Discovery of N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine, a potent apoptosis inducer and efficacious anticancer agent with high blood brain barrier penetration

Article abstract of DOI:10.1021/jm801315b

As a continuation of our structure-activity relationship (SAR) studies on 4-anilinoquinazolines as potent apoptosis inducers and to identify anticancer development candidates, we explored the replacement of the 2-Cl group in our lead compound 2-chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine (6b, EP128265, MPI-0441138) by other functional groups. This SAR study and lead optimization resulted in the identification of N-(4-methoxyphenyl)-N,2- dimethylquinazolin-4-amine (6h, EP128495, MPC-6827) as an anticancer clinical candidate. Compound 6h was found to be a potent apoptosis inducer with EC ₅₀ of 2 nM in our cell-based apoptosis induction assay. It also has excellent blood brain barrier penetration, and is highly efficacious in human MX-1 breast and other mouse xenograft cancer models.

Full text of DOI:10.1021/jm801315b

J. Med. Chem. 2009, 52, 2341–2351
2341
Discovery of N-(4-Methoxyphenyl)-N,2-dimethylquinazolin-4-amine, a Potent Apoptosis Inducer
and Efficacious Anticancer Agent with High Blood Brain Barrier Penetration
Nilantha Sirisoma,† Azra Pervin,† Hong Zhang,† Songchun Jiang,† J. Adam Willardsen,*,‡ Mark B. Anderson,‡ Gary Mather,‡
Christopher M. Pleiman,‡ Shailaja Kasibhatla,† Ben Tseng,† John Drewe,† and Sui Xiong Cai*,†
EpiCept Corporation, 6650 Nancy Ridge DriVe, San Diego, California 92121, and Myriad Pharmaceuticals Inc.,
320 Wakara Way, Salt Lake City, Utah 84108
ReceiVed October 17, 2008
As a continuation of our structure-activity relationship (SAR) studies on 4-anilinoquinazolines as potent
apoptosis inducers and to identify anticancer development candidates, we explored the replacement of the
2-Cl group in our lead compound 2-chloro-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine (6b, EP128265,
MPI-0441138) by other functional groups. This SAR study and lead optimization resulted in the identification
of N-(4-methoxyphenyl)-N,2-dimethylquinazolin-4-amine (6h, EP128495, MPC-6827) as an anticancer clinical
candidate. Compound 6h was found to be a potent apoptosis inducer with EC50 of 2 nM in our cell-based
apoptosis induction assay. It also has excellent blood brain barrier penetration, and is highly efficacious in
human MX-1 breast and other mouse xenograft cancer models.
Introduction
another kinase inhibitor, lapatinib, was reported to be low
as determined by whole-body autoradiography.13 Currently,
there are only a few treatment options available for malignant
glioma including surgery, radiotherapy and DNA-damaging
chemotherapies such as temozolomide.14
Currently, cancer is the second leading cause of death in
the United States and could become the most common cause
in the future.1 Cancer treatment utilizing chemotherapy
agents, including paclitaxel and docetaxel, has been a
mainstay.2 Recent advancements have come with the dis-
covery and development of small molecule targeted therapies.
These include the BCR-ABL kinase inhibitor imatinib for
chronic myeloid leukemia (CML) and gastrointestinal stromal
tumor (GISTa),3 epidermal growth factor receptor (EGFR)
kinase inhibitors gefitinib and erlotinib for nonsmall cell lung
cancer (NSCLC),4 the multikinase inhibitor sorafenib for
kidney and liver cancers,5 an inhibitor of EGFR and HER-2
kinases lapatinib ditosylate for breast cancer,6 a multikinase
inhibitor sunitinib for advanced renal cell carcinoma (RCC)
and imatinib refractory/intolerant GIST,7 a proteasome
inhibitor bortezomib for multiple myeloma,8 and an inhibitor
of mTOR (mammalian target of rapamycin) temsirolimus for
advanced kidney cancer.9
Apoptosis is known to play an important role in tumor
biology.15 Improper or excessive inhibition of apoptosis could
lead to unchecked cell proliferation as well as resistance to
cancer treatment.16 It has been well-established that anticancer
agents like the camptothecins, such as topotecan and irinote-
can,17 and vinca alkaloids, such as vincristine and vinblastine,18
kill tumors at least partially through induction of apoptosis.19
Hence, many novel approaches to activate and promote apop-
tosis in cancer cells20 via targeting regulators of apoptosis are
currently being explored.21 We therefore have developed a cell-
based Anticancer Screening Apoptosis Program (ASAP) high
throughput screening (HTS) assay to identify apoptosis inducers
through measurement of caspase activation.22,23
Unfortunately, relatively little progress has been made for
the treatment of neurologic cancers. One of the reasons is
that the blood brain barrier (BBB) effectively prevents many
traditional and newer anticancer drugs from entering the brain
parenchyma.10 For example, the EGFR inhibitor erlotinib was
reported to have cerebrospinal fluid (CSF) penetration of 7%
(CSF/plasma),11 and EGFR inhibitors gefitinib and erlotinib
have been reported to show limited activity in clinical trials
in brain cancers.12 In addition, the brain concentration of
We have previously reported the discovery of several novel
series of potent apoptosis inducers, including N-phenyl nicoti-
namides (1a),24 gambogic acid (1b),25 4-aryl-4H-chromenes
(1c),26 3-aryl-5-aryl-1,2,4-oxadiazoles (1d),27 4-anilino-2-(2-
pyridyl)pyrimidines (1e),28 and N-phenyl-1H-pyrazolo[3,4-
b]quinolin-4-amines (1f)29 using our HTS assays (Chart 1). More
recently, we reported the discovery of N4-(4-methoxyphenyl)-
N4-methyl-N2-((E)-3,7-dimethylocta-2,6-dienyl)quinazoline-2,4-
diamine (6a) as a potent apoptosis inducer using our cell- and
caspase-based HTS assay and the identification of 2-chloro-N-
(4-methoxyphenyl)-N-methylquinazolin-4-amine (6b), as a highly
potent apoptosis inducer with in ViVo anticancer activity and
high BBB penetration.30 In our quest to optimize this class of
anticancer agents, the 2-Cl group in 6b, a potential liability due
to its chemical reactivity, was replaced by other functional
groups. Herein we report that this SAR study and lead
optimization resulted in the identification of N-(4-methoxyphe-
nyl)-N,2-dimethylquinazolin-4-amine (6h, EP128495, MPC-
6827, Azixa), an anticancer clinical candidate with potent
* Corresponding authors. S.X.C.: EpiCept Corporation, 6650 Nancy
Ridge Drive, San Diego, CA 92121; tel, 858-202-4006; fax, 858-202-4000;
e-mail, scai@epicept.com. J.A.W.: Myriad Pharmaceuticals, 320 Wakara
Way, Salt Lake City, UT 84108; tel, 801-883-3846; fax, 801-548-3603;
e-mail, adam.willardsen@myriadpharma.com.
†
EpiCept Corporation.
Myriad Pharmaceuticals Inc.
‡
a Abbreviations: SAR, structure-activity relationship; GIST, gastrointes-
tinal stromal tumor; EGFR, epidermal growth factor receptor; BBB, blood
brain barrier; CSF, cerebrospinal fluid; ASAP, Anticancer Screening
Apoptosis Program; HTS, high throughput screening; iv, intravenous; AUC,
area under the concentration-time curves; HBD, hydrogen bond donor;
N+O, nitrogen plus oxygen; HBA, hydrogen bond acceptors; ADMET,
absorption, distribution, metabolism, excretion and toxicity.
10.1021/jm801315b CCC: $40.75
2009 American Chemical Society
Published on Web 03/18/2009

2342 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Sirisoma et al.
Chart 1
Scheme 1a
Scheme 2a
a Conditions: (a) 6c, NaOMe; 6d, NHMe2; 6e, NH2Me; 6f, NH2NH2;
(b) AcCl, Et3N, CH2Cl2.
Scheme 3a
a Conditions: (a) RCN (2), HCl(g); (b) POCl3, DIPEA, toluene; (c)
4-MeO-PhNHMe (5a), IPA, HCl; (d) NaOH; (e) NHMe2.
apoptosis inducing activity, high BBB penetration, good phar-
macokinetics and high efficacies in several in ViVo anticancer
models.
a Conditions: (a) 7a, 4-NO2-PhNHMe, NaH, DMF; 7b, 4-F-PhNHMe,
NaAc, THF/H2O; 9a, 4-MeO-PhNH2, IPA, HCl.
