Thiophene carboxamide inhibitors of JAK2 as potential treatments for myleoproliferative neoplasms

Article abstract of DOI:10.1016/j.bmcl.2014.02.064

A series of carboxamide-substituted thiophenes demonstrating inhibition of JAK2 is described. Development of this chemical series began with the bioisosteric replacement of a urea substituent by a pyridyl ring. Issues of chemical and metabolic stability were solved using the results of both in vitro and in vivo studies, ultimately delivering compounds such as 24 and 25 that performed well in an acute PK/PD model measuring p-STAT5 inhibition.

Full text of DOI:10.1016/j.bmcl.2014.02.064

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1968–1973
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thiophene carboxamide inhibitors of JAK2 as potential treatments
for myleoproliferative neoplasms
a
a
b
c
Andrew M. Haidle a, , Anna A. Zabierek , Kaleen K. Childers , Craig Rosenstein , Anjili Mathur ,
Michael D. Altman a, Grace Chan b, Lin Xu d, Eric Bachman c, Jan-Rung Moc, Melaney Bouthillette e,
Thomas Rush a, Paul Tempest a, C. Gary Marshall f, Jonathan R. Young a
⇑
a Department of Chemistry, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
b Department of In Vitro Sciences, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
c Department of Pharmacology, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
d Department of Drug Metabolism, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
e Department of Basic Pharmaceutical Sciences, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
f Department of Oncology, Merck & Co., 33 Avenue Louis Pasteur, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of carboxamide-substituted thiophenes demonstrating inhibition of JAK2 is described. Develop-
ment of this chemical series began with the bioisosteric replacement of a urea substituent by a pyridyl
ring. Issues of chemical and metabolic stability were solved using the results of both in vitro and
in vivo studies, ultimately delivering compounds such as 24 and 25 that performed well in an acute
PK/PD model measuring p-STAT5 inhibition.
Received 13 January 2014
Revised 20 February 2014
Accepted 24 February 2014
Available online 4 March 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
JAK2
Polycythemia vera
Bioisostere
Metabolite identification
Thiophene
Protein kinase
Inhibitor
Over the past decade, the Janus kinase (JAK) family members
(JAK1, JAK2, JAK3, and TYK2) have been the focus of a large amount
of research due to their crucial cytokine-mediated signaling roles
that affect both the immune system and hematopoiesis.1 The JAKs
are cytoplasmic kinases that associate themselves with cytokine
receptors, and upon receiving a signal from specific cytokines they
phosphorylate transcription factors called STAT (Signal Transducer
and Activator of Transcription) proteins that in turn migrate to the
nucleus and influence gene transcription. Aberrant JAK–STAT sig-
naling has been linked to many myleoproliferative neoplasms
(MPNs), which include diseases like polycythemia vera (PV), essen-
tial thrombocythemia (ET), and primary myelofibrosis (PMF). In
2005, it was found that JAK2 mutations were present in the major-
ity (>95%) of PV patients, further boosting interest in developing
JAK inhibitors for this disease.2,3
INCB0184244) in 2011 for the treatment of intermediate- and
high-risk myelofibrosis (primary and post-PV/ET). JakafiÒ is a
JAK1/JAK2 dual inhibitor, and it is still an open question whether
a more selective JAK2 inhibitor might be a better treatment for
PV patients since it would avoid interacting with the JAK1-medi-
ated inflammatory cytokine signaling cascade. Medicinal chemis-
try programs at Merck and other companies/institutions5 have
sought to develop JAK2-selective inhibitors to answer this
question.
When our JAK2 inhibitor program began, it was believed that
JAK3, not JAK1, was the family member that posed the biggest risk
in terms of detrimental off-target effects. Mutations in the JAK3
gene can cause the severe combined immunodeficiency (SCID)
phenotype featuring nearly absent T cells and non-functional B
cells.6 Thus, our efforts began with a high throughput screening
campaign that delivered multiple compounds with a kinase selec-
tivity profile like 1, i.e. selective for JAK2 over JAK3 (JAK3 served as
our main selectivity counterscreen within the JAK family).
Although 1 had several hit-like properties (e.g. ligand efficiency
of 0.55),7 further optimization towards an oral drug could have
A major milestone in targeting the JAK family to treat MPNs was
achieved through the FDA’s approval of JakafiÒ (ruxolitinib,
⇑
Corresponding author. Tel.: +1 617 992 2389.
E-mail address: andrew_haidle@merck.com (A.M. Haidle).
http://dx.doi.org/10.1016/j.bmcl.2014.02.064
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

