THIENOXAZINONE DERIVATIVES USEFUL AS ANTIVIRAL AGENTS

Abstract: Herpesvirus protease inhibitor 4H-thieno (2,3-d)(1,3) oxazin-4-one derivatives of formula (I) which are 2-substituted by X(Y)NH-CHR1 - wherein X is hydrogen or a blocking group, Y is an optionally present amino acid, and R1 is hydrogen or an amino acid side chain.

Description: THIENOXAZINONE DERIVATIVES USEFUL AS ANTIVIRAL AGENTS The present invention relates to compounds which are of potential use as antiviral agents. It has now been discovered that certain thienoxazinone derivatives are potentially useful in the treatment of infection caused by herpesviruses, especially herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), cytomegalovirus, and varicella-zoster virus. Accordingly, the present invention provides herpesvirus protease inhibitor 4H-thieno[2,3-d][l,3]oxazin-4-one derivatives which are 2-substituted by X(Y)NΗ-CΗRj- wherein X is hydrogen or a blocking group, Y is an optionally present amino acid, and Ri is hydrogen or an amino acid side chain, the derivatives 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, C\. alkyl or C\. alkoxy, C\. alkylthio, amino optionally substituted by one or two Cι_6 alkyl or optionally substituted benzyl groups, hydroxyalkyl, alkylcarbonyl, alkoxycarbonyl, optionally substituted phenyl or R4ZCONΗ wherein Z is a bond, O, NH or NCOCH3, and R4CO is an acyl group where values of R4 include aryl, alkyl or aralkyl, or R3 and R2 may be joined to form C2, C3, C4, C5, Cg, Cη, or Cg polymethylene. Examples of alkyl or alkyl containing groups include C\, C2, C3, C4, C5, Cg, branched, straight chained or cyclic alkyl, as appropriate. C _a alkyl groups include methyl, ethyl n- and wo-propyl, n-, iso-, sec- and t -butyl. Cyclic alkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. Aryl includes phenyl and naphthyl optionally substituted by one or more substituents selected from halo, C\. alkyl, C g alkoxy and C^.g alkoxycarbonyl. Halo includes fluoro, chloro, bromo and iodo. Suitable X moieties are those well known in the art of peptide chemistry. Particular values include aryl, alkanoyl, (aryl)alkoxycarbonyl, (aryl)alkylaminocarbonyl, aroyl, or arylsulphonyl. Examples of X of interest are benzyloxycarbonyl, t-butoxycarbonyl and optionally substituted phenyl. 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. The amino acids may be in the D or L form, preferably the L form. Particular examples of amino acids for Y include alanine, asparagine, valine and similar amino acids. Particular aminoacyl moieties -NH-CHRj-CO- , resulting in NH-CHRi- in the compound of formula (I), include alanine (Rj = CH3), asparagine, methionine, phenylalanine, serine (R\ = CH2OH), valine, and α-aminobutyric (R 1 = CH 2 CH 3 ). 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 amino acid or dipeptide by standard peptide 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 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. If the amino acid has a functionalised side-chain with a protecting group, then a final stage is the removal of the protecting group, for example, the removal of an acid-labile group with trifluoroacetic acid. 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). 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. Other herpesviruses are also of potential interest, such as herpesvirus-6. 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 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. Standard abbreviations used for the amino acids are the single letter codes. Scheme 1: Synthesis of Example 1 CbzHN i. EI3N, methanol; ii. NaOH/ H2O; iii. N-methylmoφholine, isobutyl chloroformate, THF, DMF, Cbz-L-alanine; iv. DEC, DMF. Scheme 2: Synthesis of Examples 2 , 3 and 4 and Example 2, method 2 Example 2 Examples 2,3,4 step 2, method 2 step 2 Example 2 step 2, method 2 Example 1 (step 1) Sodium 2-aminothiophene-3-carboxyIate To a solution of methyl 2-aminothiophene-3-carboxylate (K. Gewald, Chem. Ber. 1965, 98, 3571-7; 2.5 g, 0.016 mol) in dioxan (20 ml) was added sodium hydroxide (0.64 g, 7.5 ml of a 2.13 M solution, 0.016 mol) and the mixture was stirred at 60°C for 6 h then heated under reflux for 1.5 h. The solvent was removed under reduced pressure, the residue dissolved in water and unreacted starting material extracted using dichloromethane (3 x 50 ml). The water was removed from the product which was then dried over phosphorous pentoxide at 60°C (1.56g, 59 %). δ j ^ [(CD3)2SO] 5.95 (IH, d, J 5.5 Hz, thiophene proton), 6.75 (3H, m, thiophene proton and NH2). Example 1 (step 2) (S)-2-[N-(BenzyloxycarbonyI)-l-aminoethyl]-4H-thieno[2,3-d][l,3]oxazin-4-one. To a solution of N-Benzyloxycarbonyl-L-alanine (1.68 g, 7.51 mmol) in tetrahydrofuran (50 ml) at -10 °C was added N-methylmorpholine (0.82 ml, 0.76 g, 7.51 mmol) and isobutyl chloroformate (0.974 ml, 1.025 g, 7.51 mmol) and the mixture was stirred for 0.5 h. A solution of sodium 2-aminothiophene-3-carboxylate (1.25 g, 7.51 mmol) in NN-dimethylformamide (30 ml) was then added dropwise and the mixture was stirred for a further 18 h. The excess solvent was removed under reduced pressure and the residue was purified by column chromatography (eluting with 10 % methanol in dichloromethane containing 0.5 % acetic acid) to afford 2-[(N- benzyloxycarbonyl-L-alaninyl)amino]thiophene-3-carboxylic acid (1.77 g). To a solution of this material in DMF (50 ml) at 0°C was added ϊ-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.979 g, 5.07 mmol) and the mixture was stirred at room temperature for 2.5 h. The excess solvent was removed under reduced pressure and the residue was purified by column chromatography (eluting with 5% ethyl acetate in dichloromethane). This gave an impure product which was re-purified (eluting with 10% isopropyl alcohol in hexane) to afford (S)-2-[ -(benzyloxycarbonyl)-l-aminoethyl]-4H-thieno[2,3-d][l,3]oxazin- 4-one (23 mg), mp 140-144°C; v max (KBr) 3286, 1758, and 1686 cn ; δ Η [(CD 3 ) 2 SO] 1.44 (3H, d, J 7 Hz, CH3), 4.6 (IH, m, CH), 5.05 (2H, m, CH 2 ), 7.3 (5H, m, C6H5), 7.45 (IH, d, J 5 Hz, thiophene proton), 7.7 (IH, d, J 5 Hz, thiophene proton), and 8.04 (IH, d, J 7 Hz, ΝH) (Found C, 57.84; H, 4.47; Ν, 8.57%. C 16 H 14 Ν 2 O 4 S requires C, 58.17; H, 4.27; N, 8.48 %). Examples 2 and 3 (general method for step 1) Preparation of 2-[N-(beπzyloxycarbonyl)-L-alaninyI]aminothiophene-3- carboxyiates. To a solution of Ν-(benzyloxycarbonyl)alanine (3 mmol) in dry tetrahydrofuran (25 ml) at -5°C was added 4-methylmorpholine (3 mmol) followed by isobutyl chloroformate (3 mmol). After 5 minutes, the appropriate aminothiophene ester (3 mmol) was added and the reaction mixture was stirred at - 5°C for 30 minutes and then at room temperature for 52-60 h. The precipitate was filtered off, washed with dry