Hit to lead evaluation of 1,2,3-triazolo[4,5-b]pyridines as PIM kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.12.130

PIM kinases have become targets of interest due to their association with biochemical mechanisms affecting survival, proliferation and cytokine production. 1,2,3-Triazolo[4,5-b]pyridines were identified as PIM inhibitors applying a scaffold hopping approa

Full text of DOI:10.1016/j.bmcl.2011.12.130

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1591–1597
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hit to lead evaluation of 1,2,3-triazolo[4,5-b]pyridines as PIM kinase inhibitors
Joaquín Pastor a, , Julen Oyarzabal a, , Gustavo Saluste a, Rosa María Alvarez a, Virginia Rivero a,
⇑
Francisco Ramos a, Elena Cendón a, Carmen Blanco-Aparicio a, Nuria Ajenjo a, Antonio Cebriá a,
M.I. Albarrán a, David Cebrián a, Ana Corrionero a, Jesús Fominaya a, Guillermo Montoya b, Marco Mazzorana b,à
a Experimental Therapeutics Program, Spanish National Cancer Research Centre (CNIO), C/Melchor Fernández Almagro 3, E-28029 Madrid, Spain
b Macromolecular Crystallography Group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
PIM kinases have become targets of interest due to their association with biochemical mechanisms
affecting survival, proliferation and cytokine production. 1,2,3-Triazolo[4,5-b]pyridines were identified
as PIM inhibitors applying a scaffold hopping approach. Initial exploration around this scaffold and
X-ray crystallographic data are hereby described.
Received 14 November 2011
Revised 27 December 2011
Accepted 28 December 2011
Available online 5 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
PIM inhibitors
Scaffold hopping
Triazolopyridine
Hit to lead
Phosphorylation of BAD
FLT-3
Crystal structure
Docking
hERG Binding
Mechanism of action
PIM-1, 2 and 3 are members of the PIM (provirus insertion site of
Moloney murine leukemia virus) family of serine/threonine protein
kinases belonging to the group of calcium/calmodulin-regulated
kinases (CAMK).1
The PIM proteins are serine/threonine protein kinases that do
not require an upstream activation by another kinase partner. As
shown by its X-ray crystal structure, PIM-1 is a constitutively
active enzyme and phosphorylation is not a prerequisite for its
kinase activity.2
Given this promising target profile, we started a drug discovery
program focused on PIM-1. In order to identify PIM-1 inhibitors,
compounds selected from kinases collections of commercial
sources14 were screened. Imidazopyridazines15 and triazolopyrida-
zines were identified as potent biochemical inhibitors of PIM-1
(Fig. 1).
However, these chemical series were already known as PIM
inhibitors,16–18 and could not be considered as proprietary starting
points for a hit to lead project.
PIM-1 has been shown to have diverse biological roles in cell
survival, proliferation, differentiation and apoptosis.3 Overexpres-
sion of PIM-1 has been found in various hematopoietic malignan-
cies4–6 as well as in solid tumors7 including colon,8 prostate9 and
pancreas.10 Therefore, PIM proteins are widely considered as
attractive targets for drug discovery.11,12 The fact that PIM-1
knockout mice showed no obvious phenotype suggests that side
effects for such a drug should be minimal.13
Therefore, we needed to find novel chemical series for PIM inhi-
bition. Thus, we applied a scaffold hopping strategy based on a
chemically feasible fragments database.19 In addition to the li-
gand-based approach, we used structure-based virtual screening
(VS) to prioritize among proprietary database of scaffolds and pro-
posed novel chemotypes.20
⇑
Corresponding author. Tel.: +34 917328000; fax: +34 917328051.
E-mail address: jpastor@cnio.es (J. Pastor).
Present address: Center for Applied Medical Research (CIMA), Small Molecule
Discovery, Avda. Pio XII, 31008 Pamplona, Spain.
à
Present address: Diamond Light Source Ltd Diamond House, Science & Innovation
Campus, Didcot, Oxfordshire OX11 0DE, UK.
Figure 1. Imidazopyridazines and Triazolopyridazines described as PIM inhibitors.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.130

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J. Pastor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1591–1597
Figure 2. Triazolopyridine as new scaffold for PIM inhibitors.
