Inhibition of Src kinase activity by 4-anilino-7-thienyl-3-quinolinecarbonitriles.

Article abstract of DOI:10.1016/S0960-894X(02)00302-5

Based on a screening lead from a yeast-based assay to identify Src family kinase inhibitors, a series of 4-anilino-7-thienyl-3-quinolinecarbonitriles was prepared. When the thiophene ring was substituted with a water-solubilizing group in a 2,5-, 3,5- or 2,4-pattern, potent inhibition of Src kinase activity was observed.

Full text of DOI:10.1016/S0960-894X(02)00302-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2011–2014
Inhibition of Src Kinase Activity by 4-Anilino-7-thienyl-3-
quinolinecarbonitriles
Diane H. Boschelli,* Daniel Y. Wang, Fei Ye, Ayako Yamashita, Nan Zhang,
Dennis Powell, Jennifer Weber and Frank Boschelli
Wyeth Research, Chemical Sciences and Oncology, 401 N. Middletown Road, Pearl River, NY 10965, USA
Received 11 February 2002; accepted 1 April 2002
Abstract—Based on a screening lead from a yeast-based assay to identify Src family kinase inhibitors, a series of 4-anilino-7-thienyl-
3-quinolinecarbonitriles was prepared. When the thiophene ring was substituted with a water-solubilizing group in a 2,5-, 3,5- or
2,4-pattern, potent inhibition of Src kinase activity was observed. # 2002 Elsevier Science Ltd. All rights reserved.
Protein tyrosine kinases (TKs) are enzymes that cata-
lyze the transfer of a phosphate group from ATP to a
tyrosine residue on a protein, resulting in a variety of
cell signaling events. The TK Src is a member of the Src
family of kinases (SFK) that includes among its mem-
bers Fyn and Yes.1a,b Since the activation and/or over-
expression of Src has been implicated in cancer,1c,d
osteoporosis1e,f and stroke,1g a small molecule inhibitor
of Src activity might be beneficial for the treatment of
various disease states. Several classes of SFK inhibitors
have been reported in the literature, including pyra-
zolo[3,4-d]pyrimidines,2a pyrrolo[2,3-d]pyrimidines,2bÀf
pyrido[2,3-d]pyrimidin-7(8H)-ones,2g 1,6-naphthyridin-
2(1H)-ones,2h aminopyrido[2,3-d]pyrimidin-7-yl ureas2i
and 4-anilinoquinazolines.2j
C-7, was identified. We envisioned that 3 could be con-
verted to an Src inhibitor if the 2-fluoro-4-chloroaniline
group was replaced by the preferred Src aniline. Fur-
thermore, the addition of a water-solubilizing group to
the thiophene should provide cellular activity. Interest-
ingly, a series of related 4-anilinoquinazolines with
thiophene substituents at C-7, including 4, have been
reported to be EGFr kinase inhibitors.8a,b
We recently reported that a yeast-based screen for Src
inhibitors provided 3-quinolinecarbonitrile 1 as a lead
compound.3 It was determined that 1 was an ATP
competitive inhibitor of Src kinase activity. Optimi-
zation of the C-4 aniline substituents and the addition
of a water-solubilizing group at C-7 provided 2, a very
potent inhibitor of Src cellular activity.4 3-Quinoline-
carbonitriles have been used as templates for the inhi-
bition of kinases other than Src,5 including epidermal
growth factor receptor (EGFr)6a,b and MEK.7a,b
The 3-quinolinecarbonitrile analogue of 4 with the pre-
ferred 4-anilino group for optimal Src activity was pre-
pared as shown in Scheme 1. Treatment of 3-bromoaniline
or 3-iodoaniline with ethyl ethoxymethylenecyano-
acetate followed by thermal cyclization resulted in 5a or
5b. Chlorination provided 6a or 6b with subsequent
addition of the aniline giving 7a and 7b. Treatment of
In yeast-based screens for inhibitors of Fyn and Yes, 3,
a 3-quinolinecarbonitrile with a thiophene substituent at
*Corresponding author. Tel.: +1-845-602-3567; fax: +1-845-602-
5561; e-mail: bosched@war.wyeth.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00302-5

2012
D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2011–2014
Scheme 1. (a) (1) Ethyl ethoxymethylenecyanoacetate; (2) Dowtherm;
(b) POCl3; (c) aniline, pyridine–HCl, 2-ethoxyethanol; (d) diisopropyl
[5-(morpholinomethyl)thien-3-yl]boronate, Pd(PPh3)4, NaHCO3, DME.
