Design of potent thiophene inhibitors of polo-like kinase 1 with improved solubility and reduced protein binding

Article abstract of DOI:10.1016/j.bmcl.2009.01.094

A series of thiophene PLK1 inhibitors was optimized for increased solubility and reduced protein binding through the appendage of basic amine functionality. Interesting selectivity between PLK1 and PLK3 was also obtained through these modifications.

Full text of DOI:10.1016/j.bmcl.2009.01.094

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1694–1697
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design of potent thiophene inhibitors of polo-like kinase 1 with improved
solubility and reduced protein binding
a
a
a
Kyle A. Emmitte a, , George M. Adjebang , C. Webb Andrews , Jennifer G. Badiang Alberti ,
Ramesh Bambal b, Stanley D. Chamberlain a, Ronda G. Davis-Ward a, Hamilton D. Dickson a,
Daniel F. Hassler a, Keith R. Hornberger a, Jeffrey R. Jackson b, Kevin W. Kuntz a, Timothy J. Lansing a,
Robert A. Mook Jr. a, Kristen E. Nailor a, Mark A. Pobanz a, Stephon C. Smith a, Chiu-Mei Sung b, Mui Cheung c
*
a Five Moore Drive, Research Triangle Park, NC 27709, GlaxoSmithKline, USA
b 1250 South Collegeville Road, Collegeville, PA 19426, GlaxoSmithKline, USA
c 709 Swedeland Road, King of Prussia, PA 19406, GlaxoSmithKline, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thiophene PLK1 inhibitors was optimized for increased solubility and reduced protein binding
through the appendage of basic amine functionality. Interesting selectivity between PLK1 and PLK3 was
also obtained through these modifications.
Received 8 January 2009
Revised 27 January 2009
Accepted 28 January 2009
Available online 31 January 2009
Ó 2009 Published by Elsevier Ltd.
Keywords:
Polo-like kinase
Anti-mitotic
Cell cycle inhibitor
Thiophene
Solubility
Protein binding
A relationship between cancer proliferation and an improperly
controlled cell cycle has been well established.1 Clinical use of
anti-mitotics to disrupt the cell cycle as a method of cancer therapy
has proven effective in certain instances.2 The polo-like kinases
(PLK) represent an attractive target for cancer treatment through
interference with the cell cycle.3 These proteins are an evolution-
arily conserved family of serine/threonine kinases characterized
by an amino-terminal kinase domain and carboxy-terminal polo
box domain(s) and include PLK1, PLK2 (SNK), PLK3 (PRK/FNK),
and PLK4 (SAK). PLK1 represents the most studied member of this
family and has known roles throughout mitosis, including mitotic
entry, spindle formation, chromosome segregation, and cytokine-
sis.4 Further preclinical validation of PLK1 as a cancer target has
been demonstrated through the use of small interfering RNA in cell
culture5 and antisense oligonucleotides in mouse tumor xenograft
models.6 Finally, a correlation between elevated PLK1 expression
and poor prognosis in the clinic has been established.7 In light of
the aforementioned evidence, a program directed toward the dis-
covery of small molecule inhibitors of PLK1 was initiated.
Prior work in a thiophene series of PLK inhibitors that led to the
discovery of compound 1 was recently communicated (Fig. 1).8
Further in vitro characterization of compound 1 was also recently
disclosed.9 Compound 1 is a potent inhibitor of PLK1 and PLK310
and moderately inhibits the growth of tumor cells in vitro.11 Com-
pound 1 served as a useful tool compound for understanding the
consequences of inhibiting PLK in vitro; however, the compound
lacked many of the features considered desirable in a clinical can-
didate. In order to discover a PLK1 inhibitor suitable for clinical
studies, work continued in the thiophene series and focused on im-
proved cellular potency, solubility, and protein binding.
Subsequent to the work described previously, a minor modifica-
tion to the template resulted in a significant improvement in
O
N
S
N
NH2
PLK1 IC50 = 2 nM
PLK3 IC50 = 9 nM
O
O
O
F
1
F
F
* Corresponding author. Tel.: +1 615 936 8401; fax: +1 615 322 8577.
E-mail address: kyle.a.emmitte@vanderbilt.edu (K.A. Emmitte).
Figure 1. PLK tool compound.
0960-894X/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2009.01.094

