Tyrosine kinase inhibitors. 15. 4-(Phenylamino)quinazoline and 4- (phenylamino)pyrido[d]pyrimidine acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor

Article abstract of DOI:10.1021/jm9806603

A series of 6- and 7-acrylamide derivatives of the 4- (phenylamino)quinazoline and -pyridopyrimidine classes of epidermal growth factor receptor (EGFR) inhibitors were prepared from the corresponding amino compounds by reaction with either acryloyl chloride/base or acrylic acid/1- (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. All of the 6- acrylamides, but only the parent quinazoline 7-acrylamide, were irreversible inhibitors of the isolated enzyme, confirming that the former are better- positioned, when bound to the enzyme, to react with the critical cysteine- 773. Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6- acrylamides were all irreversible inhibitors and showed similar high potencies in the enzyme assay (likely due to titration of the available enzyme). However the pyrido[3,2-d]pyrimidine analogues were 2-6-fold less potent than the others in a cellular autophosphorylation assay for EGFR in A431 cells. The quinazolines were generally less potent overall toward inhibition of heregulin-stimulated autophosphorylation of erbB2 (in MDA-MB- 453-cells), whereas the pyridopyrimidines were equipotent. Selected compounds were evaluated in A431 epidermoid and H125 non-small-cell lung cancer human tumor xenografts. The compounds showed better activity when given orally than intraperitoneally. All showed significant tumor growth inhibition (stasis) over a dose range. The poor aqueous solubility of the compounds was a drawback, requiring formulation as fine particulate emulsions.

Full text of DOI:10.1021/jm9806603

J . Med. Chem. 1999, 42, 1803-1815
1803
Tyr osin e Kin a se In h ibitor s. 15. 4-(P h en yla m in o)qu in a zolin e a n d
4-(P h en yla m in o)p yr id o[d ]p yr im id in e Acr yla m id es a s Ir r ever sible In h ibitor s of
th e ATP Bin d in g Site of th e Ep id er m a l Gr ow th F a ctor Recep tor
J eff B. Smaill,† Brian D. Palmer,† Gordon W. Rewcastle,† William A. Denny,*,† Dennis J . McNamara,‡
Ellen M. Dobrusin,‡ Alexander J . Bridges,‡ Hairong Zhou,‡ H. D. Hollis Showalter,‡ R. Thomas Winters,‡
Wilbur R. Leopold,‡ David W. Fry,‡ J ames M. Nelson,‡ Veronika Slintak,‡ William L. Elliot,‡ Billy J . Roberts,‡
Patrick W. Vincent,‡ and Sandra J . Patmore‡
Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, The University of Auckland,
Private Bag 92019, Auckland, New Zealand, and Parke-Davis Pharmaceutical Research, Division of Warner-Lambert
Company, 2800 Plymouth Road, Ann Arbor, Michigan 48106-1047
Received November 23, 1998
A series of 6- and 7-acrylamide derivatives of the 4-(phenylamino)quinazoline and -pyridopy-
rimidine classes of epidermal growth factor receptor (EGFR) inhibitors were prepared from
the corresponding amino compounds by reaction with either acryloyl chloride/base or acrylic
acid/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. All of the 6-acrylamides,
but only the parent quinazoline 7-acrylamide, were irreversible inhibitors of the isolated enzyme,
confirming that the former are better-positioned, when bound to the enzyme, to react with the
critical cysteine-773. Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6-acryl-
amides were all irreversible inhibitors and showed similar high potencies in the enzyme assay
(likely due to titration of the available enzyme). However the pyrido[3,2-d]pyrimidine analogues
were 2-6-fold less potent than the others in a cellular autophosphorylation assay for EGFR in
A431 cells. The quinazolines were generally less potent overall toward inhibition of heregulin-
stimulated autophosphorylation of erbB2 (in MDA-MB-453-cells), whereas the pyridopyrim-
idines were equipotent. Selected compounds were evaluated in A431 epidermoid and H125
non-small-cell lung cancer human tumor xenografts. The compounds showed better activity
when given orally than intraperitoneally. All showed significant tumor growth inhibition (stasis)
over a dose range. The poor aqueous solubility of the compounds was a drawback, requiring
formulation as fine particulate emulsions.
In tr od u ction
Following numerous reports that overexpression of
the epidermal growth factor receptor (EGFR) is a
marker for poor prognosis in a significant proportion of
human tumors,1,2 selective inhibitors of tyrosine phos-
phorylation by EGFR have become an important class
of potential anticancer drugs.3-5 Several classes of
inhibitors have been reported, with the most potent and
selective being the 4-(phenylamino)quinazolines6-9 and
related 4-(phenylamino)pyrido[d]pyrimidines.10-12 Ki-
netic studies7,12 show that these compounds selectively
inhibit EGF-stimulated signal transduction by revers-
ibly binding at the ATP site of EGFR. Many of these
compounds are extremely potent inhibitors of the iso-
lated enzyme and inhibit EGF-stimulated EGFR auto-
phosphorylation in cells for up to 4 h.8 Two 4-(phenyl-
inhibitors based on the 4-(phenylamino)quinazolines.
amino)quinazolines, CP 358774 (1) and ZD 1839 (2), are
We recently reported15 that the acrylamides (3a and 5a )
reported to be in clinical trial.13,14
are potent, selective, irreversible inhibitors of the EGFR
Despite the high potency and prolonged inhibition of
tyrosine kinase, with improved in vivo antitumor activ-
EGFR function reported for some of these reversible
ity compared to the related reversible analogues 10 and
inhibitors, the high intracellular concentrations of ATP
11.
make it difficult to reach sufficiently high concentrations
These molecules were designed initially to trap the
in vivo to fully shut down EGF-stimulated signal
Cys-773 of EGFR, which had been identified from
transduction for long periods. We15 and others16 have
sequence homology data as being very uncommon at
therefore been studying the development of irreversible
that position in kinases but shared by EGFR, erbB-2,
and erbB-4. Sequence homology suggested that it is the
† The University of Auckland.
‡ Parke-Davis Pharmaceutical Research.
EGFR equivalent of Asn-127 in PKA.17 As this residue
10.1021/jm9806603 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/04/1999

1804 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Smaill et al.
Sch em e 1a
a
(i) Raney Ni/H2/MeOH/25 °C/15 h; (ii) acrylic acid/EDCI‚HCl/DMF (and/or THF, DMA)/Et3N or pyridine/25 °C/20 h; (iii) Fe/AcOH/
H2O/EtOH/reflux/1 h; (iv) acryloyl chloride/THF:DMF/DMAP/Et3N/0-20 °C/20 h; (v) acrylic acid/isobutyl chloroformate/THF/DMF/Et3N/0
°C/5 h.
has been shown by X-ray crystallographic studies to
H-bond to the 2′-hydroxyl of the ATP ribose, it should
be in relatively close proximity to the hydrophobic
adenine binding pocket where we presumed that the
bicyclopyrimidine binds. The ability to trap this cysteine
was confirmed by the demonstration that 2-thioadenos-
ine is an irreversible inhibitor of EGFR, but not of
PDGFR, where the equivalent residue is not a cys-
teine.17 Our initial modeling studies of the binding of
reversible anilinoquinazoline and anilinopyridopyrimi-
dine inhibitors15,18 suggested a binding mode whereby
the 6- and 7-positions of the bicyclic chromophore point
out of the ATP binding pocket toward the solvent. This
explains why both positions can be used to add bulky
solubilizing functionalities to inhibitors without losing
binding affinity.13,14,19,20 When an acrylamido side chain
was attached to the 6-position in the model, the γ-carbon
was placed only 3.5 Å from the cysteinyl sulfur, whereas
at the 7-position the corresponding distance was nearly
8 Å.15 These distances suggest that the 6-acrylamido
side chain is optimally placed for reaction with Cys-773
in its most stable binding mode, whereas the 7-acryla-
mido side chain would require some movement in the
binding site to allow it to approach close enough to the
sulfur for Michael addition to occur. This is consistent
with the kinetics of alkylation,15 which show extremely
rapid alkylation by the 6-acrylamide and much slower
alkylation by the 7-acrylamide.
We now report the synthesis and biological activity
of a range of 6- and 7-substituted acrylamidoquinazo-
lines and acrylamidopyrido[d]pyrimidines, made to
further investigate the structure-activity requirements
for irreversible inhibition of EGFR and to provide
potentially improved analogues in terms of potency
against EGFR and erbB2, solubility, and in vivo anti-
tumor activity.
Ch em istr y
The 6- and 7-acrylamides 3-8 of Table 1 were
prepared from the corresponding 7-aminoquinazolines
13-15, 6-aminoquinazolines 23-30, 7-aminopyrido[4,3-
d]pyrimidine 16, 6-aminopyrido[3,4-d]pyrimidines 31-
34, and 6-aminopyrido[3,2-d]pyrimidines 35-37 usually
by reaction either with acryloyl chloride and a base
(pyridine or triethylamine) or with acrylic acid in the
presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride (EDCI‚HCl) (Scheme 1).
For the synthesis of 5c, the mixed anhydride from
acrylic acid and isobutyl chloroformate was used. Many
of the required amino compounds were known,8,10,11,21
and new ones were prepared by similar methods
(Schemes 1 and 2). Yields for the final acylation step
were in the range 20-60%, with varying amounts of
polymeric material also being formed. The pyridopyri-
midines were less reactive to amide formation than the
corresponding quinazolines.

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1805
Sch em e 2a
Ta ble 1. Physicochemical and EGFR Inhibitory Properties of
4-(Phenylamino)quinazoline and
4-(Phenylamino)pyrido[d]pyrimidine Acrylamides
a
(i) 4-OMePhCH2NH2/DMSO/100 °C/16 h; (ii) TFA/reflux/30
min; (iii) NH3(g)/EtOH or i-PrOH/110 °C/18 h (pressure).
isolated enzyme autophosphorylation
IC50[app] (nM)
IC50 (nM)
Resu lts a n d Discu ssion
EGFR HER
no.
R
mp (°C)
>265
EGFRa
(A431)b (MDA)c irrevd
The structures and physicochemical properties of the
acrylamides studied are listed in Table 1, together with
their potencies for inhibition of phosphorylation of a
glutamic acid/tyrosine random copolymer substrate by
isolated EGFR enzyme, inhibition of EGF-stimulated
autophosphorylation of EGFR in A431 cells, and inhibi-
tion of heregulin-stimulated autophosphorylation of
erbB2 in MDA-MB-453 cells.12 The nature of the inhibi-
tion of the EGFR enzyme was also determined for each
compound, but not by rigorous biochemical means as
this requires radiolabeling and mass spectroscopic
methods. Instead, a variation of the cellular autophos-
phorylation assay of A431 cells was used. The cells were
incubated with a fixed concentration (2 µM) of the
inhibitor for 1 h and then washed free of drug. After 8
h EGF was added; after a further 10-min incubation the
cells were lysed, and the degree of inhibition of auto-
phosphorylation was measured. With this protocol, even
the very potent (IC50 29 pM) reversible inhibitor 9
showed only 5% inhibition after 8 h, whereas the
verified15 irreversible inhibitors 3a and 5a showed 89%
and 100% inhibition, respectively.
Compounds which showed 80% or greater inhibition
after the 8-h washout were designated as irreversible
inhibitors. Those which showed 20-80% inhibition were
designated as partially irreversible under the test
conditions (although in reality they can almost certainly
fully inactivate the enzyme via alkylation given suf-
ficient time). Those which produced less than 20%
inhibition were classified as reversible. If alkylation is
slow relative to the length of time of the incubation, not
all of the enzyme may be alkylated. Also, provided
unreacted inhibitor does not remain in the cells to
alkylate newly synthesized receptor (the half-life for
turnover of EGFR in unstimulated A431 cells is ∼20
h), some activity would be expected after the washout
period from newly synthesized receptor. Conversely, if
a reversible inhibitor were unusually well-sequestered
in cells, as has been demonstrated previously for some
reversible quinazolines,8 it could show up as a false
positive in this assay. Thus the fully reversible Zeneca
clinical candidate 2, which is strongly sequestered in
cells, produced 100% inhibition after the 8-h washout
period, demonstrating that the above method can pro-
duce false positives for irreversible inhibition.
3a Br
3b Cl
3c Me
4a Br
5a 3′-Br
5b 3′-Cl
5c 3′-Me
5d 3′-CF3
5e 3′-Br, 4′-F 276-278.5
5f 3′-Cl, 4′-F 260-262
5g 4′-OPh
5h 4′-OBn
6a 3′-Br
0.45
0.25
1.6
14
53
90
73
yes
part
no
296.5-298.5
269.5-270
215-220
258-261
223-227
247-248
195-199
0.54
0.70
1.6
0.42
0.91
0.69
0.75
5.8
108
4.3
2.3
4.7
4.5
2.7
3.1
4.8
4.2
3.4
8.3
2.1
3.3
5.6
18
part
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
5.7
22
7.3
4.3
8.6
7.6
3.9
8.8
1.1
3.0
185-191
227-229
243-245
221-223
3.6
0.91
0.48
0.72
0.77
1.1
1.2
0.75
0.40
6c 3′-Me
6e 3′-Br, 4′-F 245-248
6f 3′-Cl, 4′-F >245 dec
7a 3′-Br
7e 3′-Br, 4′-F 241-243
7f 3′-Cl, 4′-F 247
226-228
26
12
18
9.9
8
214-216
a
IC50[app] (nM) to inhibit the phosphorylation of a poly(glutamic
acid)/tyrosine random copolymer by EGFR (prepared from human
A431 carcinoma cell vesicles by immunoaffinity chromatography).
See Experimental Section for details. Values are the averages from
at least two independent dose-response curves; variation was
generally (15%. b,c IC50 values (nM) for inhibition of autophos-
phorylation of EGFR (in A431 cells) or erbB2 (in MDA-MB-453
cells) in culture. Values are the average of two experiments; see
d
Experimental Section for details. Irreversible inhibition is de-
fined as no detection of phosphorylated EGFR in A431 cells 8 h
after washing cells free of the inhibitor.
The irreversibility assay should thus be viewed as a
method for distinguishing whether a particular chemical
modification permits or destroys irreversibility. Com-
pounds categorized as “partially irreversible” are likely
capable of irreversibly inhibiting the enzyme, but at
such a rate that the process is not complete within the
1-h incubation period. Because we believe that truly
erroneous results are rare, and because slow “partial”
alkylators proved no more active in vivo than ones
which show up as fully reversible, the assay is of value
as a high-throughput initial classification of candidate
compounds.
The influence of the positioning of the acrylamide, aza
atom substitution in the quinazoline chromophore, and
(in 6-acrylamidoquinazolines) the effect of substituents
in the aniline ring on both irreversibility and inhibitory
potency were evaluated. In these evaluations, it must
be borne in mind that equilibrium enzyme kinetics are

