Syntheses and EGFR kinase inhibitory activity of 6-substituted-4-anilino [1,7] and [1,8] naphthyridine-3-carbonitriles

Article abstract of DOI:10.1016/j.bmcl.2004.01.034

The syntheses and EGFR kinase inhibitory activity of a series of 6-substituted-4-anilino [1,7] and [1,8] naphthyridine-3-carbonitriles are described. Both reversible and irreversible binding inhibitors were prepared. These series were compared with each other and with the corresponding 4-anilinoquinoline-3-carbonitriles. Compounds having a 1,7-naphthyridine core structure can retain high potency while those with a 1,8-naphthyridine core are significantly less active. These results are consistent with molecular modeling observations.

Full text of DOI:10.1016/j.bmcl.2004.01.034

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1411–1416
Syntheses and EGFR kinase inhibitory activity of
6-substituted-4-anilino [1,7] and [1,8] naphthyridine-3-carbonitriles
Allan Wissner,* Philip R. Hamann, Ramaswamy Nilakantan, Lee M. Greenberger,
Fei Ye, Timothy A. Rapuano and Frank Loganzo
Chemical and Screening Sciences and Oncology Research, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
Received 20 November 2003; accepted 15 January 2004
Abstract—The syntheses and EGFR kinase inhibitory activity of a series of 6-substituted-4-anilino [1,7] and [1,8] naphthyridine-3-
carbonitriles are described. Both reversible and irreversible binding inhibitors were prepared. These series were compared with each
other and with the corresponding 4-anilinoquinoline-3-carbonitriles. Compounds having a 1,7-naphthyridine core structure
can retain high potency while those with a 1,8-naphthyridine core are significantly less active. These results are consistent with
molecular modeling observations.
# 2004 Elsevier Ltd. All rights reserved.
Receptor protein tyrosine kinases play an important
role in signal transduction pathways that regulate cell
division and differentiation. Overexpression of certain
growth factor receptors such as Epidermal Growth
Factor Receptor (EGFR) is known to predict poor
prognosis in many human cancers.1À5 Compounds that
inhibit the kinase activity of EGFR after binding of
the growth factor are of potential interest as new
therapeutic antitumor agents.6
comment on the synthesis and EGFR kinase activity of
some 6-substituted 1,7 and 1,8-naphthyridine-3-carbo-
nitriles represented by the formula shown below.
Previously, we have described our research on a series of
kinase inhibitors based on compounds with a 4-anilino-
quinoline-3-carbonitrile core structure.7 We reported
that these compounds are effective inhibitors of EGFR
and Her2 kinases. We described compounds that function
in this way as conventional reversible ATP-competitive
inhibitors as well as those that function as irreversible
binding inhibitors.8,9 One example of the latter type,
EKB-569,10 is currently in phase II clinical trial for the
treatment of EGFR dependent cancers. In additional
reports, members of our laboratory have described the
activity of a number of 4-anilinoquinoline-3-carboni-
triles that inhibit kinases other than EGFR which are
involved along the signal transduction pathway.11
The goal of this study was to determine the effect on the
EGFR kinase activity resulting from incorporation of
an additional nitrogen atom at various positions on the
left-hand ring of the cyanoquinoline core structure. The
different naphthyridine derivatives will be compared
with each other and with the corresponding quinoline-3-
carbonitriles.
Compounds in the 1,8-naphthyridine series were pre-
pared as shown in Scheme 1. 2-Chloro-5-nitronicotinic
acid, 1,12 was converted to its acid chloride in an excess
of refluxing thionyl chloride. After removal of the excess
reagent and without additional purification, 2 was
condensed with the cyanoenamine 313 using Hunig’s
base in refluxing CH2Cl2 tofurnish the intermediate 4 in
In this present report, we would like to describe our
efforts to extend this work. In particular, we will
* Corresponding author. Tel.: +1-845-602-3580; fax: +1-845-602-
5561; e-mail: wissnea@wyeth.com
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.034

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A. Wissner et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1411–1416
good yield. Cyclization of 4 tothe intermediate 5 having
the 1,8-naphthyridine core was accomplished quantita-
tively by refluxing in a mixture of EtOH and NH4OH.
