A selective, orally bioavailable 1,2,4-triazolo[1,5-a]pyridine-based inhibitor of janus kinase 2 for use in anticancer therapy: Discovery of CEP-33779

Article abstract of DOI:10.1021/jm300248q

Members of the JAK family of nonreceptor tyrosine kinases play a critical role in the growth and progression of many cancers and in inflammatory diseases. JAK2 has emerged as a leading therapeutic target for oncology, providing a rationale for the development of a selective JAK2 inhibitor. A program to optimize selective JAK2 inhibitors to combat cancer while reducing the risk of immune suppression associated with JAK3 inhibition was undertaken. The structure-activity relationships and biological evaluation of a novel series of compounds based on a 1,2,4-triazolo[1,5-a]pyridine scaffold are reported. Para substitution on the aryl at the C8 position of the core was optimum for JAK2 potency (17). Substitution at the C2 nitrogen position was required for cell potency (21). Interestingly, meta substitution of C2-NH-aryl moiety provided exceptional selectivity for JAK2 over JAK3 (23). These efforts led to the discovery of CEP-33779 (29), a novel, selective, and orally bioavailable inhibitor of JAK2.

Full text of DOI:10.1021/jm300248q

Article
pubs.acs.org/jmc
A Selective, Orally Bioavailable 1,2,4-Triazolo[1,5-a]pyridine-Based
Inhibitor of Janus Kinase 2 for Use in Anticancer Therapy: Discovery
of CEP-33779
Benjamin J. Dugan,* Diane E. Gingrich, Eugen F. Mesaros, Karen L. Milkiewicz, Matthew A. Curry,
Allison L. Zulli, Pawel Dobrzanski, Cynthia Serdikoff, Mahfuza Jan, Thelma S. Angeles, Mark S. Albom,
Jennifer L. Mason, Lisa D. Aimone, Sheryl L. Meyer, Zeqi Huang, Kevin J. Wells-Knecht, Mark A. Ator,
Bruce A. Ruggeri, and Bruce D. Dorsey
Worldwide Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380, United States
ABSTRACT: Members of the JAK family of nonreceptor tyrosine kinases play a critical role in
the growth and progression of many cancers and in inflammatory diseases. JAK2 has emerged as
a leading therapeutic target for oncology, providing a rationale for the development of a selective
JAK2 inhibitor. A program to optimize selective JAK2 inhibitors to combat cancer while reducing
the risk of immune suppression associated with JAK3 inhibition was undertaken. The structure−
activity relationships and biological evaluation of a novel series of compounds based on a 1,2,4-
triazolo[1,5-a]pyridine scaffold are reported. Para substitution on the aryl at the C8 position of
the core was optimum for JAK2 potency (17). Substitution at the C2 nitrogen position was
required for cell potency (21). Interestingly, meta substitution of C2-NH-aryl moiety provided exceptional selectivity for JAK2
over JAK3 (23). These efforts led to the discovery of CEP-33779 (29), a novel, selective, and orally bioavailable inhibitor of
JAK2.
CYT387 (1),8 TG101348 (2),9 INCB018424, ruxolitinib (3),10
INTRODUCTION
The JAK/STAT signaling pathway is constitutively activated in
■
AZD1480 (4),11 CEP-701, lestaurtinib (5),12 and SB1518,
pacritinib13 advancing to clinical development (Figure 1).
The 1,2,4-triazolo[1,5-a]pyridine heterocycle14 provided a
novel hinge binding motif for the design of small-molecule
ATP-competitive JAK2 inhibitors. The hypothesis was that
triazole nitrogen N3 and the exocyclic NH would serve as the
H-acceptor and H-donor, respectively, forming hydrogen bonds
with the NH and the carbonyl of Leu-932, leading to a strong
two-point interaction with the hinge (Figure 2A). On the basis
of knowledge from our work with other kinase inhibitor
scaffolds,15 the core would then be modified with various
substituents that would drive potency and selectivity while the
exocyclic nitrogen would be substituted with various solubiliz-
ing groups, as this portion of the molecule would be directed
toward the solvent region. This design hypothesis and working
model was confirmed later in our program when a JAK2/
inhibitor cocrystal structure was solved by X-ray (Figure 2B,
vide infra).
a wide spectrum of hematopoietic and solid1 human tumors
and high levels of activation are often associated with a more
malignant and refractory disease, suggesting an important role
in tumor growth and progression. Indeed, there is a growing
understanding that the constitutively activated JAK/STAT
signaling contributes to tumorigenesis and tumor progression
through multiple mechanisms including promoting prolifer-
ation and increased antiapoptotic signaling in tumor cells as
well as mediating tumor-promoting functions of the stromal
compartment. The latter includes tumor-associated inflamma-
tion,2 tumor immune evasion, and angiogenesis. The vast
preclinical and clinical evidence supports targeting the JAK/
STAT pathway in oncology and inflammatory diseases.3
The Janus (JAK) family of nonreceptor tyrosine kinases
consists of four members: JAK1, JAK2, JAK3, and TYK2.4
There is growing evidence demonstrating that JAK2 activation
may be particularly critical for tumor growth and progression,5
supporting its selection as a therapeutic target. In addition,
JAK3 inhibition has been shown to be immunosuppressive,6
providing further rationale for development of a highly selective
JAK2 inhibitor. JAK1 and TYK2 have been implicated in
disease and immune suppression;7 however, activity or
selectivity against these kinases coupled with inhibition of
JAK2 is not adequately understood.
CHEMISTRY
■
A number of literature16 methods to prepare the 1,2,4-
triazolo[1,5-a]pyridine core are known and were evaluated
and adapted to meet our design modifications. Initially, we had
envisioned target molecules arising from the addition of 2-
aminopyridines 6 to arylisothiocyanates 7 to give key
intermediates 8, followed by cyclization to triazolopyridines 9
Several JAK inhibitors from a range of chemical classes,
including 2-aminopyrimidines, 2,4-diaminopyrimidines, pyrro-
lopyrimidines, indolocarbazoles, and macrocycles, have been
disclosed in the literature with several inhibitors such as
Received: February 23, 2012
© XXXX American Chemical Society
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Figure 1. Examples of reported JAK inhibitors.
Figure 2. (A) Design rationale. (B) Ribbon representation of JAK2/inhibitor cocrystal structure with 28: N-terminal domain in orange, C-terminal
domain in green, hinge region in yellow, glycine rich P-loop in darker orange. Key amino acid residues: gatekeeper Met-929, hinge binder Leu-932,
and back-pocket salt-bridge of Asp-994 and Lys-882 are explicitly shown in element color-coded stick model. Inhibitor 28 is represented in a stick
model: C, green; N, blue; O, red; S, yellow.
(Scheme 1). This methodology was employed for the initial
target, 2-phenylamino-1,2,4-triazolo[1,5-a]pyridine 9 (X, R =
H) via cyclization of the corresponding commercially available
thiourea 8 (X, R = H). The core triazolopyridines 9 (where X =
Br, R = OCH3) were prepared similarly and were subjected to
Suzuki−Miyaura cross coupling with 4-methanesulfonylphenyl-
boronic acid to furnish inhibitors 22−24 (Scheme 1). However,
this synthetic route was complicated by poor coupling reactivity
for the 2-aminopyridines and arylisothiocyanates and tedious
workups and purifications required at several intermediate
steps. The thiourea moiety required activation by S-alkylation
before addition of hydroxylamine hydrochloride, limiting the
use of certain desirable heterocyclic arylisothiocyanates. The
cyclization step required the use of phosgene, as ethyl-
chloroformate did not effect this transformation.17 Ultimately,
low overall yields coupled with the scarcity of commercially
available arylisothiocyanates or appropriately substituted
B
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a
Scheme 1. Initial Synthesis of Triazolopyridine Derivatives 9 and 22−24
a
Reagents and conditions: (a) NaH, p-dioxane, 0 °C to RT, 66−96%; (b) (CH3O)2SO2, p-dioxane, 60 °C; (c) NH2OH hydrochloride, (i-Pr)2NEt or
K2CO3, p-dioxane, 90 °C; (d) 20% phosgene in toluene, K2CO3, acetonitrile, RT, 12−58%; (e) Pd(OAc)2, TPP, 4-methanesulfonylboronic acid,
Na2CO3, water, p-dioxane, 80 °C, 39−67%. See also Table 3 (22−24).
