Discovery of thiophene inhibitors of polo-like kinase

Article abstract of DOI:10.1016/j.bmcl.2008.11.041

The discovery and development of a series of thiophenes as potent and selective inhibitors of PLK is described. Identification and characterization of 2, a useful in vitro PLK inhibitor tool compound, is also presented.

Full text of DOI:10.1016/j.bmcl.2008.11.041

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1018–1021
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of thiophene inhibitors of polo-like kinase
*
Kyle A. Emmitte , C. Webb Andrews, Jennifer G. Badiang, Ronda G. Davis-Ward, Hamilton D. Dickson,
David H. Drewry, Holly K. Emerson, Andrea H. Epperly, Daniel F. Hassler, Victoria B. Knick,
Kevin W. Kuntz, Timothy J. Lansing, James A. Linn, Robert A. Mook Jr., Kristen E. Nailor,
James M. Salovich, Glenn M. Spehar, Mui Cheung
Oncology R & D, GlaxoSmithKline, Five Moore Drive, PO Box 13398, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery and development of a series of thiophenes as potent and selective inhibitors of PLK is
described. Identification and characterization of 2, a useful in vitro PLK inhibitor tool compound, is also
presented.
Received 17 September 2008
Revised 11 November 2008
Accepted 12 November 2008
Available online 18 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Thiophene
Benzimidazole
Polo-like kinase
Mitosis
Inhibitor
Cell-cycle
O
O
N
N
The lack of proper cell cycle control and regulation is a hallmark
of cancer cells.1 Inhibition of the cell cycle through the use of anti-
mitotics has been established as an effective approach to cancer
therapy.2 Polo-like kinases (PLK) are an evolutionarily conserved
family of serine/threonine kinases characterized by an amino-ter-
minal serine/threonine kinase domain and carboxy-terminal polo
box domain(s). The PLK family includes PLK1, PLK2 (SNK), PLK3
(PRK/FNK), and PLK4 (SAK). PLK1 plays important roles throughout
mitosis and is involved in the regulation of mitotic progression,
including mitotic entry, spindle formation, chromosome segrega-
tion, and cytokinesis.3 Depletion of PLK1 with small interfering
RNA in vitro has been shown to induce mitotic arrest, and can lead
to apoptosis.4 In addition, the inhibition of PLK1 using antisense
oligonucleotides has shown activity in mouse tumor xenograft
models.5 PLK1 expression is elevated in numerous cancer types
and has been correlated with poor clinical prognosis.6 Such fea-
tures make PLK1 a novel and attractive target for cancer treat-
ment.7 Herein the discovery and development of small molecule
PLK1 inhibitors is reported, as exemplified by 2 (Fig. 1). Compound
2 is a potent inhibitor of PLK1 and is a useful tool compound for
further understanding the role(s) of PLK1 in mitosis.
S
S
N
N
NH2
NH2
O
O
O
O
F
1
2
F
F
Figure 1. Evolution of PLK1 tool compound 2.
ising lead with good PLK1 enzyme potency.8 The synthesis of this
class of compounds began with methyl 2-chloro-3-oxo-2,3-dihy-
dro-2-thiophenecarboxylate 3 (Scheme 1).9 Conjugate addition of
a benzimidazole to 3 gave N-aryl benzimidazole derivative 4. In
the case of 5-substituted benzimidazoles, this reaction produced
a regioisomeric mixture of products. Alkylation with a benzyl bro-
mide or Mitsunobu coupling with the corresponding benzyl alco-
hol provided 5. Treatment of 5 with methanolic ammonia under
sealed tube conditions with heating afforded the final amide prod-
ucts 6.
A study to investigate the effects of changing the electronic nat-
ure of the benzyl ether portion of the inhibitor was undertaken
(Table 1).10 Single-point substitutions to vary the electronic char-
acter of the phenyl ring were made. Most substitutions were well
tolerated with substitution at the 2-position generally preferred.
Ongoing efforts directed toward the design of novel kinase
inhibitor templates led to the identification of thiophene 1, a prom-
* Corresponding author. Tel. +1 615 936 8401.
E-mail address: kyle.a.emmitte@Vanderbilt.edu (K.A. Emmitte).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.041

