Design and Synthesis of Potent Quinazolines as Selective β-Glucocerebrosidase Modulators

Article abstract of DOI:10.1021/acs.jmedchem.6b00930

Gaucher's disease is a common genetic disease caused by mutations in the β-glucocerebrosidase (GBA1) gene that have been also linked to increased risk of Parkinson's disease and Lewy body dementia. Stabilization of misfolded mutant β-glucocerebrosidase (G

Full text of DOI:10.1021/acs.jmedchem.6b00930

Article
pubs.acs.org/jmc
Design and Synthesis of Potent Quinazolines as Selective
β‑Glucocerebrosidase Modulators
Jianbin Zheng,†,‡ Long Chen,† Michael Schwake,† Richard B. Silverman,*,‡ and Dimitri Krainc*,†
†Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, United States
‡Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, Center for Molecular
Innovation and Drug Discovery, and Center for Developmental Therapeutics, Northwestern University, Evanston, Illinois
60208-3113, United States
S
* Supporting Information
ABSTRACT: Gaucher’s disease is a common genetic disease
caused by mutations in the β-glucocerebrosidase (GBA1) gene
that have been also linked to increased risk of Parkinson’s
disease and Lewy body dementia. Stabilization of misfolded
mutant β-glucocerebrosidase (GCase) represents an important
therapeutic strategy in synucleinopathies. Here we report a
novel class of GCase quinazoline inhibitors, obtained in a high
throughput screening, with moderate potency against wild-type
GCase. Rational design and a SAR study of this class of
compounds led to a new series of quinazoline derivatives with single-digit nanomolar potency. These compounds were shown to
selectively stabilize GCase when compared to other lysosomal enzymes and to increase N370S mutant GCase protein
concentration and activity in cell assays. To the best of our knowledge, these molecules are the most potent noniminosugar
GCase modulators to date that may prove useful for future mechanistic studies and therapeutic approaches in Gaucher’s and
Parkinson’s diseases.
iminosugar class of compounds (Figure 1).18 However,
INTRODUCTION
■
iminosugars tend to have poor selectivity and relatively short
Gaucher’s disease (GD), the most common lysosomal storage
disease, is caused by a recessively inherited deficiency in β-
half-lives in cells.19 Several different scaffolds of noniminosugar
inhibitors (2 and 3 are examples in Figure 1) have been
glucocerebrosidase (GCase) and subsequent accumulation of
glucoceramides, toxic lipid substrates.1,2 Substrate accumulation
leads to hepatosplenomegaly, bone marrow suppression, and
bone lesions.1−3 Many of the GCase mutations are missense
mutations4 that result in single amino acid substitutions of the
enzyme. Most of these mutations, including the prevalent
N370S mutation, are still functional, although with very low
residual GCase activity5 due to enzyme misfolding and
proteasome-mediated breakdown.5 Current treatments for
GD include enzyme replacement therapy (ERT) and substrate
reduction therapy (SRT).2,6 In recent years, mutations in GBA1
were also found to be a major risk factor for Parkinson’s disease
(PD) and dementia with Lewy bodies (DLB).7−11 Accumu-
lation of β-glucosylceramide, the substrate of GCase, in neurons
promotes the formation of α-synuclein oligomers, which are
considered toxic in PD.12 Enhancement of GCase activity is
thought to be a potential therapeutic strategy for GCase-
associated synucleinopathies, including PD.13,14
An emerging therapeutic approach involves the restoration of
proper folding and lysosome delivery of degradation-prone
mutant enzymes using small molecules as pharmacological
chaperones (PCs).5 Previous studies have shown that
iminosugars increase the cellular activity of the N370S mutant
form of GCase,15,16 as well as of the wild-type enzyme.5,17
Isofagomine (IFG, 1) attracted the most attention in the
Figure 1. Structures of GCase inhibitors.
Received: June 22, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b00930
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a
Scheme 1. Synthesis of 4, 6a−6k, 7a−7i, 8a−8g, and 9a−9f with Substituents on the Secondary Amine
a
Reagents and conditions: (a) (i) sulfolane, (ii) PCl5; (b) RNH2, K2CO3, DMF
reported as GCase PCs since 2007;20−24 however, the binding
site and the interaction with GCase of these noniminosugar
PCs remain unknown.
interfere with our assay results, which was also reported
recently by Berger et al.;29 therefore, we excluded taurocholate
in our screening assay. Using this approach, we discovered
several different scaffolds of GCase inhibitors and activators
with moderate activity. Among these, we identified a quinazo-
line compound (4, Figure 1) as a potent GCase inhibitor. The
quinazoline ring had previously been found as the best scaffold
for GCase inhibitors among several ring systems assayed.21
Here we describe our modifications of the substituents on the
quinazoline ring.
To examine the SAR at the amino group, a series of
substituents was introduced at the 4-position of the N-
cyclohexyl ring of 4 (Table 1). A 4-methyl substituent (6a,
cis/trans = 3/2 mixture) resulted in 3-fold higher activity (IC50
56 nM), while 4-ethyl substitution (6b, cis/trans = 3/2 mixture)
did not show a significant change of activity, suggesting that a
smaller hydrophobic group at this position may be beneficial.
Then, a trans-4-methyl compound (6c) was synthesized, which
exhibited great improvement of inhibitory activity (IC50 20
nM). Further modification by installation of 4-methoxyl (6d),
4,4-dimethyl (6e), or 4,4-difluoro (6f) groups at the same
position decreased the potency; dimethylaminocyclohexyl (6g)
and N-methylpiperidino (6i) substituents were detrimental to
activity. Replacement of the cyclohexyl group by tetrahydro-
2H-pyran-3-yl (6h) or N-substituted piperidine (6i−6k)
afforded weak or inactive compounds, supporting the
importance of the lipophilic cyclohexyl ring.
To further expand the SAR of the amino group, the
cyclohexyl ring of 4 was replaced by a series of saturated carbon
rings of different sizes. A dramatic SAR was observed with
different carbon rings (7a−7e). As shown in Table 2, the larger
cycloalkyl rings were more potent; the compound with a
cyclooctyl group (7a, IC50 27 nM) was the most potent.
However, when bulk was introduced to the cycloalkyl ring, the
potency of the compounds (7f and 7g) decreased, suggesting
that the hydrophobic binding pocket may be compact.
Introduction of one or two carbons between the cyclohexyl
and NH groups (7h and 7i) in 4 decreased the inhibitory
activity, again indicating a hydrophobic pocket with limited
volume.
In our high throughput screening effort to discover potent
GCase modulators, compound 4 (Figure 1) was identified as a
potent GCase inhibitor (IC50 0.177 μM) in a 4-methylumbelli-
feryl β-D-glucopyranoside (4MU-β-Glc) enzyme activity based
high throughput screen. The activity was confirmed with
additional synthesized compounds. To further develop potent
GCase modulators and use them to explore the properties of
the binding site, we carried out a structure−activity relationship
(SAR) study of a series of quinazoline derivatives, leading to
the discovery of single-digit nanomolar potency GCase
inhibitory modulators.
CHEMISTRY
■
The synthesis of compound 4 and its analogues for SAR
exploration is straightforward and is detailed in Schemes 1 and
2. As showed in Scheme 1, 5 was prepared from 2-amino-
benzonitrile and nicotinoyl chloride according to a known
method.25 The reaction of 5 and appropriate amines in the
presence of potassium carbonate as a base afforded 4, 6a−6k,
7a−7i, 8a−8g, and 9a−9f.
Additional analogues having modifications at position 2 of
the quinazoline ring were synthesized, employing alkylation of
2,3-dihydro-1H-inden-2-amine with 2,4-dichloroquinazoline,
followed by Suzuki coupling with appropriate boronic acids
to afford 11a−11h (Scheme 2). The structure and purity of all
the prepared compounds were confirmed by spectroscopic and
analytical techniques.
Scheme 2. Synthesis of 11a−11h with Modifications at the 2-
a
Position of the Quinazoline Ring
a
Reagents and conditions: (a) 2,3-dihydro-1H-inden-2-amine, K2CO3,
To understand the nature of the binding site, a phenyl ring
with different length linkers was introduced into the molecules
(8a−8e, Table 3). Compound 8a, by replacement of the
cyclohexyl ring in 4 with a phenyl ring, lost activity. Insertion of
a 1−4 carbon linker between the phenyl and quinazoline rings
gave 8b−8e. Interestingly, 8c, with a phenylethyl group, was
slightly more potent than 4. Extension of the linker did not
benefit activity, suggesting that a two carbon length linker
between the phenyl ring and secondary amino group of 8c may
allow optimal binding of the phenyl group. Substitution of the
phenyl group in 8 with a 2- or 3-pyridine ring (8f or 8g) sharply
DMF; (b) RB(OH)2, Pd(PPh3)4, K2CO3, 1,4-dioxane, H2O
RESULTS AND DISCUSSION
■
In our high throughput screening efforts to discover potent
GCase inhibitors/activators, we used recombinant wild-type
GCase and 4MU-β-Glc as substrate in an optimized pH 5.9
buffer.26 In previous studies, high concentrations of taurocho-
late (4−10 mM) were used to improve the signal in GCase
enzyme activity assays.16,27,28 We found that taurocholate can
B
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Table 1. Structure and Inhibitory Activity of 2-(Pyridin-3-yl)quinazoline Derivatives with Substituted Cyclohexyl and Related
a
Rings
a
Experiments were performed in triplicate, and the mean SD is shown.
diminished potency, indicating a repulsive effect of the pyridine
nitrogen atom.
other two compounds (11b and 11c) with a methyl group at
positions 5 and 6 were much more potent, comparable to that
when a 2-furanyl group replaced the pyridine ring but still not
as potent as 9c. The replacement of the 3-pyridinyl ring in 9c
with either phenyl (11d) or 3-thienyl (11g) groups, however,
gave compounds that retained the same potency as 9c,
suggesting that more groups could be introduced at position
2 of the quinazoline ring.
We also evaluated the activity of these selected compounds at
various pH values (Table 6). Interestingly, compared to the
inhibitory activity at pH 5.9, the activity of 9a, 9b, and 9c with a
3-pyridinyl ring decreased by 3-fold at pH 5.0, while the activity
of 11d, 11f, and 11g only dropped slightly at both pH
conditions. Although the cause for the change in IC50 values is
not clear, an explanation may involve the protonation state of
the compounds or the variability of the enzyme efficacy at
different pH values.
