ANTI-BACTERIAL COMPOUNDS
20180360856 ยท 2018-12-20
Assignee
Inventors
- Ian HOLMES (Stevenage, GB)
- Alan Naylor (Royston, GB)
- Dagmar ALBER (Markyate, GB)
- Jonathan Raymond POWELL (Cambridge, GB)
- Meriel Ruth Major (Cambridge, GB)
- Gabriel NEGOITA-GIRAS (Cambridge, GB)
- Daniel Rees ALLEN (Cambridge, GB)
- Lucie Juliette GUETZOYAN (Cambridge, GB)
- Nigel Paul KING (Stevenage, GB)
Cpc classification
C07F9/6524
CHEMISTRY; METALLURGY
C07F9/65844
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
C07F9/6506
CHEMISTRY; METALLURGY
C07F9/6512
CHEMISTRY; METALLURGY
C07F9/65515
CHEMISTRY; METALLURGY
C07F9/65846
CHEMISTRY; METALLURGY
C07F9/657163
CHEMISTRY; METALLURGY
C07F9/65583
CHEMISTRY; METALLURGY
C07F9/6581
CHEMISTRY; METALLURGY
International classification
Abstract
A compound of Formula (II): for use in the prevention or treatment of a bacterial infection.
##STR00001##
Claims
1. A method of preventing or treating a bacterial infection by administering a compound of Formula (II) or a pharmaceutically acceptable salt, solvate or hydrate thereof: ##STR00177## wherein P.sup.X is selected from the group consisting of (P1), (P2) and (P3); ##STR00178## wherein R.sup.P1 and R.sup.P2 are each independently selected from methyl, ethyl, isopropyl and phenyl; R.sup.P3 is selected from the group consisting of methyl and ethyl , isopropyl, cyclopentyl, t-butyl, phenyl 4-membered or 5-membered heterocycloalkyl group linked to phosphorus via a carbon atom in the ring, including a single heteroatom independently selected from NR.sup.Z, O and S, CF.sub.3, CH.sub.2CF.sub.3, CH.sub.2CF.sub.2H, CH.sub.2CH.sub.2OR.sup.PB, CH.sub.2Q and (CH.sub.2).sub.2Q; wherein Q is a C.sub.5-6 heteroaryl group, optionally substituted with one or more groups R.sup.PA; R.sup.P4 is selected from methyl and ethyl; m is an integer selected from 1, 2 or 3; R.sup.M is one or more optional substituents on the ring independently selected from R.sup.PC when attached to a carbon atom adjacent the phosphorus atom, or OH, OC.sub.1-3alkyl and R.sup.PC, when attached to other ring carbons; L.sup.B is methylene, ethylene or is absent; when L.sup.B is present, R.sup.P4 is absent and R.sup.1 is selected from N, CH and CR.sup.PC; when L.sup.B is absent, R.sup.1 is selected from the group consisting of: O, NR.sup.Z, SO.sub.2, CH.sub.2, CHF, CF.sub.2 and CHR.sup.PC; wherein R.sup.Z is selected from the group consisting of H, C.sub.1-3alkyl, COC.sub.1-3alkyl and SO.sub.2C.sub.1-3alkyl; R.sup.5 and R.sup.8 are each independently selected from H and R.sup.PC; R.sup.6 and R.sup.7 are each independently selected from H and R.sup.PC; wherein R.sup.PC is C.sub.1-3alkyl, optionally substituted with one or more groups R.sup.PD; wherein R.sup.PA is selected from the group consisting of: linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AL; F; Cl; Br; CN; OH; OR.sup.PE; CF.sub.3; CF.sub.2H; COR.sup.PE; CH.sub.2OH; CH.sub.2OR.sup.PE; COOH; COOR.sup.PE; CONH.sub.2; CONHR.sup.PE; CONR.sup.PE.sub.2; OCOR.sup.PE; OCONH.sub.2; OCONHR.sup.PE; OCONR.sup.PE.sub.2; NH.sub.2; NHR.sup.PE; NR.sup.PE.sub.2; SO.sub.2NH.sub.2; SO.sub.2NHR.sup.PE.sub.2; SO.sub.2NR.sup.PE.sub.2; SO.sub.2R.sup.PE; NHCOH; NHCOR.sup.PE; NR.sup.PECOH and NR.sup.PECOR.sup.PE; and R.sup.PB is selected from the group consisting of: linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AT; C.sub.3-6cycloalkyl, C.sub.4-6heterocycloalkyl, C.sub.5-6cycloalkenyl or C.sub.5-6heterocycloalkenyl optionally substituted with one or more groups R.sup.AT; phenyl optionally substituted with one or more groups R.sup.AR; and C.sub.5-6heteroaryl optionally substituted with one or more groups R.sup.AR; R.sup.PE is selected from linear or branched C.sub.1-4alkyl optionally substituted with one or more groups R.sup.PD; and R.sup.PD is selected from the group consisting of: F, OH and OC.sub.1-3alkyl; L.sup.A is selected from methylene optionally substituted with one or two groups R.sup.1A1, ethylene optionally substituted with one or more groups R.sup.1A1, and a single bond; R.sup.A is selected from the group consisting of (i) 5-membered heteroaromatic groups containing at least one heteroatom selected from N, O and S optionally C-substituted with one or more groups R.sup.A1, and optionally N-substituted with one or more groups R.sup.NA1, (ii) 6-membered aromatic groups or heteroaromatic groups containing 1 to 3 N atoms, substituted with one or more groups R.sup.A1, (iii) 8- to 10-membered bicyclyl or heterobicyclyl groups with the proviso that R.sup.A is not selected from the group (A3) or the groups (X3a) to (X3b) ##STR00179## wherein one of Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 is selected from CH and N, and the others are CH; and X is independently selected from NH, S and O; and (iv) the groups (C1) to (C6) ##STR00180## with the proviso that R.sup.A is not the group (C3) when L is a single bond; Z.sup.3 is selected from the group consisting of CH.sub.2, CHR.sup.AL and CR.sup.AL.sub.2; one of Z.sup.1, Z.sup.2, Z.sup.4 and Z.sup.5 is selected from the group consisting of: CH.sub.2; CHR.sup.AL; CR.sup.AL.sub.2; O; NH; NR.sup.A2; N(COR.sup.A2); N(CONHR.sup.A2); N(SO.sub.2R.sup.A2) and N(CO.sub.2R.sup.A4); the remainder of Z.sup.1, Z.sup.2, Z.sup.4 and Z.sup.5 are independently selected from the group consisting of: CH.sub.2; CHR.sup.AL; CR.sup.AL.sub.2 and O; with the provisos that the ring contains 0 or 1 oxygen atoms, that nitrogen atoms cannot be in a 1,2 or 1,3 relationship to each other, and that when Z.sup.1 or Z.sup.5 is N, L cannot be a single bond; one of Q.sup.1 to Q.sup.4 is selected from the group consisting of: O; NH; NR.sup.A2; CH.sub.2; CHR.sup.AL; CR.sup.AL.sub.2; NCOR.sup.A2; NCONHR.sup.A2; NSO.sub.2R.sup.A2 and NCO.sub.2R.sup.A4; the remainder of Q.sup.1 to Q.sup.4 are independently selected from the group consisting of: NH; NR.sup.A2; CH.sub.2; CHR.sup.AL and CR.sup.AL.sub.2; with the proviso that the ring contains 0 or 1 oxygen atoms, that the ring contains 0 or 1 nitrogen atoms, and that when Q.sup.1 or Q.sup.4 is N, L cannot be a single bond; E.sup.A is selected from the group consisting of: OR.sup.A2; NHR.sup.A2; NR.sup.A2.sub.2; NR.sup.EA1-E.sup.A1-COR.sup.EA2 and NR.sup.EA1-E.sup.A2-E.sup.A3-COR.sup.EA2; wherein E.sup.A1, E.sup.A2 and E.sup.A3 are D- or L-amino acid residues independently selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, wherein the NR.sup.EA1 and COR.sup.EA2 groups represent terminals of the alpha or pendent functionality of the amino acids respectively; wherein the amino acid residues Asp and Glu may form amide bonds from either the alpha or pendent carboxylic acid functionality; when E.sup.A1 is Pro, R.sup.EA1 is absent, otherwise R.sup.EA1 is R.sup.E1; when E.sup.A2 is Pro, R.sup.EA1 is absent, otherwise R.sup.EA1 is R.sup.E1; wherein the acid functionality of Asp and Glu not forming an amide bond may be present as the corresponding amides or esters selected from CONH.sub.2, CONHR.sup.A2, CONR.sup.A2R.sup.E1 and COOR.sup.A2; and the hydroxyl side chain groups of Ser, Thr and Tyr may be present as their corresponding alkoxy or acetate groups selected from O(C.sub.1-3alkyl) and OCOCH.sub.3; and when E.sup.A2 and E.sup.A3 are present and E.sup.A3 is not Pro the nitrogen of the