Antiviral benzodiazepine compounds

Abstract

The invention relates to compounds of formula (I), and pharmaceutical uses thereof. Particular aspects of the invention relate to methods of synthesising the compounds and the use of those compounds in treating, ameliorating, or preventing a microbial infection.

Claims

1. A compound of formula (I): ##STR00137## wherein R.sub.1 is H; R.sub.2 is H; R.sub.3 is a side chain of a naturally occurring amino acid; and R.sub.4 is H; or R.sub.3 and R.sub.4, together with the atoms to which they are attached, combine to form a five-membered heterocyclic ring; R.sub.5 is an aromatic six membered ring optionally substituted with one or more substituents, wherein the or each substituent is independently a C.sub.1-5 straight or branched alkyl or alkenyl, a halogen, SR.sub.6, SO.sub.2R.sub.6, OR.sub.6, or NR.sub.6R.sub.7; m is 0, 1, 2, 3 or 4; n is 0, 1, 2, 3, 4 or 5; the or each X.sub.1 is independently selected from a C.sub.1-5 straight or branched alkyl or alkenyl, a halogen, SR.sub.6, SO.sub.2R.sub.6, OR.sub.6 or NR.sub.6R.sub.7; the or each X2 is independently selected from a C1-5 straight or branched alkyl or alkenyl, chlorine, bromine, SR.sub.6, SO.sub.2R.sub.6, OR.sub.6 or NR.sub.6R.sub.7; the or each R.sub.6 is independently selected from the group consisting of: hydrogen, a C.sub.1-5 straight or branched alkyl or alkenyl, C.sub.3-6 cycloalkyl or cycloalkenyl, C.sub.3-6 heterocyclyl or heteroaryl, C.sub.2-4 methane sulphonyl alkyl, C.sub.2-4 dialkylaminoalkyl and ##STR00138## the or each R.sub.7 is independently selected from the group consisting of: hydrogen, a C.sub.1-5 straight or branched alkyl or alkenyl, C.sub.3-6 cycloalkyl or cycloalkenyl, C.sub.3-6 heterocyclyl or heteroaryl, C.sub.2-4 methane sulphonyl alkyl, C.sub.2-4 dialkylaminoalkyl and ##STR00139## and/or R.sub.6 and R.sub.7 together with the nitrogen atom to which they are attached independently form a 3-7 membered ring which is optionally substituted with one or more substituents, wherein the or each substituent is independently a C.sub.1-5 straight or branched alkyl or alkenyl, a halogen, C(O)R.sub.10, SR.sub.10, SO.sub.2R.sub.10, OR.sub.10 or NR.sub.10R.sub.11; the or each R.sub.10 is independently selected from the group consisting of: hydrogen, a C.sub.1-5 straight or branched alkyl or alkenyl, C.sub.3-6 cycloalkyl or cycloalkenyl, C.sub.3-6 heterocyclyl or heteroaryl, C.sub.2-4 methane sulphonyl alkyl, C.sub.2-4 dialkylaminoalkyl and ##STR00140## the or each R.sub.11 is independently selected from the group consisting of: hydrogen, a C.sub.1-5 straight or branched alkyl or alkenyl, C.sub.3-6 cycloalkyl or cycloalkenyl, C.sub.3-6 heterocyclyl or heteroaryl, C.sub.2-4 methane sulphonyl alkyl, C.sub.2-4 dialkylaminoalkyl and ##STR00141## p is 1, 2, 3, 4 or 5; and R.sub.8 and R.sub.9 together with the nitrogen atom to which they are attached independently form a 3-7 membered ring; or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof.

2. The compound according to claim 1, wherein m is 0 and n is 0.

3. The compound according to claim 1, wherein R.sub.3 is not a cysteine side chain and/or R.sub.3 is hydrogen, methyl, isopropyl or benzyl.

4. The compound according to claim 1, wherein R.sub.5 is selected from the group consisting of: ##STR00142## wherein: q is 0, 1, 2, 3 or 4; r is 0, 1, 2 or 3; s is 0, 1, 2, 3, 4 or 5; and X.sub.3 is independently a Cis straight or branched alkyl or alkenyl, a halogen, SR.sub.6, SO.sub.2R.sub.6, OR.sub.6, or NR.sub.6R.sub.7.

5. The compound according to claim 4, wherein R.sub.5 is selected from the group consisting of: ##STR00143##

6. The compound according to claim 1, wherein R.sub.5 is a phenyl group.

7. The compound according to claim 4, wherein the compound has Formula (Ij): ##STR00144##

8. The compound according to claim 7, wherein at least one X.sub.3 group is a halogen.

9. The compound according to claim 8, wherein three X.sub.3 groups are fluorine and one X.sub.3 group is selected from fluorine, OEt, SO.sub.2Me and NR.sub.6R.sub.7, wherein R.sub.6 and R.sub.7 are each ethyl or R.sub.6 and R.sub.7 together with the nitrogen atom to which they are attached independently form a 3-7 membered ring wherein the or each substituent is independently a C1-5 straight or branched alkyl or alkenyl, a halogen, C(O)R.sub.10, SR.sub.10, SO.sub.2R.sub.10, OR.sub.10 or NR.sub.10R.sub.11.

10. The compound according to claim 4, wherein the compound has Formula (IIh): ##STR00145##

11. The compound according to claim 1, wherein the compound has Formula (Im): ##STR00146##

12. The compound according to claim 1, selected from the group consisting of: ##STR00147## ##STR00148## ##STR00149##

13. A method of treating or ameliorating a microbial infection, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of a compound of formula (I), as defined by claim 1, or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof.

14. The method according to claim 13, wherein the microbial infection comprises a viral infection.

15. A pharmaceutical composition comprising a compound in accordance with claim 1, or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof, and a pharmaceutically acceptable vehicle.

16. A method of manufacturing a compound in accordance with claim 1, the method comprising contacting a compound of formula (II), or a salt or solvate thereof: ##STR00150## with a compound of formula (III), or a salt or solvate thereof: ##STR00151## wherein R.sub.1 is H; R.sub.2 is H; R.sub.3 is a side chain of a naturally occurring amino acid; and R.sub.4 is H; or R.sub.3 and R.sub.4, together with the atoms to which they are attached, combine to form a five-membered heterocyclic ring; R.sub.5 is an aromatic six membered ring optionally substituted with one or more substituents, wherein the or each substituent is independently a C.sub.1-5 straight or branched alkyl or alkenyl, a halogen, SR.sub.6, SO.sub.2R.sub.6, OR.sub.6, or NR.sub.6R.sub.7; m is 0, 1, 2, 3 or 4; n is 0, 1, 2, 3, 4 or 5; the or each X.sub.1 is independently selected from a C.sub.1-5 straight or branched alkyl or alkenyl, a halogen, SR.sub.6, SO.sub.2R.sub.6, OR.sub.6 or NR.sub.6R.sub.7; the or each X.sub.2 is independently selected from a C1-5 straight or branched alkyl or alkenyl, chlorine, bromine, SR.sub.6, SO.sub.2R.sub.6, OR.sub.6 or NR.sub.6R.sub.7; the or each R.sub.6 is independently selected from the group consisting of: hydrogen, a C.sub.1-5 straight or branched alkyl or alkenyl, C.sub.3-6 cycloalkyl or cycloalkenyl, C.sub.3-6 heterocyclyl or heteroaryl, C.sub.2-4 methane sulphonyl alkyl, C.sub.2-4 dialkylaminoalkyl and ##STR00152## the or each R.sub.7 is independently selected from the group consisting of: hydrogen, a C.sub.1-5 straight or branched alkyl or alkenyl, C.sub.3-6 cycloalkyl or cycloalkenyl, C.sub.3-6 heterocyclyl or heteroaryl, C.sub.2-4 methane sulphonyl alkyl, C.sub.2-4 dialkylaminoalkyl and ##STR00153## and/or R.sub.6 and R.sub.7 together with the nitrogen atom to which they are attached independently form a 3-7 membered ring; p is 1, 2, 3, 4 or 5; R.sub.8 and R.sub.9 together with the nitrogen atom to which they are attached independently form a 3-7 membered ring; and R.sub.12 is H or a C.sub.1-5 straight or branched alkyl or alkenyl.

17. The method according to claim 16, wherein R.sub.12 is hydrogen.

18. The compound of claim 8, wherein and the or each halogen is a fluorine.

19. The compound of claim 10, wherein X.sub.3 is a halogen or SO.sub.2R.sub.6.

20. The compound of claim 10, wherein the or each X.sub.2 is independently a Cis straight or branched alkyl or alkenyl, SR.sub.6, SO.sub.2R.sub.6, OR.sub.6 or NR.sub.6R.sub.7.

21. The method according to claim 14, wherein the viral infection is Respiratory Syncytial Virus (RSV).

Description

(1) All features described herein (including any accompanying drawings, claims and abstract), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

(2) FIG. 1 shows the structure of N-{(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl](tetrafluoropyridin-4-ylamino) acetamide.

EXAMPLES

Example 1: Synthesis of Compounds

(3) Melting points were determined in open capillaries, using a Stuart SMP30 digital melting point apparatus and are uncorrected. NMR spectra were recorded on a Bruker Avance-III-400 (1H=400.06 MHz; 19F=376.4 MHz; 13C=100.6 MHz) at ambient probe temperature (nominal 295K) using either deuterated chloroform (CDCl.sub.3) or hexadeuterated dimethylsulphoxide (DMSO-d6) as solvents. Chemical shifts (δ) are given in ppm vs. TMS (.sup.1H NMR, .sup.13C NMR) as an internal reference. Coupling constants are given in Hertz (Hz). LC ES MS (positive ion) was performed on a QToF Premier mass spectrometer equipped with an Acquity UPLC (Waters Corp.). The LC separation was achieved on a C18 BEH chromatography column (2.1 mm×100 mm and 1.7 um particle size) using a reverse phase gradient of 100% aqueous (0.1% formic acid in water) to 100% organic (0.1% formic acid in acetonitrile) at 0.6 mL/min. Silica gel plates, Supelco. S-A (Fluorescence Indicator at 254 nM) (Sigma-Aldrich Chemie GmbH Riedstr. 2D-8955T, Steinheim 497329-970, Germany) were used for TLC testing. Column chromatography was performed using silica gel (70-230 mesh) from Sigma-Aldrich (The Old Brickyard, Gillingham, SP8 4JL. UK). Reagents were also obtained from Sigma-Aldrich and used without further purification.

Intermediate 1: Methyl 2-[[tetrafluoropyridin-4-yl)amino]acetate

(4) ##STR00059##

(5) Pentafluoropyridine (0.4 ml) in DMF (2 ml) was treated with triethylamine (0.5 ml) and glycine methyl ester hydrochloride (250 mg) in a sealed vessel at room temperature and stirred there for 30 minutes. The mixture was partitioned between water and EtOAc. The layers were separated and the EtOAc layer washed with water (5×), a portion of brine, dried over sodium sulphate, decanted and concentrated in vacuo. The resulting solid was triturated with hexane to provide the title compound as a white solid. Yield: 0.221 g (87%); m.pt. 67-69° C.; .sup.1H NMR (400 MHz, CDCl.sub.3): =5.22 (bs, 1H, NH), 4.32 (bt, 2H, CH.sub.2), 3.90 (s, 3H, OCH.sub.3); .sup.19F (376 MHz, CDCl.sub.3)−93.61 (m, 2F), −163.97 (m, 2F).