A. M. Haidle et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1968–1973
1969
been hindered by the high number of hydrogen bond donors
(HBD = 5) present in the initial hit. Viewed another way, 1 has a po-
lar surface area (PSA) of 105 Å2, which doesn’t allow for much addi-
tional polarity to be added to the compound while staying in the
range of polarity exhibited by most orally bioavailable drugs.8 In
order to further explore this chemical class, alternatives to the urea
moiety were considered. Scientists at Pfizer had recently published
on a class of isothiazole TrkA inhibitors in which they were able to
replace a urea involved in kinase hinge binding with various het-
erocycles.9 Performing this bioisosteric replacement led to the 2-
pyridyl scaffold 2, which was nearly equipotent and maintained
the selectivity over JAK3 while greatly reducing the PSA to 69 Å2
and dropping the HBD count to 3 (Fig. 1). Other aryl groups that
did not feature a ring nitrogen adjacent to the linking amino group
were less potent in the JAK2 assay (e.g. analogs 3–5), and it was
hypothesized that the sulfur-nitrogen interaction keeping the thi-
ophene and pyridyl ring of 2 in the same plane is responsible for
this preference;10,11 it has been previously observed that a high de-
gree of planarity is beneficial for binding to the JAK family
enzymes,12 and the narrow binding pocket seen in the JAK2 crystal
structure provides a basis for this preference.13
To access 2-amino-3-carboxamide-2,5-substituted thiophenes
such as 2, two main synthetic strategies were used; each was sui-
ted to examining the SAR of a particular region of the molecule. To
investigate the aryl group (Ar2) appended to the thiophene 2-ami-
no substituent, access to the core with appropriate handles for sub-
stitution was achieved through two methods—a functionalization
of 2-aminothiophene-3-carboxamide or a de novo thiophene syn-
thesis via the Gewald reaction14 (Scheme 1). The Ar2 group in both
cases was appended via Pd-catalyzed cross coupling. The choice of
solvent for this reaction was very important as it greatly affected
how much the amide nitrogen participated in this reaction; we
found that 1,4-dioxane promoted reactivity at both the aniline
and amide nitrogens, while switching to tert-butanol or tert-amyl
alcohol (to avoid the freezing of tert-butanol at ambient tempera-
tures) gave the desired aniline coupling product in high yields.
Similar reaction condition-dependent variation in regiochemical
preference in a similar ortho amide/aniline system had been ob-
served previously by Buchwald and co-workers, although they var-
ied the catalytic species as well as the solvent.15 Compounds
designed for exploration of the thiophene’s 5 position (Ar1) were
made by performing this selective heteroaniline arylation first
and then subjecting the product to a halogenation/Suzuki cross
coupling sequence (Scheme 2).
Scheme 1. Synthetic routes used to explore 2-amino substituents (Ar2). Reagents
and conditions: (a) CbzCl, DIPEA, THF; (b) N-Iodosuccinimide, CH2Cl2; (c) R-B(OR)2,
PdCl2(PPh3)2, Na2CO3, 1,2-dimethoxyethane/water, 100 °C; (d) Pd/C, H2, ethanol,
80 °C; (e) S8, morpholine, ethanol, 70 °C; (f) 2-halo pyridine, Pd2(dba)3, X-Phos,
K2CO3, t-amyl alcohol, 100 °C.
Upon reverse-phase HPLC purification using trifluoroacetic acid
in the solvents or attempting to form HCl salts with only a slight
excess (3 equiv) of acid, a ring closure was observed in which the
pyridyl nitrogen attacked the amide carbonyl, ultimately eliminat-
ing ammonia and forming a fused tricycle (Scheme 3). This issue
was resolved by optimizing for both JAK2 potency and compound
stability upon incubation in an aqueous pH 2 buffer solution (with
DMSO present to solubilize the substrate); this simple assay was
designed as a rough mimic of conditions found in human stom-
achs. Two independent solutions arose from this screening effort:
either a substituent (methyl or larger) at the 6 position of the pyr-
idyl ring (e.g. compounds 6 and 13) or an electron withdrawing
group at the 5 position (e.g. compounds 8 and 12) gave molecules
in this series the necessary stability for oral dosing (Table 1). Be-
cause the latter strategy was limited in the types of functional
groups that could be employed, these groups typically possess a
Although the 2-pyridyl ring was optimal in terms of potency, it
was soon discovered that these compounds were not stable upon
treatment with acid under fairly mild and standard conditions.
Figure 1. Attempted bioisosteric replacement of a urea with aromatic rings.

