Optimization of Eliglustat-Based Glucosylceramide Synthase Inhibitors as Substrate Reduction Therapy for Gaucher Disease Type 3

Article abstract of DOI:10.1021/acschemneuro.0c00558

There remain no approved therapies for rare but devastating neuronopathic glyocosphingolipid storage diseases, such as Sandhoff, Tay-Sachs, and Gaucher disease type 3. We previously reported initial optimization of the scaffold of eliglustat, an approved therapy for the peripheral symptoms of Gaucher disease type 1, to afford 2, which effected modest reductions in brain glucosylceramide (GlcCer) in normal mice at 60 mg/kg. The relatively poor pharmacokinetic properties and high Pgp-mediated efflux of 2 prompted further optimization of the scaffold. With a general objective of reducing topological polar surface area, and guided by multiple metabolite identification studies, we were successful at identifying 17 (CCG-222628), which achieves remarkably greater brain exposure in mice than 2. After demonstrating an over 60-fold improvement in potency over 2 at reducing brain GlcCer in normal mice, we compared 17 with Sanofi clinical candidate venglustat (Genz-682452) in the CBE mouse model of Gaucher disease type 3. At doses of 10 mg/kg, 17 and venglustat effected comparable reductions in both brain GlcCer and glucosylsphingosine. Importantly, 17 achieved these equivalent pharmacodynamic effects at significantly lower brain exposure than venglustat.

Full text of DOI:10.1021/acschemneuro.0c00558

pubs.acs.org/chemneuro
Research Article
Optimization of Eliglustat-Based Glucosylceramide Synthase
Inhibitors as Substrate Reduction Therapy for Gaucher Disease Type
3
Michael W. Wilson, Liming Shu, Vania Hinkovska-Galcheva, Yafei Jin, Walajapet Rajeswaran, Akira Abe,
Ting Zhao, Ruijuan Luo, Lu Wang, Bo Wen, Benjamin Liou, Venette Fannin, Duxin Sun, Ying Sun,
James A. Shayman,* and Scott D. Larsen*
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ABSTRACT: There remain no approved therapies for rare but
devastating neuronopathic glyocosphingolipid storage diseases,
such as Sandhoff, Tay-Sachs, and Gaucher disease type 3. We
previously reported initial optimization of the scaffold of eliglustat,
an approved therapy for the peripheral symptoms of Gaucher
disease type 1, to afford 2, which effected modest reductions in
brain glucosylceramide (GlcCer) in normal mice at 60 mg/kg. The
relatively poor pharmacokinetic properties and high Pgp-mediated
efflux of 2 prompted further optimization of the scaffold. With a
general objective of reducing topological polar surface area, and
guided by multiple metabolite identification studies, we were
successful at identifying 17 (CCG-222628), which achieves remarkably greater brain exposure in mice than 2. After demonstrating
an over 60-fold improvement in potency over 2 at reducing brain GlcCer in normal mice, we compared 17 with Sanofi clinical
candidate venglustat (Genz-682452) in the CBE mouse model of Gaucher disease type 3. At doses of 10 mg/kg, 17 and venglustat
effected comparable reductions in both brain GlcCer and glucosylsphingosine. Importantly, 17 achieved these equivalent
pharmacodynamic effects at significantly lower brain exposure than venglustat.
KEYWORDS: Glucosylceramide synthase, eliglustat tartrate, Gaucher disease, blood−brain barrier
A more recent therapeutic strategy has been the use of small
molecular entities that reversibly inhibit enzymes central to
glycolipid synthesis.3 This approach, termed substrate reduction
therapy, has primarily targeted glucosylceramide (GlcCer)
synthase. This enzyme uses ceramide and UDP-glucose as
substrates to form GlcCer which accumulates in Gaucher
disease and which is the base cerebroside in glycolipids that
accumulate in Fabry disease and the gangliosidoses. In 2014,
eliglustat tartrate4 (1, Figure 1) was approved as the first stand-
alone oral agent for the treatment of Gaucher disease type 1
based on clinical studies that demonstrated comparable efficacy
in preventing and reversing key clinical features of the disease
including splenomegaly, hepatomegaly, anemia, and thrombo-
cytopenia and its noninferiority in patients previously stabilized
on imiglucerase.5,6 The safety profile of eliglustat tartrate in
INTRODUCTION
■
Glycosphingolipid storage diseases, although rare, represent a
significant fraction of all lysosomal storage disorders by
prevalence.1 In most cases, these disorders result from the loss
of an enzyme or enzyme activator that is required for the
catabolism of a glucosylceramide based glycolipid. These
diseases include Gaucher and Fabry disease, Tay-Sachs and
Sandhoff disease, and gangliosidosis GM1. Gaucher disease type
1 and Fabry disease, in their classic clinical phenotypes, lack
central nervous system manifestations, although Parkinson’s
disease may be a long-term complication of Gaucher, and stroke
is common in Fabry due to vascular accumulation of
glycolipids.2 As such, the standard of care has been the use of
enzyme replacement therapy in which a recombinant enzyme, β-
glucocerebrosidase or α-galactosidase A, is effective in lowering
lysosomal glycolipid storage due to the ability of tissues to bind,
endocytose, and traffick lysosomal enzymes through recognition
of mannose or mannose-6-phosphate groups. Unfortunately,
enzyme replacement has not proven suitable for the treatment of
diseases with central nervous system (CNS) involvement due to
the failure to deliver recombinant enzyme across the blood brain
barrier.
Received: August 26, 2020
Accepted: September 30, 2020
Published: October 9, 2020
https://dx.doi.org/10.1021/acschemneuro.0c00558
© 2020 American Chemical Society
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Figure 1. Lead glucosylceramide synthase inhibitors.
a
Scheme 1. Synthetic Route to New Eliglustat Analogues
a
Reagents and conditions: (a) (S)-5-phenylmorpholin-2-one, toluene, azeotropic reflux, 24 h; (b) R3R4NH, heat; (c) aq HCl; (d) LiAlH4; (e) BH3;
(f) H2, Pd/C; (g) R5CH2COO-Suc; (h) R5CH2COOH, EDC.
clinical studies was favorable as well. Despite its low nanomolar
potency against GlcCer synthase, eliglustat exhibits a high
degree of selectivity vs a number of closely related enzymes,
including lysosomal glucocerebrosidase (GBA1) and non-
lysosomal glucosylceramidase (GBA2).7 Unfortunately, eliglu-
stat fails to achieve significant exposure in the brain and thus is
unsuited for the treatment of those glycosphingolipidoses with
CNS involvement which include Gaucher disease types 2 and 3,
the GM2 gangliosidoses (Tay-Sachs and Sandhoff disease), and
GM1 gangliosidosis.
venglustat (3, Figure 1), has been shown to reduce brain
glycolipids and increase lifespan in murine models of both
Gaucher disease type 3 and Sandhoff.11,12 Compound 3 has
progressed to Phase II clinical trials for Fabry, and recruitment is
underway for Gaucher type 3 patients. Compound 3 is used as a
comparator in the present studies, and we were able to
successfully demonstrate in this work that our new lead has
comparable pharmacodynamic efficacy in vivo in a mouse model
of Gaucher type 3.
