WO1997048707A1

WO1997048707A1 - 4h-3,1-benzoxazin-4-one derivatives and analogs as antiviral agents

Info

Publication number: WO1997048707A1
Application number: PCT/EP1997/003158
Authority: WO
Inventors: David Jonathan Mcnair; Antony Nicholas Shaw; Andrew Derrick Gribble; Leslie John Arthur Jennings; Martin John Parratt; Richard Lewis Jarvest
Original assignee: Smithkline Beecham Plc
Priority date: 1996-06-20
Filing date: 1997-06-16
Publication date: 1997-12-24
Also published as: AU3339797A

Abstract

Herpesvirus 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 C(XY)-R1, C(XY)-CH2-R1, C(XY)-C2H4-R1, or CX=CY-R1 wherein one of X and Y is hydrogen or halo or an alkyl substituent and the other is hydrogen or an alkyl substituent, and R1 is aryl or heteroaryl substituted by ZR4 wherein Z is CO, NRxCO, NRxCOCO, NRxCOCH2, or NRxSO2 wherein Rx is hydrogen or alkyl and R4 is aryl or heteroaryl.

Description

4H-3,1-BENZOXAZIN-4-ONE DERIVATIVES AND ANALOGS AS ANTIVIRAL AGENTS

The present invention relates to compounds which are of potential use as antiviral agents.

WO 96/19482 (SmithKline Beecham plc) describes 2-substituted herpesvirus protease inhibitor 4H-thieno[2,3-d][1,3]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.

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-3,1-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

C(XY)-R₁, C(XY)-CΗ₂-R₁, C(XY)-C₂H₄-R₁, or CX=CY-R₁ wherein one of X and Y is hydrogen or halo or an alkyl substituent and the other is hydrogen or an alkyl substituent, and R₁ 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 alkyl and R₄ is aryl or heteroaryl, wherein the derivatives are hereinafter referred to as compounds of formula (I).

The 4H-thieno[2,3-d][1,3]oxazin-4-one ring system is numbered thus:

Values for X and Y when other than hydrogen include halo and alkyl.

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, C_{1 -6} alkyl or C_{1 -6} alkoxy, C_{1 -6} alkylthio, amino optionally substituted by one or two C_{1 -6} alkyl or optionally substituted benzyl groups, hydroxyalkyl, alkylcarbonyl, 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, alkyl or aralkyl, or R₃ and R₂ may be joined to form C₃, C₄, C₅, C₆, C₇, or C₈ polymethylene.

Preferably the 5-substituent is methyl and the 6-position is unsubstituted or the

6-substituent is CH₂R_a, OCH₂R_b* or NR_cR_d wherein R_a and R_b are selected from hydrogen or a substituent, R_c is alkyl and R_d is alkyl or substituted alkyl or R_c and R_d are joined to form a heterocyclic ring containing one or more heteroatoms. A partcular value for the 6-substituent is 6-thiomorpholino.

The substituents R_a and R_b may be selected from one or more of halo, trifluoromethyl, cyano, alkyl, alkoxy, aryloxy, aryl(alkyl)oxy, alkylthio, amino optionally substituted by one or two alkyl or optionally substituted benzyl groups, hydroxy, alkylcarbonyl, 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, alkyl, aralkyl, or heteroaryl-alkyl; and Y is O or NR_f wherein R_f is hydrogen, alkyl, aryl, or aralkyl, or R_e and R_f may together form C_2-6 polymethylene; or R_a/R_b may be alkyl or alkenyl substituted by one or more of the values listed above for substituents R_a and R_b.

values of R_d substituents are as defined for R_a and R₅ above. Values for R_c and R_d when joined to form a heterocyclic ring include optionally substituted pipendine, pyrrolidine, azetidine morpholine, or piperazine. Suitable substituents are as listed above for R_a and R_b, or the heterocyclic ring may contain an exocyclic optionally protected carbonyl group.

