4-anilino-6,7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor kinase and their bioisosteric relationship to the 4-anilino-6,7-dialkoxyquinazoline inhibitors

Article abstract of DOI:10.1021/jm000206a

The synthesis and SAR of a series of 4-anilino-6,7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor (EGF-R) kinase are described. Condensation of 3,4-dialkoxyanilines with ethyl (ethoxymethylene)cyanoacetate followed by thermal cyclization gave, regiospecifically, 6,7-dialkoxy-4-oxo-1,4-dihydroquinoline-3-carbonitriles. Chlorination (POCl₃) followed by the reaction with substituted anilines furnished the 4-anilino-6,7-dialkoxyquinoline-3-carbonitrile inhibitors of EGF-R kinase. An alternate synthesis of these compounds starts with a methyl 3,4-dialkoxybenzoate. Nitration followed by reduction (Fe, NH₄Cl, MeOH-H₂O) gave a methyl 2-amino-4,5-dialkoxybenzoate. Amidine formation using DMF-acetal followed by cyclization using LiCH₂CN furnished a 6,7-dialkoxy-4-oxo-1,4-dihydroquinoline-3-carbonitrile, which was transformed as before. Compounds containing acid, ester, amide, carbinol, and aldehyde groups at the 3-position of the quinoline ring were also prepared for comparison, as were several 1-anilino-6,7-dimethoxyisoquinoline-4-carbonitriles. The compounds were evaluated for their ability to inhibit the autophosphorylation of the catalytic domain of EGF-R. The SAR of these inhibitors with respect to the nature of the 6,7-alkoxy groups, the aniline substituents, and the substituent at the 3-position was studied. The compounds were further evaluated for their ability to inhibit the growth of cell lines that overexpress EGF-R or HER-2. It was found that 4-anilinoquinoline-3-carbonitriles are effective inhibitors of EGF-R kinase with activity comparable to the 4-anilinoquinazoline-based inhibitors. A new homology model of EGF-R kinase was constructed based on the X-ray structures of Hck and FGF receptor-1 kinase. The model suggests that with the quinazoline-based inhibitors, the N3 atom is hydrogen-bonded to a water molecule which, in turn, interacts with Thr 830. It is proposed that the quinoline-3-carbonitriles bind in a similar manner where the water molecule is displaced by the cyano group which interacts with the same Thr residue.

Full text of DOI:10.1021/jm000206a

3244
J . Med. Chem. 2000, 43, 3244-3256
4-An ilin o-6,7-d ia lk oxyqu in olin e-3-ca r bon itr ile In h ibitor s of Ep id er m a l Gr ow th
F a ctor Recep tor Kin a se a n d Th eir Bioisoster ic Rela tion sh ip to th e
4-An ilin o-6,7-d ia lk oxyqu in a zolin e In h ibitor s
Allan Wissner,* Dan M. Berger, Diane H. Boschelli, M. Brawner Floyd, J r., Lee M. Greenberger,
Brian C. Gruber, Bernard D. J ohnson, Nellie Mamuya, Ramaswamy Nilakantan, Marvin F. Reich, Ru Shen,
Hwei-Ru Tsou, Erik Upeslacis, Yu Fen Wang, Biqi Wu, Fei Ye, and Nan Zhang
Wyeth-Ayerst Research, A Division of American Home Products, 401 North Middletown Road,
Pearl River, New York 10965-1215
Received May 15, 2000
The synthesis and SAR of a series of 4-anilino-6,7-dialkoxyquinoline-3-carbonitrile inhibitors
of epidermal growth factor r eceptor (EGF-R) kinase are described. Condensation of 3,4-
dialkoxyanilines with ethyl (ethoxymethylene)cyanoacetate followed by thermal cyclization gave,
regiospecifically, 6,7-dialkoxy-4-oxo-1,4-dihydroquinoline-3-carbonitriles. Chlorination (POCl3)
followed by the reaction with substituted anilines furnished the 4-anilino-6,7-dialkoxyquinoline-
3-carbonitrile inhibitors of EGF-R kinase. An alternate synthesis of these compounds starts
with a methyl 3,4-dialkoxybenzoate. Nitration followed by reduction (Fe, NH4Cl, MeOH-H2O)
gave a methyl 2-amino-4,5-dialkoxybenzoate. Amidine formation using DMF-acetal followed
by cyclization using LiCH2CN furnished a 6,7-dialkoxy-4-oxo-1,4-dihydroquinoline-3-carboni-
trile, which was transformed as before. Compounds containing acid, ester, amide, carbinol,
and aldehyde groups at the 3-position of the quinoline ring were also prepared for compari-
son, as were several 1-anilino-6,7-dimethoxyisoquinoline-4-carbonitriles. The compounds
were evaluated for their ability to inhibit the autophosphorylation of the catalytic domain of
EGF-R. The SAR of these inhibitors with respect to the nature of the 6,7-alkoxy groups, the
aniline substituents, and the substituent at the 3-position was studied. The compounds were
further evaluated for their ability to inhibit the growth of cell lines that overexpress EGF-R or
HER-2. It was found that 4-anilinoquinoline-3-carbonitriles are effective inhibitors of EGF-R
kinase with activity comparable to the 4-anilinoquinazoline-based inhibitors. A new homology
model of EGF-R kinase was constructed based on the X-ray structures of Hck and FGF
receptor-1 kinase. The model suggests that with the quinazoline-based inhibitors, the N3 atom
is hydrogen-bonded to a water molecule which, in turn, interacts with Thr 830. It is proposed
that the quinoline-3-carbonitriles bind in a similar manner where the water molecule is
displaced by the cyano group which interacts with the same Thr residue.
In tr od u ction
cytoplasmic domain of the receptor in gliomas,3 prostate
cancer,4 breast cancer,5 and non-small-cell lung cancer;6
this mutation makes the receptor constitutively active.7
In addition, the receptor need not be overexpressed or
mutated to be activated, since the ligands for the
receptor (EGF or transforming growth factor-R (TGFR))
can be produced within the same cancerous tissue or
cell that expresses EGF-R. This suggests that paracrine
or autocrine loops stimulate hyperproliferation as in
prostate cancer,8 head and neck cancer,9 ovarian can-
cer,10 non-small-cell lung cancer,11 and bladder cancer.12
EGF-R hyperactivation has also been implicated in
other diseases including polycystic kidney disease,13
psoriasis,14 and asthma.15
Protein tyrosine kinases play a role in normal cell
growth. Many of the growth factor receptor proteins
have intracellular domains that function as tyrosine
kinases, and it is with this that they effect signaling.
The interaction of growth factors with these receptors
is a necessary event in normal regulation of cell growth.
However, under certain conditions, as a result of over-
expression, mutation, or coexpression of the ligand and
the receptor, these receptors can become hyperactivated;
the result of this is uncontrolled cell proliferation.1
Among the growth factor receptor kinases that have
been identified as being important in cancer is epider-
mal growth factor r eceptor (EGF-R) kinase (also known
as erb-B1 or HER-1) and the related h uman epidermal
growth factor r eceptor HER-2 (also known as erbB-2 or
neu). EGF-R is overexpressed in numerous tumors2
including those derived from the brain, lung, bladder,
head, and neck. Such overexpression has been cor-
related with poor prognosis in some of these diseases.
The gene product is frequently mutated in the extra-
Since in many types of proliferative diseases, the
phosphorylation event mediated by EGF-R or HER-2
kinases is a necessary signal for cell division to occur
and since hyperactivation of these kinases has been
associated with these diseases, an inhibitor of this event,
an EGF-R or HER-2 kinase inhibitor, may have poten-
tial therapeutic value. Since these two receptor kinases
have a high sequence homology in their catalytic
* To whom correspondence should be addressed. Tel: 914-732-3580.
Fax: 914-732-5561. E-mail: wissnea@war.wyeth.com.
10.1021/jm000206a CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/09/2000

Inhibitors of EGF-R Kinase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3245
Sch em e 1a
domains,16 one might expect that an inhibitor of one
would have similar effects on the other.
The middle of the past decade was marked by the
discovery of the 4-anilinoquinazolines, a series of potent
and selective ATP-competitive inhibitors of EGF-R
kinase.17 Several pharmaceutical firms,18 including
ourselves,19 established programs based on these inhibi-
tors, and now at least three such compounds have
entered or will be entering clinical trials.20 In extending
our work with this series of inhibitors, we realized that
the nitrogen atom located at the 3-position of these
quinazoline inhibitors is an important feature needed
for good activity. Replacing this atom with a carbon
leads to a significant loss in the ability of the compound
to inhibit the enzyme.21 We reasoned that given the
importance of this nitrogen atom for activity, it is likely
that it has a specific interaction with the enzyme.
Presumably, it functions as a hydrogen bond acceptor.
In designing our inhibitors, we made use of a homology
model of the enzyme that is similar to the model
previously constructed.22 More recently we have devel-
oped a refined model as discussed below. With either
model, docking experiments using our inhibitors did not
reveal any residues close by to which the N3 atom could
directly interact. We therefore made the hypothesis that
this nitrogen atom could be interacting with a water
molecule and that this water molecule could then serve
as a bridge between the drug and enzyme. Such water
bridging is a well-known phenomenon.23 Since the N3
atom of the quinazoline inhibitor, in solution, surely
forms a hydrogen bond with a water molecule, retaining
this interaction after binding to the enzyme should have
a minimal effect on the entropy of binding. In addition,
it occurred to us that if this nitrogen atom were removed
and replaced with a carbon atom that had an attached
electron-withdrawing group (such as a cyano group),
such a molecule should retain a similar overall shape
and charge distribution compared to the quinazoline
hydrogen-bonded to a water molecule. In this com-
munication we would like to report that applying the
above concept has resulted in a new series of 4-anili-
noquinoline-3-carbonitriles that are potent inhibitors of
EGF-R kinase.
a
(a) For 2a : C2H5OCHdC(CO2C2H5)2, neat, 100 °C; for 2b,c:
C2H5OCHdC(CO2C2H5)CN, toluene, reflux; (b) Dowtherm, 258 °C;
(c) POCl3, reflux; (d) C2H5O(CH2)2OH, reflux; for 35: NaH, DMF,
reflux; (e) (i) NaNO2, HOAc, H2O, 0 °C, (ii) NaN3; (f) (CH3CO)2O,
CH3CO2H.
products can be isolated as the hydrochloride salt by
filtering directly from the reaction mixture, or alterna-
tively, the free base can be isolated after neutralization
of the salt with base. Due to the hindered nature of
2-bromoaniline, it was necessary to prepare compound
35 using sodium hydride in refluxing DMF. Compound
40 was prepared by acetylation of 16 with acetic
anhydride in acetic acid. Compound 39 was prepared
by a two-step process involving formation the diazonium
salt of 16 using sodium nitrite and acetic acid followed
by the reaction with sodium azide.
Ch em istr y
Since earlier work by the Parke-Davis group with the
quinazoline-based inhibitors of EGF-R established that
6,7-dialkoxy substitution is compatible with good activ-
ity,24 we decided to retain this feature in our initial
compounds. One of the methods we used to prepare the
intermediate quinolones 3a -c follows the method of
Bredereck25 as shown in Scheme 1. Condensation of
anilines 1a ,b with diethyl ethoxymethylenemalonate or
ethyl (ethoxymethylene)cyanoacetate followed by ther-
mal cyclization in refluxing Dowtherm gave, regiospe-
cifically, 3a -c. These quinolones were converted, in
good yield, to the corresponding chloroquinolines by
refluxing in an excess of POCl3. The preparations of
3c25b and 4a 26 have been described previously. In the
final step of the sequence, refluxing a solution of a
chloroquinoline and a substituted aniline derivative in
ethoxyethanol gave the 4-anilino-6,7-dialkoxyquinoline-
3-carbonitriles 5-38 (see Table 1). In most cases, the
An alternate synthesis of some of these compounds
is shown in Scheme 2. Compounds containing unsym-
metrical 6,7-dialkoxy substitution were prepared from
the isomeric methylbenzoates 41a ,b by alkylation with
ethyl iodide and potassium carbonate in refluxing DMF.
Compound 42c was prepared as already described.27
Nitration of 42a -c in acetic acid at 50 °C gave the nitro
derivatives 43a -c. Reduction using iron and NH4Cl in
a water-methanol mixture at reflux followed by ami-
dine formation using N,N-dimethylformamide dimethyl-
acetal in DMF at reflux gave 45a -c which were used
without additional purification. The lithium anion of
acetonitrile was prepared in THF by the addition of
n-butyllithium at -78 °C. Addition of the amidines to

