WO1998005332A1 - Compositions heterocycliques - Google Patents

Compositions heterocycliques Download PDF

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Publication number
WO1998005332A1
WO1998005332A1 PCT/US1997/013686 US9713686W WO9805332A1 WO 1998005332 A1 WO1998005332 A1 WO 1998005332A1 US 9713686 W US9713686 W US 9713686W WO 9805332 A1 WO9805332 A1 WO 9805332A1
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Prior art keywords
composition
compounds
heterocycle
alkyl
aryl
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PCT/US1997/013686
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English (en)
Inventor
Phillip Dan Cook
Andrew M. Kawasaki
Pei Pei Kung
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Isis Pharmaceuticals, Inc.
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Priority claimed from US08/691,139 external-priority patent/US5922872A/en
Priority claimed from US08/691,185 external-priority patent/US5731438A/en
Priority claimed from US08/688,993 external-priority patent/US5798360A/en
Priority claimed from US08/691,149 external-priority patent/US5817489A/en
Application filed by Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Priority to AU39069/97A priority Critical patent/AU3906997A/en
Publication of WO1998005332A1 publication Critical patent/WO1998005332A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/125—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/13—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
    • C07D261/08—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles 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 in position 2
    • C07D277/82—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to combinations of di-nitrogen heterocycles and wet ⁇ -benzylic and alpha-amido compounds, and their uses, inter ⁇ li ⁇ in pharmaceuticals.
  • a further object is to provide methods for the identification of useful drugs and reagents.
  • the present invention is directed to compounds having a di- nitrogen-containing heterocyclic portion to which pluralities of substituents are appended.
  • compositions are prepared comprising a plurality of such compounds, preferably more than three and even more preferably, more than six, such that such compositions can be seen to be mixtures of species having a di- nitrogen heterocyclic framework.
  • the substituents appended to the framework preferably the di-nitrogen heterocyclic frameworks as discussed hereinafter, be varied in terms of size, hydrophobicity, charge, chemistry, orientation, and subsequent reactivity.
  • the resulting compositions are useful er se, as, for example, antibiotics, reagents for use in scientific research, and otherwise. Certain preferred compositions, comprising mixtures of such compounds, have been shown to have antibiotic activity and otherwise to be useful.
  • This invention is also directed to constituent portions of novel compounds, which components can be combined to form compositions having utility er se as well as in the preparation and identification of more complex pharmaceutical and other compositions.
  • compositions comprising the reaction products of di-nitrogen heterocycles with chemically diverse substituents.
  • Compounds of one such type have Formula I:
  • n, x and y is 1, 2 or 3;
  • R is halogen, cyano, C,-C 6 alkyl, perhalo C,-C 3 alkyl, nitro, nitroso or carboxylate
  • R 2 is hydrogen, C,-C 6 alkyl, C 6 -C ]5 aryl, C 7 -C 20 alkaryl, C 4 -C I5 heterocycle or a moiety of the formula where m is O or 1;
  • R 4 is -O-R 5 , -N(R j )(R 6 ), -NCR ⁇ -O-R,, C,-C 6 alkyl, C 6 -C 15 aiyl or C A - C ]5 heterocycle where R 5 and Rg are independently hydrogen, C,-C 6 alkyl,
  • compositions comprise a plurality of compounds having Formula I (or other formulas set forth herein). Such compositions have been shown to have antibiotic activity and, hence, to be useful in the formulation of pharmaceuticals and otherwise.
  • the compounds in accordance with Formula I are such that n is 1 or 2, and each of x and y is 2 or 3. More preferably n is 1 , and x and y are 2.
  • R is halogen, cyano, C,- C 6 alkyl, perhalo C,-C 3 alkyl, nitro, or carboxylate.
  • Exemplary C,-C 6 alkyls include, methyl, ethyl, propyl, butyl, /-butyl, pentyl and hexyl.
  • Exemplary perhalo C r C 3 alkyls include, trifluoromethyl, trichloromethyl, triiodomethyl, pentafluoroethyl, pentachloroethyl, and heptafluoropropyl.
  • Exemplary carboxylates include methyl carboxylate, ethyl carboxylate, /-butyl carboxylate, and sodium carboxylate. More preferably, R, is fluorine, cyano, methyl, trifluoromethyl or methyl carboxylate. In yet another preferred embodiment of the invention, R 2 is hydrogen, C 6 -
  • Heterocycles are ring systems which contain one or more of heteroatoms, such as O, N or S.
  • a heterocycle may be aromatic or contain one or more non-aromatic unsaturated bonds, i.e., ⁇ -bonds.
  • Exemplary C 4 -C,, heterocycles include pyran, pyrrolidone, benzothiazole, pyridine, benzoxazole, piperidine, piperazine, furan, thiofuran, pyrimidine and carbazole.
  • Exemplary C 6 -C, 5 aryls include phenyl, aminophenyl, halophenyl, methoxyphenyl, hydroxyphenyl, toluyl, trifluoromethylphenyl, xylyl, ethylphenyl, propylphenyl, naphthyl, bromonaphthyl, chloronaphthyl, methylnaphthyl, hydroxynaphtyl, anthracyl, bromoanthracyl, and chloroanthracyl.
  • Halophenyls are phenyl groups with one or more halogens substituted on the phenyl ring.
  • Exemplary halophenyls include chlorophenyl, bromophenyl, chlorophenyl, fluorophenyl, dichlorophenyl, chlorofluorophenyl and dibromophenyl.
  • a preferred halophenyl is ⁇ -chlorophenyl.
  • Exemplary C 7 -C 20 alkaryl include benzyl, methoxybenzyl, bromobenzyl, fluorobenzyl, naphthylmethyl, methoxyphenylmethyl, acetoxyphenylmethyl, 2-phenylethyl, 2-naphthylethyl, 3- phenylpropyl and 2-phenylpropyl.
