Optimization of 4-phenylamino-3-quinolinecarbonitriles as potent inhibitors of Src kinase activity

Article abstract of DOI:10.1021/jm0102250

Subsequent to the discovery of 4-[(2,4-dichlorophenyl)amino] -6,7-dimethoxy-3-quinolinecarbonitrile (1a) as an inhibitor of Src kinase activity (IC₅₀ = 30 nM) several additional analogues were prepared. Optimization of the C-4 anilino group of la led to lc which contains a 2,4-dichloro-5-methoxy- substituted aniline. Replacement of the methoxy group at C-7 of 1c with a 3-(morpholin-4-yl)propoxy group provided 2c resulting in increased inhibition of both Src kinase activity and Src-mediated cell proliferation. Analogues of 2c with other trisubstituted anilines at C-4 were also potent Src inhibitors and the propoxy group of 2c was preferred over ethoxy butoxy or pentoxy. Replacement of the morpholine group of 2c with a 4-methylpiperazine group provided 31a which had an IC₅₀ of 1.2 nM in the Src enzymatic assay an IC₅₀ of 100 nM for the inhibition of Src-dependent cell proliferation and was selective for Src over non-Src family kinases. Compound 31a which had higher 1 and 4 h plasma levels than 2c effectively inhibited tumor growth in xenograft models.

Full text of DOI:10.1021/jm0102250

J . Med. Chem. 2001, 44, 3965-3977
3965
Op tim iza tion of 4-P h en yla m in o-3-qu in olin eca r bon itr iles a s P oten t In h ibitor s of
Sr c Kin a se Activity
Diane H. Boschelli,*,† Fei Ye,† Yanong D. Wang,† Minu Dutia,† Steve L. J ohnson,† Biqi Wu,† Karen Miller,†,‡
Dennis W. Powell,† Deanna Yaczko,§ Mairead Young,§ Mark Tischler,§ Kim Arndt,| Carolyn Discafani,|
Carlo Etienne,| J ay Gibbons,| J anet Grod,| J udy Lucas,| J ennifer M. Weber,| and Frank Boschelli|
Chemical Sciences, Discovery Analytical Chemistry, and Oncology, Wyeth-Ayerst Research, 401 North Middletown Road,
Pearl River, New York 10965
Received May 21, 2001
Subsequent to the discovery of 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbo-
nitrile (1a ) as an inhibitor of Src kinase activity (IC50 ) 30 nM), several additional analogues
were prepared. Optimization of the C-4 anilino group of 1a led to 1c, which contains a 2,4-
dichloro-5-methoxy-substituted aniline. Replacement of the methoxy group at C-7 of 1c with
a 3-(morpholin-4-yl)propoxy group provided 2c, resulting in increased inhibition of both Src
kinase activity and Src-mediated cell proliferation. Analogues of 2c with other trisubstituted
anilines at C-4 were also potent Src inhibitors, and the propoxy group of 2c was preferred over
ethoxy, butoxy, or pentoxy. Replacement of the morpholine group of 2c with a 4-methylpiper-
azine group provided 31a , which had an IC50 of 1.2 nM in the Src enzymatic assay, an IC50 of
100 nM for the inhibition of Src-dependent cell proliferation and was selective for Src over
non-Src family kinases. Compound 31a , which had higher 1 and 4 h plasma levels than 2c,
effectively inhibited tumor growth in xenograft models.
In tr od u ction
ment of the C-7 methoxy group with a 3-(morpholin-4-yl)-
propoxy group to provide 2a resulted in enhanced
inhibition of both Src enzymatic and cell activities, this
substitution at C-6 was detrimental. It was also reported
that 3a and 4a , the corresponding quinazoline and
quinoline analogues of 1a , were less potent Src inhibi-
tors.
Tyrosine kinases (TKs) are enzymes that catalyze the
specific phosphorylation of tyrosine residues on proteins.
The TK Src is the prototype member of the Src family
of kinases (SFKs), which share a common structural
organization and are highly homologous in their ATP-
binding regions.1-4 Since Src is involved in several cell
signaling pathways, inhibition of Src activity could be
efficacious for the treatment of various diseases, includ-
ing cancer.5,6 Src is overexpressed in several types of
human tumors, and increased Src activity is observed
in metastatic tumors.7-10 In addition, since Src has been
implicated in VEGF (vascular endothelial growth factor)
signaling in endothelial cells, Src inhibitors might
inhibit tumor growth via an anti-angiogenesis mecha-
nism.11 Src inhibitors may also prevent secondary injury
that results from VEGF-mediated increase in vascular
permeability, including the brain damage that often
follows stroke.12 Lastly, since osteoclastic bone resorp-
tion requires Src activity, Src inhibitors could prove
effective in the treatment of osteoporosis.13-15
We recently reported that a high-throughput yeast-
based assay identified 4-[(2,4-dichlorophenyl)amino]-6,7-
dimethoxy-3-quinolinecarbonitrile (1a ) as a Src inhibi-
tor.16 Further studies showed that this compound
inhibited the kinase activity of Src with an IC50 of 30
nM. This earlier work demonstrated 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 were all
crucial for optimal activity. In addition, while replace-
Other reports from our laboratories demonstrated
that altering the substituents on the aniline group at
C-4 of the 3-quinolinecarbonitrile resulted in compounds
with selectivity for other kinases, including EGFr
(epidermal growth factor receptor)17 and MAPKK (mi-
togen-activated protein kinase kinase).18 In the hope of
improving the activity and selectivity for Src, we
investigated other aniline groups at C-4.19 To this end,
several commercially available 2-chloroanilines were
added to 4-chloro-6,7-dimethoxy-3-quinolinecarbonitrile
(5).17 When compared to 1a , increased inhibition of Src
* To whom correspondence should be addressed. Phone: (845) 602-
3567. Fax: (845) 602-5561. E-mail: bosched@war.wyeth.com.
† Chemical Sciences.
‡ Summer intern from Dartmouth College.
§ Discovery Analytical Chemistry.
| Oncology.
10.1021/jm0102250 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/17/2001

3966 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Boschelli et al.
Sch em e 1a
Sch em e 3a
a
Reagents and conditions: (a) aniline, pyridine hydrochloride,
2-ethoxyethanol, reflux, or aniline, sodium hydride, tetrahydro-
furan, reflux.
Sch em e 4a
a
Reagents and conditions: (a) aniline, pyridine hydrochloride,
2-ethoxyethanol, reflux, or aniline, sodium hydride, tetrahydro-
furan or N,N-dimethylformamide, reflux; (b) 2,4-dichloro-5-meth-
oxyaniline, ethanol, reflux; (c) 2,4-dichloro-5-methoxyaniline, tris-
(dibenzylidene)acetonedipalladium, 2-(cyclohexylphosphino)-2′-
(N,N-dimethylamino)biphenyl, potassium phosphate, ethylene
glycol dimethyl ether, reflux.
Sch em e 2a
a
Reagents and conditions: (a) 3-chloropropyl p-toluenesulfonate,
potassium carbonate, tricaprylmethylammonium chloride, acetone,
reflux; (b) 70% nitric acid, acetic acid, reflux; (c) iron, ammonium
chloride, aqueous methanol, reflux; (d) (1) dimethylformamide
dimethylacetal, reflux; (2) n-butyllithium, acetonitrile, tetrahydro-
furan, -78 °C to room temperature; (e) phosphorous oxychloride,
reflux; (f) 2,4-dichloro-5-methoxyaniline, pyridine hydrochloride,
2-ethoxyethanol, reflux; (g) morpholine, sodium iodide, 90 °C.
drochloride in 2-ethoxyethanol or sodium hydride in
tetrahydrofuran. The quinazoline 3b was obtained by
reaction of 4-chloro-6,7-dimethoxyquinazoline (6)22 with
2,4-dichloro-5-methoxyaniline23 in ethanol. All of these
conditions were ineffective in the reaction of 4-chloro-
6,7-dimethoxyquinoline (7)24 with this aniline, and it
was necessary to use a palladium-mediated coupling
condition reported by Buchwald25 to prepare 4b.
None of the trisubstituted anilines used to prepare
1c-1m were commercially available. Those anilines not
previously reported in the literature were prepared as
shown in Scheme 2. Reaction of 2,4-dichloro-5-hydroxy-
acetanilide26 with ethyl iodide followed by removal of
the acetyl group provided 8a . Similar reaction conditions
employing n-propyl bromide as the alkylating agent led
to 8b. The corresponding hydroxy derivative 8c was
obtained by treatment of 2,4-dichloro-5-methoxy-
aniline23 with boron tribromide. Alkylation of 2-chloro-
4-methyl-5-nitrophenol23 with ethyl iodide followed by
reduction of the nitro group led to 9. An analogous
sequence was used to convert 2,4-dimethyl-5-nitro-
phenol27 to 10.
a
Reagents and conditions: (a) (1) ethyl iodide or n-propyl
bromide, potassium carbonate, acetone, reflux; (2) 5.0 N sodium
hydroxide, aqueous ethanol, reflux; (b) boron tribromide, dichloro-
methane, -78 °C to room temperature; (c) (1) ethyl iodide,
potassium carbonate, acetone, reflux; (2) iron, ethanol, aqueous
hydrochloric acid, reflux; (d) (1) methyl iodide, potassium carbon-
ate, acetone, reflux; (2) palladium on carbon, methanol, hydrogen
at 45 psi.
kinase activity was observed with 1b (IC50 ) 10 nM),
which contains a 2-chloro-5-methoxyaniline group at
C-4. Combination of the groups on 1a and 1b provided
1c, which had an IC50 of 4.3 nM. We present here a full
structure-activity relationship (SAR) study of ana-
logues of 1c as Src kinase inhibitors.20,21
As shown in Scheme 3, several of these trisubstituted
anilines were added to the 4-chloro group of 1128 to
provide compounds 2c-2g, which have a 3-(morpholin-
4-yl)propoxy group at C-7. The isomer of 2c, with the
3-(morpholin-4-yl)propoxy group at C-6, namely, 18, was
prepared as shown in Scheme 4. Alkylation of methyl
3-hydroxy-4-methoxybenzoate with 3-chloropropyl p-
toluenesulfonate29 provided 12. Nitration of 12 gave 13,
Ch em istr y
As depicted in Scheme 1, compounds 1b-1m were
prepared by treatment of 517 with the corresponding
aniline. Two protocols were used, either pyridine hy-

Quinolinecarbonitriles as Inhibitors of Src Kinase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3967
Sch em e 5a
a
Reagents and conditions: (a) triphenylphosphine, diethyl azodicarboxylate, 0 °C to room temperature, Cl(CH2)nCH2OH, tetrahydrofuran;
(b) 2,4-dichloro-5-methoxyaniline, pyridine hydrochloride, 2-ethoxyethanol, reflux, or 2,4-dichloro-5-methoxyaniline, sodium hydride,
tetrahydrofuran, reflux; (c) morpholine, sodium iodide, either neat at reflux or with ethylene glycol dimethyl ether as cosolvent at 90 °C;
(d) 2,4-dichloro-5-methoxyaniline, pyridine hydrochloride, 2-ethoxyethanol, reflux; (e) R′′R′NH, sodium iodide, either neat or with ethylene
glycol dimethyl ether as cosolvent at elevated temperatures.
with subsequent iron reduction of the nitro group
resulting in 14. Conversion of 14 to the amidine deriva-
tive, followed by cyclization with the anion of aceto-
nitrile, provided the 3-quinolinecarbonitrile 15. Chlori-
nation of 15 with phosphorus oxychloride led to 16,
which was converted to 17 upon treatment with 2,4-
dichloro-5-methoxyaniline. Reaction of 17 with morpho-
line provided the desired 18.
The preparation of analogues of 2c wherein the length
of the propoxy chain at C-7 was varied is shown in
Scheme 5. Treatment of 1919 under Mitsunobu condi-
tions using 2-chloroethanol, 4-chlorobutanol, and 5-chloro-
pentanol provided 20, 21, and 22, respectively. Addition
of the 2,4-dichloro-5-methoxyaniline headpiece to these
intermediates provided 23, 24, and 25, with subsequent
reaction with morpholine leading to the desired 26, 27,
and 28.
Additional analogues of 2c where the morpholine
group was replaced by other amines were prepared
using the route shown in Scheme 5. Reaction of 2918,19,28
with 2,4-dichloro-5-methoxyaniline provided 30. The
alkyl chloro group of 30 was then displaced by a variety
of amines, under the conditions used to prepare 18, to
yield 31a -31n .
most a log order more potent Src kinase inhibitor than
the 2,4-dichloro derivative 1a . Interestingly, replace-
ment of the 2,4-dichloroaniline group of both the quinazo-
line 3a and the quinoline 4a by a 2,4-dichloro-5-
methoxyaniline group to provide 3b and 4b did not
result in increased inhibition of Src kinase activity.
Further investigation of the 2,4-dichloro-5-methoxy
headpiece determined that the 2,4-dichloro-5-ethoxy
analogue 1d was much less active, having an IC50 of
only 1 µM. Decreased activity was also seen with the
5-propoxy derivative 1e, implying a size constraint at
the binding site of the anilino group. The 5-hydroxy
derivative 1f was also less active than 1c. To determine
the requirement for the chloro group at C-2 of the
trisubstituted aniline headpiece, analogues varying the
group at C-2 and retaining the 4-chloro and 5-methoxy
substituents were prepared. Reduced activity in the Src
enzymatic assay was seen with the 2-fluoro derivative
1g, while the larger 2-bromo and 2-methyl derivatives
1h and 1i retained the activity of 1c. A large decrease
in activity was also observed with the 4-chloro-3-
methoxy derivative 1j. We earlier reported that the
4-chloro-2-iodo derivative 1k was a more potent Src
inhibitor than 1a .16 By analogy, the corresponding
4-chloro-2-iodo-5-methoxy derivative could be expected
to be more active than 1c. Unfortunately, attempts to
replace the 4-chloro group of 5 with 4-chloro-2-iodo-5-
methoxyaniline resulted in loss of the 2-iodo group, and
only 1j was isolated.
Resu lts a n d Discu ssion
SAR for Sr c Kin a se a n d Sr c Cellu la r Assa ys. As
shown in Table 1, 3-quinolinecarbonitrile 1c, which has
a 2,4-dichloro-5-methoxyaniline group at C-4, was al-