amide bond between E.sup.A2 and E.sup.A3 may be optionally substituted with R.sup.E1; R.sup.EA2 is selected from OR.sup.E7, NH.sub.2, NHR.sup.A2 and NR.sup.A2R.sup.E1; R.sup.E1 is selected from H and linear or branched C.sub.1-3alkyl; E.sup.B is selected from: E.sup.BA; CO-E.sup.B1-NR.sup.EAR.sup.E2 and CO-E.sup.B2-E.sup.B3-NR.sup.EBR.sup.E2; wherein E.sup.B1, E.sup.B2 and E.sup.B3 are D- or L-amino acid residues independently selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, wherein the CO, NR.sup.EAR.sup.E2 and NR.sup.EBR.sup.E2 groups represent terminals of the alpha or pendent functionality of the amino acids; wherein the amino acid residues Asp and Glu may form amide bonds from either the alpha or pendent carboxylic acid functionality when E.sup.B1 is Pro, R.sup.EA is absent, otherwise R.sup.EA is R.sup.E1; when E.sup.B3 is Pro, R.sup.EB is absent, otherwise R.sup.EB is R.sup.E1; wherein the acid functionality of Asp and Glu not forming an amide bond may be present as the corresponding amides or esters selected from CONH.sub.2, CONHR.sup.A2, CONR.sup.A2R.sup.E1 and COOR.sup.A2; and the hydroxyl side chain groups of Ser, Thr and Tyr may be present as their corresponding alkoxy or acetate groups selected from O(C.sub.1-3alkyl) and OCOCH.sub.3; and when E.sup.B2 and E.sup.B3 are present and E.sup.B2 is not Pro the nitrogen of the amide bond between E.sup.B2 and E.sup.B3 may be optionally substituted with R.sup.E1; when E.sup.B is E.sup.BA, R.sup.E1 and E.sup.BA together with the nitrogen atom to which they are attached form a group selected from: 5- or 6-membered saturated heterocyclyl optionally substituted with one or more groups R.sup.AL, and 5- or 6-membered heteroaryl optionally substituted with one or more groups R.sup.A1; E.sup.C is selected from: OH; OR.sup.A2; NH.sub.2; NHR.sup.A2; NR.sup.A2.sub.2 and NR.sup.EC1-E.sup.C1-COR.sup.EC2; wherein E.sup.C1 is a D- or L-amino acid residue selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, wherein the NR.sup.EC1 and COR.sup.EC2 groups represent terminals of the alpha or pendent functionality of the amino acids; wherein the amino acid residues Asp and Glu may form amide bonds from either the alpha or pendent carboxylic acid functionality; when E.sup.C1 is Pro, R.sup.EC1 is absent, otherwise R.sup.EC1 is R.sup.E1; wherein the acid functionality of Asp and Glu not forming an amide bond may be present as the corresponding amides or esters selected from CONH.sub.2, CONHR.sup.A2, CONR.sup.A2R.sup.E1 and COOR.sup.A2; and the hydroxyl side chain groups of Ser, Thr and Tyr may be present as their corresponding alkoxy or acetate groups selected from O(C.sub.1-3alkyl) and OCOCH.sub.3; R.sup.EC2 is selected from OR.sup.E9, NH.sub.2, NHR.sup.A2 and NR.sup.A2R.sup.E1; R.sup.E3 and R.sup.E4 are independently selected from H and CH.sub.3; when R.sup.E1 is H and E.sup.C is OC.sub.1-3alkyl, NH.sub.2 or NHC.sub.1-3alkyl, E.sup.D is selected from H, and CO-E.sup.D1-NR.sup.EDR.sup.E6 otherwise, E.sup.D is selected from: R.sup.E5, and CO-E.sup.D1-NR.sup.EDR.sup.E6; wherein E.sup.D1 is a D- or L-amino acid residue selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, wherein the NR.sup.EDR.sup.E6 and CO groups represent terminals of the alpha or pendent functionality of the amino acids; wherein the amino acid residues Asp and Glu may form amide bonds from either the alpha or pendent carboxylic acid functionality; wherein the acid functionality of Asp and Glu not forming an amide bond may be present as the corresponding amides or esters selected from CONH.sub.2, CONHR.sup.A2, CONR.sup.A2R.sup.E1 and COOR.sup.A2; and the hydroxyl side chain groups of Ser, Thr and Tyr may be present as their corresponding alkoxy or acetate groups selected from O(C.sub.1-3alkyl) and OCOCH.sub.3; when E.sup.D1 is Pro, R.sup.ED is absent, otherwise R.sup.ED is R.sup.E1; R.sup.E2, R.sup.E5 and R.sup.E6 are independently selected from H and COCH.sub.3; R.sup.E7, R.sup.E8 and 8 and R.sup.E9 are each independently selected from H and R.sup.A2; Z.sup.6 is selected from NCOR.sup.RA2, NCONHR.sup.A2, NSO.sub.2R.sup.A2; R.sup.Z6 is one or two optional methyl substituents; R.sup.A1 is selected from the group consisting of: linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AL, F, Cl, Br, CN OH, OR.sup.A2, CF.sub.3, CF.sub.2H, COR.sup.A2, CH.sub.2OH, CH.sub.2OR.sup.A2, COOH, COOR.sup.A2, CONH.sub.2, CONHR.sup.A2, CONR.sup.A2.sub.2, OCOR.sup.A2, OCONH.sub.2, OCONHR.sup.A2, OCONR.sup.A2.sub.2, NH.sub.2, NHR.sup.A2, NR.sup.A2.sub.2, SO.sub.2NH.sub.2, SO.sub.2NHR.sup.A2.sub.2, SO.sub.2NR.sup.A2.sub.2, SO.sub.2R.sup.A2, NHCOH, NHCOR.sup.A2, NR.sup.A2COH and NR.sup.A2COR.sup.A2; R.sup.A2 is selected from the group consisting of: linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AT, wherein the alkyl chain is optionally interrupted by one or more atoms selected from O and S; OC.sub.1-6alkyl; C.sub.3-6cycloalkyl, C.sub.4-6heterocycloalkyl, C.sub.5-6cycloalkenyl or C.sub.5-6heterocycloalkenyl optionally substituted with one or more groups R.sup.AT; phenyl optionally substituted with one or more groups R.sup.AR, and C.sub.5-6heteroaryl optionally substituted with one or more groups R.sup.AR; where N is substituted by 2 R.sup.A2 groups, the N and the R.sup.A2 groups may together form a N-containing C.sub.5-6 heterocycloalkyl group, optionally substituted with one or two groups selected from linear unsubstituted C.sub.1-6 alkyl; R.sup.NA1 is selected from linear or branched C.sub.1-4alkyl; R.sup.1A1 is selected from linear or branched unsubstituted C.sub.1-3alkyl; R.sup.A3 is selected from H and unbranched unsubstituted C.sub.1-3alkyl; R.sup.A4 is selected from linear or branched unsubstituted C.sub.1-4alkyl; R.sup.AL is selected from the group consisting of: F, CN OH, OR.sup.A2, CF.sub.3, CF.sub.2H. COR.sup.A2, COOH, COOR.sup.A2, CONH.sub.2, CONHR.sup.A2, CONR.sup.A2.sub.2, OCOR.sup.A2, OCONH.sub.2, OCONHR.sup.A2, OCONR.sup.A2.sub.2, NH.sub.2, NHR.sup.A2, NR.sup.A2.sub.2, SO.sub.2NH.sub.2, SO.sub.2NHR.sup.A2.sub.2, SO.sub.2NR.sup.A2.sub.2, SO.sub.2R.sup.A2, NHCOH, NHCOR.sup.A2, NR.sup.A2COH and NR.sup.A2COR.sup.A2; and wherein R.sup.AR is selected from the group consisting of linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AL, F, Cl, Br, CN OH, OR.sup.1A1, CF.sub.3, CF.sub.2H, COR.sup.1A1, CH.sub.2OH, CH.sub.2OR.sup.1A1, CHR.sup.1A1OH, CHR.sup.1A1OR.sup.1A1 COOH, COOR.sup.1A1, CONH.sub.2, CONHR.sup.1A1, CONR.sup.1A1.sub.2, OCOR.sup.1A1, OCONH.sub.2, OCONHR.sup.1A1, OCONR.sup.1A1.sub.2, NH.sub.2, NHR.sup.1A1, NR.sup.1A1.sub.2, SO.sub.2NH.sub.2, SO.sub.2NHR.sup.1A1.sub.2, SO.sub.2NR.sup.1A1.sub.2, SO.sub.2R.sup.1A1, NHCOH, -NHCOR.sup.1A1, NR.sup.1A1COH and NR.sup.1A1COR.sup.1A1; R.sup.AT is selected from the group consisting of F, CN OH, OC.sub.1-3alkyl, CF.sub.3, CF.sub.2H, COC.sub.1-3alkyl, COOH, COOC.sub.1-3alkyl, CONH.sub.2, CONHC.sub.1-3alkyl, CON(C.sub.1-3alkyl).sub.2, OCOC.sub.1-3alkyl, OCONH.sub.2, OCONHC.sub.1-3alkyl, OCON(C.sub.1-3alkyl).sub.2, NH.sub.2, NHC.sub.1-3alkyl, N(C.sub.1-3alky).sub.2, SO.sub.2NH.sub.2, SO.sub.2NH(C.sub.1-3alkyl).sub.2, SO.sub.2N(C.sub.1-3alkyl).sub.2, SO.sub.2(C.sub.1-3alkyl), NHCOH, NHCO(C.sub.1-3alkyl), N(C.sub.1-3alkyl)COH and N(C.sub.1-3alkyl)CO(C.sub.1-3alkyl).