Intermediate 2: 2-[(tetrafluoropyridin-4-yl)amino]acetic acid

(6) ##STR00060##

(7) Methyl 2-[(tetrafluoropyridin-4-yl)amino]acetate (intermediate 1) (119 mg) in THF (9 ml) was treated with lithium hydroxide (164 mg) in water (9 ml) at room temperature. After 18 hours, 9 ml of 3M HCl was added followed by salt to saturation. This mixture was extracted with ethyl acetate (3 times), the combined extracts were dried over sodium sulphate before being decanted and concentrated in vacuo.

Compound 1: N-{(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl](phenylamino)acetamide

(8) ##STR00061##

(9) (3S)-2-Oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-ylamine (126 mg) was combined with triethylamine (190 ul), HBTU (190 mg), N-phenylglycine (76 mg) in DMF (2.5 ml) and stirred at room temperature for 18 hours. Water (5 ml) was added and the resulting solid filtered. Chromatography on silica (200:8:1, DCM, ethanol, ammonia) provided the desired compound as a pale cream solid. Yield: 0.045 g (23%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ=10.92 (s, 1H, NH), 8.94 (d, 1H, C(═O)NH), 7.66 (m, 1H, ArH), 7.54-7.43 (m, 5H, ArH), 7.10 (m, 2H, ArH), 6.60 (d, 2H, ArH), 6.50 (m, 1H, ArH), 6.06 (t, 1H, CH.sub.2NHAr), 5.26 (d, 1H, C═NCH(C═O)N), 3.84 (2H, m, CH.sub.2C═O)). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=171.2, 168.1, 167.3, 148.8, 139.1, 138.5, 132.6, 131.1, 130.9, 129.9, 129.3, 128.8, 126.6, 123.8, 122.0, 117.1, 113.0, 56.5, 47-3);

Compound 2: N-{(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl](tetrafluoropyridin-4-ylamino) acetamide

(10) ##STR00062##

(11) The above compound was prepared in an analogous manner to compound 1, substituting N-phenylglycine with 2-[(tetrafluoropyridin-4-yl)amino]acetic acid (intermediate 2). Yield: 0.11 g (48%); .sup.1H NMR (400 MHz, CDCl.sub.3): δ=9.43 (s, 1H, CONH), 8.25 (d, 1H, CHNHC═O) 7.51 (m, 3H, ArH), 7.44 (m, 1H, ArH), 7.36 (m, 3H, ArH), 7.22 (m, 2H, ArH), 5.65 (d, 1H, C═NCH(C═O)N), 5.55 (m, 1H, CH.sub.2NHAr), 4.38 (2H, m, NHCH.sub.2C═O). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=168.9, 168.7, 168.5, 145.2, 142.8, 138.2, 137.3, 137.0, 132.5, 132.47, 131.5, 130.9, 130.0, 129.8, 128.3, 127.5, 124.4, 121.6, 67.6: .sup.19F (376 MHz, CDCl.sub.3)−94.07 (m, 1F), −163.77 (m, 1F).

Compound 3: N-{2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl](phenylamino) acetamide

(12) ##STR00063##

(13) The above compound was prepared in an analogous manner to compound 1, substituting N-phenylglycine with 2-[(tetrafluoropyridin-4-yl)amino]acetic acid (Intermediate 2) and (3S)-2-oxo-5-phenyl-2,3-dihydro-H-1,4-benzodiazepin-3-ylamine with the racemate thereof. .sup.1H NMR (400 MHz, CDCl.sub.3): δ=10.90 (s, 1H, CONH), 9.34 (d, 1H, CHNHC═O) 7.65 (d, 1H, ArH), 7.55 (m, 5H, ArH), 7.48 (m, 3H, ArH)), 5.25 (d, 1H, C═NCH(C═O)N), 4.25 (2H, m, NHCH.sub.2C═O). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=168.9, 168.7, 168.5, 145.2, 142.8, 138.2, 137.3, 137.0, 132.5, 132.47, 131.5, 130.9, 130.0, 129.8, 128.3, 127.5, 124.4, 121.6, 67.6:19F (376 MHz, CDCl.sub.3)−97.0 (m, 1F), −163.54 (m, 1F).

Compound 4: 2-[(2-ethoxy-3,5,6-trifluoropyridin-4-yl)amino]-N-{(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetamide

(14) ##STR00064##

(15) N-{(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl](tetrafluoropyridin-4-ylamino) acetamide (Compound 2) (100 mg) was dissolved in ethanol (2 ml) and treated with sodium ethoxide (135 ul) at room temperature. The mixture was heated to 76° C. After 12 hours at this temperature the mixture was cooled to room temperature and a further portion (135 ul) of sodium ethoxide was added. The mixture was heated to 76° C. for a further 18 hours. Dilute HCl (2N, 3 ml) was added and the mixture partitioned between ethyl acetate and water. The aqueous phase was extracted with ethyl acetate (3×50 ml) and combined extracts were dried over sodium sulphate before being decanted and concentrated in vacuo. Chromatography on silica (150:8:1; CH.sub.2Cl.sub.2:EtOH:NH.sub.4OH) gave isolation of the desired compound as a pale tan solid. Yield 0.04 g, (39%). .sup.1H NMR (400 MHz, CDCl.sub.3): δ=10.90 (s, 1H, CONH), 9.34 (d, 1H, CHNHC═O) 7.65 (d, 1H, ArH), 7.55 (m, 5H, ArH), 7.48 (m, 3H, ArH)), 5.25 (d, 1H, C═NCH(C═O)N), 4.25 (2H, m, NHCH.sub.2C═O). .sup.13C NMR (100 MHz, CDCl.sub.3): δ=168.9, 168.7, 168.5, 145.2, 142.8, 138.2, 137.3, 137.0, 132.5, 132.47, 131.5, 130.9, 130.0, 129.8, 128.3, 127.5, 124.4, 121.6, 67.6: .sup.19F (376 MHz, CDCl.sub.3) −97.0 (m, 1F), −163.54 (m, 1F):

Example 2: Antiviral and Cell Toxicity Assays

(16) Cells were obtained from ATCC and the virus preparation was subjected to one round of centrifugation through a 40% (V/V) glycerol to remove any interferon produced from infected cells. Growth medium was Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) foetal calf serum (FCS), Viral maintainance medium was DMEM+2% (v/v) FCS.

(17) Cytopathic Effect (CPE) Assay—IC50:

(18) A549 cells and HeLa cells were used to seed a set of 96 well plates grown until 50% confluent, a 2 fold serial dilution of the compound was performed on each cell line with DMSO (only) as control starting with 100 μM of the compound. Medium used was DMEM+2% FCS (normal medium for viral growth).

(19) Cells were allowed to grow for 6-8 days to mimic a TCID50 assay after which cell viability was determined by adding Almar blur (similar to XT) and reading at 600 nm (excitation 540 nm). The results obtained are shown in table 1.

(20) TABLE-US-00001 TABLE 1 Results of cytopathic effect (CPE) assay Compound CPE IC50 (μM) 1 2.5 2 0.625 4 2.5

(21) The results show that all three compounds are effective at treating at treating RSV. Due to the low IC50 value, compound 2 is the most effect compound at treating RSV. The fact that compound 2 is substantially more effective at treating RSV than compound 4 (the racemate) indicates that there is a chiral preference, and that therefore the compounds bind directly to a protein site within the virus.

(22) Cell Cytotoxicity—CC50 (Concentration at which 50% Cell Toxicity is Observed):

(23) A549 cells and HeLa cells were used to seed a set of 96 well plates grown until 50% confluent, a 2 fold serial dilution of the compound was performed on each cell line with DMSO (only) as control starting with 100 μM of the compound. Medium used was DMEM+2% FCS (normal medium for viral growth).

(24) Cells were allowed to grow for 6-8 days to mimic a TCID50 assay after which cell viability was determined by adding Almar blur (similar to XT) and reading at 600 nm (excitation 540 nm).

(25) The results obtained are shown in table 2.

(26) TABLE-US-00002 TABLE 2 Results of cell cytotoxicity assay Compound CC50 (μM) 1 25 2 100 4 >100

(27) CC50 for all three of the compounds was significantly higher than the CPE IC50 value. This suggests that the compounds are not toxic at concentrations which may be used to treat RSV.

(28) Plaque Reduction Assay

(29) Vero cells were seeded in 96-well plates in a volume of 100 μL of Optimem supplemented with 3% FCS at a concentration of 4×10.sup.4 cells per well. After an overnight incubation at 37° C. in a humidified 5% CO.sub.2 atmosphere, the monolayer of cells should be approximately 90% confluent. Compound 2 was titrated in pre-warmed Serum Free (SF) Optimem in a U-bottom 96 well plate. For compounds in a DMSO solution, titration in 100% DMSO was performed first and each concentration added individually to a 2× final concentration at 4% DMSO in SF media before mixing with virus (2% final DMSO with virus). Media was then removed from cells and replaced with PBS (100˜l/well). RSV stock was thawed and diluted in SF Optimem media to 4000 PFU/mLl. An equal volume of virus was added to compounds on the titration plate. PBS was removed from cells which were then inoculated with the virus/compound solution (50 μL/well). Cells were incubated for 2 h in a 37° C.+5% CO.sub.2 humidified incubator to allow infection. Inoculum was removed and media (Optimem+1% FCS) added to cells (100 l/well). Cells were subsequently incubated for 48 h at 37° C.+5% CO.sub.2 in a humidified incubator.

(30) Immunostaining Procedure:

(31) Media was removed from cells and the monolayer washed with PBS. Cells were fixed with ice cold 80% Acetone in PBS (100 l/well) for 20 mins at −20° C. Fixative was removed and cells are dried for 30 mins with plates inverted. Blocking solution (5% skim milk powder in PBS-T) was added to cells (150 μL/well) and plates were incubated for 30 mins at room temperature. Blocking solution was removed and plates washed once with PBS-T. Primary antibody in blocking solution was added to plates (50l/well) and incubated for 1 h at 37° C. Plates were then washed 3 times with PBS-T. Secondary antibody in blocking solution was added to plates (50 μL/well) and incubated for 1 h at 37° C. in the dark. Plates were washed as above and then dried for 10 mins. Plates were scanned on the Odyssey Imager (Li-Cor Biosciences) at a resolution of 42 μM, medium quality and level 5 intensity in the 800 nM channel.

(32) Data Analysis:

(33) Images obtained were saved and plaque numbers counted with the aid of computer imaging software. IC.sub.50 values for compounds were derived from dose response curves [three variable log(inhibitor) vs response] obtained using Graphpad Prism software.