3968 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Boschelli et al.
Ta ble 2. Inhibition of Src Kinase Activity and Cell
Ta ble 1. Inhibition of Src Kinase Activity by
6,7-Dimethoxy-4-(phenylamino)-3-quinolinecarbonitriles
Proliferation by 6-Methoxy-7-(3-morpholinopropoxy)-4-
(phenylamino)-3-quinolinecarbonitriles
compd
R
Srca IC50 (nM)
1a 16
1b
1c
1d
1e
1f
1g
1h
1i
2,4-di-Cl
2-Cl, 5-OMe
2,4-di-Cl, 5-OMe
2,4-di-Cl, 5-OEt
2,4-di-Cl, 5-O-n-Pr
2,4-di-Cl, 5-OH
2-F, 4-Cl, 5-OMe
2-Br, 4-Cl, 5-OMe
2-Me, 4-Cl, 5-OMe
4-Cl, 3-OMe
30
10
4.3
Srca
IC50 (nM)
cellsb
IC50 (nM)
compd
R
2a 16
2b
2c
2d
2e
2f
2g
18
2,4-di-Cl
3.8
1.6
0.80
0.95
1.2
1.7
1.1
28
940
150
140
71
150
160
100
1200
1200
>5000
190
66
2-Cl, 5-OMe
2,4-di-Cl, 5-OMe
2-Br, 4-Cl, 5-OMe
2-Me, 4-Cl, 5-OMe
2,4-di-Me, 5-OMe
2-Cl, 4-Me, 5-OMe
6-O(CH2)3-morpholine,
7-OMe isomer of 2c
3.0
4.0
120
6.2
2.9
1j
1k 16
1l
2-I, 4-Cl
2-Cl, 4-Me, 5-OMe
2-Cl, 4-Me, 5-OEt
2,4-di-Me, 5-OMe
quinazoline analogue of 1a
quinoline analogue of 1a
quinazoline analogue of 1c
quinoline analogue of 1c
1m
1n
>5000
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
cellular assay. IC50 values reported represent the means of at least
two separate determinations.
4.6
3a 16
4a 16
3b
250
84
390
81
b
4b
Ta ble 3. Inhibition of Src Kinase Activity and Cell
Proliferation by Various C-7-Substituted 4-[(2,4-Dichloro-
5-methoxyphenyl)amino]-6-methoxy-3-quinolinecarbonitriles
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.
As was seen with the 2-chloro group, replacement of
the 4-chloro group on the 2,4-dichloro-5-methoxyaniline
headpiece by a methyl group was not detrimental. While
1l retained the activity of 1c, the corresponding 5-ethoxy
analogue of 1l, namely, 1m , was much less active. This
loss of activity parallels that seen with 1d and 1e.
Replacement of both the 2- and 4-chloro groups of 1c
with methyl groups, as in derivative 1n , retained the
Src inhibitory activity.
Several 3-quinolinecarbonitriles with a methoxy group
at C-6, a 3-(morpholin-4-yl)propoxy group at C-7, and
trisubstituted aniline groups at C-4, namely, 2c-2g,
showed similar activity in the Src enzymatic assay,
having IC50 values ranging from 0.80 to 1.7 nM (Table
2). These compounds were also tested for their ability
to inhibit the proliferation of c-Src-transformed rat
fibroblasts growing in suspension.16 The trisubstituted
aniline containing compounds were all more potent than
2a , with the IC50 values for 2c-2g falling within the
range of 71-160 nM. As expected from our previous
work,16 18, the isomer of 2c with the 3-(morpholin-4-
yl)propoxy group at C-6, was considerably less active
in both the enzyme (IC50 ) 28 nM) and cell (IC50 ) 1.2
µM) assays.
Attention was next focused on the length of the alkoxy
side chain at C-7 of 2c. As shown in Table 3, the
corresponding ethoxy, butoxy, and pentoxy analogues,
namely, 26, 27, and 28, were all less potent than 2c in
the enzymatic assay. While 26 was roughly comparable
to 2c in the cell assay, 27 and 28 were about 2-fold less
active than 2c. Since the propoxy group was preferred
over other chain lengths, additional analogues of 2c
were prepared wherein the morpholine group was
replaced by other amines. The 4-alkylpiperazines 31b,
31c, and 31d had good activity in both the enzymatic
and cellular assays. The unsubstituted piperazine de-
rivative 31e was much less active in the cell assay,
Srca
cellsb
compd
n
R
IC50 (nM) IC50 (nM)
2c
26
27
28
3
2
4
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
morpholine
morpholine
morpholine
morpholine
0.8
2.3
1.3
2.5
1.2
0.77
1.4
1.1
1.1
0.76
1.4
1.2
0.64
9.3
1.9
2.1
3.0
2.3
140
190
330
320
100
130
220
160
1700
610
350
150
840
1900
920
1400
1200
650
31a
31b
31c
31d
31e
31f
31g
31h
31i
31j
31k
31l
31m
31n
N-methylpiperazine
N-ethylpiperazine
cis-3,4,5-tri-Me-piperazine
4-n-Pr-piperazine
piperazine
4-(CH2)2OH-piperazine
4-Me-homopiperazine
piperidine
4-OH-piperidine
2-(1,2,3-triazole)
1-(1,2,3-triazole)
1-imidazole
NH(CH2)2-morpholine
NMe(CH2)2NMe2
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 two
separate determinations.
b
although it had comparable activity in the enzyme
assay. The 4-(2-ethanol)piperazine analogue 31f had
reduced activity in both assays compared to the other
4-alkylpiperazines. The methylene homologue of 31a ,
31g, had comparable enzyme activity but was about
3-fold less active in cells. While the piperidine analogue
31h had cell activity comparable to that of 31a , the
4-hydroxypiperidine analogue 31i had reduced activity.
This parallels the result seen with 31f, which also

Quinolinecarbonitriles as Inhibitors of Src Kinase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3969
Ta ble 4. Nude Mouse Plasma Levels Following a 50 mg/kg ip
Dose
plasma concn
plasma concn
compd
R
at 1 h (µg/mL) at 4 h (µg/mL)
2c
31a
31k
morpholine
N-methylpiperazine
1-(1,2,3-triazole)
0.10 ((0.10)
3.0 ((1.2)
1.3 ((1.1)
0.10 ((0.10)
0.90 ((0.10)
0.30 ((0.20)
contains a hydroxyl group. Three derivatives with
aromatic amines, namely, 31j, 31k , and 31l, all had
reduced activity compared to 31a . Extension of the
morpholine group of 2c by an ethylamine group, as in
31m , also led to reduced activity. In addition, 31n ,
which contains an N,N,N′-trimethylethylenediamine
group, and can be viewed as an “opened piperazine”
analogue, also had reduced activity compared to 31a .
P h a r m a cok in etic An a lysis. As shown in Tables 2
and 3, several compounds had similar activities in the
enzymatic assay and also in the cellular assay. To select
a compound for further study, including in vivo testing,
an evaluation of the plasma levels of three representa-
tive compounds was performed. An ip dose of 50 mg/kg
2c, 31a , and 31k was administered to nude mice, and
blood samples were drawn after 1 and 4 h. As shown in
Table 4, the blood levels at both the 1 and 4 h time
points for the N-methylpiperazine analogue 31a were
substantially higher than those of 2c. Although 31k was
not as potent in the enzymatic or cell assay as 2c or
31a , this compound was chosen for pharmacokinetic
evaluation because it contains a 1-(1,2,3-triazolyl)alkoxy
group. In a series of quinazoline VEGFr TK inhibitors
reported by Astra-Zeneca,30 the 1-(1,2,3-triazolyl) group
provided superior blood levels compared to a morpholine
group. While 31k had enhanced plasma levels compared
to 2c, these levels were lower than those of 31a .
F igu r e 1. Reversion of Src-transformed fibroblasts to a
nontransformed appearance by 31a . Photomicrographs of rat
fibroblasts transformed by a chimeric Prague C v-Src/human
c-Src protein. (A) Cells with no compound added. (B) Cells after
1 h of exposure to 1 µM 31a .
En zym e Selectivity. The lead compound 1a was
previously reported to be selective for Src over several
non-Src family kinases, including ErbB-2 and cyclin-
dependent kinase 4 (cdk4).16 When 31a was tested
against ErbB-2, an IC50 of 2.6 µM was obtained. In
addition, 31a provided only 20% inhibition of cdk4
activity when tested at a concentration of 10 µg/mL.
Further kinase selectivity studies were done in cell-
based systems with rat fibroblasts stably transfected
with plasmids expressing activated insulin-like growth
factor receptor TK (IGFr), platelet-derived growth factor
receptor TK (PDGFr), and fibroblast growth factor
receptor TK (FGFr). When tested in proliferation assays
with these cell lines, 31a had an IC50 of 4.0 µM for the
inhibition of PDGFr-mediated cell growth and IC50
values greater than 10 µM for the inhibition of IGFr-
and FGFr-mediated cell growth. While 31a was selective
for Src over non-SFKs, this compound had an IC50 of
410 nM for the inhibition of Fyn-dependent cell prolif-
eration. Therefore, in cell-based assays the selectivity
for Src over Fyn, another SFK, is only about 4-fold. This
selectivity ratio is similar to that earlier reported for
2a .16
F igu r e 2. Inhibition of HT29 colony formation in soft agar
by 31a . Cells were plated in soft agar as described in the
Experimental Section. (A) Colony formation in agar with no
compound added. (B) An equivalent number of cells were
plated, and 31a was added to the medium layered on top of
the soft agar layer containing the cells. The concentration of
31a in the liquid medium was 1 µM, and the liquid medium
layer comprised one-third of the total volume of the agar and
medium.
Ad d ition a l Cellu la r Assa ys. In an examination of
extracts of Src-transformed fibroblasts treated with 31a
by anti-phosphotyrosine immunoblotting, a dose-
response relationship for the inhibition of tyrosine
phosphorylation was observed that mirrored the IC50
for proliferation of these cells in suspension (data not
shown). This effect was similar to that previously
reported for 2a .16 Also, the Src-transformed fibroblasts
reverted to a nontransformed morphology after exposure
to 1 µM 31a for 1 h (Figure 1).
The efficacy of anti-sense Src expression in inhibiting
the growth of HT29 colon tumors in a nude mouse

