EP1178800A1 - Traitement des maladies neurodegeneratives par des inhibiteurs de l'aspartyl protease - Google Patents

Traitement des maladies neurodegeneratives par des inhibiteurs de l'aspartyl protease

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Publication number
EP1178800A1
EP1178800A1 EP00916643A EP00916643A EP1178800A1 EP 1178800 A1 EP1178800 A1 EP 1178800A1 EP 00916643 A EP00916643 A EP 00916643A EP 00916643 A EP00916643 A EP 00916643A EP 1178800 A1 EP1178800 A1 EP 1178800A1
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European Patent Office
Prior art keywords
substituted
group
member selected
alkyl
aryl
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German (de)
English (en)
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EP1178800A4 (fr
Inventor
Jonathan A. Ellman
Gary Lynch
Irwin D. Kuntz
Xiaoning Bi
Christina E. Lee
A. Geoffrey Skillman
Tasir Haque
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University of California
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University of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/4035—Isoindoles, e.g. phthalimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4465—Non condensed piperidines, e.g. piperocaine only substituted in position 4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4525—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • RO1 GM53696 and RO1 GM50353 awarded by the National Institutes of Health. The Government has certain rights in this invention.
  • Alzheimer's disease is the most common form of both senile and presenile dementia in the world and is recognized clinically as relentlessly progressive dementia that presents with increasing loss of memory, intellectual function and disturbances in speech (Merritt, 1979, A Textbook of Neurology, 6th edition, 484-489 Lea & Febiger, Philadelphia). The disease itself usually has a slow and insidious progress that affects both sexes equally, worldwide.
  • Alzheimer's disease afflicts an estimated 4 million human beings in the United States alone at a cost of 35 billion dollars a year (Hay and Ernst, Am. J. Public Health, 77:1169-1175 (1987)).
  • Amyloidogenic A ⁇ peptides are the principle component of the amyloid plaques that accumulate intracellularly and extracellularly in the neuritic plaques in the brain in AD.
  • a ⁇ is a 4.5 kD protein, about 40-42 amino acids long, that is derived from the C-terminus of amyloid precursor protein (APP).
  • APP is a membrane- spanning glycoprotein that, in the normal processing pathway, is cleaved inside the A ⁇ protein to produce ⁇ -sAPP, a secreted form of APP.
  • alpha ⁇ -sAPP precludes formation of A ⁇ . It has been proposed that A ⁇ accumulates by virtue of abnormal processing of APP, so that compounds that inhibit the activity of the enzymes responsible for A ⁇ production are desirable (see, e.g. , Wagner, et al , Biotech. Report, 106-107 (1994/1995); and Selkoe, TINS, 16:403-409 (1993)). In addition to the accumulation of amyloid plaques, neurons in AD brains exhibit specific alterations in r, a family of phosphoproteins that bind tubulin (Weingarten, et al , Proc. Natl Acad. Sci.
  • T proteins adopt an altered form and comprise the dominant component of abnormal cytosketal fibers known as paired helical filaments (PHFs) (see, Kosik, et al , Proc. Natl. Acad. Sci. USA, 83:4044-4088 (1986); Lee, et al , Science, 251:675-678 (1991); and Mann, et al , Neuropathol. Appl. NeurobioL , 13: 123-139 (1987)).
  • PHFs paired helical filaments
  • PHF- ⁇ proteins maintain an excessively phosphorylated state throughout postmortem intervals (Matsuo, et al , Neuron, 13:989- 1002 (1994)).
  • the present invention relates to (i) non-peptide aspartyl protease inhibitors; (ii) methods for modulating the processing of an amyloid precursor protein (APP); (iii) methods for modulating the processing of a tau protein ( ⁇ -protein); and (iv) methods for treating neurodegenerative diseases.
  • APP amyloid precursor protein
  • ⁇ -protein tau protein
  • the present invention provides a method for modulating the processing of an amyloid precursor protein (APP), the method comprising contacting a composition containing the APP with an aspartyl protease inhibitor having the general formula:
  • R R 2 and R 3 are members independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, aryloxyalkyl, substituted aryloxyalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycles, substituted heterocycles, heterocyclicalkyl and substituted heterocyclicalkyl.
  • R 5 and ⁇ are independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, aryloxyalkyl and substituted aryloxyalkyl.
  • R 5 and R 6 and the carbons to which they are bound join to form an optionally substituted 9- or 10-ring atom carbocyclic or heterocyclic fused ring system.
  • Typical 9- or 10-atom fused ring systems include, but are not limited to, napthalyl, 1,3- benzodioxolyl, 2,3-benzofuranyl, 1,4-benzodioxanyl, benzimidazoyl, benzothiazolyl etc.
  • R is a functional group including, but not limited to, substituted arylalkyl, substituted aryl, substituted alkyl and substituted heterocyclic groups.
  • R is a functional group including, but not limited to, substituted arylalkyl, substituted aryl, substituted alkyl and substituted heterocyclic groups. Examples of such functional groups include, but are not limited to, the following:
  • R 2 is a functional group including, but not limited to, substituted alkyl, heterocyclic and substituted heterocyclic groups.
  • functional groups include, but are not limited to, the following:
  • R 2 is a functional group other than a nitrogen-bonded cyclic ⁇ -amino acid or ester thereof.
  • R 3 is a functional group including, but not limited to, substituted alkyl and substituted aryl groups. Examples of such functional groups include, but are not limited to, the following:
  • R 5 and R 6 and the carbons to which they are bound join to form an optionally substituted napthalene ring.
  • R 5 and R ⁇ 5 are both hydrogen or R 5 is hydrogen and R 6 is a meta or para substituent.
  • the aspartyl protease inhibitor is selected from the group consisting of:
  • the modulation of APP can be demonstrated in a variety of ways.
  • aspartyl protease inhibitors can be evaluated for the ability to modulate generation of A ⁇ or ⁇ -sAPP.
  • the formation of A ⁇ is decreased compared to the amount formed in the absence of the aspartyl protease inhibitor.
  • formation of ⁇ -sAPP is increased compared to the amount formed in the absence of the asparty protease inhibitor.
  • the composition is a body fluid.
  • the body fluid is cerebral spinal fluid (CSF).
  • the present invention provides a method for modulating the processing of a tau-protein ( ⁇ -protein), the method comprising contacting a composition containing the ⁇ -protein with an aspartyl protease inhibitor of Formula I.
  • the modulation of 7-protein can be demonstrated in a variety of ways.
  • aspartyl protease inhibitors can be evaluated for the ability to modulate generation of T- fragments.
  • the formation of ⁇ -fragments is decreased compared to the amount formed in the absence of the aspartyl protease inhibitor.
  • the composition is a body fluid.
  • the body fluid is cerebral spinal fluid (CSF).
  • the present invention provides a method of treating a neurodegenerative disorder, the method comprising: administering to a mammal a therapeutically effective amount of an aspartyl protease inhibitor of Formula I and a pharmaceutically acceptable carrier or excipient.
  • the neurodegenerative disorder is characterized by the accumulation of amyloid plaques.
  • the neurodegenerative disorder is characterized by the accumulation of T- fragments.
  • the aspartyl protease inhibitors of the present invention can be used to treat all amyloid-pathology related diseases and all tau pathology-related diseases. Examples of such neurodegenerative diseases include, but are not limited to, the following: Alzheimer's disease, Parkinson's disease, cognition deficits, Downs
  • dementia e.g. , dementia pugilistica
  • head trauma cerebral hemorrhage with amyloidosis, dementia (e.g. , dementia pugilistica) and head trauma.
  • FIG. 1 illustrate isostere-based inhibitor design.
  • FIG. 2 illustrates components employed to prepare the libraries targeting cathepsin D.