Compounds shown to act as pharmacological chaperones for
GCase (or other lysosomal enzymes) also stabilize the enzyme
against thermal denaturation. A fluorescent thermal shift assay
was developed to evaluate the binding affinity of ligands with
protein.30 To evaluate their abilities to stabilize GCase, the
most potent compounds, 9a, 9b, 11d, 11f, and 11g, were
accessed in a wild-type GCase fluorescent thermal shift assay
with a negative control (8a) and a positive control (IFG) at pH
5.0. The selected compounds increased the GCase melting
To enhance the binding affinity of the compounds with
GCase, a new series of compounds was designed to integrate
both the hydrophobic interaction of the A ring and the π−π
interaction of the B ring by fusing the cyclohexyl ring (4) with a
phenyl ring (Figure 2 and Table 4). These derivatives (9a−9c)
exhibited single-digit nanomolar inhibitory activity against
GCase. Stereochemistry did not seem to be important (9a
and 9b). The compound with an indane ring (9c) gave
comparable activity to that of the tetralin ring (9a). Attachment
of the quinazoline ring to the tetralin ring (9d) and indane ring
(9e) at position 1 instead of position 2 (9a and 9c)
dramatically increased the IC50 values to the low micromolar
range, indicating the importance of the orientation of this
substituent for binding activity of these inhibitors. The
introduction of an oxygen atom to give a chromane (9f) did
not significantly affect the potency. These results suggest
important hydrophobic and π−π interactions in the binding of
this series of compounds to GCase.
Finally, we examined the substituent effect on the pyridine
ring. A methyl group was introduced at different positions of
the 3-pyridinyl ring of 9c to give 11a−11c (Table 5).
Methylation of the pyridine ring decreased the potency.
Whereas the compound with a methyl group at position 4 of
the pyridinyl ring (11a) showed only moderate potency, the
C
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a
Table 2. Structure and Inhibitory Activity of 2-(Pyridin-3-yl)quinazoline Derivatives with Saturated Alkyl Rings
a
Experiments were performed in triplicate, and the mean SD is shown.
a
Table 3. Structure and Inhibitory Activity of 2-(Pyridin-3-yl)quinazoline Derivatives with Aromatic Rings
a
Experiments were performed in triplicate, and the mean SD is shown.
point in a dose-dependent manner (Figure 3), while inactive
compound 8a did not change the melting points significantly.
Most compounds exhibited greater ability to stabilize GCase
than IFG at lower concentrations, and 11g showed the
maximum thermal shift up to around 11 °C. It should be
noted that the increment in melting point has a direct
correlation with the compound’s binding affinity.31 The
maximum thermal shift of our compounds corresponded to
their inhibitory activity at pH 5.0, suggesting a close correlation
of the binding affinity and the inhibitory activity for these
compounds.
The selected compounds (9a, 9b, 11d, 11g, and 11f) were
further evaluated against two other lysosomal hydrolases, acid
α-glucosidase (GAA) and α-galactosidase A (GLA). The
activities of tested enzymes were not significantly changed by
D
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CONCLUSIONS
■
In this paper, we describe the design and SAR of a series of
quinazoline GCase inhibitors having single-digit nanomolar
potency. The SAR suggested that a hydrophobic interaction
and a π−π interaction may be involved in compound binding to
GCase. These quinazoline derivatives also stabilized GCase, as
indicated by thermal shift assays, and exhibited high selectivity
against other lysosomal hydrolases. Furthermore, the most
potent compound (11g) increased the mature post-ER form of
GCase and the enzyme activity in patient-derived N370S
fibroblasts. It will be of interest to further test these compounds
in other biological assays and models of Gaucher’s and
Parkinson’s disease.
Figure 2. Rational design of a new series of potent quinazoline
inhibitors.
compound treatments up to 100 μM (representative results
shown for 11g in Figure 4).
We further tested compounds 4 and 11g by measuring
GCase activity at various substrate concentrations (30−150
μM) and in the absence or presence of increasing
concentrations of GCase inhibitors. Similarly to reported
noniminosugar inhibitors,20 both of our inhibitors exhibited
linear mixed inhibition, with an increase in Km and decrease in
Vmax values upon increasing inhibitor concentrations (Figure
5A,B)
Finally, we tested 11g in patient-derived N370S fibroblasts.
The fibroblasts were treated with 11g and IFG for 3 days, and
levels of GCase protein were determined by immunoblot. We
found that 11g treatments (2, 5, and 10 μM) increased the level
of GCase, while IFG showed a similar effect only at the higher
concentrations (20 and 50 μM) (Figure 6, top). We also used
endoH and PNGase F to digest the cell lysates to determine
whether 11g treatment affected GCase maturation. The
immunoblot (Figure 6, middle) showed that most of the
GCase bands were resistant to endoH digestion, indicating that
the major GCase signal after treatment is a post-ER form.
PNGase F digestion gave a single band with increased GCase
concentration after 11g treatment (Figure 6, bottom). In
addition to this finding, we also found that a 3-day treatment of
compound 11g (2 μM) increased 50% of GCase activity in a
separate enzyme activity assay, suggesting that 11g increased
both GCase levels and the activity of patient-derived N370S
fibroblasts.
EXPERIMENTAL SECTION
■
Materials and Methods. Chemistry. Commercially available
reagents and solvents were used without further purification. All
reactions were monitored by thin-layer chromatography (TLC) using
0.25 mm Silicycle extra hard 250 μM TLC plates (60 F254).
Purification of reaction products was carried out by flash
chromatography using an Agilent 971-FP flash purification system
with Silicycle silica gel columns. The yields are not optimized. The
purity of all compounds was over 95% as analyzed with an Agilent
1260 Infinity HPLC system and an Agilent Poroshell 120 EC-C18 (4.6
mm × 50 mm, 2.7 μm) reverse phase column, detecting with UV
1
absorbance (254 nm). H NMR and 13C NMR spectra were obtained
1
using a Bruker Avance III 500 MHz system (500 MHz for H NMR
and 125 MHz for 13C NMR) spectrometer. Chemical shifts are
1
reported relative to chloroform (δ = 7.26 for H NMR and δ = 77.16
for 13C NMR spectra) or dimethyl sulfoxide (δ = 2.50 for 1H and δ =
39.52 for 13C NMR spectra). Data are reported as br = broad, s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Mass
spectra were obtained using a Bruker AmaZon SL system. High
resolution mass spectra (HRMS) were performed using an Agilent
6210A LC-TOF instrument with a dual spray ESI source, with a high
resolution time-of-flight (TOF) mass analyzer and collecting in a 2
GigHz detector mode coupled with an Agilent 1200 HPLC.
Preparation of 4-Chloro-2-(pyridin-3-yl)quinazoline (5).25 To a
solution of 2-aminobenzonitrile (5.90 g, 50 mmol) in sulfolane (20
mL) was added nicotinoyl chloride hydrochloride (12.0 g, 67.4 mmol),
and the mixture was stirred at 100 °C for 16 h. PCl5 (18.2 g, 87.5
a
Table 4. Structure and Inhibitory Activity of 2-(Pyridin-3-yl)quinazoline Derivatives with Fused Rings
a
Experiments were performed in triplicate, and the mean SD is shown.
E
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a
Table 5. Structure and Inhibitory Activity of 4-(2,3-Dihydro-1H-2-indenamino)quinazoline Derivatives with Aromatic Rings
a
Experiments were performed in triplicate, and the mean SD is shown.
mmol) was added in one portion and stirred at 100 °C for 10 h. The
mixture was cooled to room temperature and carefully poured into
400 mL of saturated sodium bicarbonate solution cooling in an ice
bath. The solid was filtered, washed with water, dried, and purified by
flash chromatography to give 5 as a pale-yellow solid (5.50 g, 46%);
mp 160−163 °C. 1H NMR (400 MHz, CDCl3) δ 9.76 (d, J = 1.3 Hz,
1H), 8.82 (dt, J = 8.0, 1.9 Hz, 1H), 8.73 (dd, J = 4.7, 1.4 Hz, 1H), 8.26
(dd, J = 8.4, 0.8 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.95 (ddd, J = 8.4,
7.0, 1.4 Hz, 1H), 7.69 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.44 (dd, J = 7.5,
4.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 162.9, 158.3, 151.8,
151.7, 150.3, 136.1, 135.2, 132.4, 129.1, 128.9, 126.0, 123.5, 122.8. ESI-
MS m/z: 242 (M + H)+.
General Procedure for Compounds 4, 6a−6k, 7a−7i, 8a−8g,
and 9a−9f. A mixture of 4-chloro-2-(pyridin-3-yl)quinazoline 5 (72
mg, 0.3 mmol), amine (0.3 mmol), and potassium carbonate (69 mg,
0.3 mmol) in DMF (3 mL) was stirred at room temperature or 60 °C
overnight. Water (20 mL) was added, and the formed solid was
filtered, washed with water, and dried in vacuo to give product. The
products were usually pure (>95% purity). Those products without
sufficient purity were purified by flash chromatography.
Analytical Data for Compounds. N-Cyclohexyl-2-(pyridin-3-
yl)quinazolin-4-amine (4). Off-white solid (64 mg, 70%); mp 108−
111 °C. 1H NMR (500 MHz, CDCl3) δ 9.74 (s, 1H), 8.84 (d, J = 6.6
Hz, 1H), 8.70 (s, 1H), 7.95 (d, J = 7.1 Hz, 1H), 7.74 (t, J = 7.7 Hz,
2H), 7.49−7.35 (m, 2H), 5.77 (s, 1H), 4.48−4.32 (m, 1H), 2.28−2.20
(m, 2H), 1.93−1.79 (m, 2H), 1.79−1.67 (m, 1H), 1.61−1.46 (m, 2H),
1.46−1.23 (m, 3H). 13C NMR (125 MHz, CDCl3) δ 158.9, 158.8,
150.7, 150.5, 150.3, 135.7, 134.6, 132.7, 129.0, 125.8, 123.2, 120.5,
113.9, 50.1, 33.0, 25.9, 25.1. HRMS (ESI): calcd for C19H21N4 [M +
H]+, 305.1761; found, 305.1764.