(34) The results are given in Table 3.

(35) TABLE-US-00003 TABLE 3 Standard plaque reduction assay data and time of addition study plaque reduction assay data for compound 2 Plaque Plaque IC50 μM Plaque IC50 μM IC50 μM (during infection only) (post infection only) 1.1 10.9 2

(36) The plaque reduction assay supports the data showing that the compound may be used to treat RSV. In addition to this standard plaque reduction assay, the compounds were tested against RSV in two further formats: first compounds were added during infection only and removed during the replication phase and in a second format the compounds were added only during the replication phase, post infection Since the IC50 value of the second plaque reduction is much higher than the standard plaque reduction assay this suggests that the compound is not effective in the first two hours of infection. Similarly, since the IC50 values for the standard and second format plaque reduction assays are similar, it strongly suggests that the compound acts relatively late in the viral life cycle. In turn, this suggests that the compound is a viral replication inhibitor. Although they do not wish to be bound by any hypothesis, the inventors believe that the compound of the invention bind to the N-protein of RSV, and so inhibits virus replication, and is unlikely to be viral-host cell fusion inhibitors, which may have been the case had the compounds acted earlier in the viral life cycle.

(37) Cytotoxicity

(38) MTT Cell Toxicity Assay

(39) Vero cells were seeded in 96-well plates in a volume of 100 μL Optimem media supplemented with 1% FBS at a concentration of 1×10.sup.4 cells per well. After an overnight incubation at 37° C. in a humidified 5% CO.sub.2 atmosphere, the monolayer of cells should be approximately 90% confluent. Compound 2 was titrated in pre-warmed Serum Free (SF) Optimem in a U-bottom 96 well plate. For compounds in a DMSO solution, titration in 100% DMSO was performed first and each concentration added individually to a 2× final concentration at 4% DMSO. Media was then removed from cells and replaced with titrated compounds (100 μl/well). Cells were incubated at 37° C. in a humidified 5% CO.sub.2 atmosphere for 48 hours.

(40) The MTT solution on Optimem (4 mg/ml) was prepared and 2 ul added to each well before it was incubated for 2 hours at 37° C. in a humidified 5% CO.sub.2 atmosphere. Media was then removed from the cells and DMSO added (50 ul/well). Plates were placed on a shaker prior to reading in a Spectromax at 570 nm. Data was normalised relative to untreated controls (1% DMSO only) and CC50 values determined using non-linear regression analysis with Graphpad Prism software.

(41) Using this method compound 2 was found to have a CC50 value of 63 μM. The difference in cytotoxicity values observed for compound 2 may be due to a cell line difference (Vero versus A549) coupled with biological variability. It should also be noted that the different experiments were performed in different labs at different times, which could also lead to a degree of variability.

(42) Meanwhile, a CC50 value of 63 μM is still significantly higher than the CPE IC50 value. This further supports the conclusion that compound 2 is not toxic at concentrations which may be used to treat RSV.

(43) Results and Discussion

(44) The results show that compounds 1, 2 and 4 may be used to treat RSV. Furthermore, the compounds are effective at non-toxic concentrations. It is thought that the compounds bind directly to a protein site and inhibits replication of the virus.

Example 3: Permeability Assays

(45) MDR1-MDCK cells obtained from the NIH (Rockville, Md., USA) are used between passage numbers 6-30. Cells are seeded onto Millipore Multiscreen Transwell plates at 3.4×10.sup.5 cells/cm.sup.2. The cells are cultured in DMEM and media is changed on day 3. On day 4 the permeability study is performed. Cell culture and assay incubations are carried out at 37° C. in an atmosphere of 5% CO2 with a relative humidity of 95%. On the day of the assay, the monolayers are prepared by rinsing both basolateral and apical surfaces twice with Hanks Balanced Salt Solution (HBSS) at the desired pH warmed to 37° C. Cells are then incubated with HBSS at the desired pH in both apical and basolateral compartments for 40 min to stabilise physiological parameters.

(46) The dosing solutions are prepared by diluting compound 2 with assay buffer to give a final compound 2 concentration of 10 μM (final DMSO concentration of 1% v/v). The fluorescent integrity marker lucifer yellow is also included in the dosing solution. Analytical standards are prepared from test compound DMSO dilutions and transferred to buffer, maintaining a 1% v/v DMSO concentration.

(47) For assessment of A-B permeability, HBSS is removed from the apical compartment and replaced with compound 2 dosing solution. The apical compartment insert is then placed into a companion plate containing fresh buffer (containing 1% v/v DMSO). For assessment of B-A permeability, HBSS is removed from the companion plate and replaced with compound 2 dosing solution. Fresh buffer (containing 1% v/v DMSO) is added to the apical compartment insert, which is then placed into the companion plate.

(48) At 60 min the apical compartment inserts and the companion plates are separated and apical and basolateral samples diluted for analysis.

(49) Compound 2 permeability is assessed in duplicate. Compounds of known permeability characteristics are run as controls on each assay plate.

(50) Compound 2 and control compounds are quantified by LC-MS/MS cassette analysis using an 8-point calibration with appropriate dilution of the samples. The starting concentration (C.sub.o) is determined from the dosing solution and the experimental recovery calculated from C.sub.o and both apical and basolateral compartment concentrations.

(51) The integrity of the monolayer throughout the experiment is checked by monitoring lucifer yellow permeation using fluorimetric analysis. Lucifer yellow permeation is high if monolayers have been damaged.

(52) The permeability data is shown in Table 4.

(53) TABLE-US-00004 TABLE 4 Permeability data for compound 2 Permeability A2B Permeability B2A Efflux ratio (cm/s) (cm/s) B2A/A2B 0.553 76.3 138

(54) This data indicates that compound 2 is a permeable compound but with a susceptibility for an efflux mechanism, possibly via a transporter such as PGP.

Example 4: Clearance Assays

(55) Pooled human liver microsomes (pooled male and female), pooled rat liver microsomes (male Sprague Dawley rats) and pooled dog liver microsomes (male Beagle dog) are purchased from a reputable commercial supplier and stored at −80° C. prior to use.

(56) Microsomes (final protein concentration 0.5 mg/mL), 0.1 M phosphate buffer pH 7.4 and compound 2 (final substrate concentration 3 μM; final DMSO concentration 0.25%) are pre-incubated at 37° C. prior to the addition of NADPH (final concentration 1 mM) to initiate the reaction. The final incubation volume is 50 μL. A control incubation is included for each compound tested where 0.1 M phosphate buffer pH 7.4 is added instead of NADPH (minus NADPH). Two control compounds are included with each species. All incubations are performed singularly for each test compound.

(57) Compounds are incubated for 0, 5, 15, 30 and 45 min. The control (minus NADPH) is incubated for 45 min only. The reactions are stopped by transferring 25 μL of incubate to 50 μL methanol at the appropriate time points. The termination plates are centrifuged at 2,500 rpm for 20 min at 4° C. to precipitate the protein. Following protein precipitation, the sample supernatants are combined in cassettes of up to 4 compounds, internal standard is added and samples analysed by LC-MS/MS. From a plot of ln peak area ratio (compound peak area/internal standard peak area) against time, the gradient of the line is determined. Subsequently, half-life and intrinsic clearance are calculated, and this is shown in Table 5.

(58) TABLE-US-00005 TABLE 5 Microsomal clearance data for compound 2 Intrinsic Intrinsic Intrinsic clearance - t½ - clearance - t½ - clearance - t½ - rat rat dog dog human human (μl/min/mg) (mins) (μl/min/mg) (mins) (μl/min/mg) (mins) 29.2 47.5 7.05 197 52.6 26.3

(59) This data indicates the potential of the example to undergo phase 1 metabolism. Moderate and low clearances are observed for the example in rat and dog microsomal preparations with a higher clearance observed in human microsomal preparations.

Example 5: Mouse In Vivo Pharmacokinetic Assay

(60) Pharmacokinetic evaluation of compound 2 was performed in male CD-1 mice following intravenous (IV) and oral (PO) administration. For intravenous administration the compound was formulated in solution in 40:60 DMA:saline and was administered at 1 mg/kg via the tail vein. For oral administration the compound was formulated in suspension in water containing 1% methylcellulose and 0.1% Tween80, and was administered at 10 mg/kg by oral gavage. Blood samples were taken by cardiac puncture under terminal anaesthesia (isoflurane) at ten timepoints between 1 minute and 24 hours following IV administration, and at nine timepoints between 5 minutes and 24 hours following PO administration (n=3 per timepoint). Plasma was immediately obtained by centrifugation, and the resulting samples frozen until analysis. Samples, along with a calibration curve prepared by spiking the compound into control plasma, were subsequently prepared for quantitative analysis by precipitation of plasma proteins with acetonitrile. Analysis was by ultra high performance liquid chromatography (Agilent 1290 system) coupled to time of flight mass spectrometry, using electrospray ionisation (Agilent 6550 system). Pharmacokinetic parameters were determined by non-compartmental analysis of mean concentration data using Phoenix WinNonlin v6.4. The data obtained is shown in Table 6.

(61) TABLE-US-00006 TABLE 6 In vivo mouse pharmacokinetic data for compound 2 IV Clearance IV t½ Vd PO t½ PO cmax F (ml/min/kg) (mins) (L/kg) (mins) (ng/ml) (%) 63.1 59 3.65 132 349 54.8

(62) This data indicates the example 2 is orally bioavailable with moderate clearance.

Example 6: Synthesis of Further Compounds

(63) Reagents were obtained from commercial sources and were used without further purification. Anhydrous reactions were carried out in oven-dried glassware under a nitrogen atmosphere. TLC was performed on aluminium backed silica gel plates with fluorescence indicator at 254 nM (median pore size 60 Å). Flash column chromatography was performed using a Biotage Isolera One system using KP-Sil, Ultra or KP-NH columns. NMR spectra were recorded on a 400 MHz spectrometer at ambient probe temperature (nominal 295K). Chemical shifts (δ) are given in ppm and calibrated by using the residual peak of the solvent as the internal standard (CDCl.sub.3, δ.sub.H=7.26 ppm, δ.sub.C=77.16 ppm; DMSO-d.sub.6, δ.sub.H=2.50 ppm, δ.sub.C=39.52 ppm). Coupling constants are given in Hertz (Hz). LRMS were recorded using an Advion Plate Express expression.sup.L compact mass spectrometer equipped with either an APCI or ESI ion source.

Preparatory Examples

Intermediates

3A: tert-Butyl 2-[(2,3,5,6-tetrafluoropyridin-4-yl)amino]acetate

(64) ##STR00065##

(65) Pentafluoropyridine (3.293 mL, 30 mmol) was added to a cooled (0° C.) solution of glycine tert-butyl ester hydrochloride (2.514 g, 15 mmol) and triethylamine (4.39 mL, 31.5 mmol) in DMF (45 mL) and stirred at 0° C. for 6 h. The reaction was allowed to attain rt and stirred for 18 h. The volatiles were removed under reduced pressure and the residue purified by flash chromatography (SiO.sub.2, 0-45% EtOAc in heptane) to afford a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3): δ 5.16 (br s, 1H), 4.20-4.15 (m, 2H), 1.50 (s, 9H). LRMS (APCI−) m/z 278.6 [M−H].sup.−.

