Synthesis and inhibition of Src kinase activity by 7-ethenyl and 7-ethynyl-4-anilino-3-quinolinecarbonitriles

Article abstract of DOI:10.1016/j.bmcl.2004.02.035

A series of 7-ethynyl and 7-ethenyl-4-anilino-3-quinolinecarbonitriles were synthesized and tested for Src inhibition. Derivatives bearing a C-6 methoxy group and 2,4-dichloro-5-methoxyaniline at C-4 showed optimal inhibition of Src enzymatic and cellular activity. The ethenyl and ethynyl groups were incorporated at C-7 utilizing a Stille, Heck, or Sonogashira coupling reaction.

Full text of DOI:10.1016/j.bmcl.2004.02.035

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2155–2158
Synthesis and inhibition of Src kinase activity by 7-ethenyl and
7-ethynyl-4-anilino-3-quinolinecarbonitriles
Ana Carolina Barrios Sosa,a,* Diane H. Boschelli,a Fei Ye,a Jennifer M. Golasb and
Frank Boschellib
aWyeth Research, Chemical and Screening Sciences, 401 N. Middletown Road, Pearl River, NY 10965, USA
bWyeth Research, Oncology, 401 N. Middletown Road, Pearl River, NY 10965, USA
Received 20 September 2003; revised 9 February 2004; accepted 9 February 2004
Abstract—A series of 7-ethynyl and 7-ethenyl-4-anilino-3-quinolinecarbonitriles were synthesized and tested for Src inhibition.
Derivatives bearing a C-6 methoxy group and 2,4-dichloro-5-methoxyaniline at C-4 showed optimal inhibition of Src enzymatic and
cellular activity. The ethenyl and ethynyl groups were incorporated at C-7 utilizing a Stille, Heck, or Sonogashira coupling reac-
tion.
Ó 2004 Elsevier Ltd. All rights reserved.
Protein tyrosine kinases constitute approximately 20%
of known and predicted protein kinases. These enzymes
play a central role in signal transduction and cellular
mechanisms.1 Src, a nonreceptor tyrosine kinase, is the
founding member of a group of structurally homo-
logous proteins referred to as the Src family of kinases
(SFKs). Elevated Src activity/expression has been asso-
ciated with several disease states including cancer and
osteoporosis.2 As a result, Src has been recognized as an
important therapeutic target and several classes of
inhibitors have been synthesized.3
phenyl derivatives 2a and 2b constitute potent Src
inhibitors.
Cl
Cl
O
Cl
Cl
HN
HN
O
O
CN
O
O
CN
X
N
N
n
N
1b: n = 3; X = N
1c: n = 1; X = CH
1a
In earlier work we identified 4-anilino-6,7-dialkoxy-3-
quinolinecarbonitrile 1a as an ATP competitive inhibi-
tor of Src kinase activity, with no significant inhibition
against other kinases, including EGFr, FGFr, and
Cdk4.4 After optimization of the substituents at C-4 and
C-7, superior Src inhibitory activity was seen with
derivatives 1b5 and 1c,6 which bear a 2,4-dichloro-5-
methoxy aniline group at C-4 and a 3-(4-methyl-1-
piperazinyl)-propoxy or 1-methylpiperidinemethoxy
substituent at C-7, respectively.
H
Cl
Cl
N
HN
O
HN
CN
N
Y
N
R
N
N
N
O
N
2a: R =
HC CH
C C
3a: Y =
3b: Y =
S
We have also shown that groups other than alkoxy are
tolerated at C-7.7 For example, 7-thienyl and 7-
N
2b: R =
N
Keywords: Src kinase; Src inhibitors; Kinase inhibitors; Tyrosine kin-
ase; Quinolinecarbonitriles.
Interestingly, related 4-heteroaryl-6-ethenyl quinazo-
lines and their 6-ethynyl derivatives (3a, 3b) have been
* Corresponding author. Tel.: +1-845-602-1703; fax: +1-845-602-5561;
e-mail: barrioa1@wyeth.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.035

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A. C. Barrios Sosa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2155–2158
claimed to be EGFr inhibitors.8 As part of our ongoing
investigations, we sought to introduce an ethenyl or
ethynyl group at C-7 of the 3-quinolinecarbonitrile.
