Conversion of Quinazoline Modulators from Inhibitors to Activators of β-Glucocerebrosidase

Article abstract of DOI:10.1021/acs.jmedchem.8b01294

Gaucher's disease is a lysosomal disease caused by mutations in the β-glucocerebrosidase gene (GBA1 and GCase) that have been also linked to increased risk of Parkinson's disease (PD) and Diffuse Lewy body dementia. Prior studies have suggested that mutant GCase protein undergoes misfolding and degradation, and therefore, stabilization of the mutant protein represents an important therapeutic strategy in synucleinopathies. In this work, we present a structure-activity relationship (SAR) study of quinazoline compounds that serve as inhibitors of GCase. Unexpectedly, we found that N-methylation of these inhibitors transformed them into GCase activators. A systematic SAR study further revealed that replacement of the key oxygen atom in the linker of the quinazoline derivative also contributed to the activity switch. PD patient-derived fibroblasts and dopaminergic midbrain neurons were treated with a selected compound (9q) that partially stabilized GCase and improved its activity. These results highlight a novel strategy for therapeutic development of noninhibitory GCase modulators in PD and related synucleinopathies.

Full text of DOI:10.1021/acs.jmedchem.8b01294

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Conversion of Quinazoline Modulators from Inhibitors to Activators
of β‑Glucocerebrosidase
Jianbin Zheng,†,‡ Sohee Jeon,‡ Weilan Jiang,‡ Lena F. Burbulla,‡ Daniel Ysselstein,‡ Kristine Oevel,‡
Dimitri Krainc,*,‡ and Richard B. Silverman*,†
†Department of Chemistry, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, and Center
for Developmental Therapeutics, Northwestern University, Evanston, Illinois 60208, United States
‡Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, United States
S
* Supporting Information
ABSTRACT: Gaucher’s disease is a lysosomal disease caused by mutations in the β-glucocerebrosidase gene (GBA1 and
GCase) that have been also linked to increased risk of Parkinson’s disease (PD) and Diffuse Lewy body dementia. Prior studies
have suggested that mutant GCase protein undergoes misfolding and degradation, and therefore, stabilization of the mutant
protein represents an important therapeutic strategy in synucleinopathies. In this work, we present a structure−activity
relationship (SAR) study of quinazoline compounds that serve as inhibitors of GCase. Unexpectedly, we found that N-
methylation of these inhibitors transformed them into GCase activators. A systematic SAR study further revealed that
replacement of the key oxygen atom in the linker of the quinazoline derivative also contributed to the activity switch. PD
patient-derived fibroblasts and dopaminergic midbrain neurons were treated with a selected compound (9q) that partially
stabilized GCase and improved its activity. These results highlight a novel strategy for therapeutic development of noninhibitory
GCase modulators in PD and related synucleinopathies.
INTRODUCTION
■
Gaucher’s disease (GD), an autosomal recessive lysosomal
storage disorder, is caused by mutations of the GBA1 gene
encoding the lysosomal enzyme β-glucocerebrosidase
(GCase). Mutations in GBA1 were also found as a major
risk factor for Parkinson’s disease (PD) and dementia with
Lewy bodies (DLB).1−5 Most of these mutations, including the
prevalent N370S mutation, are still functional, although with
low residual GCase activity.6 Loss of function of GCase leads
to accumulation of glucosylceramide (GluCer),7,8 which also
promotes the formation and stabilization of α-synuclein
oligomers.9 Enhancement of GCase activity is thought to be
a potential therapeutic strategy for GCase-associated synuclei-
nopathies, including PD.10,11
Several different scaffolds of non-iminosugar GCase
inhibitors have been reported as GCase pharmacological
chaperones (PCs) since 200712−16 (Figure 1). We found
Figure 1. Structures of GCase inhibitors and activators.
that a noninhibitory GCase PC NCGC00188758 (2)17 can
enhance GCase activity specifically within the lysosomal
compartment, reduce GluCer and hexosylsphingosine sub-
strates, and subsequently enhance the clearance of pathological
α-synuclein.18 These findings suggested that reduction of
Received: August 15, 2018
GluCer may provide benefit in PD and strengthen the notion
© XXXX American Chemical Society
A
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that GCase is a valuable target for the treatment of
synucleinopathies.
2-phenoxyethanamine with 2,4-dichloroquinazoline, followed
by Suzuki coupling with appropriate boronic acids to afford
11b−11i (Scheme 2). Compound 11a was synthesized by
reaction of commercially available 4-chloro-2-(pyridin-2-yl)-
quinazoline and N-methyl-2-phenoxyethanamine. The struc-
ture and purity of all of the prepared compounds were
confirmed by spectroscopic and analytical techniques.
In our previous work,19 we discovered a series of quinazoline
inhibitors with nanomolar potency (see representative
compounds 3 and 4, Figure 1). Here, we unexpectedly found
that N-methylation of some of the quinazoline analogues
transformed quinazoline inhibitors into activators. We describe
a broader structure−activity relationship (SAR) study of
quinazoline derivatives to better understand the pharmaco-
phoric requirements for this unusual activity switch as a result
of such a minor structural change and to design novel
noninhibitory modulators.
RESULTS AND DISCUSSION
■
Because of our unexpected observation that N-methylation of
inhibitor 5a to 6a (R2 = CH3) transformed its activity into an
activator (Figure 2), we explored a broader SAR of quinazoline
derivatives in an attempt to uncover the pharmacophoric basis
for this finding and to identify novel noninhibitory modulators.
First, we examined a series of moderate to potent quinazoline
inhibitors that we previously reported19 to see if they could
also be switched to activators by N-methylation (Table 1).
Interestingly, we were able to switch the moderately active
inhibitors (5a−5f) to activators (6a−6f) by N-methylation,
suggesting that these inhibitors and activators may share an
allosteric binding site. According to our recent findings, the
N−H forms a H-bond with Ser345 in the allosteric site;21 this
interaction may be important for attaining an inhibitory
conformation, and N-methylation disrupted the H-bond and
may change that conformation to one that promotes activation.
However, for the most potent inhibitors (5g−5j), N-
methylation only produced weak inhibitory or inactive
compounds (6g−6j). As we noted in our inhibitor SAR
study,20 the most potent inhibitors (5h−5j), having fused
substituents (R groups), have additional hydrophobic inter-
actions (and π−π stacking with Phe347) with loop 2 residues
of GCase, which stabilizes the GCase dimer form. The removal
of this presumed NH H-bond interaction with Ser345 is
apparently not sufficient to completely change the conforma-
tion and/or break dimer formation so that these compounds
retain partial inhibitory activity, and are not activators.
Therefore, substituents of the secondary amines of inhibitors
might also affect an activity switch.
CHEMISTRY
■
The synthesis of target compounds for SAR exploration is
similar to our previous synthesis route19 and is detailed in
Schemes 1 and 2. As shown in Scheme 1, the intermediate 4-
a
Scheme 1. Synthesis of N-Methyl Quinazoline Derivatives
a
Reagents and conditions: (a) (i) sulfolane (ii) PCl5; 46%; (b)
R1NH2, K2CO3, DMF; 34−73%; (c) (i) NaH, DMF (ii) R2I; (d)
R1R2NH, K2CO3, DMF; 33−96%; (e) RH, K2CO3, DMF; 26−89%.
Scheme 2. Synthesis of 11a−11i with Modifications at the 2-
a
Position of the Quinazoline Ring
To further explore the SAR, a methylene group in the
phenylpropyl substituent of 5a was modified to an oxygen
atom to give ether 7a, which was a moderate activator even
without N-methylation (Figure 3). Compared to 7a, the
compounds with a longer substituent (7b and 7c) or with a
methyl group at the α- or β-position of 7a (7d and 7e)
retained their inhibitory activity (Table 2). This finding
suggests that the oxygen atom in 7a may be involved in an
intramolecular H-bond interaction with the NH group to avoid
the intermolecular H-bond with Ser345, but an appropriate
distance between the oxygen atom and quinazoline ring with
no steric hindrance was required for proper H-bonding and to
achieve this activation. Installation of an N-methyl group on
the secondary amine of 7a afforded 8a, having the greatest
maximum activation (Emax 761%) with an AC50 value (3.55
μM) similar to that of 7a (Figure 3). Likewise, N-methylation
of 7b−7e switched these inhibitors to moderate activators 8b−
8e (Table 2). Activation of 8f−8h was decreased with a longer
substituent or with increased steric hindrance of the carbon
substituents (Table 2), suggesting a small hydrophobic pocket
for the alkyl groups.
a
Reagents and conditions: (a) N-methyl-2-phenoxyethanamine,
K2CO3, DMF, 80%; (b) ArB(OH)2, Pd(PPh3)4, K2CO3, 1,4-dioxane,
H2O; 40−82%; (c) N-methyl-2-phenoxyethanamine, K2CO3, DMF,
73%.
chloro-2-(pyridin-3-yl)quinazoline was prepared from 2-
amino-benzonitrile and nicotinoyl chloride according to a
published synthesis route.20 The reaction of 4-chloro-2-
(pyridin-3-yl)quinazoline and appropriate amines in the
presence of potassium carbonate as a base afforded 5a−5j
and 7a−7e. Compounds 6a−6j, 8a−8h, and 9a−9v were
obtained either by deprotonation and N-methylation of the
corresponding secondary amines (5a−5j and 7a−7e), or by
alkylation of 4-chloro-2-(pyridin-3-yl)quinazoline with appro-
priate secondary amines under basic conditions.
To further explore the conformation required for the
activation activity, some conformationally restricted substitu-
ents were introduced in the modulators (9a−9k, Table 3). As
we expected, these compounds, without a free NH group,
Additional analogues, having modifications at position 2 of
the quinazoline ring, were synthesized by reaction of N-methyl-
B
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Figure 2. GCase enzyme activity switch by N-methylation of quinazoline derivatives. (A) Example of the change in activity by N-methylation; (B)
plot demonstrating the change in activity from inhibition (5a) to activation (6a).
also observed the key interaction of the aromatic ring in the
activators. For example, compared to 9j, compound 9k with a
fused phenyl ring gave a 5-fold lower AC50 value.
Table 1. Structure and Enzyme Activity of Quinazoline
Derivatives without and with the N-Methyl Substituent
a
To pursue potent activators with greater maximum
activation, we further designed and synthesized derivatives
9l−9v having a conformationally restricted chain or a fused
ring (Table 3). Most of the compounds showed similar
activation activity as 8a, except for 9n, 9u, and 9v. Similar to
9b and 9c, compound 9q has slightly better binding potency
than its enantiomer (9r).
Finally, we examined the aromatic ring effect at position 2 of
the quinazoline ring (Table 4). As we found in our SAR of
quinazoline inhibitors, the 3-pyridinyl ring could be replaced
by other aromatic rings, such as phenyl and 3-thienyl groups,
without losing activity.19 Unlike the inhibitors, the activator
series modulators were not tolerant of modification of the 3-
pyridinyl ring, except for 11b, 11c, and 11h, which were
comparable or enhanced in activity relative to 8a. It should be
noted that compound binding to the GCase allosteric site may
not induce either inhibitory or activation activity because the
readout of compound activity was based on the enzyme
activity. A binding affinity-based or competitive-binding assay
may be developed for accurate evaluation of the compounds
without inhibitory or activation activity.
