Discovery of 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines as potent PIM inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.12.091

PIM kinases are a family of Ser/Thr kinases that are implicated in tumorigenesis. The discovery of a new class of PIM inhibitors, 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines, is discussed with optimized compounds showing excellent potency against all three PIM isoforms.

Full text of DOI:10.1016/j.bmcl.2014.12.091

Accepted Manuscript
Discovery of 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines as potent PIM inhib-
itors
Bin Wu, Hui-Ling Wang, Victor Cee, Brian Lanman, Thomas Nixey, Liping
Pettus, Anthony B. Reed, Ryan P. Wurz, Nadia Guerrero, Christine Sastri, Jeff
Winston, J. Russell Lipford, Matthew R. Lee, Christopher Mohr, Kristin
Andrews, Andrew S. Tasker
PII:
S0960-894X(14)01399-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.12.091
BMCL 22336
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 November 2014
23 December 2014
29 December 2014
Please cite this article as: Wu, B., Wang, H-L., Cee, V., Lanman, B., Nixey, T., Pettus, L., Reed, A.B., Wurz, R.P.,
Guerrero, N., Sastri, C., Winston, J., Russell Lipford, J., Lee, M.R., Mohr, C., Andrews, K., Tasker, A.S., Discovery
of 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines as potent PIM inhibitors, Bioorganic & Medicinal Chemistry
Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2014.12.091
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Discovery of 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines as potent PIM inhibitors
Bin Wu,*,a Hui-Ling Wang,a Victor Cee,a Brian Lanman,a Thomas Nixey,a Liping Pettus,a
Anthony B. Reed,a Ryan P. Wurz,a Nadia Guerrero,b Christine Sastri,b Jeff Winston,b J.
Russell Lipford,b Matthew R. Lee,c Christopher Mohr,c Kristin Andrews,c and Andrew S.
Tasker.a
Amgen Inc. One Amgen Center Drive, Thousand Oaks, California 91320-1799, USA
a) Department of Therapeutic Discovery; b) Department of Oncology; c) Department of Molecular
Structure and Characterization.
* To whom correspondence should be addressed:
Bin Wu: Tel +1-805-313-5661; Fax 805-480-3016; email:binw@amgen.com
Abstract: PIM kinases are a family of Ser/Thr kinases that are implicated in
tumorigenesis. The discovery of a new class of PIM inhibitors, 5-(1H-indol-5-yl)-1,3,4-
thiadiazol-2-amines, is discussed with optimized compounds showing excellent potency
against all three PIM isoforms.
PIM kinases are a family of serine/threonine kinases in the CAMK group
(calcium/calmodulin-dependent protein kinase) that are constitutively active and are
regulated at the level of transcription and translation, as opposed to the more classical
post-translational phosphorylation. 1 There are three isoforms in the family, PIM1, PIM2
and PIM3, which share high homology and exhibit some functional redundancy. PIM
kinases are widely expressed and are involved in a variety of biological processes,
including cell survival, proliferation, differentiation and apoptosis.2 Over-expression of
PIM kinases has been reported in hematological and solid tumors such as diffuse large B-
cell lymphomas (DLBCL) and prostate cancer.3 Other evidence has also suggested that
PIM kinases play a role in tumorigenesis, making it an attractive target for cancer
therapy.4 There have been several reports of both PIM1-selective and pan-PIM
inhibitors, including three clinical compounds, SGI-1776, AZD1208, (structures shown
in Figure 1) and LGH447 (structure not disclosed).5,6 Considering the compensatory