Results and Discussion
Chemistry. Compounds 6h-6k were prepared in three steps
as shown in Scheme 1. 4-Chloro-2-methyl-quinazoline (4a)31
was prepared via reaction of 2-aminobenzoic acid methyl ester
and acetonitrile (2a) in the presence of HCl to produce 2-methyl-
quinazolin-4-ol (3a), followed by treatment of 3a with freshly
distilled POCl3 in anhydrous toluene and diisopropylethylamine
(Scheme 1). Reaction of 4a with N-methyl-4-methoxyaniline
(5a) in anhydrous isopropanol (IPA) in the presence of
concentrated HCl produced N-(4-methoxyphenyl)-N,2-dimeth-
ylquinazolin-4-amine (6h) as its hydrochloride salt. In a similar
fashion, compounds 6i-6k were prepared in three steps via
replacing acetonitrile with propiononitrile, fluoroacetonitrile and
chloroacetonitrile, respectively (Scheme 1). Compounds 6l, 6m
were prepared via reaction of 6k with sodium hydroxy and
dimethylamine, respectively (Scheme 1).
Compounds 6c-6f were prepared via displacement of the
chloro group in 6b with the corresponding nucleophiles
(NaOMe, NHMe2, NH2Me, NH2NH2); reaction of 6e with acetyl
chloride subsequently produced the amide 6g (Scheme 2).
Compounds 7a and 7b were prepared from reaction of 4a
with the corresponding N-Me-aniline 5b and 5c in the presence
of a base such as NaH or NaAc. Compound 9a was prepared
from reaction of 4a with 4-methoxyaniline 8a (Scheme 3).
N-(4-Ethoxyphenyl)-2-methylquinazolin-4-amine (9b) was
prepared via reaction of 4a with 4-ethoxyaniline 8b. Reaction
of 9b with methyl iodide in the presence of sodium hydride
Scheme 4a
a Conditions: (a) ArNH2 (8), NaAc, THF/H2O; (b) MeI, NaH, DMF.
produced N-(4-ethoxyphenyl)-N,2-dimethylquinazolin-4-amine
(7c) (Scheme 4). In a similar fashion the following compounds
were prepared. Reaction of 4a with substituted aniline 8c-8g;
or substituted pyridyl-amine (8i, 8j); or substituted pyrimidyl-
amine 8k or pyrazinyl-amine 8l produced compounds 9c-9m.
Reaction of 9c-9m with methyl iodide in the presence of
sodium hydride smoothly produced compounds 7d-7f and
7l-7s (Scheme 4, and see Tables 2 and 3 for structures). The
4-hydroxyl analogue 7h was prepared from demethylation of
6h via treatment with boron tribromide.
4-Amino compound 7i was prepared from hydrogenation of
4-nitro analogue 7a. Treatment of 7i with formaldehyde and
sodium cyanoborohydride produced the dimethylamino analogue
7g. Reaction of 7i with acetic anhydride produced the acetamide

N-(4-Methoxyphenyl)-N,2-dimethylquinazolin-4-amine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2343
Scheme 5a
Table 1. SAR of 4-(4-Methoxyanilino)quinazolines in the Caspase
Activation Assay
EC50 (µM)b
entry R1
R2
T47D
HCT116
SNU-398
6a
6b
6c
6d
6e
6f
6g
6h
6i
Me NHRa
Me Cl
0.29 ( 0.020
0.46 ( 0.098
0.27 ( 0.03
0.002 ( 0.0001 0.002 ( 0.0001 0.002 ( 0.0001
0.004 ( 0.0002 0.004 ( 0.0005 0.003 ( 0.0002
0.015 ( 0.001 0.043 ( 0.006 0.033 ( 0.004
0.008 ( 0.001 0.009 ( 0.002 0.005 ( 0.0003
0.023 ( 0.004 0.036 ( 0.007 0.020 ( 0.002
0.009 ( 0.0004 0.009 ( 0.001 0.010 ( 0.001
0.002 ( 0.0001 0.003 ( 0.0002 0.002 ( 0.0003
0.009 ( 0.0002 0.013 ( 0.001 0.008 ( 0.001
0.002 ( 0.0002 0.002 ( 0.0001 0.001 ( 0.0001
0.048 ( 0.007 0.048 ( 0.011 NDc
Me OMe
Me NMe2
Me NHMe
Me NHNH2
Me NMeAc
Me Me
Me Et
a Conditions: (a) H2, Pd/C, EtOH; (b) HCHO, sodium cyanoborohydride;
(c) Br2, AcOH; (d) T2, Pd/C, EtOH; (e) NaNO2, HCl, then NaN3.
6j
6k
6l
Me CH2F
Me CH2Cl
Me CH2OH
0.002 ( 0.0002 0.004 ( 0.0001 0.002 ( 0.0001
6m Me CH2NMe2
9a Me
1.8 ( 0.06
6.4 ( 0.38
2.6 ( 0.02
6.1 ( 0.32
2.0 ( 0.09
5.3 ( 0.04
7k. The azido analogue 7j was prepared via reaction of 7i with
sodium nitrite in aqueous HCl followed by treatment with
sodium azide. The corresponding tritium labeled analogue 7v
was prepared in three steps. Reaction of 7i with bromine
produced the dibromo analogue 7t, which was treated with
tritium gas under hydrogenation conditions to produce the
ditritium analogue 7u. Diazotization of 7u followed by treatment
with sodium azide, using procedures similar to the preparation
of the cold azido analogue 7j, produced tritium analogue 7v
(Scheme 5).
HTS and SAR Studies. The apoptosis inducing activities of
4-anilinoquinozalines were measured using our proprietary cell-
and caspase-based HTS assay in human breast cancer cells
T47D, human colorectal carcinoma cells HCT116 and hepato-
cellular carcinoma cancer SNU-398 cells as described previ-
ously.24 In brief, the cancer cells were placed in a 384-well
microtiter plate containing various concentration of test com-
pound and incubated at 37 °C for 24 h. After incubation, the
samples were treated with a fluorogenic substrate N-(Ac-
DEVD)-N’-ethoxycarbonyl-R110. The caspase activation activ-
ity (EC50) is summarized in Tables 1-3.
We have found that the methyl group on the nitrogen linker
was essential for the apoptosis inducing activity, and substitution
in the 6- and 7-positions of the quinazoline core structure
resulted in decreased apoptosis inducing activity.30 Since the
Cl group in the 2-position of our lead compound 6b could be
a potential liability due to its chemical reactivity, we expanded
our SAR study by replacing the 2-Cl group with other functional
groups. As expected, the Cl group in 6b was replaced by
nucleophiles to produce compounds 6c-6f. These analogues
in general were less active than 6b in the caspase activation
assay. The 2-OMe analogue 6c and the 2-NHMe analogue 6e,
with a small hydrophobic group at the 2-position, were the more
potent ones giving EC50 values of 4 and 8 nM, respectively.
The more polar hydrazino analogue 6f was the least potent one
of this cluster of analogues, with an EC50 value of 23 nM while
the amide 6g was more active with an EC50 value of 9 nM.
Since methyl is a small hydrophobic group with good
chemical stability, we decided to replace the 2-chloro group in
6b with a methyl group. Compound 6h was found to be highly
potent with an EC50 value of 2 nM, a potency similar to that of
6b in the caspase activation assay. Other alkyl and substituted
alkyl groups were also introduced in the 2-position, including
CH2F, CH2Cl, Et, CH2OH and CH2NMe2 (6i-6m). Several of
H
a R )
.
b Data are the mean of three or more experiments
and are reported as mean ( standard error of the mean (SEM). c ND, not
determined.
these compounds were also highly potent in our caspase
activation assay. For example, the 2-CH2F analogue 6j and
2-CH2OH analogue 6l had potencies similar to that of 6h.
Similarly as observed above, compounds with large or polar
groups in the 2-position, such as 6m, were much less potent as
inducers of apoptosis. Compound 9a, with a hydrogen in the
linker nitrogen instead of a Me group as in 6h, was found to
have an EC50 value of 6400 nM, which is >3000-fold less active
than 6h, confirming our previous observation that a methyl
group on the linker nitrogen was critical for the apoptosis
inducing activity of 6h and related compounds.30
We then explored the SAR of compound 6h through altering
the substitutions in the anilino ring (Table 2), while keeping
the 2-methyl-quinazoline ring as a “fixed” unit. The 4-nitro
analogue 7a and 4-fluoro analogue 7b were >100-fold less potent
than 6h, indicating that an electron-withdrawing group was not
preferred, in agreement with what we reported previously.30 The
4-OEt 7c, 4-OCHF2 7d, 4-SMe 7e and 4-NMe2 7g analogues
were all highly potent, only slightly less active than 6h,
indicating that a small hydrophobic and electron-donating group
was preferred at the 4-position. The 4-OCHF2 group in 7d, a
potentially more metabolically stable group, was introduced
since the demethylation metabolite of 6h, the 4-OH analogue
7h, was significantly less active (see below). The basic 4-NMe2
group in 7g was intended to increase the aqueous solubility of
the compound by using suitable salt forms. Interestingly, the
4-Et analogue 7f was about 10-fold less potent than 6h,
indicating that a heteroatom was preferred in that position. The
4-OH analogue 7h and 4-NH2 analogue 7i were >40-fold less
potent than 6h, indicating that a hydrophilic group was not
preferred at the 4-position. The 4-azido analogue 7j was found
to have good activity with an EC50 value of 16 nM, which
allowed us to make a tritium labeled version 7v that was used
to identify tubulin as the primary molecular target of these
apoptosis inducing 4-anilinoquinazolines.32 The aromatic di-
substituted analogues 7l-7n were found to be highly potent
with EC50 values of 2-10 nM. The electron-withdrawing fluoro
group in 7l and 7m could increase the metabolic stability of
the phenyl ring.

