Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
  • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
  • C07D205/06—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
  • C07D205/08—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member with one oxygen atom directly attached in position 2, e.g. beta-lactams
• • 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
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes viruses
• • 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

Definitions

• This invention relates to azetidinone derivatives having activity against herpes infections. More specifically, the invention relates to azetidin-2-one derivatives exhibiting antiherpes activity, to pharmaceutical compositions comprising the derivatives, and methods of using the derivatives to inhibit the replication of herpes virus and to treat herpes infections.
• Herpes viruses inflict a wide range of diseases against humans and animals. For instance, herpes simplex viruses, types 1 and 2 (HSV-1 and HSV-2), are responsible for cold sores and genital lesions, respectively; varicella zoster virus (VZV) causes chicken pox and shingles ; and the human cytomegalovirus (HCMV) is a leading cause of opportunistic infections in immunosuppressed individuals.
• HSV-1 and HSV-2 herpes simplex viruses
• VZV varicella zoster virus
• HCMV human cytomegalovirus
• nucleoside analogs Over the past two decades, a class of compounds known as the purine and pyrimidine nucleoside analogs has received the most attention by investigators in the search for new therapeutic agents for treatment of herpes virus infections. As a result, several nucleoside analogs have been developed as antiviral agents. The most successful to date is acyclovir which is the agent of choice for treating genital HSV infections. Another nucleoside analog, ganciclovir, has been used with some success in treating HCMV infections .
• the present application discloses a group of azetidin-2-one derivatives particularly active against cytomegalovirus. This activity coupled with a wide margin of safety, renders these derivatives desirable agents for combating herpes infections.
• Azetidin-2-one derivatives have been reported in the literature as having variety of biological activities; mainly antibacterial, antiinflammatory, antidegenerative, etc. However, azetidin-2-one derivatives have not been reported to be antiviral agents against herpes viruses.
• the present azetidin-2-one derivatives are distinguished from the prior art compounds in that they possess different chemical structures and biological activities.
• the azetidin-2-one derivatives are represented by formula 1 :
• R- is hydrogen, methyl, ethyl, methoxy or methylthio
• R 2 and R 3 each independently is hydrogen or C ⁇ _ 3 alkyl ;
• R 4 is hydrogen, lower alkyl, methoxy, ethoxy, or benzyloxy;
• R 5 is lower alkyl, lower cycloalkyl, (CH 2 ) m C (0) 0R 6 wherein m is the integer 1 or 2 and R 6 is lower alkyl or phenyl (lower alkyl); phenyl, phenyl monosubstituted, disubstituted or trisubstituted with a substituent selected independently from the group consisting of lower alkyl, lower alkoxy, lower alkylthio, halo, hydroxy and amino; phenyl (lower alkyl), phenyl (lower alkyl) monosubstituted or disubstituted on the phenyl portion thereof with a substituent selected independently from the group consisting of lower alkyl, lower alkoxy, lower alkylthio, halo, hydroxy, nitro, amino, lower alkylammo, di (lower alkyl)amino, lower acylamino, di ( lower alkyl ) aminocarbonyl ,
• R 4 and R 5 together with the nitrogen atom to which they are attached form a piperidino, morpholino, thiomorpholino, piperazino, N-methylpiperazino, 1-
• Y is Ci-io non-cyclic or cyclic alkyl; phenyl (lower alkyl), said phenyl ring optionally mono- or di-substituted with a lower alkyl or lower alkoxy, said phenyl ring being optionally fused with an aromatic ring to form a bicyclic ring, said aromatic ring optionally containing a heteroatom selected from the group consisting of N, 0 and S; Het or Het (lower alkyl) containing one or more heteroatom selected from the group consisting of N,
• said Het optionally mono- or di-substituted with a lower alkyl or lower alkoxy group; said heterocyclic ring being optionally fused with an aromatic ring to form a bicyclic ring, said aromatic ring optionally containing one or more heteroatom selected from the group consisting of N, 0 and S; C(0)R 9 wherein R 9 is lower alkyl or phenyl (lower alkyl ) ; or when X is NR 8 , wherein R 8 is lower alkyl and Y is lower alkyl or lower alkoxy, X and Y are joined together to form a morpholino or piperidino ring;
