Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/06—Peri-condensed systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/08—Bridged systems

Definitions

• This invention relates to polycyclic ⁇ -amino-e-caprolactams and related compounds which are useful as synthetic intermediates in the preparation of inhibitors of ⁇ -amyloid peptide release and/or its synthesis.
• AD Alzheimer's Disease
• AD 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.
• AD is a very common cause of progressive mental failure (dementia) in aged humans and is believed to represent the fourth most common medical cause of death in the United States.
• AD has been observed in 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 in the United States alone. AD is at present incurable. No treatment that effectively prevents AD or reverses its symptoms and course is currently known.
• the brains of individuals with AD exhibit characteristic lesions termed senile (or amyloid) plaques, amyloid angiopathy (amyloid deposits in blood vessels) and neurofibrillary tangles.
• senile or amyloid
• amyloid angiopathy amyloid deposits in blood vessels
• neurofibrillary tangles Large numbers of these lesions, particularly amyloid plaques and neurofibrillary tangles, are generally found in several areas of the human brain important for memory and cognitive function in patients with AD. Smaller numbers of these lesions in a more restrictive anatomical distribution are also found in the brains of most aged humans who do not have clinical AD.
• Amyloid plaques and amyloid angiopathy also characterize the brains of individuals with Trisomy 21 (Down's Syndrome) and Hereditary Cerebral
• HCVWA-D Hemorrhage with Amyloidosis ofthe Dutch Type
• the principal chemical constituent ofthe amyloid plaques and vascular amyloid deposits (amyloid angiopathy) characteristic of AD and the other disorders mentioned above is an approximately 4.2 kilodalton (kD) protein of about 39-43 amino acids designated the ⁇ -amyloid peptide ( ⁇ AP) or sometimes A ⁇ , A ⁇ P or ⁇ /A4.
• ⁇ AP ⁇ -amyloid peptide
• ⁇ -Amyloid peptide was first purified and a partial amino acid sequence was provided by Glenner, et al. 1 The isolation procedure and the sequence data for the first 28 amino acids are described in U.S. Patent No.
• ⁇ - amyloid peptide is a small fragment of a much larger precursor protein termed the amyloid precursor protein (APP), that is normally produced by cells in many tissues of various animals, including humans.
• APP amyloid precursor protein
• Knowledge of the structure ofthe gene encoding APP has demonstrated that ⁇ -amyloid peptide arises as a peptide fragment that is cleaved from APP by protease enzyme(s).
• protease enzyme(s) The precise biochemical mechanism by which the ⁇ -amyloid peptide fragment is cleaved from APP and subsequently deposited as amyloid plaques in the cerebral tissue and in the walls ofthe cerebral and meningeal blood vessels is currently unknown.
• a mutation at amino acid 693 ofthe 770-amino acid isoform of APP has been identified as the cause ofthe ⁇ -amyloid peptide deposition disease, HCHWA-D, and a change from alanine to glycine at amino acid 692 appears to cause a phenotype that resembles AD is some patients but HCHWA-D in others.
• the discovery of these and other mutations in APP in genetically based cases of AD prove that alteration of APP and subsequent deposition of its ⁇ -amyloid peptide fragment can cause AD.
• the treatment methods would advantageously be based on drugs which are capable of inhibiting ⁇ -amyloid peptide release and/or its synthesis in vivo.
• This invention is directed to the discovery of a class of intermediates which are useful in the preparation the cycloalkyl, lactam, lactone and related compounds described in U.S. Patent Application Serial No. 08/996,422, which compounds inhibit ⁇ -amyloid peptide release and/or its synthesis. Accordingly, in one of its composition aspects, this invention is directed to a compound of formula I:
• ring A together with the atoms ofthe e-caprolactam to which it is attached, forms a carbocyclic or heterocyclic ring selected from the group consisting of aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic;
• ring B together with the atoms of the e-caprolactam to which it is attached, forms a carbocyclic or heterocyclic ring selected from the group consisting of aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic;
• ring C together with the atoms ofthe e-caprolactam to which it is attached, forms a heteroaryl or heterocyclic ring;
• R 1 is selected from the group consisting of hydrogen and an amino- blocking group
• each R 2 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, cycloalkyl, heteroaryl and heterocyclic;
• R 3 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic; each R 4 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic; m is an integer from 0 to 2; n is an integer from 0 to 2; and salts thereof.
• R 1 is hydrogen or tert-butoxycarbonyl (Boc).
• R 2 substituents include, by way of example, hydrogen, methyl, ethyl, rt-propyl, isopropyl, H-butyl, isobutyl, sec-butyl, tert- butyl, -CH 2 CH(CH 2 CH 3 ) 2 , 2-methyl- «-butyl, 6-fluoro-n-hexyl, phenyl, benzyl, cyclohexyl, cyclopentyl, cycloheptyl, allyl, wo-but-2-enyl, 3-methylpentyl, -CH 2 -cyclopropyl, -CH 2 -cyclohexyl, -CH 2 CH 2 -cyclopropyl, -CH 2 CH 2 -cyclohexyl, -CH 2 -indol-3 -yl, ?-(phenyl)phenyl, ⁇ -fluorophenyl, m-fluorophenyl,
• R 3 is selected from the group consisting of hydrogen, alkyl, substituted alkyl and cycloalkyl.
• Particularly preferred R 3 substituents include, by way of example, hydrogen, methyl, 2-methypropyl, hexyl, methoxycarbonylmethyl, 3,3-dimethyl- 2-oxobutyl, 4-phenylbutyl, cyclopropylmethyl, 2,2,2-trifluoroethyl, cyclohexyl, and the like.
• R 4 is preferably alkyl or substituted alkyl.
• n is 0 or 1. More preferably, m is 0.
• W is preferably a substituted e-caprolactam selected from the group consisting of:
• A, B, R , R and m are as defined herein.
• Wx ' s a substituted e-caprolactam ofthe formula:
• this invention is also directed to compounds of formula I wherein W is independently selected from each ofthe substituted e-caprolactams illustrated above.
• Rings A and B may be the same or different and are preferably independently selected from the group consisting of aryl, cycloalkyl, cycloalkenyl, heteroaryl and heterocyclic. More preferably, rings A and B are independently selected from the group consisting of aryl and cycloalkyl. Still more preferably, rings A and B are independently aryl.
• Particularly preferred A and B rings include, by way of example, phenyl, substituted phenyl, including fluoro-substituted phenyl, cyclohexyl and the like.
• the A and B rings are fused to one another, they preferably form a naphthyl or sustituted naphthyl ring.
• Particularly preferred C rings include, by way of example, pyrrolidinyl, piperidinyl, mo ⁇ holino and the like.
• each R 5 is independently selected from the group consisting of acyl, acylamino, acyloxy, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, alkyl, substituted alkyl, alkynyl, substituted alkynyl, amino, substituted amino, aminoacyl, aryl, aryloxy, carboxyl, carboxyalkyl, cyano, cycloalkyl, substituted cycloalkyl, halo, heteroaryl, heterocyclic, nitro, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl, and
• R 7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic; p is an integer from 0 to 4; q is an integer from 0 to 4; and salts thereof.
