WO1998045298A1

WO1998045298A1 - Antiviral agents

Info

Publication number: WO1998045298A1
Application number: PCT/GB1998/001021
Authority: WO
Inventors: Ashley Edward Fenwick; Andrew Derrick Gribble; David Haigh; Brian Peter Slingsby
Original assignee: Smithkline Beecham Plc
Priority date: 1997-04-10
Filing date: 1998-04-07
Publication date: 1998-10-15

Abstract

Herpesvirus protease inhibitor 4H-3,1-benzoxazin-4-one, 4H-thieno[3,2-d][1,3]oxazin-4-one, and 4H-thieno[2,3-d][1,3]oxazin-4-one derivatives which are 2-substituted by -C(XY)-R1, -C(XY)-CH2-R1, -C(XY)-C2H4-R1, or -CX=CY-R1 wherein one of X and Y is hydrogen, halo or C1-6 alkyl and the other is hydrogen or C1-6 alkyl, and R1 is aryl or heteroaryl, substituted by ZR4 wherein Z is CO, NRxCO, NRxCOCO, NRxCOCH2, or NRxSO2 wherein Rx is hydrogen or C1-6 alkyl, and R4 is aryl or heteroaryl, substituted by NRzOH wherein Rz is hydrogen or C1-6 alkyl; and pharmaceutically acceptable salts thereof.

Description

ANTIVIRAL AGENTS

The present invention relates to compounds which are of potential use as antiviral agents, pharmaceutical compositions containing them and their use in therapy. WO 96/19482 (SmithKline Beecham pic) and WO 97/27200 (SmithKline

Beecham pic, published after the priority date of this application) describe 2-substituted herpesvirus protease inhibitor 4H-thieno[2,3-d][l,3]oxazin-4-one derivatives.

WO 96/37485 (G.D. Searle & Co.) describes 2-amino-benzoxazinones as herpesvirus protease inhibitors. Jarvest et al, Bioorg. Med. Chem. Lett., (1996), 6(20), 2463-2466, describes benzoxazinone derivatives as herpesvirus protease inhibitors.

WO 97/48707 (SmithKline Beecham pic, published after the priority date of this application) describes 2-substituted herpesvirus protease inhibitor 4H-3,l-benzoxazin-4- one, 4H-thieno[3,2-d][l,3]oxazin-4-one, and 4H-thieno[2,3-d][l,3]oxazin-4-one derivatives.

It has now been discovered that certain benzoxazinone and thienoxazinone derivatives are potentially useful in the treatment of infection caused by herpesviruses.

Accordingly, the present invention provides herpesvirus protease inhibitor 4H-3,l-benzoxazin-4-one, 4H-thieno[3,2-d][l,3]oxazin-4-one, or 4H-thieno[2,3-d][l,3]oxazin-4-one derivatives which are 2-substituted by -C(XY)-R_j, -C(XY)-CΗ₂-Rι, -C(XY)-C2H₄-R_l5 or -CX-CY-Ri wherein one of X and Y is hydrogen, halo or C\. alkyl and the other is hydrogen or C\ .$ alkyl, and R is aryl or heteroaryl, substituted by ZR4 wherein Z is CO, NR_xCO, NR_xCOCO, NR_xCOCH2, or NR_xSO2 wherein R_x is hydrogen or C g alkyl, and R4 is aryl or heteroaryl, substituted by NR_zOH wherein R_z is hydrogen or Cj.g alkyl; and pharmaceutically acceptable salts thereof; the derivatives are hereinafter referred to as compounds of formula (I).

The 4H-thieno[2,3-d][l,3]oxazin-4-one ring system is numbered thus:

There may be substituents in the 5- and 6-positions (designated R3 and R respectively) as well as the 2-substituent. These may be selected from halo, C\. alkyl, C\-6 alkoxy, C^.g alkylthio, amino optionally substituted by one or two C^.g alkyl or optionally substituted benzyl groups, hydroxy C\.β alkyl, C\_6 alkylcarbonyl, C\. alkoxycarbonyl, optionally substituted phenyl or R5ZCONH wherein Z is a bond, O, NH or NCOCH3, and R5CO is an acyl group where values of R5 include aryl, C\.β alkyl or aryl C\. alkyl; or R3 and R may be joined to form C3..8 polymethylene.

Preferably the 5-substituent is methyl, and the 6-position is unsubstituted or the 6- substituent is CH^Ra, OCH2Rb_> or NRςRd wherein R_a and Rtø are selected from hydrogen or a substituent, Rς is \. alkyl and R_f is C\_6 alkyl or substituted . alkyl or Rς and R^ are joined to form a heterocyclic ring, for example containing one or more heteroatoms selected from N, O and S. A particular value for the 6-substituent is 6- thiomorpholino. A further particular value for the 6-substituent is methyl.