  • R 2 is hydrogen, phenyl, halophenyl, m- trifluoromethylphenyl, benzyl, m-methylbenzyl, m-nitrobenzyl, w-fluorobenzyl, m- cyanobenzyl, w-trifluoromethylbenzyl, Aw-methylcarboxylbenzyl, 6-trifluoromethyl-2- pyridyl or a moiety of the formula -(CH 2 ) m -R 3 -R 4 .
  • R 4 is -O-R 5 , -N(R 5 )(R 6 ), -NfR ⁇ -O-R,, C,-C 6 alkyl, C 6 -C !5 aryl or C 4 -C, 5 heterocycle.
  • R is -O-R 5 , -N(R 5 )(R 6 ), -NCR ⁇ -O-R*, C 6 -C 15 aryl or C 4 -C I5 heterocycle.
  • Preferred C 6 -C 15 aryl groups for R are phenyl, m-methylphenyl, m- nitrophenyl, m-fluorophenyl, m-cyanophenyl, m-trifluoromethylphenyl or m- methylcarboxylphenyl.
  • a preferred C 4 -C l5 heterocycle for R 4 is 3-(2',4'-dichlorophenyl)- 5-isoxazolyl.
  • R 5 and Rg are independently hydrogen, C,-C 6 alkyl, C 6 -C, 5 aryl, C 3 -C 12 cycloalkyi, C 4 -C l5 heterocycle or C 7 -C 20 alkaryl.
  • R 5 is hydrogen, C,-C 6 alkyl or C 7 -C 20 alkaryl.
  • Preferred C,-C 6 alkyl groups for R 5 are methyl and /-butyl.
  • Preferred C 7 -C 20 alkaryl groups for R 5 are benzyl, m-methylbenzyl, m-nitrobenzyl, /M-fluorobenzyl, n-cyanobenzyl, /w-trifluoromethylbenzyl and m-methylcarboxylbenzyl.
  • R ⁇ - is hydrogen, C,-C 6 alkyl, C 3 -C 12 cycloalkyi, C 4 -C 15 heterocycle or C 7 -C 20 alkaryl.
  • Cycloalkyi groups can be monocyclic, bicyclic, bridged cyclic or polycyclic. Cycloalkyi groups also can be substituted.
  • Exemplary cycloalkyi groups include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclononane, cyclodecane, cycloundecane, cyclododecane, adamantane, norcamphor, [3,3,0]bicyclooctane and norborneol.
  • a preferred C 3 -C 12 cycloalkyi for ⁇ is cycloheptyl.
  • a preferred C r C 6 alkyl for 1 ⁇ is methyl.
  • a preferred C 4 -C )5 heterocycle for R ⁇ is 2-benzothiazolyl, and a preferred C 7 -C 20 alkaryl for 1 ⁇ is benzyl.
  • compositions comprising compounds having Formula II or III:
  • p and s are independently 0, 1 or 2; x and y are independently 1 , 2 or 3; Qi and Q 2 are independently -N(R, 5 )-, -O- or -S-;
  • R 2 is hydrogen, C,-C 6 alkyl, -C l 5 aryl, C 7 -C 20 alkaryl, C 4 -C, 5 heterocycle or a moiety of the formula -(CH 2 ) m -R 3 -R 4 where: m is 0 or 1 ;
  • R,, and R I4 are independently hydrogen, C r C 6 alkyl, C 7 -C 20 alkaryl, C 6 -C 20 aryl, C 4 -C 15 heterocycle or C 3 -C 20 cycloalkyi;
  • N-(aminoalkyl)- and/or N- (amidoalkyl)-di-nitrogen heterocycles may be seen to relate to N-(aminoalkyl)- and/or N- (amidoalkyl)-di-nitrogen heterocycles.
  • p and s are independently 0 or 1
  • x and y are independently 2 or 3. More preferably, p and s are 1
  • x and y are 2.
  • Q, and Q 2 are -N(R )5 )-.
  • 4 be independently C,-C 6 alkyl, C 7 -C 20 alkaryl, C 6 -C 20 aryl, C 4 -C 15 heterocycle or C 3 - C 20 cycloalkyi. More preferably, R ⁇ and R, 4 are independently C 7 -C 20 alkaryl, -C 20 aryl, C 4 -C 15 heterocycle or C 3 -C 20 cycloalkyi. Still more preferably, R, , and R 14 are independently C 6 -C 20 aryl, C 4 -C ]5 heterocycle or C 3 -C 20 cycloalkyi.
  • R n and R 14 are independently selected from the group consisting of substituted or unsubstituted phenyl, furanyl, pyridyl, benzothiazolyl, thiofuranyl, naphthyl, pyrimidyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and cyclododecyl. Still even more preferably, R ⁇ and R 14 are independently 2- benzothiazolyl, cycloheptyl or -methoxyphenyl.
  • R 15 is hydrogen, such that Q ⁇ and Q 2 are -NH-.
  • compositions which comprise mixtures of compounds of the invention. Surprisingly, it has been found that such compositions have antibacterial activity, in some cases against both Gram negative and Gram positive bacteria.
  • compositions comprising compounds disclosed herein are useful as antibiotics as well as in other therapeutic areas including treatment of fungal infections, viral infections, various type of neoplastic disease, cardiovascular diseases, central nervous system disorders, inflammation and immune disorders.
  • Compositions of the present invention can inhibit both Gram positive bacteria, exemplified by Escherichia Coli (E. Col ⁇ ), and Gram negative bacteria, such as Streptococcus Pyogenes (S. Pyogenes). Accordingly, the present invention provides therapeutic regimes against bacterial infection employing compositions set forth herein.
  • compounds of the present invention are useful in other pharmaceutical areas and as intermediates for preparation and discovery of pharmaceutically active agents.
