-(N-
cycloamino)phenylquinazolines (4a−c and 5a−m) were
designed, efficiently synthesized, and evaluated in cellular and
tubulin inhibition assays, resulting in the discovery of a new
class of antitumor agents as tubulin-polymerization inhibitors.
The most potent compound, 5f, showed low nanomolar
antiproliferative activity in cellular assays with GI50 values of
1.9−3.2 nM, significant inhibition of tubulin assembly with an
IC50 value of 0.77 μM, and exceptionally potent inhibition of
colchicine binding to tubulin (99% at 5 μM and 93% at 1 μM
with colchicine at 5 μM). As a probe in mechanism studies, 5f
arrested most cells in the G2/M phase of the cell cycle and
disrupted cellular microtubules, thus providing evidence that
these active compounds are a new kind of tubulin-polymer-
ization inhibitors acting at the colchicine site. After druglike
property assessments of several active compounds (GI50 < 33
nM in the HTCL panel), 5f and its N-methylated derivative 5g
were evaluated in parallel with 1a and 1b in nude mouse MCF7
a
Data presented as mean from three separate experiments with or
b
without
standard deviation (SD). Data reported previously.10
c
Propranolol has moderate metabolic stability with t1/2 of 3−5 h in
d
vivo. Terfenadine has low metabolic stability with t1/2 of <3 h in vivo.
comparison, the druglike parameters of most of the new 2-
methylquinazoline compounds were better than those of 1a
and similar to those of 1b (i.e., moderate aqueous solubility
(1−10 μg/mL), lower log P values (<3, except 5k), and greater
metabolic stability (t1/2 20−55 min, human liver microsome
assay)). Among them, the most active compound, lactam 5f,
displayed better metabolic stability in vitro than propranolol
(t1/2 54.81 versus 40.82), a drug with moderate metabolic
stability. Therefore, we postulate that new compound 5f might
be a good potential drug candidate for further development on
the basis of its new structural scaffold, high potency in cellular
and tubulin assays, and improved druglike properties.
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Figure 6. Antitumor activity of 5g in vivo. MCF7 cells were injected into the flanks of nude mice. When the tumor volume reached about 100 mm3,
the mice were sorted into two groups (n = 7) and administration started. (A) Growth difference of tumor volumes was significant from day 10
onward. * indicates a significant difference from control (Student’s t test, p < 0.05). (B) At the end of experiment, tumors were resected and
●
weighed. indicates the weight value of each tumor; the red line indicates the average value of the tumor weights.
rt for 1−12 h or refluxed for 1−5 h) or condition B (in the presence of
xenograft models to determine their antitumor activity.
Compound 5g displayed strong in vivo antitumor activity,
suppressing tumor growth by 51% at a dose of 4 mg/kg with no
obvious signs of toxicity, whereas 5f resulted in the death of all
treated mice on the same dose schedule. SAR studies revealed
that (1) the cyclic N-linker (B-ring) can be modified by
introducing a polar group at the β and/or γ position to improve
activity and physicochemical properties, (2) a suitable torsional
angle between the two aromatic rings is important for
enhancing molecular affinity for tubulin, (3) the six- or
seven-membered cyclic N-linker rings yield active compounds,
but the five-membered ring substantially decreased antitumor
potency, and (4) the 4-methoxyphenyl ring and the quinazoline
moiety are necessary for antitumor activity. Molecular modeling
results suggested that the presence of a lactam ring might be
responsible for the high potency of 5f and 5g by producing
additional interactions with the colchicine site on tubulin.
NaHCO3 (126 mg, 1.5 mmol) in anhydrous EtOH (5 mL) refluxed
for 1−4 h). The reaction was monitored by TLC until the reaction was
complete. The mixture was poured into ice water and extracted three
times with EtOAc. The combined organic phases were washed with
water and brine successively and dried over anhydrous Na2SO4. After
removal of solvent in vacuo, the crude product was purified by flash
column chromatography (gradient elution: EtOAc/petroleum ether,
0−70%) to obtain the pure product.
2-Chloro-4-(N-(6-methyl-3,4-dihydroquinolinyl))quinazoline
(4a). Using condition B starting with 2a (200 mg, 1.0 mmol) and 3a
(176 mg, 1.2 mmol), the mixture was refluxed for 4 h to produce 248
1
mg of 4a: 80% yield, yellow solid, mp 134−136 °C. H NMR δ 2.12
(2H, m, 3′-CH2), 2.33 (3H, s, CH3), 2.86 (2H, t, J = 6.8 Hz, 4′-CH2),
4.07 (2H, t, J = 6.8 Hz, 2′-CH2), 6.64 (1H, d, J = 8.0 Hz, ArH-8′), 6.78
(1H, dd, J = 8.4 and 1.6 Hz, ArH-7′), 7.07 (1H, s, ArH-5′), 7.14 (1H,
m, ArH-6), 7.36 (1H, dd, J = 8.0 and 0.8 Hz, ArH-5), 7.65 (1H, m,
ArH-7), 7.80 (1H, d, J = 8.0 and 0.8 Hz, ArH-8). MS m/z (%) 310 (M
+ 1, 100), 312 (M + 3, 30). HPLC purity 99.5%.
