The discovery and optimization of aminooxadiazoles as potent Pim kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.12.067

High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers. These findings suggest that inhibition of Pim signaling by a small molecule Pim-1,2 inhibitor could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal starting from the highly Pim-selective indole-thiadiazole compound (1), which was derived from a nonselective hit identified in a high throughput screening campaign. Optimization of this compound's potency and its pharmacokinetic properties resulted in the discovery of compound 29. Cyclopropane 29 was found to exhibit excellent enzymatic potency on the Pim-1 and Pim-2 isoforms (K_i values of 0.55 nM and 0.28 nM, respectively), and found to inhibit the phosphorylation of BAD in the Pim-overexpressing KMS-12 cell line (IC₅₀ = 150 nM). This compound had moderate clearance and bioavailability in rat (CL = 2.42 L/kg/h; %F = 24) and exhibited a dose-dependent inhibition of p-BAD in KMS-12 tumor pharmacodynamic (PD) model with an EC₅₀ value of 6.74 μM (18 μg/mL) when dosed at 10, 30, 100 and 200 mg/kg po in mice.

Full text of DOI:10.1016/j.bmcl.2014.12.067

Accepted Manuscript
The discovery and optimization of aminooxadiazoles as potent Pim kinase in-
hibitors
Ryan P. Wurz, Liping H. Pettus, Claire Jackson, Bin Wu, Hui-Ling Wang, Brad
Herberich, Victor Cee, Brian A. Lanman, Anthony B. Reed, Frank Chavez Jr.,
Thomas Nixey, Jimmy Laszlo III, Paul Wang, Yen Nguyen, Christine Sastri,
Nadia Guerrero, Jeff Winston, J. Russell Lipford, Matthew R. Lee, Kristin L.
Andrews, Christopher Mohr, Yang Xu, Yihong Zhou, Darren L. Reid, Andrew
S. Tasker
PII:
S0960-894X(14)01375-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.12.067
BMCL 22312
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 November 2014
16 December 2014
19 December 2014
Please cite this article as: Wurz, R.P., Pettus, L.H., Jackson, C., Wu, B., Wang, H-L., Herberich, B., Cee, V., Lanman,
B.A., Reed, A.B., Jr., F.C., Nixey, T., Laszlo, J. III, Wang, P., Nguyen, Y., Sastri, C., Guerrero, N., Winston, J.,
Russell Lipford, J., Lee, M.R., Andrews, K.L., Mohr, C., Xu, Y., Zhou, Y., Reid, D.L., Tasker, A.S., The discovery
and optimization of aminooxadiazoles as potent Pim kinase inhibitors, Bioorganic & Medicinal Chemistry Letters
(2015), doi: http://dx.doi.org/10.1016/j.bmcl.2014.12.067
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The discovery and optimization of aminooxadiazoles as potent Pim kinase
inhibitors.
Ryan P. Wurz,*,a Liping H. Pettus,a Claire Jackson,a Bin Wu,a Hui-Ling Wang,a Brad Herberich,a Victor
Cee,a Brian A. Lanman,a Anthony B. Reed,a Frank Chavez Jr.,a Thomas Nixey,a Jimmy Laszlo III,e Paul
Wang,c Yen Nguyen,c Christine Sastri,b Nadia Guerrero,b Jeff Winston,b J. Russell Lipford,b Matthew R.
Lee,c Kristin L. Andrews,c Christopher Mohr,c Yang Xu,d Yihong Zhou,d Darren L. Reid,f 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; d) Department of Pharmacokinetics and Drug Metabolism; e) Department of Discovery Technologies;
f)Department of Pharmaceutics R&D.
* To whom correspondence should be addressed:
Ryan P. Wurz: Tel +1-805-313-5400; Fax 805-480-1337; email: rwurz@amgen.com
This is where the receipt/accepted dates will go; Received Month XX, 2014; Accepted Month XX, 2014 [BMCL RECEIPT]
Abstract—High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers. These findings
suggest that inhibition of Pim signaling by a small molecule Pim-1,2 inhibitor could provide patients with therapeutic benefit. Herein,
we describe our progress towards this goal starting from the highly Pim-selective indole-thiadiazole compound (1), which was derived
from a nonselective hit identified in a high throughput screening campaign.
Optimization of this compound’s potency and its pharmacokinetic properties
resulted in the discovery of compound 29. Cyclopropane 29 was found to
exhibit excellent enzymatic potency on the Pim-1 and Pim-2 isoforms (Ki values
of 0.55 nM and 0.28 nM, respectively), and found to inhibit the phosphorylation
of BAD in the Pim-overexpressing KMS-12 cell line (IC50 = 150 nM). This
compound had moderate clearance and bioavailability in rat (CL = 2.42 L/kg/h;
%F = 24) and exhibited a dose-dependent inhibition of p-BAD in KMS-12
tumor pharmacodynamic (PD) model with an EC50 value of 6.74 µM (18 µg/mL)
when dosed at 10, 30, 100 and 200 mg/kg P.O. in mice.
Proviral Integration site of Moloney (Pim) murine leukemia virus kinases are serine/threonine kinases
that are involved in cell survival and proliferation as well as a number of other signal transduction
pathways.1,2 The Pim-1, -2 and -3 isoforms share a high level of sequence homology and appear largely
redundant in function. High levels of Pim expression have been implicated as oncogenic drivers in
1

