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WO2004007445A2 - Protein kinase c modulators, and methods of use thereof - Google Patents

Protein kinase c modulators, and methods of use thereof Download PDF

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Publication number
WO2004007445A2
WO2004007445A2 PCT/US2003/022038 US0322038W WO2004007445A2 WO 2004007445 A2 WO2004007445 A2 WO 2004007445A2 US 0322038 W US0322038 W US 0322038W WO 2004007445 A2 WO2004007445 A2 WO 2004007445A2
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Prior art keywords
compound
alkyl
cycloalkyl
aralkyl
absent
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PCT/US2003/022038
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French (fr)
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WO2004007445A3 (en
Inventor
Alan P. Kozikowski
Peter M. Blumberg
Henry Hennings
Ireneusz Nowak
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Georgetown University
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Priority to AU2003256538A priority Critical patent/AU2003256538A1/en
Publication of WO2004007445A2 publication Critical patent/WO2004007445A2/en
Publication of WO2004007445A3 publication Critical patent/WO2004007445A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/18—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
    • C07D207/22—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/24—Oxygen or sulfur atoms
    • C07D207/26—2-Pyrrolidones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D245/00—Heterocyclic compounds containing rings of more than seven members having two nitrogen atoms as the only ring hetero atoms
    • C07D245/04—Heterocyclic compounds containing rings of more than seven members having two nitrogen atoms as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • C07D245/06—Heterocyclic compounds containing rings of more than seven members having two nitrogen atoms as the only ring hetero atoms condensed with carbocyclic rings or ring systems condensed with one six-membered ring

Definitions

  • Neuronal dysfunction includes cognitive decline, which is characterized by concentration loss, memory-acquisition loss, and information-storage or retrieval loss. Neuronal dysfunction can result from central nervous system (“CNS”) injury, such as stroke, spinal-cord injury, and peripheral-nerve injury. Cognitive decline is symptomatic of neuronal disorders, such as cognitive decline associated with aging and minimal cognitive impairment (also known as minimal cognitive disorder) as well as severe neurodegenerative disorders, such as Alzheimer's disease. Neuronal dysfunction is also associated with disorders that result in loss of motor skills, such as Parkinson's disease and amyotrophic lateral sclerosis.
  • CNS central nervous system
  • cognitive decline is symptomatic of neuronal disorders, such as cognitive decline associated with aging and minimal cognitive impairment (also known as minimal cognitive disorder) as well as severe neurodegenerative disorders, such as Alzheimer's disease. Neuronal dysfunction is also associated with disorders that result in loss of motor skills, such as Parkinson's disease and amyotrophic lateral sclerosis.
  • Alzheimer's disease Approximately 5-15% of the population of the United States over age 65 (1.24 million) has Alzheimer's disease. This disease is the most frequent cause of institutionalization for long-term care, hi 1983, more than $27 billion was spent in the U.S. in health care for Alzheimer's afflicted individuals.
  • Six basic research areas into Alzheimer's disease have b een defined by R. J. Wurtman, Scientific A merican 1985, 62. These areas include faulty genes, accumulations of amyloid protein, infectious agents, environmental toxins (e g., aluminum and certain unusual amino acids), inadequate blood flow and energy metabolism, and cholinergic deficits. A number of possible therapeutic interventions for treatment of Alzheimer's disease are currently under study.
  • NGF nerve growth factors
  • AChE acetylcholinesterase
  • GABA-inverse agonists GABA-inverse agonists
  • NMDA modulators NMDA modulators
  • PKC protein kinase C
  • protein kinase C represents a family of at least 12 serine/threonine kinases that are involved in signal transduction in response to a host of hormonal, neuronal, and growth factor stimuli.
  • the N-terminal regulatory domains of the classical and novel PKCs contain a conserved Cl domain comprised of two cysteine-rich zinc fingers that bind to the natural PKC activator diacylglycerol (DAG) as well as to certain natural products, such as the phorbol esters and the i ndolactams.
  • DAG PKC activator diacylglycerol
  • the c lassical and n ovel P KC i sozymes are c ytosolic and are translocated to the membrane upon interaction with Ca 2+ , whereby they are activated through interaction with membrane phospholipids and DAG.
  • PKC has been shown to play a major role in a variety o f disease states including diabetes, heart disease, c aiicer, and Alzheimer's disease, and thus b oth i sofonn s elective activators and inhibitors may provide novel leads in the development of therapeutic agents.
  • Bryostatin a macrocyclic lactone from the bryozoan Bugula n eritina, binds to the DAG regulatory site of PKC, yet is not a tumor promoter; rather, it acts as an antineoplastic agent that has been used to treat murine melanoma. Hennings, H.; Hennings, H.; Blumberg P. M.; Pettit, G. R.; Herald, C. L.; Shores, R.; Yuspa, S. H., Carcinogenesis 1987, 8, 1343- 1346.
  • PKC amyloid precursor protein
  • This enzyme cleaves just outside the A ⁇ sequence, leaving a large membrane-bound fragment that is later cleaved by a yet to be identified ⁇ -secretase at positions 711 and 713, thereby generating A ⁇ 1-40 and 1-42, respectively.
  • PKC activators belonging to the phorbol family have been shown to dramatically enhance ⁇ -secretase activity, thus leading to enhanced secretion of sAPP- .
  • the present invention relates to a compound represented by
  • X represents NR 5 , O or S
  • Y represents O or S
  • R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R 1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl, acylaminoalkyl, or sulfonylaminoalkyl;
  • R 3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or aryls
  • R 5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in A is R, S, or a mixture thereof.
  • a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR 5 .
  • a compound of the present invention is represented by A and the attendant definitions, wherein Y represents O.
  • a compound of the present invention is represented by A and the attendant definitions, wherein R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl.
  • a compound of the present invention is represented by A and the attendant definitions, wherein R 1 represents alkyl or cycloalkyl. i certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
  • a compound of the present invention is represented by A and the attendant definitions, wherein R 3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl.
  • a compound of the present invention is represented by A and the attendant definitions, wherein R 4 is absent.
  • a compound of the present invention is represented by A and the attendant definitions, wherein R 5 is alkyl.
  • a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR 5 ; and Y represents O.
  • a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR 5 ; Y represents O; and R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl.
  • a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR 5 ; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; and R 1 represents alkyl or cycloalkyl.
  • a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR 5 ; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R 1 represents alkyl or cycloalkyl; and R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
  • a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR 5 ; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R 1 represents alkyl or cycloalkyl; R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R 3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl.
  • a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR 5 ; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R 1 represents alkyl or cycloalkyl; R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; R 3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl; and R 4 is absent.
  • a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR 5 ; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R 1 represents alkyl or cycloalkyl; R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; R 3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl; R 4 is absent; and R 5 is alkyl.
  • a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR 5 ; Y represents O; R represents
  • R represents isopropyl; R represents hydroxylmethyl; R represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl; R 4 is absent; and R 5 is methyl.
  • certain compounds according to structure A In an assay based on mammalian PKC ⁇ , certain compounds according to structure A have an IC50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM. hi an assay based on mammalian PKC ⁇ , certain compounds according to structure A have an EC50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM.
  • the present invention relates to a compound represented by B:
  • X represents NR 5 , O or S
  • Y represents O or S
  • R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R 1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, ⁇ . alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl, acylaminoalkyl, or sulfonylaminoalkyl;
  • R 3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
  • R 4 is absent or present from 1 to 3 times inclusive;
  • R 5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in B is R, S, or a mixture thereof.
  • certain compounds according to structure B have an IC 50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM.
  • certain compounds according to structure B have an EC 50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM.
  • the present invention relates to a compound represented by
  • X represents NR 5 , O or S;
  • Y represents O or S;
  • R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R 1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylaminoalkyl;
  • R 3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
  • R 4 is absent or present from 1 to 3 times inclusive
  • certain compounds according to structure C have an IC 50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM.
  • certain compounds according to structure C have an EC 50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM.
  • the present invention relates to a compound represented by
  • Y represents O or S
  • R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R 1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl
  • R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylaminoalkyl;
  • R 3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
  • R 4 is absent or present from 1 to 3 times inclusive
  • R 5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in D is R, S, or a mixture thereof.
  • an assay based on mammalian PKC ⁇ certain compounds according to structure D have an IC 50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM.
  • an assay based on mammalian PKC ⁇ certain compounds according to structure D have an EC 50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM.
  • the present invention relates to a compound represented by
  • L represents NR 4 , O, or S
  • W represents a divalent alkyl, alkenyl, alkynyl or glycol chain
  • X represents NR 4 , O or S
  • Y represents O or S
  • R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R 1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylarninoalkyl;
  • R 3 is absent or present from 1 to 3 times inclusive
  • R 4 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in E is R, S, or a mixture thereof.
  • a compound of the present invention is represented by E and the attendant definitions, wherein L represents NR 4 . h certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W is a divalent alkyl or glycol chain. In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein X represents NR 4 .
  • a compound of the present invention is represented by E and the attendant definitions, wherein Y represents O.
  • a compound of the present invention is represented by E and the attendant definitions, wherein R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl.
  • a compound of the present invention is represented by E and the attendant definitions, wherein R 1 represents alkyl or cycloalkyl.
  • a compound of the present invention is represented by E and the attendant definitions, wherein R 2 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl. i certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein R 3 is absent.
  • a compound of the present invention is represented by E and the attendant definitions, wherein R 4 is H or alkyl.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR 4 ; L represents NR 4 ; and Y represents O.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR 4 ; L represents NR 4 ; Y represents O; and R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl.
  • W represents a divalent alkyl or glycol chain; X represents NR 4 ; L represents NR 4 ; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; and R 1 represents alkyl or cycloalkyl.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR 4 ; L represents NR 4 ; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R 1 represents alkyl or cycloalkyl; and R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR 4 ; L represents NR 4 ; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R 1 represents alkyl or cycloalkyl; R 2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R 3 is absent.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR 4 ; L represents NR 4 ; Y represents O; R represents independently for each
  • R represents alkyl or cycloalkyl
  • R represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl
  • R is absent
  • R 4 is H or alkyl.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH 2 ) 4 -; X represents NMe; L represents NH; Y represents O; R represents H; R 1 represents isopropyl; R 2 represents hydroxylmethyl; and R 3 is absent.
  • W represents -(CH 2 ) 6 -; X represents NMe; L represents NH; Y represents O; R represents H; R 1 represents isopropyl; R 2 represents hydroxylmethyl; and R is absent.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH 2 ) 8 -; X represents NMe; L represents NH; Y represents O; R represents H; R 1 represents isopropyl; R 2 represents hydroxylmethyl; and R 3 is absent.
  • W represents -(CH 2 ) 1 o-; X represents NMe; L represents NH; Y represents O; R represents H; R 1 represents isopropyl; R 2 represents hydroxylmethyl; and R 3 is absent.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH 2 ) 12 -; X represents NMe; L represents NH; Y represents O; R represents H; R 1 represents isopropyl; R 2 represents hydroxylmethyl; and R 3 is absent.
  • W represents -(CH 2 ) 14 -; X represents NMe; L represents NH; Y represents O; R represents H; R 1 represents isopropyl; R 2 represents hydroxylmethyl; and R 3 is absent.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH 2 )2Q-; X represents NMe; L represents NH; Y represents O; R represents H; R represents isopropyl; R represents hydroxylmethyl; and R 3 is absent.
  • W represents -CH 2 -(OCH 2 CH2) 4 O-CH2-; X represents NMe; L represents NH; Y represents O; R represents H; R 1 represents isopropyl;
  • R represents hydroxylmethyl; and R is absent.
  • a compound of the present invention is represented by E and the attendant definitions, wherein W represents -CH2-(OCH 2 CH2) 5 O-CH2-; X represents NMe; L represents NH; Y represents O; R represents H; R 1 represents isopropyl;
  • R represents hydroxylmethyl; and R is absent.
  • an assay based on mammalian PKC ⁇ certain compounds according to structure E have an IC 50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM.
  • an assay based on mammalian PKC ⁇ certain compounds according to structure E have an EC 50 value less than 1 ⁇ M, preferably less than 100 nM, and more preferably less than 10 nM.
  • the present invention relates to a compound represented by
  • W represents a divalent alkyl, alkenyl, alkynyl or glycol chain;
  • X represents independently for each occurrence NR, O or S;
  • R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • R 1 represents independently for each occurrence hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylaminoalkyl;
  • R 2 is absent or present from 1 to 6 times inclusive
  • R 3 represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in F is R, S, or a mixture thereof.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain.
  • a compound of the present invention is represented by F and the attendant definitions, wherein X represents NR.
  • a compound of the present invention is represented by F and the attendant definitions, wherein R 1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein R is absent.
  • a compound of the present invention is represented by F and the attendant definitions, wherein R represents alkyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain;
  • X represents NR; and
  • R 1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; and R 2 is absent.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; and R 3 represents alkyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; R 1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; R 1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R 3 represents alkyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; R 1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH 2 ) 4 -; X represents NH; R 1 represents hydroxylmethyl; R 2 is absent; and R 3 represents isopropyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH 2 ) 6 -; X represents NH; R 1
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH 2 ) 8 -; X represents NH; R 1
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH 2 ) 10 -; X represents NH; R 1 represents hydroxylmethyl; R 2 is absent; and R 3 represents isopropyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH 2 ) 1 2-; X represents NH; R 1 represents hydroxylmethyl; R 2 is absent; and R 3 represents isopropyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH 2 ) 14 -; X represents NH; R 1 represents hydroxylmethyl; R 2 is absent; and R 3 represents isopropyl.
  • W represents -(CT ⁇ o X represents NH; R 1 represents hydroxylmethyl; R 2 is absent; and R 3 represents isopropyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents -CH2-(OCH 2 CH2) O-CH 2 -; X represents NH; R 1 represents hydroxylmethyl; R 2 is absent; and R 3 represents isopropyl.
  • a compound of the present invention is represented by F and the attendant definitions, wherein W represents -CH 2 -(OCH 2 CH 2 )5 ⁇ -CH 2 -; X represents NH; R 1 represents hydroxylmethyl; R 2 is absent; and R 3 represents isopropyl.
  • W represents -CH 2 -(OCH 2 CH 2 )5 ⁇ -CH 2 -;
  • X represents NH;
  • R 1 represents hydroxylmethyl;
  • R 2 is absent; and
  • R 3 represents isopropyl.
  • the present invention relates to a compound represented by any of the structures outlined above, wherein said compound is a single stereoisomer. In certain embodiments, the present invention relates to a composition, comprising a compound represented by any of the structures outlined above; and a pharmaceutically acceptable excipient.
  • the present invention relates to compounds that modulate the activity of protein kinase C in a mammal, wherein the compounds are represented by any of the structures outlined above, and any of the sets of definitions associated with one of those structures, hi certain embodiments, the compounds of the present invention are antagonists or agonists of protein kinase C in a mammal, hi any event, the compounds of the present invention preferably exert their effect on protein kinase C in a mammal at a concentration less than about 1 micromolar, preferably at a concentration less than about 100 nanomolar, and more preferably at a concentration less than 10 nanomolar.
  • the present invention contemplates pharmaceutical formulations comprising a compound of the present invention.
  • the pharmaceutical formulations will comprise a compound of the present invention that selectively effects a particular isoform of protein kinase C, and thereby has a therapeutic effect on an acute or chronic ailment, disease or malady that is at least in part due to biochemical or physiological processes associated with that isoform of protein kinase C.
  • the Background of the Invention teaches examples o f acute or chronic ailments, diseases or maladies that are caused or exacerbated by biochemical or physiological processes associated with various isoforms of protein kinase C.
  • the present invention contemplates that pharmaceutical formulations comprising a compound of the present invention will be of medicinal value against the aforementioned acute or chronic ailments, diseases or maladies.
  • the present invention relates to a method of modulating the activity of protein kinase C in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention.
  • the present invention relates to a method of increasing secretion of sAPP ⁇ in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention.
  • the present invention relates to methods of treating a mammal suffering from neuronal dysfunction, cognitive decline, neurodegeneration, stroke, Alzheimer's disease, Parkinson's disease, diabetes, heart disease, or cancer, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention.
  • the present invention relates to methods of treating a mammal suffering from Alzheimer's disease, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention.
  • the present invention relates to a method of inducing hyperplasia in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention.
  • the methods of the invention can be prophylactic, therapeutic, or curative. When the methods are practiced prior to an individual showing any clinical sign or symptom of a disease or disorder, they are considered prophylactic.
  • Prophylactic treating can be practiced, for example, on individuals suspected of having a disease or disorder, or on individuals suspected of being at high risk of developing a disease or disorder, i embodiments, prophylactic methods reduce or eliminate the risk of developing a disease or disorder characterized by neuronal dysfunction, hi embodiments, prophylactic methods reduce or eliminate the risk of developing a disease or disorder characterized by cognitive decline. In embodiments, prophylactic methods reduce or eliminate the risk of developing a disease or disorder characterized by aberrant activity of protein kinase C.
  • prophylactic methods reduce or eliminate the risk of developing Alzheimer's disease.
  • the methods of treating are practiced on an individual already showing at least one clinical sign or symptom of a disease or disorder, the methods can be therapeutic or curative.
  • Therapeutic methods are those methods that result in a detectable change in at least one symptom of the disease or disorder. Preferably, the detectable change is an improvement in the symptom.
  • therapeutic methods reduce or eliminate neuronal dysfunction
  • hi embodiments therapeutic methods reduce or eliminate cognitive decline.
  • therapeutic methods reduce or eliminate a disease or disorder characterized by aberrant activity of protein kinase C.
  • therapeutic methods reduce or eliminate Alzheimer's disease.
  • Curative methods are those therapeutic methods that result in elimination of at least one symptom of a disease or disorder.
  • curative methods eliminate the cause of the disease or disorder.
  • curative methods eliminate neuronal dysfunction, hi embodiments, curative methods eliminate cognitive decline.
  • curative methods eliminate a disease or disorder characterized by aberrant activity of protein kinase C.
  • curative methods eliminate Alzheimer's disease.
  • the methods of treating can include a single administration to an individual, or can include multiple administrations. Treatment and dosing regimens can be designed and implemented in accordance with those that are well-known and widely practiced in the art.
  • each regimen will be tailored to the individual to be treated and the disease(s), disorder(s), and/or symptom(s) involved.
  • individual tailoring is well within the skill of those in the art and does not involve undue or excessive experimentation.
  • the compounds or compositions of the present invention may be used in the manufacture of a medicament to treat any of the foregoing protein kinase C related conditions or diseases.
  • the present invention is directed to a method for formulating compounds of the present invention in a pharmaceutically acceptable carrier or excipient.
  • kits containing a compound of the present invention also provides kits containing a compound of the present invention.
  • a compound of the present invention is provided in the kit as the sole component of the kit. In embodiments, it is present as part of a composition.
  • the kits of the invention can contain all the necessary compounds, solutions, and equipment for administration of the compounds and compositions contained therein to an individual, or the kits can be designed for in vitro use of a compound of the present invention.
  • Figure 1 depicts the secretion of sAPP ⁇ as determined by densitometry analyses of immunoblots.
  • Figure 2 depicts representative immunoblotting blots (A and B) showing the signal obtained with various compounds used at 1 and 0.1 ⁇ M.
  • the two panels represent distinct experiments and blots obtained by conventional immunoblotting techniques to detect secreted sAPP ⁇ .
  • Figure 3 depicts hydrogen bonding interactions between PKC ⁇ Clb domain and ligands BL (A) and generic 5 (B).
  • Figure 4 depicts the correlation of binding affinities of 7a-7g with the spacer length in A.
  • Cis configurations are often labeled as (Z) configurations.
  • trans is art-recognized and refers to the arrangement of two atoms or groups around a double bond such that the atoms or groups are on the opposite sides of a double bond. Trans configurations are often labeled as (E) configurations.
  • combinatorial library or “library” are art-recognized and refer to a plurality of compounds, which may be termed “members,” synthesized or otherwise prepared from one or more starting materials by employing either the same or different reactants or reaction conditions at each reaction in the library. There are a number of other terms of relevance to combinatorial libraries (as well as other technologies).
  • identifier tag is art-recognized and refers to a means for recording a step in a series of reactions used in the synthesis of a chemical library.
  • immobilized is art- recognized and, when used with respect to a species, refers to a condition in which the species is attached to a surface with an attractive force stronger than attractive forces that are present in the intended environment of use of the surface, and that act on the species.
  • solid support is art-recognized and refers to a material which is an insoluble matrix, and may (optionally) have a rigid or semi-rigid surface.
  • linker is art- recognized and refers to a molecule or group of molecules connecting a support, including a solid support or polymeric support, and a combinatorial library member.
  • polymeric support is art-recognized and refers to a soluble or insoluble polymer to which a chemical moiety can be covalently bonded by reaction with a functional group of the polymeric support.
  • functional group of a polymeric support is art-recognized and refers to a chemical moiety of a polymeric support that can react with an chemical moiety to form a polymer-supported amino ester.
  • synthetic is art-recognized and refers to production by in vitro chemical or enzymatic synthesis.
  • meso compound is art-recognized and refers to a chemical compound which has at least two chiral centers but is achiral due to a plane or point of symmetry.
  • chiral is art-recognized and refers to molecules which have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.
  • a "prochiral molecule” is a molecule which has the potential to be converted to a chiral molecule in a particular process.
  • stereoisomers is art-recognized and refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups i n s pace. I n p articular, " enantiomers” r efer t o t wo s tereoisomers o f a compound which are non-superimposable mirror images of one another. "Diastereomers”, on the other hand, refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.
  • a “stereoselective process” is one which produces a particular stereoisomer of a reaction product in preference to other possible stereoisomers of that product.
  • An “enantioselective process” is one which favors production of one of the two possible enantiomers of a reaction product.
  • regioisomers is art-recognized and refers to compounds which have the same molecular formula but differ in the connectivity of the atoms. Accordingly, a "regioselective process" is one which favors the production of a particular regioisomer over others, e.g., the reaction produces a statistically significant increase in the yield of a certain regioisomer.
  • esters are art-recognized and refers to molecules with identical chemical constitution and containing more than one stereocenter, but which differ in configuration at only one of these stereocenters.
  • ED 50 is art-recognized. In certain embodiments, ED 50 means the dose of a drug which produces 50% of its maximum response or effect, or alternatively, the dose which produces a pre-determined response in 50% of test subjects or preparations.
  • LD 50 is art-recognized, hi certain embodiments, LD50 means the dose of a drug which is lethal in 50% of test subjects.
  • therapeutic index is an art-recognized tenn which refers to the therapeutic index of a drug, defined as LD 5 0/ED 5 0.
  • ligand refers to a compound that binds at the receptor site.
  • active agent pharmaceutically active agent
  • drug drug
  • active agent when used, “pharmacologically active agent” and “drug” are used, or when a particular drug, such as oxycodone, is identified, it is to be understood as including the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
  • structure-activity relationship or "(SAR)” is art-recognized and refers to the way in which altering the molecular structure of a drug or other compound alters its interaction with a receptor, enzyme, nucleic acid or other target and the like.
  • agonist is art-recognized and refers to a compound that mimics the action of natural transmitter or, when the natural transmitter is not known, causes changes at the receptor complex in the absence of other receptor ligands.
  • antagonist is art-recognized and refers to a compound that binds to a receptor site, but does not cause any physiological changes unless another receptor ligand is present.
  • competitive antagonist i s art-recognized and refers to a compound or that binds to a receptor site; its effects may be overcome by increased concentration of the agonist.
  • partial agonist refers to a compound or that binds to a receptor site but does not produce the maximal effect regardless of its concentration.
  • a “target” shall mean a site t o which t argeted c onstructs b ind.
  • a t arget m ay b e either in vivo or in vitro.
  • a target may be a tumor (e.g., tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast and colon as well as other carcinomas and sarcomas), hi other embodiments, a target may be a site of infection (e.g., by bacteria, viruses (e.g., HIV, herpes, hepatitis) and pathogenic fungi (Candida sp.).
  • a target may refer to a molecular structure to which a targeting moiety binds, such as a hapten, epitope, receptor, dsDNA fragment, carbohydrate or enzyme.
  • a target may be a type of tissue, e.g., neuronal tissue, intestinal tissue, pancreatic tissue etc.
  • targeting moiety refers to any molecular structure which assists the construct in localizing to a particular target area, entering a target cell(s), and/or binding to a target receptor.
  • lipids including cationic, neutral, and steroidal lipids, virosomes, and liposomes
  • antibodies, lectins, ligands, sugars, steroids, hormones, nutrients, and proteins may serve as targeting moieties.
  • modulation is art-recognized and refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart.
  • treating is art-recognized and refers to curing as well as ameliorating at least one symptom of any condition or disease.
  • prophylactic or therapeutic treatment refers to administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom).
  • a "patient,” “subject” or “host” to be treated by the subject method may mean either a human or non-human animal.
  • mammals include humans, primates, bovines, porcines, canines, felines, equines and rodents (e.g., mice and rats).
  • bioavailable is art-recognized and refers to a form of the subject invention that allows for it, or a portion of the amount administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.
  • systemic administration refers to the administration of a subject composition, therapeutic o r o ther m aterial other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • parenteral administration and “administered parenterally” are art- recognized and refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articulare, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion.
  • hyperplasia is art recognized and refers to an increase in, or excessive growth of, the normal elements of any part. As one example, hyperplasia may refer to an abnormal increase in the number of cells in an organ.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups, hi preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer.
  • L ikewise, preferred cycloalkyls have from 3 -10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
  • aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • alkaryl refers to an aryl group substituted with an alkyl group; for example, (phenyl)methyl is an aralkyl moiety.
  • heteroaralkyl refers to an alkyl group substituted with a heteroaryl group; for example, (2-pyridyl)methyl is a heteroaralkyl moiety.
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • alkenyl refers to an alkenyl group substituted with an aryl group.
  • aryl as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, naphthalene, anthracene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, p yridine, p yrazine, p yridazine and pyrimidine, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or “heteroaromatics.”
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
  • ortho, meta scad para apply to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively.
  • 1,2-dimethylbenzene and ortAo-dimethylbenzene are synonymous.
  • heterocyclyl or “heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles.
  • Heterocyclyl groups include, for example, azetidine, azepine, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, pheno
  • the heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic o r h eteroaromatic m oiety, - CF3, - CN, o r t he like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,
  • polycyclyl or “polycyclic group” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • E ach ofthe rings o fthe polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • nitro means -NO2; the term “halogen” designates -F, -Cl, -Br or -I; the term “sulfhydryl” means -SH; the term “hydroxyl” means -OH; and the term “sulfonyl” means -SO2-.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:
  • R9, R1 Q and R'10 each independently represent a group pennitted by the rules of valence.
  • acylamino is art-recognized and refers to a moiety that can be represented by the general formula:
  • R 9 is as defined above, and R'j 1 represents a hydrogen, an alkyl, an alkenyl or -(CH2) m -R8 5 where m and Rg are as defined above.
  • amido is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula: wherein R9, R1 Q are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.
  • lactam is art-recognized and refers to a cyclic amide, and includes compounds represented by the general formula:
  • alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto, hi preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2) m -Rg, wherein m and Rg are defined above.
  • Representative alkylthio groups include methylthio, ethyl thio, and the like.
  • carbonyl is art recognized and includes such moieties as can be represented by the general formula:
  • X is a bond or represents an oxygen or a sulfur
  • R ⁇ ⁇ represents a hydrogen, an alkyl, an alkenyl, -(CH2) m -R or a pharmaceutically acceptable salt
  • R'n represents a hydrogen, an alkyl, an alkenyl or -(CH2)m-R8 > where m and Rg are as defined above.
  • X is an oxygen and R ⁇ ⁇ or R'I 1 is not hydrogen
  • the formula represents an "ester”.
  • X is an oxygen, and R is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when Ri j is a hydrogen, the formula represents a "carboxylic acid".
  • alkoxyl or "alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • An "ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O- alkenyl, -O-alkynyl, -O-(CH2) m - 8 > where m and Rg are described above.
  • Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, ?-toluenesulfonyl and methanesulfonyl, respectively.
  • a more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.
  • Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.
  • each expression e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
  • substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds, hi a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein above.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
  • protecting group means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
  • protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
  • the field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2 nd ed.; Wiley: New York, 1991).
  • Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis- and tr ⁇ s-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
  • a particular enantiomer of a compound of the present invention may be prepared by asymmetric synthesis, it may be isolated using chiral chromatography methods, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl
  • diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.
  • Contemplated equivalents of the compounds described above include compounds which o therwise c orrespond t hereto, a nd w hich h ave t he s ame general properties thereof (e.g., functioning as analgesics), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound in binding to opioid receptors, hi general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures, hi these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.
  • octahydromezerein is a tumor promoter, while mezerein itself acts as a non-promoting inflammatory agent.
  • 12-O-acetylphorbol 13-(2,4-decadienoate) has been shown to have practically no effect as a tumor promoter in contrast to its saturated counterpart, 12-O-acetylphorbol 13-decanoate.
  • terminal aryl and fluoroalkylaryl groups will serve to increase the ClogP, as well as to modify the association of the protein-ligand complex with the membrane.
  • linkage of the side chain to the benzolactam core by an amide bond was chosen for synthetic convenience.
  • the reaction produces exclusively the 8-nitro derivative 2 in an excellent yield of 92%, provided that the reaction is promptly terminated after completion. Prolonged exposure to the nitration reagent leads to a mixture of incompletely characterized products, with overnitration probably being the main process. Catalytic reduction of the nitro group over Pd C afforded the desired amino derivative 3 which was then reacted without purification with acyl chlorides 4a-f. The resulting amides were subjected to deacetylation under basic conditions to obtain the final products 5a-c, 5e, 5g, and 5i in 31-42% yield over the three steps.
  • Reagents and conditions (a) HNO 3 , Ac 2 O, 5 min, 0 °C, 92%; (b) Pd/C, H 2 , 3 h, rt; (c) RCOCl, Et 3 N, 4 h, rt; (d) Na 2 CO 3 , MeOH, 1.5 h, rt; (e) Pd/C, H 2 , 3 h, rt; 90%.
  • R -(CH 2 ) 12 -
  • the free acid 12 was coupled with 8 -amino benzolactam in the presence of 1 -[3 '-(dimethyl amino) propyl]-3-ethyl carbodiimide hydrochloride to give the monodentate benzolactam derivative 13.
  • Reagents and conditions (a) Methyl 9-(Chlorocarbonyl)nonanoate (Zayed, S.; Sorg, B.; Hecker, E., Planta Medica 1984, 4, 65-69), Et 3 N, THF, rt; (b) LiOH, MeOH, rt; (c) Ac 2 O, K 2 CO 3 , CH 2 C1 2 ; (d) 8-amino benzolactam, Et 3 N, rt; (e) Na 2 CO 3 , MeOH, rt.
  • Reagents and conditions (a) Diacid chloride, pyridine, rt; (b) 1,2-ethanedithiol (10 equiv.), BF 3 .Et 2 O (2 equiv.), CH 2 C1 2 , rt.
  • Ki values are mean ⁇ SEM for three independent experiments. Data taken from Qiao, L.; Zhao, L. Y.; Rong, S. B.; Wu, X. W.; Wang, S.; Fujii, T.; Karanietz, M. G.; Rauser, L.; Savage, J.; Roth, B. L.; Anderson, J. F.; Kozikowski, A. P., Bioorg. Med. Chem. Lett. 2001, 11, 955-959.
  • the nature ofthe benzolactam side chain has a substantial effect on the ability ofthe benzolactams to increase the formation of sAPP ⁇ . This is most clearly seen in comparing the decynyl-substituted benzolactam with the benzolactams 5c, e-g. Of particular note is the improved activity of 5f in comparison to 8-(l-decynyl)-benzolactam, although the ClogP of the former is one log unit lower while their Ki values are very similar. On the basis ofthe present modeling studies, it is believed that the side chain amide group of these new benzolactams is able to form an additional H-bonding interaction with serine residue 110 present in loop A.
  • C ompounds 5 e and 5f are relatively potent PKC ligands exhibiting binding affinities that are only 40 times poorer than that of PDBU (phorbol 12,13- dibutyrate).
  • PDBU phorbol 12,13- dibutyrate
  • PKC delta inserts into the plasma and nuclear membranes in response to the tumor promoting derivative 12-deoxyphorbol 13-tetradecanoate; in contrast, it goes to the nuclear membrane and punctate aggregates, but not the plasma membrane, after treatment with 12- deoxyphorbol 13 -acetate, a derivative which is inflammatory but which acts to inhibit tumor promotion.
  • the important influence of hydrophobicity in the translocation of PKC delta was similarly shown for a series of homologous, symmetrically substituted phorbol 12,13- diesters, where both the kinetics and ultimate position of translocation of PKC delta depended on the hydrophobicity.
