Discovery, structure - Activity relationship, and biological evaluation of noninhibitory small molecule chaperones of glucocerebrosidase

Article abstract of DOI:10.1021/jm300063b

A major challenge in the field of Gaucher disease has been the development of new therapeutic strategies including molecular chaperones. All previously described chaperones of glucocerebrosidase are enzyme inhibitors, which complicates their clinical development because their chaperone activity must be balanced against the functional inhibition of the enzyme. Using a novel high throughput screening methodology, we identified a chemical series that does not inhibit the enzyme but can still facilitate its translocation to the lysosome as measured by immunostaining of glucocerebrosidase in patient fibroblasts. These compounds provide the basis for the development of a novel approach toward small molecule treatment for patients with Gaucher disease. This article not subject to U.S. Copyright. Published 2012 by the American Chemical Society.

Full text of DOI:10.1021/jm300063b

Article
pubs.acs.org/jmc
Discovery, Structure−Activity Relationship, and Biological Evaluation
of Noninhibitory Small Molecule Chaperones of Glucocerebrosidase
Samarjit Patnaik,† Wei Zheng,† Jae H. Choi,‡ Omid Motabar,‡ Noel Southall,† Wendy Westbroek,‡
Wendy A. Lea,† Arash Velayati,‡ Ehud Goldin,‡ Ellen Sidransky,‡ William Leister,† and Juan J. Marugan*,†
†NIH Chemical Genomic Center, National Center for Advancing Translation Sciences, National Institutes of Health, 9800 Medical
Center Drive, Rockville, Maryland, United States
‡Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892,
United States
S
* Supporting Information
ABSTRACT: A major challenge in the field of Gaucher disease has been
the development of new therapeutic strategies including molecular
chaperones. All previously described chaperones of glucocerebrosidase
are enzyme inhibitors, which complicates their clinical development
because their chaperone activity must be balanced against the functional
inhibition of the enzyme. Using a novel high throughput screening
methodology, we identified a chemical series that does not inhibit the
enzyme but can still facilitate its translocation to the lysosome as
measured by immunostaining of glucocerebrosidase in patient fibroblasts.
These compounds provide the basis for the development of a novel
approach toward small molecule treatment for patients with Gaucher disease.
with side effects that include weight loss, diarrhea, tremors, and
peripheral nerve damage.9 Both ERT and SRT are ineffective
against types 2 and 3 neuronopathic forms of the disease.10
It has been shown that in many lysosomal disorders, like
Gaucher disease, the mutant enzymes often retain catalytic
activity but fold improperly in the endoplasmic reticulum
(ER).11−14 This triggers ER accumulation of the mutant
protein, which is eventually tagged for proteosome degradation
by ubiquitination, avoiding the transport of the enzyme to the
lysosome.15,16 This mechanism has led to the proposal of an
emerging form of therapy with chemical chaperones, small
molecules able to bind and stabilize mutant proteins, facilitating
their folding and eventually increasing their transport to the
lysosome.17−19 Improved trafficking of the mutant protein from
the ER and to the lysosome results in the reduction of lysosome
size and correction of the storage. Small molecule chaperones
may also increase the stability of mutant enzymes toward
degradation in the lysosome.20 Ironically, most small molecule
chaperones for lysosomal storage disorders reported in the
literature are inhibitors of the enzyme, with the majority being
iminosugars.21 Unfortunately iminosugars have poor selectivity
and exhibit a narrow therapeutic window between improving
translocation and inhibiting enzyme activity.22
INTRODUCTION
■
Gaucher disease, the most common of the lipid storage disorders,
results from a deficiency of the lysosomal enzyme glucocere-
brosidase (GCase).1,2 This enzyme catalyzes the hydrolysis of
glucosylceramides (glucocerebrosides) (1, GC, Figure 1) in the
lysosome. In the disease state, macrophages become engorged
with GC, leading to the characteristic appearing Gaucher cell. In
the most common form of Gaucher disease (type 1) clinical
manifestations include enlarged spleen and liver, platelet
deficiency, anemia, and bone disease. Types 2 and 3 Gaucher
disease are neuronopathic forms. Patients with type 2 have a
median life span of 9 months, while type 3 is more variable.
Classified as a rare autosomal recessive disorder, Gaucher disease
affects about 1 in every 60 000 individuals.3 It is especially
prevalent in the Ashkenazi Jewish population where the
frequency is 1:800.4
Approved treatments for this lysosomal storage disease
include enzyme replacement therapy (ERT) and substrate
inhibition therapy (SRT).5 The ERT approach involves long-
term treatment via injection of a recombinant enzyme
(imiglucerase) into patients with type I disease.6 Introduced
by Genzyme Therapeutics in 1994, it is quite effective in
reducing and reversing the clinical symptoms of the disease.
However, the treatment cost is $100000 to $400000 per year.7
SRT is generally indicated for the treatment of adult patients
with mild to moderate type I Gaucher disease for whom ERT is
not a therapeutic option.8 The prescribed drug, an iminosugar
miglustat (4, Figure 2), developed by Actelion Pharmaceuticals
inhibits glucosylceramide synthetase, reducing the production
of GC in the lysosome. SRT with miglustat is often associated
One of the first chaperone molecules reported to increase the
cellular activity of GCase was N-(n-nonyl)deoxynojirimycin
(NN-DNJ, 5, Figure 2).23 Subsequently other imino-
sugar inhibitors have been characterized as pharmacological
Received: January 14, 2012
This article not subject to U.S. Copyright.
Published XXXX by the American Chemical
Society
A
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Figure 1. Chemical reaction catalyzed by glucocerebrosidase (GCase).
Figure 2. Clinically relevant small molecules for Gaucher disease.
Figure 3. Representative chaperone inhibitors discovered at the NIH Chemical Genomics Center.
chaperones of GCase as well.24−28 Out of these, isofagomine
tartrate (6) has received the most attention, with Amicus
Therapeutics advancing it to phase 2 clinical trials.29 While all
enrolled patients experienced an increased level of the target
enzyme (GCase) in white blood cells, clinically meaningful
improvements in key measures of disease were observed in just
one of the 18 patients who completed the study.30 On the basis
of these results, the advancement of isofagomine was halted,
thereby highlighting the difficulty of developing hydrolase
inhibitors as chaperone molecules.
High throughput screens of small molecule libraries have led
to the identification of novel non-iminosugar small molecule
chaperones of GCase (Figure 3).31−33 Compounds 7, 8, and 9
(Figure 3) were the first non-iminosugar GCase chaperone
inhibitors disclosed by the NCGC. The primary screening assay
monitored the hydrolysis of the fluorogenic substrate 4-
methylumbelliferyl β-D-glucopyranoside (4MU-Glc) with puri-
fied wild type (wt) GCase. From a structural viewpoint, non-
iminosugar structures have the potential advantage of reducing
the risk of cross-inhibition of other glycosidases. In subsequent
studies, when the GCase activity was examined with wt enzyme
present in human spleen tissue homogenates, a >10-fold shift in
activity was observed. In fact, inhibitors 7 and 9 were
completely inactive against the N370S variant of the enzyme
in spleen extracts from a Gaucher patient. This was a highly
relevant observation, as the N370S mutation is the most
common mutation observed in Gaucher patients.2 The
chaperone activity of inhibitors 7 and 9 was demonstrated via
a translocation assay (vide infra) in fibroblasts derived from
normal tissue and patients. However, the potency in this assay
was 100−1000 weaker compared to the wt enzymatic activity.
Such differences in activity spurred the development of a novel
high throughput assay that used spleen homogenates from
patients with the N370S/N370S genotype. The new screening
paradigm did not use a disease relevant mutant, but now the
enzyme also operated in the presence of natural activators
present in the spleen. This approach led to the discovery of
non-iminosugar GCase inhibitors represented by 10 and 11
(Figure 3).34 While compound 10 displayed potent inhibition
of the enzyme derived from tissue extracts, there was a
substantial loss of potency in the purified enzyme with
complete inactivity against the N370S mutant (Table 1).35
On the other hand, the chemical series represented by 11
demonstrated enzyme inhibition in all four assays represented
in Table 1.36 Compounds 10 and 11 displayed comparable
chaperone activity to isofagomine at 1 and 10 μM, respectively.
Interestingly, isofagomine, a more potent inhibitor of GCase
(IC50 = 30 nM), shows chaperone activity at micromolar
concentrations in advanced translocation assays. On other
hand, the enantiomeric L-isomer of isofagomine is the first
example of a noncompetitive inhibitor of GCase (Ki = 5.7 μM)
that shows chaperoning activity in patient fibroblasts causing a
1.6-fold increase in specific activity at 500 μM.37 These facts
highlight the difficulty of extrapolating results from activity-
based screening assays to the actual therapeutic potential for
the treatment of lysosomal storage disorders.
B
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activity.43 However, at the outset, it is important to realize that
specific activators of hydrolases are most likely allosteric
binders, as it is not possible to increase the rate of hydro-
lysis by competing for the active site with the substrate. The
binding affinity of such activators and their influence on the
hydrolytic reaction may vary with different GCase conforma-
tions that present themselves as the enzyme accommodates
various substrates and cofactors present under different assay
conditions. Additionally, the capacity of a molecule to modulate
the hydrolytic function of an enzyme does not necessarily
correlate with its capacity to bind, induce, and accelerate folding
into a particular conformation that improves protein trans-
location. Therefore, modulators of enzymatic hydrolysis may
not demonstrate chaperone capacity and vice versa. At the same
time, if a particular chemical series shows both enzymatic
modulation and the capacity to increase translocation, we
expect that compounds showing greater enzymatic modulation
(inhibitors or activators) will also exhibit improved trans-
location capacity. This follows since increments in the AC50
imply higher affinity toward the enzyme, which often correlates
with greater refolding capacity. This rationale was used to drive
the SAR using the activity in the functional hydrolysis assay as a
surrogate measure of affinity toward the enzyme. Subsequently,
more labor-intensive translocation assays with patient fibro-
blasts were performed on select compounds to confirm
chaperone behavior. The following account details the use of
such a strategy to progress the aforementioned set of
pyrazolopyrimidine activators and their development as the
first publicly disclosed noninhibitory small molecule chaper-
ones of GCase. The discussion includes a detailed description
of the SAR, characterization of optimized compounds in
orthogonal assays, and preliminary evaluation of pharmacoki-
netic properties.
