Discovery and Optimization of a Novel Series of Highly Selective JAK1 Kinase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00076

Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-m

Full text of DOI:10.1021/acs.jmedchem.8b00076

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Article
Discovery and Optimization of a Novel Series
of Highly Selective JAK1 Kinase Inhibitors
Neil P. Grimster, Erica Anderson, Marat Alimzhanov, Geraldine Bebernitz, Kirsten Bell, Claudio
Chuaqui, Tracy Deegan, Andrew D. Ferguson, Thomas Gero, Andreas Harsch, Dennis Huszar,
Aarti Kawatkar, Jason Grant Kettle, Paul D Lyne, Jon A. Read, Caroline Rivard Costa, Linette
Ruston, Patricia Schroeder, Jie Shi, Qibin Su, Scott Throner, Dorin Toader, Melissa Marie
Vasbinder, Richard Woessner, Haixia Wang, Allan Wu, Minwei Ye, Weijia Zheng, and Michael Zinda
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00076 • Publication Date (Web): 01 Jun 2018
Downloaded from http://pubs.acs.org on June 1, 2018
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Discovery and Optimization of a Novel Series of Highly Selective JAK1
Kinase Inhibitors
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Neil P. Grimster,1* Erica Anderson,1 Marat Alimzhanov,1 Geraldine Bebernitz,1 Kirsten Bell,1
Claudio Chuaqui,1 Tracy Deegan,1 Andrew D. Ferguson,2 Thomas Gero,1 Andreas Harsch,3
Dennis Huszar,3 Aarti Kawatkar,1 Jason G. Kettle,3 Paul Lyne,1 Jon A. Read,4 Caroline Rivard
Costa,1 Linette Ruston,5 Patricia Schroeder,1 Jie Shi,1 Qibin Su,1 Scott Throner,1 Dorin Toader,1
Melissa Vasbinder,1 Richard Woessner,1 Haixia Wang,1 Allan Wu,2 Minwei Ye,1 Weijia Zheng,1
Michael Zinda.1
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1Oncology, IMED Biotech Unit, AstraZeneca, Waltham, USA. Discovery Sciences, IMED
Biotech Unit, AstraZeneca, Waltham, USA. 3Oncology, IMED Biotech Unit, AstraZeneca,
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Cambridge, UK. Discovery Sciences, IMED Biotech Unit, AstraZeneca, Cambridge, UK.
5Pharmaceutical Sciences, IMED Biotech Unit, AstraZeneca, Macclesfield, UK.
Abstract
Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling, and have thus
been implicated in both cancer and inflammatory diseases. The JAK family consists of 4 highly
homologous members: JAK1,2,3 and TYK2. The development of small molecule inhibitors that
are selective for a specific family member would represent highly desirable tools for
deconvoluting the intricacies of JAK family biology.
Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays ~1000 fold
selectivity over the other highly homologous JAK family members (determined by biochemical
assays), while also possessing good selectivity over other kinases (determined by panel
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screening). Moreover, this compound was demonstrated to be orally bioavailable, and possesses
acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose
dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1
inhibition).
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Introduction
Janus kinases (JAKs) are critical in cytokine signaling through type I and II receptors, and have
been linked to both cancer1 and inflammatory diseases.2 The JAK family consists of four
members: JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2), and there are currently two FDA
approved JAK family inhibitors on the market: tofacitinib (1, a JAK1/3 inhibitor from Pfizer)3
for rheumatoid arthritis, and ruxolitinib (2, a selective JAK1/2 inhibitor from Incyte/Novartis)4
for myelofibrosis (Figure 1).
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Figure 1. FDA approved JAK family inhibitors. a Measured in our inꢀhouse assays at 5 mM ATP
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concentration. All values represent the geometric mean of at least two independent experiments.
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Both compounds display good efficacy in a number of disease areas, but have known on target
toxicities, e.g., increased risk of infection, anemia (platelets, red and white blood cells),
hypercholesterolemia, and increased transglutaminases and creatinine levels. As such, second
generation inhibitors with increased levels of selectivity for JAK1 are currently under clinical
evaluation.5 The expectation is that these agents will enable better prognosis upon chronic usage,
because their selectivity over JAK2 will lead to less severe hematological side effects.6
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In oncology, the JAK/STAT pathways have been associated with a wide variety of malignancies
upon constitutive activation. This can occur through autocrine and paracrine cytokine
production, as well as specific activating mutations on the signaling pathway (e.g. cytokine
receptors, JAKs, or STATs). Increased activation of STAT3 via phosphorylation at Y705
(pSTAT3) is reported to be elevated in a wide variety of cancers, including breast, liver, prostate,
colorectal, head and neck, esophageal, pancreatic, bladder, and nonꢀsmall cell lung;7 it has also
been implicated in the pathogenesis of diffuse large Bꢀcell lymphoma8 and nasopharyngeal
carcinomas.9 Overall, persistent elevated STAT3 activity is present in ~70% of human tumors,10
and has been linked to poorer prognosis in many of these settings.11 Moreover, elevation of
STAT3 signaling is observed in response to both chemotherapy and treatment with oncogenic
signaling pathway inhibitors e.g. EGFR, MAPK and Akt.12 JAK1 is often the primary kinase that
phosphorylates STAT3, and thus drives downstream signaling.13 Taken together, these findings
suggest that small molecule inhibition of JAK1 may offer a potential therapeutic approach
toward the treatment of these resistance or “escape” mechanisms. The highly complex interplay
of the JAK/STAT pathways, however, means that elucidating the precise role of JAK1 in these
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processes has proved challenging. One approach to address these challenges is through the
development of small molecule inhibitors of JAK1 that display a high degree of selectivity vs.
the other family members.
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Herein, we describe the discovery of a highly potent ATPꢀcompetitive JAK1 inhibitor. This
compound displays remarkable selectivity against the other JAK family members, as well as
excellent selectivity against all other kinases tested. A detailed biological and pharmacokinetic
evaluation of this compound will also be described.
