7-(4H-1,2,4-Triazol-3-yl)benzo[c][2,6]naphthyridines: A novel class of Pim kinase inhibitors with potent cell antiproliferative activity

Article abstract of DOI:10.1016/j.bmcl.2011.09.059

A novel class of pan-Pim kinase inhibitors was designed by modifying the CK2 inhibitor CX-4945. Introduction of a triazole or secondary amide functionality on the C-7 position and 2′-halogenoanilines on C-5 resulted in potent inhibitors of the Pim-1 and Pim-2 isoforms, with many analogs active at single digit nanomolar concentrations. The molecules inhibited the phosphorylation at Serine 112 of the apoptosis effector BAD, and had potent antiproliferative effects on the AML cell line MV-4-11 (IC₅₀ <30 nM). This work delivers an excellent lead-optimization platform for Pim targeting anticancer therapies.

Full text of DOI:10.1016/j.bmcl.2011.09.059

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6687–6692
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
7-(4H-1,2,4-Triazol-3-yl)benzo[c][2,6]naphthyridines: A novel class
of Pim kinase inhibitors with potent cell antiproliferative activity
⇑
Fabrice Pierre , Eric Stefan, Anne-Sophie Nédellec, Marie-Claire Chevrel, Collin F. Regan,
Adam Siddiqui-Jain, Diwata Macalino, Nicole Streiner, Denis Drygin, Mustapha Haddach,
Sean E. O’Brien, Kenna Anderes, David M. Ryckman
Cylene Pharmaceuticals, 5820 Nancy Ridge Drive, Suite 200, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of pan-Pim kinase inhibitors was designed by modifying the CK2 inhibitor CX-4945. Intro-
duction of a triazole or secondary amide functionality on the C-7 position and 20-halogenoanilines on C-5
resulted in potent inhibitors of the Pim-1 and Pim-2 isoforms, with many analogs active at single digit
nanomolar concentrations. The molecules inhibited the phosphorylation at Serine 112 of the apoptosis
effector BAD, and had potent antiproliferative effects on the AML cell line MV-4-11 (IC50 <30 nM). This
work delivers an excellent lead-optimization platform for Pim targeting anticancer therapies.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 31 August 2011
Revised 14 September 2011
Accepted 15 September 2011
Available online 22 September 2011
Keywords:
Pim
Kinase inhibitor
Cancer
Leukemia
AML
The Pim kinases (Proviral Integration site of moloney Murine
leukemia virus)1,2 constitute a family of oncogenic serine/threo-
nine protein kinases overexpressed in a number of tumors of
hematological and epithelial origin and often associated with
strongly elevated MYC levels. The three isoforms Pim-1, Pim-2
and Pim-3 are constitutively active and largely regulated at the
transcriptional and translational levels, mainly through the
JAK/STAT signaling pathway. Pim kinases mediate cell survival
through the direct phosphorylation of Bcl-2 antagonist of cell
death (BAD) and other substrates.3–5 In acute myeloid leukemia
(AML) cell lines carrying the receptor tyrosine kinase FLT3-ITD
mutation, Pim-1 has been shown to be one of the principal kinases
mediating the anti-apoptotic function of FLT3-ITD signaling.6,7
A large body of work has proposed the pharmacological target-
ing of Pim kinases for anti-cancer therapy and several classes of
inhibitors have already been described.8–18 However, with a few
notable exceptions,19–23 most of these inhibitors potently inhibited
only Pim-1 and there is increasing evidence that several protein
isoforms should be concurrently modulated for optimal therapeu-
tic efficiency. In particular, simultaneous targeting of Pim-1 and
Pim-2 is necessary to overcome compensatory up-regulation
mechanisms in protein tyrosine kinase-mediated leukemia.24 The
recent withdrawal from development of the first clinical stage
Pim kinase inhibitor SGI-1776,25 due to cardiac QTc prolongation,26
stimulates the discovery of novel inhibitor chemotypes that inhib-
its multiple Pim isoforms and have fewer off-target toxicities.
