Structure-based drug design of RN486, a potent and selective Bruton's tyrosine kinase (BTK) inhibitor, for the treatment of rheumatoid arthritis

Article abstract of DOI:10.1021/jm500305p

Structure-based drug design was used to guide the optimization of a series of selective BTK inhibitors as potential treatments for Rheumatoid arthritis. Highlights include the introduction of a benzyl alcohol group and a fluorine substitution, each of which resulted in over 10-fold increase in activity. Concurrent optimization of drug-like properties led to compound 1 (RN486) (J. Pharmacol. Exp. Ther. 2012, 341, 90), which was selected for advanced preclinical characterization based on its favorable properties.

Full text of DOI:10.1021/jm500305p

Brief Article
pubs.acs.org/jmc
Structure-Based Drug Design of RN486, a Potent and Selective
Bruton’s Tyrosine Kinase (BTK) Inhibitor, for the Treatment of
Rheumatoid Arthritis
Yan Lou,* Xiaochun Han, Andreas Kuglstatter, Rama K. Kondru, Zachary K. Sweeney, Michael Soth,
Joel McIntosh, Renee Litman, Judy Suh, Buelent Kocer, Dana Davis, Jaehyeon Park, Sandra Frauchiger,
Nolan Dewdney, Hasim Zecic, Joshua P. Taygerly, Keshab Sarma, Junbae Hong, Ronald J. Hill,
Tobias Gabriel, David M. Goldstein, and Timothy D. Owens
pRED, Pharma Research & Early Development, Small Molecule Research, Discovery Chemistry, Hoffmann-La Roche Inc., 3431
Hillview Avenue, Palo Alto, California 94304, United States
S
* Supporting Information
ABSTRACT: Structure-based drug design was used to guide the optimization of a series of selective BTK inhibitors as potential
treatments for Rheumatoid arthritis. Highlights include the introduction of a benzyl alcohol group and a fluorine substitution,
each of which resulted in over 10-fold increase in activity. Concurrent optimization of drug-like properties led to compound 1
(RN486) (J. Pharmacol. Exp. Ther. 2012, 341, 90), which was selected for advanced preclinical characterization based on its
favorable properties.
concept studies in RA patients are yet to be reported. This
manuscript describes our structure-based design of compound
1, a potent and selective BTK inhibitor (Chart 1).8
INTRODUCTION
■
Rheumatoid arthritis (RA) is a serious autoimmune disease
with an unknown etiology. Despite the discovery of many
efficacious biological agents for the treatment of this condition,
there is still a critical need for novel agents with different
mechanisms of action for patients unresponsive to current
therapies. Furthermore, orally bioavailable small molecules
would be particularly desirable in the treatment of RA given
both their convenience of administration and ability to clear
more rapidly from the body than their biological counterparts
in the event of a serious infection during treatment.
CHEMISTRY
■
The synthesis of compound 1 is described in Scheme 1.
Intermediate 7 was assembled from nitropyridine 4 through
aromatic substitution with N-methyl piperazine, nitro reduc-
tion, selective mono Buchwald/Hartwig amination with N-
methyl dibromopyridone, and subsequent conversion to the
Chart 1
The most notable recent advance in developing an oral drug
for RA was the introduction of tofacitinib,1−4 Pfizer’s selective
pan-JAK inhibitor.
The successful development of tofacitinib demonstrated that
small molecule oral therapies for RA could achieve similar
efficacy to biological agents.
Additional efforts to introduce novel RA treatments have
focused on the development of inhibitors of B-cell function. For
example, the SYK inhibitor fostamatinib2,5 from Rigel/
AstraZeneca advanced to phase IIb trials. BTK, a downstream
kinase of SYK, has also been shown to play crucial roles in B
cell and mast cell activation. The selective BTK inhibitors,
PCI32765 (2)6 and CGI1746 (3),7 have shown excellent
activity in preclinical animal models for RA. However, proof-of-
Special Issue: New Frontiers in Kinases
Received: March 3, 2014
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
essentially the same as that later reported for 3 (Figure 1A).7
Both inhibitors form bidentate hydrogen bonding interactions
a
Scheme 1
a
Reactions and conditions: (a) N-methyl piperizine, K2CO3, Bu4NI,
DMSO, 120 °C, 83%; (b) Pd/C, MeOH, rt, 98%; (c) Pd2dba3,
Xantphos, Cs2CO3, dioxane, 120 °C, 28%; (d) Pd(OAc)2, X-Phos,
pinacolato diboron, KOAc, 100 °C, 84%; (e) carbonyldiimidazole,
NH4OH, THF, rt, 87%; (f) Pd2dba3, cyclopropylboronic acid, K2CO3,
toluene/water, reflux, 82%; (g) (i) (CH3)2NCH(OCH3)2, MeTHF, 60
°C, (ii) tBuOK, THF, 60 °C, 77%; (h) CuI, K2CO3, DMF, 120 °C,
60%; (i) NaBH4, CH2Cl2/i-PrOH, 4 °C, 87%; (j) 7, Pd(OAc)2, PCy3,
K2CO3, dioxane, 88 °C, 83%.