Chemistry
2,4ꢀDichloropyrimidines, differentially substituted at the Cꢀ5 position, were reacted with anilines
under basic conditions to selectively displace the Cꢀ4 chloride. A subsequent acid catalyzed
aniline displacement yielded the desired bisꢀanilino pyrimidines (For an example see Scheme 1).
Scheme 1. Example Synthesis of Compound 7a
aReagents and conditions: (a) NaHCO3, EtOH, 80 °C, 16 h, 63 %; (b) S14, HCl, EtOH, 120 °C (microwave), 2 h, 36 %.
To override the inherent Cꢀ4 reactivity of the pyrimidine core, formylated anilines were
deprotonated with sodium hydride and then reacted with Cꢀ2 methyl sulfone pyrimidines.14
A
subsequent basic workꢀup cleaved the formyl group and yielded the desired 2ꢀanilinoꢀ4ꢀ
chloropyrimidines. Substitution at the 4ꢀposition was carried out under acidic conditions in the
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case of anilines, and basic conditions when alkylamines were used (For an example see Scheme
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Scheme 2. Example Synthesis of Compound 24a
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aReagents and conditions: (a) NaH, THF, 40 °C, 16 h, 83% (b) (R)ꢀbutanꢀ2ꢀamine, DIPEA, butanꢀ1ꢀol, 125 °C, 24 h, 69%.
Results and Discussion
SAR Development. To identify selective JAK1 inhibitors, we first screened a 50,000 membered
subset of our compound collection, which was biased toward motifs known to bind the ATP
binding site of kinases. Initially, three point IC50s were determined, and approximately 1,400
compounds were identified as possessing an IC50 <100 nM against JAK1 (Km ATP
concentration, 55 µM). These compounds were subsequently reꢀscreened in a 5 point IC50 format
vs. JAK1, 2, and 3, at the corresponding Km ATP concentrations (55 µM, 15 µM, and 3µM;
respectively) in order to identify potent and selective starting points for further optimization.
Through these efforts, we identified compound 3 from our previously described EphB4
program.15 Compound 3 displayed excellent JAK1 biochemical potency, as well as moderate
selectivity over the other JAK family members tested (Table 1). Interestingly, we observed that
the degree of selectivity toward JAK1 appeared to be, in part, determined by the Cꢀ2 aniline,
with other bisꢀ2,4ꢀanilinoꢀpyrimidines, such as 4, showing a preference for JAK2 inhibition
(Table 1).
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Table 1. Initial Screening Hits
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a
Measured at Km ATP concentration. All values represent the geometric mean of at least two independent experiments. For experimental
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details, see the Supporting Information. Fold selectivity derived from JAK2/JAK1 enzyme IC50. For experimental details, see the Supporting
Information.
We believed that both 3 and 4 were ATP competitive inhibitors that accessed the same binding
mode as previously reported,15 i.e. the Cꢀ2 aniline of the pyrimidine was positioned toward the
solvent channel. While the ATP binding sites of JAK1, 2, and 3 are highly homologous, there are
key structural differences in this region; notably, the solvent channel in JAK1 is flanked by a
positively charged arginine while the analogous residues in JAK2 and 3 are neutral (glutamine
and serine, respectively). We hypothesized that the interaction of the methylꢀsulfone of 3 with
the arginine may account for its observed selectivity (JAK1 Vs. JAK2).
To test this hypothesis, we combined the Cꢀ2 aniline of 3 with the Cꢀ4 aniline of 4, and
synthesized compound 5. Initially, we observed that under our assay conditions (Km ATP
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concentration) 5 maintained JAK1 potency (Table 2). Increasing the ATP concentration to a
more physiologically relevant level (5 mM), reduced the IC50 of 5 to 100 nM vs. JAK1 (Table
2). Utilizing the same high ATP conditions to probe JAK2 and JAK3 activity revealed 5 to be
139 and >300 fold selective for JAK1 over JAK2 and JAK3, respectively. (Table 2). Moreover,
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replacing the highly lipophilic 3ꢀchloroꢀ2,4ꢀdifluoroaniline group from
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3ꢀhydroxymethylaniline from 4 had the effect of significantly lowering logD (3.11 vs. 1.75) and
thus generating a more favorable starting point to begin medicinal chemistry efforts. Initially, we
focused on identifying an optimal Cꢀ5 substituent of the pyrimidine ring (5ꢀ9, Table 2).
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Table 2. C-5 Pyrimidine Substitution
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b
a Measured at 5 mM ATP concentration. All values represent the geometric mean of at least two independent experiments. Fold selectivity
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derived from JAK2/JAK1 enzyme IC50. For experimental details, see the Supporting Information. Measured at Km ATP concentration. For
experimental details, see the Supporting Information.
As can been seen in Table 2, increasing the size of the substituent at the Cꢀ5 position led to an
increase in potency for JAK1. While potency was also increased vs. JAK2, selectivity was
improved for the 5ꢀMe (7) and 5ꢀCl (8) substitution. We elected to continue our studies
maintaining a constant Cꢀ5 methyl group, due to its acceptable balance of potency, selectivity
and lipophilicity.
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Figure 2. Xꢀray crystal structures of 7 (PDB ID 6ELR) bound to the human JAK1 kinase domain. Carbon atoms for compounds are shown in
green. The protein backbone cartoon is represented in yellow. Selected atoms for the JAK1 side chains are shown as sticks. Close polar contacts
are represented as dotted cyan lines. Refined (2fofc) electron density contoured at 1.0 σ is represented as a wire mesh. Some atoms of the active
site covering loop have been removed for clarity.
Subsequently, a coꢀcrystal structure of 7 bound in the kinase domain of JAK1 was obtained
(Figure 2). As expected, the aminopyrimidine core forms a hydrogen bond donorꢀacceptor motif
with the backbone amides of the hinge region, Phe958 and Leu959; while the 5ꢀMe substituent is
positioned proximal to gatekeeper residue, Met956. The NꢀH of the Cꢀ4 aniline appears to
interact with Asp1021 via a bridging water molecule, and the hydroxymethyl group forms a
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hydrogen bond to the amide side chain of Asn1008. In the solvent channel, the Nꢀmethyl
piperazine base and methyl sulfone generate a twoꢀpoint binding interaction; the base forms a
salt bridge with Glu966, while the sulfone forms a bidentate interaction with Arg879, as
hypothesized. We believed that these structural features in the solvent channel may account for
the observed selectivity of 7 (JAK1 vs. JAK2,3).
