Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • 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
  • A61P25/28—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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
  • C07D223/14—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
  • C07D223/18—Dibenzazepines; Hydrogenated dibenzazepines

Definitions

• This invention relates to ⁇ -ammoacid containing compounds which inhibit ⁇ -amyloid peptide release and/or its synthesis and are useful m treating Alzheimer's disease.
• Alzheimer's Disease is a degenerative brain disorder characterized clinically by progressive loss of memory, cognition, reasoning, judgment and emotional stability that gradually leads to profound mental deterioration and ultimately death.
• Alzheimer's disease is a very common cause of progressive mental failure (dementia) m aged humans and is believed to represent the fourth most common medical cause of death m the United States.
• Alzheimer's disease has been observed m races and ethnic groups worldwide and presents a major present and future public health problem. The disease is currently estimated to affect about two to three million individuals m the United States alone. Alzheimer's disease is at present incurable. No treatment that effectively prevents Alzheimer's disease or reverses its symptoms and course is currently known.
• the brains of individuals with Alzheimer's disease exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits m blood vessels) and neurofibrillary tangles.
• senile or amyloid
• amyloid angiopathy amyloid deposits m blood vessels
• neurofibrillary tangles Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found m several areas of the human brain important for memory and cognitive function m patients with Alzheimer's disease. Smaller numbers of these lesions m a more restrictive anatomical distribution are also found m the brains of most aged humans who do not have clinical Alzheimer's disease.
• Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral Hemorrhage with Amyloidosis of the Dutch Type (HCH A-D) .
• a definitive diagnosis of Alzheimer's disease usually requires observing the aforementioned lesions m the brain tissue of patients who have died with the disease or, rarely, m small biopsied samples of brain tissue taken during an invasive neurosurgical procedure.
• amyloid angiopathy The principal chemical constituent of the amyloid plaques and vascular amyloid deposits (amyloid angiopathy) characteristic of Alzheimer's disease and the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 39-43 ammo acids designated the ⁇ - amyloid peptide ( ⁇ AP) or sometimes A ⁇ , A ⁇ P or ⁇ /A4 ⁇ - Amylo d peptide was first purified and a partial ammo acid sequence was provided by Glenner, et al . Biochem. Biophys . Res . Commun. , 120:885-890, (1984). The isolation procedure and the sequence data for the first 28 ammo acids are described in U.S. Patent No. 4,666,8292.
• ⁇ -amyloid peptide is a small fragment of a much larger precursor protein termed the amyloid precursor protein (APP) , that is normally produced by cells m many tissues of various animals, including humans.
• APP amyloid precursor protein
• s protease enzyme
• a mutation at ammo acid 693 of the 770-ammo acid isoform of APP has been identified as the cause of the ⁇ -amyloid peptide deposition disease, HCHWA-D, and a change from alanme to glycme at am o acid 692 appears to cause a phenotype that resembles Alzheimer's disease is some patients but HCHWA-D others.
• the discovery of these and other mutations m APP m genetically based cases of Alzheimer's disease prove that alteration of APP and subsequent deposition of its ⁇ -amyloid peptide fragment can cause Alzheimer's disease.
• the treatment methods would advantageously be based on drugs which are capable of inhibiting ⁇ -amyloid peptide release and/or its synthesis m SUMMARY OF THE INVENTION
• This invention provides ⁇ -aminoacid containing compounds of formula I :
• Ri is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, aryl, heteroaryl and heterocyclic ;
• R 2 is selected from the group consisting of hydrogen, alkyl, cycloalkyl, and aryl;
• R is selected from the group consisting of hydrogen, alkyl, cycloalkyl, and aryl;
• Z is represented by the formula -CX'X"- wherein
• X' is selected from the group consisting of hydrogen, hydroxy, and fluoro
• X" is selected from the group consisting of hydrogen, hydroxy, and fluoro
• X' and X" together form an oxo group
• W is a cyclic group selected from the group consisting of
• Q ' is oxygen or sulfur
• each V is independently selected from the group consisting of hydroxy, acyl , acyloxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, aminoacyl , alkaryl, aryl, aryloxy, carboxyl, carboxylalkyl , cyano, halo, nitro, heteroaryl, thioalkoxy, substituted thioalkoxy, trihalomethyl ;
• Ra is selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, amino, carboxyl, carboxyl alkyl, cyano, halo;
• Rb is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, aryl, heteroaryl, heterocyclic;
• Rc is selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, heteroaryl, heterocyclic, cycloalkyl, and substituted cycloalkyl;
• t is an integer from 0 to 4.
• w is an integer from 0 to 4.
• This invention also provides for novel pharmaceutical compositions comprising a compound of the formula I and a pharmaceutically acceptable diluent. Additionally, this invention provides a method for inhibiting ⁇ -amyloid peptide release and/or its synthesis in a cell which method comprises administering to such a cell an amount of a compound or a mixture of compounds of formula I above effective in inhibiting the cellular release and/or synthesis of ⁇ -amyloid peptide.
