Selective Inhibitors of G2019S-LRRK2 Kinase Activity

Article abstract of DOI:10.1021/acs.jmedchem.0c01243

Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.

Full text of DOI:10.1021/acs.jmedchem.0c01243

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Selective Inhibitors of G2019S-LRRK2 Kinase Activity
́
Albert W. Garofalo,* Jessica Bright, Stephane De Lombaert, Alyssa M. A. Toda, Kerry Zobel,
Daniele Andreotti, Claudia Beato, Silvia Bernardi, Federica Budassi, Laura Caberlotto, Peng Gao,
Cristiana Griffante, Xinying Liu, Luisa Mengatto, Marco Migliore, Fabio Maria Sabbatini, Anna Sava,
Elena Serra, Paolo Vincetti, Mingliang Zhang, and Holly Carlisle
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ABSTRACT: Pathogenic variants in the leucine-rich repeat kinase
2 (LRRK2) gene have been identified that increase the risk for
developing Parkinson’s disease in a dominantly inherited fashion.
These pathogenic variants, of which G2019S is the most common,
cause abnormally high kinase activity, and compounds that inhibit
this activity are being pursued as potentially disease-modifying
therapeutics. Because LRRK2 regulates important cellular
processes, developing inhibitors that can selectively target the
pathogenic variant while sparing normal LRRK2 activity could offer
potential advantages in heterozygous carriers. We conducted a
high-throughput screen and identified a single selective compound
that preferentially inhibited G2019S-LRRK2. Optimization of this
scaffold led to a series of novel, potent, and highly selective
G2019S-LRRK2 inhibitors.
variants within LRRK2 have been identified. Of these, the
G2019S variant occurring within the kinase domain is the most
common, accounting for 4% of familial PD and up to 1% of
apparently sporadic PD worldwide.2 Interestingly, while some
of the pathogenic variants are found within the kinase domain,
all pathogenic variants have been reported to show increased
autophosphorylation at S1292 when expressed in cells,3
implicating abnormal kinase activity as the likely cause of
pathogenicity. LRRK2 kinase inhibition has therefore become
a promising therapeutic strategy for PD, and several small-
molecule LRRK2 kinase inhibitors are currently in clinical
development.
While the therapeutic potential of LRRK2 kinase inhibitors
is clear, this strategy is complicated by the potential for on-
target safety liabilities associated with the loss of physiological
LRRK2 activity. It has been found that LRRK2 has a role in
mouse kidney and lung homeostasis, as implied in knockout
and kinase-dead mutants.4 Genetic knockout of LRRK2 in
rodents causes vacuolization in specific cell types in kidney and
lung, suggesting that normal LRRK2 plays a critical and
INTRODUCTION
■
Parkinson’s disease (PD) is the most common form of
parkinsonism and the second most common age-related
neurodegenerative disease. It is estimated to affect about 1%
of the population over age 65 with prevalence increasing with
increasing age. It is a chronic disease marked by progressive
disability and diminished quality of life. PD is characterized by
motor symptoms that include resting tremor, rigidity, postural
instability, impaired speech, and bradykinesia, which are
predominantly driven by loss of dopaminergic neurons in the
substantia nigra pars compacta. Non-motor symptoms
including hyposmia, dysautonomia, and changes in sleep,
cognition, and mood also occur with varying frequency.
Current therapeutic strategies for PD are largely focused on
controlling motor symptoms with dopamine replacement and
by enhancing the activity of the remaining dopaminergic
neurons. At present, no disease-modifying therapies exist that
address the underlying neuropathological drivers of disease;
this remains a significant unmet medical need for PD patients.
It has long been known that family members of PD patients
have an increased risk of developing the disease compared to
the general population. In 2004, variants in the leucine-rich
repeat kinase 2 (LRRK2) gene were shown to co-segregate
with disease in familial PD.1 LRRK2 is a large, multidomain
protein that is unusual for having two functional enzymatic
domains: a Ras-like GTPase domain and a serine−threonine
kinase domain. At least seven dominantly inherited pathogenic
Received: July 31, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01243
© XXXX American Chemical Society
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Figure 1. Homology model of the LRRK2 kinase domain depicting compound 1 docked into the ATP pocket. Principal hydrogen-bonding
interactions involve both N-1 and N-2 of the indazole.
nonredundant role in vesicular trafficking in those cell types in
rodents.5 Supporting these interpretations, pharmacological
inhibition of LRRK2 kinase activity in rodents causes
morphological changes in type II pneumocytes of the lung
and proximal tubules in the kidney that are similar to those
observed in knockout animals.6 In nonhuman primates, strong
inhibition of LRRK2 kinase activity also causes vacuolization in
lung cells, although the effect is reversible and without
apparent functional consequence over the duration tested.7
However, the safety risks associated with chronic LRRK2
kinase inhibition in humans cannot be fully assessed without
long-term clinical observation. Because the majority of PD
patients carrying the LRRK2-G2019S variant are hetero-
zygous,8 a precision medicine approach of selectively targeting
only the pathogenic activity while sparing the physiological
LRRK2 activity could offer efficacy and safety advantages for
this patient population.
tions. While not all of the structure−activity relationships
described below were predicted by this model, certain aspects
of the SAR were consistent with the general pose.
Docking compound 1 in our homology model suggested
that the indazole binds to the hinge, as shown in Figure 1. This
pose agreed with our expectations as indazole is a privileged
structure for kinase inhibitors and numerous crystal structures
containing indazole ligands bound in the ATP site of kinases
are available in the protein data bank (PDB). In the majority of
these structures, the indazole functions as a hinge binder as
demonstrated with the known LRRK2 inhibitor MLi-2.11 In
our model, N-1 of the indazole hydrogen bond donates to
Glu1948 and N-2 accepts a hydrogen bond from Ala1950
within the ATP pocket. Our confidence in this binding mode
was bolstered by the complete loss of activity upon N-
methylation of the indazole or replacement of the indazole
with benzo[d]isoxazole (not shown). This binding pose
suggested that substituents at the indazole C-3′ position
would be partially solvent-exposed and might therefore be used
to modulate the properties of an inhibitor series. As shown in
Table 1, substitution of the available valence at C-3′ (R1) was
tolerated and afforded a number of subnanomolar inhibitors.
The addition of a methyl group (2) tripled the potency while
maintaining selectivity. Larger groups afforded potencies of less
than a nanomolar. The lower limit of reliable quantification of
potency in the biochemical assay was ∼0.2 nM, and therefore,
the actual IC50 values for 4, 6, and 7 on G2019S-LRRK2 were
likely lower than 0.2 nM. For compound 4, potency against
both mutant and WT was such that selectivity could not be
accurately determined in the biochemical assays. In general,
the preferred substitution at C-3′ tended to be an aromatic or
heteroaromatic ring as demonstrated with analogues 3−7.
Other substituents such as bromine (8), carboxamide (9), and
benzoylamine (10) also improved potency relative to
compound 1. However, not all substituents at C-3′ were
found to increase potency, as is apparent with butanoylamine
11.
RESULTS AND DISCUSSION
■
We conducted a high-throughput screening campaign
searching for inhibitors of G2019S-LRRK2 kinase activity on
a 50K compound subset of the Aptuit library that was
prioritized based on ligand efficiency.9 This set had generally
lower molecular weight and better CNS multiparameter
optimization (MPO) scores10 as compared with the overall
collection. Compounds that met a specified potency threshold
in the primary screen were progressed to IC50 determination in
an 11-point biochemical concentration−response assay against
both wild-type (WT) and G2019S-LRRK2 to confirm potency
and assess selectivity. Out of this effort emerged indazole 1, the
single hit that exhibited a preference for inhibiting the G2019S
mutant protein over WT protein by >10-fold.
Lacking an X-ray crystal structure of LRRK2, we constructed
a proprietary homology model of the kinase domain based on
chimeric Aurora kinase A crystal structures where point
mutations were introduced to mimic the LRRK2 hinge and
glycine-rich loop regions combined. Mutated residues in the
chimera included L210M, Y212L, P214S, and L215K in the
hinge region and R137Q, P138L, K141D, K143S, and N146S
in the glycine-rich loop. The homology model based on this
chimera was then optimized to accommodate SAR observa-
We also varied the methyl substituents on the dihydrote-
trazolopyrimidine during our initial investigations. Removal of
the C-5 methyl as in compound 12 completely abolished
activity on both WT and G2019S-LRRK2. Single carbon
B
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Table 1. Biochemical Inhibitory Potency and Selectivity of C-7-Unsubstituted 4,7-Dihydrotetrazolo[1,5-a]pyrimidines
a
In vitro biochemical (IVB) potency values are the geometric mean of a minimum of two IC50 values determined in independent experiments. ND
= not determined.
homologation at C-5 (compound 13) also had a detrimental
effect on potency, as well as the preference for G2019S
inhibition over WT. Similarly, homologation to the N-
ethyltetrazolopyrimidine (compound 14) decreased potency;
however, in this case, selectivity was preserved. Though
additional substituents were tried at N-4 (R3), none exhibited
an improved combination of potency and selectivity over the
methyl group found in 1 (cf. compounds 15−18).
In an effort to better understand key interacting elements in
these inhibitors, we examined the role of the amide by
preparing the N-methylamide 19 and thioamide 20 (Figure 2).
N-Methylation of the amide resulted in complete loss of
activity, suggesting a potentially important hydrogen-bonding
Figure 2. Amide substitutions.
interaction with the amide NH occurring within the binding
pocket. Of course, other factors such as a change in molecular
conformational or an unfavorable steric interaction cannot be
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Table 2. Biochemical Inhibitory Potency and Selectivity of C-7-Substituted 4,7-Dihydrotetrazolo[1,5-a]pyrimidines
a
In vitro biochemical (IVB) potency values are the geometric mean of a minimum of two IC50 values determined in independent experiments. ND
= not determined.
Figure 3. Determination of absolute stereochemistry from chiral carboxylic acids. (a) Chiral HPLC separation using an (R,R)-Whelk-O 1 column
(25 cm × 2 cm), 10 μm, eluting with 40:60 n-hexane:EtOH.
ruled out. Since thioamides are known to be weaker hydrogen
bond acceptors than amides,12 the lack of any reduction in
activity observed for compound 20 coupled with maintenance
of selectivity for G2019S-LRRK2 inhibition suggested that the
carbonyl oxygen is not involved in critical hydrogen bonding.
We further elaborated the dihydrotetrazolopyrimidine by
incorporating substituents at C-7 (R2 and R3), as shown in
Table 2. Addition of a single methyl group at C-7 of (R)-
configuration, compound 21, improved inhibition of both WT
and G2019S-LRRK2 but most importantly increased the
G2019S over WT selectivity to 43-fold. The (S)-enantiomer,
22, had the opposite effect on both potency and selectivity.
The same pattern with respect to potency was also observed
with compounds 23 and 24. The previously demonstrated
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increases in potency with 3-(pyridin-4-yl) and 3-(2-morpho-
linopyridin-4-yl) indazole were preserved in compounds 25−
27, although biochemical selectivity could not be accurately
determined due to the above-described assay limitations.
The synthesis of compounds 21−27 employed commercially
available, racemic 4,7-dihydro-4,5,7-trimethyltetrazolo[1,5-a]-
pyrimidine-6-carboxylic acid (32), which was separated into its
individual enantiomers by chiral high-performance liquid
chromatography (HPLC). To determine the absolute config-
uration of these inhibitors, we prepared 21 and 22 from the
resolved carboxylic acids. A small-molecule X-ray crystallo-
graphic analysis of the carboxylic acid used to prepare the
eutomer, 21, was obtained and indicated the (R)-stereo-
chemistry assigned to this compound (Figure 3).
Table 3. Biochemical Potency and Selectivity of LRRK2
Inhibitors with Alternative Hinge Binders
We also looked at geminal substitution on the dihydropyr-
imidine, which afforded compounds with greatly increased
preference for inhibition of the G2019S protein. As shown in
Table 2, compound 28 is 108-fold more potent against
G2019S-LRRK2 than WT. The selectivity obtained with
geminal substitution appears to result from decreased affinity
for binding in the WT ATP-binding pocket rather than a
significantly increased affinity for the G2019S ATP-binding
pocket. As before, adding substituents to the indazole, as in
compounds 29−31, boosted the potency of these inhibitors. In
the case of compound 29, methyl substitution at the indazole
C-3 afforded a subnanomolar inhibitor with 190-fold
selectivity. Aryl substituted compounds, 30 and 31, were too
potent in our assay to gauge their biochemical selectivity.
However, selectivity of these two analogues was confirmed by
cellular activity assays (vide infra).
During the course of our investigations, we also evaluated
the effects of replacing the indazole with other known hinge
binders and related bicycles (Table 3). Replacing the indazole
with either indole, as in compound 33, or benzimidazole,
compound 34, resulted in complete loss of activity against both
WT and G2019S-LRRK2. Imidazopyridines 35 and 36 were
also ineffective replacements for indazole (cf. compound 2) as
was indolinone 37. Interesting results were observed with
benzimidazolones 38−40. Analogue 38 exhibited >36-fold
selectivity but lost potency relative to compound 1. Alkylation
of the cyclic urea afforded compounds with quite different
profiles. Analogue 39 lost all activity against both WT and
G2019S-LRRK2, likely due to an unfavorable steric interaction
with the hinge, while analogue 40 was both potent against
G2019S-LRRK2 and very selective. Phthalimide 41 had a
potency and selectivity profile similar to 40, as did
isoindolinone 42. The most successful indazole replacement
was 1-aminoisoquinoline. Compound 43 had equivalent
selectivity compared with compound 21 with a G2019S-
LRRK2 IC50 = 12 nM.
We profiled a number of these inhibitors for potency and
selectivity in cellular assays (Table 4). Inhibition of
phosphorylation at two sites on LRRK2, S935, and S1292
was measured in HEK293 cell lines stably overexpressing
human WT or G2019S-LRRK2. S1292 is an autophosphor-
ylation site that is positively correlated with LRRK2 kinase
activity.13 Although this is not the case with S935, which is
more highly phosphorylated on the inactive fraction of LRRK2
in cells, S935 nonetheless becomes dephosphorylated in
response to inhibitor binding and is therefore a useful
pharmacodynamic marker.14 Inhibitor IC50 values determined
using pS935 and pS1292 on G2019S-LRRK2 were well
correlated. For WT-LRRK2, IC50 values measured on pS935
a
In vitro biochemical (IVB) potency values are the geometric mean of
a minimum of two IC50 values determined in independent
experiments. ND = not determined.
were consistently higher than those measured on pS1292,
resulting in a trend toward greater selectivity on pS935.
