Tyrosine kinase inhibitors. 18. 6-Substituted 4-anilinoquinazolines and 4-anilinopyrido[3,4-d]pyrimidines as soluble, irreversible inhibitors of the epidermal growth factor receptor

Article abstract of DOI:10.1021/jm000372i

4-Anilinoquinazoline- and 4-anilinopyrido[3,4-d]pyrimidine-6-acrylamides are potent pan-erbB tyrosine kinase inactivators, and one example (CI-1033) is in clinical trial. A series of analogues with a variety of Michael acceptor units at the 6-position wer

Full text of DOI:10.1021/jm000372i

J . Med. Chem. 2001, 44, 429-440
429
Tyr osin e Kin a se In h ibitor s. 18. 6-Su bstitu ted 4-An ilin oqu in a zolin es a n d
4-An ilin op yr id o[3,4-d ]p yr im id in es a s Solu ble, Ir r ever sible In h ibitor s of th e
Ep id er m a l Gr ow th F a ctor Recep tor
J eff B. Smaill,† H. D. Hollis Showalter,# Hairong Zhou,# Alexander J . Bridges,# Dennis J . McNamara,#
David W. Fry,# J ames M. Nelson,# Veronika Sherwood,# Patrick W. Vincent,# Bill J . Roberts,#
William L. Elliott,# and William A. Denny*,†
Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, The University of Auckland, Private Bag
92019, Auckland, New Zealand, and Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road,
Ann Arbor, Michigan 48106-1047
Received August 28, 2000
4-Anilinoquinazoline- and 4-anilinopyrido[3,4-d]pyrimidine-6-acrylamides are potent pan-erbB
tyrosine kinase inactivators, and one example (CI-1033) is in clinical trial. A series of analogues
with a variety of Michael acceptor units at the 6-position were prepared to define the structural
requirements for irreversible inhibition. A particular goal was to determine whether additional
functions to increase solubility could be appended to the Michael acceptor. Substituted
acrylamides were prepared by direct acylation of the corresponding 6-amines with the requisite
acid or acid chloride. Vinylsulfonamide derivatives were obtained by acylation of the amines
with chloroethylsulfonyl chloride followed by base-promoted elimination. Vinylsulfone and
vinylsulfine derivatives were prepared by oxidation and base elimination of a hydroxyethylthio
intermediate. The compounds were evaluated for their inhibition of phosphorylation of the
isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in
A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells.
Substitution at the nitrogen of the acrylamide was tolerated only with a methyl group; larger
substituents were dystherapeutic, and no substitution at all was tolerated at the acrylamide
R-carbon. In contrast, while electron-donating groups at the acrylamide â-carbon were not
useful, even quite large electron-withdrawing groups (which increase its electrophilicity) were
tolerated. A series of derivatives with solubility-enhancing substituents linked to the acrylamide
â-carbon via amides were potent irreversible inhibitors of isolated EGFR (IC50s ) 0.4-1.1 nM),
with weakly basic morpholine and imidazole derivatives being the best. Vinylsulfonamides
were also potent and irreversible inhibitors, but vinylsulfones and vinylsulfines were reversible
and only poorly active. Two compounds were evaluated against A431, H125, and MCF-7
xenografts in nude mice but were inferior in these assays to the clinical trial compound CI-
1033.
In tr od u ction
Overexpression of the epidermal growth factor recep-
tor (EGFR) has been reported in a significant number
of human tumors and is associated with poor progno-
sis.1,2 Inhibition of growth signal pathways mediated
through EGFR tyrosine autophosphorylation is thus of
therapeutic interest, and inhibitors of this process have
to shut-down EGF-stimulated autophosphorylation for
been widely sought as potential anticancer drugs.3-5
long periods, we10,11 and others12 have been exploring
4-Anilinoquinazolines and related 4-anilinopyrido[d]-
pyrimidines have been shown to be potent and selective
the use of irreversible inhibitors. We have recently
reported13-15 that 6- and (to a lesser extent) 7-acryl-
reversible inhibitors of both isolated EGFR and EGF-
amide analogues of the 4-anilinoquinazolines and py-
stimulated EGFR autophosphorylation in cells, via
rido[d]pyrimidines (e.g., 3 and 4) act at the ATP binding
domain of EGFR, specifically alkylating an adjacent
Cys-773 residue and irreversibly shutting down kinase
activity. The 6-acrylamides are irreversible inhibitors
of both EGFR and erbB2 autophosphorylation and show
significantly improved in vivo antitumor activity com-
competitive binding to the ATP site,6,7 and two com-
pounds of this type, CP-358,774 (1) and ZD 1839 (Iressa)
(2), are in clinical trial.8,9
In expectation that the high levels of intracellular
ATP in some cell lines may make it difficult to achieve
sufficiently high intracellular levels of such inhibitors
pared to closely related reversible analogues.13 They
tolerate a wide range of structural variations in the
molecule with retention of irreversibility and potency,
including substitution at the vacant 7-position of the
quinazoline nucleus with a range of amine-bearing side
* To whom correspondence should be addressed. Tel: 64 9 3737 599,
ext. 6144. Fax: 64 9 3737 502. E-mail: b.denny@auckland.ac.nz.
† The University of Auckland.
#
Pfizer Global Research and Development.
10.1021/jm000372i CCC: $20.00 © 2001 American Chemical Society
Published on Web 12/30/2000

430 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Smaill et al.
Sch em e 1a
Sch em e 2a
a
(i) CH2dCHCO2H/EDCl‚HCl/pyridine; (ii) CH2dCHCOCl/
DMAP (cat.)/Et3N.
a
(i) R3CHdC(R2)CO2H/EDCl‚HCl/pyridine/THF/DMA; (ii) R3-
Sch em e 3a
CHdC(R2)COCl/DMAP (cat.)/THF/Et3N; (iii) ClCH2CH2SO2Cl/
DMAP (cat.)/THF/Et3N.
chains, and provide a class of soluble, orally active,
potent, selective, and irreversible inhibitors of the EGFR
family of tyrosine kinases.15 This novel class of com-
pounds is exemplified by CI-1033 (5) which has recently
begun phase I clinical trials.15,16
Attempts to develop a soluble pyrido[d]pyrimidine
analogue of 5, by introducing amine-bearing soluble side
chains at the 7-position of a pyrido[3,2-d]pyrimidine
nucleus, met with only limited success.15 While these
compounds (e.g., 6) showed excellent potency for inhibi-
tion of isolated EGFR enzyme, they were considerably
less potent in cellular assays when compared to the
analogous quinazolines. This lack of potency has in part
been attributed to the increased reactivity of the acryl-
amide moiety toward cellular glutathione.15
In further development of this class, we now report
the synthesis and biological activity of a range of
4-anilinoquinazolines and pyrido[3,4-d]pyrimidines sub-
stituted at the 6-position with a variety of Michael
acceptors apart from the parent acrylamide. This work
sought to define the structural requirements of the
Michael acceptor necessary to provide irreversible in-
hibition of EGFR, with the aim of developing one that
can tolerate substitution with a solubility-enhancing
side chain while retaining irreversible inhibition, po-
tency, and selectivity for EGFR.
a
(i) Morpholine/p-TsOH/THF; (ii) BH3‚DMS/THF; (iii) CH2dCH-
CO2H/EDCl‚HCl/Et3N/DMF.
fonyl chloride, followed by base-promoted in situ elimi-
nation of HCl, to generate the vinyl moiety (Scheme 1).
The pyrido[3,4-d]pyrimidines 21 and 22 of Table 1 were
obtained by acylation of the known amines 40 and 41
with acrylic acid (EDCI‚HCl-promoted) and acryloyl
chloride, respectively (Scheme 2).
Synthesis of the quinazoline 23 required first the
synthesis of amine 43. This was obtained by the acid-
catalyzed Michael addition of morpholine to acrylamide
4, followed by borane-dimethyl sulfide reduction of the
amide functionality. Amine 43 was then coupled with
acrylic acid using EDCI‚HCl to give acrylamide 23 of
Table 1 (Scheme 3). Compounds 24 and 25 of Table 1,
bearing an acrylamide moiety substituted in the 3-posi-
tion with a solubility-enhancing ester and amide func-
tionality, respectively, were obtained from the reaction
of 3-(N,N-dimethylamino)propan-1-ol and N,N-dimethyl-
1,3-propanediamine. The key intermediate was acid
chloride 44, in turn prepared from acylation of amine
39 with fumaryl chloride (Scheme 4).
Ch em istr y
The substituted acrylamides 7-20 of Table 1 were
obtained by direct acylation of the known 6-amino
derivatives 37-39, with either the requisite acid under
EDCI‚HCl-promoted coupling or with the acid chloride
in the presence of catalytic DMAP and a base such as
triethylamine or pyridine. The vinylsulfonamide deriva-
tives 32 and 33 of Table 1 were similarly obtained by
acylation of the amines 38 and 39 with chloroethylsul-
EDCI‚HCl-promoted coupling of acids 47a -d with the
amines 37, 38, and 48 provided compounds 26-31 of
Table 1. The acids 47a -d needed for this work were
synthesized from (E)-but-2-enedioic acid monoethyl
ester (45). Conversion to the acid chloride with oxalyl
chloride and then condensation with the required amine

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 431
Sch em e 4a
Sch em e 6a
a
(i) ClOCCHdCHCOCl/THF; (ii) Me2N(CH2)3OH/THF; (iii)
Me2N(CH2)3NH2/THF.
Sch em e 5a
a
(i) HSCH2CH2OH/Cs2CO3/DMSO/50 °C/2 h; (ii) MCPBA/CHCl3/
20 °C/4 h; (iii) MsCl/Et3N/CH2Cl2/0-5 °C/2.5 h; (iv) Davis’ reagent/
CHCl3.
can almost certainly fully inactivate the enzyme via
alkylation given enough time). Those that produced less
than 20% inhibition were classified as reversible. For
compounds capable of rapid and complete alkylation of
the enzyme, the IC50 values derive essentially from
titrating the enzyme activity in a stoichiometric manner
and for this reason are designated as apparent IC50s
(IC50[app]).14,15 The concentration of EGFR in the isolated
enzyme assays is calculated at 1.18 nM and was held
as constant as possible (<10% variation). The IC50[app]
values are an average of at least two separate deter-
minations.
The present study investigated several different types
of Michael acceptors in addition to the original acryl-
amides 3 and 4. Compounds 7-20 represent 11 modi-
fications of the parent acrylamide itself, using both
quinazoline and pyrido[3,4-d]pyrimidine chromophores,
seeking acceptable positions for substitution that would
allow attachment of a solubility-enhancing function. The
N-methyl analogue 7 showed irreversible inhibition of
the isolated enzyme with high potency (IC50[app] ) 0.17
nM compared with 0.91 nM for 3). Although there was
a small loss of potency in the cellular assay, this position
appeared suitable for further substitution (but see later).
In contrast, the R-methylacrylamides 8 and 9 showed
lower potencies in both the enzyme and cellular assays
compared to 3 and 4 and a complete loss of irrevers-
ibility, indicating that substitution at this position of
the Michael acceptor is not tolerated.
A larger number of different substitutions were
investigated at the acrylamide â-position (compounds
10-20). Compounds 10 and 11 show that a â-methyl
substituent gives a small (2-3-fold) reduction in potency
in the autophosphorylation assay, but more importantly
resulted in a reduction in the rate of enzyme alkylation,
providing only partially irreversible inhibition of EGFR.
This is consistent with introduction of a small amount
of steric bulk and electron donation to the Michael
acceptor double bond, reducing its electrophilicity and
therefore its alkylating ability. This is supported by the
data for the cis-chloro and trifluoromethyl analogues 12
and 13. These groups have similar steric properties to
the methyl groups in 10 and 11, but these more electron-
withdrawing groups increase the electrophilicity of the
double bond of the Michael acceptor, resulting in fully
a
(i) Oxalyl chloride/THF/20 °C/1 h, then RNH2; (ii) aq Et3N/60
°C/45 min or LiOH/aq MeOH/20 °C/2 h; (iii) EDCl‚HCl/pyridine/0
°C/1 h and 37, 38, or 48.
side chains gave the amide esters 46a -d , which were
subsequently converted to the acids 47a -d by alkaline
hydrolysis (Scheme 5). Synthesis of the vinylsulfone 35
and vinylsulfine 36 of Table 1 was achieved by base-
catalyzed elimination of the respective mesylates of the
hydroxyethyl compounds 34 and 51. These were in turn
prepared from the reaction of 2-mercaptoethanol with
6-fluoropyrido[3,4-d]pyrimidine (49) to give the hy-
droxyethyl sulfide 50, which was oxidized with either
MCPBA to give 34 or Davis’ reagent to give 51 (Scheme
6).
Resu lts a n d Discu ssion
Alter n a tive Mich a el Accep tor s. Table 1 lists the
structures and physicochemical properties of a series
of 4-(3-bromoanilino)quinazolines and pyrido[3,4-d]py-
rimidines substituted at the 6-position with a variety
of Michael acceptors. We have previously shown14 that
quinazoline- and pyrido[3,4-d]pyrimidine-6-acrylamides
(e.g., 3 and 4) show broadly similar results in these
assays, and the anilino ring was kept constant in order
to facilitate intercomparisons of the different Michael
acceptors. Potencies (IC50[app] in nM) were determined
for both inhibition of phosphorylation of a random
glutamic acid/tyrosine copolymer substrate by isolated
EGFR enzyme and inhibition of EGF-stimulated auto-
phosphorylation of EFGR in A431 cells. The type of
inhibition of the isolated EGFR enzyme is also listed.
Irreversible inhibition is defined13-15 as 80% or greater
inhibition after a 10-min exposure to drug, followed by
drug washout and re-stimulation by EGF 8 h later.
Drugs that produced 20-80% inhibition were desig-
nated as partially irreversible (although in reality they