The outcome of this process was the identification of the 1,2,3-
triazolo[4,5-b]pyridine as an alternative to the scaffolds already
known (Fig. 2).
another explicit interaction between the NH from piperidine and
the side chain of Asp128.
Based on the structural data generated, we can evaluate the
docking studies performed for this chemical series. Proposed bind-
ing mode perfectly fits with the experimental data; in fact, the hea-
vy atom RMS deviation between the docked and crystal structure
measured 1.80 Å (Fig. 3b). This low RMS deviation, together with
the identification of the two main interactions (Lys67 and
Glu121), indicates a good performance for docking studies, which
were utilized as part of the scaffold hopping strategy,20 and vali-
dates our structure-based working hypothesis for this chemical
series.
The results obtained from the exploration with piperidinyl-
methyl amines pointed out that the presence of an unsubstituted
NH in the amine fragment might be required for high PIM-1 inhib-
itory potency. With this idea, we made a selection of amines, vary-
ing the distance of a free NH from the amino group attached to the
triazolopyridine scaffold including different degree of chemical
complexity. As a result, compounds 7a–j were synthesized and
evaluated for PIM-1 inhibition (Table 3).
Compounds 7a and 7b, having a methylene unit less than the
parent compound 6b, have shown inhibitory activities in the nano-
molar range, whereas derivative 7c, with a piperazine, was less po-
tent. It was of special interest the subseries of compounds
containing the structurally more complex spiroamines (7d–j). This
exploration led to highly active compounds, showing some of them
inhibitory activities toward PIM-1 in the low nanomolar range
(7d–f).
For SAR comparison, we also prepared compound 7k bearing an
alkoxy group attached to the bicyclic core instead of an amine (7a).
The activity data indicates that this replacement is detrimental for
PIM-1 activity.
As a proof-of-concept, we applied all the available SAR (struc-
ture–activity relationship) information generated from internal
HTS (high throughput screening) data. Fragments from imidazopy-
ridazines and triazolopyridazines hits were transferred to the new
triazolopyridine scaffold. Then, compounds 5a–d and 5e–h were
prepared using, respectively, triazolopyridazines (I) and imidazo-
pyridazines (II) as a reference (Table 1). In order to complete this
first exploration compounds 5i–l were also included.
The synthesis of compounds 5a–l (R4 = H) is depicted in Scheme
1. The amination of the commercially available 2,6-dichloro-3-
nitropyridine 1, followed by the reduction of the nitro group of 2
gave the diamine 3. Treatment of 3 with sodium nitrite21 yielded
triazole 4. Finally, a second amination afforded the desired com-
pounds 5.
Most compounds (5a and 5e–j) showed activities for PIM-1
inhibition lower than 0.5 lM (Table 1), with compound 5h being
the most potent (IC50 = 69 nM). These results validate the scaffold
hopping strategy and encouraged us to further explore this core.
Replacement of the p-hydroxy group by p-methoxy in compounds
5h and 5i led to inactive compounds (5k, 5l).
In general, the first triazolopyridine analogs synthesized were
less potent than their corresponding imidazopyridazine and/or
triazolopyridazine counterparts. In search for more potent inhibi-
tors on this series, we decided to incorporate to our exploration
SuperGen’s preferred amines for the imidazopyridazine series23
as well as a set of tertiary amines, to evaluate a range of nitrogen
pKa’s. We selected the 3-trifluoromethoxy-phenyl moiety as an
aryl group which was present in the hits found during our first
exploration. We avoid using the phenol group of our most potent
compound 5h, due to its potential in vivo glucuronidation.
Thus, we synthesized compounds 6a–i (Table 2), bearing pipe-
ridinyl-methyl amines. In general, compounds 6a–d showed PIM-
1 inhibition in the same range of the analogous imidazopyridazine
compounds, with derivative 6b being the most active in our triaz-
olopyridine series (IC50 = 6 nM). The substitution of the piperidine
NH with alkyl chains results in a slight decrease in potency (6a and
6c–e). Incorporating a fluorine atom (6f), methoxy (6g), keto (6h)
or amide (6i) groups to the alkyl chain leads to less active com-
pounds against the PIM-1 kinase.
