Inhibition of Src kinase activity by 7-ethynyl-4-phenylamino-3-quinolinecarbonitriles: Identification of SKS-927

Article abstract of DOI:10.1016/j.bmcl.2006.11.077

Of a series of 7-ethynyl-3-quinolinecarbonitriles, the most potent Src inhibitory activity was observed with 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[4-(4-methylpiperazi n-1-yl)but-1-ynyl]-3-quinolinecarbonitrile (SKS-927). Variation of the so

Full text of DOI:10.1016/j.bmcl.2006.11.077

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1358–1361
Inhibition of Src kinase activity by 7-ethynyl-4-phenylamino-3-
quinolinecarbonitriles: Identification of SKS-927
Diane H. Boschelli,a,* Ana Carolina Barrios Sosa,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 24 October 2006; revised 21 November 2006; accepted 30 November 2006
Available online 3 December 2006
Abstract—Of a series of 7-ethynyl-3-quinolinecarbonitriles, the most potent Src inhibitory activity was observed with 4-[(2,4-dichlo-
ro-5-methoxyphenyl)amino]-6-methoxy-7-[4-(4-methylpiperazin-1-yl)but-1-ynyl]-3-quinolinecarbonitrile (SKS-927). Variation of
the solubilizing amine tail or removal of the methoxy group from either C-6 of the quinoline core or C-5 of the aniline headpiece
led to reduced activity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Src, a non-receptor tyrosine kinase, plays a key role in
several cell signaling pathways. Studies with Src
knock-out mice showed that Src is required for bone
resorption,1 and one of the early indications for small
molecule Src inhibitors was the treatment of osteoporo-
sis.2,3 In addition, Src is over-expressed or upregulated
in a variety of tumors and therefore Src inhibitors are
also of interest for the treatment of cancer.4–6
acute stroke.18 When SKI-606 was tested in a similar
model in the rat, an iv dose of 10 mg/kg, as a solution
in D5W/lactic acid at pH 4.5, provided a reduction in in-
farct volume along with decreased neurological
deficits.19,20
Replacement of the 7-alkoxy group of SKI-606 with an
ethynyl group, as exemplified by 1a–b, retained the Src
inhibitory activity of the lead compound.21 Unfortu-
nately these analogs had very poor solubility even at
acidic pH. In the hope of increasing the water solubility,
7-ethynyl-3-quinolinecarbonitriles were targeted where
the pyridine ring of 1a–b was replaced with an alkyl
chain.
Diverse templates have been used to develop small mol-
ecule Src inhibitors including pyrido[2,3-d]pyrimidines7
pyrrolo[2,3-d]pyrimidines,8 purines,9 and quinazo-
lines.10,11 BMS-354825, dasatinib, a Src inhibitor with
an aminothiazole core, was recently approved by the
FDA for the treatment of Gleevec-resistant CML.12,13
SKI-606, bosutinib, a Src inhibitor based on a 3-quino-
linecarbonitrile template, is currently in oncology clini-
cal trials.14–16
Cl
Cl
O
Cl
Cl
O
HN
HN
O
CN
O
CN
Another potential use of a small molecule Src inhibitor
is for the treatment of stroke. Src regulates VEGF med-
iated vascular permeability17 and both Src knock-out
mice and mice treated with the Src kinase inhibitor
PP1 demonstrated reduced brain injury in a model of
N
O
N
N
N
N
R'RN
SKI-606
1a: NRR' = N-Me-piperazine
1b: NRR' = NMe2
Keywords: Src; 3-Quinolinecarbonitrile.
*
The preparation of the analog of 1a with a methylene
linker is shown in Scheme 1. Coupling of 222 with 1-
(2-propynyl)-4-methylpiperazine23 in the presence of tet-
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 ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.077