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Having identified this twoꢀpoint interaction in the solvent channel, we decided to probe the
contributions of the individual components (NꢀMe piperazine and methylsulfone). To this end,
compounds 10ꢀ11 were synthesized (Table 3) and screened against JAK1, JAK2 and JAK3.
Consistent with our model that both interactions are required for potent and selective JAK1
inhibitors, replacement of the basic nitrogen with an oxygen, 10, or removal of the sulfone
moiety, 11, resulted in a reduction in both potency and selectivity.
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Table 3. Probing the Solvent Tail Interactions
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a
Measured at 5 mM ATP concentration. All values represent the geometric mean of at least two independent experiments. For experimental
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details, see the Supporting Information. Fold selectivity derived from JAK2/JAK1 enzyme IC50. Represents cellular mode of action assay
measuring inhibition of pSTAT3 in NCIꢀH1975 cells. For experimental details, see the Supporting Information. N. D. = not determined.
Having generated a potent JAK1 inhibitor, 7, we were intrigued to examine whether it would
show pathway modulation in a cellular setting. To this end, we developed a mode of action assay
in NCIꢀH1975 cells, and were pleased to observe that 7 inhibited the phosphorylation of STAT3,
a direct substrate of JAK1, with an EC50 of 99 nM (Table 3 and Supporting Information). With
a compound in hand capable of modulating the JAK1ꢀSTAT3 pathway, and an understanding of
the structural features that govern selectivity over closely related family members, we turned our
attention to its metabolic stability. We noted that 7 suffered from high in vitro metabolism
(human liver microsomes: CLint = 59.5 ꢀl/min/mg (Table 4)), and believed that this may be due
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to demethylation of the NꢀMe piperazine base.16 In an attempt to address this possible metabolic
hotspot, we first synthesized secondary amine 12. While 12 maintained potency and selectivity
in our biochemical assay (Table 4), we observed a large reduction in cellular potency. We
attributed this to its poor cellular permeability, a problem that was not alleviated upon steric
modulation of the secondary amine’s pKA through incorporation of flanking cisꢀmethyl groups,
13 (Table 4). Next, we removed the basic center entirely by capping it as an acetamide, 14, and
somewhat surprisingly observed only a minor drop in potency for JAK1. Unfortunately, 14
displayed a significant reduction in selectivity over JAK2. Compounds 15 and 16 attempted to
modulate the basicity of the NꢀMe piperazine, but introduction of an extra hydrogen bond donor,
15, resulted in a substantial disconnect between biochemical and cellular activity; while 16 lost
potency against JAK1. Finally, we introduced a bicyclic base, 17, which yielded similar levels of
potency (biochemical and cellular) and selectivity to 7, but was found to be more metabolically
stable (human liver microsomes: CLint = 7.3 vs. 59.5 ꢀl/min/mg, respectively, Table 4).
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Table 4. Base Optimization
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a
Measured at 5 mM ATP concentration. All values represent the geometric mean of at least two independent experiments. For experimental
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details, see the Supporting Information. Represents cellular mode of action assay measuring inhibition of pSTAT3 in NCIꢀH1975 cells. For
experimental details, see the Supporting Information. N. D. = not determined.
During optimization of the base region, we had observed that small structural changes could
result in a disconnect between cellular and biochemical potency, and believed that the
compounds’ high polar surface area (PSA – Table 4) reduced their ability to traverse the cell
membrane.17 To address this issue, we synthesized 18, which contained a diꢀfluoro methyl group
as an isostere18 for the highly polar methyl sulfone (Table 5). Pleasingly, despite observing a
reduction in JAK1 biochemical potency, 18 displayed improved cellular activity (Table 5).
Unfortunately, this came at the expense of selectivity over other JAK family members. In an
attempt to lock the conformation of 18 at the base of the solvent channel, and thereby rigidify its
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twoꢀpoint binding mode, we inserted an orthoꢀfluorine atom, 19. Remarkably, 19 was found to
be completely inactive in our biochemical assay against both JAK2 and JAK3. Unfortunately,
the removal of the sulfone motif and the addition of three fluorine atoms resulted in an ~2 log
unit increase in lipophilicity (measured logD 17: 0.82 vs. 19: 2.80), which we believed may lead
to poor overall kinase selectivity. In light of this, we replaced the diꢀfluoro methylphenyl group
with a more polar pyridine motif, 20, which we believed may interact with Arg879 via a bridging
water molecule. Pleasingly, 20 displayed good JAK1 biochemical potency and excellent JAK
family selectivity. It should be noted that removal of pyridine nitrogen, 21, maintained JAK
family selectivity but at the expense of JAK1 potency, providing some evidence for the proposed
water mediate interaction with Arg879, and suggesting that the placement of the fluorine atom is
key to JAK family selectivity.
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Table 5. Sulfone Isosteres
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a
Measured at 5 mM ATP concentration. For experimental details, see the Supporting Information. b Represents cellular mode of action assay
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measuring inhibition of pSTAT3 in NCIꢀH1975 cells. Represents cellular antiꢀproliferation assay in A549 cells. For experimental details, see
the Supporting Information. N.D. = not determined.
Having designed out the sulfone group, while maintaining potency and selectivity for JAK1, we
were keen to assess the offꢀtarget activity of 20. When we evaluated it an A549 cellular
proliferation assay (a cell line in which JAK1 knockdown had no effect on cellular viability or
proliferation)19, we noted that 20 potently inhibited proliferation (GI50 = 0.253 ꢀM).
We believed that to generate a truly unique tool molecule capable of probing JAK1 biology in
vivo required further improvement in overall kinase selectivity.5 To this end, we synthesized a
diverse library of compounds which varied the Cꢀ4 substituent of 20, installing both alkyl amines
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and anilines. These compounds were evaluated for JAK1 biochemical potency and their
antiproliferative effects in A549 cells. This exercise revealed several compounds which
possessed high levels of JAK1 potency, but appeared inactive against our A549 assay (Graph 1
– boxed compounds).
