Alkynyl pyrimidines as dual EGFR/ErbB2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.01.111

Anilinoalkynylpyrimidines were prepared and evaluated as dual EGFR/ErbB2 kinase inhibitors. A preference was found for substituted phenyl and heteroaromatic rings attached to the alkyne. In addition, the presence of a potential hydrogen bond donor appende

Full text of DOI:10.1016/j.bmcl.2006.01.111

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2419–2422
Alkynyl pyrimidines as dual EGFR/ErbB2 kinase inhibitors
Alex G. Waterson,* Kirk L. Stevens, Michael J. Reno, Yue-Mei Zhang,
Eric E. Boros, Frederic Bouvier, Abdullah Rastagar, David E. Uehling,
Scott H. Dickerson, Bryan Reep, Octerloney B. McDonald, Edgar R. Wood,
David W. Rusnak, Krystal J. Alligood and Sharon K. Rudolph
GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709-3398, USA
Received 16 December 2005; revised 25 January 2006; accepted 26 January 2006
Available online 17 February 2006
Abstract—Anilinoalkynylpyrimidines were prepared and evaluated as dual EGFR/ErbB2 kinase inhibitors. A preference was found
for substituted phenyl and heteroaromatic rings attached to the alkyne. In addition, the presence of a potential hydrogen bond
donor appended to this ring was favored. Selected molecules in the series demonstrated some activity against human tumor cell lines.
Ó 2006 Elsevier Ltd. All rights reserved.
F
Protein tyrosine kinase inhibitors are a source of tremen-
dous interest due to their promise as therapeutic agents
O
NH
N
Cl
NH
N
for the treatment of a variety of disease states, particularly
cancer.1 One of the first kinases to be successfully targeted
is the epidermal growth factor receptor (EGFR), with
some inhibitors already demonstrating clinical benefit.2
EGFR is one of a family of four related kinases, collec-
tively called the ErbB family, that also includes ErbB2
(Her2/neu), kinase inactive ErbB3, and ErbB4.3 Inhibi-
tion of multiple kinases within this family has emerged
as a promising strategy for cancer treatment.2b,4 A wide
range of structural classes has been employed as kinase
inhibitors.1 The most commonly utilized template in inhi-
bition of the ErbB family is the anilinoquinazoline.
N
O
O
N
O
O
N
O
O
1, Gefitinib
2, Erlotinib
O
F
NH
Cl
O
N
N
H
SO2Me
N
3, Lapatinib
Figure 1. Examples of clinical ErbB family inhibitors.
Examples include the launched molecules gefitinib (1)5
and erlotinib (2),6 as well as the clinical candidate lapat-
inib (3) (GW572016)7 (Fig. 1). While gefitinib and erloti-
nib are selective inhibitors of EGFR over ErbB2,
lapatinib is a potent inhibitor of both.7
alkyne to the 5 position of the pyrimidine nucleus would
place functional groups into a similar orientation as the
substituted furan of lapatinib (Fig. 2). In initial studies,
the fluorobenzyloxy-chloroaniline present in lapatinib
was conserved in order to further maximize the chances
of identifying dual inhibitors.8
In this paper, dual EGFR/ErbB2 inhibitors based upon
truncated quinazolines, specifically the pyrimidine sub-
structure, are described. It appeared that attaching an
Alkynyl pyrimidyl derivatives were synthesized accord-
ing to the sequence outlined in Scheme 1. Treatment
of pyrimidone 4 with sodium hydroxide and iodine
was followed by chlorination with POCl3 to give iodo-
chloropyrimidine 5. Displacement of the chloride with
the appropriate aniline was performed in isopropanol
in the presence of catalytic acid to give 6. Analogs were
constructed from this key intermediate in two ways.
Keywords: Receptor tyrosine kinase; Kinase inhibition; EGFR; ErbB2;
Medicinal chemistry.
*
Corresponding author. Tel.: +1 919 483 6262; fax: +1 919 483
6053; e-mail: Alex.G.Waterson@gsk.com
Present address: J&J Pharm, 1000 Route 202, Raritan, NJ 08869,
USA.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.111

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A. G. Waterson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2419–2422
Table 1. Substituted anilinoalkynylpyrimidines and selected other
compounds
a
EGFR IC50 ErbB2 IC50
a
Compound
R
3
Des-iodo 6
—
—
0.010
0.009
21.0
0.320
7
H
–Ph
0.085b
0.079b
0.063b
0.042b
0.030
3.80
8a
8b
8c
8d
0.78b
0.091b
0.044b
0.036
2-Aminophenyl
3-Aminophenyl
4-Aminophenyl
O
Figure 2. Overlay of anilinoquinazoline nucleus (gray) with ani-
linoalkynylpyrimidine (green).
