Discovery of a New Class of Anilinoquinazoline Inhibitors with High Affinity and Specificity for the Tyrosine Kinase Domain of c-Src

Article abstract of DOI:10.1021/jm030317k

Deregulated activity of the nonreceptor tyrosine kinase c-Src is believed to result in signal transduction, cytoskeletal and adhesion changes, ultimately promoting a tumor-invasive phenotype. We report here the discovery of a new class of anilinoquinazoline inhibitors with high affinity and specificity for the tyrosine kinase domain of the c-Src enzyme. Special attention was directed toward finding inhibitors selective against KDR tyrosine kinase in order to ensure that the in vivo profile of a specific Src inhibitor could be determined. The 4-aminobenzodioxole quinazoline series gave compounds with excellent potency and selectivity. The most interesting compounds were evaluated in vivo and displayed good pharmacokinetics following oral dosing. Compounds such as the aminobenzodioxoles were shown to be potent inhibitors of tumor growth in a c-Src-transformed 3T3 xenograft model in vivo, resulting in more than 90% growth inhibition at doses as low as 6 mg/kg po once daily. Src tyrosine kinase inhibitors such as these may provide a novel therapeutic modality for targeting cancer invasion and metastasis.

Full text of DOI:10.1021/jm030317k

J . Med. Chem. 2004, 47, 871-887
871
Discover y of a New Cla ss of An ilin oqu in a zolin e In h ibitor s w ith High Affin ity
a n d Sp ecificity for th e Tyr osin e Kin a se Dom a in of c-Sr c
Patrick A. Ple´,*,† Tim P. Green,‡ Laurent F. Hennequin,† J on Curwen,‡ Michael Fennell,‡ J ack Allen,‡
Christine Lambert-van der Brempt,† and Gerard Costello‡
AstraZeneca, Centre de Recherches, Z.I.S.E. La Pompelle B.P.1050, 51689 Reims Cedex 2, France, and
AstraZeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.
Received J une 30, 2003
Deregulated activity of the nonreceptor tyrosine kinase c-Src is believed to result in signal
transduction, cytoskeletal and adhesion changes, ultimately promoting a tumor-invasive
phenotype. We report here the discovery of a new class of anilinoquinazoline inhibitors with
high affinity and specificity for the tyrosine kinase domain of the c-Src enzyme. Special attention
was directed toward finding inhibitors selective against KDR tyrosine kinase in order to ensure
that the in vivo profile of a specific Src inhibitor could be determined. The 4-aminobenzodioxole
quinazoline series gave compounds with excellent potency and selectivity. The most interesting
compounds were evaluated in vivo and displayed good pharmacokinetics following oral dosing.
Compounds such as the aminobenzodioxoles were shown to be potent inhibitors of tumor growth
in a c-Src-transformed 3T3 xenograft model in vivo, resulting in more than 90% growth
inhibition at doses as low as 6 mg/kg po once daily. Src tyrosine kinase inhibitors such as
these may provide a novel therapeutic modality for targeting cancer invasion and metastasis.
In tr od u ction
data suggest that deregulated c-Src tyrosine kinase
activity is associated predominantly with adhesion and
cytoskeletal changes in tumor cells, ultimately resulting
in a change to a motile, invasive phenotype. c-Src
tyrosine kinase activity has been shown to be an
important component in the epithelial to mesenchymal
transition that occurs in the early stages of invasion of
carcinoma cells.4 Increased c-Src tyrosine kinase activity
results in breakdown of E-cadherin-mediated epithelial
cell:cell adhesion,5,6 which can be restored by Src
inhibition.6 c-Src activity is also known to be essential
in the turnover of focal adhesions,7 a critical cell motility
component. In in vivo models of metastasis, c-Src
inhibition markedly reduced the rate of lymph and liver
metastasis.6,8 Evidence from the clinic also supports a
link between deregulated c-Src activity and increased
invasive potential of tumor cells. In colon tumors,
increased c-Src kinase activity has been shown to
correlate with tumor progression, with the highest
activity found in metastatic tissue.3c Increased Src
activity in colon tumors has also been shown to be an
indicator of poor prognosis.9 In breast3d,f and ovarian10
cancers, enhancement of Src kinase activity has been
reported, and in transitional cell carcinoma of the
bladder, c-Src activity peaked as superficial tumors
became muscle invasive.11
The identification, more than 25 years ago, of v-Src
tyrosine kinase as the transforming element of the
oncogenic Rous Sarcoma retrovirus, and the subsequent
findings demonstrating the oncogenic potential of the
normal cellular homologue c-Src,1 played a major role
in the understanding of signal transduction pathways
and oncogenic transformation. c-Src kinase, a non-
receptor tyrosine kinase, is the best understood member
of a family of closely related kinases. Three members
of this family, c-Src, c-Yes, and Fyn are ubiquitously
expressed while other family members (six known) have
a more restricted expression. c-Src is expressed at low
levels in most cell types and, in the absence of appropri-
ate extracellular stimuli, maintained in an inactive
conformation through phosphorylation of a regulatory
tyrosine domain at Tyr530. Activation of c-Src occurs
through dephosphorylation of the Tyr530 site and
phosphorylation of a second tyrosine, Tyr416, which is
present within the kinase domain of the enzyme.
Platelets, osteoclasts, and neural cells are the only
normal cell types known to contain high levels of Src
kinase. c-Src gene knock-out experiments in mice have
shown that the only phenotypic consequence is osteo-
petrosis, a defect in osteoclast function.2 In contrast to
its highly regulated role in normal cells, there is
significant evidence demonstrating deregulated, in-
creased kinase activity of c-Src in several human tumor
types, most notably colon and breast tumors.3 The
consequence of this deregulated activity in the genesis
of cancer is still to be tested in clinical trials. Recent
Src kinase has been, and still is, one of the most
studied cellular protein tyrosine kinases, yet no inhibi-
tor has reached the market either for osteoporosis by
inhibiting osteoclast function12 or for cancer by targeting
tumor growth,13 cell adhesion, and mobility. In this
paper, we describe the synthesis and structure-activity
relationships of several new series of anilinoquinazo-
lines as potent inhibitors of tyrosine phosphorylation
by the c-Src kinase, with potential as therapeutic agents
in invasive cancers.
* To whom correspondence should be addressed. Phone:
+33 03 26 61 68 68. Fax: +33 03 26 61 68 54. E-mail: patrick.ple@
astrazeneca.com.
† AstraZeneca, Centre de Recherches.
‡ AstraZeneca Pharmaceuticals.
10.1021/jm030317k CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/14/2004

872 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Ple´ et al.
Sch em e 1a
Sch em e 4a
a
Reagents: (a) hexamethylenetetramine/TFA/115 °C micro-
waves; (b) diethyl bromomalonate/K2CO3/Bu4NBr reflux; (c) KOH/
EtOH reflux; (d) CuO/quinoline 210 °C; (e) Raney Ni/hydrazine
hydrate 55 °C.
a
Reagents: (a) PPh3/(1-(3-hydroxypropyl)morpholine 30/DEAD
rt; (b) NH3/methanol rt; (c) SOCl2/DMF 80 °C; (d) Ar-NH2/iPrOH/
HCl gas 80 °C or Ar-NH2/NaHMDS rt.
Sch em e 2a
Sch em e 5a
a
Reagents: (a) 2-chloro-5-methoxyaniline/iPrOH/HCl gas 80 °C.
Sch em e 3a
a
Reagents: (a) NaH/EtI; (b) concd H2SO4/NaNO2/water 0 °C;
(c) allyl bromide/base rt; (d) 230 °C; (e) ozone/0 °C then H3PO4
100 °C; (f) KOH/water/reflux; (g) DPPA/Et3N/tBuOH reflux then
TFA 0 °C.
Sch em e 6a
a
Reagents: (a) DPPA/Et3N/tBuOH reflux; (b) TFA 0 °C.
Ch em istr y
Most of the anilinoquinazolines (1-6, 8-24) described
here were prepared as shown in Scheme 1. The basic
side chain in the C-7 position of the quinazoline was
generally added on the quinazoline 3114 by Mitsunobu
reaction followed by removal of the POM protecting
group with ammonia. Chlorination of the quinazoline
33 gave the versatile 4-chloroquinazoline 34, which was
extensively used for optimization of the aniline. Intro-
duction of the desired aniline was accomplished using
a secondary alcohol as solvent and HCl gas as catalyst.
The chloroquinazoline 36,15 having an N-methylpiperi-
dine side chain, was reacted similarly with 2-chloro-5-
methoxyaniline to give 7 (Scheme 2).
Ben zofu r a n Syn th eses. The 4-aminobenzofuran 39
was easily prepared from the known benzofuran-4-
carboxylic acid 3716 by Curtius rearrangement followed
by cleavage of the Boc group (Scheme 3). 4-Amino-5-
chlorobenzofuran 45 was prepared by formylation of
4-chloro-3-nitrophenol 40 followed by cyclization with
diethylbromomalonate, saponification of the remaining
ester, decarboxylation, and reduction of the nitro group
(Scheme 4). The 6-chloro-7-amino-benzofuran 54 was
made by esterification of the 2-amino-6-chlorobenzoic
acid 47 followed by diazotisation to give the phenol 49,
which was alkylated with allyl bromide before being
submitted to a Claisen rearrangement to give 51.
Ozonolysis of the double bond gave the aldehyde, which
was cyclized into the benzofuran 53. Curtius rearrange-
ment of 53 followed by deprotection of the Boc group
gave the desired aniline 54 (Scheme 5). 3-Chloro-7-
aminobenzofuran 58 was synthesized from the
7-nitrobenzofuran 5517 by addition of chlorine followed
a
Reagents: (a) AcOH/Cl2 18 °C; (b) KOH/EtOH rt; (c) Raney
Ni/hydrazine hydrate 60 °C.
Sch em e 7a
a
Reagents: (a) NaH/MeI rt; (b) 10% Pd/C/H2.
by elimination of HCl and reduction of the nitro group
(Scheme 6).
In d ole Syn th eses. Nitroindole 59 was simply N-
methylated and hydrogenated to give 4-amino-1-meth-
ylindole 61 (Scheme 7). The other indoles used were
commercially available or have been previously de-
scribed in the literature (see Experimental Section).
Ben zodioxole Syn th eses. 2,3-Dihydroxybenzoic acid
62 was esterified with methanol and the catechol
cyclized into a benzodioxole ring with dibromomethane.
Saponification followed by Curtius rearrangement and
deprotection of the Boc group gave the 2,3-methylene-
dioxyaniline 67 (Scheme 8). Similar routes were used
for the 5-halogeno-3-benzodioxol-4-amines 71, 75, and
83.
Qu in a zolin e Syn th eses. 7-Fluoro-3,4-dihydroquin-
azolin-4-one 8418 was reacted with sodium benzylate
and then with phosphorus pentasulfide to give the
thione 86. Alkylation with methyl iodide followed by
removal of the benzyl group and Mitsunobu coupling

A New Class of Anilinoquinazoline Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 873
Sch em e 8a
a
Reagents: (a) H2SO4/MeOH 60 °C; (b) KF/CH2Br2; (c) NaOH rt; (d) DPPA/Et3N/tBuOH reflux; (e) HCl or TFA 0 °C; (f) LDA/CO2
-78 °C; (g) AcOH/HBr 130 °C; (h) SOCl2/MeOH; (i) CsCO3/CH2BrCl 110 °C; (j) KOH aq rt.
Sch em e 9a
Sch em e 11a
a
Reagents: (a) Na/BnOH 80 °C; (b) P2S5/pyridine reflux; (c)
NaOH/MeI rt; (d) TFA reflux; (e) DEAD/PPh3/4-(3-hydroxypro-
pyl)morpholine rt.
a
Reagents: (a) 1-(3-chloropropyl)piperidine/K2CO3 90 °C; (b)
Sch em e 10a
NH3/MeOH rt; (c) SOCl2 DMF reflux; (d) NaHMDS/aniline 71 rt.
Sch em e 12a
a
Reagents: (a) Bu4NF rt; (b) HCl/iPrOH/aniline 71; (c) TFA
reflux; (d) DEAD/PPh3/96 rt.
a
Reagents: (a) HCl reflux; (b) SOCl2 80 °C; (c) aniline 71/iPrOH/
HCl gas 100 °C.
chloride, and aniline coupling under basic conditions
using NaHMDS as a base. Finally, coupling the aniline
71 onto the quinazoline 9814 followed by deprotection
of the benzyl group gave 100 (Scheme 12). Deprotection
of the TBDMS group on the cyanopyridine 9622 gave 97,
which was coupled with 100 via a Mitsunobu reaction
to give 29.
Biologica l Eva lu a tion Over view . Compounds were
tested sequentially in our in vitro cascade and had to
meet the following criteria to progress to the next test:
Src enzyme <0.1 µM, KDR g1 µM (vide infra), and Src-
3T3 and A549 cells <1 µM. The Src-3T3 proliferation
test uses a mouse fibroblast cell line that has been
transfected with constitutively active human c-Src ki-
with 4-(3-hydroxypropyl)morpholine19 gave the desired
quinazoline intermediate 89 ready for coupling with an
aniline (Scheme 9). The 4-chloro-7-methoxy-6-(3-mor-
pholinopropoxy)quinazoline 92 was prepared most con-
veniently by hydrolysis of compound 9020 to get the
quinazolone 91 followed by chlorination with thionyl
chloride (Scheme 10). Reaction of 92 with the 5-chloro-
3-benzodioxol-4-amine 71 gave the desired anilino-
quinazoline 26. One of our lead compounds 28 (Scheme
11) was made by alkylation of the 7-hydroxyquinazolone
3114 with 1-(3-chloropropyl)piperidine21 followed by
deprotection of the POM group, treatment with thionyl

874 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Ta ble 1. Initial Lead Src Inhibitors
Ple´ et al.
IC50 (µM)a
enzyme inhibition
R
cell
no.
2′
3′
4′
6′
side chains
Src
KDR
Src 3T3
A549
1.05
1
2
3
4
5
6
7
MeO
MeO
Me
0.253
0.037
0.560
0.477
0.010
0.963
0.010
4-morpholinyl-(CH2)3
4-morpholinyl-(CH2)3
4-morpholinyl-(CH2)3
4-morpholinyl-(CH2)3
4-morpholinyl-(CH2)3
4-N-methylpiperidinyl-CH2
1.11
0.69
MeO
MeO
MeO
EtO
MeO
Cl
Cl
Cl
1.18
0.67
0.31
0.53
0.31
0.48
a
Values are mean from at least three independent dose-response curves; variation was generally (15% for Src and KDR enzymes
and (20% in 3T3 and A549 cells.
nase, allowing it to grow in a low serum concentration
(0.5% fetal calf serum) incompatible with the growth of
wild-type 3T3. In the second cell test, A549 (human non-
small-cell lung cancer) cells were used in a migration
assay. To prove that inhibition of cell migration was not
due to a simple cytotoxic effect, cytotoxicity studies
using MTT as a marker were routinely performed (see
Experimental Section).
allow investigation of the in vivo effects of Src inhibition.
Interestingly, the replacement of the methoxy group by
an ethoxy group (6, 0.963 µM) substantially decreased
the activity of the molecule. The enzymatic activity of
5 transposed well into our proliferation and migration
cell assays (0.3 µM against both cell lines). C-7 side
chain variations around 5 led to 7, which gave high
blood levels in mice up to 24 h (12 µM at 24 h from a
50 mg/kg oral dose). Compound 7 (also known as
AZM475271) was used extensively to develop our in vivo
cascade and to provide a benchmark profile for Src
kinase inhibition.
Resu lts a n d Discu ssion
For clarity of discussion, data on only a limited,
representative set of compounds are used to describe
the structure-activity relationship. Where trends are
exemplified by a single pair of compounds, it is to be
understood that more examples23 exist to support the
structure-activity relationship described below. To
make the relationship clearer we have deliberately
chosen compounds with only one type of side chain
(morpholinopropoxy) in the C-7 position of the quinazo-
line. Several of the compounds tested in our in vitro
cascade met our criteria of Src enzyme <0.1 µM, KDR
g1 µM (vide infra), and Src-3T3 and A549 cells <1 µM.
When performing proliferation and migration cell as-
says, no toxicity was observed with any of our inhibitors
at 10-20 µM.
Meth oxy An ilin es Ser ies. High-throughput screen-
ing gave us our first lead (1, Table 1) belonging to the
anilinoquinazoline family from which the EGFR-TKI
(epidermal growth factor receptor-tyrosine kinase in-
hibitor) gefitinib (IRESSA, ZD1839)24 and the angio-
genesis inhibitor ZD647415 had previously been devel-
oped. Replacement of the methoxy group in the C-7
position of the quinazoline by polar side chains, such
as a morpholinopropoxy (2), gave a significant increase
in enzyme potency (1, 0.253 µM; 2, 0.037 µM) and
confirmed the m-methoxyanilino-quinazoline as being
an interesting starting point. Directed robotic syntheses
allowed us to explore the substitution pattern as well
as the influence of other substituents on the aniline ring.
The methoxy group in the meta position proved optimal
(compare 2 with 3 and 4), while the addition of a
chlorine atom in the ortho position gave 5, a very potent
Src inhibitor selective versus KDR (0.010 µM, 100-fold
selective versus KDR). A mixed Src-KDR inhibitor could
be very advantageous, but only a selective agent could
Bicyclic An ilin es. In an attempt to gain insight
into the structural basis of the inhibitory activity of
our methoxy aniline series, we performed molecular
modeling and docking studies using the available 3D
structure of activated Lck, a closely related Src family
member.25,26 On the basis of in-house studies and
published experimental data, we found a binding mode
consistent with the structure-activity relationship data
(Figure 1).14,27 In this model, the quinazoline ring
occupies the adenine binding site. The highly conserved
hydrogen bond is created by N1 of the quinazoline,
which binds to the backbone amide of Met319 (Met341
in Src). The 3′-methoxy anilino group is buried in the
hydrophobic pocket adjacent to the adenine site. It is
clear from the model that the methoxy group adopts a
particular conformation, coplanar with the aromatic
ring and directed toward position 2′ rather than 4′ of
the aniline. This latter orientation results in steric
clashes with the proximal Ile314 and Lys273 (Ile336 and
Lys295 in Src). From this observation, it was therefore
tempting to make cyclic analogues such as benzofurans.
The simple 4-anilinobenzofuran 8 (Table 2) proved to
be more potent than the corresponding methoxy aniline
2, but the introduction of a chlorine atom in position 5′
of this heterocycle (9) did not give the increase in
activity observed between 2 and 5. Interestingly, the
7-anilinobenzofuran 11 had the same level of activity
as its regioisomer 9, even though the oxygen atom was
no longer in the same place as in the methoxyaniline 5.
This could reflect the favorable environment created by
the proximal Thr316 (Thr338 in Src), which lies at the
entrance to the selectivity pocket (Figure 1). Reduction
of the chlorobenzofuran gave 12, which showed reduced