3970 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Boschelli et al.
tions, however, the animals exhibited abdominal bloat-
ing. When 31a was dosed at 25 mg/kg ip bid, a T/C of
30% was observed, with reduced bloating. A decrease
in activity was observed with a 10 mg/kg ip bid dose of
31a (T/C of 44% (p < 0.01) at day 21), and no activity
was observed with a 3 mg/kg ip bid dose of 31a . When
dosed orally at 50 mg/kg bid, 31a inhibited tumor
growth (T/C of 35% (p < 0.01) at day 21) with no
associated bloating, but increasing the oral dose to 100
mg/kg bid resulted in 40% mortality by day 21. There-
fore, while 31a has significant antitumor activity, this
compound does not have a wide therapeutic window in
this xenograft model.
Con clu sion . Optimization of the C-4 anilino group
of a 6,7-dimethoxy-3-quinolinecarbonitrile with a trisub-
stituted aniline provides enhanced Src inhibitory activ-
ity over disubstituted aniline groups. Replacement of
the 7-methoxy group of these compounds with an alkoxy
group terminating with a basic amine results in in-
creased Src enzymatic and cellular inhibition. Analogue
31a , which contains a 3-(4-methylpiperazin-1-yl)propoxy
group, has an IC50 of 1.2 nM in the Src enzymatic assay
and an IC50 of 100 nM for the inhibition of Src-
dependent cell proliferation and is selective for Src over
non-Src family kinases. In this series of compounds, the
4-methylpiperazine group provides superior blood levels
over a morpholine or 1-(1,2,3-triazole) group. Compound
31a effectively inhibited tumor growth in xenograft
models employing Src-transformed fibroblasts and also
showed activity against HT29 xenografts.
F igu r e 3. Antitumor activity of 31a vs a Src-transformed
fibroblast xenograft. (A) Unstaged model where mice were
treated with 31a at 30 mg/kg ip bid with dosing beginning
just prior to injection of cells (9), vehicle alone (2). (B) Staged
model where mice were treated with 31a at 25 mg/kg ip bid
after the tumors reached 100 mg or more in volume (9), vehicle
alone (2).
Exp er im en ta l Section
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
xenograft model suggested that the growth of these
tumor cells in vivo is dependent upon Src activity.31,32
When 31a was tested in an HT29 cell proliferation
assay, under adherent conditions, an IC50 of only 5 µM
was observed, but colony formation in agar was inhib-
ited at submicromolar concentrations (Figure 2). These
results imply that 31a is a potent inhibitor of Src
activity in cells and that while HT29 cells do not exhibit
a strong dependence for Src activity for their growth in
monolayer culture, their growth in agar is Src depend-
ent.
Xen ogr a ft Stu d ies. The Src inhibitor 31a was
chosen for evaluation in xenograft models employing the
Src-transformed fibroblasts. In an unstaged model, a 30
mg/kg dose of 31a administered ip twice a day (bid) for
14 days provided a T/C of 18% (Figure 3A). In a staged
model, where the tumors were implanted 3 days prior
to dosing, a 25 mg/kg dose of 31a administered ip bid
for 10 days provided a T/C of 25% (Figure 3B). These
results suggest that 31a is sufficiently bioavailable to
inhibit Src in vivo.
Although 31a was not a potent inhibitor of the
proliferation of HT29 cells during monolayer growth, the
ability of 31a to inhibit HT29 cell growth in agar implied
that some aspect of the tumorigenic phenotype of these
cells was affected. Therefore, 31a was tested in a staged
xenograft model with HT29 cells. When 31a was dosed
at 30 mg/kg ip bid for 21 days, a statistically significant
reduction in tumor growth rate was observed (T/C of
30% (p < 0.01) on day 21). Under these dosing condi-
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-Ch lor o-5-m eth oxyp h en yl)a m in o]-6,7-d im eth oxy-
3-qu in olin eca r bon itr ile (1b). A mixture of 5 17 (120 mg, 0.48
mmol), 6-chloro-m-anisidine hydrochloride (103 mg, 0.53 mmol),
and pyridine hydrochloride (61 mg, 0.53 mmol) in 6 mL of
2-ethoxyethanol was heated at reflux for 6 h and then
concentrated in vacuo. The residue was treated with water and
neutralized by addition of sodium carbonate (112 mg). After
being stirred for 1.5 h, the aqueous suspension was extracted
with dichloromethane. The combined organic phase was
washed with brine, dried over sodium sulfate, and then
filtered. Removal of the solvent gave a dark residue that was
purified by preparative thin-layer chromatography, developing
with 5% methanol in dichloromethane, to provide 35 mg (20%)
of 1b as a light brown solid: mp 143-145 °C; 1H NMR (DMSO-
d6) δ 3.77 (s, 3H), 3.86 (s, 3H), 3.93 (s, 3H), 6.98 (dd, J ) 9, 3
Hz, 1H), 7.07 (d, J ) 3 Hz, 1H), 7.33 (s, 1H), 7.48 (d, J ) 9 Hz,
1H), 7.83 (s, 1H), 8.41 (s, 1H), 9.56 (s, 1H); MS (ES) m/z 370.2,
372.2 (M + 1).
Anal. (C19H16ClN3O3·1.0H2O) C, H, N.
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6,7-d im eth -
oxy-3-qu in olin eca r bon itr ile (1c). A mixture of 2,4-dichloro-
5-methoxyaniline (771 mg, 4.0 mmol)23 and 60% sodium
hydride (160 mg, 4.0 mmol) in 25 mL of tetrahydrofuran was
heated at reflux for 1 h. After the mixture was cooled to room
temperature, 5 (500 mg, 2.0 mmol) was added, and the reaction
mixture was heated at reflux overnight. The reaction mixture

Quinolinecarbonitriles as Inhibitors of Src Kinase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3971
was cooled to room temperature, and 40 mg of sodium hydride
was added. After being heated at reflux for 5.5 h, the reaction
mixture was cooled to room temperature and partitioned
between ethyl acetate and water. The organic layer was
washed with water, dried over magnesium sulfate, filtered,
and concentrated in vacuo. Diethyl ether was added to the
residue, and the solid was collected by filtration to provide 556
mg (69%) of 1c as an off-white solid: mp 258-259 °C; 1H NMR
(DMSO-d6) δ 3.86 (s, 3H), 3.94 (s, 3H), 3.95 (s, 3H) 7.32 (s,
2H), 7.73 (s, 1H), 7.83 (s, 1H), 8.41 (s, 1H), 9.63 (br s, 1H); MS
(ES) m/z 404.2, 406.2 (M + 1).
matography, eluting with a gradient of 1:1 hexane/ethyl
acetate to all ethyl acetate, followed by trituration with diethyl
ether and hexane, 1i in 39% yield as an off-white solid: mp
1
softens at 95 °C; H NMR (DMSO-d6) δ 2.10 (s, 3H), 3.81 (s,
3H), 3.94 (s, 3H) 3.95 (s, 3H), 7.10 (s, 1H), 7.32 (s, 1H), 7.42
(s, 1H), 7.84 (s, 1H), 8.36 (s, 1H), 9.40 (br s, 1H); MS (ES) m/z
384.1, 386.2 (M + 1).
Anal. (C20H18ClN3O3‚0.1(CH3CH2)2O) C, H, N.
6,7-Dim e t h oxy-4-[(2,4-d im e t h yl-5-m e t h oxyp h e n yl)-
a m in o]-3-qu in olin eca r bon itr ile (1j). According to the pro-
cedure used to prepare 1g, reaction of 5 with 10 provided 1j
1
Anal. (C19H15Cl2N3O3) C, H, N.
in 56% yield as an off-white solid: mp 138-140 °C; H NMR
(DMSO-d6) δ 2.07 (s, 3H), 2.17 (s, 3H), 3.75 (s, 3H), 3.93 (s,
3H), 3.94 (s, 3H), 6.84 (s, 1H), 7.09 (s, 1H), 7.29 (s, 1H), 7.85
(s, 1H), 8.32 (s, 1H), 9.38 (s, 1H); MS (ES) m/z 364.3 (M + 1).
Anal. (C21H21N3O3‚1.0H2O) C, H, N.
4-[(2-Ch lor o-5-m et h oxy-4-m et h ylp h en yl)a m in o]-6,7-
d im eth oxy-3-qu in olin eca r bon itr ile (1k ). According to the
procedure used to prepare 1g, reaction of 5 with 2-chloro-5-
methoxy-4-methylaniline23 provided 1k in 34% yield as a white
solid: mp 203-204 °C; 1H NMR (DMSO-d6) δ 2.20 (s, 3H), 3.79
(s, 3H), 3.94 (s, 3H) 3.95 (s, 3H), 7.07 (s, 1H), 7.32 (s, 1H),
7.37 (s, 1H), 7.85 (s, 1H), 8.37(s, 1H), 9.60 (br s, 1H); MS (ES)
m/z 384.2, 386.2 (M + 1).
4-[(2,4-Dich lor o-5-eth oxyph en yl)am in o]-6,7-dim eth oxy-
3-qu in olin eca r bon itr ile (1d ). According to the procedure
used to prepare 1c, reaction of 5 with 8a provided 1d in 60%
yield as a light yellow solid: mp 254-255 °C; 1H NMR (DMSO-
d6) δ 1.35 (t, J ) 7 Hz, 3H), 3.94 (s, 3H), 3.95 (s, 3H), 4.11 (q,
J ) 7 Hz, 2H), 7.33 (s, 2H), 7.74 (s, 1H), 7.83 (s, 1H), 8.42 (s,
1H), 9.60 (br s, 1H); MS (ES) m/z 417.9, 419.9 (M + 1).
Anal. (C20H17Cl2N3O3‚0.20H2O) C, H, N.
4-[(2,4-Dich lor o-5-p r op oxyp h en yl)a m in o]-6,7-d im eth -
oxy-3-qu in olin eca r bon itr ile (1e). According to the proce-
dure used to prepare 1c, reaction of 5 and 8b provided 1e in
57% yield as a white solid: mp 262-265 °C; 1H NMR (DMSO-
d6) δ 0.98 (t, J ) 7 Hz, 3H), 1.76 (m, 2H), 3.94 (s, 3H), 3.95 (s,
3H) 4.02 (t, J ) 7 Hz, 2H), 7.33 (s, 2H), 7.74 (s, 1H), 7.83 (s,
1H), 8.42 (s, 1H), 9.60 (br s, 1H); MS (ES) m/z 431.9, 433.9 (M
+ 1).
Anal. (C20H18ClN3O3‚0.5H2O‚0.2CH3C(O)OCH2CH3) C, H, N.
4-[(2-Ch lor o-5-e t h oxy-4-m e t h ylp h e n yl)a m in o]-6,7-
d im eth oxy-3-qu in olin eca r bon itr ile (1l). According to the
procedure used to prepare 1g, reaction of 5 with 9 provided 1l
1
Anal. (C21H19Cl2N3O3) C, H, N.
in 62% yield as a white solid: mp 240-242 °C dec; H NMR
(DMSO-d6) δ 1.35 (t, J ) 6 Hz, 3H), 2.19 (s, 3H), 3.93 (s, 3H),
3.94 (s, 3H), 4.02 (d, J ) 6 Hz, 2H), 7.06 (s, 1H), 7.31 (s, 1H),
7.37 (s, 1H), 7.84 (s, 1H), 8.36 (s, 1H), 9.53 (s, 1H); MS (ES)
m/z 398.0 (M + 1).
4-[(2,4-Dich lor o-5-h yd r oxyp h en yl)a m in o]-6,7-d im eth -
oxy-3-qu in olin eca r bon itr ile (1f). A mixture of 5 (250 mg,
1.0 mmol), 8c (0.2 g, 1.1 mmol), and pyridine hydrochloride
(130 mg, 1.1 mmol) in 20 mL of 2-ethoxyethanol was heated
at reflux for 6 h. The mixture was diluted with ethyl acetate,
washed with saturated aqueous sodium bicarbonate, dried over
sodium sulfate, and concentrated in vacuo. The solids were
collected by filtration, washing with diethyl ether, to provide
Anal. (C21H20ClN3O3) C, H, N.
4-[(4-Ch lor o-3-m eth oxyp h en yl)a m in o]-6,7-d im eth oxy-
3-qu in olin eca r bon itr ile (1m ). According to the procedure
used to prepare 1g, reaction of 5 with 4-chloro-m-anisidine,32
using N,N-dimethylformamide as the solvent, provided, after
flash column chromatography, eluting with a gradient of 25-
50% ethyl acetate in hexane, 1m in 66% yield as a white
solid: mp 136-137 °C; 1H NMR (DMSO-d6) δ 3.85 (s, 3H), 3.93
(s, 3H), 3.96 (s, 3H), 6.82 (dd, J ) 8, 2 Hz, 1H), 7.04 (d, J ) 2
Hz, 1H), 7.36 (s, 1H), 7.41 (d, J ) 8 Hz, 1H), 7.74 (s, 1H), 8.52
(s, 1H), 9.56 (s, 1H); MS (ES) m/z 369.9.
1
110 mg (28%) of 1f as a light tan solid: mp 145-150 °C; H
NMR (DMSO-d6) δ 3.93 (s, 3H), 3.95 (s, 3H) 6.99 (s, 1H), 7.34
(s, 1H), 7.62 (s, 1H), 7.79 (s, 1H), 8.44 (br s, 1H), 9.50 (br s,
1H); MS (ES) m/z 389.9, 391.9 (M + 1).
Anal. (C18H13Cl2N3O3‚1.0H2O) C, H, N.
4-[4-Ch lor o-2-flu or o-5-m e t h oxyp h e n yl)a m in o]-6,7-
d im et h oxy-3-qu in olin eca r b on it r ile (1g). A mixture of 5
(300 mg, 1.20 mmol), 4-chloro-2-fluoro-5-methoxyaniline (340
mg, 1.94 mmol),23 and pyridine hydrochloride (135 mg, 1.16
mmol) in 10 mL of 2-ethoxyethanol was heated at reflux for 3
h. After being cooled, the reaction mixture was partitioned
between ethyl acetate and saturated sodium bicarbonate. The
organic layer was washed with 1.0 N sodium hydroxide, dried
over magnesium sulfate, filtered, and concentrated in vacuo.
The residue was treated with diethyl ether, and the resultant
solid was collected by filtration, washing with diethyl ether,
to provide 318 mg (68%) of 1g as a light brown solid: mp 231-
Anal. (C19H16ClN3O3‚1.8 H2O) C, H, N.
4-[(2-Ch lor o-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-7-[3-
(4-m or p h olin yl)p r op oxy]-3-q u in olin eca r b on it r ile (2b ).
According to the procedure used to prepare 1g, reaction of 11
with 6-chloro-m-anisidine hydrochloride provided, after flash
column chromatography, eluting with a gradient of 2% metha-
nol in dichloromethane to 5% methanol in dichloromethane,
1
2b in 28% yield as a white solid: mp 179-190 °C; H NMR
(DMSO-d6) δ 1.91-2.01 (m, 2H), 2.43-2.49 (m, 6H), 3.59 (t, J
) 5 Hz, 4 H), 3.75 (s, 3H), 3.93 (s, 3H), 4.20 (t, J ) 6 Hz, 2H),
6.98 (dd, J ) 9, 3 Hz, 1H), 7.07 (d, J ) 3 Hz, 1H), 7.32 (s, 1H),
7.46 (d, J ) 9 Hz, 1H), 7.82 (s, 1H), 8.40 (s, 1H), 9.55 (s, 1H);
MS (ES) m/z 482.9 (M + 1).
1
233 °C dec; H NMR (DMSO-d6) δ 3.33 (s, 3H), 3.85 (s, 3H),
3.95 (s, 3H), 7.24 (d, J ) 7 Hz, 1H), 7.34 (s, 1H), 7.60 (d, J )
10 Hz, 1H), 7.81 (s, 1H), 8.47 (s, 1H), 9.55 (s, 1H); MS (ES)
m/z 388.2 (M + 1).
Anal. (C25H27ClN4O4‚0.15CH2Cl2) C, H, N.
Anal. (C19H15ClFN3O3) C, H, N.
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-
7-[3-(4-m or ph olin yl)pr opoxy]-3-qu in olin ecar bon itr ile (2c).
According to the procedure used to prepare 1c, reaction of 11
with 2,4-dichloro-5-methoxyaniline23 provided 2c in 88% yield
as an off-white solid: mp 160-162 °C; 1H NMR (DMSO-d6) δ
1.92-2.04 (m, 2H), 2.35-2.50 (m, 6H), 3.58 (t, J ) 5 Hz, 4H),
3.86 (s, 3H), 3.95 (s, 3H), 4.21 (t, J ) 6 Hz, 2H), 7.34 (br s,
2H), 7.75 (s, 1H), 7.83 (s, 1H), 7.82 (s, 1H), 8.42 (s, 1H), 9.62
(s, 1H); MS (ES) m/z 517.0, 518.9 (M + 1).
4-[(2-Br om o-4-ch lor o-5-m e t h oxyp h e n yl)a m in o]-6,7-
d im eth oxy-3-qu in olin eca r bon itr ile (1h ). According to the
procedure used to prepare 1c, reaction of 5 and 2-bromo-4-
chloro-5-methoxyaniline,23 in N,N-dimethylformamide, pro-
vided, after flash column chromatography, eluting with a
gradient of 25-50% ethyl acetate in hexane, 1h in 22% yield
as an off-white solid: mp 254-255 °C; 1H NMR (DMSO-d6) δ
3.86 (s, 3H), 3.94 (s, 3H), 3.95 (s, 3H), 7.33 (s, 1H), 7.36 (s,
1H), 7.85 (s, 1H), 7.86 (s, 1H), 8.41 (s, 1H), 9.64 (s, 1H); MS
(ES) m/z 447.8, 449.7 (M + 1).
Anal. (C19H15BrClN3O3‚0.5H2O‚0.5HC(O)NH2) C, H, N.
4-[(4-Ch lor o-5-m et h oxy-2-m et h ylp h en yl)a m in o]-6,7-
d im eth oxy-3-qu in olin eca r bon itr ile (1i). According to the
procedure used to prepare 1g, reaction of 5 with 4-chloro-5-
methoxy-2-methylaniline23 provided, after flash column chro-
Anal. (C25H26Cl2N4O4) C, H, N.
4-[(2-Br om o-4-ch lor o-5-m eth oxyph en yl)am in o]-6-m eth -
oxy-7-[3-(4-m or p h olin yl)p r op oxy]-3-q u in olin e ca r b o-
n itr ile (2d ). According to the procedure used to prepare 1c,
reaction of 11 with 2-bromo-4-chloro-5-methoxyaniline23 pro-
vided, after flash column chromatography, eluting with a
gradient of dichloromethane to 5% methanol in dichloro-