Table 1. Inhibitory Profile of GCase Chaperone Inhibitors
Illustrated in Figure 3
a
purified GCase
IC50, μM
spleen homogenate
IC50, μM
compd
wt
N370S
wt
N370S
7
0.40
0.03
0.10
8.9
11
7.9
2.2
3.2
inactive
8
0.56
1.6
3.2−12.6*
inactive
9
10
11
inactive
2.5
0.28
0.36
0.33
0.58
0.71
a
The data represent inhibition of turnover of 4-methylumbelliferyl β-
D-glucopyranoside (4MU-Glc) except where indicated with an asterisk
(∗), where resorufin β-D-glucopyranoside is the substrate.
A potential reason for the discrepancy in activity observed in
assays using the purified enzyme versus the spleen homogenate
could be the presence of additional activating factors in the
tissue. For instance, while in cells GCase activity is modulated
through the binding of the allosteric activators such as saposin
C,38,39 the addition of the bile salt sodium taurocholate is
required to activate the enzyme in the purified enzyme assay.40
Therefore, the variation in inhibitory activity could be due to
differences in binding with the active GCase conformation
induced by detergent versus that induced by saposin C. These
conformational differences could also explain why a chemical
series with the capacity to activate the enzyme has not yet been
identified via conventional screening assays using isolated
enzyme. In fact, the amount of sodium taurocholate added to
the assay is optimized to achieve a maximum signal, and
therefore, further activation by a small molecule cannot be
observed. In contrast, the tissue homogenate conditions do not
require the addition of activating components because natural
activators like saposin C are already present in the homogenate.
Moreover, the Vmax of the N370S mutant enzyme is lower than
that of the wt enzyme.41 These advantages with the new N370S
spleen homogenate screening conditions have led to the
discovery of a series of pyrazolo[1,5-a]pyrimidine-3-carbox-
amides that displayed GCase activation at micromolar
concentrations.42
RESULTS AND DISCUSSION
■
A total of 326 770 compounds were screened using the qHTS
format in titrations from 90 nM to 57 μM final concentration
using spleen homogenate from a Gaucher patient with
genotype N370S/N370S.44,45 From this primary screen, we
identified several series of compounds that modulated the
turnover of the 4MU-Glc substrate. Several iminosugar
molecules, such as isofagomine, previously reported to inhibit
GCase, were also found, validating the tissue homogenate
The development of GCase activators, especially in the
context of chaperone therapy, is ideal because this could lead to
small molecules that not only facilitate the translocation of
mutant GCase to the lysosome but also increase its specific
a
Table 2. GCase Activation Profile with 4MU-Glc as Substrate under Spleen Homogenate and Purified Enzyme Conditions
a
Values are average of N = 2.
C
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Scheme 1. Analogue Synthesis
approach. Further confirmatory efforts, including the ordering
of additional analogues, identified a set of pyrazolopyrimidines
as activators from the screen (Table 2). Additional studies
revealed that the activity in assays using N370S mutant or wild
type spleen homogenate preparations (Table 2) was 3- to 10-
fold better than purified enzyme conditions. Assays using
resorufin β-D-glucopyranoside as the substrate failed to show
consistent activation among lead molecules of this series,
underscoring the importance of proper substrate choice for
assay development and SAR within a particular chemical
series.46 These pyrazolopyrimidines were inactive in the
absence of GCase as well as against α-glucosidase (EC
3.2.1.20) and α-galactosidase (EC 3.2.1.22) in assays following
the turnover of the corresponding 4-MU-α-glucose and 4-MU-
α-galactose substrates (data not shown), confirming selectivity
for GCase.
This discovery of these selective GCase activators prompted
the synthesis of additional analogues in order to improve
activity and to evaluate the key structural features present
within the chemotype critical for activity. The synthetic
sequence (Scheme 1) used to generate the analogues started
with the condensation of a 1,3-diketone 16 with ethyl 3-amino-
1H-pyrazole-4-carboxylate 17 in acetic acid.47,48 Initial efforts to
simplify the evaluation of the amide SAR centered on the use of
acetylacetone as the diketone to eliminate the formation of
regioisomers (R1 = R2 = CH3). With unsymmetrical ketones, a
mixture of regioisomers (18, 19) was formed, which were
typically separated by chromatography. Then ester hydrolysis
provided an acid that was coupled with amines or anilines to
generate compounds (20, 21, Scheme 1) for biological analysis.
Medicinal chemistry efforts with R1 = R2 = CH3 led to the
production of the compounds presented in Table 3. The
subsequent activity tables enumerate the SAR observed toward
an increase in the hydrolysis of 4MU-Glc. The SAR and activity
observed with spleen homogenates derived from controls or
patients with genotype N370S/N370S were comparable, and
thus, for brevity, the N370S data are presented here.
activation of GCase. The N-phenyl derivative 31 prompted an
exploration of substitutions around the phenyl ring.
A simple methyl group at the para position (32) led to a 5-
fold improvement in activity compared to the plain phenyl
derivative, while a moderate improvement was noticed at the
meta position (34). The o-methyl substitution led to loss of
activity in 33 and in the 2,4-dimethylated compound 36 as well.
The 3,4-dimethylated compound 35, as well as its cyclic indane
variant 37, showed reasonable activity, underscoring the
presence of an aliphatic substituent at the para position of
the phenyl ring. This was further substantiated with 38 and 39,
which had AC50 of 3.3 and 2.3 μM with p-ethyl and tert-butyl
groups, respectively. However, submicromolar activity was
achieved by 40 with a terminal alkyne at the para position. The
correlation of para substitution and better activity was seen with
the inductively electron withdrawing aliphatic trifluoromethyl
(41), bromo (43), and chloro (44) groups. However, a simple
pyridyl ring (46) showed complete lack of activity. The same
trend was seen with the electron withdrawing methylsulfone
(47), cyano (48, 49), methyl ketone (50, 51), and acetamido
(52, 53) containing congeners at 3- and/or 4- positions.
The electron donating methoxy group at the para position
resulted in compound 54 with an AC50 of 8.2 μM. However,
the presence of an additional methoxy at one (56) or both meta
positions (55) led to inactive compounds. Dioxolane 57 and
dioxane 58, which have conformational constraints, had similar
activities compared to the p-methoxy compound 54, implying
that the freely rotating m-methoxy group present in 56 was
deleterious toward activity. In conjunction with the p-methoxy
group, the presence of a m-fluoro (59) or m-chloro atom (60)
led to improved potencies of 1.46 and 2.06 μM, respectively.
As the SAR of the amide was being explored, a parallel
approach was taken to evaluate the substitutions at the 5 (R1)
and 7 (R2) positions of the pyrazolo[1,5-a]pyrimidine core
using the chemistry described above (Scheme 1). A phenyl
group at R2 was not tolerated, while a methyl group was. This
was seen consistently in the context of four different substituted
phenyl amides when compared to compounds with R1 = R2 =
CH3 (32/62, 54/63, 44/64, 66/68, Tables 4 and 5). However,
the regioisomers with R1 = Ph and R2 = CH3 retained activity as
observed with the 3,4-dichloro 65, indane70, and 4-ethyl 73
derivatives. The replacement of the R1 methyl with a
cyclopropyl group led to compounds with almost equal
potency (13, 67, 69, and 71). Similarly, the R2 methyl could
be replaced with di- (13, 69, 71, and 72) or trifluoromethyl
(67) substituents to produce equipotent compounds. This SAR
is well suited to ameliorate P450 mediated hepatic metabolism
of the R2 CH3 if it becomes an issue during future in vivo
studies.
Compounds derived from the coupling of 5,7-dimethyl-
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (18 or 19 with R1
= R2 = CH3, Scheme 1) with primary aliphatic amines like
methyl-, isobutyl-, or isopropylamine and pyrrolidine proved to
be inactive (22−25, Table 3). However, the cyclohexylamide
26 had an AC50 of 10 μM, which was further improved upon
the addition of a 4-tert-butyl group to the cyclohexyl core as in
27. The other isomer 28 derived from 4-tert-butylcyclohex-
amine (the cis and trans forms have not been determined yet)
was 2.5-fold less active. While arylmethylamides 29 and 30
were not that active, the amide derived from simple aniline 31
had an AC50 of 8.2 μM. Thus, it appeared that the presence of a
six-membered cyclic aliphatic moiety at R3 was critical for the
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Table 3. GCase Activation of the Glycolysis of 4MU-Glc in
Homogenate Derived from the Spleen of Patients with the
N370S Mutation: Evaluation of the R3 Group
Table 4. GCase Activation of the Glycolysis of 4MU-Glc in
Homogenate Derived from the Spleen of Patients with the
N370S Mutation: Evaluation of the R1 and R2 Groups
a
a
a
AC50 values are average of N = 2.