tetrahydrofuran (2 x 10 ml), the combined filtrates and the washings were evaporated to dryness and the residue was coevaporated with toluene (2 x 50 ml). The product was purified by column chromatography on silica gel eluting with ethyl acetate-hexane mixtures. Example 2 (step 1) Ethyl 2-[N-(benzyloxycarbonyI)-L-alaninyI]amino-4-methylthiophene-3- car boxy late Yield 73 %; v max (KBr) 3305, 1696, 1670, 1551, 1521, and 1452 cm"!; δ H [(CD 3 ) 2 SO] 1.17 (6H, m, 2 x CH 3 ), 2.32(3H, d, J = 1.1 Hz, CH 3 ), 4.29 (3H, m, CH and CH 2 ), 5.08 (2H, m, CH 2 Ph), 6.71 (IH, d, J = 1.1 Hz, 5-H), 7.36 (5H, m, aromatic protons), 8.15 (H, d, J = 6.32 Hz, D2O exchangeable, ΝH), and 11.58 (H, s, D 2 O exchangeable, ΝH) (Found: C, 58.34; H, 5.67; Ν, 7.65%. C19H22Ν2O5S requires C, 58.45; H, 5.68; N, 7.17%.) Example 3 (step 1) Ethyl 2-[(N-benzyloxycarbonyI)-L-alaninyI]amino-4,5-dimethylthiophene-3- carboxylate Yield 48%; v max (KBr) 3276, 2985, 1687, 1565, 1529, and 1452 cm " 1 . δ H [(CD 3 ) 2 SO] 1.32 (6H, m, 2 x CH3), 2.21 (3H, s, CH3), 2.24 (3H, s, CH3), 4.26 (3H, m, CH and CH2), 5.08 (2H, m, CH2Ph), 7.36 (5H, m, aromatic protons), 8.12 (IH, d, J = 6.6 Hz, D2O exchangeable, ΝH), and 11.5 (IH, s, D2O exchangeable, ΝH) (Found: C, 59.40, H, 5.91, Ν, 7.05%. C20H24Ν2O5S requires C, 59.39, H, 5.98, N, 6.93%.) Example 4 (step 1) Ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-cyclopentan[b]thiophene-3- carboxylate. To a solution of ethyl 2-aminocyclopentan[b]thiophene-3-carboxylate (1 g, 4.8 mmol) in dry NN-dimethylformamide (20 ml) was added l-hydroxy-7-azabenzotriazole (5.7 mmol, 0.77g), Ν-(benzyloxycarbonyl)alanine (5.7 mmol, 1.27 g) and l-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5,7 mmol, 1.1 g). The resulting reaction mixture was stirred at room temperature for 52 h. The solvent was removed and the residue was dissolved in dichloromethane (200 ml). The solution was washed with 10% aqueous citric acid (2 x 50 ml), saturated aqueous sodium bicarbonate (2 x 50 ml), brine (1 x 50 ml) and dried (MgSO4). The solvent was removed and the product was purified by column chromatography on silica gel (eluting with 5-10% ethyl acetate in chloroform) to give ethyl 2-[N- (benzyloxycarbonyl)-L-alaninyl]amino-cyclopentan[b]thiophene-3-carboxylate as a colourless solid (0.61 g, 31%); v max (KBr) 3286, 2976, 2934, 1700, 1670, 1559, 1527, and 1453 cm " 1 ; δ H [(CD 3 ) 2 SO] 1.33 (6 H, m, 2 x CH 3 ), 2.32 (2 H, m, CH 2 ), 2.83 (4H, m, 2 x CH 2 ), 4.26 (3H, m, CH 2 and CH), 5.09 (2H, m, CH 2 Ph), 7.36 (5H, m, aromatic protons), 8.14 (IH, d, J = 6.04 Hz , D2O exchangeable, ΝH,), and 11.36 (IH, br s, D 2 O exchangeable, ΝH) (Found: C, 59.92; H, 5.79; Ν, 6.62%. C2iH 4Ν 2 O 5 S.0.2 H2O requires C, 59.99; H, 5.85; N, 6.67%.) Examples 2, 3 and 4 (general method for step 2) Preparation of (S)-2-[N-(benzyloxycarbonyl)-l-aminoethyI]-4H- thieno[2,3-d][l,3]oxazin-4-ones To a solution of the appropriate amidothiophene ester (0.5 mmol) in dry dichloromethane (10 ml) was added triphenylphosphine (2 mmol) and carbon tetrachloride (3 mmol). The resulting reaction mixture was refluxed at 70°C for 3.0- 3.5 h, then the mixture was diluted with dichloromethane (100 ml), washed with saturated aqueous sodium bicarbonate (2 x 20 ml) and dried (MgSO4). The solvent was removed and the product was purified by column chromatography on silica gel eluting with ethyl acetate-hexane mixtures. Example 2 (step 2) (S)-2-[N-Benzyloxycarbonyl)-l-aminoethyI]-5-methyl-4H- thieno[2,3-d][l,3]oxazin-4-one - Yield 41 %, mp 145-150°C; v ma χ (KBr) 3425, 3289,1756, 1691, 1604, 1553, 1540, and 1453 cm " 1 ; δ H [(CD 3 ) 2 SO] 1.43 (3H, d, J = 7.15 Hz, CH3), 2.42 (3H, d, J = 0.82 Hz, CH3), 4.56 (IH, m, CH), 5.06 (2H, m, CH2Ph), 7.32 (IH, d, J = 1.1 Hz, 6- H), 7.36 (5H, m, aromatic protons), and 8.02 (IH, d, J = 7.15 Hz, D2O exchangeable, NH) (Found: C, 59.20, H, 4.69, N, 8.19%. C17H16N2O4S requires C, 59.29, H, 4.68, N, 8.13%.) Example 3 (step 2) (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-5,6-dimethyl-4H- thieno[2,3-d][l,3]oxazin-4-one Yield 90 %, mp 122-125°C; v max (KBr) 3413, 3302, 1757, 1724, 1685, 1601, 1539, and 1455 cm " 1 ; δ H [(CD 3 ) 2 SO] 1.42 (3H, d, J = 7.15 Hz, CH3), 2.33 (3H, s, CH3), 2.40 (3H, s, CH3), 4.56 (IH, m, CH), 5.06 (2H, m, CH2Ph), 7.36 (5H, m, aromatic protons), and 8.01 (IH, d, J = 7.43 Hz, D2O exchangeable, ΝH) (Found: C, 60.61, H, 5.29, Ν, 7.62%. C18H18Ν2O4S requires C, 60.32, H, 5.06, N, 7.82%.) Example 4 (step 2) (S)-2-[N-(BenzyloxycarbonyI)-l-aminoethyl]-6,7-dihydro-4H,5H- cyclopenta[4,5]thieno[2,3-d][l,3]oxaziπ-4-one Yield 28 %; Vmax (KBr) 3414, 3295, 2986, 1775, 1687, 1600, 1537, and 1453 cm " 1 . δ Η [(CD 3 ) 2 SO] 1.42 (3H, d, J = 6.87 Hz, CH3), 2.43 (2H, m, CH2), 3.1 (4H, m, 2 x CH2), 4.57 (IH, m, CH), 5.06 (2H, m, CH2Ph), 7.36 (5H, m, aromatic protons), and 8.01 (IH, d, J = 7.43 Hz, D2O exchangeable, ΝH) (Found: C, 60.00, H, 4.91, Ν, 7.36%. C19H18O4Ν2S requires C, 61.61, H, 4.90, N, 7.56%.) Example 2, (step 2 - method 2) (S)-2-[N-Benzyloxycarbonyl)-l-aminoethyl]-5-methyl-4H- thieno[2,3-d][l,3]oxazin-4-one To a solution of ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4- methylthiophene-3-carboxylate (0.51 mmol, 0.2 g) in dioxane (2 mL) was added 1 M aqueous sodium hydroxide (0.52 ml). The resulting reaction mixture was stirred at room temperature for 8 h, further 1 M aqueous sodium hydroxide (0.3 ml) was added and the stirring was continued for 12 h. The solution was neutralised with dilute hydrochloric acid, the solvent was removed and the residue was coevaporated with toluene (2 x 20 ml). The residue was taken up in dry N,N-dimethylformamide (10 ml), to the resulting solution was added l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (1 mmol, 0.19 g) and the solution was stirred at room temperature for 60 h. The solvent was removed, the residue was dissolved in dichloromethane (100 ml), washed with 10% aqueous citric acid (1 x 10 ml), saturated aqueous sodium bicarbonate (1 x 10 ml), brine (1 x 10 ml) and dried (MgSO4). The product was purified by column chromatography on silica gel (eluting with 20-25% ethyl acetate in hexane) to give (S)-2-[N-(benzyloxycarbonyl)-l- aminoethyl]-5-methyl-4H-thieno[2,3-d][l,3]oxazin-4-one (15 mg, 8%); v m ax (KBr) 3417, 3288, 1756, 1691, 1604, 1553, 1539, and 1453 cm " 1 . δ Η [(CD 3 ) 2 SO] 1.43 (3H, d, J = 7.15 Hz, CH3), 2.42 (3H, d, J = 1.1 Hz, CH3), 4.56 (IH, m, CH), 5.05 (2H, m, CH2Ph), 7.32 (IH, d, J = 1.1 Hz, 6-H), 7.36 (5H, m, aromatic protons), and 8.02 (IH, d, J = 7.42 Hz, D2O exchangeable, ΝH) (Found: C, 59.41, H, 4.76, Ν, 8.08%. C17H16Ν2O4S requires C, 59.29, H, 4.68, N, 8.13%.) The following compounds were prepared in a similar way to Examples 2 and 3: Example 5 (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-5,6,7,8-tetrahydro-4H- [l]benzothieno[2,3-d][l-3]oxazin-4-one