432 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Smaill et al.
Ta ble 1. EGFR Inhibitory Properties of 6-Substituted 4-Anilinoquinazolines and 4-Anilinopyrido[3,4-d]pyrimidines Bearing Various
Michael Acceptors
IC50[app] (nM)a
EGFR
IC50 (nM)b
A431
no.
family
X
R1
R2
R3
irrev inhibc
3d
4d
I
I
N
C
H
H
H
H
H
H
0.91
0.70
1.5
0.17
1.6
1.2
3.4
2.7
7.4
13
yes
yes
5e
yes
yes
no
no
7
I
N
N
C
N
C
N
C
N
C
N
C
C
C
N
N
N
C
C
C
N
N
N
N
N
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8
I
H
44
9
I
H
16
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
I
Me
0.50
0.55
0.69
1.75
1.1
7.7
8.7
20
partially
partially
yes
I
Me
I
cis-Cl
CF3
CHdCH2
dCH2
Ph
I
35
27
120
77
yes
I
partially
partially
partially
partially
yes
I
1.6
I
9.1
I
COMe
COOH
COOEt
COOEt
H
H
H
1.2
1039
>500
64
I
0.37
2.7
I
partially
yes
I
1.5
51
I
(CH2)2NMe2
4.2
2282
156
194
108
59
partially
no
I
(CH2)3-N-morpholinyl
2.7
I
(CH2)3-N-morpholinyl
3.3
no
I
H
COO(CH2)3NMe2
2.4
yes
I
H
CONH(CH2)3NMe2
0.44
1.1
yes
I
H
CONH(CH2)3NMe2
57
21
8.8
14
193
14
yes
I
H
CONH(CH2)3NEt2
0.73
0.81
0.56
1.45
0.61
0.76
1.4
93.5
0.43
4.6
yes
I
H
CONH(CH2)3-N-morpholinyl
CONH(CH2)3-N-imidazolyl
CONH(CH2)3NMe2
yes
I
H
yes
I
Me
partially
yes
II
III
III
III
III
III
N
C
N
N
N
NHSO2CHdCH2
NHSO2CHdCH2
SO2CH2CH2OH
SO2CHdCH2
2.4
2.7
yes
yes
no
>500
340
yes
SOCHdCH2
no
a
Concentration to inhibit by 50% the phosphorylation of a polyglutamic acid/tyrosine random copolymer by EGFR enzyme (prepared
from human A431 carcinoma cell vesicles by immunoaffinity chromatography). Values are the averages from at least two independent
b
dose-response curves; variation was generally (15%. Concentration to inhibit by 50% the autophosphorylation of EGFR in A431 cells
(detected by immunoblotting). c Irreversible inhibition is defined as >80% inhibition of formation of phosphorylated EGFR in A431 cells
d
8 h after washing cells free of the inhibitor. Data from ref 14. e Data from ref 15.
irreversible compounds. We have previously reported15
that increased reactivity of the Michael acceptor can
result in increased background alkylation of cellular
thiols, such as glutathione, and therefore a reduction
in cellular potency for inhibition of EGFR. It is likely
therefore that a balance is required, between increased
reactivity of the Michael acceptor and increased steric
bulk, to provide compounds that are still capable of
rapid alkylation of the target Cys-773 without display-
ing significantly increased background alkylation. The
irreversibility assay in conjunction with the cellular
assay for inhibition of EGFR autophosphorylation pro-
vides a measure of the success of this balancing act.
Substitution at the â-position with an unsaturated
double bond, an allene functionality, or a phenyl group
(compounds 14-16, respectively) resulted in only par-
tially irreversible inhibition, suggesting the mild electron-
withdrawing abilities of these groups was not sufficient
to overcome their respective steric hindrances. Com-
pounds possessing more strongly electron-withdrawing
â-carbonyl substituents such as methyl ketone (17), acid
(18), and ethyl ester (19 and 20) groups at the 3-position
showed more promise, with 18 and 20 possessing potent
activity against the isolated enzyme (IC50[app] ) 0.37 and
1.5 nM, respectively) while also being fully irreversible.
However, acid 18 showed a large loss of potency in the
cellular assay (IC50 > 500 nM), presumably due to its
lack of ability to permeate the cell membrane. Ester 20
also showed a 15-fold loss of potency when compared to
the parent acrylamide 3 in the cellular assay, possible
due to partial ester hydrolysis to the nonpotent acid.
Atta ch m en t of Solu ble Sid e Ch a in s a t th e Acr yl-
a m id e Nitr ogen . The N-methyl analogue 7 retained
potent irreversible inhibitory activity against the EGFR
enzyme. However, substitution of the acrylamide nitro-
gen with the larger N,N-dimethylaminoethyl or mor-
pholinopropyl groups (compounds 21-23, respectively)
resulted in a much larger attenuation of potency (46-
671-fold), with an associated loss of irreversible inhibi-
tion of cellular autophosphorylation. This suggests that
there is only minimal steric tolerance (no bigger than a
methyl group) for substitution at the acrylamide nitro-
gen.
Atta ch m en t of Solu ble Sid e Ch a in s a t th e Acr yl-
a m id e â-Ca r bon . The tolerance for carbonyl substit-
uents at the acrylamide â-position led to the synthesis
of a small subset of such carbonyl-linked soluble side
chain derivatives (24-31). The ester-linked quinazoline

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 433
Ta ble 2. Comparative Inhibition of Autophosphorylation of
EGFR and erbB2 by Selected Analogues
Ta ble 3. In Vivo Antitumor Properties of Selected
4-Anilinoquinazolines and 4-Anilinopyrido[3,4-d]pyrimidine-
6-acrylamides
IC50 (nM)
IC50 (nM)
dose
wt change T/C (%) last T-Cd
no.
EGFRa
7.4
108
59
57
erbB2b
no.
EGFRa
21
8.8
14
14
erbB2b
no. tumor (mg/kg)
schedulea
(g)b
therapy dayc (days)
5c
24
25
26
9.0
110
207
13
27
28
29
31
14
5e A431
A431
18f
days 10-24
-1.0
-0.1
-0.4
-0.3
-0.1
-0.3
+
0
62
56
88
70
41.3
5.2
7.3
1.5
5.2
7.9
6.8
7
200 HDTg days 12-26
5.0
8.1
24
H125 200 HDT days 15-29
MCF-7 200 HDT days 11-25
31 A431
200 HDT days 12-26
a
Concentration to inhibit by 50% the EGF-stimulated auto-
phosphorylation of EGFR in A431 cells. Values are the averages
from at least two independent dose-response curves; variation
H125 200 HDT days 15-29
MCF-7 200 HDT days 17-31
23
124
a
b
Compound 5 was administered as a solution of the isethionate
was generally (15%. Concentration to inhibit by 50% the he-
salt in 50 mM sodium lactate buffer, pH 4.0. Compounds 7 and
31 were administered as suspensions in 0.5% methylcellulose in
water. All three compounds were administered orally on the
indicated schedule. Therapy was initiated when tumor masses in
the respective experiments reached 100-150 mg. Differences in
treatment schedules indicate data from separate experiments. It
is important to initiate therapy in different experiments at an
equivalent tumor mass rather than fix the days of therapy.
regulin-stimulated autophosphorylation of erbB2 in MDA-MB 453
cells. Values are the averages from at least two independent dose-
response curves; variation was generally (15%. c Data from ref
15.
24 was fully irreversible but not particularly potent in
the cellular assay (IC50 ) 108 nM). This loss of potency
may be due to partial ester hydrolysis to the (nonpotent)
parent acid 18. The more stable amide derivatives (25-
29, 31), containing a variety of cationic side chains, were
all fully irreversible, displayed good potency against
isolated EGFR (IC50[app] ) 0.4-1.1 nM), and were more
potent than the ester analogue 24. The most potent of
these were the weakly basic morpholine and imidazole
derivatives 28 and 29 (IC50s for inhibition of autophos-
phorylation ) 8.8 and 14 nM, respectively). A compari-
son of the 3-bromo- and 3-chloro-4-fluoroanilino (the
anilino substituents employed in 5), in analogues 28 and
31, showed that the 3-bromo side chain gives a slight
advantage in outright potency in the cellular assay (IC50
) 8.8 compared to 14 nM). Interestingly, the soluble
N-methyl analogue 30 showed a large loss of potency
(IC50 ) 193 nM) and a partial loss of irreversibility in
the cellular assay compared to the N-methyl compound
7 (which was fully irreversible). Presumably the intro-
duction of the methyl group at the acrylamide nitrogen,
in combination with the bulky soluble side chain at the
acrylamide 3-position, is too much of a steric impedi-
ment, preventing the positioning of the Michael acceptor
in a conformation where it can alkylate the enzyme
effectively. The solubility-enhanced analogues showing
irreversible inhibition (24-29, 31) were compared for
their ability to inhibit both EGF-stimulated autophos-
phorylation of EGFR in A431 cells and heregulin-
stimulated autophosphorylation of erbB2 in MDA-MB
453 cells (Table 2). With the exception of 25, the
compounds were equipotent inhibitors of both receptors,
with IC50 values comparable to those of 5. Such broad-
spectrum inhibition of different erbB family members
is a potential advantage, since signaling through this
pathway occurs via both homo- and heterodimers.
Com p a r ison of Qu in a zolin e a n d P yr id o[3,4-d ]-
p yr im id in e Ch r om op h or es. Several pairs of quinazo-
lines and pyrido[3,4-d]pyrimidines (8/9, 10/11, 19/20, 22/
23, and 32/33) were evaluated to determine the influence
of the 7-aza atom. We have previously14 shown little
activity difference between these chromophore classes,
and the present results confirm this (Table 1).
b
Maximum therapy-induced weight loss. A net weight gain is
indicated by a “+”. c Ratio of median treated tumor mass/median
d
control tumor mass × 100. The difference in days for the treated
(T) and control (C) tumors to reach a fixed evaluation size of 750
mg. e Data from ref 15. f Maximum tolerated dose (eLD10). g HDT,
highest dose tested.
) 2.4 and 2.7 nM, respectively). However, subsequent
in vivo experiments with vinylsulfonamide 32 suggested
that it was unstable in biological systems, and this
avenue was therefore not pursued. The vinylsulfone 35
was an irreversible inhibitor but showed very poor
potency in the cellular assay (IC50 > 500 nM), while its
hydroxyethyl precursor 34 and the vinylsulfine 36 were
poorly active and were reversible inhibitors.
The above results show that both N-methyl-substi-
tuted and â-substituted acrylamides retain good potency
and ability for irreversible inhibition of EGFR and that
the latter are suitable templates for the introduction of
a wide range of solubility-enhancing groups.
In Vivo Stu d ies. The N-methyl- and â-morpholino-
propylbutenediamide analogues (7 and 31, respectively)
were evaluated against A431 epidermoid, H125 non-
small-cell lung, and MCF-7 estrogen-dependent breast
xenografts in mice, and the results are given in Table
3. The N-methyl compound 7 was selected as it was the
most active against the isolated EGFR enzyme, while
31 was selected for direct comparison with the clinical
candidate 5 (same anilino ring substitution pattern).
Both compounds proved ineffective against the A431
xenograft, while the clinical candidate 5 (CI-1033) was
highly active. Neither 7 nor 31 showed any meaningful
antitumor effects against the H125 or MCF-7 xe-
nografts. Other than the weight loss noted in Table 3,
there were no clinical signs of toxicity associated with
the 200 mg/kg dosage level for either compound. The
fact that 200 mg/kg doses were tolerated by the tumor-
bearing animals may indicate that these compounds
were not highly bioavailable when dosed as suspensions.
The pyrido[3,4-d]pyrimidine 31 is clearly inferior15,16 to
the clinical evaluation compound 5, a quinazoline which
has the same anilino substitution pattern but bears the
solubility-enhancing group separately off the 7-position,
rather than off the end of the acrylamide. Since we have
shown above and previously14 that the chromophore has
little effect, it appears that the positioning of the
solubility-enhancing group is an important factor.
Non -Acr yla m id e Mich a el Accep tor s. The vinyl-
sulfonamide was studied as an alternate Michael ac-
ceptor that would allow subsequent substitution with
soluble cationic side chains. Compounds 32 and 33 were
both able to irreversibly inhibit EGFR autophosphory-
lation and were very potent in the cellular assay (IC50s