2. The method according to claim 1, wherein P.sup.X is P1, and either R.sup.P3 is methyl; or R.sup.P3 is ethyl; or R.sup.P3 is oxetanyl or tetrahydrofuranyl; or R.sup.P3 is selected from the group consisting of CF.sub.3, CH.sub.2CF.sub.3, CH.sub.2CF.sub.2H and CH.sub.2CH.sub.2OR.sup.PB, where R.sup.PB is a linear or branched C.sub.1-6 alkyl; or R.sup.P3 is CH.sub.2Q.
3-9. (canceled)
10. The method according to claim 1, wherein P.sup.X is P2, and either R.sup.P4 is methyl; or R.sup.P4 is ethyl.
11-15. (canceled)
16. The method according to claim 1, wherein P.sup.X is P3, L.sup.B is methylene or ethylene and either R.sup.1 is N; or R.sup.1 is CH; or R.sup.1 is CR.sup.PC, wherein R.sup.PC is unsubstituted C.sub.1-3 alkyl.
17-21. (canceled)
22. The method according to claim 1, wherein P.sup.X is selected from the group consisting of: ##STR00181##
23-29. (canceled)
30. The method according to claim 1, wherein R.sup.A is a 5-membered heteroaromatic group containing up to 4 heteroatoms selected from N, O and S, at least one of which being N.
31-46. (canceled)
47. The method according to claim 1, wherein R.sup.A is a 6-membered heteroaryl group containing one or two nitrogen atoms, substituted with one or more groups R.sup.A1.
48-53. (canceled)
54. The method according to claim 1, wherein R.sup.A is a 8- to 10-membered heterobicyclyl group containing one or more heteroatoms independently selected from N, O and S.
55. (canceled)
56. The method according to claim 1, wherein either R.sup.A is the group (C1): ##STR00182## wherein Z.sup.3 is selected from the group consisting of CH.sub.2, CHF and CF.sub.2; one of Z.sup.1, Z.sup.2, Z.sup.4 and Z.sup.5 is selected from the group consisting of: CH.sub.2; CHR.sup.AL; CR.sup.AL.sub.2; O; NH; NR.sup.A2; N(COR.sup.A2); N(CONHR.sup.A2); N(SO.sub.2R.sup.A2) and N(CO.sub.2R.sup.A4); and the remainder of Z.sup.1, Z.sup.2, Z.sup.4 and Z.sup.5 are independently selected from the group consisting of: CH.sub.2; CHR.sup.AL; CR.sup.AL.sub.2; and O; with the provisos that the ring contains 0 or 1 oxygen atoms, that nitrogen atoms cannot be in a 1,2 or 1,3 relationship to each other, and that when Z.sup.1 or Z.sup.5 is N, L cannot be a single bond; or R.sup.A is the group (C.sub.2) ##STR00183## wherein one of Q.sup.1 to Q.sup.4 is selected from the group consisting of: O; NH; NR.sup.A2; CH .sub.2; CHR.sup.AL; CR.sup.AL.sub.2; NCOR.sup.A2; NCONHR.sup.A2; NSO.sub.2R.sup.A2 and NCO.sub.2R.sup.A4; and the remainder of Q.sup.1 to Q.sup.4 are independently selected from the group consisting of: CH.sub.2; CHR.sup.AL and CR.sup.AL.sub.2. with the proviso that the ring contains 0 or 1 oxygen atoms, that the ring contains 0 or 1 nitrogen atoms, and that when Q.sup.1 or Q.sup.4 is N, L cannot be a single bond.
57-92. (canceled)
93. The method according to claim 1, wherein R.sup.A is selected from the group consisting of: ##STR00184## ##STR00185## ##STR00186##
94. The method according to claim 1, wherein R.sup.A is the group (C6): ##STR00187## wherein Z.sup.6 is selected from NCOR.sup.A2 and NCONHR.sup.A2; and R.sup.Z6 is one or two optional methyl substituents.
95. A method of preventing or treating a bacterial infection by administering a compound according to Formula (I) or a pharmaceutically acceptable salt, solvate or hydrate thereof: ##STR00188## wherein P.sup.Y is independently selected from the group consisting of (P1), (P2) and (P3); ##STR00189## wherein L.sup.C is methylene, ethylene or is absent; R.sup.P1 and R.sup.P2 are each independently selected from methyl; when L.sup.C is absent R.sup.P3 is selected from the group consisting of 4-membered or 5-membered heterocycloalkyl group linked to phosphorus via a carbon atom in the ring, including a single heteroatom independently selected from NR.sup.Z, O and S, CF.sub.3, CH.sub.2CF.sub.3, CH.sub.2CF.sub.2H, CH.sub.2CH.sub.2OR.sup.PB, CH.sub.2Q and (CH.sub.2).sub.2Q; when L.sup.C is methylene or ethylene R.sup.P3 is selected from the group consisting of methyl and ethyl, 4-membered or 5-membered heterocycloalkyl group linked to phosphorus via a carbon atom in the ring, including a single heteroatom independently selected from NR.sup.Z, O and S, CF.sub.3, CH.sub.2CF.sub.3, CH.sub.2CF.sub.2H, CH.sub.2CH.sub.2OR.sup.PB, CH.sub.2Q and -(CH.sub.2).sub.2Q; wherein Q is a C.sub.5-6 heteroaryl group, optionally substituted with one or more groups R.sup.PA; R.sup.P4 is selected from methyl and ethyl; m is an integer selected from 1, 2 or 3; R.sup.M is one or more optional substituents on the ring independently selected from R.sup.PC when attached to a carbon atom adjacent the phosphorus atom, or OH, OC.sub.1-3alkyl and R.sup.PC, when attached to other ring carbons; L.sup.B is methylene, ethylene or is absent; when L.sup.B is present, R.sup.P4 is absent and R.sup.1 is selected from N, CH and CR.sup.PC; when L.sup.B is absent, R.sup.1 is selected from the group consisting of O , NR.sup.Z, SO.sub.2, CH.sub.2, CHF, CF.sub.2 and CHR.sup.P; wherein R.sup.Z is selected from the group consisting of H, C.sub.1-3alkyl, COC.sub.1-3alkyl and SO.sub.2C.sub.1-3alkyl; R.sup.5 and R.sup.8 are each independently selected from H and R.sup.PC; R.sup.6 and R.sup.7 are each independently selected from H and R.sup.PC; wherein R.sup.PC is selected from the group consisting of C.sub.1-3alkyl, optionally substituted with one or more groups R.sup.PD; wherein R.sup.PA is selected from the group consisting of linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AL, F, Cl, Br, CN OH, OR.sup.PE, CF.sub.3, CF.sub.2H, COR.sup.PE, CH.sub.2OH, CH.sub.2OR.sup.PE, COOH, COOR.sup.PE, CONH.sub.2, CONHR.sup.PE, CONR.sup.PE.sub.2, OCOR.sup.PE, OCONH.sub.2, OCONHR.sup.PE, OCONR.sup.PE.sub.2, NH.sub.2, NHR.sup.PE, NR.sup.PE.sub.2, SO.sub.2NH.sub.2, SO.sub.2NHR.sup.PE.sub.2, SO.sub.2NR.sup.PE.sub.2, SO.sub.2R.sup.PE, NHCOH, NHCOR.sup.PE, NR.sup.PECOH and NR.sup.PECOR.sup.PE; and R.sup.PB is selected from the group consisting of linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AT, C.sub.3-6cycloalkyl, C.sub.4-6heterocycloalkyl, C.sub.5-6cycloalkenyl or C.sub.5-6heterocycloalkenyl optionally substituted with one or more groups R.sup.AT, phenyl optionally substituted with one or more groups R.sup.AR, and C.sub.5-6heteroaryl optionally substituted with one or more groups R.sup.AR; R.sup.PE is selected from linear or branched C.sub.1-4alkyl optionally substituted with one or more groups R.sup.PD; and R.sup.PD is selected from the group consisting of F, OH and OC.sub.1-3alkyl. R.sup.B is independently selected from the groups (A1) to (A5) ##STR00190## wherein each of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 is independently selected from CH or N; wherein at least three of Y.sub.1, Y.sup.2, Y.sup.3, Y4 and Y.sup.9 are independently CH; V is independently selected from O, CHOR.sup.O1, NCOR.sup.C8, NCONHR.sup.C8, NSO.sub.2R.sup.C8, NCO.sub.2R.sup.C2 and NR.sup.N2; one of Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 is selected from CH and N, and the others are CH; X is independently selected from NH, S and O; R.sup.C1 is selected from OR.sup.O2 or NHR.sup.N1; R.sup.O1 is selected from H and C.sub.1-3 unbranched alkyl; R.sup.O2 is selected from H and C.sup.1-3 unbranched alkyl; R.sup.N1 is selected from H and C.sup.1-3 unbranched alkyl; R.sup.N2 is C.sub.1-3 unbranched alkyl; R.sup.C2 and R.sub.C8 are each independently selected from C.sup.1-3 unbranched alkyl and C.sub.34 branched alkyl; R.sup.C3 is selected from C.sub.1-3 unbranched alkyl and C.sub.2H.sub.4CO.sub.2H; R.sup.C4 is either H or Me; R.sup.C5 is either H or Me; R.sup.C6 represents one or two optional methyl substituents; R.sup.C7 is selected from H and COCH.sub.3; and n is an integer selected from 2 to 8.