We previously reported on the use of the eliglustat scaffold as
the basis for the design of CNS-permeant glucosylceramide
synthase inhibitors.8 Based on a computational comparison of
eliglustat to CNS-active FDA approved compounds, modifica-
tions to the carboxamide N-acyl group of eliglustat were made to
lower topological polar surface area (TPSA) and rotatable bond
number. One analogue CCG-203586 (2, Figure 1) was
identified that inhibited glucosylceramide synthase at low
nanomolar concentrations and was able to lower glucosylcer-
amide in wild-type mouse brain. We subsequently demonstrated
that this compound could partially prevent the accumulation of
ganglioside GM2 in a mouse model of Sandhoff disease.9 While
conceptually promising, 2 is characterized by rapid clearance
and relatively poor brain exposure in mice. The present work
reports on the further optimization of eliglustat analogues with a
continued focus on properties that determine CNS exposure,
combined with a metabolite-guided optimization of pharmaco-
kinetics. We now report the identification of an analogue of
eliglustat with significantly greater CNS exposure and activity.
Sanofi/Genzyme has reported CNS-active GlcCer synthase
inhibitors based on a novel pharmacophore.10 One compound,
RESULTS AND DISCUSSION
■
Chemistry. All new compounds were prepared by the route
shown in Scheme 1, which has been previously reported.13
Commercial benzaldehydes 4 were condensed with (S)-5-
phenylmorpholin-2-one under Dean−Stark conditions to
produce aminals 5 as single diastereomers. Lactone opening
with a secondary amine, followed by acidic hydrolysis provided
amides 6. After hydride-mediated reduction of the amide, the
chiral auxiliary was removed by catalytic hydrogenation to give
primary amines 8. Final N-acylation with N-hydroxysuccini-
midyl esters or carbodiimide-mediated coupling to carboxylic
acids afforded the desired final compounds 9, which were
isolated as single diastereomers within the limits of NMR
detection, thereby ruling out racemization of either chiral center.
Assessment of In Vitro Biological Activity. All new
analogues were assayed for inhibitory activity against GlcCer
synthase in two different formats: broken cell membrane
preparations (“crude enzyme”) and intact MDCK cells, and
results are reported in Tables 1-3. The broken cell assay is a
direct measurement of the glycolipid synthase activity in an assay
optimized for the detection of glucosylceramide synthesis. The
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the para-methoxy analogue 15 was more potent than the para-
fluoro 16. Unfortunately, the addition of the fluorine failed to
improve stability to metabolism, suggesting that the majority of
metabolism was due to O-demethylation. A metabolite
identification study on 15 indeed confirmed this hypothesis
(Figure 3). Interestingly, this study also showed that a minor
metabolite is the dealkylated primary amine, with no indication
of indane oxidation occurring.
Table 1. Modification of the Indane
Following up on these results, we examined several analogues
that replace the O-methyl with more metabolically stable
moieties (Table 2). Gratifyingly, the simple 4-trifluoromethoxy
analogue 17 retained excellent potency in our whole cell assay while
improving the half-life in mouse liver microsomes by over 10-fold (37
min vs 2.5 min). Importantly, this improvement extended to
human liver microsomes (HLM), where we observed a 3-fold
increase in half-life. Closely related analogues difluoromethoxy
(18) and 2,2,2-trifluoroethoxy (19) also retained potency, but
were not as stable to metabolism in MLM or HLM as 17. This
could be due to either oxidation of the available CH moieties on
the side chains, or to increased electron density on the aromatic
ring, or both. The poor metabolic stability of the cyclo-
propylmethyl analogue 20 is consistent with this hypothesis.
Moving the trifluoromethoxy group to the meta position (21)
greatly diminished activity, as did replacement by trifluor-
omethyl (22). Overall the SAR results in Table 2 are consistent
with the well-established requirement for an electron donating
atom at the 4-position (R2) of the aromatic ring for good
inhibition of GlcCer synthase.15,17
Further follow up of the metabolite id results in Figure 2
entailed replacement of the pyrrolidine of 2 with 3-
fluoropyrrolidine (23) and azetidine (24), both of which were
expected to be less susceptible to N-dealkylation (Table 3). The
more active of the two new analogues, fluoro-pyrrolidine 23
unfortunately offered no improvement in stability to MLM,
confirming that N-dealkylation is secondary to oxidation of the
benzodioxane ring.
crude enzyme
MDCKs cell
IC50 (nM)
MLM
a
no.
G
R6
R7
IC50 (nM)
T
1/2 (min)
2
CH2
H
H
H
F
H
H
F
27
193
14
15.3
2.5
b
10 CO
11 CH2
12 CH2
>30
1.3
7.2
1.1
2.6
H
36.5
a
b
Half-life when incubated with mouse liver microsomes. 28%
inhibition at 30 nM.
MDCK cell assay measures the ability of a compound to lower
the cellular content of glucosylceramide over 24 h in a viable cell
in the setting of its endogenous glycolipid metabolism. We have
previously described these assays in detail.8,14
Structure−Activity Relationships. Although in our
previous work we demonstrated that 2 can induce measurable
decreases of GlcCer in the brains of normal mice and prevent the
accumulation of ganglioside GM2 in a mouse model of Sandhoff
disease, subsequent pharmacokinetic studies revealed that
systemic clearance in mice is rapid and overall brain exposure
low (Table 4). To guide the design of more metabolically stable
analogues, we subjected 2 to a metabolite identification study to
pinpoint the specific sites at which CYP450-mediated oxidative
metabolism was occurring (Figure 2). After incubation with
mouse liver microsomes (MLM) for 60 min, analysis by LC/
MS/MS indicated that the two major routes of metabolism
entail oxidation of the benzodioxane ring, along with a minor
route of indane oxidation.