  • the nitrogen heterocycles of the invention are likely to be useful in a number of therapeutic arenas, including muscle relaxants (as, for example, pipercurium bromide), anthelminthic drugs (as, for example, piperazine and its analogues), antineoplastic agents (as, for example, piposulfan), biological buffers (as, for example, piperazine derivatives such as piperazinediethanesulfonic acid), anti-ulcerative agents (as, for example, pirenzepine), antihypertensive agents (as, for example, prazosin), and anti-inflammatory agents (as, for example, protacine (proglumetacin)).
  • muscle relaxants as, for example, pipercurium bromide
  • anthelminthic drugs as, for example, piperazine and its analogues
  • antineoplastic agents as, for example, piposulfan
  • biological buffers as, for example, piperazine derivatives such as piperazinediethanes
  • compositions of matter of this invention can also be used in or as an intermediate for preparing or discovering drugs useful in the treatment of neoplastic diseases, immune disorders, cardiovascular diseases, central nervous system disorders and inflammation, among others.
  • routes of drug administration and dosage levels for particular compositions of matter of this invention in view of the objects thereby to be attained.
  • the dosage forms of the present invention can be administered orally, rectally, parenterally, or transdermally, alone or in combination with other psychostimulants, antidepressants, and the like to a patient in need of treatment.
  • Oral dosage forms include tablets, capsules, dragees, and other conventional, pharmaceutical forms.
  • Isotonic saline solutions conveniently containing about 1-200 milligrams of drug per milliliter can be used for parenteral administration which includes intramuscular, intrathecal, intravenous and intra- arterial routes.
  • Rectal administration can conveniently be effected through the use of suppositories such as can easily be formulated from conventional carriers such as cocoa butter.
  • Transdermal administration can be effected through the use of transdermal patch delivery systems and the like. The preferred routes of administration are oral and parenteral.
  • the dosage employed should be carefully titrated to the patient, considering age, weight, severity of the condition, and clinical-profile. The actual decision as to dosage will depend upon the exact drug being employed and will be made by the attending physician as a matter of routine. Such physician can, however, determine an appropriate regime employing well-known medical considerations.
  • Unit dosage forms are selected as a matter of routine depending upon the selected route of administration. For oral administration, formulation into tablets using tableting excipients are conveniently employed, although capsular and other oral forms are also useful.
  • pharmaceutical pharmaceutically active
  • pharmaceutically useful are used interchangeably herein and refer to ability of the compounds of the present invention to provide some therapeutic or physiological beneficial, effect.
  • the terms include any physiologically or pharmacologically activity that produces a localized or systemic effect or effects in animals, including warm blooded mammals such as humans.
  • Pharmaceutically active agents may act on the peripheral nerves, adrenergic receptors, cholinergic receptors, the skeletal muscles, the cardiovascular system, smooth muscles, the blood circulatory system, synoptic sites, neuroeffector junctional sites, endocrine and hormone systems, the immunological system, the reproductive system, the skeletal system, autocoid systems, the alimentary and excretory systems, the histamine system and central nervous systems as well as other biological systems.
  • the compounds of the present invention may be used as sedatives, psychic energizers, tranquilizers, anticonvulsants, muscle relaxants, antiparkinson agents, analgesics, antiinflammatories, local anesthetics, muscle contractants, antibiotic, antiviral, antiretroviral, antimalarials, diuretics, lipid regulating agents, antiandrogenic agents, antiparasitics, neoplasties, antineoplastics and chemotherapy agents.
  • the compounds of the present invention could further be used to treat cardiovascular diseases, central nervous system diseases, cancer, metabolic disorders, infections and dermatological diseases as well as other biological disorders and infections.
  • uses of the compositions and compounds of the present invention are uses in scientific research as research reagents.
  • Exemplary uses of this type are those described in Fodor et al, United States patent 5,489,678; Pirrung et ⁇ /.United States patent 5,143,854; Lerner et al, PCT patent application WO 93/20242; Lebl et al. PCT patent application WO 94/28028; Hollis et al. PCT patent application WO 93/22678; Brennan United States patent 5,472,672, Nishioka United States patent 5,449,754 and Ecker et al, PCT patent application WO 93/04204.
  • compositions can be obtained by preparing mixtures of compounds formed from the constituent moieties forming the present compounds.
  • compounds formed by reacting reactive appendage compounds such as meta benzylic compounds, alpha-amide compounds or other compounds having a reactive group thereupon, with one or a family of scaffold molecules having a plurality of reactive functionalities thereupon have great utility as pharmaceuticals.
  • the scaffold molecules have at least one reactive functionality which can react with the reactive functionality on the appendage molecules, together with at least one additional reactive moiety for reaction with other appendages or functional groups.
  • the scaffold molecules conveniently have nucleophilic functionalities while the appendage molecules are comprise one or more leaving groups. The reverse can also be true, however, such that the scaffold molecules have electrophilic centers and leaving groups while the appendages are nucleophilic. Other reactions may also be employed in this context in addition to nucleophilic substitution reactions.
  • the reactive functions reside on the benzylic carbon atom and that the same comprise a leaving group.
  • the reactive functional group is also a leaving group, but may conveniently reside alpha to the carbonyl.
  • the leaving group is a halogen, such that the groups are alpha haloamides.
  • Other appendage molecules and a wide variety of functional groups thereupon may be employed in accordance with the spirit of this invention.
  • Preferred scaffold molecules are those which possess at least two functional groups, at least one of which can react with appendage molecules. It is preferred that two or more functional groups be present such that great diversity of resulting species can be attained. Thus, scaffolds having two, three and more functional groups reactive with appendage molecules — either in the same chemical way or in different ways — are highly useful in the practice of this invention.
  • Preferred scaffold species are di-nitrogen heterocycles as disclosed herein. Many other scaffold species can be used, however.
  • compositions can be seen to be mixtures of reaction species.
  • One preferred use for such mixtures is in the identification of chemical species which have biological activity, especially pharmaceutical activity. Such mixtures can be screened for activity and active molecular species determined.
