In Vivo and Mechanistic Studies on Antitumor Lead 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one and Its Modification as a Novel Class of Tubulin-Binding Tumor-Vascular Disrupting Agents

Article abstract of DOI:10.1021/acs.jmedchem.7b00273

7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (2), a promising anticancer lead previously identified by us, inhibited tumor growth by 62% in mice at 1.0 mg/kg without obvious signs of toxicity. Moreover, compound 2 exhibited extremely high antiproliferative activity in the NIH-NCI 60 human tumor cell line panel, with low to sub-nanomolar GI₅₀ values (10^-10 M level). It also showed a suitable balance between aqueous solubility and lipophilicity, as well as moderate metabolic stability in vivo. Mechanistic studies using Mayer's hematoxylin and eosin and immunohistochemistry protocols on xenograft tumor tissues showed that 2 inhibited tumor cell proliferation, induced apoptosis, and disrupted tumor vasculature. Moreover, evaluation of new synthetic analogues (6a-6t) of 2 indicated that appropriate 2-substitution on the quinazoline ring could enhance antitumor activity and improve druglike properties. Compound 2 and its analogues with a 4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one scaffold thus represent a novel class of tubulin-binding tumor-vascular disrupting agents (tumor-VDAs) that target established blood vessels in tumors.

Full text of DOI:10.1021/acs.jmedchem.7b00273

Article
pubs.acs.org/jmc
In Vivo and Mechanistic Studies on Antitumor Lead 7‑Methoxy-4-(2-
methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)‑one and Its
Modification as a Novel Class of Tubulin-Binding Tumor-Vascular
Disrupting Agents
Mu-Tian Cui,† Li Jiang,† Masuo Goto,‡ Pei-Ling Hsu,‡ Linna Li,∥ Qi Zhang,∥ Lei Wei,† Shou-Jun Yuan,∥
Ernest Hamel,§ Susan L. Morris-Natschke,‡ 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, 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
⊥Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 40402, Taiwan
S
* Supporting Information
ABSTRACT: 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihy-
droquinoxalin-2(1H)-one (2), a promising anticancer lead
previously identified by us, inhibited tumor growth by 62% in
mice at 1.0 mg/kg without obvious signs of toxicity. Moreover,
compound 2 exhibited extremely high antiproliferative activity in
the NIH-NCI 60 human tumor cell line panel, with low to sub-
nanomolar GI50 values (10−10 M level). It also showed a suitable
balance between aqueous solubility and lipophilicity, as well as
moderate metabolic stability in vivo. Mechanistic studies using Mayer’s hematoxylin and eosin and immunohistochemistry
protocols on xenograft tumor tissues showed that 2 inhibited tumor cell proliferation, induced apoptosis, and disrupted tumor
vasculature. Moreover, evaluation of new synthetic analogues (6a−6t) of 2 indicated that appropriate 2-substitution on the
quinazoline ring could enhance antitumor activity and improve druglike properties. Compound 2 and its analogues with a 4-(2-
methylquinazolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one scaffold thus represent a novel class of tubulin-binding tumor-vascular
disrupting agents (tumor-VDAs) that target established blood vessels in tumors.
INTRODUCTION
■
During the past decade, a novel class of antitumor agents
termed tumor-vascular disrupting agents (tumor-VDAs) have
been investigated intensively.1,2 Unlike angiogenesis inhibitors
(AIs), tumor-VDAs target established tumor blood vessels and
exert an almost immediate effect on these vessels, thus leading
to a rapid vascular collapse and subsequent tumor cell death via
extensive necrosis and apoptosis.3 Due to the morphological
vasculature difference between normal tissues and solid tumors,
Figure 1. Colchicine and some clinical trial VDA candidates.
tumor-VDAs preferentially block the blood flow of solid
tumors, while the blood flow in normal tissues remains
relatively intact.4 Currently, more than a dozen tumor-VDA
drug candidates are undergoing clinical trials,5,6 and the most
mechanism(s) of action of tumor-VDAs are still not known,
most of these compounds show common pharmacological
advanced candidate, CA-4P, a phosphate prodrug of
charateristics, including inhibition of microtubule polymer-
combretastatin A-4 (CA-4, Figure 1), is in phase III trials for
ization, competitive binding to the colchicine binding site on
anaplastic thyroid cancer and Phase II trials for non-small-cell
lung cancer (NSCLC).7 Meanwhile, many diverse small
molecule tumor-VDAs with high antiproliferative activity have
Received: February 21, 2017
also been reported recently.8,9 Even though the exact
© XXXX American Chemical Society
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tubulin, and disruption of the tumor cell cytoskeleton, thus
classifing them as tubulin-binding tumor-VDAs,4 such as CA-4.
In our prior studies on novel antitumor agents,10−12 we
discovered a series of new N-aryl-1,2,3,4-tetrahydroquinoline
compounds with high antiproliferative activity in cellular assays,
exemplified by prior leads 1a and 1b11 shown in Figure 2.
confirmed to be important for enhanced antitumor activity.
Apart from the scaffold hopping, our prior structure
optimizations on lead 1a12 also revealed that the 2-substituent
on the quinazoline ring was modifiable to improve druglike
properties without loss of antitumor potency, especially with
the introduction of an alkylamino side chain. Our prior
modeling studies further demonstrated that both 1a and 2
could dock within the tubulin−colchicine binding site and
superimposed well with DAMA-colchicine (the bound ligand in
the tubulin crystal structure, PDB code 1SA0).12,13 In addition,
the docking study showed that the 2-substituent on the
quinazoline ring in series-1 analogues overlapped well with the
7-side chain in DAMA-colchicine (Figure 1), thus indicating
that the quinazoline 2-position has sufficient chemical space for
structural modification. Consequently, in our current study, a
series of new 4-(2-substituted quinazolin-4-yl)-3,4-dihydroqui-
noxalin-2(1H)-one derivatives (6a−6t) were designed by
maintaining the new scaffold in 2 and introducing various
substituents on the quinazoline ring 2-position. Our aims were
to simultaneously enhance antitumor activity and improve
molecular drug-like properties. All synthesized new compounds
were first evaluated in a human tumor cell line (HTCL) panel.
Subsequently, new active compounds were further tested in
tubulin assembly assays and assessed for their essential drug-like
properties.
Figure 2. Leads and new derivatives.
Compounds 1a and 1b show high antiproliferative activity, with
GI50 values of 1.5−1.7 and 11−18 nM, respectively, and they
also inhibit tubulin polymerization with IC50 values of 0.93 and
1.0 μM, respectively. On the other hand, compared with 1a,
compound 1b displays improved aqueous solubility, a better log
P value, and greater metabolic stability. Subsequent lead
optimization on the C-ring (skeleton hopping) led to the
discovery of 7-methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihy-
droquinoxalin-2(1H)-one (2) as a new lead compound.13
Compared with the corresponding prior lead 1b, compound 2,
which has a novel structural scaffold with an altered C-ring,
exhibits more potent activities in cytotoxicity and tubulin assays
and better drug-like properties (Figure 2), including its log P
value (2.97, pH 7.4), aqueous solubility (8.28 μg/mL, pH 7.4),
and metabolic stability in vitro (t1/2 55 min in human liver
microsomes).13 In the current study, we screened 2 in an
extensive cellular panel (NCI-60 cell lines) to confirm the
antitumor activity and determine its antitumor spectrum.
Moreover, we also evaluated 2 in several in vivo systems,
including a H-460 xenograft model and vascular disruption
assays. Based on these results, the new scaffold in 2 was
CHEMISTRY
■
New compounds 6a−6p with various 2-alkylamino or 2-alkoxy
substituents on the quinazoline ring were synthesized as shown
in Scheme 1. The commercially available 2,4-dichloroquinazo-
line was coupled with 4-methoxy-2-nitroaniline (3) in i-PrOH
in the presence of a catalytic amount of HCl to obtain 2-chloro-
4-(4-methoxy-2-nitrophenyl)aminoquinazoline (4) in 85%
yield. The nitro group was then reduced to an amino group
by using zinc powder in acetic acid at 0 °C. Reaction of this
intermediate with 2-chloroacetyl chloride produced 5. Next,
compound 5 underwent an intramolecular cyclization in DMF
in the presence of K2CO3 to yield 4-(2-chloroquinazolin-4-yl)-
7-methoxy-3,4-dihydroquinoxalin-2(1H)-one (6a). Displace-
ment of the 2-chloro group on the quinazoline ring of 6a
with various amines led to a series of 2-alkylamino-derivatives
6b−6n. The reactions were performed in anhydrous i-PrOH
a
Scheme 1
a
Reagents and conditions: (i) i-PrOH/HCl, rt, 5−6 h; (ii) Zn/CH3COOH, CH2Cl2, 0.5 h; (iii) ClCH2COCl/K2CO3, 0 °C, 1 h; (iv) K2CO3/DMF,
100 °C, 0.5 h; (v) amine, microwave (MW), i-PrOH, 60−120 °C, 15−30 min (for 6b−d, 6f−g, 6k), or with H2SO4, 120 °C, 20 min (for 6e, 6j, 6l,
6m), or with K2CO3, 60−120 °C, 15−20 min (for 6h, 6i, 6n); (vi) EtOH or cyclopropylcarbinol/NaH at 0 °C, then MW, 95 °C, 5 min (for 6o, 6p).
B
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a
Scheme 2
a
Reagents and conditions: (i) ClCH2CN/HCl (conc), 90 °C, 24 h; (ii) TsCl, DMAP, Et3N, rt, 3 h; (iii) i-PrOH, 45 °C, 6 h; (iv) KOAc, 55 °C, 6 h;
(v) Pd/C, H2, rt, 2 h; (vi) ClCH2COCl/K2CO3, acetone, 0 °C, 1 h; (vii) K2CO3/DMF, 100 °C, 1 h; (viii) NaOH (10%), rt, 1 h; (ix)
Br(CH2)3COOEt/Cs2CO3, 90 °C, 4 h; (x) NH2OH·HCl, 0 °C, 4 h.
b
a
Table 1. Antiproliferative Potencies (GI50, nM) of 2 against the NCI-60 Panel
Leukemia
CCRF-CEM
Colon Cancer
COLO205
Ovarian Cancer
IGROV1
CNS Cancer
0.551
0.361
0.359
0.605
0.603
0.301
0.322
0.371
0.369
0.413
0.307
0.353
0.487
0.830
0.266
2.43
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
0.788
0.318
0.298
0.655
0.360
0.437
HL-60(TB)
K562
HCC-2998
HCT-116
HCT-15
HT29
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
MOLT-4
RPMI-8226
SR
>100
0.524
0.301
0.409
KM12
Non-Small-Cell Lung Cancer
SW-620
Prostate Cancer
Breast Cancer
A549/ATCC
EKVX
0.588
0.632
0.690
Melanoma
Renal Cancer
PC-3
0.636
0.409
LOX IMVI
MALME-3M
M14
0.526
786-0
3.46
DU-145
HOP-62
>100
A498
0.376
0.926
0.497
0.246
1.01
HOP-92
>100
0.336
0.208
ACHN
CAKI-1
RXF-393
SN12C
TK-10
UO-31
MCF7
0.363
0.914
0.601
2.10
c
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
3.03
MDA-MB-435
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
MDA-MB-231/ATCC
HS 578T
0.458
0.904
0.397
0.294
>100
0.400
>100
0.432
BT-549
>100
10.0
T-47D
MDA-MB-468
0.415
a
b
The data were provided by the NCI, USA. The TGI and LC50 values are >1.0 × 10−7 M in most tested cell lines in the same assays (see SI). The
c
concentration that corresponds to 50% growth inhibition (GI50). Not available.
under microwave (MW) heating at less than 120 °C for 20 min
in the presence of acidic or weakly basic catalysts, for example,
H2SO4 for 6e, 6l, and 6m and K2CO3 for 6h, 6i, 6k, and 6n.