Inhibition of LRRK2 phosphotransferase activity to an
endogenously expressed substrate, Rab10, was assessed for
three of the most potent and selective inhibitors (23, 30, and
31) in cell lines overexpressing either WT-LRRK2 or G2019S-
LRRK2 (Table 4). Potency and selectivity in the pThr-Rab10
assay were well aligned with the inhibition of pS935-LRRK2 in
the same cell lines. Compound 31 (EB-42486) exhibited a
WT/G2019S selectivity ratio of >300-fold on pS935-LRRK2
and on the substrate pThr73-Rab10 (Figure 4). In contrast, the
nonselective inhibitor, MLi-2 (Figure 4), showed no
preference for the G2019S-LRRK2 variant. The level of
selectivity exhibited by compound 31 is remarkable, given that
G2019S-LRRK2 differs from WT-LRRK2 by only a single
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Table 4. Cellular Potencies for Inhibition of pS935-LRRK2, pS1292-LRRK2, and pThr73-Rab10
b
c
d
pS935-LRRK2
pS1292-LRRK2
pThr73-Rab10
WT IC50
a
G2019S IC50
ratio WT/
G2019S
WT IC50
a
G2019S IC50
ratio WT/
G2019S
WT IC50
a
G2019S IC50
ratio WT/
G2019S
a
a
a
cmpd
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
4
7
21
23
377
3300
>10 000
5220
(8743)
8.3
21
189
45
157
>53
74.4
242
2180
380
9.2
21
104
19
8
ND
ND
ND
9736
ND
ND
ND
137
ND
ND
ND
71
12
21
20
e
e
46.1 (106)
113 (82)
e
25
26
30
31
1140
65
145 (259)
1060
(2092)
14.7
2.4
1.1 (1.5)
78
27
272
20.9
33.7
117
12.1
1.2
1.0
22
18
34
71
ND
ND
473
3924
ND
ND
4.7
ND
ND
101
344
e
e
e
132 (173)
339 (837)
e
e
3.1 (2.5)
1.7
11.4
e
a
b
Cellular potencies are reported as the geometric mean of a minimum of two IC50 values determined in independent experiments. Potency on
c
pS935-LRRK2 was measured with a fluorescence resonance energy-transfer (FRET)-based assay. pS1292-LRRK2 was measured with a Meso Scale
d
e
Discovery (MSD) electrochemiluminescence assay. pThr73-Rab10 inhibition was measured with an immunoblot-based assay. pS935 IC50 and
selectivity values shown in parentheses were determined with an immunoblot-based assay performed on the same cell lysates that were evaluated in
the pThr73-Rab10 assay. ND = not determined.
Figure 4. Inhibition of pThr73-Rab10 in HEK293 cell lines overexpressing either WT-LRRK2 or G2019S-LRRK2. (A) Compound 31 inhibited
phosphorylation of the LRRK2 substrate, Rab10, 344-fold more potently in the G2019S-LRRK2 overexpression line (IC50 = 11.4 1.1 nM, n = 3)
compared to that in the WT-LRRK2 overexpression line (IC50 = 3,924 1.6 nM, n = 3). (B) MLi-2 showed similar potency on pThr73-Rab10 in
both cell lines (HEK/G2019S-LRRK2 IC50 = 8.5 1.3 nM, n = 3; HEK/WT-LRRK2 IC50 = 12.5 1.1 nM, n = 3). (C) Compound 31 inhibited
pS935-LRRK2 837-fold more potently in the G2019S-LRRK2 line (IC50 = 2.5 1.1 nM, n = 3) compared to that in the WT-LRRK2 line (IC50
=
2,092 1.4 nM, n = 3). (D) MLi-2 showed similar potency on pS935-LRRK2 in both cell lines (HEK/G2019S-LRRK2 IC50 = 2.8 1.2 nM, n = 3;
HEK/WT-LRRK2 IC50 = 3.9 1.1 nM, n = 3). Potency is reported as the geometric mean of IC50 values geometric standard deviation. Error
bars show standard deviation.
amino acid within the ATP-binding pocket. This compound
was also profiled at 100 nM (>500-fold biochemical G2019S
IC50) against a panel of 409 kinases using a commercial, active
site-directed competition binding assay to determine its
kinome selectivity. Even at this high concentration relative to
its on-target potency, compound 31 exhibited impressive
kinome selectivity in this panel, with only six kinases being
highly inhibited: HPK1, JNK1, JNK2, JNK3, TTK, and YSK4.
In general, the compounds described herein were found to
have disappointing in vitro absorption, distribution, metabo-
lism, and excretion (ADME) properties (e.g., low solubility,
substrates for efflux transporters). However, it was desirable to
F
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develop in vitro−in vivo correlations during the early stage of
our explorations, and based on the exceptional potency and
selectivity for G2019S-LRRK2 measured in cellular activity
assays, analogue 31 was selected for pharmacokinetic (PK)
profiling in CD-1 mice (summarized in Table 5). Following a
coupling of aminoindazole 46 and carboxylic acid 49 afforded
inhibitor 3. Compounds 6 and 9−11 were prepared as shown
in Scheme 2 for compound 6. Compound 18 was prepared as
shown in Scheme 3. Ester 48 was treated with allyl bromide
and hydrolyzed to give carboxylic acid 53. Coupling of 53 with
1H-indazol-5-amine followed by deprotection and acylation
gave 18.
The N-methylamide, 19, and the thioamide, 20, were
prepared as shown in Schemes 4 and 5. Amide bond coupling
between 5-methylamino indazole, 56, and carboxylic acid 49
gave 19 and treatment of compound 1 with Lawesson’s reagent
afforded 20.
Compounds 21−25 were prepared by coupling either (R)-
32 or (S)-32 with commercially available 5-aminoindazoles.
The synthesis of compounds 26 and 27 is shown in Scheme
6. The pinacolborane, 58, was prepared in two steps from 3-
bromo-5-nitro-1H-indazole (44). Suzuki−Miyaura cross-cou-
pling with 4-(4-bromopyridin-2-yl)morpholine followed by
nitro group reduction gave amine 60. Amide bond coupling
with (R)-32 afforded 61. Treatment of 61 with 4 M HCl
successfully removed the SEM protecting group but also
caused racemization of the chiral center. The enantiomeric
pairs, 26 and 27, were then isolated using chiral chromatog-
raphy.
Table 5. Pharmacokinetic Parameters of 31 (EB-42486) in
Fasted CD-1 Mice
a
b
IV administration
(0.5 mg/kg)
oral administration
(2 mg/kg)
parameter
AUC (μM/h)
Cmax (μM)
Tmax (h)
0.528 0.023
0.615 0.029
0.847 0.060
0.299 0.024
0.50 0.0
c
T1/2 (h)
MRT (h)
Cl (mL/min/
kg)
0.87 0.09
1.14 0.12
38.1 1.7
1.85 0.07
2.89 0.11
F (%)
40.1
a
Dosed as a solution of the free base (0.25 mg/mL) in a mixture of
b
dimethyl sulfoxide (DMSO), solutol, and water (5:5:90). Dosed as a
fine suspension of the free base (0.2 mg/mL) in a mixture of DMSO,
c
solutol, and water (5:5:90). Initial concentration. MRT, mean
residence time; F, bioavailability.
single 2 mg/kg oral dose in fasted mice, a mean maximum
plasma concentration of 0.299 μM was measured approx-
imately 30 min post dose. The mean residence time (MRT)
after oral dosing was 2.89 h. The compound exhibited
moderate clearance, which was commensurate with an IV
half-life of 0.87 h, and reasonable oral bioavailability of ∼40%
was observed. Bioanalytical assessment of brain tissue after 2
mg/kg oral dosing showed no compound exposure in the
brain. This observation was consistent with in vitro experi-
ments in MDCKII-MDR1 cells indicating that 31 is subject to
P-glycoprotein-mediated efflux.
Synthetic Chemistry. Compounds 1 and 2 were prepared
by coupling commercially available aminoindazoles with
carboxylic acid 49. Compounds 3−5, 7−8, and 13−17 were
prepared as shown for compound 3 in Scheme 1. Suzuki−
Miyaura cross-coupling of 3-bromo-5-nitroindazole (44) with
4-pyridylboronic acid, followed by reduction of the nitro group
afforded aminoindazole 46. Separately, the Biginelli reaction15
was employed to form tetrazolo[1,5-a]pyrimidine 48, which
was hydrolyzed to give carboxylic acid 49. T3P-mediated
The gem-dimethyl-4,7-dihydrotetrazolopyrimidines 28−31
were prepared as shown for compound 28 in Scheme 7.
Condensation of aminotetrazole with methyl 2-acetyl-3-
methyl-2-butenoate afforded tetrazolopyrimidine 64. N-Meth-
ylation with MeI followed by LiOH hydrolysis gave carboxylic
acid 66, which was coupled with N-(1)-Boc-5-aminoindazole
and deprotected to afford compound 28.
Compounds 33, 34, 37, 38, 39, and 40 were similarly
prepared using commercially available amine and carboxylic
acid (R)-32. Compound 35 was prepared as shown in Scheme
8. Bromination of 2-methyl-5-nitropyridine (68) gave bromide
69. Formation of amine 70 with NH4OH followed by
cyclization with Ac2O and nitro group reduction afforded 3-
methylimidazo[1,5-a]pyridin-6-amine (72), which was then
coupled with carboxylic acid 49 to yield compound 35.
Compound 36 was prepared as shown in Scheme 9 starting
from 4-bromopyridine-2-carbonitrile (73). Grignard addition
followed by concomitant reduction of the imine with NaBH4
gave amine 74. Formation of the formamide and cyclization
a
Scheme 1. Synthesis of Inhibitor 3
a
Reagents: (a) 4-pyridylboronic acid, KOAc, Pd(amphos)Cl2, EtOH, 100 °C; (b) Zn, NH4Cl, EtOH, 80 °C, 56%, two steps; (c) EtOAc,
formaldehyde (37 wt % in H2O), AcOH, EtOH, 120 °C, microwave, 62%; (d) MeI, Cs2CO3, 50 °C; (e) LiOH, EtOH, tetrahydrofuran (THF),
H2O, 55 °C, 59%, two steps; and (f) T3P, EtOAc, Et3N, 15 °C or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), pyridine, 30 °C, 5%.
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a
Scheme 2. Synthesis of Inhibitor 6
a
Reagents: (a) 49, T3P, Et3N, EtOAc, 60 °C, 16%; and (b) (4-sulfamoylphenyl)boronic acid, Pd(PPh3)4, Na2CO3, N,N-dimethylformamide
(DMF), H2O, 100 °C, 16%.
a
Scheme 3. Synthesis of Inhibitor 18
a
Reagents: (a) allyl bromide, NaH, THF, 15 °C, 27%; (b) LiOH·H2O, EtOH, H2O, 15 °C, 66%; (c) 1H-indazol-5-amine, T3P, EtOAc, Et3N, 20
°C, 50%; (d) 1,3-dimethylbarbituric acid, Pd(PPh3)4, DCM, EtOH, 55 °C, 57%; and (e) CH3COCl, Et3N, DCM, 15 °C, 6%.
a
measured in a biochemical and confirmed by several cellular
assays. To the best of our knowledge, this is the first
identification of small molecules that can precisely target a
prominent pathogenic mutation in LRRK2. Though only a
single amino acid within the ATP-binding pocket differentiates
WT from G2019S-LRRK2, selectivity of >200-fold for
biochemical kinase activity and >300-fold for cellular activity
on an endogenously expressed substrate have been demon-
strated. While compounds disclosed herein were found to be
poorly brain-penetrant, the initial SAR emerging from the
investigation of this class of compounds constitutes a
promising starting point for the continued, focused develop-
ment of additional G2019S-LRRK2 selective compounds with
improved pharmacokinetic properties.
Scheme 4. Synthesis of N-Methyl Amide 19
a
Reagents: (a) 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), DMF,
Et3N, 21%.
a
Scheme 5. Synthesis of Thioamide 20
EXPERIMENTAL SECTION
■
Materials and Methods. Reagents and solvents were obtained
from commercial sources and used as supplied. 1H NMR spectra were
recorded on either a Bruker Avance 400, a Bruker Avance III 400, or
an Agilent Direct Drive spectrometer. The spectra were acquired in
the stated solvent, and chemical shifts (δ) are reported in ppm relative
to the residual solvent peak. Chemical shifts are given in parts per
million using conventional abbreviations for designation of major
peaks, e.g., s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of
doublets; dt, doublet of triplets; br, broad. Mass spectrometry was
performed using an ultraperformance liquid chromatography/mass
spectrometry (UPLC/MS) AcquityTM system equipped with a PDA
detector and coupled to a Waters single quadrupole mass
spectrometer. Where thin-layer chromatography (TLC) has been
used, Rf is the distance traveled by the compound divided by the
distance traveled by the solvent on a TLC plate. Chiral SFC
separation was performed using a Thar SFC Prep 80 instrument and a
Phenomenex Lux Cellulose-2 column (250 mm ×25 mm, 10 μm).
Purities of all target compounds were greater than 95% as determined
by HPLC analysis. Compounds were analyzed for and found not to
contain pan assay interference (PAINS) substructural features.
a
Reagents: (a) Lawesson’s reagent, dioxane, 18 h, 100 °C, 3%.
with POCl3 gave imidazopyridine 76. Coupling of 76 with
amide 77 afforded compound 36.
The synthesis of compound 43 is summarized in Scheme 10.
Boc protection of 6-nitroisoquinolin-1-amine (78) followed by
reduction of the nitro group, coupling with carboxylic acid
(R)-32, and deprotection yielded target 43.
CONCLUSIONS
■
Our efforts to find G2019S-LRRK2 selective inhibitors for a
targeted therapeutic approach in a genetically defined PD
patient population have resulted in the discovery of compound
31 (EB-42486). Employing a ligand-based drug design
approach starting from a single HTS hit led to a series of
potent and highly selective G2019S-LRRK2 inhibitors, as
H
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a
Scheme 6. Synthesis of Inhibitors 26 and 27
a
Reagents: (a) SEM-Cl, NaH, DMF, 0 °C - r.t., 60%; (b) bis(pinacolato)diboron, KOAc, 1,4-dioxane, Pd(dppf)Cl2, 100 °C; (c) 4-(4-
bromopyridin-2-yl)morpholine, Cs2CO3, THF, water, Pd(dppf)Cl2, 100 °C, 44%, two steps; (d) NH4Cl, Fe, EtOH, water, 80 °C, 80%; (e) (R)-32,
Et3N, HATU, DMF, 0 °C - r.t., 33%; (f) 4 M HCl, dioxane, THF, r.t., 18%; and (g) chiral chromatography.
a
Scheme 7. Synthesis of Inhibitor 28
a
Reagents: (a) EtOH, 4 Å molecular sieves, reflux, 56%; (b) MeI, Cs2CO3, DMF, 97%; (c) LiOH, THF; (d) N-(1)-Boc-5-aminoindazole, HATU,
Et3N, DMF, r.t.; and (e) trifluoroacetic acid (TFA), DCM, 0 °C - r.t., 4%, three steps.
a
Scheme 8. Synthesis of Inhibitor 35
a
Reagents: (a) 2-methyl-5-nitropyridine, N-bromosuccinimide (NBS), benzoyl peroxide, CCl4, 80 °C, 31%; (b) NH4OH, dioxane, 25 °C; (c)
Ac2O, PTSA, 100 °C, 32%, two steps; (d) H2, 10% Pd/C, MeOH, 25 °C; and (e) 49, HATU, i-Pr2NEt, DMF, 25 °C, 16%, two steps.
All experiments utilizing animals were conducted under protocols
approved by the WuXi Institutional Animal Care and Use Committee
(IACUC).
silica gel chromatography using a 0−10% MeOH/EtOAc gradient
1
eluent to afford 1 as a pale pink solid (4.1 mg, 2%). H NMR (400
MHz, DMSO-d6) δ 12.99 (br s, 1H), 9.99 (s, 1H), 8.14 (s, 1H), 8.04
(s, 1H), 7.44−7.55 (m, 2H), 5.28 (s, 2H), 3.43 (s, 3H), 2.25 (s, 3H).
MS-ESI (m/z) calcd for C14H15N8O [M + H]+: 311.1. Found 311.0.
