New heterocyclic analogues of 4-(2-chloro-5-methoxyanilino)quinazolines as potent and selective c-Src kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.08.106

A series of 5,7-disubstituted quinazolines, bearing 4-heteroaryl substituents such as 2-pyridinylamine or 2-pyrazinylamine, has been synthetised and evaluated as c-Src kinase inhibitors. Highly potent inhibition, high selectivity and physical properties suitable for oral dosing were achieved within this series: 23d and 42 were identified as sub-0.1 μM inhibitors in a c-Src-driven cell proliferation assay and displayed adequate rat pharmacokinetics after oral administration.

Full text of DOI:10.1016/j.bmcl.2005.08.106

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5446–5449
New heterocyclic analogues of
4-(2-chloro-5-methoxyanilino)quinazolines as potent
and selective c-Src kinase inhibitors
b
a
b
a
Bernard Barlaam,a, Mike Fennell, Herve Germain, Tim Green, Laurent Hennequin,
*
´
Remy Morgentin, Annie Olivier, Patrick Ple, Michel Vautier and Gerard Costello
a
a
a
a
b
´
´
aAstraZeneca, Centre de Recherches, Z.I.S.E. La Pompelle B.P.1050, 51689 Reims, Cedex 2, France
bAstraZeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK
Received 21 July 2005; revised 23 August 2005; accepted 30 August 2005
Available online 3 October 2005
Abstract—A series of 5,7-disubstituted quinazolines, bearing 4-heteroaryl substituents such as 2-pyridinylamine or 2-pyrazinyl-
amine, has been synthetised and evaluated as c-Src kinase inhibitors. Highly potent inhibition, high selectivity and physical prop-
erties suitable for oral dosing were achieved within this series: 23d and 42 were identified as sub-0.1 lM inhibitors in a c-Src-driven
cell proliferation assay and displayed adequate rat pharmacokinetics after oral administration.
Ó 2005 Elsevier Ltd. All rights reserved.
The non-receptor tyrosine kinase c-Src is the best-under-
stood member of a family of closely related kinases, that
includes among its members c-Yes, Fyn and Lck. c-Src
is expressed at low levels in most cell types and, in
the absence of appropriate extracellular stimuli, is
maintained in an inactive conformation. c-Src gene
knock-out experiments in mice have shown that the only
phenotypic consequence is osteopetrosis, a defect in
osteoclast function.1
Evidence from the clinic also supports a link between
deregulated c-Src activity and increased invasive poten-
tial of tumour cells. In colon tumours, increased c-Src
kinase activity has been shown to correlate with tumour
progression, with the highest activity found in metastatic
tissue.2 Increased Src activity in colon tumours has also
been shown to be an indicator of poor prognosis.7
We have reported data on 6,7-substituted8 and
5,7-substituted9 4-anilinoquinazolines bearing a 2-chlo-
ro-5-methoxy aniline or a 4-amino-5-chloro-1,3-benzo-
dioxole (e.g., structures 1–5 pictured below in Fig. 1)
as potent and selective c-Src inhibitors. In this commu-
nication, we describe the synthesis of heterocyclic
In contrast to its highly regulated role in normal cells,
there is significant evidence demonstrating deregulated,
increased kinase activity of c-Src in several human tu-
mour types, most notably colon and breast tumours.2
Recent data suggest deregulated c-Src tyrosine kinase
activity is associated predominantly with adhesion and
cytoskeletal changes in tumour cells, ultimately resulting
in a change to a motile, invasive phenotype.3 Increased
c-Src tyrosine kinase activity results in break down of
E-cadherin-mediated epithelial cell/cell adhesion,4,5
which can be restored by Src inhibition.5 c-Src activity
is also known to be essential in the turnover of focal
adhesions,6 a critical cell motility component.
Cl
Cl
HN
O
HN
O
O
O
O
O
O
N
N
N
N
N
2
1
N
Cl
Cl
O
R
O
HN
N
O
O
HN
O
O
N
N
O
N
O
N
O
N
3
4
5
R= 4-tetrahydropyranyl
R= i Pr
Keywords: c-Src; Kinase; Inhibitor; 5,7-Disubstituted quinazoline.