A reasonable starting point for the design of Pim inhibitory
agents is protein kinase CK2 inhibitors. There have been several re-
ports of small molecules interacting with both proteins due to the
structural similarity of their ATP binding site.12,27 We recently de-
scribed CX-4945 (1) (Fig. 1),28,29 the first ATP-competitive inhibitor
of CK2 to enter clinical trials for cancer. CX-4945 was also found to
moderately inhibit Pim-1 in
(IC50 = 0.046 M), albeit inactive at 10
cell-based assay.29
a
biochemical kinase screen
l
lM in a functional Pim
SAR and structural studies28,30 revealed that the carboxylic acid
moiety on C-8 was necessary for binding to CK2 and that its re-
moval or displacement to the adjacent sites C-7 and C-9 sup-
pressed inhibition of CK2. This observation led us to further
HN
Cl
4
5
3
N
CK2
IC50 = 0.001 µM
1
Pim-1 IC50 = 0.046 µM
N
7
8
1
CO2H
9
⇑
Corresponding author. Tel.: +1 858 875 5113; fax: +1 858 875 5101.
E-mail address: fpierre@cylenepharma.com (F. Pierre).
Figure 1. Structure of CX-4945 (1) and inhibitory activity against CK2 and Pim-1 in
enzyme assays.29
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.059

6688
F. Pierre et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6687–6692
explore the Pim SAR of the scaffold lower ring, intuitively speculat-
ing that the structural changes responsible for the loss of activity of
the molecules against acidophilic CK2 might not be as detrimental
for their ability to inhibit the Pim kinases. Herein, we report our
preliminary exploration efforts and the ensuing discovery of novel
Pim kinase inhibitors with potent cellular antiproliferative
properties.
The synthesis of compounds 1–3, 8 (Scheme 1), 10 and 11
(Table 1) has been already described.28 Carboxylic acids 1–3 were
easily converted to carboxamides 4a, 5 and 6 using amide coupling
reagents and ammonium chloride as a source of nitrogen. Com-
pounds 4b–e bearing extended amides on R1 were obtained in a
similar manner. Carboxamide 4a (–NR4R5 = –NH2) was reacted
with N,N-dimethylformamide dimethylacetal (DMF–DMA) and
subsequently condensed with hydrazine in acetic acid to form tri-
azole 7. An oxadiazole ring could be introduced at the same posi-
tion by reacting ester 8 with hydrazine and converting the
resulting hydrazide to 9 using triethyl orthoformate.
The promising activity of triazole 7 (Table 1) prompted us to de-
sign chemistries allowing the straightforward introduction of
amines on C-5 to study the SAR of that position (Scheme 2).
For that purpose, methyl 5-oxo-5,6-dihydrobenzo[c][2,6]naph-
thyridine-7-carboxylate 1228 was converted to triazole 13 in three
steps using chemistries described above. Reaction with phospho-
rus oxychloride afforded the corresponding crude 5-chloro-
benzo[c][2,6]naphthyridine, which reacted with various amine
reagents to form compounds 14a–p and 15a–p, that were isolated
after purification by preparative HPLC. More amide analogs 17a–j,
diversely substituted on C-5 were prepared in three steps from 12
by first substituting the chlorine of 16 with various anilines and
then converting the methyl ester to amides in two steps.
increase in IC50 value for CK2 greater than 500-fold, while the
IC50 values for Pim-1 (7- and 2-fold increase) and Pim-2 (up to
3-fold decrease) were comparatively less affected. A methyl amide
in 4b enhanced affinity for the Pim kinases to a greater degree
(Pim-1 IC50 = 0.055 lM and Pim-2 IC50 = 0.018 lM) stimulating
further exploration of that position of the scaffold. Tertiary amide
4c was totally inactive against both isoforms, indicating the neces-
sity for one H-bond donor to be present at that position for inhibi-
tion of the protein. Extension of the amides with bulkier groups (4d
and 4e) decreased activity.
