3-(3,5-Dimethoxyphenyl)-1,6-naphthyridine-2,7-diamines and related 2-urea derivatives are potent and selective inhibitors of the FGF receptor-1 tyrosine kinase

Article abstract of DOI:10.1021/jm000161d

A series of 3-aryl-1,6-naphthyridine-2,7-diamines and related 2-ureas were prepared and evaluated as inhibitors of the FGF receptor-1 tyrosine kinase. Condensation of 4,6-diaminonicotinaldehyde and substituted phenylacetonitriles gave intermediate naphthy

Full text of DOI:10.1021/jm000161d

4200
J . Med. Chem. 2000, 43, 4200-4211
3-(3,5-Dim eth oxyp h en yl)-1,6-n a p h th yr id in e-2,7-d ia m in es a n d Rela ted 2-Ur ea
Der iva tives Ar e P oten t a n d Selective In h ibitor s of th e F GF Recep tor -1 Tyr osin e
Kin a se
Andrew M. Thompson,† Cleo J . C. Connolly,‡ J ames M. Hamby,‡ Stacey Boushelle,‡ Brian G. Hartl,‡
Aneesa M. Amar,‡,# Alan J . Kraker,‡ Denise L. Driscoll,‡ Randall W. Steinkampf,‡ Sandra J . Patmore,‡
Patrick W. Vincent,‡ Bill J . Roberts,‡ William L. Elliott,‡ Wayne Klohs,‡ Wilbur R. Leopold,‡
H. D. Hollis Showalter,‡ and William A. Denny*,†
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland,
Private Bag 92019, Auckland 1000, New Zealand, and Parke-Davis Pharmaceutical Research, Division of
Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48106-1047
Received April 6, 2000
A series of 3-aryl-1,6-naphthyridine-2,7-diamines and related 2-ureas were prepared and
evaluated as inhibitors of the FGF receptor-1 tyrosine kinase. Condensation of 4,6-diamino-
nicotinaldehyde and substituted phenylacetonitriles gave intermediate naphthyridine-2,7-
diamines, and direct reaction of the monoanion of these (NaH/DMF) with alkyl or aryl
isocyanates selectively gave the 2-ureas in varying yields (23-93%). For the preparation of
more soluble 7-alkylamino-2-ureas, a number of protecting groups for the 2-amine were
evaluated (phthaloyl, 4-methoxybenzyl) following selective blocking of the 7-amine (trityl), but
these were not superior to the (required) 2-tert-Bu-urea group itself. Direct alkylation of the
anion of the (unprotected) 7-amino group with excess 4-(3-chloropropyl)morpholine in DMF
gave low (10%) yields of the desired product, but alkylation of the 7-acetamido anion, followed
by mild alkaline hydrolysis, raised this to 64%. 3-Phenyl analogues were nonspecific inhibitors
of isolated c-Src, FGFR, and PDGFR tyrosine kinases, whereas 3-(2,6-dichlorophenyl) analogues
were most effective against c-Src and FGFR, and 3-(3,5-dimethoxyphenyl) derivatives showed
high selectivity for FGFR alone. A water-soluble (7-morpholinylpropylamino) analogue retained
high FGFR potency (IC50 31 nM) and selectivity. Pairwise comparison of the 1,6-naphthyridines
and the corresponding known pyrido[2,3-d]pyrimidine analogues showed little differences in
potency or patterns of selectivity, suggesting that the 1-aza atom of the latter is not important
for activity. A 7-acetamide derivative inhibited the growth of FGFR-expressing tumor cell lines
and was particularly potent against HUVECs (IC50 4 nM). This compound was also a very
potent inhibitor of HUVEC microcapillary formation (IC50 0.01 nM) and Matrigel invasion (IC50
7 nM) and showed significant in vivo antitumor effects in a highly vascularized mammary
adenocarcinoma 16/c model at nontoxic doses. The compounds are worthy of further evaluation
as antiangiogenesis agents.
In tr od u ction
abnormal expression has been shown to accelerate the
tumorigenicity of prostate epithelial cells in vivo.12
Crystal structures of this receptor alone or in complex
with ATP or with inhibitors of the kinase have been
published.9,13 To date, few selective inhibitors of FGFR
tyrosine kinases have been reported.13,14 One of the
members of the thioindole class prepared in our earlier
studies, PD 145709 (1), specifically inhibited bFGF-
mediated tyrosine phosphorylation with moderate po-
tency (IC50 4.5 µM) as well as FGFR autophosphoryla-
tion and was a potent inhibitor of protein synthesis.15
Recently two compounds from the indolinone class were
reported to have moderate activity against the FGF
receptor-1 tyrosine kinase (IC50s of 10-20 µM), but only
one of these (2) displayed some selectivity against other
kinases.9 The Parke-Davis group has described16 studies
on the 1-oxo-3-aryl-1H-indene-2-carboxylic acid deriva-
tives which are also selective inhibitors (e.g. 3, IC50 5.1
µM).
Angiogenesis is essential for the growth and survival
of solid tumors.1 Fibroblast growth factors (FGFs) are
major angiogenic factors for some tumors.2,3 The inhibi-
tion of FGF receptor (FGFR) tyrosine kinases may
therefore be an effective strategy to prevent this inap-
propriate vascularisation.4,5 Overexpression of FGFRs,
their ligands, or other aberrant kinase function has been
implicated in various diseases, including psoriasis,
rheumatoid arthritis, atherosclerosis,6 and restenosis,7
as well as several human tumors8,9 (e.g. breast, pan-
creatic, ovarian, and prostate cancers), and in some
cases this correlates with poor survival.10
The FGF receptor-1 tyrosine kinase is the most
predominant FGFR subtype in vascular cells,11 and its
* To whom correspondence should be addressed. Tel: (64 9) 373
7599, ext 6144. Fax: (64 9) 373 7502. E-mail: b.denny@auckland.ac.nz.
† The University of Auckland.
‡ Parke-Davis Pharmaceutical Research.
#
Current address: School of Medicine, University of Virginia,
Charlottesville, VA 22908.
10.1021/jm000161d CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/10/2000

Selective Inhibitors of FGFR-1 TK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4201
Sch em e 1a
Recently the Parke-Davis group has also reported17
the discovery (through compound library screening) of
the novel pyrido[2,3-d]pyrimidine urea PD 089828 (4)
as a potent broad-spectrum inhibitor of the PDGFR,
FGFR, EGFR, and c-Src tyrosine kinases. Further
structure-activity relationship (SAR) studies18 resulted
in the identification of PD 166866 (5), as a potent and
very selective ATP competitive inhibitor of the FGF
receptor-1 tyrosine kinase,11 and developed compounds
with improved potency, solubility, and bioavailabil-
ity.13,19,20 In studies designed to determine the impor-
tance of the pyrimidine ring aza atoms, we describe here
the synthesis and biological evaluation of 3-aryl-1,6-
naphthyridine-2,7-diamines and related 2-ureas (as
1-deaza analogues of the pyrido[2,3-d]pyrimidines) (6-
20). Among these, the 7-acetamido derivative 17 shows
potent in vivo activity. Also reported are synthetic
strategies aimed at the preparation of more soluble
derivatives (e.g., the 7-[(3-morpholinylpropyl)amino]
derivative 20).
a
(i) XPhCH2CN/Na/2-EtO(CH2)2OH/135 °C/30 min (ref 23); (ii)
NaH/DMF, then RNCO/DMF or RNCO/0-20 °C/1-24 h; (iii) NaH/
DMSO, then RNCO/DMSO/20 °C/24 h.
Ta ble 1. Synthetic Yields for the Preparation of
N-(7-Amino-3-aryl-1,6-naphthyridin-2-yl)-N′-(alkyl or aryl)ureas
(Table 2) from Diamines 6, 9, and 12 by Various Methods
scale time
yield
(%)
diamine isocyanate methoda
(g)
(h)
product
6
6
9
9
9
EtNCO
tBuNCO
EtNCO
B
A
B
C
A
Ac
B
A
A
B
B
A
0.10
0.10
0.11
0.13
0.10
0.11
0.10
0.07
0.25
0.14
5.0
24
4
24 10
24 10
1
1
7
8
66
66
<37b
50
EtNCO
tBuNCO
MeNCO
EtNCO
tBuNCO
tBuNCO
tBuNCO
tBuNCO
PhNCO
11
13
23
47
56
51
70
89
93 (2)
45
12
12
12
12
12
12
12
20 14
15
1
18 15
24 15
24 15 (+19)
1
0.10
16
a
Method A: NaH (1.0-1.2 equiv)/DMF/20 °C/10 min, then
RNCO (1.0-1.2 equiv)/DMF/20 °C/time. Method B: NaH (1.2-
1.3 equiv)/DMF/20 °C/10-15 min, then RNCO (1.1-1.25 equiv)/
0-20 °C/time. Method C: NaH (1.4 equiv)/DMSO/40-50 °C/5 min,
Ch em istr y
The 3-aryl-1,6-naphthyridine-2,7-diamines and re-
lated 2-ureas were prepared by the general method
shown in Scheme 1. To start, the known21 4,6-diamino-
nicotinaldehyde (21) (derived from Raney nickel reduc-
tion of the corresponding nitrile22) was condensed with
substituted phenylacetonitriles in refluxing 2-ethoxy-
ethanol under basic conditions (the alkoxide generated
from addition of sodium to the solvent) as previously
reported21-23 to give the corresponding 3-aryl-1,6-naph-
thyridine-2,7-diamines 6, 9, and 12.
b
then RNCO (1.1 equiv)/DMSO/20 °C/time. Impure. c 0.6 equiv of
MeNCO added, to minimize bis-urea formation.
acylated NH and both C-2 and C-3 resonances. Selectiv-
ity toward urea formation at the more hindered C-2
amine position was seen in all three 3-aryl-1,6-naph-
thyridine-2,7-diamines examined and has also been
reported for the corresponding pyrido[2,3-d]pyrim-
idines,19 suggesting the formation of a thermodynami-
cally preferred anion.
Various conditions were then examined for the intro-
duction of the urea substituents (Table 1). In general
(method A), reaction of the diamines with sodium
hydride in dry DMF, followed by a solution of the
isocyanate in DMF at 20 °C for 1-24 h, selectively gave
2-mono-ureas in moderate yield, together with starting
material and small amounts of bis-ureas. The latter
were generally not isolated, but 19 was obtained (2%
yield) from a larger-scale preparation of 15. In the
3-(2,6-dichlorophenyl) series, the tert-butylurea 11 was
formed in much lower yield (23%) than was the corre-
sponding tert-butylurea 15 in the 3-(3,5-dimethoxyphen-
yl) series (51%), presumably due to the increased steric
hindrance in the former case. The structure of phenyl-
urea 16 was conclusively proved by 2-D NMR (HMQC,
HMBC) experiments from long-range correlations be-
tween the NH2 and C-8 resonances and between the
Method B, similar to that described by Hamby,19
employed the addition of the neat isocyanate at 0 °C to
the anions generated with sodium hydride in DMF at
20 °C. This method usually gave higher yields (up to
93%) but failed for the 3-(2,6-diClPh)ethylurea 10,
giving a low yield (<37%) and an inseparable bis-urea
impurity. A further modification (method C), forming
the anion from excess sodium hydride in DMSO at
higher temperature, then adding a solution of the
isocyanate in DMSO at 20 °C, mostly overcame these
difficulties, giving 10 in 50% purified yield.
Various routes were considered for the preparation
of a soluble 7-alkylamino derivative of the FGFR-
selective lead compound 15. We have shown23 that
selective diazotization of diamines 6 and 9 in concen-
trated HCl or 50% HBF4, followed by amine displace-

4202 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Thompson et al.
Sch em e 2a
Sch em e 3a
a
(i) NaH/DMF, then phthaloyl dichloride/20 °C/8 h; (ii) NaH/
DMF, then 4-OMePhCH2Cl/20 °C/24 h; (iii) TFA/50 °C/5 min; (iv)
NaH/DMF, then TrCl/20 °C/24 h or TrCl/Et3N/THF/50-60 °C/3
days; (v) NaOH/MeOH/water/CH2Cl2/40 °C/2.5 h; (vi) silica gel/
CH2Cl2/20 °C/3-4 days; (vii) NaH/DMF, then 4-OMePhCH2Cl/20
°C/2 h; (viii) TFA/70 °C/8 h or HCO2H/20 °C/20 h.
a
(i) NaH/DMF, then MeI/DMF/20 °C/2.5 h; (ii) 4-(3-chloro-
propyl)morpholine‚HCl/NaH/DMF/20 °C/7 days; (iii) TFAA/py/20
°C/16 h; (iv) AcCl/Et3N/THF/20 °C/8 days or Ac2O/py/20 °C/24 h;
(v) NaOH/MeOH/water/20 °C/30 min; (vi) step ii/52 °C/26 h; (vii)
step v/43 h.
ment of the resulting 7-halides, is a useful synthetic
route to 7-alkylamino derivatives. However, we encoun-
tered considerable difficulty with diazotization of the
more electron-rich diamine 12, due to competing nitro-
sation/oxidation reactions.23 We therefore considered
selective alkylation of the 7-amino group of 12, by first
selectively protecting the 2-amine, which forms an anion
preferentially (see above). The various protecting group
strategies examined are summarized in Scheme 2.
Phthaloylation of 12 under anionic conditions (NaH/
DMF, then ca. 0.45 equiv of phthaloyl dichloride) was
explored by analogy with the urea chemistry above. This
gave a crude mixture in which the desired 22 was a
major product, but there was much recovered starting
material, and both the 2- and 7-phthalimides (22 and
23) were isolated in extremely low yield (5%). Further-
more, surprisingly, purified 22 was relatively unstable
(on silica gel and on standing in solution), and this route
was therefore not suitable.
A second strategy used 4-methoxybenzyl (PMB) pro-
tecting groups, seeking initial double protection of both
amino groups, followed by selective deprotection at the
less hindered 7-position. Alkylation of 12 with excess
PMB chloride gave 24 in moderate yield (62%), but
treatment of this with TFA gave nonselective cleavage
at both positions to form 25 and 26 as the major
products. Use of the bulky trityl group to selectively
block the 7-position was then investigated. Tritylation
of 12 under anionic conditions (TrCl/NaH/DMF) unex-
pectedly gave the DMF-derived 7-tritylamino-2-form-
amide derivative 27 as a significant byproduct along
with the desired 28 (but 27 was easily hydrolyzed to 28
in good yield). Column chromatography of these com-
pounds on silica gel resulted in considerable detrityla-
tion, as reported for trityl ethers.24 Tritylation under
more typical conditions (e.g. TrCl/Et3N/THF) followed
by flash chromatography on silica gel gave 28 in good
yield (78-80%), along with small amounts of the ditri-
tylated derivative 29. The structure of 28 was confirmed
by comparison of its NMR data with that for the
2-tritylamino derivative 30, obtained in low yield from
the ditritylated derivative 29 by partial detritylation on
silica gel.24
Alkylation of 28 with PMB chloride as above gave 31
in good yield (83%), and selective removal of the trityl
group with silica gel24 gave 32 (69% after two cycles).
However, removal of the PMB groups unexpectedly
proved impossible. Many reagents (TFA, AcOH, HCO2H,
CAN, H2/10% Pd-C) removed one PMB group to give
26, but only refluxing HCO2H and hydrogenolysis gave
the diamine 12 (in low yield as part of a complex
mixture). Therefore this route was also abandoned in
favor of a more direct approach, using the readily
accessible 2-t-Bu-urea group as the only protection for
the 2-amine (Scheme 3).
Methylation of 15 (NaH/DMF, then MeI/DMF) gave
many products, the major one being 33 (36% yield),