  • the same disconnections provide scaffold 2.
  • Isocyanates and sulfonyl chlorides which can be used to incorporate R 2 and R 3 , provide ureas and sulfonamides, respectively.
  • FIG. 3 illustrates the used of BUILDERopt in designing the combinatorial library:
  • FIGS. 5A-5C illustrates the components used to prepare the Diverse Library. Diverse library components are labeled by lower case letter code as for the directed library.
  • Thirty-nine compounds incorporating these sidechains were synthesized on resin as described previously, EFD, EHD, FFD, FHD, KFD, KHD, LFD, LHD, MFD, MHD, NFD, NHD, OFD, OHD, PFD, PHD, QFD, QHD, RFD, RHD, SFD, SHD, TFD, THD, UFD, UHD, NFD, VHD, EHA, EHJ, EHK, EHL, EHM, EH ⁇ , EHO, EHP, EHQ, EHR, EHS.
  • the compounds were assayed at 333 nM, 100 nM and 33 nM in high-throughput screening.
  • the most active compounds were synthesized on large scale and the K x values were determined (Table 3
  • FIG. 7 illustrates structural diversity being introduced via Grignard addition to solid support-bound x - alkoxy pyrrolidine amide.
  • FIG. 8 illustrates synthesis of solid phase aspartyl protease inhibitor synthesis.
  • FIG. 9 illustrates components to generate library diversity in a 204 compound library.
  • FIG. 10 illustrates that the cathepsin D inhibitor, i.e. , CEL5-172, by itself, did not detectably change the concentration of either the tau fragment or the APP fragment, but it did block most, if not all, of the increases in the tau and APP fragments produced by ZDAP.
  • the cathepsin D inhibitor i.e. , CEL5-172
  • FIG. 11 illustrates that like CEL5-172, the cathepsin D inhibitor EA-1, by itself, did not detectably change the concentration of either the phosphorylated taus fragment, but it exhibited a much higher blocking effect than CEL5-172.
  • FIG. 12 illustrates the structures of three inhibitors used in the experiments set forth in Example III, all of which have molecular weights of 650-800 Daltons and Ki's for cathepsin D of between 1-15 nM.
  • FIG. 13 illustrates the morphological and physiological effects of cathepsin inhibitors. Semi-thin sections through the cell body layer of field CA1 of cultured hippocampal slices given no treatment (A), a 6-day exposure to an inhibitor of cathepsins B and L (B), or a-6 day exposure to an inhibitor of cathepsin D (C). Note the presence in (B) of large numbers of small, dense bodies that in some cases are clustered into torpedo shaped expansions (arrows).
  • IPSCs were well developed in treated slices (iii) as can be seen in the Schaffer- commissural responses collected with the membrane potential set to -50mV. A negative going EPSC recorded at -70mV is also shown.
  • FIG. 14 illustrates the effects of cathepsin inhibitors on concentrations of phosphorylated tau fragments.
  • Cultured slices were incubated for 6 days with an inhibitor of cathepsins B and L (ZPAD), an inhibitor of cathepsin D, or both.
  • ZPAD an inhibitor of cathepsin D
  • Western blots were then prepared from slice homogenates using an antibody against the hyperphosphorylated tau found in human neurofibrillary tangles.
  • the top panels show immunostaining in the 25-35 kDa region of the blots.
  • ZPAD increased the concentrations of phosphorylated bands in this region over the levels found in controls.
  • the bottom panels summarize analysis of AT8 staining from five separate experiments with all values expressed as percent of yoked controls. *, P ⁇ 0.05; **, P ⁇ 0.01; error bars, standard errors.
  • FIG. 15 illustrates the time course and dose dependency for suppression of phosphorylated tau fragments by a cathepsin D inhibitor.
  • A Cultured hippocampal slices were incubated for 2, 4, or 6 days with the cathepsin B/L inhibitor-ZPAD, the cathepsin D inhibitor-EA-1, or both. Western blot analyses for phosphorylated tau fragments were carried out at the end of the incubation with densitometric values expressed as percent of concentrations in yoked controls. ZPAD induced increases were detectable after 48 hrs and continued to grow thereafter.
  • the cathepsin D inhibitor had no apparent effect but blocked the increases produced by ZPAD at all time points.
  • (B) Slices were incubated with ZPAD, EA-1, or ZPAD plus the indicated concentrations of EA-1 for six days.
  • the cathepsin D inhibitor had no detectable effects on concentrations of phosphorylated tau fragments at the concentrations tested.
  • a dose of 1 ⁇ M caused a sizeable decrease in the effect of ZPAD while 5 ⁇ M completely suppressed it.
  • FIG. 16 illustrates the effects of cathepsin inhibitors on tau and cathepsin D isoforms.
  • Slices were incubated with ZPAD, EA-1, or both for 6 days after which Western blots were used to assess the concentrations of the target proteins with tau 1 antibodies (A), or anti-cathepsin D antisera (B). Densitometeic values were expressed as percent change from the concentrations in yoked control slices.
  • A ZPAD caused sizeable reductions in four unphosphorylated isoforms of native tau; EA-1 was without effect itself and did not block the changes produced by ZPAD. ZPAD also generated a large increase in a 29 kDa tau fragment; this was completely blocked by EA-1.
  • B ZPAD resulted in modest increases in procathepsin D and larger increases in the active, heavy chain variant of the protease. EA-1 suppressed the second of these effects.
  • the present invention relates to (i) non-peptide aspartyl protease inhibitors; (ii) methods for modulating the processing of an amyloid precursor protein (APP); (iii) methods for modulating the processing of a tau protein ( ⁇ -protein); and (iv) methods for treating neurodegenerative diseases.
  • APP amyloid precursor protein
  • ⁇ -protein tau protein
  • R R 2 and R 3 independently selected are identical or different (e.g. , R,, R 2 and R 3 may all be substituted alkyls or Ri and R 2 may be a substituted alkyl and R 3 may be an aryl, etc.).
  • alkyl is used herein to refer to a branched or unbranched, saturated or unsaturated, monovalent hydrocarbon radical having from 1-12 carbons and preferably, from 1-6 carbons. When the alkyl group has from 1-6 carbon atoms, it is referred to as a "lower alkyl. " Suitable alkyl radicals include, for example, methyl, ethyl, n-propyl, i-propyl, 2-propenyl (or allyl), n-butyl, t-butyl, i-butyl (or 2- methylpropyl), etc. As used herein, the term encompasses "substituted alkyls. "
  • Substituted alkyl refers to alkyl as just described including one or more functional groups such as lower alkyl, aryl, substituted aryl, acyl, halogen (i.e. , alkylhalos, e.g. , CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl, mercapto, thia, aza, oxo, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like. These groups may be attached to any carbon of the alkyl moiety. Additionally, these groups may be pendent from, or integral to, the alkyl chain.
  • aryl is used herein to refer to an aromatic substituent which may be a single aromatic ring or multiple aromatic rings which are fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety.
  • the common lmking group may also be a carbonyl as in benzophenone.
  • the aromatic ring(s) may include phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone among others.
  • aryl encompasses "arylalkyl. "
  • arylalkyl is used herein to refer to a subset of “aryl” in which the aryl group is attached to the nucleus shown in Formula 1 by an alkyl group as defined herein.
  • substituted aryl refers to aryl as just described including one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g.
  • cyclic hydrocarbons optionally substituted with one or more heteroatoms, which are fused to the aromatic ring(s), linked covalently or linked to a common group such as a methylene or ethylene moiety.
  • the linking group may also be a carbonyl such as in cyclohexyl phenyl ketone.
  • substituted aryl encompasses "substituted arylalkyl. " “Substituted arylalkyl” defines a subset of "substituted aryl” wherein the substituted aryl group is attached to the nucleus shown in Formula 1 by an alkyl group as defined herein.
