Discovery of ((S)-5-(methoxymethyl)-7-(1-methyl-1 H -indol-2-yl)-2- (trifluoromethyl)-4,7-dihydropyrazolo[1,5- a ]pyrimidin-6-yl)((S)-2-(3- methylisoxazol-5-yl)pyrrolidin-1-yl)methanone as a potent and selective I Kur inhibitor

Article abstract of DOI:10.1021/jm201386u

Previously disclosed dihydropyrazolopyrimidines are potent and selective blockers of I_Kur current. A potential liability with this chemotype is the formation of a reactive metabolite which demonstrated covalent binding to protein in vitro. When substituted at the 2 or 3 position, this template yielded potent I_Kur inhibitors, with selectivity over hERG which did not form reactive metabolites. Subsequent optimization for potency and PK properties lead to the discovery of ((S)-5-(methoxymethyl)-7-(1-methyl-1H-indol- 2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S) -2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone (13j), with an acceptable PK profile in preclinical species and potent efficacy in the preclinical rabbit atrial effective refractory period (AERP) model.

Full text of DOI:10.1021/jm201386u

Article
pubs.acs.org/jmc
Discovery of ((S)-5-(Methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-
2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-
2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone As a Potent
and Selective IKur Inhibitor
Heather J. Finlay,*,† John Lloyd,*,† Wayne Vaccaro,† Alexander Kover,† Lin Yan,† Gauri Bhave,†
Joseph Prol,† Tram Huynh,† Rao Bhandaru,† Yolanda Caringal,† John DiMarco,‡,# Jinping Gan,∥
Tim Harper,∥ Christine Huang,∥ Mary Lee Conder,§ Huabin Sun,§ Paul Levesque,§ Michael Blanar,§
Karnail Atwal,†,⊥ and Ruth Wexler†
‡
§
∥
†Departments of Discovery Chemistry, Crystallography, Biology and Preclinical Candidate Optimization, Bristol-Myers Squibb,
Research and Development, P.O. Box 5400, Princeton, New Jersey 08543-5400, United States
S
* Supporting Information
ABSTRACT: Previously disclosed dihydropyrazolopyrimi-
dines are potent and selective blockers of IKur current. A
potential liability with this chemotype is the formation of a
reactive metabolite which demonstrated covalent binding to
protein in vitro. When substituted at the 2 or 3 position, this
template yielded potent IKur inhibitors, with selectivity over
hERG which did not form reactive metabolites. Subsequent
optimization for potency and PK properties lead to the dis-
covery of ((S)-5-(methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)-
((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone (13j), with an acceptable PK profile in preclinical species and potent
efficacy in the preclinical rabbit atrial effective refractory period (AERP) model.
Selective inhibition of IKur leads to a prolongation in effective
refractory period and should terminate AF without being pro-
arrhythmic in the ventricle, leading to a potentially safer treat-
ment for patients with AF.11
INTRODUCTION
■
Atrial fibrillation (AF) is a condition in which the normal heart
rhythm is disrupted by rapid activity in areas of the atria. AF is
the most common form of sustained cardiac arrhythmia, and
the prevalence is increasing as the population continues to age.1
AF is projected to affect an estimated 5.6 million patients in the
US by 2050.2 In addition to significantly affecting quality of life,
AF is also associated with a 3-fold higher incidence of stroke
and a 2-fold increase in mortality.3 Current therapies for treat-
ment of AF include antithrombotic, rate control, or rhythm
control. The termination of AF and restoring of normal sinus
rhythm (rhythm control) is typically achieved with antiar-
rhythmic drugs.4 Currently available antiarrhythmic drugs
target ion channels which are expressed in the human atrium
and ventricle, for example, amiodarone/dronedarone,5 dofeti-
lide,6 flecainide,7 and sotalol.8 Inhibition of ventricular ion
channels can lead to prolongation of ventricular effective refrac-
tory period and the potentially life threatening arrhythmia
torsades de pointe.9 Initial administration of nonselective
rhythm control drugs is, therefore, often limited to a hospital
setting where monitoring of ventricular effects are required.
Thus, there is currently an unmet medical need for safe and
efficacious treatment of AF.
We have disclosed dihydropyrazolopyrimidines 1, 2, and 3 as
potent and selective blockers of IKur (Figure 1).12−14
Substitution at C7 indicated that a 2,3-dichloro or 3,4-dichloro
aryl group was preferred to maintain potency and selectivity for
Kv1.5. Metabolite identification studies on compounds 1 and 2
established the major routes of hepatic clearance as hydroxy-
lation and aromatization of the dihydropyrazolopyrimidine
core. Significantly, in the course of in vitro metabolite identi-
fication studies, the formation of covalently bound glutathione
(GSH) adducts were observed when these compounds were
incubated with liver microsomes in the presence of GSH. The
formation of a reactive metabolite was confirmed when the
covalently bound protein adduct of radio-labeled compound 2
was subsequently identified.15 This was of concern as com-
pounds which form reactive intermediates, (for example, those
containing unsubstituted thiophenes)16 and which irreversibly
bind to protein in the liver can be hepatotoxic and have the
additional liability of potential idiosyncratic toxicity when
administered to a diverse patient population.17 The site of
IKur is a delayed rectifier repolarization potassium current
encoded by the Kv1.5 gene in humans10 which is function-
ally expressed in the human atrium and not in the ventricle.
Received: October 26, 2011
Published: March 12, 2012
© 2012 American Chemical Society
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Figure 1. Dihydropyrazolopyrimidines which form reactive intermediates.
a
Scheme 1
a
Reagents and conditions: yields (a) DMF, NaOAc, 65 °C, 14 h, yield range 27−60%.
a
Scheme 2
reactive intermediate formation was determined using MS/MS
fragmentation analysis, indicating oxidation of the fused pyra-
zole ring.
We therefore focused our optimization efforts on blocking
the reactive intermediate formation by substitution at the C2
and C3 positions on the fused pyrazole ring.
The first series of C2 substituted analogues containing a C6
phenylpiperazine amide were obtained from the general sequence
utilizing the three-component, one-pot Biginelli reaction and
commercially available amino-pyrazoles (Scheme 1). Synthesis
of additional C2 and C3 substituted dihydropyrazolopyrimi-
dines required initial preparation of noncommercially available
amino-pyrazoles. Noncommercially available 3-substituted-1H-
pyrazol-5-amines were synthesized from the corresponding amino
pyrazoles using a multistep sequence, for example, compound 8,
Scheme 2.18−21 We had previously disclosed SAR in the pipe-
razine amide series, including characterization of the C7 antipodes
and determined that Kv1.5 potency was retained in one antipode
only.14 Additional compounds in the piperazine amide series were
synthesized and screened as racemates (Table 1) and the SAR
utilized in pyrrolo amide series where the diastereomers were
separated and characterized (Table 2 and Table 5).
A modified route was also subsequently used to allow instal-
lation of the C6 amide functionality at the final step (Scheme 3).
Compounds containing C2 and C3 alkyl and halogen
substituents were also prepared wherein the C6 phenylpipe-
razine amide substituent was replaced with the phenyl pyrrolo
amide analogous to that found in compound 2 by the same
general methods described in Schemes 1, 2, and 3. Additionally,
C2 and C3 substituted compounds wherein the C6 substituent
is a heterocyclo-pyrrolo group and the C7 substituent was
replaced by a heterocycle were also prepared by essentially the
same method utilizing a protection and deprotection sequence
to improve the yield in the amide formation step (Scheme 4).
a
Reagents and conditions: (a) toluene, 120 °C, 4 h, 84% yield; (b)
HCl, dioxane, isoamyl nitrate, −10 °C, 98% yield; (c) Cu(II)Cl2
Cu(I)Cl, ether, MeOH, −10 °C, 86% yield; (d) AcOH, 90% HNO3,
acetic anhydride, 0 °C, 32% yield; (e) anisole, 130 °C, 24 h, 100%
yield; (f) Pd/C, hydrogen, 50 psi, 95% yield; (g) NaHCO3, DMF,
55 °C, 72 h, 24% yield.
In the case wherein the C6 substituent was (S)-3-methyl-5-
(pyrrolidin-2-yl) isoxazole amide, the noncommercial pyrrolo
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evaluated for selectivity over the hERG channel,23 (representa-
tive examples are shown in Tables 1, 2, and 5). Kv1.5 and hERG
channel activity for standard 1 is also included in Table 1 for
comparison.
Table 1. C2 and C3-Substituted Phenylpiperazine Amides
We observed that direct analogues with a C2 (R1) methyl
group on the dihydropyrazolopyrimidine were within 1−2-fold
for Kv1.5 potency (for example, 5a compared to 1, 13a com-
pared to 2, and 13d compared to 13c). Substitution on the C2
(R1) position with chlorine resulted in a 4-fold loss of potency
for Kv1.5 (example 5d compared to 1) and, at the C3 (R2)
position, a significant decrease in potency (example 5c com-
pared to 1). Trifluoromethyl substitution at C2 (R1) reduced
Kv1.5 inhibition in the piperazine and phenylpyrrolidine amide
series significantly as shown for examples 5c and 13b compared
to the unsubstituted analogues 1 and 2, respectively. However,
we were surprised to observe that the C2-trifluoromethyl com-
pound was equipotent with the 3-methylisoxazol-5-yl pyrroli-
dine amide analogues (compound 13e compared to 13c).
Compound 10b with a methyl substituent at C3 (R2) showed
reduced Kv1.5 inhibition and was not profiled further.
Compounds 5a, 13a, and 13d with the methyl substituent at
C2, and compounds 13b and 13e with a trifluoromethyl
substituent at C2 and C3 substituted analogues 5d and 10b were
subsequently assayed to determine if reactive intermediates were
formed.14
The potential for reactive intermediate formation was com-
pared to the C2 and C3 unsubstituted analogues 2 and 13c.
The formation of reactive intermediates was quantified as
fluorescent GSH adducts (Table 3). Dihydropyrazolopyrimi-
dine C6 amides without substitution at positions 2 or 3 on the
fused pyrazole showed significant incorporation of labeled GSH
(2 and 13c). We were gratified to see that corresponding
analogues with substituents at either C2 or C3 significantly
reduced reactive intermediate formation, confirming that this is
the principal site of oxidation on the dihydropyrazolopyr-
imidine template, (C2 substituted examples 5a, 13a, 13d, and
13e and C3 substituted analogues 5d and 10b). Potent Kv1.5
inhibitors with acceptable selectivity versus hERG and <1% of
detected GSH adduct were advanced to in vitro liability screen-
ing and liver microsomal stability testing. C2 methyl analogues
5a and 13a and 13d had poor liver microsome stability (human
% inhibition of hERG
b
compd R1
R2
X
Kv1.5 IC50, μM
current at 10 μM
a
1
H
H
H
H
H
Cl
H
F
F
H
F
F
F
0.070
0.124
0.149
0.419
42 2.7
60 1.9
29 10
NT
5a
5b
5c
5d
8
CH3
CH3
CF3
H
c
24%
NT
Cl
0.209
NT
b
a
C7 enantiomers separated and active antipode included. Inhibition
is measured in duplicate at 3 concentrations and the mean values used
to calculate IC50 values. % Inhibition of current in L-929 cells at
c
0.3 μM, 2−4 point determinations.
isoxazole was synthesized as shown in Scheme 4. The pyrrolo
isoxazole was then coupled with the BOC protected dihy-
dropyrazolopyrimidine carboxylic acids such as 11, followed by
deprotection of the BOC group, to yield the product as a mix-
ture of diastereomers, which were then separated (Scheme 4).
Utilizing the chemistry described in Schemes 1−4, a series of
C6 amide analogues 5a to 12i were synthesized. Selected
mixtures of enantiomers were separated using chiral preparative
chromatography (Chiralcel AD). Diastereomers were separated
using normal phase silica gel chromatography. Enantiotopically
pure compounds are indicated with an asterisk.
RESULTS AND DISCUSSION
■
All compounds were assayed for block of IKur current in patch
clamped mammalian L-929 cells which were injected with
human Kv1.5 mRNA and stably expressed IKur protein.22 Inhibi-
tion and IC50 data for compounds 1−13f are shown in Tables 1
and 2. Compounds which were potent for Kv1.5 were also
Table 2. C6 Pyrrolo Amides
b
compd
R1
R2
R3
CH3
R5
Kv1.5 IC50, μM
% inhibition of hERG current at 10 μM
a
2
H
H
4-fluorophenyl
0.053
0.070
30
a
10a
H
H
CH2OCH3
CH3
3-methylisoxazol-5-yl
4-fluorophenyl
14 3.3
NT
a
c
10b
H
CH3
H
45%
a
13a
CH3
CF3
H
CH3
4-fluorophenyl
0.100
10 1.2
NT
a
c
13b
H
CH3
4-fluorophenyl
37 4.1%
a
13c
H
CH3
3-methylisoxazol-5-yl
3-methylisoxazol-5-yl
3-methylisoxazol-5-yl
3-methylisoxazol-5-yl
0.083
0.165
0.101
0.227
41 0.1
13 3.4
47 4.1
NT
a
13d
CH3
CF3
H
CH3
a
13e
H
CH3
13f
cyclopropyl
H
CH3
a
b
C7 diastereomers separated and active antipode included. Inhibition is measured in duplicate at 3 concentrations and the mean values used to
c
calculate IC50 values. Percent inhibition of current in L-929 cells at 0.3 μM, 2−4 point determinations.