  • mixtures conventionally denominated "libraries" are useful per se, and are well known to be useful in the chemical and pharmaceutical industry, where the preparation and exchange of libraries for screening is a common undertaking.
  • THF refers to tetrahydrofuran
  • DMF dimethylformamide
  • 2-Mercapto-l-ethanesulfonic acid (sodium salt), 3-mercapto-l- propanesulfonic acid (sodium salt), 1-phenylpiperazine, THF, DMF, diisopropylethylamine, and 2-aminoethanesulfonic acid were purchased from Aldrich (Milwaukee, WI), 2-aminobenzothiazole was purchased from Lancaster (Windham, NH), and bromoacetyl bromide was purchased from Fluka (Ronkonkoma, NY).
  • tert-Butyl 4-benzyI-l-piperazinecarboxylate (1), tert-Butyl 4-(3'-methylbenzyl)-l- piperazinecarboxylate (2), tert-Butyl 4-(3 , -nitrobenzyl)-l-piperazinecarboxylate (3), ter-'-Butyl 4-(3'-fluorobenzyl)-l-piperazinecarboxylate (4), tert-Butyl 4-(3'- cyanobenzyl)-l-piperazinecarboxylate (5), tert-Butyl 4-(3'-trif-uoromethylbenzyI)-l- piperazinecarboxylate (6) and tert-Butyl 4-(3'-methylcarboxylbenzyI)-l- piperazinecarboxylate (7) To a solution of /er/-butyl 1 -piperazinecarboxylate (prepared as per the procedures of Essien, H.
  • the reaction mixture was stirred at room temperature for 12 hours and then poured into an aqueous mixture of 3-mercapto-l - propanesulfonic acid, sodium salt (7.5 g, 42 mmol) and potassium carbonate (12 g, 84 mmol).
  • the resulting mixture was stirred at room temperature for about 2 hours, concentrated in vacuo and partitioned between ether and water.
  • the aqueous layer was separated and extracted with ether (2x30 mL). The organic layers were combined, dried over Na 2 SO 4 , filtered and concentrated in vacuo to afford a mixture of the title compounds (970 mg).
  • Example 2 To the mixture from Example 1 (3 mmol) in ethanol (20 mL) was added 6 M HC1 in ethanol (30 mL, 180 mmol). The reaction mixture was stirred at room temperature for about 12 hours and concentrated in vacuo. The resulting residue was dissolved in water (20 mL), made basic with NaOH and extracted with ethyl acetate (2x30 mL). The organic layers were combined, dried over Na 2 SO 4 , filtered and concentrated in vacuo to afford the title mixture of deprotected compounds (570 mg, 2.72 mmol, 91%).
  • the title compound was prepared via a modification of the literature procedure of Yuan, J.; Zhang, M., Beijing Daxue Xuebao, Ziran Kexueban, 1988, 24 , 504-506.
  • the resulting mixture was cooled to -20 °C, and bromoacetyl bromide (4.78 mL, 55.0 mmol) was added slowly.
  • the reaction mixture was warmed to room temperature over 30 minutes and stirred for an additional 30 minutes.
  • the reaction mixture was diluted with water (100 mL), stirred for 30 minutes and further diluted with ethyl acetate (500 mL). The organic layer was separated, washed with water (2x 100 mL), washed with brine (100 mL), dried over magnesium sulfate and concentrated in vacuo to afford a purple solid (14.96 g). Recrystallization of the crude product from ethyl acetate provided 8.30 g (61%) the title compound as a purple solid.
  • the reaction mixture was stirred at room temperature for 12 hours and then poured into a methanol-water solution containing 3- mercapto-1 -propanesulfonic acid, sodium salt (0.125 g, 0.7 mmol) and potassium carbonate (0.2 g, 1.4 mmol).
  • the resulting mixture was stirred at room temperature for 2 hours and concentrated in vacuo and partitioned between water and ether.
  • the aqueous layer was separated and extracted with ether (2x30 mL). The organic layers were combined, dried over Na 2 SO 4 , filtered and concentrated to afford 160 mg (0.41 mmol, 90%) of the title mixture.
  • the reaction mixture was stirred at room temperature for 12 hours and then poured into a methanol -water solution containing 3-mercapto-l-propanesulfonic acid, sodium salt (0.5 g, 3.15 mmol) and potassium carbonate (1 g, 7 mmol). The mixture was stirred at room temperature for 2 hours and concentrated. The resulting residue was partitioned between ether and water. The aqueous layer was separated and extracted with ether (2x30 mL). The organic layers were combined, washed with brine, dried over Na ⁇ SO,,, filtered and concentrated to afford 130 mg of the title library as an oil.
  • reaction mixture was warmed to room temperature over 20 minutes and stirred additional 30 minutes.
  • the reaction mixture was diluted with water (100 mL), stirred for 30 minutes and the organic layer was separated.
  • the organic layer was washed with water (2x100 mL), brine (100 mL), dried over magnesium sulfate and concentrated in vacuo to afford a beige solid (9.68 g) which was recrystallized from ethyl acetate to provide bromo-N-(4'-methoxyphenyl) acetamide as a white crystal (6.31 g, 65%).
  • Staphylococcus aureus is known to cause localized skin infections as a result of poor hygiene, minor trauma, psoriasis or eczema. It also causes respiratory infections, pneumonia, toxic shock syndrome and septicemia. It is a common cause of acute food poisoning. It exhibits rapid emergence of drug resistance to penicillin, cephalosporin, vancomycin and nafcillin.
  • the strain S. aureus ATCC 25923 (American Type Culture Collection) is used in the bioassay.
  • BBL typtocase soy broth
  • This bacteria is then used to reinoculate sample wells of 96-well microtiter plates.
  • the assays are carried out in the 96-well microtiter plates in 150 ⁇ L volume with approximately 1 x 10 6 cells per well.
