Tyrosine kinase inhibitors. 14. Structure-activity relationships for methyl-amino-substituted derivatives of 4-[3-bromophenyl)amino]-6- (methylamino)-pyrido[3,4-d]pyrimidine (PD 158780), a potent and specific inhibitor of the tyrosine kinase activity of receptors for the EGF family of growth factors

Article abstract of DOI:10.1021/jm970641d

The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a very potent in vitro inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (IC₅₀ 0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of these signal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing functions off the 6-methylamino substituent were prepared by reaction of the 6-fluoro derivatives with appropriate amine nucleophiles. These were evaluated for their ability to inhibit the tyrosine phosphorylating action of EGF- stimulated full-length EGFR enzyme and for inhibition of autophosphorylation of the EGFR in A431 human epidermoid carcinoma cells in culture. The most effective analogues were those bearing weakly basic substituents through a secondary amine linkage, which proved water-soluble (> 10 mM) and potent (IC₅₀s generally < 1 nM). No clear SAR could be discerned for these compounds with respect to amine base strength or the distance of the cationic center from the chromophore, suggesting that 6-substituents are in a favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerged for the ability of the compounds to inhibit EGFR autophosphorylation in A431 cells, where analogues bearing lipophilic weak bases were preferred. Representative analogues were evaluated for antitumor effectiveness against four in vivo tumor models. Significant in vivo activity was observed in estrogen-dependent MCF-7 breast and A431 epidermoid tumors. Marginal activity was seen in an EGFR-transfected tumor model, suggesting that while this cell line requires EGF for clone formation in soft agar, other growth factors may be able to replace EGF in vivo. Also, no activity was seen against the SK-OV-3 ovarian cancer model, which is known to express other EGF receptor family members (although it is not clear whether these are absolutely required for growth in vivo). While substantial growth delays were seen in A431 and MCF-7 tumor models, the treated tumors remained approximately the same size throughout therapy, suggesting that the compounds are cytostatic rather than cytotoxic under these test conditions. It remains to be determined if more prolonged therapy has cytotoxic effects in vivo, resulting in net tumor cell kill.

Full text of DOI:10.1021/jm970641d

742
J . Med. Chem. 1998, 41, 742-751
Tyr osin e Kin a se In h ibitor s. 14. Str u ctu r e-Activity Rela tion sh ip s for Meth yl-
a m in o-Su bstitu ted Der iva tives of 4-[(3-Br om op h en yl)a m in o]-6-(m eth yla m in o)-
p yr id o[3,4-d ]p yr im id in e (P D 158780), a P oten t a n d Sp ecific In h ibitor of th e
Tyr osin e Kin a se Activity of Recep tor s for th e EGF F a m ily of Gr ow th F a ctor s
Gordon W. Rewcastle,† Donna K. Murray,† William L. Elliott,‡ David W. Fry,‡ Curtis T. Howard,‡
J ames M. Nelson,‡ Billy J . Roberts,‡ Patrick W. Vincent,‡ H. D. Hollis Showalter,‡ R. Thomas Winters,‡ 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 September 26, 1997
The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a very potent in vitro
inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (IC50
0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of
these signal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing
functions off the 6-methylamino substituent were prepared by reaction of the 6-fluoro derivatives
with appropriate amine nucleophiles. These were evaluated for their ability to inhibit the
tyrosine phosphorylating action of EGF-stimulated full-length EGFR enzyme and for inhibition
of autophosphorylation of the EGFR in A431 human epidermoid carcinoma cells in culture.
The most effective analogues were those bearing weakly basic substituents through a secondary
amine linkage, which proved water-soluble (>10 mM) and potent (IC50s generally <1 nM). No
clear SAR could be discerned for these compounds with respect to amine base strength or the
distance of the cationic center from the chromophore, suggesting that 6-substituents are in a
favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerged for
the ability of the compounds to inhibit EGFR autophosphorylation in A431 cells, where
analogues bearing lipophilic weak bases were preferred. Representative analogues were
evaluated for antitumor effectiveness against four in vivo tumor models. Significant in vivo
activity was observed in estrogen-dependent MCF-7 breast and A431 epidermoid tumors.
Marginal activity was seen in an EGFR-transfected tumor model, suggesting that while this
cell line requires EGF for clone formation in soft agar, other growth factors may be able to
replace EGF in vivo. Also, no activity was seen against the SK-OV-3 ovarian cancer model,
which is known to express other EGF receptor family members (although it is not clear whether
these are absolutely required for growth in vivo). While substantial growth delays were seen
in A431 and MCF-7 tumor models, the treated tumors remained approximately the same size
throughout therapy, suggesting that the compounds are cytostatic rather than cytotoxic under
these test conditions. It remains to be determined if more prolonged therapy has cytotoxic
effects in vivo, resulting in net tumor cell kill.
The epidermal growth factor receptor (EGFR) has
become an important enzyme target for cancer
chemotherapy.1-3 It plays a central role in growth
signaling4,5 and is overexpressed in a significant propor-
tion of human tumors.6,7 A major step forward in the
development of EGFR-targeted drugs was the discovery
of the 4-anilinoquinazoline class of compounds.8-12
These are potent and selective inhibitors of the tyrosine
kinase activity of the EGFR via competitive binding at
the ATP site of the enzyme. Potent inhibition of the
enzyme is associated with small lipophilic electron-
withdrawing groups at the 3-position of the aniline ring
and with electron-donating groups at the 6- and/or
7-positions of the quinazoline.9-11 For example, 1 has
We have recently shown15 that related 4-(3-bromoa-
nilino)pyrido[d]pyrimidines are also potent and selective
inhibitors of both substrate phosphorylation by the
isolated EGFR enzyme and its autophosphorylation in
cells. The most potent subclass of this series of regioi-
an IC50 of 0.025 nM for inhibition of substrate phos-
phorylation by the isolated EGFR enzyme.9 Two ex-
amples of this class (2 and 3) that exemplify these
structure-activity relationships (SAR) are reported to
be in clinical trial.13,14
S0022-2623(97)00641-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/03/1998

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5 743
Sch em e 1a
somers was the pyrido[3,4-d]pyrimidines, and in par-
ticular the 6-methylamino derivative 5b (PD 158780).
This compound had an IC50 of 0.008 nM for inhibition
of substrate phosphorylation,15 compared with an IC50
of 7 nM for the corresponding 6-(methylamino)quinazo-
line analogue (4).11
A detailed study16 of 5b showed that it was a
competitive inhibitor of the EGFR with respect to ATP
and inhibited EGFR autophosphorylation in A431 hu-
man epidermoid carcinoma cells with an IC50 of 13 nM.
While the onset of inhibition was immediate, recovery
of receptor autophosphorylation was slow, requiring 8
h to complete. Compound 5b was also active against
other members of the EGFR family, with IC50s of 49 and
52 nM respectively for inhibition of heregulin-stimulat-
ed autophosphorylation in SK-BR-3 and MDA-MB-453
breast carcinomas. SK-BR-3 cells express EGFR, erbB2,
and erbB3, while MDA-MB-453 expresses erbB2, erbB3,
and erbB4; heregulin is a specific ligand for erbB4 but
also binds and activates heterodimers of erbB3, erbB2,
and erbB4.16-18 Studies of the inhibition of clone
formation in soft agar of a series of transformed
fibroblasts and established human breast carcinoma
lines confirmed that 5b had good activity against
members of the EGFR family, while not influencing the
function of related tyrosine kinases.16 The above prop-
erties of 5b make it an attractive lead compound for
further development.
a
(i) SOCl2/dichloroethane/DMF/reflux/2.5 h; (ii) 3-X-aniline/2-
propanol/reflux/45 min; (iii) RRNH/DMSO/85-95 °C/2 h-5 d.
Sch em e 2a
a
(i) MeNO2/DBU/DMSO/25 °C/24 h; (ii) 4-(2-aminoethyl)mor-
pholine/DMSO/80 °C/22 h.
Sch em e 3a
The broad SAR reported for the 4-anilinoquinazolines
and pyrido[d]pyrimidines as EGFR inhibitors are con-
sistent19 with the compounds binding to the ATP site
of the EGFR. In this model,19 the N-1 atom accepts an
H-bond from Met-769 and N-3 from the side chain of
Thr-766 on strand 5 deep in the binding cleft, and the
anilino side chain binds in an adjacent hydrophobic
pocket. This pocket is exceptionally large in the EGFR,
formed in part by three additional sulfur-containing
amino acids (Cys-751, Met-769, and Met-742). This
binding model suggests that the only positions on the
model where substantial property-modulating groups
can be placed are the 6- and 7-positions of the bicyclic
chromophore, which are situated at the entrance of the
adenine binding cleft. We have shown previously20 that
a variety of bulky side chains on the 7-position of pyrido-
[4,3-d]pyrimidines are acceptable, but show a distinct
SAR.
In this paper we report the synthesis and evaluation
of a series of 6-substituted pyrido[3,4-d]pyrimidines,
representing analogues of 5b substituted on the 6-me-
thylamino group. We also compare SAR for these
compounds with that of the related 7-substituted pyrido-
[4,3-d]pyrimidines for inhibition of isolated EGFR.
Selected analogues were also evaluated for autophos-
phorylation in A431 cells and in vivo against a range of
human tumor xenografts in nude mice.
a
(i) aqueous HCN; (ii) LiAlH4.
6-fluorine atom from these intermediates with amine
nucleophiles was normally conducted in DMSO at 80-
95 °C for 1-5 days (in a pressure vessel for the more
volatile amines) followed by purification by direct re-
crystallization or chromatography (method A). With
amino acid nucleophiles, it was necessary to preform
the sodium salts prior to reaction (method B).20
The 2-methyl derivative 10n was prepared by reaction
of 13 with nitromethane to give 17 followed by fluorine
displacement with 4-(2-aminomethyl)morpholine (Scheme
2). Most of the required amines were commercially
available. 2-[(2-Aminoethyl)methylamino]ethanol was
prepared by the general method of Kohn et al.22 (Scheme
3).