2-Methyl-4-(N-(6-methyl-1,2,3,4-tetrahydroquinolinyl))-
quinazoline (4b). Using condition A starting with 2b (89 mg, 0.5
mmol) and 3a (74 mg, 0.5 mmol), the mixture was refluxed for 1 h to
produce 108 mg of 4b: 75% yield, yellow solid, mp 136−138 °C. H
EXPERIMENTAL SECTION
■
Chemistry. The proton nuclear magnetic resonance (1H NMR)
spectra were measured on a JNM-ECA-400 (400 MHz) spectrometer
using tetramethylsilane (TMS) as the internal standard. The solvent
was CDCl3 unless otherwise indicated. Mass spectra (MS) were
measured on an API-150 mass spectrometer with an electrospray
ionization source from ABI, Inc. Melting points were measured by a
SGW X-4 micro melting-point detector without correction. The
microwave (MW) reactions were performed on a MW reactor from
Biotage, Inc. Medium-pressure column chromatography was per-
formed using a CombiFlash Companion system from ISCO, Inc. Thin-
layer chromatography (TLC) was performed on silica gel GF254 plates.
Silica gel GF254 and H (200−300 mesh) from Qingdao Haiyang
Chemical Company were used for TLC and column chromatography,
respectively. All commercial chemical reagents were purchased from
Beijing Chemical Works or Sigma-Aldrich. Reagents NADPH, MgCl2,
KH2PO4, and K2HPO4 and reference compounds propranolol and
terfenadine were purchased from Sigma-Aldrich. HPLC grade
acetonitrile for LC−MS analysis was purchased from VWR. Pooled
human liver microsomes (lot no. 28831) were purchased from BD
Biosciences. Purities of target compounds reached at least 95% and
were determined by HPLC using the following instruments and
conditions: Agilent HPLC-1200 with UV detector and Agilent Eclipse
XDB-C18 column (150 × 4.6 mm, 5 μm), flow rate 0.8 mL/min, UV
detection at 254 nm, and injection volume of 15 μL. Mobile elution
was conducted with a mixture of solvents A and B (condition 1:
acetonitrile (ACN)/H2O 60:40; condition 2: MeOH/H2O 70:30).
For some compounds, solvent B contained 0.025 mM ammonium
acetate.
1
NMR δ 2.12 (2H, m, 3′-CH2), 2.30 (3H, s, CH3), 2.75 (3H, s, CH3),
2.88 (2H, t, J = 2.4 Hz, 4′-CH2), 4.04 (2H, t, J = 2.4 Hz, 2′-CH2), 6.55
(1H, d, J = 8.4 Hz, ArH-8′), 6.73 (1H, dd, J = 8.4 and 2.4 Hz, ArH-7′),
7.03 (1H, d, J = 2.4 Hz, ArH-5′), 7.13 (1H, m, ArH-6), 7.32 (1H, dd, J
= 8.4 and 0.8 Hz, ArH-5), 7.63 (1H, m, ArH-7), 7.81 (1H, d, J = 8.4
Hz, ArH-8). MS m/z (%) 290 (M+1, 100). HPLC purity 100.0%.
4-(N-(6-Bromo-1,2,3,4-tetrahydroquinolinyl))-2-methylqui-
nazoline (4c). Using condition A starting with 2b (89 mg, 0.5 mmol)
and 3b (106 mg, 0.5 mmol), the mixture was refluxed for 1 h to
1
produce 142 mg of 4c: 80% yield, yellow solid, mp 129−130 °C. H
NMR δ 2.13 (2H, m, 3′-CH2), 2.76 (3H, s, CH3), 2.91 (2H, t, J = 6.8
Hz, 4′-CH2), 4.04 (2H, t, J = 6.8 Hz, 2′-CH2), 6.49 (1H, d, J = 8.8 Hz,
ArH-8′), 7.01 (1H, dd, J = 8.8 and 2.4 Hz, ArH-7′), 7.22 (1H, m, ArH-
6), 7.34 (1H, d, J = 2.4 Hz, ArH-5′), 7.46 (1H, dd, J = 8.4 and 0.8 Hz,
ArH-5), 7.69 (1H, m, ArH-7), 7.85 (1H, d, J = 8.4 Hz, ArH-8). MS m/
z (%) 354 (M+1, 60), 356 (M+3, 100). HPLC purity 98.1%.
2-Chloro-4-(N-(6-methoxy-2,3-dihydro-4-oxoquinolinyl))-
quinazoline (5a). Using condition A starting with 2a (100 mg, 0.5
mmol) and 3c (89 mg, 0.5 mmol), the mixture was kept at rt for 12 h
to produce 61 mg of 5a: 36% yield, yellow solid, mp 218−220 °C. 1H
NMR δ 2.98 (2H, t, J = 6.4 Hz, 3′-CH2), 3.87 (3H, s, 6′-OCH3), 4.51
(2H, t, J = 6.4 Hz, 2′-CH2), 6.68 (1H, d, J = 8.8 Hz, ArH-8′), 6.88
(1H, dd, J = 8.8 and 3.2 Hz, ArH-7′), 7.34 (1H, m, ArH-6), 7.52 (1H,
d, J = 3.2 Hz, ArH-5′), 7.59 (1H, d, J = 8.4 Hz, ArH-5), 7.78 (1H, m,
ArH-7), 7.91 (1H, d, J = 9.2 Hz, ArH-8). MS m/z (%) 340 (M + 1,
100), 342 (M + 3, 31). HPLC purity 96.9%.