• R 5 and R 6 are independently selected from the group consisting of alkoxy, substituted alkoxy, alkyl, substituted alkyl, amino, substituted amino, carboxyl, carboxyalkyl, cyano, halo, nitro, thioalkoxy and substituted thioalkoxy.
• R 5 and R 6 are fluoro.
• R 7 is preferably selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, aryl, cycloalkyl and substituted cycloalkyl. More preferably, R 7 is selected from the group consisting of hydrogen, alkyl, substituted alkyl and cycloalkyl.
• R 7 substituents include, by way of example, hydrogen, methyl, 2-methypropyl, hexyl, methoxycarbonylmethyl, 3,3-dimethyl- 2-oxobutyl, 4-phenylbutyl, cyclopropylmethyl, 2,2,2-trifluoroethyl, cyclohexyl, and the like.
• Wis a substituted e- caprolactam of the formula:
• R 5 , R 6 , and ? are as defined herein and r is an integer from 0 to 3; and salts thereof.
• R 5 , and p are as defined herein; and salts thereof.
• R 5 and/? are as defined herein; and salts thereof.
• Preferred substituted e-caprolactams include, by way of example, 5,7-dihydro-6H-dibenz[b,d]azepin-6-one-5-yl, 7-methyl-5,7-dihydro-6H- dibenz[b,d]azepin-6-one-5-yl, 7-(2-methylpropyl)-5,7-dihydro-6H- dibenz[b,d]azepin-6-one-5-yl, 7-(methoxyacetyl)-5,7-dihydro-6H- dibenz[b,d]azepin-6-one-5-yl, 7-(3,3-dimethylbutan-2-onyl)-5,7-dihydro-6H- dibenz[b,d]azepin-6-one-yl, 7-phenbutyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one- yl, 7-cyclopropymethyl-5
• Compounds of this invention include, by way of example, the following: 5-amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one
• Preferred compounds include those defined by the formulas as set forth in Tables I, II, III, IN and V below and salts thereof.
• this invention is directed a compound of formula II:
• W is a substituted e-caprolactam selected from the group consisting of:
• ring A together with the atoms ofthe e-caprolactam to which it is attached, forms a carbocyclic or heterocyclic ring selected from the group consisting of aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic;
• ring B together with the atoms ofthe e-caprolactam to which it is attached, forms a carbocyclic or heterocyclic ring selected from the group consisting of aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic;
• ring C together with the atoms of the e-caprolactam to which it is attached, forms a heteroaryl or heterocyclic ring;
• R 1 is selected from the group consisting of hydrogen and an amino- blocking group
• Y is represented by the formula:
• each R 2 is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, cycloalkyl, heteroaryl and heterocyclic;
• R 3 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic; each R 4 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl and heterocyclic; a is an integer from 2 to 6; m is an integer from 0 to 2; n is an integer from 0 to 2; and salts thereof.
• Y in formula II is the group -(CHR 2 ) a -NH-, the integer a is preferably 2, 3 or 4, more preferably 2 or 4, and still more preferably a is equal to 2.
• Y has the formula -CHR -CH,-NH-, where R 2 is as defined herein.
• R 1 , R 2 , R 3 , R 4 , A, B, C, W, m, n and the like in formula II are the same as those described herein for compounds of formula I.
• Preferred compounds include those defined by the formulas as set forth in Tables VI below and salts thereof.
• ⁇ -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 ofthe protein described by Glenner, et al. 1 including mutations and post- translational modifications ofthe normal ⁇ -amyloid peptide.
• the ⁇ -amyloid peptide is an approximate 39-43 amino acid fragment of a large membrane-spanning glycoprotein, referred to as the ⁇ -amyloid precursor protein (APP). Its 43-amino acid sequence is:
• 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, «-propyl, zs ⁇ -propyl, «-butyl, wo-butyl, n-hexyl, decyl and the like.
• Substituted alkyl refers to an alkyl group, preferably of from 1 to 20 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, amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO- substitute
• Alkylene refers to divalent alkylene groups preferably having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), the propylene isomers (e.g., -CH 2 CH 2 CH 2 - and -CH(CH 3 )CH 2 -) and the like.
• Substituted alkylene refers to an alkylene group, preferably of from 1 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, -SO 2 -alkyl, -SO 2 -substituted alkyl, -
• substituted alkylene groups include those where 2 substituents on the alkylene group are fused to form one or more cycloalkyl, aryl, heterocyclic or heteroaryl groups fused to the alkylene group.
• fused cycloalkyl groups contain from 1 to 3 fused ring structures.
• 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, -SO 2 -alkyl, -SO 2 -substituted alkyl,
• 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, ⁇ -propoxy, wo-propoxy, n-butoxy, tert-butoxy, sec-butoxy, «-pentoxy, H-hexoxy, 1,2-dimethylbutoxy, and the like.
• Substituted alkoxy refers to the group “substituted alkyl-O-" where substituted alkyl is as defined above.
• Alkylalkoxy refers to the group “-alkylene-O-alkyl” which includes by way of example, methylenemethoxy (-CH 2 OCH 3 ), ethylenemethoxy (-CH 2 CH 2 OCH 3 ), n-propylene-wo-propoxy (-CH 2 CH 2 CH 2 OCH(CH 3 ) 2 ), methylene- t-butoxy (-CH 2 -O-C(CH 3 ) 3 ) and the like.
• Alkylthioalkoxy refers to the group “-alkylene-S-alkyl” which includes by way of example, methylenethiomethoxy (-CH 2 SCH 3 ), ethylenethiomethoxy (-CH 2 CH 2 SCH 3 ), «-propylene-thio-t5o-propoxy (-CH 2 CH 2 CH 2 SCH(CH 3 ) 2 ), methylenethio-t-butoxy (-CH 2 SC(CH 3 ) 3 ) and the like.
• 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, thiol, thioalkoxy, substituted thioalkoxy, aryl, heteroaryl, heterocyclic, heterocyclooxy, nitro -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 - substituted alkyl, -SO 2 -aryl, and -SO
• 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 (-C ⁇ CH), propargyl (-CH 2 C ⁇ CH) 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, 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, -SO 2 -alkyl, -SO 2 - substituted alkyl, -SO 2 -aryl, and
• Acyl refers to the groups alkyl-C(O)-, substituted alkyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclic-C(O)- where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• Acylamino refers to the group -C(O)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.
• Amino refers to the group -NH 2 .
• Substituted amino refers to the group -N(R) 2 where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, heterocyclic and where both R groups are joined to form a heterocyclic group.
• both R groups are hydrogen
• -N(R) 2 is an amino group.
• substituted amino groups include, by way of illustration, mono- and di-alkylamino, mono- and di-(substituted alkyl)amino, mono- and di-arylamino, mono- and di-heteroarylamino, mono- and di- heterocyclic amino, and unsymmetric di-substituted amines having different substituents selected from alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic, and the like.
