Overcoming mutagenicity and ion channel activity: Optimization of selective spleen tyrosine kinase inhibitors

Article abstract of DOI:10.1021/jm5018169

Development of a series of highly kinome-selective spleen tyrosine kinase (Syk) inhibitors with favorable druglike properties is described. Early leads were discovered through X-ray crystallographic analysis, and a systematic survey of cores within a selected chemical space focused on ligand binding efficiency. Attenuation of hERG ion channel activity inherent within the initial chemotype was guided through modulation of physicochemical properties including log D, PSA, and pK_a. PSA proved most effective for prospective compound design. Further profiling of an advanced compound revealed bacterial mutagenicity in the Ames test using TA97a Salmonella strain, and subsequent study demonstrated that this mutagenicity was pervasive throughout the series. Identification of intercalation as a likely mechanism for the mutagenicity-enabled modification of the core scaffold. Implementation of a DNA binding assay as a prescreen and models in DNA allowed resolution of the mutagenicity risk, affording molecules with favorable potency, selectivity, pharmacokinetic, and off-target profiles.

Full text of DOI:10.1021/jm5018169

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Article
Overcoming Mutagenicity and Ion Channel Activity:
Optimization of Selective Spleen Tyrosine Kinase Inhibitors
J. Michael Ellis, Michael D Altman, Alan Bass, John W Butcher, Alan J Byford, Anthony Donofrio, Sheila
Galloway, James Jewell, Andrew M. Haidle, Nancy Kelly, Erica K Leccese, Sandra Lee, Matthew L Maddess,
J Richard Miller, Lily Y Moy, Ekundayo Osimboni, Ryan D Otte, M Vijay Reddy, Kerrie Spencer, Binyuan Sun,
Stella H. Vincent, Gwendolyn J Ward, Grace HC Woo, Chiming Yang, Hani Houshyar, and Alan B. Northrup
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 27 Jan 2015
Downloaded from http://pubs.acs.org on January 28, 2015
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Overcoming Mutagenicity and Ion Channel Activity:
Optimization of Selective Spleen Tyrosine Kinase
Inhibitors
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J. Michael Ellis,a* Michael D. Altman,a Alan Bass, f John W. Butcher, a Alan J. Byford, d Anthony
Donofrio, a Sheila Galloway, f Andrew M. Haidle, a James Jewell, a Nancy Kelly,d Erica K.
Leccese, d Sandra Lee, a Matthew Maddess,b J. Richard Miller, d Lily Y. Moy, d Ekundayo
Osimboni, a Ryan D. Otte, a M. Vijay Reddy, f Kerrie Spencer, a Binyuan Sun, a Stella H. Vincent, e
Gwendolyn J. Ward, f Grace H. C. Woo, a Chiming Yang, g Hani Houshyar, c Alan B. Northrup a
Departments of Discovery Chemistry, Immunology, Pharmacology, Pharmacokinetics,
Pharmacodynamics, and Drug Metabolism, Safety Assessment and Laboratory Animal
Resources, and Discovery Pharmaceutical Sciences, Merck & Co., Inc., 33 Avenue Louis
Pasteur, Boston, MA 02115
aDepartment of Discovery Chemistry
bDepartment of Process Research
cDepartment of Immunology
dDepartment of Pharmacology
eDepartment of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism
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fDepartment of Safety Assessment and Laboratory Animal Resources
gDepartment of Discovery Pharmaceutical Sciences
Spleen Tyrosine Kinase. Ames. Mutagenicity. Ion Channels.
Abstract
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Development of a series of highly kinome selective Spleen Tyrosine Kinase (Syk) inhibitors with
favorable drug-like properties is described. Early leads were discovered through X-ray
crystallographic analysis, and a systematic survey of cores within a selected chemical space
focused on ligand binding efficiency. Attenuation of hERG ion channel activity inherent within
the initial chemotype was guided through modulation of physicochemical properties including
logD, PSA, and pKa. PSA proved most effective for prospective compound design. Further
profiling of an advanced compound revealed bacterial mutagenicity in the Ames test using
TA97a Salmonella strain, and subsequent study demonstrated that this mutagenicity was
pervasive throughout the series. Identification of intercalation as a likely mechanism for the
mutagenicity enabled modification of the core scaffold. Implementation of a DNA binding assay
as a pre-screen and models in DNA allowed resolution of the mutagenicity risk, affording
molecules with favorable potency, selectivity, pharmacokinetic, and off-target profiles.