The 4H-thieno[3,2-d][1,3]oxazin-4-one ring system is numbered thus:

The substituents may be as described above for the

4H-thieno[2,3-d][1,3]oxazin-4-one ring system, except that the description for the 5-substituent applies to the 7-substituent.

The 4H-3,1-benzoxazin-4-one ring system is numbered thus:

The substituents may be as described above for the

4H-thieno[2,3-d][1,3]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 alkyl or alkyl containing groups include C₁, C₂, C₃, C₄, C₅, C₆, branched, straight chained and/or cyclic and/or primary, secondary or tertiary alkyl, as appropriate. C_1-4 alkyl groups include methyl, ethyl n- and iso-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, C_1-6 alkyl, C_1-6 alkoxy, C_1-6 alkoxycarbonyl, trifluoromethyl, trifluoromethoxy, nitro, C_1-6 alkylcarbonyl, C_1-6 alkylcarbonyloxy, amino optionally substituted by one or two C_1-6 alkyl groups, and acetylamino.

Heteroaryl includes 5 or 6 membered monocyclic heteroaryl or 9 or 10 membered fused bicyclic heteroaryl linked through carbon. 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 isøquinolyl (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.

Examples of C(XY) include CH₂, CH(CH₃), CH(C₂H₅), C(CH₃)₂, and CCl(CH₃).

Examples of R₁ include phenyl ortho, meta, or para substituted by ZR₄, or thienyl 2- or 3-substituted by ZR₄.

Examples of R₄ include phenyl, 2- or 3-thienyl or furanyl, 2-, 3-, or 4-pyridyl, or thiazolyl. R₄ when phenyl may be optionally substituted as described hereinbefore for aryl substituents. Alternative or additional optional substituents for R₄ when aryl and heteroaryl include aryloxy, heteroaryloxy, aroyl, heteroaroyl, aralkylcarbonyl, heteroarylalkylcarbonyl, and polar substituents such as hydroxy, formyl, amino, alkyl-, aryl-, heteroaryl-, aralkyl-, or heteroarylalkyl-carbonylamino, carboxyl, alkoxy-, aryloxy-, heteroaryloxy-, aralkyloxy-, or heteroarylalkyloxy-carbonyl, aminocarbonyl, alkyl-, aryl-, heteroaryl-, aralkyl-, or heteroarylalkyl-aminocarbonyl, alkyl-, aryl-, heteroaryl-, aralkyl-, or heteroarylalkyl-sulphonylamino, and CR_pR_qOH wherein R_p and R_q are independently hydrogen or C_1-6 alkyl.

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 4H-thieno[2,3-d][1,3]oxazin-4-one derivatives are prepared by activating an R₁ containing or R₁ 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 wøbutyl chloroformate 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 R₁ 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 R₁ precursors when Z is

NR_xCO, NR_xCOCO, NR_xCOCH₂, or NR_xSO₂ are the corresponding compounds of formula (I) wherein R₁ is R₁' which is substituted aryl or heteroaryl. Examples of

R\' 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 R₁' 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 Example 67 hereinafter. Similarly, the substituent on R₄ in the compound of formula (I) is convertible, for example, a nitro substituent may be converted to amino by reduction, a formyl substituent may be converted to hydroxymethyl by reduction.

The 4H-3,1-benzoxazin-4-one derivatives and the the

4H-thieno[3,2-d][1,3]oxazin-4-one derivatives are prepared in a similar manner and/or in an analogous manner to that described in the aforementioned reference publications describing compounds of these classes.

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 an, 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 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.

The following Examples illustrate the invention. All compounds wherein X and Y are different were prepared as a racemic mixture of enantiomers (RS form).