• amino-blocking group or “amino-protecting group” refers to any group which, when bound to an amino group, prevents undesired reactions from occurring at the amino group and which may be removed by conventional chemical and or enzymatic procedures to reestablish the amino group. Any known amino- blocking group may be used in this invention. Typically, the amino-blocking group is selected so as to render the resulting blocked-amino group unreactive to the particular reagents and reaction conditions employed in a subsequent predetermined chemical reaction or series of reactions. After completion ofthe reaction(s), the amino-blocking group is selectively removed to regenerate the amino group.
• Suitable amino-blocking groups include, by way of illustration, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), acetyl, 1-(1 '- adamantyl)-l-methylethoxycarbonyl (Acm), allyloxycarbonyl (Aloe), benzyloxymethyl (Bom), 2- ?-biphenylisopropyloxycarbonyl (Bpoc), tert- butyldimethylsilyl (Bsi), benzoyl (Bz), benzyl (Bn), 9-fluorenyl- methyloxycarbonyl (Fmoc), 4-methylbenzyl, 4-methoxybenzyl, 2- nitrophenylsulfenyl (Nps), 3-nitro-2-pyridinesulfenyl (NPys), trifluoroacetyl (Tfa), 2,4,6-trimethoxybenzyl (Tmob),
• Aminoacyl refers to the group -NRC(O)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(O)OR 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(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclic-C(O)O- 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, -SO 2 -alkyl
• Aryloxy refers to the group aryl-O- wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
• Carboxyalkyl refers to the groups “-C(O)Oalkyl” and "-C(O)O- 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 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, amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, - SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-hetero
• 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, amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2
• Halo or halogen refers to fluoro, chloro, bromo and iodo and preferably is either fluoro or chloro.
• 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, 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,
• heteroaryl groups can have a single ring
• 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, amino, substituted amino, aminoacyl, aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, keto, thioketo, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO
• heterocycles and heteroaryls include, but are not limited to, pyrrole, furan, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, mo ⁇ holino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitro
• Heterocyclooxy refers to the group “-O-heterocycle”.
• Oxyacylamino refers to the group -OC(O)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.
• Thiol refers to the group -SH.
• 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.
• 4,5,6,7-tetrahydro-3,7-methano-3H-3-benzazonin-2(lH)-one refers to a polycyclic e-caprolactam ring system having the formula:
• salt(s) refers to salts of a compound of formula I which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
• protecting group or “blocking group” refers to any group which when bound to one or more hydroxyl, thiol, carboxyl or other protectable functional group ofthe compound prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical and/or enzymatic steps to reestablish the unprotected functional group.
• removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, tert- butyldiphenylsilyl and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature ofthe product.
• substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, tert- butyldiphenylsilyl and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature ofthe product.
• Preferred carboxyl protecting groups include esters such as methyl, ethyl, propyl, tert-butyl, etc. which can be removed by mild hydrolysis conditions compatible with the nature of the product.
• polycyclic ⁇ -amino-e-caprolactarns and related compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
• protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions.
• the choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, 8 and references cited therein.
• the compound of this invention are prepared by first aminating a polycyclic e-caprolactam to provide a polycyclic ⁇ - amino-e-caprolactam. If desired, the amino group ofthe polycyclic ⁇ -amino-e- caprolactam can then be coupled with a mono- or diamino acid derivative to provide compounds of formula I wherein n is 1 or 2.
• polycyclic e-caprolactams employed as starting materials in this invention are either commercially available or can be prepared from commercially available materials using conventional procedures and reagents.
• 5,7-dihydro-6H-dibenz[b,d]azepin-6-one may be prepared by cyclizing a chloromethyl amide intermediate using the procedures set forth in R. F. C. Brown et al., Tetrahedron Letters 1971, 8, 667-670 9 and references cited therein.
• This reaction is typically conducted by treating 1 with about 1.0 to about 2.1 equivalents of an alkyl lithium reagent, preferably sec-butyl lithium or tert-butyl lithium, in an inert diluent, such as THF, at a temperature ranging from about -80 °C to about -60 °C for about 0.25 to about 1 hour.
• an alkyl lithium reagent preferably sec-butyl lithium or tert-butyl lithium
• an inert diluent such as THF
• the resulting methyl boronate ester is typically not isolated, but is preferably converted in situ into the pinacol ester by treating the reaction mixture with an excess, preferably about 2.0 equivalents, of pinacol.
• This reaction is typically conducted at ambient temperature for about 12 to about 24 hours to afford the 2- methylphenylboronate ester, 2, in which both R a groups are preferably joined together to form -C(CH 3 ) 2 C(CH 3 ) 2 -.
• the amino group of a 2-bromoaniline derivative, 3, is converted into the N-Boc derivative 4 by treating 3 with about 1.0 to about 1.5 equivalents of di-tert-butyl-dicarbonate.
• this reaction is conducted at a temperature ranging from 25 °C to about 100°C for about 12 to 48 hours to afford the N-Boc-2-bromoaniline derivative 4.
• the 2-methylphenylboronate ester, 2, and the N-Boc-2-bromoaniline derivative 4 can then be coupled to form the biphenyl derivative 5.
• This reaction is typically conducted by contacting 4 with about 1.0 to about 1.2 equivalents of 2 and about 1.0 to about 1.2 equivalents of potassium carbonate in the presence of a pallidium catalyst, preferably tetrakis(triphenylphosphine)pallidium(0).
• a pallidium catalyst preferably tetrakis(triphenylphosphine)pallidium(0).
• this coupling reaction is conducted in a diluent, preferably 20% water/dioxane, under an inert atmosphere at a temperature ranging from about 50°C to about 100°C for about 6 to 24 hours.
• Biphenyl derivative 5 is then readily converted into the 5,7-dihydro-6H- dibenz[b,d]azepin-6-one 6 by carboxylation ofthe 2-mefhyl group, followed by cyclization to form the e-caprolactam.
• the carboxylation reaction is typically conducted by contacting 5 with about 2.0 to about 2.5 equivalents of a suitable base, such as sec-butyllithium, tert-butyllithium and the like, in an inert diluent, such as THF, at a temperature ranging from about -100°C to about -20 °C for about 0.5 to 6 hours.
• a suitable base such as sec-butyllithium, tert-butyllithium and the like
• an inert diluent such as THF
• 5,7-dihydro-6H-dibenz[b,d]azepin-6-one, 6, is optionally N-alkylated using conventional reagents and conditions to provide a 7- alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one derivative, 7.
• this reaction is conducted by first contacting 6 with about 1.0 to 1.5 equivalents of a suitable base, such as sodium hydride, sodium bis(trimethysilyl)amide and the like, in an inert diluent, such as DMF, THF and the like, at a temperature ranging from about -78 °C to about 50°C for about 0.25 to about 6 hours.