A New Class of Anilinoquinazoline Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 875
F igu r e 1. Stereoview of compound 5 docked into the ATP-binding site of Lck. The protein is represented as a ribbon diagram
colored by secondary structure, with side chains of some selected residues shown in stick representation. The inhibitor is represented
with van der Waals spheres colored by element. The quinazoline ring occupies the adenine binding site. The H-bond interaction
between the quinazoline N1 and backbone NH of Met319 (Met341 in Src) is represented as a dotted line. The anilino moiety of
5 is buried into the hydrophobic pocket, characterized by a Thr residue at its entry (Thr316 in Lck, Thr338 in Src). It is clear from
the model that the methyl of the 3′-OCH3 points toward position 2′ rather than 4′ of the aniline, this latter orientation leading
to steric clashes with the proximal Ile314 and Lys273 (Ile336 and Lys295 in Src).
Ta ble 2. Benzofuran and Indole Inhibitors
F igu r e 2. 3′-Chlorobenzofuran derivative 13. The benzofuran
and the benzodioxole series have been docked into the ATP
binding site of Lck and fit well. The binding site is represented
by its molecular surface,28 and the inhibitors are illustrated
in stick representation and colored by element.
pocket further suggested that small substituents at the
3′-position of the 7-anilinobenzofurans might be allowed
(Figure 2), and indeed, introduction of the 3-chloro-7-
IC50 (µM)a
aminobenzofuran on the quinazoline nucleus gave a
remarkably potent molecule (13), still quite potent
against KDR as well.
enzyme
inhibition
cell
Benzofurans were then replaced by indoles (Table 2).
The 4-aminoindole 14 gave a moderate level of Src
inhibition, but N-methylation (15), to mimic the chlorine
atom on the benzofuran 13, significantly improved the
potency. The 7-aminoindole regioisomer 16 behaved
similarly to 14, and the 3-chloro-7-aminoindole 17, the
most potent of the indole series, fell short of the activity
found for the 3-chloro-7-aminobenzofuran 13. Moreover,
its IC50 against A549 cells was found to be higher than
expected.
The work on the aminobenzofurans taught us two
things: first, that the heterocyclic ring must be planar
(cf. 12 vs 10) and, second, that the oxygen atom was
equally good in the 1′- or 3′-position (8 vs 10 and 9 vs
11). This encouraged the design of a benzodioxole
heterocycle with two oxygen atoms and a pseudoplanar
conformation. As expected, modeling of this compound
no.
H2N-Ar
Src
KDR Src 3T3 A549
8
9
benzofuran A, no substituent
bnenzofuran A, 5-chloro
0.016 0.25
0.010 1.23
0.030 0.41
0.010 1.38
0.433
<0.004 0.10
0.177
0.040 0.42
0.260
0.020 2.32
0.31
0.34
0.23
0.26
10 benzofuran B, no substituent
11 benzofuran B, 6-chloro
12 benzofuran B, 2,3-dihydro
13 benzofuran B, 3-chloro
14 indole A, R ) H
15 indole A, R ) Me
16 indole B, no substituent
17 indole B, 3-chloro
0.38
0.98
a
Values are mean from at least three independent dose-
response curves; variation was generally (15% for Src and KDR
enzymes and (20% in 3T3 and A549 cells.
activity compared with 10, proving the need for a planar
geometry in that region as suggested by the shape of
the corresponding hydrophobic pocket. The study of this

876 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Ple´ et al.
chlorine in the 5′-position lies in a hydrophobic pocket
lined by an alanine residue (Ala381 in Lck, Ala403 in
Src), whereas in KDR this residue is a cysteine
(Cys1045), which would be sterically and electronically
less favorable.
Replacement of chlorine by bromine (20) again gave
good results, but fluorine (21), being smaller, gave
results similar to the parent benzodioxole (18). Replace-
ment of the methylene bridge of the benzodioxole by a
difluoromethylene (22) or changing the position of the
amino group from the 4′- to the 5′-position (23) resulted
in decreased activity. Enlargement of the dioxole into a
dioxane ring (24) was also detrimental due to the loss
of the planarity of the ring, similar to what we observed
between the benzofuran 10 and the dihydrobenzofuran
12. Removal of the C-6 methoxy group on the quinazo-
line (25) or switching around the C-7 side chain with
the C-6 methoxy group on the quinazoline (26) gave
compounds that were generally less potent against both
Src enzyme and in cells.
F igu r e 3. 5′-Chlorobenzodioxole derivative 19. The binding
site is represented by its molecular surface,28 the inhibitors
are illustrated in stick representation and colored by element.
Ta ble 3. Benzodioxole Inhibitors
The C-7 quinazoline side chain was then varied
in order to optimize the in vitro and in vivo potency
and physical properties while the chlorobenzodioxole
aniline was kept constant. Good in vitro potency, as
well as selectivity, could be achieved with basic or
nonbasic side chains and some representative exam-
ples are given in Table 4. As expected, basic side chains
(27, 28) gave compounds with a good solubility. The
aminobenzodioxole series gave particularly high free
drug levels in blood. For example, protein binding for
the chloromethoxyaniline 5 results in 4.6% free drug in
rat serum, whereas the chlorobenzodioxole 19 is 10.5%
free. As previously observed in our research program
on VEGF (vascular endothelial growth factor) tyrosine
kinase inhibitors, the basic piperidines gave good and
sustained blood levels in rats dosed orally at 20 mg/kg
coupled with a good to excellent bioavailability (Table
5). Following iv administration, clearance of the com-
pounds was moderate (equivalent to 50% and 68%
hepatic blood flow), while the elimination half-life values
(∼5.8 h) were determined mainly by the high volume
of distribution (15-20 L/kg).
The best compounds were further investigated in
a rat xenograft model based on the same 3T3 Src
transfected cell line as in the in vitro assay. Results
obtained with 28 provide a good example of the level
of activity achieved with such compounds in rat
xenografts. Inhibition of tumor growth was dose-
dependent with almost complete inhibition achieved at
doses as low as 6 mg/kg po once daily (Figure 4). Some
of these compounds have also proven to be potent
inhibitors of metastasis formation in several animal
models and these results will be published elsewhere.
IC50 (µM)a
enzyme
inhibition
Src KDR Src 3T3 A549
18 benzodioxole A, no substituent 0.014 4.96 0.31
cell
no.
H2N-Ar
0.21
0.18
0.14
0.45
19 benzodioxole A, 5-chloro
20 benzodioxole A, 5-bromo
21 benzodioxole A, 5-fluoro
22 benzodioxole A, 2,2-difluoro
23 benzodioxole B
24 benzodioxane
<0.004 17.03 0.10
<0.004 42.64 0.06
0.028 8.16 0.30
0.270
0.760
0.370
25
26
0.010 91.35 0.45
0.050 73.82 1.16
0.67
1.20
a
Values are mean from at least three independent dose-
response curves; variation was generally (15% for Src and KDR
enzymes and (20% in 3T3 and A549 cells.
suggested it would fit particularly well into the active
site (Figure 3). The first benzodioxole (18) made gave
very encouraging results (Table 3). It was as potent as
5 and more selective for Src. The introduction of a
chlorine atom in the 5′-position (19) increased the
activity against Src still further and gave us even higher
selectivity (KDR:Src ratio > 4000). Moreover, 19 was
very potent in cells (3T3, 0.09 µM; A549, 0.18 µM).
Superimposing our Lck model onto the crystal structure
of KDR and comparing their amino acid sequences
allowed us to rationalize the selectivity profile of this
series.29 In KDR the residue at the entrance to the
selectivity pocket is a Val916, which gives less favorable
contacts with the benzodioxole oxygens than the corre-
sponding Thr316 in Lck or Thr338 in Src. Moreover, the
Con clu sion s
We have discovered several new anilinoquinazoline
series that are potent Src inhibitors. Among these, the
aminobenzodioxole quinazolines have been identified as
providing the optimum profile of selectivity, enzyme
inhibition, and cellular potency. These compounds gave
high and prolonged blood levels when dosed orally and
have been shown to markedly reduce the rate of Src-
driven tumor cell growth in a rat xenograft model.

A New Class of Anilinoquinazoline Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 877
Ta ble 4. Optimization of the C-7 Side Chain in the Chlorobenzodioxole Series
a
Values are mean from at least three independent dose-response curves; variation was generally (15% for Src and KDR enzymes
and (20% in 3T3 and A549 cells.
Ta ble 5. Rat Pharmacokinetic Parameters in the
Chlorobenzodioxole Series
po (20 mg/kg)
iv (2 mg/kg)
Cl
Cmax AUC0-∞ C24h term t1/2
Vdss
F
no. (µM) (µg h/mL) (µM)
iv (h)
(mL/min/kg) (L/kg) (%)
27 0.91
28 2.96
2.5
7.9
0.06
0.08
5.7
5.8
49.0
36.0
17.0 37
14.4 86
Exp er im en ta l Section
All experiments were carried out under an inert atmosphere
and at room temperature unless otherwise stated. Flash
chromatography was carried out on Merck Kieselgel 50
(Art. 9385). The purities of compounds for biological testing
were assessed by analytical HPLC on a Hichrom S5ODS1
Spherisorb Column System set to turn isocratically with
60-70% MeOH + 0.2% CF3COOH in water as eluent. TLCs
were performed on precoated silica gel plates (Merck
Art. 5715), and the resulting chromatograms were visualized
under UV light at 254 nm. Purification by preparative HPLC/
MS was done on a Waters LC/MS system using a Waters
Symmetry column (C18, 5 µm, 19 mm diameter, 100 mm
length) using a mixture of water with 1% acetic acid and
acetonitrile (gradient from 5% to 100%) as solvent. The NMR
spectra were obtained on a J EOL J NM EX 400 (400 MHz)
spectrometer. Chemical shifts are expressed in unit of δ (ppm),
and peak multiplicities are expressed as follows: s, singlet; d,
doublet; dd, doublet of doublet; t, triplet; br s, broad singlet;
m, multiplet. Mass spectrometry was done on an analytical
Waters LC/MS system with positive and negative ion data
collected automatically. NMR and mass spectra were run on
isolated intermediates and final products and are consistent
with the proposed structures. For the microanalysis, all the
adducts mentioned were measured: water was measured by
the Karl-Fisher method using a Mettler DL 18; HCl content
was determined on a Metrohm 686 by titration using silver
nitrate solution, and the organic adducts were measured
by 1H NMR. The following abbreviations have been used:
ADDP, 1,1′-(azodicarbonyl)dipiperidine; Boc, tert-butoxycar-
bonyl; DEAD, diethyl-azodicarboxylate; DMF, N,N-dimethyl-
formamide; DMSO, dimethyl sulfoxide; DPPA, diphenylphos-
phoryl azide; Gold’s reagent, [3-(dimethylamino)-2-azaprop-
2-en-1-ylidene]dimethylammonium chloride; NaHMDS, sodium
F igu r e 4. Compound 28 dosed orally once daily from day 0
to Src 3T3 tumor-bearing nude rats. Calliper measurements
started on day 4.
bis(trimethylsilyl)amide; POM, pivaloyloxymethyl; TFA, tri-
fluoracetic acid.
6,7-Dim e t h oxy-N -(3-m e t h oxyp h e n yl)q u in a zolin -4-
a m in e (1). A mixture of 4-chloro-6,7-dimethoxyquinazoline30
(0.177 g, 0.8 mmol), 3-methoxyaniline (0.106 mL, 0.95 mmol),
2-propanol (6 mL), and a saturated solution of HCl gas in
2-propanol (0.016 mL, 5 N solution) was stirred and heated
at 90 °C for 2 h. Upon cooling to room temperature the formed
precipitate was collected by filtration and washed with a
mixture of ether:2-propanol (1:1) and then with ether. The solid
was dissolved in dichloromethane and washed with a saturated
aqueous solution of sodium bicarbonate. The organic phase was
dried over magnesium sulfate and purified by flash chroma-
tography using dichloromethane:methanol (98.5:1.5 then
98:2) as eluent. Evaporation of the solvent and trituration of
the solid under ether gave 0.15 g of 1 (61%). 1H NMR
(CDCl3): δ 3.85 (s, 3H), 4.05 (s, 6H), 6.70 (dd, 1H), 7.0 (s, 1H),
7.10 (s, 1H), 7.20 (d, 1H), 7.30 (t, 1H), 7.45 (s, 1H), 8.70 (s,
1H). MS-ESI m/z 312 [MH]+. Anal. (C17H17N3O3) C, H, N.
N-(3-Hyd r oxyp r op yl)m or p h olin e (30). Morpholine (94 g,
1.08 mol) was added dropwise to a solution of 3-bromo-1-
propanol (75 g, 0.54 mol) in toluene (750 mL) and the reaction
mixture heated at 80 °C for 4 h. Upon cooling, a solid was
removed by filtration, the volatiles were evaporated, and the

