Classifications

• • 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
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • 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
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
  • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
  • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
  • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
  • C07D239/47—One nitrogen atom and one oxygen or sulfur atom, e.g. cytosine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
  • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
  • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
  • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
  • C07D239/48—Two nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

• the present invention concerns pyrimidine derivatives for the prevention of HIV infection.
• the present invention concerns the use of pyrimidine derivatives for the manufacture of a medicament for the prevention of HIV (Human Immunodeficiency Virus) infection via sexual intercourse and related intimate contact between partners, more in particular the prevention of HIV infection via vaginal sex.
• HIV Human Immunodeficiency Virus
• AIDS immunodeficiency Syndrome
• Sexual transmission is the prevalent mode of transmission of HIV. Said sexual HIV transmission is perfectly preventable by consistent and correct condom use.
• condoms are still not systematically used, especially in Third World cultures and these cultures are heavily affected by the AIDS/HIV epidemic.
• microbicides for topical use.
• a microbicide is a chemical entity that can prevent or reduce transmission of sexually transmitted infections when applied to the site where the transmission takes place.
• microbicides have already been evaluated for their use in preventing HIV transmission: products which have a detergent-surfactant like mode of action (e.g. nonoxynol-9), but said products may cause damage to the vaginal epithelium; acid buffers; Lactobacilli; negatively charged natural or synthetic products which interfere with HIV binding to target cells (e.g. sulfated polysaccharides); HIV multiplication inhibiting agents.
• the present invention relates to the use of pyrimidine derivatives to prevent HIV infection, to prevent the transmission of HIV infection via sexual intercourse and related intimate contact between partners.
• the pyrimidine compounds exhibit HIV replication inhibiting activity in HIV infected warm-blooded animals. They are particularly characterized by an improved ability to inhibit the replication of mutant strains, i.e. strains which have become resistant to art-known drug(s) (drug or multidrug resistant HIV strains).
• the compounds are also able to prevent the transmission of HIV infection in warm-blooded animals, particularly in humans, via sexual intercourse and related intimate contact between partners.
• the compounds have the ability to work prophylactic, thus preventing that the warm-blooded animals get infected; they are also able to provide for post-exposure protection, meaning that when the present compounds are applied after the sexual intercourse and related intimate contact between partners has taken place, they are still able to prevent HIV infection. Furthernore, the compounds have little or no immunosuppressive activity at a therapeutic effective dose.
• WO 99/50250 and WO 00/27825 disclose substituted aminopyrimidines having HIV replication inhibiting properties.
• WO 97/19065 discloses substituted 2-anilinopyrimidines useful as protein kinase inhibitors.
• WO 00/62778 concerns cyclic protein tyrosine kinase inhibitors.
• WO 98/41512 describes substituted 2-anilinopyrimidines useful as protein kinase inhibitors.
• WO 00/78731 describes 5-cyano-2-aminopyrimidine derivatives as KDR kinase or FGFr kinase inhibitors useful in the prophylaxis and treatment of diseases associated with angiogenesis.
• one aspect of the invention relates to the use of a compound for the manufacture of a medicament for the prevention of HIV infection via sexual intercourse or related intimate contact between partners, wherein the compound has the formula a N-oxide, a pharmaceutically acceptable addition salt, a quaternary amine or a stereochemically isomeric form thereof, wherein
• C 1-4 alkyl as a group or part of a group defines straight or branched chain saturated hydrocarbon radicals having from 1 to 4 carbon atoms such as methyl, ethyl, propyl, 1-methylethyl, butyl;
• C 1-6 alkyl as a group or part of a group defines straight or branched chain saturated hydrocarbon radicals having from 1 to 6 carbon atoms such as the group defined for C 1-4 alkyl and pentyl, hexyl, 2-methylbutyl and the like;
• C 2-6 alkyl as a group or part of a group defines straight or branched chain saturated hydrocarbon radicals having from 2 to 6 carbon atoms such as ethyl, propyl, 1-methylethyl, butyl, pentyl, hexyl, 2-methylbutyl and the like;
• C 1-4 alkanediyl defines straight or branched chain saturated bivalent hydrocarbon radicals having from 1 to 4 carbon atoms
• a monocyclic, bicyclic or tricyclic saturated heterocycle represents a ring system consisting of 1, 2 or 3 rings and comprising at least one heteroatom selected from O, N or S, said ring system containing only single bonds
• a monocyclic, bicyclic or tricyclic partially saturated heterocycle represents a ring system consisting of 1, 2 or 3 rings and comprising at least one heteroatom selected from O, N or S, and at least one double bond provided that the ring system is not an aromatic ring system
• a monocyclic, bicyclic or tricyclic aromatic heterocycle represents an aromatic ring system consisting of 1, 2 or 3 rings and comprising at least one heteroatom selected from O, N or S.
• monocyclic, bicyclic or tricyclic saturated carbocycles are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[4,2,0]octanyl, cyclononanyl, cyclodecanyl, decahydronapthalenyl, tetradecahydroanthracenyl and the like.
• monocyclic, bicyclic or tricyclic partially saturated carbocycles are cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, bicyclo[4,2,0]octenyl, cyclononenyl, cyclodecenyl, octahydronaphthalenyl, 1,2,3,4-tetrahydronaphthalenyl, 1,2,3,4,4a,9,9a,10-octahydro-anthracenyl and the like.
