Synthesis and evaluation of aniline headgroups for alkynyl thienopyrimidine dual EGFR/ErbB-2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.01.080

Aniline 'headgroups' were synthesized and incorporated into an alkynyl thienopyrimidine series of EGFR and ErbB-2 inhibitors. Potent inhibition of enzyme activity and cellular proliferation was observed. In certain instances, protein binding was reduced a

Full text of DOI:10.1016/j.bmcl.2009.01.080

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1332–1336
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of aniline headgroups for alkynyl thienopyrimidine
dual EGFR/ErbB-2 kinase inhibitors
*
Alex G. Waterson , Kimberly G. Petrov, Keith R. Hornberger, Robert D. Hubbard, Douglas M. Sammond,
Stephon C. Smith, Hamilton D. Dickson, Thomas R. Caferro, Kevin W. Hinkle, Kirk L. Stevens,
Scott H. Dickerson, David W. Rusnak, Glenn M. Spehar, Edgar R. Wood, Robert J. Griffin, David E. Uehling
GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709-3398, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Aniline ‘headgroups’ were synthesized and incorporated into an alkynyl thienopyrimidine series of EGFR
and ErbB-2 inhibitors. Potent inhibition of enzyme activity and cellular proliferation was observed. In cer-
tain instances, protein binding was reduced and oral exposure was found to be somewhat improved rel-
ative to compounds containing the reference aniline.
Received 10 November 2008
Revised 16 January 2009
Accepted 20 January 2009
Available online 7 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Kinase inhibitor
EGFR
ErbB-2
Medicinal chemistry
The inhibition of ErbB family receptor tyrosine kinases is an
effective strategy for cancer therapy.1 The ErbB family is comprised
of four members, EGFR (ErbB-1), ErbB-2 (Her2/neu), catalytically
inactive ErbB-3, and ErbB-4.2 In addition to antibody therapy,3 sev-
eral small molecules have been approved or are under clinical
investigation to treat diseases that are a result of aberrant signaling
from one or more of these kinases. There are two clinically ap-
proved anilinoquinazoline inhibitors selective for EGFR inhibition
(erlotinib4 and gefitinib5) and one anilinoquinazoline (lapatinib6)
which is an equipotent inhibitor of both EGFR and ErbB-2. Simulta-
neous inhibition of multiple ErbB family receptors may have signif-
icant therapeutic advantage, due to differing receptor expression
patterns in human cancers and the cooperation of the ErbB recep-
tors in inducing transformation.7
It is known that the binding mode of quinazoline and related
ErbB family inhibitors places the aniline portion (referred to here-
after as the ‘headgroup’) deep in the ATP binding site of the kinase,
and that this can affect kinase selectivity.10 In this communication,
we report the effect of aniline ‘headgroup’ substitution on the
activity of alkynyl thienopyrimidine ErbB family inhibitors.11,12
Headgroup design centered on three variations of the 4-(3-flu-
orobenzyloxy)-3-chloroaniline present in lapatinib: (1) additional
benzyloxy substituted anilines, varying halo substitutions and
incorporating heterocycles into the ring systems, (2) biaryl ether
anilines and related compounds, removing the benzyl methylene,
(3) fused bicyclic anilines. These changes were intended to produce
dual EGFR/ErbB-2 inhibitors with reduced lipophilicity and protein
All of the small molecules approved to date for ErbB family inhi-
bition are reversible kinase inhibitors. Irreversible inhibitors of the
ErbB family have also been disclosed.8 These inhibitors typically
include a Michael acceptor in the molecule. We have recently re-
ported potent alkynyl thienopyrimidine based ErbB family inhibi-
tors that do not possess a typical Michael acceptor (Fig. 1) but
nonetheless form a covalent bond with a conserved cysteine resi-
due (C733 in EGFR, C805 in ErbB-2).9
"headgroup"
O
Cl
F
HN
N
H
S
N
R'
N
R
* Corresponding author at present address: Vanderbilt Institute of Chemical
Biology, 12410-D MRB-IV, Nashville, TN 37232-0412, USA. Tel.: +1 615 936 8595.
