WO2012061597A1

WO2012061597A1 - Alpha-glucosidase binders and methods of their use

Info

Publication number: WO2012061597A1
Application number: PCT/US2011/059141
Authority: WO
Inventors: Juan J. Marugan; Ellen Sidransky; Noel Southall; Jingbo Xiao; Wei Zheng
Original assignee: The United States Of America As Represented By The Secretary, Department Of Health And Human Services Office Of Technology Transfer, National Institutes Of Health
Priority date: 2010-11-03
Filing date: 2011-11-03
Publication date: 2012-05-10

Abstract

Compounds that bind to acid alpha glucosidase are described. Methods of using these compounds for the treatment of diabetes and Pompe disease are also described.

Description

ALPHA-GLUCOSIDASE BINDERS AND METHODS OF THEIR USE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S Provisional Application No. 61/409,697, filed November 3, 2010, the entirety of which is incorporated herein. TECHNICAL FIELD

The present invention is directed to compounds that bind to alpha glucosidase. The compounds are useful for the treatment of diabetes and/or Pompe disease.

BACKGROUND

Alpha-glucosidase is an enzyme that can catalyze the exohydro lysis of a- 1,4- and cc-1,6- glucosidic linkages in the lysosome to release glucose. Alpha-glucosidase inhibitors are useful for limiting the impact of carbohydrate consumption on blood glucose levels in patients with diabetes mellitus type 2, and several alpha-glucosidase inhibitors are commercially available for the treatment of diabetes, including acarbose, miglitol, and voglibose. New diabetes treatments are needed.

Pompe disease is an autosomal recessive disorder caused by the deficiency or dysfunction of alpha-glucosidase. Epidemiological studies have estimated its frequency to be 1 in every 40,000 births. This deficiency or dysfunction results in glucosidic linkages not being cleaved, resulting in an accumulation of glycogen in the lysosome, leading to lysosomal enlargement. This accumulation is especially severe in cardiac and skeletal muscle, affecting breathing and mobility.

The only approved treatment of Pompe disease is enzyme replacement therapy with recombinant acid alpha-glucosidase produced in a Chinese hamster ovary cell line. While enzyme replacement therapy is efficacious, it is expensive, costing approximately $300,000 per year.

Moreover, the development of infusion-related reactions is common and the majority of patients test positive for IgG antibodies to acid alpha-glucosidase.

Small molecules chaperones, compounds that can bind to mutant or misfolded enzymes and assist in proper folding, have been reported for the treatment of Pompe disease. The compounds, generally iminosugars, are also inhibitors of alpha glucosidase. One iminosugar compound, deoxynojirimycin, is being clinically evaluated as a treatment for Pompe disease. This compound, however, has poor selectivity and has a small therapeutic window between improving the folding of alpha glucosidase, thereby improving translocation and the proper function of the enzyme, and inhibiting the enzyme's activity.

As such, new treatments for Pompe disease are needed. SUMMARY

The invention is directed methods comprising contacting at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula I, or a pharmaceutically acceptable salt thereof

wherein Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci-₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OC_1-6alkyl, - C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂; R₂ and R₃ are each independently H, halogen, or Ci_ ₆alkyl; and R4 is H, halogen, or Ci_₆alkyl. Also within the scope of the invention are methods for the treatment of diabetes by administering to the patient a compound of formula I. Additionally, methods for the treatment of Pompe disease are described, the methods including administering to the patient a compound of formula I.

The invention is also directed to methods comprising contacting at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula II, or a pharmaceutically acceptable salt thereof

wherein n is 0 or 1; Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_ ₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH, -COOH, - C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂; R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; andR₄ is H, halogen, or Ci-₆alkyl. Also within the scope of the invention are methods for the treatment of diabetes by administering to the patient a compound of formula II. Additionally, methods for the treatment of Pompe disease are described, the methods including administering to the patient a compound of formula II.

Novel compounds that bind to at least one of wild type alpha glucosidase, mutant alpha glucosidase, and misfolded alpha glucosidase are also described. These compounds will have use in the treatment of, for example, diabetes and/or Pompe disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the thermal stabilization of alpha glucosidase activity upon incubation with compounds of the invention.

FIG. 2 depicts Western blot analysis of of protein extracts from HEK cells over-expressing

GAA (lane 1), HEK cells (lane 2) and WT primary fibroblasts (lane 3). Tubulin was used as a loading control.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is directed to compounds that bind to alpha glucosidase. Compounds that can bind to alpha glucosidase can decrease the activity of the enzyme, preferably wild type alpha glucosidase, thereby decreasing the conversion of carbohydrates into monosaccharides. Such compounds are useful for the treatment of diabetes mellitus type 2. Compounds of the invention decrease the activity of alpha glucosidase, for example wild type alpha glucosidase, and can be useful for the treatment of diabetes.

Compounds that bind to alpha glucosidase can also be useful for the treatment of other diseases, for example, Pompe disease. Pompe disease is associated with dysfunctional alpha glucosidase. The dysfunctional alpha glycosidase is generally a mutated alpha glucosidase, wherein the mutations are such that the enzyme does not fold or acquire the necessary shape to function properly. It has been determined that the compounds of the invention can bind to acid alpha- glucoside and act as "chaperones" by correcting the misfolding of mutant alpha glucosidase enzymes. Correction of the misfolding of mutant alpha-glucosidase can result in an increase of enzyme translocation from the endoplasmic reticulum to the lysosome which translates to an overall increase in the activity of the mutant alpha glucosidase to hydro lyze terminal a- 1,4- and cc-1,6- glucosidic linkages of glycogen in the lysosome. Importantly, even though compounds of the invention exhibit binding activity against wild type alpha glucosidase, this binding is not so great as to result in complete inactivation of misfolded enzymes. As such, compounds of the invention will be useful for the treatment of Pompe disease.

The invention is directed to methods comprising contacting at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula I, or a pharmaceutically acceptable salt thereof.

wherein

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-0] -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and P3 are each independently H, halogen, or Ci_₆alkyl; and

P4 is H, halogen, or C_halky!

Alternatively, Ri is -C(0)Ci_₆alkyl or phenyl optionally substituted with

-OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R4 is H, halogen, or C_halky!

In some embodiments, the compound of formula I binds to wild type alpha glucosidase. In those embodiments, the binding of the compound of formula I to the wild type alpha glucosidase reduces the activity of the wild type alpha glucosidase.

In other embodiments, the compound of formula I binds to mutant alpha glucosidase. In those embodiments, the binding of the compound of formula I to the mutant alpha glucosidase stabilizes the mutant alpha glucosidase. Preferably, the binding of the compound of formula I to mutant alpha glucosidase results in an increase in the lysosomal activity of the mutant alpha glucosidase.

In yet other embodiments, the compound of formula I binds to misfolded alpha glucosidase. In those embodiments, the binding of the compound of formula I to the misfolded alpha glucosidase stabilizes the misfolded alpha glucosidase. Preferably, the binding of the compound of formula I to the misfolded alpha glucosidase results in an increase in the lysosomal activity of the misfolded alpha glucosidase.

In still other embodiments, the compound of formula I binds to recombinant alpha glucosidase. In those embodiments, the binding of the compound of formula I to the recombinant alpha glucosidase increases the half life of the recombinant alpha glucosidase. Preferably, the recombinant alpha glucosidase is recombinant human alpha glucosidase, also known as rhGAA. One exemplary recombinant human alpha glucosidase is MYOZYME (Genzyme Corporation, Cambridge, MA).

Also within the scope of the invention are methods of treating diabetes in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof

wherein

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R₄ is H, halogen, or C_halky!

Alternatively, Ri is -C(0)Ci_₆alkyl or phenyl optionally substituted with Ci-₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R₄ is H, halogen, or C_halky!

Other embodiments of the invention are directed to methods of treating Pompe disease in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof:

wherein

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with

Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R₄ is H, halogen, or C_halky!

Alternatively, Ri is -C(0)Ci_₆alkyl or phenyl optionally substituted with

-OH,

Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R₄ is H, halogen, or C_halky! Other embodiments of the invention are directed to methods for enhancing the stability of at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase comprising contacting the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula I, or a pharmaceutically acceptable salt thereof

wherein

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci-₆alkyl; and P4 is H, halogen, or C_halky!

Alternatively, Ri is -C(0)Ci_₆alkyl or phenyl optionally substituted with

-OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OC_1-6alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R₄ is H, halogen, or C_halky!

In preferred methods of these embodiments of the invention, the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase. Preferably, the half life is increased by at least 5%, more preferably 10%, and even more preferably by at least 20%. In exemplary embodiments of the invention, the compounds of the invention will enhance the stability of a recombinant alpha glucosidase, preferably a recombinant human alpha glucosidase, for example, a recombinant human alpha glucosidase such as MYOZYME® (Genzyme Corporation, Cambridge, MA). In such embodiments, the half life of the recombinant alpha glucosidase will be increased, preferably by at least 5%, more preferably by at least 10%, and even more preferably by at least 20%.

In preferred embodiments of the methods of the invention using compounds of formula I, R₂ is H. In other embodiments, R₃ is H. In still other embodiments, R₄ is H. Preferably, R₂, R₃, and R₄ are each H.

In other embodiments of the invention using compounds of formula I, R₂ is halogen. In yet other embodiments, R₃ is halogen. Preferably, R₂ and R₃ are each halogen. In those embodiments, R₂ and R₃ may be the same or different halogens. In some preferred embodiments, R₂ and R₃ are each CI. In some embodiments, R₄ is halogen. In some preferred embodiments, R₄ is CI.

In some embodiments of the invention of the invention using compounds of formula I, Ri is -C(0)Ci_₆alkyl, for example -C(0)CH₃, -C(0)CH₂CH₃, -C(0)C(CH₃)₃, and the like. Preferred embodiments include those where Ri is -C(0)CH_3.

In other embodiments of the invention of the invention using compounds of formula I, Ri is a substituted phenyl. While the phenyl group can be substituted at position 2, 3, or 4 of the phenyl ring, substitution at the 4-position is preferred.