Compound 6f was prepared in N,N-dimethylacetamide (DMA)
at 155 °C. The syntheses of 2-alkoxy-compounds 6o and 6p
were performed in the presence of NaH, used to deprotonate
and enhance the nucleophilicity of the alcohol reactant.
Moreover, compounds 6q−6t, which have a methylene
(CH2) group inserted between the quinazoline ring and the
remainder of the 2-side chain, were synthesized as shown in
Scheme 2. Using a literature method,14 2-chloromethyl-3H-
quinazolin-4-one (8) was prepared from the commercial
reagents methyl 2-aminobenzoate and 2-chloroacetonitrile in
85% yield. Treatment of 8 with p-toluenesulfonyl chloride in
CH2Cl2 in the presence of trimethylamine and a catalytic
amount of dimethylaminopyridine (DMAP) produced the
quinazoline sulfonate ester 9 in 88% yield. A substitution
reaction between 9 and 3 gave intermediate 10. The 2-
chloromethyl on the quinazoline ring in 10 was converted to
the 2-acetoxymethyl in intermediate 11. Next, compound 6q
was obtained from 11 by successive catalytic hydrogenation,
amidation, and intramolecular cyclization. Notably, the
cyclization of intermediate 13 was performed in DMA, rather
than DMF, in 99% yield. Compound 6q was then hydrolyzed
C
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under basic conditions to produce hydroxymethyl compound
6r in 98% yield. Subsequently, compound 6r was reacted with
ethyl 4-bromobutanoate15 in the presence of Cs2CO3 to yield
6s with an extended side chain on the quinazoline ring. Finally,
compound 6s was treated with hydroxylamine to produce
hydroxamide compound 6t. Compounds 6s and 6t were
purified by recrystallization from EtOAc.
RESULTS AND DISCUSSION
■
Testing of Compound 2. NCI-60 Cell Line Screening. To
determine its antitumor spectrum, compound 2 was tested in
the National Cancer Institute’s cellular panel of 60 human
tumor cell lines (NCI-60). As shown in Table 1, compound 2
exhibited excellent potency with low to subnanomolar GI50
values (at 10−10 M level) against most tumor cell lines,
including many multidrug resistant phenotypes, in the standard
NCI testing protocol.16 However, the inhibitory potency of 2
was much reduced against six tumor cell lines (HOP-92,
MALME-3M, SK-MEL-28, UACC-257, OVCAR-5, and TK-
10). Interestingly, MALME-3M and TK10 cell lines were also
significantly resistant toward a tubulin polymerization inhib-
itory 4-substituted-5-methyl-furo[2,3-d]pyrimidine derivative.17
These cell lines may be less sensitive to polymerization
inhibitors, due to certain biological properties, such as
overexpression or ligand binding site mutations on tubulin.
Thus, 2 is an extremely potent antitumor agent with a broad
spectrum of activity.
Antitumor Activity in Vivo. Next, compound 2 was
evaluated in vivo due to its remarkable antiproliferative activity
in vitro, as well as strong inhibitory effects on tubulin assembly,
moderate in vitro metabolic stability (t1/2 55 min in human liver
microsomes), and good aqueous solubility and lipophilicity.
Pilot experiments showed that 4 mg/kg (one dose treatment)
was the lethal dose of 2 in nude mice bearing MCF-7 xenograft
tumors.13 This result allowed us to better assess the in vivo
pharmacokinetic properties of 2. Consequently, the assessment
was performed in SD rats (n = 3) by intravenous (iv)
administration at a single-dose of 1.0 mg/kg.18 Compound 2
was relatively stable metabolically in rats with a terminal half-
life of 52 h, providing an experimental basis for establishing
administration schedules in a subsequent pharmacodynamic
study. We established nude mouse subcutaneous xenograft
models of NCI-H460 lung cancer and sorted the mice into five
groups (n = 8 animals per group) with almost equal mean
tumor size (100 mm3). The mice in three groups were treated
with 2 by iv injection at doses of 1.0, 0.5, or 0.25 mg/kg every 5
days for 3 weeks. Control mice and paclitaxel-treated mice were
treated in the same way, receiving vehicle solution or paclitaxel
(15 mg/kg), respectively (Figure 3). All of the animals survived
through the entire experiment, and no obvious toxic effects
were observed, although there was some effect on animal
weights with the two higher doses of 2 and with paclitaxel. At
the end of treatment, the mice were sacrificed, and the tumors
were recovered and weighed. As shown in Table 2 and Figure 3,
compound 2 significantly inhibited the growth of the NCI-
H460 xenograft tumors in a dose-dependent manner. The
average tumor weights of 2-treated groups were less than those
of control mice (statistical significance). The tumor growth
inhibition rates were 17.8%, 36.8%, and 61.9% at doses of 0.25,
0.5, and 1.0 mg/kg, respectively. Meanwhile, the reference
group treated with paclitaxel at a 15 mg/kg dose showed a
tumor inhibition rate of 60.4%, comparable to what was
Figure 3. Dose-dependent anticancer effects of compound 2 on NCI-
H460 lung cancer xenografts in nu/nu mice. Treatments started when
tumors reached a mean volume of around 100 mm3. Mice were treated
with 2 at an iv dose of 0.25, 0.5, or 1 mg/kg or paclitaxel at 15 mg/kg
as reference or the vehicle as control (n = 8), every 5 days for 3 weeks.
(A) Growth difference of tumor volumes at different time points. (B)
Images of resected NCI-H460 xenograft tumors. (C) Tumors were
●
resected and weighed at the end of experiment, ( ) indicates the
weight value of each tumor; () indicates average value of tumor
weights. *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle controls (one-
way analysis of variance with Tukey’s post hoc method).
observed with 2 at the 1.0 mg/kg dose. Therefore, compound 2
had significant in vivo antitumor effects.
Testing of New Derivatives (6a−6t) of Compound 2.
Antiproliferative Activity in Cellular Assays and SAR Analysis.
The newly synthesized N4-(2-substituted quinazolin-4-yl)-7-
methoxy-3,4-dihydroquinoxalin-2(1H)-one compounds (6a−
D
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a
Table 2. Tumor Growth Inhibition of 2 on Human Lung H460 Xenograft Tumors in Nude Mice
body weight change
antitumor activity
dose (mg/kg)
animals start/end
start (g)
end (g)
%
tumor weight (g)
inhibition rate (%)
vehicle
8/8
8/8
8/8
8/8
8/8
22.50 1.23
22.44 1.27
22.28 1.33
22.13 1.24
22.50 1.29
26.40 1.43
25.96 1.54
23.85 1.57
20.50 3.27
23.59 2.66
17.42
15.71
7.30
2.71 1.07
2
0.25
0.5
2.23 0.57
17.8
36.8
61.9
60.4
2
1.72 0.78*
1.03 0.71***
1.08 0.78**
2
1.0
−7.23
4.99
paclitaxel
15
a
Administration by iv. Schedule: Q5D × 4, that is, every 5 days for 3 weeks. Data are presented as the mean SD, *p < 0.05, **p < 0.01, ***p <
0.001 vs control.
6t) with the same scaffold as 2 were initially evaluated in a
HTCL panel, including A549 (lung carcinoma), KB (originally
isolated from epidermoid carcinoma of the nasopharynx),
multidrug-resistant (MDR) cell line KB-VIN (vincristine-
resistant KB subline), MDA-MB-231 (triple-negative breast
cancer), and MCF-7 (estrogen receptor-positive breast
cancer),19,20 in parallel with paclitaxel as an experimental
control. The activity in the cellular assays was determined using
the established sulforhodamine B (SRB) method.21 The
antiproliferative activities (GI50) and structures are summarized
in Table 3.
Table 3. Antiproliferative Activity of New Series Compounds
(6a−6t) against Human Tumor Cell Lines
As expected, 4-(2-chloroquinazo-4-yl)-7-methoxy-3,4-dihy-
droquinoxalin-2(1H)-one (6a) exhibited significant antiproli-
ferative activity with low nanomolar GI50 values of 0.53−2.01
nM (cf, 1a, GI50 1.5−1.7 nM, Figure 2). After the scaffold
change in the C-ring from piperidine to piperazin-2-one, the
potency increased 3−6-fold (compare prior leads 1a and 1b
with new compounds 6a and 2, respectively), which supported
our previous hypothesis that the lactam C-ring (3,4-
dihydropyrazin-2(1H)-one) might be an important moiety to
enhance antiproliferative potency.
In addition, other series-6 compounds with the same new
scaffold but different substituents at the quinazoline 2-position
showed significant antiproliferative activities in the tested
HTCL panel with GI50 values ranging from subnanomolar to
submicromolar levels (Table 3). Among them, except for 6h
and 6k, most 2-alkylamino-compounds with either a linear side
chain (6a−6e) or a small cycloalkylamino substituent (6f, 6g,
6i, and 6j) were highly potent with GI50 values of 0.54−10.6
nM, with comparable or greater potency than paclitaxel in the
same assays against chemosensitive cell lines. With GI50 values
of >10−80 nM, the 2-(N-pyrrolidinyl) (6h) and 2-(N-(3-
methoxyazetidinyl) (6k) compounds exhibited lower potency
than the above-mentioned compounds.
Moreover, differing results were obtained when a 2-
arylamino substituent was introduced at the quinazoline 2-
position. Compound 6l with a 2-(4-cyanophenyl)amino
substitutent displayed high potency with low nanomolar GI50
values (<10 nM), but compound 6m with a 2-(3-cyanophenyl)-
amino group displayed at least ten times decreased potency
(GI50 61.5−384 nM), suggesting that the bulkiness of the 2-
substituent, for example, a m-substituted phenyl moiety, might
obstruct the small molecule from fitting into the binding pocket
on its biological target. Additionally, compound 6n, another 2-
arylamino [2-(4-pyridinylmethyl)amino] substituted com-
pound, showed high potency only against A549 and KB cell
growth (GI50 6.62 and 7.50 nM, respectively) but decreased
potency toward drug-resistant cell lines KB-VIN (GI50 48.8
nM) and MDA-MB-231 (GI50 73.6 nM), as well as MCF-7
(GI50 71.8 nM).
a
The GI50 values are the concentrations corresponding to 50% cell
growth inhibition and are expressed as the mean SD from the dose−
response curves of at least three independent experiments. Data
published in ref 13. NT: not tested. Paclitaxel served as positive
control in the same assays.
b
c
d
Next, we evaluated the presence of a 2-alkoxy or 2-
alkoxymethyl substituent on the quinazoline ring. The
corresponding 2-ethoxy (6o), 2-cyclopropylmethoxy (6p), 2-
acetoxymethyl (6q), and 2-hydroxymethyl (6r) substituted
compounds all exhibited high potency against tumor cell
growth, generally showing single to double digit nanomolar
GI50 values in the HTCL panel. However, the presence of a
longer linear 2-substituent (more than six atoms) reduced
antiproliferative potency, see compounds 6s and 6t with GI50
values of 0.2−1.28 and 0.31−1.21 μM, respectively.