• the compounds of formula I can also be employed in conjunction with a pharmaceutical composition to prophylactically and/or therapeutically prevent and/or treat Alzheimer's disease. Accordingly, the present invention provides a prophylactic method for preventing the onset of Alzheimer's disease in a patient at risk for developing Alzheimer's disease which method comprises administering to said patient a pharmaceutical composition comprising a pharmaceutically inert carrier and an effective amount of a compound or a mixture of compounds of formula I above.
• the present invention also provides a therapeutic method for treating a patient with Alzheimer's disease in order to inhibit further deterioration in the condition of that patient which method comprises administering to said patient a pharmaceutical composition comprising a pharmaceutically inert carrier and an effective amount of a compound or a mixture of compounds of formula I above.
• ⁇ -amyloid peptide refers to a 39-43 amino acid peptide having a molecular weight of about 4.2 kD, which peptide is substantially homologous to the form of the protein described by Glenner, et al . Biochem. Biophys . Res . Commun. (1984) 120:885-890, including mutations and post- translational modifications of the normal ⁇ -amyloid peptide.
• the ⁇ -amyloid peptide is an approximate 39-43 ammo acid fragment of a large membrane-spanning glycoprotem, referred to as the ⁇ -amyloid precursor protein (APP) . Its 43 -ammo acid sequence is:
• Glu Val His His Gin Lys Leu Val Phe Phe 21 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala 31 lie lie Gly Leu Met Val Gly Gly Val Val 41 lie Ala Thr (SEQ ID NO : 1) or a sequence which is substantially homologous thereto .
• Alkyl refers to monovalent alkyl groups preferably having from 1 to 20 carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, n-pentyl , n-hexyl, and the like. It is understood that the term alkyl includes C 1 -C 4 alkyl. "C 1 -C 4 alkyl” refers to monovalent alkyl groups preferably having from 1 to 4 carbon atoms.
• This term is exemplified by groups such as methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl .
• Substituted alkyl refers to an alkyl group, preferably of from 1 to 10 carbon atoms, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, ammo, substituted ammo, ammoacyl , ammoacyloxy, oxyacylammo, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl , keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy , heterocyclic, heterocyclooxy, hydroxyamino , alkoxyamino, nitro, -SO-alkyl, -
• Substituted alkenylene refers to an alkenylene group, preferably of from 2 to 10 carbon atoms, having from 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxyl , acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl , aminoacyloxy, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, heteroaryl, heterocyclic, heterocyclooxy, nitro -SO-alkyl, - SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -S0 2 -alkyl, -S0 2 -substituted
• Alkaryl refers to -alkylene-aryl groups preferably having from 1 to 8 carbon atoms in the alkylene moiety and from 6 to 10 carbon atoms in the aryl moiety. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.
• Alkoxy refers to the group “alkyl-O-”. Preferred alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1 , 2-dimethylbutoxy, and the like.
• Substituted alkoxy refers to the group “substituted alkyl-O-" where substituted alkyl is as defined above.
• alkenyl refers to alkenyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkenyl unsaturation .
• Substituted alkenyl refers to an alkenyl group as defined above having from 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxyl , acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl , keto, thioketo, thioi, thioalkoxy, substituted thioalkoxy, aryl, heteroaryl, heterocyclic, heterocyclooxy, nitro -SO-alkyl, -SO-substituted alkyl, -SO- aryl, -SO-heteroaryl, -S0-alkyl, -S0 2 -substituted alkyl, - S0 -aryl
• Alkynyl refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-2 sites of alkynyl unsaturation.
• Preferred alkynyl groups include ethynyl, propargyl, and the like.
• Substituted alkynyl refers to an alkynyl group as defined above having from 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkoxy, substituted cycloalkoxyl, acyl, acylamino, acyloxy, ammo, substituted amino, aminoacyl, aminoacyloxy, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, heteroaryl, heterocyclic, heterocyclooxy, nitro -SO-alkyl, -SO-substituted alkyl, -SO- aryl, -SO-heteroaryl, -S0 2 -alkyl, -S0-substituted alkyl, -S0 2 -
• Acyl refers to the groups alkyl-C(O)-, substituted alkyl-C(O)-, cycloalkyl-C (0) - , substituted cycloalkyl-C (0) - , aryl-C(O)-, heteroaryl-C (0) - and heterocyclic-C (0) - where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• Acylamino refers to the group -C(0)NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, heterocyclic and where both R groups are joined to form a heterocyclic group, wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• Substituted ammo refers to the group -N(R) 2 where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, and where both R groups are joined to form a heterocyclic group.
• R groups are hydrogen, - N(R) 2 is an ammo group.
• substituted ammo groups include, by way of illustration, mono- and di- alkylamino, mono- and di- (substituted alkyl)ammo, mono- and di-arylammo, mono- and di-heteroarylammo, mono- and di- heterocyclic ammo, and unsymmet ⁇ c di-substituted amines having different substituents selected from alkyl, substituted alkyl, aryl, and the like.
• blocking group or "protecting group” refers to any group which prevents undesired reactions from occurring at the protected functionality and which may be removed by conventional chemical and/or enzymatic procedures. Selection and use of protecting groups is well understood and appreciated m the art. For example see,
• a protecting group may also be a covalently attached to a solid support as is well known and appreciated m the art of peptide synthesis and combinatorial chemistry.
