Investigation of the effect of varying the 4-anilino and 7-alkoxy groups of 3-quinolinecarbonitriles on the inhibition of Src kinase activity

Article abstract of DOI:10.1016/j.bmcl.2003.07.001

Several 7-alkoxy-4-anilino-3-quinolinecarbonitriles were synthesized and evaluated for Src kinase inhibitory activity. Optimal inhibition of both Src enzymatic and cellular activity was seen with analogues having a 2,4-dichloro-5-methoxyaniline group at C-4. Compound 18, which has a 1-methylpiperidinemethoxy group at C-7, showed in vivo activity in a xenograft model.

Full text of DOI:10.1016/j.bmcl.2003.07.001

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3797–3800
Investigation of the Effect of Varying the 4-Anilino and
7-Alkoxy Groups of 3-Quinolinecarbonitriles on the
Inhibition of Src Kinase Activity
Diane H. Boschelli,a,* Fei Ye,a Biqi Wu,a Yanong D. Wang,a Ana Carolina Barrios Sosa,a
Deanna Yaczko,a Dennis Powell,a Jennifer M. Golas,b Judy Lucasb 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 20 May 2003; revised 25 July 2003; accepted 29 July 2003
Abstract—Several 7-alkoxy-4-anilino-3-quinolinecarbonitriles were synthesized and evaluated for Src kinase inhibitory activity.
Optimal inhibition of both Src enzymatic and cellular activity was seen with analogues having a 2,4-dichloro-5-methoxyaniline
group at C-4. Compound 18, which has a 1-methylpiperidinemethoxy group at C-7, showed in vivo activity in a xenograft model.
# 2003 Elsevier Ltd. All rights reserved.
Signaling via protein tyrosine kinases (TKs), including
Src, is central to many cellular events.1 Src acts in the
regulation of many biological pathways and therefore
small molecule Src inhibitors may be useful for the
treatment of a variety of disease states, including cancer
and osteoporosis.2 The relevance of Src as a therapeutic
target is highlighted by the variety of known classes of
Src kinase inhibitors described in the literature including
pyrrolo[2,3-d]pyrimidines,3a pyrido[2,3-d]pyrimidines3b
and oxindoles.3c
The 4-(3,4,5-trimethoxyanilino) derivative 48 was pre-
pared from the known intermediate4 as shown in
Scheme 1. As was observed with 3, 4 showed decreased
Src cell activity compared to 2 (Table 1).
We previously reported that the 4-anilino-6,7-dime-
thoxy-3-quinolinecarbonitriles of formula 1a–c were
potent Src inhibitors and that these analogues had
greater activity than the corresponding 4-anilino-6,7-
dimethoxyquinazolines.4À6 For compounds 1a–c, super-
ior Src inhibitory activity was seen with 1c which has a
2,4-dichloro-5-methoxyaniline group at C-4.5 Addition
of various 3-(dialkylamino)-propoxy groups at C-7 of
1c increased the inhibition of Src cell activity, with the
optimal derivative being 2, SKI-606.6,7 While addition
of a 3-(4-methyl-1-piperazinyl)-propoxy substituent at
C-7 also increased the Src cell activity of 1b, this analo-
gue 3, was not as potent as 2.5
We next looked at the effect on Src inhibitory activity
upon retaining these three anilino groups at C-4 and
adding solubilizing groups other than 3-(4-methyl-1-
piperazinyl)-propoxy at C-7. A survey of various 4-ani-
linoquinazoline kinase inhibitors currently in clinical
trials suggested the use of two rather disparate groups.
*Corresponding author. Tel.:+1-845-602-3567; fax:+1-845-602-5561;
e-mail: bosched@wyeth.com
Scheme 1. (a) 1-Methylpiperazine, NaI, ethylene glycol dimethyl ether.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.07.001