• monocyclic, bicyclic or tricyclic aromatic carbocycles are phenyl, naphthalenyl, anthracenyl.
• monocyclic, bicyclic or tricyclic saturated heterocycles are tetrahydrofuranyl, pyrrolidinyl, dioxolanyl, imidazolidinyl, thiazolidinyl, tetrahydrothienyl, dihydrooxazolyl, isothiazolidinyl, isoxazolidinyl, oxadiazolidinyl, triazolidinyl, thiadiazolidinyl, pyrazolidinyl, piperidinyl, hexahydropyriridinyl, hexahydropyrazinyl, dioxanyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, decahydroquinolinyl, octahydroindolyl and the like.
• monocyclic, bicyclic or tricyclic partially saturated heterocycles are pyrrolinyl, imidazolinyl, pyrazolinyl, 2,3-dihydrobenzofuranyl, 1,3-benzodioxolyl, 2,3-dihydro-1,4-benzodioxinyl, indolinyl and the like.
• monocyclic, bicyclic or tricyclic aromatic heterocycles are azetyl, oxetylidenyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, pyranyl, benzofuryl, isobenzofuryl, benzothienyl, isobenzothienyl, indolizinyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzisoxazolyl, benzisothiazolyl, benzopyrazolyl
• ( ⁇ O) forms a carbonyl moiety when attached to a carbon atom, a sulfoxide moiety when attached to a sulfur atom and a sulfonyl moiety when two of said terms are attached to a sulfur atom.
• halo is generic to fluoro, chloro, bromo and iodo.
• polyhalomethyl as a group or part of a group is defined as mono- or polyhalosubstituted methyl, in particular methyl with one or more fluoro atoms, for example, difluoromethyl or trifluoromethyl;
• polyhaloC 1-4 alkyl or polyhaloC 1-6 alkyl as a group or part of a group is defined as mono- or polyhalosubstituted C 1-4 alkyl or C 1-6 alkyl, for example, the groups defined in halomethyl, 1,1-difluoro-ethyl and the like.
• more than one halogen atoms are attached to an alkyl group within the definition of polyhalomethyl, polyhaloC 1-4 alkyl or polyhaloC 1-6 alkyl, they may be the same or different.
• heterocycle in the definition of R 7 or R 7a is meant to include all the possible isomeric forms of the heterocycles, for instance, pyrrolyl comprises 1H-pyrrolyl and 2H-pyrrolyl.
• the carbocycle or heterocycle in the definition of R 7 or R 7a may be attached to the remainder of the molecule of formula (I) through any ring carbon or heteroatom as appropriate, if not otherwise specified.
• the heterocycle when the heterocycle is imidazolyl, it may be 1-imidazolyl, 2-imidazolyl, 4-imidazolyl and the like, or when the carbocycle is naphthalenyl, it may be 1-naphthalenyl, 2-naphthalenyl and the like.
• salts of the compounds of formula (I) are those wherein the counterion is pharmaceutically acceptable.
• salts of acids and bases which are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. All salts, whether pharmaceutically acceptable or not are included within the ambit of the present invention.
• the pharmaceutically acceptable addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid addition salt forms which the compounds of formula (I) are able to form.
• the latter can conveniently be obtained by treating the base form with such appropriate acids as inorganic acids, for example, hydrohalic acids, e.g.
• hydrochloric, hydrobromic and the like sulfuric acid; nitric acid; phosphoric acid and the like; or organic acids, for example, acetic, propanoic, hydroxy-acetic, 2-hydroxypropanoic, 2-oxopropanoic, oxalic, malonic, succinic, maleic, fumaric, malic, tartaric, 2-hydroxy-1,2,3-propanetricarboxylic, methanesulfonic, ethanesulfonic, benzenesulfonic, 4-methylbenzenesulfonic, cyclohexanesulfamic, 2-hydroxybenzoic, 4-amino-2-hydroxybenzoic and the like acids.
• the salt form can be converted by treatment with alkali into the free base form.
• the compounds of formula (I) containing acidic protons may be converted into their therapeutically active non-toxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases.
• Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g.
• primary, secondary and tertiary aliphatic and aromatic amines such as methylamine, ethylamine, propylamine, isopropylamine, the four butylamine isomers, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline, the benzathine, N-methyl-D-glucamine, 2-amino-2-(hydroxymethyl)-1,3-propanediol, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.
• the salt form can be converted by treatment with acid into the free acid form.
• addition salt also comprises the hydrates and solvent addition forms which the compounds of formula (I) are able to form. Examples of such forms are e.g. hydrates, alcoholates and the like.
• quaternary amine as used hereinbefore defines the quaternary ammonium salts which the compounds of formula (I) are able to form by reaction between a basic nitrogen of a compound of formula (I) and an appropriate quaternizing agent, such as, for example, an optionally substituted alkylhalide, arylhalide or arylalkylhalide, e.g. methyliodide or benzyliodide.
• Other reactants with good leaving groups may also be used, such as alkyl trifluoromethanesulfonates, alkyl methanesulfonates, and alkyl p-toluenesulfonates.
• a quaternary amine has a positively charged nitrogen.
• Pharmaceutically acceptable counterions include chloro, bromo, iodo, trifluoroacetate and acetate. The counterion of choice can be introduced using ion exchange resins.