Preferred embodiments of the invention using compounds of formula I include those where Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂. Other embodiments of the invention using compounds of formula I, include those where Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

Still other embodiments of the invention using compounds of formula I include those where Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci_ ₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

More preferred embodiments of the invention using compounds of formula I, include those where Ri is phenyl substituted with -C(0)Ci_₆alkyl.

In preferred embodiments of the invention using compounds of formula I, Ri is phenyl substituted with Ci-₆alkoxy, for example, methoxy, ethoxy, tert-butoxy, and the like, with methoxy being an exemplary embodiment.

In some embodiments of the invention using compounds of formula I, Ri is phenyl substituted with -OH.

In some embodiments of the invention using compounds of formula I, Ri is phenyl substituted with Ci-₆alkyl, for example, methyl, ethyl, propyl, isopropyl, pentyl, and the like, with methyl being preferable. In other embodiment, Ri is phenyl substituted with Ci_₆alkyl substituted with 1-3 halogen, for example, -CH₂F, -CHF₂, -CF₃, -CH₂CF₃, and the like, with -CF₃ being preferable.

In some embodiments of the invention using compounds of formula I, Ri is phenyl substituted with Ci_₆alkylene-OH, for example, -CH₂OH, -CH₂CH₂OH, CH₂CH₂CH₂OH, and the like, with -CH₂OH being preferred.

In some embodiments of the invention using compounds of formula I, Ri is phenyl substituted with -COOH.

In some embodiments of the invention using compounds of formula I, Ri is phenyl substituted with -C(0)OCi_₆alkyl, for example -C(0)OCH₃, -C(0)OCH₂CH₃, -C(0)OC(CH₃)₃, and the like, with -C(0)OCH₃ being preferred.

In some embodiments of the invention using compounds of formula I, Ri is phenyl substituted with -C(0)H.

In some embodiments of the invention using compounds of formula I, Ri is phenyl substituted with halogen, for example, F, CI, Br, or I, with F, CI, and Br, especially CI, being preferred. In some embodiments of the invention using compounds of formula I, Ri is phenyl substituted with -CN.

In some embodiments of the invention using compounds of formula I, Ri is phenyl substituted with -N0₂.

In other embodiments of the invention using compounds of formula I, Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_ ₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

Preferred compounds for use in the methods of the invention include the compounds listed the following table:

Comp. No Ri R₂ R₃ R₄

1 -C(0)-CH₃ H H H

2 4-OCH₃-Ph H H H

3 4-OH-Ph H H H

4 4-CH₃-Ph H H H

5 4-CH₂-OH-Ph H H H

6 4-C(0)OH-Ph H H H

7 4-C(0)OCH₃-Ph H H H

8 4-CN-Ph H H H

9 4-C(0)H-Ph H H H

10 4-N0₂-Ph H H H

11 4-F-Ph H H H

12 4-Cl-Ph H H H

13 4-Br-Ph H H H

14 4-CF₃-Ph H H H

15 3-CF₃-Ph H H H

16 4-C(0)CH₃-Ph CI CI H

Preferred compounds for use in the methods of the invention include:

Other compounds of formula I envisioned for use in the invention include those having pyridinyl, pyrazyl, pyrimidyl, or pyridazyl ring, which can be prepared using the knowledge in art, along with this description. Those compounds include, for example:

While the above compounds of formula I depict the pyridinyl, pyrazyl, pyrimidyl, and pyridazyl ring substituted with -C(0)-CH₃, the pyridinyl, pyrazyl, pyrimidyl, and pyridazyl rings of the invention can alternatively be substituted with

-OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

Also within the scope of the invention are compounds of formula I, or a pharmaceutically acceptable salt thereof:

wherein

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_₆alkoxy at the 4-position, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)H, halogen at the 2-position, halogen at the 4-position, -CN, or -N0₂; and when at least one of R₂, R₃, and R₄ is halogen, then the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl can further be optionally substituted with -C(0)Ci_₆alkyl at the 4-position.

R₂ and R₃ are each independently H or halogen; and

R₄ is H, halogen, or C_halky!

Alternatively, Ri is -C(0)Ci_₆alkyl or phenyl optionally substituted with

at the 4- position, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH, -COOH, - C(0)OCi_₆alkyl, -C(0)H, halogen at the 2-position, halogen at the 4-position, -CN, or -N0₂; and when at least one of R₂, R₃, and R₄ is halogen, then the phenyl can further be optionally substituted with -C(0)Ci_₆alkyl at the 4-position.

R₂ and R₃ are each independently H or halogen; and

R₄ is H, halogen, or C_halky!

In preferred embodiments, R₂ is H. In other embodiments R₃ is H. Most preferred compounds are those wherein R₂ and R₃ are each H. In other embodiments R₂ is halogen, preferably CI. In other embodiments, R₃ is halogen, preferably CI. More preferably, R₂ and R₃ are each halogen, preferably CI.

Preferred embodiments of the invention include those embodiments wherein R₄ is H. Other preferred embodiments include those wherein R₄ is halogen, preferably CI. Yet other embodiments include those wherein R₄ is Ci-₆alkyl, for example, methyl, ethyl, isopropyl, and the like.

In some embodiments, Ri is -C(0)Ci_₆alkyl, for example, -C(0)CH₃, -C(0)CH₂CH₃, and the like, preferably -C(0)CH₃.

In other embodiments, Ri is a substituted phenyl. Preferably, the substituent is at the 4 position of the phenyl ring, although substitutions at the 2- and 3-positions are also envisioned.

In some embodiments, R¹ is phenyl substituted with Ci-₆alkoxy, for example, methoxy, ethoxy, tert-butoxy, preferably methoxy, at the 4-position.

In other embodiments, Ri is phenyl substituted with -OH.

In still other embodiments, Ri is phenyl substituted with Ci_₆alkyl substituted with 1-3 halogen, for example, -CH₂F, -CHF₂, -CF₃, -CH₂CF₃, and the like, with -CF₃ being preferable. In some embodiments, Ri is phenyl substituted with Ci_₆alkylene-OH, for example, -CH₂OH, -CH₂CH₂OH, CH₂CH₂CH₂OH, and the like, with -CH₂OH being preferred.

In some embodiments, Ri is phenyl substituted with -COOH.

In some embodiments, Ri is phenyl substituted with -C(0)OCi_₆alkyl, for example -C(0)OCH₃, -C(0)OCH₂CH₃, -C(0)OC(CH₃)₃, and the like, with -C(0)OCH₃ being preferred.

In some embodiments, Ri is phenyl substituted with -C(0)H.

In some embodiments, Ri is phenyl substituted with halogen at the 2-positions of the 4- position, for example, with F, CI, Br, or I, with F, CI, and Br, especially CI, being preferred.

In some embodiments, Ri is phenyl substituted with -CN.

In some embodiments, Ri is phenyl substituted with -NO₂.

Preferred compounds of the invention include those listed in the following table:

Comp. No. Ri R2 R₃ R₄

1 -C(0)-CH₃ H H H

2 4-OCH₃-Ph H H H

3 4-OH-Ph H H H

4 4-CH₃-Ph H H H

5 4-CH₂-OH-Ph H H H

6 4-C(0)OH-Ph H H H

7 4-C(0)OCH₃-Ph H H H

8 4-CN-Ph H H H

9 4-C(0)H-Ph H H H

10 4-N0₂-Ph H H H

11 4-F-Ph H H H

12 4-Cl-Ph H H H

13 4-Br-Ph H H H

14 4-CF₃-Ph H H H

15 3-CF₃-Ph H H H

16 4-C(0)CH₃-Ph CI CI H Comp. No. i R₂ R₃ R₄

17 4-C(0)CH₃-Ph H H 2-Cl

More preferably, compounds of the invention include those listed in the following table:

Other particularly preferred compounds include:

The invention is also directed to methods comprising contacting at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula II, or a pharmaceutically acceptable salt thereof.

wherein

n is 0 or 1 ;

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH,

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R4 is H, halogen, or C_halky!

In some embodiments, the compound of formula II binds to wild type alpha glucosidase. In those embodiments, the binding of the compound of formula II to the wild type alpha glucosidase reduces the activity of the wild type alpha glucosidase. In other embodiments, the compound of formula II binds to mutant alpha glucosidase. In those embodiments, the binding of the compound of formula II to the mutant alpha glucosidase stabilizes the mutant alpha glucosidase. Preferably, the binding of the compound of formula II to mutant alpha glucosidase results in an increase in the lysosomal activity of the mutant alpha glucosidase.

In yet other embodiments, the compound of formula II binds to misfolded alpha glucosidase. In those embodiments, the binding of the compound of formula II to the misfolded alpha

glucosidase stabilizes the misfolded alpha glucosidase. Preferably, the binding of the compound of formula II to the misfolded alpha glucosidase results in an increase in the lysosomal activity of the misfolded alpha glucosidase.

In other embodiments, the compound of formula II binds to recombinant alpha glucosidase. Preferably, the binding of the compound of formula II to the recombinant alpha glucosidase results in an increase in the half life of the recombinant alpha glucosidase. Preferably, the recombinant alpha glucosidase is recombinant human alpha glucosidase, also known as rhGAA. One exemplary recombinant human alpha glucosidase is MYOZYME (Genzyme Corporation, Cambridge, MA).

Also within the scope of the invention are methods of treating diabetes in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula II, or a pharmaceutically acceptable salt thereof

wherein

n is 0 or 1 ;

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and P3 are each independently H, halogen, or Ci_₆alkyl; and

P4 is H, halogen, or C_halky!. Other embodiments of the invention are directed to methods of treating Pompe disease in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula II, or a pharmaceutically acceptable salt thereof:

wherein

n is 0 or 1 ;

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R₄ is H, halogen, or C_halky!

Other embodiments of the invention are directed to methods for enhancing the stability of at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase comprising contacting the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula II, or a pharmaceutically acceptable salt thereof

wherein

n is 0 or 1 ;

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci-₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH, -COOH,

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R₄ is H, halogen, or C_halky!. In preferred methods of these embodiments of the invention, the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase. Preferably, the half life is increased by at least 5%, more preferably 10%, and even more preferably by at least 20%. In exemplary embodiments of the invention, the compounds of the invention will enhance the stability of a recombinant alpha glucosidase, preferably recombinant human alpha glucosidase, for example, a recombinant human alpha glucosidase such as MYOZYME® (Genzyme, Cambridge, MA). In such embodiments, the half life of the recombinant alpha glucosidase will be increased, preferably by at least 5%, more preferably by at least 10%, and even more preferably by at least 20%.