8e
8f
0.012
0.013
1.82
N
H
H
N
25.1
O
O
0.11c
0.045
0.032
0.052b
0.032
0.029
O
O
Cl
8g
8h
8i
S
N
F
I
O
H
a,b
N
c
N
NH
Cl
I
N
N
H
N
N
0.079
0.060b
0.050
0.031
4
5
N
O
g
6
d,e
H
N
O
Cl
F
O
Ar
N
NH
R
NH
N
O
O
f
N
8j
N
H
S
O
O
N
8
O
7
8k
N
H
O
Ar = 3-chloro-4-(3-fluorobenzyloxy)aniline
a Mean values in micromolar, at least two determinations, standard
deviation less than 0.025.
Scheme 1. General synthetic route. Reagents and conditions: (a)
NaOH, I2, 64%; (b) POCl3, 88%; (c) ArNH2, i-PrOH, cat HCl, 90 °C,
86%; (d) trimethylsilyl acetylene, PdCl2(PPh3)2, CuI, Et3N, THF,
65 °C, 83%; (e) TBAF, THF, 0 °C, 94%; (f) RBr, PdCl2(PPh3)2, CuI,
Et3N, THF, 65 °C, 20–75%; (g) R-alkyne, PdCl2(PPh3)2, CuI, Et3N,
THF, 65 °C, 19–78%.
b One determination.
c Deviation <0.030.
parent unsubstituted analog 8a. Acetamide analogs were
also prepared by acylation with acetic anhydride and
demonstrated striking SAR. The para-acetamide 8f
was greater than 1 lM, while the meta-acetamide 8e
had a further 3-fold increase in dual EGFR/ErbB2
potency compared with 8c, resulting in IC50’s approach-
ing 10 nM for both enzymes. Based on homology mod-
eling, the meta-acetamide may occupy roughly the same
physical space as the amine in lapatinib. It is possible
that a heteroatom in this position is important for suc-
cessful dual kinase inhibition in these compounds. It is
speculated that the acetamide may be functioning as a
hydrogen bond donor, interacting with Asp808 of
ErbB2. In an attempt to optimize the character of this
functionality, the simple acetamide was replaced with
a number of related functional groups, including sulfon-
amide 8g and reversed amide 8h. Neither of these com-
pounds improved potency. Analog 8i, bearing an
additional methylene unit between the aryl ring and
the acetamide, was constructed by performing the
Sonogashira coupling reaction with N-[(3-iodophe-
nyl)methyl]acetamide, which was constructed from
3-iodobenzyl amine. This homolog likewise had
decreased activity compared to 8e. Amides 8j and k,
bearing extended alkyl chains, were generated by
reacting 8c with acyl chlorides. Both amides had
EGFR/ErbB2 dual potencies lower than 8e.
Alkyne 7 was obtained by a Sonogashira coupling with
trimethylsilyl acetylene, followed by removal of the silyl
group with TBAF. A second Sonogashira coupling was
then employed to introduce desired substituents. Alter-
natively, derivatives could be directly constructed from
iodide 6 using an appropriately substituted alkyne. In
some cases, additional functionality was introduced
using known chemical transformations (vida infra).
The potency of several aryl ring derivatives is reported
in Table 1. EGFR and ErbB2 enzyme inhibition values
were obtained as previously described.9 For compari-
son, lapatinib shows 10 nM potency against EGFR
and 9 nM potency against ErbB2. Compounds in the
alkynyl pyrimidine series that exhibit 20 nM or better
potency on both kinases were desired. The parent anili-
nopyrimidine (des-iodo 6) showed only modest inhibi-
tion of EGFR (0.32 lM) and was 21 lM against
ErbB2. Unfunctionalized alkyne 7 showed a 4-fold
improvement in EGFR potency, but was still only
3.8 lM against ErbB2. Placing a phenyl ring on the al-
kyne gave 8a, with sub-micromolar potency on both en-
zymes. ortho-, meta-, and para-Amino phenyl analogs
(8b–d) were prepared and were found to have a 9- to
20-fold increase in ErbB2 potency compared with the

A. G. Waterson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2419–2422
2421
In addition to phenyl-substituted alkynes, heterocyclic
analogs were also evaluated (Table 2). The 3-pyridyl
analog 8l is similar to 8a against EGFR and 5-fold bet-
ter against ErbB2. The 2-pyridyl compound 8m exhibit-
ed very good affinity for EGFR and also demonstrated a
>10-fold improvement in ErbB2 potency compared with
the phenyl derivative 8a. Pyrimidine and pyrazole
groups (8n and 8o, respectively) were also synthesized
and, despite similar ErbB2 potencies to 8m, were less po-
tent on EGFR than the pyridyl compound 8m. In the
phenyl series, the presence of a putative hydrogen bond
donor in the meta position was important. Several het-
erocycles with small hydrogen bond donating substitu-
tions were evaluated to determine if this was also
important with the heterocyclic analogs. The aminopyr-
imidine 8p demonstrated 4-fold improved activity
against ErbB2 compared with 8m. However, the pyridyl
meta-acetamide 8q was less effective in contrast to the
phenyl series. The hydrogen bonding potential of the
acetamide may be compromised due to the lower elec-
tron density of the pyridyl ring or by the proximity of
the heterocyclic nitrogen’s lone pair of electrons. Other
heterocylic derivatives likewise met with mixed success.