The substituents R_a and Rtø may be selected from one or more of halo, trifiuoromethyl, cyano, Cj.6 alkyl, C^.β alkoxy, aryloxy, aryl(Cι_6 alkyl)oxy,

Cι_6 alkylthio, amino optionally substituted by one or two \. alkyl or optionally substituted benzyl groups, hydroxy, C\. alkylcarbonyl, C\. alkoxycarbonyl, trifluoromethylcarbonyl, optionally substituted phenyl or RgZCOY wherein Z is a bond, O, NH or NCOCH3, and R_eCO is an acyl group where values of R_e include aryl, C\.β alkyl, aryl C\. alkyl, or heteroaryl C\.β alkyl; and Y is O or NRf wherein Rf is hydrogen, Cj.g alkyl, aryl, or aryl C\. alkyl, or R_e and Rf may together form C2-6 polymethylene; or R_a and Rtø may be selected from Cj.g alkyl or C2-6 alkenyl substituted by one or more of the values listed above for substituents R_a and Rtø.

Values of R^ substituents are as defined for R_a and Rtø above. Values for R and R4 when joined to form a heterocyclic ring include optionally substituted piperidine, pyrrolidine, azetidine, morpholine, and piperazine. Suitable substituents are as listed above for R_a and R^,, or the heterocyclic ring may contain an exocyclic optionally protected carbonyl group.

The 4H-thieno[3,2-d][l,3]oxazin-4-one ring system is numbered thus:

1

The substituents may be as described above for the 4H-thieno[2,3-d][l,3]oxazin- -one ring system, except that the description for the 5-substituent applies to the -substituent.

The 4H-3,l-benzoxazin-4-one ring system is numbered thus:

The substituents may be as described above for the 4H-thieno[2,3-d][l,3]oxazin- 4-one ring system, except that the description for the 5- and 6-substituents applies to substituents selected from 5-, 6-, 7-, and 8-. Examples of C \ _g alkyl or C \ _β alkyl containing groups include C\.β, particularly C1.4, branched, straight chained and/or cyclic and/or primary, secondary or tertiary alkyl, as appropriate. C1.4 alkyl groups include methyl, ethyl, n- and wø-propyl, n-, iso-, sec- and tert-butyl. Cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Alkenyl includes all suitable values including E and Z forms.

Aryl includes phenyl and naphthyl optionally substituted by one or more substituents. Such substituents may be selected from halo, Cj.g alkyl, C\.β alkoxy, C\. alkoxycarbonyl, trifluoromethyl, trifluoromethoxy, nitro, Cj.g alkylcarbonyl, C\. alkylcarbonyloxy, amino optionally substituted by one or two C\. alkyl groups, and acetylamino.

Heteroaryl includes 5- or 6-membered monocyclic heteroaryl or 9- or 10- membered fused bicyclic heteroaryl linked through carbon, for example containing 1, 2 or 3 heteroatoms selected from N, O and S. Monocyclic heteroaryl groups include pyridyl, pyrimidyl, pyrazinyl, pyrryl, imidazolyl, thienyl, furanyl, oxazole and thiazole (all possible isomers). Bicyclic heteroaryl groups include benzofuranyl, benzothiophenyl, indolyl and indazolyl, quinolyl and woquinolyl (all possible isomers).

Heteroaryl may be optionally substituted by one or more substituents. These may be selected from those described above for aryl substituents. Halo includes fluoro, chloro, bromo and iodo.

A particular 2-substituent which may be mentioned is -C(XY)-R_j . Examples of -C(XY)- include CH₂, CH(CH₃), CH(C H₅), C(CH₃)₂, and CC1(CH3), a particular example being CH2-

Examples of R\ include phenyl ortho, meta, ox para substituted by ZR4, and thienyl 2- or 3-substituted by ZR4, a particular example being phenyl ortho, meta, or para substituted by ZR4.

Particular examples of Z are CO and NR_xCO. An example of R_x is hydrogen.

Examples of R4 include phenyl, 2- or 3-thienyl or furanyl, 1-, 3- or 4-pyridyl, and thiazolyl, substituted by NR_zOH, a particular example being phenyl substituted by NR_zOH.

R_z is preferably hydrogen.

R4 when phenyl may also be optionally substituted as described hereinbefore for aryl substituents. Examples of pharmaceutically acceptable salts of the compound of formula (I) are included in the invention, as appropriate.