  • Examples include: phorbol 12,13-dihexanoate versus phorbol 12,13-dihexa-2,4-dienoate; phorbol 12-tetradecanoate 13-acetate versus phorbol 12- tetradeca-2,4,6,8-tetraenoate 13-acetate; and 12-deoxyphorbol 13-decanoate versus 12- deoxyphorbol 13-deca-2,4-dienoate.
  • Such compounds could be used alone or in combination with ⁇ -secretase inhibitors to decrease the formation of neurotoxic ⁇ -amyloid peptide, thereby slowing the progression ofthe disease process.
  • the nature ofthe spacer and its length play an important role in the potencies ofthe compounds toward the isozymes PKC ⁇ and PKC ⁇ £ This is clearly seen from the K ⁇ values of 7a-7i (Table 2).
  • a graphical representation between the correlation of spacer length and binding affinity to PKC ⁇ is depicted in Figure 4. The activities of 7a and 7b are slightly reduced compared to the reference compound l. 20 Kozikowski, A. P.; Nowak, I.; Petukhov, P.
  • the K ⁇ value for binding to the Clb domain ofthe parent benzolactam (un-substituted) is 334.0 ⁇ 14.0 x 10 "9 M. Kozikowski, A. P.; Wang, S.; Ma, D.; Yao, J.; Ahmad, S.; Glazer, R.
  • the compound 7e was selected for testing with different isozymes of PKC, namely ⁇ , ⁇ l, ⁇ , ⁇ , and ⁇ (Table 4). Its affinity was almost equal for all of these isozymes, with a slightly higher affinity for PKCs- and lower affinity for PKC& Compounds 7f and 7g showed very high affinity toward the entire series and are comparable to PDBU (phorbol 12,13-dibutyrate).
  • PDBU phorbol 12,13-dibutyrate
  • the derivatives 7h and 7i in which the ohgomethylene chain is replaced by an oligoethylene glycol chain exhibit drastically reduced binding affinities (Table 2). This shows that the nature of the spacer is very important to achieve good binding to the PKCs.
  • hydrophobic moieties increase the ability of the compound to form a stable association with the membrane, which is known to be essential for inducing downregulation of PKC.
  • a hydrophobic side chain preferably 8- 10 carbon
  • Assaying processes are well known in the art in which a reagent is added to a sample, and measurements ofthe sample and reagent are made to identify sample attributes stimulated by the reagent.
  • one such assay process concerns determining in a chromogenic assay the amount of an enzyme present in a b iological s ample or solution. Such assays are based on the development of a colored product in the reaction solution. The reaction develops as the enzyme catalyzes the conversion of a colorless chromogenic substrate to a colored product.
  • Another assay useful in the present invention concerns determining the ability of a ligand to bind to a biological receptor utilizing a technique well known in the art referred to as a radioligand binding assay.
  • This assay accurately determines the specific binding of a radioligand to a targeted receptor through the delineation of its total and nonspecific binding components.
  • Total binding is defined as the amount of radioligand that remains following the rapid separation ofthe radioligand bound in a receptor preparation (cell homogenates or recombinate receptors) from that which is unbound.
  • the nonspecific binding component is defined as the amount of radioligand that remains following separation of the reaction mixture consisting of receptor, radioligand and an excess of unlabeled ligand. Under this condition, the only radioligand that remains represents that which is bound to components other that receptor.
  • the specific radioligand bound is determined by subtracting the nonspecific from total radioactivity bound. For a specific example of radioligand binding assay for ⁇ -opioid receptor, see Wang, J. B. et al. FEBS Letters 1994, 338, 217.
  • Assays useful in the present invention concern determining the activity of receptors the activation of which initiates subsequent intracellular events in which intracellular stores of calcium ions are released for use as a second messenger.
  • Activation of some G-protein- coupled receptors stimulates the formation of inositol triphosphate (IP3, a G-protein- coupled receptor second messenger) through phospholipase C-mediated hydrolysis of phosphatidylinositol, Berridge and Irvine (1984). Nature 312:315-21.
  • IP3 in turn stimulates the release of intracellular calcium ion stores.
  • G-protein-coupled receptor function A change in cytoplasmic calcium ion levels caused by release of calcium ions from intracellular stores is used to determine G-protein-coupled receptor function. This is another type of indirect assay.
  • G-protein-coupled receptors are muscarinic acetylcholine receptors (mAChR), adrenergic receptors, sigma receptors, serotonin receptors, dopamine receptors, angiotensin receptors, adenosine receptors, bradykinin receptors, metabotropic excitatory amino acid receptors and the like.
  • Cells expressing such G-protein-coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it maybe desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores.
  • Another type of indirect assay involves determining the activity of receptors which, when activated, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP, cGMP. For example, activation of some dopamine, serotonin, metabotropic glutamate receptors and muscarinic acetylcholine receptors results in a decrease in the cAMP or cGMP levels ofthe cytoplasm.
  • cyclic nucleotide-gated ion channels e.g., rod photoreceptor cell channels and olfactory neuron channels [see, Altenhofen, W. et al. (1991) Proc. Natl. Acad. Sci U.S.A. 88:9868-9872 and Dhallan et al. (1990) Nature 347:184-187] that are permeable to cations upon activation by binding of cAMP or cGMP.
  • a change in cytoplasmic ion levels caused by a change in the amount of cyclic nucleotide activation of photo-receptor or olfactory neuron channels is used to determine function of receptors that cause a change in cAMP or cGMP levels when activated, hi cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it may be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e .g., forskolin, prior to adding a receptor-activating compound to the cells in the assay.
  • agents that increase intracellular cyclic nucleotide levels e .g., forskolin
  • Cell for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-gated ion channel and a DNA encoding a receptor (e.g., certain metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors and the like, which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.
  • a receptor e.g., certain metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors and the like, which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.
  • Any cell expressing a receptor protein which is capable, upon activation, of directly increasing the intracellular concentration of calcium, such as by opening gated calcium channels, or indirectly affecting the concentration of intracellular calcium as by causing initiation of a reaction which utilizes Ca ⁇ 2+> as a second messenger (e.g., G-protein- coupled receptors), may form the b asis o f an assay.
  • C ells endogenously expressing such receptors or ion channels and cells which may be transfected with a suitable vector encoding one or more such cell surface proteins are known to those of skill in the art or may be identified by those of skill in the art.
  • HEK human embryonic kidney
  • CRL1573 Chinese hamster ovary (CHO) cells (ATCC Nos. CRL9618, CCL61, CRL9096), PC12 cells (ATCC No. CRL1721) and COS-7 cells (ATCC No. CRL1651).
  • Preferred cells for heterologous cell surface protein expression are those that can be readily and efficiently transfected.
  • Preferred cells include HEK 293 cells, such as those described in U.S. Pat. No. 5,024,939.
  • Any compound which is known to activate ion channels or receptors of interest may be used to initiate an assay. Choosing an appropriate ion channel- or receptor-activating reagent depending on the ion channel or receptor of interest is within the skill of the art.
  • Direct depolarization of the cell membrane to determine calcium channel activity may be accomplished by adding a potassium salt solution having a concentration of potassium ions such that the final concentration of potassium ions in the cell-containing well is in the range of about 50-150 mM (e.g., 50 mM KC1).
  • ligands are known which have affinity for and activate such receptors.
  • nicotinic acetyloholine receptors are known to be activated by nicotine or acetylcholine; similarly, muscarinic and acetylcholine receptors may be activated by addition of muscarine or carbamylcholine.
  • Agonist assays may be carried out on cells known to possess ion channels and/or receptors to determine what effect, if any, a compound has on activation or potentiation of ion channels or receptors of interest. Agonist assays also may be carried out using a reagent known to possess ion channel- or receptor-activating capacity to determine whether a cell expresses the respective functional ion channel or receptor of interest.
  • a functional receptor or ion channel with agonist typically activates a transient reaction; and prolonged exposure to an agonist may desensitize the receptor or ion channel to subsequent activation.
  • assays for determining ion channel or receptor function should be initiated by addition of agonist (i.e., in a reagent solution used to initiate the reaction).
  • the potency of a compound having agonist activity is determined by the detected change in some observable in the cells (typically an increase, although activation of certain receptors causes a decrease) as compared to the level ofthe observable in either the same cell, or substantially identical cell, which is treated substantially identically except that reagent lacking the agonist (i.e., control) is added to the well.
  • an agonist assay is performed to test whether or not a cell expresses the functional receptor or ion channel of interest
  • known agonist is added to test-cell-containing wells and to wells containing control cells (substantially identical cell that lacks the specific receptors or ion channels) and the levels of observable are compared.
  • cells lacking the ion channel and/or receptor of interest should exhibit substantially no increase in observable in response to the known agonist.
  • a substantially identical cell may be derived from the same cells from which recombinant cells are prepared but which have not been modified by introduction of heterologous DNA. Alternatively, it may be a cell in which the specific receptors or ion channels are removed.
  • any statistically or otherwise significant difference in the level of observable indicates that the test compound has in some manner altered the activity of the specific receptor or ion channel or that the test cell possesses the specific functional receptor or ion channel.
  • individual wells or duplicate wells, etc.
  • individual wells contain a distinct cell type, or distinct recombinant cell line expressing a homogeneous population of a receptor or ion channel of interest, so that the compound having unidentified activity may be screened to determine whether it possesses modulatory activity with respect to one or more of a variety of functional ion channels or receptors.
  • each of the individual wells may contain the same cell type so that multiple compounds (obtained from different reagent sources in the apparatus or contained within different wells) can be screened and compared for modulating activity with respect to one particular receptor or ion channel type.
  • Antagonist assays may be carried out by incubating cells having functional ion channels and/or receptors in the presence and absence of one or more compounds, added to the solution bathing the cells in the respective wells of the microtiter plate for an amount of time sufficient (to the extent that the compound has affinity for the ion channel and/or receptor of interest) for the compound(s) to bind to the receptors and/or ion channels, then activating the ion channels or receptors by addition of known agonist, and measuring the level of observable in the cells as compared to the level of observable in either the same cell, or substantially identical cell, in the absence of the putative antagonist.
  • assays are thus useful for rapidly screening compounds to identify those that modulate any receptor or ion channel in a cell, hi particular, assays can be used to test functional ligand-receptor or ligand-ion channel interactions for cell receptors including ligand-gated ion channels, voltage-gated ion channels, G-protein-coupled receptors and growth factor receptors.
  • assays may encompass measuring a detectable change of a solution as a consequence of a cellular event which allows a compound, capable of differential characteristics, to change its characteristics in response to the cellular event. By selecting a particular compound which is capable of differential characteristics upon the occurrence of a cellular event, various assays may be performed.
  • assays for determining the capacity of a compound to induce cell injury or cell death may be carried out by loading the cells with a pH-sensitive fluorescent indicator such as BCECF (Molecular Probes, hie, Eugene, Oreg. 97402, Catalog #B1150) and measuring cell injury or cell death as a function of changing fluorescence over time.
  • BCECF pH-sensitive fluorescent indicator
  • the function of receptors whose activation results in a change in the cyclic nucleotide levels of the cytoplasm may be directly determined in assays of cells that express such receptors and that have been injected with a fluorescent compound that changes fluorescence upon binding cAMP.
  • the fluorescent compound comprises cAMP-dependent-protein kinase in which the catalytic and regulatory subunits are each labelled with a different fluorescent-dye [Adams et al. (1991) Nature 349:694-697].
  • cAMP binds to the regulatory subunits, the fluorescence emission spectrum changes; this change can be used as an indication of a change in cAMP concentration.
  • the function of certain neurotransmitter transporters which are present at the synaptic cleft at the junction between two neurons may be determined by the development of fluorescence in the cytoplasm of such neurons when conjugates of an amine acid and fluorescent indicator (wherein the fluorescent indicator ofthe conjugate is an acetoxymethyl ester derivative e.g., 5-(aminoacetamido)fluorescein; Molecular Probes, C atalog #A1363) are transported by the neurotransmitter transporter into the cytoplasm of the cell where the ester group is cleaved by esterase activity and the conjugate becomes fluorescent.
  • the fluorescent indicator ofthe conjugate is an acetoxymethyl ester derivative e.g., 5-(aminoacetamido)fluorescein; Molecular Probes, C atalog #A1363
  • a reporter gene construct is inserted into an eukaryotic cell to produce a recombinant cell which has present on its surface a cell surface protein of a specific type.
  • the cell surface receptor may be endogenously expressed or it may be expressed from a heterologous gene that has been introduced into the cell.
  • Methods for introducing heterologous DNA into eukaryotic cells are-well known in the art and any such method may be used.
  • DNA encoding various cell surface proteins is known to those of skill in the art or it may be cloned by any method known to those of skill in the art.
  • the recombinant cell is contacted with a test compound and the level of reporter gene expression is measured.
  • the contacting may be effected in any vehicle and the testing may be by any means using any protocols, such as s erial dilution, for assessing specific molecular interactions known to those of skill in the art.
  • the level of gene expression is measured.
  • the amount of time to effect such interactions may be empirically determined, such as by running a time course and measuring the level of transcription as a function of time.
  • the amount of transcription may be measured using any method known to those of skill in the art to be suitable.
  • specific mRNA expression may be detected using Northern blots or specific protein product may be identified by a characteristic stain.
  • the amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test, compound or it may be compared with the amount of transcription in a substantially identical cell that lacks the specific receptors.
  • a substantially identical cell may be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Alternatively, it may be a cell in which the specific receptors are removed. Any statistically or otherwise significant difference in the amount of transcription indicates that the test compound has in some manner altered the activity ofthe specific receptor.
  • the assay may be repeated and modified by the introduction of a step in which the recombinant cell is first tested for the ability of a known agonist or activator of the specific receptor to activate transcription if the transcription is induced, the test compound is then assayed for its ability to inhibit, block or otherwise affect the activity ofthe agonist.
  • the transcription based assay is useful for identifying compounds that interact with any cell surface protein whose activity ultimately alters gene expression, hi particular, the assays can be used to test functional ligand-receptor or ligand-ion channel interactions for a number of categories of cell surface-localized receptors, including: ligand-gated ion channels and voltage-gated ion channels, and G protein-coupled receptors.
  • Any transfectable cell that can express the desired cell surface protein in a manner such the protein functions to intracellularly transduce an extracellular signal may be used.
  • the cells may be selected such that they endogenously express the cell surface protein or may be genetically engineered to do so. Many such cells are known to those of skill in the art.
  • Such cells include, but are not limited to Ltk ⁇ - > cells, PC12 cells and COS-7 cells.
  • the preparation of cells which express a receptor or ion channel and a reporter gene expression construct, and which are useful for testing compounds to assess their activities, is exemplified in the Examples provided herewith by reference to mammalian Ltk ⁇ - > and COS-7 cell lines, which express the Type I human muscarinic (HM1) receptor and which are transformed with either a c-fos promoter-CAT reporter gene expression construct or a c- fos promoter-luciferase reporter gene expression construct.
  • HM1 human muscarinic
  • the cell surface protein may be endogenously expressed on the selected cell or it may be expressed from cloned DNA.
  • Exemplary cell surface proteins include, but are not limited to, cell surface receptors and ion channels.
  • Cell surface receptors include, but are not limited to, muscarinic receptors (e.g.,, human M2 (GenBank accession #M16404); rat M3 (GenBank accession #M16407); human M4 (GenBank accession #M16405); human M5 (Bonner et al.
  • Neuronal nicotinic acetylcholine receptors e.g., the alpha 2, alpha 3 and beta 2 subtypes disclosed in U.S. Ser. No. 504,455 (filed Apr. 3, 1990), hereby expressly incorporated by reference herein in its entirety
  • the rat alpha 2 subunit Wanga et al. (1988) Science 240:330-334
  • the rat alpha 3 subunit Bodet al. (1986) Nature 319:368-374
  • the rat alpha 4 subunit Goldman et al.
  • GABA receptors e.g., the bovine alpha 1 and beta 1 subunits (Schofield et al. (1987) Nature 328:221-227); the bovine alpha 2 and alpha 3 subunits (Levitan et al. (1988) Nature 335:76-79); the gamma - subunit (Pritchett et al. (1989) Nature 338:582-585); the beta 2 and beta 3 subunits (Ymer et alo (1989) EMBO J. 8:1665-1670); the delta subunit (Shivers, B.D.
  • GABA receptors e.g., the bovine alpha 1 and beta 1 subunits (Schofield et al. (1987) Nature 328:221-227); the bovine alpha 2 and alpha 3 subunits (Levitan et al. (1988) Nature 335:76-79); the gamma - subunit (Pritchett et al. (1989) Nature 3
  • glutamate receptors e.g., receptor isolated from rat brain (Holhnann et al. (1989) Nature 342:643-648); and the like
  • adrenergic receptors e.g., human beta 1 (Frielle et al. (1987) Proc. Natl. Acad. Sci. 84.:7920-7924); human alpha 2 (Kobilka et al. (1987) Science 238:650-656); hamster beta 2 (Dixon et al. (1986) Nature 321:75-79); and the like
  • dopamine receptors e.g., human D2 (Stormann et al.
  • NGF receptors e.g., human NGF receptors (Johnson et al. (1986) Cell 47:545-554); and the like
  • serotonin receptors e.g., human 5HTla (Kobilka et al. (1987) Nature 329:75-79); rat 5HT2 (Julius et al. (1990) PNAS 87:928-932); rat 5HTlc (Julius et al. (1988) Science 241:558-564); and the like).
  • Reporter gene constructs are prepared by operatively linking a reporter gene with at least one transcriptional regulatory element. If only one transcriptional regulatory element is included it must be a regulatable promoter. At least one of the selected transcriptional regulatory elements must be indirectly or directly regulated by the activity of the selected cell-surface receptor whereby activity of the receptor can be monitored via transcription of the reporter genes.
  • the construct may contain additional transcriptional regulatory elements, such as a
  • FIRE sequence or other sequence, that is not necessarily regulated by the cell surface protein, but is selected for its ability to reduce background level transcription or to amplify the transduced signal and to thereby increase the sensitivity and reliability ofthe assay.
  • reporter genes and transcriptional regulatory elements are known to those of skill in the art and others may be identified or synthesized by methods known to those of skill in the art.
  • a reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein.
  • Preferred reporter genes are those that are readily detectable.
  • the reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties.
  • reporter genes include, but are not limited to CAT
  • Transcriptional control elements include, but are not limited to, promoters, enhancers, and repressor and activator binding sites.
  • Suitable transcriptional regulatory elements may be derived from the transcriptional regulatory regions of genes whose expression is rapidly induced, generally within minutes, of contact between the cell surface protein and the effector protein that modulates the activity of the cell surface protein. Examples of such genes include, but are not limited to, the immediate early genes (see, Sheng et al. (1990) Neuron 4: 477-485), such as c-fos.
  • Immediate early genes are genes that are rapidly induced upon binding of a ligand to a cell surface protein.
  • the transcriptional control elements that are preferred for use in the gene constructs include transcriptional control elements from immediate early genes, elements derived from other genes that exhibit some or all ofthe characteristics ofthe immediate early genes, or synthetic elements that are constructed such that genes in operative linkage therewith exhibit such characteristics.
  • the characteristics of preferred genes from which the transcriptional control elements are derived include, but are not limited to, low or undetectable expression in quiescent cells, rapid induction at the transcriptional level within minutes of extracellular simulation, induction that is transient and independent of new protein synthesis, subsequent shut-off of transcription requires new protein synthesis, and mRNAs transcribed from these genes have a short half-life. It is not necessary for all of these properties to be present.
  • compositions which comprise a therapeutically effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarect
  • terapéuticaally-effective amount means that amount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing acid (e.g., lubricant, talc magnesium, calcium stearate, zinc stearate, or stearic acid) or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • manufacturing acid e.g., lubricant, talc magnesium, calcium stearate, zinc stearate, or stearic acid
  • solvent encapsulating material involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
  • certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids.
  • pharmaceutically-acceptable salts refers to the relatively non-toxic, inorganic and organic a cid a ddition s alts o f c ompounds o f the p resent i nvention.
  • T hese salts can be prepared in situ in the admimstration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound ofthe invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.
  • sulfate bisulfate
  • phosphate nitrate
  • acetate valerate
  • oleate palmitate
  • stearate laurate
  • benzoate lactate
  • phosphate tosylate
  • citrate maleate
  • fumarate succinate
  • tartrate naphthate
  • mesylate glucoheptonate
  • lactobionate lactobionate
  • laurylsulphonate salts and the like See, for example,
  • the pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids.
  • such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
  • pharmaceutically-acceptable s alts refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention.
  • salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the p urified c ompound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge e
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention.
  • an aforementioned formulation renders orally bioavailable a compound ofthe present invention.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • a compound ofthe present invention may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrohdone, sucrose and/or acacia; (3) huniectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption
  • pharmaceutically-acceptable carriers such as sodium citrate or dicalcium phosphate
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture ofthe powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions ofthe present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more ofthe above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs, hi addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetiahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing o ne o r m ore compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
  • dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may c ontain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood ofthe intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, hi addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature ofthe particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • the preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the compounds of the present invention which may be used in a suitable hydrated form, and or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels ofthe active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of admimstration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of admimstration, the time of administration, the rate of excretion or metabolism ofthe particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable daily dose of a compound ofthe invention will be that amount ofthe compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated desired effects will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the preferred dosing is daily.
  • compositions which comprise a therapeutically-effective amount of one or more of the subject compounds, as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin, lungs, o r o ral c avity; or (4) intravaginally or intravectally, for example, as a pessary, cream or foam; (5) sublingually;
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue
  • parenteral administration for example, by subcutaneous, intramuscular
  • the compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals .
  • treatment is intended to encompass also prophylaxis, therapy and cure.
  • the p atient receiving this treatment i s any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
  • the compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides.
  • Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered.
  • the addition of the active compound of the invention to animal feed is preferably accomplished by preparing an appropriate feed premix containing the active compound in an effective amount and incorporating the premix into the complete ration.
  • an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed.
  • feed premixes and complete rations are described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A., 1969 or
  • a combinatorial library for the purposes ofthe present invention is a mixture of chemically related compounds which may be screened together for a desired property; said libraries may be in solution or covalently linked to a solid support.
  • the preparation of many related compounds in a single reaction greatly reduces and simplifies the number of screening processes which need to be carried out. Screening for the appropriate biological, pharmaceutical, agrochemical or physical property may be done by conventional methods.
  • the substrate aryl groups used in a combinatorial approach can be diverse in terms of the core aryl moiety, e.g., a variegation in terms of the ring structure, and or can be varied with respect to the other substituents.
  • a library of substituted diversomers can be synthesized using the subject reactions adapted to the techniques described in the Still et al. PCT publication WO 94/08051, e.g., being linked to a polymer bead by a hydrolyzable or photolyzable group, e.g., located at one of the positions of substrate.
  • a hydrolyzable or photolyzable group e.g., located at one of the positions of substrate.
  • the library is synthesized on a set of beads, each bead including a set of tags identifying the particular diversomer on that bead, hi one embodiment, which is particularly suitable for discovering enzyme inhibitors, the beads can be dispersed on the surface of a permeable membrane, and the diversomers released from the beads by lysis of the bead linker. The diversomer from each bead will diffuse across the membrane to an assay zone, where it will interact with an enzyme assay. Detailed descriptions of a number of combinatorial methodologies are provided below.
  • MS mass spectrometry
  • a compound selected from a combinatorial library can be irradiated in a MALDI step in order to release the diversomer from the matrix, and ionize the diversomer for MS analysis.
  • the libraries of the subject method can take the multipin library format.
  • Geysen and co-workers (Geysen et al. (1984) PNAS 81 :3998-4002) introduced a method for generating compound libraries by a parallel synthesis on polyacrylic acid-grated polyethylene pins arrayed in the microtitre plate format.
  • the Geysen technique can be used to synthesize and screen thousands of compounds per week using the multipin method, and the tethered compounds may be reused in many assays.
  • Appropriate linker moieties can also be appended to the pins so that the compounds may be cleaved from the supports after synthesis for assessment of purity and further evaluation (c.f, Bray et al. (1990) Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem 197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166V
  • a variegated library of compounds can be provided on a set of beads utilizing the strategy of divide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135; and U.S. Patents 4,631,211; 5,440,016; 5,480,971).
  • the beads are divided into separate groups equal to the number of different substituents to be added at a particular position in the library, the different substituents coupled in separate reactions, and the beads recombined into one pool for the next iteration.
  • the divide-couple-recombine strategy can be carried out using an analogous approach to the so-called "tea bag” method first developed by Houghten, where compound synthesis occurs on resin sealed inside porous polypropylene bags (Houghten et al. (1986) PNAS 82:5131-5135). Substituents are coupled to the compound- bearing resins by placing the bags in appropriate reaction solutions, while all common steps such as resin washing and deprotection are performed simultaneously in one reaction vessel. At the end ofthe synthesis, each bag contains a single compound.
  • a scheme of combinatorial synthesis in which the identity of a compound is given by its locations on a synthesis substrate is termed a spatially-addressable synthesis.
  • the combinatorial process is carried out by controlling the addition of a chemical reagent to specific locations on a solid support (Dower et al. (1991) Annu Rep Med Chem 26:271-280; Fodor, S.P.A. (1991) Science 251:767; Pirrung et al. (1992) U.S. Patent No. 5,143,854; Jacobs et al. (1994) Trends Biotechnol 12:19-26).
  • the spatial resolution of photolithography affords miniaturization. This technique can be carried out through the use of protection/deprotection reactions with photolabile protecting groups.
  • a synthesis substrate is prepared for coupling through the covalent attachment of photolabile nitroveratryloxycarbonyl (NVOC) protected amino linkers or other photolabile linkers.
  • Light is used to selectively activate a specified region of the synthesis support for coupling. Removal of the photolabile protecting groups by light (deprotection) results in activation of selected areas. After activation, the first of a set of amino acid analogs, each bearing a photolabile protecting group on the amino terminus, is exposed to the entire surface. Coupling only occurs in regions that were addressed by light in the preceding step.
  • the reaction is stopped, the plates washed, and the substrate is again illuminated through a second mask, activating a different region for reaction with a second protected building block.
  • the pattern of masks and the sequence of reactants define the products and their locations. Since this process utilizes photolithography techniques, the number of compounds that can be synthesized is limited only by the number of synthesis sites that can be addressed with appropriate resolution. The position of each compound is precisely known; hence, its interactions with other molecules can be directly assessed. hi a light-directed chemical synthesis, the products depend on the pattern of illumination and on the order of addition of reactants. By varying the lithographic patterns, many different sets of test compounds can be synthesized simultaneously; this characteristic leads to the generation of many different masking strategies.
  • the subject method utilizes a compound library provided with an encoded tagging system.
  • a recent improvement in the identification of active compounds from combinatorial libraries employs chemical indexing systems using tags that uniquely encode the reaction steps a given bead has undergone and, by inference, the structure it carries.
  • this approach mimics phage display libraries, where activity derives from expressed peptides, but the structures of the active peptides are deduced from the corresponding genomic DNA sequence.
  • the first encoding of synthetic combinatorial libraries employed DNA as the code.
  • a variety of other forms of encoding have been reported, including encoding with sequenceable bio-oligomers (e.g., oligonucleotides and peptides), and binary encoding with additional non-sequenceable tags.
  • a combinatorial library of nominally 7 7 ( 823,543) peptides composed of all combinations of Arg, Gin, Phe, Lys, Val, D-Val and Thr (three-letter amino acid code), each of which was encoded by a specific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively), was prepared by a series of alternating rounds of peptide and ohgonucleotide synthesis on solid support, hi this work, the amine linking functionality on the bead was specifically differentiated toward peptide or ohgonucleotide synthesis by simultaneously preincubating the beads with reagents that generate protected OH groups for ohgonucleotide synthesis and protected NH2 groups for peptide synthesis (here, in a ratio of 1:20).
  • the tags each consisted of 69-mers, 14 units of which carried the code.
  • the bead-bound library was incubated with a fluorescently labeled antibody, and beads containing bound antibody that fluoresced strongly were harvested by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • the DNA tags were amplified by PCR and sequenced, and the predicted peptides were synthesized.
  • compound libraries can be derived for use in the subject method, where the ohgonucleotide sequence of the tag identifies the sequential combinatorial reactions that a particular bead underwent, and therefore provides the identity ofthe compound on the bead.
  • ohgonucleotide tags permits extremelyly sensitive tag analysis. Even so, the method requires careful choice of orthogonal sets of protecting groups required for alternating co-synthesis of the tag and the library member. Furthermore, the chemical lability of the tag, particularly the phosphate and sugar anomeric linkages, may limit the choice of reagents and conditions that can be employed for the synthesis of non-oligomeric libraries.
  • the libraries employ linkers permitting selective detachment of a library member for assaying. Peptides have also been employed as tagging molecules for combinatorial libraries. Two exemplary approaches are described in the art, both of which employ branched linkers to solid phase upon which coding and ligand strands are alternately elaborated.
  • orthogonality in synthesis is achieved by employing acid-labile protection for the coding strand and base- labile protection for the compound strand.
  • branched linkers are employed so that the coding unit and the test compound can both be attached to the same functional group on the resin, hi one embodiment, a cleavable linker can be placed between the branch point and the bead so that cleavage releases a molecule containing both code and the compound (Ptek et al. (1991) Tetrahedron Lett 32:3891-3894). In another embodiment, the cleavable linker can be placed so that the test compound can be selectively separated from the bead, leaving the code behind. This last construct is particularly valuable because it permits screening of the test compound without potential interference of the coding groups. Examples in the art of independent cleavage and sequencing of peptide library members and their corresponding tags has confirmed that the tags can accurately predict the peptide structure.
  • An alternative form of encoding the test compound library employs a set of non- sequencable electrophoric tagging molecules that are used as a binary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926).
  • Exemplary tags are haloaromatic alkyl ethers that are detectable as their trimethylsilyl ethers at less than femtomolar levels by electron capture gas chromatography (ECGC).
  • the compound would be attached to the solid support via the photocleavable linker and the tag is attached through a catechol ether linker via carbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem 59:4723-4724).
  • This orthogonal attachment strategy permits the selective detachment of library members for assays in solution and subsequent decoding by ECGC after oxidative detachment ofthe tag sets.
  • amide-linked libraries in the art employ binary encoding with the electrophoric tags attached to amine groups, attaching these tags directly to the bead matrix provides far greater versatility in the structures that can be prepared in encoded combinatorial libraries.
  • Successive photoelution also permits a very high throughput iterative screening strategy: first, multiple beads are placed in 96-well microtiter plates; second, compounds are partially detached and transferred to assay plates; third, a metal binding assay identifies the active wells; fourth, the corresponding beads are rearrayed singly into new microtiter plates; fifth, single active compounds are identified; and sixth, the structures are decoded.
  • any compositions ofthe present invention will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the subject composition. Any of the subject formulations may be administered in a single dose or in divided doses. Dosages for the compositions of the present invention may be readily determined by techniques known to those of skill in the art or as taught herein.
  • the dosage of the subject compounds will generally be in the range of about 0.01 ng to about 10 g per kg body weight, specifically in the range of about 1 ng to about 0.1 g per kg, and more specifically in the range of about 100 ng to about 10 mg per kg.
  • An effective dose or amount, and any possible affects on the timing of administration of the formulation may need to be identified for any particular composition ofthe present invention. This may be accomplished by routine experiment as described herein, using one or more groups of animals (preferably at least 5 animals per group), or in human trials if appropriate.
  • the effectiveness of any subject composition and method of treatment or prevention may be assessed by administering the composition and assessing the effect ofthe administration by measuring one or more applicable indices, and comparing the post-treatment values of these indices to the values ofthe same indices prior to treatment.
  • the precise time of admimstration and amount of any particular subject composition that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a subject composition, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like.
  • the guidelines presented herein may be used to optimize the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.
  • the health of the patient may be monitored by measuring one or more of the relevant indices at predetermined times during the treatment period.
  • Treatment including composition, amounts, times of administration and formulation, m ay b e o ptimized a ccording t o t he r esults of such monitoring.
  • the patient may be periodically reevaluated to determine the extent of improvement by measuring the same parameters. Adjustments to the amount(s) of subject composition administered and possibly to the time of administration may be made based on these reevaluations.
  • Treatment may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is attained.
  • compositions may reduce the required dosage for any individual agent contained in the compositions because the onset and duration of effect of the different agents maybe complimentary.
  • Toxicity and therapeutic efficacy of subject compositions may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50.
  • the data obtained from the cell culture assays and animal studies may be used in formulating a range of dosage for use in humans.
  • the dosage of any subject composition lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose may be estimated initially from cell culture assays.
  • kits for conveniently and effectively implementing the methods of this invention comprise any subject composition, and a means for facilitating compliance with methods of this invention. Such kits provide a convenient and effective means for assuring that the subject to be treated takes the appropriate active in the correct dosage in the correct manner.