m.p. 131-133°C Example 6 (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-6-ethoxycarbonyl-5- methylthieno[2,3-d][l,3]oxazin-4-one m.p. 100-102°C Example 7 (step 1) Ethyl 2-[N-(benzyloxycarbonyl-L-alaninyl]amino-4-methyl-5-nitrothiophene-3- carboxylate To a stirred solution of ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4- ethyl thiophene- 3-carboxy late (1 g, 2.56 mmol) and copper nitrate (0.1 g) in acetic anhydride (20 ml) at 45°C was added copper nitrate (0.5 g) over 2 h. The reaction mixture was cooled to RT, poured into ice (500 g) and extracted with dichloromethane (3 x 100 ml). The solvent was evaporated, the residue was coevaporated with toluene (2 x 50 ml) and the product was purified by column chromatography on silica gel, eluting with ethyl acetate-dichloromethane (2:98), (0.61 g, 55% yield); δ Η (DMSO-d 6 ) 1.35 (6H, m, 2 x CH,), 2.78 (3H, s, CH,), 4.37 (3H, m, CH and CH,), 5.08 (2H, m, CH.Ph), 7.14-7.38 (5H, m, aromatic protons), 8.18 (IH, br. d, J = 6.6 Hz, D 2 O exchangeable, ΝH), and 11.86 (IH, br. s, D 2 O exchangeable, NH) (Found : C, 52.59; H, 4.80; N, 9.65%. C.,H,.N,O 7 S requires C, 52.41; H, 4.86; N, 9.65%.) Example 7 (step 2) Ethyl 2-[(N-benzyloxycarbonyl-L-alaninyl)amino]-4-methyl-5-aminothiophene- 3-carboxylate A mixture of the 5-nitrothiophene derivative (0.43 g, 1 mmol), iron (0.3 g,) and 1 M ammonium chloride (5 ml) in dioxane-ethanol (20 ml, 1:1) was sonicated at 40°C for lh 40 min. Then the iron was filtered off and washed with dioxane-ethanol (1:1, 2 x 20 ml). The filtrate and washings were combined and concentrated to a small volume. Water (50 ml) was added to this mixture and the product was extracted with dichloromethane (5 x 15 ml). The combined extracts were evaporated and the residue was coevaporated with toluene (3 x 20 ml) to give the product (0.40 g) which was used without purification in the next step; 5 (DMSO-d 6 ) 1.31 (6H, m, 2 x CH,), 2.07 (3H, s, CH,), 4.22 (3H, m, CH and CH,), 4.88 ( 2H, br. s, ΝH,), 5.08 (2H, m, CH j Ph), 7.14-7.39 (5H, m, aromatic protons), 8.08 (IH, d, J = 6.6 Hz, ΝH), and 11.35 (IH, s, ΝH). Example 7 (step 3) Ethyl 2-[(N-benzyloxycarbonyl-L-aIaninyl)amino]-4-methyI-5-aminothiophene- 3-carboxylate and Ethyl 2-[(N-benzyloxycarbonyI-L-alaninyl)amino]-4-methyl-5- acetamidothiophene-3-carboxylate A mixture of 5-nitrothiophene derivative (0.53 mmol, 0.230 g) and iron (100 mg) in acetic acid (15 ml) was sonicated at 30-40°C for 30 min. A further amount of iron (100 mg) was then added and the sonication was continued at 30-40°C for another hour. Water (50 ml) was added to this mixture and it was allowed to stand at RT for 30 min. The products were extracted with dichloromethane (5 x 50 ml), the combined extracts were evaporated and the residue was coevaporated with toluene (3 x 50ml). The products were separated by column chromatography on silica gel, eluting with ethyl acetate-hexane (2:8) to give 5-aminothiophene derivative (50 mg , 23% yield) and then with ethyl acetate-hexane (1 :1) to give the 5-acetamidothiophene derivative (40 mg, 17% yield); Η ΝMR (DMSO-d 6 ) δ: 1.30 (6H, m, 2 x CH,), 2.06 (3H, s, CH,), 4.23 (3H, m, CH, and CH), 4.88 (2H, br. s, D,O exchangeable, ΝH,), 5.08 (2H, m, CH j Ph), 7.22-7.39 (5H, m, aromatic protons), 8.08 (IH, d, J = 6.32 Hz, D,O exchangeable, NH), 11.34 (IH, br s, D,O exchangeable, NH). HRMS calcd for Ca j H a N j O j S 405.1358, found 405.1359. Η NMR (DMSO-d 6 ) δ:1.32 (6H, m, 2 x CH,,), 2.09 (3H, s ,CH,), 2.24 (3H, s, CH,CO), 4.26 (3H, m, CH, and CH), 5.08 (2H, m, CH^h), 7.34-7.39 (5H, m, aromatic protons), 8.12 (IH, d, /=6.32 Hz, D,O exchangeable, NH), 9.98 (IH, br. s, D 2 O exchangeable, NH), 11.49 (IH, br. s, D,O exchangeable, NH). HRMS calcd for 447.1463, found 447.1464. Example 7 (step 4) (5)-2-[N-(BenzyloxycarbonyI)-l-aminoethyI]-5-methyl-6-acetamido-4H- thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 2, step 2, yield 16%; 6 H (DMSO-d 6 ) 1.41(3H, d, J = 7.15 Hz, CH,), 2.18 (3H, s, CH,), 2.49 (3H, s, CH,CO), 4.53 (IH, m, CH), 5.06 (2H, m, CH,Ph), 7.31-7.36 (5H, m, aromatic protons), 8.00 (IH, d, J = 7.69 Hz, D,O exchangeable, ΝH), 10.67 (IH, br. s, D,O exchangeable, ΝH). HRMS calcd for C 19 H 19 Ν,O 3 S 401.1046, found 401.1048. Example 8 (S)-2-[N-(BenzyloxycarbonyI)-l-aminoethyl]-5-methyl-6-(N- isobutyryIamino)-4H-thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 9; Η (DMSO-d 6 ) 1.13 (6Η, d, J = 6.87 Hz, 2 x CH,), 1.42 (3H, d, J = 7.15 Hz, CH,), 2.43 (3H, s, CH,), 2.86 (IH, q, / = 6.87 Hz, CH), 4.56 (IH, m, CH), 5.06 (2H, m, CH,Ph), 7.13-7.41 (5H, m, aromatic protons), 8.0 (IH, d, J = 7.43 Hz, D,O exchangeable, ΝH), 10.56 (IH, s, D,O exchangeable, ΝH). HRMS calcd for .H^O.S 429.1359, found 429.1359. Example 9 (step 1) Ethyl -2-[(N-benzyloxycarbonyl-L-alaninyl)amino]-4-methyI-5-(N- hexanoylamino)thiophene-3-carboxylate A solution of ethyl 2-[(N-benzyloxycarbonyl-L-alaninyl)amino]-4-methyl-5- aminothiophene-3-carboxylate (0.4 g, 1 mmol), hexanoyl chloride (0.17ml, 1.2mmol) and 4-methylmorpholine (0.13ml, 1.2 mmol) in tetrahydrofuran (8 ml) was stirred at 0 - 5°C for 1.5 hr. The precipitate was filtered off, washed with tetrahydrofuran (2 x 20ml). The combined filtrates and washings were evaporated to dryness, the residue was dissolved in dichloromethane (80 ml), washed with saturated aqueous sodium hydrogen carbonate (2 x 20ml) and dried (MgSO . The solvent was removed and the residue was coevaporated with toluene (1 x 20ml). The product was purified by column chromatography on silica gel eluting with ethyl acetate- dichloromethane (5:95), (0.264 g, 53%); δ H (DMSO-d 6 ) 0.88 (3H, t, J = 6.6 Hz, CH,), 1.22-1.39 (10H, m, 2 x CH,, 2 x CH,), 1.57 (2H, , CH,), 2.23 (3H, s, CH,), 2.36 (2H, t, J = 7.15 Hz, CH,), 4.20-4.32 (3H, m, CH and CH,), 5.08 (2H, m, CH,Ph), 7.30-7.39 (5H, m, aromatic protons), 8.12 (IH, ά, J = 6.33 Hz, D 2 O exchangeable, NH), 9.89 (IH, s, D 2 O exchangeable, NH), and 11.49 (IH, s, D 2 O exchangeable, NH). HRMS calcd for C a H,,N,O 6 S 503.209, found 503.2104. Example 9 (step 2) (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-5-methyI-6-(N-hexanoylamino)- 4H-thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 2, step 2, yield 50%; (DMSO-d 6 ) 0.88 (3Η, t, J = 6.6 Hz, CH,), 1.30 (4H, m, 2 x CH,), 1.41 (3H, d, / = 7.15 Hz, CH,), 1.61 (2H, t, J = 1.1 Hz, CH,), 2.42 (3H, s, CH,), 2.45 (2H, m, CH,), 4.55 (IH, m, CH), 5.05 (2H, m, CH,Ph), 7.13-7.43 (5H, m, aromatic protons), 8.00 (IH, d, J = 7.7 Hz), D,O exchangeable, ΝH), 10.6 (IH, s, D,O exchangeable, ΝH). HRMS calcd for C„H^Ν,O 3 S 457,1672, found 457.1670. Example 10 (step 1) Ethyl 2-[N-(benzyloxycarbonyI)-L-alaninyl]amino-4-methyI-5- [N-(benzyIoxycarbonyl)-β-aIaninyl)]thiophene-3-carboxylate To a stirred solution of N-(benzyloxycarbonyl)-β-alanine (0.27g 1.15 mmol) and 4-methylmorpholine (0.13ml, 1.15 mmol) in tetrahydrofuran (10 ml) at 0°C was added isobutyl chloroformate (0.15 ml, 1.15 mmol). After 15 min. the resulting mixture was added dropwise during 10 min to a solution of ethyl 2-[(N- benzyloxycarbonyl)-L-alaninyl]amino-4-methyl-5-aminothiophene-3-carboxylate (1 mmol, crude material) in tetrahydrofuran (10ml) at 0°C and the reaction mixture was stirred at 0-5°C for 1 hr. A further amount of the mixed anhydride (1.15 mmol in 6ml of tetrahydrofurane) prepared as described above, was then added to this mixture. After stirring it for 1 hr the precipitate was filtered off, washed with tetrahydrofurane (20 ml). The combined washings and filtrates were evaporated to dryness and the product was purified by column chromatography on silica gel, eluting with ethyl acetate-dichloromethane (1:1): (0.370g, 53% yield after two steps); 'H NMR (DMSO-d 6 ) δ: 1.33 (6H, m, 2 x CH,), 2.23 (3H, s, CH,), 2.58 (2H, t, J = 6.9 Hz, CH,), 3.39 (2H, t, J = 6.9 Hz, after D,O exchange, CH,), 4.27 (3H, m, CH, and CH), 5.05 (4H, m, 2 x O-LPh), 7.17-7.39 (1 IH, m, aromatic protons and D,O exchangeable NH), 8.12 (IH, br. d, J = 5.78 Hz, D,O exchangeable, NH), 9.99 (IH, br. s, D,O exchangeable, NH), 11.50 (IH, br. s, D,O exchangeable, NH). HRMS calcd for C^N.O.S 611.2175, found 611.2175. Example 10 (step 2) 2-[N-(BenzyloxycarbonyI)-(lS)-l-(aminoethyI)]-5-methyl-6- [(N-benzyloxycarbonyl-β-alaninyl)amino]-4H-thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 2, step 2, yield 22%; Η NMR (DMSO-d 6 ) δ: 1.41 (3H, d, J = 6.9 Hz, CH,), 2.49 (3H, s, CH,), 2.68 (2H, t, J = 6.9 Hz, CH,), 3.32 (2H, m, CH,), 4.55 (IH, m, CH), 5.02 (2H, s, CH,Ph), 5.05 (2H, m, CH,Ph), 7.33 (10H, m, aromatic protons), 7.43 (IH, m, D,O exchangeable, NH), 8.01 (IH, br. d, D,O exchangeable, NH), 10.7 ( IH, s, D,O exchangeable, NH). HRMS calcd for 564,168, found 564.1678. Example 11 2-[N-(BenzyloxycarbonyI)-(lS)-l-(aminoethyl)]-5-methyl-6-[[6-N-(benzyloxy carbonyl)aminohexanoyI]amino]-4H-thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 10, yield 5%; δ H (DMSO-d.) 1.23-1.38 (2H, m, CH,), 1.41 (3H, d, J = 6.88 Hz, CH,), 1.46- 1.63 (2H, m, CH,), 2.42 (3H, s, CH,), 2.49 (2H, m, CH,), 3.01 (2H, m, CH,), 4.55 (IH, m, CH), 5.00 (2H, s, CH,Ph), 5.05 (2H, m, CH,Ph), 7.25-7.42 (6H, m, D,O exchangeable NH and aromatic protons), 8.01 (IH, d, / = 7.7 Hz, D,O exchangeable, NH), 10.6 (IH, s, D,O exchangeable, NH). HRMS calcd for C^N^S 607.2227, found 607.2230. Example 12 (step 1) Ethyl 2-[N-(benzyloxycarbonyl-L-alaninyl]amino-4-methyl-5- (Ν-benzyloxycarbonylamino)thiophene-3-carboxylate To a stirred solution of ethyl 2-[(N-benzyloxycarbonyl)-L-alaninyl]amino-4-methyl- 5-aminothiophene-3-carboxylate (110 mg, 0.27 mmol) and 4-methyl morpholine (0.038ml, 0.35mmol) in tetrahydrofuran (4 ml) at 0°C was added benzyl chloroformate (0.1ml, 0.35 M solution in tetrahydrofuran). The reaction mixture was stirred at 0-5°C for 1 hr , the precipitate was filtered off, and washed with tetrahydrofuran (2 x 5ml). The filtrate and the washings were combined, and the solvent was removed. The product was purified by column chromatography on silica gel, eluting with ethyl acetate-dichloromethane (11:89): (85 mg, yield 57% after two steps); Η NMR (DMSO-d 6 ) δ: 1.32 (6H, m, 2 x CH,), 2.16 ( 3H, s, CH,), 4.26 (3H, m, CH and CH,), 5.08 ( 2H, s, CH^h), 7.12-7. 1 (10H, m, aromatic protons), 8.13 (IH, d, J = 6.32 Hz, NH), 9.57 (IH, s, NH), 11.55 (IH, br. s, NH). (Found C, 59.96, H, 5.30, N, 7.52 %. C„H,,N,O 7 S requires C, 60.10, H, 5.42, N, 7.79 %.) Example 12 (step 2) (S)-2-[N-(BenzyIoxycarbonyl)-l-aminoethyI]-5-methyl-6- (triphenylphosphoranylideneamino)-4H-thieno[2,3-d][l,3]oxazin-4-one. A solution of thiophene 5-benzylcarbamate (70 mg, 0.13 mmol), triphenylphosphine (68mg , 0.26 mmol) and carbon tetrachloride (0.05 ml, 0.5 mmol) in dichloromethane (5 ml) was heated under reflux at 60-70°C for 1 hr. A further amount of triphenylphosphine (30 mg, 0.11 mmol) was then added and the reflux at 60-70°C was continued for another hour. The solution was diluted with dichloromethane (50 ml), washed with aqueous saturated sodium hydrogen carbonate (2 x 20 ml), and dried (MgSO . The product was purified by column chromatography on silica gel, eluting with ethyl acetate-dichloromethane (1:9) (58.8 mg, yield 73%); Η NMR (DMSO-d 6 ) δ: 1.33 (3Η, d, J = 6.88 Hz, CH,), 2.33 (3H, d, J = 1.1 Hz, CH,), 4.44 (IH, m, CH), 5.02 (2H, m, CH,Ph), 7.08- 7.31(5H, m, aromatic protons), 7.59-7.80 (15H, m, aromatic protons), 7.86 (IH, d, J = 7.70 Hz, NH). (Found: C, 67.12, H, 4.71 , N, 6.50%. C^H^N .PS requires C, 67.84, H, 4.88, N, 6.78 %.) Example 12 (step 3) (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-5-methyI-6-(N-benzoylamino)-4H- thieno[2,3-d][l,3]oxazin-4-one A solution of iminophosphorane derivative (0.230g, 0.37 mmol), benzoyl chloride (0.05ml, 0.45 mmol) and triethylamine (0.1ml, 0.74mmol) in dichloromethane (5ml) was stirred at RT for 3 days. The reaction mixture was diluted with dichloromethane (80 ml), washed with saturated aqueous sodium hydrogen carbonate (2x10 ml) and dried (MgSO4). The solvent was removed and the residue was purified by column chromatography on silica gel eluting with ethyl acetate-dichloromethane (35:65) to give the product ( 45 mg, yield 26%): Η NMR (DMSO-d 6 ) δ: 1.44 (3H, d, J = 6.87 Hz, CH,), 2.51 (3H, s, CH,), 4.58 (IH, m, CH), 5.07 (2H, m, OLPh), 7.30-7.42 (5H, m, aromatic protons), 7.54-7.69 (3H, m, aromatic protons), 7.98-8.04 (3H, m, D,O exchangeable, NH and aromatic protons), 10.93 (IH, s, D,O exchangeable, NH). HRMS calcd for C^.N^S 463.1202, found 463.1202. Example 13 (S)-2-[N-(BenzyloxycarbonyI)-l-aminoethyl]-5-methyl-6- [(Ν-isopropoxycarbonyl)amino]-4H-thieno[2,3-d][l,3]oxazin-4-one Ethyl 2-[N-(benzyloxycarbonyl-L-alaninyl]amino-4-methyl-5- (N-isopropoxycarbonylamino)thiophene-3-carboxylate was prepared in a similar way to Example 12, step 1 and cyclised in a similar way to Example 2, step 2; yield 48%; Η ΝMR (DMSO-d 6 ) δ: 1.28 (6H, d, J = 6.05 Hz, 2 x CH,), 1.41 (3H, d, J = 7.15 Hz, CH,), 2.33 (3H, s, CH,), 4.54 (IH, m, CH), 4.95 (IH, q, J = 6.05 Hz, CH), 5.05 (2H, m, CH,Ph), 7.14-7.63 (5H, aromatic protons), 8.00 (IH, d, J = 7.42 Hz, D,O exchangeable, ΝH), 10.34 (IH, s, D,O exchangeable, ΝH). HRMS calcd for C^Ν j O-S 445.1308, found 445.1310. Example 14 )-2-[N-(BenzyIoxycarbonyI)-l-aminoethyl]-5-methyl-6- [(N-isobutoxycarbonyl)amino]-4H-thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 13 yield 48%; δ H (DMSO-d 6 ) 0.94 (6H, d, J = 6.6 Hz, 2 x CH,), 1.41 (3H, d, J = 6.88 Hz, CH,), 1.96 (IH, m, CH), 2.34 (3H, s, CH,), 3.94 (2H, d, J = 6.7 Hz, CH,), 4.55 (IH, m, CH), 5.05 (2H, m, CH,Ph), 7.30- 7.36 (5H, m, aromatic protons), 8.0 (IH, d, J = 7.69 Hz, D,O exchangeable, ΝH), 10.32 (IH, br. s, D,O exchangeable.