  • acyl is used to describe a ketone substituent, — C(O)R, where R is alkyl or substituted alkyl, aryl or substituted aryl as defined herein.
  • halogen is used herein to refer to fluorine, bromine, chlorine and iodine atoms.
  • hydroxy is used herein to refer to the group —OH.
  • amino is used to describe primary amines, R— NH 2 .
  • alkoxy is used herein to refer to the —OR group, where R is a lower alkyl, substituted lower alkyl, aryl, substituted aryl, arylalkyl or substituted arylalkyl wherein the alkyl, aryl, substituted aryl, arylalkyl and substituted arylalkyl groups are as described herein.
  • alkoxy radicals include, for example, methoxy, ethoxy, phenoxy, substituted phenoxy, benzyloxy, phenethyloxy, t-butoxy, etc.
  • alkylamino denotes secondary and tertiary amines wherein the alkyl groups may be either the same or different and are as described herein for “alkyl groups.
  • acylamino describes substituents of the general formula RC(O)NR', wherein R' is a lower alkyl group and R represents the nucleus shown in Formula 1 or an alkyl group, as defined herein, attached to the nucleus.
  • acyloxy is used herein to describe an organic radical derived from an organic acid by the removal of the acidic hydrogen.
  • Simple acyloxy groups include, for example, acetoxy, and higher homologues derived from carboxylic acids such as ethanoic, propanoic, butanoic, etc.
  • the acyloxy moiety may be oriented as either a forward or reverse ester (i.e. , RC(O)OR' or R'OC(O)R, respectively, wherein R comprises the portion of the ester attached either directly or through an intermediate hydrocarbon chain to the nucleus shown in claim 1).
  • aryloxy denotes aromatic groups which are linked to the nucleus shown in FIG. 1 directly through an oxygen atom. This term encompasses “substituted aryloxy” moieties in which the aromatic group is substituted as described above for “substituted aryl. "
  • aryloxyalkyl defines aromatic groups attached, through an oxygen atom to an alkyl group, as defined herein.
  • the alkyl group is attached to the nucleus shown in FIG. 1.
  • aryloxyalkyl encompasses "substituted 13 aryloxyalkyl” moieties in which the aromatic group is substituted as described for “substituted aryl. "
  • mercapto defines moieties of the general structure R— S— R' wherein R and R' are the same or different and are alkyl, aryl or heterocyclic as described herein.
  • saturated cyclic hydrocarbon denotes groups such as the cyclopropyl, cyclobutyl, cyclopentyl, etc. , and substituted analogues of these structures. These cyclic hydrocarbons can be single- or multi-ring structures.
  • unsaturated cyclic hydrocarbon is used to describe a monovalent non-aromatic group with at least one double bond, such as cyclopentene, cyclohexene, etc. and substituted analogues thereof. These cyclic hydrocarbons can be single- or multi-ring structures.
  • heteroaryl refers to aromatic rings in which one or more carbon atoms of the aromatic ring(s) are substituted by a heteroatom such as nitrogen, oxygen or sulfur.
  • Heteroaryl refers to structures which may be a single aromatic ring, multiple aromatic ring(s), or one or more aromatic rings coupled to one or more non-aromatic ring(s). In structures having multiple rings, the rings can be fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety.
  • the common linking group may also be a carbonyl as in phenyl pyridyl ketone.
  • rings such as thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan, etc. or benzo-fused analogues of these rings are defmed by the term "heteroaryl. "
  • Heteroarylalkyl defines a subset of “heteroaryl” wherein an alkyl group, as defined herein, links the heteroaryl group to the nucleus shown in FIG. 1.
  • Substituted heteroaryl refers to heteroaryl as just described wherein the heteroaryl nucleus is substituted with one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g. , CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc.
  • substituted analogues of heteroaromatic rings such as thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan, etc. or benzo-fused analogues of these rings are defined by the term "substituted heteroaryl.
  • Substituted heteroarylalkyl refers to a subset of "substituted heteroaryl” as described above in which an alkyl group, as defined herein, links the heteroaryl group to the nucleus shown in FIG. 1.
  • heterocyclic is used herein to describe a monovalent saturated or unsaturated non-aromatic group having a single ring or multiple condensed rings from 1-12 carbon atoms and from 1-4 heteroatoms selected from nitrogen, sulfur or oxygen within the ring.
  • Such heterocycles are, for example, tetrahydrofuran, morpholine, piperidine, pyrrolidine, etc.
  • substituted heterocyclic as used herein describes a subset of “heterocyclic” wherein the heterocycle nucleus is substituted with one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g. , CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc.
  • heterocyclicalkyl defines a subset of "heterocyclic” wherein an alkyl group, as defined herein, links the heterocyclic group to the nucleus shown in FIG.
  • optionally substituted napthylene ring describes a naphthalene ring which may be unsubstituted or may be substituted with one or more functional groups including lower alkyl, halogen, acyl, hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy or aryl.
  • substituted heterocyclicalkyl defines a subset of “heterocyclic alkyl” wherein the heterocyclic nucleus is substituted with one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g. , CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc.
  • functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g. , CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc.
  • amyloid precursor protein or "APP” is used herein to refer to the progenitor of deposited amyloidogenic A ⁇ peptides (A ⁇ ) found in senile plaques in patients with diseases, such as Alzheimer's disease (AD), that are characterized by such deposition.
  • a ⁇ amyloidogenic A ⁇ peptides
  • AD Alzheimer's disease
  • ⁇ -sAPP is an alternative cleavage product of APP; its formation precludes formation of A ⁇ .
  • contacting is used herein interchangeably with the following: combined with, added to, mixed with, passed over, incubated with, flowed over, etc.
  • aspartyl protease inhibitors of present invention can be "administered" by any conventional method such as, for example, parenteral, oral, topical and inhalation routes as described herein.
  • An amount sufficient or “an effective amount” is that amount of a given aspartyl protease inhibitor which exhibits the binding/inhibitory activity of interest or, which provides either a subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • the present invention relates to the identification of a number of small-molecule compounds which are capable of binding to and inhibiting aspartyl proteases and, in particular, cathepsin D employing a combined combinatorial library (see, e.g., Thompson, et al, Chemical Reviews, 96, 555-600 (1996)) and structure based design approach (see, e.g. , Kuntz, I.D., Science, 257, 1078-1082 (1992)).
  • the libraries of potential aspartyl protease inhibitors e.g. , cathepsin D inhibitors
  • the Pj sidechain (R 4 ) was held constant as a benzyl substituent based upon X-ray crystallographic data of cathepsin D complexed with the peptide-based natural product pepstatin as reported by Erickson (Baldwin, et al, Proc. Natl. Acad. Sci. USA, 90, 6796-6800 (1993)). As illustrated in FIG. 2, diversity was introduced at three positions: a primary amine introduced the R t substituent, and acylating agents serve to introduce the R 2 and R 3 substituents.
  • the libraries were screened to identify compounds capable of binding to and inhibiting aspartyl proteases and, in particular, cathepsin D.
  • the present invention provides compounds having the general formula:
  • R,, R 2 and R 3 are members independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, aryloxyalkyl, substituted aryloxyalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycles, substituted heterocycles, heterocyclicalkyl and substituted heterocyclicalkyl.
  • R 5 and $ are independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, aryloxyalkyl and substituted aryloxyalkyl.
  • R 5 and R 6 and the carbons to which they are bound join to form an optionally substituted 9- or 10-ring atom carbocyclic or heterocyclic fused ring system.
  • Typical 9- or 10-atom fused ring systems include, but are not limited to, napthalyl, 1,3- benzodioxolyl, 2,3-benzofuranyl, 1,4-benzodioxanyl, benzimidazoyl, benzothiazolyl etc.