In preferred embodiments of the methods of the invention using compounds of formula II, n is 0. In other embodiments, n is 1.

In preferred embodiments of the methods of the invention using compounds of formula II, R₂ is H. In other embodiments, R₃ is H. In still other embodiments, R₄ is H. Preferably, R₂, R₃, and R₄ are each H.

In other embodiments using compounds of formula II, R₂ is halogen. In yet other embodiments, R₃ is halogen. Preferably, R₂ and R₃ are each halogen. In those embodiments, R₂ and R₃ may be the same or different halogens. In some preferred embodiments, R₂ and R₃ are each CI. In some embodiments, R₄ is halogen. In some preferred embodiments, R₄ is CI.

In some embodiments of the invention using compounds of formula II, Ri is -C(0)Ci_₆alkyl, for example -C(0)CH₃, -C(0)CH₂CH₃, -C(0)C(CH₃)₃, and the like. Preferred embodiments include those where Ri is -C(0)CH_3.

In other embodiments of the invention using compounds of formula II, Ri is a substituted phenyl. While the phenyl group can be substituted at position 2, 3, or 4 of the phenyl ring, substitution at the 4-position is preferred.

Preferred embodiments of the invention using compounds of formula II include those where

Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

Other embodiments of the invention using compounds of formula II include those where Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂. Still other embodiments of the invention using compounds of formula II include those where Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

More preferred embodiments of the invention using compounds of formula II include those where R¹ is phenyl substituted with -C(0)Ci_₆alkyl.

In preferred embodiments of the invention using compounds of formula II, Ri is phenyl substituted with Ci-₆alkoxy, for example, methoxy, ethoxy, tert-butoxy, and the like, with methoxy being an exemplary embodiment.

In some embodiments of the invention using compounds of formula II, Ri is phenyl substituted with -OH.

In some embodiments of the invention using compounds of formula II, Ri is phenyl substituted with Ci-₆alkyl, for example, methyl, ethyl, propyl, isopropyl, pentyl, and the like, with methyl being preferable. In other embodiment, Ri is phenyl substituted with Ci_₆alkyl substituted with 1-3 halogen, for example, -CH₂F, -CHF₂, -CF₃, -CH₂CF₃, and the like, with -CF₃ being preferable.

In some embodiments of the invention using compounds of formula II, Ri is phenyl substituted with Ci_₆alkylene-OH, for example, -CH₂OH, -CH₂CH₂OH, CH₂CH₂CH₂OH, and the like, with -CH₂OH being preferred.

In some embodiments of the invention using compounds of formula II, Ri is phenyl substituted with -COOH.

In some embodiments of the invention using compounds of formula II, Ri is phenyl substituted with -C(0)OCi_₆alkyl, for example -C(0)OCH₃, -C(0)OCH₂CH₃, -C(0)OC(CH₃)₃, and the like, with -C(0)OCH₃ being preferred.

In some embodiments of the invention using compounds of formula II, Ri is phenyl substituted with -C(0)H.

In some embodiments of the invention using compounds of formula II, Ri is phenyl substituted with halogen, for example, F, CI, Br, or I, with F, CI, and Br, especially CI, being preferred.

In some embodiments of the invention using compounds of formula II, Ri is phenyl substituted with -CN. In some embodiments of the invention using compounds of formula II, Ri is phenyl substituted with -N0₂.

In other embodiments of the invention using compounds of formula II, Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene- OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

Preferred compounds of the invention include those of formula II

wherein

n is 0 or 1 ;

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R4 is H, halogen, or C_halky!

Particularly preferred compounds of formula II for use in the invention include

Other compounds envisioned for use in the invention of formula II include those having pyridinyl, pyrazyl, pyrimidyl, or pyridazyl ring, which can be prepared using the knowledge in art, along with this description. Those compounds include, for example:

o , and O

While the above compounds of formula II depict the pyridinyl, pyrazyl, pyrimidyl, and pyridazyl ring substituted with -C(0)-CH₃, the pyridinyl, pyrazyl, pyrimidyl, and pyridazyl rings of the invention can alternatively be substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂. As used herein, "alkyl" refers to an optionally substituted, saturated straight, or branched, hydrocarbon radical having from about 1 to about 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein).

As used herein, the terms "treatment" or "therapy" (as well as different word forms thereof) includes preventative (e.g., prophylactic), curative or palliative treatment.

As used herein, "contacting" refers to bringing together, either directly or indirectly, a compound of the invention into physical proximity to an alpha glucosidase, for example, wild type, mutant, or misfolded alpha glucosidase. The alpha glucosidase can be present in any number of buffers, salts, solutions, etc. Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the alpha glucosidase.

As used herein, "wild type" refers to the phenotype of the typical form of alpha glucosidase.

As used herein, "mutant" refers to a version of alpha glucosidase that is not wild type.

As used herein, "misfolded" refers to a version of alpha glucosidase that does fold properly or does not acquire the necessary shape to exhibit a particular activity of the enzyme. Misfolded alpha glucosidase can result in the enzyme's inability to hydro lyze terminal alpha 1,4- and alpha 1,6- glucosidic linkages of glycogen in the lysosome.

As used herein, the term "binding" means the physical or chemical interaction between a compound of the invention and an alpha glucosidase, for example wild type, mutant, or misfolded alpha glucosidase. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc. Binding may be detected in many different manners, including the methods described herein. Other methods of detecting binding are well known to those of skill in the art.

As used herein, the term "activity" refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e., having a measurable effect in response to some exposure or stimulus, including, for example, the affinity of a compound of the invention for directly binding an alpha glucosidase, for example, wild type, mutant, or misfolded alpha glucosidase. For example, the activity of an alpha glucosidase can be measured by contacting the alpha glucosidase, with or without the presence of a compound of the invention, with 4- methylumbelliferyl cc-D-glucopyranoside. Activity of the alpha glucosidase can be correlated to the amount of hydrolysis of the 4- methylumbelliferyl cc-D-glucopyranoside. Upon hydrolysis by alpha glucosidase, the blue fluorescent dye 4-methylumbelliferone is liberated, producing a fluorescent emission at 440 nm when excited at 370 nm. In addition, as a control for autofluorescence, a second substrate can be used, resorufin cc-D-glucopyranoside, which liberates, upon hydrolysis by alpha glucosidase, the red dye resorufin at an emission wavelength of 590 nm when excited at 530 nm.

As used herein, "reduces the activity" refers to a decrease in the amount, quality, or effect of a particular activity of an alpha glucosidase.

As used herein, "increase the activity" refers to refers to an increase in the amount, quality, or effect of a particular activity of an alpha glucosidase.

As used herein, "stabilizes" refers to the ability of a compound of the invention to assist in the folding of the misfolded or mutant alpha glucosidase such that the alpha glucosidase achieves a folding and/or shape that facilitates the activity of the alpha glucosidase.

As used herein, "enhance" refers to an improvement in the amount, quality, or effect of a particular activity of an alpha glucosidase.

As employed above and throughout the disclosure the term "effective amount" refers to an amount effective, at dosages, and for periods of time necessary, to achieve the desired result with respect to the treatment of the relevant disorder, condition, or side effect. It will be appreciated that the effective amount of components of the present invention will vary therapy to therapy (i.e., diabetes or Pompe disease) and from patient to patient not only with the particular compound, component or composition selected, the route of administration, and the ability of the components to elicit a desired response in the individual, but also with factors such as the disease state or severity of the condition to be alleviated, hormone levels, age, sex, weight of the individual, the state of being of the patient, and the severity of the pathological condition being treated, concurrent medication or special diets then being followed by the particular patient, and other factors which those skilled in the art will recognize, with the appropriate dosage ultimately being at the discretion of the attendant physician. Dosage regimens may be adjusted to provide the improved therapeutic response. An effective amount is also one in which any toxic or detrimental effects of the components are outweighed by the therapeutically beneficial effects. As an example, the compounds useful in the methods of the present invention are administered at a dosage and for a time such that the level of activation and adhesion activity of platelets is reduced as compared to the level of activity before the start of treatment.

"Pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio.

Within the present invention, the disclosed compounds may be prepared in the form of pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional nontoxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. These physiologically acceptable salts are prepared by methods known in the art, e.g., by dissolving the free amine bases with an excess of the acid in aqueous alcohol, or neutralizing a free carboxylic acid with an alkali metal base such as a hydroxide, or with an amine.

The compounds of this invention may be administered orally or parenterally, neat or in combination with conventional pharmaceutical carriers, diluents, or excipients, which may be liquid or solid. As such, also within the scope of the invention are compositions comprising one or more compounds of formula I and at least one carrier or excipient. The applicable solid carrier, diluent, or excipient may function as, among other things, a binder, disintegrant, filler, lubricant, glidant, compression aid, processing aid, color, sweetener, preservative, suspensing/dispersing agent, tablet- disintegrating agent, encapsulating material, film former or coating, flavors, or printing ink. Of course, any material used in preparing any dosage unit form is preferably pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations. Parenteral administration in this respect includes administration by, inter alia, the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelial including transdermal, ophthalmic, sublingual and buccal; topically including ophthalmic, dermal, ocular, rectal and nasal inhalation via insufflation, aerosol, and rectal systemic.

In powders, the carrier, diluent, or excipient may be a finely divided solid that is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier, diluent or excipient having the necessary compression properties in suitable proportions and compacted in the shape and size desired. For oral therapeutic administration, the active compound may be incorporated with the carrier, diluent, or excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound(s) in such therapeutically useful compositions is preferably such that a suitable dosage will be obtained. The therapeutic compositions preferably contain up to about 99% of the active ingredient.

Liquid carriers, diluents, or excipients may be used in preparing solutions, suspensions, emulsions, syrups, elixirs, and the like. The active ingredient of this invention can be dissolved or suspended in a pharmaceutically acceptable liquid such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fat. The liquid carrier, excipient, or diluent can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmo -regulators.