To verify compound activity in cells, the most active
compounds underwent preliminary screening in healthy
control fibroblast cell assays. Compound 9q, showing the
most promising effect, was further tested in healthy control and
compound heterozygous N370S/84GG GBA1 mutant fibro-
blasts derived from a GD patient. Compound treatment
increased the post-ER form (resistant to Endo H digestion) of
wild-type GCase protein levels and improved enzyme activity
at a concentration of 15 μM in control fibroblasts, while
control compound isofagomine (IFG) did not change post-ER
GCase levels and enzyme activity at a concentration of 25 μM
(Figures 4A and 5A). Similarly, we observed an increase of
GCase protein levels and improved enzyme activity upon 9q
treatment in N370S/84GG GBA1 mutant fibroblasts (Figures
4B and 5B).
To confirm that the increase of GCase activity observed in
fibroblast lysates occurred because of an increase in lysosomal
GCase, we measured GCase activity in live cells. Fibroblasts
from a healthy control and fibroblasts containing a
homozygous N370S or L444P GBA1 mutation were treated
with 9q and IFG and assayed for lysosomal GCase activity.
Lysosomal specificity was achieved by calculating only the
bafilomycin-sensitive hydrolysis of the substrate. We found that
treatment with 9q led to a significant, dose-dependent, increase
in lysosomal GCase activity in healthy control and GBA1
mutant fibroblasts with a 55 and 85% increase in the 15 μM
a
Experiments were performed in triplicate, and the mean
shown.
SD is
displayed activation activity (except for 9g and 9h).
Interestingly, the chiral center of the steroisomers (9b and
9c) did not have a striking effect on the activity. We had
previously demonstrated the important role of an aromatic ring
in the N-substituents of our quinazoline inhibitors;19 here, we
C
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Figure 3. Modification of the N-substituent by oxygen replacement (7a) and N-methylation (8a).
activators of GCase by N-methylation. Treatment of wild-
type and GBA1 mutant fibroblasts, as well as dopaminergic
neurons with modulator 9q, led to increased GCase protein
levels and lysosomal enzyme activity, suggesting that further
development of these compounds as GCase allosteric
activators is warranted. It will be important to analyze the
GCase-compound complex by crystallography to elucidate the
mechanism of enzyme activation for future drug design.
Table 2. Structures and Inhibition or Activation Activity of
Quinazoline Derivatives Having an Oxygen Isostere and
Methylation in the N-Substituent
a
EXPERIMENTAL SECTION
■
Chemistry. Materials, and Methods. 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 >95% as analyzed with an Agilent 1260
Infinity HPLC system. 1H 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) or Agilent DD MR-400 system
1
(400 MHz for H NMR and 100 MHz for 13C NMR) spectrometer.
1
Chemical shifts are reported relative to chloroform (δ = 7.26 for H
NMR and δ = 77.16 for 13C NMR spectra) or dimethyl sulfoxide
(DMSO) (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-time of flight (TOF)
instrument with a dual spray electrospray ionization (ESI) source
with a high resolution TOF mass analyzer and collecting in a 2 GHz
detector mode coupled with an Agilent 1200 HPLC.
General Procedure for Compounds 5a−5j, 7a−7e, 9a−9v, and
11a. A mixture of 4-chloro-2-(pyridin-3-yl)quinazoline19,22 (72 mg,
0.3 mmol), amine (0.3 mmol), and potassium carbonate (69 mg, 0.3
mmol) in dimethylformamide (DMF, 2 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 the products, which were usually pure (>95% purity). Those
products without sufficient purity were further purified by flash
chromatography.
General Procedure for Compounds 6a−6j and 8a−8h. To a
solution of 5a−5j in dry DMF was added NaH (60% in mineral oil)
under an argon atmosphere at room temperature, and the mixture was
stirred for 15 min. Iodomethane or an appropriate halogenated alkane
was added dropwise to the mixture, and the mixture was allowed to
stir at room temperature overnight. Water was added, and the formed
solid was filtered, washed with water, and dried in vacuo to give the
desired product.
a
Experiments were performed in triplicate, and the mean
shown.
SD is
treated N370S and L444P fibroblast, respectively (Figure 5C−
E). In contrast, treatment with IFG showed little or no effect
on GCase activity.
Similar effects were observed in induced pluripotent stem
cell (iPSC)-derived healthy control neurons and heterozygous
N370S GBA1 mutant dopaminergic neurons from a PD patient
(see Supporting Information Figure for the characterization of
iPSCs and differentiated neurons) treated with increasing
concentrations of 9q for 10 days and digested with Endo H or
PNGase. 9q increased GCase protein levels and enzyme
activity in both wild-type and mutant neurons (Figures 6 and
7), suggesting the potential application of these GCase
modulators in PD patients with/without GBA1 mutations.
Preparation of 2-Chloro-N-methyl-N-(2-phenoxyethyl)-
quinazolin-4-amine (10). A mixture of 2,4-dichloroquinazoline
(1.99 g, 10 mmol), N-methyl-2-phenoxyethanamine (1.51 g, 10
mmol), and potassium carbonate (1.38 g, 10 mmol) in DMF (20 mL)
were stirred at room temperature overnight. Water (120 mL) was
added, and the formed solid was filtered, washed with water, and dried
to give 10 as a white solid (2.5 g, 80%).
CONCLUSIONS
1H NMR (500 MHz, CDCl3): δ 8.10 (d, J = 8.5 Hz, 1H), 7.77 (dd,
J = 8.4, 0.8 Hz, 1H), 7.71−7.65 (m, 1H), 7.37 (ddd, J = 8.4, 7.0, 1.3
Hz, 1H), 7.30−7.25 (m, 2H), 6.97−6.91 (m, 3H), 4.38 (t, J = 5.5 Hz,
■
Our SAR study revealed a remarkable finding that previously
reported quinazoline inhibitors could be transformed to
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Table 3. Structure and Activation Activity of Quinazoline Derivatives with Conformationally Restricted Substituents and
a
Fused Aromatic Ring Substituents
a
b
Experiments were performed in triplicate, and the mean SD is shown. Inhibitor.
2H), 4.18 (t, J = 5.4 Hz, 2H), 3.61 (s, 3H). 13C NMR (125 MHz,
CDCl3): δ 163.7, 158.3, 156.1, 153.6, 133.0, 129.6, 127.8, 125.7,
124.8, 121.1, 114.4, 65.5, 52.6, 42.2. MS (ESI) m/z: [M + H]+ calcd,
314.11; found, 314.49.
General Procedure for Compounds 11b−11h. A mixture of 2-
chloro-N-methyl-N-(2-phenoxyethyl)quinazolin-4-amine (10, 126
mg, 0.4 mmol), boronic acid (0.5 mmol), Pd(PPh3)4 (50 mg, 0.05
mmol), potassium carbonate (276 mg, 2.0 mmol) in dioxane (10
mL), and water (1.5 mL) was heated at 100 °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 chromatography to give the product.
1.7 Hz, 1H), 8.66 (d, J = 4.4 Hz, 1H), 7.92−7.84 (m, 2H), 7.71−7.63
(m, 1H), 7.37 (dd, J = 7.9, 4.8 Hz, 1H), 7.31−7.25 (m, 3H), 7.21−
7.16 (m, 3H), 3.86−3.80 (m, 2H), 3.41 (s, 3H), 2.74 (t, J = 7.5 Hz,
2H), 2.25−2.16 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 163.3,
157.3, 152.9, 150.6, 150.2, 141.2, 135.5, 134.3, 132.1, 128.7, 128.5,
128.4, 126.1, 125.2, 124.4, 123.1, 115.0, 52.7, 40.0, 33.3, 28.8. HRMS
(ESI): calcd for C23H23N4 [M + H]+, 355.1917; found, 355.1928.
N-Methyl-N-phenethyl-2-(pyridin-3-yl)quinazolin-4-amine (6b).
Pale-yellow solid (yield: 81%). 1H NMR (500 MHz, CDCl3): δ
9.74 (s, 1H), 8.80−8.56 (m, 2H), 7.94 (d, J = 8.2 Hz, 1H), 7.89 (d, J
= 8.3 Hz, 1H), 7.70−7.62 (m, 1H), 7.41−7.30 (m, 2H), 7.30−7.16
(m, 5H), 4.07−3.97 (m, 2H), 3.39 (s, 3H), 3.17−3.07 (m, 2H). 13C
NMR (125 MHz, CDCl3): δ 163.0, 157.4, 152.9, 150.6, 150.1, 138.9,
135.6, 134.5, 132.2, 128.8, 128.7, 128.7, 126.5, 125.3, 124.5, 123.2,
115.0, 54.9, 40.7, 33.6. HRMS (ESI): calcd for C22H21N4 [M + H]+,
341.1761; found, 341.1770.
Analytical Data of the Novel Quinazoline Derivatives (6a−6j,
7a−7e, 8a−8h, 9a−9v, and 11a−11h). N-Methyl-N-(3-phenyl-
propyl)-2-(pyridin-3-yl)quinazolin-4-amine (6a). Yellow solid (yield:
72%). 1H NMR (500 MHz, CDCl3): δ 9.69 (s, 1H), 8.70 (dt, J = 7.9,
E
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1.78−1.70 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 163.3, 157.4,
152.9, 150.5, 150.1, 141.9, 135.6, 134.4, 132.2, 128.7, 128.3, 125.9,
125.3, 124.4, 123.3, 115.1, 53.1, 40.0, 35.6, 28.7, 26.9. HRMS (ESI):
calcd for C24H25N4 [M + H]+, 369.2074; found, 369.2080.
Table 4. Structure and Activation Activity of N-Methyl-N-
(2-phenoxyethyl)quinazolin-4-amine Derivatives
a
Containing Aromatic Rings (11a−11g)
N-Cyclopentyl-N-methyl-2-(pyridin-3-yl)quinazolin-4-amine
(6d). Yellow solid (yield: 76%). 1H NMR (500 MHz, CDCl3): δ 9.71
(s, 1H), 8.77 (dt, J = 7.9, 1.8 Hz, 1H), 8.65 (dd, J = 4.7, 1.5 Hz, 1H),
7.98 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.68 (t, J = 7.1 Hz,
1H), 7.44−7.31 (m, 2H), 5.04−4.92 (m, 1H), 3.25 (s, 3H), 2.15−
2.02 (m, 2H), 1.86−1.60 (m, 6H). 13C NMR (125 MHz, CDCl3): δ
164.4, 157.4, 152.9, 150.5, 150.2, 135.6, 134.4, 132.1, 128.6, 125.4,
124.4, 123.1, 115.5, 61.6, 34.4, 28.8, 24.6. HRMS (ESI): calcd for
C19H21N4 [M + H]+, 305.1761; found, 305.1762.
N-Cyclohexyl-N-methyl-2-(pyridin-3-yl)quinazolin-4-amine (6e).