Table 1. Inhibition of Src enzymatic and Src cellular activity for
compounds 8–12 and 14–20
Scheme 2. (a) Morpholine, NaCNBH3, EtOH, AcOH; (b) 7a,
Pd(PPh3)4, NaHCO3, DME; (c) Br2, AlCl3; (d) morpholine, EDCI,
DMAP; (e) BH3–Me2S; (f) n-BuLi, DMF.
Compd
Thiophene
isomer
X
Src IC50
(nM)
Cell IC50
(nM)
8
9
3
O
O
2.7
2.5
5.7
440
240
3.8
1.4
3.8
2.0
4.2
4.4
280
1.2
200
220
240
2
2,4 isomer of 8
3,2 isomer of 8
3,4 isomer of 8
10
11
12
14
15
16
17
18
19
20
2
>10,000
>10,000
64
3
3
2
2
NMe
CH(OH)
NMe
CH(OH)
CH2
120
69
120
190
630
3600
100
2
2
C-6 isomer of 8
S
7a with diisopropyl [5-(morpholinomethyl)thien-3-yl]-
boronate8a under Suzuki coupling conditions provided
8.9,10 As shown in Table 1, 8 was a potent inhibitor of
Src activity in both the enzymatic and cell assays,
showing comparable activity to 2.11
Scheme 3. (a) Tributyl[5-(1,3-dioxolan-2-yl)-2-thienyl]stannane or tri-
butyl[5-(1,3-dioxolan-2-yl)-3-thienyl]stannane, (PPh3)2PdCl2, dioxane;
(b) 1 N HCl, THF; (c) amine, NaB(OAc)3H, CH2Cl2, DMF, AcOH.
In compound 8, the thiophene ring has a 3,5-substi-
tution pattern. Four of the five possible thiophene
regioisomers of 8, namely 9–12, were prepared by the
routes depicted in Scheme 2.
observed with 11 and 12 is not surprising since we pre-
viously reported that analogues of 1 with a mono
methoxy group at either the C-5 or C-8 position had
greatly reduced Src inhibitory activity compared to the
mono C-6 or C-7 methoxy analogues.3
As shown in Table 1, compounds 9 and 10 wherein the
thiophene is substituted in a 2,5- or 2,4-fashion showed
Src inhibitory activity comparable to that of 8, the 3,5-
regioisomer, in both the enzymatic and cell assays.
These compounds have the solubilizing group pointing
away from the 3-quinolinecarbonitrile core and can be
considered to be ‘linear’ analogues as opposed to the
‘angular’ analogues 11 and 12. The decreased activity
We next turned our attention to replacing the morpho-
line group of 8 and 9 with other basic amines. In order
to facilitate the preparation of these additional

D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2011–2014
2013
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Scheme 4. (a) (1) DMF–DMA; (2) n-BuLi, CH3CN, THF; (b) (1) POCl3;
(2) aniline; (c) (1) tributyl[5-(1,3-dioxolan-2-yl)-3-thienyl]stannane,
(PPh3)2PdCl2, dioxane; (2) 1 N HCl, THF; (3) morpholine,
NaB(OAc)3H, CH2Cl2, DMF, AcOH.
analogues, we used the thiophene carboxaldehydes 13a
and 13b as common intermediates. Palladium catalyzed
coupling of tributyl[5-(1,3-dioxolan-2-yl)-3-thienyl]-
stannane and tributyl[5-(1,3-dioxolan-2-yl)-2-thienyl]-
stannane12 with 7b followed by acid hydrolysis of the
acetal group provided 13a and 13b. Reductive amina-
tion with various heterocyclic amines resulted in 14–19
(Scheme 3).13
As shown in Table 1, analogues 14–19 had IC50s of
1.4–4.4 nM in the Src enzymatic assay. When tested in
the Src cell assay, two compounds with an N-methyl-
piperazine group, namely 14 and 16, had IC50s that were
3-fold lower than those of the corresponding morpho-
line analogues 8 and 9. In addition, 14 and 16 were
more potent inhibitors of Src-dependent cell prolifera-
tion (IC50s of 64 and 69 nM, respectively) than of Fyn-
dependent cell proliferation (IC50s of 580 and 630 nM,
respectively).3
3. Boschelli, D. H.; Wang, D. Y.; 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.
In our earlier work on 4-anilino-6,7-dialkoxy-3-quino-
linecarbonitriles, it was shown that while addition of a
water solubilizing group at C-7 was advantageous,
addition of the same group at C-6 was detrimental.3 To
determine if this same effect is observed in this series,
the C-6 thiophene isomer of 8, namely 20, was prepared
from 5-bromoanthranilic acid as shown in Scheme 4. As
predicted, 20 had greatly reduced activity compared to 8
(see Table 1).
