Journal of Medicinal Chemistry
Article
experiments described involving animals, all procedures were carried
out in compliance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals (1985), under approval of the
Institutional Animal Care and Use Committee (IACUC).
CONCLUSIONS
■
Herein, we describe our strategy to discover an orally available
covalent Btk inhibitor with sufficient selectivity over other
similar Src and Tek family kinases. Our approach was to start
with a highly efficient fragment and incrementally grow it using
the tenants of FBDD along with the aid of cocrystal structures,
namely, keeping a close watch on the heavy-atom count and
lipophilicity constraints (LE and LLE) to rapidly arrive at our
lead molecule 3. The SAR (covered in Table 2) from the
initiation of effort until identification of TAK-020 spanned 6
months of synthetic work. The efficiency of TAK-020 and its
rapid identification highlighted the advantages of FBDD to the
team. Further thorough in vitro and in vivo profiling of 3
followed by CIA models demonstrated both pharmacodynamic
(PD) and efficacy endpoints (paw swelling in a rat CIA) being
met. Examination of target engagement in vitro and
extrapolation toward humans (efficacious dose and target
occupancy) revealed that compound 3 could be efficacious at a
very low human dose.22 This led to successful transfer of the
program into the clinic; in vivo human PK, PD, and target
engagement data have been recently revealed in separate
publications.23
Preparation of (S)-5-(1-((1-Acryloylpyrrolidin-3-yl)oxy)-
isoquinolin-3-yl)-2,4-dihydro-3H-1,2,4-triazol-3-one (3). Step
1: (S)-tert-Butyl 3-((3-chloroisoquinolin-1-yl)oxy)pyrrolidine-1-car-
boxylate. To (S)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (1.134
g, 6.06 mmol) in NMP (10 mL) at 0 °C was added NaH (60%) (202
mg, 5.05 mmol). The mixture was stirred for 5 min, and 1,3-
dichloroisoquinoline (1.000 g, 5.05 mmol) was added. The reaction
mixture was stirred at RT for 5 min and then heated at 135 °C for 30
min in a microwave reactor. The mixture was diluted with water (400
mL) and extracted with EtOAc (3 × 125 mL). The organic layers
were combined, washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The crude product was purified by silica
column chromatography eluting with a gradient of 25−50% EtOAc in
1
hexane to give the title compound (5.29 g, 75%). H NMR (500
MHz, DMSO-d6): δ 1.40 (d, J = 14.16 Hz, 9 H), 2.12−2.34 (m, 2 H),
3.42−3.58 (m, 3 H), 3.69 (td, J = 12.33, 4.64 Hz, 1 H), 5.63−5.76
(m, 1 H), 7.59 (s, 1 H), 7.64 (ddd, J = 8.30, 7.08, 1.22 Hz, 1 H), 7.81
(td, J = 7.57, 1.46 Hz, 1 H), 7.87−7.92 (m, 1 H), 8.11−8.19 (m, 1
H); ESI-MS m/z: [M + H-tert-butyl]+, 293.5.
Step 2: (S)-tert-Butyl 3-((3-cyanoisoquinolin-1-yl)oxy)-
pyrrolidine-1-carboxylate. A solution of (S)-tert-butyl 3-((3-chlor-
oisoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate (4.430 g, 12.70
mmol), zinc cyanide (2.980 g, 25.40 mmol), and Pd(PPh3)4 (1.468
g, 1.27 mmol) in DMF (36.3 mL) was heated at 160 °C for 20 min in
a microwave reactor. The reaction mixture was filtered, diluted with
water (400 mL), and extracted with EtOAc (2 × 100 mL). The
organic layers were combined, washed with brine, dried over Na2SO4,
and concentrated in vacuo. The crude product was purified by silica
column chromatography to give the title compound as a white-to-
pale-yellow solid (3.570 g, 83%). 1H NMR (500 MHz, DMSO-d6): δ
1.40 (d, J = 13.18 Hz, 9 H), 2.23 (d, J = 11.23 Hz, 2 H), 3.42−3.59
(m, 3 H), 3.65−3.75 (m, 1 H), 5.68−5.80 (m, 1 H), 7.82−7.89 (m, 1
H), 7.91−7.98 (m, 1 H), 8.06 (d, J = 8.79 Hz, 1 H), 8.21−8.30 (m, 2
H); ESI-MS m/z: [M + H-tert-butyl]+, 284.6.
Step 3: (S)-tert-Butyl 3-((3-(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-
yl)isoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate. (S)-tert-Butyl 3-
((3-cyanoisoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate (4.670 g,
13.76 mmol), ethyl hydrazinecarboxylate (7.160 g, 68.80 mmol),
DBU (1.037 mL, 6.88 mmol), and NMP (34.6 mL) were mixed in a
200 mL high-pressure reaction vessel. The resulting suspension was
heated at 170 °C overnight and was then cooled to room temperature.
