Dual EGFR/ErbB-2 inhibitors from novel pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines

Article abstract of DOI:10.1016/j.bmcl.2008.09.090

A novel class of substituted pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines has been identified that are potent and selective inhibitors of both EGFR/ErbB-2 receptor tyrosine kinases. The inhibitors are found to display a range of enzyme and cellular potency and also to display a varying level of covalent modification of the kinase targets. Selected molecules, including compound 15h, were found to be potent in enzymatic and cellular assays while also demonstrating exposure in the mouse from an oral dose.

Full text of DOI:10.1016/j.bmcl.2008.09.090

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5738–5740
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dual EGFR/ErbB-2 inhibitors from novel pyrrolidinyl-acetylenic
thieno[3,2-d]pyrimidines
Robert D. Hubbard , Scott H. Dickerson, Holly K. Emerson, Robert J. Griffin, Michael J. Reno,
*
Keith R. Hornberger, David W. Rusnak, Edgar R. Wood, David E. Uehling, Alex G. Waterson
GlaxoSmithKline Research & Development, PO Box 13398, 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:
Received 30 July 2008
Revised 22 September 2008
Accepted 23 September 2008
Available online 27 September 2008
A novel class of substituted pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines has been identified that are
potent and selective inhibitors of both EGFR/ErbB-2 receptor tyrosine kinases. The inhibitors are found to
display a range of enzyme and cellular potency and also to display a varying level of covalent modifica-
tion of the kinase targets. Selected molecules, including compound 15h, were found to be potent in enzy-
matic and cellular assays while also demonstrating exposure in the mouse from an oral dose.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Epidermal growth factor receptor
EGFR
ErbB-2
Inhibitor
Thieno[3,2-d]pyrimidines
Overexpression of the epidermal growth factor receptor tyro-
sine kinases1 EGFR (ErbB-1) and/or HER-2 (ErbB-2) has been impli-
cated in a variety of cancers.2 The ability to disrupt the ErbB-family
signaling pathway either by monoclonal antibody therapy3 or
small molecule ATP-competitive kinase inhibitors has yielded no-
vel anticancer agents.4 A common chemical class of the initial TK
inhibitors is the 4-anilinoquinazolines. This class has provided
the reversible ErbB-family inhibitors gefinitib,5 erlotinib,6 and
lapatinib.7 The latter possesses a novel anilino-aryl head-group
that yields dual EGFR/ErbB-2 potency (Fig. 1).8 The quinazoline
ErbB-family inhibitor canertinib9 contains an acrylamide Michael
acceptor that forms a covalent bond with a Cys residue (Cys733 in
EGFR and Cys805 in ErbB-2).10 A potential advantage of a covalent
agent is the prolonged inhibition of the enzyme, thereby compet-
ing with the high intracellular concentrations of ATP to effectively
impair the ErbB-signaling pathway.11
In an effort to find effective dual inhibitors of EGFR and ErbB-2
from a distinct structural class relative to lapatinib, we discovered
that pyrrolidinyl-acetylenic thienopyrimidines (1) are potent and
selective EGFR/ErbB-2 enzyme inhibitors, with the ability to react
irreversibly with the Cys733 of EGFR.12 Previous SAR exploration
had indicated that analogs with a basic propargylic nitrogen tend
to show greater covalent reactivity.12 This paper describes our fur-
ther synthetic explorations and the in vitro characterization of se-
lected members of this class of receptor tyrosine kinase inhibitors.
As shown in Scheme 1, the synthesis of the analogs begins with
the known acetylenic pyrrolidines (2, 3)13 which were separately
coupled to the known thienopyrimidinyl bromide 414 via Sono-
gashira reaction to afford the desired alkynes 5, 6. Subsequent
deprotection with TFA in CH2Cl2 yielded the desired analogs 7
and 8. In order to assess the role of the basic pyrrolidine nitrogen
Cl
O
S
O
O
HN
N
HN
O
N
F
Ar
NH
R1
S
lapatinib
N
Cl
N
R2
N
F
1
O
HN
N
HN
N
N
O
O
* Corresponding author. Tel.: +1 919 483 6262; fax: +1 919 483 6053.
E-mail address: Alex.G.Waterson@gsk.com (A.G. Waterson).
canertinib
Figure 1. Quinazoline and thienopyrimidine-based ErbB-family inhibitors.
Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.090

