Soluble 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas. Structure-activity relationships against selected tyrosine kinases and exploration of in vitro and in vivo anticancer activity

Article abstract of DOI:10.1021/jm0004291

In continuing our search for medicinal agents to treat proliferative diseases, we have discovered 2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas as a novel class of soluble, potent, broadly active tyrosine kinase (TK) inhibitors. An efficient route

Full text of DOI:10.1021/jm0004291

J . Med. Chem. 2001, 44, 1915-1926
1915
Solu ble 2-Su bstitu ted Am in op yr id o[2,3-d ]p yr im id in -7-yl Ur ea s.
Str u ctu r e-Activity Rela tion sh ip s a ga in st Selected Tyr osin e Kin a ses a n d
Exp lor a tion of in Vitr o a n d in Vivo An tica n cer Activity
Mel C. Schroeder,*,§ J ames M. Hamby,§ Cleo J . C. Connolly,§ Patrick J . Grohar,§ R. Thomas Winters,§
Mark R. Barvian,§ Charles W. Moore,# Stacey L. Boushelle,# Sheila M. Crean,# Alan J . Kraker,#
Denise L. Driscoll,# Patrick W. Vincent,# William L. Elliott,# Gina H. Lu,† Brian L. Batley,† Tawny K. Dahring,†
Terry C. Major,† Robert L. Panek,† Annette M. Doherty,§ and H. D. Hollis Showalter§
Departments of Chemistry, Cancer Research, and Vascular and Cardiac Diseases, Pfizer Global Research & Development,
Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received October 2, 2000
In continuing our search for medicinal agents to treat proliferative diseases, we have discovered
2-substituted aminopyrido[2,3-d]pyrimidin-7-yl ureas as a novel class of soluble, potent, broadly
active tyrosine kinase (TK) inhibitors. An efficient route was developed that enabled the
synthesis of a wide variety of analogues with substitution on several positions of the template.
From the lead structure 1, several series of analogues were made that examined the C-6 aryl
substituent, a variety of water solublizing substitutents at the C-2 position, and urea or other
acyl functionality at the N-7 position. Compounds of this series were competitive with ATP
and displayed submicromolar to low nanomolar potency against a panel of TKs, including
receptor (platelet-derived growth factor, PDGFr; fibroblast growth factor, FGFr;) and nonre-
ceptor (c-Src) classes. Several of the most potent compounds displayed submicromolar inhibition
of PDGF-mediated receptor autophosphorylation in rat aortic vascular smooth muscle cells
and low micromolar inhibition of cellular growth in five human tumor cell lines. One of the
more thoroughly evaluated members, 32, with IC50 values of 0.21 µM (PDGFr), 0.049 µM
(bFGFr), and 0.018 µM (c-Src), was evaluated in in vivo studies against a panel of five human
tumor xenografts, with known and/or inferred dependence on the EGFr, PDGFr, and c-Src
TKs. Compound 32 produced a tumor growth delay of 14 days against the Colo-205 colon
xenograft model.
In tr od u ction
in order to arrest aggressively proliferating cells. There-
fore, given the complex nature of signal transduction,
i.e., redundancies and crosstalk between signaling
pathways, absolute selectivity may not be desirable
when the need arises to simultaneously inhibit multiple
growth signals. Within this latter context, we have
reported on a strategy for uncovering broadly acting,
nonselective small molecule inhibitors of the PDGFr,
FGFr, and c-Src TKs, which might serve to overcome
these redundancies in growth signaling. Our initial ef-
forts led us to the development of a class of ATP-
competitive 6-arylpyrido[2,3-d]pyrimidine ureas that
are active against a range of TKs, including EGFr,
PDGFr, FGFr, and c-Src. We profiled the initial screen-
ing lead in this area, urea compound 1 (Figure 1), which
possesses low micromolar potency toward inhibition of
PDGFr, FGFr, and c-Src TKs.13 Subsequent SAR studies
led to the synthesis of analogues with improved potency,
solubility, and bioavailability relative to the initial
lead.14 More recently, we have developed an extensive
SAR around the related mass screen hit, 2-amino-6-(2,6-
dichlorophenyl)pyrido[2,3-d]pyrimidin-7(8H)-one (2),15
which shows a similar profile toward the above kinases,
and have disclosed related work within the naphthyri-
dine congeners 316 and 4,17 respectively, of both series.
Signaling for a large number of cellular functions,
including cell growth, differentiation, and migration,1
is modulated by a variety of transmembrane growth
factor receptor and cytoplasmic protein tyrosine kinases
(TKs) that catalyze the specific phosphorylation of
tyrosine residues on proteins.2 Abnormal signaling via
tyrosine kinases has been linked to a number of patho-
physiological states including cancer, cardiovascular,
and immunoinflammatory diseases. Since distinct TKs
are implicated in such diverse conditions as angiogen-
esis,3 restenosis,4 atherosclerosis,5 and tumor growth,6
selective TK inhibitors are deemed highly desirable.
Despite the large number of TKs that have been
described and the structural homology between the TKs,
especially in their ATP-binding regions,7 several selec-
tive inhibitors have been identified including those of
the EGFr,8 FGFr,9 PDGFr,10 VEGFr,11 and more re-
cently c-Src TKs.12
While selective TK inhibitors should be less likely to
affect normal cells and thus produce fewer side effects,
broadly acting, nonselective inhibitors may be required
to overcome redundancies in growth signaling pathways
* Correspondence: Mel C. Schroeder. Phone: 734-622-7803. Fax:
734-622-1407. E-mail: mel.schroeder@pfizer.com.
§ Department of Chemistry.
In this paper, we extend our earlier work within the
urea series14 and present the synthesis and structure-
activity relationships (SAR) toward several TKs for
#
Department of Cancer Research.
† Department of Vascular and Cardiac Diseases.
10.1021/jm0004291 CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/05/2001

1916 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Schroeder et al.
Preparation of the disubstituted guanidine, 74, with
N,N′-bis(BOC)-N-ethyl-S-ethylisothiourea20 gave the
product, although in poor yield. Recently we reported
that trisubstituted guanidines can be prepared via
amine anion additions to disubstituted carbodiimides.
Further, if one of the substituents of the carbodiimide
is trityl, it can be removed under acidic conditions to
give unsymmetrical disubstitued guanidines.21 Using
these conditions, the guanidines 8021 and 81 were
prepared in good yields (66% and 81%, respectively;
method I, Scheme 2). Last, the sulfonamide 79 was
prepared in a manner similar to the amides by refluxing
a suspension of 10 and phenylsulfonyl chloride in
dioxane (method J , Scheme 2).
F igu r e 1.
analogues of 1 in which a wide range of solubilizing
alkyl substituents have been introduced at the C-2
nitrogen in combination with selected variations on the
urea moiety at the C-7 position. We also report on
cellular effects and in vivo anticancer activity for
selected compounds drawn from this series.18
SAR vs Isola ted Tyr osin e Kin a ses
The structures of the 6-arylpyrido[2,3-d]pyrimidines
(1 and 11-83) synthesized for this study are shown in
Tables 1-4. They were evaluated for their ability to
prevent phosphorylation of a synthetic glutamate-ty-
rosine polymer by isolated mouse PDGF-â, human FGF-
1, and EGF receptor TKs, as well as the avian c-Src TK.
At least two dose-response curves were determined for
each compound, and averaged IC50 values are listed.
As reported earlier, the initial SAR of 1, wherein
variation about the C-6 aryl and N-7 urea moieties was
explored, showed that compounds with the 2,6-dichlo-
rophenyl substituent (e.g., compound 1, Table 1) at C-6
were the most potent inhibitors against a range of tested
TKs while the 3,5-dimethoxyphenyl (e.g., compound 23,
Table 1) and 2,3,5,6-tetramethylphenyl (e.g., compound
25, Table 1) substituents were found to impart FGFr
selectivity.14 Unfortunately, the poor solubility of 1 and
its analogues precluded the possibility of further in vivo
studies. Three positions of the template, namely the C-6
aryl, the N-7 urea, and the N-2 amino substituents,
offered potential sites for structural changes to increase
potency and solubility. In light of our previous studies,
we decided to complete an initial survey around the C-6
position of 1 toward determining its effect on TK
selectivity and then hold the optimal substituent con-
stant while focusing on developing the SAR of the N-7
urea and N-2 amine positions.
The initial SAR survey for compounds of this class,
built upon the lead structure 1, is shown in Table 1.
Initially we examined the attributes of the tert-butyl
urea at the N-7 position. The diamine, 11, which shows
poor aqueous solubility, is 10- and 20-fold less potent
toward PDGFr and FGFr TK, respectively, than its urea
congener 1. Similar to 1, the nonselective TK inhibitors
14 and 18 showed dramatic decreases in PDGFr and
FGFr activity and moderate decreases in c-Src relative
to their 7-ureas, 15 and 19, respectively, while the FGFr
selective inhibitors showed a substantial decrease in
FGFr potency with removal of the tert-butyl urea (23
vs 22 and 25 vs 24). The necessity of the urea function-
ality for good enzyme potency was independent of the
C-6 aryl substituent.
Ch em istr y
Most of the compounds reported in this paper were
prepared via the generalized route outlined in Scheme
1.14,19 Aldehyde 5 was condensed with an arylacetoni-
trile under basic conditions to give the 2,7-diaminopy-
ridopyrimidine, 6 (method A). Treating the diamine with
1 equiv of NaH followed by acylation with 1 equiv of an
isocyanate gave mostly urea 7 with traces of bis-2,7-
urea byproduct (method B). Alternatively, the N-2
amine of 6 could first be selectively displaced under
acidic conditions with refluxing amine side chain as the
solvent (method C). The resulting pyridopyrimidine, 8,
was treated with 1 equiv of NaH followed by quenching
with 1 equiv of isocyante as described for method B
above to give urea 9. In cases where the isocyanate was
unavailable, the urea was formed by refluxing 8 with
1,1′-carbonyldiimidazole in dry dioxane, followed by
quenching with the desired amine (method D; 50, 58
and 59), or simply from reaction of the anion of 8 with
an appropriate carbamoyl chloride (method E; 60).
While method C is extremely simple and efficient for
introducing simple dialkyl aminoalkyl side chains at the
N-2 position, it suffers from the disadvantage that the
alkylamine must be used in large excess. The amine
displacement also must be carried out at elevated
temperatures under acidic conditions, which could pose
problems for sensitive substrates or products. An alter-
nate pathway that utilizes a methyl sulfoxide as the
leaving group at C-2 has been developed and will be
disclosed in future publications on compounds from this
series.
In addition to urea moieties, we also incorporated
amide, thiourea, guanidine, amidine, and sulfone func-
tionality at N-7 for comparative purposes, as shown in
Schemes 1 and 2. Preparation of amide 77, by treating
the anion of 8 with tert-butyl acetyl chloride (method
E, Scheme 1), gave product in low yield (14%). In
contrast, simply refluxing a suspension of 10 and the
acyl chloride in dry dioxane gave the amides 71, 76, and
78 in good yield (58-64%; method F, Scheme 2). The
amidine, 72, was prepared by refluxing a solution of 10
and DMF dimethyl acetal in DMF (method G, Scheme
2), while thioureas 73, 75, 82, and 83 were prepared in
a manner similar to their oxygen counterparts by anion
formation followed by quenching with the desired
isothiocyanate (method H, Scheme 2).
Recently the crystal structure of PD 173074, an FGFr
selective inhibitor within the same series of ureas, was
determined in complex with the tyrosine kinase domain
of FGFr1.22 Two significant features surrounding the
urea functionality were observed. The first is that the
hydrogen on the terminal nitrogen of the N-7 urea forms

