Discovery of a novel noniminosugar acid α glucosidase chaperone series

Article abstract of DOI:10.1021/jm3005543

Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA). Many disease-causing mutated GAA retain enzymatic activity but are not translocated from endoplasmic reticulum

Full text of DOI:10.1021/jm3005543

Article
pubs.acs.org/jmc
Discovery of a Novel Noniminosugar Acid α Glucosidase Chaperone
Series
Jingbo Xiao,† Wendy Westbroek,‡ Omid Motabar,† Wendy A. Lea,† Xin Hu,† Arash Velayati,‡
Wei Zheng,† Noel Southall,† Ann Marie Gustafson,‡ Ehud Goldin,‡ Ellen Sidransky,‡ Ke Liu,†
Anton Simeonov,† Rafael J. Tamargo,‡ Antonia Ribes,§ Leslie Matalonga,§ Marc Ferrer,†
and Juan J. Marugan*,†
†NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, National Center for Advancing Translational
Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, United States
‡Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Building 35 Room 1A213, 35
Convent Drive, Bethesda, Maryland 20892, United States
§Enfermedades Metabol
́ ́
icas Hereditarias, Institut de Bioquímica Clínica, Servicio de Bioquímica y Genetica Molecular, Hospital
Clínic y Provincial de Barcelona, Barcelona, Spain
S
* Supporting Information
ABSTRACT: Pompe disease is an autosomal recessive lysosomal storage disorder (LSD)
caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA). Many disease-
causing mutated GAA retain enzymatic activity but are not translocated from endoplasmic
reticulum (ER) to lysosomes. Enzyme replacement therapy (ERT) is the only treatment for
Pompe disease but remains expensive, inconvenient, and does not reverse all disease
manifestations. It was postulated that small molecules which aid in protein folding and
translocation to lysosomes could provide an alternate to ERT. Previously, several
iminosugars have been proposed as small-molecule chaperones for specific LSDs. Here we identified a novel series of
noniminosugar chaperones for GAA. These moderate GAA inhibitors are shown to bind and thermostabilize GAA and increase
GAA translocation to lysosomes in both wild-type and Pompe fibroblasts. AMDE and physical properties studies indicate that
this series is a promising lead for further pharmacokinetic evaluation and testing in Pompe disease models.
children with this disease is ERT with recombinant human GAA
(alglucosidase alfa), a recombinant human GAA produced in
Chinese hamster ovary cells.5 Although recombinant human
GAA (alglucosidase alfa) is proven to be clinically efficacious,
treatment is not optimal. Many patients test positive for IgG
antibodies to GAA, which reduces the clinical utility,6 its
intravenous administration is inconvenient, the cost can be over
$300000/year, and adverse side effects can occur.7 This
reinforces the need for developing alternative treatments for
Pompe disease.
Protein translocation from ER to the site of action is a dynamic
process involving distinct transporters that interact with low
energy conformations of the protein, a thermodynamically
driven process and kinetically accelerated by ER chaperones.8
Treatment with appropriate small-molecule chaperones could
kinetically accelerate the protein folding process and promote
translocation of mutant enzyme.9 For GAA, improving
trafficking of the mutant protein between the ER and the
lysosome by small-molecule chaperone treatment might reduce
glycogen storage and lysosome size.10 Iminosugars are well-
known small-molecule inhibitors of glycosidases11 that bind to
the active site of the enzyme, mimicking the transition state of the
INTRODUCTION
■
Lysosomal storage disorders (LSDs) represent over 50 different
rare diseases and result from mutations in lysosomal enzymes.
Phenotypically, cells affected by LSDs are characterized by
lysosomal enlargement due to accumulation of disease-specific
substrates.1 Pompe disease, also known as glycogen storage
disease type II or acid maltase deficiency, is an autosomal
recessive LSD caused by mutations in the lysosomal enzyme
GAA2 with a frequency of approximately 1 in every 40000 births.3
Disease progression is variable, and clinical symptoms can
include progressive muscle weakness and loss of motor,
respiratory, and cardiac function. In most patients, premature
death occurs due to respiratory complications. GAA hydrolyzes
the terminal α-1,4-glucosidic linkages of glycogen in the
lysosome. Mutations in GAA result in lysosomal enlargement
due to glycogen accumulation, especially in cardiac and skeletal
muscle.2 There are more than 100 different disease-causing GAA
mutations identified in Pompe patients affecting enzyme
expression, conformation, and activity.4 As in other LSDs,
many of the mutant proteins retain residual enzyme activity in
vitro but are unable to function because of impaired translocation
from the ER to the lysosome. Consequently, mutant enzyme
accumulates in the ER, is ubiquinated, and will eventually
undergo endoplasmic reticulum-associated protein degradation
(ERAD). Currently, the only FDA-approved treatment for
Received: April 18, 2012
© XXXX American Chemical Society
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Figure 1. Known iminosugars as inhibitors of α-glucosidase.
glycolytic hydrolysis (Figure 1).12 This accelerates the folding
process, and therefore iminosugar inhibitors have been proposed
for the treatment of various LSDs.13 Although the chaperone
activity of iminosugars is established, their use has several pitfalls.
They exhibit poor selectivity among several glycosidases and
need to be actively transport, and therefore differences in
bioavailability and body distribution among patients must be
considered. Additionally, due to their inhibitory activity, the
therapeutic window between translocation and enzyme function
efficacy is small and pharmacokinetics must be taken into
consideration for modulation of the concentration at the site of
action. Currently all small-molecule GAA chaperones reported in
the literature are iminosugar inhibitors, with 1-deoxynojirimycin
(DNJ) being at the moment evaluated in a phase II clinical trial as
a therapy for Pompe disease by Amicus Therapeutics Inc.14
Other than DNJ and NB-DNJ, no other reported inhibitors have
been shown to have GAA chaperone capacity or utility in the
treatment of Pompe disease.10
glucopyranoside, which liberates the red dye resorufin at an
emission wavelength of 590 nm when excited at 530 nm
(Figure 2).
Our group has developed several new screening method-
ologies to identify novel noniminosugar series impacting
enzymatic activity in LSD assays. We have focused on testing
enzymes in a context as native as possible, including measuring
the hydrolytic capacity of GAA in tissue homogenate.15 Many
isolated glucosidases require allosteric activation to be func-
tional,16 so we attempted to avoid using purified enzyme
preparations which depend upon the use of detergents to induce
the active conformation and catalysis the activity of the enzyme.
While it is common to find compounds that can inhibit isolated
enzymes, these molecules are often inactive in cellular lysates.
This is likely due to conformational differences between
detergent-induced enzymatic systems and the physiological
enzyme in cells or problems with nonspecific protein binding.
Another limitation of reconstituted assays is the inability to
detect enzyme activators, presumably because the detergent used
in reconstituted assays overactivates the enzyme in a non-
physiological way. One way to overcome these problems is to
screen the enzyme directly from tissue homogenate using a probe
specific for GAA activity such as 4-methylumbelliferyl α-D-
glucopyranoside.15 Upon hydrolysis, the blue fluorescent dye 4-
methylumbelliferone is liberated, producing a fluorescent
emission at 440 nm when excited at 370 nm. To control for
autofluorescence, we also used a second substrate, resorufin α-D-
Figure 2. Hydrolytic reactions of the red and blue dyes.
In this study, we present the first noniminosugar inhibitor
chaperone series for GAA identified, screening a novel method
using tissue homogenate instead of purified enzyme prepara-
tions. This series inhibited GAA activity in both tissue
homogenate and purified enzyme assays and showed trans-
location capacity to the lysosomal compartment in wild-type and
Pompe fibroblasts in a cell-based immunostaining assay.17 This
promising series merits further evaluation as a potential new
therapy for Pompe disease.
RESULTS AND DISCUSSION
■
Hit from qHTS. First, 244319 compounds from the NIH
Molecular Libraries−Small Molecule Repository (ML-SMR)
were screened.18 The Z′ across the entire screen was 0.82 0.04.
Only one noniminosugar inhibitor series, represented by
compound 1, was identified based on selectivity against related
enzymes including α-galactosidase and glucocerebrosidase (data
in Supporting Information). In addition, compound 1 displayed
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similar activity in both purified GAA and tissue homogenate
assays, and it was not autofluorescent as determined by spectral
profiling (Figure 3). Then the inhibitory activity of compound 1
prevent this loss of activity. Moreover, compounds able to
prevent thermal destabilization usually also promote proper
cellular folding and translocation and therefore can be good
chaperones. Figure 4 clearly demonstrates that compound 1 can
thermostabilize GAA.