The compounds wherein X and Y are different exist in more than one enantiomeric form. The invention extends to both of these forms and to mixtures thereof, including racemates. The compounds of formula (I) including their pharmaceutically acceptable salts may form solvates such as hydrates and these are included wherever a compound of formula (I) or a salt thereof is herein referred to.

The compounds are prepared by activating an R^ containing or R\ precursor containing carboxylic acid by standard coupling procedures such as: i) formation of an activated ester with a carbodiimide or other coupling reagent and a moiety such as 1-hydroxybenzotriazole, or ii) formation of a mixed anhydride with a reagent such as wobutyl chloro formate and reacting with a 2-aminothiophene 3-carboxylic acid or ester.

The intermediate amide may be isolated or the crude reaction product cyclised directly. Reagents suitable for the cyclisation of the thiophene acid derivatives include coupling agents or dehydrating agents such as carbodiimides, acetic anhydride or sulphonyl chlorides. Reagents suitable for the cyclisation of the thiophene ester derivatives include triphenylphosphine/carbon tetrachloride. It will be appreciated that according to the nature of the 5- and 6- substituents in the required product, the cyclisation may occur prior to or after introduction modification of the relevant substituent(s).

It will be appreciated that R may be modified after the oxazinone formation. Such modifications include alkylation of X or Y when hydrogen and interconversion of aryl or heteroaryl substituents. Suitable R precursors when Z is NR_xCO, NR_xCOCO, NR_xCOCH2, or NR_xSO2 are the corresponding compounds of formula (I) wherein R is Ri' which is substituted aryl or heteroaryl. Examples of Rj' include phenyl substituted by nitro, which is then reduced, followed by conversion to the appropriate value of ZR4 by conventional methods. When Z is CO a further example of Ri ' is phenyl substituted by halo, such as iodo, followed by conversion to the appropriate value of ZR4 by conventional methods, such as the boronic acid route described in Examples 2 and 3 hereinafter. Similarly, the substituent on R4 in the compound of formula (I) is convertible, for example, a compound of formula (I) in which R4 is substituted by NHOH may be prepared by reduction of the corresponding compound in which R4 is substituted by nitro.

The compounds which are derivatives of 4H-3,l-benzoxazin-4-one and 4H-thieno[3,2-d][ 1 ,3]oxazin-4-one may be prepared by analogous methods and/or the methods described in the aforementioned references.

Pharmaceutically acceptable salts may be prepared in conventional manner, for example, in the case of acid addition salts, by reaction with the appropriate organic or inorganic acid. The compounds of the invention are of potential use in the treatment of infections caused by herpesviruses such as herpes simplex types 1 and 2, varicella-zoster virus, Epstein-Barr virus and cytomegalovirus, especially cytomegalovirus and/or herpes simplex 2 (HSV-2).

The compounds of the invention may be formulated for use in a pharmaceutical composition. Accordingly, in a further aspect of the invention, there is provided a pharmaceutical composition which comprises a compound of formula (I) or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier or excipient.

A composition which may be administered by the oral route to humans may be compounded in the form of a syrup, tablet or capsule. When the composition is in the form of a tablet, any pharmaceutical carrier suitable for formulating such solid compositions may be used, for example magnesium stearate, starch, lactose, glucose, rice, flour and chalk. The composition may also be in the form of an ingestible capsule, for example of gelatin, to contain the compound, or in the form of a syrup, a solution or a suspension. Suitable liquid pharmaceutical carriers include ethyl alcohol, glycerine, saline and water to which flavouring or colouring agents may be added to form syrups. The compounds may also be presented with a sterile liquid carrier for injection.

The composition may possibly also be formulated for topical application to the skin or eyes.

For topical application to the skin, the composition may be in the form of a cream, lotion or ointment. These formulations may be conventional formulations well known in the art, for example, as described in standard books of pharmaceutics and cosmetics, such as Harry's Cosmeticology published by Leonard Hill Books and the British Pharmacopoeia.

The composition for application to the eyes may be a conventional eye-drop composition well known in the art, or an ointment composition.

Preferably, the composition of the invention is in unit dosage form or in some other form that may be administered in a single dose. A suitable dosage unit might contain from 50 mg to 1 g of active ingredient, for example 100 to 500 mg.

Such doses may be administered 1 to 4 times a day or more usually 2 or 3 times a day. An effective dose of compound of formula (I) will generally be in the range of from 1.0 to 20 mg/kg of body weight per day or more usually 2.0 to 10 mg/kg per day. No unacceptable toxicological effects are indicated at the above described dosage levels.

The invention also provides the use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of viral diseases caused by herpesviruses.

The invention also provides a method of treating viral infections in a human or non-human animal, which comprises administering to the animal an effective, non-toxic amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

The invention also provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use as an active therapeutic substance, in particular for the treatment of viral infections.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.