1
Pale-yellow solid (yield: 59%). H NMR (500 MHz, CDCl3): δ 9.72
(s, 1H), 8.79 (d, J = 7.8 Hz, 1H), 8.67 (d, J = 3.8 Hz, 1H), 7.94 (d, J =
8.3 Hz, 2H), 7.70 (t, J = 7.6 Hz, 1H), 7.43−7.33 (m, 2H), 4.58−4.46
(m, 1H), 3.28 (s, 3H), 2.01 (d, J = 11.5 Hz, 2H), 1.92 (d, J = 13.2 Hz,
2H), 1.79−1.63 (m, 3H), 1.53−1.41 (m, 2H), 1.26−1.15 (m, 1H).
13C NMR (125 MHz, CDCl3): δ 163.8, 157.3, 153.0, 150.7, 150.3,
135.7, 134.4, 132.2, 128.7, 125.5, 124.4, 123.2, 115.4, 59.9, 34.3, 30.1,
26.0, 25.8. HRMS (ESI): calcd for C20H23N4 [M + H]+, 319.1917;
found, 319.1921.
N-Cycloheptyl-N-methyl-2-(pyridin-3-yl)quinazolin-4-amine (6f).
1
Pale-yellow solid (yield: 72%). H NMR (500 MHz, CDCl3): δ 9.71
(s, 1H), 8.77 (d, J = 7.9 Hz, 1H), 8.65 (d, J = 3.5 Hz, 1H), 7.94 (d, J =
8.3 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.73−7.60 (m, 1H), 7.44−7.31
(m, 2H), 4.77−4.65 (m, 1H), 3.27 (s, 3H), 2.13−2.02 (m, 2H),
1.93−1.74 (m, 4H), 1.74−1.52 (m, 6H). 13C NMR (125 MHz,
CDCl3): δ 163.4, 157.4, 153.0, 150.5, 150.2, 135.6, 134.5, 132.0,
128.6, 125.4, 124.2, 123.1, 115.3, 61.2, 34.5, 32.2, 27.7, 25.5. HRMS
(ESI): calcd for C21H25N4 [M + H]+, 333.2074; found, 333.2082.
N-Cyclooctyl-N-methyl-2-(pyridin-3-yl)quinazolin-4-amine (6g).
Yellow solid (yield: 86%). 1H NMR (500 MHz, CDCl3): δ 9.71 (d, J
= 1.6 Hz, 1H), 8.77 (dt, J = 7.9, 1.9 Hz, 1H), 8.65 (dd, J = 4.7, 1.6 Hz,
1H), 7.96 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.72−7.63
(m, 1H), 7.41−7.31 (m, 2H), 4.99−4.87 (m, 1H), 3.27 (s, 3H),
a
Experiments were performed in triplicate, and the mean
shown.
SD is
N-Methyl-N-(4-phenylbutyl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(6c). Pale-yellow oil (yield: 67%). H NMR (500 MHz, CDCl3): δ
9.72 (s, 1H), 8.75 (d, J = 7.9 Hz, 1H), 8.68 (s, 1H), 7.94 (d, J = 7.7
Hz, 1H), 7.91 (d, J = 8.3 Hz, 1H), 7.71−7.66 (m, 1H), 7.42−7.33 (m,
2H), 7.28−7.23 (t, J = 7.6 Hz, 2H), 7.19−7.12 (m, 3H), 3.85−3.79
(m, 2H), 3.39 (s, 3H), 2.68 (t, J = 7.6 Hz, 2H), 1.93−1.84 (m, 2H),
Figure 4. Activator 9q increases GCase protein levels in (A) healthy control fibroblasts and (B) compound heterozygous GBA1 mutant fibroblasts
(N370S/84GG) derived from a GD patient. Cell lysates from fibroblasts treated with vehicle (DMSO), 9q (5, 15 μM), or IFG were analyzed by
immunoblotting after no digestion (top), Endo H digestion (middle), and PNGase F digestion (bottom); n = 4.
F
DOI: 10.1021/acs.jmedchem.8b01294
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Figure 5. GCase enzyme activity in cell lysates and in the lysosome of 9q-treated cells. GCase activity was measured in lysates from (A) healthy
control fibroblasts and (B) compound heterozygous GBA1 mutant fibroblasts (N370S/84GG). Lysosomal GCase was measured using a live-cell
assay in (C) healthy control, (D) homozygous N370S, and (E) homozygous L444P mutant fibroblasts after 3 day treatment with 9q (5, 15 μM), 5
μM IFG, or vehicle (DMSO). The data are presented as the mean SEM, n = 3−4; *p < 0.05, **p < 0.01, ***p < 0.001 vs vehicle treatments;
one-way ANOVA was followed by the Tukey’s multiple comparisons post hoc test.
Figure 7. GCase enzyme activity in protein lysates of (A) wild-type
control and (B) patient-derived heterozygous N370S GBA1 mutant
dopaminergic neurons after 10 days consecutive treatment with
vehicle (DMSO) or 9q (day 70 of differentiation). The data are
presented as the mean SEM, n = 4−7; *p < 0.05, **p < 0,01 versus
vehicle treatments; one-way ANOVA was followed by the Tukey’s
multiple comparisons post hoc test.
Figure 6. Activator 9q increases GCase protein levels and enzyme
134.5, 132.0, 128.6, 125.5, 124.2, 123.1, 115.3, 59.5, 34.7, 31.6, 29.7,
26.4, 25.2. HRMS (ESI): calcd for C22H27N4 [M + H]+, 347.2230;
found, 347.2238.
activity in (A) wild-type control and (B) patient-derived heterozygous
N370S GBA1 mutant dopaminergic neurons at day 70 of differ-
entiation. Cell lysates from neurons treated with vehicle (DMSO) or
9q (5, 15 μM) for 10 consecutive days were analyzed by
immunoblotting after no digestion (top), Endo H digestion (middle),
and PNGase F digestion (bottom); n = 3.
N-(2,3-Dihydro-1H-inden-2-yl)-N-methyl-2-(pyridin-3-yl)-
1
quinazolin-4-amine (6h). Yellow solid (yield: 60%). H NMR (500
MHz, CDCl3): δ 9.74 (d, J = 1.5 Hz, 1H), 8.77 (dt, J = 7.9, 1.9 Hz,
1H), 8.68 (dd, J = 4.7, 1.5 Hz, 1H), 8.04 (d, J = 7.9 Hz, 1H), 7.96 (d,
J = 8.3 Hz, 1H), 7.78−7.69 (m, 1H), 7.45−7.41 (m, 1H), 7.39 (dd, J
= 7.9, 4.8 Hz, 1H), 7.29 (dd, J = 5.2, 3.4 Hz, 2H), 7.25−7.20 (m, 2H),
5.66−5.58 (m, 1H), 3.47 (dd, J = 16.4, 8.6 Hz, 2H), 3.35−3.26 (m,
2.02−1.87 (m, 4H), 1.87−1.77 (m, 2H), 1.77−1.52 (m, 8H). 13C
NMR (125 MHz, CDCl3): δ 163.2, 157.4, 153.0, 150.5, 150.2, 135.6,
G
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5H). 13C NMR (125 MHz, CDCl3): δ 164.7, 157.5, 153.1, 150.8,
150.4, 141.3, 135.7, 134.3, 132.5, 129.0, 126.9, 125.5, 124.8, 124.7,
123.3, 115.7, 60.2, 36.4, 35.6. HRMS (ESI): calcd for C23H21N4 [M +
H]+, 353.1761; found, 353.1772.
7.8, 4.8 Hz, 1H), 7.32−7.25 (m, 2H), 6.99−6.90 (m, 3H), 6.50 (s,
1H), 4.18 (t, J = 5.6 Hz, 2H), 4.02−3.95 (m, 2H), 2.28 (p, J = 5.9 Hz,
2H). 13C NMR (125 MHz, CDCl3): δ 159.7, 158.6, 158.5, 150.6,
150.2, 150.2, 135.6, 134.5, 132.7, 129.6, 128.8, 125.8, 123.1, 121.1,
120.6, 114.4, 113.9, 66.8, 39.7, 28.5. HRMS (ESI): calcd for
C22H21N4O [M + H]+, 357.1710; found, 357.1723.
(S)-N-Methyl-2-(pyridin-3-yl)-N-(1,2,3,4-tetrahydronaphthalen-
1
2-yl)quinazolin-4-amine (6i). Pale-yellow oil (yield: 63%). H NMR
(500 MHz, CDCl3): δ 9.70 (s, 1H), 8.77 (dt, J = 7.9, 1.9 Hz, 1H),
8.65 (s, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.77−
7.63 (m, 1H), 7.45−7.30 (m, 2H), 7.16−7.10 (m, 4H), 5.07−4.90
(m, 1H), 3.34 (s, 3H), 3.21 (d, J = 8.4 Hz, 2H), 3.12−2.91 (m, 2H),
2.29−2.17 (m, 1H), 2.11 (qd, J = 11.9, 5.9 Hz, 1H). 13C NMR (125
MHz, CDCl3): δ 164.1, 157.3, 153.0, 150.6, 150.2, 135.6, 135.6,
135.2, 134.3, 132.3, 129.4, 128.8, 126.1, 126.0, 125.3, 124.6, 123.2,
115.4, 56.5, 34.2, 32.0, 29.5, 27.1. HRMS (ESI): calcd for C24H23N4
[M + H]+, 367.1917; found, 367.1932.
N-Methyl-N-(3-phenoxypropyl)-2-(pyridin-3-yl)quinazolin-4-
amine (8c). Yellow solid (77%). 1H NMR (500 MHz, CDCl3): δ 9.72
(s, 1H), 8.77 (dt, J = 7.9, 1.9 Hz, 1H), 8.66 (d, J = 3.7 Hz, 1H), 8.06
(dd, J = 8.4, 0.8 Hz, 1H), 7.91 (dd, J = 8.4, 0.8 Hz, 1H), 7.70 (ddd, J
= 8.3, 7.0, 1.3 Hz, 1H), 7.42−7.33 (m, 2H), 7.29−7.24 (m, 2H), 6.93
(t, J = 7.3 Hz, 1H), 6.88 (d, J = 7.8 Hz, 2H), 4.10 (t, J = 5.8 Hz, 2H),
4.07−4.02 (m, 2H), 3.48 (s, 3H), 2.40−2.32 (m, 2H). 13C NMR (125
MHz, CDCl3): δ 163.2, 158.7, 157.3, 152.9, 150.6, 150.1, 135.6,
134.3, 132.2, 129.5, 128.8, 125.4, 124.5, 123.1, 120.8, 115.1, 114.4,
65.2, 50.5, 40.5, 27.2. HRMS (ESI): calcd for C23H22N4O [M + H]+,
371.1866; found, 371.1879.
(R)-N-Methyl-2-(pyridin-3-yl)-N-(1,2,3,4-tetrahydronaphthalen-
1
2-yl)quinazolin-4-amine (6j). Pale-yellow oil (yield: 69%). H NMR
(500 MHz, CDCl3): δ 9.70 (s, 1H), 8.77 (dt, J = 7.9, 1.9 Hz, 1H),
8.65 (s, 1H), 7.97 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.77−
7.63 (m, 1H), 7.45−7.30 (m, 2H), 7.16−7.10 (m, 4H), 5.07−4.90
(m, 1H), 3.34 (s, 3H), 3.21 (d, J = 8.4 Hz, 2H), 3.12−2.91 (m, 2H),
2.29−2.17 (m, 1H), 2.11 (qd, J = 11.9, 5.9 Hz, 1H). 13C NMR (125
MHz, CDCl3): δ 164.1, 157.3, 153.0, 150.6, 150.2, 135.6, 135.6,
135.2, 134.3, 132.3, 129.4, 128.8, 126.1, 126.0, 125.3, 124.6, 123.2,
115.4, 56.5, 34.2, 32.0, 29.5, 27.1. HRMS (ESI): calcd for C24H23N4
[M + H]+, 367.1917; found, 367.1927.
