Optimization of a novel kinase inhibitor scaffold for the dual inhibition of JAK2 and FAK kinases

Article abstract of DOI:10.1016/j.bmcl.2011.11.049

The elaboration of a novel scaffold for the inhibition of JAK2 and FAK kinases was targeted in order to provide a dual inhibitor that could target divergent pathways for tumor cell progression.

Full text of DOI:10.1016/j.bmcl.2011.11.049

Bioorganic & Medicinal Chemistry Letters 22 (2012) 133–137
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of a novel kinase inhibitor scaffold for the dual inhibition
of JAK2 and FAK kinases
⇑
⇑
Craig A. Zificsak , Diane E. Gingrich , Henry J. Breslin, Derek D. Dunn, Karen L. Milkiewicz, Jay P. Theroff,
Tho V. Thieu, Ted L. Underiner, Linda R. Weinberg, Lisa D. Aimone, Mark S. Albom, Jennifer L. Mason,
Lisa Saville, Jean Husten, Thelma S. Angeles, James P. Finn, Mahfuza Jan, Teresa M. O’Kane,
Pawel Dobrzanski, Bruce D. Dorsey
Worldwide Discovery Research, Cephalon, Inc., 145 Brandywine Parkway, West Chester, PA 19380, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The elaboration of a novel scaffold for the inhibition of JAK2 and FAK kinases was targeted in order to
provide a dual inhibitor that could target divergent pathways for tumor cell progression.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 4 October 2011
Revised 11 November 2011
Accepted 14 November 2011
Available online 25 November 2011
Keywords:
Kinase
JAK2
FAK
Inhibitors
Janus kinase 2 (JAK2) has received a great deal of attention upon
the identification of the JAKV617F mutation in patients with
myeloproliferative neoplasms (MPNs).1 In subsequent years follow-
ing this discovery, the dysregulation of the JAK/signal transducers
and activators of transcription (STAT) signaling pathway has been
implicated in hematopoietic malignancies and solid tumors. Consti-
tutive activation of the JAK/STAT pathway has been detected in a
wide spectrum of human cancers and correlated with a particularly
malignant and metastatic phenotype.2,3 The JAK/STAT signaling
pathway is involved in mechanisms of tumorigenesis including cell
proliferation, survival, angiogenesis, and immune evasion and sur-
veillance. The JAK family of kinases is comprised of four members:
JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2). Several potent JAK
inhibitors have been identified including CEP-701,4 AZD14805 and
INCB-184246 (Ruxolitinib) which is likely to be approved for
myelofibrosis in the US later in 2011.7
would provide a powerful treatment for a wide spectrum of malig-
nancies. Activated FAK promotes tumor cell motility, invasiveness/
metastasis, and angiogenesis while activated JAK2 plays a critical
role in tumor cell proliferation, anti-apoptotic signaling, chemo-
resistance to cytotoxic therapies, and immune evasion. Therefore,
dual FAK–JAK2 inhibition offers the potential for simultaneous
targeting of all major mechanisms of tumor pathogenesis and
progression. The research objective for the team was to optimize
a chemotype for both potent FAK and JAK2 inhibition in order to
provide a more beneficial and effective chemotherapeutic agent.
Cephalon has recently disclosed the synthesis8 and utility of a
novel series of 2-anilino-7-aryl-pyrrolo[2,1-f][1,2,4]triazines as
ALK inhibitors (Fig. 1).9,10 Screening of 1 showed a subnanomolar
IC50 in the JAK2 enzyme assay (IC50 = 0.2 nM) with good potency
in the FAK assay (IC50 = 17 nM) (Table 1). The bioavailable ALK
inhibitor 2 showed equipotent FAK enzymatic activity, with modest
inhibition of JAK2. These results compared well with the known
Novartis inhibitor 4, featuring a closely related heterocyclic system,
previously disclosed to be active in enzymatic assays for both FAK
(IC50 = 200 nM)11 and JAK2 (IC50 = 5.9 nM).12
One strategy for the development of improved treatments in
oncology is the development of kinase inhibitors targeting multi-
ple pathways of tumorigenesis. Coupling JAK2 inhibition with
inhibitory activity toward another kinase such as focal adhesion
kinase (FAK), which plays a role in tumor invasion and metastasis,
Knowing that the o-methoxy group in the aniline at C2 of 2 pro-
vides pan-kinase selectivity13,14 and may be detrimental to the
JAK2 potency, we embarked on an SAR campaign wherein the ani-
line present in 2 was profiled as its desmethoxy congener paired
with a variety of R-groups at C7 (Fig. 1,3). The desmethoxy C2 ani-
line was prepared from 4-phenylpiperidine in three steps
⇑
Corresponding authors. Tel.: +1 610 738 6745; fax: +1 610 738 6643 (C.A.Z.);
tel.: +1 610 738 6591; fax: +1 610 738 6643 (D.E.G.).
E-mail addresses: czificsa@cephalon.com (C.A. Zificsak), dgingric@cephalon.com
(D.E. Gingrich).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.049