D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1358–1361
1359
Cl
Cl
O
To prepare these analogs, a more flexible approach was
investigated where 3-butynol was coupled to 2 to
provide the alcohol 5. In situ conversion of 5 to the
corresponding mesylate, followed by addition of
1-methylpiperazine, provided 4. This route was then
used to prepare analogs 6–8 by substituting other
amines for the 1-methylpiperazine in the reaction with
5. As shown in Table 1, while these analogs had similar
activity in the Src enzyme assay, 4 was the most potent
by a factor of greater than 2 in the Src cell assay.
Cl
Cl
O
HN
HN
O
CN
O
I
CN
a
N
N
N
N
2
(CH2)n
3: n = 1
4: n = 2
b
Cl
Cl
O
Cl
Cl
HN
HN
O
We previously reported examples where removal of the
C-6 methoxy group from a series of 7-ethynyl-3-quino-
linecarbonitriles led to reduced inhibition of Src activi-
ty.25,26 The des 6-methoxy analog of 4, namely 10, was
prepared as shown in Scheme 2. Reaction of 927 with
3-butynol followed by mesylation and addition of 1-
methylpiperazine provided 10. As shown in Table 1, 10
was a weaker Src inhibitor than 4 having IC50s of only
28 and 1700 nM in the enzyme and cell assays, respec-
tively. The analog of 10 with the ethynyl tail at C-6
was prepared by coupling 1127 with 1-(3-butynyl)-4-
methylpiperazine. Consistent with earlier findings where
moving the ethynyl tail to C-6 resulted in decreased
activity, 12 had reduced activity compared to 10.25
O
CN
c
O
CN
N
N
HO
R'RN
5
4: RR'N = N-Me-piperazine
6: RR'N = Me2N
7: RR'N = morpholine
8: RR'N = piperazine
Scheme 1. Reagents and conditions: (a) for 3: 1.5 equiv 1-(2-
propynyl)-4-methylpiperazine, (Ph3P)4Pd, CuI, Et3N, DMF, 100 ꢁC,
3.5 h; for 4: 3.5 equiv 1-(3-butynyl)-4-methylpiperazine, (Ph3P)2PdCl2,
CuI, Ph3P, Et3N, NMP, 70 ꢁC, 4 h; (b) 1.7 equiv 3-butynol, (Ph3P)4Pd,
CuI, Et3N, dioxane, reflux, 1.5 h; (c) 1—3.0 equiv MsCl, 5.0 equiv
Et3N, DMF, THF, 0 ꢁC to room temperature, 1.25 h; 2—4–12 equiv
RR’NH, room temperature, 1–2 days.
The analog of 4 lacking the methoxy at C-5 of the aniline
headpiece, namely 16, was prepared as shown in Scheme
3. Reaction of 2,4-dichloroaniline with cyanoacetic acid
and 1,2-diisopropylcarbodiimide provided the cyanoace-
tamide derivative 13. Treatment of 13 with 3-iodo-4-
methoxyaniline and triethylorthoformate gave 14 which
upon treatment with phosphorus oxychloride cyclized
to the 3-quinolinecarbonitrile 15. Coupling of 15 with
1-(3-butynyl)-4-methylpiperazine under the standard
conditions provided 16. The finding that 16 was a weaker
Src inhibitor than 4 was expected based on the reduced
activity of related 3-quinolinecarbonitriles with a C-7
rakis(triphenylphosphine)palladium dichloride provided
3. In an analogous fashion, treatment of 2 with 1-(3-
butynyl)-4-methylpiperazine24 gave the ethylene-linked
analog 4. When tested in a Src enzymatic assay 3
and 4 had IC50s of 11 and 3.9 nM, respectively (see
Table 1). A larger difference in activity was seen in a cell
proliferation assay employing Src transformed rat
fibroblasts. In this assay 3 had an IC50 of 740 nM, while
that of 4 was 73 nM. Since the ethylene linker provided
more potent Src inhibition than the methylene linker,
additional ethylene analogs were targeted.
Table 1. Src enzyme and cell inhibitory activity of 7-ethynyl-3-quinolinecarbonitriles
RAr
HN
R
CN
N
R'
RAr
Compound32
R0
R
Src enzyme IC50 (nM)
SD
Src cell IC50 (nM)
SD
SKI-606
3.833
4.221
4.521
11
10014
12021
12021
740
1a
1b
3
N–Me-piperazine
OMe
OMe
OMe
OMe
OMe
H
2,4-di-Cl, 5-OMe
2,4-di-Cl, 5-OMe
2,4-di-Cl, 5-OMe
2,4-di-Cl, 5-OMe
2,4-di-Cl, 5-OMe
2,4-di-Cl, 5-OMe
2.9
230
3.8
8.4
16
4
CH2–N–Me-piperazine
CH2–NMe2
CH2-morpholine
3.9
4.9
0.50
0.21
0.07
0.78
4.2
73
250
6
7
4.1
5.4
190
520
8
CH2-piperazine
CH2–N–Me-piperazine
C-6 isomer of 10
120
610
370
31
10
12
16
21
24
25
28
1700
3900
430
230
8.7
28
CH2–N–Me-piperazine
CH2–N–Me-piperazine
CH2–N–Me-piperazine
CH2–N–Me-piperazine
OMe
OEt
OMe
H
2,4-di-Cl
0.90
0.17
5.0
2,4-di-Cl, 5-OMe
3,4,5-tri-OMe
3,4,5-tri-OMe
4.1
21
130
590
42
120
28
220
93
2100