4-(N-(6-Methoxy-2,3-dihydro-4-oxoquinolinyl))-2-methyl-
quinazoline (5b). Using condition A starting with 2b (89 mg, 0.5
mmol) and 3c (89 mg, 0.5 mmol), the mixture was refluxed for 1 h to
General Procedure of the Nucleophilic Substitution Re-
action for 4a−c, 5a−b, 5d−e, 5h−m, and 6. A solution of 2,4-
dichloroquinazoline (2a, 0.5 mmol) or 4-chloro-2-methylquinazoline
(2b, 0.5 mmol) and an amine (0.5 mmol) under condition A
(anhydrous i-PrOH (5 mL) with a drop of concentrated HCl stirred at
1
produce 109 mg of 5b: 64% yield, yellow solid, mp 213−214 °C. H
NMR δ 2.79 (1H, s, CH3), 2.97 (2H, t, J = 6.4 Hz, 3′-CH2), 3.85 (3H,
s, OCH3), 4.43 (2H, t, J = 6.4 Hz, 2′-CH2), 6.57 (1H, d, J = 8.8 Hz,
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1
ArH-8′), 6.84 (1H, dd, J = 8.8 and 3.2 Hz, ArH-7′), 7.29 (1H, m, ArH-
6), 7.50 (1H, d, J = 3.2 Hz, ArH-5′), 7.65 (1H, dd, J = 8.4 and 0.8 Hz,
ArH-5), 7.75 (1H, m, ArH-7), 7.91 (1H, d, J = 8.4 Hz, ArH-8). MS m/
z (%) 320 (M + 1, 100). HPLC purity 100.0%.
159−160 °C. H NMR δ 2.17 (2H, m, 4′-CH2), 3.04 (2H, t, J = 1.6
Hz, 5′-CH2), 3.81 (3H, s, OCH3), 4.13 (2H, t, J = 1.6 Hz, 3′-CH2),
4.96 (2H, s, 1′-CH2), 6.77 (2H, m, ArH-6′ and 8′), 7.28 (1H, m, ArH-
9′), 7.35 (1H, m, ArH-6), 7.68 (1H, m, ArH-7), 7.76 (1H, d, J = 8.4
Hz, ArH-5), 7.93 (1H, d, J = 8.4 Hz, ArH-8). MS m/z (%) 161 (M−
158, 100), 340 (M + 1, 43), 342 (M + 3, 12).
2-Chloro-4-(N-(7-methoxy-3,4-dihydro-2H-benzo[b][1,4]-
oxazinyl))quinazoline (5d). Using condition B starting with 2a (100
mg, 0.5 mmol) and 3d (91 mg, 0.55 mmol) in EtOH, the mixture was
refluxed for 1 h to produce 88 mg of 5d: 54% yield, yellow solid, mp
4-(N-(7-Methoxy-2,3,4,5-tetrahydro-1H-benzo[c]azepinyl))-
2-methylquinazoline (5m). Using condition B starting with 2b (178
mg, 1.0 mmol) and 3h (212 mg, 1.2 mmol) in EtOH, the mixture was
refluxed for 2 h to produce 236 mg of 5m: 74% yield, yellow solid, mp
1
173−174 °C. H NMR δ 3.79 (3H, s, 7′-OCH3), 4.23 (2H, t, J = 4.8
Hz, 2′-CH2), 4.51 (2H, t, J = 4.8 Hz, 3′-CH2), 6.31 (1H, dd, J = 8.8
and 2.8 Hz, ArH-6′), 6.54 (1H, d, J = 2.8 Hz, ArH-8′), 6.72 (1H, d, J =
8.8 Hz, ArH-5′), 7.29 (1H, m, ArH-6), 7.73 (1H, m, ArH-7), 7.81
(1H, d, J = 8.4 Hz, ArH-5), 7.84 (1H, d, J = 8.4 Hz, ArH-8). MS m/z
(%) 328 (M + 1, 100), 330 (M + 3, 50). HPLC purity 100.0%.
2-Methyl-4-(N-(7-methoxy-3,4-dihydro-2H-benzo[b][1,4]-
oxazinyl)quinazoline (5e). Using condition A starting with 2b (89
mg, 0.5 mmol) and 3d (83 mg, 0.5 mmol), the mixture was refluxed
for 1 h to produce 112 mg of 5e: 73% yield, yellow solid, mp 139−140
°C. 1H NMR δ 2.73 (3H, s, CH3), 3.78 (3H, s, OCH3), 4.17 (2H, t, J
= 4.4 Hz, 3′-CH2), 4.50 (2H, t, J = 4.4 Hz, 2′-CH2), 6.28 (1H, dd, J =
8.8 and 2.8 Hz, ArH-6′), 6.53 (1H, d, J = 2.8 Hz, ArH-8′), 6.67 (1H, d,
J = 8.8 Hz, ArH-5′), 7.26 (1H, m, ArH-6), 7.71 (1H, m, ArH-7), 7.82
(1H, dd, J = 8.4 and 0.8 Hz, ArH-5), 7.85 (1H, d, J = 8.4 Hz, ArH-8).
MS m/z (%) 308 (M + 1, 100). HPLC purity 100.0%.
1
125−127 °C. H NMR δ 2.14 (2H, m, 4′-CH2), 2.65 (3H, s, CH3),
3.03 (2H, t, J = 5.6 Hz, 5′-CH2), 3.81 (3H, s, OCH3), 4.09 (2H, t, J =
5.2 Hz, 3′-CH2), 4.89 (2H, s, 1′-CH2), 6.73 (1H, dd, J = 8.4 and 2.8
Hz, ArH-8′), 6.77 (1H, d, J = 2.8 Hz, ArH-6′), 7.24 (1H, d, J = 8.4 Hz,
ArH-9′), 7.30 (1H, m, ArH-6), 7.65 (1H, m, ArH-7), 7.76 (1H, d, J =
8.4 Hz, ArH-5), 7.89 (1H, d, J = 8.4 Hz, ArH-8). MS m/z (%) 161 (M
− 158, 100), 320 (M + 1, 55).
