5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)indolin-2-one Analogs as Inhibitors of Acid alpha-Glucosidase for Potential Chaperone Treatment of Pompe Disease or Intervention for Diabetes Mellitus Type 2

Xiao J, Marugan JJ, Zheng W, et al.

Publication Details

In the field of Lysosomal Storage Disorders, small molecule chaperones are a promising new therapeutic approach that are able to impact the accumulation of functional mutant in the Endoplasmic Reticulum, increasing translocation and ameliorating the disease phenotype. Here, we present the discovery, structure activity relationship (SAR) and initial biological results of a new series of acid alpha glucosidase non-iminosugar inhibitors with chaperone capacity, as exemplified by ML201. In addition, these compounds could potentially be useful for Diabetes type 2 treatment.

Screening Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Assay Submitter & Institution: Wei Zheng, NIH Chemical Genomics Center

PubChem Summary Bioassay Identifier (AID): 1473

Probe Structure & Characteristics

ML201.

ML201

Recommendations for Scientific Use of the Probe

Acid alpha-glucosidase is an enzyme that catalyzes the exohydrolysis of 1,4-alpha-glucosidic linkages to release glucose. Alpha-glucosidase inhibitors are not only useful for limiting the impact of carbohydrate consumption on blood glucose levels in patients with diabetes mellitus type 2, but may also be useful as small molecule chaperones for correcting the misfolding and mistrafficking of mutant alpha-glucosidase in Pompe disease. Herein, we describe a new class of inhibitors of alpha-glucosidase based upon a substituted 5-(4-phenylpiperazin-1-ylsulfonyl)indolin-2-one scaffold identified from a qHTS campaign. We investigate the synthesis of these agents, structure activity relationships (SAR), analysis of activity using a functional, thermal destabilization assay and ADME properties, including in vivo pharmacokinetic analysis of the probe compound. These agents represent a novel non-iminosugar chemotype of inhibitors of alpha-glucosidase, and may be useful as chaperones of acid alpha-glucosidase for the treatment of Pompe disease or as enzyme inhibitors in the context of diabetes mellitus disorder type 2.

1. Introduction

Pompe disease, also called glycogen storage disease type II or acid maltase deficiency, is an autosomal recessive disorder caused by the deficiency or dysfunction of the lysosomal enzyme acid alpha-glucosidase (GAA)1. Epidemiological studies have estimated its frequency to be 1 in every 40,000 births1,2. GAA can hydrolyze terminal alpha-1,4- and alpha-1,6-glucosidic linkages of glycogen in the lysosome. Mutations in this enzyme result in lysosomal enlargement due to glycogen accumulation. Accumulation is especially severe in cardiac and skeletal muscle, affecting breathing and mobility1. The only FDA-approved treatment for children with this disease is currently enzyme replacement therapy (Myozyme), which is recombinant GAA produced in a Chinese hamster ovary cell line3,4. Although Myozyme has been proven to be clinically efficacious, the development of infusion related reactions is common. Furthermore, the majority of the patients (89%) test positive for IgG antibodies to acid alpha-glucosidase, reducing clinical utility5. In addition, some health plans have refused to subsidize Myozyme for adult patients because the treatment is not approved for adults, and it also has a high cost ($300,000/yr for life). Commonly observed adverse side effects to Myozyme treatment are pneumonia, respiratory complications, infections, and fever. More serious reactions reported include heart, lung failure, and allergic shock6. These findings reinforce the need to develop new treatments for Pompe disease.

There are more than 100 different GAA mutations that can induce Pompe disease symptoms7,8. Most of the mutants reported in literature retain enzymatic activity in vitro, but are not transported to the lysosome. These proteins accumulate in the endoplasmic reticulum (ER), presumably due to an inability to fold properly or because they do not acquire the necessary shape to be recognized for transportation to the lysosome9,10. A general strategy for lysosomal storage diseases (LSDs) has been to search for small molecule chaperones that are able to bind to these mutant enzymes and assist in their folding and transport to the lysosome. Improved trafficking of the mutant protein between the ER and the lysosome can reduce lysosome size and correct the phenotype11. In that sense, potentially, the majority of GAA mutant could benefit from the development of proper small molecule chaperones. Paradoxically, all of the small molecule chaperones reported in the literature are iminosugar inhibitors of the enzyme (Figure 1). One of these, 1-deoxynojirimycin (DNJ), is currently being studied under a phase II clinical trial as a chaperone therapeutic agent for Pompe disease by Amicus Therapeutics Inc12,13. Iminosugars are problematic due to poor selectivity and their small therapeutic window between improving translocation and inhibiting enzyme activity11,14. Therefore, alternative non-iminosugar inhibitory series with chaperone activity are highly desired.

Figure 1. Prior art: known alpha-glucosidase inhibitors.

Figure 1

Prior art: known alpha-glucosidase inhibitors.

Therapeutically, inhibitors of alpha glucosidase are also used as oral anti-diabetic drugs for the treatment of diabetes mellitus type 213. For this indication, there are three FDA approved alpha glucosidase inhibitors, all of them containing a sugar-based motif: acarbose (Precose), miglitol (Glyset) and voglibose (Figure 1). All of them act through the same mechanism, although there are some differences in clinical use among them. Acarbose is an oligosaccharide and not well-absorbed. Moreover, acarbose inhibits pancreatic alpha-amylase in addition to alpha-glucosidase. Miglitol resembles a monosaccharide and is well-absorbed by the body. Voglibose has a better side effect profile than both of the others, but it is less efficacious than acarbose. None of these inhibitors of alpha glucosidase have been shown to have chaperone capacity, or utility in the treatment of Pompe disease.

At the NCGC, we have developed several new screening methodologies to identify novel non-iminosugar series with activity in LSD assays. We have focused on testing enzymes in a context that is as native as possible, including testing the hydrolytic capacity of GAA in tissue homogenate16. Many isolated glucosidases require allosteric activation to be functional17,18, so we wanted to avoid using purified enzyme preparations, which depend on detergents to induce the active conformation and catalytic activity of the enzyme. We have observed that it is common to find compounds that can inhibit isolated enzymes but are inactive in cellular lysates. This is likely due to differences between detergent-induced enzymatic systems and physiological enzyme in cells or problems with non-specific protein binding. Another limitation of reconstituted assays is an inability to detect enzyme activators, presumably because the detergent used in reconstituted assays activates 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-glucopyranoside16. Upon hydrolysis, the blue fluorescent dye 4-methylumbelliferone is liberated, producing a fluorescent emission at 440 nm when excited at 370 nm. As a control for autofluorescence, we also used a second substrate, resorufin α-D-glucopyranoside, which liberates the red dye resorufin at an emission wavelength of 590 nm when excited at 530 nm (Figure 2).

Figure 2. Hydrolytic reactions of the red and blue dyes.

Figure 2

Hydrolytic reactions of the red and blue dyes.

2. Materials and Methods

Unless otherwise stated, all reactions were carried out under an atmosphere of dry argon or nitrogen in dried glassware. Indicated reaction temperatures refer to those of the reaction bath, while room temperature (RT) is noted as 25°C. All solvents were of anhydrous quality, purchased from Aldrich Chemical Co. and used as received. Commercially available starting materials and reagents were purchased from Aldrich, TCI and Acros and were used as received. Analytical thin layer chromatography (TLC) was performed with Sigma Aldrich TLC plates (5 × 20 cm, 60 Å, 250 μm). Visualization was accomplished by irradiation under a 254 nm UV lamp. Chromatography on silica gel was performed using forced flow (liquid) of the indicated solvent system on Biotage KPSil pre-packed cartridges and using the Biotage SP-1 automated chromatography system. 1H NMR spectra were recorded on a Varian Inova 400 MHz spectrometer. Chemical shifts are reported in ppm with the solvent resonance as the internal standard (CDCl3 7.27 ppm, DMSO-d6 2.49 ppm, for 1H NMR). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, quin = quintet, br = broad, m = multiplet), coupling constants, and number of protons. Low resolution mass spectra (electrospray ionization) were acquired on an Agilent Technologies 6130 quadrupole spectrometer coupled to an Agilent Technologies 1200 series HPLC. The HPLC retention times were recorded through standard gradient 4% to 100% acetonitrile (0.05% TFA) over 7 minutes using a Luna C18 3 micron 3 × 75 mm column with a flow rate of 0.800 ml/min. High resolution mass spectral data were collected in-house using an Agilent 6210 time-of-flight mass spectrometer, also coupled to an Agilent Technologies 1200 series HPLC system.