4 , 5 - D i m e t h y l - N - ( 3 - m e t h y l - 2 H - i n d a z o l -5 -y l ) - 4 , 7 -
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (2). To a sol-
ution of carboxylic acid 49 in 2 mL of DCM were added (COCl)2 (98
mg, 770 μmol) and one drop of DMF. The mixture was stirred at 25
°C for 30 min and concentrated to afford 4,5-dimethyl-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carbonyl chloride as a yellow
solid. Next, a solution of 3-methyl-1H-indazol-5-amine (70 mg,
475.62 μmol) in pyridine (1 mL) was cooled to 0 °C and then 4,5-
N-(1H-Indazol-5-yl)-4,5-dimethyl-4H,7H-[1,2,3,4]tetrazolo[1,5-
a]pyrimidine-6-carboxamide (1). Carboxylic acid 49 (110 mg, 0.43
mmol) was dissolved in 3 mL of DMF. Et3N (119 μL, 0.85 mmol),
1H-indazol-5-amine (113 mg, 0.85 mmol), and HATU (194 mg, 0.51
mmol) were added at 0 °C, and the reaction mixture was stirred at 0
°C for 2 h. The solvent was evaporated, and the residue was taken up
in CH3CN (1 mL with 0.1% formic acid) and then purified by
reversed-phase column chromatography using a 2−10% CH3CN/
H2O (0.1% formic acid) gradient eluent to give a purple solid (22
mg) of insufficient purity. The material was then further purified by
I
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a
Scheme 9. Synthesis of Inhibitor 36
a
Reagents: (a) MeMgBr, NaBH4, THF, MeOH, 25 °C, 94%; (b) ethyl formate, AlMe3, toluene, 90 °C, 10%; (c) POCl3, toluene, 80 °C, 93%; (d)
CDI, NH3·H2O, THF, 20 °C, 5%; and (e) Cs2CO3, Pd2(dba)3, Xantphos, dioxane, 90 °C, 2%.
a
(m, 2H), 7.28 (s, 1H), 7.22 (d, J = 5.3 Hz, 1H), 5.30 (s, 2H), 3.72−
3.77 (m, 4H), 3.50−3.55 (m, 4H), 3.44 (s, 3H), 2.27 (s, 3H). MS-ESI
(m/z) calcd for C23H25N10O2 [M + H]+: 473.2. Found: 473.3.
4,5-Dimethyl-N-(3-(6-morpholinopyrimidin-4-yl)-1H-indazol-5-
yl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (5). A
mixture of 3-(6-morpholinopyrimidin-4-yl)-1H-indazol-5-amine (150
mg, 507 μmol), carboxylic acid 49 (66 mg, 338 μmol), Et3N (103 mg,
1.01 mmol, 141 μL), and T3P (50 wt % in EtOAc, 258 mg, 406 μmol,
241 μL) in DCM (2 mL) was degassed and purged with N2 (3×);
then, the mixture was stirred at 20 °C for 12 h under a N2
atmosphere. The reaction mixture was concentrated under reduced
pressure to remove the solvent. The residue was purified by
preparative HPLC (0.1% TFA) to afford 5 (11 mg, 5%) as a solid,
red TFA salt. 1H NMR (400 MHz, DMSO-d6) δ 13.81 (s, 1H), 10.12
(s, 1H), 8.74−8.69 (m, 2H), 7.74−7.68 (m, 1H), 7.66−7.60 (m, 1H),
7.44−7.40 (m, 1H), 5.30 (s, 2H), 3.76 (d, J = 8.6 Hz, 8H), 3.43 (s,
3H), 2.27 (s, 3H). MS-ESI (m/z) calcd for C22H23N11O2 [M + H]+:
474.2. Found 474.2.
Scheme 10. Synthesis of Inhibitor 43
a
Reagents: (a) (Boc)2O, 4-dimethylaminopyridine (DMAP),
CH3CN, 70 °C, 70%; (b) H2, 10% Pd/C, EtOH, r.t., 97%; (c)
(R)-32, EDCI, DMAP, pyridine, 70 °C, 93%; and (d) TFA, DCM, r.t,
51%.
4,5-Dimethyl-N-(3-(4-sulfamoylphenyl)-1H-indazol-5-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (6). To a sol-
ution of bromoindazole 51 (50.0 mg, 128 μmol) and (4-
sulfamoylphenyl)boronic acid (25.8 mg, 128 μmol) in 2 mL of
DMF and 0.5 mL of H2O were added tetrakis(triphenylphosphine)-
palladium(0) (14.8 mg, 12.9 μmol) and Na2CO3 (41 mg, 385 μmol).
The mixture was stirred at 100 °C for 12 h under a N2 atmosphere.
The reaction mixture was filtered, and the filtrate was concentrated to
give a residue. The residue was purified by preparative HPLC (0.1%
dimethyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carbonyl chloride
(122 mg, 571 μmol) in CH3CN (0.2 mL) was added dropwise to
the solution. The resulting mixture was stirred at 25 °C for 12 h. The
reaction mixture was concentrated under reduced pressure to remove
the solvent. The residue was purified by preparative HPLC (0.1%
TFA) to afford 2 (36.9 mg, 17%) as a solid, pale yellow TFA salt. 1H
NMR (400 MHz, DMSO-d6) δ 12.58 (br s, 1H), 9.95 (s, 1H), 8.08
(s, 1H), 7.46−7.38 (m, 2H), 5.28 (s, 2H), 3.43 (s, 3H), 2.45 (s, 3H),
2.25 (s, 3H). MS-ESI (m/z) calcd for C15H17N8O [M + H]+: 325.1.
Found 325.1.
4,5-Dimethyl-N-(3-(pyridin-4-yl)-1H-indazol-5-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (3). To a sol-
ution of carboxylic acid 49 (100 mg, 512 μmol) in DCM (5 mL) was
added T3P (50 wt % in EtOAc, 0.457 mL, 768 μmol) and Et3N (156
mg, 1.54 mmol) followed by amine 46 (118 mg, 564 μmol). The
reaction mixture was stirred at 15 °C for 12 h and concentrated. The
mixture was purified by preparative HPLC (neutral) and further
purified by additional preparative HPLC (0.1% TFA) to afford 3 (14
mg, 5%) as a light-yellow, solid TFA salt. 1H NMR (400 MHz,
DMSO-d6) δ 13.99 (s, 1H), 10.16 (s, 1H), 8.88 (d, J = 6.0 Hz, 2H),
8.68 (s, 1H), 8.26 (d, J = 6.0 Hz, 2H), 7.59−7.76 (m, 2H), 5.32 (s,
2H), 3.46 (s, 3H), 2.29 (s, 3H). MS-ESI (m/z) calcd for C19H18N9O
[M + H]+: 388.2. Found: 388.1.
1
TFA) to afford 6 (12 mg, 16%) as a solid, white TFA salt. H NMR
(400 MHz, DMSO-d6) δ 13.44 (s, 1H), 10.06 (s, 1H), 8.50 (s, 1H),
8.10 (m, J = 8.6 Hz, 2H), 7.97 (m, J = 8.6 Hz, 2H), 7.61 (s, 2H), 7.42
(s, 2H), 5.30 (s, 2H), 3.44 (s, 3H), 2.25−2.29 (m, 3H). MS-ESI (m/
z) calcd for C20H20N9O3S [M + H]+: 466.1. Found: 466.1.
N-(3-(1H-Benzo[d]imidazol-2-yl)-1H-indazol-5-yl)-4,5-dimethyl-
4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (7). To a
solution of 3-(1H-benzo[d]imidazol-2-yl)-1H-indazol-5-amine (60.0
mg, 240.7 μmol) and carboxylic acid 49 (46.98 mg, 240.7 μmol) in
pyridine (1 mL) was added EDCI (69.21 mg, 361.05 μmol). The
reaction mixture was stirred at 30 °C for 12 h and then concentrated.
The residue was purified by prep-HPLC (neutral) to afford the
1
product (27.6 mg, 25%) as a pale yellow solid. H NMR (400 MHz,
DMSO-d6) δ 13.75−13.47 (m, 1H), 13.11−12.85 (m, 1H), 10.15 (s,
1H), 8.76 (s, 1H), 7.82−7.46 (m, 4H), 7.27−7.17 (m, 2H), 5.33 (s,
2H), 3.44 (s, 3H), 2.29 (s, 3H). MS-ESI (m/z) calcd for C21H19N10O
[M + H]+: 427.2. Found 427.1.
N-(3-Bromo-1H-indazol-5-yl)-4,5-dimethyl-4,7-dihydrotetrazolo-
[1,5-a]pyrimidine-6-carboxamide (8). To a solution of 3-bromo-1H-
indazol-5-amine (30 mg, 141 μmol) and carboxylic acid 49 (28 mg,
141 μmol) in 2 mL of EtOAc were added T3P (50 wt % in EtOAc,
270 mg, 424 μmol, 252 μL) and Et3N (57 mg, 565 μmol, 79 μL). The
mixture was stirred at 60 °C for 12 h. The reaction mixture was then
concentrated under reduced pressure to remove the solvent. The
residue was purified by preparative HPLC (0.1% TFA) to afford 8 (9
4,5-Dimethyl-N-(3-(2-morpholinopyridin-4-yl)-1H-indazol-5-yl)-
4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (4). To a
solution of carboxylic acid 49 (100 mg, 512 μmol) in DCM (3
mL) were added T3P (50 wt % in EtOAc, 424 mg, 666 μmol), Et3N
(156 mg, 1.54 mmol), and 3-(2-morpholinopyridin-4-yl)-1H-indazol-
5-amine (182 mg, 615 mmol). The mixture was stirred at 15 °C for 12
h. The residue was purified by preparative HPLC to afford 4 (41 mg,
1
15%) as a white solid. H NMR (400 MHz, DMSO-d6) δ 13.44 (s,
1H), 10.06 (s, 1H), 8.52 (s, 1H), 8.28 (d, J = 5.1 Hz, 1H), 7.58−7.66
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mg, 15%) as a yellow, solid TFA salt. H NMR (400 MHz, DMSO-
(m, 2H), 5.31 (s, 2H), 3.43 (s, 3H), 2.63 (q, J = 7.60 Hz, 2H), 1.17
(t, J = 7.45 Hz, 3H). MS-ESI (m/z) calcd for C15H17N8O [M + H]+:
325.1. Found 325.3.
d6) δ 13.18−13.54 (m, 1H), 10.09 (s, 1H), 8.08 (s, 1H), 7.51−7.59
(m, 2H), 5.29 (s, 2H), 3.43 (s, 3H), 2.26 (s, 3H). MS-ESI (m/z)
calcd for C14H14BrN8O [M + H]+: 389.0. Found: 389.0.
4-Ethyl-N-(1H-indazol-5-yl)-5-methyl-4,7-dihydrotetrazolo[1,5-
a]pyrimidine-6-carboxamide (14). Ethyl 4-ethyl-5-methyl-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (136 mg, 0.573
mmol) and 1H-indazol-5-amine (153 mg, 1.15 mmol) were added
to anhydrous DMF (1.5 mL) at 0 °C, and HATU (262 mg, 0.688
mmol) and Et3N (160 μL, 1.15 mmol) were added. The mixture was
stirred for 3 h and then allowed to warm to r.t. The temperature was
then increased to 40 °C, and another portion of HATU (131 mg) and
1H-indazol-5-amine (76.5 mg) was added. The reaction was then
stirred at 40 °C for an additional 3 h. Water was added, and the
mixture was extracted with EtOAc. The combined organic phases
were concentrated, and the residue was triturated with water and then
N-(3-Carbamoyl-1H-indazol-5-yl)-4,5-dimethyl-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (9). To a sol-
ution of 5-amino-1H-indazole-3-carboxamide (120 mg, 681 μmol) in
DCM (4 mL) were added T3P (50 wt % in EtOAc, 650 mg, 1.02
mmol, 607 μL) and carboxylic acid 49 (133 mg, 681 μmol). The
reaction mixture was stirred at 15 °C for 30 min and then Et3N (344
mg, 3.41 mmol, 474 μL) was added, and the reaction mixture was
stirred at 15 °C for 12 h. The reaction mixture was concentrated to
give a residue, and the residue was purified by preparative HPLC
1
(0.1% TFA) to afford 9 (23 mg, 7%) as a white, solid TFA salt. H
NMR (400 MHz, DMSO-d6) δ 13.48 (s, 1H), 10.02 (s, 1H), 8.50 (s,
1H), 7.75−7.61 (m, 2H), 7.56 (d, J = 8.8 Hz, 1H), 7.32 (s, 1H), 5.29
(s, 2H), 3.43 (s, 3H), 2.26 (s, 3H). MS-ESI (m/z) calcd for
C15H16N9O2 [M + H]+: 354.13. Found 354.1.
1
with MeOH to afford 14 (53 mg, 29%) as a pale purple solid. H
NMR (400 MHz, DMSO-d6) δ 13.00 (br s, 1H), 10.00 (br s, 1H),
8.15 (br s, 1H), 8.05 (s, 1H), 7.50 (br s, 2H), 5.28 (br s, 2H), 3.92 (d,
J = 7.26 Hz, 2H), 2.26 (s, 3H), 1.24 (t, J = 6.93 Hz, 3H). MS-ESI (m/
z) calcd for C15H17N8O [M + H]+: 325.2. Found 325.3.
N-(3-Benzamido-2H-indazol-5-yl)-4,5-dimethyl-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (10). To a
stirred solution of N-(5-amino-1H-indazol-3-yl)benzamide (100 mg,
396 μmol) and carboxylic acid 49 (77.4 mg, 396 μmol) in 2 mL of
DCM were added T3P (50 wt % in EtOAc, 757 mg, 1.19 mmol, 707
μL) and Et3N (160.45 mg, 1.59 mmol, 220.70 μL). The reaction
mixture was then stirred at 25 °C for 12 h and concentrated. The
residue was purified by preparative HPLC (neutral) to afford 10
(11.51 mg, 6%) as a colorless gum. 1H NMR (400 MHz, DMSO-d6) δ
9.97 (s, 1H) 7.94−8.09 (m, 3H) 7.43−7.62 (m, 5H) 5.24 (s, 2H)
3.39 (s, 3H) 2.21 (s, 3H). MS-ESI (m/z) calcd for C21H20N9O2 [M +
H]+: 430.2. Found 430.2.
N-(3-Butyramido-2H-indazol-5-yl)-4,5-dimethyl-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (11). To a sol-
ution of N-(5-amino-1H-indazol-3-yl)butyramide (70 mg, 320 μmol)
in 5 mL of DCM were added T3P (50 wt % in EtOAc, 408 mg, 641
μmol, 381 μL), carboxylic acid 49 (68.9 mg, 352 μmol), and Et3N
(97.4 mg, 962 μmol, 134 μL). The mixture was stirred at 40 °C for 12
h and concentrated. The residue was dissolved in DMF (2 mL) and
purified by preparative HPLC (0.04% NH4OH/10 mM NH4HCO3)
N-(1H-Indazol-5-yl)-5-methyl-4-[2-(morpholin-4-yl)ethyl]-4H,7H-
[1,2,3,4]tetrazolo[1,5-a]pyrimidine-6-carboxamide (15). 5-Methyl-
4-(2-morpholinoethyl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-car-
boxylic acid (40 mg, 0.12 mmol) was dissolved in 1.5 mL of DMF.
Et3N (32 μL, 0.23 mmol), 1H-indazol-5-amine (18 mg, 0.14 mmol),
and HATU (52 mg, 0.14 mmol) were added at 0 °C, and the reaction
mixture was stirred for 3 h. The temperature was brought to 40 °C,
and additional HATU (52 mg, 0.14 mmol) and 1H-indazol-5-amine
(18 mg, 0.14 mmol) were added. Stirring at 40 °C was continued for
3 h. H2O (15 mL) was added followed by EtOAc (15 mL). The
organic layer was separated, concentrated, and the residue was
purified by reversed-phase chromatography using a 0−10% MeOH/
EtOAc gradient eluent to give a solid, which was further purified on
reversed-phase preparative TLC using the same gradient to afford 15
1
(4.5 mg, 10%) as a white solid. H NMR (400 MHz, DMSO-d6) δ
12.91 (br s, 1H), 10.02 (br s, 1H), 8.14 (s, 1H), 8.04 (s, 1H), 7.41−
7.57 (m, 2H), 5.27 (s, 2H), 3.97 (t, J = 6.7 Hz, 2H), 3.52 (t, J = 4.4
Hz, 4H), 2.56 (t, J = 6.7 Hz, 2H), 2.41−2.48 (m, 4H), 2.26 (s, 3H).