ΝH); HRMS calcd for C^H^Ν .S 459.1464, found 459.1474. General method for Examples 15 to 22 A solution of (S)-2-[N-(benzyloxycarbonyl)-l-aminoethyl]-5-methyl-6- (triphenylphosphoranylideneamino)-4H-thieno[2,3-d] [ 1 ,3]oxazin-4-one (0.16 mmol) and the appropriate isocyanate (5.0-5.6 equivalent) in dichloromethane (3 ml) or dichloromethane-acetonitrile (1:1, 5 ml) was stirred at RT for 3-64 h. a) alkyl ureas: The solvent was removed, the residue was coevaporated with toluene (2 x 5 ml) and then treated with dioxane- 10% aqueous citric acid (2:1, 5ml)) at RT for 6 hr. The mixture was concentrated to a small volume, and the product was extracted with dichloromethane (4 x 15 ml). The extracts were combined, dried (MgSO.4) and concentrated. The residue was purified by column chromatography on silica gel , eluting with ethyl acetate-dichloromethane mixtures. In some cases, the reaction mixture was concentrated, the residue was coevaporated with toluene (2 x 5 ml) and purified by column chromatography on silica gel to give thiophene diimides and/or thiophene ureas. The diimides were treated with glacial acetic acid in dichloromethane (1:9, 2 ml) at RT for 45 min- 130 min. The solvent was removed and the residue was coevaporated with toluene (2 x 5 ml). The alkyl ureas were purified by column chromatography on silica gel, eluting with ethyl acetate-hexane or ethyl acetate-dichloromethane mixtures. b) aryl ureas: The solvent was removed and the residue was coevaporated with toluene (2 x 5 ml) to give the crude product. In some cases, the reaction mixture was diluted with dichloromethane (50 ml), washed with 10% aqueous citric acid (2 x 10 ml), aqueous sodium hydrogen carbonate (1 x 10 ml), dried (MgSO. ) or the reaction mixture was concentrated, the residue was coevaporated with toluene (2 x 5 ml), dissolved in dichloromethane (50 ml) and worked up further as described above. The solvent was removed and each residue was purified by column chromatography on silica gel eluting with ethyl acetate-dichloromethane mixtures. Example 15 (S)-2-[N-(BenzyIoxycarbonyl)-l-aminoethyl]-5-methyl-6-(3-isopropylureido)-4H- thieno[2,3-d][l,3]oxazin-4-one Yield 42%; δ„ [(CD,)SO) 1.12 (6H, d, J = 6.6 Hz, 2 x CH,), 1.41 (3H, d, J = 7.15 Hz, CH,), 2.31 (3H, s, CH,), 3.77 (IH, m, CH), 4.54 (IH, , CH), 5.05 (2H, m, CH,Ph), 6.49 (IH, d, J = 7.42 Hz, D,O exchangeable, ΝH), 7.30-7,39 (5H, m, aromatic protons), 7.98 (IH, d, J = 7.7 Hz, D,O exchangeable), and 9.04 (IH, s, D,O exchangeable, ΝH). FABMS (positive ion, ): 445 (MH * ), 467 (MΝa * ). Example 16 (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyI]-5-methyl-6-(3-butylureido)-4H- thieno[2,3-d][l,3]oxazin-4-one Yield 44%; δ H [(CD,)SO] 0.9 (3H, t, J = 7.3 Hz , CH,), 1.27-1.36 (4H, m, 2 x CH,), 1.40 (3H, d, J = 6.88 Hz, 3H), 2.31 (3H, s, CH,), 3.13 (2H, m, CH,), 4.54 (IH, m, CH), 5.05 (2H, m, CH,Ph), 6.56 (IH, t, J = 5.4 Hz, D,O exchangeable, ΝH), 7.30- 7.40 (5H, m, aromatic protons), 7.98 (IH, d, J = 7.14Hz, D,O exchangeable, ΝH), and 9.14 (IH, s, D,O exchangeable, ΝH). HRMS calcd for C^H^Ν.O.S 459.1702, found 459.1722. Example 17 (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyI]-5-methyl-6-(3-benzylureido)-4H- thieno[2,3-d][l,3]oxazin-4-one Yield 19%; δ H (CDC1,) 1.52 (3H, d, J = 6.87 Hz, CH,), 2.20 (3H, br. s, CH,), 4.45 (2H, br. s, CH,Ph), 4.65 (IH, , CH), 5.10 (2H, m, CH,Ph), 5.51-5.56 (2H, m, 2 x ΝH),.6.92-7.19 (IH, v. br. s, ΝH),and 7.26-7.43 (10H, m, aromatic protons). HRMS calcd for C M H,.N 4 O.S 493.1546, found 493.1544. Example 18 (S)-2-[N-(BenzyIoxycarbonyl)-l-aminoethyI]-5-methyl-6-(3-phenylureido)-4H- thieno[2,3-d][l,3]oxazin-4-one Yield 40%; «5„ [(CD,)SO] 1.41(3H, d, J = 6.87 Hz, CH,), 2.39 (3H, s, CH,), 4.56 (IH, m, CH), 5.05 (2H, m, CH,Ph), 7.03 (IH, t, J = 7.7 Hz, aromatic proton), 7.29-7.36 (7H, m, aromatic protons), 7.47 (2H, d, J = 7.7 Hz, aromatic protons), 8.01 (IH, d, J = 7.15 Hz, D,O exchangeable, ΝH), 9.02 ( IH, s, D,O exchangeable, ΝH), 9.40 (IH, s, D,O exchangeable,ΝH). HRMS calcd for C^H^N.O.S 478.1311, found 478.1316. Example 19 (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyI]-5-methy!-6-[3-(4- chlorophenyl)ureido]-4H-thieno[2,3-d][l,3]oxazin-4-one Yield 30%; δ Η [(CD,)SO] 1.42 (3H, d, J = 6.87Hz, CH,), 2.38 (3H, s, CH,), 4.56 (IH, m, CH), 5.06 (2H, m, CH,Ph), 7.17-7.48 (7H, m, aromatic protons), 7.51 (2H, d, J = 8.80Hz, aromatic protons), 8.01 (IH, d, J = 7.15Hz, ΝH), 9.15 (IH, s, ΝH), 9.45 (lH, s, ΝH). HRMS calcd for C,.H,.N.O-SCl 512.0922, found 512.0928. Example 20 (S)-2-[N-(BenzyloxycarbonyI)-l-aminoethyI]-5-methyI-6-[3-(4- methoxyphenyl)ureido]-4H-thieno[2,3-d][l,3]oxazin-4-one Yield 18%; δ Η f(CD,)SO] 1.42 (3H, d, J = 7.15 Hz), 2.38 (3H, s, CH,), 3.73 (3H, s, CH,O), 4.55 (IH, m, CH), 5.06 (2H, m, CH,Ph), 6.91 (2H, d, J = 9.07 Hz, aromatic protons), 7.17-7.41 (7H, m, aromatic protons), 8.00 (IH, d, J = 7.4 Hz, D,O exchangeable, ΝH), 8.84 (IH, s, D,O exchangeable, ΝH), 9.34 (IH, s, D 2 O exchangeable, ΝH). HRMS calcd for C 25 H M Ν.O 6 S 508,1417, found 508,1416. Example 21 (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-5-methyl-6-[3-(2- methoxycarbonylphenyl)ureido]-4H-thieno[2,3-d][l,3]oxazin-4-one Yield 14%; δ Η [(CD,)SO] 1.42 (3H, d, J = 7.15 Hz, CH,), 2.44 (3H, s, CH,), 3.90 (3H, s, CH,O), 4.55 (IH, m, CH), 5.06 (2H, m, CH,Ph), 7.15 (IH, dt, aromatic proton), 7.36 (5H, m, aromatic protons), 7.61 (IH, dt, aromatic proton), 7.96 (IH, dd, aromatic proton), 8.01 (IH, d, J = 7.7 Hz), D,O exchangeable, ΝH), 8.25 (IH, dd, aromatic proton), 10.18 (IH, s, D,O exchangeable, ΝH), 10.53 (IH, s, D,O exchangeable, ΝH). FABMS (positive ion ): 537 (MH * ), 559 (MΝa * ). Example 22 (S)-2-[N-(BenzyloxycarbonyI)-l-aminoethyl]-5-methyl-6-(3-acetyl,3- benzylureido)-4H-thieno[2,3-d][l,3]oxazin-4-one Yield 42%; δ„ [(CD,)SO] 1.41 (3Η, d, J = 7.15 Hz, CH,), 2.18 (3H, s, CH,CO), 2.43 (3H, s, CH,), 4.55 (IH, m, CH), 4.59 (2H, s, CH,Ph), 5.05 (2H, m, CH.Ph), 7.28-7.43 (10H, m, aromatic protons), 7.99 (IH, d, J = 5.91 Hz, ΝH), 10.68 (IH, s, ΝH). HRMS calcd for C^Ν.O.S 535.1652, found 535.1652. Example 23 (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-5-methyI-6-amino-4H- thieno[2,3-d][l,3]oxazin-4-one A solution of ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-methyl-5-[/V- (benzyloxycarbonyl)-3-aminobutyryl)]thiophene-3-carboxylate (prepared in a similar way to Example 10, step 1; (0.35 g, 0.56 mmol), triphenylphosphine (0.32 g 1.23 mmol) and carbon tetrachloride (0.3 ml, 3.1 mmol) in dichloromethane (10 ml) was heated under reflux at 60-70°C for 3 hr. A further amount of triphenylphosphine (0.16 g, 0.6 mmol) and carbon tetrachloride (0.15 ml, 1.5 mmol) was added and the reflux was continued for another 2 h. The reaction mixture was left standing at RT for 53 h, diluted with