a
Scheme 2. Improved Synthesis of Triazolopyridine Derivatives 12−21 and 25−41
a
Reagents and conditions: (a) ethoxycarbonyl isothiocyanate, p-dioxane, RT; (b) NH2OH hydrochloride, (i-Pr)2NEt, EtOH, MeOH, 60 °C, 70%;
(c) Pd(OAc)2, TPP, R1-boronic acid/ester, Na2CO3, water, p-dioxane, 80 °C, 8−95%; (d) Pd(OAc)2, ligand, arylbromide, Cs2CO3, p-dioxane, 80
°C, 6−77%. See also Table 1 (12−18), Table 2 (19−21), and Table 3 (25−41).
thioureas precluded further SAR optimization by this method-
ology.
Table 1. Effect of C8 Substitution on C2-NH2
Triazolopyridines
Therefore, a convergent synthesis, suitable for medicinal
chemistry purposes, was brought to fruition. Preparation of a
core building block substituted with orthogonal synthetic
handles (cf. 11 in Scheme 2), in which diversity could be built
upon to develop SAR in a timely manner, was accomplished.
The core 2-amino-8-bromo-1,2,4-triazolo[1,5-a]pyridine18 11
was prepared in a “one-pot” process from 2-amino-3-
bromopyridine 10. This synthetic methodology did not require
the use of phosgene or ethylchloroformate to effect cyclization.
The ethoxycarbonyl isothiocyanate reagent coupled with
various aminopyridines to provide activated thioureas that
readily reacted with hydroxylamine hydrochloride. Subsequent,
in situ, cyclization with concomitant loss of carbon dioxide
yielded desired intermediate in good yields.14 The core was
sequentially decorated utilizing a Suzuki−Miyaura cross-
coupling reaction with diverse arylboronic acids or esters,
followed by subjecting the intermediates 12−18 (Scheme 2) to
Buchwald−Hartwig coupling with arylbromides to furnish the
desired N-arylated products 19−21 and 25−41 in moderate
overall yields and in relatively few synthetic steps.
RESULTS AND DISCUSSION
■
The 1,2,4-triazolo[1,5-a]pyridine scaffold was quickly shown to
exhibit inherent activity against JAK2, as demonstrated by
compound 9 (JAK2 IC50 = 1089 nM; X, R = H), a molecule
that attained moderate potency despite its structural simplicity:
a C2 NH-phenyl scaffold. Initial structure−activity relationship
studies focused on the C8 position of the triazolopyridine.
Substitution in this position greatly influenced JAK2 binding
potency, even without an aryl group at the C2 nitrogen (i.e.,
C2-NH2, see Table 1). An electron rich aryl such as the ortho
methoxy 12 showed moderate loss of JAK2 activity compared
to 9 (X, R = H). Surprisingly, an electron deficient heterocycle
a
IC50 values are reported as the average of at least two separate
determinations; for ≥ three determinations, standard deviations are
b
included. see ref 23 for JAK2 assay information.
like the 3-pyridyl 13 was essentially devoid of activity at the
highest concentration tested; however, activity was regained
upon addition of a methoxy containing pyridyl moiety 14.
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Table 2. Effect of C8 Substitution of C2-NH-Phenyl Derivatives
a
IC50 values are reported as the average of at least three separate determinations, standard deviations are included.
Some electron-deficient heterocycles such as the 4-pyridyl (15)
and substituted aryl moieties containing electron-withdrawing
groups (16−18) showed promising activity against JAK2
especially when substituted at the para position (cf. 17 and
18). Selectivity for JAK2 over JAK3 was observed for 17 and
18. However, cellular potency was poor for compounds
unsubstituted at the C2 nitrogen (e.g., 17, IC50 >10 μM).
Improving cellular activity was targeted next in order for this
scaffold to receive further consideration. On the basis of
knowledge developed on other internal programs, it was
hypothesized that an increase in lipophilicity would positively
impact cellular activity. Efforts to increase the cell membrane
permeability focused on appending an aryl group on C2-NH2
molecules to raise lipophilicity.
Table 2 outlines the activity of N-phenyl molecules: C2-
NHPh substitution was combined with selected C8 substituents
(cf. Table 1), which caused a >20-fold boost in JAK2 potency
(compare 19−21 to 9, and 12, 16, 17). Notably, 21 showed
excellent JAK2 potency and selectivity. This molecule also
exhibited activity, albeit modest, in the cellular assay. The
combination of the para-substituted aryl at the C8 position and
N-arylation at the C2 position displayed the favorable
properties of potent JAK2 enzyme inhibition and submicro-
molar cellular potency and was explored further.
A methoxy scan was undertaken to optimize the C2-NH-aryl
moiety (Table 3). While ortho substitution was not favored
(22), either meta or para substitution showed a positive
response, both 23 and 24 having single-digit nanomolar activity
against JAK2 with selectivity against JAK3. An increase in cell
potency was also observed. Optimization was continued by
varying the substituents in the meta and para positions.
Addition of the electron withdrawing methanesulfonyl moiety
(25 and 26) resulted in potent JAK2 inhibitors with selectivity
against JAK3 but at the expense of JAK2 cell potency with 25.
Saturated heterocycles, especially those containing basic
functionality, were considered to be good in improving cell
permeability and pharmacokinetics. A systematic probe was
initiated to evaluate this hypothesis. Addition of a morpholine
moiety in compounds 27 and 28 provided subnanomolar
inhibitors with selectivity against JAK3 and good cell potency.
Importantly, greater selectivity over JAK3 was observed for
compound 27. This improved selectivity for JAK2 over JAK3
with a meta substituted aryl group was observed throughout the
optimization process. The addition of the basic N-methyl-
piperazine moiety was beneficial: molecules 29 and 30
exhibited good JAK2 enzyme and cell potency. The meta
derivative 29 was superior to 30 in terms of selectivity versus
JAK3 and JAK1 (JAK1 IC50 = 81 and 22 nM, respectively),
suggesting that further SAR refinement of the meta
regioisomers should be targeted. Moving the basic functionality
away from the aryl group by inserting a methylene− (31, 32) to
the saturated heterocycles yielded potent JAK2 inhibitors.
Unfortunately, this modification had a deleterious effect on
JAK2 cell potency compared to 29. The basic functionality was
extended toward the solvent to give morpholinyl-piperidine 33
and piperidinyl-piperidine 34. Both compounds were selective
against JAK3, and 33 showed good cell activity. C-linked
piperidine 35 exhibited excellent cell potency and good
selectivity against JAK3 and JAK1 (JAK1 IC50 = 43 nM).
Investigations into the role of the para-methanesulfonyl
group at C8 with bioisosteric electron withdrawing group
replacements lead to some interesting results (Table 4).
Increasing the size of the alkyl group from methyl to isopropyl
(36) decreased selectivity against JAK3. Replacement of the
sulfone with trifluoromethyl (37) led to a potent inhibitor;
however, a disappointing loss of cell activity was observed. The
fluoro derivative 38 showed a significant loss of potency when
compared to the methanesulfonyl derivative 29. Bioisosteric
replacement of methanesulfonyl with dimethylphosphine
oxide19 generated 39, which exhibited good potency and
JAK3 selectivity; however, cellular activity dropped 5-fold.
On the basis of their in vitro activity and selectivity profile,
compounds 29, 33, and 35 were selected for evaluation of off-
target liabilities. Compound 33 showed an unfavorable CYP
3A4 inhibition (IC50 = 0.2 μM) and was deprioritized.