• N-oxide forms of the present compounds are meant to comprise the compounds of formula (I) wherein one or several tertiary nitrogen atoms are oxidized to the so-called N-oxide.
• the compounds of formula (I) may be converted to the corresponding N-oxide forms following art-known procedures for converting a trivalent nitrogen into its N-oxide form.
• Said N-oxidation reaction may generally be carried out by reacting the starting material of formula (I) with an appropriate organic or inorganic peroxide.
• Appropriate inorganic peroxides comprise, for example, hydrogen peroxide, alkali metal or earth alkaline metal peroxides, e.g. sodium peroxide, potassium peroxide;
• appropriate organic peroxides may comprise peroxy acids such as, for example, benzenecarboperoxoic acid or halo substituted benzenecarboperoxoic acid, e.g.
• 3-chlorobenzenecarboperoxoic acid peroxoalkanoic acids, e.g. peroxoacetic acid, alkylhydroperoxides, e.g. tert.butyl hydro-peroxide.
• Suitable solvents are, for example, water, lower alcohols, e.g. ethanol and the like, hydrocarbons, e.g. toluene, ketones, e.g. 2-butanone, halogenated hydrocarbons, e.g. dichloromethane, and mixtures of such solvents.
• stereochemically isomeric forms as used hereinbefore defines all the possible stereoisomeric forms which the compounds of formula (I), and their N-oxides, addition salts, quaternary amines or physiologically functional derivatives may possess.
• chemical designation of compounds denotes the mixture of all possible stereochemically isomeric forms, said mixtures containing all diastereomers and enantiomers of the basic molecular structure as well as each of the individual isomeric forms of formula (I) and their N-oxides, salts, solvates or quaternary amines substantially free, i.e.
• stereogenic centers may have the R- or S-configuration; substituents on bivalent cyclic (partially) saturated radicals may have either the cis- or trans- configuration.
• Compounds encompassing double bonds can have an E (ent ought) or Z (zusammen)-stereochemistry at said double bond.
• the terms cis, trans, R, S, E and Z are well known to a person skilled in the art.
• the absolute stereochemical configuration was not experimentally determined.
• the stereoisomeric form which was first isolated is designated as “A” and the second as “B”, without further reference to the actual stereochemical configuration.
• said “A” and “B” stereoisomeric forms can be unambiguously characterized by for instance their optical rotation in case “A” and “B” have an enantiomeric relationship.
• a person skilled in the art is able to determine the absolute configuration of such compounds using art-known methods such as, for example, X-ray diffraction.
• stereoisomeric mixtures they can be further separated whereby the respective first fractions isolated are designated “A1” and “B13” and the second as “A2” and “B2”, without further reference to the actual stereochemical configuration.
• Pure stereochemically isomeric forms of the present compounds and the intermediates which intervene in the chemical synthesis thereof, can be obtained by the application of art-known procedures.
• diastereoisomers can be separated by physical methods such as selective crystallization or chromatographic techniques, e.g. counter current distribution, liquid chromatography and the like methods.
• Enantiomers can be obtained from racemic mixtures by first converting said racemic mixtures with suitable resolving agents such as, for example, chiral acids, to mixtures of diastereomeric salts or compounds; then physically separating said mixtures of diastereomeric salts or compounds by, for example, selective or fractional crystallization or chromatographic techniques, e.g. liquid chromatography and the like methods; and finally converting said separated diastereomeric salts or compounds into the corresponding enantiomers, for instance by treatment with alkali.
• Pure stereochemically isomeric forms may also be obtained from the pure stereochemically isomeric forms of the appropriate intermediates and starting materials, provided that the intervening reactions occur stereospecifically.
• said compound will be synthesized by stereospecific methods of preparation. These methods will advantageously employ enantiomerically pure starting materials.
