Inhibition of Src kinase activity by 7-[(2,4-dichloro-5-methoxyphenyl) amino]-2-heteroaryl-thieno[3,2-b]pyridine-6-carbonitriles

Article abstract of DOI:10.1016/j.bmcl.2005.07.061

7-[(2,4-Dichloro-5-methoxyphenyl)amino]thieno[3,2-b]pyridine-6- carbonitriles with various heteroaryl groups at C-2 are inhibitors of Src kinase activity. Of these new analogs, compounds substituted at C-2 by a 3,5-furan or a 2,5-pyridine had the best activity in the Src enzyme and cell assays.

Full text of DOI:10.1016/j.bmcl.2005.07.061

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4681–4684
Inhibition of Src kinase activity by
7-[(2,4-dichloro-5-methoxyphenyl)-
amino]-2-heteroaryl-thieno[3,2-b]pyridine-6-carbonitriles
Diane H. Boschelli,a,* Biqi Wu,a Ana Carolina Barrios Sosa,a, Joan J. Chen,a
Jennifer M. Golasb and Frank Boschellib
aChemical and Screening Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
bOncology, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
Received 14 July 2005; revised 28 July 2005; accepted 28 July 2005
Available online 24 August 2005
Abstract—7-[(2,4-Dichloro-5-methoxyphenyl)amino]thieno[3,2-b]pyridine-6-carbonitriles with various heteroaryl groups at C-2 are
inhibitors of Src kinase activity. Of these new analogs, compounds substituted at C-2 by a 3,5-furan or a 2,5-pyridine had the best
activity in the Src enzyme and cell assays.
Ó 2005 Elsevier Ltd. All rights reserved.
The non-receptor tyrosine kinase Src is a member of a
family of highly homologous kinases known as the
SFKs (Src family kinases). Since overexpression or over-
activation of Src is implicated in various diseases, small
molecule Src inhibitors are being pursued for several
therapeutic indications.1,2 Initial efforts were focused
on new agents for the treatment of cancer and osteo-
porosis, and later diversified to incorporate ischemic
diseases, such as stroke and myocardial infarction.3–6
substituent at C-2, additional analogs were prepared
varying the heteroaryl ring at this position.
Cl
Cl
O
HN
O
O
CN
N
N
N
SKI-606
4-Phenylamino-3-quinolinecarbonitriles
have
been
Cl
O
Cl
Cl
Cl
O
extensively studied as kinase inhibitors,7,8 with a com-
pound from this class, SKI-606, being a potent inhibitor
of Src kinase.9,10 It was recently reported that this activ-
ity is shared by the structurally related 7-(phenylami-
no)thieno[3,2-b]pyridine-6-carbonitriles.11 The C-2
phenyl thieno[3,2-b]pyridine-6-carbonitrile 1a had an
IC50 of 13 nM for the inhibition of Src kinase activity.
This 1,4 substituted derivative was a more potent Src
inhibitor than the 1,3 substituted isomer 1b, while the
1,2 substituted isomer had greatly reduced activity. A
difference in activity was also observed with the C-2
thiophene analogs, where 2a was a more potent Src
inhibitor than 2b.12 To further study the effect of the
HN
HN
3
CN
CN
S
S
3
4
N
N
N
2
5
S
N
2a: 3,5-isomer
2b: 2,5-isomer
1a: 1,4-isomer
1b: 1,3-isomer
N
N
The preparation of two C-2 furan analogs is shown in
Scheme 1. Treatment of the previously reported 2-iodo
intermediate 311 with commercially available 2-formyl-
4-furanboronic acid provided the aldehyde derivative
4. Reaction of 3 with tributyl[5-(1,3-dioxolan-2-yl)-2-
furanyl]stannane13, followed by acid hydrolysis of the
Keywords: Src; Thieno[3,2-b]pyridine-6-carbonitrile.
*
Corresponding author. Tel.: +1 845 602 3567; fax: +1 845 602
5561; e-mail: bosched@wyeth.com
Present address: Roche Carolina, Florence, SC, USA.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.061