2344 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Sirisoma et al.
Table 2. SAR of N,2-Dimethyl-4-anilinoquinazolines in the Caspase Activation Assay
EC50 (µM)a
entry
R2
R3
R4
T47D
HCT116
SNU-398
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
F
H
OMe
NO2
F
OEt
OCHF2
SMe
Et
NMe2
OH
NH2
N3
NHAc
OMe
OMe
OMe
0.74 ( 0.05
1.3 ( 0.01
0.63 ( 0.001
0.004 ( 0.0004
0.011 ( 0.002
0.003 ( 0.0004
0.026 ( 0.002
0.019 ( 0.004
0.13 ( 0.007
0.25 ( 0.01
0.012 ( 0.001
0.083 ( 0.009
0.002 ( 0.0001
0.004 ( 0.0003
0.014 ( 0.0002
0.62 ( 0.01
0.39 ( 0.038
0.004 ( 0.001
0.010 ( 0.001
0.003 ( 0.0003
0.021 ( 0.002
0.019 ( 0.002
0.088 ( 0.015
0.17 ( 0.03
0.011 ( 0.001
0.066 ( 0.006
0.002 ( 0.0001
0.004 ( 0.001
0.013 ( 0.001
0.42 ( 0.001
0.004 ( 0.0001
0.009 ( 0.0002
0.004 ( 0.0004
0.031 ( 0.003
0.016 ( 0.002
0.086 ( 0.007
0.18 ( 0.02
0.011 ( 0.002
0.059 ( 0.003
0.002 ( 0.0001
0.004 ( 0.0001
0.010 ( 0.001
H
a Data are the mean of three or more experiments and are reported as mean ( standard error of the mean (SEM).
Table 3. SAR of N,2-Dimethyl-4-arylaminoquinazolines in the Caspase
Activation Assay
Table 4. Inhibition of Cell Growth of 4-Arylaminoquinazolines
GI50 (µM)a
entry
T47D
HCT116
SNU-398
6c
6h
6i
9a
7o
7s
0.006 ( 0.001
0.006 ( 0.001
0.007 ( 0.001
5.2 ( 0.60
0.039 ( 0.013
0.76 ( 0.03
0.006 ( 0.001
0.006 ( 0.001
0.012 ( 0.002
6.5 ( 0.79
0.046 ( 0.010
1.0 ( 0.04
0.003 ( 0.001
0.003 ( 0.0003
0.008 ( 0.001
4.2 ( 0.51
0.019 ( 0.003
0.39 ( 0.05
EC50 (µM)a
a Data are the mean of three or more experiments and are reported as
mean ( standard error of the mean (SEM).
entry
A
B
D
R
T47D
HCT116
SNU-398
7o
7p
7q
7r
7s
N
C
N
N
C
C
N
C
C
N
C
C
C
N
N
OMe 0.011 ( 0.001 0.015 ( 0.001 0.010 ( 0.001
OMe 0.016 ( 0.001 0.044 ( 0.007 0.018 ( 0.001
NMe2 0.015 ( 0.001 0.033 ( 0.006 0.020 ( 0.002
OMe 0.053 ( 0.003 0.075 ( 0.008 0.049 ( 0.002
run in a 96-well microtiter plate as described previously.24
Briefly, the cancer cells in a 96-well microtiter plate were treated
with serial concentrations of test compound for 48 h. The
survival profiles of the cells were quantitated using the CellTiter-
Glo (Promega, Madison, WI) according to the manufacturer’s
protocol. The GI50 values are summarized in Table 4. Compound
6h was found to be one of the most potent inhibitors of cancer
cell growth among the compounds tested, with GI50 values of
3-6 nM in T47D, HCT116 and SNU-398 cells. Compounds
6c and 6i also were highly active with GI50 values similar to
that of 6h. Compound 9a was much less potent in the growth
inhibition assay, with GI50 values of 4200 to 6500 nM. This
again confirmed that the N-Me group was important for both
the apoptosis inducing and growth inhibiting activity of 6h and
related compounds.30
Characterization of 6h and Related Compounds. Utilizing
the tritium labeled compound 7v, the primary molecular target
of 6h has been identified as tubulin.32 Mechanistically, 6h was
found to inhibit tubulin polymerization with potency equal to
that of vinblastine and to bind at or near the colchicine site.32
Compound 6h was also found to be effective in multi drug
resistant cancer cell lines including P388/ADR, NCI/ADR-RES,
MCF-7/MX, MCF-7/VP, with similar GI50 values against cancer
cell lines and drug resistant cancer cell lines,32 suggesting that
it may be useful for the treatment of drug resistant cancers.
The apoptosis inducing activity of 6h was also confirmed in
a flow cytometry assay. T47D cells were treated with 5 nM of
6h for 24 or 48 h, stained with propidium iodide, and analyzed
by flow cytometry. An increase in the G2/M DNA content in
cells treated with compound 6h was observed, as shown in
OMe
0.15 ( 0.008 0.15 ( 0.008 0.13 ( 0.004
a Data are the mean of three or more experiments and are reported as
mean ( standard error of the mean (SEM).
We also explored the replacement of the anilino group in 6h
by pyridylamino and other heterocyclic-amino groups (Table
3). The pyridyl analogues 7o-7q were 5-8-fold less potent
than 6h but still highly active with EC50 values in the 11-16
nM range. These pyridyl analogues, especially compound 7q,
were intended to be more hydrophilic and, thus, more soluble
in aqueous media than 6h. Unfortunately, the pirazinyl analogue
7r and pyrimidyl analogue 7s were about 25- and 75-fold less
active than 6h, respectively. Overall, these data indicated that
the more hydrophobic phenyl ring was preferred over the
nitrogen containing heterocycles.
The activities of these compounds toward the HCT116 and
SNU-398 cells were roughly parallel to their activity toward
T47D cells. Compound 6h was highly active in HCT116 and
SNU-398 cells with EC50 values of 3 nM and 2 nM, respectively,
similar to its high activity in T47D cells. Compound 9a was
much less potent with EC50 values of 6100 nM and 5300 nM
in HCT116 and SNU-398 cells, respectively. This further
confirmed the importance of the N-Me group for apoptosis
inducing activity.30
Selected compounds were further evaluated in the traditional
growth inhibition assay to confirm that potent compounds in
the ASAP assay can inhibit cancer cell growth. The growth
inhibition assays in T47D, HCT116 and SNU-398 cell lines were

N-(4-Methoxyphenyl)-N,2-dimethylquinazolin-4-amine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2345
Figure 2. Compound 6h inhibited the growth of established (∼100
mm3) MX-1 tumor xenografts in Crl:Nu/Nu-nuBR mice. Compound
6h was dosed iv at 5, 7.5 or 10 mg/kg once weekly for three weeks.
Figure 1. Drug-induced apoptosis in T47D cells as measured by flow
cytometric analysis. The x-axis is the fluorescence intensity, and the
y-axis is the number of cells with that fluorescence intensity. A: Control
cells showing most of the cells in G1 phase of the cell cycle. B: Cells
treated with 5 nM of compound 6h for 24 h showing most of the cells
arrested in G2/M phase. C: Cells treated with 5 nM of compound 6h
for 48 h showing a progression from G2/M to cells with subdiploid
DNA content, which are apoptotic cells with fragmented nuclei.
Figure 3. PK of compound 6h in brain and plasma after a single iv
dose of 2.5 mg/kg in mice. Median brain concentrations (open circles)
and median plasma concentrations (open squares).
F1 allograft and OVCAR-3, MIAPaCa-2, MCF-7, HT-29 and
MDA-MB-435 xenograft models as reported previously.32
Using plasma from blood samples and homogenized whole
brain samples, the brain to plasma ratio of the area under the
concentration-time curves (AUC) of 6h after a single intrave-
nous (iv) dose of 2.5 mg/kg in mice was determined via
LC-MS/MS to be about 16 (Figure 3), indicating that 6h
maintained the high BBB penetration observed for 6b.30 In
comparison, many anticancer drugs have been reported to have
low BBB penetrations, including erlotinib,11 lapatinib,13 imatinib
(CSF penetration of 5%),33 gefitinib,34 ABT-751 (CSF penetra-
tion of 1.1%),35 oxaliplatin, cisplatin, and carboplatin (CSF
penetration of 2.0%, 3.6%, and 3.8%).36 Since currently there
are few active treatments available for brain cancer and one of
the reasons is limited BBB penetration of many anticancer
drugs,10 the high BBB penetration of 6h suggests that it could
potentially be developed as an effective treatment for brain
cancer.
