Novel compounds that reverse the disease phenotype in Type 2 Gaucher disease patient-derived cells

Article abstract of DOI:10.1016/j.bmcl.2019.126806

Gaucher disease (GD) results from inherited mutations in the lysosomal enzyme β-glucocerobrosidase (GCase). Currently available treatment options for Type 1 GD are not efficacious for treating neuronopathic Type 2 and 3 GD due to their inability to cross the blood-brain barrier. In an effort to identify small molecules which could be optimized for CNS penetration we identified tamoxifen from a high throughput phenotypic screen on Type 2 GD patient-derived fibroblasts which reversed the disease phenotype. Structure activity studies around this scaffold led to novel molecules that displayed improved potency, efficacy and reduced estrogenic/antiestrogenic activity compared to the original hits. Here we present the design, synthesis and structure activity relationships that led to the lead molecule Compound 31.

Full text of DOI:10.1016/j.bmcl.2019.126806

Journal Pre-proofs
Novel compounds that reverse the disease phenotype in Type 2 Gaucher disease
patient-derived cells
Wayne Childers, Rong Fan, Rogelio Martinez, Dennis J. Colussi, Edward
Melenski, Yuxiao Liu, John Gordon, Magid Abou-Gharbia, Marlene A.
Jacobson
PII:
S0960-894X(19)30775-9
DOI:
https://doi.org/10.1016/j.bmcl.2019.126806
BMCL 126806
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 October 2019
30 October 2019
1 November 2019
Please cite this article as: Childers, W., Fan, R., Martinez, R., Colussi, D.J., Melenski, E., Liu, Y., Gordon, J., Abou-
Gharbia, M., Jacobson, M.A., Novel compounds that reverse the disease phenotype in Type 2 Gaucher disease
patient-derived cells, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.126806
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Novel compounds that reverse the diseased phenotype in
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Wayne Childers*, Rong Fan, Rogelio Martinez, Dennis J. Colussi, Edward Melenski, Yuxiao Liu, John Gordon, Magid
Abou-Gharbia and Marlene A. Jacobson*

Bioorganic & Medicinal Chemistry Letters
Novel compounds that reverse the disease phenotype in Type 2 Gaucher disease
patient-derived cells
Wayne Childers*, Rong Fan , Rogelio Martinez, Dennis J. Colussi, Edward Melenski, Yuxiao Liu, John
Gordon, Magid Abou-Gharbia and Marlene A. Jacobson*
Moulder Center for Drug Discovery Research, Department of Pharmaceutical Sciences, Temple University School of Pharmacy, 3307 N. Broad Street,
Philadelphia, PA, 19140, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Gaucher disease (GD) results from inherited mutations in the lysosomal enzyme
-glucocerobrosidase (GCase). Currently available treatment options for Type 1
GD are not efficacious for treating neuronopathic Type 2 and 3 GD due to their
inability to cross the blood-brain barrier. In an effort to identify small molecules
which could be optimized for CNS penetration we identified tamoxifen from a
high throughput phenotypic screen on Type 2 GD patient-derived fibroblasts
which reversed the disease phenotype. Structure activity studies around this
scaffold led to novel molecules that displayed improved potency, efficacy and
reduced estrogenic/antiestrogenic activity compared to the original hits. Here we
present the design, synthesis and structure activity relationships that led to the
lead molecule Compound 31.
Keywords:
Keyword_1 Gaucher disease
Keyword_2 Lysosomal storage disease
Keyword_3 Phenotypic screening
Keyword_4 Diphenylethanono
Keyword_5 Neuronopathic
2009 Elsevier Ltd. All rights reserved.
Type 2 GD (acute neuronopathic) is the most severe, having rapid
and progressive neurodegeneration during the first years of life.
Type 3 GD also has neurological manifestations and exhibits a
later onset and slower progression.
Lysosomal storage disorders (LSDs) are inheritable metabolic
diseases with deficiencies in enzymes that function within the
glycosphingolipid biosynthetic and metabolic pathway. As a
consequence, non-degraded substrates accumulate and normal
lysosomal function is compromised, resulting in impaired
autophagy, disruption of cell signaling and eventually cell death.
A group of more than 70 diseases have been classified as LSDs,
including Gaucher, Tay-Sachs, Sandhoff and Fabry disease,
Niemann-Pick type C and GM1 gangliosidosis.1,2 Two thirds of
LSDs have central nervous system involvement resulting in a
progressive neurodegeneration and the leading cause of patient
death. The most common LSD is Gaucher disease, with a
prevalence of 1 in 57,000 births.3
Current treatment options for GD patients include enzyme
replacement therapy (ERT) with recombinant GCase (Cerezyme®,
imiglucerase) and substrate reduction therapy (SRT) with small
molecule inhibitors of glucosylceramide synthase (GCS) to reduce
production of substrate which is not degraded due to the reduced
GCase activity.5 Two GCS inhibitors (Figure 1) are FDA
approved for treatment of Type
1 GD, the sugar-based
glucocerebrosidase inhibitor ZavescaTM (miglustat)6 and the
benzodioxan derived GCS inhibitor Cerdelga® (eliglustat).7
Gaucher disease (GD) is an autosomal recessive disorder
resulting from mutations in the gene GBA1, encoding the
lysosomal enzyme -glucocerebrosidase (GCase). Reduced or loss
of GCase activity leads to accumulation of glucosylceramide and
glucosylsphingosine. GD is clinically defined into three types
based on the age of disease onset and clinical manifestations of
organ involvement.4 Type 1 GD is non-neuronopathic while types
2 and 3 GD have CNS involvement.
________________
* Corresponding Authors: Email Addresses: wayne.childers@temple.edu (W.
Childers) marlene.jacobson@temple.edu (M. Jacobson)
Figure 1. Small molecules for substrate reduction therapy.