MS-ESI (m/z) calcd for C19H23N9O2 [M + H]+: 410.2. Found 410.3.
N6-(1H-Indazol-5-yl)-N4,N4,5-trimethyltetrazolo[1,5-a]-
pyrimidine-4,6(7H)-dicarboxamide (16). To a solution of 4-
(dimethylcarbamoyl)-5-methyl-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carboxylic acid (85.0 mg, 337 μmol) and 1H-indazol-5-
amine (45.0 mg, 337 μmol) in DCM (5 mL) were added T3P (50 wt
% in EtOAc, 322 mg, 506 μmol, 301 μL) and Et3N (102 mg, 1.01
mmol, 141 μL). The mixture was stirred at 15 °C for 2 h and
concentrated to give a residue. The residue was purified by
preparative HPLC (with 0.1% TFA) to afford 16 (38 mg, 23%) as
a light pink, solid TFA salt. 1H NMR (400 MHz, CD3OD) δ 8.14 (s,
1H), 8.05 (s, 1H), 7.58−7.49 (m, 2H), 5.32 (s, 2H), 3.17 (s, 3H),
3.14 (s, 3H), 2.24 (s, 3H). MS-ESI (m/z) calcd for C16H18N9O2 [M +
H]+: 368.15. Found: 368.2.
1
to afford 11 (14.5 mg, 11%) as a yellow gum. H NMR (400 MHz,
DMSO-d6) δ 12.60 (s, 1H), 10.22 (s, 1H), 9.97 (s, 1H), 7.99 (s, 1H),
7.55 (br d, J = 8.68 Hz, 1H) 7.40 (d, J = 8.93 Hz, 1H), 5.28 (s, 2H),
3.43 (s, 3H), 2.34−2.40 (m, 2H), 2.25 (s, 3H), 1.66 (sxt, J = 7.29 Hz,
2H), 0.97 (t, J = 7.34 Hz, 3H). MS-ESI (m/z) calcd for C18H22N9O2
[M + H]+: 396.2. Found 396.1.
N-(1H-Indazol-5-yl)-4-methyl-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carboxamide (12). To a solution of 4-methyl-7H-
tetrazolo[1,5-a]pyrimidine-6-carboxylic acid (90 mg, 496.82 μmol)
and 1H-indazol-5-amine (66.15 mg, 496.82 μmol) in 2 mL of DMF
were added DIEA (192.63 mg, 1.49 mmol, 259.61 μL), EDCI (142.86
mg, 745.23 μmol), and HOBt (100.70 mg, 745.23 μmol). The
mixture was stirred at 15 °C for 15 h. The mixture was then purified
by preparative HPLC to afford 12 (37.45 mg, 18%) as a gray, solid
TFA salt. 1H NMR (400 MHz, DMSO-d6) δ 13.01 (br s, 1H), 9.63 (s,
1H), 8.09 (s, 1H), 8.03 (s, 1H), 7.60 (s, 1H), 7.50 (d, J = 0.7 Hz,
2H), 5.26 (s, 2H), 3.43 (s, 3H). MS-ESI (m/z) calcd for C13H13N8O
[M + H]+: 297.1. Found 297.1.
N-(1H-Indazol-5-yl)-5-methyl-4-phenyl-4,7-dihydrotetrazolo-
[1,5-a]pyrimidine-6-carboxamide (17). To a solution of 5-methyl-4-
phenyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylic acid (130
mg, 505 μmol) and 1H-indazol-5-amine (81 mg, 606 μmol) in EtOAc
(2 mL) were added T3P (50 wt % in EtOAc, 965 mg, 1.52 mmol)
and Et3N (204 mg, 2.02 mmol). The mixture was stirred at 60 °C for
12 h and concentrated to give a residue. The residue was purified by
preparative HPLC (with 0.1% TFA) to afford 17 (31 mg, 16%) as a
5-Ethyl-N-(1H-indazol-5-yl)-4-methyl-4H,7H-[1,2,3,4]tetrazolo-
[1,5-a]pyrimidine-6-carboxamide (13). A solution of 5-ethyl-4-
methyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylic acid (34
mg, 0.16 mmol) and 1H-indazol-5-amine (43 mg, 0.32 mmol) in
1.5 mL of anhydrous DMF was stirred at 0 °C. HATU (74 mg, 0.2
mmol) was then added followed by Et3N (45 μL, 0.32 mmol). The
reaction mixture was stirred for 30 min at 0 °C and concentrated to
give a residue. The residue was taken up in CH3CN (with 0.1% TFA)
and purified by reversed-phase chromatography using a 2−10%
CH3CN/H20 (0.1% formic acid) gradient eluent to afford 13 as a red
solid of insufficient purity. The compound was then repurified on
silica gel using a 0−20% MeOH/EtOAc gradient eluent to give 13
1
red, gummy TFA salt. H NMR (400 MHz, DMSO-d6) δ 10.07 (s,
1H), 8.16 (s, 1H), 8.05 (s, 1H), 7.43−7.61 (m, 7H) 5.41 (s, 2H),
1.88 (s, 3H). MS-ESI (m/z) calcd for C19H18N8O [M + H]+: 373.14.
Found: 373.1.
4-Acetyl-N-(1H-indazol-5-yl)-5-methyl-4,7-dihydrotetrazolo[1,5-
a]pyrimidine-6-carboxamide (18). To a solution of N-(2H-indazol-
5-yl)-5-methyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide
(50 mg, 169 μmol) and Et3N (34.2 mg, 337 μmol, 47.0 μL) in DCM
(6 mL) was added acetyl chloride (19.9 mg, 253 μmol, 18.1 μL) at 15
°C and the reaction mixture was stirred for 0.5 h. The reaction
1
(32 mg, 62%) as a white solid. H NMR (400 MHz, DMSO-d6) δ
13.00 (br s, 1H), 9.97 (s, 1H), 8.15 (s, 1H), 8.04 (s, 1H), 7.33−7.70
K
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mixture was concentrated under reduced pressure to give a residue.
The residue was purified by preparative HPLC to give 18 (4.1 mg,
6%) as a gummy, yellow TFA salt. 1H NMR (400 MHz, DMSO-d6) δ
13.01 (br s, 1H), 10.41 (s, 1H), 8.18 (s, 1H), 8.06 (s, 1H), 7.55−7.46
(m, 2H), 5.22 (d, J = 1.3 Hz, 2H), 2.59 (s, 3H), 2.33−2.30 (m, 3H).
MS-ESI (m/z) calcd for C15H15N8O2 [M + H]+: 339.1. Found 339.1.
N-(1H-Indazol-5-yl)-N,4,5-trimethyl-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carboxamide (19). Carboxylic acid 49 (70 mg, 0.36
mmol) and N-methyl-1H-indazol-5-amine (56) (79 mg, 0.54 mmol)
were dissolved in anhydrous DMF (2 mL). Then, the solution was
cooled to 0 °C with an ice−water bath and Et3N (0.1 mL, 0.72 mmol)
and HATU (164 mg, 0.43 mmol) were added. The mixture was
stirred at 0 °C for 30 min and then at r.t. overnight. The solution was
loaded directly on a C18 column and purified by reversed-phase
chromatography using a 5−30% CH3CN/H2O (0.1% formic acid)
gradient eluent. The purest fractions were combined, evaporated
under reduced pressure, and subjected to an additional purification by
silica gel chromatography using a 0−20% MeOH/EtOAc gradient
acid (R)-32 (35 mg, 0.17 mmol) and 3-methyl-1H-indazol-5-amine
(48 mg, 0.33 mmol) were dissolved in 2 mL of anhydrous DMF. The
solution was then cooled to 0 °C in an ice−water bath, and Et3N
(0.05 mL, 0.33 mmol) and HATU (76 mg, 0.20 mmol) were added.
The reaction mixture was stirred at 0 °C for 5 min and then at r.t. for
6 h. The material was purified by reversed-phase chromatography
using a 5−25% CH3CN/H2O (0.1% formic acid) gradient eluent.
Product-containing fractions were combined and further purified by
silica gel column chromatography using a 0−10% MeOH/EtOAc
1
gradient eluent to afford 23 (33 mg, 57%) as a white solid. H NMR
(400 MHz, DMSO-d6) δ 12.58 (s, 1H), 10.15 (s, 1H), 8.11 (s, 1H),
7.43 (s, 2H), 5.74 (d, J = 6.16 Hz, 1H), 3.43 (s, 3H), 2.47 (s, 3H),
2.20 (d, J = 0.88 Hz, 3H), 1.57 (d, J = 6.38 Hz, 3H). MS-ESI (m/z)
calcd for C16H19N8O [M + H]+: 339.3. Found 339.2.
(S)-4,5,7-Trimethyl-N-(3-methyl-1H-indazol-5-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (24). To a
stirred solution of methyl 4,5,7-trimethyl-7H-tetrazolo[1,5-a]-
pyrimidine-6-carboxylate (25 g, 112 mmol) in 1 L of toluene were
added 3-methyl-1H-indazol-5-amine (14.67 g, 99.67 mmol) and
AlMe3 (2 M, 112 mL). The reaction mixture was then stirred at 120
°C for 12 h. After cooling to 0 °C, the reaction mixture was quenched
with saturated aqueous potassium sodium tartrate (1 L) and filtered.
The solid obtained was washed with water (2 L) and petroleum ether
(2 L) and then concentrated. This process was repeated an additional
3×, and material from all four batches was combined and purified by
silica gel chromatography using a 0.5−1.7% MeOH/DCM gradient
eluent to give the racemic product (60 g, 40%) as an off-white solid.
Thirty grams of the solid was dissolved in a mixture of MeOH (1L),
CH3CN (2 L), i-PrOH (3 L), and CHCl3 (0.5 L) and stirred at 20 °C
for 0.5 h. The mixture was separated by SFC to give 24 (14 g, 46%) as
1
eluent to afford 19 (25 mg, 21%) as a white solid. H NMR (400
MHz, DMSO-d6) δ 13.15 (br s, 1H), 8.06 (s, 1H), 7.77 (d, J = 1.54
Hz, 1H), 7.52 (d, J = 8.80 Hz, 1H), 7.34 (dd, J = 8.80, 1.98 Hz, 1H),
4.94 (br s, 2H), 3.31 (s, 3H), 3.19 (s, 3H), 1.96 (s, 3H). MS-ESI (m/
z) calcd for C15H17N8O [M + H]+: 325.2. Found 325.2.
N-(1H-Indazol-5-yl)-4,5-dimethyl-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carbothioamide (20). Lawesson’s reagent (782 mg,
1.93 mmol) was added to a solution of compound 1 (300 mg, 0.95
mmol) in 8 mL of anhydrous dioxane. The solution was stirred at 100
°C for 2 h. Additional Lawesson’s reagent (782 mg, 1.93 mmol) was
added, and stirring was continued at 100 °C for 18 h. The solvent was
removed to afford the crude product that was purified by preparative
HPLC to afford 108 mg of 20, which was insufficiently pure (∼80%).
Thirty milligrams of this material was taken up in DMSO and further
purified by reversed-phase chromatography using a 0−50% CH3CN/
H2O (0.1% formic acid) gradient as eluent to give 20 (10 mg, 3%) as
1
an off-white solid. H NMR (400 MHz, DMSO-d6) δ 12.58 (s, 1H),
10.15 (s, 1H), 8.10 (s, 1H), 7.42 (s, 2H), 5.73 (q, J = 6.0 Hz, 1H),
3.42 (s, 3H), 2.47−2.43 (m, 3H), 2.19 (s, 3H), 1.56 (d, J = 6.4 Hz,
3H). MS-ESI (m/z) calcd for C16H19N8O [M + H]+: 339.1. Found
339.2.
1
a yellow solid. H NMR (400 MHz, DMSO-d6) δ 13.17 (br s, 1H),
11.80 (s, 1H), 8.38 (s, 1H), 8.13 (s, 1H), 7.49−7.66 (m, 2H), 5.30 (s,
2H), 3.45 (s, 3H), 2.18 (s, 3H). MS-ESI (m/z) calcd for C14H15N8O
[M + H]+: 327.1. Found 327.2.
(R)-4,5,7-Trimethyl-N-(3-(pyridin-4-yl)-1H-indazol-5-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (25). (R)-N-(3-
Bromo-1H-indazol-5-yl)-4,5,7-trimethyl-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carboxamide (50 mg, 0.12 mmol), (pyridin-4-yl)boronic
acid (31 mg, 0.25 mmol), and Na2CO3 (39 mg, 0.37 mmol) were
suspended in 2 mL of DMF and 0.5 mL of water. The mixture was
purged with N2 for 5 min, and then Pd(PPh3)4 (7 mg, 0.006 mmol)
was added. The reaction mixture was stirred at 100 °C for 6 h under
N2, and then it was irradiated using a microwave at 100 °C for 30 min.
The mixture was partitioned between water and EtOAc; the phases
were separated, and the aqueous layer was extracted with EtOAc. The
combined organic layers were washed with water, dried over
anhydrous Na2SO4, and the solvent was removed under reduced
pressure. The material was purified by silica gel chromatography
eluting with a 0−100% EtOAc/cyclohexane gradient followed by a 0−
10% MeOH/EtOAc gradient to afford 25 of insufficient purity. The
material was then further purified by reversed-phase chromatography
using a 0−25% CH3CN/H2O (0.1% formic acid) gradient eluent to
afford 25 (5 mg, 10%) as a yellow solid. 1H NMR (400 MHz, DMSO-
d6) δ 13.58 (br s, 1H), 10.30 (s, 1H), 8.67−8.78 (m, 2H), 8.59 (s,
1H), 7.87−7.97 (m, 2H), 7.64 (s, 2H), 5.62−5.95 (m, 1H), 3.45 (s,
3H), 2.22 (s, 3H), 1.58 (d, J = 6.38 Hz, 3H). MS-ESI (m/z) calcd for
C20H20N9O [M + H]+: 402.2. Found 402.2.
rac-4,5,7-Trimethyl-N-(3-(2-morpholinopyridin-4-yl)-1H-indazol-
5-yl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (26
and 27). To a solution of carboxamide 61 (35 mg, 0.06 mmol) in
2 mL of THF was added 0.5 mL of 4 M HCl in dioxane. The reaction
mixture was stirred at r.t. for 1 h. At this point, reaction monitoring
indicated degradation of the starting material in addition to the
formation of the desired product. The reaction mixture was quenched
with water and extracted with EtOAc. The combined organic layers
were washed with water, dried over Na2SO4, and evaporated to
dryness. The residue was redissolved in THF (2 mL) and tetra-n-
butylammonium fluoride (TBAF, 1 M in THF, 1 mL). The reaction
mixture was stirred at r.t. over the weekend and then at 70 °C for 3 h.
rac-N-(1H-Indazol-5-yl)-4,5,7-trimethyl-4,7-dihydrotetrazolo-
[1,5-a]pyrimidine-6-carboxamide (21 and 22). A solution of
compound 32 (70 mg, 0.34 mmol) and 1H-indazol-5-amine (89
mg, 0.67 mmol) in 2 mL of anhydrous DMF was stirred at 0 °C, and
HATU (153 mg, 0.402 mmol) was added followed by Et3N (94 μL,
0.67 mmol). The resulting mixture was then stirred for 2 h at r.t.
Water (15 mL) was added, and the mixture was extracted with EtOAc
(15 mL). The organic layer was concentrated, and the residue was
purified by reversed-phase chromatography using a 2−10% CH3CN/
H2O (0.1% formic acid) gradient eluent to give a solid, which was
triturated with MeOH to afford a racemic mixture of compounds 21
and 22 (21 mg, 19%) as a pale purple solid.