Figures 1B and 1C. The subdiploid DNA content (apoptotic
sub-G1 area) of cells increased from 1% in the control cells
(Figure 1A) to 15% upon compound treatment for 24 h (Figure
1B) and 34% after 48 h treatment (Figure 1C), indicating the
presence of apoptotic cells which have undergone DNA
degradation and nuclear fragmentation. Previously we reported
that when Jurkat cells were treated with 10 nM of 6h for 18 h,
68% of the cells were apoptotic with subdiploid DNA content
as measured by flow cytometry. The apoptotic cells were
decreased to 11% in the presence of a specific caspase inhibitor,
confirming the apoptotic nature of the subdiploid DNA con-
tent.32
Importantly, compound 6h was found to be highly active in
several in ViVo anticancer models. Figure 2 shows that 6h was
highly efficacious in a MX-1 breast tumor model. No tumor
growth was observed when 6h was dosed at 7.5 and 10 mg/kg
once weekly for three weeks. At 5 mg/kg 6h also effectively
inhibited the growth of MX-1 tumors. In addition, compound
6h also significantly (p < 0.05) inhibited tumor growth in B16-
The brain/plasma AUC ratios of several compounds related
with 6h also were measured using the same method at dose of
2.5 mg/kg (iv) and the results are summarized in Table 5. It
has been suggested that hydrogen bond donor (HBD) of <3,
ClogP of 2-5 and molecular weight (MW) of <500 are preferred
for potential BBB penetration.37 In addition, two simple rules

2346 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Sirisoma et al.
Table 5. The Brain/Plasma Ratio of AUC of 4-Arylaminoquinazolines
the desirable properties of a potent apoptosis inducing agent
with good BBB penetration and high efficacy in multiple
anticancer in ViVo models. The primary molecular target of 6h
has been identified as tubulin by using a tritium labeled
photoaffinity labeling reagent. From our extensive SAR and
ADMET (absorption, distribution, metabolism, excretion and
toxicity) lead optimizations efforts, we discovered N-(4-meth-
oxyphenyl)-N,2-dimethylquinazolin-4-amine (6h, EP128495,
MPC-6827, Azixa), as an anticancer clinical candidate with
potent apoptosis inducing activity. Azixa is currently under
clinical development and reports concerning its progress in the
clinic will be published in due course.
brain/plasma
ratio of
AUC
no. of no. of # of
EC50 (nM)
T47Db
entry
MW HBA HBD N+O ClogPa
6h
7o
6d
7r
7i
16.0
5.8
4.0
1.9
0.90
0.74
279
280
308
281
264
306
4
5
5
6
4
5
0
0
0
0
2
1
4
5
5
6
4
5
4.18
3.61
4.59
2.82
3.02
3.27
2
11
15
53
59
7k
180
a Calculated using ChemDraw. b Data from Tables 1, 2 and 3.
have been proposed that if the number of nitrogen plus oxygen
(N+O) is five or less in a molecule, then it has a high chance
of entering the brain; and if log P - (N+O) > 0, then log BB
(brain/blood) is positive.38 Introduction of more polar groups
as well as nitrogen and oxygen into the structure of 6h therefore
was expected to reduce BBB penetration and the brain/plasma
AUC ratio. Compound 6d, with the 2-Me group replaced by a
more polar NMe2 group and one additional nitrogen than that
of 6h, reduced the brain/plasma AUC ratio from 16 to 4.0.
Similarly, introduction of 1 or 2 more nitrogens via replacing
the phenyl group in 6h with a pyridyl (7o) or pyrazinyl (7r)
decreased the brain/plasma AUC ratio to 5.8 and 1.9, respec-
tively. These data confirmed that, in this series of compounds,
increasing the number of hydrogen bond acceptors (HBA) or
the total number of N+O resulted in less BBB penetration.
Replacing the 4-OMe group in the phenyl ring by a more polar
group such as the amino group in 7i and acetamido in 7k also
reduced the ratio to 0.90 and 0.74, respectively, indicating that
introduction of HBD effectively reduced the BBB penetration.
Our data are in agreement with the low BBB penetration of
4-anilinoquinazoline based kinase inhibitors due to large
numbers of N+O and HBD in the structures, including gefitinib
(10a, 7 N+O, 1 HBD), erlotinib (10b, 7 N+O, 1 HBD) and
lapatinib (10c, 8 N+O, 2 HBD), as well as ABT-751 (10d, 7
N+O, 3 HBD), which like 6h is a tubulin inhibitor that binds
to the colchicine site35 (Chart 2). Compound 6h appears to be
optimized for BBB penetration while other potent compounds
(Table 5) with less BBB penetration could potentially be
developed for peripheral indications.
Experimental Section
General Methods and Materials. Commercial-grade reagents
and solvents obtained from Acros, Aldrich, Apin Lancaster, TCI
or VWR were used without further purification except as indicated.
All reactions were stirred magnetically; moisture-sensitive reactions
were performed under argon in oven-dried glassware. Thin-layer
chromatography (TLC), usually using ethyl acetate/hexane as the
solvent system, was used to monitor reactions. Solvents were
removed by rotary evaporation under reduced pressure; where
appropriate, the compound was further dried using a vacuum pump.
The 1H NMR spectra were recorded at 300 MHz. All samples were
prepared as dilute solutions in deuteriochloroform (CDCl3) with
v/v 0.05% tetramethylsilane (TMS). Chemical shifts are reported
in parts per million (ppm) downfield from TMS (0.00 ppm), and J
coupling constants are reported in hertz. Purity of tested compounds
6c-6m, 7a-7s and 9a was determined by elemental analysis,
HPLC or SFC to be >95% pure. Elemental analyses were performed
by Numega Resonance Laboratories, Inc. (San Diego, CA). LCMS
was run in the ESI mode using an Xterra MS C18 (Waters) 4.6 ×
50 mm 5 µm column; HPLC purity was performed using a 4.6 ×
150 mm Xterra C18 5 µm column. Both LCMS and LC were
reverse phase with an acetonitile/water containing 0.01%v/v TFA
gradient. SFC was run using a normal phase Luna 5u silica 4.6 ×
250 mm column. Mobile phase was liquid CO2 with methanol as
the modifying solvent. A gradient was used in which the concentra-
tion of methanol was ramped from 5% to 50% over a course of 12
min with a 200 bar back-pressure at 40 °C. Human breast cancer
cells T47D, human colorectal carcinoma cells HCT116 and
hepatocellular carcinoma cancer cells SNU-398 were purchased
from the American type Culture Collection (Manasas, VA).
2-Methylquinazolin-4-ol (3a). To a solution of 2-aminobenzoic
acid methyl ester (15.0 g, 99.3 mmol) in 500 mL of acetonitrile in
a 1000 mL three necked round bottomed flask equipped with a
mechanical stirrer, condenser and a dispersion tube was bubbled
In conclusion, through SAR studies and lead optimization,
compound 6h has been identified as an anticancer development
candidate. In comparison with our original lead 6b, compound
6h does not have the potentially reactive 2-Cl, but still maintains
Chart 2

N-(4-Methoxyphenyl)-N,2-dimethylquinazolin-4-amine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2347
through HCl gas for 15 min with vigorous stirring, and the resulting
mixture was refluxed overnight, then cooled to room temperature.
The resulting precipitates were collected by filtration. The solid
was dissolved in water (250 mL), the solution was neutralized with
saturated NaHCO3 (100 mL), and the precipitates were collected
by filtration. The solid was washed with water (50 mL), dried at
room temperature for 8 h and dried further at 70 °C under high
2-Methoxy-N-(4-methoxyphenyl)-N-methylquinazolin-4-
amine (6c). To a solution of 2-chloro-N-(4-methoxyphenyl)-N-
methylquinazolin-4-amine (6b, 50 mg, 0.167 mmol) in 2 mL
methanol was added sodium methoxide (500 µL, 25% by wt. in
methanol). The solution was stirred at 80 °C for 1 h, and it was
diluted with 50 mL ethyl acetate. The solution was washed with
water, dried and concentrated. The residue was purified using small
silica column (acetate and hexane, 1:9) to give 6c as a white solid
1
vacuum overnight to give 3a as white solid (14.32 g, 90%). H
1
NMR (DMSO-d6) 12.21 (s, broad, 1H), 8.06-8.09 (m, 1H),
7.74-7.79 (m, 1H), 7.56-7.59 (m, 1H), 7.43-7.45 (m, 1H), 2.35
(s, 3H).
(22 mg, 54%). H NMR (CDCl3): 7.89 (d, J ) 8.4 Hz, 1H), 7.53
(ddd, J ) 8.7, 5.4 and 2.4 Hz, 1H), 7.19- 7.14 (m, 2H), 6.99-6.93
(m, 2H), 6.90-6.85 (m, 2H), 4.14 (s, 3H), 3.86 (s, 3H), 3.64 (s,
3H). Anal. (C17H17N3O2) C, H, N.