2-Substituted Aminopyrido[2,3-d]pyrimidin-7-yl Ureas
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1917
Sch em e 1
Sch em e 2
a six-membered ring by intramolecular hydrogen bond-
ing to N-8 of the pyridopyrimidine template. Second, the
carbonyl of the urea is bound to a water molecule. In
the case of PD 173074, this bound water in turn is
hydrogen bonded to Lys514 and Asp641, conserved
residues for this kinase. These two aspects of the N-7
urea correlated well with small molecule crystal struc-
tures of other molecules within this series14,23 as well
as a previously reported binding model wherein the
carbonyl oxygen of the urea forms an electrostatic
hydrogen bond to Lys295 of c-Src.23
15, and 19, respectively, and also provided for an
increase in aqueous solubility. In summary, the SAR
shown in Table 1 revealed the importance of the tert-
butyl urea at N-7 for maintaining good tyrosine kinase
potency. In addition, the data showed that it was indeed
possible to introduce substituents at the C-2 position
to increase aqueous solubility, while at the same time
maintaining good enzyme activity. Out of those substit-
uents examined, the data revealed that for nonselective
inhibitors the 2,6-dichlorophenyl was optimal for the
6-position.
To improve the solubility of the 2,7-diamino series of
compounds, we decided to install basic aliphatic side
chains at C-2 of the pyridopyrimidine template. As was
the case for pyridopyrimidinones, 2,15 the introduction
of a single distal basic functionality on an aliphatic
chain improved aqueous solubility (vide infra) but did
not impart greater potency toward the TKs evaluated
(11 vs 12, 14 vs 16, and 18 vs 20). However, reintroduc-
tion of the essential tert-butyl urea onto the N-7 amine
(12 vs 13, 16 vs 17, and 20 vs 21) increased potency
against all three kinases to a level similar to that of 1,
Having established the essential requirement of N-7
tert-butyl urea substitution, we next synthesized a series
of analogues where the C-2 position was substituted
with a number of aliphatic amine side chains to ex-
plore the SAR at this position. Within this series of
compounds, an initial survey of smaller aliphatic
ureas, similar to the tert-butyl urea of 1, and one aro-
matic urea was carried out. These compounds are listed
in Table 2.
Analogues incorporating ten side chains with the
potential for increasing aqueous solubility were pre-

1918 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Ta ble 1. Initial SAR Survey around the N-2, C-6, and N-7 Positionsa
Schroeder et al.
IC50, µM
no.
Ar
R1
R2
method
PDGFr
FGFr
c-Src
1
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
2,6-(Cl)2Ph
2,6-(Cl)2Ph
2,6-(Cl)2Ph
2,6-(Cl)2Ph
-NH2
-NH2
-NH(CH2)3NEt2
-NH(CH2)3NEt2
-NH2
t-BuNHCO
H
H
t-BuNHCO
H
t-BuNHCO
H
t-BuNHCO
H
t-BuNHCO
H
t-BuNHCO
H
t-BuNHCO
H
ref 14
ref 14
ref 14
ref 14
ref 14
ref 14
C
B
A
B
C
1.2
16
46
0.66
29
0.34
25
0.80
>50
1.4
0.14
3.0
2.4
0.082
13
0.40
18
0.34
13
0.29
8.3
0.19
>50
0.71
0.23
0.048
0.22
0.21
0.75
0.073
0.43
0.11
0.45
0.098
1.6
0.21
0.76
2,6-(Me)2Ph
2,6-(Me)2Ph
2,6-(Me)2Ph
2,6-(Me)2Ph
2,6-(Br)2Ph
2,6-(Br)2Ph
2,6-(Br)2Ph
2,6-(Br)2Ph
2,3,5,6-(Me)4Ph
2,3,5,6-(Me)4Ph
3,5-(MeO)2Ph
3,5-(MeO)2Ph
-NH2
-NH(CH2)3NEt2
-NH(CH2)3NEt2
-NH2
-NH2
-NH(CH2)3NEt2
-NH(CH2)3NEt2
-NH2
-NH2
-NH2
>50
B
1.1
0.097
>50b
>50b
>50
ref 14
ref 14
ref 14
ref 14
>50
>50
>50
>50
-NH2
t-BuNHCO
>50
a
IC50 values reported for kinase inhibition represent the means of at least two separate determinations done in triplicate with typical
b
variations of less than 30% between replicate values. IC50 values represent a single determination.
Ta ble 2. Further Exploration around the C-2 Amine and C-7 Urea Positionsa
IC50, µM
no.
R1
R2
method
PDGFr
FGFr
c-Src
13
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
-NH(CH2)3NEt2
-NH(CH2)2NEt2
-NH(CH2)2NEt2
-NH(CH2)2NEt2
-NH(CH2)3NEt2
-NH(CH2)3NEt2
-NH(CH2)4NEt2
-NH(CH2)4NEt2
-NH(CH2)4NEt2
-NH(CH2)4NEt2
-NH(CH2)4NEt2
-NH(CH2)3NMe2
-NH(CH2)3NMe2
-NMe(CH2)3NMe2
-NMe(CH2)3NMe2
-NHCH2CMe2CH2NMe2
-NHCH2CMe2CH2NMe2
-NH(CH2)3(morpholin-1-yl)
-NH(CH2)3(morpholin-1-yl)
-NH(CH2)3(2-methylpiperidin-1-yl)
-NH(CH2)3(2-methylpiperidin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)4(N-methylpiperazin-1-yl)
-NH(CH2)4(N-methylpiperazin-1-yl)
t-BuNHCO
H
ref 14
C
B
B
B
B
ref 14
B
ref 14
B
B
C
B
C
B
C
0.66
ntb
12
9.0
1.3
1.1
nt
0.21
0.36
0.33
0.45
nt
0.68
nt
16
nt
3.2
nt
0.082
nt
1.3
0.073
nt
2.6
EtNHCO
t-BuNHCO
EtNHCO
i-PrNHCO
H
EtNHCO
t-BuNHCO
cyclohexylNHCO
PhNHCO
H
t-BuNHCO
H
t-BuNHCO
H
t-BuNHCO
H
t-BuNHCO
H
1.8
4.1
0.13
0.077
nt
0.049
0.048
0.043
0.11
nt
0.075
nt
2.0
nt
0.21
nt
0.072
nt
0.060
0.45
0.051
nt
0.094
0.078
nt
0.018
0.0074
0.012
0.0075
nt
0.12
nt
1.1
nt
3.5
nt
0.10
nt
0.016
0.18
0.032
nt
B
C
B
C
0.84
nt
0.73
9.6
0.47
nt
t-BuNHCO
H
t-BuNHCO
H
B
ref 14
ref 14
C
t-BuNHCO
B
0.28
0.035
0.010
a
IC50 values reported for kinase inhibition represent the means of at least two separate determinations done in triplicate with typical
b
variations of less than 30% between replicate values. nt ) not tested.
pared. With regard to the effect of side chain length,
for a given urea, shortening the alkylamino chain length
by one carbon (see 13 vs 28; 29 vs 27) resulted in a
dramatic decrease in inhibition against all three TKs.
In contrast, extension of the alkylamino side chain by
one carbon resulted in a 2-10-fold increase in inhibition
of PDGFr, FGFr, and c-Src kinases (see 13 vs 33; 29 vs
32; 47 vs 49).
Disubstitution of the C-2 amine (see 37 vs 39) resulted
in a 15-20-fold loss of enzyme potency. This result and
others reported within the pyridopyrimidinone series15
support the essential requirement of a 2-NHR substitu-
tion pattern, which is shown in the crystal structure to
form a hydrogen bond to the carbonyl oxygen of Ala564
in the FGFr TK (or the corresponding amino acid in
c-Src and PDGFr TKs).23 Similarly, branching along the
carbon backbone of the alkylamino side chain led to
decreased activity (37 vs 41).
Having established the effect of side chain length, we
looked next at modification of the vicinal nitrogen.