Considering the impact that structural information may provide
to the drug discovery iterative process, compound 6b was selected
and successfully crystallized with PIM-1 (Fig. 3a). Coordinates for
the PIM-1-inhibitor complex have been deposited in the protein
databank (access code 4A7C). The triazolopyridine 6b binds to
the ATP binding site of PIM-1 in its typical inactive conformation
(i.e., Phe49 pointing to the inner side of the ATP binding site).
Compound 6b forms multiple interactions with the residues
within the catalytic site of PIM-1. As expected, there is a hydrogen
bond between the side chain of Lys67 and the nitrogen in position
1 in the triazole ring of the compound. Another bond is established
between one hydrogen atom of the phenyl ring and the main chain
carbonyl of Glu121.
This exploration was completed with two amines containing a
terminal hydroxyl group (7l, m). Both compounds were found to
be highly potent (17 and 27 nM, respectively), thereby reinforcing
the proposed exploration around R1 using H-bond donating chem-
ical structures.
Once highly potent R1 substituents for this series were identi-
fied, we carried out a limited exploration of aryl groups looking
for alternatives to the trifluoromethoxy group. Compounds 8a–d
(Table 4), bearing a dimethylamino or trifluoromethyl group,
showed activity values similar to their corresponding parent com-
pounds (6b, 7e and 7f). Both substituents can be then used as
replacements for the trifluoromethoxy group, likely adding differ-
ent physicochemical properties to these molecules.
The replacement of the aryl group by a number of heteroaryls
(8e–g), was allowed but a decrease in the inhibitory activity was
observed, with the pyridyl derivative 8e being the most active
compound of the set (133 nM).
In order to complete the biochemical profile of our best PIM
inhibitors, we evaluated them for their inhibitory activity against
isoforms PIM-2 and PIM-3 (Table 5). During the course of this
work, compound SGI-177624 (Fig. 4) from SuperGen entered Phase
I clinical trials. This molecule showed inhibitory values for PIM-1,
PIM-2 and PIM-3 of 7, 363 and 79 nM, respectively. In addition
to PIM, SGI-1776 also potently targets FLT-3 (Fms-like tyrosine
kinase receptor) (IC50 = 44 nM). Taking this fact into account, we
decided to test our compounds for their FLT-3 inhibition (Table 5).
Compound 6b also forms a number of hydrophobic contacts
with PIM-1, in particular with residues Phe49, Val52 and Leu174.
In addition to these interactions, this crystal structure described

J. Pastor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1591–1597
1593
Table 1
Inhibition of PIM-1: 1,2,3-triazolo[4,5-b]pyridine derivatives (5) compared with 1,2,4-triazolo[4,3-b]pyridazine (I) and imidazo[1,2-b]pyridazine (II) analogous
N
N
N
N
N
N
N
N
R1
N
R1
N
R1
N
R2
R2
R2
5
I
II
R1
R2
PIM-1a (5a–l)
0.282 (5a)
PIM-1a,b (scafold I or II)
H
N
0.014 (I)
0.096 (I)
N
3.910 (5b)
0.852 (5c)
2.600 (5d)
H
F
N
0.065 (I)
0.371 (I)
NH2SO2
N
O
O
F
H
N
0.316(5e)
0.433 (5f)
0.400 (5g)
0.057 (II)
0.025 (II)
0.217 (II)
OCF3
H
N
OCF3
OCF3
N
H
N
F
N
0.069 (5h)
0.162 (5i)
0.320 (5j)
3.260 (5k)
>10 (5l)
0.130 (II)
H
OH
N
—
—
—
—
OH
H
N
OH
N
OMe
F
N
H
OMe
a
IC50 (lM), the values reported are an average of two independent data points.
