Discovery of N-substituted 7-azaindoles as PIM1 kinase inhibitors – Part I

Article abstract of DOI:10.1016/j.bmcl.2017.08.069

Novel N-substituted azaindoles have been discovered as PIM1 inhibitors. X-ray structures have played a significant role in orienting the chemistry effort in the initial phase of hit confirmation. Disclosure of an unconventional binding mode for 1 and 2, as demonstrated by X-ray crystallography, is presented and was an important factor in selecting and advancing a lead series.

Full text of DOI:10.1016/j.bmcl.2017.08.069

Accepted Manuscript
Discovery of N-substituted 7-azaindoles as PIM1 kinase inhibitors - Part I
Claude Barberis, Neil Moorcroft, Chris Arendt, Mikhail Levit, Sandra Moreno-
Mazza, Joseph Batchelor, Ingrid Mechin, Tahir Majid
PII:
S0960-894X(17)30870-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.069
BMCL 25263
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 May 2017
25 July 2017
31 August 2017
Please cite this article as: Barberis, C., Moorcroft, N., Arendt, C., Levit, M., Moreno-Mazza, S., Batchelor, J.,
Mechin, I., Majid, T., Discovery of N-substituted 7-azaindoles as PIM1 kinase inhibitors - Part I, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.069
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Discovery of N-substituted 7-azaindoles as
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Claude Barberis*, Neil Moorcroft, Chris Arendt, Mikhail Levit, Sandra Moreno-Mazza, Joseph Batchelor,
Ingrid Mechin, Tahir Majid
Cl
Pim1 IC50= 0.043 uM
NH
N
O
N
Pim2 IC50= 1.27 uM
Pim3 IC50= 0.29 uM
N
NH2

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery of N-substituted 7-azaindoles as PIM1 kinase inhibitors - Part I
Claude Barberisa, Neil Moorcrofta,e, Chris Arendtb,f, Mikhail Levitb, Sandra Moreno-Mazzab,h, Joseph
Batchelorc, Ingrid Mechinc,g, Tahir Majida,d
a IDD Medicinal Chemistry, Sanofi Genzyme, 153 Second Avenue, Waltham MA 02451, USA
b Oncology Biochemistry/Biology, Sanofi Genzyme, 270 Albany Street, Cambridge, MA 02139, USA
c IDD In Vitro Biology, Sanofi, 153 Second Avenue, Waltham MA 02451
d Program management, Sanofi Genzyme, 49 New York Avenue, Framingham MA 01701, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Novel N-substituted azaindoles have been discovered as PIM1
inhibitors. X-Ray structures have played a significant role in
orienting the chemistry effort in the initial phase of hit confirmation.
Disclosure of an unconventional binding mode for 1 and 2, as
demonstrated by X-Ray crystallography, is presented and was an
important factor in selecting and advancing a lead series.
Keywords:
PIM1
X-Ray crystallography
Inhibitors
Cancer
The three members of the PIM family of serine/threonine kinases (PIM1, PIM2 and PIM3) were originally
identified as Proviral Integration sites in Moloney leukemia virus linked to the survival and proliferation of
cancers of hematopoietic origin.1 They are characterized by a constitutive serine/threonine activity that does
not depend upon post-translational modifications for activation.2 PIM is capable of supporting in vivo
tumor proliferation and survival through modification of common, as well as isoform-specific substrates.3
In normal cells, cytokine signaling through the JAK/STAT pathway strictly regulates the transcriptional
activation of the PIM genes and their expression as constitutively active protein kinases. In numerous
———
 Corresponding author. Tel.: +1-781-464-3838; fax: +1-781-672-5822; e-mail: claude.barberis2@sanofi.com
e Present address: Maxsar Biopharma, 18 Roberts Road, Warren, NJ, 07059
f Present address: Takeda, 40 Landsdowne street, Cambridge, MA 02139
g Present address: Icagen, Tucson Research Center 2090 E. Innovation Park drive, Oro Valley, AZ 85755
h Present address: PTC therapeutics, 100 Corporate court, South Plainfield NJ, 07080