Resu lts a n d Discu ssion
The structures of the 6-substituted 4-(phenylamino)-
pyrido[3,4-d]pyrimidines studied (5-10) are recorded in
Table 1. Inhibition of tyrosine phosphorylation of a
random tyrosine/glutamic acid copolymer (Sigma) by
EGF-stimulated full-length EGFR enzyme isolated from
A431 cells was measured by a filter binding assay.8 At
least two complete dose-response curves were deter-
mined for each compound, and averaged IC50s are listed
in Table 1. Most compounds were also evaluated for
their ability to inhibit autophosphorylation of the EGF
receptor in A431 human epidermoid carcinoma cells.
Ch em istr y
The 6-substituted 4-(phenylamino)pyrido[4,3-d]pyri-
midines of Table 1 were prepared by the general method
of Scheme 1. Activation of the known21 6-fluoropyrido-
[3,4-d]pyrimidin-4(3H)-one (11) with SOCl2/DMF gave
4-chloro-6-fluoropyrido[3,4-d]pyrimidine (12), and reac-
tion of this with appropriate 3-substituted anilines gave
the key intermediates 13-16. Displacement of the

744 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5
Rewcastle et al.
Ta ble 1. Structural and Biological Properties of 6-Substituted 4-(Phenylamino)pyrido[3,4-d]pyrimidines
IC50 (nM)
no.
R
X
mp (°C)
ref 13
ref 13
ref 13
solubilitya (nM)
enzymeb
autophosc
5a
5b
5c
5d
5e
5f
5 g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
NH2
NHMe
NMe2
Br
0.13
0.008
0.006
0.19
0.22
0.18
0.56
1.1
1.2
1.8
4.6
1.7
16
13
21
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
H
0.10
0.06
0.02
0.11
0.21
0.18
>48
30
38
36
>45
39
10
>44
32
36
20
12
17
NHCH2CH2OH
209-210
236-237
186.5-188
159-160
250-252
160-161
176-177
189-192
182-189
260-262
267 dec
177-178
182-183
210-211
205-207
260-262
170-171.5
200-201
195.5-197
219.5-222
234-239
225-227
276-280
ref 13
N(Me)CH2CH2OH
NHCH2CH(OH)CH2OH
N(Me)CH2CH(OH)CH2OH
NH(CH2)2NMe2
NH(CH2)3NMe2
NH(CH2)4NMe2
NHCH2CH(OH)CH2NEt2
NH(CH2)2N(Me)CH2CH2OH
N(Me)(CH2)2NMe2
NH(CH2)2morpholinyl
NH(CH2)3morpholinyl
NH(CH2)3NMepiperazinyl
NH(CH2)2N(CH2CH2OH)2
NH(CH2)3N(CH2CH2OH)2
NHCH2(3-pyridyl)
NHCH2CH2(2-pyridyl)
NH(CH2)2(4-imidazolyl)
NH(CH2)3(1-imidazolyl)
4-Mepiperazinyl
43
16
19
28
27
8.1
0.65
1.0
46
7.9
5.9
8.9
71
230
13
8.1
6.3
9.1
102
3.9
0.93
0.35
1.5
1.2
0.78
1.7
5s
5t
5u
5v
5w
5x
5y
5z
6b
7b
8b
9a
9b
9c
9n
9o
9u
10n
15
23
2.5
5
44
6.4
NHCH2COOH
N(Me)CH2COOH
NH(CH2)2COOH
0.28
0.44
0.27
9
0.19
1.1
3.1
0.45
2.8
1.5
1.8
1.3
117
38
449
NHMe
NHMe
NHMe
NH2
NHMe
NMe2
Cl
185.5-187
172-173
235.5-237
189-190
239-241
168-170
200-203
207-209
>260
CF3
Me
Me
Me
Me
Me
Me
Br
0.4
23
20
0.02
>40
>35
>30
NH(CH2)2morpholinyl
NH(CH2)3morpholinyl
NH(CH2)2(4-imidazolyl)
NH(CH2)2morpholinyld
a
o
Solubility in water at 20 C, determined by HPLC (see text). Values are for the hydrochloride or dihydrochloride salt form of amines
b
and the sodium salt form of acids. IC50, concentration of drug (nM) to inhibit the phosphorylation of a random tyrosine/glutamic acid
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 IC50s for inhibition
of autophosphorylation of EGFR in A431 cells in culture. Values are the average of two experiments; see Experimental Section for details.
d
2-Me analogue.
Compound aqueous solubilities were determined by
HPLC following sonication for 30 min at 20 °C. Lactate
buffer (0.05 M) was used for compounds with neutral
side chains and water for hydrochloride salts of amines
and sodium salts of acids.
stituent in developing the 6-substituted series, although
a small number of 3-Me analogues were also prepared
for comparison purposes.
Compounds 5d -g, bearing hydroxyl substituents,
although considerably less potent than the parent 5b
against the isolated enzyme, retained acceptable levels
of inhibition (IC50s <1 nM). However, they did not show
improved aqueous solubility over 5b. In contrast, all
but one of the basic analogues 5h -w had aqueous
solubilities of g10 mM; the exception was 5w , with a
rigid, directly attached piperazine substituent. All of
the weak bases were evaluated for inhibition of isolated
enzyme and of autophosphorylation. Compounds 5h ,m
compared the utility of a secondary versus tertiary
amine linkage; the tertiary amine 5m was less effective
against isolated enzyme, but no difference was seen in
the autophosphorylation assay. Because of this result,
and a more extensive evaluation of this aspect (which
The initial set of 6-methylamino compounds (6b, 7b,
8b, and 9b) was prepared to compare with 5b and
evaluate SAR for the anilino side-chain substituent. A
number of small lipophilic substituents have been used
at the 3′-position in anilinoquinazolines9,12-14 and cor-
responding pyrido[4,3-d]pyrimidines,23 including Cl,
CF3, Me, and ethynyl. We have previously shown11 that
the 3′-Br derivative was the most potent enzyme-
inhibitory analogue in the anilinoquinazoline series, and
this proved also to be the case with the pyrido[3,4-d]-
pyrimidines. The 3′-Br analogue 5b (IC50 0.008 nM)
proved by far to be the most potent inhibitor of the
isolated enzyme, and we focused mainly on this sub-

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5 745
Ta ble 2. Inhibition (IC50 Values, µM) of EGF-Dependent Verus
showed no advantage) in the related pyrido[4,3-d]-
pyrimidines,20 tertiary amine linkages were not inves-
tigated further.
Heregulin-Dependent Tyrosine Autophosphorylation
no.
EGFa
heregulinb
For the 14 compounds with NH-linked amine side
chains (5h -l, 5n -v), no clear SAR emerges for inhibi-
tion of substrate phosphorylation by the isolated enzyme
(Table 1). These 6-substituents vary in amine base
strength (from about pKa 7 to 10), the distance of the
cationic center from the chromophore (from 3 to 5
atoms), and the overall lipophilicity of the side chain
(containing from one to two additional OH groups).
However, nearly all of have IC50 values of around 1 nM,
varying by less than 10-fold (except for two outliers,
5k ,p , by less than 5-fold). There is no variation with
positioning of the cation (5h -j), with side-chain lipo-
philicity (for example, 5h ,q), or with base strength
(several examples). This is in contrast to the 7-sub-
stitituted pyrido[4,3-d]pyrimidines,20 where IC50s be-
tween analogues with cationic side chains varied by
about 100-fold, improving with distance from the chro-
mophore and for weak bases. While the modeling
studies19 show that both 6- and 7-substituents are in
an area of bulk tolerance in the binding site, at the
entrance of the ATP pocket, the above results suggest
that 6-substituents are in a more favorable environ-
ment.
5b
5h
5k
5n
13
43
28
7.9
52
379
1100
219
a
EGF-dependent receptor autophosphorylation in A431 cells.
b Heregulin-dependent receptor autophosphorylation in MDA-MB-
453 cells.
Ta ble 3. Selectivity of Inhibition of Isolated EGFR Over Other
Receptor Tyrosine Kinases by Selected Analogues (IC50 Values,
µM)
no. EGFRa
c-Srcb
FGFRc
PDGFRd
IRe
5h 0.0011 >50 (42%) f >50 (41%) >50 (14%) >50 (0%)
5k 0.0046 >50 (26%)
5n 0.0007 >50 (14%)
>50 (24%) >50 (12%) >50 (0%)
>50 (39%) >50 (17%) >50 (12%)
See footnote b, Table 1. b For conditions, see ref 36. c Fibroblast
a
d
growth factor receptor; for conditions, see ref 36. Platelet-derived
growth factor receptor; for conditions, see ref 36. e Insulin receptor;
for conditions, see ref 36. f Percentage inhibition at 50 µM.
phorylation in cells (Table 2), suggesting they are broad-
spectrum inhibitors across the erbB family. At the same
time, as also shown previously for 5b,16 these com-
pounds had little or no activity against a series of other
receptor tyrosine kinases (Table 3), indicating a high
degree of specificity for the erbB family.
These compounds were evaluated in four xenograft
tumor model systems in nude mice (Table 4), and the
3′-methyl analogue 9b was also studied in the EGFR
line. The A431 epidermoid xenograft was selected based
on its in vitro mitogenic responsiveness to EGF and
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.26 The
MCF-7 breast and SK-OV-3 ovarian tumor models were
selected based on their in vivo expression of other EGF
receptor family members.
Compound 5b had the best therapeutic effect against
the A431 epidermoid carcinoma when administered
either intraperitoneally or orally (Table 4) compared to
compounds 5h ,k ,n at equitoxic doses. The latter three
compounds produced only marginal responses in this
model. All three compounds (5b,h ,k ) produced measur-
able, significant effects against a mouse fibroblast
transfected with human EGFR. The antitumor effects
of these compounds were lost when the dose route was
changed from intraperitoneal to peroral. Compounds
5n and 9b were also evaluated against this tumor model
but were ineffective.
Neither compound 5b nor 5n was effective against
the SK-OV-3 ovarian xenograft. This was unexpected,
since this cell line overexpresses erbB2. The lack of
effectiveness may indicate that although this cell line
overexpresses erbB2 it may not be dependent on this
receptor for growth in vivo. Compounds 5b,n produced
a significant therapeutic effect against the estrogen-
dependent MCF-7 breast carcinoma at equitoxic dose
levels, whereas 5k was ineffective against this tumor
model. Compound 5b was the most active against the
human tumor xenografts A431 and MCF-7 when evalu-
ated at the equitoxic doses reported in the table.