4-Chloro-2-methylquinazoline (4a). A mixture of 3a (10.0 g,
62.6 mmol) in 190 mL of anhydrous toluene and anhydrous
diisopropylethylamine (DIPEA, 17.0 mL, 97.4 mmol) in a 500 mL
round bottomed flask equipped with a condenser and a drying tube
was refluxed for 1 h. To this warm solution was added freshly
distilled POCl3 (9.0 mL, 97 mmol) and the mixture was heated at
80 °C for 2 h, and cooled to room temperature. The mixture was
diluted with 500 mL of ethyl acetate, washed with 200 mL of ice
cold water, 200 mL of saturated NaHCO3, 200 mL of water, citric
acid (1 N, 4 × 200 mL), 200 mL of water, 200 mL of saturated
NaHCO3 and 200 mL of saturated NaCl. The organic layer was
dried over anhydrous Na2SO4, filtered and concentrated to obtain
N4-(4-Methoxyphenyl)-N2,N2,N4-trimethylquinazolin-2,4-di-
amine (6d). A mixture of 6b (150 mg, 0.5 mmol) and 2.0 M
dimethylamine in methanol (2.0 mL, 4 mmol) in a sealed tube was
stirred at 70-80 °C overnight. The mixture was filtered, and the
filtration was concentrated by vacuum. The residue was mixed with
ethyl acetate (10 mL) and was washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated. The crude product
was purified by chromatography on silica gel with ethyl acetate
and hexane (1:9) as eluent, yielding 6d as a solid (128 mg, 83%).
1H NMR (CDCl3): 7.44 (d, J ) 7.8 Hz, 1H), 7.36-7.30 (m, 1H),
7.11-7.08 (m, 2H), 6.90-6.85 (m, 3H), 6.65-6.59 (m, 1H), 3.82
(s, 3H), 3.51 (s, 3H), 3.30 (s, 6H). Anal. (C18H20N4O) C, H, N.
1
an off-white solid (9.69 g, 87%). H NMR (CDCl3): 8.21-8.25
(m, 1H), 7.89-7.99 (m, 2H), 7.66 (ddd, J ) 8.7, 6.6 and 1.8 Hz,
N4-(4-Methoxyphenyl)-N2,N4-dimethylquinazolin-2,4-di-
amine (6e). Compound 6e was prepared from reaction of 6b with
2.0 M methylamine in THF using a procedure similar to that of 6d
1H), 2.87 (s, 3H).
N-(4-Methoxyphenyl)-N,2-dimethylquinazolin-4-amine hy-
drochloride (6h). To a solution of 4a (2.31 g, 12.9 mmol) and
N-methyl-4-methoxyaniline (5a, 2.00 g, 14.6 mmol) in 35 mL of
anhydrous isopropanol (IPA) was added 0.6 mL of concentrated
HCl, and the mixture was stirred at room temperature overnight.
The yellow precipitates were collected by filtration, washed with
cold isopropanol, and dried at room temperature for 3 h under house
vacuum. The solid was further dried under high vacuum at 70 °C
for 48 h to remove residual isopropanol, provided compound 6h
1
and was isolated as a solid (53.7%). H NMR (CDCl3): 7.45 (d, J
) 7.8 Hz, 1H), 7.39-7.33 (m, 1H), 7.11-7.07 (m, 2H), 6.90-6.87
(m, 3H), 6.69-6.64 (m, 1H), 4.95 (brs, 1H), 3.82 (s, 3H), 3.50 (s,
3H), 3.11 (d, J ) 5.1 Hz, 3H). HRMS calcd for C17H19N4O (M +
H+), 295.1560; found, 295.1542.
2-Hydrazino-N-(4-methoxyphenyl)-N-methylquinazolin-4-
amine (6f). To a solution of 6b (100 mg, 0.33 mmol) in 2 mL of
1,4-dioxane was added 0.4 mL of hydrazine. The reaction mixture
was stirred at room temperature overnight. The solvent was removed
by evaporation, and the residue was diluted with ethyl acetate (20
mL), washed with saturated NaHCO3 aq, brine, dried over Na2SO4,
filtered and concentrated by vacuum, yielding 6f as yellow solids
1
as yellow solids (2.90 g, 71%). H NMR (CDCl3): 8.53 (dd, J )
8.1 and 0.6 Hz, 1H), 7.7 (ddd, J ) 8.4, 7.2 and 1.2 Hz, 1H), 7.22
(m, 2H), 7.13 (ddd, J ) 8.7, 7.2 and 1.2 Hz, 1H), 7.05 (m, 2H),
6.76 (d, J ) 8.7 Hz, 1H), 3.91 (s, 3H), 3.78 (s, 3H), 2.96 (s, 3H).
Anal. (C17H17N3O·HCl) C, H, N.
1
(20 mg, 21%). H NMR (CDCl3): 7.52-7.49 (m, 1H), 7.44-7.39
The following compounds were prepared by a procedure similar
to that described for the preparation of compound 6h.
(m, 1H), 7.12-7.08 (m, 2H), 6.93-6.88 (m, 3H), 6.77-6.71 (m,
1H), 6.01 (brs, 1H), 4.11 (brs, 2H), 3.83 (s, 3H), 3.50 (s, 3H). Anal.
(C16H17N5O) C, H, N.
2-Ethyl-N-(4-methoxyphenyl)-N-methylquinazolin-4-amine (6i).
Compound 6i was prepared from 4-chloro-2-ethylquinazoline and
N4-(4-Methoxyphenyl)-N2-acetyl-N2,N4-dimethylquinazolin-
2,4-diamine (6g). To a solution of 6e (100 mg, 0.34 mmol) in 5
mL of dichloromethane were added triethylamine (0.071 mL, 0.51
mmol) and acetyl chloride (0.036 mL, 0.51 mmol) followed by 2
mg of DMAP at 0 °C. The reaction mixture was allowed to warm
to room temperature and stirred for 2 h. The solvent was removed
by evaporation. The residue was dissolved in ethyl acetate (20 mL),
washed with water and brine, dried over Na2SO4, filtered and
concentrated by vacuum. The crude product was purified by
chromatography on silica gel with ethyl acetate, hexane and
methanol (1:3 to 1:1:0.05) as eluent, yielding 6g as a white solid
1
5a and was isolated as a solid (70%). H NMR (CDCl3): 7.76 (d,
J ) 8.4 Hz, 1H), 7.55-7.49 (m, 1H), 7.13-7.09 (m, 2H),
7.03-6.89 (m, 4H), 3.83 (s, 3H), 3.60 (s, 3H), 2.97 (q, J ) 7.5
Hz, 2H), 1.44 (t, J ) 7.8 Hz, 3H). HRMS calcd for C18H20N3O (M
+ H+), 294.1608; found, 294.1597.
2-Fluoromethyl-N-(4-methoxyphenyl)-N-methylquinazolin-4-
amine (6j). Compound 6j was prepared from 4-chloro-2-fluorom-
ethylquinazoline and 5a and was isolated as a solid (67%). 1H NMR
(CDCl3): 7.87-7.84 (m, 1H), 7.60-7.54 (m, 1H), 7.14-7.10 (m,
2H), 7.04-7.01 (m, 2H), 6.95-6.91 (m, 2H), 5.60 (s, 1H), 5.44
(s, 1H), 3.85 (s, 3H), 3.60 (s, 3H). HRMS calcd for C17H17FN3O
(M + H+), 298.1357; found, 298.1343.
1
(36 mg, 32%). H NMR (CDCl3): 7.70-7.67 (m, 1H), 7.56-7.52
(m, 1H), 7.17-7.14 (m, 2H), 6.97-6.93 (m, 4H), 3.86 (s, 3H),
3.57 (s, 6H), 2.52 (s, 3H). Anal. (C19H20N4O2) C, H, N.
2-Chloromethyl-N-(4-methoxyphenyl)-N-methylquinazolin-4-
amine (6k). Compound 6k was prepared from 4-chloro-2-chlo-
romethylquinazoline and 5a and was isolated as a solid (65%). 1H
NMR (CDCl3): 7.82 (d, J ) 8.7 Hz, 1H), 7.59-7.53 (m, 1H),
7.15-7.12 (m, 2H), 7.03-7.00 (m, 2H), 6.95-6.91 (m, 2H), 4.73
(s, 2H), 3.85 (s, 3H), 3.62 (s, 3H). HRMS calcd for C17H17ClN3O
(M + H+), 314.1062; found, 314.1042.
2-Hydroxymethyl-N-(4-methoxyphenyl)-N-methylquinazolin-
4-amine (6l). To a solution of 6k (67 mg, 0.19 mmol) in
1,4-dioxane (3 mL) was added 2 N NaOH aqueous solution (1 mL).