2-Substituted Aminopyrido[2,3-d]pyrimidin-7-yl Ureas
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1919
Ta ble 3. Extensive Exploration around the C-7 Urea Positiona
Our next focus was on the nature of the terminus of
the amine side chain. To examine the steric constraints
around the distal amine, we compared the N,N-dieth-
ylamino moiety in the side chain of 13 with the N-
(morpholino) and N-(2-methylpiperidino) moieties. No
change in potency was observed for either modification
(13 vs 43; 13 vs 45). Earlier work on the pyridopyrimi-
dinones15 showed that incorporating a second basic
amine into the side chain is beneficial. Attempting to
improve enzyme potency even further, we incorporated
the N-(4-methylpiperazino) moiety in the side chain of
13 and 33. In this case, we observed a moderate increase
in TK inhibition (see 13 vs 47; 33 vs 49). In summary,
the data in Table 2 show that the optimal alkylamino
side chain at C-2 is four carbons in length and un-
branched along the backbone and possesses a terminal
N-(4-methylpiperazino) group. It should be noted, how-
ever, that while four carbons are preferred over three
and N-(4-methylpiperazino) is preferred over N,N-
diethylamino, combinations of three carbons terminat-
ing in N-(4-methylpiperazino) or four carbons terminat-
ing in N,N-diethylamino were essentially equipotent
against the range of kinases evaluated.
IC50, µM
no.
R2
method PDGFr FGFr
c-Src
50 H2NCO
51 EtNHCO
52 allylNHCO
53 i-PrNHCO
D
B
B
B
ref 14
B
B
B
B
D
D
E
B
B
B
B
B
B
2.4
0.14
0.061
0.024
0.022
0.019
0.032
0.12
0.030
0.031
0.12
0.42 0.053
0.76 0.035
0.55 0.034
0.47 0.051
0.42
0.062
0.37 0.029
47 t-BuNHCO
54 n-octylNHCO
55 benzylNHCO
56 cyclohexylNHCO
57 adamantylNHCO
58 BOCNH(CH2)2NHCO
59 Me2N(CH2)2NHCO
60 Et2NCO
6.2
2.5
2.8
4.2
2.4
0.12
0.067
0.075
5.5
0.15
0.020
>50
>50b
61 PhNHCO
0.57 0.084
0.015
0.040
0.033
0.19
0.092
0.036
0.016
0.029
0.033
0.13
62 4-ClPhNHCO
63 4-BrPhNHCO
64 4-CF3PhNHCO
65 3,4-(Cl)2PhNHCO
66 4-MePhNHCO
67 2-MeOPhNHCO
68 3-MeOPhNHCO
69 4-MeOPhNHCO
70 1-naphthylNHCO
1.8
1.5
5.5
5.0
0.12
0.11
0.61
0.60
A preliminary survey on the effect of N-7 urea
substitution on enzyme potency is also contained in
Table 2. Examination of the urea functionality when the
substitution at C-2 was held constant showed that
replacing the tert-butyl urea of 13 with other smaller
alkyl ureas had no effect on potency (28 vs 27; 13 vs
29 and 30; 33 vs 32 and 34). Interestingly, even the
aryl urea analogue 35 possessed activity comparable
to its tert-butyl urea counterpart 33 in the inhibition
of PDGFr and c-Src. Both of these compounds are the
most potent inhibitors of c-Src (0.007 µM) for analogues
listed in Table 2. In summary, the data in Table 2
show that aliphatic ureas comprising e6 carbons are
comparable in potency to the tert-butyl urea congener
of 33.
0.84 0.11
0.66 0.067
0.91 0.063
0.68 0.074
B
B
B
B
2.6
0.30
a
IC50 values reported for kinase inhibition represent the means
of at least two separate determinations done in triplicate with
typical variations of less than 30% between replicate values. IC50
b
values represent a single determination.
a 2-fold increase in PDGFr and FGFr and a 4-fold
increase in c-Src TK potencies are obtained.
Continuing our survey of ureas we observed that
while smaller aliphatic ureas at N-7 gave compounds
with consistently good activity, increasing their size or
steric bulk tended to decrease inhibition toward the
kinases evaluated. In particular, the compounds suf-
fered from a 5-10-fold decrease in inhibition against
PDGFr (47 vs 54, 55, and 57). We also incorporated urea
moieties possessing a basic side chain off the distal
nitrogen as another means of enhancing aqueous solu-
bility. This, too, brought about a 5-fold decrease in
PDGFr potency (47 vs 59). In completing our study of
aliphatic ureas, we looked at the effect of disubstitution
of the urea distal nitrogen, effectively eliminating any
hydrogen bonding with N-8 of the pyridopyrimidine
template. As reported above, this hydrogen bond has
been observed in several small molecule crystal struc-
tures of these compounds14,23 as well as in the cocrystal
of the PD 173074/FGFr-1 complex.22 Not surprisingly,
the disubstituted urea was found to be inactive against
the PDGFr and c-Src TKs, and its potency against FGFr
decreased 2 orders of magnitude (47 vs 60), possibly due
to an entropic effect and/or a repositioning of the urea
alkyl substituent away from a small hydrophobic pocket
of the enzyme.
Since a number of aliphatic substituents were well
tolerated at N-7, and 35 with its aryl urea was one of
our most potent compounds against the c-Src kinase,
we decided to investigate the nature of the urea at N-7
in more detail. First, we held the C-2 position fixed
using one of our better side chains, the N-(4-methylpip-
erazino)propylamine, and constructed a SAR with 22
different ureas at N-7. These compounds are shown in
Table 3. We began our study by taking a closer look at
the effect of the size of the alkyl urea. Replacing the
tert-butyl substituent with hydrogen on the urea re-
sulted in lowering PDGFr potency by 5-fold while FGFr
and c-Src suffered a 2-3-fold decrease in potency (47
vs 50). As observed above, compounds with the smaller
aliphatic ureas, (e.g., ethyl, allyl, isopropyl, and cyclo-
hexyl) were essentially equipotent to 47 against the
three kinases (47 vs 51, 52, 53, and 56). Parenthetically,
it should be noted that the data for 51 and 53 reinforce
the observation made above regarding the attributes of
the N-(4-methylpiperazino) group. In the case of the tert-
butyl urea, only a marginal improvement in activity was
observed when the N,N-diethylamino substituent was
replaced with N-(4-methylpiperazino) (Table 2; 13 vs 47
and 33 vs 49). Comparing the data in Table 2 and Table
3, a more pronounced effect is seen in the case of the
ethyl and isopropyl ureas (51 vs 29 and 53 vs 30) where
Another area of SAR investigation was to look at the
effect of aromatic ureas at N-7. As described in the
initial example above (33 vs 35; Table 2) the phenyl
urea, 61, was equal in potency to the tert-butyl urea,
47. A brief look at the effect of substitution of the phenyl
group showed that analogues with electron-rich rings,

1920 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Ta ble 4. Other C-7 Functionalitya
Schroeder et al.
IC50, µM
no.
R1
R2
method
PDGFr
FGFr
c-Src
0.073
19
4.0
0.13
1.4
0.022
2.7
8.4
0.032
3.3
0.022
0.86
0.85
0.22
0.022
13
71
72
73
74
75
76
77
47
78
79
80
81
82
83
-NH(CH2)3NEt2
-NH(CH3)3NEt2
-NH(CH3)3NEt2
-NH(CH3)3NEt2
-NH(CH3)3NEt2
-NH(CH2)4NEt2
t-BuNHCO
t-BuCH2CO
Me2NCH
EtNHCS
EtNHCNH
ref 14
F
G
H
I
H
F
E
ref 14
F
J
I (ref 21)
I
H
H
0.66
>50b
>50
5.0
0.082
27
10
0.26
2.7
0.13
3.7
8.0
0.051
6.8
0.068
2.8
2.5
>50
(morpholin-1-yl)(CH2)3NHCS
EtCH2CO
1.1
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
-NH(CH2)3(N-methylpiperazin-1-yl)
>50
>50
0.47
>50
6.9
t-BuCH2CO
t-BuNHCO
PhCH2CO
PhSO2
PhNHCNH
37
>50
i-PrNHCN-i-Pr
t-BuNHCS
1.9
2.2
0.10
0.32
PhNHCS
a
IC50 values reported for kinase inhibition represent the means of at least two separate determinations done in triplicate with typical
b
variations of less than 30% between replicate values. IC50 values represent a single determination.
such as 2- or 4-methoxyphenyl substitution, were equi-
potent with the unsubstituted phenyl urea (61 vs 67 and
69, respectively). In contrast, analogues with electron-
deficient rings, such as 4-(trifluoromethyl) or 3,4-
dichloro substituents, or the bulky naphthyl urea re-
sulted in a decreased potency when compared to the
simple phenyl congener (61 vs 64, 65; 61 vs 70).
Although the inhibitory activity patterns tended to
follow the electronic effects of the phenyl substituents,
substitution of the phenyl ring in general resulted in
only minor to moderate changes in potency. In sum-
mary, the SAR data in Table 3 indicate that smaller
alkyl or unsubstituted phenyl ureas are optimal, whereas
larger, bulkier ureas lead to compounds with lower
potency. Furthermore, the intramolecular hydrogen
bond between the N-7 urea and N-8 of the pyridopyri-
midine template appears essential toward maintaining
good activity.
We wished to further explore the importance of this
hydrogen bonding as it relates to the urea and other
functional groups incorporated at the N-7 position. The
compounds prepared for this purpose are shown in
Table 4. As with the disubstituted urea described above,
an amide functionality at N-7 still allows for hydrogen
bonding between the carbonyl group of the amide and
the kinase conserved lysine but vitiates the possibility
for forming an intramolecular hydrogen bond with the
N-8 of the pyridopyrimidine. Thus amide installation
resulted in a total loss in PDGFr inhibitory activity, and
FGFr and c-Src activity decreased 2 orders of magnitude
(13 vs 71; 51 vs 76; 47 vs 77; 61 vs 78; see also Table
3). In contrast to the bis-substituted urea, 60, and the
amide derivatives, both thiourea and guanidine ana-
logues have the potential to form the apparent requisite
hydrogen bond between the N-7 substituent and N-8 of
the pyridopyrimidine nucleus. Comparison of urea to
corresponding thiourea congeners showed a 4-fold de-
crease in PDGFr potency, and a 2-4-fold decrease in
FGFr TK potency (29 vs 73; 47 vs 82; 61 vs 83; see also
Tables 2 and 3). This decrease may be due to the fact
that the sulfur atom of the thiourea is a poorer hydrogen
bond acceptor relative to the oxygen of the urea.24,25 This
hydrogen bond has been shown to be important for
enzyme binding in crystallogaphic22 and binding model
studies.23 Alternatively, the decrease in in vitro enzyme
potency may result from a repositioning of the thiourea
due to different steric demands. Similarly, the guanidine
congeners showed poor activity, being essentially inac-
tive vs PDGFr and suffering greater than a 10-fold
decrease in FGFr and c-Src potency (29 vs 74; 61 vs
80; see also Tables 2 and 3). One possible explanation
for this may be the guanidine’s inability to offer a second
hydrogen bonding possibility through the hydrogen of
the imine nitrogen. To accomplish this, the ethyl group
of 74 or the phenyl group of 80 must be rotated into a
spatial position distal to that occupied by the same
groups in the urea congeners, 29 and 61, respectively.
Supportive of this notion is the disubstituted guanidine,
81, where the isopropyl group on the imine nitrogen is
fixed into a space normally left vacant with urea
substitution. The final replacement for the N-7 urea
examined was the sulfonamide functionality. While 79
possessed good potency vs FGFr and c-Src, the activity
against PDGFr was similar to that seen with the
thioureas. In summary, the data in Table 4 show once
again the importance of the intramolecular hydrogen
bond between the hydrogen on the terminal amine of
the urea and N-8 of the pyridopyrimidine ring. None of
the analogues with alternate functional groups, includ-
ing thioureas and guanidines, that are capable of
forming this critical hydrogen bond increased enzyme
potency over that found in the ureas.
Having optimized the N-7 position and C-2 positions,
we selected four of our best compounds, based on their
potency against our primary panel of kinases, to look
at their selectivity against other kinases. Compounds
32, 33, 35, and 47 (Table 2) were evaluated against the
expanded panel shown in Table 5. While displaying good
potency toward PDGFr, FGFr, and c-Src TKs, they were
marginally active against the epidermal growth factor
receptor (EGFr) and inactive against the insulin recep-
tor (IR), two other tyrosine kinases. These compounds

