Novel 4-anilinoquinazolines with C-7 basic side chains: Design and structure activity relationship of a series of potent, orally active, VEGF receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1021/jm011022e

We have previously shown that 4-anilinoquinazolines can be potent inhibitors of vascular endothelial growth factor (VEGF) receptor (Flt-1 and KDR) tyrosine kinase activity. A novel subseries of 4-anilinoquinazolines that possess basic side chains at the C-7 position of the quinazoline nucleus have been synthesized. This subseries contains potent, nanomolar inhibitors of KDR (median IC₅₀ 0.02 μM, range 0.001-0.04 μM), which are comparatively less potent vs Flt-1 tyrosine kinase (median IC₅₀ 0.55 μM, range 0.02-1.6 μM). The compounds also retain some inhibitory activity against the tyrosine kinase associated to the endothelial growth factor receptor (EGFR) (median IC₅₀ 0.2 μM, range 0.075-0.8 μM) but demonstrate selectivity vs that associated to the FGF receptor 1 (median IC₅₀ 2.5 μM, range 0.9-19 μM). This selectivity profile is also evident in a growth factor-stimulated human endothelial cell (HUVEC) proliferation assay (i.e., inhibition of VEGF > EGF > FGF), with inhibition of VEGF-induced proliferation being achieved at nanomolar concentrations (median IC₅₀ 0.06 μM). Further examination of compound 2 (ZD6474) in recombinant enzyme assays revealed excellent selectivity for the inhibition of KDR tyrosine kinase (IC₅₀ 0.04 μM) vs the kinase activity of erbB2, MEK, CDK-2, Tie-2, IGFR-1R, PDK, PDGFRβ, and AKT (IC₅₀ range: 1.1 to > 100 μM). Anilinoquinazolines possessing basic C-7 side chains exhibited markedly improved aqueous solubility over previously described anilinoquinazolines possessing neutral C-7 side chains (up to 500-fold improvement at pH 7.4). In addition, aqueous solubility of the neutral fraction present at pH 7.4 of the basic subseries of anilinoquinazoline proved to be higher than that of the neutral analogue 1 (ZD4190). Oral administration of representative compounds to mice (50 mg/kg) produced plasma levels between 0.2 and 3 μM at 24 h after dosing. Our development candidate 2 demonstrated a very attractive in vitro profile combined with excellent solubility (330 μM at pH 7.4) and good oral bioavailability in rat and dog (> 80 and > 50%, respectively). This compound demonstrated highly significant, dose-dependent, antitumor activity in athymic mice. Once daily oral administration of 100 mg/kg of compound 2 for 21 days inhibited the growth of established Calu-6 lung carcinoma xenografts by 79% (P < 0.001, Mann Whitney rank sum test), and substantial inhibition (36%, P < 0.02) was evident with 12.5 mg/kg/day.

Full text of DOI:10.1021/jm011022e

1300
J . Med. Chem. 2002, 45, 1300-1312
Novel 4-An ilin oqu in a zolin es w ith C-7 Ba sic Sid e Ch a in s: Design a n d Str u ctu r e
Activity Rela tion sh ip of a Ser ies of P oten t, Or a lly Active, VEGF Recep tor
Tyr osin e Kin a se In h ibitor s
Laurent F. Hennequin,*,† Elaine S. E. Stokes,‡ Andrew P. Thomas,‡ Craig J ohnstone,‡ Patrick A. Ple´,†
Donald J . Ogilvie,‡ Michael Dukes,‡ Stephen R. Wedge,‡ J ane Kendrew,‡ and J on O. Curwen‡
AstraZeneca, Centre de Recherches, Z.I. La Pompelle, B.P. 1050, Chemin de Vrilly, 51689 Reims, Cedex 2, France, and
AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
Received August 15, 2001
We have previously shown that 4-anilinoquinazolines can be potent inhibitors of vascular
endothelial growth factor (VEGF) receptor (Flt-1 and KDR) tyrosine kinase activity. A novel
subseries of 4-anilinoquinazolines that possess basic side chains at the C-7 position of the
quinazoline nucleus have been synthesized. This subseries contains potent, nanomolar inhibitors
of KDR (median IC50 0.02 µM, range 0.001-0.04 µM), which are comparatively less potent vs
Flt-1 tyrosine kinase (median IC50 0.55 µM, range 0.02-1.6 µM). The compounds also retain
some inhibitory activity against the tyrosine kinase associated to the endothelial growth factor
receptor (EGFR) (median IC50 0.2 µM, range 0.075-0.8 µM) but demonstrate selectivity vs
that associated to the FGF receptor 1 (median IC50 2.5 µM, range 0.9-19 µM). This selectivity
profile is also evident in a growth factor-stimulated human endothelial cell (HUVEC)
proliferation assay (i.e., inhibition of VEGF > EGF > FGF), with inhibition of VEGF-induced
proliferation being achieved at nanomolar concentrations (median IC50 0.06 µM). Further
examination of compound 2 (ZD6474) in recombinant enzyme assays revealed excellent
selectivity for the inhibition of KDR tyrosine kinase (IC50 0.04 µM) vs the kinase activity of
erbB2, MEK, CDK-2, Tie-2, IGFR-1R, PDK, PDGFRâ, and AKT (IC50 range: 1.1 to >100 µM).
Anilinoquinazolines possessing basic C-7 side chains exhibited markedly improved aqueous
solubility over previously described anilinoquinazolines possessing neutral C-7 side chains (up
to 500-fold improvement at pH 7.4). In addition, aqueous solubility of the neutral fraction
present at pH 7.4 of the basic subseries of anilinoquinazoline proved to be higher than that of
the neutral analogue 1 (ZD4190). Oral administration of representative compounds to mice
(50 mg/kg) produced plasma levels between 0.2 and 3 µM at 24 h after dosing. Our development
candidate 2 demonstrated a very attractive in vitro profile combined with excellent solubility
(330 µM at pH 7.4) and good oral bioavailability in rat and dog (>80 and >50%, respectively).
This compound demonstrated highly significant, dose-dependent, antitumor activity in athymic
mice. Once daily oral administration of 100 mg/kg of compound 2 for 21 days inhibited the
growth of established Calu-6 lung carcinoma xenografts by 79% (P < 0.001, Mann Whitney
rank sum test), and substantial inhibition (36%, P < 0.02) was evident with 12.5 mg/kg/day.
In tr od u ction
Several approaches, currently being evaluated in the
clinic, are directed at blocking either the interaction of
VEGF with its receptors (VEGF-Ab,10 VEGF-receptor-
Ab)11,12 or the intracellular signaling process that follows
receptor activation (RTK inhibitors).
Angiogenesis is an important component of certain
normal physiological processes such as embryogenesis,
wound healing, and the female reproductive cycle but
also contributes to some pathological disorders and in
particular to tumor growth.1,2 VEGF-A (vascular endot-
helial growth factor A) has been identified as a key
factor promoting neovascularization of many tumors.3-7
VEGF activates endothelial cells by signaling through
two high affinity receptors, the fms-like tyrosine kinase
receptor, Flt-1, and the kinase insert domain-containing
receptor, KDR.8,9 These signaling responses are criti-
cally dependent upon receptor dimerization and activa-
tion of intrinsic receptor tyrosine kinase (RTK) activity.
Three orally bioavailable small molecules that can
interfere with VEGF RTK activity, the phthalazine
CGP79787/PTK787 (Flt-1 ∼ KDR),13,14 the indolinone
SU666815,16 (predominantly a c-kit, PDGFRâ inhibi-
tor; PDGFR . KDR), and the anilinoquinazoline 2
(ZD6474),17 are being examined in patients with several
types of cancer (Figure 1). The discovery and properties
of compound 2 are described in this paper.
We recently reported18,19 that 4-anilinoquinazolines
can be potent nanomolar inhibitors of the Flt-1 and KDR
enzymes. We showed that both of these enzymes are
quite tolerant with regard to the nature and size of the
substituent present at the C-7 position of the quinazo-
line nucleus (Figure 2). In this paper, we focus on the
* Corresponding author. Current address: AstraZeneca Pharma-
ceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, United
Kingdom. Tel: (44)1625 230836. Fax: (44)1625 513910. E-mail:
laurent.hennequin@astrazeneca.com.
† AstraZeneca, Centre de Recherches.
‡ AstraZeneca Pharmaceuticals.
10.1021/jm011022e CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/19/2002

4-Anilinoquinazolines with C-7 Basic Side Chains
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1301
F igu r e 1.
3. The N-protected 4-piperidine methanol side chains
52 and 5320 or their tosylate-activated counterparts 5421
and 5522 were first coupled at the C-7 position of the
N-3-POM-protected quinazolone 5118 to give 56 and 57.
Subsequent selective deprotection of the t-Boc group of
56 and 57 led to 58 and 59. N-methylation of the
piperidine nitrogen using formaldehyde under reducing
conditions led, respectively, to 60 and 61. The quinazolo-
ne moiety of 60 and 61 was then unmasked using
ammonia in methanol to give 62 and 63, and the C-4
position of these quinazolones was subsequently chlo-
rinated using thionyl chloride and DMF to give 64 and
65 (Scheme 3). Reaction of 64 and 65 with a range of
aniline under either acid-catalyzed or basic conditions
as described previously led to 2, 15-20, and 26 (Scheme
3). Compound 27 was obtained directly from 28 by
methylating the piperidine nitrogen under Eschweiler-
Clarke conditions (HCOOH, HCHO, reflux; Scheme 4).
The anilinoquinazolines 4 and 5 were synthesized from
the pivotal intermediates 3918 and 40.18 Alkylation of
the C-7 position of 3918 and 4018 with 1-bromo-3-
chloropropane gave 66 and 67, respectively. Subsequent
nucleophilic displacement of the aliphatic chlorine of 66
and 67 by heating in an excess of N-methylpiperazine
led to the expected anilinoquinazolines (Scheme 5).