• Another interesting group of compounds are those compounds of formula (I) having the formula the N-oxides, the pharmaceutically acceptable addition salts, the quaternary amines or the stereochemically isomeric forms thereof, wherein
• a preferred embodiment of the present compounds encompasses those compounds of formula (I), (I′), (I′′) or (I′′′) wherein R 3 is NHR 13 ; NR 13 R 14 ; —C( ⁇ O)—NHR 13 ; —C( ⁇ O)—NR 13 R 14 ; —C( ⁇ O)—R 15 ; —CH ⁇ N—NH—C( ⁇ O)—R 16 ; C 2-6 alkyl substituted with cyano or aminocarbonyl; C 1-6 alkyl substituted with NR 9 R 10 , —C( ⁇ O)—NR 9a R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 ; C 1-6 alkyl substituted with two or more substituents each independently selected from cyano, NR 9 R 10 , —C( ⁇ O)—NR 9 R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 ; C 1-6 alkyl substituted with one or more substituents each independently selected
• a further interesting group of compounds are those compounds of formula (I), (I′), (I′′) or (I′′′) wherein R 3 is NHR 13 ; NR 13 R 14 ; —C( ⁇ O)—NHR 13 ; —C( ⁇ (O)—NR 13 R 14 ; —C( ⁇ O)—R 15 ; —CH ⁇ N—NH—C( ⁇ O)—R 16 ; C 1-6 alkyl substituted with NR 9 R 10 , —C( ⁇ O)—NR 9a R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 ; C 1-6 alkyl substituted with two or more substituents each independently selected from cyano, NR 9 R 10 , —C( ⁇ O)—NR 9 R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 ; C 1-6 alkyl substituted with one or more substituents each independently selected from cyano, NR 9 R 10 , —C( ⁇ O)—
• R 3 is —CH ⁇ N—NH—C( ⁇ O)—R 16 ; C 1-6 alkyl substituted with NR 9 R 10 , —C( ⁇ O)—NR 9a R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 ; C 1-6 alkyl substituted with two or more substituents each independently selected from cyano, NR 9 R 10 , —C( ⁇ O)—NR 9 R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 ; C 1-6 alkyl substituted with one or more substituents each independently selected from cyano, NR 9 R 10 , —C( ⁇ O)—NR 9 R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 and wherein 2 hydrogen atoms bound at the same carbon atom are replaced by C 1-4 alkanediyl; C
• R 3 is NHR 13 , NR 13 R 14 , —C( ⁇ O)—R 15 , C 1-6 alkyl substituted with one or more substituents each independently selected from cyano, NR 9 R 10 , —C( ⁇ O)—NR 9 R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 ; C 1-6 alkyl substituted with one or more substituents each independently selected from cyano, NR 9 R 10 , —C( ⁇ O)—NR 9 R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 and wherein 2 hydrogen atoms bound at the same carbon atom are replaced by C 1-4 alkanediyl; C 1-6 alkyl substituted with hydroxy and a second substituent selected from cyano, NR 9 R 10 , —C( ⁇ O)—NR 9 R 10 , ——C( ⁇ O)—NR 9 R 10 , —
• R 3 is C 1-6 alkyl substituted with NR 9 R 10 , —C( ⁇ O)—NR 9a R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 ; C 1-6 alkyl substituted with two or more substituents each independently selected from cyano, NR 9 R 10 , —C( ⁇ O)—NR 9 R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 ; C 1-6 alkyl substituted with one or more substituents each independently selected from cyano, NR 9 R 10 , —C( ⁇ O)—NR 9 R 10 , —C( ⁇ O)—C 1-6 alkyl or R 7 and wherein 2 hydrogen atoms bound at the same carbon atom are replaced by C 1-4 alkanediyl; C 1-6 alkyl substituted with hydroxy and a second substituent selected from cyano,
• R 3 is C 1-6 alkyl substituted with one or more substituents each independently selected from cyano, NR 9 R 10 or R 7 ; C 2-6 alkenyl substituted with one or more substituents each independently selected from cyano, NR 9 R 10 or R 7 ; C 1-6 alkyloxyC 1-6 alkyl substituted with cyano; C 1-6 alkyl substituted with hydroxy and a second substituent selected from cyano or R 7 ; —C( ⁇ N—O—R 8 )—C 1-4 alkyl; R 7 or —X 3 —R 7 .
• Another interesting group of compounds are those compounds of formula (I), (I′), (I′′) or (I′′′) wherein R 3 is R 7 .
• Still another interesting group of compounds are those compounds of formula (I), (I′), (I′′) or (I′′′) wherein R 3 is C 1-6 alkyl substituted with cyano, in particular C 2-6 alkyl substituted with cyano, more in particular ethyl or propyl substituted with cyano; or C 2-6 alkenyl substituted with cyano.
• R 3 is C 1-6 alkyl substituted with cyano, in particular C 2-6 alkyl substituted with cyano, more in particular ethyl or propyl substituted with cyano; or C 2-6 alkenyl substituted with cyano.
• Preferred is C 2-6 alkenyl substituted with cyano.
• a further interesting group of compounds are those compounds of formula (I), (I′), (I′′) or (I′′′) wherein R 3 is C 1-6 alkyl substituted with R 7 .
• Still a further interesting group of compounds are those compounds of formula (I), (I′), (I′′) or (I′′′) wherein R 3 is —C( ⁇ N—O—R 8 )—C 1-4 alkyl.
• Another interesting group of compounds are those compounds of formula (I), (I′), (I′′) or (I′′′) wherein m is 2 or 3 and X 1 is —NR 5 —, —O—, —C( ⁇ O)—, —CH 2 —, —CHOH—, —S—, —S( ⁇ O) p —, in particular wherein X 1 is —NR 5 —, or —O—.
• Preferred compounds of formula (I), (I′), (I′′) or (I′′′) are compounds 1, 25, 84, 133, 152, 179, 233, 239, 247, 248, 255 (see Tables 1, 2 and 3), their N-oxides, pharmaceutically acceptable addition salts, quaternary amines and stereochemically isomeric forms thereof.
• the compounds of formula (I) are disclosed in WO 2003/016306. Their preparation is also described therein. Some of the intermediates and starting materials are known compounds and may be commercially available or may be prepared according to art-known procedures or some of the compounds of formula (I) or the described intermediates may be prepared according to the procedures described in WO 99/50250 and WO 00/27825.
• the present invention also relates to a novel compound, i.e. 4-[[4-[4-(2-cyanoethenyl)-2,6-dimethylphenoxy]-2-pyrimidinyl]amino]benzonitrile (E) (compound 255); a N-oxide, a pharmaceutically acceptable addition salt, a quaternary amine or a stereochemically isomeric from thereof.