Chemistry and SAR Studies. These data encouraged us to
embark on structural modifications to provide a better
understanding of SAR for this series. Schemes 1 and 2 showed
the general methodology used for the synthesis of analogues with
modifications in several regions of the molecule. Direct
chlorosulfonylation of 2-indolinone at the 5-position,19 followed
by piperazine or substituted aniline displacements, yielded
analogues 1−15 and 34 with modifications on the aromatic ring
attached to the piperazine moiety. Analogue 15 with para-methyl
ester substituent was readily converted to the corresponding
carboxylic acid or methyl alcohol analogues 16 and 17 via acidic
hydrolysis or DIBAL reduction. Alternatively, reaction of 1-(4-
(piperazin-1-yl)phenyl)ethanone with a variety of sulfonyl
chlorides in the presence of a suitable base such as triethylamine
gave analogues with modifications at the indolinone ring
(analogues 18−31). Furthermore, reaction of 1-(4-(piperazin-
1-yl)phenyl)ethanone with carboxylic acid under EDC coupling
conditions resulted in analogues 32 and 33 with replacement of
sulfonamide moiety with a carbonamide (Scheme 2).
Each synthesized analogue was assayed for GAA inhibition
using the tissue homogenate with two different pro-fluorescent
substrates, a blue-shifted dye substrate that was used in the
primary screen and a red-shifted dye substrate. The inhibitory
activity of the analogues was consistent in assays with both the
blue- and red-shifted dye substrates. Tables 1−3 demonstrate the
SAR of synthesized analogues with modifications at three areas of
the hit compound 1. Table 1 shows that a broad range of
substituents were tolerated within the aromatic ring attached to
the piperazine portion of the molecule, including para-hydroxyl
Figure 3. (A) Chemical structure of compound 1 identified from qHTS.
(B) The GAA inhibitory activity of compound 1 as demonstrated by the
hydrolysis of red and blue substrates using isolated GAA enzyme or
tissue homogenates.
was further evaluated using purified enzyme with its native
substrate, glycogen. Glycogen from bovine liver was used as the
substrate and recombinant human GAA as the enzyme
preparation. Upon hydrolysis of the substrate, the glucose
product could be detected using the Amplex Red Glucose
Oxidase assay kit, and the product of this reaction was detected in
a fluorescence plate reader. Compound 1 had a potency of 330
nM in this assay (data not shown), consistent with its activity in
the primary screen using pro-fluorescent substrates.
(analogue 5, IC50 = 1.88 μM), para-cyano (analogue 7, IC50
=
In addition to the previously described secondary assays, we
tested the ability of compound 1 to protect GAA activity upon
exposure to thermal defunctionalization conditions (Figure 4).
Briefly, hydrolytic enzymes lose their catalytic activity over time
when exposed to elevated temperatures below their melting
point due to progressive denaturation and/or aggregation of the
protein from solution. Incubating GAA at 66 °C for 60 min
reduces the enzyme activity by about 75%. Small molecules with
the capacity to bind and stabilize the enzyme are expected to
2.91 μM), para-nitro (analogue 8, IC50 = 3.66 μM), para-
formaldehyde (analogue 14, IC50 = 3.66 μM), and para-
carboxylic acid (analogue 16, IC50 = 1.30 μM) functional groups.
However, para-methoxy, para-methyl ester, and para-methyl
alcohol derivatives showed a 25−50-fold loss of activity
(analogue 4, IC50 = 23.69 μM; analogue 15, IC50 = 25.90 μM;
analogue 17, IC50 = 12.98 μM). Other functional groups, like
halo (analogues 9−11), methyl (analogue 6), and trifluor-
omethyl (analogues 12 and 13) were either inactive or had much
less activity. Surprisingly, elimination of the phenyl ring, while
keeping the methylketone moiety, resulted in derivative 3 with
decent activity (IC50 = 11.87 μM), while further elimination of
the methylketone group gave an inactive analogue 2. These data
indicate that the electronic nature of phenyl substituents has
minor effect on the inhibitory activity and that an oxygen or
nitrogen at the para-position of the phenyl group might be
involved in hydrogen bonding interactions enhancing activity.
The SAR for the indolinone group was found to be very
narrow (Table 2). Most modifications such as the introduction of
more heteroatoms (analogues 18−21, 24, 31), increasing the
size of the aliphatic portion of the indolin-2-one (analogues 25
and 26), or elimination of the aliphatic ring to obtain the
acetamide derivatives (analogues 27−30) all yielded inactive
compounds. The only modification on this part of the molecule
that did not have significant impact on activity was the
introduction of a chlorine substituent on the 6-position of the
indolinone ring (analogue 22, IC50 = 1.63 μM). The 3,3-dichloro
Figure 4. Thermostabilization of GAA functional activity. Time
indicates length of incubation at 66 °C. Ratio is the ratio of enzymatic
activity after incubation at 66 °C compared to room temperature
incubation. Black, DMSO (duplicated); red, DNJ (2.5 μM); green,
qHTS sample of compound 1 (50 μM); blue, resynthesized compound
1 (50 μM).
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Scheme 1. Synthesis of Analogues with Modifications of Phenylpiperazine Moiety of the Molecule
Scheme 2. Synthesis of Analogues with Modifications of Indolinone Ring or Sulfonamide Portion of the Molecule
substituted indolinone analogue (23, IC50 = 32.61 μM) was also
active, but much less potent.
tion of GAA, validating their potential value as small-molecule
chaperones.
Another way to test the capacity of a chemical series to stabilize
a protein is to measure the impact of the compounds on the
melting temperature (Tm) of the protein. For some chemical
series, there is a direct correlation between the binding affinities
of the compounds toward a protein and their capacity to increase
the protein’s Tm.20 Analogues 1 and 22 demonstrated a
concentration dependent ability to raise the Tm of GAA (Figure
6A). To further evaluate the binding interaction between these
two analogues and GAA, we also measured the corresponding
Table 3 demonstrates the effect of replacing the sulfonamide
with a carbonamide (analogues 32 and 33) or a reversed phenyl
and piperazine ring analogue (34). All three analogues were
found to be inactive. Therefore, the sulfonamide moiety also
contributed significantly to the inhibitory activity of this chemical
scaffold.
Biological Evaluation. With the SAR assessment estab-
lished for this scaffold, we evaluated the ability of selected
analogues with reasonable potency to stabilize GAA against
thermal defunctionalization. As shown in Figure 5, in the
presence of tested GAA inhibitors, GAA was able to maintain its
function compared to DMSO control. The stabilization observed
correlated directly with the inhibitory activity of the compounds
in the inhibition assay. In other words, the best inhibitors were
the best stabilizers; importantly, analogue 22 showed a strong
ability to stabilize GAA against thermal denaturation, similar to
the hit compound 1. These data demonstrated that these
inhibitors could stabilize the preferred 3-dimensional conforma-
binding affinity (Kd) by microscale thermophoresis (MST).21
A
direct interaction between small molecules and GAA was indeed
observed for both analogues 1 and 22 (Figure 6B). Because of
solubility limits, concentrations above 250 μM could not be
tested (thus saturation binding could not be determined for
analogue 22). Nevertheless, an apparent dissociation constant
(Kd) for analogues 1 and 22 was observed to be in a range of 20−
60 μM, with analogue 1 showing a stronger binding than
analogue 22. These calculated Kd’s are 20−40 times higher than
their reported IC50s, possibly due to differences in affinity as a
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Table 1. GAA Inhibitory Activity of Analogues with Modifications of the Aromatic Ring Attached to Piperazine Moiety
consequence of labeling the enzyme and the impact in enzymatic
conformation and in compound solubility of isolated-tissue
homogenated conditions.
primary wild-type fibroblasts, GAA staining was observed in
about 15% of the cells, and the GAA stain (green) colocalized
with the lysosomal marker cathepsin D (red) (Figure 7B, a).
Wild-type fibroblasts stained with secondary antibodies Alexa-
488 and Alexa-555 showed no signal at the same laser settings
(Figure 7B, b), indicating that the green signal from the GAA
antibody was specific and not due Alexa-488 secondary antibody
background. It should also be noted that GAA staining and
translocation to lysosomes in fibroblasts significantly decreased
with increasing cell passage number; wild-type fibroblast with
passage number 7 showed GAA staining in lysosomes in about
15% of the cells, while passage 8 cells showed translocation in
10% of the cells (data not shown). It is known that inhibitory
chaperones have a small therapeutic window in which the
molecule displays translocation without complete elimination of
the enzymatic activity. For this reason, good chaperone
molecules should be weak inhibitors, allowing its displacement
in the lysosome by the natural substrate. In vivo, this is resolved
by titrating the optimal dose for maximal activity. In cell-based
assays, this is addressed by varying the compound concentration
and length of exposure.