F igu r e 2.
physicochemical properties, in vitro profile, and in vivo
activity of a series of anilinoquinazolines that possess
basic side chains at the C-7 position of the quinazoline
nucleus (Figure 2).
Ch em istr y
The compounds described in Table 1 were usually
prepared by two main routes described in Schemes 1-3.
The first route involves the coupling of 7-hydroxy
anilinoquinazoline 39,18 40,18 44,18 and 41-43 with a
range of alcohols R2-H (see Table 1) under Mitsunobu
conditions. The second route is based on displacing the
chlorine atom of the C-4-activated quinazoline 64 and
65 (Scheme 3) with different anilines (see Table 1).
The 7-benzyloxy-4-chloro-6-methoxyquinazoline (32)18
was reacted with anilines under acid catalysis in a protic
solvent in the case of anilines with high or moderate
nucleophilic character (2′-F, 4′-Cl; 2′-F, 4′-Br; 2′-F, 4′-
Me). In the case of weakly nucleophilic anilines (2′,6′-
F2, 4′-Cl; 2′,6′-F2, 4′-Br; 2′-F, 4′-CN), the sodium salts
of the anilines were preformed in situ (NaH, dimethyl-
formamide (DMF)) and then reacted with 3218 to give
the corresponding C-7-benzyloxyanilinoquinazolines 33,18
34,18 38,18 and 35-37 (Scheme 1). Deprotection of the
C-7-benzyl moiety was subsequently achieved using
trifluoroacetyl (TFA) and led to the key intermediate
7-hydroxy-4-anilinoquinazolines 39,18 40,18 44,18 and
41-43 (Scheme 1). Reaction of 39-43 with a range of
alcohols under Mitsunobu conditions using either di-
ethyl or di-tert-butyl azodicarboxylate or the more
hydrophilic 1,1′-(azodicarbonyl)dipiperidine led to 6,
10-12, and 29-31 (Scheme 2). Using similar reaction
conditions, the N-protected piperidines 5220 and 5320
were coupled with 39-41, 43, and 4418 to give 21 and
46-49. Compound 45 was obtained by coupling the
7-hydroxy-anilinoquinazoline 4018 with the tosylate
piperidine 5421 under alkylation conditions (K2CO3,
DMF). The t-BOC protecting group of 45-49 was
subsequently removed by treatment with TFA to release
the basic piperidine nitrogen (Scheme 2). Compounds
2, 15-20, and 26 were prepared as described in Scheme
Resu lts a n d Discu ssion
For clarity of discussion, data on only a limited,
representative set of compounds are presented in Table
1 and used to describe the structure-activity relation-
ship (SAR). When some trends are exemplified by single
pairs of compounds, it is to be understood that more
examples23-25 exist to support the SAR described below.
Where cited, selectivity ratios refer to the quotients of
the respective IC50 values for enzyme (or cell growth)
inhibition.
i. In h ib it ion of R ecep t or Kin a se Act ivit y in
Vitr o. As previously reported,18 one of the preferred
patterns of substitution of the aniline ring to optimize
inhibition of the target enzymes consists of a small
lipophilic, electron-deficient substituent at the C-2′
position combined with a larger lipophilic, electron-
withdrawing atom at the C-4′ position such as chlorine
or bromine. The same patterns were apparent with basic
C-7 side chains. As shown by comparison of the 4′-cyano
and 4′-bromo derivatives 12 and 11 (Table 1), replace-
ment of hydrophilic electron-withdrawing substituents
that possess marked π-character, by more lipophilic
substituents with moderate electron-withdrawing char-

1302 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Hennequin et al.

4-Anilinoquinazolines with C-7 Basic Side Chains
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1303
Sch em e 1a,b
a
b
Reagents: (a) R1-Ar-NH2/iPrOH/HCl/reflux (procedure A or G) or R1-Ar-NH2/NaH/DMF (procedure H). TFA (procedure L or J ).
Sch em e 2a
a
Reagents: (a) DMF/K2CO3 (procedure M). (b) i. Ph3P/DEAD; ii. TFA (procedure I). (c) R2OH (see Table 1)/n-Bu3P/ADDP (procedure
C) or Ph3P/DEAD (procedure E or F). (d) 1-(3-Chloropropyl)pyrrolidine/DMF/K2CO3 (procedure D). (e) TFA (procedure J or L).
acter, led to marked improvement in both Flt-1 and
KDR inhibition (up to 350-fold). A previous development
candidate (1, ZD4190;18 Table 1) possessed one of the
preferred substitution patterns (C-2′-F, C-4′-Br) and
proved to be a very potent inhibitor of KDR (Table 1).
It also inhibited, but to a lesser extent, the TK associ-
ated with the endothelial growth factor receptor (EGFR)
(KDR:EGFR selectivity ratio ∼ 13). This pattern of
substitution also led in the C-7 neutral series to high
selectivity for KDR against FGFR-1 (fibroblast growth
factor receptor TK) (1 and 3: selectivity ratios > 1000;
Table 1). Introduction of a hydroxyl substituent at the
meta position of the aniline led to an improvement in
Flt-1 inhibition by 20-100-fold (comparison of 15-18)
while improving KDR inhibition by only 2-4-fold.
Introduction of an additional fluorine atom at the C-6′
position of the aniline in general increased the activity
against Flt-1 over KDR (2-7-fold; comparison of 19, 20,
and 24 and 15, 2, and 21).
The 1,2,3-triazole-ethoxy side chain present at the C-7
position of 1 is a typical neutral side chain commonly
used in medicinal chemistry. This side chain confers on
1 high and sustained plasma levels in mouse following
oral administration (13 µM at 24 h following 100 mg/
kg).18,19 Such plasma levels combined with nanomolar
inhibition of growth factor-stimulated endothelial cell
proliferation led to profound inhibition of growth of
human tumor xenografts in athymic mice with once
daily chronic oral administration.18,19 However, the
neutral nature of the 1,2,3-triazole-ethoxy side chain
conferred on 1 suboptimal physicochemical properties
that we believe were principally responsible for variable
pharmacokinetic properties across species (see the sec-
tions on physicochemical properties and PK properties,
Tables 3-5). We therefore turned our attention to C-7
side chains that might provide VEGF RTK inhibitors
with improved physical properties.
Introduction of nitrogen-containing, basic residues in

1304 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Hennequin et al.
Sch em e 3a
a
Reagents: (a) 54: Ph3P/CH2Cl2/DEAD. (b) 55: K2CO3/DMF. (c) TFA. (d) MeOH/THF/HCHO/NaBH3CN. (e) HCHO/NaB(OAc)3/AcOH.
(f) MeOH/NH3. (g) SOCl2/DMF. (h) R1-Ar-NH2/iPrOH/HCl (procedure A or G). (i) NaH/DMF (procedure H).
Sch em e 4a
the C-7 side chain covering a range of pKa’s from 7 to
9.5 (Table 3), as illustrated by the morpholines (7 and
8),18 the pyrrolidine (9), and the piperidine (30, 2),
retained an excellent level of KDR inhibition (median
IC50 0.02 µM, range 0.001-0.03 µM; Table 1). The KDR
enzyme proved to be consistently more sensitive than
Flt-1 (median IC50 0.55 µM, range 0.02-1.6 µM) with
Flt-1:KDR selectivity ratios ranging from 5 to 500. The
most marked separation was observed with the cyclic
N-methylpiperidine, and in particular piperidine meth-
oxy derivative 30, due to a slightly weaker activity
against Flt-1 (comparison of 15 and 2 with 9 and 24).
C-7 side chains containing nitrogens that can be pro-
tonated or hydrated tend to be slightly more potent
against Flt-1 (comparison of 1 and 3 with 7, 4, 9, and
24). Basic heteroaromatic nuclei, as illustrated by the
pyridine-containing derivative 13,18 were also potent
inhibitors of KDR.
a
HCOOH, HCHO, reflux (procedure K).

4-Anilinoquinazolines with C-7 Basic Side Chains
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1305
Ta ble 2. Selectivity Profile in a Panel of Kinase Enzymes of Compounds 1 and 2
enzyme inhibitiona (IC50 µMb)
no.
Flt-1
0.7 ( 0.06 0.03 ( 0.004 0.4 ( 0.1 >100
1.6 ( 0.4 0.04 ( 0.01 0.5 ( 0.1 2.5 ( 1.2
KDR
EGFR
Tie-2
FGFR-1
>100
3.6 ( 0.9 1.1 ( 0.3
PDGFRâ erbB2 MEK CDK2 IGF-1R AKT PDK
1
2
3.4 ( 0.3 >100
>20
>10
>20
>10
>20
>20
>20
>20
>20
>20
>20
a
b
ATP concentrations used are all <Km and ranged from 0.2 to 8 µM; for details for each enzyme, see Experimental Section. Values
are averages of at least three independent dose-response curves.
Ta ble 3. Physicochemical Parameters of Representative Anilino-Quinazolines
C7 side chain
functionality
aqueous solubility
relative calculated solubility
of the neutral forme
no.
pKa’s
5.3
7.2-5.2
7.9-5.4
9.4-5.3
LogDa
LogPb
at pH 7.4c,d (µM)
1
7
4
2
triazole
4.6
4.6
3.6
3.3
5.0
0.7
20
1
morpholine
piperazine
piperidine
3.4
2.4f
2.6f
60g
330
17h
a
b
Measured at pH 7.4 unless otherwise stated. Calculated from LogD and pKa. c Average of at least three measurements using the
free base. Measured at 3 days unless otherwise stated. e Solubility of the neutral form normalized to the solubility of the neutral form
d
of 1. f Measured at pH 7. Measured at 1 day. Solubility of the neutral form of compound 2 was measured at pH 12.7.
g
h
Sch em e 5a
Ta ble 4. Mouse Plasma Levels Following Administration of 50
or 100 mg/kg Po
plasma levels
(µM)a
dose
no. R1
R2
(mg/kg) at 6 h at 24 h
1
2
3
4
7
8
Br 1-(1,2,3-triazolyl)-(CH2)2O
Br MeN(CH2CH2)2CH-CH2O
Cl MeO-(CH2)2-O
Cl 4-Me-piperazinyl-(CH2)3O
Cl 4-morpholinyl-(CH2)3O
Cl 4-morpholinyl-(CH2)2O
50
50
100
50
100
100
100
50
14
5.5
12
5
4
12
4.6
3
5.5
0.2
3
<0.1
3
0.8
0.5
2
2
2.5
13 Cl 4-pyridyl-N(Me)-(CH2)2O
27 Br MeN(CH2CH2)2CH-CH2CH2O
30 Br (R)-MeN(CH2)(CH2)3CH-CH2O
50
a
Variation was generally (15%.