There were clear SARs for the ability of the basic
compounds to inhibit EGFR autophosphorylation in
A431 cells (Table 1). The hydrophilic OH-containing
analogues, and especially the diols 5q,r , were relatively
inactive, despite high potency against the isolated
enzyme. This is almost certainly due to poor cellular
uptake characteristics. With the exception of the (CH2)2-
NMe2 analogue 5h , where the cationic charge is posi-
tioned closest to the chromophore, all the other NH-
linked analogues were at least as potent as the parent
5b. Overall, the weaker bases (e.g., 5u ) appeared to
show better activity. A small series of analogous
3′-methyl derivatives with amine side chains was also
studied (compounds 9n ,o,u ) but these were less
active than their 3′-Br analogues in both assays, as
shown previously12,23-25 in several series of related
EGFR inhibitors. The 2-methyl compound 10n was
prepared to check bulk tolerance at this position, but it
showed very poor activity, as suggested both from
previous studies with 4-anilinoquinazolines11 and from
the binding model.19
Three anionic derivatives (5x-z) were also examined.
These were more potent than any of the amine ana-
logues against the isolated enzyme, but 5z showed very
poor activity in the cell-based autophosphorylation
assay. A similar result was seen for the 7-substituted
pyrido[4,3-d]pyrimidines20 and is again attributed to
poor cellular uptake.
From the above results, three compounds (5h ,k , n ),
which represent the range of structural variations
studied, were selected for comparison with the parent
5b in xenograft assays in vivo. They all had much
better aqueous solubility than 5b and showed acceptable
activity in the two screening assays (IC50s for inhibition
of the isolated enzyme of ∼1 nM and IC50s comparable
to that of the parent 5b for inhibition of autophospho-
rylation). Like 5b,16 all showed an ability to shut down
both EGF and heregulin-stimulated receptor autophos-

746 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5
Rewcastle et al.
Ta ble 4. In Vivo Activity of Selected Analogues against Tumor Xenografts in Nude Mice
dose
(mg/kg)
weight
change (g)
T/C(%) on last
therapy dayc
T-C
log net
no.
tumora
scheduleb
(days)d
cell kille
5b
A431
A431
75
100
38
ip, b.i.d. days 7-21
-0.8
-1.3
-0.1
+
41
38
38
67
27
14
67
8.4g
16.0
3.7g
1.2
-0.3
+0.1
-1.0
-1.3
+0.1
-0.1
-0.7
po, days 7-21
EGFR
EGFR
MCF-7
MCF-7
SK-OV-3
ip, b.i.d. days 1-15
po, days 1-15
ip, b.i.d. days 1-15
ip, b.i.d. days 1-15
ip, b.i.d. days 10-14, 17-21, 24-28
400f
50
+
15.2g
11.2g
0.8
25
30
-0.2
-1.6
5h
5k
A431
EGFR
EGFR
25
25
200
ip, b.i.d. days 7-21
ip, b.i.d. days 1-15
po, days 1-15
-1.4
-1.1
+
66
43
47
4.7
3.3g
1.1
-0.5
-1.0
-1.3
-0.5
-0.9
A431
25
25
200
25
ip, b.i.d. days 7-21
ip, b.i.d. days 1-15
po, days 1-15
-0.7
-0.9
+
62
44
94
77
5.0
5.0g
0
EGFR
EGFR
MCF-7
ip, b.i.d. days 1-15
-0.3
3.3
-0.6
5n
A431
A431
EGFR
EGFR
MCF-7
SKOV-3
12
25
ip, b.i.d. days 7-21
-0.2
-1.1
-1.3
-2.0
+
93
98
77
15
34
1.9
2.1
1.2
2.8
11.7g
0
-0.7
-0.3
-1.2
-1.1
-0.1
ip, b.i.d. days 13-26
ip, b.i.d. days 1-15
25
400f
25
po, days 1-15
ip, b.i.d. days 1-15
25
ip, b.i.d. days 10-14, 17-21, 24-28
-2.1
100
9b
EGFR
38
ip, b.i.d. days 1-15
-0.9
68
2.0
-1.2
a
b
The indicated tumor fragments were implanted sc into the right axilla of mice on day 0. Compounds were administered
intraperitoneally or orally on the indicated schedules. The maximum tolerated dose (LD10) from a complete dose-response is shown for
individual experiments. Reporting the maximum tolerated dose allows comparison of the antitumor effectiveness of test compounds at
c
d
equitoxic dose levels. Ratio of median treated tumor mass/median control tumor mass × 100%. The difference in days for the treated
(T) and control (C) tumors to reach 750 mg. e The net reduction in tumor burden, in log, between the first and last treatments. f Highest
g
dose tested. Significantly different from control (t-test, p < 0.05).
Compound 5n was active only against the MCF-7 tumor
model. In the A431 and MCF-7 models the tumors
treated with equitoxic doses of these compounds did not
increase substantially in size over the course of therapy
but resumed growth at the cessation of therapy.
analogue (9b). Meaningful in vivo activity against
human xenografts was observed only in two model
systems: the estrogen-dependent MCF-7 breast and
A431 epidermoid tumors. The small but significant in
vivo growth delay effects of these compounds against
the EGFR tumor model are reflective of the fact that
this manufactured cell line doubles in volume each day
and hence progresses very rapidly. These results may
also indicate that while this cell line requires EGF for
clone formation in soft agar, other growth factors may
be able to replace EGF in vivo.
No activity was seen for compounds 5b,n against the
SK-OV-3 ovarian tumor. While both the MCF-7 and
SK-OV-3 tumors are known to express other EGF
receptor family members in vivo, these may not be
absolutely required for growth in vivo. However, two
of the three compounds evaluated against MCF-7 (5b,n )
showed significant antitumor effects. These two com-
pounds also had the lowest relative IC50 values for the
inhibition of heregulin-dependent tyrosine phosphory-
lation in vitro (Table 2). It is notable that in the human
tumor xenografts A431 and MCF-7, where responses
were observed, the treated tumors remained approxi-
mately the same size throughout therapy, suggesting
that these compounds are cytostatic rather than cyto-
toxic under these test conditions. It remains to be
determined if more prolonged therapy would have
cytotoxic effects in vivo, resulting in high positive net
cell kill values.
Con clu sion s
This study was designed to determine the effects of
water-soluble groups attached at the 6-NHMe substitu-
ent of the extremely potent EGFR inhibitor 5b (PD
158780). A small series of 3′-substituted analogues of
5n confirmed that a 3′-bromo atom provided the most
potent derivative, and this was primarily used in the
main study. While compounds bearing neutral hy-
droxylated substituents at the 6-position retained rela-
tively good inhibitory potencies, aqueous solubility was
not improved, while anionic substituents resulted in
poor cellular uptake (as judged by relatively low poten-
cies in the autophosphorylation assay). The most suit-
able substituents were weak bases attached through a
secondary amine linkage; the corresponding compounds
had aqueous solubilities of g10 mM and IC50s for
inhibition of the isolated enzyme of ca. 1 nM. No clear
SAR could be discerned for amine base strength or the
distance of the cationic center from the chromophore,
suggesting that 6-substituents are in a favorable area
of bulk tolerance in the enzyme binding site. More
distinct SAR emerged for the ability of the compounds
to inhibit EGFR autophosphorylation in A431 cells,
where analogues bearing lipophilic weak bases were the
best.
Exp er im en ta l Section
Analyses were performed by the Microchemical Laboratory,
University of Otago, Dunedin, New Zealand. Melting points
were determined using an Electrothermal model 9200 digital
melting point apparatus and are as read. NMR spectra were
measured on Bruker AC-200 or DRX-400 spectrometers and
referenced to Me4Si. Mass spectra were recorded on a Varian
VG 7070 spectrometer at nominal 5000 resolution. HPLC was
Three analogues (5h ,k ,n ), with potent activity against
the isolated enzyme and possessing soluble basic side
chains where the above important parameters of lipo-
philicity and base strength were varied, were evaluated
for antitumor effectiveness against four in vivo tumor
model systems, along with the parent 5b and a 3′-Me

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5 747
carried out using a Bondclone 10 C18 column, with a Phillips
PU4100M gradient elution pump and a Phillips PU 4120 diode
array detector, eluting with the appropriate ratios of 80%
acetonitrile/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).
7.02 (d, J ) 7.4 Hz, 1 H, H-4′), 2.36 (s, 3 H, CH3). Anal.
(C14H11FN4) C, H, N.
4-[(3-Br om oph en yl)am in o]-6-(m eth ylam in o)pyr ido[3,4-
d ]p yr im id in e (5b), Gen er a l Meth od A of Sch em e 1. A 2-L
stainless steel reactor was flushed with dry N2 and charged
with 13 (35.0 g, 108 mmol) and anhydrous MeNH2 (57.5 g,
1.85 mol) in DMSO (1 L). The reactor was sealed and heated
at 80 °C for 24 h and then cooled. After venting off the excess
MeNH2, the mixture was concentrated to ca. 500 mL and
poured slowly into 2.5 L of water with vigorous stirring. The
precipitate was collected, washed well with water, and dried
over P2O5 to afford 5b: mp (2-propanol) 181-183 °C (lit.15 mp
172-173 °C from MeOH/H2O). Total yield after chromatog-
raphy of the mother liquor on silica gel: 32.5 g (91%).
The following compounds were prepared similarly.
2-[(2-Am in oeth yl)m eth yla m in o]eth a n ol (20), Sch em e
3. Condensation of 3-methyloxazolidine (18) and aqueous
HCN as reported22 gave [N-(2-hydroxyethyl)methylamino]-
acetonitrile (19) (93%) as an oil: bp 70-72 °C/0.03 mmHg (lit.27
1
bp 62-63 °C/0.05 mmHg); H NMR (CDCl3) δ 3.70-3.64 (br
m, with D2O wash collapses to t, J ) 5.3 Hz at δ 3.67, 2 H),
3.61 (s, 2 H), 2.69 (t, J ) 5.3 Hz, 2 H), 2.43 (s, 3 H), 2.19 (br
s, exchanges with D2O); CIMS m/z (relative intensity) 115
(MH+, 46), 88 (100).