  • the compliance means of such kits includes any means which facilitates administering the actives according to a method of this invention. Such compliance means include instructions, packaging, and dispensing means, and combinations thereof. Kit components may be packaged for either manual or partially or wholly automated practice ofthe foregoing methods, h other embodiments involving kits, this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use.
  • a well characterized cell line from an AD patient was obtained from the Coriell Cell Repository (Ca den, NJ), then cultured and maintained as described, except that the plastic material was a 6 cm diameter petri dish. Ibarreta, D.; Duchen, M.; Ma, D.; Qiao, L.; Kozikowski, A. P.; Etcheberrigaray, R., NeuroReport 1999, 10, 1035-1040. Cells were typically grown to confluence, which took approximately 4-5 days. Cells were maintained in a complete medium until 2 h prior to treatment. At that point, the medium was replaced by DMEM without serum and left undisturbed for 2 h.
  • the cells were treated with 0.1 or 1 ⁇ M of the compounds to be tested.
  • 8-(l- Decynyl)benzolactam V (BL) and DMSO were used as positive and negative controls, respectively.
  • the concentration of DMSO was maintained at less than 1% in all cases.
  • the medium was collected after 3 h to measure the sAPP- ⁇ secretion.
  • the concentration of secreted sAPP ⁇ was measured using conventional immunoblotting techniques, following with minor modifications the protocol described elsewhere. Dumbar, G. S. Protein Blotting - A Practical Approach. J-RL Press: Oxford, 1994; Ibarreta, D.; Duchen, M.; Ma, D.; Qiao, L.; Kozikowski, A.
  • Analytical Techniques Analytical and preparative thin-layer chromatography (TLC) was performed in a solvent-vapor-saturated chamber on EM Science silica gel 60 F-254 plates.
  • Example 1 (2S,5S)-0-Acetyl-8-nitrobenzoIactam V (2).
  • To this solution was added dropwise over 5 min a chilled mixture of Ac2 ⁇ (1.2 mL) and 70% HNO3 (268 mg, 3.11 mmol). The reaction was allowed to proceed for 4-5 min at 0 °C and then was stopped by adding water (50 mL) in one portion to the vigorously stirred solution.
  • the nitro compound 8 (100 mg, 0.287 mmol) was dissolved in ethanol (15 mL), and 10% Pd-C (15 mg, 0.014 mmol) was added to the reaction mixture. The mixture was vigorously stirred under a H atmosphere overnight, then filtered, and the filtrate was concentrated to give a residue, which was dried under vacuum for 1 h at 60 °C. To a solution of the residue in anhydrous tetrahydrofuran (4 mL) under nitrogen was added anhydrous triethylamine (0.5 mL) and diacid chloride (0.143 mmol). The reaction mixture was stirred at room temperature for 4 h, then filtered and concentrated.
  • Preparative TLC (10/1 ethyl acetate/ethanol as developing solvent) provided the acetyl-protected diamide, which was dissolved in ethanol (30 mL). A solution of sodium carbonate (60 mg) in water (6 mL) was added with vigorous stirring. After 1.5 h at room temperature, the solution was concentrated and an additional amount of water (20 mL) added. Extraction with ethyl acetate (4 x 30 mL) provided the diamide with free hydroxyl groups which was purified by preparative TLC (10/1 ethyl acetate/ethanol as developing solvent).
  • Example 17 Decanedioic Acid Bis-[N-(2S,5S)-l,2,3,4,5,6-hexahydro-5-(hydroxymethyl)-2- isopropyl-l-methyl-3-oxo-benzo[e][l,4]diazocin-8-yl]amide (7c). Yield 38%.
  • Example 23 3,6,9,12,15,18-Hexaoxadocosane-l,20-dioic A cid B is-[N-(2S,5S)-l,2,3,4,5,6-hexahydro- 5-(hydroxymethyl)-2-isopropyl-l-methyl-3-oxo-benzo[e][l,4]diazocin-8-yl]amide (7i).
  • Example 27 Decanedioic Acid Bis-[4-[(6R, 7R, 7aS)-5,6,7,7a-tetrahydro-(6-isopropyI-3,3-dimethyl- 5-oxo-lZT-pyrrolo[l,2-c]oxazol-7-yl)]-l-naphthyl] ester (lie).
  • Example 28 Dodecanedioic Acid Bis-[4-[(6R, 7R, 7aS)-5,6,7,7a-tetrahydro-(6-isopropyI-3,3- dimethyI-5-oxo-127-pyrrolo[l-2- ⁇ xazol-7-yl)]-l-naphthyl] ester (lid).
  • Example 41 9-[N-(4-Hydroxy-l-naphthyl)carbamoyl]nonanoic Acid iV-[(2S,5S)-l,2,3,4,5-6- hexahydro-5-(hydroxymethyl)-2-isopropyl-l-methyl-3-oxobenzo[e][l,4]diazocin-8-yI]- amide (13).
  • the nitro compound 8 50 mg, 0.144 mmol
  • 10% Pd-C 7.5 mg, 0.007 mmol

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Abstract

One aspect of the present invention relates to amide-bearing substituted benzolactams, and individual stereoisomers thereof. A second aspect of the present invention relates to combinatorial libraries of the aforementioned amide-bearing substituted benzolactams, or individual stereoisomers thereof. Another aspect of the present invention relates to compositions, comprising an amide-bearing substituted benzolactam,and a pharmaceutically acceptable excipient. The present invention also encompasses methods of modulating the activity of PKC or enhancing the secretion of sAPPα or inducing hyperplasia in a mammal. Further, the present invention relates to methods of treating a mammal suffering from neuronal dysfunction, cognitive decline, neurodegeneration, stroke, Alzheimer’s disease, Parkinson’s disease, diabetes, heart disease, or cancer.

Description

PROTEIN KINASE C MODULATORS, AND METHODS OF USE THEREOF
Background of the Invention
Neuronal dysfunction includes cognitive decline, which is characterized by concentration loss, memory-acquisition loss, and information-storage or retrieval loss. Neuronal dysfunction can result from central nervous system ("CNS") injury, such as stroke, spinal-cord injury, and peripheral-nerve injury. Cognitive decline is symptomatic of neuronal disorders, such as cognitive decline associated with aging and minimal cognitive impairment (also known as minimal cognitive disorder) as well as severe neurodegenerative disorders, such as Alzheimer's disease. Neuronal dysfunction is also associated with disorders that result in loss of motor skills, such as Parkinson's disease and amyotrophic lateral sclerosis.
Approximately 5-15% of the population of the United States over age 65 (1.24 million) has Alzheimer's disease. This disease is the most frequent cause of institutionalization for long-term care, hi 1983, more than $27 billion was spent in the U.S. in health care for Alzheimer's afflicted individuals. Six basic research areas into Alzheimer's disease have b een defined by R. J. Wurtman, Scientific A merican 1985, 62. These areas include faulty genes, accumulations of amyloid protein, infectious agents, environmental toxins (e g., aluminum and certain unusual amino acids), inadequate blood flow and energy metabolism, and cholinergic deficits. A number of possible therapeutic interventions for treatment of Alzheimer's disease are currently under study. These include the use of nerve growth factors (NGF), muscarinic and nicotinic agonists, acetylcholinesterase (AChE) inhibitors, GABA-inverse agonists, NMDA modulators, and others. The protein kinases serve a regulatory function which is crucial for all aspects of cellular development, differentiation and transformation. One of the largest gene families of non-receptor serine-threonine protein kinases is protein kinase C (PKC). Since the discovery of PKC more than a decade ago by Nishizuka and coworkers, and its identification as a major receptor for phorbol esters, a multitude of physiological signaling mechanisms have been ascribed to this enzyme. Kikkawa et al., J. Biol. Chem., 1982, 257, 13341; Ashendel et al., Cancer Res., 1983, 43, 4333. Importantly, protein kinase C represents a family of at least 12 serine/threonine kinases that are involved in signal transduction in response to a host of hormonal, neuronal, and growth factor stimuli. Newton, A. C, J. Biol. Chem. 1995, 270, 28495-28498; Quest, A. F. G., Enzyme Protein 1996, 49, 231-261; Mellor, H.; Parker, P. J., Biochem. J. 1998, 332, 281-292; Ron, D.; Kazanietz, M. G., FASEB J. 2000, 13, 1658-1676. Differences in structure as well as substrate requirements have led to the general classification of the isoforms into three groups, termed the classical, novel and atypical PKCs. The N-terminal regulatory domains of the classical and novel PKCs contain a conserved Cl domain comprised of two cysteine-rich zinc fingers that bind to the natural PKC activator diacylglycerol (DAG) as well as to certain natural products, such as the phorbol esters and the i ndolactams. N ormally, the c lassical and n ovel P KC i sozymes are c ytosolic and are translocated to the membrane upon interaction with Ca2+, whereby they are activated through interaction with membrane phospholipids and DAG. Newton, A. C; Keranen, L. M., Biochemistry 1994, 33, 661-6658; Newton, A. C, Curr. Opin. Cell. Biol. 1997, 9, 161- 167. PKC has been shown to play a major role in a variety o f disease states including diabetes, heart disease, c aiicer, and Alzheimer's disease, and thus b oth i sofonn s elective activators and inhibitors may provide novel leads in the development of therapeutic agents. Bryostatin, a macrocyclic lactone from the bryozoan Bugula n eritina, binds to the DAG regulatory site of PKC, yet is not a tumor promoter; rather, it acts as an antineoplastic agent that has been used to treat murine melanoma. Hennings, H.; Hennings, H.; Blumberg P. M.; Pettit, G. R.; Herald, C. L.; Shores, R.; Yuspa, S. H., Carcinogenesis 1987, 8, 1343- 1346. hi view of the fact that bryostatin-1 is in clinical evaluation as an anticancer agent, it is likely that other non-tumor-promoting activators of PKC will find use in treating neoplastic conditions. There has been a growing interest in studying the relationship between Alzheimer's disease (AD) and PKC. Numerous researchers have found d efective P KC in brains and peripheral tissues of AD patients. Cole, G.; Dobkins, K. R.; Hansen, L. A.; Terry, R. D.; Saitoh, T., Brain Res. 1988, 452, 165-174; Masliah, E.; Cole, G.; Shimohada, S.; Hansen, L.; DeTeresa, R; Terry, R. D.; Saitoh, T., J. Neurosci. 1990, 10, 2113-2124; Shimohama, S.; Narita, M. A.; Matsushima, H.; Kimura, J.; Kameyama, M.; Hagiwara, M.; Hidaka, H.; Taniguchi, T.; Neurology 1993, 43, 1407-1413; Wang, H.-Y.; Pisano, M. R.; Friedman, E., Neurobiology of Aging 1994, 15, 293-298; Masliah, R; Cole, G. M.; Hansen, L. A.; Mallory, M.; Albright, T.; Terry, R. D.; Saitoh, T., J. Neurosci. 1991, 11, 2759-2767; Chachin, M.; Shimohama, S.; Kunugi, Y.; Taniguchi, T., Jpn. J. Pharmacol. 1996, 71, 75- 177; Huynh, T. V.; Cole, G.; Katzman, R.; Huang, K.-P.; Saitoh, T., Arch. Neurol 1989, 46, 1195-1199; Govoni, S.; Bergamaschi, S.; Racchi, M.; Battaini, F.; Binetti, G.; Bianchetti, A., Neurology 1993, 43, 258-2586; Racchi, M.; Wetsel, W. C; Trabucchi, M.; Govoni, S.; Battaini, F.; Binetti, G.; Bianchetti, A.; Bergamaschi, S., Neurology 1994, 44, A164; Govoni, S.; Racchi, M.; Bergamaschi, M.; Trabucchi, M.; Battaini, F.; Bianchetti, A.; Binetti, G., Ann. N. Y. Acad. Set 1996, 777, 332-337. PKC is known to participate in the processing of the amyloid precursor protein (APP), including the amyloidogenic fragments Aβ 1-40 and 1-42. Gandy, S.; Greengard, P., Int. Rev. Neurobiol. 1994, 36, 29- 50. There are three well-characterized proteolytic routes of amyloid processing, all of which seem to occur in normal and pathological states. -Secretase, a metalloprotease neither completely identified nor characterized, cleaves APP within the Aβ sequence (between residues K and L), generating a large secreted fragment termed sAPPα and a smaller intracellular fragment P3; both fragments are of no pathological significance. Buxbaum, j. D.; Liu, K.-N.; Luo, Y.; Slack, j. L.; Stocking, K. L.; Peschon, j. j.; Johnson, R. S.; Castner, B. J.; Ceretti, D. P.; Black, R, J. Biol. Chem. 1998, 273, 27766-27767. The process leading to Aβ formation or "amyloidogenic" processing and ultimately Aβ deposition and plaque formation in AD, involves the participation of another enzyme, β- secretase. Cai, H.; Wang, Y.; McCarthy, D.; Wen, H.; Borchelt, D. R; Price, D. L.; Wong, P.C., Nature Neuroscience 2001, 4, 233-234; Vassar, R.; Citron, M., Neuron 2000, 27, 419- 422. This enzyme cleaves just outside the Aβ sequence, leaving a large membrane-bound fragment that is later cleaved by a yet to be identified γ-secretase at positions 711 and 713, thereby generating Aβ 1-40 and 1-42, respectively. Wolfe, M. S.; Haass, C, J. Biol. Chem. 2001, 276, 5413-5416. In depth description and discussion of APP processing can be found elsewhere. Selkoe, D. J., Nature 1999, 399, A23-A31; Wiltfang, ; Esselmann, H.; Maler, j. M.; Bleich, S.; Huther, G.; Kornhuber, j., Gerontology 2001, 47, 65-71; Selkoe, D. j., Physiol. Rev. 2001, 81, 741-766; Coughlan, CM; Breen, K.C., Pharmacol. Ther. 2000, 86, 111-144.
PKC activators belonging to the phorbol family have been shown to dramatically enhance α-secretase activity, thus leading to enhanced secretion of sAPP- . Gandy, S.; Greengard, P., Int. Rev. Neurobiol. 1994, 36, 29-50; Desdouits, F.; Buxbaum, J. D.; Desdouits-Magnen, J.; Nairn, A. C; Greengard, P. A., J. Biol. Chem. 1996, 271, 24670- 24674; Efthimiopoulos, S.; Puηj, S.; Manopoulos, V.; Pangalos, M.; Wang, G. P.; Refolo, L. M.; Robakis, N. K, J. Neurochem. 1996, 67, 872-875; Kinouchi, T.; Sorimachi, H.; Maruyama, K.; Mizuno, K.; Ohno, S.; Ishiura, S.; Suzuki, K., FEBS Lett. 1995, 364, 203- 206; Jolly-Tomietta, C; Wolf, B. A., Biochemistry 2000, 39, 7428-7435; Yeon, S. W.; Jung, M. W.; Ha, M. J.; Kim, S. U.; Huh, K.; Savage, M. J.; Masliah, E.; Mook-Joung, I., Biochem. Biophys. Res Commun. 2001, 280, 782-787; RoBner, S.; Mendla, K; Schliebs, R.; Bigl, V., Eur. J. Neurosci. 2001, 13, 1644-1648. Because the secretases seem to compete for a single pool of APP, enhancing α-secretase activity would result in a decrease of amyloidogenic products of β-secretase. Vassar, R.; Bennet, B. D.; Babu-Khan, S.; Khan, S.; Mendiaz, E. A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.; Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M. A; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J. C; Collins, F.; Treanor, J.; Rogers, G.; Citron, M., Science 1999, 286, 735-741; Skovronsky, D. M.; Moore, D. B.; Milla, M. E.; Doms, R. W.; Lee, V. M. L., J. Biol. Chem. 2000, 275, 2568-2575. hi fact, direct decrease of Aβ 1-40 has been documented after PKC activation. Desdouits, F.; Buxbaum, J. D.; Desdouits-Magnen, J.; Nairn, A. C; Greengard, P., J. Biol. Chem. 1996, 271, 24670-24674; Efthimiopoulos, S.; Punj, S.; Manopoulos, V.; Pangalos, M.; Wang, G. P.; Refolo, L. M.; Robakis, N. K., J. Neurochem. 1996, 67, 872-875.
A few other studies have failed to show a direct relationship between enhanced sAPPα and decreased Aβ 1-40. However, they still show that PKC activation has
"positive" effects as reflected in an increase in the non-pathogenic sAPPα or a decrease in β-amyloid. Fuller, S. J.; Storey, E.; Li, Q.-X.; Smith, A. I.; Beyreuther, K.; Masters, C. L.,
Biochemistry 1995, 34, 8091-8098; LeBlanc, A. C; Koutroumanis, M.; Goodyer, C. G., J.
Neurosci. 1998, 18, 2909-2913; Savage, M. J.; Trusko, S. P.; Howland, D. S.; Pinsker, L.
P.; Mistretta, S.; Reaume, A. G.; Greenberg, B. D.; Siman, R.; Scott, R. W., J. Neurosci.
1998, 18, 1743-1752. 8-(l-Decynyl)benzolactam has been shown to restore an otherwise abnormal K channel activity in fϊbroblasts from AD patients, linked to enhanced translocation of PKCα. Kozikowski, A. P.; Wang, S.; Ma, D.; Yao, J.; Ahmad, S.; Glazer, R. I.; Bogi, K.; Acs, P.; Modarres, S.; Lewin, N. E.; Blumberg, P. M., J. Med. Chem. 1997, 40, 1316-1326; Etcheberrigaray, R; Ito, E.; Kim, C. S.; Alkon, D. L., Science 1994, 264, 276-279; Bhagavan, S.; Ibarreta, D.; Ma, D.; Kozikowski, A. P.; Etcheberrigaray, R., Neurobiology of Disease 1998, 5, 177-187. Further, the same compound was later shown to significantly enhance sAPPα secretion. Ibarreta, D.; Duchen, M.; Ma, D.; Qiao, L.; Kozikowski, A. P.; Etcheberrigaray, R, NeuroReport 1999, 10, 1035-1.
Summary of the Invention i certain embodiments, the present invention relates to a compound represented by
A:
Figure imgf000006_0001
wherein
X represents NR5, O or S;
Y represents O or S;
R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl, acylaminoalkyl, or sulfonylaminoalkyl; R3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
R4 is absent or present from 1 to 3 times inclusive; R4, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylO2C-, heteroarylO2C-, aralkylO2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in A is R, S, or a mixture thereof.
hi certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR5.
In certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein Y represents O.
In certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl.
In certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein R1 represents alkyl or cycloalkyl. i certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl. In certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein R3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl.
In certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein R4 is absent.
In certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein R5 is alkyl. hi certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR5; and Y represents O. In certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR5; Y represents O; and R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl. hi certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; and R1 represents alkyl or cycloalkyl. hi certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; and R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl. i certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl. hi certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; R3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl; and R4 is absent. hi certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; R3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl; R4 is absent; and R5 is alkyl.
In certain embodiments, a compound of the present invention is represented by A and the attendant definitions, wherein X represents NR5; Y represents O; R represents
1 0 independently for each occurrence H; R represents isopropyl; R represents hydroxylmethyl; R represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl; R4 is absent; and R5 is methyl.
In an assay based on mammalian PKCα, certain compounds according to structure A have an IC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM. hi an assay based on mammalian PKCα, certain compounds according to structure A have an EC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM.
i certain embodiments, the present invention relates to a compound represented by B:
Figure imgf000010_0001
B wherein
X represents NR5, O or S;
Y represents O or S; R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, <. alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylaminoalkyl, arylaminoalkyl, acylaminoalkyl, or sulfonylaminoalkyl;
R3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl; R4 is absent or present from 1 to 3 times inclusive;
R4, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylO2C-, heteroarylO2C-, aralkylO2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in B is R, S, or a mixture thereof.
In an assay based on mammalian PKCα, certain compounds according to structure B have an IC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM. h an assay based on mammalian PKCα, certain compounds according to structure B have an EC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM.
i certain embodiments, the present invention relates to a compound represented by
Figure imgf000011_0001
wherein
X represents NR5, O or S; Y represents O or S; R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylaminoalkyl;
R3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
R4 is absent or present from 1 to 3 times inclusive;
R4, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylO2C-, heteroarylO2C-, aralkylO2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl; R5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in C is R, S, or a mixture thereof.
i an assay based on mammalian PKCα, certain compounds according to structure C have an IC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM.
i an assay based on mammalian PKCα, certain compounds according to structure C have an EC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM. In certain embodiments, the present invention relates to a compound represented by
D:
Figure imgf000013_0001
D wherein X represents NR5, O or S;
Y represents O or S;
R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylaminoalkyl;
R3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
R4 is absent or present from 1 to 3 times inclusive;
R4, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylO2C-, heteroarylO2C-, aralkylO2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in D is R, S, or a mixture thereof. an assay based on mammalian PKCα, certain compounds according to structure D have an IC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM. hi an assay based on mammalian PKCα, certain compounds according to structure D have an EC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM.
In certain embodiments, the present invention relates to a compound represented by
E:
Figure imgf000014_0001
E wherein
L represents NR4, O, or S;
W represents a divalent alkyl, alkenyl, alkynyl or glycol chain;
X represents NR4, O or S;
Y represents O or S; R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylarninoalkyl;
R3 is absent or present from 1 to 3 times inclusive;
R3, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylOaC-, heteroarylO2C-, aralkylO2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R4 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in E is R, S, or a mixture thereof.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein L represents NR4. h certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W is a divalent alkyl or glycol chain. In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein X represents NR4.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein Y represents O. i certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl. h certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein R1 represents alkyl or cycloalkyl.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein R2 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl. i certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein R3 is absent.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein R4 is H or alkyl. i certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; and Y represents O.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; and R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl. i certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; and R1 represents alkyl or cycloalkyl.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; and R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R3 is absent.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; R represents independently for each
1 9 occurrence H, alkyl, cycloalkyl, or aralkyl; R represents alkyl or cycloalkyl; R represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; R is absent; and R4 is H or alkyl.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH2)4-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R3 is absent. hi certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH2)6-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R is absent. hi certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH2)8-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R3 is absent. h certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH2)1o-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R3 is absent.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH2)12-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R3 is absent. hi certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH2)14-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R3 is absent.
In certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents -(CH2)2Q-; X represents NMe; L represents NH; Y represents O; R represents H; R represents isopropyl; R represents hydroxylmethyl; and R3 is absent. h certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents -CH2-(OCH2CH2)4O-CH2-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl;
9 -a
R represents hydroxylmethyl; and R is absent. hi certain embodiments, a compound of the present invention is represented by E and the attendant definitions, wherein W represents -CH2-(OCH2CH2)5O-CH2-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl;
9 "
R represents hydroxylmethyl; and R is absent. hi an assay based on mammalian PKCα, certain compounds according to structure E have an IC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM. i an assay based on mammalian PKCα, certain compounds according to structure E have an EC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM.
In certain embodiments, the present invention relates to a compound represented by
F:
Figure imgf000019_0001
F wherein
W represents a divalent alkyl, alkenyl, alkynyl or glycol chain; X represents independently for each occurrence NR, O or S;
R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents independently for each occurrence hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylaminoalkyl;
R2 is absent or present from 1 to 6 times inclusive;
R2, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylO2C-, heteroarylO2C-, aralkylθ2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R3 represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in F is R, S, or a mixture thereof.
i certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain. i certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein X represents NR. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
In certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein R is absent. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein R represents alkyl. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; and R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; and R2 is absent. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; and R3 represents alkyl. i certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or
9 ■ sulfonyloxylalkyl; and R is absent. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R3 represents alkyl.
In certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents a divalent alkyl or glycol chain; X represents NR; R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or
9 sulfonyloxylalkyl; R is absent; and R represents alkyl. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH2)4-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH2)6-; X represents NH; R1
9 represents hydroxylmethyl; R is absent; and R represents isopropyl. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH2)8-; X represents NH; R1
9 ^ represents hydroxylmethyl; R is absent; and R represents isopropyl. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH2)10-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl. In certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH2)12-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
In certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CH2)14-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl. hi certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents -(CT^o X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
In certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents -CH2-(OCH2CH2) O-CH2-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
In certain embodiments, a compound of the present invention is represented by F and the attendant definitions, wherein W represents -CH2-(OCH2CH2)5θ-CH2-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl. h an assay based on mammalian PKCα, certain compounds according to structure F have an IC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM. hi an assay based on mammalian PKCα, certain compounds according to structure F have an EC50 value less than 1 μM, preferably less than 100 nM, and more preferably less than 10 nM.
In certain embodiments, the present invention relates to a compound represented by any of the structures outlined above, wherein said compound is a single stereoisomer. In certain embodiments, the present invention relates to a composition, comprising a compound represented by any of the structures outlined above; and a pharmaceutically acceptable excipient. hi certain embodiments, the present invention relates to compounds that modulate the activity of protein kinase C in a mammal, wherein the compounds are represented by any of the structures outlined above, and any of the sets of definitions associated with one of those structures, hi certain embodiments, the compounds of the present invention are antagonists or agonists of protein kinase C in a mammal, hi any event, the compounds of the present invention preferably exert their effect on protein kinase C in a mammal at a concentration less than about 1 micromolar, preferably at a concentration less than about 100 nanomolar, and more preferably at a concentration less than 10 nanomolar.
The present invention contemplates pharmaceutical formulations comprising a compound of the present invention. In certain embodiments, the pharmaceutical formulations will comprise a compound of the present invention that selectively effects a particular isoform of protein kinase C, and thereby has a therapeutic effect on an acute or chronic ailment, disease or malady that is at least in part due to biochemical or physiological processes associated with that isoform of protein kinase C. For example, the Background of the Invention (see above) teaches examples o f acute or chronic ailments, diseases or maladies that are caused or exacerbated by biochemical or physiological processes associated with various isoforms of protein kinase C. One of ordinary skill in the art will be able to accumulate, by reference to the scientific literature, a more comprehensive list of acute or chronic ailments, diseases or maladies that are caused or exacerbated by biochemical or physiological processes associated with various isoforms of protein kinase C. The present invention contemplates that pharmaceutical formulations comprising a compound of the present invention will be of medicinal value against the aforementioned acute or chronic ailments, diseases or maladies. certain embodiments, the present invention relates to a method of modulating the activity of protein kinase C in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention. hi certain embodiments, the present invention relates to a method of increasing secretion of sAPPα in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention. i certain embodiments, the present invention relates to methods of treating a mammal suffering from neuronal dysfunction, cognitive decline, neurodegeneration, stroke, Alzheimer's disease, Parkinson's disease, diabetes, heart disease, or cancer, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention. hi certain embodiments, the present invention relates to methods of treating a mammal suffering from Alzheimer's disease, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention.
In certain embodiments, the present invention relates to a method of inducing hyperplasia in a mammal, comprising administering to said mammal a therapeutically effective amount of a compound of the present invention.
The methods of the invention can be prophylactic, therapeutic, or curative. When the methods are practiced prior to an individual showing any clinical sign or symptom of a disease or disorder, they are considered prophylactic. Prophylactic treating can be practiced, for example, on individuals suspected of having a disease or disorder, or on individuals suspected of being at high risk of developing a disease or disorder, i embodiments, prophylactic methods reduce or eliminate the risk of developing a disease or disorder characterized by neuronal dysfunction, hi embodiments, prophylactic methods reduce or eliminate the risk of developing a disease or disorder characterized by cognitive decline. In embodiments, prophylactic methods reduce or eliminate the risk of developing a disease or disorder characterized by aberrant activity of protein kinase C. hi embodiments, prophylactic methods reduce or eliminate the risk of developing Alzheimer's disease. When the methods of treating are practiced on an individual already showing at least one clinical sign or symptom of a disease or disorder, the methods can be therapeutic or curative. Therapeutic methods are those methods that result in a detectable change in at least one symptom of the disease or disorder. Preferably, the detectable change is an improvement in the symptom. In embodiments, therapeutic methods reduce or eliminate neuronal dysfunction, hi embodiments, therapeutic methods reduce or eliminate cognitive decline. In embodiments, therapeutic methods reduce or eliminate a disease or disorder characterized by aberrant activity of protein kinase C. hi embodiments, therapeutic methods reduce or eliminate Alzheimer's disease.
Curative methods are those therapeutic methods that result in elimination of at least one symptom of a disease or disorder. Preferably, curative methods eliminate the cause of the disease or disorder. In embodiments, curative methods eliminate neuronal dysfunction, hi embodiments, curative methods eliminate cognitive decline. In embodiments, curative methods eliminate a disease or disorder characterized by aberrant activity of protein kinase C. In embodiments, curative methods eliminate Alzheimer's disease. The methods of treating can include a single administration to an individual, or can include multiple administrations. Treatment and dosing regimens can be designed and implemented in accordance with those that are well-known and widely practiced in the art. It is contemplated that each regimen will be tailored to the individual to be treated and the disease(s), disorder(s), and/or symptom(s) involved. However, such individual tailoring is well within the skill of those in the art and does not involve undue or excessive experimentation. In another aspect of the present invention, the compounds or compositions of the present invention may be used in the manufacture of a medicament to treat any of the foregoing protein kinase C related conditions or diseases. In certain embodiments, the present invention is directed to a method for formulating compounds of the present invention in a pharmaceutically acceptable carrier or excipient.
The present invention also provides kits containing a compound of the present invention. In embodiments, a compound of the present invention is provided in the kit as the sole component of the kit. In embodiments, it is present as part of a composition. In embodiments, i t i s p rovided in combination with other compounds, solutions, or devices necessary or desirable for use of the compounds and/or compositions contained therein. Thus, the kits of the invention can contain all the necessary compounds, solutions, and equipment for administration of the compounds and compositions contained therein to an individual, or the kits can be designed for in vitro use of a compound of the present invention. Brief Description of the Figures
Figure 1 depicts the secretion of sAPPα as determined by densitometry analyses of immunoblots.
Figure 2 depicts representative immunoblotting blots (A and B) showing the signal obtained with various compounds used at 1 and 0.1 μM. The two panels represent distinct experiments and blots obtained by conventional immunoblotting techniques to detect secreted sAPPα.
Figure 3 depicts hydrogen bonding interactions between PKCα Clb domain and ligands BL (A) and generic 5 (B).
Figure 4 depicts the correlation of binding affinities of 7a-7g with the spacer length in A. Detailed Description of the Invention
Definitions
For convenience, certain terms employed in the specification, examples, and appended claims are collected here. The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.
The term "cis" is art-recognized and refers to the arrangement of two atoms or groups around a double bond such that the atoms or groups are on the same side of the double bond. Cis configurations are often labeled as (Z) configurations. The term "trans" is art-recognized and refers to the arrangement of two atoms or groups around a double bond such that the atoms or groups are on the opposite sides of a double bond. Trans configurations are often labeled as (E) configurations.
The terms "combinatorial library" or "library" are art-recognized and refer to a plurality of compounds, which may be termed "members," synthesized or otherwise prepared from one or more starting materials by employing either the same or different reactants or reaction conditions at each reaction in the library. There are a number of other terms of relevance to combinatorial libraries (as well as other technologies). The term "identifier tag" is art-recognized and refers to a means for recording a step in a series of reactions used in the synthesis of a chemical library. The term "immobilized" is art- recognized and, when used with respect to a species, refers to a condition in which the species is attached to a surface with an attractive force stronger than attractive forces that are present in the intended environment of use of the surface, and that act on the species. The term "solid support" is art-recognized and refers to a material which is an insoluble matrix, and may (optionally) have a rigid or semi-rigid surface. The term "linker" is art- recognized and refers to a molecule or group of molecules connecting a support, including a solid support or polymeric support, and a combinatorial library member. The term "polymeric support" is art-recognized and refers to a soluble or insoluble polymer to which a chemical moiety can be covalently bonded by reaction with a functional group of the polymeric support. The term "functional group of a polymeric support" is art-recognized and refers to a chemical moiety of a polymeric support that can react with an chemical moiety to form a polymer-supported amino ester.
The term "synthetic" is art-recognized and refers to production by in vitro chemical or enzymatic synthesis. The term "meso compound" is art-recognized and refers to a chemical compound which has at least two chiral centers but is achiral due to a plane or point of symmetry.
The term "chiral" is art-recognized and refers to molecules which have the property of non-superimposability of the mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner. A "prochiral molecule" is a molecule which has the potential to be converted to a chiral molecule in a particular process.
The term "stereoisomers" is art-recognized and refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups i n s pace. I n p articular, " enantiomers" r efer t o t wo s tereoisomers o f a compound which are non-superimposable mirror images of one another. "Diastereomers", on the other hand, refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.