Suitable solid carriers, diluents, and excipients may include, for example, calcium phosphate, silicon dioxide, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, ethylcellulose, sodium carboxymethyl cellulose, microcrystalline cellulose,

polyvinylpyrrolidine, low melting waxes, ion exchange resins, croscarmellose carbon, acacia, pregelatinized starch, crospovidone, HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar-agar, tragacanth, or mixtures thereof.

Suitable examples of liquid carriers, diluents and excipients for oral and parenteral administration include water (particularly containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil), or mixtures thereof.

For parenteral administration, the carrier, diluent, or excipient can also be an oily ester such as ethyl oleate and isopropyl myristate. Also contemplated are sterile liquid carriers, diluents, or excipients, which are used in sterile liquid form compositions for parenteral administration.

Solutions of the active compounds as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. A dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.

Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is preferably sterile and fluid to provide easy syringability. It is preferably stable under the conditions of manufacture and storage and is preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier, diluent, or excipient may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion, and by the use of surfactants. The prevention of the action of

microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions may be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compounds in the required amounts, in the appropriate solvent, with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions may be prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation may include vacuum drying and the freeze drying technique that yields a powder of the active ingredient or ingredients, plus any additional desired ingredient from the previously sterile- filtered solution thereof. The compounds of the invention may be administered in an effective amount by any of the conventional techniques well-established in the medical field. The compounds employed in the methods of the present invention including, for example, the compounds of formula I may be administered by any means that results in the contact of the active agents with the agents' site or sites of action in the body of a patient. The compounds may be administered by any conventional means available.

Preferably the pharmaceutical composition is in unit dosage form, e.g. as tablets, buccal tablets, troches, capsules, elixirs, powders, solutions, suspensions, emulsions, syrups, wafers, granules, suppositories, or the like. In such form, the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient; the unit dosage forms can be packaged compositions, for example packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids. The unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form. In addition, dosage forms of the present invention can be in the form of capsules wherein one active ingredient is compressed into a tablet or in the form of a plurality of microtablets, particles, granules or non-perils. These microtablets, particles, granules or non-perils are then placed into a capsule or compressed into a capsule, possibly along with a granulation of the another active ingredient.

The compounds of the invention may also be administered in combination with other therapies, for example, for Pompe disease, compound of the invention may be administered in conjunction with enzyme replacement therapy. Alternatively, the compounds of the invention may be administered in conjunction with other compounds useful for the treatment of diabetes or Pompe disease.

Synthesis of Compounds of the Invention

Schemes 1 and 2 show the general methodology used for the synthesis of compounds of the invention. Direct chlorosulfonylation of 2-indolone at the 5 position followed by piperidine displacement yield analogues with modifications on the aromatic ring attached to the piperidine. Alternatively, reaction of l-(4-(piperazin- l-yl)phenyl)ethanone with sulfonyl clorides in the presence of a suitable base such us triethylamine or its reaction with carboxylic acid in coupling conditions yields analogues with modifications at the sulfonamide portion of the molecule. Scheme 1

Scheme 2

P /⁼ f— \ ^EDC. ^{DMAp Q}- -N N-

OH / r~ ( ^\⁴— y // H ' ,^NH DMF, rt ~

R₃

Chemistry general 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 x 20 cm, 60 A, 250 μιη). 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 (CDC1₃ 7.27 ppm, DMSO-J₆ 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 time were recorded through standard gradient 4% to 100% acetonitrile (0.05% TFA) over 7 minutes using Luna Ci8 3 micron 3 x 75 mm column with a flow rate of 0.800 mL/min. High resolution mass spectral data was collected in-house using and Agilent 6210 time-of- flight mass spectrometer, also coupled to an Agilent Technologies 1200 series HPLC system.

2- Oxo indo line- 5 - sulfo nyl chloride . Bouchikhi, F; Anizon, F; Moreau, P. Eur. J. Med. Chem., 2008, 43, 755-762, incorporated by reference in its entirety. 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 h and heated at 70 °C for 1 h. The reaction was slowly quenched with water, the light pink solid was filtered and dried to give 5.33 g (yield 61%) pink solid which was used directly in the next reaction without further purification. 1H NMR (400 MHz, DMSO-de) 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 l-(4-(piperazin-l-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 for over night. The crude mixture was filtered through a frit and purified by preparative HPLC to give the final product.

General protocol C. A solution of l-(4-(piperazin-l-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 for over night. 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 l-(4-(piperazin- l-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 (0.043 g, 0.350 mmol). The reaction mixture was stirred at room temperature for over night. The crude mixture was filtered through a frit and purified by preparative HPLC to give the final product.

5-(4-(4-Acetylphenyl)piperazin- l-ylsulfonyl)indolin-2-one (18). A solution of l-(4-(piperazin- l- yl)phenyl)ethanone (485 mg, 2.374 mmol), triethylamine (0.602 mL, 4.32 mmol) in DMF (10.0 mL) was treated at room temperature with 2-oxoindoline-5-sulfonyl chloride (500 mg, 2.158 mmol). The reaction mixture was stirred at room temperature for over night. 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 C₂oH₂₂N₃0₄S[M+H⁺] 400.1331, found 400.1327.

5-(Piperazin- l-ylsulfonyl)indolin-2-one (19). 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 for over night. 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-de) 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⁺] .

5 - ( 4- Acetylpiperazin- 1 - ylsulfo n yl) indo lin-2- one ( 1 ) . 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⁺] .

5- ( 4- (4-Hydroxyphenyl)piperazin- 1 - ylsulfo nyl)indolin-2-one ( 2) . 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, 7=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⁺] .

5-(4-(4-Hydroxyphenyl)piperazin- l-ylsulfonyl)indolin-2-one (3). 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, 7=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⁺] .

5-(4-p-Tolylpiperazin- 1 -ylsulfo nyl indolin-2-one (4). 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⁺].

4- (4- (2- Oxoindo lin- 5 - ylsulfo nyDpiperazin- 1 - yPbenzonitrile (8) . The title compound was prepared according to the general protocol A. 1H NMR (400 MHz, DMSO-de) 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⁺] .

5-(4-(4-Nitrophenyl)piperazin- 1 -ylsulfo nyl)indolin-2-one (10). The title compound was prepared according to the general protocol A. 1H NMR (400 MHz, DMSO-de) 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⁺] .

5 - (4- (4-Fluorophenyl)piperazin- 1 - ylsulfo nyl) indo lin-2- one (11). The title compound was prepared according to the general protocol A. 1H NMR (400 MHz, DMSO-de) 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-de) δ ppm - 126.55 - - 123.72 (m); LCMS RT = 5.148 min, m/z 376.1 [M+H⁺] .

5-(4-(4-Chlorophenyl)piperazin- l-ylsulfonyl)indolin-2-one (12). The title compound was prepared according to the general protocol A. 1H NMR (400 MHz, DMSO-de) 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⁺] .

5-(4-(4-Bromophenyl)piperazin- l-ylsulfonyl)indolin-2-one (13). The title compound was prepared according to the general protocol A. 1H NMR (400 MHz, DMSO-de) 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⁺] .

5-(4-(4-(Trifluoromethyl)phenyl)piperazin- l-ylsulfonyl)indolin-2-one (14). The title compound was prepared according to the general protocol A. 1H NMR (400 MHz, DMSO-de) 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-de) δ ppm -59.64 (s); LCMS RT = 5.782 min, m/z 426.1 [M+H⁺].

5-(4-(3-(Trifluoromethyl)phenyl)piperazin- l-ylsulfonyl)indolin-2-one (15). The title compound was prepared according to the general protocol A. 1H NMR (400 MHz, DMSO-de) 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-de) δ ppm -61.14 (s); LCMS RT = 5.776 min, m/z 426.1 [M+H⁺] .

4- (4- (2- Oxoindo lin- 5 - ylsulfo nyPpiperazin- 1 - yDbenzaldehyde (9) . A solution of 4-(piperazin- l- 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 h. 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⁺] .

Methyl 4-(4-(2-oxoindolin-5-ylsulfonyl)piperazin- l-yl)benzoate (7). A solution of methyl 4- (piperazin- l-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⁺] .

4-(4-(2-Oxoindolin-5-ylsulfonyl)piperazin- l-yl)benzoic acid (6). A suspension of methyl 4- (4- (2- oxoindolin-5-ylsulfonyl)piperazin- l-yl)benzoate (15) (240 mg, 0.578 mmol) in 6 N HC1 (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-de) 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⁺] .

5-(4-(4-(Hydroxymethyl)phenyl)piperazin- l-ylsulfonyl)indolin-2-one (5). DIBAL-H (0.325 mL, 1.0m in THF, 0.325 mmol) was added drop wise to a solution of methyl 4-(4-(2-oxoindolin-5- ylsulfonyl)piperazin- l-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-de) 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⁺] .

5-(4-(4-Acetylphenyl)piperazin- l-ylsulfonyl)-6-chloroindolin-2-one (17). The title compound was prepared according to the general protocol B. 1H NMR (400 MHz, DMSO-de) 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⁺] .

5-(4-(4-Acetylphenyl)piperazin- l-ylsulfonyl)-3,3-dichloroindolin-2-one (16). The title compound was prepared according to the general protocol B. 1H NMR (400 MHz, DMSO-de) 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⁺] .

5-(4-(4-Acetylphenyl)piperazine- l-carbonyl)indolin-2-one (32). The title compound was prepared according to the general protocol D. 1H NMR (400 MHz, DMSO-Je) δ ppm 10.55 (s, 1 H), 7.70 - 7.88 (m, 2 H), 7.23 - 7.40 (m, 2 H), 6.98 (d, J=9.2 Hz, 2 H), 6.85 (d, J=8.0 Hz, 1 H), 3.63 (br. s., 4 H), 3.51 (s, 2 H), 3.37 - 3.46 (m, 4 H), 2.45 (s, 3 H); LCMS RT = 4.14 min, m/z 364.1 [M+H⁺]; HRMS (ESI) m/z calcd for C₂₁H₂₂N₃O₃S [M+H⁺] 364.1656, found 364.1661.