We then studied the effect of replacing the 3-cyano
substituent with an ester (12) or acid group (13). For the
synthesis of these derivatives, ethyl 7-bromo-1,4-dihy-
dro-4-oxo-3-quinolinecarboxylate 9, synthesized by
We began our synthetic studies by reacting the previ-
ously reported intermediate 7-bromo-4-chloro-3-quino-
linecarbonitrile7a with 2,4- and 2,4,5-substituted
anilines, 2,4-dichlorobenzylamine, and 2,4-dichlorophe-
nol to give the 4-substituted derivatives 4–8 (Scheme 1).
Subsequent treatment of 4–8 with 4-vinylpyridine under
Heck coupling conditions provided C-7 ethenyl deriva-
tives 4a–8a. Similarly, treatment of intermediates 4 and
57a with 3-ethynylpyridine under Sonogashira coupling
conditions yielded C-7 ethynyl derivatives 4b and 5b.
a
known route,9 was treated with phosphorous
oxychloride to give ethyl 7-bromo-4-chloro-3-quinoline-
carboxylate 10 (Scheme 2). Reaction of 10 with 2,4-
dichloroaniline using pyridine hydrochloride gave
intermediate 11. Heck coupling reaction of 11 with
4-vinylpyridine provided ethyl 3-quinolinecarboxylate
12, which was hydrolyzed to give carboxylic acid 13.
Derivatives 12 and 13 showed no significant Src inhibi-
tory activity, implying that the 3-cyano group is
required for good Src inhibition.
As previously observed with the 7-alkoxy, 7-thienyl, and
7-phenyl series, 2,4-dichloro-5-methoxy aniline (5a) was
found to be the preferred aniline for Src inhibition
(Table 1). Although removal of the aniline C-5 methoxy
substituent (4a) resulted in a decrease in inhibitory
activity, replacement of this group with an ethoxy sub-
stituent (6a) was more detrimental. Similarly, extending
the C-4 NH linker of 4a with a methylene group (7a) or
replacing it with an oxygen (8a) decreased the Src
inhibitory activity 24- and 9-fold, respectively, in the
enzyme assay. Loss of activity was also observed in the
Src-dependent cell proliferation assay.
Following the synthesis of derivatives with varying
groups at C-3, we proceeded to replace the pyridine ring
linked to C-7, with other aryl or heteroaryl groups. As
shown in Scheme 3, intermediate 57a was reacted with
several ethenyl10 and ethynyl reagents under Heck and
Sonogashira coupling conditions, respectively. For the
synthesis of the unsubstituted vinyl derivative 14, 5 was
reacted with tributyl(vinyl)tin under Suzuki conditions.
Ethyl derivative 24 was synthesized by treatment of 23
with Pd/C under a hydrogen atmosphere.
Of the different aryl and heteroaryl groups tested in the
7-ethenyl series, 3- and 4-ethenylpyridine derivatives 19
and 5a showed superior Src inhibitory activity.
Although the IC50 value obtained for 2-ethenylpyridine
18 in an enzyme assay did not differ significantly from
the values found for 19 and 5a, compound 18 was much
less potent in a cell proliferation assay. In contrast to the
ethenyl series, in the C-7 ethynyl series, 3-ethynylpyr-
idine 5b was found to inhibit Src-dependent cell prolif-
eration with an IC50 value of 0.65 lM, while the
4-ethynylpyridine 23 had an IC50 of only 2.7 lM and the
IC50 for the 2-ethynylpyridine 22 was >10 lM. The IC50
values obtained from the enzyme assay showed a similar
correlation. Saturation of the ethynyl linker (24) resulted
in a large decrease in Src inhibition.