Introduction
The utility of kinase inhibitors in treating oncological disorders was affirmed by the approval
of imatinib in 2001.1 In recent years, small molecule inhibitors of protein kinases have been
examined for use in various autoimmune disorders, including rheumatoid arthritis (RA).2 RA is a
systemic inflammatory disorder that affects ~1% of the worldwide population with women three
times more likely than men to develop the disease.3 Disease progression is characterized by
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synovial hyperplasia, inflammation, and irreversible joint destruction. This commonly leads to
disability and consequent reduction in the quality of life and, if untreated, reduces life
expectancy in patients. The pathogenesis of RA consists of a cascade of pro-inflammatory
cytokines and chemokines leading to the recruitment of inflammatory cells. Traditionally, T
cells, macrophages, neutrophils, and synovial fibroblasts were thought to be associated with
RA;4 however, current evidence suggests that autoreactive B cells, immune complexes,
osteoclasts, mast cells, and basophils also contribute to the disease.5
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Current therapies used to treat RA generally fall into two broad categories: palliative therapies
and disease-modifying agents. Palliative therapies are prescribed to provide symptomatic relief
from the pain and inflammation characteristic of RA. Disease-modifying therapies including
conventional disease-modifying antirheumatic drugs (DMARDs) and biologic agents are
prescribed to halt the destructive course of RA and prevent debilitating joint damage. Despite
their success, the use of conventional DMARDs and biologics is limited by non-response in a
subset of patients, safety concerns, and the high cost and discomfort of continuous injections
with biologics. The 2012 approval of tofacitinib, an oral pan-Janus kinase (JAK) inhibitor, for
treatment of RA cases that are inadequately responsive to methotrexate provides proof of
concept for the therapeutic approach of kinase inhibition for the treatment of RA.6
Spleen tyrosine kinase (Syk) is a key enzyme involved in Fc and B cell receptor (BCR)
signaling in multiple cell types involved in the immune response.7 Inhibition of Syk activity is
expected to slow the destructive course of RA by affecting the initiation phase via modulation of
signaling downstream of the BCR, the effector phase via blockade of the Fc receptors (FcεR,
FCγR) in various immune cells in the synovium, and the ensuing tissue damage via inhibition of
osteoclast maturation.8 Multiple preclinical studies have shown that genetic depletion of Syk or
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inhibition of Syk with small molecule inhibitors (including published R788 (1), PRT-062607 (2),
RO9021 (3), and GS-9973 (4)) attenuates inflammation, tissue damage and bone destruction in
preclinical RA models (Figure 1).9,10,11,12 Human proof-of-concept has been achieved in RA
clinical trials with the poorly selective oral Syk inhibitor 1 (52% of 100 kinases tested >100X
Syk IC50).13 Additional human proof-of-biology has been established with the B-cell depletion
therapy Rituximab, approved for the treatment of RA.14 Dosing with 1 in humans was limited by
hypertension and diarrhea as the most common adverse events. We hypothesized that the
hypertension observed was driven by off-target activity15 and sought to develop a selective Syk
inhibitor for the treatment of various autoimmune and hematological diseases, including RA,
oncology,16,17 and asthma,18,19 devoid of this undesired pharmacology. This manuscript describes
the preparation of diaminocarboxamides that are highly selective, potent Syk inhibitors as well as
details regarding the attenuation of inherent hERG ion channel activity and mutagenicity risk
within the chemotype to ultimately afford novel Syk inhibitors with favorable overall profiles.
Figure 1. Diverse Syk Inhibitor Motifs from the Literature.
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Chemistry
The main two points of structure activity relationship diversity for the
aminodipyridylcarboxamides were in the basic amine region and the aminoheterocycle (solvent
front) region. We developed modular approaches allowing late stage introduction of each of
these functionalities (Scheme 1). Namely, the basic amine portion of the molecule could be
introduced first starting from a dihalopyridyl nitrile under SNAr or palladium-mediated
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conditions. As an example of the former method, the synthesis of picolinamide 5 commenced via
reaction of 3-bromo-5-fluoropyridine-2-carbonitrile (6) and tert-butyl [(1S,2R)-2-
aminocyclohexyl]carbamate mediated by base to afford bromide 7 in 82% yield. Introduction of
2-amino-4,6-dimethylpyridine was accomplished under Buchwald–Hartwig conditions in high
yield.20 Alkaline peroxide promoted the hydrolysis of nitrile 8 to the corresponding amide, which
was subsequently deprotected under acidic conditions and free-based with ammonia in methanol
to provide carboxamide 5 in 84% yield over three steps.