The Compound of Example 2 was separated into R and S forms by chromatography. Synthetic Scheme for Preparation of Compound Examples

Preparation of intermediate thiophene acid

2-Amino-4-methylthiophene-3-carboxyiic acid

To a solution of ethyl 2-amino-4-methylthiophene-3-carboxylate (10.0 g, 54.0 mmol) 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 h 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 × 500 ml, 1 × 250 ml). The combined organics were dried (MgSO₄), filtered and evaporated to give 2-amino-4-methylthiophene-3-carboxylic acid as a tan coloured solid (5.9 g, 69%); d_H

[(CD₃)₂SO] 2.16 (3H, s, CH₃), 5.89 (1H, s, 5-H), 7.26 (2H, br s, D₂O exchangeable, NH₂), 11-90 (1H br s, D₂O exchangeable, CO₂H).

Example 1

5-Methyl-2-[1-[4-(benzoyl)phenyl]ethyl]-4H-thieno[2,3-d][1,3]oxazin-4-one

To a solution of 2-[4-(benzoyl)phenyl]propanoic acid (0.90 g, 3.50 mmol) and 1-hydroxy-7-azabenzotriazole (0.48 g, 3.50 mmol) in dry DMF (25 ml) at 0°C was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.01 g, 10.5 mmol). The mixture was stirred at 0°C for 0.5 h before 2 -amino-4-methylthiophene-3-carboxylic acid (0.55 g, 3.50 mmol) was added. The mixture was stirred at room temperature for 18 h. The solvent was removed and the residue was purified by column chromatography on silica gel (eluting with 20-33% acetone in hexane) to give 5-methyl-2-[1-[4-(benzoyl)phenyl]ethyl]-4H-thieno[2,3-d][3,1]oxazin-4-one as a pale yellow waxy solid (0.437g, 33%); d_H [(CD₃)₂SO] 1.61 (3H, d, J 7 Hz, CHCH₃), 2.41 (3Η, s, CH₃), 4.37 (1H, q, J 7 Hz, CHCH₃), 7.31 (1H, s, 6-H), 7.50-7.60 (9H, m, C₆H₅ and C₆H₄). Examples 2 and 3

5-Methyl-2-[1-[4-(thien-2-ylcarbonyl)phenyl]ethyl]-4H-thieno[2,3-d][1,3]oxazin-4-one and

5-Methyl-2-[1-chloro-1-[4-(thien-2-ylcarbonyl)phenyl]ethyl]-4H-thieno[2,3-d][1,3]oxazin-4-one

Stage 1, Method 1 To a solution of 2-[4-(thien-2-ylcarbonyl)phenyl]propanoic acid (0.70 g, 2.69 mmol) and N-methylmorpholine (0.27 g, 2.69 mmol) in dry THF (15 ml) at 0°C was added isobutyl chloroformate (0.40 g, 2.96 mmol). The mixture was stirred at 0°C for 0.5 h before ethyl 2-amino-4-methylthiophene-3-carboxylate (0.50 g, 2.69 mmol) was added. The mixture was stirred at room temperature for 18 h. The solvent was removed and the residue was purified by column chromatography (eluting with dichloromethane) to give ethyl 4-methyl-2-[2-[4-(thien-2-ylcarbonyl)phenyl]propionyl]aminothiophene-3-carboxylate as a colourless gum (0.267 g, 23%); d_H (CDCI₃) 1.36 (3H, t, J 7 Hz, CH₂CH₃), 1.69 3Η, d, J 7 Hz, CHCH₃), 2.33 (3Η, d, J 1 Hz, CH₃), 3.92 (1H, q, J 7 Hz, CHCH₃), 4.29 (2H, q, J 7 Hz, CH₂CH₃), 6.37 (1H, s, 5-H), 7.15 (1H, dd, J 4 Hz, 5 Hz, 4'-H), 7.52 (2H, d, J 8 Hz, 2H of C₆H₄), 7.67 (1H, dd, J 1 Hz, 4 Hz, 3'-H/5'-H), 7.72 (1H, dd, J 1 Hz, 5 Hz, 3'-H/5'-H), 7.87 (2H, d, J 8 Hz, 2H of C₆H₄).

