Synthesis and tyrosine kinase inhibitory activity of a series of 2- amino-8H-pyrido[2,3-d]pyrimidines: Identification of potent, selective platelet-derived growth factor receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1021/jm980398y

Screening of a compound library led to the identification of 2-amino-6- (2,6-dichlorophenyl)-8-methylpyrido[2,3-d]pyrimidine (1) as a inhibitor of the platelet-derived growth factor receptor (PDGFr), fibroblast growth factor receptor (FGFr), and c-src tyrosine kinases (TKs). Replacement of the primary amino group at C-2 of 1 with a 4-(N,N-diethylaminoethoxy)phenylamino group yielded 2a, which had greatly increased activity against all three TKs. In the present work, variation of the aromatic group at C-6 and of the alkyl group at N-8 of the pyrido[2,3d]pyrimidine core provided several analogues that retained potency, including derivatives that were biased toward inhibition of the TK activity of PDGFr. Analogues of 2a with a 3-thiophene or an unsubstituted phenyl group at C-6 were the most potent inhibitors. Compound 54, which had IC₅₀ values of 31, 88, and 31 nM against PDGFr, FGFr, and c-src TK activity, respectively, was active in a variety of PDGF- dependent cellular assays and blocked the in vivo growth of three PDGF- dependent tumor lines.

Full text of DOI:10.1021/jm980398y

J . Med. Chem. 1998, 41, 4365-4377
4365
Syn th esis a n d Tyr osin e Kin a se In h ibitor y Activity of a Ser ies of
2-Am in o-8H-p yr id o[2,3-d ]p yr im id in es: Id en tifica tion of P oten t, Selective
P la telet-Der ived Gr ow th F a ctor Recep tor Tyr osin e Kin a se In h ibitor s
Diane H. Boschelli,*,† Zhipei Wu,† Sylvester R. Klutchko,† H. D. Hollis Showalter,† J ames M. Hamby,†
Gina H. Lu,‡ Terry C. Major,‡ Tawny K. Dahring,‡ Brian Batley,‡ Robert L. Panek,‡ J oan Keiser,‡
Brian G. Hartl,# Alan J . Kraker,# Wayne D. Klohs,# Bill J . Roberts,# Sandra Patmore,# William L. Elliott,#
Randy Steinkampf,# Laura A. Bradford,§ Hussein Hallak,§ and Annette M. Doherty†
Departments of Medicinal Chemistry, Cancer Research, Vascular and Cardiac Diseases, and Pharmacokinetics and Drug
Metabolism, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road,
Ann Arbor, Michigan 48105
Received J uly 6, 1998
Screening of a compound library led to the identification of 2-amino-6-(2,6-dichlorophenyl)-8-
methylpyrido[2,3-d]pyrimidine (1) as a inhibitor of the platelet-derived growth factor receptor
(PDGFr), fibroblast growth factor receptor (FGFr), and c-src tyrosine kinases (TKs). Replace-
ment of the primary amino group at C-2 of 1 with a 4-(N,N-diethylaminoethoxy)phenylamino
group yielded 2a , which had greatly increased activity against all three TKs. In the present
work, variation of the aromatic group at C-6 and of the alkyl group at N-8 of the pyrido[2,3-
d]pyrimidine core provided several analogues that retained potency, including derivatives that
were biased toward inhibition of the TK activity of PDGFr. Analogues of 2a with a 3-thiophene
or an unsubstituted phenyl group at C-6 were the most potent inhibitors. Compound 54, which
had IC50 values of 31, 88, and 31 nM against PDGFr, FGFr, and c-src TK activity, respectively,
was active in a variety of PDGF-dependent cellular assays and blocked the in vivo growth of
three PDGF-dependent tumor lines.
In tr od u ction
importance of PDGF and its receptors in cancer.10-17
Several years ago it was found that PDGF was the
human homologue of the simian oncoprotein v-sis.39,40
Later studies showed that both PDGF and its receptor
are expressed in several types of human gliomas.41-44
Furthermore, a shortened form of the PDGF receptor
inhibited the growth of C6 glioma cells.45 Therefore a
small molecule capable of suppressing PDGFr TK
activity has potential utility as an anticancer therapeu-
tic agent.
The tyrosine kinases (TKs) are a key group of en-
zymes that catalyze the specific phosphorylation of
tyrosine residues on proteins. The TKs can be divided
into two classes, the cytoplasmic TKs such as c-src and
the transmembrane growth factor receptor TKs (RTKs).
When growth factors such as platelet-derived growth
factor (PDGF), fibroblast growth factor (FGF), epidermal
growth factor (EGF), or vascular endothelial growth
factor (VEGF) bind their receptors, the receptors dimer-
ize and become active TKs. The subsequent autophos-
phorylation of the receptors leads to a signal transduc-
tion cascade which triggers a wide variety of events
including proliferation and migration.1,2
Since distinct TKs are implicated in such diverse
conditions as angiogenesis,3,4 restenosis,5,6 atheroscle-
rosis,7 and tumor growth,8 selective TK inhibitors are
highly desirable. The obstacles to identifying selective
inhibitors are the large number of TKs that have been
described and the structural homology between the TKs,
especially in their ATP-binding regions.9 Despite these
difficulties several selective TK inhibitors have been
identified10-38 including those of EGFr27-35 and FGFr
TK36-38 activity reported by Parke-Davis. We were also
interested in identifying compounds that could selec-
tively modulate the activity of PDGFr TK due to the
We previously reported that screening of a compound
library led to the identification of 2-amino-6-(2,6-dichlo-
rophenyl)-8-methyl-8H-pyrido[2,3-d]pyrimidin-7-one (1)
as an inhibitor of PDGFr, FGFr, and c-src TK activity.46
Optimization of the various groups at C-2 of 1 deter-
mined that aromatic amino substituents were greatly
preferred over primary or alkyl amino groups. This
finding was exemplified by the broad-spectrum TK
inhibitor 2a which has a 4-(N,N-diethylaminoethoxy)-
phenylamino group at C-2 and had IC50 values of 79,
43, and 9 nM against PDGFr, FGFr, and c-src TK
activity, respectively.47 While the initial focus was on
variation of the C-2 amino group, it was also determined
that replacement of the 8-methyl group of 2a with an
ethyl group provided 2b, which was roughly equipotent
to 2a but more PDGFr-selective. It was considered that
further modification at the N-8 position of the pyrido-
[2,3-d]pyrimidin-7-one could possibly lead to more active
and/or more selective congeners.
* Address for correspondence: Wyeth-Ayerst Research, 401 N.
Middletown Rd., Pearl River, NY 10965. Phone: 914-732-3567. Fax:
914-732-5561. E-mail: bosched@war.wyeth.com.
† Department of Medicinal Chemistry.
In the initial screen that identified 1 as a TK inhibi-
tor, similar activity was found for the related pyrido-
[2,3-d]pyrimidine 3a .48 This nonspecific kinase inhibi-
‡ Department of Vascular and Cardiac Diseases.
#
Department of Cancer Research.
§ Department of Pharmacokinetics and Drug Metabolism.
10.1021/jm980398y CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/17/1998

4366 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Boschelli et al.
Sch em e 1
tor was converted into the selective FGFr TK inhibitor
3b by replacement of the 2,6-dichlorophenyl group at
C-6 of 3a with a 3,5-dimethoxyphenyl group.36,37 In
addition, a model of the binding of these two classes of
pyrido[2,3-d]pyrimidines to the kinase domains of TKs
proposed that modification of the group at C-6 could
confer selectivity.49 All of these findings encouraged us
to utilize 2a as a template for the preparation of
potential selective inhibitors, through variation of the
C-6 and N-8 moieties.50
ammonia or methylamine in a sealed tube provided 30
and 31, respectively (listed in Table 2).
A more direct route to the preparation of analogues
retaining the methyl group at N-8 and a phenylamino
group at C-2 while varying the aromatic group at C-6
is shown in Scheme 3. Treatment of 4-chloro-5-cyano-
2-methylsulfanyl-pyrimidine52 with methylamine pro-
vided 32. Displacement of the 2-methyl sulfide group
of 32 with aniline in the presence of a catalytic amount
of concentrated hydrochloric acid gave 33. Reduction
of the nitrile group of 33 with diisobutylaluminum
hydride followed by hydrolysis gave the corresponding
aldehyde 34. It should be noted that while the desired
product was obtained by this protocol, it was necessary
to stop the reaction while some 33 remained since
extended reaction time led to the formation of additional
products. Condensation of 34 with 2-thiopheneaceto-
nitrile or 4-biphenylacetonitrile in 2-ethoxyethanol and
sodium hydride gave good yields of the pyridopyrimidin-
7-imines 35 and 36, respectively. Acetylation of the
imines followed by acid hydrolysis provided the corre-
sponding 7-ones 37 and 38.
2-Phenylamino analogues with an ethyl group at N-8
group were prepared from 44, the 4-ethylamino ana-
logue of 34. While a similar route to that used to
prepare 34 could be adapted, a more reproducible route
to 44 starting with commercially available 39 was
employed (Scheme 4). Thus, the chloro group of 39 was
displaced with aqueous ethylamine to give 40. Oxida-
tion of the 2-methyl sulfide group of 40 with m-
chloroperbenzoic acid gave mixtures of products thought
to arise due to the instability of the corresponding
sulfone. It was found that use of (()-trans-2-phenyl-
sulfonyl-3-phenyloxaziridine53 as the oxidant provided
Ch em istr y
Additional N-8 alkyl derivatives of 1 were obtained
by alkylation of 446 with various halides employing
sodium hydride as the base. The minor O-alkylation
product was readily separated from the major product
by chromatography. Compounds 5-10 and 12-15 were
prepared by this route as shown in Scheme 1. Cleavage
of the tert-butyl ester group of 10 with trifluoroacetic
acid provided the acid derivative 11. Catalytic hydro-
genation of 15 removed the benzyl group to give 16. We
were not able to find conditions to convert 15 to 16
without some concomitant dechlorination.
Analogues of 2a with various aromatic groups at C-6
of the pyrido[2,3-d]pyrimidin-7-one could be prepared
as shown in Scheme 2. To this end, condensation of the
2-methyl sulfide derivative 1746 with phenylacetonitrile
or 3-thiopheneacetonitrile in DMF in the presence of
potassium carbonate gave the pyridopyrimidin-7-imines
18 and 19 that were then hydrolyzed to the 7-ones 20
and 21. Oxidation of the methyl sulfide groups of 20
and 21 with m-chloroperbenzoic acid provided the
sulfones 22 and 23. The sulfone group was readily
displaced by arylamines such as aniline, converting 22
and 23 to the 2-phenylamino derivatives 24 and 25. In
a similar fashion treatment of 22 and 23 with 4-ami-
nopyridine provided 26 and 27, while 4-(N,N-diethyl-
aminoethoxy)aniline51 provided 28 and 29, respectively.