2-Substituted Aminopyrido[2,3-d]pyrimidin-7-yl Ureas
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1921
Ta ble 5. Expanded Panel of Kinase Enzyme Inhibitiona
IC50, µM
MEK/
no. PDGFr FGFr c-Src EGFr ERKb,c IRb CDK4 PKCb
32
33
35
47
0.21
0.36
0.45
0.49
0.049 0.018
1.3b
>5
>50 >40
>50
23
>50
22
0.048 0.0074 0.39
2.8 >50 >40
5.0 >50 >40
7.0 >50 >40
0.11 0.0075 1.0b
0.051 0.032
0.15
a
IC50 values reported for kinase inhibition represent the means
of at least two separate determinations done in triplicate with
typical variations of less than 30%. b IC50 values represent a single
determination. c MEK/ERK was run as a cascade assay.
were also inactive toward the inhibition of cyclin de-
pendent kinase (CDK4) and protein kinase C (PKC),
both serine/threonine kinases, and possessed poor activ-
ity in a cascade assay for MEK1 (MAP kinase kinase)
and ERK2. Thus, the data in Table 5 suggest that the
compounds of this series are selective toward a range
of tyrosine kinases, in particular PDGFr, FGFr, and
c-Src.
While the aqueous solubility of 1 was insufficient for
further in vivo studies (<1 µg/mL), optimization of the
alkylamino substituent at N-2 and the urea substituent
at N-7 resulted in compounds with a tremendous
increase in aqueous solubility. In addition, we observed
that compounds containing an ethyl urea, e.g., 32
(>5200 µg/mL) and 51 (>4500 µg/mL), were more
soluble than the corresponding compounds bearing a
tert-butyl urea, e.g., 33 (760 µg/mL) and 47 (210 µg/mL),
or phenyl urea 35 (47 µg/mL). Within the series shown
in Tables 1-4, 32 showed excellent potency toward all
TKs surveyed with IC50 values of 0.21 µM (PDGFr),
0.049 µM (FGFr), and 0.018 µM (c-Src). On the basis of
its excellent potency toward the kinases profiled, as well
as its physiochemical properties, it was selected for
additional functional, cellular, and animal studies. We
also profiled other closely related congeners of 32 in
selected in vitro studies.
F igu r e 2. Growth delay assay for compound 47 using
RAVSMC P6.
Ta ble 6. PDGF Receptor Autophosphorylation and Growth
Delay Effects on Human Colon Carcinoma Cell Lines, C-6 Rat
Glioma, and NIH3T3/PDGF Mouse Transfected Fibroblasts
IC50, µM
PDGF
C-6 rat
NIH3T3/
no. autophosa HCT-8b SW-620b HT-29b gliomab PDGF MTFb
32
33
35
47
51
61
0.24
0.45
1.2
0.27
nt
4.02
16.3
3.2
>15.2
9.3
2.76
>25
2.6
1.17
0.9
8.8
5.8
8.1
4.0
6.7
6.7
1.01
ntd
nt
1.08
nt
0.8
>25
11.1c
0.9
2.5
4.1
0.37
4.0
1.5
nt
a
b
Results are the mean of at least two experiments. Results
are the mean of at least two experiments done in duplicate. c IC50
value represents a single determination. nt, not tested.
d
in RAVSMCs26 (Table 6). Stimulation of the PDGFr TK
with its ligand (PDGF) results in phosphorylation of the
intracellular cytoplasmic domain. Compound inhibition
is determined by lysing the cells and quantifying the
level of the 190 Kd tyrosine phosphorylated receptor
protein after Western blotting with an anti-phosphoty-
rosine antibody. The IC50 values for the inhibition of
autophosphorylation of the PDGF receptor found in
RAVSMCs were similar to those found for the inhibition
of substrate phosphorylation in isolated PDGFr. The
IC50 for inhibition of PDGFr tyrosine kinase activity was
0.47 µM for compound 47. This value correlated with
both inhibition of vascular smooth muscle cell PDGFr
autophosphorylation (IC50 0.27 µM) and growth (IC50
0.35 µM). These results indicate that the selected
inhibitors permeate the cell membrane well and that
RAVSMC growth was arrested due to inhibition of
PDGFr phosphorylation.
In another set of cellular assays which served as a
primary, front line screen, the same compounds plus 51
were evaluated in cell culture against five tumor cell
lines, and the growth of these cells was monitored for 3
days (Table 6).27 The first three cell lines, HCT-8, SW-
620, and HT-29, are comprised of human colon carci-
noma cells whose cellular growth is regulated by an
unknown number of kinases. In the colon carcinoma cell
lines, HCT-8, SW-620, and HT-29, compounds 32, 35,
51, and 61 were more potent than 33 and 47, possessing
low micromolar activity. This potency is comparable to
that observed for cisplatin in these cell lines. Although
Cellu la r Stu d ies
Reputedly, the abnormal proliferation of vascular
smooth muscle cells plays a critical role in some diseases
such as restenosis and atherosclerosis.4,5 We evaluated
one of our better compounds, 47, for its ability to inhibit
PDGF-stimulated growth of rat aortic vascular smooth
muscle cells (RAVSMCs) over an 5 day period. In this
assay,26 growth arrested RAVSMCs were treated with
vehicle (control) or 47 (at concentrations of 0.1, 0.3, or
1.0 µM) and stimulated to grow in serum. The compound
was washed out and replaced with fresh drug daily, and
cells were counted on days 1-5. The results shown in
Figure 2 indicate that 47 inhibits RAVSMC growth in
a dose-dependent fashion with an IC50 of 0.35 µM on
day 5. This result is similar to that reported earlier for
33 (IC50 0.30 µM).14 Additionally, when the treatment
of RAVSMCs with 47 at 1 µM concentration was
discontinued after 3 days, normal cellular growth
resumed, paralleling that of the control (see 1 µM
washout in Figure 2). These results indicate that 47 is
not cytotoxic and is a potent inhibitor of vascular smooth
muscle cell proliferation in vitro.
Looking further at the mechanism of inhibition, five
compounds, 32, 33, 35, 47, and 61, were tested for their
effect on PDGF-mediated receptor autophosphorylation

1922 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Schroeder et al.
Ta ble 7. In Vivo Anticancer Activity of Compound 32 against Six Human Tumor Xenografts and a Mouse Fibroblast Line
Transfected with PDGFa
dose
(mg/kg)
weight
change (g)
T/C (%) on last
T - C
net cell kill
(log10)
d
tumor
A431
tissue type
schedule
therapy dayb
(days)c
epidermoid
glioma
colon
colon
breast
30
30
26
26
30
26
40
days 7-21
days 1-15
days 9-23
days 15-29
days 1-15
days 15-29
days 1-15
-2.2
-1.5
-6.0
-4.0
-1.1
-3.0
-3.2
103
76
26
127
46
85
0.5
0.0
14
0.0
2.5
0.0
2.8
-0.8
0.0
C6
Colo-205
HT-29
MCF-7
SK-OV-3
PDGF
-0.5
-1.8
ovarian
fibroblast
51
a
Tumor fragments (approximately 30 mg) were implanted sc into the right axilla of mice on day 0. All treatments were ip on the
b
indicated schedules. The maximum tolerated dose (eLD10) from a complete dose response is shown. Ratio of median treated tumor
mass (mg)/median control tumor mass × 100% on the last day of therapy. c The difference (in days) for the treated (T) and the control (C)
d
tumors to reach 750 mg. The net reduction in tumor burden (in logs) between the first and last treatments.
32 was slightly less potent than 47 in the growth delay
of C-6 rat glioma cells, both compounds were equipotent
(IC50 ) 1 µM) toward the inhibition of the NIH3T3/
PDGF transfected cell line. The growth delay data in
Table 6 show that the selected inhibitors, which potently
inhibit receptor and nonreceptor tyrosine kinases, are
also capable of inhibiting normal and transfected cell
proliferation, albeit at concentrations greater than those
necessary for enzyme inhibition. Although the source
of differing potency for isolated enzyme vs cellular
inhibition was not characterized, these may reside in
uptake and distribution mechanisms within cells. Given
that these are broadly active TK inhibitors, it would also
seem plausible that this could reflect different potencies
for kinases that were not examined.
F igu r e 3. Effect of compound 32 on the growth of Colo-205
(colon adenocarcinoma xenograft) in nude mice. Tumors were
implanted sc on day 0. Intraperitoneal treatments were
In Vivo Stu d ies
Compound 32 was evaluated in vivo against six
human tumor xenografts and a mouse fibroblast line
transfected with PDGF (Table 7). The Colo-205, HT-29,
and SK-OV-3 in vivo tumor models were selected on the
basis of their endogenous c-Src activity.28 Colo-205, HT-
29, and SK-OV-3 are reported to have in vitro c-Src
activities of 6, 11, and 19 pmol/min/mg protein with poly
glu-tyr as substrate. These activity levels were not the
highest nor the lowest reported for other cell lines in
the NCI 60-cell line panel, but they were deemed to be
representative of the cell lines in this panel. The A431
epidermoid xenograft and MCF-7 breast xenografts
were selected based on the purported involvement of
c-Src in the EGF signaling pathway and in breast
tumors, respectively.29 The C6 glioma was evaluated for
sensitivity to 32 based on the importance of PDGF for
the in vivo growth of this tumor model.30 The fibroblast
line transfected with PDGF was included due to its high
tumorigenic potential as compared to the parent fibro-
blast line. Compound 32 produced tumor stasis against
only the Colo-205 colon xenograft model as reflected in
a tumor growth delay of 14 days for 15 days of therapy.
Tumor growth resumed once therapy was terminated
(Figure 3). The maximum tolerated dose of 32 produced
varying degrees of animal weight loss from 4 to greater
than 20%, with the greatest weight loss in the evalua-
tion against the Colo-205 tumor model. There was no
evidence of any antitumor effect against any of the other
tumor models.
administered on days 9-23. Tumors were approximately 100-
120 mg on day 9 when the first treatment was administered.
Data are the median tumor mass on each measurement day
and the interquartile range.
inhibitors. These compounds inhibit selected kinases at
the ATP binding site and have been shown to exert
growth delay effects on rapidly proliferating cells, such
as RAVSMCs and various tumor cell lines. One com-
pound, 32, demonstrated measurable in vivo activity
against one of the murine models, the Colo-205 colon
tumor xenograft. Due to their level of c-Src activity, we
were surprised that none of the other tumor models
responded. However, this could indicate that the inhibi-
tion of c-Src and/or the PDGF receptor in the nonre-
sponding tumor models might not be sufficient to
produce an antitumor effect due to the redundancy
present in the signaling pathways in vivo. The growth
delay observed for 32 against Colo-205 suggests that in
sensitive tumors compounds of this type may produce
tumor stasis in vivo and that prolonged treatment
periods could maintain complete suppression of tumor
growth.
Exp er im en ta l Section
Gen er a l Meth od s. See Supporting Information.
Meth od A. 6-(2,6-Dibr om o-p h en yl)-p yr id o[2,3-d ]p yr i-
m id in e-2,7-d ia m in e (18). To cold (5 °C) 2-ethoxyethanol (10
mL) was cautiously added NaH (0.139 g, 5.80 mmol) in por-
tions with stirring. The mixture was allowed to warm to room
temperature. To the reaction was added 2,6-dibromophenyl-
acetontrile (4.18 g, 15.2 mmol), followed by aldehyde 514,19 (2.00
g, 14.48 mmol). The reaction was heated at reflux for 4 h,
allowed to cool to room temperature, and poured into water.
Su m m a r y
In conclusion, we have reported the synthesis and
SAR of a number of soluble, potent tyrosine kinase

