Structure-based design of 3-aryl-6-amino-triazolo[4,3-b]pyridazine inhibitors of Pim-1 kinase

Article abstract of DOI:10.1016/j.bmcl.2009.04.061

A series of substituted 3-aryl-6-amino-triazolo[4,3-b]pyridazines were identified as highly selective inhibitors of Pim-1 kinase. Initial exploration identified compound 24 as a potent, selective inhibitor, limited in its utility by poor solubility and permeability. Understanding the unusual ATP-binding site of the Pim kinases and X-ray crystallographic data on compound 24 led to design improvements in this class of inhibitor. This resulted in compound 29, a selective, soluble and permeable inhibitor of Pim-1.

Full text of DOI:10.1016/j.bmcl.2009.04.061

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3019–3022
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based design of 3-aryl-6-amino-triazolo[4,3-b]pyridazine
inhibitors of Pim-1 kinase
Ron Grey a, Albert C. Pierce a, Guy W. Bemis a, Marc D. Jacobs a, Cameron Stuver Moody a, Rahul Jajoo b,
Narinder Mohal b, Jeremy Green a,
*
a Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, MA 02139, USA
b GVK Biosciences, Kundanbagh, Begumpet, Hyderabad 500 016, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 February 2009
Revised 3 April 2009
Accepted 7 April 2009
Available online 20 April 2009
A series of substituted 3-aryl-6-amino-triazolo[4,3-b]pyridazines were identified as highly selective
inhibitors of Pim-1 kinase. Initial exploration identified compound 24 as a potent, selective inhibitor, lim-
ited in its utility by poor solubility and permeability. Understanding the unusual ATP-binding site of the
Pim kinases and X-ray crystallographic data on compound 24 led to design improvements in this class of
inhibitor. This resulted in compound 29, a selective, soluble and permeable inhibitor of Pim-1.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Pim-1 kinase
Pim-1 kinase inhibitors
Triazolo[4,3-b]pyridazine
Solubility
Permeability
The Pim kinases are a small sub-family of three Ser/Thr protein
kinases within the CAMK (calmodulin-dependent protein kinase-
related) family, comprising Pim-1, -2 and -3.1 The Pim kinases
are relatively small enzymes (ca. 34 kDa) containing a kinase do-
main, but no other recognizable domain structures, hence regula-
tion is controlled at the transcriptional and translational levels.2
Homology between the Pim kinases is high: Pim-2 and Pim-3 are
61% and 71% identical in sequence to Pim-1, respectively. Pim-1
is widely expressed, however overexpression has been observed
in a range of human hematopoietic malignancies, such as leuke-
mias and lymphomas3 as well as in prostate cancers.4 Additionally,
Pim-1 expression is up-regulated in response to a wide variety of
cytokine and growth factor stimuli signaling events.1 Pim-1 knock-
out mice show no obvious phenotype, suggesting redundancy in
the Pim signaling pathways.5 Pim-1,2,3-triple knockout mice are
viable, but are significantly smaller in size than wild-type, indicat-
ing impairment of growth factor signaling.6 In vitro assays also
show a dramatic reduction in growth-factor dependent hemato-
poiesis in colony-forming assays6 in triple knockout bone marrow
cells. Furthermore, mice overexpressing Pim-1 show an increased
susceptibility to chemical and radiation induced tumorigenesis.2
Taken together this information suggests that the Pim kinases
may be appropriate targets for cancer intervention.
Recent publications have described the unusual structural fea-
tures of the Pim-1 kinase domain.7 Most notably, the hinge region
presents two features of interest: an insertion residue as well as a
proline residue (Pro123), which combine to form an ATP- and
inhibitor-binding region quite distinct from other protein kinases.
In a typical protein Ser/Thr or Tyr kinase, ATP forms two hydrogen
bonds with the hinge region: one as donor from the adenine NH2 to
a hinge carbonyl, and a second as an acceptor contact between the
adenine N-1 and a backbone NH of the kinase. In the Pim family
this residue is proline so the same H-bonding interaction cannot
exist. The insertion and the proline together cause the hinge region
to expand and create greater space for inhibitor occupancy.
Several recent reports have disclosed inhibitors of Pim-18–14
:
exemplary compounds are illustrated in Figure 1. Bullock et al.13
have reported imidazo[1,2-b]pyrimidine (e.g., compound 6) and
similar pyrazolo[1,5-a]pyrimidine (e.g., compound 7) inhibitors of
Pim-1 kinase. Here we report our discovery of triazolo[4,3-b]pyrid-
azine-based inhibitors and development of this series as soluble,
potent and selective Pim-1 inhibitors.
To identify inhibitors of Pim-1 kinase we undertook a high
throughput screen of our compound collection and identified triaz-
olo[4,3-b]pyridazine compound 8 as an ATP-competitive Pim-1
inhibitor, with Ki = 320 nM. This inhibitor was sufficiently distinct
from other hit classes to warrant further inspection.
To further explore this scaffold we prepared a small library of
analogs according to the method shown in Scheme 1.
* Corresponding author. Tel.: +1 6174446315.
E-mail address: jeremy_green@vrtx.com (J. Green).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.061