hematologic and solid malignancies3 including multiple myeloma,4 acute myeloid leukemia, prostate
cancer, and gastric and liver carcinomas. These findings suggest that inhibition of Pim signaling by a
small molecule pan-Pim inhibitor could provide patients with therapeutic benefit since the upregulation
of Pim kinases correlates with a poor prognosis.1 In addition to cancer, Pim kinases have been reported
to play a role in several autoimmune diseases.5
All three Pim isoforms share the unique feature of being the only enzymes in the kinome with a
proline residue in the hinge, which results in only one hydrogen bond interaction with ATP.6 This
unique property can be exploited to derive selectivity over other kinases. In addition, the KM,ATP for Pim-
2 is 136-fold lower than that for Pim-1.7 As a consequence of this phenomenon, Pim inhibitors that
have potent cellular activities in pan-Pim expressing cell lines has been very challenging. Pim directly
phosphorylates Bcl-2-associated death promoter (BAD), which becomes sequestered in p-BAD-(14-3-3)
protein heterodimers. Inhibition of Pim-mediated BAD phosphorylation is expected to lead to high
levels of free BAD, which can bind to the antiapoptotic Bcl-2 protein, causing release of the
proapoptotic Bax and BAK.8 To evaluate the cell potency of novel Pim inhibitors, a multiple myeloma
KMS-12 BM cell line9 with a readout of the inhibition of phosphorylation of BAD (p-BAD) was chosen
to interrogate chemical matter in this manuscript.
Numerous research groups have investigated small molecule Pim inhibitors10,11 which resulted in
several clinical compounds such as SGI-177612 and AZD-120813 (Fig. 1). Herein, we describe our
progress towards the development of a novel class of Pim-1,2 inhibitors based on an aminooxadiazole-
indole scaffold.
INSERT FIGURE 1 HERE
Compound 1 resulted from an effort to improve Pim potency and kinase off-target selectivity of the
indazole-thiazole high-throughput screening (HTS) hit.14 While compound 1 exhibited a favorable
selectivity profile, the Pim potency and oxidative stability of the molecule were seen as major
limitiations. Improvements in potency could be achieved upon replacement of the aminothiadiazole
2

