Stereochemical Differences in Fluorocyclopropyl Amides Enable Tuning of Btk Inhibition and Off-Target Activity

Article abstract of DOI:10.1021/acsmedchemlett.0c00249

Bruton's tyrosine kinase (Btk) is thought to play a pathogenic role in chronic immune diseases such as rheumatoid arthritis and lupus. While covalent, irreversible Btk inhibitors are approved for treatment of hematologic malignancies, they are not approved for autoimmune indications. In efforts to develop additional series of reversible Btk inhibitors for chronic immune diseases, we sought to differentiate from our clinical stage inhibitor fenebrutinib using cyclopropyl amide isosteres of the 2-aminopyridyl group to occupy the flat, lipophilic H2 pocket. While drug-like properties were retained - and in some cases improved - a safety liability in the form of hERG inhibition was observed. When a fluorocyclopropyl amide was incorporated, Btk and off-target activity was found to be stereodependent and a lead compound was identified in the form of the (R,R)- stereoisomer.

Full text of DOI:10.1021/acsmedchemlett.0c00249

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Letter
Stereochemical differences in fluorocyclopropyl amides
enable tuning of Btk inhibition and off-target activity
James J. Crawford, Wendy Lee, Adam R. Johnson, Kelly J Delatorre, Jacob Chen, Charles Eigenbrot,
Julia Heidmann, Satoko Kakiuchi-Kiyota, Arna Katewa, James R. Kiefer, Lichuan Liu, Joseph W. Lubach,
Dinah Misner, Hans Purkey, Karin Reif, Jennifer Vogt, Harvey Wong, Christine Yu, and Wendy B. Young
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00249 • Publication Date (Web): 13 Jul 2020
Downloaded from pubs.acs.org on July 13, 2020
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Stereochemical differences in
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fluorocyclopropyl amides enable tuning of
Btk inhibition and off-target activity
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James J. Crawford*, Wendy Lee*, Adam R. Johnson, Kelly J. Delatorre, Jacob Chen, Charles
Eigenbrot, Julia Heidmann, Satoko Kakiuchi-Kiyota, Arna Katewa, James R. Kiefer, Lichuan
Liu, Joseph W. Lubach, Dinah Misner, Hans Purkey, Karin Reif, Jennifer Vogt, Harvey Wong,
Christine Yu, Wendy B. Young
Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080
ABSTRACT:
Bruton’s tyrosine kinase (Btk) is thought to play a pathogenic role in chronic immune
diseases such as rheumatoid arthritis and lupus. While covalent, irreversible Btk inhibitors
are approved for treatment of hematologic malignancies, they are not approved for
autoimmune indications. In efforts to develop additional series of reversible Btk inhibitors
for chronic immune diseases, we sought to differentiate from our clinical stage inhibitor
fenebrutinib using cyclopropyl amide isosteres of the 2-aminopyridyl group to occupy the
flat, lipophilic H2 pocket. While drug-like properties were retained—and in some cases
improved—a safety liability in the form of hERG inhibition was observed. When a
fluorocyclopropyl amide was incorporated, Btk and off-target activity was found to be
stereo-dependent and a lead compound was identified in the form of the (R,R)- stereoisomer.
KEYWORDS: Kinase inhibitor, Btk, Rheumatoid arthritis, Cardiomyocytes, hERG
Bruton’s tyrosine kinase (Btk) is a nonreceptor tyrosine kinase that is expressed in all
hematopoietic cells except T cells.1,2 Antigen binding to the B-cell antigen receptor (BCR) on
B lymphocytes and immune complex binding to Fc receptors (e.g., FcγR, FcR) on myeloid
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cells activates Btk.1,3−5 Preclinical and emerging clinical data support the hypothesis that
inhibition of Btk may be effective for the treatment of immunological disorders such as
rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), diseases in which B cells
and/or myeloid cells foster an excessive autoimmune response.4-17 Thus, significant efforts
have been expended over the past two decades toward the goal of developing Btk inhibitors
for treatment of chronic inflammatory diseases and hematologic malignancies.18 Irreversible
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Btk inhibitors ibrutinib
1 2 (ACP-196,
(PCI-32765, Imbruvica),19 acalabrutinib
Calquence),20,21 and zanubrutinib 3 (BGB-3111)22 are approved for treatment of B-cell
malignancies. Given the potential concern with the chronic use of covalent inhibitors, our
efforts focused on non-covalent inhibitors to achieve a clinical profile appropriate for the
treatment of immune diseases.
