Discovery of a JAK1/3 Inhibitor and Use of a Prodrug to Demonstrate Efficacy in a Model of Rheumatoid Arthritis

Article abstract of DOI:10.1021/acsmedchemlett.8b00508

The four members of the Janus family of nonreceptor tyrosine kinases play a significant role in immune function. The JAK family kinase inhibitor, tofacitinib 1, has been approved in the United States for use in rheumatoid arthritis (RA) patients. A number

Full text of DOI:10.1021/acsmedchemlett.8b00508

Letter
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Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Discovery of a JAK1/3 Inhibitor and Use of a Prodrug To
Demonstrate Efficacy in a Model of Rheumatoid Arthritis
Steven H. Spergel,* Michael E. Mertzman, James Kempson, Junqing Guo, Sylwia Stachura,
Lauren Haque, Jonathan S. Lippy, Rosemary F. Zhang, Michael Galella, Sidney Pitt, Guoxiang Shen,
Aberra Fura, Kathleen Gillooly, Kim W. McIntyre, Vicky Tang, John Tokarski, John S. Sack, Javed Khan,
Percy H. Carter, Joel C. Barrish, Steven G. Nadler, Luisa M. Salter-Cid, Gary L. Schieven,
Stephen T. Wrobleski, and William J. Pitts
Research and Development, Bristol-Myers Squibb Company, Route 206 and Provinceline Road, Princeton, New Jersey 08543-4000,
United States
S
* Supporting Information
ABSTRACT: The four members of the Janus family of
nonreceptor tyrosine kinases play a significant role in immune
function. The JAK family kinase inhibitor, tofacitinib 1, has
been approved in the United States for use in rheumatoid
arthritis (RA) patients. A number of JAK inhibitors with a
variety of JAK family selectivity profiles are currently in clinical
trials. Our goal was to identify inhibitors that were functionally
selective for JAK1 and JAK3. Compound 22 was prepared with the desired functional selectivity profile, but it suffered from
poor absorption related to physical properties. Use of the phosphate prodrug 32 enabled progression to a murine collagen
induced arthritis (CIA) model. The demonstration of a robust efficacy in the CIA model suggests that use of phosphate
prodrugs may resolve issues with progressing this chemotype for the treatment of autoimmune diseases such as RA.
KEYWORDS: JAK1, JAK2, JAK3, TYK2, prodrug, mouse CIA model
he Janus Kinase (JAK) family is made up of four
required to obtain potent inhibitors of γc chain cytokine
Tstructurally related kinases, JAK1, JAK2, JAK3, and TYK2 signaling.9 A detailed analysis of the JAK family members Km
for ATP helped explain this observation.10 This limitation
(Tyrosine Kinase-2). The JAK family kinases, through the
toward selectively targeting JAK3 has recently been overcome
by pursuing a covalent inhibition strategy, which has identified
inhibitors that selectively block γc chain cytokine signal-
ing.11−16 PF-06651600 is one such covalent inhibitor that has
advanced to clinical trials. As a result of these considerations
and our own difficulty in identifying potent selective
noncovalent JAK3 inhibitors, we chose to pursue a JAK1/3
selective strategy in the hope of accessing a ‘JAK1 selective
profile’.