Ta ble 5. Pharmacokinetic Parameters in Rat and Dog of 1 and
2
a
doga
IV t1/2 (h)
a
rat
IV t1/2 (h)
Reagents: (a) Cl-(CH2)3-Br/K2CO3/DMF. (b) N-Methylpipera-
zine/100 °C (procedure B).
no.
F (%)
F (%)
1
2
∼1.3 ( 0.2b
>80b
>50b
1.1 ( 0.2c
8.3 ( 1.8b
<5c
The N-methylpiperidine nucleus proved to be par-
ticularly interesting. When directly linked to the C-7
position of the quinazoline, it resulted in a weaker
inhibitor of both Flt-1 and KDR (compound 14:18 IC50
1.5 µM). An increase in flexibility of the side chain,
resulting from the insertion of a single methylene
between the piperidine ring and the C7-O atom,
provided a 38-100-fold increase in potency against KDR
with no enhancement of activity against Flt-1 (compari-
son of 1418 with 2 and 15); insertion of two methylenes
(27) increased potency against KDR 250-fold and Flt-1
by 10-fold. 3-Piperidinyl isomers were more potent than
their 4-isomers (compare 2, 30, and 31), and chirality
of the 3-piperidine side chain also influenced potency,
the (R)-enantiomer 30 being 10-fold more potent of an
inhibitor than the (S)-enantiomer 31. In the 2′-F, 4′-Cl,
or 4′-Br series, alkylation of the C-7 side chain piperi-
dine-nitrogen suggests an increase in selectivity of 3-7-
fold for KDR over Flt-1 (Flt-1/KDR IC50 ratio’s, respec-
tively, 40- and 13-fold for 2 and 22; 133- and 20-fold for
15 and 21; and 25- and 5-fold for 27 and 28). This effect
∼15 ( 0.2b
>50b
a
Measured on the free base. For dose and vehicles, see
Experimental Section. Average of three males. c Average of four
b
males.
is not apparent in the difluorinated series (2′-F, 4′-F,
4′-Cl, or 4′-Br series: comparison of 19 and 24 and 20
and 25).
All of the compounds described in this paper are also
inhibitors of the EGFR enzyme (median IC50 0.2 µM,
range 0.075-0.8 µM), but in all cases, potency against
KDR was greater (KDR:EGFR selectivity ratios 3-200).
Inhibition of the FGFR-1 enzyme was consistently
weaker (median IC50 2.5 µM, range 0.9-19 µM; KDR:
FGFR selectivity ratios 33-1800; Table 1), but by
comparison with the C-7 neutral side chain series (e.g.,
1 and 3), selectivity with respect to FGFR is slightly
reduced in the basic side chain series.
Selectivity of representative compounds, and 2 in
particular, against a broad panel of kinases, has also
been investigated. As shown in Table 2, compounds such

1306 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Hennequin et al.
as 2 and 1 possess good selectivity profiles and are
devoid of significant activity against kinases such as
MEK, AKT, and erbB2 or kinases involved in cell cycle
regulation such as CDK-2. Similarly, no significant
activity was detected against the kinase enzymes as-
sociated with the angiopoietin receptor (Tie-2) and the
IGF-1 receptor.
as 1. As shown in Table 3, the 4-methylpiperidine-
methoxy side chain in 2 also provided a 17-fold improve-
ment in aqueous solubility of the neutral form as
compared to 1. At intestinal pH (∼6.5), the solubility of
2 is ∼650 µM (310 µg/mL).
iv. Ser u m P r ot ein Bin d in g (SP B) in Differ en t
Sp ecies. Introduction of a basic function at the C-7
position of the anilinoquinazoline nucleus, such as the
4-methylpiperidine-methoxy (pKa, 9.4) of 2, led to good
levels of free drug in rodent plasma, in part by virtue
of the positive charge present on the C-7 side chain at
physiological pH. Comparison of the neutral (1) and
basic (2) anilinoquinazolines indicates a 2-fold higher
level of unbound basic drug despite a slight (∼0.4 unit)
increase in logP (Mouse SPB: 1, 98 ( 0.5%; 2, 96 ( 0.5%.
Rat SPB: 1, 95 ( 0.5%; 2,26 90 ( 0.5%; mean ( SE, n )
5). Free plasma levels were consistently about 2-fold
higher in rat than in mouse.
ii. In h ibition of En d oth elia l Cell P r olifer a tion .
Selectivity for the VEGF, EGF, and FGF pathways in
whole endothelial cells was investigated using growth
factor-stimulated human endothelial cells (HUVECs).
Inhibition of proliferation was again achieved with
nanomolar concentrations (median IC50 vs VEGF 0.06
µM, range 0.003-0.5 µM), and the selectivity profile
observed in the kinase assays was reasonably conserved
in HUVECS (VEGF > EGF; Table 1), the median IC50
vs EGF being 0.15 µM (range 0.07-1.4 µM). The best
selectivity ratios were observed with the meta-hydroxy
derivatives 17 and 18 (VEGF:EGF ratios of 23 and 66,
respectively). Selectivity for VEGF vs FGF inhibition
was also confirmed in these cells. In the case of 1 and
2, concentrations of 1.5 and 0.8 µM were necessary to
reduce FGF-stimulated growth by 50% giving VEGF:
FGF selectivity ratios of 30 and 13, respectively. The
slight changes in selectivity occasionally observed be-
tween recombinant enzyme and whole cell data can be
attributed to differences (e.g., ATP concentration) be-
tween the two assays.
v. Blood Levels in Mice F ollow in g Or a l a n d
In tr a ven ou s Ad m in istr a tion . We reported that anili-
noquinazolines with neutral or weakly basic C-7 side
chains and 1 in particular were orally bioavailable in
mice.18 Anilinoquinazolines possessing more basic C-7
moieties also yielded good plasma levels in mice follow-
ing oral administration (Table 4), which declined more
slowly than those with neutral or weakly basic side
chains (compare 24 h levels of 1, 3, and 8 with those of
2, 4, 13,18 and 30). As we previously reported,19 pro-
longed exposure combined with good intrinsic potency
was particularly important for good efficacy in the
Calu-6 tumor xenograft model with once daily oral
administration.
vi. Bioa va ila b ilit y a n d H a lf-Lives in R a t a n d
Dog. The pharmacokinetic profile of 1 in the mouse
following oral dosing at 50 mg/kg suggested rapid
elimination in this species. This was confirmed in the
rat where rapid clearance (CL ∼ 3.2 L/hr/kg), moderate
plasma half-life (t1/2 ∼ 1.3 h), and a low volume of
distribution (∼1.2 L/kg) were observed. The half-life of
1 was also short in the dog (Table 5), and its bioavail-
ability was low (<5%) and variable. Attempts to increase
the aqueous solubility and bioavailability of 1 by making
salts was successful in these respects but introduced
stability and hygroscopicity issues that did not auger
well for development.
In the case of the more soluble 4-anilinoquinazolines
possessing C-7 basic side chains such as 2, the improve-
ment in physicochemical properties, in particular better
aqueous solubility, was accompanied by good (>50%)
and more consistent bioavailability in rat and dog (Table
5). Moreover, and probably as a consequence of proto-
nation of the C-7 basic moiety, plasma half-lives in rat
and dog proved to be longer than those observed in the
neutral side chain series. For example, in rat and dog,
2 had half-lives 12- and 8-fold, respectively, higher than
1.
vii. In Vivo Effica cy in Mou se. We have previously
reported that 1 was effective at reducing the growth of
a number of human tumor xenografts grown subcutane-
ously in athymic mice.18,19 The antitumor activity of the
anilinoquinazolines reported here was routinely exam-
ined using a Calu-6 lung tumor xenograft model. As
shown in Table 6, once daily oral administration of 2
for 21 days inhibited the growth of established (∼0.2
The compounds presented in this paper do not sig-
nificantly affect basal, unstimulated growth of HUVECs
(IC50 range: 1 to >10 µM; Table 1), which clearly
suggests that activity is specifically directed to the
receptor-activated signaling pathways and that the
compounds do not impart any direct cytostatic or
cytotoxic effect.
iii. P h ysicoch em ica l P r op er ties. As shown in
Table 3, the lipophilic character of 1 as well as the
neutral nature of its C-7 side chain (1-(1,2,3-triazolyl)-
ethoxy) confers low aqueous solubility on this compound
(∼0.7 µM). Nevertheless, good plasma levels were seen
in mouse following oral dosing.18,19 Its bioavailability
in rats was also good, but in higher species, it was found
to be low and variable. The low aqueous solubility of 1
was thought to be the major factor contributing to this
inconsistency (see the section on pharmacokinetics). C-7
basic side chains proved extremely effective for increas-
ing both solubility and dissolution rate. As expected,
aqueous solubility increased proportionally to the pKa
of the basic function present at C-7. Side chains such
as the morpholinyl-propoxy and the N-methylpiperazi-
nyl-propoxy (compounds 4 and 7, Table 3) led to 30- and
∼80-fold, respectively, increases in solubility relative to
1. This is attributed to partial protonation at pH 7.4
and a reduction in overall lipophilicity of these mol-
ecules as compared to 1. Introducing more basic func-
tions, such as N-methylpiperidine methoxy (compound
2, Table 3), that are almost completely protonated at
physiological pH increased aqueous solubility even more
markedly. For compound 2, solubility (>5 mM at pH
3.6 and ∼330 µM at pH 7.4) was ∼470-fold that of 1. As
compounds are absorbed through the gut wall predomi-
nantly in the nonprotonated form, it was also advanta-
geous if the solubility of the neutral form present at the
site of absorption was improved over compounds such

4-Anilinoquinazolines with C-7 Basic Side Chains
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1307
Ta ble 6. Growth Inhibition of Established Calu-6 Tumor
Xenografts Implanted Subcutaneously in Athymic Mice,
Measured after 21 Days of Dosing of 2
mL) was heated at reflux for 1.5 h. The mixture was cooled,
and the solid was filtered, washed with 2-propanol (2 mL)
followed by diethyl ether (2 mL), and dried under vacuum
overnight at 50 °C to give 304 mg of 2 as an hydrochloride
salt (90%).