96. The method according to claim 95, wherein P.sup.Y is P1, and either R.sup.P3 is methyl; or R.sup.P3 is ethyl; or R.sup.P3 is oxetanyl or tetrahydrofuranyl; or R.sup.P3 is selected from the group consisting of CF.sub.3, CH.sub.2CF.sub.3, CH.sub.2CF.sub.2H and CH.sub.2CH.sub.2OR.sup.PB where R.sup.PB is a linear or branched C.sub.1-6 alkyl; or R.sup.P3 is CH.sub.2Q.
97-103. (canceled)
104. The method according to claim 95, wherein P.sup.Y is P2, and either R.sup.P4 is methyl; or R.sup.P4 is ethyl.
105-109. (canceled)
110. The method according to claim 95, wherein P.sup.Y is P3 and either R.sup.1 is N; or R.sup.1 is CH; or R.sup.1 is CR.sup.PC, wherein R.sup.PC is unsubstituted C.sub.1-3 alkyl.
111-115. (canceled)
116. The method according to claim 95, wherein P.sup.Y is selected from the group consisting of: ##STR00191##
117. The method according to claim 95, wherein R.sup.B is A1: ##STR00192## and either one of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 is N; or two of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 are N; or R.sup.B is phenyl.
118-120. (canceled)
121. The method according to claim 95, wherein R.sup.B is A2: ##STR00193## and either V is O; or V is CHOR.sup.O1; or V is NOC.sub.2R.sup.C2; or V is NR.sup.N2.
122-131. (canceled)
132. The method according to claim 95, wherein R.sup.B is A3: ##STR00194## and either X is O and one of Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 is N; or X is NH and Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 are CH.
133-134. (canceled)
135. The method according to claim 95, wherein either R.sup.B is A4: ##STR00195## wherein either R.sup.C1 is OR.sup.O2 where R.sup.O2 is methyl; or R.sup.C1 is NHR.sup.N1, and R.sup.N1 is H; or R.sup.B is A5: ##STR00196##
136-145. (canceled)
146. The method according to claim 1, wherein the bacterial infection prevented and/or treated is infection by one or more Gram-positive bacteria; or wherein the bacterial infection prevented and/or treated is infection by one or more Gram-negative bacteria.
147-166. (canceled)
167. A compound of either Formula (I) or Formula (II): ##STR00197## or a pharmaceutically acceptable salt, solvate or hydrate thereof wherein P.sup.X is selected from the group consisting of (P1), (P2) and (P3); ##STR00198## wherein R.sup.P1 and R.sup.P2 are each independently selected from methyl, ethyl, isopropyl and phenyl; R.sup.P3 is selected from the group consisting of methyl and ethyl , isopropyl, cyclopentyl, t-butyl, phenyl 4-membered or 5-membered heterocycloalkyl group linked to phosphorus via a carbon atom in the ring, including a single heteroatom independently selected from NR.sup.Z, O and S CF.sub.3, CH.sub.2CF.sub.3, CH.sub.2CF.sub.2H, CH.sub.2CH.sub.2OR.sup.PB, CH.sub.2Q and (CH.sub.2).sub.2Q; wherein Q is a C.sub.5-6 heteroaryl group, optionally substituted with one or more groups R.sup.PA; R.sup.P4 is selected from methyl and ethyl; m is an integer selected from 1, 2 or 3; R.sup.M is one or more optional substituents on the ring independently selected from R.sup.PC when attached to a carbon atom adjacent the phosphorus atom, or OH, OC.sup.1-3alkyl and R.sup.PC, when attached to other ring carbons; L.sup.B is methylene, ethylene or is absent when L.sup.B is present, R.sup.P4 is absent and R.sup.1 is selected from N, CH and CR.sup.PC; when L.sup.B is absent, R.sup.1 is selected from the group consisting of: O, NR.sup.Z, SO.sub.2 CH.sub.2, CHF, CF.sub.2 and CHR.sup.PC; wherein R.sup.Z is selected from the group consisting of H, C.sup.1-3alkyl, COC.sup.1-3alkyl and SO.sub.2C.sub.1-3alkyl; R.sup.5 and R.sup.8 are each independently selected from H and R.sup.PC; R.sup.6 and R.sup.7 are each independently selected from H and R.sup.PC; wherein R.sup.PC is C.sup.1-3alkyl, optionally substituted with one or more groups R.sup.PD; wherein R.sup.PA is selected from the group consisting of: linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AL; F; Cl; Br; CN; OH; OR.sup.PE; CF.sub.3; CF.sub.2H; COR.sup.PE; CH.sub.2OH; CH.sup.2OR.sup.PE; COOH; COOR.sup.PE; CONH.sub.2; CONHR.sup.PE; CONR.sup.PE.sub.2; OCOR.sup.PE; OCONH.sub.2; OCONHR.sup.PE; OCONR.sup.PE.sub.2; NH.sub.2; NHR.sup.PE; NR.sup.PE.sub.2; SO.sub.2NH.sub.2; SO.sub.2NHR.sup.PE.sub.2; SO.sub.2NR.sup.PE.sub.2; SO.sub.2R.sup.PE; NHCOH; NHCOR.sup.PE; NR.sup.PECOH and NR.sup.PECOR.sup.PE; and R.sup.PB is selected from the group consisting of: linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AT; C.sub.3-6cycloalkyl, C.sub.4-6heterocycloalkyl, C.sub.5-6cycloalkenyl or C.sub.5-6heterocycloalkenyl optionally substituted with one or more groups R.sup.AT; phenyl optionally substituted with one or more groups R.sup.AR; and C.sub.5-6heteroaryl optionally substituted with one or more groups R.sup.AR; R.sup.PE is selected from linear or branched C.sub.1-4alkyl optionally substituted with one or more groups R.sup.PD; and R.sup.PD is selected from the group consisting of: F, OH and OC.sup.1-3alkyl; L.sup.A is selected from methylene optionally substituted with one or two groups R.sup.1A1, ethylene optionally substituted with one or more groups R.sup.1A1, and a single bond; R.sup.A is selected from the group consisting of (i) 5-membered heteroaromatic groups containing at least one heteroatom selected from N, O and S optionally C-substituted with one or more groups R.sup.A1, and optionally N-substituted with one or more groups R.sup.NA1, (ii) 6-membered aromatic groups or heteroaromatic groups containing 1 to 3 N atoms, substituted with one or more groups , (iii) 8- to 10-membered bicyclyl or heterobicyclyl groups with the proviso that R.sup.A is not selected from the group (A3) or the groups (X3a) to (X3b) ##STR00199## wherein one of Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 is selected from CH and N, and the others are CH; and X is independently selected from NH, S and O; and (iv) the groups (C1) to (C6) ##STR00200## with the proviso that R.sup.A is not the group (C3) when L is a single bond; Z.sup.3 is selected from the group consisting of CH.sub.2, CHR.sup.AL and CR.sup.AL.sub.2; one of Z.sup.1, Z.sup.2, Z.sup.4 and Z.sup.5 is selected from the group consisting of: CH.sub.2; CHR.sup.AL; CR.sup.AL.sub.2; O; NH; NR.sup.A2; N(COR.sup.A2); N(CONHR.sup.A2); N(SO.sub.2R.sup.A2) and N(CO.sub.2R.sup.A4); the remainder of Z.sup.1, Z.sup.2, Z.sup.4 and Z.sup.5 are independently selected from the group consisting of: CH.sub.2; CHR.sup.AL; CR.sup.AL.sub.2 and O; with the provisos that the ring contains 0 or 1 oxygen atoms, that nitrogen atoms cannot be in a 1,2 or 1,3 relationship to each other, and that when Z.sup.1 or Z.sup.5 is N, L cannot be a single bond; one of Q.sup.1 to Q.sup.4 is selected from the group consisting of: O; NH; NR.sup.A2; CH.sub.2; CHR.sup.AL; CR.sup.AL.sub.2; NCOR.sup.A2; NCONHR.sup.A2; NSO.sub.2R.sup.A2 