N-(2-Phenoxypropyl)-2-(pyridin-3-yl)quinazolin-4-amine (7d).
Off-white solid (yield: 34%). H NMR (500 MHz, CDCl3): δ 9.76
1
(s, 1H), 8.79 (d, J = 7.9 Hz, 1H), 8.71 (s, 1H), 7.92 (d, J = 7.9 Hz,
1H), 7.75 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H),
7.50−7.36 (m, 2H), 7.31−7.26 (m, 2H), 7.02−6.91 (m, 3H), 6.35−
6.20 (m, 1H), 4.87−4.75 (m, 1H), 4.31 (ddd, J = 13.9, 6.8, 3.5 Hz,
1H), 3.85 (ddd, J = 13.9, 7.5, 4.7 Hz, 1H), 1.44 (d, J = 6.2 Hz, 3H).
13C NMR (125 MHz, CDCl3): δ 159.6, 158.5, 157.6, 150.7, 150.3,
150.2, 135.6, 134.3, 132.8, 129.7, 128.9, 126.0, 123.2, 121.4, 120.6,
116.2, 113.8, 72.8, 46.1, 17.6. HRMS (ESI): calcd for C22H21N4O [M
+ H]+, 357.1710; found, 357.1720.
N-(2-Phenoxyethyl)-2-(pyridin-3-yl)quinazolin-4-amine (7a).
1
Off-white solid (yield: 67%). H NMR (500 MHz, CDCl3): δ 9.75
(d, J = 1.6 Hz, 1H), 8.81 (dt, J = 7.9, 1.9 Hz, 1H), 8.70 (dd, J = 4.8,
1.6 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.79−7.73 (m, 2H), 7.50−7.46
(m, 1H), 7.42 (dd, J = 7.9, 4.8 Hz, 1H), 7.34−7.28 (m, 2H), 7.01−
6.95 (m, 3H), 6.28−6.17 (m, 1H), 4.39−4.29 (m, 2H), 4.28−4.23
(m, 2H). 13C NMR (125 MHz, CDCl3): δ 159.7, 158.6, 150.8, 150.4,
150.3, 135.7, 134.4, 132.9, 129.7, 129.0, 126.0, 123.3, 121.3, 120.8,
114.6, 114.0, 66.4, 40.8. HRMS (ESI): calcd for C21H19N4O [M +
H]+, 343.1553; found, 343.1564.
N-Methyl-N-(2-phenoxyethyl)-2-(pyridin-3-yl)quinazolin-4-
amine (8a). Off-white solid (yield: 96%). 1H NMR (500 MHz,
CDCl3): δ 9.69 (s, 1H), 8.75 (dt, J = 7.9, 1.9 Hz, 1H), 8.67 (d, J = 3.5
Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.71
(ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.44−7.34 (m, 2H), 7.28−7.24 (m,
2H), 6.96−6.87 (m, 3H), 4.45 (t, J = 5.4 Hz, 2H), 4.28 (t, J = 5.4 Hz,
2H), 3.63 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 163.1, 158.5,
157.2, 153.0, 150.7, 150.1, 135.5, 134.2, 132.3, 129.5, 128.8, 125.5,
124.6, 123.2, 121.1, 115.1, 114.5, 65.9, 52.5, 41.9. HRMS (ESI): calcd
for C22H21N4O [M + H]+, 357.1710; found, 357.1722.
N-Methyl-N-(2-phenoxypropyl)-2-(pyridin-3-yl)quinazolin-4-
amine (8d). Off-white solid (yield: 67%). 1H NMR (500 MHz,
CDCl3): δ 9.67 (s, 1H), 8.74 (dt, J = 7.9, 1.7 Hz, 1H), 8.67 (d, J = 3.8
Hz, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.73−
7.65 (m, 1H), 7.38 (dd, J = 7.9, 4.8 Hz, 1H), 7.37−7.32 (m, 1H),
7.21−7.13 (m, 2H), 6.91−6.79 (m, 3H), 5.06−4.97 (m, 1H), 4.36
(dd, J = 14.1, 3.2 Hz, 1H), 3.90 (dd, J = 14.1, 8.1 Hz, 1H), 3.59 (s,
3H), 1.42 (d, J = 6.2 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 163.0,
157.8, 157.1, 153.0, 150.6, 150.0, 135.6, 134.3, 132.3, 129.5, 128.6,
125.6, 124.5, 123.2, 121.0, 115.7, 115.0, 72.9, 58.4, 42.9, 18.1. HRMS
(ESI): calcd for C23H22N4O [M + H]+, 371.1866; found, 371.1879.
N-(1-Phenoxypropan-2-yl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(7e). Pale-yellow solid (yield: 60%). H NMR (500 MHz, CDCl3): δ
10.01 (s, 1H), 9.08 (d, J = 7.9 Hz, 1H), 8.96 (s, 1H), 8.21 (d, J = 8.2
Hz, 1H), 8.09−7.96 (m, 2H), 7.76−7.65 (m, 2H), 7.58−7.50 (m,
3H), 7.26−7.21 (m, 2H), 6.44 (d, J = 7.1 Hz, 1H), 5.39−5.29 (m,
1H), 4.51 (dd, J = 9.4, 4.3 Hz, 1H), 4.46 (dd, J = 9.3, 3.7 Hz, 1H),
1.84 (d, J = 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 159.0,
158.6, 158.4, 150.5, 150.1, 150.0, 135.8, 134.3, 132.9, 129.6, 128.7,
126.0, 123.3, 121.3, 120.7, 114.6, 113.8, 70.3, 46.2, 17.5. HRMS
(ESI): calcd for C22H21N4O [M + H]+, 357.1710; found, 357.1726.
N-Methyl-N-(1-phenoxypropan-2-yl)-2-(pyridin-3-yl)quinazolin-
N-(2-(Benzyloxy)ethyl)-2-(pyridin-3-yl)quinazolin-4-amine (7b).
1
Pale-yellow solid (51%). H NMR (500 MHz, CDCl3): δ 9.72 (s,
1H), 8.77 (d, J = 7.9 Hz, 1H), 8.68 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H),
7.78−7.71 (m, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.46−7.41 (m, 1H),
7.41−7.37 (m, 1H), 7.36−7.30 (m, 4H), 6.27−6.16 (m, 1H), 4.59 (s,
2H), 4.01 (q, J = 5.2 Hz, 2H), 3.84−3.80 (m, 2H). 13C NMR (125
MHz, CDCl3): δ 159.6, 158.5, 150.6, 150.2, 150.1, 137.8, 135.6,
132.7, 128.8, 128.5, 127.9, 127.8, 125.8, 123.2, 120.7, 113.9, 73.3,
68.4, 41.0. HRMS (ESI): calcd for C22H21N4O [M + H]+, 357.1710;
found, 357.1721.
1
4-amine (8e). Pale-yellow oil (yield: 78%). H NMR (500 MHz,
CDCl3): δ 9.71 (s, 1H), 8.76 (d, J = 7.9 Hz, 1H), 8.67 (s, 1H), 8.06
(d, J = 8.4 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.73−7.66 (m, 1H),
7.43−7.32 (m, 2H), 7.22 (dd, J = 8.6, 7.5 Hz, 2H), 6.91 (t, J = 7.4 Hz,
1H), 6.84 (d, J = 7.8 Hz, 2H), 5.27−5.16 (m, 1H), 4.28 (dd, J = 9.8,
7.6 Hz, 1H), 4.16 (dd, J = 9.9, 5.1 Hz, 1H), 3.34 (s, 3H), 1.51 (d, J =
6.9 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ 164.5, 158.4, 157.3,
153.0, 150.6, 150.2, 135.6, 134.3, 132.3, 129.5, 128.7, 125.4, 124.6,
123.2, 121.1, 115.4, 114.6, 69.2, 54.4, 33.9, 14.3. HRMS (ESI): calcd
for C23H22N4O [M + H]+, 371.1866; found, 371.1882.
N-(2-(Benzyloxy)ethyl)-N-methyl-2-(pyridin-3-yl)quinazolin-4-
1
amine (8b). Pale-yellow oil (87%). H NMR (500 MHz, CDCl3): δ
9.63 (s, 1H), 8.68 (dt, J = 7.9, 1.9 Hz, 1H), 8.61 (d, J = 3.7 Hz, 1H),
8.07 (dd, J = 8.4, 0.8 Hz, 1H), 7.85 (dd, J = 8.4, 0.8 Hz, 1H), 7.63
(ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.34−7.28 (m, 2H), 7.24−7.16 (m,
5H), 4.50 (s, 2H), 4.01 (t, J = 5.5 Hz, 2H), 3.88 (t, J = 5.5 Hz, 2H),
3.46 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 163.2, 157.2, 152.9,
150.6, 150.1, 138.0, 135.5, 134.3, 132.2, 128.7, 128.4, 127.7, 127.5,
125.6, 124.5, 123.1, 115.1, 73.3, 68.1, 53.1, 41.2. HRMS (ESI): calcd
for C23H22N4O [M + H]+, 371.1866; found, 371.1880.
N-Ethyl-N-(2-phenoxyethyl)-2-(pyridin-3-yl)quinazolin-4-amine
1
(8f). Off-white solid (yield: 68%). H NMR (500 MHz, CDCl3): δ
9.69 (d, J = 1.5 Hz, 1H), 8.74 (dt, J = 7.9, 1.9 Hz, 1H), 8.67 (dd, J =
4.8, 1.6 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H),
7.74−7.67 (m, 1H), 7.43−7.34 (m, 2H), 7.26−7.22 (m, 2H), 6.95−
6.87 (m, 3H), 4.42 (t, J = 5.6 Hz, 2H), 4.21 (t, J = 5.6 Hz, 2H), 3.94
(q, J = 7.1 Hz, 2H), 1.53 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz,
CDCl3): δ 162.8, 158.5, 157.2, 152.9, 150.7, 150.1, 135.5, 134.2,
132.3, 129.5, 129.0, 124.9, 124.8, 123.2, 121.0, 115.2, 114.5, 65.7,
N-(3-Phenoxypropyl)-2-(pyridin-3-yl)quinazolin-4-amine (7c).
1
Off-white solid (yield: 73%). H NMR (500 MHz, CDCl3): δ 9.75
(s, 1H), 8.79 (d, J = 7.9 Hz, 1H), 8.66 (d, J = 3.4 Hz, 1H), 7.89 (d, J =
8.4 Hz, 1H), 7.77−7.63 (m, 2H), 7.47−7.39 (m, 1H), 7.37 (dd, J =
H
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49.7, 47.7, 13.7. HRMS (ESI): calcd for C23H22N4O [M + H]+,
371.1866; found, 371.1882.