K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1694–1697
1695
cellular potency (Table 1). Installation of a methyl group at the
benzylic carbon in the R-configuration (3) resulted in a more than
40-fold improvement in the ability to inhibit the growth of HCT116
(colorectal carcinoma) cells over the unsubstituted analog (2). The
enantiomer (4) was a less potent inhibitor of PLK1 at the enzyme
level; however, its potency in the cellular assay was similar to 2.
The effect of benzylic substitution on PLK3 potency was similar
to that observed with PLK1. An explanation for the improvement
in cell potency remains unclear. Since the concentration of PLK1
in the enzyme assay is 2 nM, the potency of 3 may have been
underestimated in that assay. It was also noted that compound 3
maintained an excellent kinase selectivity profile among more
than 40 kinases tested in house, which is comparable to 1.
are residues such as Glu140 and Ser137, which are potentially dis-
posed to favorably interact with such polar functional groups.
In order to effectively prosecute this hypothesis, efficient syn-
theses of versatile phenol intermediates 14 and 15 were desired
(Scheme 1). The syntheses began with Buchwald coupling of aryl
bromide 5 with amines 6 or 7.14 Reduction of nitro-anilines 8
and 9 was accomplished by hydrogenation using a sulfided plati-
num catalyst. Upon treatment with pyridinium p-toluensulfonate
and trimethyl orthoformate, diamines 10 and 11 cyclized to afford
benzimidazoles 12 and 13. Subsequent reaction with trifluoroace-
tic acid cleanly removed the 4-methoxybenzyl protecting group to
provide phenols 14 and 15. These phenols served as versatile inter-
mediates for evaluation of solubility enhancing groups at this posi-
tion. Functionalization was typically accomplished through
Mitsunobu couplings15 or standard SN2 alkylations, followed by
additional routine transformations.16
Methyl ether analogs 16 and 17 were both highly protein bound
and poorly soluble (Table 2). Although a modest improvement in
cellular potency was observed, incorporation of a diol (18) only
minimally improved solubility. The installation of flexible amine
chains (19–22) improved solubility and reduced protein binding
relative to 16 and 17. While compounds such as 21 and 22 were
quite potent, potency in the cellular assay could be further
enhanced by elimination of the methylene spacer between the
oxygen atom and the piperidine ring (23–25). These analogs exhib-
ited an excellent combination of potency, solubility, and reduced
protein binding. The azepine analogs (26 and 27) were similar in
potency and solubility to their piperidine counterpart (25); how-
ever, protein binding increased in each case. It should also be noted
that methylation of the piperidine nitrogen led to both improved
solubility and reduced protein binding (24 vs 23).
With this marked improvement in cellular potency, attention
was next directed to additional areas for lead optimization. Since
the target product profile for a PLK1 inhibitor included an intrave-
nous formulation, solubility was an important attribute in any po-
tential clinical candidate. Compounds were routinely screened in a
high throughput manner for their solubility in a phosphate buf-
fered solution at pH 7.4 and were generally poorly soluble
(3 = 2 l
M).12 It was also noted that compounds in this series were
typically highly bound to human plasma proteins.13 As a means to
improve the solubility and reduce the protein binding of com-
pounds, the installation of more polar functional groups was exam-
ined. Analysis of a homology model of 3 bound in the ATP-binding
site of PLK1 indicated that the 6-position of the benzimidazole
might be a good location for appending such groups (Fig. 2). With
the nitrogen of the benzimidazole contacting the b-strand at
Cys133, the 6-position is oriented toward solvent. In addition, there
Perhaps the most interesting observation with regard to these
new analogs with basic amine functionality was their improved
selectivity versus PLK3. Examination of 23 in a homology model
bound in the ATP-binding site of PLK1 provided some rationale
Table 1
Benzylic methyl group
O
N
S
N
NH2
O
O
O
O
O
R
NO2
O2N
H
N
S
S
H2N
+
Br
a
O
Cl
O
O
X
a
a
a
O
X
Compound
R
PLK1 IC50 (nM)
PLK3 IC50 (nM)
HCT116 IC50 (nM)
OPMB
OPMB
2
3
4
H
2
0.8
30
9
6
44*
1140
25
994
Me (R)
Me (S)
8 X = Cl
9 X = CF3
5
6 X = Cl
7 X = CF3
a
Data are the average of n P 2, except where noted with (*), which is n = 1.
b
O
O
N
H2N
H
N
S
S
N
12 X = Cl
c
O
O
13 X = CF3
O
O
X
OPMB
OPMB
10 X = Cl
X
d
11 X = CF3
O
N
S
N
O
14 X = Cl
O
15 X = CF3
OH
X
Scheme 1. Reagents and conditions: (a) Pd2(dba)3, Cs2CO3, XANTPHOS, dioxane,
60 °C (80% for 8, 74% for 9); (b) H2 (1 atm), Pt(sulfided)/C, EtOAc (97% for 10); (c)
PPTS, CH(OMe)3, Et2O (97% over 2 steps for 12); (d) TFA, CH2Cl2, 0 °C (75% for 14,
92% for 15 over 2 steps).
Figure 2. Homology model of 3 in PLK1.