liquid and solid tumors4, PIM kinases are over-expressed and this has been associated with drug resistance
mechanisms.4h
The observation that PIM kinases are selectively over-expressed in certain cancers, while apparently
playing non-essential roles in normal cells (as inferred from studies on mice deficient in all three PIM
kinases), prompted numerous research groups5 to initiate medicinal chemistry efforts to identify small
molecule inhibitors of PIM kinases. With limited understanding of the exact role of these kinases in human
disease, we initiated a high-throughput screening campaign against PIM1 using our proprietary compound
collection.
The screening campaign was fruitful, with over thirty confirmed distinct chemical scaffolds inhibiting
significantly PIM1 kinase activity at 10 uM. Among these identified scaffolds, a few showed a non-
classical ATP-competitive binding mode as confirmed by X-ray crystallography.
Our initial objective in the early phase of the project was to identify compounds for in vitro credentialing
studies. To this end, we selected hits on the basis of their chemical originality, potential for derivatization,
binding affinity and selectivity profiles. We also wanted to establish a structure-based drug design
optimization strategy.
PIM1, 2 and 3 are highly conserved kinases with unique structural properties.6 Interested by the Pro-123
insertion in the hinge region of the ATP-binding cleft of the preceding kinase, we investigated the
molecular interactions of the selected hits with PIM1 by X-ray crystallography. Among co-crystallized
structures collected, we were particularly interested by the unexpected interaction observed with
compound A (light orange, Figure 1) which is an azaindole derivative. It’s binding mode is different from
the one reported for 7-azaindole B co-crystallized in the PAK1 kinase active site (Blue, Figure 1).
NH
N
N
Cl
N
HN
N
O
O
N
HN
O
N
N
Cl
Compound B
Compound A
Figure 1. X-ray crystal structures of 7-azaindoles A and B respectively bound to PIM1 (orange/5TEL) and
PAK1 (Blue/5KBR)
In the ATP-binding pocket of PAK1 (Figure 1, Blue), compound B binds as a typical type I ATP-
competitive binder with the donor-acceptor motif interacting directly with the hinge. As shown in Figure 1
(orange), the donor-acceptor motif of compound A is facing away from the hinge site and it is the C4-
chlorine atom of the aromatic ring that points to this part of the binding pocket. With the exception of a
weak and non well-defined interaction with Asp-131via the piperazine motif, compound A does not show
any obvious direct contact with PIM1. This binding mode was further confirmed with other 7-azaindole hits
such as compound 1 in Figure 2 exhibiting this “reverse” binding mode in PIM1 as shown in Figure 3.
Knowing that the azaindole N1-H was not involved in a classical bidentate H-bond interaction with PIM1,
we decided to modify this part of the molecule with the objective to improve potency and reduce off-target
kinase activity. To this end, compounds 1 and 3 (Figure 2) which had modest potencies on PIM1 (1.03/2.5
uM) and ligand efficiencies (LE = -0.37), and cross-reacting against cell cycle kinases such as CDK9 and
CDC7 was converted to compounds 2 and 4 (Figure 2) using the synthetic chemistry approach described in
Scheme 1 and 2.

The new derivatives 2 and 4 maintained the original potencies towards PIM1 (IC50 ~1 uM) and also
retained the original binding mode (Figure 3), but showed a much improved selectivity against CDK9 and
CDC7. The binding mode of compounds 1-4 confirmed that cross-reactivity with the kinome would not be
an issue. This enabled us to synthesize highly selective tool compounds to interrogate PIM biology.7
R
R
NH
N
NH
N
N
N
N
N
H
2
1
PIM1 IC50 = 1.5 uM
CDK9 IC50 = 5 uM
CDC7 IC50 = 10 uM
PIM1 IC50 = 1.03 uM
CDK9 IC50 = 0.02 uM
CDC7 IC50 = 0.04 uM
R = Cl
4
3
PIM1 IC50 = 2.5 uM
CDK9 IC50 = 0.15 uM
CDC7 IC50 = 0.06 uM
PIM1 IC50 = 0.9 uM
CDK9 IC50 = 6.1 uM
CDC7 IC50 > 10 uM
R = CN
Figure 2. Comparison of the selectivity of 4-cyano/chloro-7-azaindoles 1-4 towards CDK9 and CDC7.
Encouraged by these initial results, and given that the 7-azaindole scaffold offered interesting possibilities
for a multi-dimensional optimization approach, we initiated medicinal chemistry around the 4-chloro-7-
azaindole chemotype. Scheme 1 outlines our synthetic strategy to introduce chemical diversity. We desired
a flexible synthetic approach for developing a spectrum of analogs for this series. The synthesis of
intermediate 8 (Scheme 1), which proved to be accessible in large quantities, was key to our strategy.
Figure 3. Superimposition of two X-ray crystal structures of compounds 3 (Blue/5TOE) and 4
(purple/5TUR) bound to PIM1.
Commercially available 7-azaindole was treated with mCPBA to provide compound 5 as a salt. The free
base N-oxide was released at pH 9 by treatment with an aqueous solution of K2CO3. The N-oxide 5 was
treated with POCl3 to provide Cl-azaindole 6 regioselectively in reasonable yields. The N1 nitrogen was
protected with a MOM group to produce 7. The directing group helped provide 8 in reproducibly high
yield. Intermediate 8 was used as a central template to diversify chemistry at C2, C3 and N1 positions, as
needed.
Cl
Cl
Cl
a
b
c
d
I
N+
O
N
N
N
N
N
H
N
H
N
N
N
H
O
O
5
6
7
8
Scheme 1. Reagents and conditions: (a) mCPBA, DME/heptane, 89%; (b) POCl3, 100oC, 80%; (c)
Bu4NHSO4, NaOH, MOM-Cl, MeCN, 75%; (d) LDA, THF, I2, 91%.