E
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Table 4. Drug-like Properties and Inhibition of Tubulin Polymerization and Colchicine Binding of Active Compounds 6a−6g,
6i−6j, and 6q−6r
b
parameters (pH 7.4)
aq. sol. (μg/mL)
colchicine binding inhibition (%)
a
log P
tubulin assembly IC50 (μM) SD
5 μM
1 μM
6a
6b
6c
6d
6e
6f
13.4 0.98
28.6 7.35
2.74 0.11
1.34 0.10
80.9 17.8
67.8 10.3
1.45 0.12
0.46 0.01
0.58 0.00
8.07 0.47
19.1 1.58
11.1 0.30
2.94 0.15
2.77 0.40
2.56 0.10
3.91 0.03
2.60 0.27
2.26 0.13
2.33 0.00
2.48 0.17
2.14 0.01
2.70 0.01
2.14 0.01
2.63 0.00
1.60 0.04
2.80 0.3
0.63 0.09
0.42 0.01
1.90 0.04
2.90 0.3
0.59 0.01
0.97 0.1
0.48 0.02
0.62 0.05
0.58 0.01
0.77 0.07
0.54 0.06
99
1
88
3
5
4
88 0.9
53
78
96
96
1
1
83 0.8
77 0.5
87 0.7
c
47
c
4
4
6g
6i
70
90
97
2
1
2
43
6j
85 0.3
6q
6r
95 0.9
97 0.7
99 0.02
67
75
1
4
d
2
93 0.8
82
e
CA-4
98
1
2
a
b
The tubulin assembly assay measured the extent of assembly of 10 μM tubulin after 20 min at 30 °C. Inhibition of [3H]colchicine binding was
c
d
performed by incubating with tested compounds at 5 or 1 μM for 10 min at 37 °C, with tubulin at 1.0 μM. Not determined. Tubulin assay data see
ref 13. Reference compound is a drug candidate in phase II/III clinical trials.
e
Figure 4. Predicted binding mode of 2 (brown stick) and 6e (green stick) with tubulin (PDB code 5LYJ) and overlapping with CA-4 (pink, the
bound of ligand of 5LYJ). 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.
In general, most 6-series compounds showed similar potency
against A549, MCF-7, KB, and resistant KB-VIN cell lines, but
were less potent against the triple-negative breast cancer cell
line MDA-MB-231. Because antiproliferative activity was
mostly comparable against the KB and MDR KB-VIN cell
lines, the new compounds are likely not substrates of P-
glycoprotein (Table 3).
Overall, the current results demonstrated that the new
structural scaffold, 4-(quinazolin-4-yl)-3,4-dihydroquinoxalin-
2(1H)-one, is very important for high anticancer activity,
while a suitable 2-substituent on the quinazoline ring can
enhance or maintain pharmacological activity. The new SAR
correlations revealed that (1) linear alkyamino substituents are
more favorable than alkoxy and arylamino groups, which is a
consistent finding with our prior results,12 (2) H-bond donor
groups (i.e., NH or OH) favor high antiproliferative potency,
and (3) the length and bulky volume of the 2-substituent
should be limited.
Assessments of Physicochemical Properties. Because
potential drug candidates should have a good balance between
potency and druglike properties, molecular physicochemical
properties of 11 (6a−6g, 6i, 6j, 6q, and 6r) of our highly potent
compounds (GI50 ≤ 10 nM) were further assessed in parallel
with 2. Aqueous solubility and log P values were measured at
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pH 7.4 according to methods described previously.22 As shown
in Table 4, six compounds (6a, 6b, 6e, 6f, 6q, and 6r) showed
moderate aqueous solubility ranging from 8 to 87 μg/mL,
which were similar to or improved compared with that of 2 (11
μg/mL). Among them, 6b, 6e, 6f, and 6r have at least one H-
bond donor group (NH or OH) in the quinazoline 2-
substituent, thus indicating that H-bond donor groups favor
increased aqueous solubility. On the other hand, lipophilicity is
another major determinant of many ADME/Tox properties, as
well as of pharmacological activity. Log P values were also
measured at pH 7.4 to estimate molecular lipophilicity. Except
for 6d, all compounds in Table 4 showed lower log P values
than those of 1a and 1b (4.13 and 3.65, respectively) and fell
into a desireable druglike range (1 < log P7.4 < 3), which might
lead to increased intestinal absorption. Thus, compound 2 and
its new derivatives reach an acceptable balance between
solubility and passive diffusion permeability, as well as increased
metabolic stability.23 Therefore, our current results indicate that
the new structural scaffold in 2 and the series-6 compounds
makes a major contribution to reduced molecular lipophilicity,
while the changeable quinazoline 2-substituents can enhance
anticancer activity and improve aqueous solubility.
Figure 5. Effects of compounds on cell cycle. A549 cells were treated
for 24 h with 6d or 6e at 15 nM. CA-4 (20 nM) or DMSO was used as
a tubulin polymerization inhibitor or control, respectively. Fixed and
propidium iodide (PI)-stained cells were analyzed by flow cytometry.
Inhibition of Tubulin Polymerization and Colchicine
Binding. To measure effects of the new compounds on
microtubule polymerization, the same 11 highly active
compounds (6a-g, 6i−j, and 6q−r; GI50 ≤ 10 nM) were
further tested in tubulin assembly and colchicine binding
assays24 in parallel with CA-4, a clinical trial drug candidate and
highly potent competitive inhibitor of the binding of colchicine
to tubulin.25 Consistent with our previous results, all of the
compounds were highly active in the tubulin assembly assay
with low to submicromolar inhibitory IC50 values (0.42−2.90
μM), comparable with those of 2 (0.77 μM) and CA-4 (0.54
μM) in the same assay. However, an obvious difference was
seen in the competitive colchicine−tubulin binding assay. With
more than 97% and 83% inhibition at 5 and 1 μM, respectively,
compounds 6a, 6d, and 6j showed similar potency to CA-4.
The eight remaining series-6 compounds were less potent than
CA-4. Moreover, compound 2 was more potent (99% at 5 μM
and 93% at 1 μM) than CA-4 in the colchicine binding
inhibition assay. These results demonstrated that active series-6
compounds, with the same scaffold as 2, also inhibit tubulin
polymerization by targeting the tubulin−colchicine binding
site; however, different 2-substituents on the quinazoline affect
the molecular affinity with tubulin. Next, compounds 6e and 2
were docked at the colchicine binding site with the tubulin
crystal structure (PDB code 5LYJ). As shown in Figure 4,
molecules 6e and 2 superimposed well with the original ligand
CA-4 in the binding site, while the outspread C2-side chain in
6e formed two hydrogen bonds with a.a. Tyr202 and Asp251.
These findings support the postulate that, similar to CA-4, this
compound series targets the tubulin−colchicine binding site.
As representatives, compounds 6d and 6e were further
investigated for their effects on the cell cycle (Figure 5).
Similarly to CA-4, both compounds induced cell cycle arrest at
the G2/M phase. Therefore, this series of 4-(quinazolin-4-yl)-
3,4-dihydroquinoxalin-2(1H)-one derivatives, including 2 and
active series-6, have been identified to have characteristics
similar to those of CA-4 and, like CA-4, have potential as new
antitumor agents.
series-6 derivatives not only exhibit high antiproliferative
activity but also inhibit tubulin polymerization and competitive
colchicine binding to tubulin (Table 4), as well as arrest cells in
the G2/M phase of the cell cycle, indicating that this
compound type has characteristics similar to those of CA-4.
Thus, we postulated that they might act as new tubulin-binding
tumor-VDAs. The antivascular activity of 2 was evaluated on a
xenograft tumor tissue section by staining with hematoxylin−
eosin (HE) and by immunohistochemistry (IHC) using
antibodies to CD31, cleaved caspase-3, and Ki67 (Figure 6).
Figure 6. Compound 2 treatment resulted in apoptosis and vascular
disruption of NCI-H460 xenograft tumors. Representative images of
HE staining and IHC staining of CD31 (endothelial marker), cleaved
caspase-3 (apoptosis marker), and Ki67 (proliferation marker) in
different treatment groups. Sections were counterstained with
hematoxylin. Red arrows in CD31 staining images indicate micro-
vessels. Scale bar, 100 μm.
Testing of Compound 2 and New Derivatives (6a−t)
for Antivascular Effects in Vivo. Our prior and current
studies have revealed that compound 213 and its analogous
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mm × 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, ACN/H2O; condition 2,
MeOH/H2O). Target compounds were found to be at least 95% pure
in the above two HPLC analytical conditions.
HE staining showed that the nuclei of tumor cells in vehicle
controls were large and hyperchromatic, while the nuclei of 2-
treated tumor cells became pyknotic. IHC staining of CD31
(endothelial marker) further demonstrated that the numbers
and, especially, the sizes of the vessels in the 2-treated groups
were reduced (Figure 6). In addition, 2-treated tumors had
stronger cleaved caspase-3 (apoptosis marker) staining and
weaker Ki67 (proliferation marker) staining, compared to
control xenograft tumors. All of these results confirmed that 2
can inhibit proliferation, induce apoptosis, and disrupt tumor
vasculature. Therefore, our current study results validate that,
like CA-4, compound 2 is a new tubulin-binding tumor-VDA.
Most likely, the 6-series compounds, which are analogues of 2,
should belong to the same class of new antitubulin agents.
2-Chloro-4-(4-methoxy-2-nitro-phenyl)aminoquinazoline (4). A
mixture of 2,4-dichloro-quinazoline (1 g, 5.1 mmol) and 4-methoxy-2-
nitroaniline (3, 0.86 g, 5.1 mmol) in anhydrous i-PrOH (50 mL) with
a catalytic amount of HCl (conc, 10 drops) was stirred at room
temperature (rt) for 5−6 h and monitored by TLC until the reaction
was complete. The light yellow solid was removed by filtration and
washed with a small amount of i-PrOH. Next, the collected solid was
stirred in a saturated aqueous NaHCO3 solution, refiltered, washed
with water until pH = 7, and dried to obtain 4 as an orange solid (1.42
g, 85% yield). Mp 214−215 °C; 1H NMR δ 3.93 (3H, s, OCH3), 7.44
(1H, dd, J = 9.0 and 2.8 Hz, ArH-5′), 7.57 (1H, d, J = 2.8 Hz, ArH-3′),
7.59 (1H, d, J = 9.0, ArH-6′), 7.65 (1H, t, J = 7.6 Hz, ArH-6), 7.70
(1H, d, J = 7.6 Hz, ArH-5), 7.92 (1H, t, J = 7.6 Hz, ArH-7), 8.46 (1H,
d, J = 7.6 Hz, ArH-8), 10.61 (1H, s, NH); MS m/z (%) 331 (M + 1,
100), 333 (M + 3, 33).