6-(4-(4-Acetylphenyl)piperazine-l-carbonyl)-3,4-dihydroquinolin-2(lH)-one (33). The title compound was prepared according to the general protocol D. 1H NMR (400 MHz, DMSO-de) δ ppm 10.22 (s, 1 H), 7.69 - 7.88 (m, 2 H), 7.26 (d, J=1.8 Hz, 1 H), 7.22 (dd, J=8.0, 2.0 Hz, 1 H), 6.90 - 6.98 (m, 2 H), 6.86 (d, J=8.2 Hz, 1 H), 3.60 (br. s., 4 H), 3.51 (br. s., 4 H), 2.88 (t, J=7.5 Hz, 2 H), 2.44 - 2.45 (m, 1 H), 2.40 - 2.43 (m, 4 H); LCMS RT = 4.30 min, m/z 378.2 [M+H⁺]; HRMS (ESI) m/z calcd for C₂₂H₂₄N₃O₃S [M+H⁺] 378.1812, found 378.1815.

5-r4-(4-Acetyl-phenyl)-piperazine-l-carbonyll-6-chloro-l,3-dihydro-indol-2-one (35). A solution of l-(4-(piperazin-l-yl)phenyl)ethanone (86.0 mg, 0.420 mmol; available from Aldrich), 6- chloro-2-oxoindoline-5-carboxylic acid (74.1 mg, 0.350 mmol; available from Ryan Scientific) in DMF (2.00 mL) will be treated at room temperature with EDC (67.0 mg, 0.350 mmol) and DMAP (43.0 mg, 0.350 mmol). The reaction mixture will be stirred at room temperature for overnight. The crude mixture will be filtered through a frit and purified by preparative HPLC to give the final product.

EXPERIMENTAL SECTION

Compounds of the invention were tested against enzymes in a context as native as possible, including testing the hydrolytic capacity of acid alpha glucosidase in tissue homogenate. Many isolated glucosidases require allosteric activation to be functional, so purified enzyme preparations, which depend upon the use of detergents to induce the active conformation and catalytic activity of the enzyme, were avoided. It had been observed that it is common to find compounds that can bind isolated enzymes but are inactive in cellular lysates, 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 acid alpha glucosidase activity, such as 4-methylumbelliferyl CC-D-glucopyranoside. Upon hydrolysis, the blue fluorescent dye 4-methylumbelliferone is liberated, producing a fluorescent emission at 440 nm when excited at 370 nm. In addition, as a control for auto fluorescence, a second substrate was used, resorufin cc-D-glucopyranoside, which liberates the red dye resorufin at an emission wavelength of 590 nm when excited at 530 nm.

a- -g ucopyranos e

Compounds of the invention were screened against the acid alpha glucosidase purified enzyme, as well as acid alpha glucosidase enzyme obtained directly from tissue homogenate. A probe specific for acid alpha glucosidase activity, such as 4-methylumbelliferyl a-D- glucopyranoside was used. Upon hydrolysis, the blue fluorescent dye 4-methylumbelliferone is liberated, producing a fluorescent emission at 440 nm when excited at 370 nm. In addition, as a control for auto fluorescence, a second substrate, resorufin a -D-glucopyranoside, was used that liberates the red dye resorufin at an emission wavelength of 590 nm when excited at 530 nm. Data for compounds of the invention with purified enzyme as substrate is set forth in Table 1. Data for compounds of the invention with spleen homogenate as substrate is set forth in Table 2.

Table 1

Comp. i R₂ R₃ R₄ AC50 (μΜ) AC50 (μΜ) No. Blue Red

3 4-OH-Ph H H H 1.88 2.36

4 4-CH₃-Ph H H H >100 >100

5 4-CH₂-OH-Ph H H H 12.98 12.98

6 4-C(0)OH-Ph H H H 1.29 1.45

7 4-C(0)OCH₃-Ph H H H 25.90 36.60

8 4-CN-Ph H H H 2.90 3.26

9 4-C(0)H-Ph H H H 3.65 4.10

10 4-N0₂-Ph H H H 3.65 3.26

11 4-F-Ph H H H 236 188

12 4-Cl-Ph H H H 94.31 94.31

13 4-Br-Ph H H H 236 188

14 4-CF₃-Ph H H H 236 236

15 3-CF₃-Ph H H H 14.94 14.94

16 4-C(0)CH₃-Ph CI CI H 32.60 41.05

17 4-C(0)CH₃-Ph H H 2-Cl 1.63 1.63

18 4-C(0)CH₃-Ph H H H 0.65 0.51

Table 2

Comp. i R₂ R₃ R₄ AC50 (μΜ) AC50 (μΜ) No. Spleen Blue Spleen Red

3 4-OH-Ph H H H 6.67 11.87

4 4-CH₃-Ph H H H >100 >100

5 4-CH₂-OH-Ph H H H 25.90 12.98

6 4-C(0)OH-Ph H H H 2.59 1.63

7 4-C(0)OCH₃-Ph H H H 20.57 1.15

8 4-CN-Ph H H H 1.15 1.63

9 4-C(0)H-Ph H H H 4.60 5.16

10 4-N0₂-Ph H H H 3.65 5.79

11 4-F-Ph H H H >100 >100

12 4-Cl-Ph H H H >100 >100

13 4-Br-Ph H H H >100 >100

14 4-CF₃-Ph H H H >100 >100

15 3-CF₃-Ph H H H 18.81 8.40

16 4-C(0)CH₃-Ph CI CI H 32.60 20.57

17 4-C(0)CH₃-Ph H H 2-Cl 1.83 4.60

18 4-C(0)CH₃-Ph H H H 1.29 1.03

To evaluate the potential capacity of compounds of the invention to stabilize acid alpha glucosidase and thus be useful in the treatment of Pompe disease, preferred compounds of the invention were evaluated on a thermo-shift assay to identify a compound's ability to prevent the loss of enzyme function when the enzyme is warmed to 66 °C for 60 minutes. It is well known that hydrolytic enzymes lose their catalytic function upon temperature elevation below the denaturation point due to a progressive aggregation and loss of activation state. Thus, when warming acid alpha glucosidase at 66 °C over 60 minutes, a progressive lost of function is observed with about 40% of original hydrolytic capacity remaining after 1 hour. Compounds able to bind to the enzyme and avoid the loss hydrolytic function demonstrate a capacity to stabilize the enzyme. It has been shown that compounds able to maintain enzyme stability also promote folding, and therefore have a potential capacity of being "chaperone" molecules and thus useful in the treatment of Pompe disease.

The results of this experiment are shown in FIG. 1. As shown in FIG. 1, in the absence of compound, the enzyme loses most of its hydrolytic capacity within 1 hour. In the presence of preferred compounds of the invention, the enzyme is able to maintain its function. qHTS Assay for Binders of 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, 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). In 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).

1536-well assay protocol for the human alpha-glucosidase:

(1) Add 2 μΐ/well of enzyme (4 nM final)

(2) Add 23 nL compounds in DMSO solution. The final titration was 0.7 nM to 77 μΜ.

(3) Add 1 μΐ of substrate (400 μΜ final)

(4) Incubate at room temperature for 20 min.

(5) Add 2 μΐ 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 Binders 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, 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 (Table 1) 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)

1536-well assay protocol for the human alpha-glucosidase:

(1) Add 2 ul/well of enzyme (4 nM final)

(2) Add 23 nL compounds in DMSO solution. The final titration was 0.7 nM to 77 uM.

(3) Add 1 ul of substrate (400 uM final)

(4) Incubate at room temperature for 20 min.

(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)

(6) Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex=365 nm and

Em=440nm.

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

This is a fluorogenic enzyme assay with resorufin- alpha-D-pyrano side 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 (Table 1) values were determined from concentration-response data modeled with the standard Hill equation.

1536-well assay protocol for the human alpha-glucosidase:

( 1 ) Add 2 ul/well of enzyme (4 nM final)

(3) Add 1 ul of substrate (400 uM final)

(4) Incubate at room temperature for 20 min.

(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)

Em=440nm. qHTS Assay for Binders 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 (Table 2) 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)

1536-well assay protocol for the alpha-glucosidase from human spleen homogenate:

(1) Add 2 ul/well spleen homogenate (1 ug)

(2) Add 23 nL compounds in DMSO solution. The final titration was 0.5 nM to 58 uM.

(3) Add 2 ul of substrate ( 1 mM final)

(4) Incubate at 37 C for 40 min

(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)

Em=440nm.

Confirmation of Binders and Activators of Human alpha-Glucosidase From Spleen Homogenate

(1) Add 2 ul/well spleen homogenate (1 ug) (2) Add 23 nL compounds in DMSO solution. The final titration was 0.5 nM to 58 uM.

(3) Add 2 ul of substrate (1 mM final)

(4) Incubate at 37 C for 40 min

(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)

Em=440nm.

Confirmation of Binders 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 (Table 2) were determined from concentration-response data modeled with the standard Hill equation.

(1) Add 2 ul/well spleen homogenate (1 ug)

(3) Add 2 ul of substrate (1 mM final)

(4) Incubate at 37 C for 40 min

(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)

Em=440nm. qHTS Assay for Binders and Activators of Human alpha-Glucosidase Cleavage of Glycogen!

This will be 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 will be detected using the Amplex Red Glucose Oxidase Assay Kit (Invitrogen catalog #: A22189). The product of this reaction will be read with a fluorescence plate reader with an excitation at 573 nm and an emission at 610 nm. Data will be normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values will be determined from concentration-response data modeled with the standard Hill equation. Assay buffer for enzyme reaction will be: 50 mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20 (pH 5.0)

Assay buffer for Amp lex Red reaction: Tris-HCl, pH 7.5

1536-well assay protocol for the alpha-glucosidase assay will be:

(1) Add 2 ul/well alpha-glucosidase enzyme solution (4 nM final)

(2) Add 23 nL compounds in DMSO solution. The final titration will be 0.7 nM to 77 uM.