and NCO.sub.2R.sup.A4; the remainder of Q.sup.1 to Q.sup.4 are independently selected from the group consisting of: NH; NR.sup.A2; CH.sub.2; CHR.sup.AL and CR.sup.AL.sub.2; with the proviso that the ring contains 0 or 1 oxygen atoms, that the ring contains 0 or 1 nitrogen atoms, and that when Q.sup.1 or Q.sup.4 is N, L cannot be a single bond; E.sup.A is selected from the group consisting of: OR.sub.A2; NHR.sup.A2; NR.sup.A2.sub.2; NR.sup.EA1-E.sup.A1-COR.sup.EA2 and NR.sup.EA1-E.sup.A2-E.sup.A3-COR.sup.EA2; wherein E.sup.A1, E.sup.A2 and E.sup.A3 are D- or L-amino acid residues independently selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, wherein the NR.sup.EA1 and COR.sup.EA2 groups represent terminals of the alpha or pendent functionality of the amino acids respectively; wherein the amino acid residues Asp and Glu may form amide bonds from either the alpha or pendent carboxylic acid functionality; when E.sup.A1 is Pro, R.sup.EA1 is absent, otherwise R.sup.EA1 is R.sup.E1; when E.sup.A2 is Pro, R.sup.EA1 is absent, otherwise R.sup.EA1 is R.sup.E1; wherein the acid functionality of Asp and Glu not forming an amide bond may be present as the corresponding amides or esters selected from CONH.sub.2, CONHR.sup.A2, CONR.sup.A2R.sup.E1 and COOR.sup.A2; and the hydroxyl side chain groups of Ser, Thr and Tyr may be present as their corresponding alkoxy or acetate groups selected from O(C.sub.1-3alkyl) and OCOCH.sub.3; and when E.sup.A2 and E.sup.A3 are present and E.sup.A3 is not Pro the nitrogen of the amide bond between E.sup.A2 and E.sup.A3 may be optionally substituted with R.sup.E1; R.sup.EA2 is selected from OR.sub.E7, NH.sub.2, NHR.sup.A2 and NR.sup.A2R.sup.E1; R.sup.E1 is selected from H and linear or branched C.sup.1-3alkyl; E.sup.B is selected from: E.sup.BA; CO-E.sup.B1-NR.sup.EAR.sup.E2 and CO-E.sup.B2-E.sup.B3-NR.sup.EB-R.sup.E2. wherein E.sup.B1, E.sup.B2 and E.sup.B3 are D- or L-amino acid residues independently selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, wherein the CO, NR.sup.EAR.sup.E2 and NR.sup.EBR.sup.E2 groups represent terminals of the alpha or pendent functionality of the amino acids; p1 wherein the amino acid residues Asp and Glu may form amide bonds from either the alpha or pendent carboxylic acid functionality when E.sup.B1 is Pro, R.sup.EA is absent, otherwise R.sup.EA is R.sup.E1; when E.sup.B3 is Pro, R.sup.EB is absent, otherwise R.sup.EB is R.sup.E1; wherein the acid functionality of Asp and Glu not forming an amide bond may be present as the corresponding amides or esters selected from CONH.sub.2, CONHR.sup.A2, CONR.sup.A2R.sup.E1 and COOR.sup.A2; and the hydroxyl side chain groups of Ser, Thr and Tyr may be present as their corresponding alkoxy or acetate groups selected from O(C.sup.1-3alkyl) and OCOCH.sub.3; and when E.sup.B2 and E.sup.B3 are present and E.sup.B2 is not Pro the nitrogen of the amide bond between E.sup.B2 and E.sup.B3 may be optionally substituted with R.sup.E1; when E.sup.B is E.sup.BA, R.sup.E1 and E.sup.BA together with the nitrogen atom to which they are attached form a group selected from: 5- or 6-membered saturated heterocyclyl optionally substituted with one or more groups R.sup.AL, and 5- or 6-membered heteroaryl optionally substituted with one or more groups R.sup.A1; E.sup.C is selected from: OH; OR.sup.A2; NH.sub.2; NHR.sup.A2; NR.sup.A2.sub.2and NR.sup.EC1-E.sup.C1-COR.sup.EC2; wherein E.sup.C1 is a D- or L-amino acid residue selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, wherein the NR.sup.EC1 and COR.sup.EC2 groups represent terminals of the alpha or pendent functionality of the amino acids; wherein the amino acid residues Asp and Glu may form amide bonds from either the alpha or pendent carboxylic acid functionality; when E.sup.C1 is Pro, R.sup.EC1 is absent, otherwise R.sup.EC1 is R.sup.E1; wherein the acid functionality of Asp and Glu not forming an amide bond may be present as the corresponding amides or esters selected from CONH.sub.2, CONHR.sup.A2, CONR.sup.A2R.sub.E1 and -COOR.sup.A2; and the hydroxyl side chain groups of Ser, Thr and Tyr may be present as their corresponding alkoxy or acetate groups selected from O(C.sub.1-3alkyl) and OCOCH.sub.3; R.sup.EC2 is selected from OR.sup.E9, NH.sub.2, NHR.sup.A2 and NR.sup.A2R.sup.E1; R.sup.E3 and R.sup.E4 are independently selected from H and CH.sub.3; when R.sup.E1 is H and E.sup.C is OC.sub.1-3alkyl, NH.sub.2 or NHC.sub.1-3alkyl, E.sup.D is selected from H, and CO E.sup.D1-NR.sup.EDR.sup.E6 otherwise, E.sup.D is selected from: R.sup.E5, and CO-E.sup.D1-NR.sup.EDR.sup.E6; wherein E.sup.D1 is a D- or L-amino acid residue selected from Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, wherein the NR.sup.EDR.sup.E6 and CO groups represent terminals of the alpha or pendent functionality of the amino acids; wherein the amino acid residues Asp and Glu may form amide bonds from either the alpha or pendent carboxylic acid functionality; wherein the acid functionality of Asp and Glu not forming an amide bond may be present as the corresponding amides or esters selected from CONH.sub.2, CONHR.sup.A2, CONR.sup.A2R.sup.E1 and COOR.sup.A2; and the hydroxyl side chain groups of Ser, Thr and Tyr may be present as their corresponding alkoxy or acetate groups selected from O(C.sub.1-3alkyl) and OCOCH.sub.3; when E.sup.D1 is Pro, R.sup.ED is absent, otherwise R.sup.ED is R.sup.E1; R.sup.E2, R.sup.E5 and E.sup.6 are independently selected from H and COCH.sub.3; R.sup.E7, R.sup.E8 and R.sup.E9 are each independently selected from H and R.sup.A2; Z.sup.6 is selected from NCOR.sup.A2, NCONHR.sup.A2, NSO.sub.2R.sup.A2; R.sup.Z6 is one or two optional methyl substituents; R.sup.A1 is selected from the group consisting of: linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AL, F, Cl, Br, CN OH, OR.sup.A2, CF.sub.3, CF.sub.2H, COR.sup.A2, CH.sub.2OH, CH.sub.2OR.sup.A2, COOH, COOR.sup.A2, CONH.sub.2, CONHR.sup.A2, CONR.sup.A2.sub.2, OCOR.sup.A2, OCONH.sub.2, OCONHR.sup.A2, OCONR.sup.A2.sub.2, NH.sub.2, NHR.sup.A2, NR.sup.A2.sub.2, SO.sub.2NH.sub.2, SO.sub.2NHR.sup.A2.sub.2, SO.sub.2NR.sup.A2.sub.2, SO.sub.2R.sup.A2, NHCOH, NHCOR.sup.A2, NR.sup.A2COH and NR.sup.A2COR.sup.A2; R.sup.A2 is selected from the group consisting of: linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AT, wherein the alkyl chain is optionally interrupted by one or more atoms selected from O and S; OC.sub.1-6alkyl; C.sub.3-6cycloalkyl, C.sub.4-6heterocycloalkyl, C.sub.5-6cycloalkenyl or C.sub.5-6heterocycloalkenyl optionally substituted with one or more groups R.sup.AT; phenyl optionally substituted with one or more groups R.sup.AR, and C.sub.5-6heteroaryl optionally substituted with one or more groups R.sup.AR; where N is substituted by 2 