• a suitable base such as sodium hydride, sodium bis(trimethysilyl)amide and the like
• an inert diluent such as DMF, THF and the like
• the resulting anion is then treated in situ with an excess, preferably about 1.1 to about 2.0 equivalents, of an alkyl, substituted alkyl, cycloalkyl halide, etc., typically a chloride, bromide or iodide. This reaction is typically conducted at a temperature ranging from about
• the 7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one 7 is then oximated by contacting 7 with an excess, preferably with about 1.0 to 1.5 equivalents of a suitable base, such as sodium bis(trimethysilyl)amide and the like, in the presence of about 1.0 to about 2.0 equivalents of an alkyl nitrite.
• a suitable base such as sodium bis(trimethysilyl)amide and the like
• This reaction is typically conducted in an inert diluent, such as THF and the like, at a temperature ranging from about -10°C to about 20 °C for about 0.5 to about 6 hours to afford the 7-alkyl-5-oximo-5,7-dihydro-6H-dibenz[b,d]azepin-6- one derivative 8.
• an inert diluent such as THF and the like
• this reduction reaction is conducted by hydrogenating the oxime 8 in the presence of a catalyst, such as Raney nickel.
• a catalyst such as Raney nickel.
• This reaction is typically conducted under about 200 psi to about 600 psi of hydrogen at a temperature of about 70 °C to about 120°C for about 8 to 48 hours in a diluent, preferably a mixture of ethanol and ammonia (about 20:1).
• the oxime may be reduced using 10% Pd/C and between about 30 to about 60 psi of hydrogen at a temperature ranging from about 20°C to about 50°C for about 4 hours.
• the resulting 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one 9 is generally purified using well known procedures, such as recrystallization and/or chromatography.
• the reaction is stirred at a temperature ranging from about 0°C to about 20°C for about 2 to about 4 hours to afford 5-iodo-5,7-dihydro-6H- dibenz[b,d]azepin-6-one, 10.
• Displacement of iodide from 10 using an alkali metal azide then affords 5- azido-5,7-dihydro-6H-dibenz[b,d]azepin-6-one, 11.
• this reaction is conducted by contacting 10 with about 1.1 to about 1.5 equivalents of sodium azide in an inert diluent, such as DMF, at a temperature ranging from about 0°C to about
• the azido derivative 11 is then reduced to the corresponding amino derivative 12 using conventional procedures and reagents.
• the azido group is preferably reduced by contacting 11 with an excess, preferably with about 3 equivalents, of triphenylphosphine in a diluent, preferably a mixture of THF and water.
• This reduction reaction is typically conducted at a temperature ranging from about 0°C to about 50°C for about 12 to 48 hours to afford 5-amino-5,7-dihydro- 6H-dibenz[b,d]azepin-6-one, 12.
• the amino group of 12 is then protected or blocked using a conventional amino blocking group.
• compound 12 is treated with about 1.0 to about 1.1 equivalents of di-tert-butyl dicarbonate in the presence of an excess, preferably about 2 to about 3 equivalents, of a trialkylamine, such as triethylamine.
• This reaction is typically conducted in an inert diluent, such as THF, at a temperature ranging from about 0°C to about 50°C for 3 to about 24 hours to provide 5-(N- Boc-amino)-5,7-dihydro-6H-dibenz[b,d]azepin-6-one, 13.
• Compound 13 is then optionally N-alkylated to afford, after de-blocking of the amino group, a 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one, 9.
• the N-alkylation reaction is typically conducted by treating 13 with about 1.0 to 1.5 equivalents of an alkyl halide, a substituted alkyl halide or a cycloalkyl halide in the presence of about 1.0 to about 1.5 equivalents of a suitable base, such as cesium carbonate and the like.
• This reaction is generally conducted in an inert diluent, such as DMF and the like, at a temperature ranging from about 25 °C to about 100°C for about 12 to about 48 hours.
• alkyl, substituted alkyl and cycloalkyl halides suitable for use in this N-alkylation reaction include, by way of illustration, l-iodo-2- methylpropane, methyl bromoacetate, l-chloro-3,3-dimethyl-2-butanone, 1-chloro- 4-phenylbutane, bromomethylcyclopropane, 1 -bromo-2,2,2-trifluoroethane, bromocyclohexane, 1-bromohexane and the like.
• the N-Boc protecting group is then removed using conventional procedures and reagents to afford the 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6- one, 9.
• This deblocking reaction is typically conducted by treating the N-Boc compound 13 with anhydrous hydrogen chloride in an inert diluent, such as 1,4- dioxane, at a temperature ranging from about 0°C to about 50 °C for about 2 to about 8 hours.
• the resulting 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin- 6-one 9 is generally purified using well known procedures, such as recrystallization and/or chromatography.
• the 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-ones, 9, can also be prepared via an azide transfer reaction as illustrated in Scheme 3.
• the 7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one 7 is then reacted with an azide transfer reagent to afford 5-azido-7-alkyl-5,7-dihydro-6H- dibenz[b,d]azepin-6-one 14.
• this reaction is conducted by first contacting 7 with an excess, preferably with about 1.0 to 1.5 equivalents of a suitable base, such as lithium diisopropylamine and the like, in an inert diluent such as THF, at a temperature ranging from about -90°C to about -60°C for about 0.25 to about 2.0 hours.
• the resulting anion is then treated with an excess, preferably with about 1.1 to about 1.2 equivalents, of an azide transfer reagent, such as 2,4,6-triisopropylbenzenesulfonyl azide (trisyl azide).
• an azide transfer reagent such as 2,4,6-triisopropylbenzenesulfonyl azide (trisyl azide).
• This reaction is typically conducted at a temperature ranging from about -90 °C to about -60 °C for about 0.25 to about 2.0 hours.
• the reaction mixture is then typically treated with an excess of glacial acetic acid and the mixture is allowed to warm to ambient temperature and then heated at about 35 °C to about 50 °C for about 2 to 4 hours to afford the 5-azido-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one derivative 14.
• the aryl rings of 5-amino-7-alkyl-5,7-dihydro-6H- dibenz[b,d]aze ⁇ in-6-ones, 9, and related compounds may be partially or fully saturated by treatment with hydrogen in the presence of a hydrogention catalyst.
• this reaction is conducted by treating 9 with hydrogen at a pressure of about 10 to about 100 psi in the presence of a catalyst, such as rhodium on carbon.
• a catalyst such as rhodium on carbon.
• This reaction is typically conducted at a temperature ranging from about 20°C to about 100°C for about 12 to 96 hours in a suitable diluent, such as ethyl acetate/acetic acid (1:1) and the like.
• a suitable diluent such as ethyl acetate/acetic acid (1:1) and the like.
• the ⁇ -amino group may be coupled with a mono- or dipeptide derivative (i.e., an amino acid derivative) to prepared compounds of formula I, wherein n is 1 or 2.
• Scheme 4 illustrates the coupling of a representative polycyclic ⁇ -amino-e-caprolactam, i.e., 9, with a mono- or dipeptide derivative 15, wherein R 2 and n are as defined above and R 1' is an amino-blocking group.