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a
Scheme 3
a
Reagents and conditions: (a) DMF, NaHCO3, 65 °C, 24 h; (b) TMSOTf, DCM, RT; (c) EDCI, DCM, TEA, RT, 14 h.
a
Scheme 4
a
Reagents and conditions: (a) heptane, catalytic piperidine, 75 °C, 120 h; (b) di-tert-butyl carbonate, THF, DMAP, RT; (c) LiOH, THF; (d) EDCI,
DCM; (e) AcOH, microwave 150 °C, 2 min; (f) acetone oxime, nBuLi, THF, −15 °C to RT; (g) H2SO4, −15 °C to RT, 51% yield, 2 steps; (h)
TfOH, DCM RT, 42% yield.
and rat) compared with C2 CF3 analogue 13e. Compound 13e
had the best combination of potency, selectivity, and micro-
somal stability and was advanced to PK profiling in rat and dog
(Table 4). Compound 13e had an acceptable PK profile in rat
and dog with moderate clearance and good bioavailability and
was further evaluated for efficacy in the rabbit pharmacody-
namic model.25
Like humans, rabbits express the IKur current in atrium but
not ventricle and compound 13e demonstrated a dose depen-
dent increase in atrial effective refractory period (AERP) with-
out increasing ventricular effective refractory period (VERP)
(Figure 2).24 At a dose of 1 mg/kg, compound 13e achieved a
20% increase in AERP at a plasma concentration of 1.7 μM.
This is noteworthy, as an increase of AERP by 10−20% in this
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Table 3. Percentage Parent Trapped As Fluorescent GSH
Adduct
Table 5. Examples of C7 Substituted Methylisoxazolopyrrolo
Amides
%fluorescent adducts as %
initial parent
%fluorescent adducts as %
initial parent
compd
compd
2
33
<1
4
10b
13c
13d
13e
<1
21
<1
<1
5a
5d
13a
5
Table 4. PK Summary for Compound 13e in Rat and Dog
rat
dog
dose (μmol/kg)
Vss (L/kg)
Cl (mL/min/kg)
t1/2 (h)
12 iv; 26 po
5.6 iv, 5.2 po
3.0
27
1
1.7
11
1.8
53
%F
68
a
Inhibition is measured in duplicate at three concentrations and the
b
mean values used to calculate IC50 values. Percent Inhibition of
current in L-929 cells at 0.3 μM, 2−4 point determinations. *C7
diastereomers separated and active antipode included.
c
Figure 2. Effect on AERP and VERP in rabbit for compound 13e.
Dose (mg/kg IV) with plasma concentration in μM at each dose (n = 3)
(PEG/EtOH/H2O) and subsequently the vehicle used for the
slow infusion PD studies (DMF).
Compound 13j also showed good selectivity over other
cardiac ion channels (Table 6) and was evaluated for reactive
model is considered sufficient to produce a clinically relevant
effect.11 Concomitant with evaluating compound 13e in vivo,
we sought to identify additional compounds which maintain the
improved liability profile and further increase the potency in
this series.
Table 6. Cardiac Ion Channel Inhibition for Compound 13j
After optimizing the substitution on the pyrazole ring to
address the problem of reactive intermediate formation, we
attempted to further optimize these compounds by modifica-
tion of the substituent at the C7 position on the pyrazolo-
dihydropyrimidine core (Table 5). We reasoned that a bicyclic
heterocycle may fill the same space as the dichlorophenyl group
in the previous compounds. The 2-benzothiazole (13g) was 2−
3-fold less potent than the corresponding 3,4-dichlorophenyl
analogue, and 2-N-methyl indole (13i) showed similar potency
to the dichlorophenyl analogue (13e). The N-methyl benzi-
midazole (13h) was significantly less potent than the isosteric
N-methyl indole, possibly due to the greater basicity. Because of
superior potency, the N-methylindole compound (13i) was
chosen for further optimization of the 5-substituent. We had
discovered that replacing the 5-methyl substituent in 13c with
the 5-methoxymethyl substituent was well tolerated when the
7-substituent was 3,4-dichlorophenyl as illustrated by com-
pound 10a compared to compound 13c. Likewise, potency
was maintained within 2-fold when the 5-methyl substituent
was replaced with the 5-methoxymethyl substituent when the
7-substituent was N-methylindole as illustrated by compound
13j compared to compound 13i. In addition, we observed that
compounds substituted with a methoxymethyl group at C5
had improved solubility in the vehicles used for PK dosing
channel/current
IC50 (μM)
Kv1.5
hERG
ICaL
0.15
>10 (14% inhibition at 10 μM)
>10 (37% inhibition at 10 μM)
>10 (10% inhibition at 10 μM)
5.8
INa
Kv4.3
intermediate formation in the same assay using fluorescently
labeled glutathione. Like compound 13e, compound 13j
showed less than 1% GSH adduct formation quantified by
the detection of fluorescently labeled material.
Compound 13j was evaluated for pharmacokinetic properties
in rats and dogs (Table 7) and showed acceptable half-life and
Table 7. PK Summary for Compound 13j in Rat, Dog, and
Rabbit
rat
dog
rabbit
dose (μmol/kg)
Vss (L/kg)
Cl (mL/min/kg)
t1/2 (h)
12 iv; 25.5 po
5.6 iv, 5.2 po
5.6 iv
5.0
1.9
12
3.7
29
47
2.5
61
1.8
31
2.0
%F
ND
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monitored by TLC using 0.25 mm E. Merck silica gel plates (60 F254
)
bioavailability with moderate clearance. The pharmacodynamic
effect of 13j was tested in the rabbit PD model, which mea-
sured the effective refractory period (ERP) in both atrium and ven-
tricle.25 The compound was dosed at 0.3, 1.0, 3.0, and 10 mg/kg,
and at a dose of 1 mg/kg, compound 13j achieved a 20%
increase in AERP at a plasma concentration of 0.7 μM. There
was no effect on VERP for either 13e or 13j reflecting the selec-
tivity for Kv1.5 over ventricular ion channels (Figures 1 and 3).
and were visualized with UV light, with 5% phosphomolybdic acid in
95% EtOH, or by sequential treatment with 1N HCl/MeOH followed
by ninhydrin staining. LC MS data were recorded on a Shimadzu
LC-10AT equipped with a SIL-10A injector, a SPD-10AV detector,
normally operating at 220 nm, and interfaced with a Micromass ZMD
mass spectrometer. LC MS or HPLC retention times, unless otherwise
noted, are reported using a Phenomenex Luna C-18 4.6 mm × 50 mm
column eluted with a 4 min gradient from 0 to 100% B, where A =
10% MeOH−90% H2O−0.1% TFA and B = 90% MeOH−10% H2O−
0.1% TFA. All solvents were removed by rotary evaporation under
vacuum using a standard rotovap equipped with a dry ice condenser.
All filtrations were performed with a vacuum unless otherwise stated.
Purity of all intermediates and final compounds was determined to
be >95% by HPLC (Phenomenex Luna C-18 4.6 mm × 50 mm
column eluted with a 4 min gradient from 0 to 100% B, where A =
10% MeOH−90% H2O−0.1% TFA and B = 90% MeOH−10% H2O−
0.1% TFA and detection at 220 nm (method A), Xbridge C18 3.5 μm,
4.6 mm × 150 mm, 1.0 mL/min gradient 10−100% 95:5 AcCN in
H2O (0.05% TFA) in 5:95 AcCN in H2O (0.05% TFA) (method B)
and Xbridge Phenyl 3.5 μm, 4.6 mm × 150 mm, 1.0 mL/min gradient
10−100% 95:5 AcCN in H2O (0.05% TFA) in 5:95 AcCN in H2O
(0.05% TFA) (method C)) and/or elemental analyses.
(7-(3,4-Dichlorophenyl)-2,5-dimethyl-4,7-dihydropyrazolo-
[1,5-a]pyrimidin-6-yl)(4-phenylpiperazin-1-yl)methanone (5a).
1-(4-Phenylpiperazin-1-yl)butane-1,3-dione (4a) (0.40 g, 1.5 mmol)
and 3,4-dichlorobenzaldehyde (0.32 g, 1.8 mmol) was dissolved in
DMF (10 mL). NaOAc (0.37 g, 4.0 mmol) and 3-methyl-1H-pyrazol-
5-amine (0.26 g, 1.9 mmol) were added and the solution heated to
55 °C for 24 h. The cooled solution was diluted with EtOAc and the
organic portion washed with water and satd NaCl, dried over Na2SO4,
decanted, and concentrated. The residue was purified by silica gel
chromatography elution with 1:1 hexane:acetone to yield 5a as a pale-
yellow solid (450 mg, 60% yield). 1H NMR (400 MHz, MeOD) δ 7.47
(d, J = 8.3 Hz, 1H), 7.22 (d, J = 2.0 Hz, 1H), 6.95 (m, 4H), 6.84 (m,
2H), 6.13 (s, 1H), 5.49 (s, 1H), 4.23 (br s, 2H), 3.31 (br s, 4H), 2.87
(br s, 2H), 2.12 (s, 3H). 13C NMR (101 MHz, MeOD) δ 169.66,
163.15, 160.50, 158.09, 155.36, 151.27, 149.22, 143.53, 141.83, 133.83,
133.01, 132.18, 129.87, 127.72, 120.37, 120.29, 116.55, 116.33, 100.60,
87.71, 61.27, 16.32, 13.19. LCMS: [M + 1] 500.1. HPLC: purity 94%,
retention time 7.11 min, method B; purity 94%, retention time 7.18 min,
method C.
(7-(3,4-Dichlorophenyl)-2,5-dimethyl-4,7-dihydropyrazolo-
[1,5-a]pyrimidin-6-yl)(4-phenylpiperazin-1-yl)methanone (5b).
(7-(3,4-Dichlorophenyl)-2,5-dimethyl-4,7-dihydropyrazolo[1,5-a]-
pyrimidin-6-yl)(4-phenylpiperazin-1-yl)methanone was prepared from
4a and 3-methyl-1H-pyrazol-5-amine as described for example 5a in
29% yield, Rf 0.29 5% MeOH in EtOAc. 1H NMR (400 MHz, MeOD)
δ 7.49 (d, J = 8.3 Hz, 1H), 7.21−7.29 (m, 3H), 7.00 (dd, J = 8.3,
2.1 Hz, 1H), 6.92−6.82 (m, 3H), 6.16 (s, 1H), 5.51 (s, 1H), 4.29
(s, 2H), 3.38 (s, 4H), 2.97 (s, 2H),2.14 (s, 3H), 1.94 (s, 3H). 13C
NMR (101 MHz, MeOD) δ 169.66, 152.54, 151.27, 143.50, 141.83,
133.87, 133.00, 132.16, 130.14, 129.84, 127.70, 122.03, 118.43, 100.62,
87.70, 61.69, 16.71, 13.66. HPLC: purity 96%, retention time 7.52 min,
method B; purity 96%, retention time 7.58 min, method C. LCMS:
[M + 1] 482.1, [M + 3] 484.1.
Figure 3. Effect on AERP and VERP in rabbit for compound 13j.
Because of the superior effect in the pharmacodynamic assay
and the acceptable projected human dose derived from allo-
metric scaling of the rat and dog PK properties, compound 13j
was chosen for additional characterization in preclinical toxi-
cology studies.
CONCLUSIONS
■
In conclusion, we identified and blocked the site(s) of reactive
intermediate formation in the described dihydropyrazolopyr-
imidine Kv1.5 inhibitors. In general, although 1−2-fold less
potent, the 2-methyl and 2-trifluoromethyl substituted analo-
gues offered a profile worthy of follow up. Compound 13e was
identified with less than 1% GSH adduct formation with an
improved PK profile and equivalent PD efficacy to lead com-
pound 2. Subsequent concomitant optimization at the C7 and
C5 positions resulted in discovery of compound 13j, a potent
Kv1.5 inhibitor which showed less than 1% GSH adduct
formation and had an improved PK profile and increased PD
efficacy compared to compounds 1 and 13e. Compound 13j
was advanced to preclinical single dose toxicology studies in
rats and dog, and there were no clinical observations related to
liver toxicity observed.