Furthermore, a "stereoselective process" is one which produces a particular stereoisomer of a reaction product in preference to other possible stereoisomers of that product. An "enantioselective process" is one which favors production of one of the two possible enantiomers of a reaction product.
The term "regioisomers" is art-recognized and refers to compounds which have the same molecular formula but differ in the connectivity of the atoms. Accordingly, a "regioselective process" is one which favors the production of a particular regioisomer over others, e.g., the reaction produces a statistically significant increase in the yield of a certain regioisomer.
The term "epimers" is art-recognized and refers to molecules with identical chemical constitution and containing more than one stereocenter, but which differ in configuration at only one of these stereocenters.
The term "ED50" is art-recognized. In certain embodiments, ED50 means the dose of a drug which produces 50% of its maximum response or effect, or alternatively, the dose which produces a pre-determined response in 50% of test subjects or preparations. The term "LD50" is art-recognized, hi certain embodiments, LD50 means the dose of a drug which is lethal in 50% of test subjects. The term "therapeutic index" is an art-recognized tenn which refers to the therapeutic index of a drug, defined as LD50/ED50.
The term "ligand" refers to a compound that binds at the receptor site.
The terms "active agent", "pharmacologically active agent" and "drug" are used interchangeably herein to refer to a chemical compound that induces a desired pharmacological, physiological effect. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs, and the like. When the terms "active agent", "pharmacologically active agent" and "drug" are used, or when a particular drug, such as oxycodone, is identified, it is to be understood as including the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
The term "structure-activity relationship" or "(SAR)" is art-recognized and refers to the way in which altering the molecular structure of a drug or other compound alters its interaction with a receptor, enzyme, nucleic acid or other target and the like.
The term "agonist" is art-recognized and refers to a compound that mimics the action of natural transmitter or, when the natural transmitter is not known, causes changes at the receptor complex in the absence of other receptor ligands. The term "antagonist" is art-recognized and refers to a compound that binds to a receptor site, but does not cause any physiological changes unless another receptor ligand is present.
The term "competitive antagonist" i s art-recognized and refers to a compound or that binds to a receptor site; its effects may be overcome by increased concentration of the agonist.
The term "partial agonist" is art-recognized and refers to a compound or that binds to a receptor site but does not produce the maximal effect regardless of its concentration.
A "target" shall mean a site t o which t argeted c onstructs b ind. A t arget m ay b e either in vivo or in vitro. In certain embodiments, a target may be a tumor (e.g., tumors of the brain, lung (small cell and non-small cell), ovary, prostate, breast and colon as well as other carcinomas and sarcomas), hi other embodiments, a target may be a site of infection (e.g., by bacteria, viruses (e.g., HIV, herpes, hepatitis) and pathogenic fungi (Candida sp.). h still other embodiments, a target may refer to a molecular structure to which a targeting moiety binds, such as a hapten, epitope, receptor, dsDNA fragment, carbohydrate or enzyme. Additionally, a target may be a type of tissue, e.g., neuronal tissue, intestinal tissue, pancreatic tissue etc.
The term "targeting moiety" refers to any molecular structure which assists the construct in localizing to a particular target area, entering a target cell(s), and/or binding to a target receptor. For example, lipids (including cationic, neutral, and steroidal lipids, virosomes, and liposomes), antibodies, lectins, ligands, sugars, steroids, hormones, nutrients, and proteins may serve as targeting moieties.
The term "modulation" is art-recognized and refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart.
The term "treating" is art-recognized and refers to curing as well as ameliorating at least one symptom of any condition or disease.
The term "prophylactic" or "therapeutic" treatment is art-recognized and refers to administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom). A "patient," "subject" or "host" to be treated by the subject method may mean either a human or non-human animal.
The term "mammal" is known in the art, and exemplary mammals include humans, primates, bovines, porcines, canines, felines, equines and rodents (e.g., mice and rats).
The term "bioavailable" is art-recognized and refers to a form of the subject invention that allows for it, or a portion of the amount administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.
The terms "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" are art-recognized and refer to the administration of a subject composition, therapeutic o r o ther m aterial other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
The terms "parenteral administration" and "administered parenterally" are art- recognized and refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articulare, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion.
The term "hyperplasia" is art recognized and refers to an increase in, or excessive growth of, the normal elements of any part. As one example, hyperplasia may refer to an abnormal increase in the number of cells in an organ.
The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium. The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups, hi preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), and more preferably 20 or fewer. L ikewise, preferred cycloalkyls have from 3 -10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group). The term "alkaryl", as used herein, refers to an aryl group substituted with an alkyl group; for example, (phenyl)methyl is an aralkyl moiety.
The term "heteroaralkyl", as used herein, refers to an alkyl group substituted with a heteroaryl group; for example, (2-pyridyl)methyl is a heteroaralkyl moiety.
The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
The term "aralkenyl", as used herein, refers to an alkenyl group substituted with an aryl group.
The term "aryl" as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, naphthalene, anthracene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, p yridine, p yrazine, p yridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
The terms ortho, meta scad para apply to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ortAo-dimethylbenzene are synonymous.
The terms "heterocyclyl" or "heterocyclic group" refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, azetidine, azepine, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic o r h eteroaromatic m oiety, - CF3, - CN, o r t he like.
The terms "polycyclyl" or "polycyclic group" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. E ach ofthe rings o fthe polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like. The term "carbocycle", as used herein, refers to an aromatic or non-aromatic ring in which each atom ofthe ring is carbon.
As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -Cl, -Br or -I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO2-.
The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:
Figure imgf000033_0001
wherein R9, R1 Q and R'10 each independently represent a group pennitted by the rules of valence. The tenn "acylamino" is art-recognized and refers to a moiety that can be represented by the general formula:
Figure imgf000033_0002
wherein R9 is as defined above, and R'j 1 represents a hydrogen, an alkyl, an alkenyl or -(CH2)m-R85 where m and Rg are as defined above.
The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:
Figure imgf000034_0001
wherein R9, R1 Q are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.
The term "lactam" is art-recognized and refers to a cyclic amide, and includes compounds represented by the general formula:
Figure imgf000034_0002
wherein R is as defined above.
The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto, hi preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2)m-Rg, wherein m and Rg are defined above. Representative alkylthio groups include methylthio, ethyl thio, and the like.
The term "carbonyl" is art recognized and includes such moieties as can be represented by the general formula:
Figure imgf000034_0003
wherein X is a bond or represents an oxygen or a sulfur, and R\ \ represents a hydrogen, an alkyl, an alkenyl, -(CH2)m-R or a pharmaceutically acceptable salt, R'n represents a hydrogen, an alkyl, an alkenyl or -(CH2)m-R8> where m and Rg are as defined above. Where X is an oxygen and R\ \ or R'I 1 is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when Ri j is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen, and R'1 j is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where X is a sulfur and Rj ι or R'i \ is not hydrogen, the formula represents a "thiolester." Where X is a sulfur and Ri \ is hydrogen, the formula represents a "thiolcarboxylic acid." Where X is a sulfur and R'i \ is hydrogen, the formula represents a "thiolformate." On the other hand, where X is a bond, and Rj \ is not hydrogen, the above formula represents a "ketone" group. Where X is a bond, and R is hydrogen, the above formula represents an "aldehyde" group.
The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O- alkenyl, -O-alkynyl, -O-(CH2)m- 8> where m and Rg are described above.
The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, ?-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.
Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.
As used herein, the definition of each expression, e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds, hi a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
The phrase "protecting group" as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).
Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trα s-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention. If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, it may be isolated using chiral chromatography methods, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.
Contemplated equivalents of the compounds described above include compounds which o therwise c orrespond t hereto, a nd w hich h ave t he s ame general properties thereof (e.g., functioning as analgesics), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound in binding to opioid receptors, hi general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures, hi these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.
For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table ofthe Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.
Design and Synthesis
An important role has been ascribed to the nature of the side chains present in certain PKC activators, because unsaturation in these side chains tends to increase inflammatory activity while decreasing tumor promoting activity. For example, octahydromezerein is a tumor promoter, while mezerein itself acts as a non-promoting inflammatory agent. Sharkey, N. A.; Hennings, H.; Yuspa, S. H.; Blumberg, P. M., Carcinogenesis 1989, 10, 1937-1941. Likewise, 12-O-acetylphorbol 13-(2,4-decadienoate) has been shown to have practically no effect as a tumor promoter in contrast to its saturated counterpart, 12-O-acetylphorbol 13-decanoate. von Fϋrstenberger, G.; Hecker, E., Planta Medica 1972, 22, 241-266. Guided by these observations, comparison of the properties of benzolactam derivatives having unsaturated and saturated side chains of the same length was studied. Aliphatic side chains, both trans, trans-die c and saturated, linked to the benzolactam scaffold by means of an amide bond and terminating with a methyl, phenyl, p- trifluorophenyl, bis(3,5-trifluoromethyl)phenyl, and τ (3,3,4,4,5,5,6,6,7,7,8,8,8- tridecafluoro(oct-l-enyl)phenyl or j9-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro(octanyl)phenyl group were attached. The terminal aryl and fluoroalkylaryl groups will serve to increase the ClogP, as well as to modify the association of the protein-ligand complex with the membrane. Hiyama, T. Ed., Springer-Verlag: Berlin, 2000; pp 137-183. hi preferred embodiments, linkage of the side chain to the benzolactam core by an amide bond was chosen for synthetic convenience.
The synthesis of these 8-substituted b enzolactam derivatives c ontaining an amide moiety commences from the unsubstituted benzolactam, the synthesis of which has previously been published. Kozikowski, A. P.; Wang, S.; Ma, D.; Yao, J.; Ahmad, S.; Glazer, R. I.; Bogi, K.; Acs, P.; Modarres, S.; Lewin, N. E.; Blumberg, P. M., J. Med. Chem. 1997, 40, 1316-1326. After O-acetylation, the benzolactam 1 can be nitrated under mild conditions with a solution of nitric acid in acetic anhydride. The reaction produces exclusively the 8-nitro derivative 2 in an excellent yield of 92%, provided that the reaction is promptly terminated after completion. Prolonged exposure to the nitration reagent leads to a mixture of incompletely characterized products, with overnitration probably being the main process. Catalytic reduction of the nitro group over Pd C afforded the desired amino derivative 3 which was then reacted without purification with acyl chlorides 4a-f. The resulting amides were subjected to deacetylation under basic conditions to obtain the final products 5a-c, 5e, 5g, and 5i in 31-42% yield over the three steps. Catalytic reduction of the unsaturated side chain appendages of 5c, 5e, 5g, and 5i over Pd/C provided the saturated analogues 5d, 5f, 5h, and 5j. The synthesis of the required trifluoromethylated acid chlorides was carried out using a modification of Huang's method, involving the reaction between methoxycarbonylallylidenetriphenylarsorane generated in situ and the requisite benzaldehyde. Huang, Y.; Shen, Y.; Zheng, J.; Zhang, S., Synthesis 1985, 57-58. Subsequent base hydrolysis of the ester and reaction with thionyl chloride provided the requisite acylating agent. For the synthesis of 5i, the required acid chloride was prepared by coupling of the E,E-isomer of methyl 5-(4-iodophenyl)-2,4-pentadienoate and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-l-octene in the presence of Pd(OAc)2, NaHCO3, n- Bu4NHSO4 in DMF under nitrogen, followed by base hydrolysis and acid chloride formation. Dorses, S.; Pucheault, M.; Genet, J. P., Eur. J. Org. Chem. 2001, 1121-1128. See Scheme 1. Scheme 1. Synthetic scheme for certain compounds ofthe invention.
Figure imgf000039_0001
5a-c, 5e, 5g, and 5i
4, RC(O)Cl
Figure imgf000039_0002
4c: R = ph^^^55^1"
Figure imgf000039_0003
4e: R =
3,5-(CF3)2Ph'
Figure imgf000039_0004
Reagents and conditions: (a) HNO3, Ac2O, 5 min, 0 °C, 92%; (b) Pd/C, H2, 3 h, rt; (c) RCOCl, Et3N, 4 h, rt; (d) Na2CO3, MeOH, 1.5 h, rt; (e) Pd/C, H2, 3 h, rt; 90%.
Success with the results of the above compounds led to the synthesis of dimers of benzolactams with oligomethylene tethers of lengths differing from n = 4 to n = 20. Once the optimum chain length was established, in order to lower the ClogP values of the derivatives, modifying the spacer by substituting the saturated chain with a p olyethylene glycol chain of similar length was explored. In order to assess whether the realized binding affinity was due to the bidentate nature ofthe molecule, a monodentate molecule 13 with a benzolactam unit at one end of the chain and a 4-hydroxy naphthyl unit (which does not bind to PKC) at the other end was synthesized.
The synthesis of the diamides 7a-i (Scheme 2) started with the 8-nitro benzolactam acetate (8), the synthesis of which has been previously published. Kozikowski, A. P. Nowak, L; Petukhov, P. A.; Etcheberrigaray, R.; Mohamed, A.; Tan, M.; Levin, N. Hennings, H.; Pearce, L. L.; Blumberg, P. M., J Med. Chem. 2003, 46, 364-373; Zhang, G. Kazanietz, M. G.; Blumberg, P. M.; Hurley, J. H., Cell 1995, 81, 917-924. Catalytic reduction of the nitro group with Pd/C gave the intermediate 8-amino benzolactam, which was utilized without purification for further reaction with the diacid chlorides. The diamides formed were then subjected to deacetylation with potassium carbonate in ethanol to form the required products in 18-38% yield over three steps. The saturated diacid chlorides were prepared by treatment of the respective diacids with an excess of thionyl chloride or oxalyl chloride. Wittmann, V.; Takayama, S.; Gong, K. W.; Schmidt, G. W.; Wong, C. H., J Org. Chem. 1998, 63, 5137-5143.
Scheme 2. Synthetic scheme for certain compounds ofthe invention.
Figure imgf000040_0001
7a: R = -(CH2)4- 7f: R = -(CH2)14-
7b: R = -(CH2)6- 7g: R = -(CH2)20-
7c: R = -(CH2)8- 7h: R = -(CH2)-(OCH2CH2)4O-CH2-
7d: R = -(CH2)10- 7i: R = -CH2-(OCH2CH2)5O-CH2-
7e: R = -(CH2)12-
Reagents and conditions: (a)Pd/C, H2, MeOH, rt; (b) Et3N, diacid chloride, rt; (c) Na CO3,
MeOH, rt. The monodentate derivative 13 of benzolactam was synthesized as shown in Scheme 3. The monomethyl ester of sebacic acid was refluxed with oxalyl chloride to obtain the acid chloride, which was reacted with 4-hydroxy-l-naphthylamine hydrochloride in the presence of triethylamine t o g ive t he a mide. T he m ethyl e ster w as t hen h ydrolyzed w ith lithium hydroxide and the resulting product was treated with acetic anhydride in the presence of one equivalent of potassium carbonate to selectively protect the phenolic hydroxyl as the acetate. The free acid 12 was coupled with 8 -amino benzolactam in the presence of 1 -[3 '-(dimethyl amino) propyl]-3-ethyl carbodiimide hydrochloride to give the monodentate benzolactam derivative 13.
Scheme 3. Synthetic scheme for certain compounds ofthe invention.
Figure imgf000041_0001
Reagents and conditions: (a) Methyl 9-(Chlorocarbonyl)nonanoate (Zayed, S.; Sorg, B.; Hecker, E., Planta Medica 1984, 4, 65-69), Et3N, THF, rt; (b) LiOH, MeOH, rt; (c) Ac2O, K2CO3, CH2C12; (d) 8-amino benzolactam, Et3N, rt; (e) Na2CO3, MeOH, rt.
The synthesis of the dimers of naphthylpyrrolidone 9 -g was accomplished by the esterification of the parent phenols 10 with the diacid chlorides. Qiao, L.; Wang, S.; George C; Lewin N. E.; Blumberg, P. M.; Kozikowski, A. P., J Am. Chem. Soc. 1998, 120, 6629-6630. The esters 11 were subsequently subjected to acetonide cleavage by treatment with 1,2-ethanedithiol and boron triflouride-diethyl ether to give the required dimers (Scheme 4). Scheme 4. Synthetic scheme for certain compounds ofthe invention.
Figure imgf000042_0001
Figure imgf000042_0002
9a-g
9a: R = -(CH2)2- 9e: R = -(CH2)ιo-
9b: R = -(CH2)4- 9f: R = -(CH2)i2-
9c: R = -(CH2)6- 9 : R = -(CH2)ι8-
9d: R= -(CH2)8-
Reagents and conditions: (a) Diacid chloride, pyridine, rt; (b) 1,2-ethanedithiol (10 equiv.), BF3.Et2O (2 equiv.), CH2C12, rt.
Bindins Studies
The interaction of the benzolactams 5a-5j with PKC was assessed by determining their ability to displace bound [20-3H]phorbol-12,13-dibutyrate (PDBU) from recombinant PKCα in the presence of phosphatidylserine. The partition coefficients (ClogP) were calculated according to the fragment-based program KOWWIN 1.63. Meylan, W. M.; Howard, P. H., J. Pharm. Sci. 1995, 84, 83. The results are presented in Table 1. The K; values of all compounds are in the nanomolar range, and the calculated log P values are in the range of 2-11. Dienic ligands and their saturated counterparts generally display comparable binding affinities, except in the case of 5a and 5b.
Figure imgf000043_0001
Table 1. ClogP and K; Values of BL and Amides 5a-5j.
Compound Appendage R = ClogP K;
BL 6.0 14.7 ± 1.3
5a 2.0 1967 ± 8.0
Figure imgf000043_0002
Figure imgf000044_0001
The interaction of all dimers with PKC was appraised by evaluating their ability to displace [20-3H] phorbol 12,13-dibutyrate (PDBU) binding from recombinant bovine PKCα, murine PKC<5and the Clb domain of murine PKC£ Preparation of these constructs has been described previously, as has the [3H]PDBu binding assay. Kazanietz, M. G.; Areces, L. B.; Bahador, A.; Mischak, H.; Goodnight, J.; Mushinski, F.; Blumberg, P. M. Characterisation of ligand and substrate specificity for the calcium-dependent and calcium- independent PKC isozymes. Mol. Pharmacol. 1993, 44, 298-307; Kazanietz, M. G.; Wang, S., Milne; G. W. A., L ewin; N . E ., B lumberg, P . M ., J. B iol. Chem. 1995, 2 70, 2 1852- 21859; Lewin, N.E.; Blumberg, P. M. [3H]phorbol 12,13-dibutyrate binding assay for protein kinase C and related proteins. In: protein kinase C protocols, Vol. 233, ed. Alexandra C. Newton; Humana Press, Totowa, N.J., pp. 129-156, 2003. The results for the dimers of benzolactam are given in Table 2 and those for the dimers of naphthylpyrrolidones in Table 3. Table 2. K and ClogP Values of Dimers 2a-2i and 4."
Figure imgf000045_0002
a Ki
Figure imgf000045_0001
Kozikowski, A. P.; Nowak, I.; Petukhov, P. A.; Etcheberrigaray, R.; Mohamed, A.; Tan, M.; Levin, N.; Hennings, H.; Pearce, L. L.; Blumberg, P. M., J. Med. Chem. 2003, 46, 364- 373.
Table 3. ζ and ClogP Values of Dimers of Naphthylpyrrolidones 13a-g.β
Figure imgf000045_0003
"Ki values are mean ± SEM for three independent experiments. Data taken from Qiao, L.; Zhao, L. Y.; Rong, S. B.; Wu, X. W.; Wang, S.; Fujii, T.; Karanietz, M. G.; Rauser, L.; Savage, J.; Roth, B. L.; Anderson, J. F.; Kozikowski, A. P., Bioorg. Med. Chem. Lett. 2001, 11, 955-959.
All compounds gave full inhibition of [3H]PDBU binding. The K\ values of all analogs are in the nanomolar range, and some ofthe dimers of benzolactam show very high potency towards both PKCα and PKC<X To test for isozyme selectivity, the dimer 7e was assessed for inhibitory activity against the human isoforms of PKCα, PKC/5I, PKC γ, PKC δ, and PKC-r (Pan Vera, Madison, WI). The results are listed in Table 4. The K\ values for all PKCs were in the nanomolar range and comparable.
Figure imgf000046_0001
Table 4. Human PKC Isozyme Data for Compound 7e.α
Figure imgf000046_0002
aKi values are mean ± SEM for three independent experiments. sAPPa Secretion
The ability of our compounds to enhance sAPPα secretion using a cell line derived from an AD patient are summarized as bar graphs in Figure 1. Each bar represents a different treatment (all 1 μM). Results are from 4 to 6 independent experiments, except 5f (triplicate), 5 i-j (duplicates), and 12 repeats for BL (BL is the "+" reference or control). The units are relative to the signal obtained from cells treated with solvent (DMSO) alone, which reflects non-stimulated or basal secretion of sAPPα. The normalization was applied to each blot. Increased sAPPα was particularly marked after stimulation with 5e, 5f, and 5g (Kruskal-Wallis and Dunn's post-test). Noticeable increases were also achieved with 5c, 5d, and 5h, and to a lesser extend BL. The inset shows that 5e and 5f also have a significant effect at 0.1 μM. Compounds 5c, 5g, and 5h also induce noticeable secretion. Most experiments for 0.1 μM are duplicates. Six repeats were carried our for 5g and 5h and single experiments for 5a and 5b. A substantial effect was observed at 1 μM for all compounds tested with the exception of compound 5i. The effect on sAPPα secretion correlates reasonably well with the K. for inhibition of PDBU. Thus compounds 5a and 5b exhibit only modest effects in this assay, as their K,s are between 200 and 2000 nM. Of interest is the absence of activity found for the highly lipophilic ligand 5i, which has a K. of 11 nM. On the other hand, its saturated counterpart 5j, which possesses a comparable K,- and ClogP, retains activity in the secretase activity. The enhancement of sAPPα secretion by compounds 5c-5h is higher than that observed for 8-(l-decynyl)benzolactam. Compounds 5c and 5e-5h enhanced sAPPα secretion even at 0.1 μM, although to a lesser degree than at the higher concentration. A noticeable difference is observed between most of the compounds and the ligands 5e and 5f which are both lipophilic and bind potently to PKCα. Their activity in the sAPPα assay dropped by merely one-half when the concentration was decreased 10-fold. A representative Western blot illustrates the sAPPα signal analyzed for all ofthe compounds (Figure 2).
Hyperplasia Assays
Compounds 5e and 5f were evaluated for induction of hyperplasia after topical application to the shaved backs of outbred Sencar mice (NCI-Frederick, Frederick, MD). The extent of hyperplasia was estimated in terms of the number of cell layers in the epidermis (Table 5). The potencies of 5e and 5f were compared with those of TPA and of mezerein. Detectable hyperplasia was observed after a single application of 1 μg of TPA or after a single application of 3 μg of mezerein. 5f showed a modest response at 100 μg and 5e at 300 μg, suggesting that 5f was approximately 30-fold less potent than mezerein and 100-fold less potent than TPA. 5e was approximately 3-fold less active than 5f. Similar relationships were observed after four applications, although the extent of hyperplasia was somewhat more marked.
Table 5. Hyperplasia induced by compounds 5e and 5f in comparison to TPA and mezerein, estimated by the number of cell layers in the epidermis.
Compound Dose Epidermal Cell Layers
(μg per application) Single Application Four Applications
Acetone Control 1-2 1-2
2 2-5 5-7 TPA 1 2-4 3-7
0.3 1-3 1-4
10 3-6 3-7
Mezerein 3 2-6 2-6
1 1-2 1-3
300 1-4 2-4
100 1-2 2-5
5e 30 1-2 1-4
10 1-2 1-2
3 1-2 1-2 300 2-6 3-7 100 1-5 2-4 30 1-2 1-3
5f 10 1-2 1-3 3 1-2 1-2 1 1-2 1-2
Docking and Molecular Dynamics Simulations
The truncated version of 8-(l-decynyl)benzolactam (BL) bearing only an acetylene group at position 8 and genereic ligand 5 containing -NH(CO)-CH=CHMe (E double bond) as a side chain appendage were docked to the binding domain of PKCα Clb, and the region within an 8 A sphere around the ligand was subjected to molecular dynamics simulations. Molecular modeling revealed that truncated BL forms four highly polpulated (close to 100%) hydrogen bonds with amino acid residues Ser 111, Thr 113, Leu 122, and Gly 124. It also forms a less populated hydrogen bond with Tyr 109 (21%) (Figure 3, a table of the hydrogen bond interactions between PKCα Clb domain and the modeled ligands can be found in Kozikowski, A.P. et al., J. Med. Chem., 2003, 46, 364-373). Generic ligand 5 also forms four hydrogen bonds with a n occupancy close to 100% with Ser 111, Thr 113, Leu 122, and Gly 124. Like BL, ligand 5 forms a low occupancy bond with Tyr 109 (16%). However, in comparison to BL, the hydrogen atom from the amido group located at position 8 of 5 is able to form one additional hydrogen bond with Ser 110, which shows an occupancy of 67%.
Discussion
The nature ofthe benzolactam side chain has a substantial effect on the ability ofthe benzolactams to increase the formation of sAPPα. This is most clearly seen in comparing the decynyl-substituted benzolactam with the benzolactams 5c, e-g. Of particular note is the improved activity of 5f in comparison to 8-(l-decynyl)-benzolactam, although the ClogP of the former is one log unit lower while their Ki values are very similar. On the basis ofthe present modeling studies, it is believed that the side chain amide group of these new benzolactams is able to form an additional H-bonding interaction with serine residue 110 present in loop A. The effect of this extra H bond on the orientation ofthe side chain coupled with the possible interaction of the side chain amide carbonyl with complementary groups present in the membrane phospholipids may better anchor the PKC-ligand complex to the membrane, allowing it to be a more effective mediator of the events resulting in α- secretase activation. C ompounds 5 e and 5f are relatively potent PKC ligands exhibiting binding affinities that are only 40 times poorer than that of PDBU (phorbol 12,13- dibutyrate). Ma, D.; Tang, W.; Kozikowski, A. P.; Lewin, N. E.; Blumberg, P., J. Org. Chem. 1999, 64, 6366-6373. The in vitro assays show the most dramatic increase in the secretion of non-pathogenic sAPPα in the case of 5e and 5f.
Concerning the hyperplasia results, our findings with 5e and 5f are consistent with the structure-activity relations that have been shown for other classes of DAG-mimetics derived from phorbol and related diterpenes. Here, the concept that has emerged is that interaction with the binding site on protein kinase C is relatively independent ofthe pattern of side chain substitution. This independence can be understood from the X-ray crystallographic analysis ofthe complex between the Clb binding domain of protein kinase C and phorbol 13-acetate, in which the primary hydroxyl group (C20) and several of the secondary hydroxy groups ofthe constrained diterpene ring system insert into a hydrophilic pocket in an otherwise hydrophobic face of the Cl domain, whereas the ester side chains project away from the Cl domain and presumably interact with the phospholipid bilayer. Zhang, G. G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J. H., Cell 1995, 81, 917-924.
Computer modeling and site directed mutagenesis have permitted extrapolation of this model to other classes of constrained ligands, including the resiniferonol derivative thymeleatoxin (Pak, Y.; Enyedy, I.; Varady, J.; Kung, J. W.; Lorenzo, P. S.; Blumberg, P.
M.; Wang, S. Structural basis of binding of high affinity ligands to protein kinase C: prediction of the binding modes through a new molecular dynamics method and evaluation by site-directed mutagenesis. J. Med. Chem. 2001, 44, 1690-1701), the ingenol 3- monoesters (id.), constrained diacylglycerol lactones (Nacro, K.; Bienfait, B.; Lee, J.; Han,
K. C; Kang, J. H.; B enzaria, S .; Lewin, N. E .; Bhattacharyya, D . K.; Blumberg, P . M .;
Marquez, V. E. Conformationally constrained analogues of diacylglycerol (DAG). 16. How much structural complexity is necessary for recognition and high binding affinity to protein kinase C. J. Med. Chem. 2000, 43, 921-944), and the indole alkaloids (Wang, S.; Liu, M.; Lewin, N. E.; Lorenzo, P. S.; Bhattacharyya, D.; Qiao, L.; Kozikowski, A. P.; Milne, G. W.
A.; Blumberg, P. M. Probing the binding of indolactam-V to protein kinase C through site directed mutagenesis and computational docking simulations. J. Med. Chem. 1999, 42,
3426-3446). In all of these cases, the side chains project away from the Cl domain.
Although the side chains do not directly interact with the Cl domain, studies at the cellular level clearly indicate that the side chains can influence into which membranes PKC can insert. This influence is most evident in the case of PKC delta. Wang, Q. J.; Bhattacharyya, D. K.; Garfield, S.; Marquez, V. E.; Blumberg, P. M. "Differential localization of protein kinase C delta by phorbol esters and related compounds using a fusion protein with green fluorescent protein" J. Biol. Chem. 1999, 274, 37233-37239. PKC delta inserts into the plasma and nuclear membranes in response to the tumor promoting derivative 12-deoxyphorbol 13-tetradecanoate; in contrast, it goes to the nuclear membrane and punctate aggregates, but not the plasma membrane, after treatment with 12- deoxyphorbol 13 -acetate, a derivative which is inflammatory but which acts to inhibit tumor promotion. The important influence of hydrophobicity in the translocation of PKC delta was similarly shown for a series of homologous, symmetrically substituted phorbol 12,13- diesters, where both the kinetics and ultimate position of translocation of PKC delta depended on the hydrophobicity. Wang, Q. J.; Fang, T. W.; Fenick, D.; Garfield, S.; Bienfait, B.; Marquez, V. E.; Blumberg, P. M. J. Biol. Chem. 2000, 275, 12136-12146. Since subcellular location should drive the availability of substrates for PKC, the ability of side chains on PKC ligands to control localization could plausibly contribute to differences in their biological activities. hi the case of derivatives which differ by being either unsaturated on saturated in their side chains, the biological effect has been consistent but the mechanism remains unresolved. In early studies, Hecker and co-workers described several series of diterpene esters in which unsaturation was associated with retention of inflammatory activity but loss oftumor promoting activity. Examples include: phorbol 12,13-dihexanoate versus phorbol 12,13-dihexa-2,4-dienoate; phorbol 12-tetradecanoate 13-acetate versus phorbol 12- tetradeca-2,4,6,8-tetraenoate 13-acetate; and 12-deoxyphorbol 13-decanoate versus 12- deoxyphorbol 13-deca-2,4-dienoate. Hergenhahn, M.; Furstenberger, G.; Opferkuch, H. J.; Adolf, W.; Mack, H.; Hecker, E. "Biological assays for irritant tumor-initiating and - promoting activities I; Kinetics ofthe irritant response in relation to the initiation-promoting activity of polyfunctional diterpenes representing tigliane and some daphnane types" J. Cancer Res. Clin. Oncol. 1982, 104, 31-39. It has also been demonstrated that mezerein, a phorbol-related diterpene with a 5-phenyl-penta-2,4-dienoate side chain at C12 that is only weak as a tumor promoter, regained tumor promoting activity upon hydrogenation to generate octahydromezerein. Sharkey, N. A.; Hennings, H.; Yuspa, S. H.; Blumberg, P. M. "Comparison of octahydromezerein and mezerein as protein kinase C activators and as mouse skin tumor promoters" Carcinogenesis 1989, 10, 1937-1941. Mezerein did not induce any striking difference in the pattern of PKC translocation, however, suggesting that unsaturation may have a more subtle effect on PKC function, such as positioning PKC relative to cholesterol-rich rafts in the plasma membrane. With respect to 5e and 5f, similar affinities for PKCα and only a modest, 3-fold decrease in potency for skin hyperplasia for the unsaturated derivative were discovered. On the basis of the effect of unsaturation for other potent PKC ligands, one may predict that 5e would retain biological activity in most assays but would show a marked loss of tumor-promoting activity.
