Substituted 4-anilino-7-phenyl-3-quinolinecarbonitriles as Src kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00577-2

A series of substituted 4-anilino-7-phenyl-3-quinolinecarbonitriles has been prepared as Src kinase inhibitors. Optimal activity is observed with compounds that have basic amines attached via the para position of the 7-phenyl ring, and a hydrogen atom at the C-6 position. The best compounds are low nanomolar inhibitors of Src kinase, and have potent activity against Src-transformed fibroblast cells.

Full text of DOI:10.1016/S0960-894X(02)00577-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2989–2992
Substituted 4-Anilino-7-phenyl-3-quinolinecarbonitriles as Src
Kinase Inhibitors
Dan Berger,a,* Minu Dutia,a Dennis Powell,a Allan Wissner,a Frenel DeMorin,a
Yuri Raifeld,a Jennifer Weberb and Frank Boschellib
aChemical Sciences, Wyeth Research, Pearl River, NY 10965, USA
bDiscovery Oncology, Wyeth Research, Pearl River, NY 10965, USA
Received 2 April 2002; accepted 26 June 2002
Abstract—A series of substituted 4-anilino-7-phenyl-3-quinolinecarbonitriles has been prepared as Src kinase inhibitors. Optimal
activity is observed with compounds that have basic amines attached via the para position of the 7-phenyl ring, and a hydrogen
atom at the C-6 position. The best compounds are low nanomolar inhibitors of Src kinase, and have potent activity against Src-
transformed fibroblast cells.
# 2002 Elsevier Science Ltd. All rights reserved.
Protein tyrosine kinases (TKs) play an important role in
cell growth and differentiation. These enzymes catalyze
the transfer of a phosphate group from ATP to a tyrosine
residue on an appropriate substrate, thereby bringing
about cell signaling events. Several nonreceptor TKs
have been identified, among them the Src family of
cytoplasmic protein TKs.1 The TK Src has been impli-
cated in several disease states, including cancer,2a,b
osteoporosis,3a,b and stroke.4 Therefore, inhibition of
Src kinase could prove useful in the treatment of these
and other diseases. A number of Src family kinase inhib-
itors have been reported in the literature, including 4-
anilinoquinazolines,5 pyrazolo[3,4-d]pyrimidines,6 pyrrolo
[2,3-d]pyrimidines,7aꢀe 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
As part of our research effort to explore the optimal aryl
substitution at the C-7 position, we synthesized a series
of phenyl substituted compounds possessing different
water-solubilizing groups. With the phenyl ring spacer,
the attached water-solubilizing groups are oriented dif-
ferently as compared to those on the C-7 thiophene
substituted quinolinecarbonitriles. Compounds were
synthesized with these substituents at the ortho, meta,
and para positions. Finally, we also looked at the effect of
these 7-phenyl compounds with an additional methoxy
group attached at the C-6 position. This substitution
enhances the activity of compounds with 7-alkoxy sub-
stituents,12a and thus it was of interest to determine the
effects on the aryl-linked compounds.
A series of 4-anilino-3-quinolinecarbonitrile compounds
has been reported to be potent inhibitors of EGFr,11
Src12aꢀc and MEK13a,b kinases. While earlier synthetic
efforts were directed at 4-anilino-6,7-dialkoxy-3-quinoline-
carbonitrile Src kinase inhibitors, such as compound
1,12c recent efforts have focused on 4-anilino-7-thienyl-
3-quinolinecarbonitriles (e.g., 2) as potent Src kinase
inhibitors.14
The synthesis of the substituted phenyl intermediates 5a–c
is shown in Scheme 1. Reductive amination of cyclic
amines 4a,b with 3-bromobenzaldehyde or 4-bromo-
benzaldehyde provided the substituted benzylamines.
A two-step reaction sequence was utilized to synthesize
intermediates 7a–c, which have the amine substituents
*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)00577-2

2990
D. Berger et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2989–2992
converted to the corresponding arylboronic acids by
reaction with dipinacoldiboron using catalytic [1,10-
bis(diphenylphosphino)ferrocene]dichloropalladium(II)
complex with dichloromethane.15 Reaction of the
resulting boronic acids with triflate 13, or bromide 1414
in the presence of catalytic tetrakis triphenylphosphine
palladium(0) provided the final products.16
Scheme 1. (a) NaCNBH3, HOAc, EtOH.