  • Bacteria in typtocase soy broth (75 ⁇ L) is added to the compound mixtures in solution in 75 ⁇ L water/4% DMSO in the individual well of the microtiter plate. Final concentrations of the compound mixtures are 25 ⁇ M, 10 ⁇ M and l ⁇ M. Each concentration of the compound mixtures are assayed in triplicate. The plates are incubated at 37°C and growth monitored over a 24 hour period by measuring the optical density at 595 nm using a BioRad model 3550 UV microplate reader. The percentage of growth relative to a well containing no compound is determined. Ampicillin and tetracycline antibiotic positive controls are concurrently tested in each screening assay. PROCEDURE 2
  • the strain S. aureus ATCC 14289 (American Type Culture Collection) is used in the bioassay.
  • a sample of bacteria is grown overnight at 37 °C in lx Todd-Hewitt broth. This bacteria is then used to reinoculate sample wells of 96-well microtiter plates.
  • the assays are carried out in the 96-well microtiter plates in 150 ⁇ L volume with approximately 1 x 10 6 cells per well. Bacteria in lx Todd-Hewitt broth (75 ⁇ L) is added to the compound mixtures in solution in 75 ⁇ L water in the individual well of the microtiter plate.
  • Final concentrations of the compound mixtures are 25 ⁇ M, 10 ⁇ M and 1 ⁇ M.
  • concentration of the compound mixtures are assayed in triplicate.
  • the plates are incubated at 37 °C and growth monitored over a 24 hour period by measuring the optical density at 595 nm using a BioRad model 3550 UV microplate reader. The percentage of growth relative to a well containing no compound is determined. Ampicillin and tetracycline antibiotic positive controls are concurrently tested in each screening assay.
  • B. E. coli imp- In this assay, the strain E. coli imp- obtained from Spenser Bensen (Sampson, B.A., Misra, R. & Benson, S.A. (1989), Genetics, 122, 491-501, Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability) is used. To initiate the exponential phase of bacterial growth prior to the assay, a sample of bacteria is grown overnight at 37 °C in Luria broth. This bacteria is then used to reinoculate sample wells of 96-well microtiter plates. The assays are carried out in the 96-well microtiter plates in 150 ⁇ L volume with approximately 1 x 10 6 cells per well.
  • Bacteria in Luria broth (75 ⁇ L) is added to the compound mixtures in solution in 75 ⁇ L water in the individual well of the microtiter plate. Final concentrations of the compound mixtures are 25 ⁇ M, 10 ⁇ M and l ⁇ M. Each concentration of the compound mixtures are assayed in triplicate. The plates are incubated at 37 °C and growth monitored over a 24 hour period by measuring the optical density at 595 nm using a BioRad model 3550 UV microplate reader. The percentage of growth relative to a well containing no compound is determined. Ampicillin and tetracycline antibiotic positive controls are concurrently tested in each screening assay.
  • Combinatorial libraries in accordance with the present invention have been tested for antibacterial activity utilizing assays that determine the minimum inhibitory concentration (MIC).
  • the antibacterial assays utilize streptococcus pyogenes and escherichia coli imp-. Activity has been detected in a number of libraries of the present invention.
  • the MIC exhibited by a mixture of compounds containing l-[2'-(N-cycloheptyl)amino]ethyl-4-alkaryl piperazine is 50-100 ⁇ M for gram-positive bacteria, E. Coli and 25-50 ⁇ M for gram-negative bacteria, S. Pyogenes.
  • the MIC exhibited by a mixture of compounds containing l-[2'-(N- cycloheptyl-N-alkaryl)amino]ethyl-4-alkaryl piperazine is 25-50 ⁇ M for gram-positive bacteria, E. Coli and 12.5-25 ⁇ M for gram-negative bacteria, S. Pyogenes.
  • the strain C. albicans ATCC 10231 (American Type Culture Collection) is used in the bioassay.
  • a sample of yeast is grown overnight at 37 °C in YM media. This yeast is then used to reinoculate sample wells of 96-well microtiter plates.
  • the assays are carried out in the 96-well microtiter plates in 150 ⁇ L volume with approximately 1 x 10 6 cells per well.
  • Yeast in YM media (75 ⁇ L) is added to the compound mixtures in solution in 75 ⁇ L water in the individual well of the microtiter plate. Final concentrations of the compound mixtures are 25 ⁇ M, 10 ⁇ M and l ⁇ M.
  • Each concentration of the compound mixtures are assayed in triplicate.
  • the plates are incubated at 37 °C and growth monitored over a 24 hour period by measuring the optical density at 595 nm using a BioRad model 3550 UV microplate reader. The percentage of growth relative to a well containing no compound is determined. Amphotericin B positive control is concurrently tested in each screening assay.
  • PROCEDURE 4 RNA Binding Assay The effect of libraries on tat/TAR interactions
  • the effects of combinatorial libraries on tat TAR, RNA protein interactions are examined using a rapid and reproducible binding assay.
  • the assay consists of a biotinylated truncated version of the HIV-l TAR stem-loop, which is anchored to the wells of a 96 well ELISA plate which has been coated with streptavidin.
  • the TAR RNA is recognized by the HTV-l protein tat and the amount of tat bound is quantitated using an antibody raised against tat and a secondary antibody conjugated to an alkaline phosphatase or HRP enzyme to produce a colorimetric reaction.
  • a 39 residue tat peptide (aa 49-85 of HIV tat protein). This is the C terminal basic binding domain of the tat protein. This peptide was synthesized by a contract lab. A 30 base RNA oligonucleotide consisting of the bulge and stem/loop structure of HIV TAR which has also been Biotin conjugated. This RNA oligonucleotide was synthesized in house.
  • Protein A G alkaline phosphatase (Pierce).