Our initial report described the discovery of pyrrolopyr-
idazine-3-carboxamides (PPZ) as potent inhibitors of the JAK
kinase family.17 Subsequently, we discovered that addition of
an aryl or heteroaryl group at the C-6 position significantly
increased JAK family potency. Compound 2 (Figure 1)
demonstrated efficacy in a pseudoestablished mouse colla-
gen-induced arthritis (mouse CIA) model; however, a dose
response was not obtained.18 Further progression of 2 was also
hampered by multiple cardiovascular liabilities, which included
hemodynamic effects attributed to inhibition of Rho kinase
(ROCK1 IC50 = 50 nM) and the potential for QTc
actions of specific cytokines binding to their receptors, are key
drivers of immune system and inflammatory responses.1 Once
these receptors are activated, the kinases phosphorylate
cytokine receptors and subsequently activate signal transducers
and activators of transcription (STATs). The STATs dimerize
and are translocated to the nucleus where they bind to regions
of genes that promote cytokine production leading to a
propagation of inflammatory and immune signals. As a result of
their function, the JAK family kinases have been of significant
interest to the pharmaceutical industry, and various inhibitor
profiles have been explored as possible treatments for
autoimmune disease. Tofacitinib is a pan-JAK family inhibitor
that has been approved for the treatment of rheumatoid
arthritis (RA),2 psoriatic arthritis,3 and ulcerative colitis.4
Baricitinib is a selective JAK1/2 inhibitor that has been
recently approved for the treatment of RA.5 Selectively
targeting JAK1 would be expected to not only inhibit γc
chain cytokine signaling (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21),
but also block signaling from other pro-inflammatory cytokines
such as IL-6 and Type I interferon. Selective inhibitors of JAK1
such as figlotinib,6 upadicitnib,7 and abrocitinib8 are currently
in clinical trials for the treatment of RA and other autoimmune
or inflammatory diseases. Early attempts to identify JAK3
selective inhibitors suggested that JAK1 inhibition was also
Received: October 26, 2018
Accepted: February 7, 2019
© XXXX American Chemical Society
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indication of the level of protein binding and suggested a
minimal concentration to target for PK/PD and in vivo efficacy
studies. Selected compounds were further profiled for cellular
JAK2 activity (SET2 proliferation assay20 and an EPO
phosphoSTAT5 assay) and cellular TYK2 activity (IL23
KIT225 STAT reporter assay).
Initial efforts involved fixing the C-6 substituent as phenyl
while varying the C-4 position using aliphatic amines as
represented by compounds 11−15 (Table 1). These analogs
Figure 1.
a b
,
Table 1. Secondary Alkyl Amines 11−15
prolongation due to significant inhibition of the hERG ion
channel.
Our chemistry library plan centered on utilizing intermediate
6, 6-bromo-4-chloro-pyrrolopyridazine-3-carboxamide, to rap-
idly expand SAR at the 4 and 6 position in this series (Scheme
1). Reaction of 6 with amines provided 7, which could be
1
Scheme 1. Synthetic Strategy for C-4 Diversity
a
Assay protocols are described in the Supporting Information.
Values in table represent n = 1. Synthesized in library format.
b
c
were potent nanomolar inhibitors of the JAK family,
particularly JAK3. However, 11−15 showed poor inhibition
in the IL2 IFNγ hWB assay (IC50 > 4 μM), poor metabolic
stability (<50% remaining after 10 min incubation with mouse
microsomes), and poor aqueous solubility (<1 μg/mL). The
overall lipophilicity of the molecules (cLogP 2.9−3.7, LogP 12
= 3.94, 13 = 5.08) was suspected to be a contributing factor to
the observed liabilities.
Subsequent efforts focused on reducing lipophilicity and
improving solubility within the series,21,22 with the hope of
improving whole blood potency, by incorporating a more polar
4-methoxy-3-pyridyl group at C-6 and exploring amino alcohol
side chains at C-4 (Table 2). Given the potent hERG activity
of 2, we avoided incorporation of basic amines as part of this
SAR effort. Compounds 16, 17, 19, 21, and 22 were potent
inhibitors of JAK3 and gratifyingly showed submicromolar
potency in the IL-2 driven T-cell proliferation assay (IL-2 T-
cell) with the more potent c-propyldiol diastereomer 19
1
Experimental details described in the Supporting Information.
Scheme conditions: (a) (i) NaH, DMF; (ii) Chloramine solution in
MTBE (90%); (b) (i) 3,3-diethyoxypropionitrile, IPA, 85 °C; (ii)
DBU, DCE, 85 °C (c) POCl3, 75 °C. 29%, 2 steps); (d) Conc.