R.sup.A2 groups, the N and the R.sup.A2 groups may together form a N-containing C.sub.5-6 heterocycloalkyl group, optionally substituted with one or two groups selected from linear unsubstituted C.sub.1-6 alkyl; R.sup.NA1 is selected from linear or branched C.sub.1-4alkyl; R.sup.1A1 is selected from linear or branched unsubstituted C.sup.1-3alkyl; R.sup.A3 is selected from H and unbranched unsubstituted C.sup.1-3alkyl; R.sup.A4 is selected from linear or branched unsubstituted C.sub.1-4alkyl; R.sup.AL is selected from the group consisting of: F, CN OH, OR.sup.A2, CF.sub.3, CF.sub.2H, CORA.sup.2, COOH, COOR.sup.A2, CONH.sub.2, CONHR.sup.A2, CONR.sup.A2.sub.2, OCOR.sup.A2, OCONH.sub.2, OCONHR.sup.A2, OCONR.sup.A2.sub.2, NH.sub.2, NHR.sup.A2, NR.sup.A2.sub.2, SO.sub.2NH, SO.sub.2NHR.sup.A2.sub.2, SO.sub.2NR.sup.A2.sub.2, SO.sub.2R.sup.A2, NHCOH, NHCOR.sup.A2, NR.sup.A2COH and NR.sup.A2COR.sup.A2; and wherein R.sup.AR is selected from the group consisting of linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AL, F, Cl, Br, CN OH, OR.sup.1A1, CF.sub.3, CF.sub.2H, COR.sup.1A1, CH.sub.2OH, CH.sub.2OR.sup.1A1, CHR.sup.1A1OH, CHR.sup.1A1OR.sup.1A1 COOH, COOR.sup.1A1, CONH.sub.2, CONHR.sup.1A1, CONR.sup.1A1.sub.2, OCOR.sup.1A1, OCONH.sub.2, OCONHR.sup.1A1, OCONR.sup.1A1.sub.2, NH.sub.2, NHR.sup.1A1, NR.sup.1A1.sub.2, SO.sub.2NH, SO.sub.2NHR.sup.1A1.sub.2, SO.sub.2NR.sup.1A1.sub.2, SO.sub.2R.sup.1A1, NHCOH, NHCOR.sup.1A1, NR.sup.1A1COH and NR.sup.1A1COR.sup.1A1; R.sup.AT is selected from the group consisting of F, CN OH, OC.sup.1-3alkyl, CF.sub.3, CF.sub.2H, COC.sup.1-3alkyl, COOH, COOC.sup.1-3alkyl, CONH.sub.2, CONHC.sub.1-3alkyl, CON(C.sup.1-3alkyl).sub.2, OCOC.sub.1-3alkyl, OCONH.sub.2, OCONHC.sub.1-3alkyl, OCON(C.sup.1-3alkyl).sub.2, NH.sub.2, NHC.sub.1-3alkyl, N(C.sup.1-3alkyl).sub.2; SO.sub.2NH.sub.2, SO.sub.2NH(C.sup.1-3alkyl).sub.2, SO.sub.2N(C.sup.1-3alkyl).sub.2; SO.sub.2(C.sub.1-3alkyl), NHCOH, NHCO(C.sub.1-3alkyl), N(C.sub.1-3alkyl)COH and N(C.sub.1-3alkyl)CO(C.sub.1-3alkyl); with the proviso that when P.sup.X is PMe.sub.3 and L.sup.A is a single bond, R.sup.A is not selected from the groups ##STR00201## P.sup.Y is independently selected from the group consisting of (P1), (P2) and (P3), ##STR00202## wherein L.sup.C is methylene, ethylene or is absent; R.sup.P1 and R.sup.P2 are each independently selected from methyl; when L.sup.C is absent R.sup.P3 is selected from the group consisting of 4-membered or 5-membered heterocycloalkyl group linked to phosphorus via a carbon atom in the ring, including a single heteroatom independently selected from NR.sup.Z, O and S, CF.sub.3, CH.sub.2CF.sub.3, CH.sub.2CF.sub.2H, CH.sub.2CH.sub.2OR.sup.PB, CH.sub.2Q and (CH.sub.2).sub.2Q; when L.sup.C is methylene or ethylene R.sup.P3 is selected from the group consisting of methyl and ethyl, 4-membered or 5-membered heterocycloalkyl group linked to phosphorus via a carbon atom in the ring, including a single heteroatom independently selected from NR.sup.Z, O and S CF.sub.3, CH.sub.2CF.sub.3, CH.sub.2CF.sub.2H, CH.sub.2CH.sub.2OR.sup.PB, CH.sub.2Q and (CH.sub.2).sub.2Q; wherein Q is a C.sub.5-6heteroaryl group, optionally substituted with one or more groups R.sup.PA; R.sup.P4 is selected from methyl and ethyl; m is an integer selected from 1, 2 or 3; R.sup.M is one or more optional substituents on the ring independently selected from R.sup.PC when attached to a carbon atom adjacent the phosphorus atom, or OH, OC.sup.1-3alkyl and R.sup.PC, when attached to other ring carbons; L.sup.B is methylene, ethylene or is absent; when L.sup.B is present, R.sup.P4 is absent and R.sup.1 is selected from N, CH and CR.sup.PC; when L.sup.B is absent, R.sup.1 is selected from the group consisting of O, NR.sup.Z, SO.sub.2, CH.sub.2, CHF, CF.sub.2 and CHR.sup.PC, wherein R.sup.Z is selected from the group consisting of H, C.sup.1-3alkyl, COC.sup.1-3alkyl and SO.sub.2C.sub.1-3alkyl; R.sup.5 and R.sup.8 are each independently selected from H and R.sup.PC; R.sup.6 and R.sup.7 are each independently selected from H and R.sup.PC; wherein R.sup.PC is selected from the group consisting of C.sub.1-3alkyl, optionally substituted with one or more groups R.sup.PD; wherein R.sup.PA is selected from the group consisting of linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AL, F, Cl, Br, CN OH, OR.sup.PE, CF.sub.3, CF.sub.2H, COR.sup.PE, COOH, COOR.sup.PE, CONH.sub.2, CONHR.sup.PE, CONR.sup.PE.sub.2, OCOR.sup.PE, OCONH.sub.2, OCONHR.sup.PE, OCONR.sup.PE.sub.2, NH.sub.2, NHR.sup.PE, NR.sup.PE.sub.2, SO.sub.2NH.sub.2, SO.sub.2NHR.sup.PE.sub.2, SO.sub.2NR.sup.PE.sub.2, SO.sub.2R.sup.PE, NHCOH, NHCOR.sup.PE, NR.sup.PECOH and NR.sup.PECOR.sup.PE; and R.sup.PB is selected from the group consisting of linear or branched C.sub.1-6alkyl, C.sub.2-6alkenyl or C.sub.2-6alkynyl optionally substituted with one or more groups R.sup.AT, C.sub.3-6cycloalkyl, C.sub.4-6heterocycloalkyl, C.sub.5-6cycloalkenyl or C.sub.5-6heterocycloalkenyl optionally substituted with one or more groups R.sup.AT, phenyl optionally substituted with one or more groups R.sup.AR, and C.sub.5-6heteroaryl optionally substituted with one or more groups R.sup.AR; R.sup.PE is selected from linear or branched C.sub.1-4alkyl optionally substituted with one or more groups R.sup.PD; and R.sup.PD is selected from the group consisting of F, OH and OC.sup.1-3alkyl. R.sup.B is independently selected from the groups (A1) to (A5) ##STR00203## wherein each of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 is independently selected from CH or N; wherein at least three of Y.sup.1, Y.sup.2, Y.sup.3, Y.sup.4 and Y.sup.9 are independently CH; V is independently selected from O, CHOR.sup.O1, NCOR.sup.C8, NCONHR.sup.C8, NSO.sub.2R.sup.C8, NCO.sub.2R.sup.C2 and NR.sup.N2; one of Y.sup.5, Y.sup.6, Y.sup.7 and Y.sup.8 is selected from CH and N, and the others are CH; X is independently selected from NH, S and O; R.sup.O1 is selected from OR.sup.O2 or NHR.sup.N1; R.sup.O1 is selected from H and C.sub.1-3 unbranched alkyl; R.sup.O2 is selected from H and C.sub.1-3 unbranched alkyl; R.sup.N1 is selected from H and C.sub.1-3 unbranched alkyl; R.sup.N2 is C.sub.1-3 unbranched alkyl; R.sup.C2 and R.sup.C8 are each independently selected from C.sub.1-3 unbranched alkyl and C.sub.3-4 branched alkyl; R.sup.C3 is selected from C.sub.1-3 unbranched alkyl and C.sub.2H.sub.4CO.sub.2H; R.sup.C4 is either H or Me; R.sup.C5 is either H or Me; R.sup.C6 represents one or two optional methyl substituents; R.sup.C7 is selected from H and COCH.sub.3; and n is an integer selected from 2 to 8
168. (canceled)
169. A pharmaceutical composition comprising a compound according to claim 167 or a pharmaceutically acceptable salt, solvate or hydrate thereof, and a pharmaceutical acceptable diluent or excipient.