Deprotection of 8 (Scheme 2) using bromo-catecholborane proved to be a slow 2-stage process to
ultimately give 9. Alkylation at N1 gave N-methyl derivative 10, which underwent a smooth cross-coupling
at C2 to provide intermediate 11. Iodination at C3 to give 12 followed by Suzuki coupling yielded 13. Final
targets were accessed from intermediates 7, 8, and 12 as needed.8
Cl
N
Cl
Cl
N
Cl
N
a
b
c
I
I
I
N
NBoc
N
N
N
H
N
N
O
8
9
10
11
d
X
Cl
N
Cl
N
I
e
N
NBoc
N
H
N
N
N
13
12
Scheme 2. Reagents and conditions: (a) Br-catecholborane, DCM, followed by NaOH; (b) NaH, DMF,
MeI, 80% two steps; (c) Pd(PPh3)4, boronate, Cs2CO3, DME/H2O, 75%; (d) THF/MeOH, NIS, 95%; (e) i)
step c); ii) EtOH, HCl.
Biochemical IC50 measurements1 for PIM1, 2 and 3 were obtained using a TR-FRET assay with full-length
human BAD substrate. While most of the compounds showed double-digit nanomolar activity against
PIM1, potency against PIM2 was in comparison significantly lower. This can be explained by the
difference in Km for ATP between these enzymes. We found using a radioactive filter-binding assay ATP
Km = 67, 5.4, 97 uM for PIM1, 2 and 3 respectively2. Surprisingly, PIM3 inhibition was comparable to
PIM2 for this set of compounds which could not be explained by structural biology.
In fact, while this initial exploration led to PIM1 biochemical potencies below 100 nM, it was only
achieved using large basic moieties at C3 and/or C2 reaching water exposed-region, and was usually
accompanied by a two log difference for PIM2 and PIM3, as shown in Scheme 3 for representative
compounds.
Having identified a series of PIM1-specific inhibitors, we next performed the evaluation for a few of them
in a panel of cancer cell lines to try to understand the biological impact of PIM1 inhibition.
———
1
IC50 against PIM1, PIM2, and PIM3 kinases was routinely tested using an in vitro TR-FRET assay based on
detection of phosphorylation of specific Ser112 residue in Bad protein. Final reaction mixtures (40 μL) contained 2
nM PIM proteins, 50 nM Bad, 40 µM ATP, and dilutions of the tested compounds in reaction buffer.
2 Apparent Km(ATP) for PIM1, PIM2, and PIM3 were measured using radiolabeling filter-binding assay. Reaction
mixtures containing 2 nM Pim proteins, 100 µM substrate peptide Pim2tide representing amino-acids 107-117 of
mouse BAD, and varying concentrations of 33P-ATP in reaction were incubated at ambient temperature. The level of
33P-Pim2tide was quantified using a TopCount plate reader (PerkinElmer).

N
N
O
N
H
Cl
N
Cl
N
N
N
N
N
N
N
15
PIM1 IC50 = 28 nM
14
PIM1 IC50 = 23 nM
PIM2 IC50 = 872 nM
PIM3 IC50 = 492 nM
PIM2 IC50 = 3300 nM
PIM3 IC50 = 1400 nM
Scheme 3. Representative hits obtained after initial analoging
The proliferative inhibition induced by compounds 14 and 15 across a number of liquid and solid tumor
cancer cell lines is shown in Table 1. Results were modest, with the best potency observed in the colon
cancer cell lines HCT-116 and HT-29.
Compound 15 was selected to be screened against an internal panel of 49 representative kinases at 10 uM
which demonstrated a selective series (data not shown) with weak cellular potency. Given the additional
expression of PIM2 and/or PIM3 in numerous cancers, we surmised that selective inhibition of PIM1 may
be insufficient to efficiently inhibit cellular growth, and that a pan-PIM inhibitor may be required to
overcome PIM pathway-mediated pro-survival processes associated with tumorogenesis.
Table 1. Cancer cell line proliferative inhibition by 14 and 15
EOL-1 MOLM-13 MV4-11 K-562 HCT-116 HT-29 H460 PC3
EC50 14 (uM)
EC50 15 (uM)
0.8
0.5
1.1
0.6
1.3
0.6
1.3
1.1
0.2
0.1
0.3
0.1
1.1
1.0
1.0
0.8
At this point, we turned our attention to the last unexplored position of the 7-azaindole, namely position 6.
This position was initially set aside due to the lack of optimizable space, based on the X-ray data. However,
this position was proximal to Glu-121 carbonyl so compounds 19 and 20 were designed to target it as
shown in Scheme 4.
Cl
NH
N
O
N
N
NH2
19
e, f
Cl
N
Cl
Cl
N
a
b
c, d
I
9
I
N
NBoc
N+
O
N
N
N
H
H
N
N
18
16
17
f
Cl
N
NH
N
N
N
20
Scheme 4. Reagents and conditions: (a) mCPBA, AcOEt; (b) TMSCN, Et3N, ACN; (c) NaH, DMF, MeI;
(d) Pd(PPh3)4, Boronate, Cs2CO3, DME/H2O; (e) TMSOK, THF; (f) TFA, DCM.
While compound 20 was found inactive with a PIM1 IC50 above 10 uM, we observed that 19 had a PIM1
IC50 of 250 nM, about four-fold more potent than compound 2. Of note, this was achieved without a
substituent at C3, previously found to improve potency at least ten fold.