Stage 1, Method 2 To a solution of 2-[4-(thien-2-ylcarbonyl)phenyl]propanoic acid (0.70 g, 2.69 mmol), ethyl 2-amino-4-methylthiophene-3-carboxylate (0.50 g, 2.69 mmol) and 1-hydroxy-7-azabenzotriazole (0.37 g, 2.69 mmol) in dry DMF (15 ml) at 0°C was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.62 g, 3.23 mmol). The mixture was stirred at room temperature for 18 h. The solvent was removed and the residue was purified by column chromatography (eluting with 0-5% ethyl acetate in dichloromethane) to give ethyl 4-methyl-2-[2-[4-(thien-2-ylcarbonyl)phenyl]propionyl]aminothiophene-3-carboxylate as a colourless gum (0.302 g, 26%); d_H (CDCI₃) 1.36 (3H, t, J 7 Hz, CH₂CH₃), 1.69 3H, d, J 7 Hz, CHCH₃), 2.33 (3Η, d, J 1 Hz, CH₃), 3.92 (1H, q, J 7 Hz, CHCH₃), 4.29 (2H, q, J 7 Hz, CH₂CH₃), 6.37 (1H, s, 5-H), 7.15 (1H, dd, J 4 Hz, 5 Hz, 4'-H), 7.52 (2H, d, J 8 Hz, 2H of C₆H₄), 7.67 (1H, dd, J 1 Hz, 4 Hz, 3'-H/5'-H), 7.72 (1H, dd, J 1 Hz, 5 Hz, 3'-H/5'-H), 7.87 (2H, d, J 8 Hz, 2H of C₆H₄). Stage 2 To a solution of ethyl 4-methyl-2-[2-[4-(thien-2-ylcarbonyl)phenyl]propionyl]aminothiophene-3-carboxylate (0.44 g, 1.04 mmol) in dry dichloromethane (20ml) was added triphenylphosphine (1.09 g, 4.14 mmol) and carbon tetrachloride (0.96 g, 6.22 mmol). The mixture was heated at 70°C for 3 h before being diluted with dichloromethane (100 ml) and washed with saturated aqueous sodium bicarbonate solution (2 × 20 ml) and dried (MgSO₄). The solvent was removed and the residue was purified by column chromatography (eluting with dichloromethane) to give 5-methyl-2-[1-[4-(thien-2-ylcarbonyl)phenyl]ethyl]-4H-thieno[2,3-d][3,1]oxazin-4-one as a waxy white solid (0.209 g, 53%); d_H

[(CD₃)₂SO] 1.62 (3H, d, J 7 Hz, CHCH₃), 2.40 (3Η, s, CH₃), 4.36 (1H, q, J 7Hz, CHCH₃), 7.28 (1H, m, 4'-H), 7.32 (1H, s, 6-H), 7.56 (2H, J 8 Hz, 2H of C₆H₄), 7.75 (1H, m, 3'-H/5'-H), 7.84 (2H, J 8 Hz, 2H of C₆H₄), 8.11 (1H, d, J 5Hz, 3'-H/5'-H), together with 5-methyl-2-[1-chloro-1-[4-(thien-2-ylcarbonyl)phenyl]ethyl]-4H-thieno[2,3-d][3,1]oxazin-4-one as a white solid (0.132 g, 31%); d_H [(CD₃)₂SO] 2.30 (3H, s, CH₃CCI), 2.44 (3H, s, CH₃), 7.30 (1H, dd, J 4 Hz, 5 Hz, 4'-H), 7.48 (1H, s, 6-H), 7.78 (1H, m, 3'-H/5'-H), 7.80 (2H, d, J 8 Hz, 2H of C₆H₄), 7.90 (2H, d, J 8 Hz, 2H of C₆H₄), 8.15 (1H, d, J 5 Hz, 3'-H/5'-H).