H
O
O
O
N
N
NH2
N
O
NH2
N
H2N
N
N
N
N
N
N
O
N
HN
N
N
N
O
O
3, Zanubrutinib
1, Ibrutinib
2, Acalabrutinib
O
N
N
O
N
N
N
N
NH
N
O
O
HO
NH
NH
N
N
O
O
HO
N
N
N
N
H
N
N
N
N
O
S
O
O
N
F
4, GDC-0834
5, G-278
6, GDC-0853
Figure 1. Ibrutinib, acalabrutinib, zanubrutinib and Genentech’s published Btk inhibitors.
We previously reported the discovery and structure-activity relationship (SAR) of several
series of non-covalent Btk inhibitors. In those efforts we optimized beyond our first clinical
inhibitor, GDC-0834 (4),23,24 to molecules such as G-278 (5) by incorporating tricyclic ring
systems.25 However, 5 and analogs had low safety margins in rat and dog,26 in line with a
cytotoxicity observed in primary human hepatocytes.27 Subsequent efforts focused on
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modifying the partially solvent-exposed aryl piperazine and lowering both the logD and the
predicted human dose.27,28 These efforts led to the identification of the highly potent and
selective, non-covalent Btk inhibitor fenebrutinib (GDC-0853, 6), currently in late-stage
clinical development for the treatment of autoimmune diseases.
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In parallel, we set out to identify additional series with structural diversity from 6, a
strategy that might also avoid potential bioactivation of the 1,4-disubstituted aromatic ring
that has been implicated in many toxicities including drug induced liver injury.28,29 Since the
H3 tricyclic motif and the hydroxymethyl pyridyl ring linker were extensively optimized, we
explored the partly solvent-exposed H2 region of the molecule. We hypothesized that
replacing the aromatic ring with a suitably-substituted amide might offer a viable starting
point.30 In particular, we hoped that a small cyclopropane carboxamide might replicate the
sp2 character of the pyridyl  system (Figure 2); others have reported such substitutions in
different kinases.31,32 Molecular modeling of cyclopropane carboxamide 8 in Btk (Figure 3)
suggested that the carbonyl oxygen overlaps with the pyridine nitrogen of 6, orienting the
amine N-H towards the hinge. We hypothesised that by removing an aromatic ring, amide
analogues would benefit from lower molecular weight and lipophilicity, lack of a basic amine,
and improved aqueous solubility.33,34
'H2'
Target: cyclopropylamide
Flat, lipophilic pocket
replacement
O
N
N
Hinge
binder
O
NH
N
NH
HO
O
'H1'
HO
O
N
N
N
N
N
N
O
N
'H3' /
Selectivity
pocket
O
N
6
8
GDC-0853
Fenebrutinib
Figure 2. Schematic representation of the hinge binding (H1), lipophilic pocket (H2), and
selectivity pocket (H3) elements of Btk inhibitor fenebrutinib (6, GDC-0853), and the
disconnection of the proposed isosteric cyclopropylamide in the H2 pocket with 8.
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Figure 3. Model of a cyclopropane carboxamide as an isostere for 2-aminopyridine.
Superposing computationally docked 8 (cyan) on the Btk crystal structure with 6 (tan)
indicates that replacing the pyridine tail of 6 with cyclopropylamide retains pyridone
hydrogen bonding with the hinge and forms an additional interaction with the polarized
cyclopropyl C-H.