The enantiomers were then separated using semipreparative chiral
HPLC (column: Whelk O1 (R,R) (25 cm × 2.0 cm), 10 μm; mobile
phase: n-hexane/EtOH 40/60% v/v; flow rate: 17 mL/min. DAD
detection: 220 nm; loop: 3000 μL). Solubilization: 13 mg in 3 mL of
1:1 hexafluoro-2-propanol/EtOH.
(R)-N-(1H-Indazol-5-yl)-4,5,7-trimethyl-4,7-dihydrotetrazolo[1,5-
1
a]pyrimidine-6-carboxamide (21). Second eluting enantiomer. H
NMR (400 MHz, DMSO-d6) δ 12.99 (br s, 1H), 10.17 (br s, 1H),
8.16 (s, 1H), 8.05 (s, 1H), 7.29−7.63 (m, 2H), 5.74 (d, J = 6.38 Hz,
1H), 3.43 (s, 3H), 2.19 (s, 3H), 1.57 (d, J = 6.16 Hz, 3H). MS-ESI
(m/z) calcd for C15H17N8O [M + H]+: 325.1. Found 325.2.
(S)-N-(1H-Indazol-5-yl)-4,5,7-trimethyl-4,7-dihydrotetrazolo[1,5-
a]pyrimidine-6-carboxamide (22). First eluting enantiomer. 1H
NMR (400 MHz, DMSO-d6) δ 12.99 (br s, 1H), 10.17 (s, 1H),
8.16 (s, 1H), 8.05 (d, J = 0.75 Hz, 1H), 7.43−7.57 (m, 2H), 5.74 (q, J
= 6.02 Hz, 1H), 3.43 (s, 3H), 2.19 (d, J = 1.00 Hz, 3H), 1.57 (d, J =
6.53 Hz, 3H). MS-ESI (m/z) calcd for C15H17N8O [M + H]+: 325.1.
Found 325.2.
(R)-4,5,7-Trimethyl-N-(3-methyl-1H-indazol-5-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (23). Carboxylic
L
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After cooling to r.t., the mixture was diluted with EtOAc, washed with
water, and concentrated. The material was purified by reversed-phase
column chromatography using a 5−25% CH3CN/H2O (0.1% formic
acid) gradient eluent to afford a racemic mixture of 26 and 27 (5 mg,
18%).
The enantiomers were then separated using chiral HPLC (column:
Chiralpak AD-H (25 cm × 2.0 cm), 5 μm; mobile phase: n-hexane/
EtOH/MeOH 70/15/15% v/v; flow rate: 18 mL/min. DAD
detection: 220 nm; loop: 500 μL). Solubilization: 3 mg in 1 mL of
EtOH.
ution of ester 65 (70 mg, 0.3 mmol) and 3-phenyl-1H-indazol-5-
amine (93 mg, 0.44 mmol) in anhydrous toluene (3 mL) was added
Al(CH3)3 (2 M in toluene, 0.44 mL, 0.89 mmol). The reaction
mixture was stirred for 18 h at 90 °C. The reaction was cooled to r.t.,
quenched with water, and extracted with DCM. The organic phase
was passed through a phase separator and concentrated under
reduced pressure. The crude material was purified by reversed-phase
column chromatography using a 5−50% CH3CN/H2O (0.1% formic
acid) gradient eluent to afford material of insufficient purity. The
material was then further purified by chiral semipreparative HPLC
(Chiralcel OD-H column; 25 cm × 2.0 cm) to afford 30 (11 mg, 9%)
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.22 (br s, 1H),
10.32 (s, 1H), 8.51 (s, 1H), 7.93 (d, J = 7.26 Hz, 2H), 7.52−7.63 (m,
4H), 7.38−7.47 (m, 1H), 3.44 (s, 3H), 2.14 (s, 3H), 1.79 (s, 6H).
MS-ESI (m/z) calcd for C22H23N8O [M + H]+: 415.2. Found 415.2.
4,5-Dimethyl-N-(3-methyl-1H-indol-5-yl)-4,7-dihydrotetrazolo-
[1,5-a]pyrimidine-6-carboxamide (33). To a solution of 3-methyl-
1H-indol-5-amine (90 mg, 615 μmol) and carboxylic acid 49 (120
mg, 615 μmol) in 2 mL of pyridine was added EDCI (177 mg, 923
μmol). The mixture was stirred at 25 °C for 4 h and concentrated.
The residue was purified by preparative HPLC to afford 33 (12 mg,
6%) as a pale pink solid. 1H NMR (400 MHz, DMSO-d6) δ 10.68 (br
s, 1H) 9.79 (s, 1H) 7.84 (s, 1H) 7.21−7.30 (m, 2H) 7.10 (s, 1H) 5.27
(s, 2H) 3.43 (s, 3H) 2.25 (s, 3H) 2.23 (s, 3H). MS-ESI (m/z) calcd
for C16H18N7O [M + H]+: 324.1. Found 324.1.
(R)-4,5,7-Trimethyl-N-(3-(2-morpholinopyridin-4-yl)-1H-indazol-
5-yl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (26).
1
Second eluting enantiomer. H NMR (400 MHz, MeOD) δ 8.57
(s, 1H), 8.27 (d, J = 5.28 Hz, 1H), 7.55−7.66 (m, 2H), 7.39 (s, 1H),
7.31−7.36 (m, 1H), 5.76 (q, J = 6.24 Hz, 1H), 3.82−3.93 (m, 4H),
3.57−3.65 (m, 4H), 3.52 (s, 3H), 2.30 (d, J = 1.10 Hz, 3H), 1.70 (d, J
= 6.38 Hz, 3H). MS-ESI (m/z) calcd for C24H27N10O2 [M + H]+:
487.2. Found 487.8.
(S)-4,5,7-Trimethyl-N-(3-(2-morpholinopyridin-4-yl)-1H-indazol-
5-yl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (27).
1
First eluting enantiomer. H NMR (400 MHz, MeOD) δ 8.57 (s,
1H), 8.27 (d, J = 5.28 Hz, 1H), 7.55−7.66 (m, 2H), 7.39 (s, 1H),
7.31−7.36 (m, 1H), 5.76 (q, J = 6.24 Hz, 1H), 3.82−3.93 (m, 4H),
3.57−3.65 (m, 4H), 3.52 (s, 3H), 2.30 (d, J = 1.10 Hz, 3H), 1.70 (d, J
= 6.38 Hz, 3H). MS-ESI (m/z) calcd for C24H27N10O2 [M + H]+:
487.2. Found 487.8.
N - ( 1 H -B e nz o [ d ] i m i d az o l - 5 - y l ) - 4 , 5 - d i m e t hy l -4 , 7 -
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (34). Carboxylic
acid 49 (70 mg, 0.36 mmol) and 1H-benzo[d]imidazol-5-amine (96
mg, 0.72 mmol) were dissolved in anhydrous DMF (2 mL). Then, the
solution was cooled to 0 °C with an ice−water bath and Et3N (0.10
mL, 0.72 mmol) and HATU (164 mg, 0.43 mmol) were added. The
mixture was stirred at 0 °C for 30 min and then at r.t. overnight. The
mixture was then diluted with water and concentrated under reduced
pressure to remove some of the DMF before purifying the material by
reversed-phase chromatography using a 2−15% CH3CN/H2O (0.1%
formic acid) gradient eluent. Product-containing fractions were
combined, evaporated to dryness, and subjected to an additional
purification by reversed-phase chromatography using a 2−25%
CH3CN/H2O (0.1% ammonium hydroxide) gradient eluent to afford
34 (20 mg, 18%). 1H NMR (400 MHz, DMSO-d6) δ 9.77 (br s, 1H)
8.20 (s, 1H) 8.02 (s, 1H) 7.53 (d, J = 8.60 Hz, 1H) 7.31−7.41 (m,
1H) 5.25 (s, 2H) 3.43 (s, 3H) 2.25 (d, J = 1.32 Hz, 3H). MS-ESI (m/
z) calcd for C14H15N8O [M + H]+: 311.1. Found 311.0.
N-(1H-Indazol-5-yl)-4,5,7,7-tetramethyl-4H,7H-[1,2,3,4]-
tetrazolo[1,5-a]pyrimidine-6-carboxamide (28). Carboxamide 67
(7.5 mg, 0.017 mmol) was dissolved in 2 mL of DCM. TFA (0.5 mL)
was added dropwise at 0 °C, and the reaction mixture was stirred at
r.t. for 3 h. The solvent was evaporated under reduced pressure, and
the material was purified by preparative HPLC to afford 28 (2.1 mg,
1
4%, three steps) as a white solid. H NMR (400 MHz, DMSO-d6) δ
8.15 (d, J = 1.10 Hz, 1H), 8.06 (s, 1H), 7.47−7.63 (m, 2H), 3.46−
3.54 (m, 3H), 2.23 (s, 3H), 1.90 (s, 6H). MS-ESI (m/z) calcd for
C16H19N8O [M + H]+: 339.2. Found 339.8.
4,5,7,7-Tetramethyl-N-(3-methyl-2H-indazol-5-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (29). To a sol-
ution of methyl ester 65 (50 mg, 339 μmol) and 3-methyl-1H-
indazol-5-amine (100 mg, 421 μmol) in 2 mL of toluene was added
Al(CH3)3 (2 M in toluene, 679.45 μL), and the mixture was stirred at
90 °C for 12 h. The reaction was quenched with MeOH (2 mL), and
then the mixture was concentrated. The residue was purified by
preparative HPLC to afford 29 (20 mg, 16%) as a pale yellow solid.
1H NMR (400 MHz, DMSO-d6) δ 8.08 (s, 1H) 7.46 (s, 2H) 3.49 (s,
4,5-Dimethyl-N-(3-methylimidazo[1,5-a]pyridin-6-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (35). To a sol-
ution of carboxylic acid 49 (65 mg, 333 μmol) in 4 mL of DMF were
added amine 72 (49.0 mg, 333 μmol) and i-Pr2NEt (129 mg, 999
μmol). A solution of HATU (190 mg, 499 μmol) in 1 mL of DMF
was then added to the mixture dropwise at 0 °C. The mixture was
stirred at 0 °C for 1 h and then at 25 °C for 11 h. The reaction
mixture was concentrated and purified by preparative HPLC (neutral
3H) 2.55 (s, 3H) 2.22 (s, 3H) 1.88 (s, 6H). MS-ESI (m/z) calcd for
C17H21N8O [M + H]+: 353.2. Found 353.2.
4,5,7,7-Tetramethyl-N-(3-(2-morpholinopyridin-4-yl)-1H-inda-
zol-5-yl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide
(30). Carboxylic acid 66 (210 mg, 21% pure by NMR, 0.31 mmol
theoretical) and 3-(2-morpholinopyridin-4-yl)-1H-indazol-5-amine
(109 mg, 0.37 mmol) were dissolved in 2.5 mL of anhydrous
DMF. The solution was cooled to 0 °C with an ice−water bath, and
Et3N (87 μL, 0.62 mmol) and HATU (143 mg, 0.38 mmol) were
sequentially added. The mixture was stirred at 0 °C for 5 min and
then at r.t. for 18 h. The mixture was partitioned between EtOAc and
water and extracted. The combined organic layers were dried over
Na2SO4, filtered, and concentrated. The material was purified by
column chromatography (silica gel 110 NH2) using a 0−10% MeOH/
EtOAc gradient eluent to afford material of insufficient purity. The
material was then further purified by chiral semipreparative HPLC
(Chiralcel OD-H column; 25 cm × 2.0 cm) to afford 30 (10 mg, 7%)
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 13.45 (br s, 1H),
10.34 (s, 1H), 8.56 (s, 1H), 8.30 (d, J = 5.06 Hz, 1H), 7.61 (d, J =
1.10 Hz, 2H), 7.29 (s, 1H), 7.23 (dd, J = 5.28, 1.10 Hz, 1H), 3.68−
3.82 (m, 4H), 3.50−3.61 (m, 4H), 3.45 (s, 3H), 2.14 (s, 3H), 1.79 (s,
6H). MS-ESI (m/z) calcd for C25H29N10O2 [M + H]+: 501.2. Found
501.3.
1
condition) to afford 35 (21 mg, 16%, two steps) as a gray solid. H
NMR (400 MHz, DMSO-d6) δ 9.95 (s, 1H) 8.71 (s, 1H) 7.51 (d, J =
10 Hz, 1H) 7.23 (s, 1H) 6.74−6.78 (m, 1H) 5.27 (s, 2H) 3.43 (s,
3H) 2.53 (s, 3H) 2.25 (s, 3H). MS-ESI (m/z) calcd for C15H17N8O
[M + H]+: 325.1. Found 325.0.
4,5-Dimethyl-N-(1-methylimidazo[1,5-a]pyridin-7-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (36). To a sol-
ution of 4,5-dimethyl-7H-tetrazolo[1,5-a]pyrimidine-6-carboxamide
(77) (10 mg, 52 μmol) and 7-bromo-1-methyl-imidazo[1,5-a]-
pyridine (76) (10.9 mg, 51.5 μmol) in 2 mL of dioxane were
added Cs2CO3 (33.6 mg, 103 μmol), Pd2(dba)3 (4.7 mg, 5.2 μmol),
and Xantphos (4.2 mg, 7.2 μmol). The reaction mixture was then
stirred at 90 °C under N2 for 12 h and concentrated to give a residue.
The residue was purified by preparative TLC using 33% EtOH/
EtOAc as eluent followed by preparative HPLC to afford 36 (2.2 mg,
1
2%) as a yellow solid. H NMR (400 MHz, DMSO-d6) δ 9.96 (s,
1H), 8.19 (d, J = 7.5 Hz, 1H), 8.16 (s, 1H), 7.92 (s, 1H), 6.68 (dd, J =
2.0, 7.6 Hz, 1H), 5.27 (s, 2H), 3.44 (s, 3H), 2.35 (s, 3H), 2.25 (s,
4,5,7,7-Tetramethyl-N-(3-phenyl-1H-indazol-5-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (31). To a sol-
M
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3H). MS-ESI (m/z) calcd for C15H17N8O [M + H]+: 325.1. Found
325.1.
mL, 2.3 mmol) in DMF (3.3 mL) at 0 °C was added T3P (50%
solution in DMF, 0.417 mL, 0.688 mmol). The reaction mixture was
stirred at r.t. for 18 h. The mixture was diluted with H2O (20 mL) and
extracted with EtOAc (2 × 20 mL). The organic phases were
combined, dried over Na2SO4, filtered, and then concentrated. The
material was purified by preparative HPLC to afford racemic N-(1,3-
dioxoisoindolin-5-yl)-4,5,7-trimethyl-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carboxamide (29 mg, 0.082 mmol). 24.4 mg of this
racemic mixture was separated by chiral preparative HPLC to afford
4,5-Dimethyl-N-(2-oxoindolin-5-yl)-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carboxamide (37). To a solution of 5-aminoindolin-2-
one (73 mg, 492 μmol) in 2 mL of DCM were added carboxylic acid
49 (80 mg, 410 μmol), T3P (50 wt % in EtOAc, 783 mg, 1.23 mmol),
and Et3N (166 mg, 1.64 mmol, 228 μL). The reaction mixture was
then stirred at 25 °C for 12 h. The mixture was concentrated, washed
with MeOH (3 mL), filtered, and dried under vacuum to afford 37
1
1
(54 mg, 37%) as a white solid. H NMR (400 MHz, DMSO-d6) δ
41 (second eluting enantiomer, 7.2 mg) as a white solid. H NMR
10.32 (s, 1H) 9.83 (s, 1H) 7.53 (s, 1H) 7.37 (br d, J = 8.38 Hz, 1H)
6.75 (d, J = 8.38 Hz, 1H) 5.22 (s, 2H) 3.47 (s, 2H) 3.40 (s, 3H) 2.20
(s, 3H). MS-ESI (m/z) calcd for C15H16N7O2 [M + H]+: 326.1.