878 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Ple´ et al.
resulting yellow oil was purified by distillation at 0.4-0.7
mmHg to give 30 (40 g, 50%) as a colorless oil. bp 68-70 °C
(∼0.5 mmHg). 1H NMR (DMSO-d6): δ 1.65-1.78 (m, 2H), 2.50
(t, 4H), 2.60 (t, 2H), 3.68 (t, 4H), 3.78 (t, 2H), 4.90 (br d, 1H).
1H), 9.10 (s, 1H). MS-ESI m/z 425 [MH]+. Anal. (C23H28N4O4‚
0.2H2O) C, H, N.
6-Meth oxy-N-(4-m eth oxyp h en yl)-7-(3-m or p h olin -4-yl-
p r op oxy)qu in a zolin -4-a m in e (4) was prepared as described
for the synthesis of 1. 4-Chloro-6-methoxy-7-(3-morpholino-
propoxy)quinazoline 34 (0.15 g, 0.44 mmol) was reacted with
p-anisidine (0.06 mL, 0.53 mmol) in the presence of HCl in
2-propanol (0.1 mL, 0.49 mmol, 5 N solution) to give 0.135 g
of 4 (72%). 1H NMR (DMSO-d6): δ 1.95 (m, 2H), 2.35 (m, 4H),
2.45 (m, 2H), 3.6 (m, 4H), 3.80 (s, 3H), 3.95 (s, 3H), 4.15 (t,
2H), 6.95 (d, 2H, J ) 9.0 Hz), 7.15 (s, 1H), 7.60 (d, 2H, J ) 9.0
Hz), 7.8 (s, 1H), 8.35 (s, 1H), 9.40 (s, 1H). MS-ESI m/z 425
[MH]+. Anal. (C23H28N4O4) C, H, N.
[6-Meth oxy-7-(3-m or ph olin -4-ylpr opoxy)-4-oxoqu in azo-
lin -3(4H)-yl]m eth yl P iva la te (32). 7-Hydroxy-6-methoxy-3-
((pivaloyloxy)methyl)-3,4-dihydroquinazolin-4-one 3114 (12 g,
39.2 mmol) was dissolved in dichloromethane (125 mL) under
argon, and triphenylphosphine (13.3 g, 51 mmol) and 1-(3-
hydroxypropyl)morpholine (6.25 g, 43 mmol) were added
followed by dropwise addition of DEAD (8 mL, 51 mmol) in
dichloromethane (20 mL). After being stirred for 3 h, the
solvent was evaporated and the residue purified by flash
chromatography using increasingly polar solvent mixtures
starting with dichloromethane, then ethyl acetate:dichlo-
romethane (95:5), then pure ethyl acetate, and ending with
dichloromethane:ethyl acetate:methanol (8:1:1). Evaporation
of the solvent gave 17 g (100%) of 32 as a white solid. 1H NMR
(DMSO-d6): δ 1.15 (s, 9H), 1.95 (m, 2H), 2.40 (m, 4H), 2.50 (t,
2H), 3.60 (m, 4H), 3.90 (s, 3H), 4.20 (t, 2H), 5.90 (s, 2H), 7.15
(s, 1H), 7.50 (s, 1H), 8.35 (s, 1H).
N-(2-Ch lor o-5-m et h oxyp h en yl)-6-m et h oxy-7-(3-m or -
p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (5). A mixture of
4-chloro-6-methoxy-7-(3-morpholinopropoxy)quinazoline 34 (6.9
g, 20 mmol), 2-chloro-5-methoxyaniline (3.9 g, 25 mmol),
2-propanol (100 mL), and a saturated solution of HCl gas in
2-propanol (4 mL) was stirred and heated at 80 °C for 2.5 h.
Upon cooling to room temperature the formed precipitate was
collected by filtration and washed with a mixture of ether:
isopropyl alcohol (1:1) and then with ether. The solid was dried
under vacuum overnight at 40 °C to give 10 g of 5 as a
6-Me t h oxy-7-(3-m or p h olin -4-ylp r op oxy)q u in a zolin -
4(3H)-on e (33). A mixture of [6-methoxy-7-(3-morpholin-4-
ylpropoxy)-4-oxoquinazolin-3(4H)-yl]methyl pivalate 32 (17 g,
39 mmol), methanol (65 mL) and a saturated solution of
ammonia gas in methanol (350 mL) was stirred overnight. The
white precipitate that formed was dissolved with dichlo-
romethane, the solution was filtered to remove some insoluble
material, the filtrate was concentrated, and the residue was
triturated under ether:dichloromethane (95:5) and dried under
1
dihydrochloride (94%). H NMR (DMSO-d6 and CD3CO2D): δ
2.35 (m, 2H), 3.1 (m, 2H), 3.3 (m, 2H), 3.5 (m, 2H), 3.8 (s, 3H),
3.9 (m, 2H), 3.95 (m, 2H), 4.05 (s, 3H), 4.35 (m, 2H), 7.05 (dd,
1H, J 1 ) 3.1, J 2 ) 9.0 Hz), 7.15 (d, 1H, J ) 3.0 Hz), 7.45 (s,
1H), 7.55 (d, 1H, J ) 9.0 Hz), 8.3 (s, 1H), 8.8 (s, 1H). MS-ESI
m/z 459 [MH]+. Anal. [C23H27ClN4O4‚0.15H2O‚0.08(2-propanol)‚
2HCl] C, H, N.
1
vacuum to give 12.5 g of 33 (100%) as a white solid. H NMR
N-(2-Ch lor o-5-eth oxyp h en yl)-6-m eth oxy-7-(3-m or p h o-
lin -4-ylp r op oxy)qu in a zolin -4-a m in e (6) was prepared as
described for the synthesis of 1 and purified as described for
23. 4-Chloro-6-methoxy-7-(3-morpholinopropoxy)quinazoline
34 (0.12 g, 0.35 mmol) was reacted with 2-chloro-5-ethoxy-
aniline (0.072 g, 0.42 mmol) in the presence of HCl in
2-propanol (0.08 mL, 0.40 mmol, 5 N solution) to give 0.105 g
of 6 (63%). 1H NMR (CDCl3; CF3CO2D): δ 1.45 (t, 3H), 2.50
(m, 2H), 3.10 (m, 2H), 3.45 (m, 2H), 3.75 (m, 2H), 3.95-4.40
(m, 9H), 4.40 (m, 2H), 6.90 (dd, 1H, J 1 ) 8.5, J 2 ) 3.0 Hz),
7.35 (d, 1H, J ) 3.0 Hz), 7.45 (d, 1H, J ) 8.5 Hz), 7.55 (s, 1H),
(DMSO-d6): δ 1.95 (m, 2H), 2.4 (m, 4H), 2.5 (t, 2H), 3.6 (m,
4H), 3.90 (s, 3H), 4.2 (t, 2H), 7.15 (s, 1H), 7.45 (s, 1H), 8.0 (s,
1H).
4-Ch lor o-6-m e t h oxy-7-(3-m or p h olin -4-ylp r op oxy)-
qu in a zolin e (34). A mixture of 6-methoxy-7-(3-morpholin-4-
ylpropoxy)quinazolin-4(3H)-one 33 (12.5 g, 39 mmol), thionyl
chloride (107 mL), and DMF (0.9 mL) was heated to 80 °C for
1.5 h. The volatiles were removed under vacuum and the
remaining traces of thionyl chloride were eliminated by
azeotropic distillation with toluene using a rotary evaporator.
Dichloromethane was added and the solution was cooled to
0 °C prior to the addition of water and a saturated sodium
bicarbonate solution until pH 8 was reached. The aqueous
phase was extracted with dichloromethane, the organic phase
was combined, washed in turn with water and brine, and dried
over magnesium sulfate. The solvent was evaporated under
vacuum and the residue was purified by column chromatog-
raphy on silica using a dichloromethane:methanol (92:8) as
eluent to give 34 as a white solid (6.9 g, 54%). 1H NMR (DMSO-
d6): δ 1.95 (m, 2H), 2.40 (m, 4H), 2.50 (t, 2H), 3.60 (m, 4H),
4.05 (s, 3H), 4.30 (t, 2H), 7.40 (s, 1H), 7.45 (s, 1H), 8.9 (s, 1H).
7.70 (s, 1H), 8.70 (s, 1H). MS-ESI m/z 473 [MH]+. Anal. (C24H29
-
ClN4O4) C, H, N.
N-(2-Ch lor o-5-m eth oxyp h en yl)-6-m eth oxy-7-[(1-m eth -
ylpiper idin -4-yl)m eth oxy]qu in azolin -4-am in e (7). 4-Chloro-
6-methoxy-7-(1-methylpiperidine-4-ylmethoxy)quinazoline 3615
(49 g, 152 mmol) and 2-chloro-5-methoxyaniline (35.5 g, 183
mmol) were reacted together using the same procedure as the
1
one described for 2 to give 60 g of 7 (89%). H NMR (DMSO-
d6): δ 1.3-1.4 (m, 2H), 1.78 (m, 3H), 1.9 (dd, 2H), 2.15 (s, 3H),
2.8 (d, 2H), 3.8 (s, 3H), 3.95 (s, 3H), 4.0 (d, 2H), 6.9 (d, 1H),
7.15 (m, 2H), 7.48 (d, 1H), 7.8 (s, 1H), 8.3 (s, 1H), 9.5 (bs, 1H).
MS-ESI m/ z 442 [MH]+. Anal. (C23H27ClN4O3) C, H, N.
6-Meth oxy-N-(3-m eth oxyp h en yl)-7-(3-m or p h olin -4-yl-
p r op oxy)qu in a zolin -4-a m in e (2). Using a procedure similar
to the one described for compound 1, 4-chloro-6-methoxy-7-
(3-morpholinopropoxy)quinazoline 34 (0.1 g, 0.3 mmol) and
3-methoxyaniline (0.04 mL, 0.35 mmol) were reacted to give
after workup and purification 0.088 g of 2 (70%). 1H NMR
(DMSO-d6): δ 1.95 (m, 2H), 2.35 (m, 4H), 2.45 (m, 2H), 3.6
(m, 4H), 3.80 (s, 3H), 3.95 (s, 3H), 4.20 (t, 2H), 6.70 (dd, 1H,
J 1 ) 1.8, J 2 ) 7.8 Hz), 7.20 (s, 1H), 7.3 (t, 1H, J 1 ) J 2 ) 7.9
ter t-Bu tyl 1-Ben zofu r a n -4-ylca r ba m a te (38). Benzofu-
ran-4-carboxylic acid 3716 (0.5 g, 2.8 mmol), DPPA (1.2 mL,
5.6 mmol), triethylamine (0.79 mL, 5.6 mmol), and tert-butyl
alcohol (1.5 mL) were heated to reflux for 18 h. Upon cooling
to room temperature the mixture was poured into water and
extracted with ethyl acetate. The organic phase was washed
with water and brine, dried over magnesium sulfate, and
filtered, and the solvent was evaporated. The resulting oil was
purified by flash chromatography using dichloromethane as
solvent. Evaporation of the solvent gave 0.8 g of 38 as an oil
which still contained some DPPA. MS-ESI m/z 256 [MNa]+.
Hz), 7.40 (d, 1H, J 1 ) 1.9, J 2 ) 8.0 Hz), 7.50 (s, 1H, J 1 ) J 2
)
2.2 Hz), 7.85 (s, 1H), 8.45 (s, 1H), 9.45 (s, 1H). Anal.
(C23H28N4O4‚0.3H2O) C, H, N.
6-Meth oxy-N-(2-m eth oxyp h en yl)-7-(3-m or p h olin -4-yl-
p r op oxy)qu in a zolin -4-a m in e (3) was prepared as described
for the synthesis of 1. 4-Chloro-6-methoxy-7-(3-morpholino-
propoxy)quinazoline 34 (0.15 g, 0.44 mmol) was reacted with
o-anisidine (0.06 mL, 0.53 mmol) in the presence of HCl in
2-propanol (0.1 mL, 0.49 mmol, 5 N solution) to give 0.125 g
of 3 (66%). 1H NMR (DMSO-d6): δ 1.95 (m, 2H), 2.35 (m, 4H),
2.45 (m, 2H), 3.6 (m, 4H), 3.80 (s, 3H), 3.95 (s, 3H), 4.20 (t,
2H), 7.0 (t, 1H, J 1 ) J 2 ) 7.8 Hz), 7.15 (m, 2H), 7.25 (t, 1H, J 1
) J 2 ) 7.8 Hz), 7.50 (d, 1H, J ) 7.7 Hz), 7.80 (s, 1H), 8.30 (s,
4-Am in oben zofu r a n (39). tert-Butyl 1-benzofuran-4-yl-
carbamate 38 (0.65 g, 2.7 mmol) was dissolved in dichloro-
methane (20 mL), TFA (2.5 mL) was added at 0 °C, and the
mixture stirred for 1 h at the same temperature and then
1.5 h at room temperature. The volatiles were removed, and
the residue was taken up in a saturated solution of sodium
bicarbonate and extracted with ethyl acetate. The organic
phase was dried over magnesium sulfate and filtered and
the solvent evaporated. The resulting oil was purified by
flash chromatography using dichloromethane:petroleum ether

A New Class of Anilinoquinazoline Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 879
(80:20) as solvent. Evaporation of the solvent gave 0.35 g of
39 (95% over the last two steps). H NMR (DMSO-d6): δ 5.50
(br s, 2H), 6.35 (d, 1H, J ) 8.0 Hz), 6.70 (d, 1H, J ) 8.0 Hz),
6.95 (dd, 1H, J 1 ) 8.0, J 2 ) 8.0 Hz), 7.0 (d, 1H, J ) 2.4 Hz),
7.70 (d, 1H, J ) 2.4 Hz). MS m/z 133 [M]+.
solvent evaporated. The crude product was purified using
dichloromethane:petroleum ether (4:6) as eluent. Evaporation
of the solvent gave 0.037 g of 45 (45%). H NMR (DMSO-d6):
δ 5.80 (br s, 2H), 6.80 (d, 1H, J ) 8.6 Hz), 7.10 (d, 1H, J ) 8.7
Hz), 7.15 (d, 1H, J ) 2.3 Hz), 7.80 (d, 1H, J ) 2.0 Hz).
1
1
N-(1-Ben zofu r a n -4-yl)-6-m eth oxy-7-(3-m or p h olin -4-yl-
p r op oxy)qu in a zolin -4-a m in e (8) was prepared as described
for the synthesis of 1 and purified as described for 23. 4-Chloro-
6-methoxy-7-(3-morpholin-4-ylpropoxy)quinazoline 34 (0.1 g,
0.3 mmol) was reacted with 4-aminobenzofuran 39 (0.059 g,
N-(5-Ch lor o-1-b en zofu r a n -4-yl)-6-m et h oxy-7-(3-m or -
p h olin -4-ylp r op oxy)q u in a zolin -4-a m in e Dih yd r och lo-
r id e (9). 4-Chloro-6-methoxy-7-(3-morpholin-4-ylpropoxy)-
quinazoline 34 (0.059 g, 0.17 mmol), 4-amino-5-chlorobenzofuran
45 (0.035 g, 0.2 mmol), 2-pentanol (4 mL), and HCl in
2-propanol (6 N, 0.038 mL, 0.19 mmol) were heated at 110 °C
under anhydrous conditions for 5 h. Upon cooling, the pre-
cipitate was collected by filtration and washed with ether:2-
propanol (1:1) and then with ether only. The solid was
dissolved in methanol, MP carbonate resin (140 mg) was
added, and the mixture stirred for 2 h. The resin was discarded
and the methanol was evaporated. The crude product was
purified by flash chromatography using first dichloromethane,
then dichloromethane:methanol (95:5), and finally dichlo-
romethane:methanol (92.5:7.5). The solvent was evaporated
and dichloromethane was added followed by addition of a
solution of HCl in ether. The precipitate was collected by
filtration and dried under vacuum to give 0.15 g of 9 (24%).
1H NMR (DMSO-d6 and CF3CO2D): δ 2.35 (m, 2H), 3.15 (m,
2H), 3.35 (t, 2H), 3.55 (d, 2H), 3.70 (t, 2H), 4.05 (m, 5H), 4.35
(t, 2H), 6.95 (dd, 1H, J 1 ) 2.4, J 2 ) 0.9 Hz), 7.40 (s, 1H), 7.60
(d, 1H, J ) 8.8 Hz), 7.80 (dd, 1H, J 1 ) 8.8, J 2 ) 0.9 Hz), 8.15
(d, 1H, J ) 2.4 Hz), 8.25 (s, 1H), 8.80 (s, 1H). MS-ESI m/z 469
[MH]+. Anal. (C24H25ClN4O4‚0.4H2O) C, H, N.
7-Am in oben zofu r a n (46). 7-Nitrobenzofuran17 (0.5 g, 3
mmol), methanol (9 mL), and Raney nickel (20 mg) were
heated to 55 °C. Hydrazine hydrate (0.45 mL, 9 mmol) was
added dropwise and the mixture heated to reflux for 30 min.
The catalyst was removed by filtration, the solvent evaporated,
water added, and the product extracted with dichloromethane.
The organic phase was dried over magnesium sulfate and the
solvent evaporated to give 0.4 g of 46 (100%) as an oil. 1H NMR
(DMSO-d6): δ 5.25 (br s, 2H), 6.55 (d, 1H, J ) 7.5 Hz), 6.80
(m, 2H), 6.9 (t, 1H, J ) 7.5 Hz), 7.85 (d, 1H, J ) 1.6 Hz).
1
0.44 mmol) to give 0.99 g of 8 (76%). H NMR (DMSO-d6 and
CF3CO2D): δ 2.35 (m, 2H), 3.15 (t, 2H), 3.35 (t, 2H), 3.50 (d,
2H), 3.80 (t, 2H), 4.0 (m, 2H), 4.05 (s, 3H), 4.35 (t, 2H), 6.90
(s, 1H), 7.40 (m, 3H), 7.65 (d, 1H, J ) 8.2 Hz), 8.05 (d, 1H, J
) 2.3 Hz), 8.35 (s, 1H), 8.80 (s, 1H). MS-ESI m/z 435 [MH]+.
Anal. (C24H26N4O4) C, H, N.
2-Nitr o-3-ch lor o-6-h ydr oxyben zaldeh yde (41). 4-Chloro-
3-nitrophenol 40 (1 g, 5.8 mmol), TFA (9 mL) and hexameth-
ylenetetramine (0.8 g, 5.8 mmol) were placed in a Teflon-lined
reactor. Six identical reactors were placed in a microwave oven
and heated at 115 °C for 1.5 h. Each reactor was then
processed as follows: 4 N HCl (20 mL) was added and the
mixture was extracted with dichloromethane. The organic
phase was washed twice with brine and dried over magnesium
sulfate and the solvent evaporated. Purification of the pooled
samples by flash chromatography using dichloromethane gave
3 g of 41 (43%). 1H NMR (DMSO-d6): δ 7.25 (m, 1H), 7.85 (m,
1H), 10.25 (s, 1H).
2-(Eth oxyca r bon yl)-5-ch lor o-4-n itr oben zofu r a n (42). A
mixture of 2-nitro-3-chloro-6-hydroxybenzaldehyde 41 (3 g,
14.8 mmol), diethyl bromomalonate (2.78 mL, 16.3 mmol),
potassium carbonate (3.1 g, 22.2 mmol), tetrabutylammonium
bromide (0.477 g, 1.4 mmol), and toluene (50 mL) was heated
to reflux in a Dean-Stark apparatus for 20 h. Upon cooling,
the precipitate was removed by filtration and the solvent was
evaporated. The residue was solubilized in ethyl acetate,
washed with water and brine, dried over magnesium sulfate,
and filtered, and the solvent was evaporated. The resulting
oil was purified by flash chromatography using dichlo-
romethane as solvent. Evaporation of the solvent gave 3 g of
42 (77%). 1H NMR (DMSO-d6): δ 3.35 (t, 3H), 4.45 (q, 2H),
7.85 (s, 1H), 7.90 (d, 1H, J ) 8.1 Hz), 8.20 (d, 1H, J ) 8.1 Hz).
MS m/z 269 [M]+.
2-Ca r boxy-5-ch lor o-4-n itr oben zofu r a n (43). 2-(Ethoxy-
carbonyl)-5-chloro-4-nitrobenzofuran 42 (3 g, 11.1 mmol) was
suspended in ethanol (17 mL). KOH (2 N, 11.1 mL, 22.2 mmol)
was added and the mixture refluxed for 1 h. The ethanol was
evaporated and the residue redissolved in water. HCl (6 N)
was added to adjust the pH to 2 and the resulting precipitate
collected by filtration, washed with water, and dried under
vacuum over phosphorus pentoxide to give 2.65 g of 43 (98%).
1H NMR (DMSO-d6): δ 7.25 (s, 1H), 7.70 (d, 1H, J ) 8.8 Hz),
8.05 (d, 1H, J ) 8.8 Hz).
N-(1-Ben zofu r a n -7-yl)-6-m eth oxy-7-(3-m or p h olin -4-yl-
p r op oxy)qu in a zolin -4-a m in e (10). 7-Aminobenzofuran 46
(0.047 g, 0.35 mmol) was reacted with 4-chloro-6-methoxy-7-
(3-morpholin-4-ylpropoxy)quinazoline 34 (0.1 g, 0.3 mmol)
using a procedure similar to the one described for the synthesis
of 5 and purified as described for 23 to give 0.11 g of 10 (84%).
1H NMR (DMSO-d6 and CF3CO2D): δ 2.35 (m, 2H), 3.15 (t,
2H), 3.35 (t, 2H), 3.55 (d, 2H), 3.75 (t, 2H), 4.05 (t, 3H), 4.10
(m, 2H), 4.35 (t, 2H), 7.10 (d, 1H, J ) 1.9 Hz), 7.45 (m, 3H),
7.75 (dd, 1H, J 1 ) 1.3, J 2 ) 7.7 Hz), 8.05 (d, 1H, J ) 1.9 Hz),
8.25 (s, 1H), 8.85 (s, 1H). MS-ESI m/z 435 [MH]+. Anal.
(C24H26N4O4) C, H, N.
Eth yl 2-Am in o-6-ch lor oben zoa te (48). 2-Amino-6-chlo-
robenzoic acid 47 (18 g, 100 mmol) was dissolved in DMF,
sodium hydride (4.6 g, 110 mmol, 60% in oil) was added, and
the mixture was stirred for 30 min. Ethyl iodide (10 mL, 125
mmol) was added and the reaction mixture stirred for 2 days
at room temperature. The solvent was evaporated, water was
added, and the product was extracted with ethyl acetate. The
organic phase was washed with water and brine, dried over
magnesium sulfate, and filtered, and the solvent was evapo-
rated. The crude product was purified by flash chromatography
using ethyl acetate:petroleum ether (2:8) as solvent. Evapora-
5-Ch lor o-4-n itr oben zofu r a n (44). 2-Carboxy-5-chloro-4-
nitrobenzofuran 43 (1.6 g, 6.6 mmol) and copper oxide (0.080
g, 1.0 mmol) were suspended in quinoline (14 mL) and heated
at 210 °C for 30 min. Copper (0.020 g, 0.3 mmol) was then
added and heating was increased to 230 °C for another 20 min.
Upon cooling, the copper and unreacted starting material were
removed by filtration, the filtrate was diluted with ether,
washed with 2 N HCl (3×), and dried over magnesium sulfate,
and the solvent was evaporated. The residue was purified by
flash chromatography using ether:petroleum ether (15:85) as
1
tion of the solvent gave 15.8 g of 48 (80%) as an oil. H NMR
1
(DMSO-d6): δ 1.30 (t, 3H), 4.30 (q, 2H), 5.70 (br s, 2H), 6.60
(d, 1H), 6.70 (d, 1H), 7.10 (t, 1H).
eluent. Evaporation of the solvent gave 0.36 g of 44 (28%). H
NMR (DMSO-d6): δ 7.20 (d, 1H, J ) 1.4 Hz), 7.70 (d, 1H, J )
8.8 Hz), 8.05 (d, 1H, J ) 8.8 Hz), 8.35 (d, 1H, J ) 1.4 Hz).
Eth yl 2-Hyd r oxy-6-ch lor oben zoa te (49). Ethyl 2-amino-
6-chlorobenzoate 48 (12.7 g, 63.6 mmol) was suspended in a
mixture of water (38 mL), concentrated sulfuric acid (27.9 mL),
and ice (76 g). Sodium nitrite (4.5 g in 100 mL water) was
added dropwise over 5 min to this suspension. The reaction
mixture was stirred at 0 °C for an additional 20 min, heated
to 120 °C for 1 h, and poured into ice-water, and the product
was extracted with ether. The organic phase was washed with
water and brine, dried over magnesium sulfate, filtered, and
4-Am in o-5-ch lor oben zofu r a n (45). Methanol (7 mL),
Raney nickel (0.020 g), and hydrazine hydrate (0.097 mL, 2
mmol) were warmed to 55-60 °C and 5-chloro-4-nitrobenzo-
furan 44 (0.1 g, 0.5 mmol) was added portionwise. The reaction
mixture was then heated to reflux for 30 min. The catalyst
was removed by filtration, the solvent evaporated, the residue
dissolved in water and extracted with dichloromethane. The
organic phase was dried over magnesium sulfate and the