HTS results.
b
Compounds with a similar profile to SGI-177625 (PIM-1/PIM-3/
Selected compounds were evaluated for their ability to inhibit
the phosphorylation of the apoptosis effector BAD at Serine 112
in the H1299 cell line (Table 5). All PIM kinases family members
have been shown to phosphorylate BAD at S112. However, in order
to make the phosphorylation of this substrate at S112 mostly
FLT-3) were found (7f and 8c), while other compounds (6b, 7a, 7e,
8a) were selective versus FLT-3 (PIM-1/PIM-3 profile). Addition-
ally, compounds with a pan-PIM like profile and selective versus
FLT-3 were identified (7l,m).

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J. Pastor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1591–1597
Scheme 1. Reagents and conditions: (a) Aromatic or heteroaromatic amine,
NaHCO3, EtOH, 23–92%; (b) H2, Ni-Raney, EtOAc, 50–100%; (c) NaNO2, AcOH, H2O,
11–93%; (d) Amine,22 TEA, EtOH, 27–99%.
Table 2
PIM-1 Inhibition results for piperidinyl-methyl amines exploration: 1,2,3-triazol-
o[4,5-b]pyridine derivatives (6) compared with imidazo[1,2-b]pyridazine (III)
analogous
N
N
N
N
N
R1
N
R1
N
OCF3
OCF3
6
III
R1
PIM-1a (6a–i)
PIM-1b (III)
0.007/0.138
N
0.155 (6a)
N
H
HN
0.006 (6b)
0.035 (6c)
0.077 (6d)
0.127
N
H
N
N
0.018/0.111
0.079
N
H
N
H
N
Figure 3. (a) Triazolopyridine ligand 6b, bound to PIM-1. Key PIM-1 aminoacids
involved in binding are highlighted; (b) overlapping between crystallized (carbon
atoms colored in orange) and docked ligand.
0.226 (6e)
0.266 (6f)
0.238 (6g)
—
—
—
N
H
N
N
F
H
Compound 6b, bearing the piperidinyl-methyl amine and the
trifluoromethoxy group as aryl substitution, displayed the best cel-
lular value (45 nM). Compound 8a, containing a dimethylamino
instead of the trifluoromethoxy substituent was less active
(140 nM), but with a potency comparable to the reference com-
pound in our assay. A drop in cellular potency versus their bio-
chemical activity was observed with compounds containing
spiroamines (7e, 7f, 8c, 8d) or 4-hydroxycyclohexylamines
(7l,m). These results could be due to cellular permeability issues.
Taking into account their cellular activity, compounds 6b and
8a were selected to further explore R3 and R4 with additional sub-
stitutions in the triazolopyridine core. Hence, we synthesized27,28
and tested compounds 9a–c (Table 6).
N
N
H
O
O
0.436 (6h)
0.844 (6i)
—
—
N
N
H
O
N
N
N
H
a
IC50
(
lM), the values reported are an average of two independent data points.
b
Reported data by SuperGen, IC50 M), (WO2008/058126).
(l
Compound 9a showed improved PIM activity when compared
to its parent compound (6b), with an enhanced PIM-2 inhibitory
activity. Moreover, compound 9a showed a higher selectivity for
PIM with respect to FLT-3 than 6b. Compound 9b presented a sim-
ilar biochemical profile to its parent compound 8a, but better
dependent on PIM-1 we have overexpressed human PIM-1 in
H1299 (NSCLC cell line that endogenously has very low levels of
expression of PIM-1, 2 and 3).26

J. Pastor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1591–1597
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Table 3
Table 4
PIM-1 inhibition results for broad amine exploration
PIM-1 inhibition results for aryl exploration
N
N
N
N
N
N
R1
N
R1
N
R2
Compd
R1
R2
PIM-1a
OCF3
Compd
R1
PIM-1a
0.038
HN
N
H
8a
0.005
H
HN
7a
N
N
N
N
H
7b
0.087
HN
HN
HN
HN
HN
N
H
8b
8c
8d
8e
0.014
0.009
0.014
0.133
N
7c
7d
7e
0.336
0.010
0.011
CF3
CF3
CF3
HN
HN
HN
N
N
H
H
N
H
N
HN
7f
0.004
N
H
7g
7h
7i
HN
HN
0.068
0.126
0.108
N
N
H
CF3
S
N
N
N
N
N
H
HN
HN
8f
0.317
F3C
N
7j
0.165
2.42
HN
HN
HO
HO
O
7k
7l
N
H
8g
0.405
N
H
N
0.017
0.027
NH2
IC50 (lM), the values reported are an average of two independent data points.