170-174. (canceled)
Description
EXAMPLES
[1130] Analytical Methods
[1131] Analysis of products and intermediates has been carried out using reverse phase analytical HPLC-MS using the parameters set out below.
[1132] HPLC Analytical Methods:
[1133] AnalpH2_MeOH_4min: Phenomenex Luna C18 (2) 3 m, 504.6 mm; A=water+0.1% formic acid; B=MeOH+0.1% formic acid; 45 C.; % B: 0.0 min 5%, 1.0 min 37.5%, 3.0 min 95%, 3.5 min 95%, 3.51 min 5%, 4.0 min 5%; 2.25 mL/min.
[1134] Preparative HPLC Methods
[1135] Reverse Phase Preparative HPLC-MS: Mass-directed purification by preparative LC-MS using a preparative C-18 column (Phenomenex Luna C18 (2), 10021.2 mm, 5 m).
[1136] Generic Acidic Conditions:
[1137] A=water +0.1% formic acid; B=MeOH+0.1% formic acid; 20 C.; % B: 0.0 min Initial between 2% and 50%, 0.1 min % as per Initial, 7.0 min between 40% and 95%, 9.0 min 95%, 10.0 min 95%, 10.1 min back to Initial %; 12.0 min Initial %; 20.0 mL/min.
[1138] Generic Basic Conditions:
[1139] A=water pH 9 (Ammonium Bicarbonate 10 mM); B=MeOH; 20 C.; % B: 0.0 min Initial between 2% and 50%, 0.1 min % as per Initial, 7.0 min between 40% and 95%, 9.0 min 95%, 10.0 min 95%, 10.1 min back to Initial %; 12.0 min Initial %; 20.0 mL/min.
[1140] NMR was also used to characterise final compounds. NMR spectra were obtained Bruker Advance 400 or Bruker DRX 400 at room temperature unless otherwise stated. 1H NMR spectra are reported in ppm and referenced to either tetramethylsilane (0.00 ppm), DMSO-d6 (2.50 ppm), CDCl.sub.3 (7.26 ppm) or CD.sub.3OD (3.31 ppm).
[1141] Abbreviations Used
[1142] For the examples below as well as throughout the application, the following abbreviations have the following meanings. If not defined, the terms have their generally accepted meanings.
[1143] C. Degrees Centigrade
[1144] Ac Acetyl
[1145] app Apparent
[1146] aq. Aqueous
[1147] br Broad
[1148] d Doublet
[1149] DABCO 1,4-Diazabicyclo[2,2,2]octane
[1150] DCM Dichloromethane
[1151] DIPEA N,N-Diisopropylethylamine
[1152] DMA Dimethylacetamide
[1153] DMF Dimethylformamide
[1154] DMSO Dimethyl sulfoxide
[1155] Et Ethyl
[1156] EtOAc Ethyl acetate
[1157] EtOH Ethanol
[1158] Et.sub.2O Diethyl ether
[1159] FA Formic acid
[1160] g Gram
[1161] h Hour(s)
[1162] HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
[1163] HMPA Hexamethylphosphoramide
[1164] HPLC High-performance liquid chromatography
[1165] .sup.iPr Isopropyl
[1166] J Coupling constant
[1167] LC-MS Liquid chromatography-mass spectrometry
[1168] Me Methyl
[1169] MeCN Acetonitrile
[1170] MeOH Methanol
[1171] mg Milligram
[1172] min Minute(s)
[1173] mL Millilitre
[1174] mmol Millimole
[1175] Ms Mesyl
[1176] O/N Overnight
[1177] ppm Parts per million
[1178] ppt Precipitate
[1179] q Quartet
[1180] quint Quintet
[1181] rt Room temperature
[1182] Rochelle Salt Potassium sodium tartrate tetrahydrate
[1183] s Singlet
[1184] TCEP.HCl Tris(2-carboxyethyl)phosphine hydrochloride
[1185] TEA Triethylamine
[1186] TFA Trifluoroacetic acid
[1187] THF Tetrahydrofuran
[1188] TLC Thin layer chromatography
[1189] TMS Trimethylsilyl
[1190] t Triplet
[1191] WIPE Water/isopropanol/Ethyl acetate (1:2:9)
[1192] XantPhos 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene
[1193] Synthesis of Key Intermediates
[1194] A number of the requisite precursors III, IV, V, VI, and IX, necessary for coupling with gold(I) phosphine chloride complexes VII, required synthesis from commercial starting materials. Other precursors used were commercially available.
[1195] (S)-2-Acetylamino-4-[(R)-1-(ethoxycarbonylmethyl-carbamoyl)-2-mercapto-ethylcarbamoyl]-butyric acid ethyl ester I-3
##STR00056##
[1196] (a) (4S,9R,14R,19S)-ethyl19-acetamido-9,14-bis((2-ethoxy-2-oxoethyl)carbamoyl)-4-(ethoxycarbonyl)-2,7,16-trioxo-11,12-dithia-3,8,15-triazaicosan-20-oate I-2
[1197] Oxidised L-glutathione I-1 (980 mg, 1.6 mmol) was suspended in dry EtOH (40 mL). The reaction mixture was cooled to 0 C. and AcCl (6.82 mL, 96 mmol) was added dropwise over 5 min. The reaction mixture was heated to 50 C. O/N, followed by evaporation to dryness to afford a pink crude solid. The crude material was resuspended in dry THF (50 mL), DIPEA (600 L, 3.44 mmol) was added, followed by dropwise addition of acetic anhydride (4.9 mL, 51.84 mmol). The reaction mixture was stirred at rt O/N. The solvent was evaporated and the crude material was purified by preparative HPLC (acidic conditions) to afford the title compound (90 mg, 0.11 mmol, 7%).
[1198] (b) (4S,9R,14R,19S)-ethyl19-acetamido-9,14-bis((2-ethoxy-2-oxoethyl)carbamoyl)-4-(ethoxycarbonyl)-2,7,16-trioxo-11,12-dithia-3,8,15-triazaicosan-20-oate I-3 I-2 (90 mg, 0.11 mmol) was dissolved in a mixture of MeOH (0.5 mL) and water (0.5 mL). TCEP (165 mg, 0.58 mmol) was added and the reaction mixture was stirred at rt O/N. The solvent was evaporated, the residual solid was dissolved in water and extracted with DCM (3). The combined organic fractions were dried and evaporated to yield the title compound as a white solid (90 mg, 0.11 mmol, quantitative).
[1199] 2-(1-Methyl-1H-tetrazol-5-ylmethyl)-isothiourea I-6
##STR00057##
[1200] (a) 5-Chloromethyl-1-methyl-1H-tetrazole I-5
[1201] N-Methylchloroacetamide I-4 (1.0 g, 9.3 mmol) was dissolved in dry toluene (30 mL). PCl.sub.5 (2.13 g, 10.23 mmol) was added in one portion and the reaction mixture was stirred at rt for 1 h under an atmosphere of N.sub.2. Trimethylsilyl azide (1.85 mL, 13.95 mmol) was added dropwise over 15 min and the resulting reaction mixture was stirred at rt O/N. The reaction mixture was diluted with EtOAc and washed with water, 2M NaOH (aq.) and brine. The organic fraction was concentrated to dryness and purified by flash column chromatography (Biotage Isolera Four, 25 g KP-Sil column eluting with a gradient from isohexane to EtOAc) to yield the desired product (283 mg, 2.14 mmol, 23%).