While for arylamines it was necessary to employ the
more labile sulfone group, in the case of the more
nucleophilic alkylamines the sulfide group was suf-
ficiently reactive. Therefore, treatment of 26 with

2-Aminopyridopyrimidines as PDGFr TK Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4367
Sch em e 2a
a
(a) RCH2CN, K2CO3, DMF; (b) (1) Ac2O, reflux, (2) concd HCl; (c) m-CPBA, CHCl3; (d) aniline, 150 °C; (e) 4-aminopyridine, 175 °C;
(f) 4-(N,N′-diethylaminoethoxy)aniline, 175 °C; (g) RNH2, DMF, high pressure.
Sch em e 3a
a
(a) Methylamine; (b) aniline, concd HCl, 170 °C; (c) DiBAL, THF, CH2Cl2; (d) RCH2CN, NaH, EtO(CH2)2OH; (e) (1) Ac2O, reflux; (2)
concd HCl, reflux.
Sch em e 4a
a
(a) Aqueous ethylamine, triethylamine, THF; (b) trans-2-phenylsulfonyl-3-phenyloxaziridine, CHCl3; (c) aniline, 130 °C; (d) LiAlH4,
THF; (e) MnO2, CHCl3; (f) RCH2CN, NaH, EtO(CH2)2OH; (g) (1) Ac2O, (2) concd HCl.
the corresponding sulfoxide 41 in good yield. Displace-
ment of the sulfoxide group of 41 with aniline at
elevated temperature gave the desired 2-phenylamino
derivative 42. The ester group at C-5 was sequentially
reduced to the alcohol 43 with lithium aluminum
hydride and then oxidized to the aldehyde 44 with
manganese oxide. Condensation of 44 with phenylac-
etonitrile in the presence of sodium hydride in ethoxy-
ethanol gave the pyrido[2,3-d]pyrimidin-7-imine 45
which was hydrolyzed to the 7-one derivative 47 as
described previously. The 7-imine (46) and 7-one (48)
derivatives containing a 3-thiophene group at C-6 were
prepared in an analogous fashion from 44.
The more flexible route employing the 2-methyl
sulfide group was then used to allow for variation of the
amino group at C-2 as shown in Scheme 5. Aldehyde
4946 was condensed with phenylacetonitrile, and the
resultant imine 50 was hydrolyzed to the 7-one 51.
Oxidation of the 2-methyl sulfide group of 51 with
m-chloroperbenzoic acid gave sulfone 52 that was
treated with aniline to provide 47 by an alternate route
to that used initially (Scheme 5). The additional 2-ary-

4368 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Boschelli et al.
Sch em e 5a
a
(a) PhCH2CN, K2CO3, DMF; (b) (1) Ac2O, reflux, (2) concd HCl; (c) m-CPBA, CHCl3; (d) ArNH2, 175 °C.
Ta ble 1. Inhibition of Tyrosine Kinase Activity by
against all three TKs. Although none of the additional
N-8-substituted analogues (12-16) showed improved
PDGFr TK inhibition, 14, which contains a N,N-dim-
ethylaminopropyl group, was the most potent inhibitor
of c-src TK activity observed in this series (IC50 ) 0.05
µM). In summary, for PDGFr TK inhibition the best
group at N-8 was ethyl.
8-Alkyl-2-amino-6-(2,6-dichlorophenyl)pyridopyrimidin-7-onesa
Certain analogues of 1 with a methyl group at N-8
and a phenylamino group at C-2, namely, 24, 25, and
37 where the group at C-6 was phenyl, 3-thiophene, and
2-thiophene, respectively, showed selectivity for PDGFr
(Table 2). In contrast 38, which contains a biphenyl
group at C-6, was inactive against all three TKs,
suggesting that size is critical at the C-6 position also.
The 4-pyridinylamino analogues of 24 and 25, 26 and
27, respectively, had basically unchanged activity against
PDGFr but were relatively more potent inhibitors of
c-src. The direct analogues of 2a with a phenyl or a
3-thiophene group at C-6, namely, 28 and 29, were still
very potent inhibitors of PDGFr with IC50 values of 42
and 54 nM, respectively. However these compounds
were weaker inhibitors of both FGFr and c-src than was
2a . The most dramatic difference was seen in the c-src
activity which decreased from an IC50 of 9 nM for 2a to
IC50 values of 140 and 210 nM for 28 and 29, respec-
tively. As was seen in the 6-(2,6-dichlorophenyl) series,
for compounds with a 6-(3-thiophene) substituent the
corresponding 2-amino and 2-methylamino derivatives
30 and 31 were less active than the 2-phenylamino
derivative 29. The decrease in activity seen with the
primary amino group at C-2, however, was much greater
when the substituent at C-6 was 3-thiophene. Com-
pared to 1 which had IC50 values of 3.8, 1.3, and 0.26
µM against PDGFr, FGFr, and c-src, the IC50 values for
30 were 24, >50, and >50 µM, respectively.
IC50 (µM)
no.
R8
PDGFr
FGFr
c-src
0.26
0.16
0.63
0.33
0.89
0.34
0.56
1
5
6
7
8
Me
Et
n-Pr
n-Bu
i-Bu
3.8
0.98
1.2
1.2
1.6
1.3
0.54
0.51
0.58
0.50
1.7
1.7
7.9
1.4
9
CH2COOMe
CH2COO-t-Bu
CH2COOH
5.0
7.2
10
11
12
13
14
15
16
>50
>50
CH2Ph
4.2
1.2
1.8
6.8
0.16
0.63
0.05
1.4
CH2-4-pyridinyl
(CH2)3NMe2
(CH2)3OCH2Ph
(CH2)3OH
0.72
0.88
3.2
1.1
0.37
0.18
a
IC50 values reported for TK inhibition represent the means of
at least two separate determinations done in triplicate with typical
variations of less than 30% between replicate values.
lamines 53 and 54 were obtained from sulfone 52 by
the standard conditions.
Resu lts a n d Discu ssion
SAR a ga in st Tyr osin e Kin a ses. Beginning with an
examination of the effect of varying the N-8 alkyl group,
it was determined that replacement of the methyl group
at N-8 of 1 with ethyl, 5, provided a 4-fold increase in
potency against PDGFr and 2.5- and 1.5-fold increased
inhibition of FGFr and c-src TK activity, respectively
(Table 1). However, no substantive advantage was seen
with the additional N-8 alkyl derivatives 6-8 when
compared to 5. The ester analogues 9 and 10 retained
the c-src TK inhibitory activity of 1 but were much less
potent inhibitors of PDGFr and FGFr TK activity. The
acetic acid analogue 11 had greatly decreased activity
In the C-6 2,6-dichlorophenyl series, for compounds
with a C-2 primary amino group, replacing the methyl
group at N-8 with an ethyl resulted in increased
inhibition (1 vs 5), while this same change at N-8 for
compounds with a C-2 aromatic amino group did not
alter the biological activity to such a large extent (2a

2-Aminopyridopyrimidines as PDGFr TK Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4369
Ta ble 2. Inhibition of Tyrosine Kinase Activity by 8-Alkylpyrido[2,3-d]pyrimidinesa
IC50 (µM)
no.
R6
R2
R8
PDGFr
FGFr
c-src
2a
24
25
26
27
28
29
30
31
37
38
2b
47
48
53
54
2,6-di-ClPh
phenyl
3-thiophene
phenyl
3-thiophene
phenyl
3-thiophene
3-thiophene
3-thiophene
2-thiophene
biphenyl
2,6-di-ClPh
phenyl
3-thiophene
phenyl
Ph-4-O(CH2)2NEt2
phenyl
phenyl
4-pyridine
4-pyridine
Ph-4-O(CH2)2NEt2
Ph-4-O(CH2)2NEt2
H
Me
phenyl
phenyl
Ph-4-O(CH2)2NEt2
phenyl
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
0.079
0.29
0.80
0.32
0.48
0.042
0.054
24.4
0.043
5.0
0.009
2.4
8.1
0.47
1.7
0.14
0.21
>50
4.7
22.6
0.35
2.9
>50
>50
>50
>50
0.030
1.5
>50
>50
>50
>50
>50
0.95
>50
0.15
0.18
0.41
0.074
0.031
0.017
1.8
41
0.41
0.031
Et
Et
Et
Et
phenyl
4-pyridine
Ph-4-O(CH2)2NEt2
>50
1.1
0.088
phenyl
a
IC50 values reported for TK inhibition represent the means of at least two separate determinations done in triplicate with typical
variations of less than 30% between replicate values.
Ta ble 3. Effect of Tyrosine Kinase Inhibitors on PDGFr
Autophosphorylation and C6 Proliferation
vs 2b). In comparing those compounds with a phenyl-
amino group at C-2 and a 3-thiophene group at C-6, the
N-8 ethyl analogue 48 was 2-fold more potent in
inhibiting PDGFr than the N-8 methyl analogue 25.
While 48 was a 5-fold less potent c-src inhibitor than
25, neither compound inhibited FGFr activity. In
examining the effect of varying the N-8 group from
methyl to ethyl for analogues with an unsubstituted
phenyl group at C-6, the general trend was that the N-8
ethyl group resulted in increased activity against all
three TKs. When compared to 2b, the corresponding
C-6 2,6-dichlorophenyl analogue 54 was 3 times more
potent against PDGFr and 2-3-fold weaker against both
FGFr and c-src TK activity. With an IC50 of 31 nM
against PDGFr TK activity, 54 is the most potent
inhibitor of this TK identified in this work. When tested
against a panel of other kinases, 54 showed moderate
activity against EGFr TK (IC50 ) 0.34 µM) and much
weaker activity against cdk4, MAPK, and the insulin
receptor (IC50 values of 2.4, 10, and 50 µM).54
inhibition of PDGFr
autophos/IC50 (µM)
soft agar clonogenic
IC50 (µM)
C6
C6 proliferationd contin
drug
no. RAVSMCSa
cellsc
IC50 (µM)
expe
removedf
2a
29
47
48
54
53
0.0065
0.054
0.0046
NTb
0.005
NTb
>0.025
>0.025
0.058
0.003
0.34
NTb
NTb
NTb
7.2
0.12
NTb
NTb
NTb
4.9
1.80
NTb
NTb
NTb
13.2
0.15
NTb
0.0015
1.2
0.12
a
See ref 47 for assay conditions. b NT, not tested. c C6 cells were
serum-starved for 24 h, treated for 2 h with various concentrations
of the indicated compound, and stimulated with 25 ng/mL PDGF
for 5 min. Anti-phosphotyrosine Western blots were performed as
d
described in the Experimental Section. C6 cells were treated for
4 days with various concentrations of compound in 96-well plates,
and the effect of each compound was determined using sulfor-
hodamine B staining. e C6 cells were treated for 3 days with
various concentrations of compound in 6-well tissue culture plates
and cloned in soft agar in the continued presence of drug. f C6 cells
were treated as in footnote e but were cloned in soft agar in the
absence of drug.