1360
D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1358–1361
O
I
Cl
Cl
O
HO
I
Cl
Cl
O
a
NH2
NO2
HN
HN
17
CN
CN
a
Cl
Cl
O
O
N
Br
N
b
N
H
N
9
CN
10
N
18
Cl
Cl
Cl
O
O
I
Cl
O
HN
O
c
N
Cl
Cl
O
I
CN
Cl
Cl
N
N
H
N
H
N
CN
HN
O
HN
O
Br
CN
19
20
b
CN
Cl
Cl
O
N
d
N
HN
11
12
O
CN
Scheme 2. Reagents and conditions: (a) 1—1.5 equiv 3-butynol,
(Ph3P)4P, CuI, Et3N, DMF, 98 ꢁC, 3 h; 2—3.0 equiv MsCl, 5.0 equiv
Et3N, DMF, THF, 0 ꢁC to room temperature, 1.25 h; 3—6.0 equiv 1-
methylpiperazine, rt, overnight; (b) 4.0 equiv 1-(3-butynyl)-4-methyl-
piperazine (Ph3P)2PdCl2, CuI, Ph3P, Et3N, NMP, 70 ꢁC, 4 h.
N
N
N
21
Scheme 4. Reagents and conditions: (a) 1—6.0 equiv EtI, 3.7 equiv
K2CO3, DMF, 70 ꢁC, 3 h; 2—3.5 equiv Fe, 5.0 equiv NH4Cl, EtOH,
H2O, reflux, 1 h; (b) 2.6 equiv (EtO)3CH, i-PrOH, reflux, overnight; (c)
6.0 equiv POCl3, butyronitrile, reflux, overnight; (d) 1—1.5 equiv 3-
butynol, (Ph3P)4Pd, CuI, Et3N, dioxane, 95 ꢁC, 3 h; 2—3.0 equiv
MsCl, 5.0 equiv Et3N, DMF, THF, 0 ꢁC to room temperature, 1.25 h;
3—6.0 equiv 1-methylpiperazine, room temperature, overnight, fol-
lowed by 45 ꢁC, 7 h.
Cl
Cl
O
Cl
Cl
a
N
H
H2N
CN
13
O
I
b
NH2
Cl
Cl
Cl
Reaction of 17 with 2-cyano-N-(2,4-dichloro-5-meth-
oxyphenyl)acetamide, 18,22 and triethylorthoformate
resulted in 19 with subsequent phosphorus oxychloride
mediated ring closure leading to the formation of 20.
Coupling of 20 with 3-butynol, conversion to the mesy-
late, and displacement with 1-methylpiperazine provid-
ed 21. Interestingly, although 21 had very similar
activity to 4 in the Src enzyme assay, having an IC50
of 4.1 nM, this C-6 ethoxy analog was less potent than
4 in the Src cell assay, having an IC50 of 130 nM.
O
Cl
HN
O
c
O
I
CN
I
N
H
N
H
CN
N
14
15
Cl
Cl
d
HN
O
CN
N
Replacement of the C-4 (2,4-dichloro-5-methoxyphe-
nyl)amino group of 1b with a (3,4,5-trimethoxyphe-
nyl)amino group led to a sixfold reduction in Src
enzymatic activity and a threefold reduction in Src cell
activity.21 As depicted in Scheme 5, coupling of the
C-7 triflate derivative 2225 with 1-(3-butynyl)-4-methyl-
piperazine provided the (3,4,5-trimethoxyphenyl)amino
analog of 4, namely 24.
N
16
N
Scheme 3. Reagents and conditions: (a) 1.0 equiv cyanoacetic acid,
1.0 equiv 1,2-diisopropylcarbodiimide, THF, reflux, 15 min; (b)
1.0 equiv 3-iodo-4-methoxyaniline, 1.0 equiv (EtO)3CH, i-PrOH,
reflux, overnight; (c) 7.0 equiv POCl3, butyronitrile, reflux, overnight;
(d) 3.6 equiv 1-(3-butynyl)-4-methylpiperazine, (Ph3P)2PdCl2, CuI,
Ph3P, Et3N, NMP, 70 ꢁC, 4 h.
O
3-pyridinylethynyl substitutent upon removal of the C-5
methoxy group from the aniline.26
O
O
O
HN
N
O
HN
N
O
In a Src enzymatic assay, 4-[(2,4-dichlorophenyl)amino]-
6,7-diethoxy-3-quinolinecarbonitrile was about three-
fold more potent than the corresponding 6,7-dimethoxy
analog.28 The C-6 ethoxy analog of 4 was therefore tar-
geted. The prerequisite aniline 17 was prepared as
shown in Scheme 4. Alkylation of 2-iodo-4-nitrophe-
nol29 with ethyl iodine in the presence of K2CO3, fol-
lowed by reduction of the nitro group, provided 17.
X
CN
X
Y
CN
a
N
24: X = OMe
25: X = H
22: X = OMe, Y = OTf
23: X= H, Y = Br
N
Scheme 5. Reagents and conditions: (a) 3.0–4.0 equiv 1-(3-butynyl)-4-
methylpiperazine, (Ph3P)2PdCl2, CuI, Ph3P, Et3N, NMP, 70 ꢁC, 4 h.