434 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Smaill et al.
pressure and passed through a crude column of silica gel before
preparative layer chromatography on silica gel eluting with
EtOAc/CH2Cl2 (1:1), gave 8 (18 mg, 6%): mp (CH2Cl2/hexane)
Con clu sion s
These results show that a range of Michael acceptors,
apart from the unsubstituted acrylamide at the 6-posi-
tion of 4-anilinoquinazolines and pyrido[3,4-d]pyrim-
idines, provide irreversible inhibitors of the EGFR
enzyme. Of the non-acrylamide Michael acceptors stud-
ied, only the vinylsulfonamide provided comparably
potent irreversible inhibitors, but these were less stable.
Within the modified acrylamides, there was very limited
bulk tolerance for substitution at the acrylamide nitro-
gen, with only the N-methyl analogue 7 retaining
irreversible activity, and there was no tolerance at all
for substitution at the acrylamide R-carbon (compounds
8/9). In contrast, quite large electron-withdrawing
groups (which increase acrylamide electrophilicity) were
acceptable at the â-carbon. These amide-derived soluble
analogues were potent irreversible inhibitors of isolated
EGFR and effective inhibitors of both EGF-stimulated
autophosphorylation of EGFR and heregulin-stimulated
autophosphorylation of erbB2 in cellular assays, with
activity profiles comparable to that of the clinical agent
5. However, the best of these (31) was not nearly as
active as 5 in vivo. Thus positioning the solubility-
enhancing group off the â-carbon of the acrylamide may
not be as useful as positioning it separately off the
7-position, since it may raise the general alkylating
ability of the inhibitor.
1
177-178 °C; H NMR [(CD3)2SO] δ 10.61 (s, 1 H, NH), 10.29
(s, 1 H, NH), 9.06 (s, 1 H), 8.93 (s, 1 H), 8.67 (s, 1 H), 8.19 (t,
J ) 1.6 Hz, 1 H, H-2′), 7.91 (dt, J ) 7.6, 1.6, 1.6 Hz, 1 H, H-6′),
7.38 (t, J ) 7.9 Hz, 1 H, H-5′), 7.34 (dt, J ) 8.1, 1.4, 1.4 Hz, 1
H, H-4′), 6.04 (s, 1 H, CH2C(CH3)CO), 5.64 (s, 1 H, CH2C(CH3)-
CO), 2.03 (s, 1 H, CH2C(CH3)CO); HRMS (DEI) C17H1481BrN5O
requires 385.03613, found 385.03595.
N-[4-(3-Br om oa n ilin o)qu in a zolin -6-yl]-2-m eth yla cr yl-
a m id e (9): Exa m p le of Meth od of Sch em e 1. To a stirred
solution of 6-amino-4-(3-bromoanilino)quinazoline18 (39) (0.50
g, 1.59 mmol) in THF (20 mL) under nitrogen were added Et3N
(excess, 1.0 mL), a catalytic amount of DMAP and methacry-
loyl chloride (171 µL, 1.75 mmol) dropwise. The reaction was
stirred at room temperature for 1.5 h over which time two
further amounts (50 µL) of methacryloyl chloride were added.
Workup as above followed by chromatography on silica gel
eluting with CH2Cl2/EtOAc (1:1) to MeOH/CH2Cl2/EtOAc (5:
45:50) and recrystallization from EtOAc gave 9 (195 mg,
1
32%): mp 244-245 °C; H NMR [(CD3)2SO] δ 10.15 (s, 1 H,
NH), 9.90 (s, 1 H, NH), 8.80 (br s, 1 H, H-5), 8.60 (s, 1 H, H-2),
8.20 (br s, 1 H, H-2′), 7.97 (br d, J ) 8.6 Hz, 1 H, H-7), 7.89
(br d, J ) 7.7 Hz, 1 H, H-6′), 7.80 (d, J ) 8.9 Hz, 1 H, H-8),
7.35 (t, J ) 8.0 Hz, 1 H, H-5′), 7.30 (br d, J ) 7.5 Hz, 1 H,
H-4′), 5.94 (s, 1 H, CH2C(CH3)CO), 5.62 (s, 1 H, CH2C(CH3)-
CO), 2.02 (s, 3 H, CH2C(CH3)CO). Anal. (C18H15BrN4O) C, H,
N.
(2E)-N-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-
yl]-2-bu ten a m id e (10). EDCI‚HCl (98 mg, 0.5 mmol) was
added to a stirred solution of 38 (32 mg, 0.1 mmol) and trans-
crotonic acid (35 mg, 0.4 mmol) in pyridine (0.4 mL) under N2
at 0-5 °C. Cooling was removed and the mixture was stirred
at 25 °C for 2 h, then diluted with water and the resulting
suspension stirred for 15 min and filtered. The solid was
dissolved in EtOAc, washed with 5% aqueous NaHCO3, dried
(MgSO4) and filtered through a silica gel column. The filtrate
was concentrated, and the resulting solid was triturated in
hot EtOAc to give 10 (11 mg, 28%): mp >260 °C dec; 1H NMR
[(CD3)2SO] δ 10.87 (s, 1 H, NH), 10.31 (s, 1 H, NH), 9‚03 (s, 1
H), 9.00 (s, 1 H), 8.65 (s, 1 H), 8.17 (s, 1 H), 7.89 (d, J ) 7.5
Hz, 1 H), 7.39-7.33 (m, 2 H), 6.99-6.90 (m, 1 H), 6.39 (dd, J
) 15.4, 1.7 Hz, 1 H), 1.91 (dd, J ) 7.0, 1.4 Hz, 3 H); MS (APCI)
m/z (relative %) 381.8 (74), 382.8 (27), 383.8 (100), 384.8 (30),
385.9 (10). Anal. (C17H14BrN5O‚0.25H2O) C, H, N.
(2E)-N-[4-(3-Br om oa n ilin o)-6-q u in a zolin yl]-2-b u t en -
a m id e (11). Excess trans-crotonyl chloride was added to a
solution of 39 (316 mg, 1.0 mmol) in THF (6 mL) stirred under
N2 at 0 °C. After 2.5 h the resulting yellow solid was collected
by filtration and sonicated with EtOAc to give 11 (216 mg,
52%): mp 279-281 °C; 1H NMR [(CD3)2SO] δ 11.55 (br s, 1 H,
NH), 10.78 (s, 1 H, NH), 9.17 (d, J ) 1.9 Hz, 1 H, H-5), 8.97
(s, 1 H, H-2), 8.12 (dd, J ) 9.1, 2.0 Hz, 1 H, H-7), 8.05 (t, J )
1.9 Hz, 1 H, H-2′), 7.99 (d, J ) 9.0 Hz, 1 H, H-8), 7.76 (dd, J
) 8.1, 2.0 Hz, 1 H, H-6′), 7.58 (dd, J ) 8.6, 1.7 Hz, 1 H, H-4′),
7.52 (t, J ) 8.1 Hz, 1 H, H-5′) 7.03-6.94 (m, 1 H, (CO)CHd),
6.34 (dd, J ) 15.1, 1.7 Hz, 1 H, CHdCHCH3), 1.98 (dd, J )
6.8, 1.4 Hz, 3 H, CH3); MS (CI) 385 (89, 81BrMH+), 384 (51,
81BrM+), 383 (100, 79BrMH+), 382 (37, 79BrM+). Anal. (C18H15N4-
BrO‚HCl) C, H, N.
(2Z)-N-[4-(3-Br om oa n ilin o)-6-p yr id o[3,4-d ]p yr im id in -
6-yl]-3-ch lor o-2-p r op en a m id e (12). A stirred solution of 38
(128 mg, 0.4 mmol) and cis-3-chloroacrylic acid (172 mg, 1.6
mmol) in pyridine (2 mL) under N2 was treated at -20 °C with
EDCI‚HCl (392 mg, 1.5 mmol). After 4.5 h at -20 °C,
additional cis-3-chloroacrylic acid (57 mg) and EDCI‚HCl (130
mg) were added, and the temperature was brought to -10 °C.
After a total reaction time of 7 h, the viscous mixture was
diluted with DMF and the resulting solution was poured into
EtOAc/water (1:1). The aqueous phase was further extracted
with EtOAc (2×), and the combined organic phases were
washed with brine (2×), dried (MgSO4) and filtered through a
column of flash silica gel. The filtrate was concentrated to a
Exp er im en ta l Section
Analyses were performed by the Microchemical Laboratory,
University of Otago, Dunedin, NZ, or by the Analytical
Department, Pfizer Global Research and Development, Ann
Arbor Laboratories. 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 AC-200 or AM-400 or Varian Unity 400-MHz
spectrometers and referenced to Me4Si. Mass spectra were
recorded either on a Varian VG 7070 spectrometer at nominal
5000 resolution or on a Finnigan MAT 900Q spectrometer.
N-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-yl]-N-
m eth yla cr yla m id e (7): Exa m p le of Meth od of Sch em e
1. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDCI‚HCl) (294 mg, 1.5 mmol) was added in one portion
to a stirred solution of 4-(3-bromoanilino)-6-methylaminopy-
rido[3,4-d]pyrimidine17 (37) (100 mg, 0.3 mmol), redistilled
acrylic acid (75 µL, 1.05 mmol) and pyridine (0.3 mL) in THF/
DMA (3:2, 1.8 mL) under N2 at 0 °C. After 30 min the reaction
was warmed to 25 °C, and after 3.75 h further acrylic acid (25
µL) was added. The mixture was stirred for an additional 3 h,
then quenched with water. The precipitate was collected, air-
dried, and triturated in hot CH2Cl2/EtOAc to give 7 (67 mg,
1
56%): mp 215-223 °C dec; H NMR [(CD3)2SO] δ 10.11 (s, 1
H, NH), 9.14 (s, 1 H), 8.80 (s, 1 H), 8.45 (s, 1 H), 8.22 (s, 1 H),
7.91 (br d, J ) 7.7 Hz, 1 H), 7.43-7.36 (m, 2 H), 6.36-6.23
(m, 2 H), 5.66 (dd, J ) 9.5, 3.0 Hz, 1 H), 3.44 (s, 3 H, CH3);
CIMS m/z (relative %) 383 (23), 384 (100), 385 (40), 386 (99),
387 (20). Anal. (C17H14BrN5O‚0.5H2O) C, N; H: found 3.5, calcd
4.1.
N-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-yl]-2-
m eth yla cr yla m id e (8): Exa m p le of Meth od of Sch em e
1. To a solution of 6-amino-4-(3-bromoanilino)pyrido[3,4-d]-
pyrimidine17 (38) (250 mg, 0.82 mmol), Et3N (excess, 2.0 mL)
and DMAP (catalytic) in THF (30 mL) under nitrogen was
added methacryloyl chloride (88 µL, 0.90 mmol). The reaction
was stirred at room temperature for 1.5 h over which time
two further amounts (88 µL) of methacryloyl chloride were
added. The reaction was then diluted with saturated NaHCO3
and extracted with EtOAc. The combined organic extracts were
dried over anhydrous Na2SO4, concentrated under reduced