Compounds 29 and 35 did not exhibit CYP 3A4 concerns
(IC50 = 11 and 24 μM, respectively). Both molecules exhibited
acceptable in vitro liver microsome stability in mouse, rat and
human (all >15 min) while stability in dog was less than
desirable (Table 5). Inhibitors 29 and 35 showed excellent
kinase selectivity20 (S(90) = 0.090 and 0.097, respectively) and
were evaluated in vivo (vide infra).
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Table 3. Effect of Aryl Substitution in C2-NH-Aryl Derivatives
a
IC50 values are reported as the average of at least three separate determinations, standard deviations are included.
X-Ray Crystallographic Data. A cocrystal structure
obtained with 28 in JAK2 catalytic domain (Figure 2B)
confirmed the initial hypothesis on the docking of triazolopyr-
idine into the JAK2 ATP-binding site. Intermolecular hydrogen
bonding was observed as designed: triazolopyridine nitrogen
N(3) and exocyclic N(2)-H established interactions with the
E
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Table 4. Optimization of the para Position
a
IC50 (nM)
ex
R1
JAK2
JAK3
JAK2 Cell
JAK3/2 ratio
cell/enz ratio
29
36
37
38
39
SO2CH3
SO2-i-propyl
CF3
1.8 0.6
150 37
38 11
61 12
85 10
476 70
83
29
55
21
95
34
65
1.27 0.09
4
26
3
1
6
1
218 27
547 119
286 122
119
F
(PO)(CH3)2
318 49
106
a
IC50 values are reported as the average of at least three separate determinations, standard deviations are included.
profile in nude mouse, JAK2 enzyme selectivity of 83 and the
cellular to enzyme ratio of 34, 29 was selected for in vivo PK/
PD evaluation in a single dose study. JAK2 inhibition was
monitored by determining levels of STAT3 phosphorylation.
The results from the single dose PK/PD experiments with 29
in CWR225a prostate carcinoma tumor xenografts in nude
mouse are shown in Figure 3. The 55 mg/kg oral dose
Table 5. In Vitro Liver Microsome Stability Expressed as
Half-Lives (t1/2
)
t1/2 (min)
ex
mouse
rat
dog
human
29
35
20
40
27
40
5
8
40
40
NH and the carbonyl, respectively, of JAK2 hinge residue Leu-
932. The heterocyclic-aromatic aniline fragment at position 2
was exposed to solvent region. The C(8) substituent was
located in the binding pocket adjacent to the Lys-882−Asp-994
salt bridge and extended toward the glycine rich P-loop where
the methanesulfonyl moiety was involved in water mediated
hydrogen bonding interactions. The C(6)-H portion of the
core was in the proximity of the gate keeper residue Met-929.
Indeed, substitution at the C6 position of the triazolopyridine
core in 30 to give derivatives 40 (R(C6) = CF3) and 41
(R(C6) = F) was accompanied by a severe drop in intrinsic
potency for 40 (JAK2 IC50 = >10 μM), likely from a steric clash
of the large −CF3 fragment with Met-929, and only by a
moderate loss of potency in case of 41 (JAK2 IC50 = 32 nM),
with a significantly smaller fluorine substituent.
Lead Evaluation/Pharmacokinetics/Pharmacodynam-
ics/Efficacy. On the basis of their intrinsic and cellular
potency, JAK3/2 selectivity, kinase selectivity, CYP450
inhibition profile, and liver microsome stability, lead molecules
29 and 35 were selected to advance to single dose
pharmacokinetic (PK) experiments in mouse. Plasma exposure
post oral dosing in CD-1 mouse of each molecule was
evaluated. At a single 30 mg/kg, po dose, plasma levels
observed for 29 and 35 at 6 h were 535 and 257 ng/mL,
respectively. Compound 29 was also tested in nude mouse
(Table 6) and exhibited a favorable PK profile, an iv half-life of
1 h, moderate distribution (Vd = 2.6 L/kg), and measurable oral
exposure with an estimated bioavailability of 33%. On the basis
of the 2-fold higher plasma exposure in CD-1 mouse, the PK
Figure 3. Results of PK/PD experiment in CWR22 xenograft in nude
mouse: (A) percent inhibition of STAT3 activation with a single dose
of 29 (55 mg/kg po as a solution in PEG400), (B) plasma and tumor
compound concentration.
exhibited a pharmacodynamic effect in CWR22 xenografts over
24 h postdose with ≥90% inhibition of STAT3 phosphor-
ylation at the 2 and 6 h time points (Figure 3A). The levels in
plasma reached 4−5 μM for up to 6 h, with levels dropping
below the minimum quantifiable level (MQL) at 24 h (Figure
3B). Tumor levels were approximately 3-fold higher than
plasma levels out to 6 h and were still measurable 24 h
postdose. As shown in Figure 4, compound 29 demonstrated
antitumor efficacy in the CWR22 xenograft model; oral dosing
for 14 days at 30 mg/kg bid resulted in tumor stasis and partial
regressions in 5/10 animals. No body weight loss or overt
toxicity was noted with this dosing regimen.
In addition to solid tumors, the JAK/STAT pathway is
implicated in the tumorigenesis of multiple hematopoietic
cancers. Classical Hodgkin lymphoma (cHL) is characterized
Table 6. Single Dose Pharmacokinetic Profiles of 29 in Nude Mouse (Nu/Nu)
a
species
nude
dose (mg/kg)
t1/2 (h)
AUC0→∞ (ng·h/mL)
Vd (L/kg)
CL ((mL/min)/kg)
28
Cmax (ng/mL)
F (%)
1 iv
1.0
1.9
590
2.6
30 po
5791
1331
33
a
Formulated as solutions in 25% hydroxyl-propyl-β-cyclodextrin (iv) and 100% PEG400 (po).
F
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the para to the meta position on the solvent exposed side of the
molecule was key to reducing the potential risk of immune
suppression related to undesired JAK3 inhibition. The N-
methylpiperazine heterocycle resulted in excellent intrinsic and
cellular potency. The moderate basicity of the heterocycle
enhanced the physiochemical properties of this scaffold to
achieve oral bioavailability. CEP-33779,22 29, exhibited
excellent JAK2 potency, selectivity, and oral bioavailability in
mouse. Additionally, 29 has demonstrated antitumor efficacy in
multiple JAK2-driven tumor xenografts and efficacy in
inflammation models in mouse and has potential as a preclinical
development candidate.
Figure 4. Antitumor efficacy of 29 in CWR22 xenograft model, dosed
po as a solution in PEG400.
EXPERIMENTAL SECTION
■
by a frequent amplification of the JAK2 locus and high levels of
constitutive JAK2/STAT signaling. Inhibitor 29 was evaluated
in HDLM-221 (cHL) tumor xenografts in SCID/NOD mouse.
As shown in Figure 5A, compound 29 exhibited dose related
tumor growth inhibition with oral dosing at 30 mg/kg bid for
19 days and 55 mg/kg bid for 7 days; the 55 mg/kg group was
switched to a single daily dose schedule (qd) at day 7 due to
moderate body weight loss (<10% average). Despite switching
to qd dosing with the 55 mg/kg group, a dose-related response
was maintained in the efficacy study up to day 19. The increase
of tumor volume in the treated groups beginning around day 12
was likely due to a clonal selection of more resistant cells in the
tumors in response to treatment. A dose related increase in
plasma (7 and 9 μM, respectively) and tumor levels (6 and 10
μM, respectively) were observed at 3 h post last dose, Figure
5B.
