Identification of N-{cis-3-[Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (PF-04965842): A Selective JAK1 Clinical Candidate for the Treatment of Autoimmune Diseases

Article abstract of DOI:10.1021/acs.jmedchem.7b01598

Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.

Full text of DOI:10.1021/acs.jmedchem.7b01598

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Article
Identification of N-{cis-3-[Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-
yl)amino]cyclobutyl}propane-1-sulfonamide (PF-04965842): A Selective
JAK1 Clinical Candidate for the Treatment of Autoimmune Diseases
Michael L Vazquez, Neelu Kaila, Joseph W Strohbach, John D Trzupek, Matthew F. Brown, Mark E.
Flanagan, Mark J MItton-Fry, Timothy A Johnson, Ruth E TenBrink, Eric P Arnold, Arindrajit Basak,
Steven E Heasley, Soojin Kwon, Jonathan Langille, Mihir D. Parikh, Sarah H Griffin, Jeffrey M. Casavant,
Brian A Duclos, Ashley E Fenwick, Thomas M Harris, Seungil Han, Nicole L. Caspers, Martin E.
Dowty, Xin Yang, Mary Ellen Banker, Martin Hegen, Peter T. Symanowicz, Li Li, Lu Wang, Tsung
H. Lin, Jason Jussif, James D Clark, Jean-Baptiste Telliez, Ralph P. Robinson, and Ray Unwalla
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 03 Jan 2018
Downloaded from http://pubs.acs.org on January 3, 2018
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Robinson, Ralph; Pfizer Inc, PGRD
Unwalla, Ray; Pfizer Research and Development, World wide Medicinal
Chemistry
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Identification of Nꢀ{cisꢀ3ꢀ[Methyl(7Hꢀpyrrolo[2,3ꢀd]pyrimidinꢀ4ꢀ
yl)amino]cyclobutyl}propaneꢀ1ꢀsulfonamide (PFꢀ04965842): A Selective JAK1 Clinical
Candidate for the Treatment of Autoimmune Diseases
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Michael L. Vazquez , Neelu Kaila , Joseph W. Strohbach , John D. Trzupek , Matthew F.
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Brown , Mark E. Flanagan , Mark J. MittonꢀFry , Timothy A. Johnson, Ruth E. TenBrink, Eric
‡
‡
‡
‡
P. Arnold , Arindrajit Basak, Steven E. Heasley , Soojin Kwon , Jonathan Langille , Mihir D.
‡
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‡
Parikh , Sarah H. Griffin , Jeffrey M. Casavant , Brian A. Duclos, Ashley E. Fenwick, Thomas
‡
‡
‡
║
‡
‡
M. Harris , Seungil Han , Nicole Caspers , Martin, E. Dowty , Xin Yang , Mary Ellen Banker ,
#
#
#
#
#
#
Martin Hegen , Peter T. Symanowicz , Li Li , Lu Wang , Tsung H. Lin , Jason Jussif , James D.
#
#
‡
†
Clark , JeanꢀBaptiste Telliez , Ralph P. Robinson * and Ray Unwalla *
†
Medicine Design, Pfizer Inc, 610 Main Street, Cambridge, MA 02139
‡
Medicine Design, Pfizer Inc, Eastern Point Road, Groton, CT 06340
§
Chemical Research Development, Pfizer Inc, Eastern Point Road, Groton, CT 06340
║
Medicine Design, Pfizer Inc, 1 Burtt Rd, Andover, MA 01810
#
Inflammation and Immunology, Pfizer Inc, 610 Main Street, Cambridge, MA 02139
*
Corresponding authors: ralph.p.robinson@pfizer.com and ray.unwalla@pfizer.com
ABSTRACT: Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling
of numerous cytokines and growth factors involved in the regulation of immunity, inflammation
and hematopoiesis. As JAK1 pairs with JAK2, JAK3 and TYK2, a JAK1ꢀselective inhibitor
would be expected to inhibit many cytokines involved in inflammation and immune function,
while avoiding inhibition of the JAK2 homodimer regulating erythropoietin (EPO) and
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thrombopoietin (TPO) signaling. Our efforts began with tofacitinib, an oral JAK inhibitor
approved for the treatment of rheumatoid arthritis (RA). Through modification of the 3ꢀ
aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with
nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and
selectivity were achieved via structural modifications suggested by Xꢀray crystallographic
analysis. After demonstrating efficacy in a rat adjuvantꢀinduced arthritis (rAIA) model, PFꢀ
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4965842 (25) was nominated as a clinical candidate for the treatment of JAK1ꢀmediated
autoimmune diseases.
INTRODUCTION
Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous
cytokines and growth factors involved in the regulation of immunity, inflammation and
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hematopoiesis. There are four members of the Janus kinase family: JAK1, JAK2, JAK3 and
TYK2. A cytokine binding to its receptor initiates the dimerization or multimerization of
receptor units. JAKs associated with the receptor subunits are brought into proximity resulting in
their phosphorylation and activation. The activated JAKs in turn phosphorylate specific tyrosine
residues within the intracellular domain of the cytokine receptor. These phosphorylated tyrosine
residues create docking sites for the recruitment and phosphorylation of signal transducers and
activators of transcription (STAT) proteins. Subsequently, the STATs dimerize and translocate to
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the cell nucleus where they modulate gene expression.
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JAK1 has the broadest cytokine signaling profile amongst the JAK family members and is the
only isoform that pairs with the other three JAKs. In combination with JAK3, which pairs only
with JAK1, it regulates the signaling of the gamma common (γ ) cytokines. JAK1 pairing
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specifically with JAK2 regulates signaling of type II interferon (IFNγ). JAK1 paired with TYK2
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regulates signaling of type I interferons (IFNα, IFNβ) and the ILꢀ10 family of cytokines.
Additionally, when paired with JAK2 or TYK2, JAK1 regulates signaling of gp130ꢀcontaining
receptors (including the prototypic cytokine ILꢀ6 receptor) as well as signaling through the
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granulocyte macrophage colonyꢀstimulating factor (GMꢀCSF) receptor. JAK1ꢀindependent
cytokines include ILꢀ12 and ILꢀ23 whose receptors signal via JAK2 and TYK2. JAK2 is the only
member of the family that pairs with itself, regulating signals for various cytokines and growth
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factors including ILꢀ3, ILꢀ5, GMꢀCSF, EPO and TPO. There are no redundant pathways known
for cytokines that signal through the JAKs and many of these cytokines have been implicated in
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a number of autoimmune diseases, making them attractive drug targets.
Tofacitinib (1) (Figure 1), a JAK1/JAK3 inhibitor with moderate activity on JAK2 and
baricitinib (2) a JAK1/JAK2 inhibitor are approved for the treatment of rheumatoid arthritis
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(
RA). Ruxolitinib (3), which inhibits both JAK1 and JAK2, is approved for the treatment of
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primary myelofibrosis (PMF). Tofacitinib and baricitinib are also in clinical trials for other
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autoimmune indications. Since these compounds have activity against JAK2, the potential for
anemia and thrombocytopenia exists due to interference with EPO and TPO signaling. In Phase 2
dose rangingꢀstudies, reduction in hemoglobin was observed in patients treated with baricitinib
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0,11
and tofacitinib.