CONCLUSION
■
In this study, we have identified 7-methoxy-4-(2-methylquina-
zolin-4-yl)-3,4-dihydroquinoxalin-2(1H)-one (2) as a new
tubulin-binding tumor-VDA. Compound 2 showed significant
dose-dependent antitumor efficacy in vivo in nude mouse H460
subcutaneous xenograft models, suppressing tumor growth by
61.9% at a dose of 1 mg/kg by iv injection every 5 days for 3
weeks without obvious signs of toxicity. It also displayed high
antitumor potency in the NIH-NCI60 panel in vitro with low
nanomolar GI50 values and reasonable druglike properties, such
as an acceptable balance between aqueous solubility and
lipophilicity, and moderate metabolic stability in vitro and in
vivo. Therefore, compound 2 should be a promising antitumor
drug candidate as a new tumor-VDA and merits further
development for clinical treatment of various tumors and
cancers. With the same scaffold, a new compounds series (6)
also exhibited high antiproliferative potency (GI50 < 10 nM)
and inhibited tubulin polymerization comparably to paclitaxel
and CA-4 in corresponding assays. Thus, these compounds
most likely also act as a new class of tubulin-binding tumor-
VDAs. New SAR results indicated that (1) the 2-substituent on
the quinazoline ring can be modified to enhance antitumor
activity and improve druglike properties, (2) the presence of a
H-bond donor in the 2-substituent is beneficial to antitumor
activity and aqueous solubility, and (3) the length and bulk of
2-substituents should be restricted.
4-(2-Chloroacetylamino-4-methoxyphenyl)amino-2-chloroqui-
nazoline (5). A mixture of 4 (330 mg, 1 mmol) and zinc powder (660
mg, 1 mmol) in 50 mL of CH2Cl2 in the presence of 0.4 mL of HOAc
was stirred at 0 °C for 0.5 h. After the solid was removed, the filtrate
was concentrated to obtain the amine product. It was immediately
dissolved in acetone (25 mL), anhydrous K2CO3 (138 mg, 1 mmol)
was added, and the mixture was cooled to 0 °C. Chloroacetyl chloride
(0.1 mL, excess) was dropped slowly into the mixture, which was kept
at 0 °C with stirring for another 1−2 h. Then the mixture was poured
into ice−water. The precipitated solid was removed by filtration,
washed successively with water to neutral and brine, and dried to
1
obtain 5 as a pink solid (210 mg, 56% yield). Mp 199−200 °C; H
NMR δ 3.78 (3H, s, OCH3), 4.23 (2H, s, CH2), 6.83 (1H, dd, J = 8.4
and 2.8 Hz, PhH-5′), 7.34 (1H, d, J = 2.8 Hz, PhH-3′), 7.48 (1H, d, J
= 8.4 Hz, PhH-6′), 7.59 (1H, t, J = 7.6 Hz, ArH-6), 7.67 (1H, d, J = 7.6
Hz, ArH-5), 7.83 (1H, t, J = 7.6 Hz, ArH-7), 8.41 (1H, d, J = 7.6 Hz,
ArH-8), 9.57 (1H, s, NH), 10.87 (1H, br, NH); MS m/z (%) 377 (M
+ 1, 100), 379 (M + 3, 58).
4-(2-Chloroquinazolin-4-yl)-7-methoxy-3,4-dihydroquinoxalin-
2(1H)-one (6a). A mixture of 5 (380 mg, 1 mmol) and anhydrous
K2CO3 (138 mg, 1 mmol) in DMF (10 mL) was heated to 100 °C for
1 h until the reaction was complete as judged by TLC monitoring. The
mixture was poured into ice−water, and the solid product 6a was
removed by filtration, washed with water, and dried to give 333 mg in
1
a 98% yield. Yellow solid; mp 250−251 °C; H NMR δ 3.74 (3H, s,
OCH3), 4.01 (2H, s, CH2), 6.42 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6′),
6.65 (1H, d, J = 2.8 Hz, ArH-8′), 6.84 (1H, d, J = 8.8 Hz, ArH-5′),
7.35 (1H, t, J = 8.4 Hz, ArH-6), 7.41 (1H, d, J = 8.4 Hz, ArH-5), 7.77
(1H, t, J = 8.4 Hz, ArH-7), 7.78 (1H, d, J = 8.4 Hz, ArH-8), 10.87 (1H,
s, NH); 13C NMR (DMSO-d6) δ 51.7, 55.9, 102.6, 108.2, 115.6, 122.2,
122.8, 126.3, 126.6, 128.0, 132.7, 134.7, 153.4, 155.8, 157.8, 161.2,
167.8; MS m/z (%) 341 (M + 1, 100), 343 (M + 3, 34); HRMS m/z
calcd for C17H14ClN4O2 [M + H]+ 341.0805, found 341.0867,
343.0843; HPLC purity 98.2%.
EXPERIMENTAL SECTION
■
Chemistry. The proton and carbon nuclear magnetic resonance
(1H NMR and 13C NMR) spectra were measured on a JNM-ECA-400
(400 MHz) spectrometer using tetramethylsilane (TMS) as internal
standard and DMSO as solvent unless otherwise indicated. Mass
spectra (MS) and high resolution mass spectra (HRMS) were
measured on an API-150 mass spectrometer (ABI, Inc.) and Waters
Xevo G2 with an electrospray ionization source, respectively. Melting
points were measured with a SGW X-4 Micro-Melting point detector
without correction. The MW reactions were performed on a MW
reactor from Biotage, Inc. Medium-pressure column chromatography
was performed using a CombiFlash Companion system from ISCO,
Inc. Thin-layer chromatography (TLC) and preparative TLC were
performed on silica gel GF254 plates. Silica gel GF254 was from Qingdao
Haiyang Chemical Company. All commercial chemical reagents were
purchased from Beijing Chemical Works or InnoChem, Inc. Pooled
human liver microsomes (lot no. 20150323H) were purchased from
iPhase Bioscience (Beijing) Ltd. Reagents. NADPH, MgCl2, KH2PO4,
K2HPO4, reference compounds, and propranolol, as well as HPLC
grade acetonitrile (ACN) and methanol (MeOH), were purchased
from Sigma-Aldrich. Purities of target compounds were determined by
HPLC using the following instruments and conditions: Agilent HPLC-
1200 with UV detector and an Agilent Eclipse XDB-C18 column (150
7-Methoxy-4-(2-(methylamino)quinazolin-4-yl)-3,4-dihydroqui-
noxalin-2(1H)-one (6b). A mixture of 6a (100 mg, 0.29 mmol) and
methylamine (1 mL) was heated via MW to 80 °C for 20 min. The
mixture was poured into ice−water, and the pH was adjusted to 7 with
dilute aq. HCl. The solid was collected, washed with water, and dried
to give pure 6b as a yellow solid (59 mg, 61% yield). Mp 274−275 °C;
1H NMR δ 2.85 (3H, d, J = 4.8 Hz, NCH3), 3.67 (3H, s, OCH3), 4.30
(2H, s, CH2), 6.38 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6), 6.52 (1H, d, J
= 8.8 Hz, ArH-5), 6.61 (1H, d, J = 2.8 Hz, ArH-8), 6.85 (1H, t, J = 8.0
Hz, ArH-6′), 7.05 (1H, q, J = 4.8 Hz, NH-2′), 7.18 (1H, d, J = 8.0 Hz,
ArH-5′), 7.39 (1H, d, J = 8.0 Hz, ArH-8′), 7.47 (1H, t, J = 8.0 Hz,
ArH-7′), 10.73 (1H, s, NH); 13C NMR (DMSO-d6) δ 28.5, 51.5, 55.8,
102.6, 108.0, 120.8, 124.9, 126.2(x2), 131.7, 133.4 (x2), 154.5, 156.4
(x2), 159.9, 160.5, 168.4; MS m/z (%) 336 (M + 1, 100); HRMS m/z
calcd for C18H18N5O2 [M + H]+ 336.1460, found 336.1501; HPLC
purity 97.5%.
H
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General Procedure for Preparing Compounds 6c−6n under MW
Irradiation. A mixture of 6a and an amine (excess) in anhydrous i-
PrOH was heated under MW irradiation for 15−30 min. The reaction
was monitored by TLC or LC-MS until complete. The mixture was
poured into ice−water and was neutralized with dilute HCl (ca.10%)
to pH 7. Precipitated solid was collected, washed with water, and dried
to obtain the corresponding products.
4-(2-(Ethylamino)quinazolin-4-yl)-7-methoxy-3,4-dihydroqui-
noxalin-2(1H)-one (6c). A mixture of 6a (50 mg, 0.14 mmol) and
ethylamine (65−75%, 1 mL, excess) in anhydrous i-PrOH (1 mL) was
heated to 80 °C under MW irradiation for 30 min to obtain pure 6c as
a yellow solid (25 mg, 48% yield). Mp 277−278 °C; 1H NMR δ 1.13
(3H, t, J = 7.2 Hz, CH3), 3.34 (2H, q, J = 7.2 Hz, NCH2), 3.67 (3H, s,
OCH3), 4.31 (2H, s, CH2), 6.37 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6),
6.51 (1H, d, J = 8.8 Hz, ArH-5), 6.61 (1H, d, J = 2.8 Hz, ArH-8), 6.84
(1H, t, J = 8.0 Hz, ArH-6′), 7.07 (1H, bs, NH), 7.16 (1H, d, J = 8.0
Hz, ArH-5′), 7.36 (1H, d, J = 8.0, ArH-8′), 7.46 (1H, t, J = 8.0 Hz,
ArH-7′); 13C NMR (DMSO-d6) δ 15.3, 36.0, 51.5, 55.8, 102.6, 108.0,
112.7, 120.7, 120.8, 124.8, 126.2(x2), 131.7, 133.4, 154.4, 156.4, 159.2,
160.6, 168.4; MS m/z (%) 350 (M + 1, 100); HRMS m/z calcd for
C19H20N5O2 [M + H]+ 350.1617, found 350.1626; HPLC purity
96.2%.