The encouraging results from the GAA thermostabilization
and direct binding experiments led us to explore whether this
chemical series had an impact on GAA translocation. Most LSDs
are biochemically diagnosed by measuring the specific activity of
the hydrolase on patient cell samples. However, Pompe-
associated glycogen accumulation primarily impacts cardiac
and skeletal muscle, and these tissues are often not available.
Therefore, in many LSDs, including Pompe disease, the enzyme
activity is analyzed on skin fibroblasts. Thus, we decided to
evaluate our inhibitors in fibroblasts. In contrast to other LSDs,
there is no common mutation in Pompe disease, therefore we
initially chose wild-type human fibroblasts to evaluate the
chaperone and translocation capacity of analogues 1 and 22.
First, we tested the specificity of the mouse monoclonal anti-
GAA antibody. On Western blot, the antibody recognized the
GAA protein (kDa) in protein lysate from human embryonic
kidney (HEK) cells electroporated with a pCMV6XL6 plasmid
containing the GAA cDNA (accession no. NM_000152.2); non-
electroporated HEK cells and wild-type fibroblast protein lysates
(Figure 7A) did not show a GAA specific signal. However, wild-
type fibroblasts express low levels of GAA. Next, we performed a
cell-based translocation assay for GAA. In DMSO-treated
Compound 1 was toxic for both wild-type and Pompe
fibroblasts (passage number 7) at 10, 15, and 20 μM after a 6-day
treatment, suggesting that this molecule has too much inhibitory
capacity, resulting in total suppression of enzyme activity.
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Table 2. GAA Inhibitory Activity of Analogues with Modifications of the Indolinone Ring
Moreover, 6-day ablation of GAA activity in fibroblasts induces
cell death. After 5 days of treatment, both wild-type cells and
Pompe fibroblast were viable but did not show an increase of
GAA translocation upon exposure to 1 or 5 μM of compound 1,
indicating that this compound might have a poor therapeutic
window or that our GAA antibody is not sensitive enough to
detect minor changes in this enzyme. In contrast, a 6-day
treatment of wild-type fibroblasts (passage number 7) with 15
μM (Figure 7B, c) or 5 μM (Figure 7B, d) of compound 22
significantly up-regulated the translocation of GAA to the
lysosomes. About 40% of the cells showed translocation of GAA
with treatment at 15 μM concentration and 30% with 5 μM.
Pompe fibroblasts treated with DMSO showed no GAA signal
(green) in lysosomes (Figure 7C, a). Six days of treatment with
compound 22 in Pompe fibroblasts resulted in cell toxicity,
probably due to excessive inhibition of the enzyme. Reducing the
time of exposure provided better results. Thus, a 5-day treatment
with 5 μM of compound 22 showed translocation in all three
Pompe cell lines (Figure 7C, b; data only shown for the F2845
Pompe cell line), while treatment with 1 μM did not induce
translocation, indicating that analogue 22 is able to increase the
translocation of GAA in both wild-type and Pompe cell lines and
showing that this chemical series has potential as a GAA
chaperone for the treatment of Pompe disease. It should be
noted that the concentration necessary for reasonable trans-
location in vivo could be considerably lower due to the high
expression of GAA in affected tissues.
Last, we also measured the elevation of GAA specific activity
upon treatment. Figure 8 discloses that upon five days of
treatment, inhibitors 1 and 22 are able to increase GAA activity of
wt and one of our mutant cell lines, confirming the results
observed in our translocation experiment. Importantly, incre-
ments in activity can be observed at lower concentrations of their
described IC50s, indicating a potentially good therapeutic
window.
In Vitro ADME Properties. Five selected analogues with
IC50 less than 3.0 μM in the GAA inhibitory assay using blue dye
were profiled for PBS aqueous solubility, mouse liver microsomal
stability, and cell permeability in Caco-2 cells (Table 4). The
aqueous solubility of the hit compound 1 in PBS buffer was 10-
fold above its IC50 value. The solubility was significantly
increased for analogues with polar functionalities such as
hydroxyl or carboxylic acid groups. Interestingly, the introduc-
tion of a chlorine atom at the 6-position of indolinone moiety
also enhanced its aqueous buffer solubility. The microsomal
stability tests disclosed that all five analogues showed excellent
metabolic stability in mouse liver microsomes. In addition, this
metabolic transformation was NADPH-dependent (data not
shown), indicating that the major metabolic process occurs
through cytochrome P450-dependent oxidation. Moreover, the
data in Table 4 indicates that hit compound 1 had very good cell
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Table 3. GAA Inhibitory Activity of Analogues with Modifications of Sulfonamide and Piperazine Moieties
both wild-type and Pompe cells. Therefore, analogue 22 is a
promising lead candidate for further development for the
treatment of Pompe disease.
CONCLUSIONS
■
In summary, we present the first noniminosugar GAA chaperone
series identified from a qHTS campaign. This series of
compounds inhibit GAA activity in both tissue homogenate
and purified enzyme assays using native or artificial substrates. In
addition, these inhibitors are highly selective against the related
lysosomal hydrolases α-galactosidase and glucocerebrosidase.
SAR studies produced several optimized compounds that are
able to stabilize GAA in thermofunctional and thermal
denaturation assays. Furthermore, MST studies display that
these molecules are indeed binders of GAA, with their apparent
Kds tracking well with their inhibitory activities. Improved
compounds display good physical and ADME properties while
maintaining GAA inhibitory activity. Importantly optimized
analogue 22 showed an appropriate balance between inhibitory
and translocation capacity in both wild-type and Pompe cells,
Figure 5. Capacity of inhibitors at 50 μM to maintain the function of
GAA after incubation at 66 °C for 60 min.
permeability, with reasonable efflux (ratio = 1.9, [B→A/A→B]).
The chloro analogue 22 has even better permeability, with a
reduced efflux ratio (0.57). As expected, the carboxylic acid
analogue 16 displayed very poor cell permeability. Overall,
analogue 22 demonstrated improved aqueous solubility,
excellent microsomal stability, and cell permeability in addition
to its capacity of increasing GAA translocation and activity in
Figure 6. (A) ΔTm versus concentrations for analogues 1 and 22. (B) Microscale thermophoresis study of analogues 1 and 22 with GAA.
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Figure 7. Evaluation of compound 22 in GAA translocation assays using wild-type and Pompe fibroblasts. (A) Western blot analysis of protein extracts
from HEK cells overexpressing GAA (lane 1), HEK cells (lane 2), and wild-type primary fibroblasts (lane 3). Tubulin was used as a loading control. (B,
a) DMSO-treated primary wild-type fibroblasts. (B, b) DMSO-treated primary wild-type fibroblasts stained with mouse Alexa-488 and goat Alexa-
555(red) as a negative staining control. (B, c,d) Primary wild-type fibroblasts treated with 15 μM (c) and 5 μM (d) of compound 22 for 6 days. (C, a)
DMSO-treated primary Pompe fibroblasts. (C, b) Pompe fibroblasts (F2845) treated with 5 μM of compound 22 for 5 days. All stainings were
performed with the anti-GAA mouse monoclonal antibody (green) and the anti-Cathepsin D goat polyclonal antibody (red); a DAPI stain was
performed to visualize the nucleus (blue). Scale bar = 20 μm (B) and 50 μm (C).
Figure 8. Relative inhibition of GAA in WT (A) and Pompe F2845 fibroblasts (B) in a whole cell assay with treatment of compound 22 and compound 1
at pH 4 with no wash-out and after 18 and 40 h wash-out. Data points have been normalized to GAA activity in vehicle treated cells (100%).
Luna C18 (5 μm, 30 mm × 75 mm; Phenomenex, Inc., Torrance, CA) at
a flow rate of 45.0 mL/min. The mobile phase consisted of acetonitrile
and water (each containing 0.1% trifluoroacetic acid). A gradient of 10−
50% acetonitrile over 8 min was used during the purification. Fraction
collection was triggered by UV detection at 220 nM. Analytical analysis
was performed on an Agilent LC/MS (Agilent Technologies, Santa
Clara, CA) using a method of a 7 min gradient of 4−100% acetonitrile
(containing 0.025% trifluoroacetic acid) in water (containing 0.05%
trifluoroacetic acid) with an 8 min run time at a flow rate of 1.0 mL/min.
A Phenomenex Luna C18 column (3 μm, 3 mm × 75 mm) was used at a
temperature of 50 °C. Purity determination was performed using an
making it a promising lead for further pharmacological
development.