  • Rj is a functional group including, but not limited to, substituted arylalkyl, substituted aryl, substituted alkyl and substituted heterocyclic groups.
  • Rj is a functional group including, but not limited to, substituted arylalkyl, substituted aryl, substituted alkyl and substituted heterocyclic groups. Examples of such functional groups include, but are not limited to, the following:
  • R 2 is a functional group including, but not limited to, substituted alkyl, heterocyclic and substituted heterocyclic groups.
  • functional groups include, but are not limited to, the following:
  • R 2 is a functional group other than a nitrogen-bonded cyclic c--amino acid or ester thereof.
  • R 3 is a functional group including, but not limited to, substituted alkyl and substituted aryl groups.
  • functional groups include, but are not limited to, the following:
  • R 5 and R 6 and the carbons to which they are bound join to form an optionally substituted napthalene ring.
  • R 5 and R 6 are both hydrogen or R 5 is hydrogen and R 6 is a meta or para substituent.
  • R ⁇ can be a member selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, aryloxyalkyl, substituted aryloxyalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycles, substituted heterocycles, heterocyclicalkyl and substituted heterocyclicalkyl.
  • the compounds of Formula I can be a racemic mixture (mixtures of diastereomers or enantiomers) or as stereochemically distinct compounds.
  • the compounds of the present invention have the following stereochemistry:
  • Tables I and II set forth compounds in accordance with the present invention that are particularly preferred.
  • the compounds in Table I and throughout this specification are often referred to by code numbers, which are used for convenience only, and are strictly arbitrary for purposes of this invention.
  • the compounds of the present invention can be synthesized in a variety of ways, using conventional synthetic chemistry techniques.
  • the compounds of the present invention are prepared according to the reaction scheme set forth in FIG. 2, wherein R R 2 and R 3 are as defined above.
  • the use of appropriate organic solvents, temperature and time conditions for ranning the reactions are within the level of skill in the art. Reactions of this type are generally described by E.K. Kick and J.A. Ellman, J. Med. Chem. 38, 1427-1430 (1995), the teachings of which are hereby incorporated by reference.
  • the compounds of the present invention have been found to be potent inhibitors of aspartyl proteases and, in particular, cathepsin D.
  • the present invention contemplates using the compounds of the present invention to inhibit cathepsin D, either in vivo or in vitro.
  • the present invention provides a method of inhibiting cathepsin D, the method comprising contacting cathepsin D with an aspartyl protease inhibitor having the general formula:
  • R R 2 and R 3 are members independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, aryloxyalkyl, substituted aryloxyalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycles, substituted heterocycles, heterocyclicalkyl and substituted heterocyclicalkyl.
  • R x , R 2 and R 3 and their preferred embodiments are fully applicable to the aspartyl protease inhibitors used in this method of the present invention and, thus, will not be repeated with respect to this particular method.
  • R 5 and R 6 are as defined above.
  • the present invention provides a method of inhibiting protein processing by cathepsin D in living cells, the method comprising contacting the cells with an effective amount of a compound having the general formula:
  • the reactants are mixed, the reaction is allowed to proceed for a specific period of time and the fluorescence of the reaction products is monitored to determine the extent to which the peptide substrate has been cleaved.
  • Compounds found to exhibit inhibitory activity towards cathepsin D using the foregoing assay can be synthesized on a larger scale and a more detailed kinetic analaysis can be carried out using an assay similar to that set forth in Table IV, infra, and described in greater detail by G. A. Kraft, et al, Methods Enzymol 241, 70-86 (1994). As such, following the methods of the present invention, compounds can be readily synthesized and screened to identify compounds that inhibit cathepsin D.
  • the aspartyl protease inhibitors of the present invention modulate the processing of numerous proteins, such as amyloid precursor protein (APP), involved in diseases.
  • the aspartyl proteases of the present invention are used to modulate the processing of APP.
  • the present invention provides a method for modulating the processing of an amyloid precursor protein (APP), the method comprising contacting a composition containing the APP with an aspartyl protease inhibitor having the general formula:
  • the modulation of APP can be demonstrated in a variety of ways.
  • aspartyl protease inhibitors can be evaluated for the ability to modulate generation of A ⁇ or ⁇ -sAPP.
  • the formation of A ⁇ is decreased compared to the amount formed in the absence of the aspartyl protease inhibitor.
  • formation of ⁇ -sAPP is increased compared to the amount formed in the absence of the asparty protease inhibitor.
  • the composition is a body fluid.
  • the body fluid is cerebral spinal fluid (CSF).
  • CSF cerebral spinal fluid
  • Numerous in vitro and in vivo animal models can be used to screen a given aspartyl protease inhibitor for its ability to modulate APP processing.
  • Exemplar assays are set forth below, in the Example Section and in, for example, Hoffman, et al , Neuroscience Letters, 250:75-78 (1998); Bahr, et al , Experimental Neurology, 129:81-94 (1994); and U.S. Patent No. 5,872,101, the teachings of which are inco ⁇ orated herein by reference.
  • a number of commercially available tests can be used to detect A ⁇ in a composition (e.g. , CSF).
  • the ADmark Assay which is commercially available from Athena Neurosciences, Inc. , can be used to detect A ⁇ in CSF.
  • in vitro assays The aspartyl protease inhibitors provided herein yield a positive result in one or more in vitro assays that assess the effects of test compounds on processing of APP.
  • in vitro assay systems for identifying such compounds are provided herein. These assays evaluate the effects of a test compound on processing of APP and use cultured human glioblastoma cell lines that have been transfected with DNA encoding either a wild-type 695 amino acid isoform of APP or a mutein of APP that contains changes (in this case two or three amino acid changes have been made) that appear to make the molecule more susceptible to proteolytic cleavage that results in increased production of A ⁇ (see, e.g. , Mullan, et al , Nature Genet. , 1:345-347 (1992)).
  • a test compound is added to the culture medium and, after a selected period of time, the culture medium and/or cell lysates are analyzed using immunochemical assays to detect the relative amounts of A ⁇ , total soluble APP (sAPP), a portion of sAPP designated ⁇ -sAPP, and C-terminal fragments of APP.
  • sAPP total soluble APP
  • ⁇ -sAPP a portion of sAPP designated ⁇ -sAPP
  • C-terminal fragments of APP C-terminal fragments of APP.
  • the culture medium and cell lysates are analyzed by immunoblotting coupled with laser scanning densitometry and ELISAs using several different antibodies.
  • a positive test occurs when: (1) there is a decrease in the approximately equal to 4-kDa amyloid jS-protein (A ⁇ ) in the medium relative to control cultures (4-kDa assay); and/ or (2) the relative amount of sAPP in the medium increases; and/or (3) there is a decrease in the amount of C-terminal amyloidogenic fragments larger than 9 kDa and smaller than 22 kDa in the cell lysate as a result of differential processing; and/ or (4) there is an increase in the amount of ⁇ -sAPP in the medium relative to control cultures.
  • Control cultures can be cultures that have not been contacted with the compound.
  • the A ⁇ assay is done using cells (e.g.
  • HGB 717/Swed that have been transfected with DNA encoding the mutein APP; the other assays are performed using cells, such as HGB695 cells, that have been transfected with DNA encoding a wild-type APP.
  • the relative amount of ⁇ -sAPP and the ratio of ⁇ -sAPP to total sAPP in CSF are known to be useful markers in the detection of neurodegenerative disorders characterized by cerebral deposition of amyloid (e.g. , AD) and in monitoring the progression of such disease. Furthermore, assay systems inco ⁇ orating these markers can be used in monitoring therapeutic intervention of these diseases.