(500 MHz, CDCl3) δ 9.75 (s, 1H), 8.80 (d, J = 7.8 Hz, 1H), 8.69 (s,
1H), 7.92 (d, J = 8.3 Hz, 1H), 7.77−7.70 (m, 2H), 7.48−7.39 (m,
2H), 5.63 (d, J = 5.3 Hz, 1H), 4.38−4.26 (m, 1H), 2.28 (d, J = 9.9 Hz,
2H), 1.86−1.81 (m, 2H), 1.54−1.40 (m, 1H), 1.40−1.30 (m, 2H),
1.30−1.14 (m, 2H), 0.98 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ 159.1, 158.8, 150.8, 150.5, 150.4, 135.8, 134.5, 132.8, 129.0,
125.9, 123.3, 120.7, 114.0, 50.5, 34.1, 33.1, 32.3, 22.4. HRMS (ESI):
calcd for C20H23N4 [M + H]+, 319.1917; found, 319.1921.
N-(4-Ethylcyclohexyl)-2-(pyridin-3-yl)quinazolin-4-amine (cis/
trans = 3/2) (6b). Pale-yellow solid (38 mg, 30%); mp 62−65 °C.
1
Cis isomer: H NMR (500 MHz, CDCl3) δ 9.74 (s, 1H), 8.81 (d, J =
7.7 Hz, 1H), 8.68 (s, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.77−7.71 (m,
1H), 7.48−7.39 (m, 2H), 5.85 (d, J = 5.8 Hz, 1H), 4.67−4.61 (m,
1H), 2.02−1.70 (m, 5H), 1.38−1.15 (m, 4H), 0.93 (t, J = 7.4 Hz, 3H).
13C NMR (125 MHz, CDCl3) δ 159.0, 158.8, 150.8, 150.4, 135.8,
134.5, 132.8, 128.9, 125.9, 123.3, 120.5, 113.9, 47.5, 39.0, 30.8, 29.7,
29.3, 28.1, 11.8. Trans isomer: 1H NMR (500 MHz, CDCl3) δ 9.74 (s,
1H), 8.81 (d, J = 7.7 Hz, 1H), 8.68 (s, 1H), 7.92 (d, J = 8.2 Hz, 1H),
7.77−7.71 (m, 1H), 7.48−7.39 (m, 2H), 5.63 (brs, 1H), 4.38−4.30
(m, 1H), 2.28 (d, J = 9.9 Hz, 2H), 1.99−1.70 (m, 2H), 1.54−1.40 (m,
1H), 1.40−1.30 (m, 2H), 1.30−1.15 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H).
13C NMR (125 MHz, CDCl3) δ 159.1, 158.8, 150.8, 150.4, 135.8,
134.5, 132.8, 129.0, 125.9, 123.3, 120.7, 114.0, 50.8, 37.7, 33.1, 31.7,
28.3, 11.8. HRMS (ESI): calcd for C21H25N4 [M + H]+, 333.2074;
found, 333.2080.
Trans-N-(4-Methylcyclohexyl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(6c). White solid (52 mg, 55%); mp 173−175 °C. H NMR (500
MHz, CDCl3) δ 9.75 (s, 1H), 8.80 (d, J = 7.8 Hz, 1H), 8.69 (s, 1H),
7.92 (d, J = 8.3 Hz, 1H), 7.77−7.71 (m, 1H), 7.70 (d, J = 8.2 Hz, 1H),
7.53−7.33 (m, 2H), 5.63 (d, J = 5.3 Hz, 1H), 4.38−4.26 (m, 1H), 2.28
(d, J = 9.9 Hz, 2H), 1.84 (d, J = 12.6 Hz, 2H), 1.54−1.40 (m, 1H),
1.40−1.30 (m, 2H), 1.30−1.14 (m, 2H), 0.98 (d, J = 6.5 Hz, 3H). 13C
NMR (125 MHz, CDCl3) δ 159.0, 158.8, 150.8, 150.5, 150.4, 135.7,
134.6, 132.7, 129.0, 125.7, 123.2, 120.5, 113.9, 50.4, 34.1, 33.1, 32.3,
22.3. HRMS (ESI): calcd for C20H23N4 [M + H]+, 319.1917; found,
319.1921.
N-(4-Methylcyclohexyl)-2-(pyridin-3-yl)quinazolin-4-amine (cis/
trans = 3/2) (6a). White solid (54 mg, 43%); mp 130−134 °C. Cis
1
isomer: H NMR (500 MHz, CDCl3) δ 9.75 (s, 1H), 8.80 (d, J = 7.8
Hz, 1H), 8.69 (s, 1H), 7.92 (d, J = 8.3 Hz, 1H), 7.77−7.71 (m, 2H),
7.48−7.39 (m, 2H), 5.87 (d, J = 5.8 Hz, 1H), 4.65−4.58 (m, 1H),
2.02−1.94 (m, 2H), 1.86−1.81 (m, 2H), 1.76−1.62 (m, 3H), 1.36−
1.21 (m, 2H), 1.00 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz, CDCl3)
δ 159.0, 158.8, 150.8, 150.4, 150.3, 135.8, 134.5, 132.8, 128.8, 125.9,
123.3, 120.5, 113.9, 47.2, 30.8, 30.4, 29.2, 21.3. Trans isomer: 1H NMR
N-(4-Methoxycyclohexyl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(6d). White solid (50 mg, 50%); mp 163−165 °C. H NMR (500
MHz, CDCl3) δ 9.73 (s, 1H), 8.81 (d, J = 7.7 Hz, 1H), 8.70 (d, J = 3.4
F
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a
Table 6. GCase Inhibitory Activity of 9a, 9b, 9c, 11d, 11f, and 11g at pH 5.0, pH 5.9, and pH 7.0, Respectively
a
Experiments were performed in triplicate, and the mean SD is shown.
Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.81−7.57 (m, 2H), 7.52−7.32 (m,
2H), 5.69 (d, J = 0.8 Hz, 1H), 4.52−4.27 (m, 1H), 3.40 (s, 3H), 3.31−
3.13 (m, 1H), 2.35 (d, J = 12.5 Hz, 2H), 2.18 (d, J = 11.3 Hz, 2H),
1.61−1.22 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 159.1, 158.7,
150.8, 150.3, 135.8, 134.3, 132.8, 128.9, 125.9, 123.3, 120.6, 113.8,
78.4, 56.0, 49.7, 30.6, 30.4. HRMS (ESI): calcd for C20H23N4O [M +
H]+, 335.1866; found, 335.1866.
125.8, 123.2, 120.6, 113.9, 50.3, 37.9, 31.8, 29.9, 28.7, 25.2. HRMS
(ESI): calcd for C21H25N4 [M + H]+, 333.2074; found, 333.2076.
N-(4,4-Difluorocyclohexyl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(6f). White solid (52 mg, 51%); mp 196−197 °C. H NMR (500
MHz, CDCl3) δ 9.72 (s, 1H), 8.80 (d, J = 7.8 Hz, 1H), 8.70 (d, J = 2.6
Hz, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.80−7.75 (m, 1H), 7.73 (d, J = 8.1
Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.43 (dd, J = 7.7, 4.8 Hz, 1H), 5.70
(d, J = 6.2 Hz, 1H), 4.60−4.45 (m, 1H), 2.32 (d, J = 12.6 Hz, 2H),
2.28−2.12 (m, 2H), 2.12−1.93 (m, 2H), 1.85−1.70 (m, 2H). 13C
NMR (125 MHz, CDCl3) δ 159.1, 158.5, 150.8, 150.3, 150.1, 135.8,
134.3, 133.1, 129.0, 126.2, 123.6 (q, JC−F = 241 Hz), 123.4, 120.5,
113.8, 48.1, 32.5 (t, JC−F = 24.7 Hz), 28.6 (d, JC−F = 9.2 Hz). HRMS
(ESI): calcd for C19H19F2N4 [M + H]+, 341.1572; found, 341.1577.
N-(4,4-Dimethylcyclohexyl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(6e). White solid (65 mg, 65%); mp 171−172 °C. H NMR (500
MHz, CDCl3) δ 9.74 (s, 1H), 8.80 (d, J = 7.9 Hz, 1H), 8.69 (d, J = 3.7
Hz, 1H), 7.90 (s, 1H), 7.79−7.63 (m, 2H), 7.45 (s, 1H), 7.41 (d, J =
3.1 Hz, 1H), 5.66 (d, J = 5.5 Hz, 1H), 4.43−4.25 (m, 1H), 2.16−2.00
(m, 2H), 1.64−1.38 (m, 6H), 1.01 (d, J = 3.0 Hz, 6H). 13C NMR (125
MHz, CDCl3) δ 159.0, 158.8, 150.8, 150.4, 135.7, 134.5, 132.7, 129.0,
G
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Figure 3. Fluorescent thermal shift analysis of selected compounds.
Compound 9a, 9b, 11d, 11f, 11g, and IFG showed their ability to
stabilize wild-type GCase in a dose-dependent manner. Data represent
the results of three independent experiments performed with three
replicates per sample.
Figure 5. Lineweaver−Burk plots of the enzyme kinetics of GCase
inhibitors (A) 4 and (B) 11g. Each inhibitor was tested in triplicate in
two independent assays at the concentrations shown in the legend box
○
of each graph, with ( ) indicating the absence of inhibitor. (A)
Compounds 4 and (B) 11g showed an increase in Km and a decrease
in Vmax, indicating linear mixed inhibition.
Figure 4. Selectivity of inhibitor 11g with related hydrolases. 11g was
tested on GCase, acid α-glucosidase (GAA), and α-galactosidase A
(GLA). Data represent the results of three independent experiments
performed with three replicates per sample.
1H), 8.68 (dd, J = 4.6, 1.2 Hz, 1H), 7.91 (d, J = 8.3 Hz, 1H), 7.77−
7.73 (m, 1H), 7.71 (d, J = 8.2 Hz, 1H), 7.49−7.43 (m, 1H), 7.41 (dd, J
= 7.8, 4.8 Hz, 1H), 5.68 (d, J = 7.1 Hz, 1H), 4.49−4.35 (m, 1H), 2.99
(d, J = 11.1 Hz, 2H), 2.90−2.79 (m, 1H), 2.47 (t, J = 11.6 Hz, 2H),
2.28 (d, J = 11.5 Hz, 2H), 1.80−1.66 (m, 2H), 1.13 (d, J = 6.4 Hz,
6H). 13C NMR (125 MHz, CDCl3) δ 159.0, 158.7, 150.8, 150.5, 150.4,
135.7, 134.5, 132.8, 129.0, 125.9, 123.3, 120.6, 113.9, 54.9, 48.7, 47.8,
32.3, 18.4. HRMS (ESI): calcd for C21H26N5 [M + H]+, 348.2183;
found, 348.2184.