motif with an aminooxadiazole (2 versus 1, Table 1).15 Although rat liver microsomal turnover was
modest, rat pharmacokinetics of 2 yielded high clearance with low bioavailability (CL 3.9 (L/kg)/h; %F
= 3.5, Table 5). The parent compound, lacking substitution at the 3-position of the indole ring (R = H,
3), displayed greatly diminished activity in the Pim-1 and Pim-2 enzyme assays (Ki values >1 µM).
Removal of the pyridine nitrogen present in compound 2 was also greatly detrimental (4). Replacement
of the iso-propoxy group with a morpholine (compound 5) resulted in erosion in enzymatic and cellular
potency as compared to compound 2 with a small improvement in microsomal stability. Introduction of
an additional nitrogen atom into the pyridine ring of compound 2 resulted in compound 6 which was
roughly equipotent to compound 2 but whose microsomal stability was somewhat diminished.
Replacement of the iso-propoxy group with either a difluoropiperidine or a difluoropyrrolidine
(compounds 7-8) resulted in an improvement in potency, however, these substitution patterns led to a
decline in microsomal stability. Introduction of a 4-cyclopropylpyrimidin-2-yl group (compound 9)
was one substitution that resulted in a modest improvement in Pim-2 enzymatic potency along with a 2-
fold gain in cellular potency (IC50 = 319 nM) as compared to compound 2. Furthermore, this
substitution pattern also resulted in a favorable improvement in microsomal activity.
Rat
pharmacokinetics demonstrated a nominal improvement in rat iv clearance (CL 2.9 (L/kg)/h) compared
to compound 2, however, the problem of low bioavailability persisted (%F = 1.2, Table 5). The
cyclopropyl group was an important driver of potency as its removal (compound 10) led to a large
decrease in enzyme potency. The positions of the two nitrogen atoms in the 4-cyclopropylpyrimidin-2-
yl ring were somewhat important as introduction of a 6-cyclopropylpyrazin-2-yl ring (compound 11)
was slightly inferior to compound 9 both in terms of cellular activity and microsomal activity. Other
substitution patterns on the pyrimidine ring were briefly explored (see compounds 12-17), however, the
combination of good cellular potency and microsomal stability did not surpass compound 9.
INSERT TABLE 1 HERE
3

We explored replacements for the aminooxadiazole motif in an attempt to further drive cellular potency
and improve PK properties. These modifications included triazolopyridine and triazolopyridazine rings
(compounds 18-19, Table 2) which preserved the two hydrogen bond acceptors of the oxadiazole ring.
Presumably the introduction of the second nitrogen in compound 19 allowed the compound to adopt a
planar conformation and this proved beneficial for the enzyme and cellular potency of this compound.
Unfortunately, 19 exhibited higher microsomal turnover compared to compound 9. Introduction of
pyrimidin-4-one or cyclopropylpyrazine16 motifs were also explored (compounds 20-21), but these
compounds were found to lack sufficient enzyme potency.17
INSERT TABLE 2 HERE
In an attempt to remedy the rapid clearance of compounds 2 and 9, we returned to explore the
introduction of various substitutions on the oxadiazole ring. Replacement of the amino-group with
three all-carbon-based substitutions with increasing size including a cyclopropyl, iso-butyl or a phenyl
ring led to a loss in enzymatic potency (compounds 22-24, Table 3). A more promising avenue of
exploration resulted from introduction of small alkyl groups to the primary amine of the
aminooxadiazole motif thereby preserving a single hydrogen bond donor. In particular, compound 25,
which contains a 1,1,1-trifluoroethyl group, exhibited subnanomolar potency in the Pim-1 and Pim-2
enzyme assays, however, this improvement did not translate into improved cellular potency.
Unfortunately, this substitution also led to erosion in microsomal stability. Rat pharmacokinetics of this
compound again showed high iv clearance (CL = 2.98 (L/kg)/h) but a promising improvement in
bioavailability (%F = 25) when compared to compound 9 (Table 5). Introduction of an iso-propyl
group (26) again gave excellent enzymatic potency in both Pim-1 and Pim-2 enzyme assays but with a
smaller cell-shift than observed with the 1,1,1-trifluoroethyl group. This compound also showed
similar microsomal stability to compound 9. Rat pharmacokinetics showed a slight improvement in iv
clearance compared to compound 9 with a similar increase in bioavailability (CL 2.33 (L/kg)/h; %F =
4

