Inhibitors of Memapsin 2 and use thereof

• US 7,678,760 B2
• Filed: 06/14/2007
• Issued: 03/16/2010
• Est. Priority Date: 06/28/1999
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of The University of Illinois, Oklahoma Medical Research Foundation
• Original Assignee: Board of Trustees of The University of Illinois, Oklahoma Medical Research Foundation
• Inventors: Ghosh, Arun K., Tang, Jordan J. N.
• Primary Examiner(s): Tsang; Cecilia
• Assistant Examiner(s): HEARD, THOMAS SWEENEY
• Application Number:
  US11/763,342
• Publication Number:
  US 20080021196A1
• Time in Patent Office: 1,006 Days
• Field of Search: None
• US Class Current: 514/21.91
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 1/1136 by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• CPC Class Codes: C07K 5/06026 the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
• CPC Class Codes: C07K 5/06043 Leu-amino acid
• CPC Class Codes: C07K 5/0806 the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
• CPC Class Codes: C07K 5/1021 with the first amino acid being acidic
• CPC Class Codes: C12N 9/6421 from mammals
• CPC Class Codes: C12N 9/6478 Aspartic endopeptidases (3.4.23)
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Methods for the production of purified, catalytically active, recombinant memapsin 2 have been developed. The substrate and subsite specificity of the catalytically active enzyme have been determined. The substrate and subsite specificity information was used to design substrate analogs of the natural memapsin 2 substrate that can inhibit the function of memapsin 2. The substrate analogs are based on peptide sequences, shown to be related to the natural peptide substrates for memapsin 2. The substrate analogs contain at least one analog of an amide bond which is not capable of being cleaved by memapsin 2. Processes for the synthesis of two substrate analogues including isosteres at the sites of the critical amino acid residues were developed and the substrate analogues, OMR99-1 and OM99-2, were synthesized. OM99-2 is based on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with the Leu-Ala peptide bond substituted by a transition-state isostere hydroxyethylene group (FIG. 1). The inhibition constant of OM99-2 is 1.6×10⁻⁹ M against recombinant pro-memapsin 2. Crystallography of memapsin 2 bound to this inhibitor was used to determine the three dimensional structure of the protein, as well as the importance of the various residues in binding. This information can be used by those skilled in the art to design new inhibitors, using commercially available software programs and techniques familiar to those in organic chemistry and enzymology, to design new inhibitors to memapsin 2, useful in diagnostics and for the treatment and/or prevention of Alzheimer'"'"'s disease.

Claims

1. A method of preparing a Leu* Ala dipeptide isostere, comprising the steps of:

a) reacting ethyl propiolate and N-(tert-butoxycarbonyl)-leucinal in the presence of n-butyl lithium or lithium diisopropyl amine to form ethyl-5-{(tert-butoxycarbonyl)amino}-4-hydroxy-7-methyloct-2-ynoate represented by the following structural formula;

b) reacting the ethyl-5-{(tert-butoxycarbonyl)amino}-4-hydroxy-7-methyloct-2-ynoate with hydrogen in the presence of Pd/BaSO₄ to form an intermediate;

c) reacting the intermediate with an acid to form 5-{1′

-{(tert-butoxycarbonyl)amino}-3′

-methylbutyl}-dihydrofuran-2(3H)-one represented by the following structural formula;

d) reacting iodomethane with 5-{1′

-{(tert-butoxycarbonyl)amino}-3-methylbutyl}-dihydrofuran-2(3H)-one in the presence of hexamethyldisilazane to form 5-{1′

-{(tert-butoxycarbonyl)amino}-3′

-methylbutyl}-3-methyl-dihydrofuran-2(3H)-one represented by the following structural formula;

e) reacting 5-{1′

-{(tert-butoxycarbonyl)amino}-3′

-methylbutyl}-3-methyl-dihydrofuran-2(3H)-one with a base to form a Leu* Ala dipeptide isostere represented by the following structural formula;

2. The method of claim 1, further comprising the steps of:

a) reacting N-{tert-butyloxycarbonyl)-leucine with N,O-dimethylhydroxyamine hydrochloride in the presence of an aprotic base to form N-(tert-butoxycarbonyl)-leucine-N′

-methoxy-N′

-methylamide represented by the following structural formula;

b) reacting N-(tert-butoxycarbonyl)-leucine-N′

-methoxy-N′

-methylamide with lithium aluminum hydride to form N-(tert-butoxycarbonyl)-leucinal represented by the following structural formula;

3. The method of claim 1, further comprising the step of reacting the Leu* Ala dipeptide isostere with tert-butyldimethylchlorosilane in the presence of a base to form a hydroxy protected Leu* Ala dipeptide isostere represented by the following structural formula:

4. The method of claim 2, further comprising the step of reacting the Leu* Ala dipeptide isostere with tert-butyldimethylchlorosilane in the presence of a base to form a hydroxy protected Leu* Ala dipeptide isostere represented by the following structural formula:

5. The method of claim 3, further comprising the steps of:

a) treating the hydroxy protected Leu* Ala dipeptide isostere with an acid to form a Leu* Ala dipeptide isostere having a deprotected amine group represented by the following structural formula;

b) reacting the amine deprotected Leu* Ala dipeptide isostere with N-(9-fluorenylmethyoxycarbonyl-succinimide (FMOC) in the presence of a base to form an FMOC protected Leu* Ala dipeptide isostere represented by the following structural formula;

6. The method of claim 4, further comprising the steps of:

a) treating the hydroxy protected Leu* Ala dipeptide isostere with an acid to form a Leu* Ala dipeptide isostere having a deprotected amine group represented by the following structural formula;

b) reacting the amine deprotected Leu* Ala dipeptide isostere with N-(9-fluorenylmethyoxycarbonyl-succinimide (FMOC) in the presence of a base to form an FMOC protected Leu* Ala dipeptide isostere represented by the following structural formula;

7. A compound of the formula

8. A composition comprising a compound of claim 7.