1806 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Smaill et al.
not valid for irreversible compounds and that IC50
values for both the isolated enzyme and cellular data
depend on the rate and completeness of alkylation
(which in turn depend on the reversible binding affinity
of each acrylamide-bearing template) as well as on the
concentration of enzyme present. In fact, for irreversible
inhibitors the IC50 values derive essentially from titrat-
ing the enzyme activity in a stoichiometric manner and
for this reason are designated as apparent IC50’s
(IC50[app]). The concentration of EGFR in the isolated
enzyme assays is calculated at 1.18 nM and was held
as constant as possible (<10% variation). All isolated
enzyme assays were carried out identically over a 10-
min incubation period and used the same enzyme
preparation. The data in Table 1 are thus not kinetic
parameters, but comparative data for each compound
under precisely defined conditions. For rapid alkylators,
where the addition reaction goes to completion within
the incubation period, the IC50[app] values should all be
similar (approximately one-half the concentration of the
enzyme). However, it is still possible to obtain low-
nanomolar IC50[app] values in the isolated enzyme assay
for partially irreversible (slow alkylators) and reversible
inhibitors due to their intrinsically high reversible
binding affinity.
Br to 3′-Cl to 3′-Me, alkylation becomes slow enough on
the time scale of the experiment to become unimportant.
Aza Su b st it u t ion in t h e Qu in a zolin e Ch r om o-
p h or e. Five sets of compounds were prepared where
the position of the acrylamide and the nature of the
aniline substituents were unchanged (within a particu-
lar set), while the central chromophore was varied from
quinazoline to pyrido[3,4-d]pyrimidine, pyrido[3,2-d]-
pyrimidine, and pyrido[4,3-d]pyrimidine (Table 1). We
have previously shown11 that all of these pyrido[d]-
pyrimidine chromophores bind reversibly at the ATP
site of EGFR.
In the 7-acrylamide set (3a and 4a ), changing the
quinazoline to pyrido[4,3-d]pyrimidine resulted in loss
of irreversible inhibition and a corresponding loss in
potency in the autophosphorylation assay (although
retaining comparable IC50[app] values against isolated
EGFR). This reinforces the above view that the 7-acryl-
amide is not well-placed to react with Cys-773, espe-
cially as the aza substituent would be expected to
increase the reactivity of the acrylamide as a Michael
acceptor. The remaining sets were 6-acrylamide ana-
logues that all retained irreversible inhibition of EGFR,
indicating a broad tolerance for structural variation
when the acrylamide is optimally situated. Compounds
5c and 6c, which compare quinazoline and pyrido[3,4-
d]pyrimidine analogues with a 3′-Me aniline substitu-
ent, had similar IC50[app] values against the isolated
enzyme (close to the maximum expected), consistent
with rapid, irreversible inhibition. The remaining three
sets of three (5a , 6a , 7a ; 5e, 6e, 7e; and 5f, 6f, 7f)
compare quinazoline, pyrido[3,4-d]pyrimidine, and py-
rido[3,2-d]pyrimidine chromophores with 3′-bromo, 3′-
bromo-4′-fluoro, and 3′-chloro-4′-fluoro aniline substit-
uents, respectively. Again, IC50[app] values for inhibition
of isolated enzyme were similar for all three chro-
mophores and consistent with rapid irreversible inhibi-
tion.
P osition in g of th e Acr yla m id e Mich a el Accep -
tor . We have previously reported,15 from studies with
3a and 5a , that a 6-acrylamide appears better posi-
tioned than a 7-acrylamide (4 Å closer to the Cys-773
thiol) to form the required bond. The present work
provides additional support for this proposal, by a
comparison of the properties of two more pairs of 6- and
7-acrylamides (3b and 5b, 3c and 5c). All three 6-acryl-
amides (5a -c) are completely irreversible inhibitors of
autophosphorylation in A431 cells (Table 1) and have
isolated enzyme IC50[app] values close to those expected
for compounds capable of rapid and complete alkylation
of the enzyme (see above). In contrast, only the original
3′-Br analogue 3a in the 7-acrylamide series proved
irreversible in the 8-h washout assay. The 3′-Cl and 3′-
Me analogues 3b and 3c were partial irreversible and
reversible inhibitors, respectively. Although the IC50[app]
values of these two sets of compounds against the
isolated enzyme were not very different, the fully
irreversible 6-acrylamides were much more potent
inhibitors than the partially irreversible 7-acrylamides
in the cellular autophosphorylation assays. These re-
sults suggest that while high potency in the isolated
enzyme assay is desirable, it does not necessarily
distinguish between compounds capable of rapid and
complete alkylation of the enzyme and compounds
capable of potent enzyme inhibition via high-affinity
reversible binding. However there appears to be a
correlation between irreversible binding (as determined
by the 8-h washout assay) and potency in the cellular
autophosphorylation assays, suggesting that this assay
may be more useful in ranking compounds for advanced
evaluation and development. These results are consis-
tent with the idea that the ATP binding pocket does not
have enough flexibility to allow an optimal distance
between the 7-acrylamide and Cys-773 and that as the
intrinsic binding to the enzyme decreases (as seen from
the SAR for reversible inhibition) when going from 3′-
However, the latter two pyrido[3,2-d]pyrimidines
were somewhat less potent than the other analogues
in the EGFR autophosphorylation assay. One possible
explanation for this is that the aza substituent increases
the electron deficiency of the Michael acceptor, making
it too reactive to retain selectivity and allowing loss by
indiscriminate alkylation. The aniline side chain is also
known to have a different favored conformation when
a 5-aza atom is present.22 The quinazolines were gener-
ally slightly less potent inhibitors of erbB2 than EGFR
in the cellular autophosphorylation assays, but the
pyridopyrimidines proved equipotent in these assays.
The latter compounds thus form a group of novel and
very potent generic inhibitors of the erbB family of
receptor TKs. We have previously reported15 that 14C-
labeled 3a binds covalently to both EGFR and erbB2,
and it is thus very likely that the excellent activity
shown by the pyridopyrimidines against erbB2 is largely
due also to irreversible inhibition of this enzyme. Among
reversible compounds, the pyridopyrimidines are more
active than the corresponding quinazolines against
erbB2 autophosphorylation.23
Va r ia tion s in Sid e Ch a in Su bstitu en ts. A series
of 6-acrylamidoquinazolines (5a -5h ) with variations in
the aniline were investigated. The 3′-substituents were
known from previous work to assist binding by interac-

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1807
Ta ble 2. In Vivo Anticancer Activity of Selected 4-(Phenylamino)quinazoline and 4-(Phenylamino)pyrido[d]pyrimidine Acrylamides
against Tumor Xenografts in Nude Mice
weight
change
(g)
T/C (%)
on last
dose
T-C
net cell
no.
tumora
(mg/kg)b
schedulec
therapy dayd
(days)e
kill (log10)f
5a
A431
A431
H125
H125
A431
A431
H125
H125
A431
H125
A431
A431
H125
H125
A431
H125
A431
H125
47
ip, b.i.d. days 7-11, 14-18, 21-25
po, b.i.d. days 10-24
ip, b.i.d. days 11-15, 18-22, 25-29
po, days 21-35
-1.0
2
2
15
8
11
3
24
15
11
16
17
11
26
20
13
19
12
27
29.0
28.6
13.9
15.7
32.3
28.2
10.5
11.9
20.8
27.4
12.0
24.3
8.7
+0.6
+1.0
-0.2
+0.1
+0.9
+0.8
-0.2
-0.3
+0.4
+0.9
150 (hdt)
50 (hdt)
50 (hdt)
75 (hdt)
260 (hdt)
250 (hdt)
50 (hdt)
40 (hdt)
200 (hdt)
12 (ldt)
200 (hdt)
9.6
200 (hdt)
200 (hdt)
200 (hdt)
200 (hdt)
200 (hdt)
-1.7
-1.5
+
5c
ip, b.i.d. days 14-28
po, days 10-23
-2.2
-1.3
-0.1
0
po, days 21-35
ip, b.i.d. days 11-15, 18-22, 25-29
po, days 10-23
5f
0
po, days 21-35
ip, b.i.d. days 13-26
po, days 10-24
-0.3
-2.4 (2/6)g
+
-1.3
+
+
-1.8
-0.3
-1.8
6a
+0.9
-0.5
+0.1
+1.7
+0.2
+0.4
-0.1
ip, b.i.d. days 11-15, 18-22, 25-29
po, days 21-35
16.5
51.1
18.2
18.3
12.7
6e
6f
po, days 10-24
po, days 15-29
po, days 10-24
po, days 15-29
a
b
The indicated tumor fragments were implanted sc into the right axilla of mice on day 0. hdt, highest dose tested; ldt, lowest dose
tested. c Compounds were administered intraperitoneally or orally on the indicated schedules. The maximum tolerated dose (LD10) from
d
a complete dose response is shown for individual experiments, unless otherwise noted. Ratio of median treated tumor mass/median
control tumor mass × 100%. e The difference in days for the treated (T) and the control (C) tumors to reach 750 mg. f The net reduction
g
in tumor burden, in logs, between the first and last treatments. 2/6, 2 toxic deaths in a group of 6 animals.
tion with a lipophilic pocket in the ATP binding domain,
while a 4′-F group has been shown24 to extend plasma
half-life by slowing down metabolism in the dianili-
nophthalimide EGFR inhibitors. All except the last two
(5g and 5h ), with the more bulky side chains (4′-OPh
and 4′-OBn), had similar IC50[app] values against the
isolated enzyme (∼0.8 nM), consistent with irreversible
inhibition. These showed lower potency against the
isolated enzyme (IC50[app] ) 5.8 and 3.6 nM, respec-
tively), despite being fully irreversible in the 8-h wash-
out assay. This suggests they have a lower binding
affinity for EGFR than the other analogues (5a -5f)
(presumably due to steric bulk in the 4′-position) and
therefore a rate of alkylation that falls between the
incubation times for the isolated enzyme and irrevers-
ibility assays (10 min and 1 h, respectively). These
compounds also dramatically illustrate the apparent
advantage of irreversible inhibition for cellular erbB2-
stimulated autophosphorylation activity. The corre-
sponding(reversible)and EGFR-selective25 6,7-dimethoxy-
quinazoline inhibitors have reported IC50 values of 19
and 11 nM against isolated EGFR, respectively, but IC50
values of 700 and 1200 nM in the erbB2 autophospho-
rylation assay.26 Thus the EGFR (isolated enzyme)
potencies are only increased about 3-fold, whereas the
erbB2 (cellular) potencies are increased around 100-fold.
The bromoindoline analogue 8, a tethered aniline, was
also fully irreversible and possessed potent isolated
enzyme (IC50[app] ) 0.40 nM) and cellular autophospho-
rylation activity.
versible 4-(phenylamino)quinazoline inhibitors.20 The
compounds were dosed as suspensions, both ip and po.
Ip dosing was generally more toxic, as evidenced by
generally greater treatment-related weight loss, gross
morphological liver changes, and attainment of maxi-
mum tolerated doses for 5a and 6a . With po dosing in
particular, toxicity was not reached at the highest doses
tested (Table 2), and no liver alterations were observed.
All of the compounds tested showed robust in vivo
activity. On po dosing, all showed significant tumor
growth inhibition over a dose range and activity at the
highest doses tested without lethality or significant
treatment-associated weight loss (>10%), indicating
acceptable therapeutic indices. Net tumor cell kill values
were around -0.5 to +0.5 log unit, indicating tumor
stasis. Upon cessation of therapy treated tumor growth
resumed, but in po dosing all of the compounds produced
a growth delay at least as long as the dosing period
against A431, and four of the six compounds did the
same with the more refractory H125 tumors. However,
it was not possible to discern major differences between
the compounds, aside from the fact that ip dosing was
apparently more toxic than po dosing.
Con clu sion s
The above results show that 6-acrylamides of 4-(phe-
nylamino)quinazolines and -pyridopyrimidines are ro-
bust, irreversible inhibitors of the epidermal growth
factor receptor, presumably by reaction at Cys-773, as
previously shown15 for the parent compound 5a. Quinazo-
line, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimi-
dine 6-acrylamides all showed irreversible inhibition
and in general similar potencies in the isolated enzyme
assay, suggesting titration of the available EGFR. The
fact that the pyrido[3,2-d]pyrimidine analogues were
somewhat less potent in the cellular autophosphoryla-
tion assays suggests another factor, possibly increased
reactivity, altered absorption or metabolism. The quinazo-
lines were generally less potent against erbB2 than
EGFR in the cellular assays, but the pyridopyrimidines
In Vivo Stu d ies. Compounds 5a , 5c, 5f, 6a , 6e, and
6f were evaluated in vivo against the A431 epidermoid
and H125 non-small-cell lung cancer human tumor
xenograft models. As rapid, irreversible inhibitors, all
these compounds had essentially similar in vitro poten-
cies (Table 1), so that any differences revealed in vivo
might reflect differences in pharmacokinetics and me-
tabolism. The tumor models were selected because they
express at least two members of the erbB receptor
family20,27 and because of their responsiveness to re-