E-mail address: Alex.G.Waterson@vanderbilt.edu (A.G. Waterson).
Figure 1. Previously reported covalent alkynyl thienopyrimidine EGFR/ErbB-2
inhibitors.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.080

A. G. Waterson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1332–1336
1333
acid. The bromides (13) were then subjected to a Sonogashira reac-
tion with alkynes that have been described previously.14 Final BOC
deprotection provided the 6-ethynylthieno-[3,2-d]pyrimidin-4-
anilines (15). Alternatively, the order of the reactions was
switched, with the thienopyrimidine core (12) subjected to an ini-
tial Sonogashira coupling and then elaborated using a nucleophilic
displacement as described above. Once again, a final deprotection
of the pyrrolidine nitrogen generated the target molecules (15).
The target 6-ethynylthieno[3,2-d]-pyrimidin-4-anilines (15)
were evaluated for EGFR and ErbB-2 potency using assays that
have been described previously.6,9,10 All molecules were also tested
in cell proliferation assays, using cell lines that overexpress EGFR
or ErbB-2.15 These analogues have the potential to be irreversible
inhibitors9 by alkylating the conserved cysteine residue in the en-
zyme active site.16 As a result of the time-dependent nature of
these inhibitors, the IC50 values can change as a function of time.
Therefore, the IC50 values presented are the apparent IC50s based
on the reaction time of the enzyme assay (40 min) and should
not be used to draw conclusions about absolute potency. No con-
sensus exists in the literature concerning the use of covalent inhib-
itors. Although toxicity risks may exist, it has been suggested that
selective covalent binding may be a positive contributor to com-
pound potency, due to prolonged inhibition of the enzyme and
the ability to effectively compete with high concentrations of a
natural ligand, in this case ATP.17
YH
X
a
O2N
O2N
Y
X
Ar
Y
X
Ar
n
(
)
n
b
2
4
(
)
O2N
or
H2N
3
5
F
X
c
n = 0,1
X = F, Cl
Y = O, S
n = 0,1
Scheme 1. Reagents and conditions: (a) 1—NaH, THF; 2—for n = 1: BrCH2Ar, for
n = 0: XAr (opt. Cu salt). (b) H2, Pt/C, EtOH. (c) HYCH2Ar or HYAr, DMF, base.
R
N
R
H
N
N
b
a
X
X
X
O2N
O2N
H2N
8, X = N
11, X = CH
7, X = N
10, X = CH
6, X = N
9, X = CH
Scheme 2. Reagents: (a) ArCH2X, K2CO3 or ArSO2Cl, Et3N. (b) Pt/C, H2 or Na2S, EtOH.
binding relative to molecules containing the lapatinib headgroup.
It was also felt that the benzyloxy group may be a potential meta-
bolic liability.
A morpholinocarbamate at the pyrrolidine C4 was chosen as a
standard group for this study due to its favorable contribution to
enzyme and cellular potency, coupled with a reduced degree of
covalent reactivity and moderate pharmacokinetics.14b Compound
15a, containing the reference fluorobenzyloxychloro-aniline, dem-
onstrated potent inhibition of EGFR and ErbB-2 and displayed 3-
fold better anti-proliferative activity on the ErbB-2 overexpressing
BT474 cell line versus the EGFR driven HN5 line (Table 1). In gen-
eral, headgroup substitution had only a small effect on the level of
covalent reactivity toward the kinase. Most compounds showed
rather modest covalent modification of EGFR enzyme, less than
20% after 3 h. However, notable exceptions were observed. For
example, the napthyl derived compound 15e displayed nearly
complete modification of EGFR after only 3 h, and 15d, 15g, and
15k were also significant covalent modifiers in this time frame.