The following Examples illustrate the invention and the following assay description illustrates the herpes protease inhibition activity.

Intermediate Preparation 1 2-Amino-4-methyIthiophene-3-carboxyIic acid

To a solution of ethyl 2-amino-4-methylthiophene-3-carboxylate (10.0 g, 54.0 mrnol) in a mixture of dioxane (37.5 ml), methanol (25 ml) and water (12.5 ml) was added lithium hydroxide hydrate (11.3 g, 270 mmol). The mixture was heated at 70°C for 14 hrs then partitioned between water (500 ml) and dichloromethane (500 ml). The aqueous phase was acidified by addition of 10% citric acid solution (500 ml) and the product was extracted into dichloromethane (2 x 500 ml, 1 x 250 ml). The combined organic solutions were dried (MgSO4), filtered and evaporated to give 2-amino-4- methylthiophene-3-carboxylic acid as a tan coloured solid (5.9 g, 69%). ^lH NMR δ (DMSO-d₆): 2.16 (3H, s, CH₃), 5.89 (1H, s, 5-H), 7.26 (2H, br s, D₂O exchangeable, NH ), 11.90 (1H, br s, D2O exchangeable, CO2H). Intermediate Preparation 2 4-(3-Nitrobenzoyl)phenylacetic acid a) Methyl 4-iodophenylacetate (16.0 g, 58 rnmol), 3-nitrophenylboronic acid (9.67 g, 58 mmol), bis(triphenylphosphine)palladium(II) dichloride (1.40 g) and potassium carbonate (24.0 g, 174 mmol) in anisole (250 ml) were heated at 90 - 95°C in an atmosphere of carbon monoxide for 22 hrs. The mixture was cooled to room temperature and filtered through CELITE, washing well with ether. The solvents were evaporated to give a red oil which was purified by chromatography on silica gel (petroleum ether / ethyl acetate) to give methyl 4-(3-nitrobenzoyl)phenylacetate as a light brown solid (6.15 g, 35%) mp 73-5°C.

^!H NMR δ (CDCI3): 3.74 (5H, s), 7.42 (2H, d, J=8Hz), 7.6 (3H, m), 8.13 (1H, m), 8.46 (1H, s), 8.62 (1H, s). b) Methyl 4-(3-nitrobenzoyl)phenylacetate (3.29 g, 11 mmol) was dissolved in warm methanol (100 ml), cooled to 30°C and IN NaOH (22 ml) added. The solution was stirred at ambient temperature for 2 hrs, the methanol evaporated and water (50 ml) added. The solution was washed with ether, filtered through CELITE then acidified to pH3 with dilute HCl. The precipitated solid was filtered off, washed with water and dried to give the title compound as a copper coloured solid (2.91 g, 93%) mp 142-4°C. 1H NMR δ (DMSO-d₆): 3.83 (3H, s), 7.7 (2H, d, J=8Hz), 7.75 (2H, d, J=8Hz), 7.87 (1H, m,), 8.15 (1H, m), 8.43 (1H, s), 8.5 (1H, m).

Example 1

5-MethyI-2-[4-[3-(N-hydroxyamino)benzoyI]benzyl]-4H-thieno-[2,3-d][l,3]-oxazin-4- one (El) {a 4H-thieno[2,3-d][l,3]oxazin-4-one wherein R₂=Η, R3=CH₃, X=H, Y=H, Ri = phenyl para substituted by 3-(N-hydroxyamino)benzoyl} a) 4-(3-Nitrobenzoyl)phenylacetic acid, (2.85 g, 10 mmol), hydroxybenzotriazole (1.35 g, 10 mmol) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.92 g, 10 mmol) were stirred in dry DMF (50 ml) for 30 min. To the resulting solution was added 2-amino-4-methylthiophene-3-carboxylic acid (1.57 g, 10 mmol) and triethylamine (1.4 ml, 10 mmol) and the mixture stirred for 6 hrs. Further l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (1.92 g) was added and after 24 hrs a second addition (1.92 g) made. After 18 hrs the mixture was added to water (200 ml) and EtOAc (200 ml), separated and the aqueous extracted with EtOAc (200 ml). The combined organic solutions were washed with saturated NaHCO3, water, brine, dried (MgSO4) and evaporated to a brown oil which was purified by chromatography on silica gel (CH2CI2). Trituration with EtOAc gave 5-methyl-2-[4-(3-nitrobenzoyl)benzyl]-4H- thieno-[2,3-d][l,3]-oxazin-4-one as a pale yellow solid (2.22 g, 55%) mp 173-4°C. *H NMR δ (CDCI3): 2.51 (3H, s), 4.09 (3H, s), 6.87 (1H, s), 7.56 (2H, d, J=8Hz), 7.7 (1H, m), 7.80 (2H, d, J=8Hz), 8.13 (1H, m), 8.46 (1H, m), 8.5 (1H, s). b) 5-Methyl-2-[4-(3-nitrobenzoyl)benzyl]-4H-thieno-[2,3-d]-[l,3]-oxazin-4-one (773 mg, 1.9 mmol) was dissolved in EtOAc (300 ml) and 10% palladium on charcoal (250 mg) added. The mixture was hydrogenated under 50psi of hydrogen at ambient temp for 5 hrs. The mixture was filtered through CELITE then evaporated to an orange oil which was purified by chromatography on silica gel (dichloromethane / ether - gradient elution from 0-12% ether) to give the title compound as a yellow solid (435 mg, 60%) mp 136- 7°C.