4H-1,2,4-triazoles and 1,3,4-oxadiazoles are well known het-
erocyclic bioisosteres of amides.37 Their straightforward chemical
syntheses from carboxamides and esters led us to prepare com-
pounds 7 and 9 from 4a and 8, respectively. 7-(4H-1,2,4-Triazol-
3-yl)benzo[c][2,6]naphthyridine
inhibitor of Pim-1 (IC50 = 0.027
7
was found to be
a
potent
M),
l
M) and Pim-2 (IC50 = 0.018 l
10-fold more active than 1,3,4-oxadiazole 9 (Pim-1 IC50 = 0.228
and Pim-2 IC50 = 0.117 M), confirming the need for a H-bond do-
nor in this area of the inhibitors.
lM
l
The chemistry described in the upper part of Scheme 2 allowed
a convenient and facile examination of the SAR of 7-(4H-1,2,4-
triazol-3-yl)benzo[c][2,6]naphthyridines analogs bearing various
amine substituents on C-5. The inhibitory activities against Pim-1
and Pim-2 of the resulting compounds are reported in Tables 2
and 3. A series of analogs bearing diverse cycloalkyl amines
(14a–c, Table 2) showed a reasonable double digit nanomolar
activity, indicating a good potential for further optimization. An
extended primary alkylamine (14d) or tertiary alkyl amine (14e)
lowered activity.
Overall, aniline moieties on C-5 induced good activities against
the Pim kinases (from 3 nM to 92 nM, 14f–p, 7, Table 2). A series of
analogs mono-substituted at various positions revealed a notewor-
thy increase in potency against both isoforms of the enzyme when
introducing on the aniline 20-position a chlorine (14k), a fluorine
(14o) or a methyl (14p) group. Analogs substituted in the meta
and para position with the same substituents (7, 14j,m,n) or others
(14g,i) were generally less potent. Activity was lowered when a
methoxy (14h) was in the ortho position, suggesting that substitu-
ents at this position were likely to interact with a small hydropho-
bic pocket. Finally, alkylation of the aniline amino group (14l)
induced a modest reduction in activity.
Following these results, a series of analogs disubstituted at the
aniline and bearing 20-chlorine (15a–g), 20-methyl (15h,i) or
20-fluorine (15j–p) moieties were prepared (Table 3). The data con-
firmed the potent activity of the 20-chloroaniline analogs, five of
them showing single digit nanomolar inhibitions of at least one
isoform. A preliminary exploration showed that the aniline moiety
could sustain large solubilizing groups in 40-position of the aniline
and retain potent activity (15e, 15f). Certain substituents, such as
alkylsulfone (15c), trifluoromethoxy (15d) or fluorine (15a) de-
creased activity at the same position. Finally 30-methoxy analog
15g displayed good activity, indicating another possible position
for the introduction of solubilizing groups. 20-Methyl and 20-fluo-
rine analogs (15h,i, 15j–p) were also very potent, notably com-
pound 15j, bearing two fluorine atoms in the ortho positions of
A number of disubstituted 2-halogenoanilines 18a–b and 19a–c
(Scheme 3) bearing solubilizing groups in the para position were
not commercially available and required synthesis using proce-
dures derived from published documents.31–34
The compounds35 described on Scheme 1 were tested in a
radiometric enzymatic assay using recombinant human CK2 holo-
enzyme (aabb) and recombinant human Pim-1 and Pim-2.36 Preli-
minary SAR data (Table 1, compounds 1–3, 4a, 5, 8, 10–11) showed
that most of the structural changes on the C ring that reduced
inhibitory activity against CK2 also affected Pim-1 and Pim-2 inhi-
bition. However, the magnitude of change was significantly lower
for the latter enzymes in compounds bearing on R1 a carboxylate
(2) or a carboxamide (4a) moiety. When compared with CX-4945
1, the structural modifications leading to 2 and 4a resulted in an
2
1
3
R1 = CO2H, R2 = H, R3 = H
R1 = H, R2 = CO2H, R3 = H
R1 = H, R2 = H, R3 = CO2H
a
4a-e R1 = CONR4R5, R2 = H, R3 = H
5
6
HN
Cl
R1 = H, R2 = CONH2, R3 = H
R1 = H, R2 = H, R3 = CONH2
N
b
c
N
R1
R2
N
N
the aniline and potently inhibiting Pim-1 (IC50 = 0.002 lM) and
R1 =
R2 = H, R3 = H
7
N
Pim-2 (IC50 = 0.001 lM). Large solubilizing groups could also be
H
R3
placed on the 20-fluoro-anilines resulting in a minimum loss of
activity (15n–p).
