WO1998054192A1

WO1998054192A1 - Thienoxazinone derivatives as antiviral agents

Info

Publication number: WO1998054192A1
Application number: PCT/EP1998/003181
Authority: WO
Inventors: David Haigh; David Glynn Smith; Robert William Ward
Original assignee: Smithkline Beecham P.L.C.
Priority date: 1997-05-29
Filing date: 1998-05-25
Publication date: 1998-12-03
Also published as: GB9710928D0

Abstract

Herpes protease inhibitor 4H-3,1-benzoxazin-4-one, 4H-thieno[3,2-d][1,3]oxazin-4-one, or 4H-thieno[2,3-d][1,3]oxazin-4-one derivatives which are 2-substituted by XNH-CHR1- wherein X is R4CO wherein R4 is (aryl or heteroaryl substituted by NRZOH, wherein RZ is hydrogen or C1-6alkyl)-CR6=CH- wherein R6 is hydrogen or a halo, C1-6alkyl or aryl substituent; and R1 is hydrogen or an amino acid side chain; and pharmaceutically acceptable salts thereof.

Description

THIENOXAZINONE DERIVATIVES AS ANΗVIRAL 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 herpes protease inhibitors. Jarvest et al, Bioorg. Med. Chem. Lett., (1996), 6(20), 2463-2466, describes benzoxazinone derivatives as herpes protease inhibitors.

WO97/48707 (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 XNH-CHR^- wherein X is R4CO wherein R4 is (aryl or heteroaryl substituted by R_zOH, wherein R_z is hydrogen or C g alkyl)-CR6=CH- wherein R6 is hydrogen or a halo, Ci _g alkyl or aryl substituent; and R\ is hydrogen or an amino acid side chain; 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 R2 respectively) as well as the 2-substituent. These may be selected from halo, Cμg alkyl or C\. alkoxy, C\. alkylthio, amino optionally substituted by one or two C\. alkyl or optionally substituted benzyl groups, hydroxy Cj, .5 alkyl, C\. 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 R2 may be joined to form C3, C4, C5, Cg, C7, or Cg polymethylene.

Preferably the 5-substituent is methyl and the 6-position is unsubstituted or the 6- substituent is CH2R_a, OCH-2Rb_> or NR_CR^ wherein R_a and Rtø are selected from hydrogen or a substituent, -Rς is Cγ. alkyl and R^ is C1 _g alkyl or substituted alkyl or R^ and Rd are joined to form a 5 or 6 membered heterocyclic ring containing one or more heteroatoms. A particular value for the 6-substituent is 6-thiomorpholino.

Particular compounds which may be mentioned are those in which the 6-position is unsubstituted.

The substituents R_a and Rtø may be selected from one or more of halo, trifluoromethyl, cyano, C\. alkyl, C\. alkoxy, aryloxy, aryl(Ci_g alkyl)oxy, C\. alkylthio, amino optionally substituted by one or two C\. alkyl or optionally substituted benzyl groups, hydroxy, C\. alkylcarbonyl, Cj.g alkoxycarbonyl, trifluoromethylcarbonyl, optionally substituted phenyl or R_eZCOY wherein Z is a bond, O, NH or NCOCH3, and R_eCO is an acyl group where values of R_e include aryl, C1 _g alkyl, aryl Ci _g alkyl, or heteroaryl- γ. alkyl; and Y is O or NRf wherein Rf is hydrogen, C\.β alkyl, aryl, or aryl Cμg alkyl, or R_e and Rf may together form C2-6 polymethylene; or R_a/Rb may be C\. alkyl or C2-6 alkenyl substituted by one or more of the values listed above for substituents R_a and Rj,.

Values of R^ substituents are as defined for R_a and Rtø above. Values for R_c and Rr\ when joined to form a heterocyclic ring include optionally substituted piperidine, pyrrolidine, azetidine morpholine, or piperazine. Suitable substituents are as listed above for R_a and Rtø, 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\. alkyl or C\. alkyl containing groups include C\, C2, C3, C4, C5, Cg, particularly C\_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 z^'-sO-propyl, n-, iso-, sec- and tert-butyl. Cyclic alkyl includes cyclopropyl, cyclobutyl, cyclopentyl, 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, Cι _g alkyl, Cι _g alkoxy, C1 _g alkoxycarbonyl, trifluoromethyl, trifluoromethoxy, nitro, Cι _6 alkylcarbonyl, Cι_6 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 include pyridyl, pyrimidyl, pyrazinyl, pyrryl, imidazolyl, thienyl, furanyl, oxazole or thiazole (all possible isomers). Bicyclic heteroaryl 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. The amino acid side chain may be of one or more amino acids. Suitable examples of amino acids are as described in the literature relating to synthetic peptides and includes natural and 'unnatural' amino acids. The amino acids may be in the D or L form, preferably the L form. Particular aminoacyl moieties -NH-CHRi -CO- , resulting in NH-CHR}- in the compound of formula (I), include alanine (Rj = CH3), serine ( \ = CH2OH), and α-aminobutyric (Ri = CH2CH3). Examples of X include R4CO wherein R4 is phenyl-CRg=CH-, 2- or 3-thienyl-

CR6=CH- or furanyl-CR6=CH-, 2-, 3-, or 4-pyridyl-CRg=CH-, or thiazolyl-CRg=CH-, substituted by NR_zOH, a particular example being phenyl-CRg=CH- substituted by NR_ZOH.