As noted in the data in Tables 2−4, the nature of enzyme
activation in the newly discovered activator series was
demonstrated by measuring the fluorescence of 4-methyl-
umbelliferone (4MU) released during the hydrolysis of 4MU-
Glc (labeled as HTS in Figure 5 and Figure 4a) . In order to
dispel any notion that the chemical series may be a fluorescent
artifact, it was imperative to demonstrate the activation by an
alternative technique. To that end, an assay was developed
to measure the release of 4-MU by high performance liquid
chromatography−mass spectrometry (labeled as HPLC in
Figure 5).49 Four representative active molecules from this
chemical series (Table 5) were analyzed using this assay, and all
exhibited a dose-dependent activation of substrate turnover.
Thus, the capacity of this series to increase the turnover of
the 4MU-Glc substrate was established by two separate
methods. However, unlike the inhibitor isofagomine (Table 5,
a
AC50 values are average of N = 2. The assignment of relative
stereochemistry of the 4-tert-butylcyclohexanamide is tentative and
deduced from the 1H NMR spectra of the individual racemic
compounds (see Supporting Information for additional details).
E
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Table 5. GCase Activation with Representative Pyrazolopyrimidines and Inhibition with Isofagomine in Homogenates from a
a
N370S/N370S Genotype Gaucher Patient Spleen
a
Activation was measured by monitoring the (a) fluorescence of 4MU released on hydrolysis of 4MU-Glc, (b) release of 4MU from 4MU-Glc with
HPLC and (c) release of C12-BODIPY-Cer from fluorescent C12-BODIPY-GlcCer with HPLC. AC50 values are average of N = 2.
Figure 4. Two GCase assays. (a) “Blue” GCase assay. The profluorescent substrate 4MU-Glc is hydrolyzed to form glucose and 4MU (excitation
peak at 365 nm;emission peak at 440 nm). This assay was used for the primary screen and can be followed by HPLC/MS. (b) “Fluorescent
ceramide” GCase assay. The glucosyl ceramide fluorescent substrate (C12-BODIPY-GlcCer) is hydrolyzed to form glucose and C12-BODIPY-Cer
(excitation peak at 506 nm; emission peak at 540 nm), and their ratio was detected upon separation by HPLC.
increase the translocation of GCase to the lysosome was
evaluated by laser scanning confocal microscopy.50,51 Dermal
fibroblasts derived from a Gaucher patient with genotype
N370S/N370S were incubated for 6 days with compound 40,
or with closely related congeners at three different concen-
trations (1, 5, 25 μM), and the known chaperone isofagomine.
After treatment, cells were fixed and stained with a custom-
made GCase antibody labeled with Alexa555 (Figures 6 and 7,
red), an antibody for the lysosomal marker LAMP-2 labeled
with Alexa488 (Figures 6 and 7, green), and a DAPI stain for
visualization of the nucleus (Figures 6 and 7, blue). In cell-
based translocation assays, compounds able to enhance GCase
trafficking from the ER to the lysosome should increase the
overall GCase amount in the lysosomes, which will result in
Figure 5. Concentration response profiles for 40, 41, 43, 66, and
isofagomine for the assays a and b indicated in the footnote of Table 5.
increased colocalization between GCase and the lysosomal
marker. In this study DMSO, which was the vehicle (Figure 6A),
and isofagomine (Figure 6B) were used as negative and
positive controls. Compound 40 increased the amount of
GCase protein, as evident by the brighter red color (parts C
and D of Figure 6, upper left quadrant) compared to DMSO
vehicle treatment (Figure 6A). Also, the increase in the
intensity of the bright yellow color (parts C and D of Figure 6,
lower right quadrant) indicated that the amount of GCase
protein (red) was increased in the lysosomes (green) after
treatment with compound 40. Furthermore, cells incubated
IC50 = 2.7 μM), these compounds did not show an increase in
substrate turnover in the analogous HPLC assay with the
fluorescent glucosylceramide-like C12-BODIPY-GlcCer sub-
strate (Figure 4b), suggesting that compounds from this series
may not activate the hydrolysis of the natural substrate.
The disparate behavior toward activation of the 4MU-Glc
and not the fluorescent ceramide substrate necessitated an
evaluation of chaperone activity. The capacity of compounds to
F
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Figure 6. Translocation studies of endogenous N370S GCase enzyme in Gaucher N370S/N370S dermal fibroblasts. Fibroblasts were treated for 6
days with (A) DMSO, (B) 1 μM isofagomine, (C) 5 μM 40, (D) 1 μM 40. Cells were stained for LAMP-2 (green), GCase (red), and the nucleus
(DAPI, blue). The bottom right quadrant in each image is an overlay of all stains; bright yellow (overlap of red and green) indicates increased GCase
in the lysosome. All laser settings were held constant across all of the fields imaged. Scale bar shows 50 μm.
Figure 7. Translocation activity of compound 40 (C), isofagomine (B), and DMSO vehicle control (A) in N370S/N370S Gaucher fibroblasts. The
red, green, and blue stains represent GCase, the lysosomal marker LAMP2, and nucleus (DAPI), receptively. All laser settings were constant across
all of the fields imaged. Shown are high magnification images with scale bar indicating 10 μm.
with 1 μM isofagomine (Figure 6B) or compound 40 (parts C
and D of Figure 6) resulted in increased amounts GCase in the
lysosome compared to DMSO vehicle treatment, confirming
that the positive control worked in our assay (Figure 6A). The
high magnification images of treatment with compound 40
(Figure 7C, yellow, bottom right quadrant) clearly showed the
presence of CGase (red) in lysosomes (green), even in the
periphery of the cell.
The translocation experiment proved that our chemical series
exemplified by the pyrazolopyrimidine 40 was able to increase
G
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levels of GCase in N370S/N370S patient fibroblasts. Whether
this translated to an increase in specific activity was yet to be
verified. To that end we incubated 40 with fibroblasts derived
from four different cell lines (control, N370S carrier, N370S/
N370S, L444P/L444P) and measured GCase activity after 6
days (Figure 8). Gratifyingly, irrespective of genotype, an
homogenate conditions. In contrast, the negative control 22
failed to display significant binding within the same titration
range.
By use of these novel GCase noninhibitor chaperones,
ADME studies were performed for the selection of possible
candidates for in vivo studies. The stability of representative
compounds from this chemical series was examined in mouse
liver microsomes. As observed in Table 6, compound 40 and
the closely related p-trifluoro compound 41 displayed good
stability. The permeability of the most potent compound 40
was also analyzed in a standard Caco-2 permeability assay. The
A to B and B to A apparent permeability were determined as
6.0 and 2.0 (10−6 cm s−1). The values of these numbers below
10 × 10−6 cm s−1 reflect possible limitations in solubility. The
efflux ratio of 0.3 suggests that the compound is not recognized
by the ABC transporters expressed in the Caco-2 monolayer
and therefore is expected to have reasonable good oral
absorption and perhaps penetration through the blood−brain
barrier, which is especially relevant for the treatment of types 2
and 3 Gaucher disease.
Following the encouraging potency and in vitro ADME
properties for compound 40, a mouse PK study was initiated. A
50 mpk ip single administration of 40 in male mice resulted in a
Cmax of 1.03 μM in plasma and a half-life of 19.3 h (Table 7,
Figure 10). The total exposure in liver, which is characteristi-
cally enlarged in type 1 Gaucher disease, was quite high (>200
times higher than plasma). The compound crossed the blood−
brain barrier and had ∼3 times the exposure observed in the
plasma. Thus, 40 appeared to reach the target organs affected in
all three forms of Gaucher disease, rendering it an ideal lead
molecule for preliminary in vivo proof-of-principle studies.
Figure 8. GCase activity, as measured by hydrolysis of 4MU-Glc, after
6 days of treatment with 5 μM 40 or no compound (NT = no
treatment) in four fibroblast cell lines (WT = control, WT/370S =
carrier mutation, N370S/N370S = derived from patients homozygous
for N370S mutation, L444P/L444P = derived from patients
homozygous for L444P mutation).
increase in activity was observed at 5 μM across all lines compared
to control (NT = no treatment) without any evidence of toxicity.
The compound’s effect on the L444P/L444P line is significant, as
this missense mutation is commonly found in the neuronopathic
forms of the disease.52 This increase in activity was not observed
with the inactive control 22 (data not shown).
Microscale thermophoresis (MST), a recently developed
technology that measures molecule movements under a
controlled temperature gradient, was applied to determine if
the putative activator series physically interacts with GCase.53
MST uses fluorescently labeled protein targets that, on binding
to ligands, can show changes in the movement of the protein
molecule along the temperature gradient. This technique was
best suited for binding analysis because of its low protein
requirements and its sensitivity, which is critical as these
allosteric activators had AC50 values in the micromolar range in
the functional assay.54 Figure 9 shows binding affinity data with
CONCLUSIONS
■
The use of spleen homogenate for HTS followed by medicinal
chemistry optimization identified the lead pyrazolopyrimidine
40, which displayed GCase activation by hydrolyzing 4MU-β-
Glc at submicromolar AC50 concentrations. Compound 40 also
demonstrated GCase activation of purified wt and N370S
enzyme with AC50 of 5.2 and 6.5 μM, respectively, a 10-fold
attenuation in potency consistent with the data in Table 2,
likely due to the absence of necessary cofactors present in the
spleen homogenate. While the activation of GCase with 40 and
other congeners was also observed in an orthogonal HPLC
assay, the series was inactive toward the hydrolysis of a
fluorescent ceramide-like substrate in the same assay. In
addition, these compounds failed to show GCase activation
in the primary fluorescence assay with resorufin β-D-
glucopyranoside as the substrate. Previously, we have observed
such discrepancies in the activation of a glycolytic enzyme
toward the hydrolysis of different substrates.55 The enzyme
kinetics of the hydrolysis and the outcome of the reaction
depend on the reaction substrate as well as the assay conditions
(for example, isolated enzyme or tissue homogenate). Thus, a
compound may activate the hydrolysis of two different
substrates with different efficiencies because the substrate
binding is different in each case, and the compound might
stabilize one particular conformation more than the other.