(3) Add 1 ul of glycogen substrate solution (30 ug)

(4) Incubate at 37 C for 40 min

(5) Add 2 ul 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=610nm. qHTS Selectivity for alpha glucosidase: Assay for Binders and Activators ofN370S

glucocerebrosidase

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. This assay determines whether the compounds of the invention are selective for alpha glucosidase. 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.

1536-well assay protocol:

(1) Add 2 ul/well of spleen homogenate (27 ug final)

(2) Add 23 nL compounds in DMSO solution. The final titration was 0.5 nM to 58 uM. (3) Add 2 ul of substrate (1 mM final)

(4) Incubate at 37C for 40 min.

(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)

Em=440nm.

The compounds of the invention did not inhibit beta-glucosidase when tested in this assay. qHTS Selectivity for alpha glucosidase: Assay for Binders 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. This assay determines whether the compounds of the invention are selective for alpha glucosidase. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 1. 4-Methyllumbelliferone, can be excited at 365 nm and emits at 440 nm. This fluorescence 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). In 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 4.5), 0.005% Tween-20, pH 4.5. (pH 4.5 is an optimal condition for this enzyme assay)

1536-well assay protocol for the human alpha-galactosidase:

(1) Add 2 ul/well of enzyme (12 nM final)

(3) Add 1 ul of substrate (40080 uM final)

(4) Incubate at room temperature for 20 min.

(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)

Em=440nm

The compounds of the invention did not inhibit alpha-galactosidase when tested in this assay. Selectivity for alpha glucosidase: Confirmation of Binders 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.

1536-well assay protocol for the alpha-galactosidase from human spleen homogenate:

(1) Add 2 ul/well spleen homogenate (1 ug)

(3) Add 2 ul of substrate (1 mM final)

(4) Incubate at 37C for 40 min

(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)

Em=440nm.

The compounds of the invention did not inhibit alpha-galactosidase when tested in this assay.

Selectivity for alpha Glucosidase: Confirmation of Binders 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, 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). In 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) 1536-well assay protocol for the human alpha-glucosidase:

(1) Add 2 ul/well of enzyme (4 nM final)

(3) Add 1 ul of substrate (400 uM final)

(4) Incubate at room temperature for 20 min.

(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)

Em=440nm.

The compounds of the invention did not inhibit alpha-galactosidase when tested in this assay. qHTS Assay for Binders of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Stabilizers of Alpha-Glucosidase Under Thermal Defunctionalization Conditions

This assay involves heating purified 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 only DMSO or 50x IC50 of compound before being exposed to 68 °C heat inactivation. The heating 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. See FIG. 1.

Temperature Denature Protocol for alpha-Glucosidase

Assay buffer: 50 mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20

1. Prepare 80X solution 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 50X IC50:

50x IC50 1-DNJ = 2.5 uM

50x IC50 of the test compounds = 50 uM

4. Incubate 10 min RT after compound addition.

5. Distribute 10 ul from each tube into PCR tubes. 6. Time course of heating: Heat solutions at 68C for 60 min, taking a set of tubes out at 10, 20, 30, 45, and 60 min, while keeping one set on ice whole time.

7. After heating, take out tubes and place on ice for a minimum of 5 min.

8. Transfer 10 ul solution to 790 ul (1:80 dilution) buffer in 1.5 ml tube.

9. Vortex and take three 20 ul aliquots and put in 384 plate.

10. Add 20 ul substrate solution to each well (100 uM 4-methylumbelliferyl alpha-D- glucopyranoside).

11. Incubate 20 min.

12. Add 25 ul stop solution (1 M NaOH, 1 M glycine pH 10).

13. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex=365 nm and

Em=440nm.

In Pompe disease there is not a single common mutation; therefore, compounds of the invention were evaluated for chaperone and translocation capacity using wild type human

fibroblasts. The specificity of the mouse monoclonal anti-GAA antibody was tested. On western blots, the antibody recognized the GAA protein (kDa) in protein lysate from human embryonic kidney (HEK) cells electroporated with a pCMV6XL6 plasmid containing the GAA cDNA (Acc No. NM_000152.2); non-electroporated HEK cells or WT fibroblast protein lysates (Figure 2) did not show a GAA specific signal. In general, WT fibroblasts expressed low levels of GAA. Thus, DMSO-treated WT fibroblasts showed GAA staining in about 15 % of the cells and the GAA stain co-localized with the lysosomal marker cathepsin D. Although, only 15 % of the cells stained positive, that signal was due to GAA since the antibody used was specific (Figure 2). Moreover, WT fibroblasts stained with Alexa-488 and Alexa-555 secondary antibodies showed no signal at the same laser settings, indicating that the signal from the GAA antibody is specific and not due Alexa- 488 secondary antibody background.

Treatment of WT fibroblasts with 15 μΜ or 5 μΜ of compound 17 up-regulated the translocation of GAA to the lysosomes significantly. About 40 % of the cells showed translocation of GAA with treatment at 15 μΜ concentration and 30 % with 5 μΜ. Compound 17 also induced the enlargement of lysosomes when compared to DMSO treated fibroblasts. Surprisingly, the non- inhibitory analogue compound 32 also displayed very good chaperone activity, confirming that the inhibitory and chaperone activities do not always correlate. Fibroblasts treated with 15 μΜ of compound 32 showed translocation of GAA to the lysosomes in about 50% of the cells while treatment with 5 μΜ resulted in translocation of GAA in about 40 % of the cells. Treatment with compound 32 did not cause lysosomal enlargement. It should be noted that GAA staining and translocation to lysosomes in cells significantly decreased with increasing cell passage number; WT fibroblast with passage number 7 showed GAA staining in lysosomes in about 15 % of the cells while those with passage number 8 showed only 10 % of the cells positive for GAA translocation to the lysosomes, this was reduced to 5 % at passage number 9. This was also the case for compound treated cells, where translocation of GAA to lysosomes was significantly reduced with increased passage number.

Microscale thermophoresis

Alpha glucosidase was labeled with a fluorescent dye NT-495 (Nano Temper Technologies) and the final concentration of the protein applied in equilibrium binding experiments was estimated to be 50 nM. A 16-point titration series of selected compounds was prepared and mixed with the protein at a 1: 1 ratio in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl₂, pH 7.5. Depending on the solubility of each compound, the final concentration range varied. For example, compound 32 started at 2 mM and was titrated down to 61.04 nM. Monolith NT™ hydrophilic capillaries (Nano Temper Technologies) were filled with the samples after a 15-min incubation.

Samples were subsequently scanned to locate capillaries on a Nano Temper Monolith NT.115 instrument, and thermophoresis was successively measured in each capillary. Measurement took place at room temperature in a range of IR- laser powers: 20%, 40% and 80%, with the blue LED power set at 100%. Specifically, a laser-on time of 30 seconds and a laser-off time of 5 seconds were applied at each IR-laser power. Data normalization and curve fitting were performed using Nano Temper Analysis 1.2.101. The change in normalized fluorescence was used for Kd

determination.

Using this protocol, compound 32 exhibited an average Kd of 121 μΜ and compound 17 exhibited an average Kd of 20.67 μΜ with the control compound DNJ showing an average Kd of 1.87 μΜ. In vitro ADME

Assessement of the permeability and metabolic stability of compound 18 was also analyzed in microsomal stability (Table 3) and Caco-2 permeability assays (Table 4). Compound 18 has a very low intrinsic clearance and a half life of more than 1 hour in mouse microsomes. After incubation with compound 18 in mouse liver microsomes for 60 minutes in the presence of

NADHP, the mean parent molecule remaining was more than 60%. In the absence of NADHP, the parent compound was almost intact after 60 minutes. This indicates that the major metabolic process is likely through cytochrome P450-dependent oxidation. See Table 3.

Table 3

Plus NADPH Minus NADPH

Parent

Parent remaining remaining

test 2 nd

test 1^st mean 1^st 2^nd

mean

ID cone

species

(μ ) (%) (%) (%) (%) (%) (%)

Verapamil mouse 1 0.3 0.3 0.3 106.4 1 10.7 108.5

Warfarin mouse 1 104.2 94.7 99.5 131 .9 1 1 1 .6 121 .7

mouse 1 53.4 67.9 60.6 1 17.7 95.3 106.5

Caco-2 data indicated that compound 18 displayed very good cell permeability in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)). See Table 4. The B-A permeability was higher than the corresponding A-B permeability (efflux ratio - 1.9 [B-A/ A-B]. This suggests that compound 18 has potential to remain at high concentrations in the gastrointestinal track. Table 4

test

mean A->B P_a mean B->A P_a

cone. Efflux

ID comment

ratio^b

(μΜ) (10⁶ cm s ¹) (10⁶ cm s ¹)

Low

Ranitidine 1 0 0.6 5.3 8.5 permeablility

control High

Warfarin 1 0 61 .7 5.7 0.1 permeability

control

Quinidine 1 0 2.7 48.4 1 7.9 P-gp substrate

A full pharmacokinetic analysis of compound 18 was carried out. The plasma and intestine concentration of compound 18 in male Swiss Albino Mice after single oral gavage administration of compound 18 at a dose of 30 mg/kg were measured. As data shown in Table 5, the compound 18 displayed a very high concentration in intestine which is greater than plasma confirming our hypothesis from the Caco-2 data. The pharmacokinetic parameters of compound 18 in plasma and intestine were calculated and AU o-t) of plasma and intestine used for determination of intestine to plasma ratio were summarized in Table 6. The intestine to plasma ratio of compound 18 in male Swiss Albino Mice was found to be 78.7. Compound 18 possessed a reasonable long intestine half- life (f 1/2 = 5.04 h) in comparison with half- life in plasma (tm = 0.51 h). The concentration of compound 18 in intestine reached at 47.65 μιηοΐ/kg in only 5 minutes and at 156.35 μιηοΐ/kg as C_max in 30 minutes (IC₅₀ of 18 was 0.52 μΜ).