An example of NR_zOH is NHOH. Rg is preferably H.

R4 when phenyl- CRg=CH- 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 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 isobutyl 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 X may be modified after the oxazinone formation. Such modifications include interconversion of aryl or heteroaryl substituents. Suitable R4 precursors are the corresponding compounds of formula (I) wherein R4 is R4' which is substituted aryl-CRg=CH- or heteroaryl-CRg=CH-. Examples of R4' include phenyl- CRg=CH- substituted by nitro, which is then reduced.

The compounds which are derivatives of 4H-3,l-benzoxazin-4-one, 4H-thieno[3,2-d][l,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 (ΗSV-2). 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 Pharmacopaeia.

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 this 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. The effective dose of compound will in general 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.

Example 1 {a 4H-thieno[2,3-d][l,3]oxazin-4-one wherein R2=H, R3 IΗ3, RJ=CΗ3, R₄=(phenyl meta substituted by NΗOΗ)-CΗ=CΗ-} a) (S)-2-[N-(t-Butoxycarbonyl)-l-aminoethyl]-5-methyl-4H^r- thieno[2,3-d][l,3]oxazin-4-one A solution of N-(t-butoxycarbonyl)alanine (495 mg, 2.6 mmol) in dry NN- dimethylformamide (5 ml) at 0-5°C was treated with 1-hydroxybenzotriazole (390 mg, 2.87 mmol) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (550 mg, 2.87 mmol) and the reaction mixture was stirred for 15 min at 0-5°C. 2-Amino-4- methylthiophene-3-carboxylic acid (410 mg, 2.6 mmol) was then added in one portion, the mixture was allowed to come to room temperature and stirred for 18 h. A further portion of l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (550 mg, 2.87 mmol) was then added and the reaction was stirred at room temperature for a further 18 h. The solvent was evaporated under reduced pressure and the residue was dissolved in ethyl acetate and washed with water (2 x 50 ml) and saturated sodium bicarbonate (1 x 50 ml) and the organic layer was dried (MgSO₄) and filtered. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography on silica gel eluting with ethyl acetate-hexane (15-85%) to give a yellow solid which was crystallised from acetone/hexane to give yellow crystals (410 mg, 50%), mp 109-111°C; υ_max (KBr) 3347, 3103, 2986, 1759, 1693, 1607, 1533, 1454, 1345, 1266, 1249, 1175 and 1054 cm"¹; δ_H [(CD₃)₂SO] 1.38 (12H, m, (CH₃)₃C, + CH₃CH ), 2.42 (3H, s, CH₃ ), 4.46 (1H, m, CHCH₃ ), 7.30 (1H, s, 5-H), 7.54 (1H, d, J = 7.4 Hz. CHΝH). (C,₄H₁₈Ν₂O₄S requires: C, 54.18; H, 5.85; N, 9.03 %. Found: C, 53.81; H, 5.74; N, 8.90%. b) (S)-2-(l-Aminoethyl)-5-methyl-4H-thieno[2,3-d][l,3]oxazin-4-one, trifluoroacetic acid salt

A solution of (S)-2-[N-(t-butoxycarbonyl)-l -aminoethyl]-5-methyl-4H- thieno[2,3-d][l,3]oxazin-4-one (200 mg, 0.61 mmol) in dry dichloromethane (10 ml) was treated with anisole (0.075 ml, 0.73 mmol) and trifluoroacetic acid (1.0 ml) and allowed to stand at RT for 1 h. The solvent was evaporated under reduced pressure and the residue was azeotroped with toluene (2 x 50 ml). The residue was purified by column chromatography on silica gel, eluting with 3% methanol/dichloromethane to give the product as a pale gum; δ_Η (DMSO-d₆) 1.54 (3H, d, J = 6.9Hz, CH₃CH), 2.45 (3H, d, J = 1.4Hz, CH₃), 4.52 (1H, m, CHCH₃), 7.42 (1H, d, J = 1.1Hz, 6-H), and 8.60-8.80 (3H, v. broad, ΝH₃ ⁺); FABMS (+ve ion) 211 (MH⁺). c) (S)-2-[N-2-(trø/J5-3-nitrocinnamoyl)-l-aminoethyl]-5-methyl-4H-thieno[2,3- d] [l,3]oxazin-4-one