1970
A. M. Haidle et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1968–1973
Table 1
The effect of pyridyl substituents on potency and chemical stability
Compound
R
JAK2 IC50 (nM)
65
t1/2 @ PH 2
2
<1 h
6
7
275
398
>18 h
<1 h
Scheme 2. Synthetic route used to explore phenyl substituents (Ar1). Reagents and
conditions: (a) 2-halo pyridine, Pd2(dba)3, X-Phos, K2CO3, t-amyl alcohol, 100 °C; (b)
N-Iodosuccinimide, CH2Cl2/DMF; (c) R-B(OR)2, PdCl2(PPh3)2, Na2CO3, 1,2-dime-
thoxyethane/water, 100 °C.
8
9
146
161
>18 h
1 h
Scheme 3. Acidic conditions promote a self-condensation reaction.
10
11
227
178
<1 h
<1 h
large amount of PSA, and the acceptable functionality was gener-
ally fairly planar, most of the analogs of the Ar2 ring made subse-
quently relied on the former strategy, that is encumbering the 6
position to avoid the cyclization event. This allowed for the use
of groups known to impart solubility (basic amines, ethereal side
chains) to otherwise planar molecules (vide infra).
With the chemical stability issue under control, our attention
turned to metabolic stability as it had been discovered that the
parent compounds were also rapidly metabolized. A metabolite
identification study performed with rat hepatocytes demonstrated
the major metabolic event was an oxidative modification of the
phenyl ring at the 5-position of the thiophene (Fig. 2). In response
to these data, a library of thiophene carboxamides with substituted
phenyl rings (as well as some heterocyclic replacements for the
phenyl, compounds not shown) was generated and then these
compounds were tested for microsomal stability, which should
capture the phase I metabolism seen in the metabolite identifica-
tion study (Table 2). Halogen substituents on the phenyl ring had
a pronounced effect on both JAK2 potency and microsomal stabil-
ity; unfortunately, the most potent substituents were also the least
stable. A general trend that emerged was placing a fluorine or chlo-
rine atom in the ortho position hurts stability while placing a sub-
stituent in the para position was beneficial.
12
13
16
>18 h
>18 h
891
This microsomal screening effort was done in parallel with
in vivo rat PK screening in order to build an in vitro-in vivo corre-
lation. A breakthrough came when the rat PK properties of 14 and
23 were compared: addition of the benzylic morpholine, originally
done to increase solubility as discussed above, gave a 4 fold in-
crease in exposure (Table 3). Since the microsomal data showed
the ortho fluoro was one of the worst substituents on the phenyl
ring for metabolic stability, it was hypothesized that switching to
some of the preferred substituents would afford compounds with
even more favorable pharmacokinetic properties. SAR exploration
had shown that a tertiary alcohol at the para position of Ar1 was
Figure 2. Metabolic ID experiments on prototypical compound 2.
tolerated in terms of potency, and as seen in Table 2, para substi-
tution was relatively beneficial for microsomal stability. Moreover,
the tertiary alcohol’s higher polarity relative to substituents like
trifluoromethyl helped in general to decrease the logD to a more