3246 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
Wissner et al.
Sch em e 2a
Sch em e 3a
a
(a) DIBAL, toluene; (b) PCC, NaOAc, CH2Cl2; (c) NaOH, H2O,
C2H5OH, reflux; (d) (i) CDl, DMF, (ii) NH3; (e) pyridine‚HCl, 210
°C; (f) C2H5I, K2CO3, DMF; (g) CH3OCH2Cl, K2CO3, DMF; (h)
CH3O(CH2)2Br, K2CO3, DMF; (i) BrCH2Cl, Cs2CO3, DMF; (j)
Br(CH2)3Br, K2CO3, DMF; (k) substituted aniline, isopropanol,
reflux.
a
(a) C2H5I, K2CO3, DMF, 100 °C; (b) HNO3, HOAc, 50 °C; (c)
Fe, NH4Cl, CH3OH, H2O, reflux; (d) DMA-DMF, DMF, reflux;
(e) (i) n-BuLi, CH3CN, THF, -78 °C, (ii) HOAc, -78 to 25 °C; (f)
for 47a ,c: POCl3, reflux; for 47b: (COCl)2, CH2Cl2, reflux; (g)
C2H5O(CH2)2OH or C2H5OH, reflux.
Cs2CO3 in DMF or 1,3-dibromopropane and potassium
carbonate in DMF, respectively.
this solution containing 2 equiv of the anion followed
by the addition of acetic acid and warming to 25 °C
furnished the quinolones 46a -c. These were then
converted to the 4-anilino-6,7-dialkoxyquinoline-3-car-
bonitriles 48-50 as described above.
Compounds containing varying substitution at the
3-position were prepared by simple functional group
manipulations as shown in Scheme 3. Reduction of the
ester 5 with DIBAL gave the carbinol derivative 51
which, in turn, was oxidized to the aldehyde 52 using
pyridinium chlorochromate buffered with sodium ace-
tate in methylene chloride. Base hydrolysis of 5 fur-
nished the carboxylic acid 53 which was then converted
to the amide 54 using a two-step procedure consisting
of first activating the carboxylate group as the acyl
imidazole derivative followed by the addition of am-
monia.
Finally, we were interested in seeing if replacement
of the N1 atom of a quinazoline inhibitor with a carbon
bearing a cyano group would also lead to an active
EGF-R inhibitor. Accordingly, we prepared the 1-anili-
noisoquinoline-4-carbonitrile derivatives 61 and 62 from
the corresponding known28 chloro derivative 60 by
treating it with the substituted aniline in refluxing
2-propanol.
Molecu la r Mod elin g
In a prior publication,19 we discussed modeling work
on EGF-R kinase using a homology model similar to that
described by Knighton et al.22 This model was based on
the first crystal structure of a protein kinase, viz. cAMP-
dependent protein kinase, a serine/threonine kinase.
While the sequence homology between cAMP-dependent
protein kinase and EGF-R kinase is 41%, the sequence
identity is only 27%. Since then, a number of additional
kinase structures have been solved, including several
tyrosine kinases. We therefore decided to build a homol-
ogy model of EGF-R kinase using more closely related
crystal structures as templates. We used the BLAST
server at NCBI to search the Protein Data Bank. The
top-ranked structure was fibroblast growth factor re-
ceptor-1 kinase,29 FGF receptor-1 (PDB code: 1fgk),
with a sequence identity of 33%. However, several
residues in the activation loop are missing from this
Additional variation of the 6,7-alkoxy groups was
accomplished as shown in Scheme 3 by first demethy-
lating 8 by heating in neat pyridine hydrochloride at
210 °C to give the 6,7-dihydroxy derivative 55. Bis-
alkylation of 55 with ethyl iodide, chloromethyl methyl
ether, or bromoethyl methyl ether using K2CO3 in
refluxing DMF gave compounds 6, 56, and 57, respec-
tively. Compounds 58 and 59 which have, respectively,
a fused five- or seven-membered diether ring were
prepared by alkylation using bromochloromethane and

Inhibitors of EGF-R Kinase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3247
F igu r e 1. Proposed binding models for (a) quinazoline 63 and (b) 3-cyanoquinoline 8 at the ATP site in EGF-R based on the
homology model.
structure. Another high-ranked hit in the BLAST
search, h ematopoietic cell k inase,30 Hck (PDB code:
1qcf) was also explored as a possible template. However,
its alignment with EGF-R involved a major insertion
in the glycine loop which is close to the ATP binding
site. The resulting model had major distortions in the
ATP binding site. We therefore decided to use a com-
bination of these two templates: FGF receptor-1 for the
N-terminal lobe and Hck for the C-terminal lobe. We
used the homology modeling facility within the Molec-
ular Operating Enviroment (MOE) software to do this.
The resulting model was minimized using Quanta/
CHARMm and used for subsequent studies.
A recent paper from a group at Glaxo Wellcome31
revealed the crystal structures of the 4-anilinoquinazo-
line class of compounds complexed with two kinases:
CDK2 and p38-MAP kinase. The binding mode of the
quinazoline ring in both structures is similar, the N1
atom of the quinazoline being hydrogen-bonded to the
backbone nitrogen of a residue on the domain connector
strand (Leu 83 in CDK2 and Met 109 in p38). In the
p38 structure, the N3 atom forms a hydrogen bond with
a water molecule, which, in turn, makes an interaction
with the hydroxyl group of a Thr residue. These two
crystal structures strongly suggest that there is a single
binding orientation of the quinazoline ring in kinases.
We therefore revised our earlier binding model to reflect
these new data from the crystal structures. The water
molecules found in the Hck structure were added to the
homology model. We also used a pentapeptide model
substrate AEYLR by positioning it so that the tyrosine
hydroxyl is close to the catalytic aspartate (Asp 813) and
close to the γ-phosphate of ATP. The enzyme-ATP-
substrate model was built and optimized separately. We
then removed the ATP molecule and replaced it with
quinazoline inhibitor 63 (PD 153035),24 using the figures
and descriptions of the published X-ray structures as a
guide to orient the molecule. The entire complex was
optimized by several cycles of molecular dynamics and
minimization using Quanta/CHARMm.
In the final model with the quinazoline 63 (see Figure
1a), the N1 atom of the quinazoline forms a hydrogen
bond with the backbone NH of Met 769 and the N3 atom
forms a hydrogen bond to a water molecule. This water
molecule, in turn, forms a hydrogen bond with the
hydroxyl group of Thr 830. The C2 atom of the quinazo-
line is 3.28 Å from the backbone carbonyl oxygen of Gln
767 and the C8 atom is 4.20 Å from the backbone
carbonyl oxygen of Met 769.
The corresponding model with the quinoline-3-carbo-
nitrile 8 (see Figure 1b) was generated in a similar
manner. The orientation of the molecule and its interac-
tions with the protein are similar to that observed in
the quinazoline model. The hydrogen bond of the N1
atom to the backbone NH of Met 769 is retained. In this
model the 3-cyano group displaces the water molecule
that was previously hydrogen-bonded to the N3 atom
of the quinazoline and instead makes its own interaction
with the hydroxyl group of Thr 830.
As indicated by these models, the similar manner in
which 63 and 8 bind suggests that the carbon with its
attached cyano group (C-CN) of 8 is bioisosteric with
the N3 atom of 63 hydrogen-bonded to a water molecule.
To lend more credence to this idea, we performed single-
point AM1 calculations on 63 and 8 each in their binding
conformations as extracted from the models. The results
of these calculations are shown in Figure 2. It is evident
from these calculations that both systems are similar
in terms of their overall shape and the location of
charge.
After this work was completed, a report appeared that
described a series of quinazoline and benztriazine
inhibitors of Scytalone dehydratase along with the
3-cyanoquinoline and 3-cyanocinnoline inhibitors de-
rived from them, respectively.32 This report compared
the X-ray structures of a benztriazine and its corre-

3248 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
Wissner et al.
ences in the nature of the enzyme and substrate as well
as to the overall assay conditions. We use a solid-phase
ELISA-based assay. Our enzyme consists of the purified
cytoplasmic domain of EGF-R, and we measure the
inhibition of autophosphorylation of this protein. Other
workers have measured inhibition using, in a soluble
format, the entire enzyme, purified from A431 cells,
along with an exogenous peptide substrate. To further
address this issue, two compounds, 63 and its corre-
sponding quinoline-3-carbonitrile analogue 8, were ad-
ditionally evaluated in EGF-R kinase assays using a
soluble format, purified cytoplasmic domain, and exog-
enous substrate as described previously.19
In the ELISA-based EGF-R assay 63 is only about 3
times more potent than 8. The difference in potency
between the two compounds is similar when using the
alternate protocol with the same enzyme preparation
but in a soluble format and with an exogenous peptide
substrate where the IC50 values for 63 and 8 are 0.0022
and 0.0075 µM, respectively. As expected, the IC50
values are lower in the latter assay. These results
establish that the quinoline-3-carbonitriles are poten-
tially comparable to the quinazolines in their ability to
inhibit EGF-R kinase.
F igu r e 2. (a) Electrostatic potential surfaces (0.005 contour
level) calculated for 63 hydrogen-bonded to the water molecule
and (b) the corresponding surface for 8. Single-point calcula-
tions of the structures extracted from the binding models were
accomplished using the semiempirical AM1 method as imple-
mented in MacSpartan.
sponding 3-cyanocinnoline bound at the active site of
this enzyme. Although this protein is not a kinase, the
manner in which the molecules bind has relevance to
the discussion at hand. In this structure, the N3 atom
of the benztriazine forms a hydrogen bond with a
crystallographic water molecule. The water molecule,
in turn, forms a bridge to the hydroxyl groups of two
Tyr residues. In addition, the authors prepared a
corresponding 3-cyanocinnoline, and in the resulting
X-ray structure, the cyano group occupies a position
corresponding to the position of the water molecule in
the benztriazine structure where the nitrogen atom of
the cyano groups bridges the same two Tyr residues.
This is analogous to the situation we are now proposing
for the binding of the quinazoline and quinoline-3-
carbonitrile inhibitors at the ATP binding pocket of
EGF-R.
Our homology model of EGF-R differs somewhat from
the model proposed by the Parke-Davis group33 in that
while the orientation of the bound quinazoline is similar
and has the same interaction between N1 and Met 769,
the latter model has a direct hydrogen bond between
N3 and the hydroxyl group of Thr 766; it would seem
that the EGF-R inhibition activity exhibited by the
quinoline-3-carbonitriles, as discussed below, is not
easily explained by this latter model.
We were interested in determining the effect on
activity of replacing the 3-cyano group with other
functionalities. With respect to EGF-R inhibition, re-
placement with an ester (5), carboxylic acid (53),
carbinol (51), or amide (54) group leads to significant
(79-fold to greater than 530-fold) loss in potency. While
the aldehyde derivative 52 was closest in activity to 8,
it is still about 18-fold less potent. A possible rational-
ization of these data is that one might expect for 5, 53,
54, and 52 that the low-energy conformations would be
characterized by an intramolecular hydrogen bond
between the carbonyl oxygen of the 3-substituent and
the NH group of the aniline moiety. Our binding model
would suggest that this hydrogen bond would have to
be sacrificed for these compounds to adopt an appropri-
ate binding conformation; this will result in a com-
mensurate loss in binding energy. In addition, the sp-
hybridization of the cyano substituent could result in a
more favorably directed interaction with Thr 830 than
when the substituents are sp2-hybridized.
Since replacement of the N3 atom of the quinazoline
with a carbon atom bearing a cyano group is compatible
with good activity, it was obviously of interest to see
what would happen when the same replacement was
made with the N1 atom. Accordingly, we found that the
isoquinoline-4-carbonitruiles 61 and 62 are, respec-
tively, 368- and 496-fold less potent than their corre-
sponding quinoline-3-carbonitrile isomers 8 and 9. This
is an expected result in view of the binding model, which
indicates a direct interaction of N1 of the quinoline, and
quinazoline-based inhibitors with the backbone NH of
Met 769.
Resu lts a n d Discu ssion
The compounds shown in Table 1 were evaluated for
their ability to inhibit EGF-R kinase. To compare these
data with similar data already reported for the quinazo-
line-based inhibitors, the inhibitory activity of the
standard compound, 63, under our assay conditions, is
incorporated into the data set. First, it is immediately
evident that the IC50 value we obtain with 63 for
inhibition of EGF-R is significantly higher than was
found by previous workers.24 Additionally, for our
compounds, we do not see the extremely large range in
potency found by these workers for the quinazoline
inhibitors. We attribute these observations to differ-
We surveyed a variety of substituents at different
positions on the aniline ring of these quinoline-3-
carbonitriles. Our molecular model suggests that the
aniline ring resides in a large, mostly lipophilic, pocket
buried deep within the active site. Good activity is seen
when the aniline ring is monosubstituted with a bro-
mine (8) or chlorine (9) atom at the meta-position. A
similar observation was reported for the quinazoline