4-[(3-Br om op h en yl)a m in o]-6-[(2-h yd r oxyeth yl)a m in o]-
p yr id o[3,4-d ]p yr im id in e (5d ): from 13 and ethanolamine
(35% yield after two recrystallizations from MeOH); mp 209-
Reduction of 19 with LiAlH4 gave 20 as an oil: bp 81-82
°C/0.42 mmHg (lit.28 bp 115-117 °C/16 mmHg, lit.29 bp 103-
104 °C/8 mmHg); 1H NMR (CDCl3) δ 3.61 (t, J ) 5.3 Hz, 2 H),
2.81 (t, J ) 5.8 Hz, 2 H), 2.55 (t, J ) 5.5 Hz, 2 H), 2.50 (t, J )
6.3 Hz, 2 H), 2.35 (br s, 3 H), 2.29 (s, 3 H); CIMS m/z (relative
intensity) 119 (MH+, 100), 102 (97), 88 (91).
1
210 °C; H NMR [(CD3)2SO] δ 9.72 (s, 1 H, NH), 8.75 (s, 1 H,
H-8), 8.40 (s, 1 H, H-2), 8.20 (br s, 1 H, H-2′), 7.91 (br d, J )
8.0 Hz, 1 H, H-6′), 7.37 (t, J ) 8.0 Hz, 1 H, H-5′), 7.31 (br d,
J ) 8.4 Hz, 1 H, H-4′), 7.15 (s, 1 H, H-5), 6.66 (t, J ) 5.7 Hz,
1 H, NH), 4.80 (t, J ) 5.3 Hz, 1 H, OH), 3.65 (q, J ) 5.8 Hz,
2 H, CH2), 3.38 (q, J ) 6.1 Hz, 2 H, CH2). Anal. (C15H14BrN5)
C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-[N-(2-h yd r oxyet h yl)-N-
m eth yla m in o]p yr id o[3,4-d ]p yr im id in e (5e). A mixture of
13 (0.20 g, 0.63 mmol) and 2-(methylamino)ethanol (2.4 g, 31
mmol, 50 equiv) in EtOH (50 mL) was heated at 95 °C for 18
h in a sealed pressure vessel. The solvent was removed under
reduced pressure, and the residue was triturated with water
and recrystallized from MeOH to give 5e (0.21 g, 89%): mp
236-237 °C; 1H NMR [(CD3)2SO] δ 9.73 (s, 1 H, NH), 8.82 (s,
1 H, H-8), 8.41 (s, 1 H, H-2), 8.18 (br s, 1 H, H-2′), 7.93 (br d,
J ) 8.0 Hz, 1 H, H-6′), 7.38 (t, J ) 8.0 Hz, 1 H, H-5′), 7.33 (br
d, J ) 8.4 Hz, 1 H, H-4′), 7.24 (s, 1 H, H-5), 4.73 (t, J ) 5.3
Hz, 1 H, OH), 3.76 (t, J ) 6.1 Hz, 2 H, CH2), 3.63 (dd, J ) 6.1,
5.6 Hz, 2 H, CH2), 3.19 (s, 3 H, CH3). Anal. (C16H16BrN5O)
C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-flu or op yr id o[3,4-d ]p yr i-
m id in e (13), Sch em e 1. A stirred suspension of 6-fluoropy-
rido[3,4-d]pyrimidin-4(3H)-one21 (11) (30.0 g, 182 mmol) in 1,2-
dichloroethane (182 mL) was treated successively with SOCl2
(182 mL) and then ca. 1 mL of DMF. The mixture was heated
at reflux for 2.5 h, then concentrated to a solid, and coevapo-
rated twice with 1,2-dichloroethane. The residue was dis-
solved in CH2Cl2 and filtered through a short pad of silica gel,
eluting with CH2Cl2 to give 4-chloro-6-fluoropyrido[3,4-d]-
pyrimidine (12) (30.5 g, 91%). A sample was crystallized from
tert-butyl methyl ether: mp 75-76 °C; 1H NMR (CDCl3) δ 9.29
(s, 1 H, H-2), 9.16 (s, 1 H, H-8), 7.65 (dd, J ) 0.7, 2.0 Hz, 1 H,
H-5); 19F NMR δ -68.9 (s); 13C NMR δ 162.1 (d, J C-F ) 7 Hz),
161.8 (d, J C-F ) 242 Hz), 154.1 (d, J C-F ) 2 Hz), 153.4 (d, J C-F
) 15.3 Hz), 143.7 (d, J C-F ) 3 Hz), 130.7 (d, J C-F ) 9 Hz),
100.9 (d, J C-F ) 39.7 Hz). Anal. (C7H3N3ClF) C, H, N.
A mechanically stirred solution of 12 (30.0 g, 163 mmol) and
3-bromoaniline (33.75 g, 196 mmol) in 2-propanol (400 mL)
was heated at reflux for 45 min. The resulting suspension
was concentrated to ca. 150 mL, and the resulting precipitate
was collected, washed successively with 2% aqueous NaOH
to neutral pH, water, and 2-propanol, and dried over P2O5 to
give 13 (49.1 g, 94%): mp (2-propanol) 224-226 °C (lit.15 mp
4-[(3-Br om op h en yl)a m in o]-6-[(2,3-d ih yd r oxyp r op yl)-
a m in o]p yr id o[3,4-d ]p yr im id in e (5f): from 13 and 2,3-
1
dihydroxypropylamine (42%); mp (MeOH) 186.5-188 °C; H
NMR [(CD3)2SO] δ 9.74 (s, 1 H, NH), 8.75 (s, 1 H, H-8), 8.41
(s, 1 H, H-2), 8.21 (br s, 1 H, H-2′), 7.91 (br d, J ) 7.9 Hz, 1 H,
H-6′), 7.37 (t, J ) 8.0 Hz, 1 H, H-5′), 7.31 (br d, J ) 8.4 Hz, 1
H, H-4′), 7.16 (s, 1 H, H-5), 6.46 (t, J ) 5.5 Hz, 1 H, NH), 4.93
(d, J ) 4.9 Hz, 1 H, OH), 4.68 (t, J ) 5.5 Hz, 1 H, OH), 3.77
(sextet, J ) 5.5 Hz, 1 H, CH), 3.47-3.39 (m, 2 H, CH2), 3.27-
3.20 (m, 2 H, CH2). Anal. (C16H16BrN5O2‚0.5H2O) C, H; N:
found, 18.1; calcd, 18.5.
1
219.5-221 °C); H NMR [(CD3)2SO] δ 10.06 (br s, 1 H, NH),
8.94 (s, 1 H, H-8), 8.73 (s, 1 H, H-2), 8.23 (br s, 2 H, H-5,2′),
7.90 (br d, J ) 7.5 Hz, 1 H, H-6′), 7.39 (t, J ) 7.8 Hz, 1 H,
H-5′), 7.35 (d, J ) 8.0 Hz, 1 H, H-4′); 19F NMR δ -74.12 (s);
13C NMR δ 159.8 (d, J C-F ) 233 Hz, C-6), 156.6 (d, J C-F ) 5
Hz), 154.9 (C-2), 150.7 (d, J C-F ) 15 Hz, C-8), 142.8, 140.0,
130.5, 126.7, 124.1, 123.2 (d, J C-F ) 9 Hz), 121.2, 120.6, 99.6
(d, J C-F ) 40 Hz, C-5); CIMS m/z (relative intensity) 321 (MH
+ 2+, 65), 319 (MH+, 100).
4-[(3-Br om oph en yl)am in o]-6-[N-(2,3-dih ydr oxypr opyl)-
N-m eth yla m in o]p yr id o[3,4-d ]p yr im id in e (5g): from 13
and 2,3-dihydroxypropyl-N-methylamine20 (72%); mp (MeOH)
159-160 °C; 1H NMR [(CD3)2SO] δ 9.71 (s, 1 H, NH), 8.82 (s,
1 H, H-8), 8.41 (s, 1 H, H-2), 8.18 (t, J ) 1.9 Hz, 1 H, H-2′),
7.93 (br d, J ) 8.2 Hz, 1 H, H-6′), 7.39 (t, J ) 8.0 Hz, 1 H,
H-5′), 7.33 (br d, J ) 8.4 Hz, 1 H, H-4′), 7.22 (s, 1 H, H-5),
4.75 (d, J ) 4.9 Hz, 1 H, OH), 4.59 (t, J ) 5.7 Hz, 1 H, OH),
3.84-3.79 (m, 2 H, CH2), 3.59 (dd, J ) 8.7, 6.7 Hz, 1 H, CH),
The following compounds were prepared similarly.
4-[(3-Ch lor op h en yl)a m in o]-6-flu or op yr id o[3,4-d ]p yr i-
m id in e (14): from 12 and 3-chloroaniline (89%); mp (MeOH/
1
H2O) 224-225 °C; H NMR [(CD3)2SO] δ 10.11 (s, 1 H, NH),
8.97 (s, 1 H, H-8), 8.76 (s, 1 H, H-5), 8.28 (s, 1 H, H-2), 8.14 (t,
J ) 2.0 Hz, 1 H, H-2′), 8.26 (dd, J ) 8.2, 1.3 Hz, 1 H, H-6′),
7.47 (t, J ) 8.1 Hz, 1 H, H-5′), 7.24 (dd, J ) 8.0, 1.4 Hz, 1 H,
H-4′). Anal. (C14H12ClN5) C, H, N.
3.41-3.35 (m, 2 H, CH2), 3.19 (s, 3 H, CH3). Anal. (C17H18
BrN5O2) C, H, N.
-
4-[(3-Br om op h e n yl)a m in o]-6-[[2-(d im e t h yla m in o)-
eth yl]a m in o]p yr id o[3,4-d ]p yr im id in e (5h ): from 13 and
2-(dimethylamino)ethylamine (63%); mp (CH2Cl2) 114-118 °C;
1H NMR [(CD3)2SO] δ 9.69 (s, 1 H, NH), 8.76 (s, 1 H, H-8),
8.41 (s, 1 H, H-2), 8.22 (t, J ) 1.9 Hz, 1 H, H-2′), 7.91 (br d, J
) 8.0 Hz, 1 H, H-6′), 7.37 (t, J ) 8.0 Hz, 1 H, H-5′), 7.31 (br d,
J ) 8.4 Hz, 1 H, H-4′), 7.15 (s, 1 H, H-5), 6.50 (t, J ) 5.4 Hz,
1 H, NH), 3.38 (dd, J ) 6.6, 5.7 Hz, 2 H, CH2), 2.53 (t, J ) 6.6
Hz, 2 H, CH2), 2.23 (s, 6 H, CH3). Dihydrochloride salt (from
EtOH): mp 250-252 °C. Anal. (C17H19BrN6‚2HCl) C, H, N,
Cl.