Ongoing studies are evaluating the addition of saposin C, an
allosteric activator for GCase, to assays where specific chemical
series fail to show inhibition or activation.30 The hydrolysis
assay still remains a useful surrogate assay for developing SAR.
The pyrazolopyrimides series demonstrated activity by
increasing the translocation of GCase to the lysosome in wild
Figure 9. Change in fluorescence observed with 40 (Kd = 8.91 μM)
and an inactive analogue 22 with increasing concentration in a
microscale thermophoresis experiment.
GCase for the potent activator 40 and an inactive analogue 22.
In this experiment, 40 binds to fluorescently labeled GCase in a
dose-dependent manner, with an apparent Kd of ∼9 μM. This
binding response has been recapitulated upon multiple testing.
This study was performed with isolated enzyme where the
series exhibited lower potency compared to the tissue
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a
Table 6. Stability of Representative Compounds at 1 μM in Mouse Liver Microsomes after 60 min
a
The data represent the percentage of parent compound remaining.
Table 7. PK Parameters for 40 Determined in Plasma, Brain, and Liver after a 50 mpk ip Dose in Male C57BL/6 Mice
PK parameter
Tmax
unit
plasma
0.250
brain
0.250
liver
0.250
h
Cmax
ng/mL
298
802
5020
(1.03 μM)
19.3
(2.76 μM)
18.2
(17.29 μM)
28.9
T1/2
h
AUClast
h·ng/mL
h·ng/mL
%
434
1260
1390
290
54300
AUCINF
474
105000
12512
AUCtissue/AUCplasma
Figure 10. Mean concentration−time profiles for 40 in plasma, brain, and liver after 50 mpk ip dose in male C57BL/6 mice (N = 3).
type (not shown) and patient fibroblasts, with 40 exhibiting
chaperone activity very similar to isofagomine at 1 μM. The
chaperone activity increased in a dose-dependent manner and
was not observed with a structurally related congener (61) that
was inactive in the primary assay (not shown). Furthermore,
this increase in translocation was mirrored by a consistent
increase in GCase activity in fibroblasts derived from four
different cell lines that included the N370S/N370S and L444P/
L444P genotypes derived from Gaucher patients. Preliminary
binding experiments revealed association of the compound with
the wt enzyme. 40 also displayed promising pharmacokinetic
properties.
The class of pyrazolpyrimidines described in this report
demonstrated biochemical activation of CGase, as well as
chaperone activity. These first-in-class noninhibitory chaper-
ones have the unique advantage over existing chaperones of not
being inhibitors of the GCase enzyme, and therefore, they do
not need to be displaced by the natural substrate in the
lysosome for the resumption of catalytic activity. These
molecules are promising new candidates for chaperone therapy
toward Gaucher disease and are currently being evaluated with
the aim of advancing toward clinical development.
MATERIALS AND METHODS
■
Biology. The recombinant wild-type enzyme imiglucerase
(Cerezyme) was obtained from Genzyme Corporation (Cambridge,
MA). N370S recombinant glucocerebrosidase was a gift from Dr. Tim
Edmunds at Genzyme. Spleen samples were obtained during
splenectomies with informed consent under an NIH-IRB approved
clinical protocol. Control spleens were obtained under an NIH
protocol. 4-Methylumbelliferyl-β-D-glucopyranoside (4MU-Glc), a
blue fluorogenic substrate, resorufin β-D-glucopyranoside (Res-glc), a
red fluorogenic substrate, sodium taurocholate, and the buffer
components were purchased from Sigma-Aldrich (St. Louis, MO).
Isofagomine and N-nonyldeoxynojirimycin (NN-DNJ) were pur-
chased from Toronto Research Biochemicals (Ontario, Canada).
The human spleen tissue was homogenized using a food blender at
the maximal speed for 5 min, followed by 10 passes in a motor-driven
50 mL glass-Teflon homogenizer. The homogenate was centrifuged
at 1000g for 10 min. The supernatant was then filtered using a
40 μm filter, and aliquots of resultant spleen homogenate were frozen
at −80 °C until use.
Enzyme Assay in 1536-Well Plate Format. In black 1536-well
plates, a BioRAPTR FRD Microfluidic workstation (Beckman Coulter,
Inc., Fullerton, CA) was used to dispense 2 μL/well spleen
homogenate (27 μg final) or pure enzyme solution (5 nM final)
into 1536-well plates. The assay buffer for the spleen homogenate was
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50 mM citric acid, 115 mM K2HPO4, 110 mM KCl, 10 mM NaCl,
1 mM MgCl2, and 0.01% Tween-20 at pH 5. The buffer for the
purified enzyme was 50 mM citric acid, KH2PO4 (titrated to pH 5.9
for recombinant wt enzyme and pH 7 for N370S variant), and 0.01%
Tween-20. An automated pin-tool station (Kalypsys, San Diego, CA)
was used to transfer 23 nL/well compound (the final titration was
0.5 nM to 58 μM) to the assay plate. After 5 min of incubation at
room temperature, the enzyme reaction was initiated by the addition
of 2 μL/well substrate. Final concentrations of the blue substrate
(4MU-Glc) and red substrate (Res-Glc) were 1 mM and 15 μM,
respectively. After 30−45 min of incubation at 37 °C, the reaction was
terminated by the addition of 2 μL/well stop solution (1 M NaOH
and 1 M glycine mixture, pH 10). Then 1 M Tris-HCl at pH 8.0 was
used as the stop solution for the red substrate assay. The fluorescence
was then measured in the Viewlux, a CCD-based plate reader (Perkin-
Elmer, Waltham, MA), with Ex = 365 nm and Em = 440 nm for the
blue substrate (4MU) and Ex = 573 nm and Em = 610 nm for the red
substrate (resorufin).
LC−MS Hydrolysis Experiment. This assay uses liquid
chromatography linked to a mass spectrometer to assess the ability
of glucocerebrosidase in the spleen homogenate to cleave the
profluorescent substrate 4MU-Glc or C12-BODIPY-GlcCer. Chroma-
tography was performed using an Agilent HPLC instrument on
stopped enzymatic reactions. The Agilent 1200 LC instrument was
equipped with a quaternary pump, a G1315 diode array detector, and a
G1321 fluorescent detector. A 4.6 mm × 250 mm Agilent Eclipse Plus
C18 (5 μm) instrument at ambient temperature was used at a flow rate
of 1.8 mL/min with a gradient of 85/15 (methanol/0.1% formic acid
in water) to 100% methanol over 10 min. Compounds were
monitored using fluorescence detection with Ex = 365 nm and
Em = 440 nm for 4MU or Ex = 506 nm and Em = 540 nm for C12-
BODIPY. We verified that the mass of the fluorescent peaks matched
the expected ones for the substrate and product of the reaction. The
concentrations of the compounds used were from 0 to 20 nM to
50 μM (1:2 dilutions from 50 μM, 9 concentrations). 140 μg/well of
spleen homogenate was used. Final concentrations of 2 mM for the 4-
MU-Glc and 25 μM for C12-BODIPY-Cer were used. The assay
conditions were the same as the enzyme assay.
Thermophoresis Assay. Glucocerebrosidase (GC, Protalix) was
labeled with a NT-647 dye (NanoTemper Technologies) and was
used in the thermophoresis experiment at a final concentration of
∼20 nM. A 16-point dilution series was made for selected compounds
in DMSO. Each compound dilution series was subsequently
transferred to protein solutions in 50 mM phosphate buffer, pH 7.2,
0.05% Tween-20. After a 15-min incubation of the labeled GC with
each dilution point (1:1 mix) at room temperature, samples were filled
into standard capillaries (NanoTemper Technologies). Measurements
were taken on a Monolith NT.115 microscale thermophoresis system
(NanoTemper) under a setting of 50% LED and 50% IR laser. Laser-
on time was set at 30 s, and laser-off time was set at 5 s.
Immunocytochemistry and Laser Scanning Confocal Mi-
croscopy. Primary dermal fibroblasts derived from skin biopsies from
previously described N370S/N370S Gaucher patients were seeded in
Lab-Tek 4 chamber slides (Fisher Scientific, Pittsburgh, PA). After
compound treatment, fibroblasts were fixed in 3% paraformaldehyde.
The cells were permeabelized with 0.1% Triton-X for 10 min and
blocked in PBS containing 0.1% saponin, 100 μM glycine, 0.1% BSA,
and 2% donkey serum followed by incubation with mouse monoclonal
LAMP-2 (1:100, Developmental Studies Hybridoma Bank, University
of Iowa, Iowa City, IA) and the rabbit polyclonal anti-GCase R386
antibody (1:500). The cells were washed and incubated with
secondary donkey anti-mouse or anti-rabbit antibodies conjugated to
ALEXA-488 or ALEXA-555, respectively (Invitrogen, Carlsbad, CA),
washed again, and mounted in VectaShield with DAPI (Vector
Laboratories, Burlingame, CA).
Plan-Apochromat 100×/1.4 oil DIC objective, respectively. Images
were taken with the same laser settings.