*H NMR δ (DMSO-dg): 2.41 (3H, s), 4.20 (2H, s), 7.09 (2H, m), 7.21 (1H, s), 7.3 (2H, m), 7.55 (2H, d), 7.72 (2H, d), 8.49 (1H, m), 8.55 (1H, s).

Example 2 5,6-Dimethyl-2-[4-[3-(N-hydroxyamino)benzoyl]benzyl]-4H-thieno-[2,3-d][l,3]- oxazin-4-one

(E2) {a 4H-thieno[2,3-d][l,3]oxazin-4-one wherein R2=CΗ₃, R3=CH₃, X=H, Y=H,

R\ = phenyl para substituted by 3-(N-hydroxyamino)benzoyl} a) A solution of 4-iodophenylacetic acid (5.24 g, 20 mmol), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (DEC.HC1, 3.83 g, 20 mmol) and hydroxybenzotriazole (2.71 g, 20 mmol) in dry DMF (100 ml) was stirred at room temperature under argon for 35 min prior to the addition of 2-amino-4,5-dimethyl- thiophene-3-carboxylic acid (3.42 g, 20 mmol) [prepared from the appropriate ethyl ester which was hydrolysed with lithium hydroxide in aqueous methanolic dioxan in an analogous procedure to that described for Intermediate Preparation 1] and triethylamine (2.8 ml, 20 mmol). The mixture was stirred at room temperature for 3.5 hrs prior to the addition of a further aliquot of DEC.HC1 (7.67 g, 40 mmol). The solution was stirred for 16 hrs before being diluted with water (2 L) and extracted with ethyl acetate (3 x 500 ml). The combined organic solutions were washed with water (2 L) and brine (2 L), dried (MgSO4) and evaporated. The residual solid was chromatographed on silica gel using dichloromethane as eluent to afford 5,6-dimethyl-2-(4-iodobenzyl)-4H-thieno- [2,3-d][l,3]-oxazin-4-one as a solid, mp 146-7°C, following trituration with hexane. *H NMR δ (CDC1₃): 2.38 (6H, s), 3.89 (2H, s), 7.13 (2H, d), 7.66 (2H, d). b) A mixture of 5,6-dimethyl-2-(4-iodobenzyl)-4H-thieno-[2,3-d][l,3]-oxazin-4-one (1.985 g, 5 mmol), 3-mtrobenzeneboronic acid (0.915 g, 5 mmol), potassium carbonate (2.075 g, 15 mmol) and bis(triphenylphosphine)palladium(II) chloride (175 mg, 5 mol %) was suspended in anisole (30 ml) and degassed under vacuum. The flask was flushed with carbon monoxide and the degassing cycle repeated several times, finally leaving the reaction mixture under 1 atmosphere (balloon) of carbon monoxide. The mixture was heated at 80°C for 28 hrs, then cooled, diluted with water (500 ml) and extracted with dichloromethane (3 x 500 ml). The combined organic solutions were washed with water and brine, dried (MgSO4) and concentrated. The residue was chromatographed on silica gel (ethyl acetate-dichloromethane, 2:98 v/v) as eluent to afford 5,6-dimethyl-2-[4-(3- nitrobenzoyl)benzyl]-4H-thieno-[2,3-d][l,3]-oxazin-4-one, mp 182-3°C. !H NMR δ (CDCI3): 2.40 (6H, s), 4.07 (2H, s), 7.55 (2H, d), 7.70 (1H, t), 7.80 (2H, d), 8.10 (1H, d), 8.42 (1H, dm), 8.60 (lH,m). c) The title compound was prepared from 5,6-dimethyl-2-[4-(3- nitrobenzoyl)benzyl]-4H-thieno-[2,3-d][l,3]-oxazin-4-one (0.294 g, 0.7 mmol) by a procedure analogous to that described for Example lb) and purified by chromatography on silica gel (ether-dichloromethane, 5:95 v/v) as eluent. iH NMR δ (DMSO-d₆): 2.32 (3H, s), 2.38 (3H, s), 4.18 (2H, s), 7.05-7.75 (8H, m), 8.40- 8.60 (2H, br, exchanged with D2O).