The following compounds were prepared wherein R₁ is phenyl substituted by 4 and R₂ and R₃ are as in Example 1:

The following compounds were prepared wherein R₁ is 2- or 3-thienyl which is 5-substituted by phenylcarbonyl, and R₂ and R₃ are as in Example 1:

The following compounds were prepared wherein X is hydrogen, R₁ is phenyl substituted by ZR₄ and R₂ and R₃ are as in Example 1:

Exampies 25 to 71 and 75 to 85

A = B = H except where indicated

The corresponding compound to Example 23, but wherein NHCO is replaced by N(Me)CO is also prepared.

A corresponding compound to any one of the Examples and compounds listed above, but wherein R₂ is thiomorpholino, is also prepared. Exampies 5 and 7

5-Methyl-2-(1-(4-(2-thiophenecarbonyl)phenyl)-1-methylethyl)-4H-thieno[23-d][1,3]oxazin-4-one

A solution of (±)-5-Methyl-2-(1-(4-(2-thiophene carbonyl)phenyl)ethyl)-4H-thieno[2,3-d][1,3]oxazin-4-one (Example 2) (0.27g, 0.000708 mol) in 1,3 dimethyl-2-imidazolidinone (4ml) was added dropwise to a stirred suspension of sodium hydride (60% dispersion in mineral oil, 0.055g, 0.00138 mol) in 1,3 dimethyl-2-imidazolidinone (2ml) at 15°C and under argon. After addition the mixture was stirred at room temperature for 90 minutes then methyl iodide (0.13ml,0.00207mol) was added by syringe in a single portion and the reaction left to stir overnight. The reaction mixture was poured into 15% aqueous NaH₂PO₄ and extracted with toluene(3x). The combined toluene extracts were washed with water, dried (MgSO4) and concentrated onto silica gel. Silica gel chromatography (80% CΗ₂Cl₂/40°-60°C petroleum spirit then 100% CΗ₂Cl₂) was used to yield title compound as a white solid (0.10g, 37%).

MP 135°C-136°C

NMR, MS consistent

(±)-5-Methyl-2-(1-(4-(2-thiophenecarbonyl)phenyI)propyl)-4H-thieno[2,3-d][1,3]oxazin-4-one was synthesised by similar procedure from the compound of Example 4, which was prepared in an analogous manner to the compound of Example 2.

Examples 8-13

The compounds were prepared by a similar procedure to Examples 1 and 2.

Examples 24, 21 and 23

(±)-5-Methyl-2-(3-(2-thiophenecarboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one

Solid 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (0.18g, 0.000939 mol) was added in a single portion to a stiιτed solution of 2-thiophene carboxylic acid (0.12g, 0.000918 mol) and 1-hydroxy-7-azabenzotriazole (0.12g, 0.000918 mol) in DMF (15ml) at room temperature and under argon. After 40 minutes solid 2-(1-(3-aminophenyl)-ethyl)-5-methyl-4H- thieno[2,3-d][1,3]oxazin-4-one (0.25g, 0.000918mol) was added in a single portion, followed by triethylamine (0.093g, 0.000918mol) and the reaction stirred for 8 hours. Solid

(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride (0.35g, 0.00182 mol) was added in a single portion and the mixture stirred for 16 hours. The mixture was poured into dilute aqueous sodium chloride and extracted with ethyl acetate (3x). Combined organic fractions were washed with dilute aqueous sodium chloride (1x), dried (MgSO₄) and concentrated onto silica gel. Silica gel chromatography (CΗ₂Cl₂ then 2% methanol/CΗ₂Cl₂) was used to yield title compound as a yellow solid (0.32g, 91%).

MP 167°C-168°C

NMR, MS(EI) consistent

(±)-5-Methyl-2-(4-(3-thiophenecarboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one and (±)-5-methyl-2-(3-(phenyl

carboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one MP 160ºC-161ºC, were synthesised by similar procedure using appropriate starting materials. Examples 18, 19, and 20

(±)-5-Methyl-2-(4-(phenylcarboxamido)phenyl)ethyI-4H- thieno[23-d][1,3]oxazin-4-one

Benzoyl chloride (0.08ml, 0.000681 mol) was added by syringe to a stirred solution of 2-(1-(4-aminophenyl)-ethyl)-5-methyl-4H-thieno[2,3-d][1,3]oxazin-4-one (0.15g, 0.000524mol) in CΗ₂Cl₂ (7ml) under argon, then stirred for 2 hours. The mixture was poured into aqueous HCl (1M) and extracted with CΗ₂Cl₂(3x).