3972 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Boschelli et al.
methane, 2d in 40% yield as an off-white solid: mp 153-155
°C; 1H NMR (DMSO-d6) δ 1.97 (m, 2H), 2.38 (t, J ) 4 Hz, 4H),
2.44 (t, J ) 7 Hz, 2H), 3.59 (t, J ) 4 Hz, 4H), 3.83 (s, 3H), 3.94
(s, 3H), 4.21 (t, J ) 6 Hz, 2H), 7.31 (s, 1H), 7.32 (s, 1H), 7.84
(s, 1H), 7.86 (s, 1H), 8.40 (s, 1H), 9.63 (s, 1H); MS (ES) m/z
561.1, 563.1 (M + 1).
layer was dried over sodium sulfate and concentrated in vacuo
to provide 1.66 g (98%) of 2,4-dichloro-5-ethoxyacetanilide as
a white solid.
A mixture of 2,4-dichloro-5-ethoxyacetanilide (1.66 g, 6.6
mmol) and 3 mL of 5 N sodium hydroxide in 5 mL of ethanol
and 7 mL of water was heated at reflux for 2 h and then stirred
at room temperature for 18 h. After the mixture was heated
at reflux for 1 h, the ethanol was removed and the residue
was extracted with dichloromethane. The organic layer was
dried over sodium sulfate and concentrated to give 1.2 g (88%)
of 8a as a light yellow oil: 1H NMR (DMSO-d6) δ 1.33 (t, J )
7 Hz, 3H), 3.98 (q, J ) 7 Hz, 2H), 5.47 (br s, 2H), 6.53 (s, 1H),
7.14 (s, 1H); MS (ES) m/z 205.8, 207.7 (M + 1).
2,4-Dich lor o-5-p r op oxya n ilin e (8b). According to the
procedure used to prepare 8a , reaction of 2,4-dichloro-5-
hydroxyacetanilide26 with n-propyl bromide, followed by basic
hydrolysis, provided 8b in 66% yield as a light yellow oil: 1H
NMR (DMSO-d6) δ 0.98 (t, J ) 7 Hz, 3H), 1.70 (m, 2H), 3.86
(t, J ) 7 Hz, 2H), 5.46 (br s, 2H), 6.54 (s, 1H), 7.21 (s, 1H); MS
(ES) m/z 219.7, 221.8 (M + 1).
Anal. (C25H26BrClN4O4‚1.0H2O) C, H, N.
4-[(4-Ch lor o-5-m eth oxy-2-m eth ylph en yl)am in o]-6-m eth -
oxy-7-[3-(4-m or p h olin yl)p r op oxy]-3-q u in olin e ca r b o-
n itr ile (2e). According to the procedure used to prepare 1g,
reaction of 11 with 4-chloro-5-methoxy-2-methylaniline23 pro-
vided, after flash column chromatography, eluting with a
gradient of 5% methanol in dichloromethane to 10% methanol
in dichloromethane, 2e in 40% yield as an off-white solid: mp
1
205-206 °C; H NMR (DMSO-d6) δ 1.93-1.99 (m, 2H), 2.10
(s, 3H), 2.33-2.52 (m, 6H), 3.53-3.62 (m, 4H), 3.81 (s, 3H),
3.94 (s, 3H), 4.02 (t, J ) 6 Hz, 2H), 7.10 (s, 1H), 7.30 (s, 1H),
7.42 (s, 1H), 7.83 (s, 1H), 8.35 (s, 1H), 9.41 (s, 1H); MS (ES)
m/z 497.3 (M + 1).
Anal. (C26H29ClN4O4‚0.20H2O) C, H, N.
4-[(2,4-Dim eth yl-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-
7-[3-(4-m or ph olin yl)pr opoxy]-3-qu in olin ecar bon itr ile (2f).
According to the procedure used to prepare 1g, reaction of 11
with 10 provided 2f in 90% yield as an off-white solid: mp
softens 83-85 °C; 1H NMR (DMSO-d6) δ 1.92 (m, 2H), 2.06 (s,
3H), 2.17 (s, 3H), 2.38 (t, J ) 5 Hz, 4H), 2.43 (t, J ) 7 Hz, 2H),
3.59 (t, J ) 5 Hz, 4H), 3.74 (s, 3H), 3.93 (s, 3H), 4.19 (t, J ) 6
Hz, 2H), 6.84 (s, 1H), 7.09 (s, 1H), 7.29 (s, 1H), 7.84 (s, 1H),
8.31 (s, 1H), 9.37 (s, 1H); MS (ES) m/z 477.3 (M + 1).
Anal. (C27H32N4O4‚1.2 H2O) C, H, N.
2,4-Dich lor o-5-h yd r oxya n ilin e (8c). A solution of 2,4-
dichloro-5-methoxyaniline (800 mg, 4.0 mmol)23 in 10 mL of
dichloromethane was cooled to -78 °C, and 7.5 mL of 1.0 M
boron tribromide in dichloromethane (7.5 mmol) was added.
After being stirred at room temperature overnight, the reaction
mixture was cooled to -78 °C, and an additional 4.0 mL of
1.0 M boron tribromide in dichloromethane (4.0 mmol) was
added. The reaction mixture was stirred at room temperature
overnight and then quenched by the addition of 10 mL of
water. The mixture was extracted with diethyl ether, and the
product was extracted into 2 N sodium hydroxide. The aqueous
layer was neutralized (pH 7) with 1 N hydrochloric acid, and
the product was extracted into diethyl ether. The organic layer
was dried over sodium sulfate, filtered, and concentrated in
vacuo to provide 280 mg (39%) of 8c as an off-white solid: mp
4-[(2-Ch lor o-5-m eth oxy-4-m eth ylph en yl)am in o]-6-m eth -
oxy-7-[3-(4-m or p h olin yl)p r op oxy]-3-q u in olin e ca r b o-
n itr ile (2g). According to the procedure used to prepare 1g,
reaction of 11 with 2-chloro-5-methoxy-4-methylaniline23 pro-
1
vided 2g in 43% yield as a white solid: mp 146-148 °C; H
1
115-116 °C; H NMR (DMSO-d6) δ 5.34 (s, 2H), 6.41 (s, 1H),
7.10 (s, 1H), 9.89 (s, 1H); MS (ES) m/z 176.07, 178.07 (M + 1).
Anal. (C6H5Cl2NO) C, H, N.
NMR (DMSO-d6) δ 1.92-2.04 (m, 2H), 2.20 (s, 3H), 2.33-2.55
(m, 6H), 3.59 (t, J ) 5 Hz, 4H), 3.79 (s, 3H), 3.94 (s, 3H), 4.20
(t, J ) 6 Hz, 2H), 7.07 (s, 1H), 7.31 (s, 1H), 7.37 (s, 1H), 7.84
(s, 1H), 8.36 (s, 1H), 9.54 (s, 1H); MS (ES) m/z 497.1 (M + 1).
Anal. (C26H29ClN4O4‚0.50H2O) C, H, N.
2-Ch lor o-5-eth oxy-4-m eth yla n ilin e (9). 4-Chloro-2-meth-
yl-5-nitrophenol (3.80 g, 20.2 mmol)26 was dissolved in 60 mL
of acetone, and potassium carbonate (3.0 g, 21.7 mmol) and
ethyl iodide (2.1 mL, 26.3 mmol) were added. The reaction
mixture was heated at reflux for 3.5 h, cooled, and concen-
trated in vacuo. The residue was partitioned between water
and ethyl acetate. The organic layer was washed with water
and 2.0 N sodium hydroxide, dried over magnesium sulfate,
filtered, and concentrated in vacuo. Diethyl ether and hexanes
were added to the residue, and the resultant solid was collected
by filtration to give 2.70 g (62%) of 2-chloro-5-ethoxy-4-
methylnitrobenzene as an off-white solid.
A mixture of 2-chloro-5-ethoxy-4-methylnitrobenzene (2.96
g, 13.7 mmol) and iron (1.64 g, 29.4 mmol) in 90 mL of 10%
aqueous ethanol and 2 mL of concentrated hydrochloric acid
was heated at reflux for 5 h. Additional iron (1.00 g, 18.2 mmol)
and concentrated hydrochloric acid (1 mL) were added, and
the mixture was heated at reflux for an additional 2 h. The
mixture was filtered while warm, washing with ethyl acetate.
The filtrate was concentrated in vacuo, and the residue was
partitioned between ethyl acetate and aqueous saturated
sodium bicarbonate. The organic layer was washed with water,
dried over magnesium sulfate, filtered, and concentrated in
vacuo to provide 2.20 g (86%) of 9 as a waxy yellow-brown
solid: 1H NMR (DMSO-d6) δ 1.31 (t, J ) 7 Hz, 3H), 1.98 (s,
3H), 3.85 (q, J ) 7 Hz, 2H), 5.04 (br s, 2H), 6.39 (s, 1H), 6.90
(s, 1H); MS (ES) m/z 186.0, 188.0 (M + 1).
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6,7-d im eth -
oxyqu in a zolin e (3b). According to the procedure used to
prepare 1c, reaction of 622 with 2,4-dichloro-5-methoxyaniline23
provided 3b in 25% yield as a light yellow solid: mp 223-225
1
°C; H NMR (DMSO-d6) δ 3.86 (s, 3H), 3.94 (s, 6H), 7.19 (s,
1H), 7.35 (s, 1H), 7.70 (s, 1H), 7.82 (s, 1H), 8.34 (s, 1H), 9.59
(s, 1H); MS (ES) m/z 380.2, 382.2 (M + 1).
Anal. (C17H15Cl2N3O3‚0.50 H2O) C, H, N.
N-(2,4-Dich lor o-5-m et h oxyp h en yl)-6,7-d im et h oxy-4-
qu in olin a m in e (4b). 2,4-Dichloro-5-methoxyaniline23 (175
mg, 0.89 mmol) and 724 (100 mg, 0.45 mmol) were added to a
mixture of tris(dibenzylidene)acetonedipalladium (41 mg,
0.045 mmol), 2-(dicyclohexylphosphino)-2′-(N,N-dimethyl-
amino)biphenyl (25) (54 mg, 0.14 mmol), and potassium
phosphate (142 mg, 0.67 mmol) in 4.5 mL of ethylene glycol
dimethyl ether. The mixture was heated at reflux for 2.5 h,
cooled to room temperature, and partitioned between saturated
aqueous sodium bicarbonate and ethyl acetate. The combined
organic extracts were dried over sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by flash
column chromatography, eluting with a gradient of dichloro-
methane to 3% methanol in dichloromethane, followed by
recrystallization from acetone and hexane, to provide 130 mg
(76%) of 4b as a white solid: mp 165-167 °C; 1H NMR (DMSO-
d6) δ 3.86 (s, 3H), 3.91 (s, 3H), 3.93 (s, 3H), 6.20 (d, J ) 5 Hz,
1H), 7.22 (s, 1H), 7.26 (s, 1H), 7.70 (s, 1H), 7.74 (s, 1H), 8.25
(d, J ) 5 Hz, 1H), 8.67 (s, 1H); MS (ES) m/z 379.3.
Anal. (C9H12ClNO) C, H, N.
2,4-Dim et h yl-5-m et h oxya n ilin e (10). 2,4-Dimethyl-5-
nitrophenol (4.40 g, 26.3 mmol)27 was dissolved in acetone (100
mL), and iodomethane (5.51 g, 38.8 mmol) and potassium
carbonate (4.14 g, 30.0 mmol) were added. The reaction
mixture was heated at reflux for 2 h, cooled to room temper-
ature, and partitioned between water and ethyl acetate. The
organic extract was evaporated, and the residue was recrystal-
lized from diethyl ether and hexanes to provide 3.75 g (79%)
Anal. (C18H16Cl2N2O3‚0.7CH3COCH3) C, H, N.
2,4-Dich lor o-5-eth oxya n ilin e (8a ). A mixture of 2,4-
dichloro-5-hydroxyacetanilide (1.50 g, 6.8 mmol),26 ethyl iodide
(0.81 mL, 10.2 mmol), and potassium carbonate (1.41 g, 10.2
mmol) in 25 mL of acetone was heated at reflux for 4 h. The
reaction mixture was concentrated in vacuo, and the residue
was partitioned between ethyl acetate and water. The organic