EXPERIMENTAL SECTION
■
General Chemistry Methods. All air or moisture sensitive
reactions were performed under positive pressure of nitrogen with
oven-dried glassware. Anhydrous solvents such as dichloromethane,
N,N-dimethylformamide (DMF), acetonitrile, methanol, and triethyl-
amine were purchased from Sigma-Aldrich (St. Louis, MO). Preparative
purification was performed on a Waters semipreparative HPLC system
(Waters Corp., Milford, MA). The column used was a Phenomenex
H
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Table 4. ADME Profile of Five Selected Analogues with IC50 Less than 3.0 μM in the GAA Inhibitory Assay Using Blue Dye
Agilent diode array detector, and all of the analogues tested in the
biological assays have a purity of greater than 95%. Mass determination
was performed using an Agilent 6130 mass spectrometer with
5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)indolin-2-one
(1). A solution of 1-(4-(piperazin-1-yl)phenyl)ethanone (485 mg, 2.37
mmol) and triethylamine (0.602 mL, 4.32 mmol) in DMF (10.0 mL)
was treated at room temperature with 2-oxoindoline-5-sulfonyl chloride
(500 mg, 2.16 mmol). The reaction mixture was stirred overnight at
room temperature. The mixture was poured into water, and the solid
fraction was crushed out, filtered, and dried to give 658 mg (yield 76%)
1
electrospray ionization in the positive mode. H NMR spectra were
recorded on Varian 400 MHz spectrometers (Agilent Technologies,
Santa Clara, CA). Chemical shifts are reported in ppm with
undeuterated solvent (DMSO-d6 at 2.49 ppm) as internal standard for
DMSO-d6 solutions. High resolution mass spectrometry was recorded
on Agilent 6210 time-of-flight (TOF) LC/MS system. Confirmation of
molecular formula was accomplished using electrospray ionization in the
positive mode with the Agilent Masshunter software (version B.02).
General Protocol A. A solution of 1-substituted piperazine (0.216
mmol) and triethylamine (0.120 mL, 0.863 mmol) in DMF (1.50 mL)
was treated at room temperature with 2-oxoindoline-5-sulfonyl chloride
(50.0 mg, 0.216 mmol). The reaction mixture was stirred overnight at
room temperature. The crude mixture was filtered and purified by
preparative HPLC to give the final product.
General Protocol B. A solution of 1-(4-(piperazin-1-yl)phenyl)-
ethanone (71.5 mg, 0.350 mmol) and triethylamine (0.098 mL, 0.700
mmol) in DMF (2.00 mL) was treated at room temperature with
sulfonyl chloride (0.350 mmol). The reaction mixture was stirred
overnight at room temperature. The crude mixture was filtered and
purified by preparative HPLC to give the final product.
General Protocol C. A solution of 1-(4-(piperazin-1-yl)phenyl)-
ethanone (71.5 mg, 0.350 mmol) and triethylamine (0.098 mL, 0.700
mmol) in DMF (2.00 mL) was treated at room temperature with
sulfonyl chloride (0.350 mmol). The reaction mixture was stirred
overnight at room temperature. The mixture was poured into water, and
the solid fraction was crushed out. The solid fraction was filtered and
dried to give the final product.
General Protocol D. A solution of 1-(4-(piperazin-1-yl)phenyl)-
ethanone (0.086 g, 0.420 mmol) and carboxylic acid (0.350 mmol) in
DMF (2.00 mL) was treated at room temperature with EDC (0.067 g,
0.350 mmol) and DMAP (0.043 g, 0.350 mmol). The reaction mixture
was stirred overnight at room temperature. The crude mixture was
filtered and purified by preparative HPLC to the final product.
2-Oxoindoline-5-sulfonyl Chloride. Indolin-2-one (5.00 g, 37.6
mmol) was added at 30 °C to hypochlorous sulfonic anhydride (10.2
mL, 153 mmol) in portions. The reaction mixture was stirred at room
temperature for 1.5 h and heated to 70 °C for 1 h. The reaction was
slowly quenched with water, and the light-pink solid precipitation was
filtered and dried to give 5.33 g (yield 61%) of product, which was used
in the next reaction without further purification. 1H NMR (400 MHz,
DMSO-d6) δ ppm 10.40 (s, 1 H), 7.3−7.46 (m, 2 H), 6.71 (dd, J = 7.8,
0.6 Hz, 1 H), 3.45 (s, 2 H).
1
of product as a white solid. H NMR (400 MHz, DMSO-d6) δ ppm
10.83 (s, 1 H), 7.67−7.95 (m, 2 H), 7.47−7.71 (m, 2 H), 7.01 (d, J = 8.2
Hz, 1 H), 6.88−6.97 (m, 2 H), 3.59 (s, 2 H), 3.38−3.50 (m, 4 H), 2.92−
3.05 (m, 4 H), 2.43 (s, 3 H). LCMS RT = 4.46 min, m/z 400.1 [M + H+].
HRMS (ESI) m/z calcd for C20H22N3O4S [M + H+] 400.1326, found
400.1330.
5-(Piperazin-1-ylsulfonyl)indolin-2-one (2). A solution of
piperazine (1.67 g, 19.4 mmol) and triethylamine (2.71 mL, 19.4
mmol) in DMF (10.0 mL) was treated at 0 °C with 2-oxoindoline-5-
sulfonyl chloride (1.50 g, 6.48 mmol) in DMF (10.0 mL). The reaction
mixture was stirred overnight at room temperature. DMF was removed,
and dichloromethane was added to precipitate out the product. The
solid fraction was filtered and washed with dichloromethane to give the
final product as a brown solid. 1H NMR (400 MHz, DMSO-d6) δ ppm
10.89 (s, 1 H), 8.47 (br s, 1 H), 7.51−7.74 (m, 2 H), 6.97−7.09 (m, 1
H), 3.62 (s, 2 H), 3.11−3.25 (m, 4 H), 2.99−3.11 (m, 4 H). LCMS RT =
2.66 min, m/z 282.1 [M + H+]. HRMS (ESI) m/z calcd for
C12H16N3O3S [M + H+] 282.0907, found 282.0912.
5-(4-Acetylpiperazin-1-ylsulfonyl)indolin-2-one (3). The title
1
compound was prepared according to general protocol A. H NMR
(400 MHz, DMSO-d6) δ ppm 10.79 (s, 1 H), 7.35−7.60 (m, 2 H), 6.96
(d, J = 8.2 Hz, 1 H), 3.55 (s, 2 H), 3.46 (q, J = 5.2 Hz, 4 H), 2.80 (ddd, J =
19.8, 5.2, 4.9 Hz, 4 H), 1.89 (s, 3 H). LCMS RT = 3.36 min, m/z 346.1
[M + Na+]. HRMS (ESI) m/z calcd for C14H18N3O4S [M + H+]
324.1013, found 324.1019.
5-(4-(4-Methoxyphenyl)piperazin-1-ylsulfonyl)indolin-2-one
(4). The title compound was prepared according to general protocol A.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.84 (s, 1 H), 7.48−7.71 (m, 2
H), 7.02 (d, J = 8.2 Hz, 1 H), 6.70−6.94 (m, 4 H), 3.66 (s, 3 H), 3.61 (s, 2
H), 3.02−3.11 (m, 4 H), 2.91−3.02 (m, 4 H). LCMS RT = 4.45 min, m/
z 388.1 [M + Na+]. HRMS (ESI) m/z calcd for C19H22N3O4S [M + H+]
388.1326, found 388.1330.
5-(4-(4-Hydroxyphenyl)piperazin-1-ylsulfonyl)indolin-2-one
(5). The title compound was prepared according to general protocol A.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.80 (s, 1 H), 8.88 (br s, 1 H),
7.42−7.68 (m, 2 H), 6.98 (d, J = 8.0 Hz, 1 H), 6.74 (d, J = 7.6 Hz, 2 H),
6.45−6.67 (m, 2 H), 3.57 (s, 2 H), 2.82−3.09 (m, 8 H). LCMS RT =
3.49 min, m/z 374.1 [M + H+]. HRMS (ESI) m/z calcd for
C18H20N3O4S [M + H+] 374.1169, found 374.1173.
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5-(4-p-Tolylpiperazin-1-ylsulfonyl)indolin-2-one (6). The title
compound was prepared according to general protocol A. H NMR
HRMS (ESI) m/z calcd for C19H20N3O4S [M + H+] 386.1169, found
386.1169.