2.1. Assays

AID 1466: qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-pyranoside as the substrate and human alpha-glucosidase as the enzyme preparation. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 1, 4-methyllumbelliferone, can be excited at 365 nm and emits at 440 nm, which can be detected by a standard fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50mM citric acid (titrated with potassium phosphate to pH 5.0), 0.005% Tween-20, pH 5.0 (pH 5.0 is an optimal condition for this enzyme assay).

See qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease Assay Protocol in Appendix 1.

AID 2112: qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase from Spleen Homogenate

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-glucopyranoside as the substrate and human spleen homogenate containing alpha-glucosidase as the enzyme preparation. Upon hydrolysis of this fluorogenic substrate, the resulting product, 4-methylumbelliferone (which excites at 365 nm and emits at 440 nm) can be detected by a fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50 mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20 (pH 5.0 is an optimal condition for this enzyme assay).

See qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase from Spleen Homogenate Assay Protocol in Appendix 1.

AID 2100: qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase Cleavage of Glycogen

This is an enzyme assay using glycogen from bovine liver (Sigma catalog #: G0885) as the substrate and recombinant human alpha-glucosidase as the enzyme preparation. Upon hydrolysis of the substrate, the glucose product can be detected using the Amplex Red Glucose Oxidase Assay Kit (Invitrogen catalog #: A22189). The product of this reaction can be read with a fluorescence plate reader with an excitation at 573 nm and an emission at 610 nm. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer for enzyme reaction: 50mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20 (pH 5.0 is an optimal condition for this enzyme assay). Assay buffer for Amplex Red reaction: Tris-HCl, pH 7.5.

See qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase Cleavage of Glycogen Assay Protocol in Appendix 1.

AID 2101: qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate and N370S glucocerebrosidase from human spleen homogenate as the enzyme preparation. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 4-methyllumbelliferone, can be excited at 365 nm and emits at 440 nm, which can be detected by a standard fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50 mM citric acid (titrated with potassium phosphate to pH 5.0), 100 mM potassium chloride, 10 mM sodium chloride, 1 mM magnesium chloride, 0.01% Tween-20.

See qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease Assay Protocol in Appendix 1.

AID 1467: qHTS Assay for Inhibitors of Human alpha-Galactosidase at pH 4.5

This is a fluorogenic enzyme assay with 4-Methylumbelliferyl alpha-D-galactopyranoside as the substrate and human alpha-galactolucosidase as the enzyme preparation. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 4-Methyllumbelliferone, can be excited at 365 nm and emits at 440 nm. This fluorescence can be detected by a standard fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50mM citric acid (titrated with potassium phosphate to pH 4.5), 0.005% Tween-20, pH 4.5. (pH 4.5 is an optimal condition for this enzyme assay).

See qHTS Assay for Inhibitors of Human alpha-Galactosidase at pH 4.5 Assay Protocol in Appendix 1.

AID 2107: qHTS Assay for Inhibitors and Activators of Human alpha-Galactosidase from Spleen Homogenate

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-galactopyranoside as the substrate and human spleen homogenate containing alpha-galactosidase as the enzyme preparation. Upon hydrolysis of this fluorogenic substrate, the resulting product, 4-methylumbelliferone (which excites at 365 nm and emits at 440 nm) can be detected by a fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20 (pH 5.0 is an optimal condition for this enzyme assay).

See qHTS Assay for Inhibitors and Activators of Human alpha-Galactosidase from Spleen Homogenate Assay Protocol in Appendix 1.

AID 2115: Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-pyranoside as the substrate and human alpha-glucosidase as the enzyme preparation. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 4-methyllumbelliferone, can be excited at 365 nm and emits at 440 nm, which can be detected by a standard fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50mM citric acid (titrated with potassium phosphate to pH 5.0), 0.005% Tween-20, pH 5.0. (pH 5.0 is an optimal condition for this enzyme assay).

See Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase Assay Protocol in Appendix 1.

AID 2113: Confirmation of Inhibitors and Activators of Human alpha-Glucosidase from Spleen Homogenate

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-glucopyranoside as the substrate and human spleen homogenate containing alpha-glucosidase as the enzyme preparation. Upon hydrolysis of this fluorogenic substrate, the resulting product, 4-methylumbelliferone (which excites at 365 nm and emits at 440 nm) can be detected by a fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20 (pH 5.0 is an optimal condition for this enzyme assay).

See Confirmation of Inhibitors and Activators of Human alpha-Glucosidase from Spleen Homogenate Assay Protocol in Appendix 1.

AID 2110: Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase Using an Alternate Red Fluorescent Substrate

This is a fluorogenic enzyme assay with resorufin-alpha-D-pyranoside as the substrate and recombinant human alpha-glucosidase as the enzyme preparation. Upon the hydrolysis of this fluorogenic substrate, the resulting product, resorufin, can be excited at 573 nm and emits at 610 nm, which can be detected by a standard fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50 mM citric acid (titrated with potassium phosphate to pH 5.0), 0.005% Tween-20, pH 5.0. (pH 5.0 is an optimal condition for this enzyme assay).

See Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase Using an Alternate Red Fluorescent Substrate Assay Protocol in Appendix 1.

AID 2111: Confirmation of Inhibitors and Activators of Human alpha-Glucosidase From Spleen Homogenate Using an Alternate Red Fluorescent Substrate

This is a fluorogenic enzyme assay with Resorufin-alpha-D-glucopyranoside as the substrate and human spleen homogenate containing alpha-glucosidase as the enzyme preparation. Upon hydrolysis of this fluorogenic substrate, the resulting product, resorufin (which excites at 573 nm and emits at 610 nm) can be detected by a fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20 (pH 5.0 is an optimal condition for this enzyme assay).

See Confirmation of Inhibitors and Activators of Human alpha-Glucosidase From Spleen Homogenate Using an Alternate Red Fluorescent Substrate Assay Protocol in Appendix 1.

AID 2108: Confirmation of Inhibitors of Human alpha-Galactosidase Using Spleen Homogenate

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-galactopyranoside as the substrate and human spleen homogenate containing alpha-galactosidase as the enzyme preparation. Upon hydrolysis of this fluorogenic substrate, the resulting product, 4-methylumbelliferone (which excites at 365 nm and emits at 440 nm) can be detected by a fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20 (pH 5.0 is an optimal condition for this enzyme assay).

See Confirmation of Inhibitors of Human alpha-Galactosidase Using Spleen Homogenate Assay Protocol in Appendix 1.

AID 2109: Confirmation of Inhibitors and Activators of Purified Human alpha-Galactosidase

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-pyranoside as the substrate and human alpha-glucosidase as the enzyme preparation. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 4-methyllumbelliferone, can be excited at 365 nm and emits at 440 nm, which can be detected by a standard fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation. Assay buffer: 50mM citric acid (titrated with potassium phosphate to pH 5.0), 0.005% Tween-20, pH 5.0. (pH 5.0 is an optimal condition for this enzyme assay).

See Confirmation of Inhibitors and Activators of Purified Human alpha-Galactosidase Assay Protocol in Appendix 1.

AID 2641: qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Functional, Thermal Destabilization Assay of Alpha-Glucosidase

This assay involves heating purified acid alpha-glucosidase (Myozyme) in the presence of inhibitors to observe potential stabilization of the enzyme. This binding assay is an indirect measure of chaperone activity, as stabilization may imply proper folding and trafficking of the enzyme to its functional site. Alpha-glucosidase was pre-incubated with either DMSO or 50-fold IC50 of compound before being exposed to 68°C heat inactivation. The enzyme’s activity was measured over time, and it was observed that untreated alpha-glucosidase lost more of its activity over time than enzyme treated with compounds. These results demonstrate that compound binding stabilizes alpha-glucosidase against thermal denaturation, and imply that these compounds may help promote folding and trafficking of the enzyme to the lysosomes. Temperature Destabilization Protocol for alpha-Glucosidase. Assay buffer: 50mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20.

See qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Functional, Thermal Destabilization Assay of Alpha-Glucosidase Assay Protocol in Appendix 1.