This compound was chlorinated by refluxing in an
excess of POCl3, and after removal of the excess reagent
and aqueous KOH extractive work up at 0 ꢀC, 6 was
obtained as a yellow solid. Condensation of 6 with
either aniline 7a or 7b gave the desired 4-anilino-1,8-
naphthyridine-3-carbonitriles 8a and 8b, respectively, as
the hydrochloride salts. The nitro groups of these inter-
mediates were reduced by using iron powder in a
refluxing mixture of aqueous NH4Cl and MeOH. Inhi-
bitors 10 and 11 bearing the butynamide Michael
acceptor side chain were prepared by the reaction of the
mixed anhydride of butynoic acid, prepared from the
acid using isobutyl chloroformate and N-methylmor-
pholine in THF at 0 ꢀC, and adding this solution to a
mixture of 9a or 9b in pyridine, containing a catalytic
amount of DMAP. The inhibitor 12 with the acrylamide
side chain was prepared in poor yield by acylation with
acryloyl chloride in a THF–DMF mixture. The inhi-
bitor with the 4-dimethylaminocrotonamide side chain,
14, was prepared in low yield by acylation with acid
chloride 1314 at 0 ꢀC using Hunig’s base in THF, and
adding enough dry N-methyl pyrolidone to dissolve the
solids. This was followed by the addition of 15 equiv of
2M dimethylamine in THF todisplace the bromide and
incorporate the dimethylamino group. The product was
purified by chromatography.
Compounds in the 1,7-naphthyridine series were
prepared as shown in Scheme 2. 2-Chloro-5-nitropyridine
Scheme 2. (a) KF, DMSO, 70 ꢀC, 18 h; (b) Raney Ni, H2, EtOAc; (c)
(Boc)2O, t-BuOH, 40 ꢀC, 4 h; (d) 1. n-BuLi, TMEDA, ether, À78 ꢀC,
2. Dry CO2, 3. TMSCHN2 in hexanes, MeOH–CHCl3 (1:3), 0 ꢀC then
rt 2 h; (e) 1. n-BuLi, CH3CN, THF, À78 ꢀC, 1 h, 2. HOAc, warm tort;
(f) DMF–DMA, DMF (1:1), 5 h; (g) CO2Cl2, DMF (cat), CH2Cl2,
reflux 2 h; (h) EtOH reflux 8 h; (i) NaOCH2CH2N(CH3)2, THF, reflux
2 h; (j) p-MeO-PhCH2NH2, EtOH, reflux 8 days; (k) TFA–CH2Cl2
(1:1), rt 20 h; (l) 1. i-Pr2NEt, THF, 0 ꢀC, 2. NaBr, THF–DMF (10:1), 3
days and then 2M Me2NH in THF.
Scheme 1. (a) SOCl2, reflux, 5 h; (b) i-Pr2NEt, CH2Cl2, reflux, 16 h; (c)
NH4OH, EtOH, reflux, 4 h; (d) POCl3, reflux, 24 h; (e) i-PrOH, reflux,
3 h; (f) NH4Cl, Fe, H2O, MeOH, reflux, 1 h; (g) 1. butynoic acid,
i-ButOCOCl, N-methylmorpholine, THF, 0ꢀC and 2. 9a or 9b, DMAP
(cat), pyridine, rt, 1 h.; (h) acryloyl chloride, N-methylmorpholine,
THF, DMF, 3 h; (i) i-Pr2NEt, THF, N-methyl pyrolidone, 0 ꢀC, 1.5 h
and then 2M Me2NH in THF, 30 min.

