Tyrosine kinase inhibitors. 13. Structure - Activity relationships for soluble 7-substituted 4-[(3-bromophenyl)amino]pyrido[4,3-d]pyrimidines designed as inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor

Article abstract of DOI:10.1021/jm970366v

The general class of 4-(phenylamino)quinazolines are potent (some members with IC₅₀ values << 1 nM) and selective inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), via competitive binding at the ATP site of the enzyme, but many of the early analogues had poor aqueous solubility (<<1 mM). A series of 7-substituted 4- [(3-bromophenyl)-amino]pyrido[4,3-d]pyrimidines, together with selected (3- methylphenyl)amino analogues, were prepared by reaction of the analogous 7- fluoro derivatives with appropriate amine nucleophiles in 2-BuOH or aqueous 1-PrOH. All of the compounds were evaluated for their ability to inhibit the tyrosine-phosphorylating action of EGF-stimulated full-length EGFR enzyme. Selected analogues were also evaluated for their inhibition of autophosphorylation of the EGF receptor in A431 human epidermoid carcinoma cells in culture and against A431 tumor xenografts in mice. Analogues bearing a wide variety of polyol, cationic, and anionic solubilizing substituents retained activity, but the most effective in terms of both increased aqueous solubility (>40 mM) and retention of overall inhibitory activity (IC₅₀'s of 0.5-10 nM against isolated enzyme and 8-40 nM for inhibition of EGFR autophosphorylation in A431 cells) were weakly basic amine derivatives. These results are broadly consistent with a proposed model for the binding of these compounds to EGFR, in which the 6- and 7-positions of the pyridopyrimidine ring are in a largely hydrophobic binding region of considerable steric freedom, at the entrance of the adenine binding cleft. The most active cationic analogues have a weakly basic side chain where the amine moiety is three or more carbon atoms away from the nucleus. Two of the compounds (bearing weakly basic morpholinopropyl and strongly basic (dimethylamino)butyl solubilizing groups) produced in vivo tumor growth delays of 13-21 days against advanced stage A431 epidermoid xenografts in nude mice, when administered ip twice per day on days 7-21 posttumor implant. Treated tumors did not increase in size during therapy and resumed growth at the termination of therapy, indicating an apparent cytostatic effect for these compounds under these treatment conditions. The data suggest that continuous long-term therapy with these compounds may result in substantial tumor growth inhibition.

Full text of DOI:10.1021/jm970366v

J . Med. Chem. 1997, 40, 3915-3925
3915
Tyr osin e Kin a se In h ibitor s. 13. Str u ctu r e-Activity Rela tion sh ip s for Solu ble
7-Su bstitu ted 4-[(3-Br om op h en yl)a m in o]p yr id o[4,3-d ]p yr im id in es Design ed a s
In h ibitor s of th e Tyr osin e Kin a se Activity of th e Ep id er m a l Gr ow th F a ctor
Recep tor
Andrew M. Thompson,† Donna K. Murray,† William L. Elliott,‡ David W. Fry,‡ J ames A. Nelson,‡
H. D. Hollis Showalter,‡ Bill J . Roberts,‡ Patrick W. Vincent,‡ and William A. Denny*,†
Cancer Society Research Laboratory, Faculty of Medicine and Health Science, The University of Auckland,
Private Bag 92019, Auckland, New Zealand, and Parke-Davis Pharmaceutical Research, Division of Warner-Lambert
Company, 2800 Plymouth Road, Ann Arbor, Michigan 48106-1047
Received J une 4, 1997X
The general class of 4-(phenylamino)quinazolines are potent (some members with IC50 values
, 1 nM) and selective inhibitors of the tyrosine kinase activity of the epidermal growth factor
receptor (EGFR), via competitive binding at the ATP site of the enzyme, but many of the early
analogues had poor aqueous solubility (,1 mM). A series of 7-substituted 4-[(3-bromophenyl)-
amino]pyrido[4,3-d]pyrimidines, together with selected (3-methylphenyl)amino analogues, were
prepared by reaction of the analogous 7-fluoro derivatives with appropriate amine nucleophiles
in 2-BuOH or aqueous 1-PrOH. All of the compounds were evaluated for their ability to inhibit
the tyrosine-phosphorylating action of EGF-stimulated full-length EGFR enzyme. Selected
analogues were also evaluated for their inhibition of autophosphorylation of the EGF receptor
in A431 human epidermoid carcinoma cells in culture and against A431 tumor xenografts in
mice. Analogues bearing a wide variety of polyol, cationic, and anionic solubilizing substituents
retained activity, but the most effective in terms of both increased aqueous solubility (>40
mM) and retention of overall inhibitory activity (IC50’s of 0.5-10 nM against isolated enzyme
and 8-40 nM for inhibition of EGFR autophosphorylation in A431 cells) were weakly basic
amine derivatives. These results are broadly consistent with a proposed model for the binding
of these compounds to EGFR, in which the 6- and 7-positions of the pyridopyrimidine ring are
in a largely hydrophobic binding region of considerable steric freedom, at the entrance of the
adenine binding cleft. The most active cationic analogues have a weakly basic side chain where
the amine moiety is three or more carbon atoms away from the nucleus. Two of the compounds
(bearing weakly basic morpholinopropyl and strongly basic (dimethylamino)butyl solubilizing
groups) produced in vivo tumor growth delays of 13-21 days against advanced stage A431
epidermoid xenografts in nude mice, when administered ip twice per day on days 7-21
posttumor implant. Treated tumors did not increase in size during therapy and resumed growth
at the termination of therapy, indicating an apparent cytostatic effect for these compounds
under these treatment conditions. The data suggest that continuous long-term therapy with
these compounds may result in substantial tumor growth inhibition.
The 4-(phenylamino)quinazolines (e.g., 1, 2) are known
to be potent and selective inhibitors of the tyrosine
kinase activity of the epidermal growth factor receptor
(EGFR), via competitive binding at the ATP site of the
enzyme.1-7 These compounds are of interest as poten-
tial anticancer drugs. The EGFR and related members
of this family such as erbB2 constitute the starting
points for many signal transduction pathways in cells,
and their overexpression is an indicator of a poor
rido[4,3-d]pyrimidines (e.g., 3a -c), were also potent
prognosis in a number of human cancers, including
mammary,8,9 ovarian,10 esophageal,11 and squamous cell
head and neck carcinomas.12 Structure-activity rela-
tionships (SAR) for this class of compounds showed the
desirability of a 3-bromo substituent on the phenyl ring
and of small electron-donating groups off the long axis
(at positions 6 and/or 7) of the quinazoline.2,3
inhibitors of the isolated enzyme but with significantly
different SAR for enzyme inhibition. In particular,
while the 7-aminopyrido[4,3-d]pyrimidine (3a ) was con-
siderably less potent than the corresponding 7-amino-
quinazoline (2a ) against the isolated enzyme, (IC50’s of
10 and 0.1 nM, respectively), the 7-(methylamino)-
pyrido[4,3-d]pyrimidine (3b) was much more effective
than the corresponding 7-(methylamino)quinazoline
analogue (2b) (IC50’s of 0.13 and 4 nM, respectively).
The impressive enzyme inhibitory potency of these
compounds made them of interest for in vivo evaluation,
but their poor aqueous solubility (similar to that of the
original quinazolines) has made this difficult.14
We later showed13 that related 4-[(3-bromophenyl)-
amino]pyrido[d]pyrimidines, including 7-substituted py-
† The University of Auckland.
‡ Parke-Davis Pharmaceutical Research.
X Abstract published in Advance ACS Abstracts, November 1, 1997.
S0022-2623(97)00366-X CCC: $14.00 © 1997 American Chemical Society

3916 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Thompson et al.
Sch em e 1a
Sch em e 2a
a
(i) BnNHMe/MeOH/reflux/2 h; (ii) Pd/C/H2/MeOH/20 °C/18 h.
Sch em e 3a
a
(i) SOCl2/reflux/3.5 h, then 3-bromoaniline or 3-methylaniline/
CH2Cl2/2-PrOH/20 °C/16 h; (ii) R1R2NH/2-BuOH or n-PrOH-
water/90-100 °C/16 h-8 days.
Model-building studies15 suggest that the adenine of
ATP binds in a cleft between the N- and C-terminal
lobes, with the adenine forming H-bonds between N-1
and the backbone amide of Met-769 and between N-6
and the backbone carbonyl of Gln-767. The 4-(phenyl-
amino)quinazolines and pyridopyrimidines are sug-
gested to bind similarly, placing the bicyclic chro-
mophore in the adenine pocket, with N-1 H-bonding
again to Met-769 and N-3 to the side chain of Thr-766
on strand 5 deep in the binding cleft. The high potency
and selectivity of these compounds for EGFR may be
due to an increased hydrophobic binding of the phenyl-
amino side chain to a unique hydrophobic region in the
enzyme, adjacent to the ATP binding pocket and formed
in part by three additional sulfur-containing amino
acids (Cys-751, Met-769, and Met-742). In this model,
the 6- and 7-positions of the quinazoline or pyridopyri-
midine ring lie in the entrance of the adenine binding
cleft, providing some steric freedom.
With the above indications for steric tolerance at the
7-position of the 4-[(3-bromophenyl)amino]pyrido[4,3-
d]pyrimidines, we sought to develop soluble analogues
of 3b,c by the attachment of solubilizing functions via
an alkylamine at this position and report here the
synthesis, aqueous solubilities, and SAR for these
compounds, together with in vivo data for two ana-
logues.
a
(i) Morpholine/EtOH/Et3N/reflux/19 h, then NH2NH2‚H2O/
EtOH/reflux/6 h, then BnOCOCl/THF/Et3N/20 °C/15 h; (ii) Pd/C/
H2/MeOH/20 °C/1 day.
out of the reaction mixture in good purity directly. This
compound was slightly unstable in solution and partly
decomposed during conversion to its hydrochloride salt.
The N-oxide 3t was obtained in excellent yield from 3r
by selective oxidation of the side chain aliphatic tertiary
nitrogen using Davis’ reagent, 2-(phenylsulfonyl)-3-
phenyloxaziridine.16
Most of the required amines were commercially
available. 3-(Methylamino)propane-1,2-diol (10) was
prepared by amination of glycidol (8) with N-benzyl-
methylamine followed by catalytic hydrogenolysis of the
benzyl group (5% Pd/C) (Scheme 2). 4-(4-Aminobutyl)-
morpholine17 (13) was prepared by alkylation of mor-
pholine with 4-bromobutylphthalimide (11) followed by
standard cleavage of the phthalimide group with hy-
drazine and purification of the resulting amine as its
benzyloxycarbonyl derivative 12 (Scheme 3).
Resu lts a n d Discu ssion
The structures of the 7-substituted 4-[(3-bromophe-
nyl)amino]pyrido[4,3-d]pyrimidines (3a -ee) prepared
are recorded in Table 1. They were evaluated for their
ability to inhibit tyrosine phosphorylation of a 14-
residue polypeptide (a portion of phospholipase C-γ1)
by EGF-stimulated full-length EGFR enzyme isolated
from A431 cells.1 At least two complete dose-response
curves were determined for each compound, and aver-
aged IC50’s are listed in Table 1. Selected compounds
were also evaluated for their ability to inhibit autophos-
phorylation of the EGF receptor in A431 human epi-
dermoid carcinoma cells.
Three types of solubilizing 7-substituents were
investigated: neutral (alcohols and polyols, 3d -k ),
cationic (amines, 3l-z), and anionic (acids, 3a a -ee).
Because both the methylamino and dimethylamino
parent compounds 3b,c proved equally effective, three
pairs of compounds with NHR and corresponding N(Me)R
neutral side chains were evaluated (3d /3e, 3f/3g, and
3j/3k ). In the first two cases, while the NHR compounds
had IC50’s below 1 nM, the N(Me)R derivatives were
significantly less effective, and in the third case both
compounds were similarly less effective (IC50’s of 3-5
nM). These three pairs explored the use of mono-, di-,
and pentols, respectively. The diols 3f,g proved the
most soluble, but only 10-15-fold more than the parent
compounds. Surprisingly, the pentols 3j,k were slightly
less soluble than the parent compounds, possibly due
to tight internal H-bonding. The other two examples
(3h ,i) possess branched diol side chains of much larger
Ch em istr y
The 7-substituted 4-[(3-bromo- and 3-methylphenyl)-
amino]pyrido[4,3-d]pyrimidines (3 and 4) of Table 1
were prepared from the known13 4-[(3-bromophenyl)-
amino]-7-fluoropyrido[4,3-d]pyrimidine, 6, or its methyl
analogue 7 (made by reaction of the known13 7- fluoro-
pyrido[4,3-d]pyrimidin-4-one, 5, with thionyl chloride
followed by 3-methylaniline) (Scheme 1). Displacement
of the fluorine atom in 6 and 7 with appropriate amine
nucleophiles was normally conducted in 2-BuOH at 95
°C for 1-5 days, although for very polar amines (e.g.,
N-methyl-D-glucamine), aqueous 1-PrOH was a more
effective solvent. The products from reactions with
amino alcohols were generally solids which could be
isolated and crystallized directly (method A). Crude
products from reactions with diamines were generally
oils which required basic workup and chromatography
(method B). In the case of amino acid nucleophiles, it
was necessary to preform their sodium salts prior to
reaction (method C).
The above conditions were not successful with 6 and
hydrazine as the nucleophile, since its greater reactivity
led to the formation of a complex mixture of products.
However, by allowing the reaction to proceed over
several days at 20 °C, the desired product 3x crystallized

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3917
Ta ble 1. Structural and Biological Properties of Soluble 7-Substituted 4-[(3-Bromophenyl)amino]pyrido[4,3-d]pyrimidines and
Selected (3-Methylphenyl)amino Analogues
IC50 (nM)
no.