EXPERIMENTAL SECTION
■
All reactions were carried out under a static atmosphere of argon or
nitrogen and stirred magnetically unless otherwise stated. All reagents
used were of commercial quality and were obtained from Aldrich
Chemical Co., Sigma Chemical Co., Lancaster Chemical Co.,
(7-(3,4-Dichlorophenyl)-5-methyl-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)(4-(4-fluorophenyl)-
piperazin-1-yl)methanone (5c). (7-(3,4-Dichlorophenyl)-5-methyl-
2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)(4-(4-
fluorophenyl)piperazin-1-yl)methanone was prepared from 1-(4-(4-
fluorophenyl)piperazin-1-yl)butane-1,3-dione (4b) and 3,3-(trifluoro-
methyl)-1H-pyrazol-5-amine as described for example 5a in 46%
1
Oakwood Chemical Co., and Matrix Chemical Co. H (400 MHz)
and 13C (100 MHz) NMR spectra were recorded on a JEOL GSX400
spectrometer using tetramethylsilane as an internal standard unless
1
otherwise stated. H (500 MHz) and 13C (125 MHz) NMR spectra
were recorded on a JEOL JNM-ECP500 spectrometer. Chemical shifts
are given in parts per million (ppm) downfield from internal reference
tetramethylsilane in δ units, and coupling constants (J values) are
given in hertz (Hz). Selected data are reported in the following
manner: chemical shift, multiplicity, coupling constants. All reactions
were carried out using commercially available anhydrous solvents from
Aldrich Chemical Co. or EM Science Chemical Co. unless otherwise
stated. All flash chromatographic separations were performed using
E. Merck silica gel (particle size 0.040−0.063 mm). Reactions were
1
yield, Rf 0.38 10% MeOH in DCM. H NMR (400 MHz, MeOD) δ
8.00 (s, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.29 (dd, J = 12.4, 2.1 Hz, 1H),
7.09−6.93 (m, 3H), 6.89−6.79 (m, 2H), 6.28 (s, 1H), 5.95 (s, 1H),
4.24 (s, 1H), 3.87−3.68 (m, 1H), 3.58−3.16 (m, 4H), 1.96 (s, 3H).
13C NMR (101 MHz, MeOD) δ 169.04, 164.90, 160.53, 158.07,
149.22, 144.35, 143.88, 142.52, 142.20, 133.98, 133.46, 132.35, 130.05,
127.83, 120.37, 120.30, 116.56, 116.34, 101.09, 86.13, 62.52, 49.33,
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49.55, 16.72. HPLC: purity 96%, retention time 10.32 min, method B;
purity 95%, retention time 9.37 min, method C. LCMS: [M + 1]
554.1.
(2.7 mL, 16 mmol). The resulting slurry was stirred at 0 °C for 90 min,
diluted with ether (100 mL), and the solid collected by filtration and
washed further with ether, yielding intermediate 5-diazo-1H-pyrazole
(2.0 g, 98% yield). 5-Diazo-1H-pyrazole (1.0 g, 7.3 mmol) was
suspended in concentrated HCl (10 mL), ether (10 mL), and MeOH
(10 mL) at −10 °C, and then CuCl (0.038 g, 0.38 mmol) and CuCl2
(0.50 g, 3.7 mmol) were added. The reaction mixture was stirred at
0 °C for 48 h and then poured into saturated ammonium hydroxide
(37%, 30 mL) and then extracted with EtOAc. The combined organic
portions were dried over Na2SO4, decanted, and concentrated under
reduced pressure to yield 5-chloro-1H-pyrazole (0.64 g, 86% yield). 1H
NMR (400 MHz, CDCl3) δ 7.53 (broad s, 1H), 6.28 (broad s, 1H). 5-
Chloro-1H-pyrazole (0.65 g, 6.3 mmol) was dissolved directly in acetic
acid (0.91 mL) and then HNO3 (90%, 0.91 mL) was added dropwise
at 0 °C followed by acetic anhydride (2.3 mL). The reaction mixture
allowed to warm to room temperature and stirred for 14 h and then
poured cautiously into water (50 mL). The pH of the solution was
adjusted to pH 7−8 by the addition of Na2CO3 and the aqueous
solution extracted with ether. The combined ether portions were dried
over Na2SO4, decanted, concentrated, and the residue purified by
column chromatography eluting with 4:1 hexane:EtOAc, yielding 3-
chloro-1-nitro-1H-pyrazole as a white solid (0.30 g, 32% yield, Rf 0.65
(3-Chloro-7-(3,4-dichlorophenyl)-5-methyl-4,7-dihydropyrazolo-
[1,5-a]pyrimidin-6-yl)(4-(4-fluorophenyl)piperazin-1-yl)-
methanone (5d). 7-(3,4-Dichlorophenyl)-5-methyl-4,7-dihydropyrazolo-
[1,5-a]pyrimidin-6-yl)(4-(4-fluorophenyl)piperazin-1-yl)methanone
was prepared by the method described for 5a from 4b and pyrazol-
5-amine in 27% yield. HPLC: purity 99%, retention time 8.59 min,
1
method B; purity 99%, retention time 8.24 min, method C. H NMR
(400 MHz, MeOD) δ 7.59 (s, 1H), 7.54 (d, J = 8.3 Hz, 1H), 7.32 (d,
J = 2.1 Hz, 1H), 7.08−6.98 (m, 3H), 6.97−6.88 (m, 3H), 6.32 (s, 1H),
4.25 (brs, 1H), 3.44 (brs, 3H), 2.99 (brs, 1H), 2.65 (brs, 1H), 1.99 (s,
3H), 1.60 (brs, 1H). 13C NMR (101 MHz, MeOD) δ 169.08, 161.03,
160.30, 159.90, 158.65, 148.09,142.33, 142.01, 140.61, 134.07, 133.59,
132.41, 130.05, 127.84, 120.82, 120.74, 118.13, 116.81, 116.59, 115.34,
100.82, 61.98, 16.69. LCMS: [M + 1] 485.9, [M + 3] 487.7. 7-(3,4-
Dichlorophenyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-
yl)(4-(4-fluorophenyl)piperazin-1-yl)methanone (59 mg, 0.12 mmol)
was dissolved in DCM (4 mL) and NCS (18 mg, 0.13 mmol) was
added at 0 °C. After 30 min, the crude solution was applied directly to
a prep TLC plate (25 cm × 25 cm, 1 mm thickness of silica) and
eluted with 1:1 hexane:acetone. Compound 5d was isolated as a white
solid (38 mg, 61% yield). 1H NMR (400 MHz, DMSO) δ 9.52 (s, 1H),
7.57 (d, J = 8.4 Hz, 1H), 7.44 (s, 1H), 7.26 (d, J = 1.8 Hz, 1H), 7.00−
7.05 (m, 3H), 6.95 (d, J = 8.4 Hz, 1H), 6.83 (m, 2H), 6.08 (s, 1H), 4.00
(s, 2H), 3.20 (s, 4H), 2.90 (s, 2H), 1.90 (s, 3H). 13C NMR (101 MHz,
MeOD) δ 181.55, 168.80, 157.93, 148.72, 142.56, 139.51, 137.46,
134.06, 133.09, 132.29, 130.10, 127.92, 120.39, 120.31, 116.56, 116.34,
101.43, 90.86, 62.53, 49.33, 49.12, 30.63, 16.59. HPLC: purity 94%,
retention time 9.67 min, method B; purity 94%, retention time 8.89 min,
method C. LCMS: [M + 1] 520.1.
1
in 4:1 hexane:EtOAc). H NMR (400 MHz, CDCl3) δ 8.31 (d, J =
4.4 Hz, 1H), 6.47 (d, J = 4.4 Hz, 1H). 3-Chloro-1-nitro-1H-pyrazole
(0.30 g, 2.0 mmol) was dissolved in anisole (4 mL) and the solution
heated at 130 °C for 48 h. The cooled reaction mixture was poured
into 1:1 hexane:water (100 mL), the aqueous phase separated and the
organic portion extracted with NaOH (10% aqueous solution, 4 ×
10 mL). The combined aqueous extracts were acidified to pH 2 by the
addition of concentrated HCl and extracted with EtOAc. The
combined EtOAc extracts were dried over Na2SO4, decanted, and
concentrated to yield 3-chloro-5-nitro-1H-pyrazole as a white solid
(0.30 g, 100% yield, Rf 0.2 in 4:1 hexane:EtOAc) 1H NMR (400 MHz,
CDCl3) δ 6.81 (s, 1H). HPLC: purity 97%, retention time 6.18 min,
Xbridge C18 3.5 μm, 4.6 mm × 150 mm, 1.0 mL/min gradient 10−
100% 95:5 AcCN in H2O (0.05% TFA) in 5:95 AcCN in H2O (0.05%
TFA); purity 97%, retention time 5.38 min, Xbridge Phenyl 3.5 μm,
4.6 mm × 150 mm, 1.0 mL/min gradient 10−100% 95:5 AcCN in
H2O (0.05% TFA) in 5:95 AcCN in H2O (0.05% TFA). A Parr thick
walled reaction vessel was charged with chloro-5-nitro-1H-pyrazole
(0.66 g, 4.5 mmol), Pd/C (33 mg, 5% by weight), and MeOH
(40 mL). The reaction mixture was hydrogenated at 50 psi (3.6 atm)
for 2 h 15 min. The reaction mixture was flushed with nitrogen, diluted
with MeOH, and filtered through a pad of Celite. The solvents were
removed to yield 3-chloro-1H-pyrazol-5-amine as brown needles (0.48 g,
95% yield, Rf 0.2 in 2:1 hexane:acetone). 1H NMR (400 MHz, CDCl3) δ
5.50 (s, 1H), 4.30 (broad s, 2H). 13C NMR (101 MHz, MeOD) δ 153.75,
142.43, 91.48. HPLC: purity 95%, retention time 3.20 min, method B;
purity 95%, retention time 2.95 min, method C.
(2-Chloro-7-(3,4-dichlorophenyl)-5-methyl-4,7-dihydropyrazolo-
[1,5-a]pyrimidin-6-yl)(4-phenylpiperazin-1-yl)methanone (8).
A Teflon screw capped vial was charged with 2-(1-(3,4-dichlorophenyl)-
ethylidene)-1-(4-phenylpiperazin-1-yl)butane-1,3-dione (0.13 g,
0.31 mmol), 3-chloro-1H-pyrazol-5-amine (0.040 g, 0.34 mmol),
NaHCO3 (0.078 g, 0.93 mmol), and DMF (1.5 mL). The sealed
reaction vessel was heated at 55 °C for 72 h and the cooled solu-
tion poured into LiCl (10% aqueous solution). The solution was
extracted with EtOAc, and the combined organic portions were dried
over Na2SO4, decanted, and concentrated. The residue was purified by
prep HPLC (YMC S5 ODS 30 mm × 100 mm, gradient 40−100% B
where A = 10% MeOH−90% H2O−0.1% TFA and B = 90%
MeOH−10% H2O−0.1% TFA and detection at 220 nm, retention
time 10.1 min) to yield (2-chloro-7-(3,4-dichlorophenyl)-5-methyl-
4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)(4-phenylpiperazin-1-yl)-
methanone (38 mg, 24% yield, 95% purity, retention time 4.27 min,
method A). 1H NMR (400 MHz, MeOD) δ 7.50 (d, J = 8.3 Hz, 1H),
7.27 (d J = 2.2 Hz, 1H), 7.01 (dd, J = 8.3 Hz and J = 2.2 Hz, 1H),
6.94−6.98 (m, 2H), 6.86 (m, 2H), 6.12 (s, 1H), 5.64 (s, 1H), 4.25 (s,
2H), 3.40 (s, 4H), 2.90 (s, 2H), 1.92 (s, 3H). 13C NMR (101 MHz,
MeOD) δ 169.15, 159.53, 158.62, 142.19, 133.11, 132.31, 130.01,
2-(1-(3,4-Dichlorophenyl)ethylidene)-1-(4-phenylpiperazin-
1-yl)butane-1,3-dione (6). To a solution of tert-butyl 3-
oxobutanoate (1.2 g, 7.7 mmol) in toluene (10 mL) was added 1-
(4-fluorophenyl)piperazine (1.4 g, 7.7 mmol). The reaction mixture
was heated to 120 °C for 4 h and then the cooled solution was
extracted with HCl (1N aqueous solution, 3 × 20 mL). Satd NaHCO3
was added to the combined aqueous portions until the pH was
adjusted to pH 8. A white precipitate formed which was extracted into
EtOAc. The combined EtOAc portions were dried over Na2SO4,
decanted, and concentrated to yield intermediate 1-(4-(4-
fluorophenyl)piperazin-1-yl)butane-1,3-dione (4a), which was used
directly without further purification (1.7 g, 84% yield, 98% purity
retention time 2.50 min). 1-(4-(4-Fluorophenyl)piperazin-1-yl)butane-
1,3-dione (200 mg, 0.76 mmol) was dissolved in DMF (2 mL). To the
reaction mixture was added 3,4-dichlorobenzaldehyde (0.13 g, 0.76
mmol), acetic acid (20 mg), piperidine (30 mg), and powdered 3A
molecular sieves (100 mg). The resulting solution was heated to 55 °C
for 14 h and then the cooled slurry was diluted with EtOAc (50 mL)
and poured into LiCl (10% aqueous solution, 50 mL). The organic
portion was separated and washed further with LiCl (10% aqueous
solution), dried over Na2SO4, decanted, and concentrated. The residual
oil was purified by column chromatography elution with 5:1 hexane:
acetone to yield 2-(1-(3,4-dichlorophenyl)ethylidene)-1-(4-phenyl-
piperazin-1-yl)butane-1,3-dione (0.25 g, 79% yield, 98% purity
1
retention time 4.16 min method A). H NMR (400 MHz, CDCl3) δ
7.53 (d, J = 2.0 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.31 (dd, J = 2.0 and
J = 8.4 Hz, 1H), 6.87 (m, 2H), 6.73 (m, 2H), 3.84 (t, J = 5.2 Hz, 2H),
3.30 (broad s, 1H), 3.20 (broad s, 1H), 3.05 (broad s, 1H), 2.95 (broad
s, 1H), 2.90 (broad s, 1H), 2.50 (broad s, 1H), 2.37 (s, 3H). LCMS
[M + 1] 421.07.