3020
R. Grey et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3019–3022
H
N
O
N
O
OH
O
O
O
O
F
CN
OH
HO HN
O
OH
HO
HO
O
O
N
Cl
H
OH
N
N
Br
OH
Ru
C
2
IC50 = 50 nM
1
Ki = 22 nM
3
IC50 = 340 nM
IC50 = 0.22 nM
4
N
N
N
N
O
S
F3C
N
N
N
NH
N
H
N
N
N
H
N
N
N
H
O
O
Cl
CF3
5
7
8
IC50 = 24 nM
IC50 = 61 nM
6
Figure 1. Inhibitors of Pim-1. inhibition data from sources.
O
O
NHNH2
H
N
(a)
(b)
Cl
R1
+
N
H
N
R1
Cl
Cl
N
N
Cl
N
N
9
10
11
N
N
N
(c)
R2
N
N
N
N
H
N
R1
R1
12
13-31
Scheme 1. Synthesis of N-substituted-3-aryl-[1,2,4]triazolo[4,3-b]pyridazin-6-amines. Reagents and conditions: (a) MeCN, rt, 2 h; (b) POCl3, MeCN, 80 °C, 16 h (49%, R = m-CF3);
(c) R2–NH2, 70 °C, 4 h (11–66%).
In this scheme, substituted aroyl chlorides (10) and 3-chloro-6-
hydrazinylpyridazine (9) were combined in MeCN and mixed for
2 h at RT. After confirming the conversion to the acylhydrazine
11 by HPLC, POCl3 was added to effect the ring closure by heating
to 80 °C. Compound 12 was treated with neat amine at 70 °C to
convert to the desired products. Yields were highly dependent on
the particular amine and products were isolated by preparative
HPLC (>95% purity).
A brief evaluation of both N-substituents and phenyl ring sub-
stituents revealed interesting trends in Pim-1 inhibition summa-
rized in Table 1. ortho-Aromatic substituents (compounds 14, 16,
19) were significantly less potent than the screening hit (8), as
was the unsubstituted ring system (13). By moving the CF3 group
from the para position of the initial hit (8), to the meta position
(15) an 18-fold improvement in inhibition was obtained. For the
6-amino substituent (R2 in Table 1), extension of the cyclopropyl
group with a methylene insertion (22), analogous to published
compound 6, did not improve inhibitory activity. Replacement of
the N-cyclopropyl with a cyclohexyl group afforded the most po-
tent inhibitor of this initial series, 24. An X-ray crystal structure
of 24 bound to Pim-1 was obtained14 and revealed an unusual
binding mode consistent with that of compound 6 previously re-
ported by Pogacic et al.13b In the structure of 24 bound to Pim-1
shown in Figure 2, the m-CF3-phenyl ring makes contact with the
hinge Glu121 by way of a bifurcated H-bond between the aromatic
protons on C-5 and C-6 and the backbone carbonyl of Glu121.16
This observation helps to explain the weaker activity of the
ortho-substituted compounds, as ortho substitution disrupts this
interaction by causing the phenyl ring to twist significantly out
Table 1
N-Substituted-3-aryl-[1,2,4]triazolo[4,3-b]pyridazin-6-amine inhibitors of Pim-1
N
N
R2
N
N
H
N
R1
Compound
R1
H
2-CF3
3-CF3
4-CF3
2-F
R2
PIM-1, Ki (nM)15
13
14
15
8
16
17
18
19
20
21
22
23
24
cPr
cPr
cPr
cPr
cPr
cPr
cPr
cPr
cPr
cPr
1800
>3300
18
320
>3300
430
210
>3300
410
980
50
3-F
4-F
2-OMe
3-OMe
4-OMe
3-CF3
3-CF3
3-CF3
cPrCH2
cBu
cHex
54
11