• a novel compound i.e. 4-[[4-[4-(2-cyanoethenyl)-2,6-dimethylphenoxy]-2-pyrimidinyl]amino]benzonitrile (E) (compound 255); a N-oxide, a pharmaceutically acceptable addition salt, a quaternary amine or a stereochemically isomeric from thereof.
• Said novel compound can be prepared as follows:
• the compounds of formula (I), (I′), (I′′), (I′′′) show antiretroviral properties (reverse transcriptase inhibiting properties) in HIV infected warm-blooded animals, in particular against Human Immunodeficiency Virus (HIV), which is the aetiological agent of Acquired Immune Deficiency Syndrome (AIDS) in humans.
• HIV Human Immunodeficiency Virus
• the HIV virus preferentially infects human T-4 cells and destroys them or changes their normal function, particularly the coordination of the immune system.
• an infected patient has an ever decreasing number of T-4 cells, which moreover behave abnormally.
• the immunological defense system is unable to combat infections and neoplasms and the HIV infected subject usually dies by opportunistic infections such as pneumonia, or by cancers.
• Other conditions associated with HIV infection include thrombocytopaenia, Kaposi's sarcoma and infection of the central nervous system characterized by progressive demyelination, resulting in dementia and symptoms such as, progressive dysarthria, ataxia and disorientation. HIV infection further has also been associated with peripheral neuropathy, progressive generalized lymphadenopathy (PGL) and AIDS-related complex (ARC).
• PDL progressive generalized lymphadenopathy
• ARC AIDS-related complex
• the compounds of formula (I), their N-oxides, pharmaceutically acceptable addition salts, quaternary amines and stereochemically isomeric forms thereof are useful in the treatment of individuals infected by HIV.
• the HIV replication inhibiting effect of the compounds of formula (I) is described in WO 2003/016306.
• Compound 255 has a pIC 50 value of 9.00 when tested in the test described under the heading “C. Pharmacological example” of WO 2003/016306.
• the compounds of formula (I) can not only be used to treat HIV infected warm-blooded animals, but that they can also be used to prevent that warm-blooded animals, including humans, get HIV infected via sexual intercourse or related intimate contact between partners.
• the present invention relates to the use of a compound of formula (I) for the manufacture of a medicament for the prevention of HIV infection via sexual intercourse or related intimate contact between partners, in particular to prevent HIV-1 infection and further in particular to prevent HIV or HIV-1 infection with (multi) drug resistant HIV strains, i.e. HIV strains, especially HIV-1 strains, that have acquired resistance to one or more art-known non-nucleoside reverse transcriptase inhibitors.
• Art-known non-nucleoside reverse transcriptase inhibitors are those non-nucleoside reverse transcriptase inhibitors other than the present compounds and in particular commercial non-nucleoside reverse transcriptase inhibitors.
• the invention also relates to a method of preventing HIV infection via sexual intercourse or related intimate contact between partners comprising administering to a subject in need thereof an effective amount of a compound of formula (I).
• sexual intercourse or related intimate contact between partners comprises vaginal sex, anal sex, oral sex and contact of body sites with HIV infected fluids of the sexual partner, in particular semen.
• sexual intercourse or related intimate contact between partners constitutes vaginal, anal or oral sex, more particularly vaginal sex.
• the contact sites believed to be most responsible for the transmission of HIV via sexual intercourse or related intimate contact between partners are the genitals, rectum, mouth, hands, lower abdomen, upper thighs.
• partners as mentioned hereinbefore or hereinafter defines two or more warm-blooded animals, in particular humans, who are sexually active with each other, ie. who have sexual intercourse with each other or who have intimate contact with each other related to sexual activities.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically acceptable carrier and as active ingredient a therapeutically effective amount of a compound of formula (I) characterized in that the composition is in a form adapted to be applied to the site where the sexual intercourse or related intimate contact takes place, such as the genitals, rectum, mouth, hands, lower abdomen, upper thighs, especially the vagina and mouth.
• compositions there may be cited all compositions usually employed for being applied to the vagina, rectum, mouth and skin such as for example gels, jellies, creams, ointments, films, sponges, foams, intravaginal rings, cervical caps, suppositories for rectal or vaginal application, vaginal or rectal or buccal tablets, mouthwashes.
• compositions of this invention an effective amount of the particular compound, optionally in addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of administration.
• a pharmaceutically acceptable carrier which carrier may take a wide variety of forms depending on the form of administration.
• any of the usual pharmaceutical media may be employed such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as mouthwashes in the form of a suspension, emulsions and solutions; or solid carriers such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents and the like in the case of tablets.
• compositions suitable for cutaneous administration the carrier optionally comprises a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin. Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions.
• suitable wetting agent optionally combined with suitable additives of any nature in minor proportions, which additives do not introduce a significant deleterious effect on the skin.
• Said additives may facilitate the administration to the skin and/or may be helpful for preparing the desired compositions.
• These compositions may be administered in various ways, e.g., as a cream or gel.
• a bioadhesive in particular a bioadhesive polymer.
• a bioadhesive may be defined as a material that adheres to a live biological surface such as for example a mucus membrane or skin tissue.
• the term bioadhesive is well-known to the person skilled in the art.