Cellu la r Stu d ies. The best inhibitors of PDGFr TK
activity in the in vitro assay were tested for their ability
to inhibit PDGF-stimulated autophosphorylation of the
PDGF receptor in cells. Rat aortic vascular smooth
muscle cells (RAVSMCs) that express the receptor for
PDGF were pretreated for 2 h with compound and then
exposed to PDGF. The extent of autophosphorylation
was measured by anti-phosphotyrosine immunoblotting
as reported previously.47 Compounds 29, 47, and 54,
which had in vitro IC50 values of 54, 180, and 31 nM,
had IC50 values of 54, 4.6, and 1.5 nM, respectively, in
this autophosphorylation assay (Table 3). In this assay
the C-6 phenyl derivatives 47 and 54 were not substan-
tially different from 2a , which had an IC50 of 6.5 nM,47
while 29, the C-6 3-thiophene analogue of 54, was much
less active.
analogue of 54, was 1 log order less active (Table 3).
Further studies showed that 2a , 53, and 54 strongly
inhibited the proliferation of C6 cells in culture while
47 and 48 were much less active (IC50 > 25 nM). The
three most active compounds were next examined in soft
agar clonogenic assays. While 2a and 53 had a de-
creased effect on cell growth when the compound was
removed from the growth media as opposed to continu-
ous exposure, no decrease in activity was seen with 54.
In Vivo Stu d ies. The TK inhibitor 54 was next
evaluated for efficacy in various tumor model systems.
The C6 glioma was chosen based on the importance of
PDGF for the in vivo growth of this tumor.45 The NIH
3T3 fibroblast line transfected with PDGF was included
due to its high tumorigenic potential as compared to the
parent fibroblast line implicating the potential for an
autocrine loop via the ligand PDGF. In these studies
the C6 glioma and PDGF-transfected NIH 3T3 cell lines
In addition to RAVSMCs, C6 rat glioma cells also
express the PDGF receptor. In a similar PDGF auto-
phosphorylation assay in these cells, 54 was again
equipotent to 2a , while 53, the 2-(4-pyridinyl)amino

4370 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Boschelli et al.
Ta ble 4. In Vivo Activity of 54 against Tumor Xenografts in Nude Mice
dose
(mg/kg)
weight
change (g)
T/C (%) on last
therapy dayc
T - C
net cell
kill (log10)
e
tumora
scheduleb
(days)d
C6 glioma
PDGF transfect
DU-145
40
20
48
days 1-15
-0.7
+
+
0
1
40
9.6
6.9
11.4
-0.3
-1.2
-0.4
days 1-15
days 16-20, 23-27, 30-34
a
b
The indicated tumor fragments were implanted sc into the right axilla of mice on day 0. Compounds were administered orally on
the indicated schedules. The maximum tolerated dose (LD10) from a complete dose-response is shown. c Ratio of median treated tumor
mass/median control tumor mass × 100%. The difference, in days, for the treated (T) and the control (C) tumors to reach 750 mg. e The
d
net reduction in tumor burden, in logs, between the first and last treatments.
Ta ble 5. Mean Oral Pharmacokinetic Parameter Values in
Rat following a Single Oral Gavage Dose of 53 or 54a
parameter
54
53
no. of animals
dose (mg/kg)
Cmax
tmax
t1/2
3
3
5
5
78.3
2.67
6.65
525
730
1.50
3.72
4764
5240
AUC(0-tldc
AUC(0-∞)
)
828
a
Cmax, maximum observed concentration (ng/mL); tmax, time to
reach Cmax (h); t1/2, apparent terminal phase elimination half-life
(h); AUC(0-tldc), area under concentration-time curve from time
0 to time of last detectable concentration (ng‚h/mL); AUC(0-∞),
area under concentration-time curve from time 0 to time infinity
(ng‚h/mL).
F igu r e 1. In vivo effectiveness of 54 against the C6 rat
glioma. Tumor fragments were implanted sc on day 0, and po
therapy with 54 was initiated on day 1 and continued through
day 15.
F igu r e 3. Individual animal plasma concentration-time
profiles of 54 (closed symbols) and 53 (open symbols) following
a single 5 mg/kg oral gavage dose.
F igu r e 2. In vivo effectiveness of 54 against an NIH 3T3
fibroblast transfected with PDGF. Tumor fragments were
implanted sc on day 0, and po therapy with 54 was initiated
on day 1 and continued through day 15.
other human tumor xenografts (Table 4). As in the C6
and PDGF-transfected NIH 3T3 studies, there was no
significant weight loss in the DU-145 tumor-bearing
animals nor was any other gross toxicity noted at the
maximum tolerated dosage level.
had tumor volume doubling times of 4.4 and 1.8 days,
respectively. At tolerated doses 54 completely inhibited
the progression of both tumor models as shown in the
growth curves (Figures 1 and 2) and the very low zero
or near zero T/C values on the last day of therapy (Table
4). Weight loss in these trials as a measure of drug
toxicity was minimal, and no other gross toxicity was
noted at the maximum tolerated dose. Tumor growth
delays in the range of 7-9 days were measurable in both
models but were more significant in the PDGF-trans-
fected NIH 3T3 tumor model due to its short tumor
volume doubling time and hence rapid growth.
P h ar m acokin etics, Or al Bioavailability, an d Dose
P r op or tion a lity Stu d ies in Ra ts. In an oral pharm-
acokinetic study, a single 5 mg/kg oral gavage dose of
53 or 54 was administered to three male Wistar rats.
Both compounds were rapidly absorbed with a tmax value
of less than 3 h (Table 5, Figure 3). Compound 53
achieved higher systemic exposure than 54 as expressed
by Cmax and AUC values. The differences are likely
attributed to metabolism on the side chain of 54.
Previous studies with compound 2a , which has the
identical side chain as 54, showed that the terminal
dialkylamine is metabolized to the N-oxide; then further
metabolism results in the formation of the terminal
primary amine. The pyridine functionality of 53 is not
susceptible to this metabolism.
Compound 54 was also effective against the DU-145
prostate epithelial cell line xenograft. The 11.4-day
tumor growth delay observed in this study suggests that
54 may have the potential to produce activity against

2-Aminopyridopyrimidines as PDGFr TK Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4371
2H), 7.87 (s, 1H), 8.69 (s, 1H); MS (CI) m/z 381 (M + 1). Anal.
(C16H12Cl2N4O3) C, H, N.
Con clu sion
We have demonstrated that a screening lead, 1, could
be converted into a potent inhibitor of the activity of
PDGFr TK. By replacement of the 2-amino group with
4-(N,N-diethylaminoethoxy)phenylamino, the C-6 2,6-
dichlorophenyl group with phenyl, and the N-8 methyl
group with ethyl, 1 was converted into 54. The IC50
values for the inhibition of the TK activity of PDGFr,
FGFr, and c-src improved from 3.8, 1.3, and 0.26 µM
for 1 to 0.031, 0.088, and 0.031 µM for 54. This agent
was active in several PDGF-dependent cellular assays
including PDGFr autophosphorylation and inhibition of
tumor cell growth. In addition, 54 exhibited meaningful
in vivo activity against three tumor model systems.
Studies to evaluate the potential use of 54 as an
anticancer therapeutic agent are continuing.
[2-Am in o-6-(2,6-d ich lor op h en yl)-7-oxo-7H-p yr id o[2,3-
d ]p yr im id in -8-yl]a cetic a cid ter t-bu tyl ester (10): yield
30%; mp 171-173 °C; 1H NMR (DMSO-d6) δ 1.38 (s, 9H), 4.88
(s, 2H), 7.47 (br s, 2H), 7.45 (t, J ) 8 Hz, 1H), 7.57 (d, J ) 8
Hz, 2H), 7.85 (s, 1H), 8.68 (s, 1H); MS (CI) m/z 423 (M + 1).
Anal. (C19H18Cl2N4O3) C, H, N.
[2-Am in o-6-(2,6-d ich lor op h en yl)-7-oxo-7H-p yr id o[2,3-
d ]p yr im id in -8-yl]a cetic Acid (11). To a solution of 10 (157
mg, 0.37 mmol) in 4 mL of CH2Cl2 was added 2 mL of
trifluoroacetic acid. The solution was stirred at room temper-
ature for 5 h and then concentrated. The resultant oil was
partitioned between CH2Cl2 and brine. The aqueous layer was
washed with EtOAc, and the organic layers were combined,
dried over MgSO4, filtered, and concentrated to give a gummy
solid. Diethyl ether was added, and the resultant precipitate
was collected. Hexane was added to the filtrate, and the
resultant precipitate was collected. The solids were combined
to give 71 mg (52%) of 11 that was >97% pure by HPLC: mp
297-300 °C dec; 1H NMR (DMSO-d6) δ 4.93 (s, 2H), 7.45 (t, J
) 8 Hz, 1H), 7.48 (br s, 2H), 7.57 (d, J ) 8 Hz, 2H), 7.85 (s,
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were determined with
a Thomas-Hoover capillary melting point apparatus or a MEL-
TEMP melting point apparatus and are uncorrected. 1H NMR
spectra were recorded using a Varian Unity 400-MHz spec-
trometer. Chemical shifts are in parts per million (δ) refer-
enced to Me4Si. Chemical ionization mass spectra (CI) were
recorded on a VG Trio 2 mass spectrometer instrument using
a reagent gas of 1% NH3 in CH4. Atmospheric presssure
chemical ionization (APCI) and electrospray mass (ES) spectra
were recorded using a VG Trio 2000 mass spectrometer. Flash
chromatography was performed with silica gel 60 (230-400
mesh; E. Merck Darmstad). Combustion analyses (CHN) were
performed by the Parke-Davis Pharmaceutical Research Ana-
lytical Department or Robertson Microlit (Madison, NJ ).