[Figure 1]
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Based on human genetic data, the loss of JAK3 function through mutation results in severe
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combined immunodeficiency (SCID) due to the loss in signaling of the γ cytokines. The SCID
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clinical phenotype resulting from JAK3 inactivating mutations essentially identical to the Xꢀ
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linked SCID phenotype resulting from inactivating mutations in the γ receptor chain. As such,
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JAK3 has been a target of interest for the treatment of transplant rejection and other immune
disorders including RA. Since inhibition of transplant rejection was a focus during the initial
drug discovery program leading to the discovery of tofacitinib, JAK3 was of particular interest
due to its role in controlling common γꢀchain cytokine signaling and because of its known
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expression, which is restricted to lymphoid cells. However, with the development of improved
kinase assays, the panꢀJAK (i.e., nonꢀselective) inhibitory nature of tofacitinib within the JAK
family was ultimately revealed and with it the recognition that JAK1 inhibition is a major
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contributor to its pharmacology. In light of these findings, we initiated a program to discover
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and develop a potent and selective JAK1 inhibitor.
As JAK1 pairs with JAK2, JAK3 and
TYK2, a JAK1ꢀselective inhibitor would be expected to inhibit many cytokines involved in
inflammation and immune function, while avoiding the inhibition of the JAK2 homodimer
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regulating EPO and TPO signaling and other cytokines. Herein we describe the identification
and characterization of a novel series of potent and orally bioavailable small molecule inhibitors
of JAK1, along with our efforts at achieving the desired JAK1/JAK2 selectivity that allowed
identification of a clinical candidate.
RESULTS AND DISCUSSION
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Screening strategy: Literature accounts reporting kinase selectivity typically use the ratio of
biochemical IC ’s determined at the K for ATP. These selectivity ratios are misleading for
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kinases with dissimilar ATP K values and when extrapolating data to cellular settings. JAK1
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(
K = 40 ꢀM) has a K for ATP about 10ꢀfold higher than that for JAK2 (4 ꢀM) and JAK3 (4
m
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ꢀ
M) and about 3ꢀfold higher than that for TYK2 (12 ꢀM). When tofacitinib is assayed at a
physiologically relevant concentration of ATP (1 mM), JAK1 and JAK2 potencies are decreased
when compared to data obtained at ATP concentrations equal to their respective K ’s. Due to
m
these differences in K for ATP, there is a greater potency reduction for JAK2 with its higher
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affinity for ATP and, as a result, tofacitinib becomes 5ꢀfold more potent for JAK1 than JAK2 at
physiological ATP levels. Importantly, the higher potency of tofacitinib inhibition of JAK1 over
JAK2 also translates into more potent inhibition of cytokines such as IFNα, which are JAK1ꢀ
dependent and JAK2ꢀindependent, over cytokines such as EPO that are solely JAK2ꢀ
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dependent. Differences in the K for ATP may also come into play when assessing selectivity
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across the kinome as kinases with lower K values may appear to have greater activity
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(biochemical) but may not translate into functional (cellular) assays which are determined at
physiologic ATP levels.
When assessing JAK selectivity, the human cytokine inhibition profile in patients is most
relevant. However, determination of this required modeling of human whole blood (HWB)
potencies onto projected human pharmacokinetics, not a quick and inexpensive analysis to
routinely conduct on all project compounds. Clinical data suggested that maximizing JAK1
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selectivity over JAK2 in HWB is required to reduce the risk of hematopoietic effects.
Thus,
we routinely generated IC ’s for IFNα stimulation in HWB (JAK1) and CD34+ cells spiked into
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stimulation. These values correlated well with the biochemical enzyme assays conducted at 1
mM ATP.
To identify compounds with a high probability of demonstrating in vivo selectivity for JAK1
versus JAK2, we aimed for an in vitro selectivity window (biochemical or HWB) of ~25ꢀfold
and human liver microsome (HLM) stability of ~8 µL/min/mg or lower for desirable in vitro
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clearance. These values were chosen to allow for uncertainties in the cellular assays and in the
translation of the dynamic range of HLM measurements to in vivo clearance. Compounds which
met these criteria were selected for advancement into pharmacokinetic and efficacy studies.
Lead analogs – replacing the tofacitinib 3ꢀaminopiperidine group. In our efforts to develop
highly selective JAK1 inhibitors, we noted that tofacitinib (1) has excellent overall kinome
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selectivity and attributed much of this beneficial characteristic to the pyrrolopyrimidine hingeꢀ
binding motif. Therefore, we opted to retain the pyrrolopyrimidine while a variety of diamine
linkers were systematically evaluated as alternatives to the tofacitinib 3ꢀaminopiperidine group.
The terminal amino group was capped to obtain a variety of amide and sulfonamide derivatives.
As a consequence of this survey, we observed that sulfonamides bearing a cisꢀ1,3ꢀcyclobutane
diamine linker tended to confer not only excellent potency in the low nanomolar range, but more
importantly, excellent selectivity within the JAK family. Interestingly, unlike the 3ꢀ
aminopiperidine group in 1, capping the cisꢀ1,3ꢀcyclobutane diamine with an amide group
resulted in compounds possessing JAK1 activity in the micromolar range (data not shown),
significantly less active than the sulfonamide analogs. One of the hits, phenylsulfonamide 4
(Table 1), possessed remarkable potency (JAK1 IC50 = 6 nM) and selectivity (69ꢀfold over
JAK2) when assayed at an ATP concentration of 1 mM. Although HLM clearance of this
compound was high (58 ꢀL/min/mg), we felt that we would be able to improve metabolic
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stability by appropriate substitution and reduction of lipophilicity (LogD). The initial cohort of
alkyl sulfonamides 5ꢀ8 demonstrated increased potency and, importantly, a concomitant increase
in JAK1 selectivity as the sulfonamide alkyl group increased in size. Even the simple
methanesulfonamide 5 possessed good potency and selectivity for JAK1 (IC = 90 nM; 14ꢀfold
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selective over JAK2) suggesting that the sulfonamide series has an inherent potency and
selectivity advantage for JAK1. Additionally, the alkyl sulfonamides 5ꢀ8 possessed reasonable
metabolic stability as measured by HLM. The trifluoroethyl analog 8, was interesting in that
JAK1 potency was ~8ꢀfold higher than the ethyl analog 6, but selectivity over JAK2 was not
improved. Analog 9, bearing a pꢀfluorophenyl sulfonamide exhibited high JAK1 potency and
selectivity versus JAK2 (JAK1 IC = 3 nM; 80ꢀfold selectivity over JAK2), but unfortunately,
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like 4, metabolic stability (39 ꢀL/min/mg) was poor. Potency was somewhat reduced for the
benzylsulfonamide 10 (JAK1 IC = 92 nM) and, like the phenyl sulfonamides, this compound
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suffered from high HLM (24 ꢀL/min/mg) clearance.