  • the amount of ⁇ -sAPP and the ratio of ⁇ -sAPP to total sAPP in CSF samples can be used as an indicator of Alzheimer's Disease and other neurodegenerative disorders. For pu ⁇ oses herein, this amount and/ or the ratio can also be used to assess the effectiveness of compounds provided herein in treating Alzheimer's Disease and neurodegenerative disorders.
  • Alzheimer's disease (as diagnosed by other indicia, or confirmed by autopsy) have a statistically significant lower ratio of ⁇ -sAPP to total sAPP in CSF and also have statistically significant lower levels of ⁇ -sAPP. Therefore, by comparison with non- Alzheimer's disease controls or by existence of a ratio lower than a predetermined standard, based, for example, on averages in samples from large numbers of unafflicted individuals, or an amount of ⁇ - sAPP lower than a predetermined standard, Alzheimer's disease or, depending upon other indications, another neurodegenerative disease is indicated.
  • the ability of compounds to modulate processing of APP can also be evaulated using in vivo assays (See, e.g. , Lamb, et al , Nature Genet. , 5:22-29 (1993); Pearson, et al. Proc. Natl. Acad. Sci. U.S.A. 90: 10578-10582 (1993); Kowall, et al , Proc. Natl Acad. Sci. U.S.A. , 88:7247-7251 (1991)).
  • Compounds can be administered through a canula implanted in the cranium of a rat or other suitable test animal. After a predetermined period of administration the rats are sacrificed.
  • the hippocampi are evaluated in immunoblot assays or other suitable assays to determine the relative level of ⁇ -sAPP and C-terminal fragments of APP compared to untreated control animals. Aspartyl protease inhibitors that result in relative increases in the amount of ⁇ -sAPP are selected.
  • the present invention provides a method for modulating the processing of a tau-protein ( ⁇ -protein), the method comprising contacting a composition containing the ⁇ -protein with an aspartyl protease inhibitor having the general formula:
  • the modulation of ⁇ -protein can be demonstrated in a variety of ways.
  • aspartyl protease inhibitors can be evaluated for the ability to modulate generation of T- fragments.
  • the formation of ⁇ -fragments is decreased compared to the amount formed in the absence of the aspartyl protease inhibitor.
  • the composition is a body fluid.
  • the body fluid is cerebral spinal fluid (CSF).
  • CSF cerebral spinal fluid
  • Numerous in vitro and in vivo animal models can be used to screen a given aspartyl protease inhibitor for its ability to modulate the processing of ⁇ -protein. Exemplar assays are set forth below, in the
  • the supernatant is made to 35% ammonium sulfate and kept on ice for 30 minutes.
  • the slurry is centrifuged for 20 minutes at 10,000 g; supernatant is saved, made to 45 % ammonium sulfate, and incubated on ice for 30 minutes.
  • the pellet is resuspended in ⁇ 4 mL of buffer A and made to 2.5% perchloric acid.
  • the slurry is centrifuged for 15 minutes at 15,000 g.
  • the supernatant is made to 20% trichloroacetic acid, ice for 25 minutes and centrifuged for 15 minutes at 15,000 g.
  • the pellet is resuspended in 95% ethanol and dried under vacuum.
  • protease, test compound and substrate are combined and incubated at 37 °C for various durations.
  • Partially purified T is first resuspended in assay buffer (50 mM citric acid/sodium citrate buffer, pH 4.0). Reactions are initiated by the addition of human liver cathepsin D (1 U; Calbiochem, San Diego, Ca, U.S.A.) and test compound to 0.1 mg of T and terminated by removing aliquots at the designated time, adding SDS and 2-mercaptoethanol, and boiling for 5 minutes.
  • cathepsin D is defined as the amount of enzyme that generates an increase in absorbance (at 280 nm) of 1.0 per hour when co-incubated with hemoglobin in 10% trichloroacetic acid.
  • the specific activity of the enzyme is 300 U/mg of protein, and its purity is greater than 98 % by SDS-PAGE.
  • Brains from 3-month-old Sprague-Dawley rats are removed and dissected in artificial cerebrospinal fluid (124 mM NaCl, 20 mM glucose, 5mM HEPES, 3 mM KC1, 1.25 mM KH 2 PO 4 , 2.8 mM MgSO 4 , 2 mM CaCl 2 , mM NaHCO 3 , 0.5 mM ascorbate, Ph 7.4).
  • Frontal cortices are homogenized (Teflon to glass, 10 strokes) in 7 mM HEPES buffer, pH 7.35, additionally containing 135 mM NaCl, 2mM EDTA, 2mM EGTA, and 2.0 ⁇ M Okadaic acid. Slurries are centrifuged at 1,000 g for five minutes at 4°C. The supernatant is collected, sonicated, and subjected to two freeze/thaw cycles.
  • Proteolytic assays are conducted by co-incubating 0.1 mg of the supernatant described above with 0.35 U of human liver cathepsin D and the test compound.
  • the enzyme-to-substrate ratio should be about 1:86 (wt/wt).
  • the reaction is allowed proceeded at constant pH for 5 hours at 37 °C and is terminated by adding SDS and 2-mercaptoethanol and boiling the samples for five minutes.
  • aspartyl proteases e.g. , cathepsin D
  • cathepsin D are enzymes that plays an important role in protein metabolism, catabolism and antigen processing.
  • the compounds of the present invention can be used for a number of therapeutic applications.
  • the present invention provides a method of treating a neurodegenerative disorder, the method comprising: administering to a mammal a therapeutically effective amount of an aspartyl protease inhibitor and a pharmaceutically acceptable carrier or excipient, the aspartyl protease inhibitor having the general formula:
  • the neurodegenerative disorder is characterized by the accumulation of amyloid plaques. In another embodiment, the neurodegenerative disorder is characterized by the accumulation of ⁇ -fragments.
  • the aspartyl protease inhibitors of the present invention can be used to treat all amyloid-pathology related diseases and all tau pathology-related diseases. Examples of such neurodegenerative diseases include, but are not limited to, the following: Alzheimer's disease, Parkinson's disease, cognition deficits, Downs Syndrome, cerebral hemorrhage with amyloidosis, dementia (e.g. , dementia pugilistica) and head trauma.
  • the compounds, i.e. , aspartyl protease inhibitors, of the present invention can be inco ⁇ orated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc. , administration.
  • Suitable formulations for use in the present invention are found in Remington 's Pharmaceutical Sciences (Mack Publishing Company, Philadelphia, PA, 17fh ed. (1985)), which is inco ⁇ orated herein by reference.
  • Remington 's Pharmaceutical Sciences (Mack Publishing Company, Philadelphia, PA, 17fh ed. (1985)), which is inco ⁇ orated herein by reference.
  • Langer, Science 249: 1527-1533 (1990) which is inco ⁇ orated herein by reference.
  • the compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g. , other protease inhibitors).
  • the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting. It should be noted that since the compounds of the present invention are non-peptidic in nature, they tend to have better pharmacokinetic properties (e.g. , better oral availability and increased circulating half- lives) than compounds that are peptidic in nature.
  • the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stearate
  • the compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • the compounds can be utilized in aerosol formulation to be administered via inhalation.
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • the compounds of the present invention can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention.
  • unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • Preferred formulations of the compounds are oral preparations, particularly capsules or tablets containing each from about 10 milligrams up to about 1000 milligrams of active ingredient.
  • the compounds are formulated in a variety of physiologically compatible matrixes or solvents suitable for ingestion or injection.
  • the library synthesis was designed to use commercially available compounds for inco ⁇ oration of the functionality at R R 2 , and R 3 . Exhaustive combination of available materials would provide a library of over 10 billion compounds.
  • version 93.2 of the Available Chemical Directory (ACD) from MDL Information Systems (San Leandro, CA) was used to search for all amines, carboxylic acids, sulfonyl chlorides and isocyanates with MW ⁇ 275 daltons. Compounds with functionality obviously incompatible with the synthesis were eliminated.