General Methods. All commercial reagents and solvents were
used as received unless otherwise indicated. H and 13C NMR spectra
1
were recorded on a Bruker Avance spectrometer at 400 or 100 MHz,
respectively, in the deuterated solvent noted with tetramethylsilane as
the internal standard. Mass spectrometry (LC/MS) data were
collected on either an Agilent 1100 series HPLC coupled to Bruker
Esquire 2000 ion trap mass spectrometer (2.1 mm × 30 mm Agilent
Eclipse XBD C8 3.5 μm column, elution with 10−100% acetonitrile
and water with 0.1% formic acid solvent gradient, over 5 min) or a
Waters Acquity Ultra Performance LC coupled to a Waters Micromass
ZQ quadrapole mass spectrometer (2.1 mm × 30 mm Acquity BEH
C18 1.7 μm column, elution with 5−95% acetonitrile and water with
0.1% formic acid solvent gradient, over 2 min) and are reported as (M
+ H)+, i.e., molecular ion plus hydrogen. Analytical HPLC data was
collected using an Agilent 1100 series HPLC coupled with an Agilent
Eclipse XDB C18 5 μm (4.6 mm × 150 mm) column, elution with
10−100% acetonitrile and water (with 0.1% trifluoroacetic acid as
modifier) solvent gradient, over 5 min. Reverse phase chromatography
was performed with a Gilson apparatus coupled to a Phenomenex
Gemini-NX C18 5 μm column, either (150 mm × 30 mm) or (100
mm × 21 mm) using an acetonitrile and water (with 0.1%
trifluoroacetic acid as modifier) solvent gradient. Automated normal
phase chromatography was performed with a Teledyne ISCO
CombiFlash Companion apparatus using RediSep Rf flash columns
(SiO2 or amine modified SiO2). Melting point temperatures were
collected using a Mel-Temp device and are uncorrected. Compounds
have ≥95% purity except where indicated and compound purity is
listed as the area percentage (A%) [peak mAU·s/total mAU·s] at a
wavelength of 254 nm.
CONCLUSION
■
Optimization of the 1,2,4-triazolo[1,5-a]pyridine scaffold as a
kinase hinge binding element led to the discovery of a novel,
selective JAK2 inhibitor. The optimization process was greatly
enhanced by the straightforward synthetic sequence developed.
The readily prepared, multisubstituted 1,2,4-triazolo[1,5-a]-
pyridine core was sequentially appended via Suzuki−Miyaura
cross coupling followed by a Buchwald−Hartwig coupling to
provide desired targets.
The developed SAR led to the understanding that para
substitution of the aryl ring at the C8-position of the core was
optimal, with the methanesulfonyl moiety displaying excellent
JAK2 potency. The serendipitous discovery of enhanced JAK2
selectivity based solely on moving a saturated heterocycle from
Select Examples. Phenyl-[1,2,4]triazolo[1,5-a]pyridin-2-yl-
amine (9, X and R = H). To a suspension of 1-phenyl-3-pyridin-2-
yl-thiourea (1.00 g, 4.40 mmol) in acetonitrile (6 mL) was added
dimethyl sulfate (0.45 mL, 4.8 mmol) and heated at reflux for 4 h,
cooled to room temperature, and the volatiles were evaporated to yield
Figure 5. Results of PK and efficacy experiment in HDLM-2 xenograft in SCID/NOD mouse: (A) antitumor efficacy of 29, dosed po as a solution in
PEG400, 55 mg/kg bid switched to qd dosing at day 7, (B) plasma and tumor compound levels at 3 h post last dose.
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viscous yellow oil. To a solution of the yellow oil in dichloromethane
(8 mL) was added hydroxylamine hydrochloride (0.67 g, 9.6 mmol)
followed by N,N-diisopropylethylamine (3.3 mL, 19.0 mmol). The
mixture was stirred at room temperature for 18 h. Water (10 mL) was
added, and a nitrogen sparge tube connected to a bleach scrubber was
fitted to the reaction flask. The mixture was sparged with nitrogen.
The resulting oily liquid was partitioned between ethyl acetate (100
mL) and water (20 mL). The organic layer was washed with water (3
× 20 mL), dried over magnesium sulfate, filtered, and evaporated to a
waxy solid. To a cooled solution of waxy solid in acetonitrile (10 mL)
at −5 °C was added potassium carbonate (1.5 g, 0.011 mol) followed
by dropwise addition of 20% phosgene in toluene (2.6 mL, 4.9 mmol).
The mixture was stirred for 1 h at −5 °C and then warmed to room
temperature. Additional 20% phosgene in toluene (1.0 mL) and
potassium carbonate (1.0 g) was added to the mixture and stirred for
48 h. The volatiles were evaporated, and the residue was partitioned
between ethyl acetate (100 mL) and water (20 mL). The organic layer
was washed with water (3 × 20 mL), dried over magnesium sulfate,
filtered, and evaporated to an orange waxy solid. The product (0.17 g,
18% yield) was isolated via column chromatography (silica gel 40 g,
166.17, 148.91, 148.84, 135.40, 131.17, 127.31, 126.90, 123.32, 121.18,
111.46; mp 185−187 °C. LC/MS (ESI+) m/z 211.99 (M + H)+.
HPLC: 91 A%.
8-Pyridin-4-yl-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (14). 1H
NMR (DMSO-d6) δ 8.69 (dd, J = 4.8, 1.4 Hz, 2H), 8.65 (dd, J =
6.5, 0.8 Hz, 1H), 8.18 (dd, J = 4.7, 1.6 Hz, 2H), 7.94 (dd, J = 7.5, 0.9
Hz, 1H), 7.03 (t, J = 6.8 Hz, 1H), 6.24 (s, 2H). 13C NMR (DMSO-d6)
δ 166.23, 149.82, 148.85, 142.48, 128.16, 127.44, 122.27, 120.94,
111.34; mp 209−215 °C. LC/MS (ESI+) m/z 212.00 (M + H)+.
HPLC: 92 A%.
8-(6-Methoxy-pyridin-3-yl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl-
1
amine (15). H NMR (DMSO-d6) δ 8.91 (d, J = 2.5 Hz, 1H), 8.54
(dd, J = 6.6, 0.9 Hz, 1H), 8.44 (dd, J = 8.7, 2.5 Hz, 1H), 7.73 (dd, J =
7.6, 1.0 Hz, 1H), 6.97 (t, J = 6.9 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H),
6.12 (s, 2H), 3.92 (s, 3H). 13C NMR (DMSO-d6) δ 166.01, 163.15,
148.81, 146.32, 138.69, 126.54, 125.74, 124.74, 121.29, 111.46, 109.96,
53.28; mp 157−158 °C. LC/MS (ESI+) m/z 241.95 (M + H)+.
HPLC: 89 A%.
8-(3-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl-
1
amine (16). H NMR (DMSO-d6) δ 8.64−8.60 (m, 1H), 8.59 (t, J =
1
1.6 Hz, 1H), 8.50−8.46 (m, 1H), 7.98−7.94 (m, 1H), 7.86−7.83 (m,
1H), 7.78 (dd, J = 7.8, 7.8 Hz, 1H), 7.02 (dd, J = 7.1, 7.1 Hz, 1H), 6.18
(s, 1H), 3.29 (S, 3H). 13C NMR (DMSO-d6) δ 166.17, 148.91, 141.17,
136.59, 133.11, 129.51, 127.49, 127.36, 126.30, 126.21, 122.39, 111.44,
43.48; mp 173−183 °C. LC/MS (ESI+) m/z 289.03 (M + H)+.
HPLC: 85 A%.
5%→100% ethyl acetate/heptane). H NMR (CDCl3) δ 8.46−8.42
(m, 1H), 7.63−7.59 (m, 2H), 7.51−7.41 (m, 2H), 7.38−7.33 (m, 2H),
7.31 (br s, 1H), 7.02−6.97 (m, 1H), 6.90−6.85 (m, 1H). 13C NMR
(CDCl3) δ 162.51, 150.24, 140.38, 129.60, 129.16, 127.85, 121.37,
117.19, 113.83, 111.96; mp 180−184 °C. High resolution mass
spectrum (ESI+) m/z 211.0990 [(M + H)+ calcd for C12H10N4:
211.0984]. HPLC: 96 A%.