A. M. Haidle et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1968–1973
1971
Table 2
The effect of Ar1 phenyl substituents on potency and microsomal stability (rat and human)
Compound
R
JAK2 IC50 (nM)
270
RKM t1/2 (min)
HLM t1/2 (min)
6
8
24
19
14
36
2
15
16
17
155
140
178
13
29
23
32
39
24
18
19
20
80
5
17
11
33
93
3
312
19
21
22
690
380
29
53
36
61
needed; 23 exhibited strong hERG channel inhibition in vitro17
(IC50 = 161 nM), and the superior analog 24’s IC50 value of 1410
nM was still deemed too low relative to the JAK2 potency. To in-
crease this window, efforts to both lower the on-target and raise
the off-target potency were pursued. Through the addition of fluo-
rines to Ar1 (JAK2 potency increase) and replacing the morpholine
with an ethereal chain (decreasing hERG inhibition),18 an accept-
able difference (>1000Â) in these values was achieved with com-
pound 25 (Table 4).
Table 3
Addition of a benzylic morpholine improves pharmacokinetic properties
To exemplify how these thiophene-based JAK2 inhibitors might
bind to the enzyme, compound 25 was docked into a JAK2 crystal
structure (Fig. 3).19 As discussed previously, the sulfur of the thio-
phene and nitrogen of the pyridine were constrained to be copla-
nar during docking, providing a fairly flat surface for binding
near the hinge. Both the primary amide N–H and O as well as the
biaryl nitrogen’s N–H are making critical interactions with the
hinge backbone residues Glu 930 and Leu 932. The tertiary hydro-
xyl appears to be able to hydrogen bond with Asn 981 or alterna-
tively to Asp 994, while the gem-dimethyl group can pack under
the glycine-rich loop. The ethereal side chain is predicted to project
out toward solvent, although its exact location is unknown; the
docking experiments revealed many reasonable alternatives, and
the interaction with Gln 853 shown in Figure 3 is just one possibil-
JAK2 (IC50
Rat CIp
Rat AUC (Norm)
Rat VD
Rat t1/2
PSA
)
36 nM
181 mL/min/kg
34 nM
38 mL/min/kg
1.05 lM h kg/mg
4.7 L/kg
3.5 h
85 Å2
3
0.28
lM h kg/mg
11.8 L/kg
0.9 h
68 Å2
3
H-bond donors
drug-like level,16 leading to compound 24 that had greatly im-
proved pharmacokinetic properties. Further optimization in other
respects, chiefly on-target potency and hERG inhibition, was also

1972
A. M. Haidle et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1968–1973
Table 4
Comparison of two thiophene-based JAK2 inhibitors with acceptable pharmacokinetic
parameters
JAK2 (IC50
JAK3 (IC50
JAK2 pSTAT5 cell (IC50
HPLC logD pH 7.4
Rat CIp
)
)
20 nM
7.5 nM
763 nM
90 nM
1350 nM
520 nM
2.2
)
2.6
1.3 mL/min/kg
4.5 mL/min/kg
Rat AUC (Norm)
Rat VD
Rat t1/2
Rat %F
PSA
28
l
M h kg/mg
7.6 lM h kg/mg
1.1 L/kg
10.3 h
72%
3.3 L/kg
5.3 h
100%
117 Å2
5
105 Å2
4
HBD
hERG inhibition (IC50
)
1.4
lM
12.0 lM
Figure 4. Results for compounds (a) 24 and (b) 25 tested in an acute PK/PD model.
Numbers in red on the bar graph represent plasma concentrations at a given time
point (lM).
Figure 3. Docking model of 25 with the JAK2 enzyme.
ity. This flexibility correlates with the permissive SAR observed in
this region of the molecule, which was useful for the fine-tuning of
physical properties in this series.
Based on the favorable potency and pharmacokinetic profiles of
compounds 24 and 25, including reasonable activity in our cell-
based JAK2 assay that monitors for inhibition of STAT5 phosphor-
ylation,20 these two molecules were further profiled. An acute PK/
PD mouse model,21 in which the inhibition of STAT5 phosphoryla-
tion serves as a read-out, was used as a filter for compounds before
they were progressed into either a chronic wild-type mouse model
of PCV or a more intensive JAK2V617F genetic knock-in mouse
model.22 The acute model involves treatment of mice with a high
dose of recombinant erythropoietin (AranespÒ) to induce many
of the clinical features seen in polycythemia vera such as elevated
hematocrit, splenomegaly, and elevated levels of phosphorylated
STAT5 (pSTAT5). The results of interrogating the acute model with
compounds 24 and 25 are shown in Figure 4. Both compounds
Figure 5. Radar plot indicating the in vitro kinase selectivity of compound 25. The
dotted circles represent inhibitor concentrations at 10-fold intervals above the JAK2
IC50
.
more potent than 24 (IC50 = 5.4 lM) and showed a longer lasting
effect. The discrepancy between the difference in their cell poten-
cies (ꢀ6-fold) and in vivo potencies (ꢀ3-fold) may be related to dif-
ferences in the free drug concentrations; although the mouse
plasma protein binding for these compounds was not measured,
the higher logD of compound 25 and lack of a basic amine implies
that it has a lower free fraction in vivo. In addition to sufficient
demonstrated in vivo activity; however, 25 (IC50 = 2.0 lM) was