Selective Inhibitors of FGFR-1 TK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4203
Ta ble 2. Structure and Kinase Inhibitory Activities of 1,6-Naphthyridines as FGFR Inhibitors
IC50 (µM)
no.
Fm
X
Y
FGFa
17
1.3
1.9
PDGFa
c-Srca
6
7
8
A
A
A
B
B
B
C
C
C
C
C
C
C
C
C
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NHAc
NH2
17
7.7
2.6
35
11
23
5.1
4.4
NHCONHEt
NHCONHtBu
NH2
NHCONHEt
NHCONHtBu
NH2
NHCONHMe
NHCONHEt
NHCONHtBu
NHCONHPh
NHCONHtBu
NH2
9
2.8
0.68
0.10
0.17
>50
>50
>50
>50
>50
3.8
10
11
12
13
14
15
16
17
18
19
20
0.15
0.12
0.20
0.095
0.029
0.042
0.51
0.025
0.061
0.16
0.031
1.8
>50
>50
>50
38
>50
4.7
NHCOCF3
>50
>50
45
>50
>50
>50
NHCONHtBu
NHCONHtBu
NHCONHtBu
NH(CH2)3Nmorphb
a
IC50: concentration of drug (µM) to inhibit the phosphorylation of a random glutamate-tyrosine (4:1) copolymer by FGFR, PDGFR,
or c-Src proteins. For active compounds, values are an average of two or more separate determinations; variation was generally (30%.
N-Morpholinyl.
b
resulting from N2-methylation and cleavage of the urea
group. The structure of 33 was deduced from the
similarity of its NMR to that of the 2-PMB-amino
derivative 26 above and by HSQC and HMBC 2-D NMR
(long-range correlations between the NH2 and C-8
resonances, between the alkylated NH and both C-2 and
C-3 resonances, and between the NCH3 and C-2 reso-
nances). Alkylation of 15 with excess 4-(3-chloropropyl)-
morpholine hydrochloride/NaH/DMF gave a low yield
of the desired 20 (10%), together with the N-1 alkylated
product 34 (1.5%) and recovered 15 (48%). The structure
of 34 is proposed from its distinctive UV spectrum and
hydrolysis30 converted the mixture cleanly to the 7-acet-
amide 17 (72% yield overall). Reactions involving Ac2O
and Et3N or NaH gave undesired products, probably due
to deprotonation of the 2-urea. However, Ac2O/pyridine
gave an excellent yield of the desired product 17 directly
(92%).
Alkylation of 17 using excess 4-(3-chloropropyl)-
morpholine hydrochloride/NaH/DMF gave a 1:1 mixture
of the 7-NH- and 7-NAc-alkyl derivatives 20 and 36 in
good yield (70%). This demonstrates high selectivity for
alkylation of the assumed dianion (from deprotonation
of the 2-urea and 7-acetamide) at the least hindered (7-
N) position. Mild alkaline hydrolysis of this mixture
selectively cleaved the N,N-disubstituted acetamide in
the presence of the 2-urea to give 20 in 64% overall
yield. However, alkylation of 17 appears to be very
sensitive to traces of moisture, since repetition (on twice
the scale) gave a much lower yield of 20 (16%) and no
36, together with the 7,7-bis derivative 37 (14%), a
mixture of 17 and the 7-amino derivative 15 (ca. 32%),
and more polar components.
1
strong upfield shifts in the H NMR, similar to those
observed for 1,6-naphthyridin-2(1H)-ones22 in compari-
son to the corresponding 2-t-Bu-ureas. This was further
supported by the observation that strong base hydrolysis
of 34 did not yield a 2-NH-alkyl derivative (TLC, NMR),
whereas loss of the 2-urea to give 33 above was
evidently very facile.
These alkylation results are consistent with those
reported for reactions on 2-aminopyridine by Dome,25
where mixtures of mono- and dibenzylated products
were obtained in moderate yield (35% and 23%, respec-
tively), and by Whitmore,26 who prepared the 2-(mor-
pholinylpropyl)amino derivative (18%) by heating the
preformed anion with 0.5 equiv of the alkyl chloride in
toluene. However, selective monoalkylation of 2-amino-
pyridine in better yield has been reported27,28 via the
corresponding 2-formamide or acetamide (whereas 2-sul-
fonamides reportedly29 gave alkylation on the ring
nitrogen).
Trifluoroacetylation of 15 with TFAA/pyridine gave
one major product, 18 (78%), which had lost the 2-t-Bu-
urea functionality (possibly due to acylation, which
could activate it toward hydrolysis). Because 18 was also
somewhat unstable, we instead examined the more
stable acetamide. Acetylation of 15 with AcCl/Et3N/THF
gave a mixture of the 7-acetamido and 7-diacetylamino
derivatives 17 and 35 in good yield, and mild alkaline
Resu lts a n d Discu ssion
The compounds listed in Table 2 were evaluated for
their ability to prevent phosphorylation of a model
glutamate-tyrosine copolymer substrate by isolated
avian c-Src, human FGF receptor-1 (FGFR), and mouse
PDGF-â receptor (PDGFR) tyrosine kinase enzymes,
using published methods.11,17,31 The IC50 values were
defined as the concentration of inhibitor to reduce by
50% the level of 32P (from added [32P]ATP) incorporated
into the copolymer substrate. The 3-phenyl compounds
6-8 were nonspecific inhibitors, with about equal
activity in the three assays, similar to the pattern seen
with the corresponding pyrido[2,3-d]pyrimidines.19 For-
mation of the 2-urea increased potency but not selectiv-
ity. The corresponding 3-(2,6-dichlorophenyl) analogues
9-11 were significantly more potent than 6-8 for
inhibition of the FGFR and c-Src kinases only but

4204 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Thompson et al.
Ta ble 3. Comparison of the Kinase Inhibitory Activities of
1,6-Naphthyridines (A) and Pyrido[2,3-d]pyrimidines (B)
showed that the naphthyridine was considerably more
potent in all the lines. Overall, the compounds displayed
similar, modest potencies toward both the PDGF-
dependent C6 rat glioma line32 (expressing moderate
FGFR levels33) and the FGFR-overexpressing human
ovarian A90 line34 but much higher potencies toward
human umbilical vein endothelial cells (HUVECs),
whose growth has been shown to be FGF-dependent.35
The 7-acetamide 17 was particularly potent against
HUVECs (IC50 4 nM), and this compound was also an
extremely effective inhibitor of HUVEC microcapillary
formation and Matrigel invasion (Table 4).
One compound (17) was selected for in vivo evaluation
against three murine tumor model systems: mammary
adenocarcinoma 16/c, M5076 reticulum cell sarcoma,
and Lewis lung carcinoma. These models were selected
due to their high degree of vascularization and have
been used by a number of other investigators in the
analysis of angiogenesis inhibitors. While we expected
that 17 would produce measurable antitumor effects in
all three models, it was effective only against the
mammary adenocarcinoma 16/c (Table 5). Although the
drug had to be administered as a suspension, enough
was apparently absorbed from the gastrointestinal tract
following oral dosing to produce good antitumor activity.
Similar results were obtained for both once and twice
daily treatment schedules, and at the doses tested, there
was no toxicity or significant clinical signs (animal
weight loss was minimal). The reasons for the differ-
ences in the effectiveness of 17 among the three highly
vascularized tumor models are not known. It could be
due to redundancy in the tyrosine kinase receptor
signaling pathways with respect to angiogenesis in these
tumor models and is an area of current investigation.
IC50 (µM)
no. Fm
X
R
FGFa PDGFa c-Srca
8
38b
9
A
B
A
B
A
B
A
B
A
B
A
B
H
H
NHCONHtBu 1.9
NHCONHtBu 3.7
2.6
4.7 >50
35
21
11
1.3
1.8
1.1
4.4
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
2,6-diCl
NH2
2.8
0.68
0.21
0.10
0.08
0.17
0.22
39c
10
40b
11
4b
NH2
3.0
NHCONHEt
NHCONHEt
0.15
0.13
NHCONHtBu 0.12
NHCONHtBu 0.13
12
41b
15
5b
3,5-diOMe NH2
3,5-diOMe NH2
3,5-diOMe NHCONHtBu 0.042
0.20
0.23
>50
>50
>50
>50
>50
>50
38
3,5-diOMe NHCONHtBu 0.060 >50
a
b
As for Table 2. Ref 19. c Data from ref 21.
Ta ble 4. Growth Delay and in Vitro Antiangiogenesis
Activities of 1,6-Naphthyridines and Related
Pyrido[2,3-d]pyrimidines
IC50 (µM)a
no.
C6b
>25
A90c
HUVECd
microcape
0.1
invasione
1.1
5
12
14
15
17
18
19
20
15
17
9.3
1.2
12
17
11
0.92
0.58
0.82
0.17
0.0055
0.004
4.9
0.017
0.085
8.9
8.0
8.5
0.65
9.0
0.1
0.00001
>10
0.007
13
3.1
0.01
0.001
a
IC50: concentration of drug (µM) to inhibit in vitro cell growth
or HUVEC microcapillary formation or invasion. For active
compounds, values are an average of two or more separate
Con clu sion s
b
determinations. PDGF-dependent rat glioma. c FGFR overex-
The broadly similar SARs found above for the 1,6-
naphthyridines and the previously reported19 pyrido-
[2,3-d]pyrimidines suggest a very similar binding mode
to the enzymes, with the 1-aza atom of the pyrido[2,3-
d]pyrimidines not being required for this binding. In
both series, substituents on the appended phenyl group
have a major impact on the pattern of inhibition of
different kinases, suggesting that the geometry of this
ring is a critical factor in binding site selectivity.19
Computer modeling studies36 and crystal structure
data13 of the closely related 6-phenylpyrido[2,3-d]pyrim-
idine 7-ureas show the need for bifurcated H bonds
between the N-2 exocyclic and N-3 endocyclic atoms and
a residue (Ala564) on the extended coil stretch of the
FGFR enzyme. The 7-NHR 1,6-naphthyridine-2-ureas
also contain this required structural motif. The 3-(3,5-
dimethoxyphenyl) compounds showed particular selec-
tivity for FGFR over PDGFR and c-Src, presumably
because the methoxy residues fit better into the larger
hydrophobic pocket of FGFR.13 The potent inhibition by
17 of HUVEC growth and microcapillary formation and
invasion suggests that these compounds are worthy of
further evaluation as antiangiogenesis agents. It is
notable that the in vivo tumor growth delay values
obtained for 17 against mammary 16/c were approxi-
mately equal to the duration of therapy (9 days), which
suggests that this compound is cytostatic rather than
cytotoxic under these test conditions. It remains to be
d
pressing human ovarian carcinoma. FGF-dependent human
umbilical vein endothelial cells. e Matrigel assay.
remained nonselective between these two kinases. In
contrast, the 3-(3,5-dimethoxyphenyl) derivatives 12-
20 showed both high potency and excellent selectivity
for FGFR versus the other kinases. Both PDGFR and
in particular c-Src activity was completely lost, resulting
in c-Src/FGFR selectivities of >1000-fold for many of
the compounds. The three alkyl ureas 13-15 did not
differ markedly in potency, but the phenyl urea 16 was
significantly less effective. This is consistent with results
reported by Hamby et al.19 for the related pyrido[2,3-
d]pyrimidines. Acetamide 17 was the most potent FGFR
inhibitor (IC50 25 nM) but was less selective against
PDGFR and c-Src. The bis-urea 19 was less potent, but
the more soluble analogue 20 retained high potency and
selectivity. Table 3 provides some pairwise comparisons
of 1,6-naphthyridines and pyrido[2,3-d]pyrimidines.
Overall no significant differences can be seen between
the two chromophore series, either in absolute potency
or in patterns of selectivity.
Certain of the FGFR-selective 3-(3,5-dimethoxy-
phenyl) derivatives were evaluated for growth inhibition
in a variety of serum-stimulated cell lines, together with
the pyrido[2,3-d]pyrimidine 5 (Table 4). A comparison
of 5 and its direct 1,6-naphthyridine analogue 15