20, Table 5) as seen with compound 25. A cyclopropyl substitution (compound 27) was found to have
decreased microsomal stability and was slightly less potent in the cellular assay. Introduction of
additional polarity in an attempt to lower the cLogP of the compound using an oxetane ring (compound
28) lead to erosion in the enzyme potency, however, the shift in the cellular assay was not a large as
was the case with previous compounds. Expanding the size of the substituent on the amino-group to a
tert-butyl group (29) gave a small improvement in microsomal stability compared to compound 26 and
a small gain in cellular potency (KMS-12 IC50 = 150 nM). The pharmacokinetic properties of this
compound were virtually identical to compound 26 (CL 2.43 (L/kg)/h; %F = 19, Table 5). Finally,
larger groups such as a 3-fluorophenyl substitution (30) were not well tolerated in terms of potency.
INSERT TABLE 3 HERE
In a further attempt to address the poor pharmacokinetic properties of this series, we took two
different approaches: introduction of fluorine atoms to protect potential metabolic soft spots, and
introduction of polarity to reduce cLogP. We believed that introduction of a 5-fluoro-substitution
extending from the 4-cyclopropylpyrimidin-2-yl ring into the solvent exposed portion of the inhibitor
should be tolerated and could possibly improve the microsomal stability. Unfortunately, compound 31
bearing this substitution displayed a moderate loss of enzymatic potency as compared to 26 with little
improvement on microsomal stability. In an attempt to reduce the cLogP of the comopund, an amide
function was introduced at the 5- and 6-positions of the 4-cyclopropylpyrimidin-2-yl ring (compounds
32-33). The 6-substitution (compound 33) was preferred in terms of cellular potency, however, this
compound had inferior microsomal stability to its regioisomer in the 5-position (32). Removal of the
cyclopropyl group (compound 34) resulted in improved microsomal stability but also resulted in a
dramatic loss in enzyme potency. Modifications to the indole ring in an attempt to improve PK
prompted the synthesis a 6-fluoro-indole18 analog (compound 35). This substitution was tolerated in
terms of cellular potency and led to a small improvement in rat iv clearance (CL = 2.16 (L/h)/kg),
however, this modification was detrimental to oral bioavailability (%F = 6). Introduction of a nitrogen
5

atom into the indole ring at the 6-position (compound 36)19 was beneficial for microsomal stability;
however, its introduction ablated cellular potency.
INSERT TABLE 4 HERE
The X-ray co-crystal structure of compound 29 in the Pim-1 protein was obtained (Fig. 2). This
structure revealed a number of key interactions with the Pim-1 protein that likely contributed to
potency. The N−H of the indole engages in a hydrogen bonding interaction (3.1 Å) with the carbonyl
of the Glu121 residue of the linker region of the ATP binding pocket. The benzene ring of the indole
sits against the gatekeeper residue Leu120 and potentially gains van der Waals interactions. Additional
favorable hydrophobic contacts are observed between the pyrimidine ring and Val126 and Leu174
residues along with the tert-butyl group and Ile185. One of the nitrogen atoms in the oxadiazole ring
engages in a hydrogen bonding (2.9 Å) interaction with the conserved catalytic Lys67 while the tert-
butylamino group donates a hydrogen bond to form what is likely a weak hydrogen bonding interaction
with the Asp186 residue (3.4 Å). The cyclopropyl group and tert-butyl group are facing each other (3.4
Å and 3.7 Å) to give the inhibitor a horseshoe like shape. Finally, the Phe49 residue is displaced from
the binding pocket to accommodate the ligand.
INSERT FIGURE 2 HERE
Glu121
A representative synthesis of this class of Pim inhibitors is depicted in Scheme 1 where compound
29 is prepared in a seven step sequence.20 Commercially available 1H-indole-5-carboxylic acid (37) was
selectively iodinated at the 3-position in high yield using iodine and potassium hydroxide in DMF to
afford the corresponding 3-iodoindole 38.21 Protection of the indole with a tosyl group yielded
compound 39. The hydrazide 40 was then prepared using a HOBt/EDAC mediated coupling which was
then converted into the desired tert-butylaminooxadiazole 41 upon heating with tert-butyl-isothiocyanate
followed by treatment with EDAC.22 Transformation of the protected 3-iodoindole 41 into its
Lys67
6

corresponding pinacolatoboron reagent was accomplished using standard borylation conditions23
affording the key intermediate for analoging (compound 42). A Pd-catalyzed Suzuki coupling with
commercially available 2-bromo-4-cyclopropylpyrimidine then afforded the fully elaborated inhibitor 43.24
Tosyl group deprotection with 1N NaOH in dioxane afforded compound 29 in moderate yield (55%).23
INSERT SCHEME 1 HERE
INSERT TABLE 5 HERE
Based on the best-in-series rat PK properties and cellular potency, compound 29 was selected for
further evaluation in an in vivo mouse tumor pharmacodynamic (PD) model. Female CD1 nu/nu mice
were implanted with KMS-12 BM tumor cells (5 x 106 cells) and dosed orally with 29 at 10, 30, 100 and
200 mg/kg. At 3, 6, 12, 24 h time points post dosing, tumor cells and plasma were harvested. Total p-
BAD levels in the tumor were measured with a quantitative MSD assay. Compound 29 showed a dose-
dependent inhibition of phosphorylation of BAD, indicating that it effectively inhibited Pim in vivo (Fig.
3). The total plasma concentration for 50% tumor PD reduction (EC50) in this experiment was calculated
to be 6.74 µM (18 µg/mL).
INSERT FIGURE 3 HERE
The selectivity of compound 29 against a panel of 112 protein and lipid kinases was examined in the
DiscoveRX KINOMEscan platform.25 In this panel, the competitive binding of 29 at 1 µM was
measured as a percentage of control (POC). For all of the kinases assayed, competitive binding (POC
<35%) by 29 was observed for 7 kinases including GAK (0.3% POC), CLK4, (4.3% POC), CLK1 (11%
POC), CLK2 (11% POC), DYRK1B (20% POC), DYRK1A (27% POC) and CSNK2A1 (34% POC).26
Compound 29 showed moderate permeability and low efflux (19.8 x 10-6 cm/s and ER = 1.8 and 2.5,
respectively in mouse and rat LLC-PK1 cell line transfected with a MDR1 gene), and exhibited moderate
7