Found 326.2.
(400 MHz, DMSO-d6) δ 11.24 (br s, 1H), 10.71 (br s, 1H), 8.19 (d, J
= 1.51 Hz, 1H), 7.93 (dd, J = 8.28, 1.76 Hz, 1H), 7.81 (d, J = 8.28 Hz,
1H), 5.78 (d, J = 6.27 Hz, 1H), 3.45 (s, 3H), 2.20 (d, J = 0.75 Hz,
3H), 1.55 (d, J = 6.27 Hz, 3H). MS-ESI (m/z) calcd for C16H16N7O3
[M + H]+: 354.1. Found 354.2.
4,5-Dimethyl-N-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-
4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (38). To a
solution of 5-amino-1,3-dihydro-2H-benzo[d]imidazol-2-one (76 mg,
512 μmol) in 1 mL of DCM were added carboxylic acid 49 (100 mg,
512 μmol) and T3P (50 wt % in EtOAc, 489 mg, 768 μmol). The
reaction mixture was then stirred at 20 °C for 30 min. Et3N (153 mg,
1.54 mmol, 214 μL) was added, and the reaction mixture was stirred
at 20 °C for 12 h. The reaction mixture was washed with CH3CN (1
mL), saturated aqueous NaHCO3 (1 mL), and H2O (5 mL) and then
(R)-4, 5, 7-Trimethyl-N-(1-oxoisoindolin-5-yl)-4, 7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (42). To a
stirred solution of carboxylic acid (R)-32 (120 mg, 0.573 mmol), 5-
aminoisoindolin-1-one (102 mg, 0.688 mmol), and Et3N (0.32 mL,
2.3 mmol) in DMF (3.3 mL) at 0 °C was added T3P (50% solution in
DMF, 0.417 mL, 0.688 mmol). The reaction mixture was stirred at r.t.
for 18 h. The mixture was diluted with H2O (20 mL) and extracted
with EtOAc (20 mL). The organic phase was dried over Na2SO4,
filtered, and then concentrated. The material was purified by
preparative HPLC. The product-containing fractions were collected
and lyophilized to give racemic 4,5,7-trimethyl-N-(1-oxoisoindolin-5-
yl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (13 mg,
0.038 mmol) as a white solid. 7.34 mg of this racemic mixture was
separated by chiral preparative HPLC to give 42 (second eluting
1
concentrated to afford 38 (28 mg, 16%) as a white solid. H NMR
(400 MHz, DMSO-d6) δ 10.52 (br s, 3H), 7.46 (s, 1H), 7.08 (br d, J
= 7.5 Hz, 1H), 6.84 (br d, J = 7.9 Hz, 1H), 5.24 (br s, 2H), 3.41 (s,
3H), 2.21 (s, 3H). MS-ESI (m/z) calcd for C14H15N8O2 [M + H]+:
327.1. Found 327.2.
(R)-4,5,7-Trimethyl-N-(1-methyl-2-oxo-2,3-dihydro-1H-benzo[d]-
imidazol-5-yl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxa-
mide (39). Carboxylic acid (R)-32 (50 mg, 0.24 mmol) and 5-amino-
1-methyl-1,3-dihydro-2H-benzimidazol-2-one (78 mg, 0.48 mmol)
were dissolved in 2.5 mL of anhydrous DMF. The solution was then
cooled to 0 °C with an ice−water bath, and Et3N (0.07 mL, 0.48
mmol) and HATU (109 mg, 0.29 mmol) were sequentially added.
The mixture was stirred at 0 °C for 5 min and then at r.t. for 18 h.
The mixture was diluted with water (20 mL) and extracted with
EtOAc. The combined organic layers were dried over Na2SO4,
filtered, and concentrated. The material was purified by reversed-
phase column chromatography using a 5−60% CH3CN/H2O (0.1%
formic acid) gradient eluent to afford 39 (32 mg, 37%) as a beige
solid. 1H NMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H), 10.07 (s, 1H),
7.53 (d, J = 1.32 Hz, 1H), 7.17 (dd, J = 8.36, 1.76 Hz, 1H), 7.03 (d, J
= 8.36 Hz, 1H), 5.71 (q, J = 5.87 Hz, 1H), 3.42 (s, 3H), 3.26 (s, 3H),
2.17 (s, 3H), 1.54 (d, J = 6.38 Hz, 3H). MS-ESI (m/z) calcd for
C16H19N8O2 [M + H]+: 355.2. Found 355.2.
1
enantiomer, 1.7 mg, 0.01 mmol) as a white solid. H NMR (400
MHz, DMSO-d6) δ 10.46 (s, 1H), 8.41 (s, 1H), 8.00 (s, 1H), 7.58−
7.68 (m, 2H), 5.75 (q, J = 5.80 Hz, 1H), 4.36 (s, 2H), 3.44 (s, 3H),
2.18 (d, J = 1.00 Hz, 3H), 1.55 (d, J = 6.27 Hz, 3H). MS-ESI (m/z)
calcd for C16H18N7O2 [M + H]+: 340.1. Found 340.2.
(R)-N-(1-Aminoisoquinolin-6-yl)-4,5,7-trimethyl-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (43). Bis-carba-
mate 81 (40 mg, 0.073 mmol) was dissolved in 3 mL of DCM. TFA
(1 mL) was then added to the solution, and the reaction mixture was
stirred for 1.5 h at r.t. Volatiles were removed under reduced pressure,
and the crude material was purified by reversed-phase column
chromatography using a 5−50% CH3CN/H2O (0.1% formic acid)
gradient eluent. The white solid obtained was passed through a strong
cation-exchange column eluting with 1 M NH3 in MeOH to afford 43
1
(13 mg, 51%) as a white solid. H NMR (400 MHz, DMSO-d6) δ
10.39 (s, 1H), 8.07−8.18 (m, 2H), 7.75 (d, J = 5.94 Hz, 1H), 7.55
(dd, J = 9.02, 1.98 Hz, 1H), 6.83 (d, J = 5.72 Hz, 1H), 6.67 (s, 2H),
5.78 (q, J = 6.16 Hz, 1H), 3.45 (s, 3H), 2.20 (d, J = 0.88 Hz, 3H),
1.56 (d, J = 6.38 Hz, 3H). MS-ESI (m/z) calcd for C17H19N8O [M +
H]+: 351.2. Found 351.2.
5-Nitro-3-(pyridin-4-yl)-1H-indazole (45). A mixture of indazole
44 (450 mg, 1.86 mmol), 4-pyridylboronic acid (274 mg, 2.23 mmol),
KOAc (547 mg, 5.58 mmol), and Pd(Amphos)Cl2 (132 mg, 186
μmol, 132 μL) in 6 mL of EtOH and 1.5 mL of H2O was degassed
and purged with N2 (3×). The mixture was then stirred at 100 °C for
16 h under N2. The residue was diluted with 2 M HCl (40 mL) and
EtOAc (20 mL). A yellow solid formed, which was collected and
dried under vacuum to afford 45 (350 mg), which was used without
further purification. MS-ESI (m/z) calcd for C12H9N4O2 [M + H]+:
241.1. Found 240.8.
3-(Pyridin-4-yl)-1H-indazol-5-amine (46). To a solution of 45
(350 mg, 1.46 mmol) in 4 mL of EtOH and 1 mL of H2O were added
Zn (476 mg, 7.29 mmol) and NH4Cl (390 mg, 7.29 mmol), and the
mixture was stirred at 80 °C for 12 h. The mixture was then filtered,
and the solid that was collected was dissolved in 10 mL of DMF and
filtered. The filtrate was concentrated to yield 46 (220 mg, 56% two
steps) as a yellow gum, which was used without further purification.
1H NMR (400 MHz, DMSO-d6) δ 13.19 (br s, 1 H), 8.62 (br d, J = 6
Hz, 2H), 7.90 (br d, J = 6 Hz, 2H), 7.34 (d, J = 9 Hz, 1 H), 7.18 (s,
1H), 6.86 (br d, J = 9 Hz, 1 H), 5.02 (s, 2H). MS-ESI (m/z) calcd for
C12H11N4 [M + H]+: 211.1. Found 211.1.
(R)-4,5,7-Trimethyl-N-(3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]-
imidazol-5-yl)-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxa-
mide (40). 5-Amino-3-methyl-1H-benzimidazol-2-one hydrochloride
(200 mg, 1 mmol) was passed through a strong cation-exchange
column eluting with MeOH followed by 1 M NH3 in MeOH to afford
the free base. The free base (78.mg, 0.48 mmol) and carboxylic acid
(R)-32 (50 mg, 0.24 mmol) were dissolved in 2.5 mL of anhydrous
DMF. The solution was then cooled to 0 °C with an ice−water bath,
and Et3N (70 μL, 0.48 mmol) and HATU (109 mg, 0.29 mmol) were
added. The mixture was stirred at 0 °C for 5 min and then at r.t. for
18 h. The reaction mixture was diluted with water (20 mL) and
extracted with EtOAc. The combined organic layers were dried over
Na2SO4, filtered, and concentrated. The material was purified by
reversed-phase column chromatography using a 5−50% CH3CN/
1
H2O gradient eluent to afford 40 (37 mg, 43%) as a beige solid. H
NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H), 10.09 (s, 1H), 7.54 (d,
J = 1.32 Hz, 1H), 7.13 (dd, J = 8.36, 1.76 Hz, 1H), 6.93 (d, J = 8.36
Hz, 1H), 5.72 (q, J = 6.16 Hz, 1H), 3.43 (s, 3H), 3.26 (s, 3H), 2.18
(d, J = 0.88 Hz, 3H), 1.55 (d, J = 6.38 Hz, 3H). MS-ESI (m/z) calcd
for C16H19N8O2 [M + H]+: 355.2. Found 355.2.
(R)-N-(1,3-Dioxoisoindolin-5-yl)-4,5,7-trimethyl-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (41). To a
stirred solution of carboxylic acid (R)-32 (120 mg, 0.573 mmol), 5-
aminoisoindoline-1,3-dione (111 mg, 0.688 mmol), and TEA (0.32
N
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Ethyl 4,5-Dimethyl-4H,7H-[1,2,3,4]tetrazolo[1,5-a]pyrimidine-6-
carboxylate (48). To a mixture of 5-aminotetrazole monohydrate
(36, 606 mg, 5.88 mmol), formaldehyde aqueous solution (36.5−38%
in H2O; 477 mg, 5.88 mmol), and EtOAc (742 μL, 5.88 mmol) in
EtOH (1.5 mL) was added a catalytic amount of acetic acid (84 μL,
1.5 mmol). The mixture was then heated under microwave irradiation
at 120 °C for 10 min. Volatiles were removed under reduced pressure,
and the residue was purified on Biotage (C18, 30 g cartridge, reverse
phase, H2O/CH3CN as eluent, 95:5−60:40) to afford 48 as a white
4-Allyl-N-(1H-indazol-5-yl)-5-methyl-4,7-dihydrotetrazolo[1,5-
a]pyrimidine-6-carboxamide (54). To a stirred solution of carboxylic
acid 53 (300 mg, 1.36 mmol) in DCM (3 mL) were added 1H-
indazol-5-amine (181 mg, 1.36 mmol) and T3P (50 wt % in EtOAc,
1.29 g, 2.03 mmol), and the reaction mixture was stirred at 20 °C for
0.5 h. Et3N (412 mg, 4.07 mmol) was then added, and the reaction
mixture was stirred at 20 °C for 12 h. The reaction mixture was
concentrated, and the residue was purified by preparative HPLC
(0.04% NH4OH/10 mM NH4HCO3) to give 54 (230 mg, 50%) as a
1
1
purple solid. H NMR (400 MHz, DMSO-d6) δ 13.04 (br s, 1 H),
solid (765 mg, 62%). H NMR (400 MHz, DMSO-d6) δ 10.89 (s,
10.07 (s, 1H), 8.14 (s, 1H), 8.02 (s, 1H), 7.44−7.51 (m, 2H), 5.88−
5.99 (m, 1 H), 5.14−5.25 (m, 2H), 4.51 (br d, J = 4 Hz, 2H), 3.15 (d,
J = 5 Hz, 2H), 2.19 (s, 3H). MS-ESI (m/z) calcd for C16H17N8O [M
+ H]+: 337.1. Found: 337.1.
N-(1H-Indazol-5-yl)-5-methyl-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carboxamide (55). Carboxamide 54 (200 mg, 594.62
μmol), 1,3-dimethylbarbituric acid (186 mg, 1.19 mmol), and
Pd(PPh3)4 (68.7 mg, 59.5 μmol) in 10 mL of DCM and 5 mL of
EtOH were degassed and then heated to 55 °C for 12 h under N2.
After cooling to 20 °C, the reaction mixture was filtered and the
filtrate was concentrated. The residue was purified by prep-HPLC
(0.04% NH4OH/10 mM NH4HCO3) to give 55 (100 mg, 57%) as a
white solid. MS-ESI (m/z) calcd for C13H13N8O [M + H]+: 297.1.
Found: 297.3.
3-Bromo-5-nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-inda-
zole (57). To a solution of 3-bromo-5-nitro-1H-indazole (44) (400
mg, 1.65 mmol) in DMF (7 mL) at 0 °C was added NaH (60% w/w,
79 mg, 1.98 mmol), and the mixture was stirred for 15 min. SEM-Cl
(0.35 mL, 1.98 mmol) was then added, and the reaction mixture was
warmed to r.t. and stirred for 2 h. The reaction was carefully
quenched with a sat. aq. NH4Cl, and the mixture was extracted with
EtOAc. The combined organic extracts were concentrated to dryness
under reduced pressure and purified by silica gel chromatography
using a 0−40% EtOAc/cyclohexane gradient eluent to afford 57 (372
mg, 60%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.66 (d, J =
1.8 Hz, 1H), 8.38 (dd, J = 2.0, 9.2 Hz, 1H), 7.68 (d, J = 9.2 Hz, 1H),
5.76 (s, 2H), 3.68−3.52 (m, 2H), 0.95−0.87 (m, 2H), −0.03 (s, 9H).
MS-ESI (m/z) calcd for C13H19BrN3O3Si [M + H]+: 372.0, 374.0.
Found 372.2, 374.1.
1H) 5.10 (d, J = 0.66 Hz, 2H) 4.14 (q, J = 7.04 Hz, 2H) 2.35 (s, 3H)
1.25 (t, J = 7.04 Hz, 3H). MS-ESI (m/z) calcd for C8H12N5O2 [M +
H]+: 210.1. Found 210.2.
4,5-Dimethyl-4H,7H-[1,2,3,4]tetrazolo[1,5-a]pyrimidine-6-car-
boxylic Acid (49). To a solution of ester 48 (200 mg, 0.96 mmol) in
DMF (4 mL) were added Mel (119 μL, 1.91 mmol) and Cs2CO3
(405 mg, 1.24 mmol), and the mixture was stirred at 50 °C for 1 h.
Cooled H2O (15 mL) was then added, and the mixture was extracted
with EtOAc (15 mL). The organic layer was separated, dried over
Na2SO4, filtered, and concentrated to afford a residue. The residue
was purified by silica gel chromatography using a 0−20% EtOAc/
cyclohexane gradient eluent to give ethyl 4,5-dimethyl-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (95 mg) as a color-
less oil. MS-ESI (m/z) calcd for C9H13N5O2 [M + H]+: 224.1. Found
224.2.
LiOH (54 mg, 1.28 mmol) was added to a solution of ester 48 (95
mg, 0.43 mmol) in an EtOH/THF/H2O mixture (4:1:0.6, 4.15 mL).