4-(2-(n-Butylamino)quinazolin-4-yl)-7-methoxy-3,4-dihydroqui-
noxalin-2(1H)-one (6d). A mixture of 6a (60 mg, 0.18 mmol) and n-
butylamine (1 mL, excess) in anhydrous i-PrOH (1 mL) was heated to
120 °C under MW irradiation for 20 min. The mixture was poured
into ice−water and was neutralized with dilute HCl (ca.10 mL) to pH
7. The mixture was extracted with EtOAc three times. The combined
organic phases were washed with water and brine successively. After
solvent was removed under reduced pressure, residue was washed with
a small amount of cold acetone to give pure 6d as a yellow solid (20
mg, 29% yield). Mp 204−205 °C; 1H NMR δ 0.87 (3H, t, J = 7.6 Hz,
CH3), 1.32 and 1.52 (each 2H, m, J = 7.6 Hz, CH2), 3.31 (2H, m,
NCH2), 3.67 (3H, s, OCH3), 4.31 (2H, s, CH2), 6.38 (1H, dd, J = 8.8
and 2.8 Hz, ArH-6), 6.51 (1H, d, J = 8.8 Hz, ArH-5), 6.61 (1H, d, J =
2.8 Hz, ArH-8), 6.83 (1H, t, J = 8.0 Hz, ArH-6′), 7.08 (1H, m, NH),
7.16 (1H, d, J = 8.0 Hz, ArH-5′), 7.36 (1H, d, J = 8.0 Hz, ArH-8′),
7.46 (1H, t, J = 8.0 Hz, ArH-7′), 10.73 (1H, s, NH); MS m/z (%) 378
(M + 1, 100); HRMS m/z calcd for C21H24N5O2 [M + H]+ 378.1930,
found 378.2017; HPLC purity 97.8%.
4-(2-(3-Hydroxypropylamino)quinazolin-4-yl)-7-methoxy-3,4-di-
hydroquinoxalin-2(1H)-one (6e). A mixture of 6a (100 mg, 0.29
mmol) and 3-aminopropanol (1 mL, excess) in anhydrous i-PrOH
(0.5 mL) with one drop of H2SO4 (conc) was heated to 120 °C under
MW irradiation for 20 min to obtain 82 mg of 6e (75% yield, white
solid), mp 179−180 °C; 1H NMR δ 1.69 (2H, m, CH2), 3.38 and 3.45
(each 2H, m, OCH2 and NCH2), 3.67 (3H, s, OCH3), 4.31 (2H, s,
CH2), 4.59 (1H, br, OH), 6.38 (1H, dd, J = 9.2 and 2.8 Hz, ArH-6),
6.53 (1H, d, J = 9.2 Hz, ArH-5), 6.60 (1H, d, J = 2.8 Hz, ArH-8), 6.85
(1H, t, J = 8.0 Hz, ArH-6′), 7.06 (1H, t, J = 5.6 Hz, NH), 7.17 (1H, d,
J = 8.0 Hz, ArH-5′), 7.35 (1H, d, J = 8.0 Hz, ArH-8′), 7.47 (1H, t, J =
8.0 Hz, ArH-7′); MS m/z (%) 380 (M + 1, 100); HRMS m/z calcd for
C20H22N5O3 [M + H]+ 380.1723, found 380.1730; HPLC purity
99.1%.
°C under MW irradiation for 30 min to give pure 6g as a yellow solid
(70 mg, 62% yield). Mp 268−269 °C; 1H NMR δ 1.51 (4H, m, CH2 ×
2), 1.66 (2H, m, CH2), 1.90 (2H, m, CH2), 3.67 (3H, s, OCH3), 4.27
(1H, m, NCH), 4.31 (2H, s, CH2), 6.36 (1H, dd, J = 8.8 and 2.8 Hz,
ArH-6), 6.51 (1H, d, J = 8.8 Hz, ArH-5), 6.61 (1H, d, J = 2.8 Hz, ArH-
8), 6.84 (1H, t, J = 8.0 Hz, ArH-6′), 7.07 (1H, br, NH), 7.16 (1H, d, J
= 8.0 Hz, ArH-5′), 7.31 (1H, d, J = 8.0 Hz, ArH-8′), 7.48 (1H, t, J =
8.0 Hz, ArH-7′), 10.72 (1H, br, NH); 13C NMR (DMSO-d6) δ
24.0(x2), 32.9(x2), 51.5, 52.7, 55.8, 102.6, 108.0, 112.5, 120.7, 120.8,
124.9, 126.1(x2), 131.7, 133.4, 154.4, 156.4, 158.9, 160.5, 168.4; MS
m/z (%) 390 (M + 1, 100); HRMS m/z calcd for C22H24N5O2 [M +
H]+ 390.1930, found 390.1984; HPLC purity 99.1%.
7-Methoxy-4-(2-(pyrrolidin-1-yl)quinazolin-4-yl)-3,4-dihydroqui-
noxalin-2(1H)-one (6h). A mixture of 6a (50 mg, 0.15 mmol), 4-
picolylamine (1 mL, excess), and K2CO3 (30 mg, 0.21 mmol) in i-
PrOH (2 mL) was heated to 60 °C under MW irradiation for 15 min
to obtain 6h as a white solid (42 mg, 58% yield). Mp 217−218 °C; 1H
NMR δ 1.90 (4H, m, CH2 × 2), 3.55 (4H, m, NCH2 × 2), 3.67 (3H, s,
OCH3), 4.36 (2H, s, CH2), 6.38 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6),
6.51 (1H, d, J = 2.8 Hz, ArH-8), 6.62 (1H, d, J = 8.8 Hz, ArH-5), 7.17
(1H, t, J = 8.0 Hz, ArH-6′), 7.19 (1H, d, J = 8.0 Hz, ArH-5′), 7.38
(1H, d, J = 8.0 Hz, ArH-8′), 7.47 (1H, t, J = 8.0 Hz, ArH-7′), 10.72
(1H, s, NH); 13C NMR (DMSO-d6) δ 25.6(x2), 46.9(x2), 51.4, 55.9,
102.6, 107.9, 111.8, 120.7(x2), 124.8, 126.2, 126.3, 131.7, 133.4, 154.7,
154.4, 157.4, 160.2, 168.5; MS m/z (%) 376 (M + 1, 100); HRMS m/
z calcd for C21H22N5O2 [M + H]+ 376.1773, found 376.1784; HPLC
purity 95.4% (MeOH/H2O).
4-(2-(3,3-Difluoropyrrolidin-1-yl)quinazolin-4-yl)-7-methoxy-3,4-
dihydroquinoxalin-2 (1H)-one (6i). A mixture of 6a (50 mg, 0.15
mmol) and 3,3-difluoropyrrolidine hydrochloride (30 mg, 0.29 mmol)
and K2CO3 (30 mg, 0.21 mmol) in i-PrOH (2 mL) was heated to 120
°C under MW irradiation for 20 min to obtain 6i as a brown solid (52
mg, 85% yield). Mp 247−248 °C; 1H NMR δ 2.51 (2H, m, CH2), 3.68
(3H, s, OCH3), 3.79 (2H, t, J = 7.2 Hz, NCH2), 3.98 (2H, t, JH−F
=
12.8 Hz, NCH2CF2), 4.41 (2H, s, 3′-CH2), 6.38 (1H, dd, J = 8.8 and
2.8 Hz, ArH-6), 6.56 (1H, d, J = 8.8 Hz, ArH-5), 6.63 (1H, d, J = 2.8
Hz, ArH-8), 6.92 (1H, t, J = 8.0 Hz, ArH-6′), 7.23 (1H, d, J = 8.0 Hz,
ArH-5′), 7.45 (1H, d, J = 8.0 Hz, ArH-8′), 7.53 (1H, t, J = 8.0 Hz,
ArH-7′), 10.87 (1H, s, NH); 13C NMR (DMSO-d6) δ 33.8, 44.5, 51.4,
53.6, 55.8, 102.7, 108.0, 112.2, 120.9, 121.6, 124.4, 126.2, 126.5, 128.7,
132.0, 133.7, 154.2, 156.6, 157.1, 160.4, 168.4; MS m/z (%) 412 (M +
1, 100); HRMS m/z calcd for C21H20 F2N5O2 [M + H]+ 412.1585,
found 412.1664; HPLC purity 95.6%.
4-(2-(3,3-Difluoroazetidin-1-yl)quinazolin-4-yl)-7-methoxy-3,4-
dihydroquinoxalin-2(1H) -one (6j). A mixture of 6a (100 mg, 0.29
mmol) and 3,3-difluoroazetidine hydrochloride (45 mg, 1.1 mmol) in
i-PrOH (2 mL) in the presence of one drop H2SO4 (conc) was heated
to 120 °C under MW irradiation for 20 min. The crude product was
dissolved in MeOH and left uncovered overnight. The precipitated
solid was collected to obtain pure 6j as a yellow solid (40 mg, 35%
yield). Mp 280−281 °C; 1H NMR δ 3.68 (3H, s, OCH3), 4.41(2H, s,
CH2), 4.51 (4H, t, JH−F = 12.8 Hz, NCH2 × 2), 6.39 (1H, dd, J = 8.8
and 2.8 Hz, ArH-6), 6.61 (1H, d, J = 8.8 Hz, ArH-5), 6.63 (1H, d, J =
2.8 Hz, ArH-8), 6.99 (1H, t, J = 8.0 Hz, ArH-6′), 7.25 (1H, d, J = 8.0
Hz, ArH-5′), 7.51 (1H, d, J = 8.0 Hz, ArH-8′), 7.58 (1H, t, J = 8.0 Hz,
ArH-7′), 10.78 (1H, s, NH); MS m/z (%) 398 (M + 1, 100); HRMS
m/z calcd for C20H18 F2N5O2 [M + H]+ 398.1429, found 398.1441 (M
+ 1, 100); HPLC purity 97.8%.
4-(2-(Cyclopropylamino)quinazolin-4-yl)-7-methoxy-3,4-dihydro-
quinoxalin-2(1H)-one (6f). A mixture of 6a (100 mg, 0.29 mmol) and
cyclopropylamine (1 mL, excess) in DMA (2 mL) was heated to 155
°C under MW irradiation for 20 min to give pure 6f as a yellow solid
(68 mg, 65% yield). Mp 269−270 °C; 1H NMR δ 0.49 and 0.65 (each
2H, m, CH2), 2.81 (1H, m, CH), 3.67 (3H, s, OCH3), 4.32 (2H, s,
CH2), 6.38 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6), 6.54 (1H, d, J = 8.8
Hz, ArH-5), 6.61 (1H, d, J = 2.8 Hz, ArH-8), 6.87 (1H, t, J = 8.0 Hz,
ArH-6′), 7.15 (1H, d, J = 8.0 Hz, ArH-5′), 7.29 (1H, m, NH), 7.41
(1H, d, J = 8.0 Hz, ArH-8′), 7.48 (1H, t, J = 8.0 Hz, ArH-7′), 10.72
(1H, s, NH); MS m/z (%) 362 (M + 1, 100); HRMS m/z calcd for
C20H20N5O2 [M + H]+ 362.1617, found 362.1658; HPLC purity
95.0%.