N-(4-Dimethylaminocyclohexyl)-2-(pyridin-3-yl)quinazolin-4-
1
amine (6g). Pale-yellow solid (63 mg, 60%); mp 231−235 °C. H
NMR (500 MHz, CDCl3) δ 9.70 (s, 1H), 8.76 (d, J = 7.8 Hz, 1H),
8.65 (d, J = 3.6 Hz, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 8.1 Hz,
1H), 7.75−7.66 (m, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.37 (dd, J = 7.5, 4.9
Hz, 1H), 6.02 (d, J = 6.7 Hz, 1H), 4.64 (s, 1H), 2.38 (s, 6H), 2.30 (s,
1H), 2.05 (s, 2H), 1.83 (s, 6H). 13C NMR (125 MHz, CDCl3) δ
159.0, 158.6, 150.6, 150.3, 150.2, 135.7, 134.6, 132.7, 128.8, 125.9,
123.2, 120.8, 114.0, 62.2, 46.0, 42.5, 27.9, 25.6. HRMS (ESI): calcd for
C21H26N5 [M + H]+, 348.2183; found, 348.2184.
N-(1-Benzylpiperidin-4-yl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(6k). Yellow solid (67 mg, 57%); mp 180−182 °C. H NMR (500
MHz, CDCl3) δ 9.72 (d, J = 1.6 Hz, 1H), 8.78 (dt, J = 7.9, 1.9 Hz,
1H), 8.69 (dd, J = 4.8, 1.6 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.77−
7.72 (m, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.50−7.43 (m, 1H), 7.41 (dd, J
= 7.8, 5.0 Hz, 1H), 7.39−7.27 (m, 5H), 5.66 (d, J = 7.1 Hz, 1H),
4.49−4.37 (m, 1H), 3.62 (s, 2H), 2.98 (d, J = 10.6 Hz, 2H), 2.38−2.29
(m, 2H), 2.24 (d, J = 11.4 Hz, 2H), 1.81−1.69 (m, 2H). 13C NMR
(125 MHz, CDCl3) δ 159.0, 158.7, 150.8, 150.5, 150.3, 135.6, 134.5,
132.8, 129.4, 129.0, 128.4, 127.4, 125.9, 123.2, 120.6, 113.9, 63.2, 52.5,
48.4, 32.0. HRMS (ESI): calcd for C25H26N5 [M + H]+, 396.2183;
found, 396.2184.
N-(Tetrahydro-2H-pyran-3-yl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(6h). White solid (48 mg, 53%); mp 112−113 °C. H NMR (500
MHz, CDCl3) δ 9.72 (s, 1H), 8.80 (dt, J = 7.9, 1.9 Hz, 1H), 8.68 (d, J
= 3.7 Hz, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H),
7.78−7.73 (m, 1H), 7.50−7.44 (m, 1H), 7.41 (dd, J = 7.8, 4.8 Hz,
1H), 6.18 (d, J = 7.1 Hz, 1H), 4.68−4.59 (m, 1H), 4.01 (dd, J = 11.5,
2.7 Hz, 1H), 3.89−3.80 (m, 1H), 3.80−3.66 (m, 2H), 2.08−2.00 (m,
3H), 1.95−1.83 (m, 1H), 1.73−1.61 (m, 1H). 13C NMR (125 MHz,
CDCl3) δ 159.0, 158.6, 150.7, 150.3, 150.2, 135.9, 134.4, 133.0, 128.9,
126.1, 123.3, 120.7, 113.9, 71.5, 68.6, 46.5, 27.8, 23.0. HRMS (ESI):
calcd for C18H19N4O [M + H]+, 307.1553; found, 307.1556.
N-Cyclooctyl-2-(pyridin-3-yl)quinazolin-4-amine (7a). White solid
(65 mg, 66%); mp 140−142 °C. 1H NMR (500 MHz, CDCl3) δ 9.75
(d, J = 1.4 Hz, 1H), 8.82 (d, J = 7.9 Hz, 1H), 8.69 (dd, J = 4.7, 1.4 Hz,
1H), 7.92 (d, J = 8.3 Hz, 1H), 7.76−7.72 (m, 1H), 7.70 (d, J = 8.1 Hz,
1H), 7.51−7.35 (m, 2H), 5.73 (d, J = 6.2 Hz, 1H), 4.73−4.56 (m,
1H), 2.18−2.00 (m, 2H), 2.00−1.43 (m, 12H). 13C NMR (125 MHz,
CDCl3) δ 158.8, 158.6, 150.7, 150.3, 150.2, 135.8, 134.5, 132.7, 128.9,
125.8, 123.3, 120.5, 114.0, 51.0, 32.7, 27.2, 26.1, 24.2. HRMS (ESI):
calcd for C21H25N4 [M + H]+, 333.2074; found, 333.2076.
N-Cycloheptyl-2-(pyridin-3-yl)quinazolin-4-amine (7b). Off-white
solid (70 mg, 74%); mp 158−159 °C. 1H NMR (500 MHz, CDCl3) δ
9.75 (s, 1H), 8.82 (d, J = 7.9 Hz, 1H), 8.68 (s, 1H), 7.93 (d, J = 8.3
Hz, 1H), 7.78−7.65 (m, 2H), 7.50−7.37 (m, 2H), 5.76 (d, J = 5.6 Hz,
1H), 4.64−4.46 (m, 1H), 2.25−2.12 (m, 2H), 1.83−1.52 (m, 10H).
N-(1-Methylpiperidin-4-yl)-2-(pyridin-3-yl)quinazolin-4-amine
(6i). Pale-yellow solid (34 mg, 35%); mp 132−133 °C. 1H NMR (500
MHz, CDCl3) δ 9.72 (s, 1H), 8.78 (d, J = 7.5 Hz, 1H), 8.68 (d, J = 3.4
Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.73 (t, J = 7.4 Hz, 1H), 7.49−7.34
(m, 3H), 5.73 (d, J = 6.1 Hz, 1H), 4.48−4.30 (m, 1H), 2.91 (d, J = 9.1
Hz, 2H), 2.36 (s, 3H), 2.32−2.17 (m, 4H), 1.72 (m, 2H). 13C NMR
(125 MHz, CDCl3) δ 159.0, 158.7, 150.8, 150.4, 150.3, 135.6, 134.5,
132.8, 129.0, 125.9, 123.2, 120.7, 113.9, 54.7, 48.0, 46.3, 32.1. HRMS
(ESI): calcd for C19H22N5 [M + H]+, 320.1870; found, 320.1872.
N-(1-Isopropylpiperidin-4-yl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(6j). Yellow solid (49 mg, 47%); mp 191−192 °C. H NMR (500
MHz, CDCl3) δ 9.73 (d, J = 1.2 Hz, 1H), 8.79 (dt, J = 7.8, 1.5 Hz,
H
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Figure 6. Modulator 11g increases GCase protein levels in patient-derived Gaucher’s disease fibroblasts. Top: Western blot of N370S fibroblast
treated with 11g or IFG for 3 days. Middle: Western blot of endoH digestion of N370S fibroblast after a 3-day treatment of 11g, IFG, or DMSO;
endoH resistance of proteins indicates their post-ER localization; Bottom: Western blot of PNGase F digestion of N370S fibroblast after a 3-day
treatment of 11g, IFG, or DMSO. The GAPDH signal was used for the loading control.
13C NMR (125 MHz, CDCl3) δ 158.7, 158.6, 150.7, 150.3, 150.2,
135.8, 134.5, 132.7, 128.8, 125.9, 123.3, 120.6, 113.9, 52.3, 34.9, 28.3,
24.6. HRMS (ESI): calcd for C20H23N4 [M + H]+, 319.1917; found,
319.1918.
N-Cyclopentyl-2-(pyridin-3-yl)quinazolin-4-amine (7c). Off-white
solid (52 mg, 60%); mp 147−148 °C. 1H NMR (500 MHz, CDCl3) δ
9.76 (d, J = 1.5 Hz, 1H), 8.81 (dt, J = 7.9, 1.9 Hz, 1H), 8.68 (dd, J =
4.8, 1.7 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.76−7.71 (m, 1H), 7.70 (d,
J = 8.2 Hz, 1H), 7.46−7.42 (m, 1H), 7.40 (dd, J = 7.9, 4.8 Hz, 1H),
5.73 (d, J = 6.1 Hz, 1H), 4.80−4.72 (m, 1H), 2.34−2.25 (m, 2H),
1.86−1.71 (m, 4H), 1.67−1.59 (m, 2H). 13C NMR (125 MHz,
CDCl3) δ 159.4, 158.8, 150.7, 150.4, 150.3, 135.7, 134.6, 132.7, 129.0,
125.8, 123.2, 120.6, 113.9, 53.2, 33.4, 24.1. HRMS (ESI): calcd for
C18H19N4 [M + H]+, 291.1604; found, 291.1604.
N-Cyclobutyl-2-(pyridin-3-yl)quinazolin-4-amine (7d). Off-white
solid (47 mg, 57%); mp 173−174 °C. 1H NMR (500 MHz, CDCl3) δ
9.75 (s, 1H), 8.87−8.79 (m, 1H), 8.69 (d, J = 4.2 Hz, 1H), 7.93 (dd, J
= 8.0, 4.1 Hz, 1H), 7.74 (t, J = 8.2 Hz, 2H), 7.47−7.43 (m, 1H), 7.43−
7.39 (m, 1H), 6.03 (s, 1H), 4.96−4.97 (m, 1H), 2.66−2.54 (m, 2H),
2.16−2.03 (m, 2H), 1.96−1.85 (m, 2H). 13C NMR (125 MHz,
CDCl3) δ 158.8, 150.7, 150.5, 150.4, 135.7, 134.5, 132.8, 129.0, 125.9,
123.2, 120.6, 113.7, 46.8, 31.4, 15.6. HRMS (ESI): calcd for C17H17N4
[M + H]+, 277.1448; found, 277.1449.