A. Wissner et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1411–1416
1413
was converted to the fluoro derivative 16 by displace-
ment, using KF in hot DMSO. The nitro group was
then reduced with Raney nickel togive 17. The amino
group was then protected as the Boc derivative. Lithia-
tion of 18 in ether at À78 ꢀC was regioselective and was
accomplished with n-BuLi and tetramethyethylene-
diamine (TMED). The lithiated species was then quen-
ched with dry CO2 and the resulting acid was
methylated using (trimethylsilyl)diazomethane to give
19. Cyclization of 19 tointermediate 21 having a 1,7-
naphthyridine core structure was accomplished in good
yield using a multi-step process beginning with the for-
mation of the lithium anion of acetonitrile in THF at
À78 ꢀC using n-BuLi. A solution of 19 was added toan
excess (3 equiv) of the anion solution and after 1 h, the
mixture was quenched with an excess of acetic acid and
then warmed to room temperature giving 20. The reac-
tion of 20 with dimethylformamide dimethylacetal
(DMA–DMF) at room temperature for 5 h gave the
1,7-naphthyridine derivative 21. This compound was
chlorinated using oxalyl chloride in refluxing CH2Cl2 in
the presence of a catalytic amount of DMF. Condensa-
tion of 22 with either aniline 7a or 7b gave the desired 4-
anilino-3-cyano 1,7-naphthyridines 23a and 23b,
respectively. Inhibitors 24 and 25 with the 6-(2-dime-
thylaminoethoxy) side chain were prepared by refluxing
a THF solution of 23a or 23b with sodium 2-dimethyl-
amino-ethoxide. The inhibitor 28 in the 1,7-naphthyr-
idine series, having the 4-dimethylamino crotonamide
Michael acceptor side chain, was prepared by the reac-
tion of 23a with 4-methoxy-benzylamine in refluxing
EtOH for 8 days to give 26. The deprotection of the
amino group was accomplished in good yield using
TFA in CH2Cl2.
earlier studies that the 3-bromoanilino and the 3-chloro-
4-fluoroanilino substituents at the 4-position of the qui-
nazoline and quinoline-3-carbonitrile cores are among
the optimal choices when targeting EGFR and, conse-
quently, only these substituents have been used in the
present work.
These naphthyridine derivatives have been evaluated for
their ability toinhibit EGFR kinase. These data are
shown in Table 1. For comparison, we have also inclu-
ded similar data for several quinoline-3-carbonitriles
(29–32)8 that correspond in structure to some of the
naphthyridines. In addition, data is provided for our
clinical lead EKB-569.8 We measured the ability of
these compounds to inhibit the phosphorylation of a
peptide substrate by EGFR kinase, as well as the ability
to inhibit EGFR autophosphorylation, using a protocol
and enzyme source that is different than that described
in our earlier studies.17 We find that the IC50 for inhi-
biting autophosphorylation is always significantly
greater than the IC50 for inhibiting phosphorylation of a
peptide substrate. However, the relative activities of the
inhibitors are highly correlated between these different
measurements. In earlier reports,8,14 we already com-
mented on some of the pitfalls involved in the inter-
pretation of IC50 measurements when it comes to
irreversible binding inhibitors. Such measurements
reflect a combination of the ability of an inhibitor to fit
at the active site as well as its reactivity towards the
target Cys residue.
It is evident that in the 1,7-naphthyridine series, the 6-
(2-dimethylaminoethoxy) side-chain is not conducive
for good enzyme inhibitory activity as shown by the
results obtained for 24 and 25. In contrast, the com-
pound in this series, 28, which was designed tofunction
as an irreversible binder, is an extremely potent inhi-
bitor of the enzyme with an IC50 of 1.9 nM for inhibit-
ing phosphorylation of the substrate. This compound
appears to be more potent than the corresponding inhi-
bitor, 32, in the quinoline-3-carbonitrile series. It is also
evident that 28 is much more potent than the corres-
ponding analogue 14 in the 1,8-naphthyridine series,
where the IC50 values differ by 1000-fold. In comparing
14 and 32, it is clear that placing a nitrogen atom at the
8-position is detrimental to activity. This is also evident
when compounds with the other types of Michael
acceptors are compared. In each case, the 1,8-naph-
thyridine 10, 11, o r12 are significantly (11- to37-fold)
less potent than the respective quinoline-3-carbonitrile
29, 30, o r31.
The side chain was introduced by acylation of 27 with
acid chloride 13 as described above. A mixture of the 4-
bromo and 4-chloro butenamides was obtained. This
was treated with NaBr in THF–DMF for 3 days to
convert the chloro derivative to the bromide. The mixture
was quenched with a large excess of (CH3)2NH togive
28 in modest yield after chromatographic purification.
