Potent, selective, and orally available benzoisothiazolone phosphomannose isomerase inhibitors as probes for congenital disorder of glycosylation Ia

Article abstract of DOI:10.1021/jm101401a

We report the discovery and validation of a series of benzoisothiazolones as potent inhibitors of phosphomannose isomerase (PMI), an enzyme that converts mannose-6-phosphate (Man-6-P) into fructose-6-phosphate (Fru-6-P) and, more importantly, competes wit

Full text of DOI:10.1021/jm101401a

ARTICLE
pubs.acs.org/jmc
Potent, Selective, and Orally Available Benzoisothiazolone
Phosphomannose Isomerase Inhibitors as Probes for Congenital
Disorder of Glycosylation Ia
Russell Dahl,†,‡ Yalda Bravo,†,‡ Vandana Sharma,§ Mie Ichikawa,§ Raveendra-Panickar Dhanya,†,‡
Michael Hedrick,‡ Brock Brown,‡ Justin Rascon,‡ Michael Vicchiarelli,‡ Arianna Mangravita-Novo,‡
Li Yang,†,‡ Derek Stonich,‡ Ying Su,‡ Layton H. Smith,‡ Eduard Sergienko,‡ Hudson H. Freeze,§ and
Nicholas D. P. Cosford*,†,‡
†Apoptosis and Cell Death Research Program, ‡Conrad Prebys Center for Chemical Genomics, and §Sanford Children's Health Research
Center, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, United States
ABSTRACT: We report the discovery and validation of a series
of benzoisothiazolones as potent inhibitors of phosphoman-
nose isomerase (PMI), an enzyme that converts mannose-6-
phosphate (Man-6-P) into fructose-6-phosphate (Fru-6-P)
and, more importantly, competes with phosphomannomutase
2 (PMM2) for Man-6-P, diverting this substrate from critical
protein glycosylation events. In congenital disorder of glycosy-
lation type Ia, PMM2 activity is compromised; thus, PMI
inhibition is a potential strategy for the development of therapeutics. High-throughput screening (HTS) and subsequent chemical
optimization led to the identification of a novel class of benzoisothiazolones as potent PMI inhibitors having little or no PMM2
inhibition. Two complementary synthetic routes were developed, enabling the critical structural requirements for activity to be
determined, and the compounds were subsequently profiled in biochemical and cellular assays to assess efficacy. The most
promising compounds were also profiled for bioavailability parameters, including metabolic stability, plasma stability, and
permeability. The pharmacokinetic profile of a representative of this series (compound 19; ML089) was also assessed,
demonstrating the potential of this series for in vivo efficacy when dosed orally in disease models.
’ INTRODUCTION
Congenital disorder of glycosylation type Ia (CDG-Ia) is a genetic
disorder caused by mutations in the pmm2 gene that leads to reduced
phosphomannomutase 2 (PMM2) enzyme activity. PMM2 plays an
important role in N-glycosylation by competing with phosphoman-
nose isomerase (PMI) for a common substrate, mannose-6-phos-
phate (Man-6-P), and converting it to mannose-1-phosphate
(Man-1-P), which ultimately enters the N-glycosylation pathway
(Figure 1). Any aberration in PMM2 results in underglycosylation of
Figure 1. PMI and phosphomannomutase (PMM2) are important
regulators of glycosylation. Inhibitors were designed to inhibit PMI
but not PMM2, facilitating the accumulation of Man-6-P to drive
glycosylation (HK = hexokinase).
proteins in CDG-Ia patients, and this leads to multiorgan symptoms
including neurological problems. There is currently no therapy for
CDG-Ia patients, and the prognosis is extremely poor.1
Unfortunately, mannose therapy alone does not benefit CDG-
Ia patients since most of the supplied mannose is catabolized by
over PMM2 has been reported for any PMI inhibitors prior to this
PMI and is therefore unavailable for glycosylation. We hypothesized
work. Herein, we disclose the discovery and validation of benzoi-
sothiazolone derivatives that are selective inhibitors of human PMI.
that CDG-Ia patients might benefit from dietary mannose supple-
mentation combined with inhibition of PMI using small molecule
This class shows favorable absorption, distribution, metabolism,
inhibitors selective for PMI over PMM2, thus diverting metabolic
flux toward the glycosylation pathway (Figure 1).1a,2 Small mole-
excretion (ADME) profiles, and a representative compound is
orally available when dosed in mice. These compounds are viable
cule inhibitors of PMI are scarce. The only inhibitors reported are
either substrate based or show very weak inhibition (Figure 2).3 In
leads for the development of novel therapeutics to treat CDG-Ia.
contrast, many of our newly discovered inhibitors have more than a
10-fold increase in potency over the most potent of the previously
reported inhibitors. In addition, no cell-based efficacy or selectivity
Received: April 26, 2010
Published: May 03, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Benzisothiazolones via
(a) Acylnitrenium Ion-Mediated Cyclization or
(b) Copper-Catalyzed N-Arylationa
a PIFA = phenyliodine bis(trifluoroacetate).
goal of developing a compound with a profile suitable for in vivo
proof-of-concept studies.
Chemical Synthesis. To facilitate the generation of analogues,
two chemical routes were developed to enable parallel synthesis of
chemical libraries around the benzisothiazolone scaffold (Scheme 1).
The first route utilized chemistry developed by Correa et al.4 and
involved a key cyclization step using phenyliodine bis(trifluoro-
acetate) (PIFA) to generate a N-acylnitrenium ion followed by intra-
molecular trapping by sulfur (Scheme 1a). This synthetic methodol-
ogy allowed for substitution of both aromatic portions of the
molecule and was utilized particularly to probe the effects of
substituents on the core benzisothiazolone ring.