Interestingly, the compounds with the highest levels of covalent
modification tended to be among the less potent compounds in
the enzyme assays. The time-dependent nature of the inhibitor
has the potential to complicate the interpretation of enzyme and
cellular inhibition results. As the cellular assays are performed over
3 days, it is possible that all compounds may, in fact, be significant
covalent modifiers of the kinases in this longer time frame, leading
The synthetic route to benzyloxy substituted aniline head-
groups and biaryl ether headgroups began with a sodium hydride
mediated benzylation or arylation of nitro substituted phenols or
thiophenols (2) (Scheme 1). Alternately, the nitro aromatic precur-
sors were produced via a SNAr displacement of the fluoride (4) with
a benzyl alcohol or phenol. Selective reduction of the nitro group
versus the aromatic halogen was accomplished by utilizing a cata-
lytic hydrogenation with platinum on carbon to produce the de-
sired aniline (5).
Fused bicyclic anilines were prepared by the route shown in
Scheme 2. The appropriate nitroindazole (6) or nitroindole (9)
was alkylated, acylated, or sulfonylated to afford substituted het-
erocycles (7, 10). The nitro group was reduced using standard plat-
inum catalyzed hydrogenation or treatment with sodium sulfide to
give the desired bicyclic anilines (8, 11).
The target molecules were constructed using one of two routes
illustrated in Scheme 3. In the more commonly used method the
aniline headgroups were combined with the known 6-bromo-4-
chlorothieno[3,2-d]pyrimidine (12)13 in the presence of catalytic
Ar
HN
Cl
S
S
N
N
a
Br
Br
N
N
12
13
b
b,c
O
O
N
O
N
Ar
N
O
Cl
HN
N
O
S
N
O
S
a,c
N
BOC
N
H
N
15
14
Scheme 3. Reagents and condition: (a) ArNH2, HCl, i-PrOH, 80 °C. (b) RCCH, PdCl2(PPh3)2, CuI, Et3N, THF, 60 °C. (c) TFA, CH2Cl2.

1334
A. G. Waterson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1332–1336
Table 1 (continued)
Table 1
In vitro evaluation of inhibitors
Compound Ar
EGFRa,b
ErbB-
2a
HN5c BT474c
Compound Ar
EGFRa,b
80 (3)
ErbB-
2a
HN5c BT474c
N
N
O
15m
15n
80 (4)
50
30
70
70
20
30
F
15a
80
260
240
90
60
N
HN
HN
HN
HN
Cl
O
15b
15c
50e (7)
120 (2)
30e
30 (6)
F
N
N
Cl
F
S
O
N
60
440
490
70
15o
15p
15q
33
35
130
85
250
10
130
110
N
N
F
F
(NDd)
HN
HN
Cl
N
N
HN
HN
O
F
15d
2600
(43)
450
250
120 (0)
230 (0)
70
N
F
O
S
O
N
15e
15f
1050
(89)
260
60e
120
280
100
80
O
O
540
N
HN
HN
N
HN
HN
O
N
O
S
N
S
190e
(14)
Cl
15r
15s
15t
15u
150e
(16)
120e
630
190
120
110
O
15g
15h
400 (74)
280 (0)
370 1450
130
140
N
O
HN
Cl
N
90 (10)
60
F
HN
180
390
O
N
3160e
(23)
2660e 2020 4850
HN
HN
Cl
N
N
HN
HN
O
15i
15j
180 (0)
87 3470 2600
S
N
680 (0)
180
210
550
320
120
120
240 (14)
60
270
70
HN
N
15v
240 (10)
S
N
F
HN
15k
15l
1650
(48)
220
800
380
310
100
150
F
HN
HN
a
Apparent IC50 in nM, HTRF (Homogeneous Time-Resolved Fluorescence) assay
format; mean values, n P 2.
S
Number in parentheses is % covalent modification of EGFR after 3 h. See Ref. 14
IC50 in nM; mean values, n P 2.
.
b
c
d
e
630 (0)
Cl
ND = not determined.