While the compounds such as 24 and 25 with non-
reactive side chains at the 6-position were designed to
function as conventional reversible binding ATP-com-
petitive inhibitors, those compounds with Michael
acceptor functionality at the 6-position such as 10, 11,
12, 14, and 28 were designed tofunction as irreversible
binding inhibitors. It is assumed that these latter com-
pounds will function in this manner by virtue of the fact
that they can form a covalent bond to a Cys residue
(Cys 773) located in the ATP binding pocket of EGFR.
Other groups,15 as well as ourselves,8,14,16 have pre-
viously shown that 4-anilinoquinazolines and 4-anilino-
quinoline-3-carbonitriles with appropriate Michael
acceptor functionality at the 6-position do bind cova-
lently to the target protein. In this earlier work, we
found that compounds bearing 2-butynamide, acrylam-
ide, or 4-dimethylamino crotonamide groups at the 6-
position were particularly effective in this regard; it is
for this reason that these functionalities were chosen for
the present study. In addition, we also know from our
It was alsoof interest tocompare the cellular activities
of some of these inhibitors, in particular, those with the
4-dimethylaminocrotonamide side-chains. These data
are shown in Table 1. Three human carcinoma cell lines
were used: A431 (epidermoid) which over-expresses
EGFR, SKBR3 (breast) which over-expresses HER-2
and to a lesser extent EGFR, and SW620 (colon) which
is believed not to express either EGFR or HER-2 to a
significant extent. Full experimental details for these
assays have already been reported.14 EGFR and HER-2
show a very high sequence homology in the region of

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A. Wissner et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1411–1416
Table 1. Inhibition of EGFR kinase17 and inhibition of cell growth
Compda
R1 R2
X1
X2
R3
Substrateb
Autophosc
A431d
SKBR3d
SW620d
IC50 (ÆSD) nM IC50 (ÆSD) nM IC50 (mg/mL) IC50 (mg/mL) IC50 (mg/mL)
10
11
12
14
H
F
Br
Cl
Br
Br
CH
CH
CH
CH
N
N
N
N
6266.2
3762.4
>10,000
6794.8
975.7
H
H
374.8Æ4.8
2043.6
4128.6
2.76
4.83
>5
24
25
H
F
Br
Cl
N
N
CH
CH
(H3C)2NCH2CH2O
(H3C)2NCH2CH2O
>10,000
>10,000
>10,000
>10,000
28
H
H
F
Br
Br
Cl
Br
Br
N
CH
CH
CH
CH
CH
1.9Æ0.6
258.9Æ212.9
330.2Æ289
9.7Æ2.9
8.6Æ4.1
>1000
0.088
0.036
0.356
29
CH
CH
CH
CH
30
1027.8Æ195
86.9Æ50.6
122.9Æ98.1
97.6Æ84.1
31
H
H
F
32
37.2Æ26.5
0.230
0.089
2.80
EKB-569
Cl C-OEt CH
14.5Æ15.7
a All new compounds were fully characterized by NMR and MS. Purity was assessed by elemental analysis.
b Concentration in nM needed to inhibit the phosphorylation of a peptide substrate by 50% as determined from the dose–response curve. Where
more than one test was run, IC50’s are averages of multiple determinations and the standard deviation (SD) is reported.
c Concentration in nM needed to inhibit the autophosphorylation of EGFR by 50% as determined from the dose–response curve. Where more than
one test was run, IC50’s are averages of multiple determinations and the standard deviation (SD) is reported.
d Dose–response curves were determined at 7 concentrations. The IC50 values are the concentrations in mg/mL needed toinhibit cell growth by 50%
as determined from these curves.
the active site. The Cys residue targeted in EGFR
kinase is conserved in HER-2 kinase and, therefore, we
expect (and earlier work has confirmed) that our inhi-
bitors will be effective in inhibiting the growth of cell
lines that express either of these growth factor receptors.