Caco-2 Permeability and Microsomal Stability

Detailed protocols are given in Appendix 1. Briefly, Analytical signal was optimized for each compound. Samples were analyzed by LC/MS/MS. CaCo-2 cells grown under standard tissue culture conditions. The test agent was alternately added to the apical (A) side and amount of permeation was determined on the basolateral (B) side; for Basolateral to Apical (B>A) permeability, the test agent was added to the B side and the amount of permeation was determined on the A side. For microsomal stability testing, the test agent is incubated in duplicate with microsomes at 37 ºC. The samples are centrifuged to remove precipitated protein, and the supernatants are analyzed by LC/MS/MS to quantitate the remaining parent. Data are reported as % remaining by dividing by the time zero concentration value.

Single Oral Dose Pharmacokinetic Study in Male Swiss Albino Mice

ML201 (1, see Table 1) was dosed orally to mice, and plasma and intestine concentrations were determined pre-dose and at 0, 5, 15, 30, 60, 120, 240, 480, 720, and 1440 minutes, with 3 animals per time point. Male Swiss Albino mice used in this experiment were procured from National Institute of Nutrition (NIN), Hyderabad, India. Animals were acclimatized for three days in an animal holding room. Test article ML201 (1) was dissolved in DMA, TEG, and water for injection in the ratio of 20:40:40, and vortexed. Compound was administered orally using stainless steel gavage needles at a dose level of 30 mg/kg at dose volume of 10 ml/kg. After dosing of each animal, animals were observed for any abnormal behavioral signs exhibited after drug administration.

Table 1. Activity of analogs with modifications on the aromatic ring attached to the piperazine.

Table 1

Activity of analogs with modifications on the aromatic ring attached to the piperazine.

Each mouse was anesthetized using isoflurane. Blood was collected through a capillary, guided into the retro-orbital plexus. The blood samples were collected in pre-labeled heparin coated tubes (BD, cat. No.365965). 0.3 ml of blood was collected from each mouse at their respective time points. After collection of blood samples at each time point, the blood samples were stored on wet ice prior to centrifugation. Blood samples were centrifuged within 15 minutes to separate plasma at 5000 rpm, 4°C for 10 minutes. The plasma was separated and transferred to pre-labeled tubes and promptly frozen at −80 ± 10°C until the bio-analysis could take place. Immediately after blood withdrawal for PK estimation, a laparotomy was performed on the animal to expose the abdominal cavity and collect the small intestine. The abdomen of the mouse was exposed, the pyloric part of stomach was grasped with a pair of curved forceps and duodenum was severed. The ileum was cut from the ileocaecal junction in the same manner. The intestine was then flushed with ice cold saline to flush out all the intestinal contents. Separated intestine was dried on a tissue paper and immediately weighed and freezed at −80 ± 10°C until homogenization. PK parameters are calculated for mean concentration by the non-compartmental model, trapezoid rule (linear interpolation method) using WinNonlin Software Version 4.1.

2.2. Probe Chemical Characterization

Structural verification information for the probe, ML201

  • 5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)indolin-2-one (ML201). 1H 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).
  • LC/MS (Agilent system): Retention time t1 (long) = 4.671 min;
  • HRMS (ESI): m/z calcd for C20H22N3O4S [M+H+] 400.1331, found 400.1327;
  • Purity: UV220 > 95%, UV254 > 95%; MS m/z 400.1 (M+H);
  • Column: 3 x 75 mm Luna C18, 3 micron;
  • Run time: 4.671 min (long);
  • Gradient: 4% to 100% over 7 min;
  • Mobile phase: Acetonitrile (0.05% TFA), water (0.05% TFA);
  • Flow rate: 0.800ml;
  • Temperature: 50°C;
  • UV wavelength: 220 nm, 254 nm.

Summary of probe properties

ML201 is soluble at 10mM concentration in DMSO. The compound is not fluorescent at blue excitation wavelengths (~340 nm).

Solubility: 20 μM (7.9 μg/ml) in phosphate buffered saline (PBS), pH 7.4, at room temperature (23°C). Experimental Log D 1.70.

Stability in phosphate buffered saline (PBS) at room temperature (23ºC): The probe is stable in PBS over 48 hrs. (Figure 3).

Figure 3. Stability of ML201 over time.

Figure 3

Stability of ML201 over time.

  • PubChem CID: 20969430; MLS#: MLS001030468; SID: 49731601
  • Molecular Weight: 399.46
  • Molecular Formula: C20H21N3O4S
  • ClogP: 2.0501
  • H-Bond Donor: 1.0
  • H-Bond Acceptor: 4.0
  • Rotatable Bond Count: 4.0
  • Exact Mass: 399.1253
  • Topological Polar Surface Area: 86.79
  • IUPAC Name: 5-(4-(4-acetylphenyl)piperazin-1-ylsulfonyl)indolin-2-one

Canonical SMILES

O=C1NC2=CC=C(S(N3CCN(C4=CC=C(C(C)=O)C=C4)CC3)(=O)=O)C=C2C1

MLSMR Numbers for Probe and Analogs

2.3. Probe Preparation

Schemes 1 and 2 show the general methodology used for the synthesis of analogs with modifications in several areas of the molecule. Direct chlorosulfonylation of 2-indolinone at the 5-position followed by piperazine displacement yielded analogs with modifications on the aromatic ring attached to the piperazine. The para-methyl ester group was readily converted to the corresponding carboxylic acid or methyl alcohol via saponification or DIBAL reduction (Scheme 1). 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, or its reaction with carboxylic acid under the EDC coupling condition, gave analogs with modifications at the indolinone ring or sulfonamide portion of the molecule, respectively (Scheme 2).

Scheme 1. Synthesis of analogs with modifications on the aromatic ring attached to the piperazine.

Scheme 1

Synthesis of analogs with modifications on the aromatic ring attached to the piperazine.

Scheme 2. Synthesis of analogs with modifications at indoline ring or sulfonamide portion of the molecule.

Scheme 2

Synthesis of analogs with modifications at indoline ring or sulfonamide portion of the molecule.

A detail synthetic methodology of all described analogs can be found in Appendix 2.

3. Results

244,319 compounds were screened16, with a Z’ across the entire run of 0.82 ± 0.04. Only one non-iminosugar inhibitor series confirmed out of the cherry-picks selected for confirmation. MLS001030468 (CID 20969430/ML201, 1, see table 1), the primary representative of the series, was cherry-picked from the MLSMR for confirmation based on selectivity against the related enzyme alpha-galactosidase. After its activity was confirmed, it was ordered from a commercial vendor and re-synthesized in-house. This series inhibits purified acid alpha-glucosidase activity, with similar activity in tissue homogenate assays. The compound was not auto-fluorescent by spectral profiling and did not inhibit the related enzymes acid alpha-galactosidase and glucocerebrosidase (acid beta-glucosidase). In addition, several compounds of the series were further validated in a functional thermal destabilization assay.

3.1. Summary of Screening Results

Due to concerns that the recombinant, purified assay might be missing important protein factors, and the previous difficulty in translating hits against other purified lysosomal targets into hits in a cellular context, the primary screen was formulated to measure alpha-glucosidase specific activity in tissue homogenate. This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-glucopyranoside as the substrate and human tissue homogenate containing alpha-glucosidase as the enzyme preparation. Upon hydrolysis of this fluorogenic substrate, the resulting product, 4-methylumbelliferone (which excites at 365 nm and emits at 440 nm) can be detected by a fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The IC50 values were determined from concentration-response data modeled with the standard Hill equation. Signal to background ratios above 40 are expected. Z's for the screen average 0.8.

Select compounds were also assayed using a red-fluorescent substrate, which is used to assess whether the observed activity is uniquely substrate dependent. The hit from the primary screen and its analogs all showed very similar activity with both the blue-fluorescent and red-fluorescent substrates, and also between the purified enzyme assay and the assay run in tissue homogenate, indicating robust activity. The hit series was also screened against the related enzymes acid alpha-glactosidase and glucocerebrosidase (acid beta-glucosidase) for selectivity. The compounds from this series were completely inactive at the highest concentrations tested in these assays and are selective inhibitors of acid alpha-glucosidase.

ML201 was further assayed using purified enzyme with its native substrate, glycogen. Glycogen from bovine liver was used as the substrate and recombinant human alpha-glucosidase as the enzyme preparation. Upon hydrolysis of the substrate, the glucose product can be detected using the Amplex Red Glucose Oxidase Assay Kit, the product of which can be read with a fluorescence plate reader. The compound had a potency of 330 nM in this assay, consistent with its activity in the primary screening assay.