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Graph 1. Plot of JAK1 biochemical potency vs. cellular antiꢀproliferation assay in A549 cells.
Seven compounds in the black box possess potent JAK1 activity while proving inactive (>10
ꢀM) against A549 proliferation.
Of these seven compounds, four contained highly lipophilic anilines and we thus focused our
attention on the remaining three compounds (22ꢀ24, Table 6). Interestingly, all three were highly
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homologous, with 22 and 2420 sharing a common R configuration at the stereogenic center.
Further profiling these compounds revealed remarkably similar profiles, as may be expected.
Compound 24, however, possessed a lower logD (24: 2.26 vs. 22: 2.70 23: 2.70), and we
therefore elected to examine whether it would make an appropriate in vivo probe molecule.
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Table 6. C-5 Alkylamine Compounds
a Measured at 5 mM ATP concentration. All values represent the geometric mean of at least two independent experiments. b Represents cellular
c
mode of action assay measuring inhibition of pSTAT3 in NCIꢀH1975 cells. Represents cellular antiꢀproliferation assay in A549 cells. For
experimental details, see the Supporting Information.
Selectivity profile
Having already established that 24 displayed excellent selectivity against both JAK2 and JAK3,
we determined its potency against the final family member TYK2, under high ATP conditions.
Pleasingly, 24 also showed no activity (>30 ꢀM) against TYK2. With a compound in hand that
we were confident had ~1000 fold selectivity over closely related family members, we were
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interested in assessing its broader kinase selectivity. As such, we screened it against a panel of
386 kinases at a single concentration (1 ꢀM). The kinome profile of compound 24 is available as
Supporting Information (Table 8). In line with our inꢀhouse data, 24 displayed 100 % inhibition
at 1 ꢀM for JAK1, while appearing far less active against JAK2 (31 %) and JAK3 (46 %). For all
other kinases tested, only PRKD1 (73 %), NUAK1 (71 %) and LRRK2 (71 %) displayed >70%
inhibition, while 9 others showed >50% inhibition.
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Pharmacokinetic Properties of 24
Having established that 24 was both a highly potent and selective JAK1 inhibitor, we performed
a pharmacokinetic study in mice to evaluate its potential as an in vivo tool compound (Table 7).
Table 7. Pharmacokinetic Properties of 24
a 10% HPbCD + 5% dextrose in WFI at pH 4. b 5% SBECD in WFI at pH 2. N. D. = not determined. For experimental details, see the Supporting
Information.
While 24 displayed moderately high clearance but acceptable bioavailability at lower dose
(10 mg/kg), the plasma concentrations reached at the higher oral dose of 50 mg/kg were
anticipated to achieve target coverage (Free plasma concentration over cellular EC50, Mouse PPB
= 15 % free) for ~12 hours. We proceeded to test this theory in an in vivo xenograft model.
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24 Demonstrates a Clear Pharmacodynamic (PD) Effect in NCI-H1975 Mouse Xenograft
Model.
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As previously discussed, STAT3 is a direct phosphorylation substrate of JAK1 in NCIꢀH1975
cells. Therefore, we measured pSTAT3 levels in tumors from an NCIꢀH1975 mouse xenograft
model. Single doses of 24 at 1, 3, 10, 30, 50 and 100 mg/kg were given by oral gavage and
animals were sacrificed at 2 h. An additional 6 h group was added for the 50 mg/kg dose.
Interestingly, we observed a dose dependent decrease in pSTAT3 across the doses examined,
which correlated with the plasma concentrations of 24 (Graph 2). In addition, after 6 h the 50
mg/kg dose showed a reduction in both the plasma concentration of 24 as well as suppression of
pSTAT3 (Graph 2).
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Graph 2. Plot of %inhibition of pSTAT3 in an NCIꢀH1975 mouse xenograft model and the
corresponding plasma concentrations of 24. Student’s tꢀtest calculated versus vehicle control; p
<0.05 for doses 100, 50 (2 and 6 hr), and 30 mg/kg.
Conclusion
In summary, we have described the discovery of a highly potent and selective JAK1 ATP
competitive inhibitor 24, which displays almost 1,000 fold selectivity over the highly
homologous JAK family members (as determined by high ATP biochemical assays), as well as
good selectivity against other kinases tested (as determined by panel screening). Compound 24
also possesses favorable PK parameters, and was demonstrated to be orally efficacious at
inhibiting the phosphorylation of STAT3 in tumors in an in vivo model. In the future, we believe
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that 24 will prove a valuable probe molecule for deconvoluting the intricacies of the JAK family
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biology.
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Experimental Section
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All reactions were performed under inert conditions (nitrogen) unless otherwise stated.
Temperatures are given in degrees Celsius (°C); operations were carried out at room or ambient
temperature, that is, at a temperature in the range of 18 to 25°C. All solvents and reagents were
purchased from commercial sources and used without further purification. For coupling
reactions, all solvents were dried and degassed prior to reaction. Reactions performed under
microwave irradiation utilized either a Biotage Initiator or CEM Discover Microwave. Upon
work up, organic solvents were typically dried prior to concentration with anhydrous MgSO4 or
Na2SO4. Flash silica chromatography was typically performed on an Isco Companion, using
Silicycle silica gel, 230ꢀ400 mesh 40ꢀ63 ꢁm cartridges, Grace Resolv silica cartridges or Isolute
Flash Si or Si II cartridges. Reverse phase chromatography was performed using a Waters
XBridge Prep C18 OBD column, 5ꢁ silica, 19 mm diameter, 100 mm length), using decreasingly
polar mixtures of either water (containing 0.2% NH4OH) and acetonitrile, or water (containing
0.1% formic acid) and acetonitrile, as eluents. Analytical LCꢀMS was performed on a Waters
2790 LC with a 996 PDA and 2000 amu ZQ Single Quadrupole Mass Spectrometer using a
Phenomenex Gemini 50 x 2.1mm 5 ꢁm C18 column, or UPLCꢀMS was performed on a Waters
Acquity UPLC and Waters SQD mass spectrometer. UV detection = 210ꢀ400nm, mass spec =
ESI with positive/negative switching. The column used was a Waters Acquity HSS T3, 1.8 µm,
o
2.1 x30 mm, at temperature 30 C. Flow rate was 1mL/min using a solvent gradient of 2 to 98%
B over 1.5 minutes (total runtime with equilibration back to starting conditions = 2 min), where
A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile. Purities were measured
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by UV absorption at 254 nm or TIC and are ≥ 95% unless otherwise stated. NMR spectra were
recorded on a Bruker AV300, AV400 and DRX500 spectrometer at 300 or 500 MHz at 303K
unless otherwise indicated. 1H NMR spectra are reported as chemical shifts in parts per million
(ppm) relative to an internal solvent reference. Yields are given for illustration only and are not
necessarily those which can be obtained by diligent process development; preparations were
repeated if more material was required. Purity of final compounds was assessed by reversedꢀ
phase UHPLC with UV diode array detection at 210ꢀ400 nM; all tested compounds were >95%
purity.