dichloromethane (80 ml), washed with saturated aqueous sodium hydrogen carbonate (2 x 15 ml), water (15ml) and dried (MgSO.). The solvent was removed and the residue was purified by column chromatography eluting with 6-9% ethyl acetate-dichloromethane to give the product (0.10 g, 50% yield): δn (DMSO-d 6 ) 1.38 (3H, ά, J = 6.88 Hz, CH,), 2.16 (3H, s, CH,), 4.51 (IH, m, CH), 5.06 (2H, m, CH,Ph), 5.95 (2H, s, D,O exchangeable, ΝH,), 7.31-7.41 (5H, m, aromatic protons), 7.93 (IH, d, J = 7.7 Hz, D,O exchangeable, NH). HRMS calcd for C 17 H. 7 N,O S 359.094, found 359.0941. Example 24 (step 1) Ethyl 2-[N-(benzyloxycarbonyI)-L-alaninyl]amino-4-methyI-5-(N,N- dimethylamino)thiophene-3-carboxylate To a stirred solution of ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-methyl- 5-aminothiophene-3-carboxylate (0.2 g, 0.5 mmol) and 37% aqueous formaldehyde (0.2 ml) in acetonitrile (5 ml) at RT was added sodium cyanoborohydride (50 mg, 0.8 mmol) in acetonitrile (2 ml). The reaction mixture was stirred for 15 min and then neutralized with glacial acetic acid. Stirring at RT was continued for another 45 min. The mixture was poured into diethyl ether (50 ml) and then washed with aqueous IN potassium hydroxide, (3 x 10 ml), brine (1 x 10 ml), and water (1 x 10 ml). The solvent was evaporated to give the product (0.2 g) which was used without purification in the next step; (DMSO-d 6 ) 1.31 (6H, m, 2 x CH,), 2.20 (3H, s, CH,), 2.61 (6H, s, (CH,),Ν), 4.27 (3H, m, CH and CH,), 5.07 (2H, m, CH,Ph), 7.33-7.39 (5H, m, aromatic protons), 8.12 (IH, v. br. d, D,O exchangeable, NH), 11.58 ( IH, br. s, D,O exchangeable, NH). HRMS calcd for C 21 H n N,O s S 434.1750, found 434.1758. Example 24 (step 2) (S)-2-[N-(BenzyIoxycarbonyI)-l-aminoethyl]-5-methyI-6-(N^-dimethylamino)- 4H-thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 2, step 2, yield 36%; (DMSO-d 6 ) 1.39 (3Η, d, J = 7.15 Hz, CH,), 2.28 (3H, s, CH,), 2.69 (6H, s, (CH,),Ν), 4.50 (IH, m, CH), 5.07 (2H, m, CH,Ph), 7.10-7.39 (5H, m, aromatic protons), 8.00 (IH, br.d, D,O exchangeable, NH). HRMS calcd for C.-H^O.S 387.1253, found 387.1253. Example 25 (S)-2-[N-(Benzyloxycarbonyl)-l-amiπoethyI]-5-methyl-6-(N,N-dipropyIamino)- 4H-thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 24, yield 50%; C-H (DMSO-d 6 ) 0.86 (3H, t, / = 7.15 Hz, 2 x CH,), 1.40 (7H, m, 2 x CH, and CH,), 2.30 (3H, s, CH,), 2.86 (3H, t, J = 7.15 Hz, 2 x CH,), 4.52 (IH, m, CH), 5.07 (2H, m, CH,Ph), 7.11-7.41 (5H, m, aromatic protons), 8.03 (IH, ά, J = 7.42 Hz, D,O exchangeable, NH). HRMS calcd for C^H^N j O.S 443.1879, found 443.1881. Example 26 (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-5-methyI-6-(N-benzyl-N- methylamino)-4H-thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 24, yield 40%; Η ΝMR (DMSO-d 6 ) δ: 1.39 (3H, d, J = 6.87 Hz, CH,), 2.31 (3H, s, CH,), 2.70 (3H, s, CH,), 4.09 (2H, s, θy h), 4.51 (IH, m, CH), 5.04 (2H, , CH,Ph), 7.26-7.40 (10H, m, aromatic protons), 7.98 (IH, d, J = 7.42 Hz, D,O exchangeable, ΝH). HRMS calcd for C^H^Ν j O.S 464.1644, found 464.1661. Example 27 (step 1) Ethyl 2-[(N-benzyloxycarbonyl)-L-alaninyI]amino-4-methyl-5-bromothiophene- 3-car boxy late To a solution of ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4- methylthiophene-3-carboxylate (0.61 g, 1.56 mmol) in dioxane-water (18 ml, 5:1) at 55°C was added dropwise a solution of bromine (0.25 g) in dioxane (30 ml) over 2.5 hr. The solution was neutralized to pH 7.0 with Amberlite IR-45(OH), the resin was filtered off, and washed with dioxane (3 x 50 ml). The filtrate and washings were combined and evaporated to dryness to give the product, (0.71 g, 97% yield); δH(DMSO-d 6 ) 1.33 (6H, , 2 x CH,), 2.30 (3H, s, CH,), 4.31 (3H, m, CH, CH,), 5.09 (2H, m, CH,Ph), 7.35-7.39 (5H, m, aromatic protons), 8.15 (IH, d, J = 6.32 Hz, ΝH), 11.62 (IH, s, ΝH). Anal, calcd. for C.,H,.Ν,O 3 SBr C, 48.62; H, 4.51; N, 5.97. Found C, 49.23; H, 4.51; N, 5.88. HRMS calcd for C. 9 H,.N,O.SBr 468.0355, found 468.0357. Example 27 (step 2) (S)-2-[N-(BenzyloxycarbonyI)-l-aminoethyl]-5-methyl-6-bromo-4H-thieno[2,3- d][l,3]oxazin-4-one Prepared in a similar way to Example 2, step 2, yield 47%; δj-i (DMSO-d 6 ) 1.42 (3H, d, J = 7.15 Hz, CH,), 2.38 (3H, s, CH,), 4.56 (IH, m, CH), 5.05 (2H, m, CH,Ph), 7.14-7.41 (5H, m, aromatic protons), 8.04 (IH, d, / = 7.42 Hz, D,O exchangeable, ΝH). Anal, calcd. for C n H.aN,O 4 SBr C, 48.24; H, 3.57; N, 6.62. Found C, 48.59; H, 3.57; N, 6.23. Example 28 (step 1) Preparation of ethyl -2-[(N-benzyloxycarbonyl-L-alaninyI)amino]-4-methyl-5- phenyl thiophene-3-car boxy late. A mixture of ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-methyl-5- bromothiophene-3-carboxylate (0.150g, 0.320g), phenylboronic acid (70 mg, 0.57 mmol) and bis(triphenylphosphine)palladium chloride (10 mg) in tetrahydrofuran-2M sodium carbonate (2 ml-0.3 ml) was stirred for 2 h 15 min. The mixture was diluted with dichloromethane (50 ml), washed with saturated aqueous sodium chloride (20 ml), and dried (MgSO 4 ). The solvent was removed and the product was purified by column chromatography on silica gel (eluting with ethyl acetate-dichloromethane (2:98), (74.5mg, 50% yield); 6H (DMSO-d 6 ) 1.34 (6H, m, 2 x CH,), 2.34 (3H, s, CH,), 4.43 (3H, m, CH, and CH), 5.10 (2H, m, CH,Ph), 7.30-7.51 (10H, m, aromatic protons), 8.17 (IH, d, J = 5.22 Hz, D,O exchangeable, ΝH), 11.63 (IH, s, D,O exchangeable, ΝH). HRMS calcd for C^Ν^S 466.1563, found 466.1565. Example 28 (step 2) (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyI]-5-methyl-6-phenyI-4H- thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 2, step 2, yield 43%; δμ (DMSO-d 6 ) 1.47 (3H, d, J = 7.06 Hz, CH,), 2.49 (3H, s, CH,), 4.58 (IH, m, CH), 5.07 (2H, , CH,Ph), 7.13-7.39 (5H, m, aromatic protons), 7.46-7.55 (5H, m, aromatic protons), 8.04 (IH, d, J = 6.61 Hz, D.O exchangeable, ΝH). HRMS calcd for C^H^O.S 420.1 144, found 420.1148. Example 29 (step 1) Ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-methyI-5-iodothiophene-3- carboxylate Ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-methylthiophene-3-carboxylate was iodinated by an adaption of the method of Y. Kobayashi, I. Kumadaki and T. Yoshida [J Chem Res (S), 1977, 215], mp 104-105°C; δ H (CDC1,) 1.35 (3H, t, J = 7Hz, CH,CH 3 ), 1-5 (3H, d, J = 7Hz, CH,CH,), 2.35 (3H, s, CH,), 4.3 (2H, q, J = 7Hz, CH,CH,), 4.5 (IH, m, CH,CH),), 5.15 (2H, s, CH,Ph), 5.5 (IH, br, ΝH), and 7.3 (5H, br, C^), 11.8 (IH, br, ΝH) (Found: C, 44.35; H, 3.87; Ν, 5.21%. C.,H,.