4682
D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4681–4684
Cl
O
Cl
Cl
Cl
O
HN
HN
CN
S
CN
S
a
I
OHC
N
O
N
4: 3,5-isomer
5: 2,5-isomer
3
Cl
Cl
b
O
HN
3
CN
N
4
5
S
N
2
N
O
6: 3,5-isomer
7: 2,5-isomer
N
Cl
Cl
O
N
HN
N
Br
Br
3
N
b
CN
N
N
S
N
OHC
c
S
S
S
N
8
9
Scheme 1. Reagents: (a) for 4: 2-formyl-4-furanboronic acid, (Ph3P)4Pd, DME, satd aq NaHCO3; for 5: (1) tributyl[5-(1,3-dioxolan-2-yl)-2-
furanyl]stannane, (Ph3P)2PdCl2, dioxane, (2) 2N HCl, THF; (b) 1-methylpiperazine, Na(OAc)3BH, CH2Cl2, NMP, HOAc; (c) (1) (Me3Sn)2,
(Ph3P)4Pd, dioxane, (2) (Ph3P)4Pd, dioxane.
intermediate acetal, provided aldehyde 5. Reductive
amination of 4 and 5 with 1-methylpiperazine in the
presence of sodium triacetoxyborohydride led to the
desired derivatives 6 and 7.
IC50 of 13 nM in the Src enzyme assay and an IC50 of
260 nM in the Src cell assay. These results correspond
to those observed with the thiophene isomers 2a and
2b. Since the C-2 3,5-thiophene substituent showed good
Src inhibitory activity, the C-2 4,2-thiazole 9 was target-
ed. This compound has a N in place of the CH group at
C-4 of the thiophene ring of 2a.
As shown in Table 1, 6, the 3,5-furan isomer, was more
potent than 7, the 2,5-furan isomer, with 6 having an
Table 1. Inhibition od Src kinase activity
Cl
Cl
Ar
R
HN
CN
S
Linker
R
N
Compound
Linker
R
RAr
Src Enzyme
IC50 nM18
Src Cell
IC50 nM18
SKI-606
3.819
13
10020
720
1a
1b
2a
2b
6
1,4-Phenyl
1,3-Phenyl
3,5-Thiophene
2,5-Thiophene
3,5-Furan
CH2-N-Me-piperazine
CH2-N-Me-piperazine
CH2-N-Me-piperazine
CH2-N-Me-piperazine
CH2-N-Me-piperazine
CH2-N-Me-piperazine
CH2-N-Me-piperazine
CH2-N-Me-piperazine
CH2-N-Me-piperazine
CH2-N-Me-piperazine
CH2NMe2
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
21
1200
430
7.2
25
2400
260
13
52
7
2,5-Furan
3100
2900
420
9
4,2-Thiazole
2,5-Pyridine
2,6-Pyridine
Alkyne
56
13
12
13
14
16
18
20
51
1900
>5000
650
300
13
2,5-Pyridine
2,5-Pyridine
2,5-Pyridine
CO-N-Me-piperazine
CH2-N-Me-piperazine
14
47
360
>5000