8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl-
1
amine Hydrochloride (17). H NMR (DMSO-d6) δ 8.69−8.65 (m,
8-(2-Methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine
(12). To a solution of 3-bromo-pyridin-2-ylamine (10.0 g, 57.8 mmol)
in 1,4-dioxane (100 mL) was added dropwise ethoxycarbonyl
isothiocyanate (6.8 mL, 58 mmol). The mixture was stirred under
nitrogen for 18 h and then volatiles were evaporated. The recovered
material was triturated with hexane (250 mL) and then filtered. The
off-white solid was added to a stirred suspension of hydroxylamine
hydrochloride (20.0 g, 288 mmol) and N,N-diisopropylethylamine
(30.0 mL, 172 mmol) in a mixture of methanol (80 mL) and ethanol
(80 mL). A nitrogen sparge tube connected to a bleach scrubber was
fitted to the reaction flask. The mixture was stirred at room
temperature for 2 h, heated to 60 °C for 18 h, and then cooled to
room temperature. The suspension was filtered, rinsed with methanol
and then water and methanol again. 8-Bromo-[1,2,4]triazolo[1,5-
a]pyridin-2-ylamine (11) was isolated as an off-white solid (9.66 g,
1H), 8.37−8.32 (m, 2H), 8.07−8.02 (m, 2H), 7.88−7.85 (m, 1H),
7.11−7.06 (m, 1H), 5.32 (br s, 3H), 3.29 (s, 3H). 13C NMR (DMSO-
d6) δ 165.40, 148.23, 140.14, 140.03, 128.95, 128.21, 127.90, 127.03,
122.31, 111.98, 43.51; mp 237−239 °C. High resolution mass
spectrum (ESI+) m/z 289.0767 [(M + H)+ calcd for C13H12N4O2S:
289.0759]. HPLC: 98 A%.
8-(4-Trifluoromethyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl-
amine (18). 1H NMR (DMSO-d6) δ 8.62 (d, J = 6.3 Hz, 1H), 8.34 (d,
J = 8.0 Hz, 2H), 7.88−7.80 (m, 3H), 7.02 (t, J = 6.8 Hz, 1H), 6.18 (s,
2H); mp 186−187 °C. LC/MS (ESI+) m/z 279.05 (M + H)+. HPLC:
91 A%.
[8-(2-Methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-phenyl-
amine (19). 1H NMR (CDCl3) δ 8.42 (dd, J = 6.6, 0.9 Hz, 1H), 7.62
(dd, J = 7.5, 1.7, 1H), 7.58−7.52 (m, 3H), 7.44−7.38 (m, 1H), 7.36−
7.30 (m, 2H), 7.10 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.97
(t, J = 7.4 Hz, 1H), 6.93 (t, J = 7.1 Hz, 1H), 6.87 (s, 1H), 3.82 (s, 3H);
mp 141−145 °C. LC/MS (ESI+) m/z 317.06 (M + H)+. HPLC: 89 A
%.
[8-(3-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
phenyl-amine (20). 1H NMR (CDCl3) δ 8.68 (t, J = 1.8 Hz, 1H), 8.49
(dd, J = 6.7, 1.3 Hz, 1H), 8.38 (m, 1H), 8.01 (m, 1H), 7.74 (dd, J =
7.8, 7.8 Hz, 1H), 7.70−7.60 (m, 5H), 7.58−7.51 (m, 1H), 7.49−7.43
(m, 2H), 7.40−7.34 (m, 2H), 7.04−6.99 (m, 2H), 6.91 (s, 1H), 3.14
(s, 3H); mp 184−191 °C. LC/MS (ESI+) m/z 365.04 (M + H)+.
HPLC: 82 A%.
1
78%). H NMR (DMSO-d6) δ 8.58 (d, J = 6.5 Hz, 1H), 7.73 (d, J =
7.5 Hz, 1H), 6.80 (dd, J = 7.0, 7.0 Hz, 1H), 6.25 (br s, 2H); mp 219−
222 °C. LC/MS (ESI+) m/z = 213.05, 214.80 (M + H)+. HPLC: 85 A
%
A dry tube was charged with palladium acetate (0.10 g, 0.47 mmol),
triphenylphosphine (0.31 g, 1.2 mmol), and 1,4-dioxane (5 mL) and
then purged under vacuum then backflushed and stirred under
nitrogen for 10 min. 8-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine
(11) (0.50 g, 2.3 mmol), 2-methoxybenzeneboronic acid (0.71 g, 4.7
mmol), N,N-dimethylformamide (5 mL), and 1.50 M of sodium
carbonate in water (4.7 mL, 7.0 mmol) were added, respectively. The
tube was sealed, and the reaction mixture was heated at 80 °C for 18 h.
The mixture was cooled to room temperature and transferred to a
round-bottom flask, and the volatiles were evaporated under reduced
pressure. 8-(2-Methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl-
amine (12) (0.41 g, 34%) was isolated via column chromatography
(amine modified silica gel 47 g, 0%→10% methanol/dichloro-
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
1
phenyl-amine (21). H NMR (CDCl3) δ 8.51 (dd, J = 6.6, 0.9 Hz,
1H), 8.26−8.21 (m, 2H), 8.11−8.06 (m, 2H), 7.66 (dd, J = 7.3, 0.9
Hz, 1H), 7.61−7.57 (m, 2H), 7.39−7.32 (m, 2H), 7.05−6.99 (m, 2H),
6.94 (s, 1H), 3.10 (s, 3H); mp 197−201 °C. LC/MS (ESI+) m/z
365.11 (M + H)+. HPLC: 79 A%.
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
(2-methoxy-phenyl)-amine (22). 1H NMR (CDCl3) δ 8.53−8.49 (m,
1H), 8.38 (dd, J = 8.0, 1.3 Hz, 1H), 8.24 (d, J = 8.4 Hz, 2H), 8.10 (d, J
= 8.5 Hz, 2H), 7.66−7.62 (m, 1H), 7.61 (s, 1H), 7.08−6.89 (m, 4H),
3.92 (s, 3H), 3.11 (s, 3H); mp 211−213 °C. LC/MS (ESI+) m/z
395.10 (M + H)+. HPLC: 88 A%.
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
(3-methoxy-phenyl)-amine (23). 1H NMR (CDCl3) δ 8.50 (d, J = 6.1
Hz, 1H), 8.24 (d, J = 7.8 Hz, 2H), 8.08 (d, J = 7.7 Hz, 2H), 7.66 (d, J
= 7.4 Hz, 1H), 7.37 (s, 1H), 7.28−7.22 (m, 1H), 7.08−7.00 (m, 2H),
6.92 (s, 1H), 6.58 (d, J = 7.6 Hz, 1H), 3.86 (s, 3H), 3.10 (s, 3H); mp
204−206 °C. LC/MS (ESI+) m/z 395.15 (M + H)+. HPLC: 90 A%.
1
methane). H NMR (DMSO-d6) δ 8.50 (dd, J = 6.6,1.0 Hz, 1H),
7.51 (dd, J = 7.5,1.6 Hz, 1H), 7.43−7.36 (m, 2H), 7.13 (d, J = 8.2 Hz,
1H), 7.03 (t, J = 7.3 Hz, 1H), 6.91 (t, J = 6.8 Hz, 1H), 5.97 (br s, 2H),
3.73 (s, 3H). 13C NMR (DMSO-d6) δ 165.92, 156.57, 149.81, 131.10,
129.46, 129.01, 126.29, 124.65, 122.73, 120.02, 111.53, 110.78, 55.47;
mp 200−202 °C. LC/MS (ESI+) m/z 240.98 (M + H)+. HPLC: 97 A
%.