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Alternatively, picolinamide 9 was prepared using the latter method of early-stage
aminoheterocycle introduction. Anionic addition of 4,6-dimethylpyridin-2-amine to 5-bromo-3-
fluoropicolinonitrile (10) gave bromopyridine 11. Palladium-mediated coupling of tert-butyl (1-
(aminomethyl)cyclopropyl)carbamate gave diaminopyridine 12, which was further
functionalized via acidic deprotection and hydrolysis to afford carboxamide 9.
Scheme 1. Synthesis of N-linked heterocycles.
(a) tert-butyl [(1S,2R)-2-aminocyclohexyl]carbamate, DIEA, 110 °C; (b) 2-amino-4,6-
dimethylpyridine, Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 80 °C; (c) NaOH, 35% aq. H2O2,
DMSO, rt; (d) HCl, dioxane; (e) NH3, MeOH, rt.
Scheme 2. Alternative approach to the synthesis of N-linked heterocycles.
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(a) 2-amino-4,6-dimethylpyridine, KO-t-Bu, THF,
(aminomethyl)cyclopropyl)carbamate, Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 80 °C; (c) TFA,
CH2Cl2, rt (d) NaOH, 35% aq. H2O2, DMSO, rt.
0
°C;
(b) tert-butyl (1-
7
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9
For the later generation C-linked carboxamides, a modified synthetic approach was employed.
As an exemplar, preparation of 13 commenced via SNAr addition of tert-butyl ((1S,2R)-2-
aminocyclohexyl)carbamate and 2,6-dichloropyrazine (14) to give chloropyrazine 15 in 88%
yield. Iodination para to the newly installed amine was accomplished using N-iodosuccinimide
to afford dihalopyrazine 16 in 91% yield. Carefully monitored cyanation under palladium-
mediated conditions provided nitrile 17 in 83% yield, which was subsequently functionalized via
a Suzuki–Miyaura coupling to install the indole moiety.21 Deprotection and hydrolysis of the
nitrile 18 delivered carboxamide 13 in 48% yield over three steps.
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Scheme 3. Synthesis of C-linked heterocycles.
(a) tert-butyl [(1S,2R)-2-aminocyclohexyl]carbamate, DIEA, 115 °C; (b) NIS, DMF, 65 °C; (c)
Zn(CN)2, Pd(PPh3)4, DMF, 120 °C; (d) 1H-indole-2-boronic acid pinacol ester, Pd(OAc)2, PCy3,
K2CO3, THF, 65 °C; (e) TFA, CH2Cl2, rt; (f) KOH, 35% aq. H2O2, DMSO, rt.
Results and Discussion
Our primary medicinal chemistry goal was to identify a highly kinome selective molecule that
demonstrated favorable properties for further development activities. Carboxamide scaffolds
(e.g. PRT-062607) were known in the literature at the outset of our efforts as potent Syk
inhibitors. Examining the available structural and crystallographic data for Syk revealed Asp-512
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as a key residue to target for selectivity enhancement.22 Namely, an appropriately positioned
array of hydrogen bond donors, such as the diamine present in YM-70220, had been
demonstrated to recruit Asp-512 toward the ATP site, affording improved kinome selectivity as
this motion of the catalytic Asp is unfavorable for a number of kinases (Figure 3).23 Having
settled upon Asp-512 as a key point of interaction, we next undertook a systematic core scan of
diaminocarboxamides (Table 1). Our objective was to ensure a high ligand efficiency for Syk,
while obviating the presence of a potentially reactive aniline substructure, which has been
reported to increase risk for toxicity.24 Generally, ligand efficiencies were in an acceptable range
for all permutations tested (0.42–0.56); structure activity relationships revealed that aryl
carboxamides possessing nitrogen ortho to the carboxamide were beneficial to potency
(picolinamide 5 vs. benzamide 19), while a nitrogen at the meta position was deleterious to
potency (benzamide 19 vs. nicotinamide 20).25 We hypothesized that an intramolecular
hydrogen bond between the amide NH and the heteroaryl nitrogen served to pre-organize the
ground state conformation in a planar geometry stabilizing the bioactive conformation as well as
lower the penalty for desolvation (23 versus 24, Figure 2). In contrast, an antagonistic steric
interaction between the NH of the amide and the CH ortho to the amide is present in chemotypes
lacking this heteroaryl nitrogen. Further, we proposed that the meta-nitrogen present in
nicotinamide 20 and pyridazinecarboxamide 21 favors a ground state diamine conformation that
requires a 180° rotation for recruitment of Asp-512 in the bioactive conformation (25 versus 26).