R. Grey et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3019–3022
3021
ously reported for compound 6 bound to Pim-1. The cyclohexyl-
amino group attached at position-6 of the core bicyclic
heterocycle lies within the active site and points outwards to
solvent.
Although compound 24 was found to be a potent and selective
Pim-1 kinase inhibitor, its physical properties limit its utility. Sol-
ubility at pH7.4 was less than 0.3 lM, while c log P and c log D7.4
are 4.9 and 5.0, respectively. To improve upon the physical charac-
teristics, solubility-enhancing features were designed into a subse-
quent library of analogs, prepared according to the method shown
in Scheme 1. Data for these compounds are presented in Table 2.
As shown in Table 2, introduction of solubility-enhancing fea-
tures, such as ethers, basic amines or hydroxyl groups into the
cyclohexyl ring N-substituent all serve to significantly improve sol-
ubility and calculated physical properties, without compromising
Pim-1 kinase inhibition activity. The most significant improve-
ments in pH7.4 solubility were obtained by substituting 3- or 4-
piperidyl rings in place of the cyclohexyl ring. Compound 27
Figure 2. Crystal structure of compound 24 bound to Pim-1.
showed >200 lM solubility at pH 7.4, while maintaining low nano-
molar potency. The most potent inhibitor, compound 29, contains a
trans-4-hydroxycyclohexylamine substituent and inhibits Pim-1
kinase below the limit of detection of our assay (<5 nM). In addi-
tion to the improvements in physical properties, enhanced cellular
permeability was also observed for compounds 27 and 29, relative
to compound 24, as measured by MDCK permeability assay.18
of plane. The superiority of the m-CF3 relative to p-CF3 is clear from
the structure. The m-CF3 creates hydrophobic interaction with the
hinge Pro123, while the p-CF3 group disrupts the interaction with
the hinge. The bicyclic triazolo[4,3-b]pyridazine ring system forms
an H-bond between the triazole N-1 and the catalytic lysine (Lys
67). These structural observations are consistent with those previ-
Table 2
Pim-1 inhibition and physical properties
N
N
N
R
N
CF3
Compound
R
PIM-1 Ki, (nM)15
c log P
c log D7.4
5.0
Solubility (
l
M)17
MDCK permeability18 A–B/B–A
0.6/0.2
24
11
4.9
<0.3
N
H
O
25
26
44
94
2.5
3.1
3.2
1.1
46
NA
NA
N
H
H
N
>200
N
H
HN
27
28
29
30
21
2.5
2.9
2.8
5.1
0.2
1.6
3.9
5.7
>200
>200
170
1.4/13.2
NA
N
H
MeN
HO
160
<5
N
H
35.3/49.0
NA
N
H
49
<0.3
N
Me
MeN
31
100
3.1
2.4
>200
NA
N
Me