While the availability of ERT and SRT has had significant impact
on the treatment of GD, their effectiveness is limited to patients
with non-neuronopathic Type 1 GD, as neither the enzyme nor the
small molecules are able to cross the blood-brain barrier. Recently
a GCS inhibitor, Venglustat (ibiglustat, Figure 1) with improved
properties to penetrate the CNS has been reported with potential to
overcome the limitations of existing SRT and is currently in
clinical trials.8,9
screening strategies.12 Disruption of calcium homeostasis and
impaired lysosomal function are measurable, phenotypic
characteristics of LSD patient-derived cells, including GD.13-16
We discovered that Type
2 GD patient-derived cells
(L444P/L444P genotype) display reduced calcium release from
lysosomal acidic stores in response to Gly-Phe--napthylamide
(GPN) compared to normal cells (WT) (Figure 2). GPN is a
substrate of cathepsin C, which upon hydrolysis, produces osmotic
lysis and release of calcium from lysosomal acidic stores into the
cytosol.17 On the basis of this phenotypic difference between GD
and WT cells, we developed a high throughput fluorescence-based
assay to screen for compounds with activity to reverse the
lysosomal calcium release deficit found in GD patient-derived
cells. We have previously described this HTS assay and its
application to Tay-Sachs patient-derived cells.18 Briefly, GPN-
induced calcium release into the cytosol is detected with a
fluorescent calcium indicator (Fluo-8AM) and monitored in real-
time on a fluorescence kinetic plate reader (Hamamatsu FDSS
Cell). A pilot screen on 1,200 known FDA approved drugs
(Prestwick Chemical Library®), 10 M screening concentration,
was completed. One of the hits identified was tamoxifen (Figure
3), a selective estrogen receptor modulator (SERM) used to treat
estrogen-sensitive breast cancers. Tamoxifen exhibited modest
functional potency, EC50 = 10 M and 148% activation over the
basal response to GPN-induced lysosomal calcium release
measured on GD cells in the absence of compound treatment
(Figure 2). Subsequent testing of related analogs identified
idoxifene (2, Figure 3), which showed similar activity as
tamoxifen. The known drug-like properties of these molecules
make them reasonable starting points for hit-to-lead activities.
However, their potent estrogenic activity would be considered
unacceptable as a chronic therapy, especially in the younger
patient population affected by Type 2 GD. We therefore carried
out structure-activity studies to enhance the potency and efficacy
of this chemical scaffold while reducing the estrogen activity. Our
results are presented below.
Figure 2. (A) GPN-induced calcium release is reduced in Gaucher (GD)
patient-derived cells compared with normal (WT) patients; Tamoxifen
(10 M) partially restores lysosomal calcium in GD patient-derived
cells. (B) Tamoxifen dose response on GD and WT patient-derived cells.
Table 1. SAR of tamoxifen scaffold.
Estrogen†
Receptor
% Activity
@ 10 M
Lysosomal
Ca+2 Assay*
The application of high throughput screening (HTS)
approaches to identify small molecule treatments for GD have
targeted GCase to either identify chaperones which improve or
enhance enzyme activity or inhibitors of GCS to reduce substrate
burden into the defective enzyme pathway.10,11 With the goal to
discover small molecule therapeutics for neuronopathic GD, we
chose a phenotypic screening approach utilizing patient-derived
cells to identify small molecules with activity to reverse the
diseased lysosomal phenotype as an alternative to target-based
Cd #
EC50,
Emax,
%
M
N(CH3)2
O
3
5.8
73%
80%
19%
35%
43%
N(CH3)2
O
4
5
15.5
4.3
N(CH3)2
O
154%
Figure 3. Structures and activity of hits tamoxifen and idoxifene.