134
C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 133–137
OMe
OMe
HN
HN
a
MeO
MeO
MeO
MeO
N
N
N
N
N
N
H
5
6
NO2
N
N
H
N
4
b, c
1
H2N
H2N
N
N
O
O
N
N
NH2
N
OMe
N
7
H
OMe
2
N
N
e
N
d
N
N
N
X
N
S
N
S
N
R
Br
R
H2N
8
10a-q
9a-q
N
O
N
f, g
N
N
N
H
R
H2N
3, R = aryl, heteroaryl
N
O
N
Figure 1. Cephalon pyrrolotriazine analogs and Novarti’s related pyrrolopyrimidine.
N
N
N
H
R
3a-q
Scheme 1. Reagenta and conditions: (a) HNO3, H2SO4, AcOH, 40%; (b) iodoaceta-
mide, Cs2CO3, MeCN, 95%; (c) H2, Pd/C, MeOH, 85%; (d) boronic acid/ester,
Pd(PPh3)4; (e) m-CPBA (X = SO) or H2O2, Na2WO4 (X = SO2); (f) NaOH; (g) PhN(Tf)2
then 7.
Table 1
Screening of initial pyrrolo[2,1-f][1,2,4]triazine leads versus Novartis’ pyrrolopyrim-
idine
a,b
Compd
IC50 (nM)
JAK2 enzyme
FAK enzyme
The electron rich C5 of the pyrrolo[2,1-f][1,2,4]triazine system
was previously shown10 to be a metabolic liability and was
investigated in this series using the 2-(N-methyl)methanesulfon-
amidophenyl substituent at C7. Utilizing the regiomeric bromide
11,8 the C5 bromide was converted (Scheme 2) into either the
methyl analog (via Suzuki coupling) or the chloride (using NiCl2).16
Subsequent halogenation at C7 and Suzuki coupling afforded inter-
mediates 12a–b. Following methylation of the sulfonamide, the
methyl sulfide at C2 was oxidized and displaced to furnish the hy-
droxyl analogs, which were activated for displacement and finally
reacted with aniline 7 to furnish 14a–b.
1
2
4
0.20
250
5.9
17
21
200
a
IC50 values are reported as the average of at least two separate determinations.
Values for 4 are literature values; see Refs. 11 (FAK) and 12 (JAK2).
b
(Scheme 1). Nitration gave a mixture of ortho and para nitration,
but the desired para derivative was selectively crystallized from
cyclohexane. Capping of the piperidine nitrogen with iodoaceta-
mide and hydrogenation of the nitro group furnished the desired
aniline 7.
While C5 methyl analog 14a lost activity in the cellular assays
(Table 3), 14b exhibited slight improvement in both FAK and JAK2
cellular assays. As expected, the stability of 14b in liver microsomes
improved in all four species as compared to 3b (14b: Mouse:
26 min, Rat: 21 min, Dog: 17 min, Human: 11 min vs 3b: Mouse:
15 min, Rat: 6 min, Dog: 11 min, Human: <5 min). Analog 14b
was profiled in Sprague–Dawley rats (Table 4) and exhibited a
desirable profile, with an iv half-life of 1.0 h, moderate distribution
(Vd = 1.3 0.3 L/kg) and measurable oral exposure, resulting in a
bioavailability of 12 2%. Internal selectivity screening showed
14b to be equipotent against JAK3 (enzyme IC50 = 2.9 nM) and
selective against the insulin receptor (enzyme IC50 = 380 nM). Fur-
ther profiling was conducted at Invitrogen; 14b displayed >90%
inhibition against 19 of 53 oncology-related kinases (36%) when
Heterocyclic bromide 88 readily undergoes arylation at C7 when
coupled with a variety of boronic acids or esters to furnish 9a–q.
Oxidation of 9a–q with either m-CPBA or a hydrogen peroxide/so-
dium tungstate system proceeded efficiently to activate C2 for
hydrolysis with aqueous sodium hydroxide from either the sulfox-
ide or sulfone. As previously described,8 in situ formation of the
triflate followed by displacement with aniline 7 gave the targeted
analogs 3a–q.
The enzymatic activity of analogs 3a–q showed a wide range of
aromatic and heteroaromatic substitution that was tolerated for
inhibition of JAK2 (Table 2). In the cellular assay, analog 3b showed
the highest potency (IC50 = 15 nM) with most of the remaining
analogs meeting program criteria of IC50 <100 nM.15 In the FAK
assays, analog 3c, featuring a m-tert-butylsulfonamide, showed
the greatest inhibition (IC50 = 5.7 nM) with analog 3b again show-
screened at 1 l
M.17
A PK/PD experiment was conducted, dosing 14b as a single oral
dose (55 mg/kg) and monitoring pSTAT3 and pFAK phosphoryla-
tion in CWR22 tumor xenografts (Fig. 2). pSTAT3 signaling was
inhibited >75% out to 12 h, with return of signal at 24 h, indicative
of robust inhibition of JAK2. The high plasma protein binding of
14b (99.4% in mouse) is likely contributing to strong inhibition of
pSTAT3 at the 12 h timepoint. The inhibition of pFAK at 2 h was
>75%, but decreased at both the 6—and 12-h timepoints, before
returning to that of vehicle at 24 h. This short inhibition of FAK
ing good potency (IC50 = 14 nM). Derivatives bearing
a basic
heterocyle at C7 (3k–m, 3q) or a basic alkylamine (3o) showed
the least activity, as did fused ring systems (3o, 3p). Inhibition of
FAK in the cellular assay was not as robust in this series as it
was for JAK2. Cellular IC50 values ranged from 610 nM to >10
with analogs 3a, 3b, 3e and 3j showing IC50 values below 1
lM,
lM.