Our first amide analogue cyclopropanyl 8 inhibited Btk with an IC50 of 7.1 nM vs. 2.3 (±0.7)
nM for 6. A targeted library incorporating small amide substituents (Table 1, 8–17) was
prepared from advanced intermediate 7. Cyclopropyl substitutions retained the greatest
inhibitory potency against Btk (Table 1; assay protocols in Supporting Information). Kinetic
solubility gave an initial gauge of intrinsic solubility. 35 The initial cyclopropane amide 8 was
the most potent amide in this series. Similarly-sized short alkyl and cyclobutyl analogs (9–
11, 13–14) were less potent than the cyclopropyl. Branched alkyl i-Pr (12) was even less
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potent, and was likely too bulky to fit into the narrow pocket previously occupied by a flat,
aryl group. Added polarity with oxetane, methoxy, or tetrahydrofuran substitution (15–17,
respectively) drastically decreased potency.
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Cognizant of the tight SAR and seeking to improve upon the potency of 8, we postulated
that the incorporation of fluorine atoms on the cyclopropane might be tolerated and could
improve physicochemical properties.36,37 Fluoro substitution at the tertiary carbon in 18
resulted in a significant loss in Btk potency (IC50=60 nM), possibly due to a then-
hypothesised loss of a non-classical H-bond between the cyclopropyl C-H and the hinge
(Figure 3). In contrast, the cis-2-fluoro cyclopropane showed improved potency, with the
(S,S)-enantiomer 19 (IC50=2.3 nM) slightly more active than the (R,R)-enantiomer 20
(IC50=3.8 nM). Additionally, both 19 and 20 demonstrated improved kinetic solubility over
8. Illustrative of the improved potency and decreased lipophilicity, the LLE increased by a
log unit. This is indicative of a specific interaction with the protein rather than the simple
lipophilic partitioning that might have been expected from the introduction of the
cyclopropyl motif.38
Table 1: Amide-based 2-aminopyridine replacements in the Btk H2 pocket.
cglogDd/logD
Cmpd
R
Btk IC50 (nM)a LLEb
K. sol.c (M)
(pH 7.4)e
8
7.1±1.1
15±1.5
14±4.2
5.5
6.0
5.7
9
2.5/3.0
9
82
105
1.8/2.1
2.3/2.5
Me
Et
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44±20
200±3.8
12±1.6
22±2.4
74±11
74±12
320±78
5.1
3.8
4.9
4.6
5.2
5.9
4.7
83
2.6/2.6
2.7/3.2
2.6/3.3
2.8/3.4
1.8/2.2
1.8/1.5
2.3/2.1
Pr
85
iPr
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68
84
O
80
O
122
107
Me
O
F
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19
60±14
5.0
6.5
85
93
2.4/2.5
2.3/2.5
F
2.3±0.5
H
F
H
H
H
20
3.8±0.6
6.5
95
2.3/2.3
IC50 values (mean±SD) were determined in at least triplicate. See Supporting Information for assay
details. bLLE=pIC50(Btk)–cgLogD. cKinetic solubility at pH 7.4. dCalculated logD using MoKa program
v2 (www.moldiscovery.com). eLogD measured using a high throughput microscale shake flask with
LC tandem MS quantitation.
Seeking to improve our preclinical inhibitors by optimally balancing physicochemical
properties and potency, we synthesized a set of analogs containing the simple cyclopropyl
amide combined with potent H3 selectivity pocket motifs (Table 2).25,39 Compound 21 with
a
more polar H3 motif lost potency (IC50=98 nM) while pyridazinone-fused
tetrahydrobenzothiophene 22 (IC50=7.7 nM) was equipotent to 8 (IC50=7.1 nM). However,
the potency of 22 to prevent BCR-dependent B cell activation in human whole blood,
assessed by monitoring anti-IgM-induced CD69 expression on B cells (CD69 IC50=1100 nM)
was ~8-fold less than 8 (CD69 IC50=140 nM). In contrast, t-butyl phthalazinone 23 possessed
improved biochemical (IC50=4.8 nM) and whole blood (CD69 IC50=78 nM) potency.
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Additionally, 23 had improved solubility (76 μM) over 8 (9 μM) and was stable in human and
5
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rat liver microsomes (HLM 3.2, RLM 13 mL/min/kg).
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Table 2: H3 Selectivity Pocket SAR.