8
9
R1 = CO2Me, R2 = H, R3 = H
The robust activity of 20-halogenoaniline inhibitors encouraged
the synthesis and testing of more amide compounds bearing these
moieties (17a–j, Table 4). Although the compounds were generally
as effective as their triazole counterpart in inhibiting the Pim-2 iso-
form, they were slightly less potent against Pim-1. On average,
amides were fivefold selective for Pim-2 as measured by Pim-1/
Pim-2 IC50 ratios (from 2- to 9-fold). Extending the amide function
N
N
R1 =
R2 = H, R3 = H
O
Scheme 1. Reagents and conditions: (a) NH4Cl or HNR4R5 (for structure of R4 and R5
see Table 1), NMP, EDCI, HOBtꢀH2O, DIEA or NEt3, 70 °C, 5–83%; (b) (R4 = R5 = H)
(MeO)2CHNMe2 neat, 80 °C then NH2NH2ꢀH2O, AcOH, 80 °C, 20%; (c) NH2NH2ꢀH2O,
MeOH, 60 °C then (EtO)3CH, 120 °C, 20%.

F. Pierre et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6687–6692
6689
Table 1
CK2, Pim-1, Pim-2 inhibitory activity of various substituted 5-(3-chlorophenylamino) benzo[c][2,6]naphthyridines
HN
B
Cl
N
A
N
R1
R2
C
R3
Compd
R1
R2
R3
CK2
IC50
Pim-1
IC50
Pim-2
IC50 (lM)
(l
M)
(
l
M)
1
10
5
11
3
6
H
H
H
H
H
H
CO2H
CO2Me
CONH2
H
H
H
H
0.001
>0.5
0.417
>0.5
0.350
>5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
>0.5
0.048
>2.5
0.798
>2.5
0.577
>2.5
0.186
>2.5
1.25
>2.5
0.294
>2.5
0.894
0.094
0.054
0.018
H
H
H
H
H
H
H
H
H
H
H
H
CO2H
CONH2
8
2
CO2Me
CO2H
CONH2
CONHMe
H
H
H
H
H
H
H
H
H
2.22
0.342
0.100
0.055
4a
4b
4c
4d
4e
7
CONMe2
>2.5
>2.5
CONH(c-Pr)
CONH(CH2)2OMe
C-(4H-1,2,4-Triazol-3-yl)
C-(1,3,4-Oxadiazole)
0.107
0.114
0.027
0.228
0.074
0.158
0.018
0.117
9
R10
H2N
R9
R6
R7
O
N
N
N
N
N
NH
N
a, b, c
d, e
O
O
O
19a R10 = F, R9=
19b R10= F, R9=
18a R10 = Cl, R9 =
18b R10 = Cl, R9 =
O
O
N
NMe2
N
N
N
N
N
H
H
N
NMe
13
14a-p
15a-p
O
19c R10= F, R9=
OMe
NH
Scheme 3. Structure of anilines 18a–b and 19a–c.
N
CO2Me
12
R6
R7
N
the top 5 of the kinases affected, while Pim-3 was inhibited at a
lower level (88%).
N
Cl
O
g, h
At the concentration tested, 14k exerted potent inhibition of
Flt3 (95%). Because Flt-3 regulates the Pim kinases in leukemia,7,6
its level of inhibition is important information for the interpreta-
tion of cellular experiments intended to study Pim targeted drugs.
Compound 14k, eight additional representative triazoles 14f,o,
15b,e,l–o and the amide 17b were therefore subjected to Flt3
IC50 determination (Table 5). Additionally, Pim-3 IC50 values were
measured as well as Pim-1 and Pim-2 under the same Millipore
conditions.38 The molecules generally affected Flt3 with double di-
git nanomolar IC50 values, with some degree of enzyme selectivity
(IC50 Flt3/Pim-1 >20) observed for triazoles 15b and 14k. The data
confirmed our measures of potent inhibition of Pim-1 and Pim-2,
and revealed that the molecules inhibited the Pim-3 isoform with
IC50 values generally higher by one to two orders of magnitude
than for the two other isoforms. The most potent Pim-3 inhibitor
15o had an IC50 of 35 nM. The Pim-3 isoform has been shown to
be aberrantly expressed in pancreatic, gastric and colon can-
cer,39–41 therefore appropriate optimization of Pim-3 inhibition
could lead to new therapies for these malignancies.