In addition to the previously described secondary assays, we tested compounds’ ability to prevent loss of enzyme function following thermal destabilization of alpha-glucosidase (Figure 3). Briefly, hydrolytic enzymes lose their catalytic activity over time after exposure to elevated temperatures below their melting point. This is due to progressive denaturation and/or aggregation of the protein from solution. Warming acid alpha glucosidase to 66°C for 60 minutes reduces enzyme activity by about 75%. Compounds that bind to the enzyme may prevent this loss of activity. Compounds that prevent thermal destabilization have also been shown to promote cellular folding and translocation, and therefore have the potential capacity of being chaperone molecules9a. Results for ML201 shown in Figure 4 demonstrate that this compound can stabilize alpha-glucosidase.

Figure 4. Thermal stabilization of alpha-glucosidase functional activity following incubation at destabilizing temperatures.

Figure 4

Thermal stabilization of alpha-glucosidase functional activity following incubation at destabilizing temperatures. Time indicates length of incubation at 66°C. Ratio is the ratio of enzymatic activity after incubation at 66°C compared (more...)

3.2. Dose Response Curves for Probe

Figure 5. Concentration response curve for probe ML201 and the iminosugar DNJ, CID 1374 (SID 50111045), in the primary screening assay for acid alpha-glucosidase activity using tissue homogenate.

Figure 5Concentration response curve for probe ML201 and the iminosugar DNJ, CID 1374 (SID 50111045), in the primary screening assay for acid alpha-glucosidase activity using tissue homogenate

IC50 for probe ML201 is 520 +/− 60nM based on triplicate data.

3.3. Scaffold/Moiety Chemical Liabilities

This scaffold does not have any functional groups with potential chemical liabilities. Metabolic liabilities will be commented on in the in vitro ADME part of Section 3.6.

3.4. SAR Tables

Tables 1 to 3 disclose the SAR of all synthesized analogs with modifications at three areas of the probe ML201 in the tissue homogenate assay of acid alpha-glucosidase using two different pro-fluorescent substrates – the blue-shifted dye substrate that was used in the primary screen and a red-shifted dye substrate. The inhibitory activity of analogs was consistent in assays with both blue and red-shifted dye substrates. Table 1 shows that a broad range of substituents were tolerated within the aromatic ring attached to the piperazine portion of the molecule, such as hydroxyl (analog 5, IC50 = 1.88 μM), cyano (analog 7, IC50 = 2.91 μM), nitro (analog 8, IC50 = 3.66 μM), aldehyde (analog 14, IC50 = 3.66 μM) and carboxylic acid (analog 16, IC50 = 1.30 μM) functional groups. Other functional groups like halo, methyl and trifluoromethyl were inactive or had much less activity, which indicated that the oxygen or nitrogen at the para-position of the phenyl group involved in hydrogen bonding interactions is essential for activity. In addition, the methylketone moiety contributed more towards activity than the phenyl ring itself (analog 3, IC50 = 11.87 μM).

Table 3. Activity of analogs with modifications on sulfonamide and piperazine moieties.

Table 3

Activity of analogs with modifications on sulfonamide and piperazine moieties.

Surprisingly, the SAR around indolin-2-one was found to be very narrow (Table 2). Most modifications at the indolin-2-one ring portion yielded inactive compounds, such as increasing the size of the aliphatic portion of the indolin-2-one (analogs 25 and 26), introducing heteroatoms in the 3-position (analogs 1821, 24, 31) or eliminating of the aliphatic ring to obtain the acetamide analogue (analogs 2730). The only modification on this part of the molecule that did not have a deep impact on the activity was the introduction of a chlorine substituent on the 6 position of the indolinone ring (analog 22, IC50 = 1.30 μM). Furthermore, analog 22 later showed a strong ability to stabilize acid alpha glucosidase from thermal destabilization similar to the probe compound ML201 (1) (figure 6). The 3,3-dichloro substituted indolin-2-one analog (23, IC50 = 32.61 μM) was also active, but much less potent.

Table 2. Activity of analogs with modifications on the indolin-2-one ring.

Table 2

Activity of analogs with modifications on the indolin-2-one ring.

Figure 6. Capacity of inhibitors to maintain the function of acid alpha glucosidase after incubation at 66°C for 60 minutes.

Figure 6

Capacity of inhibitors to maintain the function of acid alpha glucosidase after incubation at 66°C for 60 minutes.

Table 3 shows the replacement of sulfonamide with carboxylic amide (analogs 32, 33) and a reversed phenyl and piperazine ring analog (34). Unfortunately, all 3 analogs were inactive. Therefore, the sulfonamide moiety also contributes significantly to the activity of this series.

The ability of our best inhibitors to stabilize acid alpha glucosidase from thermal destabilization was evaluated to further validate their potential as chaperone molecules. As described above, this assay measured the ability of our probe and several analogs to prevent the loss of function when the enzyme was incubated at 66°C for 60 minutes (Figure 6). In the absence of compound, the enzyme lost most of its hydrolytic capacity within 1 hour. In contrast, in the presence of certain compounds, the enzyme was able to maintain its function. The stabilization observed correlates directly with the inhibitory activity of the compounds in the inhibition assay. In other words, the best inhibitors were the best stabilizers. These data indicated that our inhibitors were able to stabilize the conformational structure of acid alpha glucosidase and therefore, they may be useful as chemical chaperones although this activity had not yet been directly demonstrated for these compounds in cells.

3.5. Cellular Activity

For measuring the capacity of our compounds to activate ER-lysosome translocation of GAA, we follow the previously described protocol by treating Pompe fibroblast with potential chaperone compounds for five days, followed by immunostaining (GAA and Cathepsin D as lysosomal marker), repetitive wash and image analysis.

Pompe fibroblasts are difficult to obtain, and in general, they are of slow growth and can only be expanded for a few passages. For this reason, an enzyme activity assay was performed to confirm the Pompe status of our patient fibroblasts (Pompe A and B cells). Both cell lines have the mutations p.Y455C/p.G638W and are derived from the same patient. Figure 7 shows that the activity of this particular mutant is around 3% of the displayed by GAA wt.

Figure 7. GAA specific activity of this patient having p.Y455C/p.G638W mutations.

Figure 7

GAA specific activity of this patient having p.Y455C/p.G638W mutations.

Although analysis of the specific activity of GAA in fibroblast (skin biopsies) is the method currently used for Pompe diagnosis, in general, fibroblasts do not express GAA in abundant amounts, and with increased passage number, a decrease in GAA expression is observed. Figure 8 shows this observation.

Figure 8. Images of WT fibroblasts treated with DMSO at passage 7 (left panels) and passage 8 (right panels).

Figure 8

Images of WT fibroblasts treated with DMSO at passage 7 (left panels) and passage 8 (right panels). Levels of GAA decrease with each cell passage.

Immunostaining imaging assays shows the undetectable levels of GAA in patient fibroblast. GAA signal was never observed in the lysosomes of fibroblasts from Pompe Disease patients, while GAA could be observed in 5% of the WT cells (passage 8, Figure 9).

Figure 9. Images of WT fibroblasts (left panels) and Pompe fibroblasts (right panels) treated with DMSO show no detectable levels of GAA in the lysosom of patient cells.

Figure 9

Images of WT fibroblasts (left panels) and Pompe fibroblasts (right panels) treated with DMSO show no detectable levels of GAA in the lysosom of patient cells.

DNJ is an non-selective iminosugar currently in clinical trials for Pompe Disease. The chaperone activity of DNJ is GAA mutant dependent, and there is no previous literature data indicating whether DNJ is active for treating p.Y455C/p.G638W GAA mutations. We tested the chaperone effect of DNJ in GAA translocation in patient fibroblasts and were not able to detect any activity (Figure 10) at concentrations 1, 5, and 15 μM. At 20 μM, translocation of GAA to the lysosomes occurred in 3% of the cells. The images in Figure 10 are representative of the data obtained in the experiment. DNJ did also not show translocation of GAA at concentrations 1, 5, and 15 μM. At 20 μM, translocation of GAA to the lysosomes occurred in 3% of the cells. As it was disclosed in Figure 9, none of the Pompe cells showed GAA translocation with DMSO treatment. These values are in the same range as those reported for other GAA mutants, where DNJ increased GAA translocation at 20 μM. Opposite to other reported iminosugars of lysosomal hydrolases, DNJ does not increase the translocation of wt GAA (data not shown).

Figure 10. DNJ did not show GAA translocation activity in Pompe fibroblasts (left panels are untreated and right panels are DNJ 20 μM).