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O
NH
HO
O
R
N
N
Kinetic
Solubility
(M)c
HLM/RLM
CLhep
Btk
IC50 WB CD69
IC50 (nM)b
LLEd/logD
(pH 7.4)e
Cmpd
R
(nM)a
(mL/min/kg)f
140±20
n=2
N
N
O
8
7.1±1.1
98±9.9
9
5.5/3.0
6.1/1.2
5.3/7.2
9.8/20
>4300
n=1
N
N
21
27
O
N
1100
n=1
N
22
23
7.7±1.8
9
6.3/2.1
6.2/2.5
7.4/8.4
3.2/13
S
O
N
N
4.8
78±26
n=3
76
O
n=1
F
a-e See footnotes from Table 1. Human and rat liver microsome-predicted hepatic clearance.
f
The t-butyl phthalazinone H3 was combined with all four stereoisomers of the 2-fluoro
substituted cyclopropyl amide (Table 3). The cis-fluoro isomers 24 and 25 were nearly
equipotent against Btk (IC50=3.2 and 2.4 nM, respectively) relative to unsubstituted
cyclopropane 23 (IC50=4.8 nM). Conversely, the trans-fluoro isomers 26 and 27 were less
potent against Btk and in human whole blood CD69 and offered no advantages over 23. In
whole blood the (S,S)-enantiomer 25 was twice as potent as the (R,R)-enantiomer 24. Of the
four diastereomers, cis (S,S)-isomer 25 offered the best LLE (7.3) and kinetic solubility (107
M) with high permeability (MDCK Papp A:B 12.5 10–6 cm/s) and good stability in both rat
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(RLM 11 mL/min/kg) and human liver microsomes (HLM 8 mL/min/kg). As a final
optimisation step we studied the effect of changes to the key hinge-binding heterocycle.
Replacement of the pyridone with pyrazinone 28 and pyridazinone 29 could influence
physicochemical properties such as logD,40 but these changes were deleterious to both
biochemical and whole blood potency.
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Table 3: Single stereoisomers of 2-fluorocyclopropyl amides with the t-butyl-phthalazinone
H3 motif and core heterocycle SAR.
R
NH
OH
O
N
N
X
N
Y
F
O
N
WB
HLM/
MDCK
Btk IC50 CD69
LLEd/logD RLM CLhep Papp A:B
K. sol.
Cmpd
R
X
Y
(nM)a
IC50
(pH 7.4)e
6.2/2.5
6.9/2.0
(mL/min (10-6
(M)c
(nM)b
/kg)f
cm/s)g
O
4.8
78±26
n=3
23
24
25
26
CH CH
76
74
3.2/13
13.5
6.4
n=1
H
H
180±73
n=2
O
(R)
(R)
CH CH 3.2±0.5
CH CH 2.4±0.4
5.5/11
8/11
F
O
H
78±57
n=3
F
107
85
7.3/1.7
4.8/3.0
12.5
10.8
H
(S)
(S)
F
H
32
CH CH
n=1
326
n=1
O
(S)
(R)
8.5/14
H
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O
H
7.2
n=1
110
n=1
H
27
28
29
CH CH
4.6/15
<3.9/13
7.4/16
51
77
6.0/2.5
6.6/1.3
14
--
F
(R)
(S)
(S)
(S)
O
H
F
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N
CH 26±3.2
--
H
(S)
O
H
214
n=1
F
CH
N
5.1±1.2
127
6.9/1.7
--
H
(S)
a-fSee footnotes from Tables 1 & 2. gPermeability in Madin-Darby Canine Kidney (MDCK) cells, apical-
to-basolateral flux.
We solved a 1.6-Å resolution X-ray co-crystal structure of 25 bound to Btk (Figure 4). The
compound formed extensive van der Waals and hydrogen bonding interactions within the
active site. As predicted, 25 formed hydrogen bonds between the pyridone carbonyl and the
Met477 amide backbone NH of the hinge. Additionally, the participation of the
cyclopropane’s polarized, tertiary hydrogen in a dipole-induced dipole to the carbonyl of
Met477 (Figure 4a) was confirmed. As shown by our modeling, a cyclopropane fits well in
the H2 region, bisecting the plane previously occupied by the pyridine ring and resulting in
favorable van der Waals interactions with the protein surface (Figure 4b).