D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1358–1361
1361
12. Shah, N. P.; Tran, C.; Lee, F. Y.; Chen, P.; Norris, D.;
Sawyers, C. L. Science 2004, 305, 399.
This change resulted in a fivefold reduction in Src enzy-
matic activity and an eightfold reduction in Src cell
activity. The des C-6 methoxy analog of 24 was pre-
pared by coupling of the C-7 bromo analog 2325 with
1-(3-butynyl)-4-methylpiperazine. This modification led
to a further decrease in activity with 25 having IC50s
of only 220 and 2100 nM for the inhibition of Src enzy-
matic and cell activity, respectively.
13. Lombardo, L. J.; Lee, F. Y.; Chen, P.; Norris, D.; Barrish,
J. C.; Behnia, K.; Castaneda, S.; Cornelius, L. A.; Das, J.;
Doweyko, A. M.; Fairchild, C.; Hunt, J. T.; Inigo, I.;
Johnston, K.; Kamath, A.; Kan, D.; Klei, H.; Marathe, P.;
Pang, S.; Peterson, R.; Pitt, S.; Schieven, G. L.; Schmidt,
R. J.; Tokarski, J.; Wen, M. L.; Wityak, J.; Borzilleri, R.
M. J. Med. Chem. 2004, 47, 6658.
14. 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.;
Discafani, C.; Etienne, C.; Gibbons, J.; Grod, J.; Lucas,
J.; Weber, J. M.; Boschelli, F. J. Med. Chem. 2001, 44,
3965.
15. 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.
16. Golas, J. M.; Lucas, J.; Etienne, C.; Golas, J.; Discafani,
C.; Sridharan, L.; Boghaert, E.; Arndt, K.; Ye, F.;
Boschelli, D. H.; Li, F.; Titsch, C.; Huselton, C.; Chau-
dhary, I.; Boschelli, F. Cancer Res. 2005, 65, 5358.
17. Eliceiri, B. P.; Paul, R.; Schwartzberg, P. L.; Hood, J. D.;
Leng, J.; Cheresh, D. A. Mol. Cell 1999, 4, 915.
18. 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.
When tested in a panel of kinases, 4 had an IC50 of
720 nM for the inhibition of EGFR and IC50s of greater
than 5 lM for the inhibition of AKT, CDK4, IGFR,
KDR, and PDK1. Pharmaceutical profiling showed 4
had
good
permeability
(PAMPA
value
of
6.8 · 10ꢀ6 cm/s) and while the solubility was low at neu-
tral pH (5 lg/mL), the solubility increased dramatically
at pH 4.5 to greater than 100 lg/mL. This increased sol-
ubility compared to that of 1b allowed for successful iv
formulation of 4 for testing in the rat stroke model. Pre-
liminary results of these in vivo studies with 4 (SKS-927)
were recently reported.30,31
Acknowledgments
19. Zaleska, M. M.; Liang, S.; Xiao, Y.; Gonzales, C.; Dilks,
D.; Ye, F.; Boschelli, D.; Boschelli, F.; Pong, K. 33rd
Annual Society for Neuroscience Conference, Abstract
741.14 2003.
20. Gonzales, C.; Xiao, Y.; Liang, S.; Dilks, D.; Ye, F.;
Boschelli, D.; Boschelli, F.; Pong, K.; Zaleska, M. M. 33rd
Annual Society for Neuroscience Conference, Abstract
741.15 2003.
We thank the Wyeth Chemical Technology department
for compound characterization and the pharmaceutical
profiling results. We also thank members of the Wyeth
Oncology department for the kinase profiling data and
Drs. Dennis Powell and Tarek Mansour for their
support.
21. Wu, B.; Barrios Sosa, A. C.; Boschelli, D. H.; Boschelli,
F.; Honores, E. E.; Golas, J. M.; Powell, D. W.; Wang, Y.
D. Bioorg. Med. Chem. Lett. 2006, 16, 3993.
References and notes
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Lucas, J.; Boschelli, F. J. Med. Chem. 2006, accepted for
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