Crushed ice was added, and the mixture was stirred. A yellow
precipitate was collected by vacuum filtration, washed with additional
water, and dried in a vacuum oven at 45 °C overnight to give the title
compound, which was used in the next step without further
purification (5.47 g). 1H NMR (500 MHz, DMSO-d6): δ 1.33−
1.51 (m, 9 H), 2.09−2.38 (m, 2 H), 3.39−3.60 (m, 3 H), 3.75 (dd, J
= 12.20, 4.88 Hz, 1 H), 6.03−6.22 (m, 1 H), 7.62−7.71 (m, 1 H),
7.81 (td, J = 7.57, 1.46 Hz, 1 H), 7.95−8.05 (m, 1 H), 8.11−8.29 (m,
2 H), 11.78 (s, 1 H), 12.03 (br s, 1 H).
Step 4: (S)-5-(1-(Pyrrolidin-3-yloxy)isoquinolin-3-yl)-2,4-dihydro-
3H-1,2,4-triazol-3-one. To a 200 mL round-bottom flask charged
with crude (S)-tert-butyl 3-((3-(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-
yl)isoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate (5.47 g) and diox-
ane (27.5 mL) was added 4 M HCl in dioxane (13.76 mL, 55.1
mmol). The suspension was stirred at RT with periodic monitoring by
HPLC. Upon completion, the reaction mixture was concentrated in
vacuo to give a HCl salt of the title compound as a light tan powder
that was dried and used without further purification. ESI-MS m/z: [M
+ H]+, 298.6.
Step 5: (S)-5-(1-((1-Acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-
2,4-dihydro-3H-1,2,4-triazol-3-one. To a suspension of (S)-5-(1-
(pyrrolidin-3-yloxy)isoquinolin-3-yl)-2,4-dihydro-3H-1,2,4-triazol-3-
onehydrochloride (4.29 g) in DCM (48.1 mL) was added 2,6-
dimethylpyridine (3.19 mL, 27.4 mmol). Upon cooling the
suspension to 0 °C, acryloyl chloride (1.3 mL, 15.9 mmol) was
EXPERIMENTAL SECTION
General Methods for Chemistry. All commercially obtained
■
solvents and reagents were used as received. Microwave-assisted
reactions were run in a Biotage initiator. H and 13C NMR spectra
1
were recorded on a Bruker Advance 400 or Varian 500. Chemical
shifts are reported in parts per million (ppm, δ) using the residual
solvent line as a reference. Splitting patterns are designated using the
following abbreviations: s, singlet; d, doublet; t, triplet; dd, doublet of
doublet; m, multiplet; br, broad. Coupling constants (J) are reported
in hertz (Hz). The purity of all final compounds was determined by
HPLC, and the compounds are at least ≥95% pure. HPLC purity
determination used the following equipment: an Agilent 1200
quaternary pump, an Agilent Autosampler 1200 series, and an Agilent
1200 series DAD. The purity of the test compounds was determined
to be ≥95% using one of the following two HPLC conditions. For
HPLC method A, a Phenomenex Kinetix C18 column at 200C was
used, 2.1 mm × 100 mm, 2.6 μm, eluting with mobile phase A 0.05%
TFA in water and mobile phase B 0.0375% TFA in acetonitrile with a
flow rate of 1 mL min−1 with a gradient of 5−90% B over 6 min. UV
detection was performed (λ = 220/254 nm). For HPLC method B, a
Phenomenex Kinetix C18 column at 400C was used, 2.1 mm × 100
mm, 2.6 μm, eluting with mobile phase A 0.05% TFA in water and
mobile phase B 0.035% TFA in acetonitrile with a flow rate of 1 mL
min−1 with a gradient of 5−90% B over 6 min. UV detection was
performed (λ = 220/254 nm). LC−MS data were acquired on a
Waters Acquity UPLC/MS system equipped with a UPLC binary
pump, an SQD 3100 mass spectrometer with an electrospray
ionization (ESI) source, a PDA detector (210−400 nm), and an
evaporative light scattering detector (ELSD). LC was performed using
a Waters BEH C18,1.7 μm, ID 2.1 mm × 50 mm at a 55 °C column
eluting with mobile phase A 0.05% TFA in water and mobile phase B
0.035% TFA in acetonitrile with a flow rate of 0.8 mL min−1 with a
gradient of 5−90% B over 1.5 min. The UV absorbance wavelength
was sampled at a rate of 20 points s−1 between 210 and 400 nm at a
resolution of 1.2 nm. High-resolution mass measurements were
obtained on a Waters Xevo G2-XS QTof quadrupole time-of-flight
mass spectrometer (Waters Corporation, Milford, MA 01757) in
positive and negative electrospray modes. The samples were separated
using a Waters Acquity UPLC I-Class PLUS system on a BEH 2.1 ×
50 mm, 1.7 μm column eluted with mobile phase A 0.01% formic acid
in water and mobile phase B methanol, with a flow rate of 0.3 mL
min−1 with a gradient of 5−90% B over 9.2 min at 60 °C. The mass
accuracy was calculated for major observed isotopes against the
theoretical mass ions derived from the chemical formula. For
G
J. Med. Chem. XXXX, XXX, XXX−XXX