Quinolinecarbonitriles as Inhibitors of Src Kinase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3973
of 2,4-dimethyl-5-methoxynitrobenzene as an off-white solid:
1H NMR (DMSO-d6) δ 2.20 (s, 3H), 2.43 (s, 3H), 3.86 (s, 3H),
7.28 (s, 1H), 7.52 (s, 1H); MS (ES) m/z 182.1 (M + 1).
(2.50 g, 61 mmol) in 142 mL of tetrahydrofuran was added
dropwise. After the solution was stirred for 15 min, a solution
of methyl 5-(3-chloropropoxy)-2-{[(dimethylamino)methylene]-
amino}-4-methoxybenzoate in 75 mL of tetrahydrofuran was
added dropwise over 30 min. The reaction mixture was allowed
to stir for 25 min at -78 °C. Acetic acid (5.22 g, 87 mmol) was
added, and the reaction was allowed to warm to room tem-
perature. After being stirred overnight, the heterogeneous
mixture was concentrated in vacuo and diluted with 250 mL
of water. The resulting suspension was stirred at room
temperature for 20 min. The solids were collected by filtration
and washed with diethyl ether to provide 4.0 g (47%) of 15 as
a pale yellow solid: mp > 260 °C; 1H NMR (DMSO-d6) δ 2.19-
2.25 (m, 2H), 3.81 (t, J ) 5 Hz, 2H), 3.89 (s, 3H), 4.18 (t, J )
5 Hz, 2H), 7.05 (s, 1H), 7.47 (s, 1H), 8.57 (s, 1H); MS (ES) m/z,
292.8 (M + 1).
A mixture of 2,4-dimethyl-5-methoxynitrobenzene (1.97 g,
11 mmol) and 10% palladium on carbon (197 mg) in 30 mL of
methanol was hydrogenated at 45 psi overnight. The reaction
mixture was filtered through a short pad of Celite, and the
filtrate was concentrated to provide 1.54 g (93%) of 10 as a
1
light tan solid: mp 80-81°C; H NMR (DMSO-d6) δ 1.94 (s,
3H), 1.95 (s, 3H), 3.65 (s, 3H), 4.58 (br s, 2H), 6.24 (s, 1H),
6.63 (s, 1H); MS (ES) m/z 152.1 (M + 1).
Anal. (C9H13NO) C, H, N.
Meth yl 3-(3-Ch lor op r op oxy)-4-m eth oxyben zoa te (12).
A mixture of potassium carbonate (20.1 g, 150 mmol), methyl
3-hydroxy-4-methoxybenzoate (20.0 g, 110 mmol), 3-chloro-
propyl p-toluenesulfonate (27.2 g, 110 mmol),29 and tricapryl-
methylammonium chloride (0.45 g, 1.1 mmol) in 240 mL of
acetone was heated at reflux overnight. The solids were
removed by filtration and washed with acetone. The filtrate
was concentrated, and the residue was dissolved in dichloro-
methane and washed with 1 N sodium hydroxide, 10% aqueous
potassium hydroxide, saturated aqueous sodium bicarbonate,
and brine. The organic layer was dried over magnesium
sulfate, filtered, and concentrated in vacuo to provide 22.85 g
Anal. (C14H13ClN2O3) C, H, N.
4-Ch lor o-6-(3-ch lor op r op oxy)-7-m eth oxy-3-qu in olin e-
ca r bon itr ile (16). A mixture of 15 (2.00 g, 6.84 mmol) and
phosphorus oxychloride (9.44 g, 61 mmol) was heated at reflux
for 1 h. The solution was concentrated, and saturated aqueous
sodium bicarbonate was added to the residue. The resultant
precipitate was collected by filtration to provide 1.93 g (91%)
of 16 as a gray solid: mp 138-140 °C; 1H NMR (DMSO-d6) δ
2.26-2.33 (m, 2H), 3.85 (t, J ) 5 Hz, 2H), 4.03 (s, 3H), 4.33 (t,
J ) 5 Hz, 2H), 7.43 (s, 3H), 7.53 (s, 3H), 8.97 (s, 3H); MS (ES)
m/z 310.8, 312.8 (M + 1).
1
(77%) of 12 as a white solid: mp 46-48 °C; H NMR (CDCl3)
δ 2.27-2.33 (m, 2H), 3.77 (t, J ) 5 Hz, 2H), 3.89 (s, 3H), 3.91
(s, 3H), 4.22 (t, J ) 5 Hz, 2H), 6.89 (d, J ) 6 Hz, 1H,) 7.58 (d,
J ) 2 Hz, 1H), 7.69 (dd, J ) 6, 2 Hz, 1H); MS (ES) m/z 259.2
(M + 1).
Anal. (C14H12Cl2N2O2) C, H, N.
6-(3-Ch lor opr opoxy)-4-[(2,4-dich lor o-5-m eth oxyph en yl)-
a m in o]-7-m eth oxy-3-qu in olin eca r bon itr ile (17). According
to the procedure used to prepare 1f, reaction of 16 with 2,4-
dichloro-5-methoxyaniline23 provided, after flash column chro-
matography, eluting with 5% methanol in diethyl ether, 17 in
19% yield: mp 100-105 °C; 1H NMR (DMSO-d6) δ 2.25-2.31
(m, 2H), 3.75-3.86 (m, 5H), 3.96 (s, 3H), 4.26 (t, J ) 6 Hz,
2H), 7.35 (br s, 2H), 7.75 (s, 1H), 7.87 (s, 1H), 8.42 (s, 1H),
9.62 (s, 1H); MS (ES) m/z 466.3, 468.3 (M + 1).
Anal. (C12H15ClO4) C, H.
Met h yl 5-(3-Ch lor op r op oxy)-4-m et h oxy-2-n it r ob en -
zoa te (13). To a solution of 12 (20.63 g, 80 mmol) in 60 mL of
acetic acid was added 20 mL of 70% nitric acid dropwise over
20 min. The reaction mixture was heated at reflux for 2 h,
poured into ice-water, and extracted with dichloromethane.
The organic layer was washed with water, followed by 0.5 N
aqueous sodium hydroxide, dried over magnesium sulfate,
filtered, and concentrated in vacuo to provide 19.12 g (79%) of
Anal. (C21H18Cl3N3O3) C, H.
1
13 as a white solid: mp 63-64 °C; H NMR (CDCl3) δ 2.32-
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-7-m eth oxy-
6-[3-(4-m or ph olin yl)pr opoxy]-3-qu in olin ecar bon itr ile (18).
A mixture of 17 (200 mg, 0.43 mmol) and a catalytic amount
of sodium iodide in 2 mL of morpholine was heated at 90 °C
overnight. The reaction mixture was partitioned between brine
and ethyl acetate. The organic layer was dried over sodium
sulfate, filtered, and concentrated in vacuo until a solid began
to precipitate out. The solid was collected by filtration to
provide 165 mg (74%) of 18 as an off-white solid: mp 181-
2.38 (m, 2H), 3.77 (t, J ) 5 Hz, 2H), 3.90 (s, 3H), 3.95 (s, 3H),
4.26 (t, J ) 5 Hz, 2H), 7.11 (s, 1H), 7.45 (s, 1H); MS (APCI)
m/z 304.1 (M + 1).
Anal. (C12H14ClNO6) C, H, N.
Meth yl 2-Am in o-5-(3-ch lor op r op oxy)-4-m eth oxyben -
zoa te (14). A solution of ammonium chloride (16.83 g, 315
mmol) and iron (11.60 g, 208 mmol) in 55 mL of water and
135 mL of methanol was heated at reflux under mechanical
stirring for 10 min. The solution was allowed to cool slightly,
and 13 (19.12 g, 63 mmol) dissolved in 140 mL of methanol
and 60 mL of water was added in portions. The reaction
mixture was heated at reflux for 4 h. After the reaction mixture
was cooled to room temperature, dilute aqueous sodium
bicarbonate was added, and the insoluble material was
removed by filtration, washing with dichloromethane. The
aqueous filtrate was extracted with dichloromethane, and the
organic extracts were combined and washed with brine. The
organic layer was dried over sodium sulfate, filtered, and
concentrated in vacuo. Addition of 10:1 hexane/diethyl ether
to the residue provided 15.96 g (93%) of 14 as a white solid:
1
185 °C; H NMR (DMSO-d6) δ 1.94-2.02 (m, 2H), 2.34-2.43
(m, 4H), 2.46 (t, J ) 5 Hz, 2H), 3.57 (t, J ) 3 Hz, 4 H), 3.86 (s,
3H), 3.95 (s, 3H), 4.16 (t, J ) 6 Hz, 2H), 7.32 (br s, 2H), 7.73
(s, 1H), 7.82 (s, 1H), 7.82 (s, 1H), 8.41 (s, 1H), 9.60 (s, 1H); MS
(ES) m/z 516.8, 518.8 (M + 1).
Anal. (C25H26Cl2N4O4) C, H, N.
4-Ch lor o-7-(2-ch lor oet h oxy)-6-m et h oxy-3-q u in olin e-
ca r bon itr ile (20). To a mixture of 19 (185 mg, 0.79 mmol),19
triphenylphosphine (373 mg, 1.42 mmol), and 2-chloroethanol
(200 µL, 2.98 mmol) in 20 mL of tetrahydrofuran at 0 °C was
added dropwise diethyl azodicarboxylate (230 µL, 1.45 mmol).
The reaction mixture was kept at 0 °C for 15 min and then
allowed to warm to room temperature. The reaction mixture
was concentrated, and the residue was purified by flash
column chromatography, eluting with 2:1 hexane/ethyl acetate
to provide 166 mg (71%) of 20 as a white solid: 174-176 °C;
1H NMR (DMSO-d6) δ 4.01-4.09 (m, 2H), 4.05 (s, 3H), 4.54 (t,
J ) 5 Hz, 2H), 7.48 (s, 1H), 7.61 (s, 1H), 9.00 (s, 1H); MS (ES)
m/z 297.1, 299.1 (M + 1).
1
92-93 °C; H NMR (DMSO-d6) δ 2.07-2.13 (m, 2H), 3.74 (s,
3H), 3.75 (s, 3H), 3.77 (t, J ) 5 Hz, 2H), 3.93 (t, J ) 5 Hz, 2H),
6.37 (s, 1H), 6.48 (s, 2H), 7.18 (s, 1H); MS (ES) m/z 273.8 (M
+ 1).
Anal. (C12H16ClNO4) C, H, N.
6-(3-Ch lor op r op oxy)-4-h yd r oxy-7-m et h oxy-3-q u in o-
lin eca r bon itr ile (15). A mixture of 14 (8.00 g, 29.0 mmol)
and dimethylformamide dimethylacetal (5.18 g, 44.0 mmol)
was heated at reflux for 8 h. The reaction was concentrated
in vacuo to provide methyl 5-(3-chloropropoxy)-2-{[(dimethyl-
amino)methylene]amino}-4-methoxybenzoate that was used
directly without purification.
To an oven-dried flask containing 95 mL of tetrahydrofuran
was added 38 mL of 1.6 M n-butyllithium (61 mmol) in
hexanes. The solution was cooled to -78 °C, and acetonitrile
Anal. (C13H10Cl2N2O2) C, H, N.
4-Ch lor o-7-(4-ch lor ob u t oxy)-6-m et h oxy-3-q u in olin e-
ca r bon itr ile (21). According to the procedure used to prepare
20, reaction of 19 with 4-chlorobutanol provided 21, after flash
column chromatography, eluting with 2:1 hexane/ethyl acetate,
in 65% yield as a white solid: mp 134-136 °C; 1H NMR
(DMSO-d6) δ 1.88-1.89 (m, 4H), 3.76 (t, J ) 6 Hz, 2H), 4.02