Picolinamide 22 demonstrated an antagonistic interaction between the aniline solvent front and
the picolinamide core resulting in a 100-fold intrinsic potency loss versus picolinamide 5 (27
versus 28). The potency advantage of the aminodipyridine was aligned with our strategy to avoid
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anilines. With this data set, we chose to focus our efforts on the 3,5-diaminopicolinamides
represented by compound 5.
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Table 1. Carboxamide Core Scan: Enzyme Potency and Ligand Binding Efficiency (LBE).
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Analog
19
Structure
Syk IC50 (nM) LBE
2.8
0.47
0.42
0.56
0.45b
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aSee supporting information for details. bSee reference 25.
Figure 2. Rationale for Intrinsic Potency Differences Across Cores.
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Figure 3. Interaction of Basic Amine with Asp-512 for Syk Potency and Selectivity. X-ray Co-
crystal Structure of YM-70220 in Syk (Yellow) Overlayed with AMP-PMP Structure with Syk
(Cyan).
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Further profiling of representative 3,5-diaminopicolinamide 5 revealed a number of favorable
aspects; high enzymatic (Syk IC50 = 60 pM) and human whole blood potency (hWB CysLT IC50
= 58 nM) had been achieved, along with exquisite kinome selectivity (100% of 265 kinases
tested >100X Syk IC50). Carboxamide 5 also demonstrated high enzymatic selectivity versus the
homologous enzyme Zeta-chain-associated protein kinase 70 (Zap70) with an IC50 of 5.3 nM
(Zap70 IC50/Syk IC50 = 90X).26 Further, the physicochemical properties (HPLC logD = 2.1, PSA
= 118) and oral pharmacokinetic profile (Rat Plasma Clp (Clu) = 33 (710) mL/min/kg, Rat T1/2
=
2.7 h, F = 19%) of this molecule were in a desirable range.27 Picolinamide 5 was profiled in the
rat collagen induced arthritis model and demonstrated therapeutic efficacy at doses as low as 3
mg/kg QD PO (Cmax = 0.2 μM, Cmin = 0.007 μM, Figure 4). However, a key off-target activity of
compound 5 was inhibition of the human Ether-a-go-go Related Gene (hERG, IKr IC50 = 1.6
μM). Several strategies have proven effective in the medicinal chemistry literature for
attenuating delayed rectifying potassium channel activity including modulation of basicity and
polarity.28 We targeted the diamine and aminoheterocycle directed toward the solvent front for
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modification in an attempt to merge the favorable properties of carboxamide 5 with weaker ion
channel activity.
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Figure 4. Rat Collagen Induced Arthritis Efficacy of 3,5-diamino picolinamide 5.
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Table 2 details a systematic survey of the diamine portion of the molecules while holding the
2-amino-4,6-dimethyl pyridine constant. A number of calculated and measured properties were
then leveraged to examine correlations between each category of properties and the
corresponding hERG ion channel activities. A good correlation has been established in the
literature, and was recapitulated with these chemotypes, between an automated patch clamp
system, PatchXpress, and a higher throughput binding assay for measuring or estimating IKr IC50
values.29,30 It is well-precedented that attenuation of amine basicity can result in weaker delayed
rectifying potassium channel binding.31 To maintain good Syk potency, it was important to avoid
rendering the diamine non-basic at physiological pH (e.g. 29 and 33 ). Calculated and/or
measured pKa’s above 7.0 showed little to no correlation with ion channel activity as measured
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via PatchXpress (Figure 5). There was also a lack of correlation between measured HPLC logD
and IKr activity within a logD range of 0.7 and 2.3, though it did trend slightly in the expected
direction with more polar molecules showing lower ion channel binding (Figure 5). The most
robust correlation was found between polar surface area (PSA) and hERG ion channel activity,
as a higher PSA biased molecules toward weaker activity, while maintaining Syk potency (32).
The diamino-tetrahydrothiopyran dioxide identified from this survey and exemplified in
picolinamide 32 emerged as a preferred moiety for interacting with Asp-512 while maintaining
potency (Syk IC50 = 0.1 nM), reasonable kinome selectivity (100% of kinases >100X, 265
kinases tested), and an improved off-target profile (hERG, IKr IC50 = 29 μM).