N
f
N
R8
N
CO2Me
N
H
17a-j
16
Scheme 2. Reagents and conditions: (a) NaOH, H2O, EtOH, reflux, 93%; (b) NH4Cl,
NMP, EDCI, HOBtꢀH2O, DIEA, 80 °C, 96%; (c) (MeO)2CHNMe2 neat, 80 °C then
NH2NH2ꢀH2O, AcOH, 80 °C, 92%; (d) POCl3, ACN, 100 °C, >100% (crude); (e) HNR6R7
(for structure of R6 and R7 see Tables 2 and 3), NMP,
lwave 120–160 °C, preparative
HPLC, 2–93%; (f) POCl3, reflux, 92%; (g) HNR6R7 (for structure of R6 and R7 see
Table 4), NMP, 80 °C or
lwave 140 °C, 85–93% then NaOH, H2O, EtOH, 80 °C, 56–
98%; (h) NH4Cl or R8NH2 (for structure of R8 see Table 4), dioxane, EDCI, HOBtꢀH2O,
DIEA, 80 °C, 39–100%.
with bulkier alkyl groups (17d–f), did not induce any substantial
loss of activity suggesting that further derivatization at this posi-
tion was possible.
Representative triazole 14k was chosen for a biochemical
screen against 107 kinases to assess its selectivity profile (Per-
formed at Millipore, Dundee, UK; see Supplementary data). Using
Several potent molecules (Pim-1 or Pim-2 IC50 <5 nM) were
evaluated for their ability to inhibit the phosphorylation of the
apoptosis effector BAD at Ser1126,5 in MV-4-11,42 a leukemia cell
line carrying the FLT3-ITD activating mutation (Table 6). All of the
a concentration of 0.5
1), only 10 of the 107 kinases were inhibited by more than 90%.
Pim-1 (99% inhibition at 0.5 M) and Pim-2 (98%) were within
lM (500-fold greater than the IC50 of Pim-
l

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F. Pierre et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6687–6692
Table 2
Table 4
Pim-1 and Pim-2 inhibitory activity of various substituted 7-(4H-1,2,4-triazol-3-
yl)benzo[c][2,6]naphthyridines
Pim-1 and Pim-2 inhibitory activity of various substituted 5-(phenylamino)-
benzo[c][2,6]naphthyridine-7-carboxamides
3'
R6
R7
N
R10
4'
N
4
1
R9
5
3
N
5'
N
HN
N
R11
N
N
O
H
R8
N
N
H
Compd
–NR6R7
Pim-1
IC50
Pim-2
IC50 (lM)
(
l
M)
Compd
R11
R10
R9
R8
Pim-1
IC50
Pim-2
IC50 (lM)
14a
14b
14c
14d
14e
14f
14g
14h
14i
–HN(c-Pr)
–HN(c-Bu)
–HN(c-Pen)
–HN(CH2)2OH
Pyrrolidin-1-yl
–HN-Phenyl
–HN(30-MeO-Phenyl)
–HN(20-MeO-Phenyl)
0.013
0.025
0.039
0.052
0.194
0.017
0.023
0.041
0.028
0.027
0.028
0.005
0.021
0.074
0.018
0.005
0.007
0.008
0.020
0.026
0.055
0.174
0.010
0.026
0.092
0.010
0.018
0.021
0.006
0.015
0.059
0.016
0.003
0.006
(lM)
17a
17b
17c
17d
17e
17f
17g
17h
17i
H
H
H
H
H
H
H
H
F
Cl
Cl
Cl
Cl
Cl
Cl
F
F
F
F
H
H
H
0.016
0.009
0.010
0.008
0.016
0.021
0.041
0.024
0.025
0.013
0.008
0.001
0.005
0.002
0.002
0.005
0.010
0.004
0.004
0.002
Me
Me
Et
i-Pr
sec-Bu
H
Me
H
Me
30-OMe
H
H
H
H
H
H
H
–HN(30-Acetylenyl-phenyl)
–HN(30-Cl-Phenyl)
–HN(40-Cl-Phenyl)
–HN(20-Cl-Phenyl)
–(Me)N(20-Cl-Phenyl)
–HN(30-F-Phenyl)
7
14j
14k
14l
14m
14n
14o
14p
17j
F
–HN(40-F-Phenyl)
–HN(20-F-Phenyl)
–HN(20-Me-Phenyl)
Table 5
Inhibitory activity of selected molecules in Flt3, Pim-3, Pim-1 and Pim-2 Millipore