3974 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Boschelli et al.
(s, 3H), 4.28 (t, J ) 6 Hz, 2H), 7.42 (s, 1H), 7.54 (s, 1H), 8.97
(s, 1H); MS (ES) m/z 325.1, 327.1 (M + 1).
Anal. (C15H14Cl2N2O2) C, H, N.
J ) 6 Hz, 2H), 7.33 (s, 1H), 7.38 (s, 1H), 7.75 (s, 1H), 7.83 (s,
1H), 8.42 (s, 1H), 9.61 (s, 1H); MS (ES) m/z 503.2, 505.2 (M +
1).
Anal. (C24H24Cl2N4O4‚0.60H2O) C, H, N.
4-Ch lor o-7-[(5-ch lor op en t yl)oxy]-6-m et h oxy-3-q u in o-
lin eca r bon itr ile (22). According to the procedure used to
prepare 20, reaction of 19 with 5-chloropentanol provided, after
flash column chromatography, eluting with 2:1 hexane/ethyl
acetate, 22 in 67% yield as a white solid: mp 139-140 °C; 1H
NMR (DMSO-d6) δ 1.53-1.63 (m, 2H), 1.77-1.90 (m, 4H), 3.68
(t, J ) 7 Hz, 2H), 4.01 (s, 3H), 4.23 (t, J ) 6 Hz, 2H), 7.41 (s,
1H), 7.53 (s, 1H), 8.96 (s, 1H); MS (ES) m/z 339.1, 341.1 (M +
1).
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-
7-[4-(4-m or ph olin yl)bu toxy]-3-qu in olin ecar bon itr ile (27).
According to the procedure used to prepare 26, reaction of 24
and morpholine provided, after recrystallization from ethyl
acetate, 27 as a light yellow solid in 47% yield: mp 158-161
1
°C; H NMR (DMSO-d6) δ 1.56-1.69 (m, 2H), 1.75-1.90 (m,
2H), 2.29-2.40 (m, 6H), 3.57 (t, J ) 5 Hz, 4H), 3.86 (s, 3H),
3.94 (s, 3H), 4.18 (t, J ) 6 Hz, 2H), 7.33 (s, 2H), 7.74 (s, 1H),
7.83 (s, 1H), 8.42 (s, 1H), 9.60 (s, 1H); MS (ES) m/z 531.1, 533.2
(M + 1).
Anal. (C16H16Cl2N2O2) C, H, N.
7-(2-Ch lor oeth oxy)-4-[(2,4-d ich lor o-5-m eth oxyp h en yl)-
a m in o]-6-m eth oxy-3-qu in olin eca r bon itr ile (23). A mix-
ture of 2,4-dichloro-5-methoxyaniline (153 mg, 0.80 mmol),
pyridine hydrochloride (75 mg, 0.65 mmol), and 20 (300 mg,
0.66 mmol) in 30 mL of 2-ethoxyethanol was heated at reflux
for 4 h. The reaction mixture was partitioned between ethyl
acetate and saturated aqueous sodium bicarbonate. The
organic layer was washed with saturated aqueous sodium
bicarbonate and brine, then dried over sodium sulfate, filtered,
and concentrated in vacuo. The residue was purified by flash
column chromatography, eluting with 7% methanol in di-
chloromethane to provide 210 mg (70%) of 23 as a pale yellow
solid: mp 218-220 °C; 1H NMR (DMSO-d6) δ 3.86 (s, 3H), 3.96
(s, 3H), 4.05 (t, J ) 5 Hz, 2H), 4.46 (t, J ) 5 Hz, 2H), 7.33 (s,
1H), 7.37 (s, 1H), 7.74 (s, 1H), 7.86 (s, 1H), 8.42 (s, 1H), 9.65
(br s, 1H); MS (ES) m/z 452.2, 454.2 (M + 1).
Anal. (C26H28Cl2N4O4 ‚ 0.20 H2O) C, H, N.
4-[(2,4-Dich lor o-5-m e t h oxyp h e n yl)a m in o]-6-m e t h -
oxy-7-{[5-(4-m or p h olin yl)p en tyl]oxy}-3-qu in olin eca r bo-
n itr ile (28). A mixture of 25 (200 mg, 0.40 mmol) and sodium
iodide (70 mg, 0.47 mmol) in 4 mL of morpholine was heated
at reflux for 2.5 h and then stirred at room temperature
overnight. The reaction mixture was concentrated in vacuo,
and the residue was partitioned between ethyl acetate and
water. The organic layer was washed with saturated aqueous
sodium bicarbonate, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The residue was purified by flash
column chromatography, eluting with 3:1 ethyl acetate/
methanol followed by recrystallization from ethyl acetate and
diethyl ether to provide 40 mg (18%) of 28 as an off-white
1
solid: mp 144-146 °C; H NMR (DMSO-d6) δ 1.49-1.60 (m,
4H), 1.75-1.87 (m, 2H), 2.25-2.40 (m, 6H), 3.56 (t, J ) 4.5
Hz, 4H), 3.85 (s, 3H), 3.93 (s, 3H), 4.15 (t, J ) 6 Hz, 2H), 7.31
(br s, 2H), 7.73 (s, 1H), 7.82 (s, 1H), 8.40 (s, 1H), 9.65 (br s,
1H); MS (ES) m/z 545.1, 547.1 (M + 1).
Anal. (C20H16Cl3N3O3‚0.5H2O) C, H.
7-(4-Ch lor obu toxy)-4-[(2,4-d ich lor o-5-m eth oxyp h en yl)-
a m in o]-6-m eth oxy-3-qu in olin eca r bon itr ile (24). According
to the procedure used to prepare 23, reaction of 21 with 2,4-
dichloro-5-methoxyaniline provided 24 in 43% yield as an off-
white solid: mp 191-195 °C; 1H NMR (DMSO-d6) δ 1.89-1.98
(m, 4H), 3.72-3.79 (m, 2H), 3.86 (s, 3H), 3.95 (s, 3H), 4.17-
4.24 (m, 2H), 7.33 (s, 1H), 7.35 (s, 1H), 7.74 (s, 1H), 7.83 (s,
1H), 8.42 (s, 1H), 9.60 (s, 1H); MS (ES) m/z 480.1, 482.1 (M +
1).
Anal. (C27H30Cl2N4O4‚0.50H2O) C, H, N.
7-(3-Ch lor opr opoxy)-4-[(2,4-dich lor o-5-m eth oxyph en yl)-
a m in o]-6-m eth oxy-3-qu in olin eca r bon itr ile (30). A mix-
ture of 2,4-dichloro-5-methoxyaniline (1.35 g, 7.0 mmol),
pyridine hydrochloride (690 mg, 6.1 mmol), and 2928 (2.00 g,
6.4 mmol) in 20 mL of 2-ethoxyethanol was heated at reflux
for 2.5 h. The reaction mixture was partitioned between ethyl
acetate and saturated aqueous sodium bicarbonate. The
organic layer was washed with water, filtered, and concen-
trated in vacuo until a solid began to appear. This solid was
collected by filtration to provide 1.30 g (46%) of 30 as an off-
white solid: mp 183-186 °C; 1H NMR (DMSO-d6) δ 2.22-2.33
(m, 2H), 3.83 (t, J ) 6 Hz, 2H), 3.86 (s, 3H), 3.95 (s, 3H), 4.29
(t, J ) 6 Hz, 2H), 7.34 (s, 1H), 7.37 (s, 1H), 7.75 (s, 1H), 7.85
(s, 1H), 8.42 (s, 1H), 9.64 (s, 1H); MS (ES) m/z 465.8, 467.8 (M
+ 1).
Anal. (C22H20Cl3N3O3‚0.2H2O) C, H, N.
7-[(5-Ch lor op en t yl)oxy]-4-[(2,4-d ich lor o-5-m et h oxy-
p h en yl)a m in o]-6-m eth oxy-3-qu in olin eca r bon itr ile (25).
A mixture of 2,4-dichloro-5-methoxyaniline (840 mg, 4.37
mmol) and 60% sodium hydride (176 mg, 4.59 mmol) in 30
mL of tetrahydrofuran was heated at reflux for 30 min. The
mixture was cooled, and 22 (854 mg, 2.5 mmol) was added.
The reaction mixture was heated at reflux overnight and then
cooled to room temperature. The residue was partitioned
between ethyl acetate and water. The organic layer was
washed with water, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The residue was purified by flash
column chromatography, eluting with 1:1 hexane/ethyl acetate
to provide 317 mg (25%) of 25 as a pale yellow solid: mp 173-
Anal. (C21H18Cl3N3O3) C, H, N.
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m 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 bo-
n itr ile (31a ). A mixture of 30 (656 mg, 1.40 mmol) and sodium
iodide (210 mg, 1.40 mmol) in 4 mL of N-methylpiperazine was
heated at 80 °C for 20 h. The reaction mixture was concen-
trated in vacuo and partitioned between ethyl acetate and
saturated aqueous sodium bicarbonate. The organic layer was
washed with brine, dried over sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography, eluting with 30% methanol in dichloro-
methane. The fractions containing product were collected and
concentrated in vacuo. Diethyl ether was added to the residue,
and the light pink solid was collected by filtration to provide
560 mg (75%) of 31a : mp 116-120 °C; 1H NMR (DMSO-d6) δ
1.89-2.05 (m, 2H), 2.15 (s, 3H), 2.29-2.52 (m, 10H), 3.86 (s,
3H), 3.94 (s, 3H), 4.19 (t, J ) 6 Hz, 2H), 7.31 (br s, 2H), 7.73
(s, 1H), 7.82 (s, 1H), 8.40 (s, 1H), 9.60 (s, 1H); MS (ES) m/z
530.2, 532.2 (M + 1).
1
175 °C; H NMR (DMSO-d6) δ 1.49-1.67 (m, 2H), 1.75-1.90
(m, 4H), 3.69 (t, J ) 7 Hz, 2H), 3.85 (s, 3H), 3.94 (s, 3H), 4.16
(t, J ) 6 Hz, 2H), 7.32 (br s, 2H), 7.73 (s, 1H), 7.82 (s, 1H),
8.40 (s, 1H), 9.26 (br s, 1H); MS (ES) m/z 494.1, 496.0 (M +
1).
Anal. (C23H22Cl3N3O3) C, H, N.
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-
7-[2-(4-m or p h olin yl)eth oxy]-3-qu in olin eca r bon itr ile (26).
A mixture of 23 (300 mg, 0.66 mmol), sodium iodide (87 mg,
0.6 mmol), and 1 mL of morpholine in 3 mL of ethylene glycol
dimethyl ether was heated at 90 °C overnight. The reaction
mixture was partitioned between ethyl acetate and saturated
aqueous sodium bicarbonate. The organic layer was washed
with saturated aqueous sodium bicarbonate and brine, then
dried over sodium sulfate, filtered, and concentrated in vacuo.
The residue was purified by preparative thin-layer chroma-
tography, eluting with 15% methanol in dichloromethane to
provide 26 as an off-white solid in 45% yield: mp 199-201
Anal. (C26H29Cl2N5O3‚2.0H2O) C, H, N.
4-[(2,4-D ic h lo r o -5-m e t h o x y p h e n y l)a m in o ]-7-[3-(4-
e t h yl-1-p ip e r a zin yl)p r op oxy]-6-m e t h oxy-3-q u in olin e -
ca r bon itr ile (31b). A mixture of 30 (3.50 g, 7.50 mmol),
sodium iodide (1.12 g, 7.50 mmol), and 4.8 mL of N-ethyl-
piperazine in 5 mL of ethylene glycol dimethyl ether was
1
°C; H NMR (DMSO-d6) δ 2.49-2.58 (m, 4H), 2.73-2.84 (m,
2H), 3.59 (t, J ) 7 Hz, 4H), 3.86 (s, 3H), 3.94 (s, 3H), 4.29 (t,