(66) The following intermediate compounds were prepared by the same general procedure.

(67) TABLE-US-00007 TABLE 7 Intermediate compounds of formula (3) (3) Prep .sup.1H NMR δ TLC Example R Name (400 MHz) LRMS R.sub.f value 3B tert-Butyl (2S)- 2-[(2,3,5,6- tetrafluoropyridin- 4-yl)amino] propanoate (CDCl.sub.3): δ 5.12 (br s, 1H), 4.52-4.43 (m, 1H), 1.52 (d, J = 7.0 Hz, 3H), 1.49 (s, 9H) APCI− 292.7 [M − H].sup.− 3C tert-Butyl (2R)-2- [(2,3,5,6- tetrafluoropyridin- 4-yl)amino] propanoate (CDCl.sub.3): δ 5.12 (br s, 1H), 4.52-4.43 (m, 1H), 1.52 (d, J = 7.0 Hz, 3H), 1.48 (s, 9H) APCI− 292.8 [M − H].sup.− 3D tert-Butyl (2S)- 3-methyl-2- [(2,3,5,6- tetrafluoropyridin- 4-yl)amino] butanoate (CDCl.sub.3): δ 5.09 (d, J = 9.0 Hz, 1H), 4.38- 4.32 (m, 1H), 2.27- 2.17 (m, 1H), 1.49 (9H), 1.04 (d, J = 6.9 Hz, 3H), 1.01 (d, J = 6.9 Hz, 3H) APCI+ 322.8 [M + H].sup.+ 3E 0 tert-butyl (2R)- 3-methyl-2- [(2,3,5,6- tetrafluoropyridin- 4-yl)amino] butanoate (CDCl.sub.3): δ 5.09 (d, J = 9.0 Hz, 1H), 4.38- 4.32 (m, 1H), 2.27- 2.17 (m, 1H), 1.49 (9H), 1.04 (d, J = 7.0 Hz, 3H), 1.01 (d, J = 6.9 Hz, 3H) APCI+ 322.8 [M + H].sup.+ 3F tert-butyl (2S)- 3-phenyl-2- [(2,3,5,6- tetrafluoropyridin- 4-yl)amino] propanoate APCI+ 371.1 [M + H].sup.+ 0.69 (EtOAc: heptane; 1:2) 3G tert-butyl (2S)- 1-(2,3,5,6- tetrafluoropyridin- 4-yl)pyrrolidine- 2-carboxylate (CDCl.sub.3): δ 4.69- 4.63 (m, 1H), 3.99- 3.91 (m, 1H), 3.90- 3.81 (m, 1H), 2.35- 2.24 (m, 1H), 2.09- 1.91 (m, 3H), 1.44 (s, APCI+ 320.8 [M + H].sup.+ 9H) 3H tert-butyl (2R)- 1-(2,3,5,6- tetrafluoropyridin- 4-yl)pyrrolidine- 2-carboxylate (CDCl.sub.3): δ 4.69- 4.63 (m, 1H), 4.00- 3.91 (m, 1H), 3.89- 3.81 (m, 1H), 2.35- 2.24 (m, 1H), 2.10- 1.91 (m, 3H), 1.44 (s, APCI+ 320.8 [M + H].sup.+ 9H) 3J 4-(2,3,5,6- Tetrafluoropyridin- 4-yl)morpholine (CDCl.sub.3): δ 3.94-3.80 (m, 4H), 3.52-3.46 (m, 4H) — 0.55 (EtOAc: heptane; 1:2) 3K 4-(2,3,5,6- Tetrafluoropyridin- 4-yl) thiomorpholine (CDCl.sub.3): δ 3.71- 3.67 (m, 4H), 2.78- 2.74 (m, 4H) — 0.4 (EtOAc: heptane; 1:9). 3L 1-[4-(2,3,5,6- tetrafluoropyridin- 4-yl)piperazin- 1-yl]ethan-1-one (CDCl.sub.3): δ 3.80- 3.58 (m, 4H), 3.53- 3.43 (m, 4H), 2.16 (s, 3H) APCI+ 278.0 [M + H].sup.+ 3M N,N-Diethyl- 2,3,5,6- tetrafluoropyridin- 4-amine (DMSO-d.sub.6) δ 3.42 (qt, J = 7.0, 1.5 Hz, 4H), 1.17 (t, J = 7.0 Hz, 6H) APCI+ 223.1 [M + H]+ 3N 4-(4,4- Difluoropiperidin- 1-yl)- 2,3,5,6- tetrafluoropyridine (DMSO-d.sub.6) δ 3.55 (t, J = 5.8 Hz, 4H), 2.18- 2.04 (m, 4H). — 0.78 (EtOAc: heptane; 1:1)

4A: Ethyl 2-{[3,5,6-trifluoro-4-(thiomorpholin-4-yl]pyridin-2-yl)amino}acetate

(68) ##STR00079##

(69) A solution of 4-(2,3,5,6-tetrafluoropyridin-4-yl)thiomorpholine (intermediate 3K) (856 mg, 3.39 mmol), glycine ethyl ester hydrochloride (947 mg, 6.79 mmol) and K.sub.2CO.sub.3 (1.641 g, 11.88 mmol) in N-methyl-2-pyrrolidinone (NMP, 10 mL) was heated in a sealed tube at 100° C. for 17 h. After cooling to rt, the reaction was quenched with saturated aqueous NaHCO.sub.3 solution (20 mL) and extracted with EtOAc (3×20 mL). The combined organic extracts were washed successively with saturated aqueous NaHCO.sub.3 solution, water then brine (20 mL each), dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography (SiO.sub.2, 0-30% EtOAc in heptane) afforded a white solid (541 mg, 48%0). .sup.1HNMR (400 MHz, DMSO-d.sub.6) δ: 7.11 (t, J=6.3 Hz, 1H), 4.09 (d, J=7.1 Hz, 2H), 3.92 (d, J=6.2 Hz, 2H), 3.54-3.48 (m, 4H), 2.72-2.67 (m, 4H), 1.17 (t, J=7.1 Hz, 3H). LRMS (APCI+) m/z 336.0 [M+H].sup.+.

(70) The following intermediate compounds were prepared by the same general procedure.

(71) TABLE-US-00008 TABLE 8 Intermediate compounds of formula (4) (4) 0 Prep LRMS TLC Ex R Name APCI+ R.sub.f value 4B Ethyl 2-{[4-(4-acetylpiperazin-1-yl)-3,5,6- trifluoropyridin-2-yl]amino}acetate 361.1 [M + H].sup.+ 0.59 (EtOAc) 4C Ethyl 2-{[4-(diethylamino)-3,5,6- trifluoropyridin-2- yl]amino}acetate 306.2 [M + H].sup.+ 0.77 (EtOAc: heptane; 1:1) 4D Ethyl 2-{[4-(4,4-difluoropiperidin-1-yl)-3,5,6- trifluoropyridin-2-yl]amino}acetate 354.1 [M + H].sup.+ 0.63 (EtOAc: heptane; 1:1)

5-A: tert-Butyl 2-{[2,3,5-trifluoro-6-(morpholin-4-yl)pyridin-4-yl]amino}acetate

(72) ##STR00084##

(73) A solution of tert-butyl 2-[(2,3,5,6-tetrafluoropyridin-4-yl)amino]acetate (intermediate 3A) (200 mg, 0.710 mmol) and morpholine (71.8 μL, 0.820 mmol) in DMF (4 mL) was heated at 80° C. for 7 h. Extra morpholine (15.6 μL, 0.178 mmol) was added and the reaction heated at 85° C. for a further 18 h. After cooling to rt, the reaction was diluted with water (15 mL) and extracted with EtOAc (3×15 mL). The combined organic extracts were washed successively with water (3×20 mL) and brine (20 mL), dried (MgSO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography (SiO.sub.2, 3-50% EtOAc:heptane) afforded the product as a white solid (120 mg, 48%). .sup.1H NMR (400 MHz, CDCl.sub.3): 4.84 (br s, 1H), 4.13-4.10 (m, 2H), 3.81-3.77 (m, 4H), 3.35-3.30 (m, 4H), 1.49 (s, 9H). LRMS (APCI+) m/z 347.8 [M+H].sup.+.

6A: tert-Butyl 2-{[2,3,5-trifluoro-6-(methylsulfanyl)pyridin-4-yl]amino}acetate

(74) ##STR00085##

(75) Sodium methanethiolate (58 mg, 0.820 mmol) was added to a solution of tert-butyl 2-[(2,3,5,6-tetrafluoropyridin-4-yl)amino]acetate (intermediate 3A) (200 mg, 0.710 mmol) in DMF (4 mL) and heated at 80° C. for 7 h. The reaction mixture was cooled to rt, diluted with water (20 mL) and extracted with EtOAc (3×20 mL). The combined organic extracts were washed successively with water (4×30 mL) and brine (15 mL), dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography (SiO.sub.2, 0-100% EtOAc:heptane) afforded the product as a yellow oil (182 mg, 83%). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.89 (s, 1H), 4.15-4.11 (m, 2H), 2.49 (s, 3H), 1.49 (s, 9H). LRMS (APCI+) m/z 308.8 [M+H].sup.+

7A: tert-Butyl 2-[(2,3,5-trifluoro-6-methanesulfonylpyridin-4-yl)amino]acetate

(76) ##STR00086##

(77) meta-Chloroperoxybenzoic acid (m-CPBA) (˜77% pure, 384 mg, 1.710 mmol) was added to a cooled (0° C.) solution of tert-butyl 2-[(2,3,5-trifluoro-6-methylsulfanylpyridin-4-yl)amino]acetate (intermediate 3A) (176. mg, 0.570 mmol) in CH.sub.2Cl.sub.2 (5 mL) and stirred for 3 h. The reaction was diluted with saturated aqueous NaHCO.sub.3 solution (15 mL) and extracted with CH.sub.2Cl.sub.2 (3×20 mL). The combined organic extracts were washed successively with NaOH (0.5 M aqueous solution), saturated aqueous NaHCO.sub.3 solution (2×) and brine (20 mL each), dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography (SiO.sub.2, 0-70% EtOAc in heptane) gave the product as a white solid (149 mg, 76%). .sup.1H NMR (400 MHz, CDCl.sub.3): 5.31 (br s, 1H), 4.22-4.18 (m, 2H), 3.26 (s, 3H), 1.51 (s, 9H). LRMS (APCI−) m/z 338.7 [M−H].sup.−.