H
N
a
7m
a
IC50 (lM), the values reported are an average of two independent data points.
cellular activity. Finally, compound 9c, bearing a chlorine atom at
the R3 position, was less selective towards FLT-3 and less active
in the cellular assay than 6b.
At this stage, SGI-1776 was discontinued from clinical Phase I
due to cardiotoxicity.29 In order to check if we could face similar
issues, we decided to evaluate the activity of our best PIM inhibi-
tors with respect to the hERG ion channel (Table 7).
Compounds 6b and 9a exhibited a similar pIC50 value for hERG
inhibition30 when compared to SGI-1776. The replacement of the
trifluoromethoxy substituent by a dimethylamino group (9b)
slightly decreased hERG binding affinity. Unfortunately, introduc-
tion of a chlorine atom at R3 position (9c) increased hERG affinity.
Compounds 6b, 9a and 9b were also tested in the hERG Patch
Clamp31 cellular assay (Table 7). Although more moderated hERG
Table 5
PIM-1,2,3 and FLT-3 inhibition resultsa,b and phosphorylation of BADb,c
Compd
PIM-1
PIM-2
PIM-3
FLT-3
p-BAD
6b
7a
7e
7f
7l
7m
8a
8c
6
38
11
4
17
27
5
9
14
7
230
640
218
404
69
152
>1000
853
358
363
7
66
23
40
55
83
11
28
18
69
231
>1000
778
45
700
590
740
405
550
140
810
380
200e
12
>1000
>1000
>1000
28
314
44
8d
SGI-1776d
a
b
c
IC50 (nM).
The values reported are an average of two independent data points.
EC50 (nM).
Published in Ref. 24
In house generated data.
inhibition values (less or equal to 10 lM) were observed in the
functional assay, these data could be affected by the permeability
of the compounds.
d
e
To assess the kinase selectivity of this series, a selection of com-
pounds was further profiled in a panel of 24 kinases.32The intro-
duction of a methoxy group at the R4 position, clearly improves
selectivity (inhibition <13%) for all kinases tested when compared
to R4-unsubstituted compounds.
Overall, compound 9a had the best profile, including good
microsomal stability33 and human cytochrome P450’s inhibition34
(Table 8), it was therefore selected for pharmacokinetic studies.
The pharmacokinetic parameters estimated for 9a after IV
administration (5 mg/Kg) in BALB/C mice: plasmatic half life
Figure 4. Chemical structure of SGI-1776.

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J. Pastor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1591–1597
Table 6
(0.95 h) and plasmatic clearance (3.28 L/h/Kg), indicated that opti-
mization of this compound was needed. Nonetheless, we used it for
in vivo proof-of-concept. The mechanism of action in Jeko human
mantle lymphoma xenograft SCID mice, showed a 40% reduction
of phosphorylation of BAD levels on Serine 112 compared to con-
trol group receiving vehicle (Fig. 5).
In summary, we have presented a series of 1,2,3-triazolo[4,5-
b]pyridine with potent biochemical and diverse cellular PIM activ-
ities. Hit validation as well as hit to lead phase progress has been
described. Compounds with different isoform profiles PIM-1/PIM-
3 or pan-PIM, and with FLT-3 activity or selectivity, have been
obtained.
Compound 9a is a potent pan-PIM inhibitor and selective
against FLT-3, showing a different profile to that of SGI-1776
(PIM-1/PIM-3/FLT-3). However, taking into account the hERG data,
we could face with our compounds a cardiotoxicity issue similar to
the SuperGen compound. Nevertheless, compound 9a has been se-
lected as a lead compound for further optimization in particular of
this liability. The results of this work will be reported in due
course.
PIM-1,2,3 and FLT-3 inhibitiona,b and pBADb,c results for R3/R4 exploration
R4
R3
N
N
N
R2
N
N
H
HN
R3 R4
Compd R2
PIM1 PIM2 PIM3 FLT3
pBAD
70
9a
H
H
Cl
OMe
1
29
752
0.8
2.5
16
4000
7000
OCF3
N
9b
OMe 1.5
48
9c
H
6
1000
125 420
OCF3
References and notes
a
b
c
IC50 (nM).