With respect tothe cell assays, it is evident that each
compound is a much better inhibitor of the A431 and
SKBR3 cell lines than the SW620 line. This is consistent
with the mechanism of cell growth inhibition being reli-
ant, toa degree, on the target kinases. The fact that
some of these compounds do inhibit the growth of the
SW620 line at higher concentrations could suggest that
this line has some dependence on EGFR or HER-2,
even though these receptors are not expressed to a sig-
nificant degree, or that these compounds inhibit cell
growth by an undefined mechanism at these higher
concentrations. We find that the order of activity in
inhibiting cell growth is 28>32> >14. It can be seen
that this order parallels the potency we see in the
enzyme assay.
previous EGFR kinase modeling efforts,8,9,14 we made
use of a homology model of the catalytic domain of the
enzyme that was constructed using a combination of
twodifferent crystal structures as templates, FGF
Receptor-1 for the N-terminal lobe, and Hematopoietic
Cell Kinase (Hck) for the C-terminal lobe. Recently the
X-ray crystal structure of EGFR kinase having a bound
quinazoline-based ligand was reported.18 For the pre-
sent study, we decided to use the published coordi-
nates19 from this X-ray structure as the starting point
for the modeling studies. The publication of this struc-
ture also allows us to retrospectively evaluate our earlier
homology model. Overall, our model and the reported
structure agree with respect to most of the important
features. We find a RMS difference of 4.95 A when
comparing Ca atoms of the full catalytic domains. In the
region surrounding the ATP-binding site, the RMS
difference is 1.68 A.
The ligand was removed from the published X-ray
structure and replaced with inhibitor 28. The inhibitor
was oriented in a manner similar to the original ligand,
and similarly to the orientation that we used in some of
The above observations can be nicely rationalized from
the results of molecular modeling experiments. In our

A. Wissner et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1411–1416
1415
our earlier modeling studies of the quinazolines and
quinoline-3-carbonitriles. The complex was energy
minimized using the CharmM forcefield as implemented
in QuantaTM software.20 As in our previous work, we
allowed the entire system of protein, ligand, and water
molecules to move during minimization. In the final
model (Fig. 1) the N1 atom of 28 is hydrogen-bonded to
the backbone NH of Met 769. The 3-cyano group of 28
has displaced the water molecule that bridged the N3
atom of the original quinazoline ligand to the hydroxyl
group of Thr 766. The nitrogen atom of the 3-cyano
group now interacts with that same residue. The 4-(3-
bromoaniline) moiety lies in a hydrophobic pocket con-
taining Leu 820, Thr 830, Asp 831, Val 702, Ala 719, Ile
720, Lys 721, Thr 766, and Leu 764. Most significantly,
the b-carbon atom of the Michael acceptor side chain of
28 is located 4.13A from the sulfhydryl group of Cys
773, and should be easily accessible for covalent inter-
action. Additionally, the N-atom of the dimethylamino
group on 28 is located 3.56 A away from the sulfhydryl
hydrogen of Cys 773. In our earlier work on inhibitors
in the quinazoline and quinoline-3-carbonitrile series,
we proposed (and presented experimental evidence in
support of the fact) that this dimethylamino group can
serve as an intramolecular base catalyst for Michael
additions to these types of inhibitors. We are now pro-
posing that the dimethylamino group of 28 is playing a
similar role. Also, given the arrangement of functional-
ities predicted by this binding model, we suggest that
this intramolecular catalysis operates after 28 binds at
the active site of EGFR and that the Michael addition
reaction of the sulfhydryl group of Cys 773 to bound 28
is accelerated (relative to reactions of other nucleophiles
that may be in the cytosol) due to the close proximity of
the reactive center, nucleophile, and base catalyst.
interaction of the hydrogen atom attached to C8 and
the carbonyl oxygen of Met 769 is expected to be stabi-
lizing21À23 via a weak CH–O hydrogen bond. If the 1,8-
naphthyridine analogue 14 adopted a similar binding
orientation compared to 28, not only would the favor-
able aromatic hydrogen–carbonyl interaction be lost,
but it would be replaced by an unfavorable interaction
resulting from the repulsion of the lone pairs on N8 of
the inhibitor and the carbonyl oxygen of Met 769. In
reality, it is likely that 14 would bind in a manner that
would mitigate this unfavorable interaction, but this
might orient the molecule in a less favorable position for
the covalent interaction to occur at an efficient rate.