We performed one final metabolite id study on 17 to
determine if the major site of metabolism had shifted due to
oxidation of the aromatic ring being largely blocked by the
trifluoromethoxy group (Figure 4). Although all metabolites
were minor relative to the parent drug under the conditions of
the assay, both of the major metabolites were the result of
oxidation proximal to the pyrrolidine nitrogen, indicating a clear
shift away from the alkoxy aromatic ring. We therefore also
prepared the azetidine and 3-fluoropyrrolidine analogues of 17
to determine if stability could be improved even further (25 and
26, Table 3). Although azetidine 26 ultimately proved to be our
most stable analogue to date in MLM, both of these new amine
analogues suffered unacceptable losses in potency.
Based on these results, we first explored modification of the
indane (Table 1). Blocking oxidation of the cyclopentyl ring of
the indane (10) with a carbonyl resulted in an unacceptable loss
of potency. Fluorination of the aromatic ring at the two possible
locations (11, 12) enhanced potency, especially at the meta
position, but did not improve metabolic stability.
We then turned our focus to the left-hand aromatic ring
(Table 2). Because the ethylenedioxy moiety of the benzodiox-
ane ring donates significant electron density into the aromatic
ring, as well as being highly susceptible to metabolism itself, we
first replaced it with simple regioisomeric (and lower TPSA)
monomethoxys (13 and 14). Although both of these analogues
retained excellent potency, they did not significantly improve the
half-life in MLM. We next added ortho-fluorines to the methoxy
regioisomers (15 and 16). Consistent with SAR precedent,15−17
Included in Table 2 for comparison to 17 is the Sanofi/
Genzyme clinical lead 3. Our optimized compound is 10-fold
Figure 2. Identification of metabolites of 2 formed by incubation with mouse liver microsomes.
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Table 2. Modification of the Aromatic Ring
a
b
no.
R1
R2
−OCH2CH2O−
crude enzyme IC50 (nM)
MDCK cells IC50 (nM)
MLM T1/2 (min)
HLM T1/2 (min)
2
27
47.6
54.1
27
>300
110
123
56
46
>1000
>300
15.3
3.4
5.5
2.7
72.7
3.6
9.6
0.87
1.0
52.4
35.0
37
2.5
5.5
4
28
13
14
15
16
17
18
19
20
21
22
3
H
OMe
F
OMe
H
H
H
H
OCF3
H
NA
OMe
H
OMe
F
OCF3
OCHF2
OCH2CF3
OCH2c-Pr
H
2.7
37
18
10
<3
84
33
21
CF3
NA
240
>60
>60
a
b
Half-life when incubated with mouse liver microsomes. Half-life when incubated with human liver microsomes.
Figure 3. Identification of metabolites of 15 formed by incubation with mouse liver microsomes.
Figure 4. Identification of metabolites of 17 formed by incubation with mouse liver microsomes.
Table 3. Modification of the Amine
a
no.
R1
R2
NR3R4
pyrrolidine
(R)-3-F-pyrrolidine
azetidine
pyrrolidine
(R)-3-F-pyrrolidine
azetidine
crude enzyme IC50 (nM)
MDCK cells IC50 (nM)
MLM T1/2 (min)
2
−OCH2CH2O−
−OCH2CH2O-
−OCH2CH2O−
H
H
H
27
43.6
112
110
>300
>300
15.3
6.6
21.1
3.6
23.2
23.9
2.5
1.4
23
24
17
25
26
OCF3
OCF3
OCF3
37
>60
a
Half-life when incubated with mouse liver microsomes.
more potent in our MDCK cell assay (3.6 nM vs 37 nM), and
now has more comparable stability in liver microsomes, in
contrast to our original lead 2. It is also important to note that 17
has stability in HLM (T1/2 = 84 min) that is much greater than
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that of the approved oral drug from this class, eliglustat (HLM
4). In this model, the B/P ratio of 17 (0.88) was actually higher
than that of venglustat (0.64).
T1/2 = 12.5 min).
Pharmacokinetic Profiles. Prior to in vivo efficacy testing
in mice, 17 was compared with 2 and 3 in mouse
pharmacokinetics (PK) studies (Table 4). Following single IP
Pharmacodynamic Studies in Mice. To compare the in
vivo efficacy of 17 with our previous lead 2, we first evaluated it
in normal C57BL/6 mice. Mice (6−8-weeks old) were injected
IP with 17 or vehicle for 3 days in two separate dose response
assays (Figure 5). Collectively, these data indicate a significant
Table 4. In Vitro and In Vivo Pharmacokinetic Data
MDR1-MDCK cell permeability
Mouse PK (IP, 10 mg/kg)
P
app A-B app B-A efflux brain AUC plasma AUC
P
no. (×10−6 cm/s) (×10−6 cm/s) ratio
(h·ng/g)
(h·ng/mL)
a
a
2
17
0.14 (9.8)
40.8 (6.9)
56.6 (8.6)
49 (13.4)
298
60
36
1046
317
1308
a
a
a
0.95 (15.8)
a
3
0.58 (19.8)
84
6188
7164
a
Values in parentheses were measured in the presence of the Pgp
inhibitor, valspodar.
doses of drug (10 mg/kg), 17 exhibited a nearly 30-fold increase
in overall brain exposure vs 2 (AUC = 1046 h·ng/g vs 36 h·ng/
g). Furthermore, the cumulative B/P ratio (as estimated from
the simple ratio of brain AUC/plasma AUC) improved from
0.11 to 0.80. The B/P AUC value of 17 is now comparable to 3
(0.86), although the overall brain exposure of 17 remains much
less.
Figure 5. Effect of 17 on brain glucosylceramide in wild-type mice.
Compound or vehicle was administered IP at the indicated doses for 3
days. Data shown is a merge of two separate dose response experiments
(Exp 1: 30, 10, 1 mg/kg; Exp 2: 1.0, 0.30, 0.10, 0.03 mg/kg). ns: change
is not significant.
There are at least two rationales for why 17 is so much more
brain-permeable than 2. First, its physical properties are more
aligned with those of CNS-permeable drugs, namely, a higher
ClogP (4.8 vs 2.9) and lower TPSA (62 vs 71), although its MW
is somewhat higher (463 vs 437). We also hypothesized that 17
might be less susceptible to efflux by P-glycoprotein (Pgp). This
was tested using a standard MDR1-MDCK cell monolayer
(Table 4), and indeed revealed a markedly lower susceptibility
of 17 to efflux than 2 (efflux ratio 60 vs 298). Confirmation that
efflux was primarily by the Pgp transporter was achieved by
parallel experiments in the presence of Pgp inhibitor valspodar,
which resulted in nearly equivalent apical (A-B) and basal (B-A)
permeabilities. Since these two analogues possess the same basic
amine, a known Pgp recognition element, it is possible that the
ethylendioxy moiety of 2 is also recognized by Pgp, perhaps due
to its high TPSA. Furthermore, the TPSA of the single remaining
aryl oxygen atom of 17 may be effectively masked by the strongly
electron-withdrawing trifluoromethyl moiety. Interestingly,
despite its high brain exposure in mice, clinical comparator 3
also exhibited an efflux ratio in our assay that is much higher than
typical CNS permeant drugs and that was comparable to 17 (84,
Table 4). This suggests that this particular attribute may not
itself be a barrier to clinical development of our series for
neuronopathic disease.