6-F lu or o-4-[[3-(tr iflu or om eth yl)p h en yl]a m in o]p yr id o-
[3,4-d ]p yr im id in e (15): from 12 and 3-(trifluoromethyl)-
aniline (95%); mp (MeOH/H2O) 209-211 °C; 1H NMR [(CD3)2-
SO] δ 10.25 (s, 1 H, NH), 8.99 (s, 1 H, H-8), 8.78 (s, 1 H, H-5),
8.35 (s, 1 H, H-2), 8.30 (s, 1 H, H-2′), 8.26 (d, J ) 8.3 Hz, 1 H,
H-6′), 7.68 (t, J ) 8.0 Hz, 1 H, H-5′), 7.53 (d, J ) 7.7 Hz, 1 H,
H-4′). Anal. (C15H12F3N5) C, H, N.
6-F lu or o-4-[(3-m eth ylp h en yl)a m in o]p yr id o[3,4-d ]p yr i-
m id in e (16): from 12 and m-toluidine (92%); mp (MeOH/H2O)
190-192 °C; 1H NMR [(CD3)2SO] δ 9.99 (s, 1 H, NH), 8.93 (s,
1 H, H-8), 8.68 (d, J H-F ) 1.8 Hz, 1 H, H-5), 8.29 (s, 1 H, H-2),
7.72-7.68 (m, 2 H, H-2′,6′), 7.32 (t, J ) 8.0 Hz, 1 H, H-5′),
4-[(3-Br om op h en yl)a m in o]-6-[[3-(d im eth yla m in o)p r o-
p yl]a m in o]p yr id o[3,4-d ]p yr im id in e (5i): from 13 and

748 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5
Rewcastle et al.
3-(dimethylamino)propylamine (80%); mp (CH2Cl2/hexane)
160-161 °C; 1H NMR [(CD3)2SO] δ 9.72 (s, 1 H, NH), 8.75 (s,
1 H, H-8), 8.40 (s, 1 H, H-2), 8.20 (t, J ) 1.8 Hz, 1 H, H-2′),
7.91 (br d, J ) 8.2 Hz, 1 H, H-6′), 7.37 (t, J ) 8.0 Hz, 1 H,
H-5′), 7.31 (br d, J ) 8.4 Hz, 1 H, H-4′), 7.08 (s, 1 H, H-5),
6.84 (t, J ) 5.4 Hz, 1 H, NH), 3.29 (q, J ) 6.2 Hz, 2 H, CH2),
2.35 (t, J ) 6.9 Hz, 2 H, CH2), 2.15 (s, 6 H, CH3), 1.78 (pentet,
J ) 6.9 Hz, 2 H, CH2). Anal. (C18H21BrN6) C, H, N.
was dried (Na2SO4), and the solvent was removed under
reduced pressure. Chromatography of the residue on silica
gel, eluting with EtOAc/MeOH (99:1), gave 5n (0.19 g, 71%
1
yield): mp (MeOH) 185-187 °C; H NMR [(CD3)2SO] δ 9.69
(s, 1 H, exchangeable with D2O, NH), 8.76 (s, 1 H, H-8), 8.41
(s, 1 H, H-2), 8.21 (t, J ) 1.9 Hz, 1 H, H-2), 7.91 (br d, J ) 8.1
Hz, 1 H, H-6), 7.37 (t, J ) 8.0 Hz, 1 H, H-5), 7.31 (br d, J )
7.9 Hz, 1 H, H-4), 7.15 (s, 1 H, H-5), 6.55 (t, J ) 5.5 Hz, 1 H,
exchangeable with D2O, NH), 3.60 (t, J ) 4.6 Hz, 4 H, CH2O),
3.41 (dd, J ) 6.5, 5.9 Hz, 2 H, CH2N), 2.60 (t, J ) 6.7 Hz, 2 H,
CH2N), 2.46 (m, 4 H, CH2N). Anal. (C19H21BrN6O) C, H, N.
Dihydrochloride salt: mp (MeOH/EtOH) 267 °C dec. Anal.
(C19H21BrN6O‚2HCl) C, H, N, Cl.
4-[(3-Br om oph en yl)am in o]-6-[[4-(dim eth ylam in o)bu tyl]-
a m in o]p yr id o[3,4-d ]p yr im id in e (5j): from 13 and 4-(di-
methylamino)butylamine (98%); mp (CH2Cl2/hexane) 176-177
°C; 1H NMR [(CD3)2SO] δ 9.68 (s, 1 H, NH), 8.75 (s, 1 H, H-8),
8.39 (s, 1 H, H-2), 8.21 (t, J ) 1.8 Hz, 1 H, H-2′), 7.92 (br d, J
) 8.3 Hz, 1 H, H-6′), 7.37 (t, J ) 8.0 Hz, 1 H, H-5′), 7.31 (dd,
J ) 8.4, 1.2 Hz, 1 H, H-4′), 7.06 (s, 1 H, H-5), 6.87 (t, J ) 5.6
Hz, 1 H, NH), 3.27 (q, J ) 6.4 Hz, 2 H, CH2), 2.24 (t, J ) 7.1
Hz, 2 H, CH2), 2.12 (s, 6 H, CH3), 1.65 (pentet, J ) 7.1 Hz, 2
4-[(3-Br om op h en yl)a m in o]-6-[[3-(4-m or p h olin o)p r op y-
l]a m in o]p yr id o[3,4-d ]p yr im id in e (5o): from 13 and 4-(3-
aminopropyl)morpholine (87%); mp (MeOH/H2O) 177-178 °C;
1
H NMR [(CD3)2SO] δ 9.70 (s, 1 H, NH), 8.75 (s, 1 H, H-8),
H, CH2), 1.53 (pentet, J ) 7.2 Hz, 2 H, CH2). Anal. (C19H23
BrN6) C, H, N.
-
8.40 (s, 1 H, H-2), 8.20 (t, J ) 1.9 Hz, 1 H, H-2′), 7.91 (ddd, J
) 8.0, 1.8, 1.2 Hz, 1 H, H-6′), 7.37 (t, J ) 8.0 Hz, 1 H, H-5′),
7.31 (dd, J ) 8.0, 1.2 Hz, 1 H, H-4′), 7.08 (s, 1 H, H-5), 6.86 (t,
J ) 5.5 Hz, 1 H, NH), 3.58 (t, J ) 4.6 Hz, 4 H, CH2), 3.31 (q,
J ) 6.4 Hz, 2 H, CH2), 2.41 (t, J ) 7.0 Hz, 2 H, CH2), 2.36 (br
m, 4 H, CH2), 1.81 (pentet, J ) 6.9 Hz, 2 H, CH2). Anal.
(C20H23BrN6O) C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-[[3-(d ieth yla m in o)-2-h y-
d r oxyp r op yl]a m in o]p yr id o[3,4-d ]p yr im id in e (5k ). A so-
lution of 13 (5.74 g, 18 mmol) and 1-amino-3-(diethylamino)-
2-propanol30 (26.3 g, 180 mmol) in DMSO (27 mL) was heated
at 80 °C under N2 for 23 h. The DMSO was evaporated under
reduced pressure (0.1 mm) at 100 °C, and the residue was
purified by flash chromatography on silica gel. Elution with
a gradient of MeOH in EtOAc containing 2% Et3N and pooling
of appropriate fractions gave 5k (4.87 g). Rechromatography
of the mother liquor gave an additional 0.48 g (total yield
67%): mp (dihydrochloride salt from iPrOH/CH2Cl2/HCl)
(EtOAc) 189-192 °C; 1H NMR [free base, (CD3)2SO] δ 9.72 (s,
exchanges with D2O, 1 H), 8.75 (s, 1 H), 8.41 (s, 1 H), 8.21 (t,
J ) 1.9 Hz, 1 H), 7.91 (d, J ) 7.0 Hz, 1 H), 7.39-7.29 (m, 2
H), 7.14 (s, 1 H), 6.63 (t, J ) 5.3 Hz, exchanges with D2O, 1
H), 4.77 (d, J ) 4.3 Hz, exchanges with D2O, 1 H), 3.87-3.83
(m, 1 H), 3.39-3.28 (m, 2 H), 2.58-2.41 (m, 6 H), 0.96 (t, J )
7.0 Hz, 6 H); CIMS m/z (relative intensity) 445 (MH+, 13), 447
(13). Anal. (C20H25BrN6O‚2HCl‚0.5H2O) C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-[[2-[N-(2-h yd r oxyeth yl)-
m eth yla m in o]eth yl]a m in o]p yr id o[3,4-d ]p yr im id in e (5l).
Reaction of 13 with 2-[(2-aminoethyl)methylamino]ethanol
(20), followed by column chromatography, gave 5l (60% as the
dihydrochloride salt): mp (CH2Cl2/2-propanol) 182-189 °C dec;
1H NMR [(CD3)2SO] δ 11.63 (br s, 1 H, exchanges with D2O),
10.11 (br s, 1 H, exchanges with D2O), 8.92 (s, 1 H), 8.72 (s, 1
H), 8.21 (s, 1 H), 7.94 (d overlapping s, 2 H, with D2O wash
collapses to δ 7.85, d, J ) 8.2 Hz, 1 H, δ 7.68, s, 1 H), 7.60-
7.40 (br s overlapping m, 3 H, with D2O wash collapses to d,
2 H), 3.84-3.75 (m, 4 H), 3.53-3.40 (m, 1 H), 3.40-3.27 (m, 2
H), 3.26-3.16 (m, 1 H), 2.90 (d, J ) 4.8 Hz, 3 H, with D2O
wash collapses to s); CIMS m/z (relative intensity) 417 (MH+,
26), 419 (25). Anal. (C18H21BrN6O‚2HCl‚0.5 H2O) C, H, N,
Cl.