3a
3b
3c
R
mp
ref 13
ref 13
ref 13
formula
Parent
anal.
solubilitya (mM)
Eb
10
0.13
0.09
Ac
NH2
NHMe
NMe2
110
16
14
0.15
0.13
Alcohols
C15H14BrN5O
C16H16BrN5O
C16H16BrN5O2
C17H18BrN5O2
C16H16BrN5O2
C17H18BrN5O2‚H2O
C19H22BrN5O5‚H2O
C20H24BrN5O5
3d
3e
3f
3g
3h
3i
NHCH2CH2OH
N(Me)CH2CH2OH
NHCH2CH(OH)CH2OH
N(Me)CH2CH(OH)CH2OH
NHCH(CH2OH)2
N(CH2CH2OH)2
NHCH2(CHOH)4CH2OH
N(Me)CH2(CHOH)4CH2OH
218-219
232-235
222.5-224.5
232-233
168-171
199-201
207-208
224.5 dec
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
0.40
0.06
1.6
1.9
2.2
0.96
0.10
0.12
0.24
2.6
0.92
3.2
14
12
4.8
3.2
227
3j
3k
Amines
C17H19BrN6
C18H21BrN6
C19H23BrN6‚H2O
C20H25BrN6
3l
NH(CH2)2NMe2
NH(CH2)3NMe2
NH(CH2)4NMe2
NH(CH2)5NMe2
207-208
196-198
171-174
123-126
195-196
250-253
173-174
149-151
189-190
111-112.5
121.5-124.5
144-146
220 dec
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
H,N;Cg
C,H,N
C,H,N
C,H,N
C,H,N
H,N;Cg
C,H,N
C,H,N
C,H,N
C,H,N
>40
>40
>40
37
>40
>40
>40
>40
33
>40
36
>40
<18
>40
35
45
3m
3n
3o
3p
3q
3r
8.8
7.4
8.4
40
3.2
1.9
5.4
0.74
4.9
9.2
38
36
N(Me)(CH2)2NMe2
NH(CH2)2Nmorphd
NH(CH2)3Nmorphd
NH(CH2)4Nmorphd
NH(CH2)3N(O)morphd
NH(CH2)3NMepipe
NH(CH2)2N(CH2CH2OH)2
NH(CH2)3N(CH2CH2OH)2
NHNH2
C18H21BrN6
C19H21BrN6O
C20H23BrN6O
C21H25BrN6O
C20H23BrN6O2‚2H2O
C21H26BrN7‚H2O
C19H23BrN6O2‚H2O
C20H25BrN6O2
C13H11BrN6
8.1
3s
3t
>1000
3u
3v
3w
3x
3y
3z
1.2
7.1
0.51
0.91
NH(CH2)3(1-imid)f
NH(CH2)2(4-imid)f
231-232.5
246-249
C19H18BrN7
C18H16BrN7
Acids
C15H12BrN5O2
C16H14BrN5O2
C17H16BrN5O2‚1.5H2O
C16H14BrN5O2‚H2O
C15H14BrN5O3S
3a a
3bb
3cc
3d d
3ee
NHCH2COOH
230-236 dec
241-243
H,N;Cg
C,H,N
C,N;Hh
C,H,N
C,H
7.4
>40
32
28
>40
1.5
0.61
0.28
16
1.4
NHCH2CH2COOH
NH(CH2)3COOH
N(Me)CH2COOH
NHCH2CH2SO3H
11400
230-233
215 dec
338-345 dec
>1000
3′-Me Amines
4b
4n
4r
4u
4y
4z
NHMe
275-277
C
15H15N5
C,H,N
C,H,N
C,H,N
C,H,N
C,N;Hi
C,H,N
1.3
>40
>40
>40
>34
>39
1.4
5.4
9.3
5.6
3.5
7.2
NH(CH2)4NMe2
NH(CH2)3Nmorphd
NH(CH2)3NMepipe
NH(CH2)3(1-imid)f
NH(CH2)2(4-imid)f
178-180
C20H26N6‚0.25H2O
C21H26N6O
C22H29N7‚0.25H2O
C20H21N7‚0.25H2O
C19H19N7
181.5-182.5
142-145
158-159
216-216.5
a
Solubility in water or aqueous buffer at 20 °C, determined by HPLC (see text). Values are for the hydrochloride salt form of amines
b
and the sodium salt form of acids. IC50, concentration of drug (nM) to inhibit the phosphorylation of a polyglutamic acid/tyrosine random
copolymer by EGFR (prepared from human A431 carcinoma cell vesicles by immunoaffinity chromatography). See Experimental Section
for details. Values are the averages from at least two independent dose-response curves; variation was generally (15%. c IC50’s for inhibition
of autophosphorylation of EGFR in A431 cells in culture. Values are the average of two experiments; see Experimental Section for details.
d
g
h
i
N-Morpholine derivative. e 4-Methylpiperazin-1-yl. f Imidazolyl. C out by 0.5. H out by 0.5. H out by 0.6.
cross-sectional area than the other examples. While the
NHCH(CH2OH)2 derivative 3h showed the highest
solubility of all the polyol side chain analogues (2.2 mM),
3h ,i were the least potent of the polyols (IC50’s of 14
and 12 nM, respectively), suggesting some steric limita-
tion due to the width of the binding site. Diol 3f, which
was one of the more potent in the isolated enzyme assay,
was evaluated in the autophosphorylation assay but was
relatively ineffective (IC50 ) 227 nM), possibly because
of poor cellular uptake.
salts), with all but five being >40 mM (Table 1).
Compounds 3l-o explore a homologous series of strongly
basic side chains (pKa ca. 10),18 where activity against
the isolated enzyme improves steadily as the cationic
charge is shifted away from the ring for a certain
distance, leveling off only after the chain is four-carbon
atoms long. In this case the pair of NHR and N(Me)R
compounds (3l,p ) shows similar (albeit relatively low)
potency. The more weakly basic morpholide analogues
3q-s (pKa ca. 7)18 proved much more effective against
the isolated enzyme. Potency against the isolated
enzyme again increased as the charge was shifted away
As expected, the cationic derivatives 3l-z showed
much greater aqueous solubility (as the hydrochloride

3918 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Thompson et al.
Ta ble 2. In Vivo Anticancer Activity of 3n ,r against the A431 Epidermoid Carcinoma and a Mouse Fibroblast Line Transfected with
the h-EGF Receptor
tumor systema
compd
dose (mg/kg)
scheduleb
wt loss (g)
T/Cc (%)
T-C (days)d
net log cell kille
A431
A431
EGFR
EGFR
3r
3n
3r
25
25
25
25
b.i.d. days 7-21
b.i.d. days 7-21
b.i.d. days 3-17
b.i.d. days 1-15
1.7
1.0
0.9
1.0
34
11
85
69
12.8
20.5
0
-0.1
-0.1
3n
2.3
-1.1
a
b
Tumor fragments of A431 or EGFR were implanted sc into the right axilla of mice on day 0. All treatments were ip on the indicated
schedules. The maximum tolerated dose (ELD10) from a complete dose-response is shown. c Ratio of median treated tumor mass (mg) on
d
the last day of therapy divided by the median control tumor mass × 100%. The difference in days for the treated (T) and the control (C)
tumors to reach 750 mg. e The net reduction in tumor burden, in logs, between the first and last treatments.
from the ring, with the C3 analogue 3r being the best.
The morpholide N-oxide derivative 3t was prepared as
an example of a neutral dipolar compound and proved
the most potent of the morpholide derivatives against
the isolated enzyme (IC50 ) 0.74 nM). The piperazine
derivative 3u employed a different weak base (pKa ca.
8)18 but was less effective than the morpholide of similar
chain length. The amine diols 3v,w were prepared to
further increase water solubility using a weak base (pKa
ca. 8.5),18 and the longer side-chain analogue (3w ) was
again the more effective against the isolated enzyme.
The hydrazine 3x was investigated as an example of a
nonbulky weak base but had relatively poor solubility
and stability and only moderate potency. The imida-
zoles 3y,z (pKa’s ca. 7.5)18 were the most potent of all
the amine analogues, with IC50’s below 1 nM for
inhibition of the isolated enzyme.
F igu r e 1. Effect of 3n ,r on the growth of the A431 epidermoid
xenograft in nude mice. Tumors were implanted sc on day 0.
Intraperitoneal treatments were administered b.i.d. on days
7-21. Both compounds were dosed at 25 mg/kg/injection,
which was the maximum tolerated dose (ELD10) obtained from
a complete dose-response.
Representative analogues were tested in the cellular
autophosphorylation assay in A431 cells. The mor-
pholide 3r was the most potent (IC50 ) 8 nM; 2 times
as effective as the parent compound 3b, despite the fact
that 3b is 15-fold more potent than 3r in the isolated
enzyme assay). The more weakly basic but dipolar
morpholine N-oxide 3t had low activity in this assay
(IC50 > 1000 nM), again suggesting poor uptake by cells.
Compounds 3a a -ee were prepared to study the
utility of anionic side chains. All but the glycine
(NHCH2COOH) analogue 3a a had adequate aqueous
solubility as the sodium salts. The NH(CH2)nCOOH
series (3a a -cc) showed excellent activity against the
isolated enzyme, with 3cc being one of the most potent
7-NH-substituted compounds (IC50 ) 0.28 nM). As
shown above for the alcohols, the NMe analogue 3d d
was much less effective than the corresponding NH
derivative 3a a . The sulfonic acid 3ee, made as an
example of a fully ionized acid (pKa ca. 1),19 maintained
high potency in the isolated enzyme assay. However,
the low activity of both 3bb (IC50 ) 11 400 nM) and 3ee
(IC50 > 1000 nM) in the cellular autophosphorylation
assay suggests that these acids have difficulty getting
into cells. A similar phenomenon was reported earlier
for indolinethione acids20 and lavendustin analogues21
evaluated as EGFR inhibitors.
9-fold more soluble than 3b. A recent study of analo-
gous pyrimido[5,4-d]pyrimidines showed similar fea-
tures.23
Overall, the cationic derivatives (3l-z) were the most
effective. Accordingly, two of these (the strongly basic
(dimethylamino)butyl analogue 3n and the weakly basic
morpholinopropyl analogue 3r ) were selected for pre-
liminary in vivo studies against two different tumor
xenografts in nude mice. The A431 epidermoid xe-
nograft was selected based on its in vitro mitogenic
responsiveness to EGF and the inhibition of growth on
plastic by anti-EGF receptor monoclonal antibodies. The
EGFR cell line was selected due to its expression of the
transformed phenotype upon transfection with the
human EGF receptor and its EGF requirement for clone
formation in soft agar. Both compounds were measur-
ably active against the A431 xenograft, as evidenced by
tumor growth delays in the range of 13-21 days (Table
2 and Figure 1). There was, however, no apparent
tumor burden reduction against this model, based on
the near-zero net kill values. In contrast, both com-
pounds were ineffective against the EGFR tumor model
on the basis of tumor growth delay, percent T/C, and
net log cell kill (Table 2). Weight loss data indicated
that at maximum tolerated doses both compounds
induced a similar amount of treatment-related weight
loss.
Finally, a small series of (3-methylphenyl)amino
analogues (4b,n ,r ,u ,y,z) were prepared for comparison
with their 3-bromophenyl counterparts. This substitu-
ent group is moderately effective in (phenylamino)-
quinazoline5 and (phenylamino)pyrrolopyrimidine EGFR
inhibitors,22 resulting in more soluble compounds, but
with 5-20-fold lower potency. Of the six 3′-Me deriva-
tives, two (4n ,u ) had IC50’s similar to their bromo
counterparts, but four (4b,r ,y,z) were 5-10-fold less
potent. The parent NHMe derivative 4b was, however,
Con clu sion s
The above results show that a wide variety of solu-
bilizing substituents are tolerated at the 7-position of
the 4-[(3-bromophenyl)amino]pyrido[4,3-d]pyrimidine
nucleus, as determined by the ability of the compounds

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3919
morpholine (2.00 mL, 23.0 mmol), and Et3N (5.00 mL, 35.9
mmol) in absolute EtOH (50 mL) was refluxed for 19 h and
then cooled. Hydrazine hydrate (2.00 mL, 41.2 mmol) and
absolute EtOH (50 mL) were added; then the mixture was
refluxed for 6 h, cooled, and filtered, washing the precipitate
with absolute EtOH. The solvent was removed from the
filtrate and the residue suspended in dry THF (100 mL) and
Et3N (20 mL) and cooled in ice. Benzyl chloroformate (15 mL
of a 50% solution in toluene, 52.5 mmol) was added dropwise
with stirring; then the mixture stirred at 20 °C for 15 h.
Excess reagent was quenched with MeOH; then the solvents
were removed under reduced pressure. The residue was
diluted with water, and the resulting solution was acidified
to pH 1 (dilute HCl), washed with CH2Cl2, then treated with
excess Na2CO3 (to pH 10), and extracted with EtOAc (4 × 100
mL). Removal of the solvent and chromatography of the
residue on silica gel, eluting with 1-3% MeOH/CH2Cl2, gave
N-(benzyloxycarbonyl)-4-(4-aminobutyl)morpholine (12) (3.04
g, 59% overall): mp (CH2Cl2/light petroleum) 45-46.5 °C; 1H
NMR [(CD3)2SO] δ 7.37-7.28 (m, 5 H, ArH), 5.58 (br s, 1 H,
NH), 5.09 (s, 2 H, OCH2), 3.70 (t, J ) 4.6 Hz, 4 H, (CH2)2O),
3.20 (q, J ) 6.0 Hz, 2 H, NHCH2), 2.41 (m, 4 H, (CH2)2N), 2.34
(t, J ) 6.7 Hz, 2 H, NCH2), 1.55 (m, 4 H, 2CH2); 13C NMR δ
156.43 (s, OCONH), 136.68 (s, Ar), 128.47 (d, 2 C, Ar), 128.08
(d, Ar), 128.03 (d, 2 C, Ar), 66.82 (t, 2 C, (CH2)2O), 66.50 (t,
OCH2), 58.46 (t, NCH2), 53.61 (t, 2 C, N(CH2)2), 40.95 (t,
CH2N), 27.89, 23.92 (2 t, 2CH2). Anal. (C16H24N2O3) C, H, N.