N-Cyclopropyl-2-(pyridin-3-yl)quinazolin-4-amine (7e). Pale-yel-
low solid (30 mg, 38%); mp 180−181 °C. 1H NMR (500 MHz,
CDCl3) δ 9.81 (s, 1H), 8.89 (d, J = 7.9 Hz, 1H), 8.70 (d, J = 3.7 Hz,
1H), 7.96 (d, J = 8.3 Hz, 1H), 7.77−7.73 (m, 1H), 7.71 (d, J = 8.2 Hz,
1H), 7.47−7.36 (m, 2H), 6.14 (s, 1H), 3.17 (td, J = 6.9, 3.1 Hz, 1H),
1.07−0.95 (m, 2H), 0.79−0.67 (m, 2H). 13C NMR (125 MHz,
CDCl3) δ 160.9, 158.9, 150.8, 150.5, 150.4, 135.8, 134.5, 132.8, 129.0,
126.0, 123.2, 120.6, 113.9, 24.5, 7.5. HRMS (ESI): calcd for C16H15N4
[M + H]+, 263.1291; found, 263.1291.
CDCl3) δ 9.73 (d, J = 1.6 Hz, 1H), 8.83 (d, J = 7.8 Hz, 1H), 8.69 (dd,
J = 4.7, 1.5 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.80−7.69 (m, 1H), 7.66
(d, J = 8.1 Hz, 1H), 7.51−7.32 (m, 2H), 5.58 (s, 1H), 2.38 (d, J = 2.3
Hz, 6H), 2.25−2.18 (m, 3H), 1.88−1.74 (m, 6H). 13C NMR (125
MHz, CDCl3) δ 159.0, 158.1, 150.7, 150.3, 135.8, 134.5, 132.6, 128.9,
125.9, 123.3, 120.6, 114.2, 53.6, 41.7, 36.7, 29.7. HRMS (ESI): calcd
for C23H25N4 [M + H]+, 357.2074; found, 357.2074.
N-(Bicyclo[2.2.1]heptan-2-yl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(7g). White solid (47 mg, 50%); mp 145−146 °C. H NMR (500
MHz, CDCl3) δ 9.75 (s, 1H), 8.82 (dt, J = 7.9, 1.8 Hz, 1H), 8.68 (d, J
= 3.9 Hz, 1H), 7.91 (d, J = 8.3 Hz, 1H), 7.75−7.71 (m, 1H), 7.69 (d, J
= 8.2 Hz, 1H), 7.49−7.35 (m, 2H), 5.64 (d, J = 5.5 Hz, 1H), 4.27−
4.16 (m, 1H), 2.51 (d, J = 4.1 Hz, 1H), 2.39 (s, 1H), 2.05 (ddd, J =
13.2, 7.9, 2.2 Hz, 1H), 1.73−1.61 (m, 1H), 1.61−1.53 (m, 1H), 1.51
(d, J = 10.1 Hz, 1H), 1.48−1.36 (m, 2H), 1.34−1.20 (m, 2H). 13C
NMR (125 MHz, CDCl3) δ 158.9, 158.7, 150.7, 150.3, 150.2, 135.8,
134.5, 132.7, 128.9, 125.8, 123.3, 120.5, 113.9, 55.1, 42.1, 41.0, 36.0,
35.9, 28.3, 26.7. HRMS (ESI): calcd for C20H21N4 [M + H]+,
317.1761; found, 317.1763.
N-(Cyclohexylmethyl)-2-(pyridin-3-yl)quinazolin-4-amine (7h).
1
Pale-yellow solid (67 mg, 71%); mp 121−123 °C. H NMR (500
MHz, CDCl3) δ 9.75 (s, 1H), 8.81 (d, J = 7.8 Hz, 1H), 8.68 (d, J = 3.8
Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.79−7.65 (m, 2H), 7.50−7.34 (m,
2H), 5.98 (s, 1H), 3.65 (t, J = 6.1 Hz, 2H), 1.87 (d, J = 12.6 Hz, 2H),
1.82−1.61 (m, 4H), 1.32−1.18 (m, 3H), 1.14−1.04 (m, 1H). 13C
NMR (125 MHz, CDCl3) δ 159.9, 158.8, 150.7, 150.3, 135.7, 134.6,
132.7, 128.9, 125.8, 123.2, 120.5, 114.0, 47.6, 38.0, 31.3, 26.5, 26.0.
HRMS (ESI): calcd for C20H23N4 [M + H]+, 319.1917; found,
319.1920.
N-(2-Cyclohexylethyl)-2-(pyridin-3-yl)quinazolin-4-amine (7i).
1
White solid (48 mg, 48%); mp 153−154 °C. H NMR (500 MHz,
CDCl3) δ 9.76 (s, 1H), 8.86−8.78 (m, 1H), 8.69 (d, J = 4.1 Hz, 1H),
7.92 (dd, J = 7.9, 5.4 Hz, 1H), 7.79−7.67 (m, 2H), 7.48−7.37 (m,
2H), 5.84 (s, 1H), 3.86−3.75 (m, 2H), 1.83 (d, J = 12.3 Hz, 2H),
N-(Adamantan-1-yl)-2-(pyridin-3-yl)quinazolin-4-amine (7f).
1
White solid (37 mg, 35%); mp 114−116 °C. H NMR (500 MHz,
I
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1.76−1.60 (m, 5H), 1.50−1.38 (m, 1H), 1.33−1.12 (m, 3H), 1.08−
0.96 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 159.7, 158.8, 150.7,
150.4, 150.3, 135.8, 135.7, 134.5, 132.8, 132.7, 128.9, 128.9, 125.9,
123.2, 120.6, 114.0, 39.4, 37.0, 35.7, 33.4, 26.6, 26.3. HRMS (ESI):
calcd for C21H25N4 [M + H]+, 333.2074; found, 333.2074.
N-Phenyl-2-(pyridin-3-yl)quinazolin-4-amine (8a). Off-white solid
(55 mg, 62%); mp 179−180 °C. 1H NMR (500 MHz, CDCl3) δ 9.73
(d, J = 1.8 Hz, 1H), 8.82 (dt, J = 7.9, 1.9 Hz, 1H), 8.70 (dd, J = 4.8, 1.6
Hz, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.95 (d, J = 8.1 Hz, 1H), 7.86 (d, J
= 7.6 Hz, 2H), 7.84−7.80 (m, 1H), 7.69 (brs, 1H), 7.61−7.52 (m,
1H), 7.50−7.45 (m, 2H), 7.43 (dd, J = 7.9, 4.8 Hz, 1H), 7.21 (t, J = 7.4
Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 158.4, 157.6, 150.8, 150.7,
150.2, 138.4, 136.1, 134.2, 133.3, 129.3, 129.2, 126.8, 124.6, 123.4,
121.6, 120.5, 114.1. HRMS (ESI): calcd for C19H15N4 [M + H]+,
299.1291; found, 299.1292.
N-Benzyl-2-(pyridin-3-yl)quinazolin-4-amine (8b). Off-white solid
(65 mg, 70%); mp 140−141 °C. 1H NMR (500 MHz, CDCl3) δ 9.56
(s, 1H), 8.61 (d, J = 7.9 Hz, 1H), 8.47 (d, J = 3.6 Hz, 1H), 7.74 (d, J =
8.3 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H), 7.58−7.44 (m, 1H), 7.28−7.18
(m, 4H), 7.18−7.13 (m, 2H), 7.13−7.07 (m, 1H), 6.39 (s, 1H), 4.78
(d, J = 5.4 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 159.6, 158.7,
150.6, 150.4, 150.2, 138.5, 135.8, 134.5, 132.8, 128.8, 128.0, 127.7,
126.0, 123.2, 120.9, 113.9, 45.4. HRMS (ESI): calcd for C20H17N4 [M
+ H]+, 313.1448; found, 313.1453.
N-Phenethyl-2-(pyridin-3-yl)quinazolin-4-amine (8c). White solid
(43 mg, 44%); mp 140−141 °C. 1H NMR (500 MHz, CDCl3) δ 9.79
(s, 1H), 8.85 (d, J = 7.1 Hz, 1H), 8.70 (s, 1H), 7.94 (d, J = 8.4 Hz,
1H), 7.78−7.69 (m, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.46−7.39 (m,
2H), 7.38−7.31 (m, 2H), 7.30−7.25 (m, 3H), 5.98 (s, 1H), 4.11−3.99
(m, 2H), 3.11 (t, J = 6.9 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ
159.6, 158.7, 150.8, 150.3, 150.2, 139.0, 135.8, 134.4, 132.9, 129.0,
128.9, 126.8, 126.0, 123.3, 120.5, 113.9, 42.6, 35.4. HRMS (ESI): calcd
for C21H19N4 [M + H]+, 327.1604; found, 327.1603.
(m, 2H), 2.98 (t, J = 6.9 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ
159.6, 158.8, 150.7, 150.3, 145.1, 135.8, 134.5, 132.8, 129.8, 128.9,
128.8, 125.9, 123.3, 120.6, 115.6, 114.0, 42.8, 34.5. HRMS (ESI): calcd
for C20H18N5 [M + H]+, 328.1557; found, 328.1560.
(S)-2-(Pyridin-3-yl)-N-(1,2,3,4-tetrahydronaphthalen-2-yl)-
quinazolin-4-amine (9a). Off-white solid (74 mg, 70%); mp 150−152
°C. 1H NMR (500 MHz, CDCl3) δ 9.75 (s, 1H), 8.82 (d, J = 7.9 Hz,
1H), 8.68 (d, J = 3.7 Hz, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.75 (t, J = 7.7
Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.47−7.42 (m, 1H), 7.41 (dd, J =
7.9, 4.8 Hz, 1H), 7.24−7.11 (m, 4H), 5.87 (d, J = 5.2 Hz, 1H), 5.00−
4.87 (m, 1H), 3.43 (dd, J = 16.2, 5.0 Hz, 1H), 3.12−2.88 (m, 3H),
2.40−2.30 (m, 1H), 2.13−2.01 (m, 1H). 13C NMR (125 MHz,
CDCl3) δ 159.2, 158.7, 150.8, 150.2, 135.8, 135.7, 134.3, 134.1, 132.9,
129.7, 129.1, 128.9, 126.4, 126.2, 126.0, 123.3, 120.7, 113.9, 47.1, 35.8,
28.5, 27.5. HRMS (ESI): calcd for C23H21N4 [M + H]+, 353.1761;
found, 328.1762.