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 435
solid that was dissolved in warm EtOAc and chromatographed
on flash silica gel, eluting with EtOAc. The appropriate
fractions were pooled and evaporated. The product was
triturated in EtOAc/tert-butyl methyl ether (1:1), dried at 0.1
mm/25 °C and recrystallized from EtOAc to give 12 (30 mg,
(2E)-N-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-
yl]-3-p h en yl-2-p r op en a m id e (16). trans-Cinnamic acid (60
mg, 0.4 mmol) was added to a stirred solution of 38 (32 mg,
0.1 mmol) in pyridine (0.4 mL). EDCI‚HCl (98 mg, 0.5 mmol)
was added and the mixture was stirred under N2 at 25 °C for
2 h. The mixture was diluted with water, and the solids were
collected and dissolved in EtOAc. The solution was washed
with 5% aqueous NaHCO3, dried (MgSO4) and filtered through
silica gel. The residue from removal of solvent was triturated
with hot EtOAc to give 16 (23 mg, 51%): mp 253-256 °C; 1H
NMR [(CD3)2SO] δ 11.07 (s, 1 H, NH), 10.36 (s, 1 H, NH), 9.06
(s, 1 H), 9.02 (s, 1 H), 8.67 (s, 1 H), 8.19 (s, 1 H), 7.90 (d, J )
7.7 Hz, 1 H), 7.72-7.65 (m, 3 H), 7.51-7.34 (m, 5 H), 7.14 (d,
J ) 15.7 Hz, 1 H). Anal. (C22H16N5OBr‚0.25H2O) C, H, N.
(2E)-N-[4-(3-Br om oan ilin o)-6-qu in azolin yl]-4-oxo-2-pen -
ten a m id e (17). N-Ethyldiisopropylamine (0.26 mL, 1.5 mmol)
and 39 (0.23 g, 0.75 mmol) were added to a stirred solution of
(E)-4-oxopent-2-enoic acid (171 mg, 1.5 mmol) and EDCI‚HCl
(288 mg, 1.5 mmol) in THF/DMF (3:1, 4 mL) under N2 at 25
°C. The ice bath was then removed, and the reaction mixture
was stirred at 25 °C for 4 h, when further N-ethyldiisopropy-
lamine (0.13 mL, 0.75 mmol), (E)-4-oxopent-2-enoic acid (86
mg, 0.75 mmol) and EDCI‚HC1 (144 mg, 0.75 mmol) were
added. The reaction mixture was stirred for a further 14 h at
25 °C, then added dropwise to stirred cold water (100 mL).
The solid was collected, dissolved in MeOH (50 mL) and
evaporated onto silica gel (3 g). Flash chromatography on silica
gel, eluting with 10% MeOH/CH2Cl2 (1:9), gave 17 (0.14 g,
45%): mp 230 °C dec; 1H NMR [(CD3)2SO] δ 10.91 (s, 1 H,
NH), 9.99 (s, 1 H, NH), 8.87 (d, J ) 1.9 Hz, 1 H, H-5), 8.60 (s,
1 H, H-2), 8.17 (t, J ) 1.9 Hz, 1 H, H-2′), 7.85 (m, 3 H, H-7,8,6′),
7.37 (m, 2 H, H-5′,4′), 7.15 (d, J ) 15.7 Hz, 1 H, pentenyl H-3),
6.99 (d, J ) 15.7 Hz, 1 H, pentenyl H-2), 2.40 (s, 3 H, CH3);
MS (APCI) 412.7 (100, 81BrMH+), 410.8 (98, 79BrMH+). Anal.
(C19H15BrN4O2) C, H, N.
(2E)-4-{[4-(3-Br om oa n ilin o)-6-qu in a zolin yl]a m in o}-4-
oxo-2-bu ten oic Acid (18). Maleic anhydride (0.266 g, 2.7
mmol) was added to a solution of 39 (0.78 g, 2.5 mmol) in DMF
(8 mL), and the mixture was heated with stirring in a 70 °C
oil bath for 2.5 h. The resulting suspension was cooled to room
temperature and then diluted with water. The solid was
collected, washed sequentially with a mixture of toluene/DMF
(1:1), water, and IPA. The solid was dried under high vacuum
at 60 °C for 16 h to give 18 (0.87 g, 86%): mp 224-225 °C
dec; 1H NMR [(CD3)2SO] δ 13.00 (br s, 1 H, COOH), 10.85 (br
s, 1 H, NH), 9.96 (br s, 1 H, NH), 8.73 (d, J ) 1.8 Hz, 1 H,
H-5), 8.54 (s, 1 H, H-2), 8.11 (br s, 1 H, (CH3)2NCHO), 7.91-
7.75 (m, 4 H), 7.32-7.24 (m, 2 H), 6.46 (d, J ) 12.0 Hz, 1 H,
CHdCH), 6.35 (d, J ) 12.0 Hz, 1 H, CHdCH), 2.84 (s, 3 H,
(CH3)2NCHO), 2.68 (s, 3 H, (CH3)2NCHO); MS (APCI) 412.8
(100, 81BrM+), 410.8 (96, 79BrM+), 413.8 (26, 81BrMH+), 411.8
(24, 79BrMH+). Anal. (C18H13BrN4O3‚DMF) C, H, N.
Eth yl (2E)-4-{[4-(3-Br om oa n ilin o)-6-qu in a zolin yl]a m i-
n o}-4-oxo-2-bu ten oa te (19). N-Ethyldiisopropylamine (0.26
mL, 1.5 mmol) and 39 (0.23 g, 0.75 mmol) were added to a
solution of (E)-4-ethoxy-4-oxobut-2-enoic acid (216 mg, 1.5
mmol) and EDCI‚HC1 (288 mg, 1.5 mmol) in THF/DMF (3:1,
4 mL) stirred under N2 at 25 °C. The ice bath was removed,
and the reaction mixture was stirred at 25 °C for 4 h, when
further N-ethyldiisopropylamine (0.13 mL, 0.75 mmol), (E)-
4-ethoxy-4-oxobut-2-enoic acid (108 mg, 0.75 mmol), and EDCI‚
HC1 (144 mg, 0.75 mmol) were added. After stirring a further
14 h at 25 °C, the reaction mixture was added dropwise to
stirred cold water (100 mL). The solid was collected, dissolved
in MeOH (50 mL), and dried onto silica gel (3 g) and flash
chromatographed on silica gel, eluting with MeOH/CH2C12 (1:
9). Concentration of pure fractions under reduced pressure
gave 19 (0.19 g, 58%): mp >255 °C; 1H NMR [(CD3)2SO] δ
10.93 (s, 1 H, NH), 9.99 (s, 1 H, NH), 8.89 (d, J ) 1.9 Hz, 1 H,
H-5), 8.60 (s, 1 H, H-2), 8.16 (t, J ) 1.9 Hz, 1 H, H-2′), 7.85
(m, 3 H, H-7,8,6′), 7.33 (m, 3 H, H-5′,4′, pentenyl H-3), 6.79
(d, J ) 15.4 Hz, 1H, pentenyl H-2), 4.24 (q, J ) 7.1 Hz, 2 H,
CH2), 1.29 (t, J ) 7.1 Hz, 3 H, CH3); MS (APCI) 442.8 (99,
81BrMH+), 440.8 (100, 79BrMH+). Anal. (C20H17BrN4O3) C, H,
N.
1
18%): mp 165-175 °C dec; H NMR [(CD3)2SO] δ 11.09 (s, 1
H, NH), 10.38 (s, 1 H, NH), 9‚04 (s, 1 H), 9.00 (s, 1 H), 8.66 (s,
1 H), 8.16 (t, J ) 1.9 Hz, 1 H), 7.88 (dt, J ) 7.7, 1.7 Hz, 1 H),
7.40-7.33 (m, 2 H), 7.07 (d, J ) 8.0 Hz, 1 H), 6.77 (d, J ) 8.0
Hz, 1 H); MS (APCI) m/z (relative %) 365.8 (29), 366.8 (36),
367.8 (35), 368.8 (35), 401.8 (82), 402.8 (18), 403.8 (100), 404.8
(20), 405.8 (29). Anal. (C16H11BrClN5O‚0.25C4H8O2) C, H, N.
(2E)-N-[4-(3-Br om oa n ilin o)-6-q u in a zolin yl]-4,4,4-t r i-
flu or o-2-bu ten a m id e (13). A stirred solution of 39 (158 mg,
0.5 mmol) and 4,4,4-trifluorobut-2-enoic acid (153 mg, 1.1
mmol) in THF/DMF (4:1, 2.5 mL) was treated with EDCI‚HCl
(192 mg, 1.0 mmol) under N2 at 0 °C. After 1 h the mixture
was diluted with water (10 mL), and the resulting precipitate
was collected, washed with water (2 × 5 mL) and ether (10
mL) and air-dried. The solid was suspended in EtOAc (10 mL),
refluxed briefly, and sonicated for 10 min, then collected by
filtration, washed with EtOAc (5 mL) and dried in a vacuum
oven at 75 °C for 1.5 h to give 13 as the hydrochloride salt (76
mg, 33%): mp 273-278 °C; 1H NMR [(CD3)2SO] δ 11.09 (br s,
1 H, NH), 10.43 (s, 1 H, NH), 8.90 (s, 1 H, H-2), 8.70 (s, 1 H,
H-5), 8.11 (s, 1 H, H-2′), 7.97 (dd, J ) 2.5, 9.2 Hz, 1 H, H-7),
7.87 (d, J ) 9.0 Hz, 1 H, H-8), 7.81 (d, J ) 6.9 Hz, 1 H, H-6′),
7.41-7.33 (m, 2 H, H-5′,H-4′), 7,11 (d, J ) 16.4 Hz, 1 H, CHd
CHCF3), 7.03 (dq, J d ) 16.4 Hz, J q ) 6.4 Hz, 1 H, CHdCHCF3);
MS (CI) 439 (78 81BrM+), 437 (100 79BrM+). Anal. (C18H13
-
BrF3N4O‚0.5HCl) C, H, N.
(2E)-N-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-
yl]-2,4-p en ta d ien a m id e (14). A stirred solution of 38 (160
mg, 0.5 mmol), 80% trans-2,4-pentadienoic acid (245 mg, 2
mmol), and pyridine (0.5 mL) in THF/DMA (2:1, 3 mL) under
N2 was cooled to 0-5 °C and treated with one portion of EDCI‚
HCl (490 mg, 2.5 mmol). Cooling was removed, and the viscous
mixture was stirred at 25 °C for 23 h, then charged with
additional trans-2,4-pentadienoic acid (125 mg), EDCI‚HCl
(240 mg) and THF/DMA (2:1, 2 mL). After a further 19 h the
mixture was diluted with water and EtOAc. The biphasic
mixture was warmed, then filtered through Celite, and the
filter pad washed well with water and hot EtOAc. The filtrate
was extracted with EtOAc (3×) and the combined organic
phases were washed with brine, dried (MgSO4) and concen-
trated. The resulting solid was dissolved in hot EtOAc and
chromatographed on silica gel, eluting with EtOAc. The
product was triturated in warm EtOAc to give 14 (27 mg,
1
13%): mp 210-215 °C; H NMR [(CD3)2SO] δ 11.04 (s, 1 H,
NH), 10.34 (s, 1 H, NH), 9.04 (s, 1 H), 9.02 (s, 1 H), 8.66 (s, 1
H), 8.17 (t, J ) 1.9 Hz, 1 H), 7.89 (dt, J ) 7.7, 1.7 Hz, 1 H),
7.40-7.27 (m, 3 H), 6.60 (dt, J ) 16.9, 10.6 Hz, 1 H), 6.53 (d,
J ) 15.2 Hz, 1 H), 5.75 (d, J ) 16.9 Hz, 1 H), 5.56 (d, J ) 11.1
Hz, 1 H); MS (APCI) m/z (relative %) 395.9 (89), 396.9 (20),
397.9 (100), 398.9 (20). Anal. (C18H14BrN5O) C, H.
N-[4-(3-Br om oa n ilin o)-6-q u in a zolin yl]-2,3-b u t a d ien -
a m id e (15). EDCI‚HCl (384 mg, 2.0 mmol) was added to a
stirred solution of 39 (316 mg, 1.0 mmol), and 2,3-butadienoic
acid (173 mg, 2.06 mmol) in DMF (5 mL) stirred under N2 at
0 °C. After 1.5 h the reaction was quenched with 0.1 M HCl
(10 mL), and the resulting precipitate was collected and
washed successively with water and Me2CO, then dissolved
into Me2CO with the addition of Et3N. The solution was filtered
through a short column of silica gel in Me2CO/CH2Cl2 (1:1) to
1
give 15 (247 mg, 56%): mp 268-270 °C; H NMR [(CD3)2SO]
δ 10.39 (s, 1 H, NH), 9.93 (s, 1 H, NH), 8.76 (d, J ) 2.2 Hz, 1
H, H-5), 8.58 (s, 1 H, H-2), 8.18 (s, 1 H, H-2′), 7.87 (dt, J )
9.0, 1.9 Hz, 2 H, H-7,8), 7.79 (d, J ) 8.8 Hz, 1 H, H-6′), 7.34 (t,
J ) 7.9 Hz, 1 H, H-5′), 7.29 (d, J ) 8.3 Hz, 1 H, H-4′), 6.07 (t,
J ) 6.5 Hz, 1 H, CHdCdCH2), 5.49 (d, J ) 6.6 Hz, 2 H, dCd
CH2); MS (APCI) 382.8 (88, 81BrMH+), 381.8 (19, 81BrM+),
380.7 (100, 79BrMH+). Anal. (C18H13BrN4O‚0.8C3H6O‚0.5H2O)
C, H, N.