  • the resulting list included approximately 700 amines and 1900 acylating agents. However, this list still provided access to more than 1 billion compounds.
  • additional selection criteria were required, and a computational screening process was turned to in an effort to enhance selection.
  • the structure-based design process began with coordinates for pepstatin in a complex with cathepsin D (E. T. Baldwin, et al , Proc. Natl Acad. Sci. , U.S.A. 90, 6796-6800 (1993)).
  • the scaffold is identical to pepstatin on the P r P 3 side, but differs on the B r -P 3 . side and cannot form the same hydrogen bonds with the enzyme (FIG. 3A).
  • the pepstatin positions for the P r P 3 side were used and the three scaffold torsion angles on the P r -P 3 - side were systemically rotated. Each rotation was followed by energy minimization within the cathepsin D active site, using the AMBER (S. J.
  • a diverse library which was set at the same size as the directed library, was prepared to provide a "hit" rate when structure-based methods were not employed.
  • the diverse library was designed to maximize the variety of functional groups and structural motifs of the library components.
  • the sidechains for this library were selected by clustering the original list of components based on their similarity to each other. Components were clustered with the Jarvis-Patrick algorithm (R. A. Jarvis, et al, IEEE Comput C22, 1025-1034 (1973)) using the Daylight connectivity measure of similarity (Daylight Clustering Toolkit, Daylight Chemical Information Systems, Inc., Santa Fe, NM) and a binary Tanimoto metric (P.
  • the R j (amine) components were clustered directly as the primary amines.
  • the R 2 and R 3 acylating agents were each attached to a portion of the scaffold before clustering to yield the proper chemical context at the linkage site.
  • the directed and diverse libraries (1000 compounds each) were prepared using diastereomer 1 of the hydroxyethylamine scaffold with the components used in library syntheses shown in FIGS. 4 and 5, respectively. Because the pilot study with R and S epimers only showed activity at 1 ⁇ M inhibitor concentration for the S epimers, only the S epimers of the directed and diverse library were synthesized. All libraries were synthesized in a spatially separate format, and were screened in a high-throughput fluorometric assay for inhibitory activity against cathepsin D (G. A. Krafft, et al, Methods Enzymol 241, 70-86 (1994))
  • R, B, C, E, F, a, e, h, i, j
  • R 2 B, C, D, E, H, a, e, f
  • R 3 A, D E H, a, b, e, g, h, i (FIGS. 4 and 5).
  • the remaining components were assumed to be compatible with the synthesis sequence.
  • the library synthesis was performed on polystyrene beads (20-40 mesh).
  • the library was synthesized in a spatially separate array using a 96-well filter apparatus. Transfer of the resin to the individual wells was performed using an isopycnic mixture of NN-dimethylformamide (DMF) and 1,2-dichloroethane. Inco ⁇ oration of R ! was carried out using 1.0 M free amine in N-methylpyrrolidinone ( ⁇ MP) at 80°C for 36 h.
  • DMF NN-dimethylformamide
  • ⁇ MP N-methylpyrrolidinone
  • Inco ⁇ oration of R 2 was carried out using stock solutions of 0.3 M carboxylic acid, 0.3 M benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), 0.3 M 7-aza-l-hydroxybenzotriazole (HOAt), and 0.9 M .Pr2Et ⁇ in NMP overnight. The coupling reactions were performed twice to ensure that complete coupling had occurred. After azide reduction with SnCl 2 , PhSH and Et 3 N, inco ⁇ oration of R 3 was carried out as reported above for R 2 .
  • the cleavage mixture was removed from the resin via filtration, followed by rinsing the resin and concentration of the filtrates using a Jouan 10.10 centrifugation concentrator. Toluene was added to form an azeotrope with trifluoroacetic acid during the concentration step. After concentration, the libraries were stored at -20° C. Compounds from each library, picked by random number generation, were analyzed by mass spectrometry in a matrix of ⁇ -cyano cinnamic acid on a Perseptive Biosystems MALDI instrument. For the diverse library the expected molecular ion peaks were observed for 46 of 49 compounds (poor ionization was obtained for the other three) .
  • the assay was performed in DYNATECH Microfluor fluorescence microtiter plates, and readings were taken on a Perkin-Elmer LS-50B with an attached 96-well plate reader.
  • the excitation wavelength was 340 nm.
  • a 340 nm interference filter (Hoya, U-340) for excitation and a 430 nm cut-off filter for emission were used.
  • DMSO (10%) was used to ensure complete dissolution of the inhibitors.
  • the fluorescent unit readings were taken at three time points within the linear region of the substrate cleavage, and percent activity of the enzyme was determined by comparing the change of fluorescent units (FU) for each well to the average change in FU for six control wells without inhibitor.
  • FU fluorescent units
  • Each library was screened at approximately 1 ⁇ M inhibitor with the concentration based on the assumption that 50% of the theoretical yield was obtained for each inhibitor. All wells that showed ⁇ 50% cathepsin D activity were screened at subsequent three-fold dilutions. All active compounds that showed ⁇ 60% enzyme activity in 1 ⁇ M or lower inhibitor concentrations were assayed in duplicate).
  • the directed library yielded 67 compounds that inhibited cathepsin D activity > 50%(G. A. Krafft, et al, Methods Enzymol. 241, 70-86 (1994)). Further dilution of 333 nM and 100 nM inhibitor concentrations afforded 23 and 7 compounds, respectively, that inhibited cathepsin D activity > 50% (see, Table III).
  • the data for the diverse library are also in Table III, infra. There are many uncertainties that can influence the results of a high-throughput fluorescence assay, including the purity of each compound, the concentration of the compounds, and the experimental errors associated with the microtiter fluorescence assay. From repetitive experiments, these errors were estimated to be approximately 30%, ex ressed as enz me activity. Table III. Number of Compounds with ⁇ 50% Inhibition of Cathepsin D in Library Screen"
  • An additional six compounds provided 40-50% inhibition of cathepsin D. tEAA, EFA, EHA, FAA, FFA, FHA, EHB, EFD, EHD, EEF, EHF, FHF, EFH, EHH, FAH, FFH, EH, EHI, EAJ, EFJ, EGJ, EHJ, FHJ.
  • An additional thirty compounds provided 40-50% inhibition of cathepsin D. ⁇ One hundred compounds were selected by random number generation for testing at 10 ⁇ M.
  • the K t values were calculated from IC 50 determinations (see, Table IV). From the compounds that were fully characterized, one compound was obtained from the directed library with a K ⁇ below 100 nM, whereas the diverse library contained inhibitors that were 3-4 times less potent. Table TV. Inhibition Constants for a Number of the Compounds That Are Potent Inhibitors 0
  • Structural diversity may be derived through Grignard addition to a solid support-bound -alkoxy pyrrolidine amide 3 (see, FIG. 7).
  • the source of diversity is derived from aromatic and alkyl Grignard reagents.
  • the Grignard reagents that are not commercially available can be synthesized using activated magnesium turnings, or a magnesium anthracene THF complex and the corresponding aromatic and alkyl halides.
  • Grignard reagents are a suitable source to introduce diversity in the R ⁇ site of potential aspartyl protease inhibitors, since the Sj protease surface tends to be hydrophobic. The resulting ketone is reduced using chelation and non-chelation conditions to provide the desired diastereomer.
  • the pyrrolidine amide 4 prepared in 3 steps in an overall 76% yield from commercially available methyl (s)-(-)-2,2-dimethyl-l,3-dioxolane-4-carboxylate, was coupled to benzyloxybenzyl bromide resin 5 using sodium hydride, tetrabutylammonium iodide, and catalytic 18-Crown-6 in THF for 2 hours at 45 °C (see, FIG. 8).