Based on their excellent potency and high selectivity, further optimization efforts focused on the
phenylsulfonamides. However, in spite of their impressive activity, extensive modifications to
the aryl ring did not result in analogs with acceptable clearance and they were not pursued
further (data not shown). All compounds were assessed against JAK3 and TYK2 in a
biochemical assay. The majority of the sulfonamide analogs did not possess significant activity
for JAK3 (IC ’s ~8 uM to >10 uM) and showed only modest activity against TYK2 (IC ’s
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>200 nM) (Table S1, Supporting Information).
Analogs 4ꢀ10 were evaluated in a HWB assay using IFNα as stimulant to assess JAK1 potency
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in a cellular system. We also evaluated 5ꢀ9 in a HWB assay to which we added CD34 cells and
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14,18
used EPO as stimulant to assess JAK2 potency.
A general observation was made that the
HWB potency was rightꢀshifted 5ꢀ to 35ꢀfold from the biochemical assay and the shift roughly
correlated with LogD of the compounds. In general, we attributed this to plasma protein binding
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(
Figure S2). In addition, the JAK2/JAK1 selectivity ratios in HWB were similar to those derived
from the biochemical assays, but with a trend towards being slightly more JAK1ꢀselective. Also
potentially contributing to the potency shift was use of the truncated kinase domain in the
biochemical assay, whereas in the HWB assay, the full length kinase is present in its native
cellular environment at physiologic ATP levels. For these reasons we decided that the HWB
assays were most relevant for translating selectivity from in vitro to in vivo.
With these results and the initial evaluation of sulfonamides 4ꢀ10 in hand, it became apparent
that smaller, lower LogD compounds would be more likely to possess all of the properties
required to advance a molecule to the clinic and we therefore focused our efforts on analogs with
smaller alkyl analogs in place of the aryl group in 10.
[
Table 1]
Sulfonamide replacements. Beyond exploration of different sulfonamide derivatives, we also
evaluated variants of the sulfonylꢀcontaining linker, namely sulfamides, sulfones and “reverse
sulfonamides” (Table 2).
Sulfamide analogs 11ꢀ15 possessed smaller alkyl groups and exhibited JAK1 potencies
(
IC ’s) ranging from 51ꢀ144 nM with 8 to 24ꢀfold selectivity over JAK2. The ethyl (11), propyl
50
(12) and cyclopropyl (13) analogs had modest potency but their selectivities were lower,
suggesting that shorter alkyl groups in sulfamides do not make efficient interactions with JAK1.
Extending the cyclopropyl group by a methylene gave 14, which improved the JAK1 potency
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(
IC = 51 nM) and increased JAK2 selectivity to 21ꢀfold. HLM clearances for 12 and 14 were
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good, but reduced metabolic stability was noted when larger, more lipophilic alkyl groups were
employed to increase potency and selectivity. Thus, analog 15 incorporating a nitrile was
prepared to introduce polarity into the alkyl chain and lower LogD. The choice of nitrile as a
polar function was based on the reported favorable interaction of the nitrile in 1 with the
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20b
backbone atoms of the residues of the Pꢀloop i.e. Gly831, Gly834 of JAK3. However,
although 15 exhibited a good metabolic stability and selectivity profile, no enhancement in JAK1
potency was observed.
Sulfones 16ꢀ20 (Table 2) exhibited improved JAK1 potencies relative to the sulfamides,
however selectivities against JAK2 were not improved. The ethyl analog 16 (JAK1 IC = 57
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nM) was approximately 3ꢀfold more potent than the related sulfamide 11 and about equipotent to
the cyclopropyl analog 17. While an improvement in potency was observed with the nꢀpropyl
analog 18, a more significant increase in potency was observed with the longer nꢀbutyl analog 19
(
IC = 9 nM), which possessed an improved JAK2 selectivity of 24ꢀfold. This was the first
50
analog to show significant JAK1 selectivity while also having high potency and modest HLM
clearance (14 ꢀL/min/mg). We assessed the potency of the sulfones in the HWB assays and,
although there was some variability in the right shift, HWB activities were largely in line with
biochemical activity. The nꢀbutyl analog 19 displayed excellent activity against IFNα (IC =
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155 nM) and good selectivity versus EPO (IC = 4089 nM). We prepared phenyl analog 20 to
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assess potency and clearance within an aryl sulfone motif. As previously noted with
phenylsulfonamide 4, the JAK1 potency was exceptional (10 nM), but HLM clearance was high
(20 ꢀL/min/mg). Unlike 4, which showed good selectivity over JAK2 (69ꢀfold), 20 displayed no
advantage in terms of selectivity, which was reduced to 16ꢀfold.
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The low nanomolar potency previously observed with the sulfonamides and sulfones was again
observed with reverse sulfonamides 21ꢀ24 (Table 2). For a similar LogD range, the clearance
was comparable to the other analogs. However, desirable potency and selectivity levels required
introduction of larger alkyl groups as in 23 for which HLM clearance increased from moderate to
a high level.
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Observations from compounds 4ꢀ24 indicated that, for all variants of the sulfonylꢀcontaining
linker, selectivity generally improved as the side chains grew larger. Although based on a limited
number of examples, the sulfamide subset was of lower interest as these compounds possessed
the poorest overall JAK1 potency. A comparison of the JAK1/JAK2 selectivity ratios of
sulfonamides to sulfones and reverse sulfonamides suggested that, the sulfonamides were
capable of achieving the best selectivities for JAK1: 39ꢀfold for isobutyl analog 7 and 80ꢀfold for
pꢀfluorophenyl analog 9. Selectivity peaked at about 25ꢀfold for the other series containing
similar alkyl and aryl groups. We also observed a trend that suggested HLM clearance values
were often >8.0 ꢀL/min/mg when LogD7.4 exceeded 2.0. Based on these observations, the alkyl
sulfonamide series appeared to provide the best balance of potency and clearance. We thus
prepared an additional cohort of alkyl sulfonamides (Table 3).
In order to determine the impact of stereochemistry on potency and selectivity, the 1,3ꢀtransꢀ
cyclobutyl analog of 25 was prepared and evaluated. Whereas the cisꢀconfigured isomer 25
showed good potency (JAK1 IC = 29 nM), the trans isomer 26 suffered a ~20ꢀfold loss in
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potency. Since the JAK2 IC of 26 was greater than that of the top screening concentration, we
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were unable to determine its JAK2/JAK1 selectivity ratio.