880 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Ple´ et al.
the solvent was evaporated. The resulting oil was purified by
flash chromatography using dichloromethane:petroleum ether
(2:8) as solvent. Evaporation of the solvent gave 9.8 g of 49
extracted with ethyl acetate. The organic phase was dried over
magnesium sulfate and filtered, and the solvent was evapo-
rated. The resulting oil was purified by flash chromatography
using dichloromethane:petroleum ether (25:75) as solvent.
1
(77%). H NMR (DMSO-d6): δ 1.30 (t, 3H), 4.30 (q, 2H), 6.90
1
Evaporation of the solvent gave 0.376 g of 54 (45%). H NMR
(d, 1H, J ) 8.4 Hz), 6.95 (d, 1H, J ) 8.4 Hz), 7.25 (t, 1H, J )
(DMSO-d6): δ 5.50 (br s, 2H), 6.85 (m, 2H), 7.10 (d, 1H, J )
8.4 Hz), 10.45 (br s, 1H).
8.4 Hz), 7.95 (d, 1H, J ) 2.2 Hz). MS m/z 167 [M]+.
Eth yl 2-Allyloxy-6-ch lor oben zoa te (50). Ethyl 2-hy-
droxy-6-chlorobenzoate 49 (9.8 g, 48.8 mmol) was dissolved in
acetonitrile (250 mL), and allyl bromide (5.5 mL, 63 mmol)
was added followed by 1,5,7-triazabicyclo[4.4.0]dec-5-ene (10.4
g, 73 mmol). The reaction mixture was stirred for 20 h, the
solvent was evaporated, and the crude product was purified
by flash chromatography using petroleum ether:ether (85:15)
as solvent. Evaporation of the solvent gave 10.3 g of 50 (88%).
1H NMR (DMSO-d6): δ 1.30 (t, 3H), 4.35 (q, 2H), 4.65 (d, 2H),
5.25 (d, 1H), 5.40 (d, 1H), 6.0 (m, 1H), 7.15 (m, 2H), 7.45 (t,
1H, J ) 8.3 Hz).
2-Hyd r oxy-3-a llyl-6-ch lor oben zoa te (51). Ethyl 2-allyl-
oxy-6-chlorobenzoate 50 (10.3 g, 42.8 mmol) was heated at 230
°C for 1 h and then purified by flash chromatography using
dichloromethane:petroleum ether (20:80) as solvent. Evapora-
tion of the solvent gave 7.3 g of 51 (71%). 1H NMR (DMSO-
d6): δ 1.30 (t, 3H), 3.30 (m, 2H), 4.35 (q, 2H), 5.05 (m, 2H),
5.95 (m, 1H), 6.95 (d, 1H, J ) 8.2 Hz), 7.15 (d, 1H, J ) 8.2
Hz), 9.70 (br s, 1H).
N-(6-Ch lor o-1-b en zofu r a n -7-yl)-6-m et h oxy-7-(3-m or -
p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (11). NaHMDS
(0.592 mL, 0.59 mmol, 1 M solution in THF, Aldrich) was
added to 6-chloro-7-amino-benzofuran 54 (0.099 g, 0.59 mmol)
in solution in DMF (5 mL) under argon. After 30 min at room
temperature, 4-chloro-6-methoxy-7-(3-morpholin-4-ylpropoxy)-
quinazoline 34 (0.1 g, 0.3 mmol) was added. The reaction
mixture was stirred for 3 h and then water was added followed
by ethyl acetate extraction. The organic phase was washed
with water and brine, dried over magnesium sulfate, and
filtered, and the solvent was evaporated. The crude product
was purified as described for 23 to give 0.105 g of 11 (67%).
1H NMR (DMSO-d6 and CF3CO2D): δ 2.35 (m, 2H), 3.15 (m,
2H), 3.35 (m, 2H), 3.55 (d, 2H), 3.75 (t, 2H), 4.0 (m, 2H), 4.05
(s, 3H), 4.35 (t, 2H), 7.15 (d, 1H, J ) 2.2 Hz), 7.45 (s, 1H), 7.55
(d, 1H, J ) 8.5 Hz), 7.80 (d, 1H, J ) 8.3 Hz), 8.10 (d, 1H, J )
2.2 Hz), 8.25 (s, 1H), 8.80 (s, 1H). MS-ESI m/z 469 [MH]+. Anal.
(C24H25ClN4O4) C, H, N.
N-(2,3-Dih ydr o-1-ben zofu r an -7-yl)-6-m eth oxy-7-(3-m or -
p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (12). 2,3-Dihydro-
1-benzofuran-7-amine31 (0.072 g, 0.53 mmol) was reacted with
4-chloro-6-methoxy-7-(3-morpholin-4-ylpropoxy)quinazoline 34
(0.15 g, 0.44 mmol) using a procedure similar to the one
described for the synthesis of 5 and purified as described for
6-Ch lor o-7-(eth oxyca r bon yl)ben zofu r a n (52). Two dif-
ferent batches of ethyl 2-hydroxy-3-allyl-6-chlorobenzoate 51
were combined (8 g, 33 mmol), dissolved in methanol, and
cooled at -78 °C. Ozone was bubbled through the solution for
30 min. Dimethyl sulfide was added (13 mL) and the solution
was left to reach ambient temperature. The solvent was
evaporated, and ether and water were added. The ether phase
was washed with water and brine, dried over magnesium
sulfate, and filtered and the solvent was evaporated. Evapora-
tion of the solvent gave the desired aldehyde, which was
immediately suspended in phosphoric acid (85%, 35 mL), and
the mixture was heated to 100 °C for 20 min. Upon cooling to
room temperature the acid solution was diluted with water
and extracted with ether. The ether phase was washed with
water and brine, dried over magnesium sulfate, and filtered,
and the solvent was evaporated. The crude product was
purified by flash chromatography using dichloromethane:
petroleum ether (3:7) as solvent. Evaporation of the solvent
gave 5.9 g of 52 (79%). 1H NMR (DMSO-d6): δ 1.35 (t, 3H),
4.45 (q, 2H), 7.10 (d, 1H, J ) 2.3 Hz), 7.45 (d, 1H, J ) 8.5 Hz),
7.85 (d, 1H, J ) 8.7 Hz), 8.15 (d, 1H, J ) 2.3 Hz).
1
23 to give 0.155 g of 12 (81%). H NMR (DMSO-d6 and CF3-
CO2D): δ 2.35 (m, 2H), 3.15 (m, 2H), 3.30 (m, 4H), 3.55 (d,
2H), 3.80 (t, 2H), 4.0 (s, 3H), 4.05 (m, 2H), 4.35 (t, 2H), 4.6 (t,
2H), 6.95 (t, 1H, J ) 7.8 Hz), 7.20 (d, 1H, J ) 7.8 Hz), 7.30 (d,
1H, J ) 7.8 Hz), 7.40 (s, 1H), 8.20 (s, 1H), 8.80 (s, 1H). MS-
ESI m/z 437 [MH]+. Anal. (C24H28N4O4) C, H, N.
cis- a n d tr a n s-2,3-Dich lor o-2,3-d ih yd r o-7-n itr oben zo-
fu r a n (56). 7-Nitrobenzofuran 5517 (1.2 g, 7.3 mmol) was
dissolved in AcOH (12 mL), and chlorine was bubbled through
the solution for 30 min. The temperature was maintained at
18 °C during the reaction. The AcOH was evaporated and the
residue was purified by flash chromatography using ethyl
acetate:petroleum ether (1:1). Evaporation of the solvent gave
0.77 g of 56 (45%). MS m/z 233 [M]+.
3-Ch lor o-7-n itr oben zofu r a n (57). Two different batches
of cis- and trans-2,3-dichloro-2,3-dihydro-7-nitrobenzofuran 56
(0.85 g, 3.6 mmol) were combined and dissolved in ethanol (35
mL). A solution of KOH in ethanol (45.5 mL, 36 mmol, 0.8M
solution) was added and the mixture stirred for 1.25 h. The
mixture was concentrated to 1:10, water was added, the pH
adjusted to 2 with 6 N HCl, and the product was extracted
with ether. The organic phase was washed with water and
brine, dried over magnesium sulfate, and filtered, and the
solvent was evaporated to give 0.7 g of 57 (98%) as a white
solid. 1H NMR (DMSO-d6): δ 7.65 (t, 1H, J ) 8.1 Hz), 8.15 (d,
1H, J ) 8.1 Hz), 8.30 (d, 1H, J ) 8.4 Hz), 8.65 (s, 1H). MS m/z
197 [M]+.
3-Ch lor o-7-a m in oben zofu r a n (58). A mixture of hydra-
zine hydrate (0.81 mL, 16.7 mmol), Raney nickel (0.16 g), and
methanol (30 mL) was heated to 60 °C. 3-Chloro-7-nitro-
benzofuran 57 (0.66 g, 3.3 mmol) in solution in methanol (25
mL) was added dropwise over 5 min. The reaction mixture was
heated to reflux for 5 min and cooled to room temperature,
and the catalyst was removed by filtration. The solvent was
evaporated, and the residue was diluted with water and
extracted with dichloromethane. The organic phase was
washed with water, dried over magnesium sulfate, and filtered,
and the solvent was evaporated. The resulting oil was purified
by flash chromatography using ethyl acetate:petroleum ether
(1:1) as solvent. Evaporation of the solvent gave 0.41 g of 58
(75%). 1H NMR (DMSO-d6): δ 5.50 (br s, 2H), 6.65 (d, 1H, J )
7.7 Hz), 6.75 (d, 1H, J ) 7.7 Hz), 7.05 (t, 1H, J ) 7.7 Hz), 8.20
(s, 1H).
6-Ch lor o-7-ca r boxyben zofu r a n (53). 6-Chloro-7-(ethoxy-
carbonyl)benzofuran 52 (5.9 g, 26.1 mmol) was dissolved in
methanol, KOH (12.7 mL, 35% in water) was added, and the
reaction mixture refluxed for 1 h. The methanol was evapo-
rated, water was added, and the pH was adjusted to 2 with 6
N HCl. The precipitate was collected by filtration, washed with
water, and dried under vacuum over phosphorus pentoxide to
1
give 4.6 g of 53 (90%). H NMR (DMSO-d6): δ 7.05 (d, 1H, J
) 2.2 Hz), 7.40 (d, 1H, J ) 8.2 Hz), 7.75 (d, 1H, J ) 8.5 Hz),
8.10 (d, 1H, J ) 2.0 Hz). MS m/z 196 [M]+.
6-Ch lor o-7-a m in oben zofu r a n (54). 6-Chloro-7-carboxy-
benzofuran 53 (1 g, 5 mmol), DPPA (2.2 mL, 10 mmol),
triethylamine (1.4 mL, 10 mmol), and tert-butyl alcohol
(2.7 mL) were heated to reflux for 18 h. Upon cooling to room
temperature the mixture was poured into water and ex-
tracted with ethyl acetate. The organic phase was washed with
water and brine, dried over magnesium sulfate, and filtered,
and the solvent was evaporated. The resulting oil was purified
by flash chromatography using neutral alumina and increas-
ingly polar solvent mixtures starting with dichloromethane:
petroleum ether, then dichloromethane, then dichloromethane:
ethyl acetate (9:1), and ending with dichloromethane:ethyl
acetate (8:2). Evaporation of the solvent gave a mixture of
6-chloro-7-aminobenzofuran as well as the desired amide. This
mixture was treated with TFA (1.2 mL) in dichloromethane
(15 mL) at 0 °C for 1 h. The solvent was evaporated, and the
residue was taken up in a saturated solution of NaHCO3 and