1
Methyl 4-(4-(2-Oxoindolin-5-ylsulfonyl)piperazin-1-yl)-
benzoate (15). A solution of methyl 4-(piperazin-1-yl)benzoate
(0.687 g, 3.12 mmol) and triethylamine (0.870 mL, 6.24 mmol) in
dichloromethane (15.0 mL) was treated at room temperature with 2-
oxoindoline-5-sulfonyl chloride (0.723 g, 3.12 mmol). The reaction
mixture was stirred overnight at room temperature. The yellow solid was
filtered and dried to the final product. 1H NMR (400 MHz, DMSO-d6) δ
ppm 10.80 (s, 1 H), 7.66−7.82 (m, 2 H), 7.46−7.63 (m, 2 H), 6.79−7.09
(m, 3 H), 3.72 (s, 3 H), 3.55 (s, 2 H), 3.32−3.45 (m, 4 H), 2.85−3.05
(m, 4 H). LCMS RT = 5.14 min, m/z 416.1 [M + H+]. HRMS (ESI) m/
z calcd for C20H22N3O5S [M + H+] 416.1275, found 416.1282.
4-(4-(2-Oxoindolin-5-ylsulfonyl)piperazin-1-yl)benzoic Acid
(16). A suspension of methyl 4-(4-(2-oxoindolin-5-ylsulfonyl)-
piperazin-1-yl)benzoate (15) (240 mg, 0.578 mmol) in 6.0 N HCl
(75.0 mL) was refluxed for 1 h. The reaction mixture was concentrated
and purified by preparative HPLC to the final product. 1H NMR (400
MHz, DMSO-d6) δ ppm 12.25 (br s, 1 H), 10.80 (br s, 1 H), 7.63−7.79
(m, 2 H), 7.38−7.63 (m, 2 H), 6.70−7.08 (m, 3 H), 3.56 (s, 2 H), 3.32−
3.43 (m, 4 H), 2.84−3.05 (m, 4 H). LCMS RT = 4.46 min, m/z 402.1
[M + H+]. HRMS (ESI) m/z calcd for C19H20N3O5S [M + H+]
402.1118, found 402.1126.
5-(4-(4-(Hydroxymethyl)phenyl)piperazin-1-ylsulfonyl)-
indolin-2-one (17). DIBAL (0.325 mL, 1.0 M in THF, 0.325 mmol)
was added dropwise to a solution of methyl 4-(4-(2-oxoindolin-5-
ylsulfonyl)piperazin-1-yl)benzoate (15) (45.0 mg, 0.108 mmol) in THF
(5.00 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min. The
reaction was quenched by addition of methanol, concentrated as a
yellow oil, which was purified by preparative HPLC to the final product.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.80 (s, 1 H), 7.48−7.72 (m, 2
H), 7.10 (d, J = 8.8 Hz, 2 H), 6.98 (d, J = 8.2 Hz, 1 H), 6.74−6.86 (m, 2
H), 4.32 (s, 2 H), 3.57 (s, 2 H), 3.05−3.22 (m, 4 H), 2.88−3.04 (m, 4
H). LCMS RT = 4.06 min, m/z 388.1 [M + H+]. HRMS (ESI) m/z calcd
for C19H22N3O4S [M + H+] 388.1326, found 388.1330.
(400 MHz, DMSO-d6) δ ppm 10.82 (s, 1 H), 7.45−7.66 (m, 2 H), 7.00
(dd, J = 8.0, 5.9 Hz, 3 H), 6.74−6.85 (m, 2 H), 3.59 (s, 2 H), 3.06−3.21
(m, 4 H), 2.89−3.06 (m, 4 H), 2.17 (s, 3 H). LCMS RT = 5.08 min, m/z
372.1 [M + H+]. HRMS (ESI) m/z calcd for C19H22N3O3S [M + H+]
372.1376, found 372.1380.
4-(4-(2-Oxoindolin-5-ylsulfonyl)piperazin-1-yl)benzonitrile
(7). The title compound was prepared according to general protocol A.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.79 (s, 1 H), 7.40−7.66 (m, 4
H), 6.82−7.13 (m, 3 H), 3.54 (s, 2 H), 3.36−3.43 (m, 4 H), 2.88−2.97
(m, 4 H). LCMS RT = 4.89 min, m/z 383.1 [M + H+]. HRMS (ESI) m/
z calcd for C19H19N4O3S [M + H+] 383.1172, found 383.1173.
5-(4-(4-Nitrophenyl)piperazin-1-ylsulfonyl)indolin-2-one (8).
The title compound was prepared according to general protocol A. 1H
NMR (400 MHz, DMSO-d6) δ ppm 10.79 (s, 1 H), 8.00 (d, J = 9.6 Hz, 2
H), 7.47−7.64 (m, 2 H), 6.88−7.04 (m, 3 H), 3.45−3.68 (m, 6 H),
2.85−3.01 (m, 4 H). LCMS RT = 5.02 min, m/z 425.1 [M + Na+].
HRMS (ESI) m/z calcd for C18H19N4O5S [M + H+] 403.1071, found
403.1071.
5-(4-(4-Fluorophenyl)piperazin-1-ylsulfonyl)indolin-2-one
(9). The title compound was prepared according to general protocol A.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.80 (s, 1 H), 7.41−7.69 (m, 2
H), 6.93−7.08 (m, 3 H), 6.77−6.92 (m, 2 H), 3.56 (s, 2 H), 3.05−3.16
(m, 4 H), 2.87−3.00 (m, 4 H). 19F NMR (376 MHz, DMSO-d6) δ ppm
−126.55−123.72 (m). LCMS RT = 5.15 min, m/z 376.1 [M + H+].
HRMS (ESI) m/z calcd for C18H19FN3O3S [M + H+] 376.1126, found
376.1138.
5-(4-(4-Chlorophenyl)piperazin-1-ylsulfonyl)indolin-2-one
(10). The title compound was prepared according to general protocol A.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.82 (s, 1 H), 7.49−7.74 (m, 2
H), 7.13−7.31 (m, 2 H), 7.01 (d, J = 8.2 Hz, 1 H), 6.78−6.96 (m, 2 H),
3.59 (s, 2 H), 3.13−3.24 (m, 4 H), 2.87−3.03 (m, 4 H). LCMS RT =
5.57 min, m/z 392.1 [M + H+]. HRMS (ESI) m/z calcd for
C18H19ClN3O3S [M + H+] 392.0830, found 392.0842.
5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-1H-benzo[d]-
imidazol-2(3H)-one (18). The title compound was prepared
5-(4-(4-Bromophenyl)piperazin-1-ylsulfonyl)indolin-2-one
(11). The title compound was prepared according to general protocol A.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.80 (s, 1 H), 7.48−7.72 (m, 2
H), 7.19−7.37 (m, 2 H), 6.98 (d, J = 8.2 Hz, 1 H), 6.77−6.87 (m, 2 H),
3.56 (s, 2 H), 3.10−3.21 (m, 4 H), 2.88−2.98 (m, 4 H). LCMS RT =
5.67 min, m/z 436.0 [M + H+]. HRMS (ESI) m/z calcd for
C18H19BrN3O3S [M + H+] 436.0325, found 436.0338.
1
according to general protocol B. H NMR (400 MHz, DMSO-d6) δ
ppm 11.15 (d, J = 1.2 Hz, 1 H), 10.99 (s, 1 H), 7.59−7.87 (m, 2 H), 7.33
(dd, J = 8.2, 1.8 Hz, 1 H), 7.04−7.22 (m, 2 H), 6.79−6.97 (m, 2 H),
3.33−3.51 (m, 4 H), 2.84−3.03 (m, 4 H), 2.39 (s, 3 H). LCMS RT =
4.40 min, m/z 401.1 [M + H+]. HRMS (ESI) m/z calcd for
C19H21N4O4S [M + H+] 401.1278, found 401.1286.
5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-1,3-dimethyl-
1H-benzo[d]imidazol-2(3H)-one (19). The title compound was
prepared according to general protocol C. 1H NMR (400 MHz, DMSO-
d6) δ ppm 7.65−7.78 (m, 2 H), 7.40−7.54 (m, 2 H), 7.34 (d, J = 8.6 Hz,
1 H), 6.79−6.96 (m, 2 H), 3.38−3.44 (m, 4 H), 3.36 (s, 3 H), 3.33 (s, 3
H), 2.92−3.04 (m, 4 H), 2.39 (s, 3 H). LCMS RT = 5.07 min, m/z 429.1
[M + H+]. HRMS (ESI) m/z calcd for C21H25N4O4S [M + H+]
429.1591, found 429.1592.
6-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)benzo[d]oxazol-
2(3H)-one (20). The title compound was prepared according to general
protocol C. 1H NMR (400 MHz, DMSO-d6) δ ppm 12.14 (br s, 1 H),
7.70−7.78 (m, 2 H), 7.63 (d, J = 1.4 Hz, 1 H), 7.53 (dd, J = 8.2, 1.6 Hz, 1
H), 7.26 (d, J = 8.2 Hz, 1 H), 6.85−6.94 (m, 2 H), 3.35−3.45 (m, 4 H),
2.92−3.03 (m, 4 H), 2.39 (s, 3 H). LCMS RT = 5.07 min, m/z 402.1 [M
+ H+]. HRMS (ESI) m/z calcd for C19H20N3O5S [M + H+] 402.1118,
found 402.1130.