Of considerable interest in understanding the complexity of these side chain interactions is the complete absence of activity observed in the sAPPα assay for the highly fluorinated analogue 5i. Its binding affinity for PKCα is good, and its calculated lipophilicity is extremely high. It is unclear as to what is happening in this case, since its saturated c ounterpart i s s till e ffective, and t hus s olubility i ssues m ay b e e liminated from consideration. However, it is hypothesized that the somewhat more rigid side chain of the ligand 5i is responsible for the lack of activity observed in the sAPPα assay. Particularly, since all double bonds in 5i are trans and the orientation of the amido group in 5i is fixed because of the presence of a hydrogen bond interaction with Ser 110, the overall rigidity of the long lipophilic side chain of 5i is greater than that of 5j. Although, the amido group in position 8 of 5j also forms a hydrogen bond with Ser 110, unlike 5i, the side chain of 5j is much more flexible because of its saturated nature. These differences in side chain rigidity may accordingly alter the subcellular localization of PKC and contribute to the observed differences in their effects on secretase activity. h light of the present findings, it is believed that compounds such as 5e that are capable of enhancing sAPPα secretion are worth exploring further as possible therapeutics in the treatment of Alzheimer's disease. Such compounds could be used alone or in combination with β -secretase inhibitors to decrease the formation of neurotoxic β -amyloid peptide, thereby slowing the progression ofthe disease process. h the case ofthe dimers of benzolactam, the nature ofthe spacer and its length play an important role in the potencies ofthe compounds toward the isozymes PKCα and PKC<£ This is clearly seen from the K\ values of 7a-7i (Table 2). A graphical representation between the correlation of spacer length and binding affinity to PKCα is depicted in Figure 4. The activities of 7a and 7b are slightly reduced compared to the reference compound l.20 Kozikowski, A. P.; Nowak, I.; Petukhov, P. A.; Etcheberrigaray, R; Mohamed, A.; Tan, M.; Levin, N.; Hennings, H.; Pearce, L. L.; Blumberg, P. M., J Med. Chem. 2003, 46, 364- 373. As the spacer length is increased from 4 carbons to 6 carbons there is an initial decrease in binding affinity. When the length is increased further by another 2 carbons, there is a dramatic enhancement in binding affinity (and a correspondingly smaller K). Marginal further enhancements are observed with additional increments in chain length from the 8-carbon chain to the 10-, 12-, and 14-carbon chain. For a 20-carbon chain, there is a slight decrease in affinity to PKCα, but the affinity for PKC remains unchanged. Indeed, the activities of 7d, 7e, 7f and 7g are comparable, within 3-fold difference of each other. Compound 7f has an almost 100-fold higher affinity for PKCα than does the n- pentyl amide derivative 5b. All compounds showed a modest selectivity for PKCc) vs. PKCα. hi turn, the affinities for PKC£paralleled the affinities for the Clb domain of PKC (Clb of PKCcvTable 2). On the basis of the two-site model that depicts a single ligand molecule bound simultaneously to the two target sites Cla and Clb on a single molecule of PKC, the binding affinity of the dimer can be defined in terms of the binding affinities of individual ligands and a contribution by the spacer (S) to the binding affinity: ϋ-i(dimer) = iζ(binding to Cla)* i(binding to Clb)*S. Jencks, W. P. On the attribution and additivity of binding energies. Proc. Nat. Acad. Sci. USA 1981, 78, 4046-4050; Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W., Science 1996, 274, 1531-1541; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W., Science 1997, 278, 497-503; Qin, D.; Sullivan, R.; Berkowitz, W. F.; Bittman, R.; Rotenberg, S. A., J. Med. Chem. 2000, 43, 1413-1417. The K{ value for binding to the Clb domain ofthe parent benzolactam (un-substituted) is 334.0 ± 14.0 x 10"9 M. Kozikowski, A. P.; Wang, S.; Ma, D.; Yao, J.; Ahmad, S.; Glazer, R. I.; Bogi, K.; Acs, P.; Modarres, S.; Lewin, N. E.; Blumberg, P. M., J. Med. Chem. 1997, 40, 1316-1326. Assuming a similar binding affinity to the Cla domain, the value for i(dimer) can be calculated as: [334.0 x 10"9 M [334.0 x 10"9 M] = 1.0 x 10"13 M. For S = 1, the observed affinity is much weaker than the calculated one. From these results it is believed that these dimers do not bind simultaneously to both Cl domains on the same molecule of protein kinase C, presumably due to steric constraints, and that the compounds do not show a bidentate character. This is consistent with the result for the monodentate derivative 13, which had comparable affinities (within a 1-4 fold range) to that of 7c (having the same spacer length) for all three biological systems, namely PKCα, PKCc> and the Cl domain of PKC-5 ( Table 2 ). I t s hould o f c ourse be n oted t hat for a b identate 1 igand, e ach m ole of ligand contains two moles of binding moiety, and potencies should therefore be corrected by a factor of two for appropriate comparison with a monodentate ligand. Since the bidentate compounds were able to fully compete for [3H]PDBu binding to PKCα and δ, it is clear that they can bind to both the Cla and Clb domains of PKC and their failure to show the predicted enhancement in affinity cannot be explained by their activity on only one of these Cl domains. The naphthylpyrrolidone dimers 9a-g showed moderate activity in the high nanomolar range for PKCα (Table 3). The activity of these dimers was in the same range as that of our earlier synthesized monomer, thus showing that the presence of two binding moieties in a single molecule did not yield any significant improvement. Qiao, L.; Wang, S.; George C; Lewin N. E.; Blumberg, P. M.; Kozikowski, A. P., J. Am. Chem. Soc. 1998, 120, 6629-6630. There is a slight improvement in activity as the length of the spacer is increased from 10- to 12- and 14-carbon atoms. A further increase in the chain length to 16 and 22-carbon atoms reduces the binding affinity. All differences in affinity are within a 2- fold range. The results from the dimers of both benzolactam and naphthylpyrrolidones illustrate that the presence of two binding moieties does not result in any synergistic binding with the Cl domains of PKCs.
The compound 7e was selected for testing with different isozymes of PKC, namely α, βl, δ, γ, and ε (Table 4). Its affinity was almost equal for all of these isozymes, with a slightly higher affinity for PKCs- and lower affinity for PKC& Compounds 7f and 7g showed very high affinity toward the entire series and are comparable to PDBU (phorbol 12,13-dibutyrate). The derivatives 7h and 7i in which the ohgomethylene chain is replaced by an oligoethylene glycol chain exhibit drastically reduced binding affinities (Table 2). This shows that the nature of the spacer is very important to achieve good binding to the PKCs.
It has been shown that the lipophilicity of molecules plays a crucial role in determimng their biological activity and potency. In the case of phorbol esters the phorbol 12,13-diC8 esters and the 12-myristoyl derivative are among the most potent tumor promoters, whereas the 12-deoxy-13-phenylacetate derivative actually inhibits tumor promotion. Zayed, S.; Sorg, B.; Hecker, E., Planta Medica 1984, 4, 65-69; Szallasi, Z.; Krsmanovic, L.; Blumberg, P. M., Cancer Res. 1993, 53, 2507-2512; Schmidt, R.; Hecker, E. h Carcinogenesis and Biological Effects of Tumor Promoters: Hecker, E., Fϋsenig, N. E., Kunz, W., Marks, F., Thielmann, H. W., Eds): Schattauer, Stuttgart, 1982, pp. 171-179. The length of the side chain is important for the ligand to rapidly equilibrate in aqueous solution and for the translocation of the complex to the plasma membrane. Wang, Q. J.; Fang, T. W.; Fenick, D.; Garfield, S.; Bienfait, B.; Marquez, V. E.; Blumberg, P. M., J. Biol. Chem. 2000, 275, 12136-12146. In the case of phorbol esters, longer chain lengths resulted in loss of tumor promoting activity due to non-equilibration in aqueous solution whereas very short chain lengths showed decrease in affinity for ligand added directly to the lipid phase, h the case ofthe dimers ofthe benzolactam this relationship is not seen as 7e, 7f and 7g (containing 12C, 14C and 20C spacers) show very high binding affinity for both PKCα and R Cδ. It should be noted, however, that in these experiments the compounds were added directly to the lipids so as to avoid problems of equilibration. Little effect of linker length on activity was seen for the naphthylpyrrolidone dimers 9a-9g.
Even though the compounds 7d-7g showed no isozyme specificity, their affinities were much higher than those reported earlier for the monomeric benzolactam amide series. Kozikowski, A. P.; Nowak, I.; Petukhov, P. A.; Etcheberrigaray, R.; Mohamed, A.; Tan, M.; Levin, N.; Hennings, H.; Pearce, L. L; Blumberg, P. M., J. Med. Chem. 2003, 46, 364- 373. This increase in affinity could be due to the extensive hydrophobic facade presented by the spacer chain. It is generally accepted that hydrophobic moieties increase the ability of the compound to form a stable association with the membrane, which is known to be essential for inducing downregulation of PKC. Earlier studies by our group on both phorbol and lyngbyatoxin A have shown that the presence of a hydrophobic side chain (preferably 8- 10 carbon) provides for a tight association of the enzyme to the membrane. Basu, A.; Kozikowski, A. P.; Lazo, J. S., Biochem. 1992, 31, 3824-3830. hi this connection it is noted that certain lyngbyatoxin A analogs have been explored previously in which, for example, incorporation of a short, more polar 4-hydroxybutyl chain at the 7-position led to a reduction in PKC activation, presumably due to poorer membrane interaction. The ability of this compound to act as a PKC antagonist could not be observed under our experimental conditions. Kozikowski, A. P.; Shum, P. W.; Basu, A.; Lazo, J. S., J. Med. Chem. 1991, 34, 2420-2430; Kozikowski, A. P.; Ma, D.; Du, L.; Lewin, N. E.; Blumberg, P. M., J. Am. Chem. Soc. 1995, 117, 6666-6672. More recently, Shibasaki and co-workers following a similar logic have described a phorbol ester derivative comprised of ethylene glycol units that is capable of acting as a weak PKC antagonist. Wada, R.; Suto, Y.; Kanai, M.; Shibasaki, M., J. Am. Chem. Soc. 2002, 124, 10658-10659; Sodeoka, M.; Arai, M. A.; Adachi, K. U.; S hibasaki, M ., J. A m. Chem. Soc. 1998, 120, 457-458. T his antagonist activity was again ascribed to reduced membrane interaction and inability to bring about PKC translocation. While the increased activity of the bivalent ligands 7d-7g described presently is likely due to more favorable membrane interaction as mentioned, one can not rule out the possibility that the increase in affinity may also stem from additional interactions ofthe second binding unit with other sites on the protein. The present findings thus add to the storehouse of knowledge on ligand interactions with PKC. Such information will be valuable to achieving a better understanding of how these side chain appendages can be manipulated so as to influence certain cellular events coupled to PKC activation.
Biochemical Activity at Cellular Receptors, and Assays to Detect That Activity
Assaying processes are well known in the art in which a reagent is added to a sample, and measurements ofthe sample and reagent are made to identify sample attributes stimulated by the reagent. For example, one such assay process concerns determining in a chromogenic assay the amount of an enzyme present in a b iological s ample or solution. Such assays are based on the development of a colored product in the reaction solution. The reaction develops as the enzyme catalyzes the conversion of a colorless chromogenic substrate to a colored product. Another assay useful in the present invention concerns determining the ability of a ligand to bind to a biological receptor utilizing a technique well known in the art referred to as a radioligand binding assay. This assay accurately determines the specific binding of a radioligand to a targeted receptor through the delineation of its total and nonspecific binding components. Total binding is defined as the amount of radioligand that remains following the rapid separation ofthe radioligand bound in a receptor preparation (cell homogenates or recombinate receptors) from that which is unbound. The nonspecific binding component is defined as the amount of radioligand that remains following separation of the reaction mixture consisting of receptor, radioligand and an excess of unlabeled ligand. Under this condition, the only radioligand that remains represents that which is bound to components other that receptor. The specific radioligand bound is determined by subtracting the nonspecific from total radioactivity bound. For a specific example of radioligand binding assay for μ-opioid receptor, see Wang, J. B. et al. FEBS Letters 1994, 338, 217.
Assays useful in the present invention concern determining the activity of receptors the activation of which initiates subsequent intracellular events in which intracellular stores of calcium ions are released for use as a second messenger. Activation of some G-protein- coupled receptors stimulates the formation of inositol triphosphate (IP3, a G-protein- coupled receptor second messenger) through phospholipase C-mediated hydrolysis of phosphatidylinositol, Berridge and Irvine (1984). Nature 312:315-21. IP3 in turn stimulates the release of intracellular calcium ion stores.
A change in cytoplasmic calcium ion levels caused by release of calcium ions from intracellular stores is used to determine G-protein-coupled receptor function. This is another type of indirect assay. Among G-protein-coupled receptors are muscarinic acetylcholine receptors (mAChR), adrenergic receptors, sigma receptors, serotonin receptors, dopamine receptors, angiotensin receptors, adenosine receptors, bradykinin receptors, metabotropic excitatory amino acid receptors and the like. Cells expressing such G-protein-coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both intracellular stores and via activation of ion channels, in which case it maybe desirable although not necessary to conduct such assays in calcium-free buffer, optionally supplemented with a chelating agent such as EGTA, to distinguish fluorescence response resulting from calcium release from internal stores. Another type of indirect assay involves determining the activity of receptors which, when activated, result in a change in the level of intracellular cyclic nucleotides, e.g., cAMP, cGMP. For example, activation of some dopamine, serotonin, metabotropic glutamate receptors and muscarinic acetylcholine receptors results in a decrease in the cAMP or cGMP levels ofthe cytoplasm.
Furthermore, there are cyclic nucleotide-gated ion channels, e.g., rod photoreceptor cell channels and olfactory neuron channels [see, Altenhofen, W. et al. (1991) Proc. Natl. Acad. Sci U.S.A. 88:9868-9872 and Dhallan et al. (1990) Nature 347:184-187] that are permeable to cations upon activation by binding of cAMP or cGMP. A change in cytoplasmic ion levels caused by a change in the amount of cyclic nucleotide activation of photo-receptor or olfactory neuron channels is used to determine function of receptors that cause a change in cAMP or cGMP levels when activated, hi cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it may be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e .g., forskolin, prior to adding a receptor-activating compound to the cells in the assay. Cell for this type of assay can be made by co-transfection of a host cell with DNA encoding a cyclic nucleotide-gated ion channel and a DNA encoding a receptor (e.g., certain metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, serotonin receptors and the like, which, when activated, causes a change in cyclic nucleotide levels in the cytoplasm.
Any cell expressing a receptor protein which is capable, upon activation, of directly increasing the intracellular concentration of calcium, such as by opening gated calcium channels, or indirectly affecting the concentration of intracellular calcium as by causing initiation of a reaction which utilizes Ca<2+> as a second messenger (e.g., G-protein- coupled receptors), may form the b asis o f an assay. C ells endogenously expressing such receptors or ion channels and cells which may be transfected with a suitable vector encoding one or more such cell surface proteins are known to those of skill in the art or may be identified by those of skill in the art. Although essentially any cell which expresses endogenous ion channel and/or receptor activity may be used, it is preferred to use cells transformed or transfected with heterologous DNAs encoding such ion channels and/or receptors so as to express predominantly a single type of ion channel or receptor. Many cells that may be genetically engineered to express a heterologous cell surface protein are known. Such cells include, but are not limited to, baby hamster kidney (BHK) cells (ATCC No. CCL10), mouse L cells (ATCC No. CCLI.3), DG44 cells [see, Chasin (1986) Cell. Molec. Genet. 12:555] human embryonic kidney (HEK) cells (ATCC No. CRL1573), Chinese hamster ovary (CHO) cells (ATCC Nos. CRL9618, CCL61, CRL9096), PC12 cells (ATCC No. CRL1721) and COS-7 cells (ATCC No. CRL1651). Preferred cells for heterologous cell surface protein expression are those that can be readily and efficiently transfected. Preferred cells include HEK 293 cells, such as those described in U.S. Pat. No. 5,024,939.
Any compound which is known to activate ion channels or receptors of interest may be used to initiate an assay. Choosing an appropriate ion channel- or receptor-activating reagent depending on the ion channel or receptor of interest is within the skill of the art. Direct depolarization of the cell membrane to determine calcium channel activity may be accomplished by adding a potassium salt solution having a concentration of potassium ions such that the final concentration of potassium ions in the cell-containing well is in the range of about 50-150 mM (e.g., 50 mM KC1). With respect to ligand-gated receptors and ligand- gated ion channels, ligands are known which have affinity for and activate such receptors. For example, nicotinic acetyloholine receptors are known to be activated by nicotine or acetylcholine; similarly, muscarinic and acetylcholine receptors may be activated by addition of muscarine or carbamylcholine.
Agonist assays may be carried out on cells known to possess ion channels and/or receptors to determine what effect, if any, a compound has on activation or potentiation of ion channels or receptors of interest. Agonist assays also may be carried out using a reagent known to possess ion channel- or receptor-activating capacity to determine whether a cell expresses the respective functional ion channel or receptor of interest.
Contacting a functional receptor or ion channel with agonist typically activates a transient reaction; and prolonged exposure to an agonist may desensitize the receptor or ion channel to subsequent activation. Thus, in general, assays for determining ion channel or receptor function should be initiated by addition of agonist (i.e., in a reagent solution used to initiate the reaction). The potency of a compound having agonist activity is determined by the detected change in some observable in the cells (typically an increase, although activation of certain receptors causes a decrease) as compared to the level ofthe observable in either the same cell, or substantially identical cell, which is treated substantially identically except that reagent lacking the agonist (i.e., control) is added to the well. Where an agonist assay is performed to test whether or not a cell expresses the functional receptor or ion channel of interest, known agonist is added to test-cell-containing wells and to wells containing control cells (substantially identical cell that lacks the specific receptors or ion channels) and the levels of observable are compared. Depending on the assay, cells lacking the ion channel and/or receptor of interest should exhibit substantially no increase in observable in response to the known agonist. A substantially identical cell may be derived from the same cells from which recombinant cells are prepared but which have not been modified by introduction of heterologous DNA. Alternatively, it may be a cell in which the specific receptors or ion channels are removed. Any statistically or otherwise significant difference in the level of observable indicates that the test compound has in some manner altered the activity of the specific receptor or ion channel or that the test cell possesses the specific functional receptor or ion channel. hi an example of drug screening assays for identifying compounds which have the ability to modulate ion channels or receptors of interest, individual wells (or duplicate wells, etc.) contain a distinct cell type, or distinct recombinant cell line expressing a homogeneous population of a receptor or ion channel of interest, so that the compound having unidentified activity may be screened to determine whether it possesses modulatory activity with respect to one or more of a variety of functional ion channels or receptors. It is also contemplated that each of the individual wells may contain the same cell type so that multiple compounds (obtained from different reagent sources in the apparatus or contained within different wells) can be screened and compared for modulating activity with respect to one particular receptor or ion channel type.
Antagonist assays, including drug screening assays, may be carried out by incubating cells having functional ion channels and/or receptors in the presence and absence of one or more compounds, added to the solution bathing the cells in the respective wells of the microtiter plate for an amount of time sufficient (to the extent that the compound has affinity for the ion channel and/or receptor of interest) for the compound(s) to bind to the receptors and/or ion channels, then activating the ion channels or receptors by addition of known agonist, and measuring the level of observable in the cells as compared to the level of observable in either the same cell, or substantially identical cell, in the absence of the putative antagonist. The assays are thus useful for rapidly screening compounds to identify those that modulate any receptor or ion channel in a cell, hi particular, assays can be used to test functional ligand-receptor or ligand-ion channel interactions for cell receptors including ligand-gated ion channels, voltage-gated ion channels, G-protein-coupled receptors and growth factor receptors. Those of ordinary skill in the art will recognize that assays may encompass measuring a detectable change of a solution as a consequence of a cellular event which allows a compound, capable of differential characteristics, to change its characteristics in response to the cellular event. By selecting a particular compound which is capable of differential characteristics upon the occurrence of a cellular event, various assays may be performed. For example, assays for determining the capacity of a compound to induce cell injury or cell death may be carried out by loading the cells with a pH-sensitive fluorescent indicator such as BCECF (Molecular Probes, hie, Eugene, Oreg. 97402, Catalog #B1150) and measuring cell injury or cell death as a function of changing fluorescence over time.
In a further example of useful assays, the function of receptors whose activation results in a change in the cyclic nucleotide levels of the cytoplasm may be directly determined in assays of cells that express such receptors and that have been injected with a fluorescent compound that changes fluorescence upon binding cAMP. The fluorescent compound comprises cAMP-dependent-protein kinase in which the catalytic and regulatory subunits are each labelled with a different fluorescent-dye [Adams et al. (1991) Nature 349:694-697]. When cAMP binds to the regulatory subunits, the fluorescence emission spectrum changes; this change can be used as an indication of a change in cAMP concentration.
The function of certain neurotransmitter transporters which are present at the synaptic cleft at the junction between two neurons may be determined by the development of fluorescence in the cytoplasm of such neurons when conjugates of an amine acid and fluorescent indicator (wherein the fluorescent indicator ofthe conjugate is an acetoxymethyl ester derivative e.g., 5-(aminoacetamido)fluorescein; Molecular Probes, C atalog #A1363) are transported by the neurotransmitter transporter into the cytoplasm of the cell where the ester group is cleaved by esterase activity and the conjugate becomes fluorescent. hi practicing an assay of this type, a reporter gene construct is inserted into an eukaryotic cell to produce a recombinant cell which has present on its surface a cell surface protein of a specific type. The cell surface receptor may be endogenously expressed or it may be expressed from a heterologous gene that has been introduced into the cell. Methods for introducing heterologous DNA into eukaryotic cells are-well known in the art and any such method may be used. In addition, DNA encoding various cell surface proteins is known to those of skill in the art or it may be cloned by any method known to those of skill in the art.
The recombinant cell is contacted with a test compound and the level of reporter gene expression is measured. The contacting may be effected in any vehicle and the testing may be by any means using any protocols, such as s erial dilution, for assessing specific molecular interactions known to those of skill in the art. After contacting the recombinant cell for a sufficient time to effect any interactions, the level of gene expression is measured. The amount of time to effect such interactions may be empirically determined, such as by running a time course and measuring the level of transcription as a function of time. The amount of transcription may be measured using any method known to those of skill in the art to be suitable. For example, specific mRNA expression may be detected using Northern blots or specific protein product may be identified by a characteristic stain. The amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test, compound or it may be compared with the amount of transcription in a substantially identical cell that lacks the specific receptors. A substantially identical cell may be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Alternatively, it may be a cell in which the specific receptors are removed. Any statistically or otherwise significant difference in the amount of transcription indicates that the test compound has in some manner altered the activity ofthe specific receptor.
If the test compound does not appear to enhance, activate or induce the activity of the cell surface protein, the assay may be repeated and modified by the introduction of a step in which the recombinant cell is first tested for the ability of a known agonist or activator of the specific receptor to activate transcription if the transcription is induced, the test compound is then assayed for its ability to inhibit, block or otherwise affect the activity ofthe agonist. The transcription based assay is useful for identifying compounds that interact with any cell surface protein whose activity ultimately alters gene expression, hi particular, the assays can be used to test functional ligand-receptor or ligand-ion channel interactions for a number of categories of cell surface-localized receptors, including: ligand-gated ion channels and voltage-gated ion channels, and G protein-coupled receptors. Any transfectable cell that can express the desired cell surface protein in a manner such the protein functions to intracellularly transduce an extracellular signal may be used. The cells may be selected such that they endogenously express the cell surface protein or may be genetically engineered to do so. Many such cells are known to those of skill in the art. Such cells include, but are not limited to Ltk< - > cells, PC12 cells and COS-7 cells. The preparation of cells which express a receptor or ion channel and a reporter gene expression construct, and which are useful for testing compounds to assess their activities, is exemplified in the Examples provided herewith by reference to mammalian Ltk< - > and COS-7 cell lines, which express the Type I human muscarinic (HM1) receptor and which are transformed with either a c-fos promoter-CAT reporter gene expression construct or a c- fos promoter-luciferase reporter gene expression construct. Any c ell s urface p rotein that i s known t o those o f skill in the art or that may be identified by those of skill in the art may be used in the assay. The cell surface protein may be endogenously expressed on the selected cell or it may be expressed from cloned DNA. Exemplary cell surface proteins include, but are not limited to, cell surface receptors and ion channels. Cell surface receptors include, but are not limited to, muscarinic receptors (e.g.,, human M2 (GenBank accession #M16404); rat M3 (GenBank accession #M16407); human M4 (GenBank accession #M16405); human M5 (Bonner et al. (1988) Neuron 1:403-410); and the like); neuronal nicotinic acetylcholine receptors (e.g., the alpha 2, alpha 3 and beta 2 subtypes disclosed in U.S. Ser. No. 504,455 (filed Apr. 3, 1990), hereby expressly incorporated by reference herein in its entirety); the rat alpha 2 subunit (Wada et al. (1988) Science 240:330-334); the rat alpha 3 subunit (Boulter et al. (1986) Nature 319:368-374); the rat alpha 4 subunit (Goldman et al. (1987) c ell 48:965-973); the rat alpha 5 subunit (Boulter et al. (1990) J. Biol. Chem. 265:4472-4482); the rat beta 2 subunit (Deneris et al. (1988) Neuron 1:45-54); the rat beta 3 subunit (Deneris et al. (1989) J. Biol. Chem. 264: 6268-6272); the rat beta 4 subunit (Duvoisin et al. (1989) Neuron 3:487-496); combinations ofthe rat alpha subunits, beta subunits and alpha and beta subunits; GABA receptors (e.g., the bovine alpha 1 and beta 1 subunits (Schofield et al. (1987) Nature 328:221-227); the bovine alpha 2 and alpha 3 subunits (Levitan et al. (1988) Nature 335:76-79); the gamma - subunit (Pritchett et al. (1989) Nature 338:582-585); the beta 2 and beta 3 subunits (Ymer et alo (1989) EMBO J. 8:1665-1670); the delta subunit (Shivers, B.D. (1989) Neuron 3:327- 337); and the like); glutamate receptors (e.g., receptor isolated from rat brain (Holhnann et al. (1989) Nature 342:643-648); and the like); adrenergic receptors (e.g., human beta 1 (Frielle et al. (1987) Proc. Natl. Acad. Sci. 84.:7920-7924); human alpha 2 (Kobilka et al. (1987) Science 238:650-656); hamster beta 2 (Dixon et al. (1986) Nature 321:75-79); and the like); dopamine receptors (e.g., human D2 (Stormann et al. (1990) Molec. Pharm.37:l- 6); rat (Bunzow et al. (1988) Nature 336:783-787); and the like); NGF receptors (e.g., human NGF receptors (Johnson et al. (1986) Cell 47:545-554); and the like); serotonin receptors (e.g., human 5HTla (Kobilka et al. (1987) Nature 329:75-79); rat 5HT2 (Julius et al. (1990) PNAS 87:928-932); rat 5HTlc (Julius et al. (1988) Science 241:558-564); and the like).
Reporter gene constructs are prepared by operatively linking a reporter gene with at least one transcriptional regulatory element. If only one transcriptional regulatory element is included it must be a regulatable promoter. At least one of the selected transcriptional regulatory elements must be indirectly or directly regulated by the activity of the selected cell-surface receptor whereby activity of the receptor can be monitored via transcription of the reporter genes. The construct may contain additional transcriptional regulatory elements, such as a
FIRE sequence, or other sequence, that is not necessarily regulated by the cell surface protein, but is selected for its ability to reduce background level transcription or to amplify the transduced signal and to thereby increase the sensitivity and reliability ofthe assay.
Many reporter genes and transcriptional regulatory elements are known to those of skill in the art and others may be identified or synthesized by methods known to those of skill in the art. A reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Preferred reporter genes are those that are readily detectable.
The reporter gene may also be included in the construct in the form of a fusion gene with a gene that includes desired transcriptional regulatory sequences or exhibits other desirable properties. Examples of reporter genes include, but are not limited to CAT
(chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase
(deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and
Silverman (1984), PNAS 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663- 3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al.
(1983) J. Mol. Appl. Gen. 2: 101).
Transcriptional control elements include, but are not limited to, promoters, enhancers, and repressor and activator binding sites. Suitable transcriptional regulatory elements may be derived from the transcriptional regulatory regions of genes whose expression is rapidly induced, generally within minutes, of contact between the cell surface protein and the effector protein that modulates the activity of the cell surface protein. Examples of such genes include, but are not limited to, the immediate early genes (see, Sheng et al. (1990) Neuron 4: 477-485), such as c-fos. Immediate early genes are genes that are rapidly induced upon binding of a ligand to a cell surface protein. The transcriptional control elements that are preferred for use in the gene constructs include transcriptional control elements from immediate early genes, elements derived from other genes that exhibit some or all ofthe characteristics ofthe immediate early genes, or synthetic elements that are constructed such that genes in operative linkage therewith exhibit such characteristics. The characteristics of preferred genes from which the transcriptional control elements are derived include, but are not limited to, low or undetectable expression in quiescent cells, rapid induction at the transcriptional level within minutes of extracellular simulation, induction that is transient and independent of new protein synthesis, subsequent shut-off of transcription requires new protein synthesis, and mRNAs transcribed from these genes have a short half-life. It is not necessary for all of these properties to be present.
Pharmaceutical Compositions h another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.
The phrase "therapeutically-effective amount" as used herein means that amount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing acid (e.g., lubricant, talc magnesium, calcium stearate, zinc stearate, or stearic acid) or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.
As set out above, certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term "pharmaceutically-acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic a cid a ddition s alts o f c ompounds o f the p resent i nvention. T hese salts can be prepared in situ in the admimstration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound ofthe invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19)
The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
In o ther c ases, t he c ompounds o f t he p resent i nvention m ay c ontain o ne o r m ore acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable s alts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the p urified c ompound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra)
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound ofthe present invention.
Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound ofthe present invention may also be administered as a bolus, electuary or paste. hi solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrohdone, sucrose and/or acacia; (3) huniectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents, h the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture ofthe powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions ofthe present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more ofthe above-described excipients.
Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs, hi addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetiahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing o ne o r m ore compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention. Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may c ontain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood ofthe intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, hi addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature ofthe particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels ofthe active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of admimstration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of admimstration, the time of administration, the rate of excretion or metabolism ofthe particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses ofthe compounds ofthe invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. h general, a suitable daily dose of a compound ofthe invention will be that amount ofthe compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated desired effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.
If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. However, the preferred dosing is daily.
While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). h another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the subject compounds, as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin, lungs, o r o ral c avity; or (4) intravaginally or intravectally, for example, as a pessary, cream or foam; (5) sublingually;
(6) ocularly; (7) transdermally; or (8) nasally.
The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals . The term "treatment" is intended to encompass also prophylaxis, therapy and cure.
The p atient receiving this treatment i s any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
The compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered. The addition of the active compound of the invention to animal feed is preferably accomplished by preparing an appropriate feed premix containing the active compound in an effective amount and incorporating the premix into the complete ration.
Alternatively, an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed. The way in which such feed premixes and complete rations can be prepared and administered are described in reference books (such as "Applied Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A., 1969 or
"Livestock Feeds and Feeding" O and B books, Corvallis, Ore., U.S.A., 1977).
Combinatorial Libraries
The subject compounds readily lend themselves to the creation of combinatorial libraries for the screening of pharmaceutical, agrochemical or other biological or medically- related activity or material-related qualities. A combinatorial library for the purposes ofthe present invention is a mixture of chemically related compounds which may be screened together for a desired property; said libraries may be in solution or covalently linked to a solid support. The preparation of many related compounds in a single reaction greatly reduces and simplifies the number of screening processes which need to be carried out. Screening for the appropriate biological, pharmaceutical, agrochemical or physical property may be done by conventional methods.
Diversity in a library can be created at a variety of different levels. For instance, the substrate aryl groups used in a combinatorial approach can be diverse in terms of the core aryl moiety, e.g., a variegation in terms of the ring structure, and or can be varied with respect to the other substituents.
A variety of techniques are available in the art for generating combinatorial libraries of small organic molecules. See, for example, Blondelle et al. (1995) Trends Anal. Chem.
14:83; the Affymax U.S. Patents 5,359,115 and 5,362,899: the Ellman U.S. Patent 5,288,514: the Still et al. PCT publication WO 94/08051; Chen et al. (1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCT publications WO92/10092, WO93/09668 and W 091/07087; and the Lerner et al. PCT publication WO93/20242). Accordingly, a variety of libraries on the order of about 16 to 1,000,000 or more diversomers can be synthesized and screened for a particular activity or property. hi an exemplary embodiment, a library of substituted diversomers can be synthesized using the subject reactions adapted to the techniques described in the Still et al. PCT publication WO 94/08051, e.g., being linked to a polymer bead by a hydrolyzable or photolyzable group, e.g., located at one of the positions of substrate. According to the Still et al. technique, the library is synthesized on a set of beads, each bead including a set of tags identifying the particular diversomer on that bead, hi one embodiment, which is particularly suitable for discovering enzyme inhibitors, the beads can be dispersed on the surface of a permeable membrane, and the diversomers released from the beads by lysis of the bead linker. The diversomer from each bead will diffuse across the membrane to an assay zone, where it will interact with an enzyme assay. Detailed descriptions of a number of combinatorial methodologies are provided below.
A. Direct Characterization
A growing trend in the field of combinatorial chemistry is to exploit the sensitivity of techniques such as mass spectrometry (MS), e.g., which can be used to characterize sub- femtomolar amounts of a compound, and to directly determine the chemical constitution of a compound selected from a combinatorial library. For instance, where the library is provided on an insoluble support matrix, discrete populations of compounds can be first released from the support and characterized by MS. In other embodiments, as part of the MS sample preparation technique, such MS techniques as MALDI can be used to release a compound from the matrix, particularly where a labile bond is used originally to tether the compound to the matrix. For instance, a bead selected from a library can be irradiated in a MALDI step in order to release the diversomer from the matrix, and ionize the diversomer for MS analysis.