• aminoacyl refers to the group -NRC(0)R where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• Aminoacyloxy refers to the group -NRC(0)0R where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• Alkyloxy refers to the groups alkyl-C (0) 0- , substituted alkyl-C (0)0- , cycloalkyl-C (0) 0- , substituted cycloalkyl-C (0) -, aryl-C(0)0-, heteroaryl-C (0) 0- , and heterocyclic-C (0) 0- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• Aryl refers to an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl) .
• Preferred aryls include phenyl, naphthyl and the like.
• such aryl groups can optionally be substituted with from 1 to 5 substituents selected from the group consisting of acyloxy, hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heterocyclic, aminoacyloxy, oxyacylamino , thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO- alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -S0 : -
• Aryloxy refers to the group aryl-0- wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
• Carboxyalkyl refers to the groups " -C (0) Oalkyl " and "-C (0) 0-substituted alkyl” where alkyl is as defined above.
• Cycloalkyl refers to cyclic alkyl groups of from 3 to 12 carbon atoms having a single cyclic ring or multiple condensed rings
• Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as qummclidme, adamantanyl, and the like.
• Substituted cycloalkyl refers to cycloalkyl groups having from 1 to 5 (preferably 1 to 3 ) substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, ammo, substituted ammo, aminoacyl, aminoacyloxy, oxyacylammo, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyammo , alkoxyammo, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl,
• Cycloalkenyl refers to cyclic alkenyl groups of from 4 to 8 carbon atoms having a single cyclic ring and at least one point of internal unsaturation.
• suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl , cyclopent-3-enyl , cyclooct-3-enyl and the like.
• Substituted cycloalkenyl refers to cycloalkenyl groups having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, ammo, substituted ammo, aminoacyl, aminoacyloxy, oxyacylammo, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyammo, alkoxyammo, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-hetero
• Heteroaryl refers to an aromatic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring) .
• heteroaryl groups can be optionally substituted with 1 to 5 substituents selected from the group consisting of acyloxy, hydroxy, acyl, alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, ammo, substituted ammo, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heterocyclic, aminoacyloxy, oxyacylammo, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO- aryl, -SO-heteroaryl, -S0 2
• heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizmyl or benzothienyl) .
• Preferred heteroaryls include pyridyl, pyrrolyl and furyl .
• Heteroaryloxy refers to the group “-O-heteroaryl” .
• Heterocycle or “heterocyclic” refers to a monovalent saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 15 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur or oxygen within the ring.
• heterocyclic groups can be optionally substituted with 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, ammo, substituted ammo, aminoacyl, aminoacyloxy, oxyacylammo, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyammo, alkoxyammo, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heter
• heterocyclic groups can have a single ring or multiple condensed rings.
• Preferred heterocyclics include morpholmo, pipe ⁇ dmyl, and the like.
• Examples of heterocycles and heteroaryls include, but are not limited to, pyrrole, furan, lmidazole, pyrazole, pyridme, pyrazme, pyrimidme, py ⁇ dazme, mdolizme, isoindole, mdole, mdazole, purme, qumolizme, lsoqu olme, qumol e, phthalaz e, naphthylpy ⁇ dme, qumoxalme, qumazoline, cmnolme, pteridme, carbazole, carbol e, phenanthridine, acridme, phenanthroline, isothiazole, phenazme, isoxazole, phenoxa
• Heterocyclooxy refers to the group “-O-heterocycle” .
• Oxyacylammo refers to the group -OC(0)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein .
• Thioalkoxy refers to the group -S-alkyl.
• Substituted thioalkoxy refers to the group -S- substituted alkyl.
• Thioaryloxy refers to the group aryl-S- wherein the aryl group is as defined above including optionally substituted aryl groups also defined above.
• Thioheteroaryloxy refers to the group heteroaryl-S- wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.
• pharmaceutically-acceptable addition salt refers to an acid addition salt
• the compound of formula I and the intermediates described herein form pharmaceutically acceptable acid addition salts with a wide variety of organic and inorganic acids and include the physiologically acceptable salts which are often used m pharmaceutical chemistry Such salts are also part of this invention.
• a pharmaceutically-acceptable addition salt is formed from a pharmaceutically-acceptable acid as is well known m the art Such salts include the pharmaceutically acceptable salts listed m Journal of Pharmaceutical Science, 66 , 2-19 (1977) which are known to the skilled artisan.
• Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hyd ⁇ odic, nitric, sulfuric, phosphoric, hypophospho ⁇ c , metaphospho ⁇ c , pyrophospho ⁇ c , and the like.
• Salts derived from organic acids such as aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfomc acids, may also be used.
• Such pharmaceutically acceptable salts thus include acetate, phenylacetate, t ⁇ fluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dmitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, ⁇ -hydroxybutyrate, butyne-1, 4-d carboxylate, hexyne-1 , 4-d ⁇ carboxylate, caprate, caprylate, cmnamate, citrate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate maleate, hydroxymaleate malonate, mandelate, mesylate, nicotmate, isonicotmate, nitrate, oxalate, phthalate teraphthalate, propiolate, propionate, pheny
• compounds of formula I exist as stereoisomers .
• the present invention relates to the stereoisomers of the compounds of formula I.