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Table 2. Amine Region hERG Ion Channel SAR.
Analog
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Amine
Syk IC50
(nM)a
Measured
pKa
HPLC
LogD
PSAb
IKr IC50
(µM)
0.06
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124
135
157
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1.6
29
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0.9
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>60d
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b
c
d
aSee supporting information for details. Polar surface area. Calculated pKa. MK-499
displacement IC50 (μM).
Figure 5. Physicochemical Properties versus hERG Ion Channel Activity.
Efforts were next directed toward examining the impact of the aminoheterocycle directed
toward the solvent front on hERG ion channel activity. In this case, the cyclohexyldiamino group
was held constant to ensure a sufficient dynamic range for assessing the impact of the
heterocycle. Again, polarity emerged as the key driver, as both measured HPLC logD and
calculated PSA demonstrated strong correlations with hERG interaction. Namely lower HPLC
logD (<1.0) and higher PSA (>145) effectively ablated potent IKr activity in the absence of
attenuated diamine basicity. Having demonstrated the impacts of PSA across two distinct
portions of the molecules on ion hERG activity, planning of future molecules was guided by
calculation of this physicochemical property. This prospective analysis allowed rapid triage and
prioritization of novel inhibitor designs. In short order, picolinamide 42 was synthesized and
demonstrated a favorable overall profile. Carboxamide 42 was a potent and selective Syk
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inhibitor (Syk IC50 = 0.6 nM, 99% of kinases >100X, 265 kinases tested, BLK IC50 = 32 nM,
ROS IC50 = 43 nM) that demonstrated good cell and functional activity (hWB CysLT IC50 = 56
nM) lacking the ion channel activity of previous prototypes (hERG, IKr IC50 >30 μM). Though 42
has a higher calculated PSA (PSA = 167) than many drug-like molecules (typically PSA ≤140
for oral bioavailability), a favorable pharmacokinetic profile in preclinical species was obtained,
including oral exposure (Rat Plasma Clp (Clu) = 19 (270) mL/min/kg, Rat T1/2 = 2.8 h, 30 %F).
The calculated PSA is believed to be an overestimate due to the extensive intramolecular
hydrogen bonding network within the ground state of the molecule that serves to mask some of
the inherent polarity from a topological assessment.
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Table 3. Solvent Front hERG Ion Channel SAR.
Analog
Solvent
Group
Syk IC50 (nM)a
HPLC
LogD
PSAb
118
IKr IC50
(µM)
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35
2.2
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1.4
130
138
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0.01
aSee supporting information for details. bPolar surface area.
Figure 6. Physicochemical Properties versus hERG Ion Channel Activity
Figure 7. Profile of Picolinamide 42.
While preparing carboxamide 42 to move toward development, a report appeared in the
literature of mutagenicity in the bacterial reverse mutation (Ames) test for a molecule of a
similar chemotype, GSK-143 (43).32 The report described a positive Ames test result for
pyrimidinecarboxamide 43 in Salmonella typhimurium strain TA1537 without metabolic
activation (Figure 8). Mutation in this strain often reflects intercalation of the test molecule into
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DNA, and such activity can also be detected in Salmonella strains TA97 and TA97a which are
considered equally acceptable with TA1537 under international test guidelines.33 Since TA97a is
the strain we use routinely, we selected this strain when carrying out a minimal screen, and
performed the mutation testing in the presence of rat S-9 because the mutagenicity of 43 and
some other candidates was enhanced by S-9 in our hands, and potential mutagenicity of aromatic
amine metabolites (see below) would require metabolic activation. Our lead compound 42,
induced mutation in strain TA97a in the presence of rat S9.. Similarly, picolinamide 5 was
tested, and showed a positive result at ≥ 300 µg/plate. Speculating that a metabolite could be the
culprit in these results, we prepared and tested two potential degradative products.
Diaminopyridine 45 had been found as a N-dealkylative metabolite in rat and human hepatocyte
MetID studies. This molecule was predicted by DEREK for Windows (version 12.5.4, Lhasa
Ltd., Leeds, UK) and Multi-CASE (MC4PC version 2.3.0.15, Multi-CASE Inc., Ohio, USA) to
be positive in the Ames test due to the known mutagenic substructure of the primary aromatic
amine in 45 (anilines 46 and 47 are known Ames positive compounds that feature this motif).