2-Substituted Aminopyrido[2,3-d]pyrimidin-7-yl Ureas
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1923
After standing at room temperature for 1 h, the suspension
was filtered and the precipitated crude product was washed
sequentially with H2O, CH3CN, and Et2O. Drying the solid in
vacuo at 60 °C overnight gave 18 (3.62 g, 63% yield, mp 284
°C dec): 1H NMR (DMSO-d6) δ 6.58 (br s, 2H), 6.73 (br s, 2H),
7.29 (t, J ) 8.2 Hz, 1H), 7.54 (s, 1H), 7.78 (d, J ) 8.2 Hz, 2H),
8.64 (s, 1H); CIMS m/ z 422 (M + C2H5)+, 394 (M + H)+. Anal.
(C13H9N5Br2) C, H, N.
APCIMS m/ z 489 (M + H)+. Anal. (C22H26N8Cl2O) C, H, N,
Cl, H2O.
Meth od E. 3-{6-(2,6-Dich lor o-p h en yl)-2-[3-(4-m eth yl-
p ip er a zin -1-yl)-p r op yla m in o]-p yr id o[2,3-d ]p yr im id in -7-
yl}-1,1-d ieth yl-u r ea (60). A solution of pyridopyrimidine 4614
(0.50 g, 1.12 mmol) in DMF (5 mL) was treated with NaH
(0.059 g, 2.46 mmol). After being stirred at room temperature
for 1 h, the suspension was cooled to 0 °C and diethylcarbamyl
chloride (0.15 mL, 1.20 mmol) was added dropwise. The bath
was removed, and the reaction mixture was stirred at room
temperature for 18 h and then concentrated in vacuo. Purifica-
tion of the residue by medium-pressure chromatography,
eluting with a solution of EtOAc/MeOH/Et3N (90:10:1), gave
60 (0.46 g, 75% yield, mp 66 °C dec): 1H NMR (CDCl3) δ 0.72-
0.89 (m, 3H), 1.08-1.22 (m, 3H), 1.68-1.92 (m, 2H), 2.17-
2.94 (m, 12H), 3.00-3.19 (m, 2H), 3.22-3.42 (m, 2H), 3.43-
3.67 (m, 2H), 6.19-6.39 (m, 1H), 7.13-7.49 (m, 5H), 8.30-
8.47 (m, 1H), 13.80-14.11 (m, 1H); CIMS m/ z 504 (M + H)+.
Anal. (C26H34N8Cl2O‚1.0H2O‚0.3EtOAc) C, H, N.
N-{6-(2,6-Dich lor o-p h en yl)-2-[3-(4-m et h yl-p ip er a zin -
1-yl)-p r op yla m in o]-p yr id o[2,3-d ]p yr im id in -7-yl}-3,3-d i-
m eth yl-bu tyr a m id e (77). Using the procedure described for
the synthesis of 60, reaction of the sodium salt of pyridopyri-
midine 4614 and tert-butyl acetyl chloride for 48 h followed by
medium-pressure chromatography, eluting with a solution of
EtOAc/MeOH/Et3N (9:2:1), gave 77 (0.17 g, 14% yield, mp 84-
100 °C dec): 1H NMR (DMSO-d6) δ 0.78 (s, 9H), 1.72-1.80
(m, 2H), 2.08 (s, 2H), 2.15 (s, 3H), 2.21-2.54 (m, 8H), 3.19-
3.50 (m, 3H), 7.19-7.40 (m, 1H), 7.49-7.54 (m, 2H), 7.90-
7.99 (m, 1H), 8.11 (s, 1H), 9.09 (s, 1H), 10.17-10.29 (m, 2H);
ESMS m/ z 544.4 (M + H)+. Anal. (C27H35N7Cl2O‚0.4H2O‚
0.6EtOH) C, H, N, Cl.
Meth od F. N-[6-(2,6-Dich lor o-ph en yl)-2-(3-dieth ylam in o-
p r op yla m in o)-p yr id o[2,3-d ]p yr im id in -7-yl]-3,3-d im eth yl-
bu tyr a m id e (71). To a suspension of 1214 (42.0 mg, 0.20
mmol) in p-dioxane (0.5 mL) was added tert-butyl acetyl
chloride (34.0 mg, 0.25 mmol). The reaction was heated at
reflux for 1 h then concentrated in vacuo to a semisolid. This
residue was dissolved in CH2Cl2, washed with cold 5% aque-
ous NaOH, dried (MgSO4), and concentrated to a thick oil.
Medium-pressure chromatography, eluting with a solution of
EtOAc/MeOH/Et3N (90:10:1), gave 71 (30.0 mg, 58% yield, mp
82 °C dec): 1H NMR (DMSO-d6) δ 0.58-1.13 (m, 15H), 1.60-
1.83 (m, 2H), 1.97-2.13 (m, 2H), 2.29-2.71 (m, 4H), 3.14-
3.56 (m, 4H), 7.28-7.42 (m, 1H), 7.46-7.60 (m, 2H), 7.90-
8.03 (m, 1H), 8.11 (s, 1H), 9.08 (s, 1H), 10.17-10.30 (m, 1H);
CIMS m/ z 517 (M + H)+. Anal. (C26H34N6Cl2O‚0.6H2O‚
0.4EtOAc) C, H, N.
Meth od G. N′-[6-(2,6-Dich lor op h en yl)-2-{3-(d ieth yla m i-
n o)p r op yla m in o}p yr id o[2,3-d ]p yr im id in -7-yl]-N,N-d im -
eth ylfor m a m id in e (72). To a suspension of pyridopyrimidine
1214 (210 mg, 1.00 mmol) in DMF (0.80 mL) was added DMF
dimethyl acetal (0.80 mL). The mixture was stirred at room
temperature for 5.5 h and then concentrated in vacuo. The
residual oil was partitioned between CH2Cl2 and H2O. The
organic phase was dried (MgSO4) and then concentrated to a
glass that was crystallized from CH3CN to give 72 (160 mg,
68% yield, mp 100-104 °C): 1H NMR (CDCl3) δ 1.04 (t, J )
7.0 Hz, 6 H), 1.81 (t, J ) 6.4 Hz, 2 H), 2.60-2.52 (m, 6 H),
2.80, (s, 3 H), 3.08 (s, 3 H), 3.68 (br t, 1 H), 6.55 (br s, 1 H),
7.20 (t, J ) 8 Hz, 1 H), 7.36 (d, J ) 8 Hz, 2 H), 7.62 (s, 1 H),
8.73 (s, 1 H), 8.82 (s, 1 H); CIMS m/ z 474 (M + H)+. Anal.
(C23H29N7Cl2‚0.4H2O) C, H, N.
Meth od B. 1-[2-Am in o-6-(2,6-d ibr om o-p h en yl)-p yr id o-
[2,3-d ]p yr im id in -7-yl]-3-ter t-bu tyl-u r ea (19). A suspension
of diamine 18 (0.25 g, 0.63 mmol) in DMF (5 mL) was treated
with NaH (0.017 g, 0.73 mmol). After the mixture was stirred
at room temperature for 1 h, tert-butyl isocyanate (0.077 mL,
0.68 mmol) was added, and the reaction was stirred at room
temperature for 18 h. The insoluble salts were removed by
filtration and rinsed with DMF. The filtrate was concentrated
in vacuo and H2O added to the wet residue to give a yellow
precipitate. Purification of the precipitate by medium-pressure
chromatography, eluting with a solution of EtOAc/CHCl3 (1:
1), gave 19 (0.18 g, 58% yield, mp > 300 °C dec): 1H NMR
(DMSO-d6) δ 1.39 (s, 9H), 7.27 (br s, 2H), 7.33-7.44 (s, 1H),
7.48 (s, 1H), 7.79-7.90 (m, 2H), 7.93 (s, 1H), 8.94 (s, 1H), 10.17
(s, 1H); CIMS m/ z 495 (M + H)+. Anal. (C18H18N6Br2O‚0.4H2O)
C, H, N, Br.
1-ter t-Bu tyl-3-[6-(2,6-d ich lor o-p h en yl)-2-(2-d ieth yla m i-
n o-eth yla m in o)-p yr id o[2,3-d ]p yr im id in -7-yl]-u r ea (28).
To a solution of pyridopyrimidine 26 (1.00 g, 2.47 mmol) in
DMF (10 mL) was added NaH (0.059 g, 2.47 mmol). After the
mixture was stirred for 1 h at room temperature, tert-butyl
isocyanate (0.244 g, 2.47 mmol) was added. After being stirred
for 1 h, the reaction mixture was concentrated in vacuo and
the residue partitioned between EtOAc and H2O. The aqueous
layer was extracted three times with EtOAc. The combined
EtOAc layers were washed with a saturated solution of NaCl,
dried (MgSO4), and concentrated. Chromatography of the
residue, eluting with a solution of EtOAc/EtOH/Et3N (18:2:1),
gave 28 (0.76 g, 61% yield, mp 94.5-96.5 °C): 1H NMR
(DMSO-d6) δ 0.99 (t, J ) 7.1 Hz, 6H), 1.39 (s, 9H), 2.49-2.58
(m, 4H), 2.58-2.68 (m, 2H), 3.43-3.53 (m, 2H), 7.50-7.58 (m,
1H), 7.62-7.69 (m, 2H), 7.69-7.82 (br m, 2H), 7.96 (s, 1H),
8.90 (s, 1H), 10.45 (s, 1H); CIMS m/ z 504 (M + H)+. Anal.
(C24H31N7Cl2O) C, H, N.
Meth od C. N2-(3-Dieth yla m in o-pr op yl)-6-(2,6-d im eth yl-
p h en yl)-p yr id o[2,3-d ]p yr im id in e-2,7-d ia m in e (16). A mix-
ture of diamine 1414 (3.00 g, 11.31 mmol), sulfamic acid (2.20
g, 22.61 mmol), and 3-(diethylamino)propylamine (30 mL) was
heated at reflux for 18 h. The cooled reaction mixture was
poured into 500 mL of ice H2O. The resultant precipitate was
filtered and washed with water. Crystallization of the crude
product from EtOAc gave 16 (3.50 g, 80% yield, mp 216-219
°C): 1H NMR (DMSO-d6) δ 0.85-1.08 (t, 6H), 1.57-1.80 (m,
2H), 2.02 (s, 6H), 2.33-2.61 (m, 6H), 3.22-3.47 (m, 4H), 7.07-
7.22 (m, 4H), 7.46 (s, 1H), 8.60 (br s, 1H); ESMS m/ z 379.5
(M + H)+. Anal. (C22H30N6‚0.2H2O) C, H, N.
Meth od D. {6-(2,6-Dich lor o-p h en yl)-2-[3-(4-m eth yl-p ip -
er a zin -1-yl)-p r op yla m in o]-p yr id o[2,3-d ]p yr im id in -7-yl}-
u r ea (50). Into a suspension of pyridopyrimidine 4614 (1.00 g,
2.24 mmol) in dry dioxane (6 mL) was added 1,1′-carbonyldi-
imidazole (0.44 g, 2.69 mmol). After heating at reflux over-
night, the reaction was cooled to room temperature and poured
into a saturated solution of NH3 gas in dry dioxane. The
reaction was concentrated to a residue that was partitioned
between EtOAc and H2O. The EtOAc layer was washed twice
each with H2O and brine, dried (MgSO4), and concentrated.
The crude product was purified by chromatography, eluting
first with a solution of EtOAc/EtOH/Et3N (9:2:1) then EtOAc/
EtOH/Et3N (9:3:1). Further purification of the product by
crystallization from EtOAc/EtOH, followed by drying the
product at 55 °C in vacuo, gave 50 (0.35 g, 32% yield, mp 166-
168 °C): 1H NMR (DMSO-d6) δ 1.64-1.82 (m, 2H), 2.07-2.60
(m, 13H), 3.36-3.49 (m, 2H), 7.36 (br s, 1H), 7.49-7.57 (m,
1H), 7.60-7.68 (m, 2H), 7.85-7.93 (m, 1H), 7.96-8.11 (m, 2H),
8.91(s) + 9.00 (s) - total integration 1H, 9.42-9.61 (m, 1H);
Meth od H. 1-[6-(2,6-Dich lor o-ph en yl)-2-(3-dieth ylam in o-
p r op yla m in o)-p yr id o[2,3-d ]p yr im id in -7-yl]-3-eth yl-th io-
u r ea (73). To a solution of 1214 (420 mg, 1.00 mmol) in DMF
(5 mL) was added NaH (0.034 g, 1.40 mmol). After the mixture
was stirred at room temperature for 0.5 h, ethyl isothiocyanate
(113 mg, 1.20 mmol) was added. The resulting mixture was
stirred for 16 h, poured into a saturated aqueous solution of
NaHCO3, extracted three times with CH2Cl2, dried (Na2SO4),
and concentrated. Chromatography of the residue, eluting with
a solution of EtOAc/MeOH/Et3N (85:15:2), gave 73 (362 mg,
77% yield, mp 172-173 °C): 1H NMR (CHCl3/D2O) δ 0.99 (t,