Figure 2. Structures and activities of previously reported PMI
inhibitors.3
To efficiently assess the effects of substituents on the N-phenyl
ring, we developed a copper-mediated Ullman type N-arylation
reaction, based on a variation of methods reported by Buchwald
and others.5 Beginning from commercially available benzoisothia-
zolone, this reaction employs catalytic amounts of copper iodide
and N,N0-dimethylethylenediamine as the ligand (Scheme 1b).
These conditions allowed rapid access to a diverse set of analogues
in one step. It should also be noted that other ligands and copper
sources were also screened. Generally, diamines and CuI gave the
best overall yields (yield range 6ꢁ42%), although CuCl and Cu2O
were almost as effective. This reaction was also performed using
microwave irradiation with acceptable product recovery in minutes.
This is the first example of N-arylation chemistry of this heterocycle
type and allowed rapid interrogation of the N-aryl species. Using a
combination of both routes, the relevant sites of SAR were
investigated to afford optimal substitutions with respect to PMI
potency, PMM2 selectivity, and cellular efficacy.
Biochemical Evaluation. The potency and selectivity of the
synthesized compounds were assessed by in vitro enzymatic
assays using purified human PMI and PMM2. The effects of
substitution on the pendant N-phenyl ring on PMI and PMM2
inhibition are shown in Table 1. Like the lead compound 1, all of
the synthesized derivatives showed selectivity for PMI over
PMM2, with the exception of the 4-trifluoromethylphenyl deri-
vative 6, which was inactive against both enzymes. In general,
para substitution was favored over meta, with a 2ꢁ3-fold increase
in PMI inhibition seen. The exceptions to this trend included
trifluoromethyl substitution (compounds 5 and 6) and ester
substitution (9 and 10). The effect of ortho substitution was
explored with compound 16. In comparison to compound 3, the
des-methyl variant of this compound, little effect on PMI activity
was seen. However, a measurable inhibition of PMM2 was
observed (Table 1). Some notable examples for overall potency
and selectivity include compound 8 with a PMI IC50 of 1.8 μM
and compound 12, which showed comparable activity. These
Figure 3. Benzoisothiazolone PMI inhibitor HTS hit.
’ RESULTS AND DISCUSSION
High-throughput screening (HTS) of 196,000 compounds
from the NIH Molecular Libraries Small Molecule Repository
(MLSMR) was conducted to identify small molecule inhibitors
of PMI. Because compounds of interest were expected to show
efficacy in an environment rich in Man-6-P content resulting
from PMM2 deficiency and a supply of external mannose, we
were specifically interested in identifying compounds with non-
or uncompetitive modes of inhibition. To help select the most
promising scaffolds, parallel primary screening was performed
in the presence of 2 ꢀ KM and 10 ꢀ KM concentrations of Man-
6-P. Compounds of interest were expected to be efficacious in
both assays. Among the confirmed hits that inhibited in both
assays was a novel class of benzoisothiazolones showing robust
PMI inhibition, PMI/PMM2 selectivity, and a structure amen-
able to optimization of both biological activity and drug
properties. This series is exemplified by compound 1
(Figure 3), a PMI inhibitor that was among the HTS hits
chosen for follow up. While this compound was selective for
PMI over PMM2 when tested up to 20 μM, it was recognized as
a relatively weak inhibitor of PMI (IC50 = 6.4 μM). Thus,
because of the observed PMI/PMM2 selectivity and the
potential for parallel synthesis, we initiated structureꢁactivity
relationship (SAR) studies around this scaffold to optimize
potency, selectivity, and cellular PMI inhibitory activity. In
addition, the most promising compounds were evaluated for
their ADME and pharmacokinetic (PK) properties with the
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Table 1. SAR of N-Phenyl Ring Substituentsa
Scheme 2. Replacement of Heteroatoms of the
Benzisothiazolone Abolished All PMI Inhibition
IC50 (μM)
compd
Ar
PMI
PMM2
1
2
Ph
6.4
9.4
6.0
5.2
3.4
>20
4.8
1.9
4.9
7.2
8.5
1.9
4.3
5.0
3.7
6.6
>20
>20
>20
>20
13.9
>20
>20
>20
15.4
>20
>20
13.3
>20
>20
>10
17.9
2-naphthyl
3-Me-Ph
3
4
4-Me-Ph
5
3-CF3ꢁPh
4-CF3ꢁPh
3-Cl-Ph
6
7
8
4-Cl-Ph
9
3-COOMe-Ph
4-COOEt-Ph
3-N(Me)2-Ph
4-N(Me)2-Ph
3-I-Ph
10
11
12
13
14
15
16
PMM2. For example, compound 19, which has an unsubstituted
phenyl ring and fluorine at the 5-position of the fused aryl ring,
had a PMI IC50 of 1.3 μM, as compared to the analogous des-
fluoro derivative 1, which had a PMI IC50 of 6.4 μM. In addition,
both of these compounds were inactive against PMM2 when
tested up to 20 μM. An exception to this trend was seen with the
4-chloro-substituted compounds 24 and 8, with PMI IC50 values
of 1.8 and 1.9 μM, respectively. The most potent compounds
were in this series, specifically the dimethyl substituted 17 with
fluorine in the 6-position and the 4-methoxy derivative 22 with
fluorine in the 5-position. These derivatives both showed PMI
inhibition of 1.0 μM, representing a full fold-better potency than
the most potent derivatives from the previous des-methyl series.