H2SO4, 55 °C (89%); (e) R-NH2; (f) Ar−B(OH)2/Suzuki
conditions; (g) EtSH, K2CO3, DMF, rt, 18 h (65%); (h) ArB(OH)2,
X-Phos, Pd(OAc)2, 2 M K3PO4, dioxane 125 °C ; (i) oxone, acetone,
water; (j) R-NH2, DMF, 85 °C.
further elaborated at C-6 via a Suzuki-Miyaura coupling
procedure. However, when we attempted to conduct the
Suzuki-Miyaura coupling as the first step on 6, we obtained
undesired 8 as the major product. To circumvent this
regiochemical outcome, 6 was reacted with ethanethiol to
provide the more versatile C-6 ethylsulfide intermediate, which
enabled regio-controlled installation of the aryl group at C-6
followed by oxidation of the sulfide with Oxone to afford 9a
and 9b. Reaction of 9a and 9b with amines then provided the
desired analog 10.
displaying submicromolar potency in IL2 INFγ hWB (IC50
=
780 nM). Truncating the alkyl side chain in 20 resulted in a
significant decrease in JAK family potency. We postulated that
the cyclopropyl group might be projecting into the tofacitinib
“methyl pocket”.23
Compounds were evaluated in JAK1, JAK2, JAK3, and
TYK2 enzymatic assays and an IL-2 dependent T-cell
proliferation assay.19 Compounds of interest were also
evaluated in a human whole blood IL-2 driven IFNγ
production assay (IL-2 IFNγ hWB), which provided an
Proceeding on this assumption we decided to explore a
change of the cyclopropyl group to a methyl group to provide
21 and 22, both of which were in the same potency range as
the cyclopropyl analog 19, and significantly more potent than
20. All four possible diol isomers were prepared and 22,
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a c
,
Table 2. C-4 Amino Alcohol Derivatives
a
b
c
Assay protocols are described in the Supporting Information. Synthesized in library format. Values in table represent n = 1 unless otherwise
d
e
noted. T-cell proliferation assay. Octanol−water (pH 6.5) HPLC determination.
derived from the natural amino acid, threonine, proved to be
the most potent against JAK1/3, and more importantly the
most potent in the human whole blood assay. On examination
of the JAK3 inhibitor complex with 22, we were somewhat
surprised to see that the methyl group of the secondary alcohol
did not fully occupy the “methyl pocket” observed in the
tofacitinib JAK3 crystal structure and the pocket was altered
due to a change in the orientation of Leu956 (see Figure 2).
Additionally we noticed the primary alcohol formed a
hydrogen bond with the glycine rich P-loop (Leu828). It is
interesting to note that diasterioisomers 23 and 25 are
significantly less potent than 22. The secondary alcohol of 22
did not appear to make any specific interaction with the
protein; however, other crystal structures of JAK3 (e.g., 3LXK)
showed the existence of a water network in this region of the
Figure 2. Crystal structure of compound 22 bound to the kinase
protein.
Compound 22 was chosen for additional profiling. 22
catalytic domain of JAK3. The carbon atoms are colored green/
magenta, oxygens are colored red, and nitrogens are colored blue.
Hydrogen bonds are indicated with dashed lines.24
demonstrated reduced hERG channel activity compared to 2
(>10 fold improvement) and was deemed to have an
acceptable kinome profile. Additionally, 22 was stable in
human, mouse, and rat liver microsomes and was permeable in
the PAMPA assay. On the basis of this profile, 22 was
advanced to an oral mouse PK study. A 10 mg/kg oral dose of
22 (formulated with 5% EtOH, 5% TPGS, 90% PEG 300)
provided a plasma Cmax of >3 μM and a time above the IL2-
IFNγ mouseWB IC50 of about 3 h. This appeared to be a
promising result, although we wanted to explore higher dose
PK to allow for greater exposure during the dosing period.