Ν,O.SI requires C, 44.20; H, 4.10; N, 5.43%.) Example 29 (step 2) (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-5-methyl-6-iodo-4H- thieno[2,3-d][l,3]oxazin-4-one Ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-methyl-5-iodothiophene-3- carboxylate was cyclised in a similar way to Example 2, step 2. Mp 142-144°C; 6H(CDC1,) 1.55 (3H, d, J = 7Hz, CH 3 CH), 2.5 (3H, s, CH,), 4.75 (IH, m, CH,CH), 5.1 (2H, s, CH,Ph), 5.45 (IH, br, ΝH) and 7.35 (5H, br s, C 6 H 5 ) (Found: M * 469.9797. C I7 H 13 Ν,O 4 SI requires 469.9798.) Example 30 (step 1) Methyl 2-amino-4-ethyl-5-methylthiophene-3-carboxylate Prepared by the method of K. Gewald, E. Schinke and H. Boucher [Chem. Berichte, 1966, 99, 94-100] Example 30 (step 2) Methyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-ethyl-5-methyIthiophene- 3-carboxylate To a solution of N-(benzyloxycarbonyl)-L-alanine (1.12 g, 5 mmol) in dry DMF (10 ml.) at 0 to -5 a was added N-methylmorpholine (0.5 ml, 5 mmol) followed by isobutyl chloroformate (0.65 ml, 5 mmol) dropwise with stirring. The reaction mixture was then stirred for 15 min. Methyl 2-amino-4-ethyl-5-methylthiophene-3- carboxylate (1.0 g, 5 mmol) was then added at 0 to -5 2 and the reaction was allowed to come to RT and stirred for 18 h. The solvent was evaporated under reduced pressure, and the residue was partitioned between ethyl acetate (50 ml) and water (3 x 20 ml) and the organic layer was dried (MgSO4), filtered and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with 20% diethyl ether/hexane, followed by crystallisation from acetone/hexane to give a colourless product (1.1 g, 55%). Example 30 (step 3) (5)-2-{N-(Benzyloxycarbonyl)-l-aminoethyl]-5-ethyl-6-methyl-4H- thieno[2,3-d][l,3]oxazin-4-one Methyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-ethyl-5-methylthiophene-3- carboxylate was cyclised in a similar way to Example 2, step 2. Mp 90-2°C; OH(DMSO-d 6 ) 1.08 (3H, t, J = 7.4Hz, CH 3 CH,), 1.42 (3H, d, J = 7.2Hz, CH,CH), 2.42 (3H, S, CH,), 2.79 (2H, q, J = 7.4Hz, CH j CH,), 4.55 (IH, quin, J = 7.2Hz, CH 3 CHNH), 5.06 (2H, m, CH^h), 7.25-7.40 (5H, m, C 6 H 5 ), and 8.01 (IH, d, J = 7.4Hz, NH) (Found: C, 61.53; H, 5.52; N, 7.45%. C.^NAS requires C, 61.27; H, 5.41; N, 7.52%.) Example 31 (5)-2-[N-(BenzyIoxycarbonyl)-l-aminoethyl]-5-isopropyl-4H-thieno[2,3-d][l,3]- oxazin-4-one Prepared in a similar way to Example 30. Mp 139-41 °C; δ H (DMSO-d 6 ) 1.24 (6H, d, J = 6.9Hz, (CH,),CH), 1.43 (3H, d, J = 7.2Hz, CH,CH), 3.41 (IH, sept, J = 6.6Hz, (CH,),CH), 4.57 (IH, quin, J = 7.2Hz, CH,CHNH), 5.00-5.20 (2H, m, CH,Ph), 7.25- 7.50 (6H, m, C 6 H 5 and 6-H), and 8.03 (IH, d, J = 7.1Hz, NH) (Found: C, 61.19; H, 5.29; N, 6.99%. C^H^N^S requires C, 61.27; H, 5.41; N, 7.52%.) Example 32 (S)-2-[N-(Benzyloxycarbonyl)-l-aminoethyl]-5-isobutyl-4H-thieno[2,3-d][l,3]- oxazin-4-one Prepared in a similar way to Example 30. Mp 137-9°C; δ Η (DMSO-d 6 ) 0.88 (6H, J = 6.6Hz, (CH,),CH), 1.43 (3H, d, J = 7.2Hz, CH,CH), 1.90 (IH, sept, J = 6.6Hz, (CH,),CH), 2.70 (2H, d, J = 6.2Hz, CH,CH), 4.56 (IH, quin, J = 7.2Hz, CH,CHΝH), 5.00-5.15 (2H, m, CH,Ph), 7.25-7.50 (6H, m, C 6 H 5 and 6-H), and 8.02 (IH, d, J = 7.15Hz, NH) (Found: C, 62.08; H, 5.82; N, 7.35%. C-^N .S requires C, 62.16; H, 5.74; N, 7.25%.) Example 33 (S)-2-[N-(BenzyloxycarbonyI)-l-aminoethyI]-5-cyclopropyl-4H- thieno[2,3-d][l,3]oxazin-4-one Prepared in a similar way to Example 30. Mp 138-40°C; δ H (DMSO-d 6 ) 0.70 (2H,_m, CH,), 0.95 (2H, m, CH,), 1.43 (3H, d, J = 7.1Hz, CH,CH), 2.41 (IH, m, CH), 4.56 (IH, quin, J = 6.0Hz, CH,CHΝH), 5.06 (2H, d, J = 2.5Hz, CH,Ph), 7.12 (IH, s, 6-H), 7.35 (5H, m, C 6 H 5 ), and 8.03 (IH, d, J = 7.2Hz, NH) (Found: C, 61.46; H, 4.86; N, 7.50%. C.,H.,N,O 4 S requires C, 61.61; H, 4.90; N, 7.56%.) Example 34 (step 1) Ethyl 2-amino-4-methoxythiophene-3-carboxylate A solution of ethyl 2-amino-4-hydroxythiophene-3-carboxylate (750 mg, 4.0 mmol) in dry dichloromethane was treated with trimethyloxonium tetrafluoroborate (882 mg, 6.0 mmol) and stirred for 1 h at RT. The solvent was then evaporated under reduced pressure and the residue was purified by column chromatography on silica gel, eluting with 2% methanol/dichloromethane, followed by crystallisation from acetone to give the product as yellow crystals, mp 103-5°C; δ H (DMSO-d 6 ) 1.22 (3h, t, J = 7.1Hz, CH,CH,), 3.64 (3H, s, OCH,), 4.15 (2H, q, J = 6.8Hz, CH^CH,), 5.29 (IH, s, 5-H), 7.34 (2H, s, NH,) ( Found: C, 47.43; H, 5.42; N,6 .90%. C,H π NO,S requires C, 47.75; H, 5.51; N, 6.96%.) Example 34 (step 2) Ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-methoxythiophene-3- carboxylate Ethyl 2-amino-4-hydroxythiophene-3-carboxylate was acylated in a similar way to Example 30, step 2. Example 34 (step 3) (S)-2-[N-(BenzyloxycarbonyI)-l-aminoethyI]-5-methoxy-4H-thieno[2,3-d][l,3]- oxazin-4-one Ethyl 2-[N-(benzyloxycarbonyl)-L-alaninyl]amino-4-methoxythiophene-3- carboxylate was cyclised in a similar way to Example 2, step 2. Mp 173-5°C; δ-f- j (DMSO-d 6 ) 1.42 (3Η, d, J = 7.2Hz, CH 3 CH), 3.85 (3H, s, OCH,), (IH, m, CH,CHΝH), 5.05 (2H, m, CH,Ph), 6.72 (IH, s, 6-H), 7.25-7.50 (5H, m, C 6 H 5 ), and 8.04 (IH, d, J = 7.4Hz, NH) (Found: C, 56.67; H, 4.32; N, 7.69%. C I7 H 16 N,O 3 S requires C, 56.66; H, 4.48; N, 7.77%.) Example 35 (S)-2-[N-(Benzyloxycarbonyl)aminomethyl]-5-methyl-4H-thieno[2,3-d][l,3]- oxazin-4-one A solution of N-(benzyloxycarbonyl)glycine (209 mg, 1 mmol) in dry DMF (10 ml) at 0 to -5° was treated with 1-hydroxybenzotriazole (148 mg, 1.1 mmol) and l-(3-dimethylaminopiOpyl)-3-ethylcarbodiimide hydrochloride (210 mg, 1.1 mmol) and stirred for 15 min. 2-Amino-4-methylthiophene-3-carboxylic acid (157 mg, 1 mmol) was then added and the reaction was allowed to come to RT and stirred for 18 h. A further portion of DEC (210 mg, 1.1 mmol) was then added and the reaction mixture was stirred at RT for 18 h. The solvent was then evaporated under reduced pressure, and the residue was partitioned between ethyl acetate (50 ml) and water (3 x 20 ml) and the organic layer was dried (MgSO4), filtered and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel eluting with 50% ethyl acetate/hexane, followed by crystallisation from acetone/hexane to give the product as colourless needles (130 mg, 39%), mp 120-122°C; v%. (DMSO-d 6 ) 2.42 (3H, d, J = 1.1Hz, CH,), 4.22 (2H, d, J = 6.05Hz, CH^NH), 5.08 (2H, s, CH,Ph), 7.25-7.50 (6H, m, C 6 H 3 and 6-H), 7.97 (IH, t, J = 6.1Hz, NH) (Found: C, 58.19; H, 4.26; N, 8.50%. C l6 H 14 N,O 4 S requires C, 58.17; H, 4.27; N, 8.48%.) Example 36 (S)-2-[N-(BenzyIoxycarbonyl)-l-aminopropyI]-5-methyl-4H-thieno[2,3-d][l,3]- oxazin-4-one Prepared in a similar way to Example 35. Mp 129-130°C; δ Η (DMSO-d 6 ) 0.94.