• the coupling of 5-amino-7-alkyl-5,7-dihydro- 6H-dibenz[b,d]azepin-6-ones, 9, with a mono- or dipeptide 15 affords the amide 16.
• This reaction is typically conducted by reacting at least a stoichiometric amount ofthe amino compound 9 and the mono- or dipeptide 15 with a standard coupling reagent, typically in the presence of a trialkylamine, such as ethyldiisopropylamine, under conventional coupling reaction conditions.
• a trialkylamine such as ethyldiisopropylamine
• well-known coupling promoters such N-hydroxysuccinimide, 1- hydroxybenzotriazole and the like, may be employed in this reaction.
• this coupling reaction is conducted at a temperature ranging from about 0°C to about 60 °C for about 1 to about 72 hours in an inert diluent, such as THF, to afford the
• Suitable coupling reagents include, by way of example, carbodiimides, such as ethyl-3-(3-dimethylamino)propylcarboiimide (EDC), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) and the like, and other well-known coupling reagents, such as N,N' -carbonyl diimidazole, 2-ethoxy-l-ethoxycarbonyl- 1,2-dihydroquinoline (EEDQ), benzotriazol-1-yloxy- tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and the like.
• the coupling reagent may also be bound to a solid support. For example, a polymer supported form of EDC is described in Tetrahedron Letters 1993, 34(48), 7685.”
• PEPC l-(3-(l-pyrrolidinyl)propyl-3-ethylcarbodiimide
• PEPC l-(3-(l-pyrrolidinyl)propyl-3-ethylcarbodiimide
• PEPC can be prepared by first reacting ethyl isocyanate with l-(3- aminopropyl)pyrrolidine. The resulting urea is treated with 4-toluenesulfonyl chloride to provide PEPC. The polymer supported form is prepared by reacting
• the mono- or dipeptide derivatives employed in the coupling reaction are commercially available or can be prepared using conventional procedures and reagents from commercially available starting materials.
• the mono- and dipeptide derivatives may be derived from naturally-occurring and non-natural amino acids.
• suitable monopeptide derivatives include, by way of illustration, N-Boc-glycine, N-Boc-L-alanine, N-Boc-L-valine, N-Boc-L-leucine, N-Boc-L-isoleucine, N-Boc-tert-L-leucine, N-Boc-L-methionine, N-Boc-L-phenylalanine, N-Boc-L-phenylglycine, N-Boc-L-aspartic acid ⁇ -tert- butyl ester, N-Boc-L-glutamic acid ⁇ -tert-butyl ester, N-Boc- ⁇ e-Cbz-L-lysine, N- Boc-norleucine and the like.
• Exemplary dipeptides include, for pu ⁇ oses of illustration only, ⁇ -Boc-glycinyl-L-alanine, ⁇ -Boc-L-alaninyl-L-alanine, ⁇ -Boc- L-alaninyl-L-valine, ⁇ -Boc-glycinyl-L-phenylglycine, ⁇ -Boc-L-phenylglycinyl-L- valine and the like.
• the amino-blocking group R 1 is typically removed to reestablish the amino group.
• the N-Boc group can be removed by treating 16 with anhydrous hydrogen chloride in an inert diluent, such as 1 ,4-dioxane. This reaction is typically conducted at a temperature ranging from about -10°C to about 15°C while hydrogen chloride gas is introduced into the reaction mixture, and then at a temperature ranging from about 10°C to about 60 °C for about 1 to about 24 hours.
• Other amino-blocking groups can be removed using well-known art recognized procedures.
• the deoxy derivatives of formula II can also be prepared using conventional reagents and procedures.
• the synthesis of such compounds is described more fully in U.S. Patent Application Serial No. / , filed on even date herewith (Attorney Docket No. 002010-136) and entitled "Deoxyamino Acid Compounds, Pharmaceutical Compositions Comprising Same, and Methods for Inhibiting ⁇ -Amyloid Peptide Release, and/or its Synthesis by Use of Such Compounds," the disclosure of which is inco ⁇ orated herein by reference in its entirety, and in the Examples set forth below.
• the polycyclic ⁇ -amino-e- caprolactams of this invention may contain one or more chiral centers.
• such compounds will be prepared as a racemic mixture.
• such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) ofthe polycyclic ⁇ -amino-e-caprolactams of formula I are included within the scope of this invention.
• Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well known in the art.
• racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.
• the compounds ofthe invention are useful as synthetic intermediates in the preparation of inhibitors of ⁇ -amyloid peptide release and/or its synthesis. Accordingly, the intermediates of this inventions have utility in the preparation of compounds which are useful, for example, for diagnosing and treating Alzheimer's disease in mammals, including humans.
• Boc tert-butoxycarbonyl
• HOBt 1-hydroxybenzotriazole hydrate
• Hunig's base diisopropylethylamine
• PEPC l-(3-(l-pyrrolidinyl)propyl)-3-ethylcarbodiimide
• UV ultraviolet
• Step A To a stirred solution of 5,7-dihydro-6H-dibenz[b,d]azepin-6-one
• Step B The 7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one (1 eq.) from Step A was dissolved in THF and isoamylnitrite (1.2 eq.) was added. The mixture was cooled to 0°C in an ice bath.
• Step C The resulting oxime from Step B was dissolved in EtOH/NH 3 (20: 1) and hydrogenated in a bomb using Raney nickel and hydrogen (500 psi) at
• the resolved di-/?-toluoyl-D-tartaric salt was then dissolved in EtOAc and saturated NaHCO 3 until pH 9-10 was reached.
• the layers were separated and the organic layer was washed again with saturated NaHCO 3 , H 2 O, and brine.
• the organic layer was dried over MgSO 4 and the drying agent was removed by filtration.
• the filtrate was concentrated in vacuo.
• the free amine was dissolved in MeOH and HCl (12M, 1.0 eq.) was added.
• the salt was concentrated in vacuo and the resulting film was triturated with EtOAc.
• the HCl salt was filtered and rinsed with EtOAc.
• the ee was determined by chiral HPLC. This General Procedure can also be used to resolve other 5-amino-7-alkyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-ones.
• a stream of anhydrous HCl gas was passed through a stirred solution ofthe N-Boc compound in 1,4-dioxane (0.03-0.09 M), chilled in a ice bath to ⁇ 10°C under ⁇ 2 , for 10-15 minutes.
• the cooling bath removed and the solution was allowed to warm to room temperature with stirring for 2-24 hours, monitoring by TLC for the consumption of starting material.
• the solution was concentrated (and in some instances dissolved in CH 2 C1 2 then re-concentrated and placed in vacuum oven at 60-70 °C to remove most ofthe residual dioxane). The residue was typically used without further purification.
• Step B Synthesis of 7-Methyl-5-oximo-5,7-dihydro-6H- dihenz[b,d]azepin-6-one
• the product from Step A (0.700 g, 3.14 mmol) was dissolved in 20 mL of toluene and treated with butyl nitrite (0.733 mL, 6.28 mmol).