While inhibition of PMM2 was seen in these most potent
examples, they still maintained a 7ꢁ9-fold selectivity for PMI.
Finally, the importance of heteroatoms in the benzisothiazo-
lone ring was investigated. To accomplish this, selected analo-
gues were synthesized with the nitrogen and sulfur atoms in the
benzisothiazolone ring either removed or replaced (compounds
27ꢁ29, Scheme 2). As can be seen, all of these modifications
abrogated all PMI activity when tested up to 10 μM in the
enzyme assay. These data support the importance of the benzi-
sothiazolone ring system for PMI inhibition.
4-tBu-Ph
3-OMe-Ph
2,5-di-Me-Ph
a PMI and PMM assay data are the mean of at least three determinations.
See the Experimental Section for assay details.
Table 2. SAR of Fluorine-Substituted Benzisothiazolonesa
IC50 (μM)
compd
R
Ar
PMI
PMM2
17
18
19
20
21
22
23
24
25
26
6-F
6-F
5-F
5-F
5-F
5-F
5-F
5-F
5-F
6-F
2,5-di-Me-Ph
4-OMe-Ph
Ph
1.0
3.1
1.3
1.9
3.6
1.0
4.3
1.8
8.3
2.9
7.3
12.9
>80
31.3
>80
9.1
Cellular Evaluation. A cellular assay was developed to eval-
uate the biological and functional efficacy of the PMI inhibitors.
[2-3H]-Mannose is a specific label, which is used to measure the
amount of mannose that is incorporated into glycoproteins ver-
sus that which is catabolized (Figure 1). In the presence of PMI
2,5-di-Me-Ph
4-F-Ph
4-OMe-Ph
2-F-Ph
>20
>20
>20
>20
4-Cl-Ph
3
inhibitors, more H-mannose will be diverted toward PMM2,
3-F-Ph
resulting in more of the 3H radiolabel present in glycoproteins as
compared with untreated cells. Representative PMI inhibitors
that display reproducible cellular activity are shown in Figure 4.
Briefly, cells were incubated with inhibitor and labeled with 3H-
2-Me-Ph
a PMI and PMM2 assay data are the mean of at least three determina-
tions. See the Experimental Section for assay details.
3
mannose and 35S-Met/Cys. The amounts of H- and 35S radi-
compounds were identified as promising leads for further
optimization.
Next, the effects of fluorine substitution at positions 5 and 6 on
the core benzisothiazolone aryl ring were assessed with respect to
PMI potency and PMM2 selectivity (Table 2). Generally, fluo-
rine substitution at these positions afforded an overall increase in
PMI potency with all of the examples maintaining selectivity over
olabel were then determined in the precipitated proteins. The 35S
label was included to determine the protein synthesis capacity of
the cells as well as the toxicity. Decreased 35S-Met/Cys incor-
poration at high concentration was observed for some com-
pounds. This was attributed to off-target activity because similar
effects were observed in PMI null cells (data not shown).
Similarly, the toxicity was unlikely to be due to PMM2 inhibition
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Figure 4. Cell-based PMI inhibitor data. Shown is the 3H-mannose incorporation normalized to 35S protein synthesis in the presence of increasing
inhibitor concentrations.
Table 3. ADME Profiles of Selected PMI Inhibitors
solubility pH
plasma stabilityb microsomal stabilityb
a
compd 7.4 (μg/mL) Log Pe
(mouse)
(mouse)
14
17
19
20
2.5
22.5
3.8
ꢁ2.8
ꢁ2.9
ꢁ3.2
ꢁ2.9
10
62
48
82.3
85
99
11
ND
81
a Permeability determined by PAMPA assay. b Percent of compound
remaining after incubation for 3 (plasma) or 1 h (microsomes) at 37 °C.
because PMM2-deficient CDG-Ia patient fibroblasts with 20%
or less residual PMM2 activity grow slowly but normally and do
not show less 35S-incorporation into the glycoproteins. Com-
pound 6, which was inactive against both PMI and PMM2, was
included as an example of a compound that exhibits cellular
toxicity in the assay. While all of the PMI inhibitors display
enhanced glycosylation as indicated by increased 3H-mannose
incorporation, compounds 14 and 19 both show a favorable
combination of cellular efficacy with lessened toxicity. While
compound 19 is the most potent compound in vitro, the
maximal efficacy in cells appeared to be slightly lower than
other derivatives (Figure 4). This may be due to the membrane
permeability of compound 19, as it is predicted to be moder-
ately permeable when assessed in vitro (Table 3). However, on
the basis of its overall profile, compound 19 was selected for
evaluation at an extended range of inhibitor concentrations and
displayed a dose-dependent increase in mannose incorporation
(Figure 5).
In Vitro ADME Profiling. The PMI inhibitors with the most
favorable cellular efficacy were also profiled in in vitro ADME
assays to assess their drug-likeness6 and potential for systemic
activity in animal models. Many of the benzisothiazolones were
shown to have suitable properties for oral administration includ-
ing acceptable metabolic and plasma stabilities, good permeabil-
ity across artificial lipid membranes, and good solubility. The
results of the specific in vitro ADME profiling assays are shown in
Table 3. Although the profiles of many of the synthesized ana-
logues had druglike ADME profiles, it was clear that in addition
to showing acceptable aqueous solubility at physiological pH, the
mouse plasma and microsomal stabilities of 19 were superior
(Table 3). Compound 19 was therefore selected for additional
studies, including its propensity to generate glutathione (GSH)-
Figure 5. Cell-based efficacy of compound 19. Shown is the 3H-
mannose incorporation normalized to 35S protein synthesis in the
presence of increasing concentrations of 19.