Selective Inhibitors of FGFR-1 TK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4205
Ta ble 5. In Vivo Activity of 17 against Murine Tumor Models
tumora
dose (mg/kg)b
schedulec
wt change (g)
T/C (%) on last therapy dayd
T-C (days)e
mammary 16/c
mammary 16/c
M5076 sarcoma
Lewis lung
80 hdt
200 hdt
200 hdt
200 hdt
po, q12hx2, days 1-9
po, days 1-9
-0.7
+
0
0
78
10.8
11.3
1.0
po, days 8-16
+
po, days 4-12
-0.4
109
0.0
a
b
The indicated tumor fragments were implanted sc into the right axilla of mice on day 0. hdt, highest dose tested. c Compound was
administered orally on the indicated schedules. Ratio of median treated tumor mass/median control tumor mass × 100%. e The difference
d
in days for the treated (T) and control (C) tumors to reach 750 mg.
N-[7-Am in o-3-(3,5-dim eth oxyph en yl)-1,6-n aph th yr idin -
2-yl]-N′-m eth ylu r ea (13). Similar reaction of a stirred solu-
tion of 3-(3,5-dimethoxyphenyl)-1,6-naphthyridine-2,7-diamine23
(12) (106 mg, 0.358 mmol) in dry DMF (4 mL) with 60% NaH
(14 mg, 0.35 mmol) under N2 at 20 °C for 10 min, then with a
solution of methyl isocyanate (12 µL, 0.204 mmol) in dry DMF
(1 mL) at 20 °C for 1 h, followed by chromatography of the
resulting product on silica gel (eluting with 1.5% MeOH/CH2-
Cl2) gave 13 (59 mg, 47%): mp (MeOH/CH2Cl2/hexane) 134-
137 °C; 1H NMR [(CD3)2SO] δ 9.73 (br q, J ) 4.6 Hz, 1 H,
NHCH3), 8.66 (s, 1 H, H-5), 7.99 (s, 1 H, H-4), 7.24 (br s, 1 H,
NH), 6.63 (s, 3 H, H-2′,4′,6′), 6.62 (s, 1 H, H-8), 6.31 (br s, 2 H,
NH2), 3.80 (s, 6 H, 2OCH3), 2.84 (d, J ) 4.6 Hz, 3 H, NHCH3);
13C NMR δ 161.09 (s, 2 C, C-3′,5′), 160.23 (s, C-7), 154.12,
152.11 (2 s, CONH, C-2), 151.39 (d, C-5), 149.66 (s, C-8a),
137.51 (d+s, 2 C, C-4,1′), 121.14 (s, C-3), 113.36 (s, C-4a),
107.07 (d, 2 C, C-2′,6′), 100.16 (d, C-4′), 96.26 (d, C-8), 55.37
(q, 2 C, 2OCH3), 26.09 (q, CH3). Anal. (C18H19N5O3‚1.25H2O)
C, H, N.
determined if more prolonged therapy would have
cytotoxic effects in vivo, resulting in the regression of
established tumors.
Exp er im en ta l Section
Analyses were performed by the Microchemical Laboratory,
University of Otago, Dunedin, NZ. Melting points were
determined using an Electrothermal model 9200 digital melt-
ing point apparatus and are as read. NMR spectra were
measured on a Bruker DRX-400 spectrometer and referenced
to Me4Si. Mass spectra were recorded on a Varian VG-70SE
spectrometer at nominal 5000 resolution.
N-(7-Am in o-3-p h en yl-1,6-n a p h th yr id in -2-yl)-N′-ter t-bu -
tylu r ea (8): Exa m p le of Gen er a l Meth od A. A solution of
3-phenyl-1,6-naphthyridine-2,7-diamine23 (6) (103 mg, 0.436
mmol) in dry DMF (5 mL) was treated with 60% NaH (20 mg,
0.50 mmol); then the reaction flask was immediately sealed
with a rubber septum, degassed (water pump vacuum) and
filled with dry N2 (balloon), and the mixture stirred at 20 °C
for 10 min. A solution of tert-butyl isocyanate (58 µL, 0.509
mmol) in dry DMF (1 mL, then 1 mL to rinse) was added
(dropwise via syringe); then the mixture was stirred at 20 °C
for 4 h. The resulting mixture was cooled in ice, then treated
with ice/aqueous NaHCO3 (50 mL) and extracted with EtOAc
(5 × 50 mL). The extracts were evaporated to dryness and the
residue was then chromatographed on silica gel. Elution with
25-30% EtOAc/light petroleum gave foreruns; then further
elution with 33-40% EtOAc/light petroleum gave 8 (97 mg,
66%): mp (CH2Cl2/hexane) 174-175.5 °C; 1H NMR [(CD3)2-
SO] δ 10.26 (br s, 1 H, NH), 8.67 (s, 1 H, H-5), 7.98 (s, 1 H,
H-4), 7.58 (t, J ) 7.2 Hz, 2 H, H-3′,5′), 7.51 (t, J ) 7.3 Hz, 1 H,
H-4′), 7.50 (d, J ) 6.7 Hz, 2 H, H-2′,6′), 6.91 (br s, 1 H, NH),
6.49 (s, 1 H, H-8), 6.37 (br s, 2 H, NH2), 1.41 (s, 9 H, C(CH3)3);
13C NMR δ 160.36 (s, C-7), 152.45, 151.99 (2 s, CONH, C-2),
151.49 (d, C-5), 149.42 (s, C-8a), 137.73 (d, C-4), 135.48 (s, C-1′),
129.42, 129.11 (2 d, 2 × 2 C, C-2′,3′,5′,6′), 128.52 (d, C-4′),
121.25 (s, C-3), 113.34 (s, C-4a), 95.73 (d, C-8), 49.93 (s,
C(CH3)3), 28.64 (q, 3 C, C(CH3)3). Anal. (C19H21N5O) C, H, N.
Further elution of the column with EtOAc gave a mixture;
then further elution with 10% MeOH/CH2Cl2 gave crude
recovered 6 (25 mg, 24%) as an oil.
Further elution of the column with 2-4% MeOH/CH2Cl2
gave a mixture; then further elution with 4-10% MeOH/CH2-
Cl2 gave crude recovered 12 (57 mg, 54%) as an oil.
N-[7-Am in o-3-(3,5-dim eth oxyph en yl)-1,6-n aph th yr idin -
2-yl]-N′-p h en ylu r ea (16). Similar reaction of a stirred solu-
tion of 12 (100 mg, 0.338 mmol) in dry DMF (5 mL) with 60%
NaH (13 mg, 0.325 mmol) under N2 at 20 °C for 10 min, then
with a solution of phenyl isocyanate (35 µL, 0.322 mmol) in
dry DMF (1 mL) at 20 °C for 1 h, followed by chromatography
of the resulting product on silica gel (eluting with 0.25-1%
MeOH/CH2Cl2) gave 16 (63 mg, 45%): mp (MeOH/CH2Cl2/
1
hexane) 205-207 °C; H NMR [(CD3)2SO] δ 12.68 (br s, 1 H,
NH), 8.73 (s, 1 H, H-5), 8.10 (s, 1 H, H-4), 7.61 (d, J ) 7.6 Hz,
2 H, H-2′′,6′′), 7.61 (br s, 1 H, NH), 7.39 (t, J ) 7.9 Hz, 2 H,
H-3′′,5′′), 7.12 (t, J ) 7.4 Hz, 1 H, H-4′′), 6.72 (s, 1 H, H-8),
6.69 (d, J ) 2.2 Hz, 2 H, H-2′,6′), 6.64 (t, J ) 2.2 Hz, 1 H,
H-4′), 6.41 (br s, 2 H, NH2), 3.82 (s, 6 H, 2OCH3); 13C NMR δ
161.12 (s, 2 C, C-3′,5′), 160.50 (s, C-7), 152.09 (s, C-2), 151.75
(d, C-5), 151.08 (s, CONH), 149.14 (s, C-8a), 138.31 (s, C-1′′),
138.19 (d, C-4), 137.26 (s, C-1′), 129.07 (d, 2 C, C-3′′,5′′), 123.28
(d, C-4′′), 121.40 (s, C-3), 119.07 (d, 2 C, C-2′′,6′′), 113.53 (s,
C-4a), 107.19 (d, 2 C, C-2′,6′), 100.25 (d, C-4′), 95.87 (d, C-8),
55.41 (q, 2 C, 2OCH3). Anal. (C23H21N5O3‚0.5H2O) C, H, N.
N-[7-Am in o-3-(2,6-d ich lor op h en yl)-1,6-n a p h t h yr id in -
2-yl]-N′-ter t-bu tylu r ea (11). Similar reaction of a stirred
solution of 3-(2,6-dichlorophenyl)-1,6-naphthyridine-2,7-di-
amine23 (9) (100 mg, 0.328 mmol) in dry DMF (5 mL) with
60% NaH (16 mg, 0.40 mmol) under N2 at 20 °C for 10 min,
then with a solution of tert-butyl isocyanate (45 µL, 0.395
mmol) in dry DMF (1 mL) at 20 °C for 1 h, followed by
chromatography of the resulting product on silica gel (eluting
with 0.25-0.5% MeOH/CH2Cl2) gave 11 (30 mg, 23%): mp
(CH2Cl2/hexane) 165-167 °C; 1H NMR [(CD3)2SO] δ 10.35 (br
s, 1 H, NH), 8.65 (s, 1 H, H-5), 7.95 (s, 1 H, H-4), 7.65 (d, J )
8.4 Hz, 2 H, H-3′,5′), 7.53 (dd, J ) 8.7, 7.4 Hz, 1 H, H-4′), 7.43
(br s, 1 H, NH), 6.48 (s, 1 H, H-8), 6.44 (br s, 2 H, NH2), 1.40
(s, 9 H, C(CH3)3); 13C NMR δ 160.69 (s, C-7), 152.45, 152.37 (2
s, CONH, C-2), 151.60 (d, C-5), 149.80 (s, C-8a), 139.48 (d, C-4),
135.48 (s, 2 C, C-2′,6′), 132.56 (s, C-1′), 131.51 (d, C-4′), 128.77
(d, 2 C, C-3′,5′), 116.34 (s, C-3), 112.87 (s, C-4a), 95.57 (d, C-8),
49.91 (s, C(CH3)3), 28.64 (q, 3 C, C(CH3)3). Anal. (C19H19Cl2N5O)
C, H, N.
Further elution of the column with 2.5% MeOH/CH2Cl2 gave
a mixture; then further elution with 5-8% MeOH/CH2Cl2 gave
crude recovered 12 (55 mg, 55%) as an oil.
N-[7-Am in o-3-(3,5-dim eth oxyph en yl)-1,6-n aph th yr idin -
2-yl]-N′-ter t-bu tylu r ea (15) a n d N-(ter t-Bu tyl)-N′-[7-(3-
ter t-bu tylu r eido)-3-(3,5-dim eth oxyph en yl)-1,6-n aph th yr i-
d in -2-yl]u r ea (19): Exa m p le of Gen er a l Meth od B. A
solution of 12 (5.01 g, 16.9 mmol) in dry DMF (100 mL) was
treated with 60% NaH (0.83 g, 20.8 mmol); then the mixture
was sealed under N2 (as above) and stirred at 20 °C for 15
min and then at 0 °C for 30 min. tert-Butyl isocyanate (2.40
mL, 21.0 mmol) was added (dropwise via syringe); then the
mixture was stirred at 20 °C for 1 day. The resulting mixture
was cooled in ice, then treated with ice/aqueous NaHCO3 (500
mL) and extracted with EtOAc (10 × 400 mL). The extracts
were evaporated to dryness and the residue was then chro-
matographed on silica gel. Elution with 33% EtOAc/light
petroleum gave first N-(tert-butyl)-N′-[7-(3-tert-butylureido)-
3-(3,5-dimethoxyphenyl)-1,6-naphthyridin-2-yl]urea (19) (186
mg, 2%): mp (EtOAc/light petroleum) 208-210 °C dec; 1H
NMR [(CD3)2SO] δ 10.10 (br s, 1 H, NH), 9.04 (br s, 1 H, NH),
Further elution of the column with 0.5-2% MeOH/CH2Cl2
gave a mixture; then further elution with 2-5% MeOH/CH2-
Cl2 gave crude recovered 9 (66 mg, 66%) as an oil.