1808 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Smaill et al.
9.0, 2.2 Hz, 1 H, H-6), 7.57 (m, 2 H, H-2′,6′), 7.38 (dd, J ) 7.6,
8.6 Hz, 1 H, H-5′), 7.15 (d, J ) 7.6 Hz, 1 H, H-4′), 2.37 (s, 3 H,
Me). Anal. (C15H12N4O2‚HCl‚0.5C3H6O) C, H, N.
were equipotent in both assays, providing an interesting
class of generic inhibitors of the erbB family. The
compounds showed encouraging in vivo activity, being
cytostatic rather than cytotoxic, but with good thera-
peutic indices. Their poor aqueous solubility was a
drawback, requiring formulation as fine particulate
emulsions. While variation in aniline substitution im-
proves solubility somewhat (in the order 3′-Br to 3′-Cl,
3′-CF3, and 3′-CH3), further substantial improvements
are desirable, and this is the focus of continuing work.
A solution of 12 (338 mg, 1.07 mmol) in MeOH (75 mL) was
hydrogenated at 50 psi over Ra Ni (0.3 g) at 25 °C for 14.5 h.
After isolation the crude product was dissolved in water and
neutralized with dilute NaOH solution. The solid was collected
and purified by column chromatography on silica gel, eluting
with 5-10% MeOH in CHCl3, to give 7-amino-4-[(3-meth-
ylphenyl)amino]quinazoline (15) (135 mg, 50%) as a pale-
yellow solvated glass that was used directly (lit.32 mp 196-
197 °C): 1H NMR [(CD3)2SO] δ 9.24 (brs, 1 H, NH), 8.35 (s, 1
H, H-2), 8.18 (d, J ) 9.0 Hz, 1 H, H-5), 7.64 (m, 2 H, H-4′, 8),
7.22 (t, J ) 7.8 Hz, 1 H, H-5′), 6.89-6.83 (m, 2 H, H-6, H6′),
6.69 (d, J ) 2.2 Hz, 1 H, H-2′), 6.02 (brs, 2 H, NH2), 2.32 (s, 3
H, Me).
Coupling of 15 with acrylic acid and EDCI.HCl in DMF and
Et3N (1.1 mmol) as above for 20 h and trituration of the crude
product by sonication in a mixture of CH2Cl2/EtOAc/MeOH
gave 3c (75 mg, 49%): mp 269.5-270 °C; 1H NMR [(CD3)2SO]
δ 10.63 (s, 1 H, CONH), 9.68 (s, 1 H, NH), 8.58 (s, 1 H, H-2),
8.54 (d, J ) 9.3 Hz, 1 H, H-6), 8.25 (d, J ) 2.2 Hz, 1 H, H-8),
7.83 (dd, J ) 9.0, 1.9 Hz, 1 H, H-5), 7.71 (m, 2 H, H-2′,6′), 7.32
(t, J ) 8.3 Hz, 1 H, H-5′), 6.99 (d, J ) 7.1 Hz, 1 H, H-4′), 6.56,
(dd, J ) 16.8, 10.0 Hz, 1 H, CHdCH2), 6.40 (dd, J ) 17.1, 5.0
Hz, 1 H, CHdCH2), 5.9 (dd, J ) 10.3, 2.0 Hz, 1 H, CHdCH2),
2.39 (s, 3 H, CH3). Anal. (C18H16N4O‚0.5H2O) C, H, N.
Exp er im en ta l Section
Analyses were performed by the Microchemical Laboratory,
University of Otago, Dunedin, NZ, or by Parke-Davis Phar-
maceutical Research Analytical Department. Melting points
were determined using an Electrothermal model 9200 or
Gallenkamp digital melting point apparatus and are as read.
NMR spectra were measured on Bruker DRX-400 or Varian
Unity 400-MHz spectrometers and referenced to Me4Si. Mass
spectra were recorded either on a Varian VG 7070 spectrom-
eter at nominal 5000 resolution or an a Finnigan MAT 900Q
spectrometer. 2-Bromo-1-fluoro-4-nitrobenzene was prepared
by bromination of 1-fluoro-4-nitrobenzene according to the
literature procedure.28,29 Iron dust reduction then gave 3-bromo-
4-fluoroaniline30 as an oil, which was used directly.
N-[4-[(3-Br om op h en yl)a m in o]p yr id o[4,3-d ]p yr im id in -
7-yl]a cr yla m id e (4a ). A stirred solution of 7-amino-4-[(3-
bromophenyl)amino]pyrido[4,3-d]pyrimidine10 (16) (140 mg,
0.46 mmol), DMAP (14 mg), and Et3N (excess, 2.0 mL) in THF/
DMF (7:1, 17 mL) at 0 °C under N2 was treated dropwise over
4 h with acryloyl chloride (4.8 mol equiv, 182 µL). The reaction
was then stirred at 20 °C overnight before being worked up
as above and chromatographed on silica gel. Elution with
MeOH/CH2Cl2/EtOAc (5:45:50) gave 4a (12 mg, 7%): mp (CH2-
Cl2/hexane) 215-220 °C dec; 1H NMR [(CD3)2SO] δ 11.15 (s, 1
H, CONH), 10.25 (s, 1 H, NH), 9.67 (s, 1 H, H-5), 8.71 (s, 1 H,
H-2), 8.40 (s, 1 H, H-8), 8.21 (t, J ) 1.9 Hz, 1 H, H-2′), 7.88
(dt, J ) 7.6, 1.5 Hz, 1 H, H-6′), 7.38 (t, J ) 7.7 Hz, 1 H, H-5′),
7.36 (dt, J ) 7.7, 1.5 Hz, 1 H, H-4′), 6.68 (dd, J ) 17.1, 10.2
Hz, 1 H, CHdCH2), 6.39 (dd, J ) 17.0, 1.8 Hz, 1 H, CHdCH2),
5.86 (dd, J ) 10.1, 1.8 Hz, 1 H, CHdCH2). Anal. (C16H12BrN5O)
C, H.
N-[4-[(3-Br om op h en yl)a m in o]q u in a zolin -6-yl]a cr yla -
m id e (5a ). Acrylic acid (12.7 mmol, 0.87 mL) was added to a
solution of 6-amino-4-[(3-bromophenyl)amino]quinazoline21 (23)
(2.0 g, 6.35 mmol) in dry DMF (20 mL) under N2. The resulting
solution was cooled to 0 °C, and EDCI‚HCl (7.62 mmol, 1.46
g) was added. The reaction was stirred at 0 °C for 15 min and
then allowed to warm to room temperature and stirred for a
further 2 h, after which additional acrylic acid (0.30 mL) and
EDCI.HCl (0.30 g) were added. After a further 2 h, the reaction
was complete by TLC. Solvent was removed under reduced
pressure, and the resulting residue was diluted with saturated
NaHCO3 and repeatedly extracted with EtOAc. The combined
organic extracts were washed with brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. Column
chromatography on grade III alumina, eluting with EtOAc/
MeOH (95:5), followed by recrystallization from EtOAc/hexane
gave a spongy white solid, which upon several hours under
high vacuum gave pure 5a (1.06 g, 45%): mp 258-261 °C; 1H
NMR [(CD3)2SO] δ 10.51 (s, 1 H, CONH), 9.93 (s, 1 H, NH),
8.83 (br s, 1 H, H-5), 8.59 (s, 1 H, H-2), 8.18 (br s, 1 H, H-2′),
7.94-7.78 (m, 3 H, H-6′,8,5′), 7.40-7.27 (m, 2 H, H-7&4′), 6.54
(dd, J ) 17.0, 9.8 Hz, 1 H, CHdCH2), 6.36 (dd, J ) 16.9, 2.1
Hz, 1 H, CHdCH2), 5.85 (dd, J ) 9.7, 2.0 Hz, 1 H, CHdCH2).
Anal. (C17H13BrN4O) C, H, N.
N-[4-((3-Br om op h en yl)a m in o)q u in a zolin -7-yl]a cr yla -
m id e (3a ). An ice-cold solution of 7-amino-4-[(3-bromophenyl)-
amino]quinazoline21 (13) (0.158 mg, 0.5 mmol) and acrylic acid
(0.108 g, 1.5 mmol) in dry DMF (5 mL) was treated with 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI‚
HCl; 0.288 g, 1.5 mmol). The mixture was stirred for 5 min at
0 °C, during which time it became homogeneous, and then at
20 °C for a further 3 h. The mixture was then poured into ice/
water and basified with saturated aqueous NaHCO3. The
mixture was extracted with EtOAc (3×), and the combined
extracts were dried (MgSO4), filtered, and concentrated under
reduced pressure. The solid residue was dissolved in MeOH
(100 mL) and filtered, and the filtrate was concentrated under
reduced pressure to approximately 10 mL. The resulting
precipitate was dried under vacuum at 80 °C to give 3a (0.050
g, 28%): mp >265 °C; 1H NMR [(CD3)2SO] δ 10.61 (s, 1 H,
CONH), 9.79 (s, 1 H, NH), 8.61 (s, 1 H, H-2), 8.50 (d, J ) 8.9
Hz, 1 H, H-5), 8.26-8.23 (m, 2 H, H-2′, H-8), 7.91 (d, J ) 8.0
Hz, 1 H, H-6′), 7.82 (dd, J ) 9.2, 2.2 Hz, 1 H, H-6), 7.36 (t, J
) 8.1 Hz, 1 H, H-5′), 7.30 (m, 1 H, H-4′), 6.51 (dd, J ) 16.9,
10.1 Hz, 1 H, CHdCH2), 6.36 (dd, J ) 17.0, 1.9 Hz, 1 H, CHd
CH2), 5.86 (dd, J ) 10.1, 1.9 Hz, 1 H, CHdCH2). Anal. (C17H13
-
BrN4O) C, H, N.
N-[4-((3-Ch lor op h en yl)a m in o)qu in a zolin -7-yl]a cr yla -
m id e (3b). Similar reaction of 7-amino-4-[(3-chlorophenyl)-
amino]quinazoline8 (14) with acrylic acid and EDCI‚HCl in
DMF for 18 h gave a crude product that was dissolved in the
minimum amount of MeOH at 25 °C and then concentrated
under reduced pressure at 25 °C to one-sixth the volume. The
resulting precipitate was recrystallized from MeOH below 0
°C to give 3b (20%): mp 296.5-298.5 °C; 1H NMR [(CD3)2SO]
δ 10.61 (br s, 1 H, CONH), 9.80 (s, 1 H, NH), 8.62 (s, 1 H,
H-2), 8.50 (d, J ) 9.0 Hz, 1 H, H-5), 8.25 (d, J ) 2.0 Hz, 1 H,
H-8), 8.13 (t, J ) 2.0 Hz, 1 H, H-2′), 7.87-7.78 (m, 2 H, H-6,6′),
7.42 (t, J ) 8.2 Hz, 1 H, H-5′), 7.16 (dd, J ) 7.9, 2.2 Hz, 1 H,
H-4′), 6.51 (dd, J ) 17.1, 10.2 Hz, 1 H, CHdCH2), 6.35 (dd, J
) 17.1, 1.8 Hz, 1 H, CHdCH2), 5.86 (dd, J ) 10.1, 1.8 Hz, 1
H, CHdCH2). Anal. (C17H13ClN4O‚0.25H2O) C, H, N.
N-[4-((3-Meth ylp h en yl)a m in o)qu in a zolin -7-yl]a cr yla -
m id e (3c). A mixture of 3-methylaniline (223 mg, 2.1 mmol)
and 4-chloro-7-nitroquinazoline31 (420 mg, 2.0 mmol) in i-PrOH
(10 mL) was refluxed for 16 h. On cooling, the solid was
collected by Buchner filtration and air-dried to give the
hydrochloride salt of 4-[(3-methylphenyl)amino]-7-nitroquinazo-
line31 (12) as an acetone hemisolvate (401 mg, 58%): mp 230-
N-[4-[(3-Ch lor op h en yl)a m in o]qu in a zolin -6-yl]a cr yla -
m id e (5b). A mixture of 3-chloroaniline (5.10 g, 40 mmol) and
4-chloro-6-nitroquinazoline hydrochloride31 (10 mmol) in i-
PrOH (20 mL) was swirled at 25 °C for 10 min, producing a
noticeable exotherm and a bright-yellow precipitate. The
mixture was then stirred and refluxed for 3 h. On cooling, the
1
233 °C; H NMR [(CD3)2SO] δ 9.06 (d, J ) 9.0 Hz, 1 H, H-5),
8.95 (s, 1 H, H-2), 8.65 (d, J ) 2.5 Hz, 1 H, H-8), 8.52 (dd, J )

