8-Anilinoimidazo[4,5-g]quinoline-7-carbonitriles as Src kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00524-3

A series of 8-anilinoimidazo[4,5-g]quinoline-7-carbonitriles was synthesized and evaluated as Src kinase inhibitors. Several aniline substituents were surveyed, as well as water-solubilizing groups at the C-2 and N-3 positions. Potent Src inhibitors were

Full text of DOI:10.1016/S0960-894X(02)00524-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2761–2765
8-Anilinoimidazo[4,5-g]quinoline-7-carbonitriles as
Src Kinase Inhibitors
Dan Berger,a,* Minu Dutia,a Dennis Powell,a Biqi Wu,a Allan Wissner,a
Frenel DeMorin,a Jennifer Weberb and Frank Boschellib
aChemical Sciences, Wyeth-Ayerst Research, Pearl River, NY 10965, USA
bDiscovery Oncology, Wyeth-Ayerst Research, Pearl River, NY 10965, USA
Received 16 April 2002; accepted 12 June 2002
Abstract—A series of 8-anilinoimidazo[4,5-g]quinoline-7-carbonitriles was synthesized and evaluated as Src kinase inhibitors. Sev-
eral aniline substituents were surveyed, as well as water-solubilizing groups at the C-2 and N-3 positions. Potent Src inhibitors were
identified, with N-3 providing the best position for an additional water-solubilizing group.
# 2002 Elsevier Science Ltd. All rights reserved.
Protein tyrosine kinases (TKs) catalyze the phosphor-
ylation of a tyrosine residue on substrate proteins, a
process important for regulation of cell growth and dif-
ferentiation. The Src family of TKs belong to a class of
nonreceptor TKs, which are present in the cytoplasm.1
Src itself participates in signaling pathways controlling
proliferation, migration and angiogenesis. Small mole-
cule inhibitors of Src have thus been considered to be
potentially useful agents for therapeutic intervention in
cancer2a,b and other disease states, such as osteopor-
osis3a,b and stroke.4
Thus, for example, structure 1 is a highly potent inhib-
itor of EGFr.14 An 8-anilinoimidazo[4,5-g]-quinazoline
(2) had been shown to be even more effective as an
EGFr kinase inhibitor.15 In this work it was shown that
water-solubilizing groups attached at the N-1or N-3
positions decreased enzyme activity (although these
compounds were still very potent EGFr inhibitors), but
the C-2 position was not similarly explored.
Several different structural types of compounds have been
reported to be inhibitors of Src family TKs, including 4-
anilinoquinazolines,5 pyrazolo[3,4-d]pyrimidines,6 pyr-
rolo[2,3-d]pyrimidines,7aÀe pyrido[2,3-d]pyrimidin-7(8H)-
ones,8 1,6-naphthyridin-2(1H)-ones,9 and aminopyrido-
[2,3-d]pyrimidin-7-yl ureas.10
In recent publications, it has been shown that 6,7-
dialkoxy substituted 4-anilino-3-quinolinecarbonitriles
are particularly potent EGFr,11 Src,12aÀc and MEK13a,b
kinase inhibitors. These compounds are known to bind
at the site normally occupied by ATP and parallel the
activity seen with 4-anilinoquinazolines, which also
have activity as tyrosine kinase inhibitors.