3-Chloro-1H-pyrazol-5-amine (7). HCl (4.0 M in dioxane,
6.0 mL, 24 mmol) was added to a stirred solution of 1H-pyrazol-5-
amine (1.5 g, 18 mmol) in MeOH (15 mL) at RT. After 3 h, the
solvents were removed in vacuo and the residue redissolved in MeOH
(10 mL). A further 3 mL of HCl (4.0 M in dioxane, 12 mmol) was
added and the turbid solution stirred for 2 h and then concentrated,
yielding a pale-yellow solid. The residue was redissolved in MeOH
(15 mL) at 0 °C, and then isoamylnitrate was added dropwise
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Journal of Medicinal Chemistry
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127.84, 120.63, 118.62, 116.74, 116.52, 101.58, 87.11, 61.87, 49.68,
49.47, 49.26, 49.04, 48.83, 48.61, 48.40, 16.70. LCMS: [M + 1] 520.0,
[M + 3] 522.0
10.52 min, method B; purity 95%, retention time 9.11 min, method C.
LCMS [M + 1] 394.46. Compound 11b (0.20 g, 0.51 mmol) was
dissolved in DCM (3 mL), and TMSOTf was added dropwise (0.20 g).
The resulting slurry was stirred at room temperature for 1 h, the
DCM was removed by pipet, and the residual solid dissolved in THF
and treated with NaHCO3 (0.5 g). The organic layer was purified by
silica gel chromatography elution with EtOAc (1% AcOH), yielding
7-(3,4-dichlorophenyl)-3,5-dimethyl-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylic acid as a white powder (0.15 g, 86% yield,
100% purity, retention time 3.89 min). HPLC: purity 99%, retention
time 10.47 min, method B; purity 97%, retention time 9.65 min,
((R)-7-(3,4-Dichlorophenyl)-5-(methoxymethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-
5-yl)pyrrolidin-1-yl)methanone (10a). Intermediate tert-butyl
7-(3,4-dichlorophenyl)-5-(methoxymethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylate (9a). To a solution of 1H-pyrazol-5-amine
(5.4 g, 63 mmol) in DMF (50 mL) was added 3,4-dichlorobenzalde-
hyde (9.6 g, 53 mmol) and tert-butyl 4-methoxy-3-oxobutanoate (10 g,
53 mmol). NaHCO3 (18 g, 252 mmol) was added, and the solution
was heated at 65 °C for 24 h. The cooled reaction mixture was diluted
with 10% LiCl solution in water (800 mL) and the precipitate
collected, dissolved in DCM, and transferred to a separation funnel.
The organic portion was further washed with 10% LiCl solution and
the solvents evaporated. The residue was triturated with methanol to
yield 9a as a white powder (8.5 g, 39% yield, purity 91%, retention
time 3.82 min). The material was separated into the corresponding
enantiomers using a chiral cell AS column elution (5 cm × 50 cm,
20 μm, Chiral Technologies) with 100% IPA (1% TEA) at 60 mL/min.
Isomer 1 eluted at a retention time of 56 min, and isomer 2 eluted at a
retention time of 98 min. Analytical data for isomer 1: HPLC purity
95%, retention time 11.16 min, method B; purity 95%, retention time
1
method C. H NMR (400 MHz, THF) δ 9.89 (s, 1H), 7.86 (s, 1H),
7.48 (d, J = 2.1 Hz, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.31 (dd, J = 8.3,
2.2 Hz, 1H), 6.52 (s, 1H), 2.49 (s, 3H), 2.06 (s, 3H). 13C NMR (101
MHz, THF) δ 164.22, 144.05, 139.23, 138.99, 134.74, 131.72, 131.35,
129.86, 128.38, 126.55, 100.96, 97.76, 57.87, 16.47, 5.08. LCMS [M + 1]
338.27, [M + 3] 340.41.
7-(3,4-Dichlorophenyl)-3,5-dimethyl-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylic acid and (S)-2-(4-fluorophenyl)pyrrolidine
were coupled under the conditions described for the preparation of
example 13a to yield (7-(3,4-dichlorophenyl)-3,5-dimethyl-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(4-fluorophenyl)-
pyrrolidin-1-yl)methanone as a 1:1 mixture of diastereomers, which
was separated by prep TLC plate (25 cm × 25 cm, 1 mm thickness)
elution with 1:1 hexane:EtOAc (5% IPA). The Rf values for the
diastereomers were 0.55 for ((S)-7-(3,4-dichlorophenyl)-3,5-dimethyl-4,
7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(4-fluorophenyl)-
pyrrolidin-1-yl)methanone and 0.45 for ((R)-7-(3,4-dichlorophenyl)-
3,5-dimethyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(4-
fluorophenyl)pyrrolidin-1-yl)methanone, which were isolated as white
solids in yields of 4% and 5%, respectively. Analytical data for ((R)-7-
(3,4-dichlorophenyl)-3,5-dimethyl-4,7-dihydropyrazolo[1,5-a]-
pyrimidin-6-yl)((S)-2-(4-fluorophenyl)pyrrolidin-1-yl)methanone.
HPLC: purity 95%, retention time 10.50 min, method B; purity 92%,
1
9.67 min, method C. H NMR (400 MHz, MeOD) δ 7.43 (d, J =
8.3 Hz, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.34 (d, J = 2.1 Hz, 1H), 7.11
(dd, J = 8.3, 2.1 Hz, 1H), 6.23 (d, J = 9.9 Hz, 1H), 5.82 (d, J = 2.0 Hz,
1H), 4.76 (s, 2H), 3.50 (s, 3H), 1.35 (d, J = 10.0 Hz, 9H). 13C
NMR (101 MHz, MeOD) δ 166.39, 147.84, 144.93, 141.56, 138.99,
133.04, 132.54, 131.58, 130.59, 127.92, 96.60, 90.21, 81.89, 70.70,
60.39, 59.49, 28.59, 27.97. LCMS [M + 1] 410.13. tert-Butyl 7-(3,4-
dichlorophenyl)-5-(methoxymethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylate (isomer 1) was treated with TMSOTf to yield
7-(3,4-dichlorophenyl)-5-(methoxymethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylic acid in 93% yield, purity 94%, retention time
3.02 min, method A. 7-(3,4-Dichlorophenyl)-5-(methoxymethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidine-6-carboxylic acid was coupled to
(S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole (12) using the conditions
described for example 13a to yield ((R)-7-(3,4-dichlorophenyl)-5-
(methoxymethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-
methylisoxazol-5-yl)pyrrolidin-1-yl)methanone as a white solid (66%
yield). HPLC: purity 100%, retention time 8.12 min, method B; purity
100%, retention time 7.75 min, method C. 1H NMR (400 MHz,
MeOD) δ 7.47 (d, J = 7.8 Hz, 1H), 7.44 (d, J = 1.8 Hz, 1H), 7.21 (s,
1H), 6.99 (d, J = 8.1 Hz, 1H), 5.80 (brs, 1H), 5.69 (s, 1H), 5.70 (d, J =
2.2 Hz, 1H), 5.48 (s, 1H), 5.12 (m, 1H), 4.03 (m, 1H), 3.91 (pentet,
J = 6.6 Hz, 2H), 3.64 (m, 1H), 3.34 (s, 3H), 3.20 (s, 1H), 2.24 (s, 3H),
1.95 (s, 2H). 13C NMR (101 MHz, MeOD) δ 173.43, 168.41, 161.41,
142.81, 141.56, 141.02, 133.68, 133.07, 131.92, 130.11, 127.95, 103.62,
103.18, 88.53, 69.87, 61.30, 59.04, 11.28. LCMS: [M + 1] 511.41.
LCMS [M + 1] 487.9. HRMS calcd 488.126, obsd 488.126. The relative
stereochemistry at the C7 postion was inferred from NMR analysis of
the diagnostic proton shift for the methylene proton at C7.
((R)-7-(3,4-Dichlorophenyl)-3,5-dimethyl-4,7-dihydropyrazolo-
[1,5-a]pyrimidin-6-yl)((S)-2-(4-fluorophenyl)pyrrolidin-1-yl)-
methanone (10b). Intermediate tert-butyl-7-(3,4-dichlorophenyl)-
3,5-dimethyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate
(9b). To a solution of 4-methyl-1H-pyrazol-5-amine (0.36 g, 3.7 mol)
in IPA (20 mL) was added 3,4-dichlorobenzaldehyde (0.43 g, 2.4 mol)
and methyl 3-oxobutanoate (0.36 g, 2.4 mol). Heptane (4 mL)
and piperidine (0.080 mg, 0.93 mmol) was added, and the solu-
tion was heated to 95 °C for 48 h. The cooled reaction mixture was
concentrated in vacuo and the residue redissolved in THF and
purified by silica gel chromatography gradient elution with 4:1
hexane:EtOAc to 2:1 hexane:EtOAc. tert-Butyl-7-(3,4-dichlorophenyl)-
3,5-dimethyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate
was isolated as a pale-yellow powder (0.49 g, 51% yield, 99% purity,
retention time 3.95 min, method A). 1H NMR (400 MHz, THF-d8) δ
8.68 (s, 1H), 7.39 (d, J = 1.6 Hz, 1H), 7.36 (d, J = 10.0 Hz, 1H), 7.12
(dd, J = 1.8 Hz and J = 8.4 Hz, 1H), 7.02 (s, 1H), 6.20 (s, 1H), 2.45
(s, 3H), 1.95 (s, 3H), 1.34 (s, 9H). HPLC: purity 95%, retention time
1
retention time 10.19 min, method C. H NMR (400 MHz, THF) δ
7.96 (s, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.29 (s, 1H), 7.06 (d, J = 8.3
Hz, 1H), 7.02−6.91 (m, 4H), 5.81 (d, J = 27.5 Hz, 1H), 4.91 (m, 1H),
3.74 (m, 3H), 3.44 (s, 2H), 2.18 (s, 3H), 1.92 (s, 3H), 1.58 (d, J = 13.2
Hz, 1H). LCMS [M + 1] 484.9.
((R)-7-(3,4-Dichlorophenyl)-2,5-dimethyl-4,7-dihydropyrazolo-
[1,5-a]pyrimidin-6-yl) ((S)-2-(4-fluorophenyl)pyrrolidin-1-yl)-
methanone (13a). Intermediate (R)-4-tert-butyl-6-methyl-7-(3,4-
dichlorophenyl)-2,5-dimethylpyrazolo[1,5-a]pyrimidine-4,6(7H)-
dicarboxylate was prepared as follows. To a solution of 3-methyl-
1H-pyrazol-5-amine (3.0 g, 0.031 mol) in THF (15 mL) was added
3,4-dichlorobenzaldehyde (6.0 g, 0.034 mol) and methyl 3-oxobutanoate
(4.0 g, 0.034 mol). Heptane (4 mL) and piperidine (0.080 mg,
0.93 mmol) were added, and the solution was heated to 69 °C for 12 h. A
white precipitate formed and the slurry diluted with hexane (25 mL),
filtered, and the filter cake washed further with hexane and dried in
vacuo to yield methyl-7-(3,4-dichlorophenyl)-2,5-dimethyl-4,7-dihydro-
pyrazolo[1,5-a]pyrimidine-6-carboxylate as a white powder (8.3 g, 76%
yield, 95% purity, retention time 3.43 min, method A). LCMS: [M + 1]
352.0. To a slurry of methyl 7-(3,4-dichlorophenyl)-2,5-dimethyl-4,7-
dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate (4.0 g, 0.011 mol) in
THF (37 mL) was added BOC anhydride (3.2 g, 0.014 mol) and
DMAP (67 mg, 0.55 mol). The solution was stirred at RT for 2 h and
then the solvents removed under reduced pressure. The residual oil was
dissolved in DCM (100 mL), washed successively with 1 M HCl, satd
NaHCO3, and satd NaCl, dried over Na2SO4, decanted, and concen-
trated, yielding racemic 4-tert-butyl-6-methyl-7-(3,4-dichlorophenyl)-
2,5-dimethylpyrazolo[1,5-a]pyrimidine-4,6(7H)-dicarboxylate (5.6 g,
100%, purity 95%, retention time 4.21 min). The racemate was
separated into the corresponding enantiomers using a Chiral cell AD
column (50 mm × 500 mm) elution with 1% anhydrous EtOH
in heptane at a flow rate of 40 mL/min with detection at 254 nm.