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General Procedure: Displacement of Cꢀ2 Pyrimidine Chlorides with Anilines
To an alcoholic solution (0.128 mmol) of 5ꢀsubsituted (3ꢀ((2ꢀchloropyrimidinꢀ4ꢀ
yl)(methyl)amino)phenyl)methanol (1.2 eq) in a microwave reaction vessel was added 3ꢀ(4ꢀ
methylpiperazinꢀ1ꢀyl)ꢀ5ꢀ(methylsulfonyl)aniline (1 eq), and acid (3 eq) and heated to 80ꢀ125 °C
in a microwave for 1ꢀ2 h. Solvent was evaporated under reduced pressure and the resultant
residue was partitioned between ethyl acetate and sat. NaHCO3 aq. The organic layer was taken,
washed with brine, and dried over MgSO4, filtered and evaporated. The residue was purified as
indicated per example.
N4-(3-chloro-2,4-difluorophenyl)-N2-(3-(4-methylpiperazin-1-yl)-5-
(methylsulfonyl)phenyl)pyrimidine-2,4-diamine (3).
Scale: 0.56 mmol. Acid: pTSA. Solvent: IPA. Temp: 125 °C. Time: 1 h. Purification column
chromatography (silica gel, 0ꢀ2% MeOH (1% 7M NH3 in MeOH)/DCM). Sample: 3 as a light
brown solid (25 mg, 14%); 1H NMR (300 MHz, DMSOꢀd6) δ 9.14 ꢀ 9.42 (m, 2 H), 8.10 (d, 1 H),
7.91 ꢀ 8.07 (m, 1 H), 7.70 (s, 1 H), 7.56 (s, 1 H), 7.26 (td, 1 H), 6.92 (s, 1 H), 6.37 (d, 1 H), 2.96 ꢀ
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3.17 (m, 8 H), 2.35 ꢀ 2.44 (m, 3 H), 2.21 (s, 3 H); m/z (ES+) (M+H)+ = 509.13.
5
6
7
(3-(2-(3,5-Dimorpholinophenylamino)-5-methylpyrimidin-4-ylamino)phenyl)methanol (4).
8
9
Scale: 0.20 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: EtOH. Temp: 100 °C. Time: 3 h.
Purification: column chromatography (silica gel, 0ꢀ2% MeOH (1% 7M NH3 in MeOH)/DCM).
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1
Sample: 4 as an off white solid (38 mg, 14%); H NMR (300 MHz, DMSOꢀd6) δ 8.63 (s, 1 H),
8.22 (s, 1 H), 7.88 (s, 1 H), 7.65 (s, 1 H), 7.55 (s, 1 H), 7.21 (t, 1 H), 6.97 (d, 1 H), 6.84 (s, 2 H),
6.02 (s, 1 H), 5.12 (t, 1 H), 4.46 (d, 2 H), 3.56 ꢀ 3.68 (m, 8 H), 2.83 ꢀ 2.95 (m, 8 H), 2.08 (s, 3 H);
m/z (ES+) (M+H)+ = 477.26.
(3-(2-(3-(4-Methylpiperazin-1-yl)-5-(methylsulfonyl)phenylamino)pyrimidin-4-
ylamino)phenyl)methanol (5).
Scale: 0.26 mmol. Acid: HCl, 2M in diethyl ether. Solvent: EtOH. Temp: 80 °C. Time: 2.5 h.
Purification: reverseꢀphase chromatography (25ꢀ35% MeCN/H2O 0.2% NH4OH, 4 min).
Sample: 5 as an offꢀwhite solid (5 mg, 4%); 1H NMR (300 MHz, DMSOꢀd6) δ 9.31 (br. s., 1 H),
9.22 (br. s., 1 H), 7.98 (d, 1 H), 7.66 ꢀ 7.90 (m, 2H), 7.62 (s, 1 H), 7.38 (s, 1 H), 7.20 (t, 1 H),
6.73 ꢀ 7.04 (m, 2 H), 6.21 (d, 1 H), 5.08 (br. s., 1 H), 4.42 (s, 2 H), 3.07 (m, 7 H), 2.36 (m, 4 H),
2.12 (s, 3 H); m/z (ES+) (M+H)+ = 469.31.
(3-(5-fluoro-2-(3-(4-methylpiperazin-1-yl)-5-(methylsulfonyl)phenylamino)pyrimidin-4-
ylamino)phenyl)methanol (6).
Scale: 0.48 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: EtOH. Temp: 120 °C. Time: 0.5 h.
Purification: reverseꢀphase chromatography (30ꢀ60% MeCN/H2O 0.2% NH4OH, 10 min).
1
Sample: 6 as an offꢀwhite solid (100 mg, 43%); H NMR (300 MHz, DMSOꢀd6) δ 9.23 ꢀ 9.48
(m, 2 H), 8.16 (d, 1 H), 7.79 (d, 1 H), 7.55 ꢀ 7.73 (m, 3 H), 7.29 (t, 1 H), 7.04 (d, 1 H), 6.94 (s, 1
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H), 5.15 (br. s., 1 H), 4.50 (s, 2 H), 2.94 ꢀ 3.27 (m, 8 H), 2.40 (m, 3 H), 2.21 (s, 3 H); m/z (ES+)
5
6
(M+H)+ = 487.21.