Inhibitors of EGF-R Kinase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3249
Ta ble 1. Inhibition of EGF-R Kinase and Inhibition of Cell Proliferation
IC50 (µM)
compda
X
Y
R1
R2
R′′
EGF-Rb
A431c
SKBR3c
SW620c
5
6
C-CO2Et
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CN
C-CH2OH
C-CHO
C-CO2H
C-CONH2
C-CN
C-CN
C-CN
C-CN
C-CN
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
CH3
C2H5
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
CH3
CH3
C2H5
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
-CH2CH2-
CH3
CH3
CH3
CH3
H
3-Br
3-Br
3-Cl, 4-F
3-Br
3-Cl
3-CF3
3,4-di-OCH3
3-F
3-CN
4-F
3-COCH3
3-NH2
3-N(CH3)2
3-NO2
>81.3
0.42
3.01
0.11
0.27
0.78
0.59
2.53
5.28
3.07
3.51
1.43
7.37
6.62
0.65
2.14
0.70
0.818
1.52
0.96
5.91
0.75
3.01
3.136
0.94
1.451
0.93
0.89
3.02
13.2
4.35
0.76
21.03
7.72
3.64
0.73
1.48
2.62
0.57
0.14
0.42
7.91
1.95
18.23
4.65
2.23
14.50
0.71
2.62
2.08
18.76
26.13
1.52
6.21
0.09
0.49
0.48
1.76
13.42
13.16
8.29
25.97
0.74
5.12
2.45
0.36
2.00
0.47
0.54
0.95
0.64
8.44
0.55
1.29
2.017
1.19
1.54
0.94
1.69
2.91
16.12
6.74
1.47
0.80
0.52
0.39
1.38
1.16
1.55
0.64
0.06
0.27
15.36
2.97
35.71
7.78
2.74
14.20
0.56
1.94
5.07
24.96
27.67
0.82
6.93
0.16
0.49
3.72
7.06
35.63
8.76
13.52
43.29
2.87
6.30
14.33
0.99
17.27
2.3
7
0.98
8
9
0.19e
0.18
1.45
50.49
0.74
10
11
12
13
14
15
16
17d
18
19
20
21
22
23
24
25
26
27d
28d
29d
30d
31
32
33
34
35
36d
37
38
39
40
48
49
50
51
52
53
54
55
56
57
58
59
61d
62
63
14.18
47.94
7.38
0.82
0.85
0.87
3-Cl, 4-F
4-Cl, 2-F
3-OH
0.54
13.85
6.36
5.34
7.8
4-CH3
2.73
60.43
6.29
2.89
2.75
3.43
3.01
4.98
24.96
19.17
3.51
4.98
3.80
3.76
17.02
29.53
11.83
3.09
4.74
2.74
4.24
0.06
0.43
23.15
5.24
53.82
9.87
4.52
9.79
4.32
4.18
2.90
48.49
64.45
13.94
3-CONH2
3-Br, 4-CH3
3-Cl, 4-OH
3,5-di-Cl, 4-OH
5-Cl, 2-OH
3-SCH3
3-Cl, 4-CH3
2-F, 4-Br
4-CH(CH3)2
2-CH(CH3)2
3-CH(CH3)2
4-Br
7.69
1.79
9.04
3.15
17.02
0.76
1.48
>91.2
0.23
0.36
62.32
6.65
2-Br
4.18
3-CF3, 4-F
2-CH3, 3-Br
3-CH3, 4-Br
3-N3
0.16
0.84
0.62
2.21
3-NHCOCH3
3-Br
69.40
21.19
33.02
>104.7
>102.2
3.37
3-Br
3-Br
CH3
CH3
CH3
CH3
H
CH3OCH2
CH3O(CH2)2
3-Br
3-Br
3-Br
34.22
15.17
0.35
3-Br
3-Br
CH3OCH2
3-Br
6.75
CH3O(CH2)2
3-Br
1.10
-CH2-
-(CH2)3-
CH3
3-Br
6.79
3-Br
5.15
C-CN
C-CN
N
CH3
CH3
CH3
3-Br
70.84
88.29
0.07f
N
CH3
CH3
3-Cl
N
3-Br
a
b
Unless indicated compounds were tested as the free base. Concentration needed to inhibit the autophosphorylation of the cytoplasmic
domain of EGF-R by 50% as determined from the dose-response curve. Determinations were done in duplicate and repeat values agreed,
on average, within 40%. c Dose-response curves were determined at five concentrations. The IC50 values are the concentrations needed
to inhibit cell growth by 50% as determined from these curves. Compound tested as the HCl salt. e Using the assay protocol described
d
in ref 19 and purified cytoplasmic domain, the IC50 for inhibition is 0.0075 µM. f Using the assay protocol described in ref 19 and purified
cytoplasmic domain, the IC50 for inhibition is 0.0022 µM.
series.24 Moving the bromine atom to the ortho- or para-
position as in 35 and 34 results in a 22- and 35-fold
decrease, respectively, in the ability of the compound
to inhibit EGF-R. Surprisingly, with the weakly electron-
donating isopropyl group, this trend is reversed; the
meta-substituted derivative 33 is by far less potent than
either the para- or ortho-isomers 31 and 32, respec-
tively. Modeling offers no immediate explanation for this
trend. For the meta-trifluoromethyl-substituted com-
pounds 10 and 36, adding an additional para-fluoro
substituent leads to a 9-fold increase in potency, while
for the meta-chloro derivatives 9 and 19 this same
modification leads to a modest (3-fold) decrease. Com-
pounds 21 and 25-27, having a hydroxyl group in
various positions on the aniline ring, each showed poor
activity, while compounds 16 and 17, which bear amino
groups at the meta-position, had only a moderate loss
in potency.
We made a number of modifications to the 6,7-
dialkoxy substituents. For both compounds 8 and 19,

3250 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
Wissner et al.
replacing 6,7-dimethoxy groups with ethoxy groups (6
and 7, respectively) leads, in each case, to a small (2-
fold) decrease in EGF-R inhibitory activity. This is in
contrast to what was reported in the quinazoline series,
where replacing the 6,7-dimethoxy groups with 6,7-
diethoxy groups led to a gain in potency.24 Surprisingly,
the 7-ethoxy-6-methoxy and 6-ethoxy-7-methoxy deriva-
tives 48 and 49, respectively, are both very much less
potent than 8; we can offer no explanation for this
result. The 6,7-dihydroxy derivative 55 has comparable
activity to 8. Incorporation of the 6,7-alkoxy substituents
into a fused ring as in 50, 58, and 59 results in
significant loss of activity. Likewise, placing polyether
chains at the 6,7-positions as in 56 and 57 decreases
potency compared to 8.
tors that is consistent with much of the observed
activities. In future reports, we will attempt to show the
versatility of this new series of tyrosine kinase inhibi-
tors.
Of additional significance is our proposal that a
carbon atom bearing a cyano group can sometimes be
bioisosteric with an azomethine group that is hydrogen-
bonded to a water molecule. Potentially, this concept
may have utility in other areas of drug design.
Exp er im en ta l Section
Biology. P r ep a r a tion a n d p u r ifica tion of EGF -R DNA
con str u cts: A 1.6-kb cDNA for the EGF-R cytoplasmic
domain (amino acids 45-1186) was cloned in baculoviral
expression vectors pBlueBacHis2B (Invitrogen, Carlsbad, CA)
and pFASTBacHTc (GIBCO, Rockville, MD). A sequence that
encodes (His)6 is located 5′ upstream to the EGF-R sequence.
Sf-9 cells were infected at moi ) 10 for 3 days for protein
expression. Sf-9 cell pellets were solubilized at 0 °C in a buffer
at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1% Triton,
10 µM ammonium molybdate, 100 µM sodium vanadate, 10
µg/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL pepstatin, and
16 µg/mL benzamidine HCl for 20 min followed by 20-min
centrifugation. Crude extract supernatant was passed through
an equilibrated Ni-NTA superflow packed column (Qiagen,
Valencia, CA) and washed with 10 and 100 mM imidazole to
remove nonspecifically bound material. Histidine-tagged pro-
teins were eluted with 250 and 500 mM imidazole and dialyzed
against 50 mM NaCl, 20 mM HEPES, 10% glycerol and 1 µg/
mL each of aprotinin, leupeptin and pepstatin for 2 h. The
entire purification procedure was performed at 4 °C or on ice.
Purified materials were subjected to electrophoresis followed
by Coomassie blue stain to determine the purity of the
preparation and Western blot for the identity of protein.
Enzyme preparations were stored at -80 °C.
EGF -R k in a se a u top h osp h or yla tion a ssa y by DELF IA/
tim e-r esolved flu or om etr y: The EGF-R kinase assay was
set up to assess the level of autophosphorylation based on
DELFIA/time-resolved fluorometry. Compounds were dis-
solved in 100% DMSO and diluted to the appropriate concen-
trations with 25 mM HEPES at pH 7.4. In each well, 10 µL of
compound was incubated with 10 µL of recombinant enzyme
(1:80 dilution in 100 mM HEPES) for 10 min at room
temperature (>90% EGF-R binds to maxisorp plate within 1
h of incubation). Then, 10 µL of 5x buffer (containing 20 mM
HEPES, 2 mM MnCl2, 100 µM Na3VO4 and 1mM DTT) and
20 µL of 0.1 mM ATP-50 mM MgCl2 was added and the
mixture was incubated for 1 h. Positive and negative controls
were included in each plate by incubation of enzyme with or
without ATP-MgCl2. At the end of incubation, liquid was
aspirated and plates were washed three times with wash
buffer. A 75-µL (400 ng) sample of europium-labeled anti-
phosphotyrosine antibody was added to each well for another
1 h of incubation. After washing, enhancement solution was
added and the signal was detected by Victor (Wallac Inc.) with
excitation at 340 nm and emission at 615 nm. All reagents
were supplied by Wallac Inc. (Wallac/Perkin-Elimer, Gaith-
ersburg, MD). The percentage of autophosphorylation inhibi-
tion by the compound was calculated using the equation: 100
- [(test - negative control)/(positive control - negative
control)]. The IC50 was obtained from curves of percentage
inhibition with 8 concentrations of compound. As the purity
of EGF-R enzyme preparation is greater than 80%, the
majority of the signal detected by the anti-phosphotyrosine
antibody is from EGF-R. The IC50 values reported in Table 1
are averages of duplicate determinations.
The compounds were also evaluated for their ability
to inhibit the growth of certain cell lines. Three human
carcinoma cell lines were used: A431 (epidermoid)
which highly overexpresses EGF-R, SKBR3 (breast)
which highly overexpresses HER-2 and, to a lesser
extent, EGF-R, and SW620 (colon) which serves as a
control line expressing low levels of EGF-R and HER-
2. It is immediately obvious from the data shown in
Table 1 that inhibition of cell proliferation exhibited by
these compounds is likely the result of multiple mech-
anisms; cell lines such as SW620 that have little
dependence on EGF-R or HER-2 are inhibited. It is
possible that this result reflects a lack of selectivity and
that these compounds inhibit other kinases. In fact, we
know that the selectivity of these compounds for a
particular kinase can be modulated by changing the
substitution pattern on the aniline ring; this will be the
subject of future communications. While multiple kinase
activities would make a straightforward interpretation
of these results difficult, some trends are evident. There
is an approximate correlation between IC50 values
observed for growth inhibition of the A431 and SKBR3
lines. In addition, for the majority of the compounds,
we find that they are better inhibitors of proliferation
of the A431 and SKBR3 lines than the control SW620
line suggesting that, at least in part, inhibition of
proliferation of the these lines is the result of EGF-R
or HER-2 kinase inhibition. In comparing the quinoline-
3-carbonitrile 8 with its quinazoline counterpart 63, we
find that while 8 is about twice as potent as 63 in
inhibiting the growth of both A431 and SKBR3 lines, it
is also a more potent inhibitor of the SW620 control line.
Some of the most potent inhibitors of cell growth are
compounds that contain an ethoxy group at the 7-posi-
tion such as 6, 7, and 49, but these are also some of the
least selective inhibitors since they inhibit the control
line at comparable levels suggesting that with these
compounds, a major component of the inhibition is
unrelated to the EGF-R or HER-2 kinase activity.
Con clu sion s
The above results demonstrate that 4-anilino-6,7-
dialkoxyquinoline-3-carbonitriles are effective inhibitors
of EGF-R kinase. The SAR profile exhibited by these
compounds is, in a number of respects, similar to that
shown by the quinazoline series of EGF-R inhibitors.
In addition, we constructed a homology model of EGF-R
with which we proposed a binding model for the
quinazoline- and quinoline-3-carbonitrile-based inhibi-
Cell p r olifer a tion a ssa y: The cell proliferation assays
were done with three human carcinoma cell lines: A431
(epidermoid carcinoma), SKBR3 (breast carcinoma), and SW620
(colon carcinoma), as previously described.19 All cell lines were
obtained from the American Type Culture Collection. Cells
were plated in 96-well plates at densities of 5.0 × 104/mL in
RPMI-1640 medium supplemented with 5% fetal bovine