• Preferred compounds of the invention include compounds of formula (1) wherein R x is hydrogen or C ⁇ -2 alkyl;
• R 2 and R 3 each independently is hydrogen, methyl or ethyl ;
• R 4 is hydrogen or lower alkyl
• R 5 is phenyl optionally substituted with a substituent selected independently from the group consisting of lower alkyl, lower alkoxy; phenyl (lower alkyl) optionally mono- or disubstituted on the phenyl portion thereof with a substituent selected independently from the group consisting of lower alkyl, lower alkoxy, nitro, halo, cyano, trifluoromethyl, and C(0)OR 7 wherein R 7 is lower alkyl or phenyl (lower alkyl); He (lower alkyl) wherein Het represents a five or six-membered, monovalent heterocyclic ring containing a heteroatom selected from the group consisting of N, 0, or S, said ring being optionally substituted with lower alkyl or lower alkoxy;
• R 4 and R 5 together with the nitrogen atom to which they are attached form a pyrrolidino optionally substituted with benzyloxycarbonyl or phenyl said phenyl ring optionally mono- or di-substituted with halo, nitro, cyano or trifluoromethyl;
• X is selected from the group consisting of O, S, NR 8 , wherein R 8 is H or lower alkyl;
• Y is Ci-io non-cyclic or cyclic alkyl; phenylcarbonyl; phenyl or benzyl optionally mono- or di-substituted with lower alkyl or lower alkoxy, said phenyl ring being optionally fused with an aromatic ring to form a bicyclic ring, said aromatic ring optionally containing a heteroatom selected from the group consisting of N, O and S; and
• More preferred compounds of the invention include compounds of formula 1 wherein R l f R 2 and R 3 each independently is hydrogen, methyl or ethyl; R 4 is hydrogen or C ⁇ - 3 alkyl; R 5 is phenyl optionally substituted with a substituent selected independently from the group consisting of lower alkyl or lower alkoxy;
• (C ⁇ - 2 alkyl) phenyl optionally mono- or di-substituted on the phenyl portion thereof with a substituent selected independently from the group consisting of lower alkyl, lower alkoxy, nitro, halo, cyano, trifluoromethyl, and C(0)OR 7 wherein R 7 is lower alkyl or (lower alkyl ) phenyl ;
• X is selected from the group consisting of O, S, NR 8 , wherein R 8 is H or lower alkyl;
• Y is lower non-cyclic or cyclic alkyl; phenyl optionally mono- or di-substituted with lower alkyl or lower alkoxy; or
• Most preferred compounds of the invention include compounds of formula (1) wherein R x , R 2 and R 3 each independently is hydrogen;
• R 4 is hydrogen or methyl
• R 5 is benzyl optionally mono-substituted on the phenyl portion thereof with nitro or trifluromethyl, or 1 (R) -phenylethyl ;
• X is S; and Y is pyrimidine optionally substituted with lower alkyl; pyridine; N-Me-tetrazole; or benzoxazole.
• compositions for treating cytomegalovirus infections in a human comprising a compound of formula 1, or a therapeutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
• the scope of the invention also includes a method for treating cytomegalovirus infections in a human comprising administering thereto an effective amount of the compound of formula 1, or a therapeutically acceptable salt thereof.
• Also included within the scope is a method for protecting human cells against cytomegalovirus pathogenesis comprising treating said cells with an anti-cytomegalovirus effective amount of a compound of formula 1, or a therapeutically acceptable salt thereof .
• Compounds of formula 1 according to the present invention may also be used in co-therapies, with other conventional anti-herpes compounds, such as but not limited to ganciclovir, foscarnet, acyclovir, valaciclovir, famciclovir, cidofovir, penciclovir and lobucavir .
• other conventional anti-herpes compounds such as but not limited to ganciclovir, foscarnet, acyclovir, valaciclovir, famciclovir, cidofovir, penciclovir and lobucavir .
• Compounds of formula 1 according to the present invention may also be used in co-therapies with anti- retroviral compounds such as reverse transcriptase inhibitors (i.e. AZT, 3TC) or protease inhibitors. Processes for preparing the compounds of formula 1 are described hereinafter.
