Carboxamide Spleen Tyrosine Kinase (Syk) Inhibitors: Leveraging Ground State Interactions To Accelerate Optimization

Article abstract of DOI:10.1021/acsmedchemlett.6b00353

Optimization of a series of highly potent and kinome selective carbon-linked carboxamide spleen tyrosine kinase (Syk) inhibitors with favorable drug-like properties is described. A pervasive Ames liability in an analogous nitrogen-linked carboxamide series was obviated by replacement with a carbon-linked moiety. Initial efforts lacked on-target potency, likely due to strain induced between the hinge binding amide and solvent front heterocycle. Consideration of ground state and bound state energetics allowed rapid realization of improved solvent front substituents affording subnanomolar Syk potency and high kinome selectivity. These molecules were also devoid of mutagenicity risk as assessed via the Ames test using the TA97a Salmonella strain.

Full text of DOI:10.1021/acsmedchemlett.6b00353

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Letter
Carboxamide Spleen Tyrosine Kinase (Syk) Inhibitors:
Leveraging Ground State Interactions to Accelerate Optimization
J. Michael Ellis, Michael D. Altman, Brandon D. Cash, Andrew M. Haidle,
Rachel L. Kubiak, Matthew L Maddess, Youwei Yan, and Alan B. Northrup
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00353 • Publication Date (Web): 07 Oct 2016
Downloaded from http://pubs.acs.org on October 8, 2016
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Carboxamide Spleen Tyrosine Kinase (Syk) Inhibitors: Leveraging
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J. Michael Ellis,* Michael D. Altman,* Brandon Cash, Andrew M. Haidle, Rachel L. Kubiak, Matthew
L. Maddess, Youwei Yan, Alan B. Northrup
Department of Discovery Chemistry, Merck & Co., Inc., 33 Avenue Louis Pasteur, Boston, MA 02115
Spleen tyrosine kinase, intramolecular hydrogen bond, structure-based drug design
ABSTRACT: Optimization of a series of highly potent and kinome selective carbon-linked carboxamide Spleen Tyrosine Kinase
(Syk) inhibitors with favorable drug-like properties is described. A pervasive Ames liability in an analogous nitrogen-linked car-
boxamide series was obviated by replacement with a carbon-linked moiety. Initial efforts lacked on-target potency, likely due to
strain induced between the hinge binding amide and solvent front heterocycle. Consideration of ground state and bound state ener-
getics allowed rapid realization of improved solvent front substituents affording subnanomolar Syk potency and high kinome selec-
tivity. These molecules were also devoid of mutagenicity risk as assessed via the Ames test the using the TA97a Salmonella strain.
The advancement of kinase inhibitors as oncology therapeu-
tics has resulted in approval of a number of effective drugs
over the past two decades. Recently, efforts have been directed
toward the treatment of autoimmune disorders, particularly
rheumatoid arthritis (RA), via kinase inhibition. RA is a
chronic inflammatory disease that presents with pathophysiol-
ogy including synovial hyperplasia, inflammation, and joint
destruction. RA is characterized by up-regulation of cytokines
and chemokines ultimately leading to inflammation. Treat-
ment paradigms have consisted of palliative therapies to miti-
gate symptoms including pain and disease-modifying an-
tirheumatic drugs (DMARDs) or biologics with the goal to
slow or stop disease progression. Existing therapies are not
universally effective for all RA patients, underscoring the need
for additional treatment options.
Spleen tyrosine kinase (Syk) is a well-studied enzyme that
plays a critical role in immune signaling via Fc and B cell
receptors (BCR).1 Preclinical studies demonstrated that phar-
macological modulation of Syk (R788 (1), PRT-062607 (2),
RO9021 (3), and GS-9973 (4)) is efficacious in preclinical RA
, , ,
models (Figure 1).2 3 4 5 Further, poorly selective Syk inhibitor
1 (52% of 100 kinases tested >100X Syk IC50) has demon-
Picolinamide 5 was designed via analysis of X-ray crystal-
lographic data with a focus on ligand binding efficiency and
ensuring favorable physicochemical properties (Figure 2).