N(CH3)2
O
6
7
3.0
53%
43%
75%
N(CH3)2
O
> 20
0
Scheme 1. Synthesis of compounds 17-24. Reagents and Conditions:
(a) Trifluoroacetic acid, r.t., overnight; (b) NaH, R1-X; (c) aq. 40%
dimethylamine, reflux.
N(CH3)2
O
8
9
3.1
2.3
5.4
67%
40%
70%
69%
47%
70%
N(CH3)2
O
N(CH3)2
Cl
O
10
N(CH3)2
F
O
11
12
13
> 20
> 20
4.3
0
0
46%
23%††
Scheme 2. Synthesis of compounds 25-29. Reagents and Conditions:
(a) Trifluoroacetic acid, r.t., overnight; (b) NaH, R-X; (c) aq. 40%
dimethylamine, reflux; (d) R2-tetramethyl-dioxoboralane, K2CO3,
Pd(PPh3)4, 120oC; (e) 1 atm. H2, 10% Pd/C.
N(CH3)2
NC
O
47%
32%
25%
Published SAR studies suggested that the anti-estrogenic
activity of the tamoxifen scaffold can be modulated by substitution
on the aryl rings not containing the dimethylaminoethoxy group
group.19,20 The greatest effect was seen when 4-substitution was
modified on the aryl ring occupying the 2-position of the double
bond (carbon 26)21. Therefore, we initiated a short structure-
activity relationship (SAR) study to examine the effects of aryl
substitutions, ring deletions, positioning of the alkyl group and
saturation of the double bond on the potency of the scaffold as an
estrogen modulator versus potency in the GPN-induced lysosomal
calcium release assay in Type 2 GD fibroblasts (Table 1). Detailed
synthetic methods are provided in the Supplementary Material.
14
15
> 20
> 20
0
0
46%†
(8%††)
N(CH3)2
O
Removing one of the phenyl rings reduced estrogen receptor
activity with a switch in functional activity from inhibitor to
activator. However, most of the analogs lost potency in the
calcium release assay. The one exception was compound 5, which
was somewhat more potent than and equally efficacious as
tamoxifen in the calcium assay. Homologating the one phenyl ring
to a benzyl group (compounds 8, 9) did not enhance the desired
potency and efficacy or reduce the estrogenic activity.
Substitution on the aryl ring lacking the dimethylaminoethoxy
16
1
7.2
10
56%
98%
148%
100%††
* EC50 units - M; Emax = percent activation of basal GPN-induced calcium
response measured in the absence of compound.
† Percent agonist response relative to the response with -estradiol EC100
.
†† Percent inhibition of EC100 response to -estradiol.