The mixture was stirred at 55 °C for 1 h. Subsequently, the mixture
was acidified with 1 M HCl and extracted with DCM (10 mL × 3).
The pH of the aqueous layer was brought to pH = 7 with 1 M NaOH
and extracted with DCM (10 mL). The combined organic layers were
concentrated in vacuo to afford 49 (110 mg, 59%, two steps) as a
1
white solid, which was used without further purification. H NMR
(400 MHz, DMSO-d6) δ 12.55 (br s, 1 H), 5.06 (s, 2H), 3.42 (s, 3H),
2.52 (s, 3H). MS-ESI (m/z) calcd for C9H13N5O2 [M + H]+: 196.1.
Found 196.1.
N-(3-Bromo-1H-indazol-5-yl)-4,5-dimethyl-4,7-dihydrotetrazolo-
[1,5-a]pyrimidine-6-carboxamide (51). To a solution of 3-bromo-
1H-indazol-5-amine (50) (30 mg, 141 μmol) and carboxylic acid 49
(27.6 mg, 141 μmol) in 2 mL of EtOAc were added T3P (50 wt % in
EtOAc, 270 mg, 424 μmol) and Et3N (78.8 μL, 566 μmol). The
mixture was then stirred at 60 °C for 12 h and concentrated. The
residue was purified by preparative HPLC (with 0.1% TFA) to afford
5-Nitro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-indazole (58). Indazole 57 (372
mg, 1.0 mmol), bis(pinacolato)diboron (279 mg, 1.1 mmol), and
KOAc (294 mg, 3.0 mmol) were suspended in 3 mL of 1,4-dioxane.
The mixture was purged with N2 for 5 min, and then Pd(dppf)Cl2 (36
mg, 0.05 mmol) was added. The resulting mixture was heated to 100
°C for 1 h under a N2 atmosphere. The mixture was partitioned
between water and EtOAc and extracted. The combined organic
layers were washed with water, dried over anhydrous Na2SO4, and the
solvent was removed under reduced pressure to afford 58, which was
used without further purification.
1
51 (8.7 mg, 16%) as a yellow solid TFA salt. H NMR (400 MHz,
DMSO-d6) δ 13.18−13.54 (m, 1 H), 10.09 (s, 1H), 8.08 (s, 1H),
7.51−7.59 (m, 2H), 5.29 (s, 2H), 3.43 (s, 3H), 2.26 (s, 3H). MS-ESI
(m/z) calcd for C14H14BrN8O [M + H]+: 389.0, 391.0. Found 389.0,
390.9.
Ethyl 4-Allyl-5-methyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-
carboxylate (52). To a solution of ester 48 (2.0 g, 9.6 mmol) in
THF (15 mL) was added NaH (574 mg, 14.3 mmol, 60% purity) at 0
°C, and the mixture was stirred at 15 °C for 0.5 h. Allyl bromide (1.50
g, 12.4 mmol, 2.20 mL) was then added at 0 °C. The reaction mixture
was stirred at 15 °C for 16 h, quenched with H2O (100 mL) at 0 °C,
and extracted with EtOAc (25 mL × 3). The combined organic layers
were washed with brine (30 mL), dried over Na2SO4, filtered, and
concentrated to give a residue. The residue was purified by silica gel
chromatography using a 0−20% EtOAc/petroleum ether gradient
eluent to give 52 (820 mg, 27%) as a yellow oil. MS-ESI (m/z) calcd
for C11H16N5O2 [M + H]+: 250.1. Found: 250.3.
4-Allyl-5-methyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-car-
boxylic Acid (53). To a solution of compound 52 (820 mg, 2.57
mmol) in EtOH (10 mL) and H2O (10 mL) was added LiOH·H2O
(324 mg, 7.71 mmol). The mixture was stirred at 15 °C for 16 h and
concentrated. The reaction mixture was then acidified with 1 N HCl
to pH = 3. The resulting precipitate was collected by filtration to give
the product (510 mg, 66%) as a white solid, which was used without
further purification. MS-ESI (m/z) calcd for C9H12N5O2 [M + H]+:
222.1. Found: 222.3.
1H NMR (400 MHz, CDCl3) δ 9.05 (d, J = 2.20 Hz, 1 H) 8.32
(dd, J = 9.24, 2.20 Hz, 1 H) 7.72 (d, J = 9.24 Hz, 1 H) 5.89 (s, 2H)
3.54−3.61 (m, 2H) 1.46 (s, 12 H) 0.86−0.91 (m, 2H) −0.05 (s, 9H).
MS-ESI (m/z) calcd for C13H19BrN3O3Si [M+H-C6H10]+: 338.1.
Found 338.2.
4-(4-(5-Nitro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-indazol-3-
yl)pyridin-2-yl)morpholine (59). Boronic ester 58 (crude, 1.0 mmol),
4-(4-bromopyridin-2-yl)morpholine (292 mg, 1.2 mmol), and
Cs2CO3 (977 mg, 3.0 mmol) were suspended in 5 mL of THF and
1 mL of water. The mixture was purged with N2 for 5 min, and then
Pd(dppf)Cl2 (73 mg, 0.10 mmol) was added. The reaction mixture
was stirred at 100 °C for 1 h under N2. The mixture was partitioned
between water and EtOAc and extracted. The combined organic
layers were washed with water, dried over anhydrous Na2SO4, and the
solvent was removed under reduced pressure. The material was
purified by silica gel chromatography using a 0−30% EtOAc/
cyclohexane gradient eluent to afford 59 (200 mg, 44%, two steps) as
a yellow oil. 1H NMR (400 MHz, CDCl3) δ 8.99 (d, J = 2.0 Hz, 1H),
8.44−8.35 (m, 2H), 7.74 (d, J = 9.2 Hz, 1H), 7.24 (dd, J = 1.3, 5.1
Hz, 1H), 7.21 (s, 1H), 5.85 (s, 2H), 3.94−3.88 (m, 4H), 3.70−3.61
O
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(m, 6H), 0.99−0.88 (m, 2H), −0.03 (s, 9H). MS-ESI (m/z) calcd for
C22H30N5O4Si [M + H]+: 456.2. Found 456.4.
(m, 6H). MS-ESI (m/z) calcd for C9H14N5O2 [M + H]+: 224.1.
Found 224.2.
3-(2-Morpholinopyridin-4-yl)-1-((2-(trimethylsilyl)ethoxy)-
methyl)-1H-indazol-5-amine (60). A mixture of nitroindazole 59
(200 mg, 0.44 mmol), NH4Cl (26 mg, 0.48 mmol), and iron powder
(98 mg, 1.8 mmol) in 4 mL of EtOH/H2O (1:1) was stirred at 80 °C
for 30 min. The solids were filtered through a celite pad, washing with
EtOH. Volatiles were removed under vacuum, and the residue was
redissolved in EtOAc. Water was added, and the two phases were
separated. The aqueous layer was extracted with EtOAc, and the
combined organic layers were washed with water, dried over Na2SO4,
and the solvent was removed under reduced pressure to afford 60
tert-Butyl 5-(4,5,7,7-Tetramethyl-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carboxamido)-1H-indazole-1-carboxylate (67). Car-
boxylic acid 66 (60 mg, 0.27 mmol) was dissolved in 2 mL of DMF,
and Et3N (0.075 mL, 0.54 mmol), N-(1)-Boc-5-aminoindazole (53.8
mg, 0.4 mmol), and HATU (103 mg, 0.27 mmol) were added. The
reaction mixture was then stirred at r.t. for 1 h. The solvent was
evaporated, and the residue was extracted with EtOAc and water. The
organic layer was separated, dried over Na2SO4, filtered, and
concentrated. The material was purified by preparative HPLC to
afford 67 (7.5 mg) as a white solid. 1H NMR (400 MHz, DMSO-d6)
δ 8.44 (br s, 1H), 8.04−8.22 (m, 3H), 7.45−7.57 (m, 1H), 3.52 (s,
3H), 1.90−1.99 (m, 3H), 1.72−1.77(m, 9H), 1.59 (s, 6H). MS-ESI
(m/z) calcd for C21H27N8O3 [M + H]+: 439.2. Found 439.8.
2-(Bromomethyl)-5-nitropyridine (69). To a solution of 2-methyl-
5-nitropyridine (68) (5.0 g, 36 mmol) in CCl4 (75 mL) were added
benzoyl peroxide (1.75 g, 7.24 mmol) and NBS (7.09 g, 39.82 mmol).
The mixture was stirred at 80 °C for 12 h and then concentrated. The
material was purified by column chromatography using a 0−10%
EtOAc/petroleum ether gradient eluent to afford 69 (2.5 g, 31%) as a
1
(150 mg, 80%). H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 5.1 Hz,
1H), 7.47 (d, J = 8.8 Hz, 1H), 7.26−7.18 (m, 3H), 6.96 (dd, J = 2.0,
8.8 Hz, 1H), 5.74 (s, 2H), 3.94−3.87 (m, 4H), 3.77 (br s, 2H), 3.66−
3.60 (m, 6H), 0.99−0.85 (m, 2H), −0.04 (s, 9H). MS-ESI (m/z)
calcd for C22H32N5O2Si [M + H]+: 426.2. Found 426.4.
(R)-4,5,7-Trimethyl-N-(3-(2-morpholinopyridin-4-yl)-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-indazol-5-yl)-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamide (61). Carboxylic
acid (R)-32 (35 mg, 0.17 mmol) and amine 60 (71 mg, 0.17 mmol)
were dissolved in 2 mL of anhydrous DMF. Then, the solution was
cooled to 0 °C with an ice−water bath and Et3N (0.05 mL, 0.33
mmol) and HATU (76 mg, 0.20 mmol) were added. The mixture was
stirred at 0 °C for 5 min, at room temperature overnight, heated at 50
°C for 2 h, and then at 70 °C for an additional 2 h. The mixture was
partitioned between water and EtOAc and extracted. The combined
organic layers were washed with water, dried over Na2SO4, filtered,
and the solvent was removed under vacuum. The material was
purified by silica gel chromatography using a 50−100% EtOAc/
cyclohexane gradient eluent to afford material of insufficient purity.
The material was then further purified by silica gel chromatography
(silica gel 110 NH2) using a 0−80% EtOAc/cyclohexane gradient
1
yellow oil. H NMR (400 MHz, CDCl3) δ 9.38 (d, J = 3 Hz, 1 H),
8.49 (dd, J = 9, 3 Hz, 1 H), 7.67 (d, J = 9 Hz, 1 H), 4.61 (s, 2H). MS-
ESI (m/z) calcd for C6H6BrN2O2 [M + H]+: 217.0, 219.0. Found
217.0, 219.0.
(5-Nitropyridin-2-yl)methanamine (70). To a mixture of NH3·
H2O (10 mL) and dioxane (30 mL) was added nitropyridine 69 (2.45
g, 11.3 mmol) in dioxane (10 mL). The resulting mixture was stirred
at 25 °C for 2 h. The reaction mixture was then concentrated to afford
70 (1.70 g) as a brown oil, which was used without further
purification. MS-ESI (m/z) calcd for C6H8N3O2 [M + H]+: 154.1.
Found 154.1.
3-Methyl-6-nitroimidazo[1,5-a]pyridine (71). To a solution of
methanamine 70 (1.70 g, 11 mmol) in Ac2O (30 mL) was added
PTSA (1.91 g, 11.1 mmol). The mixture was stirred at 100 °C for 2 h.
The reaction mixture was cooled to 25 °C, poured into ice water (100
mL), and extracted with EtOAc (50 mL × 3). The combined organic
layers were dried over Na2SO4, filtered, and concentrated. The
material was purified by column chromatography using a 0−50%
EtOAc/petroleum ether gradient eluent to afford 71 (650 mg, 32%,
two steps) as a red solid. 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H),
7.36−7.45 (m, 2H), 7.27−7.35 (m, 1 H), 2.70 (s, 3H). MS-ESI (m/z)
calcd for C8H8N3O2 [M + H]+: 178.1. Found 178.1.
3-Methylimidazo[1,5-a]pyridin-6-amine (72). To a solution of
nitroimidazopyridine 71 (70 mg, 395 μmol) in 60 mL of MeOH was
added 10% Pd/C (110 mg). The mixture was degassed and purged
with H2 (3×) and stirred at 25 °C for 0.5 h under a H2 atmosphere
(15 psi). The reaction mixture was filtered, and the filtrate was
concentrated to afford 72 (50 mg) as a green oil, which was used
without further purification. MS-ESI (m/z) calcd for C8H10N3 [M +
H]+: 148.1. Found 148.3.
1-(4-Bromopyridin-2-yl)ethan-1-amine (74). Into a 100 mL three-
necked, round-bottom flask, purged and maintained with an inert
atmosphere of nitrogen, was placed a solution of MeMgBr (3 M,
16.39 mL) in 25 mL of THF. A solution of 4-bromopyridine-2-
carbonitrile (73) (3 g, 16.39 mmol) in 15 mL of THF was added
dropwise at 25 °C over 30 min. MeOH (10 mL) was then added
dropwise followed by addition of NaBH4 (3.10 g, 81.96 mmol) in five
portions. The resulting mixture was stirred at 25 °C for 2 h and
diluted with EtOAc (40 mL) and H2O (30 mL) at 0 °C. The pH was
brought to 9 with 1 M NaOH, and the solid was removed by filtration.
The filtrate was extracted with EtOAc, and the combined organic
layers were concentrated to afford 74 (3.1 g, 94%) as a yellow oil,
which was used without further purification. MS-ESI (m/z) calcd for
C7H10BrN2 [M + H]+: 201.0, 203.0. Found 201.0, 203.0.
1
eluent to afford 61 (35 mg, 33%). H NMR (400 MHz, CDCl3) δ
8.52 (s, 1H), 8.36 (d, J = 5.1 Hz, 1H), 8.28 (s, 1H), 7.69−7.57 (m,
2H), 7.28−7.26 (m, 1H), 5.81 (s, 2H), 5.61 (q, J = 5.9 Hz, 1H),
3.93−3.83 (m, 4H), 3.70−3.56 (m, 6H), 3.49 (s, 3H), 2.31 (s, 3H),
1.74 (d, J = 6.4 Hz, 3H), 1.02−0.86 (m, 2H), 0.00−0.07 (m, 9H).
MS-ESI (m/z) calcd for C30H41N10O3Si [M + H]+: 617.3. Found
617.4.
Methyl 5,7,7-Trimethyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-
carboxylate (64). A mixture of 5-aminotetrazole monohydrate (63)
(66 mg, 0.64 mmol) and methyl 2-acetyl-3-methyl-2-butenoate16
(62) (100 mg, 0.64 mmol) was heated in 5 mL of EtOH in the
presence of molecular sieves for 4 h at reflux. The reaction was cooled
to r.t., filtered, and concentrated to afford 64 (80 mg, 56%) as a white
1
solid, which was used without further purification. H NMR (400
MHz, DMSO-d6) δ 10.70 (s, 1H), 3.79−3.88 (m, 3H), 2.38−2.48 (m,
3H), 1.94−2.02 (m, 6H). MS-ESI (m/z) calcd for C9H14N5O2 [M +
H]+: 224.1. Found 224.3.
Methyl 4,5,7,7-Tetramethyl-4,7-dihydrotetrazolo[1,5-a]-
pyrimidine-6-carboxylate (65). To a solution of methyl ester 64
(73 mg, 0.33 mmol) in DMF (5 mL) were added MeI (0.12 mL, 2.0
mmol) and Cs2CO3 (699 mg, 2.0 mmol), and the mixture was stirred
at 50 °C for 0.5 h. The solvent was evaporated, and the material was
extracted with water and EtOAc. The organic layer was separated,
dried over Na2SO4, filtered, and concentrated to afford 65 (75 mg,
97%) as a white solid, which was used without further purification. 1H
NMR (400 MHz, DMSO-d6) δ 4.84 (s, 1H), 3.76−3.88 (m, 3H),
3.47−3.58 (m, 3H), 2.21−2.32 (m, 3H), 1.78−1.92 (m, 6H). MS-ESI
(m/z) calcd for C10H16N5O2 [M + H]+: 238.1. Found 238.2.