B Multipin Synthesis
The libraries of the subject method can take the multipin library format. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS 81 :3998-4002) introduced a method for generating compound libraries by a parallel synthesis on polyacrylic acid-grated polyethylene pins arrayed in the microtitre plate format. The Geysen technique can be used to synthesize and screen thousands of compounds per week using the multipin method, and the tethered compounds may be reused in many assays. Appropriate linker moieties can also be appended to the pins so that the compounds may be cleaved from the supports after synthesis for assessment of purity and further evaluation (c.f, Bray et al. (1990) Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem 197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166V
O Divide-Couple-Recombine hi yet another embodiment, a variegated library of compounds can be provided on a set of beads utilizing the strategy of divide-couple-recombine (see, e.g., Houghten (1985) PNAS 82:5131-5135; and U.S. Patents 4,631,211; 5,440,016; 5,480,971). Briefly, as the name implies, at each synthesis step where degeneracy is introduced into the library, the beads are divided into separate groups equal to the number of different substituents to be added at a particular position in the library, the different substituents coupled in separate reactions, and the beads recombined into one pool for the next iteration. hi one embodiment, the divide-couple-recombine strategy can be carried out using an analogous approach to the so-called "tea bag" method first developed by Houghten, where compound synthesis occurs on resin sealed inside porous polypropylene bags (Houghten et al. (1986) PNAS 82:5131-5135). Substituents are coupled to the compound- bearing resins by placing the bags in appropriate reaction solutions, while all common steps such as resin washing and deprotection are performed simultaneously in one reaction vessel. At the end ofthe synthesis, each bag contains a single compound.
D Combinatorial Libraries by Light-Directed. Spatially Addressable Parallel Chemical Synthesis
A scheme of combinatorial synthesis in which the identity of a compound is given by its locations on a synthesis substrate is termed a spatially-addressable synthesis. In one embodiment, the combinatorial process is carried out by controlling the addition of a chemical reagent to specific locations on a solid support (Dower et al. (1991) Annu Rep Med Chem 26:271-280; Fodor, S.P.A. (1991) Science 251:767; Pirrung et al. (1992) U.S. Patent No. 5,143,854; Jacobs et al. (1994) Trends Biotechnol 12:19-26). The spatial resolution of photolithography affords miniaturization. This technique can be carried out through the use of protection/deprotection reactions with photolabile protecting groups.
The key points of this technology are illustrated in Gallop et al. (1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared for coupling through the covalent attachment of photolabile nitroveratryloxycarbonyl (NVOC) protected amino linkers or other photolabile linkers. Light is used to selectively activate a specified region of the synthesis support for coupling. Removal of the photolabile protecting groups by light (deprotection) results in activation of selected areas. After activation, the first of a set of amino acid analogs, each bearing a photolabile protecting group on the amino terminus, is exposed to the entire surface. Coupling only occurs in regions that were addressed by light in the preceding step. The reaction is stopped, the plates washed, and the substrate is again illuminated through a second mask, activating a different region for reaction with a second protected building block. The pattern of masks and the sequence of reactants define the products and their locations. Since this process utilizes photolithography techniques, the number of compounds that can be synthesized is limited only by the number of synthesis sites that can be addressed with appropriate resolution. The position of each compound is precisely known; hence, its interactions with other molecules can be directly assessed. hi a light-directed chemical synthesis, the products depend on the pattern of illumination and on the order of addition of reactants. By varying the lithographic patterns, many different sets of test compounds can be synthesized simultaneously; this characteristic leads to the generation of many different masking strategies.
E) Encoded Combinatorial Libraries
In yet another embodiment, the subject method utilizes a compound library provided with an encoded tagging system. A recent improvement in the identification of active compounds from combinatorial libraries employs chemical indexing systems using tags that uniquely encode the reaction steps a given bead has undergone and, by inference, the structure it carries. Conceptually, this approach mimics phage display libraries, where activity derives from expressed peptides, but the structures of the active peptides are deduced from the corresponding genomic DNA sequence. The first encoding of synthetic combinatorial libraries employed DNA as the code. A variety of other forms of encoding have been reported, including encoding with sequenceable bio-oligomers (e.g., oligonucleotides and peptides), and binary encoding with additional non-sequenceable tags.
1) Tagging with sequenceable bio-oligomers
The principle of using oligonucleotides to encode combinatorial synthetic libraries was described in 1992 (Brenner et al. (1992) PNAS 89:5381-5383), and an example of such a library appeared the following year (Needles et al. (1993) PNAS 90:10700-10704). A combinatorial library of nominally 77 (= 823,543) peptides composed of all combinations of Arg, Gin, Phe, Lys, Val, D-Val and Thr (three-letter amino acid code), each of which was encoded by a specific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively), was prepared by a series of alternating rounds of peptide and ohgonucleotide synthesis on solid support, hi this work, the amine linking functionality on the bead was specifically differentiated toward peptide or ohgonucleotide synthesis by simultaneously preincubating the beads with reagents that generate protected OH groups for ohgonucleotide synthesis and protected NH2 groups for peptide synthesis (here, in a ratio of 1:20). When complete, the tags each consisted of 69-mers, 14 units of which carried the code. The bead-bound library was incubated with a fluorescently labeled antibody, and beads containing bound antibody that fluoresced strongly were harvested by fluorescence-activated cell sorting (FACS). The DNA tags were amplified by PCR and sequenced, and the predicted peptides were synthesized. Following such techniques, compound libraries can be derived for use in the subject method, where the ohgonucleotide sequence of the tag identifies the sequential combinatorial reactions that a particular bead underwent, and therefore provides the identity ofthe compound on the bead.
The use of ohgonucleotide tags permits exquisitely sensitive tag analysis. Even so, the method requires careful choice of orthogonal sets of protecting groups required for alternating co-synthesis of the tag and the library member. Furthermore, the chemical lability of the tag, particularly the phosphate and sugar anomeric linkages, may limit the choice of reagents and conditions that can be employed for the synthesis of non-oligomeric libraries. In preferred embodiments, the libraries employ linkers permitting selective detachment of a library member for assaying. Peptides have also been employed as tagging molecules for combinatorial libraries. Two exemplary approaches are described in the art, both of which employ branched linkers to solid phase upon which coding and ligand strands are alternately elaborated. In the first approach (Kerr JM et al. (1993) J Am Chem Soc 115:2529-2531), orthogonality in synthesis is achieved by employing acid-labile protection for the coding strand and base- labile protection for the compound strand.
In an alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170), branched linkers are employed so that the coding unit and the test compound can both be attached to the same functional group on the resin, hi one embodiment, a cleavable linker can be placed between the branch point and the bead so that cleavage releases a molecule containing both code and the compound (Ptek et al. (1991) Tetrahedron Lett 32:3891-3894). In another embodiment, the cleavable linker can be placed so that the test compound can be selectively separated from the bead, leaving the code behind. This last construct is particularly valuable because it permits screening of the test compound without potential interference of the coding groups. Examples in the art of independent cleavage and sequencing of peptide library members and their corresponding tags has confirmed that the tags can accurately predict the peptide structure.
2) Non-sequenceable Tagging: Binary Encoding
An alternative form of encoding the test compound library employs a set of non- sequencable electrophoric tagging molecules that are used as a binary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926). Exemplary tags are haloaromatic alkyl ethers that are detectable as their trimethylsilyl ethers at less than femtomolar levels by electron capture gas chromatography (ECGC). Variations in the length of the alkyl chain, as well as the nature and position of the aromatic halide substituents, permit the synthesis of at least 40 such tags, which in principle can encode 2^0 (e.g., upwards of 1012) different molecules, hi the original report (Ohlmeyer et al., supra) the tags were bound to about 1% of the available amine groups of a peptide library via a photocleavable o-nitrobenzyl linker. This approach is convenient when preparing combinatorial libraries of peptide-like or other amine-containing molecules. A more versatile system has, however, been developed that permits encoding of essentially any combinatorial library. Here, the compound would be attached to the solid support via the photocleavable linker and the tag is attached through a catechol ether linker via carbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem 59:4723-4724). This orthogonal attachment strategy permits the selective detachment of library members for assays in solution and subsequent decoding by ECGC after oxidative detachment ofthe tag sets. Although several amide-linked libraries in the art employ binary encoding with the electrophoric tags attached to amine groups, attaching these tags directly to the bead matrix provides far greater versatility in the structures that can be prepared in encoded combinatorial libraries. Attached in this way, the tags and their linker are nearly as unreactive as the bead matrix itself. Two binary-encoded combinatorial libraries have been reported where the electrophoric tags are attached directly to the solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031) and provide guidance for generating the subject compound library. Both libraries were constructed using an orthogonal attachment strategy in which the library member was linked to the solid support by a photolabile linker and the tags were attached through a linker cleavable only by vigorous oxidation. Because the library members can be repetitively partially photoeluted from the solid support, library members can be utilized in multiple assays. Successive photoelution also permits a very high throughput iterative screening strategy: first, multiple beads are placed in 96-well microtiter plates; second, compounds are partially detached and transferred to assay plates; third, a metal binding assay identifies the active wells; fourth, the corresponding beads are rearrayed singly into new microtiter plates; fifth, single active compounds are identified; and sixth, the structures are decoded.
Dosages
The dosage of any compositions ofthe present invention will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the subject composition. Any of the subject formulations may be administered in a single dose or in divided doses. Dosages for the compositions of the present invention may be readily determined by techniques known to those of skill in the art or as taught herein.
In certain embodiments, the dosage of the subject compounds will generally be in the range of about 0.01 ng to about 10 g per kg body weight, specifically in the range of about 1 ng to about 0.1 g per kg, and more specifically in the range of about 100 ng to about 10 mg per kg.
An effective dose or amount, and any possible affects on the timing of administration of the formulation, may need to be identified for any particular composition ofthe present invention. This may be accomplished by routine experiment as described herein, using one or more groups of animals (preferably at least 5 animals per group), or in human trials if appropriate. The effectiveness of any subject composition and method of treatment or prevention may be assessed by administering the composition and assessing the effect ofthe administration by measuring one or more applicable indices, and comparing the post-treatment values of these indices to the values ofthe same indices prior to treatment. The precise time of admimstration and amount of any particular subject composition that will yield the most effective treatment in a given patient will depend upon the activity, pharmacokinetics, and bioavailability of a subject composition, physiological condition of the patient (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage and type of medication), route of administration, and the like. The guidelines presented herein may be used to optimize the treatment, e.g., determining the optimum time and/or amount of administration, which will require no more than routine experimentation consisting of monitoring the subject and adjusting the dosage and/or timing.
While the subject is being treated, the health of the patient may be monitored by measuring one or more of the relevant indices at predetermined times during the treatment period. Treatment, including composition, amounts, times of administration and formulation, m ay b e o ptimized a ccording t o t he r esults of such monitoring. The patient may be periodically reevaluated to determine the extent of improvement by measuring the same parameters. Adjustments to the amount(s) of subject composition administered and possibly to the time of administration may be made based on these reevaluations.
Treatment may be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage may be increased by small increments until the optimum therapeutic effect is attained.
The use of the subject compositions may reduce the required dosage for any individual agent contained in the compositions because the onset and duration of effect of the different agents maybe complimentary.
Toxicity and therapeutic efficacy of subject compositions may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50. The data obtained from the cell culture assays and animal studies may be used in formulating a range of dosage for use in humans. The dosage of any subject composition lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For compositions of the present invention, the therapeutically effective dose may be estimated initially from cell culture assays.
Kits
This invention also provides kits for conveniently and effectively implementing the methods of this invention. Such kits comprise any subject composition, and a means for facilitating compliance with methods of this invention. Such kits provide a convenient and effective means for assuring that the subject to be treated takes the appropriate active in the correct dosage in the correct manner. The compliance means of such kits includes any means which facilitates administering the actives according to a method of this invention. Such compliance means include instructions, packaging, and dispensing means, and combinations thereof. Kit components may be packaged for either manual or partially or wholly automated practice ofthe foregoing methods, h other embodiments involving kits, this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use.
Exemplification
The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. Reagents and General Procedures
Analysis of Inhibition of [3H]PDBU Binding by Nonradioactive Ligands. Enzyme-ligand interactions were analyzed by competition with [3H]PDBU binding to the single isozyme PKCα as described previously. Nacro, K.; Bienfait, B.; Lee, J.; Han, K.-C; Kang, J.-H.; Benzaria, S.; Lewin, N. E.; Bhattacharyya, D. K.; Blumberg, P. M.; Marquez, V. E., J. Med. Chem.2000, 43, 921-944.
Cells and Cell Culture. A well characterized cell line from an AD patient (AG06848) was obtained from the Coriell Cell Repository (Ca den, NJ), then cultured and maintained as described, except that the plastic material was a 6 cm diameter petri dish. Ibarreta, D.; Duchen, M.; Ma, D.; Qiao, L.; Kozikowski, A. P.; Etcheberrigaray, R., NeuroReport 1999, 10, 1035-1040. Cells were typically grown to confluence, which took approximately 4-5 days. Cells were maintained in a complete medium until 2 h prior to treatment. At that point, the medium was replaced by DMEM without serum and left undisturbed for 2 h. Then, the cells were treated with 0.1 or 1 μM of the compounds to be tested. 8-(l- Decynyl)benzolactam V (BL) and DMSO were used as positive and negative controls, respectively. The concentration of DMSO was maintained at less than 1% in all cases. The medium was collected after 3 h to measure the sAPP-α secretion. sAPPα Determinations. The concentration of secreted sAPPα was measured using conventional immunoblotting techniques, following with minor modifications the protocol described elsewhere. Dumbar, G. S. Protein Blotting - A Practical Approach. J-RL Press: Oxford, 1994; Ibarreta, D.; Duchen, M.; Ma, D.; Qiao, L.; Kozikowski, A. P.; Etcheberrigaray, R., NeuroReport 1999, 10, 1035-1040. Precipitated protein extracts form each dish treatment were loaded to freshly prepared 8% acrylamide TrisΗCl minigels and separated by SDPAGE. The volume of sample loaded was corrected for total cell protein per dish. Proteins were then electrophoretically transferred to PVDF membranes. Membranes were saturated with 5% non-fat dry milk to block non-specific binding. Blocked membranes were incubated overnight at 4 °C with the commercially available antibody 6E10 (1:500), which recognizes sAPP-alpha in the conditioned medium (SENETEK).41,52 After washing, the membranes were incubated at room temperature with horseradish peroxidase conjugated anti-mouse IgG secondary antibody (Jackson's Laboratories). The signal was then detected using enhanced chemiluminescence followed by exposure of Hyperfϊlm ECL (Amersham). The band intensities were quantified by densitometry using a BioRad GS-800 calibrated scanning densitometer and Multianalyst software (BioRad). Hyperplasia Studies. Mice were 7 weeks old at the beginning ofthe treatments and were in the resting phase of the hair cycle. Compounds were dissolved in 0.2 mL acetone and either were applied once or else were applied twice weekly for a total of four applications. 2 animals were treated at each dose of compound. Seventy-two hours after the last application, the animals were euthanized and two portions of treated skin were removed from each animal, fixed in neutral buffered formalin, and stained with hematoxylin and eosin for histological analysis (staining of sections was performed by American Histolabs, Gaithersburg, MD). TPA (12-O-tetradecanoylphorbol 13-acetate) and mezerein were from LC Laboratories (Woburn, MA).
Analytical Techniques. Analytical and preparative thin-layer chromatography (TLC) was performed in a solvent-vapor-saturated chamber on EM Science silica gel 60 F-254 plates.
Spots were visualized by UV. Melting points (uncorrected) were determined in open capillaries on a Thomas Hoover apparatus. Infrared spectra were obtained on an ATI
Mattson Genesis Series spectrometer in KBr pellets. NMR spectra were recorded on a
Varian instrument ( 1 H frequency: 300 MHz) using TMS as an internal standard ( 1 H and
13 C) or CFCI3 as internal or external standard. Elemental analyses were performed by
Micro-Analysis, hie, and the results were within 0.4% of the theoretical value. Mass spectra were obtained on a Shimadzu QP-5000 mass spectrometer using a direct inlet probe and an electron beam energy of 70 eV. Determinations of purity by HPLC were performed with a Shimadzu LC-10 AD pump and a Waters 484 tunable absorbance detector using the following conditions: A) Supelco Discovery RP Amide Ci6 250 mm x 3.0 mm; flow rate = 0.5 mL/min; detection at 280 nm; 0-30 min, 40-80% acetonitrile in water; 30-60 min, 80% acetonitrile in water. B) Waters μBondapak Cl8 300 mm x 7.8 mm; flow rate = 2.8 mL/min; detection at 280 nm; 0-30 min, 40-80 % acetonitrile in water; 30-60 min, 80% acetonitrile in water. C) Supelco Discovery RP Amide Ci6250 mm x 3.0 mm; flow rate = 0.4 mL/min; detection at 280 nm; 0-15 min, 0-100 % methanol in water; 15-50 min, methanol.
Example 1 (2S,5S)-0-Acetyl-8-nitrobenzoIactam V (2). A mixture of (2S,5S)-O-acetylbenzolactam V (1) (280 mg, 1.06 mmol) in Ac2θ (1.2 mL) was stirred at 0 °C. To this solution was added dropwise over 5 min a chilled mixture of Ac2θ (1.2 mL) and 70% HNO3 (268 mg, 3.11 mmol). The reaction was allowed to proceed for 4-5 min at 0 °C and then was stopped by adding water (50 mL) in one portion to the vigorously stirred solution. Extraction with EtOAc (4 x 40 mL), followed by evaporation and column chromatography on silica gel (1/1 EtOAc/hexane as eluent) provided 340 mg (92%) of the product: mp 180-182 °C, yellow prisms (EtOH); [α]2°D -1017 (c 1.44, CHCI3); IR (KBr) 1743, 1670, 1505, 1326, 1231 cm"
l; lH NMR (CDCI3) δ 0.77 (d, 3H, J= 6.8 Hz), 1.06 (d, 3H, J= 6.3 Hz), 2.13 (s, 3H), 2.50
(m, IH), 2.93 (s, 3H), 2.96 (d, IH, J= 17.5 Hz), 3.44 (dd, IH, J = 7.2, 17.5 Hz), 3.63 (d, IH, J= 10.3 Hz), 3.68 (m, IH), 3.94 (dd, IH, J= 9.4, 11.1 Hz), 4.29 (dd, IH, J= 4.1, 11.2
Hz), 5.97 (s, IH), 6.90 (d, IH, J= 9.0 Hz), 8.01 (d, IH, J= 2.4 Hz), 8.08 (dd, IH, J= 2.7,
9.0 Hz); 13C NMR (CDCI3) δ 18.9, 20.5, 27.5, 33.5, 37.5, 51.5, 66.8, 66.9, 116.3, 123.8,
128.0, 139.2, 156.3, 170.2, 171.5; MS m/z 349 (M+, 26%), 332, 306, 278, 203, 177, 133, 43 (100%). Anal. (C17H23N3O5) C, H, N.
Example 2
General Procedure for the Synthesis of (2S,5S)-8-(AcyIamino)benzolactams V (5a-d).
A mixture of nitro compound 2 (100 mg, 287 μmol) and 10% Pd-C (45 mg) in ethanol (20 mL) was shaken in a Parr apparatus under 4.0 atm of hydrogen at room temperature for 3 h. The mixture was concentrated, and the residue was dried under vacuum for 1 h at 60 °C. To the crude amine 3, dry THF (4 mL) was added under inert atmosphere, followed by dry Et3N (0.5 mL) and the acyl chloride (1.6 eq). The mixture was stirred at room temperature for 4 h, then filtered and concentrated. Preparative TLC (ethyl acetate as developing solvent) provided an intermediate, which was dissolved in ethanol (30 mL). A solution of sodium carbonate (60 mg) in water (6 mL) was added with vigorous stirring. After 1.5-2 h at room temperature, the solution was concentrated and water (20 mL) added. Extraction with ethyl acetate (4 x 30 mL) provided the crude amides 5a-d which were purified by preparative TLC as above.
Example 3
(2S,5S)-(J- ^E)-8-(2,4-Hexadienoylamino)benzolactam V (5a). The title compound was obtained according to the general procedure m 38% yield as yellow oil: [α] 20 £> -253° (c
0.80, MeOH); IR (KBr) 1646, 1539, 1506 cm"1; NMR (CDCI3) δ 1.06 (d, 3H, J= 6.8 Hz), 1.11 (d, 3H, J= 6.8 Hz), 1.85 (d, 3H, J = 6.1 Hz), 2.38 (m, IH), 2.60 (s, 3H), 2.63, 2.85 (ABq, 2 H, J= 15.6 Hz, both parts d with J= 10.5 and 5.4 Hz, resp.), 3.32 (d, IH, J= 5.6 Hz), 3.47-3.56 (m, IH), 3.64-3.73 (m, IH), 4.06 (br s, IH), 4.94 (br s, IH), 5.96 )d, 1 H, J= 15.1 Hz), 6.03-6.25 (m, 2H), 6.37 (s, IH), 6.96 (d, IH, J= 8.8 Hz), 7.23 (dd, IH, J = 10.3, 14.9 Hz), 7.43 (br d, IH, J= 7.3 Hz), 7.61 (dd, IH, J= 2.0, 8.3 Hz), 8.02 (br s, IH);
13C NMR (CDCI3) δ 18.2, 18.6, 20.9, 28.9, 37.0, 38.9, 52.5, 64.2, 78.1, 119.7, 122.4,
122.6, 123.0, 129.9, 134.3, 135.3, 137.9, 141.6, 147.7, 164.7, 174.6; MS m/z 371 (M+, 13%), 340, 276, 255, 95, 44 (100%); HPLC retention time 9.5 min (96.1% purity) using conditions A, 29.6 min (96.9% purity) using conditions B.
Example 4
(2S,5S)-8-(Hexanoylamino)benzolactam V (5b). The title compound was obtained according to the general procedure in 35% yield as colorless oil: [α] 20 D -166 (c 0.85,
MeOH); IR (KBr) 3290 (br), 1650, 1540, 1506 cm'1; *H NMR (CDCI3) δ 0.92 (t, 3H, J= 6.8 Hz), 1.02 (d, 3H, J= 6.8 Hz), 1.10 (d, 3H, J= 6.8 Hz), 1.27-1.42 ( , 4H), 1.68 (m, 2H), 2.27 (t, 2H, J= 7.4 Hz), 2.38 (m, IH), 2.64 (s, 3H), 2.72, 2.88 (ABq, 2H, J= 15.6 Hz, both parts d with J = 9.8 and 4.8 Hz, resp.), 3.32 (d, IH, J= 5.9 Hz), 3.46-3.56 (m, IH), 3.64- 3.73 (m, IH), 4.01 (br s, IH), 4.85 (br s, IH), 6.50 (s, IH), 7.00 (d, IH, J= 8.8 Hz), 7.31 (d, IH, J= 7.1 Hz), 7.48 (dd, IH, J= 2.2, 8.8 Hz), 7.69 (s, IH); 13C NMR (CDCI3) δ 14.0, 18.4, 20.7, 22.5, 25.4, 28.8, 31.5, 37.1, 37.4, 38.6, 52.6, 64.3, 77.3, 119.7, 122.6, 134.2,
134.8, 147.8, 171.9, 174.4; MS m/z 375 (M+, 14%), 344, 304, 259, 147, 44 (100%); HPLC retention time 11.6 min (98.5% purity) using conditions A, 29.6 min (98.9% purity) using conditions C.
Example 5
(2S,5S)-(J5 B)-8-(5-Plιenyl-2,4-pentadienoylamino)benzolactam V (5c). The title compound was obtained according to the general procedure in 31% yield as a yellow oil: [α]20D -187 (c 0.56, MeOH); IR (KBr) 1653, 1540, 1507 cm"1; lJi NMR (CDCI3) δ 1.07 (d, 3H, J= 6.8 Hz), 1.12 (d, 3H, J= 6.8 Hz), 2.40 (m, IH), 2.63 (s, 3H), 2.70, 2.88 (ABq, IH, J = 15.8 Hz, both parts d with J= 10.3 and 5.2 Hz, resp.), 3.36 (d, IH, J = 5.6 Hz), 3.48-3.58 (m, IH), 3.66-3.76 (m, 2H), 4.88 (br s, IH), 6.18 (d, IH, J= 14.9 Hz), 6.51 (s, IH), 6.81-6.95 (m, 2H), 7.00 (d, IH, J= 8.8 Hz), 7.25-7.49 (m, 7H), 7.58 (d, IH, J = 7.6 Hz), 7.92 (s, IH); 13C NMR (CDCI3) δ 18.4, 20.9, 28.8, 37.1, 38.6, 52.7, 64.4, 77.5, 119.8, 122.6, 122.8, 125.0, 126.6, 127.0, 128.7, 128.8, 134.2, 134.9, 136.3, 139.3, 141.3, 147.9,
164.3, 174.6; MS m/z 433 (M+, 46%), 402, 362, 317, 276, 157, 128, 44 (100%); HPLC retention time 20.3 min (98.3% purity) using conditions A, 26.0 min (96.4% purity) using conditions B.
Example 6
(2S,5S)-8-(5-Phenylpentanoylamino)benzolactam V (5d). A mixture of 5c (40.0 mg, 92 μ ol), Pd/C (10%) (20 mg), and methanol (20 mL) was shaken under 3.3 atm of H2 at room temperature for 19 h. Filtration from the catalyst, evaporation, and thin-layer
25 chromatography (EtOAc as eluent) provided 5d (36.1 mg, 90%) as a colorless oil: [α] -
129 (c 1.8, MeOH); IR (KBr) 1652, 1541, 1507 cm"1; lR NMR (CDCI3) δ 1.02 (d, 3H, J= 6.8 Hz), 1.09 (d, 3H, J= 6.6 Hz), 1.61-1.79 (m, 4H), 2.20-2.42 (m, 3H), 2.60 (s, 3H), 2.61- 2.68 (m, 2H), 2.68, 2.85 (ABq, 2H, J = 16.0 Hz, both parts d with J = 10.0 and 5.0 Hz, resp.), 3.28 (d, IH, J= 5.6 Hz), 3.45-3.54 (m, IH), 3.62-3.70 (m, IH), 4.06 (br s, IH), 4.85 (br s, IH), 6.46 (s, IH), 6.93 (d, IH, J= 8.5 Hz), 7.13-7.21 (m, 3 H), 7.24-7.34 (m, 3H),
7.45 (dd, IH, J = 1.6, 8.7 Hz), 7.72 (s, IH); 13C NMR (CDCI3) δ 18.3, 20.8, 25.3, 28.8, 31.2, 35.7, 37.1, 37.2, 38.6, 52.6, 64.2, 77.5, 119.7, 122.5, 122.6, 125.7, 128.3, 128.4,
134.3, 134.8, 142.2, 147.8, 171.6, 174.5; MS m/z 437 (M+, 12%), 406, 366, 321, 261, 147, 91, 44 (100%); HPLC retention time 19.9 min (97.9% purity) using conditions A, 30.4 min (97.3% purity) using conditions C.
Example 7
(2S-5S)-(E E -8-[5-[4-(Trifluoromethyl)phenyl]-2,4-pentadienoylamino]benzolactam V (5e). A mixture of 6 (0.91 g, 3.55 mmol), KOH (0.60 g, 10.7 mmol), methanol (30 mL) and water (2 mL) was stirred at room temperature for 22 h. Solvents were evaporated, and the residue was dissolved in a minimum amount of water. The resulting solution was acidified with 3M HCl. The precipitate which formed immediately was separated by filtration and dried under vacuum. This product (0.74 g, 3.06 mmol) and SOCI2 (6.6 mL) were heated at
70 °C for 3 h. Evaporation ofthe excess of SOCI2 provided the crude acid chloride, which was used in the synthesis of compound 5e (see general procedure above) without
9ft purification. Compound 5e: yield 42%; yellow oil; [α] υD -165 (c 0.52, MeOH); IR (KBr)
1653, 1541, 1507, 1324 cm"1; ^ NMR ^DCls) δ 1.11 (d, 3H, J= 7.1 Hz), 1.14 (d, 3H, J
= 6.8 Hz), 2.41 (m, IH), 2.62 (s, 3H), 2.67, 2.90 (ABq, 2H, J= 15.8 Hz, both parts d withJ = 5.7 and 10.2 Hz, resp.), 3.35 (d, IH, J= 5.4 Hz), 3.52-3.61 (m, IH), 3.69-3.78 (m, IH), 3.93 (br s, IH), 5.00 (br s, IH), 6.26 (d, IH, J= 14.9 Hz), 6.33 (s, IH), 6.80-7.02 (m, 3H),
7.38-7.68 (m, 7 H), 8.05 (br s, IH); 13C NMR (CDCI3) 18.1, 21.0, 28.9, 37.0, 39.1, 52.5, 64.1, 78.6, 119.8, 122.4, 123.2, 124.0 (q, J= 272 Hz), 125.6 (q, J= 3.5 Hz), 126.7, 127.0,
128.9, 130.1 (q, J= 32.7 Hz), 134.7, 137.2, 139.7, 140.4, 147.9, 164.1, 174.8; MS m/z 501
(M+, 94%), 470, 430, 415, 385, 276, 225 (100%), 177; HPLC retention time 26.0 min (98.9% purity) using conditions A, 20.2 min (97.3% purity) using conditions B. Example 8
25 (2S,55)-8-[5-[4-(Trifluoromethyl)phenyl]pentanoylamino]benzolactam V (5f): [α] D -
127 (c 0.70, MeOH); IR (KBr) 3290 (br), 1649, 1504, 1326, 1121, 1068 cm"1; XH NMR (CDCI3) δ 1.02 (d, 3H, J= 6.8 Hz), 1.10 (d, 3H, J= 6.8 Hz), 1.63-1.82 (m, 4H), 2.21-2.43 (m, 3H), 2.60 (s, 3H), 2.65-2.78 (m, 3H), 2.87 (dd, IH, J= 5.0, 15.7 Hz), 3.28 (d, IH, J = 5.6 Hz), 3.47-3.59 (m, IH), 3.65-3.75 (m, IH), 3.90 (br s, IH), 4.88 (br s, IH), 6.38 (br s, IH), 6.94 (d, IH, J= 8.5 Hz), 7.29 (d, 2H, J= 8.1 Hz), 7.36 (br s, IH), 7.47 (d, IH, J= 8.8
Hz), 7.52 (d, 2H, J= 8.3 Hz), 7.59 (br s, IH); 13C NMR (CDCI3) δ 18.2, 20.8, 25.2, 28.8, 30.8, 35.5, 37.0, 38.9, 52.5, 64.1, 78.1, 119.6, 122.5, 122.7, 124.3 (q, J= 272 Hz), 125.2 (q,
J= 3.5 Hz), 128.1 (q, J= 32.2 Hz), 128.7, 134.5, 135.0, 146.3, 147.8, 171.4, 174.5; MS m/z
505 (M+, 32%), 474, 462, 434, 419, 389, 261, 159, 44 (100%); HPLC retention time 24.9 min (99.7% purity) using conditions A, 19.0 min (99.5% purity) using conditions B.
Figure imgf000091_0001
(2S,5S)-(^^)-8-[5-[3,5-Bis(trifluoromethyl)phenyl]-2,4- pentadienoylamino]benzolactam V (5g). The title compound was obtained according to
9ft 1 the general procedure m 42% yield as yellow oil: [α] D -149 (c 0.25, MeOH); ΛH NMR (CDCI3) δ 1.11 (d, 3H, J = 6.8 Hz), 1.14 (d, 3H, J = 6.8 Hz), 2.43 (m, IH), 2.63 (s, 3H),
2.70, 2.91 (ABq, 2H, J= 15.8 Hz, both parts d with J= 10.3 and 5.5 Hz, resp.), 3.36 (d, IH,
J= 5.4 Hz), 3.53-3.62 (m, IH), 3.76 (br d, IH, J= 10.5 Hz), 3.88 (br s, IH), 5.01 (br s, IH),
6.31 (d, IH, J= 14.9 Hz), 6.35 (br s, IH), 6.83-7.08 (m, 3H), 7.43 (dd, IH, J = 10.5, 14.9
Hz), 7.55 (br s, IH), 7.65 (d, IH, J= 8.3 Hz), 7.76 (s, IH), 7.83 (s, 2H), 8.04 (br s, IH); 19F
NMR (CDCI3) δ -63.5 (m); 13C NMR (CDCI3) δ 18.1, 21.0, 29.0, 37.0, 39.2, 52.4, 64.0, 78.9, 119.8, 121.7 (sept, J= 3.4 Hz), 122.4, 123.1 (q, J= 273 Hz), 123.3, 126.5 (narrow m), 127.8, 130.2, 132.1 (q, J= 33.4 Hz), 134.8, 135.2, 135.3, 138.4, 139.7, 148.1, 163.7, 174.9;
MS m/z 569 (M+, 3%), 453, 279, 245, 167, 149, 44 (100%), 177; HPLC retention time 31.3 min (98.4% purity) using conditions A, 32.1 min (96.7% purity) using conditions C.