8A: Ethyl 2-{[3,5,6-trifluoro-4-(morpholin-4-yl]pyridin-2-yl]amino}acetate

(78) ##STR00087##

(79) A solution of 4-(2,3,5,6-tetrafluoropyridin-4-yl)morpholine (intermediate 3J) (180 mg, 0.760 mmol), ethyl 2-aminoacetate hydrochloride (128 mg, 0.910 mmol) and K.sub.2CO.sub.3 (253 mg, 1.830 mmol) in DMF (2.5 mL) was heated at 60° C. for 4 h, then at 100° C. for 44 h. The reaction mixture was cooled to rt, diluted with water (20 mL) and extracted with EtOAc (3×15 mL). The combined organic extracts were washed successively with water (4×15 mL) and brine (15 mL), dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography (0-100% EtOAc in heptane) afforded the product as a white solid (84 mg, 34%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.88 (br s, 1H), 4.23 (q, J=7.1 Hz, 2H), 4.12 (d, J=5.6 Hz, 2H), 3.82-3.77 (m, 4H), 3.41-3.36 (m, 4H), 1.29 (t, J=7.1 Hz, 2H). LRMS (APCI+) m/z 319.8 [M+H].sup.+

9A: (2S)-2-[(2,3,5,6-Tetrafluoropyridin-4-yl)amino]propanoic acid

(80) ##STR00088##

(81) A solution of tert-butyl (2S)-2-[(2,3,5,6-tetrafluoropyridin-4-yl)amino]propanoate (intermediate 3B) (212 mg, 0.720 mmol) was stirred in CH.sub.2Cl.sub.2/trifluoroacetic acid (TFA; 1:1, 2 mL) at rt for 4 h. The volatiles were removed under reduced pressure to afford the crude product as a white solid (171 mg, 99.5%). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 4.98 (d, J=6.9 Hz, 1H), 4.74-4.65 (m, 1H), 1.64 (d, J=7.1 Hz, 3H). LRMS (APCI−) m/z 236.9 [M−H].sup.−.

(82) The following intermediate compounds were prepared by the same general procedure.

(83) TABLE-US-00009 TABLE 8 Intermediate compounds of formula (9) (9) Prep .sup.1H NMR δ TLC Ex R Name (400 MHz) LRMS R.sub.f value 9B 0 (2R)-2- [(2,3,5,6- Tetrafluoropyridin- 4-yl)amino] propanoic acid (CDCl.sub.3): δ 4.98 (d, J = 7.5 Hz, 1H), 4.74-4.65 (m, 1H), 1.64 (d, J = 7.1 Hz, 3H) APCI− 236.7 [M − H].sup.− 9C (2S)-3- Methyl-2- [(2,3,5,6- tetrafluoropyridin- 4-yl)amino] butanoic acid (CDCl.sub.3): δ 4.96 (d, J = 9.4 Hz, 1H), 4.57-4.51 (m, 1H), 2.38-2.27 (m, 1H), 1.11-1.06 (m, 6H). APCI+ 266.8 [M + H].sup.+ 9D (2R)-3- Methyl-2- [(2,3,5,6- tetrafluoropyridin- 4-yl)amino] butanoic acid (CDCl.sub.3): δ 4.95 (d, J = 9.4 Hz, 1H), 4.56-4.52 (m, 1H), 2.39-2.26 (m, 1H), 1.11-1.06 (m, 6H). APCI− 264.7 [M − H].sup.− 9E (2S)-1- (2,3,5,6- Tetrafluoropyridin- 4-yl)pyrrolidine- 2-carboxylic acid (CDCl.sub.3): δ 4.88- 4.82 (m, 1H), 4.03- 3.93 (m, 1H), 3.92-3.83 (m, 1H), 2.24-2.15 (m, 1H), 2.29- APCI+ 264.8 [M + H].sup.+ 2.15 (m, 1H), 2.11- 1.95 (m, 2H). 9F (2R)-1- (2,3,5,6- Tetrafluoropyridin- 4-yl)pyrrolidine- 2-carboxylic acid (CDCl.sub.3): δ 4.88- 4.82 (m, 1H), 4.03- 3.94 (m, 1H), 3.92-3.83 (m, 1H), 2.44-2.33 (m, 1H), 2.24- APCI+ 264.8 [M + H].sup.+ 2.15 (m, 1H), 2.10- 1.97 (m, 2H). 9G (2S)-3- Phenyl-2- [(2,3,5,6- tetrafluoropyridin- 4-yl)amino] propanoic acid (DMSO-d.sub.6) δ 13.25 (s, 1H), 7.49-7.45 (m, 2H), 7.43- 7.32 (m, 3H), 7.28 (d, J = 7.3 Hz, 1H), 5.58 (d, J = 7.2 Hz, 1H), 3.65-3.40 (m, 2H) 0.51 (EtOAc: heptane; 1:2)

10A: 2-[(2,3,5-Trifluoro-6-methanesulfonylpyidin-4-yl)amino]acetic acid

(84) ##STR00096##

(85) Prepared by the general procedure described for intermediate 9A from intermediate 7A. R.sub.f=0.17 (EtOAc).

11A: 2-{[2,3,5-Trifluoro-6-(morpholin-4-yl)pyridin-4-yl]amino}acetic acid hydrochloride

(86) ##STR00097##

(87) tert-Butyl 2-{[2,3,5-trifluoro-6-(morpholin-4-yl)pyridin-4-yl]amino}acetate (intermediate 5A) (119 mg, 0.340 mmol) was suspended in a mixture of HCl (1 M aqueous) and Et.sub.2O (1:1, 6 mL) and stirred at rt for 24 h. Further HCl (2 M in Et.sub.2O, 3 mL) was added and the reaction stirred for 24 h. The volatiles were removed under reduced pressure to afford the crude product as a pale brown solid (97 mg, 86%). R.sub.f=0.66 (EtOAc). LRMS (APCI+) m/z 292.2 [M+H].sup.+

12A: Lithium (2-{[3,5,6-trifluoro-4-(morpholin-4-yl)pyridin-2-yl]amino}acetate

(88) ##STR00098##

(89) LiOH (1 M aqueous, 392 μL, 0.392 mmol) was added to a solution of ethyl 2-[(3,5,6-trifluoro-4-morpholin-4-ylpyridin-2-yl)amino]acetate (intermediate 8A) (63 mg, 0.200 mmol) in THF (2.5 mL) and stirred at rt for 20 h. The volatiles were removed under reduced pressure and the residue triturated with Et.sub.2O. The residue was then dissolved in CH.sub.2Cl.sub.2:iPrOH:EtOH (˜4:1:1), filtered, washing with EtOH and the solvent removed under reduced pressure to afford the crude lithium salt as a white solid. .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 5.92 (br s, 1H), 3.70-3.65 (m, 2H), 3.42-3.26 (m, 8H). LRMS (APCI+) m/z 291.7 [M+H].sup.+

13A: 2-{[4-(4-Acetylpiperazin-1-yl)-3,5,6-trifluoropyridin-2-yl]amino}acetic acid

(90) ##STR00099##

(91) LiOH (1 M aqueous, 1.49 mL, 1.490 mmol) was added to a solution of ethyl 2-[[4-(4-acetylpiperazin-1-yl)-3,5,6-trifluoropyridin-2-yl]amino]acetate (intermediate 4B) (269 mg, 0.750 mmol) in THF (5 mL) at rt and stirred for 17 h. The reaction mixture was acidified with aqueous HCl (1 M) to ca. pH 6-7 and extracted with EtOAc (3×15 mL), then acidified with aqueous HCl (1 M) to ca. pH 4-5 and extracted with EtOAc (3×15 mL). The combined organic extracts were washed with water and brine (15 mL each), dried (Na.sub.2SO.sub.4), and the solvent removed under reduced pressure. The residue was triturated with heptane (3×), and dried under reduced pressure to afford a white solid (189 mg, 76%). .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ 12.48 (br s, 1H), 7.00 (t, J=6.2 Hz, 1H), 3.85 (d, J=6.1 Hz, 2H), 3.58-3.50 (m, 4H), 3.28-3.22 (m, 2H), 2.03 (s, 3H). LRMS (APCI+) m/z 333.1 [M+H].sup.+.

(92) The following intermediate compounds were prepared by the same general procedure.

(93) TABLE-US-00010 TABLE 9 Intermediate compounds prepared by the same procedure as compound 13A Prep .sup.1H NMR δ LRMS Ex R Name (400 MHz, DMSO-d.sub.6) APCI+ 13B 00 Ethyl 2-{[4- (diethylamino)- 3,5,6- trifluoropyridin- 2- yl]amino}acetate 12.48 (s, 1H), 6.87 (t, J = 6.0 Hz, 1H), 3.84 (d, J = 6.2 Hz, 2H), 3.29 (q, J = 7.0 Hz, 4H), 1.09 (t, J = 7.0 Hz, 6H) 277.9 [M + H].sup.+ 13C 01 2-{[4-(4,4- Difluoropiperidin- 1-yl)-3,5,6- trifluoropyridin- 2-yl]amino}acetic acid 12.40 (s, 1H), 7.00 (t, J = 6.3 Hz, 1H), 3.84 (d, J = 6.1 Hz, 2H), 3.41 (t, J = 5.7 Hz, 4H), 2.08 (tt, J = 14.1, 5.7 Hz, 4H) 325.9 [M + H].sup.+

14A: (2S)—N-[(3S)-1-[(4-methoxyl)phenyl)methyl]-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]-1-(2,3,5,6-tetrafluoropyridin-4-yl)pyrrolidine-2-carboxamide

(94) ##STR00102##

(95) (3S)-3-Amino-1-[(4-methoxyphenyl)methyl]-5-phenyl-3H-1,4-benzodiazepin-2-one (60 mg, 0.16 mmol) was produced according to the procedure described in WO2005/090319. (3S)-3-Amino-1-[(4-methoxyphenyl)methyl]-5-phenyl-3H-1,4-benzodiazepin-2-one (60 mg, 0.16 mmol) and (2S)-1-(2,3,5,6-tetrafluoropyridin-4-yl)pyrrolidine-2-carboxylic acid (intermediate 9E) (64 mg, 0.24 mmol) were dissolved in DMF (1 mL). Triethylamine (0.07 mL, 0.48 mmol) was added followed by 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (123 mg, 0.32 mmol) and the mixture stirred at rt for 17 h. Water (15 mL) was added and the mixture extracted with EtOAc (3×10 mL). The combined organics were washed successively with water (3×10 mL), brine (10 mL) and dried (MgSO.sub.4). Flash chromatography on SiO.sub.2 (35-60% EtOAc:heptane) provided the desired compound (79 mg, 79%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.90 (d, J=8.5 Hz, 1H), 7.61-7.56 (m, 1H), 7.54-7.48 (m, 2H), 7.38 (t, J=7.8 Hz, 2H), 7.33-7.29 (m, 2H), 7.22 (d, J=4.1 Hz, 2H), 6.95-6.84 (m, 2H), 6.67-6.55 (m, 2H), 5.65 (d, J=8.4 Hz, 1H), 5.61 (d, J=14.9 Hz, 1H), 4.91-4.80 (m, 1H), 4.67 (d, J=14.9 Hz, 1H), 4.21-4.10 (m, 1H), 3.93-3.81 (m, 1H), 3.68 (s, 3H), 2.48-2.39 (m, 1H), 2.37-2.29 (m, 1H), 2.25-2.14 (m, 1H), 2.09-1.99 (m, 1H); LRMS APCI+618.7 [M+H].sup.+.