4-[(3-Br om op h en yl)a m in o]-6-[[3-(4-m eth yl-1-p ip er a zi-
n yl)p r op yl]a m in o]p yr id o[3,4-d ]p yr im id in e (5p ): from 13
and 3-(4-methylpiperazyl)propylamine (83%); mp (CH2Cl2/
hexane) 182-183 °C; 1H NMR [(CD3)2SO] δ 9.70 (s, 1 H, NH),
8.75 (s, 1 H, H-8), 8.40 (s, 1 H, H-2), 8.20 (t, J ) 1.9 Hz, 1 H,
H-2′), 7.92 (br d, J ) 8.2 Hz, 1 H, H-6′), 7.37 (t, J ) 8.0 Hz, 1
H, H-5′), 7.31 (dd, J ) 8.3, 1.2 Hz, 1 H, H-4′), 7.07 (s, 1 H,
H-5), 6.85 (t, J ) 5.5 Hz, 1 H, NH), 3.30 (q, J ) 6.3 Hz, 2 H,
CH2), 2.40 (t, J ) 7.0 Hz, 2 H, CH2), 2.33 (br m, 4 H, CH2),
2.13 (s, 3 H, CH3), 1.79 (pentet, J ) 7.2 Hz, 2 H, CH2). Anal.
(C21H26BrN7) C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-[[2-[N,N-b is(2-h yd r oxy-
e t h y l)a m in o ]e t h y l]a m in o ]p y r id o [3,4-d ]p y r im id in e
(5q): from 13 and N,N-bis(2-hydroxyethyl)ethylamine (41%);
1
mp (MeOH) 210-211 °C; H NMR [(CD3)2SO] δ 9.70 (s, 1 H,
NH), 8.76 (s, 1 H, H-8), 8.41 (s, 1 H, H-2), 8.22 (br s, 1 H, H-2′),
7.92 (br d, J ) 8.1 Hz, 1 H, H-6′), 7.37 (t, J ) 8.0 Hz, 1 H,
H-5′), 7.31 (br d, J ) 8.0 Hz, 1 H, H-4′), 7.14 (s, 1 H, H-5),
6.59 (t, J ) 5.4 Hz, 1 H, NH), 4.44 (t, J ) 5.4 Hz, 2 H, OH),
3.46 (dd, J ) 5.9, 5.7 Hz, 4 H, CH2), 3.34 (q, J ) 6.2 Hz, 2 H,
CH2), 2.78 (t, J ) 6.3 Hz, 2 H, CH2), 2.62 (t, J ) 6.1 Hz, 4 H,
CH2). Anal. (C19H23BrN6O2) C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-[[3-[N,N-b is(2-h yd r oxy-
e t h yl)a m in o]p r op yl]a m in o]p yr id o[3,4-d ]p yr im id in e
(5r ): from 13 and N,N-bis(2-hydroxyethyl)propylamine (48%);
1
H NMR [(CD3)2SO] δ 9.68 (s, 1 H, NH), 8.75 (s, 1 H, H-8),
8.40 (s, 1 H, H-2), 8.21 (t, J ) 1.8 Hz, 1 H, H-2′), 7.91 (br d, J
) 8.4 Hz, 1 H, H-6′), 7.37 (t, J ) 8.0 Hz, 1 H, H-5′), 7.31 (br d,
J ) 8.3 Hz, 1 H, H-4′), 7.07 (s, 1 H, H-5), 6.87 (t, J ) 5.5 Hz,
1 H, NH), 4.40 (t, J ) 4.9 Hz, 2 H, OH), 3.45 (dd, J ) 5.9, 5.3
Hz, 4 H, CH2), 3.31 (dd, J ) 6.2, 5.9 Hz, 2 H, CH2), 2.61 (t, J
) 6.9 Hz, 2 H, CH2), 2.55 (t, J ) 6.3 Hz, 4 H, CH2), 1.76 (pentet,
J ) 6.7 Hz, 2 H, CH2). Dihydrochloride salt (from EtOH): mp
205-207 °C. Anal. (C20H25BrN6O2‚2HCl) C, H, N, Cl.
A higher Rf component, eluted from the column with EtOAc/
MeOH/Et3N (90:10:1) (29 mg), was identified as 5e, resulting
from nucleophilic addition of the tertiary amine of the side
chain followed by extrusion of aziridine.
4-[(3-Br om op h en yl)a m in o]-6-[N-[2-(d im et h yla m in o)-
eth yl]-N-m eth ylam in o]pyr ido[3,4-d]pyr im idin e (5m ): from
13 and 2-(dimethylamino)-N-methylethylamine (72%); 1H
NMR [(CD3)2SO] δ 9.72 (s, 1 H, NH), 8.83 (s, 1 H, H-8), 8.41
(s, 1 H, H-2), 8.18 (br s, 1 H, H-2′), 7.92 (br d, J ) 7.9 Hz, 1 H,
H-6′), 7.38 (t, J ) 8.0 Hz, 1 H, H-5′), 7.33 (br d, J ) 8.0 Hz, 1
H, H-4′), 7.21 (s, 1 H, H-5), 3.80 (t, J ) 6.8 Hz, 2 H, CH2), 3.14
(s, 3 H, CH3), 2.45 (t, J ) 6.8 Hz, 2 H, CH2), 2.19 (s, 6 H, CH3).
Dihydrochloride salt (from EtOH): mp 260-262 °C. Anal.
(C18H21BrN6‚2HCl) C, H, N, Cl.
4-[(3-Br om op h en yl)a m in o]-6-[[2-(4-m or p h olin o)eth yl]-
a m in o]p yr id o[3,4-d ]p yr im id in e (5n ). A mixture of 13 (0.20
g, 0.63 mmol) and 4-(2-aminoethyl)morpholine (4.1 g, 31 mmol,
50 equiv) in DMSO (40 mL) was heated at 95 °C for 18 h. Most
of the DMSO was removed under reduced pressure at 80 °C,
and the residue was diluted with water and extracted into
EtOAc. After being washed twice with water, the organic layer
4-[(3-Br om oph en yl)am in o]-6-[(3-pyr idyl)m eth ylam in o]-
p yr id o[3,4-d ]p yr im id in e (5s): from 13 and 3-(aminometh-
yl)pyridine (37%); 1H NMR [(CD3)2SO] δ 9.72 (s, 1 H, NH), 8.78
(s, 1 H, H-8), 8.64 (d, J ) 1.7 Hz, 1 H, H-2′′), 8.44 (dd, J ) 4.7,
1.4 Hz, 1 H, H-4′′), 8.41 (s, 1 H, H-2), 8.21 (t, J ) 1.9 Hz, 1 H,
H-2′), 7.89 (br d, J ) 7.9 Hz, 1 H, H-6′), 7.79 (br d, J ) 6.2 Hz,
1 H, H-6′′), 7.47 (t, J ) 6.3 Hz, 1 H, NH), 7.37 (t, J ) 8.0 Hz,
1 H, H-5′), 7.35-7.30 (m, 2 H, H-4′,5′′), 7.25 (s, 1 H, H-5), 4.60
(d, J ) 4.4 Hz, 2 H, CH2). Dihydrochloride salt (from EtOH):
mp 260-262 °C. Anal. (C19H16BrN6‚2HCl) C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-[[2-(2-p yr id yl)eth yl]a m i-
n o]p yr id o[3,4-d ]p yr im id in e (5t): from 13 and 3-(2-amino-
ethyl)pyridine (87%); mp (MeOH/H2O) 170-171.5 °C; 1H NMR
[(CD3)2SO] δ 9.72 (s, 1 H, NH), 8.76 (s, 1 H, H-8), 8.53 (dd, J
) 4.2, 0.9 Hz, 1 H, H-6′′), 8.41 (s, 1 H, H-2), 8.20 (t, J ) 1.9
Hz, 1 H, H-2′), 7.90 (br d, J ) 7.7 Hz, 1 H, H-6′), 7.73 (td, J )

Tyrosine Kinase Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5 749
7.6, 1.7 Hz, 1 H, H-4′′), 7.38-7.34 (m, 2 H, H-5′,3′′), 7.31 (br
d, J ) 8.1 Hz, 1 H, H-4′), 7.24 (ddd, J ) 8.2, 4.9, 0.8 Hz, 1 H,
H-5′′), 7.12 (s, 1 H, H-5), 6.88 (t, J ) 5.4 Hz, 1 H, NH), 3.65
(dd, J ) 7.0, 5.5 Hz, 2 H, CH2), 3.12 (t, J ) 7.2 Hz, 2 H, CH2).
Anal. (C20H17BrN6) C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-[[2-(4-im id a zolyl)eth yl]-
a m in o]p yr id o[3,4-d ]p yr im id in e (5u ): from 13 and 2-(imi-
dazol-4-yl)ethylamine (66%); mp (MeOH) 200-201 °C; 1H
NMR [(CD3)2SO] δ 11.87 (br, 1 H, NH), 9.71 (s, 1 H, NH), 8.76
(s, 1 H, H-8), 8.41 (s, 1 H, H-2), 8.20 (t, J ) 1.8 Hz, 1 H, H-2′),
7.91 (br d, J ) 8.0 Hz, 1 H, H-6′), 7.57 (d, J ) 0.8 Hz, 1 H,
H-2′′), 7.37 (t, J ) 8.0 Hz, 1 H, H-5′), 7.31 (br d, J ) 8.2 Hz, 1
H, H-4′), 7.13 (s, 1 H, H-5), 6.89 (br s, 1 H, H-5′′), 6.85 (t, J )
5.4 Hz, 1 H, NH), 3.51 (dd, J ) 7.2, 5.7 Hz, 2 H, CH2), 2.88 (t,
J ) 7.3 Hz, 2 H, CH2). Anal. (C19H18BrN7) C, H, N.
H, CH2), 2.47 (br m, 4 H, CH2), 2.35 (s, 3 H, CH3). Anal.
(C20H24N6O‚0.5 H2O) C, H, N.