7-Methoxy-4-(2-(3-methoxyazetidin-1-yl)quinazolin-4-yl)-3,4-di-
hydroquinoxalin-2(1H)-one (6k). A mixture of 6a (50 mg, 0.15
mmol), methoxyazetidine (50 mg, 0.41 mmol), and K2CO3 (50 mg,
0.36 mmol) in anhydrous i-PrOH (2 mL) was heated to 120 °C under
MW irradiation for 15 min to obtain pure 6k as a gray solid (23 mg,
1
39% yield) after recrystallization from MeOH. Mp 221−222 °C; H
NMR δ 3.23 (3H, s, OCH3), 3.68 (3H, s, OCH3), 3.88 (2H, d, J = 7.2
Hz, NCH2), 4.25 (2H, d, J = 7.2 Hz, NCH2), 4.28 (1H, m, CH), 4.36
(2H, s, CH2), 6.38 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6), 6.54 (1H, d, J
= 8.8 Hz, ArH-5), 6.62 (1H, d, J = 2.8 Hz, ArH-8), 6.90 (1H, t, J = 8.0
Hz, ArH-6′), 7.20 (1H, d, J = 8.0 Hz, ArH-5′), 7.42 (1H, d, J = 8.0 Hz,
ArH-8′), 7.51 (1H, t, J = 8.0 Hz, ArH-7′), 10.72 (1H, s, NH); 13C
4-(2-(Cyclopentylamino)quinazolin-4-yl)-7-methoxy-3,4-dihydro-
quinoxalin-2(1H)-one (6g). A mixture of 6a (100 mg, 0.29 mmol) and
cyclopentylamine (1 mL, excess) in i-PrOH (1 mL) was heated to 120
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NMR (DMSO-d6) δ 51.6, 55.8, 55.9, 57.4, 69.8(x2), 102.6, 108.0,
112.5, 120.9, 121.5, 126.2, 127.4, 131.8, 133.6(x2), 154.1, 156.6, 160.0,
160.4, 168.4; MS m/z (%) 392 (M + 1, 100); HRMS m/z calcd for
C21H22N5O2 [M + H]+ 392.1723, found 392.1729; HPLC purity
96.0%.
(260 mg, 11 mmol), and 6a (100 mg, 0.29 mmol) was stirred at 0 °C
for about 1.5 h and then heated to 95 °C under MW irradiation for 5
min to obtain pure 6p as a yellow solid (90 mg, 83% yield). Mp 215−
216 °C; 1H NMR δ 0.34 (2H, m, CH2), 0.55 (2H, m, CH2), 0.81 (1H,
m, CH), 3.71 (3H, s, OCH3), 4.17 (2H, d, J = 7.2 Hz, OCH2), 4.41
(2H, s, CH2), 6.39 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6), 6.64 (2H, m,
ArH-5 and -8), 7.13 (1H, m, ArH-6′), 7.35 (1H, d, J = 8.0 Hz, ArH-
5′), 7.63 (2H, m, ArH-8′ and -7′), 10.77 (1H, br, NH); 13C NMR
(DMSO-d6) δ 3.74(x2), 10.5, 51.6, 55.8, 71.8, 102.6, 108.1, 114.2,
121.4, 123.7, 123.9, 126.2, 127.3, 132.2, 134.0, 153.2, 157.0, 161.9,
162.2, 168.2; MS m/z (%) 377 (M + 1, 100); HRMS m/z calcd for
C21H21N4O3 [M + H]+ 377.1614, found 377.1615; HPLC purity
95.1%.
4-(2-Acetoxymethylquinazolin-4-yl)-7-methoxy-3,4-dihydroqui-
noxalin-2(1H)-one (6q). A mixture of 13 prepared as described below
(390 mg, 0.94 mmol) and anhydrous K2CO3 (260 mg, 1.88 mmol) in
DMA (3 mL) was heated at 100 °C for 1 h. After the reaction was
complete as judged by LC-MS monitoring, the mixture was poured
into ice water and extracted with EtOAc three times. The combined
organic phases were washed successively with water and brine and
dried over anhydrous Na2SO4. After removal of solvent in vacuo, the
residue was purified by flash column chromatography (gradient
elution, petroleum ether/EtOAc, 0−30%) to afford 6q as a yellow
solid (355 mg, 99% yield). Mp 192−194 °C; 1H NMR (CDCl3) δ 2.26
(3H, s, CH3), 3.80 (3H, s, OCH3), 4.66 (2H, s, NCH2), 5.35 (2H, s,
OCH2), 6.40 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6), 6.54 (1H, d, J = 2.8
Hz, ArH-8), 6.63 (1H, d, J = 8.8 Hz, ArH-5),7.25 (1H, t, J = 8.0 Hz,
ArH-6′), 7.48 (1H, d, J = 8.0 Hz, ArH-5′), 7.72 (1H, t, J = 8.0 Hz,
ArH-7′), 7.92 (1H, d, J = 8.0 Hz, ArH-8′), 8.37 (1H, s, NH); 13C
NMR (400 Hz, CDCl3) δ 21.0, 51.1, 55.7, 66.4, 102.4, 108.6, 115.7,
120.8, 123.9, 125.6, 125.8, 128.8, 130.4, 133.2, 152.1, 157.2, 159.5,
160.4, 169.3, 170.9; MS m/z (%) 379 (M + 1, 100); HRMS m/z calcd
for C20H19N4O4 [M + H]+ 379.1406, found 379.1431; HPLC purity
95.5%.
4-(2-(4-Cyanophenyl)aminoquinazolin-4-yl)-7-methoxy-3,4-dihy-
droquinoxalin-2(1H)-one (6l). A mixture of 6a (50 mg, 0.15 mmol)
and 4-aminobenzonitrile (30 mg, 0.25 mmol) in i-PrOH (2 mL) in the
presence of H2SO4 (conc, 1 drop) was heated to 120 °C under MW
irradiation for 20 min to obtain pure 6l as a yellow solid (50 mg, 79%
1
yield) after being recrystallized from MeOH. Mp 183−184 °C; H
NMR δ 3.71 (3H, s, OCH3), 4.57 (2H, s, CH2), 6.47 (1H, dd, J = 8.8
and 2.8 Hz, ArH-6), 6.66 (1H, d, J = 8.8 Hz, ArH-5), 6.89 (1H, d, J =
2.8 Hz, ArH-8), 7.16 (1H, t, J = 8.0 Hz, ArH-6′), 7.37 (2H, d, J = 8.2
Hz, 2′-PhH), 7.65 (1H, d, J = 8.0 Hz, ArH-5′), 7.70 (1H, t, J = 8.0 Hz,
ArH-7′), 7.75 (2H, d, J = 8.2 Hz, 2′-PhH), 7.95 (1H, d, J = 8.0 Hz,
ArH-8′), 10.58 (1H, brs, NH), 10.87 (1H, s, NH); MS m/z (%) 423
(M + 1, 100); HRMS m/z calcd for C24H19N6O2 [M + H]+ 423.1569,
found 423.1649; HPLC purity 95.3%.
4-(2-(3-Cyanophenyl)aminoquinazolin-4-yl)-7-methoxy-3,4-dihy-
droquinoxalin-2(1H)-one (6m). A mixture of 6a (50 mg, 0.15 mmol)
and 3-aminobenzonitrile (30 mg, 0.25 mmol) in i-PrOH (2 mL) in the
presence of H2SO4 (conc,1 drop) was heated to 120 °C under MW
irradiation for 20 min to obtain 6m as a yellow solid (55 mg, 88%
yield). Mp 262−263 °C; 1H NMR δ 3.71 (3H, s, OCH3), 4.57 (2H, s,
CH2), 6.50 (1H, dd, J = 8.8 and 2.8 Hz, ArH-6), 6.65 (1H, d, J = 2.8
Hz, ArH-8), 7.00 (1H, d, J = 8.8 Hz, ArH-5), 7.22 (1H, m, ArH-6′),
7.41 (1H, s, ArH-3″), 7.56 (2H, m, ArH-5′ and ArH-4″), 7.65 (1H, d,
J = 7.6 Hz ArH-8′), 7.76 (1H, t, J = 7.6 Hz ArH-7′), 7.92 (1H, m,
ArH-5″), 8.13 (1H, m, ArH-6″), 10.44 (1H, br, NH), 10.84 (1H, s, 1-
NH); 13C NMR (DMSO-d6) δ 52.2, 56.0, 102.6, 108.6, 112.3, 119.1,
121.3, 124.1, 124.3, 124.5, 125.2, 127.0, 127.1, 127.3, 128.6, 130.9,
133.3, 135.9, 139.0, 151.9, 152.1, 158.8, 161.6, 167.1; MS m/z (%) 423
(M + 1, 100); HRMS m/z calcd for C24H19N6O2 [M + H]+ 423.1569,
found 423.1571; HPLC purity 96.6%.
4-(2-(Hydroxymethyl)quinazolin-4-yl)-7-methoxy-3,4-dihydro-
quinoxalin-2(1H)-one (6r). To a solution of 6q (355 mg, 0.94 mmol)
in MeOH (8 mL) was added aqueous NaOH (10%, ca.1.0 mL, excess)
at rt. The mixture was stirred for 1.5 h. Then, the pH was adjusted to 7
with aq. HCl (10%), and the mixture was extracted with EtOAc three
times. The combined organic layer was washed successively with water
and brine and dried over anhydrous Na2SO4. After removal of solvent
in vacuo, 6r was obtained as a yellow powder (310 mg, 98% yield). Mp
7-Methoxy-4-(2-((pyridin-4-ylmethyl)amino)quinazolin-4-yl)-3,4-
dihydroquinoxalin-2(1H)-one (6n). A mixture of 6a (35 mg, 0.1
mmol), pyridin-4-ylmethanamine (1 mL, excess), and K2CO3 (30 mg,
0.21 mmol) in i-PrOH (2 mL) was heated to 120 °C under MW
irradiation for 1 h to obtain 6n as a yellow solid (20 mg, 47% yield).
1
Mp 219−220 °C; H NMR δ 3.71 (3H, s, OCH3), 4.31 (2H, s, 3-
CH2), 4.55 (2H, d, J = 6.0 Hz, CH2), 6.36 (1H, dd, J = 8.8 and 2.8 Hz,
ArH-6), 6.55 (1H, d, J = 8.8 Hz, ArH-5), 6.60 (1H, d, J = 2.8 Hz, ArH-
8), 6.67 (1H, t, J = 7.6 Hz, ArH-6′), 7.18 (1H, d, J = 7.6 Hz, ArH-5′),
7.33 (3H, m, ArH-8′ and PyH), 7.47 (1H, t, J = 7.6 Hz, ArH-7′), 7.73
(1H, m, NH), 8.43 (2H, m, PyH), 10.73 (1H, br, 1-NH); MS m/z (%)
413 (M + 1, 100); HRMS m/z calcd for C23H21N6O2 [M + H]+
413.1726, found 413.1799; HPLC purity 97.8%.