C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 133–137
135
Table 2
H2N
N
Biological data for compounds 3a–k
O
N
N
N
N
H
Ar
a
Compd
Ar
IC50 (nM)
JAK2 enzyme
0.60
JAK2 cell
41
FAK enzyme
55
FAK cell
O
N
3a
3b
690
610
SO2Me
2.0
15
90
14
3c
0.64
5.7
1340
1190
SO2NHt-Bu
3d
0.77
0.22
62
60
39
21
N
OMe
3e
660
SO2Me
N
H
3f
0.09
0.27
45
27
48
33
>10,000
1390
SO2Me
N
3g
OH
3h
3i
0.22
0.52
29
58
114
25
>10,000
1010
SO2Me
SO2Me
N
SO2Me
3j
2.1
16
38
14
750
N
OMe
N
N
N
3k
3l
1140
NT
2040
>10,000
551
>10,000
NT
N
85
3m
8.9
282
NT
N
O
O
F
F
3n
3o
13
>1000
NT
122
NT
NT
2900
>1000
Me2N
O
O
3p
3q
16
NT
205
167
NT
NT
N
S
N
6.3
170
a
IC50 values are reported as the average of at least two separate determinations.

136
C. A. Zificsak et al. / Bioorg. Med. Chem. Lett. 22 (2012) 133–137
R
Br
R
N
N
d, e
N
a, b, c
N
N
N
H
N
X
N
S
N
S
N
N
SO2Me
SO2Me
11
13a-b
f, g
12a-b
H2N
R
N
O
N
N
N
N
H
N
SO2Me
14a-b
Scheme 2. Reagents and conditions: (a) R = Me: trimethylboroxine, Pd(PPh3)4; R = Cl: NiCl2; (b) R = Me: NBS; R = Cl: NIS; (c) boronic acid, Pd(PPh3)4; (d) methyl sulfate;
(e) R = Me: m-CPBA (X = SO); R = Cl: H2O2, Na2WO4 (X = SO2); (f) NaOH; (g) PhN(Tf)2 then 7.
Table 3
Biological data for compounds 3b, 14a–b
H2N
R
N
O
N
N
N
N
H
N
SO2Me
a
Compd
R
IC50 (nM)
JAK2 enzyme
JAK2 cell
FAK enzyme
FAK cell
3b
14a
14b
H
Me
Cl
2.0
3.6
2.5
15
39
12
14
11
5.0
610
1230
420
a
IC50 values are reported as the average of at least two separate determinations.
Table 4
Pharmacokinetic parameters of 14b in Sprague–Dawley ratsa
PK parameters
iv
Dose (mg/kg)
t1/2 (h)
1
1.0
0
AUC0-t (ng h/mL)
Vd (L/kg)
CL (mL/min/kg)
1320 299
1.3 0.3
Figure 2. Inhibition of pFAK and pSTAT3 phosphorylation in CWR22 tumor
xenografts in mice by 14b with a single oral dose (55 mg/kg).
14
4
po
Dose (mg/kg)
Cmax (ng/mL)
tmax (h)
AUC0-1 (ng h/mL)
F%
5
185 24
2.2
832 121
12
1
2
a
Values are the average SEM from three animals.
in vivo is consistent with the cellular potency of 14b
(IC50 = 420 nM) and observed levels in tumor and plasma (Fig. 3).
In summary, progress toward a dual inhibitor of both FAK and
JAK2 was achieved, providing a bioavailable analog that demon-
strated knockdown of pSTAT3 in vivo over 12 h and to a lesser
extent, reduction in FAK phosphorylation. Further refinement of
this or another chemical series is necessary to provide a more
potent dual inhibitor that would be capable of validating the
FAK–JAK2 dual inhibition hypothesis.
Figure 3. PK data for 14b from PK/PD study in mice with a single oral dose
(55 mg/kg).
References and notes
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