(3H, t, J = 7.4Hz, CH,CH,), 1.70-1.90 (2H, m, CH,CH,), 2.42 (3H, s, CH,), 4.35 (IH, m, CHCH,), 5.04 (2H, m, CH 2 Ph), 7.25-7.50 (5H, m, C 6 H 3 ), 7.32 (IH, s, 6-H), 8.00 (IH, d, J = 7.7Hz, ΝH) (Found: C, 60.15; H, 5.13; Ν, 7.68%. C 18 H lg Ν,O 4 S requires C, 60.32; H, 5.06; N, 7.82%.) Example 37 (step 1) (S)-2-[N-(Benzyloxycarbonyl)-l-amino-2-(t-butoxy)ethyl]-4,5-dimethyl-4H- thieno[2,3-d][l,3]oxazin-4-one was prepared in a similar way to Example 30, step 2. Example 37 (step 2) (S)-2-[N-(BenzyIoxycarbonyl)-l-amino-2-hydroxyethyl]-4,5-dimethyl-4H- thieno[2,3-d][l,3]oxazin-4-one A solution of (5)-2-[/V-(benzyloxycarbonyl)-l-amino-2-(t-butoxy)ethyl]-4,5- dimethyl-4H-thieno[2,3-d][l,3]oxazin-4-one (300 mg, 0.69 mmol) in dry dichloromethane (5 ml) was treated with trifluoroacetic acid (1 ml) and allowed to stand at RT for 18 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 30% ethyl acetate/hexane to give the product as a pale gum; δ^ (DMSO-d 6 ) 2.33 (3H, s, CH,), 2.40 (3H, s, CH,), 3.75 (2H, m, CH,), 4.50 (IH, m, CH), 5.05 (3H, m, CH^Ph and OH), 7.25-7.50 (5H, m, C β H 3 ), and 7.85 (IH, d, J = 7.7Hz, NH). Example 38 (S)-2-[N-(t-Butoxycarbonyl)-l-aminoethyl]-5-methyl-4H-thieno[2,3-d][l^]- oxazin-4-one Prepared in a similar way to Example 35. Mp 109-111°C; δ^ (DMSO-d 6 ) 1.38.(12H, m, (CH,),C + CH,CH), 2.42 (3H, s, CH,), 4.46 (IH, m, CHCH,), 7.30 (IH, s, 6-H), and 7.54 (IH, d, J = 7.4Hz, ΝH) (Found: C, 53.81; H, 5.74; Ν, 8.90%. C 1 H„Ν,O 4 S requires C, 54.18; H, 5.85; N, 9.03%.) Example 39 (S)-2-(l-Aminoethyl)-5-methyI-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-d 6 ) 1.54 (3H, d, J = 6.9Hz, CH 3 CH), 2.45 (3H, d, J = 1.4Hz, CH,), 4.52 (IH, m, CHCH,), 7.42 (IH, d, J = 1.1Hz, 6-H), and 8.60-8.80 (3H, v. broad, ΝH, * ); FABMS (+ve ion) 211 (MH * ). Example 40 (S)-2-[N-(4-methoxyphenoxycarbonyI)-l-aminoethyI]-5-methyl-4H- thieno[2,3-d] [l,3]oxazin-4-one A solution of (S)-2-(l-aminoethyl)-5-methyl-4H-thieno[2,3-d][l,3]oxazin-4-one, trifluoroacetic acid salt (100 mg, 0.37 mmol) in dry dichloromethane was treated with NN-diisopropylethylamine (0.065 ml, 0.37 mmol) and 4-methoxyphenyl chloroformate (0.56 ml, 0.37 mmol) and stirred at RT for 2 h. The solvent was then evaporated under reduced pressure and the residue was purified by column chromatography on silica gel, eluting with 20% ethyl acetate/hexane, followed by crystallisation from acetone/hexane to give the product as colourless crystals, mp. 173-5°C; δπ (DMSO-d 6 ) 1.49 (3H, d, J = 6.9Hz, CH,CH), 2.43 (3H, d, J = 0.8Hz, CH,), 3.73(3H, s, OCH,), 4.61 (IH, m, CHCH,), 6.90 (2H, d, J = 9Hz, 2 x ArH), 7.02 (2H, d, J = 9.1Hz, 2 x ArH), 7.33 (IH, d, J = 1.1Hz, 6-H), and 8.42 (IH, d, J = 7.2Hz, NH) (Found: C, 56.75; H, 4.25; N, 7.45%. C I7 H 16 N,O 3 S requires C, 56.66; H, 4.48; N, 7.77%.) The following compound of the invention is also prepared: The compound wherein R3 is methyl, the 6-substituent R2 is Cbz-Ala-NH- and the 2-substituent is CHMeNHCbz. Peptidolytic Assay for Proteases Compounds are assayed in the following way: A solution of inhibitor is added to a solution of enzyme (0.5 - 1 uM) diluted in buffer (50 mM sodium phosphate, 150 mM sodium chloride, 0.001 mM EDTA, 0.01% PEG 3400; pH 8.0) to give a final inhibitor concentration of 0.01 to 300 uM in a total volume of 25 microliters. Following a 15 minute preincubation, 15 microliters of a solution of the 14-mer peptide substrate, Ac-HTYLQASEKFKMWG, is added to give a final substrate concentration of 250 uM. The sample is incubated at 27°C for 1 hour and reaction is by the addition of 40 microliters of 5% trifluoroacetic acid in water. The sample is analyzed by HPLC to quantitate the amounts of the substrate peptide and of the N-terminal and C-terminal cleavage fragments. The percentage cleavage is calculated and expressed as a fraction of the value obtained for an uninhibited control sample. 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) with the addition of an amino terminal MGHHHHHHSSA. 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 the addition of an amino terminal MGHHHHHHHHSSGHIDDDDK. The polypeptide has cleaved beween A143 and A144 (natural UL 80 numbering) to give an active heterodimer. 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). The results were as follows: The compounds of the Examples showed activity in one or more of the above tests at a concentration of lOuM or luM. The compounds of Examples 1-4, 7-10, 12-18, 21-22, 24-29 and 35 afforded > 50% inhibition of one or more of the proteases at a concentration of 1 uM, the Compounds of Examples 10, 17, 18, 21, 27; 2, 3, 15, 21, 28; and 8, 15, 21, 28; showing preferred activity for HSV-2; CMV; and VZV; respectively.

Claims: Claims 1. Herpesvirus protease inhibitor 4H-thieno[2,3-d][l,3]oxazin-4-one derivatives of formula (I) which are 2-substituted by X(Y)NΗ-CΗRι- wherein X is hydrogen or ' < a blocking group, Y is an optionally present amino acid, and Rj is hydrogen or an amino acid side chain. 2. A compound according to claim 1 wherein the 4H-thieno[2,3-d][l,3]oxazin-4- one 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\. alkyl or C\. alkoxy, Cj.g alkylthio, amino optionally substituted by one or two C\. alkyl or optionally substituted benzyl groups, hydroxyalkyl, alkylcarbonyl, alkoxycarbonyl, optionally substituted phenyl or R4ZCONΗ wherein Z is a bond, O, NH or NCOCH3, and R4CO is an acyl group where values of R4 include aryl, alkyl or aralkyl, or R3 and RT may be joined to form C2, C3, C4, C5, C6, Cη, or Cg polymethylene. 4. A compound according to any one of claims 1 to 3 claim 1 wherein X is selected from aryl, alkanoyl, (aryl)alkoxycarbonyl, (aryl)alkylaminocarbonyl, aroyl, or arylsulphonyl. 5. A compound according to claim 4 wherein X is selected from benzyloxycarbonyl, t-butoxycarbonyl and optionally substituted phenyl. 6. A compound according to any one of claims 1 to 5 wherein Y when present is alanine, asparagine, valine or a similar amino acid. 7. A compound according to any one of claims 1 to 6 wherein the aminoacyl moieties -NH-CHRi-CO- , resulting in NH-CHRj- in the compound of formula (I), include alanine (Rj = CH3), asparagine, methionine, phenylalanine, serine (R j = CH2OH), valine, and α-aminobutyric (Rj = CH2CH3). 8. A compound selected from the compounds of Examples 1 to 40, as described herein. 9. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier. 8. A compound according to any one of claims 1 to 8 for use as an active therapeutic substance. 9. A compound according to any one of claims 1 to 8 for use in treating viral diseases. 10. Use of a compound according to any one of claims 1 to 8 in the manufacture of a medicament for use in the treatment of viral diseases caused by herpesviruses. 11. 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 6.

Inventors: JARVEST RICHARD LEWIS, PARRATT MARTIN JOHN, SERAFINOWSKA HALINA TERESA, JARVEST, RICHARD, LEWIS, PARRATT, MARTIN, JOHN, SERAFINOWSKA, HALINA, TERESA
Applicants: JARVEST RICHARD LEWIS, PARRATT MARTIN JOHN, SERAFINOWSKA HALINA TERESA, SMITHKLINE BEECHAM PLC, JARVEST, RICHARD, LEWIS, PARRATT, MARTIN, JOHN, SERAFINOWSKA, HALINA, TERESA
Application year: 1995
Main classification: C07D498
IPC codes: A61K31, A61P31, C07D498