In this communication, we describe the activity of sub-
stituted 8-anilinoimidazo[4,5-g]quinoline-7-carbonitriles
(3) as Src kinase inhibitors. We chose to attach water-
solubilizing substituents at the C-2 and N-3 positions of
the 8-anilinoimidazo[4,5-g]quinoline-7-carbonitriles, since
it has been shown that attachment of these groups at the
C-7 position of the 4-anilinoquinoline-3-carbonitrile
series provides optimal activity.12aÀc
*Corresponding author. Tel.:+1-845-602-3435; fax:+1-845-602-5561;
e-mail: bergerd@wyeth.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00524-3

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D. Berger et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2761–2765
An optimized route to the 8-anilinoimidazo[4,5-g]quin-
oline-7-carbonitriles is outlined in Scheme 1. The reac-
substituted anilines12c in refluxing ethoxyethanol in the
presence of pyridine hydrochloride to provide target
compounds 11a–d.16
tion of
4 with diethyl ethoxymethylenemalonate,
followed by cyclization in refluxing Dowtherm provided
intermediate 5 as a single isomer. Hydrolysis to the
corresponding carboxylic acid 6 was readily accom-
plished in refluxing aqueous sodium hydroxide. Con-
version of the acid to an amide was accomplished using
CDI as an activating agent, followed by reaction with
ammonia. Cyanuric chloride was utilized to convert the
resulting amide to the 3-cyano substituent, providing 7
(compound 7 could also be prepared directly from the
reaction of 4 with ethyl (ethoxymethylene)cyanoacetate,
but this reaction provided two regioisomeric cyclization
products as an inseparable mixture). Protection of the
quinoline nitrogen with SEM chloride provided 8,
which more readily underwent a nucleophilic displace-
ment of the chloro substituent at C-7 and was more
soluble in common organic solvents than 7.
Scheme 2 outlines the chemistry utilized to synthesize
compounds with water-solubilizing groups attached via
a C-2 amino group. Cyanoquinolone 7 was converted to
the corresponding 4-chloro intermediate by reaction
with oxalyl chloride, followed by displacement with
substituted anilines to provide 12a,b. Azide displace-
ment of the chloro group, followed by catalytic hydro-
genation provided the diamino intermediates 13a,b.
Reaction of the diamine with morpholinoethylisothio-
cyanate, followed by cyclization with mercuric oxide,
afforded products 14a,b.
Scheme 3 outlines the chemistry utilized to synthesize a
compound with a water-solubilizing group attached at
C-2 via a methylene spacer. The reaction of 13a with
chloroacetyl chloride utilizing diethylaniline as a base
Displacement of the chloro substituent by sodium azide
at 60 ꢀC produced a 7-azido intermediate that was
readily reduced by catalytic hydrogenation to provide
the oxidatively unstable diamine 9 in excellent overall
yield. Treatment of 9 with refluxing formic acid simul-
taneously removed the SEM group and formed the imi-
dazole ring of 10 in one step. It was necessary to add at
least 4 equiv of imidazole to this reaction to scavenge
the formaldehyde generated by the deprotection. In the
absence of a scavenging nucleophile, significant amounts
of dimerized 10 were formed, bonded by a methylene
linkage. Treatment of 10 with oxalyl chloride provided
the corresponding chloride, which was displaced by
Scheme 2. (a) Oxalyl chloride; (b) aniline, pyridine–HCl, ethoxy-
ethanol; (c) NaN3; (d) H2, Pd/C; (e) morpholinoethylisothiocyanate;
(f) HgO.
Scheme 1. (a) Diethyl ethoxymethylenemalonate; (b) Dowtherm; (c)
NaOH; (d) (1) CDI/DMF, (2) NH3; (e) cyanuric chloride; (f) (1) NaH
(2) SEMCl; (g) NaN3; (h) H2, Pd/C; (i) HCO2H, imidazole; (j) oxalyl
chloride; (k) pyridine–HCl, aniline, ethoxyethanol.
Scheme 3. (a) Chloroacetyl chloride, diethylaniline; (b) acetic acid; (c)
morpholine.

D. Berger et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2761–2765
2763
provided mixtures of isomers 15a,b. Cyclization to the
imidazocyanoquinoline 16 was achieved by refluxing in
acetic acid. The reaction of 16 with morpholine pro-
vided target compound 17. Attempts to utilize similar
chemistry to attach water-solubilizing groups with
longer spacers were unsuccessful.
substituted at the 2, 4, and 5 positions, with only slightly
diminished activity for 2,5-substituted anilines. As
shown in Table 1, compounds 11a–c show a similar
trend, with 11b being the most potent compound with
an IC50 of 16 nM, and 11c being a little less potent
(IC50: 30 nM).17 For 4-anilino-3-quinolinecarbonitriles,
3,4,5-trimethoxyaniline substituents12b provide potent
Src kinase activity. In this series, the 3,4,5-trimethoxy
substituted 11d was less potent than 11a. Compounds
11a–c possessed moderate cell activity, the best being
11b, which inhibited Src transformed rat fibroblasts
with an IC50 of 2.8 mM.