8-Pyridin-3-yl-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (13). 1H
NMR (DMSO-d6) δ 9.28 (d, J = 2.1 Hz, 1H), 8.62−8.58 (m, 2H),
8.49 (m, 1H), 7.82 (dd, J = 7.4, 0.7 Hz, 1H), 7.53 (dd, J = 8.0, 4.7 Hz,
1H), 7.01 (t, J = 7.0 Hz, 1H), 6.18 (br s, 2H). 13C NMR (DMSO-d6) δ
H
dx.doi.org/10.1021/jm300248q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
(4-methoxy-phenyl)-amine (24). 1H NMR (CDCl3) δ 8.47 (d, J = 6.6
Hz, 1H), 8.23 (d, J = 8.5 Hz, 2H), 8.09 (d, J = 8.5 Hz, 2H), 7.64 (d, J
= 7.4 Hz, 1H), 7.00 (dd, J = 6.8, 6.8 Hz, 1H), 6.95−6.90 (m, 2H), 6.71
(s, 1H), 3.82 (s, 3H), 3.10 (s, 3H); mp 208−212 °C. LC/MS (ESI+)
m/z 395.13 (M + H)+. HPLC: 89 A%.
δ 8.47 (dd, J = 6.7, 1.1 Hz, 1H), 8.25−8.20 (m, 2H), 8.10−8.06 (m,
2H), 7.63 (dd, J = 7.4, 1.1 Hz, 1H), 7.51−7.46 (m, 2H), 7.01−6.95
(m, 2H), 6.67 (s, 1H), 3.20−3.15 (m, 4H), 3.10 (s, 3H), 2.62−2.58
(m, 4H), 2.36 (s, 3H); mp 242−244 °C. LC/MS (ESI+) m/z 463.13
(M + H)+. HPLC: 91 A%.
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
[3-(4-methyl-piperazin-1-ylmethyl)-phenyl]-amine (31). 1H NMR
(CDCl3) δ 8.51 (d, J = 6.6 Hz, 1H), 8.24 (d, J = 8.5 Hz, 2H), 8.09 (d, J
= 8.5 Hz, 2H), 7.66 (dd, J = 7.4, 0.9 Hz, 1H), 7.58 (dd, J = 7.8, 1.9 Hz,
1H), 7.46 (s, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H),
6.98 (d, J = 7.5 Hz, 1H), 6.90 (s, 1H), 3.54 (s, 2H), 3.10 (s, 3H),
2.70−2.32 (m, 8H), 2.29 (s, 3H). LC/MS (ESI+) m/z 477.18 (M +
H)+. HPLC: 90 A%.
(3-Methanesulfonyl-phenyl)-[8-(4-methanesulfonyl-phenyl)-
1
[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amine (25). H NMR (CDCl3) δ
8.55 (d, J = 6.9 Hz, 1H), 8.47 (s, 1H), 8.26 (d, J = 7.9 Hz, 2H), 8.12
(d, J = 7.5 Hz, 2H), 7.74−7.69 (m, 2H), 7.58−7.50 (m, 2H), 7.17 (s,
1H), 7.12−7.07 (m, 1H), 3.11 (s, 6H); mp 230−234 °C. LC/MS (ESI
+) m/z 443.11 (M + H)+. HPLC: 94 A%.
(4-Methanesulfonyl-phenyl)-[8-(4-methanesulfonyl-phenyl)-
1
[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amine (26). H NMR (CDCl3) δ
N-{3-[(1,1-Dioxidothiomorpholin-4-yl)methyl]phenyl}-8-[4-
(methylsulfonyl)phenyl][1,2,4]triazolo[1,5-a]pyridin-2-amine (32).
1H NMR (CDCl3) δ 8.52 (dd, J = 6.7, 1.0 Hz, 1H), 8.23 (d, J =
8.56 (d, J = 5.7 Hz, 1H), 8.22 (d, J = 8.0 Hz, 2H), 8.10 (d, J = 7.6 Hz,
2H), 7.92 (d, J = 7.8 Hz, 2H), 7.77 (d, J = 7.3 Hz, 2H), 7.72 (d, J = 7.3
Hz, 1H), 7.32 (s, 1H), 7.14−7.09 (m, 1H), 3.11 (s, 3H), 3.06 (s, 3H);
mp 280−282 °C. LC/MS (ESI+) m/z 443.10 (M + H)+. HPLC: 92 A
%.
8.H Hz, 2H), 8.09 (d, J = 8.4 Hz, 2H), 7.68 (dd, J = 7.4,0.9 Hz, 1H),
7.62−7.60 (m, 1H), 7.48 (dd, J = 8.1, 1.8 Hz, 1H), 7.32 (t, J = 7.7 Hz,
1H), 7.05 (t, J = 7.1 Hz, 1H), 6.97−6.93 (m, 2H), 3.70 (s, 2H), 3.13−
3.01 (m, 11H). LC/MS (ESI+) m/z 512.08 (M + H)+. HPLC: 94 A%.
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
[3-(4-morpholin-4-yl-piperidin-1-yl)-phenyl]-amine (33). 1H NMR
(CDCl3) δ 8.49 (d, J = 6.4 Hz, 1H), 8.26 (d, J = 8.0 Hz, 2H), 8.08 (d, J
= 7.1 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.35 (s, 1H), 7.24−7.18 (m,
1H), 7.01 (d, J = 6.7 Hz, 1H), 6.96 (d, J = 7.9 Hz, 1H), 6.84 (s, 1H),
6.60 (d, J = 8.4 Hz, 1H), 3.81 (d, J = 11.7 Hz, 2H), 3.77−3.72 (m,
4H), 3.10 (s, 3H), 2.78 (t, J = 11.8 Hz, 2H), 2.63−2.58 (m, 4H),
2.40−2.30 (m, 1H), 1.97 (d, J = 12.7 Hz, 2H), 1.74−1.62 (m, 2H); mp
230−236 °C. LC/MS (ESI+) m/z 533.16 (M + H)+. HPLC: 94 A%.
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
{3-[4-(4-methyl-piperazin-1-yl)-piperidin-1-yl]-phenyl}-amine (34).
1H NMR (CDCl3) δ 8.49 (d, J = 6.5 Hz, 1H), 8.25 (d, J = 6.9 Hz,
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
(3-morpholin-4-yl-phenyl)-amine (27). 1H NMR (CDCl3) δ 8.50
(dd, J = 6.6, 1.1 Hz, 1H), 8.26−8.22 (m, 2H), 8.10−8.05 (m, 2H),
7.66 (dd, J = 7.3, 1.1 Hz, 1H), 7.36 (t, J = 2.2 Hz, 1H), 7.25 (t, J = 8.0
Hz, 1H), 7.05−6.98 (m, 2H), 6.85 (s, 1H), 6.59 (dd, J = 8.3, 1.9 Hz,
1H), 3.91−3.87 (m, 4H), 3.24−3.20 (m, 4H), 3.10 (s, 3H); mp 234−
236 °C. LC/MS (ESI+) m/z 450.17 (M + H)+. HPLC: 94 A%.
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
(4-morpholin-4-yl-phenyl)-amine (28). To an oven-dried tube was
added 8-(4-methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-
ylamine (100.0 mg, 0.3468 mmol), 4-(4-bromo-phenyl)-morpholine
(100.0 mg, 0.4130 mmol), palladium acetate (10.0 mg, 0.0445 mmol),
and 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (75.0 mg,
0.130 mmol), cesium carbonate (240.0 mg, 0.7366 mmol), and 1,4-
dioxane (5 mL). The mixture was purged under vacuum and
backflushed with nitrogen three times. The tube was sealed and
heated at 80 °C for 72 h. The mixture was cooled to room temperature
and diluted with dichloromethane (10 mL), filtered, and the filtrate
was evaporated. The product (0.063 g, 40%) was isolated via column
chromatography (silica gel 40 g, 20%→100% ethyl acetate/hexane).