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1809
solid was collected by Buchner filtration, rinsed with i-PrOH
(10 mL), and dried in a vacuum oven at 80 °C to give 4-[(3-
chlorophenyl)amino]-6-nitroquinazoline (17) (2.35 g, 78%) as
a yellow solid that was used directly (lit.31 mp 272-274 °C):
1H NMR [(CD3)2SO] δ 10.47 (brs, 1 H, NH) 9.64 (d, J ) 2.2
Hz, 1 H, H-5), 8.77 (s, 1 H, H-2), 8.55 (dd, J ) 2.1, 9.3 Hz, 1
H, H-7), 8.07 (t, J ) 2.1 Hz, 1 H, H-2′), 7.94 (d, J ) 9.3 Hz, 1
H, H-8), 7.84 (dd, J ) 1.0, 8.1 Hz, 1 H, H-6′), 7.45 (t, J ) 8.1
Hz, 1 H, H-5′), 7.23 (ddd, J ) 1.0, 1.7, 8.1 Hz, 1 H, H-4′).
phase was extracted again with EtOAc. The combined organic
extracts were washed with water and saturated brine, dried
(MgSO4), and adsorbed onto silica gel (150 g) by evaporation
of the solvent. Flash chromatography of this on 700 g of silica
gel, eluting with Me2CO/CH2Cl2 (gradient from 25% to 40%
Me2CO), gave a crude product that was triturated in hot EtOAc
by sonication (20 min at 60 °C). The resulting solid was
collected by filtration and dried at 75 °C under vacuum for 16
h to give 5c (11.38 g, 35%): mp 247-248 °C; 1H NMR δ 10.49
(br s, 1 H, NH), 9.76 (br s, 1 H, NH), 8.75 (d, J ) 2.5 Hz, 1 H,
H-5), 8.52 (s, 1 H, H-2), 7.89 (dd, J ) 9.2, 2.0 Hz, 1 H, H-7),
7.77 (d, J ) 8.9 Hz, 1 H, H-8), 7.64-7.60 (m, 2 H, H2′,H-6′),
7.26 (td, J ) 7.5, 1.4 Hz, 1 H, H-5′), 6.94 (d, J ) 7.2 Hz, 1 H,
H-4′), 6.53 (dd, J ) 16.9, 10.1 Hz, 1 H, CHdCH2), 6.34 (dd, J
) 16.9, 1.9 Hz, 1 H, CHdCH2), 5.84 (dd, J ) 10.1, 1.9 Hz, 1
H, CHdCH2), 2.34 (s, 3 H, CH3). Anal. (C18H16N4O‚0.25H2O)
C, H, N.
A solution of 17 (2.05 g, 6.8 mmol) in THF/MeOH (3:1, 100
mL) was hydrogenated at 52.5 psi over Ra Ni (4 g) at 25 °C
for 22 h. Solvent was removed rigorously under reduced
pressure, and the residual solid was dried under vacuum at
80 °C to give 6-amino-4-[(3-chlorophenyl)amino]quinazoline
(24) (1.80 g, 98%) as a brown solid. An analytical sample was
purified by preparative TLC (7% MeOH/CHCl3): mp 186-189
1
°C (lit.32 mp >150 °C dec; H NMR [(CD3)2SO] δ 9.47 (brs, 1
H, NH), 8.39 (s, 1 H, H-2), 8.12 (t, J ) 2.0 Hz, 1 H, H-2′), 7.83
(ddd, J ) 0.7, 1.9, 8.2 Hz, 1 H, H-6′), 7.55 (d, J ) 8.7 Hz, 1 H,
H-5), 7.38 (t, J ) 8.1 Hz, 1 H, H-5′), 7.34 (d, J ) 2.1 Hz, 1 H,
H-5), 7.26 (dd, J ) 2.3, 8.8 Hz, 1 H, H-7), 7.10 (ddd, J ) 0.7,
1.9, 7.9 Hz, 1 H, H-4′), 5.64 (br s, 2 H, NH2); MS CI (1% NH3/
MeOH) 273 (29, 37ClMH+), 271 (100, 35ClMH+). Anal. (C14H11N4-
Cl) H, N; C: found, 61.7; required, 62.1.
N-[4-[(3-Tr iflu or om et h ylp h en yl)a m in o]q u in a zolin -6-
yl]a cr yla m id e (5d ). A solution of 6-amino-4-[(3-trifluoro-
methylphenyl)amino]quinazoline8 (26) and acrylic acid in
DMF/pyridine were treated with EDCI.HCl as above for 1 h.
The solution was then cooled to 0 °C and treated with dilute
HCl, and the resulting precipitate was collected by filtration,
washed with water, and dried at 75 °C under vacuum for 12
h to give 5d as the hydrochloride (87 mg, 45%): mp 195-199
A stirred solution of 24 (136 mg, 0.5 mmol), acrylic acid (74
mg, 1.0 mmol), and pyridine (200 mg, 2.5 mmol) in THF/DMF
(4:1, 2.5 mL) was treated at 0 °C with EDCI.HCl (190 mg, 1
mmol) and then stirred at 25 °C for 3 h. The mixture was
poured into water and extracted with EtOAc, and the organic
layer was separated and washed with dilute HCl. The resulting
precipitate was collected by filtration and washed with water
1
°C; H NMR δ 11.59 (br s, 1 H, NH), 10.99 (s, 1 H, NH), 9.17
(d, J ) 2.0 Hz, 1 H, H-5), 8.92 (s, 1 H, H-2), 8.12 (s, 1 H, H-2′),
8.10 (dd, J ) 9.2, 2.0 Hz, 1 H, H-7), 8.04 (d, J ) 8.0 Hz, 1 H,
H-6′), 7.98 (d, J ) 9,0 Hz, 1 H, H-8), 7.74 (t, J ) 7.9 Hz, 1 H,
H-5′), 7.68 (d, J ) 7.8 Hz, 1 H, H-4′), 6.60 (dd, J ) 16.9, 10.1
Hz, 1 H, CHdCH2), 6.38 (dd, J ) 16.9, 1.6 Hz, 1 H, CHdCH2),
5.89 (dd, J ) 10.1, 1,6 Hz, 1 H, CHdCH2). Anal. (C18H13F3N4O‚
HCl‚0.5H2O) C, H, N.
1
to give 5b as the HCl salt (93 mg, 48%): mp 223-227 °C; H
NMR [(CD3)2SO] δ 11.46 (br s, 1 H, NH), 11.05 (s, 1 H, NH),
9.13 (d, J ) 9.0, 2.0 Hz, 1 H, H-5), 8.90 (s, 1 H, H-2), 8.12 (dd,
J ) 9.0, 2.0 Hz, 1 H, H-7), 7.99 (d, J ) 9.0 Hz, 1 H, H-8), 7.88
(t, J ) 2.0 Hz, 1 H, H-2′), 7.68 (dd, J ) 6.1, 1.0 Hz, 1 H, H-6′),
7.51 (t, J ) 8.0 Hz, 1 H, H-5′), 7.37 (dd, J ) 8.1, 1.2 Hz, 1 H,
H-4′), 6.63 (dd, J ) 17.1, 10.3 Hz, 1 H, CHdCH2), 6.37 (dd, J
) 17.1, 1.6 Hz, 1 H, CHdCH2), 5.87 (dd, J ) 10.1, 1.7 Hz, 1
H, CHdCH2); MS CI (1% NH3/MeOH) 327 (8, 37ClMH+), 325
(37, 35ClMH+). Anal. (C18H13ClN4O‚HCl‚1.5H2O) C, H, N.
N-[4-[(3-Br om o-4-flu or op h en yl)a m in o]qu in a zolin -6-yl]-
a cr yla m id e (5e). Reaction of 4-chloro-6-nitroquinazoline31
with 3-bromo-4-fluoroaniline according to the standard cou-
pling procedure21 gave 4-[(3-bromo-4-fluorophenyl)amino]-6-
1
nitroquinazoline (19) (95%): mp (i-PrOH) 257-258.5 °C; H
NMR [(CD3)2SO] δ 10.50 (s, 1 H, NH), 9.63 (d, J ) 2.3 Hz, 1
H, H-5), 8.77 (d, J ) 1.9 Hz, 1 H, H-2), 8.57 (dd, J ) 9.2, 2.4
Hz, 1 H, H-7) 8.28 (td, J ) 6.2, 2.6 Hz, 1 H, H-2′), 7.96 (d, J )
9.2 Hz, 1 H, H-8), 7.94-7.88 (m, 1H, H-6′), 7.46 (t, J ) 8.8 Hz,
H-5′). Anal. (C14H8BrFN4O2) C, H, N.
N-[4-[(3-Meth ylp h en yl)a m in o]qu in a zolin -6-yl]a cr yla -
m id e (5c). A mixture of 3-methylaniline (16.0 g, 150 mmol)
and 4-chloro-6-nitroquinazoline hydrochloride31 (12.3 g, 50
mmol) in i-PrOH (100 mL) was swirled at 25 °C for 10 min.
After the exotherm, the mixture was stirred under reflux for
2 h. On cooling, the solid was collected by Buchner filtration,
rinsed with i-PrOH (2 × 25 mL), and dried in a vacuum oven
at 60 °C to give 4-[(3-methylphenyl)amino]-6-nitroquinazoline32
(18) (9.28 g, 66%) as a yellow solid: mp 250-252 °C; 1H NMR
[(CD3)2SO] δ 10.40 (brs, 1 H, NH) 9.68 (d, J ) 2.2 Hz, 1 H,
H-5), 8.71 (s, 1 H, H-2), 8.56 (dd, J ) 2.3, 9.1 Hz, 1 H, H-7),
7.93 (d, J ) 9.1 Hz, 1 H, H-8), 7.68-7.62 (m, 2 H, H-2′, 6′),
7.31 (t, J ) 8.0 Hz, 1 H, H-5′), 7.02 (d, J ) 7.6 Hz, 1 H, H-4′),
2.36 (s, 3 H, Me); MS CI (1% NH3/MeOH) 281 (100, MH+).
Anal. (C15H12N4O2) C, H, N.
Iron dust reduction of 19 according to the general proce-
dure21 gave 6-amino-4-[(3-bromo-4-fluorophenyl)amino]quinazo-
line (27) (85%): mp (i-PrOH) 224-225.5 °C; 1H NMR [(CD3)2-
SO] δ 9.47 (s, 1 H, NH), 9.47 (s, 1 H, NH), 8.36 (s, 1 H, H-2),
8.31 (dd, J ) 6.3, 2.4 Hz, 1 H, H-2′), 7.90-7.86 (m, 1 H, H-6′),
7.55 (d, J ) 8.8 Hz, 1 H, H-8), 7.38 (t, J ) 8.8 Hz, 1 H, H-5′),
7.31 (d, J ) 1.7 Hz, 1 H, H-5), 7.31 (dd, J ) 8.8, 1.9 Hz, 1 H,
H-7), 5.63 (s, 2 H, NH2). Anal. (C14H10BrFN4) C, H, N.
A mixture of 27 (0.50 g, 1.5 mmol) and acrylic acid (0.325
g, 4.5 mmol) in DMA (15 mL) was cooled to 0 °C under
nitrogen, and EDCI.HCl (0.865 g, 4.5 mmol) was added. The
mixture was stirred at 0 °C for 15 min and allowed to warm
to room temperature overnight. The crude product was ex-
tracted with EtOAc and eluted through a short column of silica
gel with EtOAc to give 5e (0.26 g, 45%) as a white solid: mp
Hydrogenation of 18 (9.27 g, 33 mmol) at 52.5 psi over Ra
Ni (4 g) in THF/MeOH (3:2, 250 mL) at 25 °C for 3.5 h as above
gave 6-amino-4-[(3-methylphenyl)amino]quinazoline (25) (8.14
mg, 97%) as a beige solid: mp 173-175.5 °C (lit.32 mp 205-
1
(MeOH) 276-278.5 °C; H NMR [(CD3)2SO] δ 10.51 (s, 1 H,
1
NH), 9.95 (s, 1 H, NH), 8.82 (d, J ) 1.8 Hz, 1 H, H-5), 8.57 (s,
1 H, H-2), 8.25 (td, J ) 6.4, 2.6 Hz, 1 H, H-2′), 7.90-7.85 (m,
2 H, H-7, 6′), 7.81 (d, J ) 8.9 Hz, 1 H, H-8), 7.41 (t, J ) 8.8
Hz, 1 H, H-5′), 6.53 (dd, J ) 17.0, 10.0 Hz, 1 H, CHdCH2),
6.35 (dd, J ) 17.0, 1.9 Hz, 1 H, CHdCH2), 5.85 (dd, J ) 10.1,
1.9 Hz, 1 H, CHdCH2). Anal. (C17H12BrFN4O) C, H, N.
206 °C); H NMR [(CD3)2SO] δ 9.24 (brs, 1 H, NH), 8.35 (s, 1
H, H-2), 8.18 (d, J ) 9.0 Hz, 1 H, H-5), 7.64 (m, 2 H, H-4′, 8),
7.22 (t, J ) 7.8 Hz, 1 H, H-5′), 6.89-6.83 (m, 2 H, H-6, H-6′),
6.69 (d, J ) 2.2 Hz, 1 H, H-2′), 6.02 (br s, 2 H, NH2), 2.32 (s,
3 H, CH3). Anal. (C15H14N4) C, H, N.
Isobutyl chloroformate (20.35 g, 0.15 mol) was added drop-
wise over 20 min to a stirred solution of acrylic acid (10.82 g,
0.15 mol) and Et3N (30.19 g, 0.30 mol) in THF (400 mL) under
N2 at 0 °C. The slurry was stirred at 0 °C for 30 min; then 25
(27.71 g, 107 mmol) in DMF (80 mL) was added dropwise over
45 min. After a further 4 h, an additional 0.05 mol of mixed
anhydride was added in one portion, and the mixture was
stirred for a further 15 min and then poured onto ice/water (1
L). The mixture was extracted with Et2O, and the aqueous
N-[4-[(3-Ch lor o-4-flu or op h en yl)a m in o]qu in a zolin -6-yl]-
a cr yla m id e (5f). Reaction of 4-chloro-6-nitroquinazoline31
with 3-chloro-4-fluoroaniline according to the standard cou-
pling procedure21 gave 4-[(3-chloro-4-fluorophenyl)amino]-6-
nitroquinazoline (20) as its HCl salt (55%): mp (i-PrOH)
274.5-277 °C; 1H NMR [(CD3)2SO] δ 9.86 (d, J ) 2.3 Hz, 1 H,
H-5), 9.0 (s, 1 H, H-2), 8.76 (dd, J ) 9.1, 1.6 Hz, 1 H, H-7) 8.15
(d, J ) 9.0 Hz, 1 H, H-8), 8.09 (d, J ) 7.6 Hz, H-2′), 7.82-7.75