• anti- retroviral compounds such as reverse transcriptase inhibitors (i.e. AZT, 3TC) or protease inhibitors.
• reduct with reference to an amino acid or amino acid derivative means a radical derived from the corresponding ⁇ -amino acid by eliminating the hydroxyl of the carboxy group and one hydrogen of the ⁇ -amino group.
• the terms Gin, Ala, Gly, lie, Arg, Asp, Phe, Ser, Leu, Cys , Asn, Sar and Tyr represent the "residues" of L-glutamine, L-alanine, glycine, L-isoleucine, L-arginine, L-aspartic acid, L-phenylalanine, L-serine, L-leucine, L-cysteine, L- asparagine, sarcosine and L-tyrosine, respectively.
• side chain with reference to an amino acid or amino acid derivative means a residue attached to the ⁇ -carbon atom of the ⁇ -amino acid.
• R-group side chain for glycine is hydrogen, for alanine it is methyl, for valine it is isopropyl.
• specific R-groups or side chains of the ⁇ - amino acids reference is made to A.L. Lehninger's text on Biochemistry (see chapter 4) .
• halo as used herein means a halo radical selected from bromo, chloro, fluoro or iodo.
• lower alkyl or (lower alkyl) as used herein, either alone or in combination with another radical, means straight or branched chain alkyl radicals containing up to six carbon atoms and includes methyl, ethyl, propyl, butyl, hexyl, 1- methylethyl, 1-methylpropyl , 2-methylpropyl and 1,1- dimethylethyl .
• lower alkoxy as used herein means straight chain alkoxy radicals containing one to four carbon atoms and branched chain alkoxy radicals containing three to four carbon atoms and includes methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1- dimethylethoxy.
• the latter radical is known commonly as fcer ⁇ -butoxy.
• lower alkanoyl as used herein, either alone or in combination with another radical, means a straight chain 1-oxoalkyl containing from one to six carbon atoms or a branched chain 1-oxoalkyl containing from four to six carbon atoms; for example, acetyl, propionyl (1-oxopropyl) , 2- methylpropionyl and 2-ethylbutyryl .
• lower cycloalkyl as used herein, either alone or in combination with another radical, means saturated cyclic hydrocarbon radicals containing from three to seven carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
• amino as used herein means an amino radical of formula -NH2.
• lower alkylamino as used herein means alkylamino radicals containing one to six carbon atoms and includes methylamino, propylamino, (1-methylethyl) amino and (2- methylbutyl) amino.
• di (lower alkyl) amino means an amino radical having two lower alkyl substituents each of which contains one to six carbon atoms and includes dimethylamino, diethylamino, ethylmethylamino and the like.
• Het as used herein means a monovalent radical derived by removal of a hydrogen from a five- or six-membered saturated or unsaturated heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur.
• the heterocycle may bear one or two substituents; for example, N-oxido , lower alkyl, (C ⁇ _ 3 ) alkylphenyl, lower alkoxy, halo, amino or lower alkylamino.
• the five- or six-membered heterocycle can be fused to a phenyl.
• heterocycles and optionally substituted heterocycles include pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, lH-imidazole, 1-methyl-lH-imidazole, pyrazole, furan, thiophene, oxazole, isoxazole, thiazole, 2-methylthiazole, 2-aminothiazole, 2- (methylamino) -thiazole, piperidine, 1- methylpiperidine, 1-methylpiperazine, 1,4-dioxane, morpholine, pyridine, pyridine N-oxide , pyrimidine, 2 , 4-dihydroxypyrimidine, 2 , 4-dimethylpyrimidine, 2,6- dimethylpyridine, 1-methyl-lH-tetrazole, 2-methyl-2H- tetrazole, benzothiazole, benzoxazole and thiazolo [4, 5-b] -pyridine.
• pharmaceutically acceptable carrier means a non-toxic, generally inert vehicle for the active ingredient which does not adversely affect the ingredient.
• an effective amount means a predetermined antiviral amount of the antiviral agent, i.e. an amount of the agent sufficient to be effective against the virus in vivo .