A New Class of Anilinoquinazoline Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 881
N-(3-Ch lor o-1-b en zofu r a n -7-yl)-6-m et h oxy-7-(3-m or -
p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (13). 3-Chloro-7-
aminobenzofuran 58 (0.062 g, 0.37 mmol) was reacted with
4-chloro-6-methoxy-7-(3-morpholin-4-ylpropoxy)quinazoline 34
(0.1 g, 0.3 mmol) using a procedure similar to the one described
for the synthesis of 5 and purified as described for 23 to give
0.103 g of 13 (76%). 1H NMR (DMSO-d6 and CF3CO2D): δ 2.3
(m, 2H), 3.15 (t, 2H), 3.35 (t, 2H), 3.55 (d, 2H), 3.75 (t, 2H),
4.0 (s, 3H), 4.05 (m, 2H), 4.35 (t, 2H), 7.30 (s, 1H), 7.55 (t, 1H,
J ) 7.8 Hz), 7.60 (dd, 1H, J 1 ) 7.8, J 2 ) 1.2 Hz), 7.70 (dd, 1H,
J 1 ) 7.8, J 2 ) 1.2 Hz), 8.20 (s, 1H), 8.40 (s, 1H), 8.85 (s, 1H).
MS-ESI m/z 469 [MH]+. Anal. (C24H25ClN4O4) C, H, N.
3H), 4.35 (t, 2H), 7.25 (m, 2H), 7.4 (s, 1H), 7.55 (m, 2H), 8.25
(s, 1H), 8.75 (s, 1H). MS-ESI m/z 468 [MH]+. Anal. (C24H26
ClN5O3‚1.37H2O) C, H, N.
-
Meth yl 2,3-Dih yd r oxyben zoa te (63). A mixture of 2,3-
dihydroxybenzoic acid 62 (5 g, 32 mmol), methanol (50 mL),
and concentrated sulfuric acid (10 drops) was stirred and
heated at 60 °C for 24 h. The mixture was evaporated and the
residue was taken up in ethyl acetate. The organic solution
was washed with a saturated solution of sodium bicarbonate,
dried over magnesium sulfate, and evaporated to give 2.19 g
1
of 63 (40%). H NMR (CDCl3): δ 3.95 (s, 3H), 5.7 (s, 1H), 6.8
(t, 1H, J ) 8.1 Hz), 7.15 (dd, 1H, J 1 ) 8.1, J 2 ) 1.5 Hz), 7.25
(s, 1H), 7.35 (dd, 1H, J 1 ) 8.1, J 2 ) 1.5 Hz).
N-(1H -in d ol-4-yl)-6-m et h oxy-7-(3-m or p h olin -4-ylp r o-
p oxy)qu in a zolin -4-a m in e (14). 4-Aminoindole (0.041 g, 31
mmol) was reacted with 4-chloro-6-methoxy-7-(3-morpholin-
4-ylpropoxy)quinazoline 34 (0.08 g, 0.24 mmol) using a pro-
cedure similar to the one described for the synthesis of 5 and
purified as described for 23 to give 0.075 g of 14 (72%). 1H
NMR (DMSO-d6 and CF3CO2D): δ 2.35 (m, 2H), 3.15 (m, 2H),
3.35 (m, 2H), 3.55 (t, 2H), 3.80 (m, 2H), 4.0 (m, 2H), 4.05 (s,
3H), 4.35 (t, 2H), 6.35 (d, 1H), 7.15 (d, 1H, J ) 7.8 Hz), 7.20
(t, 1H, J ) 7.8 Hz), 7.40 (s, 1H), 7.42 (s, 1H), 7.5 (d, 1H, J )
7.8 Hz), 8.30 (s, 1H), 8.75 (s, 1H). MS-ESI m/z 434 [MH]+. Anal.
(C24H27N5O3) C, H, N.
Meth yl 2,3-Meth ylen ed ioxyben zoa te (64). After repeti-
tion of the previous reaction, a mixture of methyl 2,3-
dihydroxybenzoate 63 (2.8 g, 16 mmol), potassium fluoride (4.8
g, 83 mmol), and DMF (45 mL) was stirred at ambient
temperature for 30 min. Dibromomethane (1.28 mL, 18 mmol)
was added and the mixture was heated to 120 °C for 3 h. The
mixture was cooled to ambient temperature, poured into water,
and extracted with diethyl ether. The organic phase was
washed with water and then brine, dried over magnesium
sulfate, and evaporated. The residue was purified by column
chromatography using a 9:1 mixture of petroleum ether (bp
40-60 °C) and ethyl acetate as eluent. Evaporation of the
1-Meth yl-4-n itr oin d ole (60). Sodium hydride (0.135 g) was
added to a solution of 4-nitroindole 59 (0.5 g) in DMF (10 mL)
cooled in an ice bath. Methyl iodide (0.19 mL) was added
dropwise and the reaction stirred for 2 h. The solvent was
removed under vacuum and the residue partitioned between
ethyl acetate and water. The organic phase was dried over
magnesium sulfate and the solvent evaporated to give 0.49 g
1
solvents gave 2.3 g of 64 (80%). H NMR (CDCl3): δ 3.95 (s,
3H), 6.1 (s, 2H), 6.85 (t, 1H, J ) 8.1 Hz), 7.0 (dd, 1H, J 1 ) 8.1,
J 2 ) 1.5 Hz), 7.45 (dd, 1H, J 1 ) 8.1, J 2 ) 1.5 Hz).
2,3-Meth ylen ed ioxyben zoic Acid (65). Methyl 2,3-me-
thylenedioxybenzoate 64 (2.8 g, 15 mmol), a 2 N aqueous
potassium hydroxide solution (15.5 mL), and methanol (40 mL)
were stirred at ambient temperature for 2 h. The solution was
concentrated to about one-quarter of the original volume and
cooled in an ice bath. The mixture was acidified to pH 3.5 by
the addition of 2 N HCl. The resultant precipitate was collected
by filtration, washed in turn with water and diethyl ether, and
dried under vacuum to give 1.87 g of 65 (73%). 1H NMR
(DMSO-d6): δ 6.1 (s, 2H), 6.9 (t, 1H, J ) 7.9 Hz), 7.15 (dd,
1H, J 1 ) 1.1, J 2 ) 7.9 Hz), 7.3 (dd, 1H, J 1 ) 1.1, J 2 ) 7.9 Hz),
13.0 (br s, 1H).
1
of 60 (98%). H NMR (DMSO-d6): δ 3.80 (s, 3H), 7.00 (d, 1H),
7.34 (dd, 1H), 7.73 (d, 1H), 7.96 (d, 1H), 8.06 (d, 1H).
4-Am in o-1-m eth ylin d ole (61). 1-Methyl-4-nitroindole 60
(0.49 g) and 10% Pd/C (49 mg) in EtOH (20 mL) were stirred
under an atmosphere of hydrogen overnight. The catalyst was
filtered and concentrated under vacuum to give 0.43 g of 61
1
(99%) as an oil. H NMR (DMSO-d6): δ 3.63 (s, 3H), 5.10 (br
s, 2H), 6.16 (dd, 1H), 6.23 (d, 1H), 6.55 (d, 1H), 6.80 (t, 1H),
7.00 (d, 1H).
ter t-Bu tyl 2,3-Meth ylen ed ioxyp h en ylca r ba m a te (66).
2,3-Methylenedioxybenzoic acid 65 (1.8 g, 10.8 mmol) was
suspended in anhydrous dioxane (30 mL), and DPPA (2.45 mL,
11 mmol), triethylamine (1.6 mL, 11 mmol), and tert-butyl
alcohol (9 mL) were added. The mixture was heated to reflux
for 5 h. The mixture was cooled to ambient temperature,
concentrated by evaporation, and diluted with ethyl acetate.
The organic phase was washed in turn with a 5% aqueous
citric acid solution, water, an aqueous sodium bicarbonate
solution, and brine and dried over magnesium sulfate. The
solvent was evaporated and the residue was purified by column
chromatography on silica using a 19:1 mixture of petroleum
ether (bp 40-60 °C) and ethyl acetate as eluent. Evaporation
of the solvent gave 1.98 g of 66 (77%) as a solid. 1H NMR
(CDCl3): δ 1.55 (s, 9H), 5.95 (s, 2H), 6.4 (br s, 1H), 6.55 (dd,
1H, J 1 ) 8.1, J 2 ) 1.1 Hz), 6.8 (t, 1H, J ) 8.1 Hz), 7.45 (d, 1H,
J ) 8.1 Hz).
6-Meth oxy-N-(1-m eth yl-1H-in d ol-4-yl)-7-(3-m or p h olin -
4-ylp r op oxy)qu in a zolin -4-a m in e (15). 4-Chloro-6-methoxy-
7-(3-morpholin-4-ylpropoxy)quinazoline 34 (0.08 g, 0.24 mmol)
was reacted with 4-amino-1-methylindole 61 (0.045 g, 0.31
mmol) using a procedure similar to the one described for the
synthesis of 5 and purified as described for 23 to give 0.057 g
of 15 (53%). 1H NMR (DMSO-d6 and CF3CO2D): δ 2.35 (m,
2H), 3.15 (m, 2H), 3.35 (t, 2H), 3.55 (d, 1H), 3.8 (m, 2H), 3.85
(s, 3H), 4.0 (m, 2H), 4.05 (s, 3H), 4.35 (t, 2H), 7.20 (d, 1H, J )
7.8 Hz), 7.30 (t, 1H, J ) 7.8 Hz), 7.38 (s, 1H), 7.41 (s, 1H),
7.45 (d, 1H, J ) 7.8 Hz), 8.30 (s, 1H), 8.75 (s, 1H). MS-ESI
m/z 448 [MH]+. Anal. (C25H29N5O3‚0.01CH2Cl2‚0.07EtOAc‚
0.2H2O) C, H, N.
N-(1H -in d ol-7-yl)-6-m et h oxy-7-(3-m or p h olin -4-ylp r o-
p oxy)q u in a zolin -4-a m in e (16). 4-Chloro-6-methoxy-7-(3-
morpholin-4-ylpropoxy)quinazoline 34 (0.15 g, 0.3 mmol) was
reacted with 7-aminoindole (0.073 g, 0.56 mmol) using a
procedure similar to the one described for the synthesis of 5
2,3-Meth ylen ed ioxya n ilin e (67). HCl (5 N, 30 mL) was
added to a solution of tert-butyl 2,3-methylenedioxyphenyl-
carbamate 66 (1.9 g, 8 mmol) in ethanol (38 mL), and the
reaction mixture was stirred at ambient temperature for 20
h. The ethanol was evaporated and the residual aqueous phase
was washed with diethyl ether and neutralized to pH 7 by the
addition of solid potassium hydroxide. The resultant mixture
was filtered and the aqueous phase was extracted with diethyl
ether. The organic phase was washed with brine and dried
over magnesium sulfate, and the solvent was evaporated to
1
and purified as described for 23 to give 0.18 g (93%) of 16. H
NMR (DMSO-d6 and CF3CO2D): δ 2.35 (m, 2H), 3.15 (m, 2H),
3.35 (m, 2H), 3.55 (d, 2H), 3.8 (t, 2H), 4.0 (m, 2H), 4.05 (s,
3H), 4.35 (t, 2H), 6.55 (d, 1H, J ) 3.2 Hz), 7.12 (t, 1H, J ) 7.6
Hz), 7.18 (d, 1H, J ) 7.6 Hz), 7.35 (d, 1H, J ) 3.2 Hz), 7.4 (s,
1H), 7.6 (d, 1H, J ) 7.6 Hz), 8.3 (s, 1H), 8.75 (s, 1H). MS-ESI
m/z 434 [MH]+.
N-(3-Ch lor o-1H-in d ol-7-yl)-6-m eth oxy-7-(3-m or p h olin -
4-ylp r op oxy)qu in a zolin -4-a m in e (17). 4-Chloro-6-methoxy-
7-(3-morpholin-4-ylpropoxy)quinazoline 34 (0.15 g, 0.44 mmol)
was reacted with 7-amino-3-chloroindole32 (0.110 g, 0.55 mmol)
using a procedure similar to the one described for 5 and
purified as described for 23 to give 0.166 g of 17 (81%). 1H
NMR (DMSO-d6 and CF3CO2D): δ 2.35 (m, 2H), 3.15 (m, 2H),
3.35 (m, 2H), 3.55 (d, 2H), 3.8 (t, 2H), 4.0 (m, 2H), 4.05 (s,
1
give 1.0 g of 67 (91%). H NMR (CDCl3): δ 3.0 (br s, 2H), 5.9
(s, 2H), 6.3 (m, 2H), 7.25 (t, 1H, J ) 8.1 Hz).
N-(1,3-Ben zod ioxol-4-yl)-6-m eth oxy-7-(3-m or p h olin -4-
ylp r op oxy)qu in a zolin -4-a m in e (18) was prepared as de-
scribed for the synthesis of 5. 4-Chloro-6-methoxy-7-(3-
morpholin-4-ylpropoxy)quinazoline 34 (0.1 g, 0.3 mmol) was
reacted with 2,3-methylenedioxyaniline 67 (0.048 g, 0.36