1696
K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1694–1697
Table 2
Analogs with solubility enhancing groups
O
N
S
N
NH2
O
X
OR
Solubility PBS, pH 7.4a
(lM)
Eq. dialysis PBa (%)
a
a
a
Compound
X
R
PLK1 IC50 (nM)
PLK3 IC50 (nM)
HCT116 IC50 (nM)
16
17
Cl
CF3
Me
Me
1
3
35*
41*
321
476
3*
1*
99.5*
99.5*
OH
18
19
Cl
Cl
1
4
61*
73
32
15*
—
OH
OH
N
N
460*
161*
97.4*
20
21
22
23
24
25
26
Cl
3
7
6
3
2
2
3
4
610*
550
45
30
34
15
12
11
17
13
170*
109
96.2*
97.7
CF3
Cl
N
N
1100*
350
P172
104
97.9*
96.6
CF3
CF3
Cl
NH
270*
630*
240*
250
179
94.8*
91.5*
96.9*
98.0*
N
N
P190
P200*
200*
N
Cl
N
27
Cl
a
Data are the average of n P 2, except where noted with (*), which is n = 1.
Table 3
Expanded cellular proliferation panel
for this observation (Fig. 3). In this model, it appears that there is a
hydrogen bond interaction between Glu140 and the piperidine
nitrogen. The corresponding residue in both PLK3 and PLK2 is a his-
tidine. The interaction of the charged amine with the histidine res-
idue is likely not favorable, resulting in the reduced potency
observed against PLK3.
Cell line
Tumor type
IC50 (nM)a
24
25
COLO205
HT29
A549
Colon
Colon
Lung
5
5
3
3
14
10
32
11
9
11
MX-1
SKOV-3
Breast
Ovarian
a
Data are the average of n P 2.
Compounds 24 and 25 were considered to have a good balance
of potency, selectivity, solubility, and lower protein binding. Each
was evaluated against a broader panel of cell lines in order to fur-
ther determine its ability to inhibit cellular proliferation (Table
3).11 Both proved to be potent inhibitors of cell growth in vitro
across a wide range of cell lines representing multiple tumor types.
The kinase selectivity of early compounds within this chemical
series was considered excellent.8 Examination of 24 and 25 against
an expanded panel of more than 50 human kinases showed these
new analogs have maintained good selectivity (Table 4).17 Both
compounds have activity against Nek2, which is also a serine/thre-
onine kinase with important roles throughout mitosis, including
centrosome separation, microtubule organization, and mitotic
exit.18 Selectivity against other kinases compared to PLK1 is greater
Figure 3. Homology model of 23 in PLK1.