K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1018–1021
1019
H
N
O
O
N
S
b
R1
N
S
O
N
O
Cl
a
OH
R1
O
4
3
X
R2
O
N
O
N
S
S
N
N
NH2
c
O
O
R1
O
R1
6
5
R2
R2
Figure 2. Homology model of 1 docked into PLK1.
Scheme 1. Reagents and conditions: (a) CHCl3, NaHCO3 or N-methylimidazole; (b)
K2CO3, DMF (X = Br) or PPh3, DIAD, CH2Cl2 (X = OH); (c) 7 N ammonia in methanol,
80 °C, sealed tube.
Table 2
Benzimidazole substitution SAR
O
N
Table 1
Initial SAR of the benzyl ether
S
N
NH2
2
5
R1
O
N
O
S
6
N
NH2
2
3
O
4
R2
3
Compound
R1
R2
PLK1 IC50 (nM)
HCT116 IC50 (nM)
4
19
20
21
22
23
24
25
26
2
5-Cl
6-Cl
2-Me
2-Me
2-Br
21
4
300
9
250
12
8
>28,000
1690
>30,000
3590
>30,000
2960
2700
R
5-CF3
6-CF3
5-SO2Me
6-SO2Me
5-OMe
Compound
R
PLK1 IC50 (nM)
2-Br
1
7
8
H
61
12
12
70
12
28
35
12
22
100
15
11
14
2-CF3
2-CF3
2-CF3
2-CF3
2-CF3
2-Me
3-Me
4-Me
2-OMe
3-OMe
4-OMe
2-Cl
3-Cl
4-Cl
2-CF3
2-Br
2-F
9
6-OMe
5,6-Di-OMe
2
2
1230
699
10
11
12
13
14
15
16
17
18
At this point, the effort returned to the benzyl ether to further
evaluate this portion of the molecule (Table 3). Halogens at the
2-position (28 and 29) provided similar potency to 2 at the cellular
level although the effect was subtle compared to the unsubstituted
benzyl ether (27). Larger groups at the 2-position (31 and 32) gave
analogs that were less potent. Substitution at either the 3- or 4-po-
sition also resulted in a reduction in potency regardless of the size
of the substituent (35–39).
Based on 1 docked into a homology model of PLK1 (Fig. 2), it
was believed that the benzimidazole portion of the molecule of-
fered an area for further SAR exploration. The benzimidazole nitro-
gen was thought to contact the hinge at cysteine residue 133.
Analysis of the model indicated that there was likely room for
the attachment of functional groups at both the 5- and 6-position
of the benzimidazole. In search of a more significant potency boost,
attention was turned to modification of this area (Table 2). Com-
pounds with promising enzyme data were also screened in a 3-
day cellular proliferation assay using the human colon carcinoma
cell line HCT116.11 Benzimidazoles substituted at the 6-position
were more potent than the corresponding five substituted analogs.
Comparison of the PLK1 kinase data for 20 and 26 indicated that
the electronic nature of the 6-position substituent may not be of
primary importance for potency. Overall, the dimethoxy analog 2
proved to be the most potent compound from this set with respect
to both the enzyme and cellular assay. In addition, the symmetrical
benzimidazole was desirable from a synthetic standpoint since it
did not produce regioisomers in the previously described conju-
gate addition reaction. Therefore, the SAR around the 5,6-dimeth-
oxybenzimidazole was further explored.
To expand the SAR of this template, a series of analogs was pre-
pared in which the benzyl ether moiety was replaced with other
ethers (Table 4). This series included saturated alkyl-, aryl-, and
heteroaryl-containing ethers. Extending the length of the alkyl
chain between the oxygen and the aryl ring was not effective at
enhancing potency (40 and 41). Although saturated alkyl ethers
42 and 43 maintained similar enzyme potency to benzyl ether
27, there was an approximately threefold drop in the cellular po-
tency. Thiophenes linked by a methylene unit were tolerated
(44–46), while furans proved less effective (47 and 48). Finally,
while an unsubstituted pyridine analog (49) had reduced potency,
incorporation of a bromide at the 2-position (50) restored potency.
To determine if the primary amide was the optimal group for
the 2-position of the thiophene, modifications were made at this
position (Table 5). Only the acid 52 and the thioamide 55 main-
tained similar enzyme potency to 2; however, both molecules suf-
fered from poor cellular potency. It may be that these compounds
lack the necessary properties to penetrate the cell. It is clear that