882 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Ple´ et al.
mmol) to give 0.138 g of 18 (90%) as a dihydrochloride salt.
1H NMR (DMSO-d6 and CF3CO2D): δ 2.35 (m, 2H), 2.5 (m,
2H), 3.15 (m, 2H), 3.3 (m, 2H), 3.55 (d, 2H), 3.8 (t, 2H), 4.05
(s, 3H), 4.35 (t, 2H), 6.1 (s, 2H), 7.0 (m, 3H), 7.4 (s, 1H), 8.2 (s,
1H), 8.85 (s, 1H). MS-ESI m/z 439. Anal. (C23H26N4O5‚2HCl‚
0.5H2O) C, H, N.
5-Ch lor o-1,3-ben zod ioxole-4-ca r boxylic Acid (69). Di-
isopropylamine (4.92 mL, 35 mmol) in THF (100 mL) was
cooled to -78 °C, n-butyl lithium (14 mL, 35 mmol, 2.5M) was
added dropwise, and 15 min later 5-chloro-1,3-benzodioxole 68
(3.73 mL, 32 mmol) was added dropwise at the same temper-
ature. Thirty minutes later, dry CO2 was bubbled into the
reaction mixture for another 30 min. The solution was left to
reach ambient temperature, and 1 h later the reaction mixture
was quenched with water. The solvent was evaporated off and
the solution acidified to pH 2 with 2 N HCl. The solid was
collected by filtration and washed with water and ether to give
5.4 g of 69 (84%). 1H NMR (DMSO-d6): δ 6.15 (s, 2H), 6.95 (d,
1H, J ) 8.2 Hz), 7.0 (d, 1H, J ) 8.5 Hz), 13.7 (br s, 1H). MS-
ESI m/z 199 [M - H]-.
ter t-Bu tyl 5-Ch lor o-1,3-ben zodioxol-4-ylcar bam ate (70).
5-Chloro-1,3-benzodioxole-4-carboxylic acid 69 (1 g, 5 mmol)
was dissolved in 1,4-dioxane (15 mL). Anhydrous tert-butyl
alcohol (4 mL), DPPA (1.12 mL, 5.2 mmol), and triethylamine
(0.73 mL, 5.2 mmol) were added in turn under argon, and the
mixture was heated to 100 °C for 4 h. The solvent was
evaporated, and the residue was taken up in ethyl acetate and
washed with 5% citric acid, water, saturated bicarbonate
solution, and brine. The solid was removed by filtration and
the solution dried over magnesium sulfate, filtered, and
evaporated. The crude product was purified by flash chroma-
tography using petroleum ether:ethyl acetate (9:1) as eluent
and gave 1.1 g of 70 (81%). 1H NMR (DMSO-d6): δ 1.45 (s,
9H), 6.1 (s, 2H), 6.85 (d, 1H, J ) 8.4 Hz), 6.95 (d, 1H, J ) 8.4
Hz), 8.75 (s, 1H).
N-(5-Br om o-1,3-ben zod ioxol-4-yl)-6-m eth oxy-7-(3-m or -
p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (20) was prepared
as described for the synthesis of 1. 4-Chloro-6-methoxy-7-(3-
morpholin-4-ylpropoxy)quinazoline 34 (0.1 g, 0.3 mmol) was
reacted with 5-bromo-1,3-benzodioxol-4-amine 75 (0.070 g, 0.32
mmol) to give 0.45 g of 20 (29%). 1H NMR (DMSO-d6 and CF3-
CO2D): δ 2.3 (m, 2H), 3.15 (m, 2H), 3.35 (t, 2H), 3.55 (d, 2H),
3.7 (t, 2H), 4.0 (s, 3H), 4.05 (m, 2H), 4.35 (t, 2H), 6.15 (s, 2H),
7.05 (d, 1H, J ) 8.3 Hz), 7.3 (d, 1H, J ) 8.3 Hz), 7.4 (s, 1H),
8.15 (s, 1H), 8.9 (s, 1H). MS-ESI m/z 517 [MH]+. Anal. (C23H25
BrN4O5‚0.3H2O) C, H, N.
-
6-F lu or o-2,3-d im eth oxyben zoic Acid (77). In a flask
containing diisopropylamine (49.6 g, 0.49 mol) in dry THF
(1200 mL) cooled at -78 °C under nitrogen n-butyl lithium
(196 mL, 2.5N solution in hexane, 0.49 mol) was added
dropwise, keeping the internal temperature below -40 °C. The
solution was stirred for an additional 20 min and then cooled
to -60 °C. 1-Fluoro-3,4-dimethoxybenzene 76 (69.5 g, 0.445
mol) was added dropwise, the mixture was stirred at -60 °C
for 1 h, and dry carbon dioxide was bubbled through the
solution for 30 min. The temperature was left to rise to -5 °C
and water was slowly added. The solvents were evaporated
under vacuum, the residue was acidified to pH 1.5 with 2 N
HCl and extracted twice with ethyl acetate. The organic layer
was washed with brine and concentrated. The thick solid was
triturated with pentane, filtered, washed with more pentane,
1
and dried under vacuum to give 57.6 g of 77 (64%). H NMR
(DMSO-d6): δ 3.46 (s, 3H), 3.50 (s, 3H), 6.53 (dd, 1H, J 1 ∼ J 2
) 9 Hz), 6.54 (dd, 1H, J 1 ) 9, J 2 ) 5.3 Hz).
6-F lu or o-2,3-d ih yd r oxyben zoic Acid (78). To a solution
of acetic acid (750 mL) and aqueous hydrobromic acid (47%,
850 mL) heated at 130 °C (oil bath) was added 6-fluoro-2,3-
dimethoxybenzoic acid 77 (57.6 g, 0.288 mol). The mixture was
heated at 140 °C for 1 h. After cooling, the mixture was diluted
with cold water (2.5 L) and extracted with ethyl acetate. The
organic layer was washed with water and brine, dried over
magnesium sulfate, and evaporated. The residue was azeo-
troped with toluene to give 61 g of 78 (quant.). 1H NMR
(DMSO-d6): δ 6.55 (dd, 1H, J 1 ) 10, J 2 ) 8.6 Hz), 6.9 (dd, 1H,
J 1 ) 8.6, J 2 ) 5.6 Hz), 9.3 (br s, 2H).
5-Ch lor o-1,3-ben zodioxol-4-am in e (71). tert-Butyl 5-chloro-
1,3-benzodioxol-4-yl carbamate 70 (1.1 g, 4.0 mmol) was
dissolved in dichloromethane (20 mL), TFA (6 mL) was added,
and the solution was stirred for 3 h at room temperature. The
solvent was evaporated off and the residue resolubilized in
ethyl acetate. Water was added and the pH adjusted to 6.5
with a saturated solution of sodium bicarbonate. The organic
phase was washed with brine, dried over magnesium sulfate,
and filtered, and the solvent was evaporated to give 0.642 g
Meth yl 6-F lu or o-2,3-d ih yd r oxyben zoa te (79). Thionyl
chloride (62.7 g, 0.527 mmol) was added dropwise to a solution
of 6-fluoro-2,3-dihydroxybenzoic acid 78 (60.5 g, 0.351 mol) in
methanol (520 mL) cooled at 0 °C. The temperature was
allowed to increase to room temperature and the mixture was
then heated at 70 °C for 18 h. The mixture was cooled and
the solvents evaporated under vacuum. The residue was
dissolved in ethyl acetate, washed with saturated aqueous
sodium bicarbonate and brine, and dried over magnesium
sulfate, and the solvent was evaporated. Purification over a
short pad of silica using dichloromethane as eluent gave 39 g
1
of 70 (93%). H NMR (DMSO-d6): δ 5.15 (s, 2H), 6.0 (s, 2H),
6.25 (d, 1H, J ) 8.4 Hz), 6.75 (d, 1H, J ) 8.5 Hz).
N-(5-Ch lor o-1,3-ben zod ioxol-4-yl)-6-m eth oxy-7-(3-m or -
p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (19) was prepared
as described for the synthesis of 1. 4-Chloro-6-methoxy-7-(3-
morpholin-4-ylpropoxy)quinazoline 34 (0.1 g, 0.3 mmol) was
reacted with 5-chloro-1,3-benzodioxol-4-amine 71 (0.056 g, 0.32
mmol) to give 0.072 g of 19 (51%). 1H NMR (DMSO-d6 and
CF3CO2D): δ 2.35 (m, 2H), 3.15 (m, 2H), 3.35 (m, 2H), 3.5 (m,
2H), 3.8 (m, 2H), 4.0 (m, 2H), 4.05 (s, 3H), 4.35 (m, 2H), 6.15
(s, 2H), 7.05 (d, 1H), 7.15 (d, 1H), 7.45 (s, 1H), 8.25 (s, 1H),
1
of 79 (60%). H NMR (CDCl3): δ 4.0 (s, 3H), 5.55 (s, 1H), 6.55
(dd, 1H, J 1 ) 10.5, J 2 ) 9 Hz), 7.0 (dd, 1H, J 1 ) 9, J 2 ) 5 Hz),
11.45 (s, 1H).
Meth yl 5-F lu or o-1,3-ben zod ioxole-4-ca r boxyla te (80).
Bromochloromethane (40 g, 0.306 mol) and cesium carbonate
(100 g, 0.306 mol) were added to a solution of methyl 6-fluoro-
2,3-dihydroxybenzoate 79 (38 g, 0.204 mol) in dry DMF (450
mL) under nitrogen. The mixture was heated at 110 °C for
1.5 h. The mixture was cooled, the solids were filtered off, and
the solvents were evaporated under vacuum. The residue was
dissolved in ethyl acetate, washed with water, and dried over
magnesium sulfate. After evaporation of the solvent, the
residue was purified by flash chromatography using ethyl
acetate:petroleum ether (2:8 up to 3:7) as eluent. Evaporation
of the solvent gave 32.2 g of 80 (80%). 1H NMR (CDCl3): δ
3.92 (s, 3H), 6.09 (s, 2H), 6.57 (dd, 1H, J 1 ) 11.4, J 2 ) 8.8 Hz),
6.83 (dd, 1H, J 1 ) 8.8, J 2 ) 4 Hz).
8.9 (s, 1H). MS-ESI m/z 471 and 473 [MH]+. Anal. (C23H25
ClN4O5‚0.6H2O) C, H, N.
-
5-Br om o-1,3-ben zod ioxole-4-ca r boxylic a cid (73) was
prepared as described for the synthesis of 69. 5-Bromo-1,3-
benzodioxole 72 (1 g, 5 mmol) gave 0.88 g of 73 (72%). 1H NMR
(DMSO-d6 and CF3CO2D): δ 6.15 (s, 2H), 6.95 (d, 1H, J ) 8.3
Hz), 7.1 (d, 1H, J ) 8.3 Hz). MS-ESI m/z 243 [M - H]-.
ter t-Bu tyl 5-br om o-1,3-ben zod ioxol-4-ylca r ba m a te (74)
was prepared as described for the synthesis of 70. 5-Bromo-
1,3-benzodioxole-4-carboxylic acid 73 (0.88 g, 3.6 mmol) gave
1
1.1 g of 74 (96%). H NMR (DMSO-d6): δ 1.45 (s, 9H), 6.1 (s,
2H), 6.80 (d, 1H, J ) 8.4 Hz), 7.1 (d, 1H, J ) 8.4 Hz), 8.70 (s,
1H).
5-Br om o-1,3-ben zod ioxol-4-a m in e (75) was prepared as
described for the synthesis of 71. tert-butyl 5-bromo-1,3-
benzodioxol-4-ylcarbamate 74 (1.1 g, 3.4 mmol) gave 0.6 g of
75 (81%). 1H NMR (DMSO-d6): δ 5.05 (s, 2H), 6.0 (s, 2H), 6.25
(d, 1H, J ) 8.4 Hz), 6.9 (d, 1H, J ) 8.4 Hz). MS-ESI m/z 216
and 218 [MH]+.
5-F lu or o-1,3-ben zod ioxole-4-ca r boxylic Acid (81). An
aqueous solution of potassium hydroxyde (2 N, 162 mL, 0.325
mmol) was added to a suspension of methyl 5-fluoro-1,3-
benzodioxole-4-carboxylate 80 (32.2 g, 0.162 mol) in methanol
(415 mL). The mixture was stirred for 3 h, and the solvents
were evaporated under vacuum. Water was added and the pH

A New Class of Anilinoquinazoline Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 883
was adjusted to 2. The mixture was extracted with ethyl
acetate, and the organic layer was washed with brine, dried
over magnesium sulfate, and evaporated to give 29.1 g of 81
3.60 (m, 4H), 3.95 (s, 3H), 4.15 (t, 2H), 4.25 (m, 4H), 6.80 (dd,
1H, J 1 ) 1.7, J 2 ) 8.4 Hz), 6.85 (t, 1H, J ) 8.0 Hz), 7.05 (dd,
1H, J 1 ) 1.7, J 2 ) 8.4 Hz), 7.15 (s, 1H), 7.80 (s, 1H), 8.20 (s,
1H), 9.15 (s, 1H). MS-ESI m/z 453 [MH]+. Anal. (C24H28N4O5‚
0.5H2O) C, H, N.
(97%). 1H NMR (CDCl3): δ 6.15 (s, 2H), 6.70 (dd, 1H, J 1
11.3, J 2 ) 8.6 Hz), 7.05 (dd, 1H, J 1 ) 8.6, J 2 ) 4.2 Hz).
)
ter t-Bu tyl 5-Flu or o-1,3-ben zodioxol-4-ylcar bam ate (82).
Dry tert-butyl alcohol (123 mL), triethylamine (16.7 g, 0.65
mol), and DPPA (45.5 g, 0.165 mol) were added to a solution
of 5-fluoro-1,3-benzodioxole-4-carboxylic acid 81 (29 g, 0.157
mol) in dioxane (430 mL) under nitrogen. The mixture was
heated at 100 °C for 4.5 h. Upon cooling, the cloudy mixture
was filtered. The filtrate was evaporated under vacuum,
diluted in ethyl acetate, washed with a 5% aqueous citric acid,
a 5% aqueous sodium bicarbonate, water, and brine, dried over
magnesium sulfate, and concentrated under vacuum to give
7-Ben zyloxy-3,4-d ih yd r oqu in a zolin -4-on e (85). Sodium
metal (4.4 g, 191 mmol) was added to benzyl alcohol (100 mL)
and the resultant mixture was stirred at ambient temperature
for 30 min and then heated to 80 °C for 1 h. The mixture was
cooled to 40 °C and 7-fluoro-3,4-dihydroquinazolin-4-one 84
(7.8 g, 47.6 mmol) was added. The reaction mixture was stirred
at 130 °C for 4 h. The mixture was allowed to cool and was
stirred for a further 18 h. The solution was quenched with
water (800 mL) and acidified to pH 3 by the addition of
concentrated HCl. The resultant precipitate was collected by
filtration, washed with water and diethyl ether, and dried
under vacuum for 4 h at 60 °C to give 7.02 g of 85 (59%).
1
37.6 g of 82 (93%). H NMR (CDCl3): δ 1.5 (s, 9H), 5.95 (br s,
1H), 6.0 (s, 2H), 6.55 (m, 2H).
5-F lu or o-1,3-b en zod ioxol-4-a m in e (83). Trifluoroacetic
acid (150 mL) was added to a solution of tert-butyl 5-fluoro-
1,3-benzodioxol-4-ylcarbamate 82 (37.5 g, 0.147 mol) in dichlo-
romethane (550 mL). The mixture was stirred for 3 h. After
evaporation of the solvents, the residue was dissolved in ethyl
acetate, washed with 5% aqueous sodium bicarbonate and
brine, dried over magnesium sulfate, and concentrated. The
residue was chromatographed on silica gel using dichlo-
romethane as solvent to give 21.6 g of 83 (94%). 1H NMR
(CDCl3): δ 3.60 (m, 2H), 5.95 (s, 2H), 6.20 (dd, 1H, J 1 ) 8.3,
J 2 ) 3.5 Hz), 6.50 (dd, 1H, J 1 ) 11.3, J 2 ) 8.3 Hz).
N-(5-F lu or o-1,3-ben zod ioxol-4-yl)-6-m eth oxy-7-(3-m or -
p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (21) was prepared
as described for the synthesis of 1. 4-Chloro-6-methoxy-7-(3-
morpholin-4-ylpropoxy)quinazoline 34 (0.2 g, 0.6 mmol) was
reacted with 5-fluoro-1,3-benzodioxol-4-amine 83 (0.1 g, 0.65
mmol) to give 0.214 g of 21 (79%). 1H NMR (DMSO-d6 and
CF3CO2D): δ 2.3 (m, 2H), 3.15 (m, 2H), 3.35 (m, 2H), 3.45 (m,
2H), 3.75 (t, 2H), 4.0 (s, 3H), 4.05 (m, 2H), 4.35 (t, 2H), 6.15
(s, 2H), 6.90 (dd, 1H, J 1 ) 8.5, J 2 ) 10.3 Hz), 7.0 (dd, 1H, J 1
) 8.5, J 2 ) 4.4 Hz), 7.40 (s, 1H), 8.15 (s, 1H), 8.90 (s, 1H).
MS-ESI m/z 455 [M - H]-. Anal. (C23H25FN4O5‚0.4H2O) C, H,
N.
7-Ben zyloxy-3,4-dih ydr oqu in azolin -4-th ion e (86). 7-Ben-
zyloxy-3,4-dihydroquinazolin-4-one 85 (7.0 g, 28 mmol), phos-
phorus pentasulfide (12.5 g, 56 mmol), and pyridine (350 mL)
were stirred under reflux for 8 h. After cooling, the mixture
was poured into water (1 L), and the precipitate was collected
by filtration and washed with water. The solid was dissolved
in 6 N NaOH and the solution was filtered. The filtrate was
acidified to pH 2 with 6 N HCl. The resultant precipitate was
collected, washed with water, and dried under vacuum at 60
°C to give 7.42 g of 86 (99%). 1H NMR (DMSO-d6): 5.32 (s,
2H), 7.25 (d, 1H), 7.32 (m, 1H), 7.4 (m, 1H), 7.45 (t, 2H), 7.55
(d, 2H), 8.15 (s, 1H), 8.5 (d, 1H).
7-Ben zyloxy-4-m eth ylth ioqu in azolin e (87). 7-Benzyloxy-
3,4-dihydroquinazolin-4-thione 86 (3.45 g, 12.9 mmol) was
dissolved in THF (13 mL) and 1 N NaOH (25.7 mL, 25.7 mmol)
was added. Methyl iodide (0.97 mL, 15.6 mmol) was added
dropwise and the mixture was stirred at ambient temperature
for 30 min. The mixture was neutralized by the addition of 2
N HCl and the mixture was diluted by the addition of water.
The resultant solid was collected, washed with water, and
dried under vacuum to give 3.3 g of 87 (90%). 1H NMR (DMSO-
d6): δ 2.67 (s, 3H), 5.32 (s, 2H), 7.3-7.45 (m, 5H), 7.5 (d, 2H),
8.05 (d, 1H), 8.9 (s, 1H).
N-(2,2-Diflu or o-1,3-b en zod ioxol-4-yl)-6-m et h oxy-7-(3-
m or p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (22) was pre-
pared as described for the synthesis of 1 and purified as
described for 23. 4-Chloro-6-methoxy-7-(3-morpholin-4-ylpro-
poxy)quinazoline 34 (0.15 g, 0.44 mmol) was reacted with 2,2-
difluoro-1,3-benzodioxol-4-ylamine (0.092 g, 0.53 mmol) to give
0.10 g (48%) of 22. 1H NMR (DMSO-d6 and CF3CO2D): δ 2.35
(m, 2H), 3.15 (m, 2H), 3.35 (m, 2H), 3.55 (d, 2H), 3.8 (t, 2H),
3.9 (m, 2H), 4.0 (s, 3H), 4.35 (t, 2H), 7.35 (m, 2H), 7.45 (m,
2H), 8.3 (s, 1H), 8.95 (s, 1H). MS-ESI m/z 475 [MH]+. Anal.
(C23H24F2N4O5) C, H, N.
7-Hyd r oxy-4-m eth ylth ioqu in a zolin e (88). 7-Benzyloxy-
4-methylthioquinazoline 87 (3 g, 10.6 mmol) and TFA (30 mL)
were heated to reflux for 5 h. The acid was evaporated and
the residue was suspended in water. Solid sodium bicarbonate
was added until complete dissolution. The solution was
extracted with diethyl ether and the aqueous layer was
acidified to pH 2 by the addition of 2 N HCl. The obtained
precipitate was collected by filtration, washed with water and
diethyl ether, and dried under vacuum to give 2 g of 88 (97%).
1H NMR (DMSO-d6): δ 2.7 (s, 3H), 7.15 (d, 1H), 7.25 (m, 1H),
8.0 (d, 1H), 8.9 (s, 1H).
N-(1,3-Ben zod ioxol-5-yl)-6-m eth oxy-7-(3-m or p h olin -4-
ylp r op oxy)qu in a zolin -4-a m in e (23) was prepared as de-
scribed for the synthesis of 1. 4-Chloro-6-methoxy-7-(3-
morpholin-4-ylpropoxy)quinazoline 34 (0.074 g, 0.22 mmol)
was reacted with 3,4-(methylenedioxy)aniline (0.032 g, 0.24
mmol) and purified by preparative HPLC/MS on a Waters/
ZMD Micromass with a Beta Basic Hypercil 21 × 100 mm 5
µm column using an ammonium carbonate (2 g/L) aqueous
solution:acetonitrile solvent gradient (8:2 up to 100% aceto-
nitrile over a 7.5 min period, 25 mL/min). The solvents were
evaporated under vacuum, the residue was dissolved into 1
mL of dichloromethane:methanol (8:2), and 10 mL of ether was
added to precipitate the product, which was then collected by
filtration and dried in a vacuum oven at 50 °C to give 0.082 g
of 23 (76%). MS-ESI m/z 439 [MH]+. Anal. (C23H25N4O5‚
0.75H2O) C, H, N.
4-Meth ylth io-7-(3-m or ph olin opr opoxy)qu in azolin e (89).
DEAD (2.92 g, 17 mmol) was added dropwise to a stirred
mixture of 7-hydroxy-4-methylthioquinazoline 88 (2.5 g, 12.9
mmol), 4-(3-hydroxypropyl)morpholine19 (2.47 g, 17 mmol),
triphenylphosphine (4.45 g, 17 mmol), and methylene chloride
(65 mL). The reaction mixture was stirred at ambient tem-
perature for 1 h. The mixture was evaporated and the residue
was partitioned between a 1:1 mixture of ethyl acetate and
diethyl ether and 1 N HCl. The aqueous layer was adjusted
to pH 9 with solid sodium bicarbonate and extracted with
methylene chloride. The organic layer was separated, washed
with water and brine, dried over magnesium sulfate, and
evaporated. The residue was purified by column chromatog-
raphy on silica using increasingly polar mixtures of methylene
chloride, ethyl acetate, and methanol (from 6:3:1 to 5:3:2 to
75:0:25) as eluent. Evaporation of the solvents gave 2.03 g of
1
89 (49%). H NMR (DMSO-d6 and CF3CO2D): δ 2.2-2.3 (m,
N-(2,3-Dih yd r o-1,4-b en zod ioxin -5-yl)-6-m et h oxy-7-(3-
m or p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (24) was pre-
pared as described for the synthesis of 1. 4-Chloro-6-methoxy-
7-(3-morpholin-4-ylpropoxy)quinazoline 34 (0.1 g, 0.3 mmol)
was reacted with 2,3-dihydro-1,4-benzodioxin-5-amine hydro-
2H), 2.7 (s, 3H), 3.05-3.25 (m, 2H), 3.35 (t, 2H), 3.55 (d, 2H),
3.7 (t, 2H), 4.05 (d, 2H), 4.32 (t, 2H), 7.38 (d, 1H), 7.4 (s, 1H),
8.1 (d, 1H), 9.05 (d, 1H). MS-ESI m/z 320 [MH]+.
N-(5-Ch lor o-1,3-ben zod ioxol-4-yl)-7-(3-m or p h olin -4-yl-
p r op oxy)qu in a zolin -4-a m in e (25). 5-Chloro-1,3-benzodioxol-
4-amine 71 (0.295 g, 1.72 mmol) was reacted with 4-methylthio-
1
chloride (0.068 g, 0.36 mmol) to give 0.075 g of 24 (55%). H
NMR (DMSO-d6): δ 1.95 (m, 2H), 2.35 (m, 4H), 2.45 (t, 2H),