• the reaction temperature was lowered to 0°C and the solution was treated with KHMDS (9.42 mL, 0.5 M) under N 2 atmosphere. After stirring for 1 h the reaction was quenched with a saturated solution of NaHSO 4 , diluted with CH 2 C1 2 and separated.
• the organic layer was dried over Na 2 SO 4 and the title compound purified by chromatography (SiO 2 , 98:2 CHCl 3 /MeOH) giving 0.59 g (80 %) as a colorless solid.
• Step B Synthesis of 5- A7ido-5.7-dihvdro-6H-diben7[h.d]a7epin-6-
• Step C Synthesis of 5-(N-Boc-AminoY- 7-Hihyrim-fiW 7W- dihen7[h,d]a7epin-6-one
• the product from Step B was dissolved in THF/H 2 O and stirred at 23 °C for
• Step B Synthesis of 5-Amino-7-(2-rnethylpropy1)-5,7-dihydro-6H- dihen7[h,d]azepin-6-one Hydrochloride
• the product from Step A was deprotected using General Procedure E to provide the title compound as a slightly colored solid after neutralization and extraction with ethyl acetate, drying over Na 2 SO 4 and vacuum drying.
• Step B Synthesis of 5-Amino-7-fmethoxycarhony1methyn- 1 7- dihyriro-6H-dihenz[b.d]azepin-6-one HyHror.h1r>ri e
• Example 3 (0.2 g, 0.617 mmol) (Example 3) in DMF was treated with Cs 2 CO 3 (0.3 g, 0.925 mmol) and warmed to 60 °C. To this reaction mixture was added l-chloro-3,3- dimethyl-2-butanone (0.096 mL, 0.74 mmol) (Aldrich) and stirring was continued for 17 h. After cooling to 23 °C, the mixture was diluted with CH 2 C1 2 , washed with several portions of brine and dried over ⁇ a ⁇ O,,. The title compound was isolated as a colorless solid.
• Step C Synthesis of 5- Amino-7-phenhuty1-5 r 7-dihydro-6H- dibenz[h,d]azepin-6-one
• Step B Synthesis of 5-Hydroxyirmno-7-cyclopropy1rnethyl-S,7- dihydro-6H-dihenz[b,d]azepin-6-one
• Step B Synthesis of ⁇ /-Boc-2-bromoaniline 2-Bromoaniline (1 eq.) (Aldrich) and di-tert-butyl-dicarbonate (1.1 eq.)
• N-Boc-2-bromoaniline (1 eq.) (Step B), the arylboronate ester (1.1 eq.) (Step A), K 2 CO 3 (1.1 eq.) and tetrakis(triphenylphosphine)palladium(0) (0.02 eq.) were stirred in 20% water/dioxane under nitrogen. The solution was heated at reflux for 10 hours. The mixture was allowed to cool then was concentrated. The resulting residue was partitioned between water and chloroform. The organic portion was dried and concentrated to yield an oil which was purified by silica gel chromatography using 1 : 1 CH 2 Cl 2 /hexanes.
• Step D Synthesis of 9-Fh ⁇ oro-5 -dihydro-6H-riihen7[h,d]a7epin-6-
• Step E Synthesis of 9-Flnoro-7-methy1-5,7-dihyHrn-r H- dihen7[b,d]azepin-6-one 9-Fluoro-5,7-dihydro-6H-dibenz[b,d]azepin-6-one (1 eq.) (Step D), cesium carbonate (1.1 eq.) (Aldrich) and methyl iodide (1.1 eq.) (Aldrich) were stirred in dry DMF at ambient temperature for 16 hours. The mixture was concentrated under reduced pressure to provide a residue which was partitioned between EtOAc and water. The organic portion was dried and concentrated to yield an oil which was purified by silica gel chromatography to provide the title compound. Physical data were as follows:
• Step F Synthesis of 5- Amino-9-fluoro-7-methyl-5,7-dihydro-6H- riihenz[b,d]a7ftnin-6-one
• Step E the title compound was prepared.
• Step F Synthesis of 5- Amino- 10-fiuoro-7-methyl-5,7-dihydro-6H- dihen7.[b r d]a7epin-6-one
• Step D Synthesis of 1 -Fluoro-5.7-dihydro-6H-dihen7[h,d]a7epin-6- one
• Step F Synthesis of 5-Amino-l 3-fluoro-7-methy1-5,7-dihydro-6H- dihen7[b,d]a7e ⁇ in-fi-one Gen. Proc.
• Example 1 Example 1 was dissolved in a 1:1 mixture of EtOAc/HOAc. Rhodium on carbon (5%) was added and the mixture was stirred at 60°C under 60 psi of hydrogen for 3 days. The reaction mixture was then filtered and the filtrate was concentrated to provide an oil which was purified by SCX-cation exchange chromatography to yield the title compound.
• N-Boc-L-Alanine (0.429 g, 2.26 mmol) (Aldrich) was dissolved in THF and treated with HOBt hydrate (0.305 g, 2.26 mmol), and 5-amino-7-methyl-5,7- dihydro-6H-dibenz[b,d]azepin-6-one (0.45 g, 1.89 mmol) (Example 1). The temperature was lowered to 0°C and the reaction mixture was treated with EDC
• Step B Synthesis of (S - and ⁇ -5-fL-A1aniny1)am ⁇ no-7-mt » rhy1-
• Step A Synthesis of (S and ⁇ tt-5-(N-Boc-T ⁇ Va1iny1)ami ⁇ n-7- methy1-5,7-dihydrn-fiH-dihen7[b r d]azepin-fi-nne N-Boc-L-Valine (0.656 g, 3.02 mmol) (Aldrich) was dissolved in THF and treated with HOBt hydrate (0.408, 3.02 mmol), DLPEA (1.05 mL, 6.05 mmol) and 5-amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one hydrochloride (0.75 g, 2.75 mmol) (Example 1).
• Step B Synthesis of fSV and fRV5-rL-Va1iny1Vaminn-7-methy1- ,7- dihydrn-6H-dibenz[b r d]azepin-6-one Hydroch1nriHe
• each ofthe isomers from Step A was dissolved in dioxane and treated with excess HCl(g). After stirring for 17 hours, the title compounds were isolated as colorless solids after evaporation and vacuum drying.
• Step A Synthesis of (S)- and (R)-5-(V-Boc-L-tert-Leuciny1)-amino- 7-methy1-5 r 7-Hihydrn-6H-diben7[b.d]a7epin-6-nne
• N-Boc-L-tert-Leucine (0.698 g, 3.02 mmol) (Fluka) was dissolved in THF and treated with HOBt hydrate (0.408, 3.02 mmol), DLPEA (1.05 mL, 6.05 mmol) and 5-amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one hydrochloride
• Example 1 (0.75 g, 2.75 mmol) (Example 1). The temperature was lowered to 0°C and the reaction mixture was treated with EDC (0.601 g, 3.02 mmol) (Alrich) and stirred for 17 hours under ⁇ 2 . The reaction mixture was then evaporated and the residue diluted with EtOAc/H 2 O, washed 1.0 N HCl, sat. NaHCO 3 , brine and dried over Na ⁇ O ⁇ The diastereomers were separated on a Chiralcel OD column using 10% IP A/heptane at 1.5 mL/minute.