A. M. Haidle et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1968–1973
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kinases, including the JAK and Src family kinases; against an
in vitro panel of 100 kinases, it showed 100% selectivity at 10-fold
the JAK2 IC50 concentration, 97% selectivity at 30-fold, and 86%
selectivity at 100-fold (Fig. 5).23 Thus 25 was further progressed
as a compound of interest for the program (results not shown).
In conclusion, we have described the discovery of novel and
selective trisubstituted thiophene inhibitors of the JAK2 enzyme
for the treatment of polycythemia vera. The urea of HTS hit 1
was rapidly replaced with a pyridyl substituent in an effort to re-
duce the PSA/HBD count of this series. This ring system was then
held constant as physical and metabolic stability was optimized
using an array of in vitro and in vivo assays. This effort eventually
delivered compounds such as 25 that had excellent pharmacoki-
netic properties and showed PK/PD modulation of the JAK2 path-
way in vivo.
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19. First, an ensemble of low-energy conformations for 22 was generated using the
MCMM torsional sampling algorithm within MacroModel (BatchMin v9.7,
Schrödinger, LLC). Planarity of the core was enforced by constraining the
dihedral angles between the thiophene and pyridine rings as well as between
the thiophene and amide during the search. Conformations retained were
within 5 kcal/mol of the identified global minimum. Next, these conformations
were rigidly docked into the ATP site of a JAK2 crystal structure (PDB 2B7A,13
prepared for calculation using the PrepWizard functionality within MAESTRO
v8.5, Schrödinger, LLC) using GLIDE v55110 (Schrödinger, LLC) and the SP
scoring function (all other settings at default), requiring hydrogen bonds to be
made to the backbone of Glu 930 and Leu 932. The resulting poses were
subsequently filtered by visual inspection.
20. Detection of STAT phosphorylation was performed using AlphaScreen™
SureFire™ p-STAT5 assay (Perkin–Elmer and TGR Biosciences) using both
biotinylated anti-phospho-STAT5 antibody, which is captured by Streptavidin-
coated donor beads, and anti-total STAT5 antibody, which is captured by
Protein A conjugated acceptor beads. IC50 values are reported as the averages of
at least two independent determinations; standard deviations are within 25–
50% of IC50 values. For further details see: WO2008156726.
21. Mathur, A.; Mo, J.; Kraus, M.; O’Hare, E.; Sinclair, P.; Young, J.; Zhao, S.; Wang,
Y.; Kopinja, J.; Qu, X.; Reilly, J.; Walker, D.; Xu, L.; Aleksandrowicz, D.; Marshall,
G.; Scott, M. L.; Kohl, N. E.; Bachman, E. Biochem. Pharmacol. 2009, 78, 382.
22. Kraus, M.; Wang, Y.; Aleksandrowicz, D.; Bachman, E.; Szewczak, A. A.; Walker,
D.; Xu, L.; Bouthillette, M.; Childers, K. M.; Dolinski, B.; Haidle, A. M.; Kopinja,
J.; Lee, L.; Lim, L.; Little, K. D.; Ma, Y.; Mathur, A.; Mo, J.; O’Hare, E.; Otte, R. D.;
Taoka, B. M.; Wang, W.; Hong, Y.; Zabierek, A. A.; Zhang, W.; Zhao, S.; Zhu, J.;
Young, J.; Marshall, C. G. PLoS ONE 2012, 7, e37207.
23. Kinases with a <30-fold potency difference relative to JAK2 in the kinase panel:
JAK1, SRC, and TYK2; additional kinases with a <100-fold potency difference
relative to JAK2 in the kinase panel: ABL1, CSF1R, DAPK1, FLT3, HCK, JAK3, KDR,
LCK, NTRK1, NTRK3, and NAUK1.