4-[(3-Met h ylp h en yl)a m in o]-6-[[3-(4-m or p h olin o)p r o-
p yl]a m in o]p yr id o[3,4-d ]p yr im id in e (9o): from 16 and 1-(3-
aminopropyl)morpholine (81%); mp 200-203 °C; 1H NMR
[(CD3)2SO] δ 9.57 (s, 1 H, NH), 8.71 (s, 1 H, H-8), 8.33 (s, 1 H,
H-2), 7.67 (br d, J ) 8.1 Hz, 1 H, H-6′), 7.63 (br s, 1 H, H-2′),
7.28 (t, J ) 7.8 Hz, 1 H, H-5′), 7.10 (s, 1 H, H-5), 6.96 (br d, J
) 7.4 Hz, 1 H, H-4′), 6.79 (q, J ) 5.6 Hz, 1 H, NH), 3.58 (t, J
) 4.6 Hz, 4 H, CH2), 3.30 (q, J ) 6.4 Hz, 2 H, CH2), 2.42 (t, J
) 7.1 Hz, 2 H, CH2), 2.37 (br m, 4 H, CH2), 2.35 (s, 3 H, CH3),
1.80 (pentet, J ) 6.9 Hz, 2 H, CH2). Anal. (C21H26N6O‚0.5H2O)
C, H, N.
6-[[2-(4-Im id a zolyl)eth yl]a m in o]-4-[(3-m eth ylp h en yl)-
a m in o]p yr id o[3,4-d ]p yr im id in e (9u ): from 16 and 2-(imi-
dazol-4-yl)ethylamine (70%); mp 207-209 °C; 1H NMR [(CD3)2-
SO] δ 11.85 (br, 1 H, NH), 9.58 (s, 1 H, NH), 8.72 (s, 1 H, H-8),
8.34 (s, 1 H, H-2), 7.67 (br d, J ) 8.1 Hz, 1 H, H-6′), 7.63 (br
s, 1 H, H-2′), 7.56 (br s, 1 H, H-2′′), 7.28 (t, J ) 7.8 Hz, 1 H,
H-5′), 7.15 (s, 1 H, H-5), 6.96 (br d, J ) 7.2 Hz, 1 H, H-4′),
6.90 (m, 1 H, NH), 6.78 (t, J ) 5.0 Hz, 1 H, H-5′′), 3.50 (dd, J
) 7.2, 5.7 Hz, 2 H, CH2), 2.88 (t, J ) 6.8 Hz, 2 H, CH2), 2.34
(s, 3 H, CH3). Anal. (C19H19N7‚0.25H2O) C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-[(2-ca r boxyeth yl)a m in o]-
pyr ido[3,4-d]pyr im idin e (5z), Gen er al Exam ple of Meth od
B of Sch em e 1. A mixture of 13 (0.20 g, 0.63 mmol) and
sodium 2-aminopropanoate (3.4 g, 31 mmol, 50 equiv) (pre-
pared from â-alanine and Na in EtOH) in EtOH (50 mL) was
heated at 95 °C for 48 h in a sealed pressure vessel. The
solvent was removed under reduced pressure, the residue was
dissolved in water and filtered to remove insolubles, and the
filtrate was acidified (AcOH) to give 5z (0.11 g, 45%): mp
(MeOH) 276-280 °C; 1H NMR [(CD3)2SO] δ 12.25 (br, 1 H,
CO2H), 9.74 (s, 1 H, NH), 8.76 (s, 1 H, H-8), 8.41 (s, 1 H, H-2),
8.20 (t, J ) 1.9 Hz, 1 H, H-2′), 7.90 (br d, J ) 8.0 Hz, 1 H,
H-6′), 7.37 (t, J ) 8.0 Hz, 1 H, H-5′), 7.31 (br d, J ) 8.0 Hz, 1
H, H-4′), 7.14 (s, 1 H, H-5), 6.80 (t, J ) 5.5 Hz, 1 H, NH), 3.51
(dd, J ) 6.8, 5.7 Hz, 2 H, CH2), 2.63 (t, J ) 7.0 Hz, 2 H, CH2).
Anal. (C16H14BrN5O2) C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-[[3-(1-im id a zolyl)p r op yl]-
a m in o]p yr id o[3,4-d ]p yr im id in e (5v): from 13 and 3-(imi-
dazol-1-yl)propylamine (64%); mp (MeOH/H2O) 195.5-197 °C;
1
H NMR [(CD3)2SO] δ 9.71 (s, 1 H, NH), 8.77 (s, 1 H, H-8),
8.41 (s, 1 H, H-2), 8.20 (t, J ) 1.9 Hz, 1 H, H-2′), 7.90 (br d, J
) 8.1 Hz, 1 H, H-6′), 7.66 (s, 1 H, H-2′′), 7.37 (t, J ) 8.0 Hz, 1
H, H-5′), 7.32 (br d, J ) 8.2 Hz, 1 H, H-4′), 7.22 (s, 1 H, H-5′′),
7.08 (s, 1 H, H-5), 6.98 (t, J ) 5.4 Hz, 1 H, NH), 6.91 (s, 1 H,
H-4′′), 4.11 (t, J ) 7.0 Hz, 2 H, CH2), 3.25 (q, J ) 6.1 Hz, 2 H,
CH2), 2.09 (pentet, J ) 6.8 Hz, 2 H, CH2). Anal. (C18H16BrN7)
C, H, N.
4-[(3-Br om op h en yl)a m in o]-6-(4-m eth yl-1-p ip er a zin yl)-
p yr id o[3,4-d ]p yr im id in e (5w ): from 13 and 1-methylpip-
erazine (25%); mp (CH2Cl2) 219.5-222 °C; 1H NMR [(CD3)2SO]
δ 9.75 (s, 1 H, NH), 8.86 (s, 1 H, H-8), 8.48 (s, 1 H, H-2), 8.19
(t, J ) 1.7 Hz, 1 H, H-2′), 7.92 (br d, J ) 8.1 Hz, 1 H, H-6′),
7.50 (s, 1 H, H-5), 7.39 (t, J ) 8.0 Hz, 1 H, H-5′), 7.33 (br d, J
) 8.3 Hz, 1 H, H-4′), 3.63 (t, J ) 4.8 Hz, 4 H, CH2), 2.49 (t, J
) 4.8 Hz, 4 H, CH2), 2.26 (s, 3 H, CH3). Anal. (C18H19BrN6)
C, H, N.
4-[(3-Ch lor op h en yl)a m in o]-6-(m et h yla m in o)p yr id o-
[3,4-d ]p yr im id in e (7b): from 14 and MeNH2 (82%); mp
1
(MeOH/H2O) 185.5-187 °C; H NMR [(CD3)2SO] δ 9.72 (s, 1
H, NH), 8.76 (s, 1 H, H-8), 8.42 (s, 1 H, H-2), 8.11 (t, J ) 2.0
Hz, 1 H, H-2′), 7.87 (dd, J ) 7.9, 2.0 Hz, 1 H, H-6′), 7.43 (t, J
) 8.1 Hz, 1 H, H-5′), 7.18 (dd, J ) 7.8, 1.8 Hz, 1 H, H-4′), 7.07
(s, 1 H, H-5), 6.83 (q, J ) 4.9 Hz, 1 H, NH), 2.89 (d, J ) 4.8
Hz, 3 H, CH3). Anal. (C14H12ClN5) C, H, N.
The following compounds were prepared similarly.
4-[(3-Br om op h en yl)a m in o]-6-[(ca r boxym eth yl)a m in o]-
p yr id o[3,4-d ]p yr im id in e (5x): from 13 and the sodium salt
1
of glycine (34%); mp (MeOH) 234-239 °C; H NMR [(CD3)2-
6-(Meth ylam in o)-4-[[3-(tr iflu or om eth yl)ph en yl]am in o]-
p yr id o[3,4-d ]p yr im id in e (8b): from 15 and MeNH2 (72%);
mp (MeOH/H2O) 172-173 °C; 1H NMR [(CD3)2SO] δ 9.85 (s, 1
H, NH), 8.77 (s, 1 H, H-8), 8.42 (s, 1 H, H-2), 8.31 (br s, 1 H,
H-2′), 8.27 (br d, J ) 8.2 Hz, 1 H, H-6′), 7.64 (t, J ) 8.0 Hz, 1
H, H-5′), 7.47 (br d, J ) 7.7 Hz, 1 H, H-4′), 7.08 (s, 1 H, H-5),
6.85 (q, J ) 4.9 Hz, 1 H, NH), 2.90 (d, J ) 5.0 Hz, 3 H, CH3).
Anal. (C15H12F3N5) C, H, N.
6-(Met h yla m in o)-4-[(3-m et h ylp h en yl)a m in o]p yr id o-
[3,4-d ]p yr im id in e (9b): from 16 and MeNH2 (74%); mp
(MeOH/H2O) 189-190 °C; 1H NMR [(CD3)2SO] δ 9.57 (s, 1 H,
NH), 8.72 (s, 1 H, H-8), 8.34 (s, 1 H, H-2), 7.69 (br d, J ) 7.9
Hz, 1 H, H-6′), 7.65 (br s, 1 H, H-2′), 7.29 (t, J ) 7.8 Hz, 1 H,
H-5′), 7.08 (s, 1 H, H-5), 6.96 (br d, J ) 7.6 Hz, 1 H, H-4′),
6.76 (q, J ) 4.9 Hz, 1 H, NH), 2.88 (d, J ) 4.9 Hz, 3 H, CH3),
2.35 (s, 3 H, CH3). Anal. (C15H15N5) C, H, N.
6-(Dim eth yla m in o)-4-[(3-m eth ylp h en yl)a m in o]p yr id o-
[3,4-d ]p yr im id in e (9c): from 16 and dimethylamine (68%);
mp 239-241 °C; 1H NMR [(CD3)2SO] δ 9.61 (s, 1 H, NH), 8.81
(s, 1 H, H-8), 8.36 (s, 1 H, H-2), 7.69 (br d, J ) 7.9 Hz, 1 H,
H-6′), 7.64 (br s, 1 H, H-2′), 7.30 (m, 1 H, H-5′), 7.30 (s, 1 H,
H-5), 6.98 (br d, J ) 7.7 Hz, 1 H, H-4′), 3.17 (s, 6 H, CH3), 2.35
(s, 3 H, CH3). Anal. (C16H17N5) C, H, N.