Hydrogenation of 12 with 5% Pd/C in MeOH at 60 psi for
24 h, followed by workup as above, gave 13 as an oil (lit.17),
which was used without further purification: 1H NMR [(CD3)2-
SO] δ 3.55 (t, J ) 4.7 Hz, 4 H, (CH2)2O), 2.54 (br t, J ) 6.6 Hz,
2 H, NH2CH2), 2.31 (br t, J ) 4.6 Hz, 4 H, (CH2)2N), 2.23 (t, J
) 7.2 Hz, 2 H, NCH2), 1.47-1.30 (m, 4 H, 2CH2).
7-F lu or o-4-[(3-m eth ylp h en yl)a m in o]p yr id o[4,3-d ]p yr i-
m id in e (7) (Sch em e 1). A suspension of 7-fluoropyrido[4,3-
d]pyrimidin-4(3H)-one13 (5) (457 mg, 2.77 mmol) in SOCl2 (25
mL) containing 2 drops of DMF was stirred under reflux for
3.5 h and then concentrated under reduced pressure to give
crude 4-chloro-7-fluoropyrido[4,3-d]pyrimidine as an oil. This
was cooled in ice, and a solution of 3-methylaniline (1.0 mL,
9.35 mmol) in CH2Cl2 (50 mL) was added followed by dry
2-PrOH (40 mL). The mixture was stirred at 20 °C for 16 h,
then solvents were removed under reduced pressure, and the
residue was diluted with aqueous Na2CO3 (100 mL) and
extracted with EtOAc (3 × 100 mL). Chromatography of this
extract on silica gel, eluting with 1% MeOH/CHCl3, gave 7 (648
mg, 92%): mp (MeOH/CHCl3/light petroleum) 212-213.5 °C;
1H NMR [(CD3)2SO] δ 10.35 (s, 1 H, NH), 9.61 (s, 1 H, H-5),
8.66 (s, 1 H, H-2), 7.60 (m, 2 H, H-2′,6′), 7.36 (s, 1 H, H-8),
7.32 (dd, J ) 8.4, 7.8 Hz, 1 H, H-5′), 7.03 (d, J ) 7.6 Hz, 1 H,
H-4′), 2.35 (s, 3 H, 3′-CH3); 13C NMR δ 164.72 (d, J C-F ) 237
Hz, C-7), 159.35 (d, C-2), 158.01 (s, C-4), 157.32 (d, J C-F ) 13
Hz, C-8a), 148.37 (dd, J C-F ) 19 Hz, C-5), 137.85, 137.78 (2 s,
C-1′,3′), 128.38 (d, C-5′), 125.44 (d, C-4′), 123.47 (d, C-2′),
120.21 (d, C-6′), 110.56 (d, J C-F ) 3 Hz, C-4a), 102.87 (dd, J C-F
) 35 Hz, C-8), 21.05 (q, CH3). Anal. (C14H11FN4) C, H, N.
4-[(3-Br om op h en yl)a m in o]-7-[(2,3-d ih yd r oxyp r op yl)-
a m in o]p yr id o[4,3-d ]p yr im id in e (3f). Exa m p le of Gen -
er a l Meth od A. A solution of 4-[(3-bromophenyl)amino]-7-
fluoropyrido[4,3-d]pyrimidine13 (6) (101 mg, 0.32 mmol) and
3-aminopropane-1,2-diol (0.60 mL, 7.74 mmol) in 2-BuOH (10
mL) was stirred at 100 °C for 3 days. The resulting solution
was concentrated under reduced pressure and cooled to give
a solid, which was collected by filtration and washed well with
water. Recrystallization from DMSO/water gave 3f (100 mg,
81%): mp (DMSO/water) 222.5-224.5 °C; 1H NMR [(CD3)2-
SO] δ 9.89 (br s, 1 H, NH), 9.35 (s, 1 H, H-5), 8.45 (s, 1 H,
H-2), 8.19 (s, 1 H, H-2′), 7.84 (d, J ) 8.0 Hz, 1 H, H-6′), 7.34
(t, J ) 8.0 Hz, 1 H, H-5′), 7.29 (br d, J ) 8.1 Hz, 1 H, H-4′),
7.05 (br t, J ) 5.7 Hz, 1 H, 7-NHCH2), 6.50 (s, 1 H, H-8), 4.88
(d, J ) 4.9 Hz, 1 H, CHOH), 4.64 (t, J ) 5.7 Hz, 1 H, CH2OH),
3.67 (m, 1 H, CHOH), 3.5-3.15 (m, 4 H, 7-NHCH2CH(OH)-
CH2OH); 13C NMR δ 160.99 (s, C-7), 157.94 (d, C-2), 157.71
(s, C-4), 154.57 (s, C-8a), 148.23 (d, C-5), 140.72 (s, C-1′), 130.39
(d, C-5′), 126.03 (d, C-4′), 124.27 (d, C-2′), 121.15 (s, C-3′),
120.79 (d, C-6′), 103.76 (s, C-4a), 95.80 (br d, C-8), 70.23 (d,
to inhibit tyrosine phosphorylation of a substrate by
EGF-stimulated full-length EGFR enzyme. While poly-
hydroxy (neutral), amino (cationic), and carboxylic acid
(anionic) groups were all tolerated, the most effective
in terms of aqueous solubility and retention of both
enzyme and cellular activity were weakly basic amine
derivatives. These results are broadly interpretable in
terms of the proposed binding model,15 which indicates
considerable bulk tolerance (in the entrance of the
adenine binding cleft of the enzyme) for substituents
at the 7-position. The above SAR for cationic deriva-
tives suggests that this is largely a hydrophobic binding
region, with preferred activity for analogues where the
amine group is either sufficiently far away from the
nucleus (e.g., 3n ) or a sufficiently weak base that a
significant proportion of neutral form may be present
(e.g., 3r ).
The soluble analogues 3n ,r gave substantial growth
delays in A431 xenografts, but not in the EGFR tumor
model, despite the fact that the EGFR cell line has a
lower number of EGFR targets per cell than the A431
(200 000-300 000 versus several million). Thus, overall
receptor number is probably not responsible for the lack
of in vivo effectiveness against the EGFR cell line. It
is possible that, while this cell line requires EGF for
clone formation in soft agar, it may not absolutely
require EGF in vivo, where other growth factors may
take over. This is supported by a similar lack of an
effect with anti-EGFR monoclonal antibodies against
this tumor model in vivo.24 The treated A431 tumors
began to increase in size at the end of therapy, and it is
not clear whether longer periods of therapy would
produce greater apparent antitumor effects by main-
taining tumor growth suppression. The A431 tumors
treated with 3n did not appear to decrease in size during
treatment, indicating that these compounds are cyto-
static rather than cytotoxic under these conditions. It
remains to be determined if prolonged therapy will
ultimately result in a cytotoxic effect in vivo (reflected
by a positive net cell kill).
Exp er im en ta l Section
Analyses were performed by the Microchemical Laboratory,
University of Otago, Dunedin, NZ. Melting points were
determined using an Electrothermal model 9200 digital melt-
ing point apparatus and are as read. NMR spectra were
measured on Bruker AC-200 or DRX-400 spectrometers and
referenced to Me4Si. HPLC was carried out using a Bondclone
10 C18 reverse-phase silica gel column, with
a Phillips
PU4100M gradient elution pump and a Phillips PU 4120 diode
array detector, eluting with the appropriate ratios of 80%
MeCN/20% water (solvent A) and ammonium formate buffer
(solvent B; 28 g of ammonium formate + 2.55 mL of formic
acid, made up to 1 L in deionized water, pH 4.5).
3-(Meth yla m in o)p r op a n e-1,2-d iol (10) (Sch em e 2). A
solution of racemic glycidol (8) (5.00 mL, 0.075 mol) and
N-benzylmethylamine (9.25 mL, 0.072 mol) in MeOH (100 mL)
was heated under reflux for 2 h and then cooled. The resulting
solution of crude 3-(N-benzyl-N-methylamino)propane-1,2-diol
(9) was hydrogenated over 5% Pd/C (2 g) at 50 psi for 18 h
and then filtered through Celite, washing with MeOH. Evapo-
ration of the combined filtrates gave 10 as a viscous liquid,
which was used without further purification: 1H NMR (CDCl3)
δ 3.79 (ddt, J ) 7.2, 5.1, 3.9 Hz, 1 H, CHOH), 3.70 (dd, J )
11.4, 3.7 Hz, 1 H, CHOH), 3.59 (dd, J ) 11.4, 5.2 Hz, 1 H,
CHOH), 2.73 (dd, J ) 12.1, 4.0 Hz, 1 H, NCH), 2.66 (dd, J )
12.2, 7.3 Hz, 1 H, NCH), 2.44 (s, 3 H, CH3).
4-(4-Am in obu tyl)m or p h olin e (13) (Sch em e 3). A solu-
tion of 4-bromobutylphthalimide (11) (5.00 g, 17.7 mmol),

3920 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Thompson et al.
CHOH), 63.83 (t, CH2OH), 44.78 (t, NCH2). Anal. (C16H16
BrN5O2) C, H, N.
The following analogues were prepared similarly.
-
(br d, J ) 8.0 Hz, 1 H, H-4′), 7.01 (br s, 1 H, NHCH2), 6.50 (s,
1 H, H-8), 4.92 (d, J ) 4.7 Hz, 1 H, CHOH), 4.50 (d, J ) 5.1
Hz, 1 H, CHOH), 4.46 (d, J ) 5.6 Hz, 1 H, CHOH), 4.38 (d, J
) 6.7 Hz, 1 H, CHOH), 4.35 (t, J ) 5.6 Hz, 1 H, CH2OH), 3.82,
3.70, 3.60 (3 m, 3 × 1 H, 3CH), 3.50 (m, 3 H, 3CH), 3.41, 3.27
(2 m, 2 × 1 H, 2CH); 13C NMR δ 160.92 (s, C-7), 157.86 (d,
C-2), 157.62 (s, C-4), 154.58 (s, C-8a), 148.19 (d, C-5), 140.72
(s, C-1′), 130.32 (d, C-5′), 125.91 (d, C-4′), 124.16 (d, C-2′),
121.09 (s, C-3′), 120.67 (d, C-6′), 103.69 (s, C-4a), 95.50 (br d,
C-8), 72.04 (d, CHOH), 71.42 (d, 2 C, 2CHOH), 69.62 (d,
4-[(3-Br om op h en yl)a m in o]-7-[(2-h yd r oxyeth yl)a m in o]-
p yr id o[4,3-d ]p yr im id in e (3d ): 78%, by reaction of 6 with
2-aminoethanol (20 equiv) in 2-BuOH (95 °C for 4 days),
followed by chromatography of the product on silica gel, eluting
with 5-7% MeOH in CH2Cl2; mp (MeOH/CH2Cl2) 218-219 °C;
1H NMR [(CD3)2SO] δ 9.89 (br s, 1 H, NH), 9.36 (s, 1 H, H-5),
8.45 (s, 1 H, H-2), 8.19 (br s, 1 H, H-2′), 7.84 (br d, J ) 7.9 Hz,
1 H, H-6′), 7.34 (t, J ) 7.9 Hz, 1 H, H-5′), 7.29 (dt, J ) 7.9, 1.2
Hz, 1 H, H-4′), 7.13 (br t, J ) 5.7 Hz, 1 H, NHCH2), 6.46 (s, 1
H, H-8), 4.78 (t, J ) 5.5 Hz, 1 H, CH2OH), 3.59 (q, J ) 5.8 Hz,
2 H, CH2OH), 3.38 (br q, J ) 5.8 Hz, 2 H, NHCH2); 13C NMR
δ 160.86 (s, C-7), 157.84 (d, C-2), 157.62 (s, C-4), 154.51 (s,
C-8a), 148.25 (d, C-5), 140.70 (s, C-1′), 130.30 (d, C-5′), 125.91
(d, C-4′), 124.17 (d, C-2′), 121.08 (s, C-3′), 120.67 (d, C-6′),
103.69 (s, C-4a), 95.85 (br d, C-8), 59.61 (t, CH2OH), 43.92 (t,
NCH2). Anal. (C15H14BrN5O) C, H, N.
4-[(3-Br om op h en yl)a m in o]-7-[N-(2-h yd r oxyet h yl)-N-
m eth yla m in o]p yr id o[4,3-d ]p yr im id in e (3e): 99%, by reac-
tion of 6 with 2-(methylamino)ethanol (20 equiv) in 2-BuOH
(100 °C for 2 days); mp (2-BuOH) 232-235 °C; 1H NMR [(CD3)2-
SO] δ 9.91 (br s, 1 H, NH), 9.39 (s, 1 H, H-5), 8.47 (s, 1 H,
H-2), 8.19 (t, J ) 1.9 Hz, 1 H, H-2′), 7.84 (dt, J ) 8.1, 1.7 Hz,
1 H, H-6′), 7.34 (t, J ) 7.9 Hz, 1 H, H-5′), 7.29 (dt, J ) 8.1, 1.4
Hz, 1 H, H-4′), 6.53 (s, 1 H, H-8), 4.75 (t, J ) 5.4 Hz, 1 H,
CH2OH), 3.73 (t, J ) 6.0 Hz, 2 H, NCH2), 3.61 (q, J ) 5.8 Hz,
2 H, CH2OH), 3.14 (s, 3 H, NCH3); 13C NMR δ 159.81 (s, C-7),
157.89 (d, C-2), 157.63 (s, C-4), 154.74 (s, C-8a), 147.66 (d, C-5),
140.67 (s, C-1′), 130.31 (d, C-5′), 125.94 (d, C-4′), 124.17 (d,
C-2′), 121.09 (s, C-3′), 120.56 (d, C-6′), 103.15 (s, C-4a), 95.58
(d, C-8), 58.45 (t, CH2OH), 51.88 (t, NCH2), 37.11 (q, NCH3).