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The addition of a methyl to give linear (27) butyl chain resulted in a ~3ꢀfold increase in
potency relative to 25, the expected increase for a nonꢀspecific hydrophobic interaction of methyl
2
1
with a protein. However, this addition increased LogD to 2.4, resulting in a concomitant
increase in HLM clearance to 17 ꢀL/min/mg. The incorporation of cyclopropyl rings as in 28
and 29 did not afford an improvement in potency or selectivity over the simple linear analogs 25
and 27, but the reduced LogD of the cyclopropyl group did improve HLM clearance. In contrast,
cyclobutylmethyl analog 30, which did not afford a reduction in LogD, demonstrated improved
potency and selectivity, but displayed higher clearance. The incorporation of trifluoromethyl
within the side chain to improve metabolic stability was successful as analogs 31 and 32
maintained JAK1 potency and low clearance (<8.0 ꢀL/min/mg) while displaying increased
selectivity (54ꢀfold and 61ꢀfold respectively). The ether analogs 33 and 34 were weaker in
potency, but retained low clearance when compared to the trifluoromethyl analogs.
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In the HWB assays, the simple alkyl analogs 25ꢀ30 had potency shifts compared to the
biochemical assay similar to the earlier sulfonamide cohort, while the groups containing
heteroatoms 31ꢀ34 appeared to have greater right shifts than would be expected based on LogD
alone. For example, compound 31, which has a similar LogD value and JAK1 biochemical
potency comparable to 30, shows a larger right shift (63 vs. 20 fold for 30) in the HWB IFNα
assay. The right shift in the HWB CD34+ EPO assay was more variable, but generally agreed
with the magnitude of the right shift observed in the IFNα assay.
At this stage we were pleased that several compounds, especially sulfonamides 25, 28, and 29,
exhibited promising overall profiles including good JAK1 potency (biochemical and HWB INFα
IC ≤ 0.05 ꢁM and ≤ 0.3 ꢁM respectively), good JAK1/JAK2 selectivity (≥ 25ꢀfold), and high in
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vitro metabolic stability (HLM ≤ 9 ꢁL/min/mg). Although HLM clearance for sulfone 19 was
somewhat higher (~14 ꢁL/min/mg), its profile was also attractive in terms of potency (JAK1 IC50
= 9 nM) and selectivity (24ꢀfold). Comparing sulfonamides 25, 28, and 29, compound 25 was the
most selective based on ratios determined using HWB IC s (38ꢀfold vs. 22ꢀ and 29ꢀfold for 28
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and 29, respectively). Thus, representing two structural series (sulfones and sulfonamides),
compounds 19 and 25 were selected for further profiling from among the analogs shown in
Tables 1ꢀ3 (vide infra).
[
Table 3]
Xꢀray crystallographic studies – understanding JAK1/JAK2 selectivity. While initial
compound synthesis and evaluation was in progress, we developed a robust crystallographic
22
soaking method enabling the routine determination of JAK1 inhibitor structures. Notably, we
determined the Xꢀray structure of compound 25 in complex with the kinase domains of JAK1
(2.1 Å) and JAK2 (1.8 Å). This effort built upon our knowledge of these important tyrosine
kinases and provided invaluable insight into the observed JAK1/JAK2 selectivity (28ꢀfold) of
compound 25.
2
3
The overall folds of the two kinase domains are similar to those previously reported
(R.M.S.D ~0.80 Å. Although the JAK1 kinase domain shares only 53% overall sequence identity
with JAK2, most of the residues in the ATPꢀbinding site are conserved between the two
enzymes. The residue differences (within 5 Å radius from the ligand) are located in the hinge
region, phosphateꢀbinding region, i.e., Pꢀloop (Table 4) and in the solvent exposed regions
towards the periphery of the binding site. While at first these differences may seem sufficient for
a ligand to achieve a high level of selectivity, these residues point away from the ligand, and
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hence they are not amenable for direct hydrogen bonding contacts with the ligand. There is one
exception, the Glu966(JAK1)/Asp939(JAK2) residue difference that lies towards the solventꢀ
exposed region of the binding site (Figures 2 and 3). This difference has been specifically
targeted in the literature for a differential hydrogen bonding interaction with favorable
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24
improvement in JAK1 selectivity (36 fold). This observation was supported by coꢀcrystal
structures of both JAK1 and JAK2 that indeed revealed differential interaction between the two
isoforms. Additionally, Vasbinder and coworkers observed waterꢀmediated hydrogen bonds
25
between Glu966 and a potent JAK1ꢀselective inhibitor.
[
Table 4]
Comparison of the crystal structures of 25 bound to both JAK1 and JAK2 showed that,
although the ligand binding mode is similar in both cases, there are subtle differences in
hydrogen bonding pattern with residues within the binding site and in the orientation of the
propyl tail group in the Pꢀloop region that could contribute to the JAK1 selectivity. The core
pyrrolopyrimidine ring of the ligand in both structures makes two critical hinge binding
interactions, the pyrrole NH with the carbonyl backbone of Glu957(JAK1)/Glu930(JAK2) and
the pyrimidine N1 nitrogen with the NH backbone of Leu959(JAK1)/Leu932(JAK2) residues.
There are other differences in the structures that we believe contribute to overall ligand
selectivity, for example a water molecule is in position to bridge a hydrogen bond interaction
between the N3 of the pyrimidine ring and the side chain Glu966 residue in JAK1. In JAK2, due
to the shorter Asp939 residue at this position, a direct water mediated bridge interaction is not
possible and requires an extended network of water molecules in the solventꢀexposed region to
make interaction with the N3 atom. Although the extent to which this differential interaction
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contributes to selectivity is difficult to predict, these differences in the hydrogen bonding pattern
could have influence on the JAK1 selectivity of 25.
[Figures 2 and 3]
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In both structures, the observed conformation of the sulfonamide group has the HꢀNꢀSꢀO
26,27
dihedral at 1ꢀ10°. A search of this fragment in the Cambridge Crystallographic Database
indicated that this is a preferred conformation for this group. It is believed that this conformation
allows the nitrogen lone pair to favorably interact with the sulfur dꢀorbitals. The cis
configuration of the puckered cyclobutyl ring allows the NH of the sulfonamide to be in a
position to pick up hydrogen bond interactions with both the backbone carbonyl of
Arg1007(JAK1)/Arg980(JAK2) and also the side chain of Asn1008(JAK1) /Asn981(JAK2) in
both structures. We did observe differences in the rotamer states of the Arg residue, for example,
in the JAK2 Xꢀray structure, a single rotamer was observed for the Arg980 residue which allows
a hydrogen bond from the side chain of Arg980 to the SO group of the ligand. In JAK1, this
2
particular rotamer is only observed as a minor state. Additionally in JAK2, there is a water
mediated hydrogen bond observed between oxygen of the SO group and NH backbone of the
2
Lys857 residue in the Pꢀloop which could influence the flexibility of the Pꢀloop. This will be
discussed in detail in the next section.