4,5,7,7-Tetramethyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-
carboxylic Acid (66). To a solution of methyl ester 65 (75 mg, 0.32
mmol) in 2.0 mL of THF was added a solution of LiOH (39 mg, 0.93
mmol) in H2O (2 mL). The mixture was stirred at 50 °C for 15 h.
The solvent was evaporated, and the remaining aqueous solution was
acidified with 1 M HCl to pH = 1, extracted with EtOAc, dried over
Na2SO4, filtered, and evaporated to afford 66 (118 mg) as a colorless
N-(1-(4-Bromopyridin-2-yl)ethyl)formamide (75). To a solution
of 1-(4-bromopyridin-2-yl)ethan-1-amine (74) (3.1 g, 15 mmol) and
ethyl formate (1.14 g, 15.4 mmol) in 30 mL of toluene was added
AlMe3 (2.0 M in toluene, 7.7 mL). The mixture was stirred at 90 °C
for 5 h and quenched by the addition of 15 mL of H2O at 0 °C. The
1
oil, which was of sufficient purity for further elaboration. H NMR
(400 MHz, DMSO-d6) δ 3.34−3.39 (m, 3H), 1.90 (s, 3H), 1.64−1.71
P
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Article
mixture was then concentrated, and the residue was diluted with
MeOH (50 mL) and filtered. The filtrate was concentrated and
purified by silica gel chromatography using a 0−100% EtOAc/
petroleum ether gradient eluent to afford 75 (410 mg, 10%) as a
fluorescence (HTRF) assay. Briefly, a 2 nM nominal concentration of
enzymes was preincubated for 15 min with inhibitors before the
addition of 0.4 μM LRRKtide and 25 μM ATP. Assays were carried
out in a 384-well plate in a final volume of 10 μL. After 1 h
incubation, the reaction was stopped with a LanthaScreen time-
resolved (TR-FRET) Dilution Buffer containing 10 mM ethylenedi-
amine tetraacetic acid (EDTA) and 2 nM Tb-anti-pERM-
(pLRRKtide) Ab, and HTRF signal was read in an Envision reader.
pS935-LRRK2 Cellular Assays. HEK293 cells stably transfected
with human LRRK2 or the G2019S variant of human LRRK2 (HEK/
WT-LRRK2 and HEK/G2019S-LRRK2 lines) were seeded into 384-
well poly-D-lysine-coated plates at a density of 5K cell/well in 50 μL
of DMEM medium supplemented with 1% serum. LRRK2 inhibitors
(0.25 μL) in DMSO were added using an acoustic dispenser. After 2 h
incubation at 37 °C, the medium was removed, and phospho-LRRK2
(Ser935) kit reagents from CisBio were added following the
manufacturer’s instructions. HTRF signal was read using an Envision
reader.
pS1292-LRRK2 Cellular Assays. HEK/WT-LRRK2 or HEK/
G2019S-LRRK2 cells were seeded into 96-well poly-D-lysine-coated
plates at a density of 30−50K cell/well in 100 μL of DMEM medium
supplemented with 1% serum. After addition of 1 μL of test article in
100% DMSO, an additional 100 μL of medium was added to each
well. After 2 h incubation at 37 °C, the medium was removed, and
after washing with phosphate-buffered saline (PBS), Lysis Buffer plus
phosphatase inhibitors were added. After shaking for 1 h at 4 °C,
lysates were transferred into MSD assay plates precoated with mouse
monoclonal anti-LRRK2 antibody at a concentration of 1 μg/mL for
1 h at r.t. Plates were sealed and incubated overnight at 4 °C, with
shaking. The unbound proteins were removed by washing plates with
TBST. Rabbit anti-pS1292 antibody diluted 1:400 in 1% Blocker A/
TBST was added and incubated for 2 h at r.t. with shaking. The
antibody was removed by washing, and MSD Sulfo-Tag labeled
antirabbit diluted 1:500 in 3% Blocker A/TBST was added and
incubated in the dark for 1 h at RT, with shaking. The antibody
solution was removed by washing plates, and Read Buffer was added.
Plates were read immediately in an MSD reader (e.g., MSD Sector
6000).
pThr73-Rab10 and pS935-LRRK2 Cellular Immunoblot-
Based Assays. HEK/WT-LRRK2 or HEK/G2019S-LRRK2 cells
were seeded into 24-well plates (Corning 353047) at 100 000 cells/
well in 500 μL EMEM media containing 1% FBS. Approximately 24 h
after plating, cells were treated with 250 μL EMEM media containing
1% FBS and 3.75 μL test article in 100% DMSO. After a 2 h
incubation, cells were washed with DPBS, and 50 μL of lysis buffer
was added to each well. Lysis buffer contained 1% sodium dodecyl
sulfate (SDS), 25 μM Tris pH 7.4, 137 μM NaCl, and 2.7 μM KCl in
water supplemented with protease inhibitors (Thermo Scientific,
78425) and phosphatase inhibitors (G Biosciences, 786−452; G
Biosciences, 786−782). After incubating the plate on an orbital shaker
for 5 min at 800 rpm at RT, the cell lysates were collected, heated at
65 °C for 10 min, and load dye (Cell signaling, 7722S) was added
according to the manufacturer’s instructions. Levels of pThr73-Rab10,
total Rab10, pS935-LRRK2, and total LRRK2 were quantified from
immunoblots. For pS935 and total LRRK2, samples were diluted 1:30
and 8 μL of sample was separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). For pThr73-Rab10
and total Rab10, 10 μL of sample was separated by SDS-PAGE.
Antibodies were used at the following dilutions: LRRK2 total
antibody (1:10 000, Antibodies Incorporated, cat #75−253), pS935
antibody (1:2000, Abcam, ab133450), rab10 total antibody (1:2000,
United States Biological, 030583), and pRab10 antibody (1:500,
Abcam, ab230261).
1
brown oil. H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 5.2 Hz, 1H),
8.21 (s, 1H), 7.45−7.38 (m, 2H), 7.02−7.00 (m, 1H), 5.21−5.14 (m,
1H), 1.49 (d, J = 6.8 Hz, 3H). MS-ESI (m/z) calcd for C8H10BrN2O
[M + H]+: 229.0, 231.0. Found 228.9, 230.8.
7-Bromo-1-methylimidazo[1,5-a]pyridine (76). To a solution of
N-(1-(4-bromopyridin-2-yl)ethyl)formamide (75) (410 mg, 1.8
mmol) in 4 mL of toluene was added POCl3 (549 mg, 3.58 mmol,
333 μL) under N2, and the mixture was stirred at 80 °C for 2 h. The
pH was brought to 10 carefully with saturated aqueous Na2CO3 at 0
°C and extracted with EtOAc (30 mL × 3). The combined organic
layers were concentrated in vacuo to afford 76 (350 mg, 93%) as a
brown solid, which was used without further purification. MS-ESI (m/
z) calcd for C8H8BrN2 [M + H]+: 211.0, 213.0. Found 210.9, 213.0.
4,5-Dimethyl-4,7-dihydrotetrazolo[1,5-a]pyrimidine-6-carboxa-
mide (77). To a solution of carboxylic acid 49 (2.0 g, 10 mmol) in 40
mL of THF was added CDI (1.83 g, 11.3 mmol). The mixture was
stirred at 20 °C for 1.5 h and then NH3·H2O (7.18 g, 51.2 mmol, 7.89
mL, 25% purity) was added, and the reaction mixture was stirred at 20
°C for 0.25 h. The reaction mixture was concentrated and purified by
preparative HPLC to give 77 (0.1 g, 5%) as a white solid. MS-ESI (m/
z) calcd for C7H11N6O [M + H]+: 195.1. Found 195.0.
tert-Butyl (tert-Butoxycarbonyl)(6-nitroisoquinolin-1-yl)-
carbamate (79). A suspension of 6-nitroisoquinolin-1-amine (78)
(110 mg, 0.54 mmol), di-tert-butyl dicarbonate (335 mg, 1.53 mmol),
and DMAP (3.5 mg) in 3.0 mL of CH3CN was stirred at 70 °C for 1
h. Volatiles were then removed under reduced pressure, and the
residue was purified by silica gel chromatography using a 0−20%
EtOAc/cyclohexane gradient eluent. Product-containing fractions
were combined to afford 79 (160 mg, 70%) as a pale yellow solid.
1H NMR (400 MHz, CDCl3) δ 8.84 (d, J = 2.0 Hz, 1H), 8.64 (d, J =
5.5 Hz, 1H), 8.41 (dd, J = 2.2, 9.0 Hz, 1H), 8.17 (d, J = 9.2 Hz, 1H),
7.88 (d, J = 5.7 Hz, 1H), 1.36 (s, 18H). MS-ESI (m/z) calcd for
C19H24N3O6 [M + H]+: 390.2. Found 390.2.
tert-Butyl (6-Aminoisoquinolin-1-yl)(tert-butoxycarbonyl)-
carbamate (80). Carbamate 79 (160 mg, 0.41 mmol) was dissolved
in 5.0 mL of EtOH, and 10% Pd/C (50 mg) was added. The mixture
was left to react under H2 (1 atm) at room temperature for 90 min.
The catalyst was then removed by filtration, washing with EtOH. The
filtrate was concentrated and dried under reduced pressure to afford
80 (143 mg, 97%) as an off-white solid. 1H NMR (400 MHz, DMSO-
d6) δ 8.04 (d, J = 5.7 Hz, 1H), 7.50 (d, J = 9.0 Hz, 1H), 7.38 (d, J =
5.7 Hz, 1H), 7.05 (dd, J = 2.1, 8.9 Hz, 1H), 6.78 (d, J = 2.0 Hz, 1H),
6.06 (s, 2H), 1.31 (s, 18H). MS-ESI (m/z) calcd for C19H26N3O4 [M
+ H]+: 360.2. Found 360.4.
tert-Butyl (R)-(tert-Butoxycarbonyl)(6-(4,5,7-trimethyl-4,7-
dihydrotetrazolo[1,5-a]pyrimidine-6-carboxamido)isoquinolin-1-
yl)carbamate (81). Carboxylic acid (R)-32 (55 mg, 0.26 mmol) and
bis-carbamate 80 (114 mg, 0.32 mmol) were dissolved in 0.5 mL of
pyridine (0.5 mL). EDCI (61 mg, 0.32 mmol) and DMAP (3 mg,
0.025 mmol) were then added. The resulting solution was stirred at
70 °C for 16 h. The mixture was diluted with EtOAc and washed with
water (3×) and brine (1×). The organic layer was dried over
anhydrous Na2SO4 and evaporated to dryness under reduced
pressure. The crude material was purified by reversed-phase column
chromatography using a 0−60% CH3CN/H2O gradient eluent (0.1%
formic acid) followed by silica gel chromatography (silica gel 110
NH2) using a 50−100% EtOAc/cyclohexane gradient eluent to afford
81 (43 mg, 29%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ
10.63 (s, 1H), 8.49 (d, J = 1.8 Hz, 1H), 8.33 (d, J = 5.7 Hz, 1H),
7.91−7.77 (m, 3H), 5.81 (q, J = 6.2 Hz, 1H), 3.45 (s, 3H), 2.22 (s,
3H), 1.57 (d, J = 6.4 Hz, 3H), 1.31 (s, 18H). MS-ESI (m/z) calcd for
C27H35N8O5 [M + H]+: 551.3. Found 551.3.
Pharmacokinetics. Following intravenous bolus and oral gavage
administration of compound to fasted male CD-1 mice, blood
samples were collected at 0.25, 0.5, 1, 2, 4, 8, and 12 h from the
saphenous vein into polypropylene tubes at each time point, blood
was transferred to EDTA-K2 tubes, and plasma was prepared by
centrifugation. Brain samples were homogenized with 4 volumes (w/
v) of 15 mM PBS/ACN (2:1). Levels of EB-42486 in plasma and
LRRK2 Biochemical Assays. Biochemical tests were carried out
using purified truncated LRRK2 enzymes (WT and G2019S) and
reagents from LifeTechnologies. Inhibition of LRRKtide substrate
phosphorylation was assessed using a homogeneous time-resolved
Q
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Article
brain samples were determined by liquid chromatography and tandem
mass spectrometry analyses (LC-MS/MS). Plasma PK profile was
analyzed by noncompartmental approaches using Phoenix WinNonlin
6.3 software program.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01243
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01243.
■
sı
ABBREVIATIONS USED
■
CDI, 1,1′-carbonyldiimidazole; Cl, clearance; DMEM, Dul-
becco’s modified Eagle’s medium; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; HPK1, hematopoietic
progenitor kinase 1; IVB, in vitro biochemical; JNK1, c-Jun
N-terminal kinase 1; JNK2, c-Jun N-terminal kinase 2; JNK3,
c-Jun N-terminal kinase 3; LRRK2, leucine-rich repeat kinase
2; MPO, multiparameter optimization; MRT, mean residence
time; ND, not determined; PD, Parkinson’s disease; PTSA, p-
toluenesulfonic acid; T3P, propylphosphonic anhydride; TTK,
dual specificity protein kinase TTK; YSK, serine/threonine-
protein kinase 25
Compound 1 homology model (PDB)
Molecular formula strings (CSV)
Kinase panel for compound 31 (CSV)
Crystal data for compound (R)-32; 1H NMR and HPLC
traces for compounds 1, 4, 7, 19, 20, 21, 22, 23, 25, 26,
30, and 31; and table of geometric mean and geometric
standard deviation of biochemical potency (PDF)
AUTHOR INFORMATION
Corresponding Author
Albert W. Garofalo − ESCAPE Bio, South San Francisco,
California 94080, United States; orcid.org/0000-0002-
1837-417X; Phone: 650-431-0140; Email: garofaloa@
escapebio.com
■
REFERENCES
■
́
́
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Daniele Andreotti − Aptuit, an Evotec Company, Verona
37135, Italy
Claudia Beato − Aptuit, an Evotec Company, Verona 37135,
Italy
Silvia Bernardi − Aptuit, an Evotec Company, Verona 37135,
Italy
Federica Budassi − Aptuit, an Evotec Company, Verona
37135, Italy
Laura Caberlotto − Aptuit, an Evotec Company, Verona
37135, Italy
Peng Gao − WuXi AppTec, Tianjin 300456, P. R. China
Cristiana Griffante − Aptuit, an Evotec Company, Verona
37135, Italy
Xinying Liu − WuXi AppTec, Tianjin 300456, P. R. China
Luisa Mengatto − Aptuit, an Evotec Company, Verona 37135,
Italy
Marco Migliore − Aptuit, an Evotec Company, Verona 37135,
Italy
Fabio Maria Sabbatini − Aptuit, an Evotec Company, Verona
37135, Italy
Anna Sava − Aptuit, an Evotec Company, Verona 37135, Italy
Elena Serra − Aptuit, an Evotec Company, Verona 37135,
Italy
Paolo Vincetti − Aptuit, an Evotec Company, Verona 37135,
Italy
Mingliang Zhang − WuXi AppTec, Tianjin 300456, P. R.
China
Holly Carlisle − ESCAPE Bio, South San Francisco,
California 94080, United States
R
https://dx.doi.org/10.1021/acs.jmedchem.0c01243
J. Med. Chem. XXXX, XXX, XXX−XXX

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pubs.acs.org/jmc
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́
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S
https://dx.doi.org/10.1021/acs.jmedchem.0c01243
J. Med. Chem. XXXX, XXX, XXX−XXX