Gen er a l P r oced u r e for th e P r ep a r a tion of 8-N-Su b-
st it u t ed -2-a m in o-6-(2,6-d ich lor op h en yl)-8H -p yr id o[2,3-
d ]p yr im id in -7-on es fr om 4, Used To P r ep a r e 5-10, 12,
a n d 15. 2-Am in o-6-(2,6-d ich lor op h en yl)-8-eth yl-8H-p y-
r id o[2,3-d ]p yr im id in -7-on e (5). To a suspension of NaH
(60% in mineral oil, 27 mg) in 5 mL of DMF was added 4 (172
mg, 0.56 mmol). The mixture was heated at 50 °C for 1 h
resulting in a clear solution. Ethyl iodine (60 µL, 0.75 mmol)
was added, and the solution was stirred at 50 °C for 3.5 h,
then cooled to room temperature, and poured onto 30 mL of
ice water. The resulting precipitate was collected by filtration,
washed with water, and purified by flash chromatography,
eluting with 1:1 hexane/EtOAc to provide 104 mg (55%) of 6:
mp 207-209 °C; 1H NMR (DMSO-d6) δ 1.20 (t, J ) 7 Hz, 3H),
4.30 (q, J ) 7 Hz, 2H), 7.41-7.46 (m, 3H), 7.57 (d, J ) 8 Hz,
2H), 7.70 (s, 1H), 8.65 (s, 1H); MS (CI) m/z 335 (M + 1). Anal.
(C15H12Cl2N4O) C, H, N.
1H), 8.68 (s, 1H); MS (CI) m/z 367 (M + 1). Anal. (C15H10
-
C12N4O3) Calcd: C, 49.34; H, 2.76; N, 15.34. Found: C, 46.01;
H, 2.77; N, 13.28.
2-Am in o-8-b en zyl-6-(2,6-d ich lor op h en yl)-8H -p yr id o-
[2,3-d ]p yr im id in -7-on e (12): yield 43%; mp 220-222 °C; 1H
NMR (DMSO-d6) δ 5.48 (s, 2H), 7.21-7.35 (complex m, 5H),
7.43-7.47 (complex m, 3H), 7.57 (d, J ) 8 Hz, 2H), 7.84 (s,
1H), 8.67 (s, 1H); MS (CI) m/z 399 (M + 1). Anal. (C20H14
Cl2N4O) C, H, N.
-
2-Am in o-6-(2,6-dich lor oph en yl)-8-(pyr idin -4-ylm eth yl)-
8H-p yr id o[2,3-d ]p yr im id in -7-on e (13). To a suspension of
NaH (60% in mineral oil, 32 mg) in 6 mL of DMF was added
4 (200 mg, 0.65 mmol), and the mixture was heated to 70 °C.
In a second flask containing Et3N (220 µL, 1.59 mmol) in 4
mL of DMF was added 4-picolyl chloride hydrochloride (137
mg, 0.84 mmol). This dark red mixture was added to the
solution of the sodium salt of 4. The mixture was heated to
70 °C, then cooled to room temperature, and poured into 20
mL of ice water. The resulting precipitate was collected by
filtration and washed with water followed by 10% MeOH in
EtOAc to provide 96 mg of crude product. The organic filtrate
was concentrated to provide an additional 77 mg of crude 13.
An analytical sample was obtained by flash chromatography,
eluting with a gradient of EtOAc to 10% MeOH in EtOAc to
1
provide 13: mp 268-270 °C dec; H NMR (DMSO-d6) δ 5.49
(s, 2H), 7.22 (dd, J ) 4.5, 1.5 Hz, 2H), 7.44 (t, J ) 8 Hz, 1H),
7.48 (br s, 2H), 7.57 (d, J ) 8 Hz, 2H), 7.90 (s, 1H), 8.49 (dd,
J ) 4.5, 1.5 Hz, 2H), 8.70 (s, 1H); MS (CI) m/z 400 (M + 1).
Anal. (C19H13Cl2N5O) C, H, N.
2-Am in o-6-(2,6-d ich lor op h en yl)-8-(3-d im et h yla m in o-
p r op yl)-8H-p yr id o[2,3-d ]p yr im id in -7-on e (14). To a sus-
pension of NaH (60% in mineral oil, 50 mg) in 8 mL of DMF
was added 4 (319 mg, 1.04 mmol). The mixture was heated
at 70 °C for 1.5 h resulting in a clear solution. In a second
flask containing NaH (60% in mineral oil, 68 mg) in 6 mL of
DMF was added 3-dimethylaminopropyl chloride hydrochlo-
ride (248 mg, 1.56 mmol). This suspension was stirred at room
temperature for 30 min, then heated at 70 °C for 10 min, and
added to the solution of the sodium salt of 4. The resultant
suspension was heated at 70 °C for 3 h, then cooled to room
temperature, and filtered, washing with EtOAc. The filtrate
was concentrated, and EtOAc and hexane were added. The
resulting solid was collected by filtration to provide 216 mg
(53%) of 14: mp 136-141 °C; 1H NMR (DMSO-d6) δ 1.76 (pent,
J ) 7 Hz, 2H), 2.11 (s, 6H), 2.26 (t, J ) 7 Hz, 2H), 4.29 (t, J
) 7 Hz, 2H), 7.39 (br s, 2H), 7.44 (t, J ) 8 Hz, 1H), 7.57 (d, J
) 8 Hz, 2H), 7.77 (s, 1H), 8.64 (s, 1H); MS (CI) m/z 394 (M +
1). Anal. (C18H19Cl2N5O) C, H, N.
2-Am in o-6-(2,6-d ich lor op h en yl)-8-p r op yl-8H -p yr id o-
[2,3-d ]p yr im id in -7-on e (6): yield 68%; mp 196-197 °C; H
NMR (DMSO-d6) δ 0.89 (t, J ) 7 Hz, 3H), 1.66 (m, 2H), 4.23
(t, J ) 7 Hz, 2H), 7.41-7.46 (m, 3H), 7.57 (d, J ) 8 Hz, 2H),
7.77 (s, 1H), 8.64 (s, 1H); MS (CI) m/z 351 (M + 1). Anal.
(C16H14Cl2N4O) C, H, N.
1
2-Am in o-8-bu tyl-6-(2,6-d ich lor op h en yl)-8H-p yr id o[2,3-
d ]p yr im id in -7-on e (7): yield 64%; mp 202-205 °C; 1H NMR
(DMSO-d6) δ 0.91 (t, J ) 7 Hz, 3H), 1.31 (m, 2H), 1.62 (m,
2H), 4.26 (t, J ) 7 Hz, 2H), 7.39 (br s, 2H), 7.44 (t, J ) 8 Hz,
1H), 7.56 (d, J ) 8 Hz, 2H), 7.76 (s, 1H), 8.63 (s, 1H); MS (CI)
m/z 365 (M + 1). Anal. (C17H16Cl2N4O‚0.08EtOAc) C, H, N.
2-Am in o-6-(2,6-d ich lor op h en yl)-8-isobu tyl-8H-p yr id o-
1
[2,3-d ]p yr im id in -7-on e (8): yield 51%; mp 193-195 °C; H
NMR (DMSO-d6) δ 0.87 (t, J ) 6.5 Hz, 6H), 2.24 (m, 1H), 4.13
(d, J ) 7.5 Hz, 2H), 7.37 (br s, 2H), 7.44 (t, J ) 7.5 Hz, 1H),
7.57 (d, J ) 7.5 Hz, 2H), 7.77 (s, 1H), 8.64 (s, 1H); MS (CI)
m/z 365 (M + 1). Anal. (C17H16Cl2N4O) C, H, N.
[2-Am in o-6-(2,6-d ich lor op h en yl)-7-oxo-7H-p yr id o[2,3-
d ]p yr im id in -8-yl]a cetic a cid m eth yl ester (9): yield 61%;
mp 188-190 °C; H NMR (DMSO-d6) δ 3.68 (s, 3H), 5.03 (s,
2H), 7.50 (br s, 2H), 7.45 (t, J ) 8 Hz, 1H), 7.57 (d, J ) 8 Hz,
2-Am in o-8-(3-ben zyloxyp r op yl)-6-(2,6-d ich lor op h en yl)-
8H-p yr id o[2,3-d ]p yr im id in -7-on e (15): yield 71%; mp un-
defined (softens at 63 °C); 1H NMR (DMSO-d6) δ 1.94 (pent, J
) 7 Hz, 2H), 3.50 (t, J ) 7 Hz, 2H), 4.36 (t, J ) 7 Hz, 2H),
1

4372 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Boschelli et al.
4.42 (s, 2H), 7.23-7.34 (complex m, 5H), 7.40 (br s, 2H), 7.44
(t, J ) 8 Hz, 1H), 7.56 (d, J ) 8 Hz, 2H), 7.76 (s, 1H), 8.63 (s,
1H); MS (CI) m/z 457 (M + 1). Anal. (C23H20Cl2N4O2‚
0.2EtOAc) Calcd: C, 60.44; H, 4.60; N, 11.85. Found: C, 60.90;
H, 4.77; N, 11.91.
2-[4-(2-Dieth yla m in oeth oxy)p h en yla m in o]-8-m eth yl-6-
p h en yl-8H-p yr id o[2,3-d ]p yr im id in -7-on e (28). A mixture
of 22 (197 mg, 0.63 mmol) and 4-(2-diethylaminoethoxy)aniline
(260 mg, 1.25 mmol) was heated at 175 °C for 5 min. The
reaction was cooled to room temperature, and the residue was
purified by flash chromatography eluting with 5% MeOH in
2-Am in o-6-(2,6-d ich lor op h en yl)-8-(3-h yd r oxyp r op yl)-
8H-p yr id o[2,3-d ]p yr im id in -7-on e (16). A solution of 15
(379 mg, 0.83 mmol) in 75 mL of acetic acid was treated with
increasing amounts of 5-20% palladium on carbon over 12 h.
The catalyst was filtered off, and the filtrate was concentrated.
The residue was partitioned between EtOAc and 1 N NaOH.
The organic layer was dried over MgSO4, filtered, and con-
centrated. Flash chromatography gave 94 mg (31%) of 16: mp
undefined (softens at 98 °C); 1H NMR (DMSO-d6) δ 1.78 (pent,
J ) 7 Hz, 2H), 3.45 (dt, J ) 7, 5.5 Hz, 2H), 4.31 (t, J ) 7 Hz,
2H), 4.46 (t, J ) 5.5 Hz, 1H), 7.42 (br s, 2H), 7.44 (t, J ) 8 Hz,
1H), 7.57 (d, J ) 8 Hz, 2H), 7.78 (s, 1H), 8.65 (s, 1H); MS (CI)
m/z 367 (M + 1). Anal. (C16H14Cl2N4O2‚0.2 H2O) C, H, N.