The mixture was heated at 80 °C for 24 h and then was cooled to
room temperature. The reaction mixture was diluted with ethyl
acetate (20 mL) and then was washed with water and dried with
NaSO4. The solvent was evaporated, and the residue was purified
by column chromatography on silica gel with ethyl acetate and
N-(4-Methoxyphenyl)-2-methylquinazolin-4-amine (9a). Com-
pound 9a was prepared from 4a and 4-methoxyaniline and was
1
1
isolated as a solid (60%). H NMR (DMSO-d6): 11.28 (brs, 1H),
hexane (1:1) as eluent, yielding 6l as a solid (25 mg, 44%). H
8.73 (d, J ) 8.4 Hz, 1H), 8.06 (t, J ) 7.5 Hz, 1H), 7.85-7.78 (m,
2H), 7.66 (d, J ) 9 Hz, 2H), 7.06 (d, J ) 8.7 Hz, 2H), 3.81 (s,
3H), 2.60 (s, 3H). HRMS calcd for C16H16N3O (M + H+), 266.1295;
found, 266.1289.
NMR (CDCl3): 7.78-7.75 (m, 1H), 7.59-7.53 (m, 1H), 7.15-7.12
(m, 2H), 7.02-7.00 (m, 2H), 6.94-6.91 (m, 2H), 4.79 (s, 2H),
3.85 (s, 3H), 3.59 (s, 3H). HRMS calcd for C17H18N3O2 (M + H+),
296.1382; found, 296.1389.

2348 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Sirisoma et al.
2-(Dimethylaminomethyl)-N-(4-methoxyphenyl)-N-meth-
ylquinazolin-4-amine (6m). Compound 6m was prepared from
reaction of 6k and dimethylamine by a procedure similar to that of
compound 6l and was isolated as a solid (56%). 1H NMR (DMSO-
d6): 7.71 (d, J ) 8.7 Hz, 1H), 7.60 (t, J ) 8.4 Hz, 1H), 7.20 (d, J
) 8.4 Hz, 2H), 7.09 (t, J ) 8.1 Hz, 1H), 7.00-6.96 (m, 3H), 3.78
(s, 3H), 3.63 (s, 2H), 3.50 (s, 3H), 2.33 (s, 6H). Anal. (C19H22N4O)
C, H, N.
N-(3,4-Dimethoxyphenyl)-2-methylquinazolin-4-amine (9h).
Compound 9h was prepared from 4a (178.6 mg, 1.0 mmol) and
3,4-dimethoxyaniline (153 mg, 1.0 mmol) and was isolated as off
1
white solids (295 mg, 100%). H NMR (CDCl3): 7.85-7.70 (m,
4H), 7.51-7.46 (m, 1H), 7.38 (brs, 1H), 7.16 (dd, J ) 8.7 and 2.1
Hz, 1H), 6.90 (d, J ) 8.7 Hz, 1H), 3.95 (s, 3H), 3.91 (s, 3H), 2.69
(s, 3H).
N-(6-Methoxypyridin-3-yl)-2-methylquinazolin-4-amine (9i).
Compound 9i was prepared from 4a and 3-amino-6-methoxypyri-
dine and was isolated as a solid (32%). 1H NMR (CDCl3): 8.51 (d,
J ) 1.8 Hz, 1H), 8.12 (m, 1H), 7.72-7.89 (m, 3H), 7.49 (m, 2H),
6.81 (d, J ) 8.7 Hz, 1H), 3.89 (s, 3H), 2.68 (s, 3H).
N-(5-Methoxypyridin-2-yl)-2-methylquinazolin-4-amine (9j).
Compound 9j was prepared from 4a and 2-amino-5-methoxypy-
2,N-Dimethyl-N-(4-nitrophenyl)quinazolin-4-amine (7a). To
a solution of 4a (1.40 g, 7.84 mmol) and N-methyl-4-nitroaniline
(1.09 g, 7.16 mmol) in 20 mL of dimethylformamide cooled to 0
°C was added sodium hydride (0.6 g, 60 oil suspension, 15 mmol).
The reaction mixture was stirred at 0 °C for 1 h and quenched by
adding 200 µL of water. It was diluted with 150 mL of ethyl acetate,
washed with water (100 mL × 3) and saturated NaCl, dried over
anhydrous MgSO4, filtered and concentrated. The residue was
purified by chromatography (30% ethyl acetate/hexanes) to give
1
ridine and was isolated as a solid (42%). H NMR (CDCl3): 8.43
(m, 1H), 7.72-7.92 (m, 2H), 7.46-7.56 (m, 2H), 7.32-7.40 (m,
2H), 3.88 (s, 3H), 2.74 (s, 3H).
1
N-(6-Dimethylaminopyridin-3-yl)-2-methylquinazolin-4-
amine (9k). Compound 9k was prepared from 4a (182 mg, 1.02
mmol) and 3-amino-6-dimethylaminopyridine (140 mg, 1.02 mmol)
7a (1.41 g, 67%). H NMR (CDCl3): 8.14 (m, 2H), 7.91 (m, 1H),
7.71 (ddd, J ) 8.4, 6.6 and 1.8 Hz, 1H), 7.19-7.32 (m, 2H), 7.05
(m, 2H), 3.76 (s, 3H), 2.82 (s, 3H). Anal. (C16H14N4O2) C, H, N.
N-(4-Fluorophenyl)-N,2-dimethylquinazolin-4-amine (7b). A
mixture of 4a (178 mg, 1.00 mmol) and N-methyl-4-fluoroaniline
(125 mg, 2.52 mmol) and sodium acetate (248 mg, 3.02 mmol) in
6 mL of solvent (THF:water ) 1:1) was stirred at 70 °C for 1 h.
The reaction mixture was diluted with 30 mL of ethyl acetate. The
organics were washed with brine, dried over anhydrous Na2SO4,
filtered and concentrated. The crude product was purified by
chromatography on silica gel with ethyl acetate and hexane (1:5)
as eluent, yielding 7b as a solid (125 mg, 46.8%). 1H NMR (CDCl3):
7.76 (dd, J ) 8.3 and 0.9 Hz, 1H), 7.58-7.52 (m,1H), 7.16-7.00
(m, 6H), 3.60 (s, 3H), 2.73 (s, 3H). HRMS calcd for C16H15FN3
(M + H+), 268.1252; found, 268.1239.
1
and was isolated as a pale yellow solid (60 mg, 21%). H NMR
(CDCl3): 8.43 (d, J ) 2.4 Hz, 1H), 7.95 (dd, J ) 9.3 and 2.7 Hz,
1H), 7.84-7.72 (m, 3H), 7.47 (t, J ) 8.1 Hz, 1H), 7.16 (brs, 1H),
6.61 (d, J ) 9.0 Hz, 1H), 3.12 (s, 6H), 2.65 (s, 3H).
N-(5-Methoxypyrazin-2-yl)-2-methylquinazolin-4-amine (9l).
Compound 9l was prepared from 4a (150 mg, 0.84 mmol) and
2-amino-5-methoxypyrazine (105 mg, 0.84 mmol) and was isolated
as a solid (106 mg, 47%). The crude product was used as is in the
next step.
N-(5-Methoxypyrimidin-2-yl)-2-methylquinazolin-4-amine (9m).
Compound 9m was prepared from 4a (343 mg, 1.92 mmol) and
2-amino-5-methoxypyrimidine (240 mg, 1.92 mmol) and was
1
isolated as a solid (283 mg, 55%). H NMR (CDCl3): 8.69 (dd, J
The following intermediates were prepared by a procedure similar
to that described for the preparation of compound 7b.
) 8.1 and 1.5 Hz, 1H), 8.43 (s, 2H), 7.73-7.67 (m, 1H), 7.60 (d,
J ) 7.2 Hz, 1H), 7.49-7.43 (m, 1H), 3.95 (s, 3H), 2.58 (s, 3H).
N-(4-Methylthiophenyl)-N,2-dimethylquinazolin-4-amine (7e).
To a solution of 9d (263 mg, 0.94 mmol) in DMF (4 mL) at 0 °C
was added sodium hydride (56.4 mg, 1.40 mmol, 60% oil
dispersion) and followed by methyl iodide (0.09 mL, 1.4 mmol).
The mixture was stirred at 0 °C for 1 h, then allowed to warm to
room temperature and stirred for an additional 2 h. The reaction
mixture was diluted with ethyl acetate (15 mL), washed with
saturated NaHCO3 aq, brine, dried over Na2SO4, filtered and
concentrated by vacuum. The residue was purified by chromatog-
raphy on silica gel with ethyl acetate and hexane (1:2 to 1:1) as
eluent, yielding 120 mg of 7e (40.7%). 1H NMR (CDCl3): 7.76 (d,
J ) 9.0 Hz, 1H), 7.54 (t, J ) 7.5 Hz, 1H), 7.24-7.19 (m, 2H),
7.10-6.97 (m, 4H), 3.59 (s, 3H), 2.74 (s, 3H), 2.48 (s, 3H). HRMS
calcd for C17H18N3S (M + H+), 296.1223; found, 296.1207.