1
216−218 °C; H NMR (CDCl3) δ 3.80 (3H, s, OCH3), 4.00 (1H, s,
OH), 4.67 (1H, s, CH2), 4.85 (1H, s, OCH2), 6.41 (1H, dd, J = 8.8
and 2.8 Hz, ArH-6), 6.57 (1H, d, J = 2.8 Hz, ArH-8), 6.64 (1H, d, J =
8.8 Hz, ArH-5), 7.26 (1H, t, J = 8.0 Hz, ArH-6′),7.51 (1H, d, J = 8.0
Hz, ArH-5′), 7.73 (1H, t, J = 8.0 Hz, ArH-7′), 7.92 (1H, d, J = 8.0 Hz,
ArH-8′), 8.82 (1H, s, NH); 13C NMR (400 Hz, CDCl3) δ 51.2, 55.7,
64.4, 102.5, 108.7, 115.9, 120.8, 123.9, 125.6, 125.8, 128.3, 130.4,
133.4, 151.4, 157.2, 159.6, 163.8, 169.2; MS m/z (%) 337 (M + 1,
100); HRMS m/z calcd for C18H17N4O3 [M + H]+ 337.1301, found
337.1290; HPLC purity 96.3%.
4-(2-Ethoxyquinazolin-4-yl)-7-methoxy-3,4-dihydroquinoxalin-
2(1H)-one (6o). Ethanol (2 mL) was cooled to 0 °C, and NaH (260
mg, 11 mmol) was carefully added in portions with stirring over 1 h.
Compound 6a (100 mg, 0.29 mmol) was then added into the solution
with stirring and kept at the same temperature for another 0.5 h until
hydrogen was no longer formed. The mixture was then heated to 95
°C under MW irradiation for 5 min and monitored by TLC and LC-
MS until the reaction was complete. The mixture was poured into ice−
water, and the precipitated solid was collected, washed with water, and
dried to obtain pure 6o as a brown solid (85 mg, 85% yield). Mp 233−
4-(2-((4-Ethoxy-4-oxobutoxy)methyl)quinazolin-4-yl)-7-me-
thoxy-3,4-dihydroquinoxalin-2(1H)-one (6s). A mixture of 6r (165
mg, 0.5 mmol) and Cs2CO3 (325 mg, 1 mmol) in CH3CN (10 mL)
was heated to reflux for 0.5 h. Then, ethyl 4-bromobutyrate (325 mg
1.0 mmol) was added dropwise at the same temperature, and stirring
was continued for 3 h. The mixture was passed through Celite, which
was washed several times with CH3CN. The combined filtrate was
concentrated in vacuo to obtain 6s as a yellow oil, which was used in
the next reaction without further purification. However, pure 6s was
obtained as a yellow solid by crystallization from EtOAc. Mp 160−161
°C; 1H NMR (CDCl3) δ 1.26 (3H, t, J = 7.2 Hz, CH3), 2.01 (2H, m,
CH2), 2.45 (2H, t, J = 7.2 Hz, COCH2), 3.86 (3H, s, OCH3), 4.07
(2H, t, J = 7.2 Hz, OCH2), 4.15 (2H, q, J = 7.2, OCH2), 4.65 (2H, s,
CH2), 4.83 (2H, s, ArCH2O), 6.42 (1H, dd, J = 8.8 and 2.4 Hz, ArH-
6), 6.66 (1H, d, J = 8.8 Hz, ArH-5), 7.01 (1H, d, J = 2.4 Hz, ArH-8),
7.24 (1H, t, J = 8.0 Hz, ArH-6′), 7.46 (1H, d, J = 8.0 Hz, ArH-5′), 7.72
(1H, t, J = 8.0 Hz, ArH-7′), 7.92 (1H, d, J = 8.0 Hz, ArH-8′); 13C
NMR (400 Hz, CDCl3) δ 14.3, 22.1, 31.1, 41.7, 51.7, 55.9, 60.8, 64.5,
1
234 °C; H NMR δ 1.34 (3H, t, J = 7.2 Hz, CH3),3.68 (3H, s, 7′-
OCH3), 4.41 (4H, m, OCH2 and 3′-CH2), 6.39 (1H, dd, J = 8.8 and
2.8 Hz, ArH-6), 6.65 (2H, m, ArH-5 and -8), 7.12 (1H, m, ArH-6′),
7.35 (1H, d, J = 8.0 Hz, ArH-5′), 7.63 (2H, m, ArH-8′, ArH-7′), 10.78
(1H, br s, NH); 13C NMR (DMSO-d6) δ 15.0, 51.6, 55.9, 62.9, 102.7,
108.1, 114.2, 121.4, 123.7, 124.0, 126.2, 127.3, 132.3, 134.0, 153.5,
157.0, 161.9, 162.2, 167.9; MS m/z (%) 351 (M + 1, 100); HRMS m/
z calcd for C19H19N4O3 [M + H]+ 351.1457, found 351.1507; HPLC
purity 96.2%.
4-(2-(Cyclopropylmethoxy)quinazolin-4-yl)-7-methoxy-3,4-dihy-
droquinoxalin-2(1H)-one (6p). The preparation was the same as
described for 6o. The mixture of cyclopropylcarbinol (2 mL), NaH
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103.0, 108.0, 115.9, 121.0, 125.7 (×2), 126.0, 128.4, 133.0, 133.4,
151.5, 157.6, 159.4, 163.9, 167.1, 173.1; MS m/z (%) 451 (M + 1,
100); HRMS m/z calcd for C24H27N4O5 [M + H]+ 451.1981, found
451.2083; HPLC purity 95.7%.
solid (300 mg, 65% yield). Mp 159−160 °C; 1H NMR (CDCl3) δ 2.24
(3H, s, CH3), 3.90 (3H, s, OCH3), 5.35 (2H, s, OCH2), 7.33 (1H, dd,
J = 9.6 and 3.2 Hz, ArH-5′), 7.64 (1H, t, J = 8.4 Hz, ArH-6), 7.76 (1H,
d, J = 3.2 Hz, ArH-3′), 7.84 (1H, t, J = 8.4 Hz, ArH-7), 7.93 (1H, d, J =
8.4 Hz, ArH-5), 8.04 (1H, d, J = 8.4 Hz, ArH-8), 9.24 (1H, d, J = 9.6
Hz, ArH-6′), 11.24 (1H, s, NH); MS m/z (%) 369 (M + 1, 100).
2-Acetoxymethyl-4-(4-methoxy-2-aminophenyl)-
aminoquinazoline (12). To a solution of 11 (154 mg, 0.54 mmol) in
a mixed solvent of EtOAc and EtOH (25 mL, v/v 3:2) was added Pd/
C (15 mg, 10% w/w) and hydrogen gas. The mixture was shaken
under 40 psi at rt for 2 h. The Pd/C was removed by filtration and
washed with EtOAc several times. After removal of the solvent in
vacuo, 185 mg of light yellow solid 12 was obtained and used directly
in the next step without further purification.
2-Acetoxymethyl-4-(2-chloroacetylamino-4-methoxyphenyl)-
aminoquinazoline (13). Crude 12 (ca. 545 mg, 1.61 mmol) was
immediately dissolved in acetone (8 mL), and K2CO3 (667 mg, 4.83
mmol) was added. The mixture was cooled to 0 °C and chloroacetyl
chloride (361 mg, 3.22 mmol) was added dropwise. Stirring was
continued for an additional 1 h at 0 °C. When the reaction was
complete, the mixture was poured into ice water, the pH was adjusted
to neutral, and the product was extracted with EtOAc three times. The
combined organic phase was washed successively with water and brine,
and dried over anhydrous Na2SO4 overnight. After removal of solvent
in vacuo, 13 was obtained as a gray solid and used in the synthesis of
6q (400 mg, 60% yield over two steps). Mp 121−122 °C; MS m/z
(%) 415 (M + 1, 100), 417 (M + 3, 33).
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 phosphate buffer (1.0 mL, pH 7.4).
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 mm × 4.6 mm, 5
μm), and elution was with 50%−80% ACN in water. The flow rate was
0.8 mL/min, and injection volume was 20 μL. Aqueous concentration
was determined by comparison of the peak area of the saturated
solution with a standard curve plotted peak area versus known
concentrations, which were 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
was dissolved in 1.0−2.0 mL of n-octanol to obtain a 1.0 mg/mL
solution. Next, the same volume of water as n-octanol 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-octanol and in
water.
4-(2-((4-(Hydroxyamino)-4-oxobutoxy)methyl)quinazolin-4-yl)-7-
methoxy-3,4-dihydroquinoxalin-2(1H)-one (6t). A solution of 6s
(225 mg, 0.5 mmol) in CH2Cl2 and MeOH (9 mL, 1:2, v/v) was
cooled to 0 °C. Aqueous hydroxylamine, prepared from hydroxyl-
amine hydrochloride (1.04 g, 1.5 mmol) and NaOH (0.8 g, 2.0 mmol)
in 2 mL of water, was added dropwise into the solution of 6s with
stirring at 0 °C, and the mixture was stirred for an additional 1 h. After
organic solvent was removed under reduced pressure, the water phase
was adjusted to pH 7−8 with HOAc and extracted with EtOAc three
times. The combined organic phase was washed with water and brine
and dried over anhydrous Na2SO4. After solvent was removed in
vacuo, the residue was recrystallized from EtOAc to obtain pure 6t as a
1
yellow solid (50 mg), mp 196−198 °C; H NMR (CDCl3) δ 1.84
(2H, m, CH2), 2.04 (2H, t, J = 7.2 Hz, C(O)CH2), 3.77 (3H, s,
OCH3), 3.95 (2H, t, J = 7.2 Hz, OCH2), 4.60 (2H, s, CH2), 4.61 (2H,
s, ArCH2O), 5.20 (1H, s, OH), 6.48 (1H, dd, J = 8.8 and 2.4 Hz, ArH-
6′), 6.67 (1H, d, J = 8.8 Hz, ArH-5′), 7.02 (1H, d, J = 2.4 Hz, ArH-8′),
7.31 (1H, t, J = 8.0 Hz, ArH-6), 7.36 (1H, d, J = 8.0 Hz, ArH-5), 7.76
(1H, t, J = 8.0 Hz, ArH-7), 7.83 (1H, d, J = 8.0 Hz, ArH-8), 8.71 (1H,
s, NH), 10.38 (1H, s, NH); 13C NMR (400 Hz, DMSO-d6) δ 22.9,
29.6, 40.2, 51.7, 56.1, 65.4, 103.4, 108.4, 115.7, 121.2, 125.9, 126.1,
126.2, 128.6, 133.0, 133.8, 152.0, 157.4, 159.2, 165.3, 167.0, 169.1; MS
m/z (%) 438 (M + 1, 100); HRMS m/z calcd for C22H24N5O5 [M +
H]+ 438.1777, found 438.1866; HPLC purity 95.1%.