Table 4. PK Parameters of Compound 19
oral dose
Cmax
Tmax
AUC (0ꢁt)
(nM h)
t1/2
F
compd (mg/kg)
(nM)
(min)
(h)
(%)
19
20
162 ( 32 45 ( 21
305 ( 34
1.3 ( 0.64 35
trapped reactive metabolites. The S9-based GSH transferase
assay has been shown to be a reliable means of identifying
compounds known to react with GSH in vivo. When tested in the
presence of rat liver S9 fraction and GSH for 2 h, the quantity of
19 was not significantly reduced, and no GSH adducts were
observed by liquid chromatographyꢁmass spectrometry
(LCMS) analysis.
In Vivo PK. On the basis of the promising properties displayed
by compound 19, i.e. potent PMI inhibition, PMI/PMM2
selectivity, cellular efficacy with low toxicity, and a favorable
ADME profile, the PK and oral availability of this compound
were evaluated in mice to enable future advanced studies in
CDG-Ia disease models. To this end, compound 19 was dosed at
5 mg/kg i.v. and 20 mg/kg p.o. in mice. As predicted by the
favorable ADME profile, the bioavailability of 19 (F = 35%) was
satisfactory. The various PK parameters are shown in Table 4.
Overall, the compound has a good PK profile with acceptable
parameters to enable future in vivo efficacy experiments.
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’ CONCLUSION
column chromatography was carried out using prepacked silica car-
tridges from RediSep (ISCO Ltd.) and eluted using an Isco Companion
system. H NMR spectra were acquired on a Varian Inova 300 MHz
A series of novel, druglike benzisothiazolone inhibitors of PMI
were synthesized, and their ability to modulate flux toward glyco-
sylation has been demonstrated. Two synthetic routes for this
scaffold were optimized, including a novel copper-catalyzed
N-arylation reaction amenable to parallel derivitization. These
compounds are the most potent inhibitors of PMI to date, and
their dose-dependent efficacy in cells has been demonstrated.
In addition, they are selective over PMM2 at all concentra-
tions tested. The compounds possess favorable ADME and PK
profiles, including good oral bioavailability, which should enable
the further therapeutic development of these compounds.
1
instrument. Chemical shifts are reported in ppm from residual solvent
peaks (δ 7.27 for CDCl3 1H NMR). HPLC-MS analyses were per-
formed on a Shimadzu 2010EV LCMS using the following conditions:
Kromisil C18 column (reverse phase, 4.6 mm ꢀ 50 mm); a linear
gradient from 10% acetonitrile and 90% water to 95% acetonitrile and
5% water over 4.5 min; flow rate of 1 mL/min; and UV photodiode array
detection from 200 to 300 nm. High-resolution ESI-TOF mass spectra
were acquired at the Center for Mass Spectrometry at The Scripps
Research Institute (La Jolla, CA).
General Methods for the Synthesis of Benzoisothiazolone
PMI Inhibitors. General Method A. To a stirred solution of the amine
(900 mg, 5.95 mmol) in CH2Cl2 at 0 °C under nitrogen, AlMe3 (6 mL, 2
M in THF) was added dropwise, and the reaction was slowly warmed to
room temperature. The mixture was stirred continuously for an addi-
tional 30 min. Methyl thiosalicylate (500 mg, 2.97 mmol) was added,
and the reaction was heated to 60 °C and heated under reflux overnight.
The reaction was quenched with HCl (5% aq.), and CH2Cl2 was added
(50 mL). The organic layer was separated and washed with saturated
NaHCO3 solution, then brine, and dried over Na2SO4. The solvents
were removed by rotary evaporation, and the products were isolated by
flash chromatography or reverse phase HPLC and lyophilized to provide
the final compounds, which were determined to be >95% pure by
HPLC-UV, HPLC-MS, and 1H NMR.
General Method B. To a crimp top microwave vial was added the
benzoisothiazolone (76 mg, 0.5 mmol), aryl-X (1.05 mmol), K2CO3
(138 mg, 1.0 mmol), CuI (20 mol %), and DMEDA (20 mol %) in
dioxane (5 mL). The reaction mixture was heated in the microwave at
195 °C for 7 min. Following filtration and evaporation of solvents, the
products were isolated by flash chromatography or reverse phase HPLC
and lyophilized to provide the final compounds, which were determined
to be >95% pure by HPLC-UV, HPLC-MS, and 1H NMR.
2-Phenyl-2-hydrobenzo[d]isothiazol-3-one (1). Compound
1 was prepared according to general procedure A (67%). 1H NMR (300
MHz, CDCl3): δ 7.32 (m, 1H), 7.51 (m, 3H), 7.57 (m, 1H), 7.67 (m,
3H), 8.09 (m, J = 7.9, 1H). 13C NMR (100 MHz, CDCl3): 120.0, 124.6,
125.8. 127.0, 127.2, 129.3, 132.3, 137.2, 139.9, 164.1. ESI-MS m/z 227
[M þ H]þ. HRMS m/z calcd for C13H9NOS [M þ H]þ, 228.0405;
found, 228.0522.
’ EXPERIMENTAL SECTION
Compound Collection Utilized in HTS. The compound library
was supplied by the MLSMR (http://www.mli.nih.gov/mlsmr). The
MLSMR, funded by the NIH, is responsible for the selection of small
molecules for HTS screening, their purchase and QC analysis, library
maintenance, and distribution within the NIH Molecular Libraries
Screening Center Network (MLPCN, http://www.mli.nih.gov/mlpcn).