4. Boschelli, D. H.; Ye, F.; Wang, D. Y.; 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.
5. Wang, Y. D.; Miller, K.; Boschelli, D. H.; Ye, F.; Wu, B.;
Floyd, M. B.; Powell, D. W.; Wissner, A.; Weber, J. M.;
Boschelli, F. Bioorg. Med. Chem. Lett. 2000, 10, 2477.
6. (a) Wissner, A.; Berger, D. M.; Boschelli, D. H.; Floyd,
M. B.; Greenberger, L. M.; Gruber, B. C.; Johnson, B. D.;
Mamuya, N.; Nilakatan, R.; Reich, M. F.; Shen, R.; Tsou,
H. R.; Upeslacis, E.; Wang, Y. F.; Wu, B.; Ye, F.; Zhang, N.
J. Med. Chem. 2000, 43, 3244. (b) Torrance, C. J.; Jackson,
P. E.; Montgomery, E.; Kinzler, K. W.; Vogelstein, B.; Wiss-
ner, A.; Nunes, M.; Frost, P.; Discafani, C. M. Nat. Med.
2000, 6, 1024.
7. (a) Zhang, N.; Wu, B.; Powell, D.; Wissner, A.; Floyd,
M. B.; Kovacs, E. D.; Toral-Barza, L.; Kohler, C. Bioorg.
Med. Chem. Lett. 2000, 10, 2825. (b) Zhang, N.; Wu, B.;
Eudy, N.; Wang, Y.; Ye, F.; Powell, D.; Wissner, A.; Feld-
berg, L. R.; Kim, S. C.; Mallon, R.; Kovacs, E. D.; Toral-
Barza, L.; Kohler, C. A. Bioorg. Med. Chem. Lett. 2001, 11,
1407.
In conclusion, as shown previously with a series of
6,7-dialkoxy-3-quinolinecarbonitriles, 7-thienyl-3-quino-
linecarbonitriles are also potent Src inhibitors. The best
cell activity was observed with compounds containing
an N-methylpiperazine group. We are currently extend-
ing this work to include other aryl and heteroaryl
groups at C-7.
References and Notes
8. (a) Barker, A. J.; Johnstone, C. US Patent 5,814,630, Sept
29, 1998. (b) Reviewed in: Bridges, A. J. Chem. Rev. 2001, 10,
2541.
1. (a) Abram, C. L.; Courtneidge, S. A. Exp. Cell Res. 2000,
254, 1. (b) Boschelli, D. H.; Boschelli, F. Drug. Fut. 2000, 25,

2014
D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2011–2014
9. Preparation of 8: 4-(2,4-Dichloro-5-methoxyanilino)-7-[5-
(4-morpholinylmethyl) - 3 - thienyl] - 3 - quinoline - carbonitrile.
1-[(4-Bromo-2-thienyl)methyl]-morpholine (248 mg, 0.95 mmol)8a
was dissolved in 20 mL of THF and the solution was cooled to
À78 ꢀC. Tri-isopropylborate (196 mg, 1.04 mmol) was added
followed by 2.5 M n-BuLi in hexane (0.39 mL, 97 mmol). The
mixture was stirred at À78 ꢀC for 30 min and then allowed to
warm to room temperature. The solvent was removed in vacuo
to provide the intermediate boronate. A mixture of this boro-
nate, 7a (200 mg, 0.47 mmol) and (Ph3P)4Pd (96 mg,
0.08 mmol) was heated at reflux in 8 mL of DME and 5 mL of
saturated NaHCO3 for 4.5 h. The reaction was cooled to room
temperature, quenched with 5 mL of 1.0 N NaOH, and parti-
tioned between ethyl acetate and brine. The layers were sepa-
rated and the ethyl acetate layer was dried over MgSO4,
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography eluting with 20% MeOH in
CH2Cl2 to provide 98 mg (41% yield) of 8: mp 170 ꢀC; 1H
NMR (DMSO-d6) d 2.35–2.50 (m, 4H), 3.55–3.65 (m, 4H),
3.75 (s, 2H), 3.87 (s, 3H), 7.42 (s, 1H), 7.68 (s, 1H), 7.77 (s,
1H), 8.08 (d, J=9 Hz, 1H), 8.17 (s, 1H), 8.22 (s, 1H), 8.54 (d,
J=9 Hz, 1H), 8.57 (s, 1H), 9.99 (s, 1H); MS (ES) 525.1
(M+H). Analysis for C26H22Cl2N4O2S: Calcd: C, 59.43; H,
4.22; N, 10.66. Found: C, 59.28; H, 3.93; N, 10.61.