A solution of 3-nitrocinnamic acid (1.19 g, 6.2 mM) in dichloromethane (35ml) was treated with 1-hydroxybenzotriazole (1.68 g, 12.4 mM) and l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.38 g, 12.4 mM) and the reaction mixture stirred for 30 min. (S)-2-[l-aminoethyl]-5-methyl-4H- thieno[2,3-d][l,3]oxazin-4-one trifluoroacetate salt (2 g, 6.2 mM) and diisopropylethylamme (1.06 ml) was then added and the mixture stirred for a further 18 h. The reaction mixture was washed with saturated sodium bicarbonate (1 x 50ml) and brine (1 x 50ml) and the organic layer dried (Na2SO4) and filtered. The solvent was evaporated under reduced pressure and the residue purified by column chromatography eluting with ethyl acetate-hexane/methanol to give a yellow solid (0.9 g, 38%) v_max (KBr) 3260, 1755, 1659, 1602 and 1528 cm^"1; δ_Η[CDCl₃] 1.65 (3H, d, J = 6.6Hz), 2.52 (3H, s). 5.15 (IH, m), 6.61 (IH, bs), 6.66 (IH, d, J = 13 Hz), 6.88 (IH, s), 7.57 (IH, t, J = 7 Hz), 7.72 (IH, d, J = 13 Hz), , 7.79 (IH, d, J = 7 Hz), 8.20 (IH, d, J = 7 Hz), 8.40 (IH, s) Mass Spectrum API⁺ 386 (MH⁺, 100%) C₁₇H₁₅N₂O₅S requires 385. d) (S)-2-[N-2-(tr «s-3-[hydroxylamino]cinnamoyl)-l-aminoethyI]-5-methyl-4H- thieno [2,3-d] [1 ,3] oxazin-4-one A solution of (S)-2-[N-2-(tra«.s-3-nitrocinnamoyl)- 1 -aminoethyl]-5-methyl-4H- thieno[2,3-d][l,3]oxazin-4-one (250 mg, 0.9 mM) in tetrahydrofuran (7 ml) was added to a solution of sodium hypophosphite (289 mg, 2.7 mM) in water (5ml). 5% Palladium on carbon (28 mg) was added and the mixture stirred at room temperature for 1 h. The reaction mixture was filtered and the residue washed with ether. The filtrate was washed with water, the organic solutions concentrated in vacuo and the residue purified by column chromatography eluting with ethyl acetate/hexane to give a yellow powder (130 mg, 39%). δ_Η [(CD₃)₂SO] 1.50 (3H, J = 6.6 Hz), 2.42 (3H, s), 4.83 (IH, m), 6.66 (IH, d, J = 13 Hz), 6,84 (IH, d, J = 7 Hz), 6.94 (IH, d, J - 7 Hz), 7.06 (IH, s), 7.21 (IH, t, J = 7 Hz), 7.33 (IH, s), 7.35 (IH, d, J = 13 Hz), 8.45 (2H, bs), 8.79 (IH, d, J = 6.6 Hz) Mass Spectrum API⁺ 394 (M + Νa⁺), 372 (MH⁺, 20%)

Example 2 {a 4H-thieno[2,3-d][l,3]oxazin-4-one wherein R2=Η, R3=CH3, Rj=CH3, R4=(phenyl ortho substituted by NHOH)-CH=CH-} a) (S)-2-[N-2-(tr /ιs-2-nitrocinnamoyl)-l-aminoethyl]-5-methyl-4iϊ-thieno[2,3- d][l,3]oxazin-4-one

Prepared in a similar way to Example lc) using 2-nitrocinnamic acid δ_H [CDCI₃] 1.50 (3H, d, J = 6.6 Hz), 2.43 (3H, s), 4.88 (IH, m), 6.71 (IH, d, J = 13 Hz), 7.33 (IH, s), 7.62 - 7.81 (4H, m), 8.06 (IH, d, J = 6.6 Hz), 8.98 (IH, d, J = 6.6 Hz);

Mass Spectrum API⁺ 386 (MH⁺, 100%) C₁₇H₁₅N₂O₅S requires 385. b) (S)-2-[N-2-(trαns-2-[hydroxylamino]cinnamoyl)-l-aminoethyl]-5-methyl-4H- thieno [2,3-d] [1 ,3] oxazin-4-one

Prepared in a similar way to Example Id) using the material of Example 2a) above. δ_H [(CD₃)₂SO] 1.47 (3H, d, J = 6.6 Hz), 2.42 (3H, s), 4.84 (IH, m), 6.54 (IH, d, J = 13 Hz), 6.87 (IH, t, J = 7 Hz), 7.1-7.4 (4H, m), 7.59 (IH, d, J = 13 Hz), 8.27 (IH, bs), 8.47 (IH, bs), 8.69 (IH, d, J = 6.6 Hz).