A difference in the orientation of the nꢀpropyl group of the ligand towards the Pꢀloop region
was observed when comparing the two JAK structures. In both structures the electron densities
of the Pꢀloop and ligand were well resolved, which aided general conclusions on the origin of
ligand selectivity from this region. A comparison of the crystal structures of 25 bound to JAK1
and JAK2 (Figure 4) suggested that the Pꢀloop in JAK2 is clamped down slightly more onto the
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active site than the one observed in the relatively more open JAK1 Pꢀloop. It is possible that
although the side chains of residues within the Pꢀloop region are oriented away from the ligand
binding site, the residue differences between the JAK1 and JAK2 (Table 4) along the Pꢀloop may
still affect the rigidity of the Pꢀloop and allow different conformations to be accessed by JAK1
compared to JAK2. A shift in the Pꢀloop position observed between the JAK1 and JAK2 Xꢀray
structures has been previously invoked to explain the selectivity of a series of
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imidazopyrrolopyridines.
[
Figure 4]
As a consequence of the disparate Pꢀloop orientations, the ligand is forced to adopt different
conformations in the bound states for the two torsion angles, i.e., S(O )ꢀCꢀCꢀC and NꢀS(O )ꢀCꢀC,
2
2
in order to maximize its shape and electrostatic complimentarity with the Pꢀloop. To get a better
understanding of the energetics associated with these dihedrals, we calculated the torsion profile
using the simple fragment Nꢀmethylpropaneꢀ1ꢀsulfonamide in a quantum mechanical calculation
29
at B3LYP/6ꢀ31++G level (Figures 5a and 5b). For both dihedrals, two minima are observed,
i.e., anti and gauche, with the anti being lower in energy. In the JAK1 structure, the ligand
adopts a low energy anti conformation for the propyl chain, i.e., S(O )ꢀCꢀCꢀC dihedral ~175°,
2
while in JAK2 a higher energy (ꢁE = 1.2 kcal/mol) conformation exists having a dihedral of
~
95°. In contrast, the observed torsional angle NꢀS(O )ꢀCꢀC is in a high energy (ꢁE = 3.2
2
kcal/mol) conformation in JAK1 (dihedral of 126°), while a lower energy dihedral (171°) is seen
in JAK2. These calculations suggest that, in both ligand bound states, an internal energy penalty
is paid by the ligand to adopt a conformation that makes efficient van der Waals and electrostatic
interactions with the Pꢀloop. While the overall energy penalty is calculated to be higher in JAK1,
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the ligand seems able to overcome the conformational penalty from the NꢀS(O )ꢀCꢀC torsion by
2
forming more favorable van der Waals contacts with the Pꢀloop, thus improving JAK1
selectivity. This is consistent with the observed trend of increase in selectivity with larger alky
groups as these are able to make better Pꢀloop interactions in JAK1 than JAK2, i.e., 5 (14ꢀfold),
1
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(>19ꢀfold), 25 (28ꢀfold) and 27 (66ꢀfold).
[
Figure 5a and 5b]
Structureꢀenlightened “second generation” analog design
Based on insights provided from the Xꢀray crystallography studies and analysis, a number of
new analogs were prepared in the sulfamide, sulfonamide and reverse sulfonamide series (Table
).
[Table 5]
5
Sulfamides: As noted previously, the observed conformation of sulfonamide 25 has an HꢀNꢀ
30
SꢀO dihedral of ~10°, which allows for the stabilization of this rotamer by the nitrogen lone
pair interaction with the sulfur d orbitals. We reasoned that, in the case of sulfamides, the distal
NH (i.e., NH furthest from the pyrrolopyrimidine ring) would form a similar interaction with the
other S=O group, giving rise to an sꢀcis, sꢀtrans conformation (Figure 6a). Indeed, a search of the
Cambridge Crystallographic Database for acyclic sulfamides revealed a strong preference for
this conformation. All 21 CSD entries from the search displayed a preference for the sꢀcis, sꢀ
trans conformation (for example, see Figure 6b). Interestingly, this conformational preference
implies that, in the bound state, the ligand would favor the proximal NH in an sꢀcis configuration
so as to make hydrogen bond interactions with both the backbone carbonyl of
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Arg1007(JAK1)/Arg980(JAK2) and the side chain of Asn1008(JAK1) /Asn981(JAK2), while
the distal NH would be favored in an sꢀtrans conformation. This would have the undesirable
effect of orienting the Rꢀgroup away from the Pꢀloop and thus have a detrimental effect on JAK1
potency. We reasoned that this was a possible explanation for the lower potency and selectivity
observed with the sulfamides 11ꢀ15. We hypothesized that Nꢀalkylation of the distal sulfamide
nitrogen would disrupt the conformational preference of the distal NꢀHꢀSꢀO group and thereby
allow the Rꢀgroup to make an interaction with the Pꢀloop and potentially improve potency.
Indeed, Nꢀmethyl analogs 37 and 38 exhibited a 4 to 7ꢀfold increase in potency for JAK1 over
the nonꢀmethylated analogs 11 and 13 respectively and >2ꢀfold increase in JAK1/JAK2
selectivity (Table 5). This was attributed to the asymmetric nature of the substituents on the
distal nitrogen that allows the alkyl group, i.e., ethyl in 37 and cyclopropyl in 38, to be directed
towards the Pꢀloop. We also prepared symmetrical piperidine 39, which retained JAK1 potency
and demonstrated improved JAK2/JAK1 selectivity. Unfortunately, 37 and 38 still lacked the
desired selectivity, while 39 had poor HWB potency and HLM stability. Thus, in spite of the
improvements in potency and selectivity, the overall profiles were not improved.
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Based on the mixed results seen with these “second generation” sulfamides, no further
advancement of the compounds was undertaken and we focussed our efforts on the sulfonamide
and reverse sulfonamide analogs.
[
Figure 6a and 6b]
Sulfonamides: Applying insight from our structural studies indicating that the sulfonamide
NH group is an important determinant of JAK1 selectivity and that interaction of the alkyl group
with the Pꢀloop contributes to potency and selectivity, sulfonamides containing polar
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functionality were prepared (Table 5). The choice of polar groups was based on the reported
interaction of nitrile in 1 with the Pꢀloop of JAK3 to confer high potency. Compound 35
containing the nitrile functionality was potent, metabolically stable and had excellent selectivity.
Its potency in the HWB IFNα assay (IC = 216 nM) was in a desirable range and was well
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separated from HWB EPO (IC >6934 nM). The oxetane 36 had a similar profile, demonstrating
5
0
excellent potency and selectivity in the HWB assays and high metabolic stability. Although only
slightly more potent than the acyclic ether 34 in the JAK1 biochemical assay, the LogD was
reduced by 0.5 which may account in part for the 4ꢀfold improvement in HWB IFNα potency.
Reverse Sulfonamides: Based on the success with distal Nꢀmethylation in the sulfamide
series, we prepared additional Nꢀmethyl reverse sulfonamides having the potential for
improvements in potency and selectivity through interactions with the Pꢀloop. Analogs 41 and 42
(Table 5) showed the expected improvements in JAK1 potency and selectivity versus JAK2
when compared to the corresponding NH analogs 23 and 24. These results are comparable in
magnitude to those observed with the sulfamides. Cyclopropylmethyl analog 42 demonstrated
good HWB IFNα potency, but suffered from poor metabolic stability. Once again we introduced
symmetry to improve selectivity and prepared the pyrrolidine sulfonamide 43 as a cyclized
version of nꢀbutyl sulonamide 23. The potency was modestly improved, but selectivity was
reduced, likely due to the smaller size of the unsubstituted 5ꢀmembered ring and resulting
weaker interaction with the Pꢀloop. In this cohort, high selectivity was associated with poor
metabolic stability although HWB activity was acceptable for several analogs.