Combined organic fractions were dried (MgSO₄) and concentrated onto silica gel. Silica gel chromatography (40% diethyl ether/40°-60°C petroleum spirit then 70% diethyl ether/40°-60°C petroleum spirit) was used to give a gum which was crystallised from toluene/40°-60°C petroleum spirit to yield the title compound as a white solid (0.0.029g, 14%).

MP 170°C-172°C

NMR, MS(EI) consistent

(±)-5-MethyI-2-(4-(3-pyridinecarboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one MP 132°C-135°C, and (±)-5-methyl-2-(4-(4-pyridinecarboxamido)phenyI)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one MP 154°C-156°C were synthesised by similar procedure using appropriate starting materials.

Example 22

(±)-5-Methyl-2-(4-(phenyl sulphamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one

Benzene sulphonyl chloride (0.10ml, 0.000786mol) was added by syringe to a stirred solution of 2-(1-(4-aminophenyl)-ethyl)-5-methyl-4H-thieno[2,3-d][1,3]oxazin-4-one(0.15g, 0.000524mol) and triethylamine(0.16g, 0.00157mol) in CΗ₂Cl₂ (8ml) at 0°C and under argon. The reaction mixture was stirred for 15 minutes at 0°C and then for 4 hours at room temperature and finally for 4 hours at reflux. The reaction mixture was poured into 15% aqueous NaH₂PO₄ and extracted with CΗ₂Cl₂(3x). Combined organic fractions were washed with 15% aqueous NaH₂PO₄(1x), dried (MgSO₄) and concentrated onto silica gel. Silica gel chromatography (70% CΗ₂Cl₂/40°-60°C petroleum spirit then 100% CΗ₂CI₂) was used to yield title compound as a white solid (0.17g, 81%).

MP 212°C-213°C

NMR, MS (El) consistent Example 67

5-MethyI-2-(4-(3-formylbenzoyl)benzyl)-4H-thieno-[2,3-d][1,3]oxazin-4-one

I

a) 4-Iodophenylacetic acid, (5.0g, 19mM), hydroxybenzotriazole (2.58g, 19mM) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.64g, 19mM) were stirred in dry DMF (100ml) for 30mins. To the resulting solution was added 2-amino-4-methylthiophene-3-carboxylic acid (2.99g, 19mM) and triethylamine (2.7ml, 19mM) and the mixture stirred for 18hrs. Further 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.64g) was added and stirring continued for 70hrs. The mixture was added to water (300ml) and EtOAc (200ml), separated and the aqueous extracted with EtOAc (3x200ml). The combined organic solutions were washed with satd NaHCO₃, water, brine, dried over MgSO₄ and evaporated to a brown solid which was chromatographed on silica gel (CΗ₂Cl₂). Trituration with ether gave 5-methyl-2-(4-iodobenzyl)-4H-thieno-[2,3-d][1,3]-oxazin- 4-one as a pale yellow solid (3.45g). mpl48-9°C, 47% yield.

1H NMR δ (CDCI₃) 2.49 (3H,s), 3.92 (2H,s), 6.83 (1H,s), 7.14 (2H, d, J=8Hz), 7.67 (2H, d, J=8Hz). b) 5-Methyl-2-(4-iodobenzyl)-4H-thieno-[2,3-d][1,3]-oxazin-4-one (383mg,

1mM), 3-formylphenylboronic acid (150mg, ImM),

bis(triphenylphosphine)palladium(II) dichloride (21mg) and potassium carbonate (483mg, 3,5mM) in anisole (6ml) were heated at 90 - 95°C in an atmosphere of carbon monoxide for 22hrs. The mixture was cooled to room temperature and filtered through CELITE, washing well with dichloromethane. The solvents were evaporated to give a red oil which was chromatographed on silica gel