  • a Streptavidin coated 96 well ELISA plate is added 200 ⁇ l of a solution of the 30 base TAR sequence (20 nM) in binding buffer. The plate is incubated at 4 °C for 1 hour. The biotintylated HIV RRE RNA oligonucleotide is bound to selected wells as a negative control RNA. The plate is washed with binding buffer three times and 100 ⁇ l of a 100 nM solution of the 39 residue tat peptide in binding buffer is added to each well. Combinatorial libraries or deconvoluted combinatorial libraries are added to selected wells of the plate at initial concentrations of 100 ⁇ M. The plate is incubated for 1 hour at room temperature.
  • the plate is washed with binding buffer three times and blocked with binding buffer + 5% FCS.
  • 100 ⁇ l of tat antiserum diluted 1 :700 in binding buffer is added to the wells of the plate and the plate is incubated for 1.5 hours at 4 °C.
  • the plate is washed three times with binding buffer and 150 ⁇ L of a solution of protein A/G alkaline phosphatase diluted 1 :5000 in binding buffer is added to each well.
  • the plate is incubated for 1.5 hours at 4 °C followed by washing three times with binding buffer.
  • 150 ⁇ L of PNPP substrate is added to each well and the plate is incubated for 1 hour at 37 °C.
  • the absorbance of each well is read in a multiwell plate reader.
  • DNA gyrase is a bacterial enzyme which can introduce negative supercoils into DNA utilizing the energy derived from ATP hydrolysis. This activity is critical during DNA replication and is a well characterized target for antibiotic inhibition of bacterial growth.
  • libraries of compoimds are screened for inhibition of DNA gyrase. The assay measures the supercoiling of a relaxed plasmid by DNA gyrase as an electrophoretic shift on an agarose gel. Initially all library pools are screened for inhibitory activity at 30 ⁇ M and then a dose response analysis is effected with active subsets. Novobiocin, an antibiotic that binds to the ⁇ subunit of DNA gyrase is used as a positive control in the assay.
  • the sensitivity of the DNA gyrase assay was determined by titrating the concentration of the know DNA gyrase inhibitor, Novobiocin, in the supercoiling assay.
  • the IC 50 was determined to be 8 nM, sufficient to identify the activity of a single active species of comparable activity in a library having 30 ⁇ M concentration.
  • This procedure is used to identify compounds of the invention from libraries of compounds constructed to include a ring that contains an ultraviolet chromophore.
  • the chemical functional groups attached to the compounds of the invention are selected from metal binders, coordinating groups such as amine, hydroxyl and carbonyl groups, and other groups having lone pairs of electrons, such that the compounds of the invention can form coordination complexes with heavy metals and imaging agents.
  • the procedure is used to identify compounds of the invention useful for chelating and removing heavy metals from industrial broths, waste stream eluents, heavy metal poisoning of farm animals and other sources of contaminating heavy metals, and for use in identifying imaging agent carriers, such as carriers for technetium 99.
  • An aliquot of a test solution having the desired ion or imaging agent at a known concentration is added to an aliquot of standard solution of the pool under assay.
  • the UV spectrum of this aliquot is measured and is compared to the UV spectrum of a further aliquot of the same solution lacking the test ion or imaging agent.
  • a shift in the extinction coefficient is indicative of binding of the metal ion or imaging ion to a compound in the library pool being assayed.
  • PROCEDURE 7 Assay of combinatorial library for PLA 2 inhibitors
  • Phospholipases A 2 are a family of enzymes that hydrolyze the sn-2 ester linkage of membrane phospholipids resulting in release of a free fatty acid and a lysophospholipid (Dennis, E.A., The Enzymes, Vol. 16, pp. 307-353, Boyer, P.D., ed., Academic Press, New York, 1983). Elevated levels of type II PLA 2 are correlated with a number of human inflammatory diseases. The PLA 2 - catalyzed reaction is the rate-limiting step in the release of a number of pro-inflammatory mediators.
  • Arachidonic acid a fatty acid commonly linked at the sn-2 position, serves as a precursor to leukotrienes, prostaglandins, lipoxins and thromboxanes.
  • the lysophospho- lipid can be a precursor to platelet-activating factor.
  • PLA 2 is regulated by pro- inflammatory cytokines and, thus, occupies a central position in the inflammatory cascade (Dennis, ibid.; Glaser et al., TiPs Reviews 1992, 14, 92; and Pruzanski et al, Inflammation 1992, 16, 451). All mammalian tissues evaluated thus far have exhibited PLA 2 activity.
  • Type II pancreatic
  • cytosolic synovial fluid
  • Type II cytosolic
  • Type I and type II the secreted forms of PLA 2 , share strong similarity with phospholipases isolated from the venom of snakes.
  • the PLA 2 enzymes are important for normal functions including digestion, cellular membrane remodeling and repair, and in mediation of the inflammatory response.
  • Both cytosolic and type II enzymes are of interest as therapeutic targets.
  • Increased levels of the type II PLA 2 are correlated with a variety of inflammatory disorders including rheumatoid arthritis, osteoarthritis, inflammatory bowel disease and septic shock, suggesting that inhibitors of this enzyme would have therapeutic utility.
  • the type II PLA 2 enzyme from synovial fluid is a relatively small molecule (about 14 kD) and can be distinguished from type I enzymes (e.g. pancreatic) by the sequence and pattern of its disulfide bonds. Both types of enzymes require calcium for activity.
  • type I enzymes e.g. pancreatic
  • the crystal structures of secreted PLA 2 enzymes from venom and pancreatic PLA 2 , with and without inhibitors, have been reported (Scott et al, Science 1990, 250, 1541 ). Recently, the crystal structure of PLA 2 from human synovial fluid has been determined (Wery et al. Nature 1991, 352, 79). The structure clarifies the role of calcium and amino acid residues in catalysis.
  • Calcium acts as a Lewis acid to activate the scissile ester carbonyl bond of 1 ,2-diacylglycerophospholipids and binds to the lipid, and a His- Asp side chain diad acts as a general base catalyst to activate a water molecule nucleophile. This is consistent with the absence of any acyl enzyme intermediates, and is also comparable to the catalytic mechanism of serine proteases.