Table 2. SAR of diphenylethanone scaffold.
Estrogen†
Receptor
% Activity
@ 10 M
Lysosomal Ca+2
Assay
Estrogen†
Receptor
% Activity
@ 10 M
Lysosomal Ca+2
Assay
Cd #
R1
R2
Cd #
R1
R2
EC50, M
Emax, %
EC50, M
Emax, %
17
18
Et
H
H
0
0
0
0
ND
ND
25
26
n-Bu
n-Bu
2.3
177%
138%
100%
87%††
n-Pr
Et
0.65
19
i-Pr
H
4.4
54%
ND
27
n-Bu
n-Bu
2.2
257%
35%
20
21
gem-di-n-Pr
n-Bu
H
H
4.0
60%
0
25%
ND
28
29
i-Pr
i-Pr
1.4
1.7
401%
406%
45%
39%
> 20
22
H
0.9
108%
40%
30
31
1
3-i-Pr
1.5
3.1
10
80%
240%
148%
ND
0%
23
24
H
H
3.6
191%
0
35%
5%
100%††
> 20
* EC50 units - M; Emax = percent activation of basal GPN-induced calcium response measured in the absence of compound.
† Percent agonist response relative to the response with -estradiol EC100
†† Percent inhibition of EC100 response to -estradiol.
* ND = Not Determined.
.
group (10-15) reduced potency in the calcium assay in general.
One structural difference between compound 5 and most other
analogs is the ability of the distal aryl ring to more easily assume
a skewed conformation relative to the conjugated system due to a
lack of the central double bond. Hypothesizing that the ability of
this ring to assume a non-planar, unconjugated conformation
might play a role in the improved efficacy on GD cells, we
embarked on a series of novel di-phenylethanone derivatives
(compounds 17-31) that possessed substitution in the 2-position of
the aryl ring that we felt needed to be perpendicular to the rest of
the scaffold. The purpose of the ketone was to arrive at a novel
chemical scaffold and to eliminate the extend conjugation that
promotes planarity in tamoxifen and related analogs. The results
are presented in Table 2.
The general method for the syntheses of compounds 25-31 are
depicted in Schemes 2. A similar sequence to that depicted in
Scheme 1 was used to arrive at a common intermediate, which was
then subjected to a Suzuki coupling to arrive at compounds 25 and
27. Those compounds were then catalytically reduced to provide
compounds, 26 and 28-31. The order of some steps was altered
for the synthesis of compounds 30 and 31 but the same series of
reactions was used. Detailed experimental methods are provided
in the Supplementary Material.
For our initial SAR studies on the diphenylethanone series
(Table 2) we held the dimethylaminoethoxy group constant, set R2
= H and varied the alkyl substituent R1 (compounds 17 – 24).
Compounds with linear alkyl groups such as ethyl (17), n-propyl
(18), n-butyl (21) and n-hexyl (24) demonstrated little effect on
lysosomal calcium release from GD patient-derived cells.
Increasing lipophilic bulk in this region of the molecule by
installing an isopropyl group (19) or gem-di-propyl moieties (20)
provided analogs that demonstrated greater potency to that
demonstrated by tamoxifen in the GPN-stimulated calcium release
assay (EC50 = 4 - 4.4 M vs. 10 M, respectively) but these
compounds did not increase the percent of calcium release over
basal levels to the same percentage as tamoxifen (54 – 60% vs/
148%, respectively). Shifting the lipophilic bulk to further out on
the alkyl chain provided the isobutyl derivative 22, which was 10-
fold more potent but somewhat less efficacious than tamoxifen,
and the isopentyl derivative 23 which was 3-fold more potent and
The synthetic method for compounds 17-24 is depicted in
Scheme 1. Phenyl acetic acid and 2-(chloroethoxy)benzene were
condensed according to the method of Horton et al.22 to give the
desired diphenylethanone intermediate, which was then alkylated
and aminated to provide the compounds. Some final targets were
isolated as free bases, while the ones that were purified by reversed
chromatography were obtained as trifluoroacetate salts. The one
exception to this general method was the synthesis of compound
17 where 2-phenylbutanoic acid and 2-(chloroethoxy)benzene
were condensed directly to give the ethyl intermediate which was
then converted to the dimethylamine. Detailed experimental
methods are provided in the Supplementary Material.