R. D. Hubbard et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5738–5740
5739
Ar
Ar
NH
NH
H
R1
R2
S
S
N
N
a
2
+
Br
N
N
N
N
BOC
BOC
5: when C2 is (
6: when C2 is (
2: R1 = -H,
4
S
R
)
)
R
2 = -CCH
3: R1 = -CCH,
Cl
O
2 = -H
Ar
R
NH
Figure 2. Superimposition of a possible binding conformation of lapatinib (green)
and 8 (red) docked into ErbB-2.
H
S
N
b
2
Ar =
N
H
N
F
modify EGFR. Therefore, while derivatization of the pyrrolidine
nitrogen yielded potent enzyme inhibitors, these analogs were
generally less effective at inhibiting cell proliferation than the par-
ent analogs 7 and 8, particularly against the BT474 line.
An overlay of lapatinib and 8, each docked into a model of ErbB-2,
revealed that functionalization of C4 of the pyrrolidinering might ac-
cess similar chemical space as the sulfone tail present in lapatinib
(Fig. 2). We found that the 4-hydroxy group of the commercially
7: when C2 is (
8: when C2 is (
S
R
)
)
Ar
NH
9: R = -(CH2)2OCH3
10: R = -SO2CH3
11: R = -COCH3
H
S
c, d, e, or f
N
8
N
R
N
12: R = -C(O)OCH3
Scheme 1. Reagents and conditions: (a) (Ph3P)2PdCl2, CuI, Et3N, 60 °C, THF ; (b) TFA,
0 °C to rt, CH2Cl2; (c) 2-chloroethyl methyl ether, K2CO3, sealed tube, 100 °C, CH3CN;
(d) for the sulfonamide: MsCl, 0 °C, CH2Cl2; (e) for the acetamide, Ac2O, 0 °C, CH2Cl2;
(f) for the carbamate: methyl chloroformate, 5 N NaOH, 0 °C, Et2O.
available N-BOC-trans-4-hydroxy-L-proline methyl ester (13) affor-
ded access to a variety of such analogs. The ester 13 was treated
sequentially with DIBAL-H, then with the modified Seyferth–Gilbert
reagent17 under previously disclosed one-pot conditions,18 to afford
the key alkyne intermediate 14a (Scheme 2). Generation of the so-
dium alkoxide derived from 14a followed by addition of the appro-
present in 8, we performed a series of chemoselective reactions, in
which the pyrrolidine nitrogen was alkylated (9), sulfonylated (10),
acylated (11), or carbamoylated (12).
Analogs were initially assayed for their intrinsic potency against
EGFR and ErbB-2. Since these analogs possess the ability to cova-
lently modify either EGFR or ErbB-2, it should be noted that the
IC50 values are time-dependent and may vary significantly as a
function of incubation time.15 The data presented is the apparent
IC50 values obtained with a 40-min reaction. The analogs were fur-
ther progressed to assess their inhibition of cellular proliferation
against two transformed cell lines, whose proliferation was driven
by the targets of interest: EGFR (HN5) and ErbB-2 (BT474).16
Comparison of the unsubstituted pyrrolidines 7 and 8 revealed
that 8 was clearly more potent on the enzyme and also somewhat
more effective at inhibiting cellular proliferation (Table 1). Inter-
estingly, pyrrolidine 8 was also significantly more reactive, modify-
ing the EGFR kinase 47% after 3 h, versus 15% for pyrrolidine 7.
Functionalization of the pyrrolidine nitrogen with electron
withdrawing substituents yielded analogs with generally reduced
cellular efficacy (7 vs 10, 12), despite roughly similar enzyme
potencies. Interestingly, amide 11 was a more potent inhibitor of
EGFR than 7. Reducing the basicity of the pyrrolidine nitrogen in
analogs 10, 11, and 12 also abrogated their ability to covalently
HO
R1O
where:
H
H
a
b or c
14b: R1 = Et
14c: R1 = SO2Me
13
N
N
BOC
BOC
14a
R2
O
where:
O
14d: R2 = -Et
14h: R2 = -N(Me)2
14i: R2=
H
S
d or e
14e: R2 = -OEt
14a
N
N
14f: R2 = -NHCH3
14g: R2 = -NHEt
O
BOC
H3C
O
R2
HO
NH
O
H
H
H
O
c
f, g, h
N
N
N
BOC
BOC
BOC
where:
16
17
14j: R2 = -C(O)OEt
14k: R2 = -SO2Et
Cl
O
Table 1
EGFR and ErbB-2 kinase and cell proliferation inhibition
Ar =
EGFR mod.c (%)
a,b
a
Analog
Enzyme IC50 (nM)
Cellular IC50 (nM)
F
EGFR
ErbB-2
HN5
BT474
Ar
NH
R
7
109
7
130
120
58
54
13
110
88
38
120
536
238
190
550
37
205
94
110
650
670
1700
15
47
19
0
0
0
R
H
H
8
9
S
j, k
N
N
N
H
10
11
12
N
BOC
14a-k
15a-k
140
590
a
Average values, n > 2.
Since these analogs possess the ability to covalently modify either EGFR, ErbB-2
Scheme 2. Reagents and conditions: (a) 1—DIBAL-H, À78 °C, toluene; then, MeOH;
2—MeC(@O)C(@N2)P(@O)(OCH3)2, K2CO3, 0 °C to rt; (b) NaH, 0 °C, DMF; then EtI, rt;
(c) MsCl, Et3N, 0 °C to rt, CH2Cl2; (d) for 14d and 14e: CH3CH2C(O)Cl or EtOC(O)Cl,
Et3N, 0 °C to rt, CH2Cl2; (e) for 14f–i: isocyanate, DMAP, 60 °C, DMF; (f) NaN3, DMF,
70 °C; (g) Ph3P, H2O, 25 °C, THF; (h) for 14j: EtOC(O)Cl, Et3N, 0 °C to rt; (i) for 14k:
MsCl, Et3N, 0 °C to rt, CH2Cl2; (j) 4, (Ph3P)2PdCl2, CuI, Et3N, 60 °C, THF; (k) TFA, 0 °C
to rt, CH2Cl2.
b
or ErbB-4 it should be noted that the IC50 results are time-dependent and may vary
significantly as a function of incubation time. The data presented is the apparent
IC50 obtained with the 40-min reaction.
c
Value represents % covalent modification after 3 h. For experimental details, see
Ref. 12.