Enantiomer A eluted at a retention time of 70 min and entantiomer B
at 582 min. Enantiomer B corresponded to (R)-4-tert-butyl-6-methyl-
7-(3,4-dichlorophenyl)-2,5-dimethylpyrazolo[1,5-a]pyrimidine-
4,6(7H)-dicarboxylate and was isolated in 21% yield from the corresponding
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racemate in >98% ee purity, retention time 7.50 min, Chiral cell OD
column (4.6 mm × 250 mm) elution with 2% IPA in heptane. HPLC:
purity 98%, retention time 12.17 min, method B; purity 98%, retention
time 10.30 min, method C. LCMS: [M + 1] 452.0, [M + 3] 454.0. 1H
NMR (400 MHz, MeOD) δ 7.44 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 2.1
Hz, 1H), 7.13 (dd, J = 8.4, 2.2 Hz, 1H), 6.30 (s, 1H), 6.11 (s, 1H),
3.80 (s, 3H), 2.58 (s, 3H), 2.21 (s, 3H), 1.58 (s,9H). 13C NMR (101
MHz, MeOD) δ 166.47, 151.20, 150.95, 149.09, 141.20, 138.56,
133.85, 133.44, 132.15, 129.35, 127.57, 116.52, 97.42, 86.46, 59.36,
52.80, 28.28, 21.42, 13.92.
7.30 (d, J = 2.1 Hz, 1H), 7.14 (dd, J = 8.3, 2.2 Hz, 1H), 6.51 (s, 1H),
6.47 (s, 1H), 2.66 (s, 3H), 1.59 (s, 9H). 13C NMR (101 MHz, MeOD)
δ 167.30, 150.66, 148.79, 141.14, 140.15, 139.06, 136.23, 135.00,
134.15, 133.99, 133.81, 132.34, 129.36, 127.62, 117.59, 113.60,
86.98, 80.66, 60.48, 40.98, 28.17, 22.00, 21.21. LCMS: [M + 1]
491.8.
((R)-7-(3,4-Dichlorophenyl)-5-methyl-2-(trifluoromethyl)-
4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(4-
fluorophenyl)pyrrolidin-1-yl)methanone (13b). Compound 11b
and (S)-2-(4-fluorophenyl)pyrrolidine were coupled under the
conditions described for the preparation of example 13a to yield inter-
mediate tert-butyl 7-(3,4-dichlorophenyl)-6-((S)-2-(4-fluorophenyl)-
pyrrolidine-1-carbonyl)-5-methyl-2-(trifluoromethyl)pyrazolo[1,5-a]-
pyrimidine-4(7H)-carboxylate (0.53 g, 0.82 mmol) which was dis-
solved in 1:1 TFA:DCM (20 mL) and the reaction mixture stirred
at room temperature for 3 h. The reaction mixture was concentrated
and the residue azeotroped with toluene and then purified by silica
gel chromatography elution with 1:3:7 IPA:EtOAc:hexane to yield
((R)-7-(3,4-dichlorophenyl)-5-methyl-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(4-fluorophenyl)-
pyrrolidin-1-yl)methanone as the less polar diastereomer and
((S)-7-(3,4-dichlorophenyl)-5-methyl-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(4-fluorophenyl)-
pyrrolidin-1-yl)methanone as the more polar diastereomer as white
solids in yields of 29% and 17%, respectively. HPLC: purity 97%,
LiOH (10N aqueous solution, 5 mL) was added to a solution of
(R)-4-tert-butyl-6-methyl-7-(3,4-dichlorophenyl)-2,5-
dimethylpyrazolo[1,5-a]pyrimidine-4,6(7H)-dicarboxylate (0.53 g,
1.2 mmol) in THF (5 mL). The reaction mixture was stirred at RT
for 15 h and then diluted with HCl (1N aqueous solution, 20 mL) and
extracted with DCM. The combined organic portions were dried over
Na2SO4, decanted, and concentrated to yield (R)-4-(tert-butoxycar-
bonyl)-7-(3,4-dichlorophenyl)-2,5-dimethyl-4,7-dihydropyrazolo[1,5-
a]pyrimidine-6-carboxylic acid (11a) as an off-white powder (0.44 g,
yield 87%, purity 92%, retention time 4.03 min, method A). LCMS
[M + 1] 438.18. To a solution of 11a (1.2 g, 2.7 mmol) in DCM (40 mL)
was added EDCI (0.71 g, 3.7 mmol), HOBt (0.50 g, 3.7 mmol), and
(S)-2-(4-fluorophenyl)pyrrolidine (0.61 g, 3.7 mmol). The reaction
mixture was stirred at room temperature for 14 h and then diluted
further with DCM (50 mL) and the solution washed successively with
saturated NaHCO3 and satd NaCl. The organic portion was dried over
Na2SO4, decanted, and concentrated to yield intermediate (R)-tert-
butyl-7-(3,4-dichlorophenyl)-6-((S)-2-(4-fluorophenyl)pyrrolidine-1-
carbonyl)-2,5-dimethylpyrazolo[1,5-a]pyrimidine-4(7H)-carboxylate
as a tan solid (1.2 g, 75% yield, 95% purity at retention time 4.18 min).
LCMS [M + 1] 585.16. To (R)-tert-butyl-7-(3,4-dichlorophenyl)-6-
((S)-2-(4-fluorophenyl)pyrrolidine-1-carbonyl)-2,5-dimethylpyrazolo-
[1,5-a]pyrimidine-4(7H)-carboxylate (1.2 g, 2.0 mmol) was added
1:1 DCM:TFA solution (3 mL). The solution was stirred at room
temperature for 3 h and then diluted with DCM, and the organic
portion was washed with satd NaHCO3, dried over Na2SO4, decanted,
and concentrated. The residue was purified by silica gel chromatog-
raphy elution with 1:1:0.1 hexane:EtOAc:IPA to yield compound 13a
as a white powder (0.22 g, 23% yield, 99% purity, retention time 6.83
min, method B); Rf 0.30 10% IPA in 1:1 hexane:EtOAc. 13C NMR
(101 MHz, MeOD) δ 164.0, 162.5, 152.0, 145.0, 142.0, 141.0, 133.8,
133.7, 133.6, 132.0, 130.0, 128.3, 116.0, 102.8, 87.58, 62.0, 28.0, 17.0,
13.6. Due to rotomers, the proton spectra for this diastereomer had
1
retention time 3.86 min, method A. H NMR (400 MHz,) δ 7.44
(s, 1H), 7.28 (d, J = 8.2 Hz, 1H), 7.09 (s, 1H), 7.02 (s, 2H), 6.88 (t,
J = 8.5 Hz, 2H), 6.78 (d, J = 10.4 Hz, 2H), 6.23 (s, 1H), 5.56 (s, 1H),
4.86 (t, J = 7.7 Hz, 1H), 4.05−3.82 (m, 1H), 3.32 (s, 1H), 3.13 (s, 1H),
2.19 (m, 2H), 1.74 (s, 3H). 19F NMR (376 MHz) δ −62.53, −114.61.
13C NMR (101 MHz,) δ 167.43, 160.08, 138.57, 132.89, 130.77, 128.30,
127.50, 125.78, 115.45, 115.24, 103.29, 85.29, 77.32, 77.01, 76.69, 64.48,
60.62, 49.31, 34.85, 32.25, 25.32, 16.81, 0.99, −10.63. HRMS [M + 1]
obsd 539.1028, calcd 539.1029.
((R)-7-(3,4-Dichlorophenyl)-5-methyl-4,7-dihydropyrazolo-
[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-
1-yl)methanone (13c). (R)-4-(tert-Butoxycarbonyl)-7-(3,4-dichlor-
ophenyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylic
acid (11c) was coupled to (S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole
(12) using the conditions and subsequent deprotection described for
example 13a to yield ((R)-7-(3,4-dichlorophenyl)-5-methyl-4,7-dihydro-
pyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-
1-yl)methanone in 26% yield. HPLC: purity 95%, retention time 7.88
1
min, method B; purity 96%, retention time 7.59 min, method C. H
1
broad peaks as noted. H NMR (400 MHz, MeOD) δ 7.15 (s, 1H),
NMR (400 MHz, CDCl3) δ 7.39 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 1.9
Hz, 1H), 7.25 (d, J = 4.4 Hz, 1H), 7.24 (d, J = 1.9 Hz, 1H), 7.01 (dd,
J = 8.3, 1.8 Hz, 1H), 6.50 (s, 1H), 6.07 (s, 1H), 5.53 (d, J = 1.9 Hz,
1H), 5.52 (s, 1H), 3.52 (s, 1H), 3.19 (s, 1H), 2.24 (s, 3H), 2.02−1.84
(m, 5H), 2.04 (s, 2H). 13C NMR (101 MHz, MeOD) δ 173.58,
161.88, 151.20, 143.56, 141.76, 133.45, 132.88, 131.93, 130.13, 127.99,
103.10, 102.44, 87.74, 61.17, 16.88, 13.64, 11.32. LCMS: [M + 1]
458.1, [M + 3] 460.1
7.32 (s, 1H), 6.95 (t, J = 8.0 Hz, 2H), 6.73 (s, 1H), 5.95 (s, 1H), 5.41
(s, 2H), 5.05 (s, 1H), 3.69 (s, 2H), 3.47 (m, 1H), 3.30 (s, 1H), 3.19
(s, 1H), 2.30 (s, 1H), 2.06 (s, 3H), 2.00−1.61 (m, 2H). HRMS [M + 1]
obsd 485.1309, calcd 485.1311.
4-(tert-Butoxycarbonyl)-7-(3,4-dichlorophenyl)-5-methyl-2-
(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-car-
boxylic Acid (11b). Methyl 7-(3,4-dichlorophenyl)-5-methyl-2-
(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate
was prepared as described for example 11a from the condensation of
3-(trifluoromethyl)-1H-pyrazol-5-amine, 3,4-dichlorobenzaldehyde,
and methyl 3-oxobutanoate to yield intermediate methyl 7-(3,4-dichloro-
phenyl)-5-methyl-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylate as an orange solid in 52% yield, 96% purity,
((R)-7-(3,4-Dichlorophenyl)-2,5-dimethyl-4,7-dihydropyra-
zolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)-
pyrrolidin-1-yl)methanone (13d). Compound 11a was coupled to
(S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole (12) using the conditions
and subsequent deprotection described for example 13a to yield ((R)-
7-(3,4-dichlorophenyl)-2,5-dimethyl-4,7-dihydropyrazolo[1,5-a]-
pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)-
methanone in 64% yield. HPLC: purity 94%, retention time 7.22 min,
1
retention time 2.90 min. H NMR (400 MHz,) δ 7.35 (d, J = 8.0 Hz,
1H), 7.31 (d, J = 2.1 Hz, 1H), 7.13 (dd, J = 8.3, 2.1 Hz, 1H), 6.63
(s, 1H), 6.38 (s, 1H), 5.87 (s, 1H), 3.61 (s, 3H), 2.52 (s, 3H). LCMS
[M + 1] 405.95, [M + 3] 407.91. Methyl 7-(3,4-dichlorophenyl)-5-methyl-
2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate
was converted to 4-tert-butyl-6-methyl-7-(3,4-dichlorophenyl)-5-methyl-
2-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-4,6(7H)-dicarboxylate
and subsequently hydrolyzed as described to 4-(tert-butoxycarbonyl)-
7-(3,4-dichlorophenyl)-5-methyl-2-(trifluoromethyl)-4,7-dihydro-
pyrazolo[1,5-a]pyrimidine-6-carboxylic acid using the procedure
described in example 13a in 94% yield. HPLC: purity 96%, reten-
tion time 11.85 min, method B; purity 93%, retention time 10.28 min,
method C. 1H NMR (400 MHz, CD3OD) δ 7.39 (d, J = 8.3 Hz, 1H),
1
method B; purity 96%, retention time 7.32 min, method C. H NMR
(400 MHz, MeOD) δ 7.50 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 1.7 Hz,
1H), 7.02 (dd, J = 8.3, 1.9 Hz, 1H), 5.92 (s, 1H), 5.72 (s, 1H), 5.48
(s, 1H), 5.11 (s, 1H), 3.62 (dt, J = 10.8, 7.1 Hz, 1H), 3.21 (s, 1H), 2.26
(s, 3H), 2.12 (s, 3H), 1.98 (m, 4H). 13C NMR (101 MHz, MeOD)
δ 173.58, 161.41, 151.20, 143.56, 141.76, 133.45, 132.88, 131.93, 130.13,
127.99, 103.10, 102.44, 87.74, 61.17, 16.88, 13.64, 11.32. LCMS:
[M + 1] 472.1, [M + 3] 474.1
((R)-7-(3,4-Dichlorophenyl)-5-methyl-2-(trifluoromethyl)-
4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisox-
azol-5-yl)pyrrolidin-1-yl)methanone (13e). Compound 11b was
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coupled to (S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole (12) using the
conditions and subsequent deprotection described for example 13a to
yield ((R)-7-(3,4-dichlorophenyl)-5-methyl-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)-
pyrrolidin-1-yl)methanone in 36% yield. 1H NMR (400 MHz, MeOD)
δ 7.52 (d, J = 8.3 Hz, 1H), 7.30 (s, 1H), 7.08 (d, J = 8.2 Hz, 1H), 6.14
(s, 1H), 5.92 (s, 1H), 5.69 (s, 1H), 5.13 (s, 1H), 3.65 (dt, J = 10.8, 7.2
Hz, 1H), 2.17 (s, 3H), 2.00 (s, 3H), 1.31 (m, 4H). 13C NMR (101
MHz, MeOD) δ 173.55, 161.44, 142.27, 141.58, 133.58, 133.34,
132.06, 130.39, 128.80, 128.17, 126.58, 103.04, 102.77, 86.17, 61.90,
49.69, 49.47, 49.26, 49.05, 48.83, 48.62, 48.41, 16.88, 11.29. HPLC:
purity 98%, retention time 9.83 min, method B; purity 98%, retention
time 8.92 min, method C. LCMS: [M + 1] 526.2
4-(tert-Butoxycarbonyl)-2-cyclopropyl-7-(3,4-dichlorophen-
yl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carbox-
ylic Acid (11f). Intermediate methyl 2-cyclopropyl-7-(3,4-dichloro-
phenyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate.