2-Methyl-4-(2-nitro-4-methoxyphenyl)aminoquinazoline
(6). Using condition A starting with 2b (268 mg, 1.5 mmol) and 2-
nitro-4-methoxyaniline (3e, 278 mg, 1.65 mmol), the mixture was
refluxed for 5 h to produce 350 mg of 6: 75% yield, orange solid, mp
174−175 °C. 1H NMR δ 2.77 (3H, s, CH3), 3.91 (3H, s, OCH3), 7.37
(1H, dd, J = 9.6 and 3.2 Hz, ArH-5′), 7.06 (1H, t, J = 7.6 Hz, ArH-6),
6.62 (1H, d, J = 8.8 Hz, ArH-5), 7.77 (1H, d, J = 3.2 Hz,ArH-3′), 7.83
(1H, t, J = 7.6 Hz, ArH-7), 8.04 (1H, d, J = 7.6 Hz, ArH-8), 9.37 (1H,
d, J = 9.6 Hz, ArH-6′), 11.22 (1H, br s, NH). MS m/z (%) 311 (M +
1, 100).
4-(N-(4-Hydroxy-6-methoxy-1,2,3,4-tetrahydroquinolinyl))-
2-methylquinazoline (5c). To a solution of 5b (100 mg, 0.31
mmol) in 6 mL of MeOH was added NaHB4 (23 mg, 0.62 mmol) in
portions at 0 °C. The mixture was then stirred at rt for another 1 h.
After completion of the reaction, the mixture was poured into ice
water, neutralized with aqueous HCl (2 N) to pH 6, and extracted
three times with EtOAc (20 mL). The combined organic phases were
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
elution: EtOAc/petroleum ether, 0−80%) to give 62 mg of 5c: 62%
yield, yellow solid, mp 136−138 °C. 1H NMR δ 2.13 (1H, m, 3′-CH),
2.37 (1H, m, 3′-CH), 2.74 (3H, s, CH3), 3.82 (3H, s, OCH3), 4.00
(1H, m, 2′-CH), 4.21 (1H, m, 2′-CH), 4.94 (1H, m, 4′-CH), 6.60
(1H, dd, J = 9.2 and 2.8 Hz, ArH-7′), 6.66 (1H, d, J = 9.2 Hz, ArH-8′),
7.06 (1H, s, J = 2.8 Hz, ArH-5′), 7.19 (1H, m, ArH-6), 7.50 (1H, d, J =
8.4 Hz, ArH-5), 7.67 (1H, m, ArH-7), 7.84 (1H, d, J = 8.4 Hz, ArH-8).
MS m/z (%) 322 (M + 1, 100). HPLC purity 98.8%.
2-Chloro-4-(N-(5-methoxy)indolinyl)quinazoline (5h). Using
condition B starting with 2a (100 mg, 0.5 mmol) and 3f (75 mg, 0.5
mmol) in anhydrous EtOH (5 mL), the mixture was kept at rt for 1 h
to produce 83 mg of 5h: 53% yield, yellow solid, mp 142−144 °C. 1H
NMR δ 3.21 (2H, t, J = 8.0 Hz, 3′-CH2), 3.82 (3H, s, OCH3), 4.51
(2H, t, J = 8.0 Hz, 2′-CH2), 6.75 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6′),
6.86 (1H, d, J = 2.8 Hz, ArH-4′), 7.42 (1H, dd, J = 8.4 and 1.2 Hz,
ArH-6), 7.57 (1H, d, J = 8.8 Hz, ArH-7′), 7.76 (1H, dd, J = 8.4 and 1.2
Hz, ArH-7), 7.84 (1H, dd, J = 8.4 Hz and 1.2 Hz, ArH-5), 8.08 (1H, d,
J = 8.4 Hz, ArH-8). MS m/z (%) 312 (M + 1, 100), 314 (M + 3, 39).
HPLC purity 95.1%.
4-(N-(5-Methoxy)indolinyl)-2-methylquinazoline (5i). Using
condition B starting with 2b (89 mg, 0.5 mmol) and 3f (90 mg, 0.6
mmol) in anhydrous EtOH (5 mL), the mixture was kept at rt for 1 h
to produce 112 mg of 5i: 77% yield, yellow solid, mp 116−117 °C. 1H
NMR δ 2.70 (3H, s, CH3), 3.19 (2H, t, J = 8.0 Hz, 3′-CH2), 3.81 (3H,
s, OCH3), 4.45 (2H, t, J = 8.0 Hz, 2′-CH2), 6.69 (1H, dd, J = 8.8 and
2.4 Hz, ArH-6′), 6.86 (1H, d, J = 2.4 Hz, ArH-4′), 7.37 (2H, m, ArH-
7′ and ArH-6), 7.73 (1H, d, J = 7.6 Hz, ArH-7), 7.84 (1H, d, J = 8.4
Hz, ArH-5), 8.03 (1H, d, J = 8.4 Hz, ArH-8). MS m/z (%) 292 (M +
1, 100). HPLC purity 100.0%.