Carboxamide 5 possessed desirable intrinsic (Syk IC50 = 60
pM) and human whole blood potency (hWB CysLT IC50 = 58
nM). Additionally, kinome selectivity was high (99% of 265
kinases tested >100X Syk IC50),13 and the oral pharmacokinet-
ic profile (rat plasma Clp (Clu) = 33 (710) mL/min/kg, rat T1/2
= 2.7 h, F = 19%) enabled testing in the rat collagen-induced
arthritis model, where the compound demonstrated therapeutic
strated efficacy in human clinical trials, however dosing was
limited by observations of hypertension and diarrhea.6 We
aimed to develop a selective Syk inhibitor that would obviate
the clinical side effects and could be useful for autoimmune
, , ,
,
and oncological disorders.7 8 9 10 11. As previously described,12
a
picolinamide series of Syk inhibitors we developed was halted
by mutagenicity risk as indicated by positive results in the
Ames assay. Described herein are the design considerations
employed to overcome this liability via a scaffold modification
affording novel Syk inhibitors consistent with our objective.
efficacy at doses as low as 3 mg/kg QD (Cmax = 0.2 μM, Cmin
=
0.007 μM). However, this molecule, and this series of dia-
minopicolinamides, was plagued with positive results in the
Ames test using TA97a Salmonella strain. Since mutation in
this strain often reflects intercalation of the test molecule into
DNA, we developed a docking model leveraging DNA cocrys-
Figure 1. Diverse Published Syk Inhibitors.
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tals with acridine. Based upon this model, an approach we energetic penalty to adopt the bound conformation. In addi-
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pursued to ameliorate this activity involved changing the two-
dimensional shape of how the molecules are presented, aimed
at disrupting the favorable interactions with DNA.
tion, the energy minimum at the presumed bound confor-
mation is predicted to become much deeper than alternative
states, which likely reduces entropic penalties as well. This is
likely influenced by an intramolecular hydrogen bond between
the more polarized NH of the indole and the carbonyl of the
carboxamide. Gratifyingly, indole 8’s doubly-stabilized pla-
narity afforded a significantly improved intrinsic potency (Syk
IC50 = 5 nM). Interestingly, the predicted ground-state geome-
try for 8 suggests a pseudo 7-membered ring where the angles
open up to accommodate an intramolecular hydrogen bond
between the indole NH and the carboxamide carbonyl (Figure
S1).15
Figure 2. Lead Molecule Picolinamide 5.
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Figure 3. Initial C-linked carboxamides and predicted quantum
mechanical potential energy landscape for rotation about the aryl
and amide dihedral angles. (0, 0) represents the presumed planar
bioactive conformation in the orientation as drawn. White regions
represent configurations with severe intramolecular clashes and
thus no energy was computed. This figure was prepared with
MATLAB.
Our initial foray into changing two-dimensional shape fo-
cused on altering the vector at which the “solvent front” group
was presented; this strategy involved carbon-linking to replace
the previous nitrogen-linked moiety. To this end, picolinamide
6 was quickly prepared via Suzuki coupling14 of available
intermediates to replace the aminoheterocycle with a C-linked
3-methylpyrazole analog (Figure 3). However, picolinamide 6
was inactive in our enzymatic assay (Syk IC50 >10 μM, 9%
inhibition at 10 μM). We hypothesized that the 3-
methylpyrazole may introduce torsional strain with the car-
bonyl oxygen of the carboxamide, disfavoring the planar ge-
ometry desired for efficient interaction with the hinge domain
of Syk, and kinases in general. To probe this theory, we pur-
sued computational quantum mechanical approaches to esti-
mate the lowest energy dihedral angles about these two sub-
stituents and the energetic penalty required to achieve a fully
flat conformation (amide dihedral = 0 ° and aryl solvent front
dihedral = 0 °) for binding.15 For picolinamide 6, calculations
suggest that the preferred ground state conformation positions
the two critical substituents ~30 ° and -30 ° out of plane, re-
sulting in an estimated penalty of 2–4 kcal/mol to adopt the
planar bound-state conformation, which is reflected in the lack
of potency for this analog.
Considering rational structural modifications that could be
implemented to further favor the flat ground state confor-
mation, minimizing the motion and associated energetic penal-
ty required to achieve the flat bioactive state, calculations were
executed to examine the roles of different heteroatom substi-
tuted cores and solvent fronts. Among those surveyed, the
pyrazinecarboxamide core was predicted to favor a smaller
dihedral angle, and corresponding lower energetic penalty (0–
2 kcal/mol) than the picolinamide. In the picolinamide 9, there
is likely increased torsional strain resulting from the two CH’s
in close proximity compared to the pyrazine 10, where this
strain is removed and the conformation may be reinforced by
an additional intramolecular hydrogen bond (Figure 4). Pyra-
zinecarboxamide 7 was prepared to test this hypothesis and
showed improved potency (Syk IC50 = 1.6 μM). It is worth
noting that the cyclohexyldiamine generally introduces a ~10X
potency enhancement versus the propanyldiamine, though this
matched pair was never made with the methypyrazole solvent
front.
Figure 4. Rationale for picolinamide and pyrazinecarboxamide
ground state dihedral angles and resultant intrinsic potencies.