The following compounds were prepared by a procedure similar
to that described for the preparation of compound 7e.
N-(4-Ethoxyphenyl)-2-methylquinazolin-4-amine (9b). Com-
pound 9b was prepared from 4a and 4-ethoxyaniline and was
1
isolated as a solid (93%). H NMR (CDCl3): 7.83 (m, 1H), 7.81
(m, 1H), 7.42 (m, 1H), 7.65-7.71 (m, 2H), 7.46 (ddd, J ) 8.4, 6.9
and 1.5 Hz, 1H), 7.37 (s, broad, 1H), 6.92-6.97 (m, 2H), 4.06 (q,
J ) 6.9 Hz, 2H), 2.68 (s, 3H), 1.43 (t, J ) 6.9 Hz, 3H).
N-(4-Difluoromethoxyphenyl)-2-methylquinazolin-4-amine (9c).
Compound 9c was prepared from 4a (450 mg, 2.52 mmol) and
4-difluoromethoxyphenylamine (0.32 mL, 2.52 mmol) and was
1
isolated as a solid (713 mg, 94%). H NMR (CDCl3): 7.87-7.76
(m, 5H), 7.51 (t, J ) 8.4 Hz, 1H), 7.40 (brs, 1H), 7.19 (d, J ) 8.7
Hz, 2H), 6.76-6.27 (m, 1H), 2.71 (s, 3H).
N-(4-Methylthiophenyl)-2-methylquinazolin-4-amine (9d). Com-
pound 9d was prepared from 4a (178 mg, 1.00 mmol) and
4-methylthioaniline (139 mg, 1.00 mmol) and was isolated as a
solid (273 mg, 97%). 1H NMR (CDCl3): 7.86-7.82 (m, 2H),
7.79-7.73 (m, 3H), 7.50-7.45 (m, 2H), 7.34-7.26 (m, 2H), 2.7
(s, 3H), 2.51 (s, 3H).
N-(4-Ethoxyphenyl)-N,2-dimethylquinazolin-4-amine (7c). Com-
pound 7c was prepared from 9b and methyl iodide and was isolated
as a solid (67%). 1H NMR (CDCl3): 7.71-7.74 (m, 1H), 7.51 (ddd,
J ) 8.1, 6.6 and 1.5 Hz, 1H), 7.09 (m, 2H), 6.95-7.04 (m, 2H),
6.86-6.92 (m, 2H), 4.04 (q, J ) 6.9 Hz, 2H), 3.58 (s, 3H), 2.72
(s, 3H), 1.44 (t, J ) 6.9 Hz, 3H). HRMS calcd for C18H20N3O (M
+ H+), 294.1608; found, 294.1595.
N-(4-Ethylphenyl)-2-methylquinazolin-4-amine (9e). Com-
pound 9e was prepared from 4a (100 mg, 0.56 mmol) and
4-ethylaniline (0.07 mL, 0.56 mmol) and was isolated as a solid
(134 mg, 91%). 1H NMR (CDCl3): 7.85-7.78 (m, 2H), 7.76-7.71
(m, 3H), 7.49 (t, J ) 7.5 Hz, 1H), 7.36 (brs, 1H), 7.24 (s, 2H),
2.70-2.69 (m, 5H), 1.26 (t, J ) 7.5 Hz, 3H).
N-(4-Difluoromethoxyphenyl)-N,2-dimethylquinazolin-4-
amine (7d). Compound 7d was prepared from 9c (710 mg, 2.36
mmol) and methyl iodide (1.03 mL, 16.5 mmol) and was isolated
N-(3-Fluoro-4-methoxyphenyl)-2-methylquinazolin-4-amine
(9f). Compound 9f was prepared from 4a (150 mg, 0.84 mmol)
and 3-fluoro-4-methoxyaniline (118 mg, 0.84 mmol) and was
1
as a solid (301 mg, 40.8%). H NMR (CDCl3): 7.77 (dd, J ) 8.4
and 0.9 Hz, 1H), 7.59-7.53 (m, 1H), 7.17-7.10 (m, 4H),
7.06-6.99 (m, 2H), 6.78-6.29 (m, 1H), 3.62 (s, 3H), 2.75 (s, 3H).
HRMS calcd for C17H16F2N3O (M + H+), 316.1263; found,
316.1245.
1
isolated as a solid (90 mg, 34%). H NMR (CDCl3): 7.89-7.75
(m, 4H), 7.53-7.48 (m, 1H), 7.38-7.34 (m, 2H), 7.0 (t, J ) 8.7
Hz, 1H), 3.92 (s, 3H), 2.71 (s, 3H).
N-(2-Fluoro-4-methoxyphenyl)-2-methylquinazolin-4-amine
(9g). Compound 9g was prepared from 4a and 2-fluoro-4-
methoxyaniline and was isolated as a solid (79%). 1H NMR
(CDCl3): 8.54 (m, 1H), 7.83 (m, 2H), 7.65 (m, 1H), 7.50 (m, 1H),
7.47 (s, broad, 1H), 6.74-6.81 (m, 2H), 3.83 (s, 3H), 2.70 (s, 3H).
N-(4-Ethylphenyl)-N,2-dimethylquinazolin-4-amine (7f). Com-
pound 7f was prepared from 9e (122 mg, 0.46 mmol) and methyl
iodide (0.2 mL, 3.3 mmol) and was isolated as a solid (39 mg,
52%). 1H NMR (CDCl3): 7.74 (d, J ) 8.1 Hz, 1H), 7.54-7.49 (m,
1H), 7.19 (d, J ) 8.4 Hz, 2H), 7.09-6.92 (m, 4H), 3.61 (s, 3H),

N-(4-Methoxyphenyl)-N,2-dimethylquinazolin-4-amine
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2349
2.73-2.63 (m, 5H), 1.26 (d, J ) 7.5 Hz, 3H). HRMS calcd for
C18H20N3 (M + H+), 278.1659; found, 279.1640.
(m, 2H), 3.64 (s, 3H), 2.79 (s, 3H). HRMS calcd for C16H16N3O
(M + H+), 266.1295; found, 266.1279.
N-(4-Aminophenyl)-N,2-dimethylquinazolin-4-amine (7i). A
mixture of 7a (200 mg, 0.68 mmol) in 25 mL of ethyl acetate was
hydrogenated over palladium on carbon (70 mg) at 50 psi for 4 h,
and the reaction mixture was filtered through a pad of Celite and
concentrated. The resulting crude product was purified by chro-
matography (50% ethyl acetate/hexane) to give 7i as a solid (140
N-(3-Fluoro-4-methoxyphenyl)-N,2-dimethylquinazolin-4-
amine (7l). Compound 7l was prepared from 9f (250 mg, 0.88
mmol) and methyl iodide (0.39 mL, 6.2 mmol) and was isolated
1
as a solid (32 mg, 12%). H NMR (CDCl3): 7.76 (d, J ) 8.4 Hz,
1H), 7.59-7.53 (m, 1H), 7.09-6.82 (m, 5H), 3.91 (s, 3H), 3.58
(s, 3H), 2.73 (s, 3H). HRMS calcd for C17H17FN3O (M + H+),
298.1357; found, 298.1343.
1
mg, 78%). H NMR (CDCl3): 7.71 (m, 1H), 7.51 (ddd, J ) 8.4,
6.9 and 1.5 Hz, 1H), 7.09 (m, 1H), 6.93-7.05 (m, 3H), 6.68 (m,
2H), 3.74 (s, broad, 2H), 3.56 (s, 3H), 2.71 (s, 3H). HRMS calcd
for C16H17N4 (M + H+), 265.1455; found, 265.1436.
N-(2-Fluoro-4-methoxyphenyl)-N,2-dimethylquinazolin-4-
amine (7m). Compound 7m was prepared from 9g and methyl
1
iodide and was isolated as a solid (51%). H NMR (CDCl3): 7.76
N-(4-Dimethylaminophenyl)-N,2-dimethylquinazolin-4-
amine (7g). To a solution of 7i (14 mg, 0.053 mmol) in methanol
(1.5 mL), 37% aqueous formaldehyde (0.5 mL) and 10 µL of glacial
acetic was added sodium cyanoborohydride (15 mg, 0.24 mmol),
and the mixture was stirred at room temperature for 2 h. The
reaction mixture was quenched by adding 50 µL of 1 N HCl. The
mixture was diluted with 50 mL of ethyl acetate, washed with
saturated sodium bicarbonate, and followed by saturated sodium
chloride. The organic layer was dried over anhydrous Na2SO4,
filtered and concentrated. The residue was purified by column
chromatography (25% ethyl acetate/hexanes) on silica gel to give
7g as a solid (12 mg, 80%). 1H NMR (CDCl3): 7.71 (m, 1H), 7.50
(ddd, J ) 8.4, 6.9 and 1.5 Hz, 1H), 7.03-7.09 (m, 3H), 6.95 (ddd,
J ) 8.1, 6.6 and 0.9 Hz, 1H), 6.70 (m, 2H), 3.57 (s, 3H), 2.99 (s,
6H), 2.71 (s, 3H). HRMS calcd for C18H21N4 (M + H+), 293.1768;
found, 293.1755.