nature and overlapping functions exhibited by the three PIM isoforms, we believe that
pan-PIM inhibitors are of more therapeutic value. Herein, we describe our efforts in
designing such agents for cancer therapy.
Figure 1. Structures of PIM inhibitors reported in clinical trials.
A high-throughput screening revealed one hit, an indazole 1, which showed excellent
activity against all three PIM kinase isoforms in HTRF assays (PIM1: 0.017 µM, PIM2:
0.031 µM and PIM3: 0.007 µM). However, 1 was found to be a multi-kinase inhibitor in
our internal kinase screening panel; 7 of the 47 kinases (PKD2, GSK3-β, ROCK2,
DYRK1α, CDK2, FLT3 and HGK) were inhibited more than 90% at 1 µM. A co-crystal
structure of 1 and PIM1 was obtained to facilitate the design of more selective inhibitors.7
Figure 2. Screening hit 1 and its indole analog 2.
The co-crystal structure revealed three key interactions between indazole 1 and the PIM1
protein (Fig. 3). The amino group on the thiazole forms an H-bond with Asp186, and the
adjacent N1 engages the catalytic Lys67, while the NH on the indazole forms an H-bond
with the backbone carbonyl group of Glu121. The quinoline ring resides between the
glycine-rich loop and the lower hinge region with the conformation as shown. One
unique feature of the PIM kinases is the presence of Pro123 at the hinge; thus ATP uses
solely the NH2 donor in this region, as opposed to the more common donor-acceptor pair
of the adenine ring.8 We reasoned that removal of N2 of the indazole ring of 1 should
improve kinase selectivity. The resulting indole compound 2 indeed was devoid of
activity against other kinases, yet remained moderately potent against the PIM isoforms.
Thus 2 served as a useful starting point for our SAR studies with the goal to improve the
potency by at least ten fold while maintaining selectivity against other kinases.
Figure 3. The co-crystal structure of compound 1 bound in the ATP binding site of
unphosphorylated PIM-1 protein (PDB code: 4WSY).

Noting the presence of a crystallographic water molecule strongly hydrogen bonded to
the backbone NH of Asp186, we replaced the thiazole (2) with a thiadiazole (3); this
substitution resulted in improved potency against all three isoforms. Methyl amino
analog 4 maintained potency, while the corresponding thiomethyl analog 5 lost
significant activity presumably due to the absence of an H-bond donor for capturing the
interaction with Asp186. It is noteworthy that the potency against the PIM3 isoform
correlated well with IC50 values against PIM1, while the PIM2 isoform was more difficult
to inhibit.
Table 1
PIM1, PIM2 and PIM3 inhibitory activity of substituted 3-(1H-indol-3-yl)quinolinesa
H
N
R
N
PIM1
IC50(µM)
PIM2
IC50(µM)
PIM3
IC50 (µM)
Cmpd
R
N
0.312
2.71
0.267
2
S
NH2
0.163
0.272
2.83
1.01
1.63
>22
0.178
0.181
4.11
3
4
5
a Data represents an average of at least two separate determinations.

In an attempt to better engage the glycine-rich loop and with the aminothiadiazole held
constant, we turned our attention to the nature of the C3 substituent on the indoles. The
preparation of these analogs is shown in Scheme 1. The synthesis commenced with a
Suzuki coupling between boronic ester 6 and 2-bromo-5-(methylthio)-1,3,4-thiadiazole.
Subsequent iodination and protection with p-toluenesulfonyl chloride (TsCl) gave
intermediate 9. The thiomethyl group was displaced with p-methoxybenzylamine
(PMBNH2) after oxidation with H2O2 and sodium tungstate. Various aromatic and
heterocyclic groups were introduced at C3 of the indole through Stille coupling.
Removal of the Ts and PMB protecting groups under basic and acidic conditions
respectively, furnished the desired analogs.
Scheme 1. Reagents and conditions: (a) K2CO3 , Pd(PPh3)4, 1,4-dioxane, water, 50%; (b)
I2, KOH; (c) p-MeC6H4SO2Cl, NaH, DMF, 78% for two steps; (d) H2O2, NaWO4, AcOH;
(e) p-MeOC6H4CH2NH2, dioxane, 47% for two steps; (f) R-SnBu3, Pd(PPh3)4, CuI, DMF;
(g) KOH, THF; (h) TFA.
Representative substituted aromatic groups at C3 to probe potential interactions with the
glycine-rich loop are shown in Table 2. While the quinoline and substituted phenyl
analogs (3 and 13) showed only moderate activity, 2-pyridyl groups substituted in the 6-
position with bulky groups appeared to be most promising as exemplified by compounds
14 – 19. Potent compounds with IC50 less than 100 nM in the PIM1 assay were further
profiled in cellular assays measuring the inhibition of PIM1- and PIM2-dependent
phosphorylation of BAD in U2OS cells.9 Despite the high homology in the kinase
domains, the three PIM isoforms show quite different ATP Km values. In particular,
PIM2 has a much lower Km of 4 µM whereas values of 400 µM and 40 µM are observed
for PIM1 and PIM3, respectively. This property renders the identification of cell active
PIM2 inhibitors a challenge.5a-5d, 5g, 5n, 5r
The 6-aminopyridine analog 14 showed only submicromolar activity against PIM
kinases, however, the introduction of a pyrrolidine in the 6-position of the pyridine ring
(compound 15) significantly improved the potency by at least 9 fold. Further
improvement was seen with the 6-membered ring substitution, especially the morpholino
analog with an IC50 of 152 nM in the PIM1 cell assay and measurable PIM2 cell potency