Example 3

5-Methyl-2-[3-[3-(N-hydroxyamino)phenylcarboxamido]benzyl]-4H-thieno- [2,3-d] [l,3]-oxazin-4-one (E3) {a 4H-thieno[2,3-d][l ,3]oxazin-4-one wherein R₂=Η, R3=CH₃, X=H, Y=H, Ri = phenyl meta substituted by 3-(N-hydroxyamino)benzoylamino} a) 5-Methyl-2-[3-(3-nitrophenylcarboxamido)benzyl]-4H-thieno-[2,3-d] [ 1 ,3]-oxazin- 4-one was prepared from 5-methyl-2-(3-aminobenzyl)-4H-thieno-[2,3-d][l,3]-oxazin-4- one (0.58 g, 2.13 mmol) [prepared by reducing the corresponding 3-nitrobenzyl compound, which was prepared as described for compounds of formula (I), using the appropriate carboxylic acid] and 3-nitrobenzoic acid (0.356 g, 2.13 mmol) by standard EDC coupling methodology (with HOBT in dimethylformamide). The reaction mixture was diluted with water (100 ml), and extracted with ethyl acetate (3x100 ml). The combined organic solutions were washed with saturated NaHCO3 and brine, dried (Na2SO4) and evaporated to dryness. The residue was washed with methanol (20 ml) to give the required product as a pale yellow solid.

!H NMR δ (DMSO-d₆): 2.41 (3H, s), 4.08 (2H, s), 7.15 (IH, d), 7.30 (IH, s), 7.38 (IH, t), 7.70-7.90 (3H, m), 8.42 (2H, m), 8.79 (IH, s), 10.62 (IH, s). b) The title compound was prepared from 5-methyl-2-[3-(3- nitrophenylcarboxamido)benzyl]-4H-thieno[2,3-d][l,3]oxazin-4-one (0.25 g, 0.59 mmol) by a procedure similar to that described for Example lb), with a reaction time of 29 hrs.

The isolated product mixture was chromatographed on silica gel (gradient of 0-25% ether in dichloromethane) and further purified by crystallization from acetonitrile to give the title compound as a pale yellow solid.

*H NMR δ (DMSO-d₆): 2.41 (3H, s), 4.05 (2H, s), 6.95-7.15 (2H, overlapping m), 7.2- 7.45 (5H, overlapping m), 7.65-7.80 (2H, m), 8.45 (IH, d, exchanged with D2O), 8.49

(IH, s, exchanged with D2O), 10.20 (IH, s, partially exchanged with D2O).

Example 4 5-Methyl-2-[3-[4-(N-hydroxyamino)phenyIcarboxamido]benzyl]-4H-thieno- [2,3-d][l,3]-oxazin-4-one

(E4) {a 4H-thieno[2,3-d][l,3]oxazin-4-one wherein R₂=Η, R3=CH3, X=H, Y=H,

Ri = phenyl meta substituted by 4-(N-hydroxyamino)benzoylamino} a) 5-Methyl-2-[3-(4-nitrophenylcarboxamido)benzyl]-4H-thieno-[2,3-d][l,3]-oxazin-

4-one was prepared from 5-methyl-2-(3-aminobenzyl)-4H-thieno-[2,3-d][l,3]-oxazin-4- one (0.58 g, 2.13 mmol) [prepared by reducing the corresponding 3-nitrobenzyl compound, which was prepared as described for compounds of formula (I), using the appropriate carboxylic acid] and 4-nitrobenzoic acid (0.356 g, 2.13 mmol) by standard EDC coupling methodology (with HOBT in dimethylformamide). The resulting white solid was collected, washed with dichloromethane and methanol and dried in vacuo. IH NMR δ (CDCI3+CD3OD): 2.49 (3H, s), 4.01 (2H, s), 6.88 (IH, s), 7.20 (IH, d), 7.37 (IH, t), 7.64 (IH, s), 7.79 (IH, d), 8.11 (2H, d), 8.33 (2H, d). b) The title compound was prepared from 5-methyl-2-[3-(4- nitrophenylcarboxamido)benzyl]-4H-thieno-[2,3-d][l,3]-oxazin-4-one (0.25 g, 0.59 mmol) by a procedure similar to that described for Example lb), with a reaction time of 1.75 hrs. The isolated product mixture was purified by crystallization from acetonitrile and further purified by chromatography on silica gel (gradient of 0-20% ether in dichloromethane) to give the title compound as a white solid.