Cl
CN
Br
N
a
Cl
Cl
R
Cl
Cl
R
X
X
b
CN
CN
N
Finally, we investigated the importance of having an
alkoxy substituent at C-6. Previous studies on 6,7-
dialkoxy-3-quinolinecarbonitries showed that the pres-
ence of a small alkoxy group at C-6, like OMe, generally
improved Src inhibitory activity.6 Interestingly, the
opposite is true for the 7-phenyl series,7b where the best
Src inhibition was observed when C-6 was unsubsti-
tuted. As shown in Scheme 4, known intermediate
4-chloro-3-cyano-6-methoxy-7-quinolinyl-trifluorome-
thanesulfonate was prepared as previously reported.7b
6-Methoxy-7-ethenyl derivatives 25–27 and 6-methoxy-
7-ethynyl derivatives 28 and 29 were prepared as
previously described. As observed for the 6,7-dialkoxy-
3-quinolinecarbonitrile series, the presence of a methoxy
group at C-6 improved Src inhibition, in most cases,
more than 2-fold. 4-Ethenylpyridine derivative 27
exhibited an improved IC50 value of 0.08 lM in the Src
dependent cell assay and of 4.2 nM in the enzyme assay,
compared to its C-6 unsubstituted derivative 5a, which
exhibited IC50 values of 0.23 lM and 9.9 nM, respec-
tively.
Br
N
N
4: R = H, X = NH
4a: R = H, X = NH
5: R = OMe, X = NH
6: R = OEt, X = NH
7: R = H, X = NH-CH2
8: R = H, X = O
5a: R = OMe, X = NH
6a: R = OEt, X = NH
7a: R = H, X = NH-CH2
8a: R = H, X = O
Cl
Cl
R
c
HN
CN
N
N
4b: R = H
5b: R = OMe
Scheme 1. Reagents and conditions: (a) aniline, pyridine hydrochlor-
€
ide, or NaH; benzylamine, Hunigs base/DMF; phenol, K2CO3/DMF;
(b) 4-vinylpyridine, Pd(OAc)2, P(o-Tol)3, DMF, TEA; (c) 3-ethynyl-
pyridine, Pd(Ph3P)4, CuI, DMF, TEA.

A. C. Barrios Sosa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2155–2158
2157
Table 1.
Cl
Cl
X
Q
Y
L
Z
R
N
Compound11
R
L
Y
X
Q
Z
Src enzyme12
IC50 nM (SD)
Src cells13
IC50 lM (SD)
1b
4a
4b
5a
5b
6a
7a
8a
12
13
14
15
16
17
18
––
––
––
H
H
H
H
H
H
H
H
H
H
H
H
H
H
––
H
H
––
––
3.6
42 (6.0)
0.10
1.2 (0.13)
4-Pyridyl
3-Pyridyl
4-Pyridyl
3-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
4-Pyridyl
H
Ethenyl
Ethynyl
Ethenyl
Ethynyl
Ethenyl
Ethenyl
Ethenyl
Ethenyl
Ethenyl
Ethenyl
Ethenyl
Ethenyl
Ethenyl
Ethenyl
NH
NH
NH
NH
NH
NHCH2
O
CN
CN
CN
CN
CN
CN
CN
CO2Et
CO2H
CN
CN
CN
CN
CN
200 (49)
9.9 (2.9)
47 (11)
3.9 (0.049)
0.23 (0.10)
0.65 (0.093)
>10 (n ¼ 2)
>10 (n ¼ 2)
7.2 (2.3)
OMe
OMe
OEt
H
3100 (610)
1000 (180)
380(23)
H
H
NH
NH
NH
NH
NH
NH
NH
12,000 (4300)
1600 (200)
97 (28)
>10 (n ¼ 2)
>10 (n ¼ 2)
9.3 (3.2)
H
OMe
OMe
OMe
OMe
OMe
Phenyl
180(9.8)
2300 (500)
320(86)
2.7 (0.98)
4-Biphenyl
2-Naphthyl
2-Pyridyl
>10 (n ¼ 2)
>10( n ¼ 2)
50% inhibition
at 10 lM (22%)
0.23 (0.003)
>10( n ¼ 2)