N-(4-Azidophenyl)-N,2-dimethylquinazolin-4-amine (7j). To
a solution of 7i (20 mg, 0.076 mmol) in 1.2 mL of 1 N HCl was
added 100 µL of methanol and the mixture cooled to 0 °C. One
drop of concentrated HCl was added, and the solution was stirred
at 0 °C for 0.25 h. To the solution was added dropwise a solution
of sodium nitrite (25 mg, 0.36 mmol) in 200 µL of water. The
reaction mixture was stirred for 0.5 h, then was added a solution
of sodium azide (25 mg, 0.38 mmol) in 300 µL of water followed
by an additional batch of sodium azide (25 mg, 0.38 mmol). The
reaction mixture was stirred at 0 °C for 1 h, diluted with 50 mL of
ethyl acetate, and washed with saturated sodium bicarbonate
followed by saturated sodium chloride. The organic layer was dried
over anhydrous Na2SO4, filtered and concentrated. The residue was
purified by column chromatography (20-25% ethyl acetate/
hexanes) on silica gel to give 7j as a solid (20 mg, 90%). 1H NMR
(CDCl3): 7.76 (m, 1H), 7.56 (ddd, J ) 8.1, 6.3 and 1.8 Hz, 1H),
7.13 (m, 2H), 6.98-7.13 (m, 4H), 3.61 (s, 3H), 2.74 (s, 3H). HRMS
calcd for C16H15N6 (M + H+), 291.1360; found, 291.1347.
N-(4-Acetamidophenyl)-N,2-dimethylquinazolin-4-amine (7k).
To a solution of 7i (28 mg, 0.11 mmol) in 2 mL of dichloromethane
with triethylamine (50 µL, 0.36 mmol) cooled at 0 °C was added
acetic anhydride (50 µL, 0.53 mmol), followed by a few crystals
of 4-dimethylaminopyridine, and the mixture was allowed to warm
to room temperature. The reaction mixture was stirred for 0.5 h
and 25 mL of ethyl acetate was added. The solution was washed
with saturated NaHCO3, dried over anhydrous Na2SO4, filtered and
concentrated. The crude was purified by column chromatography
(80% ethyl acetate/hexane) to give 7k (32.5 mg, 100%). 1H NMR
(CDCl3): 7.75 (d, J ) 8.1 Hz, 1H), 7.5-7.57 (m, 3H), 6.94-7.12
(m, 4H), 3.60 (s, 3H), 2.72 (s, 3H), 2.42 (s, 3H). HRMS calcd for
C18H19N4O (M + H+), 307.1560; found, 307.1553.
(d, J ) 8.1 Hz, 1H), 7.55 (ddd, J ) 8.1, 6.3 and 1.8 Hz, 1H),
6.98-7.11 (m, 3H), 6.66-6.76 (m, 2H), 3.83 (s, 3H), 3.54 (s, 3H),
2.73 (s, 3H). HRMS calcd for C17H17FN3O (M + H+), 298.1357;
found, 298.1346.
N-(3,4-Dimethoxyphenyl)-N,2-dimethylquinazolin-4-amine (7n).
Compound 7n was prepared from 9h (288 mg, 0.98 mmol) and
methyl iodide (0.094 mL, 1.5 mmol) and was isolated as off white
solids (70 mg, 23%). 1H NMR (CDCl3): 7.75-7.72 (m, 1H),
7.56-7.50 (m, 1H), 7.05-6.94 (m, 2H), 6.85 (d, J ) 7.5 Hz, 1H),
6.76-6.71 (m, 2H), 3.92 (s, 3H), 3.78 (s, 3H), 3.61 (s, 3H), 2.73
(s, 3H). Anal. (C16H14N4O2) C, H, N.
N-(6-Methoxypyridin-3-yl)-N,2-dimethylquinazolin-4-
amine (7o). Compound 7o was prepared from 9i and methyl iodide
and was isolated as a solid (28%). 1H NMR (CDCl3): 8.03 (d, J )
2.7 Hz, 1H), 7.77 (m, 1H), 7.56 (ddd, J ) 8.1, 6.3 and 1.8 Hz,
1H), 7.38 (dd, J ) 8.7 and 3.0 Hz, 1H), 7.01 (m, 2H), 6.76 (d, J
) 9.0 Hz, 1H), 3.96 (s, 3H), 3.59 (s, 3H), 2.73 (s, 3H). HRMS
calcd for C16H17N4O (M + H+), 281.1404; found, 281.1400.
N-(5-Methoxypyridin-2-yl)-N,2-dimethylquinazolin-4-
amine (7p). Compound 7p was prepared from 9j and methyl iodide
and was isolated as a solid (10%). 1H NMR (CDCl3): 8.31 (d, J )
3.3 Hz, 1H), 7.80 (d, J ) 8.4 Hz, 1H), 7.58 (ddd, J ) 8.4, 6.6 and
1.5 Hz, 1H), 7.13 (dd, J ) 9.0 and 3.3 Hz, 1H), 6.99-7.10 (m,
2H), 6.82 (d, J ) 9.0 Hz, 1H), 3.87 (s, 3H), 3.70 (s, 3H), 2.76 (s,
3H). HRMS calcd for C16H17N4O (M + H+), 281.1404; found,
281.1384.
N-(6-Dimethylaminopyridin-3-yl)-N,2-dimethylquinazolin-4-
amine (7q). Compound 7q was prepared from 9k (45 mg, 0.16
mmol) and methyl iodide (0.016 mL, 0.24 mmol) and was isolated
as a pale yellow solid (22 mg, 47%). 1H NMR (CDCl3): 8.07 (d, J
) 2.4 Hz, 1H), 7.63 (dd, J ) 8.4 and 0.9 Hz, 1H), 7.56-7.51 (m,
1H), 7.27-7.18 (m, 2H), 7.05-7.00 (m, 1H), 6.50 (d, J ) 9.3 Hz,
1H), 3.55 (s, 3H), 3.12 (s, 6H), 2.72 (s, 3H). HRMS calcd for
C17H20N5 (M + H+), 294.1720; found, 294.1693.
N-(5-Methoxypyrazin-2-yl)-N,2-dimethylquinazolin-4-
amine (7r). Compound 7r was prepared from 9l (106 mg, 0.40
mmol) and methyl iodide and was isolated as a solid (63 mg, 56%).
1H NMR (CDCl3): 8.06 (d, J ) 1.2 Hz, 1H), 7.85-7.81 (m, 1H),
7.74 (d, J ) 1.5 Hz, 1H), 7.65-7.60 (m, 1H), 7.17-7.05 (m, 2H),
3.94 (s, 3H), 3.70 (s, 3H), 2.78 (s, 3H). HRMS calcd for C15H16N5O
(M + H+), 282.1356; found, 282.1355.
N-(5-Methoxypyrimidin-2-yl)-N,2-dimethylquinazolin-4-
amine (7s). Compound 7s was prepared from 9m (170 mg, 0.64
mmol) and methyl iodide and was isolated as a solid (141 mg, 50%).
1H NMR (CDCl3) 8.12 (s, 2H), 7.91 (d, J ) 8.4 Hz, 1H), 7.74-7.69
(m, 1H), 7.38-7.34 (m, 1H), 7.30-7.24 (m, 1H), 3.85 (s, 3H),
3.78 (s, 3H), 2.83 (s, 3H). Anal. (C15H15N5O·0.7H2O) C, H, N.
N-(4-Hydroxyphenyl)-N,2-dimethylquinazolin-4-amine (7 h).
To a solution of 6h (106 mg, 0.336 mmol) in dichloromethane (10
mL) at -78 °C was added slowly boron tribromide (1 M in CH2Cl2,
0.75 mL) under argon. The cold bath was removed, and the reaction
mixture was allowed to warm up slowly to 10 °C in 1.5 h. The
reaction mixture was quenched with water (10 mL), basified with
2 N NaOH to pH ) 10, and extracted with ethyl acetate (2 × 25
mL). The ethyl acetate extracts were dried and evaporated to give
a light brown residue. The crude was purified by column chroma-
tography (SiO2, ethyl acetate:hexanes/15-50%) to give 7h as a
Supporting Information Available: Table of elemental analysis
and HPLC data for the targeted compounds 6c-6m, 7a-7s and
9a. Caspase activation assays, growth inhibition assays, tubulin
inhibition assay, cell cycle analysis, MX-1 tumor model studies of
6h, and determination of brain/plasma AUC ratio of 6h and related
analogues. This material is available free of charge via the Internet
at http://pubs.acs.org.
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