plasma protein binding (97.7%, 97.8% and 97.4% bound in human, mouse and rat, respectively). The
aqueous solubility of compound 29 was quite low: 0.14 mg/mL, 0.001 mg/mL and 0.005 mg/mL in
0.01N HCl, PBS and SIF, respectively.27
In conclusion, a structurally novel class of Pim-1,2 inhibitors containing an aminooxadiazole motif has
been prepared. From this series, compound 29 was highly selective against 112 other kinases and had
superior cellular potency and PK properties over compound 1. Compound 29 was efficacious in a tumor
PD assay in mice with an EC50 value of 6.74 µM (18 µg/mL).
Supplementary data X-Ray data for the co-crystal structure of compound 29 in Pim-1 protein is
provided.
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3153. (u) Casuscelli, F.; Ardini, E.; Avanzi, N.; Casale, E.; Cervi, G.; D’Anello, M.; Donati, D.; Faiardi, D.; Ferguson, R.
D.; Fogliatto, G.; Galvani, A.; Marsiglio, A.; Mirizzi, D. G.; Montemartini, M.; Orrenius, C.; Papeo, G.; Piutti, C.; Salom,
B.; Felder, E. R. Bioorg. Med. Chem. 2013, 23, 7364-7380. (v) Suchaud, V.; Gavara, L.; Saugues, E.; Nauton, L.; Théry,
V.; Anizon, F.; Moreau, P. Bioorg. Med. Chem. 2013, 21, 4102-4111. (w) Gavara, L.; Suchaud, V.; Nauton, L.; Théry, V.;
Anizon, F.; Moreau, P. Bioorg. Med. Chem. Lett. 2013, 23, 2298-2301. (x) Burger, M. T.; Han, W.; Lan, J.; Nishiguchi,
G.; Bellamacina, C.; Lindval, M.; Atallah, G.; Ding, Y.; Mathur, M.; McBride, C.; Beans, E. L.; Muller, K.; Tamez, V.;
Zhang, Y.; Huh, K.; Feucht, P.; Zavorotinskaya, T.; Dai, Y.; Holash, J.; Castillo, J.; Langowski, J.; Wang, Y.; Chen, M.
Y.; Garcia, P. D. ACS Med. Chem. Lett. 2013, 4, 1193-1197. (y) Xu, Y.; Brenning, B. G.; Kultgen, S. G.; Foulks, J. M.;
Clifford, A.; Lai, S.; Chan, A.; Merx, S.; McCullar, M. V.; Kanner, S. B.; Ho, K.-K. ACS Med. Chem. Lett. 2014, ASAP,
DOI: 10.1021/ml500300c.
12. Mumenthaler, S. M.; Ng, P. Y.; Hodge, A.; Bearss, D.; Berk, G.; Kanekal, S.; Redkar, S.; Taverna, P.; Agus, D. B.;
Jain, A.; Mol. Cancer Ther. 2009, 8, 2882-2893.
13. Dakin, L. A.; Block, M. H.; Chen, H.; Code, E.; Dowling, J. E.; Feng, X.; Ferguson, A. D.; Green, I.; Hird, A. W.;
Howard, T. Bioorg. Med. Chem. Lett. 2012, 22, 4599-4604.
14. 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.; Lipford, R.; Lee, M. R.; Mohr, C.; Andrews, K.; Tasker, A. S. Bioorg. Med. Chem. Lett. manuscript accepted.
15. For a recent report from our laboratories on aminooxadiazoles as inhibitors of Cdc7 see: Harrington, P. E.; Bourbeau,
M. P.; Fotsch, C.; Bailis, J. M.; Bush, T.; Escobar, S.; Hickman, D.; Heller, S.; Hsieh, F.; Orf, J. N.; Rong, M.; San
Miguel, T.; Tan, H.; Zalameda, L.; Allen, J. G. Bioorg. Med. Chem. Lett. 2013, 23, 6396-6400.
16. For a structurally related example see: ref. 11k.
9