To a solution of 3-cyclopropyl-1H-pyrazol-5-amine (1.0 g, 8.1 mol)
in THF (50 mL) was added 3,4-dichlorobenzaldehyde (1.4 g, 8.1 mol)
and methyl 3-oxobutanoate (0.94 g, 8.1 mol). Heptane (10 mL) and
piperidine (0.020 mg, 0.24 mmol) were added, and the solution was
heated to 75 °C for 18 h. The cooled reaction mixture was concentrated
in vacuo and the residue redissolved in THF and purified by silica gel
chromatography gradient elution with 2:1 hexane:EtOAc. Methyl
2-cyclopropyl-7-(3,4-dichlorophenyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylate was isolated as a tan solid (1.7 g, 56% yield, Rf
0.2 in 2:1 hexane:EtOAc). HPLC: purity 99%, retention time 9.42 min,
method B; purity 100%, retention time 8.57 min, method C. 1H NMR
(400 MHz, MeOD) δ 7.41 (d, J = 8.4, 1H), 7.31 (dd, J = 8.9, 2.2 Hz,
1H), 7.09 (dd, J = 8.4, 2.2 Hz, 1H), 6.18 (s, 1H), 5.38 (s, 1H), 3.66 (s,
3H), 2.44 (s, 3H), 1.91−1.48 (m, 1H), 1.02−0.75 (m, 2H), 0.71−0.46
(m, 2H). 13C NMR (101 MHz, MeOD) δ 168.19, 158.29, 148.13, 145.17,
139.83, 133.08, 132.39, 131.51, 130.09, 127.74, 115.68, 97.20, 85.25,
59.82, 51.45, 19.43, 10.18, 8.50, 8.30. LCMS [M + 1] 378.19
Methyl 2-cyclopropyl-7-(3,4-dichlorophenyl)-5-methyl-4,7-dihydro-
pyrazolo[1,5-a]pyrimidine-6-carboxylate was treated with BOC anhydride
and the methyl ester hydrolyzed as described for example 11a to yield
4-(tert-butoxycarbonyl)-2-cyclopropyl-7-(3,4-dichlorophenyl)-5-methyl-
4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylic acid (11f) as an orange
foam (0.71 g, 82% yield for 2 steps). HPLC: purity 99%, retention time
11.18 min, method B; purity 99%, retention time 9.80 min, method C. 1H
NMR (400 MHz, MeOD) δ 7.48 (d, J = 8.4 Hz, 1H), 7.22 (t, J = 2.1 Hz,
1H), 7.10 (dd, J = 8.0, 2.0 Hz 1H), 6.32 (s, 1H), 6.00 (s, 1H), 2.70
(s, 3H), 1.97−1.76 (m, 1H), 1.58 (m, 9H), 1.02−0.83 (m, 2H), 0.78−0.60
(m, 2H). 13C NMR (101 MHz, MeOD) δ 157.76, 151.09, 141.72, 138.79,
132.13, 129.20, 127.36, 117.96, 107.21, 94.44, 89.14, 86.22, 75.48, 59.75,
28.22, 21.27, 13.35, 10.24, 8.51. LCMS: [M + 1] 478.15.
((R)-2-Cyclopropyl-7-(3,4-dichlorophenyl)-5-methyl-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxa-
zol-5-yl)pyrrolidin-1-yl)methanone (13f). Compound 11f was
coupled to (S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole (12) using the
conditions and subsequent deprotection described for example 13a to
yield a 1:1 diasteromeric mixture of (2-cyclopropyl-7-(3,4-dichlor-
ophenyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-
(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone in 51% yield. The
diastereomers were separated using prep TLC (25 cm × 25 cm × 1
mm thickness) elution with 1:1 hexane:EtOAc (10% IPA). The Rf
values for the diastereomers were 0.60 for ((R)-2-cyclopropyl-7-(3,4-
dichlorophenyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)-
((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone and 0.35 for
((S)-2-cyclopropyl-7-(3,4-dichlorophenyl)-5-methyl-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)-
pyrrolidin-1-yl)methanone, which were isolated as white solids in
yields of 31% and 22%, respectively. Analytical data for ((R)-2-cyclo-
propyl-7-(3,4-dichlorophenyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]-
pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)-
methanone. HPLC: purity 99%, retention time 8.96 min, method B;
purity 98%, retention time 8.90 min, method C. 1H NMR (400 MHz,
MeOD) δ 7.47 (d, J = 8.3 Hz, 1H), 7.17 (d, J = 1.7 Hz, 1H), 6.96
(dt, J = 6.7, 3.4 Hz, 1H), 6.07−5.56 (m, 1H), 5.26 (s, 1H), 5.02 (s, 1H),
3.96−3.84 (m, 1H), 3.59 (dd, J = 10.2, 6.9 Hz, 1H), 2.23 (s, 4H), 1.91
(s, 7H), 1.80−1.66 (m, 1H), 0.92−0.86 (m, 1H), 0.86−0.80 (m, 2H),
0.66−0.53 (m, 2H). 13C NMR (101 MHz, THF) δ 170.49, 168.43,
164.23, 158.03, 151.52, 146.01, 140.87, 139.28, 129.71, 127.87, 126.84,
124.75, 100.27, 99.18, 95.09, 80.49, 65.03, 64.81, 64.59, 64.37, 64.15,
57.39, 51.24, 28.71, 27.10, 22.90, 22.70, 22.50, 22.31, 22.11, 14.07, 8.44,
7.37, 5.27. LCMS: [M + 1] 498.17.
7-(Benzo[d]thiazol-2-yl)-4-(tert-butoxycarbonyl)-5-methyl-2-
(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-car-
boxylic Acid (11g). Intermediate methyl 7-(benzo[d]thiazol-2-yl)-5-
methyl-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-
6-carboxylate. Methyl 7-(benzo[d]thiazol-2-yl)-5-methyl-2-(trifluoro-
methyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate was
prepared from the condensation of benzo[d]thiazole-2-carbaldehyde,
3-(trifluoromethyl)-1H-pyrazol-5-amine, and methyl 3-oxobutanoate as
described in example 11a. The solvents from the reaction mixture were
removed in vacuo and the residue dissolved in MeOH. The resulting
white precipitate was collected and dried to yield methyl 7-(benzo-
[d]thiazol-2-yl)-5-methyl-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-
a]pyrimidine-6-carboxylate in 61% yield. HPLC: purity 97%; retention
time 2.31 min; method A. 1H NMR (400 MHz, CDCl3) δ 9.91 (s, 1H),
7.96−7.92 (m, 1H), 7.88−7.84 (m, 1H), 7.51−7.42 (m, 2H), 6.91 (s,
1H), 5.62 (s, 1H), 3.74 (s, 3H), 2.22 (s, 3H). LCMS [M + 1] 395.22.
Methyl 7-(benzo[d]thiazol-2-yl)-5-methyl-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate was treated with
BOC anhydride and the methyl ester hydrolyzed as described for
example 11a to yield 7-(benzo[d]thiazol-2-yl)-4-(tert-butoxycarbonyl)-
5-methyl-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-
carboxylic acid (11g) in 95% yield. HPLC: purity 100%, retention time
10.92 min, method B; purity 100%, retention time 9.69 min, method
1
C. H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 7.9 Hz, 1H), 7.80 (d,
J = 7.9 Hz, 1H), 7.48−7.42 (m, 1H), 7.40−7.34 (m, 1H), 6.97 (s, 1H),
6.59 (s, 1H), 2.67 (s, 3H), 1.59 (s, 9H). LCMS [M + 1] 481.32. 13C
NMR (101 MHz, MeOD) δ 168.14, 167.07, 153.94, 150.56, 149.98,
139.79, 136.57, 127.70, 127.08, 124.30, 123.10, 116.02, 96.51, 86.80,
59.48, 44.51, 28.21, 21.19, −1.95. LCMS [M + 1] 480.9.
((S)-7-(Benzo[d]thiazol-2-yl)-5-methyl-2-(trifluoromethyl)-
4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisox-
azol-5-yl)pyrrolidin-1-yl)methanone (13g). Compound 11g was
coupled to (S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole (12) using the
conditions and subsequent deprotection described for example 13a to
yield a 1:1 diasteromeric mixture of 7-(benzo[d]thiazol-2-yl)-5-methyl-
2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-
(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone. The diastereomers
were separated using silica gel chromatography gradient elution with
10% EtOAc in hexane to 100% EtOAc over 20 min. The retention
times for the diastereomers were 14 min for ((R)-7-(benzo[d]thiazol-
2-yl)-5-methyl-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-
6-yl)((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone and 17.5 min
for ((S)-7-(benzo[d]thiazol-2-yl)-5-methyl-2-(trifluoromethyl)-4,
7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)-
pyrrolidin-1-yl)methanone, which were isolated as white solids in yields
of 53% and 50%, respectively. Analytical data for ((S)-7-(benzo[d]-
thiazol-2-yl)-5-methyl-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)-
methanone. HPLC: purity 98%, retention time 10.05 min, method B;
purity 99%, retention time 9.35 min, method C. 1H NMR (400 MHz,
CDCl3) δ 7.93 (d, J = 7.9 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.47−7.34
(m, 2H), 6.88 (s, 1H), 6.46 (s, 1H), 6.03 (s, 1H), 5.83 (s, 1H), 5.31
(t, J = 7.7 Hz, 1H), 3.77−3.70 (m, 1H), 3.52−3.40 (m, 1H), 2.21
(m, 3H), 2.08 (m, 3H), 1.93 (m, 2H). 13C NMR (126 MHz, MeOD) δ
168.74, 150.61, 140.60, 133.00, 124.52, 123.77, 121.37, 120.20, 100.08, 97.67,
83.24, 57.62, 51.95, 21.89, 13.41, 8.23, −3.81. LCMS [M + 1] 515.31.
4-(tert-Butoxycarbonyl)-5-methyl-7-(1-methyl-1H-benzo[d]-
imidazol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylic Acid (11h). Intermediate methyl
5-methyl-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-2-(trifluoromethyl)-
4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate. Methyl 5-methyl-
7-(1-methyl-1H-benzo[d]imidazol-2-yl)-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate was prepared from the
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Journal of Medicinal Chemistry
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condensation of 1-methyl-1H-benzo[d]imidazole-2-carbaldehyde,
3-(trifluoromethyl)-1H-pyrazol-5-amine, and methyl 3-oxobutanoate
as described in example 11a. The solvents from the reaction mixture
were removed in vacuo and the residue dissolved in MeOH. The
resulting white precipitate was collected and dried to yield methyl
5-methyl-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-2-(trifluoromethyl)-
4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate in 56% yield.
Methyl 5-methyl-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-
4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate was treated with
BOc anhydride and the methyl ester hydrolyzed as described for
example 11a to yield 4-(tert-butoxycarbonyl)-5-methyl-7-(1-methyl-
1H-indol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylic acid (11i) in 73% yield. HPLC: purity 95%,
retention time 3.82 min, method A. HPLC: purity 100%, retention
time 10.95 min, method B; purity 100%, retention time 9.76 min,
method C. 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 7.91 Hz, 1H),
7.33−7.28 (m, 1H), 7.21 (t, J = 7.47 Hz, 1H), 7.04 (t, J = 7.25 Hz,
1H), 6.74 (s, 1H), 6.41 (s, 1H), 6.37 (s, 1H), 4.05 (s, 3H), 2.72
(s, 3H), 1.56 (s, 9H). 13C NMR (101 MHz, THF) δ 163.48, 147.38,
144.32, 135.91, 135.09, 125.52, 124.53, 119.84, 117.66, 117.46, 115.33,
109.52, 107.21, 92.72, 82.43, 52.11, 29.85, 25.21,18.02. LCMS: [M + 1]
477.36.