(96) The following intermediate compounds were prepared by the same general procedure.

(97) TABLE-US-00011 TABLE 10 Intermediate compounds of formula (14) (14) 03 TLC Prep .sup.1H NMR δ LRMS R.sub.f Ex R Name (400 MHz) APCI+ value 14B 04 (2R)-N-[(3S)-1- [(4- methoxyphenyl) methyl]-2-oxo-5- phenyl-2,3- dihydro-1H-1,4- benzodiazepin-3- yl]-1- (2,3,5,6- (CDCl.sub.3) δ 8.20 (d, J = 8.2 Hz, 1H), 7.73-7.64 (m, 1H), 7.62-7.55 (m, 2H), 7.45-7.39 (m, 2H), 7.35- 7.31 (m, 1H), 7.30-7.20 (m, 2H), 7.02-6.83 (m, 2H), 6.70-6.53 (m, 2H), 5.74 (d, J = 8.2 Hz, 1H), 5.67 (d, J = 14.9 Hz, 1H), 618.7 [M + H].sup.+ tetrafluoropyridin- 4.97-4.78 (m, 2H), 4.72 4-yl)pyrrolidine- (d, J = 14.9 Hz, 1H), 2- 4.22-4.05 (m, 1H), 3.98- carboxamide 3.83 (m, 1H), 3.68 (s, 3H), 2.51-2.42 (m, 2H), 2.27-2.18 (m, 1H), 2.11- 2.01 (m, 1H). 14C 05 (2S)-N-[(3S)-1- [(4- methoxyphenyl) methyl]-2-oxo-5- phenyl-2,3- dihydro-1H-1,4- benzodiazepin-3- yl]-3-methyl-2- [(2,3,5,6- 619.8 [M + H].sup.+ 0.55 (EtOAc: heptane; 1:1) tetrafluoropyridin- 4-yl)amino] butanamide 14D 06 (2R)-N-[(3S)-1- [(4- methoxyphenyl) methyl]-2-oxo-5- phenyl-2,3- dihydro-1H-1,4- benzodiazepin-3- yl]-3-methyl-2- [(2,3,5,6- 619.8 [M + H].sup.+ 0.54 (EtOAc: heptane; 1:1) tetrafluoropyridin- 4-yl)amino] butanamide 14E 07 (2S)-N-[(3S)-1- [(4- methoxyphenyl) methyl]-2-oxo-5- phenyl-3H-1,4- benzodiazepin-3- yl]-2-[(2,3,5,6- tetrafluoropyridin- 4-yl)amino] propanamide (CDCl.sub.3) δ 7.96 (d, J = 8.1 Hz, 1H), 7.67-7.49 (m, 3H), 7.45-7.32 (m, 4H), 7.31-7.22 (m, 2H), 6.93 (d, J = 8.7 Hz, 2H), 6.65 (d, J = 8.6 Hz, 2H), 5.81- 5.60 (m, 3H), 4.80-4.64 (m, 2H), 3.71 (s, 3H), 1.70 (d, J = 6.9 Hz, 3H). 591.8 [M + H].sup.+ 0.68 (CH.sub.2Cl.sub.2: EtOH: NH.sub.4OH; 100:8:1) 14F 08 (2R)-N-[(3S)-1- [(4- methoxyphenyl) methyl]-2-oxo-5- phenyl-3H-1,4- benzodiazepin-3- yl]-2-[(2,3,5,6- tetrafluoropyridin- 4-yl)amino] propanamide (CDCl.sub.3) δ 7.91 (d, J = 8.0 Hz, 1H), 7.56 (ddd, J = 32.2, 7.7, 4.4 Hz, 3H), 7.44-7.20 (m, 7H), 6.94 (d, J = 8.5 Hz, 2H), 6.70- 6.56 (m, 2H), 5.77-5.59 (m, 2H), 5.42 (s, 1H), 4.72 (t, J = 12.9 Hz, 2H), 3.71 (s, 3H), 1.74 (d, J = 6.8 Hz, 3H). 591.6 [M + H].sup.+ 0.72 (CH.sub.2Cl.sub.2: EtOH: NH.sub.4OH; 100:8:1) 14G 09 (2S)-N-[(3S)-1- [(4- methoxyphenyl) methyl]-2-oxo-5- phenyl-2,3- dihydro-1H-1,4- benzodiazepin-3- yl]-3-phenyl-2- [(2,3,5,6- — 668.9 [M + H].sup.+ 0.48 (EtOAc: heptane; 1:1) tetrafluoropyridin- 4- yl)amino] propanamide 14H 0 2-[[4-(4- acetylpiperazin-1- yl)-3,5,6- trifluoropyridin- 2-yl]amino]-N- [(3S)-1-[(4- methoxyphenyl) methyl]-2-oxo-5- phenyl-3H-1,4- benzodiazepin-3- yl]acetamide (DMSO-d.sub.6) δ 9.08 (d, J = 8.0 Hz, 1H), 7.78 (dd, J = 8.4, 1.2 Hz, 1H), 7.64 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 7.56-7.47 (m, 1H), 7.45-7.36 (m, 2H), 7.32- 7.12 (m, 4H), 6.94 (t, J = 6.3 Hz, 1H), 6.89-6.81 (m, 2H), 6.70-6.60 (m, 2H), 5.47 (d, J = 15.2 Hz, 1H), 5.38 (d, J = 8.0 Hz, 1H), 4.87 (d, J = 15.2 Hz, 1H), 4.11-3.92 (m, 2H), 3.64 (s, 3H), 3.61-3.50 (m, 4H), 3.30-3.20 (m, 2H), 2.04 (s, 3H). 686.3 [M + H].sup.+ 0.39 (CH.sub.2Cl.sub.2: EtOH: NH.sub.4OH; 100:8:1) 14J 2-[[4-(1,1-dioxo- 1,4-thiazinan-4- yl)-3,5,6- trifluoropyridin- 2-yl]amino]-N- [(3S)-1-[(4- methoxyphenyl) methyl]-2-oxo-5- phenyl-3H-1,4- benzodiazepin-3- yl]acetamide (DMSO-d.sub.6) δ 9.09 (d, J = 8.0 Hz, 1H), 7.78 (dd, J = 8.4, 1.1 Hz, 1H), 7.65 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 7.56-7.47 (m, 1H), 7.45-7.36 (m, 2H), 7.32- 7.13 (m, 4H), 7.02 (t, J = 6.0 Hz, 1H), 6.89-6.81 (m, 2H), 6.68-6.60 (m, 2H), 5.47 (d, J = 15.2 Hz, 1H), 5.38 (d, J = 8.0 Hz, 1H), 4.87 (d, J = 15.2 Hz, 1H), 4.12-3.94 (m, 2H), 3.71 (br s, 4H), 3.27 (br s, 4H). 693.2 [M + H].sup.+ 0.42 (CH.sub.2Cl.sub.2: EtOH: NH.sub.4OH; 100:8:1) 14K N-[(3S)-1-[(4- ethoxyphenyl) methyl]-2-oxo-5- phenyl- 2,3-dihydro-1H- 1,4- benzodiazepin-3- yl]-2-{[3,5,6- trifluoro-4- (morpholin-4- yl)pyridin-2- yl]amino} acetamide (DMSO-d.sub.6) δ 9.08 (d, J = 8.1 Hz, 1H), 7.78 (dd, J = 8.4, 1.1 Hz, 1H), 7.65 (ddd, J = 8.4, 7.3, 1.6 Hz, 1H), 7.54-7.48 (m, 1H), 7.45-7.36 (m, 2H), 7.33- 7.16 (m, 4H), 6.92 (t, J = 6.1 Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 6.64 (d, J = 8.7 Hz, 2H), 5.46 (d, J = 15.2 Hz, 1H), 5.37 (d, J = 8.0 Hz, 1H), 4.87 (d, J = 15.2 Hz, 1H), 4.01 (t, J = 645.7 [M + H].sup.+ 6.4 Hz, 2H), 3.75-3.66 (m, 4H), 3.64 (s, 3H), 3.34-3.29 (m, 4H). 14L 2-{[4- (Diethylamino)- 3,5,6- trifluoropyridin- 2-yl]amino}-N- [(3S)-1-[(4- methoxyphenyl) methyl]-2-oxo-5- phenyl-2,3- dihydro-1H-1,4- benzodiazepin-3- (DMSO-d.sub.6) δ 9.08 (d, J = 8.0 Hz, 1H), 7.77 (dd, J = 8.4, 1.1 Hz, 1H), 7.64 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 7.56-7.47 (m, 1H), 7.45-7.35 (m, 2H), 7.31- 7.11 (m, 4H), 6.89- 6.78 (m, 3H), 6.70-6.56 (m, 2H), 5.46 (d, J = 15.2 Hz, 1H), 5.36 (d, J = 8.1 Hz, 1H), 4.86 (d, J = 15.2 631.3 [M + H].sup.+ yl]acetamide Hz, 1H), 4.08-3.94 (m, 2H), 3.63 (s, 3H), 3.29 (q, J = 7.1 Hz, 4H), 1.09 (t, J = 7.0 Hz, 6H). 14M N-[(3S)-1-[(4- Methoxyphenyl) methyl]-2-oxo-5- phenyl- 2,3-dihydro-1H- 1,4-benzodiazepin- 3-yl]-2-[(2,3,5- trifluoro-6- methanesulfonylpyridin- 4-yl)amino] acetamide 638.5 [M + H].sup.+ 0.81 (EtOAc)

Procedure for Deprotection

Compounds: (2S)—N-[(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]-1-(2,3,5,6-tetrafluoropyridin-4-yl)pyrrolidine-2-carboxamide

(98) ##STR00115##

(99) AlCl.sub.3 (201 mg, 1.48 mmol) was added to a solution of (2S)—N-[(3S)-1-[(4-methoxyphenyl)methyl]-2-oxo-5-phenyl-3H-1,4-benzodiazepin-3-yl]-1-(2,3,5,6-tetrafluoropyridin-4-yl)pyrrolidine-2-carboxamide (intermediate 14C) (76 mg, 0.12 mmol) in anhydrous anisole (1.5 mL) and the mixture stirred at rt for 40 h. The mixture was cooled to 0° C., quenched with water (10 mL), and extracted with CH.sub.2Cl.sub.2 (4×10 mL). The organics were washed with brine (15 mL) and dried (Na.sub.2SO.sub.4). Flash chromatography on SiO.sub.2 (30-45% EtOAc:heptane) provided the desired compound as a white solid (24 mg, 39%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.59 (s, 1H), 7.67-7.47 (m, 2H), 7.45-7.34 (m, 3H), 7.26-7.18 (m, 2H), 5.55 (d, J=8.4 Hz, 1H), 4.91-4.79 (m, 1H), 4.13-4.05 (m, 1H), 3.94-3.78 (m, 1H), 2.39 (dt, J=14.9, 7.5 Hz, 1H), 2.24 (dd, J=13.1, 6.4 Hz, 1H), 2.13 (dt, J=13.1, 6.4 Hz, 1H), 2.03-1.95 (m, 1H). LRMS APCI+498.8 [M+H].sup.+.