% inhibition of tumor growth @ mg/kg/daya po
no.
100
79b
50
25
12.5
36c
The nuclear magnetic resonance (NMR) spectrum of the
protonated form of 2 hydrochloride shows the presence of two
forms (A and B) in a ratio A:B of approximately 9:1 attribut-
able to the protonation of the basic side chain. 1H NMR: δ
1.60-1.78 (m, form A 2H), 1.81-1.93 (br s, form B 4H), 1.94-
2.07 (d, form A 2H), 2.08-2.23 (br s, form A 1H), 2.29-2.37
(br s, form B 1H), 2.73 (d, form A 3H), 2.77 (d, form B 3H),
2.93-3.10 (q, form A 2H), 3.21 (br s, form B 2H), 3.27 (br s,
form B 2H), 3.42-3.48 (d, form A 2H), 4.04 (s, 3H), 4.10 (d,
form A 2H), 4.29 (d, form B 2H), 7.49 (s, 1H), 7.53-7.61 (m,
2H), 7.78 (d, 1H), 8.47 (s, 1H), 8.81 (s, 1H), 10.48 (br s, form A
1H), 10.79 (br s, form B 1H), 11.90 (br s, 1H). Anal. (C22H24N4O2-
BrF, 0.5 H2O, 1.8 HCl, 0.08 2-propanol) C, H, N.
For another NMR reading, some solid potassium carbonate
was added into the dimethyl sulfoxide (DMSO) solution of the
2 hydrochloride described above, to release the free base in
the NMR tube. The NMR spectrum was then recorded again
and showed only one form as described below. 1H NMR
(DMSO-d6; solid potassium carbonate): δ 1.30-1.45 (m, 2H),
1.75 (d, 2H), 1.70-1.90 (m, 1H), 1.89 (t, 2H), 2.18 (s, 3H), 2.80
(d, 2H), 3.98 (s, 3H), 4.0 (d, 2H), 7.20 (s, 1H), 7.48 (d, 1H),
7.55 (t, 1H), 7.68 (d, 1H), 7.80 (s, 1H), 8.35 (s, 1H), 9.75 (s,
1H).
2
67b
51b
a
Percent inhibition represents the difference between the
median of 10 control (vehicle-treated) and 9-10 compound-treated
mice, following 21 days of treatment; measured from start of
b
treatment. p < 0.001. c p < 0.02 (Mann Whitney rank sum one-
tailed test).
cm3) Calu-6 tumors in a dose-dependent manner. Tumor
growth was almost completely arrested at a dose of 100
mg/kg/day and more than halved at 25 mg/kg/day.
Compound 2 has been evaluated against a range of
human tumor xenografts including colon, lung, breast,
prostate, and ovary tumors and has demonstrated
highly significant activity in each model.
Compound 2 does not inhibit the growth of Calu-6
cells directly in vitro (IC50 14 ( 0.4 µM; mean ( SE,
n ) 3) at concentrations of compound likely to be freely
available in vivo. Hence, the tumor growth inhibition
observed in vivo, even taking into account possible
accumulation of the compound at the highest doses
evaluated, cannot be attributed to a direct cytotoxic or
cytostatic effect on the tumor cells. Furthermore, we
have shown that selective inhibitors of VEGF receptor
A sample of 2 (free base) was generated from the 2
hydrochloride, prepared as described above, as follows:
2
hydrochloride (50 mg) was suspended in methylene chloride
(2 mL) and was washed with saturated sodium hydrogen
carbonate. The methylene chloride solution was dried (MgSO4),
and the volatiles were removed by evaporation to give 2 (free
base). The NMR of 2 (free base) so generated shows only one
form, the NMR spectrum of which is identical to that described
above. 1H NMR: δ 1.30-1.45 (m, 2H), 1.76 (d, 2H), 1.70-1.90
(m, 1H), 1.90 (t, 2H), 2.19 (s, 3H), 2.80 (d, 2H), 3.95 (s, 3H),
4.02 (d, 2H), 7.20 (s, 1H), 7.48 (d, 1H), 7.55 (t, 1H), 7.68 (dd,
1H), 7.80 (s, 1H), 8.38 (s, 1H), 9.55(br s, 1H).
kinases, completely devoid of EGFR inhibition (IC50
>
10 µM), inhibit the growth of Calu-6 tumors when
administered orally,23,24,26 whereas, over a similar dose
range, selective EGFR inhibitors devoid of VEGF recep-
tor inhibition (IC50 > 10 µM) do not. The activity
observed in the Calu-6 xenograft model with a com-
pound possessing activity against VEGF and EGF
tyrosine kinase such as 2 is thus most likely attributable
to the inhibition of the VEGF signaling pathway.
For another NMR reading, some CF3COOD was added into
the NMR DMSO solution of 2 (free base) described above and
the NMR spectrum was recorded again. The spectrum of the
protonated form of the trifluoroacetate salt of 2 so obtained
shows the presence of two forms (A and B) in a ratio A:B of
Con clu sion s
Anilinoquinazolines possessing C-7 basic side chains
are potent nanomolar inhibitors of KDR tyrosine kinase,
and some are also potent inhibitors of Flt-1 tyrosine
kinase. Their enzyme inhibition profiles translate very
well into endothelial cells as demonstrated by nano-
molar inhibition of VEGF-stimulated HUVEC prolifera-
tion. The presence of a basic side chain confers excellent
physicochemical properties, in particular, good aqueous
solubility, improved bioavailability in rodent and dog,
and reduced protein binding. The plasma half-life of this
subseries of anilinoquinazolines is generally longer than
that of anilinoquinazolines with neutral side chains. In
view of its excellent physicochemical properties, selec-
tivity profile, pharmacokinetics, and antitumor activity
in preclinical models, 2 was preferred over 1 and
replaced it in development. Compound 2 is currently
undergoing phase I clinical evaluation.17
1
approximately 9:1. H NMR (DMSO-d6; CF3COOD): δ 1.50-
1.70 (m, form A 2H), 1.93 (br s, form B 4H), 2.0-2.10 (d, form
A 2H), 2.17 (br s, form A 1H), 2.35 (br s, form B 1H), 2.71 (s,
form A 3H), 2.73 (s, form B 3H), 2.97-3.09 (t, form A 2H),
3.23 (br s, form B 2H), 3.34 (br s, form B 2H), 3.47-3.57 (d,
form A 2H), 4.02 (s, 3H), 4.15 (d, form A 2H), 4.30 (d, form B
2H), 7.2 (s, 1H), 7.3-7.5 (m, 2H), 7.60 (d, 1H), 7.90 (s, 1H),
8.70 (s, 1H).
A similar procedure was used to prepare 15, 17, 18, 26, and
35.
N-(4-Ch lor o-2-flu or op h en yl)-6-m et h oxy-7-[3-(4-m et h -
ylp ip er a zin -1-yl)p r op oxy]qu in a zolin -4-a m in e (4). P r o-
ced u r e B. A suspension of 4-(4-chloro-2-fluoroanilino)-7-(3-
chloropropoxy)-6-methoxyquinazoline (66; 150 mg, 0.38 mmol)
in 1-methylpiperazine (2 mL) was heated at 100 °C for 3 h.
The mixture was cooled and was partitioned between an
aqueous solution of sodium carbonate (pH 11.5) and ethyl
acetate. The organic layer was separated, washed with brine,
and dried (MgSO4), and the volatiles were removed by evapo-
ration. The residue was dissolved in methylene chloride, and
diethyl ether was added. The precipitate was filtered, washed
with diethyl ether (2 mL), and dried. The solid was dissolved
in methylene chloride, and 2.2 M hydrogen chloride in diethyl
ether (1 mL) was added. After the solution was concentrated
to half of its initial volume, the precipitate was filtered, washed
with diethyl ether (2 mL), and dried under vacuum to give
158 mg of 4 hydrochloride (75%). 1HNMR (DMSO-d6; CF3-
COOD; 60 °C): δ 2.35 (m, 2H), 2.95 (s, 3H), 3.43 (t, 2H), 3.52-
3.70 (m, 8H), 4.03 (s, 3H), 4.34 (t, 2H), 7.41 (s, 1H), 7.45 (d,
Exp er im en ta l Section
For general procedures, see the Supporting Information
section.
N-(4-Br om o-2-flu or op h en yl)-6-m eth oxy-7-[(1-m eth ylp i-
p er id in -4-yl)m eth oxy]qu in a zolin -4-a m in e (2). P r oced u r e
A. A mixture of 4-chloro-6-methoxy-7-[(1-methylpiperidin-4-
yl)methoxy]quinazoline (64; 200 mg, 0.62 mmol) and 4-bromo-
2-fluoroaniline (142 mg, 0.74 mmol) in 2-propanol (3 mL)
containing 6 M hydrogen chloride in 2-propanol (110 µL, 0.68

1308 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Hennequin et al.