Pharmacokinetics. Based on JAK1 potency (biochemical and HWB), JAK1/JAK2 selectivity,
and acceptable in vitro metabolic stability, “second generation” sulfonamides 35 and 36 (Table
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5
) were selected for pharmacokinetic profiling along with sulfone 19 (Table 2) and sulfonamide
2
5 (Table 3). Pharmacokinetic parameters were determined in rat at 1 mg/kg i.v. or 3 mg/kg p.o.
(Table 6). Clearances of 19 and 25 were low relative to total liver blood flow, whereas 35 and 36
showed higher clearance values. Oral bioavailability was better for compounds 19 and 25,
consistent with lower clearance as well as good in vitro permeability and solubility. In addition,
volumes of distribution were moderate (0.74ꢀ1.99 L/kg) for this cohort of analogs. Accordingly
single species allometric scaling predicted more favorable human clearance values for
compounds 19 and 25 compared with 35 and 36.
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[
Table 6. PK properties in the SꢀD rat following doses of 1 mg/kg i.v. or 3 mg/kg p.o. and in
vitro physicochemical properties]
Although compounds 19 and 25 showed similar pharmacokinetic profiles and similar potencies
in JAK biochemical and cellular assays, a broad functional CEREP screen at 10 ꢀM consisting
of a diverse set of GPCR, ion channel, transporter and enzyme (64 targets in total) for offꢀtarget
effects indicated that 19 showed activity against the CBꢀ1 receptor in a binding assay (IC = 120
5
0
nM). This translated into observed motor effects consistent with CBꢀ1 inhibition in a rat
toxicokinetic study. Compound 25 showed no measurable activity (>50%) at any targets profiled
with the exception of KDR kinase (VEGFR2, IC = 1.2 ꢀM) and weak activity on monoamine
50
oxidase A (MAOꢀO, IC = 6 ꢀM). A followꢀup study in a functional Caliper whole cell KDR
50
kinase assay showed no effects on KDR kinase activity at concentrations up to 30 µM.
Sulfonamide 25 was also evaluated in a broad kinase panel (Table S3), which indicated that it
is a highly selective compound, crossing over most potently onto JAK3 with 61% inhibition at 1
uM. The lack of offꢀtarget kinase inhibition is particularly notable, given that the broad kinase
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panel was run at the K of ATP to maximize assay sensitivity for the detection of potential offꢀ
m
target activity. Although JAK3 showed up as an offꢀtarget in the broad kinase panel, 25 had an
IC of 605 nM when measured at the K of ATP and >10 ꢀM when measured at 1 mM ATP. In
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summary, compound 25 is a potent JAK1 inhibitor with 28ꢀfold selectivity over JAK2, >340ꢀ
fold over JAK3 and 43ꢀfold over TYK2 (Table S1) at 1mM ATP and an excellent selectivity
profile over the broader kinome. Based on its favorable overall properties and pharmacokinetic
profile across multiple species (Tables S4 and S5), 25 progressed into pharmacodynamic and
efficacy studies in vivo.
Rat Target Modulation. The in vivo pharmacokineticꢀpharmacodynamic relationship of 25
was characterized following single oral doses of 5, 15, and 50 mg/kg to naïve Lewis rats. Serial
blood samples were taken at 0.25, 0.5, 2, 4, 8, and 24 hours and analyzed for drug concentrations
and STAT phosphorylation (pSTAT) activities. pSTAT1 was measured following ex vivo
stimulation of blood with ILꢀ6 or IFNγ to assess JAK1/JAK2 signal inhibition, while pSTAT3
was measured following stimulation with ILꢀ21 to assess JAK1/JAK3 signal inhibition (Figure
7
). The inhibition of pSTAT formation induced by IFNγ, ILꢀ21 and ILꢀ6 was directly linked to
the plasma concentration of 25 and was independent of time. The respective calculated unbound
plasma IC values were 191, 938 and 176 nM.
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[
Figure 7 Target Modulation plots]
Rat adjuvantꢀinduced arthritis model. The effect of JAK1 inhibition by 25 in vivo was
evaluated using a therapeutic dosing paradigm in a rat adjuvantꢀinduced arthritis (AIA) disease
3
1a
model . Female Lewis rats immunized with complete Freund’s adjuvant were dosed orally with
25 or vehicle for seven consecutive days after disease onset as measured by hind paw volume
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using plethysmography. Paw volumes and weights were followed throughout the study. At the
end of seven days of dosing, plasma concentrations of 25 were assessed. Two studies were
completed. In the initial study, immunized rats were dosed QD with either: 5, 15, or 50 mg/kg of
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5 or vehicle (PO) (Figure S3). Since a significant reduction in paw swelling was observed for
all doses, a second study was conducted focused on lower doses (0.5, 1, 5, or 15 mg/kg 25 or
vehicle control) were evaluated. A significant reduction of hind paw swelling was observed
down to 1 mg/kg (Figure 8, Days 6ꢀ7). Collective pharmacodynamics modeling of both studies
2
using an Emax model fit indicated an unbound Cave of approximately 1.3 ꢁM (r ~ 0.91) (Figure
50
9
).
[
[
Figure 8 Disease activity plots]
Figure 9 Emax modeling Cave]
To determine the effect of 25 on JAK2/JAK2 dependent GMꢀCSF signaling, blood samples
were taken at peak plasma concentration (0.25 hr post dose) in the rats treated with 25 on day 7.
GMꢀCSF induced pSTAT5 in monocytes was reduced by approximately 30% at the top dose of
15 mg/kg and less than 5% at lower doses. In contrast, ILꢀ21, IFNα and IFNγ induced stat
phosphorylation was reduced >60% at 1 mg/kg.
Human prediction of pharmacokinetics and pharmacodynamics. The primary clearance
mechanisms of compound 25 were determined to be mediated by CYP450 metabolism, with
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limited renal and biliary clearance expected. Human clearance predictions were made using
human liver microsomes and hepatocyte data as well as standard single species allometric
scaling techniques, all of which suggested low to moderate human clearance would be observed
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(Table 7).
[
Table 7]
Preclinical and clinical studies with JAK inhibitors have shown the value in using average drug
1
9,31b,32
plasma concentration (Cave) in predicting antiꢀinflammatory response.