1810 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Smaill et al.
(m, 1 H, H-6′), 7.58 (t, J ) 9.0 Hz, H-5′). Anal. (C14H8ClFN4O2‚
A solution of 29 (328 mg, 1.0 mmol), acrylic acid (148 mg,
2.04 mmol), and pyridine (165 mg, 2.1 mmol) in THF (10 mL)
was treated with EDCI.HCl (385 mg, 2.0 mmol) (in one
portion), and the mixture was stirred under nitrogen at 0 °C
for 4 h and at 25 °C for 2 h. The reaction was then cooled to
0 °C, and water (2 mL) was added dropwise. This solution was
poured onto rapidly stirred ice-water (40 mL), the pH was
raised to 7 with saturated Na2CO3 solution, and the very fine
precipitate was collected by Buchner filtration. The solid was
rinsed with water (2 × 10 mL), dried in a vacuum oven at 65
°C for 4 h, and then dissolved (heating and sonication) in
EtOAc (10 mL). The solution was filtered, and the filtrate was
eluted through a small silica gel plug in EtOAc to give 5g (162
mg, 41%) as a pale-yellow glass: mp 185-191 °C; 1H NMR
[(CD3)2SO] δ 10.50 (br s, 1 H, NH), 9.86 (br s, 1 H, NH), 8.80
(d, J ) 2.0 Hz, 1 H, H-5), 8.50 (s, 1 H, H-2), 7.89 (dd, J ) 2.2,
9.0 Hz, 1 H, H-7), 7.81 (d, J ) 8.9 Hz, 2 H, H-2′,6′), 7.77 (d, J
) 9.0 Hz, 1 H, H-8), 7.39 (dd, J ) 7.6, 8.6 Hz, 2 H, H-3′′,5′′),
7.12 (t, J ) 7.4 Hz, 1 H, H-4′′), 7.06 (d, J ) 8.8 Hz, 2 H, H-3′,5′),
7.02 (d, J ) 7.8 Hz, 2 H, H-2′′,6′′), 6.53 (dd, J ) 10.1, 17.0 Hz,
1 H, CHdCH2), 6.34 (dd, J ) 1.8, 17.0 Hz, 1 H, CHdCH2),
5.84 (dd, J ) 1.8, 10.1 Hz, 1 H, CHdCH2); MS (APCI) 383.1
(100 MH+). Anal. (C23H18N4O2‚0.25HCl‚0.06C4H6O2) C, H, N.
N-[4-[(4-Ben zyloxyph en yl)am in o]qu in azolin -6-yl]acr yl-
a m id e (5h ). A suspension of crude 4-chloro-6-nitroquinazoline
hydrochloride31 (5 mmol), 4-benzyloxyaniline (999 mg, 5 mmol),
and N,N-dimethylaniline (1.209 g, 10 mmol) in 2-propanol (10
mL) was refluxed under nitrogen with stirriing for 3 h. The
mixture was cooled, and the precipitate was collected by
Buchner filtration, rinsed with 2-propanol (2 × 10 mL), and
dried to give 4-[(4-benzyloxyphenyl)amino]-6-nitroquinazoline
hydrochloride25 (22) (1.675 g, 82%) as a yellow solid: mp 246-
248 °C (lit.25 mp 222-223 °C); 1H NMR [(CD3)2SO] δ 11.98 (br
s, 1 H, NH), 9.85 (d, J ) 2.2 Hz, 1 H, H-5), 8.94 (s, 1 H, H-2),
8.76 (dd, J ) 2.3, 9.1 Hz, 1 H, H-7), 8.15 (d, J ) 9.3 Hz, 1 H,
H-8), 7.65 (d, J ) 9.0 Hz, 2 H, H-2′,6′), 7.48 (d, J ) 7.0 Hz, 2
H, H-2′′,6′′),7.41 (t, J ) 7.4 Hz, 2 H, H-3′′,5′′), 7.31 (t, J ) 7.4
Hz, 1 H, H-4′′), 7.15 (d, J ) 9.1 Hz, 2 H, H-3), 5.16 (s, 2 H,
benzylic); MS (APCl) 373 (100 MH+). Anal. (C21Hl6N4O3‚HCl)
C, H, N.
HCl) C, H, N.
The free base of 20 (1.97 g, 62 mmol) in THF:MeOH (2:1,
600 mL) was hydrogenated at 50 psi over Ra Ni (10 g) for 3.5
h. The mixture was filtered, and the solvent was removed
rigorously under reduced pressure to give 6-amino-4-[(3-chloro-
4-fluorophenyl)amino]quinazoline33 (28) (17.9 g, 100%) as a
light-orange-brown solid. An analytical sample was obtained
by preparative TLC on silica gel, eluting twice with 5% MeOH/
1
CHCl3: mp 263.5-265.5 °C; H NMR [(CD3)2SO] δ 9.47 (s, 1
H, NH), 9.48, (s, 1 H, NH, 8.36 (s, 1 H, H-2), 8.21 (dd, J ) 6.7,
2.3 Hz, 1 H, H-2′), 7.83 (ddd, J ) 2.8, 4.2, 9.0 Hz, 1 H, H-6′),
7.54 (d, J ) 8.8 Hz, 1 H, H-8), 7.40 (t, J ) 9.1 Hz, 1 H, H-5′),
7.31 (d, J ) 2.2 Hz, 1 H, H-5), 7.25 (dd, J ) 9.0, 2.1 Hz, 1 H,
H-7), 5.64 (s, 2 H, NH2). Anal. (C14H10ClFN4) C, H, N.
EDCI‚HCl (19.17 g, 0.1 mol) was added to a solution of 28
(14.435 g, 0.05 mol), acrylic acid (7.2 g, 0.1 mol), and pyridine
(7.9 g, 0.1 mol) in THF (250 mL) and stirred under nitrogen
at 0 °C. After 6 h, water (50 mL) was added to the reaction
mixture with swirling, dissolving all of the gummy precipitate.
This solution was poured slowly onto stirred ice-water, and
the residual solid was collected slowly by Buchner filtration,
rinsed with water (100 mL), and air-dried overnight. The
greenish black solid was dissolved in the minimum of DMF
(40 mL), added to a mixture of silica gel (150 g) Me2CO (150
mL) for adsorption, and then subjected to flash chromatogra-
phy on silica gel (500 g), eluting with a gradient of Me2CO
(0% to 60%) in CH2Cl2, to give 5f (6.25 g, 35%) as a pale-yellow
1
solid: 260-262 °C; H NMR [(CD3)2SO] δ 10.52 (s, 1 H, NH),
9.97 (s, 1 H, NH), 8.83 (d, J ) 1.9 Hz, 1 H, H-5), 8.57 (s, 1 H,
H-2), 8.15 (dd, J ) 7.0, 2.6 Hz, 1 H, H-2′), 7.89 (dd, 1 H, H-7),
7.81 (d, J ) 8.9 Hz, 1 H, H-8), 7.82-7.78 (m, 1 H, H-6′), 7.45
(t, J ) 9.1 Hz, 1 H, H-5′), 6.53 (dd, J ) 16.9, 10.1 Hz, 1 H,
CHdCH2), 6.35 (dd, J ) 16.9, 1.9 Hz, 1 H, CHdCH2), 5.85
(dd, J ) 10.1, 1.9 Hz, 1 H, CHdCH2). Anal. (C17H12ClFN4O)
C, H, N.
N-[4-[(4-P h en oxyp h en yl)a m in o]qu in a zolin -6-yl]a cr yl-
a m id e (5g). A suspension of crude 4-chloro-6-nitroquinazoline
hydrochloride31 (5 mmol), 4-phenoxyaniline (926 mg, 5 mmol),
and N,N-dimethylaniline (1.215 g, 10 mmol) in 2-propanol (10
mL) was refluxed under nitrogen with stirring for 3 h. The
mixture was allowed to cool to 25 °C, and the precipitate was
collected by Buchner filtration, rinsed with 2-propanol (2 ×
10 mL), and dried at 60 °C in a vacuum oven to give 6-nitro-
4-[(4-phenoxyphenyl)amino]quinazoline hydrochloride25 (21)
(1.70 g, 86%) as an orange solid: mp 293-294 °C (lit.25 mp
A solution of 22 (1.46 g, 3.57 mmol) in MeOH/THF (1:1, 100
mL) was hydrogenated over Raney nickel (1 g) at 51.5 psi and
23 °C for 18 h. The reaction mixture was filtered, and the
volatiles were removed under reduced pressure. The residue
was partially redissolved in MeOH (25 mL), and dilute aqueous
Na2CO3 solution (0.1 M, 50 mL) was added with vigorous
stirring. After 2 h the precipitate was collected by Buchner
filtration, rinsed with water (50 mL), dried in a vacuum oven
at 60 °C, and then purified by flash chromatography on silica
gel, eluting with CHCl3 and then 4% MeOH in CHCl3 to give
6-amino-4-[(4-benzyloxyphenyl)amino]quinazoline25 (30) (1.018
g, 80%) as a cream powder: mp l73-175 °C, remelt 240-245
1
>200 °C); H NMR [(CD3)2SO] δ 12.00 (br s, 1 H, NH), 9.88
(d, J ) 2.4 Hz, 1 H, H-5), 8.97 (s, 1 H, H-2), 8.76 (dd, J ) 2.3,
9.1 Hz, 1 H, H-7), 8.16 (d, J ) 9.3 Hz, 1 H, H-8), 7.76 (d, J )
9.O Hz, 2 H, H-2′,6′), 7.44 (dd, J ) 7.6, 8.8 Hz, 2 H, H-3′,5′),
7.18 (t, J ) 7.3 Hz, 1 H, H-4′), 7.14 (d, J ) 9.0 Hz, 2 H,
H-2′′,6′′), 7.08 (d, J ) 7.6 Hz, 2 H, H-3′′,5′′); MS (APCI) 359
(100 MH+). Anal. (C20H14N4O3‚HCl) C, H, N.
1
°C; H NMR [(CD3)2SO] δ 9.25 (br s, 1 H, NH), 8.25 (s, 1 H,
H-2), 7.70 (d, J ) 9.0 Hz, 2 H, H-2′,6′), 7.52-7.47 (m, 3 H,
H-2′′,6′′,5), 7.41 (t, J ) 7.5 Hz, 2 H, H-3′′,5′′), 7.38-7.35 (m, 2
H, H-7,8), 7.02 (d, J ) 9.0 Hz, 2 H, H-3′,6′), 5.54 (br s, 2 H,
NH2), 5.11 (s, 2 H, benzylic); MS (APCI) 343 (100, MH+). Anal.
(C20H16N4O‚0.67H2O) C, H, N.
A solution of 21 (1.65 g, 4.2 mmol) in MeOH/THF (1:1, 100
mL) was hydrogenated over Raney nickel (1 g) at 50 psi and
27 °C for 18 h. The reaction mixture was filtered through
Celite, and the volatiles were removed under reduced pressure.
The residue was partially redissolved in MeOH (25 mL), and
dilute aqueous Na2CO3 solution (0.1 M, 50 mL) was added with
vigorous stirring. After 2 h, the precipitate was collected by
Buchner filtration, rinsed with water (50 mL), air-dried, and
purified by flash chromatography on silica gel, eluting with
2.5% and then 4% MeOH in CH2Cl2, to give 6-amino-4-[(4-
phenoxyphenyl)amino]quinazoline25 (29) (1.113 g, 80%) as a
pale-yellow glassy foam: mp 89-90 °C, remelt 195-200 °C
EDCI‚HCl (385 mg, 2.0 mmol) was added in one portion to
a solution of 30 (342 mg, 1. 0 mmol), acrylic acid (144 mg, 2.0
mmol), and pyridine (163 mg, 2.06 mmol) in THF (10 mL) and
stirred under nitrogen at 0 °C. After 4 h at 0 °C and a further
2 h at 25 °C, the mixture was recooled to 0 °C, and water (2
mL) was added dropwise. This solution was poured onto
rapidly stirred ice-water (40 mL); the pH was raised to 7 with
saturated Na2CO3 solution. The precipitate was collected by
Buchner filtration, rinsed with water (2 × 10 mL), and dried
in a vacuum oven at 65 °C for 4 h and then dissolved in CHCl3/
Me2CO (1:1, 40 mL). This was adsorbed onto silica gel (5 g)
and used as the origin of a silica gel flash chromatography
column, eluting with 25% Me2CO/CHC13, to give 5h (152 mg,
38%): mp 227-229 °C; 1H NMR [(CD3)2SO] δ 10.47 (br s, 1 H,
NH), 9.74 (br s, 1 H, NH), 8.76 (d, J ) l.9 Hz, 1 H, H-5), 8.45
(s, 1 H, H-2), 7.86 (dd, J ) 2.0, 9.0 Hz, 1 H, H-7), 7.74 (d, J )
1
(lit.25 mp 187-191 °C); H NMR [(CD3)2SO] δ 9.38 (brs, 1 H,
NH), 8.30 (s, 1 H, H-2), 7.87 (d, J ) 9.0 Hz, 2 H, H-2′,6′), 7.40
(d, J ) 8.8 Hz, 1 H, H-8), 7.38 (dd, J ) 7.3, 8.6 Hz, 2 H,
H-3′′,5′′), 7.34 (d, J ) 2.4 Hz, 1 H, H-5), 7.23 (dd, J ) 2.4, 8.9
Hz, 1 H, H-7), 7.11 (t, J ) 7.4 Hz, 1 H, H-4′′), 7.05 (d, J ) 8.8
Hz, 2 H, H-3′,5′), 7.00 (d, J ) 7.8 Hz, 2 H, H-2′′,6′′), 5.58 (br s,
2 H, NH2); MS (APCI) 329 (100 MH+). Anal. (C20H16N4O‚
0.25H2O) C, H, N.