Quinolinecarbonitriles as Inhibitors of Src Kinase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23 3975
heated at 95 °C for 20 h. The reaction mixture was concen-
trated in vacuo and partitioned between ethyl acetate and
saturated aqueous sodium bicarbonate. The organic layer was
washed with saturated aqueous sodium bicarbonate, followed
by brine, dried over sodium sulfate, filtered, and concentrated
in vacuo. Diethyl ether was added to the residue, and the white
solid was collected by filtration to provide 1.80 g (44%) of
31b: mp 102-104 °C; 1H NMR (DMSO-d6) δ 0.98 (t, J ) 7
Hz, 3H), 1.88-2.02 (m, 2H), 2.25-2.52 (m, 12 H), 3.86 (s, 3H),
3.94 (s, 3H), 4.19 (t, J ) 6 Hz, 2H), 7.30 (br s, 2H), 7.72 (s,
1H), 7.82 (s, 1H), 8.39 (s, 1H), 9.60 (s, 1H); MS (ES) m/z 544.3,
546.4 (M + 1).
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-
7-[3-(4-p ip er id in -1-yl )p r op oxy]-3-qu in olin eca r bon itr ile
(31h ). According to the procedure used to prepare 31b,
reaction of 30 with piperidine provided, after preparative thin-
layer chromatography, eluting with 20% methanol in dichloro-
methane, 31h in 61% yield as a white solid: mp 157-161 °C;
1H NMR (DMSO-d6) δ 1.34-1.45 (m, 2H), 1.45-1.57 (m, 4H),
1.88-2.02 (m, 2H), 2.32-2.49 (m, 6H), 3.86 (s, 3H), 3.94 (s,
3H), 4.19 (t, J ) 6 Hz, 2H), 7.31 (br s, 2H), 7.73 (s, 1H), 7.83
(s, 1H), 8.40 (s, 1H), 9.62 (s, 1H); MS (ES) m/z 515.2, 517.2 (M
+ 1).
Anal. (C26H28Cl2N4O3‚2.7H2O) C, H, N.
Anal. (C27H31Cl2N5O3 ‚ 2.0 H2O) C, H, N.
4-[(2,4-Dich lor o-5-m et h oxyp h en yl)a m in o]-7-[3-(4-h y-
d r oxyp ip er id in -1-yl)p r op oxy]-6-m et h oxy-3-q u in olin e-
ca r bon itr ile (31i). According to the procedure used to prepare
31b, reaction of 30 with 4-hydroxypiperidine provided 31i as
a white solid in 75% yield: mp 122-125 °C; 1H NMR (DMSO-
d6) δ 1.30-1.46 (m, 2H), 1.65-1.78 (m, 2H), 1.88-2.09 (m, 4H),
2.43 (t, J ) 6 Hz, 2H), 2.67-2.78 (m, 2H), 3.39-3.50 (m, 1H),
3.86 (s, 3H), 3.94 (s, 3H), 4.18 (t, J ) 6 Hz, 2H), 4.53 (d, J )
3 Hz, 1H), 7.31 (br s, 2H), 7.74 (s, 1H), 7.83 (s, 1H), 8.40 (s,
1H), 9.62 (s, 1H); MS (ES) m/z 531.0, 533.1 (M + 1).
4-[(2,4-Dich lor o-5-m e t h oxyp h e n yl)a m in o]-6-m e t h -
oxy-7-[3-(cis-3,4,5-t r im e t h ylp ip e r a zin yl)p r op oxy]-3-
qu in olin eca r bon itr ile (31c). According to the procedure
used to prepare 31b, reaction of 30 and cis-1,2,6-trimethyl-
piperazine33 provided, after flash column chromatography,
eluting with 25% methanol in dichloromethane, 31c in 39%
1
yield as a white solid: mp 98-101 °C; H NMR (DMSO-d6) δ
0.96 (s, 3H), 0.98 (s, 3H), 1.65-1.77 (m, 2H), 1.90-1.98 (m,
2H), 2.07-2.18 (m, 5H), 2.34-2.41 (m, 2H), 2.69-2.78 (m, 2H),
3.86 (s, 3H), 3.94 (s, 3H), 4.19 (t, J ) 6 Hz, 2H), 7.32 (br s,
2H), 7.74 (s, 1H), 7.83 (s, 1H), 8.41 (s, 1H), 9.60 (s, 1H); MS
(ES) m/z 558.3, 560.3 (M + 1).
Anal. (C26H28Cl2N4O4‚2.0H2O) C, H, N.
4-[(2,4-Dich lor o-5-m e t h oxyp h e n yl)a m in o]-6-m e t h -
oxy-7-[3-(2H-1,2,3-tr iazol-2-yl)pr opoxy]-3-qu in olin ecar bo-
n it r ile (31j) a n d 4-[(2,4-Dich lor o-5-m et h oxyp h en yl)-
a m in o]-6-m eth oxy-7-[3-(1H-1,2,3-tr ia zol-1-yl)p r op oxy]-3-
qu in olin eca r bon itr ile (31k ). According to the procedure
used to prepare 31b, reaction of 30 with 1,2,3-triazole provided
a mixture of 31j and 31k . The mixture was separated by
preparative thin-layer chromatography, eluting with 5% metha-
nol in dichloromethane. 31j was obtained as an off-white solid
Anal. (C28H33Cl2N5O3‚2.0H2O) C, H.
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-
7-[3-(4-p r op yl-1-p ip er a zin yl)p r op oxy]-3-qu in olin eca r bo-
n itr ile (31d ). According to the procedure used to prepare 31b,
reaction of 30 and N-propylpiperazine provided, after prepara-
tive thin-layer chromatography, eluting with 20% methanol
in dichloromethane, and subsequent recrystallization from
diethyl ether and hexane, 31d in 15% yield as a white solid:
1
in 30% yield: mp 190-191 °C; H NMR (DMSO-d6) δ 2.35-
1
mp 97-101 °C; H NMR (DMSO-d6) δ 0.84 (t, J ) 7 Hz, 3H),
2.46 (m, 2H), 3.86 (s, 3H), 3.94 (s, 3H), 4.18 (t, J ) 6 Hz, 2H),
4.64 (d, J ) 7 Hz, 2H), 7.29 (s, 1H), 7.34 (s, 1H), 7.75 (s, 1H),
7.80 (s, 2H), 7.85 (s, 1H), 8.42 (s, 1H), 9.63 (s, 1H); MS (ES)
m/z 499.4, 501.3 (M + 1).
1.32-1.47 (m, 2H), 1.89-2.03 (m, 2H), 2.15-2.27 (m, 2H),
2.25-2.52 (m, 10 H), 3.86 (s, 3H), 3.94 (s, 3H), 4.19 (t, J ) 6
Hz, 2H), 7.32 (br s, 2H), 7.74 (s, 1H), 7.83 (s, 1H), 8.41 (s, 1H),
9.61 (s, 1H); MS (ES) m/z 558.2, 560.2 (M + 1). Anal.
(C28H33Cl2N5O3‚2.2H2O) C, H.
Anal. (C23H20Cl2N6O3) C, H, N.
31k was obtained as an off-white solid in 39% yield: mp
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-
7-(3-piper azin -1-ylpr opoxy)-3-qu in olin ecar bon itr ile (31e).
According to the procedure used to prepare 31b, reaction of
30 with piperazine provided, after preparative thin-layer
chromatography, eluting with 25% methanol in dichloro-
methane, 31e in 48% yield as a white solid: mp 123-125 °C;
1H NMR (DMSO-d6) δ 1.89-2.02 (m, 2H), 2.27-2.37 (m, 4 H),
2.42 (t, J ) 7 Hz, 2H), 2.72 (t, J ) 5 Hz, 4H), 3.85 (s, 3H), 3.93
(s, 3H), 4.19 (t, J ) 6 Hz, 2H), 7.28 (s, 1H), 7.30 (s, 1H), 7.72
(s, 1H), 7.82 (s, 1H), 8.38 (s, 1H), 9.61 (s, 1H); MS (ES) m/z
516.2, 518.1 (M + 1).
1
188-190 °C; H NMR (DMSO-d6) δ 2.33-2.45 (m, 2H), 3.86
(s, 3H), 3.96 (s, 3H), 4.17 (t, J ) 6 Hz, 2H), 4.60 (d, J ) 7 Hz,
2H), 7.32 (s, 1H), 7.33 (s, 1H), 7.75 (s, 2H), 7.85 (s, 1H), 8.20
(s, 1H), 8.41 (s, 1H), 9.63 (s, 1H); MS (ES) m/z 499.4, 501.3 (M
+ 1).
Anal. (C23H20Cl2N6O3‚0.3H2O) C, H, N.
4-[(2,4-Dich lor o-5-m e t h oxyp h e n yl)a m in o]-7-[3-(1H -
im id a zol-1-yl)p r op oxy]-6-m e t h oxy-3-q u in olin e ca r b o-
n itr ile (31l). A mixture of 30 (200 mg, 0.43 mmol), imidazole
(300 mg, 4.3 mmol), sodium hydroxide powder (80 mg, 2.0
mmol), and sodium iodide (30 mg, 0.2 mmol) in 3 mL of N,N-
dimethylformamide was heated at 80 °C for 2 h. The reaction
mixture was cooled, poured into water, and stirred. The
resultant solid was collected by filtration, washing with water
and diethyl ether. The solid was purified by silica gel chro-
matography, eluting with a gradient of 3% methanol in
dichloromethane to 6% methanol in dichloromethane, to
provide 180 mg (84%) of 31l as a yellow solid: mp 155-170°
C; 1H NMR (DMSO-d6) δ 2.27-2.44 (m, 2H), 3.87 (s, 3H), 3.95
(s, 3H), 4.26 (t, J ) 6 Hz, 2H), 4.42 (t, J ) 7 Hz, 2H), 7.36 (s,
1H), 7.39 (s, 1H), 7.71 (s, 1H), 7.76 (s, 1H), 7.84 (s, 1H), 7.96
(s, 1H), 8.14 (s, 1H), 8.55 (s, 1H), 9.18 (s, 1H); MS (ES) m/z
498.0, 500.1 (M + 1).
Anal. (C25H27Cl2N5O3) C, H.
4-[(2,4-Dich lor o-5-m e t h oxyp h e n yl)a m in o]-6-m e t h -
oxy-7-{3-[4-(2-h yd r oxyet h yl)p ip er a zin -1-yl]p r op oxy}-3-
qu in olin eca r bon itr ile (31f). According to the procedure used
to prepare 31b, reaction of 30 with 4-(2-hydroxyethyl)piper-
azine provided 31f in 75% yield as a white solid: mp 133-
1
137 °C; H NMR (DMSO-d6) δ 1.90-2.01 (m, 2H), 2.31-2.50
(m, 10H), 3.47 (q, J ) 6 Hz, 2H), 3.86 (s, 3H), 3.94 (s, 3H),
4.18 (t, J ) 6 Hz, 2H), 4.37 (t, J ) 5 Hz, 1H), 7.31 (br s, 2H),
7.74 (s, 1H), 7.82 (s, 1H), 8.41 (s, 1H), 9.62 (s, 1H); MS (ES)
m/z 560.1, 562.1 (M + 1).
Anal. (C27H31Cl2N5O4‚1.2H2O) C, H, N.
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-
7-[3-(4-m eth yl-[1,4]d ia zep a n -1-yl)p r op oxy]-3-qu in olin e-
ca r bon itr ile (31g). According to the procedure used to
prepare 31b, reaction of 30 with 1-methylhomopiperazine
provided, after preparative thin-layer chromatography, eluting
with 20% methanol in dichloromethane, 31g in 43% yield as
Anal. (C24H21Cl2N5O3‚1.05CH2Cl2) C, H, N.
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-6-m eth oxy-
7-{3-[(2-(4-m or ph olin yl)eth yl)am in o]pr opoxy}-6-m eth oxy-
3-qu in olin eca r bon itr ile (31m ). According to the procedure
used to prepare 31b, reaction of 30 with 4-(2-aminoethyl)-
morpholine provided, after preparative thin-layer chromatog-
raphy, eluting with 20% methanol in dichloromethane, 31m
as an orange solid in 31% yield: mp 75-80 °C; 1H NMR
(DMSO-d6) δ 1.95-2.06 (m, 2H), 2.31-2.44 (m, 6H), 2.72 (t, J
) 6.5 Hz, 2H), 2.79 (t, J ) 7 Hz, 2H), 3.86 (s, 3H), 3.94 (s,
3H), 4.23 (t, J ) 6 Hz, 2H), 4.53 (d, J ) 3 Hz, 1H), 7.30 (s,
1
a light yellow solid: mp 134-137 °C; H NMR (DMSO-d6) δ
1.65-1.77 (m, 2H), 1.86-1.97 (m, 2H), 2.25 (s, 3H), 2.50-2.69
(m, 10H), 3.86 (s, 3H), 3.94 (s, 3H), 4.19 (t, J ) 6 Hz, 2H),
7.30 (br s, 2H), 7.73 (s, 1H), 7.82 (s, 1H), 8.40 (s, 1H), 9.61 (s,
1H); MS (ES) m/z 544.2, 546.2 (M + 1). Anal.
(C27H31Cl2N5O3‚1.7 H2O) C, H, N.