  • the catalytic residues and the calcium ion are at the end of a deep cleft (ca. 14 A) in the enzyme. The walls of this cleft contact the hydrocarbon portion of the phospholipid and are composed of hydrophobic and aromatic residues.
  • the positively-charged amino-terminal helix is situated above the opening of the hydrophobic cleft.
  • N-terminal portion is the interfacial binding site Achari et al, Cold Spring Harbor Symp. Quant. Biol. 1987, 52, 441 ; Cho et al., J. Biol. Chem. 1988, 263, 11237; Yang et al, Biochem. J. 1989, 262, 855; and Noel et al, J. Am. Chem. Soc. 1990, 112, 3704).
  • PLA 2 displays a lag phase during which the enzyme adsorbs to the substrate bilayer and a process called interfacial activation occurs. This activation may involve desolvation of the enzyme/lipid interface or a change in the physical state of the lipid around the cleft opening.
  • Evidence favoring this hypothesis comes from studies revealing that rapid changes in PLA 2 activity occur concurrently with changes in the fluorescence of a membrane probe (Burack et al, Biochemistry 1993, 32, 583). This suggests that lipid rearrangement is occurring during the interfacial activation process.
  • PLA 2 activity is maximal around the melting temperature of the lipid, where regions of gel and liquid- crystalline lipid coexist. This is also consistent with the sensitivity of PLA, activity to temperature and to the composition of the substrate, both of which can lead to structurally distinct lipid arrangements separated by a boundary region. Fluorescence microscopy was used to simultaneously identify the physical state of the lipid and the position of the enzyme during catalysis (Grainger et al, FEBS Lett. 1989, 252, 73). These studies clearly show that PLA 2 binds exclusively at the boundary region between liquid and solid phase lipid.
  • PLA 2 While the hydrolysis of the secondary ester bond of 1 ,2-diacylglycerophospholipids catalyzed by the enzyme is relatively simple, the mechanistic and kinetic picture is clouded by the complexity of the enzyme-substrate interaction.
  • a remarkable characteristic of PLA 2 is that maximal catalytic activity is observed on substrate that is aggregated (i.e. phospholipid above its critical micelle concentration), while low levels of activity are observed on monomeric substrate.
  • competitive inhibitors of PLA 2 either have a high affinity for the active site of the enzyme before it binds to the substrate bilayer or partition into the membrane and compete for the active site with the phospholipid substrate.
  • compounds of the invention are selected for their potential to interact with, and preferably inhibit, the enzyme PLA 2 .
  • compounds of the invention can be used for topical and or systemic treatment of inflammatory diseases including atopic dermatitis and inflammatory bowel disease.
  • advantage can be taken of PLA 2 's preference for anionic vesicles over zwitterionic vesicles.
  • Preferred compounds of the invention for assay for PLA 2 include those having aromatic diversity groups to facilitate binding to the cleft of the PLA 2 enzyme (Oinuma et al, J. Med. Chem. 1991, 34, 2260; Marki et al, Agents Actions 1993, 38, 202; and Tanaka et al, J.
  • PLA 2 -directed compounds of the invention can further include hydrophobic functional groups such as tetraethylene glycol groups. Since the PLA 2 enzyme has a hydrophobic channel, hydrophobicity is believed to be an important property of inhibitors of the enzyme.
  • the resulting libraries or pools of compounds are screened for inhibition of human type II PLA 2 enzymatic activity. The assay is effected at the conclusion of each round of synthesis to identify the wining pool from that round of synthesis. Concurrently, the libraries additionally can be screened in other in vitro assays to determine further mechanisms of inhibition.
  • the pools of the libraries are screened for inhibition of PLA 2 in the assay using E. coli labeled with 3 H-oleic acid (Franson et al, J. Lipid Res. 1974, 15, 380; and Davidson et al., J. Biol. Chem. 1987, 262, 1698) as the substrate.
  • Type II PLA 2 originally isolated from synovial fluid, expressed in a baculovirus system and partially purified, serves as a source of the enzyme.
  • a series of dilutions of each of the library pools is done in water: 10 ⁇ l of each pool is incubated for 5 minutes at room temperature with a mixture of 10 ⁇ l PLA 2 , 20 ⁇ l 5X PLA 2 Buffer (500 mM Tris 7.0-7.5, 5 mM CaCl 2 ), and 50 ⁇ l water. Samples of each pool are run in duplicate. At this point, 10 ⁇ l of 3 H E. coli cells is added. This mixture is incubated at 37 °C for 15 minutes.
  • the enzymatic reaction is stopped with the addition of 50 ⁇ L 2M HCl and 50 ⁇ L fatty-acid-free BSA (20 mg/mL PBS), vortexed for 5 seconds, and centrifuged at high speed for 5 minutes. 165 ⁇ L of each supernate is then put into a scintillation vial containing 6 ml of scintillant (ScintiVerse) and cpms are measured in a Beckman Liquid Scintillation Counter.
  • a reaction without the combinatorial pool is run alongside the other reactions as well as a baseline reaction containing no compounds of the invention as well as no PLA 2 enzyme. CPMs are corrected for by subtracting the baseline from each reaction data point.
  • the "winner” is compared to phosphatidyl ethanolamine, the preferred substrate of human type II PLA 2 , to confirm its activity.
  • PLA 2 from other sources (snake venom, pancreatic, bee venom) and phospholipase C, phospholipase D and lysophospholipase can be used to further confirm that the inhibition is specific for human type II PLA 2 .
  • Probes for the detection of specific proteins and mRNA in biological samples For the reliable, rapid, simultaneous quantification of multiple varieties of proteins or mRNA in a biological sample without the need to purify the protein or mRNA from other cellular components, a protein or mRNA of interest from a suitable biological sample, i.e., a blood borne virus, a bacterial pathogen product in stool, urine and other like biological samples, is identified using standard microbiological techniques.