Both MLSMR and MLPCN are parts of the Molecular Libraries
Initiatives (MLI, http://nihroadmap.nih.gov/molecularlibraries) under
the NIH Roadmap Initiative (www.nihroadmap.nih.gov). MLSMR
compounds are acquired from commercial and in part from academic
and government sources and are selected based on the following criteria:
Samples are available for resupply in 10 mg quantities, are at least 90%
pure, have acceptable physicochemical properties, and contain no
functional groups or moieties, which are known to generate artifacts
in HTS (http://mlsmr.glpg.com/). Compounds are selected to repre-
sent diversified chemical space with clusters of closely related analogues
around them to aid in the HTS-based SAR analysis.
In Vitro Assays. For concentrationꢁresponse assays, 9 μL of the
substrate solution was dispensed into 384-well black plates (Greiner
784076), and either the compound (2 μL in 10% DMSO) or the 10%
DMSO alone was added for the test and control wells, respectively. The
reaction was initiated with 9 μL of the enzyme solution. Fluorescence in
the rhodamine channel (excitation, 544 nm; emission, 590 nm; dichroic
mirror, 561 nm) was measured on a Molecular Devices Analyst HT after
a 20 min incubation at room temperature.
2-(3-Methylphenyl)-2-hydrobenzo[d]isothiazol-3-one (3).
Compound 3 was prepared according to general procedure B (22%). 1H
NMR (300 MHz, CDCl3): δ 2.40 (s, 3H), 7.12 (d, J = 7.3, 1H), 7.33 (t,
J = 7.9, 1H), 7.49 (m, 4H), 7.62 (m, 1H), 8.08 (d, J = 7.9, 1H). ESI-MS
m/z 241 [M þ H]þ. HRMS m/z calcd for C14H11NOS [M þ H]þ,
242.0561; found, 242.0672.
2-(4-Methylphenyl)-2-hydrobenzo[d]isothiazol-3-one (4).
Compound 4 was prepared according to general procedure B (6%). 1H
NMR (300 MHz, CDCl3): δ 2.37 (s, 3H). 7.26 (m, 3H), 7.42 (m, 1H),
7.55 (m, 2H), 7.64 (m, 1H), 8.08 (m, 1H). ESI-MS m/z 241 [M þ H]þ.
HRMS m/z calcd for C14H11NOS [M þ H]þ, 242.0561; found,
242.0673.
The PMI substrate solution contained 50 mM Hepes, pH 7.4, 0.4 mM
Man-6-P, 1.6 IU/mL diaphorase, and 0.2 mM resazurin. The PMI enzyme
solution contained 50 mM Hepes, pH 7.4, 0.5 mM NADPþ, 10 mM
MgCl2, 4.6 μg/mL phosphoglucose isomerase, 2 μg/mL glucose-6-
phosphate dehydrogenase, 30 ng/mL PMI, and 0.011% Tween 20
(PubChem AID 1209). In the primary screening, the same assay was
also performed in the presence of saturating PMI substrate concentra-
tions. Both working solutions had the composition as above, except for
2 mM Man-6-P in the substrate solution (PubChem AID 1220).
The PMM2 substrate solution contained 50 mM Hepes, pH 7.4,
0.17 mM Man-1-P, 0.4 IU/mL diaphorase, and 56 μM resazurin. The
PMM2 enzyme solution contained 50 mM Hepes, pH 7.4, 10 mM
MgCl2, 1.1 mMNADPþ, 0.11 mMR-D-glucose 1,6-bisphosphate, 5 μg/mL
phosphoglucose isomerase, 3.33 μg/mL PMI, 2 μg/mL glucose-6-phosphate
dehydrogenase, 1.67 μg/mL PMM2, and 0.011% Tween 20 (PubChem AID
1655). Man-6-P and Man-1-P were omitted from the positive control wells
for PMI and PMM2, respectively. The IC50 values were determined using
nonlinear regression data analysis according to the Hill equation.
2-[3-(Trifluoromethyl)phenyl]-2-hydrobenzo[d]isothiazol-
3-one (5). Compound 5 was prepared according to general procedure B
(15%). 1H NMR (300 MHz, CDCl3): δ 7.46 (t, J = 7.9, 1H), 7.58 (m,
3H), 7.68 (m, 1H), 7.93 (d, J = 7.9, 1H), 8.01 (s, 1H), 8.10 (d, J = 7.9,
1H). ESI-MS m/z 295 [M þ H]þ. HRMS m/z calcd for C14H8F3NOS
[M þ H]þ, 296.0279; found, 296.0391.
General Synthetic Procedures. All solvents and chemicals used
were purchased from Sigma-Aldrich, Acros, or Chembridge and were
used as received without further purification. Purity and characterization
of compounds were established by a combination of LCMS and NMR
analytical techniques and was >95% for all tested compounds. Silica gel
2-[4-(Trifluoromethyl)phenyl]-2-hydrobenzo[d]isothiazol-
3-one (6). Compound 6 was prepared according to general procedure B
(27%). 1H NMR (300 MHz, CDCl3): δ 7.59 (m, 5H), 7.90 (m, 2H), 8.10
(d, J = 7.3, 1H). ESI-MS m/z 295 [M þ H]þ. HRMS m/z calcd for
C14H8F3NOS [M þ H]þ, 296.0279; found, 296.0390.
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2-(3-Chlorophenyl)-2-hydrobenzo[d]isothiazol-3-one (7).