(7.57 µM). Introduction of primary alkylamines at 6-position of pyridine led to further
improved potency in the PIM2 cell assay, with the exception of cyclohexylamine analog
19, possibly due to the increased steric demand imposed by this substituent.
Table 2
Enzymatic and cellular activity of substituted 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-
amines
PIM1
PIM2
PIM3
PIM1cell PIM2cell
Compd
R
IC50(uM)a IC50(uM)a IC50(uM)a IC50(uM) IC50(uM)
3
0.163
0.177
1.01
0.178
0.097
2.74
ND
>31.6
ND
13
0.348
N
14
15
0.865
0.048
0.977
0.104
0.186
0.015
ND
ND
NH2
0.526
>31.6
16
0.016
0.059
0.006
0.152
7.57

17
18
0.024
0.012
0.037
0.021
0.005
0.005
0.384
0.111
2.19
3.09
19
0.109
0.227
0.022
ND
ND
20
21
0.142
0.011
0.192
0.042
0.029
0.009
ND
ND
0.159
12.1
22
23
0.005
0.002
0.012
0.010
0.004
0.001
0.163
0.04
1.90
0.945
24
0.001
0.006
0.001
0.057
1.22

25
0.004
0.018
0.002
0.056
2.60
a Data represents an average of at least two separate determinations. *ND = not determined
The X-ray co-crystal structure of cyclopentyl amine analog 18 bound to PIM1 was
obtained.10 As illustrated in Figure 4, the overall binding mode is similar to screening hit
1. N3 and N4 of the thiadiazole form a hydrogen bond network with a water molecule,
Lys67 and Glu89. The 2-pyridyl system adopts a planar conformation with respect to the
indole, and the aliphatic side-chain forms van der Waals contacts with the glycine-rich
loop and phenyl ring of Phe49.
Figure 4. The co-crystal structure of compound 18 bound in the ATP binding site of
unphosphorylated PIM1 protein (PDB code: 4WT6).
Following these results, a series of 2-amino-substituted pyrazines was prepared. As
shown in Table 2, the pyrazine analogs demonstrated better potency against PIM1 and
PIM2 in both biochemical and cell assays than the corresponding pyridine analogs,
indicating possible more favorable water-mediated interactions between the pyrazine and
extended hinge residues. Among these, both 23 and 24 exhibited single-digit nanomolar
pan-PIM enzyme activity, an improvement of more than 100-fold over the original
compound 2.
We next examined O-substituted pyridine and pyrazine analogs. As shown in Table 3,
the optimal affinities for the three PIM kinases were achieved with isopropoxy
substitution. Smaller substituents, such as methoxy and ethoxy groups, or larger
substituents such as cyclobutoxy and cyclopentoxy led to various degrees of erosion of
PIM inhibitor potency. In addition, substitution at the C5 pyrazine position (33) resulted
in significant loss of activity, presumably due to lack of contact with the glycine-rich
loop.