• the azetidin-2-one derivatives of formula 1 can be obtained in the form of therapeutically acceptable acid addition salts.
• examples of such salts are those with organic acids, e.g. acetic, lactic, succinic, benzoic, salicylic, methanesulfonic or p- toluenesulfonic acid, as well as polymeric acids such as tannic acid or carboxymethyl cellulose, and salts with inorganic acids such as hydrohalic acids, e.g. hydrochloric acid, or sulfuric acid, or phosphoric acid.
• Step a Intermediate V is prepared according to known procedures starting from suitably protected D- aspartic acid (ref. P.E. Finke et al . , J. Med. Chem. 1995, 38, 2449) .
• Step b The acid function of intermediate V is reduced to give alcohol IV.
• Step c When X is 0 or S, and Y is aryl or Het, the primary alcohol IV is converted into intermediate II, using Mitsunobu reaction conditions (Ref . D.L. Hughes, Org . Reaction 1992, 42, 335; J.R. Dormoy, Synthesis 1982, 753).
• Step b' The alcohol IV is converted into a leaving group "LG" (e.g. mesylate, iodide, bromide) to give intermediate III.
• LG e.g. mesylate, iodide, bromide
• Step c ' The intermediate III is then reacted with a nucleophile (e.g. alkylthiolate or amine) to yield an alternative key intermediate II'.
• a nucleophile e.g. alkylthiolate or amine
• Step d Key intermediates II or II' are converted to the desired inhibitor via deprotection using fluoride ions (e.g. cesium fluoride), followed by condensation with the appropriate reagent.
• fluoride ions e.g. cesium fluoride
• the appropriate reagent is an isocyanate of formula R 5 '-NCO, wherein R 5 ' is as defined above but not pyridine, and the condensation is done in the presence of a tertiary amine (e.g. diisopropylethylamine) or preferably lithium bis ( trimethylsilyl) amide.
• a tertiary amine e.g. diisopropylethylamine
• lithium bis ( trimethylsilyl) amide e.g. diisopropylethylamine
• activated carbamate of formula VI or VI ' can be used in the condensation of intermediate II or II' when the appropriate isocyanate R 5 , -NCO is not available commercially.
• the primary alcohol 3_ was oxidized to the corresponding aldehyde using oxalyl chloride- activated dimethyl sulfoxide (K. Omura and D. Swern, Tetrahedron 1978, 34, 1651) or triacetoxy periodinane (D.B. Dess and J.C. Martin, J. Org. Chem. 1983, 48, 4155) .
• This aldehyde was then reacted with an appropriate Grignard reagent such as methylmagnesium bromide to give the addition product 1_ as a mixture of diastereoisomers .
• preactivation could be achieved via formation of the N-phenoxycarbamate derivative _15 by reacting the amine 14_ with phenyl chloroformate in the presence of a tertiary amine such as triethylamine in dichloromethane .
• non commercially available pyrrolidine derivatives such as 1_9 could be prepared as follows: a, b) Amine 1_6 was protected by reaction with di- tert-butylcarbonate in the presence of an aqueous base such as sodium hydroxide. The protected amine was reacted a benzyl halide such as benzyl bromide or chloride in presence of a base such as sodium hydride in tetrahydrofuran to give intermediate 17. c) Cyclisation of intermediate 1/7 was accomplished using strong base such as n-butyllithium in the presence of tetramethylethylenediamine in tetrahydrofuran to give pyrrolidine derivative 18.
• Sulfoxides and sulfones are readily accessible from thioether intermediates such as 2_0, by using peroxide oxidation or preferably by reaction with oxone in a mixture of methanol/water as shown.
• the antiherpes activity of the aforementioned azetidinone derivatives of formula 1 can be demonstrated by biochemical, microbiological and biological procedures .
• a biochemical procedure for demonstrating anti- cytomegalovirus activity for the azetidinone derivatives of formula 1 is described in the examples hereinafter.
• This particular assay determines the ability of a test compound to inhibit the activity (IC 50 ) of HCMV protease. More specifically, in the assay described herein, the inhibitory activity of the test compound is evaluated on the basis of its ability to interfere with the HCMV N Q protease cleavage of a fluorogenic peptide substrate which in turn is based on the maturation cleavage site of the enzyme .