Compound 7 was prepared according to general procedure B (15%). 1H
NMR (300 MHz, CDCl3): δ 7.26 (m, 1H), 7.42 (m, 2H), 7.62 (m, 3H),
7.78 (m, 1H), 8.09 (m, 1H). ESI-MS m/z 261 [M þ H]þ. HRMS m/z
calcd for C13H8ClNOS [M þ H]þ, 262.0015; found, 262.0092.
2-(4-Chlorophenyl)-2-hydrobenzo[d]isothiazol-3-one (8).
Compound 8 was prepared according to general procedure B (12%). 1H
NMR (300 MHz, CDCl3): δ 7.43 (m, 3H), 7.57 (d, J = 7.9, 1H), 7.65
(m, 3H), 8.05 (d, J = 7.9, 1H). ESI-MS m/z 261 [M þ H]þ. HRMS m/z
calcd for C13H8ClNOS [M þ H]þ, 262.0015; found, 262.0098.
2-[4-(Dimethylamino)phenyl]-2-hydrobenzo[d]isothiazol-
3-one (12). Compound 12 was prepared according to general proce-
dure B (38%). 1H NMR (300 MHz, CDCl3): δ 2.97 (s, 6H), 6.75 (m,
2H), 7.42 (m, 3H), 7.54 (m, 1H), 7.60 (m, 1H), 8.07 (d, J = 7.3, 1H). 13C
NMR (100 MHz, CDCl3): 40.5, 112.5, 120.0, 124.8, 125.4, 125.5, 126.8,
127.0, 131.9, 140.2, 149.9, 164.4. ESI-MS m/z 270 [M þ H]þ. HRMS
m/z calcd for C15H14N2OS [M þ H]þ, 271.0827; found, 271.078.
2-(3-Iodophenyl)-2-hydrobenzo[d]isothiazol-3-one (13).
Compound 13 was prepared according to general procedure B (18%).
1H NMR (300 MHz, CDCl3): δ 7.17 (t, J = 7.9, 1H), 7.44 (m, 1H), 7.66
(m, 4H), 8.08 (m, 2H). 13C NMR (300 MHz, CDCl3): 94.1, 120.1,
123.6, 125.0, 126.0, 127.3, 130.7, 132.6, 133.0, 136.0, 138.3, 139.6,
164.0. ESI-MS m/z 352 [M þ H]þ. HRMS m/z calcd for C13H8INOS
[M þ H]þ, 353.9371; found, 353.9492.
2H), 7.77 (dd, J = 2.4, 7.9, 1H). 13C NMR (100 MHz, CDCl3): 113.0,
113.3, 121.0, 121.3, 121.6, 121.7, 124.6, 127.3, 129.4, 135.1, 137.0, 160.1,
162.5. ESI-MS m/z 273 [M þ H]þ. HRMS m/z calcd for C15H12FNOS
[M þ H]þ, 274.0624; found, 274.0739.
5-Fluoro-2-(4-fluorophenyl)-2-hydrobenzo[d]isothiazol-
3-one (20). Compound 20 was prepared according to general proce-
dure A (65%). 1H NMR (300 MHz, CDCl3): 7.76 (dd, J = 2.4, 7.9, 1H),
7.67 (m, 2H), 7.54 (m, 1H), 7.44 (m, 3H), 7.31 (m, 1H). 13C NMR (100
MHz, CDCl3): 40.5, 112.5, 120.0, 124.8, 125.4, 125.5, 126.8, 127.0,
131.9, 140.2, 149.9, 164.3. ESI-MS m/z 245 [M þ H]þ. HRMS m/z
calcd for C13H8FNOS [M þ H]þ, 246.0311; found, 246.0395.
5-Fluoro-2-(4-fluorophenyl)-2-hydrobenzo[d]isothiazol-
3-one (21). Compound 21 was prepared according to general proce-
dure A (33%). 1H NMR (300 MHz, CDCl3): δ 7.15 (m, 2H), 7.42 (m,
1H), 7.54 (m, 1H), 7.62 (m, 2H), 7.76 (dd, J = 2.4, 7.9, 1H). ESI-MS
m/z 263 [M þ H]þ. HRMS m/z calcd for C13H7F2NOS [M þ H]þ,
264.0216; found, 264.0299.
5-Fluoro-2-(4-methoxyphenyl)-2-hydrobenzo[d]isothia-
zol-3-one (22). Compound 22 was prepared according to general
procedure A (80%). 1H NMR (300 MHz, CDCl3): δ 3.83 (s, 3H), 6.97
(m, 2H), 7.41 (m, 1H), 7.52 (m, 3H), 7.76 (dd, J = 2.4, 7.9, 1H). 13C
NMR (100 MHz, CDCl3): 55.6, 113.0, 113.2, 114.6, 126.8, 129.4, 135.2,
159.0, 162.4. ESI-MS m/z 275 [M þ H]þ. HRMS m/z calcd for
C14H10FNO2S [M þ H]þ, 276.0416; found, 276.0499.
5-Fluoro-2-(4-fluorophenyl)-2-hydrobenzo[d]isothiazol-
3-one (23). Compound 23 was prepared according to general proce-
dure A (47%). 1H NMR (300 MHz, CDCl3): 8.12 (m, 1H), 7.67
(m, 1H), 7.58 (m, 1H), 7.44 (m, 3H), 7.22 (m, 1H). ESI-MS m/z 263
[M þ H]þ. HRMS m/z calcd for C13H7F2NOS [M þ H]þ, 264.0216;
found, 264.0404.