4-(2-Chloroacetylamino-4-methoxyphenyl)amino-2-methyl-
quinazoline (8). A mixture of 6 (310 mg, 1.0 mmol) and Pd/C (30
mg, 10% w/w) in 25 mL of EtOAc was stirred under a hydrogen gas
atmosphere at rt for 2 h. After removal of the Pd/C by filtration and
removal of the solvent in vacuo, the product, 4-(2-amino-4-
methoxyphenyl)amino-2-methylquinazoline (260 mg), was dissolved
in acetone (20 mL), and K2CO3 (386 mg, 2.8 mmol) was added. The
mixture was cooled to 0 °C, and chloroacetyl chloride (211 mg, 1.8
mmol) was added dropwise. After addition was complete, the mixture
was stirred at the same temperature for 1 h. The mixture was poured
into ice water and extracted three times with EtOAc (30 mL). The
combined organic phases were washed with water and brine
successively and dried over anhydrous Na2SO4 overnight. After
removal of solvent in vacuo, 264 mg of product 8 was obtained: 69%
yield, yellow solid. 1H NMR δ 2.64 (3H, s, CH3), 3.84 (3H, s, OCH3),
4.15 (2H, s, CH2), 6.85 (1H, dd, J = 8.4 and 2.8 Hz, ArH-5′), 7.39
(1H, d, J = 8.4 Hz, ArH-6′), 7.52 (1H, t, J = 7.6 Hz,ArH-5), 7.83 (4H,
m, ArH-6, 7, 8, 3′), 9.41 (1H, br s, NH). MS m/z (%) 264 (M − 92,
100), 357 (M + 1, 31), 359 (M + 3, 22).
2-Chloro-4-(N-(7-methoxy-2,3,4,5-tetrahydro-1H-benzo[b]-
azepinyl))quinazoline (5j). Using condition B starting with 2a (200
mg, 1.0 mmol) and 3g (177 mg, 1.0 mmol) in anhydrous EtOH, the
mixture was refluxed for 2 h to produce 254 mg of 5j: 75% yield, pale
1
yellow solid, mp 102−103 °C. H NMR δ 3.86 (3H, s, OCH3), 6.68
(1H, dd, J = 8.4 and 2.8 Hz, ArH-8′), 6.72 (1H, d, J = 8.8 Hz, ArH-5),
6.85 (1H, d, J = 8.4 Hz, ArH-9′), 6.92 (1H, d, J = 2.8 Hz, ArH-6′),
6.97 (1H, m, ArH-7), 7.54 (1H, m, ArH-6), 7.71 (1H, d, J = 8.4 Hz,
ArH-8). 13C NMR δ 26.03 (4′C), 28.81 (3′C), 34.27 (5′C), 50.93
(2′C), 55.49 (OCH3), 112.37 (6′C), 114.97 (8′C), 116.17 (9′C and
10C), 124.88 (8C), 126.07 (11′C), 127.25 (6C), 127.65 (5C), 138.36
(10′C), 141.15 (7′C), 153.37 (9C), 158.98 (2C), 160.92 (4C). MS m/
z (%) 340 (M + 1, 100), 342 (M + 3, 32). HPLC purity 100.0%.
4-(N-(7-Methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepinyl))-
2-methylquinazoline (5k). Using condition A starting with 2b (89
mg, 0.5 mmol) and 3g (88 mg, 0.5 mmol) in i-PrOH, the mixture was
refluxed for 2 h to produce 206 mg of 5k: 64% yield, yellow solid, mp
130−131 °C. 1H NMR δ 2.70 (3H, s, CH3), 3.84 (3H, s, OCH3), 6.62
(1H, dd, J = 8.8 and 2.8 Hz, ArH-8′), 6.77 (2H, m, ArH-5 and 9′),
6.92 (2H, m, ArH-7 and 6′), 7.50 (1H, m, ArH-6), 7.71 (1H, d, J = 8.4
Hz, ArH-8). MS m/z (%) 320 (M + 1, 100). HPLC purity 98.5%.
2-Chloro-4-(N-(7-methoxy-2,3,4,5-tetrahydro-1H-benzo[c]-
azepinyl))quinazoline (5l). Using condition B starting with 2a (199
mg, 1.0 mmol) and 3h (177 mg, 1.0 mmol) in EtOH, the mixture was
refluxed for 2 h to produce 264 mg of 5l: 78% yield, yellow solid, mp
2-Methyl-4-(7-methoxy-2-oxo-3,4-dihydroquinoxalin-4(1H)-
yl)quinazoline (5f). A mixture of 8 (260 mg, 0.73 mmol) and
anhydrous K2CO3 (201 mg, 1.46 mmol) in DMF (5 mL) was heated
at 100 °C for 2 h. After the reaction was complete, the mixture was
poured into ice water, and the precipitated solid was obtained by
filtration and dried to give crude product, which was further purified
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Article
by flash column chromatography (gradient elution: EtOAc/petroleum
ether, 0−80%) to afford 161 mg of 5f: 69% yield, yellow solid, mp
232−233 °C. 1H NMR δ 2.78 (3H, s, 3-CH2), 3.81 (3H, s, 7-OCH3),
4.68 (2H, s, 3-CH2), 6.40 (1H, dd, J = 9.2 and 2.8 Hz, ArH-6′), 6.57
(1H, d, J = 2.8 Hz, ArH-8′), 6.62 (1H, d, J = 9.2 Hz, ArH-5′), 7.22
(1H, m, ArH-6), 7.48 (1H, d, J = 8.4 Hz, ArH-5), 7.70 (1H, m, ArH-
7), 7.88 (1H, d, J = 8.4 Hz, ArH-8), 8.63 (1H, br s, NH). MS m/z (%)
321 (M + 1, 100). HPLC purity 98.9%.
mg/mL (10 μM) of tubulin and varying compound concentrations
were preincubated for 15 min at 30 °C without guanosine 5′-
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 an
increase in turbidity by 50% relative to a control sample were
determined.