The percent level
of cytokine inhibition (IC ) of compound 25 for each cytokine pathway can be calculated by
xx
combining the corresponding unbound whole cell IC50 value (Table 8) with the predicted
unbound human Cave value (i.e., IC = 100*Cave/(IC + Cave)). The overall selectivity profile
xx
50
of cytokine inhibition can be visualized at an expected efficacious human dose of 200 mg QD
based on human hepatocyte scaling), which is expected to achieve a Cave for IFNα using this
(
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method of analysis (Figure 10). Selectivity of inhibition of compound 25 for cytokines signaling
through JAK1 heterodimer pairs (IFNα, IFNγ, ILꢀ6, ILꢀ10, ILꢀ15, ILꢀ21, ILꢀ23 and ILꢀ27)
(
Table 8) was evident compared to the sparing of JAK2/JAK2 (EPO) and JAK2/TYK2 (ILꢀ12
and ILꢀ23) signaling. Additional data showing the cytokine inhibition profile for compounds 19,
5, 36 in the HWB assays is shown in Table S6.
3
[
Table 8]
CHEMISTRY
Preparative routes to the sulfonamide and sulfamide analogs are shown in Scheme 1.
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Benzyl (3ꢀoxocyclobutyl)carbamate (44) was condensed with methylamine (AcOH/THF/rt) and
then treated with NaBH (0°C to rt) to afford cyclobutylamine 45 as a mixture of cis and trans
4
isomers. Hydrochloride salt formation followed by recrystallization allowed the isolation of the
major isomer, cisꢀ45·HCl, in moderate yield (63%). In addition the minor isomer transꢀ45 (used
for the preparation of 26), could be isolated by supercritical fluid chromatography. Subsequent
reaction of cisꢀ45·HCl with 4ꢀchloroꢀ7ꢀ[(4ꢀmethylphenyl)sulfonyl]ꢀ7Hꢀpyrrolo[2,3ꢀd]pyrimidine
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(
46a) in presence of DIPEA (iꢀPrOH/75°C) provided the protected intermediate 47a. Here, the
electronꢀwithdrawing tosyl protecting group allowed the nucleophilic aromatic substitution
reaction to occur readily in high yield (95%).
[Scheme 1]
Treatment of 47a with HBr (EtOAc/AcOH/rt) served to remove the pendant carbobenzyloxy
protecting group and provided 48a, isolated as the stable, freeꢀflowing dihydrobromide salt.
Conversion of 48a·2HBr to target sulfonamides was then achieved in two steps: sulfonylation
(
RSO Cl/ base) followed by removal of the tosyl group. Typically aqueous hydroxide was used
2
for the deprotection step, but occasionally, due to the potential for side reactions involving the
sulfonamide sideꢀchain, other conditions were used such as in the preparation of 35, where the
presence of the nitrile function necessitated use of Cs CO as base.
2
3
Several sulfonamide analogs were prepared starting with 2,4ꢀdichloroꢀ7Hꢀpyrrolo(2,3ꢀ
d)pyrimidine (46b). Though less robust, this route was generally useful in compound
preparation, not requiring the sometimes problematic tosyl deprotection step. Taking advantage
of the activation effect of the additional chlorine atom on the pyrrolopyrimidine ring, 46b was
combined with cisꢀ45·HCl (K CO /H O/95°C) to afford adduct 47b in high yield (98%).
2
3
2
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Exposure of 47b to catalytic hydrogenation conditions (Pd(OH) ) then gave amine 48b, which
2
was taken on either as the hydrochloride salt or as the free base.
Sulfonamide formation using 48b could be carried out directly under standard conditions
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(
RSO Cl/base) but often mixtures arose and yields were low due to competing sulfonylation of
2
the pyrrolopyrimidine NH. This problem could be avoided by in situ protection of the
pyrrolopyrimidine NH function. Thus 48b (hydrochloride) was first treated with LiHDMS
(2.2.eq.) and TMSCl (2.2 eq.) in THF to provide what was assumed to be a bisꢀTMS derivative
(
pyrrolidine NH and primary amine capped by TMS). Addition of RSO Cl then afforded the
2
desired sulfonamide target after workup, presumably via preferential sulfonylation of –NHTMS.
As shown in Scheme 1, sulfamide analogs were accessed via reaction of Nꢀ
34
sulfamoyloxazolidinone 50, prepared from 48a·2HBr by the method of Montero with primary
1
2
and secondary amines (R R NH) at elevated temperature in the presence of base (TEA or
3
5
DIPEA). As with the preparation of sulfonamides, removal of the Nꢀtosyl group was typically
carried out using aqueous LiOH or NaOH, often in the presence of a coꢀsolvent such as EtOH.
Compounds in the reverse sulfonamide and sulfone series were prepared starting with the two
step conversion of ethyl 3ꢀaminocyclobutanecarboxylate hydrochloride (cis/trans = 10:1)
3
6
(
52·HCl) to 3ꢀ(methylamino)cyclobutyl]methanol (53), also a 10:1 mixture of cisꢀ and transꢀ
isomers (Scheme 2). Reaction of 53 with 4ꢀchloroꢀ7ꢀ[(4ꢀmethylphenyl)sulfonyl]ꢀ7Hꢀpyrrolo[2,3ꢀ
d]pyrimidine (46a) (TEA/KI/acetone/56°C) gave alcohol 54 as a mixture of isomers (85%)
which were separated by supercritical fluid chromatography. The major isomer, cisꢀ54 was then
advanced in good overall yields to common intermediates 55a and 55b via Oꢀtosylation and Oꢀ
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mesylation respectively (pꢀTsCl or MsCl/DMAP/DCM). Tosylate displacement on 55a with
potassium thioacetate (DMF/55°C) provided Sꢀacetate 56.
[Scheme 2]
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Compounds in the reverse sulfonamide series (Scheme 2) were obtained in four steps from 56.
Oxidation to sulfonic acid 57a (aq. H O /HCO H) followed by reaction with thionyl chloride
2
2
2
(cat. DMF/DCM/0°C) provided sulfonyl chloride 57b which, after sulfonamide formation
3
4
(
R R NH/base) and deprotection, gave the target analogs.
Sulfone analogs could be accessed in two or three steps from either tosylate 55a or mesylate 55b.
2
Sulfonate displacement with in situ generated thiolate (R SH/DBU/NMP/rt) provided thioethers
of general structure 58a, which usually underwent cleavage of the Nꢀtosyl group under the
reaction conditions to afford deprotected thioethers 58b. In some cases, tosyl cleavage was
incomplete, necessitating a subsequent tosyl group removal step (e.g., aq. LiOH/EtOH).
®
Oxidation of 58b with Oxone then provided the target sulfones. These could also be prepared
3
8
via Sꢀacetate 56 starting with cleavage of the acetyl group (K CO /NaBH /MeOH/0°C) and in
2
3
4
situ alkylation of the resulting thiolates with alkyl halides, alkyl tosylates and alkyl mesylates
2
(
R ꢀX). The product thioethers 58a then afforded the target sulfone analogs after tosyl group
cleavage and oxidation. Sulfone synthesis via 56 became a preferred method as it obviated the
need to use thiols as starting materials.