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1811
8.9 Hz, 1 H, H-8), 7.66 (d, J ) 9.2 Hz, 2 H, H-2′,6′), 7.49 (d, J
) 7.0 Hz, 2 H, H-2′′,5′′), 7.41 (t, J ) 7.4 Hz, 2 H, H-3′′,5′′),
7.34 (t, J ) 7.2 Hz, l H, H-4′′), 7.04 (d, J ) 9.2 Hz, 2 H, H-3′,5′),
6.53 (dd, J ) 10.1, 16.9 Hz, 1 H, CHdCH2), 6.34 (dd, J ) 1.9,
16.9 Hz, 1 H, CHdCH2), 5.83 (dd, J ) 1.9, 10.1 Hz, 1 H, CHd
CH2), 5.13 (s, 2 H, benzylic); MS (APCI) 397.2 (100 MH+). Anal.
(C24H20N4O2‚0.1HCl) C, H, N.
(dd, J ) 6.4, 2.6 Hz, 1 H, H-2′), 7.89-7.85 (m, 1 H, H-6′), 7.42
(t, J ) 8.8 Hz, 1 H, H-5′), 7.33 (d, J ) 8.5 Hz, 2 H, H-2′′,6′′),
7.31 (br s, 1 H, NH), 7.19 (s, 1 H, H-5), 6.89 (br d, J ) 8.7 Hz,
2 H, H-3′′,5′′), 4.49 (d, J ) 6.2 Hz, 2 H, CH2), 3.71 (s, 3 H,
OMe).
Without further purification, 42 was dissolved in TFA (50
mL), and the solution was refluxed for 30 min. The TFA was
removed under reduced pressure; the residue was made basic
with aqueous NH3 and extracted into EtOAc. After drying and
removal of solvent, the residue was chromatographed on silica
gel, eluting with EtOAc/MeOH (98:2), to give 6-amino-4-[(3-
bromo-4-fluorophenyl)amino]pyrido[3,4-d]pyrimidine (33) (1.61
g, 91%): mp (EtOAc) 268-270 °C; 1H NMR [(CD3)2SO] δ 9.78
(s, 1 H, NH), 8.71 (s, 1 H, H-8), 8.38 (d, J ) 1.4 Hz, 1 H, H-2),
8.32 (dd, J ) 6.5, 2.6 Hz, 1 H, H-2′), 7.92-7.87 (m, 1 H, H-6′),
7.41 (t, J ) 8.8 Hz, 1 H, H-5′), 7.13 (s, 1 H, H-5), 6.31 (br s, 2
H, NH2). Anal. (C13H9BrFN5) C, H, N.
N-[4-[(3-Br om op h en yl)a m in o]p yr id o[3,4-d ]p yr im id in -
6-yl]a cr yla m id e (6a ). A solution of 6-amino-4-[(3-bromophe-
nyl)amino]pyrido[3,4-d]pyrimidine11 (31) (520 mg, 1.64 mmol),
redistilled acrylic acid (0.4 mL, 3.5 equiv), and pyridine (1.64
mL, 12.4 equiv) in THF/DMA (2:1, 6.6 mL) purged with
nitrogen was cooled to 0-5 °C and treated with EDCI.HCl
(1.61 g, 5 equiv) in one portion. The resulting suspension was
kept at 0-5 °C for 30 min and then brought to 25 °C. After
2.5 h, the solution was ice-cooled and treated slowly with an
excess of water (ca. 10-12 mL). After the thick suspension
stirred for about 30 min, the solids were collected, washed well
with water, and air-dried. The solids were suspended in ca.
15 mL of i-PrOH, and the stirred suspension was heated at
70-80 °C for 30 min. After cooling, the solids were collected,
washed with i-PrOH, and dried to give 6a (360 mg, 59%): mp
243-245 °C; 1H NMR [(CD3)2SO] δ 11.07 (s, 1 H, CONH), 10.33
(s, 1 H, NH), 9.05 (s, 1 H, aromatic), 9.03 (s, 1 H, aromatic),
8.66 (s, 1 H, aromatic), 8.18 (br s, 1 H, H-2′), 7.89 (br d, J )
7.6 Hz, 1 H, H-6′). 7.40-7.33 (m, 2 H, H-4′,5′), 6.70 (dd, J )
17.0, 10.2 Hz, 1 H, CHdCH2), 6.41 (dd, J ) 16.9, 1.2 Hz, 1 H,
CHdCH2), 5.87 (dd, J ) 10.1, 1,2 Hz, 1 H, CHdCH2); 13C NMR
δ 163.35, 156.82, 154.13, 150.87, 147.92, 141.64, 140.40,
131.25, 130.26, 127.86, 126.49, 124.76, 121.30, 121.02, 120.97,
103.43; CIMS m/z (relative %) 369 (29), 370 (100), 371 (46),
372 (95), 373 (17). Anal. (C16H12N5OBr) Calcd: C, 51.9; H, 3.3;
N, 18.9. Found: C, 51.8; H, 3.1; N, 18.6.
A stirred solution of 33 (1.705 g, 5.1 mmol) in dry pyridine
(26 mL) at 0-5 °C was treated with redistilled acrylic acid
(1.4 mL, 20.4 mmol), followed by EDCI.HCl (4.9 g, 25.5 mmol).
The mixture was stirred for 3 h; then the viscous suspension
was poured into 200 mL of ice-cold 2% aqueous NaHCO3 (using
some DMF to assist in the transfer). The precipitate was
collected, washed well with water, then suspended in water,
and sonicated for 15 min. The solids were collected, washed
with H2O, and air-dried. Upon standing overnight, the com-
bined aqueous filtrates precipitated additional solids, which
were collected as above. The two crops were combined and
suspended in 70 mL of hot EtOAc. After heating at reflux for
2 h, the suspension was cooled and the solids were collected
to give 6e (1.65 g, 80%) as a pale-yellow solid: mp (MeOH)
1
245-248 °C; H NMR [(CD3)2SO] δ 11.07 (s, 1 H, NH), 10.35
(s, 1 H, NH), 9.04 and 9.01 (2s, 2 H, H-5 and H-8), 8.64 (s, 1
H, H-2), 8.25 (dd, J ) 6.4, 2.6 Hz, 1 H, H-2′), 7.91-7.87 (m, 1
H, H-6′), 7.43 (t, J ) 8.8 Hz, 1 H, H-5′), 6.70 (dd, J ) 17.0,
10.1 Hz, 1 H, CHdCH2), 6.39 (dd, J ) 17.0, 1.9 Hz, 1 H, CHd
The filtrate was evaporated to dryness and dissolved in
EtOAc/i-PrOH (9:1), and the solution was filtered through a
pad of flash silica gel, eluting with EtOAc. Further processing
of the filtrate as above provided a second crop of 6a (57 mg,
9%): mp 240-243 °C.
CH2), 5.86 (dd, J ) 10.1, 1.9 Hz, 1 H, CHdCH2). Anal. (C16H11
BrFN5O) C, H, N.
-
N-[4-[(3-Meth ylp h en yl)a m in o]p yr id o[3,4-d ]p yr im id in -
6-yl]a cr yla m id e (6c). To a stirred solution of 6-amino-4-[(3-
methylphenyl)amino]pyrido[3,4-d]pyrimidine20 (32) (140 mg,
0.56 mmol), DMAP (14 mg), and Et3N (excess, 0.5 mL) in THF/
DMF (7:1, 20 mL) at 0 °C under N2 was added acryloyl chloride
(2.7 mol equiv, 123 µL) dropwise over 3 h. The reaction was
then stirred at 20 °C for 1 h, diluted with water, and extracted
with EtOAc. The combined organic extracts were washed with
brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure before being chromatographed on silica gel
eluting with CH2Cl2/EtOAc (1:1) to MeOH/CH2Cl2/EtOAc (2:
48:50) to give 6c (41 mg, 24%): mp (EtOAc/hexane) 221-223
°C dec; 1H NMR [(CD3)2SO] δ 11.03 (s, 1 H, CONH), 10.18 (s,
1 H, NH), 9.02 (s, 1 H, aromatic), 9.01 (s, 1 H, aromatic), 8.59
(s, 1 H, aromatic), 7.63 (m, 2 H, H-2′,6′), 7.29 (m, 1 H, H-5′),
6.89 (br d, J ) 7.5 Hz, 1 H, H-4′), 6.69 (dd, J ) 17.0, 10.2 Hz,
1 H, CHdCH2), 6.37 (dd, J ) 17.0, 1.9 Hz, 1 H, CHdCH2),
5.85 (dd, J ) 10.2, 1.9 Hz, 1 H, CHdCH2), 2.35 (s, 3 H, CH3-
Ar). Anal. (C17H15N5O) C, H, N.
N-[4-[(3-Ch lor o-4-flu or op h en yl)a m in o]p yr id o[3,4-d ]p y-
r im id in -6-yl]a cr yla m id e (6f). A mechanically stirred solu-
tion of 4-chloro-6-fluoropyrido[3,4-d]pyrimidine20 (7.62 g, 41.5
mmol) and 3-chloro-4-fluoroaniline (7.3 g, 49.9 mmol) in
i-PrOH (120 mL) was heated at reflux for 3 h. The resulting
suspension was concentrated to ca. 50 mL, and the thick yellow
precepitate was collected and washed with a little i-PrOH. The
filter cake was further washed with 2% aqueous NaOH to pH
12 and water to neutral pH and then dried at 70 °C/2 mmHg
over P2O5 for 4 days to give 4-[(3-chloro-4-fluorophenyl)amino]-
6-fluoropyrido[3,4-d]pyrimidine34 (41) (9.65 g, 79%): mp 267-
269 °C (lit.34 mp 264-266 °C); 1H NMR [(CD3)2SO] δ 10.10 (s,
1 H, NH), 8.91 (s, 1 H, H-8), 8.68 (s, 1 H, H-2), 8.18 (br s, 2 H,
H-5, H-2′), 7.79-7.75 (m, 1 H, H-6′), 7.44 (t, J ) 9.1 Hz, 1 H,
H-5′). Anal. (C13H7N4F2Cl) C, H, N, F, Cl.
A solution of 41 (9.6 g, 32.8 mmol) and 4-methoxybenzyl-
amine (17.1 mL, 131 mmol) in DMSO (66 mL) was heated at
120 °C for 16 h. Most of the DMSO and excess amine were
removed under reduced pressure at 80 °C, and the residual
yellow solid was dissolved in minimum hot EtOAc. Upon
storage in the cold overnight, the solids that had precipitated
were collected, washed with cold EtOAc, and dried to give 4-[(3-
chloro-4-fluorophenyl)amino]-6-[(4-methoxyphenyl)methylami-
no]pyrido[3,4-d]pyrimidine (43) (6.77 g, 50%): mp (EtOAc)
196-197 °C; 1H NMR [(CD3)2SO] δ 9.74 (s, 1 H, NH), 8.76 (s,
1 H, H-8), 8.38 (s, 1 H, H-2), 8.18 (dd, J ) 6.9, 2.5 Hz, 1 H,
H-2′), 7.84-7.79 (m, 1 H, H-6′), 7.46 (t, J ) 8.9 Hz, 1 H, H-5′),
7.36-7.32 (d, J ) 8.7 Hz + overlapping br s, 3 H, H-2′′,6′′ and
NH), 7.17 (s, 1 H, H-5), 6.88 (br d, J ) 8.7 Hz, 2 H, H-3′′,5′′),
4.49 (d, J ) 6.3 Hz, 2 H, CH2), 3.71 (s, 3 H, OCH3). Anal.
(C21H17N5OClF) C, H, N, Cl, F.
N-[4-[(3-Br om o-4-flu or op h en yl)a m in o]p yr id o[3,4-d ]p y-
r im id in -6-yl]a cr yla m id e (6e). Reaction of 4-chloro-6-fluo-
ropyrido[3,4-d]pyrimidine20 with 3-bromo-4-fluoroaniline, by
the standard procedure,20 gave 4-[(3-bromo-4-fluorophenyl)-
amino]-6-fluoropyrido[3,4-d]pyrimidine (40) (59%): mp (i-
1
PrOH) 269-270 °C; H NMR [(CD3)2SO] δ 10.13 s, 1 H, NH),
8.97 (s, 1 H, H-8), 8.74 (s, 1 H, H-2), 8.33 (td, J ) 6.0, 2.6 Hz,
H-2′), 8.23 (br s, 1 H, H-5), 7.93-7.87 (m, 1 H, H-6′), 7.46 (t,
J ) 8.8 Hz, 1 H, H-5′). Anal. (C13H7BrF2N4) C, H, N.
A mixture of 40 (2.03 g, 6 mmol) and 4-methoxybenzylamine
(16.5 g, 0.12 mol) in DMSO (50 mL) was heated at 100 °C for
16 h. The mixture was diluted with water and extracted with
EtOAc to give a crude solid which was chromatographed on
silica gel, eluting with EtOAc/CH2Cl2 (1:1), to give 4-[(3-bromo-
4-fluorophenyl)amino]-6-[(4-methoxyphenyl)methylamino]py-
rido[3,4-d]pyrimidine (42) (2.4 g, 88%): 1H NMR [(CD3)2SO]
δ 9.72 (s, 1 H, NH), 8.75 (s, 1H, H-8), 8.38 (s, 1 H, H-2), 8.26
Processing of the EtOAc filtrate by concentration, dissolu-
tion of the residue in a minimum amount of solvent, and
storage in the cold yielded 2.41 g (∼20%) of additional product
in two crops. TLC showed this to be predominately 35 along
with a smaller amount (10-20%) of a lower Rf spot that