8-Meth yl-2-m eth ylsu lfa n yl-6-p h en yl-8H-p yr id o[2,3-d ]-
p yr im id in -7-ylid en ea m in e (18). To a solution of 17 (1.50
g, 8.18 mmol) and phenylacetonitrile (1.2 mL, 10.36 mmol) in
10 mL of DMF was added K2CO3 (6.60 g, 47.83 mmol). The
mixture was heated at 125 °C for 1.5 h. The hot mixture was
filtered, and the solid was washed with DMF. Water was
added to the filtrate, and the black solid was removed by
filtration. The crystals that formed in the filtrate were
collected to give 449 mg (19%) of 18: mp 150-151 °C; 1H NMR
(DMSO-d6) δ 2.51 (s, 3H), 3.57 (s, 3H), 7.28 (s, 1H), 7.37-7.52
(m, 6H), 8.48 (s, 1H); MS (CI) m/z 283 (M + 1). Anal.
(C15H14N4S) C, H, N.
1
CHCl3 to give 30 mg (11%) of 28: mp 157-158 °C; H NMR
(DMSO-d6) δ 0.98 (t, J ) 7 Hz, 6H), 2.55 (q, J ) 7 Hz, 4H),
2.77 (t, J ) 6 Hz, 2H), 3.66 (s, 3H), 4.00 (t, J ) 6 Hz, 2H), 6.94
(d, J ) 9 Hz, 2H), 7.36-7.45 (m, 3H), 7.67-7.72 (m, 4H), 8.00
(s, 1H), 8.80 (s, 1H), 10.00 (s, 1H); MS (CI) m/z 444 (M + 1).
Anal. (C26H29N5O2‚0.5H2O) C, H, N.
8-Meth yl-2-m eth ylsu lfan yl-6-th ioph en e-3-yl-8H-pyr ido-
[2,3-d ]p yr im id in -7-ylid en ea m in e (19). To a solution of 17
(10.0 g, 54.6 mmol) and 3-thiopheneacetonitrile (13.52 g, 109.2
mmol) in 100 mL of DMF was added K2CO3 (37.7 g, 273.0
mmol). The mixture was heated at 125 °C for 1.5 h. The hot
mixture was filtered, and the solid was washed with EtOAc.
The solid that appeared in the filtrate was collected and
washed with 10% EtOAc in hexane followed by acetone to give
1
10.87 g (69%) of 19: mp 146-147 °C; H NMR (DMSO-d6) δ
2.57 (s, 3H), 3.64 (s, 3H), 7.27-7.28 (m, 1H), 7.41 (s, 1H), 7.71
(s, 1H), 7.76-7.78 (m, 2H), 8.53 (s, 1H); MS (CI) m/z 289 (M +
1). Anal. (C13H12N4S2) C, H, N.
8-Meth yl-2-m eth ylsu lfan yl-6-th ioph en e-3-yl-8H-pyr ido-
[2,3-d ]p yr im id in -7-on e (21). A mixture of 19 (7.39 g, 25.7
mmol) and 50 mL of Ac2O was heated at reflux for 15 min.
The cooled solution was mixed with 200 mL of diethyl ether
and concentrated. The solid was washed with diethyl ether
to provide 6.27 g (74%) of the N-acetylimine derivative which
was refluxed with concentrated HCl for 5 min. The resulting
solid was collected, washed with water, and purified by flash
chromatography eluting with CH2Cl2 to give 2.99 g (55%) of
21: mp 176-177 °C; 1H NMR (DMSO-d6) δ 2.63 (s, 3H), 3.70
(s, 3H), 7.64-7.70 (m, 2H), 8.35-8.36 (m, 1H), 8.40 (s, 1H),
8.91 (s, 1H); MS (CI) m/z 290 (M + 1). Anal. (C13H11N3OS2)
C, H, N.
8-Meth yl-2-m eth ylsu lfon yl-6-th ioph en e-3-yl-8H-pyr ido-
[2,3-d ]p yr im id in -7-on e (23). To a solution of 21 (2.01 g, 6.95
mmol) in 150 mL of CHCl3 was added 50-60% m-chloroper-
benzoic acid (5.50 g, 16.0-19.2 mmol). The solution was
stirred at room temperature for 1 h, then washed with
saturated NaHCO3, dried over MgSO4, and concentrated. The
solid was purified by flash chromatography eluting with CH2-
Cl2 to give 1.81 g (81%) of 23: 1H NMR (DMSO-d6) δ 3.50 (s,
3H), 3.76 (s, 3H), 7.70-7.76 (m, 2H), 8.48-8.49 (m, 1H), 8.58
(s, 1H), 9.28 (s, 1H); MS (CI) m/z 322 (M + 1).
8-Meth yl-2-p h en yla m in o-6-th iop h en e-3-yl-8H-p yr id o-
[2,3-d ]p yr im id in -7-on e (25). A mixture of 23 (214 mg, 0.67
mmol) and aniline (400 mg, 4.82 mmol) was heated at 150 °C
for 15 min. The reaction was cooled to room temperature and
directly purified by flash chromatography eluting with 5%
MeOH in CHCl3 to give 67 mg (30%) of 25: mp 237-238 °C;
1H NMR (DMSO-d6) δ 3.70 (s, 3H), 7.04 (t, J ) 7 Hz, 1H),
7.36 (t, J ) 8 Hz, 2H), 7.61-7.68 (m, 2H), 7.84 (d, J ) 8 Hz,
2H), 8.27 (s, 1H), 8.30 (s, 1H), 8.83 (s, 1H), 10.15 (s, 1H); MS
(CI) m/z 335 (M + 1). Anal. (C18H14N4OS‚0.25H2O) C, H, N.
8-Met h yl-2-(p yr id in -4-yla m in o)-6-t h iop h en e-3-yl-8H -
p yr id o[2,3-d ]p yr im id in -7-on e (27). A mixture of 23 (190
mg, 0.59 mmol) and 4-aminopyridine (170 mg, 1.81 mmol) was
heated at 175 °C for 5 min. The reaction was cooled to room
temperature and directly purified by flash chromatography
eluting with 5% MeOH in CHCl3 to give 61 mg (31%) of 27:
mp 310-311 °C; 1H NMR (DMSO-d6) δ 3.74 (s, 3H), 6.69-
7.63 (m, 2H), 7.84 (d, J ) 6 Hz, 2H), 8.30-8.45 (m, 4H), 8.92
(s, 1H), 10.54 (s, 1H); MS (CI) m/z 336 (M + 1). Anal.
(C17H13N5OS‚0.25H2O) C, H, N.
8-Meth yl-2-m eth ylsu lfa n yl-6-p h en yl-8H-p yr id o[2,3-d ]-
p yr im id in -7-on e (20). A mixture of 18 (745 mg, 2.64 mmol)
and 5.5 mL of Ac2O was heated at reflux for 30 min. The
cooled solution was mixed with diethyl ether and concentrated.
The solid was washed with diethyl ether to provide 718 mg
(84%) of the N-acetylimine derivative which was refluxed with
concentrated HCl for 5 min. The resulting solid was collected
and washed with water and 2-propanol to give 512 mg (87%)
of 20 that was not purified.
8-Meth yl-2-m eth ylsu lfon yl-6-th ioph en e-3-yl-8H-pyr ido-
[2,3-d ]p yr im id in -7-on e (22). To a solution of 20 (206 mg,
0.73 mmol) in 30 mL of CHCl3 was added 50-60% m-
chloroperbenzoic acid (600 mg, 1.74-2.09 mmol). The solution
was stirred at room temperature for 2 h, then washed with
saturated NaHCO3, dried over MgSO4, and concentrated.
EtOAc was added to the residue followed by hexane. The solid
was collected and washed with 4:1 hexane/EtOAc to give 139
1
mg (60%) of 22: mp 191-193 °C; H NMR (DMSO-d6) δ 3.44
(s, 3H), 3.68 (s, 3H), 7.39-7.47 (m, 3H), 7.69 (d, J ) 2 Hz,
2H), 8.26 (s, 1H), 9.26 (s, 1H); MS (CI) m/z 316 (M + 1). Anal.
(C15H13N3O3S) C, H, N.
8-Meth yl-6-p h en yl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p y-
r im id in -7-on e (24). A mixture of 22 (174 mg, 0.55 mmol)
and aniline (440 mg, 5.30 mmol) was heated at 175 °C for 10
min. The reaction was cooled to room temperature and
directly purified by flash chromatography eluting with EtOAc
to give 62 mg (34%) of 24: mp 210-211 °C; 1H NMR (DMSO-
d6) δ 3.68 (s, 3H), 7.04 (t, J ) 7 Hz, 1H), 7.34-7.39 (m, 3H),
7.44 (d, J ) 7 Hz, 2H), 7.68 (d, J ) 7 Hz, 2H), 7.85 (d, J ) 8
Hz, 2H), 8.03 (s, 1H), 8.85 (s, 1H), 10.16 (s, 1H); MS (CI) m/z
329 (M + 1). Anal. (C20H16N4O‚0.25H2O) C, H, N.
8-Met h yl-6-p h en yl-2-(p yr id in -4-yla m in o)-8H -p yr id o-
[2,3-d ]p yr im id in -7-on e (26). A mixture of 22 (146 mg, 0.46
mmol) and 4-aminopyridine (262 mg, 2.78 mmol) was heated
at 175 °C for 10 min. The reaction was cooled to room
temperature and directly purified by flash chromatography
eluting with EtOAc to give 77 mg (51%) of 26: mp 244-245
°C; HPLC purity >99%, column VYDAC C18 218TP54, mobile
phase gradient 10-76% 0.1% TFA in acetonitrile to 0.1% TFA
in water; 1H NMR (DMSO-d6) δ 3.67 (s, 3H), 7.31-7.42 (m,
3H), 7.63 (t, J ) 7 Hz, 2H), 7.78 (d, J ) 5 Hz, 2H), 8.03 (s,
1H), 8.38 (d, J ) 5 Hz, 2H), 8.88 (s, 1H), 10.49 (s, 1H); MS
(CI) m/z 330 (M + 1). Anal. (C19H15N5O) Calcd: C, 69.29; H,
4.59; N, 21.26. Found: C, 66.79; H, 5.05; N, 19.04.