Hoping to parlay the potency gain achieved moving from
the picolinamide to the pyrazinecarboxamide with a solvent
front group predicted to further favor a planar disposition,
indole 8 was synthesized.16 Calculations suggested that the
indole substituted pyrazinecarboxamide should strongly favor
a flat ground state conformation, resulting in a 0–0.5 kcal/mol
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In order to further understand the binding interactions of cokinetic (Clp (Clu) = 134 (277) mL/min/kg, rat T1/2 = 4.8 h)
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these compounds, as well as interrogate the computational
predictions, we solved the X-ray crystal structure of com-
pound 8 bound to the kinase domain of human Syk (Figure 5).
profile.
Figure 6. Advanced C-linked carboxamides.
,
,
Similar to other carboxamide-based Syk inhibitors,17 18 1920
8
4
binds to the hinge region via bidentate hydrogen bonding to
the backbone of Glu449 and Ala451. The indole region ex-
tends toward solvent and packs against the side chain of
Met450 at the top of the hinge, while NH groups of the dia-
mine region form interactions with the catalytic Asp512 and
surrounding backbone carbonyls from Arg498 and Asn499.
As predicted, the core is planar with a pseudo 7-membered
ring geometry stabilized by an intramolecular hydrogen bond
between the indole NH and the carboxamide oxygen with a
short N-O distance of 2.6 Å (Figure 5, magenta dashed line).
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Figure 5. Binding site view of the crystal structure of 8 bound to
the human Syk kinase domain. The carbon atoms of Syk protein
are shown in white and carbon atoms of 8 are in green. Modeled
water molecules are shown as red spheres, an intramolecular hy-
drogen bond in 8 is shown in magenta dashes, and polar interac-
tions between 8 and protein are shown as yellow dashes. This
figure was prepared with PyMOL.
Encouraged by the successes of exploiting intramolecular
hydrogen bonds, we investigated the use of weaker H-bond
donors and other configurations predicted to maintain a planar
geometry. Benzofuran 13 lost ~100X potency (Syk IC50 = 530
nM) versus its indole analog 8. Styrene 14 had comparable
intrinsic affinity, ~3X loss (Syk IC50 = 24 nM) versus the cor-
responding benzimidazole 12. Aryl ether 15, interestingly
predicted to prefer a planar configuration affords an equipotent
inhibitor (Syk IC50 = 9 nM) to benzimidazole 12. Alkyne 16
was readily prepared, but demonstrated a substantial potency
loss (Syk IC50 = 990 nM), likely due to negative interaction of
the phenyl moiety with the hinge region of Syk.
Table 1. Additional C-linked carboxamide SAR
Analog
Structure
Syk IC50
(nM)a
13
530
Seeking to further optimize the C-linked carboxamides, in-
troduction of the cyclohexyldiamine moiety to afford pyra-
zinecarboxamide 11 delivered the expected potency enhance-
ment (Syk IC50 = 500 pM) approaching the previously
achieved intrinsic activity of the N-linked picolinamide 5.
Further, indole 11 featured excellent kinase selectivity (99%
of 101 kinases tested >100X Syk IC50, LRRK2 IC50 = 30 nM)
and human whole blood potency (hWB CysLT IC50 = 130 nM)
with a favorable pharmacokinetic profile in rat Clp (Clu) = 44
(255) mL/min/kg, rat T1/2 = 2.8 h, F = 19%). As previously
described, all molecules tested from the C-linked carbox-
amides were negative in the mutagenicity assay in strain
TA97a Salmonella with rat S9 up to testable concentrations.12
Additional data to support the effectiveness of the shape
change in ablating interaction with DNA were gathered via
consistent results in a UV-Vis perturbation assay and a DNA
unwinding assay. We subsequently examined a nitrogen trans-
position of pyrazinecarboxamide 11 to afford benzimidazole
12. Interestingly, picolinamide 12 showed a loss in intrinsic
potency (Syk IC50 = 7 nM), but a maintained selectivity (97%
of 101 kinases tested >100X Syk IC50, LRRK2 IC50 = 130 nM,
CHK2 IC50 = 160 nM, TSSK3 IC50 = 420 nM), human whole
blood potency (hWB CysLT IC50 = 93 nM), and rat pharma-
14
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The authors declare no competing financial interest.
16
990
0.7
ACKNOWLEDGMENT
We would like to acknowledge Jaren Arbanas and Uwe Mueller
for routine In Vitro Pharmacology support, and Bruce Adams and
Bridget Becker for NMR structural support.
ABBREVIATIONS
10
11
12
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Syk, Spleen tyrosine kinase; RA, rheumatoid arthritis; DMARD,
disease-modifying antirheumatic drug; Fc, fragment, crystalliza-
ble; BCR, B cell receptor; Syk IC50, recombinant human Syk IC50;
hWB, human whole blood; PEG, polyethylene glycol.