Figure 10

DNJ did not show GAA translocation activity in Pompe fibroblasts (left panels are untreated and right panels are DNJ 20 μM).

Three compounds (ML201 [probe], CID 36649951 and CID 44825300) of our analogs were selected for further evaluation in the translocation assay using fibroblasts with p.Y455C/p.G638W GAA mutations. Figure 11 shows our results.

Figure 11. Representative image of p.Y455C/p.G638W GAA fibroblast treated with selective compounds.

Figure 11

Representative image of p.Y455C/p.G638W GAA fibroblast treated with selective compounds.

All of the compounds were toxic for cells carrying the p.Y455C/p.G638W mutations. At low concentration (1 μM), some of the cells survived but they looked very sick (big holes in the cells are indicated by the arrows in Figure 11 and enlarged lysosomes). One potential explanation is that the GAA activity of this cell line with p.Y455C/p.G638W mutations is very low (Figure 7), and therefore the addition of an inhibitor could completely eliminate the function of the enzyme, having an impact in cell survival. To test this hypothesis, we decided to evaluate the same compounds in wild type fibroblast. Our probe molecule (ML201) seemed to be toxic even for wild type, although the other compounds provided better results (Figures 12 and 13).

Figure 12. Treatment of wt fibroblast with 15 μM (left panel) and of 5 μM (right panel) CID 36649951 (compound 22, table 2).

Figure 12

Treatment of wt fibroblast with 15 μM (left panel) and of 5 μM (right panel) CID 36649951 (compound 22, table 2).

Figure 13. Treatment of wt fibroblast with 15 μM (first row, left panel), of 5 μM (first row, right panel) and 1 μM (second row, right panel) of CID 44825300 (compound 32, table 3).

Figure 13

Treatment of wt fibroblast with 15 μM (first row, left panel), of 5 μM (first row, right panel) and 1 μM (second row, right panel) of CID 44825300 (compound 32, table 3).

Figure 12 shows that CID 36649951 (compound 22, table 2) upregulates Cathepsin D and translocates wt GAA to lysosomes, increasing the percent of cells having GAA in the lysosome (from 15% to 30%). At the same time, the size of lysosomes increase, which generally is not considered healthy for a cell.

Figure 13 shows that CID 44825300 (compound 32, table 3) upregulates Cathepsin D and translocates wt GAA to lysosomes, increasing the percent of cells having GAA in the lysosome (from 15% to 50%) without increasing the size of lysosomes. Therefore, this compound could be considered an excellent chaperone for wt GAA. Interestingly, this compound does not disclose any inhibitory activity against wt GAA in our functional assay (Table 3), probing that the inhibitory activity and the translocation capacity do not correlate directly.

Although numerous inhibitors of lysosomal hydrolases have been disclosed as having chaperone activity, the capacity of a molecule to block the action of an enzyme does not always correlate with its ability to induce and accelerate the folding and translocation. Hence, inhibitors exist without chaperone activity and vice-versa. If a particular compound series demonstrates both an inhibitory activity and an ability to enhance translocation, compounds with the higher inhibitory activity normally also show greater translocation capacity. In these cases, lower IC50 values imply higher affinity toward the enzyme and correlate with a greater capacity to induce a folded conformation and better translocation ability. Still, within a series of inhibitors, it is possible to increase the binding affinity of the compound for the enzyme without incrementing its inhibitory capacity; therefore, the best chaperone molecules within a series do not necessarily have to be the most potent inhibitors. From the therapeutic point of view, an ideal chaperone series should increase the translocation of the protein without inhibiting its enzymatic function. Such non-inhibitory small molecule chaperones for GAA has not yet been described in the literature. For this reason, our results with CID 44825300 are particularly exciting. Currently, we are evaluating the action of this molecule in another GAA mutant that has greater specific activity, with the hope that we observe translocation without affecting cell viability. We will update this probe report incorporating that data as soon we complete those studies.

3.6. Profiling Assays

No profiling assays have been completed for this project yet, aside from data already shown in this report.

In vitro ADME

As an initial assessment of the permeability and metabolic stability of the series, we analyzed the probe compound ML201 in microsomal stability and Caco-2 permeability assays. Table 4 shows that the probe compound ML201 has a low intrinsic clearance and a long in vitro half life, with more than 60% of the mean parent compound remaining after one hour of incubation with mouse microsomes. The transformation that does occur is NADPH-dependent, which indicates that the major metabolic process is mostly through the cytochrome P450 dependent oxidation.

Table 4. Mouse microsomal stability assays at 60 minutes.

Table 4

Mouse microsomal stability assays at 60 minutes. ML201 shown as last row in table.

Caco-2 data in Table 5 indicates that this probe compound ML201 displayed very good cell permeability in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)), However, the B-A permeability was higher than the corresponding A-B permeability (efflux ratio = 1.9, [B-A/A-B]). This slight preference indicates active efflux by P-glycoprotein and other ABC transporters, although the value of the efflux ratio is reasonable and the permeability in every direction is high, therefore expecting in vivo saturation of the efflux transport.

Table 5. Caco-2 permeability assays.

Table 5

Caco-2 permeability assays. ML201 shown as last row in table

In vivo Pharmacokinetic Analysis

Subsequently to the in vitro ADME assays, full pharmacokinetic analysis of probe compound ML201 was carried out. The plasma and intestine concentrations of ML201 in male Swiss Albino Mice after single oral gavage administration of ML201 at a dose of 30 mg/kg was measured. As data shows in Table 6, the probe compound 1 displayed a very high concentration in intestine, greater than it was in plasma, confirming our hypothesis from the Caco-2 data. The pharmacokinetic parameters of ML201 (1) in plasma and intestine were calculated and AUC(0-t) of plasma and intestine used for determination of intestine to plasma ratio are summarized in Table 7. The intestine to plasma ratio of ML201 (1) in male Swiss Albino Mice was found to be 78.7. ML201 possesses a reasonably long intestine half-life (t1/2 = 5.04 h) in comparison with its half-life in plasma (t1/2 = 0.51 h). The concentration of ML201 in intestine reached 47.65 μmol/kg in only 5 minutes and 156.35 μmol/kg as Cmax in 30 minutes (IC50 of 1 was 0.52 μM). In addition, no behavioral changes or toxicity signs were observed in animals administered with test compound throughout the whole study period. Overall, this means that upon 30 mg/kg oral gavage administration, our probe displays intestine concentrations above its acid alpha-glucosidase IC50 for more than 24 hrs; therefore, this compound is an excellent candidate for diabetes II treatment. Regarding its potential capacity for the treatment of Pompe’s disease, the compound reaches levels in plasma above its IC50 for a short period of time. Due to the permeability and efflux in vitro values and its microsome stability, we were expecting a higher exposure in vivo and maintaining higher concentrations in plasma for a longer period of time. We are suspecting a high in vivo secretion value, but additional pharmacokinetic experiments should be carried out to corroborate this idea.

Table 6. Mice plasma and intestine levels for probe compound ML201 following oral gavage administration at 30 mg/kg in mice.

Table 6

Mice plasma and intestine levels for probe compound ML201 following oral gavage administration at 30 mg/kg in mice.

Table 7. Mice pharmacokinetic parameters for probe compound ML201 following oral gavage administration at 30 mg/kg.

Table 7

Mice pharmacokinetic parameters for probe compound ML201 following oral gavage administration at 30 mg/kg.

The therapeutic utility of a small molecule chaperone with inhibitory capacity depends on its IC50 and its pharmacokinetics. In that sense, therapeutically useful chaperones must have a low association constant, and therefore a low inhibitory activity that allow their displacement by the natural substrate. High inhibitory binders may increase the translocation, but they do not allow the function of the enzyme, having no therapeutic utility. Moreover, beside the Kd, the displacement equilibrium between the natural substrate and the inhibitor, also depends on the concentration of the inhibitor. For that reason, useful chaperone inhibitors could have a relatively short half life, allowing their elimination and restoring the function of the enzyme upon translocation. Our probe molecule, ML201, has a relatively low inhibitory activity (IC50 = 0.52 μM) and low half life in plasma. It remains to be seen if this short half life is good enough to produce a therapeutic effect in Pompe’s models in vivo. Prior to its evaluation in such a model, we need to complete our cell-based translocation experiments to have an idea if the chaperone capacity of our molecule correlates with its inhibitory capacity. In addition, we are testing other molecules of our series in the Caco-2 permeability model to see if any of them show lower reflux, and whether any of them have the potential to reach higher levels in plasma and a longer half life in plasma. Potentially, those compounds could be better or back-up candidates for Pompe model evaluation.