After resynthesis of 22, we planned a PK study at doses of 25
and 50 mg/kg. This new lot failed to dissolve in the original
formulation (even at 10 mg/kg dosing concentration). A
number of formulation options were explored, but none was
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found to be satisfactory. The new lot was found to be
crystalline and exhibited poor solubility (<1 μg/mL in 50 mM
phosphate buffer). The original amorphous lot was equally
insoluble in water. On the basis of these observations, we
explored spray dried dispersion but were unable to prevent
crystallization at higher drug loads in the matrix. Ultimately we
resorted to a microsuspension formulation at a 50 mg/kg oral
dose; however, the plasma Cmax for this study was only 0.1 μM.
A single crystal X-ray structure of 22 shows that the compound
forms a stable crystal lattice due to an extensive intermolecular
hydrogen bonding matrix between neighbor molecules within
the unit cell.25 This observation is also supported by a higher
melting point for the dihydroxy analog 22 (280 °C dec) versus
the t-butylmethyl analog 14 (204 °C dec).
and the higher dose was selected to cover the whole blood IC50
at trough over a 24 h period (mouse IL-15 WB IC50= 810
nM).26 In addition, 1 was used as a positive control and was
dosed to cover the whole blood IC50 for 50% of the time. This
level of coverage was targeted to approach the exposure
observed at the clinical dose of 10 mg BID in humans.27 The
lower dose of 32 demonstrated comparable efficacy to 1, and
we were gratified to observe increased efficacy at the high dose
of 32, which demonstrated that the phosphate prodrug strategy
successfully enabled dose escalation (see Figure 3).
Next we explored the use of a solubilizing prodrug of 22 by
preparing and evaluating a phosphate of the primary alcohol
(Scheme 2). Benzyl protected Boc-L-threonine (26) was
Scheme 2. Synthesis of Phosphate Prodrug (32)
Figure 3. Pseudo-Established Mouse Collagen-Induced Arthritis
Study with 32.* All compounds were dosed orally BID.
Drug levels of the parent 22 after dosing of 32 were close to
the initial projections discussed above with the high dose
achieving greater than 90% coverage of the whole blood IC50
over 24 h. This result clearly demonstrated the utility of a
phosphate prodrug strategy to overcome solubility issues
associated with the highly crystalline form of the parent.
In summary, we were able to identify novel pyrrolopyr-
idazine diol 22 as a potent inhibitor of JAK1 and JAK3. The
compound displayed poor PK properties, which were largely
attributed to poor kinetic solubility of a highly crystalline form.
However, this limitation was overcome by use of a phosphate
prodrug. Prodrug 32 displayed robust dose dependent efficacy
in a mouse CIA model, which demonstrated preclinically that a
JAK1/3 inhibitor profile may have utility in the treatment of
autoimmune disease.
Conditions: (a) LAH, Et2O, 0 °C (90%); (b) HCl, dioxane, rt (99%);
(c) 6, Hunig’s base, DMA, 100 °C (95%); (d) PhB(OH)2, X-Phos,
Pd(OAc)2, 2 M K3PO4, 100 °C (73%.); (e) (i) dibenzyl
diisopropylphosphoramidite, tetrazole, DCM/THF, 0 °C; (ii) 30%
H2O2, 0 °C (73%); (f) (i) H2, Pd/C, EtOH (74%); (ii) NaHCO3,
MeOH/H2O (99%).
reduced to the corresponding alcohol, 27, with LAH in 90%
yield. Treatment with anhydrous HCl afforded the amino-
alcohol hydrochloride, 28, which was treated with 6 and
Hunig’s base at elevated temperature in DMA to afford 29 in a
95% yield over two steps. Suzuki-Miyaura reaction of 29 with
phenylboronic acid afforded 30 in 73% yield. Phosphorylation
with dibenzyl diisopropylphosphoramidite and hydrogen
peroxide, followed by deprotection and salt formation, afforded
the disodium salt of the phosphate 32 in good yield over three
steps.
The phosphate prodrug (32) proved to be freely soluble in
water (>1 mg/mL). We progressed the prodrug to PK and
found that exposure of the parent was dose proportional (10
mg/kg, AUC ∼1 μM h; 40 mg/kg, AUC ∼6 μM h). On the
basis of this promising result, we designed a mouse collagen
induced arthritis study where the low dose was selected to
cover the whole blood IC50 at trough for 67% of a 24 h period,
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00508.