Cellular Assay To Measure GCase Specific Activity. Fibroblast
cell lines from controls, carriers, and Gaucher individuals (N370S,
L444P) were seeded in 96-well clear-bottom blank plates with a
density of 5000 cells/well in DMDM containing 15% FBS and
GlutaMax. After 1 day the medium is changed to OPTI-MEM
containing 5% FBS and no antibiotics. Each compound is dissolved in
the same medium to desired concentrations (1−20 μM final), and the
cells were treated with compound for 6 days. The plates were assayed
on day 6 by adding 1 volume of 0.2 M acetate buffer, pH 4, and 1
volume of PBS, pH 7.4, containing 2.5 mM 4MU-Glc. Plates were
incubated at 37 °C for 1 h. The reaction was stopped by adding 1 M
glycine and 1 M NaOH, which lyzed the cells. The fluorescence was
measured by a plate reader at (Em = 355, Ex = 460). The second set of
luminescence plates containing the same lines was used for viability
assay using CellTiter-Glo (Promega) as described by the manufac-
turer.
Microsome Stability. The test agent is incubated in duplicate with
CD-1 mouse liver microsomes at 37 °C. The mixture contains
microsomal protein in 100 mM potassium phosphate, 2 mM NADPH,
3 mM MgCl2, pH 7.4. A control is run for each test agent omitting
NADPH to detect NADPH-free degradation. At indicated times, an
aliquot is removed from each experimental and control mixture and
mixed with an equal volume of ice-cold stop solution (0.3% AcOH in
MeCN containing haloperidol, diclofenac, or other internal standard).
Stopped mixtures are incubated at least 10 min at −20 °C, and an
additional volume of water is added. The samples are centrifuged to
remove precipitated protein, and the supernatants are analyzed by
LC−MS/MS to quantitate the remaining parent. Data are reported as
percent remaining by dividing by the time zero concentration value.
Caco-2 Permeability. Caco-2 cells grown in tissue culture flasks
are trypsinized, suspended in medium, and the suspensions were
applied to wells of a collagen-coated BioCoat cell environment in 24-
well format (BD Biosciences) at 24 500 cells per well. The cells are
allowed to grow and differentiate for 3 weeks, feeding at 2-day
intervals. To verify that Caco-2 cell monolayers are properly formed,
aliquots of the cell buffers are analyzed by fluorescence to determine
the transport of the impermeable dye Lucifer yellow. For permeability,
the test agent is added to the apical (A) or basolateral (B) side and
amount of permeation to the other side is determined by LC−MS/
MS. The A-side buffer contains 100 μM Lucifer yellow dye in
transport buffer (1.98 g/L glucose in 10 mM HEPES, 1× Hank’s
balanced salt solution), pH 6.5, and the B-side buffer is transport
buffer, pH 7.4. Caco-2 cells are incubated with these buffers for 2 h.
Data are expressed as permeability (Papp), where dQ/dt is the rate of
permeation. In bidirectional permeability studies, the asymmetry index
(AI) or efflux ratio is also calculated. AI > 1 indicates a potential
substrate for PGP or other active transport.
Pharmacokinetics. C57BL/6 mice, 18−26 g, male, N = 36, were
purchased from SLAC Laboratory Animal Co. Ltd. The animals had
free access to food and water. The ip dosing solution was prepared in
20% PEG 400 + 80% (20% HP-β-CD). Brain and liver samples were
homogenized with 3 volumes of PBS (pH 7.4) before sample
extraction. The final concentration was adjusted with a dilution factor
of 4, assuming 1 g of wet brain equals 1 mL. LC−MS/MS analysis of
samples was done with an Acquity UPLC BEH C18 column, flow rate
0.6 mL/min, with a mobile phase consisting of solvent A (H2O−0.2%
FA, 10 mM NH4OAC), solvent B (MeOH−0.2%FA, 10 mM
NH4OAC). No clinical findings were observed during the entire life
study.
Chemistry. General Methods. All solvents and reagents were
purchased from Sigma Aldrich and other commercial vendors and used
as such. Purification was done with either normal phase SiO2
chromatography using Biotage purification stations or reverse phase
chromatography on a Waters semipreparative HPLC column. High
resolution mass spectrometry data were recorded on an Agilent 6210
time-of-flight LC−MS system. Confirmation of molecular formula was
accomplished using electrospray ionization in the positive mode with
Cells were imaged with a Zeiss 510 META confocal laser-scanning
microscope (Carl Zeiss, Microimaging Inc., Germany) using an argon
(458, 477, 488, 514 nm) 30 mW laser, a HeNe (543 nm) 1 mW laser,
and a laser diode (405 nm). Low and high magnification images
were acquired using a Plan-Apochromat 20×/0.75 objective and a
1
the Agilent Masshunter software (version B.02). H and 13C NMR
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spectra were recorded on a Varian Inova 400 MHz spectrometer.
Chemical shifts are reported in δ with the solvent resonance as the
internal standard (DMSO-d6 δ 2.50 for 1H, DMSO-d6 δ 39.5 for 13C).
Analytical purity analysis and retention times reported here were
performed using three methods. Method 1 involves the following:
column, Phenomenex Luna C18 (3 μm, 3 mm × 75 mm); run time,
8 min; gradient, 4−100% acetonitrile in water over 7 min; mobile
phase, acetonitrile (0.025% TFA), water (0.05% TFA); flow rate,
1 mL/min; temperature, 50 °C; UV wavelength, 220 and 254 nm.
Method 2 involves the following: detection UV, 214 nm; Gemini
column; solvent A, water; solvent B, 90% acetonitrile, 10% water, 0.1%
AcOH modifier; gradient initial condition, 100% A with hold time
0.5 min; gradient time, 3 min; gradient final concentration, 100% B;
gradient final hold, 0.5 min; run time, 5 min; flow rate, 1.5 mL/min.
Method 3 involves the following: detection UV, 220 nm;
Phenomenex Luna 2.5 μm C18 100 mm × 2.00 mm column; solvent
A, water (0.05% TFA); solvent B, acetonitrile (0.025% TFA); gradient
initial condition, 95% A, 5% B with hold time 0.1 min; gradient time,
2.1 min; gradient final concentration, 100% B, 5% A; gradient final
hold, 0.5 min and then another hold for further 0.3 min with 98% A,
2% B; run time, 3 min.; flow rate, 0.30−0.5 mL/min.
2.71 (s, 3 H), 2.77 (s, 3 H), 4.11 (s, 1 H), 7.21 (s, 1 H), 7.49 (d, J =
8.6 Hz, 2 H), 7.77 (d, J = 8.6 Hz, 2 H), 8.65 (s, 1 H), 10.31 (s, 1 H).
13C NMR (101 MHz, DMSO-d6, 100 °C) δ ppm 15.6, 23.7, 78.6, 83.1,
103.5, 109.9, 115.9, 118.6, 131.9, 138.9, 144.7, 144.8, 147.0, 159.2,
161.4.
This coupling procedure was used to synthesize all analogues.
Analogues were obtained with >95% purity by simple filtration/
washing/drying and/or by preparative purification on a Waters
semipreparative HPLC. The column used was a Phenomenex Luna
C18 (5 μm, 30 mm × 75 mm), and the flow rate was 45 mL/min. The
mobile phase consisted of acetonitrile and water (each containing 0.1%
trifluoroacetic acid). Generally a gradient of 10−50% acetonitrile over
8 min was used during the purification. The fraction collection was
triggered by UV detection (220 nm, 254 nm).
For R1 = Me, R2 = Ph, and R1 = Ph, R2 = Me. Step 1 from the
above general procedure was followed with 1-phenylbutane-1,3-dione
(1.05 g, 6.45 mmol), ethyl 3-amino-1H-pyrazole-4-carboxylate (1.00 g,
6.45 mmol), and acetic acid (15 mL). Flash SiO2 column
chromatography (10−80% EtOAc/hexanes) provided ethyl 7-meth-
yl-5-phenylpyrazolo[1,5-a]pyrimidine-3-carboxylate (0.23 g, 0.82
1
mmol, 13% yield). LC−MS (method 2): tR = 3.66 min. H NMR
All analogues were determined to have >95% purity based on the
above methods.
(400 MHz, DMSO-d6) δ ppm 1.37 (t, J = 7.0 Hz, 3 H), 2.84 (s, 3 H),
4.32 (q, J = 7.2 Hz, 2 H), 7.60 (m, 3 H), 7.94 (s, 1 H), 8.32 (m, 2 H),
8.63 (s, 1 H). Ethyl 5-methyl-7-phenylpyrazolo[1,5-a]pyrimidine-3-
carboxylate was also provided (0.95 g, 3.4 mmol, 52% yield). LC−MS
General Procedure toward Analogues.
1
(method 2): tR = 3.56 min. H NMR (400 MHz, DMSO-d6) δ ppm
1.33 (t, J = 7.0 Hz, 3 H), 2.68 (s, 3 H), 4.31 (q, J = 7.0 Hz, 2 H), 7.40
(s, 1 H), 7.62 (m, 3 H), 8.06 (m, 2 H), 8.57 (s, 1 H). The separated
pure esters were hydrolyzed to the corresponding acids (character-
ization data below).
5-Methyl-7-phenylpyrazolo[1,5-a]pyrimidine-3-carboxylic Acid
(Precursor to 62, 63, 64, 68. LC−MS (method 2): tR = 3.11 min.
1H NMR (400 MHz, DMSO-d6) δ ppm 2.65 (s, 3 H), 7.34 (s, 1 H),
7.51−7.66 (m, 3 H), 8.04 (d, J = 6.7 Hz, 2 H), 8.52 (s, 1 H) (note:
acid OH is not visible). 13C NMR (101 MHz, DMSO-d6) δ ppm 24.5,
101.8, 110.4, 128.4, 129.6, 130.1, 131.1, 146.0, 147.0, 148.0, 162.6,
163.3.