The hydroxymethyl thiazole 8r showed reduced activity
toward both kinases when compared to 8m, while the
hydroxymethyl furan 8s demonstrated ErbB2 potency
similar to that of 8m. Placing the hydroxymethyl moiety
onto a 2-pyridinyl ring with analog 8t enhanced potency
against ErbB2 with a 4-fold improvement over 8m. A
number of different functional groups were found to
be tolerated in the pseudo-benzylic pyridyl position.
The primary amine 8u10 was a 21 nM EGFR inhibitor
with 38 nM potency against ErbB2. Methyl urea 8v
was also a good EGFR inhibitor, with potency similar
to that of 8m, but fell somewhat short against ErbB2.
Adding tethered functionality resulted in sulfone analog
8w, with an IC50 <15 nM in both the EGFR and ErbB2
enzyme assays.
It seems that having a potential hydrogen bond donor in
the meta position of an aromatic ring can be important
for ErbB2 inhibition. However, it appears that the effec-
tiveness of the putative hydrogen bond can be affected
by its electronic environment, whether via substitution
around the donor or by changing the nature of the aro-
matic ring. Although no X-ray co-crystal structure yet
exists for these compounds in ErbB2, a homology model
(Fig. 3) suggests that the molecule binds in the same
fashion as lapatinib, making a hinge contact at
Met801. The aniline portion should extend past the
Thr798 ‘gatekeeper’ residue deep into a large hydropho-
bic pocket. This binding model places the potential
hydrogen bond donor substituents in the proximity of
Asp808 of ErbB2. We expect similar binding in EGFR.
Some of the more interesting compounds have been fur-
ther profiled. Compounds 8e, k, s, t, and w were greater
than 100-fold selective against a panel of non-ErbB fam-
ily kinases, including CDK2, GSK3, SRC, and
VEGFR2.
Table 2. Heteroarylalkynylpyrimidines
a
EGFR IC50 ErbB2 IC50
a
Compound R
8l
3-Pyridyl
2-Pyridyl
0.074c
0.017
0.14
8m
8n
8o
0.055c
0.060
0.066b
2-Pyrimidyl
3-Pyrazolyl
0.077c
0.042b
These same compounds were also tested in cellular pro-
liferation assays (Table 3).11 Most of the inhibitors did
not exhibit the level of cellular potency predicted by
their enzyme activity. A number of potential explana-
tions exist for this phenomenon. One possibility is a lack
of cell penetration. Further investigation revealed that
8e did not inhibit kinase activity in a cellular autophos-
phorylation assay (data not shown), possibly suggesting
N
8p
8q
8r
8s
8t
0.032
0.091
0.076
0.033
0.040
0.021
0.023
0.015
0.012
0.059
0.16
N
N
NH2
O
N
H
S
N
OH
OH
0.047
0.013
0.038c
0.069
0.009
O
OH
N
8u
8v
8w
NH2
N
N
H
N
H
N
O
H
N
H
N
O
S
N
O
O
a Mean values in micromolar, at least two determinations, standard
deviation less than 0.025.
b One determination.
c Deviation <0.050.
Figure 3. Acetamide 8e docked into an ErbB2 homology model.

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A. G. Waterson et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2419–2422
Table 3. Inhibition of cellular proliferation by dual inhibitors
2. (a) Hirsch, F. R.; Witta, S. Curr. Opin. Oncol. 2005, 17,
118, and references therein; (b) Burris, H. A. The
Oncologist 2004, 9(Suppl. 3), 10.
a
a
a
Compound
HN5 IC50
BT474 IC50
HFF IC50
8e
8k
8s
13.7
3.47
0.93
4.89
0.77
21.6
4.24
2.04
5.79
0.84
>30.0
22.4
15.8
3. Yarden, Y.; Sliwkowski, M. X. Nat. Rev. Mol. Biol. 2001,
2, 127.