D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4681–4684
4683
Known 4-bromo-1,3-thiazole-2-carbaldehyde14 was
converted to 8 via reductive amination with 1-methyl-
piperazine. In situ conversion of 8 to the tributylstann-
ane derivative, followed by coupling with 3, provided
9. Surprisingly, 9 was much less potent than 2a, having
an IC50 in the Src enzyme assay of only 56 nM. If
replacement of the CH of the thiophene with a N led
to reduced activity, would the same replacement in the
phenyl series also lead to reduced activity?
of 15, which was prepared by reductive amination
of 5-bromo-2-pyridinecarboxaldehyde with dimethyl-
amine. To prepare the amide analog, the methyl group
of 2-bromo-5-methylpyridine was first oxidized to the
corresponding acid with potassium permanganate. Sub-
sequent amide formation with N,N-carbonyldiimidazole
and 1-methylpiperazine provided 17. The trimethyl-
stannane derivative of 17 was generated and coupled
with 3 to provide 18.
To this end, Scheme 2 depicts the preparation of the C-2
2-pyridine analogs of 1a and 1b. The commercially
available 6-bromo 2- and 3-pyridinecarboxaldehydes
were converted to 10 and 11 via reductive amination
with 1-methylpiperazine. Preparation of the tributylst-
annane derivatives of 10 and 11, followed by coupling
with 3, resulted in the formation of 12 and 13. As shown
in Table 1, 12 was as potent in the Src enzyme assay as
1a and was more potent than 1a in the Src cell assay.
Therefore, as opposed to what was observed with the
thiophene to thiazole pair of analogs, replacement of
the CH group at C-2 of the phenyl ring of 1a with a
N did not lead to a reduction in activity. Corresponding
to the reduced activity of 1b compared to 1a, the 2,6-iso-
mer 13 was about 5-fold less potent than 12. The com-
pounds with greater Src inhibitory activity can be
viewed as being more linear than the less active com-
pounds. Conceivably, the most linear analog possible
would contain a triple bond at C-2 of the thieno[3,2-
b]pyridine ring. The highly rigid ethynyl analog 14 was
prepared by reaction of 3 with 1-methyl-4-(2-propy-
nyl)piperazine.15 This compound was a weak Src inhib-
itor having an IC50 of only 300 nM in the enzyme assay
and an IC50 of greater than 5 lM in the cell assay.
Therefore, to inhibit Src activity it may be necessary that
a molecule has a greater degree of flexibility at C-2 than
that conferred by the triple bond.
We had previously observed that 1a and 2a were more
potent Src inhibitors than the analogs without a 5-
OMe group on the aniline at C-7.11,12 The C-7 (2,4-
dichlorophenyl)amino analog of 12 was readily prepared
from the known 1911 using the same reaction conditions
used to convert 3 to 12. As shown in Table 1, while the
dimethylamine analog 16 and the amide analog 18 had
comparable activity to that of 12 in the enzyme assay,
and values within 2 fold in the cell assay, the C-7 (2,4-
dichlorophenyl)amino analog 20 had greatly reduced
activity especially in the Src cell assay.
Of these analogs, compound 12 was selected for further
evaluation. When tested against a panel of kinases, 12
had IC50s of 1.6 lM for the inhibition of Raf/MEK
and 2.0 lM for the inhibition of EGFR. IC50s of great-
er than 10 lM were obtained for the inhibition of
CDK4, IKK, AKT, and PDK1. When tested for inhi-
bition of Abl kinase, 12 had an IC50 of 1.3 nM, making
it a comparable Abl inhibitor to 1a and SKI-606,
which had IC50s of 2.3 and 1.0 nM, respectively. Due
to similarly active conformations of the ATP binding
sites of these two kinases, many compounds first iden-
tified as Src inhibitors were later found to also inhibit
Abl.16,17
Testing of 12 at 3 lM, resulted in less than 30% inhibi-
tion of CYPs 3A4, 2D6, and 2C9. When incubated with
nude mouse liver microsomes, 12 had a half-life of great-
er than 30 min. In addition, 24 h after administration of
a 50 mg/kg oral dose of 12 to nude mice, a plasma level
Additional analogs of 12 were prepared, as shown in
Scheme 3. The dimethylamine analog 16 was obtained
by coupling of 3 with the tributylstannane derivative
Cl
Cl
O
HN
3
4
CN
3
S
a
CHO
N
2
5
Br
N
Br
N
b
N
N
6
N
N
12: 2,5-isomer
13: 2,6-isomer
10: 2,5-isomer
11: 2,6-isomer
N
Cl
Cl
O
HN
N
CN
S
3
N
N
c
N
N
14
Scheme 2. Reagents: (a) 1-Methylpiperazine, Na(OAc)3BH, CH2Cl2, NMP, HOAc; (b) (1) n-BuLi, Bu3SnCl, THF, (2) (Ph3P)2PdCl2, dioxane;
(c) (Ph3P)4Pd, CuI, Et3N, benzene.