  • Bromide resin 5 was derived from carbon tetrabromide, triphenylphosphine, and commercially available Wang resin.
  • Secondary alcohol 7 was converted to azide 8 through the formation of a secondary nosylate using 4-nitrobenzenesulfonyl chloride and 4-pyrrolidinopyridine in chloroform followed by azide displacement with sodium azide in NN-dimethylformamide at 50°C.
  • the -methoxy trityl protecting group was selectively removed using 1 % p- toluenesulfonic acid in methylene chloride.
  • ⁇ osylation of the primary alcohol with 4- nitrobenzenesulfonyl chloride and pyridine in chloroform provided azido-nosylate 9.
  • a library of 204 compounds was derived from the components in FIG. 9.
  • the most potent inhibitors of Cathepsin D were synthesized on a larger scale, purified, and biologically assayed to determine K, values as detailed in Table VI.
  • Overall yields of these scaled-up inhibitors ranged from 46-48 % for the entire 12 step solid-phase synthesis as determined by the mass balance of desired product after column chromatography purification.
  • Novel low nanomolar inhibitors of cathepsin D were identified rapidly using combinatorial chemistry coupled with two different computational strategies.
  • the diverse and directed libraries together yielded over 90 compounds active at 1 ⁇ M and 26 active in the submicromolar range.
  • the "hit rate" for activity at 1 ⁇ M is 6-7% for the directed library and 2-3 % for the diverse library.
  • both the directed and diverse libraries are based on the "active" epimer of the scaffold, the results from the directed library are clearly superior.
  • At all concentrations ⁇ 1 ⁇ M there were more "hits” in the directed library than the diverse library.
  • the most potent inhibitors from the directed library are 3-4 fold better than those in the diverse library. It is clear from the results that the number and quality of the active compounds can be increased by using relevant information about the target.
  • a strength of the structure-based procedure is that it leads directly to testable geometrical hypotheses.
  • S epimers are predicted to bind better than the R epimers; 2) there are two energetically reasonable scaffold conformations (family 1 +2, family 3 +4), which place R groups into different pockets; 3) all the inhibitors are assumed to bind in approximately the same orientation as pepstatin.
  • the first hypothesis was directly tested in pilot experiments where no inhibitors based upon the R epimer had activity at 1 ⁇ M.
  • the R epimer of one of the most potent compounds had a K t no better than 5 ⁇ M while the K ⁇ of the S epimer was 15 nM (see, Table V).
  • Organotypic entorhinohippocampal cultures were prepared using the technique of Stoppini, et al , J. Neurosci. Methods, 37, 173-182 (1991). Briefly, the caudal pole of the cerebral hemisphere containing the entorhinal cortex and hippocampus were harvested from brains of 6-7 days old Sprague-Dawley rat pups under sterile condition.
  • Brain tissue explants were then planted onto 30 mm cell culture inserts (Illicell-CM, Millipore, Bedford, MA) that were placed in 6 well culture trays with 1 mL of growth medium (MEM with Hank's salts, Gibco, 20% horse serum, 3 mM glutamine, 25 mM HEPES, 5 mM NaHCO 3 , 25 mM glucose, 0.5 mM ascorbate, 2 mM CaCl 2 , 2.5 mM MgCl 2 , 0.5 mg/L insulin, and penicillin, pH 7.2; Bi, et al , J. Comp. Neuro. , 401, 382-394 (1998). The cultures were incubated at 35°C with a 5% CO 2 -enriched atmosphere and fed every other day until use.
  • MEM Hank's salts, Gibco, 20% horse serum, 3 mM glutamine, 25 mM HEPES, 5 mM NaHCO 3 , 25 mM glucose, 0.5 mM as
  • organotypic cultures were incubated with growth medium containing either 20 ⁇ M N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD; BACHEM Bioscience, Torrance, CA), a selective inhibitor of cathepsins B and L (Shaw and Dean, 1980), in 0.01 % DMSO, 20 ⁇ M chloroquine (Sigma) or vehicle alone for days as specified.
  • ZPAD N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone
  • EA-1 To test the effect of EA-1 on the generation of hype ⁇ hosphorylated tau fragments found in neurofibrillary tangles in Alzheimer's disease and other tau pathology-related diseases, 1 ⁇ M of EA-1 or 10 ⁇ M of CEL5-172 were applied alone or together with 20 ⁇ M ZPAD.
  • entorhinohippocampal explants were collected and sonicated in 10 mM Tris-HCl buffer (pH 7.4) containing 0.32 M sucrose, 2 mM EDTA, 2 mM EGTA, and 0.1 mM leupeptin. Aliquots of homogenate (80-100 ⁇ g protein lane) were diluted with equal amounts of 2x sample buffer [Ix sample buffer consists of 2% sodium dodecyl sulphate (SDS), 50 mM Tris-HCl (pH 6.8), 10% 2-mercaptoethanol, 10% glycerol and 0.1 % Bromophenol Blue].
  • SDS sodium dodecyl sulphate
  • proteins were subjected to SDS-PAGE performed according to the method of Laemmli (1970) using 10% poly acrylamide gel; and then transferred on to nitrocellulose membranes as described by Towbin, et al , Proc. Natl. Acad. Sci. USA, 76, 4350-4354 (1979).
  • Nitrocellulose membranes were first incubated in 3 % gelatin in Tris-buffered saline (TBS) for 1 hour at room temperature, followed by incubation with 1 % gelatin in TBS with 0.5% Tween 20 (TTBS) containing antibodies that recognize either the phosphorylated tau protein (AT8; 1:500) or unphosphorylated tau protein (tau 1, PCI C6; 1: 100, Boehringer Mannheim) at room temperature overnight.
  • TBS Tris-buffered saline
  • TTBS Tween 20
  • EA-1 Another four groups of cultured entorhinohippocampal slices were maintained for 14 days and used to test the effect of EA-1: (1) control medium; (2) 20 ⁇ M of ZPAD; (3) a new selective inhibitor (EA-1 at 1 ⁇ M) of cathepsin D; (4) ZPAD combined with EA-1. Following this, the slices ere homogenized and samples processed for immunoblotting. Like CEL5-172, EA-1 by itself did not detectably change the concentrations of hype ⁇ hosphorylated tau fragments (see, FIG. 11); however, it exhibited a much higher blocking effect than CEL5-172. It is noted that EA-1 and CEL5-172 have the following structures, respectively:
  • ACSF artificial cerebrospinal fluid
  • EDTA ethylenediaminetetraacetic acid
  • EGTA ethyleneglycol bis (
  • PBS phosphate-buffered saline
  • SDS sodium dodecyl sulphate
  • TBS Tris-buffered saline
  • ZPAD N-CBZ-L-phenylalanyl-L-alanme-diazomethylke tone.
  • Hippocampal slice cultures were exposed to medium containing one of three cathepsin D inhibitors (see, below) or to 'ZPAD' (N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone), a selective inhibitor of cathepsins B and L (Green, et al , J. Biol Chem., 256: 1923-1928 (1981); Richardson, et al , J. Cell Biol, 107:2097-2107 (1988); and Shaw, et al , Biochem. J., 186:385-390 (1980)).
  • ZPAD was used at 20 ⁇ M, and both ZPAD and cathepsin D inhibitors were dissolved first in dimethyl sulfoxide (DMSO), then diluted to the concentrations needed using culture media. Equal amount of DMSO ( ⁇ 0.1 %) was also applied to control slices.
  • DMSO dimethyl sulfoxide
  • Physiology experiments were performed on hippocampal slices kept in vitro for 2 weeks followed by being incubated with cathepsin inhibitors for an additional six days.
  • the slices were placed in a submersion chamber containing artificial cerebrospinal fluid (ACSF) and maintained at room temperature.