884 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Ple´ et al.
7-(3-morpholinopropoxy)quinazoline 89 (0.5 g, 1.57 mmol)
using a procedure similar to the one described for 11. The
crude product was purified by flash chromatography using
increasingly polar solvent mixtures of dichloromethane:metha-
nol (95:5 up to 90:10). Evaporation of the solvent gave 0.38 g
rotary evaporation, and the obtained oil placed under vacuum
overnight to give 2.8 g of 93 (100%). H NMR (DMSO-d6 and
CD3CO2D): δ 1.10 (s, 9H), 1.40 (m, 2H), 1.50 (m, 4H), 1.95
(m, 2H), 2.35 (m, 4H), 2.40 (m, 2H), 3.90 (s, 3H), 4.20 (t, 2H),
5.95 (s, 2H), 7.15 (s, 1H), 7.50 (s, 1H), 8.40 (s, 1H).
6-Meth oxy-7-(3-piper idin -1-ylpr opoxy)qu in azolin -4(3H)-
on e (94). [6-Methoxy-4-oxo-7-(3-piperidin-1-ylpropoxy)quinazo-
lin-3(4H)-yl]methyl pivalate 93 (2.8 g, 6.5 mmol) and a 7 N
solution of ammonia in methanol (50 mL) were stirred for 16
h. The volatiles were evaporated, and the residue was tritu-
rated under diethyl ether. The resultant solid was isolated,
washed in turn with diethyl ether and a 9:1 mixture of diethyl
ether and methylene chloride, and dried under vacuum to give
2.06 g of 94 (100%). 1H NMR (DMSO-d6): δ 1.40 (m, 2H), 1.50
(m, 4H), 1.90 (m, 2H), 2.35 (m, 4H), 2.4 (t, 2H), 3.9 (s, 3H),
4.15 (t, 2H), 7.11 (s, 1H), 7.44 (s, 1H), 7.9 (s, 1H).
1
1
of 25 (55%). H NMR (CDCl3): δ 1.80 (m, 2H), 2.35 (m, 4H),
2.45 (t, 2H), 3.6 (m, 4H), 4.05 (t, 2H), 5.90 (s, 2H), 6.60 (d, 1H,
J ) 8.4 Hz), 6.85 (d, 1H, J ) 8.4 Hz), 6.90 (s, 1H), 7.05 (dd,
1H, J 1 ) 9.4, J 2 ) 2.6 Hz), 7.10 (d, 1H, J ) 2.6 Hz), 7.70 (d,
1H, J ) 9.4 Hz), 8.50 (s, 1H). MS-ESI m/z 443 and 445 [MH]+.
Anal. (C22H23ClN4O4) C, H, N.
7-Meth oxy-6-(3-m or p h olin op r op oxy)-3,4-d ih yd r oqu in -
a zolin -4-on e (91). A mixture of 4-(3-chloro-4-fluoroanilino)-
7-methoxy-6-(3-morpholinopropoxy)quinazoline 9020 (6 g, 13
mmol) and 6 N HCl (120 mL) was stirred and heated to reflux
for 6 h. The mixture was cooled to 0 °C and was carefully
neutralized by the addition of concentrated ammonium hy-
droxide. The resultant precipitate was isolated, washed in turn
with dilute ammonium hydroxide and water, and dried under
4-Ch lor o-6-m eth oxy-7-(3-p ip er id in -1-ylp r op oxy)qu in -
a zolin e (95). Several batches of 6-methoxy-7-(3-piperidin-1-
ylpropoxy)quinazolin-4(3H)-one 94 (76.3 g, 240 mmol) were
pooled together and suspended in thionyl chloride (750 mL)
under anhydrous conditions. DMF (5 mL) was added and the
reaction mixture was heated to reflux for 2 h and then allowed
to cool overnight. The volatiles were evaporated, the residue
was triturated with toluene, and the mixture was evaporated.
The solid was dissolved in dichloromethane, and ice and
sodium bicarbonate were added alternatively under stirring
at 0 °C until the solution reached pH 6-7. Sodium hydroxide
2 N (∼400 mL) was then added until pH 9-10 was reached.
The aqueous phase was extracted with dichloromethane, the
organic phase was washed with water and brine, dried over
magnesium sulfate, and filtered, and the solvent evaporated.
The solid was triturated with petroleum ether:ether, collected
by filtration, and dried under vacuum to give 64.2 g of 95
1
vacuum to give 4.2 g of 91 (98%). H NMR (DMSO-d6): δ 2.4
(m, 6H), 3.59 (t, 4H), 3.75 (t, 2H), 3.9 (s, 3H), 4.12 (t, 2H), 7.12
(s, 1H), 7.43 (s, 1H), 7.98 (s, 1H), 12.0 (br s, 1H). MS-ESI m/z
320 [MH]+.
4-Ch lor o-7-m eth oxy-6-(3-m or p h olin op r op oxy)qu in a zo-
lin e (92). A solution of 7-methoxy-6-(3-morpholinopropoxy)-
3,4-dihydroquinazolin-4-one 91 (0.990 g, 3.1 mmol) in thionyl
chloride (10 mL) and DMF (0.1 mL) was heated at 80 °C for
1.5 h. The mixture was allowed to cool, toluene was added,
and the solvent was removed by evaporation. The residue was
partitioned between ethyl acetate and water and the aqueous
layer was adjusted to pH 7.5 with 2 M NaOH. The organic
layer was separated, washed with brine, dried over magnesium
sulfate, and the solvent was removed by evaporation. The
residue was purified by flash chromatography eluting with
methylene chloride:methanol (95:5). The solid was triturated
with hexane, collected by filtration, and washed with ether to
give 0.614 g of 92 (58%). 1H NMR (CDCl3): δ 2.12 (m, 2H),
2.50 (br s, 4H), 2.59 (t, 2H), 3.73 (t, 4H), 4.05 (s, 3H), 4.27 (t,
2H), 7.33 (s, 1H), 7.40 (s, 1H), 8.86 (s, 1H).
1
(80%). H NMR (DMSO-d6): δ 1.40 (m, 2H), 1.55 (m, 4H), 2.0
(m, 2H), 2.40 (m, 4H), 2.45 (t, 2H), 4.0 (s, 3H), 4.29 (t, 2H),
7.41 (s, 1H), 7.46 (s, 1H), 8.9 (s, 1H).
N-(5-Ch lor o-1,3-b en zod ioxol-4-yl)-6-m et h oxy-7-(3-p i-
p er id in -1-ylp r op oxy)q u in a zolin -4-a m in e (28). 5-Chloro-
1,3-benzodioxol-4-amine 71 (1.07 g, 6.25 mmol) was reacted
with NaHMDS (6.25 mL, 6.25 mmol, 1 M in THF) in the
presence of 4-chloro-6-methoxy-7-(3-piperidin-1-ylpropoxy)-
quinazoline 95 (1.0 g, 2.98 mmol) using a procedure similar
to the one described for the synthesis of 11 and gave 1.4 g of
N-(5-Ch lor o-1,3-ben zod ioxol-4-yl)-7-m eth oxy-6-(3-m or -
p h olin -4-ylp r op oxy)qu in a zolin -4-a m in e (26) was prepared
as described for the synthesis of 1. 4-Chloro-7-methoxy-6-(3-
morpholinopropoxy)quinazoline 92 (0.25 g, 0.67 mmol) was
reacted with 5-chloro-1,3-benzodioxol-4-amine 71 (0.14 g, 0.8
1
28 (75%). H NMR (DMSO-d6 and CD3CO2D): δ 1.5-1.64 (m,
1
2H), 1.66-1.84 (m, 4H), 2.18-2.32 (m, 2H), 3.11-3.39 (m, 6H),
3.95 (s, 3H), 4.25 (t, 2H), 6.07 (s, 2H), 6.93 (d, 1H, J ) 8.4 Hz),
7.05 (d, 1H, J ) 8.4 Hz), 7.26 (s, 1H), 7.89 (s, 1H), 8.33 (s,
1H). MS-ESI m/z 469 and 471 [MH]+. mp: 163-165 °C. Anal.
(C24H27ClN4O4 0.2H2O) C, H, N.
mmol) to give 0.08 g of 26 (25%). H NMR (DMSO-d6): δ 2.0
(m, 2H), 2.40 (m, 4H), 2.50 (t, 2H), 3.60 (m, 4H), 3.95 (s, 3H),
4.15 (t, 2H), 6.10 (s, 2H), 6.95 (d, 1H, J ) 8.4 Hz), 7.05 (d, 1H,
J ) 8.4 Hz), 7.20 (s, 1H), 7.85 (s, 1H), 8.30 (s, 1H), 9.50 (s,
1H). MS-ESI m/ z 473 and 475 [MH]+. Anal. (C23H25ClN4O5‚
0.45H2O) C, H, N.
4-(Hydr oxym eth yl)pyr idin e-2-car bon itr ile (97). 4-({[tert-
Butyl(dimethyl)silyl]oxy}methyl)pyridine-2-carbonitrile22 (3.37
g, 15 mmol) was dissolved in THF, tert-butyl ammonium
fluoride (24 mL, 1 M THF solution) was added under argon,
and the mixture was stirred for 1 h. The solvent was
evaporated, the residue was diluted with ethyl acetate, washed
with a saturated solution of ammonium chloride, water, and
brine, dried over magnesium sulfate, and filtered, and the
solvent was evaporated. The residue was purified by flash
chromatography using increasingly polar solvent mixtures
starting with petroleum ether:ethyl acetate (6:4) and ending
with ethyl acetate. Evaporation of the solvent gave 1.37 g of
97 (68%). 1H NMR (CDCl3): δ 2.25 (br s, 1H), 4.85 (s, 2H),
7.55 (d, 1H, J ) 5.1 Hz), 7.75 (s, 1H, J ) 5.1 Hz), 8.7 (d, 1H).
7-(Ben zyloxy)-N-(5-ch lor o-1,3-ben zodioxol-4-yl)-6-m eth -
oxyqu in a zolin -4-a m in e (99) was prepared using conditions
similar to those described for the synthesis of 5. 7-Benzyloxy-
4-chloro-6-methoxyquinazoline 98 (7 g, 23 mmol) was reacted
with 5-chloro-1,3-benzodioxol-4-amine 71 (4.4 g, 25 mmol) to
give 9.1 g of 99 (84%) as a hydrochloride. 1H NMR (DMSO-
d6): δ 4.0 (s, 3H), 5.35 (s, 2H), 6.15 (s, 2H), 7.05 (d, 1H, J )
8.4 Hz), 7.15 (d, 1H, J ) 8.4 Hz), 7.45 (m, 4H), 7.55 (d, 2H, J
) 7.0 Hz), 8.25 (s, 1H), 8.8 (s, 1H).
N-(5-Ch lor o-1,3-ben zodioxol-4-yl)-6-m eth oxy-7-[(1-m eth -
ylpiper idin -4-yl)m eth oxy]qu in azolin -4-am in e (27). 4-Chlo-
ro-6-methoxy-7-((1-methylpiperidin-4-yl)methoxy)quinazo-
line 3615 (0.8 g, 2.5 mmol) was reacted with 5-chloro-1,3-
benzodioxol-4-amine 71 (0.47 g, 2.7 mmol) using a procedure
similar to the one described for 2. The crude product was
purified by flash chromatography using increasingly polar
solvent mixtures starting with dichloromethane:methanol (9:
1) and ending with dichloromethane:methanol:methanol satu-
rated with ammonia (9:8:2). Evaporation of the solvent gave
0.746 g of 27 (66%). 1H NMR (DMSO-d6 and CD3CO2D): δ 1.60
(m, 2H), 2.0 (m, 2H), 2.1 (m, 1H), 2.70 (s, 3H), 2.85 (m, 2H),
3.35 (m, 2H), 3.95 (s, 3H), 4.05 (d, 2H), 6.1 (s, 2H), 6.95 (d,
1H, J ) 8.4 Hz), 7.05 (d, 1H, J ) 8.4 Hz), 7.25 (s, 1H), 7.85 (s,
1H), 8.35 (d, 1H). MS-ESI m/z 455 and 457 [M - H]-. Anal.
(C23H25ClN4O4) C, H, N.
[6-Meth oxy-4-oxo-7-(3-p ip er id in -1-ylp r op oxy)qu in a zo-
lin -3(4H)-yl]m eth yl P iva la te (93). 7-Hydroxy-6-methoxy-3-
((pivaloyloxy)methyl)-3,4-dihydroquinazolin-4-one 3114 (2 g, 6.5
mmol) was dissolved in DMF (16 mL), and potassium carbon-
ate (1.26 g, 9.1 mmol), and 1-(3-chloropropyl)piperidine21 (1.26
g, 7.8 mmol) were added. The mixture was purged from oxygen
with argon and heated at 90 °C for 1.5 h. The excess potassium
carbonate was removed by filtration, the DMF evaporated by
4-[(5-Ch lor o-1,3-b en zod ioxol-4-yl)a m in o]-6-m et h oxy-
qu in a zolin -7-ol (100). 7-(Benzyloxy)-N-(5-chloro-1,3-benzo-