Interestingly, even though PIM2 potency for 19 was still in the high micromolar range, PIM3 biochemical
IC50 was now 400 nM. Equipotency between PIM1 and PIM3, while initially expected and desired, was
not attained until now.
Cl
Pim1 IC50= 0.25 uM
Pim2 IC50= >30 uM
Pim3 IC50= 0.4 uM
NH
N
O
N
N
NH2
19
N
Cl
N
Cl
N
NH
N
N
Pim1 IC50= >30 uM
Pim2 IC50= >30 uM
NH
N
O
N
2
NH2
21
Pim1 IC50= 1 uM
Pim2 IC50= >30 uM
Cl
Pim1 IC50= 0.043 uM
Pim2 IC50= 1.27 uM
Pim3 IC50= 0.29 uM
NH
N
O
N
N
NH2
22
Scheme 5. Synthesis of C6-substituted 7-azaindoles
To further investigate the increase in PIM1 inhibitory potency of 19, we generated compounds 21 and 22
(Scheme 5). To our surprise, 21 lost PIM1 potency (IC50 > 30 uM), while 22 exhibited a further 10-fold
increase in PIM1 potency. We once again turned our attention to X-ray crystallography to understand these
results.
O
H2N
N
Cl
N
Cl
N
N
OH
Compound 24
Pim1 IC50= 0.2 uM
Pim2 IC50= >30 uM
Pim3 IC50= 0.4 uM
N
N
Compound 23
N
Pim1 IC50= 0.4 uM
Pim2 IC50= 13.8 uM
Pim3 IC50= 10.6 uM
N
H
Figure 4. X-ray crystallography of 24 (Yellow/5KCX) and analog 23 (Pink/5TEX), plus overlaid in PIM1
As shown in Figure 4, newly synthesized 24 (Yellow) had rotated 60° clockwise in the PIM1 binding
pocket, compared to analog 23 (Pink). This rotation was presumably triggered by an hydrogen bond of the
C6 carboxamide with the catalytic Lys-67. Such an interaction was not possible with any of the previous
analogs. This repositioning of the scaffold explained the biochemical results observed and opened new
opportunities to design compounds that might inhibit all three PIM enzymes.
We now had inhibitors with two distinct interactions with the protein, one of which pointed the C4-chlorine
towards Glu-121, generating an interesting halogen bonding interaction.9 The second of these interactions
was mediated by the C6 carboxamide binding to the conserved Lys-67 of PIM1. The C3 position of the
azaindole core was now facing the hinge with limited space to accommodate substituents, which explained
the lack of PIM1 potency for 21 and 23 used for crystallography . The newly oriented molecule presented a
direct access to the aspartate cluster (Asp-128, Asp-131 and Glu-171) from the N1 position, offering an
opportunity to optimize inhibitory potency on PIM2.
In summary, we report the discovery of original PIM1 kinase inhibitors. Although the initial SAR with this
chemical series was disappointing, we uncovered a change from the original binding mode, which
permitted us to re-orient our efforts towards the discovery of a lead series. The initial objectives were thus
attained, namely identifying a hit series with specific interactions with the Lys-67 and Glu-121, but in a
non-typical hinge-binding modality. We also discovered that we could access the ribose-binding pocket,
which might improve potency on PIM2. Exploitation of this new binding mode towards finding a pan-PIM
lead series is described in the accompanying Parts.

Acknowledgments
The authors thank Isabelle Morize and Kimberly Haas for their contribution to molecular modeling and
Peter Hamley’s team in Frankfurt for compound library synthesis support. We also thank Jean-Paul Nicolas
for the evaluation of compounds on solid tumors panel.
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