6-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-3-
methylbenzo[d]oxazol-2(3H)-one (21). The title compound was
prepared according to general protocol C. 1H NMR (400 MHz, DMSO-
d6) δ ppm 7.71−7.76 (m, 2 H), 7.69 (d, J = 1.4 Hz, 1 H), 7.62 (dd, J =
8.3, 1.7 Hz, 1 H), 7.45 (d, J = 8.0 Hz, 1 H), 6.78−6.95 (m, 2 H), 3.36−
3.45 (m, 4 H), 3.33 (s, 3 H), 2.92−3.04 (m, 4 H), 2.39 (s, 3 H). LCMS
RT = 5.22 min, m/z 416.1 [M + H+]. HRMS (ESI) m/z calcd for
C20H22N3O5S [M + H+] 416.1275, found 416.1284.
5-(4-(4-(Trifluoromethyl)phenyl)piperazin-1-ylsulfonyl)-
indolin-2-one (12). The title compound was prepared according to
general protocol A. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.82 (s, 1
H), 7.52−7.68 (m, 2 H), 7.48 (d, J = 8.6 Hz, 2 H), 6.76−7.13 (m, 3 H),
3.58 (s, 2 H), 3.33−3.42 (m, 4 H), 2.93−3.03 (m, 4 H). 19F NMR (376
MHz, DMSO-d6) δ ppm −59.64 (s). LCMS RT = 5.78 min, m/z 426.1
[M + H+]. HRMS (ESI) m/z calcd for C19H19F3N3O3S [M + H+]
426.1094, found 426.1101.
5-(4-(3-(Trifluoromethyl)phenyl)piperazin-1-ylsulfonyl)-
indolin-2-one (13). The title compound was prepared according to
general protocol A. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.83 (s, 1
H), 7.50−7.73 (m, 2 H), 7.40 (t, J = 7.9 Hz, 1 H), 7.11−7.22 (m, 2 H),
7.08 (d, J = 8.4 Hz, 1 H), 7.01 (d, J = 8.0 Hz, 1 H), 3.59 (s, 2 H), 3.25−
3.31 (m, 4 H), 2.94−3.04 (m, 3 H). 19F NMR (376 MHz, DMSO-d6) δ
ppm −61.14 (s). LCMS RT = 5.78 min, m/z 426.1 [M + H+]. HRMS
(ESI) m/z calcd for C19H19F3N3O3S [M + H+] 426.1094, found
426.1104.
4-(4-(2-Oxoindolin-5-ylsulfonyl)piperazin-1-yl)-
benzaldehyde (14). A solution of 4-(piperazin-1-yl)benzaldehyde,
TFA salt (58.0 mg, 0.139 mmol), and triethylamine (0.039 mL, 0.277
mmol) in dichloromethane (2.00 mL) was treated at room temperature
with 2-oxoindoline-5-sulfonyl chloride (32.1 mg, 0.139 mmol). The
reaction mixture was stirred for 3 h at room temperature. The solid was
1
filtered and dried to a yellow solid product. H NMR (400 MHz,
5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-6-chloroindo-
DMSO-d6) δ ppm 10.79 (s, 1 H), 9.67 (s, 1 H), 7.60−7.72 (m, 2 H),
7.49−7.61 (m, 2 H), 6.90−7.04 (m, 3 H), 3.55 (s, 2 H), 3.37−3.50 (m, 4
H), 2.85−2.98 (m, 4 H). LCMS RT = 4.55 min, m/z 386.1 [M + H+].
lin-2-one (22). The title compound was prepared according to general
1
protocol B. H NMR (400 MHz, DMSO-d6) δ ppm 10.88 (s, 1 H),
7.67−7.89 (m, 3 H), 6.79−7.08 (m, 3 H), 3.57 (s, 2 H), 3.36−3.46 (m, 4
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H), 3.19−3.27 (m, 4 H), 2.43 (s, 3 H). LCMS RT = 4.92 min, m/z 434.1
[M + H+]. HRMS (ESI) m/z calcd for C20H21ClN3O4S [M + H+]
434.0936, found 434.0942.
ppm 10.88 (s, 1 H), 7.71−7.78 (m, 2 H), 7.25−7.30 (m, 1 H), 7.23−7.25
(m, 1 H), 7.13 (d, J = 8.4 Hz, 1 H), 6.85−6.94 (m, 2 H), 4.67 (s, 2 H),
3.37−3.46 (m, 4 H), 2.91−3.00 (m, 4 H), 2.40 (s, 3 H). LCMS RT =
4.94 min, m/z 416.1 [M + H+]. HRMS (ESI) m/z calcd for
C20H22N3O5S [M + H+] 416.1275, found 416.1281.
5-(4-(4-Acetylphenyl)piperazine-1-carbonyl)indolin-2-one
(32). The title compound was prepared according to general protocol D.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.55 (s, 1 H), 7.70−7.88 (m, 2
H), 7.23−7.40 (m, 2 H), 6.98 (d, J = 9.2 Hz, 2 H), 6.85 (d, J = 8.0 Hz, 1
H), 3.63 (br s, 4 H), 3.51 (s, 2 H), 3.37−3.46 (m, 4 H), 2.45 (s, 3 H).
LCMS RT = 4.14 min, m/z 364.1 [M + H+]. HRMS (ESI) m/z calcd for
C21H22N3O3S [M + H+] 364.1656, found 364.1661.
6-(4-(4-Acetylphenyl)piperazine-1-carbonyl)-3,4-dihydro-
quinolin-2(1H)-one (33). The title compound was prepared according
to general protocol D. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.22 (s,
1 H), 7.69−7.88 (m, 2 H), 7.26 (d, J = 1.8 Hz, 1 H), 7.22 (dd, J = 8.0, 2.0
Hz, 1 H), 6.90−6.98 (m, 2 H), 6.86 (d, J = 8.2 Hz, 1 H), 3.60 (br s, 4 H),
3.51 (br s, 4 H), 2.88 (t, J = 7.5 Hz, 2 H), 2.44−2.45 (m, 1 H), 2.40−2.43
(m, 4 H). LCMS RT = 4.30 min, m/z 378.2 [M + H+]. HRMS (ESI) m/
z calcd for C22H24N3O3S [M + H+] 378.1812, found 378.1815.
N-(4-(4-Acetylpiperazin-1-yl)phenyl)-2-oxoindoline-5-sulfo-
namide (34). A solution of 1-(4-(4-aminophenyl)piperazin-1-yl)-
ethanone (76.0 mg, 0.345 mmol) and triethylamine (0.096 mL, 0.691
mmol) in DMF (3.00 mL) was treated at 0 °C with 2-oxoindoline-5-
sulfonyl chloride (80.0 mg, 0.345 mmol). The reaction mixture was
warmed to room temperature and stirred overnight. The crude mixture
was filtered and purified by preparative HPLC to give the final product.
1H NMR (400 MHz, DMSO-d6) δ ppm 10.72 (s, 1 H), 9.70 (s, 1 H),
5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-3,3-dichloroin-
dolin-2-one (23). The title compound was prepared according to
general protocol B. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.86 (s, 1
H), 7.95 (d, J = 1.8 Hz, 1 H), 7.83 (dd, J = 8.4, 2.0 Hz, 1 H), 7.73−7.80
(m, 2 H), 7.20 (d, J = 8.4 Hz, 1 H), 6.90−6.98 (m, 2 H), 3.38−3.48 (m, 4
H), 2.99−3.08 (m, 4 H), 2.42 (s, 3 H). LCMS RT = 5.57 min, m/z 468.0
[M + H+]. HRMS (ESI) m/z calcd for C20H20Cl2N3O4S [M + H+]
468.0546, found 468.0547.
6-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)isobenzofuran-
1(3H)-one (24). The title compound was prepared according to general
protocol C. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.11 (dd, J = 8.0, 1.6
Hz, 1 H), 8.05 (d, J = 1.0 Hz, 1 H), 7.94 (dd, J = 8.1, 0.7 Hz, 1 H), 7.69−
7.77 (m, 2 H), 6.85−6.95 (m, 2 H), 5.48 (s, 2 H), 3.36−3.46 (m, 4 H),
2.99−3.10 (m, 4 H), 2.39 (s, 3 H). LCMS RT = 5.12 min, m/z 401.1 [M
+ H+]. HRMS (ESI) m/z calcd for C20H21N2O5S [M + H+] 401.1166,
found 401.1177.