SPA (Scintillation Proximity Assay) assay format.

A. G. Waterson et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1332–1336
1335
to smaller potency differences than might have otherwise been
anticipated.
heterocyclic headgroups relative to the substituted phenyl com-
pounds. The most dramatic improvement was realized with the
inclusion of a 2-pyridyl moiety in 15b or a thiazole in 15c, both
of which lower the cLogP of the inhibitors by over 1.5 units rel-
ative to the fluorophenyl group in reference compound 15a.
Other substitutions produced similar improvements. The bicyclic
indazole containing compounds 15m and 15n exhibited 1 log
unit improvements in lipophilicity. However, the presence of
the quinoline moiety of 15h or the sulfur linker of 15f resulted
in compounds that were more lipophilic than the reference com-
pound 15a. The retention time on an HPLC column containing
immobilized HSA (human serum albumin) was used as a surro-
gate to measure the propensity of the compounds to bind to
plasma proteins.20 Several of the aniline headgroups displayed
somewhat reduced protein binding. Notably, the heterocyclic
derivatives 15b and 15c, in addition to the indazole 15n, were
improved over 15a. Indeed, the 3% improvement in protein bind-
ing for 15c and 15n represents a potential doubling of the free
fraction for these inhibitors relative to 15a. Although many of
the less lipophilic compounds did exhibit improved protein bind-
ing, there was no direct relationship observed between cLogP
and protein binding in this series.19
The distal heterocycles 15b and 15c demonstrated a relatively
similar enzymatic and cellular profile to 15a, with 15b being
slightly more potent against the enzymes. However, the use of a
pyridyl moiety as a replacement for the aniline phenyl group
(15d) produced a compound that was significantly less potent
against the kinases. A napthyl group in this position (15e) was also
a less potent inhibitor of the kinases versus 15a. Interestingly, for
both 15d and 15e the cellular proliferation results, particularly
for the EGFR driven HN5 line, showed far better potency than the
enzyme assays would have predicted. This is likely a result of the
increased covalent modification of the enzyme by these com-
pounds. Eliminating the benzylic methylene of 15b by use of a
biaryl ether (15f) produced a compound that was somewhat selec-
tive for enzymatic ErbB-2 inhibition. Isoxazole (15g) and the larger
quinoline (15h) based replacements of the pyridyl of 15f were less
successful. Interestingly, adding a methylene spacer between the
thienopyrimidine and the phenyl ring of the headgroup via use
of a benzylamine18 produced a compound, 15i, that was a rela-
tively potent enzyme inhibitor, but performed poorly in the cellu-
lar proliferation assays. Replacing the biaryl ether oxygen with a
methylene spacer (15j)18 is tolerated in the active site, but the
thioethers 15k and 15l showed significantly reduced inhibition of
the kinases relative to 15a.
Bicyclic derived aniline headgroups, 15m–v, also enabled ro-
bust inhibition of EGFR and ErbB-2 in the enzyme and cellular pro-
liferation assays (Table 1). Indeed, the indazole compounds 15m
and 15n proved superior to the reference compound 15a. Interest-
ingly, adding a second halogen to the distal ring (15o), simulta-
neously enhanced ErbB-2 driven cellular potency and reduced
EGFR driven potency, giving a 20-fold preference for ErbB-2 inhibi-
tion in the cellular assays. Changes in the benzylic linker between
the heterocycle and the distal phenyl ring met with only limited
success. A methyl group (15p) or sulfonyl group (15q and 15r) re-
duced potency relative to 15n. Indole based heterocycles were gen-
erally inferior to the indazoles. For example, indole 15s proved
inferior to 15m in the cellular assays, although the potency in
the kinase inhibition assays was similar to 15a and 15m. Extending
the distal ring away from the indole, in 15t, was very detrimental
to kinase activity. Although the use of a distal heterocycle was
quite successful in the phenyl based series (e.g., 15b and 15c),
the use of a thiazole in 15u displayed reduced potency in the in-
dole series. Finally, the inclusion of a halogen on the indole ring
(15v) also failed to improve kinase activity.