1H NMR (CDCl3) δ 8.49−8.45 (m, 1H), 8.25−8.20 (m, 2H), 8.11−
2H), 8.08 (d, J = 7.7 Hz, 2H), 7.65 (d, J = 7.1 Hz, 1H), 7.31 (s, 1H),
7.24−7.18 (m, 1H), 7.04−6.95 (m, 2H), 6.82 (s, 1H), 6.60 (d, J = 7.9
Hz, 1H), 3.81 (d, J = 11.4 Hz, 2H), 3.10 (s, 3H), 2.77 (t, J = 12.2 Hz,
2H), 2.72−2.33 (m, 9H), 2.30 (s, 3H), 1.96 (d, J = 11.2 Hz, 2H),
1.75−1.63 (m, 2H); mp 237−239 °C. LC/MS (ESI+) m/z 546.20 (M
+ H)+. HPLC: 94 A%.
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
1
[3-(1-methyl-piperidin-4-yl)-phenyl]-amine (35). H NMR (CDCl3)
δ 8.51 (d, J = 6.5 Hz, 1H), 8.08 (d, J = 8.3 Hz, 2H), 8.10 (d, J = 7.3
Hz, 2H), 7.66 (d, J = 6.6 Hz, 1H), 7.55 (s, 1H), 7.38 (d, J = 8.7 Hz,
1H), 7.31−7.25 (m, 1H), 7.02 (t, J = 7.4 Hz, 1H), 6.91−6.85 (m, 2H),
3.10 (s, 3H), 3.01 (d, J = 10.9 Hz, 2H), 2.57−2.45 (m, 1H), 2.35 (S,
3H), 2.11−2.03 (m, 2H), 1.92−1.83 (m, 4H); mp 208−210 °C. LC/
MS (ESI+) m/z 462.16 (M + H)+. HPLC: 91 A%.
[3-(4-Methyl-piperazin-1-yl)-phenyl]-{8-[4-(propane-2-sulfonyl)-
phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl}-amine (36). 1H NMR
(CDCl3) δ 8.49 (d, J = 6.7 Hz, 1H), 8.27 (d, J = 7.7 Hz, 2H), 8.01
(d, J = 7.9 Hz, 2H), 7.67 (d, J = 7.3 Hz, 1H), 7.39 (s, 1H), 7.26−7.20
(m, 1H), 7.04−6.95 (m, 2H), 6.85 (s, 1H), 6.60 (d, J = 7.7 Hz, 1H),
3.31−3.19 (m, 5H), 2.63−2.58 (m, 4H), 2.38 (s, 3H), 1.35 (d, J = 6.7
Hz, 6H). LC/MS (ESI+) m/z 491.19 (M + H)+. HPLC: 85 A%.
[3-(4-Methyl-piperazin-1-yl)-phenyl]-[8-(4-trifluoromethyl-phe-
nyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amine (37). 1H NMR
(CDCl3) δ 8.46 (d, J = 7.0 Hz, 1H), 8.15 (d, J = 7.7 Hz, 2H), 7.76
(d, J = 8.2 Hz, 2H), 7.62 (d, J = 7.2 Hz, 1H), 7.48 (s, 1H), 7.25−7.19
(m, 1H), 7.02−6.97 (m, 1H), 6.92 (d, J = 7.3 Hz, 1H), 6.83 (s, 1H),
6.59 (d, J = 7.8 Hz, 1H), 3.30−3.25 (m, 4H), 2.62−2.57 (m, 4H), 2.37
(s, 3H); mp 234−236 °C. LC/MS (ESI+) m/z 453.15 (M + H)+.
HPLC: 97 A%.
8.06 (m, 2H), 7.63 (dd, J = 7.4, 0.9 Hz, 1H), 7.53−7.48 (m, 2H), 6.99
(t, J = 7.0 Hz, 1H), 6.97−6.93 (m, 2H), 6.73 (s, 1H), 3.90−3.86 (m
4H), 3.14−3.09 (m, 7H); mp 244−246 °C. LC/MS (ESI+) m/z
450.08 (M + H)+. HPLC: 88 A%.
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
[3-(4-methyl-piperazin-1-yl)-phenyl]-amine (29). To an oven-dried
tube was added 8-(4-methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]-
pyridin-2-ylamine (50.0 mg, 0.173 mmol), 1-(3-bromo-phenyl)-4-
methyl-piperazine (53.0 mg, 0.208 mmol), palladium acetate (8.0 mg,
0.036 mmol), 2,2′-bis-dicyclohexylphosphanyl-biphenyl (20.0 mg,
0.0366 mmol), cesium carbonate (140.0 mg, 0.4297 mmol), and 1,4-
dioxane (2 mL) under an atmosphere of nitrogen. The tube was
vacuum purged and backflushed with nitrogen three times and then
the tube was sealed and the mixture was heated at 100 °C for 3 days.
The reaction mixture was cooled to room temperature diluted with
dichloromethane (10 mL), filtered, and the filtrate was evaporated.
The product was isolated (0.029 g, 36%) via column chromatography
(amine modified silica gel 47 g, 0%→10% methanol:dichloro-
1
methane). H NMR (CDCl3) δ 8.49 (dd, J = 6.6, 1.0 Hz, 1H), 8.25
(d, J = 8.4 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H), 7.66 (dd, J = 7.5, 0.9 Hz,
1H), 7.39−7.36 (m, 1H), 7.23 (t, J = 8.2 Hz, 1H), 7.02 (t, J = 7.1 Hz,
1H), 6.97 (dd, J = 7.8, 1.4 Hz, 1H), 6.88 (s, 1H), 6.60 (dd, J = 8.3, 1.8
Hz, 1H), 3.30−3.25 (m, 4H), 3.10 (s, 3H), 2.63−2.58 (m, 4H), 2.38
(s, 3H). 13C NMR (CDCl3) δ 162.65, 152.28, 148.87, 141.00, 140.91,
140.05, 129.64, 129.29, 128.18, 127.85, 127.76, 124.77, 112.03, 109.40,
108.59, 104.80, 55.19, 49.02, 46.19, 44.59; mp 208−211 °C. High
resolution mass spectrum (ESI+) m/z 463.1925 [(M + H)+ calcd for
C24H26N6O2S: 463.1916]. HPLC: 95 A%.
[8-(4-Fluoro-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-[3-(4-
1
methyl-piperazin-1-yl)-phenyl]-amine (38). H NMR (DMSO-d6) δ
9.56 (s, 1H), 8.78 (d, 1H), 8.20 (m, 2H), 7.85 (d, 1H), 7.50 (s, 1H),
7.35 (m, 2H), 7.10 (m, 3H), 6.45 (m, 1H), 3.12 (m, 4H), 2.46 (m,
4H), 2.23 (s, 3H); mp 194−196 °C. LC/MS (ESI+) m/z 403 (M +
H)+. HPLC: 95 A%.
{8-[4-(Dimethyl-phosphinoyl)-phenyl]-[1,2,4]triazolo[1,5-a]-
pyridin-2-yl}-[3-(4-methyl-piperazin-1-yl)-phenyl]-amine (39). 1H
NMR (CDCl3) δ 8.46 (d, J = 6.2 Hz, 1H), 8.15 (d, J = 8.0 Hz,
2H), 7.92−7.84 (m, 2H), 7.63−7.50 (m, 1H), 7.34 (s, 1H), 7.23 (t, J =
[8-(4-Methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-
1
[4-(4-methyl-piperazin-1-yl)-phenyl]-amine (30). H NMR (CDCl3)
I
dx.doi.org/10.1021/jm300248q | J. Med. Chem. XXXX, XXX, XXX−XXX

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Article
8.2 Hz, 1H), 7.02−6.96 (m, 2H), 6.84 (s, 1H), 6.59 (d, J = 7.8 Hz,
1H), 3.30−3.25 (m, 4H), 2.63−2.58 (m, 4H), 2.37 (s, 3H), 1.80 (s,
3H), 1.77 (s, 3H); mp 243−246 °C. LC/MS (ESI+) m/z 461.0 (M +
H)+. HPLC: 100 A%.