desired boronic ester. The synthesis of intermediate 13 was
achieved via the following sequence from benzoic acid 8: (i)
amide coupling with ammonium hydroxide, (ii) Suzuki
coupling to give cyclopropane 10, (iii) one-pot procedure of
converting primary amide in 10 to bicycle 11 through
amidination and base-induced ring closure, (iv) selective Suzuki
coupling with 2-bromo-6-chlorobenzaldehyde to yield 12, and
(v) reduction of the aldehyde group. Finally, compound 1 was
assembled through a Suzuki coupling between intermediates 7
and 13.
RESULTS AND DISCUSSION
■
Figure 1. X-ray crystal structures of BTK−inhibitor complexes in ball-
and-stick representation: (A) compound 3 at 1.8 Å resolution (PDB
accession number 3OCS); (B) compound 14 at 1.55 Å resolution
(4OT5). Inhibitors are displayed in green, selected amino acids in
yellow, and water molecules in cyan. Black dashes indicate hydrogen
bonds.
Inspired by CGI Pharmaceuticals’ pioneering work on a class of
imidazopyrazines specifically claimed as BTK inhibitors7 (i.e., 3,
14), we initiated a crystallography effort aimed at under-
standing the binding mode of these compounds. No
satisfactory docking pose with BTK was obtained using
structures available at the time. This work led to the first
novel in-house cocrystal structure of 14 (Chart 2) with BTK.
The binding mode observed for 14 to BTK kinase domain is
with the backbone of Met477 in the kinase hinge region. The
tertiary benzamide groups stack between residues Gly480 and
Leu408 and point toward solvent. The ligands’ phenyl linker
moieties are interacting with the Val416 side chain (not shown)
and stack “edge-to-face” against the three consecutive peptide
bonds L408-G409-T410-G411 of the Gly-rich loop (amino
acids 408−417). The t-butyl-benzamide group of 3 and 14
points into a selectivity pocket formed by the side chains of
Q412, F413 (both Gly-rich loop), D521, N526 (both catalytic
loop), D539 (DFG), L542, S543, V546, and Y551 (all
activation loop). The ligands secondary amide moiety forms a
direct hydrogen bond with the side chain of the conserved
K430 as well as water-mediated hydrogen bonds with the Gly-
rich loop.
Chart 2
B
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Interestingly, when the lipophilic t-butyl group is replaced
with a much more polar methyl sulfone moiety, the
corresponding analogue 15 (Chart 2) no longer occupies the
specificity pocket and instead adopts a “U” shape conformation,
putting the sulfone in the solvent exposed region (Figure 2B),
high clearance.10 Simply shifting a nitrogen atom from the
central bicycle to the aniline ring resulted in compound 18
(Chart 3). The clogP of 18 was half a unit lower (clogP = 5.1)
Chart 3
than the clogP compound 16. As coplanarity of the aniline ring
and the bicyclic group was conserved, 18 has a similar potency
(BTK IC50 of 30 nM) to 16. Subsequent truncation of the
bicyclic core to a monocyclic pyridone core led to compound
19. With a now substantially reduced clogP of 3.8, not only did
we observe an increase in BTK IC50 (10 nM) but 19 also
exhibited an improved IC50 of 700 nM in the HWB assay. To
confirm that coplanarity of the core and front aniline group is
preferred, compound 20 was prepared. This inhibitor was more
than 10-fold less potent inhibitor of BTK (IC50 of 160 nM)
relative to analogue 19.