436 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Smaill et al.
Eth yl (2E)-4-{[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im i-
d in -6-yl]a m in o}-4-oxo-2-bu ten oa te (20). Reaction of 38 (32
mg, 0.1 mmol) with fumaric acid monoethyl ester (58 mg, 0.4
mmol) and EDCI‚HCl (98 mg, 0.5 mmol) in pyridine (0.5 mL)
was carried out as described above. After 5 h, the solution was
poured into water, which formed a precipitate. The suspension
was sonicated, then the solid was collected, washed well with
water, and dried to give 20 (90 mg, 89%): mp >230 °C dec;
1H NMR [(CD3)2SO] δ 11.44 (s, 1 H, NH), 10.37 (s, 1 H, NH),
9.07 (s, 1 H, H-2), 9.05 (s, 1 H, H-8), 8.68 (s, 1 H, H-5), 8.17 (d,
J ) 2.0 Hz, 1 H, H-2′), 7.89 (dt, J ) 7.5, 1.9, 1.7 Hz, 1 H, H-4′),
7.48 (d, J ) 15.4 Hz, 1 H, fumarate H), 7.40-7.34 (m, 2 H,
H-5′,6′), 6.83 (d, J ) 15.4 Hz, 1 H, fumarate H), 4.24 (q, J )
To a stirred solution of 42 (0.85 g, 1.86 mmol) in THF (30
mL) under N2 at 0 °C was added BH3‚DMS (372 µL of a 10M
solution, 2 mol equiv) dropwise. The resulting solution was
allowed to warm to room temperature and then stirred for 2
h, before being quenched by the cautious addition of 1 N HCl
(40 mL). The reaction mixture was then stirred at 50 °C for 2
h, basified by the addition of saturated Na2CO3 and extracted
with EtOAc. The combined organic extracts were washed with
brine, dried over anhydrous Na2SO4, concentrated under
reduced pressure and chromatographed on silica gel eluting
with MeOH/CH2Cl2/EtOAc (3:8:8) to give N4-(3-bromophenyl)-
N6-[3-(4-morpholinyl)propyl]-4,6-quinazolinediamine (43) (130
mg, 16%) as a yellow glass (ca. 90% pure by NMR). This was
used without further purification: 1H NMR [(CD3)2SO] δ 9.40
(s, 1 H, NH), 8.37 (s, 1 H, H-2), 8.17 (t, J ) 1.9 Hz, 1 H, H-2′),
7.91 (br d, J ) 8.2 Hz, 1 H, H-6′), 7.54 (d, J ) 9.0 Hz, 1 H,
H-8), 7.34 (t, J ) 8.0 Hz, 1 H, H-5′), 7.27 (m, 2H, H-4′, 7), 7.16
(d, J ) 2.2 Hz, 1 H, H-5), 6.25 (t, J ) 5.1 Hz, 1 H, CH2NH),
3.59 (t, J ) 4.5 Hz, 4 H, morph. CH2), 3.22 (q, J ) 6.0 Hz, 1 H,
CH2NH), 2.45 (t, J ) 6.9 Hz, 2 H, CH2CH2CH2NH), 2.39 (br s,
4 H, morph. CH2), 1.82 (quintet, J ) 7.0 Hz, 2 H, CH2CH2-
CH2).
7.0 Hz, 2 H, CH2), 1.28 (t, J ) 7.0 Hz, 3 H, CH3). Anal. (C19H16
BrN5O3‚0.25H2O) C, H, N.
-
N-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-yl]-N-
[2-(d im et h yla m in o)et h yl]a cr yla m id e (21): E xa m p le of
Meth od of Sch em e 2. EDCI‚HCl (980 mg, 5 mmol) was added
to a stirred solution of N4-(3-bromophenyl)-N6-[2-(dimethyl-
amino)ethyl]pyrido[3,4-d]pyrimidine-4,6-diamine19 (40) (387
mg, 1 mmol) and redistilled acrylic acid (0.25 mL, 3.6 mmol)
in pyridine (5 mL) under N2 cooled to 0-5 °C. After 30 min
cooling was removed, and the solution was stirred for an
additional 45 min, then diluted with 1% aqueous NaHCO3.
The mixture was extracted with EtOAc (4×), and the combined
extracts were washed with brine, dried (MgSO4), and concen-
trated to give 21 (122 mg, 28%): mp (EtOAc, 5 °C) >160 °C
A stirred solution of 43 (133 mg, 0.30 mmol) in DMF (5.0
mL) under N2 was treated sequentially with acrylic acid (83
µL, 1.20 mmol), Et3N (excess, 0.5 mL), and EDCI‚HCl (115
mg, 0.6 mmol). Standard workup as above, followed by
chromatography on silica gel, eluting with EtOAc:CH2Cl2 (1:
1) to MeOH/CH2Cl2/EtOAc (3:7:10), gave 23 (39 mg, 26%): mp
(CH2Cl2/hexane) 171-175 °C; 1H NMR [(CD3)2SO] δ 9.86 (s, 1
H, NH), 8.70 (s, 1 H, H-2), 8.52 (d, J ) 2.0 Hz, 1 H, H-5), 8.20
(t, J ) 1.9 Hz, 1 H, H-2′), 7.91 (partially obscured br d, J )
8.6 Hz, 1 H, H-6′), 7.89 (d, J ) 8.9 Hz, 1 H, H-8), 7.79 (dd, J
) 8.8, 2.1 Hz, 1 H, H-7), 7.38 (t, J ) 7.9 Hz, 1 H, H-5′), 7.33
(dt, J ) 8.4, 1.7, 1.7 Hz, 1 H, H-4′), 6.22 (dd, J ) 16.7, 2.3 Hz,
1 H, CH2CHCO), 6.05 (br s, 1 H, CH2CHCO), 5.61 (br d, J )
8.8 Hz, 1 H, CH2CHCO), 3.87 (t, J ) 7.4 Hz, 2 H, CH2NRCO),
3.49 (t, J ) 4.5 Hz, 4 H, morph. CH2), 2.28 (partially obscured
t, J ) 7.1 Hz, 2 H, CH2CH2CH2NRCO), 2.27 (br s, 4 H, morph.
CH2), 1.69 (quintet, J ) 7.3 Hz, 2 H, CH2CH2CH2); HRMS
(DEI) (M+) calcd for C24H26Br81N5O2 497.1249, found 497.1250.
3-(Dim eth yla m in o)p r op yl (2E)-4-{[4-(3-Br om oa n ilin o)-
6-qu in a zolin yl]a m in o}-4-oxo-2-bu ten oa te (24): Exa m p le
of Meth od of Sch em e 4. A solution of 39 (158 mg, 0.5 mmol)
in THF (10 mL) was added dropwise over 15 min to a solution
of fumaryl chloride (382 mg, 2.5 mmol) in THF (10 mL) stirred
under N2 at 0 °C. After 1 h at 0 °C the suspension was allowed
to settle, and the supernatant of crude acid chloride 44 was
decanted. Fresh THF (5 mL) was added, and the suspension
was stirred at 0 °C while a solution of 3-(N,N-dimethylamino)-
propan-l-ol (1.18 mL, 10 mmol) in THF (5 mL) was added
dropwise. The suspension was stirred at 25 °C for 1 h, the
solvent was evaporated under reduced pressure, and the
residue was triturated with cold water. The solid was collected,
dissolved in a minimum volume of DMF, and absorbed onto
silica gel (2 g) and dried. Flash chromatography on silica gel,
eluting with CH2Cl2/MeOH (2:1) gave a product that was
dissolved in AcOH/water (3:2, 2.5 mL), passed through a 0.45-
µm filter, and purified by HPLC on a Vidac C18 218TP1022
reverse-phase HPLC column. Elution with 10% to 50% gradi-
ent of 0.1% TFA in water/0.l % TFA in CH3CN over 60 min
gave 24 as the tris trifluoroacetate salt (51 mg, 12%): mp 60
°C; 1H NMR [(CD3)2SO] δ 11.14 (s, 1 H, NH), 10.85 (br s, 1 H,
NH), 9.57 (br s, 1 H, NH), 9.01 (d, J ) 1.7 Hz, 1 H, H-5), 8.79
(s, 1 H, H-2), 8.07 (s, 1H, H-2′), 8.02 (dd, J ) 2.1, 9.0 Hz, 1H,
H-7), 7.89 (d, J ) 8.9 Hz, 1H, H-8), 7.78 (d, J ) 6.5 Hz, 1 H,
H-6′), 7.43 (m, 2 H, H-4′,5′), 7.34 (d, J ) 15.4 Hz, 1 H, butenyl
H-3), 6.84 (d, J ) 15.4 Hz, 1 H, butenyl H-2), 4.26 (t, J ) 6.2
Hz, 2 H, OCH2), 3.19 (m, 2 H, CH2N), 2.81 (d, J ) 4.6 Hz, 6 H,
CH3), 2.05 (m, 2 H, CH2); MS (APCI) 499.8 (100, 81BrMH+),
497.9 (97, 79BrMH+). Anal. (C23H24BrN5O3‚3CF3CO2H) C, H,
N.
1
dec; H NMR [(CD3)2SO] δ 10.16 (s, 1 H, NH), 9.15 (s, 1 H),
8.80 (s, 1 H), 8.43 (s, 1 H), 8.22 (s, 1 H), 7.93 (d, J ) 7.7 Hz, 1
H), 7.42-7.35 (m, 2 H), 6.29-6.22 (m, 2 H), 5.66 (dd, J ) 9.0,
3.5 Hz, 1 H), 4.05 (t, J ) 7.1 Hz, 2 H) 2.42 (t, J ) 7.1 Hz, 2 H),
2.11 (s, 6 H); MS (APCI) m/z (relative %) 440.9 (99), 441.8 (23),
442.8 (100), 443.9 (24). Anal. (C20H21BrN6O) C, H, N.
N-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-yl]-N-
[3-(4-m or p h olin yl)p r op yl]a cr yla m id e (22): Exa m p le of
Meth od of Sch em e 2. To a stirred solution of N4-(3-bro-
mophenyl)-N6-[3-(4-morpholinyl)propyl]pyrido[3,4-d]pyrimidine-
4,6-diamine19 (41) (400 mg, 0.90 mmol), DMAP (40 mg) and
Et3N (excess, 2.0 mL) at 0 °C under N2 was added acryloyl
chloride (89 µL, 1.08 mmol). After 1 h stirring a further two
portions of acid chloride (89 µL each) were added over the next
2 h and the procedure and workup above were followed to give,
after column chromatography on silica gel eluting with MeOH/
EtOAc (1:9) to MeOH/EtOAc (1:5), 22 (142 mg, 32%): mp (CH2-
Cl2/hexane) 178-180 °C; 1H NMR [(CD3)2SO] δ 10.15 (s, 1 H,
NH), 9.15 (s, 1 H), 8.80 (s, 1 H), 8.47 (s, 1 H), 8.21 (br s, 1 H,
H-2′), 7.92 (br d, J ) 7.6 Hz, 1 H, H-6′), 7.41 (t, J ) 8.0 Hz, 1
H, H-5′), 7.37 (dt, J ) 8.1, 1.6, 1.6 Hz, 1 H, H-4′), 6.25 (m, 2
H, CH2CHCO, CH2CHCO), 5.66 (m, 1 H, CH2CHCO), 3.98 (t,
J ) 7.5 Hz, 2 H, CH2NRCO), 3.46 (t, J ) 4.5 Hz, 4 H, morph.
CH2), 2.29 (t, J ) 7.1 Hz, 2 H, CH2CH2CH2NRCO), 2.24 (br s,
4 H, morph. CH2), 1.73 (quintet, J ) 7.2 Hz, 2 H, CH2CH2-
CH2). Anal. (C23H25BrN6O2‚H2O) C, H, N.
N-[4-(3-Br om oan ilin o)-6-qu in azolin yl]-N-[3-(4-m or ph oli-
n yl)p r op yl]a cr yla m id e (23): E xa m p le of Met h od of
Sch em e 3. A stirred solution of 414 (1.78 g, 4.82 mmol),
morpholine (excess, 4.0 mL) and p-toluenesulfonic acid (cata-
lytic) in THF (50 mL) was heated at 50 °C for 4 h before being
concentrated under reduced pressure, diluted with water and
extracted with EtOAc. The combined organic extracts were
washed with brine, dried over anhydrous Na2SO4, concentrated
under reduced pressure and chromatographed on silica gel
eluting with MeOH/CH2Cl2/EtOAc (15:40:45) to give N-[4-(3-
bromoanilino)-6-quinazolinyl]-3-(4-morpholinyl)propanamide
(42) (1.86 g, 78%): mp (EtOAc) 184-186 °C; 1H NMR [(CD3)2-
SO] δ 10.37 (s, 1 H, NH), 9.91 (s, 1 H, NH), 8.72 (d, J ) 1.9
Hz, 1 H, H-5), 8.58 (s, 1 H, H-2), 8.17 (t, J ) 2.1 Hz, 1 H,
H-2′), 7.86 (m, 2 H, H-7, 6′), 7.78 (d, J ) 8.9 Hz, 1 H, H-8),
7.35 (t, J ) 8.0 Hz, 1 H, H-5′), 7.29 (dt, J ) 1.2, 1.2, 8.0 Hz, 1
H, H-4′), 3.40 (t, J ) 4.6 Hz, 4 H, morph. CH2), 2.69 (t, J ) 6.6
Hz, 2 H, NCH2CH2CONH), 2.58 (t, J ) 6.6 Hz, 2 H, NCH2-
CH2CONH), 2.44 (br s, 4 H, morph. CH2). Anal. (C21H22BrN5O2)
C, H, N.
(2E)-N1-[4-(3-Br om oa n ilin o)-6-qu in a zolin yl]-N4-[3-(d i-
m eth yla m in o)p r op yl]-2-bu ten ed ia m id e (25). A solution of
39 (158 mg, 0.5 mmol) in THF (10 mL) was added dropwise