4206 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Thompson et al.
8.87 (s, 1 H, H-5), 8.16 (s, 1 H, H-4), 7.83 (s, 1 H, H-8), 7.27
(br s, 1 H, NH), 7.15 (br s, 1 H, NH), 6.68 (d, J ) 2.0 Hz, 2 H,
H-2′,6′), 6.66 (t, J ) 2.1 Hz, 1 H, H-4′), 3.81 (s, 6 H, 2OCH3),
1.41 (s, 9 H, C(CH3)3), 1.34 (s, 9 H, C(CH3)3); 13C NMR δ 161.15
(s, 2 C, C-3′,5′), 153.45, 153.07, 152.81, 151.77 (4 s, 2CONH,
C-2,7), 150.39 (d, C-5), 149.09 (s, C-8a), 137.15 (d, C-4), 136.89
(s, C-1′), 124.13 (s, C-3), 115.72 (s, C-4a), 107.08 (d, 2 C, C-2′,6′),
102.04 (d, C-8), 100.51 (d, C-4′), 55.43 (q, 2 C, 2OCH3), 50.03,
49.51 (2 s, 2C(CH3)3), 28.87, 28.64 (2 q, 2 × 3 C, 2C(CH3)3).
Anal. (C26H34N6O4) H, N; C: calcd, 63.1; found, 63.6.
gave 10 (82 mg, 50%): mp (MeOH/CH2Cl2/hexane) 210-212
°C; 1H NMR [(CD3)2SO] δ 10.06 (br t, J ) 5.3 Hz, 1 H, NHCH2),
8.66 (s, 1 H, H-5), 7.96 (s, 1 H, H-4), 7.81 (br s, 1 H, NH), 7.64
(d, J ) 8.1 Hz, 2 H, H-3′,5′), 7.52 (dd, J ) 8.8, 7.3 Hz, 1 H,
H-4′), 6.58 (s, 1 H, H-8), 6.41 (br s, 2 H, NH2), 3.29 (qd, J )
7.1, 5.6 Hz, 2 H, NHCH2), 1.19 (t, J ) 7.2 Hz, 3 H, CH3); 13C
NMR δ 160.61 (s, C-7), 153.81, 152.44 (2 s, CONH, C-2), 151.56
(d, C-5), 149.99 (s, C-8a), 139.54 (d, C-4), 135.50 (s, 2 C, C-2′,6′),
132.74 (s, C-1′), 131.40 (d, C-4′), 128.73 (d, 2 C, C-3′,5′), 116.49
(s, C-3), 113.01 (s, C-4a), 95.95 (d, C-8), 34.07 (t, NCH2), 14.98
(q, CH3). Anal. (C17H15Cl2N5O) C, H, N.
Further elution of the column with 40-50% EtOAc/light
petroleum gave N-[7-amino-3-(3,5-dimethoxyphenyl)-1,6-naph-
thyridin-2-yl]-N′-tert-butylurea (15) (6.19 g, 93%): mp (CH2-
2-[7-Am in o-3-(3,5-d im eth oxyp h en yl)-1,6-n a p h th yr id in -
2-yl]-1H-isoin d ole-1,3(2H)-d ion e (22) a n d 2-[2-Am in o-3-
(3,5-d im eth oxyp h en yl)-1,6-n a p h th yr id in -7-yl]-1H-isoin -
d ole-1,3(2H)-d ion e (23). A solution of 12 (102 mg, 0.345
mmol) in dry DMF (6 mL) was treated with 60% NaH (15 mg,
0.375 mmol); then the mixture was sealed under N2 (as above)
and stirred at 20 °C for 10 min. Phthaloyl dichloride (25 µL of
‘practical’, ca. 0.156 mmol) was added directly (dropwise via
syringe); then the mixture was stirred at 20 °C for 8 h. The
resulting mixture was cooled in ice, then treated with ice/
aqueous NaHCO3 (50 mL) and extracted with EtOAc (4 × 50
mL). The extracts were evaporated to dryness and the residue
was then chromatographed on silica gel. Elution with 0-1%
MeOH/CH2Cl2 gave foreruns; then further elution with 1%
MeOH/CH2Cl2 gave 2-[2-amino-3-(3,5-dimethoxyphenyl)-1,6-
naphthyridin-7-yl]-1H-isoindole-1,3(2H)-dione (23) (7 mg,
5%): mp (DMSO/water) 270-271.5 °C; 1H NMR [(CD3)2SO] δ
8.95 (s, 1 H, H-5), 8.07 (s, 1 H, H-4), 8.02, 7.95 (2 m, 2 × 2 H,
H-3′′,4′′,5′′,6′′), 7.49 (s, 1 H, H-8), 6.90 (br s, 2 H, NH2), 6.68
(d, J ) 2.3 Hz, 2 H, H-2′,6′), 6.60 (t, J ) 2.2 Hz, 1 H, H-4′),
3.82 (s, 6 H, 2OCH3); 13C NMR δ 166.66 (s, 2 C, 2CdO), 160.79
(s, 2 C, C-3′,5′), 159.04 (s, C-2), 151.83 (s, C-7), 150.71 (d, C-5),
143.98 (s, C-8a), 138.39 (s, C-1′), 135.36 (d, C-4), 134.90 (d, 2
C, C-4′′,5′′), 131.44 (s, 2 C, C-2a′′,6a′′), 126.54 (s, C-3), 123.59
(d, 2 C, C-3′′,6′′), 119.33 (s, C-4a), 116.94 (d, C-8), 106.52 (d, 2
C, C-2′,6′), 100.46 (d, C-4′), 55.27 (q, 2 C, 2OCH3); HRFABMS
calcd for C24H19N4O4 m/z (MH+) 427.1406, found 427.1401.
Anal. (C24H18N4O4‚0.5H2O) C, H, N.
Further elution of the column with 1-1.5% MeOH/CH2Cl2
gave 2-[7-amino-3-(3,5-dimethoxyphenyl)-1,6-naphthyridin-2-
yl]-1H-isoindole-1,3(2H)-dione (22) (8 mg, 5%): mp (CH2Cl2/
hexane) 213-214 °C; 1H NMR [(CD3)2SO] δ 9.06 (s, 1 H, H-5),
8.48 (s, 1 H, H-4), 7.95 (m, 4 H, H-3′′,4′′,5′′,6′′), 6.72 (s, 1 H,
H-8), 6.64 (br s, 2 H, NH2), 6.43 (d, J ) 2.4 Hz, 2 H, H-2′,6′),
6.40 (t, J ) 2.3 Hz, 1 H, H-4′), 3.59 (s, 6 H, 2OCH3); 13C NMR
δ 166.46 (s, 2 C, 2CdO), 160.26 (s, C-7), 160.22 (s, 2 C, C-3′,5′),
153.38 (d, C-5), 151.00, 147.35 (2 s, C-2,8a), 139.49 (d, C-4),
138.47 (s, C-1′), 135.42 (d, 2 C, C-4′′,5′′), 130.79 (s, 2 C, C-2a′′,-
6a′′), 128.98 (s, C-3), 123.93 (d, 2 C, C-3′′,6′′), 116.55 (s, C-4a),
105.82 (d, 2 C, C-2′,6′), 99.79 (d, C-4′), 96.39 (d, C-8), 54.99 (q,
2 C, 2OCH3). Anal. (C24H18N4O4) C, H, N.
1
Cl2/hexane) 127-130 °C; H NMR [(CD3)2SO] δ 10.24 (br s, 1
H, NH), 8.66 (s, 1 H, H-5), 7.99 (s, 1 H, H-4), 7.01 (br s, 1 H,
NH), 6.63 (m, 3 H, H-2′,4′,6′), 6.47 (s, 1 H, H-8), 6.36 (br s, 2
H, NH2), 3.81 (s, 6 H, 2OCH3), 1.40 (s, 9 H, C(CH3)3); 13C NMR
δ 161.11 (s, 2 C, C-3′,5′), 160.35 (s, C-7), 152.34, 152.02 (2 s,
CONH, C-2), 151.47 (d, C-5), 149.42 (s, C-8a), 137.41 (d, C-4),
137.37 (s, C-1′), 121.16 (s, C-3), 113.18 (s, C-4a), 107.11 (d, 2
C, C-2′,6′), 100.19 (d, C-4′), 95.74 (d, C-8), 55.39 (q, 2 C,
2OCH3), 49.93 (s, C(CH3)3), 28.66 (q, 3 C, C(CH3)3). Anal.
(C21H25N5O3‚0.25H2O) C, H, N.
N-(7-Am in o-3-p h en yl-1,6-n a p h th yr id in -2-yl)-N′-eth yl-
u r ea (7). Similar reaction of a stirred solution of 6 (102 mg,
0.432 mmol) in dry DMF (5 mL) with 60% NaH (22 mg, 0.55
mmol) under N2 at 20 °C for 10 min, then (upon cooling to 0
°C) with ethyl isocyanate (43 µL, 0.544 mmol) at 0-20 °C for
1 day, followed by chromatography of the resulting product
on silica gel (eluting with 0.9-1% MeOH/CH2Cl2), then further
chromatography on silica gel (eluting with 50-75% EtOAc/
light petroleum) gave 7 (87 mg, 66%): mp (CH2Cl2/hexane)
181-182.5 °C; 1H NMR [(CD3)2SO] δ 9.94 (br m, 1 H, NHCH2),
8.67 (s, 1 H, H-5), 7.99 (s, 1 H, H-4), 7.57 (t, J ) 7.2 Hz, 2 H,
H-3′,5′), 7.51 (m, 3 H, H-2′,4′,6′), 7.10 (br s, 1 H, NH), 6.59 (s,
1 H, H-8), 6.34 (br s, 2 H, NH2), 3.30 (qd, J ) 7.2, 5.9 Hz, 2 H,
NHCH2), 1.19 (t, J ) 7.2 Hz, 3 H, CH3); 13C NMR δ 160.28 (s,
C-7), 153.36, 152.32 (2 s, CONH, C-2), 151.45 (d, C-5), 149.62
(s, C-8a), 137.82 (d, C-4), 135.61 (s, C-1′), 129.41, 129.10 (2 d,
2 × 2 C, C-2′,3′,5′,6′), 128.48 (d, C-4′), 121.32 (s, C-3), 113.49
(s, C-4a), 96.13 (d, C-8), 34.10 (t, NCH2),15.04 (q, CH3). Anal.
(C17H17N5O‚0.25H2O) C, H, N.
N-[7-Am in o-3-(3,5-dim eth oxyph en yl)-1,6-n aph th yr idin -
2-yl]-N′-eth ylu r ea (14). Similar reaction of 12 (100 mg, 0.338
mmol) in dry DMF (5 mL) with 60% NaH (17 mg, 0.425 mmol)
under N2 at 20 °C for 10 min, and then (upon cooling to 0 °C)
with ethyl isocyanate (30 µL, 0.379 mmol) at 0-20 °C for 20
h, followed by chromatography of the resulting product on
silica gel (eluting with 1-1.25% MeOH/CH2Cl2) gave 14 (70
mg, 56%): mp (CH2Cl2/hexane) 149.5-151.5 °C; 1H NMR
[(CD3)2SO] δ 9.93 (br t, J ) 5.3 Hz, 1 H, NHCH2), 8.66 (s, 1 H,
H-5), 8.00 (s, 1 H, H-4), 7.21 (br s, 1 H, NH), 6.63 (s, 3 H,
H-2′,4′,6′), 6.58 (s, 1 H, H-8), 6.33 (br s, 2 H, NH2), 3.81 (s, 6
H, 2OCH3), 3.30 (qd, J ) 7.2, 5.6 Hz, 2 H, NHCH2), 1.19 (t, J
) 7.2 Hz, 3 H, CH3); 13C NMR δ 161.10 (s, 2 C, C-3′,5′), 160.28
(s, C-7), 153.37, 152.21 (2 s, CONH, C-2), 151.45 (d, C-5),
149.60 (s, C-8a), 137.50 (d, C-4), 137.48 (s, C-1′), 121.19 (s,
C-3), 113.33 (s, C-4a), 107.08 (d, 2 C, C-2′,6′), 100.17 (d, C-4′),
96.12 (d, C-8), 55.38 (q, 2 C, 2OCH3), 34.09 (t, NCH2), 15.04
(q, CH3). Anal. (C19H21N5O3‚H2O) C, H, N.
Further elution of the column with 1.5-2.5% MeOH/CH2-
Cl2 gave a mixture; then further elution with 3.5-6% MeOH/
CH2Cl2 gave crude recovered 12 (78 mg, 76%) as an oil.
3-(3,5-Dim eth oxyp h en yl)-N2,N2,N7,N7-tetr a k is(4-m eth -
oxyben zyl)-1,6-n a p h th yr id in e-2,7-d ia m in e (24). A solution
of 12 (51 mg, 0.172 mmol) in dry DMF (5 mL) was treated
with 60% NaH (60 mg, 1.50 mmol); then the mixture was
sealed under N2 (as above) and stirred at 20 °C for 5 min.
4-Methoxybenzyl chloride (0.183 mL, 1.35 mmol) was added;
then the mixture was stirred at 20 °C for 1 day. The resulting
solution was cooled in ice, then treated with ice/aqueous
NaHCO3 (50 mL) and extracted with EtOAc (6 × 50 mL). The
extracts were evaporated to dryness and the residue was then
chromatographed on silica gel. Elution with 50-75% CH2Cl2/
light petroleum gave foreruns; then further elution with 80%
CH2Cl2/light petroleum and CH2Cl2 gave 24 (83 mg, 62%): mp
N-[7-Am in o-3-(2,6-d ich lor op h en yl)-1,6-n a p h t h yr id in -
2-yl]-N′-eth ylu r ea (10): Exa m p le of Gen er a l Meth od C.
A solution of 9 (133 mg, 0.436 mmol) in dry DMSO (5 mL)
was treated with 60% NaH (24 mg, 0.60 mmol); then the
mixture was sealed under N2 (as above) and stirred at 40-50
°C for 5 min and then at 20 °C for 90 min. A solution of ethyl
isocyanate (38 µL, 0.481 mmol) in dry DMSO (1 mL, then 2 ×
0.5 mL to rinse) was added (dropwise via syringe); then the
mixture was stirred at 20 °C for 1 day. The resulting mixture
was cooled in ice, then treated with ice/aqueous NaHCO3 (50
mL) and extracted with EtOAc (5 × 50 mL). The extracts were
evaporated to dryness and the residue was then chromato-
graphed on silica gel. Elution with 0-0.5% MeOH/CH2Cl2 gave
foreruns; then further elution with 0.5-1.25% MeOH/CH2Cl2
1
(CH2Cl2/hexane) 143.5-145.5 °C; H NMR [(CD3)2SO] δ 8.74
(s, 1 H, H-5), 7.91 (s, 1 H, H-4), 7.20 (d, J ) 8.7 Hz, 4 H, 2H-
2′′,6′′), 7.02 (d, J ) 8.6 Hz, 4 H, 2H-2′′′,6′′′), 6.88 (d, J ) 8.7
Hz, 4 H, 2H-3′′,5′′), 6.78 (d, J ) 8.7 Hz, 4 H, 2H-3′′′,5′′′), 6.68
(d, J ) 2.2 Hz, 2 H, H-2′,6′), 6.46 (t, J ) 2.3 Hz, 1 H, H-4′),
6.40 (s, 1 H, H-8), 4.79 (s, 4 H, N(CH2)2), 4.19 (s, 4 H, N(CH2)2),