• the present invention also relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and as active ingredient a therapeutically effective amount of a compound of formula (I) characterized in that the pharmaceutical composition is bioadhesive to the site of application.
• the site of application is the vagina, rectum, mouth or skin, most preferred is the vagina.
• bioadhesives which may be used in the pharmaceutical compositions of the present invention comprise polyacrylic acid derivatives, such as for example carbopol or polycarbophil, e.g. carbopol 934P, carbopol 940, polycarbophil AA1; cellulose ether derivatives such as for example hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose, chitosan; natural polymers such as for example alginates, tragacanth, inulin; pregelatinized starch.
• polyacrylic acid derivatives such as for example carbopol or polycarbophil, e.g. carbopol 934P, carbopol 940, polycarbophil AA1
• cellulose ether derivatives such as for example hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, sodium
• An embodiment of the present invention relates to a gel containing carbopol, hydroxypropyl cellulose, hydroxyethyl cellulose or pregelatinized starch.
• cyclodextrins may be included in the compositions.
• suitable ingredients e.g. cyclodextrins
• cyclodextrins are ⁇ -, ⁇ -, ⁇ -cyclodextrins or ethers and mixed ethers thereof wherein one or more of the hydroxy groups of the anhydroglucose units of the cyclodextrin are substituted with C 1-6 alkyl, particularly methyl, ethyl or isopropyl, e.g.
• complexants and/or solubilizers are ⁇ -CD, randomly methylated ⁇ -CD, 2,6-dimethyl- ⁇ -CD, 2-hydroxyethyl- ⁇ -CD, 2-hydroxyethyl- ⁇ -CD, 2-hydroxypropyl- ⁇ -CD and (2-carboxymethoxy)propyl- ⁇ -CD, and in particular 2-hydroxypropyl- ⁇ -CD (2-HP- ⁇ -CD).
• mixed ether denotes cyclodextrin derivatives wherein at least two cyclodextrin hydroxy groups are etherified with different groups such as, for example, hydroxy-propyl and hydroxyethyl.
• the average molar substitution (M.S.) is used as a measure of the average number of moles of alkoxy units per mole of anhydroglucose.
• the average substitution degree (D.S.) refers to the average number of substituted hydroxyls per anhydroglucose unit.
• the M.S. and D.S. value can be determined by various analytical techniques such as nuclear magnetic resonance (NMR), mass spectrometry (MS) and infrared spectroscopy (IR). Depending on the technique used, slightly different values may be obtained for one given cyclodextrin derivative.
• NMR nuclear magnetic resonance
• MS mass spectrometry
• IR infrared spectroscopy
• slightly different values may be obtained for one given cyclodextrin derivative.
• the M.S. ranges from 0.125 to 10 and the D.S. ranges from 0.125 to 3.
• the compounds of formula (I) may be formulated in the pharmaceutical compositions of the present invention in the form of particles consisting of a solid dispersion comprising a compound of formula (I) and one or more appropriate pharmaceutically acceptable water-soluble polymers.
• a solid dispersion used hereinafter defines a system in a solid state (as opposed to a liquid or gaseous state) comprising at least two components, in casu the compound of formula (I) and the water-soluble polymer, wherein one component is dispersed more or less evenly throughout the other component or components (in case additional pharmaceutically acceptable formulating agents, generally known in the art, are included, such as plasticizers, preservatives and the like).
• additional pharmaceutically acceptable formulating agents generally known in the art, are included, such as plasticizers, preservatives and the like.
• a solid dispersion also comprises dispersions which are less homogenous throughout than solid solutions. Such dispersions are not chemically and physically uniform throughout or comprise more than one phase.
• the term “a solid dispersion” also relates to a system having domains or small regions wherein amorphous, microcrystalline or crystalline compound of formula (I), or amorphous, microcrystalline or crystalline water-soluble polymer, or both, are dispersed more or less evenly in another phase comprising water-soluble polymer, or compound of formula (I), or a solid solution comprising compound of formula (I) and water-soluble polymer. Said domains are regions within the solid dispersion distinctively marked by some physical feature, small in size, and evenly and randomly distributed throughout the solid dispersion.
• the solution-evaporation process comprises the following steps:
• the two components are also dissolved in an appropriate solvent and the resulting solution is then sprayed through the nozzle of a spray dryer followed by evaporating the solvent from the resulting droplets at elevated temperatures.
• the preferred technique for preparing solid dispersions is the melt-extrusion process comprising the following steps:
• melt and “melting” should be interpreted broadly. These terms not only mean the alteration from a solid state to a liquid state, but can also refer to a transition to a glassy state or a rubbery state, and in which it is possible for one component of the mixture to get embedded more or less homogeneously into the other. In particular cases, one component will melt and the other component(s) will dissolve in the melt thus forming a solution, which upon cooling may form a solid solution having advantageous dissolution properties.
• the obtained products can be optionally milled and sieved.
• the solid dispersion product may be milled or ground to particles having a particle size of less than 600 ⁇ m, preferably less than 400 ⁇ m and most preferably less than 125 ⁇ m.
• the particles prepared as described hereinabove can then be formulated by conventional techniques into the pharmaceutical dosage forms of the present invention.
• the water-soluble polymers in the particles are polymers that have an apparent viscosity, when dissolved at 20° C. in an aqueous solution at 2% (w/v), of 1 to 5000 mPa ⁇ s more preferably of 1 to 700 mPa ⁇ s, and most preferred of 1 to 100 mPa ⁇ s.