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 437
over 15 min to a solution of fumaryl chloride (382 mg, 2.5
mmol) in THF (10 mL) stirred under N2 at 0 °C. After 1 h at
0 °C, the suspension was allowed to settle, and the supernatant
was decanted. Fresh THF (5 mL) was added and the suspen-
sion was stirred at 0 °C while a solution of N1,N1-dimethyl-
1,3-propanediamine (1.26 mL, 10 mmol) in THF (5 mL) was
added dropwise. The suspension was stirred at 25 °C for 1 h,
the solvent was stripped under reduced pressure, and the
residue was triturated with cold water. The solid was collected,
dissolved in boiling MeOH (25 mL), filtered, and evaporated
under reduced pressure. The residue was dissolved in AcOH/
water (3:2, 2.5 mL) and purified by HPLC on a Vidac C18
218TP1022 reverse-phase HPLC column. Elution with a 10%
to 50% gradient of 0.1% TFA in water/0.l% TFA in CH3CN
over 60 min gave 25 as the tris trifluoroacetate salt (154 mg,
37%): mp 40 °C; 1H NMR [(CD3)2SO] δ 11.02 (s, 1 H, NH),
9.50 (br s, 1 H, NH) 9.02 (d, J ) 1.7 Hz, 1 H, H-5), 8.82 (s, 1
H, H-2), 8.74 (t, J ) 5.7 Hz, 1 H, NH), 8.05 (s, 1 H, H-2′), 8.02
(dd, J ) 2.1, 9.0 Hz, 1 H, H-7), 7.89 (d, J ) 8.9 Hz, 1 H, H-8),
7.76 (d, J ) 7.2 Hz, H-6′), 7.45 (m, 2 H, H-4′,5′), 7.17 (d, J )
14.9 Hz, 1 H, butenyl H-3), 7.05 (d, J ) 15.2 Hz, 1 H, butenyl
H-2), 3.03 (m, 2 H, NCH2), 3.08 (m, 2 H, CH2N), 2.79 (d, J )
4.8 Hz, 6 H, CH3), 1.83 (m, 2 H, CH2); MS (APCI) 498.8 (100,
81BrMH+), 496.9 (97, 79BrMH+). Anal. (C23H25BrN6O2‚3CF3-
CO2H) C, H, N.
(2E)-N1-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-
yl]-N4-[3-(4-m or p h olin yl)p r op yl]-2-b u t en ed ia m id e (28):
Exa m p le of Meth od of Sch em e 5.20 A solution of (E)-but-
2-enedioic acid monoethyl ester (45) (5.77 g 40.0 mmol) in THF
(40 mL) was treated with oxalyl chloride (7.0 mL, 80.0 mmol),
followed by two drops of DMF. The solution was stirred at room
temperature for 1 h, then concentrated. The resulting residue
was dissolved in CH2Cl2 (50 mL) and added dropwise during
20 min to a solution of 3-morpholin-4-ylpropylamine (6.43 mL,
44.0 mmol) in CH2Cl2 (200 mL) in a dry ice/acetone bath. At
the end of the addition the reaction was allowed to come to
room temperature, then poured into of 5% aqueous NaHCO3
(500 mL). The layers were separated, and the aqueous layer
was extracted with CH2Cl2, and the combined organic extracts
were dried (MgSO4) and concentrated. Flash chromatography
over silica gel, eluting with MeOH/CH2Cl2 (1:5) gave ethyl (2E)-
4-{[3-(4-morpholinyl)propyl]amino}-4-oxo-2-butenoate (46a) (8.44
g, 78%) as an oil: 1H NMR [(CD3)2SO] δ 8.53 (t, J ) 5.5 Hz, 1
H, NH), 7.76 (d, J ) 15.4 Hz, 1 H, olefinic), 6.55 (d, J ) 15.4
Hz, 1 H, olefinic), 4.20 (q, 2 H, J ) 7.0 Hz, CH2CH3), 3.56 (t,
J ) 4.7 Hz, 4 H, morph. CH2), 3.18 (q, 2 H, CH2NH, coalesces
to t on D2O wash), 2.32 (br s, 4 H, morph. CH2), 2.28 (t, J )
7.2 Hz, 2 H, morpholino-CH2CH2CH2), 1.59 (quintet, J ) 7.0
Hz, 2 H, CH2CH2CH2), 1.24 (t, J ) 7.1 Hz, 3 H, CH3); MS
(APCI) M + 1 calcd 271.2, found 271.1.
A solution of 46a (0.50 g, 1.8 mmol) and Et3N (0.50 mL, 3.6
mmol) in water (10 mL) was stirred at room temperature
overnight, concentrated, and coevaporated with EtOH to give
crude (2E)-4-{[3-(4-morpholinyl)propyl]amino}-4-oxo-2-buteno-
ic acid (47a ) as a gum (containing Et3N): 1H NMR [(CD3)2SO]
δ 8.41 (t, J ) 5.3 Hz, 1 H, NH), 6.81 (d, J ) 15.7 Hz, 1 H,
olefinic), 6.49 (d, J ) 15.4 Hz, 1 H, olefinic), 3.56 (t, J ) 4.6
Hz, 4 H, morph. CH2), 3.16 (dd, J ) 12.8, 7.0 Hz, 2 H, CH2-
NH, coalesces to t on D2O wash), 2.32 (br s, 4 H, morph. CH2),
2.27 (t, J ) 7.1 Hz, 2 H, morpholino-CH2CH2CH2), 1.59
(quintet, J ) 7.0 Hz, 2 H, CH2CH2CH2); MS (APCI) M + 1
calcd 243.1, found 243.2.
A mixture of 47a (assumed 1.8 mmol) and amine 38 (0.10
g, 0.32 mmol) in pyridine (2 mL) was treated with EDCI‚HCl
(0.34 g, 1.8 mmol), and the solution was stirred at room
temperature overnight. The reaction was then poured into
water, and the resulting solid was purified by flash chroma-
tography over silica gel, eluting with MeOH/CH2Cl2 (1:4) to
give 28 (54 mg, 30.0%): mp 237-240 °C dec; 1H NMR [(CD3)2-
SO] δ 11.3 (s, 1 H, NH), 10.4 (s, 1 H, NH), 9.05 (s, 1 H), 9.02
(s, 1 H), 8.67 (s, 1 H), 8.58 (t, J ) 5.5 Hz, 1 H, CH2NH), 8.17
(d, J ) 1.7 Hz, 1 H), 7.89 (d, J ) 6.5 Hz, 1 H), 7.36 (m, 2 H),
7.27 (d, J ) 15.2 Hz, 1 H, olefinic), 7.08 (d, J ) 15.2 Hz, 1 H,
olefinic), 3.57 (t, J ) 4.6 Hz, 4 H, morph. CH2), 3.21 (dd, J )
12.7, 6.6 Hz, 2 H, CH2NH, coalesces to t with D2O), 2.34 (br s,
4 H, morph. CH2), 2.31 (t, J ) 7.2 Hz, 2 H, morpholino-CH2-
CH2CH2), 1.63 (quintet, J ) 7 Hz, 2 H, CH2CH2CH2); MS
(APCI) M + 1 calcd 540.1, found 540.2. Anal. (C24H26BrN7O3‚
1.5H2O) C, H, N.
(2E)-N1-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-
yl]-N4-[3-(d im eth yla m in o)p r op yl]-2-bu ten ed ia m id e (26).
Similar reaction of 45 (5.75 g, 40 mmol), oxalyl chloride (7 mL)
and DMF (3 drops) gave the crude acid chloride. This was
diluted with anhydrous Et2O (200 mL) and the solution was
cooled to -78 °C with mechanical stirring. The rapidly stirring
solution was then treated dropwise with a solution of 3-di-
methylaminopropylamine (5.54 mL, 44 mmol) in Et2O (60 mL).
After addition was completed, the bath was removed and the
thick suspension was allowed to slowly warm to room tem-
perature over 1-2 h. The solid was collected and washed well
with ether. It was then dissolved in water (orange solution),
and the pH was adjusted to ca. 10.5 with sodium carbonate.
The aqueous phase was extracted with EtOAc (4×), then the
combined extracts were washed with brine, dried, and filtered
through a pad of flash silica gel. The filtrate was concentrated
to leave crude ethyl (2E)-4-{[3-(dimethylamino)propyl]amino}-
4-oxo-2-butenoate (46b) (2.75 g, 30%; low yield was likely due
to incomplete extraction due to water solubility), sufficiently
pure by NMR to use in the next reaction: 1H NMR (CDCl3) δ
7.94 (br s, 1 H, NH), 6.80 (d, J ) 15.6 Hz, 1 H, olefinic), 6.73
(d, J ) 15.6 Hz, 1 H, olefinic), 4.20 (q, J ) 7.1 Hz, 2 H, CH2-
CH3), 3.42 (q, J ) 5.8 Hz, 2 H, CH2NH), 2.43 (t, J ) 5.8 Hz, 2
H, Me2NCH2CH2CH2), 2.24 (s, 6 H, N(CH3)2), 1.68 (quintet, J
) 5.8 Hz, 2 H, CH2CH2CH2), 1.27 (t, J ) 7.1 Hz, 3 H, CH3).
A solution of 46b (2.43 g, 10.6 mmol) in deionized water
(40 mL) was heated at reflux for 4 h. The water was stripped
off and the residue was coevaporated with MeOH. The oil was
dissolved in ca. 20 mL of MeOH, then treated with excess
2-propanolic HCl. The resultant solution was concentrated, the
resulting solid was triturated in EtOH, washed with EtOH
and dried to give (2E)-4-{[3-(dimethylamino)propyl]amino}-4-
oxo-2-butenoic acid (47b) as the HCl salt (1.13 g): mp 148-
1
152 °C; H NMR [(CD3)2SO] δ 8.77 (t, J ) 5.8 Hz, 1 H, NH),
6.92 (d, J ) 15.7 Hz, 1 H, olefinic), 6.53 (d, J ) 15.7 Hz, 1 H,
olefinic), 3.23 (q, J ) 6.5 Hz, 2 H, CH2NH, coalesces to t on
D2O wash), 3.03 (m, 2 H, Me2NCH2CH2CH2), 2.72 (s, 6 H,
N(CH3)2), 1.84 (quintet, J ) 7.0 Hz, 2 H, CH2CH2CH2). Anal.
(C9H16N2O3‚HCl) C, H, N. Concentration of the filtrate gave a
second crop (573 mg; total yield 1.7 g (68%)).
A suspension of 38 (64 mg, 0.2 mmol) and 47b HCl salt (190
mg, 0.8 mmol) in pyridine (1 mL) was mixed by warming, then
cooled in an ice bath and treated with EDCI‚HCl (196 mg).
The resulting suspension was stirred under nitrogen at 0-5
°C for 2.5 h, then diluted with DMF (2 mL). The solution was
added to a 1:1 mixture of water/EtOAc, and shaken until phase
separation. The aqueous phase was further extracted with
EtOAc (3×) and the combined extracts were discarded. The
aqueous layer was made basic with 5% aqueous NaHCO3, and
then extracted with EtOAc (5×) using small amounts of MeOH
each time if necessary to bring about phase separation. The
EtOAc extracts were combined and washed with brine, dried
(MgSO4), and concentrated to leave a residue. This was
sonicated in 2-propanol to give 26 (67 mg, 66%): mp 235-38
°C; 1H NMR [(CD3)2SO] δ 11.3 (s, 1 H, NH), 10.4 (s, 1 H, NH),
9.05 (s, 1 H), 9.02 (s, 1 H), 8.67 (s, 1 H), 8.57 (t, J ) 5.5 Hz, 1
H, CH2NH), 8.17 (br s, 1 H), 7.88 (d, J ) 7.5 Hz, 1 H), 7.36
(m, 2 H), 7.27 (d, J ) 15.2 Hz, 1 H, olefinic), 7.07 (d, J ) 15.2
Hz, 1 H, olefinic), 3.20 (q, J ) 7.0 Hz, 2 H, CH2NH, coalesces
to t with D2O), 2.23 (t, J ) 7.0 Hz, 2 H, Me2NCH2CH2CH2),
2.13 (s, 6 H, N(CH3)2), 1.59 (quintet, J ) 7.0 Hz, 2 H, CH2CH2-
CH2). Anal. (C22H24BrN7O2‚0.5H2O) C, H, N.
(2E)-N1-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-
yl]-N4-[3-(d iet h yla m in o)p r op yl]-2-b u t en ed ia m id e (27).
Similar reaction of the acid chloride of 45 (from 5.75 g of 45)
with 3-diethylaminopropylamine (7.0 mL, 44.4 mmol) in CH2-
Cl2 (200 mL) gave ethyl (2E)-4-{[3-(diethylamino)propyl]amino}-
4-oxo-2-butenoate (46c) (4.07 g, 37%) as an oil: 1H NMR
(CDCl3) δ 8.47 (br s, 1 H, NH), 6.84 (d, J ) 15.4 Hz, 1 H,