• the Cahn-Prelog-Ingold designations of (R) - and (S)- and the designations of L- and D- for stereochemistry relative to the isomers of glyceraldehyde are used to refer to specific isomers where designated.
• the specific isomers of the compounds of formula I can by prepared by stereospecific synthesis.
• the compounds of formula I and the starting materials for their preparation can be resolved and recovered by techniques known the art, such as, chromatography on chiral stationary phases, and fractional recrystallization of addition salts formed by reagents used for that purpose.
• Useful methods of resolving and recovering specific stereoisomers are known m the art and described m Stereochemistry of Organic Compounds, E.L. Eliel and S.H. Wilen (Wiley-Interscience 1994), Enantiomers, Racemates, and Resolutions, J. Jacques, A. Collet, and S.H. Wilen (Wiley-Interscience 1981), and
• R 2 or R 3 are phenyl substituted with from 1 to 3 substituents selected from the group consisting of hydrogen, alkyl, alkoxy, and halo are more preferred with phenyl being most preferred.
• Rb is selected from the group consisting of hydrogen, alkyl and aryl
• Rc is selected from the group consisting of alkyl, and aryl ;
• w is 0; are more preferred.
• Reaction Scheme A.l, step a depicts the coupling reaction of an appropriate amino-protected ⁇ -amino acid of formula (1) and an appropriate compound W-NH of formula (2) .
• Appropriate amino-protected ⁇ -amino acids are ones in which R 2 and R 3 are as desired in the final product of formula I and readily available to the person skilled in the art and can be prepared as described herein.
• An appropriate compound of formula (1) may also have the stereochemistry that is desired in the final compound of formula I.
• An appropriate compound of formula (2) is one in which W is as desired in the final compound of formula I .
• An appropriate compound of formula (2) may also have the stereochemistry that is desired in the final compound of formula I.
• benzodiazepine derivatives suitable for use in this invention can be prepared using conventional procedures and reagents.
• a 2-aminobenzophenone can be readily coupled to ⁇ - ( isopropylthio) -N- (benzyloxycarbonyl) glycine by first forming the acid chloride of the glycine derivative with oxayl chloride, and then coupling the acid chloride with the 2-ammobenzophenone m the presence of a base, such as 4-methylmorpholme, to afford the 2- (oc- ( isopropylthio) -N- (benzyloxycarbonyl ) glycmyl ) -ammobenzophenone .
• a base such as 4-methylmorpholme
• 2 , 3-dihydro-5-phenyl-lH-l, 4- benzod ⁇ azepm-2-ones can be readily ammated at the 3- position using conventional azide transfer reactions followed by reduction of the resulting azido group to form the corresponding ammo group. The conditions for these and related reactions are described in the examples set forth below. Additionally, 2 , 3-dihydro-5-phenyl-lH-l , 4- benzod ⁇ azepm-2-ones are readily alkylated at the 1-pos ⁇ t ⁇ on using conventional procedures and reagents.
• this reaction is typically conducted by first treating the benzodiazepmone with about 1.1 to about 1.5 equivalents of a base, such as sodium hydride, potassium tert-butoxide, potassium 1 , 1 , 1 , 3 , 3 , 3-hexamethyld ⁇ s ⁇ lazane, cesium carbonate, m an inert diluent, such as DMF.
• a base such as sodium hydride, potassium tert-butoxide, potassium 1 , 1 , 1 , 3 , 3 , 3-hexamethyld ⁇ s ⁇ lazane, cesium carbonate
• m an inert diluent, such as DMF.
• This reaction is typically conducted at a temperature ranging from about -78 2 C to about 80 2 C for about 0.5 to about 6 hours.
• the resulting amon is then contacted with an excess, preferably about 1.1 to about 3.0 equivalents, of an alkyl halide, typically an alkyl chloride, bromide or io
• this reaction is conducted at a temperature of about 0 2 C to about 100 2 C for about 1 to about 48 hours.
• the 3-ammo-2 , 4-d ⁇ oxo-2 ,3,4, 5-tetrahydro-lH-l , 5-benzod ⁇ azepmes employed m this invention are typically prepared by first coupling malomc acid with a 1 , 2-pnenylened ⁇ amme Conditions for this reaction are well known m the art and are described, for example, PCT Application WO 96-US8400 960603 Subsequent alkylation and amination using conventional procedures and reagents affords various 3- ammo-1 , 5-bis (alkyl) -2 , 4-d ⁇ oxo-2 ,3,4, 5-tetrahydro-lH-l , 5- benzodiazepmes . Such procedures are described m detail m PCT Application No PCT/US97 /22986
• the coupling reaction depicted m Reaction Scheme A 1, step a involves a reaction which is conventionally conducted for peptide synthesis and synthetic methods used therein can also be employed.
• well known coupling reagents such as carbodnmides with or without the use of well known additives such as N-hydroxysuccmimide, 1- hydroxybenzot ⁇ azole, etc. can be used to facilitate coupling.
• the reaction is conventionally conducted m an inert aprotic polar diluent such as dimethylformamide, methylene chloride, chloroform, acetonit ⁇ le, tetrahydrofuran and the like.