6-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-3,4-dihydro-
quinolin-2(1H)-one (25). The title compound was prepared according
to general protocol C. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.46 (s,
1 H), 7.67−7.79 (m, 2 H), 7.54 (d, J = 2.0 Hz, 1 H), 7.51 (dd, J = 8.4, 2.2
Hz, 1 H), 7.00 (d, J = 8.4 Hz, 1 H), 6.86−6.95 (m, 2 H), 3.34−3.46 (m, 4
H), 2.89−3.03 (m, 6 H), 2.40 (s, 3 H) (2 protons were hidden under
DMSO-d6 peaks). LCMS RT = 4.91 min, m/z 414.1 [M + H+]. HRMS
(ESI) m/z calcd for C21H24N3O4S [M + H+] 414.1482, found 414.1491.
7-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-4,5-dihydro-
1H-benzo[b]azepin-2(3H)-one (26). The title compound was
prepared according to general protocol C. 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.88 (s, 1 H), 7.70−7.78 (m, 2 H), 7.63 (d, J = 2.3 Hz,
1 H), 7.58 (dd, J = 8.3, 2.2 Hz, 1 H), 7.13 (d, J = 8.2 Hz, 1 H), 6.85−6.95
(m, 2 H), 3.33−3.46 (m, 4 H), 2.93−3.05 (m, 4 H), 2.75 (t, J = 6.8 Hz, 2
H), 2.40 (s, 3 H), 2.03−2.22 (m, 4 H). LCMS RT = 4.93 min, m/z 428.2
[M + H+]. HRMS (ESI) m/z calcd for C22H26N3O4S [M + H+]
428.1639, found 428.1644.
N-(4-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)phenyl)-
acetamide (27). The title compound was prepared according to
general protocol C. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.34 (s, 1
H), 7.76−7.82 (m, 2 H), 7.71−7.76 (m, 2 H), 7.61−7.69 (m, 2 H),
6.87−6.94 (m, 2 H), 3.35−3.43 (m, 4 H), 2.90−2.99 (m, 4 H), 2.40 (s, 3
H), 2.04 (s, 3 H). LCMS RT = 5.04 min, m/z 402.1 [M + H+]. HRMS
(ESI) m/z calcd for C20H24N3O4S [M + H+] 402.1482, found 402.1485.
1-(4-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)phenyl)-
pyrrolidin-2-one (28). The title compound was prepared according to
general protocol C. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.87−7.97
(m, 2 H), 7.67−7.77 (m, 4 H), 6.83−6.95 (m, 2 H), 3.84 (t, J = 7.0 Hz, 2
H), 3.36−3.46 (m, 4 H), 2.89−2.99 (m, 4 H), 2.50 (t, J = 8.1 Hz, 2 H),
2.39 (s, 3 H), 2.03 (ddd, J = 15.1, 7.5, 7.3 Hz, 2 H). LCMS RT = 5.22
min, m/z 428.2 [M + H+]. HRMS (ESI) m/z calcd for C22H26N3O4S [M
+ H+] 428.1639, found 428.1644.
7.51 (dd, J = 4.0, 2.6 Hz, 2 H), 6.89−6.96 (m, 2 H), 6.84−6.89 (m, 1 H),
6.77−6.84 (m, 2 H), 3.53 (s, 6 H), 2.93−3.08 (m, 4 H), 2.00 (s, 3 H).
LCMS RT = 3.55 min, m/z 415.1 [M + H+]. HRMS (ESI) m/z calcd for
C20H23N4O4S [M + H+] 415.1435, found 415.1448.
General Biological Experiments. The recombinant wild-type
enzyme Myozyme (alglucosidase alfa), clinically approved for ERT, was
obtained from Genzyme Corporation (Cambridge, MA). Patients’
spleens were obtained from splenectomies with informed consent under
an NIH-IRB approved clinical protocol no. 86HG0096. Control spleens
were obtained under the same NIH protocol number. 4-Methyl-
umbelliferyl-α-D-glucopyranoside (4MU-α-glc), resorufin α-D-glucopyr-
anoside (res-α-glc), sodium taurocholate, and the buffer components
were purchased from Sigma-Aldrich (St. Louis, MO). 1-Deoxynojir-
imycin (DNJ) was purchased from Tocris Bioscience (Minneapolis,
MN). The human spleen tissue was homogenized using a food blender
at the maximal speed for 5 min, followed by 10 passes in a motor-driven
50 mL glass−Teflon homogenizer. The homogenate was centrifuged at
1000g for 10 min. The supernatant was then filtered using a 40 μm filter,
and aliquots of resultant spleen homogenate were frozen at −80 °C until
use. The assay buffer was composed of 50 mM citric acid titrated with
K2PO4 to make different pH solutions and 0.01% Tween-20. The spleen
homogenate assays used buffer at pH = 5, assays with recombinant wild-
type enzyme used buffer at pH = 5.9.
Enzyme Assay in 1536-Well Plate Format. In black 1536-well
plates, a BioRAPTR FRD Microfluidic workstation (Beckman Coulter,
Inc. Fullerton, CA) was used to dispense 2 μL of the enzyme solutions
into 1536-well plates, and an automated pin-tool station (Kalypsys, San
Diego, CA) was used to transfer 23 nL/well of compound to the assay
plate. After 5 min incubation at room temperature, the enzyme reaction
was initiated by the addition of 2 μL/well substrate. After 45 min
incubation at 37 °C, the reaction was terminated by the addition of 2
μL/well stop solution. The fluorescence was then measured in the
Viewlux, a CCD-based plate reader (Perkin-Elmer, Waltham, MA), with
a 365 nm excitation and 440 nm emission for the blue substrate and 573
nm excitation and 610 nm emission for the red substrate. Then 27 μg/
well of spleen homogenate was used as the enzyme solution. The final
concentrations of the blue substrate and red substrate were 1 and 15 μM,
respectively.
1-(4-(4-(1-Acetyl-2-methylindolin-5-ylsulfonyl)piperazin-1-
yl)phenyl)ethanone (29). The title compound was prepared
1
according to general protocol C. H NMR (400 MHz, DMSO-d6) δ
ppm 8.11 (br s, 1 H), 7.67−7.78 (m, 2 H), 7.47−7.63 (m, 2 H), 6.78−
6.96 (m, 2 H), 4.51−4.76 (m, 1 H), 3.32−3.46 (m, 5 H), 2.88−3.03 (m,
4 H), 2.73 (d, J = 16.8 Hz, 1 H), 2.40 (s, 3 H), 2.23 (s, 3 H), 1.18 (d, J =
6.5 Hz, 3 H). LCMS RT = 5.38 min, m/z 442.2 [M + H+]. HRMS (ESI)
m/z calcd for C23H28N3O4S [M + H+] 442.1795, found 442.1797.
1-(4-(4-(1-Acetyl-1,2,3,4-tetrahydroquinolin-6-ylsulfonyl)-
piperazin-1-yl)phenyl)ethanone (30). The title compound was
prepared according to general protocol C. 1H NMR (400 MHz, DMSO-
d6) δ ppm 7.80−7.87 (m, 1 H), 7.70−7.77 (m, 2 H), 7.52 (d, J = 2.0 Hz,
1 H), 7.48 (dd, J = 8.6, 2.3 Hz, 1 H), 6.86−6.95 (m, 2 H), 3.62−3.72 (m,
2 H), 3.36−3.44 (m, 4 H), 2.92−3.01 (m, 4 H), 2.78 (t, J = 6.5 Hz, 2 H),
2.40 (s, 3 H), 2.18 (s, 3 H), 1.85 (dq, J = 6.6, 6.3 Hz, 2 H). LCMS RT =
5.27 min, m/z 442.2 [M + H+]. HRMS (ESI) m/z calcd for
C23H28N3O4S [M + H+] 442.1795, found 442.1807.
Thermodenaturation Experiment. This assay measures the effect
of compounds on the melting temperature (Tm) of the recombinant
wild-type GAA. The protocol was developed based on a previously
reported general guideline.22 A mixture of GAA and SYPRO Orange
7-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-2H-benzo[b]-
[1,4]oxazin-3(4H)-one (31). The title compound was prepared
1
according to general protocol C. H NMR (400 MHz, DMSO-d6) δ
K
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Journal of Medicinal Chemistry
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(5000× stock concentration, Invitrogen, Carlsbad, CA) was delivered to
a 384-well full-skirted white polypropylene plate (Roche Applied
Science, Indianapolis, IN) with a final concentration of 1 μM and 5×,
respectively. GAA and SYPRO Orange were diluted in 50 mM citrate
acid buffer at pH = 5.0 supplemented with 100 mM KCl, 10 mM NaCl,
and 1 mM MgCl2. A six-point DMSO dilution series was made
separately in a 384-well polypropylene plate (Thermo Fisher Scientific,
Hudson, NH) for all analogues whose final concentrations ranged from
0.82−200 μM. Half a microliter of each dilution point of each
compound was transferred to the aforementioned GAA-SYPRO Orange
mixture, with a final DMSO concentration of 2%. DMSO alone was also
transferred to the assay plate for each dilution series as a control sample.