Compounds with morpholine groups linked via N-3 were
synthesized as outlined in Scheme 4. The SEM-protected
cyanoquinolone 8 was reacted with substituted diamines
to provide 18. Catalytic hydrogenation over palladium on
carbon, followed by reaction in refluxing formic acid (with
added imidazole) cleanly provided 19. Chlorination by
oxalyl chloride, followed by reaction with substituted
anilines12c provided compounds 20a–e.
At the outset of this work, it was anticipated that the
presence of additional amine water-solubilizing sub-
stituents would improve cell activity, and possibly
enzyme activity as well. Compound 14b was moderately
active in the enzyme assay with an IC50 of 67 nM, while
the 3,4,5-trimethoxy substituted 14a had a significantly
higher IC50. However, 14a had comparable activity to
11d, indicating that the compounds with morpholine
groups attached via an ethylamino chain at C-2 retained
similar enzyme activity as compared to compounds
unsubstituted on the imidazole moiety. Unfortunately,
the water-solubilizing group did not provide 14a or 14b
with cell activity below 10 mM. Compound 17 was the
least active of the compounds having a 3,4,5-trimethox-
yaniline group at the C-4 position, likely indicating that
the short tether to the morpholine ring is not optimal.
It has been shown12c that the most potent 4-anilino-3-
quinolinecarbonitrile Src kinase inhibitors have anilines
Overall, the most potent group of compounds in
enzyme and cell assays was the N-3 substituted series:
20a–e. As was seen for 11a–d, compounds with 2,4,5-
trisubstituted anilines were the most potent (20c,d) with
IC50s of 29 and 9 nM, respectively, followed by the 2,5-
substituted analogues (20a,b). Compound 20e, posses-
sing a 3,4,5-trimethoxyaniline, was the least active of
this series, with activity comparable to 11d and 14a. It
appears that the 2-chloro (20a) or bromo (20d) sub-
stituents on the C-4 aniline provide about 3-fold better
activity than the 2-methyl substituted analogues (20c
Scheme 4. (a) Diamine; (b) H2, Pd/C; (c) HCO2H, imidazole; (d)
oxalyl chloride; (e) pyridine–HCl, ethoxyethanol.
Table 1. Inhibition of Src enzymatic and Src cellular activity for compounds 11a–d, 14a,b, 17 and 20a–e
Compd
R
X
Src (IC50, nMa)
Cells (IC50, mMa)
11a
11b
11c
11d
14a
14b
17
20a
20b
20c
20d
20e
2-Br, 4-Cl
2-Br, 4-Cl, 5-OMe
2-Cl, 5-OMe
3,4,5-trimethoxy
3,4,5-trimethoxy
2-Me, 5-OMe
3,4,5-trimethoxy
2-Cl, 5-OMe
2-Me, 5-OMe
2-Me, 4-Cl, 5-OMe
2-Br, 4-Cl, 5-OMe
3,4,5-trimethoxy
—
—
—
75
16
30
330
180
67
620
43
120
29
9
200
10.1
2.8
10.5
>10
>10
>10
>10
1.2
3.3
3.9
0.67
>10
—
2-NH(CH2)2
À
À
2-NH(CH2)2
À
2-CH2
3-(CH2)2
À
À
3-(CH2)2
3-(CH2)2
3-(CH2)2
3-(CH2)2
À
À
À
aThe IC50 values reported represent the means of at least two separate determinations with typical variations of less than 40% between replicate values.

2764
D. Berger et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2761–2765
and 20b). This differs somewhat from the SAR observed
within the 4-anilino-6,7-dialkoxy-3-quinolinecarboni-
triles, where the 2-methyl, chloro or bromo analogues
were essentially equivalent in activity.12c
5. Myers, M. R.; Setzer, N. N.; Spada, A. P.; Zulli, A. L.;
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J. Biol. Chem. 1996, 271, 695.