• Step B Synthesis of CSV and fRV5-fL-tert-T .eucinynamino-7- methyl-5 r 7-dihydro-6H-dibenz[b r d]azepin-fi-one Hydrochloride
• each ofthe isomers from Step A was dissolved in dioxane and treated with excess HCl(g). After stirring for 17 hours, the title compounds were isolated as colorless solids after evaporation and vacuum drying.
• Step A Synthesis of 5-(/V-Boc-L-A1aniny1)amino-7-methy1-5,7-
• Step B Synthesis nf 5-tT .- Alaninynaminn ⁇ -methyl-S -dihydrn-fiH- diben7[h,d]a7epin- ⁇ -one Hydrochloride
• Step B Synthesis of - .-Valinynamino ⁇ -methyl- ⁇ -dihydrn-fiH- diben7[h,d]a7epin-fi-one Hydrochloride
• Step B Synthesis of 5-(T -A1aniny1)amino-9-fluoro-7-methy1-5,7- dihydro-6H-dihen7[h r d]azepin-6-one Hydrochloride
• Step A Synthesis of 5-(/V-Boc-L-A1aninyl nino-10-fluoro-7- methy1-5,7-dihydro-6H-diben7[b r d]a7epin-fi-one
• Step B Synthesis of 5-(T ,- A1aniny11amino-1 -flnnrn-7-methy1-5, 7- dihydro-6H-dibenz[b.d3azepin-6-one Hydroch1nridR
• Step B Synthesis of -(T -A1aninynamino-1 -f1nnrn-7-methy1-5,7- dihydro-fiH-dihen7[h.d]azepin-6-one Hydrochloride
• Step A Synthesis of 5-(A/ ' -Boc-T.-A1aninynaminn-7- cvc1npropv1methv1-5_7-dihvdro-fiH-Hihen7[h r ]a7epin-fi-nne
• Step B Synthesis of -n,- A1aninynamino-7-cyr.1npropy1methy1-5, 7- dihvdro-6H-dihenz[b.d1azepin-6-one Hydrorh1nriHe
• Step B Synthesis of 5-(T,-A1aniny1)amino-7-phenhuty1-5,7-dihydro-
• Step B Synthesis of 5-(L-Va1iny amino-7- y 1opropy1methy1- , 7- dihydro-6H-dibenz[b.d]azepin-6-one Hydrochloride
• Step B Synthesis of 5-(L-Va1inynamino-7-phenh ⁇ ty1-5,7-Hihydrn- fiH-diben7[h,d]a7epin-6-one Hydrochloride
• Step A Synthesis of -f ⁇ -Roc-T.-Valiny1 ⁇ a ⁇ ninn-7-he ⁇ y1- 7- dihydro-6H-dibenz[b r d]azepin-fi-one
• Step B Synthesis of 5-(L-Va1iny1)amino-7-hexy1-5,7-di hydro-fiH- diben7[b,d]a7epin-6-one Hydrochloride
• Step A Synthesis of 5-(V-Boc-L-Va1inynamino-9-flunrn-7-methyl- 5,7-dihydro-fiH-dihen7[b,d]azepin-fi-one
• Step B Synthesis of -(T ,-Valinyl inn-9-flnoro-7-rnethy1-5,7- dihydro-6H-dihen7.[b r d]a7epin-6-one Hydrochloride
• Step A Synthesis of 5-(V-Boc-L-Va1iny1)arnino-10-flnoro-7-methyl-
• Step B Synthesis of 5-(T,-Va1invnamino-1 0-fh ⁇ oro-7-methy1-5,7- dihydro-6H-dibenz[b,d]azepin-6-one Hydrochloride
• Step B Synthesis of 5-fT.-Valinynamino-13-flnnro-7-methy1-5,7- dihydro-6H-dibenz[b r d]azepin-6-one Hydrochloride
• Step D Synthesis of 9 r 1 -Difh ⁇ oro-5.7-dihydro-6H- dihen7[b t d]a7epin-fi-one
• Example 33
• Step D Synthesis of 1 r n-Difluoro-5,7-dihydro-fiH- dihen7[h,d]a7epin-6-one
• Step F Synthesis of 5-Amino-10.1 3-difluoro-7-methyl-5.7-dihydro-
• Step E Synthesis of 9-Oximo-5 ⁇ fi-Dihydro-4H-qnino[8 ⁇ 1 - ah][3]he ⁇ za7epin-8(9H)-one
• Step F Synthesis of 9- A ⁇ nino-5,6-Dihydro-4H-q ⁇ ino[8, 1 - ah][3]hen7a7epin-8t9H)-one
• the product from Step E (0.360 g, 1.29 mmol) was hydrogenated over Ra Ni (0.05 g) in EtOH (50 mL)/ NH 3 (anhydrous) (5.0 mL) at 100°C and 500 psi for 10 h.
• the catalyst was removed by filtration and the resulting filtrate chromatographed over SiO 2 (CHCl 3 /MeOH, 98:2) yielding the titled compound as a colorless oil which solidified upon standing.
• Step A Synthesis of 9-fN'-Boc-L- Alaninyllamino-S. ⁇ -Dihydro ⁇ H- quino[8 r 1-ah][3]ben7azepin-8(9H)-one Following General Procedure D and using N-Boc- Alanine (Aldrich) and
• Step B Synthesis of 9-fN / -L-AlaninyPamino-5.6-dihydro-4H- quino[8,1-ab][3]ben7a7epin-8(9H)-one Hydrochloride Following General Procedure E and using the product from Step A, the title compound was prepared. Physical data were as follows:
• Step B Synthesis of 1 ,3 4,7, 17, 12a-hexahvdropvrido[2.1 -b][3]ben7a7epin-
• Step C Synthesis of 7-Oximo-l .3.4.7.12.1 ?.a-heyahydropyridn[2, 1 - h][3]henza7epin-6(2H)-one Following General Procedure A (Step B) and using 1,3,4,7,12,12a- hexahydropyrido[2,l-b][3]benzazepin-6(2H)-one (from Step B), the title compound was prepared.