((S)-5-Methyl-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-
4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisox-
azol-5-yl)pyrrolidin-1-yl)methanone (13i). Compound 11i was
coupled to (S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole (12) using the
conditions and subsequent deprotection described for example 11 to
yield a 1:1 diasteromeric mixture of 5-methyl-7-(1-methyl-1H-indol-2-
yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)-
((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone. The diastereo-
mers were separated using silica gel chromatography gradient elution with
10% EtOAc in hexane to 50% EtOAc over 20 min. The retention times
for the diastereomers were 13 min for ((R)-5-methyl-7-(1-methyl-1H-
indol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)-
((S)-2-(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone and 16 min for
((S)-5-methyl-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-
yl)pyrrolidin-1-yl)methanone, which were isolated as yellow solids
in yields of 36% and 32%, respectively. Analytical data for ((S)-5-
methyl-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-
yl)pyrrolidin-1-yl)methanone. HPLC: purity 95%, retention time
9.39 min, method B; purity 94%, retention time 8.67 min, method C.
1H NMR (400 MHz, CD3CN) δ 7.65 (m, 1H), 7.53 (t, J = 10.7 Hz,
1
HPLC: purity 87%, retention time 2.99 min, method A. H NMR
(400 MHz, CDCl3) δ 12.61 (brs, 1H), 7.58 (d, J = 8.35 Hz, 1H), 7.50
(d, J = 8.35 Hz, 1H), 7.38 (t, J = 7.69 Hz, 1H), 7.29 (t, J = 7.69 Hz,
1H), 6.78 (s, 1H), 4.21 (s, 3H), 3.66 (s, 3H), 1.56 (s, 3H). LCMS:
[M + 1] 392.29.
Methyl 5-methyl-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-2-(tri-
fluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate
was treated with BOC anhydride and the methyl ester hydrolyzed as
described for example 11a to yield 11h in 50% yield. HPLC: purity
94%, retention time 3.13 min, method A. HPLC: purity 96%, retention
time 8.91 min, method B; purity 100%, retention time 8.72 min,
method C. 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.35 Hz, 1H),
7.30−7.26 (m, 2H), 7.21−7.16 (m, 1H), 6.80 (s, 1H), 6.47 (s, 1H),
3.99 (s, 3H), 2.71 (s, 3H), 1.57 (s, 9H). LCMS [M + 1] 478.36. 13C
NMR (101 MHz, THF) δ 165.45, 150.34, 149.16, 148.68, 142.77,
138.37, 135.99, 122.38, 121.57, 119.66, 114.15, 109.73, 95.21, 84.17,
52.53, 29.42, 27.22, 20.20. LCMS: [M + 1] 477.9.
((S)-5-Methyl-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-2-(tri-
fluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-
(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone (13h). Com-
pound 11h was coupled to (S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole
(12) using the conditions and subsequent deprotection described for
example 13a to yield a 1:1 diasteromeric mixture of (5-methyl-7-
(1-methyl-1H-benzo[d]imidazol-2-yl)-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)-
pyrrolidin-1-yl)methanone. The diastereomers were separated using
silica gel chromatography gradient elution with 25% EtOAc in hexane
to 100% EtOAc over 20 min. The retention times for the diastereomers
were 15 min for ((R)-5-methyl-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-
2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-
methylisoxazol-5-yl)pyrrolidin-1-yl)methanone and 21.5 min for
((S)-5-methyl-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-2-(trifluoro-
methyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methyl-
isoxazol-5-yl)pyrrolidin-1-yl)methanone, which were isolated as
white solids in yields of 30% and 28%, respectively. Analytical data for
((S)-5-methyl-7-(1-methyl-1H-benzo[d]imidazol-2-yl)-2-(trifluorometh-
yl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-
5-yl)pyrrolidin-1-yl)methanone is as follows. HPLC: purity 96%, reten-
tion time 6.87 min, method B; purity 96%, retention time 7.74 min,
1H), 7.36 (d, J = 8.3 Hz, 1H), 7.20 (ddd, J = 24.1, 12.6, 9.1 Hz,
1H), 7.07 (dt, J = 16.1, 4.5 Hz, 1H), 6.44 (s, 1H), 5.92 (s, 1H), 5.01
(s, 1H), 3.95 (m, 1H), 3.60 (s, 2H), 3.54 (s, 3H), 3.28 (s, 1H),
2.03 (s, 3H), 1.98 (s, 2H). 13C NMR (101 MHz, CD3CN) δ 185.44,
182.95, 173.53, 166.86, 162.84, 141.31, 140.33, 138.39, 132.08,
127.62, 123.07, 121.60, 120.75, 118.30, 110.61, 103.61, 102.18,
98.30, 86.00, 54.67, 47.31, 38.51, 30.31, 17.15, 11.41. LCMS: [M + 1]
511.41.
4-(tert-Butoxycarbonyl)-5-(methoxymethyl)-7-(1-methyl-1H-
indol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylic Acid (11j). To a solution of 3-(trifluor-
omethyl)-1H-pyrazol-5-amine (2.4 g, 16 mmol) in THF (8 mL) was
added 1-methyl-1H-indole-2-carbaldehyde (2.5 g, 16 mmol) and
methyl 3-oxobutanoate (2.0 g mL, 16 mmol). Heptane (2 mL) and
piperidine (78 uL, 0.79 mmol) was added, and the solution was heated
to 75 °C for 120 h. The cooled reaction mixture was purified directly
using silica gel chromatography gradient elution with 20−80% EtOAc
in hexane over 40 min to yield methyl 5-(methoxymethyl)-7-(1-
methyl-1H-indol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylate as a white powder (2.3 g, 35% yield, 95%
1
method C. H NMR (400 MHz,) δ 7.63 (d, J = 7.9 Hz, 1H), 7.44 (d,
J = 8.0 Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.27 (d, J = 7.1 Hz, 1H), 6.44−
6.16 (m, 1H), 5.47 (s, 1H), 5.23 (s, 1H), 3.83 (s, 1H), 3.69 (s, 1H), 2.25
(s, 1H), 2.04 (s, 3H), 2.11−1.94 (m, 6H), 1.58 (s, 3H). 13C NMR (101
MHz, THF) δ 120.60, 119.89, 117.59, 107.95, 51.84, 27.67, 14.27, 7.72.
LCMS: [M + 1] 512.36
4-(tert-Butoxycarbonyl)-5-methyl-7-(1-methyl-1H-indol-2-
yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-
6-carboxylic acid (11i). Intermediate methyl 5-methyl-7-(1-methyl-
1H-indol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylate. Methyl 5-methyl-7-(1-methyl-1H-indol-2-yl)-2-
(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate was
prepared from the condensation of 1-methyl-1H-indole-2-carbaldehyde,
3-(trifluoromethyl)-1H-pyrazol-5-amine, and methyl 3-oxobutanoate as
described in example 11a. The solvents from the reaction mixture were
removed in vacuo and the residue dissolved in MeOH. The resulting
white precipitate was collected and dried to yield methyl 5-methyl-7-(1-
methyl-1H-indol-2-yl)-2-(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]-
pyrimidine-6-carboxylate in 55% yield. HPLC: purity 99%, retention
time 1.94 min, method A. 1H NMR (400 MHz, CDCl3) δ 7.52 (d, J =
8.35 Hz, 1H), 7.24−7.15 (m, 2H), 7.07−7.02 (m, 2H), 6.80 (s, 1H),
6.73 (s, 1H), 5.76 (s, 1H), 3.67 (s, 3H), 3.62 (s, 3H), 2.47 (s, 3H). LCMS:
[M + 1] 391.26.
1
purity, retention time 3.43 min, method A). H NMR (400 MHz,
CDCl3) δ 7.99 (s, 1H), 7.49 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.3 Hz,
1H), 7.06 (s, 1H), 6.69 (s, 1H), 6.30 (s, 1H), 5.90 (s, 1H), 4.90
(s, 2H), 4.06 (s, 3H), 3.59 (s, 3H), 3.60 (s, 3H). To a slurry of methyl
5-(methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-
4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylate (2.3 g, 5.5 mmol)
in THF (10 mL) was added BOC anhydride (1.4 g, 6.6 mmol) and
DMAP (33 mg, 0.27 mmol). The solution was stirred at room
temperature for 2 h and then water (10 mL) and LiOH (0.23 g,
5.5 mmol) added. After 14 h, additional LiOH was added (0.46 g,
11 mmol) and the solution stirred for an additional 14 h. The reaction
mixture was neutralized by the addition of 1 M HCl and the aqueous
portion extracted with EtOAc. The combined organic portions were
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dried over Na2SO4, decanted, and concentrated to yield 4-(tert-
butoxycarbonyl)-5-(methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-2-
(trifluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidine-6-carboxylic
acid as a brown solid (2.5 g, 100% yield). 1H NMR (400 MHz,
MeOD) δ 7.46 (d, J = 7.9 Hz, 1H), 7.38 (s, 1H), 7.23−7.14 (m, 1H),
7.06−6.95 (m, 1H), 6.86 (s, 1H), 6.54 (s, 1H), 6.37 (s, 1H), 5.24 (d,
J = 15.1 Hz, 1H), 4.92 (d, J = 15.1 Hz, 1H), 4.05 (s, 3H), 3.30 (s, 3H),
1.61 (s, 9H). 13C NMR (101 MHz, MeOD) δ 167.27, 150.90, 148.29,
139.77, 138.78, 136.29, 128.24, 123.64, 121.71, 120.96, 117.60, 110.77,
101.89, 95.29, 86.40, 69.88, 58.83, 54.24, 30.66, 28.12. HPLC: purity
90%, retention time 11.38 min, method B; purity 91%, retention time
9.96 min, method C. LCMS: [M + 1] 507.0.
extracts were dried over Na2SO4, decanted, and concentrated to yield
(5-(methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-
4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-
5-yl)pyrrolidin-1-yl)methanone as a 1:1 mixture of diastereomers.
The diastereomers were separated by silica gel chromatography
gradient elution with 40−100% EtOAc in hexane over 20 min. ((S)-5-
(Methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-2-(trifluoromethyl)-4,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-(3-methylisoxazol-5-yl)-
pyrrolidin-1-yl)methanone (13j) was isolated as the more polar isomer
1
as a tan powder (0.074 g, 24% yield). H NMR (400 MHz, 55 °C,
CDCl3) δ 7.51 (d, J = 7.7 Hz, 1H), 7.27 (d, J = 8.3 Hz, 1H), 7.19 (t,
J = 7.2 Hz 1H), 7.06 (t, J = 7.2 Hz 1H), 6.81 (s, 1H), 6.49 (s, 1H),
6.48 (s, 1H), 5.84 (s, 1H), 5.50 (s, 1H), 5.00 (s, 1H), 4.11 (m, 2H),
3.66 (s, 3H), 3.61 (m, 1H), 3.41 (s, 3H), 3.40 (m, 1H), 2.16 (s, 3H),
1.91 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 171.36, 165.50, 159.72,
138.72, 138.20, 136.00, 126.88, 122.34, 121.03, 119.84, 109.46, 103.14,
102.33, 101.93, 85.90, 68.29, 58.90, 53.92, 47.02, 31.55, 31.15, 29.91,
22.64, 14.08, 11.30. HPLC: purity 98%, retention time 7.16 min,
Zorbax SB C18, 4.6 mm × 75 mm, 2.5 mL/min gradient 10−100%
95:5 MeOH in H2O (0.1% H3PO4) in 5:95 MeOH in H2O (0.1%
H3PO4). HRMS: [M + 1] obsd 541.21847 calcd 541.21695. Elemental
analysis: C, H, N, F theoretical % 59.99, 5.03, 15.54, 10.54; obsd %
60.00, 4.95, 15.50, 10.16. The absolute stereochemistry at the C7 posi-
tion was confirmed by the X-ray structure included in the Supporting
Information.