2-Chloromethyl-3H-quinazolin-4-one (8). Compound 8 was
prepared from commercially available methyl 2-aminobenzoate (7)
and 2-chloroacetonitrile according to the literature method.26 Mp 240
°C (dec.); 1H NMR δ 4.51 (2H, s, CH2), 7.51(1H, t, J = 8.0 Hz, ArH-
6), 7.64 (1H, d, J = 8.0 Hz, ArH-8), 7.80 (1H, t, J = 8.0 Hz, ArH-7),
8.08 (1H, d, J = 8.0 Hz, ArH-5), 12.57 (1H, s, NH); MS m/z (%) 195
(M + 1, 100), 197 (M + 3, 33).
2-(Chloromethyl)quinazolin-4-yl 4-methylbenzenesulfonate (9).
A mixture of 8 (500 mg 2.58 mmol), p-toluenesulfonyl chloride (880
mg, 4.6 mmol), Et3N (521 mg 5.16 mmol), and DMAP (3 mg, 0.026
mmol) in CH2Cl2 (20 mL) was stirred at rt for 3 h until the reaction
was complete as judged by TLC monitoring. Solvent was removed
under reduced pressure, and the residue was purified by flash column
chromatography (gradient elution, EtOAc/petroleum ether, 0−15%)
to obtain 9 as a pale yellow solid (790 mg, 88% yield). Mp 149−151
1
°C; H NMR (CDCl3) δ 2.46 (3H, s, CH3), 4.73 (2H, s, CH2), 7.38
(2H, d, J = 8.4 Hz, PhH), 7.68 (1H, t, J = 8.4 Hz, ArH-6), 7.93 (1H, t,
J = 8.4 Hz, ArH-7), 7.97 (1H, d, J = 8.4 Hz, ArH-5), 8.18 (2H, d, J =
8.4 Hz, PhH), 8.20 (1H, d, J = 8.4 Hz, ArH-8); MS m/z (%) 349 (M +
1, 100), 351 (M + 3, 33).
2-Chloromethyl-4-(4-methoxy-2-nitrophenyl)aminoquinazoline
(10). A mixture of 9 (500 mg 1.43 mmol) and 3 (240 mg 1.43 mmol)
in i-PrOH and CH2Cl2 (15 mL, v/v 2:1) was stirred at 45 °C for 6 h
until the reaction was complete as judged by TLC monitoring. After
solvent was removed, the residue was dissolved in 50 mL of CH2Cl2,
washed with saturated NaHCO3, dried over anhydrous Na2SO4, and
concentrated to yield crude product 10, which was used in the next
Cell-Based Assays. Antiproliferative activities of series-6 com-
pounds were assayed by the SRB method according to procedures
described previously.27−29 The panel of cell lines included A-549,
MDA-MB-231, KB, KB-VIN (P-gp-overexpressing KB subline), and
MCF-7. These cell lines were obtained from the Lineberger
Comprehensive Cancer Center (UNC−CH) or ATCC. Cells were
propagated in RPMI-1640 (Gibco) supplemented with 10% FBS
(Corning), penicillin (100 IU/mL), streptomycin (1 μg/mL), and
amphotericin B (0.25 μg/mL) (Corning) and were cultured at 37 °C
in a humidified atmosphere of 95% air and 5% CO2. The
antiproliferative effects against tumor cell lines were expressed as
GI50 values, which represent the molar drug concentrations required to
cause 50% tumor cell growth inhibition. Flow cytometry was
performed as described previously.30 Briefly, A549 cells were seeded
24 h prior to treatment in a 12-well culture plate at density of 60 000
cells/well. After 24 h treatment with 15 nM 6d, 15 nM 6e, or 20 nM
CA-4, cells were fixed and stained with PI containing RNase (BD
Biosciences). DMSO (0.001%) was used as a control. Stained cells
were analyzed by flow cytometry operated by FACSDiva software (BD
1
step without further purification. Orange solid; Mp 180−181 °C; H
NMR δ 3.90 (3H, s, OCH3), 4.59 (2H, s, CH2), 7.38 (1H, dd, J = 9.2
and 3.2 Hz, ArH-5′), 7.67 (1H, t, J = 8.0 Hz, ArH-6), 7.77 (1H, d, J =
3.2 Hz, ArH-3′), 7.87 (1H, t, J = 8.0 Hz, ArH-7), 7.95 (1H, d, J = 8.0
Hz, ArH-5), 8.64 (1H, d, J = 8.0 Hz, ArH-8), 9.39 (1H, d, J = 9.2 Hz,
ArH-6′), 11.31 (1H, s, NH); MS m/z (%) 345 (M + 1, 100), 347 (M
+ 3, 33).
2 - A c e t o x y me t h y l - 4 - ( 4 - m e t h o x y - 2 - n i t r o - p h e n y l ) -
aminoquinazoline (11). A mixture of 10 (500 mg, 1.45 mmol) and
KOAc (427 mg, 4.36 mmol) in DMF (2.0 mL) was stirred at 55 °C for
6 h. The mixture was poured into ice−water and extracted with
EtOAc. The organic layer was washed with water and brine
successively and dried over Na2SO4. After removal of solvent in
vacuo, the residue was purified by flash column chromatography
(gradient elution, CH2Cl2/ EtOAc, 0−15%) to give 11 as an orange
K
DOI: 10.1021/acs.jmedchem.7b00273
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Journal of Medicinal Chemistry
Article
LSRFortessa, BD Biosciences). DMSO was used for preparation of
compound, and the highest concentration of DMSO for the cell-based
assay was less than 0.001% (v/v).
was downloaded from the RCSB Protein Data Bank (http://www.
rcsb.org/pdb) for use in the modeling study. LIBDOCK was used to
evaluate and predict in silico binding free energy of the inhibitors and
for automated docking. The protein model was typed with the
CHARMm force field. A binding sphere with radius of 8.5 Å was
defined through the original ligand (CA-4) as the binding site for the
study. The docked ligand 6e and 2 were refined using in situ ligand
minimization with the Smart Minimizer algorithm by standard
parameters.
Tubulin Assays. Tubulin assembly was measured by turbidimetry
at 350 nm as described previously.23 Assay mixtures containing 1.0
mg/mL (10 μM) 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 GTP to a final
concentration of 0.4 mM 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 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.31
Incubation of 1.0 μM tubulin with 5.0 μM [3H]colchicine and 5.0 or
1.0 μM inhibitor was for 10 min at 37 °C, when about 40−60% of
maximum colchicine binding occurred in control samples.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.7b00273.
Xenograft Tumor Growth Inhibition in Vivo. Six-week-old
female Balb/c-nu mice were obtained from Vital River (Beijing,
China) and housed in 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.
Human lung cancer NCI-H460 cells (2 × 106) were injected
subcutaneously into the right abdominal flanks of the Balb/c-nu
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 mm3, the mice were randomly
divided into five groups (n = 8) and compound administration was
started on the second day. The control group was dosed intra-
peritoneally daily with 7 μL/g of vehicle (0.9% NaCl containing 5%
polyethylene glycol 400 and 0.5% Tween 80). Compound 2 groups
were treated at the doses of 0.25, 0.5, or 1.0 mg/kg body weight,
respectively, every 5 days by iv injection for 3 weeks. The reference
group received paclitaxel at a dose of 15 mg/kg body weight. Both
compound 2 and paclitaxel were dissolved in vehicle solution (5%
PEG400/PBS). The body weight and tumor volume were measured at
each time point. 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 follow: inhibitory rate (%) =
[1 − (mean tumor weight of treated group)/(mean tumor weight of
HPLC purity conditions and data of 6a−6t, HRMS data
of 6a−6t, data of 2 in NIH-NCI 60 cell line panel, and
representative 13C spectra of target compounds (6a, 6b,
6h, 6q, 6r, 6s) (PDF)
SMILES molecular formula strings (CSV)
AUTHOR INFORMATION
■
Corresponding Authors
*For L. Xie. Phone/fax: +86-10-66931690. E-mail: lanxieshi@
yahoo.com; lxie@email.unc.edu.
*For K. H. Lee. Phone: 919-962-0066. Fax: 919-966-3893. E-
mail: khlee@unc.edu.
ORCID
Kuo-Hsiung Lee: 0000-0002-6562-0070
Notes
The content of this paper is solely the responsibility of the
authors and does not necessarily reflect the official views of the
National Institutes of Health.
The authors declare no competing financial interest.
control group)] × 100. Data were presented as the mean
SD.
Statistical significance of differences between groups was compared by
one-way analysis of variance test followed by Tukey’s post hoc test
(GraphPad Prism 5.0; PraphPad Softeare Inc., La Jolla, CA, USA). P <
0.05 was considered to indicate a statistically significant difference.
This study was approved by the Institutional Animal Care and Use
Committee (IACUC) of the Beijing Institute of Radiation Medicine
(Beijing, China) and performed according to the institutional
guidelines.
Immunohistochemical Staining. The tumor tissues were
formalin fixed, paraffin embedded, and sectioned into 4 μm slices.
The tissue sections were deparaffinized, rehydrated, and treated with
3% H2O2 to quench the endogenous peroxidase activity. Antigen
retrieval was performed by boiling slides in sodium citrate buffer (10
mM, pH 6.0). CD31 (Thermo Fisher Scientific; cat. no. PA5-32321),
cleaved caspase-3 (Cell Signaling Technology; cat. no. 9661), or Ki67
antibody (Thermo Fisher Scientific; cat. no. MA5-14520) was used as
primary antibody, and horseradish peroxidase (HRP)-conjugated
antibody was used as secondary antibody. HRP was detected by
diaminobenzidine (DAB) (Wuhan Boster Biological Technology,
Ltd.). The immunohistochemistry reaction was developed with a DAB
substrate kit (Wuhan Boster Biological Technology, Ltd.) prior to
counterstaining the slides with hematoxylin. The immunostained
sections were examined using an EVOS X1 microscope (Advanced
Microscopy Group; Thermo Fisher Scientific, Inc.).
ACKNOWLEDGMENTS
■
We thank the Molecular Pharmacology Branch, DTP, DCTD,
NCI, for performing the NCI 60-cell cytotoxicity assay. 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 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), and Grant 81472687
from the Natural Science Foundation of China awarded to L.L.
This study was also supported in part by the Eshelman Institute
for Innovation, Chapel Hill, North Carolina, awarded to M.G.
ABBREVIATIONS USED
■
CA-4, combretastatin A-4; DAMA-colchicine, N-deacetyl-N-(2-
mercaptoacetyl)-colchicine; DMA, dimethylacetamide; DMAP,
dimethylaminopyridine; DMF, dimethylformamide; DMSO,
dimethyl sulfoxide; GI50, concentration that inhibits 50%
human tumor cell growth; HRMS, high resolution mass
spectra; HTCL, human tumor cell line; IHC, immunohisto-
chemistry; MW, microwave; PDB, Protein Data Bank; SAR,
structure−activity relationship; tumor-VDAs, tumor vascular
disrupting agents
Molecular Modeling. The molecular modeling study was
performed with Discovery Studio 3.0 (Accelrys, San Diego, USA),
also used in our prior modeling method.13 The crystal structure of
tubulin in complex with CA-4 (PDB code 5LYJ) with 2.4 Å resolution
L
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Against Multidrug Resistant Cancer Cells. J. Med. Chem. 2016, 59,
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