*
Corresponding author. Tel.: +33 3 26 61 68 97; fax: +33 3 26 61 68
42; e-mail: bernard.barlaam2@astrazeneca.com
Figure 1. Stuctures of known Src inhibitors.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.106

B. Barlaam et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5446–5449
5447
H2N
N
OMe
Cl
N
Y
N
H2N
X
N
OMe
Y
a
a
NH2
NH2
MeO
MeO
6
24
7a X= Cl, Y= H
7b X= H, Y= Cl
25a Y= H
25b Y= Cl
OMe
Cl
O
O
O
N+ OMe
O
N
OMe
b
N
N
c
O
O
O
Cl
N
O
HN
N
Cl
Cl
b
e
NH
N
26
27
N
AcO
R
O
R
8
N+ OMe
+
N
OMe
N
OMe
9
R= OAc
10 R= OH
11 R= O(CH2)3Cl
c
d
d
12 X= O(CH2)3Cl
f
13 X= O(CH2)3-morpholine
Cl
Cl
Cl
NH2
NO2
NO2
Scheme 1. Reagents and conditions: (a) NCS (1 equiv), CHCl3, 0 °C
(7a: 73%, 7b: 11%); (b) PPh3/CCl4, ClCH2CH2Cl, 70 °C; (c) NH3/
MeOH, 0 °C (69% from 8); (d) DTAD, PPh3, Cl(CH2)3OH, CH2Cl2,
0 °C to rt, 31%; (e) NaHMDS (2 equiv), 7a (1 equiv), THF, 0 °C to rt,
74%; (f) morpholine, KI, DMA, 90 °C, 70%.
30
28
29
Scheme 3. Reagents and conditions: (a) 12 N HCl, H2O2 (1.5 equiv)
added dropwise, 70 °C (25a: 27%, 25b: 9%); (b) H2O2, Ac2O,
(CF3CO)2O, 0 °C; then 26, 0–80 °C, 53%; (c) H2SO4, HNO3; 0–80 °C
(28: 11%, 29: 9%); (d) Raney Ni, EtOAc/MeOH, 77%.
analogues of the 4-(2-chloro-5-methoxyanilino)quinazo-
lines 4 and 5 such as 4-(pyridinylamino)quinazolines and
discuss their biological activities.
Cl
X
Y
R
O
HN
O
N
31 X= N, Y= CH, R= 4-tetrahydropyranyl
32 X= N, Y= CH, R= i Pr
The synthesis of the 2-aminopyridine analogue of 4
starts from 610 (Scheme 1). Chlorination of 6 with
NCS gave two separable regioisomers 7a and 7b. The
previously described intermediate 89 was chlorinated
with triphenylphosphine/carbon tetrachloride to give
the chloroquinazoline 9, which afforded 10 after depro-
tection of the 7-acetoxy group with ammonia in metha-
nol. 10 was reacted with 3-chloropropanol under
Mitsunobu conditions to give 11, which was coupled11
with the anion of 7a to give 12. The chloro derivative
12 afforded 13 after heating with excess of morpholine
in the presence of KI.
O
N
O
N
33 X= CH, Y= N, R= 4-tetrahydropyranyl
34 X= CH, Y= N, R= i Pr
Figure 2. Other regioisomeric purid as Src inhibitors.
atoms of the previously described quinazolone 149 with
alkoxides. Treatment with sodium isopropoxide fol-
lowed by reaction with potassium 2,4-dimethoxybenzyl
oxide gave the quinazolone 16, which was chlorinated
with POCl3. Reaction of 17 with the anion of 7a fol-
lowed by functional group manipulation at C-7 gave
compounds 22a–d and 23a–d.
The other regioisomeric pyridines were prepared accord-
ing to Scheme 3. Chlorination of 2412 gave the expected
chloropyridine 25a and the dichloro adduct 25b. N-Ox-
idation of pyridine 26 with peroxytrifluoroacetic acid
gave 27, which was nitrated to give the 4-nitropyri-
dine-N-oxide 28 and some 4-nitropyridine 29. This mix-
ture was reduced to the expected 4-aminopyridine 30
with Raney nickel.
Although the 5-isopropoxy analogue 22a could have
been prepared by a route similar to 13, an alternative
procedure (Scheme 2) was devised based upon the
sequential displacement of the C-5 and C-7 fluorine
F
O
N
O
O
N
O
O
a
NH
b
NH
NH
F
DMBO
N
15
F
The two anilines 25a and 30 were transformed into 31–
34 (Fig. 2) via similar procedures13 as described in
Schemes 1 and 2.
14
16
OMe
N
Cl
N
O
HN
N
O
c
We also prepared the corresponding pyrazine and
pyrimidine as other heterocyclic replacements of the 2-
chloro-5-methoxyaniline. Chlorination of 3514 gave the
expected pyrazine 36a, along with the regioisomer 36b
and the dichloro adduct 36c. Reaction of 3715 with
ammonia in methanol followed by sodium methoxide
gave the expected pyrimidine 38 (Scheme 4).
d
Cl
N
N
DMBO
R
17
18 R= ODMB
19 R= OH
e
f
g
h
20 R= OCH2CH2CH2Cl
h
21 R= OCH2CH2Cl
22a-d R= OCH2CH2CH2-amine
23a-d R= OCH2CH2-amine
DMB: 2,4-dimethoxybenzyl
The corresponding quinazolines 42 and 43 were assem-
bled as described below (Scheme 5): reaction of 15 with
potassium piperazine ethoxide gave 39, which was
acetylated to give 40. Chlorination of 40 followed by
coupling with the anion of the corresponding aminohet-
erocycle gave the expected quinazolines 42 and 43.