1H), 7.60-7.70 (m, 2H), 8.11 (s, 1H), 8.80 (s, 1H). MS-EI
m/z: 460 [MH]+. Anal. (C23H27N5O2ClF, 0.7 H2O, 2.75 HCl) C,
H, N.
N-(4-Br om o-2-flu or op h en yl)-6-m eth oxy-7-{[(2E)-4-p yr -
r olid in -1-ylbu t-2-en yl]oxy}qu in a zolin -4-a m in e (11). P r o-
ced u r e F . Diethyl azodicarboxylate (1.55 mL, 9.89 mmol),
N-(4-bromo-2-fluorophenyl)-7-hydroxy-6-methoxy-4-quinazoli-
nylamine (40;18 1.2 g, 3.3 mmol), and a solution of 68 (697 mg,
4.9 mmol) in methylene chloride (5 mL) were added succes-
sively to a solution of triphenylphosphine (2.59 g, 9.89 mmol)
in methylene chloride (150 mL) cooled at 5 °C. The mixture
was stirred at ambient temperature for 10 min, and then,
methylene chloride (100 mL) was added followed successively
by triphenylphosphine (432 mg, 1.6 mmol), (E)-4-(pyrrolidin-
1-yl)but-2-en-1-ol (232 mg, 1.6 mmol), and diethyl azodicar-
boxylate (246 µL, 1.6 mmol). The mixture was stirred at
ambient temperature for 30 min, and then, the solvent was
removed by evaporation. The residue was purified by column
chromatography eluting with methylene chloride/methanol (80/
20 followed by 70/30 and 60/40). The semipurified product was
repurified by column chromatography eluting with methylene
chloride/methanol (80/20 followed by 75/25). The purified
product was dissolved in methylene chloride, 3.7 M hydrogen
chloride in diethyl ether (3 mL) was added, and the volatiles
were removed by evaporation. The residue was triturated with
diethyl ether, collected by filtration, and dried under vacuum
A similar procedure was used to prepare 5.
N-(4-Ch lor o-2-flu or op h en yl)-6-m et h oxy-7-[2-(4-m et h -
ylp ip er a zin -1-yl)eth oxy]qu in a zolin -4-a m in e (6). P r oce-
d u r e C. A solution of 2-(4-methylpiperazin-1-yl)ethanol (112
mg, 0.78 mmol) in methylene chloride (1 mL) was added to a
stirred suspension of N-(4-chloro-2-fluorophenyl)-7-hydroxy-
6-methoxy-4-quinazolinylamine (39;18 225 mg, 0.7 mmol) and
tributylphosphine (420 mg, 2.1 mmol) in methylene chloride
(10 mL). 1,1′-(Azodicarbonyl)dipiperidine (525 mg, 2.1 mmol)
was then added in portions to the mixture. The resulting clear,
pale yellow solution was stirred for 3.5 h and then allowed to
stand overnight. The reaction mixture was quenched with
diethyl ether (8 mL), and the precipitate was filtered. The
solvent was removed from the filtrate by evaporation, the
residue was dissolved in acetone (5 mL), and 1 M hydrogen
chloride in diethyl ether was added until the hydrochloride
salt precipitated. The precipitate was collected by filtration,
dissolved in methanol (3 mL), and then basified with excess
triethylamine. The volatiles were removed by evaporation, and
the residue was purified by column chromatography eluting
with methylene chloride/methanol/aqueous ammonia (100/8/
1). The resulting oil was triturated with diethyl ether (1 mL),
collected by filtration, and dried to give 79 mg of 6 (25%) as a
white solid; mp 173-175 °C. 1H NMR: δ 2.10 (s, 3H), 2.30
(m, 4H), 2.50 (m, 4H), 2.75 (t, 2H), 3.95 (s, 3H), 4.25 (t, 2H),
7.21 (s, 1H), 7.30 (dd, 1H), 7.50 (d, 1H), 7.55 (dd, 1H), 7.75 (s,
1H), 8.30 (s, 1H), 9.50 (s, 1H). MS-ESI m/z: 446 [MH]+. Anal.
(C22H25N5O2ClF).
1
to give 600 mg of 11 hydrochloride (32%). H NMR: (DMSO-
d6; CF3COOD): δ 1.80-1.90 (m, 2H), 2.0-2.10 (m, 2H), 3.0-
3.10 (m, 2H), 3.45-3.55 (m, 2H), 3.88 (d, 2H), 4.01 (s, 3H),
4.90 (d, 2H), 6.0 (td, 1H), 6.30 (td, 1H), 7.41 (s, 1H), 7.5-7.65
(m, 2H), 7.82 (d, 1H), 8.13 (s, 1H), 8.88 (s, 1H). MS-EI m/z:
487 [M.]+. Anal. (C23H24N4O2BrF, 0.5 H2O, 2 HCl) C, H, N.
A similar procedure was used to prepare 48.
N-(4-Meth yl-2-flu or oph en yl)-6-m eth oxy-7-[(1-m eth ylpi-
p er id in -4-yl)m eth oxy]qu in a zolin -4-a m in e (16). P r oce-
d u r e G. A suspension of 4-chloro-6-methoxy-7-[(1-methylpi-
peridin-4-yl)methoxy]quinazoline (64; 200 mg, 0.622 mmol)
and 2-fluoro-4-methylaniline (94 mg, 0.764 mmol) in 2-pro-
panol (5 mL) containing 6.2 M hydrogen chloride in 2-propanol
(110 µL) was stirred at 80 °C for 1.5 h. After the solution was
cooled, the precipitate was collected by filtration, washed with
2-propanol (2 mL) followed by diethyl ether (2 mL), and dried
under vacuum. The solid was dissolved in the minimum
volume of methanol/methylene chloride/methanol (saturated
with ammonia) and stirred for 5 min at ambient temperature.
The volatiles were removed under vacuum. The solid was
purified by column chromatography, eluting with methylene
chloride/methanol (90/10) followed by methylene chloride/
methanol containing 5% ammonia (90/10). Evaporation of the
fractions containing the expected product gave 170 mg of 16
(61%). 1H NMR: δ 1.30-1.45 (m, 2H), 1.80 (d, 2H), 1.70-1.90
(m, 1H), 1.90 (t, 2H), 2.20 (s, 3H), 2.35 (s, 3H), 2.80 (d, 2H),
3.95 (s, 3H), 4.01 (d, 2H), 7.10 (d, 1H), 7.13 (d, 1H), 7.16 (s,
1H), 7.40 (t, 1H), 7.81 (s, 1H), 8.32 (s, 1H), 9.40 (s, 1H). MS-
ESI m/z: 411 [MH]+. Anal. (C23H27N4O2F, 0.3 H2O) C, H, N.
N-(4-Ch lor o-2-flu or oph en yl)-6-m eth oxy-7-(3-pyr r olidin -
1-ylp r op oxy)q u in a zolin -4-a m in e (9). P r oced u r e D. A
solution of 1-(3-chloropropyl)pyrrolidine (230 mg, 0.96 mmol)
was added to N-(4-chloro-2-fluorophenyl)-7-hydroxy-6-meth-
oxy-4-quinazolinylamine (39;18 295 mg, 0.92 mmol) and potas-
sium carbonate (130 mg, 0.94 mmol) in DMF (8 mL). The
mixture was heated at 100 °C for 90 min and allowed to cool.
The volatiles were removed by evaporation, and the residues
were partitioned between water (10 mL) and methylene
chloride (10 mL). The organic phase was separated and passed
through phase-separating paper, and the solvent was removed
under reduced pressure. The residue was dissolved in acetone
(2 mL), and 1 M hydrogen chloride in diethyl ether (2 mL)
was added. The mixture was stirred at ambient temperature
for 30 min, and the resulting precipitate was collected by
filtration and dried to give 320 mg of 9 as a hydrochloride
1
(67%). H NMR: δ 1.80-2.0 (m, 6H), 3.0-3.60 (m, 6H), 4.05
(s, 3H), 4.30 (t, 2H), 7.40 (m, 2H), 7.55 (d, 1H), 7.60 (m, 1H),
8.40 (s, 1H), 8.80 (s, 1H). MS-ESI m/z: 431 [MH]+. Anal.
(C22H24N4O2ClF, 1.8 H2O, 1.5 HCl) C, H, N.
N-(4-Ch lor o-2-flu or op h en yl)-6-m eth oxy-7-{[(2E)-4-p yr -
r olid in -1-ylbu t-2-en yl]oxy}qu in a zolin -4-a m in e (10). P r o-
ced u r e E. Diethyl azodicarboxylate (5.91 mL, 37 mmol) was
added dropwise to a stirred mixture of (E)-4-(pyrrolidin-1-yl)-
but-2-en-1-ol (68; 3.97 g, 28 mmol), 3918 (3.0 g, 9 mmol), and
triphenylphosphine (9.84 g, 38 mmol) in methylene chloride
(300 mL). The reaction mixture was stirred for 18 h at ambient
temperature. The volatiles were removed by evaporation, and
the residue was purified by column chromatography eluting
with methylene chloride/methanol (a gradient from 80/20 to
70/30). The purified product was dissolved in methylene
chloride/methanol (10 mL/10 mL), and 1 M hydrogen chloride
in diethyl ether (25 mL) was added. The precipitated product
was collected by filtration, washed with diethyl ether (2 mL),
and dried to give 1.62 g of 10 hydrochloride (33%). 1H NMR
(DMSO-d6; CF3COOD): δ 1.85-1.95 (m, 2H), 2.0-2.15 (m,
2H), 3.0-3.10 (m, 2H), 3.50-3.60 (m, 2H), 3.95 (d, 2H), 4.10
(s, 3H), 4.95 (d, 2H), 6.10 (td, 1H), 6.35 (td, 1H), 7.40 (s, 1H),
7.45 (dd, 1H), 7.60-7.70 (m, 2H), 8.15 (s, 1H), 8.90 (s, 1H).
MS-EI m/z: 443 [MH]+. Anal. (C23H24N4O2ClF, 0.6 H2O, 1.85
HCl) C, H, N.