• the acid halide of compound (1) can be employed m the reaction and, when so employed, it is typically employed the presence of a suitable base to scavenge the acid generated during the reaction Suitable bases include, by way of example, triethylamine, N,N-d ⁇ sopropylethylamme, N-methylmorpholme
• reaction is preferably conducted at from about 0 2 C to about 60 2 C until reaction completion which typically occurs within 1 to about 24 hours
• the product of formula (3) is recovered by conventional methods including precipitation, chromatography, filtration and the like or alternatively is deprotected to the corresponding amme of formula (4) without purification and/or isolation other than conventional work-up (e.g., aqueous extraction, etc.).
• Reaction Scheme A.l, step b depicts the deprotection of a compound of formula (3) to give a compound or formula (4) .
• an acid addition salt is formed using a pharmaceutically- acceptable acid.
• ac d addition salts are well known and appreciated m the art.
• step a depicts the coupling reaction of an appropriate carboxy-protected ⁇ -amino acid of formula (6) and an appropriate compound of formula (5), as described above, to give a compound of formula (7) .
• Appropriate carboxy-protected ⁇ -amino acids are ones in which R 2 and R 3 are as desired in the final product of formula I and readily available to the person skilled in the art and can be prepared as described herein.
• An appropriate compound of formula (6) may also have the stereochemistry that is desired m the final compound of formula I.
• This coupling reaction is carried out using the acid of formula (5) or the acid halide derived therefrom, m a manner similar to those taught in Reaction Scheme A.l, step a.
• Reaction Scheme A.2, step b depicts the deprotection of a compound of formula (7) to give a compound or formula (8) .
• Such deprotections of carboxy protecting groups is well known and appreciated in the art.
• step c depicts the coupling reaction of an appropriate compound of formula (2), as described above, and a compound of formula (8) .
• Appropriate compounds of formula (2) are the coupling reaction depicted in step c are taught in Reaction Scheme A.l, step a.
• an acid addition salt is formed using a pharmaceutically- acceptable acid.
• the formation of acid addition salts is well known and appreciated in the art.
• BEMP refers to 2-tert-butylimino-2-diethylamino-l, 3- dimethylperhydro-1 , 3 , 2-diazaphosphorine
• Boc refers to t- butoxycarbonyl
• BOP refers to benzotriazol-1-yloxy- t ⁇ s (d ⁇ methylam ⁇ no)phosphon ⁇ um hexafluorophosphate
• bd refers to broad doublet
• bs refers to broad smglet
• d refers to doublet
• dd refers to doublet of doublets
• DIC refers to dnsopropyl carbodiimide
• DMF refers to dimethylformamide
• DMAP refers to 4-d ⁇ methylammopyr ⁇ dme
• DMSO refers to dimethylsulfoxide
• EDC refers to ethyl-l-(3-
• EtOAc refers to ethyl acetate
• g refers to grams
• h refers to hours
• HOBT 1- hydroxybenzotriazole hydrate
• Humg's base refers to N,N- diisopropylethylamme
• L refers to liter
• m refers to multiplet
• M refers to molar
• max refers to maximum
• meq refers to milliequivalent
• mg refers to milligram
• mL refers to milliliter
• mm refers to millimeter
• mmol refers to millimol
• MOC refers to methoxyoxycarbonyl
• N refers to normal
• N/A refers to not available
• ng refers to nanogram
• nm refers to nanometers
• OD refers to optical density
• PEPC refers to 1- (3- ( 1-pyrrolidinyl )propyl) -3-ethylcarbod ⁇ mide
• the mixture was diluted with EtOAc and washed with 0.1 M HCl (1 x 10 mL) , saturated NaHC0 3 (1 x 10 mL) , H 2 0 (1 x 10 mL) , and brine and dried over MgS0 4 .
• the drying agent was removed by filtration and the filtrate was concentrated in vacuo.
• the residue was purified by flash column chromatography on silica gel followed by trituration from EtOAc and hexanes .
• the cooling bath was removed and the mixture allowed to warm to room temperature for 10-24 hours.
• the solution or mixture was diluted with EtOAc, in a 3-5 volume multiple of the initial THF volume, and washed with 0.1-1.0 M aq. HCl (1 or 2x) , dilute NaHC0 3 (1 or 2x) , and brine
• the product containing fractions are combined and evaporated in vacuo and then dried at 40-45 2 C under vacuum.
• the dried coupling product was dissolved in dioxane (5 mL) , adding methanol, if needed, and evaporated in vacuo and again dried at 40-45 2 C under vacuum.
• the coupling product was then combined with hydrochloric acid in dioxane (5 mL, 4M, 20 mmol) . After 2-3 hours the solvent was evaporated in vacuo and then dried at 40-45 2 C under vacuum.
• the deprotected coupling product was combined with 0.86 mL (150 ⁇ mol) of a stock solution of PP-HOBt prepared by combining PP-HOBt ( 0.567 g, 1.48 mmol) and 8.5 mL of .
• An additional 0.86 mL of DMF was added and the solution was partition into 4 vials.
• a 0.1 M stock solution of a carboxylic acid (about 40 ⁇ mol) in 10% DMF/methylene chloride and then 0.4 mL (about 40 ⁇ mol) of a stock solution prepared by combining EDC HCl (0.383 g, 2.0 mmol) in methylene chloride (20 mL) were added to the vials.