(dichloromethane to dichloromethane/ether 50:1) to give the title compound as a light brown solid (181mg) mp 142-4°C, 47% yield

1H NMR δ (CDCI₃) 2.51 (3H,s), 4.08 (2H,s), 6.85 (1H,s), 7.54 (2H, d, J=8Hz), 7.71 (1H, t, J= 8Hz ), 7.80 (2H, d, J=8Hz), 8.10 (2H, m), 8.27 (1H, s), 10.10 (1H, s).

Example 71

5-Methyl-2-(4-(3-hydroxymethylbenzoyI)benzyl)-4H-thieno-[2,3-d][1,3]-oxazin-4-one

5-Methyl-2-(4-(3-formylbenzoyl)benzyl)-4H-thieno-[2,3-d][1,3]-oxazin-4-one (141mg, 0.36mM) {Example 67) and sodium triacetoxy borohydride (184mg, 0.87mM) in toluene (15ml) were heated at 80°C for 18hrs. The mixture was cooled to room temperature, ether (30ml) was added and the solution washed with with saturated NaHCO₃, water, dried over MgSO₄ and evaporated to a yellow solid. This was chromatographed on silica gel (dichloromethane to dichloromethane/ether 10:1) to give the title compound as a colourless solid (88mg). mp123-4°C, 62% yield. ¹H NMR δ (CDCI₃) 1.85 (1H, br t), 2.51 (3H,s), 4.07 (2H,s), 4.77 (2H, d, J=5Hz), 6.86 (1H,s), 7.50 (3H, m), 7.61 (1H, m), 7.70 (1H, m), 7.80 (3H, m).

Example 72

A compound corresponding to the compound of Example 1, but wherein 2-[1- [4-(benzoyl)phenyl]ethyl]- is replaced by 2-[2-[4-(benzoyl)phenyl]ethyl]-was prepared.

Example 73

The compound corresponding to the compound of Example 1, but wherein

C(XY) is replaced by CH=CH, was prepared.

Example 74

The compound corresponding to the compound of Example 16, but wherein C(XY) is replaced by CH=CH, was prepared.

Preparation of Intermediates

The biaryl ketone carboxylic acid intermediates for Examples 1-13 are either known compounds or prepared by the same procedures to the known analogues, (ref P.G.H. Van Daele et al, Arzneim-Forsch (Drug Res), 1975, 25, 1495-1501).

The intermediates for Examples 14-16 were prepared by a literature procedure (F. Clemence et al, Eur. J. Med Chem - Chimica Therapeutica, 1974, 9, 390).

Examples 17-24 were prepared from an intermediate corresponding to a compound of formula (I) but wherein R₁ is replaced by nitrophenyl, prepared as described for compounds of formula (I), using the appropriate carboxylic acid. The nitro group was reduced to an amino group.

Substituted benzophenone phenylacetic acids used as intermediates in coupling with the thienoxazinone for Example 67 and compounds prepared analogously, were prepared according to the method described by T. Ishiyama et al Tetrahedron Letters 1993, 34(47), 7595.

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

A 143 V.

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 ~1hr. The protease stock is then diluted using the correct buffer to 50X 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 10uM. 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 Annelisa 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 the Examples showed activity in one or more of the tests, the compounds of Examples 2, 3, 8, 10, 16, 25 wherein Z is CO, and Examples 17,

24, 27, 31, 36, 38, 49, 50, 52, 57, 69 wherein Z is NR_xCO, NR_xCOCO,

NR_xCOCΗ₂, or NR_xSO₂, showing preferred activity against CMV with an IC₅₀ of ≤ 0.5 uM and 1 uM respectively.