Step 1 (R1 = R2 = Me). Pentane-2,4-dione (1.46 mL, 14.2 mmol) and
ethyl 3-amino-1H-pyrazole-4-carboxylate (2.00 g, 12.9 mmol) were
heated in a sealed tube with acetic acid (10 mL) at 110 °C overnight.
The reaction reached completion by LC−MS (tR = 3.08 min). The
acetic acid was removed by blowing air on the mixture overnight.
(For R1 ≠ R2, at this stage the residue was purified by flash SiO2
chromatography to separate the regioisomers 18 and 19. Each was
separately taken to the next step.) The crude residue of ethyl 5,7-
dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylate (assumed to be
12.89 mmol) was suspended in MeOH (15 mL) and treated with
7.2 M sodium hydroxide (5.37 mL, 38.7 mmol). The mixture was
heated to 80 °C (at this temperature the solid dissolved) and then
stirred for 3 h. The mixture was cooled and neutralized to pH 6−7.
7-Methyl-5-phenylpyrazolo[1,5-a]pyrimidine-3-carboxylic Acid
1
(Precursor to 65, 70, 73). LC−MS (method 2): tR = 3.13 min. H
NMR (400 MHz, DMSO-d6) δ ppm 2.82 (s, 3 H), 7.54−7.63 (m, 3 H),
7.86 (s, 1 H), 8.24−8.31 (m, 2 H), 8.58 (s, 1 H), 12.32 (br s, 1 H). 13C
NMR (101 MHz, DMSO-d6) δ ppm 17.0, 102.6, 106.6, 127.5, 129.0,
131.0, 136.2, 147.2, 147.5, 147.8, 157.4, 163.3.
The slurry was filtered via a Buchner funnel under house vacuum, and
̈
the solid residue was washed with water and then diethyl ether to
5-Cyclopropyl-7-(difluoromethyl)pyrazolo[1,5-a]pyrimidine-3-
carboxylic Acid (Precursor to 13, 69, 71) and 5-Cyclopropyl-7-
(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxylic Acid (Pre-
cursor to 67). The title compounds were purchased from Chembridge
and used directly in coupling reactions.
obtain 5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (1.4 g,
1
7.3 mmol, 57% yield). LC−MS (method 3): tR = 2.61 min. H NMR
(400 MHz, DMSO-d6) δ ppm 2.57 (s, 3 H), 2.71 (s, 3 H), 7.10 (s, 1 H),
8.50 (s, 1 H). 13C NMR (101 MHz, DMSO-d6, 100 °C) δ ppm 16.0,
23.9, 106.0, 109.2, 145.8, 146.3, 146.6, 160.3, 164.4.
Analytical Data for Compounds. 22. 1H NMR (400 MHz,
DMSO-d6) δ ppm 2.62 (s, 3 H), 2.73 (s, 3 H), 2.89 (d, J = 4.7 Hz, 3
H), 7.10 (s, 1 H), 7.95 (q, J = 4.7 Hz, 1 H), 8.48 (s, 1 H). LC−MS
(method 1): tR = 3.71 min. HRMS (m/z) calcd for C10H13N4O (M +
H)+ 205.1084, found 205.1085
23. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.95 (m, 6 H), 1.83 (m,
1 H), 2.61 (s, 3 H), 2.74 (s, 3 H), 3.22 (t, J = 6.3 Hz, 2 H), 7.12 (s, 1 H),
8.15 (t, J = 6.1 Hz, 1 H), 8.48 (s, 1 H). LC−MS (method 1): tR =
5.08 min. HRMS (m/z) calcd for C13H19N4O (M + H)+ 247.1553,
found 247.1559
24. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.22 (m, 6 H), 2.61 (s,
3 H), 2.73 (s, 3 H), 4.09 (m, 1 H), 7.10 (s, 1 H), 7.91 (d, J = 7.8 Hz, 1
H), 8.47 (s, 1 H). LC−MS (method 1): tR = 4.66 min. HRMS (m/z)
calcd for C12H17N4O (M + H)+ 233.1397, found 233.1397
25. LC−MS (method 1): tR = 3.84 min. HRMS (m/z) calcd for
C13H17N4O (M + H)+ 245.1397, found 245.1399
Step 2. 5,7-Dimethylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid
(722 mg, 3.78 mmol), 4-ethynylaniline (442 mg, 3.78 mmol), and
HATU (1436 mg, 3.78 mmol) were taken up in DMF (10 mL) and
then treated with diisopropylethylamine (1.979 mL, 11.33 mmol). The
contents were stirred at room temperature overnight. The product had
precipitated from reaction mixture. The mixture was diluted with water
26. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.35 (m, 5 H), 1.56 (m,
1 H), 1.70 (m, 2 H), 1.87 (m, 2 H), 2.61 (s, 3 H), 2.73 (s, 3 H), 3.87
(m, 1 H), 7.11 (s, 1 H), 8.08 (d, J = 7.8 Hz, 1 H), 8.48 (s, 1 H). LC−
MS (method 1): tR = 5.55 min. HRMS (m/z) calcd for C15H21N4O
(M + H)+ 273.1710, found 273.1713.
and filtered through a Buchner funnel under house vacuum. The
̈
residue was washed with water (×2), then CH2Cl2, diethyl ether, and
air-dried to obtain N-(4-ethynylphenyl)-5,7-dimethylpyrazolo[1,5-a]-
pyrimidine-3-carboxamide (500 mg, 1.72 mmol, 46% yield). LC−MS
1
(method 3): tR = 3.65 min. H NMR (400 MHz, DMSO-d6) δ ppm
K
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27. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.88 (m, 9 H), 1.12 (m,
5 H), 1.77 (m, 2 H), 2.05 (m, 2 H), 2.61 (m, 3 H), 2.73 (m, 3 H), 3.72
(m, 1 H), 7.11 (m, 1 H), 7.92 (d, J = 7.7 Hz, 1 H), 8.47 (m, 1 H).
LC−MS (method 1): tR = 6.94 min. HRMS (m/z) calcd for
C19H29N4O (M + H)+ 329.2336, found 329.2342.
(method 1): tR = 5.78 min. HRMS (m/z) calcd for C17H15N4O
(M + H)+ 291.1240, found 291.1245.
41. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.72 (s, 3 H), 2.78 (s, 3
H), 7.23 (s, 1 H), 7.74 (d, J = 8.6 Hz, 2 H), 7.98 (d, J = 8.6 Hz, 2 H),
8.68 (m, 1 H), 10.45 (m, 1 H). LC−MS (method 1): tR = 6.43 min.
HRMS (m/z) calcd for C16H14 F3N4O (M + H)+ 335.1114, found
335.1116.
28. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.89 (s, 9 H), 1.27 (m,
3 H), 1.56 (m, 2 H), 1.68 (m, 2 H), 1.81 (m, 2 H), 2.60 (s, 3 H), 2.74
(s, 3 H), 4.26 (m, 1 H), 7.13 (m, 1 H), 8.46 (m, 2 H). LC−MS
(method 1): tR = 6.82 min. HRMS (m/z) calcd for C19H29N4O (M + H)+
329.2336, found 329.2344.
1
42. H NMR (400 MHz, DMSO-d6) δ ppm 2.69 (s, 3 H), 2.75
(s, 3 H), 7.18 (s, 1 H), 7.43 (m, 1 H), 7.59 (t, J = 7.9 Hz, 1 H), 7.85
(m, 1 H), 8.30 (t, J = 2.1 Hz, 1 H), 8.63 (s, 1 H), 10.35 (s, 1 H). LC−
MS (method 1): tR = 6.36 min. HRMS (m/z) calcd for C16H14 F3N4O
(M + H)+ 335.1114, found 335.1118.
29. LC−MS (method 1): tR = 5.19 min. HRMS (m/z) calcd for
C16H17N4O (M + H)+ 281.1397, found 281.1402.
43. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (s, 3 H), 2.77 (s, 3
H), 7.21 (s, 1 H), 7.56 (d, J = 8.6 Hz, 2 H), 7.74 (d, J = 9.0 Hz, 2 H),
8.65 (s, 1 H), 10.24 (s, 1 H). LC−MS (method 1): tR = 6.21 min.
HRMS (m/z) calcd for C15H14 BrN4O (M + H)+ 345.0346, found
345.0349.
30. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.60 (s, 3 H), 2.74 (s, 3
H), 4.58 (d, J = 5.9 Hz, 2 H), 6.31 (d, J = 3.1 Hz, 1 H), 6.41 (m, 1 H),
7.12 (s, 1 H), 7.60 (m, 1 H), 8.38 (t, J = 5.9 Hz, 1 H), 8.52 (s, 1 H).
LC−MS (method 1): tR = 4.72 min. HRMS (m/z) calcd for
C14H15N4O2 (M + H)+ 279.1190, found 279.1189.
1
31. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (s, 3 H), 2.77 (s, 3
H), 7.10 (t, J = 7.4 Hz, 1 H), 7.20 (s, 1 H), 7.38 (t, J = 7.8 Hz, 2 H),
7.75 (d, J = 7.8 Hz, 2 H), 8.64 (s, 1 H), 10.19 (s, 1 H). LC−MS
(method 1): tR = 5.52 min. HRMS (m/z) calcd for C15H15N4O (M + H)+
267.1240, found 267.1241.