Additionally, one would expect that this aromatic
hydrogen–carbonyl interaction would be stronger the
more polarized the C–H bond. The C–H bond at the 8-
position is expected to be more polarized in 28 com-
pared to 32 and this might account for the slightly better
potency of 28 compared 32.
Acknowledgements
The authors wish to thank Drs. Philip Frost, Tarek
Mansour, and Janis Upeslacis for their support and
encouragement. We also would like to thank the mem-
bers of the Wyeth Discovery Analytical Chemistry
group for analytical and spectroscopic determinations.
References and notes
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Of particular significance tothe case at hand, the C8
atom of 28 is located 3.18 A from the backbone car-
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account for the large difference in activity we see
between the 1,7- and 1,8-naphthyridines. For 28, the
8. Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.;
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Figure 1. Binding model of 28 at the active site of EGFR kinase.

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MnCl2, 100 mM Na3VO4), and 9 mL of water. Master mix
(29 mL) was added to each well and compounds (1 mL)
prepared in 100% dimethyl sulfoxide (DMSO) were
added toappropriate wells as 50 Â stocks. After 15 min at
rt, ATP/MgCl2 (20 mL o f 25mM ATP, 25 mM MgCl2, 10
mM HEPES, pH 7.4) was added toeach well toinitiate
the reaction. Final concentrations of assay components
were: 10 mM ATP, 10 mM MgCl2, 1 mM MnCl2, 4 mM
HEPES, pH 7.4, 20 mM Na3VO4, 20 mg/mL BSA, 2%
DMSO. After 40 min at RT, liquid was removed and
plates washed 3x with TBST (TBS with 0.05% Tween-20).
Wells were then incubated for 1 h at rt with 75 mL o fꢁ0.1
mg/mL Europium-conjugated anti-phosphotyrosine anti-
body (PT66; PerkinElmer Life Sciences, Boston, MA)
prepared in Assay Buffer (PerkinElmer). Plates were
washed 3Â with TBST then incubated for 15 min in the
dark with 100 mL of Enhancement Solution (Perkin-
Elmer). Plates were read in a Victor-V multi-label counter
(PerkinElmer) using the default Europium detection
protocol. Percent inhibition or IC50 of compounds was
calculated by comparison with DMSO-treated control wells.
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17. EGFR kinase assay: Activity of compounds was evaluated
by DELFIA (dissociation-enhanced lanthanide fluor-
escent immunoassay) essentially as described previously
(Loganzo, F.; Hardy, C. Am. Biotech. Lab. 1998, 16, 26.)
For peptide phosphorylation assays, Nunc Maxisorb 96-
well plates were coated at RT for 1 h with 100 mL per well
o f 25mg/mL poly(Glu4-Tyr) peptide (Sigma, St. Louis,
MO) in Tris-buffered saline (TBS=25 mM Tris pH 7.2,
150 mM NaCl). Unbound peptide was washed 3Â with
TBS. For autophosphorylation assays, no peptide was
used. EGFR enzyme (E-3641, approximately 0.2 mg/mL;
Sigma) was diluted in 0.1% BSA/ 4 mM HEPES (40-fold
dilution for autophosphorylation assay and 80-fold for
peptide assay). A master mix of enzyme plus kinase buffer
was prepared: (per well) 10 mL of diluted enzyme, 10 mL
o f 5Â kinase buffer (5Â=20 mM HEPES, pH 7.4, 5 mM
19. Protein Data Bank, PDB code 1M17.
20. Molecular Simulations Inc., 9685 Scranton Road, San
Diego, CA 92121.
21. Shewchuk, L.; Hassell, A.; Wisely, B.; Rocque, W.; Holmes,
W.; Veal, J.; Kuyper, L. F. J. Med. Chem. 2000, 43, 133.
22. Thomas, K. A.; Smith, G. M.; Thomas, T. B.; Feldman,
R. J. Proc. Natl. Acad. Sci. 1982, 79, 4843.
23. Lamb, M. L.; Jorgensen, W. L. J. Med. Chem. 1998, 41,
3928.