  • a probe comprising a compound of a combinatorial library of the invention is identified by a combinatorial search as noted in the above examples.
  • Preferred for the protein probe are compounds synthesized to include chemical functional groups that act as hydrogen bond donors and acceptors, sulfhydryl groups, hydrophobic lipophilic moieties capable of hydrophobic interactions groups and groups capable of ionic interactions.
  • the probe is immobilized on insoluble CPG solid support utilizing the procedure of Pon, R.T.,
  • the mRNA on the support is labeled with ethidium bromide, biotin or a commercial radionucleotide and the amount of label immobilized on the CPG support is measured to indicate the amount of mRNA present in the biological sample.
  • a protein is also labeled and quantified.
  • LTB 4 Leukotriene B 4
  • Library subsets are screened for competitive inhibition of radiolabeled LTB 4 binding to a receptor preparation.
  • Leukotriene B 4 reagent is prepared by adding 5 mL of ligand diluent (phosphate buffer containing NaCl, MgCl 2 , EDTA and Bacitracin, pH 7.2) to 0.25 L of the radioligand.
  • the receptor preparation is made by thawing the concentrate, adding 35 mL of ligand diluent and swirling gently in order to re-suspend the receptor homogeneously. Reagents are kept on ice during the course of the experiment, and the remaining portions are stored at -20 °C.
  • Library subsets prepared as per general procedure of examples above are diluted to 5 ⁇ M, 50 ⁇ M and 500 ⁇ M in phosphate buffer (lx PBS, 0.1% azide and 0.1% BSA, pH 7.2), yielding final test concentrations of 0.5 ⁇ M, 5 ⁇ M and 50 ⁇ M, respectively. Samples are assayed in duplicate. [ 3 H] LTB 4 (25 ⁇ L) is added to 25 ⁇ L of either appropriately diluted standard (unlabeled LTB 4 ) or library subset. The receptor suspension (0.2 mL) is added to each tube. Samples are incubated at 4°C for 2 hours. Controls include [ 3 H] LTB 4 without receptor suspension (total count vials), and sample of ligand and receptor without library molecules (standard).
  • phosphate buffer lx PBS, 0.1% azide and 0.1% BSA, pH 7.2
  • the samples are filtered through GF/B paper that had been previously rinsed with cold saline.
  • the contents of each tube are aspirated onto the filter paper to remove unbound ligand from the membrane preparation, and the tubes washed (2 x 4 mL) with cold saline.
  • the filter paper is removed from the filtration unit and the filter disks are placed in appropriate vials for scintillation counting. Fluor is added, and the vials shaken and allowed to stand at room temperature for 2 to 3 hours prior to counting. The counts/minute (cpm) obtained for each sample are subtracted from those obtained from the total counts to determine the net cpm for each sample.
  • the degree of inhibition of binding for each library subset is determined relative to the standard (sample of ligand and receptor without library molecules).

Abstract

La présente invention concerne des compositions comprenant de nouveaux composés dinitro hétérocycliques contenant au moins un groupe alkaryl à substitution N-méta et/ou de nouveaux composés méta-benzyliques. Les composés de l'invention, qui conviennent comme agents antibactériens et comme autres agents pharmaceutiques, conviennent également comme intermédiaires pour la préparation d'autres agents pharmaceutiques. En outre, les composés de la présente invention conviennent comme réactifs de recherche.
PCT/US1997/013686 1996-08-01 1997-08-01 Compositions heterocycliques WO1998005332A1 (fr)

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US08/691,185 US5731438A (en) 1996-08-01 1996-08-01 N-(aminoalkyl)-and/or N-(amidoalkyl)-dinitrogen heterocycles
US08/691,149 1996-08-01
US08/688,993 1996-08-01
US08/688,993 US5798360A (en) 1996-08-01 1996-08-01 N-(aminoalkyl)- and/or N-(amidoalkyl)- dinitrogen heterocyclic compositions
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1513825A2 (fr) * 2002-05-17 2005-03-16 Department of Health and Human Services Medicament anti-tuberculeux: compositions et methodes
EP1513825A4 (fr) * 2002-05-17 2011-01-05 Dept of Health and Human Services Medicament anti-tuberculeux: compositions et methodes
US8198303B2 (en) 2002-05-17 2012-06-12 Sequella, Inc. Methods of use and compositions for the diagnosis and treatment of infectious diseases
US8268894B2 (en) 2002-05-17 2012-09-18 The United States Of America As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for the treatment of infectious diseases
EP2431029A2 (fr) 2002-06-07 2012-03-21 Kieran Francis Scott Procédé pour inhiber la prolifération de cellules de cancer de la prostate
JP2007509095A (ja) * 2003-10-24 2007-04-12 エフ．ホフマン−ラ　ロシュ　アーゲー Ｃｃｒ３受容体アンタゴニスト
CN106588911A (zh) * 2016-12-14 2017-04-26 山东大学 一种抗变形链球菌的新型噻唑类化合物xqh‑3‑6及其应用
CN106588813A (zh) * 2016-12-14 2017-04-26 山东大学 一种抗变形链球菌的新型噻唑类化合物xqh‑2‑92及其应用
CN106699751A (zh) * 2016-12-14 2017-05-24 山东大学 一种新型化合物xqh‑3‑7及其在抗变形链球菌及抑制其生物膜形成中的应用
CN106699751B (zh) * 2016-12-14 2019-01-15 山东大学 一种新型化合物xqh-3-7及其在抗变形链球菌及抑制其生物膜形成中的应用
CN106588911B (zh) * 2016-12-14 2019-01-18 山东大学 一种抗变形链球菌的新型噻唑类化合物xqh-3-6及其应用
CN108078822A (zh) * 2018-01-17 2018-05-29 山东大学 一种白加黑特型牙膏

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