To assess the potential for 17 to be an oral drug, bioavailability
was determined through standard IV/PO PK studies in CD1
mice, and compared against venglustat and eliglustat, both of
which are effective orally (Tables S-1−S-3 in the Supporting
Information). At a PO dose of 10 mg/kg, the bioavailability of
17 was 34%, lower than that of venglustat (74%) but markedly
superior to that of eliglustat (<1%) in this model. The IV half-life
of 17 is also about 4 times that of eliglustat, consistent with
reduced clearance and a greater volume of distribution at steady
state (Vss). Although the Vss of 17 is high (7549 mL/kg), it is
comparable to that of venglustat (6673 mL/kg). Importantly, in
separate studies in mice, we demonstrated the achievement of
significant brain exposure by 17 through the oral route (Table S-
reduction in brain GlcCer of about 34% at 1 mg/kg, which did
not substantially increase in magnitude up to a dose of 30 mg/kg
(−41%). By contrast, previous lead 2 in this model was only able
to achieve a 17% reduction in brain GlcCer at 60 mg/kg,8
indicating an over 60-fold improvement in in vivo potency by 17
in this model.
As a more physiologically relevant in vivo measure of efficacy,
an inducible model of Gaucher disease type 3 was next employed
using D409 V/D409 V mice of C57BL/6 background.18
Beginning at postpartum day 15, mice were injected intra-
peritoneally with conduritol B epoxide (CBE), an irreversible
inhibitor of lysosomal β-glucocerebrosidase, at a dose of 25 mg/
kg, to induce a Gaucher disease phenotype characterized by the
accumulation of the glycosphingolipid substrates: GlcCer and
glucosylsphingosine (GlcSph). Daily injections continued for 14
days total. In addition to 17, eliglustat and Genz-682452 were
included as comparators. GlcCer and GlcSph levels in brain were
quantified by LC/MS-MS and expressed as concentration of
pmol/mg of tissues. Results are summarized in Figure 6.
From these data, it is clear that 17 was able to achieve
reductions in brain GlcCer that were comparable to those of
Genz-682452 (3) (19% and 26%, respectively). Reductions in
brain GlcSph were likewise similar (39% and 35%, respectively).
As expected, eliglustat was inactive in this assay at 10 mg/kg. It is
important to note that 17 achieved these reductions at significantly
lower brain drug levels than Genz-682452. Drug levels in the
brains of the mice at sacrifice (2 h post final dose) were 134 ng/g
for 17 and 493 ng/g for Genz-682452. Our PK studies at the
same IP dose levels (Table 4) are consistent with this
observation, indicating that the total brain exposure (AUC)
for 17 after a single 10 mg/kg dose is only about 20% that of
Genz-682452. This suggests that differences in total brain
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replacement of the 3,4-ethylenedioxy moiety of the benzodiox-
ane ring with 4-trifluoromethoxy (17, CCG-222628) afforded
one of our most stable analogues when incubated with mouse
liver microsomes, and also retained excellent cell potency. In
vivo pharmacokinetics in mice confirmed that brain exposure
had improved by nearly 30-fold, which translated into almost 60-
fold greater potency at reducing glucosylceramide in the brains
of wild-type mice. One likely contributor to the increased brain
exposure is reduced susceptibility to efflux by Pgp, which was
confirmed in an MDR1-MDCK cell monolayer assay. A logical
rationale for the markedly lower efflux is a reduction in TPSA of
the ethylendioxy moiety realized by replacement with
trifluoromethoxy. Finally, we evaluated 17 in a CBE mouse
model of Gaucher disease type 3 and compared its efficacy
directly against clinical candidate Genz-682452 (venglustat) at
equal doses. In this assay, 17 and venglustat induced comparable
reductions in brain glycosphingolipids after 14 days of dosing.
Importantly, 17 was able to effect these changes at significantly
lower total brain drug exposure than venglustat. We believe the
in vitro and in vivo activity of 17 firmly establish the potential for
the eliglustat class of compounds to treat neuronopathic
glycosphingolipid storage diseases.
METHODS
■
Chemistry. General Experimental Information. All reagents and
solvents were used in the condition received from commercial sources.
Chromatographic purifications were performed using a Teledyne ISCO
Combiflash RF with Redisep columns or by standard flash
chromatography using silica gel (220−240 mesh) obtained from
Silicycle. 1H NMR were taken in CDCl3 or DMSO-d6 at room
temperature on Varian Inova 400 or 500 MHz instruments. Reported
chemical shifts are expressed in parts per million (ppm) on the δ scale
from an internal standard of tetramethylsilane (0 ppm). Mass spectra
were recorded on a Micromass LCT time-of-flight instrument utilizing
the electrospray ionization mode. An Agilent 1100 series HPLC with an
Agilent Zorbax Eclipse Plus−C18 column (3.5 um, 4.6 × 100 mm) was
used to determine “HPLC purity” of biologically tested compounds. All
tested compounds were assayed for purity using a 6 min gradient of 10−
90% acetonitrile in water followed by a 2 min hold at 90% acetonitrile
with detection at 254 nm.
Figure 6. Effect of 17 (CCG-222628) on brain glycosphingolipids in
CBE mice. Eliglustat and venglustat (3, Genz-682452) were included as
comparators. Compounds were all coadministered IP at a dose of 10
mg/kg qd for 14 days.
exposure (AUC) in the CBE mouse study were likely even more
pronounced than the 2 h drug levels. On the other hand, drug
exposures in the peripheral organs, estimated by spleen drug
levels at sacrifice, were similar (4880 and 4570 ng/g for 17 and 3,
respectively). It is also important to note that 17 apparently
retains the selectivity of eliglustat for GlcCer synthase vs
galactosylceramide synthase as evidenced by a lack of effect on
brain levels of galactosylceramide and galactosylsphingosine
(data not shown).
Eliglustat tartrate used in these studies was obtained from Genzyme
Corporation. Genz-682452 (3, venglustat) was prepared by the method
described by Zhao et al.19
Exemplary Procedures (from Scheme 1; Preparation of 15).