Table 5

Compound 18 Concentration (Mean ± SD)

Time (h) Plasma (ng/mL) μΜ Intestine (ng/g) μΜοΙ/kg

1 21.89+10.31 0.055 13988.30+14620.27 35.02

2 8.78+8.30 0.022 2065.27+1042.06 5.17

4 3.31+2.66 0.008 1410.18+1251.07 3.53

8 0.41+0.70 0.001 498.30+543.80 1.25

12 0.00+0.00 0 1436.93+1187.98 3.6

24 1.60+1.41 0.004 999.18+600.06 2.5

Table 6

t 1/2 ^A max Cma AUCl_as, AUG,.- DAUC CI MRT last

Compound 18

(1/h) (h) (h) (ng/mL) or (ng/g) (ng-h/ml) (ng-h/ml) (%) (ml/hr/kg) (h)

4618.32

Plasma 0.097 7.13 0.08 867.87 884.34 1.86 33923.75 0.51

62455.66

Intestine 0.03 21.1 0.5 68318.29 98675.75 30.76 304.03 5.04

In addition, no behavioral changes were observed in animals administered with test compound throughout the whole study period. Overall, this means that upon 30 mg/kg oral gavage administration compound 18 displays in intestine concentrations above its acid alpha-glucosidase IC₅₀ for more than 24 h.

Regarding compound 18's potential capacity for the treatment of Pompe's disease, the compound reaches levels in plasma above its IC₅₀ for a short period of time. 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 K_d, the displacement equilibrium between the natural substrate and the inhibitor also depends on the concentration of the inhibitor. For that reason, useful chaperone inhibitors should have a relatively low half life, allowing their elimination and restoring the function of the enzyme upon translocation.

Compound 18 has a relatively low inhibitory activity (IC₅₀ = 0.52 μΜ) and low half life in plasma. REFERENCES

¹ Hirschhorn, R; Reuser, AJJ. Glycogen storage disease type II: acid cc-glucosidase (acid maltase) deficiency. In: Scriver, CR; Beaudet, AL; Sly, WS; Valle, D (Eds.). The metabolic and molecular bases of inherited disease, McGraw-Hill, New York, 2001, pp 3389-3420. The entirety of this reference is incorporated herein in its entirety.

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. The entirety of this reference is incorporated herein in its entirety.

(a) 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. The entirety of this reference is incorporated herein in its entirety, (b) http ://w ww . myo z vme . com/ The entirety of this reference is incorporated herein in its entirety.

⁴ http://www.rxlist.com/myozvme-drug.htm The entirety of this reference is incorporated herein in its entirety.

5 http://en.wikipedia.org/wiki/Myozyme The entirety of this reference is incorporated herein in its entirety.

⁶ (a) Raben, N; Plotz, P; Byrne, BJ. Curr. Mol. Med., 2002, 2, 145-166. The entirety of this reference is incorporated herein in its entirety, (b) http://www.hgmd.cf.ac.uk The entirety of this reference is incorporated herein in its entirety.

7 (a) Montalvo, AL; Cariati, R; Deganuto, M; Guerci, V; Garcia, R; Ciana, G; Bembi, B; Pittis, MG. Mol. Genet. Metab., 2004, 81, 203-208. The entirety of this reference is incorporated herein in its entirety, (b) 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. The entirety of this reference is incorporated herein in its entirety, (c) Reuser, AJ; Kroos, M; Willemsen, R; Swallow, D; Tager, JM; Galjaard, H. /. Clin. Invest., 1987, 79, 1689-1699. The entirety of this reference is incorporated herein in its entirety, (d) Reuser, AJ; Kroos, M; Oude Elferink, RP; Tager, JM. /. Biol. Chem., 1985, 260, 8336-8341. The entirety of this reference is incorporated herein in its entirety.

Beck, M. Human Genetics, 2007, 121, 1-22. The entirety of this reference is incorporated herein in its entirety.

9 (a) 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. The entirety of this reference is incorporated herein in its entirety, (b) Okumiya, T; Kroos, MA; Vliet, LV; Takeuchi, H; Van der Ploeg, AT; Reuser, AJ. Mol. Genet. Metab., 2007, 96», 49-57. The entirety of this reference is incorporated herein in its entirety.

1⁰ Fan, JQ; Ishii, S; Suzuki, Y. The FEBS J., 2007, 274, 4962-4971. The entirety of this reference is incorporated herein in its entirety.

¹¹ http://en.wikipedia.org/wiki/Alpha-glucosidase inhibitor The entirety of this reference is incorporated herein in its entirety.

¹² Motabar, O; Shi, Z; Goldin, E; Liu, K; Southall, N; Sidransky, E; Austin, CP; Griffiths, GL; Zheng, Z. Anal. Biochem., 2009, 390, 79-84. The entirety of this reference is incorporated herein in its entirety.

¹³ (a) John, M; Wendeler, M; Heller, M; Sandhoff, K; Kessler, H. Biochemistry, 2006, 45, 5206- 5216. (b) Zwerschke, W; Mannhardt, B; Massimi, P; Nauenburg, S; Pirn, D; Nickel, W; Banks, L; Reuseri, AJ; Jansen-Diirr, P. /. Biol. Chem., 2000, 275, 9534-9541. The entirety of this reference is incorporated herein in its entirety.

¹⁴ Motabar, O; Shi, Z; Goldin, E; Liu, K; Southall, N; Sidransky, E; Austin, CP; Griffiths, GL; Zheng, W. Anal. Biochem., 2009, 390, 79-84. The entirety of this reference is incorporated herein in its entirety.

¹⁵ Bouchikhi, F; Anizon, F; Moreau, P. Eur. J. Med. Chem., 2008, 43, 755-762. The entirety of this reference is incorporated herein in its entirety.

¹⁶ Austin, CP; Brady, LS; Insel, TR; Collins, FS. Science, 2004, 306, 1138-1139. The entirety of this reference is incorporated herein in its entirety.

Claims

What is Claimed:

1. A method comprising

contacting at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula I, or a

pharmaceutically acceptable salt thereof

wherein

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R4 is H, halogen, or C_halky!

2. The method of claim 1, wherein the compound of formula I binds to wild type alpha

glucosidase.

3. The method of claim 1, wherein the compound of formula I binds to mutant alpha glucosidase.

4. The method of claim 1, wherein the compound of formula I binds to misfolded alpha

glucosidase.

5. The method of claim 1, wherein the compound of formula I binds to recombinant alpha

glucosidase.

6. The method of claim 1, wherein the compound of formula I binds to at least one of mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase and stabilizes the at least one of mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase.

7. The method of claim 1, wherein the compound of formula I binds to at least one of mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase and increases the activity of the at least one of mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase.

8. The method of claim 1, wherein the compound of formula I is selected from a compound in the following table:

9. The method of claim 1, wherein R₂, R₃, and R4 are each H.

10. The method of claim 1, wherein R₂ and R₃ are each halogen.

11. The method of claim 11, wherein R₂ and R₃ are each CI.

12. The method of claim 1, wherein R₄ is halogen.

13. The method of claim 12, wherein R₄ is CI.

14. The method of claim 1, wherein Ri is phenyl substituted at the 4-position.

15. The method of claim 1, wherein Ri is phenyl substituted with Ci_₆alkoxy, -OH, C_1-6alkyl

optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OC_1-6alkyl,

-C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

16. The method of claim 1, wherein Ri is phenyl substituted with

-OH, Ci-₆alkyl

optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

17. The method of claim 1, wherein Ri is phenyl substituted with -OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

18. The method of claim 1, wherein Ri is phenyl substituted with -C(0)Ci_₆alkyl.

19. The method of claim 1, wherein Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

20. The method of claim 1, wherein the compound of formula I is

21. The method of claim 1, wherein the compound of formula I is:

22. A method of treating diabetes in a patient comprising administering to the patient a

therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof

wherein

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_ 6alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci-₆alkyl; and R₄ is H, halogen, or C_halky!.

23. The method of claim 22 wherein the compound of formula I is selected from a compound in the following table:

24. The method of claim 22, wherein R₂, R₃, and R₄ are each H. 25. The method of claim 22, wherein R₂ and R₃ are each halogen. 26. The method of claim 25, wherein R₂ and R₃ are each CI.

27. The method of claim 22, wherein R₄ is halogen.

28. The method of claim 27, wherein R₄ is CI.

29. The method of claim 22, wherein Ri is phenyl substituted at the 4-position.

30. The method of claim 22, wherein Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OC_1-6alkyl,

-C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

31. The method of claim 22, wherein Ri is phenyl substituted with

-OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H,

32. The method of claim 22, wherein Ri is phenyl substituted with -OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -NO₂.

33. The method of claim 22, wherein Ri is phenyl substituted with -C(0)Ci_₆alkyl.

34. The method of claim 22, wherein Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci-₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen,

Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

35. The method of claim 22, wherein the compound of formula I is

36. The method of claim 22 wherein the compound of formula I is:

37. A method of treating Pompe disease in a patient comprising administering to the patient a

wherein

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and P3 are each independently H or halogen; and

P4 is H or halogen.

38. The method of claim 37, wherein the compound of formula I is selected from a compound in the following table:

39. The method of claim 37, wherein R₂, R₃, and R₄ are each H.

40. The method of claim 37, wherein R₂ and R₃ are each halogen.

41. The method of claim 40, wherein R₂ and R₃ are each CI.

42. The method of claim 37, wherein R₄ is halogen.

43. The method of claim 42, wherein R₄ is CI.

44. The method of claim 37, wherein Ri is phenyl substituted at the 4-position.

45. The method of claim 37, wherein Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OC_1-6alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

46. The method of claim 37, wherein Ri is phenyl substituted with

-OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

47. The method of claim 37, wherein Ri is phenyl substituted with -OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

48. The method of claim 37, wherein Ri is phenyl substituted with -C(0)Ci_₆alkyl.

49. The method of claim 37, wherein Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

50. The method of claim 37, wherein the compound of formula I is

51. The method of claim 37 wherein the compound of formula I is:

52. A compound of formula I, or a pharmaceutically acceptable salt thereof

wherein

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is substituted with Ci_₆alkoxy at the 4-position, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene- OH, -COOH, -C(0)OCi_₆alkyl, -C(0)H, halogen at the 2-position, halogen at the 4- position, -CN, or -N0₂;

R₂ and R₃ are each independently H or halogen; and

R₄ is H, halogen, or C_halky!