17. It should be noted that compound 21 bears some structural resemblance to naturally occurring meridianin C, a marine
natural product. See for example: More, K. N.; Jang, H. W.; Lee, J. Bioorg. Med. Chem. Lett. 2014, 24, 2424-2428.
18. For the preparation of fluoroindolecarboxylic acids see: Schlosser, M.; Ginanneschi, A.; Leroux, F. Eur. J. Org. Chem.
2006, 2956-2969.
19. Compound 36 bears some structural resemblance to meriolin, a marine derived 7-azaindole natural product which is
known to inhibit a range of protein kinases. See for example: Walker, S. R.; Czyz, M. L.; Morris, J. C. Org. Lett. 2014,
16, 708-711 and references cited therein.
20. Wang, H.-L.; Cee, V. C.; Herberich, B. J.; Jackson, C. L.; Lanman, B. A.; Nixey, T.; Pettus, L. H.; Reed, A. B.; Wu,
B.; Wurz, R.; Tasker, A. Patent application WO2012/129338A1, 2012.
21. Witulski, B.; Buschmann, N.; Bergsträßer, U. Tetrahedron 2000, 56, 8473-8480.
22. See for example: Yang, S.-J.; Lee, S.-H.; Kwak, H.-J.; Gong, Y.-D. J. Org. Chem. 2013, 78, 438-444.
23. Allen, J. R.; Amegadzie, A. K.; Gardinier, K. M.; Gregory, G. S.; Hitchcock, S. A.; Hoogestraat, P. J.; Jones, W. D.
Jr.; Smith, D. L. Patent application WO2005/066126 A1, 2005.
24. Billingsley, K.; Buchwald, S. L. J. Am. Chem. Soc. 2007, 129, 3358-3366.
25. For more information see: http://www.discoverx.com/technology/technology-kinomescan.php. The Ambit kinase
panel included: Abl, Akt1, Akt2, ALK, AMPK, Aur2, BMPR1A, BRAF, BRK, BTK, CAMK2, CAMK4, CDC7, CDK2,
CDK4, CDK8, CDK18, c-KIT, CHK1, CK1δ, CKΙΙα CHK2, CLK1, CLK2, CLK4, DAPK1, DDR1, DMPK, DYRK1α,
DYRK1β, EGFR, EPHA2, EPHA4, EPHB3, Erk1, Erk2, FGFR1, FLT3, FYN, GAK, GRK1, GSK3β, HGK, IGF1R,
INSR, IRAK4, IRE1, JAK2, JNK3, KDR, LCK, LIMK1, LYN, MAPK1, MAP2K3, MAP3K1, MAP3K9, MAPK4K1,
MAPKAPK2, MARK1, MARK3, MEK5, MET, MNK1b, MSK1, MST2, mTOR, NEK4, NEK6, OXSR1, p38α, p38γ
p70S6K, PAK2, PDGFRβ, PI3Kα, PI3Kδ, PIM1, PIM2, PKAα, PKAC2, PKBα, PKBβ, PKC, PKR, PKCβ2, PKD2,
PKGα, PLK1, PLK4, PRAKAA1, PRKG1, RAF, RIPK2, ROCK2, RPS6KB1, RSK1, SGK1, SRC, STK33, STK32B,
SYK, TAK1, TAO2 TBK1, TIE2, TGFBR1, TRKA, TSSK1β, TYK2, WEE1, YSK4
26. For the POC values of the full kinase panel see the Supporting Info.
27. Aqueous equilibrium solubility assay was conducted on Symyx Solubility System. 1 mg solid compound was added to
each of the three media, equilibrated at room temperature for 48-72 hours, centrifuged and the supernatants were analyzed
by HPLC and compared against a standard curve.
10