(1R,3S,5S)-1,3-Bis(3-fluoro-4-methoxyphenyl)-5-phenyltetrahydro-
3H,8H-oxazolo[4,3-c][1,4]oxazin-8-one (5). (S)-5-Phenylmorpholin-
2-one (2.1 g, 11.9 mmol) and 3-fluoro-4-methoxybenzaldehyde (5.5 g,
35.6 mmol) were placed in a 250 mL round-bottom flask and dissolved
in toluene (80 mL). The flask was fitted with a magnetic stirrer bar and a
Dean−Stark trap. The reaction mixture was heated to reflux for 24 h
under nitrogen and the solvent removed in vacuo to yield a pale yellow
oil, which solidified under drying under high vacuum. The material was
purified by flash chromatography (EtOAc/hexane) as follows: Silica gel
was placed in a 500 mL sintered glass funnel and slurried with hexane.
The material was placed on top of the silica gel and eluted with 1000 mL
of hexane, 1000 mL of 10% EtOAc/hexane, 1000 mL of 20%, and 1000
mL of 30% 1000 mL 40%. The appropriate fractions were concentrated.
The resulting solid was triturated in ether, filtered, and dried in a
vacuum oven overnight at room temperature, leaving an off-white solid.
The title compound was obtained (1.9 g, 4.1 mmol, 34.3% yield). 1H
NMR (400 MHz, chloroform-d) δ 7.36−7.15 (m, 7H), 7.16−6.96 (m,
3H), 6.92 (t, J = 8.6 Hz, 1H), 6.75 (t, J = 8.4 Hz, 1H), 5.54−5.16 (m,
2H), 4.53−4.23 (m, 2H), 4.23−3.98 (m, 2H), 3.87 (s, 2H), 3.82 (s,
3H).
CONCLUSIONS
■
Eliglustat is an approved therapy for Gaucher disease type 1, but
has not been pursued for neuronopathic glycosphingolipid
storage diseases. We previously demonstrated, however, that the
eliglustat scaffold can be modified (2) to reduce glycosphingo-
lipid levels in the brains of mice. Subsequent in vivo
pharmacokinetic studies revealed only modest brain penetration
and rapid clearance, suggesting that further optimization was
warranted. In this work, we used multiple metabolite
identification studies to define primary and secondary sites for
CYP-mediated oxidation, guiding the design of more stable
analogues. In parallel, we endeavored to lower TPSA where
possible to facilitate brain penetration. General SAR observa-
tions were that fluoro- or oxygen-substitution of the indane did
not improve metabolic stability; modification of the pyrrolidine
ring was unacceptably detrimental to potency; and electron-
density on the left-hand aromatic ring was critical to both
potency and metabolic stability. Ultimately, we found that
(2S,3R)-3-(3-Fluoro-4-methoxyphenyl)-3-hydroxy-2-(((S)-2-hy-
droxy-1-phenylethyl)amino)-1-(pyrrolidin-1-yl)propan-1-one (6).
To a solution of (1R,3S,5S)-1,3-bis(3-fluoro-4-methoxyphenyl)-5-
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phenyltetrahydrooxazolo[4,3-c][1,4]oxazin-8(3H)-one (1 g, 2.1
mmol) in CH2Cl2 was added pyrrolidine (0.89 mL, 10.7 mmol). The
resulting mixture was stirred overnight at room temperature. After
warming to 50 °C for 2 h, the reaction was cooled and concentrated in
vacuo. The residue was dissolved in MeOH, treated with 2 M HCl, and
heated to reflux for 2 h. Completion of the reaction required adding
more 2 M HCl and stirring 2 more hours. The reaction was cooled and
concentrated in vacuo. The crude material was diluted with water and
extracted with ether. The aqueous layer was treated with saturated
NaHCO3 until it was slightly basic. The mixture was extracted with 3 ×
1:1 EtOAc/ether. The combined organic extracts were washed with
water and dried over MgSO4. Filtration through flash silica gel with 5%
MeOH/CH2Cl2 eluent provided the title compound (0.6 g, 1.5 mmol,
2-(2,3-Dihydro-1H-inden-2-yl)-N-((1R,2R)-1-hydroxy-3-(pyrroli-
din-1-yl)-1-(4-(trifluoromethoxy)phenyl)propan-2-yl)acetamide
(17). Compound 17 was prepared by procedures analogous to those
described for the preparation of 15. 1H NMR (400 MHz, chloroform-
d) δ 7.40−7.35 (m, 2H), 7.21−7.19 (m, 2H), 7.12 (m, 4H), 6.03 (d, J =
7.8 Hz, 1H), 5.12 (d, J = 2.7 Hz, 1H), 4.30−4.20 (m, 1H), 2.97−2.72
(m, 8H), 2.49 (dd, J = 15.6, 6.4 Hz, 1H), 2.29−2.16 (m, 3H), 1.83 (br s,
4H). MS m/z (APCI): 463.3 [M + H]. HPLC purity: 96%.
Preparation of Monooxalate Salt of 17. To a solution of 2-(2,3-
dihydro-1H-inden-2-yl)-N-((1R,2R)-1-hydroxy-3-(pyrrolidin-1-yl)-1-
(4-(trifluoromethoxy)phenyl)propan-2-yl)acetamide (17, 0.28 g,
0.605 mmol) in isopropanol (10 mL) was added a solution of oxalic
acid (0.055 g, 0.605 mmol) in methanol (5 mL). The resulting mixture
was stirred for 30 min, and then concentrated on a rotary evaporator.
The crude solid was triturated with EtOAc/ether, and stirred for 30 min
and then allowed to set overnight. The mixture was filtered and washed
with ether. After a second trituration with isopropyl alcohol, the white
solid was dried overnight under high vacuum. Elemental analysis calcd
for C25H29N2O3·C2H2O4·0.5H2O: C, 57.75; N, 5.74; N, 4.98. Found:
C, 57.77; H, 5.55; N, 5.00.
Broken Cell Assay for Glucosylceramide Synthase Inhibition.
Enzyme activity was measured as described previously.14 Madin-Darby
canine kidney (MDCK) cell homogenates (120 μg of protein) were
incubated with uridine diphosphate-[3H]glucose (100 000 cpm) and
liposomes consisting of 85 μg of octanoylsphingosine, 570 μg of
dioleoylphosphatidylcholine, and 100 μg of sodium sulfatide in a 200
μL reaction mixture and kept for 1 h at 37 °C. Glucosylceramide
synthase inhibitors dissolved in dimethyl sulfoxide (final concentration
< 1%, which did not affect enzyme activity) were dispersed into the
reaction mixture after adding the liposomes. Seven concentrations of
each analogue were assayed in duplicate and compared to control
activity using buffer alone.