(R)-2-(Pyridin-3-yl)-N-(1,2,3,4-tetrahydronaphthalen-2-yl)-
quinazolin-4-amine (9b). Off-white solid (65 mg, 62%); mp 153−156
°C. 1H NMR (500 MHz, CDCl3) δ 9.75 (s, 1H), 8.82 (d, J = 7.9 Hz,
1H), 8.68 (d, J = 3.7 Hz, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.75 (t, J = 7.7
Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.47−7.42 (m, 1H), 7.41 (dd, J =
7.9, 4.8 Hz, 1H), 7.24−7.11 (m, 4H), 5.87 (d, J = 5.2 Hz, 1H), 5.00−
4.87 (m, 1H), 3.43 (dd, J = 16.2, 5.0 Hz, 1H), 3.12−2.88 (m, 3H),
2.40−2.30 (m, 1H), 2.13−2.01 (m, 1H). 13C NMR (125 MHz,
CDCl3) δ 159.2, 158.7, 150.8, 150.2, 135.8, 135.7, 134.3, 134.1, 132.9,
129.7, 129.1, 128.9, 126.4, 126.2, 126.0, 123.3, 120.7, 113.9, 47.1, 35.8,
28.5, 27.5. HRMS (ESI): calcd for C23H21N4 [M + H]+, 353.1761;
found, 328.1764.
N-(2,3-Dihydro-1H-inden-2-yl)-2-(pyridin-3-yl)quinazolin-4-
1
amine (9c). Gray solid (75 mg, 74%); mp 167−168 °C. H NMR
(500 MHz, CDCl3) δ 9.78 (d, J = 1.7 Hz, 1H), 8.84 (dd, J = 7.9, 1.7
Hz, 1H), 8.69 (d, J = 4.7 Hz, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.77−7.70
(m, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.47−7.37 (m, 2H), 7.32−7.27 (m,
2H), 7.25−7.20 (m, 2H), 6.04 (s, 1H), 5.37−5.29 (m, 1H), 3.60 (dd, J
= 16.2, 7.2 Hz, 2H), 3.08 (dd, J = 16.2, 4.8 Hz, 2H). 13C NMR (125
MHz, CDCl3) δ 159.4, 158.6, 150.7, 150.3, 141.1, 135.8, 134.4, 132.9,
128.9, 127.0, 126.0, 125.1, 123.3, 120.7, 113.9, 52.6, 40.3. HRMS
(ESI): calcd for C24H21NO [M + H]+, 339.1618; found, 339.1612.
N-(1,2,3,4-Tetrahydronaphthalen-1-yl)-2-(pyridin-3-yl) quinazo-
lin-4-amine (9d). Pale-yellow sticky oil (35 mg, 33%). 1H NMR
(500 MHz, CDCl3) δ 9.78 (d, J = 0.9 Hz, 1H), 8.89 (d, J = 7.8 Hz,
1H), 8.69 (d, J = 3.5 Hz, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.83−7.73 (m,
1H), 7.68 (d, J = 8.0 Hz, 1H), 7.51−7.41 (m, 2H), 7.39 (d, J = 7.6 Hz,
1H), 7.25−7.22 (m, 1H), 7.22−7.15 (m, 2H), 6.02 (s, 1H), 5.94−5.86
(m, 1H), 3.00−2.80 (m, 2H), 2.34−2.23 (m, 1H), 2.20−2.12 (m, 1H),
2.00−1.93 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 159.0, 158.7,
150.6, 150.2, 138.1, 136.8, 136.1, 133.0, 129.5, 129.1, 128.8, 127.7,
126.6, 126.1, 123.4, 120.7, 113.8, 49.2, 29.6, 29.5, 20.2. HRMS (ESI):
calcd for C23H21N4 [M + H]+, 353.1761; found, 353.1767.
N-(3-Phenylpropyl)-2-(pyridin-3-yl)quinazolin-4-amine (8d). Off-
white sticky oil (60 mg, 59%). 1H NMR (500 MHz, CDCl3) δ 9.74 (s,
1H), 8.76 (dt, J = 7.9, 1.8 Hz, 1H), 8.68 (d, J = 3.6 Hz, 1H), 7.90 (d, J
= 8.3 Hz, 1H), 7.78−7.66 (m, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.45−
7.34 (m, 2H), 7.33−7.27 (m, 2H), 7.25−7.19 (m, 3H), 5.94 (s, 1H),
3.89−3.75 (m, 2H), 2.81 (t, J = 7.4 Hz, 2H), 2.13 (p, J = 7.2 Hz, 2H).
13C NMR (125 MHz, CDCl3) δ 159.7, 158.7, 150.7, 150.3, 150.2,
141.6, 135.8, 134.5, 132.7, 128.8, 128.7, 128.5, 126.2, 125.8, 123.2,
120.6, 113.9, 41.2, 33.7, 30.7. HRMS (ESI): calcd for C22H21N4 [M +
H]+, 341.1761; found, 341.1758.
N-(4-Phenylbutyl)-2-(pyridin-3-yl)quinazolin-4-amine (8e). Pale-
1
yellow solid (73 mg, 69%); mp 108−109 °C. H NMR (500 MHz,
CDCl3) δ 9.75 (s, 1H), 8.81 (d, J = 7.9 Hz, 1H), 8.69 (d, J = 3.7 Hz,
1H), 7.93 (d, J = 8.3 Hz, 1H), 7.77−7.71 (m, 1H), 7.70 (d, J = 8.1 Hz,
1H), 7.51−7.35 (m, 2H), 7.28 (d, J = 7.7 Hz, 2H), 7.23−7.10 (m,
3H), 5.90 (s, 1H), 3.89−3.70 (m, 2H), 2.72 (t, J = 6.7 Hz, 2H), 1.90−
1.75 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 159.7, 158.7, 150.8,
150.3, 150.1, 142.1, 135.8, 134.4, 132.8, 128.8, 128.5, 128.5, 126.0,
125.9, 123.3, 120.6, 113.9, 41.4, 35.7, 29.0, 28.9. HRMS (ESI): calcd
for C23H23N4 [M + H]+, 355.1917; found, 355.1919.
N-(2,3-Dihydro-1H-inden-1-yl)-2-(pyridin-3-yl)quinazolin-4-
1
amine (9e). Brown solid (57 mg, 56%); mp 128−130 °C. H NMR
(500 MHz, CDCl3) δ 9.78 (d, J = 1.5 Hz, 1H), 8.84 (dt, J = 7.9, 1.9
Hz, 1H), 8.67 (dd, J = 4.8, 1.6 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H),
7.80−7.73 (m, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.50−7.37 (m, 3H), 7.35
(d, J = 7.5 Hz, 1H), 7.33−7.28 (m, 1H), 7.26−7.20 (m, 2H), 6.17 (q, J
= 7.4 Hz, 1H), 6.02 (d, J = 7.5 Hz, 1H), 3.12 (ddd, J = 15.8, 8.7, 4.0
Hz, 1H), 3.04 (dt, J = 16.0, 8.0 Hz, 1H), 2.92−2.83 (m, 1H), 2.16−
2.01 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 159.5, 158.8, 150.8,
150.5, 150.3, 143.9, 143.4, 135.8, 134.5, 132.9, 129.0, 128.4, 127.0,
126.0, 125.2, 124.3, 123.3, 120.7, 113.8, 56.5, 34.1, 30.5. HRMS (ESI):
calcd for C22H19N4 [M + H]+, 339.1604; found, 339.1610.
N-(2-(Pyridin-2-yl)ethyl)-2-(pyridin-3-yl)quinazolin-4-amine (8f).
1
White solid (57 mg, 58%); mp 138−139 °C. H NMR (500 MHz,
CDCl3) δ 9.75 (d, J = 1.2 Hz, 1H), 8.82 (dt, J = 7.9, 1.9 Hz, 1H),
8.70−8.66 (m, 1H), 8.51 (s, 1H), 8.47 (d, J = 4.1 Hz, 1H), 7.93 (d, J =
8.2 Hz, 1H), 7.77−7.72 (m, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.59 (d, J =
7.8 Hz, 1H), 7.46−7.38 (m, 2H), 7.24 (dd, J = 7.7, 4.9 Hz, 1H), 6.28
(t, J = 5.3 Hz, 1H), 4.10−3.96 (m, 2H), 3.13 (t, J = 7.0 Hz, 2H). 13C
NMR (125 MHz, CDCl3) δ 159.7, 158.6, 150.7, 150.2, 150.1, 148.1,
136.6, 135.8, 134.7, 134.4, 133.0, 128.9, 126.2, 123.8, 123.4, 120.7,
113.9, 42.4, 32.6. HRMS (ESI): calcd for C20H18N5 [M + H]+,
328.1557; found, 328.1561.
N-(Chroman-3-yl)-2-(pyridin-3-yl)quinazolin-4-amine (9f). White
solid (67 mg, 63%); mp 145−148 °C. 1H NMR (500 MHz, CDCl3) δ
9.74 (s, 1H), 8.80 (dt, J = 7.9, 1.9 Hz, 1H), 8.68 (d, J = 3.6 Hz, 1H),
7.90 (d, J = 7.9 Hz, 1H), 7.72 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.63 (d, J
= 7.7 Hz, 1H), 7.45−7.34 (m, 2H), 7.19−7.12 (m, 1H), 7.07 (d, J =
6.6 Hz, 1H), 6.95−6.88 (m, 2H), 6.09 (d, J = 7.3 Hz, 1H), 5.11 (ddt, J
= 7.3, 3.6, 1.7 Hz, 1H), 4.43 (ddd, J = 11.0, 3.8, 2.2 Hz, 1H), 4.35 (dd,
J = 11.0, 1.7 Hz, 1H), 3.34 (dd, J = 16.8, 5.2 Hz, 1H), 3.10 (d, J = 16.8
Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 159.0, 158.4, 153.9, 150.7,
N-(2-(Pyridin-3-yl)ethyl)-2-(pyridin-3-yl)quinazolin-4-amine (8g).