Several analogues were synthesized via an alternative
reaction sequence, as outlined in Scheme 5. Inter-
mediate 14 was first reacted with the o-, m-, and p-sub-
stituted arylboronic acids 16a–c to provide 17a–c.
Reductive amination of intermediates 17a–c with the
appropriately substituted cyclic amines provided the
target compounds 15l–o.17
attached to the phenyl ring via a two carbon chain. The
phenylacetic acid derivatives 6a and 6b were first reacted
with amines 4a or 4b to provide the corresponding
amides, followed by reduction to form the amines 7a–c
(Scheme 2).
The intermediate 3-quinolinecarbonitrile 13 was pre-
pared as shown in Scheme 3. Methyl vanillate 8 was
benzylated and nitrated to provide 9. Treatment of 9
with boron trichloride selectively removed only the
benzyl group. The resulting phenol was reacted with
triflic anhydride, then with iron/ammonium chloride to
provide aniline 10. Compound 10 was reacted with
DMF-dimethylacetal to produce an intermediate ami-
dine, which was cyclized to 3-quinolinecarbonitrile 11
with the lithium anion of acetonitrile.
The biological activity of 15a–o is summarized in Table
1.18 Several trends are evident. Compounds possessing a
C-6 methoxy group (15a–e) are generally less active (up
to 5-fold) than the corresponding compounds with
hydrogen at C-6 (15f–15j). Since a C-6 methoxy group is
beneficial for activity in the 6,7-dialkoxy-3-quinoline-
carbonitrile series,12a we speculate that the decrease in
activity observed for this series may be due to the steric
effect of the C-6 methoxy group on the rotation of the
C-7 phenyl ring. Rotating the phenyl ring further from
the plane of the quinolinecarbonitrile core might cause
less favorable steric interactions in the active site of Src.
Compound 11 was converted to 12 using refluxing
phosphorus oxychloride. The reaction of 12 with 2,4-
dichloro-5-methoxyaniline12c in the presence of pyridine
hydrochloride provided intermediate 13.
Another clear trend can be observed with regard to the
position of attachment of the water-solubilizing group.
Compounds 15a–k were synthesized by the chemistry
outlined in Scheme 4. Intermediates 5a–c and 7a–c were
Scheme 2. (a) CH2Cl2, EDCI, DMA, 4a or 4b; (b) BH3.SMe2, THF.
Scheme 4. (a) PdCl2dppf.CH2Cl2, DMSO, KOAc, dipinacoldiboron;
(b) Pd(PPh3)4, DME, NaHCO3.
Scheme 3. (a) K2CO3, PhCH2Br, DMF; (b) HNO3, HOAc; (c) BCl3,
CH2Cl2; (d) (TfO) 2O, pyridine, CH2Cl2; (e) Fe, NH4Cl, H2O/MeOH;
(f) DMF-dimethylacetal; (g) n-BuLi /CH3CN, THF; (h) POCl3; (i) 2,4-
dichloro-5-methoxyaniline,12c pyridine hydrochloride, ethoxyethanol.
Scheme 5. (a) Pd(PPh3)4, DME, NaHCO3; (b) NaB(O2CCH3)3H,
amine, HOAc, DMF.

D. Berger et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2989–2992
Table 1. Inhibition of Src enzymatic and Src cellular activity for 15a–o
2991
Compd
Phenyl subst.
R1
n
X
Src IC50, nMa
Cells IC50, nMa
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
1
m
p
p
m
p
m
p
p
m
p
p
m
p
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
1
1
1
2O
2O
1
O
O
150
15
7.0
8100
1500
2000
NCH2CH3
74
11
,000
12
1300
O
28
3.3
3.0
29
3900
520
960
3200
1
1
O
NCH2CH3
O
2
2O
2NCH
1
1
1
1
14
1000
H
H
H
H
2CH3
NCH3
NCH3
3.7
14
3.8
4.7
4000
1.3
1900
2800
390
71
p
o
N(CH2)2OH
O
H
>10,000
100
aThe IC50 values reported represent the means of at least two separate determinations with typical variations of less than 40% between replicate
values.