thienyl]stannane from furancarboxaldehyde. Yamamoto,
M.; Izukawa, H.; Saiki, M.; Yamada, K. J. Chem. Soc.
Chem. Commun. 1988, 8, 560. Tributyl[5-(1,3-dioxolan-2-yl)-3-
thienyl]stannane was prepared from 4-bromo-2-thio-
phene-carboxaldehyde by conversion of the aldehyde to
the acetal with ethylene glycol, followed by the addition of
n-BuLi to a À78 ꢀC solution of the acetal and (n-Bu)3SnCl
in THF.
13. Preparation of 14: 4-(2,4-Dichloro-5-methoxyanilino)-7-{5-
[(4-methyl-1-piperazinyl)methyl]-3-thienyl}-3-quinolinecarbo-
nitrile. A mixture of 7c (2.5 g, 5.32 mmol), tributyl[5-(1,3-
dioxolan-2-yl)-3-thienyl]stannane (2.98 g, 6.69 mmol) and a
catalytic amount of (Ph3P)2PdCl2 in 60 mL of dioxane was
heated at reflux for 4.5 h. The reaction mixture was con-
centrated in vacuo and partitioned between ethyl acetate and
water. The organic layer was dried over MgSO4, filtered and
concentrated in vacuo. The residue was suspended in a mix-
ture of 100 mL of THF and 50 mL of 1 N HCl and stirred at
room temperature overnight. The reaction mixture was slowly
added to saturated NaHCO3 and then extracted into ethyl
acetate. The organic layer was washed with brine, dried over
MgSO4, filtered and concentrated in vacuo. Ethyl acetate was
added to the residue and the solid was collected by filtration to
provide 1.20 g of 13b. To a 0 ꢀC suspension of 13b (220 mg,
0.48 mmol) in 3 mL of CH2Cl2 and 1 mL of DMF were added
N-methylpiperazine (80 mL, 0.72 mmol) and NaB(OAc)3H
(500 mg, 2.36 mmol) followed by 1 drop of AcOH. The reac-
tion mixture was stirred at room temperature for 3 h then
partitioned between ethyl acetate and saturated NaHCO3. The
organic layer was washed with brine, dried over MgSO4, fil-
tered and concentrated in vacuo. The residue was purified by
flash column chromatography eluting with 20% MeOH in
CH2Cl2 to provide 152 mg (59% yield) of 14: mp 206–209 ꢀC;
1H NMR (DMSO-d6) d 2.16 (s, 3H), 2.35 (br m, 4H), 2.49 (br
m, 4H), 3.73 (s, 2H), 3.85 (s, 3H), 7.30 (s, 1H), 7.62 (s, 1H),
7.71 (s, 1H), 8.01 (d, J=7 Hz, 1H), 8.11 (s, 2H), 8.46–8.53 (m,
2H); MS (ES) 538.2, 540.2 (M+H). Analysis for C27H25
Cl2N5OS: Calcd: C, 60.22; H, 4.68; N, 13.01. Found: C, 59.85;
H, 4.60; N, 13.23.
10. All compounds were characterized by MS, NMR and
CHN combustion analysis.
11. Compounds were tested in a modified format of the enzy-
matic assay previously reported.3À5 Peptide was bound to the
streptavidin plate prior to the kinase reaction, and peptide
phosphorylation reaction was monitored by europium fluor-
escence as recommended by the manufacturer (Perkin–Elmer).
The IC50 values reported represent the means of at least two
separate determinations with typical variations of less than
40% between replicate values. For cell assays, Costar ultra-
low binding plates were coated with Sigma-Cote to block
residual cell attachment.
12. Tributyl[5-(1,3-dioxolan-2-yl)-2-thienyl]stannane was pre-
pared from 2-thiophenecarboxaldehyde according to the
procedure used to prepare tributyl[5-(1,3-dioxolan-2-yl)-2-