The values reported are an average of two independent data points.
EC50 (nM).
1. Bachmann, M.; Moroy, T. Int. J. Biochem. Cell Biol. 2005, 37, 726.
2. (a) Kumar, A.; Mandiyan, V.; Suzuki, Y.; Zhang, C.; Rice, J.; Tsai, J.; Artis, D. R.;
Ibrahim, P.; Bremer, R. J. Mol. Biol. 2005, 348, 183; (b) Jacobs, M. D.; Black, J.;
Futer, O.; Swenson, L.; Hare, B.; Fleming, M.; Saxena, K. J. Biol. Chem. 2005, 280,
13728; (c) Qian, K. C.; Wang, L.; Hickey, E. R.; Studts, J.; Barringer, K.; Peng, C.;
Kronkaitis, A.; Li, J.; White, A.; Mische, S.; Farmer, B. J. Biol. Chem. 2005, 280,
6130; (d) Bullock, A. N.; Debreczeni, J.; Amos, A.; Knapp, S.; Turk, B. E. J. Biol.
Chem. 2005, 280, 41675.
3. Wang, Z.; Bhattacharya, N.; Weaver, M.; Petersen, K.; Meyer, M.; Gapter, L.;
Magnuson, N. S. J. Vet. Sci. 2001, 2, 167.
4. Brault, L.; Gasser, C.; Bracher, F.; Huber, K.; Knapp, S.; Schwaller, J.
Haematogologica 2010, 95, 1004.
5. Cohen, A. M.; Grinblat, B.; Bessler, H. Leuk. Lymphoma 2004, 45, 951.
6. Claudio, J. O.; Masih-Kahn, E.; Tang, H. Blood 2002, 100, 2175.
7. Chen, J.; Kobayashi, M.; Darmanin, S.; Qiao, Y.; Gully, C.; Zhao, R.; Kondo, S.;
Wang, H.; Yeung, S. C.; Lee, M. H. Am. J. Pathol. 2009, 175, 400.
8. Popivanova, B. K.; Li, Y.-Y.; Zheng, H. Cancer Sci. 2007, 98, 321.
9. Valdman, A.; Fang, X.; Pang, S. T. Prostate 2004, 60, 367.
Table 7
hERG binding and Patch Clamp data
Compd
hERG binding (pIC50
)
hERG patch clamp (lM)
6b
9a
9b
9c
5.0
5.5
4.5
6.4
5.5a
1.7
7
10
—
SGI-1776
—
a
In house generated data.
10. Li, Y.-Y.; Popivanova, B. K.; Nagai, Y. Cancer Res. 2006, 66, 6741.
11. Morwick, T. Exper. Opin. Ther. Patents 2010, 20, 193.
12. Anizon, F.; Shtil, A. A.; Danilenko, V. N.; Moreau, P. Curr. Med. Chem. 2010, 17,
4114.
Table 8
Metabolic stabilitya and P450 cytochrome inhibitionb data for compound 9a.
13. Larid, P. W.; Van der Lugt, N. M. T.; Clarke, A.; Domen, J.; Linders, K.; McWhir, J.;
Berns, A.; Hooper, M. Nucleic Acids Res. 1993, 21, 4750.
14. Compounds were purchased from BioFocus (Cambridge, UK) and ChemDiv
(San Diego, CA).
15. Blanco-Aparicio, C.; García Collazo, A. M.; Oyarzabal, J.; Leal, J. F.; Albarán, M. I.;
Ramos Lima, F.; Pequeño, B.; Ajenjo, N.; Becerra, M.; Alfonso, P.; Reymundo, M.
I.; Palacios, I.; Mateos, G.; Quiñones, H.; Corrionero, A.; Carnero, A.; Pevarello,
P.; Rodríguez López, A.; Fominaya, J.; Pastor, J., et al Cancer Lett. 2011, 300, 145.