>10( n ¼ 2)
>10( n ¼ 2)
2.7 (1.0)
19 (2.8)
19
20
21
22
23
24
25
26
27
28
29
3-Pyridyl
Phenyl
Ethenyl
Ethynyl
Ethynyl
Ethynyl
Ethynyl
Ethylene
Ethenyl
Ethenyl
Ethenyl
Ethynyl
Ethynyl
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
15 (1.6)
320(48)
420(31)
530(81)
83 (12)
440(53)
65 (20)
8.4 (2.6)
4.2 (0.7)
57 (14)
12 (4.2)
4-Biphenyl
2-Pyridyl
4-Pyridyl
4-Pyridyl
Phenyl
>10( n ¼ 2)
1.5 (0.16)
OMe
OMe
OMe
OMe
OMe
3-Pyridyl
4-Pyridyl
Phenyl
0.14 (0.051)
0.08 (0.006)
3.0(0.87)
3-Pyridyl
0.27 (0.033)
O
Cl
N
b
CO2Et
CO2Et
a
Cl
Cl
Cl
Cl
O
Br
N
H
Br
HN
O
HN
9
10
b
CN
CN
Br
N
N
R'
20: R = phenyl
5
21: R = 4-biphenyl
22: R = 2-pyridyl
23: R = 4-pyridyl
Cl
Cl
Cl
Cl
a
HN
HN
c
CO2Et
c
CO2R
Cl
Cl
Cl
Cl
Br
N
N
HN
O
HN
O
11
N
12: R = Et
13: R = H
CN
CN
d
N
R'
N
Scheme 2. Reagents and conditions: (a) phosphorous oxychloride,
DMF; (b) 2,4-dichloroaniline, pyridine hydrochloride; (c) 4-vinyl-
pyridine, Pd(OAc)2, P(o-Tol)3, DMF, TEA; (d) NaOH.
N
24
14: R = H
15: R = phenyl
16: R = 4-biphenyl
17: R = 2-naphthyl
18: R = 2-pyridyl
19: R = 3-pyridyl
With these studies we have shown that 3-quinolinecar-
bonitriles bearing ethynylpyridine and ethenylpyridine
groups at C-7 represent potent Src inhibitors. Analogous
to the results observed with the 6,7-dialkoxy-3-quino-
linecarbonitrile series, the preferred group at C-4 was
Scheme 3. Reagents and conditions: (a) alkene, Pd(OAc)2, P(o-Tol)3,
DMF, TEA; (b) alkyne, Pd(Ph3P)4, CuI, DMF, TEA; (c) Pd/C,
MeOH, H2.

2158
A. C. Barrios Sosa et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2155–2158
Kraker, A. J.; Hartl, B. G.; Amar, A. M.; Barvian, M. R.;
Showalter, H. D. H.; Moore, C. W. Biochem. Pharm.
2000, 60, 885; (c) Blake, R. A.; Broome, M. A.; Liu, X.;
Wu, J.; Gishizky, M.; Sun, L.; Courtneidge, S. A. Mol.
Cell Biochem. 2000, 20, 9018.
Cl
Cl
O
Cl
Cl
O
HN
a
HN
O
CN
CN
O
TfO
N
R
N
4. Boschelli, D. H.; Wang, Y. D.; Ye, F.; Wu, B.; Zhang, N.;
Dutia, M.; Powell, D. W.; Wissner, A.; Arndt, K.; Weber,
J. M.; Boschelli, F. J. Med. Chem. 2001, 44, 822.
5. Boschelli, D. H.; Ye, F.; Wang, Y. D.; Dutia, M.;
Johnson, S.; Wu, B.; Miller, K.; Powell, D. W.; Arndt,
K.; Discafani, C.; Etienne, C.; Gibbons, J.; Grod, J.;
Lucas, J.; Weber, J. M.; Boschelli, F. J. Med. Chem. 2001,
44, 3965.
6. Boschelli, D. H.; Ye, F.; Wu, B.; Wang, Y. D.; Barrios
Sosa, A. C.; Yaczko, D.; Powell, D. W.; Golas, J. M.;
Lucas, J.; Boschelli, F. Bioorg. Med. Chem. Lett. 2003, 13,
3797.
25: R = phenyl
26: R = 3-pyridyl
27: R = 4-pyridyl
b
Cl
Cl
O
HN
O
CN
N
R
7. (a) Boschelli, D. H.; Wang, D. Y.; Ye, F.; Yamashita, A.;
Zhang, N.; Powell, D. W.; Weber, J. M.; Boschelli, F.