roughly 30% more efficacious than tamoxifen. Estrogenic activity
for compounds 23 and 24 was reduced compared to tamoxifen and
they demonstrated partial agonist activity compared to tamoxifen,
which demonstrated antagonist activity.
curves are provided in the Supplementary Material. All four
analogs demonstrated superior potency and percent maximal
activation of GPN-stimulated calcium release compared to
tamoxifen. And while the EC50 values for activation of GPN-
stimulated calcium release were slightly lower in WT cells
compared to GD-patient cells, all four compounds showed
significantly greater efficacy (maximal response) in GD-patient
cells. Compounds 28 and 31 in particular demonstrated high
maximal activation in GD-patient cells (227% and 240%,
respectively) in comparison to the maximal activation in WT cells
(56% and 50%, respectively).
We then moved to test our hypothesis regarding the preference
of a non-conjugated distal phenyl ring in the diphenylethanone
scaffold by introducing 2-substitutents in this ring (compounds 25
– 31, Table 2).
As previously discussed, the n-butyl derivative 21 showed no
activity in the calcium release assay in GD patient-derived cells.
However, introduction of a 2-substituent into that scaffold resulted
in analogs that showed activity in that assay that was similar to
(compounds 25 and 26) or superior to (compounds 27 and 28) that
seen with tamoxifen. Greater potency was realized when the
planarity of the substituent was eliminated. This is seen by
comparing compound 25 to 26 (EC50 = 2.3 M and 0.65 M,
respectively) and compound 27 to 28 (EC50 = 2.2 M and 1.4 M,
respectively). A significant increase in efficacy was seen when
the isopropyl group was introduced into the 2-position of the distal
phenyl ring (compound 28, Emax = 401% of the basal response to
GPN). The presence of the branched alkyl group also had a
beneficial effect on estrogenic activity. The ethenyl derivative 25
showed significant estrogen receptor activity at a concentration of
10 M (100% of the EC100 response seen with -estradiol) whereas
the propenyl (27) and i-propyl (28) analogs were less potent in the
estrogen receptor assay (35% and 45%, respectively).
Interestingly, the ethyl derivative 26 showed similar potency in the
estrogen receptor assay but was found to be an antagonist rather
than an agonist like 25, 27 and 28. The reasons for this switch in
intrinsic activity are not clear, although it should be noted that the
ethyl group is present in a comparable position in the
tamoxifen/idoxifene scaffold so it is possible that the ethyl group
is a privileged substituent in this position.
To differentiate compounds 27, 28, 29 and 31 and prioritize
one to advance to in vivo studies, we examined their in vitro drug-
like properties in two assays, namely maximum aqueous solubility
in 2% DMSO/phosphate buffered saline and stability in mouse
liver microsomes in the presence of NADPH (an indicator of
liability to oxidative metabolism). The results are summarized in
Table 4.
Table 4. Drug-like properties of selected diphenylethanones.
Cd. #
Max. Aq.
Microsomal Stability (t1/2, min.)
Solubility (µM)
Mouse
9.1
Human
24.4
27
28
29
31
1
45.2
49.4
40.0
31.7
6.9*
7.9
21.6
22.3
7.7
23.7
26.7
8.9
> 60
* Value reported in reference 23.
All four phenylethanone derivatives showed superior aqueous
solubility compared to tamoxifen. All four phenylethanone
derivatives also showed moderate stability in mouse and human
liver microsomes. Compound 29 was somewhat more stable in
mouse liver microsomes than the other three. However, compound
31 stood out from the others because of its lack of any estrogenic
activity at 10 M (Table 2). For this reason we selected compound
31 for advancement.
Similar results were seen when the i-butyl group was
substituted for the n-butyl moiety. Compound 29, which
combined the i-butyl substitution on the ethanone chain and the i-
propyl group in the 2-position of the distal phenyl ring,
demonstrated reasonable potency, superior efficacy and reduced
estrogenic activity compared to tamoxifen. Moving the i-propyl
group to the 3-position of the distal phenyl ring (compound 30)
resulted in a compound with significantly reduced efficacy (80%
compared to 406% for compound 29). Constraining the i-propyl
group to give the cyclopropyl analog 31 resulted in a molecule that
showed similar potency and enhanced efficacy in the calcium
release assay compared to tamoxifen and was devoid of estrogenic
activity at a concentration of 10 M.
In conclusion, a phenotypic screen identified tamoxifen as a hit
with modest functional potency to reverse the lysosomal calcium
deficits in GD patient-derived cells. SAR aimed at reducing the
planar conformation of tamoxifen led to the identification of a
novel series of phenylethanone derivatives, ultimately resulting in
the selection of compound 31 for further evaluation. In the current
studies compound 31 was tested as a racemic mixture. Despite the
realatively high pKa predicted for the enolic proton of 31
(calculated pKa24 for the proton adjacent to the carbonyl = 15.66),
data demonstrate that drugs containing enolic hydrogens can
undergo some racemization at physiological pH, especially in the
presence of phosphate buffers.25,26 It will be important to
understand the role of chirality on in vitro and in vivo activity in
this series of compounds and understand the propensity for them
to undergo partial or full racemization at physiological pH. These
and other studies are currently in progress and will be the subject
of future publications.
Table 3. Activity of selected compounds in lysosomal calcium
assay.
Lysosomal Ca+2 Assay
Cd #
GD-patient cells
EC50, M Emax, %
WT cells
EC50, M
Emax, %
27
28
29
31
1
2.2
1.48
1.8
3.1
10
257%
227%
449%
240%
148%
1.1
0.7
1.1
1.1
2.5
109%
56%
148%
50%
28%
Declaration of Competing Interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Activity for the four most potent compounds (27, 28, 29 and
31) and tamoxifen in the lysosomal calcium assay in GD patient-
derived cells and wild-type cells are summarized in Table 3. Full

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Acknowledgments
The authors thank Dr. Michael Denison for the BG1Luc4E2
(VM7Luc4E2) cell line. This research was partially supported by
the Officer of the Director, National Institutes of Health under
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