(100) The following compounds were prepared by the same general procedure.

(101) TABLE-US-00012 TABLE 11 Benzodiazepines compounds of formula (VI) (VI) .sup.1H NMR δ LRMS Cmpd R Name (400 MHz) APCI+ 6 N(2R)-N-[(3S)-2- oxo-5-phenyl-2,3- dihydro-1H-1,4- benzomazepin-3-yl]- 1-(2,3,5,6- tetrafluoropyridin-4- yl)pyrrolidine-2- carboxamide (CDCl.sub.3) δ 7.72-7.54 (m, 4H), 7.53-7.36 (m, 4H), 7.34 (dd, J = 8.0, 1.6 Hz, 1H), 7.29-7.23 (m, 1H), 5.53 (d, J = 7.9 Hz, 1H), 4.69 (s, 1H), 4.03 (s, 1H), 3.89-3.79 (m, 1H), 2.28 (d, J = 6.8 Hz, 2H), 2.19- 2.06 (m, 2H), 2.02-1.89 (m, 2H). 498.8 [M + H].sup.+ 7 (2S)-3-Methyl-N- [(3S)-2-oxo-5- phenyl-2,3-dihydro- 1H-1,4- benzodiazepin-3-yl]- 2-[(2,3,5,6- tetrafluoropyridin-4- yl)amino] butanamide (DMSO-d.sub.6) δ 7.73-7.27 (m, 9H), 5.62 (d, J = 8.0 Hz, 1H), 5.43 (br s, 1H), 4.44 (br s, 1H), 2.24 (br s, 1H), 1.18-1.00 (m, 6H) 499.8 [M + H].sup.+ 8 (2R)-3-Methyl-N- [(3S)-2-oxo-5- phenyl-2,3-dihydro- 1H-1,4- benzodiazepin-3-yl]- 2-[(2,3,5,6- tetrafluoropyridin-4- yl)amino] butanamide (DMSO-d.sub.6) δ 7.81-7.30 (m, 9H), 5.58 (d, J = 8.2 Hz, 1H), 5.06-5.01 (m, 1H), 4.18 (br s, 1H), 2.22 (br s, 1H), 1.13-0.99 (m, 6H) 499.8 [M + H].sup.+ 9 0 (2S)-N-[(3S)-2-oxo- 5-phenyl-1,3- dihydro-1,4- benzodiazepin-3-yl]- 2-[(2,3,5,6- tetrafluoropyridin-4- yl)amino] propanamide (CDCl.sub.3) δ 7.76-7.18 (m, 11H), 5.57 (d, J = 7.9 Hz, 2H), 4.65 (t, J = 7.1 Hz, 1H), 1.61 (d, J = 6.8 Hz, 3H) 471.8 [M + H].sup.+ 10 (2R)-N-[(3S)-2-oxo- 5-phenyl-1,3- dihydro-1,4- benzodiazepin-3-yl]- 2-[(2,3,5,6- tetrafluoropyridin-4- yl)amino] propanamide (CDCl.sub.3) δ 7.65-7.29 (m, 11H), 5.57 (d, J = 7.8 Hz, 1H), 5.38 (d, J = 7.5 Hz, 1H), 4.60 (s, 1H), 1.66 (d, J = 6.9 Hz, 3H) 471.7 [M + H].sup.+ 11 ((2S)-N-[(3S)-2-oxo- 5-phenyl-2,3- dihydro-1H-1,4- benzodiazepin-3-yl]- 3-phenyl-2-[(2,3,5,6- tetrafluoropyridin-4- yl)amino] propanamide (DMSO-d.sub.6) δ 10.93 (s, 1H), 9.64 (d, J = 7.9 Hz, 1H), 7.65 (ddd, J = 8.5, 7.2, 1.7 Hz, 1H), 7.58-7.42 (m, 6H), 7.37-7.23 (m, 5H), 7.19 (t, J = 7.4 Hz, 1H), 7.02 (d, J = 9.4 Hz, 1H), 5.26 (d, J = 7.8 Hz, 1H), 4.92 (t, J = 9.7 Hz, 1H), 3.21-3.06 (m, 1H) 548.2 [M + H].sup.+ 12 2-[[4-(4- acetylpiperazin-1-yl)- 3,5,6- trifluoropyridin-2- yl]amino]-N-[(3S)-2- oxo-5-phenyl-1,3- dihydro-1,4- benzodiazepin-3- y]acetamide (DMSO-d6) δ 10.90 (s, 1H), 9.02 (d, J = 8.1 Hz, 1H), 7.64 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.57-7.40 (m, 5H), 7.37-7.18 (m, 3H), 6.93 (td, J = 6.3, 3.1 Hz, 1H), 5.24 (d, J = 8.0 Hz, 1H), 4.13-3.92 (m, 2H), 3.62-3.48 (m, 4H), 3.26 (d, J = 5.5 Hz, 2H), 2.04 (s, 3H) 566.2 [M + H].sup.+ 13 2-[[4-(1,1-dioxo-1,4- thiazinan-4-yl)- 3,5,6- trifluoropyridin-2- yl]amino]-N-[(3S)-2- oxo-5-phenyl-1,3- dihydro-1,4- benzodiazepin-3- yl]acetamide (DMSO-d6) δ 10.92 (s, 1H), 9.03 (d, J = 8.1 Hz, 1H), 7.64 (ddd, J = 8.6, 7.1, 1.7 Hz, 1H), 7.56-7.39 (m, 5H), 7.35-7.21 (m, 3H), 7.06-6.98 (m, 1H), 5.24 (d, J = 8.0 Hz, 1H), 4.10- 3.93 (m, 2H), 3.72 (br s, 4H), 3.32-3.21 (m, 4H) 573.2 [M + H].sup.+ 14 N-[(3S)-2-oxo-5- phenyl-2,3-dihydro- 1H-1,4- benzodiazepin-3-yl]- 2-{[3,5,6-trifluoro-4- (morpholin-4- yl)pyridin-2- yl]amino}acetamide yl]amino}acetamide (DMSO-d.sub.6) δ 10.91 (s, 1H), 9.02 (d, J = 8.1 Hz, 1H), 7.64 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.56-7.42 (m, 5H), 7.38-7.21 (m, 3H), 6.92 (t, J = 6.4 Hz, 1H), 5.23 (d, J = 8.1 Hz, 1H), 4.14- 3.95 (m, 2H), 3.69 (dd, J = 5.6, 3.7 Hz, 4H), 3.32 (d, J = 5.6, 3.7 Hz, 4H) 525.0 [M + H].sup.+ 15 2-{[4- (diethylamino)-3,5,6- trifluoropyridin-2- yl]amino}-N-[(3S)-2- oxo-5-phenyl-2,3- dihydro-1H-1,4- benzodiazepin-3- yl]acetamide (DMSO-d.sub.6) δ 10.90 (s, 1H), 8.99 (d, J = 8.0 Hz, 1H), 7.63 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.56-7.42 (m, 5H), 7.33-7.21 (m, 3H), 6.80 (t, J = 6.3 Hz, 1H), 5.23 (d, J = 8.0 Hz, 1H), 4.07-3.92 (m, 2H), 3.32- 3.23 (m, 4H), 1.09 (t, J = 7.0 Hz, 6H) 511.2 [M + H].sup.+ 16 N-[(3S)-2-oxo-5- phenyl-2,3-dihydro- 1H-1,4- benzodiazepin-3-yl]- 2-[(2,3,5-trifluoro-6- methanesulfonylpyridin- 4-yl)amino] acetamide (CDCl.sub.3) δ 12.02 (s, 1H), 7.91- 7.75 (m, 4H), 7.75-7.57 (m, 4H), 7.43-7.35 (m, 2H), 6.05 (s, 1H), 5.52 (s, 1H), 4.25 (s, 1H), 4.17- 4.08 (m, J = 1H) , 3.24 (s, 3H) APCI+ 518.0 [M + H].sup.+

Compound 17: 2-{[4-(4,4-Difluoropiperidin-1-yl-6-trifluoropridin-2-yl]amino}-N-[(3S)-2-oxo-1-phenyl-2-dihydro-1H-1,4-benzodiazepin-3-yl]acetamide

(102) ##STR00128##

(103) (3S)-3-Amino-5-phenyl-1,3-dihydro-1,4-benzodiazepin-2-one (90 mg, 0.36 mmol) and 2-[[4-(4,4-difluoropiperidin-1-yl)-3,5,6-trifluoropyridin-2-yl]amino]acetic acid (intermediate 13C) (128 mg, 0.39 mmol, 1.1 eq.) were dissolved in DMF (2 mL). Triethylamine (0.10 mL, 0.72 mmol, 2 eq.) was added followed by HATU (150 mg, 0.39 mmol) The mixture was stirred at rt for 17 h. Water (20 mL) was added, the mixture was extracted with EtOAc (3×15 mL). The organics were washed with water (is mL) and brine (15 mL) and dried (MgSO.sub.4). Flash chromatography on SiO.sub.2 (50-80% EtOAc:heptane) provided the desired compound as a white solid (144 mg, 72%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 10.90 (s, 1H), 9.01 (d, J=8.1 Hz, 1H), 7.64 (ddd, J=8.6, 7-1, 1.7 Hz, 1H), 7.59-7.39 (m, 4H), 7.36-7.23 (m, 3H), 6.94 (t, J=6.4 Hz, 1H), 5.24 (d, J=8.1 Hz, 1H), 4.08-3.93 (m, 2H), 3.50-3.38 (m, 4H), 2.18-2.04 (m, 4H). LRMS APCI+559.2 [M+H].sup.+.

Compound 18: 2-[(6-Chloropyrimidin-4-yl)amino]-N-[(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetamide

Tert-butyl-2{(6-chloropyrimidin-4-yl)amino]acetate

(104) ##STR00129##

(105) 4,6-Dichloropyrimidine (4.00 g, 26.9 mmol) and glycine tert butyl ester hydrochloride (5.50 g, 26.9 mmol) were dissolved in EtOH (30 mL). Diisopropylethylamine (9.35 mL, 53.7 mmol) was added and the resulting solution was heated at 70° C. for 18 h. The mixture was allowed to cool to rt and solvent was removed under reduced pressure. The mixture was diluted with EtOAc (50 mL), washed with water (2×5 mL), brine (50 mL) and dried (MgSO.sub.4). Flash chromatography on SiO.sub.2 [20-50% (50:8:1 CH.sub.2Cl.sub.2:EtOH:NH.sub.4OH in CH.sub.2Cl.sub.2)] provided the desired compound as a white solid (4.85 g, 74%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.38 (d, J=0.9 Hz, 1H), 6.41 (s, 1H), 5.53 (s, 1H), 4.04 (s, 2H), 1.49 (s, 9H). LRMS APCI+243.8 [M+H].sup.+.