Scheme 2. Reagents and conditions: (a) iPrOH, NaH, DMF, 5 °C to
rt, 71%; (b) 2,4-(MeO)BnOH (3 equiv), tBuOK (6 equiv), THF, reflux,
68%; (c) POCl3, NEtiPr2, ClCH2CH2Cl, 75 °C, 62%; (d) NaHMDS (2
equiv), 7a (1 equiv), THF, 0 °C to rt, 76%; (e) 20% TFA, CH2Cl2,
quant; (f) Cl(CH2)3Br, Cs2CO3, DMF, 60 °C, 55%; (g) ClCH2CH2Cl,
K2CO3, DMF, 60 °C, 55%; (h) amine, KI, DMA, 90 °C, 38–79%.

5448
B. Barlaam et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5446–5449
Table 2. Structure–activity relationship for the C–7 side chains
Compound Amine IC50 (lM)
Enzyme Cell inhibition
inhibition
Srca KDRb Src 3T3c A549d
H2N
OMe
N
H2N
X
OMe
Y
N
N
36a X= Cl, Y= H
36b X= H, Y= Cl
36c X= Cl, Y= Cl
a
N
35
Cl
Cl
N
Cl
H2N
Cl
N
OMe
b,c
N
N
38
37
22a
23a
22b
23b
22c
23c
22d
23d
Morpholine
<0.004 1.9
0.020 2.9
0.006 0.9
0.015 3.6
0.03
0.12
0.07
0.12
0.06
0.07
0.02
0.06
0.09
0.13
0.14
0.08
0.09
0.17
0.09
0.09
Morpholine
Pyrrolidine
Pyrrolidine
Scheme 4. Reagents and conditions: (a) NCS (1 equiv); CHCl3, rt,
(36a: 45%, b: 16%, c: 10%); (b) NH3, MeOH, rt, 76%; (c) MeONa,
MeOH, 70 °C, 97%.
Piperazine-N-Me <0.004 0.68
Piperazine-N-Me 0.007 0.57
Piperazine-N-Ac <0.004 2.7
O
O
N
Piperazine-N-Ac
0.010 6.3
O
O
N
b
NH
a
a,b,c,dSee details in Table 1.
NH
R
N
N
O
F
39 R= H
40 R= Ac
15
Table 3. Biological activity of diazines 42–43
Cl
X
Y
Compound
IC50 (lM)
Enzyme inhibition
O
HN
N
N
OMe
O
Cl
N
Cell inhibition
N
N
c
Srca
KDRb
Src 3T3c
A549d
AcN
N
AcN
N
O
O
42
43
0.008
0.015
2.8
15
0.07
0.28
0.14
0.27
42 X= N, Y=CH
43 X= CH, Y= N
41
a,b,c,dSee details in Table 1.
Scheme 5. Reagents and conditions: (a) piperazine-N-CH2CH2OH
(1.5 equiv), tBuOK (6 equiv), THF, reflux; (b) Ac2O, 44% from 15; (c)
PPh3, CCl4, ClCH2CH2Cl, 75 °C, 62%; (d) NaHMDS (2 equiv), 36a or
38 (1 equiv), THF, 0 °C to rt, 20–63%.
dine (see 34 vs 33), similar potency for the 2- or the
3-pyridine (see 22a vs 13 and 32 vs 31).
As shown in Tables 1–3, the regioisomeric pyridines dis-
play different ranges of potency versus Src and selectiv-
ity versus KDR. The 3-pyridine (31 and 32) shows good
potency at the Src level with significant activity against
KDR, while the 4-pyridine (33 and 34) shows poorer
potency for Src and yet significant activity for KDR.
On the other hand, the 2-pyridine (13 and 22a) is the
most Src potent heterocycle both at the enzymatic and
the cellular levels without significant activity for KDR.
Optimisation of the C-4 and C-5 substituents culminat-
ed with 22a, which is significantly more potent than the
parent aniline 5.
To modulate the physical properties of 22a, C-7 varia-
tions were explored. The C-7 propoxy side chains ap-
pear slightly more active than the ethoxy analogues at
the enzymatic level (22a–d vs 23a–d) and possibly at
the cellular level (Src 3T3) too. Both moderately and
highly basic side chains are tolerated at C-7. Overall,
improvements of physical properties were seen in the
2-pyridine series compared to the parent aniline series.