3976 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 23
Boschelli et al.
1H), 7.32 (s, 1H), 7.72 (s, 1H), 7.84 (s, 1H), 8.40 (s, 1H), 9.60
(s, 1H); MS (ES) m/z 560.2, 562.1 (M + 1).
Anal. (C27H31Cl2N5O4‚1.0H2O) C, H.
The vehicle used was 2% Tween-80 in 5% dextrose/water. All
suspensions were sonicated prior to dosing. Tumors were
measured on the days indicated using digital-display vernier
calipers. The tumor size was calculated using the formula
(length × width2)/2 ) tumor mass (mg). The conversion from
cubic millimeters to milligrams was made assuming unit
density. The experiment was ended when the tumors reached
15% of the body weight of the mouse, at which time the
animals were sacrificed.
HT29 Xen ogr a ft Assa y. Human colon carcinoma HT29
cells (American Type Culture Collection, Rockville, MD, no.
HTB-38) were grown in vitro. Athymic nu/nu female mice
(Charles River, Wilmington, MA), 6-7 weeks of age, were
housed in a barrier-type facility under an approved IACUC
protocol. The animals were provided ad libitum access to both
food and water. A unit of 7 × 106 HT29 cells was injected sc
into the flanks of mice. When the tumors attained a mass of
between 80 and 120 mg, the mice were randomized into
treatment groups (day 0). The mice were treated either ip (5
mice per group) or po (orally) (10 mice per group) twice daily
on days 1-20 poststaging with either vehicle control (2%
Tween-80 in 5% dextrose/water) or 31a at the doses indicated
for each experiment. The tumor mass was determined every
7 days [(length × width2)/2] for 28 days. The relative tumor
growth (mean tumor mass on days 7, 14, 21, and 28 divided
by the mean tumor mass on day 0) was determined for each
treatment group. Statistical analysis (Student’s t test) of the
log of the relative tumor growth compares the treated group
with the control group. A p value (p e 0.05) indicates a
statistically significant reduction in the relative tumor growth
of the treated group compared to the vehicle control.
4-[(2,4-Dich lor o-5-m eth oxyp h en yl)a m in o]-7-{3-[((2-(d i-
m eth yla m in o)eth yl)m eth yl)a m in o]p r op oxy}-6-m eth oxy-
3-qu in olin eca r bon itr ile (31n ). According to the procedure
used to prepare 31a , reaction of 30 with N,N,N′-trimethyl-
ethylenediamine provided, after preparative thin-layer chro-
matography, eluting with 20% methanol in dichloromethane,
31n in 31% yield as a white solid: mp 85-90 °C; 1H NMR
(DMSO-d6/TFA) δ 2.23-2.38 (m, 2H), 2.89 (s, 6H), 2.93 (s, 3H),
3.30-3.41 (m, 2H), 3.48-3.61 (m, 4H), 3.90 (s, 3H), 4.02 (s,
3H), 4.26-4.35 (m, 2H), 7.53 (s, 1H), 7.57 (s, 1H), 7.89 (s, 1H),
8.14 (s, 1H), 9.12 (s, 1H); MS (ES) m/z 532.1, 534.1 (M + 1).
Anal. (C26H31Cl2N5O3‚0.6H2O) C, H, N.
Biologica l Eva lu a tion . The protocols for the Src kinase
and Src and Fyn cell proliferation assays were previously
reported.16
P la sm a P h a r m a cok in etic Assa y. Compounds were ad-
ministered to athymic nu/nu female mice (Charles River
Laboratories). Each compound was dosed intraperitoneally (ip)
into 10 mice with a dose of 50 mg/kg prepared in 2% Tween-
80, 5% dextrose/water (D5W). Blood was collected from five
animals for each compound at 1 h postdose and from five
animals at 4 h postdose. To collect the blood, the mice were
anesthetized with Isoflurane gas and bled by cardiac puncture.
Blood was collected in heparinized tubes and placed on ice.
Blood samples were spun down at 14 000 rpm in a microfuge
at 4 °C. The plasma was collected for each sample and stored
at -70 °C. The compounds were extracted by combining 100
µL of plasma, 50 µL of an internal standard, and 300 µL of
acetonitrile. After centrifugation for 15 min, the supernatant
was transferred to a 1.5 mL tube and evaporated at 45 °C.
The residue was dissolved in 100 µL of aqueous acetonitrile.
The samples were analyzed on an Agilent HP1100 MSD LC/
MS instrument using a Prodigy ODS3 column (5 µm, 4.6 ×
150 mm) at 1.0 mL/min and 40 °C. The mobile phase consisted
of 0.02% trifluoroacetic acid/water (A) and 0.02% trifluoroacetic
acid/acetonitrile (B). The samples (25 µL injections) were run
using a gradient of 15-70% B in 10 min and were introduced
into the electrospray source without splitting to maximize
sensitivity. Standards for each compound were prepared in an
identical manner, and a calibration curve was generated. The
samples were quantitated using both UV (wavelengths ranging
from 268 to 349 nm) and single-ion monitoring (ES+). The
results are reported as the means of five samples.
Colon y F or m a tion in Aga r . HT29 cells (15 000) were
suspended in 1.5 mL of 0.33% agar/DMEM supplemented with
10% fetal bovine serum. This mixture was layered onto 2.5
mL of 0.75% agar/DMEM with 10% fetal bovine serum. After
the top agar hardened, 2 mL of medium with 31a at 1 µM
was layered onto the top agar, and the cells were then
incubated at 37 °C. The cells were incubated for 4 weeks, with
fresh medium without compound added after weeks 2 and 3.
Sr c-Tr a n sfor m ed F ibr obla st Xen ogr a ft Assa y. Src-
transformed fibroblasts were grown to confluence and then
split 1:2 the day prior to injection. Cells were harvested on
ice into growth media and resuspended to a final concentration
of 5 million cells/mL. The cells were inoculated into female
Balb/c nu/nu (athymic) mice which were 8-10 weeks of age
and housed according to the guidelines detailed by the Inter-
national Animal Care & Use Committee (IACUC). All mouse
handling was done in a laminar flow hood, and the mice were
housed in microisolator cages according to the procedures
defined for handling nude mice. A 0.2 cm3 sample of cell
suspension was injected subcutaneously (sc) into the right side
of the mouse. For the unstaged model, the animals received
compound followed by cells 30 min later, using 15-20 mice
per group to compensate for the innate variability in tumor
growth rates. In the staged model, the number of mice per
group was reduced to 10 mice per group, with initial tumor
size ranging from 100 to 150 mg. At the time of staging,
typically 3-5 days following inoculation, the mice were dosed
with compound or vehicle, 0.5 cm3 ip bid for the noted duration.
Ack n ow led gm en t. We gratefully acknowledge the
Wyeth-Ayerst Analytical Chemistry Department for the
spectral data and elemental analyses and members of
the Wyeth-Ayerst Oncology Department for testing 31a
in the ErbB2 and cdk4 kinase assays.
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