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D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3797–3800
The EGFr inhibitor Tarceva, 5,9 has 2-methoxyethoxy
groups at both C-6 and C-7, while the KDR inhibitor,
ZD6474, 6,10 has a methoxy group at C-6 and a 1-
methylpiperidinemethoxy group at C-7.
Scheme 2. (a) 2,4-Dichloro-5-methoxyaniline, NaH, THF (for 8);
aniline, pyridine hydrochloride, 2-ethoxyethanol (for 9 and 10).
The analogues of 1a–c with 2-methoxyethoxy groups at
both C-6 and C-7 were prepared from the 4-Cl deriva-
tive 711 as shown in Scheme 2. Addition of 2,4-dichloro-
5-methoxyaniline in the presence of sodium hydride
provided 8. Analogues 9 and 10 were obtained by
treatment of 7 with the corresponding aniline in the
presence of pyridine hydrochloride. Of these three, the
best Src inhibitory activity was seen with the 2,4-
dichloro-5-methoxy analogue, 8. However, this com-
pound was about 2-fold less potent in the enzyme and
cell assays than 2.
Since we had earlier shown that large groups at C-6
could be detrimental to the inhibition of Src activity,5
the analogues of 8–10 with a methoxy group at C-6
instead of the 2-methoxyethoxy group were prepared
(Scheme 3). Starting from 11,4 addition of 2,4-dichloro-
5-methoxyaniline provided 12. The phenol group of 12
was alkylated with 2-bromoethyl methyl ether to pro-
vide 13. Since the overall yield by this route was low, an
alternative approach was examined. This route utilized
a Mitsunobu reaction of 11 with 2-methoxyethanol to
provide 14. Addition of 2,4-dichloro-5-methoxy aniline
to 14 resulted in 13. This route was then also used to
prepare analogues 15 and 16 via addition of 2,4-
dichloroaniline and 3,4,5-trimethoxyaniline respectively
to 14. As shown in Table 1, the analogues with a 6-OMe
Scheme 3. (a) 2,4-Dichloro-5-methoxyaniline, pyridine hydrochloride,
2-ethoxyethanol; (b) 2-bromoethyl methyl ether, K2CO3, DMF; (c)
triphenylphosphine,
THF; (d) aniline, pyridine hydrochloride, 2-ethoxyethanol.
diethylazodicarboxylate,
2-methoxyethanol,
group were roughly 2-fold more potent in the Src
enzyme assay than those with a 6-(2-methoxyethoxy)
group. For unknown reasons 16 showed better Src cell
activity than was expected.
Intermediate 11 was next used to prepare analogues of
1a–c with the solubilizing tail of ZD6474 at C-7, as
shown in Scheme 4. Mitsunobu reaction of 11 with 1-
methyl-4-piperidinemethanol provided 17. The 4-anilino
Table 1.
Compd
R1
R2
R3
n
Src enzyme12
IC50 nM(SD)
Src Cells12
IC 50 nM(SD)
2
3
4
8
2,4-diCl, 5-OMe
2,4-diCl
3,4,5-tri-OMe
2,4-diCl, 5-OMe
2,4-diCl
3,4,5-tri-OMe
2,4-diCl, 5-OMe
2,4-diCl
3,4,5-tri-OMe
2,4-diCl, 5-OMe
2,4-diCl
Me
Me
Me-piperazine
Me-piperazine
Me-piperazine
OMe
3
3
3
2
2
2
2
2
2
1
1
1
2
3
1.26
1006
8.75
10005
Me
CH2CH2OMe
CH2CH2OMe
CH2CH2OMe
5.1 (1.2)
2.8 (0.93)
12 (0.71)
25 (8.6)
1.5 (0.52)
4.7 (2.1)
14 (1.7)
2.0 (0)
6.6 (0.56)
8.3 (1.1)
2.4 (0.74)
1.4 (0.07)
1200 (57)
190 (74)
1200 (250)
1400 (400)
370 (211)
1400 (390)
330 (97)
220 (68)
1400 (210)
1600 (320)
230 (56)
100 (42)
9
OMe
OMe
OMe
OMe
OMe
10
13
15
16
18
19
20
23
24
Me
Me
Me
Me
Me
Me
Me
Me
Me-piperidine
Me-piperidine
Me-piperidine
Me-piperidine
Me-piperidine
3,4,5-tri-OMe
2,4-diCl, 5-OMe
2,4-diCl, 5-OMe