Selective Inhibitors of FGFR-1 TK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4207
3.74, 3.72, 3.69 (3 s, 3 × 6 H, 6OCH3). Anal. (C48H48N4O6‚
chloride (301 mg, 1.08 mmol); then the mixture was sealed
under N2 (as above) and stirred at 50 °C for 20 h. Further Et3N
(1.0 mL, 7.19 mmol), trityl chloride (340 mg, 1.22 mmol) and
dry THF (5 mL) were added; then the mixture was sealed
under N2 and stirred at 60 °C for 2 days. The resulting mixture
was concentrated under reduced pressure (to ca. 1 mL) and
then treated with ice/aqueous Na2CO3 (50 mL) and extracted
with EtOAc (4 × 50 mL). The combined extracts were
evaporated to dryness and the residue was then rapidly (total
time ca. 40 min) flash chromatographed on silica gel. Elution
with 0-0.5% MeOH/CH2Cl2 gave foreruns; then further elution
with 1% MeOH/CH2Cl2 gave an oil (95 mg) which upon
crystallization from CH2Cl2/hexane gave 28 (73 mg, 80%). The
remaining liquors (22 mg) contained ditrityl derivative 29 (see
below).
3-(3,5-Dim eth oxyp h en yl)-N2,N7-d itr ityl-1,6-n a p h th yr i-
d in e-2,7-d ia m in e (29) a n d 3-(3,5-Dim eth oxyp h en yl)-N2-
tr ityl-1,6-n a p h th yr id in e-2,7-d ia m in e (30). The mother li-
quors from the crystallization of 28 above (method B) were
combined with similar material from a repeat reaction (total
45 mg) and loaded onto a narrow column of silica gel (12 g) in
CH2Cl2, then allowed to stand at 20 °C. After 1, 2 and 3 days
the column was eluted with small amounts of CH2Cl2 (<10
mL); then, after 4 days, further elution with CH2Cl2 gave crude
3-(3,5-dimethoxyphenyl)-N2,N7-ditrityl-1,6-naphthyridine-2,7-
diamine (29) (4.5 mg) as an oil: 1H NMR [(CD3)2SO] δ 8.27 (s,
1 H, H-5), 7.60 (s, 1 H, H-4), 7.4-7.1 (m, 30 H, 6H-
2′′,3′′,4′′,5′′,6′′), 6.97 (br s, 1 H, NH), 6.66 (d, J ) 2.2 Hz, 2 H,
H-2′,6′), 6.56 (t, J ) 2.2 Hz, 1 H, H-4′), 6.43 (br s, 1 H, NH),
5.68 (br s, 1 H, H-8), 3.76 (s, 6 H, 2OCH3); HRFABMS calcd
for C54H45N4O2 m/z (MH+) 781.3543, found 781.3547.
Further elution of the column with 1% MeOH/CH2Cl2 gave
an oil (16 mg) which was further chromatographed on silica
gel. Elution with 0-10% EtOAc/CH2Cl2 gave foreruns; then
further elution with 10-15% EtOAc/CH2Cl2 gave 3-(3,5-
dimethoxyphenyl)-N2-trityl-1,6-naphthyridine-2,7-diamine (30)
(6.5 mg): mp (DMSO/water) 115-118 °C; 1H NMR [(CD3)2-
SO] δ 8.38 (s, 1 H, H-5), 7.68 (s, 1 H, H-4), 7.26 (m, 12 H,
3H-2′′,3′′,5′′,6′′), 7.17 (tt, J ) 6.8, 1.9 Hz, 3 H, 3H-4′′), 6.73 (d,
J ) 2.1 Hz, 2 H, H-2′,6′), 6.60 (br s, 1 H, NH), 6.57 (t, J ) 2.2
Hz, 1 H, H-4′), 5.84 (s, 1 H, H-8), 5.80 (br s, 2 H, NH2), 3.77 (s,
6 H, 2OCH3); 13C NMR δ 161.03 (s, 2 C, C-3′,5′), 159.36 (s,
C-7), 154.07 (s, C-2), 150.97 (s, C-8a), 150.31 (d, C-5), 145.13
(s, 3 C, 3C-1′′), 139.07 (s, C-1′), 134.69 (d, C-4), 128.39, 127.44
(2 d, 2 × 6 C, 3C-2′′,3′′,5′′,6′′), 126.31 (d, 3 C, 3C-4′′), 121.88
(s, C-3), 112.72 (s, C-4a), 106.55 (d, 2 C, C-2′,6′), 100.16 (d,
C-4′), 96.58 (d, C-8), 70.44 (s, C(Ph)3), 55.25 (q, 2 C, 2OCH3);
HRFABMS calcd for C35H31N4O2 m/z (MH+) 539.2447, found
539.2460.
0.5H2O) C, H, N.
3-(3,5-Dim eth oxyp h en yl)-N2,N7-bis(4-m eth oxyben zyl)-
1,6-n a p h th yr id in e-2,7-d ia m in e (25). A solution of 24 (0.8
mg, 1.03 µmol) in TFA (1 mL) was stirred at 50 °C for 5 min
in a sealed vial. The solvent was removed by blowing with dry
N2; then the residue was treated with ice/aqueous Na2CO3 (30
mL) and extracted with EtOAc (3 × 30 mL). The extracts were
evaporated to dryness and the residue was then purified by
preparative silica gel TLC, developed twice in 1% MeOH/CH2-
Cl2, to give two bands, which were each eluted with 10%
MeOH/CH2Cl2. The more polar component was identified as
3-(3,5-dimethoxyphenyl)-N2-(4-methoxybenzyl)-1,6-naphthyri-
dine-2,7-diamine (26) (0.1 mg, 23%) (see below), while the
major, less polar component was 3-(3,5-dimethoxyphenyl)-
N2,N7-bis(4-methoxybenzyl)-1,6-naphthyridine-2,7-diamine (25)
(0.4 mg, 72%), isolated as an oil: 1H NMR [(CD3)2SO] δ 8.43
(s, 1 H, H-5), 7.61 (s, 1 H, H-4), 7.27 (d, J ) 8.2 Hz, 4 H,
H-2′′,2′′′,6′′,6′′′), 6.92 (br t, J ) 6.2 Hz, 1 H, NHCH2), 6.88 (d,
J ) 8.6 Hz, 2 H, H-3′′,5′′), 6.83 (d, J ) 8.6 Hz, 2 H, H-3′′′,5′′′),
6.65 (br t, J ) 6.0 Hz, 1 H, NHCH2), 6.56 (d, J ) 2.3 Hz, 2 H,
H-2′,6′), 6.52 (t, J ) 2.2 Hz, 1 H, H-4′), 6.22 (s, 1 H, H-8), 4.53
(d, J ) 5.3 Hz, 2 H, NHCH2), 4.41 (d, J ) 4.6 Hz, 2 H, NHCH2),
3.77 (s, 6 H, 2OCH3), 3.72, 3.70 (2 s, 2 × 3 H, 2OCH3);
HRFABMS calcd for C32H33N4O4 m/z (MH+) 537.2502, found
537.2500.
3-(3,5-Dim eth oxyp h en yl)-N7-tr ityl-1,6-n a p h th yr id in e-
2,7-d ia m in e (28). Meth od A: A solution of 12 (51 mg, 0.172
mmol) in dry DMF (3 mL) was treated with 60% NaH (14 mg,
0.35 mmol); then the mixture was sealed under N2 (as above)
and stirred at 20 °C for 25 min. Trityl chloride (192 mg, 0.688
mmol) was added; then the mixture was stirred under N2 at
20 °C for 1 day (TLC almost no starting material). The
resulting mixture was cooled in ice, then treated with ice/
aqueous Na2CO3 (50 mL) and extracted with EtOAc (3 × 50
mL). The combined extracts were evaporated to dryness and
the residue was then chromatographed on silica gel. Elution
with 67-80% CH2Cl2/light petroleum gave foreruns; then
further elution with CH2Cl2 gave an oil (24 mg), which, upon
crystallization from MeOH/CH2Cl2, gave 3-(3,5-dimethoxy-
phenyl)-7-(tritylamino)-1,6-naphthyridin-2-ylformamide (27)
(13 mg, 13%): mp (MeOH/CH2Cl2) 201-203 °C; 1H NMR
[(CD3)2SO] δ 9.54 (br d, J ) 9.4 Hz, 1 H, NHCHO), 9.11 (d, J
) 9.3 Hz, 1 H, NHCHO), 8.67 (s, 1 H, H-5), 8.04 (s, 1 H, H-4),
7.49 (br s, 1 H, NH), 7.38 (d, J ) 8.1 Hz, 6 H, 3H-2′′,6′′), 7.31
(t, J ) 7.7 Hz, 6 H, 3H-3′′,5′′), 7.22 (t, J ) 7.2 Hz, 3 H, 3H-4′′),
6.57 (d, J ) 2.2 Hz, 2 H, H-2′,6′), 6.55 (t, J ) 2.2 Hz, 1 H,
H-4′), 6.26 (br s, 1 H, H-8), 3.76 (s, 6 H, 2OCH3); 13C NMR δ
162.65 (d, NCHO), 160.61 (s, 2 C, C-3′,5′), 157.67 (s, C-7),
151.19 (s, C-2), 150.74 (d, C-5), 149.40 (s, C-8a), 144.75 (s, 3
C, 3C-1′′), 138.13 (d, C-4), 137.77 (s, C-1′), 128.67, 127.76 (2 d,
2 × 6 C, 3C-2′′,3′′,5′′,6′′), 126.56 (d, 3 C, 3C-4′′), 123.09 (s, C-3),
115.43 (s, C-4a), 107.27 (d, 2 C, C-2′,6′), 100.35 (d, C-8), 99.71
(d, C-4′), 70.32 (s, C(Ph)3), 55.22 (q, 2 C, 2OCH3). Anal.
(C36H30N4O3‚0.25H2O) C, H, N.
Hyd r olysis of F or m a m id e 27. A solution of 27 (12 mg,
21.2 µmol) in MeOH (4 mL) and CH2Cl2 (2 mL) was treated
with NaOH (60 mg, 1.50 mmol) and water (0.5 mL); then the
mixture was stirred at 40 °C for 2.5 h. A solution of aqueous
NaHCO3 (25 mL) was then added and the mixture extracted
with EtOAc (4 × 20 mL). The combined extracts were
evaporated and crystallized as above to give 28 (9 mg, 79%).
Further elution of the column with 0-1% MeOH/CH2Cl2
gave 3-(3,5-dimethoxyphenyl)-N7-trityl-1,6-naphthyridine-2,7-
diamine (28) (37 mg, 40%): mp (CH2Cl2/hexane) 166-168 °C;
1H NMR [(CD3)2SO] δ 8.38 (s, 1 H, H-5), 7.63 (s, 1 H, H-4),
7.37 (d, J ) 7.5 Hz, 6 H, 3H-2′′,6′′), 7.31 (t, J ) 7.7 Hz, 6 H,
3H-3′′,5′′), 7.21 (t, J ) 7.2 Hz, 3 H, 3H-4′′), 6.97 (br s, 1 H,
NH), 6.52 (d, J ) 2.0 Hz, 2 H, H-2′,6′), 6.50 (t, J ) 2.2 Hz, 1
H, H-4′), 6.31 (br s, 2 H, NH2), 5.86 (br s, 1 H, H-8), 3.76 (s, 6
H, 2OCH3); 13C NMR δ 160.62 (s, 2 C, C-3′,5′), 158.10 (s, C-2),
156.95 (s, C-7), 151.33 (s, C-8a), 149.44 (d, C-5), 144.87 (s, 3
C, 3C-1′′), 139.20 (s, C-1′), 135.48 (d, C-4), 128.63, 127.74 (2 d,
2 × 6 C, 3C-2′′,3′′,5′′,6′′), 126.53 (d, 3 C, 3C-4′′), 121.56 (s, C-3),
113.61 (s, C-4a), 106.49 (d, 2 C, C-2′,6′), 99.73 (d, C-4′), 99.42
(br d, C-8), 70.15 (s, C(Ph)3), 55.14 (q, 2 C, 2OCH3). Anal.
(C35H30N4O2‚0.5H2O) C, H, N.
3-(3,5-Dim eth oxyp h en yl)-N2,N2-bis(4-m eth oxyben zyl)-
N7-tr ityl-1,6-n a p h th yr id in e-2,7-d ia m in e (31). A solution of
28 (144 mg, 0.268 mmol) in dry DMF (5 mL) was treated with
60% NaH (43 mg, 1.08 mmol); then the mixture was sealed
under N2 (as above) and stirred at 20 °C for 2 min. 4-Meth-
oxybenzyl chloride (0.10 mL, 0.738 mmol) was added; then the
mixture was stirred at 20 °C for 2 h. The resulting solution
was cooled in ice, then treated with ice/aqueous NaHCO3 to
give a solid, which was isolated by filtration, washing with
water and light petroleum. The filtrate was extracted with
EtOAc (4 × 150 mL); then the extracts were combined with
the solid above, evaporated to dryness and the residue was
rapidly (total time ca. 5 min) flash chromatographed on silica
gel. Elution with CH2Cl2 gave foreruns; then further elution
with 1% MeOH/CH2Cl2 gave crude 31 (173 mg, 83%) as an
oil, which was used directly. An analytical sample was
obtained by crystallization: mp (MeOH/water) 136-140 °C;
1H NMR [(CD3)2SO] δ 8.47 (s, 1 H, H-5), 7.79 (s, 1 H, H-4),
Further elution of the column with 2-10% MeOH/CH2Cl2
gave crude 12 (28 mg) as an oil.
Meth od B: A solution of 12 (50 mg, 0.169 mmol) and Et3N
(0.5 mL, 3.59 mmol) in dry THF (5 mL) was treated with trityl