  • the flow rate of ACSF through the recording chamber was 1.2 ml/min.
  • Electrodes were positioned 120 min after the slices had been placed in the chamber.
  • Patch-clamp recordings were made from pyramidal neurons in the stratum pyramidale of area CA1.
  • the recording pipettes had resistances of 3-5 M ⁇ . Holding potentials were -70 mV. Currents were recorded using a patch amplifier with a 4-pole low-pass Bessel filter at 2 kHz and digitized at 10 kHz.
  • Hippocampal slices were collected and sonicated in 10 mM Tris-HCl buffer (pH 7.4) containing 0.32 M sucrose, 2 mM EDTA, 2 mM EGTA, and 0.1 mM leupeptin. Aliquots of homogenate (80-100 ⁇ g protein/lane) were diluted with equal amount of 2x sample buffer [lx sample buffer consists 2% sodium dodecyl sulphate (SDS), 50 mM Tris-HCl (pH 6.8), 10% 2 mercaptoethanol, 10% glycerol and 0.1 % bromophenol blue].
  • SDS sodium dodecyl sulphate
  • proteins were subjected to SDS-PAGE performed according to the method of Laemmli (Nature, 227:680-685 (1970)) using 10% polyacrylamide gel; and then transferred on to nitrocellulose membranes as described by Towbin, et al. (Proc. Natl. Acad. Sci. USA, 76:4350-4354 (1979)).
  • Nitrocellulose membranes were first incubated in 3 % gelatin in Tris-buffered saline (TBS) for 1 hr at room temperature, followed by incubation with 1 % gelatin in TBS with 0.5% Tween 20 (TTBS) containing antibodies that recognize either the phosphorylated tau protein (AT8; 1:500; Innogenetics, Belgium), unphosphorylated tau protein (tau 1, PCI C6; 1 : 100; Boehringer Mannheim, Indianapolis, IN), or anti- cathepsin D antibodies (1: 100; Oncogene Science, Cambridge, MA) at room temperature overnight.
  • TBS Tris-buffered saline
  • TTBS Tween 20
  • the three inhibitors used in the below experiments had molecular weights of 650-800 Da and Ki's for cathepsin D between 1-15 nM (see, FIG. 12). They were products of a synthesis program in which the crystal structure of cathepsin D complexed with the peptide-based natural product pepstatin served as a model with which to select building blocks for a combinatorial library. Equivalent energy conformations of a (hydroxyethyl) amine scaffold were grouped into families and computational methods (Lewis, et al , J. Mol.
  • Carboxy-terminal fragments of the amyloid precursor protein are a characteristic feature of slices treated with ZPAD, chloroquine, or exogenous amyloid (Bahr, et al , Exp Neurol, 129: 1-14 (1994); Bahr, et al , J Comp Neurol, 397: 139-147 (1998)).
  • the cathepsin D inhibitors did not induce these peptides (not shown).
  • the compounds were also without evident effect on inhibitory and excitatory synaptic currents, extracellular field potentials, or post-synaptic responses to repetitive stimulation (FIG. 13D).
  • the new inhibitors are selective in that they do not elicit anatomical and biochemical changes found with inhibitors of cathepsins B and L, or with more generalized lysosomotropic agents, and do not influence sensitive physiological indices.
  • Antibodies e.g. , 'AT8'; Goedert, et al , Proc. Natl. Acad. Sci. USA, 90:5066-70 (1993); Greenberg, et al , Proc. Natl. Acad. Sci. USA, 87:5827-31 (1990)
  • against hype ⁇ hosphorylated tau or paired helical filaments in human brain variably label a 29 kDa band in western blots from adult rat brains (Bednarski, et al, J Neurochem., 67: 1846-1855 (1996)) or 'mature' cultured slices (Bi, et al , supra (1999)).
  • FIG. 14B summarizes the results for EA-1. This compound again had no detectable effects on tau 29 concentrations (lane 3 of FIG.
  • FIG. 15 describes the time and dose dependencies of the interactions between cathepsin inhibitors.
  • ZPAD induced increases in the phosphorylated tau fragment appeared at 48 hrs — the earliest time point tested - and increased steadily thereafter (FIG. 15A).
  • the effect of the cathepsin D inhibitor was evident from the first measurement and resulted in a complete blockade of the ZPAD-elicited changes by 96 hrs.
  • the inhibitor EA-1 had dose dependent effects in slices treated with ZPAD for 6 days (FIG. 15B); threshold concentration appeared to lie between 0.05 (no detectable effect) and 1.0 ⁇ M (41 % reduction in ZPAD induced fragments). Note that the cathepsin D inhibitor by itself had no effect on tau 29 concentrations at any time point or dosage.
  • Conversion of cathepsin D into active forms may involve autocatalysis (Conner, Biochem. J., 263:601-604 (1989); Conner, et al., Biochem., 28:3530-3533 (1989); and Hasilik, et al , Eur. J. Biochem., 125:317-321 (1982)). If so, then the inhibitors used here could indirectly block the formation of tau fragments by preventing the increases in lysosomal and cytoplasmic cathepsin D that develop within hours of chemically induced lysosomal dysfunction (Bednarski, et al , supra (1998); Hoffman, et al , Neurosci. Lett., 250:75-78 (1998)).
  • cathepsin D inhibitors have any effect on the biosynthesis and maturation of cathepsin D
  • cultured hippocampal slices were treated with inhibitor alone or inhibitor plus ZPAD.
  • EA-1 by itself did not detectably alter the levels of cathepsin D isoforms at concentrations from 50 nM to 5 ⁇ M (FIG. 16B).
  • the levels of procathepsin D and single chain cathepsin D were similar to those observed in cultures treated with ZPAD alone.
  • the increase in heavy chain isoform was substantially reduced (70 % to 15% , FIG. 16) in the presence of EA-1.
  • the compounds did not cause evident physiological changes over the time courses tested and leave unchanged biochemical measures sensitive to cathepsins B/L inhibitors or to the broad-spectrum inhibitor chloroquine. It appears, then, that inhibition of cathepsin D to a degree sufficient to block specific biochemical reactions (below) has discrete consequences and, in general, is well tolerated by brain tissue for at least several days.
  • the findings also provide a direct test of the hypothesis that the rapid formation of hype ⁇ hosphorylated tau fragments occurring in association with lysosomal dysfunction is due to cathepsin D, or cathepsin D-like aspartyl proteases. Three distinct inhibitors produced near complete suppression of the increases that normally follow pharmacologically induced lysosomal dysfunction.
  • the blocking effects were in evidence from the first appearance of tau fragmentation and had threshold concentrations in the sub-micromolar range. That the inhibitors did not reduce baseline levels may indicate that the fragments have a long half-life, a point of possible significance with regard to the production of tangles.
  • the differential effects of cathepsin D inhibitors on ZPAD- induced tau 29 vs basal level tau 29 demonstrate that the blocking effect is not due to modification of the antigenic epitopes by these non-peptidic compounds.
  • Cathepsin D inhibitors markedly reduced the formation of tau 29, but did not reverse decreases in native tau, suggesting that cathepsin D is not solely responsible for the breakdown of tau protein that occurs following pharmacologically induced lysosomal dysfunction.
  • the cathepsin D inhibitor is a compound selected from the group consisting of CEL5-A, CEL5-G and EA-1, the structures of which are set forth in FIG. 12.

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Abstract

L'invention concerne (i) des inhibiteurs non peptidiques de l'aspartyl protéase; (ii) des méthodes permettant de moduler la maturation d'une protéine précurseur amyloïde (APP); (iii) des méthodes permettant de moduler la maturation d'une protéine tau (protéine τ); et (iv) des méthodes permettant de traiter les maladies neurodégénératives.
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