• suitable water-soluble polymers include alkylcelluloses, hydroxyalkyl-celluloses, hydroxyalkyl alkylcelluloses, carboxyalkylcelluloses, alkali metal salts of carboxyalkylcelluloses, carboxyalkylalkylcelluloses, carboxyalkylcellulose esters, starches, pectines, chitin derivates, di-, oligo- and polysaccharides such as trehalose, alginic acid or alkali metal and ammonium salts thereof, carrageenans, galactomannans, tragacanth, agar-agar, gummi arabicum, guar gummi and xanthan gummi, polyacrylic acids and the salts thereof, polymethacrylic acids and the salts thereof, methacrylate copolymers, polyvinylalcohol, polyvinylpyrrolidone, copolymers of polyvinylpyrrolidone with vinyl acetate, combinations of polyvinylalcohol
• cyclodextrins can be used as water soluble polymer in the preparation of the above-mentioned particles as is disclosed in WO 97/18839.
• Said cyclodextrins include the pharmaceutically acceptable unsubstituted and substituted cyclodextrins known in the art, more particularly ⁇ , ⁇ or ⁇ cyclodextrins or the pharmaceutically acceptable derivatives thereof.
• Substituted cyclodextrins which can be used to prepare the above described particles include polyethers described in U.S. Pat. No. 3,459,731. Further substituted cyclodextrins are ethers wherein the hydrogen of one or more cyclodextrin hydroxy groups is replaced by C 1-6 alkyl, hydroxyC 1-6 alkyl, carboxy-C 1-6 alkyl or C 1-6 alkyloxycarbonylC 1-6 alkyl or mixed ethers thereof.
• substituted cyclodextrins are ethers wherein the hydrogen of one or more cyclodextrin hydroxy groups is replaced by C 1-3 alkyl, hydroxyC 2-4 alkyl or carboxyC 1-2 alkyl or more in particular by methyl, ethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, carboxy-methyl or carboxyethyl.
• ⁇ -cyclodextrin ethers e.g. dimethyl- ⁇ -cyclodextrin as described in Drugs of the Future, Vol. 9, No. 8, p. 577-578 by M. Nogradi (1984) and polyethers, e.g. hydroxypropyl ⁇ -cyclodextrin and hydroxyethyl ⁇ -cyclodextrin, being examples.
• alkyl ether may be a methyl ether with a degree of substitution of about 0.125 to 3, e.g. about 0.3 to 2.
• a hydroxypropyl cyclodextrin may for example be formed from the reaction between ⁇ -cyclodextrin an propylene oxide and may have a MS value of about 0.125 to 10, e.g. about 0.3 to 3.
• Another type of substituted cyclodextrins is sulfobutylcyclodextrines.
• the ratio of the compound of formula (I) over the water soluble polymer may vary widely. For example ratios of 1/100 to 100/1 may be applied. Interesting ratios of the compound of formula (I) over cyclodextrin range from about 1/10 to 10/1. More interesting ratios range from about 1/5 to 5/1.
• the compounds of formula (I) can also be combined with another or other antiretrovirals.
• the present invention also provides for a pharmaceutical composition according to the invention comprising a compound of formula (I) and further comprising one or more additional antiretroviral compounds.
• the present invention also relates to a product containing (a) a compound of formula (I), and (b) one or more additional antiretroviral compounds, as a combined preparation for simultaneous, separate or sequential use in HIV infection prevention.
• the different drugs may be combined in a single preparation together with pharmaceutically acceptable carriers.
• Said other antiretroviral compounds may be known antiretroviral compounds such as suramine, pentamidine, thymopentin, castanospermine, dextran (dextran sulfate), foscarnet-sodium (trisodium phosphono formate); nucleoside reverse transcriptase inhibitors, e.g.
• zidovudine (3′-azido-3′-deoxythymidine, AZT), didanosine (2′,3′-dideoxyinosine; ddI), zalcitabine (dideoxycytidine, ddC) or lamivudine (2′-3′-dideoxy-3′-thiacytidine, 3TC), stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T), abacavir and the like; non-nucleoside reverse transcriptase inhibitors such as nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido-[3,2-b:2′,3′-e][1,4]diazepin-6-one), efavirenz, delavirdine, TMC-120, TMC-125 and the like; phosphonate reverse transcriptase inhibitors, e.g.
• TAT-inhibitors e.g. RO-5-3335, or REV inhibitors, and the like
• protease inhibitors e.g. indinavir, ritonavir, saquinavir, lopinavir (ABT-378), nelfinavir, amprenavir, TMC-126, BMS-232632, VX-175 and the like
• fusion inhibitors e.g. T-20, T-1249 and the like
• CXCR 4 receptor antagonists e.g.
• AMD-3100 and the like inhibitors of the viral integrase; nucleotide-like reverse transcriptase inhibitors, e.g. tenofovir and the like; ribonucleotide reductase inhibitors, e.g. hydroxyurea and the like.
• Combination therapies as described above may exert a synergistic effect in inhibiting HIV replication because each component of the combination acts on a different site of HIV replication.
• the use of such combinations may reduce the dosage of a given conventional anti-retroviral agent which would be required for a desired prophylactic effect as compared to when that agent is administered as a monotherapy.