The most active compounds are the p-substituted phenyl
compounds, with less activity for the m-substituted ana-
logues, and essentially no activity for o-substituted 15o.
This result correlates with the SAR of the 4-anilino-7-
thienyl-3-quinolinecarbonitrile series, in which it was
observed that the ‘linear’ analogues were more potent
than the corresponding ‘angular’compounds.14
In conclusion, a series of 7-phenyl substituted 3-quinoline-
carbonitriles was synthesized and evaluated as Src kinase
inhibitors. Several of these were very potent enzyme
inhibitors, with the overall most active compound in
cells being 15n, which possesses a hydroxyethyl-
piperazine water-solubilizing substituent.
Compounds with water-solubilizing groups attached via
different chain lengths (n=1 or 2) had comparable
activity against Src enzyme. However, none of the
compounds with n=2had submicromolar activity in the
Src dependent cell proliferation assay.12a,b Generally, the
different water-solubilizing groups provided similar
enzyme activity for compounds in which all other
structural features were identical. Overall, the most
potent group of compounds were the p-substituted
phenyl analogues, with C-6=H, and n=1: 15g,h and
15m,n, which only differ by the nature of the water-
solubilizing substituent. Of these, the most active
compound in cells was 15n, which was comparable to
compound 1 with an IC50 of 71 nM. These compounds
showed good selectivity for Src versus a panel non-Src
family kinases. For example, 15g was significantly less
active against EGFr (3000 nM), cyclin-dependent kinase
4 (10% inhibition at 10 mg/mL), and MEK (280 nM).
However, 15g and 15m did show some activity in an
assay measuring Fyn dependent cell proliferation12c
(1.17 and 0.83 mM, respectively). Therefore, these com-
pounds might be only marginally selective for Src over
the other Src family members. Work is in progress to
further enhance the potency and selectivity of these Src
kinase inhibitors.
Acknowledgements
We acknowledge Dr. Diane H. Boschelli for the critical
reading of this manuscript and technical assistance.
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extracted with 30 mL of ethyl acetate. The organic layers were
combined and washed with 4ꢂ40 mL water. After drying over
magnesium sulfate, removal of the solvents gave crude 4-[3-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl]morpho-
line as a dark oil.
A mixture of 110 mg (0.26 mmol) of 14, crude 4-[3-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl]morpholine and
45 mg (0.04 mmol) of tetrakis(triphenylphosphine) palla-
dium(0) was heated at reflux in 4 mL of ethylene glycol dime-
thyl ether and 2.5 mL of saturated aqueous sodium
bicarbonate for 2h. After cooling, the mixture was partitioned
between 50 mL of ethyl acetate and 40 mL of water. The lay-
ers were separated and the ethyl acetate layer was dried over
magnesium sulfate. Removal of the solvent in vacuo gave a
dark residue which was purified by flash silica gel chromato-
graphy eluting with a gradient of ethyl acetate to 95:5 ethyl
acetate/methanol, to provide 70 mg of 15f as a yellow solid,
mp 88–91 ꢁC; 1H NMR (DMSO-d6) d 10.05 (s, 1H), 8.61
(broad s, 2H), 8.19 (s, 1H), 8.08–7.97 (m, 1H), 7.85–7.72 (m,
3H), 7.51 (t, J=8 Hz, 1H), 7.43 (s, 1H), 7.41 (s, 1H), 3.87 (s,
3H), 3.61 (s, 2H), 3.59 (s, 4H), 2.42 (s, 4H). MS (ES) m/z 519.1,
521.0 (M+1). Analysis for C28H24Cl2N4O20.5 EtOAc: Calcd:
C, 63.95; H, 5.01; N, 9.94. Found: C, 63.64; H 4.93; N, 9.97.