In addition, it would be of interest to carry out pharmacokinetic experiments with our non-inhibitory analog CID 44825300, if we confirm its chaperone capacity with other GAA mutants.

4. Discussion

In summary, a series of non-iminosugar acid acid-glucosidase inhibitors were identified from a qHTS campaign, with ML201 being the primary representative of the series. This series of compounds inhibited acid alpha-glucosidase, using both tissue homogenate and purified enzyme assays using its native substrate. The compounds were not auto-fluorescent by spectral profiling and did not inhibit the related enzymes alpha-galactosidase and beta-glucosidase (glucocerebrosidase). In addition, several compounds of the series had shown activity in the functional, thermal destabilization assay. The synthesis of these agents, structure activity relationships (SAR), structure property relationships (SPR) analysis, and ADME properties were demonstrated. Initial studies with several of our compounds disclosed that our non-inhibitor analog CID 44825300 seems to be a good chaperone for wt GAA. Further studies with other mutant are in progress. Additionally, our probe molecule ML201 may be potentially useful for the treatment of the enzyme involved in diabetes type 2 disorder.

4.1. Comparison to existing art and how the new probe is an improvement

As previously mentioned in the introduction, currently described alpha-glucosidase inhibitors were based on iminosugar scaffold with variety of side effects. The current series is a novel non-iminosugar chemotype with very good selectivity versus other glycosidases. In addition, our initial studies demonstrated that the probe compound ML201 possesses several properties as a promising diabetes type 2 candidate.

4.2. Mechanism of Action Studies

Inhibitors disclosed in this report are molecules that act as competitive inhibitors of acid alpha- glucosidase. Our probe was able to inhibit the hydrolytic capacity of acid alpha-glucosidase both in purified in vitro assays as well as in tissue homogenate assays. Furthermore, this probe is able to prevent the loss of function of this enzyme in a functional thermo-shift assay.

4.3. Planned Future Studies

The future studies for this project can be divided into two sections:

  1. Development of the current probe ML201 as a candidate for the treatment of diabetes type 2 disorder:
    The current probe compound ML201 is an excellent candidate for further studies for diabetes type 2 disorder, and a diabetes type 2 disorder animal studies need to be planned. This study will require the scale-up synthesis of 50–100 grams of probe ML201 for long term study in the daily diets for mice.
  2. Development of cell chemical chaperones of acid alpha-glucosidase for the treatment of Pompe disease:
    Further development will require a compound with good chaperone capacity. For that reason, first we have to complete our Pompe translocation studies and have a sense of the AC50 of our compound in that model. Moreover, we are doing several additional Caco-2 analyses of other potent compounds, such as analogs 5, 7, 8, 16, 22, for testing their potential capacity to have better absorption and plasma half life. If necessary, more SAR studies will be explored, specifically addressing reflux. Finally, selective compounds should be evaluated in a mouse model of Pompe disease.

We will update this probe report once additional data become available.

5. References

1.
Hirschhorn R, Reuser AJJ. Glycogen storage disease type II: acid α-glucosidase (acid maltase) deficiency. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. McGraw–Hill; New York: 2001. pp. 3389–3420.
2.
Martiniuk F, Chen A, Mack A, Arvanitopoulos E, Chen Y, Rom WN, Codd WJ, Hanna B, Alcabes P, Raben N, Plotz P. Am J Med Genet. 1998;79:69–72. [PubMed: 9738873]
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Kishnani PS, Corzo D, Nicolino M, Byrne B, Mandel H, Hwu WL, Leslie N, Levine J, Spencer C, McDonald M, Li J, Dumontier J, Halberthal M, Chien YH, Hopkin R, Vijayaraghavan S, Gruskin D, Bartholomew D, van der Ploeg A, Clancy JP, Parini R, Morin G, Beck M, De la Gastine GS, Jokic M, Thurberg B, Richards S, Bali D, Davison M, Worden MA, Chen YT, Wraith JE. Neurology. 2007;68:99–109. [PubMed: 17151339]
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Raben N, Plotz P, Byrne BJ. Curr Mol Med. 2002;2:145–166. [PubMed: 11949932]
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Montalvo AL, Cariati R, Deganuto M, Guerci V, Garcia R, Ciana G, Bembi B, Pittis MG. Mol Genet Metab. 2004;81:203–208. [PubMed: 14972326]
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Hermans MM, van Leenen D, Kroos MA, Beesley CE, Van der Ploeg AT, Sakuraba H, Wevers R, Kleijer W, Michelakakis H, Kirk EP, Fletcher J, Bosshard N, Basel-Vanagaite L, Besley G, Reuser AJ. Hum Mutat. 2004;23:47–56. [PubMed: 14695532]c. Reuser AJ, Kroos M, Willemsen R, Swallow D, Tager JM, Galjaard H. J Clin Invest. 1987;79:1689–1699. [PMC free article: PMC424503] [PubMed: 3108320]d. Reuser AJ, Kroos M, Oude Elferink RP, Tager JM. J Biol Chem. 1985;260:8336–8341. [PubMed: 3159730]
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Beck M. Human Genetics. 2007;121:1–22. [PubMed: 17089160]
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Parenti G, Zuppaldi A, Pittis GM, Tuzzi MR, Annunziata I, Meroni G, Porto C, Donaudy F, Rossi B, Rossi M, Filocamo M, Donati A, Bembi B, Ballabio A, Andria G. Mol Ther. 2007;15:508–514. [PubMed: 17213836]
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Okumiya T, Kroos MA, Vliet LV, Takeuchi H, Van der Ploeg AT, Reuser AJ. Mol Genet Metab. 2007;90:49–57. [PubMed: 17095274]
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Fan JQ, Ishii S, Suzuki Y. The FEBS J. 2007;274:4962–4971. [PubMed: 17894781]
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Motabar O, Shi Z, Goldin E, Liu K, Southall N, Sidransky E, Austin CP, Griffiths GL, Zheng Z. Anal Biochem. 2009;390:79–84. [PMC free article: PMC2737366] [PubMed: 19371716]
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John M, Wendeler M, Heller M, Sandhoff K, Kessler H. Biochemistry. 2006;45:5206–5216. [PubMed: 16618109]
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Zwerschke W, Mannhardt B, Massimi P, Nauenburg S, Pim D, Nickel W, Banks L, Reuseri AJ, Jansen-Dürr P. J Biol Chem. 2000;27(5):9534–9541. [PubMed: 10734102]

Appendix 1. Assay Protocols

qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease

1536-well assay protocol:

  1. Add 2 μl/well of enzyme (4nM final)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.7 nM to 77 μM.
  3. Add 1 μl of substrate (400 μM final)
  4. Incubate at room temperature for 20 min
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm

qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase from Spleen Homogenate

1536-well assay protocol:

  1. Add 2 μl/well spleen homogenate (1 μg)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.5 nM to 58 μM.
  3. Add 2 μl of substrate (1mM final)
  4. Incubate at 37°C for 40 min
  5. Add 2μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm

qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase Cleavage of Glycogen

1536-well assay protocol:

  1. Add 2 μl/well alpha-glucosidase enzyme solution (4 nM final)
  2. Add 23nl compounds in DMSO solution. The final titration was 0.7 nM to 77 μM.
  3. Add 1 μl of glycogen substrate solution (30 μg)
  4. Incubate at 37°C for 40 min
  5. Add 2 μl Tris-HCl buffer with Amplex Red reagents
  6. Incubate 45 min at room temperature
  7. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 573 nm and Em = 610 nm

qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease

1536-well assay protocol:

  1. Add 2 μl/well of spleen homogenate (27 μg final)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.5 nM to 58 μM.
  3. Add 2 μl of substrate (1 mM final)
  4. Incubate at 37°C for 40 min.
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm

qHTS Assay for Inhibitors of Human alpha-Galactosidase at pH 4.5

1536-well assay protocol:

  1. Add 2 μl/well of enzyme (12 nM final)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.7 nM to 77 μM.
  3. Add 1 μl of substrate (80μM final)
  4. Incubate at room temperature for 20 min.
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440nm

qHTS Assay for Inhibitors and Activators of Human alpha-Galactosidase from Spleen Homogenate

1536-well assay protocol:

  1. Add 2 μl/well spleen homogenate (1 μg)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.5 nM to 58 μM.
  3. Add 2 μl of substrate (1 mM final)
  4. Incubate at 37°C for 40 min
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440nm

Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase

1536-well assay protocol:

  1. Add 2 μl/well of enzyme (4 nM final)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.7 nM to 77 μM.
  3. Add 1 μl of substrate (400 μM final)
  4. Incubate at room temperature for 20 min.
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm

Confirmation of Inhibitors and Activators of Human alpha-Glucosidase from Spleen Homogenate

1536-well assay protocol:

  1. Add 2 μl/well spleen homogenate (1 μg)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.5 nM to 58 μM.
  3. Add 2 μl of substrate (1 mM final)
  4. Incubate at 37°C for 40 min
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm

Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase Using an Alternate Red Fluorescent Substrate

1536-well assay protocol:

  1. Add 2 μl/well of enzyme (4 nM final)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.7 nM to 77 μM.
  3. Add 1 μl of substrate (400 μM final)
  4. Incubate at room temperature for 20 min.
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm.