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Figure 4. X-ray co-crystal structure of 25 bound to Btk. a) Two predicted hydrogen bonds
to the hinge are present along with an additional favourable CH-carbonyl interaction
between the c-propyl proton and the Met477 backbone carbonyl (pink dashed line). b) The
c-propyl forms complimentary van der Waals interactions with the protein surface.
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Given the excellent biochemical and whole blood potency, as well as in vitro
pharmacokinetics (PK) properties of 25, we further profiled and compared it to our (later)
clinical stage inhibitor, fenebrutinib, 6 (Table 4). Btk potency of 25 was similar to 6, while
the whole blood potencies of 25, both anti-IgM-induced CD69 expression on B cells and Btk
Y223 autophosphorylation, were ten-fold and seven-fold less potent than 6, respectively,
likely driven by the previously-reported time dependent inhibition of Btk by 6. Although not
as potent in the whole blood CD69 and pBtk assays relative to 6, cyclopropane carboxamide
25 had improved kinetic (≥100M) and thermodynamic (27M) solubility vs. 6. We next
assessed the in vivo PK of 25 in rats and dogs. In rats, 25 (0.2 mg/kg IV and 1.0 mg/kg PO,
amorphous suspension) had low plasma clearance (13 mL/min/kg), moderate
bioavailability (F=40%), and comparable unbound clearance (283 mL/min/kg) to 6 (314
mL/min/kg). At a dose of 5.0 mg/kg in rats, crystalline 25 in aqueous suspension gave just
F=7%, but this improved 10-fold to 72% when dosed as an amorphous solid dispersion
(ASD) suspension, showing promise as a potential development candidate using this
technology. Further, 25 had excellent in vivo PK in dogs (1.0 mg/kg IV and 2.0 mg/kg PO,
amorphous suspension) with low plasma clearance (2 mL/min/kg for 25) and complete oral
bioavailability (F=123%) when dosed as an ASD suspension. Furthermore, 25 showed no
CYP450 inhibition or induction liability. Cyclopropylcarboxylic acid has been identified as a
potential toxicophore, via inhibition of mitochodrial function and/or incorporation into the
TCA cycle.41,42 We conducted in vitro metabolite identification studies to assess the
metabolic stability of the amide, whereby 25 was incubated at 5 μM concentration in
cryopreserved human, rat, and dog hepatocytes (~1.0X106 cells/mL) for three hours. In each
case ≤1 % amide hydrolysis was observed.
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Table 4. Profiles of 25 and 6
5
6
7
8
25
6 (fenebrutinib)
2.3 nM/7.4
8 nM
Btk IC50/LLE
2.4 nM/7.3
78 nM
CD69 HWB IC50
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pBtk HWB IC50
78 nM
11 nM
MW/mLogD/clogP
Kin.; T. Solubility
536/1.6/1.4
107 M; 100 M
12.1
665/1.6/2.2
67 M; 2M
15.1
MDCK Papp A:B (10-6 cm/s)
LM CLhep (H/R/D) (mL/min/kg)
8/11/20
9.5/14/29
9.3/10/<21
All >10 M
22 mL/min/kg, 96%
314 mL/min/kg
12 mL/min/kg
130%/5 mg/kg
3/295
Hep CLhep (H/R/D) (mL/min/kg)
CYP inhibition (5 isoforms)
Rat PK: iv CLp, F a
Rat Clu
6.2/11/<21
All >10 M
15 mL/min/kg, 40%
326 mL/min/kg
2 mL/min/kg
123%/2 mg/kg
1/220
Dog PK: iv CLp
PO, F/dose b
Kinase Sel. (Hits>50% at 1µM)
hERG IC50
1.3M
>30M
aDosed 1 mg/kg PO was dosed as a cassette ASD suspension, made by lyophilization. Dosed
b
2 mg/kg as an ASD suspension, discrete dosing, made via spray drying.
Compound 25 demonstrated exquisite Btk selectivity when tested at 1 M against 220
human kinases (Figure 5), showing >50% inhibition of only one off-target kinase, Src, and
minimal inhibition of Fgr and Bmx, two off-targets of 6 (Tables S1, S2). Based on IC50 values,
compound 25 is greater than 550-fold selective for inhibition of Btk over Src.