Off-white solid (67 mg, 68%); mp 102−104 °C. 1H NMR (500 MHz,
CDCl3) δ 9.77 (d, J = 1.4 Hz, 1H), 8.83 (dt, J = 7.9, 1.9 Hz, 1H), 8.68
(dd, J = 4.8, 1.7 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.73 (ddd, J = 8.3,
7.0, 1.3 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.46−7.37 (m, 2H), 7.07 (d,
J = 8.3 Hz, 2H), 6.72−6.62 (m, 2H), 5.96−5.87 (m, 1H), 4.03−3.94
J
DOI: 10.1021/acs.jmedchem.6b00930
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
150.3, 150.1, 135.6, 134.2, 132.9, 130.6, 128.9, 127.8, 126.0, 123.2,
121.5, 120.7, 119.5, 117.0, 113.7, 68.0, 44.0, 30.4. HRMS (ESI): calcd
for C22H19N4O [M + H]+, 355.1553; found, 355.1551.
120.4, 113.6, 52.4, 40.2. HRMS (ESI): calcd for C23H20N3 [M + H]+,
338.1652; found, 338.1647.
N-(2,3-Dihydro-1H-inden-2-yl)-2-(pyridin-4-yl)quinazolin-4-
1
amine (11e). Brown solid (59 mg, 35%); mp 246−247 °C. H NMR
Preparation of 2-Chloro-N-(2,3-dihydro-1H-inden-2-yl)-
quinazolin-4-amine (10). A mixture of 2,4-dichloroquinazoline (398
mg, 2.0 mmol), 2,3-dihydro-1H-inden-2-amine (266 mg, 2.0 mmol),
and potassium carbonate (276 mg, 2.0 mmol) in DMF (5 mL) was
stirred at room temperature for 5 h. Water (20 mL) was added, and
the formed solid was filtered, washed with water, and solid dried to
(500 MHz, CDCl3) δ 8.74 (d, J = 5.5 Hz, 2H), 8.40 (d, J = 5.9 Hz,
2H), 7.93 (d, J = 8.2 Hz, 1H), 7.78−7.70 (m, 1H), 7.67 (d, J = 8.1 Hz,
1H), 7.48−7.39 (m, 1H), 7.29 (dd, J = 5.2, 3.4 Hz, 2H), 7.22 (dd, J =
5.4, 3.3 Hz, 2H), 5.99 (d, J = 6.7 Hz, 1H), 5.37−5.29 (m, 1H), 3.59
(dd, J = 16.2, 7.2 Hz, 2H), 3.07 (dd, J = 16.1, 4.8 Hz, 2H). 13C NMR
(125 MHz, CDCl3) δ 159.4, 158.4, 150.2, 150.2, 146.4, 141.0, 132.8,
129.2, 126.9, 126.3, 125.0, 122.3, 120.6, 114.1, 77.2, 77.0, 76.7, 52.5,
40.1. HRMS (ESI): calcd for C22H19N4 [M + H]+, 339.1604; found,
339.1608.
1
give 10 as an off-white solid (390 mg, 66%); mp 239−240 °C. H
NMR (500 MHz, CDCl3) δ 7.76 (d, J = 8.2 Hz, 1H), 7.74−7.69 (m,
1H), 7.60 (d, J = 8.2 Hz, 1H), 7.41 (t, J = 7.4 Hz, 1H), 7.30−7.26 (m,
2H), 7.24−7.19 (m, 2H), 6.08 (d, J = 6.7 Hz, 1H), 5.27−5.15 (m,
1H), 3.53 (dd, J = 16.2, 7.0 Hz, 2H), 3.00 (dd, J = 16.2, 4.0 Hz, 2H).
13C NMR (125 MHz, CDCl3) δ 160.6, 157.8, 151.0, 140.7, 133.6,
N-(2,3-Dihydro-1H-inden-2-yl)-2-(thiophen-2-yl)quinazolin-4-
1
amine (11f). Off-white solid (68 mg, 40%); mp 243−246 °C. H
NMR (500 MHz, CDCl3) δ 8.08 (d, J = 3.1 Hz, 1H), 7.85 (d, J = 8.4
Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.61 (d, J = 8.1 Hz, 1H), 7.45 (d, J =
4.9 Hz, 1H), 7.38−7.33 (m, 1H), 7.32−7.27 (m, 2H), 7.23 (dd, J =
5.3, 3.3 Hz, 2H), 7.18−7.13 (m, 1H), 5.88 (d, J = 6.3 Hz, 1H), 5.31−
5.24 (m, 1H), 3.60 (dd, J = 16.2, 7.2 Hz, 2H), 3.06 (dd, J = 16.2, 5.0
Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 159.0, 157.3, 150.4, 145.2,
141.1, 132.6, 129.0, 128.5, 128.2, 127.9, 126.8, 125.1, 124.9, 120.5,
113.5, 52.5, 40.1. HRMS (ESI): calcd for C21H18N3S [M + H]+,
344.1216; found, 344.1218.
128.0, 127.1, 126.2, 125.1, 120.8, 113.3, 52.6, 40.2. MS (ESI) m/z [M
+ H]+: calcd, 296.09; found, 296.13.
General Procedure for 11a−11h. A mixture of 2-chloro-N-(2,3-
dihydro-1H-inden-2-yl)quinazolin-4-amine 10 (148 mg, 0.5 mmol),
boronic acid (0.5 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), potassium
carbonate (276 mg, 2.0 mmol) in dioxane (10 mL), and water (1.5
mL) was heated at 85 °C under an argon atmosphere for 16 h. Water
(5 mL) was added, and the mixture was extracted with EtOAc (25 mL
× 3). The combined organic phase was washed with brine (15 mL),
dried (Na2SO4), filtered, evaporated, and purified by flash chromatog-
raphy to give product.
N-(2,3-Dihydro-1H-inden-2-yl)-2-(thiophen-3-yl)quinazolin-4-
amine (11g). Yellow solid (75 mg, 44%); mp 232−233 °C. 1H NMR
(500 MHz, CDCl3) δ 8.34 (d, J = 3.0 Hz, 1H), 8.02 (dd, J = 5.0, 0.9
Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.74−7.65 (m, 1H), 7.62 (d, J = 8.0
Hz, 1H), 7.42−7.34 (m, 2H), 7.30 (dd, J = 5.2, 3.4 Hz, 2H), 7.25−
7.21 (m, 2H), 5.85 (d, J = 6.5 Hz, 1H), 5.36−5.25 (m, 1H), 3.58 (dd, J
= 16.2, 7.2 Hz, 2H), 3.07 (dd, J = 16.1, 5.0 Hz, 2H). 13C NMR (125
MHz, CDCl3) δ 159.1, 157.8, 150.5, 143.1, 141.1, 132.5, 128.6, 127.9,
127.4, 126.8, 125.4, 125.1, 124.9, 120.5, 113.5, 52.3, 40.2. HRMS
(ESI): calcd for C21H18N3S [M + H]+, 344.1216; found, 344.1215.
N-(2,3-Dihydro-1H-inden-2-yl)-2-(furan-2-yl)quinazolin-4-amine
Analytical Data for Compounds 11a−11h. N-(2,3-Dihydro-1H-
inden-2-yl)-2-(4-methylpyridin-3-yl)quinazolin-4-amine (11a).
1
White solid (125 mg, 71%); mp 170−172 °C. H NMR (500 MHz,
CDCl3) δ 9.16 (s, 1H), 8.50 (d, J = 5.0 Hz, 1H), 7.91 (d, J = 7.9 Hz,
1H), 7.75 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.67 (d, J = 7.8 Hz, 1H),
7.49−7.42 (m, 1H), 7.29 (dd, J = 5.3, 3.4 Hz, 2H), 7.25−7.19 (m,
3H), 5.94 (d, J = 7.0 Hz, 1H), 5.26 (tdd, J = 7.1, 4.4, 2.6 Hz, 1H), 3.54
(dd, J = 16.2, 7.1 Hz, 2H), 3.04 (dd, J = 16.2, 4.4 Hz, 2H), 2.72 (s,
3H). 13C NMR (125 MHz, CDCl3) δ 161.1, 159.1, 151.3, 150.1, 149.2,
146.6, 140.9, 135.4, 132.7, 128.8, 126.8, 125.9, 124.9, 120.7, 113.2,
52.3, 40.2, 21.0. HRMS (ESI): calcd for C23H21N4 [M + H]+,
353.1761; found, 353.1765.
1
(11h). Pale-yellow solid (80 mg, 49%); mp 231−232 °C. H NMR
(500 MHz, CDCl3) δ 7.96 (d, J = 8.4 Hz, 1H), 7.73−7.67 (m, 1H),
7.67−7.61 (m, 2H), 7.41−7.32 (m, 2H), 7.32−7.26 (m, 2H), 7.25−
7.21 (m, 2H), 6.57 (dd, J = 3.3, 1.7 Hz, 1H), 5.98 (d, J = 6.8 Hz, 1H),
5.34−5.20 (m, 1H), 3.57 (dd, J = 16.1, 7.2 Hz, 2H), 3.05 (dd, J = 16.1,
5.0 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 159.1, 153.7, 153.4,
150.1, 144.6, 141.0, 132.7, 128.8, 126.8, 125.3, 124.9, 120.5, 113.7,
113.1, 111.8, 52.3, 40.1. HRMS (ESI): calcd for C21H18N3O [M + H]+,
328.1444; found, 328.1448.
N-(2,3-Dihydro-1H-inden-2-yl)-2-(5-methylpyridin-3-yl)-
quinazolin-4-amine (11b). Yellow solid (134 mg, 76%); mp 200−201
1
°C. H NMR (500 MHz, CDCl3) δ 9.59 (d, J = 1.6 Hz, 1H), 8.63 (s,
1H), 8.53 (d, J = 1.6 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.77−7.71 (m,
1H), 7.66 (d, J = 7.8 Hz, 1H), 7.45−7.38 (m, 1H), 7.30 (dd, J = 5.3,
3.4 Hz, 2H), 7.23 (dd, J = 5.5, 3.2 Hz, 2H), 5.95 (d, J = 6.8 Hz, 1H),
5.40−5.29 (m, 1H), 3.61 (dd, J = 16.2, 7.2 Hz, 2H), 3.08 (dd, J = 16.2,
4.8 Hz, 2H), 2.45 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 159.3,
158.8, 151.2, 150.3, 147.6, 141.1, 136.0, 134.0, 132.7, 132.6, 128.7,
126.8, 125.7, 124.9, 120.8, 113.9, 52.4, 40.1, 18.5. HRMS (ESI): calcd
for C23H21N4 [M + H]+, 353.1761; found, 353.1757.