Bioorg. Med. Chem. Lett. 2002, 12, 2011; (b) Berger, D.;
Dutia, M.; Powell, D.; Wissner, A.; DeMorin, F.; Raifeld,
Y.; Weber, J.; Boschelli, F. Bioorg. Med. Chem. Lett. 2002,
12, 2989.
28: R = phenyl
29: R = 3-pyridyl
Scheme 4. Reagents and conditions: (a) alkene, Pd(OAc)2, P(o-Tol)3,
DMF, TEA; (b) alkyne, Pd(Ph3P)4, CuI, DMF, TEA.
8. Sobolov-Jaynes, S. B.; Arnold, L. D. 4-Aminoquinazoline
Derivatives. US Patent 6,225,318, 2001.
9. Lin, A. J.; Loo, T. L. J. Med. Chem. 1978, 21, 268.
10. Noncommercial 3-vinylpyridine was synthesized by reac-
tion of 3-iodopyridine with tributyl(vinyl)tin under Suzuki
conditions.
2,4-dichloro-5-methoxyaniline. Substituting C-6 with a
methoxy group resulted in an increase in Src inhibition.
Derivative 27 was the most potent inhibitor in this series,
showing comparable activity to 1b in both the Src
enzyme and Src-dependent cell proliferation assays. We
are continuing to investigate the physicochemical and
biological properties of these analogs and other related
compounds. The results will be reported in due time.
11. All compounds were characterized by MS, NMR, and
CHN combustion analysis.
12. The IC50 values reported represent the means of at least 2
determinations. The standard deviations (SD) are shown
in the brackets. Src kinase assay: Recombinant human Src
enzyme was obtained from PanVera (P3044). Biotinylated
peptide corresponding to residues 6–20of Cdk1 was used
as a substrate (Biotin-KVEKIGEGTYGVVYK-COOH).
Homogeneous fluorescence resonance energy transfer
kinase assays were performed using the europium/APC
detection format (LANCE, Perkin Elmer). Src enzyme
(10ng) was mixed with biotinylated peptide (final concen-
Acknowledgements
We thank Steve Johnson for the preparation of inter-
mediate 10 and members of the Wyeth Discovery Ana-
lytical Chemistry department for the spectral data and
elemental analyses. We also thank Dr. Dennis Powell
for his support.
tration 2 lM), 50mM Hepes (pH 7.5), 10mM MgCl ,
2
20 lg/mL BSA, 0.001% Brij-35 (Sigma), 100 lM ATP, 1%
DMSO. The kinase reaction was incubated for 70min at
37 °C. The reaction was stopped with EDTA at a final
concentration of 30mM EDTA/25 mM Hepes (pH 7.5)/
10 lg/mL BSA. The mixture was combined with
Eu-labeled anti-phosphotyrosine antibody PT66 (Perkin
Elmer, AD0068) and Streptavidin Surelight-APC (Perkin
Elmer, CR130-100) in 50 mM Hepes (pH 7.5)/20 lg/mL
BSA, and incubated for 30min according to manufac-
turerÕs specifications. Fluorescence intensity at 665 nM
was used to monitor the extent of the kinase reaction.
It should be noted that the Src enzyme assay used here is
different from the ELISA assay used in our previous
publications (Refs. 4–7).
References and notes
1. (a) Cohen, P. Nature Rev. Drug Discovery 2002, 1, 309; (b)
Blume-Jensen, P.; Hunter, T. Nature 2001, 411, 355.
2. (a) Frame, M. C. Biochem. Biophys. Acta 2002, 1602, 114;
(b) Metcalf, C. A., III; van Schravendijk, M. R.; Dalgarno,
D. C.; Sawyer, T. K. Curr. Pharm. Des. 2002, 8, 2049; (c)
Courtneidge, S. A. Biochem. Soc. Trans. 2002, 30, 11; (d)
Susa, M.; Missbach, M.; Green, J. TiPS 2000, 21, 489.
3. (a) Missbach, M.; Jeschke, M.; Feyen, J.; Muller, K.;
Glatt, M.; Green, J.; Susa, M. Bone 1999, 24, 437; (b)
13. Compounds were tested according to the Src cellular assay
reported previously.4