1812 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Smaill et al.
corresponded to 6-amino-4-[(3-chloro-4-fluorophenyl)amino]-
pyrido[3,4-d]pyrimidine (34). This material was suitable for
direct use in the next reaction.
dryness directly onto silica and chromatographed. Elution with
EtOAc/petroleum ether (1:1) gave foreruns, while EtOAc gave
6-amino-4-[(3-bromo-4-fluorophenyl)amino]pyrido[3,2-d]pyri-
1
midine (36) (0.30 g, 61%): mp (EtOH) 316-317 °C; H NMR
A solution of 43 (9.0 g, 22 mmol) in trifluoroacetic acid (58
mL) was heated at 50 °C for 1 h, and the mixture was
evaporated to dryness. The residue was coevaporated three
times with MeOH and then dissolved in a minimum volume
of DMF. The cooled solution was diluted with 175 mL of ethyl
acetate and then washed with 125 mL of 5 M aqueous NH4-
OH solution. Upon standing at 25 °C, the organic phase
deposited a precipitate that was collected, washed with EtOAc,
and dried to give 6-amino-4-[(3-chloro-4-fluorophenyl)amino]-
pyrido[3,4-d]pyrimidine (34) (2.7 g, 42%): mp (EtOAc) 263-
[(CD3)2SO] δ 9.40 (s, 1H, NH), 8.51 (dd, J ) 6.45, 2.3 Hz, 1 H,
H-2′), 8.44 (s, 1 H, H-2), 7.94-7.90 (m, 1 H, H-6′), 7.84 (d, J )
9.0 Hz, 1 H, H-8), 7.39 (dd, J ) 8.8, 8.8 Hz, H-5′), 7.13 (d, J )
9.0 Hz, 1 H, H-7), 6.75 (br s, 2 H, NH2). Anal. (C13H9BrFN5)
C, H, N.
Acrylic acid (0.32 mL, 6.31 mmol) and EDCI (1.72 g, 8.97
mmol) were added to a stirred solution of 36 (0.60 g, 1.79
mmol) and pyridine (1.74 mL, 0.021 mol) in DMF (30 mL).
Stirring was continued at room temperature for 7 days, with
further quantities of acrylic acid (0.12 mL), pyridine (0.70 mL),
and EDCI (0.66 g) being added every 48 h. The solution was
concentrated to a volume of 10 mL, and water was added to
precipitate the product. The crude material was adsorbed onto
silica from a methanol solution and chromatographed. EtOAc
eluted 7e, which was slurried in ether to give pure material
1
266 °C; H NMR [(CD3)2SO] δ 9.81 (s, 1 H, NH), 8.71 (s, 1 H,
H-8), 8.38 (s, 1 H, H-2), 8.23 (dd, J ) 6.8, 2.2 Hz, 1 H, H-2′),
7.86-7.82 (m, 1 H, H-6′), 7.45 (t, J ) 9.2 Hz, 1 H, H-5′), 7.13
(s, 1 H, H-5), 6.33 (br s, 2 H, NH2); 19F NMR δ -123.0 (F-4′).
Anal. (C13H9N5FCl‚0.25H2O) C, H, N, Cl, F.
Processing of the filtrate by concentration followed by
dissolution in a minimum volume of EtOAc and crystallization
afforded 2.6 g (40%) of slightly less pure 26 in two crops.
1
(0.25 g, 39%): mp 241-243 °C; H NMR [(CD3)2SO] δ 11.10
(s, 1H, CONH), 9.63 (s, 1 H, NH), 8.68 (d, J ) 9.2 Hz, 1 H,
H-8), 8.49 (dd, J ) 6.3, 2.6 Hz, 1 H, H-2′), 8.28 (d, J ) 9.2 Hz,
1 H, H-7), 7.94-7.90 (m, 1 H, H-6′), 7.45 (dd, J ) 8.8, 8.8 Hz,
1 H, H-5′), 6.68 (dd, J ) 17.1, 10.1 Hz, 1 H, CHdCH2), 6.42
(dd, J ) 17.1, 1.7 Hz, 1 H, CHdCH2), 5.91 (dd, J ) 10.1, 1.7
Hz, 1 H, CHdCH2). Anal. (C16H11BrFN5O) C, H, N.
A stirred solution of 34 (4.64 g, 16 mmol) in dry pyridine
(82 mL) at 0-5 °C was treated with redistilled acrylic acid
(4.5 mL, 64 mmol), followed by EDCI.HCl (15.3 g, 80 mmol).
The mixture was stirred for 45 min; then the viscous suspen-
sion was poured into 500 mL of ice-cold 2% aqueous NaHCO3.
The precipitate was collected, washed well with water, then
suspended in water, and sonicated for 15 min. The solids were
collected, washed with water, and air-dried. Upon standing
overnight, the combined aqueous filtrates precipitated ad-
ditional solids which were collected as above. The two crops
were combined and suspended in hot EtOAc (200 mL). After
heating at reflux for 1.5 h, the suspension was cooled and the
solids were collected to give 6f (4.31 g, 75%) as a pale-yellow
powder: mp >245 °C dec; 1H NMR [(CD3)2SO] δ 11.03 (s, 1 H,
NH), 10.31 (s, 1 H, NH), 8.98 & 8.95 (2xs, 2 H, H-5, H-8), 8.59
(s, 1 H, H-2), 8.09 (dd, J ) 6.0, 2.1 Hz, 1 H, H-2′), 7.80-7.74
(m, 1 H, H-6′), 7.41 (t, J ) 9.0 Hz, 1 H, H-5′), 6.64 (dd, J )
16.9, 10.2 Hz, 1 H, CHdCH2), 6.33 (dd, J ) 16.9, 1.0 Hz, 1 H,
CHdCH2), 5.80 (dd, J ) 10.2, 1.0 Hz, 1 H, CHdCH2). Anal.
(C16H11N5OClF‚H2O) C, H, N.
N-[4-[(3-Br om op h en yl)a m in o]p yr id o[3,2-d ]p yr im id in -
6-yl]a cr yla m id e (7a ). To a stirred solution of 6-amino-4-[(3-
bromophenyl)amino]pyrido[3,2-d]pyrimidine11 (35) (46 mg,
0.15 mmol) and acrylic acid (6 mol equiv, 0.91 mmol, 62 µL)
in DMA (5.0 mL) under N2 was added EDCI.HCl (4.0 mol
equiv, 0.61 mmol, 116 mg). The reaction mixture was stirred
for 48 h with additional amounts of acrylic acid and EDCI.HCl
(62 µL/116 mg) being added every 12 h. It was then poured
into water and extracted with EtOAc, and the product was
chromatographed on silica gel, eluting with EtOAc:CH2Cl2 (1:
1) to MeOH/CH2Cl2/EtOAc (2:48:50), to give 7a (14 mg, 26%):
mp (CH2Cl2/hexane) 226-228 °C; 1H NMR [(CD3)2SO] δ 11.13
(s, 1 H, CONH), 9.57 (s, 1 H, NH), 8.72 (s, 1 H, aromatic),
8.69 (d, J ) 9.1 Hz, 1 H, H-5 or H-6), 8.43 (t, J ) 1.9 Hz, 1 H,
H-2′), 8.30 (d, J ) 9.1 Hz, 1 H, H-5 or H-6), 7.87 (br d, J ) 6.9
Hz, 1 H, H-6′), 7.39 (t, J ) 8.1 Hz, 1 H, H-5′), 7.33 (dt, J ) 8.2,
1.3, 1.3 Hz, 1 H, H-4′), 6.68 (dd, J ) 17.0, 10.2, Hz, 1 H, CHd
CH2), 6.43 (dd, J ) 17.0, 1.8 Hz, 1 H, CHdCH2), 5.91 (dd, J )
10.2, 1.8 Hz, 1 H, CHdCH2). Anal. (C16H12BrN5O) C, H, N.
N-[4-[(3-Ch lor o-4-flu or op h en yl)a m in o]p yr id o[3,2-d ]p y-
r im id in -6-yl]a cr yla m id e (7f). Reaction of 4-chloro-6-fluoro-
pyrido[3,2-d]pyrimidine11 with 3-chloro-4-fluoroaniline as above
gave crude [4-[(3-chloro-4-fluorophenyl)amino]-6-fluoropyrido-
[3,2-d]pyrimidine (45) (86%) which was used directly. A
solution of 45 (0.45 g, 1.70 mmol) in propan-2-ol (30 mL) was
saturated with gaseous ammonia and heated in a pressure
vessel at 100 °C for 18 h. On cooling crude product precipitated
out. This was redissolved in MeOH, adsorbed onto silica gel,
and chromatographed. EtOAc eluted foreruns, while EtOAc/
MeOH (9:1) gave 6-amino-4-[(3-chloro-4-fluorophenyl)amino]-
pyrido[3,2-d]pyrimidine (37) (0.29 g, 59%): mp (EtOAc) 300-
1
303 °C; H NMR [(CD3)2SO] δ 9.41 (s, 1 H, NH), 8.44 (s, 1 H,
H-2), 8.41 (d, J ) 6.1 Hz, 1 H, H-2′), 7.88 (m, 1 H, H-6′), 7.83
(d, J ) 8.9 Hz, 1 H, H-8), 7.42 (dd, J ) 9.0, 9.0 Hz, 1 H, H-5′),
7.13 (d, J ) 8.9 Hz, 1 H, H-7), 6.75 (br s, 2 H, NH2). Anal.
(C13H9ClFN5‚0.5H2O) C, H, N.
Reaction of 37 with acrylic acid and EDCI over 7 days as
1
described above gave 7f (57%): mp (EtOAc) 247 °C; H NMR
[(CD3)2SO] δ 11.09 (s, 1 H, NHCO), 9.61 (s, 1 H, NH), 8.68 (d,
J ) 9.2 Hz, 1 H, H-8), 8.67 (s, 1 H, H-2), 8.40-8.37 (m, 1 H,
H-2′), 8.27 (d, J ) 9.2 Hz, 1H, H-7), 7.88-7.84 (m, 1 H, H-6′),
7.48 (dd, J ) 9 0.0, 9.0 Hz, 1H, H-5′), 6.69 (dd, J ) 17.0, 10.1
Hz, 1 H, CHdCH2), 6.42 (dd, J ) 17.0, 1.9 Hz, 1 H, CHdCH2),
5.90 (dd, J ) 10.1, 1.9 Hz, 1 H, CHdCH2). Anal. (C16H11FN5O)
C, H, N.
N -[4-[1-(6-Br om oin d olin yl)]q u in a zolin -6-yl]a cr yla -
m id e (8). A suspension of 6-bromoindoline35 (490 mg, 2.47
mmol), 4-chloro-6-nitroquinazoline31 (660 mg, 2.47 mmol), and
N,N-dimethylaniline (0.63 mL, 4.9 mmol) in 2-propanol (15
mL) was heated under reflux for 1 h. The suspension was
concentrated to give a solid, which was shaken with a mixture
of EtOAc and saturated aqueous NaHCO3, collected by filtra-
tion, and recrystallized from a boiling mixture of EtOH (100
mL) and DMF (25 mL) to give 4-[1-(6-bromoindolinyl)]-6-
nitroquinazoline (38) (290 mg, 32%): mp 249-251 °C; 1H NMR
(CF3CO2D) δ 9.45 (s, 1 H, H-5), 8.90 (dd, J ) 9.2 Hz, 1.9 Hz,
1H, H-7), 8.81 (s, 1 H, H-2), 8.79 (s, 1 H, H-7′), 8.12 (d, J ) 9.4
Hz, 1 H, H-8), 7.59 (dd, J ) 8.1 Hz, 1.3 Hz; 1 H, H-5′), 7.38 (d,
J ) 8.0 Hz, 1 H, H-4′), 5.02 (t, J ) 7.1 Hz, 2 H, H-2′), 3.46 (t,
J ) 7.0 Hz, 2 H, H-3′); MS (APCI) 373 (100, 81BrMH+), 371
(96, 79BrMH+). Anal. (C16H11Br1N4O2) C, H, N.
N-[4-[(3-Br om o-4-flu or op h en yl)a m in o]p yr id o[3,2-d ]p y-
r im id in -6-yl]a cr yla m id e (7e). 4-Chloro-6-fluoropyrido[3,2-
d]pyrimidine11 (1.10 g, 82%) was dissolved in EtOH (140 mL)
containing 3-bromo-4-fluoroaniline (2.54 g, 0.013 mol) and
concentrated HCl (2 drops), and the solution was refluxed for
15 min. After concentration under reduced pressure to a
volume of ca. 40 mL, saturated NaHCO3 solution was added
to precipitate the product. This was chromatographed on silica
gel, eluting with EtOAc/petroleum ether, to give crude [4-[(3-
bromo-4-fluorophenyl)amino]-6-fluoropyrido[3,2-d]pyrimi-
dine (44) as a yellow powder (1.35 g, 58%), which was used
directly. A solution of 44 (0.47 g, 1.48 mmol) in EtOH (50 mL)
was saturated with ammonia gas and heated in a pressure
vessel at 110 °C for 18 h. The solution was concentrated to
Iron powder (washed with 1 N HCl and then water and
dried; 180 mg, 3.23 mmol), was added to a refluxing suspension
of 38 (240 mg, 6.5 mmol) and AcOH (1.04 mL, 18 mmol) in
water (75 mL) and EtOH (100 mL). After 30 min more iron
powder (320 mg, 5.73 mmol) was added, and heating was
continued for another 30 min. The reaction was filtered while