438 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Smaill et al.
olefinic), 6.77 (d, J ) 15.4 Hz, 1 H, olefinic), 4.24 (q, J ) 7.0
Hz, 2 H, OCH2CH3), 3.48 (q, J ) 5.5 Hz, 2 H, CH2NH), 2.63
(m, 6 H, Et2NCH2CH2CH2, N(CH2CH3)2), 1.75 (quintet, J )
5.5 Hz, 2 H, CH2CH2CH2), 1.31 (t, J ) 7.0 Hz, 3 H, OCH2CH3),
1.10 (t, J ) 7.1 Hz, 6 H, N(CH2CH3)2). Anal. (C13H24N2O3‚H2O)
C, H, N.
(C10H13N3O3) C, H, N. Further processing of the filtrate
afforded 420 mg (37%) of additional product (total yield 610
mg (54%)).
Reaction of 47d (156 mg, 0.7 mmol) and 38 (64 mg, 0.2
mmol) with EDCI‚HCL (196 mg) in pyridine (1 mL) was
carried out as described above. After 2 h the dark orange
solution was diluted with DMF (2 mL) and poured into 2.5%
aqueous NaHCO3. The mixture was extracted with EtOAc
(5×), and the combined EtOAc extracts were washed with
brine, dried (MgSO4), and concentrated to a residue that was
triturated in hot 2-propanol. After storage at room temperature
for 30 min, the solid was collected to give 29 (13.5 mg, 13%):
mp 244-248 °C; 1H NMR [(CD3)2SO] δ 11.3 (s, 1 H, NH), 10.4
(s, 1 H, NH), 9.06 (s, 1 H), 9.02 (s, 1 H), 8.67 (s, 1 H), 8.64 (t,
J ) 5.8 Hz, 1 H, NH), 8.17 (d, J ) 1.7 Hz, 1 H), 7.88 (dt, J )
7.5, 1.9, 1.9 Hz, 1 H), 7.65 (s, 1 H, imidazole H), 7.37 (m, 2 H),
7.29 (d, J ) 14.9 Hz, 1 H, olefinic), 7.20 (t, J ) 1.2 Hz, 1 H,
imidazole H), 7.08 (d, J ) 14.9 Hz, 1 H, olefinic), 6.90 (t, J )
1.2 Hz, 1 H, imidazole H), 4.01 (t, J ) 7.0 Hz, 2 H, CH2N),
3.14 (q, J ) 6.8 Hz, 2 H, CH2NH, coalesces to t with D2O),
A solution of 46c (4.05 g, 14.86 mmol) and LiOH monohy-
drate (1.27 g, 30.1 mmol) in MeOH:H2O (100 mL) was stirred
at room temperature for 2 h. The mixture was evaporated to
a semisolid, then diluted with warm MeOH and filtered
through a pad of flash silica gel, washing well with MeOH.
The eluate was concentrated to a solid that was triturated in
Et2O to give (2E)-4-{[3-(dimethylamino)propyl]amino}-4-oxo-
2-butenoic acid (47c) as the lithium salt (3 g). This salt (2.55
g, 10 mmol) was dissolved in water and the pH was adjusted
to 4 with dilute aqueous HCl. This solution was loaded onto a
column packed with 10 g dry weight of Dowex 50W x 8 resin
(H+ form; 200-400 mesh; 5.2 mol equiv/g), and the column
was eluted successively with 50 mL each of water, 0.1 M
aqueous NH3, 0.2 M aqueous NH3, and 100 mL 0.3 M aqueous
NH3. Product fractions were pooled and concentrated to ca.
100 mL of solution that was then lyophilized and further dried
at 5 mm/60 °C/5 h to provide the free acid (1.82 g) as a glassy
solid that was used directly in the next reaction: 1H NMR
[(CD3)2SO] δ 8.46 (t, J ) 5.3 Hz, 1 H, NH), 6.74 (d, J ) 15.4
Hz, 1 H, olefinic), 6.48 (d, J ) 15.4 Hz, 1 H, olefinic), 3.16 (q,
J ) 5.8 Hz, 2 H, CH2NH, coalesces to t on D2O wash), 2.69
(m, 6 H, Et2NCH2CH2CH2, N(CH2CH3)2), 1.66 (quintet, J )
7.0 Hz, 2 H, CH2CH2CH2), 1.04 (t, J ) 7.2 Hz, 6 H,
N(CH2CH3)2).
1.91 (quintet, J ) 7.0 Hz, 2 H, CH2CH2CH2). Anal. (C23H21
BrN8O2‚0.75H2O) C, H, N.
-
(2E)-N1-[4-(3-Ch lor o-4-flu or oa n ilin o)p yr id o[3,4-d ]p yr i-
m id in -6-yl]-N 4-[3-(4-m or p h olin yl)p r op yl]-2-b u t e n e d i-
a m id e (31). 6-Amino-4-(3-chloro-4-fluoroanilino)pyrido[3,4-d]-
pyrimidine14 (48) (1.3 g, 4.5 mmol) and 47a (4.36 g, 18 mmol)
in dry pyridine (22.5 mL) were dissolved by gentle heating,
then the solution was cooled in an ice bath under N2 and
treated with pulverized EDCI‚HCl (4.31 g, 22.5 mmol). The
reaction was stirred at 0-5 °C for 4 h, then poured onto a
mixture of EtOAc/5% aqueous NaHCO3. The precipitated solid
was collected, washed with water, then dissolved in hot MeOH.
The solution was treated with limited conc HCl and set aside
to crystallize, giving 31 (1.34 g, 51%) as a partial HCl salt:
mp 232-235 °C; 1H NMR [(CD3)2SO] δ 11.3 (s, 1 H, NH), 10.5
(br s, ∼1 H, R2N+H), 10.4 (s, 1 H, NH), 9.06 (s, 1 H), 9.01 (s,
1 H), 8.79 (br s, 1 H, CH2NH), 8.66 (s, 1 H), 8.14 (dd, J ) 2.7
Hz, J H-F ) 7.0 Hz, 1 H), 7.83 (m, 1 H), 7.48 (dd, J ) 9.0 Hz,
J H-F ) 9.0 Hz, 1 H), 7.30 (d, J ) 15.2 Hz, 1 H, olefinic), 7.20
(d, J ) 15.2 Hz, 1 H, olefinic), 3.8 (br s, 4 H, morph. CH2),
3.25 (q, J ) 6.6 Hz, 2 H, CH2NH, coalesces to t with D2O), 3.0
(br s, 6 H, morph. CH2, NCH2CH2CH2), 1.85 (br s, 2 H,
CH2CH2CH2). Anal. (C24H25ClFN7O3‚HCl‚2H2O) C, H, N.
Reaction of 38 (32 mg, 1 mmol), 47c (97 mg, 0.4 mmol),
EDCI‚HCl (98 mg) and pyridine (0.5 mL) was carried out as
described above. After 1.25 h further EDCI‚HCl (45 mg) was
added, and the mixture was stirred at room temperature for
an additional 17 h. Workup as above, followed by trituration
1
in 2-propanol, gave 27 (10.5 mg, 20%): mp 227-232 °C; H
NMR [(CD3)2SO] δ 11.3 (s, 1 H, NH), 10.4 (s, 1 H, NH), 9.05
(s, 1 H), 9.02 (s, 1 H), 8.67 (s, 1 H), 8.56 (t, J ) 5.5 Hz, 1 H,
CH2NH), 8.17 (br s, 1 H), 7.88 (d, J ) 7.5 Hz, 1 H), 7.37 (m, 2
H), 7.27 (d, J ) 15.2 Hz, 1 H, olefinic), 7.08 (d, J ) 15.2 Hz, 1
H, olefinic), 3.20 (q, J ) 6.0 Hz, 2 H, CH2NH, coalesces to t
with D2O), 2.44 (m, 6 H, Et2NCH2CH2CH2, N(CH2CH3)2), 1.57
(quintet, J ) 7.0 Hz, 2 H, CH2CH2CH2), 0.94 (t, J ) 7.2 Hz, 6
H, N(CH2CH3)2). Anal. (C24H28BrN7O2) C, H.
(2E)-N1-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-
yl]-N4-[3-(d im eth yla m in o)p r op yl]-N1-m eth yl-2-bu ten ed i-
a m id e (30). Reaction of 37 (132 mg, 0.4 mmol) and 47b (380
mg, 1.6 mmol) with EDCI‚HCl (392 mg) in pyridine (2 mL) as
above gave a crude product that was extracted with EtOAc
(2×) to remove organic impurities. The combined EtOAc
extracts were washed with water, then all aqueous phases
were combined and adjusted to pH 9 with 2% aqueous NaOH.
The resulting precipitate was collected, washed well with
water, and then triturated in 2-propanol at 0 °C for 30 min to
give 30 (64 mg, 31%): mp 228-230 °C; 1H NMR [(CD3)2SO] δ
10.1 (s, 1 H, NH), 9.15 (s, 1 H), 8.81 (s, 1 H), 8.47 (s, 1 H), 8.43
(t, J ) 5.5 Hz, 1 H, CH2NH), 8.20 (s, 1 H), 7.90 (d, J ) 7.2 Hz,
1 H), 7.39 (m, 2 H), 6.95 (d, J ) 14.9 Hz, 1 H, olefinic), 6.72
(d, J ) 14.9 Hz, 1 H, olefinic), 3.47 (s, 3 H, CONCH3), 3.09 (q,
J ) 6.0 Hz, 2 H, CH2NH, coalesces to t with D2O), 2.15 (t, J )
7.2 Hz, 2 H, Me2NCH2CH2CH2), 2.06 (s, 6 H, N(CH3)2), 1.50
(quintet, J ) 7.2 Hz, 2 H, CH2CH2CH2). Anal. (C23H26BrN7O2‚
0.25H2O) C, H, N.
(2E)-N1-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-
yl]-N4-[3-(1H-im id a zol-1-yl)p r op yl]-2-bu ten ed ia m id e (29).
Similar reaction of the acid chloride of 45 (from 5.77 g of 45)
with a solution of 3-imidazol-1-ylpropylamine (5.25 mL, 44.0
mmol) in CH2Cl2 (200 mL) in a dry ice/acetone bath, followed
by flash chromatography of the residue on silica gel, eluting
with MeOH/CH2Cl2 (1:2), gave ethyl (2E)-4-{[3-(imidazol-1-yl)-
propyl]amino}-4-oxo-2-butenoate (46d ) (5.21 g, 52%), which
solidified on standing: 1H NMR [(CD3)2SO] δ 8.60 (t, J ) 5.4
Hz, 1 H, NH), 7.62 (s, 1 H, imidazole H), 7.18 (t, J ) 1.2 Hz,
1 H, imidazole H), 6.99 (d, J ) 15.7 Hz, 1 H, olefinic), 6.89 (s,
1 H, imidazole H), 6.57 (d, J ) 15.7 Hz, 1 H, olefinic), 4.19 (q,
J ) 7.1 Hz, 2 H, CH2CH3), 3.98 (t, J ) 7.0 Hz, 2 H, CH2N),
3.11 (q, J ) 6.8 Hz, 2 H, CH2NH, coalesces to t with D2O),
1.88 (quintet, J ) 6.9 Hz, 2 H, CH2CH2CH2), 1.24 (t, J ) 7.0
Hz, 3 H, CH3); MS (APCI) M + 1 calcd 252.1, found 252.1.
Anal. (C12H17N3O3) C, H, N.
A solution of 46d (1.27 g, 5.05 mmol) and Et3N (1.42 mL,
10.2 mmol) in deionized water (25.5 mL) was heated at 60 °C
for 30 min. The water was evaporated and residue was
coevaporated with MeOH. The residue was crystallized from
MeOH/2-propanol (1:1) to give (2E)-4-{[3-(imidazol-1-yl)propyl]-
amino}-4-oxo-2-butenoic acid (47d ) (190 mg, 17%): mp 170-
N-[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-yl]eth -
ylen esu lfon a m id e (32): Exa m p le of Meth od of Sch em e
1. To a stirred solution of 38 (0.25 g, 0.82 mmol) in THF (20
mL) under nitrogen was added Et3N (230 µL), a catalytic
amount of DMAP and chloroethanesulfonyl chloride (120 µL,
1.15 mmol) dropwise. The reaction was stirred at room
temperature for 1 h and then diluted with saturated NaHCO3
and extracted with EtOAc. The combined organic extracts were
washed with brine, dried over anhydrous Na2SO4, concentrated
under reduced pressure and chromatographed on silica gel
eluting with MeOH/CH2Cl2/EtOAc (2:48:50), then recrystalized
from CH2Cl2/hexane to give 32 (53 mg, 16%): mp 261-265
1
174 °C; H NMR [(CD3)2SO] δ 8.55 (t, J ) 5.5 Hz, 1 H, NH),
7.64 (s, 1 H, imidazole H), 7.19 (t, J ) 1.2 Hz, 1 H, imidazole
H), 6.90 (d, J ) 15.4 Hz, 1 H, olefinic), 6.89 (s, 1 H, imidazole
H), 6.52 (d, J ) 15.4 Hz, 1 H, olefinic), 3.98 (t, J ) 7.0 Hz, 2
H, CH2N), 3.11 (q, J ) 6.8 Hz, 2 H, CH2NH, coalesces to t
with D2O), 1.88 (quintet, J ) 6.8 Hz, 2 H, CH2CH2CH2). Anal.