Example 3 {a 4H-thieno[2,3-d][l,3]oxazin-4-one wherein R2=Η, R3M3H3, Rι =CH3, R4=(phenyl para substituted by NHOH)-CH=CH-} a) (S)-2-[N-2-(tr «s-4-nitrocinnamoyl)-l-aminoethyl]-5-methyl-4H-thieno[2,3- d] [l,3]oxazin-4-one

Prepared in a similar way to Example lc) using 4-nitrocinnamic acid δ_H [CDCI₃] 1.50 (3H, d, J = 6.6 Hz), 2.42 (3H, s), 4.88 (IH, d, J = 6.6 Hz), 6.89 (IH, d, J = 13 Hz), 7.32 (IH, s), 7.58 (IH, d, J = 13 Hz), 7.86 (2H, d, J = 7 Hz), 8.27 (2H, d, J = 7 Hz), 8.95 (IH, d, J = 6.6 Hz); Mass Spectrum APf 386 (MH⁺, 100%)

C₁₇H₁₅N₂O₅S requires 385. b) (S)-2-[N-2-(trαns-4-[hydroxylamino]dnnamoyl)-l-aminoethyl]-5-methyl-4H- thieno [2,3-d] [1 ,3]oxazin-4-one

Prepared in a similar way to Example Id) using the material of Example 3a) above. δ_H [(CD₃)₂SO] 1.47 (3H, d, J = 6.6 Hz), 2.42 (3H, s), 4.82 (IH, m), 6.48 (IH, d, J = 13 Hz), 6.83 (2H, d, J = 7 Hz), 7.31-7.41 (4H, m), 8.50 (IH, s), 8.62 (2H, d)

Quenched fluorescence assay for protease inhibition Compounds are assayed in the following way:

The enzyme used in the assay for HSV-2 consists of the proteolytically active domain of the HSV-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 200ul 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 NaCl lmM EDTA/0.01%PEG 3,400/0.8M sodium citrate* in 30% sucrose, pH 7.5 *For CMV 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 HSV-2, VZV and CMV 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 lOuM. FQ-7 peptide is used as the substrate for HSV-2 and VZV proteases. FQ-8 peptide is used as the substrate for CMV protease.

FQ7: Dabs-DNAVEASSKAPLK-Dans

FQ8: Dabs-RGVVNASSALAKK-Dans The plate is read every 30 seconds with a Fluostar SLT fluorometric plate reader using A nelisa software, for 15min 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 compounds of Examples 1 and 2 had an IC50 of < 2uM against CMV protease, the compound of Example 1 having preferred activity.

Claims

CLAIMS:

1. Herpes protease inhibitor 4H-3,l-benzoxazin-4-one, 4H-thieno[3,2-d][l ,3 joxazin-

4-one, or 4H-thieno[2,3-d][l,3]oxazin-4-one derivatives which are 2-substituted by XN╬ù-C╬ùRi- wherein X is R4CO wherein R4 is (aryl or heteroaryl substituted by

NR_zO╬ù, wherein R_z is hydrogen or C \ _g alkyl)-CRg=CH- wherein Rg is hydrogen or a halo, C1 _g alkyl or aryl substituent; and Rj is hydrogen or an amino acid side chain; 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,

C1 _g alkyl or C1 _g alkoxy, C1 _g alkylthio, amino optionally substituted by one or two C╬╣_g alkyl or optionally substituted benzyl groups, hydroxy C1 _g alkyl, C^.g 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^.g alkyl or aryl C1 _g alkyl, or R3 and R2 may be joined to form C3, C4, C5, Cg, C7, or Cg polymethylene.

3. A compound according to claim 2, wherein the 5-substituent R3 is methyl, and the

6-position is unsubstituted.

4. A compound according to any one of the preceding claims, wherein Rg is hydrogen.

5. A compound according to any one of the preceding claims, wherein R4 is phenyl-CRg=CH-, ortho, meta or para substituted by NRzOH.

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

7. A compound selected from:

(S)-2-[N-2-(tra┬½5-3-[hydroxylamino]cinnamoyl)-l-aminoethyl]-5-methyl-4H-thieno[2,3- d] [ 1 , 3 ] oxazin-4-one ;

(S)-2-[N-2-(tr╬▒n-s-2-[hydroxylamino]cinnamoyl)-l-aminoethyl]-5-methyl-4H-thieno[2,3- d][l,3Joxazin-4-one;

(S)-2-[N-2-(tran-s^,-4-[hydroxylamino]cinnamoyl)-l-aminoethyl]-5-methyl-4H-thieno[2,3- d] [ 1 ,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 the claims 1 to 7, or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier 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 the claims 1 to 7, or a pharmaceutically acceptable salt thereof.