IH NMR δ (DMSO-d₆): 2.41 (3H, s), 4.04 (2H, s), 6.86 (2H, d), 7.06 (IH, d), 7.22-7.39 (2H, m), 7.63-7.78 (2H, m) partially overlapping with 7.82 (2H, d), 8.57 (IH, d, exchanged with D2O), 8.79 (IH, s, exchanged with D₂O), 9.96 (IH, s, partially exchanged with D2O).

Example 5

5-Methyl-2-[4-[4-(N-hydroxyamino)benzoyl]benzyl]-4H-thieno[2,3-d][l,3]oxazin-4- one

(E4) {a 4H-thieno[2,3-d][l,3]oxazin-4-one wherein R2=Η, R3=CH3, X=H, Y=H, R\ = phenyl para substituted by 4-(N-hydroxyamino)benzoylarnino}

5-Methyl-2-[4-(4-nitrobenzoyl)benzyl]-4H-thieno[2,3-d] [ 1 ,3]oxazin-4-one [prepared in a similar manner to Example la)] (406 mg, 1 mmol) was dissolved in THF (20 ml) and a solution of sodium hypophosphite (264 mg, 3 mmol) in water (10 ml) was added. 5% Palladium on charcoal (30 mg) was added under an atmosphere of argon and the mixture stirred at room temp for lhr. The mixture was filtered through CELITE then water (50 ml) and dichloromethane (100 ml) were added, the layers separated and the organic solutions washed with water, dried (MgSO4) and evaporated to a brown solid. This was boiled in dichloromethane (50 ml), cooled to RT, filtered and the mother liquor chromatographed on silica gel (dichloromethane / ether) to give the title compound as a light brown solid (79 mg, 20%) mp 169-71°C. lH NMR δ (DMSO-d₆): 2.41 (3H, s), 4.18 (2H, s), 6.86 (2H, d, J=9Hz), 7.29 (IH, s), 7.52 (2H, d, J=8Hz), 7.63 (4H, m), 8.73 (IH, m), 9.11 (IH, s). Quenched fluorescence assay for protease inhibition

Compounds are assayed in the following way:

The enzyme used in the assay for HSN-2 consists of the proteolytically active domain of the HSN-2 UL26 homologue protein (amino acid residues 1 to 247).

WO 95/06055 (SmithKline Beecham Corp.) describes the HSV-2 protease sequence. The enzyme used in the assay for CMV consists of the proteolytically active domain of the CMV UL80 protein (amino acid residues 1 to 256) with a mutation A143V. The enzyme used in the assay for VZV consists of the proteolytically active domain of the VZV gene 33 protein (amino acid residues 1 to 237).

A quenched fluorescence based assay is used to generate IC50 data for compounds screened against HSV-2, VZV and CMV proteases. The cleavage of a quenched fluorescent (QF) peptide substrate by the protease yields an increase in measured fluorescence over the time of incubation. The assay currently uses a final volume of 200 μl of assay mixture in each well. However, volume additions and dilution steps can be altered to cope with any changes in starting and/or final concentrations for each assay component. The steps described below have been configured to run using a Beckman Biomek 2000 robot. Compounds for screening are made up as stock solutions in 100% DMSO. Four compounds in duplicate are arranged per plate for screening against all three proteases. The compound stock solution, in the first well, is serially diluted 1/1 (v/v) with DMSO to produce a 1000 fold decrease in stock concentration across the plate in 11 points. Eight wells are included containing 100% DMSO only for addition to four control and four blank wells on the reaction plate. The reaction plate is an opaque 96 well plate designed for use with a fluorometric plate reader. Compound/DMSO from the dilution plate are transferred to the reaction plate which already contains assay buffer. The type of assay buffer used depends on which of the proteases is being assessed in the screen. For HSV-2 and VZV proteases this is 50mM hepes/150mM ΝaCl lmM EDTA 0.01%PEG 3,400/0.8M sodium citrate* in 30% sucrose, pH 7.5

*For CMN protease the 0.8M sodium citrate is omitted from the assay buffer. Each protease is stored at -20°C, thawed and stored on ice for ~lhr. The protease stock is then diluted using the correct buffer to 5 OX the final concentration needed. The diluted protease stock is added to all but the four wells designed to be used as blanks. Buffer only is used in these wells. The final concentrations are 500nm, 20nm and 20nm for HSN-2, NZN and CMN respectively.

The assay mixture is incubated at 27°C for 15 minutes.