enzyme assays (see note38
)
Compd
Flt3
IC50
Pim-3
IC50
Pim-1
IC50
Pim-2
IC50 (lM)
(lM)
(
l
M)
(
l
M)
Table 3
14k
14f
14o
15b
15e
15l
15m
15n
15o
17b
0.104
0.103
0.070
0.039
0.017
0.095
0.046
0.044
0.061
0.023
0.086
0.343
0.057
0.875
0.12
0.154
0.459
0.235
0.035
0.367
0.001
0.022
0.005
0.002
0.002
0.010
0.008
0.011
0.005
0.016
0.006
0.015
0.005
0.001
0.014
0.015
0.012
0.030
0.028
0.005
Pim-1 and Pim-2 inhibitory activity of 7-(4H-1,2,4-triazol-3-yl)benzo[c][2,6]naph-
thyridines bearing various disubstituted anilines
3'
R10
4'
R9
5'
HN
6'
N
N
N
N
N
H
that within this time frame, the dephosphorylation of BAD was
likely due to a direct inhibition of Pim enzymatic activity. All tested
molecules potently inhibited MV-4-11 cellular proliferation with
IC50 values 6150 nM, many being lower than 30 nM, showing
the efficiency of targeting the Pim kinases in this cell phenotype.
Other cancer cell lines were affected by the compounds at higher
submicromolar or micromolar levels, indicating their potential
therapeutic use against solid tumors in addition to hematological
malignancies.
In summary, we have discovered a novel class of potent
benzo[c][2,6]naphthyridines Pim kinase inhibitors by relocating
and modifying functional groups of the potent CK2 inhibitor
CX-4945. Introduction of a triazole or secondary amide functional-
ity on the C-7 position of the scaffold dramatically lowered affinity
for CK2, and 20-chloro or 20-fluoro anilines on C-5 resulted in single
digit nanomolar Pim kinase inhibitors. The molecules exerted a
powerful in vitro antiproliferative activity on solid and hematolog-
ical cancer cell lines. In the most sensitive leukemia cell lines
(MV-4-11), dephosphorylation of BAD at Ser112 was observed,
providing a preliminary link between the cellular antiproliferative
properties of the molecules and their enzyme inhibitory activity.
Preliminary SAR suggests a number of possible areas of explora-
tion for further optimization. We have shown that the inhibitors
could bear large solubilizing groups on the aniline moieties, and
the favorable activity of aliphatic amines analogs predicts that a
Compd
R10
R9
Pim-1
IC50
Pim-2
IC50 (lM)
(l
M)
14k
15a
15b
15c
15d
15e
15f
15g
14p
15h
15i
14o
15j
15k
15l
15m
15n
15o
15p
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Me
Me
Me
F
F
F
F
F
F
F
F
H
0.005
0.026
0.005
0.026
0.045
0.002
0.002
0.008
0.007
0.007
0.006
0.005
0.002
0.013
0.016
0.010
0.007
0.004
0.007
0.006
0.031
0.002
0.044
0.113
0.007
0.012
0.001
0.006
0.008
0.008
0.003
0.001
0.012
0.006
0.010
0.010
0.012
0.020
40-F
40-OH
40-SO2Me
40-OCF3
4’-(2-Morpholinoethoxy)
40-(4-Methylpiperazin-1-yl)
30-OMe
H
40-F
50-F
H
60-F
60-OMe
40-F
50Cl
40-O(CH2)2OMe
40-(2-(Pyrrolidin-1-yl)ethoxy)
40-O(CH2)2NMe2
compounds tested were potent in this cellular assay with half of the