• Step D Synthesis of 7- Amino-1.3.4.7.12 r 12a-hexahydropyrido[?., 1 - h][3]henza7epin-6(2H)-one
• Step B Synthesis of 4.5.6.7-Tetrahydro-3,7-methann-3H-3- benzazQnin-2(lH)-one Following General Procedure G and using N-chloroacetyl-3- phenylpiperidine, the title compound was prepared. Physical data were as follows:
• Step C Synthesis of 1 -Oximo-4.5 r 6.7-tetrahydro-3,7-methano-3H-3- benzazonin-2( 1 H)-one Following General Procedure A (Step B) and using the product from Step
• Step D Synthesis of 1 -Amino-4 r 5.fi.7-tet ⁇ ahydro-3 r 7-methano-3H-3- ben7a7onin-2(1 HVone Following General Procedure A (Step C) and using the product from Step
• Step E Synthesis of 1-r ⁇ '-Boc-L-Alaninvnamino-4.5.6.7- tetrahydro-3,7-methano-3H-3-ben7a7onin-2(1 H)-one
• reaction mixture was stirred for 5 minutes before 6.5 mL triethylamine (46.6 mmol, 6.0 eq) was added and the reaction warmed to room temperature for 10 minutes.
• the reaction was quenched with 200 mL 0.1 N aqueous HCl, and this was extracted with 2 x 100 mL CH 2 C1 2 .
• the combined organic layers were washed with 100 mL saturated NaHCO 3 followed by
• Step B Synthesis of SS-
• Step C Synthesis of 5S-(2S-aminopropyl)-arnino-7-rnethy1-5,7- dihydro-6H-dihenz[h,d]azepin-6-one HCl (g) was bubbled through a solution of 317 mg of 5S-[N'-(tert- butyloxycarbonyl)-2S-aminopropyl]amino-7-methyl-5,7-dihydro-6H- dibenz[b,d]azepin-6-one (0.8 mmol) (from Step B) in 30 mL dioxane for 15 minutes. This was stirred at room temperature for 17 hours at which time the solvent was removed in vacuo to yield 248 mg (94 %) of a white solid. This was used without further purification.
• Step D Synthesis of 5S-[N 2S-hydroxy-3-methy1hutyryn-2S- aminopropy1]amino-7-methyl-5.7-dihydro-6H- dihen7[h,d]azepin-6-one
• Example 39 Cellular Screen for the Detection of Inhibitors of ⁇ -Amyloid Production
• compounds can be assayed for their ability to inhibit ⁇ -amyloid production in a cell line possessing the Swedish mutation.
• This mutation is commonly called the Swedish mutation and the cells, designated as “293 751 SWE", are plated in Coming 96- well plates at 2-4 x 10 4 cells per well in Dulbecco's minimal essential media (Sigma, St. Louis, MO) plus 10% fetal bovine serum. Cell number is important in order to achieve ⁇ -amyloid ELISA results within the linear range ofthe assay (-0.2 to 2.5 ng per mL).
• Drug stocks are prepared in 100% dimethyl sulfoxide such that at the final drug concentration used in the treatment, the concentration of dimethyl sulfoxide does not exceed 0.5% and, in fact, usually equals 0.1%.
• the media are again removed and replaced with fresh drug containing media as above and cells are incubated for an additional two hours.
• plates are centrifuged in a Beckman GPR at 1200 ⁇ m 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 are transferred into an ELISA plate precoated with antibody 266 [P. Seubert, Nature (1992) 359:325-327 17 ] against amino acids 13-28 of ⁇ -amyloid peptide as described in International Patent Application Publication No. 94/10569 15 and stored at 4°C overnight.
• An ELISA assay employing labeled antibody 3D6 [P.
• Cytotoxic effects ofthe compounds are measured by a modification ofthe method of Hansen, et al. 18 .
• To the cells remaining in the tissue culture plate is 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 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
• results ofthe ⁇ -amyloid peptide ELISA are fit to a standard curve and expressed as ng/mL ⁇ -amyloid peptide. In order to normalize for cytotoxicity, these results are divided by the MTT results and expressed as a percentage ofthe results from a drug free control. All results are the mean and standard deviation of at least six replicate assays.
• test compounds are assayed for ⁇ -amyloid peptide production inhibition activity in cells using this assay.
• the results of this assay can be used to demonstrate that the compounds prepared from the intermediates of this invention inhibit ⁇ -amyloid peptide production by at least 30% as compared to control when employed at 10 ⁇ g/mL.
• This example illustrates how the compounds prepared from the intermediate 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 19 ].
• the compound is usually formulated at between 1 and 10 mg/mL. Because ofthe low solubility factors of the compounds, they may be formulated with various vehicles, such as com oil (Safeway, South San Francisco, CA); 10% ethanol in corn oil; 2-hydroxypropyl- ⁇ -cyclodextrin (Research Biochemicals International, ⁇ atick MA); and carboxy-methyl-cellulose
• mice are dosed subcutaneously with a 26 gauge needle and 3 hours later the animals are euthanized via CO 2 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 °C.
• the brains of the mice are then removed and the cortex and hippocampus are dissected out and placed on ice.
• 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 ° C prior to quantitation of ⁇ -amyloid.
• ice cold guanidine buffer 5.0 M guanidine-HCl, 50 mM Tris-HCl, pH 8.0
• 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°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 17 ], is specific to amino acids 13 - 28 of ⁇ -amyloid.
• the antibody 3D6 [Johnson- Wood et al., RN S USA (1997) 94:1550-1555 20 ], 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 murine ⁇ -amyloid peptide at concentrations up to 1 ng/mL.
• the configuration ofthe sandwich ELISA quantitating the level of ⁇ - amyloid (aa 1 -42) employs the mAb 2 IF 12 [Johnson- Wood et al., PNAS USA
• Biotinylated 3D6 is also the reporter antibody in this assay which has a lower limit of sensitivity of -125 pg/mL (28 pM).
• the 266 and 2 IF 12 capture mAbs are coated at 10 ⁇ g/mL into 96 well immunoassay plates (Costar, Cambidge MA) overnight at room temperature. The plates are then aspirated and blocked with 0.25% human serum albumin in PBS buffer for at least 1 hour at room temperature, then stored desiccated at 4°C until use. The plates are rehydrated with wash buffer (Tris-buffered saline, 0.05% Tween 20) prior to use. The samples and standards are added to the plates and incubated overnight at 4°C. The plates are washed ⁇ 3 times with wash buffer between each step ofthe assay.
• wash buffer Tris-buffered saline, 0.05% Tween 20
• biotinylated 3D6 diluted to 0.5 ⁇ g/mL in casein incubation buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4) is incubated in the well for 1 hour at room temperature.
• Avidin-HRP Vector, Burlingame CA
• diluted 1 :4000 in 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 SO 4 .
• Reaction product is quantified using a Molecular Devices Vmax (Molecular Devices, Menlo Park CA) measuring the difference in absorbance at 450 nm and 650 nm. 2. Blood Assay
• the EDTA plasma is diluted 1 : 1 in specimen diluent (0.2 gm/1 sodium phosphate»H 2 O (monobasic), 2.16 gm/1 sodium phosphate »7H 2 O (dibasic), 0.5gm/l thimerosal, 8.5 gm 1 sodium 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 caprure/3D6 reporter) described above for the brain assay except the specimen diluent was used instead ofthe 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% com oil or, alternatively, in a solution comtaining 80% com 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.

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