Inhibitors of EGF-R Kinase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3251
serum. On the next day, compounds were dosed at 0.5, 5, 50,
500, and 5000 ng/mL concentrations and the cells were
cultured for 2 days. At the end of incubation, cell survival was
determined by the sulforhodamine B assay as previously
described.34 The IC50 values were obtained from the growth
curves.
of an off-white solid. A sample recrystallized from CH2Cl2 gave
an off-white solid: mp 220-223 °C; 1H NMR (DMSO-d6) δ 8.98
(s, 1H), 7.54 (s, 1H), 7.42 (s, 1H), 4.02 (s, 3H), 4.01 (s, 3H).
4-(3-Br om op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r boxylic Acid Eth yl Ester (5). A stirred mixture of 4a 26
(14.8 g, 50 mmol), 3-bromoaniline (9.46 g, 55 mmol), pyridine
(4.05 mL, 50 mmol), and 150 mL of EtOH was heated at reflux
for 30 min. The EtOH was evaporated, and the residue was
partitioned between CH2Cl2 and aqueous NaHCO3. The or-
ganic layer was washed with water, dried over MgSO4, and
concentrated. The product was recrystallized from EtOH to
Molecu la r Mod elin g. Semiempirical calculations (AM1
basis) were carried out on the quinazoline and quinoline-3-
carbonitrile compounds. In the quinazoline case, the bound
molecule along with the water molecule hydrogen-bonded to
the N3 atom were extracted from the modeled complex, and a
single-point calculation was carried out. In the quinoline-3-
carbonitrile case, only the bound compound was extracted and
used for the single-point calculation. The electrostatic potential
was mapped onto the surface (electron density contoured at
the 0.005 level) and color-coded with red representing negative
potential and blue representing positive potential. These
calculations were done using MacSpartan Plus (Wavefunction
Inc., 18401 Von Karman, Suite 370, Irvine, CA 92612).
Homology modeling was done using the MOE (Molecular
Operating Environment) software (Chemical Computing Group
Inc., 1255 University St., Suite 1600, Montreal, Quebec,
Canada H3B 3X). Initial crude minimization was done within
the homology modeling function of MOE. However, the result-
ing model needed further energy minimization. This was done
using Quanta/CHARMm (Molecular Simulations Inc., 9685
Scranton Rd., San Diego, CA 92121). The crude model from
MOE was minimized with a few thousand cycles of minimiza-
tion using the ABNR (adopted-basis Newton-Raphson) method.
Ligands were modeled by positioning them in the active site
in accordance with the published crystal structures of quinazo-
line derivatives bound to CDK2 and MAP kinase (p38).31 The
entire complex was then subjected to alternate cycles of
minimization and dynamics. Each dynamics run was short,
about 3 ps. The intent was to get a satisfactory structure for
the complex that was consistent with the published crystal
structure.
1
give 15.4 g (71%) of a white solid: mp 155-158 °C; H NMR
(DMSO-d6) δ 9.44 (s, 1H), 8.82 (s, 1H), 7.37 (s, 1H), 7.30 (s,
1H), 7.25-6.90 (m, 5H), 4.03 (q, J ) 7.0 Hz, 2H), 3.95 (s, 3H),
3.71 (s, 3H), 1.16 (t, J ) 7.0 Hz, 2H); MS (ES+) m/z 431.2,
433.2 (M + H)+1. Anal. (C20H19BrN2O4) C, H, N.
4-(3-Br om op h en yla m in o)-6,7-d ieth oxyqu in olin e-3-ca r -
bon itr ile (6). To a stirred mixture of 55 (5.34 g, 15 mmol),
K2CO3 (8.29 g, 60 mmol), and 60 mL of DMF at 0 °C was added
4.8 mL (60 mmol) of iodoethane. The resulting mixture was
warmed to 25 °C and stirred for 4.5 h. The reaction mixture
was partitioned between EtOAc and H2O as 4N HCl was added
slowly with cooling until the pH reached about 8. The organic
layer was separated, washed well with H2O, dried and
concentrated. The residue was subjected to flash chromatog-
raphy on silica gel with 60:30:1 CH2Cl2-EtOAc-TEA to give
the product (3.27 g, 53%); homogeneous on TLC with the above
solvent system. Recrystallization from EtOAc gave a white
solid: mp 173-175 °C; 1H NMR (DMSO-d6) δ 9.50 (s, 1H), 8.54
(s, 1H), 7.67 (s, 1H), 7.35 (s, 1H), 7.55-7.15 (m, 4H), 4.35-
4.10 (m, 4H), 1.45-1.35 (m, 6H); MS (ES+) m/z 412.3, 414.2
(M + H)+1. Anal. (C20H18BrN3O2) C, H, N.
4-(3-Br om op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (8). A stirred mixture of 4c (0.40 g, 1.61 mmol),
3-bromoaniline (0.28 g, 1.61 mmol), and 15 mL of 2-ethoxy-
ethanol was heated at reflux for 3 h and cooled to 25 °C. The
crude HCl salt was filtered off and washed with 2-propanol
and Et2O: yield 0.62 g (92%). The free base was obtained by
partition between EtOAc and aqueous Na2CO3. The organic
layer was dried (MgSO4) and solvent was removed to give 0.58
g of crude product. Recrystallization (EtOAc-hexane) gave a
1
Ch em istr y. H NMR spectra were determined with a NT-
300 WB spectrometer at 300 MHz. Chemical shifts (δ) are
expressed in parts per million relative to the internal standard
tetramethylsilane. Electrospray mass spectra were recorded
in positive mode on a Micromass Platform spectrometer.
Electron impact and high-resolution mass spectra were ob-
tained on a Finnigan MAT-90 spectrometer. Chromatographic
purifications were by flash chromatography using Baker 40-
µm silica gel. Melting points were determined in open capillary
tubes on a Meltemp melting point apparatus and are uncor-
rected.
1
white solid: mp 224-228 °C; H NMR (DMSO-d6) δ 9.55 (s,
1H), 8.55 (s, 1H), 7.69 (s, 1H), 7.41 (s, 1H), 7.45-7.15 (m, 4H),
3.96 (s, 3H), 3.92 (s, 3H); MS (ES+) m/z 384.1, 386.1 (M +
H)+1. Anal. (C18H14BrN3O2) C, H, N, Br.
4-(3-Ch lor o-4-flu or op h en yla m in o)-6,7-d ieth oxyqu in o-
lin e-3-ca r bon itr ile (7). The compound was prepared in 88%
yield according to the procedure for 8 from 4-chloro-6,7-
diethoxyquinoline-3-carbonitrile (4b ) and 3-chloro-4-fluoro-
aniline: mp 194-198 °C (EtOAc); 1H NMR (DMSO-d6) δ 9.50
(s, 1H), 8.47 (s, 1H), 7.70 (s, 1H), 7.54-7.25 (m, 3H), 7.32 (s,
1H), 4.26-4.15 (m, 4H), 1.42 (t, 6H, J ) 6.0 Hz); MS (ES+)
m/z 386.2 (M + H)+1. Anal. (C20H17ClFN3O2) C, H, N.
4-(3-Ch lor op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (9). The compound was prepared in 55% yield
according to the procedure for 8 using 3-chloroaniline: mp
2-Cya n o-3-(3,4-d im et h oxyp h en yla m in o)a cr ylic Acid
Eth yl Ester (2c). A mixture of 3,4-dimethoxyaniline (30.6 g,
200 mmol), ethyl (ethoxymethylene)cyanoacetate (33.8 g, 200
mmol), and 80 mL of toluene was stirred at 100 °C for 1 h and
at 125 °C for 15 min. After evaporation of the toluene, the
residue was recrystallized from EtOAc to give 40.0 g (72%) of
a tan solid: mp 166-170 °C; MS (ES+) m/z 277.2 (M + H)+1
Anal. (C14H16N2O4) C, H, N.
.
1
214-217 °C (EtOAc-hexane); H NMR (DMSO-d6) δ 9.66 (s,
6,7-Dim eth oxy-4-oxo-1,4-d ih yd r oqu in olin e-3-ca r bon i-
tr ile (3c). A stirred mixture of 2c (40 g, 145 mmol) and 1.2 L
of Dowtherm A was heated at reflux under N2 for 10 h. After
cooling to 50 °C the mixture was diluted with hexane. The
product was filtered off, washed with hexane followed by
methylene chloride, and dried to give 21.1 g (63%) of a brown
solid: mp 330-350 °C dec; 1H NMR (DMSO-d6) δ 12.57 (s,
1H), 8.59 (s, 1H), 7.44 (s, 1H), 7.03 (s, 1H), 3.89 (s, 3H), 3.87
(s, 3H); MS (ES+) m/z 231.0 (M + H)+1. Anal. (C12H10N2O3) C,
H, N.
4-Ch lor o-6,7-d im eth oxyqu in olin e-3-ca r bon itr ile (4c).
A stirred mixture of 3c (20 g, 87 mmol) and 87 mL of POCl3
was heated at reflux for 2 h. Volatile materials were removed
under vacuum at about 70 °C. The residue was stirred at 0 °C
with methylene chloride and H2O as solid K2CO3 was carefully
added until the pH was 8-9. After stirring for 30 min at 25
°C the organic layer was separated, washed with H2O, dried,
filtered through Celite, and concentrated to give 19.8 g (92%)
1H), 8.58 (s, 1H), 7.58 (s, 1H), 7.39 (s, 1H), 7.52-7.20 (m, 4H),
3.97 (s, 3H), 3.92 (s, 3H); MS (ES+) m/z 340.1, 342.2 (M +
H)+1. Anal. (C18H14ClN3O2) C, H, N.
6,7-Dim e t h oxy-4-(3-t r iflu or om e t h ylp h e n yla m in o)-
q u in olin e-3-ca r b on it r ile (10). The compound was pre-
pared in 72% yield according to the procedure for 8 using
3-trifluoromethylaniline: mp 190-193 °C (EtOAc-hexane); 1H
NMR (DMSO-d6) δ 9.67 (s, 1H), 8.58 (s, 1H), 7.71 (s, 1H), 7.65-
7.45 (m, 4H), 7.39 (s, 1H), 3.97 (s, 3H), 3.92 (s, 3H); MS (ES+)
m/z 374.2 (M + H)+1. Anal. (C19H14F3N3O2) C, H, N.
4-(3,4-Dim et h oxyp h en yla m in o)-6,7-d im et h oxyq u in o-
lin e-3-ca r bon itr ile (11). The compound was prepared in 66%
yield according to the procedure for 8 using 3,4-dimethoxy-
1
aniline: mp 230-240 °C (EtOAc-hexane); H NMR (DMSO-
d6) δ 9.45 (s, 1H), 8.36 (s, 1H), 7.98 (s, 1H), 7.30 (s, 1H), 7.00-
6.83 (m, 3H), 3.94 (s, 3H), 3.92 (s, 3H) 3.78 (s, 3H), 3.76 (s, H);
MS (ES+) m/z 366.3 (M + H)+1. Anal. (C20H19N3O4) C, H, N.

3252 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
Wissner et al.
4-(3-F lu or op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (12). The compound was prepared in 74% yield
according to the procedure for 8 using 3-fluoroaniline: mp
226-230 °C (EtOAc); 1H NMR (DMSO-d6) δ 9.57 (s, 1H), 8.54
(s, 1H), 7.70 (s, 1H), 7.37 (s, 1H), 7.45-6.95 (m, 4H), 3.96 (s,
(t, J ) 8.25 Hz, 1H) 7.33 (s, 1H), 7.72 (s, 1H), 8.46 (s, 1H),
9.37 (s, 1H), 9.52 (s, 1H). Anal. (C18H15N3O3‚0.2H2O) C, H, N.
4-(4-Met h ylp h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (22). Prepared in 88% yield according to the
procedure for 8 using 4-methylaniline: mp 128-130 °C; 1H
NMR (DMSO-d6) δ 2.33 (s, 3H), 3.91 (s, 3H), 3.94 (s, 3H), 7.15
(d, J ) 6.1, 2H Hz), 7.22 (d,, J ) 6.1 Hz, 2H), 7.32 (s, 1H),
7.76 (s, 1H), 8.41 (s, 1H), 9.40 (s, 1H). Anal. (C19H17N3O2‚
0.5H2O) C, H, N.
3-(3-Cya n o-6,7-d im e t h oxyq u in olin -4-yla m in o)b e n z-
a m id e (23). Prepared in 89% yield according to the procedure
for 8 using 3-aminobenzamide: mp 253-255 °C; 1H NMR
(DMSO-d6) δ 9.56 (br s, 1H), 8.51 (s, 1H), 8.02 (s, 1H), 7.76 (s,
2H), 7.71 (d, J ) 7.8 Hz, 1H), 7.47 (t, J ) 7.8 Hz, 1H), 7.41 (s,
1H), 7.38 (d, J ) 7.8 Hz, 1H), 7.36 (s, 1H), 3.96 (s, 3H), 3.92
(s, 3H); MS (ES+) m/z 349 (M + H)+1. Anal. (C19H16N4O3‚
0.3HCl) C, H, N.
3H), 3.92 (s, 3H); MS (ES+) m/z 324.3 (M + H)+1. Anal. (C18H14
-
FN3O2) C, H, N.
4-(3-Cya n op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (13). The compound was prepared in 89% yield
according to the procedure for 8 using 3-cyanoaniline: mp
1
285-288 °C; H NMR (DMSO-d6) δ 9.66 (s, 1H), 8.58 (s, 1H),
7.58 (s, 1H), 7.75-7.50 (m, 4H), 7.39 (s, 1H), 3.97 (s, 3H), 3.92
(s, 3H); MS (ES+) m/z 331.3 (M + H)+1. Anal. (C19H14N4O2‚
0.5H2O) C, H, N.
4-(4-F lu or op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (14). The compound was prepared in 91% yield
according to the procedure for 8 using 4-fluoroaniline: mp
282-285 °C (EtOAc); 1H NMR (DMSO-d6) δ 9.49 (s, 1H), 8.42
(s, 1H), 7.76 (s, 1H), 7.40-7.23 (m, 4H), 7.32 (s, 1H), 3.95 (s,
4-(3-Br om o-4-m eth ylph en ylam in o)-6,7-dim eth oxyqu in -
olin e-3-ca r bon itr ile (24). Prepared in 72% yield according
to the procedure for 8 using 3-bromo-4-methylaniline: mp
3H), 3.92 (s, 3H); MS (ES+) m/z 324.3 (M + H)+1. Anal. (C18H14
FN3O2) C, H, N.
-
1
292-294 °C; H NMR (DMSO-d6) δ 11.05 (br s, 1H), 8.10 (s,
4-(3-Acet ylp h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (15). The compound was prepared in 69% yield
according to the procedure for 8 using 3-acetylaniline: mp
204-206 °C (acetone-hexane); 1H NMR (DMSO-d6) δ 9.62 (s,
1H), 8.53 (s, 1H), 7.77 (d, J ) 1.7 Hz, 1H), 7.74 (s, 1H), 7.65-
7.45 (m, 4H), 3.96 (s, 3H), 3.92 (s, 3H), 2.61 (s, 3H); MS (ES+)
m/z 347.9 (M + H)+1. Anal. (C20H17N3O3) C, H, N.
4-(3-Am in op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (16). A stirred mixture of 4c (3.73 g, 15.0 mmol),
1,3-phenylenediamine (4.86 g, 45 mmol), pyridine (1.21 mL,
15 mmol), and 45 mL of 2-ethoxyethanol was heated at reflux
for 30 min, cooled to 25 °C, and stirred with aqueous NaHCO3.
The resulting solid was filtered, washed with H2O, and dried.
Recrystallization from EtOH gave 4.32 g (90%) of a brown
solid: mp 222-228 °C; 1H NMR (DMSO-d6) δ 9.28 (s, 1H), 8.43
(s, 1H), 7.74 (s, 1H), 7.31 (s, 1H), 6.99 (m, 1 H) 6.43 (m, 3H),
5.21 (bs, 2H) 3.94 (s, 3H), 3.90 (s, 3H); MS (ES+) m/z 321.0
(M + H)+1. Anal. (C18H16N4O2‚0.5H2O) C, H, N.
1H), 7.71 (d, J ) 2.1 Hz, 1H), 7.48 (d, J ) 8.3 Hz, 1H), 7.44 (s,
1H), 7.38 (dd, J ) 8.3, 2.1 Hz, 1H), 4.00 (s, 3H), 3.98 (s, 3H),
2.41 (s, 3H); MS (ES+) m/z 398 (M + H)+1. Anal. (C19H16
BrN3O2‚0.3H2O) C, H, N.
-
4-(3-Ch lor o-4-h yd r oxyp h en yla m in o)-6,7-d im et h oxy-
qu in olin e-3-ca r bon itr ile (25). Prepared in 65% yield ac-
cording to the procedure for 8 using 4-amino-2-chlorophenol:
1
mp 230-232 °C; H NMR (DMSO-d6) δ 10.29 (br s, 1H), 9.39
(br s, 1H), 8.37 (s, 1H), 7.76 (s, 1H), 7.31 (d, J ) 2.4 Hz, 1H),
7.30 (s, 1H), 7.12 (dd, J ) 8.4, 2.8 Hz, 1H), 6.99 (d, J ) 8.4
Hz, 1H), 3.94 (s, 3H), 3.92 (s, 3H); MS (ES+) m/z 356 (M +
H)+1. Anal. (C19H16BrN3O2‚0.4EtOAc) C, H, N.
4-(3,5-Dich lor o-4-h ydr oxyph en ylam in o)-6,7-dim eth oxy-
qu in olin e-3-ca r bon itr ile (26). By the procedure as described
above for 8 using 3,5-dichloro-4-hydroxyaniline, the title
compound was obtained in 89% yield: mp > 250 °C; 1H NMR
(DMSO-d6) δ 3.96 (s, 3H), 3.98 (s, 3H), 7.37 (s, 1H), 7.49 (s,
2H), 7.94 (s, 1H), 8.75 (s, 1H), 10.34 (bs,1H), 10.40 (bs, 1H);
MS (ES+) m/z 389.8, 391.8 (M + H)+1. Anal. (C18H13Cl2N3O3‚
1.0H2O) C, H, N.
4-(3-Dim eth yla m in op h en yla m in o)-6,7-d im eth oxyqu in -
olin e-3-ca r bon itr ile (17). The compound was prepared ac-
cording to the procedure for
phenylenediamine dihydrochloride. The product was recrystal-
8 using N,N-dimethyl-1,3-
4-(5-Ch lor o-2-h yd r oxyp h en yla m in o)-6,7-d im et h oxy-
qu in olin e-3-ca r bon itr ile Hyd r och lor id e (27). By the pro-
cedure as described above for 8, using 5-chloro-2-hydroxy-
aniline, the title compound was obtained in 83% yield as the
hydrochloride salt directly from the reaction mixture: mp >
lized from water to give 0.4 g (18%) of a yellow solid: mp 246-
1
249 °C; H NMR (DMSO-d6) δ 8.88 (s, 1H), 8.12 (s, 1H), 7.48
(s, 1H), 7.28 (m, 1H), 6.78 (m, 2H), 6.70 (d, 1H, J ) 11.49),
3.99 (s, 3H), 3.98 (s, 3H), 2.94 (s, 6H). MS (ES+) m/z 349.2 (M
+ H)+1, 174.9 (M + 2H)+2. Anal. (C20H20N4O2‚HCl‚0.75H2O)
C, H, N.
1
250 °C; H NMR (DMSO-d6) δ 3.98 (s, 3H), 3.99 (s, 3H), 7.02
(d, J ) 9 Hz, 1H), 7.33 (dd, J ) 2.7, 8.7 Hz, 1H), 7.41 (s, 1H),
7.44 (d, J ) 2.7 Hz, 1H), 8.07 (s, 1H), 8.90 (s, 1H), 10.51 (bs,
4-(3-Nitr op h en yla m in o)-6,7-d im eth oxyqu in olin e-3-ca r -
bon itr ile (18). The compound was prepared according to the
procedure for 8 using 3-nitroaniline. Recrystallization from
EtOH gave a 69% yield of 18 as yellow crystals: mp 221-222
1H), 10.63 (bs, 1H); MS (ES+) m/z 355.8, 357.8 (M + H)+1
Anal. (C18H14ClN3O3‚0.9HCl) C, H, N.
.
6,7-Dim eth oxy-4-(3-m eth ylth ioph en ylam in o)qu in olin e-
3-ca r bon itr ile Hyd r och lor id e (28). By the procedure as
described above for 8, using 3-methylthioaniline, the title
compound was obtained in 35% yield as the hydrochloride salt
directly from the reaction mixture: mp > 250 °C; 1H NMR
(DMSO-d6) δ 2.51 (s, 3H), 3.98 (s, 3H), 4.00 (s, 3H), 7.21 (d, J
) 8.3 Hz, 1H), 7.29 (d, J ) 8.1 Hz, 1H), 7.34 (s, 1H), 7.41 (dd,
J ) 7.0, 7.8 Hz, 1H), 7.45 (s, 1H), 8.10 (s, 1H), 8.96 (s, 1H),
11.10 (bs, 1H); MS (ES+) m/z 351.9 (M + H)+1. Anal.
(C19H17N3O2S‚1HCl) C, H, N.
1
°C; H NMR (DMSO-d6) δ 3.92 (s, 3H), 3.98 (s, 3H), 7.42 (s,
1H), 7.65 (m, 3H), 7.96 (m, 2H), 8.64 (s, 1H), 9.79 (s, 1H); MS
(ES+) m/z 351.0 (M + H)+1. Anal. (C18H14N4O4) C, H, N.
4-(3-Ch lor o-4-flu or op h en yla m in o)-6,7-d im eth oxyqu in -
olin e-3-ca r bon it r ile (19). The compound was prepared in
87% yield according to the procedure for 8 using 3-chloro-4-
fluoroaniline: mp 223-225 °C; 1H NMR (DMSO-d6) δ 9.65 (s,
1H), 8.52 (s, 1H), 7.75 (s, 1H), 7.62-7.28 (m, 3H), 7.35 (s, 1H),
3.96 (s, 3H), 3.93 (s, 3H); MS (ES+) m/z 358.2 (M + H)+1. Anal.
(C18H13ClFN3O2) C, H, N.
4-(3-Ch lor o-4-m eth ylph en ylam in o)-6,7-dim eth oxyqu in -
olin e-3-ca r bon itr ile Hyd r och lor id e (29). By the procedure
as described above for 8, using 3-chloro-4-methylaniline, the
title compound was obtained in 90% yield as the hydrochloride
salt directly from the reaction mixture: mp > 250 °C; 1H NMR
(DMSO-d6) δ 2.39 (s, 3H), 4.00 (s, 3H), 4.02 (s, 3H), 7.34 (dd,
J ) 2.13, 8.1 Hz, 1H), 7.43 (s, 1H), 7.48 (d, J ) 8.3 Hz, 1H),
7.56 (d, J ) 2.0 Hz, 1H), 8.09 (s, 1H), 8.96 (s, 1H), 10.98 (bs,
4-(4-Ch lor o-2-flu or op h en yla m in o)-6,7-d im eth oxyqu in -
olin e-3-ca r bon itr ile (20). Prepared in 52% yield according
to the procedure for 8 using 4-chloro-2-fluoroaniline: mp 184-
186 °C; 1H NMR (DMSO-d6) δ 9.49 (br s, 1H), 8.47 (s, 1H),
7.79 (s, 1H), 7.60 (dd, J ) 10.7, 2.1 Hz, 1H), 7.48 (t, J ) 8.7
Hz, 1H), 7.35 (m, 2H), 3.96 (s, 3H), 3.94 (s, 3H); MS (ES+)
m/z 358 (M + H)+1. Anal. (C18H13ClFN3O2) C, H, N.
4-(3-Hyd r oxyp h en yla m in o)-6,7-d im eth oxyqu in olin e-3-
ca r bon itr ile (21). Prepared (91% yield) according to the
procedure for 8 using 3-hydroxyaniline: mp > 250 °C; 1H NMR
(DMSO-d6) δ 3.90 (s, 3H), 3.95 (s, 3H), 6.58-6.66 (m, 3H), 7.17
1H); MS (ES+) m/z 353.9, 355.8 (M + H)+1. Anal. (C19H16
ClN3O2‚1HCl) C, H, N.
-
4-(4-Br om o-2-flu or op h en yla m in o)-6,7-d im eth oxyqu in -
olin e-3-ca r bon itr ile Hyd r och lor id e (30). By the procedure
as described above for 8, using 4-bromo-2-fluoroaniline, the

Inhibitors of EGF-R Kinase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3253
title compound was obtained in 52% yield as the hydrochloride
salt directly from the reaction mixture: mp > 250 °C; 1H NMR
(DMSO-d6) δ 4.00 (s, 6H), 7.46 (s, 1H), 7.58 (m, 2H), 7.83 (d,
J ) 9.21 Hz, 1H), 8.16(s, 1H), 9.00 (s, 1H), 11.00 (bs, 1H); MS
(ES+) m/z 410.8, 403.8 (M + H)+1. Anal. (C18H13BrFN3O2‚
1HCl‚0.2H2O) C, H, N.
4-(4-Isop r op ylp h en yla m in o)-6,7-d im eth oxyqu in olin e-
3-ca r bon itr ile (31). Prepared in 79% yield according to the
procedure for 8 using 4-isopropylaniline giving a pale yellow
solid: mp 198-200 °C; 1H NMR (DMSO-d6) δ 9.41 (s, 1H), 8.42
(s, 1H), 7.72 (s, 1H), 7.32 (s, 1H), 7.27 (d, 2H, J ) 8.4 Hz, 2H),
7.17 (d, 2H, J ) 8.4 Hz, 2H), 3.94 (s, 3H), 3.89 (s, 3H), 2.50
(m, 1H), 1.21 (d, J ) 6.9 Hz, 6H); HRMS m/z 348.1705 (M +
H)+1. Anal. (C21H21N3O2‚0.2H2O) C, H, N.
d6) δ 2.35 (s, 3H), 3.91 (s, 3H), 3.95 (s, 3H), 7.01 (dd, J ) 2.67,
8.52 Hz, 1H), 7.24 (d, J ) 2.55, 1H), 7.36 (s, 1H), 7.55 (d, J )
8.49 Hz, 1H), 7.72 (s, 1H), 8.51 (s, 1H), 9.46 (bs, 1H); MS (ES+)
m/z 397.8, 399.8 (M + H)+1. Anal. (C19H16BrN3O2‚0.26C6H14
‚
0.1H2O) C, H, N.
4-(3-Azidoph en ylam in o)-6,7-dim eth oxyqu in olin e-3-car -
bon itr ile (39). Compound 16 (0.643 g, 2.00 mmol) was
dissolved in 25 mL of 80% aqueous acetic acid and chilled to
0 °C. A solution of 0.152 g (2.21 mmol) of NaNO2 in 2.2 mL of
H2O was added, followed 10 min later by a solution of 0.144 g
(2.21 mmol) of NaN3 in 2.2 mL of H2O. Volatile material was
removed after the reaction had stirred at 25 °C for 1.5 h. The
crude product was dissolved in EtOAc and filtered and the
filtrate was treated with saturated NaHCO3. The organic layer
was separated, washed with brine, dried (Na2SO4) and evapo-
rated. The residue was filtered through silica gel (40% CH2-
Cl2 in EtOAc). The eluent was evaporated and the product was
dried in vacuo (50 °C) to give 0.526 g (76%) of product as brown
crystals: 1H NMR (DMSO-d6) δ 3.91 (s, 3H), 3.96 (s, 3H), 6.98
(m, 3H), 7.43 (m, 2H), 7.69 (s, 1H), 8.54 (s, 1H), 9.55 (s, 1H);
HRMS (CI) m/z (M + H)+1 calcd for C18H15N6O2 347.1256,
found 347.1255. Anal. (C18H14N6O2) C, H, N.
4-(2-Isop r op ylp h en yla m in o)-6,7-d im eth oxyqu in olin e-
3-ca r bon itr ile (32). Prepared in 60% yield according to the
procedure for 8 using 2-isopropylaniline giving a gray solid:
mp 90-91 °C; 1H NMR (DMSO-d6) δ 9.35 (s, 1H), 8.30 (s, 1H),
7.87 (s, 1H), 7.42 (m, 2H), 7.30 (s, 1H), 7.26 (m, 2H), 3.95 (s,
3H), 3.91 (s, 3H), 2.50 (m, 1H), 1.21 (d, J ) 6.9 Hz, 6H); HRMS
m/z 348.1705 (M + H) +1. Anal. (C21H21N3O2‚0.6H2O) C, H, N.
4-(3-Isop r op ylp h en yla m in o)-6,7-d im eth oxyqu in olin e-
3-ca r bon itr ile (33). Prepared in 70% yield according to the
procedure for 8 using 3-isopropylaniline giving a yellow solid:
N-[3-(3-Cya n o-6,7-d im eth oxyqu in olin -4-yla m in o)p h en -
yl]a ceta m id e (40). A solution of 16 (0.96 g, 3.0 mmol) in 9
mL of HOAc was treated with 0.85 mL of Ac2O at 25 °C. After
2 h the solution was evaporated to dryness at 40 °C, stirred
in MeOH, and evaporated to dryness. The residue was
recrystallized from EtOH to give 0.50 g (46%) of a brown
1
mp 160-162 °C; H NMR (DMSO-d6) δ 9.47 (s, 1H), 8.45 (s,
1H), 7.75 (s, 1H), 7.33 (s, 1H), 7.32 (t, J ) 7.9 Hz, 1H), 7.11 (s,
1H), 7.08 (m, 2H), 3.93 (d, J ) 13.3 Hz, 6H), 2.50 (m, 1H);
HRMS m/z 348.1705 (M + H) +1. Anal. (C21H21N3O2‚0.2H2O)
C, H, N.
1
solid: mp 147-150 °C; H NMR (DMSO-d6) δ 10.00 (s, 1H),
9.48 (s, 1H), 8.48 (s, 1H), 7.75 (s, 1H), 7.66 (s, 1H), 7.34 (s,
1H), 7.29 (m, 2H), 6.89 (m, 1H), 3.96 (s, 3H), 3.91 (s, 3H), 2.05
(s, 3H); MS (ES+) m/z 363.2 (M + H)+1. Anal. (C20H18N4O3‚
1.5H2O) C, H, N.
4-(4-Br om op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (34). The compound was prepared according to
the procedure for 8 using 4-bromoaniline in 47% yield, light
1
yellow solid: mp 199-200 °C; H NMR (DMSO-d6) δ 9.49 (s,
1H), 8.51 (s, 1H), 7.71 (s, 1H), 7.56 (d, J ) 9 Hz, 2H), 7.36 (s,
1H), 7.19 (d, J ) 9 Hz, 2H), 3.95 (s, 3H), 3.91 (s, 3H); MS (ES+)
m/z 383.8, 385.8 (M + H)+1. Anal. (C18H14BrN3O2‚0.5H2O) C,
H, N.
3-Eth oxy-4-m eth oxyben zoic Acid Meth yl Ester (42a ).
A mixture of 24.3 g (134 mmol) of 3-hydroxy-4-methoxybenzoic
acid methyl ester, 36.8 g (267 mmol) of K2CO3 and 16 mL (31.4
g, 201 mmol) of C2H5I in 500 mL of DMF was stirred at 100
°C for 6 h. An additional 16 mL (31.4 g, 201 mmol) of C2H5I
and 18.4 g (133 mmol) of K2CO3 was added and the reaction
was heated for 3 h more. The reaction was then filtered, the
insoluble material was washed with DMF and the combined
filtrate and wash were evaporated. The residue was mixed
with H2O and filtered and the insoluble material was washed
with H2O and dried. Recrystallization from heptane gave 22.5
4-(2-Br om op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r bon itr ile (35). To a suspension of NaH (60% in mineral
oil, 90 mg, 2.25 mmol) in 5 mL of DMF was added 2-bromo-
aniline (387 mg, 2.25 mmol). The mixture was heated to reflux
then cooled and 4c (250 mg, 1.05 mmol) was added. The
mixture was heated at reflux for 40 min then cooled to room
temperature and partitioned between water and ethyl acetate.
The organic layer was washed with dilute aqueous ammonium
hydroxide, dried over MgSO4, filtered and concentrated in
vacuo. EtOAc and diethyl ether were added to the residue and
the solid was collected to provide 168 mg (43%) of 35 as a tan
solid: mp 217-219 °C; 1H NMR (DMSO-d6) δ 9.58 (s, 1H), 8.38
(s, 1H), 7.85 (s, 1H), 7.76 (d, J ) 6 Hz, 1H), 7.58-7.42 (m,
2H), 7.36-7.32 (m, 2H), 3.95 (s, 3H), 3.93 (s, 3H); MS (ES+)
m/z 383.8, 385.8 (M + H)+1. Anal. (C18H14BrN3O2) C, H, N.
4-(4-F lu o r o -3-t r i flu o r o m e t h y lp h e n y la m i n o )-6,7-
d im eth oxyqu in olin e-3-ca r bon itr ile Hyd r och lor id e (36).
The compound was prepared as a hydrochloride salt in 72%
yield according to the procedure for 8 using 4-fluoro-3-
trifluoromethylaniline: mp 240-243 °C; 1H NMR (DMSO-d6)
δ 10.21 (broad s, 1H), 8.69 (s, 1H), 7.88 (s, 1H), 7.79-7.69 (m,
2H), 7.64-7.57 (m, 1H), 7.39 (s, 1H), 3.98 (s, 3H), 3.96 (s, 3H);
MS (ES+) m/z 391.9 (M + H)+1. Anal. (C19H13F4N3O2‚0.6HCl)
C, H, N.
1
g (80%) of product as white crystals: mp 81-83 °C; H NMR
(DMSO-d6) δ 1.34 (t, J ) 6.9 Hz, 3H), 3.81 (s, 3H), 3.84 (s,
3H), 4.04 (q, J ) 6.9 Hz, 2H), 7.07 (d, J ) 8.5 Hz, 1H), 7.42 (d,
J ) 2.0, 1H), 7.58 (dd, J ) 8.5, J ) 2.0 Hz, 1H); MS (ES+) m/z
210.9 (M + H) +1. Anal. (C11H14O4) C, H.
4-Eth oxy-3-m eth oxyben zoic Acid Meth yl Ester (42b).
Using the procedure described above for 42a and a total
reaction time of 2 h gave, after recrystallization from hexane,
82% of product as white crystals: 1H NMR (DMSO-d6) δ 1.35
(t, 3H, J ) 7.0), 3.81 (s, 3H), 3.82 (s, 3H), 4.09 (q, J ) 7.0 Hz,
2H), 7.05 (d, J ) 8.5 Hz, 1H), 7.44 (d, J ) 1.95 Hz, 1H), 7.56
(dd, J ) 8.5, 1.95 Hz, 1H); MS (EI) m/z 210 (M)+1
.
5-Eth oxy-4-m eth oxy-2-n itr oben zoic Acid Meth yl Ester
(43a ). Concentrated HNO3 (15 mL) was slowly added to a
slurry of 15.0 g (71.4 mmol) of 42a in 45 mL of glacial acetic
acid. After an exotherm had subsided, the reaction was
reheated to 50-60 °C for 45 min. It was then poured into ice
H2O and extracted with CH2Cl2. The extracts were washed
with H2O and 0.5 M NaOH, dried (MgSO4) and evaporated to
give 17.8 g (98% crude yield) of product as yellow crystals. An
analytical sample was obtained from CH2Cl2-hexane: mp 85-
88 °C; 1H NMR (DMSO-d6) δ 1.36 (t, J ) 7.0 Hz, 3H), 3.83 (s,
3H), 3.91 (s, 3H), 4.18 (q, 2H, J ) 7.0 Hz, 2H), 7.30 (s, 1H),
4-(3-Br om o-2-m eth ylph en ylam in o)-6,7-dim eth oxyqu in -
olin e-3-ca r bon itr ile (37). By the procedure as described
above for 8 using 3-bromo-2-methylaniline, the title compound
was obtained in 42% yield: mp 183-185 °C; 1H NMR (DMSO-
d6) δ 2.28 (s, 3H), 3.93 (s, 3H), 3.94 (s, 3H), 7.21 (dd, J ) 7.92,
7.89 Hz, 1H), 7.31 (d, J ) 7.70, 1H), 7.33 (s, 1H), 7.63 (d, J )
7.62 Hz, 1H), 7.82 (s, 1H), 8.37 (s, 1H), 9.49 (bs, 1H); MS (ES+)
7.63 (s, 1H); MS (ES+) m/z 256.0 (M + H)+1. Anal. (C11H13
NO6) C, H, N.
-
m/z 397.8, 399.8 (M + H)+1. Anal. (C19H16BrN3O2‚0.21C6H14
C, H, N.
)
4-Eth oxy-5-m eth oxy-2-n itr oben zoic Acid Meth yl Ester
(43b). The compound was prepared from 42b using the
procedure described above for 43a and a reaction time of 1 h.
A crude yield of 87% of the product was obtained. A purified
sample (light yellow crystals) was obtained by recrystallization
4-(4-Br om o-3-m eth ylph en ylam in o)-6,7-dim eth oxyqu in -
olin e-3-ca r bon itr ile (38). By the procedure as described
above for 8 using 4-bromo-3-methylaniline, the title compound
was obtained in 61% yield: mp 168-169 °C; 1H NMR (DMSO-

3254 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
Wissner et al.
from heptane: 1H NMR (DMSO-d6) δ 1.36 (t, J ) 7.02 Hz,
3H), 3.83 (s, 3H), 3.92 (s, 3H), 4.18 (q, J ) 7.02 Hz, 2H), 7.32
J ) 6.96 Hz, 3H), 3.86 (s, 3H), 4.12 (q, J ) 6.96 Hz, 2H), 7.03
(s, 1H), 7.45 (s, 1H), 8.59 (s, 1H); MS (ES+) m/z 245.2 (M +
(s, 1H), 7.61 (s, 1H); MS (ES+) m/z 255.8 (M + H)+1
.
H)+1
.
9-Oxo-2,3,6,9-t et r a h yd r o[1,4]d ioxin o[2,3-g]q u in olin e-
8-ca r bon itr ile (46c). Starting with 44c and using the pro-
cedure described for 46a a crude yield of 72% (tan powder) of
the title compound was obtained: 1H NMR (DMSO-d6) δ 4.34
(m, 2H), 4.37 (m, 2H), 7.05 (s, 1H), 7.46 (s, 1H), 8.58 (s, 1H);
7-Nitr o-2,3-dih ydr oben zo[1,4]dioxin e-6-car boxylic Acid
Meth yl Ester (43c). This compound was prepared from 42c27
using the procedure described for 43a and a reaction time of
3 h at 70 °C. Recrystallization from heptane-toluene gave 91%
of product as yellow crystals: 1H NMR (DMSO-d6) δ 3.80 (s,
3H), 4.39 (s, 4H), 7.32 (s, 1H), 7.66 (s, 1H); MS (EI) m/z 239
MS (ES+) m/z 228.8 (M + H)+1
.
(M)+1
.
4-Ch lor o-6-e t h oxy-7-m e t h oxyq u in olin e -3-ca r b on i-
tr ile (47a ). A slurry of 12.9 g (52.7 mmol) of 46b in 200 mL of
CH2Cl2 under N2 was treated with 23.1 mL (33.5 g, 263 mmol)
of oxalyl chloride and 1 mL of DMF for 2 days. The reaction
was then diluted with CHCl3 and mixed with saturated
aqueous NaHCO3. The organic layer was separated, dried
(MgSO4), evaporated and dried in vacuo. The crude yield was
86% of a gray solid: 1H NMR (DMSO-d6) δ 1.43 (t, 3H, J )
6.99 Hz), 4.02 (s, 3H), 4.27 (q, 2H, J ) 6.99 Hz), 7.43 (s, 1H),
7.55 (s, 1H), 8.99 (s, 1H); MS (ES+) m/z 262.8, 264.8 (M +
2-Am in o-5-eth oxy-4-m eth oxyben zoic Acid Meth yl Es-
ter (44a ). A mixture of 17.0 g (66.7 mmol) of 43a , 13.1 g (233
mmol) of powdered Fe and 17.7 g (334 mmol) of NH4Cl in 95
mL of H2O and 245 mL of MeOH was heated at reflux for 4.5
h. More Fe (13.1 g, 233 mmol) was added and the reaction
was heated for 2.5 h. Additional amounts of Fe (13.1 g, 233
mmol) and NH4Cl (17.7 g, 334 mmol) were then added and
heating was continued for 12 h more. The reaction was filtered
and the insoluble material was washed with H2O and MeOH.
The combined filtrate and wash was evaporated and the
residue was partitioned between CHCl3 and H2O. The organic
material was treated with activated carbon, dried (MgSO4),
evaporated and dried in vacuo (50 °C) to give 11.0 g (73% crude
yield) of product as tan crystals. An analytical sample was
prepared by recrystallization from EtOH: mp 120-122 °C; 1H
NMR (DMSO-d6) δ 1.26 (t, J ) 5.1 Hz, 3H), 3.74 (s, 6H), 3.85
(q, 2H, J ) 5.1 Hz), 6.36 (s, 1H), 6.44 (s, 2H), 7.14 (s, 1H); MS
(ES+) m/z 225.9 (M + H)+1. Anal. (C11H15NO4) C, H, N.
2-Am in o-4-eth oxy-5-m eth oxyben zoic Acid Meth yl Es-
ter (44b). This compound was prepared from 43b in a manner
similar to 44a . Additional powdered Fe and NH4Cl were added
as necessary to drive the reaction to completion. The crude
yield was 83% as pink crystals. A purified sample was
prepared by recrystallization from Et2O: 1H NMR (DMSO-
d6) δ 1.34 (t, J ) 6.99 Hz, 3H), 3.64 (s, 3H), 3.74 (s, 3H), 3.98
(q, 2H, J ) 6.99 Hz), 6.35 (s, 1H), 6.42 (s, 2H), 7.12 (s, 1H);
H)+1
.
4-Ch lor o-7-e t h oxy-6-m e t h oxyq u in olin e -3-ca r b on i-
tr ile (47b). This compound was prepared from 46b and oxalyl
chloride as previously described. The crude yield was 86% of
a gray solid: 1H NMR (DMSO-d6) δ 1.44 (t, 3H, J ) 6.96 Hz),
4.02 (s, 3H), 4.30 (q, 2H, J ) 6.96 Hz), 7.46 (s, 1H), 7.54 (s,
1H), 8.99 (s, 1H); MS (ES+) m/z 262.8, 264.8 (M + H)+1
.
9-Ch lor o-2,3-dih ydr o[1,4]dioxin o[2,3-g]qu in olin e-8-car -
bon itr ile (47c). This compound was prepared from 46c and
POCl3 as previously described for 4c. The crude yield was 97%
of a tan solid: 1H NMR (DMSO-d6) δ 4.48 (m, 4H), 7.55 (s,
1H), 7.57 (s, 1H), 8.94 (s, 1H); MS (ES+) m/z 246.8, 248.8 (M
+ H)+1
.
4-(3-Br om oph en ylam in o)-6-eth oxy-7-m eth oxyqu in olin e-
3-ca r bon itr ile (48). This compound was prepared from 47a
and 3-bromoaniline as previously described for 8. Recrystal-
lization from EtOH gave an 86% yield of tan crystals: mp 190-
191.5 °C; 1H NMR (DMSO-d6) δ 1.40 (t, 3H, J ) 6.99 Hz), 3.96
(s, 3H), 4.16 (q, 2H, J ) 6.99 Hz), 7.22 (m, 1H), 7.34 (m, 3H),
7.40 (s, 1H), 7.67 (s, 1H), 8.54 (s, 1H), 9.50 (s, 1H); HRMS (EI)
m/z calcd for C19H16BrN3O2 397.0426, found 397.0429. Anal.
(C19H16BrN3O2‚EtOH) C, H, N.
4-(3-Br om oph en ylam in o)-7-eth oxy-6-m eth oxyqu in olin e-
3-ca r bon itr ile (49). This compound was prepared from 47b
and 3-bromoaniline as previously described for 8. The crude
yield was 73% of a tan solid: 1H NMR (DMSO-d6) δ 1.42 (t, J
) 6.93 Hz, 3H), 3.91 (s, 3H), 4.25 (q, J ) 6.93 Hz, 2H), 7.22
(m, 1H), 7.33 (m, 3H), 7.40 (s, 1H), 7.68 (s, 1H), 8.54 (s, 1H),
9.54 (s, 1H); HRMS (EI) m/z calcd for C19H16Br N3O2 397.0426,
found 397.0436. Anal. (C19H16BrN3O2‚0.5H2O) C, H, N.
9-(3-Br om op h en yla m in o)-2,3-d ih yd r o[1,4]d ioxin o[2,3-
g]qu in olin e-8-ca r bon itr ile (50). This compound was pre-
pared from 47c and 3-bromoaniline as previously described
for 8. Recrystallization from EtOAc gave a 65% yield of tan
crystals: 1H NMR (DMSO-d6) δ 4.42 (m, 4H), 7.21 (m, 1H),
7.33 (m, 3H), 7.41 (s, 1H), 7.86 (s, 1H), 8.50 (s, 1H), 9.57 (s,
1H); HRMS (FAB+) m/z (M + H)+1 calcd for C18H13BrN3O2
382.0191, found 382.0215. Anal. (C18H12BrN3O2‚0.3H2O) C, H,
N.
MS (ES+) m/z 226.2 (M + H)+1
.
7-Am in o-2,3-d ih yd r ob en zo[1,4]d ioxin e-6-ca r b oxylic
Acid Meth yl Ester (44c). This compound was prepared from
43c in a manner similar to 44a . Additional powdered Fe and
NH4Cl were added to complete the reaction. Filtration of the
crude product through silica (CHCl3) helped to remove colored
impurities. Solids washed with MeOH to give a 90% yield of
product as tan crystals: 1H NMR (DMSO-d6) δ 3.75 (s, 3H),
4.13 (m, 2H), 4.23 (m, 2H), 6.24 (s, 3H), 7.14 (s, 1H); MS (ES+)
m/z 209.9 (M + H)+1
.
6-Eth oxy-7-m eth oxy-4-oxo-1,4-d ih yd r oqu in olin e-3-ca r -
bon itr ile (46a ). A solution of 10.2 g (45.3 mmol) of 44a and
12.6 mL (10.8 g, 90.7 mmol) of dimethylformamide dimethyl-
acetal in 50 mL of DMF was heated at reflux under N2 for 2.3
h. Volatile material was removed and the residue was azeo-
troped twice with toluene and dried in vacuo (25 °C). The
formamidine product 45a was a purple syrup that crystallized
on standing.
A solution of 5.32 mL (4.18 g, 102 mmol) of CH3CN in 80
mL of THF was added over 15 min to a solution of 40 mL (100
mmol) of 2.5 M n-BuLi in hexane and 60 mL of THF at -78
°C under N2. After 20 min, a solution of the crude 45a in 80
mL of THF was added over 0.5 h. The reaction was stirred at
-78 °C for 1 h and then quenched with 13 mL (13.7 g, 227
mmol) of glacial acetic acid. The mixture was warmed to 25
°C and volatile material was removed. The residue was
slurried with H2O, collected and dried. This material was
washed twice with CHCl3 to give 7.95 g of product as yellow
crystals. An analytical sample (cream crystals) was obtained
by boiling a portion of the crude product in MeOH and drying
in vacuo (50 °C): mp > 310 °C; 1H NMR (DMSO-d6) δ 1.38 (t,
J ) 6.96 Hz, 3H), 3.89 (s, 3H), 4.11 (q, J ) 6.96 Hz, 2H), 7.04
(s, 1H), 7.43 (s, 1H), 8.59 (s, 1H); MS (ES+) m/z 243.2 (M +
H)+1. Anal. (C13H12N2O3) C, H, N.
[4-(3-Br om op h en yla m in o)-6,7-d im eth oxyqu in olin -3-yl]-
m eth a n ol (51). To a stirred mixture of 5 (1.45 g, 3.36 mmol)
and 67 mL of toluene at 0 °C was added 1.53 M diisobutyl-
aluminum hydride in toluene (11 mL, 16.8 mmol) during 10
min. After an additional 10 min, the solution was treated with
7 mL of MeOH at 0 °C then stirred at 25 °C for 10 min. The
resulting gel was diluted with THF and stirred with Na2SO4‚
10H2O for 30 min. Solids were removed by filtration, and the
filtrate was concentrated. The residue was recrystallized from
CH2Cl2-hexane to give 0.56 g (43%) of a white solid: mp 176-
1
180 °C; H NMR (DMSO-d6) δ 8.76 (s, 1H), 8.44 (s, 1H), 7.38
(s, 1H), 7.17 (s, 1H), 7.15-6.60 (m, 4H), 5.31 (t, J ) 5.3 Hz,
1H), 4.49 (d, J ) 5.3 Hz, 2H), 3.93 (s, 3H), 3.73 (s, 3H); MS
(ES+) m/z 389.1, 391.1 (M + H)+1. Anal. (C18H17BrN2O3) C,
H, N.
7-Eth oxy-6-m eth oxy-4-oxo-1,4-d ih yd r oqu in olin e-3-ca r -
bon itr ile (46b). Starting with 44b and using the procedure
described for 46a a crude yield of 83% (yellow solid) of the
title compound was obtained: 1H NMR (DMSO-d6) δ 1.40 (t,
4-(3-Br om op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-