Mouse oral exposure was also determined for several com-
pounds (measured for 6 h following
a 10 mg/kg oral dose).
Although all compounds in this series tend toward poor to moder-
ate murine exposure, aniline substitutions were found to affect the
oral exposure. The reference compound 15a had an oral DNAUC21
of 91 ng h/mL/mg/kg. Pyridyl compound 15b displayed a modest
improvement. However, eliminating the methylene group actually
led to a lower value for 15f. The fused bicyclic headgroup com-
pounds were also found to have suitable oral exposure. The inda-
zole compound 15m displayed exposure comparable to 15a,
while 15n showed somewhat reduced oral exposure relative to
15a or 15m.
In summary, we have synthesized and evaluated a number of
thieno[3,2-d]pyrimidines with changes in the aniline headgroup.
These compounds are effective dual inhibitors of EGFR and ErbB-
2 kinases and also display potent, selective inhibition of cellular
proliferation. While most compounds, including 15b and 15m,
displayed a slight preference (2- to 10-fold) for inhibition of
ErbB-2 driven cellular proliferation, selected compounds, for
example, 15e and 15p, were found to be essentially equipotent
for EGFR and ErbB-2 driven cellular proliferation. In some cases,
modest improvements in the ADMET profiles of the compounds
relative to the reference aniline were noted. These improvements
were realized by replacing phenyl rings with heterocyclic moie-
ties (i.e., 15b) to give compounds equal or superior to 15a in po-
tency, lipophilicity, and oral exposure. Bicyclic headgroups
exemplified by 15m were also found to reduce lipophilicity
and protein binding.
Selected compounds were evaluated for in vitro and in vivo
ADMET properties,19 including protein binding and oral exposure
(Table 2). Of note is the distinctly reduced lipophilicity of the
Table 2
ADMET data for selected inhibitors
Compound
cLogPa
% Protein boundb
DNAUCb,c
References and notes
15a
15b
15c
15f
15h
15j
6.11
4.46
4.31
4.83
6.42
5.79
6.98
5.02
5.16
5.96
98.0
96.9
94.9
96.7
97.0
96.4
97.6
97.3
95.0
ND
91
120
ND
52
ND
ND
ND
98
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2. Olayioye, M. A.; Neve, R. M.; Lane, H. A.; Hynes, N. E. EMBO 2000, 19, 3159.
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15l
15m
15n
15s
51
ND
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Caponigro, F.; Salomon, D. S. Curr. Drug Targets 2005, 6, 243.
a
Calculated using ACD software, version 8.
ND = not determined.
DNAUC = dose normalized area under the curve; represented in ng h/mL/mg/
8. Reported irreversible ErbB family inhibitors include canertinib: (a) Allen, L. F.;
Eiseman, I. A.; Fry, D. W.; Lenehan, P. F. Semin. Oncol. 2003, 30, 65; HKI-272 (b)
Rabindran, S. K.; Discafani, C. M.; Rosfjord, E. C.; Baxter, M.; Floyd, M. B.; Golas,
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b
c
kg; derived from murine oral PK experiment, measured for 6 h following a 10 mg/kg
oral dose.

1336
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Zacharchuk, C.; Amorusi, P.; Adjei, A. A.; Rowinsky, E. K. J. Clin. Oncol. 2006, 24,
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Hornberger, K. R.; Rusnak, D. W.; Wood, E. R.; Uehling, D. E.; Waterson, A. G.
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Emerson, H. K.; Griffin, R. J.; Hubbard, R. D.; Keith, B. R.; Mullin, R. J.; Petrov, K.