A similar procedure was used to prepare 36.
N-(4-Ch lor o-2,6-d iflu or op h en yl)-6-m eth oxy-7-[(1-m eth -
ylp ip er id in -4-yl)m eth oxy]qu in a zolin -4-a m in e (19). P r o-
ced u r e H. Under argon, sodium hydride (60%, 76.5 mg, 1.9
mmol) was added to a solution of 4-chloro-2,6-difluoroaniline
(250 mg, 1.53 mmol) in DMF (9 mL). After the solution was
stirred for 30 min at ambient temperature, 64 (246 mg, 0.764
mmol) was added and stirring was continued for a further 20
h. The mixture was poured onto water (50 mL) and extracted
with ethyl acetate (50 mL). The organic layer was washed with
water and brine and dried (MgSO4), and the volatiles were
removed by evaporation. The residue was purified by column
chromatography on silica, eluting with methylene chloride/
methanol (95/5) followed by methylene chloride/methanol
containing 1% ammonia (90/10). The fractions containing the
expected product were combined and evaporated. The residue
was triturated with diethyl ether (2 mL), filtered, washed with
diethyl ether (2 mL), and dried under vacuum at 50 °C to give
1
210 mg of 19 (61%). H NMR: δ 1.30-1.45 (m, 2H), 1.80 (d,
A similar procedure was used to prepare 12, 29-31, 46, 47,
and 49.
2H), 1.70-1.90 (m, 1H), 1.90 (t, 2H), 2.20 (s, 3H), 2.80 (d, 2H),
3.96 (s, 3H), 4.02 (d, 2H), 7.21 (s, 1H), 7.52 (s, 1H), 7.54 (s,

4-Anilinoquinazolines with C-7 Basic Side Chains
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1309
1H), 7.82 (s, 1H), 8.35 (s, 1H). MS-ESI m/z: 449-451 [MH]+.
(78%). 1H NMR: δ 1.15-1.30 (m, 2H), 1.40-1.50 (m, 1H),
1.65-1.80 (m, 4H), 1.85 (t, 2H), 2.15 (s, 3H), 2.75 (d, 2H), 3.95
(s, 3H), 4.20 (t, 2H), 7.20 (s, 1H), 7.50 (d, 1H), 7.55 (dd, 1H),
7.80 (s, 1H), 8.38 (s, 1H), 9.55 (s, 1H). MS-ESI m/z: 489
[MH]+. Anal. (C23H26N4O2BrF, 0.1 H2O, 0.15 Et2O) C, H, N.
Anal. (C22H23N4O2ClF2) C, H, N.
A similar procedure was used to prepare 20 and 37.
N-(4-Ch lor o-2-flu or op h en yl)-6-m et h oxy-7-(p ip er id in -
4-ylm eth oxy)qu in a zolin -4-a m in e (21). P r oced u r e I. tert-
Butyl 4-(hydroxymethyl)piperidine-1-carboxylate (52;20 590
mg, 2.75 mmol) followed by triphenylphosphine (1.2 g, 4.58
mmol) and diethyl azodicarboxylate (0.72 mL, 4.58 mmol) were
added to a solution of 3918 (585 mg, 1.83 mmol) in methylene
chloride (20 mL). After the solution was stirred for 1 h at
ambient temperature, further triphenylphosphine (239 mg,
0.91 mmol) and diethyl azodicarboxylate (0.14 mL, 0.91 mmol)
were added. After the solution was stirred for 1.5 h, the
volatiles were removed under vacuum and the residue was
purified by column chromatography eluting with ethyl acetate/
methylene chloride (1/1). The crude product was used directly
in the next step.
A solution of the crude product in methylene chloride (15
mL) containing TFA (4.5 mL) was stirred at ambient temper-
ature for 1.5 h. The volatiles were removed under vacuum.
The residue was partitioned between water and ethyl acetate.
The aqueous layer was adjusted to pH 9.5 with 2 M sodium
hydroxide. The organic layer was separated, washed with
water followed by brine, dried (MgSO4), and evaporated to give
21 free base. 1H NMR: δ 1.10-1.30 (m, 2H), 1.75 (d, 2H),
1.85-2.0 (br s, 1H), 2.55 (d, 2H), 2.95 (d, 2H), 3.95 (s, 3H), 4.0
(d, 2H), 7.20 (s, 1H), 7.35 (dd, 1H), 7.55 (dd, 1H), 7.60 (t, 1H),
7.80 (s, 1H), 8.35 (s, 1H), 9.55 (s, 1H). MS-ESI m/z: 417-419
[MH]+. The hydrochloride salt was made as follows: 21 free
base was dissolved in a mixture of methanol and methylene
chloride (5 mL/5 mL), and 6 M hydrogen chloride in diethyl
ether was added. The volatiles were removed under vacuum,
and the residue was triturated with diethyl ether (5 mL),
collected by filtration, washed with diethyl ether (2 mL), and
dried under vacuum to give 390 mg of 21 hydrochloride (47%
over 2 steps). Anal. (C21H22O2N4ClF, 2.25 HCl)
N-(4-Br om o-2-flu or op h en yl)-6-m eth oxy-7-(p ip er id in -4-
ylm eth oxy)qu in a zolin -4-a m in e (28). P r oced u r e L. A solu-
tion of tert-butyl 4-[2-({4-[(4-bromo-2-fluorophenyl)amino]-6-
methoxyquinazolin-7-yl}oxy)ethyl]piperidine-1-carboxylate (46;
2.5 g, 4.3 mmol; ∼80% of purity) in methylene chloride (50
mL) containing TFA (12 mL) was stirred at ambient temper-
ature for 1.5 h. The volatiles were removed under vacuum,
and the residue was partitioned between methylene chloride
(10 mL) and water (10 mL). The aqueous layer was separated,
and the pH was adjusted to 11 with 2 M aqueous sodium
hydroxide and then extracted with methylene chloride. The
combined organic layers were washed with water and brine,
dried (MgSO4), and evaporated. The residue was purified by
column chromatography eluting with methylene chloride/
methanol (90/10) followed by methylene chloride/methanol/
methanol containing ammonia (1%, 90/5/5) followed by 80/10/
10. The fractions containing the expected product were combined
and evaporated. The residue was triturated with diethyl ether
(10 mL), filtered, washed with diethyl ether (5 mL), and dried
1
under vacuum to give 840 mg of 28 (42%). H NMR (DMSO-
d6, CD3COOD): δ 1.30-1.50 (m, 2H), 1.80 (m, 3H), 1.90-2.0
(m, 2H), 2.90 (t, 2H), 3.30 (d, 2H), 3.95 (s, 3H), 4.20 (bs, 2H),
7.22 (s, 1H), 7.50 (d, 1H), 7.55 (dd, 1H), 7.65 (d, 1H), 7.80 (s,
1H), 8.38 (s, 1H). MS-ESI m/z: 475-477 [MH]+. Anal.
(C22H24N4O2BrF, 0.54 H2O, 0.26 CH4O) C, H, N.
A similar procedure was used to prepare 41.
ter t-Bu tyl 4-[({4-[(4-br om o-2-flu or op h en yl)a m in o]-6-
m et h oxyq u in a zolin -7-yl}oxy)m et h yl]p ip er id in e-1-ca r -
boxyla te (45). P r oced u r e M. Potassium carbonate (414 mg,
3 mmol) was added to a suspension of N-(4-bromo-2-fluorophen-
yl)-7-hydroxy-6-methoxy-4-quinazolinylamine (40;18 546 mg,
1.5 mmol) in DMF (5 mL). After the suspension was stirred
for 10 min at ambient temperature, tert-butyl 4-({[(4-meth-
ylphenyl)sulfonyl]oxy}methyl)piperidine-1-carboxylate (54;21
636 mg, 1.72 mmol) was added and the mixture was heated
at 95 °C for 2 h. After the mixture was cooled, the mixture
was poured onto cooled water (20 mL). The precipitate was
collected by filtration, washed with water, and dried under
vacuum to give 665 mg of 45 (79%). 1H NMR: δ 1.15-1.30
(m, 2H), 1.46 (s, 9H), 1.80 (d, 2H), 2.0-2.10 (m, 1H), 2.65-
2.90 (m, 2H), 3.95 (s, 3H), 4.02 (br s, 2H), 4.05 (d, 2H), 7.20 (s,
1H), 7.48 (d, 1H), 7.55 (t, 1H), 7.65 (d, 1H), 7.80 (s, 1H), 8.35
(s, 1H), 9.55 (br s, 1H). MS-ESI m/z: 561-563 [MH]+.
(R)-(1-Meth ylp ip er id in -3-yl)m eth a n ol (50). (R)-Ethyl
nipecotate (5.7 g, 365 mmol) was dissolved in aqueous form-
aldehyde (38%, 45 mL) and formic acid (90 mL), and the
mixture was heated at reflux for 18 h. The mixture was
allowed to cool and was added dropwise to a cooled saturated
aqueous sodium hydrogen carbonate solution. The mixture was
adjusted to pH 12 by the addition of saturated aqueous sodium
hydroxide, and the mixture was extracted with methylene
chloride (50 mL). The organic extract was washed with brine
and dried (MgSO4), and the solvent was removed by evapora-
tion to give 4.51 g of (R)-ethyl 1-methylpiperidine-3-carboxylate
(73%) as a colorless oil. MS-ESI m/z: 172 [MH]+.
A solution of (R)-ethyl 1-methylpiperidine-3-carboxylate
(5.69 g, 33 mmol) in diethyl ether (20 mL) was added dropwise
to a stirred solution of 1 M lithium aluminum hydride in THF
(36.6 mL, 36.6 mmol) in ether (85 mL) and cooled to maintain
the reaction temperature at 20 °C. The mixture was stirred
for 1.5 h at ambient temperature, and then, water (1.4 mL),
aqueous sodium hydroxide (15%, 1.4 mL), and water (4.3 mL)
were added. The precipitate was removed by filtration, and
the volatiles were removed from the filtrate by evaporation to
give 4.02 g of 50 (94%). 1H NMR: δ 1.06 (q, 1H), 1.51-1.94
(m, 5H), 2.04 (s, 3H), 2.34 (br s, 1H), 2.62 (m, 1H), 2.78 (d,
1H), 3.49 (m, 1H), 3.59 (m, 1H). MS-ESI m/z: 130 [MH]+.