17
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The combined efforts of this scaffold re-design ultimately
delivered pyrazinecarboxamide 17,21 which possessed the
overall profile we were seeking (Syk IC50 = 700 pM) including
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, indole 17 posed
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UV-Vis = Negative). In summary, careful consideration of
designs to bias toward the bioactive conformation delivered
alternative chemotypes that maintained favorable drug-like
properties while overcoming several safety issues.
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ASSOCIATED CONTENT
Supporting Information
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Currie, K. S.; Kropf, J. E.; Lee, T.; Blomgren, P.; Xu, J.; Zhao,
Experimental procedures for synthesis of final compounds and
intermediates, descriptions of assays performed, computational
methods with additional results, and crystallographic details. The
complex between the human Syk kinase domain and compound 8
has been deposited in the Protein Data Bank (PDB) with acces-
sion code 5TIU. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Maciejewski, P.; Rossi, A. M.; Rong, H.; Macaluso, J.; Barbosa, J.; Di
Paolo, J. A.; Mitchell, S. A. Discovery of GS-9973, a selective and
orally efficacious inhibitor of spleen tyrosine kinase. J. Med. Chem.
2014, 57, 3856–3873.
6 Weinblatt, M. E.; Kavanaugh, A.; Genovese, M. C.; Jones, D. A.;
Musser, T. K.; Grossbard, E. B.; Magilavy, D. B. Effects of fosta-
matinib (R788), an oral spleen tyrosine kinase inhibitor, on health-
related quality of life in patients with active rheumatoid arthritis:
analyses of patient-reported outcomes from a randomized, double-
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AUTHOR INFORMATION
Corresponding Authors
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*Phone: +1-617-992-3144 (Ellis), +1-617-992-3077 (Altman); e-
mail: michael_ellis@merck.com (Ellis), mi-
chael_altman@merck.com (Altman)
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(R788) inhibits tumor growth in the Eμ-TCL1 transgenic mouse mod-
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Blood 2010, 116, 4894–4905.
*J. Michael Ellis and Michael D. Altman: Merck Research Labor-
atories, 33 Avenue Louis Pasteur, Boston, MA 02115, United
States
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Friedberg, J. W.; Sharman, J.; Sweetenham, J.; Johnston, P. B.;
Author Contributions
Vose, J. M.; Lacasce, A.; Schaefer-Cutillo, J.; De Vos, S.; Sinha, R.;
Leonard, J. P.; Cripe, L. D.; Gregory, S. A.; Sterba, M. P.; Lowe, A.
M.; Levy, R.; Shipp, M. A. Inhibition of Syk with fostamatinib diso-
All authors have given approval to the final version of the manu-
script.
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Zap70 IC50: 5.3 nM (Zap70 IC50/Syk IC50 = 90X), which was con-
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mologous kinase
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Miyaura, N.; Yamada, K.; Suzuki, A. A new stereospecific cross-
coupling by the palladium-catalyzed reaction of 1-alkenylboranes
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15 Model compounds were employed where the diamine portion of the
molecule was truncated leaving behind only the heterocyclic ring,
primary carboxamide, and C-linked aryl substituent. To calculate
potential energy surfaces, initial geometries were generated stepping
through the two relevant dihedral angles at +/- 0, 5, 10, 30, 60, 90,
120, 150, 170, 175, and 180 degrees. Dihedral angles of (0, 0) were
defined to be the presumed planar bioactive conformation. Geome-
tries were then optimized using GAUSSIAN 09 rev. D01 at the
B3LYP/6-31G* level of theory keeping the two dihedral angles fixed
at their initial values. Relative electronic energies across the geome-
tries were then used to generate potential energy surface maps in
MATLAB. Prediction of planar ground state geometries, such as for
the compounds in Table 1, were performed similarly. Geometries of
model compounds were generated through systematic enumeration of
dihedral angles in 30 degree increments followed by unconstrained
optimization. Compounds where the lowest-energy optimized struc-
ture was planar were considered for synthesis and experimental study.
16
Predicted ground state geometry for the core of 8 depicting the
pseudo 7-membered ring formed through intramolecular hydrogen
bonding can be found in Figure S1 of the supporting information.
17
Villasenor, A.G.; Kondru, R.; Ho, H.; Wang, S.; Papp, E.; Shaw,
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graphic analysis of three inhibitor complexes. Chem. Biol. Drug Des.
2009, 73, 466–470.
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S.; Gray, M.; Hayhow, T. G.; Hobbs, C. I.; Jones, E.; Leach, S.;
Leavens, K.; Lewis, H. D.; McCleary, S.; Neu, M.; Patel, V. K.; Pres-
ton, A. G.; Ramirez-Molina, C.; Shipley, T. J.; Skone, P. A.; Smith-
ers, N.; Somers, D. O.; Walker, A. L.; Watson, R. J.; Weingarten, G.
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