Anal. (C16H16BrN5O) C, H, N.
CHOH), 63.29 (t, CH2OH), 44.45 (t, NCH2). Anal. (C19H22
-
BrN5O5‚H2O) C, H, N.
4-[(3-Br om op h en yl)a m in o]-7-(N-m eth yl-D-glu ca m in o)-
p yr id o[4,3-d ]p yr im id in e (3k ): 90%, by reaction of 6 with
N-methyl-D-glucamine (33 equiv) in 1-PrOH/water (3:1) (95 °C
for 1 day); mp (water) 224.5 °C dec; 1H NMR [(CD3)2SO] δ 9.91
(br s, 1 H, NH), 9.41 (s, 1 H, H-5), 8.47 (s, 1 H, H-2), 8.20 (s,
1 H, H-2′), 7.85 (br d, J ) 8.0 Hz, 1 H, H-6′), 7.35 (t, J ) 8.0
Hz, 1 H, H-5′), 7.29 (br d, J ) 8.0 Hz, 1 H, H-4′), 6.59 (s, 1 H,
H-8), 4.90 (d, J ) 5.1 Hz, 1 H, CHOH), 4.52 (d, J ) 4.8 Hz, 1
H, CHOH), 4.44 (d, J ) 5.5 Hz, 2 H, 2CHOH), 4.35 (t, J ) 5.6
Hz, 1 H, CH2OH), 3.93 (m, 1 H, CH), 3.82 (dd, J ) 14.2, 4.1
Hz, 1 H, NCH), 3.68-3.35 (m, 6 H, 6CH); 13C NMR δ 159.95
(s, C-7), 157.88 (d, C-2), 157.66 (s, C-4), 154.64 (s, C-8a), 147.61
(d, C-5), 140.72 (s, C-1′), 130.34 (d, C-5′), 125.91 (d, C-4′),
124.10 (d, C-2′), 121.11 (s, C-3′), 120.63 (d, C-6′), 103.13 (s,
C-4a), 95.81 (d, C-8), 72.32, 71.38, 71.07, 69.52 (4 d, 4CHOH),
63.24 (t, CH2OH), 52.94 (t, NCH2), 37.58 (q, NCH3). Anal.
(C20H24BrN5O5) C, H, N.
4-[(3-Br om op h en yl)a m in o]-7-[[2-(d im eth yla m in o)eth -
yl]a m in o]p yr id o[4,3-d ]p yr im id in e (3l). Exa m p le of Gen -
er a l Meth od B. A solution of 6 (136 mg, 0.43 mmol) and N,N-
dimethylethylenediamine (0.50 mL, 4.58 mmol) in 2-BuOH (25
mL) was stirred at 100 °C for 2 days. The solvent was removed
under reduced pressure, and the residue was diluted with
aqueous Na2CO3 and extracted with EtOAc (3 × 50 mL).
Chromatography of this extract on alumina, eluting with 3%
EtOH/CHCl3, gave 3l (120 mg, 73%): mp (CH2Cl2/light petro-
leum) 210-211 °C; 1H NMR [(CD3)2SO] δ 9.89 (br s, 1 H, NH),
9.37 (s, 1 H, H-5), 8.45 (s, 1 H, H-2), 8.20 (s, 1 H, H-2′), 7.84
(d, J ) 8.1 Hz, 1 H, H-6′), 7.34 (t, J ) 7.9 Hz, 1 H, H-5′), 7.29
(br d, J ) 8.1 Hz, 1 H, H-4′), 7.00 (br t, J ) 5.7 Hz, 1 H,
NHCH2), 6.45 (s, 1 H, H-8), 3.39 (q, J ) 6.1 Hz, 2 H, NHCH2),
2.47 (t, J ) 6.6 Hz, 2 H, NCH2), 2.21 (s, 6 H, N(CH3)2); 13C
NMR δ 160.66 (s, C-7), 157.85 (d, C-2), 157.62 (s, C-4), 154.56
(s, C-8a), 148.30 (d, C-5), 140.72 (s, C-1′), 130.30 (d, C-5′),
125.89 (d, C-4′), 124.12 (d, C-2′), 121.09 (s, C-3′), 120.63 (d,
C-6′), 103.70 (s, C-4a), 95.70 (br d, C-8), 57.74 (t, NCH2), 45.16
(q, 2 C, N(CH3)2), 39.14 (t, NCH2). Anal. (C17H19BrN6) C, H,
N.
4-[(3-Br om oph en yl)am in o]-7-[N-(2,3-dih ydr oxypr opyl)-
N-m eth yla m in o]p yr id o[4,3-d ]p yr im id in e (3g): 61%, by
reaction of 6 with 3-(methylamino)propane-1,2-diol (10) (15
equiv) in 2-BuOH (97 °C for 2 days); mp (MeOH/CH2Cl2) 232-
1
233 °C; H NMR [(CD3)2SO] δ 9.91 (br s, 1 H, NH), 9.41 (s, 1
H, H-5), 8.48 (s, 1 H, H-2), 8.20 (br s, 1 H, H-2′), 7.85 (br d, J
) 7.9 Hz, 1 H, H-6′), 7.35 (t, J ) 8.0 Hz, 1 H, H-5′), 7.30 (dt,
J ) 8.2, 1.4 Hz, 1 H, H-4′), 6.56 (s, 1 H, H-8), 4.85 (d, J ) 5.1
Hz, 1 H, CHOH), 4.69 (t, J ) 5.7 Hz, 1 H, CH2OH), 3.80 (m,
2 H, 2CH), 3.50 (m, 1 H, CH), 3.38 (t, J ) 5.4 Hz, 2 H, CH2-
OH), 3.17 (s, 3 H, NCH3); 13C NMR δ 159.93 (s, C-7), 157.92
(d, C-2), 157.65 (s, C-4), 154.67 (s, C-8a), 147.59 (d, C-5), 140.70
(s, C-1′), 130.33 (d, C-5′), 125.93 (d, C-4′), 124.13 (d, C-2′),
121.11 (s, C-3′), 120.65 (d, C-6′), 103.15 (s, C-4a), 95.74 (d, C-8),
69.84 (d, CHOH), 63.85 (t, CH2OH), 53.03 (t, NCH2), 37.69 (q,
NCH3). Anal. (C17H18BrN5O2) C, H, N. Chromatography of
the mother liquors on silica gel, eluting with 6% MeOH/CH2-
Cl2, gave further pure product (20%).
The following analogues were prepared similarly.
4-[(3-Br om op h en yl)a m in o]-7-[[3-(d im eth yla m in o)p r o-
p yl]a m in o]p yr id o[4,3-d ]p yr im id in e (3m ): 67%, by reaction
of 6 with 3-(dimethylamino)propylamine (20 equiv) in 2-BuOH
(98 °C for 1 day) followed by chromatography of the product
on alumina, eluting with 1-2% MeOH/CH2Cl2; mp (MeOH/
CHCl3/light petroleum) 196-198 °C; 1H NMR [(CD3)2SO] δ
9.89 (br s, 1 H, NH), 9.36 (s, 1 H, H-5), 8.45 (s, 1 H, H-2), 8.19
(br s, 1 H, H-2′), 7.84 (br d, J ) 8.1 Hz, 1 H, H-6′), 7.34 (t, J
) 8.0 Hz, 1 H, H-5′), 7.29 (br d, J ) 8.1 Hz, 1 H, H-4′), 7.23
(br t, J ) 5.6 Hz, 1 H, NHCH2), 6.40 (s, 1 H, H-8), 3.30 (br q,
J ) 6.3 Hz, 2 H, NHCH2), 2.31 (t, J ) 7.0 Hz, 2 H, NCH2),
2.15 (s, 6 H, N(CH3)2), 1.71 (pentet, J ) 7.0 Hz, 2 H,
NCH2CH2); 13C NMR δ 160.85 (s, C-7), 157.87 (d, C-2), 157.62
(s, C-4), 154.63 (s, C-8a), 148.33 (d, C-5), 140.74 (s, C-1′), 130.32
(d, C-5′), 125.91 (d, C-4′), 124.17 (d, C-2′), 121.10 (s, C-3′),
120.68 (d, C-6′), 103.60 (s, C-4a), 95.16 (br d, C-8), 56.76 (t,
NCH2), 45.16 (q, 2 C, N(CH3)2), 39.55 (t, NCH2), 29.59 (t, CH2).
Anal. (C18H21BrN6) C, H, N.
4-[(3-Br om op h en yl)a m in o]-7-[N,N-b is(2-h yd r oxyet h -
yl)a m in o]p yr id o[4,3-d ]p yr im id in e (3i): 24%, by reaction of
6 with diethanolamine (46 equiv) in 2-BuOH (98 °C for 5 days)
followed by column chromatography of the product on silica
gel (eluting with 1-5% MeOH/EtOAc) and then two rounds
of preparative silica gel TLC (development with 10% MeOH/
CH2Cl2 and 10% MeOH/EtOAc, respectively); mp (MeOH/
CHCl3) 199-201 °C; 1H NMR [(CD3)2SO] δ 9.99 (br s, 1 H,
NH), 9.39 (s, 1 H, H-5), 8.48 (s, 1 H, H-2), 8.18 (br s, 1 H, H-2′),
7.83 (br d, J ) 7.8 Hz, 1 H, H-6′), 7.35 (t, J ) 7.9 Hz, 1 H,
H-5′), 7.30 (dt, J ) 8.0, 1.6 Hz, 1 H, H-4′), 6.61 (s, 1 H, H-8),
4.84 (br s, 2 H, 2OH), 3.69 (t, J ) 5.4 Hz, 4 H, N(CH2)2), 3.63
(br q, J ) 5.1 Hz, 4 H, 2CH2OH); 13C NMR δ 159.41 (s, C-7),
157.69 (d, C-2), 157.64 (s, C-4), 154.22 (s, C-8a), 147.79 (d, C-5),
140.60 (s, C-1′), 130.35 (d, C-5′), 126.07 (d, C-4′), 124.26 (d,
C-2′), 121.11 (s, C-3′), 120.78 (d, C-6′), 103.13 (s, C-4a), 95.42
(d, C-8), 58.32 (t, 2 C, 2CH2OH), 51.62 (t, 2 C, N(CH2)2). Anal.
(C17H18BrN5O2‚H2O) C, H, N.
4-[(3-Br om oph en yl)am in o]-7-[[4-(dim eth ylam in o)bu tyl]-
a m in o]p yr id o[4,3-d ]p yr im id in e (3n ): 86%, by reaction of
6 with 4-(dimethylamino)butylamine (21 equiv) in 2-BuOH (95
°C for 19 h) followed by chromatography of the product on
alumina, eluting with 1-2% MeOH/CH2Cl2; mp (CHCl3/light
petroleum) 171-174 °C; 1H NMR [(CD3)2SO] δ 9.87 (br s, 1 H,
NH), 9.35 (s, 1 H, H-5), 8.44 (s, 1 H, H-2), 8.19 (br s, 1 H, H-2′),
4-[(3-Br om oph en yl)am in o]-7-D-glu cam in opyr ido[4,3-d]-
p yr im id in e (3j): 78%, by reaction of 6 with D-glucamine (31
equiv) in 1-PrOH/water (7:1) (100 °C for 1 day); mp (water)
1
207-208 °C; H NMR [(CD3)2SO] δ 9.89 (br s, 1 H, NH), 9.36
(s, 1 H, H-5), 8.45 (s, 1 H, H-2), 8.18 (br s, 1 H, H-2′), 7.84 (br
d, J ) 7.9 Hz, 1 H, H-6′), 7.34 (t, J ) 8.0 Hz, 1 H, H-5′), 7.29

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3921
7.84 (br d, J ) 8.0 Hz, 1 H, H-6′), 7.34 (t, J ) 7.9 Hz, 1 H,
H-5′), 7.29 (br d, J ) 8.1 Hz, 1 H, H-4′), 7.24 (br t, J ) 5.6 Hz,
1 H, NHCH2), 6.39 (s, 1 H, H-8), 3.26 (br q, J ) 6.2 Hz, 2 H,
NHCH2), 2.23 (t, J ) 7.1 Hz, 2 H, NCH2), 2.12 (s, 6 H, N(CH3)2),
1.59 (pentet, J ) 7.0 Hz, 2 H, CH2), 1.49 (pentet, J ) 6.9 Hz,
2 H, CH2); 13C NMR δ 160.84 (s, C-7), 157.84 (d, C-2), 157.60
(s, C-4), 154.59 (s, C-8a), 148.32 (d, C-5), 140.73 (s, C-1′), 130.31
(d, C-5′), 125.88 (d, C-4′), 124.13 (d, C-2′), 121.09 (s, C-3′),
120.64 (d, C-6′), 103.55 (s, C-4a), 95.12 (br d, C-8), 58.72 (t,
NCH2), 45.07 (q, 2 C, N(CH3)2), 41.12 (t, NCH2), 26.40, 24.52
(2 t, 2CH2). Anal. (C19H23BrN6‚H2O) C, H, N.
(s, C-1′), 130.29 (d, C-5′), 125.85 (d, C-4′), 124.11 (d, C-2′),
121.08 (s, C-3′), 120.62 (d, C-6′), 103.61 (s, C-4a), 95.24 (br d,
C-8), 66.15 (t, 2 C, (CH2)2O), 55.83 (t, NCH2), 53.30 (t, 2 C,
N(CH2)2), 39.52 (t, NCH2), 25.50 (t, CH2). Anal. (C20H23
BrN6O) C, H, N.