molecules tested displaying IC50 values below 100 nM. It is note-
worthy that Flt3 autophosphorylation was mildly affected in the
same cell line upon the same drug exposure time (4 h), indicating

F. Pierre et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6687–6692
6691
Table 6
Activity of the most potent Pim inhibitors (Pim-1 or Pim-2 IC50 <5 nM) in various cellular assays42
Compd
Inhibition of phosphorylation MV-4-11 EC50
(l
M)a
Cell viability EC50
MV-4-11
(lM)
BAD S112
Flt3 T591
K-562
PC3
1.24
0.73
0.15
MDA-MB-231
MIA PaCa-2
14k
14o
15b
15e
15f
15g
15j
15o
17b
17c
17d
17e
17f
17h
17i
0.03
0.14
0.09
0.05
0.03
1.58
0.53
0.04
1.02
0.10
0.21
0.89
0.98
0.20
0.10
0.76
4.8
>10
1.5
6.0
>10
ND
5.3
>10
2.6
4.4
ND
ND
ND
8.9
ND
ND
<0.030
<0.030
<0.030
0.050
<0.030
0.093
1.03
0.27
0.24
1.90
0.65
1.47
0.22
0.44
0.54
0.24
2.47
6.38
8.11
0.10
0.29
0.15
0.29
1.60
2.38
>10
2.94
>10
3.14
0.79
1.78
1.90
2.12
7.47
7.87
1.00
0.69
0.59
0.30
1.15
0.58
0.05
1.50
6.81
0.83
0.53
1.24
0.97
2.96
8.06
8.90
1.05
0.44
0.36
>30
3.66
3.68
2.05
9.26
1.03
0.42
2.42
>10
>10
0.47
0.032
<0.030
<0.030
<0.030
<0.030
0.224
0.14
<0.030
0.036
0.15
0.27
17j
0.030
a
Cells were treated with the molecules for 4 h.
20. Tao, Z. F.; Hasvold, L. A.; Leverson, J. D.; Han, E. K.; Guan, R.; Johnson, E. F.; Stoll,
V. S.; Stewart, K. D.; Stamper, G.; Soni, N.; Bouska, J. J.; Luo, Y.; Sowin, T. J.; Lin,
N. H.; Giranda, V. S.; Rosenberg, S. H.; Penning, T. D. J. Med. Chem. 2009, 52,
6621.
21. Qian, K.; Wang, L.; Cywin, C. L.; Farmer, B. T.; Hickey, E.; Homon, C.; Jakes, S.;
Kashem, M. A.; Lee, G.; Leonard, S.; Li, J.; Magboo, R.; Mao, W.; Pack, E.; Peng, C.;
Prokopowicz, A.; Welzel, M.; Wolak, J.; Morwick, T. J. Med. Chem. 2009, 52, 1814.
22. Akue-Gedu, R.; Nauton, L.; Thery, V.; Bain, J.; Cohen, P.; Anizon, F.; Moreau, P.
Bioorg. Med. Chem. 2010, 18, 6865.
larger pool of potent C-5 substituted analogs remain to be discov-
ered. Other areas of modifications include the amide functionality
and the scaffold A-ring, whose interaction in CX-4945 with the
hinge region of CK2 has proven critical.30 Additional optimization
of the selectivity and drug properties of these tricyclic molecules
will be the matter of future reports.
23. Tong, Y.; Stewart, K. D.; Thomas, S.; Przytulinska, M.; Johnson, E. F.; Klinghofer,
V.; Leverson, J.; McCall, O.; Soni, N. B.; Luo, Y.; Lin, N. H.; Sowin, T. J.; Giranda, V.
L.; Penning, T. D. Bioorg. Med. Chem. Lett. 2008, 18, 5206.
Supplementary data
24. Adam, M.; Pogacic, V.; Bendit, M.; Chappuis, R.; Nawijn, M. C.; Duyster, J.; Fox,
C. J.; Thompson, C. B.; Cools, J.; Schwaller, J. Cancer Res. 2006, 66, 3828.
25. Chen, L. S.; Redkar, S.; Bearss, D.; Wierda, W. G.; Gandhi, V. Blood 2009, 114,
4150.
Supplementary data (the complete kinase selectivity profile of
14k (107 kinases)) associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.059.