4-(3-Benzylpiperidin-1-yl)-2-(pyridin-3-yl)quinazoline (9e). Off-
white solid (yield: 46%). H NMR (500 MHz, CDCl3): δ 9.70 (s,
1
N-(2-Phenoxyethyl)-N-propyl-2-(pyridin-3-yl)quinazolin-4-
1H), 8.68 (d, J = 7.6 Hz, 2H), 7.92 (d, J = 8.2 Hz, 1H), 7.71−7.59
(m, 2H), 7.44−7.36 (m, 1H), 7.36−7.29 (m, 2H), 7.27−7.23 (m,
2H), 7.21 (d, J = 7.1 Hz, 2H), 4.43 (dd, J = 30.6, 13.0 Hz, 2H), 3.30−
3.14 (m, 1H), 2.91 (dd, J = 13.0, 10.7 Hz, 1H), 2.69 (dd, J = 13.5, 6.5
Hz, 1H), 2.60 (dd, J = 13.5, 8.1 Hz, 1H), 2.24−2.08 (m, 1H), 2.06−
1.95 (m, 1H), 1.95−1.84 (m, 1H), 1.84−1.67 (m, 1H), 1.48−1.30
(m, 1H). 13C NMR (125 MHz, CDCl3): δ 164.6, 157.6, 152.7, 150.7,
150.3, 139.8, 135.7, 132.5, 129.1, 128.8, 128.6, 126.3, 125.1, 125.0,
123.3, 115.5, 55.6, 50.6, 40.7, 38.6, 31.7, 25.7. HRMS (ESI): calcd for
C25H25N4 [M + H]+, 381.2074; found, 381.2089.
1
amine (8g). Pale-yellow solid (yield: 49%). H NMR (500 MHz,
CDCl3): δ 9.68 (d, J = 1.7 Hz, 1H), 8.74 (dt, J = 7.9, 1.7 Hz, 1H),
8.66 (dd, J = 4.8, 1.5 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.93 (d, J =
8.3 Hz, 1H), 7.74−7.66 (m, 1H), 7.43−7.35 (m, 2H), 7.27−7.21 (m,
2H), 6.95−6.87 (m, 3H), 4.41 (t, J = 5.6 Hz, 2H), 4.22 (t, J = 5.6 Hz,
2H), 3.84−3.78 (m, 2H), 2.02−1.93 (m, 2H), 1.02 (t, J = 7.4 Hz,
3H). 13C NMR (125 MHz, CDCl3): δ 162.9, 158.5, 157.2, 152.9,
150.7, 150.1, 135.5, 134.2, 132.3, 129.5, 129.0, 124.9, 124.7, 123.2,
121.0, 115.2, 114.5, 65.7, 54.8, 50.4, 21.6, 11.3. HRMS (ESI): calcd
for C24H25N4O [M + H]+, 385.2023; found, 385.2039.
N-(Cyclopropylmethyl)-N-(2-phenoxyethyl)-2-(pyridin-3-yl)-
quinazolin-4-amine (8h). Off-white solid (33%). 1H NMR (500
MHz, CDCl3): δ 9.70 (d, J = 1.5 Hz, 1H), 8.75 (dt, J = 7.9, 1.8 Hz,
1H), 8.67 (dd, J = 4.7, 1.4 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H), 7.93 (d,
J = 8.4 Hz, 1H), 7.78−7.65 (m, 1H), 7.45−7.34 (m, 2H), 7.23 (t, J =
15.9 Hz, 2H), 6.92 (t, J = 7.3 Hz, 1H), 6.88 (d, J = 8.0 Hz, 2H), 4.43
(t, J = 5.6 Hz, 2H), 4.34 (t, J = 5.6 Hz, 2H), 3.79 (d, J = 6.3 Hz, 2H),
1.36−1.26 (m, 1H), 0.70−0.63 (m, 2H), 0.38−0.32 (m, 2H). 13C
NMR (125 MHz, CDCl3): δ 163.3, 158.5, 157.2, 152.9, 150.7, 150.1,
135.6, 134.2, 132.3, 129.5, 129.0, 125.1, 124.9, 123.2, 121.0, 115.3,
114.5, 65.9, 57.2, 49.6, 10.1, 4.0. HRMS (ESI): calcd for C25H25N4O
[M + H]+, 397.2023; found, 397.2043.
4-(3-Phenoxypyrrolidin-1-yl)-2-(pyridin-3-yl)quinazoline (9a).
Pale-yellow solid (yield: 31%). 1H NMR (500 MHz, CDCl3): δ
9.71 (d, J = 1.5 Hz, 1H), 8.76 (dt, J = 7.9, 1.9 Hz, 1H), 8.65 (dd, J =
4.8, 1.7 Hz, 1H), 8.17 (d, J = 8.3 Hz, 1H), 7.91 (dd, J = 8.3, 0.7 Hz,
1H), 7.72−7.67 (m, 1H), 7.41−7.35 (m, 2H), 7.33−7.28 (m, 2H),
6.98 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 7.9 Hz, 2H), 5.09−5.13 (m, 1H),
4.39−4.27 (m, 2H), 4.27−4.14 (m, 2H), 2.54−2.40 (m, 1H), 2.33−
2.17 (m, 1H). 13C NMR (125 MHz, CDCl3): δ 160.0, 157.8, 156.9,
152.5, 150.6, 150.2, 135.5, 134.3, 132.2, 129.7, 128.6, 125.1, 124.6,
123.0, 121.4, 115.6, 115.3, 75.1, 55.9, 48.8, 31.4. HRMS (ESI): calcd
for C23H21N4O [M + H]+, 369.1710; found, 369.1727.
(S)-4-(3-Phenoxypiperidin-1-yl)-2-(pyridin-3-yl)quinazoline (9b).
Yellow oil (yield: 79%). 1H NMR (500 MHz, CDCl3): δ 9.71 (d, J =
1.4 Hz, 1H), 8.73 (dt, J = 7.9, 1.9 Hz, 1H), 8.68 (dd, J = 4.8, 1.7 Hz,
1H), 7.94 (s, 1H), 7.70 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.42−7.33 (m,
2H), 7.30−7.25 (m, 2H), 6.98−6.92 (m, 3H), 4.64−4.55 (m, 1H),
4.36 (dd, J = 13.1, 3.2 Hz, 1H), 4.04−3.95 (m, 1H), 3.76−3.61 (m,
2H), 2.24−2.16 (m, 1H), 2.13−2.04 (m, 1H), 1.97−1.88 (m, 1H),
1.84−1.73 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 165.0, 157.7,
157.2, 152.7, 150.7, 150.2, 135.6, 134.2, 132.6, 129.6, 128.9, 125.2,
125.0, 123.1, 121.3, 116.0, 115.4, 71.4, 53.4, 50.3, 30.1, 22.9. HRMS
(ESI): calcd for C24H23N4O [M + H]+, 383.1866; found, 383.1881.
(R)-4-(3-Phenoxypiperidin-1-yl)-2-(pyridin-3-yl)quinazoline (9c).
Yellow oil (yield: 63%). 1H NMR (500 MHz, CDCl3): δ 9.71 (d, J =
1.4 Hz, 1H), 8.73 (dt, J = 7.9, 1.9 Hz, 1H), 8.68 (dd, J = 4.8, 1.7 Hz,
1H), 7.94 (s, 1H), 7.70 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.42−7.33 (m,
2H), 7.30−7.25 (m, 2H), 6.98−6.92 (m, 3H), 4.64−4.55 (m, 1H),
4.36 (dd, J = 13.1, 3.2 Hz, 1H), 4.04−3.95 (m, 1H), 3.76−3.61 (m,
2H), 2.24−2.16 (m, 1H), 2.13−2.04 (m, 1H), 1.97−1.88 (m, 1H),
1.84−1.73 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 165.0, 157.7,
157.2, 152.7, 150.7, 150.2, 135.6, 134.2, 132.6, 129.6, 128.9, 125.2,
125.0, 123.1, 121.3, 116.0, 115.4, 71.4, 53.4, 50.3, 30.1, 22.9. HRMS
(ESI): calcd for C24H23N4O [M + H]+, 383.1866; found, 383.1883.
4-(2-Benzylpiperidin-1-yl)-2-(pyridin-3-yl)quinazoline (9d). Yel-
low solid (yield: 42%). 1H NMR (500 MHz, CDCl3): δ 9.72 (s, 1H),
8.76 (d, J = 7.9 Hz, 1H), 8.69 (d, J = 4.6 Hz, 1H), 7.90 (d, J = 8.4 Hz,
1H), 7.73 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 7.0 Hz, 1H), 7.40 (dd, J =
7.8, 4.9 Hz, 1H), 7.36 (d, J = 7.1 Hz, 1H), 7.22−7.15 (m, 4H), 7.12
(d, J = 6.4 Hz, 1H), 5.00−5.06 (m, 1H), 4.30 (d, J = 13.2 Hz, 1H),
3.68−3.55 (m, 1H), 3.26 (dd, J = 13.5, 6.6 Hz, 1H), 3.09 (dd, J =
13.5, 8.8 Hz, 1H), 2.01−1.67 (m, 6H). 13C NMR (125 MHz,
CDCl3): δ 165.2, 157.7, 152.9, 150.7, 150.4, 139.0, 135.7, 134.5,
132.4, 129.2, 128.8, 128.5, 126.4, 125.1, 124.7, 123.2, 115.6, 58.0,
45.4, 35.8, 27.3, 25.9, 19.5. HRMS (ESI): calcd for C25H25N4 [M +
H]+, 381.2074; found, 381.2084.
4-(3-Phenylpiperidin-1-yl)-2-(pyridin-3-yl)quinazoline (9f). Yel-
1
low solid (yield: 34%). H NMR (500 MHz, CDCl3): δ 9.68 (s,
1H), 8.73 (d, J = 7.9 Hz, 1H), 8.61 (d, J = 3.9 Hz, 1H), 7.87 (dd, J =
17.0, 8.3 Hz, 2H), 7.66 (s, 1H), 7.38−7.27 (m, 5H), 7.25−7.18 (m,
2H), 4.58−4.45 (m, 2H), 3.24−3.13 (m, 2H), 3.08−2.98 (m, 1H),
2.12 (d, J = 12.3 Hz, 1H), 1.96−1.73 (m, 3H). 13C NMR (125 MHz,
CDCl3): δ 165.0, 157.7, 152.8, 150.8, 150.3, 143.5, 135.7, 134.3,
132.6, 129.0, 128.8, 127.2, 126.9, 125.3, 125.2, 123.2, 115.8, 56.6,
50.8, 42.8, 32.2, 25.9. HRMS (ESI): calcd for C24H23N4 [M + H]+,
367.1917; found, 367.1930.
4-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-(pyridin-3-yl)quinazoline
(9g). Yellow solid (yield: 44%). 1H NMR (400 MHz, CDCl3): δ 9.79
(s, 1H), 8.83 (d, J = 7.9 Hz, 1H), 8.71 (s, 1H), 7.99 (dd, J = 14.1, 8.4
Hz, 2H), 7.81−7.69 (m, 1H), 7.53−7.37 (m, 2H), 7.23 (s, 4H), 5.07
(s, 2H), 4.15 (t, J = 5.7 Hz, 2H), 3.22 (t, J = 5.6 Hz, 2H). 13C NMR
(100 MHz, CDCl3): δ 164.2, 157.6, 152.8, 150.8, 150.4, 135.8, 134.8,
133.9, 132.6, 129.0, 128.9, 126.9, 126.6, 126.5, 125.2, 125.0, 123.3,
115.6, 51.4, 48.2, 29.0. HRMS (ESI): calcd for C22H19N4 [M + H]+,
339.1604; found, 339.1613.