Confirmation of Inhibitors and Activators of Human alpha-Glucosidase From Spleen Homogenate Using an Alternate Red Fluorescent Substrate

1536-well assay protocol:

  1. Add 2 μl/well spleen homogenate (1 μg)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.5 nM to 58 μM.
  3. Add 2 μl of substrate (1 mM final)
  4. Incubate at 37°C for 40 min
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm

AID 2108: Confirmation of Inhibitors of Human alpha-Galactosidase Using Spleen Homogenate

1536-well assay protocol:

  1. Add 2 μl/well spleen homogenate (1 μg)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.5 nM to 58 μM.
  3. Add 2 μl of substrate (1 mM final)
  4. Incubate at 37°C for 40 min
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm

Confirmation of Inhibitors and Activators of Purified Human alpha-Galactosidase

1536-well assay protocol:

  1. Add 2 μl/well of enzyme (4 nM final)
  2. Add 23 nl compounds in DMSO solution. The final titration was 0.7 nM to 77 μM.
  3. Add 1μl of substrate (400 μM final)
  4. Incubate at room temperature for 20 min.
  5. Add 2 μl stop solution (1 M NaOH and 1 M Glycine mixture, pH 10)
  6. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm

qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Functional, Thermal Destabilization Assay of Alpha-Glucosidase

  1. Prepare 80-fold solution of alpha-glucosidase solution (3.2 nM * 80 = 255 nM) in assay buffer
  2. Divide solution into separate 1.5 ml tubes
  3. For each compound, add 50-fold IC50: 50 * IC50 of 1-DNJ = 2.5 μM * 50-fold IC50 of the test compounds = 50 μM
  4. Incubate 10 min room temperature after compound addition
  5. Distribute 10 μl from each tube into PCR tubes
  6. Time course of heating: Heat solutions at 68°C for 60 min, taking a set of tubes out at 10, 20, 30, 45, and 60 min, while keeping one set on ice the whole time.
  7. After heating, take out tubes and place on ice for a minimum of 5 min.
  8. Transfer 10 μl solution to 790 μl (1:80 dilution) buffer in 1.5 ml tube.
  9. Vortex and take three 20 μl aliquots and put in 384-well plate
  10. Add 20 μl substrate solution to each well (100μM 4-methylumbelliferyl alpha-D-glucopyranoside)
  11. Incubate 20 min.
  12. Add 25 μl stop solution (1M NaOH, 1M glycine pH 10)
  13. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex = 365 nm and Em = 440 nm

Caco-2 Permeability and Microsomal Stability

Analytical signal was optimized for each compound by ESI positive or negative ionization mode. A MS2 SIM scan was used to optimize the precursor ion and a product ion analysis was used to identify the best fragment for analysis and to optimize the collision energy. Samples were analyzed by LC/MS/MS using either an Agilent 6410 mass spectrometer coupled with an Agilent 1200 HPLC and a CTC PAL chilled autosampler, all controlled by MassHunter software (Agilent), or an ABI2000 mass spectrometer coupled with an Agilent 1100 HPLC and a CTC PAL chilled autosampler, all controlled by Analyst software (ABI). After separation on a C18 reverse phase HPLC column (Agilent, Waters, or equivalent) using an acetonitrile-water gradient system, peaks were analyzed by mass spectrometry (MS) using ESI ionization in MRM mode.

CaCo-2 cells grown in tissue culture flasks were trypsinized, suspended in medium, and the suspensions were applied to wells of a collagen-coated BioCoat Cell Environment in 24-well format (BD Biosciences) at 24,500 cells/well. The cells are allowed to grow and differentiate for three weeks, feeding at 2-day intervals.

For Apical to Basolateral (A->B) permeability, the test agent is added to the apical (A) side and amount of permeation is determined on the basolateral (B) side; for Basolateral to Apical (B>A) permeability, the test agent is added to the B side and the amount of permeation is determine on the A side. The A side buffer contains 100μM Lucifer yellow dye, in Transport Buffer (1.98 g/l glucose in 10mM HEPES, 1x Hank’s Balanced Salt Solution) pH 6.5, and the B side buffer is Transport Buffer, pH 7.4. CaCo-2 cells were incubated with these buffers for 2 hrs, and the receiver side buffer was removed for analysis by LC/MS/MS. To verify that CaCo-2 cell monolayers were properly formed, aliquots of the cell buffers were analyzed by fluorescence to determine the transport of the impermeable dye Lucifer Yellow.

Dataare expressed as permeability (Papp):Papp=dQ/dtC0A

where dQ/dt is the rate of permeation, C0 is the initial concentration of test agent, and A is the area of the monolayer. In bi-directional permeability studies, the asymmetry index (AI) or efflux ratio is also calculated:

AI=Papp(BA)Papp(AB)

An AI > 1 indicates a potential substrate for PGP or other active transport.

For microsomal stability testing, the test agent was incubated in duplicate with microsomes at 37ºC. The reaction contained microsomal protein in 100 mM potassium phosphate, 2 mM NADPH, 3 mM MgCl2, pH 7.4. A control was run for each test agent, omitting NADPH to detect NADPH-free degradation. At indicated times, an aliquot was removed from each experimental and control reaction and mixed with an equal volume of ice-cold Stop Solution (0.3% acetic acid in acetonitrile containing haloperidol, diclofenac, or other internal standard). Stopped reactions were incubated at least ten minutes at −20ºC, and an additional volume of water was added. The samples were centrifuged to remove precipitated protein, and the supernatants were analyzed by LC/MS/MS to quantitate the remaining parent. Data are reported as % remaining by dividing by the time zero concentration value.

Appendix 2. Synthesis of Analogs

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2-Oxoindoline-5-sulfonyl chloride.16 Indolin-2-one (5.00 g, 37.6 mmol) was added in portions to hypochlorous sulfonic anhydride (10.2 ml, 153 mmol) at 30°C. The dark mixture was stirred at room temperature for 1.5 hrs and heated at 70°C for 1 hr. The reaction was slowly quenched with water, the light pink solid was filtered and dried to give 5.33g (yield 61%) pink solid, which was used directly in the next reaction without further purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.40 (s, 1 H), 7.31 – 7.46 (m, 2 H), 6.71 (dd, J=7.8, 0.6 Hz, 1 H), 3.45 (s, 2 H).

General protocol A. A solution of 1-substituted piperazine (0.216 mmol), 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 allowed to warm to room temperature and stirred for overnight. 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), 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 at room temperature overnight. The crude mixture was filtered through a frit 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), 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 at room temperature overnight. The mixture was added to water and solid was crushed out. The solid 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), 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 (0.043g, 0.350 mmol). The reaction mixture was stirred at room temperature overnight. The crude mixture was filtered through a frit and purified by preparative HPLC to give the final product.

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5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 20969430/ML201, 1). A solution of 1-(4-(piperazin-1-yl)phenyl)ethanone (485 mg, 2.374 mmol), triethylamine (0.602 ml, 4.32 mmol) in DMF (10.0ml) was treated at room temperature with 2-oxoindoline-5-sulfonyl chloride (500 mg, 2.158 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was added to water and solid was crushed out. The solid was filtered and dried to give 658 mg white solid (yield 76%). 1H 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 = 4.461 min, m/z 400.1 [M+H+]; HRMS (ESI) m/z calcd for C20H22N3O4S[M+H+] 400.1331, found 400.1327.