Solid QF peptide substrate is resuspended in 10% DMSO/water at 400mM. This is further diluted 1/10 with assay buffer and added to each well of the reaction plate to give the final concentration of 10 μM. FQ-7 peptide is used as the substrate for HSN-2 and NZN proteases. FQ-8 peptide is used as the substrate for CMN protease. FQ7: Dabs-DΝANEASSKAPLK-Dans FQ8: Dabs-RGNVΝASSALAKK-Dans

The plate is read every 30 seconds with a Fluostar SLT fluorometric plate reader using Annelisa software, for 15 min at 27°C. Ex 355/Em 495nm. Data is transferred to Graphit software where rates of fluorescence are plotted against inhibitor concentrations. IC50 values are calculated for each of the four compounds against all three proteases.

The results were as follows:

Results

The compound of the Examples had an IC50 of < luM against CMN protease, the compound of Example 1 having preferred activity.

Claims

1. Herpes protease inhibitor 4H-3,l-benzoxazin-4-one, 4H-thieno[3,2-d][l,3]oxazin- 4-one, or 4H-thieno[2,3-d][l,3]oxazin-4-one derivatives which are 2-substituted by -C(XY)-Rι , -C(XY)-CΗ₂-Rι , -C(XY)-C₂H₄-R₁ , or -CX=CY-Ri wherein one of X and Y is hydrogen, halo or C\.β alkyl and the other is hydrogen or Ci _g alkyl, and Ri is aryl or heteroaryl, substituted by ZR4 wherein Z is CO, NR_xCO, NR_xCOCO, NR_xCOCH2, or NR_xSθ2 wherein R_x is hydrogen or C\.β alkyl, and R4 is aryl or heteroaryl, substituted by NR_zOH wherein R_z is hydrogen or C\.β alkyl; and pharmaceutically acceptable salts thereof.

2. A compound according to claim 1, which is a 4H-thieno[2,3-d][l,3]oxazin-4-one derivative wherein the ring system, numbered thus:

is optionally substituted in the 5- and 6-positions (designated R3 and R2 respectively) as well as the 2-substituent, wherein R3 and R2 when present are selected from halo, C\._β alkyl, C\.β alkoxy, C\.β alkylthio, amino optionally substituted by one or two C\.β alkyl or optionally substituted benzyl groups, hydroxy C\.β alkyl, C\.β alkylcarbonyl, C\.β alkoxycarbonyl, optionally substituted phenyl or R5ZCONΗ wherein Z is a bond, O, NH or NCOCH3, and R5CO is an acyl group where values of R5 include aryl, C .β alkyl or aryl Ci .5 alkyl; or R3 and R2 may be joined to form C3.8 polymethylene.

3. A compound according to claim 2, wherein the 5-substituent R3 is methyl, and the 6-position is unsubstituted or the 6-substituent R2 is CH2R_a, OCH2R_D> or NRςRd wherein R_a and Rtø are selected from hydrogen or a substituent, R_Q is C\.β alkyl and R is C\.β alkyl or substituted C\.β alkyl or Rς and R^ are joined to form a heterocyclic ring containing one or more heteroatoms selected from N, O and S.

4. A compound according to any one of the preceding claims, wherein Ri is phenyl ortho, meta, or para substituted by ZR4.

5. A compound according to any one of the preceding claims, wherein R4 is phenyl substituted by NR_zOH.

6. A compound according to any one of the preceding claims, wherein R_z is hydrogen.

7. A compound selected from:

5-methyl-2-[4-[3-(N-hydroxyamino)benzoyl]benzyl]-4H-thieno-[2,3-d][l,3]-oxazin-4- one;

5,6-dimethyl-2-[4-[3-(N-hydroxyamino)benzoyl]benzyl]-4H-thieno-[2,3-d][l,3]-oxazin- 4-one; 5-methyl-2-[3-[3-(N-hydroxyamino⁾phenylcarboxamido]benzyl]-4H-thieno-[2,3-d][l,3]- oxazin-4-one;

5-methyl-2-[3-[4-(N-hydroxyamino)phenylcarboxamido]benzyl]-4H-thieno-[2,3-d][l,3]- oxazin-4-one; and

5-methyl-2-[4-[4-(N-hydroxyamino)benzoyl]benzyl]-4H-thieno[2,3-d][l,3]oxazin-4-one; or a pharmaceutically acceptable salt of any one thereof.

8. A pharmaceutical composition which comprises a compound according to any one of claims 1 to 7, or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable earner or excipient.

9. Use of a compound of according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of viral diseases caused by herpesviruses.

10. A method of treating viral infections in a human or non-human animal, which comprises administering to the animal an effective, non-toxic amount of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof.