Synthesis and Src kinase inhibitory activity of a series of 4-phenylamino-3-quinolinecarbonitriles

Article abstract of DOI:10.1021/jm000420z

Screening of a directed compound library in a yeast-based assay identified 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (2a) as a Src inhibitor. An enzymatic assay established that 2a was an ATP-competitive inhibitor of the kinase activity of Src. We present here SAR data for 2a which shows that the aniline group at C-4, the carbonitrile group at C-3, and the alkoxy groups at C-6 and C-7 of the quinoline are crucial for optimal activity. Increasing the size of the C-2 substituent of the aniline at C-4 of 2a from chloro to bromo to iodo resulted in a corresponding increase in Src inhibition. Furthermore, replacement of the 7-methoxy group of 2a with various 3-heteroalkylaminopropoxy groups provided increased inhibition of both Src enzymatic and cellular activity. Compound 25, which contains a 3-morpholinopropoxy group, had an IC₅₀ of 3.8 nM in the Src enzymatic assay and an IC₅₀ of 940 nM for the inhibition of Src-dependent cell proliferation.

Full text of DOI:10.1021/jm000420z

822
J . Med. Chem. 2001, 44, 822-833
Syn th esis a n d Sr c Kin a se In h ibitor y Activity of a Ser ies of
4-P h en yla m in o-3-qu in olin eca r bon itr iles
Diane H. Boschelli,* Yanong D. Wang, Fei Ye, Biqi Wu, Nan Zhang, Minu Dutia, Dennis W. Powell,
Allan Wissner, Kim Arndt, J ennifer M. Weber, and Frank Boschelli
Chemical Sciences and Oncology, Wyeth-Ayerst Research, 401 North Middletown Road, Pearl River, New York 10965
Received September 29, 2000
Screening of a directed compound library in a yeast-based assay identified 4-[(2,4-dichlorophen-
yl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (2a ) as a Src inhibitor. An enzymatic assay
established that 2a was an ATP-competitive inhibitor of the kinase activity of Src. We present
here SAR data for 2a which shows that the aniline group at C-4, the carbonitrile group at C-3,
and the alkoxy groups at C-6 and C-7 of the quinoline are crucial for optimal activity. Increasing
the size of the C-2 substituent of the aniline at C-4 of 2a from chloro to bromo to iodo resulted
in a corresponding increase in Src inhibition. Furthermore, replacement of the 7-methoxy group
of 2a with various 3-heteroalkylaminopropoxy groups provided increased inhibition of both
Src enzymatic and cellular activity. Compound 25, which contains a 3-morpholinopropoxy group,
had an IC50 of 3.8 nM in the Src enzymatic assay and an IC50 of 940 nM for the inhibition of
Src-dependent cell proliferation.
In tr od u ction
pyrido[2,3-d]pyrimidin-7(8H)-ones provided inhibitors of
either platelet-derived growth factor receptor TK (PDG-
Fr)23 or fibroblast growth factor receptor TK (FGFr).24
By varying the aniline substituent at C-4 of their
quinazolines, the group at Rhone Poulenc Rorer ob-
tained inhibitors of CSF-1R TK.25
We recently reported that 4-[(3-bromophenyl)amino]-
6,7-dimethoxy-3-quinolinecarbonitrile (1a ) was an in-
hibitor of epidermal growth factor receptor (EGFr)
TK,26,27 showing slightly reduced activity compared to
4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline (1b),
one of the most potent EGFr inhibitors known.28 Hoping
Tyrosine kinases (TKs), enzymes that catalyze the
specific phosphorylation of tyrosine residues on proteins,
can be divided into two classes: the transmembrane
growth factor receptor TKs (RTKs) and the cytoplasmic
TKs which include Src. Src is a member of the Src family
of kinases (SFKs) that share a common structural
organization and have a high degree of homology,
especially in their ATP-binding regions.1-4 In addition
to Src, the SFKs include Yes, Fyn, Fgr, Lck, Hck, Lyn,
Blk, and Yrk. While some SFKs, including Src, Yes, and
Fyn, are widely expressed, others such as Lck are
limited in their expression.
Src is a potential therapeutic target for the treatment
of diverse human disease states. Studies with Src-
deficient mice demonstrated that bone resorption by
osteoclasts requires Src activity, implying that Src
inhibitors might be useful in the treatment of osteoporo-
sis.5-7 In addition, since Src is overexpressed in colon,
breast, hepatic, and pancreatic tumors, as well as in
certain B-cell leukemias and lymphomas, Src inhibitors
may be efficacious in the treatment of cancer.3,8-13
Further evidence for an active role for Src in tumor
growth comes from experiments where anti-sense src
expression in ovarian and colon tumor cells inhibited
the growth of these cells in mouse xenograft models.14,15
Various classes of SFK inhibitors have been reported
by several companies including, 5,10-dihydropyrimido-
[4,5-b]quinolin-4(1H)-ones16 and pyrazolo[3,4-d]pyrim-
idines17 from Pfizer, pyrrolo[2,3-d]pyrimidines from
that the 3-quinolinecarbonitrile core could function as
a template for inhibitors of other kinases, we prepared
a library of 6,7-dimethoxy-3-quinolinecarbonitriles with
various anilino groups at C-4. These compounds were
screened in several assays, including a yeast-based Src
assay. Yeast strains were prepared that harbor plasmids
containing galactose-inducible src genes.29 When grown
in medium with galactose, these cells express Src and
consequently undergo rapid cell death. These yeast
strains were embedded in agar, and when the 3-quino-
Novartis,18,19 pyrido[2,3-d]pyrimidin-7(8H)-ones20 and
21
1,6-naphthyridin-2(1H)-ones
from Parke-Davis, and
4-anilinoquinazolines from Rhone Poulenc Rorer.22 In
some cases, modification of certain substituents on these
core structures led to inhibitors of other kinase families.
For example, varying the group at C-6 of Parke-Davis’s
* To whom correspondence should be addressed. Tel: 845-732-3567.
Fax: 845-732-5561. E-mail: bosched@war.wyeth.com.
10.1021/jm000420z CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/24/2001

4-Phenylamino-3-quinolinecarbonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 823
Sch em e 1
Sch em e 2a
a
(a) DIBAL, THF, 0 °C; (b) MnO2, CHCl3; (c) (1) 0.5 N NaOH,
EtOH, reflux, (2) concd HCl; (d) (1) CDI, DMF, THF, 60 °C, (2) aq
NH4OH.
groups at this position. Ethyl 4-chloro-6,7-dimethoxy-
3-quinolinecarboxylate (14)33 was treated with 2,4-
dichloroaniline in the presence of pyridine hydrochloride
to provide 15. The ester group of 15 was reduced with
diisobutylaluminum hydride, as shown in Scheme 2, to
provide the 3-hydroxymethyl derivative 16. Oxidation
of 16 with manganese(IV) oxide provided the 3-aldehyde
derivative 17. Additional analogues of 2a were prepared
by hydrolysis of the ester group of 15 to give the
3-carboxylic acid derivative 18, which was then con-
verted to the primary amide derivative 19 by treatment
with 1,1′-carbonyldiimidazole followed by aqueous am-
monium hydroxide.
The lead compound 2a has methoxy substituents at
C-6 and C-7 of the quinoline ring. The C-6 and C-7
unsubstituted analogue 2b was prepared from the
known 4-chloro compound 4b.34 The mono 5-, 6-, 7-, and
8-methoxy derivatives, 2c-2f, were prepared from the
corresponding 4-chloro compounds, 4c-4f. While 4d -
4f were previously reported in the literature,26 4c was
prepared as shown in Scheme 3. Formation of the
amidine derivative of 2-amino-6-methoxybenzoic acid35
followed by addition of the anion of acetonitrile provided
4-hydroxy-5-methoxy-3-quinolinecarbonitrile (20). Reac-
tion of 20 with phosphorus oxychloride in the presence
of a catalytic amount of N,N-dimethylformamide gave
4c. The 5,7-dimethoxy isomer of 2a , namely 2g, was
obtained from the corresponding 4-chloro derivative 4g,
which was prepared as shown in Scheme 3. Reaction of
3,5-dimethoxyaniline with ethyl (ethoxymethylene)-
cyanoacetate and subsequent thermal cyclization pro-
vided 21. Treatment of 21 with phosphorus oxychloride
gave the desired 4-chloro derivative 4g.
linecarbonitriles were spotted on the surface, a ring of
cell growth implied that the compound was blocking the
toxic effect of Src. One of the best compounds in
restoring cell proliferation in this assay was 2a , which
contains a 2,4-dichloroanilino group at C-4.30 The
structure of 2a suggested that this compound was an
inhibitor of Src kinase activity, and this was confirmed
by an enzymatic assay. The corresponding quinazoline
3 also inhibited Src kinase activity but was 8-fold less
active than 2a . These initial results led us to investigate
additional analogues of 2a as Src kinase inhibitors.
Ch em istr y
As depicted in Scheme 1, 2a was prepared by treat-
ment of 4-chloro-6,7-dimethoxy-3-quinolinecarbonitrile
(4a )26,27 with 2,4-dichloroaniline. When this reaction was
run in ethoxyethanol in the presence of pyridine hydro-
chloride a 25% yield of 2a was obtained. This yield was
increased to 67% by first generating the anion of 2,4-
dichloroaniline with sodium hydride followed by addi-
tion of 4a . The corresponding quinazoline and quinoline
analogues, namely 3 and 6, were obtained from the
reaction of 2,4-dichloroaniline with 4-chloro-6,7-dimeth-
oxyquinazoline (5)31 and 4-chloro-6,7-dimethoxyquino-
line (7),32 respectively.
The linker group at C-4 of 2a was varied to include
the phenol derivative 8, the thiophenol derivative 9, the
benzylamine derivative 10, the methylanilino derviative
11, and the carboxamide derivative 12. All of these were
prepared from 4a by standard conditions. Treatment of
2a with methyl iodide and sodium hydride provided a
readily separable mixture of predominately 13, the
1-methyl analogue, along with the 4-methyl isomer 11.
To study the effect of the carbonitrile group at C-3,
several analogues of 2a were prepared with alternative
Additional 6,7-dialkoxy analogues of 2a were pre-
pared as shown in Scheme 4. Demethylation of 2a with
pyridine hydrochloride provided the 6,7-dihydroxy ana-
logue 2h . To cleanly alkylate the hydroxy groups of 2h ,
it was necessary to first completely acylate the molecule,
then selectively deacylate the hydroxy groups to give
22. Alkylation of the hydroxyl groups of 22 with ethyl
iodide or n-butyl bromide, followed by removal of the
acyl group from the 4-nitrogen, provided the 6,7-
diethoxy and 6,7-di-n-butoxy analogues 2i and 2j,
respectively.
A large number of analogues of 2a were prepared with
various halogen substituents on the 4-anilino group.
Compounds 2k -2v were prepared by the reaction of 2a
with the desired aniline in the presence of either
pyridine hydrochloride or sodium hydride. The anilines

824 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Boschelli et al.
Sch em e 3a
a
(a) (1) DMF-DMA, reflux, (2) n-BuLi, MeCN, THF, -78 °C; (b) POCl3, cat. DMF, reflux; (c) (1) EtOCHdC(CO2Et)CN, (2) Ph-O-Ph,
Ph-Ph, reflux.
Sch em e 4a
suggesting that this compound is an ATP-competitive
inhibitor. Compound 2a was tested against several other
kinases, including EGFr-2 (ErbB-2), FGFr, and cdk4
(cyclin-dependent kinase 4). No activity (less than 20%
inhibition) was observed against ErbB-2, FGFr, and
cdk4 when 2a was tested at doses of 2, 1, and 10 µg/
mL, respectively. When PP1, 4-amino-5-(4-methylphen-
yl)-7-tert-butylpyrazolo[3,4-d]pyrimidine, a Src inhibitor
reported by Pfizer,17 was tested under our assay condi-
tions, an IC50 value of 35 nM was obtained (Table 1).
Therefore the Src inhibitory activity of 2a is comparable
to that of PP1. As mentioned previously, compared to
the corresponding quinazoline 3 (IC50 value of 250 nM),
2a exhibited an 8-fold increase in Src inhibitory activity.
This result is consistent with our earlier report that 6,7-
dimethoxy-4-[(3,4,5-trimethoxyphenyl)amino]-3-quino-
linecarbonitrile (28a ) was a more potent inhibitor of Src
activity than the corresponding quinazoline 28b.36
a
(a) Pyridine hydrochloride, 215-222 °C; (b) (1) Ac2O, DMAP,
pyridine, reflux, (2) NaHCO3, H2O, MeOH; (c) (1) K2CO3, DMF,
RX, 60 °C, (2) K2CO3, MeOH, reflux.
used included 2-chloro, 4-chloro, all five possible dichloro
isomers, and some 2,4-dihaloanilines other than 2,4-
dichloro.
Derivatives of 2a with water-solublizing groups at C-6
and/or C-7 of the quinoline ring were prepared as shown
in Scheme 5. The diol 2h was converted into the 6,7-
di(3-chloropropoxy) derivative which was then treated
with morpholine to provide 23. In this case, unlike the
preparations of 2i and 2j, the nitrogen atom was not
protected in the alkylation reaction, which resulted in
the low yield (31%) of 23 from 2h . The 6-(3-morpholi-
nopropoxy) derivative 24 was obtained by reaction of
4w 26 with 2,4-dichloroaniline. The isomeric 7-(3-mor-
pholinopropoxy) derivative 25 was obtained in a similar
fashion from 4x.26 An alternative route to 25 was also
used wherein intermediate 4y26 was treated with 2,4-
dichloroaniline to provide 2y, which was then reacted
with morpholine in the presence of NaI. By varying the
heterocyclic amine in the reaction with 2y from mor-
pholine to N-methylpiperazine and 4-hydroxypiperidine,
analogues 26 and 27 were obtained.
As shown in Table 1, the NH group at C-4 of 2a is
essential for Src inhibitory activity. Substantial de-
creases in activity were observed with compounds 8-12,
the analogues of 2a with O, S, NHCH2, NMe, and NHC-
(O) substituents at C-4. The importance of the C-4 NH
group is further demonstrated by the lack of activity of
the 1-Me isomer 13. In addition, Table 1 also illustrates
that the 3-carbonitrile group of 2a is required for good
activity. The 3-unsubstituted quinoline 6 (IC50 value of
84 nM) was less active than 2a , and while some activity
was seen with the 3-aldehyde analogue 17 (IC50 value
of 250 nM), the 3-ester, alcohol, acid, and amide deriva-
Resu lts a n d Discu ssion
The screening lead 2a had an average IC50 value of
30 nM in the Src enzymatic assay, when run with an
ATP concentration of 0.1 mM. In a study where the ATP
concentration was increased from 0.1 to 1 to 5 mM, the
IC50 for 2a increased from 23 to 130 to 270 nM,

4-Phenylamino-3-quinolinecarbonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 825
Sch em e 5a
a
(a) 3-Chloropropyl p-toluenesulfonate, tricaprylmethylammonium chloride, K2CO3, acetone, reflux; (b) morpholine, 130 °C; (c) 2,4-
dichloroaniline, pyridine hydrochloride, 2-ethoxyethanol; (d) NaI, ethylene glycol dimethyl ether, 100 °C.
tives, 15, 16, 18, and 19, all provided less than 20%
inhibition when tested at a concentration of 1 µM. These
results correspond with the 4-(3-bromoanilino)quinoline
series of EGFr inhibitors, where after the 3-carbonitrile
derivative 1a , the best activity was seen with the
3-aldehyde derivative.27
activity was seen with the 6,7-diethoxy analogue and
the 6,7-dihydroxy analogue had activity comparable to
1a .27
In the study of the 2,4-dichloroanilino group, shown
in Table 3, the 2-chloro derivative 2k was about 3-fold
less active than 2a while the 4-chloro derivative 2l was
about 1.5-fold less active than 2a . Of the five possible
dichloro isomers of 2a , only the 3,5-dichloro analogue
2p (IC50 value of 1.06 µM) had greatly reduced activity
compared to 2a . Comparing the 4-chloroanilino ana-
logues where the substituent at C-2 was varied from
fluoro to bromo to iodo, an increase in Src inhibition was
seen with the larger bromo and iodo substituents. In
fact, the best activity in this series was seen with 2t
(IC50 value of 6.2 nM), which has a 4-chloro-2-iodo-
anilino group at C-4. However 2u , the 2-chloro-4-iodo
isomer of 2t, had reduced activity (IC50 value of 95 nM)
compared to 2a , while the 2-chloro-4-bromo analogue
2v had increased activity (IC50 value of 15 nM). These
results imply that there is a greater steric constraint
Table 2 shows the effect of varying the 6,7-dimethoxy
substituents of 2a . Removal of both alkoxy groups, as
in 2b, resulted in a dramatic loss of activity. While the
6- and 7-methoxy derivatives 2d and 2e were both about
1 log order less active than 2a , further reductions in
activity were observed with the 5-methoxy derivative
2c and especially the 8-methoxy derivative 2f. In
addition, decreased activity was seen with the 6,7-
dihydroxy analogue 2h and the 6,7-dibutoxy analogue
2j, but increased activity was seen with the 6,7-diethoxy
analogue 2i (IC50 value of 11 nM). This result is in
contrast to the 4-[(3-bromophenyl)amino]-3-quinoline-
carbonitrile series of EGFr inhibitors wherein reduced

826 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Boschelli et al.
Ta ble 1. Inhibition of Src Kinase Activity by C-3 and C-4
Ta ble 3. Inhibition of Src Kinase Activity By
6,7-Dimethoxy-4-phenylamino-3-quinolinecarbonitrilesa
Substituted 6,7-Dimethoxyquinolinesa
Src: IC50 (nM) or
% inhib at 1 µM
compd
R
Src: IC50 (nM) compd
R
Src: IC50 (nM)
compd
Y
X
2a
2k
2l
2m
2n
2o
2p
2,4-diCl
2-Cl
4-Cl
2,3-diCl
2,5-diCl
2,6-diCl
3,5-diCl
30
95
53
2q
2r
2s
2t
3,4-diCl
57
53
10
6.2
95
15
2a
3
NH
CN
30
250
84
2-F, 4-Cl
2-Br, 4-Cl
2-I, 4-Cl
2-Cl, 4-I
2-Cl, 4-Br
quinazoline analogue of 2a
6
NH
H
90
40
61
2u
2v
8
9
O
S
CN
CN
CN
CN
CN
37%
2200
24%
32%
NAb
NAb
NAb
NAb
250
1100
10
11
12
13
15
16
17
18
19
PP1c
NHCH2
NMe
NHC(O)
a
IC50 values reported for Src inhibition represent the means
of at least two separate determinations with typical variations of
less than 40% between replicate values.
1-Me analogue of 2a
COOEt
NH
NH
NH
NH
NH
CH2OH
CHO
COOH
CONH2
Ta ble 4. Inhibition of Src Kinase Activity and Cell Prolifera-
tion by 4-(2,4-Dichlorophenylamino)-3-quinolinecarbonitriles
NAb
NAb
35
a
IC50 or % inhibition values reported for Src inhibition repre-
sent the means of at least two separate determinations with typical
b
variations of less than 40% between replicate values. NA, less
than 20% inhibition at 1 µM. c Purchased from Calbiochem (San
Diego, CA).
IC50 (µM)
Ta ble 2. Inhibition of Src Kinase Activity by Various C-5, C-6,
C-7, and C-8 Substituted 4-(2,4-Dichlorophenylamino)-
3-quinolinecarbonitrilesa
nM
Src
Fyn
compd
R6
R7
Srca cellsb cellsb
2a
23
24
25
26
Me
Me
30
5.2 >10
3.3 >10
(CH2)3-morpholine (CH2)3-morpholine 19
(CH2)3-morpholine Me
220 >10
3.8
8.7
>10
3.2
2.9
4.5
Me
Me
(CH2)3-morpholine
(CH2)3-N-methyl-
piperazine
(CH2)3-4-hydroxy-
piperidine
0.94
1.0
27
Me
3.5
1.4
a
IC50 values reported for Src inhibition represent the means
of at least two separate determinations with typical variations of
less than 40% between replicate values. Anchorage-independent
assay. IC50 values reported represent the means of at least four
separate determinations.
Src: IC50 (nM) or
% inhib at 1 µM
b
compd
R5
R6
R7
R8
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
OMe
H
H
H
OMe
H
H
H
OMe
H
H
OMe
H
H
OMe
H
H
H
OMe
H
OMe
OH
OEt
O-n-Bu
H
H
H
H
H
OMe
H
H
H
30
NAb
1200
320
anchorage independence that has an absolute require-
ment for Src, while anchorage-dependent growth, i.e.,
growth on plastic, does not necessarily rely on Src
activity. Therefore, an anchorage-independent growth
assay with plates that do not support cell adhesion was
used.37 In this suspension assay, an IC50 value of 5.2
µM was obtained for 2a (see Table 4).
200
NAb
52%
NAb
11
H
OH
OEt
O-n-Bu
2j
H
160
a
IC50 or % inhibition values reported for Src inhibition repre-
sent the means of at least two separate determinations with typical
There are several reports in the literature wherein
the addition of a 3-morpholinopropoxy group at either
C-6 or C-7 of various 4-anilinoquinazoline TK inhibitors
resulted in improved water solubility and cellular
activity.38-40 This basic amine tail was added at either
the C-6, the C-7, or both the C-6 and C-7 positions of
2a , to give 23-25, respectively. As shown in Table 4,
the most potent of these three compounds was 25, which
had increased activity not only in the suspension assay
(IC50 of 940 nM) but also in the enzymatic assay (IC50
of 3.8 nM). This result suggests that a basic amine tail
at C-7 leads to an additional interaction with Src, while
this same substitution at C-6 is detrimental. The dose-
response curve for 25 in the suspension assay is shown
in Figure 1. It should be noted that in the assay
measuring the inhibition of cell growth on plastic, an
IC50 value of 3.2 µM was obtained for 25. Since addition
b
variations of less than 40% between replicate values. NA, less
than 20% inhibition at 1 µM.
at C-4 of the anilino group than at C-2. Interestingly,
we previously reported that addition of a bromo group
at C-2 of the anilino group of 28a caused a decrease in
the IC50 for Src inhibition from 35 nM for 28a to 170
nM for 28c.36 This discrepancy suggests that the 3,4,5-
trimethoxyanilino derivative 28a has a binding mode
that is different from that of 2a .
Compound 2a was next evaluated for its ability to
block the proliferation of rat fibroblasts stably trans-
fected with a plasmid expressing activated Src. In a
proliferation assay measuring the inhibition of the
growth of these cells on plastic, an IC50 value of 18 µM
was obtained for 2a . Src-transformed fibroblasts exhibit

4-Phenylamino-3-quinolinecarbonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 827
F igu r e 1. Suspension growth assay with Src-tranformed
fibroblasts treated with 25. Cells were plated into 96-well
ultralow binding plates on day 0. On day 1, serial dilutions of
compound were added to the indicated concentrations, and
proliferation was scored on day 4. Relative cell proliferation
was determined using the CellTiter 96 cell proliferation assay
kit (Promega). The curve shown is the calculated fit to the data.
of the water solublizing group at C-7 was preferred to
addition at C-6, additional derivatives of 25 were
prepared, including the N-methylpiperazine and 4-hy-
droxypiperidine analogues 26 and 27. In the enzymatic
assay, 27 had comparable activity to 25, while 26
exhibited about one-half the activity of 25. However, in
the suspension assay all three compounds had similar
activity.
We next studied the effect of 25 on the phosphoryla-
tion of cellular proteins in extracts prepared from the
Src-transformed fibroblasts. Figure 2A shows a Western
blot of these whole cell extracts probed with phospho-
tyrosine antibody. Compound 25 caused a dramatic
reduction of total cell phosphotyrosine at submicromolar
concentrations. We also examined the tyrosine phos-
phorylation of the 80-kDa Src target protein cortactin
(Figure 2B).41 A Western blot of cortactin immunopre-
cipitates from the Src-transformed fibroblasts extracts
treated with 25 indicated that the phosphorylation of
cortactin is substantially reduced at 1 µM. Under these
conditions of protein loading, none of these phosphoty-
rosine-containing proteins can be detected on similar
blots of extracts from rat fibroblasts transfected with
the vector alone (data not shown). Therefore, the ty-
rosine phosphorylation observed here is Src-dependent,
and 25 inhibits Src at sub-micromolar concentrations
in cells.
F igu r e 2. Effect of 25 on Src-dependent tyrosine phospho-
rylation. Equal numbers of Src-transformed cells were exposed
to various concentrations of 25 for 5 h. Extracts were analyzed
on a 4-15% SDS-polyacrylamide gradient gel. Blots were
probed with antibody to phosphotyrosine (A, top panel). Equal
loading was verified by probing equivalent blots with antibod-
ies to Src and actin (A, bottom panel). Lane 1, no compound;
lane 2, 0.31 µM 25; lane 3, 0.62 µM 25; lane 4, 1.25 µM 25,
lane 5, 2.5 µM 25; lane 6, 5 µM 25. (B) Immunoprecipitates of
cortactin from whole cell extracts of Src-transformed fibro-
blasts treated with (lane 1) no compound, (lane 2) 1 µM 25,
(lane 3) 5 µM 25; probed with antibodies to phosphotyrosine
(top panel) and cortactin (bottom panel).
heteroatom at C-4 or the carbonitrile group at C-3 of
2a results in compounds with greatly diminished activ-
ity. We also determined that a 2,4-dihalogen substitu-
tion pattern on the 4-phenylamino group is preferred
and that larger 2-halo groups are optimal. Addition of
a 3-morpholinopropoxy group at C-7 of 2a provided 25
which has an IC50 of 3.8 nM for the inhibition of Src
kinase activity and exhibits submicromolar activity in
inhibiting both the growth of Src-transformed rat
fibroblasts in suspension and the phosphorylation of Src
substrate proteins.
Exp er im en ta l Section
While 2a is selective for inhibiting Src compared to
some RTKs, selectivity for Src over other SFKs would
also be desirable. To investigate the activity of these
compounds against Fyn, rat fibroblasts transfected with
activated Fyn were prepared. As shown in Table 4,
compounds 25-27 exhibited only about a 3-fold selec-
tivity for inhibition of Src over Fyn. This result is not
surprising since the catalytic domain of these two SFKs
is highly conserved.
Gen er a l Meth od s. Melting points were determined in open
capillary tubes on a Meltemp melting point apparatus and are
1
uncorrected. H NMR spectra were recorded using a NT-300
WB spectrometer. Chemical shifts (δ) are in parts per million
referenced to Me4Si. Electrospray (ES) mass spectra were
recorded in positive mode on a Micromass Platform spectrom-
eter. Electron impact (EI) and high-resolution mass spectra
were obtained on a Finnigan MAT-90 spectrometer. Flash
chromatography was performed with Baker 40-µm silica gel.
Reactions were carried out under an inert atmosphere, either
nitrogen or argon.
4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in o-
lin eca r bon itr ile (2a ). A mixture of 4a (249 mg, 1.0 mmol),26,27
2,4-dichloroaniline (194 mg, 1.2 mmol) and pyridine hydro-
chloride (116 mg, 1.0 mmol) in 12 mL of 2-ethoxyethanol was
heated at reflux for 7 h. After cooling, the solvent was removed
in vacuo and the residue was treated with 10 mL of a saturated
NaHCO3 solution and the suspension was stirred for 15 min.
The precipitate was collected by filtration, washing with water
Con clu sion
By employing a novel screening strategy, we identified
and then optimized potent Src kinase inhibitors. By
systematic replacement of the functional groups of the
screening lead 2a we determined that various 4-phen-
ylamino-3-quinolinecarbonitriles are potent inhibitors
of the kinase activity of Src. Modification of either the

828 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
and ether. Drying in vacuo provided 93 mg (25%) of 2a as a
Boschelli et al.
ture was concentrated in vacuo and 50 mL of water and 1.2
mL of AcOH was added to the residue. The mixture was stirred
at room temperature for 30 min. The resultant solids were
collected by filtration washing with water and Et2O. After
drying in vacuo, 415 mg (75%) of 2h was isolated as a white
1
light tan solid: mp 243-244 °C; H NMR (DMSO-d6) δ 3.93
(s, 3H), 3.95 (s, 3H), 7.33 (s, 1H), 7.50 (s, 2H), 7.78 (s, 1H),
7.81 (s, 1H), 8.41 (s, 1H), 9.59 (br s, 1H); MS (ES) m/z 374.0,
375.9 (M + 1).
1
solid: mp 295 °C dec; H NMR (DMSO-d6) δ 6.94 (br s, 1H),
Alter n a tive P r ep a r a tion of 4-[(2,4-Dich lor op h en yl)-
a m in o]-6,7-d im eth oxy-3-qu in olin eca r bon itr ile (2a ). To a
suspension of NaH (60% in mineral oil, 800 mg) in 50 mL of
DMF was added 2,4-dichloroaniline (3.24 g, 20.0 mmol) and
the mixture was stirred at room temperature for 1 h. To the
resultant green solution was added 4a (2.50 g, 10.0 mmol) and
the mixture was heated at reflux for 1 h then poured into water
and extracted with EtOAc. The organic layer was dried over
MgSO4, filtered and concentrated in vacuo. Et2O was added
to the residue and the white solid collected by filtration to
provide 2.36 g (67%) of 2a : 1H NMR (DMSO-d6) δ 3.93 (s, 3H),
3.95 (s, 3H), 7.33 (s, 1H), 7.50 (br s, 2H), 7.77-7.81 (m, 2H),
8.41 (s, 1H), 9.57 (br s, 1H); MS (ES) m/z 373.8, 375.8 (M +
1). Anal. (C18H13Cl2N3O2‚0.5H2O) C, H, N.
7.39 (br s, 1H), 8.48 (d, J ) 8 Hz, 1H), 9.82 (br s, 1H), 10.36
(br s, 1H), 12.40 (br s, 1H); MS (ES) m/z 345.8, 347.8 (M + 1).
Anal. (C16H9Cl2N3O2‚0.8H2O) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d ieth oxyqu in olin e-
3-ca r bon itr ile (2i). To a mixture of 22 (500 mg, 1.29 mmol)
and K2CO3 (390 mg, 2.82 mmol) in 5 mL of DMF was added
ethyl iodide (602 mg, 3.86 mmol). The mixture was heated at
65 °C for 3 h then diluted with water and the pH was adjusted
to 5-6 with AcOH. The mixture was extracted into EtOAc and
the organic layer was dried over MgSO4, filtered and concen-
trated in vacuo. The residue was purified by flash column
chromatography eluting with 3:1 hexane:EtOAc to provide 383
mg of a colorless syrup. This material was combined with K2-
CO3 (475 mg, 3.44 mmol) in 10 mL of MeOH and heated at
reflux for 90 min then cooled to room temperature and diluted
with CH2Cl2. The organic layer was concentrated in vacuo in
the presence of silica gel. The residue was purified by column
chromatography eluting with 2:1 hexane:EtOAc to provide 192
4-[(2,4-Dich lor op h e n yl)a m in o]-3-q u in olin e ca r b on i-
tr ile (2b). To a suspension of NaH (60% in mineral oil, 130
mg) in 10 mL of THF was added 2,4-dichloroaniline (520 mg,
3.21 mmol) and the mixture was heated to reflux. After cooling,
4b (300 mg, 1.59 mmol)34 was added and the mixture was
heated at reflux for 30 min then cooled to room temperature
and partitioned between water and EtOAc. The organic layer
was dried over MgSO4, filtered and concentrated in vacuo. The
residue was purified by flash chromatography eluting with 1:1
hexane:EtOAc to provide 263 mg (53%) of 2b as a tan solid:
1
mg (36%) of 2i as an off-white solid: mp 85-87 °C; H NMR
(DMSO-d6) δ 1.42 (t, J ) 7 Hz, 6H), 4.22 (m, 4H), 7.32 (s, 1H),
7.51 (br s, 2H), 7.80 (br s, 2H), 8.40 (s, 1H), 9.52 (s, 1H); MS
(ES) m/z 402.1, 404.1 (M + 1). Anal. (C20H17Cl2N3O3‚0.2H2O)
C, H, N.
1
mp 196-198 °C; H NMR (CDCl3) δ 6.96 (s, 1H), 7.03 (d, J )
4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d i-n -bu toxy-3-qu in -
olin eca r bon itr ile (2j). To a mixture of 22 (400 mg, 1.03
mmol) and K2CO3 (312 mg, 2.26 mmol) in 5 mL of DMF was
added n-butyl bromide (424 mg, 3.09 mmol). The mixture was
heated at 65 °C for 4 h then cooled to room temperature and
extracted with EtOAc. The organic layer was dried over
MgSO4, filtered and concentrated in vacuo. The residue was
purified by flash column chromatography eluting with 3:1
hexane:EtOAc to provide 450 mg of product. This material was
combined with K2CO3 (496 mg, 3.59 mmol) in 10 mL of MeOH
and heated at reflux for 90 min then cooled to room temper-
ature and diluted with CH2Cl2. The organic layer was concen-
trated in vacuo in the presence of silica gel. The residue was
purified by column chromatography eluting with 2:1 hexane:
EtOAc to provide 184 mg (39%) of 2j: mp 138-140 °C; 1H
NMR (DMSO-d6) δ 0.95 (t, J ) 7 Hz, 3H), 1.07 (t, J ) 7 Hz,
3H), 1.49-1.62 (m, 4H), 1.73 (m, 2H), 1.90 (m, 2H), 3.84 (t, J
) 7 Hz, 2H), 4.18 (t, J ) 7 Hz, 2H), 6.69 (s, 1H), 6.79 (d, J )
9 Hz, 1H), 6.82 (s, 1H), 7.14 (dd, J ) 9, 2 Hz, 1H), 7.26 (s,
1H), 7.37 (s, 1H), 7.51 (d, J ) 2 Hz, 1H), 8.69 (s, 1H); MS (ES)
m/z 458.0, 460.0 (M + 1). Anal. (C24H25Cl2N3O2) C, H, N.
4-(2-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 (2k ). Following the pyridine hydrochloride route
used to prepare 2a , 2k was obtained as a light tan solid in
48% yield from 4a after chromatography eluting with 1%
MeOH in CH2Cl2: mp 227-229 °C; 1H NMR (DMSO-d6) δ 3.94
(s, 3H), 3.95 (s, 3H), 7.33 (s, 1H), 7.37-7.51 (m, 3H), 7.60 (m,
1H), 7.84 (s, 1H), 8.40 (s, 1H), 9.56 (s, 1H); MS (ES) m/z 339.9,
341.9 (M + 1). Anal. (C18H14ClN3O2‚0.17CH2Cl2) C, H, N.
4-[(4-Ch lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in olin -
eca r bon itr ile (2l). Following the pyridine hydrochloride route
used to prepare 2a , 2l was obtained as a light tan solid in 48%
yield from 4a after chromatography eluting with 1% MeOH
in CH2Cl2: mp 204-206 °C; 1H NMR (DMSO-d6) δ 3.92 (s,
3H), 3.96 (s, 3H), 7.26 (d, J ) 9 Hz, 2H), 7.36 (s, 1H), 7.45 (d,
J ) 9 Hz, 2H), 7.72 (s, 1H), 8.51 (s, 1H), 9.51 (s, 1H); MS (ES)
m/z 339.9, 341.9 (M + 1). Anal. (C18H14ClN3O2) C, H, N.
8 Hz, 1H), 7.23 (dd, J ) 9, 2 Hz, 1H), 7.53-7.60 (m, 2H), 7.80-
7.87 (m, 2H), 8.12 (d, J ) 9 Hz, 1H), 8.80 (s, 1H); MS (ES) m/z
314.1, 316.0 (M + 1). Anal. (C16H9Cl2N3) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-5-m eth oxy-3-qu in olin e-
ca r bon itr ile (2c). Following the NaH route used to prepare
2a , 2c was obtained as a gray solid in 63% yield from 4c: mp
1
216-218 °C; H NMR (DMSO-d6) δ 4.06 (s, 3H), 7.20 (d, J )
8 Hz, 1H), 7.48-7.62 (m, 3H), 7.73-7.81 (m, 2H), 8.46 (s, 1H),
10.21 (br s, 1H); MS (ES) m/z 343.9, 345.8 (M + 1). Anal.
(C17H11Cl2N3O) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-6-m eth oxy-3-qu in olin e-
ca r bon itr ile (2d ). Following the route used to prepare 2b,
2d was obtained as an off-white solid in 30% yield from 4d :26
mp 160-161 °C; 1H NMR (DMSO-d6) δ 3.94 (s, 3H), 7.48-
7.62 (m, 3H), 7.81-7.91 (m, 3H), 8.45 (s, 1H), 9.73 (s, 1H); MS
(ES) m/z 343.8, 345.8 (M + 1). Anal. (C17H11Cl2N3O) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-7-m eth oxy-3-qu in olin e-
ca r bon itr ile (2e). Following the NaH route used to prepare
2a , 2e was obtained as an off-white solid in 97% yield from
4e:26 mp 192-194 °C; 1H NMR (DMSO-d6) δ 3.93 (s, 3H), 7.23-
7.35 (br s, 2H), 7.45-7.60 (br s, 2H), 7.76 (s, 1H), 8.39 (d, J )
8 Hz, 1H), 8.49 (s, 1H), 9.81 (br s, 1H); MS (ES) m/z 344.2,
346.2 (M + 1). Anal. (C17H11Cl2N3O‚0.2H2O) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-8-m eth oxy-3-qu in olin e-
ca r bon itr ile (2f). Following the NaH route used to prepare
2a , 2f was obtained as a light tan solid in 32% yield from 4f:
26 mp 201-202 °C; 1H NMR (DMSO-d6) δ 3.90 (s, 3H), 7.34 (d,
J ) 8 Hz, 1H), 7.41-7.67 (m, 3H), 7.71-8.09 (m, 2H), 8.51 (s,
1H), 9.80 (br s, 1H); MS (ES) m/z 343.7, 345.5 (M + 1). Anal.
(C17H11Cl2N3O‚0.5H2O) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-5,7-d im eth oxy-3-qu in o-
lin eca r bon itr ile (2g). Following the NaH route used to
prepare 2a , 2g was obtained as an off-white solid in 57% yield
from 4g after flash column chromatography eluting with a
gradient of 3:1 to 1:1 hexane:EtOAc: mp 238-239 °C; 1H NMR
(DMSO-d6) δ 3.92 (s, 3H), 4.03 (s, 3H), 6.74 (d, J ) 2 Hz, 1H),
6.94 (d, J ) 2 Hz, 1H), 7.48 (dd, J ) 9, 2 Hz, 1H), 7.54 (d, J )
9 Hz, 1H), 7.77 (d, J ) 2 Hz, 1H), 8.39 (s, 1H), 9.97 (s, 1H);
MS (ES) m/z 374.1 (M + 1). Anal. (C18H13Cl2N3O2) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d ih yd r oxy-3-qu in o-
lin eca r bon itr ile (2h ). A mixture of 2a (600 mg, 1.60 mmol)
and pyridine hydrochloride (270 mg, 24.06 mmol) was heated
at 215-222 °C for 30 min. After cooling to 100 °C, 3.2 mL of
concentrated aqueous NH4OH was added. The reaction mix-
4-[(2,3-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in o-
lin eca r bon itr ile (2m ). Following the route used to prepare
2b, 2m was obtained as a pale yellow solid in 66% yield from
4a : mp 227-229 °C; 1H NMR (DMSO-d6) δ 3.93 (s, 3H), 3.95
(s, 3H), 7.34 (s, 1H), 7.39-7.46 (m, 2H), 7.61 (d, J ) 6 Hz,
1H), 7.81 (s, 1H), 8.43 (s, 1H), 9.65 (s, 1H); MS (ES) m/z 374.3,
376.3 (M + 1). Anal. (C18H13Cl2N3O2‚0.5EtOAc) C, H, N.

4-Phenylamino-3-quinolinecarbonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 829
4-[(2,5-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in o-
lin eca r bon itr ile (2n ). Following the pyridine hydrochloride
route used to prepare 2a , 2n was obtained as a light tan solid
in 14% yield from 4a after chromatography eluting with 5%
MeOH in CH2Cl2: mp 219-220 °C; 1H NMR (DMSO-d6) δ 3.93
(s, 3H), 3.95 (s, 3H), 7.35 (s, 1H), 7.42-7.52 (m, 1H), 7.59-
7.70 (m, 2H), 7.79 (s, 1H), 8.45 (s, 1H), 9.75 (s, 1H); MS (ES)
m/z 373.8, 375.8 (M + 1). Anal. (C18H13Cl2N3O2‚0.05Et2O) C,
H, N.
used to prepare 2a , 2y was obtained as an off-white solid in
49% yield from 4y:26,36 mp 183-186 °C dec; 1H NMR (DMSO-
d6) δ 2.70 (m, 2H), 3.83 (t, J ) 6 Hz, 2H), 3.95 (s, 3H), 4.29 (t,
J ) 6 Hz, 2H), 7.37 (s, 1H) , 7.52 (s, 2H), 7.79 (s, 1H), 7.82 (s,
1H), 8.42 (s, 1H), 9.56 (s, 1H); MS (ES) m/z 438.2 (M + 1).
Anal. (C20H16Cl3N3O2‚0.25H2O) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d im eth oxyqu in a zo-
lin e (3). To a suspension of 531 (300 mg, 1.34 mmol) in 7 mL
of EtOH was added 2,4-dichloroaniline (238 mg, 1.47 mmol)
and the mixture was heated at reflux overnight. The reaction
mixture was cooled to room temperature and partitioned
between 0.5 N NaOH and EtOAc. The organic layer was dried
over MgSO4, filtered and concentrated in vacuo. Et2O and
hexane were added to the residue and the off-white solid was
collected by filtration to provide 230 mg (49%) of 3: mp 220-
4-[(2,6-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in o-
lin eca r bon itr ile (2o). Following the NaH route used to
prepare 2a , 2o was obtained as a pale yellow solid in 66% yield
1
from 4a : mp 264-266 °C dec; H NMR (DMSO-d6) δ 3.95 (s,
3H), 3.96 (s, 3H), 7.35 (s, 1H), 7.49 (t, J ) 8 Hz, 1H), 7.65 (d,
J ) 8 Hz, 2H), 7.88 (s, 1H), 8.39 (s, 1H), 9.66 (s, 1H); MS (ES)
m/z 373.9, 375.8 (M + 1). Anal. (C18H13Cl2N3O2) C, H, N.
4-[(3,5-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in o-
lin eca r bon itr ile (2p ). Following the pyridine hydrochloride
route used to prepare 2a , 2p was obtained as a yellow-brown
solid in 49% yield from 4a : mp 228-230 °C; 1H NMR (DMSO-
d6) δ 3.92 (s, 3H), 3.97 (s, 3H), 7.21 (bs, 2H), 7.30 (s, 1H), 7.40
(s, 1H), 7.64 (s, 1H), 8.63 (s, 1H), 9.63 (s, 1H); MS (ES) m/z
373.8, 375.8 (M + 1). Anal. (C18H13Cl2N3O2) C, H.
1
222 °C; H NMR (DMSO-d6) δ 3.93 (s, 6H), 7.19 (s, 1H), 7.49
(dd, J ) 9, 2 Hz, 1H), 7.59 (d, J ) 9 Hz, 1H), 7.75 (d, J ) 2 Hz,
1H), 7.82 (s, 1H), 8.32 (s, 1H), 9.57 (s, 1H); MS (ES) m/z 349.8,
351.9 (M + 1). Anal. (C16H13Cl2N3O2) C, H, N.
4-Ch lor o-5-m eth oxy-3-qu in olin ecar bon itr ile (4c). A mix-
ture of 20 (811 mg, 4.05 mmol) in 10 mL of phosphorus
oxychloride and 4 drops of DMF was heated at reflux for 4 h.
After cooling, the mixture was concentrated in vacuo and the
residue was diluted with ice cold CH2Cl2. The organic phase
was washed once with ice water, twice with cold saturated
sodium carbonate solution and twice with cold brine, then
dried over Na2SO4. The solution was passed through a pad of
silica gel and the filtrate was concentrated in vacuo to give
772 mg of 4c, as a yellow solid: mp 174-176 °C; 1H NMR
(DMSO-d6) δ 3.97 (s, 3H), 7.03 (d, J ) 8 Hz, 1H), 7.28 (d, J )
8 Hz, 1H), 7.77 (t, J ) 8 Hz, 1H), 8.70 (s, 1H); MS (ES) m/z
218.7, 220.8 (M + 1).
4-Ch lor o-5,7-d im eth oxy-3-qu in olin eca r bon itr ile (4g).
A mixture of 21 (500 mg, 2.27 mmol) in 2 mL of phosphorus
oxychloride and 3 drops of DMF was heated at 110 °C for 2 h.
After cooling, the mixture was poured into ice water and
neutralized with saturated NaHCO3. The solids were collected
by filtration, washing with water. This material was purified
by flash column chromatography eluting with a gradient of
3:1 to 1:1 hexane:ethyl acetate to provide 267 mg (92%) of 4g,
as an off-white solid: mp 240-242 °C; 1H NMR (DMSO-d6) δ
3.31 (s, 3H), 3.97 (s, 3H), 6.90 (d, J ) 2 Hz, 1H), 7.15 (d, J )
2 Hz, 1H), 8.99 (s, 1H); MS (ES) m/z 248.8 (M + 1). Anal.
(C12H9ClN2O2‚0.1H2O) C, H, N.
4-[(3,4-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in o-
lin eca r bon itr ile (2q). Following the pyridine hydrochloride
route used to prepare 2a , 2q was obtained as a yellow-brown
solid in 42% yield from 4a : mp 211-215 °C; 1H NMR (DMSO-
d6) δ 3.92 (s, 3H), 3.96 (s, 3H), 7.20 (dd, J ) 9, 3 Hz, 1H), 7.39
(s, 1H), 7.47 (d, J ) 3 Hz, 1H), 7.61 (d, J ) 9 Hz, 1H), 7.68 (s,
1H), 8.58 (s, 1H), 9.60 (s, 1H); MS (ES) m/z 373.8, 375.8 (M +
1). Anal. (C18H13Cl2N3O2‚0.25H2O) C, H, N.
4-[(4-Ch lor o-2-flu or op h en yl)a m in o]-6,7-d im et h oxy-3-
qu in olin eca r bon itr ile (2r ). Following the pyridine hydro-
chloride route used to prepare 2a , 2r was obtained as a tan
solid in 52% yield from 4a after chromatography eluting with
1
EtOAc: mp 184-186 °C; H NMR (DMSO-d6) δ 3.94 (s, 3H),
3.96 (s, 3H), 7.35 (m, 2H), 7.48 (t, J ) 9 Hz, 1H), 7.60 (dd, J
) 11, 2 Hz, 1H), 7.79 (s, 1H), 8.47 (s, 1H), 9.56 (s, 1H); MS
(ES) m/z 357.9, 359.9 (M + 1). Anal. (C18H13ClFN3O2) C, H,
N.
4-[(2-Br om o-4-ch lor op h en yl)a m in o]-6,7-d im eth oxy-3-
qu in olin eca r bon itr ile (2s). Following the NaH route used
to prepare 2a , 2s was obtained as an off-white solid in 58%
yield from 4a : mp 256-257 °C; 1H NMR (DMSO-d6) δ 3.94 (s,
1H), 3.95 (s, 1H), 7.34 (s, 1H), 7.54 (br s, 2H), 7.82 (s, 1H),
7.93 (s, 1H), 8.41 (s, 1H), 9.58 (s, 1H); MS (ES) m/z 417.8, 419.8
(M + 1). Anal. (C18H13BrClN3O2) C, H, N.
4-[(4-Ch lor o-2-iodoph en yl)am in o]-6,7-dim eth oxy-3-qu in -
olin eca r bon itr ile (2t). Following the NaH route used to
prepare 2a , 2t was obtained as a white solid in 77% yield from
4a after chromatography eluting with a gradient of 1:2 to 1:1
EtOAc:hexane: mp 248-249 °C dec; 1H NMR (DMSO-d6) δ
3.94 (s, 3H), 3.95 (s, 3H), 7.33 (s, 1H), 7.49 (d, J ) 8 Hz, 1H),
7.56 (dd, J ) 8, 2 Hz, 1H), 7.84 (s, 1H), 8.06 (d, J ) 2 Hz, 1H),
8.38 (s, 1H), 9.60 (s, 1H); MS (ES) m/z 465.9, 467.9 (M + 1).
Anal. (C18H13ClIN3O2) C, H, N.
(2,4-Dich lor op h e n yl)(6,7-d im e t h oxyq u in olin -4-yl)-
a m in e (6). Following the pyridine hydrochloride route used
to prepare 2a , 6 was obtained as an off-white solid in 21% yield
from 732 after chromatography eluting with EtOAc: mp 144-
1
146 °C; H NMR (DMSO-d6) δ 3.91 (s, 3H), 3.92 (s, 3H), 6.16
(d, J ) 5 Hz, 1H), 7.26 (s, 1H), 7.46 (d, J ) 8 Hz, 1H), 7.52
(dd, J ) 8, 2 Hz, 1H), 7.68 (s, 1H), 7.80 (d, J ) 2 Hz, 1H), 8.24
(d, J ) 5 Hz, 1H), 8.65 (s, 1H); MS (ES) m/z 349.3, 351.2 (M +
1). Anal. (C17H14Cl2N2O2) C, H, N.
4-(2,4-Dich lor op h en oxy)-6,7-d im et h oxy-3-q u in olin e-
ca r bon itr ile (8). To a melt of 2,4-dichlorophenol (700 mg, 4.29
mmol) and 80 mg of KOH was added 4a (200 mg, 0.89 mmol).
The mixture was heated for 30 min then cooled. Water was
added to the reaction mixture followed by EtOAc. The layers
were separated and 1 N HCl was added to the aqueous layer.
The aqueous layer was extracted with EtOAc and the organic
layers were combined, dried over MgSO4, filtered and concen-
trated in vacuo. Methanol was added to the residue and the
light tan solid was collected by filtration to provide 192 mg
(58%) of 8: mp 234-236 °C; 1H NMR (DMSO-d6) δ 3.90 (s,
3H), 4.01 (s, 3H), 7.24 (d, J ) 9 Hz, 1H), 7.38 (s, 1H), 7.44 (dd,
J ) 9, 2 Hz, 1H), 7.56 (s, 1H), 7.90 (d, J ) 2 Hz, 1H), 8.89 (s,
1H); MS (ES) m/z 374.8, 376.8 (M + 1). Anal. (C18H12Cl2N2O3‚
0.25H2O) C, H, N.
4-[(2-Ch lor o-4-iodoph en yl)am in o]-6,7-dim eth oxy-3-qu in -
olin eca r bon itr ile (2u ). Following the NaH route used to
prepare 2a , 2u was obtained as a pale yellow solid in 32% yield
1
from 4a : mp 250-253 °C dec; H NMR (DMSO-d6) δ 3.93 (s,
3H), 3.95 (s, 3H), 7.25 (d, J ) 8 Hz, 1H), 7.34 (s, 1H), 7.75 (dd,
J ) 8, 2 Hz, 1H), 7.80 (s, 1H), 7.98 (d, J ) 2 Hz, 1H), 8.43 (s,
1H), 9.50 (s, 1H); MS (ES) m/z 466.0, 468.0 (M + 1). Anal.
(C18H13ClIN3O2‚0.2EtOAc) C, H, N.
4-[(4-Br om o-2-ch lor op h en yl)a m in o]-6,7-d im eth oxy-3-
qu in olin eca r bon itr ile (2v). Following the route used to
prepare 2b, 2v was obtained as a tan solid in 63% yield from
1
4a : mp 208-210 °C dec; H NMR (DMSO-d6) δ 3.93 (s, 3H),
3.95 (s, 3H), 7.35 (s, 1H), 7.44 (d, J ) 8 Hz, 1H), 7.62 (dd, J )
8, 2 Hz, 1H), 7.81 (s, 1H), 7.89 (d, J ) 2 Hz, 1H), 8.43 (s, 1H),
4-(2,4-Dich lor op h en ylsu lfa n yl)-6,7-d im eth oxy-3-qu in o-
lin eca r bon it r ile (9). To a suspension of 4a (249 mg, 1.00
mmol) in 10 mL of DMF was added 2,4-dichlorothiophenol (215
mg, 1.20 mmol) resulting in a homogeneous solution. After
stirring at room temperature for 30 min, the off-white solid
was collected by filtration, washing with hexane, to provide
9.53 (s, 1H); MS (ES) m/z 417.7, 419.7 (M + 1). Anal. (C18H13
BrClN3O2) C, H, N.
-
7-(3-Ch lor op r op oxy)-4-[(2,4-d ich lor op h en yl)a m in o]-6-
m eth oxy-3-qu in olin eca r bon itr ile (2y). Following the route

830 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Boschelli et al.
294 mg (75%) of 9: mp 201-202 °C; 1H NMR (DMSO-d6) δ
3.83 (s, 3H), 4.01 (s, 3H), 7.01 (d, J ) 9 Hz, 1H), 7.31 (dd, J )
9, 2 Hz, 1H), 7.43 (s, 1H), 7.57 (s, 1H), 7.82 (d, J ) 2 Hz, 1H),
[4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in -
olin yl]m eth a n ol (16). To a 0 °C mixture of 15 (1.60 g, 3.8
mmol) in 60 mL of THF, 19 mL of 1.0 M diisobutyl aluminum
hydride in hexane was added dropwise. After stirring at 0 °C
for 30 min, MeOH was added and the reaction mixture was
stirred at room temperature for 30 min. The mixture was
diluted with 300 mL of THF followed by the addition of 6.12
g of Na2SO4 decahydrate. After stirring for an additional 30
min the volatiles were removed in vacuo and the residue was
partitioned between EtOAc and water. The aqueous layer was
extracted with CH2Cl2, and the organic layers were combined,
dried over Na2SO4, filtered and concentrated in vacuo. Et2O
was added to the residue and the white solid was collected by
9.04 (s, 1H); MS (ES) m/z 390.8, 392.8 (M + 1). Anal. (C18H12
Cl2N2O2S‚0.70H2O) C, H, N.
-
4-[(2,4-Dich lor op h en yla m in o)m eth yl]-6,7-d im eth oxy-
3-qu in olin eca r bon itr ile (10). A mixture of 4a (249 mg, 1.00
mmol), 2,4-dichlorobenzylamine (387 mg, 2.20 mmol) and
Hunig’s base (284 mg, 2.20 mmol) in 7 mL of DMF was heated
at reflux for 2.5 h. After cooling to room temperature, the
mixture was partitioned between EtOAc and water. The
organic layer was dried over MgSO4, filtered and concentrated
in vacuo. The residue was purified by flash column chroma-
tography eluting with a gradient of 1:3 to 1:1 EtOAc:hexane
to provide 350 mg (90%) of 10 as a light yellow solid: mp 127-
130 °C; 1H NMR (DMSO-d6) δ 3.93 (s, 6H), 5.00 (d, J ) 6 Hz,
2H), 7.25 (d, J ) 8 Hz, 1H), 7.28 (s, 1H), 7.40 (dd, J ) 8, 2 Hz,
1H), 7.70 (d, J ) 2 Hz, 1H), 7.74 (s, 1H), 7.95 (s, 1H), 8.31 (s,
1H), 8.36 (t, J ) 6 Hz, 1H); MS (ES) m/z 387.9, 389.8 (M + 1).
Anal. (C19H15Cl2N3O2‚0.7H2O‚0.6DMF) C, H, N.
4-(2,4-Dich lor op h en ylm eth yla m in o)-6,7-d im eth oxy-3-
qu in olin eca r bon itr ile (11). To a suspension of NaH (60%
in mineral oil, 60 mg) in 10 mL of THF was added 2,4-dichloro-
N-methylaniline42 (290 mg, 1.64 mmol) and the mixture was
heated to reflux. To the resultant yellow suspension was added
4a (220 mg, 0.88 mmol) and the mixture was heated at reflux
overnight. The reaction mixture was cooled to room temper-
ature and partitioned between water and EtOAc. The organic
layer was dried over MgSO4, filtered and concentrated in
vacuo. Et2O was added to the residue and the resultant light
tan solid was collected by filtration to provide 182 mg (53%)
of 11: mp 183-186 °C; 1H NMR (DMSO-d6) δ 3.62 (s, 3H),
3.72 (s, 3H), 3.96 (s, 3H), 7.10 (s, 1H), 7.43 (s, 1H), 7.50-7.58
(m, 2H), 7.65 (d, J ) 9 Hz, 1H), 8.78 (s, 1H); MS (ES) m/z 387.9,
389.9 (M + 1). Anal. (C19H15Cl2N3O2‚0.5H2O) C, H, N.
1
filtration to provide 600 mg (42%) of 16: mp 221-222 °C; H
NMR (DMSO-d6) δ 3.65 (s, 3H), 3.93 (s, 3H), 4.53 (d, J ) 5
Hz, 2H), 5.48 (t, J ) 5 Hz, 1H), 6.26 (d, J ) 8 Hz, 1H), 6.93 (s,
1H), 7.11 (dd, J ) 8, 2 Hz, 1H), 7.39 (s, 1H), 7.60 (d, J ) 2 Hz,
1H), 7.98 (s, 1H), 8.71 (s, 1H); MS (ES) m/z 379.2, 381.2 (M +
1). Anal. (C18H16Cl2N2O3) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in o-
lin eca r ba ld eh yd e (17). A mixture of 16 (560 mg, 1.48 mmol)
and manganese dioxide (2.8 g, 33.3 mmol) in 70 mL of CHCl3
was stirred at room temperature for 2 h. The mixture was
diluted with 100 mL of CHCl3 and filtered through a pad of
Celite, washing with CHCl3. The filtrate was concentrated in
vacuo to provide 440 mg (80%) of 17: mp 168-169 °C; 1H NMR
(DMSO-d6) δ 3.48 (s, 3H), 3.95 (s, 3H), 6.80 (s, 1H), 7.15 (d, J
) 9 Hz, 1H), 7.38 (s, 1H), 7.40 (dd, J ) 9, 2 Hz, 1H), 7.80 (d,
J ) 2 Hz, 1H), 8.86 (s, 1H), 10.04 (s, 1H), 10.39 (s, 1H); MS
(ES) m/z 377.3, 379.2 (M + 1). Anal. (C18H14Cl2N2O3) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in o-
lin eca r boxylic Acid Hyd r och lor id e (18). A mixture of 15
(650 mg, 1.55 mmol) and 2.0 mL of 5.0 N NaOH in 30 mL of
ethanol was heated at reflux for 1 h. The mixture was cooled
and concentrated HCl was added to provide a pH of 2. The
EtOH was removed in vacuo and the solids collected by
filtration. Washing with water followed by Et2O provided 600
2,4-Dich lor o-N-(3-cya n o-6,7-d im eth oxyqu in olin -4-yl)-
ben za m id e (12). To a suspension of NaH (60% in mineral
oil, 80 mg) in 10 mL of DMF was added 2,4-dichlorobenzamide
(380 mg, 2.0 mmol) and the mixture was stirred at room
temperature for 15 min. To the resultant yellow solution was
added 4a (250 mg, 1.0 mmol) and the mixture was heated at
reflux for 2 h. The reaction mixture was cooled and added to
a mixture of 1 N HCl and EtOAc. The mixture was filtered
and the solid washed with EtOAc to provide 323 mg (80%) of
1
mg (98%) of 18 as a yellow solid: mp 270-272 °C; H NMR
(DMSO-d6) δ 3.53 (s, 3H), 3.98 (s, 3H), 7.06 (s, 1H), 7.46 (d, J
) 9 Hz, 1H), 7.52 (s, 1H), 7.54 (dd, J ) 9, 2 Hz, 1H), 7.88 (d,
J ) 2 Hz, 1H), 9.07 (s, 1H), 11.52 (s, 1H); MS (ES) m/z 393.1,
395.1 (M + 1). Anal. (C18H14Cl2N2O4‚HCl) C, N.
4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-qu in o-
lin eca r boxa m id e (19). A mixture of 18 (200 mg, 0.51 mmol)
and N,N-carbonyldiimidazole (170 mg, 1.05 mmol) in 5 mL of
DMF was heated at 60 °C for 1.5 h. The mixture was cooled
and an additional 15 mL of DMF and 10 mL of THF were
added. The mixture was heated until a solution was obtained
and 30 mL of concentrated aqueous NH4OH was added. The
reaction was stirred overnight at room temperature and the
resulting solids were collected by filtration, washing with
water, to provide 103 mg (53%) of 19 as a white solid: mp
1
12: mp greater than 300 °C; H NMR (DMSO-d6) δ 3.93 (s,
3H), 4.01 (s, 3H), 7.50 (s, 1H), 7.54 (s, 1H), 7.60-7.75 (m, 2H),
7.88 (d, J ) 2 Hz, 1H), 9.00 (s, 1H), 11.5 (s, 1H); MS (ES) m/z
401.8, 403.7 (M + 1). Anal. (C19H13Cl2N3O3‚0.25H2O) C, H, N.