2-[4-(tert-Butyl)phenyl]-2-hydrobenzo[d]isothiazol-3-one
(14). Compound 14 was prepared according to general procedure B
(42%). 1H NMR (300 MHz, CDCl3): δ 1.33 (s, 9H), 7.46 (m, 3H), 7.61
(m, 4H), 8.10 (d, J = 7.9, 1H). 13C NMR (100 MHz, CDCl3): 31.3, 34.7,
120.0, 124.4, 125.7, 126.3, 127.1, 132.2, 134.4, 150.3, 164.2. ESI-MS m/z
283 [M þ H]þ. HRMS m/z calcd for C17H17NOS [M þ H]þ,
284.1031; found, 284.1131.
2-(4-Chlorophenyl)-5-fluoro-2-hydrobenzo[d]isothiazol-
3-one (24). Compound 24 was prepared according to general proce-
dure A (55%). 1H NMR (300 MHz, CDCl3): δ 7.42 (m, 3H), 7.54 (m,
1H), 7.63 (m, 2H), 7.76 (dd, J = 2.4, 7.9, 1H). ESI-MS m/z 278 [M þ
H]þ. HRMS m/z calcd for C13H7ClFNOS [M þ H]þ, 279.9921; found,
280.0017.
2-(3-Methoxyphenyl)-2-hydrobenzo[d]isothiazol-3-one
(15). Compound 15 was prepared according to general procedure B
(12%). 1H NMR (300 MHz, CDCl3): δ 3.84 (s, 3H), 6.81 (m, 1H), 7.25
(m,1H), 7.36 (m, 2H), 7.43 (m, 1H), 7.57 (m, 1H), 7.65 (m, 1H), 8.09
(m, 1H). 13C NMR (100 MHz, CDCl3): 55.5, 110.1, 113.1, 116.5, 120.0,
125.0, 125.8, 127.1, 130.0, 132.4, 138.3, 140.0, 160.2, 164.1. ESI-MS m/z
257 [M þ H]þ. HRMS m/z calcd for C17H17NO2S [M þ H]þ,
258.0510; found, 258.0622.
5-Fluoro-2-(3-fluorophenyl)-2-hydrobenzo[d]isothiazol-
3-one (25). Compound 25 was prepared according to general proce-
1
dure A (60%). H NMR (300 MHz, CDCl3): δ 7.02 (m, 1H), 7.43
(m, 3H), 7.54 (m, 2H), 7.75 (dd, J = 2.4, 7.9, 1H). ESI-MS m/z 263
[M þ H]þ. HRMS m/z calcd for C13H7F2NOS [M þ H]þ, 264.0216;
found, 264.0305.
2-(2,5-Dimethylphenyl)benzo[d]isothiazol-3(2H)-one (16).
Compound 16 was prepared according to general procedure B (18%).
1H NMR (300 MHz, CDCl3): δ 2.18 (s, 3H), 2.35 (s, 1H), 7.19 (m,
3H), 7.43 (m, 1H), 7.56 (m, 1H), 7.65 (m, 1H), 8.09 (d, J = 7.93, 1H).
ESI-MS m/z 256 [M þ H]þ.
6-Fluoro-2-o-tolylbenzo[d]isothiazol-3(2H)-one (26). Com-
1
pound 26 was prepared according to general procedure A (55%). H
NMR (300 MHz, CDCl3): δ 2.17 (s, 3H), 2.33 (s, 3H), 7.18 (m, 3H),
7.41 (m, 1H), 7.53 (m, 1H), 7.78 (dd, J = 2.4, 7.9, 1H). ESI-MS m/z 273
[M þ H]þ. HRMS m/z calcd for C15H12FNOS [M þ H]þ, 274.0624;
found, 274.0734.
2-(2,5-Dimethylphenyl)-6-fluorobenzo[d]isothiazol-3(2H)-
one (17). To a stirred solution of the 4-fluoro-2-mercaptobenzoic acid
(10 mmol) in ethyl acetate was added 2-propanephosphonic acid
anhydride (30 mmol), and this mixture was stirred at room temperature
for 15 min. 2,5-Dimethylaniline (10 mmol) and N-methylmorpholine
(120 mmmol) were added, and the reaction was stirred at room
temperature for 3 h, quenched with ethyl acetate, and washed with
5% potassium sulfate solution, saturated sodium bicarbonate solution,
and brine, and dried over sodium sulfate. The organic phase was
concentrated in vacuo and purified by reverse-phase preparative liquid
2-Phenyl-1H-2-hydroindazol-3-one (27). A solution of
o-nitrobenzaldehyde (242 mg, 1 mmol) in methanol (3 mL) was added
to sodiumhydroxidein water(4mL) together withzinc dust. The resulting
reaction mixture was then heated under reflux for 15 h and filtered while
hot. The filtrate was concentrated to half volume and cooled. Any
unreacted material was removed by filtration. The filtrate was diluted with
water and acidified with dilute HCl. The crude product precipitated was
collected by filtration and further purified by column chromatography
using hexanes:ethyl acetate to afford 0.076 g (36%) of indazolone as a pale
yellow solid. 1H NMR (300 MHz, CDCl3): δ 2.24 (s, 3H), 2.31 (s, 3H),
7.19 (m, 7H), 7.55 (m, 1H), 7.89 (m, 1H). 13C NMR (100 MHz, CDCl3):
17.6, 20.7, 112.3, 118.5, 122.6, 124.2, 127.8, 129.8, 130.9, 132.1, 132.9,
134.8, 136.4, 147.1, 161.7. ESI-MS m/z 238 [M þ H]þ. HRMS m/z calcd
for C15H14N2O [M þ H]þ, 239.1106; found, 239.1205.