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8
9
(3-(5-methyl-2-(3-(4-methylpiperazin-1-yl)-5-(methylsulfonyl)phenylamino)pyrimidin-4-
ylamino)phenyl)methanol (7).
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Scale: 4.00 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: EtOH. Temp: 120 °C. Time: 2 h.
Purification: reverseꢀphase chromatography (20ꢀ40% MeCN/H2O 0.2% NH4OH, 10 min).
1
Sample: 7 as an offꢀwhite solid (0.70 g, 36%). H NMR (300 MHz, DMSOꢀd6) δ 9.14 (s, 1 H),
8.29 (s, 1 H), 7.95 (s, 1 H), 7.63 ꢀ 7.81 (m, 3 H), 7.57 (s, 1H), 7.26 (t, 1 H), 6.99 (d, 1 H), 6.89 (s,
1 H), 5.13 (t, 1 H), 4.49 (d, 2 H), 2.95 ꢀ 3.15 (m, 8 H), 2.40 (s, 3 H), 2.21 (s, 3 H), 2.13 (s, 3 H);
m/z (ES+) (M+H)+ = 483.21.
(3-(5-chloro-2-(3-(4-methylpiperazin-1-yl)-5-(methylsulfonyl)phenylamino)pyrimidin-4-
ylamino)phenyl)methanol (8).
Scale: 0.56 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: EtOH. Temp: 120 °C. Time: 0.5 h.
Purification: reverseꢀphase chromatography (20ꢀ60% MeCN/H2O 0.2% NH4OH, 10 min).
1
Sample: 8 as an offꢀwhite solid (0.14 g, 51%); H NMR (300 MHz, DMSOꢀd6) δ 9.09 (s, 1 H),
8.69 (s, 1 H), 7.89 (s, 2 H), 7.76 (s, 1 H), 7.68 (s, 2 H), 7.27 (t, 1 H), 7.01 (d, 1 H), 6.89 (s, 1 H),
5.11 (t, 1 H), 4.49 (d, 2 H), 3.88 (s, 3 H), 3.03 ꢀ 3.21 (m, 4 H), 2.42 (m, 4 H), 2.22 (s, 3 H); m/z
(ES+) (M+H)+ = 503.13.
(3-(5-Bromo-2-(3-(4-methylpiperazin-1-yl)-5-(methylsulfonyl)phenylamino)pyrimidin-4-
ylamino)phenyl)methanol (9).
Scale: 0.37 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: EtOH. Temp: 125 °C. Time: 1 h.
Purification: reverseꢀphase chromatography (20ꢀ60% MeCN/H2O 0.2% NH4OH, 10 min).
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Sample: 9 as an offꢀwhite solid (109 mg, 54%); 1H NMR (300 MHz, DMSOꢀd6) δ 9.44 (s, 1 H)
8.55 (s, 1 H) 8.29 (s, 1 H) 7.55 ꢀ 7.77 (m, 3 H) 7.51 (s, 1H) 7.29 (t, 1 H) 7.07 (d, 1 H) 6.94 (s, 1
H) 4.97 ꢀ 5.29 (m, 1 H) 4.48 (d, 2 H) 2.93 ꢀ 3.14 (m, 8 H) 2.31 ꢀ 2.43(m, 3 H) 2.21 (s, 3 H); m/z
(ES+) (M+H)+ = 548.8.
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(3-(5-Methyl-2-(3-(methylsulfonyl)-5-morpholinophenylamino)pyrimidin-4-
ylamino)phenyl)methanol (10).
Scale: 0.56 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: EtOH. Temp: 125 °C. Time: 0.5 h.
Purification: column chromatography (silica gel, 0ꢀ3% MeOH/DCM). Sample: 10 as an offꢀ
1
white solid (115 mg, 63%); H NMR (300 MHz, DMSOꢀd6) δ 9.19 (s, 1 H), 8.31 (s, 1 H), 7.95
(s, 1 H), 7.60 ꢀ 7.80 (m, 3 H), 7.55 (s, 1 H), 7.26 (t, J = 7.82 Hz, 1 H), 7.00 (d, 1 H), 6.89 (s, 1
H), 5.13 (t, 1 H), 4.49 (d, 2 H), 3.53 ꢀ 3.77 (m, 4 H), 3.11 (s, 3 H), 2.87 ꢀ 3.06 (m, 4 H), 2.13 (s, 3
H); m/z (ES+) (M+H)+ = 470.3.
(3-(5-Methyl-2-(3-(4-methylpiperazin-1-yl)phenylamino)pyrimidin-4-
ylamino)phenyl)methanol (11).
Scale: 0.34 mmol. Acid: HCl, 2 M in diethyl ether. Solvent: TFE. Temp: 160 °C. Time: 1 h.
Purification: reverseꢀphase chromatography (20ꢀ50% MeCN/H2O 0.2% NH4OH, 5 min).
Sample: 11 as an offꢀwhite solid (70 mg, 50%); 1H NMR (300 MHz, DMSOꢀd6) δ 8.73 (s, 1 H),
8.22 (s, 1 H), 7.88 (s, 1 H), 7.67 (d, 1 H), 7.59 (s, 1 H), 7.17 ꢀ 7.29 (m, 3 H), 6.94 ꢀ 7.03 (m, 2 H),
6.45 (dd, 1 H), 5.12 (t, 1 H), 4.49 (d, 2 H), 2.91 ꢀ 3.03 (m, 4 H), 2.35 ꢀ 2.42 (m, 4 H), 2.20 (s, 3
H), 2.10 (s, 3 H); m/z (ES+) (M+H)+ = 403.2.
(3-(5-methyl-2-(3-(methylsulfonyl)-5-(piperazin-1-yl)phenylamino)pyrimidin-4-
ylamino)phenyl)methanol (12).
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Scale: 0.51 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: EtOH. Temp: 110 °C. Time: 1 h.