• reaction mixture was combined with PS-piperidine resin (100-124 mg, about 3.6 mmol/g, Bruce, BG8-22P-177) and mixed for 15 minutes.
• Methanol 2.5 mL was added to the vial and the contents applied to a pre- washed (methanol) 1000 mg SCX column using an 5 mL of methanol and then 5 mL of 10% methanol /chloroform.
• the coupled product is obtained by rinsing the column with 10% methanol /chloroform.
• the product containing fractions are combined and evaporated in vacuo and then dried at 40-45 2 C under vacuum to give the product.
• the oxime isolated above (0.99 g, 3.92 mmol) was hydrogenated in a Parr apparatus at 35 psi over 10 % Pd/C (0.46 g) in 3A ethanol. After 32 h the reaction mixture was filtered through a plug of Celite, the filtrate evaporated to a foam and treated with a saturated solution of HCl (g) in Et 2 0. The resulting colorless solid was filtered, rinsed with cold Et 2 0 and vacuum dried to give 0.66 g (61 %) of 5- amino-7-methyl-5 , 7-dihydro-6H-dibenz (b, d) azepin-6-one hydrochloride.
• Drug stocks were prepared in 100% dimethyl sulfoxide such that at the final drug concentration used in the treatment, the concentration of dimethyl sulfoxide did not exceed 0.5% and, in fact, usually equaled 0.1%.
• the media were again removed and replaced with fresh drug containing media as above and cells were incubated for an additional two hours.
• plates were centrifuged in a Beckman GPR at 1200 rpm for five minutes at room temperature to pellet cellular debris from the conditioned media. From each well, 100 ⁇ L of conditioned media or appropriate dilutions thereof were transferred into an ELISA plate precoated with antibody 266 (P.
• Cytotoxic effects of the compounds were measured by a modification of the method of Hansen, et al.13.
• To the cells remaining in the tissue culture plate was added 25 ⁇ L of a 3- (4 , 5-dimethylthiazol-2-yl) -2 , 5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO) stock solution (5 mg/mL) to a final concentration of 1 mg/mL.
• MTT 5-diphenyltetrazolium bromide
• results of the ⁇ -amyloid peptide ELISA were fit to a standard curve and expressed as ng/mL ⁇ -amyloid peptide. In order to normalize for cytotoxicity , these results were divided by the MTT results and expressed as a percentage of the results from a drug free control . The test compounds were assayed for ⁇ -amyloid peptide production inhibition activity in cells using this assay.
• Example Bio-2 In Vivo Suppression of ⁇ -Amyloid Release and/or Synthesis This example illustrates how the compounds of this invention could be tested for in vivo suppression of ⁇ -amyloid release and/or synthesis.
• 3 to 4 month old PDAPP mice are used (Games et al . , (1995) Nature 373:523- 527) .
• the compound is usually formulated at between 1 and 10 mg/mL.
• the compounds may be formulated with various vehicles, such as corn oil (Safeway, South San Francisco, CA) ; 10% ethanol in corn oil; 2-hydroxypropyl- ⁇ -cyclodextrin (Research Biochemicals International, Natick MA); and carboxy-methyl-cellulose (Sigma Chemical Co., St. Louis MO).
• various vehicles such as corn oil (Safeway, South San Francisco, CA) ; 10% ethanol in corn oil; 2-hydroxypropyl- ⁇ -cyclodextrin (Research Biochemicals International, Natick MA); and carboxy-methyl-cellulose (Sigma Chemical Co., St. Louis MO).
• mice are dosed subcutaneously with a 26 gauge needle and 3 hours later the animals are euthanized via C0 narcosis and blood is taken by cardiac puncture using a 1 cc 25G 5/8" tuberculin syringe/needle coated with solution of 0.5 M EDTA, pH 8.0.
• the blood is placed in a Becton- Dickinson vacutainer tube containing EDTA and spun down for 15 minutes at 1500 xg at 5 2 C.
• the brains of the mice are then removed and the cortex and hippocampus are dissected out and placed on ice. 1 . Brain As say
• each brain region is homogenized in 10 volumes of ice cold guanidine buffer (5.0 M guanidine-HCl , 50 mM Tris-HCl, pH 8.0) using a Kontes motorized pestle (Fisher, Pittsburgh PA) .
• the homogenates are gently rocked on a rotating platform for three to four hours at room temperature and stored at -20 2 C prior to quantitation of ⁇ -amyloid.
• the brain homogenates are diluted 1:10 with ice-cold casein buffer (0.25% casein, phosphate buffered saline (PBS), 0.05% sodium azide, 20 ⁇ g/ml aprotinin, 5 mM EDTA, pH 8.0, 10 ⁇ g/ml leupeptin) , thereby reducing the final concentration of guanidine to 0.5 M, before centrifugation at 16,000 xg for 20 minutes at 4 S C. Samples are further diluted, if necessary, to achieve an optimal range for the ELISA measurements by the addition of casein buffer with 0.5 M guanidine hydrochloride added.
• the ⁇ -amyloid standards (1-40 or 1-42 amino acids) were prepared such that the final composition equaled 0.5 M guanidine in the presence of 0.1% bovine ' serum albumin (BSA) .