N-(4-Br om o-2-flu or op h en yl)-6-m eth oxy-7-(p ip er id in -4-
ylm eth oxy)qu in a zolin -4-a m in e (22). P r oced u r e J . TFA (3
mL) was added to a suspension of tert-butyl 4-[({4-[(4-bromo-
2-fluorophenyl)amino]-6-methoxyquinazolin-7-yl}oxy)methyl]-
piperidine-1-carboxylate (45; 673 mg, 1.2 mmol) in methylene
chloride (10 mL). After the suspension was stirred for 1 h at
ambient temperature, the volatiles were removed under
vacuum. The residue was triturated with a mixture of water
and diethyl ether. The organic layer was separated, and the
aqueous layer was washed again with diethyl ether. The
aqueous layer was adjusted to pH 10 with 2 M aqueous sodium
hydroxide and then extracted with methylene chloride. The
combined organic layers were dried (MgSO4), and the solvent
was removed under vacuum. The solid was triturated with a
mixture of diethyl ether and petroleum ether (1/1), filtered,
washed with diethyl ether (2 mL), and dried under vacuum to
1
give 390 mg of 22 (70%). H NMR: δ 1.13-1.30 (m, 2H), 1.75
(d, 2H), 1.87-2.0 (m, 1H), 2.50 (d, 2H), 3.0 (d, 2H), 3.96 (s,
3H), 3.98 (d, 2H), 7.20 (s, 1H), 7.50 (dd, 1H), 7.55 (t, 1H), 7.68
(dd, 1H), 7.80 (s, 1H), 8.36 (s, 1H), 9.55 (br s, 1H). MS-ESI
m/z: 461-463 [MH]+. Anal. (C21H22N4O2BrF) C, H, N.
A similar procedure was used to prepare 23-25, 42, and
43.
N-(4-Br om o-2-flu or op h en yl)-6-m et h oxy-7-[2-(1-m et h -
ylp ip er id in -4-yl)eth oxy]qu in a zolin -4-a m in e (27). P r oce-
d u r e K. Thirty-seven percent aqueous formaldehyde (480 µL)
was added to a solution of N-(4-bromo-2-fluorophenyl)-6-
methoxy-7-(piperidin-4-ylmethoxy)quinazolin-4-amine (28; 480
mg, 1.01 mmol) in formic acid (4.8 mL). The mixture was
heated at 95 °C for 4 h. The volatiles were removed under
vacuum, and the residue was partitioned between water and
ethyl acetate. The pH of the aqueous layer was adjusted to 11
with an aqueous solution of 2 M NaOH. The organic layer was
separated, washed with water and brine, dried (MgSO4), and
evaporated. The residue was purified by column chromatog-
raphy, eluting with methylene chloride/methanol containing
1%ammonia (90/10). The fractions containing the expected
product were combined and evaporated to give 386 mg of 27
7-(1-(ter t-Bu t oxyca r b on yl)p ip er id in -4-ylm et h oxy)-6-
m eth oxy-3-((p iva loyloxy)m eth yl)-3,4-d ih yd r oqu in a zolin -

1310 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Hennequin et al.
4-on e (56). Triphenylphosphine (1.7 g, 6.5 mmol) was added
under nitrogen to a suspension of 7-hydroxy-6-methoxy-3-
((pivaloyloxy)methyl)-3,4-dihydroquinazolin-4-one (51;18 1.53
g, 5 mmol) in methylene chloride (20 mL), followed by the
addition of tert-butyl 4-(hydroxymethyl)piperidine-1-carboxy-
late (52;20 1.29 g, 6 mmol) and by a solution of diethyl
azodicarboxylate (1.13 g, 6.5 mmol) in methylene chloride (5
mL). After the solution was stirred for 30 min at ambient
temperature, the reaction mixture was poured onto a column
of silica and was eluted with ethyl acetate/petroleum ether (1/
1) followed by 6/5, 6/4, and 7/3. Evaporation of the fractions
containing the expected product led to an oil that crystallized
following trituration with pentane (5 mL). The solid was
collected by filtration and dried under vacuum to give 2.32 g
in methanol (30 mL) and methylene chloride (60 mL) was
added aqueous formaldehyde (37%, 2.2 mL, 28.9 mmol) fol-
lowed by acetic acid (990 µL, 17.3 mmol). Sodium borohydride
triacetate (4.6 g, 21.6 mmol) was added in portions. After the
solution was stirred for 1 h at ambient temperature, the
volatiles were removed under vacuum and the residue was
partitioned between water (50 mL) and methylene chloride (50
mL). The pH of the aqueous layer was adjusted to 7, washed
with water and brine, dried (MgSO4), and evaporated. The
solid was triturated with diethyl ether (5 mL), filtered, washed
with diethyl ether (5 mL), and dried under vacuum to give
1
4.2 g of 61 (68%). H NMR (CDCl3): δ 1.22 (s, 9H), 1.68 (br s,
3H), 1.90 (m, 4H), 2.32 (br s, 2H), 2.52 (s, 3H), 3.18 (d, 2H),
4.0 (s, 3H), 4.20 (t, 2H), 5.95 (s, 2H), 7.10 (s, 1H), 7.65 (s, 1H),
8.20 (s, 2H). MS-ESI m/z: 432 [MH]+.
1
of 56 (92%). H NMR (CDCl3): δ 1.20 (s, 9H), 1.20-1.35 (m,
2H), 1.43 (s, 9H), 1.87 (d, 2H), 2.05-2.20 (m, 1H), 2.75 (t, 2H),
3.96 (d, 2H), 3.97 (s, 3H), 4.10-4.25 (br s, 2H), 5.95 (s, 2H),
7.07 (s, 1H), 7.63 (s, 1H), 8.17 (s, 1H). MS-ESI m/z: 526
[MNa]+. Anal. (C26H37N3O7) C, H, N.
6-Meth oxy-7-[(1-m eth ylp ip er id in -4-yl)m eth oxy]-3,4-d i-
h yd r oqu in a zolin -4-on e (62). A saturated solution of am-
monia in methanol (14 mL) was added to a solution of 60 (1.38
g, 3.3 mmol) in methanol (5 mL). After the solution was stirred
for 20 h at ambient temperature, the suspension was diluted
with methylene chloride (10 mL) and filtered. The filtrate was
evaporated under vacuum, and the residue was triturated with
diethyl ether (3 mL), filtered, washed with diethyl ether (2
7-(2-(1-ter t-Bu t oxyca r b on ylp ip er id in -4-yl)et h oxy)-6-
m eth oxy-3-((p iva loyloxy)m eth yl)-3,4-d ih yd r oqu in a zolin -
4-on e (57). A suspension of 5118 (7 g, 23 mmol) and tert-butyl
4-(2-{[(4-methylphenyl)sulfonyl]oxy}ethyl)piperidine-1-carbox-
ylate (55;22 11.4 g, 30 mmol) in DMF (70 mL) containing
potassium carbonate (6.32 g, 46 mmol) was stirred at 100 °C
for 3 h. After the suspension was cooled, the volatiles were
removed under vacuum and the residue was partitioned
between diethyl ether and water. The organic layer was
separated, washed with water and brine, dried (MgSO4), and
evaporated. The solid was triturated with pentane, filtered,
1
mL), and dried under vacuum to give 910 mg of 62 (83%). H
NMR: δ 1.30-1.45 (m, 2H), 1.75 (d, 2H), 1.70-1.85 (m, 1H),
1.90 (t, 2H), 2.20 (s, 3H), 2.80 (d, 2H), 3.90 (s, 3H), 4.0 (d, 2H),
7.13 (s, 1H), 7.45 (s, 1H), 7.99 (s, 1H). MS-ESI m/z: 304
[MH]+.
A similar procedure was used to prepare 63.
4-Ch lor o-6-m eth oxy-7-[(1-m eth ylp ip er id in -4-yl)m eth -
oxy]qu in a zolin e (64). A solution of 62 (2.8 g, 9.24 mmol) in
thionyl chloride (28 mL) containing DMF (280 µL) was refluxed
at 85 °C for 1 h. After the solution was cooled, the volatiles
were removed by evaporation. The precipitate was triturated
with diethyl ether (5 mL), filtered, washed with diethyl ether
(3 mL), and dried under vacuum. The solid was dissolved in
methylene chloride (15 mL), and saturated aqueous sodium
hydrogen carbonate was added. The organic layer was sepa-
rated, washed with water and brine, dried (MgSO4), and
evaporated to give 2.9 g of 64 (98%). 1H NMR: δ 1.30-1.50
(m, 2H), 1.75-1.90 (m, 3H), 2.0 (t, 1H), 2.25 (s, 3H), 2.85 (d,
2H), 4.02 (s, 3H), 4.12 (d, 2H), 7.41 (s, 1H), 7.46 (s, 1H), 8.90
(s, 1H). MS-ESI m/z: 322 [MH]+. Anal. (C16H20N3O2Cl, 0.3
H2O, 0.1 CH2Cl2) C, H, N.
1
and dried under vacuum to give 10.5 g of 57 (88%). H NMR
(CDCl3): δ 1.20 (s, 9H), 1.15-1.25 (m, 2H), 1.48 (s, 9H), 1.65-
1.75 (m, 1H), 1.70 (d, 2H), 1.90 (dd, 2H), 2.72 (t, 2H), 4.0 (s,
3H), 4.0-4.20 (m, 2H), 4.20 (t, 2H), 5.95 (s, 2H), 7.10 (s, 1H),
7.65 (s, 1H), 8.20 (s, 1H). MS-ESI m/z: 540 [MNa]+.