Inhibition of the binding of [3H]colchicine to tubulin was measured
as described previously.34 Incubation of 1.0 μM tubulin with 5.0 μM
[3H]colchicine and 5.0 or 1.0 μM inhibitor took place for 10 min at 37
°C, the time at which about 40−60% of maximum colchicine binding
occurs in control samples.
2-Methyl-4-(1-methyl-7-methoxy-2-oxo-3,4-dihydroquinox-
alin-4-yl)quinazoline (5g). To a solution of 5f (81 mg, 0.25 mmol)
and MeI (71 mg, 0.5 mmol) in anhydrous DMF (ca. 3 mL) was slowly
added NaH (40 mg, 1.0 mmol, 60% oil suspension) at 0 °C with
stirring over about 1 h. When the reaction was complete, as monitored
by TLC, the mixture was poured into ice water and extracted with
EtOAc three times. After removal of solvent in vacuo, the crude
product was purified by flash column chromatography (gradient
elution: EtOAc/petroleum ether, 0−70%) to produce 70 mg of 5g:
Competitive Inhibition Assays. Colchicine Binding to Tubulin.
Tubulin (4 μM, prepared from fresh dog brain tissue by a literature
method35,36) in buffer containing 25 mM PIPES (pH 6.8), 1 mM
EGTA, and 3 mM MgCl2 was incubated without and with different
concentrations (0.1, 0.3, 1, 2, 3, and 10 μM) of 5f at 37 °C for 45 min
in a nontransparent black 96-well plate. Then, colchicine (10 μM) was
added to the mixture, which was incubated at 37 °C for an additional
45 min. The fluorescence intensity of the tubulin−colchicine complex
(excitation at 340 nm, emission at 435 nm)37 was measured using a
multidetection microplate reader (SpectraMax M5). The inhibition
rates of the tested compounds were expressed as the percentage (%) of
the decreased fluorescence of the tubulin−colchicine complex. The
IC50 of 5f was determined using GraphPad Prism software (V5.01,
GraphPad Software Incorporated). Each experiment was performed
independently with at least three replicates and expressed as the mean
SD.
1
84% yield, yellow solid, mp 217−218 °C. H NMR δ 2.77 (3H, s,
CH3), 3.48 (3H, s, NCH3), 3.83 (3H, s, OCH3), 4.67 (2H, s, CH2),
6.41 (1H, dd, J = 9.2 and 2.8 Hz, ArH-6′), 6.64 (1H, d, J = 9.2 Hz,
ArH-5′), 6.72 (1H, d, J = 2.8 Hz, ArH-8′), 7.20 (1H, m, ArH-7), 7.47
(1H, d, J = 8.4 Hz, ArH-5), 7.69 (1H, m, ArH-6), 7.87 (1H, d, J = 8.4
Hz, ArH-8′). MS m/z (%) 335 (M + 1, 100). HPLC purity 98.8%.
7-Methoxy-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one (10a)
and 7-methoxy-2,3,4,5-tetrahydro-1H-benzo[c]azepin-1-one
(10b). To asolution of 6-methoxy-3,4-dihydronaphthalen- 1(2H)-
one (3.52 g, 20 mmol) in 15 mL of methanesulfonic acid was added
NaN3 (1.69 g, 26 mmol) in portions at 0 °C. The mixture was stirred
at rt for 24 h and was then poured into ice water and neutralized with
10% aqueous NaOH to pH 6. The solid was filtered, washed with
water, and dried. The crude product was purified by flash column
chromatography (gradient elution: EtOAc/petroleum ether, 0−70%)
to obtain 0.48 g of 10a in 13% yield and 1.92 g of 10b in 50% yield,
Vinblastine Binding to Tubuline.26 Tubulin (4 μM) in the same
buffer was mixed with 5f and vincristine at concentrations of 10, 30,
and 100 μM, respectively, and incubated at 37 °C. After 30 min,
BODIPY FL-vinblastine (2 μM) was added, and the mixtures were
incubated for another 30 min. The fluorescence intensity of FL-vin−
tubulin complex (excitation at 470 nm, emission at 515 nm) was
measured using a multidetection microplate reader (SpectraMax M5).
Inhibition rates (%) of the tested compounds were determined by the
percent of decreased fluorescence in the same manner as that the
colchicine binding to tubulin assay.
Cell Cycle Analysis. A549 cells treated with 5f (3 nM) or
colchicine (300 nM) for 24 h were washed twice in PBS, resuspended
in 2 mL of 70% ice-cold EtOH, and kept at 4 °C for 24 h. Fixed cells
were washed once in PBS and then treated with 150 μL of a 0.05 mg/
mL RNAase solution at 37 °C for 30 min. Cell nuclei were then
stained with a PBS solution (150 μL) containing 0.05 mg/mL of
propidium iodide for 30 min at rt in the dark. Cell cycle distribution
was determined with a FACSCalibur (BD Biosciences).
Immunocytochemistry. A549 cells were grown in black clear-
bottom 96-well plates in the presence or absence of tested compounds
(5f, paclitaxel, or colchicine) for 24 h and fixed in 4%
paraformaldehyde for 20 min. After being rinsed with PBS and
permeabilized with 0.1% Triton X-100 in PBS for 30 min in the dark,
the fixed cells were rinsed again with PBS and blocked with 5% bovine
serum albumin (BSA) for 30 min at 37 °C. Then, anti-α-tubulin
antibodies (Invitrogen) (1:2000) and the secondary antibody (Alexa
Flour 549-donkey anti-mouse IgG; Invitrogen) were added
successively to each well and incubated for 2 and 1 h, respectively.