4208 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Thompson et al.
7.38 (d, J ) 7.6 Hz, 6 H, 3H-2′′′,6′′′), 7.31 (t, J ) 7.6 Hz, 6 H,
3H-3′′′,5′′′), 7.22 (t, J ) 7.1 Hz, 4 H, 3H-4′′′, NH), 6.98 (d, J )
8.5 Hz, 4 H, 2H-2′′,6′′), 6.79 (d, J ) 8.6 Hz, 4 H, 2H-3′′,5′′),
6.61 (d, J ) 2.1 Hz, 2 H, H-2′,6′), 6.44 (t, J ) 2.1 Hz, 1 H,
H-4′), 6.18 (br s, 1 H, H-8), 4.13 (s, 4 H, N(CH2)2), 3.72, 3.71 (2
s, 2 × 6 H, 4OCH3); 13C NMR δ 160.64 (s, 2 C, C-3′,5′), 159.47
(s, C-2), 158.13 (s, 2 C, 2C-4′′), 157.37 (s, C-7), 149.85 (s, C-8a),
149.67 (d, C-5), 145.08 (s, 3 C, 3C-1′′′), 142.07 (s, C-1′), 138.40
(d, C-4), 129.99 (s, 2 C, 2C-1′′), 129.36 (d, 4 C, 2C-2′′,6′′), 128.67,
127.69 (2 d, 2 × 6 C, 3C-2′′′,3′′′,5′′′,6′′′), 126.42 (d, 3 C, 3C-
4′′′), 124.18 (s, C-3), 114.24 (s, C-4a), 113.50 (d, 4 C, 2C-3′′,5′′),
105.29 (d, 2 C, C-2′,6′), 100.13 (d, C-8), 99.38 (d, C-4′), 70.14
(s, C(Ph)3), 55.13, 54.90 (2 q, 2 × 2 C, 4OCH3), 51.56 (t, 2 C,
N(CH2)2). Anal. (C51H46N4O4‚0.5H2O) C, H, N.
NHCH2), 3.78 (s, 6 H, 2OCH3), 3.70 (s, 3 H, OCH3); 13C NMR
δ 160.69 (s, 2 C, C-3′,5′), 159.64 (s, C-7), 157.90 (s, C-4′′), 156.02
(s, C-2), 152.32 (s, C-8a), 150.07 (d, C-5), 139.00 (s, C-1′), 134.99
(d, C-4), 132.35 (s, C-1′′), 128.72 (d, 2 C, C-2′′,6′′), 121.59 (s,
C-3), 113.41 (d, 2 C, C-3′′,5′′), 112.92 (s, C-4a), 106.70 (d, 2 C,
C-2′,6′), 99.82 (d, C-4′), 96.46 (d, C-8), 55.12 (q, 2 C, 2OCH3),
54.89 (q, OCH3), 43.29 (t, NHCH2); HRFABMS calcd for
C24H25N4O3 m/z (MH+) 417.1927, found 417.1923. Anal.
(C24H24N4O3) C, H.
3-(3,5-Dim eth oxyph en yl)-N2-m eth yl-1,6-n aph th yr idin e-
2,7-d ia m in e (33). A solution of 15 (50 mg, 0.127 mmol) in
dry DMF (5 mL) was treated with 60% NaH (33 mg, 0.825
mmol); then the mixture was sealed under N2 (as above) and
stirred at 20 °C for 5 min. A solution of MeI (10 µL, 0.161
mmol) in dry DMF (1 mL, then 1 mL to rinse) was added
(dropwise via syringe); then the mixture was stirred at 20 °C
for 2.5 h. The resulting solution was cooled in ice, then treated
with ice/aqueous NaHCO3 (50 mL) and extracted with EtOAc
(5 × 50 mL). The combined extracts were evaporated to
dryness and the residue was then chromatographed on silica
gel. Elution with 0-90% EtOAc/light petroleum gave minor
mixtures; then further elution with 0-2.5% MeOH/EtOAc gave
an oil (18 mg), which was further chromatographed on silica
gel, eluting with 75% EtOAc/light petroleum and EtOAc, to
give 33 (14 mg, 36%): mp (DMSO/water) 80-83 °C dec; 1H
NMR [(CD3)2SO] δ 8.38 (s, 1 H, H-5), 7.57 (s, 1 H, H-4), 6.55
(d, J ) 1.9 Hz, 2 H, H-2′,6′), 6.54 (t, J ) 2.1 Hz, 1 H, H-4′),
6.36 (s, 1 H, H-8), 6.27 (br q, J ) 4.5 Hz, 1 H, NHCH3), 5.84
(br s, 2 H, NH2), 3.79 (s, 6 H, 2OCH3), 2.87 (d, J ) 4.5 Hz, 3
H, NHCH3); 13C NMR δ 160.65 (s, 2 C, C-3′,5′), 159.63 (s, C-7),
156.96 (s, C-2), 152.57 (s, C-8a), 150.01 (d, C-5), 139.07 (s, C-1′),
134.56 (d, C-4), 121.84 (s, C-3), 112.74 (s, C-4a), 106.80 (d, 2
C, C-2′,6′), 99.75 (d, C-4′), 96.53 (d, C-8), 55.15 (q, 2 C, 2OCH3),
28.33 (q, NCH3); HREIMS calcd for C17H18N4O2 m/z (M+)
310.1430, found 310.1425.
N-[2-Am in o-3-(3,5-dim eth oxyph en yl)-1,6-n aph th yr idin -
7-yl]-2,2,2-tr iflu or oa ceta m id e (18). A solution of 15 (27 mg,
0.068 mmol) in dry pyridine (3 mL) under N2 was treated with
a solution of trifluoroacetic anhydride (65 µL, 0.46 mmol) in
pyridine (2 mL) under N2; then the mixture was stirred at 20
°C for 16 h. The resulting solution was cooled in ice, then added
slowly to a stirred mixture of ice and aqueous NaHCO3. The
resulting suspension was extracted with EtOAc (4 × 50 mL);
then the combined extracts were evaporated to dryness and
the residue chromatographed on silica gel. Elution with 25-
33% EtOAc/light petroleum gave foreruns; then further elution
with 33% EtOAc/light petroleum gave 18 (21 mg, 78%): mp
(CH2Cl2/hexane) 221-222 °C; 1H NMR [(CD3)2SO] δ 11.97 (br
s, 1 H, NH), 8.82 (s, 1 H, H-5), 7.98, 7.97 (2 s, 2 × 1 H, H-4,8),
6.80 (br s, 2 H, NH2), 6.65 (d, J ) 2.3 Hz, 2 H, H-2′,6′), 6.58 (t,
J ) 2.2 Hz, 1 H, H-4′), 3.81 (s, 6 H, 2OCH3); 13C NMR δ 160.77
(s, 2 C, C-3′,5′), 159.12 (s, C-2), 155.01 (q, J C-F ) 39 Hz, Cd
O), 152.21 (s, C-7), 149.78 (d, C-5), 148.45 (s, C-8a), 138.53 (s,
C-1′), 135.33 (d, C-4), 125.42 (s, C-3), 117.83 (s, C-4a), 115.65
(q, J C-F ) 289 Hz, CF3), 107.63 (d, C-8), 106.53 (d, 2 C, C-2′,6′),
100.33 (d, C-4′), 55.25 (q, 2 C, 2OCH3). Anal. (C18H15F3N4O3)
C, H, N.
3-(3,5-Dim eth oxyp h en yl)-N2,N2-bis(4-m eth oxyben zyl)-
1,6-n a p h th yr id in e-2,7-d ia m in e (32). Crude 31 (153 mg,
0.197 mmol) was loaded onto a narrow column of silica gel
(12 g) in CH2Cl2 and allowed to stand at 20 °C. After 1 and 2
days, the column was eluted with small amounts of CH2Cl2
(<10 mL); then, after 3 days, elution with 10% MeOH/CH2Cl2
gave an oil (0.16 g) which was chromatographed on silica gel.
Elution with 0-0.5% MeOH/CH2Cl2 gave foreruns; then
further elution with 1% MeOH/CH2Cl2 gave recovered 31 (35
mg, 23%). Further elution with 1-1.5% MeOH/CH2Cl2 gave
32 (63 mg, 60%): mp (MeOH/water) 86-91 °C; 1H NMR
[(CD3)2SO] δ 8.58 (s, 1 H, H-5), 7.87 (s, 1 H, H-4), 7.06 (dt, J
) 8.6, 2.4 Hz, 4 H, 2H-2′′,6′′), 6.82 (dt, J ) 8.7, 2.4 Hz, 4 H,
2H-3′′,5′′), 6.71 (d, J ) 2.2 Hz, 2 H, H-2′,6′), 6.46 (t, J ) 2.3
Hz, 1 H, H-4′), 6.41 (s, 1 H, H-8), 6.07 (br s, 2 H, NH2), 4.22 (s,
4 H, N(CH2)2), 3.75, 3.70 (2 s, 2 × 6 H, 4OCH3); 13C NMR δ
160.68 (s, 2 C, C-3′,5′), 159.84, 159.76 (2 s, C-2,7), 158.16 (s, 2
C, 2C-4′′), 150.89 (d+s, C-5,8a), 142.24 (s, C-1′), 138.64 (d, C-4),
130.10 (s, 2 C, 2C-1′′), 129.29 (d, 4 C, 2C-2′′,6′′), 123.32 (s, C-3),
113.86 (s, C-4a), 113.55 (d, 4 C, 2C-3′′,5′′), 105.24 (d, 2 C,
C-2′,6′), 99.41 (d, C-4′), 96.50 (d, C-8), 55.14, 54.89 (2 q, 2 × 2
C, 4OCH3), 51.56 (t, 2 C, N(CH2)2); HRFABMS calcd for
C
32H33N4O4 m/z (MH+) 537.2502, found 537.2486. Anal.
(C32H32N4O4‚0.5H2O) C, H, N.
Further elution of the column with 1.5% MeOH/CH2Cl2 gave
crude 3-(3,5-dimethoxyphenyl)-N2-(4-methoxybenzyl)-1,6-naph-
thyridine-2,7-diamine (26) (2.8 mg, 3%) as an oil (see below).
Further elution with 1.5-2.5% MeOH/CH2Cl2 gave a mix-
ture; then elution with 10% MeOH/CH2Cl2 gave crude 12 (4.2
mg, 7%) as an oil.
Treatment of the recovered 31 (35 mg) on silica gel for 4
days, as above, followed by chromatography as above gave
further 32 (9 mg, 9%).
3-(3,5-Dim et h oxyp h en yl)-N2-(4-m et h oxyb en zyl)-1,6-
n a p h th yr id in e-2,7-d ia m in e (26). Meth od A: A solution of
32 (1.3 mg, 2.43 µmol) in TFA (1 mL) was stirred at 70 °C for
8 h. The solvent was removed under a stream of dry N2; then
the residue was treated with aqueous Na2CO3 (25 mL) and
extracted with EtOAc (3 × 30 mL). The extracts were
evaporated to dryness and the residue was then purified by
preparative silica gel TLC, developed first in 2% MeOH/CH2-
Cl2 and then in 1.3% MeOH/CH2Cl2. The major band was
recovered and eluted with 7% MeOH/CH2Cl2 to give the crude
product (0.8 mg), which was further purified by preparative
silica gel TLC, developed in 90% EtOAc/light petroleum, to
give 26 (0.6 mg, 59%) as an oil (see below).
Meth od B: A solution of 32 (54 mg, 0.101 mmol) in 99%
HCO2H (5 mL) was stirred at 20 °C for 20 h. The resulting
solution was cooled in ice, then added slowly to a stirred
mixture of ice and aqueous NaHCO3/Na2CO3 (200 mL) and the
resulting suspension was extracted with CH2Cl2 (4 × 100 mL).
The extracts were evaporated to dryness and the residue was
then chromatographed on silica gel. Elution with 0-1% MeOH/
CH2Cl2 gave foreruns; then further elution with 1-1.5%
MeOH/CH2Cl2 gave 26 (36 mg, 86%): mp (MeOH/water) 85-
89.5 °C; 1H NMR [(CD3)2SO] δ 8.39 (s, 1 H, H-5), 7.61 (s, 1 H,
H-4), 7.31 (d, J ) 8.6 Hz, 2 H, H-2′′,6′′), 6.85 (d, J ) 8.7 Hz, 2
H, H-3′′,5′′), 6.66 (br t, J ) 6.1 Hz, 1 H, NHCH2), 6.57 (d, J )
2.3 Hz, 2 H, H-2′,6′), 6.53 (t, J ) 2.2 Hz, 1 H, H-4′), 6.32 (s, 1
H, H-8), 5.86 (br s, 2 H, NH2), 4.55 (d, J ) 5.6 Hz, 2 H,
N -[2-[[(t er t -B u t y la m i n o )c a r b o n y l]a m i n o ]-3-(3,5-
d im eth oxyp h en yl)-1,6-n a p h th yr id in -7-yl]a ceta m id e (17).
Meth od A: A solution of 15 (100 mg, 0.253 mmol) and Et3N
(0.15 mL, 1.08 mmol) in dry THF (5 mL) under N2 was treated
with AcCl (23 µL, 0.323 mmol); then the mixture was stirred
at 20 °C for 3 days. Further Et3N (0.5 mL, 3.59 mmol) and
THF (7 mL) were added; then the resulting solution was cooled
in ice and further AcCl (75 µL, 1.05 mmol) was added
(dropwise); then the mixture was stirred at 20 °C for 5 days.
The resulting mixture was then treated with ice/aqueous Na2-
CO3 (50 mL) and extracted with EtOAc (6 × 50 mL); then the
combined extracts were evaporated to dryness to give an oil
(0.16 g). A subsample (3.3 mg) was purified by preparative
silica gel TLC (developed twice in 50% EtOAc/light petroleum)
to give two components (each recovered by elution with 8%
MeOH/CH2Cl2). The less polar compound was crude N-acetyl-
N-[2-[[(tert-butylamino)carbonyl]amino]-3-(3,5-dimethoxyphen-