However, the assay in strain TA97a was negative for 45 up to 3000 µg/plate with rat S9. Tricycle
44 had been observed to form upon standing with acid and could have been a prime suspect for
intercalation. However, this compound was also negative in in TA97a with S9 up to 3000
µg/plate. With these results in hand, we began to formulate hypotheses to overcome the putative
intercalation liability within this series.
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Figure 8. Initial Compounds for Mutagenicity Assessment.
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NH2
NH
NH2
NH2
NH
O
NH
N
N
N
N
N
7
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N
N
N
O
N
H
H
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O
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O
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NH2
Me
NH2
NH2
N
Me
O
O
N
O
N
Me
H
N
N
H
H2N
O
H
47
45
46
Leveraging DNA co-crystals with acridine, a docking model was developed for our Syk
inhibitors (represented by picolinamide 5) in DNA (Figure 9).34 This model revealed three
distinct opportunities to disrupt DNA intercalation with our molecules. Namely, reducing the
basicity of the amine functionality should disfavor the donation of hydrogen bonds to the DNA
carbonyl. Second, growing toward, or shrinking from, the DNA backbone was predicted to
disrupt the fit of our small molecules in DNA, leading to a less thermodynamically favorable
interaction. Finally, introducing three-dimensionality could serve to disfavor the stabilizing pi-
stack with DNA, mitigating the intercalation liability. We set out to test each of these
hypotheses. Cognizant of the possibility that general cytotoxicity of molecules could confound
comparisons across results from the mutagenicity assay, we also sought to develop an orthogonal
higher throughput in vitro assay specific for DNA interaction.
We observed that the UV-Vis spectrum of some carboxamide compounds showed a saturable
perturbation upon addition of duplex DNA, as reported for other organic ligands.35 This facile
assay allowed inhibitors to be tested prior to, or concurrent with, the TA97a mutagenicity assay,
although the metabolic activation system (S9) could not be incorporated into the DNA
interaction assay. To validate this approach, picolinamides 5 and 42 were profiled, and each
showed positive data for DNA interaction, consistent with the mutagenicity results.36 Further,
metabolite 45 was negative in the UV-Vis assay, again matching the mutagenicity results for this
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compound.37 Of note, tricycle 44, which was negative in the TA97a mutagenicity, was positive
for DNA interaction in the UV-Vis assay. Thus the UV-Vis assay can give false positive results
compared with the bacterial mutagenicity assay in TA97a (though the results may be more
robust, since cytotoxicity is not a confounder in this model), but we never observed a false
negative in this assay,38 supporting its use as an orthogonal assessment of intercalation risk and
serving as a stage gate for the lower throughput bacterial mutagenicity assay.
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Figure 9. Docking Model of Picolinamide 5 in DNA (from Acridine/DNA Co-Crystals).
As shown in table 4, our first hypothesis regarding the attenuation of basicity still afforded
mutagenic/UV-Vis positive compounds (e.g. compound 48). Ablating the basicity completely, as
in carboxamide 49, afforded compounds predicted to lack intercalation, but at the expense of Syk
activity (Syk IC50 = 60 nM). As previously detailed the basic amine interaction of our
carboxamide inhibitors with Asp-512 is a critical element for potency and selectivity on the
enzyme.
We next systematically surveyed substitution at the 4-, 5-, and 6-positions of the
aminopyridine projected toward solvent. Examples 50, 51, and 52 were selected to evaluate the
effectiveness of growing toward the DNA backbone to disrupt the fit of our inhibitors in DNA.
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4-Substituted aliphatic alcohol 50 was negative in the TA97a mutagenicity assay, but positive in
the UV-Vis assay. Of note, the compound could only be tested up to 1000 μg/plate in the
bacterial assay, as higher concentration testing was precluded by general cytotoxicity. Pyrazole
51, featuring a large substituent at the 5-position of the aminopyridine, which should be pointed
directly at the DNA backbone, was negative in both the TA97a mutagenicity assay and UV-Vis
assays while maintaining Syk potency (Syk IC50 = 1 nM), representing a potential advance in our
efforts. Growing from the 6-position of the aminopyridine, as in triazole 52, gave very strong
mutagenicity and UV-Vis positive results. Though we had gained some favorable data with
pyrazole 51, its physicochemical properties led to a particularly large cell shift (hWB CysLT
IC50/Syk IC50 = 2800X for 51 versus 98X for 42). Overall, the strategy of growing the molecule
to mitigate mutagenicity did not seem the best path from an efficiency or physicochemical
properties perspective, so we chose to prioritize alternative tactics.