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Figure 5. Kinome tree representation of kinase selectivity of 25 at 1M.
Our enthusiasm for 25 was tempered by the finding that it inhibited the human ether-à-
go-go-related gene (hERG) potassium channel current (IC50=1.3 M). It is known that hERG
blockade is the most common mechanism of acquired (drug-induced) long QT syndrome and
torsades de pointes arrhythmia.43,44 We thus set out to investigate the SAR with the goal of
attenuating the observed hERG inhibition (Table 5).
Substitution on both the H2 and amide motifs dramatically decreased the inhibitory effect
of compounds on hERG. Since hERG risk often increases with increasing lipophilicity44,45 we
first examined the effect of different H3 groups on hERG inhibition at 10 M (Table 5,
compounds 8, 21–23). Most of the H3 groups on the cyclopropyl amide showed significant
hERG inhibition ( 60% inhibition) with the exception of 21, in line with its reduced
lipophilicity. Although 21 significantly reduced hERG blockade (25%@10 M), it lacked the
desired level of Btk potency (IC50=98 nM). We next evaluated different amides (13, 18, 30–
32) while utilizing the H3 lipophilic pocket motif from fenebrutinib (6). Again, hERG
inhibitory potency was reduced with this more polar motif (lower log D). Interestingly, while
compound 25 showed 88% hERG inhibition at 10 M, its enantiomer, (R,R) isomer 24,
seemed to exhibit a significantly attenuated effect (25 %@10 M). The hERG IC50 of 24 was
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confirmed to be >10 M, >6x less potent against hERG compared with 25. To confirm that
the reduction in hERG inhibition relative to 25 is biologically meaningful, 24 and 25 were
assessed by multi-electrode array using induced pluripotent stem cells (iPSC)-derived
cardiomyocytes. Consistent with strong hERG blockade, 25 demonstrated substantial
prolongation of field potential duration (FPD) (80 and 90% increase at 3.2, 10 M,
respectively). In contrast, no change in FPD was observed with up to 10M of 24 (Figure 6).
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Table 5. hERG SAR profile of amide-H2 Btk inhibitors (% inhibition @ 10 M)
hERG (%
hERG
Log D
Btk IC50 WB CD69
IC50 (nM)e
inhib @
Cmpd Structure
(pH 7.4)c (nM)d
IC50(M)b
10M)a
O
NH
OH
O
8
65%
68%
50%
25%
–
–
–
–
3.0
3.3
2.4
1.2
7.1±1.1
12±1.6
60±14
98±9.9
137
N
N
N
O
N
O
NH
13
18
21
>5960
4170
OH
O
N
N
N
O
N
F
O
NH
OH
O
N
N
N
O
N
O
NH
OH
O
N
>4330
N
N
O
N
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84%
83%
–
–
2.1
2.5
7.7±1.8
1080
78
9
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O
NH
4.8
OH
O
N
N
n=1
N
F
O
N
F
(R)
(R)
O
NH
24
25
25%
>10
1.7
3.2±0.5
2.4±0.4
180
OH
O
N
N
N
F
O
N
F
(S)
(S)
O
NH
88%
15%
1.3
1.7
1.8
78
–
OH
O
N
N
N
F
O
N
HO
O
NH
410±240
n=2
OH
O
30
31
–
N
N
N
O
N
O
[38% inh
@
N
O
NH
17%
23%
–
–
2.3
2.0
–
–
OH
O
N
111nM],
n=1
N
N
O
N
N
280
n=1
O
NH
32
OH
O
N
N
N
O
N
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F
(S)
(S)
5
6
7
8
O
NH
OH
O
33
34
–
–
1.6
2.8
2.0
2.0±0.5
1.9±0.2
37
N
N
N
F
O
9
F
10
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F
(R)
(R)
O
NH
OH
O
>10
125
N
N
N
F
O
F
aEffects (% inhibition) of 10M compounds on hERG tested in at least duplicate. hERG patch clamp IC50 values
were determined based on five concentrations tested (0.1 – 10 M) in at least duplicate. c-eSee footnotes from
Table 1.
b
Figure 6: Drug-induced field potential shift in iPSC-derived cardiomyocytes: 24 vs. 25.