(S)-3-Methyl-5-(pyrrolidin-2-yl)isoxazole (12). Propan-2-one
oxime (0.42 g, 5.7 mmol) was dissolved in THF (10 mL), and
nBuLi (4.6 mL, 2.5 M solution in hexane, 11 mmol) was added drop-
wise. The reaction mixture was stirred for 30 min, cooled to 0 °C, and
(S)-1-benzyl 2-methyl pyrrolidine-1,2-dicarboxylate (1.0 g, 3.8 mmol)
was added and the solution stirred for 1.5 h. Concentrated H2SO4 was
added dropwise (3.5 mL), and the reaction mixture was stirred for an
additional 1.5 h and poured cautiously into a 1:1 mixture of ice and
NH4OH. The aqueous portion was extracted with ether and the com-
bined organic portions dried over Na2SO4, decanted, and concentrated
to yield intermediate (S)-benzyl 2-(3-methylisoxazol-5-yl)pyrrolidine-
1-carboxylate, which was purified by silica gel chromatography elution
with hexane:acetone (3:1) as a yellow oil (0.55 g, 51% yield, 93%
purity retention time 1.99 min). (S)-Benzyl 2-(3-methylisoxazol-5-
yl)pyrrolidine-1-carboxylate (0.39 g, 1.4 mmol) was dissolved in DCM
(8 mL), and triflic acid (1.0 g, 6.8 mmol) was added dropwise and the
solution stirred for 15 min. Water was added, followed by NaOH
(1M), until the pH was adjusted to pH 8. The organic layer was
separated and the aqueous portion extracted further with DCM. The
combined organic portions were dried over Na2SO4, decanted, and
concentrated and the residue purified by silica gel chromatography
elution with DCM:MeOH (10:1) yielding (S)-3-methyl-5-(pyrrolidin-
2-yl)isoxazole as a colorless oil (0.87 g, 42% yield). To prevent
decomposition, (S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole was stored
as the NBS monosalt. 1H NMR (400 MHz, MeOD) δ 7.72 (d, J = 6.4 Hz,
1H), 7.26 (d, J = 7.9 Hz, 1H), 6.52 (s, 1H), 4.91 (t, J = 7.7 Hz, 1H),
3.47 (dd, J = 10.3, 4.2 Hz, 1H), 2.60−2.44 (m, 1H), 2.39 (s, 3H), 2.31
(s, 3H), 2.32−2.26 (m, 2H), 2.29−2.09 (m, 1H). 13C NMR (101 MHz,
MeOD) δ 167.03, 162.03, 143.53, 141.84, 129.92, 126.99, 106.35, 55.55,
47.02, 30.12, 24.66, 21.37, 11.23. HPLC: purity 99%, retention time 4.17
min, method B; purity 95%, retention time 2.12 min, method C. LCMS:
[M + 1] 152.9.
ASSOCIATED CONTENT
* Supporting Information
■
S
Absolute stereochemistry at the C7 position confirmed by the
X-ray structure. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For H.J.F.: phone, 609-818-3734; fax, 609-818-3550; E-mail,
heather.finlay@bms.com. For J.L.: phone, 609-818-5327; fax,
609-818-3550; E-mail, john.lloyd@bms.com.
Notes
The authors declare no competing financial interest.
⊥Deceased December 29, 2006.
#Deceased September 16, 2008.
((S)-5-(Methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-2-(tri-
fluoromethyl)-4,7-dihydropyrazolo[1,5-a]pyrimidin-6-yl)((S)-2-
(3-methylisoxazol-5-yl)pyrrolidin-1-yl)methanone (13j). To
compound 11j (0.44 g, 0.87 mmol) in DCM (5 mL) was added
EDCI (0.18 g, 0.95 mmol) and (S)-3-methyl-5-(pyrrolidin-2-yl)isoxazole
(0.13 g, 8.7 mmol). The reaction mixture was stirred at room temperature
for 2 h and then purified directly by silica gel chromatography gradient
elution with 0−100% EtOAc in hexane over 18 min to yield a
diastereomeric mixture of tert-butyl 5-(methoxymethyl)-7-(1-methyl-
1H-indol-2-yl)-6-((S)-2-(3-methylisoxazol-5-yl)pyrrolidine-1-carbon-
yl)-2-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-4(7H)-carboxylate
as a yellow powder (0.38 g, 69% yield, 95% purity at retention time
ACKNOWLEDGMENTS
■
We thank Purnima Khandelwal for NMR support and Carolyn
Pommier, Michael Galella, and Doree Sitkoff for crystallog-
raphy support. We also thank Robert Languish for high
resolution mass spectrometry support.
ABBREVIATIONS USED
AERP, atrial effective refractory period; VERP, ventricular effec-
tive refractory period; AF, atrial fibrillation; GSH, glutathione
■
1
2.41 min, method A). H NMR (400 MHz,) δ 7.51 (t, J = 10.2 Hz,
REFERENCES
1H), 7.32 (d, J = 8.3 Hz, 1H), 7.13−6.99 (m, 1H), 6.59 (d, J = 11.7
Hz, 1H), 6.53 (s, 1H), 6.43 (dt, J = 24.3, 12.8 Hz, 2H), 5.97 (d, J =
22.8 Hz, 1H), 5.65 (d, J = 17.0 Hz, 1H), 5.34−5.11 (m, 1H), 4.82 (dd,
J = 12.0, 7.3 Hz, 1H), 4.74 (dd, J = 13.1, 7.5 Hz, 1H), 4.37−4.19 (m,
1H), 4.16−3.93 (m, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.82−3.43 (m,
3H), 3.34 (d, J = 16.3 Hz, 2H), 3.22 (d, J = 24.1 Hz, 2H), 2.29−2.17
(m, 4H), 2.19−1.97 (m, 4H), 1.57 (s, 9H), 1.58 (s, 9H). tert-Butyl 5-
(methoxymethyl)-7-(1-methyl-1H-indol-2-yl)-6-((S)-2-(3-methylisox-
azol-5-yl)pyrrolidine-1-carbonyl)-2-(trifluoromethyl)pyrazolo[1,5-a]-
pyrimidine-4(7H)-carboxylate (0.38 g, 0.59 mmol) was dissolved in
AcOH (5 mL) and heated to 150 °C in a microwave reactor for 2 min.
The solution was neutralized by the addition of satd NaHCO3 and the
aqueous solution extracted with EtOAc. The combined organic
■
(1) Go, A.; Hylek, E.; Phillips, K.; Chang, Y.; Henault, L.; Selby, J.;
Singer, D. Prevalence of diagnosed atrial fibrillation in adults: national
implications for rhythm management and stroke prevention: the
AnTicoagulation and Risk Factors in Atrial Fibrillation (ATRIA)
Study. JAMA, J. Am. Med. Assoc. 2001, 285, 2370−2375.
(2) Miyasaka, Y.; Barnes, M. E.; Gersh, B. J.; Cha, S. S.; Bailey, K. R.;
Abhayaratna, W. P.; Seward, J. B.; Tsang, T. S. M. Secular trends in
incidence of atrial fibrillation in Olmsted County, Minnesota, 1980 to
2000, and implications on the projections for future prevalence.
Circulation 2006, 114, 119−125.
(3) Hart, R. G.; Halperin, J. L. Atrial fibrillation and stroke: concepts
and controversies. Stroke 2001, 32, 803−808.
3047
dx.doi.org/10.1021/jm201386u | J. Med. Chem. 2012, 55, 3036−3048

Journal of Medicinal Chemistry
Article
(4) Janko, S.; Dorwarth, U.; Hoffmann, E. Pharmacotherapy of atrial
fibrillation: an old option with new possibilities. Expert Opin.
Pharmacother. 2008, 9, 913−925.
(5) Sven, K.; Dierk, T.; Karle, C. A. The novel antiarrhythmic drug
dronedarone: comparison with amiodarone. Cardiovasc. Drug Rev.
2005, 23, 217−230.
(6) Chong, P. H.; Cory, F.; Yim, B. T. Treatment of Atrial Fibrillation
and Flutter: Focus on Oral Dofetilide, a New Selective Class III
Antiarrhythmic Agent. HeartDrug 2001, 1, 320−331.
(7) Wang, Z.; Page, P.; Nattel, S. Mechanism of flecainide’s
antiarrhythmic action in experimental atrial fibrillation. Circ. Res.
1992, 71, 271−287.
(8) Hohnloser, S. H.; van de Loo, A.; Baedeker, F. Efficacy and pro-
arrhythmic hazards of pharmacologic cardioversion of atrial fibrillation:
prospective comparison of sotalol versus quinidine. J. Am. Coll. Cardiol.
1995, 26, 852−858.
ments for the Pyridine Ring in Nicotine. J. Med. Chem. 1994, 37 (26),
4455−4463.
(22) Synders, D. J.; Tamakun, M. N.; Bennett, P. B. A rapidly
activating and slowly inactivating potassium channel cloned from
human heart: functional analysis after stable mammalian cell culture
expression. J. Gen. Physiol. 1993, 101, 513−543.
(23) Zhou, Z.; Vorperian, V. R.; Gong, Q.; Zhang, S. and January,
C.T. Block of HERG potassium channels by the antihistamine
astemizole and its metabolites desmethylastemizole and norastemizole.
J. Cardiovasc. Electrophysiol. 1999, 10 (6), 836−843.
(24) Li, D.; Sun, H.; Levesque, P. Antiarrhythmic drug therapy for
atrial fibrillation: focus on atrial selectivity and safety. Cardiovasc.
Hematol. Agents Med. Chem. 2009, 7 (1), 64−75.
(25) Carlsson, Leif. The anaesthetised methoxamine-sensitised rabbit
model of torsades de pointes. Pharmacol. Ther. 2008, 119 (2), 160−
167.
(9) De Bruin, M. L.; Hoes, A. W.; Leufkens, H. G. QTc-Prolonging
Drugs and Hospitalizations for Cardiac Arrhythmias. Am. J. Cardiol.
2003, 91, 59−62.
NOTE ADDED AFTER ASAP PUBLICATION
■
After this paper was published online March 21, 2012, a
correction was made to the TOC graphic and abstract graphic.
The corrected version was reposted March 23, 2012.
(10) Nattel, S. New ideas about atrial fibrillation 50 years on. Nature
2002, 415, 219−226.
(11) Ford, J. W.; Milnes, J. T. New Drugs Targeting the Cardiac
Ultra-Rapid Delayed-Rectifier Current (IKur): Rationale, Pharmacol-
ogy and Evidence for Potential Therapeutic Value. J. Cardiovasc.
Pharmacol. 2008, 52 (2), 105−120.
(12) Lloyd, J; Finlay, H. J.; Atwal, K; Kover, A; Prol, J; Yan, L;
Bhandaru, R; Vaccaro, W; Huynh, T; Huang, C. S.; Conder, M;
Jenkins-West, T; Sun, H; Li, D; Levesque, P. Dihydropyrazolopyr-
imidines containing benzimidazoles as KV1.5 potassium channel
antagonists. Bioorg. Med. Chem. Lett. 2009, 19 (18), 5469−5473.
(13) Lloyd, J; Finlay, H. J.; Vacarro, W; Hyunh, T; Kover, A;
Bhandaru, R; Yan, L; Atwal, K; Conder, M; Jenkins-West, T; Shi, H.;
Huang, C; Li, D; Sun, H; Levesque, P. Pyrrolidine amides of
pyrazolodihydropyrimidines as potent and selective KV1.5 blockers.
Bioorg. Med. Chem. Lett. 2010, 20 (4), 1436−1439.
(14) Vacarro, W.; Huynh, T.; Lloyd, J.; Atwal, K. S.; Finlay, H. J.;
Levesque, P. C.; Conder, M. L.; Jenkins-West, T.; Shi, H.; Sun, L.
Dihydropyrazolopyrimidine inhibitors of KV1.5 (I Kur). Bioorg. Med.
Chem. Lett. 2008, I18 (24), 6381−6385.
(15) Compounds incubated (100 μM) at 37 °C for 30 min in 0.1 M
potassium phosphate buffer containing 2 mM NADPH, 2 mM dansyl
glutathione, and 2 mg/mL human liver microsomes. The reaction was
quenched with 2 volumes of 5 mM dithiothreitol in methanol then
vortexed and centrifuged. 60 μL of the supernatant was analyzed by
HPLC equipped with a fluorescence detector (Ex = 340 nm, Em =
525 nm).
(16) Treiber, A.; Dansette, P. M.; Hamid, E.; Girault, J.-P.; Ginderow,
D.; Mornon, J.-P.; Mansuy, D. Chemical and Biological Oxidation of
Thiophene: Preparation and Complete Characterization of Thiophene
S-Oxide Dimers and Evidence for Thiophene S-Oxide as an
Intermediate in Thiophene Metabolism in Vivo and in Vitro. J. Am.
Chem. Soc. 1997, 119, 1565−1571.
(17) Nassar, A. F.; Lopez-Anaya, A. Strategies for dealing with
reactive intermediates in drug discovery and development. Curr. Opin.
Drug Discovery Dev. 2004, 7, 126−136.
(18) Reimlinger, H.; Van Overstraeten, A. Reactions of 3(5)-
diazopyrazole. II. Chem. Ber. 1966, 99, 3350−3357.
(19) Kirk, K. L.; Cohen, L. A. Photochemistry of diazonium salts. I.
Synthesis of 4-fluoroimidazoles, 4-fluorohistamine, and 4-fluorohisti-
dine. J. Am. Chem. Soc. 1973, 95, 4619−4624.
(20) Janssen, J. W. A. M.; Koeners, H. J.; Kruse, C. G.; Habraken,
C. L. Pyrazoles. XII. Preparation of 3(5)-nitropyrazoles by thermal
rearrangement of N-nitropyrazoles. J. Org. Chem. 1973, 38, 1777−
1782.
(21) Garvey, D. S.; Wasicak, J. T.; Elliott, R. L.; Lebold, S.; Hettinger,
A.; Carrera, G. M.; Lin, N.; He, Y.; Holladay, M. W. Ligands for Brain
Cholinergic Channel Receptors: Synthesis and in Vitro Character-
ization of Novel Isoxazoles and Isothiazoles as Bioisosteric Replace-
3048
dx.doi.org/10.1021/jm201386u | J. Med. Chem. 2012, 55, 3036−3048