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Disruption of the pi stacking interaction with DNA was pursued via incorporation of sp3
groups on the aminopyridine heterocycle (e.g. picolinamide 53). Again, this strategy was not
successful at overcoming intercalation in our hands, as carboxamide 53 was positive in both the
mutagenicity and UV-Vis studies. Having pursued a number of paths with limited success, we
decided to look at alternative substructures to mitigate the intercalation liability.
Further pursuing a change in the shape of the carboxamide chemotype to grow toward the
DNA backbone and disfavor intercalation, we envisioned a carbon-link of the solvent group to
the core carboxamide.39 This design was applied to the synthesis of benzimidazole 54, which
demonstrated proof of concept for generation of a modestly potent/selective carbon-linked Syk
inhibitor (Syk IC50 = 7 nM, 97% of kinases >100X, 101 kinases tested, LRRK2 IC50 = 130 nM,
CHK2 IC50 = 160 nM, TSSK3 IC50 = 420 nM) that filled a different chemical space than the
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previous iteration of nitrogen-linked analogs (Figure 10).40 We also pivoted this motif to
pyrazinecarboxamide 55, which delivered a highly favorable profile (Syk IC50 = 0.5 nM, 99% of
kinases >100X, 101 kinases tested, LRRK2 IC50 = 30 nM, hWB CysLT IC50 = 130 nM, Rat
Plasma Clp (Clu) = 44 (255) mL/min/kg, Rat T1/2 = 2.8 h). Further, carboxamides 54 and 55 were
negative in the UV-Vis assay for intercalation, and pyrazine 55 was also negative in the
mutagenicity assay in strain TA97a with rat S9. Cyclohexyldiamine 55 was cytotoxic at
concentrations greater than 850 μg/plate, so to mitigate cytotoxicity, the sulfonyldiamine analog
56, was prepared. Pyrazine 56 was also negative in the UV-Vis assay, as well as the TA97a
mutagenicity assay with rat S9, this time tested up to 5000 μg/plate. Delighted by these results,
the team set out to further interrogate these findings and compare them in a third assay format. A
DNA unwinding assay was utilized to study carboxamides 42, 55, and 56; the results were in full
agreement with the TA97a mutagenicity and UV-Vis assays, further supporting the viability of
the carbon-linked motif as a method for obviating intercalation. Application of the design
principles that had been used to overcome ion channel activity and mutagenicity afforded
tetrahydropyranyldiamine 57 (Figure 11). Pyrazine 57 achieved the desired profile we had
targeted from the outset with good intrinsic potency (Syk IC50 = 0.7 nM), high selectivity (99%
of kinases >100X, 101 kinases tested, LRRK2 IC50 = 33 nM), potent cell functional activity
(hWB CysLT IC50 = 62 nM) and a favorable pharmacokinetic profile in preclinical species (Rat
Plasma Clp (Clu) = 25 (100) mL/min/kg, Rat T1/2 = 3.5 h, 29 %F). Further, this molecule 57
posed low risk for ion channel activity and DNA interaction as assessed by our ion channel and
UV-Vis displacement assays (hERG IC50 = 22 μM and UV-Vis = Negative).41
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A recent report from the literature correlated Ames mutagenicity to a metric defined as
“ovality” for a series of pyridazinecarboxamide Syk inhibitors.25 Ovality was defined as the ratio
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of the molecular surface area to the minimum surface area.42 We attempted to retrospectively
apply a similar calculation to test its predictivity for our studies. We found this metric to be
highly sensitive to subtle changes in the geometry of the solvent front heteroaryl substituent.
Across our data set, we observed no correlation between ovality and bacterial mutagenicity. As
detailed, the key to circumvention of the mutagenicity issue in our case was a mechanistic
understanding of the source of the liability (intercalation) along with testable design hypotheses
to alter the shape of the carboxamide Syk inhibitors. We pursued both increasing the spherical
nature of our molecules as well as two-dimensional perturbation of the spatial fit, ultimately
affording the C-linked carboxamides that have shown no mutagenicity risk to date.
Table 4. Mutagenicity SAR.