1924 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Schroeder et al.
J ) 7.3 Hz, 3H), 1.02 (t, J ) 6.0 Hz, 6 H), 1.18 (t, J ) 5.8 Hz,
2H), 1.76-1.85 (m, 2H), 2.46-2.59 (m, 6H), 3.63 (q, J ) 7.0
Hz, 2H), 3.75-3.81 (m, 2H), 7.43 (t, J ) 8.9 Hz, 1H), 7.68 (d,
J ) 8.6 Hz, 2H) 7.56 (s, 1H), 8.70 (s, 1H); APCIMS m/ z 506
(M + H)+. Anal. (C23H29N7Cl2S‚0.3H2O) C, H, N.
(PDGFr-â,26 FGFr-1,26 EGFr,26 c-Src,26 MEK/ERK,31 IR,32
CDK4,33 and PKC34).
P DGF Recep tor Au top h osp h or yla tion . The assay in rat
aortic vascular smooth muscle cells was carried out as previ-
ously described.26
Meth od I. N-[6-(2,6-Dich lor o-ph en yl)-2-(3-dieth ylam in o-
pr opylam in o)-pyr ido[2,3-d]pyr im idin -7-yl-N′′-eth yl-gu an i-
d in e (74). To a stirred solution of 1214 (419 mg, 1.00 mmol)
in DMF (1 mL) was added NaH (52.0 mg, 1.30 mmol). After
the mixture was stirred for 0.5 h at room temperature, N,N′-
bis(tert-butoxycarbonyl)-N-(ethyl)-S-(ethyl)isothiourea20 (360
mg, 1.10 mmol) was added in one portion. After being stirred
for 18 h, the reaction mixture was diluted with CH2Cl2, washed
twice with H2O, dried (Na2SO4), and concentrated. The result-
ing oil was chromatographed, eluting with a solution of EtOAc/
MeOH/Et3N (85:15:2), to afford a 1:2 mixture of 12 and
carbamic acid [[[6-(2,6-dichlorophenyl)-2-(3-diethylamino-pro-
pylamino)-pyrido[2,3-d]pyrimidin-7-yl]imino[[1,1-dimethyl-
ethoxy)carbonyl]amino]methyl]ethylamino]-1,1-dimethyleth-
yl ester. A portion of this mixture was dissolved in anhydrous
CH2Cl2 (0.5 mL) containing 2,6-lutidine (65.0 mg, 0.60 mmol).
Trimethylsilyl trifluomethanesulfonate (100 mg, 0.440 mmol)
was added, and the mixture was stirred at room temperature
for 30 h. The mixture was then poured into a saturated
aqueous solution of NaHCO3, extracted with CH2Cl2, dried
(Na2SO4), and concentrated. Chromatography of the resulting
oil by preparative HPLC, eluting with a linear gradient of
100/0 A/B to 63/37 A/B (where A is 0.1% aqueous TFA and B
is 0.1% TFA in CH3CN), gave the tris-TFA salt of 74 (26 mg,
4% yield): 1H NMR (CDCl3/D2O) δ 1.27 (t, J ) 7.3 Hz, 6H),
2.14-2.21 (m, 2H), 3.01-3.22 (m, 6H), 3.36-3.42 (m, 2H), 7.31
(t, J ) 9.9 Hz, 1H), 7.85 (d, J ) 10.1 Hz, 2H), 7.90 (s, 1H),
9.02 (s, 1H); APCIMS m/ z 490 (M + H)+; Anal. (C23H30N8Cl2‚
1.0H2O‚3.0TFA) C, H, N.
N-{6-(2,6-Dich lor o-p h en yl)-2-[3-(4-m eth yl-p ip er a zin -1-
yl)-p r op yla m in o]-p yr id o[2,3-d ]p yr im id in -7-yl}-N′,N′′-d i-
isop r op yl-gu a n id in e (81). To a solution of pyridopyrimidine
4614 (100 mg, 0.220 mmol) in DMF (2 mL) was added NaH
(7.0 mg, 0.29 mmol) in one portion. After the mixture was
stirred at room temperature for 0.5 h, N,N′-diisopropylcarbo-
diimide (43 mg, 0.34 mmol) was added, and the resulting
mixture was stirred for 16 h. The reaction was poured into a
saturated aqueous solution of NaHCO3, extracted three times
with CH2Cl2, dried (Na2SO4), and concentrated. Chromatog-
raphy of the residue, eluting with a solution of EtOAc/MeOH/
Et3N (85:15:2), gave 81 (102 mg, 81%). An analytically pure
sample was obtained as the tetra-HCl salt following crystal-
lization from acidic 2-propanol (mp 229 °C dec): 1H NMR
(CDCl3/D2O) δ 1.06 (d, 3H), 1.09 (s, 3H), 2.22 (s, 3H) 2.31-
2.58 (m, 10H), 3.50 (t, J ) 7.1 Hz, 2H), 3.56 (q, J ) 6.1 Hz,
2H), 7.16 (d, J ) 8 Hz, 4H), 7.29 (d, J ) 8 Hz, 4H) 7.42 (s,
1H), 8.53 (s, 1H); APCIMS m/ z 572.2 (M + H)+. Anal.
(C28H39N9Cl2‚1.0H2O‚4.0HCl) C, H, N.
Cell Cu ltu r e. Previously established literature procedures
were utilized to perform the growth delay assay using rat
aortic smooth muscle cells26 and the tumor cellular growth
delay assay using the cell lines described in Table 6.27 Briefly,
for the tumor lines, cells were seeded into 96-well tissue
culture plates and incubated for 24 h to allow for cell
attachment. One hundred microliters of drug dilutions in
culture media were added to the first well in duplicate rows
followed by serial 2-fold dilution across the plate. Plates were
incubated at 37 °C in 95% humidity and 5% CO2 for 4 and 3
days for the SW-620, HCT-8, and HT-29 lines. Cell growth was
determined by staining cells with sulforhodamine B and
reading plates on a Molecular Devices Thermax microplate
reader (Sunnyvale, CA).
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 six in vivo tumor models listed in Table 7 were
established from tissue culture cell lines and maintained by
serial passage in nude mice (NCr nu/nu). Nude mice were also
used as tumor hosts for anticancer agent evaluations against
these tumor models.
In each experiment for anticancer activity evaluation, test
mice weighing 18-22 g were 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 on the days indicated in the tables. The
vehicle for the hydrochloride salt of 32 was sterile water for
injection. 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.35-38 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. We gratefully acknowledge the
assistance of Michelle Spencer, Aneesa M. Amar, Brian
G. Hartl, J ames M. Nelson, and Paul R. Keller for
carrying out enzyme assays, Anthony Blackburn and
Lanny Mihardja for solubilty determinations, and Wayne
D. Klohs and David W. Fry for helpful discussions.
Met h od J . N-{6-(2,6-Dich lor o-p h en yl)-2-[3-(4-m et h yl-
p ip er a zin -1-yl)-p r op yla m in o]-p yr id o[2,3-d ]p yr im id in -7-
yl}-ben zen esu lfon a m id e (79). A suspension of pyridopyri-
midine 4614 (1.00 g, 2.24 mmol) in p-dioxane (5 mL) was
treated with benzenesulfonyl chloride (0.36 mL, 2.80 mmol)
and heated at reflux for 18 h. The reaction mixture was
concentrated in vacuo. The resulting residue was dissolved in
9:1 CH2Cl2/MeOH, and the solution was washed with cold 5%
aqueous NaOH, dried (MgSO4), and concentrated to leave a
dark foam. Purification by preparative HPLC, eluting with a
linear gradient of 100/0 A/B to 60/40 A/B (where A is 0.1%
aqueous TFA and B is 0.1% TFA in CH3CN), followed by
lyophilization gave the tris-TFA salt of 79 (0.38 g, 18% yield):
1H NMR (DMSO-d6) δ 1.75-2.04 (m, 2H), 2.17-4.44 (m, 15H),
7.28-7.58 (m, 8H), 7.99-8.18 (m, 1H), 8.30-8.60 (m, 1H),
8.76-8.96 (m, 1H), 11.54-12.10 (m, 1H); APCIMS m/ z 586.4
(M + H)+. Anal. (C27H29N7Cl2O2S‚0.5H2O‚3.0TFA) C, H, N.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails and spectral data are available for compounds 17, 20,
21, 26, 27, 29, 30, 32, 34-45, 48, 49, 51-59, 61-70, 75, 76,
78, 82, and 83. This material is available free of charge via
the Internet at http://pubs.acs.org.
Refer en ces
(1) (a) Levitzki, A.; Gazit, A. Tyrosine Kinase Inhibition: An
Approach to Drug Development. Science 1995, 267, 1782-1788.
(b) Traxler, P. M. Protein Tyrosine Kinase Inhibitors in Cancer
Treatment. Expert Opin. Ther. Pat. 1997, 7, 571-588.
(2) Saltiel, A. R. Signal Transduction Pathways as Drug Targets.
Sci. Am. Sci. Med. 1996, 2, 58-67.
(3) (a) Shawver, L. K.; Lipson, K. E.; Fong, A. T.; McMahon, G.;
Plowman, G. D.; Strawn, L. M. Receptor Tyrosine Kinases as
Targets for Inhibition of Angiogenesis. Drug Discovery Today
1997, 2, 50-63. (b) Folkman, J . Angiogenesis in Cancer,
Vascular, Rheumatoid and Other Diseases. Nat. Med. 1995, 1,
27-31.
Recom bin a n t Tyr osin e Kin a ses a n d Assa ys. The meth-
ods for production of the tyrosine kinases used in this study
and assay conditions for each have been previously described