2-(2-(Pyridin-3-yl)quinazolin-4-yl)-2,3,4,5-tetrahydro-1H-pyrido-
1
[4,3-b]indole (9h). Light-brown solid (yield: 37%). H NMR (500
MHz, CDCl3): δ 9.78 (s, 1H), 8.81 (d, J = 7.9 Hz, 1H), 8.69 (d, J =
3.7 Hz, 1H), 8.21 (s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.94 (d, J = 8.3
Hz, 1H), 7.74 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.8 Hz, 2H), 7.41 (dd, J
= 7.8, 4.8 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.17−7.11 (m, 1H),
7.11−7.05 (m, 1H), 5.14 (s, 2H), 4.27 (t, J = 5.5 Hz, 2H), 3.29−3.17
(m, 2H). 13C NMR (125 MHz, CDCl3): δ 164.6, 157.5, 152.8, 150.7,
150.2, 135.9, 135.7, 134.2, 132.6, 132.2, 128.9, 125.5, 125.2, 125.0,
123.2, 121.7, 119.7, 117.4, 115.6, 110.8, 107.8, 47.7, 47.0, 23.4.
HRMS (ESI): calcd for C24H20N5 [M + H]+, 378.1713; found,
378.1727.
2-(2-(Pyridin-3-yl)quinazolin-4-yl)-2,3,4,9-tetrahydro-1H-pyrido-
1
[3,4-b]indole (9i). Yellow solid (yield: 62%). H NMR (500 MHz,
CDCl3): δ 9.75 (s, 1H), 8.79 (d, J = 7.9 Hz, 1H), 8.68 (d, J = 3.7 Hz,
1H), 8.38 (s, 1H), 8.02−7.91 (m, 2H), 7.73 (t, J = 7.6 Hz, 1H), 7.54
(d, J = 7.6 Hz, 1H), 7.48−7.42 (m, 1H), 7.39 (dd, J = 7.8, 4.8 Hz,
1H), 7.35 (d, J = 7.9 Hz, 1H), 7.21−7.10 (m, 2H), 4.99 (s, 2H), 4.14
(t, J = 5.3 Hz, 2H), 3.18 (d, J = 4.7 Hz, 2H). 13C NMR (125 MHz,
CDCl3): δ 164.5, 157.4, 152.6, 150.7, 150.1, 136.3, 135.7, 134.1,
132.7, 130.9, 129.0, 127.0, 125.4, 124.6, 123.2, 121.8, 119.7, 118.0,
115.5, 111.0, 108.9, 49.8, 46.8, 22.0. HRMS (ESI): calcd for
C24H20N5 [M + H]+, 378.1713; found, 378.1728.
4-(2-(Pyridin-3-yl)quinazolin-4-yl)-1,4-oxazepane (9j). White
1
solid (yield: 58%). H NMR (500 MHz, CDCl3): δ 9.71 (d, J =
1.0 Hz, 1H), 8.81−8.57 (m, 2H), 7.93 (dd, J = 12.0, 8.4 Hz, 2H),
7.74−7.66 (m, 1H), 7.43−7.34 (m, 2H), 4.18−4.10 (m, 4H), 4.03−
3.99 (m, 2H), 3.85−3.80 (m, 2H), 2.21 (p, J = 5.7 Hz, 2H). 13C
NMR (125 MHz, CDCl3): δ 163.3, 157.2, 153.1, 150.6, 150.1, 135.5,
132.3, 128.9, 125.2, 124.5, 114.9, 70.2, 70.1, 53.7, 50.3, 30.7. HRMS
(ESI): calcd for C18H19N4O [M + H]+, 307.1553; found, 307.1557.
4-(2-(Pyridin-3-yl)quinazolin-4-yl)-2,3,4,5-tetrahydrobenzo[f ]-
[1,4]oxazepine (9k). Yellow solid (yield: 55%). 1H NMR (500 MHz,
CDCl3): δ 9.62 (s, 1H), 8.72−8.59 (m, 2H), 7.94 (t, J = 8.1 Hz, 2H),
7.77−7.68 (m, 1H), 7.45−7.39 (m, 1H), 7.39−7.31 (m, 2H), 7.22 (d,
J = 7.5 Hz, 1H), 7.10 (t, J = 7.2 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H),
4.97 (s, 2H), 4.48−4.39 (m, 2H), 4.35−4.26 (m, 2H). 13C NMR (125
MHz, CDCl3): δ 164.4, 159.3, 157.4, 152.8, 150.7, 150.2, 135.6,
132.6, 129.9, 129.0, 128.9, 128.7, 125.2, 125.1, 123.1, 120.5, 115.2,
I
DOI: 10.1021/acs.jmedchem.8b01294
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
70.9, 54.5, 54.0. HRMS (ESI): calcd for C22H19N4O [M + H]+,
355.1553; found, 355.1565.
153.1, 150.8, 150.0, 143.1, 142.6, 135.5, 134.1, 132.5, 128.9, 125.5,
124.7, 123.2, 121.8, 121.5, 117.4, 117.2, 115.0, 71.8, 66.0, 53.0, 43.1.
HRMS (ESI): calcd for C23H21N4O2 [M + H]+, 385.1659; found,
385.1653.
N-Methyl-2-(pyridin-3-yl)-N-(quinoxalin-2-ylmethyl)quinazolin-
4-amine (9s). Yellow solid (yield: 47%). 1H NMR (500 MHz,
CDCl3): δ 9.65 (s, 1H), 9.02 (s, 1H), 8.70 (d, J = 7.7 Hz, 1H), 8.65
(d, J = 2.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 8.1 Hz, 2H),
7.99 (d, J = 8.3 Hz, 1H), 7.83−7.72 (m, 3H), 7.42 (t, J = 7.6 Hz, 1H),
7.35 (dd, J = 7.2, 5.0 Hz, 1H), 5.41 (s, 2H), 3.63 (s, 3H). 13C NMR
(125 MHz, CDCl3): δ 163.5, 157.4, 153.2, 153.1, 150.8, 150.1, 144.7,
142.1, 135.7, 134.0, 132.8, 130.4, 129.9, 129.4, 129.2, 129.1, 125.3,
125.2, 123.3, 115.0, 57.0, 41.4. HRMS (ESI): calcd for C23H19N6 [M
+ H]+, 379.1666; found, 379.1682.
N-Methyl-N-(naphthalen-2-ylmethyl)-2-(pyridin-3-yl)quinazolin-
4-amine (9l). Off-white solid (yield: 52%). 1H NMR (500 MHz,
CDCl3): δ 9.77 (s, 1H), 8.83 (d, J = 7.8 Hz, 1H), 8.69 (d, J = 3.5 Hz,
1H), 7.99 (d, J = 8.6 Hz, 2H), 7.95−7.78 (m, 4H), 7.78−7.67 (m,
1H), 7.58−7.44 (m, 3H), 7.41 (dd, J = 7.7, 4.9 Hz, 1H), 7.37−7.28
(m, 1H), 5.23 (s, 2H), 3.45 (s, 3H). 13C NMR (125 MHz, CDCl3): δ
164.0, 157.5, 152.9, 150.8, 150.3, 135.8, 134.9, 134.3, 133.6, 133.0,
132.6, 129.0, 128.9, 127.9, 126.6, 126.1, 125.9, 125.3, 125.2, 125.1,
123.3, 115.1, 57.4, 39.7. HRMS (ESI): calcd for C25H21N4 [M + H]+,
377.1761; found, 377.1775.
N-(Benzo[c][1,2,5]thiadiazol-5-ylmethyl)-N-methyl-2-(pyridin-3-
yl)quinazolin-4-amine (9m). Pale yellow solid (yield: 65%). 1H
NMR (500 MHz, CDCl3): δ 9.70 (d, J = 1.6 Hz, 1H), 8.77 (dt, J =
7.9, 1.8 Hz, 1H), 8.67 (dd, J = 4.8, 1.6 Hz, 1H), 8.07−8.02 (m, 2H),
8.00 (t, J = 8.1 Hz, 2H), 7.79−7.70 (m, 1H), 7.64 (dd, J = 9.1, 1.4 Hz,
1H), 7.43−7.32 (m, 2H), 5.24 (s, 2H), 3.49 (s, 3H). 13C NMR (125
MHz, CDCl3): δ 163.9, 157.5, 155.2, 154.6, 153.1, 150.9, 150.3,
139.7, 135.7, 134.1, 132.8, 129.5, 129.2, 125.2, 125.0, 123.3, 122.2,
119.2, 115.1, 56.8, 40.3. HRMS (ESI): calcd for C21H17N6S [M +
H]+, 385.1230; found, 385.1245.
N-(Benzo[d]thiazol-2-ylmethyl)-N-methyl-2-(pyridin-3-yl)-
1
quinazolin-4-amine (9t). Yellow solid (yield: 26%). H NMR (400
MHz, CDCl3): δ 9.80 (s, 1H), 8.84 (d, J = 8.0 Hz, 1H), 8.69 (d, J =
3.6 Hz, 1H), 8.13 (d, J = 8.3 Hz, 1H), 8.02 (dd, J = 16.6, 8.2 Hz, 2H),
7.81 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.48 (d, J = 7.3 Hz,
1H), 7.46−7.34 (m, 3H), 5.44 (s, 2H), 3.58 (s, 3H). 13C NMR (100
MHz, CDCl3): δ 168.5, 163.2, 157.2, 153.1, 152.8, 150.9, 150.4,
135.9, 135.8, 133.8, 132.9, 129.1, 126.2, 125.4, 125.3, 125.3, 123.3,
123.0, 121.9, 115.0, 54.8, 40.8. HRMS (ESI): calcd for C22H18N5S [M
+ H]+, 384.1277; found, 384.1293.
N-Methyl-N-((2-methylisoindolin-5-yl)methyl)-2-(pyridin-3-yl)-
1
quinazolin-4-amine (9n). Light-brown solid (yield: 61%). H NMR
(400 MHz, CDCl3): δ 9.73 (d, J = 1.5 Hz, 1H), 8.79 (dt, J = 8.0, 1.9
Hz, 1H), 8.68 (dd, J = 4.8, 1.7 Hz, 1H), 7.99−7.91 (m, 2H), 7.77−
7.68 (m, 1H), 7.39 (dd, J = 7.9, 4.8 Hz, 1H), 7.36−7.29 (m, 1H),
7.28−7.21 (m, 4H), 5.05 (s, 2H), 3.97 (d, J = 6.7 Hz, 4H), 3.38 (s,
3H), 2.64 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 163.9, 157.6,
153.0, 150.8, 150.3, 136.2, 135.7, 134.3, 132.5, 128.9, 126.1, 125.2,
125.0, 123.2, 122.8, 121.1, 115.1, 60.9, 60.8, 57.1, 42.4, 39.6. HRMS
(ESI): calcd for C24H24N5 [M + H]+, 382.2026; found, 382.2040.