Field potential duration (FPD) was measured in iPSC-derived cardiomyocytes after one hour
of compound treatment. 24 and 25 were tested at 1, 3.2, and 10M. N=4, error bars
represent SEM.
The significant reduction in hERG inhibition with the (R,R) stereoisomer 24 was
intriguing. In contrast to the observed LLE advantage obtained with (S,S)-25, stereoisomer
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24 suffered a 2-fold loss in CD69 potency. Exploiting previously reported SAR, we
anticipated that replacing the pyridyl linker of 25 with a fluoro-aryl linker (33) would
improve the potency. Indeed, 33 was twice as potent (CD69 IC50=37 nM) as 25 (CD69
IC50=78 nM) (Table 5). However, strong hERG inhibition was also observed (IC50 1.6 M) for
this (S,S)-stereoisomer 33. Hoping to improve potency of the corresponding (R,R)-isomer,
fluoroaryl 34 was prepared. Indeed, the Btk potency improved (IC50=1.9 nM for 34 vs. 3.2
nM for 24), as did the whole blood potency (CD69 IC50=125 nM for 34 vs. 180 nM for 24).
Interestingly 34 showed no discernable hERG liability (hERG IC50>10 M) or prolongation
of FPD at up to 3.2 µM (solubility limited). While differences between enantiomers in hERG
blockade have been observed,47 this example is unusual in that the parent compounds would
be expected to be neutral at physiological pH.
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To conclude, replacement of the interaction features of a pyridyl ring with a cyclopropyl
amide resulted in a structurally-differentiated sub-series of non-covalent BTK inhibitors.
SAR investigations and subsequent combination with a (t-butyl)-phthalazinone H3 motif led
to the identification of 25, a potent 2-fluoro-cyclopropylamide Btk inhibitor with attractive
physicochemical properties, excellent kinase selectivity, and favorable oral
pharmacokinetics in rat and dog. A potential liability of these inhibitors was identified in the
form of hERG blockade that was found to be sensitive to stereochemistry, where the (R,R)-
stereoisomers were found to be at least 6-fold less active when compared to their (S,S)-
isomers.
ASSOCIATED CONTENT
Supporting Information
The supporting information is available free of charge via the Internet at http://pubs.acs.org.
Experimental procedures, compound characterization, in vitro pharmacology, kinase
selectivity profiling, in vivo methods, and crystallography details (PDF).
AUTHOR INFORMATION
Corresponding Authors
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*E-mail: crawford.james@gene.com (J. J. Crawford), orcid.org/0000-0002-6408-8246
*E-mail: wendyle@gene.com (W. Lee), orcid.org/0000-0002-9959-8529
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Author Contributions
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The manuscript was written through contributions of all authors.
ORCID
Adam Johnson: 0000-0003-1459-4236
James Kiefer: 0000-0001-6317-0902
Funding
Thanks to Genentech for funding this work.
Note
The authors are, or were, employees of Genentech/Roche and hold/have held shares in
Roche.
Abbreviations
cglogD, calculated logD (Genentech); pBtk, phosphorylated Btk whole blood assay; MDCK
Paap, Madin-Darby canine kidney apparent permeability; HWB, human whole blood; kinase
sel., kinase selectivity; Bmx, bone marrow tyrosine kinase on chromosome X.
ACKNOWLEDGMENTS
We thank Zoe Zhong, Aaron Fullerton, Orlando Zuniga, Suzanne Tay, Ivy Chen, Pawan Bir Kohli
and Sergei Romanov (Nanosyn) for their assistance in gathering and reviewing data; Snahel Patel
and Drs Gampe, Parr, Grandner, Landry, and Mackenzie for helpful comments. X-ray diffraction
data were collected at beamline 21-IDF operated by the Life Sciences Collaborative Access Team
at the Advanced Photon Source, Argonne National Laboratory. This research used resources of the
Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility
operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-
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AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic
Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817).
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