17. A typical preparation of target compounds 15l–o is illu-
strated by the following procedure for 15m: By the procedure
of the final step utilized to synthesize 15f, 14 (3.00 g, 7.1 mmol)
was reacted with 4-formylphenyl boronic acid (16b) (1.27 g,
8.5 mmol) and tetrakis(triphenylphosphene) palladium (0)
(0.30 g, 0.25 mmol) in a mixture of ethylene glycol dimethyl
ether (20 mL) and a saturated aqueous solution of sodium
bicarbonate (20 mL) to provide 3.00 g (94%) of 17b as a yel-
low solid, mp 248–251 ꢁC; 1H NMR (DMSO-d6) d 10.11 (s,
1H), 10.10 (s, 1H), 8.67 (t, J=9 Hz, 2H), 8.32 (s, 1H), 8.16 (m,
3H), 8.09 (s, 1H), 7.99 (s, 1H), 7.78 (s, 1H), 7.43 (s, 1H), 3.87
(s, 3H); MS (ES) m/z 450.0 (M+1). Analysis for
C24H15Cl2N3O2- 0.5 CH2Cl2: calcd: C, 56.91; H, 5.20; N,
10.30. Found: C, 57.20; H, 5.10; N, 9.91.
4-Methylpiperidine (63 mg, 0.63 mmol) was added to a sus-
pension of 17b (200 mg, 0.44 mmol) in 4 mL of methylene
chloride and 1 mL of N,N-dimethylformamide. The mixture
was cooled to 0 ꢁC and sodium triacetoxyborohydride (500
mg, 2.36 mmol) was added followed by a drop of acetic acid.
The reaction mixture was allowed to warm to room tempera-
ture and stirred at room temperature for 2h to give a yellow
solution. The reaction was quenched by the addition of water
and partitioned between saturated sodium bicarbonate and
methylene chloride. The organic layer was dried over sodium
sulfate, filtered and concentrated in vacuo. The residue was
purified by flash chromatography, eluting with 10% methanol
in methylene chloride to provide 183 mg (82%) of 15m as an
off-white solid, mp 120–123 ꢁC; H NMR (DMSO-d6) d 10.05
1
15. Ishiyama, T.; Murata, M.; Miyoura, N. J. Org. Chem.
1995, 60, 7508.
(broad s, 1H), 8.58 (broad s, 2H), 8.20–8.11 (m, 1H), 8.06–7.97
(m, 1H), 7.90–7.78 (m, 2H), 7.77–7.63 (m, 1H), 7.47 (dd,
J=3.3, 8.1 Hz, 2H), 7.47–7.37 (m, 1H), 3.86 (s, 3H), 3.55 (s,
2H), 2.53–2.36 (m, 8H), 2.24 (s, 3H); MS (ES) m/z 531.2, 532.0
(M+1). Analysis for C29H27Cl2N5O1.5 CH2Cl2: Calcd: C,
55.52; H, 4.58; N, 10.61. Found: C, 55.17; H, 4.50; N, 10.78.
18. Compounds were tested in a modified format of the enzy-
matic assay previously reported.12aꢀc Peptide was bound to the
streptavidin plate prior to the kinase reaction, and peptide
phosphorylation reaction was monitored by europium fluo-
rescence as recommended by the manufacturer (Perkin–
Elmer). For cell assays, Costar ultra-low binding plates were
coated with Sigma-Cote to block residual cell attachment.
16. A typical preparation of target compounds 15a–k is illu-
strated by the following procedure for 15f: To a dry flask
under a nitrogen atmosphere was added 200 mg (0.78 mmol)
of 5a, 0.218 g (0.86 mmol) of bis(pinacolato)diboron, 230 mg
(2.34 mmol) of potassium acetate, 5 mL of dimethylsulfoxide
and 32mg (0.04 mmol) of [1,1 0-bis(diphenylphos-
phino)ferrocene] dichloropalladium(II), complex with di-
chloromethane. The reaction mixture was heated at 80 ꢁC for 2
h. After cooling, the mixture was partitioned between 20 mL
of toluene, 40 mL of ethyl acetate and 40 mL of water. The
layers were separated and the aqueous layer was further