53. The compound of claim 52, wherein R₂ and R₃ are each H.

54. The compound of claim 52 wherein R₂ and R₃ are each halogen.

55. The compound of claim 54, wherein R₂ and R₃ are each CI.

56. The compound of claim 52, wherein R₄ is H.

57. The compound of claim 52, wherein Ri is -C(0)Ci_₆alkyl.

58. The compound of claim 52, wherein Ri is phenyl substituted at the 4-position.

59. The compound of claim 52, wherein Ri is phenyl substituted with

at the 4-position.

60. The compound of claim 52, wherein Ri is phenyl substituted with -OH.

61. The compound of claim 52, wherein Ri is phenyl substituted with Ci_₆alkyl optionally

substituted with 1-3 halogen.

62. The compound of claim 61, wherein Ri is phenyl substituted with -CF₃.

63. The compound of claim 52, wherein Ri is phenyl substitutedwith Ci_₆alkylene-OH.

64. The compound of claim 52, wherein Ri is phenyl substituted with -COOH.

65. The compound of claim 52, wherein Ri is phenyl substituted with -C(0)OCi_₆alkyl.

66. The compound of claim 52, wherein Ri is phenyl substituted with -C(0)H.

67. The compound of claim 52, wherein Ri is phenyl substituted halogen at the 2-position or

halogen at the 4-position.

68. The compound of claim 52, wherein Ri is phenyl substituted with -CN.

69. The compound of claim 52, wherein Ri is phenyl substituted with -N0₂.

70. The compound of claim 52, wherein the compound of formula I is selected from a compound in the following table:

71. The compound of claim 52, wherein the compound of formula I is selected from the following table:

72. The compound of claim 52, wherein the compound of formula I is

73. A composition comprising one or more compounds of claim 52 and at least one carrier or excipient.

74. A method for enhancing the stability of at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase and recombinant alpha glucosidase comprising contacting the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula I, or a

pharmaceutically acceptable salt thereof

wherein

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R4 is H, halogen, or C_halky!

75. The method of claim 74, wherein the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase.

76. The method of claim 74, wherein the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase by at least 5%.

77. The method of claim 74, wherein the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase by at least 10%.

78. The method of claim 74, wherein the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase by at least 20%.

79. The method of claim 74, wherein the stabilization increases the half life of recombinant alpha glucosidase.

80. The method of claim 74, wherein the compound of formula I is selected from a compound in the following table:

81. The method of claim 74, wherein R₂, R₃, and R4 are each H.

82. The method of claim 74, wherein R₂ and R₃ are each halogen.

83. The method of claim 82, wherein R₂ and R₃ are each CI.

84. The method of claim 74, wherein R₄ is halogen.

85. The method of claim 84, wherein R₄ is CI.

86. The method of claim 74, wherein Ri is phenyl substituted at the 4-position.

87. The method of claim 74, wherein Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

88. The method of claim 74, wherein Ri is phenyl substituted with

89. The method of claim 74, wherein Ri is phenyl substituted with -OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

90. The method of claim 74, wherein Ri is phenyl substituted with -C(0)Ci_₆alkyl.

91. The method of claim 74, wherein Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH,

-COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂. 92. The method of claim 74, wherein the compound of formula I is

93. The method of claim 74 wherein the compound of formula I is:

94. A method comprising:

contacting at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula II, or a

pharmaceutically acceptable salt thereof

wherein

n is 0 or 1 ;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R4 is H, halogen, or C_halky!.

95. The method of claim 94, wherein the compound of formula II binds to wild type alpha glucosidase.

96. The method of claim 94, wherein the compound of formula II binds to mutant alpha glucosidase.

97. The method of claim 94, wherein the compound of formula II binds to misfolded alpha

glucosidase.

98. The method of claim 94, wherein the compound of formula II binds to recombinant alpha

glucosidase.

99. The method of claim 94, wherein the compound of formula II binds to at least one of mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase and stabilizes the at least one of mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase.

100. The method of claim 94, wherein the compound of formula II binds to at least one of mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase and increases the activity of the at least one of mutant alpha glucosidase, misfolded alpha

glucosidase, and recombinant alpha glucosidase.

101. The method of claim 94, wherein the compound of formula II is

102. The method of claim 94, wherein n is 0.

103. The method of claim 94, wherein n is 1.

104. The method of claim 94, wherein R₂, R₃, and R₄ are hydrogen.

105. The method of claim 94, wherein R₂ and R₃ are each halogen.

106. The method of claim 94, wherein R₂ and R₃ are each CI.

107. The method of claim 94, wherein R₄ is halogen.

108. The method of claim 94, wherein R₄ is CI.

109. The method of claim 94, wherein Ri is phenyl substituted at the 4-position.

110. The method of claim 94, wherein Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OC_1-6alkyl,

-C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

111. The method of claim 94, wherein Ri is phenyl substituted with

-OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, - CN, or -N0₂.

112. The method of claim 94, wherein Ri is phenyl substituted with -OH, -COOH,

-C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

113. The method of claim 94, wherein Ri is phenyl substituted with -C(0)Ci_₆alkyl.

114. The method of claim 94, wherein Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH, -COOH, -C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

115. A method of treating Pompe disease in a patient comprising administering to the patient a therapeutically effective amount of a compound of formula II, or a pharmaceutically acceptable salt thereof

wherein

n is 0 or 1 ;

Ri is -C(0)Ci_₆alkyl; or phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl, wherein the phenyl, pyridinyl, pyrazyl, pyrimidyl, or pyridazyl is optionally substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci-₆alkylene-OH, -COOH,

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R₄ is H, halogen, or C_halky!.

116. The method of claim 115, wherein the compound of formula II is

117. The method of claim 115, wherein n is 0.

118. The method of claim 115, wherein n is 1.

119. The method of claim 115, wherein R₂, R₃, and R₄ are hydrogen.

120. The method of claim 115, wherein R₂ and R₃ are each halogen.

121. The method of claim 115, wherein R₂ and R₃ are each CI.

122. The method of claim 115, wherein R₄ is halogen.

123. The method of claim 115, wherein R₄ is CI.

124. The method of claim 115, wherein Ri is phenyl substituted at the 4-position.

125. The method of claim 115, wherein Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH,

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

126. The method of claim 115, wherein Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, -C(0)H, -

127. The method of claim 115, wherein Ri is phenyl substituted with -OH, -COOH,

-C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

128. The method of claim 115, wherein Ri is phenyl substituted with -C(0)Ci_₆alkyl.

129. The method of claim 115, wherein Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl

substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen,

130. A method for enhancing the stability of at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase comprising contacting the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase with a compound of formula II, or a pharmaceutically acceptable salt thereof

wherein n is 0 or 1 ;

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R4 is H, halogen, or C_halky!

131. The method of claim 130, wherein the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase.

132. The method of claim 130, wherein the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase by at least 5%.

133. The method of claim 130, wherein the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase by at least 10%.

134. The method of claim 130, wherein the stabilization increases the half life of the at least one of wild type alpha glucosidase, mutant alpha glucosidase, misfolded alpha glucosidase, and recombinant alpha glucosidase by at least 20%.

135. The method of claim 130, wherein the stabilization increases the half life of recombinant alpha glucosidase.

136. The method of claim 130, wherein the compound of formula II is

137. The method of claim 130, wherein n is 0.

138. The method of claim 130, wherein n is 1.

139. The method of claim 130, wherein R₂, R₃, and R₄ are hydrogen.

140. The method of claim 130, wherein R₂ and R₃ are each halogen.

141. The method of claim 130, wherein R₂ and R₃ are each CI.

142. The method of claim 130, wherein R₄ is halogen.

143. The method of claim 130, wherein R₄ is CI.

144. The method of claim 130, wherein Ri is phenyl substituted at the 4-position.

145. The method of claim 130, wherein Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_₆alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)OC_1-6alkyl,

-C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

146. The method of claim 130, wherein Ri is phenyl substituted with

147. The method of claim 130, wherein Ri is phenyl substituted with -OH, -COOH,

-C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

148. The method of claim 130, wherein Ri is phenyl substituted with -C(0)Ci_₆alkyl.

149. The method of claim 130, wherein Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl substituted with Ci_₆alkoxy, -OH, Ci-₆alkyl optionally substituted with 1-3 halogen,

150. A compound of formula II, or a pharmaceutically acceptable salt thereof

wherein

n is 0 or 1 ;

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂;

R₂ and R₃ are each independently H, halogen, or Ci_₆alkyl; and

R4 is H, halogen, or C_halky!.

The compound of claim 150, wherein the compound of formula II is

152. The compound of claim 150, wherein n is 0.

153. The compound of claim 150, wherein n is 1.

154. The compound of claim 150, wherein R₂, R₃, and R₄ are hydrogen.

155. The compound of claim 150, wherein R₂ and R₃ are each halogen.

156. The compound of claim 150, wherein R₂ and R₃ are each CI.

157. The compound of claim 150, wherein R₄ is halogen.

158. The compound of claim 150, wherein R₄ is CI.

159. The compound of claim 150, wherein the phenyl is substituted at the 4-position.

160. The compound of claim 150, wherein Ri is phenyl substituted with Ci_₆alkoxy, -OH, Ci_ 6alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH,

-C(0)OCi_₆alkyl, -C(0)Ci_₆alkyl, -C(0)H, halogen, -CN, or -N0₂.

161. The compound of claim 150, wherein Ri is phenyl substituted with Ci-₆alkoxy, -OH, Ci_ 6alkyl optionally substituted with 1-3 halogen, Ci_₆alkylene-OH, -COOH, -C(0)Ci_₆alkyl, - C(0)H, -CN, or -NO₂.

162. The compound of claim 150, wherein Ri is phenyl substituted with -OH, -COOH,

-C(0)Ci_₆alkyl, -C(0)H, -CN, or -N0₂.

163. The compound of claim 150, wherein Ri is phenyl substituted with -C(0)Ci_₆alkyl.

164. The method of claim 150, wherein Ri is pyridinyl, pyrazyl, pyrimidyl, or pyridazyl

165. A composition comprising one or more compounds of claim 150 and at least one carrier or excipient.