• the total ⁇ -amyloid sandwich ELISA quantitating both ⁇ -amyloid (aa 1-40) and ⁇ -amyloid (aa 1-42) consists of two monoclonal antibodies (mAb) to ⁇ -amyloid.
• the capture antibody, 266 P. Seubert, Nature (1992) 359:325-327), is specific to amino acids 13 - 28 of ⁇ -amyloid.
• the antibody 3D6 Johnson-Wood et al . , PNAS USA (1997) 94:1550-1555), which is specific to amino acids 1 - 5 of ⁇ -amyloid, is biotinylated and served as the reporter antibody in the assay.
• the 3D6 biotinylation procedure employs the manufacturer's (Pierce, Rockford IL) protocol for NHS-biotin labeling of immunoglobulins except that 100 mM sodium bicarbonate, pH 8.5 buffer is used.
• the 3D6 antibody does not recognize secreted amyloid precursor protein (APP) or full-length APP but detects only ⁇ -amyloid species with an amino terminal aspartic acid.
• the assay has a lower limit of sensitivity of -50 pg/ml (11 pM) and shows no cross- reactivity to the endogenous murme ⁇ -amyloid peptide at concentrations up to 1 ng/ml
• Biotmylated 3D6 is also the reporter antibody m this assay which has a lower limit of sensitivity of -125 pg/ml (28 pM) .
• the 266 and 21F12 capture mAbs are coated at 10 ⁇ g/ml mto 96 well lmmunoassay plates (Costar, Cambridge MA) overnight at room temperature The plates are then aspirated and blocked with 0.25% human serum albumin m PBS buffer for at least 1 hour at room temperature, then stored desiccated at 4 S C until use. The plates are dehydrated with wash buffer (T ⁇ s-buffered saline, 0.05% Tween 20) prior to use. The samples and standards are added to the plates and incubated overnight at 4 2 C. The plates are washed • 3 times with wash buffer between each step of the assay.
• biotmylated 3D6 diluted to 0.5 ⁇ g/ml m casern incubation buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4) is incubated m the well for 1 hour at room temperature.
• Avid -HRP Vector, Burlmgame CA
• casein incubation buffer is added to the wells for 1 hour at room temperature.
• the colorimetric substrate Slow TMB-ELISA (Pierce, Cambridge MA) , is added and allowed to react for 15 minutes, after which the enzymatic reaction is stopped with addition of 2 N H 2 S0 Reaction product is quantified using a Molecular Devices Vmax (Molecular Devices, Menlo Park CA) measuring the difference m absorbance at 450 nm and 650 nm
• the EDTA plasma is diluted 1:1 m specimen diluent (0.2 gm/1 sodium phosphate-H 2 0 (monobasic), 2.16 gm/1 sodium phosphate -7 H 2 0 (dibasic), 0.5gm/l thimerosal, 8.5 gm/1 sodiu chloride, 0.5 ml Triton X-405, 6.0 g/1 globulin-free bovine serum albumin; and water) .
• the samples and standards in specimen diluent are assayed using the total ⁇ -amyloid assay (266 capture/3D6 reporter) described above for the brain assay except the specimen diluent was used instead of the casein diluents described.
• Formulations other than those described above can also be used for oral delivery and intravenous delivery to a mammal.
• the compound can be mixed with either 100% corn oil or, alternatively, m a solution containing 80% corn oil, 19.5% oleic acid and 0.5% labrafil.
• the compound can be mixed with the above solutions in concentrations ranging from 1 mg/mL to 10 mg/mL.
• the compound in solution is preferably administered orally to the mammal at a dose volume of 5 mL/kg of body weight.
• the compound is preferably mixed with a solution of 3% ethanol, 3% solutol HS-15 and 94% saline.
• the compound is preferably mixed with the above solution in concentrations ranging from 0.25 mg/mL to 5 mg/mL.
• the compound in solution is preferably administered by IV to the mammal at a dose volume of 2 mL/kg of body weight.
• Formulations other than those described above can also be used for oral delivery and intravenous delivery to a mammal.
• the compound can be mixed with either 100% corn oil or, alternatively, in a solution containing 80% corn oil, 19.5% oleic acid and 0.5% labrafil.
• the compound can be mixed with the above solutions in concentrations ranging from 1 mg/mL to 10 mg/mL.
• the compound in solution is preferably administered orally to the mammal at a dose volume of 5 mL/kg of body weight.
• the compound is preferably mixed with a solution of 3% ethanol, 3% solutol HS-15 and 94% saline.
• the compound is preferably mixed with the above solution in concentrations ranging from 0.25 mg/mL to 5 mg/mL.
• the compound in solution is preferably administered by IV to the mammal at a dose volume of 2 mL/kg of body weight.
• the compounds of formula I are usually administered in the form of pharmaceutical compositions . These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal . These compounds are effective as both injectable and oral compositions.
• Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
• This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds of formula I above associated with pharmaceutically acceptable carriers .
• the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container.
• a carrier which can be in the form of a capsule, sachet, paper or other container.