1
69.7% yield). H NMR (400 MHz, chloroform-d) δ 7.43−7.16 (m,
4H), 7.13 (m, 1H), 7.00 (m, 1H), 6.83 (t, J = 8.5 Hz, 1H), 4.50 (d, J =
8.6 Hz, 1H), 3.83 (s, 3H), 3.79−3.59 (m, 2H), 3.14−2.95 (m, 2H),
2.95−2.76 (m, 2H), 2.35−2.12 (m, 1H), 2.04−1.68 (m, 2H), 1.51−
0.99 (m, 4H).
(1R,2R)-1-(3-Fluoro-4-methoxyphenyl)-2-(((S)-2-hydroxy-1-
phenylethyl)amino)-3-(pyrrolidin-1-yl)propan-1-ol (7). To a 0 °C
solution of (3R)-3-(3-fluoro-4-methoxyphenyl)-3-hydroxy-2-(((S)-2-
hydroxy-1-phenylethyl)amino)-1-(pyrrolidin-1-yl)propan-1-one (0.4
g, 0.9 mmol) in dry THF (15 mL) was added lithium aluminum
hydride (0.10 g, 2.9 mmol). The resulting mixture was stirred overnight
at room temperature. After heating to reflux for 1.5 h to complete the
reaction, it was cooled to °0 C and treated dropwise with 0.11 mL of
H2O followed by 0.11 mL of 15% NaOH. The mixture was stirred for 20
min and treated with 3.3 mL of H2O. After stirring for 1 h, the mixture
was filtered through Celite with ether eluent. The eluent was
concentrated and purified by flash chromatography (MeOH/
CH2Cl2) to obtain the title compound (0.3 g, 0.6 mmol, 62.5%
1
yield). H NMR (400 MHz, chloroform-d) δ 7.36−7.21 (m, 5H),
MDCK Cell Assay for Reduction in Glucosylceramide.
Following inhibitor treatment at seven concentrations (assayed in
duplicate), whole cellular lipids of MDCK cells were extracted as
previously described in detail.8 Briefly, cells were washed with ice-cold
PBS, fixed by methanol and collected with a rubber scraper. Chloroform
was then added to yield a theoretical ratio of chloroform−methanol−
water at 1:2:0.8 (v/v/v) to form a monophase. Cell debris and proteins
were removed by centrifugation at 2200 g for 30 min. The supernatants
were portioned by adding chloroform and 0.9% NaCl. The lower
organic phases containing neutral glycosphingolipids lipids were
washed with methanol and 0.9% NaCl and subjected to base- and
acid-hydrolysis. A portion of purified glycosphingolipids normalized to
100 nmol of total phospholipids was analyzed by high-performance
TLC. The TLC separations were processed twice. The plate pretreated
with 1% sodium borate was first developed in a solvent system
consisting of chloroform−methanol (98:2, v/v). After air drying, the
plate was then developed in a solvent system containing chloroform−
methanol−water (70:30:4, v/v/v). The levels of glucosylceramide were
detected by charring with 8% CuSO4·5H2O in 6.8% phosphoric acid
and quantified by densitometric scanning using ImageJ, NIH Image.
Image data was analyzed, and the IC50 of each inhibitor was calculated
using GraphPad Prism.
Determination of Stability to Liver Microsomes. Metabolic
stability was assessed using CD-1 mouse or human liver microsomes. A
concentration of 1 μM of each compound was incubated with 0.5 mg/
mL microsomes and 1.7 mM cofactor β-NADPH in 0.1 M phosphate
buffer (pH = 7.4) containing 3.3 mM MgCl2 at 37 °C. The DMSO
concentration was less than 0.1% in the final incubation system. At 0, 5,
10, 15, 30, 45, and 60 min of incubation, 40 μL of the reaction mixture
were taken out, and the reaction was quenched by adding 3-fold excess
of cold acetonitrile containing 100 ng/mL of internal standard for
quantification. The collected fractions were centrifuged at 15 000 rpm
for 10 min to collect the supernatant for LC−MS/MS analysis, from
which the amount of compound remaining was determined. The
natural log of the amount of compound remaining was plotted against
time to determine the disappearance rate and the half-life of tested
compounds.
7.21−7.12 (m, 1H), 7.12−6.98 (m, 1H), 6.92 (t, J = 8.6 Hz, 1H), 4.51
(d, J = 4.5 Hz, 1H), 3.88 (s, 3H), 3.69 (dd, J = 8.8, 4.3 Hz, 1H), 3.62−
3.44 (m, 4H), 3.08−2.79 (m, 1H), 2.62 (dd, J = 12.5, 8.6 Hz, 1H),
2.50−2.30 (m, 3H), 2.30−2.13 (m, 1H), 1.81−1.52 (m, 3H).
(1R,2R)-2-Amino-1-(3-fluoro-4-methoxyphenyl)-3-(pyrrolidin-1-
yl)propan-1-ol (8). To a solution of (1R)-1-(3-fluoro-4-methoxyphen-
yl)-2-(((S)-2-hydroxy-1-phenylethyl)amino)-3-(pyrrolidin-1-yl)-
propan-1-ol (0.3 g, 0.7 mmol) in methanol (15 mL) and 1 M HCl 10
mL was added palladium on carbon (10% Degussa) (0.08 g, 0.8 mmol).
The resulting mixture was bubbled with N2 for 5 min then placed on a
Parr Hydrogenator, placed briefly under vacuum then filled with
hydrogen gas. The reaction was shaken overnight at room temperature.
The mixture was filtered through Celite with methanol eluent and
concentrated in vacuo. The crude product was purified by flash
chromatography (7% conc ammonia in methanol/CH2Cl2) to obtain
the title compound as an oil (0.11 g, 0.41 mmol, 53% yield). 1H NMR
(500 MHz, chloroform-d) δ 7.62−7.61 (m, 1H), 7.03−6.71 (m, 2H),
4.59 (m, 1H), 3.79 (m, 2H), 3.14 (m, 1H), 2.75−2.26 (m, 5H), 1.78 (br
s, 4H).