4-(2,4-Dich lor op h en ylim in o)-6,7-d im eth oxy-1-m eth yl-
1,4-d ih yd r o-3-qu in olin eca r bon itr ile (13). To a suspension
of NaH (60% in mineral oil, 25 mg) in 6 mL of THF was added
2a (196 mg, 0.52 mmol) and the mixture was heated to reflux.
The bright yellow suspension was cooled slightly and methyl
iodide (0.50 mL, 0.80 mmol) was added. Within 2 min a yellow
solution formed. The solution was partitioned between EtOAc
and water. The organic layer was washed with water, dried
over MgSO4, filtered and concentrated in vacuo. Et2O was
added to the residue and the bright yellow solid was collected
by filtration to provide 99 mg (49%) of 13: mp 222-225 °C;
1H NMR (DMSO-d6) δ 3.80 (s, 3H), 3.83 (s, 3H), 3.96 (s, 3H),
6.91 (d, J ) 8 Hz, 1H), 7.03 (s, 1H), 7.23 (dd, J ) 8, 2 Hz, 1H),
7.46 (d, J ) 2 Hz, 1H), 7.81 (s, 1H), 8.27 (s, 1H); MS (ES) m/z
387.9, 389.9 (M + 1). Anal. (C19H15Cl2N3O2) C, H, N.
1
247-249 °C; H NMR (DMSO-d6) δ 3.49 (s, 3H), 3.94 (s, 3H),
6.62 (d, J ) 9 Hz, 1H), 6.69 (s, 1H), 7.22 (dd, J ) 9, 2 Hz, 1H),
7.37 (s, 1H), 7.70 (d, J ) 2 Hz, 1H), 7.76 (br s, 1H), 8.36 (br s,
1H), 8.94 (s, 1H) 10.70 (s, 1H); MS (ES) m/z 392.2, 394.2 (M +
1). Anal. (C18H15Cl2N3O3‚0.4H2O) C, H, N.
4-Hyd r oxy-5-m eth oxy-3-qu in olin eca r bon itr ile (20). A
mixture of 2-amino-6-methoxybenzoic acid35 (2.48 g, 14.8
mmol) in 30 mL of dimethylformamide dimethylacetal was
heated at reflux for 2 h. After cooling, the solvent was removed
in vacuo and the residue was passed through a Magnesol
column, eluting with CH2Cl2, to afford 2.1 g of the amidine
imtermediate that was used the next step without further
purification.
Concentration of the filtrate gave a residue which by 1H
NMR analysis was a 1:1 mixture of 13 and 11.
Eth yl 4-[(2,4-Dich lor op h en yl)a m in o]-6,7-d im eth oxy-3-
qu in olin eca r boxyla te (15). Following the pyridine hydro-
chloride route used to prepare 2a , 15 was obtained as a white
solid in 40% yield from 14:33 mp 110-113 °C; 1H NMR (DMSO-
d6) δ 1.30 (t, J ) 7 Hz, 3H), 3.54 (s, 3H), 3.95 (s, 3H), 4.26 (q,
J ) 7 Hz, 2H), 6.83 (d, J ) 9 Hz, 1H), 6.88 (s, 1H), 7.27 (dd,
J ) 9, 2 Hz, 1H), 7.40 (s, 1H), 7.74 (d, J ) 2 Hz, 1H), 8.97 (s,
1H), 9.61 (s, 1H); MS (ES) m/z 421.3, 423.3 (M + 1). Anal.
(C20H18Cl2N2O4‚0.2H2O) C, H, N.
A solution of CH3CN (734 mg, 17.9 mmol) in 10 mL of THF
was added dropwise to a solution of n-BuLi (2.5 M in hexane,
5.96 mL, 14.9 mmol) in 7 mL of THF at -78 °C. The mixture
was stirred at -78 °C for 15 min. A solution of the amidine
(1.6 g, 6.8 mmol) in 13 mL of THF was added dropwise and
the mixture was stirred at -78 °C for 2 h, and then at room
temperature for 2.5 h. The mixture was cooled to -78 °C and
AcOH was added dropwise. The mixture was allowed to stir
at room temperature overnight. The precipitate was collected

4-Phenylamino-3-quinolinecarbonitriles
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 831
by filtration and washed with water and ether. After drying
1H), 9.56 (s, 1H); MS (ES) m/z 486.9, 488.9 (M + 1). Anal.
(C24H24Cl2N4O3‚0.6H2O) C, H, N.
in vacuo, 477 mg of 20 was obtained as a light tan solid: mp
1
>260 °C; H NMR (DMSO-d6) δ 3.82 (s, 3H), 6.92 (d, J ) 8
Alter n a te P r ep a r a tion of 25 fr om 2y. A mixture of 2y
(500 mg, 1.15 mmol), morpholine (2.00 g, 23 mmol) and a
catalytic amount of sodium iodide in 10 mL of ethylene glycol
dimethyl ether was heated at reflux for 6 h. The reaction
mixture was then poured into ice water and the pH was
adjusted to 8-9 by the addition of saturated NaHCO3. The
solids were collected by filtration to provide 547 mg of crude
product. A portion (240 mg) of this material was purified by
flash column chromatography eluting with 30% MeOH in ethyl
acetate to provide 127 mg of 25 as a white solid: mp 180-182
°C.
Hz, 1H), 7.09 (d, J ) 8 Hz, 1H), 7.62 (t, J ) 8 Hz, 1H), 8.55 (s,
1H); MS (ES) m/z 200.8 (M + 1). Anal. (C11H8N2O2‚0.1H2O) C,
H, N.
5,7-Dim eth oxy-4-h yd r oxy-3-qu in olin eca r bon itr ile (21).
A mixture of 3,5-dimethoxyaniline (5.00 g, 32.7 mmol) and
ethyl (ethoxymethylene)cyanoacetate (5.52 g, 32.7 mmol) was
heated at 120 °C for 45 min. Diethyl ether was added to the
cooled reaction mixture and the solids were collected by
filtration washing with additional diethyl ether to yield 7.16
g (79%) of ethyl 2-cyano-3-(3,5-dimethoxyphenylamino)acryl-
ate. A mixture of ethyl 2-cyano-3-(3,5-dimethoxyphenylamino)-
acrylate (1.00 g, 3.62 mmol) in 50 mL of a 1:1 mixture of
biphenyl and diphenyl ether was heated at reflux for 72 h.
The reaction mixture was cooled to room temperature and
poured into hexane. The solids were collected by filtration
washing with hexane to provide 698 mg (88%) of 21 as a tan
4-[(2,4-Dich lor oph en yl)am in o]-6-m eth oxy-7-[3-(4-m eth -
yl-1-p ip er a zin yl)p r op oxy]-3-qu in olin eca r bon itr ile (26).
A mixture of 2y (250 mg, 0.57 mmol), 1-methylpiperazine (1.5
mL, 13.5 mmol) and a catalytic amount of sodium iodide was
heated at 100 °C for 3 days. The reaction mixture was cooled
and partitioned between EtOAc and brine. The organic layer
was dried over Na2SO4, filtered and concentrated in vacuo. The
residue was purified by preparative thin-layer chromatogra-
phy, eluting with 30% MeOH in CH2Cl2, to provide 95 mg
1
solid: mp >300 °C; H NMR (DMSO-d6) δ 3.82 (s, 3H), 3.90
(s, 3H), 6.49 (d, J ) 2 Hz, 1H), 6.58 (d, J ) 2 Hz, 1H), 8.83 (s,
1H); MS (ES) m/z 231.2 (M + 1). Anal. (C12H10N2O3‚0.25H2O)
C, H, N.
1
(33%) of 26 as an off-white solid: mp 158-159 °C; H NMR
N-Acetyl-4-[(2,4-d ich lor op h en yl)a m in o]-6,7-d ih yd r oxy-
3-qu in olin eca r bon itr ile (22). A mixture of 2h (2.38 g, 6.88
mmol), 4-(dimethylamino)pyridine (1.01 g, 8.26 mmol) and
acetic anhydride (7.01 g, 68.8 mmol) in 14 mL of pyridine was
heated at reflux for 1.5 h. The reaction mixture was concen-
trated in vacuo and the residue was stirred with 75 mL of
MeOH, 7.5 mL of water and 2.89 g of NaHCO3 for 6.5 h. The
mixture was concentrated in vacuo, the residue was suspended
in water and AcOH was added to provide a pH of 4-5. The
resultant solid was collected by filtration and washed with
dilute AcOH followed by water to yield 2.68 g of 22 that was
not purified.
4-[(2,4-Dich lor op h en yl)a m in o]-6,7-bis(3-m or p h olin -4-
ylp r op oxy)-3-qu in olin eca r bon itr ile (23). To a mixture of
2h (305 mg, 0.88 mmol), 3-chloropropyl p-toluenesulfonate43
(482 mg, 1.94 mmol) and tricaprylmethylammonium chloride
(36 mg, 0.09 mmol) in 2 mL of acetone was added potassium
carbonate (292 mg, 2.11 mmol) The mixture was heated at
reflux for 18 h then concentrated in vacuo. The residue was
dissolved in 20% MeOH in CHCl3 and the solution was passed
through Magnesol, washing with additional solvent. The
filtrate was concentrated in vacuo and acetone was added. A
small amount of solid was removed by filtration and the filtrate
was concentrated in vacuo. The residue was dissolved in 20
mL of morpholine and heated at 130 °C for 14 h. The
morpholine was removed in vacuo and the residue was purified
by preparative thin-layer chromatography eluting with a
gradient of 2% MeOH in CH2Cl2 to 8% MeOH in CH2Cl2 to
provide 166 mg (31%) of 23 as a colorless syrup: 1H NMR
(DMSO-d6) δ 1.87-2.03 (m, 4H), 2.32-2.43 (m, 8H), 2.46-2.50
(m, 4H), 3.48-3.63 (m, 8H), 4.11-4.27 (m, 4H), 7.33 (s, 1H),
7.51 (br s, 1H), 7.80 (br s, 1H), 8.41 (s, 1H), 9.53 (s, 1H); MS
(ES) m/z 600.1, 602.0 (M + 1). Anal. (C30H35Cl2N5O4‚1.0H2O)
C, H, N.