Finally, the cells were stained with Hoechst 33342 for 1 h at rt in the
dark. Images were acquired by Incell Analyzer 1000 (GE, ) using a 20×
objective.
Aqueous Solubility Studies. Solubility was measured at pH 7.4
by using an HPLC−UV method. Test compounds were initially
dissolved in DMSO at a concentration of 1.0 mg/mL. Ten microliters
of this stock solution was added to pH 7.4 phosphate buffer (1.0 mL),
with the final DMSO concentration being 1%. The mixture was stirred
for 4 h at rt and then centrifuged at 3000 rpm for 10 min. The
saturated supernatants were transferred to other vials for analysis by
HPLC−UV. Each sample was performed in triplicate. For
quantification, a model 1200 HPLC−UV (Agilent) system was used
with an Agilent Eclipse XDB-C18 column (150 × 4.6 mm, 5 μm), and
1
respectively. Product 10a: pale yellow solid. H NMR δ 2.12 (2H, m,
4-CH2), 2.33 (2H, t, J = 6.8 Hz, 5-CH2), 2.77 (t, J = 7.2 Hz, 3-
CH2),3.81 (3H, s, OCH3), 6.75 (2H, m, ArH-6, 8), 6.90 (1H, d, J =
8.0 Hz, ArH-9), 7.25 (1H, br s, NH). MS m/z (%)192 (M−21, 100),
214 (M+ 1, 41). Product 10b: pale yellow solid. 1H NMR δ 2.01 (2H,
m, 4-CH2), 2.85 (2H, t, J = 6.8 Hz, 5-CH2), 3.13 (1H, t, J = 6.8 Hz, 3-
CH2), 3.85 (3H, s, OCH3), 6.22 (1H, br s, NH), 6.72 (1H, d, J = 2.8
Hz, ArH-6), 6.85 (1H, dd, J = 8.4 and 2.8 Hz, ArH-8), 7.68 (1H, d, J =
8.4 Hz, ArH-9). MS m/z (%) 192 (M − 21, 100), 214 (M + 1, 54).
7-Methoxy-2,3,4,5-tetrahydro-1H-benzo[b]azepine (3g). A
solution of 10a (328 mg, 1.70 mmol) in THF (10 mL) was added
dropwise to LiAlH4 (133 mg, 3.51 mmol, excess) in anhydrous THF
(10 mL) at rt under a nitrogen gas atmosphere with stirring followed
by reflux for another 16 h. After the reaction was complete, 0.13 mL of
water, 0.39 mL of 15% aqueous NaOH, and 0.39 mL of water were
added successively to the mixture with stirring for another 10 min at rt.
The mixture was filtered through Celite, the solvent was removed in
vacuo, and the residue was purified by flash column chromatography
(gradient elution: EtOAc/petroleum ether 0−40%) to produce 256
mg of 3g: 84% yield, brown oil. 1H NMR δ 1.63 (2H, m, 4-CH2), 1.79
(2H, m, 3-CH2), 2.74 (2H, m, 5-CH2), 2.98 (2H, m, 2-CH2), 3.76
(3H, s, OCH3), 6.60 (1H, dd, J = 8.4 and 2.8 Hz, ArH-8), 6.70 (2H,
m, ArH-7, 9). MS m/z (%) 136 (M − 41, 100), 178 (M + 1, 29).
7-Methoxy-2,3,4,5-tetrahydro-1H-benzo[c]azepine (3h). Pre-
pared by the same procedure as 3g and used directly in the next step
without further purification.
Antiproliferative Activity Assay. Target compounds were
assayed by the SRB method for cytotoxic activity using a HTCL
assay according to procedures described previously.30−32 The panel of
cell lines included human lung carcinoma (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.
Tubulin Assays. Tubulin assembly was measured by turbidimetry
at 350 nm as described previously.33 Assay mixtures containing 1.0
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elution was with 50−80% ACN in water. The flow rate was 0.8 mL/
min, and the injection volume was 20 μL. Aqueous concentration was
determined by comparison of the peak area of the saturated solution
with a standard curve plotted for the peak area versus known
concentrations, which was prepared by solutions of test compound in
ACN at 50, 12.5, 3.13, 0.78, and 0.20 μg/mL.
Log P Measurements. One to two milligrams of test compound
were dissolved in 1.0−2.0 mL of n-octane to obtain a 1.0 mg/mL
solution. Next, the same volume of water as n-octane was added to
each vial. The mixture was stirred at rt for 24 h and left without stirring
overnight. The aqueous and organic phases of each mixture were
transferred to separate vials for HPLC analysis. The instrument and
conditions were the same as those for water solubility determinations.
The log P was calculated by the peak area ratio in n-octane and in
water.