[8-(4-Methanesulfonyl-phenyl)-6-trifluoromethyl-[1,2,4]triazolo-
[1,5-a]pyridin-2-yl]-[4-(4-methyl-piperazin-1-yl)-phenyl]-amine
(40). 1H NMR (CDCl3) δ 8.82 (s, 1H), 8.25 (d, J = 7.8 Hz, 2H), 8.13
(d, J = 7.3 Hz, 2H), 7.79 (s, 1H), 7.49 (d, J = 8.2 Hz, 2H), 6.99 (d, J =
8.2 Hz, 2H), 6.88 (s, 1H), 3.21 (bm, 4H), 3.13 (s, 3H), 2.66 (bm,
4H), 2.40 (s, 3H); mp 126−134 °C. LC/MS (ESI+) m/z 531.0 (M +
H)+. HPLC: 98 A%.
[6-Fluoro-8-(4-methanesulfonyl-phenyl)-[1,2,4]triazolo[1,5-a]-
pyridin-2-yl]-[4-(4-methyl-piperazin-1-yl)-phenyl]-amine (41). 1H
NMR (CDCl3) δ 8.45 (s, 1H), 8.26 (d, J = 8.0 Hz, 2H), 8.11 (d, J
= 7.7 Hz, 2H), 7.58 (d, J = 9.0 Hz, 1H), 7.48 (dd, J = 11.5 Hz, 7.8 Hz,
2H), 6.98 (d, J = 7.9 Hz, 2H), 6.76 (s, 1H), 3.27 (bm, 4H), 3.12 (s,
3H), 2.62 (bm, 4H), 2.38 (s, 3H); mp 130−138 °C. LC/MS (ESI+)
m/z 481.2 (M + H)+. HPLC: 95 A%.
Enzyme Assays for JAK1, JAK2, and JAK3. The kinase activity
of baculovirus-expressed human JAK1, JAK2, or JAK3 was measured
using the time-resolved fluorescence detection system as previously
described.23 Each 96-well Costar high binding plate (Corning,
Corning, NY) was coated with 100 μL/well of 10 μg/mL neutravidin
(Pierce Biotechnology, Rockford, IL) in TBS at 37 °C for 2 h,
followed by 100 μL/well of 1 μg/mL 15-mer peptide substrate
(biotinyl-amino-hexanoyl-EQEDEPEGDYFEWLE-amide, Infinity Bio-
tech Research and Resource, Aston, PA) at 37 °C for 1 h. The kinase
assay mixture (total volume = 100 μL/well) consisting of 20 mM
HEPES (pH 7.2), ATP (0.2 μM ATP for JAK1 and JAK2 and 0.1 μM
ATP for JAK3), 1 mM MnCl2, 0.1% BSA, and test compound (diluted
in DMSO, 2.5% DMSO final in assay) was added to the assay plate.
Enzyme was added and the reaction was allowed to proceed for 20 min
at room temperature (RT). Detection of the phosphorylated product
was performed by adding 100 μL/well of diluted Eu−N1 labeled
PY100 antibody (PerkinElmer, Boston, MA). Samples were incubated
at RT for 1 h, followed by addition of 100 μL enhancement solution
(PerkinElmer). Plates were agitated for 10 min, and the fluorescence
of the resulting solution measured using the PerkinElmer EnVision
2102 or 2104 multilabel plate reader. IC50 values were determined
using the 4-parameter logistic model in XLFit 4 (IDBS, Ltd.,
Guildford, UK).
wet ice until centrifuged to separate plasma. The plasma fraction was
transferred into clean, dry tubes, frozen on dry ice, and stored at
approximately −20 °C pending analysis. Plasma samples were analyzed
for test compounds using a preliminary method, consisting of high-
performance liquid chromatography (HPLC) coupled with tandem
mass spectrometry. Pharmacokinetic parameters were estimated using
noncompartmental methods.
PD Assay. Nude mice bearing CWR22 xenografts were dosed
orally with 55 mg/kg of compound 29 or a vehicle (PEG400). At 2, 6,
and 24 h after dosing animals (3/group) were sacrificed, tumors were
excised and plasma samples were prepared. Tumor extracts were
prepared using Triton-based extraction buffer supplemented with
inhibitors of proteases and phosphatases. Equal amounts of extracts
were resolved on SDS-PAGE gels and STAT3 phosphorylation and
expression were analyzed by Western blot using specific antibodies
(Cell Signaling). Specific bands were quantified using the GelPro
program and STAT3 activation in each sample was determined as a
ratio of phosphoSTAT3/total STAT3. Results were graphed as a mean
STAT3 phophorylation per group relative to the vehicle group which
was set at 100%.
Efficacy Studies. CWR22 prostate carcinoma cells or HDLM-2
(classical Hodgkin lymphoma) cells were injected into the flanks of
nude (CWR22) or SCID/NOD mice. After tumors became palpable,
animals were grouped (10 mice/group) and dosed with a vehicle
(PEG400) or 30 mg/kg bid (CWR22) or 30 mg/kg bid and 55 mg/kg
bid (HDLM-2) of compound 29. Tumor volume was measured every
3−4 days.
ASSOCIATED CONTENT
Accession Codes
Atomic coordinates for the cocrystal structure of JAK2 with
inhibitor 28 can be accessed using PDB code 4AQC.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 610-738-6733. Fax: 610-738-6643. E-Mail: bdugan@
cephalon.com.
■
Notes
The authors declare no competing financial interest.
Cell-Based Assay for JAK2. The JAK2 cell-based assay was
performed using a modified GeneBLAzer assay for the CellSensor irf1-
bla TF-1 cell line.23 Cryopreserved cells were suspended at 1.25 × 106
cells/mL in assay media containing modified DMEM/F12 (Invitrogen,
Carlsbad, CA), 0.5% dialyzed FBS, 2 mM glutamine (Invitrogen), 100
U/mL penicillin, and 100 μg/mL streptomycin (Mediatech, Manassas,
VA). The cell suspension (40 μL/well) was dispensed into the 384-
well assay plates (Corning, Corning, NY) and incubated overnight at
5% CO2, 37 °C. Test compound/DMSO (100 nL) was then added to
the assay plates. For the negative control wells, an internal JAK2
reference inhibitor was delivered at a concentration that produced
100% inhibition; positive control wells received only DMSO. Assay
plates were incubated for 1 h prior to adding 5 μL/well of GM-CSF
(Invitrogen) diluted in assay media (1 ng/mL final concentration in
assay, equivalent to its EC80). The plates were then incubated for 5 h
at 5% CO2, 37 °C. Development was initiated by addition of 8 μL
LiveBLAzer FRET β-lactamase loading solution per well (Invitrogen),
and the plate was incubated for 4 h at RT. Detection was performed
on the EnVision 2102 plate reader (PerkinElmer). IC50 values were
determined using the 4-parameter logistic model in XLFit 4 (IDBS,
Ltd.).
ACKNOWLEDGMENTS
■
We thank the following people: Torsten Herbertz and Arup
Ghose for molecular modeling, Kelli Zeigler and Damaris
Rolon-Steele for animal studies, Katrin Przyuski, Daniel Levy,
and Joseph Sclafani for assistance in multigram scale
preparation of intermediates and CEP-33779 (29), and Ulrike
Hars of Crelux GmbH for generating the JAK2/inhibitor
cocrystal structure of 28.
ABBREVIATIONS USED
■
JAK, Janus kinase; STAT, signal transduction and transcription;
TYK, tyrosine kinase; TPP, triphenylphosphine; SCID/NOD,
severe combined immune deficiency/nonobese diabetic; MQL,
minimum quantifiable level
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