1020
K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1018–1021
Table 3
Table 5
Benzyl ether SAR with the 5,6-dimethoxybenzimidazole
Primary amide replacement SAR
O
N
N
S
S
N
R
N
NH2
F
O
O
O F
F
O
2
O
O
3
4
R
Compound
R
PLK1 IC50 (nM) HCT116 IC50
(l
M) Synthesis
Compound
R
PLK1 IC50 (nM)
HCT116 IC50 (nM)
2
51
52
53
54
55
56
57
58
CONH2
CO2Me
CO2H
CN
1H-Tetrazol-5-yl 73
CSNH2
COMe
CONHMe
CONMe2
2
699
—
7660
—
>30,000
>30,000
—
—
—
—
—
a
b
c
d
e
f
>1000
2
700
2
27
28
29
30
31
32
33
34
35
36
37
38
39
2-CF3
H
2-Br
2-Cl
2-CN
2-SO2Me
2-COMe
2-OCF3
2-OMe
3-NMe2
3-NH2
2
12
2
2
6
31
17
5
699
1090
926
999
6
85
>1000
>1000
2630
5430
2640
1150
2110
3470
2270
>30,000
12,100
>30,000
g
8
Synthesis: (a) 51, 1 N LiOH, dioxane; (b) 2, 2-Chloro-1,3-dimethylimidazolinium
chloride12, Et3N, TFA, CH2Cl2; (c) 53, NaN3, NH4Cl, DMF, microwave; (d) 2, Lawes-
son’s reagent, dioxane, 80 °C; (e) i—52, EDC, HATU, DIEA, HNMe(OMe)ÁHCl; ii—
MeMgCl, THF; (g) i—52, SOCl2, PhMe, 90 °C; ii—NH2Me, CH2Cl2; (g) i—52, SOCl2,
PhMe, 90 °C; HNMe2, CH2Cl2.
31
21
99
13
75
3-CN
4-OMe
4-SO2Me
Table 4
Table 6
Benzyl ether replacement SAR
Cellular proliferation inhibition activity for 2
O
S
N
Cell line
Tumor type
IC50 (nM)
N
A549
H460
HCT116
HN5
MCF7
N87
Lung adenocarcinoma
Lung adenocarcinoma
Colorectal carcinoma
Head and neck squamous cell carcinoma
Breast adenocarcinoma
Gastric carcinoma
Prostate adenocarcinoma
Colorectal carcinoma
411
380
699
678
558
601
6820
398
NH2
O
OR
O
Compound
R
PLK1 IC50 (nM) HCT116 IC50 (nM)
PC-3
RKO
2
27
40
41
42
43
44
45
46
47
48
49
50
2-Trifluoromethylbenzyl
benzyl
2
12
699
1090
CH2CH2Ph
CH2CH2CH2Ph
130
61
34
17
11
17
2
27
24,100
7750
3380
3090
1540
1520
1480
>25,000
19,000
21,000
1770
Cyclopentylmethyl
Cyclohexylmethyl
2-Thienylmethyl
3-Thienylmethyl
(3-Chloro)-2-thienyl)methyl
2-Furylmethyl
3-Furylmethyl
4-Pyridinylmethyl
(2-Bromo-4-pyridinyl)methyl
Table 7
Enzyme inhibition profile of 2
Kinase
IC50 (nM)
PLK1
PLK3
2
9
92
160
360
620
4800
>48,000
>15,000
>15,000
>30,000
190
130
14
VEGFR3
PDGFR1b
VEGFR2
C-FMS
AURORA A
EGFR
the primary amide is preferred over substituted amides (57 and
58).
GSK3
P38a
SRC
Compound 2 was selected for profiling against additional cancer
cell lines in a 3-day proliferation assay (Table 6).11 Most of the cell
lines profiled showed similar sensitivity to compound 2 as the
HCT116 cells, with the exception of the PC-3 cell line. The reason
for this is unclear at the moment as PLK1 expression in PC-3 cells
is roughly equivalent to that found in HCT116 and MCF7 cells as
measured using PCR.13
Compound 2 is representative of the selectivity observed with
this series (Table 7). Not surprisingly, 2 is a potent inhibitor of
PLK3, as there is a high homology in the ATP-binding site of PLK1
and PLK3. The consequences of inhibiting PLK3 are not as well
understood as those for PLK1; however, PLK3 is also involved in
the cell cycle, and its highest levels are found during S phase
through M phase. With regard to non-familial kinases, these com-
pounds possess some noteworthy activity against VEGFR2, VEG-
FR3, C-FMS, and PDGFR1b; however, the overall selectivity profile
for 2 appears excellent.14
Table 8
Mouse pharmacokinetic profile of 2
Route
iv
Dose (mg/kg)
6.7
Cl (mL/min/kg)
47.4
VSS (L/kg)
1.5
F%
AUC (ng h/mL)
489
po
10
14
A pharmacokinetic study of 2 in mice was conducted to gauge
its clearance and oral bioavailability (Table 8).15 Compound 2
proved to be a moderate clearance molecule with a moderate vol-
ume of distribution. Although an iv route of administration for a