Despite much reduced lipophilicity, compound 19 was found
to be rapidly cleared both in vitro and in vivo. Metabolite
identification of 19 in liver microsomes showed that
compounds 21 and 22 (Chart 4), arising from oxidation of
Chart 4
Figure 2. X-ray crystal structures of BTK−inhibitor complexes in
ribbon representation: (A) compound 14 at 1.55 Å resolution (PDB
accession number 4OT5); (B) compound 15 at 2.05 Å resolution
(4OT6). The sulfone-benzamide moiety of 15 is poorly resolved.
Inhibitors (green) and selected amino acid side chains are shown in
ball-and-stick representation. Black dashes indicate hydrogen bonds.
the morpholine ring and t-butyl group, are the major
metabolites. Many other analogues made without the morpho-
line group in 19 still suffered from high in vitro and in vivo
clearance. Liver microsome metabolite identification experi-
ments consistently showed t-butyl oxidation, sometimes
exclusively (i.e., 23). Therefore, many analogues with different
replacements of the t-butyl group were prepared and profiled.
One of the most interesting analogues prepared in this effort
was compound 24, which features a N,N-dimethyl group.
Although it is less potent than 20 (BTK IC50 of 20 nM, HWB
IC50 4600 nM), it has good pharmacokinetic properties in rats
with 36% bioavailability at 5 mg/kg, AUC of 3 μM·h, and a
clearance rate of 18 mL/min/kg.
With the goal of improving the potency of 20, we analyzed
the crystal structure of BTK in complex with 24 for possible
ligand modifications that would lead to additional interactions
with the protein. The methyl group in the central phenyl group
is in close proximity to both K430 and D539 (3.9 Å for C−N of
K430 and 3.6 A for C−O of D539). It was envisioned that a
hydroxyl substitution on the methyl group might be able to
form a hydrogen bond with both K430 and D539. The
designed benzyl alcohol analogue 25 (Chart 5) was found to
have a Kd of 0.4 nM for BTK as compared with 8 nM for 24. Its
This substitution results in loss of affinity for BTK (IC50 of
1200 nM) and specificity against other kinases (>90%
inhibition of 36/256 kinases in Ambit kinome panel at 10
μM concentration).9 A cocrystal structure of an ortho-methyl
analogue 16 with BTK was also obtained. Inhibitor 16 (IC50 of
10 nM) is 5-fold more potent than compound 17 (IC50 of 50
nM), which lacks the methyl group on the central phenyl ring.
The increased affinity from the methyl group may come from
favorable hydrophobic interactions with the protein as well as
increased population of the bioactive conformer.
Although 16 is a reasonably potent BTK inhibitor in vitro,
the combination of suboptimal efficacy in human whole blood
(HWB) assays (HWB IC50 for CD69 inhibition >1 μM) and
high molecular weight and high lipophilicity (MW 588/clogP
5.6) makes it a fairly unattractive starting point for lead
optimization. However, attracted by the exquisite BTK
selectivity of this inhibitor and the availability of cocrystal
structure information, we decided to engage in issue-driven
optimization of the series, focusing on addressing key issues via
structure-based optimization.
The first issue tackled was the high lipophilicity, which is
often associated with low solubility, high protein binding, and
C
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While 24 showed highly desirable PK properties, further
identification of metabolites revealed formation of primary
aniline metabolite 26. Because of the known mutagenicity risk
of many primary anilines,12 compound 26 was tested in Ames
and MNT assays and found to be positive in the presence of S9.
Because this amide bond is almost in plane with the phenyl
group off the carbonyl group and the NH is pointed into
solvent, we decided to investigate some bicyclic systems to
avoid potential aniline release. First bicyclic quinazolinone 27
(BTK IC50 of 116 nM, Chart 6) was found to be substantially
Chart 5
Chart 6
HWB IC50 improved correspondingly to 120 nM. The
increased affinity is attributed to the alcohol’s simultaneous
hydrogen bonding interactions with K430 and D539, some-
times referred as cooperative hydrogen bonds. This was
confirmed by molecular modeling and subsequent X-ray
crystallography (see Figure 3 for a methyl vs hydroxyl−methyl
comparison). These interactions are known to significantly
boost ligand affinity.11 Many other substituents were
subsequently examined, and all of them were inferior to the
benzyl alcohol group in terms of binding affinity.
less active than the corresponding amide 28 (BTK IC50 of 28
nM). After a cocrystal structure of a close analogue 29 with
BTK was obtained, it was recognized that the repulsive
interaction between the aza group of the new bicycle with
N526 may be responsible for the reduction in inhibitor potency
(Figure 3A). The reduced form of 27, dihydroquinazolinone
30, was found to be much more potent (BTK IC50 of <10 nM).