Microsomal Stability Assay. Stock solutions of test compounds
(1 mg/mL) were prepared by dissolving the pure compound in
DMSO, and the solutions were stored at 4 °C. Before performing the
assay, the stock solution was diluted with ACN to 0.1 mM. For
measurement of metabolic stability, all compounds were brought to a
final concentration of 1 μM with 0.1 M potassium phosphate buffer at
pH 7.4, which contained 0.1 mg/mL of human liver microsomes and 5
mM MgCl2. The incubation volumes were 300 μL, and the reaction
temperature was 37 °C. Reactions were started by adding 60 μL of
NADPH (final concentration, 1.0 mM) and quenched by adding 600
μL of ice-cold ACN to stop the reaction at 5, 15, 30, and 60 min time
points. Samples at the 0 min time point were prepared by adding 600
μL of ice-cold ACN first followed by 60 μL of NADPH. All samples
were prepared in duplicate. After quenching, all samples were
centrifuged at 12 000 rpm for 5 min at 0 °C. The supernatant was
collected, and 20 μL of the supernatant was directly injected into a
Shimadzu LC−MS 2010 system with an electrospray ionization source
for further analysis. The following controls were also used: (1) positive
control incubation containing liver microsomes, NADPH, and
reference compound propranolol or terfenadine, (2) negative control
incubation omitting NADPH, and (3) baseline control containing only
liver microsomes and NADPH. The peak heights of the test
compounds at different time points were converted to the percentage
of compound remaining, and the peak height values at initial time (0
min) served as 100% values. The slope of the linear regression from
log percentage remaining versus incubation time relationships (−k)
was used to calculate the in vitro half-life (t1/2) (t1/2 = 0.693/k), which
was regarded as first-order kinetics. Conversion to in vitro CLint (in
receiving vehicle solution (5% PEG400/PBS) only. The experiment
was stopped when the tumor volumes of the control mice reached
about 1500 mm3. At the end of the treatment, the mice were sacrificed
for autopsy, and the tumors were recovered and weighed. The tumor
growth inhibitory rate was calculated as follows: inhibitory rate (%) =
[1− (mean tumor weight of treated group)/(mean tumor weight of
control group)] × 100.
Molecular Modeling Studies. All molecular modeling studies
were performed with Discovery Studio 3.0 (Accelrys). The crystal
structure of tubulin in complex with DAMA-colchicine (PDB code:
1SA0) was downloaded from the RCSB Protein Data Bank (http://
www.rcsb.org/pdb) for use in the modeling study. CDOCKER was
used to evaluate and predict in silico binding free energy of the
inhibitors and for automated docking. The protein protocol was
prepared by several operations, including standardization of atom
names, insertion of missing atoms in residues and removal of alternate
conformations, insertion of missing loop regions based on SEQRES
data, optimization of short and medium size loop regions with the
Looper algorithm, minimization of remaining loop regions, calculation
of pK, and protonation of the structure. The receptor model was typed
with the CHARMM force field. A binding sphere with a radius of 8.5 Å
was defined through the original ligand (DAMA-colchicine) as the
binding site for the study. The docking protocol employed total ligand
flexibility, and the final ligand conformations were determined by the
simulated annealing molecular dynamics search method set to a
variable number of trial runs. Docked ligand 5f was further refined
using in situ ligand minimization with the Smart Minimizer algorithm
by standard parameters. The ligand and its surrounding residues within
the above-defined sphere were allowed to move freely during the
minimization, whereas the outer atoms were frozen. The implicit
solvent model of Generalized Born with Molecular Volume (GBMV)
was also used to calculate the binding energies.
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC purity data for target compounds and cell cycle assay
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
units of mL/min/mg of protein) was calculated by the formula CLint
=
*(K.-H.L.) Phone: +1-919-962-0066. Fax: +1-919-966-3893. E-
mail: khlee@unc.edu.
*(L.X.) Phone/Fax: +86-10-66931690. E-mail: lanxie4@gmail.
com.
[0.693/(in vitro t1/2)][(mL incubation)/(mg of microsomes)]. The
HPLC−MS analysis was carried out on a Shimadzu LC−MS 2010
with an electrospray ionization source. An Alltima C18 column (5 μm,
150 × 2.1 mm) was used for HPLC with gradient elution at a flow rate
of 0.2 mL/min. The elution conditions were ACN (B) in water (A) at
30% for 0−2 min, 85% for 2−6 min, 100% for 6−9 min, and 30% for
9−12 min. The MS conditions were optimized to a detector voltage of
+1.7 kV, with the acquisition mode selected as ion monitoring of the
appropriate molecular weights of the test compounds. The curved
desolvation line temperature was 250 °C, the heat block temperature
was 200 °C, and the neutralizing gas flow was 1.5 L/min. Samples
were injected by an autosampler. Electrospray ionization was operated
in positive and negative modes.
Antitumor Assay in Vivo. Six-week-old female athymic nude mice
(Balb/c nu/nu) were obtained from Vital River and housed under
specific pathogen-free conditions in conformity with the Guide for the
Care and Use of Laboratory Animals, as adopted and promulgated by
Beijing Institute of Radiation Medicine. MCF7 cells (2 × 106) were
injected subcutaneously into the right abdominal flanks of the nude
mice. Tumor growth was measured with a slide caliper, and volumes
were estimated according to the following formula: tumor volume
(mm3) = L × W2 × 0.5, where L is length and W is width. When
tumor volume reached about 100−300 mm3, the mice were sorted into
a treatment group and a control group with similar mean tumor sizes.
The mice in the treatment group received a dose of 4 mg/kg every 3
days by i.v. injection. Control mice were treated the same way,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This investigation was supported by grants 81120108022 and
30930106 from the Natural Science Foundation of China
(NSFC) awarded to L.X. and NIH grant CA177584-01 from
the National Cancer Institute awarded to K.-H.L. This study
was also supported in part by the Taiwan Department of
Health, China Medical University Hospital Cancer Research
Center of Excellence (DOH100-TD-C-111-005).
ABBREVIATIONS USED
■
CA4, combretastatin A-4; DAMA-colchicine, N-deacetyl-N-(2-
mercaptoacetyl)-colchicine; GBMV, Generalized Born with
Molecular Volume; GI50, concentration that inhibits 50%
human tumor cell growth; HTCL, human tumor cell line;
SAR, structure−activity relationship; SPR, structure−property
relationship; VDA, vascular-disrupting agents
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