2-(2,3-Dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(3-fluoro-4-methox-
yphenyl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (9,
Compound 15). To a solution of (1R,2R)-2-amino-1-(3-fluoro-4-
methoxyphenyl)-3-(pyrrolidin-1-yl)propan-1-ol (0.10 g, 0.36 mmol) in
THF 10 mL was added 2-(2,3-dihydro-1H-inden-2-yl)acetic acid (0.08
g, 0.45 mmol), HOBt (0.06 g, 0.47 mmol), EDC (0.103 g, 0.54 mmol)
followed by DIPEA (0.14 mL, 0.83 mmol). The resulting mixture was
stirred overnight at room temperature. Saturated NaHCO3 and EtOAc
were added, and the separated aqueous layer was extracted again. The
combined organic layers were washed with saturated NaCl (3×) and
dried (MgSO4). Purification by flash chromatography (gradient of 2.5%
MeOH/CH2Cl2 to 5% to 10% MeOH (with 7% NH3)/CH2Cl2)
afforded pure 15 (0.05 g, 0.11 mmol, 28% yield). 1H NMR (400 MHz,
chloroform-d) δ 7.26 (s, 2H), 7.14 (d, J = 10.3 Hz, 3H), 7.04 (d, J = 8.5
Hz, 1H), 6.92 (t, J = 8.4 Hz, 1H), 6.11 (s, 1H), 5.06 (d, J = 2.8 Hz, 1H),
4.27 (m, 1H), 3.97−3.61 (m, 3H), 3.13−2.60 (m, 7H), 2.49 (dd, J =
15.5, 6.7 Hz, 1H), 2.43−2.08 (m, 3H), 1.87 (s, 4H), 1.25 (s, 4H). MS
m/z (EI): 426.2 [M + H]. HPLC purity: 97%.
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Identification of Metabolites Induced by Mouse Liver
Microsomes. The parent compound (10 μM) was incubated with
mouse liver microsomes (MLM) and β-NADPH at 37 °C for 20 and 40
min. Then the reaction was quenched by adding a 3-fold volume of ice-
cold acetonitrile. The mixture was centrifuged at 15,000 rpm for 10 min,
and the supernatant was saved under −80 °C for analysis. The negative
control samples were prepared by a similar procedure without NADPH
or using boiled microsome. The general approach for metabolite
identification using an AB Sciex QTrap 5500 mass spectrometer
involves the following steps: (1) Obtain a product ion spectrum of the
parent compound to establish fragmentation. (2) Interpret the
spectrum to identify major fragment ion and possible neutral loss.
(3) Collect spectra of samples using both established precursor ion scan
and neutral loss scan, EMS scan of both control and samples were
acquired also. (4) Run product ion scans and MRM scans for all
possible metabolite identified from step 3 plus expected metabolite. (5)
Interpret the spectrum of the metabolites and determine the structure
with their logical fragmentation pattern.
methanol and 1 mL of chloroform, bath sonicated for 1 min, and
incubated at room temperature for 1 h. Tissue debris was removed by
centrifugation at 2400g for 30 min. The pellets were re-extracted by
mixing with 1 mL of methanol, 0.5 mL of chloroform, and 0.4 mL of
0.9% NaCl (chloroform/methanol/0.9% NaCl, 1:2:0.8), incubated at
room temperature for 1 h, and centrifuged at 2400g for another 30 min.
Two extracts were combined and mixed with 4.5 mL of chloroform and
1.2 mL of 0.9% NaCl (chloroform/methanol/0.9% NaCl, 2:1:0.8).
After centrifugation at 800g for 5 min, the lower layer was washed with 3
mL of methanol and 2.4 mL of 0.9% NaCl. A second washing was
carried with 3 mL of methanol, 2 mL of water, and 0.4 mL of 0.9% NaCl
followed by a 5 min centrifugation at 800g. The resultant lower phase
was collected and dried under a stream of N2 gas.
The analysis of neutral glycosphingolipids from mouse brain was
processed after alkaline methanolysis. Brain lipids were incubated with
2 mL of chloroform and 1 mL of 0.21 N NaOH in methanol for 7.5 h at
room temperature. The lipid extract was normalized to 2 μmol of total
phospholipid phosphate for high performance TLC analysis. After
alkaline methanolysis, the brain lipids were passed through a silica gel
column.20 Borate-impregnated TLC plates were developed in a two
solvent system. Plates were first developed in chloroform/methanol
(98:2, v/v). The plates were then developed in chloroform/methanol/
water (64:24:4, v/v/v) and were further separated in chloroform/
methanol/water (60:30:6, v/v/v). GlcCer levels were quantified by
comparison to known standards.
In Vivo Reduction of Brain Glycosphingolipids in CBE Mice.
An inducible model of Gaucher disease type 3 was employed using Gba
D409V/D409V mutant mice of C57BL/6 genetic backgrounds.18 The
Cincinnati Children’s Hospital Research Foundation (CCHRF)
Institutional Animal Care and Use Committee (IACUC) reviewed
and approved these studies under protocol IACUC2015-0050. All mice
were housed under pathogen-free conditions in the barrier animal
facility and according to IACUC standard procedures at CCHRF.
Beginning at postpartum day 15, D409V/D409V mice were injected
intraperitoneally with conduritol B epoxide (CBE), an irreversible
inhibitor of lysosomal β-glucocerebrosidase at a dose of 25 mg/kg.
Daily injections of CBE continued for 14 days total. Test compounds
(17 and 3; 10 mg/kg) were coadministered by IP in citrate-saline buffer
or buffer alone. Mice were monitored and weighed daily. GlcCer and
GlcSph levels in brain from D409V/D409V mice treated with CBE with
or without test compounds were separated from galactosylceramide
and galactosylsphingosine, respectively, and quantified by LC/MS-MS
(Lipidomics Shared Resource, Medical University of South Carolina).
The glycolipid levels were normalized to tissue weight and expressed as
the concentration (pmol/mg tissue) of the GlcCer or GlcSph of
matched samples from citrate-saline-injected vehicle control mice.
Student’s t test was used for statistical comparisons. Each group
consisted of 8−11 mice with mixed gender.
Determination of Efflux by MDR1-MDCK Cells. Permeability
through and efflux from an MDR1-MDCK cell monolayer were
determined by Absorption Systems, 436 Creamery Way, Suite 600,
Exton, PA 19341−2556.