2-Substituted Aminopyrido[2,3-d]pyrimidin-7-yl Ureas
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1925
(4) (a) Mattsson, E.; Clowes, A. W. Current Concepts in Restenosis
Following Balloon Angioplasty. Trends Cardiovasc. Med. 1995,
5, 200-204. (b) Shaw, L. A.; Rudin, M.; Cook, N. S. Pharmaco-
logical Inhibition of Restenosis: Learning from Experience.
Trends Pharm. Sci. 1995, 16, 401-404.
(5) Raines, E. W.; Ross, R. Multiple Growth Factors are Associated
with Lesions of Atherosclerosis: Specificity or Redunancy?
BioEssays 1996, 18, 271-282.
(6) Bridges, A. J . The Epidermal Growth Factor Receptor Family
of Tyrosine Kinases and Cancer: Can an Atypical Exemplar be
a Sound Therapeutic Target? Curr. Med. Chem. 1996, 3, 167-
194.
(7) Hanks, S. K.; Quinn, A. M.; Hunter, T. The Protein Kinase
Family: Conserved Features and Deduced Phylogeny of the
Catalytic Domains. Science 1988, 241, 42-52.
(12) (a) Maly, D. J .; Choong, I. C.; Ellman, J . A. Combinatorial Target-
Guided Ligand Assembly: Identification of Potent Subtype-
Selective c-Src Inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2000,
97, 2419-2424. (b) Moasser, M. M.; Srethapakdi, M.; Sachar,
K. S.; Kraker, A. J .; Rosen, N. Inhibition of Src Kinases by a
Selective Tyrosine Kinase Inhibitor Causes Mitotic Arrest.
Cancer Res. 1999, 59, 6145-6152. (c) Kraker, A. J .; Hartl, B.
G.; Amar, A. M.; Barvian, M. R.; Showalter, H. D. H.; Moore, C.
W. Biochemical and Cellular Effects of c-Src Kinase Selective
Pyrido[2,3-d]pyrimidine Tyrosine Kinase Inhibitors. Biochem.
Pharm. 2000, 60, 885-898
(13) Dahring, T. K.; Lu, G. H.; Hamby, J . M.; Batley, B. L.; Kraker,
A. J .; Panek, R. L. Inhibition of Growth Factor-Mediated
Tyrosine Phosphorylation in Vascular Smooth Muscle by PD
089828, a New Synthetic Protein Tyrosine Kinase Inhibitor. J .
Pharmacol. Exp. Ther. 1997, 281, 1446-1456.
(8) (a) Ciardiello, F.; Caputo, R.; Bianco, R.; Damiano, V.; Pomatico,
G.; De Placido, S.; Bianco, R. A.; Tortora, G. Antitumor Effect
and Potentiation of Cytotoxic Drugs Activity in Human Cancer
Cells by ZD-1839 (Iressa), an Epidermal Growth Factor Recep-
tor-Selective Tyrosine Kinase Inhibitor. Clin. Cancer Res. 2000,
6, 2053-2063. (b) Smaill, J . B.; Rewcastle, G. W.; Bridges, A.
J .; Zhou, H.; Showalter, H. D. H.; Fry, D. W.; Nelson, J . M.;
Sherwood, V.; Elliott, W. L.; Vincent, P. W.; DeJ ohn, D. E.; Loo,
J . A.; Greis, K. D.; Chan, O. H.; Reyner, E. L.; Lipka, E.; Denny
W. A. Tyrosine Kinase Inhibitors. 17. Irreversible Inhibitors of
the Epidermal Growth Factor Receptor: 4-(Phenylamino)-
quinazoline- and 4-(Phenylamino)pyrido[3,2-d]pyrimidine-6-
acrylamides Bearing Additional Solubilizing Functions. J . Med.
Chem. 2000, 43, 1380-1397.
(9) (a) Panek, R. L.; Lu, G. H.; Dahring, T. K.; Batley, B. L.;
Connolly, C.; Hamby, J . M.; Brown, K. J . In Vitro Biological
Characterization and Antiangiogenic Effects of PD 166866, a
Selective Inhibitor of the FGF-1 Receptor Tyrosine Kinase. J .
Pharmacol. Exp. Ther. 1998, 286, 569-577. (b) Barvian, M. R.;
Panek, R. L.; Lu, G. H.; Kraker, A. J .; Amar, A.; Hartl, B.;
Hamby, J . M.; Showalter, H. D. H. 1-Oxo-3-aryl-1H-indene-2-
carboxylic Acid Derivatives as Selective Inhibitors of Fibroblast
Growth Factor Receptor-1 Tyrosine Kinase. Bioorg. Med. Chem.
Lett. 1997, 7, 2903-2908. (c) Dimitroff, C. J .; Klohs, W.; Sharma,
A.; Pera, P.; Driscoll, D.; Veith, J .; Steinkampf, R.; Schroeder,
M.; Klutchko, S.; Sumlin, A.; Henderson, B.; Dougherty, T. J .;
Bernacki, R. J . Anti-Angiogenic Activity of Selected Receptor
Tyrosine Kinase Inhibitors, PD166285 and PD173074: Implica-
tions for Combination Treatment with Photodynamic Therapy.
Invest. New Drugs 1999, 17, 121-135.
(10) (a) Boschelli, D. H.; Wu, Z.; Klutchko, S. R.; Showalter, H. D.
H.; Hamby, J . M.; Lu, G. H.; Major, T. C.; Dahring, T. K.; Batley,
B.; Panek, R. L.; Keiser, J .; Hartl, B. G.; Kraker, A. J .; Klohs,
W. D.; Roberts, B. J .; Patmore, S.; Elliott, W. L.; Steinkampf,
R.; Bradford, L. A.; Hallak, H.; Doherty, A. M. 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 In-
hibitors. J . Med. Chem. 1998, 41, 4365-4377. (b) Palmer, B. D.;
Smaill, J . B.; Boyd, M.; Boschelli, D. H.; Doherty, A. M.; Hamby,
J . M.; Khatana, S. S.; Kramer, J . B.; Kraker, A. J .; Panek, R.
L.; Lu, G. H.; Dahring, T. K.; Winters, R. T.; Showalter, H. D.
H.; Denny, W. A. Structure-Activity Relationships for 1-Phen-
ylbenzimidazoles as Selective ATP Site inhibitors of the Platelet-
Derived Growth Factor Receptor. J . Med. Chem. 1998, 41, 5457-
5465.
(11) (a) Mendel, D. B.; Laird, A. D.; Smolich, B. D.; Blake, R. A.;
Liang, C.; Hannah, A. L.; Shaheen, R. M.; Ellis, L. M.; Weitman,
S.; Shawver, L. K.; Cherrington, J . M. Development of SU5416,
a Selective Small Molecule Inhibitor of VEGF Receptor Tyrosine
Kinase Activity, as an Anti-Angiogenesis Agent. Anti-Cancer
Drug Des. 2000, 15, 29-41. (b) Hennequin, L. F.; Thomas, A.
P.; J ohnstone, C.; Stokes, E. S. E.; Ple, P. A.; Lohmann, J .-J .
M.; Ogilvie, D. J .; Dukes, M.; Wedge, S. R.; Curwen, J . O.;
Kendrew, J.; Lambert-van der Brempt, C. Design and Structure-
Activity Relationship of a New Class of Potent VEGF Receptor
Tyrosine Kinase Inhibitors. J . Med. Chem. 1999, 42, 5369-5389.
(c) Fong, T. A. T.; Shawver, L. K.; Sun, L.; Tang, C.; App, H.;
Powell, T. J .; Kim, Y. H.; Schreck, R.; Wang, X.; Risau, W.;
Ullrich, A.; Hirth, K. P.; McMahon, G. SU5416 is a Potent and
Selective Inhibitor of the Vascular Endothelial Growth Factor
Receptor (Flk-1/KDR) that Inhibits Tyrosine Kinase Catalysis,
Tumor Vascularization, and Growth of Multiple Tumor Types.
Cancer Res. 1999, 59, 99-106. (d) Bold, G.; Altmann, K.-H.; Frei,
J .; Lang, M.; Manley, P. W.; Traxler, P.; Wietfeld, B.; Brueggen,
J .; Buchdunger, E.; Cozens, R.; Ferrari, S.; Furet, P.; Hofmann,
F.; Martiny-Baron, G.; Mestan, J .; Roesel, J .; Sills, M.; Stover,
D.; Acemoglu, F.; Boss, E.; Emmenegger, R.; Laesser, L.; Masso,
E.; Roth, R.; Schlachter, C.; Vetterli, W.; Wyss, D.; Wood, J . M.
New Anilinophthalazines as Potent and Orally Well Absorbed
Inhibitors of the VEGF Receptor Tyrosine Kinases Useful as
Antagonists of Tumor-Driven Angiogenesis. J . Med. Chem. 2000,
43, 2310-2323.
(14) Hamby, J . M.; Connolly, C. J . C.; Schroeder, M. C.; Winters, R.
T.; Showalter, H. D. H.; Panek, R. L.; Major, T. C.; Olsewski,
B.; Ryan, M. J .; Dahring, T.; Lu, G. H.; Keiser, J .; Amar, A.;
Shen, C.; Kraker, A. J .; Slintak, V.; Nelson, J . M.; Fry. D. W.;
Bradford, L.; Hallak, H.; Doherty, A. M. Structure-Activity
Relationships for
a Novel Series of Pyrido[2,3-d]pyrimi-
dine Tyrosine Kinase Inhibitors. J . Med. Chem. 1997, 40, 2296-
2303.
(15) Klutchko, S. R.; Hamby, J . M.; Boschelli, D. H.; Wu, Z.; Kraker,
A. J .; Amar, A. M.; Hartl, B. G.; Shen, C.; Klohs, W. D.;
Steinkampf, R. W.; Driscoll, D. L.; Nelson, J . M.; Elliott, W. L.;
Roberts, B. J .; Stoner, C. L.; Vincent, P. W.; Dykes, D. J .; Panek,
R. L.; Lu, G. H.; Major, T. C.; Dahring, T. K.; Hallak, H.;
Bradford, L. A.; Showalter, H. D. H.; Doherty, A. M. 2-Substi-
tuted Aminopyrido[2,3-d]pyrimidin-7(8H)-ones. Structure-Ac-
tivity Relationships Against Selected Tyrosine Kinases and in
Vitro and in Vivo Anticancer Activity. J . Med. Chem. 1998, 41,
3276-3292.
(16) Thompson A. M.; Connolly, C. J . C.; Hamby, J . M.; Boushelle,
S.; Hartl, B. G.; Amar,, A. M.; Kraker, A. J .; Driscoll, D. L.;
Steinkampf, R. W.; Patmore, S. J .; Vincent, P. W.; Roberts, B.
J .; Elliott, W. L.; Klohs, W.; Showalter, H. D. H.; Denny, W. A.
3-(3,5-Dimethoxyphenyl)-1,6-naphthyridine-2,7-diamines and Re-
lated 2-Urea Derivatives are Potent and Selective Inhibitors of
the FGF-1 Receptor Tyrosine Kinase, J . Med. Chem. 2000, 43,
4200-4211.
(17) Thompson, A. M.; Rewcastle, G. W.; Boushelle, S. L.; Hartl, B.
G.; Kraker, A. J .; Lu, G. H.; Batley, B. L.; Panek, R. L.;
Showalter, H. D. H.; Denny, W. A. Synthesis and Structure-
Activity Relationships of 7-Substituted 3-(2,6-Dichlorophenyl)-
1,6-naphthyridin-2(1H)-ones as Selective Inhibitors of pp60c-src
J . Med. Chem. 2000, 43, 3134-3147.
.
(18) A preliminary account of this work has been presented, cf.
Schroeder, M. C.; Hamby, J . M.; Grohar, P. J .; Winters, R. T.;
Connolly, C. J . C.; Showalter, H. D. H.; Kraker, A. J .; Amar, A.
M.; Shen, C.; Lu, G. H.; Dahring, T. K.; Panek, R. L.; Major, T.
C.; Doherty, A. M. The Synthesis and SAR of Pyrido[2,3-d]-
pyrimidine-2,7-diamines in the Development of Potent Water
Soluble Tyrosine Kinase Inhibitors. Book of Abstracts; 25th
National Medicinal Chemistry Symposium, 1996, Abstract
No. 4.
(19) Davoll, J . Heterocyclic Amine Compounds and Methods for Their
Production. U. S. Patent 3 534 039, 1970.
(20) (a) Lammin, S. G.; Pedgrift, B. L.; Ratcliffe, A. J . Conversion of
Anilines to Bis-Boc Protected N-Methylguanidines. Tetrahedron
Lett. 1996, 37, 6815-6818. (b) Poss, M. A.; Iwanowicz, E.; Reid,
J . A.; Lin, J .; Gu, Z. A Mild and Efficient Method for the
Preparation of Guanidines. Tetrahedron Lett. 1992, 33, 5933-
5936.
(21) Barvian, M. R.; Showalter, H. D. H.; Doherty, A. M. Preparation
of N, N-Bis(aryl)gaunidines from Electron Deficient Amines
Via Masked Cabodiimides. Tetrahedron Lett. 1997, 38, 6799-
6802.
(22) Mohammadi, M.; Froum, S.; Hamby, J . M.; Schroeder, M. C.;
Panek, R. L.; Lu, G. H.; Eliseenkova, A. V.; Green, D.; Schlessing-
er, J .; Hubbard, S. R. Crystal Structure of an Angiogenesis
Inhibitor Bound to the FGF Receptor Tyrosine Kinase Domain.
EMBO J . 1998, 17, 5896-5904.
(23) Trumpp-Kallmeyer, S.; Rubin, J . R.; Humblet, C.; Hamby, J . M.;
Showalter, H. D. H. Development of a Binding Model to Protein
Tyrosine Kinases for Substituted Pyrido[2,3-d]pyrimidine In-
hibitors. J . Med. Chem. 1998, 41, 1752-1763.
(24) Raevsky, O. A.; Grigor’ev, V. Y.; Kireev, D. B.; Zefirov, N. S.
Complete Thermodynamic Description of H-Bonding in the
Framework of Multiplicative Approach. Quant. Struct.-Act.
Relat. 1992, 11, 49-63.
(25) Leahy, D. E.; Morris, J . L.; Taylor, P. J .; Wait, A. R. Model
Solvent Systems for QSAR. Part 3. An LSER Analysis of
the ‘Critical Quartet.’ New Light on Hydrogen Bond Strength
and Directionality. J . Chem. Soc., Perkin Trans. 2 1992, 705-
722.