Unfortunately, 30 was not chemically stable in aqueous solution
under various conditions. However, the related, chemically
stable dihydroisoquinolone 31 retained good BTK activity with
BTK IC50 of 28 nM.
Compound 32 was prepared and profiled in order to
combine both favorable features of 25’s benzylic alcohol and
31’s bicylic moiety. This inhibitor has BTK IC50 of 7 nM and
HWB IC50 of 100 nM. However, its poor aqueous solubility
profile (0.6/2/3 μg/mL in water, SIF, and SGF, respectively)
limits its potential for further profiling. To improve aqueous
solubility, the most successful approach was to install an
ionizable group in the solvent exposed region such as the basic
amines contained in inhibitors 33−35 (Chart 7). Compound
33 was found to have a much improved aqueous solubility
Chart 7
Figure 3. X-ray crystal structures of BTK−inhibitor complexes in ball-
and-stick representation: (A) compound 29 at 1.55 Å resolution (PDB
accession number 4OTQ); (B) compound 35 at 1.95 Å resolution
(4OTR). Inhibitors are displayed in green and selected amino acids in
yellow. Black dashes indicate hydrogen bonds, organ dashes repulsive
interaction, and magenta dashes interactions involving fluorine.
D
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Journal of Medicinal Chemistry
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profile (26/11/7290 μg/mL in water, SIF, and SGF,
respectively). This analogue maintains good HWB potency
(IC50 of 45 nM) and does not significantly inhibit the hERG
channel in in vitro assays (IC20 > 50 μM).
somes; SGF, simulated gastrointestinal fluid; SIF, simulated
intestinal fluid; SYK, spleen tyrosine kinase
REFERENCES
■
Subsequent optimization of the bicyclic moiety resulted in
cyclopropyl isoquinolone derivative 34, which is devoid of N,N-
dimethyl substitution, thus circumventing the potential for
formation of primary aniline metabolites upon double
demethylation by CYP mediated oxidation. Compound 34
has BTK Kd of 4 nM and HWB IC50 of 104 nM. Fluorine
substitution on the benzene ring ortho to the carbonyl group
led to 1, which exhibited a significantly improved binding
affinity to BTK (Kd of 0.3 nM) in addition to superior HWB
potency (IC50 of 17 nM). To better understand the fluorine
effect, a crystal structure of a close analogue 35 with BTK was
obtained (Figure 3B). The electronegative fluorine atom is
within van-der-Waals distance to the primary amine of K430
and the aromatic hydrogen at the ortho position of F413. This
suggests that the approximate 10-fold increase in potency
gained with the fluorine substituent is likely due to electrostatic
interactions with the protein. More thorough SAR studies of
the effect of fluorine substitution on different bicyclic moieties
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(0.1/1/1222 μg/mL in water, SIF, and SGF, respectively) than
33, compound 1 has a favorable in vivo PK profile with F% of
26/38, AUC of 0.57/2.37 μM·h, and T1/2 of 9.8/11 h in rats
and dogs (PO administration, 2 mg/kg). Detailed preclinical
biological profiling of 1 has been described elsewhere and
demonstrates that this inhibitor has excellent efficacy in various
animal models of RA.1 Given its desirable PK and PD profile,
compound 1 was advanced into preclinical development
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures; 1HNMR, HRMS spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 408-332-6502. E-mail: yan.lou@gmail.com or ylou@
nurix-inc.com.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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We acknowledge Xingrong Liu, Jennifer Fretland, Bill Fitch,
and Bo Wen for providing drug metabolism support and Naina
Patel and Qingyan Hu for formulation support. This work was
supported exclusively by Hoffmann-La Roche.
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ABBREVIATIONS USED
■
BTK, Bruton’s tyrosine kinase; CYP, cytochrome P450
enzymes; hERG, human ether-a-go-go related gene; HWB,
human whole blood; JAK, Janus kinase; MNT, micronucleus
test; RA, rheumatoid arthritis; S9, supernatant fraction obtained
from liver homogenate by centrifuging at 9000g for 20 min in a
suitable medium, this fraction contains cytosol and micro-
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