3022
R. Grey et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3019–3022
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Kinase selectivity profile of compounds 27 and 29
Kinase
27 Ki (nM)
29 Ki (nM)
Kinase
27 Ki (nM)
29 Ki (nM)
5. Laird, P. W.; van der Lugt, N. M.; Clarke, A.; Domen, J.; Linders, K.; McWhir, J.;
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Pim-1
Aur2
CDK2
COT
ERK2
Flt3
GSK3b
IRAK4
ITK
JAK2
JAK3
21
800
>4000
NA
NA
1400
2900
>4000
>4000
>4000
>4000
<5
NA
JNK3
KDR
MAP3K7
MET
p38a
PKA
PKCh
Plk1
ROCK
Src
>4000
>4000
NA
>4000
>4000
>4000
>4000
>4000
>4000
1800
>4000
>4000
>4000
>4000
3100
>4000
>4000
2300
230
2300
>4000
2400
2300
>4000
>4000
>4000
>4000
>4000
>4000
>4000
>4000
7. (a) 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,
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V.; Leverson, J.; McCall, O.; Soni, N. B.; Luo, Y.; Lin, N.; Sowin, T. J.; Giranda, V. L.;
Penning, T. D. Bioorg. Med. Chem. Lett. 2008, 18, 5206.
Syk
9. Cheney, I. W.; Yan, S.; Appleby, T.; Walker, H.; Vo, T.; Yao, N.; Hamatake, R.;
Hong, Z.; Wu, J. Z. Bioorg. Med. Chem. Lett. 2007, 17, 1679.
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Lilly, M. B. Mol. Cancer Ther. 2007, 6, 163.
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Knapp, S.; Meggers, E. Angew. Chem., Int. Ed. 2006, 45, 1580.
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Compounds 27 and 29 were further screened against a panel of ki-
nases and showed a high level of selectivity (Table 3).
In summary we have developed a series of highly selective and
potent Pim-1 kinase inhibitors that derive selectivity from an unor-
thodox binding mode to the unique hinge region of Pim-1. X-ray
crystallographic data on an advanced molecule allowed for further
refinement in the physicochemical property profile of this series
resulting in compounds with excellent solubility and improved cel-
lular permeability.
Smith, C. D. J. Med. Chem. 2009, 52, 74.
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Knapp, S. J. Med. Chem. 2005, 48, 7604; (b) Pagacic, V.; Bullock, A. N.; Federov,
O.; Filippakopoulos, P.; Gasser, C.; Biondi, A.; Meyer-Monard, S.; Knapp, S.;
Schwaller, J. Cancer Res. 2007, 67, 6916.
14. Pierce, A. C.; Jacobs, M.; Stuver-Moody, C. J. Med. Chem. 2008, 51, 1972. PDB ID:
3bgq.
15. Compounds were assayed by the coupled assay method described in: Fox, T.,
Coll, J. T., Xie, X., Ford, P. J., Germann, U. A., Porter, M. D., Pazhanisamy, S.,
Fleming, M. A., Galullo, V., Su, M. S., and Wilson, K. P. Protein Sci. 1998, 7, 2249.
Reactions were carried out in 100 mM HEPES pH 7.5, 10 mM MgCl2, 25 mM
Acknowledgments
The authors wish to thank Craig Zetterberg and David Howe for
obtaining MDCK permeability data.
NaCl, 1 mM DTT, 20
in the assay were 120
carried out at 30 °C. using 50 nM PIM-1. Final concentrations of the
components of the coupled enzyme system were 2.5 mM
M NADH, 30 g/ml pyruvate kinase and 10 g/
l
g/ml BSA and 1.5% DMSO. Final substrate concentrations
l
M ATP and 200 M peptide (RRRLSSLRA). Assays were
l
References and notes
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l
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overnight and concentration determined by HPLC.
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