The plate was immediately centrifuged at 1000 rpm for 10 s and
subsequently sealed with sealing foils (Roche Applied Science). The
plate was then heated using a LightCycler 480 Instrument II (Roche
Applied Science) from 20 to 95 °C at a ramping rate of 4.8 °C/s. SYPRO
Orange fluorescence was monitored by a CCD camera using excitation
and emission wavelengths of 498 and 580 nm, respectively. Tm was
calculated through the LightCycler 480 II Software.
Microscale Thermophoresis. GAA was labeled with a fluorescent
dye NT-495 (NanoTemper Technologies), and the final concentration
of the protein applied in equilibrium binding experiments was ∼50 nM.
A 16-point titration series of selected compounds was prepared in
DMSO and was further transferred toprotein solutions in a buffer
containing 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2, pH = 7.5.
The final concentrations of the compounds ranged from 250 μM to 7.63
nM, with the DMSO final concentration controlled at 2.5%. Samples
were filled into Monolith NT standard treated capillaries (NanoTemper
Technologies) after a room temperature incubation of 15 min. Capillary
scan was performed on a NanoTemper Monolith NT.115 instrument,
and thermophoresis was successively measured in each capillary.
Measurement took place at room temperature with an 80% IR laser
power, and the blue LED power set at 100%. Specifically, a laser-on time
of 30 s and a laser-off time of 5 s were applied at the indicated IR-laser
power. Data normalization and curve fitting were performed using
GraphPad Prism 5.
PBS containing 0.1% saponin, 100 μM glycine, 0.1% BSA, and 2%
donkey serum followed by incubation with mouse monoclonal anti-
GAA (Abnova, Walnut, CA) or goat anti-cathepsin D (R&D Systems,
Minneapolis, MN). The cells were washed and incubated with
secondary donkey anti-mouse or anti-goat antibodies conjugated to
ALEXA-488 or ALEXA-555, respectively (Invitrogen), washed again,
and mounted in VectaShield with DAPI (Vector Laboratories,
Burlingame, CA). Cells were imaged with a Zeiss 510 META confocal
laser-scanning microscope (Carl Zeiss, Microimaging Inc., Germany)
using an argon (458, 477, 488, 514 nm) 30 mW laser, a HeNe (543 nm)
1 mW laser, and a laser diode (405 nm). Images were acquired using a
Plan-Apochromat 63×/1.4 Oil, a Plan NeoFluar 40×/1.3 oil DIC
objective, or a Plan-Apochromat 20×/0.15. Images were taken at the
same laser settings.
Specific Activity Measurement. GAA activity in WT and Pompe
fibroblasts was measured similar to the method used by Flanagan et al.14
Briefly, for each cell line, cells were seeded in three black clear flat
bottom 96-well plates (Perkin-Elmer, Waltham, MA) at 5000 cells/well.
Inhibitors were added (50 nM to 50 μM) to the cells and incubated for 5
days; the media and compounds were changed on day 3. One plate of
each cell line was assayed immediately, while for two plates of each cell
line, fibroblasts were washed with PBS and fresh medium without
compounds was added (18 and 40 h wash out). Then 30 μL of assay
buffer (30 mM sodium citrate, 40 mM sodium phosphate dibasic, 3 mM
4MUG (Sigma), 3 μM Acarbose (Sigma), pH 4.0) was added and plates
were incubated for 2 h at 37 °C and 30 μL of stop solution (1 M NaOH,
1 M glucine, pH 10) was added; a Victor plate reader (Perkin-Elmer)
was used to measure fluorescence signal. Fluorescence counts from
fibroblast assay buffer + stop solution (background counts) were
subtracted from the cell samples, and the relative GAA activity was
calculated. Measurements were done in triplicate for each sample, and
the average GAA activity SD is reported.
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional data of our translocation experiment and selectivity
assays for compound 1. This material is available free of charge
via the Internet at http://pubs.acs.org.
Cells, Plasmids, and Electroporation. Wild-type fibroblasts and
HEK cells were purchased from ATCC (Manassas, VA). Electro-
poration of the HEK cells with pCMV6XL6-GAA plasmid (Origene,
Rockville, MD) was performed in a Nucleofector electroporator
according to the manufacturer’s guidelines (Lonza, Walkersville, MD).
Briefly, a mixture of 100 μL of Nucleofector solution and 2 μg of plasmid
DNA was added to approximately 700000 HEK cells, and electro-
poration was performed with the Q_001 Nucleofector program. For our
translocation experiments besides wild-type, we used three Pompe
fibroblast cell lines isolated from patients and with low passage number
with the following mutations:
AUTHOR INFORMATION
Corresponding Author
*Phone: 301-217-9198. Fax: 301-217-5736. E-mail: maruganj@
mail.nih.gov.
■
Notes
The authors declare no competing financial interest.
The data from the primary screening as well as all the inhibitory
curves of every final compound in all the described assays are
available on line (PubChem AID’s: 1466, 2101, 2107, 2108,
2109, 2100, 2110, 2111, 2112, 2113, 2115 and 2641).
Cell line F3248 heterozygous for: p.Y455C/p.G638W
Cell line F0833 heterozygous for: p.L169P/p.D489N
Cell line F2845 has a splice site mutation in one allele: c.IVS1−
13T > G and p.G638W on the other one.
Western Blot Analysis. Equivalent amounts of total protein, as
determined by BCA assay (Pierce Biotechnology, Rockford, IL) from
HEK cells or fibroblasts were loaded onto 4−12% Tris-Glycine gels.
After blotting (iBlot PVDF, Invitrogen, CA), the PVDF membrane was
blocked in phosphate-buffered saline (PBS) containing 0.1% Tween-20
(Sigma) and 5% fat-free milk for 1 h at room temperature. The blocked
membrane was probed with a mouse monoclonal antibody against
human GAA (Abnova, Walnut, CA), followed by incubation with HRP-
labeled secondary antimouse antibody (Amersham Biosciences, Piscat-
away, NJ). The antigen−antibody complexes were detected with an
Enhanced Chemiluminescence (ECL) kit (Amersham Biosciences).
Alpha-Tubulin was used as the internal control for normalization.
Immunocytochemistry and Laser Scanning Confocal Micros-
copy. Wild-type primary dermal human fibroblasts (ATCC) and
primary Pompe fibroblasts were seeded in Lab-Tek 4 chamber slides
(Fisher Scientific, Pittsburgh, PA). After chemical compound treatment
for five or six days, fibroblasts were fixed in 3% paraformaldehyde. The
cells were permeabilized with 0.1% Triton-X for 10 min and blocked in
ACKNOWLEDGMENTS
■
We thank William Leister, Jim Bougie, Chris Leclair, Thomas
Daniel, Heather Baker, Paul Shinn, and Danielle van Leer for
assistance with compounds purification, HRMS analysis, and
compounds management. We also thank Allison Mandich and
Mercedes Taylor for critical proofreading of the manuscript. This
research was supported by the Molecular Libraries Initiative of
the NIH Roadmap for Medical Research (U54MH084681) and
the Intramural Research Program of the National Human
Genome Research Institute and National Center for Advancing
Translational Sciences, National Institutes of Health.
ABBREVIATIONS USED
LSDs, lysosomal storage diseases; GAA, acid α glucosidase; ER,
endoplasmic reticulum; ERAD, endoplasmic reticulum-associ-
■
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(10) Beck, M. New therapeutic options for lysosomal storage
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ated protein degradation; ERT, enzyme replacement therapy;
AMDE, absorption, distribution, metabolism, and excretion;
BBB, blood−brain barrier; LAMP-2, lysosomal-associated
membrane protein 2; HTS, high throughput screening; ML-
SMR, Molecular Libraries−Small Molecule Repository; DNJ, 1-
deoxynojirimycin; FDA, U.S. Food and Drug Administration;
DIBAL, diisobutylaluminium hydride; EDC, 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride; SAR, struc-
ture−activity relationship; NADPH, nicotinamide adenine
dinucleotide phosphate; TLC, thin layer chromatography;
HPLC, high performance liquid chromatography; DMF,
dimethylformamide; DMAP, 4-dimethylaminopyridine; MST,
microscale thermophoresis
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