• the HCMV protease inhibitor When the HCMV protease inhibitor is employed as an antiviral agent, it is administered orally or systemically to humans in a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard biological practice.
• a vehicle comprising one or more pharmaceutically acceptable carriers, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard biological practice.
• the compound or a therapeutically acceptable salt thereof can be formulated in unit dosage forms such as capsules or tablets each containing a predetermined amount of the active ingredient, ranging from about 50 to 500 mg, in a pharmaceutically acceptable carrier.
• the HCMV protease inhibitor is administered by either intravenous, subcutaneous or intramuscular injection, in compositions with pharmaceutically acceptable vehicles or carriers.
• a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives as well as sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic.
• Suitable vehicles or carriers for the above noted formulations are described in standard pharmaceutical texts, e.g. in "Remington's, The Science and Practice of Pharmacy", 19 th ed. , Mack Publishing Company, Easton, Penn., 1995, or in “Pharmaceutical Dosage Forms and Drug Delivery Systems", 6 th ed. , H.C. Ansel et al . , Eds., Williams & Wilkins, Baltimore, Maryland, 1995.
• the dosage of the HCMV protease inhibitor will vary with the form of administration and the particular active agent chosen. Furthermore, it will vary with the particular host under treatment. Generally, treatment is initiated with small increments until the optimum effect under the circumstance is reached. In general, the inhibitor compound is most desirably administered at a concentration level that will generally afford antivirally effective results without causing any harmful or deleterious side effects .
• the HCMV protease inhibitor is administered in the range of 20 to 200 mg per kilogram of body weight per day, with a preferred range of 25 to 100 mg per kilogram.
• the HCMV protease inhibitor is administered either topically or intraocularly (injection or implant) in a suitable preparation.
• a suitable preparation for example, an implant containing the compound in a suitable formulation can be surgically implanted in the posterior segment of the eye through a small incision.
• the HCMV protease inhibitor is administered at a dosage of 10 mg to 150 mg per kilogram of body weight per day, although the aforementioned variations will occur. However, a dosage level that is in the range of from about 10 mg to 100 mg per kilogram of body weight per day is most desirably employed in order to achieve effective results.
• the salt was suspended in CH 2 C1 2 (150 mL) , cooled at 0° and DIEA was added (30.2 mL, 173 mmol) followed by a phosgene solution in toluene (1.93 M, 55 mL, 105.7 mmol) . After 2 h at 0 ° the reaction mixture was concentrated. The resulting thick gum was extracted with Et 2 0 and evaporation of the extract gave a light yellow oil. This oil was further purified by flash chromatography (Si0 2 , eluent : 10% EtOAc in hexane) to give a pale yellow oil (16.0 g, 84% yield).
• the resulting mixture was diluted with H 2 0 and extracted with CH 2 C1 2 .
• the organic phase was washed with aqueous HCl (10%) , saturated NaHC0 3 and brine, dried (MgS0 ) , filtered and concentrated.
• the crude aldehyde was immediately used in the next step.
• To the crude aldehyde dissolved in THF (29 mL) was added dropwise MeMgBr (3M/Et 2 0, 2.7 mL, 8.18 mmol). The reaction mixture was stirred 1 h at 0° and 1 h at room temperature, followed by the addition of aqueous saturated NH 4 C1 solution.
• step B was followed to give 1- ( ert-butyldimethylsilyl) -4 (R) - [ 1- (pyrimidin- 2-ylsulfanyl) ethyl] azetidin-2-one as a mixture of isomers . Separation of the two isomers was performed by preparative HPLC (C ⁇ 8 column, 5% to 100% CH 3 CN/H 2 O/0.06% TFA) .
• step D but using 4- (phenylsulfanylmethyl) azetidin-2 -one and (pyridin-4-ylmethyl) carbamic acid phenyl ester as reactants gave 2-oxo-4(i?)-
• step D Following the same procedure as in example 4, step D, but using 4 (R) - (benzenesulfinylmethyl) azetidin-2-one and benzylisocyanate as the starting material, gave 2 (R) -benzenesulfinylmethyl-4-oxoazetidine-l- carboxylic acid benzyl amide.