K. A. Emmitte et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1694–1697
1697
Table 4
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; pp 1417–1425.
3. (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.
4. (a) Barr, F. A.; Sillje, H. H. W.; Nigg, E. A. Nat. Rev. Mol. Cell Biol. 2004, 5, 429; (b)
Fisher, R. A. H.; Ferris, 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.
Expanded kinase selectivity panel
a
Kinase
IC50 (nM)
24
25
PLK1
PLK3
Nek2
PDGFR1b
PIM-1
2
270*
25
110*
340*
390*
530*
800
2
630*
21*
420*
310*
—
440*
1200*
5. Liu, X.; Erikson, R. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5789.
6. Elez, R.; Piiper, A.; Kronenberger, B.; Kock, M.; Brendel, M.; Hermann, E.;
Pliquett, U.; Neumann, E.; Zeuzem, S. Oncogene 2003, 22, 69.
PI3K-
c
7. Takai, N.; Hamanaka, R.; Yoshimatsu, J.; Miyakawa, I. Oncogene 2005, 24, 287.
8. Emmitte, K. A.; Andrews, C. W.; Badiang, J. G.; Davis-Ward, R. G.; Dickson, H. D.;
Drewry, D. H.; Emerson, H. K.; Epperly, A. H.; Hassler, D. F.; Knick, V. B.; Kuntz,
K. W.; Lansing, T. J.; Linn, J. A.; Mook, R. A., Jr.; Nailor, K. E.; Salovich, J. M.;
Spehar, G. M.; Cheung, M. Bioorg. Med. Chem. Lett. 2009, 19, 1018.
9. 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.
PI3K-d
VEGFR2
Nuak1
Others
840*
2000*
>1000 (50 kinases)
>1000 (48 kinases)
a
Data are the average of n P 2, except where noted with (*), which is n = 1.
10. The PLK1 enzyme assay was conducted using the kinase domain only. The PLK3
assay was conducted using full length enzyme. Both were in an SPA format. For
a complete description see Ref. 9.
11. Cell proliferation assays: Cells were seeded in 96-well dishes, incubated
overnight at 37 °C, and treated with various concentrations of inhibitors for
72 h. Cell proliferation was quantified using the CellTiter-Glo Luminescent Cell
Viability Assay (Promega, Madison, WI), following the manufacturer’s
recommendations. IC50 values were determined from using a 4-parameter
curve fit software package (XLfit4).
O
O
N
N
S
S
N
N
NH2
NH2
O
X
O
O
N
N
N
F3C
12. The compound was first dissolved in DMSO in a concentrated form (10 or
20 mM) followed by spiking a fixed volume of the DMSO stock solution into the
final buffer. Two samples were prepared for each compound. One (the standard
GSK461364
24 X = CF3
25 X = Cl
sample) contained the compound at a fixed concentration of 20
aqueous/organic mixed solvent cocktail. The other (test sample) contained the
compound at a maximum total concentration of 200 M in, pH 7.4, 0.05 M
lM in an
l
Figure 4. GSK461364.
phosphate buffer. The test sample was spun for 15 min to remove any
undissolved solid. HPLC analyses were performed on these samples. The peak
areas were used for computing the solubility.
than 100-fold in all other cases, with the exception of PDGFR1b for
24, which is approximately 50-fold selective versus that enzyme.
In summary, the appendage of cyclic amine functionality to the
6-position of the benzimidazole of thiophene inhibitors was used
to afford compounds with suitable potency, selectivity, solubility,
and protein binding for progression into advanced assays. Subse-
quent work within this chemical template resulted in the discovery
of GSK461364 (Fig. 4), a close structural analog of 24 and 25.19
GSK461364 is currently under evaluation in clinical trials. Further
communications regarding the discovery and profile of GSK461364
are planned.
13. Stock solutions of compound were spiked into human plasma at target
concentrations of 2000 ng/mL. The mixtures were inverted gently several
times to insure homogeneity and triplicate 50 lL aliquots were collected to
verify initial concentrations. Following assembly of dialysis plate (HTDialysis
membrane strips, molecular weight cut off limit of 12,000–14,000 daltons),
spiked plasma (150
lL) was placed in the donor compartment of the well and
phosphate buffered saline, pH 7.4 (150
lL) in the receiver compartment. Eight
wells were set up per compound and plasma type. Plate was placed in a 37 °C
incubator on a plate shaker. Following the 6-h incubation period, the plate was
removed. Single 50 lL aliquots from each donor and receiver compartment
(per well) were analyzed. Sample analysis was by LC/MS/MS (results reported
as Drug Peak Area/Internal Standard Peak Area ratios).
14. Hornberger, K. H.; Badiang, J. G.; Salovich, J. M.; Kuntz, K. W.; Emmitte, K. A.;
Cheung, M. Tetrahedron Lett. 2008, 49, 6348.
Acknowledgments
15. Mitsunobu, O. Synthesis 1981, 1.
16. Hornberger, K.; Cheung, M.; Pobanz, Mark A.; Emmitte, Kyle A.; Kuntz, Kevin
W.; Badiang, Jennifer G. PCT Int. Appl. 2007, WO 2007030366.
17. Other kinases were screened using conditions and assays optimized for each.
18. Hayward, D. G.; Fry, A. M. Cancer Lett. 2006, 237, 155.
19. 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.
We thank Helen Gao for cell proliferation data, Xiaoliu Geng for
HTS solubility data, and Diane Talaber for human protein binding
data.
References and notes
1. Ruddon, R. W. Cancer Biology, 3rd ed.; Oxford University: New York, 1995.
Chapter 10.