5740
R. D. Hubbard et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5738–5740
Table 2
oral exposure (DNAUC = 61 ng h/mL/mg/kg) to complement its
Comparison of enzyme and cellular potency
potency. Secondary carbamates were also explored. The dimethyl
amino carbamate 15h demonstrated potent inhibition in the en-
zyme and cell assays, displayed reduced covalent modification as
compared with 7 or 15g, and also showed good oral exposure in
the mouse (DNAUC = 93 ng h/mL/kg). However, the morpholine
analog 15i showed somewhat reduced potency compared with
15g, particularly on the EGFR enzyme and the HN5 (EGFR over-
expressing) cell line. The amine attached sulfonamide and carba-
mate, 15j and 15k, respectively, were also found to be potent in
the primary assays and were more reactive in the covalent mod-
ification assay than many of the other C4-substituted analogs.
In conclusion, we describe novel acetylenic pyrrolidine thie-
no[3,2-d]pyrimidines that are potent, selective dual EGFR/ErbB-2
enzyme inhibitors with good anti-proliferative activity against
EGFR and ErbB-2 overexpressing tumor lines. In addition, we have
presented our SAR findings around both the pyrrolidine nitrogen
and the C4-substituent. The C4-carbamoyl analog 15g was found
to have desirable enzyme and cell potency while displaying good
mouse oral PK. In addition, the secondary carbamate 15h was
found to be among the most potent molecules in the series and
also demonstrated exposure from an oral dose in the mouse. On
the basis of these encouraging results, the SAR of C4-carbamates
has been further developed and the pre-clinical developability of
these compounds has been probed. The results will be disclosed
in a future communication.
a,b,c
a
Analog
R=
Enzyme IC50
EGFRc
(nM)
Cellular IC50 (nM)
ErbB-2
HN5
BT474
15a
15b
15c
–OH
–OEt
–OSO2CH3
14 (ND)d
115 (21%)
81 (ND)
11
98
40
160
99
380
39
45
86
O
15d
15e
63 (0%)
90
52
18
34
CH3
Et
O
O
141 (0%)
135
200
O
O
O
15f
15g
15h
15i
15j
50 (17%)
32 (24%)
28 (11%)
65 (5%)
20
43
68
84
58
95
28
30
30
30
CH3
O
N
H
O
O
Et
O
O
N
H
CH3
100
160
N
CH3
O
O
O
N
O
References and notes
60 (41%)
50 (26%)
78
87
113
127
40
42
Et
N
H
O
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15. The modification data was acquired using EGFR only using methods described
in Ref. 12.
reactivity.
These analogs have not been assayed versus ErbB-2 for potential
16. Furthermore, these thienopyrimidine-based analogs (7–12, 15a–15k) were
found to be selective for the transformed cell lines versus a control human
foreskin fibroblast cell line (>1
Ref. 7.
lM IC50). Methods of testing are described in
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conversion of 13–14a, simply starting with the commercially available N-BOC-
trans-4-hydroxy-D-proline methyl ester. A full examination of the possible
diastereomers and the associated synthetic details will be disclosed in a future
communication.