6-Meth oxy-7-(p ip er id in -4-ylm eth oxy)-3-((p iva loyloxy)-
m eth yl)-3,4-d ih yd r oqu in a zolin -4-on e (58). A solution of 56
(2.32 g, 4.6 mmol) in methylene chloride (23 mL) containing
TFA (5 mL) was stirred at ambient temperature for 1 h. The
volatiles were removed under vacuum. The residue was
partitioned between ethyl acetate (20 mL) and sodium hydro-
gen carbonate. The organic solvent was removed under
vacuum, and the residue was filtered. The precipitate was
washed with water and dried under vacuum. The solid was
azeotroped with toluene and dried under vacuum to give 1.7
g of 58 (92%). 1H NMR (DMSO-d6; CF3COOD): δ 1.15 (s, 9H),
1.45-1.60 (m, 2H), 1.95 (d, 2H), 2.10-2.25 (m, 1H), 2.95 (t,
2H), 3.35 (d, 2H), 3.95 (s, 3H), 4.10 (d, 2H), 5.95 (s, 2H), 7.23
(s, 1H), 7.54 (s, 1H), 8.45 (s, 1H). MS-ESI m/z: 404 [MH]+.
Anal. (C21H29N3O5, 1 H2O, 0.8 CF3COOH) C, H, N.
A similar procedure was used to prepare 65.
N-(4-Ch lor o-2-flu or op h en yl)-7-(3-ch lor op r op oxy)-6-
m eth oxyqu in a zolin -4-a m in e (66). A mixture of 3918 (957
mg, 3 mmol), 1-bromo-3-chloropropane (2.36 g, 15 mmol), and
potassium carbonate (2.1 g, 15 mmol) in DMF (20 mL) was
heated at 40 °C for 1.5 h. The mixture was cooled, diluted with
water, and extracted with ethyl acetate. The organic extracts
were combined, washed with water and brine, and dried
(MgSO4), and the volatiles were removed by evaporation. The
residue was triturated with hexane/ethyl acetate (5 mL/5 mL),
filtered, and dried under vacuum to give 650 mg of 66 (55%).
1H NMR: δ 2.26 (m, 2H), 3.82 (t, 2H), 3.95 (s, 3H), 4.26 (t,
2H), 7.20 (s, 1H), 7.32 (dd, 1H), 7.48-7.60 (m, 2H), 7.80 (s,
1H), 8.35 (s, 1H), 9.52 (s, 1H). MS-EI m/z: 396 [MH]+.
N -(4-Br om o-2-flu or op h e n yl)-7-(3-ch lor op r op oxy)-6-
m eth oxyqu in a zolin -4-a m in e (67). A solution of 4018 (0.5 g,
1.37 mmol) and 1-bromo-3-chloropropane (0.165 mL, 1.65
mmol) in DMF (7.5 mL) containing potassium carbonate (0.475
g, 3.43 mmol) was stirred at ambient temperature overnight.
The mixture was poured onto water. The precipitate was
filtered, washed with water followed by diethyl ether (3 mL),
A similar procedure was used to prepare 59.
6-Meth oxy-7-(1-m eth ylp ip er id in -4-ylm eth oxy)-3-((p iv-
aloyloxy)m eth yl)-3,4-dih ydr oqu in azolin -4-on e (60). A 37%
aqueous solution of formaldehyde (501 µL, 6 mmol) followed
by sodium cyanoborohydride (228 mg, 3.6 mmol) was added
in portions to a solution of 58 (1.21 g, 3 mmol) in a mixture of
THF and methanol (10 mL/10 mL). After the solution was
stirred for 30 min at ambient temperature, the organic solvents
were removed under vacuum and the residue was partitioned
between methylene chloride (20 mL) and water (20 mL). The
organic layer was separated, washed with water and brine,
and dried (MgSO4), and the volatiles were removed by evapo-
ration. The residue was triturated with diethyl ether (5 mL),
and the resulting solid was collected by filtration, washed with
diethyl ether (3 mL), and dried under vacuum to give 1.02 g
1
1
of 60 (82%). H NMR (CDCl3): δ 1.19 (s, 9H), 1.40-1.55 (m,
and dried under vacuum to give 425 mg of 67 (70%). H NMR
2H), 1.90 (d, 2H), 2.0 (t, 2H), 1.85-2.10 (m, 1H), 2.30 (s, 3H),
2.92 (d, 2H), 3.96 (s, 3H), 3.99 (d, 2H), 5.94 (s, 2H), 7.08 (s,
1H), 7.63 (s, 1H), 8.17 (s, 1H). MS-ESI m/z: 418 [MH]+. Anal.
(C22H31N3O5, 0.5 H2O) C, H, N.
7-(2-(1-Meth ylpiper idin -4-yl)eth oxy)-6-m eth oxy-3-((piv-
a loyloxy)m eth yl)-3,4-d ih yd r oqu in a zolin -4-on e (61). To a
solution of 7-(2-(piperidin-4-yl)ethoxy)-6-methoxy-3-((pivaloy-
loxy)methyl)-3,4-dihydroquinazolin-4-one (59; 6 g, 14.4 mmol)
(DMSO-d6; CF3COOD): δ 2.30-2.40 (m, 2H), 3.85 (t, 2H), 4.02
(s, 3H), 4.35 (t, 2H), 7.37 (s, 1H), 7.52-7.64 (m, 2H), 7.82 (d,
1H), 8.09 (s, 1H), 8.88 (s, 1H). MS-ESI m/z: 441 [M.]+.
(E)-4-(P yr r olid in -1-yl)bu t-2-en -1-ol (68). A solution of
4-pyrrolidin-1-ylbut-2-yn-1-ol27 (4.3 g, 31 mmol) in THF (20
mL) was added dropwise to a suspension of lithium aluminum
hydride (2.35 g, 62 mmol) in anhydrous THF (8 mL), and the
mixture was stirred and heated at 60 °C for 2 h. The mixture

4-Anilinoquinazolines with C-7 Basic Side Chains
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1311
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Developmental expression of binding sites and messenger ribo-
nucleic acid for vascular endothelial growth factor suggests a
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was cooled to 5 °C, and 2 M aqueous sodium hydroxide solution
(28 mL) was added dropwise. The resulting suspension was
filtered, and the filtrate was evaporated under vacuum. The
residue was dissolved in a mixture of methylene chloride/ethyl
acetate (20 mL/20 mL) and dried (MgSO4), and the solvent
was removed by evaporation. The residue was purified by
column chromatography on aluminum oxide eluting with
methylene chloride/methanol (97/3) to give 3.09 g of 68 (70%).
1H NMR (CDCl3): δ 1.82 (m, 4H), 2.61 (m, 4H), 3.17 (m, 2H),
4.13 (s, 2H), 5.84 (m, 2H).
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Biologica l Eva lu a tion . IC50 values reported are the aver-
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vitro HUVEC proliferation assay, EGF 0.3 ng/mL was used.
Adenosine 5′-triphosphate (ATP) concentrations used in the
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FGFR1, and erbB2; 0.2 µM for CDK2; 0.8 µM for AKT; 2.5
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MEK. The concentration of ATP used was below the respective
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of homology between the kinase domain of the human KDR
receptor and the murine equivalent (from mus musculus;
similarity >96% and identity > 95%) as well as between the
human Flt-1 receptor and the murine equivalent (from mus
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Ra t a n d Dog P h a r m a cok in etics. Compounds were dosed
as free bases. Rat: 1 and 2 were dosed iv and po to groups of
3 males. Compound 1 was dosed iv at 5 mg/kg using a 2 mg/
mL solution in 25% HP â cyclodextrin and po at 50 mg/kg using
a 2 mg/mL ball-milled suspension in poly(vinylpyrrolidone)
(PVP)/sodium lauryl sulfate (SLS) solution. Compound 2 was
dosed iv at 5 mg/kg using a solution in DMSO (10%), cremo-
phor (10%), and physiological saline (80%) and po at 50 mg/
kg of a suspension of 5 mg/mL in polysorbate 80 (1%). Dog: 1
was dosed iv and po to 4 males, and 2 was dosed iv and po to
3 males. Compound 1 was dosed iv at 5 mg/kg of a 3.5 mg/mL
solution in 45% HP â-cyclodextrin and po at 30 mg/kg of a 10
mg/mL suspension in PVP (1 mg/mL)/SLS (0.2 mg/mL) solu-
tion. Compound 2 was dosed iv at 5 mg/kg of a 5 mg/mL
solution in 45% (w/v) HP â-cyclodextrin in pH 7 Sorenson’s
phosphate buffer and po at 25 mg/kg of a 5 mg/mL milled
suspension in HPMC/polysorbate 80.
(13) Bold, G.; Altmann, K.-H.; Frei, J .; Lang, M.; Manley, P. W.;
Traxler, P.; Wietfeld, B.; Bruggen, J .; Buchdunger, E.; Cozens,
R.; Ferrari, S.; Furet, P.; Hofmann, F.; Martiny-Baron, G.;
Mestan, J .; Rosel, J .; Sills, M.; Stover, D.; Acemoglu, F.; Boss,
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Ack n ow led gm en t. The technical assistance of J a-
net J ackson, Brenda Curry, Cath Grundy, Claire Bar-
nes, J odie Sharples, Rosemary Chester, Sarah Boffey,
Karen Malbon, Paula Valentine, J onathan Mills, Dick
Glarvey, J anet Culshaw, Glynn Chesterton, Steve Glos-
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Francoise Magnien, Sylvie Piva-Leblanc, Nicolas Warin,
Delphine Dorison-Duval, J ean-Paul Zimmerman, and
Christian Delvare is gratefully acknowledged.
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general procedures and the complete experimental procedures
for the synthesis of as well as the corresponding 1H NMR
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15, 17, 18, 20, 23-26, 29-31, 35-37, 41-43, 46-49, 59, 63,
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