Purification: column chromatography (silica gel, 0ꢀ10% MeOH (1% 7M NH3 in MeOH)/DCM).
Sample: 12 as an off white solid (38 mg, 14%); 1H NMR (300 MHz, DMSOꢀd6) δ 9.13 (s, 1 H),
8.29 (s, 1 H), 7.95 (d, 1 H), 7.75 (d, 1 H), 7.61 ꢀ 7.72 (m, 2H), 7.57 (s, 1 H), 7.26 (t, 1 H), 7.00
(d, 1 H), 6.87 (d, 1 H), 5.13 (br. s., 1 H), 4.49 (s, 2 H), 2.92 ꢀ 3.20 (m, 7 H), 2.65 ꢀ 2.88 (m, 4H),
2.00 ꢀ 2.17 (m, 3 H); m/z (ES+) (M+H)+ = 509.13.
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(3-(2-(3-((cis)-3,5-dimethylpiperazin-1-yl)-5-(methylsulfonyl)phenylamino)-5-
methylpyrimidin-4-ylamino)phenyl)methanol (13).
To a suspension of 3ꢀ(2ꢀchloroꢀ5ꢀmethylpyrimidinꢀ4ꢀylamino)phenyl)methanol (0.10 g, 0.40
mmol), 3ꢀ((cis)ꢀ3,5ꢀdimethylpiperazinꢀ1ꢀyl)ꢀ5ꢀ(methylsulfonyl)aniline (113 mg, 0.40 mmol) and
Cs2CO3
(0.39
g,
1.20
mmol)
in
1,4ꢀdioxane
(2
mL)
were
added
tris(dibenzylideneacetone)dipalladium (0) (37 mg, 0.04 mmol) and 2ꢀdicyclohexylphosphinoꢀ
2',4',6'ꢀtriisopropylbiphenyl (57 mg, 0.12 mmol) and sealed under a nitrogen atmosphere. The
resultant reaction mixture was stirred at 100 °C under nitrogen overnight. The reaction mixture
was cooled to room temperature, diluted with DCM and filtered. The filtrate was concentrated to
dryness and purified by column chromatography (silica, 0ꢀ10% MeOH/DCM) to afford 13 as an
1
offꢀwhite solid (47 mg, 23%); H NMR (300 MHz, DMSOꢀd6) δ 9.13 (s, 1 H), 8.27 (br s, 1H),
7.95 (s, 1 H), 7.79 (s, 1 H), 7.71 (s, 1 H), 7.62 (d, 2 H), 7.26 (t, 1 H), 6.99 (d, 1 H), 6.90 (s, 1 H),
5.11 (s, 1 H), 4.49 (d, 2 H), 3.45 (d, 2 H), 3.11 (s, 3 H), 2.71 ꢀ 2.84 (m, 2 H), 2.04 ꢀ 2.17 (m, 5
H), 0.98 (d, J = 6.22 Hz, 6 H); m/z (ES+) (M+H)+ = 497.26.
1-(4-(3-(4-(3-(hydroxymethyl)phenylamino)-5-methylpyrimidin-2-ylamino)-5-
(methylsulfonyl)phenyl)piperazin-1-yl)ethanone (14).
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To a suspension of 3ꢀ(2ꢀchloroꢀ5ꢀmethylpyrimidinꢀ4ꢀylamino)phenyl)methanol (0.10 mg, 0.40
mmol), 1ꢀ(4ꢀ(3ꢀaminoꢀ5ꢀ(methylsulfonyl)phenyl)piperazinꢀ1ꢀyl)ethanone (0.12 mg, 0.40 mmol)
and Cs2CO3 (0.39 g, 1.20 mmol) in 1,4ꢀdioxane (2 mL) were added
tris(dibenzylideneacetone)dipalladium (0) (37 mg, 0.04 mmol) and 2ꢀdicyclohexylphosphinoꢀ
2',4',6'ꢀtriisopropylbiphenyl (57 mg, 0.12 mmol) and sealed under a nitrogen atmosphere. The
resultant reaction mixture was stirred at 100 °C. The reaction mixture was cooled to room
temperature, diluted with DCM and filtered. The filtrate was concentrated to dryness and
purified by column chromatography (silica gel, 0ꢀ40% MeOH/DCM) to afford 14 as a creamꢀ
colored solid (0.11 g, 96%); 1H NMR (300 MHz, DMSOꢀd6) δ 9.19 (s, 1 H), 8.32 (s, 1 H), 7.95
(s, 1 H), 7.68 ꢀ 7.76 (m, 2 H), 7.65 (s, 1 H), 7.56 (s, 1 H), 7.26 (t, 1 H), 7.00 (d, 1 H), 6.92 (s, 1
H), 5.13 (t, 1 H), 4.49 (d, 2 H), 3.50 (s, 4 H), 3.11 (s, 3 H), 2.97 ꢀ 3.10 (m, 4 H), 2.13 (s, 3 H),
2.04 (s, 3 H); m/z (ES+) (M+H)+ = 511.08.
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-(4-(3-(4-(3-(hydroxymethyl)phenylamino)pyrimidin-2-ylamino)-5-
(methylsulfonyl)phenyl)piperazin-1-yl)ethanol (15).
Scale: 0.40 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: EtOH. Temp: 80 °C. Time: 2.5 h.
Purification: column chromatography (silica gel, 0ꢀ10% MeOH/ DCM). Sample: 15 as an offꢀ
white solid (12 mg, 6%); 1H NMR (300 MHz, DMSOꢀd6) δ 9.18 (s, 1 H), 8.32 (s, 1 H), 7.95 (s,
1 H), 7.65 ꢀ 7.77 (m, 3 H), 7.56 (s, 1 H), 7.26 (t, 1 H), 7.00 (d, 1 H), 6.87 ꢀ 6.95 (m, 1 H), 5.12 ꢀ
5.21 (m, 1 H), 4.46 ꢀ 4.54 (m, 2 H), 3.52 ꢀ 3.75 (m, 2 H), 2.96 ꢀ 3.26 (m, 8 H), 2.67 (m, J = 2.00
Hz, 2 H), 2.52 ꢀ 2.64 (m, 3 H), 2.12 (s, 3 H); m/z (ES+) (M+H)+ = 513.18.