-
4-[(3-Br om op h en yl)a m in o]-7-[[4-(4-m or p h olin o)bu tyl]-
a m in o]p yr id o[4,3-d ]p yr im id in e (3s): 90%, by reaction of 6
with 4-(4-aminobutyl)morpholine (13) (10 equiv) in 2-BuOH
(95 °C for 40 h) followed by chromatography of the product on
alumina, eluting with 0.7-0.8% MeOH/CH2Cl2; mp (CH2Cl2/
1
light petroleum) 149-151 °C; H NMR [(CD3)2SO] δ 9.87 (br
4-[(3-Br om op h en yl)a m in o]-7-[[5-(d im eth yla m in o)p en -
tyl]a m in o]p yr id o[4,3-d ]p yr im id in e (3o): 86%, by reaction
of 6 with 5-(dimethylamino)pentylamine (20 equiv) in 2-BuOH
(97 °C for 21 h) followed by chromatography of the product on
alumina, eluting with 1-2% MeOH/CH2Cl2; mp (MeOH/CHCl3/
s, 1 H, NH), 9.35 (s, 1 H, H-5), 8.44 (s, 1 H, H-2), 8.19 (t, J )
1.8 Hz, 1 H, H-2′), 7.84 (br d, J ) 8.3 Hz, 1 H, H-6′), 7.34 (t,
J ) 8.0 Hz, 1 H, H-5′), 7.29 (dt, J ) 8.0, 1.4 Hz, 1 H, H-4′),
7.24 (br t, J ) 5.6 Hz, 1 H, NHCH2), 6.40 (s, 1 H, H-8), 3.57 (t,
J ) 4.6 Hz, 4 H, (CH2)2O), 3.31 (br q, J ) 5.4 Hz, 2 H, NHCH2),
2.34 (m, 4 H, (CH2)2N), 2.30 (t, J ) 7.0 Hz, 2 H, NCH2), 1.60,
1.53 (2 m, 2 × 2 H, 2CH2); 13C NMR δ 160.83 (s, C-7), 157.84
(d, C-2), 157.59 (s, C-4), 154.63 (s, C-8a), 148.32 (d, C-5), 140.73
(s, C-1′), 130.31 (d, C-5′), 125.89 (d, C-4′), 124.13 (d, C-2′),
121.09 (s, C-3′), 120.64 (d, C-6′), 103.55 (s, C-4a), 95.18 (br d,
C-8), 66.12 (t, 2 C, (CH2)2O), 57.83 (t, NCH2), 53.27 (t, 2 C,
(CH2)2N), 41.11 (t, NCH2), 26.36, 23.37 (2 t, 2CH2). Anal.
(C21H25BrN6O) C, H, N.
1
light petroleum) 123-126 °C; H NMR [(CD3)2SO] δ 9.89 (br
s, 1 H, NH), 9.36 (s, 1 H, H-5), 8.44 (s, 1 H, H-2), 8.19 (br s, 1
H, H-2′), 7.84 (br d, J ) 7.8 Hz, 1 H, H-6′), 7.34 (t, J ) 8.0 Hz,
1 H, H-5′), 7.29 (br d, J ) 8.1 Hz, 1 H, H-4′), 7.20 (br t, J ) 5.6
Hz, 1 H, NHCH2), 6.39 (s, 1 H, H-8), 3.27 (br q, J ) 6.0 Hz, 2
H, NHCH2), 2.19 (t, J ) 7.1 Hz, 2 H, NCH2), 2.11 (s, 6 H,
N(CH3)2), 1.59 (pentet, J ) 7.2 Hz, 2 H, CH2), 1.39 (m, 4 H,
2CH2); 13C NMR δ 160.85 (s, C-7), 157.85 (d, C-2), 157.63 (s,
C-4), 154.61 (s, C-8a), 148.35 (d, C-5), 140.76 (s, C-1′), 130.33
(d, C-5′), 125.90 (d, C-4′), 124.17 (d, C-2′), 121.11 (s, C-3′),
120.68 (d, C-6′), 103.57 (s, C-4a), 95.18 (br d, C-8), 59.03 (t,
NCH2), 45.11 (q, 2 C, N(CH3)2), 41.19 (t, NCH2), 28.51, 26.76,
24.38 (3 t, 3CH2). Anal. (C20H25BrN6) C, H, N.
4-[(3-Br om op h en yl)a m in o]-7-[[3-(4-m eth ylp ip er a zin -1-
yl)p r op yl]a m in o]p yr id o[4,3-d ]p yr im id in e (3u ): 98%, by
reaction of 6 with 1-(3-aminopropyl)-4-methylpiperazine (20
equiv) in 2-BuOH (97 °C for 16 h); mp (MeOH/water) 111-
1
112.5 °C; H NMR [(CD3)2SO] δ 9.87 (br s, 1 H, NH), 9.36 (s,
4-[(3-Br om op h en yl)a m in o]-7-[N-[2-(d im et h yla m in o)-
eth yl]-N-m eth ylam in o]pyr ido[4,3-d]pyr im idin e (3p): 93%,
by reaction of 6 with N,N,N′-trimethylethylenediamine (8
equiv) in 2-BuOH (100 °C for 5 days) followed by dissolution
of the crude product in dilute aqueous HCl, filtration, and
1 H, H-5), 8.45 (s, 1 H, H-2), 8.19 (t, J ) 1.8 Hz, 1 H, H-2′),
7.84 (br d, J ) 8.1 Hz, 1 H, H-6′), 7.34 (t, J ) 8.0 Hz, 1 H,
H-5′), 7.29 (dt, J ) 8.0, 1.4 Hz, 1 H, H-4′), 7.24 (br t, J ) 5.6
Hz, 1 H, NHCH2), 6.40 (s, 1 H, H-8), 3.30 (br q, J ) 6.4 Hz, 2
H, NHCH2), 2.6-2.0 (br s, 8 H, N(CH2)4N), 2.37 (t, J ) 7.0
Hz, 2 H, NCH2), 2.15 (s, 3 H, NCH3), 1.72 (pentet, J ) 6.9 Hz,
2 H, NHCH2CH2); 13C NMR δ 160.84 (s, C-7), 157.84 (d, C-2),
157.58 (s, C-4), 154.65 (s, C-8a), 148.31 (d, C-5), 140.73 (s, C-1′),
130.30 (d, C-5′), 125.87 (d, C-4′), 124.11 (d, C-2′), 121.08 (s,
C-3′), 120.61 (d, C-6′), 103.58 (s, C-4a), 95.30 (br d, C-8), 55.46
(t, NCH2), 54.72, 52.65 (2 t, 2 × 2 C, N(CH2)4N), 45.69 (q,
1
basification with Na2CO3; mp (water) 195-196 °C; H NMR
[(CD3)2SO] δ 9.91 (br s, 1 H, NH), 9.42 (s, 1 H, H-5), 8.48 (s, 1
H, H-2), 8.21 (s, 1 H, H-2′), 7.85 (d, J ) 8.0 Hz, 1 H, H-6′),
7.35 (t, J ) 8.0 Hz, 1 H, H-5′), 7.29 (br d, J ) 8.1 Hz, 1 H,
H-4′), 6.49 (s, 1 H, H-8), 3.78 (t, J ) 6.8 Hz, 2 H, NCH2), 3.10
(s, 3 H, NCH3), 2.45 (t, J ) 6.9 Hz, 2 H, NCH2), 2.20 (s, 6 H,
N(CH3)2); 13C NMR δ 159.70 (s, C-7), 157.92 (d, C-2), 157.63
(s, C-4), 154.79 (s, C-8a), 147.75 (d, C-5), 140.72 (s, C-1′), 130.33
(d, C-5′), 125.92 (d, C-4′), 124.10 (d, C-2′), 121.12 (s, C-3′),
120.61 (d, C-6′), 103.18 (s, C-4a), 95.43 (d, C-8), 56.23, 47.16
(2 t, 2 CH2N), 45.43 (q, 2 C, N(CH3)2), 36.36 (q, NCH3). Anal.
(C18H21BrN6) C, H, N.
NCH3), 39.66 (t, NCH2), 25.81 (t, CH2). Anal. (C21H26
-
BrN7‚H2O) C, H, N.
4-[(3-Br om op h en yl)a m in o]-7-[[2-[N,N-b is(2-h yd r oxy-
eth yl)a m in o]eth yl]a m in o]p yr id o[4,3-d ]p yr im id in e (3v):
39%, by reaction of 6 (103 mg, 0.32 mmol) with N,N-bis(2-
hydroxyethyl)ethylenediamine dihydrochloride (1.55 g, 7.01
mmol) in 2.1 M aqueous NaOH (5 mL) and 1-PrOH (10 mL)
(95 °C for 20 h) followed by evaporation of solvent under
reduced pressure and dilution of the residue with aqueous
Na2CO3: mp (MeOH/water) 121.5-124.5 °C; 1H NMR [(CD3)2-
SO] δ 9.89 (br s, 1 H, NH), 9.37 (s, 1 H, H-5), 8.45 (s, 1 H,
H-2), 8.19 (t, J ) 1.9 Hz, 1 H, H-2′), 7.84 (dt, J ) 8.0, 1.4 Hz,
1 H, H-6′), 7.34 (t, J ) 8.0 Hz, 1 H, H-5′), 7.29 (dt, J ) 8.0, 1.5
Hz, 1 H, H-4′), 7.04 (br t, J ) 5.3 Hz, 1 H, NHCH2), 6.44 (s, 1
H, H-8), 4.40 (t, J ) 4.9 Hz, 2 H, 2OH), 3.45 (q, J ) 5.8 Hz, 4
H, 2CH2OH), 3.35 (br q, J ) 5.8 Hz, 2 H, NHCH2), 2.72 (t, J
) 6.3 Hz, 2 H, NCH2), 2.61 (t, J ) 6.0 Hz, 4 H, N(CH2)2); 13C
NMR δ 160.73 (s, C-7), 157.87 (d, C-2), 157.61 (s, C-4), 154.58
(s, C-8a), 148.30 (d, C-5), 140.70 (s, C-1′), 130.31 (d, C-5′),
125.91 (d, C-4′), 124.15 (d, C-2′), 121.08 (s, C-3′), 120.66 (d,
C-6′), 103.70 (s, C-4a), 95.77 (br d, C-8), 59.17, 56.82 (2 t, 2 ×
2 C, N(CH2CH2OH)2), 53.50 (t, NCH2), 39.56 (t, NCH2). Anal.
(C19H23BrN6O2‚H2O) H, N; C: calcd, 49.0; found, 49.5.
4-[(3-Br om op h en yl)a m in o]-7-[[2-(4-m or p h olin o)eth yl]-
a m in o]p yr id o[4,3-d ]p yr im id in e (3q): 86%, by reaction of
6 with 4-(2-aminoethyl)morpholine (10 equiv) in 2-BuOH (100
°C for 4 days) followed by chromatography of the product on
silica gel, eluting with 6-7% MeOH/CH2Cl2; mp (MeOH/CH2-
1
Cl2) 250-253 °C; H NMR [(CD3)2SO] δ 9.89 (br s, 1 H, NH),
9.37 (s, 1 H, H-5), 8.46 (s, 1 H, H-2), 8.20 (t, J ) 1.9 Hz, 1 H,
H-2′), 7.84 (ddd, J ) 8.0, 1.8, 1.3 Hz, 1 H, H-6′), 7.34 (t, J )
8.0 Hz, 1 H, H-5′), 7.29 (dt, J ) 8.0, 1.4 Hz, 1 H, H-4′), 7.02
(br t, J ) 5.7 Hz, 1 H, NHCH2), 6.45 (s, 1 H, H-8), 3.59 (t, J )
4.6 Hz, 4 H, (CH2)2O), 3.42 (q, J ) 5.9 Hz, 2 H, NHCH2), 2.53
(t, J ) 6.7 Hz, 2 H, NCH2), 2.44 (br t, J ) 4.6 Hz, 4 H, N(CH2)2);
13C NMR δ 160.65 (s, C-7), 157.87 (d, C-2), 157.61 (s, C-4),
154.59 (s, C-8a), 148.30 (d, C-5), 140.71 (s, C-1′), 130.31 (d,
C-5′), 125.90 (d, C-4′), 124.13 (d, C-2′), 121.09 (s, C-3′), 120.64
(d, C-6′), 103.72 (s, C-4a), 95.64 (br d, C-8), 66.12 (t, 2 C,
(CH2)2O), 57.00 (t, NCH2), 53.29 (t, 2 C, (CH2)2N), 38.32 (t,
NCH2). Anal. (C19H21BrN6O) H, N; C: calcd, 53.2; found, 52.7.