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3 439
1
°C; H NMR [(CD3)2SO] δ 11.02 (s, 1 H, SO2NH), 10.25 (s, 1
[(CD3)2SO] δ 10.64 (s, 1 H, NH), 9.30 (s, 1 H), 9.25 (s, 1 H),
8.87 (s, 1 H), 8.16 (s, 1 H), 7.89-7.85 (m, 1 H), 7.39-7.33 (m,
2 H), 7.17 (dd, J ) 10.0, 16.5 Hz, 1 H), 6.46 (d, J ) 16.4 Hz,
1 H), 6.37 (d, J ) 10.0 Hz, 1 H). Anal. (C15H11BrN4O2S‚
0.25H2O) C, H, N.
H, NH), 9.02 (s, 1 H), 8.67 (s, 1 H), 8.15 (br s, 1 H, H-2′), 8.00
(s, 1 H), 7.87 (dt, J ) 7.2, 1.9, 1.9 Hz, 1 H, H-6′), 7.40 (partially
obsc. t, J ) 7.9 Hz, 1 H, H-5′), 7.37 (partially obsc. dt, J ) 7.8,
1.9, 1.9 Hz, 1 H, H-4′), 7.07 (dd, J ) 16.5, 9.9 Hz, 1 H,
CH2CHSO2), 6.30 (d, J ) 16.5 Hz, 1 H, CH2CHSO2), 6.09 (d, J
) 9.9 Hz, 1 H, CH2CHSO2). Anal. (C15H12BrN5O2S‚0.25H2O)
C, H, N.
N-[4-(3-Br om oa n ilin o)-6-qu in a zolin yl]eth ylen esu lfon -
a m id e (33). To a stirred solution of 39 (0.30 g, 0.95 mmol) in
THF (20 mL) under nitrogen were added Et3N (3.5 mol equiv,
3.33 mmol, 245 µL), a catalytic amount of DMAP and chloro-
ethanesulfonyl chloride (1.2 mol equiv, 1.14 mmol, 119 µL)
dropwise. Workup as above followed by chromatography on
silica gel eluting with MeOH/CH2Cl2/EtOAc (3:47:50) and
crystallization from CH2Cl2/hexane gave 33 (210 mg, 54%): mp
217 °C dec; 1H NMR [(CD3)2SO] δ 10.31 (s, 1 H, SO2NH), 9.96
(s, 1 H, NH), 8.60 (s, 1 H, H-2), 8.20 (d, J ) 2.0 Hz, 1 H, H-5),
8.14 (br s, 1 H, H-2′), 7.85 (br d, J ) 7.9 Hz, 1 H, H-6′), 7.81
(d, J ) 8.9 Hz, 1 H, H-8), 7.67 (dd, J ) 8.9, 2.1 Hz, 1 H, H-7),
7.37 (t, J ) 8.0 Hz, 1 H, H-5′), 7.32 (br d, J ) 8.1 Hz, 1 H,
H-4′), 6.90 (dd, J ) 16.4, 9.8 Hz, 1 H, CH2CHSO2), 6.17 (d, J
) 16.4 Hz, 1 H, CH2CHSO2), 6.06 (d, J ) 9.8 Hz, 1 H, CH2-
CHSO2). Anal. (C16H13BrN4O2S) C, H, N.
2-{[4-(3-Br om oa n ilin o)p yr id o[3,4-d ]p yr im id in -6-yl]-
su lfon yl}eth a n ol (36): Exa m p le of Meth od of Sch em e 6.
A nitrogen-purged solution of 2-mercaptoethanol (1.75 mL, 25
mmol) and 4-(3-bromoanilino)-6-fluoropyrido[3,4-d]pyrimi-
dine17 (49) (1.6 g, 5 mmol) in DMSO (10 mL) was treated with
anhydrous cesium carbonate (3.26 g, 10 mmol). The stirred
solution was heated at 50 °C for 2 h, then poured into 2%
aqueous HCl (180 mL). After stirring for 15 min, the solids
were collected, washed well with water, and dissolved in DMF.
The solution was poured into EtOAc/water (1:1) the resulting
mixture was extracted with EtOAc (3×). The combined ex-
tracts were washed with brine, dried (MgSO4) and filtered
through a column of silica gel. The filtrate was concentrated
to a solid that was triturated in EtOAc to give 2-{[4-(3-
bromoanilino)pyrido[3,4-d]pyrimidin-6-yl]sulfanyl}ethanol (50)
N-(3-Br om op h en yl)-6-(vin ylsu lfin yl)p yr id o[3,4-d ]p yr i-
m id in -4-a m in e (36): Exa m p le of Meth od of Sch em e 6. A
suspension of 50 (226 mg, 0.6 mmol) and 3-phenyl-2-(phenyl-
sulfonyl)oxaziridine (180 mg) in CHCl3 (6 mL) was stirred at
room temperature for 3 h. The mixture was diluted with tert-
butyl methyl ether (6 mL) and the precipitate was collected
to give 2-[4-(3-bromophenylamino)pyrido[3,4-d]pyrimidine-6-
sulfinyl]ethanol (51) (210 mg, 89%): mp 233-235 °C; 1H NMR
[(CD3)2SO] δ 10.6 (s, 1 H, NH), 9.25 (s, 1 H), 9.04 (s, 1 H), 8.86
(s, 1 H), 8.23 (d, J ) 1.9 Hz, 1 H), 7.95 (dt, J ) 2.2, 2.2, 7.2
Hz, 1 H), 7.39 (m, 2 H), 5.09 (dd, J ) 5.1, 5.8 Hz, 1 H, OH),
3.87 (m, 1 H, HOCH2CH2), 3.80 (m, 1 H, HOCH2CH2), 3.32
(m, 1 H, HOCH2CH2), 2.98 (dt, J ) 4.3, 4.3, 13.3 Hz, 1 H,
HOCH2CH2), 1.91 (dd, J ) 7.0, 1.4 Hz, 3 H). Anal. (C15H13
BrN4O2S) C, H, N.
-
Methanesulfonyl chloride (0.056 mL, 0.72 mmol) was added
to an ice-cold suspension of 51 (117 mg, 0.3 mmol) and Hunig’s
base (0.3 mL, 1.7 mmol) in dichloroethane (3 mL). After 1 h,
the mixture was brought to room temperature and maintained
there for 30 min. DBU (316 mg, 2 mmol) was added, and the
solution stirred for 30 min, then quenched with water. The
mixture was extracted with dichloroethane (2×) and the
combined extracts were washed with 2% aqueous HCl, dried
(Mg2SO4), and filtered through a pad of flash silica gel, eluting
with EtOAc. Concentration of the combined product eluates
and trituration of the resulting solid with MeOH gave 36 (69
1
mg, 61%): mp 213-214 °C; H NMR [(CD3)2SO] δ 10.61 (s, 1
H, NH), 9.25 (s, 1 H), 9.00 (s, 1 H), 8.86 (s, 1 H), 8.22 (s, 1 H),
7.93 (d, J ) 7.0 Hz, 1 H), 7.38 (m, 2 H), 7.12 (dd, J ) 9.6, 16.4
Hz, 1 H), 6.16 (d, J ) 16.4 Hz, 1 H), 6.06 (d, J ) 9.6 Hz, 1 H).
Anal. (C15H11BrN4OS) C, H, N.
Tyr osin e Kin a se Assa ys. EGFR tyrosine kinase was
purified as described previously.21 Enzyme assays for IC50[app]
determinations were performed in 96-well filter plates (Mil-
lipore 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 µM adenosine
triphosphate (ATP) containing 0.5 mCi of [32P]ATP, 20 mg of
polyglutamic acid/tyrosine (Sigma Chemical Co., St. Louis,
MO), 10 ng of EGFR tyrosine kinase and appropriate dilutions
of inhibitor. All components except the ATP are 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 incubated at 25 °C for 10 min. The reaction was
terminated 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 was then washed 5
times with 0.2 mL of 10% TCA and 32P incorporation 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 mM of designated
compound to be tested as an irreversible inhibitor for 2 h. One
set of cells was then stimulated with 100 ng/mL of EGF for 5
min and extracts made as described under the Western
blotting procedure. The other set of cells was washed free of
the compound with warmed serum-free media, incubated 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 made similar to the first
set of cells.
1
(1.24 g, 66%): mp 182-185 °C; H NMR [(CD3)2SO] δ 10.03
(s, 1 H, NH), 9.10 (s, 1 H), 8.69 (s, 1 H), 8.35 (s, 1 H), 8.22 (t,
J ) 1.9 Hz, 1 H), 7.91 (dt, J ) 7.7, 1.9 Hz, 1 H), 7.42-7.34 (m,
2 H), 5.04 (t, J ) 5.5 Hz, 1 H, OH), 3.68 (dd, J ) 6.8, 5.7 Hz,
2 H), 3.36 (t, J ) 6.8 Hz, 2 H); MS (APCI) m/z (relative %)
374.8 (49), 375.8 (10), 376.9 (100), 377.8 (23), 378.9 (63), 379.8
(14). Anal. (C15H13BrN4OS) C, H, N.
A stirred suspension 50 (755 mg, 2 mmol) in CHCl3 (30 mL)
at 0-5 °C was treated with MCPBA (1.27 g of 57-86%) and
slowly warmed to 25 °C over 4 h. After 14.5 and 17.5 h further
MCPBA (720 mg and 720 mg) was added. After 19.5 h total
reaction time, the suspension was cooled to 0-5 °C, treated
with DMSO (2 mL) and allowed to warm to 20 °C for 30 min.
The mixture was then partitioned between EtOAc and 5%
aqueous NaHCO3. The organic phase was washed with brine,
dried (MgSO4), concentrated to small volume and flash chro-
matographed on silica gel, eluting with EtOAc to give 34 (540
mg, 66%): mp (EtOAc) 210-212 °C; 1H NMR (CF3CO2H) δ
10.96 (s, 1 H), 10.90 (s, 1 H),) 10.42 (s, 1 H), 9.47 (s, 1H), 9.16
(d, J ) 8.2 Hz, 1 H), 9.05 (d, J ) 8.2 Hz, 1 H), 8.83 (t, J ) 8.0
Hz, 1 H), 5.81(t, J ) 5.2 Hz, 2 H), 5.43 (t, J ) 5.2 Hz, 2 H);
MS (APCI) m/z (relative %) 378.7 (39), 380.7 (45), 408.7 (100),
409.7 (15), 410.7 (97), 411.7 (17). Anal. (C15H13BrN4O3S) C,
H, N.
N-(3-Br om op h en yl)-6-(vin ylsu lfon yl)p yr id o[3,4-d ]p y-
r im id in -4-a m in e (35). Methanesulfonyl chloride (9.3 µL, 0.12
mmol) was added dropwise to a stirred suspension of 34 (41
mg, 0.1 mmol) and Et3N (31 µL, 0.22 mmol) in CH2Cl2 (0.5
mL) under N2 at 0-5 °C. Additional charges of methanesulfo-
nyl chloride (9.3 µL) were added after 45 min and 1.5 h, the
latter with additional Et3N (50 µL). After a total of 2.5 h the
cold solution was quenched with 5% aqueous NaHCO3, then
extracted with EtOAc (2×). The combined organic extracts
were dried (MgSO4) then filtered through a pad of flash silica
gel to give 35 (17 mg, 44%): mp (EtOAc) 214-217 °C; 1H NMR
In Vivo Ch em oth er a p y. Evaluation of in vivo anticancer
effectiveness was performed as described previously.15 The
A431 epidermoid, H125 non-small-cell lung, and MCF-7 breast
xenografts were maintained by serial in vivo passage in and
anticancer effectiveness evaluated in nude mice (Charles River
Breeding Laboratories). Compounds 7 and 31 were adminis-

440 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 3
Smaill et al.
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insufficient solubility at the desired dosage concentration. Both
compounds were dosed orally in a fixed volume of 0.5 mL. Host
body weight change data are reported as the maximum
treatment-related weight loss in these studies. Calculation of
tumor growth inhibition (% T/C) and tumor growth delay (T-
C) was performed as described previously.22-24
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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