2-[4-(2-Dieth yla m in oeth oxy)p h en yla m in o]-8-m eth yl-6-
th iop h en e-3-yl-8H-p yr id o[2,3-d ]p yr im id in -7-on e (29). A
mixture of 23 (132 mg, 0.43 mmol) and 4-(2-diethylamino-
ethoxy)aniline (178 mg, 0.86 mmol) was heated at 175 °C for
5 min. The reaction was cooled to room temperature, and the
residue was purified by flash chromatography eluting with 5%
MeOH in CHCl3 to give 40 mg (22%) of 29: mp 190-192 °C;

2-Aminopyridopyrimidines as PDGFr TK Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4373
1H NMR (DMSO-d6) δ 0.98 (t, J ) 7 Hz, 6H), 2.56 (q, J ) 7
Hz, 4H), 2.77 (t, J ) 6 Hz, 2H), 3.67 (s, 3H), 4.00 (t, J ) 6 Hz,
2H), 6.94 (d, J ) 9 Hz, 2H), 7.60-7.72 (m, 4H), 8.25 (t, J ) 1
Hz, 1H), 8.27 (s, 1H), 8.78 (s, 1H), 9.99 (s, 1H); MS (CI) m/z
450 (M + 1). Anal. (C24H27N5O2S‚H2O) C, H, N.
to room temperature, and the resulting precipitate was col-
lected and washed with 1:1 hexane/EtOAc. The solid was
purified by flash chromatography eluting with 10% MeOH in
1
EtOAc to give 60 mg (41%) of 35: mp 241-242 °C; H NMR
(DMSO-d6) δ 3.66 (s, 3H), 7.00 (t, J ) 7 Hz, 1H), 7.21 (two
peaks, 1H), 7.34 (t, J ) 7 Hz, 2H), 7.44 (two peaks, 1H), 7.73-
7.79 (m, 4H), 7.81 (two peaks, 1H), 8.52 (s, 1H), 9.92 (s, 1H);
MS (CI) m/z 334 (M + 1). Anal. (C18H15N5S) C, H, N.
2-Am in o-8-m et h yl-6-t h iop h en e-3-yl-8H -p yr id o[2,3-d ]-
p yr im id in -7-on e (30). A mixture of 21 (170 mg, 0.59 mmol),
ammonia, and MeOH was sealed in a glass tube and heated
at 100 °C for 86 h. The solution was concentrated and purified
by flash chromatography eluting with EtOAc. Recrystalliza-
tion from EtOAc and hexane gave 115 mg (76%) of 30: mp
8-Meth yl-2-p h en yla m in o-6-th iop h en e-2-yl-8H-p yr id o-
[2,3-d ]p yr im id in -7-on e (37). A mixture of 35 (283 mg, 0.85
mmol) and 10 mL of Ac2O was heated at reflux for 30 min.
The solution was concentrated, and the resulting N-acetyl-
imine derivative was dissolved in 10 mL of concentrated HCl
and heated at reflux for 5 min. The reaction was cooled to
room temperature, and the crude product was collected and
purified by flash chromatography eluting with 5% MeOH in
CHCl3 to provide 74 mg (26%) of 37: mp 235-236 °C; 1H NMR
(DMSO-d6) δ 3.72 (s, 3H), 7.04 (t, J ) 7 Hz, 1H), 7.16 (d, J )
5 Hz, 1H), 7.36 (t, J ) 8 Hz, 2H), 7.59 (d, J ) 5 Hz, 1H), 7.84
(d, J ) 8 Hz, 2H), 8.45 (s, 1H), 8.87 (s, 1H), 10.19 (s, 1H); MS
(CI) m/z 335 (M + 1). Anal. (C18H14N4OS‚0.25H2O) C, H, N.
[6-(Biph en yl-4-yl)-7-im in o-8-m eth yl-7,8-dih ydr opyr ido-
[2,3-d ]p yr im id in -2-yl)p h en yla m in e (36). To a suspension
of NaH (110 mg of a 60% suspension of NaH in mineral oil) in
20 mL of 2-ethoxyethanol were added 4-biphenylacetonitrile
(516 mg, 2.67 mmol) and 34 (538 mg, 2.36 mmol). The reaction
mixture was heated at reflux for 17 h and then cooled to room
temperature. The precipitate was collected and washed with
1:1 hexane/EtOAc to give 826 mg (87%) of 36: mp 259-260
°C; 1H NMR (DMSO-d6) δ 3.69 (s, 3H), 7.00 (t, J ) 7 Hz, 1H),
7.32-7.41 (m, 5H), 7.42-7.54 (m, 4H), 7.75 (d, J ) 8 Hz, 2H),
7.82 (d, J ) 8 Hz, 4H), 8.51 (s, 1H), 9.89 (s, 1H); MS (CI) m/z
404 (M + 1). Anal. (C26H21N5‚0.3H2O) C, H, N.
6-(Bip h en yl-4-yl)-8-m eth yl-2-p h en yla m in o-8H-p yr id o-
[2,3-d ]p yr im id in -7-on e (38). A mixture of 36 (261 mg, 0.65
mmol) and 12 mL of Ac2O was heated at reflux for 30 min.
The solution was concentrated, and the resulting N-acetyl-
imine derivative was dissolved in 10 mL of concentrated HCl
and heated at reflux for 5 min. The reaction was cooled to
room temperature, and the solid was collected and purified
by flash chromatography eluting with 5% MeOH in CHCl3 to
provide 152 mg (58%) of 38: mp 263-264 °C; 1H NMR (DMSO-
d6) δ 3.70 (s, 3H), 7.04 (t, J ) 8 Hz,1H), 7.35-7.41 (m, 3H),
7.49 (t, J ) 8 Hz, 2H), 7.73-7.76 (m, 4 H), 7.80-7.86 (m, 4H),
8.11 (s, 1H), 8.87 (s, 1H), 10.17 (s, 1H); MS (CI) m/z 405 (M +
1). Anal. (C26H20N4O‚0.80H2O) C, H, N.
4-Eth yla m in o-2-m eth ylsu lfa n yl-5-p yr im id in eca r boxy-
la te Eth yl Ester (40). To a room-temperature solution of 39
(10.00 g, 43.10 mmol) in 150 mL of THF was added Et3N (18.5
mL, 133 mmol) followed by 9 mL of a 70% aqueous solution of
ethylamine. The solution was stirred for 30 min, then
concentrated, and partitioned between CHCl3 and saturated
aqueous NaHCO3. The organic layer was dried over MgSO4,
filtered, and concentrated to provide 9.32 g (90%) of 40 as an
oil: 1H NMR (DMSO-d6) δ 1.17 (t, J ) 7 Hz, 3H), 1.30 (t, J )
7 Hz, 3H), 2.48 (s, 3H), 3.51 (dq, J ) 7, 5 Hz, 2H), 4.27 (q, J
) 7 Hz, 2H), 8.31 (t, J ) 5 Hz, 1H), 8.53 (s, 1H); MS (CI) m/z
242 (M + 1). Anal. (C10H15N3O2S) C, H, N.
1
241-242 °C; H NMR (DMSO-d6) δ 3.59 (s, 3H), 7.33 (s, 2H),
7.58-7.64 (m, 2H), 8.21 (s, 1H), 8.22 (s, 1H), 8.63 (s, 1H); MS
(CI) m/z 259 (M + 1). Anal. (C12H10N4OS) C, H, N.
8-Meth yl-2-m eth yla m in o-6-th iop h en e-3-yl-8H-p yr id o-
[2,3-d ]p yr im id in -7-on e (31). A mixture of 21 (125 mg, 0.43
mmol), methylamine, and DMF was sealed in a glass tube and
heated at 110 °C for 40 h. The solution was concentrated and
the solid washed with diethyl ether to provide 61 mg (52%) of
31: mp 263-265 °C; 1H NMR (DMSO-d6) δ 2.92 (d, J ) 4 Hz,
3H), 3.65 and 3.56 (two peaks, 3H), 7.58-7.65 (m, 2H), 7.83
(br s, 1H), 8.22 (s, 2H), 7.76 and 8.63 (two peaks, 1H); MS
(CI) m/z 273 (M + 1). Anal. (C13H12N4OS‚0.25H2O) C, H, N.
4-Meth yla m in o-2-m eth ylsu lfa n yl-p yr im id in e-5-ca r bo-
n itr ile (32). Methylamine gas was bubbled through a 5 oC
solution of 4-chloro-2-methylsulfanyl-pyrimidine-5-carbonitrile
(32.0 g, 170 mmol) in 700 mL of diethyl ether. The reaction
mixture was stirred at room temperature overnight. Ad-
ditional methylamine gas was added, and the mixture stirred
at room temperature overnight. The solids were collected and
suspended in water. The suspension was filtered to provide
25.8 g (85%) of product. Recrystallization from EtOAc gave
an analytical sample of 32: mp 189-190 °C; 1H NMR (CDCl3)
δ 2.54 (s, 3H), 3.12 (d, J ) 5 Hz, 3H), 5.50 (br s, 1H), 8.82 (s,
1H). Anal. (C7H8N4S) C, H, N.
4-Meth yla m in o-2-p h en yla m in o-p yr im id in e-5-ca r bon i-
tr ile (33). A suspension of 32 (10 g, 55.5 mmol) in 30 mL of
aniline containing 4 drops of concentrated HCl was heated to
170 °C for 1 h. Upon removal of the heat source a solid rapidly
formed. While the reaction mixture was still warm, 30 mL of
MeOH was added. The thick mixture was cooled to room
temperature and the solid collected washing with additional
MeOH to provide 8.73 g (70%) of product. Recrystallization
from EtOAc and MeOH gave an analytical sample of 33: mp
275-277 °C; 1H NMR (DMSO-d6) δ 2.91 (d, J ) 4 Hz, 3H),
6.99 (t, J ) 7.5 Hz, 1H), 7.29 (t, J ) 8 Hz, 2H), 7.71 (br s, 1H),
7.76 (d, J ) 8 Hz, 2H), 8.33 (s, 1H), 9.81 (br s, 1H). Anal.
(C12H11N5) C, H, N.
4-Met h yla m in o-2-p h en yla m in o-p yr im id in e-5-ca r b a l-
d eh yd e (34). To a 0 °C solution of 33 (4.5 g, 20.00 mmol) in
100 mL of THF was added 20 mL of a 1 M solution of
diisobutylaluminum hydride in CH2Cl2. The ice bath was
removed, and an additional 20 mL of a 1 M solution of
diisobutylaluminum hydride in CH2Cl2 was added. After 30
min an additional 5 mL of a 1 M solution of diisobutylalumi-
num hydride in CH2Cl2 was added. After 30 min the reaction
was quenched by the dropwise addition of 30 mL of MeOH.