N-(2,3-Dihydro-1H-inden-2-yl)-2-(6-methylpyridin-3-yl)-
quinazolin-4-amine (11c). Yellow solid (129 mg, 73%); mp 175−176
°C. 1H NMR (500 MHz, CDCl3) δ 9.66 (d, J = 1.9 Hz, 1H), 8.69 (dd,
J = 8.1, 2.2 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.73−7.68 (m, 1H), 7.63
(d, J = 7.9 Hz, 1H), 7.41−7.36 (m, 1H), 7.29−7.25 (m, 2H), 7.23−
7.19 (m, 3H), 5.91 (d, J = 6.6 Hz, 1H), 5.33−5.23 (m, 1H), 3.58 (dd, J
= 16.2, 7.2 Hz, 2H), 3.06 (dd, J = 16.1, 4.8 Hz, 2H), 2.62 (s, 3H). 13C
NMR (125 MHz, CDCl3) δ 159.8, 159.2, 158.8, 150.4, 149.7, 141.0,
136.0, 132.6, 131.6, 128.8, 126.9, 125.6, 124.9, 122.7, 120.5, 113.7,
52.5, 40.2, 24.5. HRMS (ESI): calcd for C23H21N4 [M + H]+,
353.1761; found, 353.1767.
Enzymatic Assays. 4-Methylumbelliferyl β-D-glucopyranoside
(4MU-β-Glc), 4-methylumbelliferyl α-D-glucopyranoside, 4-methyl-
umbelliferyl α-D-galactopyranoside, and buffer components were
purchased from Sigma-Aldrich (St. Louis, MO). The recombinant
wild-type GCase enzyme velaglucerase alfa (Vpriv, Shire Human
Genetic Therapies, Inc.), acid α-glucosidase enzyme alglucosidase
alpha (Lumizyme, Genzyme Corporation), α-galactosidase A enzyme
agalsidase beta (Fabrazyme, Genzyme Corporation) were used in
activity assays. The GCase activity assay buffer was composed of 50
mM citric acid, 176 mM K2HPO4, and 0.01% Tween-20 at pH5.0, pH
5.9 and pH 7.0. A solution of 1 M sodium hydroxide and 1 M glycine
(pH 10) was used as the stop solution for all three enzyme activity
assays.
GCase Enzyme Activity Assay. The compounds in DMSO
solution (0.5 μL/well) were transferred to a black 96-well plate (the
final titration started from 100 μM, a 12- or 24-point 2-fold dilution
series). Enzyme solution (33.5 μL, 7.5 nM final concentration, in pH
5.9 buffer) was transferred to the wells. After 5 min of incubation at
room temperature, the enzyme reaction was initiated by the addition
of blue substrate (4MU-β-Glc) (33 μL/well). The final concentration
of the blue substrate was 1.5 mM. The blue substrate reaction was
terminated by the addition of 33 μL/well stop solution (1 M NaOH
and 1 M glycine mixture, pH 10) after 30 min of incubation at 37 °C.
The fluorescence was then measured in a Biotek Synergy H1
multimode plate reader with Ex = 365 nm and Em = 440 nm. The
N-(2,3-Dihydro-1H-inden-2-yl)-2-phenylquinazolin-4-amine
1
(11d). White solid (84 mg, 50%); mp 221−223 °C. H NMR (500
MHz, CDCl3) δ 8.64−8.56 (m, 2H), 7.93 (d, J = 8.3 Hz, 1H), 7.72
(ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.53−7.46 (m,
3H), 7.39 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 7.31 (dd, J = 5.3, 3.3 Hz,
2H), 7.24 (dd, J = 5.5, 3.2 Hz, 2H), 5.86 (d, J = 6.6 Hz, 1H), 5.38 (tdd,
J = 7.1, 5.1, 2.1 Hz, 1H), 3.61 (dd, J = 16.1, 7.2 Hz, 2H), 3.08 (dd, J =
16.1, 5.0 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 160.4, 159.2, 150.5,
141.1, 138.9, 132.5, 130.1, 128.9, 128.4, 128.2, 126.8, 125.3, 125.0,
K
DOI: 10.1021/acs.jmedchem.6b00930
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
selected compounds were further assayed under pH 5.0 and pH 7.0 to
evaluate their selectivity under various pH conditions.
Chemidoc MP system (Bio-Rad) was used to scan the membranes and
analyze the imaging.
Enzyme Kinetic Assay.20 The substrate resorufin β-D-glucopyr-
anoside was diluted to five concentrations, ranging from 30 to 150 μM.
Seven concentrations of inhibitors (between 0.5- and 5-fold of IC50
value) and a DMSO control were added to the enzyme solution. The
final enzyme concentration was 10 nM to give a linear reaction over 10
min. Enzyme kinetics were measured by the addition of 66 μL of
substrate to a 96-well assay plate, followed by 33 μL of enzyme
solution (with or without inhibitor) using a dispense module on a
Biotek Synergy H1 multimode plate reader. The increase in product
fluorescence was measured at 1 min intervals for 10 min in the plate
reader. The rate of product formation was calculated by converting the
fluorescence units to nanomoles of product per minute using a
standard curve of the free fluorophore, resorufin.
Enzyme Selectivity Assays. The acid α-glucosidase and α-
galactosidase A enzyme activity assay methods were similar to the
GCase enzyme activity assay above with slight modifications. The
buffer for the two enzyme assays consisted of 50 mM citric acid, 176
mM K2HPO4, and 0.01% Tween-20 at pH 4.8. The final enzyme
concentrations for acid α-glucosidase and α-galactosidase A were 8 and
1 nM, respectively. The substrate concentrations for these related
enzymes were at 0.16 and 0.4 mM, respectively.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00930.
■
S
1H and 13C NMR spectra of 4, 6c, 7a, 7b, 8c, 9a, 9b, 9c,
11b, 11d, 11f, 11g, and 11h (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*For R.B.S.: phone, 847-491-5653; Fax, 847-491-7713; E-mail,
Agman@chem.northwestern.edu.
*For D.K.: phone, 312-503-3936; fax, 312-503-3950; E-mail,
krainc@northwestern.edu.
Notes
■
The authors declare no competing financial interest.
Fluorescence Thermal Shift Analysis.32 A robotic pipeline in
the High Throughput Analysis Laboratory (HTAL) was used for
protein−ligand screening by fluorescence thermal shift (FTS) analysis.
The pipeline used a Mosquito robot (TTP Labtech) for protein
dispensing and an Echo 550 (Labcyte) to add compounds. Thermal
scanning coupled with fluorescence detection was performed on a real-
time PCR machine CFX384 (Bio-Rad Laboratories). The assay was
run in 384-well PCR plates, using 10 μL citric acid/K2HPO4 buffer (50
mM citric acid, 150 mM K2HPO4, pH 5.0) per well. The assay
concentration for protein was 1 μM and that for Sypro Orange
(Invitrogen) was 5×. Protein was premixed with Sypro Orange and
dispensed to a plate first, and compounds were added. Final
concentrations of compounds ranged from 0.5 to 200 μM. Then the
plate was sealed with an optical seal, shaken, and centrifuged. The
thermal scan was from 10 to 95 °C with a temperature ramp rate of 1.5
°C/min. The fluorescence was recorded every 10 s. Data analysis and
report generation were performed using the in-house software
excelFTS. The Tm of wild-type GCase was found to follow a
logarithmic dose-dependent trend when denaturation was performed
in the presence of isofagomine or selected compounds.
N370S Cell Culture and Compound Treatment. The N370S
fibroblast cell line was obtained from Coriell, GM00372, cultured in
DMEM medium (Life Tech) including 1% v/v L-glutamine 200 mM
(Life Tech), 1% v/v pen strep (Life Tech), 10% FBS (Life Tech) at 37
°C, and 5% CO2 and treated with different compounds at indicated
concentrations. The same volume of DMSO (0.1% v/v) was used as a
control. After a 3-day treatment, cells were washed with inhibitor free
media three times and followed by 1% Triton X-100 lysis buffer to lyse
cells. Protein concentrations were measured with a Bicinchoninic Acid
Kit (Sigma), and the GCase activity was determined at pH 5.5.
Deglycosylation of Proteins/Molecular Shift Assay.33 To
study the ER and post-ER localization of the N370S GCase mutant
after incubation with increasing concentrations of 11g, EndoH and
PNGaseF digestions were performed. For both reactions, 10−20 μg of
protein was used and the experimental procedure was performed
according to the manufacturer’s handbook (New England Biolabs). A
positive digestion resulted in a shift in molecular size after the protein
was subjected to SDS/PAGE.
ACKNOWLEDGMENTS
■
This work was supported by R01NS076054 (to D.K.). J.Z. and
M.S. are also supported by the fellowships from Lysosomal
Therapeutics Inc. (Cambridge, MA) and the Deutsche
Forschungsgemeinschaft (Heisenberg Programme), respec-
tively. This work made use of the IMSERC at Northwestern
University, which has received support from the Soft and
Hybrid Nanotechnology Experimental (SHyNE) Resource
(NSF NNCI-1542205), the State of Illinois, and the Interna-
tional Institute for Nanotechnology (IIN). We thank H.
Goudarzi and S. Shafaie in IMSERC at Northwestern
University for their assistance with HRMS experiments and
C. H. Luan in Northwestern University’s High Throughput
Analysis Laboratory for his assistance with thermal denaturation
experiments.
ABBREVIATIONS USED
■
GD, Gaucher’s disease; GCase, β-glucocerebrosidase; ER,
endoplasmic reticulum; ERT, enzyme replacement therapy;
SRT, substrate reduction therapy; PC, pharmacological
chaperone; IFG, isofagomine; PD, Parkinson’s disease; DLB,
dementia with Lewy bodies; SAR, structure−activity relation-
ship; GAA, acid α-glucosidase; GLA, α-galactosidase A
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