N-(Benzo[d][1,3]dioxol-5-ylmethyl)-N-methyl-2-(pyridin-3-yl)-
N-((1H-Benzo[d]imidazol-2-yl)methyl)-N-methyl-2-(pyridin-3-yl)-
1
quinazolin-4-amine (9u). Yellow solid (yield: 41%). H NMR (400
MHz, CDCl3): δ 9.61 (s, 1H), 8.66 (dt, J = 8.0, 1.9 Hz, 1H), 8.61−
8.48 (m, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 8.3 Hz, 1H),
7.79−7.68 (m, 1H), 7.57 (br s, 2H), 7.46−7.33 (m, 1H), 7.26−7.20
(m, 3H), 5.27 (s, 2H), 3.47 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
163.4, 156.9, 152.9, 151.1, 150.7, 149.5, 135.5, 133.7, 133.2, 128.9,
125.6, 125.5, 123.4, 122.8, 114.9, 50.5, 40.7. HRMS (ESI): calcd for
C22H19N6 [M + H]+, 367.1666; found, 367.1683.
1
quinazolin-4-amine (9o). Yellow solid (yield: 43%). H NMR (400
N-(Benzo[d]oxazol-2-ylmethyl)-N-methyl-2-(pyridin-3-yl)-
quinazolin-4-amine (9v). Off-white solid (yield: 35%). 1H NMR
(500 MHz, CDCl3): δ 9.66 (d, J = 1.5 Hz, 1H), 8.73 (dt, J = 7.9, 1.8
Hz, 1H), 8.65 (dd, J = 4.8, 1.6 Hz, 1H), 8.21 (d, J = 7.9 Hz, 1H), 7.99
(d, J = 8.3 Hz, 1H), 7.82−7.75 (m, 1H), 7.75−7.70 (m, 1H), 7.59−
7.51 (m, 1H), 7.50−7.44 (m, 1H), 7.38−7.30 (m, 3H), 5.25 (s, 2H),
3.71 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 163.5, 163.1, 157.2,
153.0, 150.9, 150.7, 150.1, 141.1, 135.9, 134.0, 132.9, 129.1, 125.4,
125.3, 125.3, 124.7, 123.3, 120.2, 115.1, 110.8, 50.4, 41.7. HRMS
(ESI): calcd for C22H18N5O [M + H]+, 368.1506; found, 368.1518.
N-Methyl-N-(2-phenoxyethyl)-2-(pyridin-2-yl)quinazolin-4-
MHz, CDCl3): δ 9.74 (s, 1H), 8.80 (d, J = 8.0 Hz, 1H), 8.68 (d, J =
3.3 Hz, 1H), 7.97 (d, J = 8.8 Hz, 2H), 7.76−7.66 (m, 1H), 7.40 (dd, J
= 7.8, 4.8 Hz, 1H), 7.37−7.32 (m, 1H), 6.89 (d, J = 9.4 Hz, 2H), 6.83
(d, J = 7.8 Hz, 1H), 5.97 (s, 2H), 4.98 (s, 2H), 3.37 (s, 3H). 13C
NMR (100 MHz, CDCl3): δ 163.8, 157.5, 152.9, 150.8, 150.3, 148.4,
147.2, 135.8, 132.5, 131.2, 128.9, 125.2, 125.0, 123.3, 120.7, 115.1,
108.6, 107.8, 101.2, 56.7, 39.6. HRMS (ESI): calcd for C22H19N4O2
[M + H]+, 371.1503; found, 371.1518.
N-((2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-N-methyl-2-
(pyridin-3-yl)quinazolin-4-amine (9p). Pale-yellow solid (yield:
1
1
amine (11a). Pale-yellow solid (yield: 73%). H NMR (500 MHz,
36%). H NMR (500 MHz, CDCl3): δ 9.73 (s, 1H), 8.79 (d, J =
CDCl3): δ 8.85 (d, J = 4.7 Hz, 1H), 8.48 (d, J = 7.9 Hz, 1H), 8.16 (d,
J = 8.4 Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.81 (td, J = 7.7, 1.8 Hz,
1H), 7.71 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.40 (ddd, J = 8.3, 7.0, 1.2
Hz, 1H), 7.37−7.32 (m, 1H), 7.27−7.24 (m, 2H), 6.97−6.87 (m,
3H), 4.44 (t, J = 5.6 Hz, 2H), 4.28 (t, J = 5.6 Hz, 2H), 3.61 (s, 3H).
13C NMR (125 MHz, CDCl3): δ 163.5, 158.5, 157.9, 156.0, 153.2,
150.0, 136.6, 132.2, 129.8, 129.5, 125.2, 124.9, 124.2, 123.6, 121.1,
115.3, 114.5, 65.8, 52.4, 41.7. HRMS (ESI): calcd for C22H21N4O [M
+ H]+, 357.1710; found, 357.1724.
N-Methyl-N-(2-phenoxyethyl)-2-(pyridin-4-yl)quinazolin-4-
amine (11b). White solid (yield: 56%). 1H NMR (500 MHz): δ 9.69
(s, 1H), 8.75 (dd, J = 7.9, 1.7 Hz, 1H), 8.67 (d, J = 4.0 Hz, 1H), 8.13
(d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.73−7.68 (m, 1H),
7.41−7.35 (m, 2H), 7.26 (d, J = 8.4 Hz, 2H), 6.95−6.87 (m, 3H),
4.45 (t, J = 5.4 Hz, 2H), 4.28 (t, J = 5.4 Hz, 2H), 3.68 (s, 3H), 3.63 (s,
3H). 13C NMR (125 MHz, CDCl3): δ 163.2, 158.5, 157.0, 152.9,
150.2, 146.2, 132.4, 129.6, 129.0, 125.5, 125.0, 122.2, 121.1, 115.3,
114.5, 65.8, 52.5, 41.7. HRMS (ESI): calcd for C22H21N4O [M + H]+,
357.1710; found, 357.1721.
N-Methyl-N-(2-phenoxyethyl)-2-(pyrimidin-5-yl)quinazolin-4-
amine (11c). White solid (yield: 61%). 1H NMR (500 MHz, CDCl3):
δ 9.73 (s, 2H), 9.27 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 8.3
Hz, 1H), 7.76−7.70 (m, 1H), 7.45−7.40 (m, 1H), 7.29−7.24 (m,
2H), 6.93 (t, J = 7.4 Hz, 1H), 6.89 (d, J = 7.9 Hz, 2H), 4.44 (t, J = 5.3
Hz, 2H), 4.28 (t, J = 5.3 Hz, 2H), 3.64 (s, 3H). 13C NMR (125 MHz,
7.9 Hz, 1H), 8.67 (d, J = 4.6 Hz, 1H), 7.95 (t, J = 8.0 Hz, 2H), 7.74−
7.66 (m, 1H), 7.39 (dd, J = 7.7, 4.9 Hz, 1H), 7.36−7.30 (m, 1H), 6.94
(s, 1H), 6.89 (s, 2H), 4.95 (s, 2H), 4.27 (s, 4H), 3.37 (s, 3H). 13C
NMR (125 MHz, CDCl3): δ 163.8, 157.5, 153.0, 150.8, 150.3, 144.0,
143.1, 135.7, 134.3, 132.5, 130.6, 128.9, 125.2, 124.9, 123.2, 120.3,
117.7, 116.1, 115.1, 64.5, 64.4, 56.5, 39.5. HRMS (ESI): calcd for
C23H21N4O2 [M + H]+, 385.1659; found, 385.1683.
(S)-N-((2,3-Dihydrobenzo[b][1,4]dioxin-2-yl)methyl)-N-methyl-2-
(pyridin-3-yl)quinazolin-4-amine (9q). Pale-yellow solid (yield:
1
85%). H NMR (500 MHz, CDCl3): δ 9.65 (s, 1H), 8.72 (d, J =
7.9 Hz, 1H), 8.69−8.64 (m, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.93 (d, J
= 8.4 Hz, 1H), 7.76−7.68 (m, 1H), 7.43−7.34 (m, 2H), 6.90−6.78
(m, 4H), 4.84−4.76 (m, 1H), 4.40−4.27 (m, 2H), 4.17−4.08 (m,
2H), 3.66 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 163.0, 157.1,
153.1, 150.8, 150.0, 143.1, 142.6, 135.5, 134.1, 132.5, 128.9, 125.5,
124.7, 123.2, 121.8, 121.5, 117.4, 117.2, 115.0, 71.8, 66.0, 53.0, 43.1.
HRMS (ESI): calcd for C23H21N4O2 [M + H]+, 385.1659; found,
385.1657.
(R)-N-((2,3-Dihydrobenzo[b][1,4]dioxin-2-yl)methyl)-N-methyl-2-
(pyridin-3-yl)quinazolin-4-amine (9r). Pale-yellow solid (yield:
1
89%). H NMR (500 MHz, CDCl3): δ 9.65 (s, 1H), 8.72 (d, J =
7.9 Hz, 1H), 8.69−8.64 (m, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.93 (d, J
= 8.4 Hz, 1H), 7.76−7.68 (m, 1H), 7.43−7.34 (m, 2H), 6.90−6.78
(m, 4H), 4.84−4.76 (m, 1H), 4.40−4.27 (m, 2H), 4.17−4.08 (m,
2H), 3.66 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 163.0, 157.1,
J
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Journal of Medicinal Chemistry
Article
CDCl3): δ 162.0, 158.2, 157.4, 155.7, 154.0, 151.8, 131.5, 130.8,
128.5, 127.8, 124.6, 124.0, 120.1, 114.2, 113.4, 64.8, 51.5, 40.9.
HRMS (ESI): calcd for C21H20N5O [M + H]+, 358.1662; found,
358.1673.
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 λex = 365 nm and λem = 440
nm.
Cell Culture and Compound Treatment. The GBA N370S/84GG
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 for fibroblasts or a 10 day
treatment for dopaminergic neurons, cells were washed with PBS
twice followed by 1% Triton X-100 lysis buffer to lyse cells. Protein
concentrations were measured with a BCA kit (Thermo), and the
GCase activity was determined at pH 5.5.
N-Methyl-N-(2-phenoxyethyl)-2-phenylquinazolin-4-amine
1
(11d). White solid (yield: 82%). H NMR (500 MHz, CDCl3): δ
8.54−8.47 (m, 2H), 8.11 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.2 Hz,
1H), 7.69 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7.48−7.43 (m, 3H), 7.36
(ddd, J = 8.3, 6.9, 1.2 Hz, 1H), 7.28−7.24 (m, 2H), 6.97−6.89 (m,
3H), 4.45 (t, J = 5.6 Hz, 2H), 4.27 (t, J = 5.6 Hz, 2H), 3.60 (s, 3H).
13C NMR (125 MHz, CDCl3): δ 163.2, 159.0, 158.6, 132.1, 130.0,
129.5, 128.4, 128.3, 125.4, 124.2, 121.0, 114.5, 65.9, 52.4, 41.8.
HRMS (ESI): calcd for C21H20N6 [M + H]+, 356.1744; found,
356.1771.