Example 10
Figure imgf000092_0001
(2S,5S)-8-[5-[3,5-Bis(trifluorometb.yl)phenyl]pentanoyIamino]benzolactam V (5h). A mixture of 5g (40.0 mg, 70 μmol), Pd/C (10%) (20 mg), and methanol (20 mL) was stirred under 1 atm of H2 at room temperature for 0.5 h. Filtration from the catalyst, evaporation, and thin-layer chromatography (EtOAc as eluent) provided 5h (37.5 mg, 93%) as a colorless oil: [α]25D -101 (c 0.26, MeOH); IR (KBr) 3410 (br), 1642, 1540, 1505, 1380,
1280, 1128 cm"1; NMR (CDCI3) δ 1.01 (d, 3H, J= 6.8 Hz), 1.10 (d, 3H, J= 6.6 Hz),
1.69-1.82 (m, 4H), 2.28-2.45 (m, 3H), 2.64 (s, 3H), 2.71-2.84 (m, 3H), 2.88 (dd, IH, J = 5.3, 16.0 Hz), 3.31 (d, IH, J = 6.1 Hz), 3.48-3.60 (m, 2H), 3.66-3.78 (m, IH), 4.81 (br s,
IH), 6.48 (br s, IH), 6.97 (d, IH, J= 8.8 Hz), 7.24 (br s, IH), 7.39-7.47 (m, 2H), 7.64 (s,
2H), 7.70 (s, IH); 19F NMR (CDCI3) δ -63.2 (m); 13C NMR (CDCI3) δ 18.5, 20.7,- 25.1, 28.8, 30.7, 35.4, 36.9, 37.1, 38.4, 52.7, 64.4, 77.2, 119.7, 119.9 (sept, J = 3.8 Hz), 122.3, 122.7, 123.4 (q, J= 272 Hz), 128.5 (narrow m), 131.5 (q, J= 32.7 Hz), 134.2, 134.4, 144.6, 148.0, 171.0, 174.3; MS m/z 573 (M+, 4%), 542, 502, 403, 261, 227, 44 (100%); HPLC retention time 29.3 min (99.0% purity) using conditions A, 31.7 min (98.3% purity) using conditions C. Example 11
Figure imgf000093_0001
(2S,55)-(^^^)-8-[5-[4-(3,3-4,4-5-5,6,6-7-7,8,8,8-tridecafluorooct-l-enyl)phenyl]-2-4- pentadienoylaminojbenzolactam V (5i). The title compound was obtained according to
9ft 1 the general procedure in 41% yield as a yellow oil: [of D -87 (c 0.185, MeOH); Η NMR
(CDCI3) δ 1.09 (d, 3H, J= 6.8 Hz), 1.13 (d, 3H, J = 6.8 Hz), 2.41 (m, IH), 2.63 (s, 3H),
2.67-2.77 (m, IH), 2.90 (dd, IH, J= 5.3, 16.0 Hz), 3.36 (d, IH, J= 5.6 Hz), 3.50-3.61 (m,
IH), 3.68-3.79 (m, 2H), 4.95 (br s, IH), 6.20 (dt, IH, J= 11.8 Hz (t), 16.1 Hz (d)), 6.22 (d,
IH, J= 14.6 Hz), 6.39 (br s, IH), 6.83, 6.95 (ABq, 2H, J= 15.6 Hz, B part d withJ= 10.7 Hz), 6.95 (dd, IH, J= 10.6, 15.5 Hz), 7.00 (d, IH, J= 8.8 Hz), 7.14 (dt, IH, J= 16.1 Hz
(d), 2.4 Hz (t)), 7.38-7.54 (m, 6H), 7.58-7.66 (m, IH), 7.93 (br s, IH); 19F NMR (CDCI3) δ -126.7 (m, 2F), -123.6 (m, 2F), -123.4 (br s, 2F), -122.1 (br s, 2F), -111.6 (m, 2F), -81.3 (m,
3F); 13C NMR (CDCI3) δ 18.3, 21.0, 28.9, 37.0, 38.9, 52.6, 64.3, 78.1, 114.4 (t, J= 22.9 Hz), 119.9, 122.5, 123.0, 126.1, 127.5, 128.0, 128.1, 133.6, 134.4, 135.1, 137.9, 138.2, 139.0 (t, J - 9.1 Hz), 140.8, 147.9, 164.1, 174.7; HPLC retention time 43.8 min (96.8% purity) using conditions A, 33.3 min (99.0% purity) using conditions C.
Example 12
Figure imgf000093_0002
(2S,5S)-8-[5-[4-(3,3-4,4,5,5,6,6,7,7,8,8,8- tridecafluorooctyl)phenyl]pentanoylamino]benzolactam V (5j). A mixture of 5i (40.0 mg, 51 μmol), Pd/C (10%) (20 mg), and methanol (20 mL) was stirred under 1 atm of H2 at room temperature for 0.5 h. Filtration from the catalyst, evaporation, and thin-layer 20 chromatography (EtOAc as eluent) provided 5j (38.6 mg, 96%) as a colorless oil: [α] D -
88 (c 1.24, MeOH); !H NMR (CDCI3) δ 1.02 (d, 3H, J= 6.8 Hz), 1.10 (d, 3H, J= 6.8 Hz),
1.60-1.80 (m, 4H), 2.23-2.44 (m, 5H), 2.62 (s, 3 H), 2.62-2.66 (m, 1 H), 2.73 (dd, 1 H, J=
9.9, 16.0 Hz), 2.81-2.92 (m, 4H), 3.30 (d, IH, J= 6.1 Hz), 3.47-3.58 (m, IH), 3.65-3.82 (m, 2H), 4.84 (br s, IH), 6.45 (br s, IH), 6.95 (d, IH, J= 8.5 Hz), 7.07-7.17 (m, 4H), 7.28-7.36
19 (m, IH), 7.44 (br d, IH, J= 8.8 Hz), 7.54 (br s, IH); ι yF NMR (CDCI3) δ -126.7 (m, 2F),
124.1 (br s, 2F), -123.4 (br s, 2F), -122.4 (br s, 2F), -115.2 (m, 2F), -81.3 (m, 3F); 13C NMR (CDCI3; weak multiplets of fluorinated C omitted) δ 18.2, 20.8, 25.3, 25.9 (t, J= 4.0
Hz), 28.9, 31.2, 33.0 (t, J= 22.2 Hz), 35.3, 37.0, 37.2, 38.9, 52.5, 64.1, 78.2, 119.7, 122.4, 122.7, 128.2, 128.8, 134.5, 135.1, 136.4, 140.7, 147.7, 171.6, 174.6; HPLC retention time 44.1 min (96.6% purity) using conditions A, 33.6 min (98.9% purity) using conditions C.
Example 13
(E,E)-Methyl 5-[(4-Trifluoromethyl)phenyl]-2,4-pentadienoate (6). 3-Methoxycarbonyl allylidenetriphenylarsorane (15.0 g, 30.0 mmol), 4-(trifluoromethyl) benzaldehyde (3.92 g,
22.5 mmol), potassium carbonate (31.0 g, 225.0 mmol), methylene chloride (100 mL) and water (1.15 mL) were placed in a flask under nitrogen atmosphere. Huang, Y.; Shen, Y.;
Zheng, J.; Zhang, S., Synthesis 1985, 57-58. The mixture was stirred for 22 h at room temperature. The solid residue was collected by filtration and washed with methylene chloride. Evaporation of the filtrate followed by column chromatography on silica gel
(hexane, then 7/1 hexane/ethyl acetate as eluent) and crystallization from methanol provided ester 6 in a 57% yield. Colorless needless; mp 114-115 °C; 1H NMR (CDC13) δ 3.77 (s,
3H), 6.05 (d, IH, J= 14.9 Hz), 6.82-6.98 (m, 2H), 7.39-7.60 (m, 5H); 13C NMR (CDC13) δ
51.5, 122.3; 123.9 (q, J= 271.9 Hz), 125.6 (q, J= 3.9 Hz), 127.1, 128.3, 130.3 (q, J= 32.6 Hz), 138.3, 139.2, 143.8, 167.0; MS m/z 256 ([M*], 39%), 225, 197, 177 (100%), 128.
Anal. (Cι3HπF3θ2) C, H. Example 14 General Procedure for the Synthesis of Compounds 7:
The nitro compound 8 (100 mg, 0.287 mmol) was dissolved in ethanol (15 mL), and 10% Pd-C (15 mg, 0.014 mmol) was added to the reaction mixture. The mixture was vigorously stirred under a H atmosphere overnight, then filtered, and the filtrate was concentrated to give a residue, which was dried under vacuum for 1 h at 60 °C. To a solution of the residue in anhydrous tetrahydrofuran (4 mL) under nitrogen was added anhydrous triethylamine (0.5 mL) and diacid chloride (0.143 mmol). The reaction mixture was stirred at room temperature for 4 h, then filtered and concentrated. Preparative TLC (10/1 ethyl acetate/ethanol as developing solvent) provided the acetyl-protected diamide, which was dissolved in ethanol (30 mL). A solution of sodium carbonate (60 mg) in water (6 mL) was added with vigorous stirring. After 1.5 h at room temperature, the solution was concentrated and an additional amount of water (20 mL) added. Extraction with ethyl acetate (4 x 30 mL) provided the diamide with free hydroxyl groups which was purified by preparative TLC (10/1 ethyl acetate/ethanol as developing solvent).
Example 15 Hexanedioic Acid Bis-[N-(2S,5S)-1 ,2-3,4,5,6-hexahydro-5-(hydroxymethyl)-2- isopropyl-l-methyI~3-oxo-benzo[e][l,4]diazocin-8-yI]amide (7a). Yield 18%. [α]20 D -249 (c 0.65, EtOH); IR (KBr) 3368, 1647, 1636, 1506 cm-1; 1H NMR (CD3OD) δ 0.98 (d, 6H, J = 6.8 Hz), 1.12 (d, 6H, J= 6.8 Hz), 1.71-1.78 (m, 4H), 2.31-2.44 (m, 6H), 2.72 (s, 3H), 2.81 (dd, 2H, J= 9.3, 15.9 Hz), 3.07 (dd, 2H, J= 4.4, 16.2 Hz), 3.38 (d, 2H, J = 6.6 Hz), 3.52 (dd, 2H, J= 6.8, 11.0 Hz), 3.59 (dd, 2H, J= 4.4, 11.2 Hz), 4.69-4.78 (m, 2H), 7.17 (d, 2H, J = 8.5 Hz), 7.30 (d, 2H, J= 2.4 Hz), 7.36 (dd, 2H, J= 2.4, 8.5 Hz); 13C NMR (CD3OD) δ 19.5, 21.3, 26.7, 30.1, 37.8, 38.5, 39.0, 54.7, 65.6, 77.4, 121.1, 123.9, 124.6, 135.7, 136.1, 150.0, 174.2, 175.9; MS m/z 319, 277, 149, 72, 45; HPLC retention time 6.3 min (98.1% purity) using conditions A, 29.2 min (98.5% purity) using conditions B. Example 16 Octanedioic Acid Bis-[7V-(2S,5S)-l,2,3,4,5,6-hexahydro-5-(hydroxymethyl)-2- isopropyl-l-methyl-3-oxo-benzo[e][l,4]diazocin-8-yI]amide (7b). Yield 26%. [α]20 D -208 (c 0.13, EtOH); IR (KBr) 3374, 1658, 1642, 1502 cm"1; 1H NMR (CD3OD) δ 0.98 (d, 6H, J = 6.8 Hz), 1.12 (d, 6H, J= 6.6 Hz), 1.37-1.45 (m, 4H), 1.69 (m, 4H), 2.33 (t, 4H, J- 7.2 Hz), 2.38 (m, 2H), 2.72 (s, 6H), 2.78 (dd, 2H, J= 9.5, 16.1 Hz), 3.07 (dd, 2H, J= 4.4, 16.1 Hz), 3.38 (d, 2H, J= 6.6 Hz), 3.51 (dd, 2H, J= 6.8, 11.0 Hz), 3.59 (dd, 2H, J= 4.9, 11.0 Hz), 4.74 (m, 2H), 7.16 (d, 2H, J= 8.5 Hz), 7.31 (d, 2H, J= 2.4 Hz), 7.36 (dd, 2H, J= 2.4, 8.5 Hz); 1 3C NMR (CD3OD) δ 1 9.5, 2 1.6, 26.2, 30.0, 30.1, 37.9, 38.5, 39.0, 54.7, 65.6, 77.4, 121.0, 123.8, 124.6, 135.7, 136.1, 149.9, 174.6, 175.8; MS m/z 558, 277, 159, 147, 72, 44; HPLC retention time 8.7 min (99.2% purity) using conditions A, 11.0 min (97.0% purity) using conditions C.
Example 17 Decanedioic Acid Bis-[N-(2S,5S)-l,2,3,4,5,6-hexahydro-5-(hydroxymethyl)-2- isopropyl-l-methyl-3-oxo-benzo[e][l,4]diazocin-8-yl]amide (7c). Yield 38%. [α]20 D -203 (c 0.55, MeOH); IR (KBr) 3387, 1657, 1642, 1503 cm"1; 1H NMR (CD3OD) δ 0.97 (d, 6H, J= 6.8 Hz), 1.11 (d, H, J= 6.6 Hz), 1.30-1.41 (m, 8H), 1.67 (m, 4H), 2.31 (t, 4H, J= 7.2 Hz), 2.38 (m, 2H), 2.71 (s, 6H), 2.78 (dd, 2H, J= 9.5, 16.1 Hz), 3.07 (dd, 2H, J= 4.4, 16.1 Hz), 3.38 (d, 2H, J= 6.6 Hz), 3.51 (dd, 2H, J= 6.8, 11.0 Hz), 3.59 (dd, 2H, J= 4.6, 11.0 Hz), 4.74(m, 2H), 7.16 (d, 2H, J= 8.5 Hz), 7.31 (d, 2H, J= 2.4 Hz), 7.36 (dd, 2H, J= 2.4, 8.5 Hz); 1 3C NMR (CD3OD) δ 1 9.5, 2 1.3, 27.0, 30.1, 30.3, 30.4, 38.0, 38.5, 39.0, 54.7, 65.6, 77.3, 121.0, 123.8, 124.5, 135.7, 136.1, 149.9, 174.6, 175.8; HPLC retention time 16.2 min (96.3% purity) using conditions A, 13.6 min (97.4% purity) using conditions C.
Example 18 Dodecanedioic Acid Bis-[N-(2S,5S)-l,2,3,4,5,6-hexahydro-5-(hydroxymethyl)-2- isopropyI-l-methyI-3-oxo-benzo[e][l,4]diazocm-8-yI]amide (7d). Yield 23%. [α]20 D -200 (c 0.54, EtOH); IR (KBr) 3368, 1653, 1541, 1507 cm-1; 1H NMR (CD3OD) δ 0.97 (d, 6H, J = 6.8 Hz), 1.12 (d, 6H, J= 6.8 Hz), 1.26-1.42 (m, 12H), 1.66 (m, 4H), 2.32 (t, 4H, J= 7.3 Hz), 2.38 (m, 2H), 2.72 (s, 6H), 2.78 (dd, 2H, J= 9.5, 16.1 Hz), 3.07 (dd, 2H, J= 4.4, 16.1 Hz), 3.38 (d, 2H, J= 6.8 Hz), 3.52 (dd, 2H, J= 6.8, 11.0 Hz), 3.59 (dd, 2H, J= 4.4, 10.8 Hz), 4.74(m, 2H), 7.16 (d, 2H, J= 8.5 Hz), 7.31 (d, 2H, J= 2.4 Hz), 7.36 (dd, 2H, J= 2.4, 8.5 Hz); 1 3C NMR (CD3OD) δ 19.5, 21.3, 27.1, 30.1, 30.4, 30.5, 30.6, 38.1, 38.5, 39.0, 54.7, 65.6, 77.4, 121.1, 123.8, 124.5, 135.7, 136.1, 149.9, 174.7, 175.9; MS m/z 664, 278, 160, 147, 72, 44; HPLC retention time 24.0 min (98.0% purity) using conditions A, 29.6 min (98.2%) purity) using conditions B.
Example 19 Tetradecanedioic Acid Bis-[N-(2S,5S)-l,2,3,4,5,6-hexahydro-5-(hydroxymethyl)-2- isopropyl-l-methyl-3-oxo-benzo[e][l,4]diazocin-8-yl]amide (7e). Yield 33%. [α]20o -162 (c 0.36, MeOH); IR (KBr) 3367, 1652, 1541, 1507 cm"1; 1H NMR (CD3OD) δ 0.97 (d, 6H, J= 6.8 Hz), 1.11 (d, 6H, J= 6.6 Hz), 1.26-1.42 (m, 16H), 1.66 (m, 4H), 2.32 (t, 4H, J= 7.4 Hz), 2.39 (m, 2H), 2.72 (s, 6H), 2.78 (dd, 2H, J= 9.5, 16.1 Hz), 3.07 (dd, 2H, J= 4.2, 16.1 Hz), 3.38 (d, 2H, J= 6.6 Hz), 3.52 (dd, 2H, J= 6.8, 11.0 Hz), 3.59 (dd, 2H, J= 4.8, 11.1 Hz), 4.74 (m, 2H), 7.16 (d, 2H, J= 8.5 Hz), 7.31 (d, 2H, J= 2.4 Hz), 7.36 (dd, 2H, J= 2.2, 8.5 Hz); 1 3C NMR (CD3OD) δ 1 9.5, 2 1.3, 27.1, 30.1, 30.4, 30.6, 30.7, 30.8, 38.1, 38.5, 39.0, 54.7, 65.6, 77.4, 121.1, 123.8, 124.6, 135.7, 136.1, 149.9, 174.7, 175.8; HPLC retention time 23.6 min (98.7% purity) using conditions A, 30.7 min (98.5% purity) using conditions B.
Example 20 Hexadecanedioic Acid Bis-[N-(2S-5S)-l,2,3,4,5,6-hexahydro-5-(hydroxymethyl)-2- isopropyl-l-methyl-3~oxo-benzo[e][l,4]diazocin-8-yl]amide (7f). Yield 26%. [α]20 D - 155.7 (c 0.415, MeOH); IR (KBr) 3368, 1650, 1542, 1506 cm"1; 1H ΝMR (CD3OD) δ 0.97 (d, 6H, J=6.6 Hz), 1.11 (d, 6H, J=6.8 Hz), 1.25-1.40 (m, 20H), 1.67 (m, 4H), 2.32 (t, 4H, j=7.4 Hz), 2.38 (m, 2H), 2.72 (s, 6H), 2.78 (dd, 2H, J=9.8, 16.1 Hz), 3.07 (dd, 2H, J=4.2, 16.1 Hz), 3.38 (d, 2H, J=6.6 Hz), 3.52 (dd, 2H, J=6.7, 11.1 Hz), 3.60 (dd, 2H, J=4.8, 11.1 Hz), 4.74 (m, 2H), 7.16 (d, 2H, J=8.5 Hz), 7.31 (d, 2H, J-2.4 Hz), 7.36 (dd, 2H, J=2.2, 8.5 Hz); 13C ΝMR (CD3OD) δ 19.5, 21.3, 27.1, 30.1, 30.4, 30.6, 30.7, 30.8, 30.9, 38.1, 38.5, 39.0, 54.7, 65.6, 77.4, 121.1, 123.8, 124.6, 135.7, 136.1, 149.9, 174.7, 175.8; HPLC retention time 33.1 min (96.6% purity) using conditions A, 32.2 min (99.9% purity) using conditions B.
Example 21
Docosanedioic Acid Bis-[N-(2S,5S)-l,2,3,4,5,6-hexahydro-5-(hydroxymethyl)-2- isopropyl-l-methyI-3-oxo-benzo[e][l,4]diazocin-8-yl]amide (7g). Yield 28%. [α]20 D - 102.0 (c 0.15, MeOH); IR (KBr) 3368, 1643, 1546, 1504 cm"1; 1H NMR (CD3OD) δ 0.97 (d, 6H, J= 6.8 Hz), 1.11 (d, 6H, J= 6.6 Hz), 1.24-1.40 (m, 32H), 1.67 (m, 4H), 2.32 (t, 4H, J= 7.4 Hz), 2.38 (m, 2H), 2.72 (s, 6H), 2.78 (dd, 2H, j=10.1, 16.2 Hz), 3.07 (dd, 2H, J = 4.0, 16.0 Hz), 3.38 (d, 2H, J=6.6 Hz), 3.52 (dd, 2H, J= 6.8, 11.0 Hz), 3.60 (dd, 2H, J= 4.8, 10.9 Hz), 4.73 (m, 2H), 7.16 (d, 2H, J= 8.5 Hz), 7.31 (d, 2H, J= 2.4 Hz), 7.36 (dd, 2H, J= 2.2, 8.5 Hz); 13C NMR (CD3OD) δ 19.5, 21.3, 27.1, 30.1, 30.5, 30.6, 30.8, 30.9 (five overlapping signals), 38.1, 38.5, 39.0, 54.7, 65.6, 77.3, 121.1, 123.8, 124.6, 135.7, 136.1, 149.9, 174.7, 175.8; HPLC retention time 50.0 min (96.0% purity) using conditions A, 34.4 min (97.9% purity) using conditions B.
Example 22 3,6,9,12-15-Pentaoxaheptadecane-l,17-dioic Acid Bis-[/V-(2S,5S)-l,2-3,4,5,6- hexahydro-5-(hydroxymethyl)-2-isopropyl-l-methyl-3-oxo-benzo[e][l,4]diazocin-8- yl]amide (7h). Yield 21%. [α]20 D -93 (c 0.55, CHC13). 1H NMR (CDC13) δ 0. 99 (d, 6H, J = 6.8 Hz), 1.11 (d, 6H, J= 6.8 Hz), 2.31-2.4 (m, 2H), 2.66 (s, 6H), 2.84 (dd, 2H, J= 9.5, 15.6 Hz), 2.97 (dd, 2H, J= 5.4, 15.4 Hz), 3.25-3.31 (m, 4H), 3.4-3.46 (m, 4H), 3.57-3.74 (m, 14H), 3.78-3.88 (m, 2H), 4.02 (s, 2H), 4.03 (s, 2H), 4.73 (br s, 2H), 7.03 (d, 2H, J= 2.4 Hz), 7.14 (d, 2H, J= 8.5 Hz), 7.43 (d, 2H, J= 6.6 Hz), 7.6 (dd, 2H, J= 2.4, 8.5 Hz), 9.06 (br s, 2H). 13C NMR (CDC13) δ 8.6, 20.8, 28.8, 29.7, 36.7, 38.5, 52.8, 64.4, 70.2, 70.3, 70.6,
71.1, 71.5, 120.2, 123.2, 123.7, 133.8, 135.1, 148.9, 168.4, 174.1. HPLC retention time 9.8 min (99.9% purity) using conditions A, 10.3 min (97.8% purity) using conditions C. HRMS Calcd. for (C42H64N6Oπ+H+) 829.4711, found 829.4695. Example 23 3,6,9,12,15,18-Hexaoxadocosane-l,20-dioic A cid B is-[N-(2S,5S)-l,2,3,4,5,6-hexahydro- 5-(hydroxymethyl)-2-isopropyl-l-methyl-3-oxo-benzo[e][l,4]diazocin-8-yl]amide (7i).
Yield 30%. [α]20 D -154 (c 1.0, CHC13). 1H NMR (CDC13): δl.00 (d, 6H, J= 6.8 Hz), 1.09 (d, 6H, J= 6.8 Hz), 2.27-2.41 (m, 2H), 2.65 (s, 6H), 2.69 (dd, 2H, J= 8.8, 18.1 Hz), 2.85 (dd, 2H, J= 9.7, 15.8 Hz), 2.95-3.08 (m, 4H), 3.21-3.4 (m, 4H), 3.38-3.61 (m, 20H), 4.00 (d, 2H, J= 16.1 Hz), 4.13 (d, 2H, J= 16.3 Hz), 4.83 (br s, 2H), 7.04 (d, 2H, J = 2.2 Hz), 7.14 (d, 2H, J = 8.6 Hz), 7.56 (d, 2H, J= 7.6 Hz), 7.64 (dd, 2H, J= 2.4, 8.6 Hz), 9.06 (s, 2H). 13C NMR (CDC13) δ 18.5, 20.9, 28.9, 36.7, 38.8, 52.9, 64.4, 69.7, 69.9, 70.2, 70.3, 70.8, 71.2, 120.7, 123.3, 124.2, 134.1, 135.1, 148.9, 168.6, 174.0 HPLC retention time 15.5 min (96.5% purity) using conditions A, 17.0 min (98.6% purity) using conditions C. HRMS Calcd. for
Figure imgf000099_0001
873.4973, found 873.4942.
Example 24 General Procedure for the Preparation of the Dimers 11 :
To the solution of compound 10 (45.0 mg, 0.133 mmol) in pyridine (0.5 mL) and CH2C1 (0.5 mL) was added diacyl chloride (0.060 mmol). The reaction mixture was stirred under N2 at room temperature for 36 h. Then the resulting solution was diluted with ethyl acetate (80 mL), washed with 1 N HCl (5 mL x 2), saturated aqueous NaHCO , and brine, dried over anhydrous Na2SO4, and concentrated. The residue was chromatographed on silica gel (25/1 CH2Cl2/acetone) to afford the dimer and recovered compound 10.
Example 25 Hexanedioic Acid Bis-[4-[(6U, 7R, 7aS)-5,6,7,7a-tetrahydro-(6-isopropyl-3,3-dimethyI- 5-oxo-lfl-pyrrolo[l,2-c]oxazol-7-yl)]-l-naphthyl] ester (11a). Yield 69%; [α]D +287 (c 1.0, CHC13); 1H NMR (CDC13) δ 0.80 (d, 6H, J= 7.2 Hz), 0.87 (d, 6H, J= 6.6 Hz), 1.34- 1.45 (m, 2H), 1.60 (s, 6H), 1.79 (s, 6H), 2.03-2.12 (m, 4H), 2.84-2.94 (m, 4H), 3.25 (dd, 2H, J= 8.7, 3.6 Hz), 3.54 (t, 2H, J= 9.0 Hz), 4.15 (t, 2H, J= 9.0 Hz), 4.22 (dd, 2H, J= 8.7, 5.7 Hz), 4.99 (td, 2H, J = 9.3, 5.7Hz), 7.26 (d, 2H, J= 8.1 Hz), 7.49 (d, 2H, J = 8.1 Hz), 7.53-7.63 (m, 4H), 7.93-7.99 (m, 4H); 13C NMR (CDC13) δ 18.9, 22.5, 24.0, 24.7, 27.1, 27.3, 34.2, 46.3, 59.6, 62.7, 69.9, 91.8, 117.5, 122.3, 123.4, 123.6, 126.9, 127.3, 127.5, 130.9, 133.4, 146.4, 171.4, 171.9. Example 26 Octanedioic Acid Bis-[4-[(6R, 7R, 7aS)-5,6,7,7a-tetrahydro-(6-isopropyl-3,3-dimethyl- 5-oxo-l#-pyrroIo[l,2-φxazol-7-yl)]-l-naphthyl] ester (lib). Yield 48%; [α]D +251 (c 1.0, CHC13); 1H NMR (CDC13) δ 0.79 (d, 6H, J= 6.9 Hz), 0.87 (d, 6H, J = 6.9 Hz), 1.33- 1.45 (m, 2H), 1.55-1.67 (m, 10H), 1.78 (s, 6H), 1.87-2.00 (m, 4H), 2.80 (t, 4H, J= 7.5 Hz), 3.25 (dd, 2H, J= 8.7, 3.3 Hz), 3.54 (t, 2H, J= 8.7 Hz), 4.14 (t, 2H, J= 9.3 Hz), 4.21 (dd, 2H, J= 8.4, 5.7 Hz), 4.99 (td, 2H, J= 9.3, 5.7Hz), 7.25 (d, 2H, J= 8.4 Hz), 7.48 (d, 2H, J= 7.8 Hz), 7.53-7.63 (m, 4H), 7.92-7.99 (m, 4H); 13C NMR (CDC13) δ 18.9, 22.5, 24.0, 25.0, 27.0, 27.3, 29.1, 34.5, 46.3, 59.6, 62.7, 69.9, 91.8, 117.5, 122.3, 123.4, 123.6, 126.8, 127.4, 127.5, 130.7, 133.4, 146.5, 171.4, 172.30.
Example 27 Decanedioic Acid Bis-[4-[(6R, 7R, 7aS)-5,6,7,7a-tetrahydro-(6-isopropyI-3,3-dimethyl- 5-oxo-lZT-pyrrolo[l,2-c]oxazol-7-yl)]-l-naphthyl] ester (lie). Yield 61%; [α]D +217 (c 1.0, CHC13); 1H NMR (CDC13) δ 0.79 (d, 6H, J= 6.9 Hz), 0.87 (d, 6H, J= 6.6 Hz), 1.32- 1.59 (m, 10H), 1.60 (s, 6H), 1.79 (s, 6H), 1.82-1.96 (m, 4H), 2.76 (t, 4H, J= 7.5 Hz), 3.25 (dd, 2H, J= 8.7, 3.6 Hz), 3.54 (t, 2H, J= 9.0 Hz), 4.15 (t, 2H, J= 9.0 Hz), 4.22 (dd, 2H, J= 8.7, 5.7 Hz), 4.99 (td, 2H, J= 9.0, 5.7 Hz), 7.24 (d, 2H, J= 7.8 Hz), 7.48 (d, 2H, J= 8.1 Hz), 7.53-7.63 (m, 4H), 7.92-7.99 (m, 4H); 13C NMR (CDC13) δ 1 8.9, 22.5, 24.0, 25.2, 27.0, 27.3, 29.4, 34.6, 46.3, 59.6, 62.7, 69.9, 91.8, 117.5, 122.3, 123.4, 123.6, 126.8, 127.4, 127.5, 130.7, 133.4, 146.5, 171.5, 172.4.
Example 28 Dodecanedioic Acid Bis-[4-[(6R, 7R, 7aS)-5,6,7,7a-tetrahydro-(6-isopropyI-3,3- dimethyI-5-oxo-127-pyrrolo[l-2-φxazol-7-yl)]-l-naphthyl] ester (lid). Yield, 53%; [α]D +292 (c 1.0, CHC13); 1H NMR (CDC13) δ 0.79 (d, 6H, J= 6.9 Hz), 0.87 (d, 6H, J= 6.6 Hz), 1.33-1.56 (m, 14H), 1.60 (s, 6H), 1.79 (s, 6H), 1.80-1.93 (m, 4H), 2.75 (t, 4H, J= 7.5 Hz), 3.24 (dd, 2H, J= 8.7, 3.6 Hz), 3.53 (t, 2H, J= 8.7 Hz), 4.14 (t, 2H, J= 9.3 Hz), 4.21 (dd, 2H, J= 8.7, 5.7 Hz), 4.99 (td, 2H, J= 9.3, 5.7Hz), 7.24 (d, 2H, J= 7.8 Hz), 7.48 (d, 2H, J - 7.8 Hz), 7.53-7.63 (m, 4H), 7.92-7.99 (m, 4H); 13C NMR (CDC13) δ 18.8, 22.5, 24.0, 25.3, 27.0, 27.3, 29.4, 29.5, 29.6, 34.6, 46.3, 59.6, 62.7, 69.9, 91.8, 117.5, 122.4, 123.4, 123.6, 126.8, 127.5, 130.7, 133.4, 146.5, 171.4, 172.5.
Example 29 Tetradecanedioic Acid Bis-[4-[(6R, IR, 7aS)-5,6,7,7a-tetrahydro-(6-isopropyl-3,3- dimethyl-5-oxo-lJflr-pyrrolo[l-2-c]oxazol-7-yl)]-l-naphthyl] ester (lie). Yield 41%; [α]D
+241 (c 0.7, CHC13); 1H NMR (CDC13) δ 0.79 (d, 6H, J= 7.2 Hz), 0.87 (d, 6H, J= 6.9 Hz),
1.29-1.54 (m, 18H), 1.60 (s, 6H), 1.78 (s, 6H), 1.80-1.92 (m, 4H), 2.75 (t, 4H, J= 7.5 Hz),
3.24 (dd, 2H, J= 8.7, 3.6 Hz), 3.53 (t, 2H, J= 8.7 Hz), 4.14 (t, 2H, J= 9.3 Hz), 4.21 (dd, 2H, J= 8.7, 5.7 Hz), 4.99 (td, 2H, J= 9.3, 5.7Hz), 7.24 (d, 2H, J= 7.8 Hz), 7.48 (d, 2H, J=
7.8 Hz), 7.53-7.63 (m, 4H), 7.92-7.99 (m, 4H); 13C NMR (CDC13) δ 18.9, 22.5, 24.0, 25.3,
27.1, 27.3, 29.4, 29.5, 29.7, 29.8, 34.6, 46.3, 59.6, 62.7, 69.9, 91.8, 117.5, 122.4, 123.4, 123.6, 126.8, 127.5, 130.7, 133.4, 146.5, 171.5, 172.50.