4. Gomez, H. L. et al. A Phase II, Randomized Trial Using
the Small Molecule Tyrosine Kinase Inhibitor Lapatinib
as a First-Line Treatment in Patients with FISH Positive
Advanced or Metastatic Breast Cancer, 41st Annual
Meeting of the American Society for Clinical Oncologists,
Orlando, FL, May 14–17, 2005; Abstract 3046. See also
Abstracts 4594 and 3583.
5. Wakeling, A. E.; Guy, S. P.; Woodburn, J. R.; Ashton, S.
E.; Curry, B. J.; Barker, A. J.; Gibson, K. H. Cancer Res.
2002, 62, 5749.
6. Moyer, J. D.; Barbacci, E. G.; Iwata, K. K.; Arnold, L.;
Boman, B.; Cunningham, A.; Diorio, C.; Doty, J.; Morin,
M. J.; Moyer, M. P.; Neveu, M.; Pollack, V. A.; Pustilnik,
L. R.; Reynolds, M. M.; Slaon, D.; Theleman, A.; Miller,
P. Cancer Res. 1997, 57, 4838.
7. (a) Spector, N.; Raefsky, E.; Hurwitz, H.; Hensing, T.;
Dowlati, A.; Dees, C.; O’Neil, B.; Koch, K.; Smith, D. A.;
Mangum, S.; Burris, H. A. Proc. Am. Assoc. Clin. Oncol.
2003, 193 (Abstract 772); (b) Rusnak, D. W.; Lackey, K.;
Affleck, K.; Wood Edgar, R.; Alligood, K. J.; Rhodes, N.;
Keith, B. R.; Murray, D. M.; Knight, W. B.; Mullin, R. J.;
Gilmer, T. M. Mol. Cancer Ther. 2001, 1, 85.
8. Zhang, Y.-M.; Cockerill, S.; Guntrip, S. B.; Rusnak, D.;
Smith, K.; Vanderwall, D.; Wood, E.; Lackey, K. Bioorg.
Med. Chem. Lett. 2004, 14, 111.
9. Brignola, P. S.; Lackey, K.; Kadwell, S. H.; Hoffman, C.;
Horne, E.; Carter, H. L.; Stuart, J. D.; Blackburn, K.;
Moyer, M. B.; Alligood, K. J.; Knight, W. B.; Wood, E.
R. J. Biol. Chem. 2002, 277, 1576.
10. Obtained from mesylation of 8t, displacement with di-tert-
butylimidocarbonate, and subsequent de-protection.
Compounds 8v and w were prepared by acylation of 8u.
11. The HN5 cell line is a head and neck carcinoma line that
overexpresses EGFR. The BT474 line is a breast carci-
noma that overexpresses ErbB2. The HFF cell line is a
human foreskin fibroblast line that represents normal
cells. Methods of testing are previously described in Ref.
7b.
8t
8w
>30.0
>30.0
a Mean values in micromolar, at least two determinations.
a lack of cell penetration. Compound 8e also exhibited a
very poor permeation rate of <3 nm/s in an artificial
membrane permeation assay.12 Two compounds, 8s
and w, did exhibit sub-micromolar cell activity in both
the cellular proliferation assays (Table 3) and in our
autophosphorylation assay, with 8w active against both
EGFR- and ErbB2-driven cell lines. For reference,
lapatinib (3) has an IC50 of approximately 25 nM on
both HN5 and BT474 cell lines.
In summary, anilinoalkynylpyrimidines have been ex-
plored as dual EGFR/ErbB2 kinase inhibitors. The best
examples showed very good inhibition in enzyme assays,
with IC50s approaching 10 nM. The most active dual
inhibitors have polar functional groups attached to an
aromatic ring that might act as hydrogen bond donors
at physiological pH. This work suggests that this substi-
tution can be important for successful dual inhibition
within this series. In addition, while some compounds
may have been limited by poor cell penetration, other
compounds were found to be sub-micromolar inhibitors
of kinase-driven cellular proliferation.
Acknowledgments
The authors thank Dr. Dennis Yamashita, Dr. Rosanna
Tedesco, and Dr. Bob Mook for their helpful sugges-
tions in the preparation of this manuscript.
12. This assay is a parallel artificial membrane permeation
assay similar to those widely reported in the literature. For
leading references, see: (a) Kansey, M.; Senner, F.;
Gubernator J. Med. Chem. 1998, 44, 923; (b) Cheng, Y.
Y.; Song, J. C.; Liu, H.; Pidgeon, C. Adv. Drug Delivery
Rev. 1996, 23, 229.
References and notes
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2004, 14, 35.