G.; Gerding, R. M.; Reno, M. J.; Rheault, T. R.; Rusnak, D. W.; Sammond, D. M.;
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11. Similar work in other ErbB family inhibitors is already reported in the literature,
including the synthesis and/or use of some of the anilines in this report. For
example: (a) Barlaam, B.; Ballard, P.; Bradbury, R. H.; Ducray, R.; Germain, H.;
Hickinson, D. M.; Hudson, K.; Kettle, J. G.; Klinowska, T.; Magnien, F.; Ogilvie, D. J.;
Olivier, A.; Pearson, S. E.; Scott, J. S.; Suleman, A.; Trigwell, C. B.; Vautier, M.;
Whittaker, R. D.; Wood, R. Bioorg. Med. Chem. Lett. 2008, 18, 674; (b) Ducray, R.;
Ballard, P.; Barlaam, B. C.; Hickinson, M. D.; Kettle, J. G.; Ogilive, D. J.; Trigwell, C.
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Fink, B. E.; Gavai, A.; Han, W.-C.; Johnson, W.; Langley, D.; Lee, F. Y.; Leavitt, K.;
Marathe, P.; Norris, D.; Oppenheimer, S.; Sleczka, B.; Tarrant, J.; Tokarski, J. S.;
Vite, G. D.; Vyas, D. M.; Wong, H.; Wong, T. W.; Zhang, H.; Zhang, G. Bioorg. Med.
Chem. Lett. 2007, 17, 4947; (d) Ballard, P.; Bradbury, R. H.; Hennequin, L. F. A.;
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15. The HN5 tumor line is a head and neck carcinoma which over expresses EGFR.
The BT474 tumor line is a breast carcinoma which over expresses ErbB-2. The
HFF (human foreskin fibroblast) line is used as
cytotoxicity. The compounds 15a–v displayed adequate selectivity, with IC50
values >1 M on the HFF line.
a measure of general
l
16. Covalent modification data has been acquired using EGFR only using
methods described in reference.9 The compounds act as Michael-type
inhibitors, reacting with a cysteine residue conserved in the ErbB family
ATP binding site. In each case,
a molecule was detected whose mass
corresponds to the mass of EGFR plus the compound of interest. These
analogs have not been assayed versus ErbB-2 or ErbB-4 for potential
reactivity.
17. For a recent mini-perspective on covalent inhibition and leading references on
potential toxic liabilities, please see: Smith, A. J. T.; Zhang, X.; Leach, A. G.;
Houk, K. N. J. Med. Chem. 2009, 52, 225.
18. The amine for compound 15i and the aniline for 15j were not prepared by the
routes described in Schemes 1 and 2 as they were commercially available at
the time of this study.
12. Many of these compounds are covered in the patent literature: Dickerson, S. H.;
Emerson, H. K.; Hinkle, K. W.; Hornberger, K. R.; Sammond, D. M.; Smith, S.;
Stevens, K. L.; Hubbard, R. D.; Petrov, K. G.; Reno, M. J.; Uehling, D. E.;
Waterson, A. G. PCT Int. Appl. WO2005007083, 2005.
13. (a) Munchoff, M. J.; Sobolov-Jaynes, S. B. PCT Int. Appl. WO 9924440 A1, 1999.;
(b) Caferro, T. R.; Chamberlain, S. D.; Donaldson, K. H.; Harris, P. A.; Gaul, M. D.;
Uehling, D. E.; Vanderwall, D. E. PCT Int. Appl. WO 2003053446 A1, 2003.; (c)
Munchoff, M. J.; Beebe, J. S.; Casavant, J. M.; Cooper, B. A.; Doty, J. L.; Higdon, R.
19. For leading references and molecular properties that influence the
pharmaceutical development of molecules, please see: Gleeson, M. P. J. Med.
Chem. 2008, 51, 817.
20. This HPLC method is similar to methods known in the literature: Lie, H.; Sabus,
C.; Carter, G. T.; Tischler, M. J. Chromatogr. 2002, 955, 237. Percentage bound
numbers were generated via comparison of the retention time of test
compounds with known standards..
21. DNAUC = dose normalized area under the curve.