2{(6-Chloropyrimidin-4-yl)amino]acetic acid hydrochloride

(106) ##STR00130##

(107) HCl (2M in Et.sub.2O, 2 mL, 4.00 mmol) was added to tert-butyl 2-[(6-chloropyrimidin-4-yl)amino]acetate (100 mg, 0.41 mmol) followed by 1,4-dioxane (5 mL). Water (3 mL) was added to give a solution. The mixture was stirred at rt for 48 h. The mixture was concentrated under reduced pressure providing the desired compound crude (110 mg, 120%) as a white solid. LRMS APCI+541.9 [M+H].sup.+. R.sub.f=0.01 (EtOAc).

2{(6-Chloropyrimidin-4-yl)amino]-N{(3S)-1{(4-methoxyphenyl)methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetamide

(108) ##STR00131##

(109) Prepared by an analogous procedure to that described for intermediate 14A from 2-[(6-chloropyrimidin-4-yl)amino]acetic acid hydrochloride. LRMS APCI+541.9 [M+H].sup.+. R.sub.f=0.35 (200:8:1 CH.sub.2Cl.sub.2:EtOH:NH.sub.4OH).

2{(6-Chloropyrimidin-4-yl)amino]-N{(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetamide

(110) ##STR00132##

(111) Prepared by an analogous procedure to that described for compound 5 from 2-[(6-chloropyrimidin-4-yl)amino]-N-[(3S)-1-[(4-methoxyphenyl)methyl]-2-oxo-5-phenyl-3H-1,4-benzodiazepin-3-yl]acetamide. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 10.90 (s, 1H), 9.24 (d, J=7.8 Hz, 1H), 8.29 (s, 1H), 7.96 (t, J=5.9 Hz, 1H), 7.64 (ddd, J=8.6, 7.2, 1.7 Hz, 1H), 7.58-7.35 (m, 5H), 7.35-7.15 (m, 3H), 6.69 (s, 1H), 5.24 (d, J=8.0 Hz, 1H), 4.25-4.08 (m, 2H). LRMS APCI+422.7 [M+H].sup.+.

Compound 19: 2-{[6-(4 Methanesulfonylpiperazin-1-yl)pyrimidin-4-yl]amino-N-[(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetamide

Tert-butyl 2-{[6-(4-methanesulfonylpiperazin-1-yl)pyrimidin-4-yl]amino}acetate

(112) ##STR00133##

(113) A solution of 1-(methylsulfonyl)piperazine (172.4 mg, 1.05 mmol) and tert-butyl 2-[(6-chloropyrimidin-4-yl)amino]acetate (122 mg, 0.50 mmol) in 1-butanol (2 mL) was heated in an ACE pressure tube at 150° C. for 22 h. The mixture was cooled to rt and the solvent removed under reduced pressure. Flash chromatography on SiO.sub.2 [10-25% (50:8:1 CH.sub.2Cl.sub.2:EtOH:NH.sub.4OH in CH.sub.2Cl.sub.2)] provided the desired compound as an off-white solid (123 mg, 66%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.20 (d, J=0.9 Hz, 1H), 5.48 (s, 1H), 5.12 (s, 1H), 3.99 (d, J=5.3 Hz, 2H), 3.76-3.64 (m, 4H), 3.33-3.18 (m, 4H), 2.79 (s, 3H), 1.48 (s, 7H). LRMS APCI+371.7 [M+H].sup.+.

2-{[6-(4-Methanesulfonylpiperazin-1-yl)pyrimidin-4-yl]amino}acetic acid hydrochloride

(114) ##STR00134##

(115) HCl (2 M in Et2O; 3.38 mL, 6.76 mmol) was added to tert-butyl 2-[[6-(4-methylsulfonylpiperazin-1-yl)pyrimidin-4-yl]amino]acetate (126 mg, 0.34 mmol) and the mixture was stirred at rt for 48 h. Water (3 mL) was added to give a solution and the mixture was stirred for a further 18 h. The mixture was concentrated and used directly without purification. LRMS APCI+371.7 [M+H].sup.+. R.sub.f=0.01 (EtOAc)

2-{[6-(4-Methanesulfonylpiperazin-1-yl)pyrimidin-4-yl]amino}-N{(3S)-1{(4-methoxyphenyl)methyl]-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetamide

(116) ##STR00135##

(117) Prepared by an analogous procedure to that described for intermediate 14A from 2-[[6-(4-methylsulfonylpiperazin-1-yl)pyrimidin-4-yl]amino]acetic acid hydrochloride. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.19 (d, J=0.8 Hz, 1H), 7.96 (d, J=7.6 Hz, 1H), 7.49 (dd, J=6.5, 2.1 Hz, 1H), 7.47-7.41 (m, 2H), 7.35-7.23 (m, 4H), 7.23-7.12 (m, 3H), 6.91-6.87 (m, 2H), 6.64-6.60 (m, 2H), 6.30 (s, 1H), 5.57 (d, J=7.5 Hz, 1H), 5.51 (d, J=15.1 Hz, 1H), 4.71 (d, J=15.1 Hz, 1H), 4.22-4.00 (m, 2H), 3.87-3.67 (m, 4H), 3.67 (s, 3H), 3.35-3.16 (m, 4H), 2.80 (s, 3H). R.sub.f=0.09 (200:8:1CH.sub.2C.sub.2:EtOH:NH.sub.4OH)

2-{[6-(4-Methanesulfonylpiperazin-1-yl)pyrimidin-4-yl]amino}-N{(3S)-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]acetamide

(118) ##STR00136##

(119) Prepared by an analogous procedure to that described for compound 5 from N-[(3S)-1-[(4-methoxyphenyl)methyl]-2-oxo-5-phenyl-3H-1,4-benzodiazepin-3-yl]-2-[[6-(4-methylsulfonylpiperazin-1-yl)pyrimidin-4-yl]amino]acetamide H NMR (400 MHz, DMSO-d.sub.6) δ 10.91 (s, 1H), 9.14 (d, J=8.0 Hz, 1H), 8.13 (s, 1H), 7.70-7.62 (m, 1H), 7.56-7.50 (m, 1H), 7.50-7.43 (m, 4H), 7.38-7.21 (m, 4H), 5.23 (d, J=7.9 Hz, 1H), 4.29-4.11 (m, 2H), 3.57-3.46 (m, 4H), 3.39-3.31 (m, 1H), 3.26-3.13 (m, 4H), 2.90 (s, 3H). R.sub.f=0.03 (200:8:1 CH.sub.2Cl.sub.2:EtOH:NH.sub.4OH).

Example 7: Antiviral and Cell Toxicity Assays

(120) Cells were obtained from ATCC and the virus preparation was subjected to one round of centrifugation through a 40% (V/V) glycerol to remove any interferon produced from infected cells. Growth medium was Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) foetal calf serum (FCS), Viral maintenance medium was DMEM+2% (v/v) FCS.

(121) Second Plaque Reduction Assay

(122) The RSV plaque reduction assay is an infectivity assay which allows quantification of the number of infectious units in a distinct foci of RSV infection. This is indicated by zones of viral antigen detected by specific antibody staining within a monolayer of otherwise healthy tissue culture cells. As each plaque originates from a single infectious virus particle an accurate calculation of the anti-viral effect can be obtained by counting plaques in the presence and absence of an anti-viral compound. HEp-2 cells (ATCC, CCL23) were passaged in flasks and seeded in 96-well plates in DMEM containing antibiotics and supplemented with 10% FBS. During inoculation and subsequent incubation, cells were cultured in DMEM containing 3% FBS. 100 plaque forming unit (PFU)/well of RSV (RSV A2 VR-1540) was mixed with ten serial dilutions of compound. Subsequently, 100 μL of the virus/compound mixtures was added to confluent HEp-2 cell monolayers. The cells and virus/compound mixtures were incubated at 35° C. in a humidified 5% CO2 incubator for 1 day.

(123) Cells were washed twice with PBS before adding 50% v/v EtOH/MeOH, and then stored at −20° C. On the day of the staining, fixative was first removed from the plates. Plates were washed 3× with PBS. A pre-titrated amount of the primary antibody was added in 60 μL PBS/2% milk powder, and plates incubated for 1 h at rt. The plates were washed 3× with PBS/0.05% Tween20 before addition of goat anti-mouse horse radish peroxidase in 60 μL PBS/2% milk powder, and incubated for 1 h at rt. Following three wash steps with PBS/0.05% Tween20, 60 μL ready-to-use TrueBlue was added and plates were incubated at rt for 10-15 min before adding MilliQ water. Plates were washed once with water, incubated for 30-60 min and after removal of water, air-dried in the dark.

(124) Plates were scanned and analyzed using the Immunospot S6 UV analyzer, which is equipped with BioSpot analysis software for counting immunostained plaques (virospots). Plaque counts were used to calculate % infection relative to the mean of the spot count (SC) in the virus control wells for RSV. IC50/IC90 values were calculated as 50% or 90% reduction in signal, respectively, by interpolation of inhibition curves fitted with a 4-parameter nonlinear regression with a variable slope in GraphPad 5.0 (Prism).

(125) Cell Cytotoxicity—CC50 (Concentration at which 50% Cell Toxicity is Observed):

(126) HepG2 cells, in media supplemented with 10% fetal bovine serum (FBS), were seeded at 4×10.sup.5 cells/well into white walled 96 well plates and incubated at 37° C., 5% CO.sub.2. Twenty-four hours post seeding media was removed and replaced with media supplemented with 2% FBS and containing final compound concentrations equal to those tested in parallel virus plaque assays. Following a further 48-hour incubation cell cytotoxicity was determined using the CellTox™ Green (Promega) kit and luminescence read using an appropriate protocol on a GloMax® Explorer System (Promega). During analysis DMSO containing wells were used as a negative control and lysed cells as a positive control.

(127) TABLE-US-00013 TABLE 12 Results of second plaque reduction assay and cell cytotoxicity assay Compound Plaque IC.sub.50 Tox CC.sub.50 Stereochem 9 5.22, 3.76 >25 SS 10 3.38, 1.79 >25 SR 7 5.18, 8.68, 10   16, >25 SS 8 4.73, 8.33, 9.5 >25, 16.3 SR 11 1.69, 5.02 >25, >25  SS 6 4.39, 8.33 >25 SR 14 0.336, 1.26  >25 S 18 4.93, 9.33 >25 S

(128) Multiple values are given in Table 12 where multiple test occasions were carried out.

(129) Results and Discussion

(130) The results show that the above tested compounds may be used to treat RSV.

(131) Furthermore, the compounds are effective at non-toxic concentrations.

CONCLUSION

(132) Compounds described herein have been shown to be inhibitors of RSV. Furthermore, the compounds are effective at non-toxic concentrations. One of these compounds (compound 2) is orally bioavailable in mice.