As an example, 23d is 8% free in rat plasma, whereas
the C-7 acetylpiperazineethoxy analogue of 5 is only
0.5% free; solubility in phosphate buffer pH 7.4 is
respectively 43 lM compared to 3 lM.
The isopropoxy group in C-5 of the quinazoline shows
better or at least equal potency compared to the 4-tetra-
hydropyranyloxy group: better potency for the 4-pyri-
Table 1. Biological activity of regioisomeric pyridines
Compound
IC50 (lM)
Enzyme inhibition
Cell inhibition
Replacement of the 2-pyridine with the corresponding
pyrazine (see 42) and pyrimidine (see 43) gives also po-
tent and selective c-Src kinase inhibitors both at the
enzymatic and the cellular levels. The pyrazine 42 reach-
es comparable potency with the 2-pyridine analog 23d.
b
Srca
KDR
Src 3T3c
A549d
4
0.01
0.4
0.2
0.08
0.4
5
0.04
0.005
<0.004
0.007
0.005
0.4
0.03
5.5
0.15
0.07
0.03
0.14
0.15
n.d.
0.35
13
22a
31
32
33
34
0.16
0.09
0.29
n.d.
n.d.
n.d.
1.9
0.03
0.03
0.08
Finally, 23d and 42 were evaluated in rat for pharmaco-
kinetic properties (rat cassette dosing of 5 compounds
orally, 2 mg/kg each). Both compounds gave good blood
levels (cmax of 0.31 and 0.42 lM, and AUC0-6h of 0.64
and 0.61 lM h, respectively for 23d and 42).
0.035
<0.002
These biological tests are described in Ref. 8. n.d. stands for Ônot
determinedÕ.
a Inhibition of c-Src enzyme activity.
b Inhibition of KDR enzyme activity.
c Inhibition of constitutively active Src-transfected 3T3 cell
proliferation.
In conclusion, we have shown that 5,7-disubstituted 4-
heteroarylaminoquinazolines such as 2-pyridinylamino-
or 2-pyrazinylamino- give good c-Src inhibition both
on the enzyme and in cells with a high degree of
d Inhibition of A549 cell migration.

B. Barlaam et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5446–5449
5449
9. (a) Hennequin, L. F.; Allen, J.; Costello, G.; Curwen, J.;
Fennell, M.; Green, T. P.; Jacobs, V.; Lambert-van der
selectivity associated with suitable physical properties
for oral dosing. Further evaluation of these compounds
for inhibiting c-Src in vivo is underway.
´
Brempt, C.; Morgentin, R.; Olivier, A.; Ple, P. A.
Structure–activity relationship and in vivo activity of a
novel series of C5-substituted anilinoquinazolines with
highly potent and selective inhibition of c-Src tyrosine
kinase activity; AACR-NCI-EORTC meeting, Boston,
Acknowledgments
´
Nov 17–21, 2003; Poster B193; (b) Hennequin L. F.; Ple,
P. A. PCT Int. Appl. (2001) WO2001094341.
We would like to acknowledge the excellent contribu-
tion of the following scientists to this work: for biology,
Vivien Jacobs, Karen Malbon, and Lindsay Millard; for
NMR, Christian Delvare; for physical chemistry, Del-
phine Dorison-Duval; for robotic synthesis, Patrice
Koza and Jacques Pelleter.
10. Bickel, A. F.; Wibaut, J. P. Recl. Trav. Chim. Pays-Bas
1946, 65, 65.
11. Representative procedure for the coupling of aminohet-
erocycles to chloroquinazolines (synthesis of 18): NaH-
MDS (5 ml, 1 M in THF, 5 mmol) was added dropwise to
an ice-cooled solution of 17 (981 mg, 2.5 mmol) and 7a
(400 mg, 2.5 mmol) in THF (13 ml). The mixture was
stirred at 0 °C for 5 min, then at rt for 3 h. After
completion of the reaction, acetic acid (5 mmol) was
added. After evaporation of the solvents, the residue was
partitioned between CH2Cl2 and water and the pH
adjusted to 7. The organic layer was extracted with
CH2Cl2 and dried over MgSO4. After evaporation of the
solvents, the residue was purified by chromatography on
silica gel (eluant: 2–5% MeOH in CH2Cl2). Evaporation of
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1
the fractions gave 18 (980 mg, 76%) as a solid: H NMR
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coupled to the chloroquinazoline. Attempts of coupling
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chloropropane in the presence of Cs2CO3 in DMA (70%)
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´
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