This mixture was partitioned between 200 mL of EtOAc and
140 mL of 1 N HCl. The layers were separated, and the acid
layer was treated with 150 mL of 1 N NaOH and extracted
with EtOAc. The organic layer was dried over MgSO4, filtered,
and concentrated to give 2.20 g of a 3:1 mixture of 34:33 by
NMR. Purification by flash chromatography eluting with 1:1
hexane/EtOAc gave an analytical sample of 34: mp 210-212
°C; 1H NMR (DMSO-d6) δ 3.02 (d, J ) 5 Hz, 3H), 7.01 (t, J )
7.5 Hz, 1H), 7.31 (t, J ) 8 Hz, 2H), 7.83 (d, J ) 8 Hz, 2H),
8.46 (s, 1H), 8.59 (br s, 1H), 9.55 (s, 1H), 9.97 (br s, 1H). Anal.
(C12H13N4O) C, H, N.
4-Eth yla m in o-2-m eth ylsu lfin yl-p yr im id in e-5-ca r boxy-
lic Acid Eth yl Ester (41). To a room-temperature solution
of 40 (2.011 g, 8.34 mmol) in 70 mL of CHCl3 was added (()-
trans-2-phenylsulfonyl-3-phenyloxaziridine (2.70 g, 10.34 mmol).
The solution was stirred at room temperature for 7 h and then
concentrated. The residue was purified by flash chromatog-
raphy eluting with a gradient of EtOAc to 3% MeOH in EtOAc
to provide 2.07 g (97%) of 41: mp 54-56 °C; 1H NMR (DMSO-
d6) δ 1.18 (t, J ) 7 Hz, 3H), 1.33 (t, J ) 7 Hz, 3H), 2.84 (s,
3H), 3.55 (dq, J ) 7, 5.5 Hz, 2H), 4.33 (q, J ) 7 Hz, 2H), 8.59
(t, J ) 5.5 Hz, 1H), 8.79 (s, 1H); MS (CI) m/z 258 (M + 1).
Anal. (C10H15N3O3S) C, H, N.
(7-Im in o-8-m eth yl-6-th iop h en e-2-yl-7,8-d ih yd r op yr id o-
[2,3-d ]p yr im id in -2-yl)p h en yla m in e (35). To a suspension
of NaH (21 mg of a 60% suspension of NaH in mineral oil) in
5 mL of 2-ethoxyethanol were added 2-thiopheneacetonitrile
(53 µL, 0.50 mmol) and 34 (101 mg, 0.44 mmol). The reaction
mixture was heated at reflux for 17 h. The solution was cooled
4-Eth yla m in o-2-p h en yla m in o-p yr im id in e-5-ca r boxyl-
ic Acid Eth yl Ester (42). A solution of 41 (5.38 g, 20.9 mmol)
in 4 mL of aniline was heated at 130 °C for 1 h. The solution
was cooled to room temperature resulting in a solid; 20 mL of

4374 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22
Boschelli et al.
1:1 hexane/EtOAc was added, and the white solid was collected
by filtration providing 1.96 g (33%) of 42. The filtrate was
concentrated and purified by flash chromatography eluting
with 3:1 hexane/EtOAc to give an additional 257 mg of 42:
mp 145-147 °C; 1H NMR (DMSO-d6) δ 1.21 (t, J ) 7 Hz, 3H),
1.30 (t, J ) 7 Hz, 3H), 3.52 (dq, J ) 7, 5 Hz, 2H), 4.24 (q, J )
7 Hz, 2H), 6.98 (t, J ) 7 Hz, 1H), 7.29 (t, J ) 8 Hz, 2H), 7.79
(d, J ) 8 Hz, 2H), 8.23 (t, J ) 5 Hz, 1H), 8.56 (s, 1H), 9.77 (br
s, 1H); MS (CI) m/z 287 (M + 1). Anal. (C15H18N4O2) C, H, N.
(4-Eth yla m in o-2-p h en yla m in o-p yr im id in -5-yl)m eth a -
n ol (43). A solution of 42 (109 mg, 0.38 mmol) in 6 mL of
THF was added dropwise to a room temperature suspension
of LAH (35 mg, 0.92 mmol) in 5 mL of THF. After 25 min, an
additional 30 mg of LAH was added and stirring was continued
for 30 min. The reaction was carefully quenched with 120 µL
of water, 200 µL of 15% NaOH, and 300 µL of water. After
stirring for 1 h, the white precipitate was removed by filtration
washing with EtOAc. The filtrate was concentrated and the
residue purified by chromatography eluting with EtOAc to
provide 36 mg (39%) of 43: mp 174-176 °C; 1H NMR (DMSO-
d6) δ 1.19 (t, J ) 7 Hz, 3H), 3.44 (dq, J ) 7, 5.5 Hz, 2H), 4.28
(d, J ) 5.5 Hz, 2H), 4.93 (t, J ) 5.5 Hz, 1H), 6.53 (t, J ) 5.5
Hz, 1H), 6.84 (t, J ) 7 Hz, 1H), 7.21 (t, J ) 8 Hz, 2H), 7.74 (s,
1H), 7.77 (d, J ) 8 Hz, 2H), 8.93 (s, 1H); MS (CI) m/z 245 (M
+ 1). Anal. (C13H16N4O) C, H, N.
4-Eth yla m in o-2-p h en yla m in o-p yr im id in e-5-ca r ba ld e-
h yd e (44). To a solution of 43 (173 mg, 0.71 mmol) in 15 mL
of CHCl3 was added MnO2 (600 mg, 6.89 mmol). After stirring
at room temperature overnight the mixture was filtered
through a pad of Celite, washing with CHCl3. The filtrate was
concentrated to give 170 mg (99%) of 44: mp 155-157 °C; 1H
NMR (DMSO-d6) δ 1.21 (t, J ) 7 Hz, 3H), 3.53 (m, 2H), 7.02
(t, J ) 7 Hz, 1H), 7.31 (t, J ) 8 Hz, 2H), 7.80 (d, J ) 8 Hz,
2H), 8.47 (s, 1H), 8.65 (br s, 1H), 9.55 (s, 1H), 9.98 (br s, 1H);
MS (CI) m/z 243 (M + 1). Anal. (C13H14N4O) C, H, N.
(8-Eth yl-7-im in o-6-p h en yl-7,8-d ih yd r op yr id o[2,3-d ]p y-
r im id in -2-yl)p h en yla m in e (45). To a suspension of NaH
(60% in mineral oil, 44 mg) in 5 mL of 2-ethoxyethanol was
added phenylacetonitrile (120 µL, 1.08 mmol). After the
mixture stirred for 5 min at room temperature, 44 (230 mg,
0.95 mmol) in 2 mL of 2-ethoxyethanol was added and the
reaction mixture was heated at reflux for 5 h. The solution
was cooled to room temperature, and the resulting precipitate
was removed by filtration and washed with water to give 305
mg of product. Recrystallization from EtOAc provided 201 mg
(62%) of 45: mp 193-195 °C; 1H NMR (DMSO-d6) δ 1.29 (t, J
) 7 Hz, 3H), 4.47 (q, J ) 7 Hz, 2H), 7.00 (t, J ) 7 Hz, 1H),
7.23-7.54 (complex m, 9H), 7.82 (d, J ) 7.5 Hz, 2H), 8.48 (s,
1H), 9.86 (s, 1H); MS (CI) m/z 342 (M + 1). Anal. (C21H19N)
C, H, N.
The reaction was heated at reflux for 5 h resulting in a dark
brown solution. The solution was cooled to room temperature,
and 3 mL of water was added. The resulting precipitate was
removed by filtration and washed with water. Recrystalliza-
tion from EtOAc and MeOH gave 129 mg (51%) of 46: mp
223-225 °C; 1H NMR (DMSO-d6) δ 1.29 (t, J ) 7 Hz, 3H),
4.47 (q, J ) 7 Hz, 2H), 7.00 (t, J ) 7 Hz, 1H), 7.26 (dd, J ) 5,
1.5 Hz, 1H), 7.30-7.34 (m, 3H), 7.43 (s, 1H), 7.73-7.76 (m,
2H), 7.82 (d, J ) 7.5 Hz, 2H), 8.47 (s, 1H), 9.87 (s, 1H); MS
(CI) m/z 348 (M + 1). Anal. (C19H17N5S) C, H, N.
8-E t h yl-2-p h en yla m in o-6-t h iop h en e-3-yl-8H -p yr id o-
[2,3-d ]p yr im id in -7-on e (48). Compound was prepared from
46 (173 mg, 0.50 mmol) following the procedure used to
prepare 47. The product was purified by flash chromatogra-
phy, eluting with a gradient of 1:1 hexane/EtOAc to all EtOAc
to give 47 mg (27%) of 48: mp 225-227 °C; 1H NMR (DMSO-
d6) δ 1.31 (t, J ) 7 Hz, 3H), 4.43 (q, J ) 7 Hz, 2H), 7.04 (t, J
) 7 Hz, 1H), 7.36 (t, J ) 7.5 Hz, 2H), 7.62 (dd, J ) 5, 3 Hz,
1H), 7.66 (dd, J ) 5, 1 Hz, 1H), 7.84 (d, J ) 7.5 Hz, 2H), 8.27
(dd, J ) 3, 1 Hz, 1H), 8.29 (s, 1H), 8.83 (s, 1H), 10.15 (s, 1H);
MS (CI) m/z 349 (M + 1). Anal. (C19H16N4OS) C, H, N.
8-Eth yl-2-m eth ylsu lfa n yl-6-p h en yl-8H-p yr id o[2,3-d ]p y-
r im id in -7-ylid en ea m in e (50). To a solution of 49 (570 mg,
2.89 mmol) and phenylacetonitrile (355 mg, 3.03 mmol) in 5
mL of DMF was added K2CO3 (1.94 g, 14.0 mmol). The
mixture was heated at 125 °C for 17 h. The hot mixture was
filtered and washed with EtOAc. The filtrate was concen-
trated and the resulting solid purified by flash chromatography
eluting with
a gradient of 1:1 hexane/EtOAc to EtOAc.
Recrystallization from EtOAc and hexane gave 196 mg (23%)
1
of 50 : mp 120-121 °C; H NMR (DMSO-d6) δ 1.24 (t, J ) 7
Hz, 3H), 2.58 (s, 3H), 4.45 (q, J ) 7 Hz, 2H), 7.35 (s, 1H), 7.43-
7.55 (m, 6H), 8.56 (s, 1H); MS (CI) m/z 297 (M + 1). Anal. (C
16H16N4S) C, H, N.