• the excipient serves as a diluent, it can be a solid, semi- solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
• compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
• the active compound In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh. - 5 C
• excipients include lactose, dextrose, sucrose, sorbitol, manmtol starches, gum acacia, calcium phosphate, algmates, tragacanth, gelatin, calcium silicate, microcrystallme cellulose, polyvmylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose
• the formulations can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents, preserving agents such as methyl- and propylhydroxy-benzoates ; sweetening agents; and flavoring agents
• the compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known m the art .
• compositions are preferably formulated m a unit dosage form, each dosage containing from about 5 to about 100 mg, more usually about 10 to about 30 mg, of the active ingredient.
• unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, m association with a suitable pharmaceutical excipient
• the compound of formula I above is employed at no more than about 20 weight percent of the pharmaceutical composition, more preferably no more than about 15 weight percent, with the balance being pharmaceutically inert carrier (s) .
• the active compound is effective over a wide dosage range and is generally administered m a pharmaceutically effective amount It, will be understood, however, that the amount of the compound actually administered will be determined by a physician, m the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient the severity of the patient's symptoms, and the like
• the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention
• a solid preformulation composition containing a homogeneous mixture of a compound of the present invention
• the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided mto equally effective unit dosage forms such as tablets, pills and capsules
• This solid preformulation s then subdivided mto unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
• the tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
• the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being m the form of an envelope over the former.
• the two components can separated by enteric layer which serves to resist disintegration the stomach and permit the inner component to pass intact mto the duodenum or to be delayed m release.
• enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac cetyl alcohol, and cellulose acetate
• compositions for inhalation or insufflation include solutions and suspensions m pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
• the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra.
• the compositions are administered by the oral or nasal respiratory route for local or systemic effect.
• compositions m preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation m an appropriate manner.
• the components are blended and compressed to form tablets, each weighing 240 mg .
• Formulation Example 3 A dry powder inhaler formulation is prepared containing the following components : Ingredient Weight %
• the active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.
• the active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly.
• the solution of polyvinyl-pyrroiidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve.
• the granules so produced are dried at 50 2 to 60 S C and passed through a 16 mesh U.S. sieve.
• the sodium carboxymethyl starch, magnesium stearate, and talc previously passed through a No . 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg .
• the active ingredient, starch, and magnesium stearate are blended, passed through a No . 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities.
• Suppositories each containing 25 mg of active ingredient are made as follows:
• the active ingredient, sucrose and xanthan gum are blended, passed through a No . 10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystallme cellulose and sodium carboxymethyl cellulose in water.
• the sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.
• a subcutaneous formulation may be prepared as follows: Ingredient Quantity
• a topical formulation may be prepared as follows:
• the white soft paraffin is heated until molten.
• the liquid paraffin and emulsifying wax are incorporated and stirred until dissolved.
• the active ingredient is added and stirring is continued until dispersed.
• the mixture is then cooled until solid.
• transdermal delivery devices Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts.
• the construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent 5,023,252, issued June II, 1991, herein incorporated by reference.
• patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
• Indirect techniques usually involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs.
• Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood-brain barrier.
• the delivery of hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier.
• the compounds and pharmaceutical compositions of the invention are useful in inhibiting ⁇ -amyloid peptide release and/or its synthesis, and, accordingly, have utility in diagnosing and treating Alzheimer's disease in mammals including humans.
• the compounds described herein are suitable for use in a variety of drug delivery systems described above. Additionally, m order to enhance the in vivo serum half-life of the administered compound, the compounds may be encapsulated, introduced mto the lumen of liposomes, prepared as a colloid, or other conventional techniques may be employed which provide an extended serum half-life of the compounds.
• a variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al , U.S Patent Nos 4,235,871, 4,501,728 and 4,837,028 each of which is incorporated herein by reference
• compositions are administered to a patient already suffering from Alzheimer's disease m an amount sufficient to at least partially arrest further onset of the symptoms of the disease and its complications
• An amount adequate to accomplish this is defined as "therapeutically effective dose ' Amounts effective for this use will depend on the judgment of the attending clinician depending upon factors such as the degree or severity of Alzheimer's disease the patient, the age, weight and general condition of the patient, and the like.
• the compounds described herein are administered at dosages ranging from about 1 to about 500 mg/kg/day.
• compositions are administered to a patient at risk of developing Alzheimer's disease (determined for example by genetic screening or familial trait) m an amount sufficient to inhibit the onset of symptoms of the disease.
• An amount adequate to accomplish this is defined as “prophylactically effective dose.” Amounts effective for this use will depend on the judgment of the attending clinician depending upon factors such as the age, weight and general condition of the patient and the like
• the compounds described herein are administered at dosages ranging from about 1 to about 500 mg/kg/day
• the compounds administered to a patient are m the form of pharmaceutical compositions described above. These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered The - Q .
• resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
• the pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 and 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.
• the compounds described herein are also suitable for use in the administration of the compounds to a cell for diagnostic and drug discovery purposes. Specifically, the compounds may be used in the diagnosis of cells releasing and/or synthesizing ⁇ -amyloid peptide. In addition the compounds described herein are useful for the measurement and evaluation of the activity of other candidate drugs on the inhibition of the cellular release and/or synthesis of ⁇ -amyloid peptide.

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