1926 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Schroeder et al.
(26) Panek, R. L.; Lu, G. H.; Klutchko, S. R.; Batley, B. L.; Dahring.
T. K.; Hamby, J . M.; Hallak, A.; Doherty, A. M.; Keiser, J . A. In
Vitro Pharmacological Characterization of PD 166285, a New
Nanomolar Potent and Broadly Active Protein Tyrosine Kinase
Inhibitor. J . Pharmacol. Exp. Ther. 1997, 283, 1433-1444.
(27) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J . T.; Bokesch, H.; Kenney, S.; Boyd, M. R.;
New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screen-
ing. J . Natl. Cancer Inst. 1990, 82, 1107-1112.
(33) Connolly, C. J . C.; Hamby, J . M.; Schroeder, M. C.; Barvian, M.;
Lu, G. H.; Panek, R. L.; Amar, A.; Shen, C.; Kraker, A. J .; Fry,
D. W.; Klohs, W. D.; Doherty, A. M. Discovery and Structure-
Activity Studies of a Novel Series of Pyrido[2,3-d]pyrimidine
Tyrosine Kinase Inhibitors. Bioorg. Med. Chem. Lett. 1997, 7,
2415-2420.
(34) Martiny-Baron, G.; Kazanietz, M. G.; Mischak, H.; Blumberg,
P. M.; Kochs, G.; Hug, H.; Marme, D.; Schachtele, C. Selective
Inhibition of Protein Kinase C Isozymes by the Indolcarbazole
Goe 6976. J . Biol. Chem. 1993, 268, 9194-9197.
(35) Schable, F. M., J r.; Griswold, D. P., J r.; Laster, W. R., J r.;
Corbett, T. H.; Lloyd, H. H. Quantitative Evaluation of Anti-
cancer Agent Activity in Experimental Animals. Pharmacol.
Ther. 1977, 1, 411-435.
(36) Elliot, W. L.; Howard, C. T.; Dykes, D. J .; Leopold, W. R.
Sequence and Schedule-Dependent Synergy of Trimetrexate in
Combination With 5-Fluorouracil in Vitro and in Mice. Cancer
Res. 1989, 49, 5586-5590.
(37) Elliot, W. L.; Roberts, B. J .; Howard, C. T.; Leopold, W. R.
Chemotherapy with CI-973 ([SP-4-3-(R)]-[1,1-cyclobutanedicar-
boxylato (2-)] (2-methyl-1,4-butanediamine-N,N′)platinum, NK121)
in Combination with Standard Agents Against Murine Tumors
in Vivo. Cancer Res. 1994, 54, 4412-4418.
(38) Leopold, W. R.; Nelson, J . M.; Plowman, J .; J ackson, R. C.
Anthrapyrazoles, a New Class of Intercalating Agents with High-
Level, Broad Spectrum Activity Against Murine Tumors. Cancer
Res. 1985, 45, 5532-5539.
(28) Budde, R. J . A.; Ke, S.; Levin, V. A. Activity of pp60c-src in 60
Different Cell Lines Derived from Human Tumors. Cancer
Biochem. Biophys. 1994, 14, 171-175.
(29) (a) Osherov, N.; Levitzki, A. Epidermal Growth Factor Depend-
ent Activation of the Src Family Kinases. Eur. J . Biochem. 1994,
225, 1047-1053. (b) Luttrell, D. K.; Lee, A.; Lansing, T. J .;
Crosby, R. M.; J ung, K. D.; Willard, D. Luther, M.; Rodriguez,
M.; Berman, J .; Gilmer, T. M. Involvement of pp60c-src with Two
Major Signaling Pathways in Human Breast Cancer. Proc. Natl.
Acad. Sci. 1994, 91, 83-87.
(30) Strawn, L. M.; Mann, E.; Elliger, S. S.; Chu, L. M.; Germain, L.
L.; Neiderfellner, G., Ulrich, A.; Shawver, L. K. Inhibition of
Glioma Cell Growth by a Truncated Platelet-Derived Growth
Factor-â Receptor. J . Biol. Chem. 1994, 269, 21215-21222.
(31) Dudley, D. T.; Pang, L.; Decker, S. J .; Bridges, A. J .; Saltiel, A.
R. A Synthetic Inhibitor of the Mitogen-Activated Protein Kianse
Cascade. Proc. Natl. Acad. Sci. 1995, 92, 7686-7689.
(32) Fry, D. W.; Kraker, A. J .; Connors, R. C.; Elliot, W. L.; Nelson,
J . M.; Showalter, H. D. H.; Leopold, W. R. Strategies for the
Discovery of Novel Tyrosine Kinase Inhibitors with Anticancer
Activity. Anti-Cancer Drug Des. 1994, 9, 331-351.
J M0004291