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Analog
48
Structure
Syk IC50 Mutagenicity UV-Visc
DNA
Ovalitye
(nM)a
(μg/plate)b
Unwindingd
51
Positive
(1000)
Positive
ND
1.42
49
42
50
63
0.6
0.4
Negative
(3000)
Negative
Positive
Positive
ND
Positive
ND
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1.39
1.42
NH2
Positive
(1000)
O
S
NH
O
N
N
N
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O
H2N
O
Negative
(1000)
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Negative
ND
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0.1
2
Positive (300) Positive
Positive (300) Positive
ND
ND
ND
1.39
1.36
1.34
7
ND
Negative
Negative
Negative
55
56
0.5
2.5
Negative
(850)
Negative
Negative
1.34
1.34
NH2
Negative
(5000)
O
S
NH
N
O
N
HN
H2N
O
a aSee supporting information for details. Number represents highest scorable concentration
b
for bacterial mutagenicity negative compounds and lowest concentration a positive score was
c
d
achieved for positive compounds. UV-Vis positive defined as Kd ≤500 μM. See supporting
e
information for details and primary data. Ovality calculated using a method adapted from the
report by Lucas, M. C. et al.25
Figure 10. Overlay of N-linked Carboxamide (42, Red) with C-linked Carboxamide (56, Blue).
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Figure 11. Profile of Pyrazine 57.
Conclusions
A novel series of orally efficacious, selective carboxamide Syk inhibitors were discovered via
systematic survey of chemotypes prioritized by ligand efficiencies and structural alerts. The
critical basic amine functionality of the 3,5-diamino picolinamides afforded undesired hERG ion
channel activity, which was mitigated via modulation of physicochemical properties. PSA
emerged as the most effective calculated property in circumventing hERG activity and was used
to prospectively design Syk inhibitors devoid of this liability. A mutagenicity risk was
subsequently discovered within these chemotypes. The mechanism for mutagenesis was
identified as involving intercalation. Several structure-based design hypotheses were interrogated
to obviate this issue; modulation of the two-dimensional shape of the molecules led to the
identification of an alternative series of C-linked heterocyclic carboxamides, delivering drug-like
molecules devoid of hERG ion channel activity, mutagenicity, and intercalation risk, while
maintaining Syk potency and selectivity.
ASSOCIATED CONTENT
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Supporting Information.
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Experimental procedures for synthesis of final compounds and intermediates, and descriptions
of assays performed. This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +1-617-992-3144; e-mail: michael_ellis@merck.com
Present Addresses
*J. Michael Ellis: Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, MA 02115,
United States
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We would like to acknowledge Jaren Arbanas and Uwe Mueller for routine In Vitro
Pharmacology support, Bruce Adams and Bridget Becker for NMR structural support, and Adam
Beard for determination of experimental pKa’s.
ABBREVIATIONS
Syk, Spleen tyrosine kinase; RA, rheumatoid arthritis; DMARD, disease-modifying
antirheumatic drug; Fc, fragment, crystallizable; BCR, B cell receptor; Syk IC50, recombinant
human Syk IC50; LBE, ligand binding efficiency; hWB, human whole blood; MetID, metabolite
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identification; SNAr, nucleophilic aromatic substitution; MeOH, methanol; DIEA, N,N-
diisopropylethylamine; NIS, N-iodosuccinimide.
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REFERENCES
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(Word Style “TF_References_Section”). References are placed at the end of the manuscript.
Authors are responsible for the accuracy and completeness of all references. Examples of the
recommended format for the various reference types can be found at
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found in The ACS Style Guide, available from Oxford Press.
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Müller-Ladner, U.; Pap, T.; Gay, R. E.; Neidhart, M.; Gay S. Mechanisms of Disease: the
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J.; Mebus, C. Tofacitinib (CP-690,550) in combination with methotrexate in patients with active
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7 Geahlen, R. L. Getting Syk: spleen tyrosine kinase as a therapeutic target. Trends Pharmacol.
Sci. 2014, 35, 414–422.
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Mocsai, A.; Ruland, J.; Tybulewicz, V. L. J. The Syk tyrosine kinase: a crucial player in
diverse biological functions. Nat. Rev. Immunol. 2010, 10, 387–402.
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Pine, P. R.; Chang, B.; Schoettler, N.; Banquerigo, M. L.; Wang, S.; Lau, A.; Zhao, F.;
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Coffey, G.; DeGuzman, F.; Inagaki, M.; Pak, Y.; Delaney, S. M.; Ives, D.; Betz, A.; Jia, Z.
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11 Liao, C.; Hsu, J.;Kim, Y.; Hu, D.-Q.; Xu, D.; Zhang, J.; Pashine, A.; Menke, J.; Whittard, T.;
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