Selective Inhibitors of FGFR-1 TK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4209
yl)-1,6-naphthyridin-7-yl]acetamide (35) (1.2 mg) as an oil: 1H
NMR [(CD3)2SO] δ 9.86 (br s, 1 H, NH), 9.19 (s, 1 H, H-5),
8.41 (s, 1 H, H-4), 7.81 (s, 1 H, H-8), 7.33 (br s, 1 H, NH), 6.72
(d, J ) 2.2 Hz, 2 H, H-2′,6′), 6.70 (t, J ) 2.2 Hz, 1 H, H-4′),
3.82 (s, 6 H, 2OCH3), 2.24 (s, 6 H, 2COCH3), 1.42 (s, 9 H,
C(CH3)3).
The more polar component was N-[2-[[(tert-butylamino)-
carbonyl]amino]-3-(3,5-dimethoxyphenyl)-1,6-naphthyridin-7-
yl]acetamide (17) (1.4 mg) as an oil (see below).
CO3 (50 mL) and extracted with CH2Cl2 (4 × 50 mL) to give
N-(tert-butyl)-N′-[3-(3,5-dimethoxyphenyl)-7-[[3-(4-morpholinyl)-
propyl]amino]-1,6-naphthyridin-2-yl]urea (20) (6.4 mg, 10%)
as an oil (see below).
Meth od B: (1) A solution of 17 (63 mg, 0.144 mmol) in dry
DMF (5 mL) was treated with 4-(3-chloropropyl)morpholine
hydrochloride (64 mg, 0.32 mmol) and 60% NaH (85 mg, 2.13
mmol); then the mixture was sealed under N2 (as above) and
stirred at 20 °C for 5 min and then at 52 °C for 26 h. The
resulting solution was cooled in ice, then treated with ice/
aqueous NaHCO3 (50 mL), and extracted with EtOAc (5 × 50
mL). The combined extracts were evaporated to dryness and
the residue was then chromatographed on silica gel. Elution
with 0-2.5% MeOH/CH2Cl2 gave foreruns; then further elution
with 4-6% MeOH/CH2Cl2 gave an oil (55 mg) (a mixture of
20 and 36). This oil was dissolved in MeOH (18 mL), cooled to
0 °C and treated with NaOH (0.76 g, 19.0 mmol) and water (2
mL, added dropwise); then the mixture was stirred at 0 °C for
1 h, and then at 20 °C for 43 h. The resulting solution was
treated with excess NaHCO3 in ice-water (100 mL) and
extracted with CH2Cl2 (3 × 100 mL). The combined extracts
were evaporated to dryness; then crystallization of the residue
from CH2Cl2/hexane gave 20 (31 mg, 41%): mp (CH2Cl2/
hexane) 120-121.5 °C; 1H NMR [(CD3)2SO] δ 10.21 (br s, 1 H,
NH), 8.69 (s, 1 H, H-5), 7.98 (s, 1 H, H-4), 7.03 (br s, 1 H, NH),
6.93 (br t, J ) 5.6 Hz, 1 H, NHCH2), 6.63 (t, J ) 2.1 Hz, 1 H,
H-4′), 6.62 (d, J ) 1.8 Hz, 2 H, H-2′,6′), 6.39 (s, 1 H, H-8), 3.80
(s, 6 H, 2OCH3), 3.58 (t, J ) 4.6 Hz, 4 H, O(CH2)2), 3.32 (m, 2
H, NHCH2), 2.38 (t, J ) 7.1 Hz, 2 H, NCH2), 2.36 (m, 4 H,
N(CH2)2), 1.73 (pentet, J ) 7.0 Hz, 2 H, CH2), 1.40 (s, 9 H,
C(CH3)3); 13C NMR δ 161.11 (s, 2 C, C-3′,5′), 159.54 (s, C-7),
152.38, 152.00 (2 s, CONH, C-2), 151.35 (d, C-5), 149.34 (s,
C-8a), 137.43 (s+d, 2 C, C-4,1′), 121.06 (s, C-3), 113.11 (s, C-4a),
107.07 (d, 2 C, C-2′,6′), 100.17 (d, C-4′), 94.88 (br d, C-8), 66.15
(t, 2 C, O(CH2)2), 55.99 (t, NCH2), 55.39 (q, 2 C, 2OCH3), 53.33
(t, 2 C, N(CH2)2), 49.92 (s, C(CH3)3), 39.57 (t, NCH2), 28.64 (q,
3 C, C(CH3)3), 25.68 (t, CH2). Anal. (C28H38N6O4) C, H, N.
The remaining product mixture (157 mg) in MeOH (45 mL)
was treated with NaOH (0.20 g, 5.0 mmol) and water (5 mL,
added dropwise); then the mixture was stirred at 20 °C for 30
min. The resulting solution was treated with excess aqueous
NaHCO3, concentrated under vacuum, then extracted with
EtOAc (4 × 50 mL). The combined extracts were evaporated
to dryness and the residue was then chromatographed on silica
gel. Elution with CH2Cl2 gave foreruns; then elution with 1%
MeOH/CH2Cl2 gave N-[2-[[(tert-butylamino)carbonyl]amino]-
3-(3,5-dimethoxyphenyl)-1,6-naphthyridin-7-yl]acetamide (17)
(80 mg, 72%): mp (CH2Cl2/hexane) 148-151 °C; 1H NMR
[(CD3)2SO] δ 10.75 (br s, 1 H, NH), 10.13 (br s, 1 H, NH), 8.97
(s, 1 H, H-5), 8.33, 8.23 (2 s, 2 × 1 H, H-4,8), 7.21 (br s, 1 H,
NH), 6.69 (d, J ) 2.1 Hz, 2 H, H-2′,6′), 6.67 (t, J ) 2.2 Hz, 1
H, H-4′), 3.82 (s, 6 H, 2OCH3), 2.16 (s, 3 H, COCH3), 1.41 (s,
9 H, C(CH3)3); 13C NMR δ 169.43 (s, CONH), 161.16 (s, 2 C,
C-3′,5′), 152.84, 151.75, 151.46 (3 s, CONH, C-2,7), 150.65 (d,
C-5), 148.96 (s, C-8a), 136.99 (d, C-4), 136.77 (s, C-1′), 125.05
(s, C-3), 116.94 (s, C-4a), 107.08 (d, 2 C, C-2′,6′), 105.07 (d,
C-8), 100.55 (d, C-4′), 55.43 (q, 2 C, 2OCH3), 50.06 (s, C(CH3)3),
28.57 (q, 3 C, C(CH3)3), 23.93 (q, CH3). Anal. (C23H27N5O4‚
0.5H2O) C, H, N.
Meth od B: A solution of 15 (4.83 g, 12.2 mmol) in pyridine
(100 mL) was treated (dropwise) with acetic anhydride (11.5
mL, 122 mmol); then the mixture was stirred at 20 °C for 1
day. The resulting solution was cooled in ice, then added slowly
to a stirred mixture of ice and aqueous NaHCO3, keeping the
pH at 8 with excess NaHCO3. The resulting suspension was
extracted with CH2Cl2 (4 × 200 mL) and EtOAc (4 × 200 mL);
then the combined extracts were evaporated to dryness and
the residue crystallized directly (from warm CH2Cl2/light
petroleum) to give 17 (4.91 g, 92%).
N-(ter t-Bu tyl)-N′-[3-(3,5-dim eth oxyph en yl)-7-[[3-(4-m or -
p h olin yl)p r op yl]a m in o]-1,6-n a p h th yr id in -2-yl]u r ea (20).
Meth od A: A solution of 15 (50 mg, 0.127 mmol) in dry DMF
(5 mL) was treated with 4-(3-chloropropyl)morpholine hydro-
chloride (30 mg, 0.15 mmol) and 60% NaH (32 mg, 0.80 mmol);
then the mixture was sealed under N2 (as above) and stirred
at 20 °C for 2 days. Further 4-(3-chloropropyl)morpholine
hydrochloride (192 mg, 0.96 mmol) and 60% NaH (100 mg,
2.5 mmol) were added and the mixture sealed under N2 and
stirred at 20 °C for 5 days. The resulting solution was cooled
in ice, then treated with ice/aqueous NaHCO3 (50 mL) and
extracted with EtOAc (5 × 50 mL). The combined extracts were
evaporated to dryness and the residue was then chromato-
graphed on silica gel. Elution with 0.5% MeOH/CH2Cl2 gave
recovered 15 (24 mg, 48%). Further elution with 3-10%
MeOH/CH2Cl2 gave a crude oil (33 mg), which was further
chromatographed on silica gel. Elution with EtOAc gave
foreruns; then further elution with 2.5% MeOH/EtOAc gave
an oil (2.2 mg), which was further purified by preparative silica
gel TLC, developed in 0.75% MeOH/EtOAc. Elution of the
major band with 10% MeOH/CH2Cl2 gave N-[7-amino-3-(3,5-
dimethoxyphenyl)-1-[3-(4-morpholinyl)propyl]-1,6-naphthyri-
din-2(1H)-ylidene]-N′-tert-butylurea (34) (1 mg, 1.5%) as an
oil: 1H NMR [(CD3)2SO] δ 11.34 (br s, 1 H, NH), 8.57 (s, 1 H,
H-5), 7.90 (s, 1 H, H-4), 6.71 (d, J ) 2.4 Hz, 2 H, H-2′,6′), 6.45
(t, J ) 2.3 Hz, 1 H, H-4′), 6.41 (s, 1 H, H-8), 6.09 (br s, 2 H,
NH2), 4.18 (t, J ) 6.6 Hz, 2 H, NCH2), 3.76 (s, 6 H, 2OCH3),
3.53 (t, J ) 4.6 Hz, 4 H, O(CH2)2), 2.28 (m, 4 H, N(CH2)2), 2.26
(t, J ) 7.1 Hz, 2 H, NCH2), 1.72 (pentet, J ) 6.9 Hz, 2 H, CH2),
1.42 (s, 9 H, C(CH3)3); HRFABMS calcd for C28H39N6O4 m/z
(MH+) 523.3033, found 523.3022.
The mother liquors were further purified by chromatogra-
phy on silica gel. Elution with 0-2% MeOH/CH2Cl2 gave
foreruns; then further elution with 3% MeOH/CH2Cl2 gave
material which was treated with aqueous Na2CO3 (50 mL) and
extracted with CH2Cl2 (5 × 50 mL) to give further 20 (17 mg,
23%).
(2) A solution of 17 (126 mg, 0.288 mmol) in dry DMF (10
mL) was treated with 4-(3-chloropropyl)morpholine hydrochlo-
ride (133 mg, 0.665 mmol) and 60% NaH (173 mg, 4.33 mmol);
then the mixture was sealed under N2 (as above) and stirred
at 20 °C for 5 min and then at 54 °C for 25 h. The resulting
solution was cooled in ice, then treated with ice/aqueous
NaHCO3 (100 mL), and extracted with EtOAc (5 × 100 mL).
The combined extracts were evaporated to dryness and the
residue was then chromatographed on silica gel. Elution with
0-3% MeOH/CH2Cl2 gave foreruns; then further elution with
4% MeOH/CH2Cl2 gave material which was treated with
aqueous Na2CO3 (50 mL) and extracted with CH2Cl2 (4 × 50
mL). The combined extracts were evaporated to dryness; then
crystallization of the residue from CH2Cl2/hexane gave 20 (24
mg, 16%).
Further elution with 5-6% MeOH/CH2Cl2 gave N-[7-[bis-
[3-(4-morpholinyl)propyl]amino]-3-(3,5-dimethoxyphenyl)-1,6-
naphthyridin-2-yl]-N′-tert-butylurea (37) (26 mg, 14%) as an
oil: 1H NMR [(CD3)2SO] δ 10.21 (br s, 1 H, NH), 8.75 (s, 1 H,
H-5), 8.00 (s, 1 H, H-4), 7.06 (br s, 1 H, NH), 6.64 (t, J ) 2.1
Hz, 1 H, H-4′), 6.62 (d, J ) 2.1 Hz, 2 H, H-2′,6′), 6.43 (s, 1 H,
H-8), 3.81 (s, 6 H, 2OCH3), 3.59 (t, J ) 4.4 Hz, 8 H, 2O(CH2)2),
3.59 (m, 4 H, N(CH2)2), 2.34 (m, 8 H, 2N(CH2)2), 2.32 (t, J )
7.1 Hz, 4 H, 2NCH2), 1.76 (pentet, J ) 7.0 Hz, 4 H, 2CH2),
1.40 (s, 9 H, C(CH3)3); 13C NMR δ 161.12 (s, 2 C, C-3′,5′), 157.80
(s, C-7), 152.42, 151.99 (2 s, CONH, C-2), 150.95 (d, C-5),
149.54 (s, C-8a), 137.37 (s, C-1′), 137.25 (d, C-4), 121.29 (s,
C-3), 112.55 (s, C-4a), 107.05 (d, 2 C, C-2′,6′), 100.13 (d, C-4′),
94.16 (d, C-8), 66.14 (t, 4 C, 2O(CH2)2), 55.55 (t, 2 C, 2NCH2),
55.38 (q, 2 C, 2OCH3), 53.28 (t, 4 C, 2N(CH2)2), 49.93 (s,
C(CH3)3), 46.35 (t, 2 C, N(CH2)2), 28.60 (q, 3 C, C(CH3)3), 23.90
Further elution of the second column above with 5% MeOH/
EtOAc gave material which was treated with aqueous Na2-