Pharmacokinetic Studies in Mice. All animal experiments in this
study were approved by the University of Michigan Committee on Use
and Care of Animals and Unit for Laboratory Animal Medicine
(ULAM). The pharmacokinetics of compounds were determined in
female CD-1 mice following intraperitoneal (IP) injection of 10 mg/kg,
respectively. Compounds were first dissolved in citrate buffer at 2 mg/
mL and then diluted with sterile saline (1:1) for injection volumes of 1
mg/mL. Blood samples were collected from individual cohorts of mice
(n = 3) using heparinized calibrated pipets following sacrifice at the
given time point over 24 h (at 0.083h, 0.25h, 0.5h, 1.5h, 4h, 8h and
24h), centrifuged at 15000 rpm for 10 min. Subsequently, blood plasma
was collected from the upper layer. The plasma was frozen at −80 °C for
later analysis. Brains were taken out at the same time and weighed out
immediately. Brains were frozen at −80 °C for later preparation and
analysis, Plasma concentrations of the compounds were determined by
the LC−MS/MS method developed and validated for this study. The
LC−MS/MS method consisted of a Shimadzu HPLC system and
chromatographic separation of tested compound which was achieved
using a Waters Xbridge-C18 column (5 cm × 2.1 mm, 3.5 μm). An AB
Sciex QTrap 4500 mass spectrometer equipped with an electrospray
ionization source (ABI-Sciex, Toronto, Canada) in the positive-ion
multiple reaction monitoring (MRM) mode was used for detection. All
pharmacokinetic parameters were calculated by noncompartmental
methods using WinNonlin, version 3.2 (Pharsight Corporation,
Mountain View, CA, USA).
In Vivo Reduction of Brain Glycosphingolipids in Normal
Mice. C57BL/6 mice were maintained on regular chow in specific-
pathogen-free facilities. All animal studies were performed under the
review of the University of Michigan Committee on the Use and Care of
Animals and conformed to the National Institutes of Health Guide for
the Care and Use of Laboratory Animals. Injection solutions were
prepared from inhibitor-ethanol stock solution (100 mM). A portion of
the stock solution was evaporated under a stream of N2 gas. Dried
eliglustat tartrate was directly dissolved into 1× PBS. Compound 17
was dissolved with 250 μL of water plus 13.7 μL of 0.5 N HCl by hand
shaking and gentle vortexing resulting in a 1 or 3 mg/mL solution. The
acidic solution was neutralized by mixing 100 μL of 10× PBS with 636.3
μL of water to bring the total volume to 1 mL. Inhibitor solutions were
sterilized by passage through a 0.2 μM filter. Inhibitor recovery after
filtration was confirmed by UV spectrometry and exceeded 99%. For
control injections, PBS containing the same amount of HCl was used.
Inhibitors were given to 6−8 week old female or male C57BL/6 mice by
intraperitoneal injection volume at 1% of body weight.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00558.
IV/PO Pharmacokinetic data for 17, 3, and 1; brain/
1
plasma ratio by the oral route in mice for 17 and 3; H
NMR, MS, and HPLC purity of compounds 10−14, 16,
and 18−26 (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Scott D. Larsen − Vahlteich Medicinal Chemistry Core, College of
Pharmacy and Department of Medicinal Chemistry, University of
Michigan, Ann Arbor, Michigan 48109, United States;
orcid.org/0000-0001-8440-4990; Email: sdlarsen@
med.umich.edu
Lipid extractions of brain were performed as previously described.20
Briefly, frozen whole brain (∼0.4 g) was individually homogenized in
sucrose buffer (250 mM sucrose, pH 7.4, 10 mM Hepes and 1 mM
EDTA), at 0.2 g tissue/1 mL of sucrose buffer, with a Tri-R
homogenizer. Each 0.8 mL of homogenate was mixed with 2 mL of
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Notes
James A. Shayman − Department of Internal Medicine -
Nephrology, University of Michigan, Ann Arbor, Michigan
48109, United States; Email: jshayman@med.umich.edu
The contents of the work are solely the responsibility of the
authors and do not necessarily represent the official views of the
NIH.
Authors
The authors declare no competing financial interest.
Michael W. Wilson − Vahlteich Medicinal Chemistry Core,
College of Pharmacy, University of Michigan, Ann Arbor,
Michigan 48109, United States
Liming Shu − Department of Internal Medicine - Nephrology,
University of Michigan, Ann Arbor, Michigan 48109, United
States
Vania Hinkovska-Galcheva − Department of Internal Medicine -
Nephrology, University of Michigan, Ann Arbor, Michigan
48109, United States
Yafei Jin − Vahlteich Medicinal Chemistry Core, College of
Pharmacy, University of Michigan, Ann Arbor, Michigan 48109,
United States
Walajapet Rajeswaran − Vahlteich Medicinal Chemistry Core,
College of Pharmacy, University of Michigan, Ann Arbor,
Michigan 48109, United States
Akira Abe − Department of Internal Medicine - Nephrology,
University of Michigan, Ann Arbor, Michigan 48109, United
States
Ting Zhao − Pharmacokinetics Core, Department of
Pharmaceutical Sciences, University of Michigan, Ann Arbor,
Michigan 48109, United States
Ruijuan Luo − Pharmacokinetics Core, Department of
Pharmaceutical Sciences, University of Michigan, Ann Arbor,
Michigan 48109, United States
Lu Wang − Pharmacokinetics Core, Department of
Pharmaceutical Sciences, University of Michigan, Ann Arbor,
Michigan 48109, United States
Bo Wen − Pharmacokinetics Core, Department of Pharmaceutical
Sciences, University of Michigan, Ann Arbor, Michigan 48109,
United States
Benjamin Liou − Division of Human Genetics, Cincinnati
Children’s Hospital, Cincinnati, Ohio 45229, United States
Venette Fannin − Division of Human Genetics, Cincinnati
Children’s Hospital, Cincinnati, Ohio 45229, United States
Duxin Sun − Pharmacokinetics Core, Department of
Pharmaceutical Sciences, University of Michigan, Ann Arbor,
Michigan 48109, United States; orcid.org/0000-0002-6406-
2126
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Ying Sun − Division of Human Genetics, Cincinnati Children’s
Hospital, Cincinnati, Ohio 45229, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00558
Author Contributions
The manuscript was written primarily by S.D.L and J.A.S. The
broken cell and MDCK cell assays were run by L.S., V.H.-G., and
A.A in the laboratory of J.A.S. Compounds were synthesized by
M.W.W., Y.J., and W.R. in the laboratory of S.D.L. Metabolite id
studies were run by T.Z., R.L., and L.W. Interpretation of
metabolite id studies and oversight of in vivo pharmacokinetics
were done by B.W. and D.S. Normal mouse studies were run by
L.S. in the laboratory of J.A.S. CBE mouse studies were run by
V.F. and B.L. in the laboratory of Y.S, and data were analyzed by
Y.S.
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Shayman, J. A., Shukla, G. S., and Radin, N. S. (1992) Improved
inhibitors of glucosylceramide synthase. J. Biochem. 111 (2), 191−6.
Funding
This work was supported by NINDS and NICHD of the
National Institutes of Health under Award Numbers R21
NS065492 (S.D.L.) and R01 HD076004 (S.D.L. and J.A.S).
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ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
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