Table 3
PIM inhibitory activity of O-substituted 5-(3-(pyridin-2-yl)-1H-indol-5-yl)-1,3,4-
thiadiazol-2-amines and 5-(3-(pyrazin-2-yl)-1H-indol-5-yl)-1,3,4-thiadiazol-2-amines
PIM1
PIM2
PIM3
PIM1 cell PIM2 cell
Compd
R
IC50 (uM)a IC50 (uM)a IC50 (uM)a IC50 (uM) IC50 (uM)
26
0.017
0.007
0.022
0.010
0.003
0.002
0.141
0.106
3.73
2.22
27
28
0.015
0.041
0.041
0.114
0.005
0.018
0.479
6.90
29
30
1.02
>31.6
0.021
0.028
0.008
0.20
2.75

31
32
0.008
0.011
0.017
0.025
0.003
0.011
0.092
0.176
2.22
6.54
33
0.262
1.29
0.185
ND
ND
a Data represents an average of at least two separate determinations.
In a subsequent kinase profiling experiment using custom KINOMEscansm assay, the two
lead compounds 23 and 27 exhibited good kinase selectivity with selectivity score of 0.07
and 0.05, respectively (S(35) at1 µM, 100 kinases).11
In summary, we have discovered 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines as a novel
class of pan-PIM inhibitors. Starting from the screening hit 1, with the aid of
crystallographic information we quickly designed indole 2 with favorable kinase
selectivity profile. Focused SAR study identified compounds 23 and 27 with over 100
fold improvement in inhibitory activity against all three PIM isoforms. Further studies of
these inhibitors will be reported in due course.
Acknowledgments The authors thank Randy Hungate, Philip Tagari and Christian
Romel for their support for this research program. Thanks also go to Randy Jensen and
Chris Wilde for spectroscopic assistance.
Supplementary data X-Ray data for the co-crystal structure of compound 1 and 18 in
Pim-1 protein and PIM enzyme assays experimental details.

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bound to PIM1 can be accessed using PDB code 4WSY. Figure 3 was generated
with The PyMOL Molecular Graphics System, Version 1.7.0.1 Schrödinger, LLC.
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9
(a) The cell lines used in the assay were generated by the stable transfection
of either Pim-1 or Pim-2 into the U2OS human osteogenic sarcoma line. (b) Aho,
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FEBS Lett. 2004, 571, 43; (c) Yan, B.; Zemskova, M.; Holder, S.; Chin, V.; Kraft,
A.; Koskinen, P. J.; Lilly, M. J. Biol. Chem. 2003, 278, 45358.
10 Atomic coordinates and structure factors for crystal structures of compound 18
bound to PIM1 can be accessed using PDB code 4WT6. Figure 4 was generated
with The PyMOL Molecular Graphics System, Version 1.7.0.1 Schrödinger, LLC.
11 Assays were performed utilizing KINOMEscansm. Activity is recorded via a
competition binding assay of selected kinases that are fused to a proprietary tag.
POC values are determined by measurements of the amount of kinase bound to an
immobilized, active-site directed ligand in the presence and absence of the test
compound. Selectivity score was calculated by dividing the number of kinases
that compounds bind to by the total number of distinct kinases tested, excluding
mutant variants. S(35) = (number of non-mutant kinases with %Ctrl <35)/(number
of non-mutant kinases tested).

Figure 1. Structures of PIM inhibitors reported in clinical trials.
Figure 2. Screening hit 1 and its indole analog 2.
Figure 3. The co-crystal structure of compound 1 bound in the ATP binding site of
unphosphorylated PIM-1 protein (PDB code: 4WSY).
Figure 4. The co-crystal structure of compound 18 bound in the ATP binding site of
unphosphorylated PIM1 protein (PDB code: 4WT6).
Scheme 1. Reagents and conditions: (a) K2CO3 , Pd(PPh3)4, 1,4-dioxane, water, 50%; (b)
I2, KOH; (c) p-MeC6H4SO2Cl, NaH, DMF, 78% for two steps; (d) H2O2, NaWO4, AcOH;
(e) p-MeOC6H4CH2NH2, dioxane, 47% for two steps; (f) R-SnBu3, Pd(PPh3)4, CuI, DMF;
(g) KOH, THF; (h) TFA.