4210 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Thompson et al.
(t, 2 C, 2CH2); HRFABMS calcd for C35H52N7O5 m/z (MH+)
650.4030, found 650.4036.
Animals were treated orally with test compound 17 on the
basis of average cage weight (6 mice/dose group). Treatment
was initiated on the day indicated in Table 5 and was
continued for 9 consecutive days. Compound 17 was suspended
in 0.5% methylcellulose in water due to its low aqueous
solubility, with dosing suspensions being 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) and tumor
growth delay (T-C) was performed as described previously.37-39
HUVEC, C6, a n d A90 Cellu la r P r olifer a tion Assa ys.
Tissue culture plates (96 well) were seeded with 100 µL of cells
in rows A-G, with row H remaining empty as a blank.
HUVECs (Clonetics) were grown in EGM media (Clonetics)
containing 2% fetal bovine serum. The cell seed density for
HUVECs was 20 000/mL. C6 cells (ATCC) were seeded at
6000/mL in F10 medium supplemented with 15% horse serum,
2.5% fetal bovine serum, and 6.0 mL of 200 mM glutamine
per 600 mL of medium. A90 cells (Dr. Kent Crickard, SUNY/
AB Medical School) were also seeded at 6000/mL but grown
in RPMI1640 plus 10% fetal bovine serum. Unless noted
otherwise, tissue culture media and components were from
GIBCO. Cells were allowed to incubate at 37 °C, 5% CO2, and
100% relative humidity for 16-24 h.
Ack n ow led gm en t. This work was partially sup-
ported by the Auckland Division of the Cancer Society
of New Zealand.
Refer en ces
Stock 5 mM solutions of compounds in DMSO were diluted
to 50 µM in EGM medium and serially diluted in duplicate
wells of the previously prepared cell plates, which were then
incubated as above for an additional 4 days. Media were then
removed and the cells were fixed using 10% trichloroacetic acid
for 30 min at 4 °C. The plates were then washed with distilled
water (5×), and the wells were treated with sulforhodamine
B (100 µL of 0.75% in 1% AcOH). Following staining, excess
stain was removed, the plates were washed with 1% AcOH
(4×) and air-dried, and bound dye was solubilized with
unbuffered TRIS base (100 µL of 10 mM per well). Absorbance
was measured on a 96-well plate reader at 540 nm, using a
reference filter wavelength of 630 nm. The concentration of
(1) Folkman, J . What is the evidence that tumors are angiogenesis
dependent? J . Natl. Cancer Inst. 1990, 82, 4-6.
(2) Tanaka, A.; Furuya, A.; Yamasaki, M.; Hanai, N.; Kuriki, K.;
Kamiakito, T.; Kobayashi, Y.; Yoshida, H.; Koike, M.; Fukayama,
M. High frequency of fibroblast growth factor (FGF) 8 expression
in clinical prostate cancers and breast tissues, immunohis-
tochemically demonstrated by a newly established neutralizing
monoclonal antibody against FGF 8. Cancer Res. 1998, 58,
2053-2056.
(3) Relf, M.; LeJ eune, S.; Scott, P. A. E.; Fox, S.; Smith, K.; Leek,
R.; Moghaddam, A.; Whitehouse, R.; Bicknell, R.; Harris, A. L.
Expression of the angiogenic factors vascular endothelial cell
growth factor, acidic and basic fibroblast growth factor, tumor
growth factor â-1, platelet-derived endothelial cell growth factor,
placenta growth factor, and pleitrophin in human primary breast
cancer and its relation to angiogenesis. Cancer Res. 1997, 57,
963-969.
(4) Hamby, J . M.; Showalter, H. D. H. Small molecule inhibitors of
tumor-promoted angiogenesis, including protein tyrosine kinase
inhibitors. Pharmacol. Ther. 1999, 82, 169-193.
(5) Gastl, G.; Hermann, T.; Steurer, M.; Zmija, J .; Gunsilius, E.;
Unger, C.; Kraft, A. Angiogenesis as a target for tumor treat-
ment. Oncology 1997, 54, 177-184.
(6) Klagsbrun, M.; Edelman, E. R. Biological and biochemical
properties of fibroblast growth factors. Implications for the
pathogenesis of atherosclerosis. Arteriosclerosis 1989, 9, 269-
278.
(7) Lindner, V.; Reidy, M. A. Expression of basic fibroblast growth
factor and its receptor by smooth muscle cells and endothelium
in injured rat arteries. An en face study. Circ. Res. 1993, 73,
589-595.
(8) McLeskey, S. W.; Ding, I. Y. F.; Lippman, M. E.; Kern, F. G.
MDA-MB-134 Breast carcinoma cells overexpress fibroblast
growth factor (FGF) receptors and are growth-inhibited by FGF
ligands. Cancer Res. 1994, 54, 523-530.
(9) Mohammadi, M.; McMahon, G.; Sun, L.; Tang, C.; Hirth, P.; Yeh,
B. K.; Hubbard, S. R.; Schlessinger, J . Structures of the tyrosine
kinase domain of fibroblast growth factor receptor in complex
with inhibitors. Science 1997, 276, 955-960.
compound needed to suppress 50% of cell proliferation (IC50
was determined from the absorbance measurements.
)
HUVEC Micr oca p illa r y Assa y. Matrigel (Becton Dick-
enson) was used to coat 24-well cluster plates (Costar) (0.3
mL/well). After polymerization of the Matrigel at 37 °C for 3
h, the compounds were added in 0.5 mL of 10% EGM media
(Clonetics) at a 2× concentration. The HUVECs (Clonetics; 105-
cells/mL) were then added suspended in 0.5 mL of 10% EGM
media. After 18 h in a 37 °C 5% CO2 incubator, 200 µL of MTT
(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide,
thiazolyblue) was added, and incubation continued for 1 h at
37 °C. Images were captured with Image Analysis, and IC50
values were determined by minimum dose effect.
HUVEC In va sion Assa y. Polycarbonate filters (Costar;
8-µm pore size) were coated with Matrigel (10 µg/insert;
Beckton Dickenson) and placed in a culture hood to dry for
18-20 h. The inserts were rehydrated with serum-free EBM
media (Clonetics) for 2 h at room temperature. HUVECs were
harvested and washed twice with serum-free EBM media
containing 0.2% heat-inactivated fetal bovine serum and
adjusted to 3 × 105 cells/mL. Cells (100 µL) were added to the
top of each insert; then 10% EGM media (0.5 mL) was added
as attractant to the bottom of each well. Compounds were
made up as a 200× stock solution in DMSO and added to the
cells and the attractant. The plates were then placed in a 37
°C 5% CO2 incubator for 18-20 h. After incubation, the top of
each insert was wiped with a cotton swab and the bottom of
each well was aspirated, then rinsed once with Hank’s bal-
anced salt solution. Calcein (Am Molecular Probes; 25 µM) was
added to the bottom of each well and the plates were then
incubated in the dark for 45 min. The number of cells that
invaded the other side of the insert was counted using a
fluorescence microscope and image analysis.
(10) Nanus, D. M.; Schmitz-Drager, B. J .; Motzer, R. J .; Lee, A. C.;
Vlamis, V.; Cordon-Cardo, C.; Albino, A. P.; Reuter, V. E.
Expression of basic fibroblast growth factor in primary human
renal tumors: correlation with poor survival. J . Natl. Cancer
Inst. 1993, 85, 1597-1599.
(11) Panek, R. L.; Lu, G. H.; Dahring, T. K.; Batley, B. L.; Connolly,
C.; Hamby, J . M.; Brown, K. J . In vitro biological characteriza-
tion and antiangiogenic effects of PD 166866, a selective inhibitor
of the FGF-1 receptor tyrosine kinase. J . Pharmacol. Exp. Ther.
1998, 286, 569-577.
(12) Feng, S.; Wang, F.; Matsubara, A.; Kan, M.; McKeehan, W. L.
Fibroblast growth factor receptor
accelerates tumorigenicity of prostate epithelial cells. Cancer
Res. 1997, 57, 5369-5378.
2 limits and receptor 1
In Vivo Ch em oth er a p y. 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. Tumors were maintained and anti-
cancer efficacy determined in the inbred strain of tumor
origin: C3H for mammary adenocarcinoma 16/c, C57BL/6 for
Lewis lung carcinoma, and M5076 reticulum cell sarcoma.
(13) 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.
(14) Showalter, H. D. H.; Kraker, A. J . Small molecule inhibitors of
the platelet-derived growth factor receptor, the fibroblast growth
factor receptor, and Src family tyrosine kinases. Pharmacol.
Ther. 1997, 76, 55-71.
(15) Fry, D. W.; Nelson, J . M. Inhibition of basic fibroblast growth
factor-mediated tyrosine phosphorylation and protein synthesis
by PD 145709, a member of the 2-thioindole class of tyrosine
kinase inhibitors. Anti-Cancer Drug Des. 1995, 10, 607-622.
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.

Selective Inhibitors of FGFR-1 TK
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4211
(16) 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.
(17) 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.
(18) 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.
(19) 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]pyrimidine ty-
rosine kinase inhibitors. J . Med. Chem. 1997, 40, 2296-2303.
(20) Batley, B. L.; Doherty, A. M.; Hamby, J . M.; Lu, G. H.; Keller,
P.; Dahring, T. K.; Hwang, O.; Crickard, K.; Panek, R. L.
Inhibition of FGF-1 receptor tyrosine kinase activity by PD
161570, a new protein-tyrosine kinase inhibitor. Life Sci. 1998,
62, 143-150.
(28) Magidson, O.; Menschikoff, G. The quaternary pyridine bases.
Chem. Ber. 1926, 59, 1209-1218.
(29) Shepherd, R. G.; Bratton, A. C.; Blanchard, K. C. Properties of
the nitrogen-carbon-nitrogen system in N1-heterocyclic sulfa-
nilamides. J . Am. Chem. Soc. 1942, 64, 2532-2537.
(30) Lyon, P. A.; Reese, C. B. The structures of di-N-aroyl derivatives
of adenosine and 2-aminopyridine. J . Chem. Soc., Perkin Trans.
1 1974, 2645-2649.
(31) Palmer, B. D.; Rewcastle, G. W.; Thompson, A. M.; Boyd, M.;
Showalter, H. D. H.; Sercel, A. D.; Fry, D. W.; Kraker, A. J .;
Denny, W. A. Tyrosine kinase inhibitors. 4. Structure-activity
relationships among N- and 3-substituted 2,2′-dithiobis(1H-
indoles) for in vitro inhibition of receptor and nonreceptor protein
tyrosine kinases. J . Med. Chem. 1995, 38, 58-67.
(32) Strawn, L. M.; Mann, E.; Elliger, S. S.; Chu, L. M.; Germain, L.
L.; Niederfellner, G.; Ullrich, 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.
(33) Gross, J . L.; Herblin, W. F.; Dusak, B. A.; Czerniak, P.; Diamond,
M. D.; Sun, T.; Eidsvoog, K.; Dexter, D. L.; Yayon, A. Effects of
modulation of basic fibroblast growth factor on tumor growth
in vivo. J . Natl. Cancer Inst. 1993, 85, 121-131.
(34) Crikard, K.; Gross, J . L.; Crikard, U.; Yoonessi, M.; Lele, S.;
Herblin, W. F.; Eidsvoog, K. Basic fibroblast growth factor and
receptor expression in human ovarian cancer. Gynecol. Oncol.
1994, 55, 277-284.
(35) Garfinkel, S.; Hu, X.; Prudovsky, I. A.; McMahon, G. A.; Kapnik,
E. M.; McDowell, S. D.; Maciag, T. FGF-1-dependent prolifera-
tive and migratory responses are impaired in senescent human
umbilical vein endothelial cells and correlate with the inability
to signal tyrosine phosphorylation of fibroblast growth factor
receptor-1 substrates. J . Cell. Biol. 1996, 134, 783-791.
(36) 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 inhibi-
tors. J . Med. Chem. 1998, 41, 1752-1763.
(37) Schabel, F. M., J r.; Griswold, D. P., J r.; Laster, W. R., J r.;
Corbett, T. H.; Lloyd, H. H. Quantitative evaluation of anticancer
agent activity in experimental animals. Pharmacol. Ther. 1977,
1, 411-435.
(38) Elliott, 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.
(39) Elliott, W. L.; Roberts, B. J .; Howard, C. T.; Leopold, W. R.
Chemotherapy with [SP-4,3-(R)]-[1,1-cyclobutanedicarboxylato-
(2-)](2-methyl-1,4-butanediamine-N,N′)platinum (CI-973, NK121)
in combination with standard agents against murine tumors in
vivo. Cancer Res. 1994, 54, 4412-4418.
(21) Blankley, C. J .; Doherty, A. M.; Hamby, J . M.; Panek, R. L.;
Schroeder, M. C.; Showalter, H. D. H.; Connolly, C. Preparation
of 6-aryl pyrido[2,3-d]pyrimidines and naphthyridines for in-
hibiting protein tyrosine kinase-mediated cellular proliferation.
WO 9615128, 1996; Chem. Abstr. 1996, 125, 114688k.
(22) 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.
(23) Thompson, A. M.; Showalter, H. D. H.; Denny, W. A. Synthesis
of 7-substituted 3-aryl-1,6-naphthyridin-2-amines and 7-substi-
tuted 3-aryl-1,6-naphthyridin-2(1H)-ones via diazotization of
3-aryl-1,6-naphthyridine-2,7-diamines. J . Chem. Soc., Perkin
Trans. 1 2000, 1843-1852.
(24) Lehrfeld, J . Silica gel catalysed detritylation of some carbohy-
drate derivatives. J . Org. Chem. 1967, 32, 2544-2546.
(25) Dome, M.; Wakselman, M. Alkylation by amino- and amidoben-
zylic halides in aqueous media. II. Reaction with some nucleo-
philes. Bull. Soc. Chim. Fr. 1975, 576-582.
(26) Whitmore, F. C.; Mosher, H. S.; Goldsmith, D. P. J .; Rytina, A.
W. Heterocyclic basic compounds. I. 2-Aminoalkylamino-
pyridines. J . Am. Chem. Soc. 1945, 67, 393-395.
(27) Blicke, F. F.; Tsao, M. U. R-Alkylaminopyridines and 1,5-di-
(alkyl-R-pyridylamino)-pentanes. J . Am. Chem. Soc. 1946, 68,
905-906.
J M000161D