Claims

1. Herpesvirus 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 C(XY)-R ₁, C(X Y)-CΗ₂-R₁,

C(XY)-C₂H₄-R ₁, or CX=CY-R₁ wherein one of X and Y is hydrogen or halo or an alkyl substituent and the other is hydrogen or an alkyl substituent, and R₁ is aryl or heteroaryl substituted by ZR₄ wherein Z is CO, NR_xCO, NR_xCOCO, NR_xCOCΗ₂, or NR_xSO₂ wherein R_x is hydrogen or alkyl and R₄ is aryl or heteroaryl.

2. A compound according to claim 1 which is a 4H-thieno[2,3-d][1,3]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 by the 2-substituent.

3. A compound according to claim 2 wherein R₃ and R₂ when present are selected from halo, C_1-6 alkyl or C_1-6 alkoxy, C_1-6 alkylthio, amino optionally substituted by one or two C_1-6 alkyl or optionally substituted benzyl groups, hydroxyalkyl, alkylcarbonyl, 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, alkyl or aralkyl, or R₃ and R₂ may be joined to form C₃, C₄, C₅, C₆, C₇, or C₈ polymethylene.

4. A compound according to claim 3 wherein the 5-substituent is methyl and the 6-substituent is unsubstituted or the 6-substituent is CΗ₂R_a, OCΗ₂R_b, or NR_cR_d wherein R_a and R_b are selected from hydrogen or a substituent, R_c is alkyl and Rr\ is alkyl or substituted alkyl or R_c and R_d are joined to form a heterocyclic ring containing one or more heteroatoms.

5. A compound according to any one of claims 1 to 4, wherein C(XY) is CΗ₂, CH(CH₃), CH(C₂H₅), C(CH₃)₂, or CCl(CH₃).

6. A compound selected from the compounds of the Examples, as described herein.

7. A compound selected from: 5-methyl-2-[1-[4-(benzoyl)phenyl]ethyl]-4H-thieno[2,3-d][1,3]oxazin-4-one,

5-methyl-2-[1-[4-(thien-2-ylcarbonyl)phenyl]ethyl]-4H-thieno[2,3-d][1,3]oxazin-4-one,

5-methyl-2-[1-chloro-1-[4-(dιien-2-ylcarbonyl)phenyl]ethyl]-4H-thieno[2,3-d][1,3]oxazin-4-one,

5-methyl-2-(1-(4-(2-thiophenecarbonyl)phenyl)-1-methylethyl)-4H-thieno[2,3-d][1,3]oxazin-4-one,

5-methyl-2-(1-(4-(2-thiophenecarbonyl)phenyl)propyl)-4H-thieno[2,3-d][1,3]oxazin- 4-one,

5-methyl-2-(3-(2-thiophenecarboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one,

5-methyl-2-(4-(3-thiophenecarboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin- 4-one,

5-methyl-2-(3-(phenylcarboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one,

5-methyl-2-(4-(phenylcarboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one, 5-Methyl-2-(4-(3-pyridinecarboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4-one,

5-methyl-2-(4-(4-pyrimnecarboxamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4- one, 5-methyl-2-(4-(phenyl sulphamido)phenyl)ethyl-4H-thieno[2,3-d][1,3]oxazin-4- one,

5-methyl-2-(4-(3-formylbenzoyl)benzyl)-4H-thieno[2,3-d][1,3]oxazin-4-one,

5-methyl-2-(4-(3-hydroxymethylbenzoyl)benzyl)-4H-thieno[2,3-d][1,3]-oxazin-4- one, and pharmaceutically acceptable salts thereof.

8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

9. A compound according to any one of claims 1 to 7 for use as an active therapeutic substance.

10. A compound according to any one of claims 1 to 7 for use in treating viral diseases.

11. Use of a compound according to any one of claims 1 to 7 in the manufacture of a medicament for use in the treatment of viral diseases caused by herpesviruses.

12. A method of treatment of viral diseases caused by herpesviruses in mammals, which comprises the administration to mammal in need of such treatment, an effective amount of a compound according to any one of claims 1 to 7.