4-[(3-Br om op h en yl)a m in o]-7-[[3-(4-m or p h olin o)p r o-
p yl]a m in o]p yr id o[4,3-d ]p yr im id in e (3r ): 96%, by reaction
of 6 with 4-(3-aminopropyl)morpholine (20 equiv) in 2-BuOH
4-[(3-Br om op h en yl)a m in o]-7-[[3-[N,N-b is(2-h yd r oxy-
eth yl)am in o]pr opyl]am in o]pyr ido[4,3-d]pyr im idin e (3w):
93%, by reaction of 6 with 3-[N,N-bis(2-hydroxyethyl)amino]-
propylamine (20 equiv) in 1-PrOH/water (10:1) (95 °C for 17
h); mp (MeOH/water) 144-146 °C; 1H NMR [(CD3)2SO] δ 9.87
(br s, 1 H, NH), 9.35 (s, 1 H, H-5), 8.44 (s, 1 H, H-2), 8.18 (br
s, 1 H, H-2′), 7.83 (br d, J ) 7.8 Hz, 1 H, H-6′), 7.34 (t, J ) 8.0
Hz, 1 H, H-5′), 7.29 (br d, J ) 7.8 Hz, 1 H, H-4′), 7.24 (br t, J
) 5.4 Hz, 1 H, NHCH2), 6.40 (s, 1 H, H-8), 4.38 (br s, 2 H,
2OH), 3.44 (m, 4 H, 2CH2OH), 3.31 (br q, J ) 6.0 Hz, 2 H,
NHCH2), 2.57 (t, J ) 6.9 Hz, 2 H, NCH2), 2.53 (t, J ) 6.4 Hz,
1
(95 °C for 19 h); mp (CHCl3/light petroleum) 173-174 °C; H
NMR [(CD3)2SO] δ 9.86 (br s, 1 H, NH), 9.36 (s, 1 H, H-5),
8.45 (s, 1 H, H-2), 8.19 (br s, 1 H, H-2′), 7.84 (br d, J ) 7.9 Hz,
1 H, H-6′), 7.34 (t, J ) 8.0 Hz, 1 H, H-5′), 7.29 (dt, J ) 8.0, 1.4
Hz, 1 H, H-4′), 7.24 (br t, J ) 5.7 Hz, 1 H, NHCH2), 6.41 (s, 1
H, H-8), 3.59 (t, J ) 4.6 Hz, 4 H, (CH2)2O), 3.33 (br q, J ) 6.3
Hz, 2 H, NHCH2), 2.37 (m, 6 H, (CH2)2NCH2), 1.74 (pentet, J
) 6.9 Hz, 2 H, NCH2CH2); 13C NMR δ 160.83 (s, C-7), 157.80
(d, C-2), 157.54 (s, C-4), 154.60 (s, C-8a), 148.31 (d, C-5), 140.77
4 H, N(CH2)2), 1.70 (pentet, J ) 6.7 Hz, 2 H, NHCH2CH2); 13
NMR δ 160.84 (s, C-7), 157.78 (d, C-2), 157.55 (s, C-4), 154.57
C

3922 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Thompson et al.
(s, C-8a), 148.29 (d, C-5), 140.76 (s, C-1′), 130.29 (d, C-5′),
125.85 (d, C-4′), 124.13 (d, C-2′), 121.08 (s, C-3′), 120.64 (d,
C-6′), 103.56 (s, C-4a), 95.12 (br d, C-8), 59.20, 56.57 (2 t, 2 ×
2 C, N(CH2CH2OH)2), 52.54 (t, NCH2), 39.62 (t, NCH2), 26.30
(t, CH2). Anal. (C20H25BrN6O2) C, H, N.
6.9 Hz, 2 H, NCH2CH2); 13C NMR δ 160.75 (s, C-7), 158.08 (d,
C-2), 157.81 (s, C-4), 154.66 (s, C-8a), 148.20 (d, C-5), 138.78,
137.50 (2 s, C-1′,3′), 128.19 (d, C-5′), 124.35 (d, C-4′), 122.86
(d, C-2′), 119.61 (d, C-6′), 103.68 (s, C-4a), 95.29 (br d, C-8),
66.15 (t, 2 C, (CH2)2O), 55.85 (t, NCH2), 53.30 (t, 2 C, (CH2)2N),
39.51 (t, NCH2), 25.51 (t, CH2), 21.10 (q, CH3). Anal.
(C21H26N6O) C, H, N.
4-[(3-Br om oph en yl)am in o]-7-[[3-(im idazol-1-yl)pr opyl]-
a m in o]p yr id o[4,3-d ]p yr im id in e (3y): 80%, by reaction of
6 with 1-(3-aminopropyl)imidazole (20 equiv) in 2-BuOH (95
°C for 2 days) followed by chromatography of the product on
silica gel, eluting with 7-10% MeOH/CH2Cl2, and then on
alumina, eluting with 5% MeOH/CH2Cl2; mp (MeOH/CHCl3/
light petroleum) 231-232.5 °C; 1H NMR [(CD3)2SO] δ 9.90 (br
s, 1 H, NH), 9.38 (s, 1 H, H-5), 8.46 (s, 1 H, H-2), 8.19 (br s, 1
H, H-2′), 7.84 (br d, J ) 8.0 Hz, 1 H, H-6′), 7.68 (s, 1 H, H-2′′),
7.34 (t, J ) 8.0 Hz, 1 H, H-5′), 7.32 (br t, J ) 5.4 Hz, 1 H,
NHCH2), 7.29 (dt, J ) 8.0, 1.4 Hz, 1 H, H-4′), 7.23 (s, 1 H,
H-4′′), 6.92 (s, 1 H, H-5′′), 6.41 (s, 1 H, H-8), 4.09 (t, J ) 6.9
Hz, 2 H, NCH2), 3.25 (br q, J ) 6.1 Hz, 2 H, NHCH2), 2.02
(pentet, J ) 6.8 Hz, 2 H, NHCH2CH2); 13C NMR δ 160.69 (s,
C-7), 157.86 (d, C-2), 157.59 (s, C-4), 154.58 (s, C-8a), 148.32
(d, C-5), 140.72 (s, C-1′), 137.24 (d, C-2′′), 130.30 (d, C-5′),
128.29 (d, C-4′′), 125.90 (d, C-4′), 124.14 (d, C-2′), 121.08 (s,
C-3′), 120.65 (d, C-6′), 119.28 (d, C-5′′), 103.78 (s, C-4a), 95.57
(br d, C-8), 43.68 (t, NCH2), 38.30 (t, NCH2), 30.19 (t, CH2).
Anal. (C19H18BrN7) C, H, N.
4-[(3-Br om op h en yl)a m in o]-7-[[2-(im id a zol-4-yl)eth yl]-
a m in o]p yr id o[4,3-d ]p yr im id in e (3z): 74%, by reaction of 6
(100 mg, 0.31 mmol) with histamine dihydrochloride (0.60 g,
3.26 mmol) in 2.9 M aqueous NaOH (2 mL) and 1-PrOH (8
mL) (96 °C for 2 days); mp (MeOH/water) 246-249 °C; 1H
NMR [(CD3)2SO] δ 11.90 (br s, 1 H, NH), 9.89 (br s, 1 H, NH),
9.37 (s, 1 H, H-5), 8.45 (s, 1 H, H-2), 8.19 (br s, 1 H, H-2′),
7.84 (br d, J ) 8.0 Hz, 1 H, H-6′), 7.58 (s, 1 H, H-2′′), 7.34 (t,
J ) 8.0 Hz, 1 H, H-5′), 7.29 (d, J ) 7.9 Hz, 1 H, H-4′), 7.25 (br
t, J ) 5.6 Hz, 1 H, NHCH2), 6.87 (s, 1 H, H-5′′), 6.43 (s, 1 H,
H-8), 3.51 (br q, J ) 6.2 Hz, 2 H, NHCH2), 2.81 (t, J ) 7.3 Hz,
2 H, NHCH2CH2); 13C NMR δ 160.73 (s, C-7), 157.96 (d, C-2),
157.71 (s, C-4), 154.68 (s, C-8a), 148.42 (d, C-5), 140.75 (s, C-1′),
134.73 (d + s, 2 C, C-2′′,4′′), 130.42 (d, C-5′), 126.06 (d, C-4′),
124.31 (d, C-2′), 121.18 (s, C-3′), 120.83 (d, C-6′), 103.79 (s,
C-4a), 95.48 (br d, C-8), 41.54 (t, NCH2), 26.52 (t, CH2). Anal.
(C18H16BrN7) C, H, N.
4-[(3-Met h ylp h en yl)a m in o]-7-[[3-(4-m et h ylp ip er a zin -
1-yl)p r op yl]a m in o]p yr id o[4,3-d ]p yr im id in e (4u ): 77%, by
reaction of 7 with 1-(3-aminopropyl)-4-methylpiperazine (10
equiv) in 2-BuOH (94 °C for 2 days) followed by chromatog-
raphy of the product on silica gel, eluting with 15-20% MeOH/
CH2Cl2, treatment of this crude product with aqueous Na2CO3,
and extraction with EtOAc (3 × 50 mL); mp (CH2Cl2/light
petroleum) 142-145 °C; 1H NMR [(CD3)2SO] δ 9.73 (br s, 1 H,
NH), 9.34 (s, 1 H, H-5), 8.37 (s, 1 H, H-2), 7.61 (m, 2 H, H-2′,6′),
7.25 (dd, J ) 8.6, 7.6 Hz, 1 H, H-5′), 7.16 (br t, J ) 5.6 Hz, 1
H, NHCH2), 6.94 (d, J ) 7.6 Hz, 1 H, H-4′), 6.36 (s, 1 H, H-8),
3.29 (br q, J ) 6.6 Hz, 2 H, NHCH2), 2.6-2.0 (br s, 8 H,
N(CH2)4N), 2.37 (t, J ) 7.0 Hz, 2 H, NCH2), 2.33 (s, 3 H, 3′-
CH3), 2.15 (s, 3 H, NCH3), 1.72 (pentet, J ) 6.9 Hz, 2 H,
NHCH2CH2); 13C NMR δ 160.76 (s, C-7), 158.09 (d, C-2), 157.82
(s, C-4), 154.67 (s, C-8a), 148.22 (d, C-5), 138.79, 137.53 (2 s,
C-1′,3′), 128.21 (d, C-5′), 124.37 (d, C-4′), 122.88 (d, C-2′),
119.62 (d, C-6′), 103.68 (s, C-4a), 95.32 (br d, C-8), 55.49 (t,
NCH2), 54.72, 52.66 (2 t, 2 × 2 C, N(CH2)4N), 45.69 (q, NCH3),
39.66 (t, NCH2), 25.84 (t, CH2), 21.12 (q, 3′-CH3). Anal.
(C22H29N7‚0.25H2O) C, H, N.
7-[[3-(Im id a zol-1-yl)p r op yl]a m in o]-4-[(3-m eth ylp h en -
yl)a m in o]p yr id o[4,3-d ]p yr im id in e (4y): 88%, by reaction
of 7 with 1-(3-aminopropyl)imidazole (10 equiv) in 2-BuOH (95
°C for 4 days) followed by chromatography of the product on
silica gel, eluting with 6-8% MeOH/CH2Cl2, and then on
alumina, eluting with 5% MeOH/CHCl3; mp (CHCl3/light
petroleum) 158-159 °C; 1H NMR [(CD3)2SO] δ 9.77 (br s, 1 H,
NH), 9.37 (s, 1 H, H-5), 8.39 (s, 1 H, H-2), 7.67 (s, 1 H, H-2′′),
7.61 (m, 2 H, H-2′,6′), 7.26 (dd, J ) 8.8, 7.5 Hz, 1 H, H-5′),
7.25 (br t, J ) 5.6 Hz, 1 H, NHCH2), 7.22 (s, 1 H, H-4′′), 6.95
(d, J ) 7.4 Hz, 1 H, H-4′), 6.91 (s, 1 H, H-5′′), 6.38 (s, 1 H,
H-8), 4.09 (t, J ) 6.9 Hz, 2 H, NCH2), 3.25 (br q, J ) 6.2 Hz,
2 H, NHCH2), 2.33 (s, 3 H, 3′-CH3), 2.02 (pentet, J ) 6.9 Hz,
2 H, NHCH2CH2); 13C NMR δ 160.61 (s, C-7), 158.12 (d, C-2),
157.82 (s, C-4), 154.66 (s, C-8a), 148.22 (d, C-5), 138.76, 137.52
(2 s, C-1′,3′), 137.25 (d, C-2′′), 128.32, 128.21 (2 d, C-5′,4′′),
124.39 (d, C-4′), 122.88 (d, C-2′), 119.62 (d, C-6′), 119.27 (d,
C-5′′), 103.87 (s, C-4a), 95.64 (br d, C-8), 43.68 (t, NCH2), 38.30
7-[[4-(Dim et h yla m in o)b u t yl]a m in o]-4-[(3-m et h ylp h e-
n yl)a m in o]p yr id o[4,3-d ]p yr im id in e (4n ): 76%, by reaction
of 7 with 4-(dimethylamino)butylamine (10 equiv) in 2-BuOH
(95 °C for 2 days) followed by chromatography of the product
on silica gel, eluting with 8-15% MeOH/CH2Cl2 containing
0.3% Et3N, treatment of this crude product with aqueous Na2-
CO3, and extraction with EtOAc (3 × 50 mL); mp (CH2Cl2/
(t, NCH2), 30.23 (t, CH2), 21.11 (q, CH3). Anal. (C20H21
-
N7‚0.25H2O) C, N; H: calcd, 5.9; found, 6.5.
7-[[2-(Im id a zol-4-yl)eth yl]a m in o]-4-[(3-m eth ylp h en yl)-
a m in o]p yr id o[4,3-d ]p yr im id in e (4z): 65%, by reaction of 7
(101 mg, 0.40 mmol) with histamine dihydrochloride (0.74 g,
4.02 mmol) and NaHCO3 (83 mg, 0.99 mmol) in 3.5 M aqueous
NaOH (2 mL) and 1-PrOH (8 mL) (94 °C for 19 h) followed by
chromatography of the product on silica gel, eluting with
8-10% MeOH/CH2Cl2, and then on alumina, eluting with 10-
20% MeOH/CH2Cl2; mp (MeOH/CHCl3/light petroleum) 216-
216.5 °C; 1H NMR [(CD3)2SO] δ 11.85 (br s, 1 H, NH), 9.76 (br
s, 1 H, NH), 9.37 (s, 1 H, H-5), 8.38 (s, 1 H, H-2), 7.61 (m, 2 H,
H-2′,6′), 7.56 (d, J ) 0.9 Hz, 1 H, H-2′′), 7.26 (dd, J ) 8.6, 7.7
Hz, 1 H, H-5′), 7.18 (br t, J ) 5.7 Hz, 1 H, NHCH2), 6.95 (d, J
) 7.6 Hz, 1 H, H-4′), 6.86 (s, 1 H, H-5′′), 6.41 (s, 1 H, H-8),
3.51 (br q, J ) 6.6 Hz, 2 H, NHCH2), 2.82 (t, J ) 7.3 Hz, 2 H,
NHCH2CH2), 2.33 (s, 3 H, 3′-CH3); 13C NMR δ 160.57 (s, C-7),
158.09 (d, C-2), 157.82 (s, C-4), 154.69 (s, C-8a), 148.23 (d, C-5),
138.77, 137.51 (2 s, C-1′,3′), 134.60 (d + s, 2 C, C-2′′,4′′), 128.20
(d, C-5′), 124.38 (d, C-4′), 122.90 (d, C-2′), 119.65 (d, C-6′),
116.70 (br d, C-5′′), 103.78 (s, C-4a), 95.41 (br d, C-8), 41.48
(t, NCH2), 26.51 (t, CH2), 21.10 (q, CH3). Anal. (C19H19N7) C,
H, N.