D. H. Boschelli et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3797–3800
3799
Scheme 5. (a) (1) N-tert-Butoxycarbonyl-4-(4-toluenesulphonyloxy-
methyl)piperidine, K2CO3, DMF; (2) HCO2H, 37% aqueous formal-
dehyde; (b) (1) fuming nitric acid, TFA, CH2Cl2; (2) H2/Pd/C, MeOH;
(c) (1) N,N-dimethylformamide dimethyl acetal; (2) n-BuLi, acetoni-
trile, THF; (3) phosphorous oxychloride.
oline-4-one with phosphorous oxychloride provided 17.
This route, while only one step shorter than that utilized
earlier, proved more suitable for larger scale synthesis.
Scheme 4. (a) Triphenylphosphine, diethylazodicarboxylate, 1-
methylpiperidine-(CH2)nOH, THF; (b) 3,4,5-trimethoxyaniline, pyri-
dine hydrochloride, 2-ethoxyethanol (for 20); aniline, NaH, THF (for
18, 19, 23 and 24).
Compound 18 was tested in a xenograft assay employ-
ing Src transformed rat fibroblasts.6 In a staged model,
where the tumors were implanted in nude mice prior to
compound administration, a 50 mg/kg ip dose of 18 for
5 days gave a statistically significant inhibition of tumor
growth (T/C=35% at day 5) as depicted in Figure 1.
derivatives 18–20 were prepared by the usual reaction
conditions. Again, when tested in the Src kinase and cell
proliferation assays, the best activity of the three ana-
logues was seen with 18, which contains the 2,4-
dichloro-5-methoxyaniline at C-4. However, 18 was
about 2-fold less potent than 2. To investigate the effect
of lengthening the chain, the ethoxy and propoxy
analogues of 18 were prepared as shown in Scheme 4.
Mitsunobu reaction of 11 with 1-methyl-4-piperidine-
ethanol or 1-methyl-4-piperidinepropanol provided 21
and 22, respectively. Addition of 2,4-dichloro-5-meth-
oxyaniline to 21 and 22 resulted in 23 and 24, both of
which had Src enzyme and cell activity in the same
range as 18 and 2.
Figure 1. Antitumor activity of 18 versus a Src-transformed fibroblast
xenograft. Mice (n=10) were treated with 18 for 5 days at 50 mg/kg ip
qd (-&-); Vehicle alone (-~-).
Compounds 8, 13, 18, and 24 had fairly comparable
activity in the Src cell assay and were chosen for a PK
study. Blood samples were drawn at 0.5, 4, and 8 h after
a 50 mg/kg ip dose administered to nude mice in a
Tween/Methocel vehicle.6 As shown in Table 2, the
group at C-7 had a significant effect on the observed
plasma concentrations. Of these four analogues, 18 had
superior plasma levels and was therefore selected for
further investigation.
We have shown here that 3-quinolinecarbonitriles
substituted at C-4 with 2,4-dichloroanilino, 3,4,5-tri-
methoxyanilino or 2,4-dichloro-5-methoxyanilino groups
inhibit Src enzymatic and cell activity. The best Src
enzymatic and cell inhibitory activity was seen with
those analogues with
a 4-(2,4-dichloro-5-methoxy-
In order to prepare additional amounts of 18, an alter-
nate route to key intermediate 17 was investigated. As
shown in Scheme 5, methyl vanillate was converted to
25 which contains the desired 1-methylpiperidine-
methoxy group. Nitration and subsequent reduction of
the nitro group provided 26.13 Formation of the ami-
dine of 26 followed by addition of the anion of
acetonitrile and then chlorination of the resultant quin-
anilino) group. In addition, it was demonstrated that 18
can inhibit Src activity in vivo. We are continuing to
investigate the biological properties of 18 and other 4-
(2,4-dichloro-5-methoxyanilino)-3-quinolinecarbonitriles
with additional alkoxy groups at C-7.
Acknowledgements
We thank the members of the Wyeth Discovery Analy-
tical Chemistry department for the spectral data and
elemental analyses.
Table 2.
Compd
0.5 h
4.0 h
8.0 h
8
1.7
0.37
4.5
1.2
0.23
2.9
0.20
0.14
1.6
13
18
24
References and Notes
0.40
0.50
0.50
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following a 50 mg/kg ip dose.

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