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Small-Molecule Modulation of Read-Through (SMMRT): Modification of 2-Phenoxyacetanilides to Improve Solution Solubility. Richard G. Wilde et al.

IP.com Disclosure Number: IPCOM000019283D
Publication Date: 2003-Sep-09
Document File: 1 page(s) / 389K

Publishing Venue

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Abstract

Heterocyclic groups (furans, benzofurans and benzopyrans) were surveyed as potential surrogates for the ether likage of the phenoxyacetanilide series. Several benzofurans were equipotent with compound 1. Monocyclic furans were much less potent. A furan had lower buffer stability of the amide bond compared to compound 1, but higher serum stability. A benzofuran of similar buffer stabiity and slightly better serum stability than compound 1.

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Slide 1 of 1

1. Introduction

14. Stability Testing

11. SAR: 5-Substituted Benzofurans

8. Synthesis of 5-Phenylfuranamides

5. Compound Targets

The tied-back ring was envisioned as either a furan (A) or pyran (B) ring. In addition, phenylfuran compounds (C) were proposed.

Three compounds (amide 1, furan 4 and benzofuran 8) were dissolved in sterile PBS buffer (7.4 pH) to 0.3 mM , and mouse serum to 0.1 mM. The solutions were incubated for 34 days at 37 °C, and assayed periodically (10 min – 34 d) in triplicate. Buffer samples were analyzed by HPLC/UV, 254 nm; mouse serum samples by LC/MS (after protein precipitation with methanol).

The previous poster (MEDI 195) provided the background for an approach to treatment of genetic diseases (such as Duchenne muscular dystrophy) involving small molecule modula-tion of read through (SMMRT). Small mole-cules can interfere with premature termination signaling by mutant mRNA, thus allowing full-length, functional protein to be expressed, and thereby lessening the effects of the disease.

Ex.

R

EC2x

Mf

Cmf

8

Br

0.10

2.35

3

9

NO2

5.5

1.59

30

10

NHC(=O)CH3

§

0

300

11

NHC(=O)Ph

§

0

300

12

 

1.7

1.56

30

13

 

8.4

1.22

30

14

 

2.5

1.80

30

15

 

43

1.29

100

16

 

§

0.67

10

17

 

0.43

2.19

10

18

 

14

1.16

30

A

B

Key: a) ArN2+Cl-, cat. CuCl2, acetone; b) ArB(OH)2, cat. Pd(OAc)2, PPh3, aq. Na2CO3, DME; c) i) oxalyl chloride, cat. DMF, DCM; ii) methyl 3-aminobenzoate, TEA, THF; d) LiI, pyridine.

t½ > 34 d

C

t½ > 34 d

PBS Buffer

2. Discovery of a Lead Compound

9. Cell Culture Assay for SMMRT

t½ = 1.0 d

An HTS screen (described earlier) of more than 300,000 compounds resulted in the discovery of a series of acetanilides, an example being compound 1 (below). This compound showed modest (low micromolar) in vitro activity, as well as activity

in an mdx mouse

model.

6. Synthesis of Benzofurans

Luciferase reporter construct containing a UGA codon was stably transfected in a 293 human embryotic kidney cell line. The 293 cells were then used in a cell-culture screen. Compounds were introduced at ten concen-trations ranging from 0.03-300 mM, and the amount of luciferase expression determined by visible detection. A control was measured for the vehicle (DMSO). Three parameters were extracted from the resulting curves: EC2x, the concentration (mM) at which two-fold over DMSO was achieved; Mf, the maximum fold; and Cmf, the concentration at which Mf was achieved. Typical standard deviations for concentration values were ± 0.5 log units (i.e. threefold higher or lower).

Mouse Serum

t½ > 34 d

t½ = 10 d

t½ = 0.57 d

3. Series Optimization

15. Conclusions

12. Disubstituted Benzofurans

Heterocyclic groups (furans, benzofurans and benzopyrans) were surveyed as potential sur-rogates for the ether linkage of the previously-described phenoxyacetanilide series.

Several benzofurans were equipotent with the original lead (compound 1). Monocyclic furans were much less potent.

A furan had lower buffer stability of the amide bond compared to compound...