4684
D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4681–4684
Cl
Cl
O
HN
CN
S
a
3
Br
OHC
Br
N
N
N
b
N
N
N
16
15
Cl
Cl
HN
O
CN
O
O
S
c
3
Br
Br
N
N
N
N
b
N
N
18
17
N
N
Cl
Cl
Cl
Cl
HN
N
HN
10
CN
CN
S
S
d
I
N
N
N
20
19
N
Scheme 3. Reagents: (a) Dimethylamine, Na(OAc)3BH, CH2Cl2, NMP, HOAc; (b) (Me3Sn)2, (Ph3P)4Pd, dioxane; (c) (1) KMnO4, H2O, Aliquat 336,
(2) CDI, THF, (3) 1-methylpiperazine; (d) (1) n-BuLi, Bu3SnCl, THF, (2) (Ph3P)2PdCl2, dioxane.
8. Boschelli, D. H. Med. Chem. Rev. Online 2004, 1, 457.
9. Golas, J. M.; Arndt, K.; Etienne, C.; Lucas, J.; Nardin,
D.; Gibbons, J.; Frost, P.; Ye, F.; Boschelli, D. H.;
Boschelli, F. Cancer Res. 2003, 63, 375.
10. Boschelli, D. H.; Ye, F.; Wang, Y. D.; Dutia, M.;
Johnson, S. L.; Wu, B.; Miller, K.; Powell, D. W.;
Yaczko, D.; Young, M.; Tischler, M.; Arndt, K.; Disca-
fani, C.; Etienne, C.; Gibbons, J.; Grod, J.; Lucas, J.;
Weber, J. M.; Boschelli, F. J. Med. Chem. 2001, 44, 3965.
11. Boschelli, D. H.; Wu, B.; Barrios Sosa, A. C.; Durutlic,
H.; Ye, F.; Raifeld, Y.; Golas, J. M.; Boschelli, F. J. Med.
Chem. 2004, 47, 6666.
12. Boschelli, D. H.; Wu, B.; Barrios Sosa, A. C.; Durutlic,
H.; Chen, J. J.; Wang, Y.; Golas, J. M.; Lucas, J.;
Boschelli, F. J. Med. Chem. 2005, 48, 3891.
13. Balachari, D.; Quinn, L.; OÕDoherty, G. A. Tetrahedron
Lett. 1999, 40, 4769.
of 160 ng/mL was observed. In comparison, in a similar
PK study 1a had a plasma level of 400 ng/mL and was
subsequently found to have good activity in an HT-29
xenograft model.11 Based on these data, 12 is an accept-
able candidate for in vivo testing in xenograft models.
Acknowledgments
We thank the Wyeth Drug Safety and Metabolism
Department and Judy Lucas for the metabolic stability
and plasma level determination. We also thank members
of the Wyeth Chemical Technology department for
compound characterization and the CYP inhibition
data, and Drs. Janis Upeslacis, Dennis Powell, and
Tarek Mansour for their support.
14. Nicolaou, K. C.; He, Y.; Roschangar, F.; King, N. P.;
Vourloumis, D.; Li, T. Angew. Chem. In. Ed. 1998, 37, 84.
15. Mayrargue, J.; Vayssiere, M.; Miocque, M. Heterocycles
1985, 23, 2173.
References and notes
16. Warmuth, M.; Damoiseaux, R.; Liu, Y.; Fabbro, D.;
Gray, N. Curr. Pharm. Des. 2003, 9, 2043.
17. Boschelli, D. H. Drug Des. Rev. Online 2004, 1, 203.
18. Assay: IC50 values represent means of at least two separate
determinations.
1. Parsons, S. J.; Parsons, J. T. Oncogene 2004, 23, 7906.
2. Boggon, T. J.; Eck, M. J. Oncogene 2004, 23, 7918.
3. Yeatman, T. J. Nat. Rev. Cancer 2004, 4, 470.
4. Susva, M.; Missbach, M.; Green, J. Trends Pharmacol.
Sci. 2000, 21, 489.
19. For conditions of the Src enzyme assay see: Boschelli, D.
H.; Wang, Y. D.; Johnson, S.; Wu, B.; Ye, F.; Barrios
Sosa, A. C.; Golas, J. M.; Boschelli, F. J. Med. Chem.
2004, 47, 1599.
20. For conditions for the Src cell assay see: 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. Paul, R.; Zhang, Z. G.; Eliceiri, B. P.; Jiang, Q.; Boccia, A.
D.; Zhang, R. L.; Chopp, M.; Cheresh, D. A. Nat. Med.
2001, 7, 222.
6. Weis, S.; Shintani, S.; Weber, A.; Kirchmair, R.; Wood,
M.; Cravens, A.; McSharry, H.; Iwakura, A.; Yoon, Y.-S.;
Himes, N.; Burstein, D.; Doukas, J.; Soll, R.; Losordo, D.;
Cheresh, D. J. Clin. Invest. 2004, 113, 885.
7. Boschelli, D. H. Curr. Topics Med. Chem. 2002, 2, 1051.