■
S
Synthetic procedures and characterization data for
compounds 4−32, in vitro profile of 22, kinome
selectivity, biological methods, efficacy study terminal
bleed data (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: steven.spergel@bms.com. Phone: 215-206-1460.
■
D
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contribution of individual JAK kinase isoforms to cellular signaling.
ACS Chem. Biol. 2014, 9, 1552.
ORCID
Steven H. Spergel: 0000-0002-5190-3942
James Kempson: 0000-0002-9540-3886
Percy H. Carter: 0000-0002-5880-1164
(11) Tan, L.; Akahane, K.; McNally, R.; Reyskens, K. M. S. E.;
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Labella, K.; Johannessen, L.; Akbay, E. A.; Wong, K.-K.; Frank, D. A.;
Marto, J. A.; Look, T. A.; Arthur, S. C.; Eck, M. J.; Gray, N. S.
Development of selective covalent Janus Kinase 3 inhibitors. J. Med.
Chem. 2015, 58, 6589.
Notes
The authors declare no competing financial interest.
(12) Goedken, E. R.; Argiriadi, M. A.; Banach, D. L.; Fiamengo, B.
A.; Foley, S. E.; Frank, K. E.; George, J. S.; Harris, C. M.; Hobson, A.
D.; Ihle, D. C.; Marcotte, D.; Merta, P. J.; Michalak, M. E.; Murdock,
S. E.; Tomlinson, M. J.; Voss, J. W. Tricyclic covalent inhibitors
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Symanowicz, P.; Hegen, M.; Banker, M. E.; Vincent, F.; Unwalla,
R.; Liang, S.; Gilbert, A. M.; Brown, M. F.; Hayward, M.;
Montgomery, J.; Yang, X.; Bauman, J.; Trujillo, J. I.; Casimiro-
Garcia, A.; Vajdos, F. F.; Leung, L.; Geoghegan, K. F.; Quazi, A.;
Xuan, D.; Jones, L.; Hett, E.; Wright, K.; Clark, J. D.; Thorarensen, A.;
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Bladd, Y.; Chaudhry, C.; Salter-Cid, L. M.; fura, A.; Barrish, J. C.;
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S.; Sack, J. S.; Khan, J.; Lippy, J. S.; Zhang, R. F.; Pitt, S.; Shen, G.;
Pitts, W. J.; Carter, P. H.; Barrish, J. C.; Nadler, S. G.; Salter-Cid, L.
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(19) See Supporting Information for in vitro assay descriptions and
profiling data and in vivo model description for compound 22.
(20) Quentmeier, H.; MacLeod, R. A. F.; Zaborski, M.; Drexler, H.
G. JAK2 V617F tyrosine kinase mutation in cell lines derived from
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inhibitors approved by the FDA from 2000 to 2011: a systematic
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2013, 9, 1597.
ACKNOWLEDGMENTS
■
We gratefully acknowledge our colleagues at BBRC and
Richard A. Rampulla for the scale-up synthesis of 6-bromo-4-
chloropyrrolo[1,2-b]pyridazine-3-carboxamide and our col-
leagues in the Lead Evaluation group for their assistance
during this SAR study.
ABBREVIATIONS
■
JAK1, Janus family kinase 1; JAK2, Janus family kinase 2;
JAK3, Janus family kinase 3; TYK2, tyrosine kinase 2; IL-2,
interleukin 2; IL-4, interleukin 4; IL-6, interleukin 6; IL-7,
interleukin 7; IL-9, interleukin 9; IL-15, interleukin 15; IL-21,
interleukin 21; ATP, adenosine triphosphate; PK, pharmaco-
kinetics; hERG, human ether-a-go-go-related gene; PAMPA,
parallel artificial membrane permeability assay; SAR, structure
activity relationship; GCK, germinal center kinase; GLK,
germinal center kinase like kinase; ROCK, rho associated
protein kinase
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