Example 30
Hexadecanenedioic Acid Bis-[4-[(6R, IR, 7aS)-5,6,7,7a-tetrahydro-(6-isopropyl-3,3- dimethyI-5-oxo-ljH-pyrrolo[l,2-c]oxazol-7-yl)]-l-naphthyl] ester (llf). Yield 56%; [α]D +213 (c 1.0, CHC13); 1H NMR (CDC13) δ 0.79 (d, 6H, J= 6.9 Hz), 0.87 (d, 6H, J- 6.6 Hz), 1.25-1.55 (m, 22H), 1.60 (s, 6H), 1.79 (s, 6H), 1.80-1.92 (m, 4H), 2.75 (t, 4H, J= 7.5 Hz), 3.24 (dd, 2H, J= 8.7, 3.6 Hz), 3.54 (t, 2H, J= 8.7 Hz), 4.14 (t, 2H, J- 9.0 Hz), 4.21 (dd, 2H, J= 8.7, 5.7 Hz), 4.99 (td, 2H, J= 9.0, 5.7 Hz), 7.24 (d, 2H, J= 7.8 Hz), 7.48 (d, 2H, J= 7.8 Hz), 7.53-7.63 ( , 4H), 7.92-7.99 (m, 4H); 13C NMR (CDC13) δ 18.8, 22.5, 24.0, 25.3, 27.0, 27.3, 29.4, 29.5, 29.7, 29.8, 29.9, 34.6, 46.3, 59.6, 62.7, 69.9, 91.7, 117.5, 122.4, 123.4, 123.6, 126.8, 127.4, 130.7, 133.4, 146.5, 171.4, 172.5.
Example 31 Docosanedioic Acid Bis-[4-[(6R, 7R, 7aS)-5,6,7-7a-tetrahydro-(6-isopropyI-3,3- dimethyI-5-oxo-liϊ-pyrrolo[l,2-c]oxazol-7-yl)]-l-naphthyl] ester (llg). Yield 49%; [α]D +209 (c 1.0, CHC13); 1H NMR (CDC13) δ 0.79 (d, 6H, J= 6.9 Hz), 0.87 (d, 6H, J= 6.6 Hz), 1.20-1.55 (m, 34H), 1.60 (s, 6H), 1.79 (s, 6H), 1.80-1.92 (m, 4H), 2.75 (t, 4H, J= 7.5 Hz),
3.25 (dd, 2H, J= 8.7, 3.6 Hz), 3.54 (t, 2H, J= 8.7 Hz), 4.15 (t, 2H, J= 9.0 Hz), 4.21 (dd, 2H, J= 8.7, 5.7 Hz), 4.99 (td, 2H, J= 9.3, 5.4 Hz), 7.24 (d, 2H, J= 7.8 Hz), 7.48 (d, 2H, J= 8.1 Hz), 7.53-7.63 (m, 4H), 7.92-7.99 (m, 4H); 13C NMR (CDC13) δ 18.9, 22.5, 24.0, 25.3, 27.0, 27.3, 29.4, 29.5, 29.7, 29.8, 29.9, 30.0, 34.6, 46.3, 59.6, 62.7, 69.9, 91.8, 117.5, 122.4, 123.4, 123.6, 126.8, 127.4, 130.6, 133.4, 146.5, 171.5, 172.5.
Example 32 General Procedure for the Preparation of 9:
To the solution of 25 μmol of 11 in anhydrous methylene chloride (3 mL) was added 1,2- ethanedithiol (65 μL) and BF3-Et2O (27 μL) at room temperature under N2. After 10 mins, the reaction was quenched by adding saturated aqueous NaHCO (2 mL) and diluted with ethyl acetate (80 mL). The organic layer was washed with brine and dried over anhydrous Na2SO . After concentration, the residue was chromatographed on silica gel (10/1 EtOAc MeOH) to afford the product.
Example 33
Hexanedioic Acid Bis-[4-[(2S, 3R, 4R)-2-(hydroxymethyI)-4-isopropyI-5-oxo — 3- pyrrolidinyI]-l-naphthaIenyl] Ester (9a). Yield 93%; [α]D +188 (c 1.0, MeOH); 1H NMR (CD3OD) δ 0.78 (d, 6H, J= 6.9 Hz), 0.84 (d, 6H, J= 6.6 Hz), 1.25-1.40 (m, 2H), 2.00-2.08 (m, 4H), 2.88-3.00 (m, 6H), 3.53 (dd, 2H, J= 11.4, 5.1 Hz), 3.78 (dd, 2H, J= 11.4, 3.0 Hz), 4.31-4.42 (m, 4H), 7.26 (d, 2H, J= 7.8 Hz), 7.50-7.65 (m, 6H), 7.97 (dd, 2H, J= 8.4, 1.2 Hz), 8.18 (d, 2H, J= 8.4 Hz); 13C NMR (CD3OD) δ 19.3, 23.0, 25.7, 28.3, 34.8, 43.1, 53.1, 60.3, 64.3, 118.7, 123.2, 125.0, 125.1, 127.7, 128.3, 128.8, 133.1, 135.1, 147.7, 173.9, 180.3; Anal. Calcd for C42H48N2O8-1.25H2O: C, H, N.
Example 34
Octanedioic Acid Bis-[4-[(2S, 3R, 4R)-2-(hydroxymethyι)-4-isopropyl-5-oxo — 3- pyrrolidinyl]-l-naphthalenyl] Ester (9b). Yield 95%; [α]D +192 (c 0.9, MeOH); 1H NMR (CD3OD) δ 0.77 (d, 6H, J= 6.9 Hz), 0.83 (d, 6H, J= 6.9 Hz), 1.28-1.38 (m, 2H), 1.57-1.67 (m, 4H), 1.84-1.97 (m, 4H), 2.83 (t, 4H, J= 7.5 Hz), 2.96 (dd, 2H, J= 9.0, 3.9 Hz), 3.52 (dd, 2H, J= 11.7, 5.1 Hz), 3.77 (dd, 2H, J= 11.7, 2.7 Hz), 4.30-4.42 (m, 4H), 7.24 (d, 2H, J = 7.8 Hz), 7.52-7.64 (m, 6H), 7.94 (dd, 2H, J= 8.1, 1.5 Hz), 8.17 (d, 2H, J= 7.8 Hz); 13C NMR (CD3OD) δ 19.3, 23.0, 26.0, 28.3, 30.0, 35.0, 43.1, 53.1, 60.3, 64.3, 118.7, 123.2,
125.0, 125.1, 127.7, 128.3, 128.8, 133.1, 135.1, 147.6, 174.2, 180.3. Anal. Calcd for C44H52N2O8-1.5H2O: C, H, N.
Example 35
Decanedioic Acid Bis-[4-[(2S, 3R, 4R)~2-(hydroxymethyl)-4-isopropyI-5-oxo— 3- pyrrolidinylj-l-naphthalenyl] Ester (9c). Yield 91%; [α]D +186 (c 0.7, MeOH); 1H NMR (CD3OD) δ 0.77 (d, 6H, J= 6.9 Hz), 0.83 (d, 6H, J= 6.9 Hz), 1.27-1.39 (m, 2H), 1.44-1.60 (m, 8H), 1.80-1.92 (m, 4H), 2.79 (t, 4H, J= 7.5 Hz), 2.96 (dd, 2H, J = 9.0, 3.9 Hz), 3.52 (dd, 2H, J= 11.7, 5.4 Hz), 3.77 (dd, 2H, J= 11.7, 2.7 Hz), 4.30-4.42 (m, 4H), 7.23 (d, 2H, J = 7.8 Hz), 7.53-7.64 (m, 6H), 7.93 (dd, 2H, J= 7.8, 1.8 Hz), 8.17 (d, 2H, J= 7.8 Hz); 13C NMR (CD3OD) δ 19.3, 23.0, 26.2, 28.3, 30.3, 30.4, 35.1, 43.1, 53.1, 60.3, 64.3, 118.7, 123.2, 125.0, 125.1, 127.7, 128.3, 128.8, 133.1, 135.1, 147.7, 174.2, 180.3. Anal. Calcd for C46H56N2O8-0.5H2O: C, H, N.
Example 36 Dodecanedioic Acid Bis-[4-[(2S, 3R, 4R)-2-(hydroxymethyι)-4-isopropyl-5-oxo — 3- pyrrolidinylH-naphthalenyl] Ester (9d). Yield 89%; [α]D +194 (c 0.7, MeOH); 1H NMR (CD3OD) δ 0.77 (d, 6H, J= 6.6 Hz), 0.83 (d, 6H, J= 6.9 Hz), 1.26-1.58 (m, 14H), 1.78- 1.88 (m, 4H), 2.77 (t, 4H, J= 7.5 Hz), 2.96 (dd, 2H, J= 9.0, 3.9 Hz), 3.52 (dd, 2H, J= 11.7, 5.4 Hz), 3.77 (dd, 2H, J= 11.7, 2.7 Hz), 4.30-4.42 (m, 4H), 7.23 (d, 2H, J= 7.8 Hz), 7.53- 7.64 (m, 6H), 7.93 (dd, 2H, J= 7.8, 1.5 Hz), 8.17 (d, 2H, J= 7.8 Hz); 13C NMR (CD3OD) δ 19.3, 23.0, 26.2, 28.3, 30.4, 30.5, 30.7, 35.1, 43.1, 53.1, 60.3, 64.3, 118.7, 123.2, 125.0,
125.1, 127.7, 128.3, 128.8, 133.1, 135.1, 147.7, 174.2, 180.3. Anal. Calcd for C48H60N2O8-0.75H2O: C, H, N.
Example 37
Tetradecanedioic Acid Bis-[4-[(2S, 3R, 4R)-2-(hydroxymethyl)-4-isopropyl-5-oxo — 3- pyrrolidinyl]-l-naphthalenyl] Ester (9e). Yield 93%; [α]D +191 (c 0.4, MeOH); 1H NMR (CD3OD) δ 0.77 (d, 6H, J = 6.6 Hz), 0.83 (d, 6H, J= 6.9 Hz), 1.26-1.56 (m, 18H), 1.77-
1.88 (m, 4H), 2.77 (t, 4H, J= 7.5 Hz), 2.96 (dd, 2H, J= 9.6, 3.9 Hz), 3.52 (dd, 2H, J= 11.7, 5.1 Hz), 3.77 (dd, 2H, J= 11.7, 2.7 Hz), 4.30-4.42 (m, 4H), 7.23 (d, 2H, J= 7.8 Hz), 7.54- 7.65 (m, 6H), 7.93 (dd, 2H, J= 7.8, 1.5 Hz), 8.17 (d, 2H, J= 7.8 Hz); 13C NMR (CD3OD) δ
19.3, 23.0, 26.2, 28.3, 30.4, 30.5, 30.7, 30.8, 35.2, 43.1, 53.1, 60.3, 64.3, 118.7, 123.2, 125.0, 125.1, 127.7, 128.3, 128.8, 133.1, 135.1, 147.7, 174.2, 180.3. Anal. Calcd for C50H64N2O8-0.75H2O: C, H, N.
Example 38
Hexadecanenedioic Acid Bis-[4-[(2S, 3R, 4R)-2-(hydroxymethyϊ)-4-isopropyl-5-oxo —
3-pyrrolidinyl]-l-naphthalenyl] Ester (9f). Yield 90%; [α]D +197 (c 0.9, MeOH); 1H NMR (CD3OD) δ 0.77 (d, 6H, J= 6.9 Hz), 0.83 (d, 6H, J= 6.9 Hz), 1.25-1.55 (m, 22H),
1.77-1.88 (m, 4H), 2.77 (t, 4H, J= 7.5 Hz), 2.96 (dd, 2H, J= 9.3, 3.9 Hz), 3.52 (dd, 2H, J=
11.4, 5.1 Hz), 3.77 (dd, 2H, J= 11.4, 2.7 Hz), 4.30-4.42 (m, 4H), 7.22 (d, 2H, J= 7.8 Hz), 7.53-7.65 (m, 6H), 7.93 (dd, 2H, J = 7.8, 1 .5 Hz), 8.17 (d, 2H, J = 7.8 Hz); 1 3C NMR (CD3OD) δ 19.3, 23.0, 26.2, 28.3, 30.4, 30.5, 30.7, 30.8, 30.9, 35.2, 43.1, 53.1, 60.3, 64.3, 118.7, 1 23.2, 1 25.0, 1 25.1, 127.7, 128.3, 128.8, 133.1, 135.1, 147.7, 174.2, 180.3. Anal. Calcd for C52H68N2O8-0.5H2O: C, H, N.
Example 39 Docosanedioic Acid Bis-[4-[(2S, 3R, 42?)-2-(hydroxymethyl)-4-isopropyI-5-oxo — 3- pyrrolidinylH-naphthalenyl] Ester (9g). Yield 89%; [α]D +164 (c 0.6, MeOH); 1H NMR (CD3OD) δ 0.77 (d, 6H, J= 6.9 Hz), 0.83 (d, 6H, J= 6.9 Hz), 1.22-1.55 (m, 34H), 1.77- 1.88 (m, 4H), 2.77 (t, 4H, J= 7.5 Hz), 2.96 (dd, 2H, J= 9.3, 3.9 Hz), 3.52 (dd, 2H, J= 11.4, 5.1 Hz), 3.77 (dd, 2H, J= 11.4, 2.7 Hz), 4.30-4.42 (m, 4H), 7.22 (d, 2H, J= 7.8 Hz), 7.53- 7.65 (m, 6H), 7.93 (dd, 2H, J= 8.1, 1.2 Hz), 8.17 (d, 2H, J= 8.1 Hz); 13C NMR (CD3OD) δ 19.3, 23.0, 26.3, 28.3, 30.4, 30.6, 30.7, 30.8, 30.9, 35.2, 43.1, 53.1, 60.3, 64.3, 118.7, 123.2, 125.0, 125.1, 127.7, 128.3, 128.8, 133.1, 135.1, 147.7, 174.2, 180.3. Anal. Calcd for C58H80N2O8-0.5H2O: C, H, N.
Example 40 9-[N-(4-Acetoxy-l-naphthaIenyI)carbamoyl]-nonanoic Acid (12):
4-Hydroxy-l-naρhthylamine hydrochloride (0.45 g, 2.2 mmol) was taken up in dry tetrahydrofuran (5 mL) and cooled to 0°C, and triethylamine (0.65 mL, 4.4 mmol) was added. Then methyl 9-(chlorocarbonyl)nonanoate41 (0.5 g, 2.2 mmol) in dry tetiahydrofuran (2.5 mL), was added slowly and the reaction mixture was allowed to stir at room temperature overnight. The mixture was diluted with ethyl acetate, washed with water and brine and dried over anhydrous sodium sulfate. After evaporation, the residue was purified on silica gel (2% methanol in chloroform) to obtain the amide, which was dissolved in methanol (5 mL). Lithium hydroxide (100 mg) was added and the mixture was stirred at room temperature for 0.5 h. Acidification with 6N HCl and extraction with ethyl acetate provided the free acid (0.21 g, 28% yield). To a solution of this acid in dichloromethane was added potassium carbonate (80 mg) and acetic anhydride (0.3 mL). The mixture was stirred at room temperature overnight. Water (5 mL) was added and the mixture stirred for another 1 h. The dichloromethane layer was separated. The water layer was extracted twice more with dichloromethane and the combined extracts were concentrated to obtain the acetate (0.20 g, 91% yield). 1H NMR (CD3OD) δ 1.23-1.48 (m, 8H), 1.52-1.68 (m, 2H), 1.70-1.79 (m, 2H), 2.27 (t, 2H, J=7.2 Hz), 2.41-2.51 (m, 5H), 7.20 (d, IH, J=7.8 Hz), 7.34 (br s, IH), 7.46-7.55 (m, IH), 7.62 (d, IH, J=7.8 Hz), 7.86 (m, 2H); 13C NMR (CDC13) δ 24.9, 25.8, 29.00, 29.05, 29.07, 29.2, 341, 37.0, 5 1.5, 108.4, 121.2, 122.8, 123.5, 124.7, 125.3, 126.5, 130.1, 151.7, 174.1, 174.2.
Example 41 9-[N-(4-Hydroxy-l-naphthyl)carbamoyl]nonanoic Acid iV-[(2S,5S)-l,2,3,4,5-6- hexahydro-5-(hydroxymethyl)-2-isopropyl-l-methyl-3-oxobenzo[e][l,4]diazocin-8-yI]- amide (13). The nitro compound 8 (50 mg, 0.144 mmol) was dissolved in ethanol (8 mL), and 10% Pd-C (7.5 mg, 0.007 mmol) was added. The mixture was stirred vigorously under an H2 atmosphere overnight. The reaction mixture was filtered and the filtrate concentrated to give a residue, which was dried under vacuum for 1 h at 60 °C. To a solution of the residue in dimethylformamide was added successively the acid 12 (37 mg, 0.12 mmol) and 1 -[3 '-(dimethyl amino) propyl]-3-ethyl carbodiimide hydrochloride (22 mg, 0.12 mmol). The mixture was stirred at room temperature for 12 h. Water was added, and the mixture was extracted with dichloromethane (thrice). The combined organic phases were dried over anhydrous sodium sulfate and concentrated. The residue was dissolved in ethanol (30 mL). A solution of sodium carbonate (60 mg) in water (6 mL) was added with vigorous stirring. After 1.5 h at room temperature, the solution was concentrated, and an additional amount of water (20 mL) added. Extraction with ethyl acetate (4 x 30 mL) followed by preparative TLC (ethyl acetate as developing solvent) provided the product (23 mg; 35% yield). [α]20 D -75.2 (c 0.35, CH3OH). 1H NMR (CD3OD) δ 0.95 (d, 3H, J= 6.8 Hz), 1.08 (d, 3H, J= 6.8 Hz), 1.25-1.29 (m, 8H), 1.68-1.82 (m, 4H), 2.01-2.08 (m, IH), 2.29-2.52 (m, 4H), 2.72 (s, 3H), 2.88-2.94 (m, 2H), 3.35-3.39 (m, IH), 3.46-3.52 (m, IH), 3.61-3.68 (m, 2H), 4.51 (br s, 1 H), 6.79-6.85 (m, IH), 7.04-7.07 (m, IH), 7.29-7.33 (m, 2H), 7.44-7.53 (m, 3H), 7.82 (d, IH, J=7.2 Hz), 8.25 (d, IH, 7.2 Hz). 13C NMR (CDC13) δ 18.9, 20.1, 25.5, 25.8, 28.8, 28.9, 29.6, 37.3, 47.6, 48.1, 48.4, 48.7, 48.9, 49.3, 56.6, 64.3, 87.0, 102.5, 107.3, 119.4, 119.7, 121.6, 121.74, 124.2, 124.7, 126.4, 147.8, 174.9, 176.2, 186.2. HPLC retention time 6.8 min (99.96% purity) using conditions C. FABMS Calcd. for (C35H46N4O5 + H+) 603.36, found 603.37.
Incorporation By Reference All ofthe patents and publications cited herein are hereby incorporated by reference.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
We claim:

Claims

1. A compound represented by A:
Figure imgf000107_0001
wherein
X represents NR5, 0 or S; Y represents O or S;
R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylaminoalkyl, or sulfonylaminoalkyl;
R3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
R4 is absent or present from 1 to 3 times inclusive;
R4, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroarallcyl, formyl, acyl, acyloxy, alkylO2C-, arylOaC-, heteroarylO2C-, aralkylθ2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl; R5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in A is R, S, or a mixture thereof.
2. The compound of claim 1 , wherein X represents NR5.
3. The compound of claim 1 , wherein Y represents O.
4. The compound of claim 1 , wherein R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl.
5. The compound of claim 1, wherein R1 represents alkyl or cycloalkyl.
6. The compound of claim 1 , wherein R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
7. The compound of claim 1, wherein R3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl.
8. The compound of claim 1, wherein R is absent.
9. The compound of claim 1, wherein R5 is alkyl.
10. The compound of claim 1 , wherein X represents NR5; and Y represents O.
11. The compound of claim 1 , wherein X represents NR5; Y represents O; and R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl.
12. The compound of claim 1, wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; and R1 represents alkyl or cycloalkyl.
13. The compound of claim 1 , wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; and R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
14. The compound of claim 1 , wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl.
15. The compound of claim 1 , wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; R3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl; and R4 is absent.
16. The compound of claim 1, wherein X represents NR5; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; R3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl; R4 is absent; and R5 is alkyl.
17. The compound of claim 1, wherein X represents NR ; Y represents O; R represents independently for each occurrence H; R represents isopropyl; R represents hydroxylmethyl; R3 represents alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, or aralkenylcarbonyl; R4 is absent; and R5 is methyl.
18. A compound represented by B:
Figure imgf000110_0001
B wherein X represents NR5, O or S;
Y represents O or S;
R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylaminoalkyl; R3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
R4 is absent or present from 1 to 3 times inclusive;
R4, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, tliiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylOiC-, heteroarylO2C-, aralkylO2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-,
Figure imgf000111_0001
or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in B is R, S, or a mixture thereof.
19. A compound represented by C :
Figure imgf000112_0001
wherein
X represents NR5, O or S; Y represents O or S;
R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylammoalkyl, or sulfonylaminoalkyl;
R3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
R4 is absent or present from 1 to 3 times inclusive;
R4, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylO2C-, heteroarylOaC-, aralkylO2C-, heteroaralkylO2C-, ill acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in C is R, S, or a mixture thereof.
20. A compound represented by D :
Figure imgf000113_0001
D wherein
X represents NR5, O or S;
Y represents O or S;
R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylaminoalkyl, or sulfonylaminoalkyl; R3 represents formyl, alkylcarbonyl, cycloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, aralkylcarbonyl, aralkenylcarbonyl, alkoxylcarbonyl, alkylaminocarbonyl, aryloxylcarbonyl, or arylaminocarbonyl, alkylsulfonyl, or arylsulfonyl;
R4 is absent or present from 1 to 3 times inclusive; R4, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylOiC-, heteroarylO2C-, aralkylO2C-, heteroaralkylθ2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R , -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R5 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in D is R, S, or a mixture thereof.
21. A compound represented by E :
Figure imgf000114_0001
E wherein
L represents NR4, 0, or S;
W represents a divalent alkyl, alkenyl, alkynyl or glycol chain;
X represents NR4, 0 or S;
Y represents O or S; R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, araikyl, or heteroaralkyl;
R1 represents alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolaikyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylaminoalkyl, or sulfonylaminoalkyl;
R3 is absent or present from 1 to 3 times inclusive;
R3, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylO2C-, heteroarylO2C-, aralkylθ2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)~, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, alkylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R4 represents H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in E is R, S, or a mixture thereof.
22. The compound of claim 21 , wherein L represents NR 4.
23. The compound of claim 21 , wherein W is a divalent alkyl or glycol chain.
24. The compound of claim 21 , wherein X represents NR4.
25. The compound of claim 21 , wherein Y represents O.
26. The compound of claim 21, wherein R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl.
27. The compound of claim 21 , wherein R1 represents alkyl or cycloalkyl.
28. The compound of claim 21, wherein R2 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
29. The compound of claim 21 , wherein R3 is absent.
30. The compound of claim 21 , wherein R4 is H or alkyl.
31. The compound of claim 21 , wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; and Y represents O.
32. The compound of claim 21, wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; and R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl.
33. The compound of claim 21, wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; and R1 represents alkyl or cycloalkyl.
34. The compound of claim 21 , wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; and R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
35. The compound of claim 21 , wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R3 is absent.
36. The compound of claim 21, wherein W represents a divalent alkyl or glycol chain; X represents NR4; L represents NR4; Y represents O; R represents independently for each occurrence H, alkyl, cycloalkyl, or aralkyl; R1 represents alkyl or cycloalkyl; R2 represents hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; R is absent; and R4 is H or alkyl.
37. The compound of claim 21, wherein W represents -(CH2)4-; X represents NMe; L
1 -^ represents NH; Y represents O; R represents H; R represents isopropyl; R represents hydroxylmethyl; and R is absent.
38. The compound of claim 21 , wherein W represents -(CH2)6-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R is absent.
39. The compound of claim 21, wherein W represents -(CH2)8-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R3 is absent.
40. The compound of claim 21, wherein W represents -(CH2)10-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R3 is absent.
41. The compound of claim 21, wherein,W represents -(CH2)!2-; X represents NMe; L
1 represents NH; Y represents O; R represents H; R represents isopropyl; R represents hydroxylmethyl; and R3 is absent.
42. The compound of claim 21 , wherein W represents -(CH2)14-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R3 is absent.
43. The compound of claim 21 , wherein W represents -(CH2)2o-; X represents NMe; L
1 represents NH; Y represents O; R represents H; R represents isopropyl; R represents hydroxylmethyl; and R3 is absent.
44. The compound of claim 21 , wherein W represents -CH2-(OCH2CH2)4O-CH2-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R2 represents hydroxylmethyl; and R3 is absent.
45. The compound of claim 21, wherein W represents -CH2-(OCH2CH2)5O-CH2-; X represents NMe; L represents NH; Y represents O; R represents H; R1 represents isopropyl; R represents hydroxylmethyl; and R is absent.
46. A compound represented by F:
Figure imgf000119_0001
wherein W represents a divalent alkyl, alkenyl, alkynyl or glycol chain;
X represents independently for each occurrence NR, O or S;
R represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
R1 represents independently for each occurrence hydroxylalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, sulfonyloxylalkyl, thiolalkyl, alkylthiolalkyl, arylthiolalkyl, acylthiolalkyl, sulfonylthiolalkyl, aminoalkyl, alkylammoalkyl, arylaminoalkyl, acylaminoalkyl, or sulfonylaminoalkyl;
R2 is absent or present from 1 to 6 times inclusive;
R2, when present, represents independently for each occurrence halogen, hydroxyl, alkoxyl, aryloxyl, thiol, alkylthio, thioalkyl, hydroxylalkyl, alkylamino, amino, aminoalkyl, nitro, nitrile, carboxylate, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, acyloxy, alkylO2C-, arylO2C-, heteroarylO2C-, aralkylO2C-, heteroaralkylO2C-, acyl(R)N-, alkylOC(O)N(R)-, arylOC(O)N(R)-, aralkylOC(O)N(R)-, heteroaralkylOC(O)N(R)-, -N=C(alkyl)2, or -N=C(aryl)2, alkylsulfonyl, arylsulfonyl, allcylsulfonyloxy, arylsulfonyloxy, alkylsulfonylamino, arylsulfonylamino, alkylselenyl, or selenoalkyl;
R3 represents independently for each occurrence H, alkyl, cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at a stereogenic center in F is R, S, or a mixture thereof.
47. The compound of claim 46, wherein W represents a divalent alkyl or glycol chain.
48. The compound of claim 46, wherein X represents NR.
49. The compound of claim 46, wherein R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
50. The compound of claim 46, wherein R is absent.
51. The compound of claim 46, wherein R represents alkyl.
52. The compound of claim 46, wherein W represents a divalent alkyl or glycol chain; X represents NR; and R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl.
53. The compound of claim 46, wherein W represents a divalent alkyl or glycol chain; X represents NR; and R2 is absent.
54. The compound of claim 46, wherein W represents a divalent alkyl or glycol chain; X represents NR; and R3 represents alkyl.
55. The compound of claim 46, wherein W represents a divalent alkyl or glycol chain; X represents NR; R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R2 is absent.
56. The compound of claim 46, wherein W represents a divalent alkyl or glycol chain; X represents NR; R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; and R3 represents alkyl.
57. The compound of claim 46, wherein W represents a divalent alkyl or glycol chain; X represents NR; R1 represents hydroxyalkyl, alkoxylalkyl, aryloxylalkyl, acyloxylalkyl, or sulfonyloxylalkyl; R2 is absent; and R3 represents alkyl.
58. The compound of claim 46, wherein W represents -(CH2)4-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
59. The compound of claim 46, wherein W represents -(CH2)6-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
60. The compound of claim 46, wherein W represents -(CH2)8-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
61. The compound of claim 46, wherein W represents -(CH2)10-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
62. The compound of claim 46, wherein W represents -(CH2)12-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
63. The compound of claim 46, wherein W represents -(CH2)ι -; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
64. The compound of claim 46, wherein W represents -(CH2)2o-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
65. The compound of claim 46, wherein W represents -CH2-(OCH2CH2)4O-CH2-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
66. The compound of claim 46, wherein W represents -CH2-(OCH2CH2)5θ-CH2-; X represents NH; R1 represents hydroxylmethyl; R2 is absent; and R3 represents isopropyl.
67. The compound of claim 1, 18, 19, 20, 21, or 46, wherein said compound has an IC50 less than 1 μM in an assay based on mammalian PKCα.
68. The compound of claim 1, 18, 19, 20, 21, or 46, wherein said compound has an IC50 less than 100 nM in an assay based on mammalian PKCα.
69. The compound of claim 1, 18, 19, 20, 21, or 46, wherein said compound has an IC50 less than 10 nM in an assay based on mammalian PKCα.
70. The c ompound o f c laim 1 , 1 8, 1 9, 20, 21, o r 46, wherein said compound has an EC50 less than 1 μM in an assay based on mammalian PKCα.
71. The c ompound o f c laim 1 , 1 8, 1 9, 20, 21, o r 46, wherein said compound has an EC50 less than 100 nM in an assay based on mammalian PKCα.
72. The c ompound o f c laim 1 , 1 8, 1 9, 20, 2 1, o r 46, wherein said compound has an EC50 less than 10 nM in an assay based on mammalian PKCα.
73. The compound of claim 1, 18, 19, 20, 21, or 46, wherein said compound is a single stereoisomer.
74. A composition, comprising a compound o f claim 1 , 1 8, 1 9, 20, 2 1, or 46; and a pharmaceutically acceptable excipient.
75. A kit comprising a compound of claim 1, 18, 19, 20, 21, or 46; and instructions for use thereof.
76. A method of modulating the activity of protein kinase C in a mammal, comprising: administering to said mammal a therapeutically effective amount of a compound of claim 1, 18, 19, 20, 21, or 46.
77. A method of increasing secretion of sAPPα in a mammal, comprising: administering to said mammal a therapeutically effective amount of a compound of claim 1, 18, 19, 20, 21, or 46.
78. A method of treating a mammal suffering from neuronal dysfunction, cognitive decline, neurodegeneration, stroke, Alzheimer's disease, Parkinson's disease, diabetes, heart disease, or cancer, comprising: administering to said mammal a therapeutically effective amount of a compound of claim 1, 18, 19, 20, 21, or 46.
79. A method of treating a mammal suffering from Alzheimer ' s disease, comprising: administering to said mammal a therapeutically effective amount of a compound of claim 1, 18, 19, 20, 21, or 46.
80. A method of inducing hyperplasia in a mammal, comprising: administering to said mammal a therapeutically effective amount of a compound of claim 1, 18, 19, 20, 21, or 46.
81. The method of claim 76, 77, 78, 79, or 80, wherein said mammal is a primate, equine, canine or feline.
82. The method of claim 76, 77, 78, 79, or 80, wherein said mammal is a human.
83. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered orally.
84. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered intravenously.
85. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered sublingually.
86. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered ocularly.
87. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered transdermally.
88. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered rectally.
89. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered vaginally.
90. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered topically.
91. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered intramuscularly.
92. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered subcutaneously.
93. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered buccally.
94. The method of claim 76, 77, 78, 79, or 80, wherein said compound is administered nasally.
PCT/US2003/022038 2002-07-15 2003-07-14 Protein kinase c modulators, and methods of use thereof WO2004007445A2 (en)

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US5962504A (en) * 1997-09-08 1999-10-05 Georgetown University Substituted 2-pyrrolidinone activators of PKC

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US5962504A (en) * 1997-09-08 1999-10-05 Georgetown University Substituted 2-pyrrolidinone activators of PKC

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Title
MA ET AL: 'General and Stereospecific Route to 9-Substituted, 8,9-Disubstituted, and 9,10-Disubstituted Analogues of Benzolactam-V8' J. ORG. CHEM. vol. 64, 1999, pages 6366 - 6373, XP002978713 *

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