1
light petroleum) 178-180 °C; H NMR [(CD3)2SO] δ 9.73 (br
s, 1 H, NH), 9.34 (s, 1 H, H-5), 8.37 (s, 1 H, H-2), 7.61 (m, 2 H,
H-2′,6′), 7.25 (dd, J ) 8.7, 7.6 Hz, 1 H, H-5′), 7.16 (br t, J )
5.6 Hz, 1 H, NHCH2), 6.94 (d, J ) 7.7 Hz, 1 H, H-4′), 6.36 (s,
1 H, H-8), 3.27 (br q, J ) 6.3 Hz, 2 H, NHCH2), 2.33 (s, 3 H,
3′-CH3), 2.23 (t, J ) 7.1 Hz, 2 H, NCH2), 2.12 (s, 6 H, N(CH3)2),
1.58 (pentet, J ) 7.0 Hz, 2 H, CH2), 1.49 (pentet, J ) 7.2 Hz,
2 H, CH2); 13C NMR δ 160.76 (s, C-7), 158.07 (d, C-2), 157.83
(s, C-4), 154.66 (s, C-8a), 148.21 (d, C-5), 138.80, 137.52 (2 s,
C-1′,3′), 128.20 (d, C-5′), 124.36 (d, C-4′), 122.88 (d, C-2′),
119.62 (d, C-6′), 103.65 (s, C-4a), 95.36 (br d, C-8), 58.74 (t,
NCH2), 45.08 (q, 2 C, N(CH3)2), 41.14 (t, NCH2), 26.44, 24.56
(2 t, 2CH2), 21.12 (q, 3′-CH3). Anal. (C20H26N6‚0.25H2O) C,
H, N.
4-[(3-Met h ylp h en yl)a m in o]-7-[[3-(4-m or p h olin o)p r o-
p yl]a m in o]p yr id o[4,3-d ]p yr im id in e (4r ): 86%, by reaction
of 7 with 4-(3-aminopropyl)morpholine (10 equiv) in 2-BuOH
(93 °C for 26 h) followed by chromatography of the product on
silica gel, eluting with 6-8% MeOH/CH2Cl2, and then on
alumina, eluting with 5% MeOH/CHCl3; mp (MeOH/CHCl3/
1
light petroleum) 181.5-182.5 °C; H NMR [(CD3)2SO] δ 9.74
4-[(3-Br om op h e n yl)a m in o]-7-[(1,3-d ih yd r oxy-2-p r o-
p yl)a m in o]p yr id o[4,3-d ]p yr im id in e (3h ). Exa m p le of
Gen er a l Meth od C. Serinol oxalate (1.30 g, 9.56 mmol) was
added to a solution of sodium (0.19 g, 8.26 mmol) dissolved in
2-BuOH (12 mL). The mixture was stirred until the reaction
was complete, then 6 (122 mg, 0.38 mmol) was added, and the
mixture was stirred at 100 °C for 8 days. The solvent was
(br s, 1 H, NH), 9.35 (s, 1 H, H-5), 8.38 (s, 1 H, H-2), 7.61 (m,
2 H, H-2′,6′), 7.26 (dd, J ) 8.5, 7.8 Hz, 1 H, H-5′), 7.16 (br t,
J ) 5.6 Hz, 1 H, NHCH2), 6.94 (d, J ) 7.4 Hz, 1 H, H-4′), 6.39
(s, 1 H, H-8), 3.59 (t, J ) 4.6 Hz, 4 H, (CH2)2O), 3.31 (br q, J
) 6.2 Hz, 2 H, NHCH2), 2.38 (t, J ) 6.9 Hz, 2 H, NCH2), 2.35
(br m, 4 H, (CH2)2N), 2.33 (s, 3 H, 3′-CH3), 1.74 (pentet, J )

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24 3923
removed under reduced pressure; then the residue was diluted
with water (50 mL) and extracted with EtOAc (3 × 40 mL).
Chromatography of this extract on silica gel, eluting with
6-10% MeOH/CH2Cl2, gave 3h (120 mg, 80%): mp (MeOH/
CH2Cl2) 168-171 °C; 1H NMR [(CD3)2SO] δ 9.88 (br s, 1 H,
NH), 9.35 (s, 1 H, H-5), 8.44 (s, 1 H, H-2), 8.19 (t, J ) 1.9 Hz,
1 H, H-2′), 7.84 (dt, J ) 7.9, 1.5 Hz, 1 H, H-6′), 7.34 (t, J ) 8.0
Hz, 1 H, H-5′), 7.29 (dt, J ) 8.0, 1.5 Hz, 1 H, H-4′), 6.80 (br d,
J ) 7.5 Hz, 1 H, NHCH), 6.55 (s, 1 H, H-8), 4.73 (t, J ) 5.6
Hz, 2 H, 2CH2OH), 3.88 (br s, 1 H, NHCH), 3.55 (t, J ) 5.5
Hz, 4 H, 2CH2OH); 13C NMR δ 160.62 (s, C-7), 157.82 (d, C-2),
157.60 (s, C-4), 154.48 (s, C-8a), 148.16 (d, C-5), 140.71 (s, C-1′),
130.31 (d, C-5′), 125.91 (d, C-4′), 124.15 (d, C-2′), 121.09 (s,
C-3′), 120.66 (d, C-6′), 103.70 (s, C-4a), 96.50 (br d, C-8), 60.22
(t, 2 C, 2CH2OH), 54.88 (d, NCH). Anal. (C16H16BrN5O2) C,
H, N.
(103 mg). This was redissolved in MeOH/aqueous Na2CO3, and
the solution was filtered, acidified with AcOH, and cooled to
give 3cc (95 mg, 74%): mp (MeOH/water) 230-233 °C; 1H
NMR [(CD3)2SO] δ 9.92 (br s, 1 H, NH), 9.36 (s, 1 H, H-5),
8.45 (s, 1 H, H-2), 8.18 (br s, 1 H, H-2′), 7.83 (br d, J ) 7.9 Hz,
1 H, H-6′), 7.34 (t, J ) 7.9 Hz, 1 H, H-5′), 7.30 (dt, J ) 8.0, 1.5
Hz, 1 H, H-4′), 7.28 (br t, J ) 5.7 Hz, 1 H, NHCH2), 6.41 (s, 1
H, H-8), 3.30 (br q, J ) 6.0 Hz, 2 H, NHCH2), 2.34 (t, J ) 7.4
Hz, 2 H, CH2COOH), 1.81 (pentet, J ) 7.2 Hz, 2 H,
NHCH2CH2); 13C NMR δ 174.24 (s, COOH), 160.80 (s, C-7),
157.72 (d, C-2), 157.63 (s, C-4), 154.32 (s, C-8a), 148.38 (d, C-5),
140.68 (s, C-1′), 130.33 (d, C-5′), 125.98 (d, C-4′), 124.24 (d,
C-2′), 121.09 (s, C-3′), 120.75 (d, C-6′), 103.59 (s, C-4a), 95.18
(br d, C-8), 40.55 (t, NHCH2), 31.05, 24.02 (2 t, 2CH2). Anal.
(C17H16BrN5O2‚1.5H2O) C, N; H: calcd, 4.4; found, 3.9.
4-[(3-Br om op h en yl)a m in o]-7-[N-(ca r b oxym et h yl)-N-
m eth yla m in o]p yr id o[4,3-d ]p yr im id in e (3d d ). Sarcosine
(0.92 g, 10.3 mmol) was added to a solution of sodium (0.20 g,
8.70 mmol) dissolved in 2-BuOH (20 mL), and the mixture was
stirred until the reaction was complete. The solution was
treated with 6 (125 mg, 0.39 mmol) at 95 °C for 3 days, then
the solvent was removed under reduced pressure, and the
residue was treated with methanolic HCl to pH ca. 3 and
cooled to give 3d d (138 mg, 91%): mp (MeOH) 215 °C dec; 1H
NMR [(CD3)2SO] δ 12.60 (br s, 1 H, COOH), 10.00 (br s, 1 H,
NH), 9.41 (s, 1 H, H-5), 8.52 (s, 1 H, H-2), 8.18 (br s, 1 H, H-2′),
7.84 (br d, J ) 7.8 Hz, 1 H, H-6′), 7.36 (t, J ) 7.9 Hz, 1 H,
H-5′), 7.31 (dt, J ) 7.9, 1.4 Hz, 1 H, H-4′), 6.60 (s, 1 H, H-8),
4.43 (s, 2 H, NCH2), 3.15 (s, 3 H, NCH3); 13C NMR δ 171.54 (s,
COOH), 159.94 (s, C-7), 157.89 (d, C-2), 157.71 (s, C-4), 154.52
(s, C-8a), 147.60 (d, C-5), 140.56 (s, C-1′), 130.36 (d, C-5′),
126.13 (d, C-4′), 124.30 (d, C-2′), 121.12 (s, C-3′), 120.82 (d,
C-6′), 103.66 (s, C-4a), 96.00 (d, C-8), 51.25 (t, NCH2), 37.38
(q, NCH3). Anal. (C16H14BrN5O2‚H2O) C, H, N.
The following analogues were prepared similarly.
4-[(3-Br om op h en yl)a m in o]-7-[(ca r boxym eth yl)a m in o]-
p yr id o[4,3-d ]p yr im id in e (3a a ). Glycine (1.35 g, 18.0 mmol)
was added to a solution of sodium (0.23 g, 10.0 mmol) dissolved
in MeOH (50 mL), and the mixture was stirred until the
reaction was complete. The solvent was removed, 6 (203 mg,
0.64 mmol) and 2-BuOH (20 mL) were added, and the mixture
was stirred at 98 °C for 5 days. The solvent was removed
under reduced pressure, and the residue was dissolved in
MeOH/water and treated with dilute HCl to pH ca. 4.
Concentration under reduced pressure and cooling gave a solid
(215 mg). This was redissolved in MeOH and aqueous Na2-
CO3; then the resulting solution was concentrated under
reduced pressure and extracted with EtOAc (2 × 100 mL).
Dilution of the aqueous suspension with DMSO (5 mL), then
neutralization with excess AcOH, concentration under reduced
pressure, and cooling gave 3a a : mp (DMSO/AcOH) 230-236
1
°C dec; H NMR [(CD3)2SO] δ 12.50 (br s, 1 H, COOH), 9.93
(br s, 1 H, NH), 9.37 (s, 1 H, H-5), 8.47 (s, 1 H, H-2), 8.18 (br
s, 1 H, H-2′), 7.83 (br d, J ) 7.7 Hz, 1 H, H-6′), 7.46 (br t, J )
6.0 Hz, 1 H, NHCH2), 7.35 (t, J ) 7.9 Hz, 1 H, H-5′), 7.30 (br
d, J ) 8.1 Hz, 1 H, H-4′), 6.53 (s, 1 H, H-8), 4.07 (d, J ) 6.0
Hz, 2 H, NHCH2); 13C NMR δ 172.04 (s, COOH), 160.42 (s,
C-7), 157.71 (d + s, C-2,4), 153.83 (s, C-8a), 148.12 (d, C-5),
140.55 (s, C-1′), 130.35 (d, C-5′), 126.13 (d, C-4′), 124.35 (d,
C-2′), 121.10 (s, C-3′), 120.86 (d, C-6′), 104.05 (s, C-4a), 96.85
(br d, C-8), 42.87 (t, NCH2). Anal. (C15H12BrN5O2) H, N; C:
calcd, 48.2; found, 47.7.
4-[(3-Br om op h en yl)a m in o]-7-(su lfoeth yla m in o)p yr id o-
[4,3-d ]p yr im id in e (3ee). Reaction of 6 (102 mg, 0.32 mmol)
with taurine (1.56 g, 12.5 mmol) in 1.1 M aqueous NaOH (10
mL) and 1-PrOH (10 mL) at 95 °C for 2 days followed by
removal of solvent under reduced pressure, dilution of the
residue with hot water (50 mL), filtration, and neutralization
with AcOH gave a solid (108 mg) after cooling. This was
dissolved in aqueous Na2CO3, washed with EtOAc, filtered,
acidified with AcOH, and cooled to give 3ee (99 mg, 73%): mp
338-345 °C dec; 1H NMR [(CD3)2SO] δ 11.11 (br s, 1 H, NH),
9.43 (s, 1 H, H-5), 8.69 (s, 1 H, H-2), 7.99 (s, 1 H, H-2′), 7.91
(br s, 1 H, NHCH2), 7.69 (br d, J ) 8.0 Hz, 1 H, H-6′), 7.49 (br
d, J ) 8.1 Hz, 1 H, H-4′), 7.43 (t, J ) 8.0 Hz, 1 H, H-5′), 6.39
(s, 1 H, H-8), 3.58 (br s, 2 H, NHCH2), 2.73 (t, J ) 7.2 Hz, 2 H,
CH2S). Anal. (C15H14BrN5O3S) C, H.