• These combinations reduce potential of resistance to single agent therapies, while minimizing any associated toxicity.
• These combinations may also increase the efficacy of the conventional agent without increasing the associated toxicity.
• the compounds of the present invention may also be administered in combination with art-known microbicides. They can block the infection by creating a barrier between the pathogen, in this case the Human Immunodeficiency Virus, and the site at which transmission will take place, e.g. the vagina; they can kill or immobilize the pathogen; they can prevent a virus from replicating once it has infected the cells that line the site of transmission, e.g. the cells that line the vaginal wall. Examples of microbicides are
• the present invention also relates to a pharmaceutical composition according to the invention comprising a compound of formula (I) and further comprising one or more components wherein the components are selected from antibodies, detergents or surfactants, coatings for the site of administration of the pharmaceutical composition, peptides, pH regulators.
• the present invention also relates to a product containing (a) a compound of formula (I), and (b) one or more components selected from antibodies, detergents or surfactants, coatings for the site of administration of the pharmaceutical composition, peptides, pH regulators, as a combined preparation for simultaneous, separate or sequential use in HIV infection prevention.
• the different drugs may be combined in a single preparation together with pharmaceutically acceptable carriers.
• the present invention relates also to a pharmaceutical composition as outlined hereinabove further comprising a spermicidal compound.
• Said compositions are able to prevent at the same time conception and EIV infection.
• Suitable spermicides are for example nonoxynol-9, octoxynol-9, menfegol, benzalkonium chloride, N-docasanol.
• the present invention focuses on the use of the present compounds for preventing HIV infection via sexual intercourse or related intimate contact between partners, the present compounds may also be used as inhibitory agents for the prevention of infections caused by other viruses which depend on similar reverse transcriptases for obligatory events in their life cycle.
• Immature monocyte derived dendritic cells represent a good model for interstitial dendritic cells, which are early targets during sexual HIV transmission and important initiators of the immune response.
• immMO-DC were used in “in vitro” models to test the prevention of HIV infection via sexual intercourse or related intimate contact between partners.
• the monotropic HIV strain Ba-L is pre-incubated with the compound of formula (I) (test compound).
• immMO-DC are added and incubated for 2 hours at 37° C. After infection, cells are washed 6 times and cultured with autologous CD4(+) T cells (ratio immMO-DC/CD4(+) T:1/10).
• Test compound is re-added and remains present during 14 days of primary culture, after which cells are extensively washed and PHA/IL-2 stimulated blasts are added (secondary culture, no test compound present). Supernatants are analysed in ELISA during primary and secondary culture.
• test compound concentration able to suppress 50% of the viral replication at the end of the primary cultures is measured. Additionally, cells are harvested after 3 weeks of secondary culture and analysed for the presence of HIV proviral DNA (PCR), to check for sterilisation and exclude viral rescue.
• PCR HIV proviral DNA
• Monocyte-derived dendritic cells were co-cultured with autologous T4 cells and infected with HIV strain Ba-L at a multiplicity of infection (MOI) of 10 ⁇ 3 .
• MOI multiplicity of infection
• a serial dilution of test compound was added at the time of infection.
• 96-well plates were washed 3 times (test compound and free virus washed away) and medium (without test compound) was added.
• Half of the medium was refreshed twice a week.
• Culture supernatants were harvested after 7 and 14 days of culture. After 14 days, cultures were washed 3 times and PHA/IL-2 stimulated PBMC were added for a secondary culture to check for viral rescue. During secondary culture, half of the medium was refreshed twice weekly (IL-2 medium, without test compound).
• Monocyte-derived dendritic cells were infected for 2 hours with the monotropic HIV strain Ba-L at a multiplicity of infection (MOI) of 10 ⁇ 3 . After infection, cells were washed 6 times and resuspended in 10% BCS at 400.000 cells/ml. Autologous CD4(+) T cells were purified out of the lymphocyte fraction of the same elutration as the MO-DC and used at a concentration of 2 ⁇ 10 6 cells/ml ((ratio MO-DC/CD4(+) T:1/5).
• test compound A serial dilution of a compound of formula (I) (test compound) was added to the MO-DC/CD4(+) T cell co-cultures. Each experiment was done in 96-well plates, in which each cup contained 50 ⁇ l of MO-DC, 50 ⁇ l of CD4(+) T cells and 100 ⁇ l of test compound. Half of the culture medium, with test compound, was refreshed twice weekly, during 14 days. Supernatants were analysed by ELISA after 14 days of culture for the presence of HIV antigens. To determine antiviral activity, the test compound concentration able to suppress 50% of the viral replication at the end of the primary cultures (EC 50 ) was measured.
• the compounds of formula (I) were tested for their immune-suppressive activity (defined as ISC 50 value) in a classical MLC in which monocyte-derived dendritic cells (MO-DC) were used as stimulators and allogenic CD4(+) T cells as responders.
• ISC 50 value ISC 50 value
• MO-DC monocyte-derived dendritic cells
• ISC 50 immune suppressive concentration
• test compound was only present during the first 24 hours of the 5-day culture period. After 24 hours, the cultures were washed (three times) and culture medium without compound was added. The experimental set-up was from then on similar to the standard MLC assay described above.

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