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10 1813
hot, and the solids were washed with EtOH. The combined
filtrates were treated with concentrated NH4OH (10 mL), and
solids were removed by filtration. The filtrate was concen-
trated, and the resulting residue was purified by flash silica
gel chromatography in CH2Cl2/MeOH (10:1) to give a solid that
was freed from acetate species by dissolving it in Et2O and
washed consecutively with water, saturated aqueous NaHCO3,
and saturated brine. Evaporation gave 6-amino-4-[1-(6-bro-
moindolinyl)]quinazoline (39) (220 mg, 100%), which was used
directly: 1H NMR [(CD3)2SO] δ 8.52 (s, 1 H, H-2), 7.66 (d, J )
8.9 Hz, 1 H, H), 7.31 (dd, J ) 8.9 Hz, 2.4 Hz, 1 H, H-5′), 7.24
(d, J ) 8.0 Hz, 1 H, H-4′), 7.18 (d, J ) 1.7 Hz, 1 H, H-7′), 7.06
(dd, J ) 7.8 Hz, 1.8 Hz, 1 H, H-7) 6.96 (d, J ) 2.4 Hz, 1 H,
H-5), 5.82 (s, 2 H, exchangeable, NH2), 4.32 (t, J ) 8.2 Hz, 2
H), 3.13 (t, J ) 8.1 Hz, 2 H, H-3′); MS (APCI) 343.1 (98,
81BrMH+), 341.1 (100, 79BrMH+).
nated by addition of 0.1 mL of 20% trichloroacetic acid (TCA).
The plate was kept at 4 °C for at least 15 min to allow the
substrate to precipitate. The wells were then washed five times
with 0.2 mL of 10% TCA, and 32P incorporation was deter-
mined with a Wallac beta plate counter (Wallac, Inc., Gaith-
ersburg, PA).
Ir r ever sibility Test P r otocol. A431 human epidermoid
carcinoma cells were grown in 6-well plates to about 80%
confluency and then incubated in serum-free media for 18 h.
Duplicate sets of cells were treated with 2 µM of designated
compound to be tested as an irreversible inhibitor for 1 h. One
set of cells was then stimulated with 100 ng/mL EGF for 5
min, and extracts were made as described under the Western
blotting procedure below. The other set of cells was washed
free of the compound with warmed serum-free media, incu-
bated for 2 h, washed again, incubated another 2 h, washed
again, and then incubated a further 4 h. This set of cells was
then stimulated with EGF, and extracts were made similar
to the first set of cells.
Au top h osp h or yla tion Assa y. Inhibition of receptor au-
tophosphorylation in viable cells was determined by antiphos-
photyrosine Western blots. Cells were grown to 90% confluency
in 6-well plates, made serum-free for 18 h, and then treated
with various concentrations of compound for 1 h. The cells were
then stimulated for 5 min with EGF (100 ng/mL) or heregulin
(10 ng/mL), and extracts were made by lysing the monolayers
in 0.2 mL of boiling Laemlli buffer (2% sodium dodecyl sulfate,
5% â-mercaptoethanol, 10% glycerol, and 50 mM tris(hy-
droxymethyl)aminomethane (Tris), pH 6.8), and the lysates
were heated to 100 °C for 5 min. Proteins in the lysate were
separated by polyacrylamide gel electrophoresis and electro-
phoretically transferred to nitrocellulose. The membrane was
washed once in 10 mM Tris, pH 7.2, 150 mM NaCl, 0.01%
azide (TNA) and blocked overnight in TNA containing 5%
bovine serum albumin and 1% ovalbumin. The membrane was
blotted for 2 h with antiphosphotyrosine antibody (UBI, 1 mg/
mL in blocking buffer) and then washed twice in TNA, once
in TNA containing 0.05% Tween-20 detergent and 0.05%
nonidet P-40 detergent, and twice in TNA. The membranes
were then incubated for 2 h in blocking buffer containing 0.1
mCi/mL [125I]protein A and then washed again as above. After
the blots were dry, they were loaded into a film cassette and
exposed to X-AR X-ray film (Eastman Kodak Co., Rochester,
NY) for 1-7 days. Band intensities were determined with a
Molecular Dynamics laser densitometer.
In Vivo Ch em oth er a p y. Immune-deficient mice were
housed in microisolator cages within a barrier facility on a 12-h
light/dark cycle and received food and water ad libitum.
Animal housing was in accord with AAALAC guidelines. All
experimental protocols involving animals were approved by
the institutional animal care and use committee. The A431
epidermoid carcinoma and the H125 NSCLC tumor lines were
maintained by serial passage in nude mice (NCr nu/ nu). Nude
mice were also used as tumor hosts for anticancer agent
evaluations against these tumor models.
In each experiment for anticancer activity evaluation, test
mice weighing 18-22 g were randomized and implanted with
tumor fragments in the region of the right axilla on day 0.
Animals were treated with test compounds on the basis of
average cage weight (6 mice/dose group) initiated when tumors
reached approximately 100-150 mg in mass and continued
for the period indicated in Table 2. Whenever possible each
test compound was evaluated over a range of dose levels
ranging from toxic to ineffective. The doses reported in Table
2 are the maximum doses that could be administered without
exceeding the LD10, unless otherwise indicated. This maximum
tolerated dose (MTD) allows comparisons to be made among
the tested compounds at an equitoxic dose level. Doses above
the 200-250 mg/kg level were not tested even if nontoxic, as
compounds with even lower levels of potency would probably
not be suitable for clinical development. The vehicle for
compounds administered intraperitoneally was 6% dimethyl-
acetamide and 94% 50 mM sodium lactate buffer, pH 4.0 (fine
suspension). In later studies compounds were suspended in
Acrylic acid (0.16 mL, 2.3 mmol) followed by N-ethyldiiso-
propylamine (0.40 mL, 2.3 mmol) were added to a stirred
suspension of 39 (200 mg, 0.6 mmol) and EDCI‚HCl (450 mg,
2.3 mmol) in DMF (1 mL) and THF (3 mL), cooled in an ice
bath under nitrogen. The ice bath was removed, and the
suspension was stirred at room temperature for 18 h. The
resulting solution was poured into cold water, and the suspen-
sion was extracted with CH2Cl2 (4 × 25 mL). The extracts were
dried (MgSO4), concentrated under reduced pressure, and
purified by flash silica gel chromatography, eluting with CH2-
Cl2/MeOH (9:1) to give 8 as a light-yellow solid (110 mg,
1
41%): mp 214-216 °C; H NMR [(CD3)2SO] δ 10.59 (s, 1 H,
NH), 8.80 (d, J ) 2.2 Hz, 1 H, H-5), 8.70 (s, 1 H, H-2), 8.03
(dd, J ) 9.0 Hz, 2.3 Hz, 1 H, H-7), 7.89 (d, J ) 9.2 Hz, 1 H,
H-8), 7.73 (d, J ) 1.7 Hz, 1 H, H-7′), 7.28 (d, J ) 8.0 Hz, 1 H,
H-4′), 7.16 (dd, J ) 7.8 Hz, 1.8 Hz, 1 H, H-5′), 6.48 (dd, J )
17.0 Hz, 10.0 Hz, 1 H, CHdCH2), 6.32 (dd, J ) 16.9 Hz, 1.9
Hz, 1 H, CHdCH2), 5.83 (dd, J ) 10.1 Hz, 1.9 Hz, 1 H, CHd
CH2), 4.51 (t, J ) 8.2 Hz, 2 H, H-2′), 3.19 (t, J ) 8.1 Hz, 2 H,
H-3′); MS (APCI) 397.1 (96, 81BrMH+), 395.1 (100, 79BrMH+).
Anal. (C19H15Br1N4O1‚0.3DMF‚0.3H2O) C, H, N.
Cell Cu ltu r e. A431 human epidermoid carcinoma and
MDA-MB-453 human breast carcinoma cells were obtained
from the American Type Culture Collection, Rockville, MD,
and maintained as monolayers in RMEM (Dulbecco’s modified
Eagle medium)/F12, 50:50 (Gibco/BRL), containing 10% fetal
bovine serum.
P u r ifica tion of Ep id er m a l Gr ow th F a ctor Recep tor
Tyr osin e Kin a se. Human EGFR tyrosine kinase was isolated
from A431 human epidermoid carcinoma cells by the following
method. Cells were grown in roller bottles in RMEM/F12 media
(Gibco/BRL) containing 10% fetal calf serum. Approximately
109 cells were lysed in 2 volumes of buffer containing 20 mM
4-(2-hydroxyethyl)-1-piperazinesulfonic acid (HEPES), pH 7.4,
5 mM ethylene glycol bis(â-aminoethyl ether) N,N,N′,N′-
tetraacetic acid (EGTA), 1% Triton X-100, 10% glycerol, 0.1
mM sodium orthovanadate, 5 mM sodium fluoride, 4 mM
pyrophosphate, 4 mM benzamide, 1 mM dithiothreitol (DTT),
80 mg/mL aprotinin, 40 mg/mL leupeptin, and 1 mM phenyl-
methanesulfonyl fluoride (PMSF). After centrifugation at
25000g for 10 min, the supernatant was applied to a fast Q
Sepharose column (Pharmacia Biotech, Inc., Piscataway, NJ )
and eluted with a linear gradient from 0.1 to 0.4 M NaCl in
50 mM Hepes, 10% glycerol, pH 7.4. Enzyme active fractions
were pooled, divided into aliquots, and stored at -100 °C.
Tyr osin e Kin a se Assa ys. Enzyme assays for IC50 deter-
minations were performed in 96-well filter plates (Millipore
MADVN6550, Millipore, Bedford, MA). The total volume was
0.1 mL containing 20 mM Hepes, pH 7.4, 50 mM sodium
vanadate, 40 mM magnesium chloride, 10 mM adenosine
triphosphate (ATP) containing 0.5 mCi of [32P]ATP, 20 mg of
poly(glutamic acid)/tyrosine (Sigma Chemical Co., St. Louis,
MO), 20 ng of EGFR tyrosine kinase (for a calculated final
concentration of 1.18 nM), and appropriate dilutions of inhibi-
tor. All components except the ATP were added to the well,
and the plate was incubated with shaking for 10 min at 25
°C. The reaction was started by adding [32P]ATP, and the plate
was incubated at 25 °C for 10 min. The reaction was termi-

1814 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 10
Smaill et al.
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(16) Wissner, A.; J ohnson, B. D.; Floyd, M. B.; Kitchen, D. B.
4-Aminoquinazolines as EGFR inhibitors. U.S. Patent 5,760,
041, J une 2, 1998.
(17) Singh, J .; Dobrusin, E. M.; Fry, D. W.; Haske, T.; Whitty, A.;
McNamara, D. J . Structure-based design of a potent, selective,
and irreversible inhibitor of the catalytic domain of the erbB
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0.5% methylcellulose in water to minimize diluent effects.
Compound dosing solutions were prepared for 5 days at a time.
Host body weight change data are reported as the maximum
treatment-related weight loss in these studies. Calculation of
tumor growth inhibition (% T/C), tumor growth delay (T-C),
and net logs of tumor cell kill was performed as described
previously.37-40 A positive net cell kill indicates that the tumor
burden at the end of therapy was less than at the beginning
of therapy. A negative net log cell kill indicates that the tumor
grew during treatment. Net cell kills near 0 indicate no tumor
growth during therapy.
Ack n ow led gm en t. This work was partially sup-
ported by the Auckland Division of the Cancer Society
of New Zealand.
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