K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1018–1021
1021
8. Subsequent to work outlined here, efforts directed toward the design of IKK-
e
PLK1 inhibitor in the clinic was preferred, oral exposure data was
also gathered. The oral bioavailability of 2 was low.
inhibitors in this chemical template were conducted, see: Bamborough, P.;
Christopher, J. A.; Cutler, G. J.; Dickson, M. C.; Mellor, G. W.; Morey, J. V.; Patel,
C. B.; Shewchuk, L. M. Bioorg. Med. Chem. Lett. 2006, 16, 6236.
9. Corral, C.; El-Ashmawy, M. B.; Lissavetzky, J.; Manzanares, I. J. Heterocyclic
Chem. 1987, 24, 1301.
10. The PLK1 enzyme assay was conducted using the kinase domain only in an SPA
format. For a complete description see Ref. 16.
11. The cellular proliferation assays were analyzed using a methylene blue stain
technique. For a complete description see Ref. 16.
In summary, a novel series of thiophenes were developed as po-
tent and selective inhibitors of PLK1. This work led to the identifi-
cation of 2, a useful tool compound for evaluation of PLK1 biology.
Further description of the biological properties of 2 have recently
been disclosed supporting its role as an effective anti-mitotic agent
in vitro.16 In addition, the work within this chemical template that
resulted in the discovery of GSK461364 was recently disclosed.17
GSK461364 is currently under evaluation in clinical trials. Subse-
quent communications regarding the extension of the SAR within
this chemical series will be forthcoming.
12. Ishikawa, T.; Isobe, T. J. Org. Chem. 1999, 64, 6984.
13. Goh, K. C.; Wang, H.; Yu, N.; Zhou, Y.; Zheng, Y.; Lim, Z.; Sangthongpitag, K.;
Fang, L.; Du, M.; Wang, X.; Jefferson, A. B.; Rose, J.; Shamoon, B.; Reinhard, C.;
Carte, B.; Entzeroth, M.; Ni, B.; Taylor, M. L.; Stünkel, W. Drug Dev. Res. 2004, 62,
349.
14. Compound 2 was screened against 58 unique in-house kinases and found to
have IC50 values less than 1
PLK3.
15. The mouse pharmacokinetic study was conducted using
lM against only four kinases other than PLK1 and
References and notes
a
40%
hydroxypropyl-b-cyclodextrin solution and 0.5% HPMC/0.1% Tween 80
a
1. Ruddon, R. W. Cancer Biology, 3rd ed.; Oxford University Press: New York, 1995.
Chapter 10.
suspension formulation for iv and oral administration, respectively, with
composite sampling of plasma. Oral bioavailability was determined using
2. Chabner, B. A.; Ryan, D. P.; Paz-Ares, L.; Garcia-Carbonero, R.; Calabresi, P. In
Goodman s the Pharmacological Basis of Therapeutics; Hardman, J. G., Limbird, L.
E., Gilman, A. G., Eds., 10th ed.; McGraw-Hill: New York, 2001; p 1417.
3. (a) Barr, F. A.; Sillje, H. H. W.; Nigg, E. A. Nat. Rev. Mol. Cell Biol. 2004, 5, 429; (b)
Fisher, R. A. H.; M, D. K. Curr. Med. Chem. Immun. Endocr. Metab. Agents 2002, 2,
125; (c) Glover, D. M.; Hagan, I. M.; Tavares, A. A. M. Genes Dev. 1998, 12, 3777.
4. Liu, X.; Erikson, R. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5789.
5. Elez, R.; Piiper, A.; Kronenberger, B.; Kock, M.; Brendel, M.; Hermann, E.;
Pliquett, U.; Neumann, E.; Zeuzem, S. Oncogene 2003, 22, 69.
AUC values calculated from
administration.
16. Lansing, T. J.; McConnell, R. T.; Duckett, D. R.; Spehar, G. M.; Knick, V. B.;
Hassler, D. F.; Noro, N.; Furuta, M.; Emmitte, K. A.; Gilmer, T. M.; Mook, R. A., Jr.;
Cheung, M. Mol. Cancer Ther. 2007, 6, 450.
17. Kuntz, K.; Salovich, J.; Mook, R.; Emmitte, K.; Chamberlain, S.; Rheault, T.;
Hornberger, K.; Emerson, H.; Smith, S.; Wilson, B.; Davis-Ward, R.; Donaldson,
K.; Adjabeng, G.; Nailor, K.; Hassler, D.; Smith, G.; Lansing, T.; Duckett, D.;
Knick, V.; McConnell, R.; Jackson, J.; Cheung, M. Abstract of Papers, 98th Annual
Meeting of the American Association for Cancer Research, Los Angeles, CA;
American Association for Cancer Research: Philadelphia, PA, 2007; Abstract
4171.
0
to infinity following iv and oral
6. Takai, N.; Hamanaka, R.; Yoshimatsu, J.; Miyakawa, I. Oncogene 2005, 24, 287.
7. (a) Warner, S. L.; Stephens, B. J.; Von Hoff, D. D. Curr. Oncol. Rep. 2008, 10, 122;
(b) McInnes, C.; Mezna, M.; Fischer, P. M. Curr. Top. Med. Chem. 2005, 5, 181.