Scheme 1. General scheme for the synthesis of compound 29. Reagents and conditions: a) I2 (1.05 equiv.), KOH (2.2 equiv.), DMF,
RT, 1 h, 98%; b) NaH (2.5 equiv.), TsCl (1.1 equiv.), DMF, 0 – 10 °C, 30 min; c) NH2NH2 (5 equiv.), HOBt (1.2 equiv.), EDAC (1.2
equiv.), DMF, RT, 2 h, 98% (2 steps); d) i) t-BuNCS (3 equiv.), THF, 60 °C, 1 h ii) EDAC (1.5 equiv.), DMF, 90 °C, 1 h, 66% (2
steps); e) (BPin)2 (3 equiv.), KOAc (5.0 equiv.), Pd(dppf)Cl2 (15 mol%), DMF, 90 °C, 1 h; f) 2-Bromo-4-cyclopropylpyrimidine (1.15
equiv.), Pd2(dba)3 (3 mol%), X-Phos (6 mol%), K3PO4 (3 equiv.), 5:1 dioxane:water, wave, 130 °C, 30 min, 56% (2 steps); g) 1N
NaOH, dioxane, wave 100 °C, 15 min, 55%.1
Table 1. Enzyme and cellular assay results for compounds arising from modifications to the 3-position of
the indolea
KMS-12
RLM/HLM
L/(min*mg)b
Pim-1
Pim-2
Compd
p-BAD
R
Ki (nM)
Ki (nM)
IC50 (nM)
1c
2
10 ± 2.0
1.7 ± 0.23
2560
6.6 ± 0.1
1.0 ± 0.11
899 ± 83
3210 ± 1005
697 ± 54
ND
85/90
74/99
150/65
H
3

55 ± 0.5
116 ± 101
3.4 ± 0.9
1.2 ± 0.12
ND
158/>399
88/92
4
5
6
2.1 ± 0.03
3.0 ± 0.48
1008 ± 187
468 ± 191
134/182
1.6 ± 1.4
1.1 ± 0.92
236 ± 23
373 ± 18
147/182
156/180
7
8
0.52 ±
0.042
0.73 ±
0.014
1.4 ± 0.71
150 ± 58
2.0 ± 0.59
31 ± 7.9
0.27 ± 0.24 319 ± 45
79/16
ND
9
47 ± 0.67
1.1 ± 0.30
8.7 ± 0.98
ND
10
11
12
557 ± 108
3280 ± 396
124/70
58/40
2.1 ± 0.72
0.40 ± 0.17 371 ± 71
160/125
13

0.39 ±
0.021
2.7 ± 0.94
14.7 ± 3.5
560 ± 35
77/42
14
15
2.2 ± 0.52
1529 ± 71
100/49
2.7 ± 1.8
0.15 ± 0.02 160 ± 18
213/332
16
1.1 ± 0.38
0.42 ± 0.31 376 ± 35
614/288
17
a Data represents an average of at least two separate determinations. b Single experimental value; estimated
clearance from percent parent compound (1 M) remaining following a 30 min incubation in liver
microsomes (0.25 mg/mL) and NADPH (1 mM). c This compound contained an aminothiadiazole.
Table 2. Enzyme and cellular assay results for compounds arising from replacement of the
aminooxadiazole motif.a
KMS-12
p-BAD
RLM/HLM
L/(min*mg)b
Pim-1
Pim-2
Compd
R
Ki (nM)
Ki (nM)
IC50 (nM)
1.4 ± 0.71
52 ± 9.7
0.27 ± 0.24
22 ± 7.0
319 ± 45
ND
79/16
9
157/195
18

3.0 ± 0.14
114 ± 11
1.0 ± 0.13
60 ± 3.6
297 ± 5.7
ND
181/163
140/107
19
20
32 ± 14
76 ± 47
ND
121/ND
21
a Data represents an average of at least two separate determinations. b Single experimental value; estimated
clearance from percent parent compound (1 M) remaining following a 30 min incubation in liver
microsomes (0.25 mg/mL) and NADPH (1 mM).
Table 3. Enzyme and cellular assay results for compounds arising from SAR on the oxadiazole ring.a
KMS-12
RLM/HLM
L/(min*mg)b
Pim-1
Pim-2
Compd
p-BAD
R
Ki (nM)
Ki (nM)
IC50 (nM)
NH2
c-C3H5
i-Bu
Ph
1.4 ± 0.71
8.3 ± 3.7
251 ± 74
115 ± 33
0.27 ± 0.24
1.4 ± 0.28
177 ± 113
355 ± 191
319 ± 45
18900 ± 5798
ND
79/16
257/245
364/251
102/58
9
22
23
24
ND