4-[(2,4-Dich lor oph en yl)am in o]-7-m eth oxy-6-(3-m or ph o-
lin -4-ylp r op oxy)-3-qu in olin eca r bon itr ile (24). Following
the pyridine hydrochloride route used to prepare 2a , 24 was
obtained as an off-white solid in 7% yield from 4w 26 after
chromatography eluting with 5% MeOH in CH2Cl2: mp 168-
170 °C; 1H NMR (DMSO-d6) δ 1.91-2.00 (m, 2H), 2.38 (m, 4H),
2.45 (m, 2H), 3.57 (m, 4H), 3.95 (s, 3H), 4.16 (t, J ) 6 Hz, 2H),
7.34 (s, 1H), 7.52 (s, 2H), 7.80 (s, 2H), 8.42 (s, 1H), 9.55 (s,
1H); MS (ES) m/z 487.0, 489.1 (M + 1). Anal. (C24H24Cl2N4O3‚
0.3H2O) C, H, N.
4-[(2,4-Dich lor oph en yl)am in o]-6-m eth oxy-7-(3-m or ph o-
lin -4-ylp r op oxy)-3-qu in olin eca r bon itr ile (25). Following
the pyridine hydrochloride route used to prepare 2a , 25 was
obtained as a brown foam in 23% yield from 4x26 after
chromatography eluting with a gradient of 2% to 8% MeOH
in CH2Cl2: 1H NMR (DMSO-d6) δ 1.91-2.07 (m, 2H), 2.32-
2.49 (m, 6H), 3.53-3.65 (m, 4H), 3.94 (s, 3H), 4.21 (t, J ) 6
Hz, 2H), 7.34 (s, 1H), 7.52 (br s, 2H), 7.81 (br s, 2H), 8.42 (s,
(DMSO-d6) δ 1.92-1.98 (m, 2H), 2,16 (s, 3H), 2.27-2.53 (m,
10H), 3.93 (s, 3H), 4.19 (t, J ) 6 Hz, 2H), 7.31 (s, 1H), 7.50 (br
s, 2H), 7.80 (m, 2H), 8.41 (s, 1H), 9.56 (br s, 1H); MS (ES) m/z
500.1, 502.1 (M + 1). Anal. (C25H27Cl2N5O2‚1.0H2O) C, H, N.
4-[(2,4-Dich lor op h en yl)a m in o]-7-[3-(4-h yd r oxy-1-p ip -
er idin yl)pr opoxy]-6-m eth oxy-3-qu in olin ecar bon itr ile (27).
A mixture of 2y (300 mg, 0.70 mmol), 4-hydroxy-1-piperidine
(93 mg, 0.92 mmol) and a catalytic amount of sodium iodide
in 2 mL of ethylene glycol dimethyl ether was heated at 100
°C overnight. The reaction mixture was cooled and partitioned
between EtOAc and brine. The organic layer was dried over
Na2SO4, filtered and concentrated in vacuo. The residue was
purified by preparative thin-layer chromatography eluting
with 30% MeOH in CH2Cl2 to provide 105 mg (30%) of 27 as
an off-white solid: mp 108-110 °C; 1H NMR (DMSO-d6) δ
1.32-1.44 (m, 2H), 1.68-1.75 (m, 2H), 1.90-2.09 (m, 4H), 2.43
(t, J ) 7 Hz, 2H), 2.68-2.77 (m, 2H), 3.42 (m, 1H), 3.93 (s,
3H), 4.18 (t, J ) 6 Hz, 2H), 4.52 (d, J ) 4 Hz, 1H), 7.31 (s,
1H), 7.50 (br s, 2H), 7.80 (m, 2H), 8.40 (s, 1H), 9.55 (br s, 1H);
MS (ES) m/z 501.1, 503.1 (M + 1). Anal. (C25H26Cl2N4O3‚
1.0H2O) C, H, N.
Con str u ction of Activa ted Sr c a n d F yn P r otein s. A
PCR-based scheme was used to construct precise gene fusions
to replace the catalytic domain of Prague C v-Src with the
catalytic domains of c-Src and FynB. Briefly, the N-terminal
248 amino acids of v-Src was fused to threonine-250 in human
c-Src, while a C-terminal fusion was created by joining alanine-
517 of human c-Src to glutamine-515 of Prague C v-Src. This
fusion thus has the v-Src SH3 and SH2 domains as well as
the v-Src carboxyl terminal tail. Similarly, a v-Src/FynB fusion
was created by an N-terminal fusion of threonine-250 in v-Src
to methionine-251 in human FynB, and at the C-terminus by
a junction of serine-518 (FynB) to glutamine-515 (v-Src). These
kinases lack all negative regulatory elements in SFKs. (The
details of these constructions will be provided on request.) The
genes encoding these fusion proteins were cloned downstream
of a GAL1/10 promoter in the yeast vector pRS316 and
downstream of a tetracycline-repressible promoter in a mam-
malian expression vector (pTRE; Clontech).
Yea st Scr een . The Src gene was cloned into pRS316,44
modified by addition of an inducible GAL1/10 yeast promoter,
and transformed into the yeast strain W303a. Src was toxic
when expressed, i.e., when cells were grown in medium
containing galactose. For the compound screen, cells were
grown overnight in minimal medium (0.67% yeast nitrogen
base, with amino acid and adenine supplements) containing
the repressing carbon source glucose. 100 µL of this saturated
culture was added to 1.2 L of liquefied 2% agar minus uracil
minimal medium (50 °C) containing 0.5% galactose. The
mixture was plated in Nunc Bioassay dishes (#240835). When
the agar solidified, compounds were spotted onto the plates

832 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Boschelli et al.
(6) Soriano, P.; Montgomery, C.; Geske, R.; Bradley, A. Targeted
Disruption of the c-src Proto-Oncogene Leads to Osteopetrosis
in Mice. Cell 1991, 64, 693-702.
(7) Lowell, C. A.; Soriano, P. Knockouts of Src-Family Kinases: Stiff
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(compounds from six 96-well plates onto one assay plate).
Plates were then incubated at 30 °C for 3-5 days. Compounds
that promoted growth were scored as hits.
Sr c Kin a se Assa y. Src kinase activity was measured in
an ELISA format (Roche Diagnostics tyrosine kinase assay
kit). Src (3 units/reaction; Upstate Biotechnologies), reaction
buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 0.1 mM EGTA,
0.5 mM Na3VO4) and cdc2 substrate peptide were added to
compound and incubated at 30 °C for 10 min. The reaction
was started by the addition of ATP to a final concentration of
100 µM, incubated at 30 °C for 1 h and stopped by addition of
EDTA. Instructions from the manufacturer were followed for
subsequent steps. Compounds were tested in duplicate and
the value given is an average of at least two determinations.
Cell Cu ltu r e. Rat2 fibroblasts (ATCC CRL-1764) were
cotransfected with pTet-Off (Clontech) and pTRE, pTRE Src,
or pTRE Fyn. Stable cell lines were obtained by selection in
G418. Highly transformed cells were selected by two sequential
cloning steps in soft agar. The Src- and Fyn-transformed cells
were morphologically distinct, exhibited anchorage indepen-
dence and exhibited high levels of cellular protein phospho-
rylation on tyrosine.
P r olifer a tion Assa ys. Cells grown on plastic (anchorage-
dependent assays) were plated at a density of 1000 cells/well
in 96-well dishes (Costar 3599) on day 0. Compound was added
on day 1 and cells were incubated until day 4 when MTS
reagent (Promega CellTiter 96) was added and optical density
at 490 nm determined. Anchorage-independent (suspension)
growth was measured in ultralow binding plates (Costar 3474)
in a similar manner except that 5000 cells/well in a 96-well
dish were seeded. We observed no cell attachment under these
conditions. The parent Rat2 line grew poorly in the low-binding
plates.
P r ot ein P r ep a r a t ion s. Equal numbers of cells were
exposed to compound for 5 h to overnight. (At higher concen-
trations of compound (5 µM), the transformed morphology was
completely reverted in about 1 h.) Cells were harvested after
three washes with ice-cold phosphated-buffered saline pH 7.4
(PBS; Gibco) to which sodium orthovanadate (Fisher Scientific)
was added to 0.5 mM. Lysates were prepared in ice-cold RIPA
buffer (PBS with 1% Triton X-100, 0.5% sodium deoxycholate,
0.1% sodium dodecyl sulfate (SDS)), supplemented with apro-
tinin and protease inhibitor cocktail (Calbiochem). Lysates
were clarified by centrifugation (20 min at 14 000 rpm in an
Eppendorf 5417R microcentrifuge), and then frozen in dry ice/
ethanol. Lysates from ∼4 × 106 cells were diluted 1:10 in
Laemmli buffer (BioRad) and analyzed by SDS-PAGE. An-
tibodies to phosphotyrosine (4G10) and Src (GD11) were
obtained from Upstate Biotechnologies, and antibody to actin
was obtained from Boehringer Mannheim (Roche Diagnostic).
Ack n ow led gm en t. The authors gratefully acknowl-
edge the Wyeth-Ayerst Analytical Chemistry Depart-
ment for the spectral data and elemental analyses and
members of the Wyeth-Ayerst Oncology Department for
testing 2a in the ErbB2, FGFr, and cdk4 kinase assays.
(21) Thompson, A. M.; Rewcastle, G. W.; Boushelle, S. L.; Hartl, B.
G.; Kraker, A. J .; Lu, G. H.; Batley, B. L.; Panek, R. L.;
Showalter, H. D. H.; Denny, W. A. Synthesis and Structure-
Activity Relationships of 7-Substituted 3-(2,6-Dichlorophenyl)-
1,6-naphthyridin-2(1H)-ones as Selective Inhibitors of pp60c-src.
J . Med. Chem. 2000, 43, 3134-3147.
Su p p or tin g In for m a tion Ava ila ble: Elemental analysis
data for compounds 2a -y, 3, 4g, 6, 8-13, 15-21, 23-27. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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