26. Phase I of SGI-1776 was discontinued in Nov. 2010 (http://www.supergen.
com/investors/pressrelease.html?pressReleaseId=905).
27. Pagano, M. A.; Bain, J.; Kazimierczuk, Z.; Sarno, S.; Ruzzene, M.; Di Maira, G.;
Elliott, M.; Orzeszko, A.; Cozza, G.; Meggio, F.; Pinna, L. A. Biochem. J. 2008, 415,
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28. Pierre, F.; Chua, P. C.; O’Brien, S. E.; Siddiqui-Jain, A.; Bourbon, P.; Haddach, M.;
Michaux, J.; Nagasawa, J.; Schwaebe, M. K.; Stefan, E.; Vialettes, A.; Whitten, J.
P.; Chen, T. K.; Darjania, L.; Stansfield, R.; Anderes, K.; Bliesath, J.; Drygin, D.;
Ho, C.; Omori, M.; Proffitt, C.; Streiner, N.; Trent, K.; Rice, W. G.; Ryckman, D. M.
J. Med. Chem. 2011, 54, 635.
29. Siddiqui-Jain, A.; Drygin, D.; Streiner, N.; Chua, P. C.; Pierre, F.; O’Brien, S. E.;
Bliesath, J.; Omori, M.; Ho, C.; Proffitt, C.; Schwaebe, M. K.; Ryckman, D. M.;
Rice, W. G.; Anderes, K. Cancer Res. 2010, 70, 10288.
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Siddiqui-Jain, A.; Haddach, M.; Anderes, K.; Ryckman, D. M.; Meggio, F.; Pinna,
L. A.Biochemistry 2011, 50, 8478.
31. Galan, A. A.; CHen, J.; Du, H.; Forsyth, T.; Huynh, T. P.; Johnson, H. W. B.;
Kearney, P.; Leahy, J. W.; Lee, M. S.; Mann, G.; Ridgway, B. H.; Takeuchi, C. S.;
Zhou, P. Patent application WO2008/042282A2, 2008, 142.
32. Andrews, D. M.; Jones, C. D.; Simpson, I.; Ward, R. A. Patent application
WO2009/024824A1, 2009, 87.
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35. All final compounds were characterized by LCMS and found to be P95% pure,
except for compounds 15a, 14l and 3 (90–93% purity). Selected compounds
were characterized by NMR.
36. CK2, Pim-1 and Pim-2 inhibitions were measured in radiometric assays
using human recombinant CK2 (aabb-holoenzyme) at [ATP] = 15
l
M (sub-
M (substrate
M (substrate
strate RRRDDDSDDD), human recombinant Pim-1 at [ATP] = 30
RSRHSSYPAGT) and human recombinant Pim-2 at [ATP] = 5
l
l
RSRHSSYPAGT). The IC50 values were derived from eight concentrations of test
inhibitors.
37. Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147.
38. Millipore assays used a distinct substrate for Pim-1 (KKRNRTLTV) and different
concentrations of ATP: Pim-1, [ATP] = 90
radiometric assay for Pim-3 used RSRHSSYPAGT as
presence of [ATP] = 155 M. The Flt3 assay used EAIYAAPFAKKK and
M. The IC50 values were derived from ten concentrations of test
l
M; Pim-2, [ATP] = 15
lM. The
a
substrate in the
l
[ATP] = 200
inhibitors.
l
39. Li, Y. Y.; Popivanova, B. K.; Nagai, Y.; Ishikura, H.; Fujii, C.; Mukaida, N. Cancer
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6692
F. Pierre et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6687–6692
40. Zheng, H. C.
;
Tsuneyama, K.; Takahashi, H.; Miwa, S.; Sugiyama, T.;
conditions and levels of Phospho-Bad (Ser112) and Phospho-FLT3 (Tyr591)
were quantified using a commercial ELISA kit from Cell Signaling, according to
the manufacturer’s protocols. Cell proliferation inhibition was determined by
Alamar Blue assay, exposing various cell lines to the tested compounds for
4 days.
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42. The AML cells MV-4-11 were treated with 8 different concentrations of the
tested compounds for 4 h. Samples were lysed under non-denaturing