44. H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (s, 3 H), 2.77
(s, 3 H), 7.21 (s, 1 H), 7.65 (m, 2 H), 8.20 (d, J = 2.3 Hz, 1 H), 8.66
(s, 1 H), 10.30 (s, 1 H). LC−MS (method 1): tR = 6.46 min. HRMS
(m/z) calcd for C15H13Cl2N4O (M + H)+ 335.0461, found 335.0463.
45. LC−MS (method 1): tR = 6.72 min. HRMS (m/z) calcd for
C15H13Cl2N4O (M + H)+ 335.0461, found 335.0467.
32. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.29 (s, 3 H), 2.71 (s, 3
H), 2.77 (s, 3 H), 7.19 (m, 3 H), 7.63 (d, J = 8.6 Hz, 2 H), 8.62 (s, 1
H), 10.11 (s, 1 H). LC−MS (method 1): tR = 5.82 min. HRMS (m/z)
calcd for C16H17N4O (M + H)+ 281.1397, found 281.1399.
46. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.74 (s, 3 H), 2.79 (s, 3 H),
7.28 (s, 1 H), 8.15 (d, J = 6.3 Hz, 2 H), 8.69 (d, J = 6.3 Hz, 2 H), 8.77
(s, 1 H), 10.94 (s, 1 H). LC−MS (method 1): tR = 3.33 min. HRMS
(m/z) calcd for C14H14N5O (M + H)+ 268.1193, found 268.1198.
1
33. H NMR (400 MHz, DMSO-d6) δ ppm 2.48 (s, 3 H), 2.68 (s,
1
47. H NMR (400 MHz, DMSO-d6) δ ppm 2.73 (s, 3 H), 2.78
3 H), 2.78 (s, 3 H), 7.02 (m, 1 H), 7.26 (m, 3 H), 8.34 (d, J = 8.2 Hz,
1 H), 8.64 (s, 1 H), 10.09 (s, 1 H). LC−MS (method 1): tR = 5.73
min. HRMS (m/z) calcd for C16H17N4O (M + H)+ 281.1397, found
281.1401.
(s, 3 H), 3.19 (s, 3 H), 7.23 (s, 1 H), 7.92 (m, 2 H), 8.00 (m, 2 H),
8.69 (d, J = 2.0 Hz, 1 H), 10.52 (d, J = 0.8 Hz, 1 H. LC−MS (method 1):
tR = 4.76 min. HRMS (m/z) calcd for C16H17N4O3S (M + H)+ 345.1016,
found 345.1021.
1
34. H NMR (400 MHz, DMSO-d6) δ ppm 2.32 (s, 3 H), 2.69
48. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.72 (s, 3 H), 2.77 (s, 3 H),
7.22 (s, 1 H), 7.84 (d, J = 8.7 Hz, 2 H), 7.96 (d, J = 8.7 Hz, 2 H), 8.68
(s, 1 H), 10.50 (s, 1 H). LC−MS (method 1): tR = 5.44 min. HRMS
(m/z) calcd for C16H15N5O (M + H)+ 292.1193, found 292.1193.
(s, 3 H), 2.75 (s, 3 H), 6.90 (m, 1 H), 7.18 (m, 1 H), 7.24 (m, 1 H),
7.54 (m, 2 H), 8.60 (s, 1 H), 10.10 (s, 1 H). LC−MS (method 1): tR =
5.84 min. HRMS (m/z) calcd for C16H17N4O (M + H)+ 281.1397,
found 281.1404.
1
49. H NMR (400 MHz, DMSO-d6) δ ppm 2.72 (s, 3 H), 2.77
1
35. H NMR (400 MHz, DMSO-d6) δ ppm 2.20 (s, 3 H), 2.25
(s, 3 H), 7.21 (s, 1 H), 7.57 (m, 2 H), 8.02 (m, 1 H), 8.28 (t, J =
2.0 Hz, 1 H), 8.66 (s, 1 H), 10.36 (s, 1 H). LC−MS (method 1): tR =
5.47 min. HRMS (m/z) calcd for C16H14N5O (M + H)+ 292.1193,
found 292.1200.
(s, 3 H), 2.71 (s, 3 H), 2.77 (s, 3 H), 7.13 (d, J = 9.0 Hz, 1 H), 7.19
(s, 1 H), 7.49 (m, 2 H), 8.61 (s, 1 H), 10.04 (s, 1 H). LC−MS
(method 1):tR = 6.09 min. HRMS (m/z) calcd for C17H19N4O
(M + H)+ 295.1553, found 295.1555.
50. LC−MS (method 1): tR = 5.25 min. HRMS (m/z) calcd for
36. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.26 (s, 3 H), 2.43 (s, 3
H), 2.66 (s, 3 H), 2.77 (s, 3 H), 7.02 (dd, J = 8.2, 2.0 Hz, 1 H), 7.09
(d, J = 2.0 Hz, 1 H), 7.20 (s, 1 H), 8.18 (d, J = 8.2 Hz, 1 H), 8.62 (s,
1 H), 10.01 (s, 1 H). LC−MS (method 1): tR = 6.11 min. HRMS
(m/z) calcd for C17H19N4O (M + H)+ 295.1553, found 295.1561.
37. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.03 (quin, J = 7.4 Hz,
2 H), 2.71 (s, 3 H), 2.77 (s, 3 H), 2.84 (t, J = 7.4 Hz, 2 H), 2.89 (t, J =
7.4 Hz, 2 H), 7.20 (m, 2 H), 7.46 (dd, J = 8.2, 1.6 Hz, 1 H), 7.66 (m,
C17H14N4O (M + H)+ 291.1245, found 291.1240.
1
51. H NMR (400 MHz, DMSO-d6) δ ppm 2.61 (s, 3 H), 2.72
(s, 3 H), 2.78 (s, 3 H), 7.21 (s, 1 H), 7.54 (t, J = 7.8 Hz, 1 H), 7.71
(dt, J = 7.7, 1.3 Hz, 1 H), 7.95 (m, 1 H), 8.35 (t, J = 2.0 Hz, 1 H), 8.66
(s, 1 H), 10.30 (s, 1 H). LC−MS (method 1): tR = 5.27 min. HRMS
(m/z) calcd for C17H17N4O2 (M + H)+ 309.1346, found 309.1349.
1
52. H NMR (400 MHz, DMSO-d6) δ ppm 2.04 (s, 3 H), 2.71
(s, 3 H), 2.77 (s, 3 H), 7.20 (s, 1 H), 7.58 (m, 2 H), 7.66 (m, 2 H),
8.62 (s, 1 H), 9.92 (s, 1 H), 10.10 (s, 1 H). LC−MS (method 1): tR =
4.39 min. HRMS (m/z) calcd for C17H18N5O2 (M + H)+ 324.1455,
found 324.1459.
1 H), 8.61 (s, 1 H), 10.11 (s, 1 H). LC−MS (method 1): tR
=
6.33 min. HRMS (m/z) calcd for C18H19N4O (M + H)+ 307.1553,
found 307.1560.
1
53. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.08 (s, 3 H), 2.69 (s, 3 H),
2.77 (s, 3 H), 7.12 (m, 1 H), 7.19 (s, 1 H), 7.27 (m, 2 H), 8.18 (m,
1 H), 8.63 (s, 1 H), 9.74 (s, 1 H), 9.94 (s, 1 H. LC−MS (method 1):
tR = 4.17 min. HRMS (m/z) calcd for C17H18N5O2 (M + H)+
324.1455, found 324.1461.
38. H NMR (400 MHz, DMSO-d6) δ ppm 1.18 (t, J = 7.6 Hz,
3 H), 2.59 (q, J = 7.4 Hz, 2 H), 2.70 (s, 3 H), 2.76 (s, 3 H), 7.20 (m,
3 H), 7.64 (d, J = 8.2 Hz, 2 H), 8.61 (s, 1 H), 10.10 (s, 1 H). LC−MS
(method 1): tR = 6.23 min. HRMS (m/z) calcd for C17H19N4O (M +
H)+ 295.1553, found 295.1558.
1
1
39. H NMR (400 MHz, DMSO-d6) δ ppm 1.29 (s, 9 H), 2.70 (s,
54. H NMR (400 MHz, DMSO-d6) δ ppm 2.70 (s, 3 H), 2.77
3 H), 2.77 (s, 3 H), 7.20 (s, 1 H), 7.39 (d, J = 9.0 Hz, 2 H), 7.64 (d,
J = 8.6 Hz, 2 H), 8.62 (s, 1 H), 10.11 (s, 1 H). LC−MS (method 1):
tR = 6.75 min. HRMS (m/z) calcd for C19H23N4O (M + H)+
323.1866, found 323.1875.
(s, 3 H), 3.76 (s, 3 H), 6.96 (d, J = 9.0 Hz, 2 H), 7.19 (m, 1 H), 7.66
(d, J = 9.0 Hz, 2 H), 8.62 (s, 1 H), 10.03 (s, 1 H). LC−MS (method 1):
tR = 5.33 min. HRMS (m/z) calcd for C16H17N4O2 (M + H)+ 297.1346,
found 297.1352.
55. H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (s, 3 H), 2.77
(s, 3 H), 3.65 (s, 3 H), 3.81 (s, 6 H), 7.09 (m, 2 H), 7.19 (s, 1 H), 8.61
40. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.70 (s, 3 H), 2.76 (s, 3
H), 4.10 (s, 1 H), 7.19 (s, 1 H), 7.47 (d, J = 8.6 Hz, 2 H), 7.76 (d, J =
8.6 Hz, 2 H), 8.64 (s, 1 H), 10.29 (s, 1 H). 13C NMR (101 MHz,
DMSO-d6) δ ppm 16.5, 24.4, 79.8, 83.5, 100.5, 103.7, 116.2, 118.9,
132.6, 139.3, 145.3, 145.3, 147.6, 159.8, 162.2. 13C NMR (101 MHz,
DMSO-d6, 100 °C) δ ppm 15.6, 23.7, 78.6, 83.1, 103.5, 109.9, 115.9,
118.6, 131.9, 138.9, 144.7, 144.8, 147.0, 159.2, 161.4. LC−MS
1
(s, 1 H), 10.04 (d, J = 1.2 Hz, 1 H). LC−MS (method 1): tR
=
5.24 min. HRMS (m/z) calcd for C18H21N4O4 (M + H)+ 357.1557,
found 357.1564.