Compounds 20d and 20a were the most potent in the
cellular assay, with IC50s of 0.67 and 1.2 mM, respec-
tively. These compounds do appear to benefit from the
presence of the water-solubilizing morpholine group,
being more potent in cells than 11b and 11c (2.8 and
10.5 mM, respectively). The N-3 substituted 20b had
better cellular activity than the C-2 substituted 14b (3.3
mM and >10 mM, respectively). It is unclear whether
the poor cellular activity of 14b is due to the C-2 sub-
stitution, or whether the length and composition of the
tether group to morpholine is the major contributing
factor. However, it was not possible to synthesize C-2
ethyl-linked analogues due to their propensity to readily
undergo E2 elimination.
7. (a) Missbach, M.; Altmann, E.; Widler, L.; Susa, M.;
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Dykes, D. J.; Panek, R. L.; Lu, G. H.; Major, T. C.; Dahring,
T. K.; Hallak, H.; Bradford, L. A.; Showalter, H. D. H.;
Doherty, A. M. J. Med. Chem. 1998, 41, 3276.
9. Thompson, A. M.; Rewcastle, G. W.; Boushelle, S. L.;
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R. L.; Showalter, H. D. H.; Denny, W. A. J. Med. Chem.
2000, 43, 3134.
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11. Wissner, A.; Berger, D. M.; Boschelli, D. H.; Floyd, M. B.;
Greenberger, L. M.; Gruber, B. C.; Johnson, B. D.; Mamuya,
N.; Nilakantan, R.; Reich, M. F.; Shen, R.; Tsou, H. R.;
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Chem. 2000, 43, 3244.
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N.; Dutia, M.; Powell, D. W.; Wissner, A.; Arndt, K.; Weber,
J. M.; Boschelli, F. J. Med. Chem. 2001, 44, 822. (b) Wang,
D. Y.; 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. (c) 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.
In summary, as part of a continuing effort to discover
novel and potent Src kinase inhibitors, a series of 8-
anilinoimidazo[4,5-g]quinoline-7-carbonitriles was syn-
thesized and evaluated. A novel reaction sequence was
employed to synthesize the target compounds, the key
step utilizing an azide addition at C-7 to place a nitro-
gen atom at this position under mild reaction condi-
tions. These compounds showed promising activity in
enzyme and cell assays, comparable to initial lead com-
pounds within the 4-anilino-6,7-dialkoxy quinoline-3-
carbonitrile series. None of the compounds tested
showed significant activity against a panel of other
kinases, including EGFr, ErbB2, and MEK (IC50s>1
mM). Attachment of water-solubilizing groups at the C-
2 position proved to be a significant challenge, due to
the difficulty of adding electrophiles to the relatively
unreactive 4-anilino-6,7-diamino-3-cyanoquinoline inter-
mediates. The analogues synthesized did not have an
increased activity in cells. The N-3 substituted 20d had
the best overall enzyme and cell activity of this series,
thereby providing a potential direction for future opti-
mization of these compounds.
Acknowledgements
13. (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.
We acknowledge Dr. Diane H. Boschelli for the critical
reading of this manuscript and technical assistance.
References and Notes
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15. Rewcastle, G. W.; Palmer, B. D.; Bridges, A. J.; Sho-
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16. All final products were characterized by 1H NMR, MS
and elemental analysis. A representative example, compound
1
11d: HNMR (DMSO-d6+trifluoroacetic acid) d 3.73 (s, 3H)
3.81 (s, 6H) 6.90 (s, 2H) 8.20 (s, 1H) 8.85 (s, 1H) 9.13 (s, 1H)
9.15 (s, 1H) 11.40 (broad s, 1H). MS (ES) m/z 376.3 (M+H)+.

D. Berger et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2761–2765
2765
Analysis for C20H17N5O30.9H2O, Calcd: C 61.34; H 4.84; N
17.88, Found: C 61.03; H 4.82; N 17.76.
17. Compounds were tested in a modified format of the enzy-
matic assay previously reported.12aÀc Peptide was bound to the
streptavidin plate prior to the kinase reaction, and peptide
phosphorylation reaction was monitored by europium
fluorescence as recommended by the manufacturer (Per-
kin–Elmer). For cell assays, Costar ultra-low binding
plates were coated with Sigma-Cote to block residual cell
attachment.