N-Methyl-N-(2-phenoxyethyl)-2-(thiophen-3-yl)quinazolin-4-
1
amine (11e). Pale-yellow solid (yield: 71%). H NMR (500 MHz,
CDCl3): δ 8.26 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.94 (dd, J = 5.0, 0.9
Hz, 1H), 7.88 (d, J = 7.5 Hz, 1H), 7.69−7.63 (m, 1H), 7.37−7.30 (m,
2H), 7.26 (dd, J = 8.6, 7.5 Hz, 2H), 6.96−6.90 (m, 3H), 4.43 (t, J =
5.6 Hz, 2H), 4.23 (t, J = 5.6 Hz, 2H), 3.58 (s, 3H). 13C NMR (125
MHz, CDCl3): δ 163.2, 158.6, 156.4, 153.1, 142.8, 132.1, 129.5,
128.5, 127.8, 127.2, 125.4, 124.0, 121.0, 114.8, 114.6, 65.9, 52.4, 41.7.
HRMS (ESI): calcd for C21H20N3OS [M + H]+, 362.1322; found,
362.1336.
Measurement of Lysosomal GCase Activity. Fibroblasts were
plated into a 96-well plate and cultured and treated with compounds
as described above. After 3 days of treatment, the medium was
replaced with media containing 5-(pentafluorobenzoylamino)-
fluorescein di-β-D-glucopyranodside (PFB-FDGlu) or media contain-
ing PFB-FDGlu with 400 μM bafilomycin. Cells were incubated for 1
h to allow for loading of the substrate into the lysosome. After
loading, substrate hydrolysis was monitored by measuring fluores-
cence unquenching every 20 min at 485 nm excitation and 525 nm
emission.
Human iPSC Culture and Neural Differentiation. Human iPSC
lines were generated from skin fibroblasts through retroviral
expression or using Sendai virus based delivery of key reprogramming
vectors including four Yamanaka factors (Oct, Sox2, Klf4, and c-
Myc).23 Heterozygous N370S GBA1 mutant fibroblasts were
reprogrammed into iPSC through Northwestern University Stem
Cell Core Facility and characterized in this study (see Supporting
Information figure).
The iPSC line from one separate healthy control subject was
previously described and characterized.24 Healthy control and GBA1
mutant iPSC lines were characterized for expression of pluripotent
markers by immunocytochemistry (NANOG, OCT4, SSEA4, and
TRA1-81). Karyotype analysis was performed by Cell Line Genetics
using standard protocols for high-resolution G-banding. iPSC were
cultured in mTeSR media (Stem Cell Technologies) and passaged
every 5−7 days on matrigel (Corning) coated plates. All lines were
routinely tested for mycoplasma contamination.
N-Methyl-N-(2-phenoxyethyl)-2-(thiophen-2-yl)quinazolin-4-
1
amine (11f). Pale-yellow solid (yield: 45%). H NMR (500 MHz,
CDCl3): δ 8.06 (d, J = 8.4 Hz, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.66
(t, J = 7.4 Hz, 1H), 7.41 (d, J = 4.8 Hz, 1H), 7.36−7.29 (m, 1H),
7.28−7.24 (m, 2H), 7.14−7.11 (m, 1H), 6.95−6.89 (m, 3H), 4.46 (t,
J = 5.5 Hz, 2H), 4.22 (t, J = 5.5 Hz, 2H), 3.60 (s, 3H). 13C NMR
(125 MHz, CDCl3): δ 162.8, 158.6, 155.9, 153.0, 145.0, 132.3, 129.5,
128.8, 128.3, 128.0, 125.5, 124.0, 121.0, 114.8, 114.5, 65.9, 52.6, 41.9.
HRMS (ESI): calcd for C21H20N3OS [M + H]+, 362.1322; found,
362.1336.
2-(Furan-2-yl)-N-methyl-N-(2-phenoxyethyl)quinazolin-4-amine
1
(11g). Yellow oil (yield: 40%). H NMR (500 MHz, CDCl3): δ 8.06
(dd, J = 8.5, 0.9 Hz, 1H), 7.95 (dd, J = 8.4, 0.8 Hz, 1H), 7.67 (ddd, J
= 8.3, 6.9, 1.3 Hz, 1H), 7.61 (dd, J = 1.6, 0.8 Hz, 1H), 7.34 (ddd, J =
8.3, 6.9, 1.3 Hz, 1H), 7.29−7.22 (m, 3H), 6.96−6.90 (m, 3H), 6.53
(dd, J = 3.3, 1.7 Hz, 1H), 4.42 (t, J = 5.6 Hz, 2H), 4.20 (t, J = 5.6 Hz,
2H), 3.56 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 163.2, 158.5,
153.2, 152.8, 152.5, 144.5, 132.3, 129.5, 128.7, 125.4, 124.2, 121.0,
115.0, 114.5, 112.7, 111.7, 65.7, 52.4, 41.7. HRMS (ESI): calcd for
C21H20N3O2 [M + H]+, 346.1550; found, 346.1559.
Differentiation of iPSCs into midbrain dopaminergic neurons was
done according to published protocols,25 passaged and maintained as
described previously.26 At d25 to d30, neural blocks were passed by
accutase treatment onto PDL/laminin-coated culture dishes. Neu-
tralization growth factors were withdrawn at d40, and neurons were
maintained in Neurobasal media (Life Technologies) containing
Neurocult SM1 (Stemcell technologies). Immunocytochemistry
demonstrated the neuralization efficiency in neurons derived from
iPSC lines using neural (β-III-tubulin) and midbrain (TH, FOXA2,
LMX1a) specific markers.
2-(Furan-3-yl)-N-methyl-N-(2-phenoxyethyl)quinazolin-4-amine
1
(11h). Yellow solid (yield: 63%). H NMR (500 MHz, CDCl3): δ
8.21 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.69−
7.63 (m, 1H), 7.47 (t, J = 1.7 Hz, 1H), 7.35−7.29 (m, 1H), 7.29−
7.24 (m, 2H), 7.11 (d, J = 1.5 Hz, 1H), 6.96−6.92 (m, 1H), 6.91 (d, J
= 7.9 Hz, 2H), 4.41 (t, J = 5.6 Hz, 2H), 4.19 (t, J = 5.6 Hz, 2H), 3.55
(s, 3H). 13C NMR (125 MHz, CDCl3): δ 163.1, 158.6, 155.7, 153.0,
144.5, 143.4, 132.1, 129.5, 128.3, 127.9, 125.4, 123.9, 121.0, 114.9,
114.5, 109.9, 65.8, 52.3, 41.6. HRMS (ESI): calcd for C21H20N3O2
[M + H]+, 346.1550; found, 346.1561.
Sequencing Analysis. DNA samples were extracted from cell
pellets using Mini prep kits (Qiagen). Eluted gDNA was used as a
template for PCR amplification using Phusion Master Mix (NEB).
The PCR product was purified using the PCR Purification Kit
(Qiagen) and sent for sequencing through Northwestern University
Seq Core or University of Chicago Seq Core.
Enzyme Activity Assay. 4-Methylumbelliferyl β-D-glucopyranoside
(4MU-β-Glc) and buffer components were purchased from Sigma-
Aldrich (St. Louis, MO). Residual injection solutions of the
recombinant wild-type GCase enzyme velaglucerase alfa (Vpriv,
Shire Human Genetic Therapies, Inc.) in clinics were collected for the
activity assays. The GCase activity assay buffer was composed of 50
mM citric acid, 176 mM K2HPO4, and 0.01% Tween-20 at pH 5.9. A
solution of 1 M sodium hydroxide and 1 M glycine was used as the
stop solution.
The compounds in DMSO solution (0.5 μL/well) were transferred
to a black 96-well plate (the final titration was 48 nM to 100 μM, a
12-point dilution series). The enzyme solution (33.5 μL, 7.5 nM final
concentration) was transferred to the wells. After 5 min of incubation
at room temperature, the enzyme reaction was initiated by the
addition of a blue substrate (4MU-β-Glc) (33 μL/well). The final
concentration of the blue substrate was 1.5 mM. The blue substrate
Deglycosylation of Proteins/Molecular Shift Assay.22 To study
the subcellular localization and transport of the various GCase
mutants (ER and post-ER localization), EndoH and PNGaseF
digestions were performed. For both reactions, 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. Anti-hGCase (From Johannes Aerts, Leiden
University, Leiden, The Netherlands) was used to detect the different
forms of GCase.
K
DOI: 10.1021/acs.jmedchem.8b01294
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Journal of Medicinal Chemistry
Article
Western Blots. Proteins were denatured in 20% SDS sample buffer
at 100 °C for 10 min; 10% Tris-glycine (Life Tech) was used for gels;
a Trans-Blot Turbo PVDF kit (Bio-Rad) was used for membrane
transfer. GCase antibody (Sigma-Aldrich) and GAPDH primary
antibodies (EMD Millipore) were incubated with the membranes
overnight, and then the incubated membranes were treated with the
secondary antibody (IRDye 680RD goat anti-rabbit IgG (H + L) or
IRDye 800CW Goat anti-mouse IgG (H + L), LI-COR) for 30 min.
An Odyssey CLx Imaging System (LI-COR) was used to scan the
membranes and analyze the imaging.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b01294.
■
S
Characterization of iPSC lines and differentiated
1
dopaminergic neurons. H and 13C NMR spectra of
6a, 8a, 9l, 9o, 9q, 9s, 11a, and 11b (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: dkrainc@nm.org. Phone: 312-503-3936. Fax: 312-
503-3950 (D.K.).
■
*E-mail: Agman@chem.northwestern.edu. Phone: 847-491-
5653. Fax: 847-491-7713 (R.B.S.).
ORCID
Dimitri Krainc: 0000-0002-4716-1886
Richard B. Silverman: 0000-0001-9034-1084
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH R01NS076054,
R01NS096240, WildKat and Buckeye Research LLC (to
D.K.) and The Michael J. Fox Foundation for Parkinson’s
Research (to J.Z.). 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
International Institute for Nanotechnology (IIN). We would
like to thank H. Goudarzi and S. Shafaie in IMSERC at
Northwestern University for their assistance with HRMS
experiments. We thank the Northwestern Stem Cell Core
Facility for the generation of iPSC lines from the GBA1
mutation carrier.
ABBREVIATIONS
■
GD, Gaucher’s disease; GCase, β-glucocerebrosidase; ER,
endoplasmic reticulum; PC, pharmacological chaperone; IFG,
isofagomine; PD, Parkinson’s disease; DLB, dementia with
Lewy bodies; SAR, structure−activity relationship
(15) Tropak, M. B.; Kornhaber, G. J.; Rigat, B. A.; Maegawa, G. H.;
Buttner, J. D.; Blanchard, J. E.; Murphy, C.; Tuske, S. J.; Coales, S. J.;
Hamuro, Y.; Brown, E. D.; Mahuran, D. J. Identification of
pharmacological chaperones for Gaucher disease and characterization
of their effects on β-glucocerebrosidase by hydrogen/deuterium
exchange mass spectrometry. ChemBioChem 2008, 9, 2650−2662.
(16) Huang, W.; Zheng, W.; Urban, D. J.; Inglese, J.; Sidransky, E.;
Austin, C. P.; Thomas, C. J. N4-phenyl modifications of N2-(2-
hydroxyl)ethyl-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamines enhance
glucocerebrosidase inhibition by small molecules with potential as
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