8-Eth yl-2-m eth ylsu lfa n yl-6-p h en yl-8H-p yr id o[2,3-d ]p y-
r im id in -7-on e (51). A mixture of 50 (156 mg, 5.27 mmol)
and 12 mL of Ac2O was heated at reflux for 30 min. Diethyl
ether was added, and the mixture was concentrated to give a
solid that was washed with diethyl ether to provide 730 mg
(41%) of the N-acetylimine derivative. This material was
refluxed with concentrated HCl for 5 min. The resulting pale
yellow solid was filtered off and washed with water and then
diethyl ether to give 550 mg (92%) of 51: mp 155-157 °C; 1H
NMR (DMSO-d6) δ 1.27 (t, J ) 7 Hz, 3H), 2.63 (s, 3H), 4.42
(q, J ) 7 Hz, 2H), 7.40-7.48 (m, 3H), 7.68-7.71 (m, 2H), 8.13
(s, 1H), 8.94 (s, 1H); MS (CI) m/z 298 (M + 1).
8-Eth yl-2-m eth ylsu lfon yl-6-p h en yl-8H-p yr id o[2,3-d ]p y-
r im id in -7-on e (52). To a solution of 51 (1.83 g, 6.16 mmol)
in 150 mL of CHCl3 was added 50-60% m-chloroperoxybenzoic
acid (4.88 g, 14.2-17.0 mmol). The solution was stirred at
room temperature for 2 h, then washed with saturated
NaHCO3, dried over MgSO4, and concentrated. The solid was
purified by flash chromatography eluting with a gradient of
1:1 hexane/EtOAc to EtOAc to give 1.07 g (53%) of 52: mp
201-202 °C; 1H NMR (DMSO-d6) δ 1.30 (t, J ) 7 Hz, 3H),
3.50 (s, 3H), 4.45 (q, J ) 7 Hz, 2H), 7.46-7.53 (m, 3H), 7.73-
7.75 (m, 2H), 8.31 (s, 1H), 9.32 (s, 1H); MS (CI) m/z 330 (M +
1). Anal. (C16H15N3O3S) C, H, N.
8-E t h yl-6-p h en yl-2-p h en yla m in o-8H -p yr id o[2,3-d ]p y-
r im id in -7-on e (47). A mixture of 52 (119 mg, 0.36 mmol)
and aniline (301 mg, 3.63 mmol) was heated at 175 °C for 10
min. The reaction was cooled to room temperature and the
residue purified by flash chromatography eluting with EtOAc
to give 50 mg (40%) of 47: mp 195-196 °C; 1H NMR (DMSO-
d6) δ 1.31 (t, J ) 7 Hz, 3H), 4.41 (q, J ) 7 Hz, 2H), 7.04 (t, J
) 7 Hz, 1H), 7.34-7.39 (m, 3H), 7.44 (t, J ) 7 Hz, 2H), 7.68
(d, J ) 7 Hz, 2H), 7.84 (d, J ) 7 Hz, 2H), 8.02 (s, 1H), 8.85 (s,
1H), 10.16 (s, 1H); MS (CI) m/z 343 (M + 1). Anal. (C21H18N4O‚
0.25H2O) C, H, N.
8-E t h yl-6-p h en yl-2-p h en yla m in o-8H -p yr id o[2,3-d ]p y-
r im id in -7-on e (47). 45 (182 mg, 0.53 mmol) was added to 2
mL of Ac2O and heated at reflux for several min. The reaction
was cooled to room temperature and concentrated. The
residue was heated at reflux with 4 mL of concentrated HCl
for several minutes. The reaction was cooled, and 10 mL of
water was added resulting in the formation of a small amount
of a precipitate that was removed by filtration and washed
with water. The filtrate was made basic with 1 N NaOH, and
the aqueous layer was extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4, and concen-
trated. The residue was combined with the solid obtained
previously. Purification by flash chromatography eluting with
a gradient of 1:1 hexane/EtOAc to EtOAc gave 92 mg (51%) of
47: mp 202-204 °C; 1H NMR (DMSO-d6) δ 1.30 (t, J ) 7 Hz,
3H), 4.41 (q, J ) 7 Hz, 2H), 7.04 (t, J ) 7 Hz, 1H), 7.34-7.46
(complex m, 5H), 7.68 (d, J ) 7.5 Hz, 2H), 7.85 (d, J ) 7.5 Hz,
2H), 8.02 (s, 1H), 8.85 (s, 1H), 10.16 (s, 1H); MS (CI) m/z 343
(M + 1). Anal. (C21H18N4O) C, H, N.
(8-Eth yl-7-im in o-6-th iop h en e-3-yl-7,8-d ih yd r op yr id o-
[2,3-d ]p yr im id in -2-yl)p h en yla m in e (46). To a suspension
of NaH (60% in mineral oil, 37 mg) in 5 mL of 2-ethoxyethanol
was added 3-thiopheneacetonitrile (100 µL, 0.88 mmol) fol-
lowed by 44 (180 mg, 0.74 mmol) in 2 mL of 2-ethoxyethanol.
8-Eth yl-6-p h en yl-2-(p yr id in -4-yla m in o)-8H-p yr id o[2,3-
d ]p yr im id in -7-on e (53). A mixture of 52 (150 mg, 0.46
mmol) and 4-aminopyridine (289 mg, 3.07 mmol) was heated
at 175 °C for 1 h. The reaction was cooled to room temperature
and the residue purified by flash chromatography eluting with

2-Aminopyridopyrimidines as PDGFr TK Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 22 4375
1
EtOAc to give 70 mg (45%) of 53: mp 265-268 °C; H NMR
(DMSO-d6) δ 1.27 (t, J ) 7 Hz, 3H), 4.39 (q, J ) 7 Hz, 2H),
7.31-7.41 (m, 3H), 7.64 (d, J ) 8 Hz, 2H), 7.77 (d, J ) 6 Hz,
2H), 8.02 (s, 1H), 8.38 (d, J ) 6 Hz, 2H), 8.88 (s, 1H), 10.49 (s,
1H); MS (CI) m/z 344 (M + 1); HPLC purity 98%, column
VYDAC C18 218TP54, mobile phase gradient 10-76% 0.1%
TFA in acetonitrile to 0.1% TFA in water.
elimination half-life values (t1/2) were calculated from elimina-
tion-rate constants as t1/2 ) 0.693/λz. Area under plasma
concentration-time curve AUC(0-∞) values were calculated
from time 0 to the time of last detectable concentration (tldc
)
using the trapezoidal rule and were extrapolated to infinity.
Ack n ow led gm en t. The authors gratefully acknowl-
edge the contributions of Mark Marlatt of Chemical
Development at Parke-Davis for providing quantities of
key intermediates used in this work. In addition, we
acknowledge Don J ohnson and Norm Colbry of Chemi-
cal Development at Parke-Davis for performing the
high-pressure reactions. We thank the Parke-Davis
Analytical Chemistry Department for the spectral data
and elemental analyses.
2-[4-(2-Diet h yla m in oet h oxy)p h en yla m in o]-8-et h yl-6-
p h en yl-8H-p yr id o[2,3-d ]p yr im id in -7-on e (54). A mixture
of 52 (129 mg, 0.39 mmol) and 4-(2-diethylaminoethoxy)aniline
(163 mg, 0.78 mmol) was heated at 175 °C for 5 min. The
reaction was cooled to room temperature and the residue
purified by flash chromatography eluting with 5% MeOH in
CHCl3. The fractions containing product were collected,
concentrated, and washed with 1:1 hexane/EtOAc. The filtrate
was concentrated, and a minimum amount of EtOAc and a
large amount of hexane were added to yield a solid which was
collected to give 37 mg (21%) of 54: mp 125-126 °C; 1H NMR
(DMSO-d6) δ 0.98 (t, J ) 7 Hz, 6H), 1.29 (t, J ) 7 Hz, 3H),
2.56 (q, J ) 7 Hz, 4H), 2.76 (t, J ) 6 Hz, 2H), 4.01 (t, J ) 6
Hz, 2H), 4.38 (q, J ) 7 Hz, 2H), 6.94 (d, J ) 9 Hz, 2H), 7.34-
7.82 (m, 7H), 7.99 (s, 1H), 8.80 (s, 1H), 10.00 (s, 1H); MS (CI)
m/z 458 (M + 1). Anal. (C27H31N5O2‚0.5H2O) C, H, N.
Recom bin a n t Tyr osin e Kin a ses a n d Assa ys.46 1. 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 experi-
mental protocols involving animals were approved by the
Institutional Animal Care and Use Committee. The C6 rat
glioma, the NIH 3T3 fibroblast line transfected with human
PDGF (ââ), and the DU-145 prostatic adenocarcinoma were
maintained by serial passage in nude mice. Nude mice were
also used as tumor hosts for test agent evaluations against
these tumor models. In each experiment for anticancer
activity evaluation, test mice (6-8/treatment group) weighing
18-22 g were randomized and implanted with tumor frag-
ments in the region of the right axilla on day 0 (at least 6 mice/
group and 12 in the control). Animals were treated with test
compound on the basis of average cage weight on the days
indicated in the table. Compound 54 was delivered as a
solution in water. Compound dosing solution was prepared
for 5 days at a time. Host body weight change data are
reported as the maximum treatment-related weight loss in
these studies and are included as a gross indicator of therapy-
related toxicity. Calculation of tumor growth inhibition (%
T/C), tumor growth delay (T - C), and net logs of tumor cell
kill was performed as described previously.55-57 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.
2. Or a l P h a r m a cok in etic P r ofiles in Ra t. Oral phar-
macokinetics were evaluated in three male Wistar rats fol-
lowing a single 5 mg/kg oral gavage dose of 53 and 54. The
doses were prepared in 17% PEG 400/83% methylcellulose
(0.5%). Heparinized plasma samples were collected via a
jugular vein cannula from 0 to 48 h. Calibration curves
containing standards were prepared in methanol. Blank rat
plasma (200 µL) was spiked with 20 µL of standards and
extracted randomized with study samples. Following liquid-
liquid extraction with ethyl ether, the organic layer was dried
under nitrogen and reconstituted with 100 µL water/acetoni-
trile (50%/50% v/v). Samples were analyzed on an LC-MS/
MS instrument with electrospray positive ion mode (VG
Quattro II triple quadrupole). The assay lower limit of
quantitation was 10 ng/mL. Pharmacokinetic parameter
values were estimated by noncompartmental analysis of
individual plasma concentration-time data. Maximum plasma
concentrations (Cmax) and times for these to occur (tmax) were
recorded as observed. Apparent terminal elimination rate
constants (λz) were estimated as the absolute value of the slope
of the least-squares linear regression of the log-linear terminal
phase of concentration-time profiles. Apparent terminal
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