4-[(3-Meth ylp h en yl)a m in o]-6-[[2-(4-m or p h olin o)eth yl]-
a m in o]p yr id o[3,4-d ]p yr im id in e (9n ): from 16 and 1-(2-
aminoethyl)morpholine (78%); mp 168-170 °C; 1H NMR
[(CD3)2SO] δ 9.56 (s, 1 H, NH), 8.72 (s, 1 H, H-8), 8.34 (s, 1 H,
H-2), 7.67 (br d, J ) 8.0 Hz, 1 H, H-6′), 7.64 (br s, 1 H, H-2′),
7.29 (t, J ) 7.8 Hz, 1 H, H-5′), 7.17 (s, 1 H, H-5), 6.97 (br d, J
) 7.5 Hz, 1 H, H-4′), 6.48 (q, J ) 5.5 Hz, 1 H, NH), 3.60 (t, J
) 4.6 Hz, 4 H, CH2), 3.31 (m, 2 H, CH2), 2.60 (t, J ) 6.7 Hz, 2
SO] δ 12.54 (br, 1 H, CO2H), 9.76 (s, 1 H, NH), 8.77 (s, 1 H,
H-8), 8.42 (s, 1 H, H-2), 8.20 (t, J ) 1.9 Hz, 1 H, H-2′), 7.90 (br
d, J ) 8.0 Hz, 1 H, H-6′), 7.37 (t, J ) 8.0 Hz, 1 H, H-5′), 7.32
(br d, J ) 8.2 Hz, 1 H, H-4′), 7.31 (s, 1 H, H-5), 7.01 (t, J ) 6.0
Hz, 1 H, NH), 4.05 (d, J ) 5.9 Hz, 2 H, CH2). Anal. (C15H12
BrN5O2‚0.5H2O) C, H, N.
-
4-[(3-Br om op h en yl)a m in o]-6-[N-(ca r b oxym et h yl)m e-
th yla m in o]p yr id o[3,4-d ]p yr im id in e (5y): from 13 and the
sodium salt of N-methylglycine (79%); mp (MeOH) 225-227
1
°C; H NMR [(CD3)2SO] δ 12.59 (br, 1 H, CO2H), 9.77 (s, 1 H,
NH), 8.82 (s, 1 H, H-8), 8.45 (s, 1 H, H-2), 8.19 (t, J ) 1.9 Hz,
1 H, H-2′), 7.92 (br d, J ) 8.3 Hz, 1 H, H-6′), 7.39 (t, J ) 8.0
Hz, 1 H, H-5′), 7.35-7.32 (m, 2 H, H-5,4′), 4.43 (s, 2 H, CH2),
3.20 (s, 3 H, CH3). Anal. (C16H14BrN5O2) C, H, N.
6-Am in o-4-[(3-m eth ylp h en yl)a m in o]p yr id o[3,4-d ]p yr i-
m id in e (9a ). Reaction of 16 (0.254 g, 1 mmol) with 4-meth-
oxybenzylamine (4.1 g, 30 mmol) in DMSO at 100 °C for 2 days
gave crude 6-[(4-methoxyphenyl)amino]-4-[(3-methylphenyl)-
amino]pyrido[3,4-d]pyrimidine (0.67 g): 1H NMR [(CD3)2SO]
δ 9.54 (s, 1 H, NH), 8.72 (s, 1 H, H-8), 8.33 (s, 1 H, H-2), 7.66
(br d, J ) 8.1 Hz, 1 H, H-6′), 7.62 (br s, 1 H, H-2′), 7.34 (d, J
) 8.6 Hz, 2 H, H-2′′,6′′), 7.29 (t, J ) 7.7 Hz, 1 H, H-5′), 7.25 (t,
J ) 6.4 Hz, 1 H, NH), 7.21 (s, 1 H, H-5), 6.96 (br d, J ) 7.6
Hz, 1 H, H-4′), 6.88 (br d, J ) 8.6 Hz, 2 H, H-3′′,5′′), 4.49 (d,
J ) 6.2 Hz, 2 H, CH2), 3.71 (s, 3 H, OCH3), 2.34 (s, 3 H, CH3).
Hydrolysis of the crude material with refluxing TFA for 1
h and chromatography on alumina, eluting with CH2Cl2/MeOH
(97:3), gave 9a (0.14 g, 56%): mp 235.5-237 °C; 1H NMR
[(CD3)2SO] δ 9.59 (s, 1 H, NH), 8.68 (s, 1 H, H-8), 8.33 (s, 1 H,
H-2), 7.67 (br s, 1 H, H-2′), 7.23 (m, 1 H, H-6′), 7.26 (t, J ) 8.0

750 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 5
Rewcastle et al.
Hz, 1 H, H-5′), 7.18 (s, 1 H, H-5), 6.94 (br d, J ) 7.5 Hz, 1 H,
H-4′), 6.24 (br s, 1 H, NH), 2.34 (s, 3 H, CH3). Anal. (C14H13N5)
C, H, N.
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)
and blocked overnight in TNA containing 5% bovine serum
albumin and 1% ovalbumin. The membrane was blotted for
2 h with anti-phosphotyrosine 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 Ci/mL [125]Iprotein A and then
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.
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, NIH 3T3 fibroblast transfected with
the human EGF receptor (EGFR), estrogen-dependent MCF-7
breast, and SK-OV-3 ovarian 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.
4-[(3-Br om op h en yl)a m in o]-2-m eth yl-6-[[2-(4-m or p h oli-
n o)eth yl]a m in o]p yr id o[3,4-d ]p yr im id in e (10n ), Sch em e
3). A solution of 13 (320 mg, 1 mmol) and nitromethane (0.28
mL, 5 mmol) in DMSO (2 mL) was treated at 25 °C with DBU
(0.45 mL, 3 mmol). The mixture was stirred at 25 °C under
nitrogen for 24 h and then diluted with 15 mL of 5% aqueous
HCl. The resulting suspension was stirred for 20 min,
sonicated briefly to break up solids, filtered, and washed well
with water and then a small amount of 2-propanol to give 4-[(3-
bromophenyl)amino]-6-fluoro-2-methylpyrido[3,4-d]pyrimi-
dine hydrochloride (17) (307 mg, 84%): mp 230-240 °C dec;
1H NMR [CD3)2SO] δ 10.30 (br s, exchanges with D2O, 1 H,
NH), 8.78 (s, 1 H, H-8), 8.26 (d, J ) 1.7 Hz, 1 H, H-2′), 8.20 (s,
1 H, H-5), 7.91 (dt, J ) 2.1, 1.9 Hz, 1 H, H-6′), 7.43-7.36 (m,
2 H, H-4′,5′), 5.14 (br s, exchanges with D2O, 1 H), 2.85 (s, 3
H, CH3); 19F NMR δ -74.0; CIMS m/z (relative intensity) 333
(MH+, 100), 335 (78). Anal. (C14H10N4BrF‚HCl) C, H, N.
A solution of 17 (207 mg, 0.56 mmol) and 4-(2-aminoethyl)-
morpholine (1.1 mL, 8.4 mmol) in DMSO (1.2 mL) was heated
at 80 °C for 22 h. The DMSO was removed under reduced
pressure, and the residue was dissolved in EtOAc and purified
by flash chromatography on silica gel, eluting with EtOAc
followed by a gradient (5%, 7.5%, and 10%) of MeOH in EtOAc.
The residue from appropriate pooled fractions was dissolved
in a minimal volume of hot EtOAc and treated with excess
dry HCl in 2-propanol to give 10n as the dihydrochloride salt
(177 mg, 60%): mp >260 °C dec; 1H NMR [(CD3)2SO] δ 11.08
(br s, exchanges D2O, 1 H), 10.98 (br s, exchanges D2O, 1 H),
8.61 (s, 1 H, H-8), 8.25 (s, 1 H, H-2′), 7.96 (d, J ) 7.7 Hz, 1 H,
H-6′), 7.71 (s, 1 H, shifts to 7.49 with D2O wash, H-5), 7.46-
7.22 (m, overlapping with br exchangeable s, 3 H), 4.5 (br s,
exchanges D2O, ca. 1.5 H), 3.99 (d, J ) 11 Hz, 2 H), 3.85-3.78
(m, 4 H), 3.55 (d, J ) 11.8 Hz, 2 H), 3.36 (d, J ) 4.8 Hz, 2 H),
3.18 (q, J ) 11 Hz, 2 H), 2.76 (s, 3 H, CH3); CIMS m/z (relative
intensity) 443 (MH+, 58), 445 (53). Anal. (C20H23N6OBr‚2HCl)
N, H, C: found, 45.9; calcd 46.5.
Mea su r em en t 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 approximately
a 50 mM solution) were then sonicated for 30 min in 0.05 M
sodium lactate buffer (neutral compounds, hydrochloride 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.
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,31 and the assays were carried out as reported
previously.8 The substrate used was based on a portion of
phospholipase C1, having the sequence Lys-His-Lys-Lys-Leu-
Ala-Glu-Gly-Ser-Ala-Tyr472-Glu-Glu-Val. The reaction was
allowed to proceed for 10 min at room temperature and then
was stopped by the addition of 2 mL of 75 mM phosphoric acid.
The solution was then passed through a 2.5-cm phosphocel-
lulose disk which bound the peptide. This filter was washed
with 75 mM phosphoric acid (5×), and incorporated label was
assessed by scintillation counting in an aqueous fluor. 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%.
In each experiment for anticancer activity evaluation, test
mice weighing 18-22 g were randomized and implanted with
tumor fragments in the region of the right axilla on day 0.
Animals were treated with test compounds on the basis of
average cage weight (6 mice/dose group) on the days indicated
in the tables. In every experiment, each test compound was
evaluated over a range of dose levels ranging from toxic to
ineffective. The doses reported in Table 4 are the maximum
doses that could be administered without exceeding 10%
compound-induced mortality. This dose level, the maximum
tolerated dose, allows comparisons to be made among the
tested compounds at an equitoxic dose level. The vehicle for
5b was 6% dimethylacetamide and 94% 50 mM sodium lactate
buffer, pH 4.0 (suspension). Compounds 5h ,k ,n and 9b were
administered as isethionate salts in water. Compound dosing
solutions 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.32-35
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.
Ack n ow led gm en t. This work was partially sup-
ported by the Auckland Division of the Cancer Society
of New Zealand.
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