NH-CH2CF3 0.24 ± 0.008 0.17 ± 0.002
1002 ± 54
207 ± 116
328 ± 54
347 ± 77
150 ± 63
ND
162/88
88/87
200/73
ND
25
26
27
28
29
30
NH-i-Pr
0.84 ± 0.20
0.30 ± 0.08
0.87 ± 0.75
0.71 ± 0.26
NH-c-C3H5 1.2 ± 0.36
NH-oxetane 2.2 ± 0.31
NH-t-Bu
0.55 ± 0.052 0.28 ± 0.072
59/71
102/73
NH-3-F-Ph 69 ± 17
38 ± 1.3
a Data represents an average of at least two separate determinations. b Single experimental value; estimated
clearance from percent parent compound (1 M) remaining following a 30 min incubation in liver
microsomes (0.25 mg/mL) and NADPH (1 mM).
Table 4. Enzyme and cellular assay results for compounds arising from variation of the indole scaffold and
peripheral substitutions on the 4-cyclopropylpyrimidin-2-yl ring.a
KMS-12
Pim-2
RLM/HLM
L/(min*mg)b
Pim-1
p-BAD
Compd Structure
Ki
(nM)
Ki (nM)
IC50
(nM)

0.84 ±
0.20
0.30 ±
0.08
207 ±
118
88/87
84/60
26
31
1.3 ±
0.42
0.54 ±
0.17
490 ± 46
0.45 ±
0.024
0.34 ±
0.041
673 ±
116
35/37
32
1.6 ±
0.45
0.49 ±
0.072
193 ± 17
177/100
33
34
35
14 ±
0.75
54 ± 6.6
ND
57/28
1.2 ±
0.091
0.57 ±
0.004
484 ± 21
135/98

3.6 ±
1.1
1.7 ±
0.15
>10 000
35/45
36
a Data represents an average of at least two separate determinations. b Single experimental value; estimated
clearance from percent parent compound (1 M) remaining following a 30 min incubation in liver
microsomes (0.25 mg/mL) and NADPH (1 mM).
Table 5. Pharmacokinetic properties of selected compounds
a
Pharmacokinetic parameters following administration in male Sprague Dawley rats; mean values from 3
b
c
animals per dosing route. Dosed at 2 mg/kg as a solution in DMSO. po doses were 5 mg/kg in 1%
d
Tween 80, 2% hydroxypropyl methylcellulose (HPMC), 97% water/methanesulfonic acid pH 2.2.
po
doses were 3 mg/kg. e po doses were 2 mg/kg.
1. Compound 29 was isolated as a light yellow amorphous solid as its TFA salt. 1H NMR (400 MHz,
DMSO-d6)  ppm 11.98 - 12.07 (1 H, m), 8.97 - 9.02 (1 H, m), 8.60 (1 H, d, J=5.3 Hz), 8.32 (1 H, d, J=2.9
Hz), 7.66 - 7.73 (2 H, m), 7.63 (1 H, d, J=8.4 Hz), 7.21 (1 H, d, J=5.3 Hz), 2.15 - 2.24 (1 H, m), 1.43 (9 H,
s), 1.23 - 1.29 (2 H, m), 1.15 - 1.23 (2 H, m). 19F NMR (377 MHz, DMSO-d6)  ppm -74.99 (1 F, s).

Figure 1. Structures of publicly disclosed Pim inhibitors and Amgen’s starting point (compound 1).
Figure 2. X-ray co-crystal of compound 29 bound in the ATP binding site of unphosphorylated Pim-1 protein determined to 2.7 Å
resolution. PDB code: 4TY1. Dashed lines indicate hydrogen bonds (distances are in Angstroms). For the inhibitor, C: green; N:
Glu121
blue; O: red.

Figure 3. Effect of compound 29 in a KMS-12 tumor pharmacodynamic model measuring the inhibition of phosphorylation of BAD.
Oral dose-response study at 4 time points. Statistical significance was evaluated by Dunnett’s method. Bars represent the average ±
SD (n=3).