• ⁇ H NMR 400 MHz, CDC1 3 ) ⁇ (1:1 mixture of diastereoisomers) 7.62-7.55 (m, 4H) , 7.54-7.45 (m, 6H) , 7.31-7.24 (m, 4H) , 7.24-7.17 (m, 6H) , 6.85-6.70
• step A Following the two step procedure as in example 7 but using an excess of aqueous oxone in step A, one obtained 4 (R) -benzenesulfonylmethyl-2-oxoazetidine-l- carboxylic acid benzylamide as a white solid.
• step D Following the same procedure as in example 4, step D, but using 4 (2?) - (methylsulfanylmethyl) azetidin-2-one as starting material, gave 2(2?)- (methylsulfanyl)methyl-4-oxoazetidine-l-carboxylic acid (1-phenylpropyl) amide as yellowish oil.
• step D using 4- (2?) - (pyrimidin-2-ylsulfanylmethyl ) azetidin- 2-one and 2-phenylpyrrolidine-l-carbamoyl chloride as reactants gave the title compounds as a 1/1 mixture. Separation of the isomers was accomplished by flash chromatography (Si0 2 , 50 % EtOAc-hexane) to give isomer A (less polar, 0.047 g, 18 % yield) and isomer B (more polar, 0.059 g, 31% yield).
• a protocol adaptable to a 96-well plate format was designed for the determination of IC 50 values of inhibitors.
• HCMV N 0 was incubated for 2.5 h at 30° in the presence of the substrate with a range of sequentially diluted inhibitor concentrations (300 to 0.06 ⁇ M depending on the potency of each compound) . After this period, enzymatic hydrolysis of the fluorogenic substrate in the absence of inhibitor led to about a 30% conversion. Quenching was not required before fluorescence measurement since the total scanning time by the plate reader accessory was brief relative to the duration of the reaction.
• the aqueous incubation buffer contained 50 mM tris (hydroxymethyl) aminomethane • HCl pH 8, 0.5M
• HCMV N 0 protease expressed in terms of total monomer concentration
• substrate 100 nM and 5 ⁇ M respectively.
• IC 50 values were obtained through fitting of the inhibition curve to a competitive inhibition model using SAS NLIN procedure. The mode of inhibition was determined by measurements of the initial rates (in cuvettes) at various substrate concentrations in the buffer as described above. The IC 50 values listed in the following tables were obtained according to this assay.
• Plaque Reduction Assay Hs-68 cells (ATCC # CRL 1635) were seeded in 12-well plates at 83,000 cells/well in 1 mL of DMEM medium (Gibco Canada Inc.) supplemented with 10% fetal bovine serum (FBS, Gibco Canada Inc.) . The plates were incubated for 3 days at 37° to allow the cells to reach 80-90% confluency prior to the assay.
• DMEM medium Gibco Canada Inc.
• FBS fetal bovine serum
• the medium was removed from the cells by aspiration.
• the cells were then infected with approximately 50 PFU of HCMV (strain AD169, ATCC VR-538) in DMEM medium supplemented with 5% inactivated FBS (assay medium) .
• DMEM medium is commercially available and has been described by R. Dulbecco et al . , Virology 1959, 8, 396.
• the virus was allowed to adsorb to cells for 2 h at 37°. Following viral adsorption, the medium was removed from the wells by aspiration. The cells were then incubated with or without 1 mL of appropriate concentrations of test reagent in assay medium. Occasionally, test compounds were added 24 h post-infection.
• examples 1 to 11 can be used to prepare other compounds of formula 1. Examples of compounds thus prepared are listed in the following Tables 1, 2, 3 and 4 together with mass spectrum data for the compounds, and results from the assays A and B of example 12.
• TC 50 Cytotoxic effects noted as TC 50 in the following tables were determined according to the tetrazolium salt (MTT) metabolic assay, F. Denizot and F. Lang, J " . Immun . Meth . , 1986, 89, 271.
• MTT tetrazolium salt
• the TC 50 reported in tables 1 to 4 also indicate that these compounds are non-toxic and have a therapeutic window that allows safe use of these compounds in mammals, including humans.

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