1
56. H NMR (400 MHz, DMSO-d6) δ ppm 2.70 (s, 3 H), 2.77
(s, 3 H), 3.75 (s, 3 H), 3.79 (s, 3 H), 6.96 (d, J = 8.6 Hz, 1 H), 7.18
L
dx.doi.org/10.1021/jm300063b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(m, 2 H), 7.49 (d, J = 2.2 Hz, 1 H), 8.61 (s, 1 H), 10.01 (s, 1 H). LC−
MS (method 1): tR = 5.03 min. HRMS (m/z) calcd for C17H19N4O3
(M + H)+ 327.1452, found 327.1456.
(s, 1 H). LC−MS (method 1): tR = 6.91 min. HRMS (m/z) calcd for
C20H19F2N4O (M + H)+ 369.1521, found 369.1529.
1
57. H NMR (400 MHz, DMSO-d6) δ ppm 2.70 (s, 3 H), 2.77
ASSOCIATED CONTENT
* Supporting Information
■
(s, 3 H), 6.02 (s, 2 H), 6.92 (d, J = 8.2 Hz, 1 H), 7.08 (dd, J = 8.4, 2.2
Hz, 1 H), 7.19 (s, 1 H), 7.51 (d, J = 2.3 Hz, 1 H), 8.61 (s, 1 H), 10.07
(s, 1 H). LC−MS (method 1): tR = 5.33 min. HRMS (m/z) calcd for
C16H15N4O3 (M + H)+ 311.1139, found 311.1142
S
Stereochemical prediction of relative sterechemistry of 27 and
28 and their NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
58. H NMR (400 MHz, DMSO-d6) δ ppm 2.69 (s, 3 H), 2.76
(s, 3 H), 4.24 (m, 4 H), 6.85 (d, J = 8.6 Hz, 1 H), 7.05 (m, 1 H), 7.18
(s, 1 H), 7.43 (d, J = 2.7 Hz, 1 H), 8.60 (s, 1 H), 10.00 (s, 1 H). LC−
MS (method 1): tR = 5.30 min. HRMS (m/z) calcd for C17H17N4O2
(M + H)+ 325.1295, found 325.1299
AUTHOR INFORMATION
Corresponding Author
*Phone: 301-217-9198. Fax: 301-217-5736. E-mail: maruganj@
mail.nih.gov.
■
1
59. H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (s, 3 H), 2.77 (s,
3 H), 3.83 (s, 3 H), 7.18 (m, 2 H), 7.40 (m, 1 H), 7.79 (dd, J = 13.5,
2.5 Hz, 1 H), 8.63 (s, 1 H), 10.11 (s, 1 H). LC−MS (method 1): tR =
5.52 min. HRMS (m/z) calcd for C16H16FN4O2 (M + H)+ 315.1252,
found 315.1257.
Notes
The authors declare no competing financial interest.
1
60. H NMR (400 MHz, DMSO-d6) δ ppm 2.71 (s, 3 H), 2.77 (s,
ACKNOWLEDGMENTS
■
3 H), 3.86 (s, 3 H), 7.17 (d, J = 9.0 Hz, 1 H), 7.20 (s, 1 H), 7.59 (dd,
J = 8.6, 2.7 Hz, 1 H), 7.97 (d, J = 2.3 Hz, 1 H), 8.63 (s, 1 H), 10.08
(s, 1 H). LC−MS (method 1): tR = 5.79 min. HRMS (m/z) calcd for
C16H16ClN4O2 (M + H)+ 331.0956, found 331.0962.
This research was supported by the Molecular Libraries
Initiative of the NIH Roadmap for Medical Research and the
Intramural Research Program of the National Human Genome
Research Institute, National Institutes of Health. We thank Jim
Bougie, Thomas Daniel Paul Shinn, Danielle van Leer, and
Christopher Leclair at the NCGC compound management and
analytical groups for assistance in compound handling, plating,
and purification.
61. LC−MS (method 1): tR = 3.72. LC−MS (m/z) calcd for
C13H19N4O2(M + H)+ 263.2, found 263.1. LC−MS (m/z) calcd for
C26H36N8NaO4 (2M + Na)+ 547.3, found 547.3.
1
62. H NMR (400 MHz, DMSO-d6) δ ppm 2.30 (s, 3 H), 2.79
(s, 3 H), 7.20 (d, J = 8.4 Hz, 2 H), 7.49 (s, 1 H), 7.65 (m, 5 H), 8.13
(m, 2 H), 8.65 (s, 1 H), 10.20 (s, 1 H). LC−MS (method 1): tR =
6.59. HRMS (m/z) calcd for C21H19N4O (M + H)+ 343.1553, found
343.1560.
ABBREVIATIONS USED
■
1
63. H NMR (400 MHz, DMSO-d6) δ ppm 2.79 (s, 3 H), 3.76
GCase, glucocerebrosidase; GC, glucocerebroside; ERT,
enzymatic replacement therapy; SRT, substrate replacement
therapy; ER, endoplasmic reticulum; NN-DNJ, N-(n-nonyl)-
deoxynojirimycin; wt, wild type; NCGC, NIH Chemical
Genomics Center; IC50, half-maximal inhibitory concentration;
AC50, half-maximal activation concentration; 4MU-Glc, 4-
methylumbelliferyl β-D-glucopyranoside; 4MU, 4-methylumbel-
liferone; HTS, high throughput screening; SAR, structure−
activity relationship; MST, microscale thermophoresis; HPLC,
high performance liquid chromatography; LC−MS, liquid
chromatography−mass spectrometry; ADME, absorption,
distribution, metabolism, elimination
(s, 3 H), 6.97 (d, J = 9.0 Hz, 2 H), 7.48 (s, 1 H), 7.65 (m, 5 H), 8.14
(dd, J = 7.8, 2.0 Hz, 2 H), 8.64 (s, 1 H), 10.12 (s, 1 H). LC−MS
(method 1): tR = 6.17.
64. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.81 (s, 3 H), 7.51 (s, 1
H), 7.67 (m, 5 H), 8.13 (m, 2 H), 8.23 (d, J = 2.7 Hz, 1 H), 8.69 (s, 1
H), 10.40 (s, 1 H). LC−MS (method 1): tR = 7.21 min. HRMS (m/z)
calcd for C20H15Cl2N4O (M + H)+ 397.0617, found 397.0628.
65. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.89 (s, 3 H), 7.65 (m,
5 H), 7.97 (s, 1 H), 8.22 (d, J = 2.7 Hz, 1 H), 8.35 (dd, J = 7.6, 2.2 Hz,
2 H), 8.75 (s, 1 H), 10.36 (s, 1 H). LC−MS (method 1): tR
=
7.26.HRMS (m/z) calcd for C20H15Cl2N4O (M + H)+ 397.0617,
found 397.0622.
1
66. H NMR (400 MHz, DMSO-d6) δ ppm 2.53 (s, 3 H), 2.73
(s, 3 H), 7.17 (m, 7 H), 7.52 (m, 1 H), 7.62 (dd, J = 7.6, 1.8 Hz, 1 H),
8.60 (s, 1 H), 8.64 (m, 1 H), 10.90 (s, 1 H). LC−MS (method 1): tR =
6.59.HRMS (m/z) calcd for C21H19N4OS (M + H)+ 375.1274, found
375.1284.
67. LC−MS (method 3): tR = 2.76 min. HRMS (m/z) calcd for
C23H18 F3N4OS (M + H)+ 455.1148, found 455.1161.
68. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.62 (s, 3 H), 7.17 (m,
4 H), 7.26 (m, 2 H), 7.43 (s, 1 H), 7.54 (m, 1 H), 7.63 (m, 4 H), 8.10
(dd, J = 7.8, 2.0 Hz, 2 H), 8.67 (m, 2 H), 11.01 (s, 1 H). LC−MS
(method 1): tR = 7.28 min. HRMS (m/z) calcd for C26H21N4OS
(M + H)+ 437.1431, found 437.1441.
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69. LC−MS (method 3): tR = 2.64 min. HRMS (m/z) calcd for
C20H19F2N4O (M + H)+ 369.1521, found 369.1529.
70. 1H NMR (400 MHz, DMSO-d6) δ ppm 2.04 (m, 2 H), 2.87 (m,
7 H), 7.24 (m, 1 H), 7.44 (m, 1 H), 7.67 (m, 4 H), 7.95 (s, 1 H), 8.33
(m, 2 H), 8.70 (s, 1 H), 10.16 (br s, 1 H). LC−MS (method 1): tR =
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73. H NMR (400 MHz, DMSO-d6) δ ppm 1.20 (t, J = 7.6 Hz,
3 H), 2.61 (q, J = 7.5 Hz, 2 H), 2.89 (s, 3 H), 7.25 (d, J = 8.4 Hz, 2 H),
7.68 (m, 5 H), 7.96 (s, 1 H), 8.33 (m, 2 H), 8.70 (s, 1 H), 10.17
1
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dx.doi.org/10.1021/jm300063b | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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