1
chromatography to afford the product in 40% yield. H NMR (300
MHz, CDCl3): δ 2.19 (s, 3H), 2.35 (s, 3H), 7.18 (m, 5H), 8.09 (m, 1H).
ESI-MS m/z 274 [M þ H]þ.
6-Fluoro-2-(4-methoxyphenyl)benzo[d]isothiazol-3(2H)-
one (18). 1H NMR (500 MHz, (CD3)2SO): δ 2.18 (s, 3H), 2.35 (s,
1H), 7.19 (m, 3H), 7.43 (m, 1H), 7.56 (m, 1H), 7.65 (m, 1H), 8.09 (d,
J = 7.9, 1H). ESI-MS m/z 276 [M þ H]þ.
5-Fluoro-2-phenylbenzo[d]isothiazol-3(2H)-one (19). Com-
pound 19 was prepared according to general procedure A (51%). 1H NMR
(300 MHz, CDCl3): δ7.33 (m, 1H), 7.45 (m, 3H), 7.54 (m, 1H), 7.67 (m,
2-(2,5-Dimethylphenyl)isoindolin-1-one (28). To a stirred
solution of the phthalaldehyde (250 mg, 1.85 mmol) in CH3CN:DMF
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Journal of Medicinal Chemistry
ARTICLE
was added the amine (230 μL, 1.85 mmol) followed by TMSCl (188 μL,
1.48 mmol) and was stirred at room temperature overnight (62%). H
blood samples were collected at several time points (po: 0.5, 1, 2, 4, and
6 h; IV: 10 min and 0.5, 1, 2, 4, and 6 h). Plasma was separated from the
red cells by centrifugation and frozen at ꢁ20 °C until assayed.
Compound 19 was extracted from the plasma samples using a 2-fold
excess of acetonitrile, and the extracts were vortexed for 3 min and then
centrifuged for 15 min (7500 g) after which the supernatants were
collected to be assayed by LCMS. PK data were analyzed using Prism
and Excel.
1
NMR (300 MHz, CDCl3): δ 2.18 (s, 3H), 2.31 (s, 3H), 4.70 (s, 2H), 7.18
(m, 2H), 7.52 (m, 3H), 7.93 (m, 1H). ESI-MS m/z 237 [M þ H]þ.
HRMS m/z calcd for C16H15NO [M þ H]þ, 238.1154; found, 238.1276.
Permeability Assay. The capacity of compounds to cross cellular
membranes was determined using a parallel artificial membrane permea-
tion assay (PAMPA).7 Briefly, the effective permeability of the com-
pounds was measured at an initial concentration of 25 μM. The buffer
solution (pH 7.4) was prepared from a concentrated stock available from
pION and according to the manufacturer's protocol. The compound of
interest was dissolved in buffer solution or water (in the case of cargos
alone, evaluated by HPLC-MS) and acetonitrile (20%, cosolvent) to the
desired concentration. The sandwich plate was separated, and the donor
well was filled with 200 μL of the compound solution of interest. The
acceptor plate was placed into the donor plate, ensuring that the
underside of the membrane was in contact with buffer. Four microliters
of the mixture of phospholipids (20 mg/mL) in dodecane was added to
the filter of each well, followed by 200 μL of buffer solution. The plate
was covered and incubated at room temperature in a saturated humidity
atmosphere for 4 h under orbital agitation at 100 rpm. The concentra-
tions of the compound remaining in the donor well, diffused through the
membrane and into the acceptor well, and reference compounds were
measured by LC/MS/MS. Results were classified as high, medium, or
low predicted absorption. Test articles were run in triplicate at one
concentration (25 μM), and permeability was assessed at one time point
(4 h) and pH (7.4). The membrane consisted of phosphatidylcholine in
dodecane on a Multiscreen PVDF membrane (0.45 μm). The positive
control was verapamil (high permeability), and the negative control was
theophylline (low permeability).
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 858-646-3100. Fax: 858-795-5225. E-mail: ncosford@
sanfordburnham.org.
’ ACKNOWLEDGMENT
This work was supported by NIH Grant HG003916, by The
Rocket Williams Fund, and by NIH Grants R01DK55615 and
R21HD062914 to H.H.F., who is a Sanford Center Professor.
’ ABBREVIATIONS USED
PMI, phosphomannose isomerase; PMM2, phosphomanno-
mutase 2; CDG, congenital disorders of glycosylation; HTS,
high-throughput screening; ADME, absorption, distribution,
metabolism, excretion; PK, pharmacokinetic; i.v., intravenous;
p.o., per os; HK, hexokinase;Man, mannose; Man-6-P, mannose-
6-phosphate; Man-1-P, mannose-1-phosphate;Fru-6-P, fructose-
6-phosphate; MLSMR, NIH Molecular Libraries Small Molecule
Repository; PIFA, phenyliodine bis(trifluoroacetate); MLPCN,
NIH Molecular Libraries Probe Production Centers Network; GSH,
glutathione; LCMS, liquid chromatographyꢁmass spectrometry
Plasma and Microsomal Stability Assays. Test articles were
run in duplicate in the presence of either fresh plasma (mouse) or
0.5 mg/mL liver microsomes (mouse) at one concentration of test
compound (10 μM). Aliquots were taken at two time points, t = 0 h and
t = 1 h (microsomes) or t = 3 h (plasma). LC/MS/MS measurement of
remaining parent compound as % parent remaining at a specific time
point was assessed. For microsomal stability, the positive control was
formation of acetaminophen from phenacetin, and the negative control
was an NADPH-deficient test mixture.
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