(3-(2-(3-(4-(2-Methoxyethyl)piperazin-1-yl)-5-(methylsulfonyl)phenylamino)-5-
methylpyrimidin-4-ylamino)phenyl)methanol (16).
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Scale: 0.38 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: IPA. Temp: 125 °C. Time: 1 h.
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an offꢀwhite solid (55 mg, 33%); H NMR (300 MHz, DMSOꢀd6) δ 7.89 (s, 1 H), 7.61 ꢀ 7.70
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(m, 2 H), 7.48 ꢀ 7.57 (m, 2 H), 7.31 (t, 1 H), 7.10 (d, 1 H), 6.98 ꢀ 7.05 (m, 1 H), 4.59 (s, 2 H),
3.58 (t, 2 H), 3.36 (s, 3 H), 3.07 ꢀ 3.14 (m, 4 H), 3.05 (s, 3 H), 2.62 (dt, 6 H), 2.17 (s, 3 H); m/z
(ES+) (M+H)+ = 527.19.
(3-(5-methyl-2-(3-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)-5-
(methylsulfonyl)phenylamino)pyrimidin-4-ylamino)phenyl)methanol (17).
Scale: 0.22 mmol. Acid: HCl, 1.25 M in MeOH. Solvent: EtOH. Temp: 90 °C. Time: 1 h.
Purification: column chromatography (silica gel, 0ꢀ10% MeOH/DCM). Sample: 17 as an offꢀ
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white solid (34 mg, 38%); H NMR (300 MHz, DMSOꢀd6) δ 9.01 (s, 1 H), 8.22 (s, 1 H), 7.86
(s, 1 H), 7.64 (d, 1 H), 7.49 (s, 1H), 7.39 (s, 1 H), 7.30 (s, 1 H), 7.19 (t, 1 H), 6.93 (d, 1 H), 6.44
(s, 1 H), 5.13 (br. s., 1 H), 4.41 (s, 2 H), 3.94 (br. s., 1 H), 3.31 (br. s., 1 H), 2.93 ꢀ 3.11 (m, 4 H),
2.63 (d, 1 H), 2.37 (d, 1 H), 2.12 ꢀ 2.24 (m, 3 H), 1.93 ꢀ 2.11 (m, 3 H), 1.53 ꢀ 1.83 (m, 3 H); m/z
(ES+) (M+H)+ = 495.23.
General Procedure: Displacement of C-4 Anilino-Pyrimidine Chlorides with Anilines
To a suspension of 4ꢀchloroꢀpyrimidine (1 eq) in TFE (0.1 M) was added (3ꢀaminophenyl)
methanol (1.1 eq), and 4M HCl in 1,4ꢀDioxane (4 eq) and heated to 150 °C in a microwave for 1
h. The reaction was cooled to room temp and solvent removed under reduced pressure. The
residue was purified as indicated per example.
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(3-(2-(3-(Difluoromethyl)-5-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-
yl)phenylamino)-5-methylpyrimidin-4-ylamino)phenyl)methanol (18).
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NH4OH, 10 min). Sample: 18 as an offꢀwhite solid (25 mg, 51%); H NMR (300 MHz, MeOHꢀ
d4) δ 7.87 (s, 1 H) 7.62 ꢀ 7.71 (m, 1 H) 7.45 ꢀ 7.62 (m, 1 H) 7.34 (t, 1 H) 7.15 (dd, 1.70 Hz, 2 H)
6.99 (s, 1 H) 6.51 (s, 1 H) 6.20 ꢀ 6.42 (m, 1 H) 4.60 (s, 2 H) 4.03 (s, 1 H) 3.52 (s, 1 H) 3.12 ꢀ 3.30
(m, 2 H) 2.60 ꢀ 2.85 (m, 2 H) 2.39 (s, 3H) 2.09 ꢀ 2.23 (m, 3 H) 1.63 ꢀ 2.01 (m, 2 H); m/z (ES+)
(M+H)+ = 467.3.
3-(2-(3-(Difluoromethyl)-2-fluoro-5-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-
yl)phenylamino)-5-methylpyrimidin-4-ylamino)phenyl)methanol (19).
Scale: 0.1 mmol. Purification: reverseꢀphase chromatography (30ꢀ50% MeCN/H2O 0.2%
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NH4OH, 5 min). Sample: 19 as an offꢀwhite solid (26 mg, 53%); H NMR (300 MHz, DMSOꢀ
d6) δ 8.25 (d, 2 H), 7.87 (d, J = 0.75 Hz, 1 H), 7.62 (d, 1 H), 7.55 (s, 1 H), 7.19 ꢀ 7.25 (m, 1 H),
7.16 (t, 1 H), 7.06 (t, 1H), 6.95 (d, 1 H), 6.27 ꢀ 6.37 (m, 1 H), 5.15 (br. s., 1 H), 4.41 (s, 2 H),
3.95 (s, 1 H), 3.36 (br. s., 1 H), 3.10 (d, 1 H), 2.97 (d, 1 H), 2.65 (d, 1 H), 2.37 ꢀ 2.45 (m, 1 H),
2.22 (s, 3 H), 2.09 (s, 3 H), 1.79 (d, 1 H), 1.70 (d, J = 8.85 Hz, 1 H); m/z (ES+) (M+H)+ = 485.1.
(3-(2-(2-Fluoro-5-((1S,4S)-5-methyl-2,5-diazabicyclo[2.2.1]heptan-2-yl)pyridin-3-ylamino)-
5-methylpyrimidin-4-ylamino)phenyl)methanol (20).
Scale: 0.43 mmol. Purification: reverseꢀphase chromatography (20ꢀ40% MeCN/H2O 0.2%
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NH4OH, 10 min). Sample: 20 as an offꢀwhite solid (95 mg, 51%); H NMR (300 MHz, DMSOꢀ
d6) δ 8.29 (s, 1 H), 8.23 (s, 1 H), 7.89 (d, 1 H), 7.62 (dd, 1 H), 7.53 ꢀ 7.60 (m, 2 H), 7.19 (t, 1
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