16. Bullock, A. N.; Debreczeni, J. E.; Fedorov, O. Y.; Nelson, A.; Marsden, B. D.;
Knapp, S. J. Med. Chem. 2005, 48, 7604.
HLM MLM RLM P450 1A2 P450 2C19 P450 2C9 P450 2D6 P450 3A4
97 80 91 30 8.7 0.5 4.5
4
a
Metabolic stability in human (HLM), mouse (MLM) and rat liver microsomes
(RLM) refer to % of compound remaining after 15 min.
b
CYP’s IC50 inhibition(lM).
17. Pogacic, V.; Bullock, A. N.; Fedorov, O.; Filippakopoulos, P.; Gasser, C.; Biondi,
A.; Meyer-Monard, S.; Knapp, S.; Schwaller, J. Cancer Res. 2007, 67, 6916.
18. Grey, R.; Pierce, A. C.; Bemis, G. W.; Jacobs, M. D.; Moody, C. S.; Jajoo, R.; Mohal,
N.; Green, J. Bioorg. Med. Chem. Lett. 2009, 19, 3019.
19. Oyarzabal, J.; Howe, T.; Alcazar, J.; Andrés, J. I.; Alvarez, R. M.; Dautzenberg, F.;
Iturrino, L.; Martínez, S.; Van der Linden, I. J. Med. Chem. 2009, 52, 2076.
20. Saluste, G.; Albarrán, M.I.; Alvarez, R.M.; Rabal, O.; Ortega, M.A.; Blanco, C.;
Kurz, G.; Salgado, A.; Pevarello, P.; Bischoff, J.R.; Pastor, J. and Oyarzabal, J.
Submitted for publication.
21. Rivalle, C.; Ducrocq, C.; Lhoste, J.-M.; Wendling, F.; Bisagni, E.; Chermann, J.-C.
Tetrahedron 1981, 37, 2097.
22. For Boc-protected amines a final Boc-deprotection step was needed.
23. Bearss, D.J.; Liu, X-H.; Vankayalapati, H.; Xu, Y. WO2008/058126.
24. Chen, L. S.; Redkar, S.; Bearss, D.; Wierda, W. G.; Gandhi, V. Blood 2009, 114,
4150.
25. SGI-1776 was synthesized in house following the method described in
WO2008058126.
26. The assay was done in the absence of serum in order to avoid the contribution
to BAD phosphorylation of the other survival kinases that are induced by
growth factors, as PIM-1 is expressed as a constitutively active kinase.
27. Compounds 9a,b were prepared following Scheme 1 (R4 = OMe). The starting
Figure 5. Effect of compound 9a on pBAD in Jeko lymphoma tissue by western blot,
2 h after a single i.v. administration (10 mg/Kg). Values are means sd; n = 4–5
animals per group.
material
1 (2,6-dichloro-4-methoxypyridine) was prepared following the
procedure described in WO2007/21710.

J. Pastor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1591–1597
1597
28. Compound 9c was prepared by chlorination with NCS of the Boc-protected
precursor of 6b, followed by Boc-deprotection.
29. http://clinicaltrials.gov/ct2/show/study/NCT00848601.
32. The values reported are an average of two independent data points. Inhibitors
used at a concentration of 1
www.proQinase.com.
lM. Details of assay conditions can be found at
30. hERG membrane radioligand binding assay. CHO-K1 cell line stably expressing
hERG channel, radioligand: [3H]-astemizole. Compounds starting concentration:
33. Test compounds were incubated at 37 °C with 0.5 mg/mL human, CD-1 mouse
and SD rat microsomes at 1 M in the presence or absence of NADPH, for 0 and
l
100
l
M, 8-points, duplicates. More details at www.wuxiapptec.com.
15 min in duplicate. More details at www.wuxiapptec.com.
34. Luminescent cytochrome P450 assay was performed in 384 well plates using
the P450-GloTM screening system (www.promega.com) according to the
manufacturer’s instructions.
31. QPatch-HTX System (Sophion Inc.). Cell line stable CHO-K1 cells expressing
hERG channel. 3-point concentration-responses, replicates: n = 3. More details
at www.wuxiapptec.com.