CONCLUSION
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By replacement of the 3ꢀaminopiperidine in tofacitinib (1) with a cisꢀ1,3ꢀcyclobutyldiamine
capped with a sulfonamide, we were able to identify highly potent and selective JAK1 inhibitors
in good physicochemical space. We found that various permutations around the sulfonyl moeity
1
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(i.e., sulfamides, sulfones and reverse sulfonamides) also afforded potent and selective
molecules. Clearance was recognized as a key factor in identifying compounds suitable for
development. Therefore the observation that compounds with LogD < 2 demonstrated very low
HLM clearance enabled us to design molecules with the right balance of lipophilicity and
polarity required for good potency and metabolic stability. Profiling in HWB assays
demonstrated that the excellent potency for JAK1 translated to a cellular system and that JAK2
+
selectivity, as assessed by the inhibition of EPO in CD34 cells was confirmed relative to the
biochemical assays. Insights obtained through Xꢀray crystallography as to how analogs bind to
JAK1 allowed us to improve potency, especially within the sulfamide and reverse sulfonamide
series. Assessment of rat pharmacokinetics for the four analogs that met advancement criteria
demonstrated that two (19 and 25) had low in vivo clearance values. Compound 25 exhibited a
superior pharmacology profile compared to 19 and therefore was further advanced. Compound
2
5 was 28ꢀfold selective over JAK2, >340ꢀfold over JAK3 and 43ꢀfold over TYK2 at 1 mM ATP
and demonstrated broad kinase selectivity. In a rat target modulation study, compound 25
inhibited pSTAT formation induced by ILꢀ6, ILꢀ2 and IFNγ in a manner directly linked to the
drug plasma concentration. Similarly, compound 25 demonstrated efficacy in a doseꢀresponsive
manner in a therapeutic rat AIA model. By using allometric scaling and human hepatocyte
clearance a human dose of approximately 200 mg QD was projected to achieve Cave inhibition
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3
7
of IFNα. Following appropriate in vivo toxicological testing, compound 25 (PFꢀ04965842)
progressed into a Phase 1 clinical study, the results of which will be reported elsewhere.
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EXPERIMENTAL SECTION
Chemistry – general information. Chemicals were typically obtained from SigmaꢀAldrich or
Alfa Aesar and used as received, unless noted otherwise. Solvents were commercial anhydrous
grade, used as received. Unless otherwise stated, reactions were run under a positive pressure of
nitrogen at ambient temperature. Except where otherwise noted, “chromatographed” and “flash
chromatography” refer to normal phase chromatography using a medium pressure Biotage or
ISCO system employing commercial preꢀpacked silica gel columns. Supercritical fluid
chromatography (SFC) was performed on an Agilent SFC system in APCI negative mode.
Analytical thin layer chromatography (TLC) was performed on 60 Å F254 glass plates preꢀ
coated with a 0.25ꢀmm thickness of silica gel purchased from EMD Chemicals Inc. TLC plates
were visualized with UV light. Except where otherwise noted, purities of compounds used in
biological studies were >95%, determined by HPLC, UPLC or LC/MS. Ultraviolet (typically
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15ꢀ220 nm) and evaporative light scattering detection (ELSD) were typically employed for
detection.
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Nuclear magnetic resonance ( H NMR) spectra were recorded on a Bruker or Varian
spectrometer operating at 400 MHz, or a Bruker spectrometer operating at 500 MHz. Chemical
shifts are reported in parts per million (δ) relative to CDCl (7.26 ppm), DMSOꢀd (2.50 ppm),
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MeOHꢀd (3.31 ppm) or D O (4.79 ppm). The following abbreviations are used for peak
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multiplicites: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broadened, dd =
doublet of doublets, dt = doublet of triplets.
ACS Paragon Plus Environment
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Page 29 of 91
Journal of Medicinal Chemistry
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Low resolution mass spectroscopy was carried out using liquid chromatography−mass
spectrometry (LC/MS) on a Waters Acquity UPLC instrument using atmospheric pressure
chemical ionization (APCI) or electrospray ionization (ESI).
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Nꢀ(cisꢀ3ꢀ(Methyl(7Hꢀpyrrolo[2,3ꢀd]pyrimidinꢀ4ꢀyl)amino)cyclobutyl)benzenesulfonamide
(4). This sulfonamide analog was prepared using a procedure similar to that used to prepare
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compound 25 starting from 48a·HBr. H NMR (400 MHz, DMSOꢀd ): 12.42 (br s, 1H), 8.27 (s,
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H), 8.08 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 7.0 Hz, 2H), 7.55ꢀ7.74 (m, 3H), 7.30ꢀ7.42 (m, 1H),
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.75ꢀ6.92 (m, 1H), 4.58ꢀ4.82 (m, 1H), 3.46ꢀ3.73 (m, 1H), 3.26 (s, 3H), 2.28ꢀ2.46 (m, 2H), 1.95ꢀ
+
2.18 (m, 2H). LC/MS m/z (M + H ) calcd for C H N O S: 358. Found: 358.
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Nꢀ{cisꢀ3ꢀ[Methyl(7Hꢀpyrrolo[2,3ꢀd]pyrimidinꢀ4ꢀyl)amino]cyclobutyl}methanesulfonamide
(5). This sulfonamide analog was prepared using a procedure similar to that used to prepare
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compound 25 starting from 48a·HBr. H NMR (DMSOꢀd , 400 MHz): 11.64 (br s, 1H), 8.10 (s,
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H), 7.40 (d, J = 8.8 Hz, 1H), 7.14ꢀ7.16 (m, 1H), 6.63ꢀ6.64 (m, 1H), 4.90ꢀ4.98 (m, 1H), 3.54ꢀ
.64 (m, 1H), 3.25 (s, 3H), 2.89 (s, 3H), 2.58ꢀ2.65 (m, 2H), 2.20ꢀ2.28 (m, 2H). LC/MS m/z (M +
+
H ) calcd for C H N O S: 296. Found: 296.
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Nꢀ{cisꢀ3ꢀ[Methyl(7Hꢀpyrrolo[2,3ꢀd]pyrimidinꢀ4ꢀyl)amino]cyclobutyl}ethanesulfonamide
(6). This sulfonamide analog was prepared using a procedure similar to that used to prepare
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compound 25 starting from 48a·HBr. H NMR (DMSOꢀd , 400 MHz): 11.64 (br s, 1H), 8.10 (s,
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1H), 7.50 (d, J = 8.5 Hz, 1H), 7.15ꢀ7.16 (m, 1H), 6.63ꢀ6.65 (m, 1H), 4.87ꢀ4.94 (m, 1H), 3.53ꢀ
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.61 (m, 1H), 3.26 (s, 3H), 2.97 (q, J = 7.7 Hz, 2H), 2.57ꢀ2.62 (m, 2H), 2.21ꢀ2.27 (m, 2H), 1.21
+
(
t, J = 7.7 Hz, 3H). LC/MS m/z (M + H ) calcd for C H N O S: 310. Found: 310. Purity
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(HPLC) = 98.5% (UV), 85.7% (ELSD).
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