4-[(3-Br om op h en yl)a m in o]-7-[[3-(4-m or p h olin o)p r o-
p yl]a m in o]p yr id o[4,3-d ]p yr im id in e N1′-Oxid e (3t). A so-
lution of 4-[(3-bromophenyl)amino]-7-[[3-(4-morpholino)propyl]-
amino]pyrido[4,3-d]pyrimidine (3r ) (122 mg, 0.28 mmol) and
2-(phenylsulfonyl)-3-phenyloxaziridine (81 mg, 0.31 mmol) in
CH2Cl2 (30 mL) was stirred at 20 °C for 16 h. Evaporation
and chromatography of the residue on alumina, eluting with
3-8% MeOH/CHCl3, gave 3t (106 mg, 84%): mp (MeOH/
CHCl3/light petroleum) 189-190 °C; 1H NMR [(CD3)2SO] δ
9.94 (br s, 1 H, NH), 9.37 (s, 1 H, H-5), 8.44 (s, 1 H, H-2), 8.18
(br s, 1 H, H-2′), 8.04 (br s, 1 H, NHCH2), 7.85 (br d, J ) 8.0
Hz, 1 H, H-6′), 7.34 (t, J ) 8.0 Hz, 1 H, H-5′), 7.29 (br d, J )
8.0 Hz, 1 H, H-4′), 6.38 (s, 1 H, H-8), 4.14 (dd, J ) 11.5, 10.3
Hz, 2 H, 2OCHa), 3.67 (dd, J ) 11.7, 2.5 Hz, 2 H, 2OCHe),
3.35 (m, 6 H, NHCH2, N(O)CH2, 2N(O)CHa), 2.89 (br d, J )
11.2 Hz, 2 H, 2N(O)CHe), 2.14 (pentet, J ) 6.8 Hz, 2 H,
NHCH2CH2); 13C NMR δ 160.72 (s, C-7), 157.89 (d, C-2), 157.65
(s, C-4), 154.68 (s, C-8a), 148.38 (d, C-5), 140.75 (s, C-1′), 130.34
(d, C-5′), 125.95 (d, C-4′), 124.24 (d, C-2′), 121.11 (s, C-3′),
120.76 (d, C-6′), 103.63 (s, C-4a), 95.58 (br d, C-8), 68.71 (t,
N(O)CH2), 63.52, 60.99 (2 t, 2 × 2 C, N(O)(CH2)4O), 36.57 (t,
NCH2), 21.04 (t, CH2). Anal. (C20H23BrN6O2‚2H2O) C, H, N.
4-[(3-Br om op h en yl)a m in o]-7-h yd r a zin op yr id o[4,3-d ]-
p yr im id in e (3x). A solution of 6 (100 mg, 0.31 mmol) and
anhydrous hydrazine (0.20 mL, 6.37 mmol) in 2-BuOH (10 mL)
was stirred at 20 °C for 3 days. The resulting precipitate was
filtered and washed with CH2Cl2/light petroleum to give 3x
4-[(3-Br om op h en yl)a m in o]-7-[(2-ca r boxyeth yl)a m in o]-
p yr id o[4,3-d ]p yr im id in e (3bb). â-Alanine (0.89 g, 10.0
mmol) was added to a solution of sodium (0.19 g, 8.26 mmol)
dissolved in 2-BuOH (20 mL), and the mixture was stirred
until the reaction was complete (monitored by pH). The
solution was treated with 6 (145 mg, 0.46 mmol) at 100 °C for
1 day, then the solvent was removed under reduced pressure,
and the residue was treated with methanolic HCl to pH ca. 4,
evaporated, and chromatographed on silica gel, eluting with
10% MeOH/CH2Cl2 containing 0.5% AcOH, to give 3bb (147
1
mg, 83%): mp (MeOH/AcOH) 241-243 °C; H NMR [(CD3)2-
SO] δ 12.26 (br s, 1 H, COOH), 9.90 (br s, 1 H, NH), 9.38 (s, 1
H, H-5), 8.45 (s, 1 H, H-2), 8.19 (t, J ) 1.8 Hz, 1 H, H-2′), 7.84
(br d, J ) 8.1 Hz, 1 H, H-6′), 7.34 (t, J ) 7.9 Hz, 1 H, H-5′),
7.29 (dt, J ) 8.0, 1.4 Hz, 1 H, H-4′), 7.23 (br t, J ) 5.6 Hz, 1
H, NHCH2), 6.45 (s, 1 H, H-8), 3.52 (q, J ) 6.3 Hz, 2 H,
NHCH2), 2.56 (t, J ) 6.8 Hz, 2 H, CH2COOH); 13C NMR δ
173.02 (s, COOH), 160.52 (s, C-7), 157.80 (d, C-2), 157.64 (s,
C-4), 154.38 (s, C-8a), 148.32 (d, C-5), 140.68 (s, C-1′), 130.32
(d, C-5′), 125.98 (d, C-4′), 124.23 (d, C-2′), 121.10 (s, C-3′),
120.74 (d, C-6′), 103.80 (s, C-4a), 95.95 (br d, C-8), 37.22, 33.65
(2 t, NCH2CH2). Anal. (C16H14BrN5O2) C, H, N.
4-[(3-Br om oph en yl)am in o]-7-[(3-car boxypr opyl)am in o]-
p yr id o[4,3-d ]p yr im id in e (3cc). 4-Aminobutyric acid (1.28
g, 12.4 mmol) was added to a solution of sodium (0.24 g, 10.4
mmol) dissolved in 2-BuOH (20 mL), and the mixture was
stirred until the reaction was complete. The solution was
treated with 6 (102 mg, 0.32 mmol) at 100 °C for 1 day; then
the solvent was removed under reduced pressure. The residue
was dissolved in dilute aqueous Na2CO3 (50 mL), washed with
EtOAc (2 × 50 mL), diluted with MeOH, filtered, then treated
with concentrated HCl to pH ca. 4, and cooled to give a solid

3924 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 24
Thompson et al.
1
(64 mg, 62%): mp (2-BuOH) 220 °C dec; H NMR [(CD3)2SO]
and blocked overnight in TNA containing 5% bovine serum
albumin and 1% ovalbumin. The membrane was blotted for
2 h with antiphosphotyrosine antibody (UBI, 1 µg/mL in
blocking buffer) and then washed twice in TNA, once in TNA
containing 0.05% Tween-20 and 0.05% nonidet P-40, and twice
in TNA. The membranes were then incubated for 2 h in
blocking buffer containing 0.1 µCi/mL [125I]protein A and
washed again as above. After the blots were dry they were
loaded into a film cassette and exposed to X-AR X-ray film for
1-7 days. Band intensities were determined with a Molecular
Dynamics laser densitometer.
δ 9.89 (br s, 1 H, NH), 9.33 (s, 1 H, H-5), 8.47 (s, 1 H, H-2),
8.18 (t, J ) 1.9 Hz, 1 H, H-2′), 8.12 (br s, 1 H, NH), 7.84 (dt,
J ) 7.9, 1.5 Hz, 1 H, H-6′), 7.34 (t, J ) 8.0 Hz, 1 H, H-5′), 7.29
(dt, J ) 8.0, 1.5 Hz, 1 H, H-4′), 6.73 (s, 1 H, H-8), 4.40 (br s, 2
H, NH2); 13C NMR δ 164.22 (s, C-7), 157.96 (d, C-2), 157.63 (s,
C-4), 155.01 (s, C-8a), 148.14 (d, C-5), 140.75 (s, C-1′), 130.39
(d, C-5′), 126.02 (d, C-4′), 124.29 (d, C-2′), 121.16 (s, C-3′),
120.80 (d, C-6′), 104.05 (s, C-4a), 94.47 (d, C-8). Anal. (C13H11
-
BrN6) C, H, N.
7-(Met h yla m in o)-4-[(3-m et h ylp h en yl)a m in o]p yr id o-
[4,3-d ]p yr im id in e (4b). A mixture of 7 (100 mg, 0.39 mmol)
and 40% aqueous methylamine (5.0 mL, 58 mmol) in 2-PrOH
(30 mL) was stirred in a pressure vessel at 65 °C for 3 h. The
solvent was removed under reduced pressure; then the residue
was diluted with aqueous Na2CO3 and extracted with EtOAc
(4 × 50 mL). Chromatography of this extract on silica gel,
eluting with 3-4% MeOH/CH2Cl2, gave 4b (94 mg, 90%): mp
(MeOH/CHCl3/light petroleum) 275-277 °C; 1H NMR [(CD3)2-
SO] δ 9.76 (br s, 1 H, NH), 9.36 (s, 1 H, H-5), 8.39 (s, 1 H,
H-2), 7.61 (m, 2 H, H-2′,6′), 7.26 (dd, J ) 8.7, 7.7 Hz, 1 H,
H-5′), 7.10 (br q, J ) 4.9 Hz, 1 H, NHCH3), 6.95 (d, J ) 7.5
Hz, 1 H, H-4′), 6.33 (s, 1 H, H-8), 2.85 (d, J ) 5.0 Hz, 3 H,
NHCH3), 2.33 (s, 3 H, 3′-CH3); 13C NMR δ 161.39 (s, C-7),
158.10 (d, C-2), 157.83 (s, C-4), 154.80 (s, C-8a), 148.20 (d, C-5),
138.77, 137.51 (2 s, C-1′,3′), 128.19 (d, C-5′), 124.37 (d, C-4′),
122.90 (d, C-2′), 119.64 (d, C-6′), 103.68 (s, C-4a), 94.71 (br d,
C-8), 28.33 (q, NCH3), 21.10 (q, 3′-CH3). Anal. (C15H15N5) C,
H, N.
In Vivo Ch em oth er a p y. Immune-deficient mice were
housed in microisolator cages within a barrier facility on a 12-h
light/dark cycle and received food and water ad libitum.
Animal housing was in accord with AAALAC guidelines. All
experimental protocols involving animals were approved by
the Institutional Animal Care and Use Committee. The A431
epidermoid carcinoma and the NIH 3T3 fibroblast transfected
with the human EGF receptor (EGFR) were maintained by
serial passage in nude mice (NCr nu/nu). Nude mice were also
used as tumor hosts for anticancer agent evaluations against
these tumor models. In each experiment for anticancer
activity evaluation, test mice weighing 18-22 g were random-
ized and implanted with tumor fragments in the region of the
right axilla on day 0. Animals were treated with 3n or 3r on
the basis of average cage weight on the days indicated in Table
1. The compounds were administered as solutions of the
isethionate salts in water and were prepared for 5 days at a
time. Host body weight change data are reported as the
maximum treatment-related weight loss in these studies.
Calculation of tumor growth inhibition (% T/C), tumor growth
delay (T - C), and net logs of tumor cell kill was performed
as described previously.26-29 A positive net cell kill indicates
that the tumor burden at the end of therapy was less than at
the beginning of therapy. A negative net log cell kill indicates
that the tumor grew during treatment. Net cell kills near 0
indicate no tumor growth during therapy.
Deter m in a tion of Aqu eou s Solu bility. Stock solutions
of drugs were made up in MeOH or DMSO and used to
calibrate the HPLC (peak area in nanomoles, assuming a
linear response). Accurately weighed amounts (to give ap-
proximately a 50 mM solution) were then sonicated for 30 min
in 0.05 M sodium lactate buffer (neutral compounds, hydro-
chloride salts of amines) or in water (sodium salts of acids).
After standing for an additional 30 min, the samples were
centrifuged at 13 000 rpm for 3 min, and the concentration of
drug in the supernatant was determined by HPLC, using the
calibration determined previously.
Ack n ow led gm en t. This work was partially sup-
ported by the Auckland Division of the Cancer Society
of New Zealand.
En zym e Assa y. Epidermal growth factor receptor was
prepared from human A431 carcinoma cell shed membrane
vesicles by immunoaffinity chromatography as previously
described.25 The enzyme assay was performed in 96-well filter
plates (Millipore, MADV6550). The total volume was 0.1 mL,
containing 20 mM HEPES, pH 7.4, 50 µM sodium vanadate,
40 mM MgCl2, 10 µM ATP containing 5 µCi of [32P]ATP, 20 µg
of polyglutamic acid/tyrosine random copolymer (Sigma Chemi-
cal Co., Saint Louis, MO), 1 ng of EGFR tyrosine kinase, and
appropriate dilutions of inhibitor and/or ATP. All components
except the ATP were added to the well, and the plate was
incubated with shaking for 10 min at 25 °C. The reaction was
started by adding [32P]ATP, the plate was then incubated with
shaking for a further 10 min at 25 °C, and the reaction was
terminated by addition of 0.1 mL of 20% trichloroacetic acid
(TCA). The plate was then kept at 4 °C for at least 15 min to
allow the substrate to precipitate, and the wells were then
washed five times with 0.125 mL of 10% TCA. [32P]ATP
incorporation was determined with a Wallac betaplate counter.
Control activity (no drug) gave a count of approximately
100 000 cpm. At least two independent dose-response curves
were done and the IC50 values computed. The reported values
are averages; variation was generally (15%.
EGF R Au top h osp h or yla tion in A431 Hu m a n Ep id er -
m oid Ca r cin om a Cells. Cells were grown to confluence in
6-well plates (35 mm diameter) and exposed to serum-free
medium for 18 h. The cells were treated with compound for 2
h and then with 100 ng/mL of EGF for 5 min. The monolayers
were lysed in 0.2 mL of boiling Laemmli buffer (2% sodium
dodecyl sulfate, 5% â-mercaptoethanol, 10% glycerol, and 50
mM Tris, pH 6.8), and the lysates were heated to 100 °C for 5
min. Proteins in the lysate were separated by polyacrylamide
gel electrophoresis and electrophoretically transferred to
nitrocellulose. The membrane was washed once in a mixture
of 10 mM Tris, pH 7.2, 150 mM NaCl, and 0.01% azide (TNA)
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