A New Class of Anilinoquinazoline Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4 885
dioxol-4-yl)-6-methoxyquinazolin-4-amine 99 (9.1 g, 19 mmol)
was dissolved in TFA (150 mL) and heated to reflux for 4 h
under anhydrous conditions. The solvent was evaporated, the
residue was diluted with water and methanol, and the pH
adjusted to 7.5 with a saturated solution of sodium carbonate.
The methanol was evaporated and the solid collected by
filtration, washed with water, and dried over magnesium
The assay was stopped by washing the plates four times with
PBST (150 µL/well). Detection of resultant tyrosine phospho-
rylation was facilitated by the addition of an anti-phospho-
tyrosine monoclonal antibody conjugated to alkaline phos-
phatase (anti-pY/HRP, Santa Cruz Biotechnology Inc., Santa
Cruz, CA); this was diluted 1:5000 in PBST/B/O (PBST + 0.5%
BSA + 0.1 mM sodium orthovanadate) and added at 100 µL/
well and incubated for 1 h. The plates were again washed (6×).
One tablet of the HRP substrate 3,3′,5,5′-tetramethylbenzidine
(TMB, Sigma-Aldrich) was dissolved in 100 µL of DMSO and
added per 10 mL of phosphate-citrate buffer with sodium
perborate (supplied as soluble capsules, Sigma-Aldrich). TMB
substrate solution (100 µL/well) was added. After 5 min of color
development, the reaction was stopped by the addition of 50
µL/well of 0.8 M H2SO4, and the positive control wells now
gave an A450nm of ca. 1.2-1.5. Control and blank wells were
included on all plates, containing compound diluent and MgCl2
solution with and without ATP, respectively, to determine the
dynamic range of the assay. The curves were plotted and the
IC50 values for compound enzyme inhibition were interpolated
using KC3 Kineticalc software (Bio-Tek Instruments) following
subtraction of the blank values.
1
sulfate to give 6 g of 100 (91%). H NMR (DMSO-d6): δ 3.95
(s, 3H), 6.1 (s, 2H), 6.95 (d, 1H), 7.1 (m, 2H), 7.90 (s, 1H), 8.3
(s, 1H), 9.60 (br s, 1H), 10.5 (br s, 1H). MS-ESI m/z 346 and
348 [MH]+.
4-[({4-[(5-Ch lor o-1,3-ben zod ioxol-4-yl)a m in o]-6-m eth -
oxyqu in a zolin -7-yl}oxy)m eth yl]p yr id in e-2-ca r bon itr ile
(29). 4-[(5-Chloro-1,3-benzodioxol-4-yl)amino]-6-methoxyquinazo-
lin-7-ol 100 (0.1 g, 0.28 mmol) was suspended in dichlo-
romethane (3 mL) under argon, and triphenylphosphine (0.151
g, 0.58 mmol) and 4-(hydroxymethyl)pyridine-2-carbonitrile 97
(0.042 g, 0.35 mmol) were added followed by dropwise addition
of DEAD (0.091 mL, 0.58 mmol). The solvent was removed by
evaporation and the residue purified by flash chromatography
using dichloromethane:ethyl acetate:methanol (60:35:5). Evapo-
ration of the solvent gave 0.084 g of 29 (65%). 1H NMR (DMSO-
d6 and CF3CO2D): δ 4.05 (s, 3H), 5.55 (s, 2H), 6.15 (s, 2H),
7.05 (d, 1H, J ) 8.7 Hz), 7.15 (d, 1H, J ) 8.7 Hz), 7.35 (s, 1H),
7.85 (d, 1H, J 1 ) 1.6, J 2 ) 5.0 Hz), 8.14 (dd, 1H, J 1 ) 0.8, J 2
) 1.6 Hz), 8.18 (s, 1H), 8.85 (d, 1H, J 1 ) 0.8, J 2 ) 5.0 Hz), 8.9
(s, 1H). MS-ESI m/z 462 and 464 [MH]+.
(ii) In Vitr o KDR Kin a se In h ibition Test. This assay
determines the ability of test compounds to inhibit KDR kinase
activity and has been used as a selectivity screen. The method
is as reported previously.14
(iii) In Vitr o c-sr c3T3 P r olifer a tion Assa y. This assay
determines the ability of test compounds to inhibit the
proliferation of cells in culture. A mouse NIH3T3 fibroblast
cell line transfected to overexpress active Src kinase (c-Src3T3)
was made and provided by Sara Courtneidge and Sydonia
Rayter. This line has been shown to grow in DMEM (Gibco,
Invitrogen Corp., Paisley, Scotland) + 0.5% serum, conditions
in which the parental NIH3T3 cells do not survive. Under
these low-serum conditions the transfected cells are driven to
proliferate through their expression of active Src kinase, and
consequently, Src kinase inhibition should revert them to the
parental phenotype. c-Src3T3 cells are routinely cultured in
DMEM medium + 5% FCS. For assay they were harvested
with trypsin and plated to 96-well plates at 1.5 × 104 cells
per well. The outer wells of the plate had media added but
are not used for the assay to avoid the possibility of edge
effects. The following day a dilution series was made from the
test compounds in neat DMSO. These were further diluted into
DMEM + 5% FCS, and 100 µL of these dilutions was added
per well to the cell plates. The final DMSO concentration per
well was 0.5% in all wells. The plates were then incubated for
a further 24 h. A colorimetric 5-bromo-2′-deoxyuridine (BrdU)
Cell Proliferation ELISA kit (Roche Diagnostics GmbH) was
then used to assess proliferation according to the manufac-
turer’s instructions. Briefly, the cells were pulse labeled with
BrdU for 2 h and fixed, and the cellular DNA was denatured
with the provided solution and then incubated with Anti-BrdU-
POD for 90 min. The plates were then washed (3×) with PBS
and patted dry, the TMB substrate solution was added, and
the plates were incubated on a plate shaker for 10-30 min
until the positive control absorbance at 690 nm was ca. 1.5.
Positive control and blank wells were included on all plates,
containing cells + compound diluent and no plated cells +
compound diluent, respectively, to determine the dynamic
range of the assay. The curves were plotted and the IC50 values
for compound inhibition of cell proliferation were interpolated
using Microcalc Origin following subtraction of the blank
values.
Molecu la r Mod elin g. Modeling and docking studies have
been performed using the published crystal structure of
activated Lck as a surrogate for Src.26 Indeed, the 3D structure
of Src was also available at that time, but the kinase was in
an inactivated form.33 Owing to the high sequence homology
between the two kinases, particularly within the ATP binding
site, and in order to be consistent with the enzymatic assay
we used, we believed that the activated Lck structure was a
better tool for our modeling studies. Our inhibitors were built
in Quanta and the charges were assigned by the Quanta
charge template method.34,35 These inhibitors have been docked
manually into the ATP binding site, and the most relevant
solutions were then energy minimized with the CHARMm
force field to relieve possible unfavorable contacts.36
Biologica l Eva lu a tion . IC50 values reported are the mean
of three to five measurements.
(i) In Vitr o Sr c Kin a se In h ibition Test. This assay
determines the ability of test compounds to inhibit Src kinase
activity. A poly(Glu,Tyr) 4:1 random copolymer (Sigma-Ald-
rich, Poole, UK) was used as the tyrosine-containing substrate.
This is stored as a 10 mg/mL stock solution in PBS at -20 °C
and diluted 1:200 with PBS to coat 96-well plates (100 µL/
well). Substrate was plated the day before an assay, and the
plates were covered with adhesive seals and stored overnight
at 4 °C. On the day of the assay, the substrate solution was
discarded, and the plates were then incubated with 120 µL/
well of 5% BSA in PBS/A for 10 min. The plates were then
washed once with PBST (PBS containing 0.05% v/v Tween 20)
and then incubated with 50 mM HEPES pH 7.4 at 100 µL/
well until the next stage. Test compounds were dissolved in
DMSO at 10 mM. A dilution series was then made in doubly
distilled H2O to give solutions at four times the final required
reaction concentrations. Solutions of 40 µM ATP in 80 mM
MgCl2 and 80 mM MgCl2 alone (for -ve controls) were prepared.
Src kinase, expressed in sf9 insect cells by recombinant
baculovirus containing the human c-src gene (Upstate Bio-
technology, Lake Placid, NY), was diluted to 0.3 U/mL in
enzyme dilution buffer (100 mM HEPES, 2 mM DTT, 0.2 mM
sodium orthovanadate, 0.02% BSA). The HEPES was dis-
carded from the substrate plates and the following additions
made in order: 25 µL/well compound dilution (water in the
case of +ve and -ve controls), 25 µL/well of ATP/MgCl2 or
MgCl2 (-ve controls) alone, and finally, 50 µL/well Src kinase
in dilution buffer to start the reaction. The final reaction
concentrations were 0.15 U/mL Src kinase, 20 mM MgCl2, and
10 µM ATP (determined as the Km for ATP). The reaction time
allowed was 15 min at room temperature on a plate shaker.
(iv) In Vitr o A549 Micr od r op let Migr a tion (Ch em ok i-
n esis) Assa y. This assay is a development from the polymorph
microdroplet migration assay.37 The assay determines the
ability of test compounds to inhibit the random motility
(chemokinesis) of A549 cells (human epithelial lung carcinoma
cells, ATCC CCL 185) in 96-well plates. Difco Nobel Agar
(Difco Laboratories, Detroit, MI) was weighed, PBS/A was
added, and the mixture was autoclaved to give a 2% sterile
stock solution. While still hot this was transferred to a 42 °C
waterbath to prevent setting. A549 cells, routinely cultured

886 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 4
Ple´ et al.
(3) (a) Rosen, N.; Bolen, J . B.; Schwartz, A.; Cohen, P.; DeSeau, V.;
Israel, M. A. Analysis of pp60c-src Protein Kinase Activity in
Human Tumour Cell Lines and Tissues. J . Biol. Chem. 1986,
261, 13754-13759. (b) Cartwright, C. A.; Meisler, A. I.; Eckhart,
W. Activation of the pp60c-src Protein Kinase is an Early Event
in Human Colon Carcinoma. Proc. Natl. Acad. Sci. U.S.A. 1990,
84, 558-562. (c) Talamonti, M. S.; Roh, M. S.; Curley, S. A.;
Gallick, G. E. Increase in Activity and Level of pp60c-Src in
Progressive Stages of Human Colorectal Cancer. J . Clin. Invest.
1993, 91, 3-60. (d) Verbeek, B. S.; Vroom, T. M.; Adriaansen-
Slot, S. S.; Ottenhoff-Kalff, A. E.; Geertzema, J . G.; Hennipman,
A.; Rijksen, G. c-Src Protein Expression is Increased in Human
Breast Cancer. An Immunohistochemical and Biochemical Analy-
sis. J . Pathol. 1996, 180, 383-388. (e) Lutz, M. P.; Esser, I. B.;
Flossmann-Kast, B. B.; Vogelmann, R.; Luhrs, H.; Friess, H.;
Buchler, M. W.; Adler, G. Overexpression and Activation of the
Tyrosine Kinase Src in Human Pancreatic Carcinoma. Biochem.
Biophys. Res. Commun. 1998, 243, 503-508. (f) Reissig, D.;
Clement, J .; Sanger, J .; Berndt, A.; Kosmehl, H.; Bohmer, F. D.
Elevated Activity and Expression of Src Family Kinases in
Human Breast Carcinoma Versus Matched Non Tumour Tissue.
J . Cancer Res. Clin. Oncol. 2001, 127, 226-230.
in DMEM + 10% FCS, were harvested by trypsinization,
counted by hemocytometer or Coulter counter, centrifuged
(Heraeus Multifuge 3 L-R, 1000 rpm, 5 min), and resuspended
at 2 × 107 cells/mL in medium plus 0.3% agarose at 37 °C.
Microdroplets (2 µL) of the cell agarose suspension were
carefully pipetted centrally to the wells of nontissue culture
treated 96-well plates (Bibby Sterilin, Stone, Staffordshire,
UK). The completed plates were then placed briefly on ice to
assist gelling of the agarose. When set, 90 µL of chilled (4 °C)
RPMI 1640 medium (Gibco) + 10% FCS was slowly added per
well, and compound dilutions in medium were added as a
further 10 µL per well. The plates were transferred to a tissue
culture incubator and incubated for 72 h to allow migration
to occur. The distance of the migration front from the cell
source (agarose microdroplet) was measured as the mean of
four perpendicular axis per well assessed by microscopy using
a calibrated eyepiece graticule. Positive controls were included
on all plates containing cells + compound diluent. The curves
were plotted and the IC50 values for compound inhibition of
cell motility were interpolated using a purpose-designed Excel
spreadsheet. The addition of MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) for 1 h
at the end of the experiment allowed a parallel assessment of
the toxicity of the compounds. MTT is converted to purple/
black formazan crystals by the mitochondria of viable cells,
thus allowing a visual assessment of compound toxicity.
(v) Ra t P h a r m a cok in etics. Pharmacokinetics was deter-
mined in the mouse and rat following single intravenous (2
mg/kg) or oral administrations (20 or 50 mg/kg) of the
compound. For the iv study, the compounds were formulated
in a mixture of 25% (w/v) hydroxypropyl-â-cyclodextrin/Sor-
renson’s phosphate buffer (pH 5.5). For the oral study, the
compounds were formulated as a solution in either 1% polysor-
bate or 0.1 M citrate buffer (pH 3).
(4) Boyer, B.; Valles, A. M.; Edmen N. Induction and Regulation of
Epithelial-Mesenchymal Transitions. Biochem. Pharmacol. 2000,
60, 1091-1099.
(5) (a) Irby, R. B.; Yeatman, T. J . Increased Src Activity Disrupts
Cadherin/Catenin-mediated Homotypic Adhesion in Human
Colon Cancer and Transformed Rodent Cells. Cancer Res. 2002,
62, 2669-2674. (b) Avizienyte, E.; Wyke, A.; J ones, R. J .;
McLean, G. W.; Westhoff, M. A.; Brunton, V. G.; Frame, M. C.
Src-induced De-Regulation of E-cadherin in Colon Cancer Cells
Requires Integrin Signalling. Nature Cell Biol. 2002, 4, 632-
638.
(6) Nam, J .-S.; Ino, Y.; Sakamoto, M.; Hirohashi, S. Src Family
Kinase Inhibitor PP2 Restores the E-Cadherin/Catenin Cell
Adhesion System in Human Cancer Cells and Reduces Cancer
Metastasis. Clin. Cancer Res. 2002, 8, 2430-2436.
(7) (a) Fincham, V. J .; Frame, M. C. The Catalytic Activity of Src is
Dispensable for Translocation to Focal Adhesions but Controls
the Turnover of those Structures During Cell Motility. EMBO
J . 1998, 17, 81-92. (b) Frame, M. C. Src in Cancer: Deregulation
and Consequences for Cell Behaviour. Biochim. Biophys. Acta
2002, 1602, 114-130.
(8) Boyer, B.; Bourgeois, Y.; Poupon, M. F. Src Kinase Contributes
to the Metastatic Spread of Carcinoma Cells. Oncogene 2002,
21, 2347-2356.
(9) Allgayer, H.; Boyd, D. D.; Heiss, M. M.; Abdalla, E. K.; Curley,
S. A.; Gallick, G. E. Activation of Src Kinase in Primary
Colorectal Carcinoma: An Indicator of Poor Clinical Prognosis.
Cancer 2002, 94, 344-351.
(10) Wiener, J . R.; Widham, T. C.; Estrella, V. C.; Parikh, N. U.; Thall,
P. F.; Deavers, M. T.; Bast, R. C.; Mills, G. B.; Gallick, G. E.
Activated Src Protein Tyrosine Kinase is Overexpressed in Late-
Stage Human Ovarian Cancers. Gynecol. Oncol. 2003, 88, 73-
79.
(vi) Ra t Xen ogr a fts. Athymic rats (Hsd Han:RNUrnu, 6-8
weeks, male) were dosed po with test compound or vehicle (1%
polysorbate 80) 1 h prior to cell inoculation and once daily
thereafter. The cell inoculation, made on the left flank of each
animal, consisted of (1 × 106 cells/0.2 mL) c-src-transfected
3T3 cells in PBS. A total of 10 rats were used per group. Tumor
size was measured three times weekly with callipers and
tumor volume estimated using the formula {[(square root of
length × width) × (length × width)] × 0.5236}. Studies were
terminated once the control tumors showed signs of ulceration
or hemorrhage or had reached 10% of the body weight of the
host animal (typically at day 15).
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Ack n ow led gm en t. We would like to acknowledge
the excellent technical expertise of the following scien-
tists: for biology and in vivo work, Vivien J acobs, Karen
Malbon, Lindsey Millard, and Robin Whittaker; for
chemistry, Alain Bertrandie, Dominique Boucherot,
Myriam Didelot, Franc¸oise Magnien, Re´my Morgentin,
Marie-J eanne Pasquet, Annie Olivier, Michel Vautier,
Nicolas Warin; for NMR, Christian Delvare; for phar-
macokinetics, J ohn Swales and Tim Smith; for physical
chemistry, Delphine Dorison-Duval; for robotic synthe-
sis, Patrice Koza and J acques Pelleter.
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