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5-(Piperazin-1-ylsulfonyl)indolin-2-one (CID 4962779, 2). A solution of piperazine (1.67 g, 19.4 mmol), triethylamine (2.71 ml, 19.4 mmol) in DMF (10.0 ml) was treated at 0°C with another solution of 2-oxoindoline-5-sulfonyl chloride (1.50 g, 6.48 mmol) in DMF (10.0 ml). The reaction mixture was stirred at room temperature overnight. DMF was removed to give a dark brown oil. Dichloromethane was added to precipitate out the product. The solid was filtered and washed with dichloromethane to give 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 = 2.661 min, m/z 282.1 [M+H+].

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5-(4-Acetylpiperazin-1-ylsulfonyl)indolin-2-one (CID 9535379, 3). The title compound was prepared according to the general protocol A. 1H 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 = 3.358 min, m/z 346.1 [M+Na+].

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5-(4-(4-Hydroxyphenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 44825295, 4). The title compound was prepared according to the general protocol A. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.80 (s, 1 H), 8.88 (br. s., 1 H), 7.40 – 7.67 (m, 2 H), 6.98 (d, J=8.0 Hz, 1 H), 6.74 (d, J=7.6 Hz, 2 H), 6.49 – 6.66 (m, 2 H), 3.57 (s, 2 H), 3.03 – 2.90 (m, 8 H); LCMS RT = 3.490 min, m/z 374.1 [M+H+].

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5-(4-(4-Hydroxyphenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 44825297, 5). The title compound was prepared according to the 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 = 3.490 min, m/z 374.1 [M+H+].

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5-(4-p-Tolylpiperazin-1-ylsulfonyl)indolin-2-one (CID 44825294, 6). The title compound was prepared according to the general protocol A. 1H NMR (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 = 5.084 min, m/z 372.1 [M+H+].

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4-(4-(2-Oxoindolin-5-ylsulfonyl)piperazin-1-yl)benzonitrile (CID 44825290, 7). The title compound was prepared according to the 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 = 4.887 min, m/z 383.1 [M+H+].

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5-(4-(4-Nitrophenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 44825289, 8). The title compound was prepared according to the 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 = 5.019 min, m/z 425.1 [M+Na+].

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5-(4-(4-Fluorophenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 44825292, 9). The title compound was prepared according to the 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 = 5.148 min, m/z 376.1 [M+H+].

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5-(4-(4-Chlorophenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 44825296, 10). The title compound was prepared according to the 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 = 5.569 min, m/z 392.1 [M+H+].

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5-(4-(4-Bromophenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 44825293, 11). The title compound was prepared according to the 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 = 5.670 min, m/z 436.0 [M+H+].

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5-(4-(4-(Trifluoromethyl)phenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 44825288, 12). The title compound was prepared according to the 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 = 5.782 min, m/z 426.1 [M+H+].

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5-(4-(3-(Trifluoromethyl)phenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 44825298, 13). The title compound was prepared according to the 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 = 5.776 min, m/z 426.1 [M+H+].

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4-(4-(2-Oxoindolin-5-ylsulfonyl)piperazin-1-yl)benzaldehyde (CID 44825307, 14). A solution of 4-(piperazin-1-yl)benzaldehyde, TFA salt (58.0 mg, 0.139 mmol), 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 at room temperature for 3 hr. The solid was filtered and dried to give a yellow solid. 1H NMR (400 MHz, 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 = 4.548 min, m/z 386.1 [M+H+].

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Methyl 4-(4-(2-oxoindolin-5-ylsulfonyl)piperazin-1-yl)benzoate (CID 44825302, 15). A solution of methyl 4-(piperazin-1-yl)benzoate (0.687 g, 3.12 mmol), 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 at room temperature for over night. The yellow solid was filtered and dried to give 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 = 5.143 min, m/z 416.1 [M+H+].

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4-(4-(2-Oxoindolin-5-ylsulfonyl)piperazin-1-yl)benzoic acid (CID 44825310, 16). A suspension of methyl 4-(4-(2-oxoindolin-5-ylsulfonyl)piperazin-1-yl)benzoate (15) (240 mg, 0.578 mmol) in 6N HCl (75.0 ml) was refluxed for 1 h. The reaction mixture was concentrated as a red solid which was purified by preparative HPLC to give 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 = 4.458 min, m/z 402.1 [M+H+].

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5-(4-(4-(Hydroxymethyl)phenyl)piperazin-1-ylsulfonyl)indolin-2-one (CID 44825308, 17). DIBAL-H (0.325 ml, 1.0 m in THF, 0.325 mmol) was added drop wise 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 give 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 = 4.062 min, m/z 387.1 [M+H+].

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5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-1H-benzo[d]imidazol-2(3H)-one (CID 44825303, 18). The title compound was prepared according to the general protocol B. 1H 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 = 4.401 min, m/z 401.1 [M+H+].

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5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-1,3-dimethyl-1H-benzo[d]imidazol-2(3H)-one (CID 39886486, 19). The title compound was prepared according to the 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 = 5.068 min, m/z 429.1 [M+H+].

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6-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)benzo[d]oxazol-2(3H)-one (CID 44825306, 20). The title compound was prepared according to the 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 = 5.067 min, m/z 402.1 [M+H+].

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6-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-3-methylbenzo[d]oxazol-2(3H)-one (CID 31534174, 21). The title compound was prepared according to the 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 = 5.215 min, m/z 416.1 [M+H+].

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5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-6-chloroindolin-2-one (CID 36649951, 22). The title compound was prepared according to the general protocol B. 1H 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 H), 3.19 – 3.27 (m, 4 H), 2.43 (s, 3 H); LCMS RT = 4.922 min, m/z 434.1 [M+H+].

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5-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-3,3-dichloroindolin-2-one (CID 44825304, 23). The title compound was prepared according to the 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 = 5.565 min, m/z 468.0 [M+H+].

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6-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)isobenzofuran-1(3H)-one (CID 44825299, 24). The title compound was prepared according to the 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 = 5.115 min, m/z 401.1 [M+H+].

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6-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-3,4-dihydroquinolin-2(1H)-one (CID 8460266, 25). The title compound was prepared according to the 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 = 4.909 min, m/z 414.1 [M+H+].

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7-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)-4,5-dihydro-1H-benzo[b]azepin-2(3H)-one (CID 44825305, 26). The title compound was prepared according to the 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 = 4.927 min, m/z 428.2 [M+H+].

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N-(4-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)phenyl)acetamide (CID 8194745, 27). The title compound was prepared according to the 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 = 5.036 min, m/z 402.1 [M+H+].

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1-(4-(4-(4-Acetylphenyl)piperazin-1-ylsulfonyl)phenyl)pyrrolidin-2-one (CID 19614457, 28). The title compound was prepared according to the 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 = 5.223 min, m/z 428.2 [M+H+].

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1-(4-(4-(1-Acetyl-2-methylindolin-5-ylsulfonyl)piperazin-1-yl)phenyl)ethanone (CID 20853151, 29). The title compound was prepared according to the general protocol C. 1H 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 = 5.382 min, m/z 442.2 [M+H+].

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1-(4-(4-(1-Acetyl-1,2,3,4-tetrahydroquinolin-6-ylsulfonyl)piperazin-1-yl)phenyl)ethanone (CID 20855818, 30). The title compound was prepared according to the 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 = 5.269 min, m/z 442.2 [M+H+].

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7-(4-(4-acetylphenyl)piperazin-1-ylsulfonyl)-2H-benzo[b][1,4]oxazin-3(4H)-one (CID 44825309, 31). The title compound was prepared according to the general protocol C. 1H NMR (400 MHz, DMSO-d6) δ 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 = 4.943 min, m/z 416.1 [M+H+].

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5-(4-(4-Acetylphenyl)piperazine-1-carbonyl)indolin-2-one (CID 44825300, 32). The title compound was prepared according to the 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 = 4.144 min, m/z 364.1 [M+H+].

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6-(4-(4-Acetylphenyl)piperazine-1-carbonyl)-3,4-dihydroquinolin-2(1H)-one (CID 44825301, 33). The title compound was prepared according to the 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 = 4.297 min, m/z 378.2 [M+H+].

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N-(4-(4-Acetylpiperazin-1-yl)phenyl)-2-oxoindoline-5-sulfonamide (CID 44825291, 34). A solution of 1-(4-(4-aminophenyl)piperazin-1-yl)ethanone (76.0 mg, 0.345 mmol), 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 allowed to warm to room temperature and stirred for 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), 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 = 3.554 min, m/z 415.1 [M+H+].