Inhibitors of EGF-R Kinase
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17 3255
ca r ba ld eh yd e (52). To a stirred solution of 51 (3.20 g, 8.22
mmol) in 82 mL of CH2Cl2 were added NaOAc (0.20 g, 2.5
mmol) and pyridinium chlorochromate (2.65 g, 12.3 mmol).
After 2 h at 25 °C the mixture was stirred at 0 °C with 1 N
NaOH (50 mL). After filtration through Celite the CH2Cl2 layer
was washed with water, dried, and concentrated. The residue
was chromatographed on silica gel with CH2Cl2-EtOAc to give
1.92 g (60%) of a white solid after recrystallization from
EtOAc: mp 175-177 °C; 1H NMR (DMSO-d6) δ 10.07 (s, 1Η),
10.05 (s, 1Η), 8.83 (s, 1Η), 7.38 (s, 1Η), 7.27 (s, 1Η), 7.14 (s,
1Η), 7.30-7.08 (s, 3Η), 3.96 (s, 3Η), 3.60 (s, 3Η); MS (ES+)
m/z 387.2, 389.2 (M + H)+1. Anal. (C18H15BrN2O3) C, H, N,
Br.
4-(3-Br om op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r boxylic Acid (53). A mixture of 5 (0.86 g, 2.0 mmol), 10
N NaOH (1.0 mL, 10 mmol), and 20 mL of EtOH was stirred
at 25 °C for 2 h. The resulting solution was concentrated to
remove EtOH, and the residue was dissolved in H2O and
acidified with NaH2PO4. The resulting white solid was filtered
off, washed with water, and dried to give 0.81 g (100%) of
product: mp 282-285 °C dec; 1H NMR (DMSO-d6) δ 14.0 (bs,
1H), 9.12 (s, 1H), 7.64 (s, 1H), 8.30-7.60 (m, 3H), 7.47 (s, 1H),
7.45 (s, 1H), 7.26 (s, 1H), 4.01 (s, 6H); MS (ES+) m/z 403.1,
405.1 (M + H)+1. Anal. (C18H15BrN2O4) C, H, N.
4-(3-Br om op h en yla m in o)-6,7-d im et h oxyq u in olin e-3-
ca r boxylic Acid Am id e (54). A mixture of 53 (4.03 g, 10
mmol), 1,1′-carbonyldiimidazole (3.24 g, 20 mmol), and 100 mL
of DMF was stirred at 55 °C for 30 min. The resulting solution
was cooled to 0 °C, saturated with NH3 gas, and stirred at 25
°C for 45 min. The residue obtained after evaporation of
solvents was stirred in H2O. The solid was filtered, washed
with H2O, dried, and recrystallized from acetone to give a gray
solid (4.0 g, 100%): mp 239-242 °C; 1H NMR (DMSO-d6) δ
10.1 (s, 1H), 8.84 (s, 1H), 8.15 (s, 1H), 7.61 (s, 1H), 7.36 (s,
1H), 7.25-6.80 (m, 5H), 3.94 (s, 6H); MS (ES+) m/z 402.1,
404.2 (M + H)+1. Anal. (C18H16BrN3O3) C, H, N, Br.
washed with several portion of water, then with brine. After
drying over anhydrous MgSO4, the solution was evaporated
in vacuo to a solid. This solid was suspended in CH2Cl2 and
poured into hexanes. The product was collected to give 0.95 g
1
(42%) of a tan solid: mp 201-205 °C; H NMR (DMSO-d6) δ
9.44 (s, 1H), 8.57 (s, 1H), 7.74 (s, 1H), 7.33 (m, 4H), 7.16 (m,
1H), 6.28 (s, 2H); MS (ES+) m/z 368.1, 370.1 (M + H)+1. Anal.
(C17H10N3O2Br) C, H, N, Br.
4-(3-Br om op h en yla m in o)-3,4-d ih yd r o-2H-[1,4]d ioxep i-
n o[2,3-g]qu in olin e-3-ca r bon itr ile (59). To a stirred mixture
of 55 (0.71 g, 2.0 mmol), K2CO3 (1.10 g, 8.0 mmol), and 8 mL
of DMF at 65 °C was added a solution of 1,3-dibromopropane
(0.20 mL, 2.0 mmol) during 30 min. After an additional 30
min at 65 °C the DMF was evaporated, and the residue was
partitioned between CH2Cl2 and H2O. After filtration, the CH2-
Cl2 layer was washed with H2O, dried, and concentrated. The
residue chromatographed on silica gel, eluting the product with
60:30:1 CH2Cl2-EtOAc-HOAc to give 0.32 g (40%) of white
solid: mp 120-130 °C; 1H NMR (DMSO-d6) δ 9.68 (s, 1H), 8.55
(s, 1H), 8.02 (s, 1H), 7.45-7.12 (m, 5H), 7.35 (s, 1H), 4.40-
4.24 (m, 4H), 2.30 (s, 3H), 2.24-2.19 (m, 2H); MS (ES+) m/z
396.1, 398.1 (M + H)+1. Anal. (C19H14BrN3O‚HO2CCH3) C, H,
N.
1-(3-Br om op h en yla m in o)-6,7-d im eth oxyisoqu in olin e-
4-ca r bon itr ile (61). A mixture of 6 mmol (0.65 mL, 1.03 g)
of 3-bromoaniline and 5 mmol (1.25 g) of 6028 in 10 mL of
2-propanol was stirred and heated at reflux in an oil bath at
114 °C for 23 h. On cooling, the product was collected and
washed with cold 2-propanol and ether giving 2.04 g (97%) of
the product: mp 291-293 °C; 1H NMR (DMSO-d6) δ 10.11 (t,
J ) 0.5 Hz, 1H), 8.42 (s, 1H), 8.06 (d, J ) 12.2 Hz, 1H), 8.03
(d, J ) 1.6 Hz, 1H), 7.80 (m, 1H), 7.36 (m, 2H), 7.17 (s, 1H),
4.02 (s, 3H), 4.01 (s, 3H); MS (ES+) m/z 384.1, 386.2 (M +
H)+1. Anal. (C18H14N3O2Br‚1.0HCl) C, H, N, Br.
1-(3-Ch lor op h en yla m in o)-6,7-d im eth oxyisoqu in olin e-
4-ca r bon itr ile (62). A mixture of 1.24 g (5 mmol) of 6028 and
0.63 mL (0.76 g, 6 mmol) of 3-chloroaniline in 10 mL of
2-methoxyethanol was heated at reflux in an oil bath at 149
°C. for 1 h. On cooling the solid was collected and washed with
ether. The salt was stirred for about 2 h with 25 mL of
saturated aqueous NaHCO3. The resulting solid was filtered,
washed with water, and dried giving 1.62 g (95%) of the
product: mp 278-280 °C; 1H NMR (DMSO-d6) δ 9.64 (s, 1H),
8.42 (s, 1H), 7.92 (m, 2H), 7.75 (m, 1H), 7.41 (t, J ) 8.1 Hz,
1H), 7.16 (m, 2H), 4.00 (s, 3H), 3.99 (s, 3H); MS (ES+) m/z
340.2 (M + H)+1. Anal. (C18H14N3O2Cl) C, H, N, Cl.
4-(3-Br om op h en yla m in o)-6,7-d ih yd r oxyq u in olin e-3-
ca r bon it r ile (55). A 5.11 g (13.3 mmol) quantity of 8 and
30.74 g (266 mmol) of pyridine hydrochloride were mixed and
then heated in an oil bath at 207 °C giving a melt. This was
heated for 1 h, then cooled and treated with 100 mL of water.
The resulting solid was filtered and digested with 2-methoxy-
ethanol. This gave 3.0 g (63%) of a gray solid: mp 270-275
1
°C; H NMR (DMSO-d6) δ 12.09 (s, 1H, broad), 10.86 (s, 1H),
10.49 (s, 1H, broad), 8.94 (s, 1H), 7.94 (s, 1H), 7.68 (s, 1H),
7.57 (m, 2H), 7.48 (m, 2H); MS (ES+) m/z 356.1, 358.1 (M +
H)+1. Anal. (C16H10N3)2Br‚1.0H2O) C, H, N.
4-(3-Br om op h en yla m in o)-6,7-b is(m et h oxym et h oxy)-
qu in olin e-3-ca r bon itr ile (56). This compound was prepared
in 32% yield using chloromethyl methyl ether according to the
procedure for 6, except that the reaction was run entirely at 0
Ack n ow led gm en t. The authors thank Drs. Philip
Frost, Tarek Mansour, Dennis Powell, and Janis Upesla-
cis for their support and encouragement. We also thank
the members of the Wyeth-Ayerst Discovery Analytical
Chemistry group for analytical and spectral determina-
tions.
1
°C: mp 169-177 °C (EtOAc); H NMR (DMSO-d6) δ 9.92 (s,
1H), 8.37 (s, 1H), 7.80 (bs, 1H), 7.25 (s, 1H), 7.20-6.80 (m,
4H), 5.44 (s, 2H), 5.31(s, 2H), 3.44 (s, 3H); 3.27 (s, 3H); MS
(ES+) m/z 444.0, 446.0 (M + H)+1. Anal. (C20H18BrN3O4) C,
H, N.
Refer en ces
4-(3-Br om op h en yla m in o)-6,7-b is(2-m et h oxyet h oxy)-
qu in olin e-3-ca r bon itr ile (57). This compound was prepared
in 24% yield using bromoethyl methyl ether according to the
procedure for 6, except the reaction was run at 50 °C: mp 135-
(1) Plowman, G. D.; Ullrich, A.; Shawver, L. K. Receptor Tyrosine
Kinases as Targets for Drug Intervention. Drug News Perspect.
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Epidermal Growth Factor-related Peptides and Their Receptors
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of the Epidermal Growth Factor Receptor in Human Cancers.
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1
138 °C (MeOH); H NMR (DMSO-d6) δ 9.50 (s, 1H), 8.54 (s,
1H), 7.71 (s, 1H), 7.40 (s, 1H), 7.36-7.05 (m, 4H), 4.33-4.23
(m, 4H), 3.77-3.73 (m, 4H), 3.35-3.30 (m, 6H); MS (ES+) m/z
472.0, 473.9 (M + H)+1. Anal. (C22H22BrN3O4) C, H, N.
8-(3-Br om op h en yla m in o)[1,3]d ioxolo[4,5-g]qu in olin e-
7-ca r bon itr ile (58). A solution of 2.17 g (6.09 mmol) of 55,
0.59 mL (1.18 g, 9.14 mmol) of bromochloromethane, and 2.98
g (9.14 mmol) of Cs2CO3 in 20 mL of DMF was stirred and
heated in an oil bath at 111 °C for 2 h. The reaction was poured
into 75 mL of water and the resulting mixture was extracted
with four 50-mL portions of CH2Cl2. The combined extracts
were dried over anhydrous MgSO4, then concentrated with the
addition of hexanes until an oil precipitated. This oil was
separated and taken up in ethyl acetate, and this solution was

3256 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 17
Wissner et al.
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