[1202] (b) 2-(1-Methyl-1H-tetrazol-5-ylmethyl)-isothiourea I-6
[1203] A mixture of 5-chloromethyl-1-methyl-1H-tetrazole I-5 (240 mg, 1.81 mmol) and thiourea (138 mg, 1.81 mmol) in EtOH (10 mL) was heated to reflux O/N. The formation of a white ppt was observed. The reaction mixture was cooled to rt, the ppt filtered and dried under high vacuum O/N to afford the title compound (311 mg, 1.81 mmol, quantitative).
[1204] 1 H-Tetrazole-5-thiol I-8
##STR00058##
[1205] 1-[(4-Methoxyphenyl)methyl]-1H-1,2,3,4-tetrazole-5-thiol I-7 (200 mg, 0.90 mmol) was dissolved in a mixture of TFA (1.7 mL) and anisole (0.3 mL). The reaction mixture was heated to 100 C. for 2 h in a microwave reactor. A white ppt had formed, which was filtered, triturated with TFA (21 mL) and dried under high vacuum O/N to afford the title compound as a white solid (60 mg, 0.59 mmol, 65%).
[1206] 6-Mercapto-nicotinamide 1-10
##STR00059##
[1207] 6-Chloronicotinamide 1-9 (400 mg, 2.55 mmol) and thiourea (214 mg, 2.81 mmol) were suspended in EtOH (30 mL) and the reaction mixture heated at reflux for 2 days. A yellow ppt had formed, which was filtered and dried. LC-MS analysis (AnalpH2_MeOH_4min) of the ppt indicated partial hydrolysis to the thiol. As a consequence, the ppt was suspended in a mixture of EtOH (30 mL) and aq. NaOH and the reaction mixture heated to reflux O/N. The reaction mixture was evaporated to dryness, redissolved in water, acidified to pH1 with conc. aq. HCl and extracted with DCM (3). A yellow solid precipitated in the aqueous fraction which was filtered and triturated with MeOH (3 ) to yield the desired product as a yellow solid (10 mg, 0.06 mmol, 3%).
[1208] S-[3-(Methylsulfonyl)phenyl]carbamothioic acid dimethyl ester I-13
##STR00060##
[1209] (a) O-[3-(Methylsulfonyl)phenyl]carbamothioic acid dimethyl ester I-12
[1210] 3-Methanesulfonylphenol I-11 (1.0 g, 5.81 mmol) was dissolved in dry DMF (10 mL). NaH (60% dispersion in mineral oil, 255 mg, 6.39 mmol) was added at which point effervescence was observed and the reaction mixture was stirred at rt for 10 min. N,N-Dimethylthiocarbamoyl chloride (790 mg, 6.39 mmol) was added and the reaction mixture was heated to 80 C. for 1 h, cooled to rt and stirred O/N. The reaction mixture was poured into brine and was extracted with DCM several times. The combined organic fractions were dried, evaporated and purified by flash column chromatography (Biotage Isolera Four, 25 g KP-Sil column eluting with a gradient from isohexane to 40% EtOAc/isohexane) to afford the title compound (1.42 g). This was used crude [80% by LC-MS (AnalpH2_MeOH_4min)] in the successive step.
[1211] (b) S-[3-(Methylsulfonyl)phenyl]carbamothioic acid dimethyl ester I-13
[1212] O-[3-(Methylsulfonyl)phenyl]carbamothioic acid dimethyl ester I-12 (crude 490 mg, 1.51 mmol based on 80% purity) was dissolved in DMSO (11 mL). The reaction was heated to 180 C. for 4 h in a microwave reactor. Purification was carried out by preparative HPLC (acidic conditions) to afford the title compound (20 mg, 0.08 mmol, 5%).
[1213] 3, 4-Diacetoxybenzenethiol I-17
##STR00061##
[1214] (a) 4-[(3,4-Dimethoxyphenyl)disulfanyl]-1,2-dimethoxy-benzene I-15
[1215] To a solution of 3,4-dimethoxybenzenethiol I-14 (1.0 g, 6.3 mmol) in EtOH (10 mL) was added 5 drops of 35% hydrogen peroxide solution under vigorous stirring. After stirring at rt for 18 h, the resulting precipitate was collected and washed with cold EtOH to afford the title compound as an off white solid (600 mg, 1.77 mmol, 56%).
[1216] (b) 4-[(3,4-Acetoxyphenyl)disulfanyl]-1,2-dimethoxy-benzene I-16
[1217] To a solution of 4-[(3,4-dimethoxyphenyl)disulfanyl]-1,2-dimethoxy-benzene I-15 (356 mg, 1.05 mmol) in dry DCM (20 mL) at 0 C. was added dropwise a 1M solution of boron tribromide in DCM (6.3 mL, 6.3 mmol). The reaction mixture was stirred for 1 h at 0 C., followed by 1 h at rt, and was then adsorbed onto silica and purified by column chromatography (Biotage SP1, 10 g KP-Sil column, 30% EtOAc/isohexane to 80% EtOAc/isohexane) to afford a brown oil (83 mg, 28%) which was used directly. The brown oil was solubilised in pyridine (30 4). Acetic anhydride (65 L, 0.66 mmol) was added and the resulting mixture was heated for 3 h at 60 C. Upon cooling to rt, the reaction mixture was diluted with DCM (15 mL), washed with water (215 mL) and brine, before passing through a phase separator cartridge (Biotage) and concentrated in vacuo. The residue was purified by column chromatography (Biotage SP1, 10 g KP-Sil column, 25% EtOAc/isohexane to 50% EtOAc/isohexane) to afford the title compound as a brown oil (115 mg, 0.26 mmol, 24% over two steps).
[1218] (c) 3,4-Diacetoxybenzenethiol I-17
[1219] To a solution of 4-[(3,4-acetoxyphenyl)disulfanyl]-1,2-dimethoxy-benzene I-16 (37 mg, 0.081 mmol) in MeOH/water 1:1 (1 mL) was added TCEP.HCl (116 mg, 0.41 mmol). The resulting reaction mixture was stirred for 18 h at rt and concentrated under reduced pressure. The residue was dissolved in water (10 mL) and extracted with DCM (310 mL). The organics extracts were combined, washed with brine and passed through a phase separator cartridge (Biotage) and concentrated in vacuo to afford the title compound as a pink oil (11 mg, 0.05 mmol, 60%).
[1220] 4-Sulfanylbenzene-1, 2-diol I-19
##STR00062##
[1221] To a solution of 3,4-dimethoxybenzenethiol I-18 (150 mg, 0.66 mmol) in dry DCM (10 mL) at 0 C. was added dropwise a 1M solution of boron tribromide in DCM (2 mL, 2 mmol). The reaction mixture was stirred for 1 h at 0 C., followed by 1 h at rt. The reaction mixture was diluted with DCM (10 mL), washed with water (210 mL), brine (10 mL), passed through a phase separator cartridge (Biotage) and concentrated in vacuo to afford a ca. 2:1 (by NMR) mixture of the title compound and the corresponding disulfide as a pink oil (48 mg).
[1222] Thioacetic acid 4,4-difluoro-cyclohexylester I-23
##STR00063##
[1223] (a) 4,4-Difluoro-cyclohexanol I-21
[1224] To a solution of 4,4-difluoro-cyclohexanone I-20 (205 mg, 1.53 mmol) in MeOH (4 mL) at 0 C. was added sodium borohydride (116 mg, 3.0 mmol). The reaction mixture was stirred at 0 C. for 3 h. The reaction was quenched with saturated aq. ammonium chloride (5 mL), MeOH was removed in vacuo and the aqueous layer was extracted with DCM (310 mL). The combined organic fractions were passed through a phase separator cartridge (Biotage) and concentrated in vacuo to afford the crude title compound as a colourless oil (216 mg)).
[1225] (b) Methanesulfonic acid 4,4-difluoro-cyclohexyl esterI-22
[1226] To a solution of 4,4-difluoro-cyclohexanol I-21 (216 mg) in DCM (5 mL) was added mesyl chloride (148 L, 1.9 mmol) and triethylamine (442 L, 3.1 mmol). The reaction mixture was stirred at 0 C. for 2 h. The reaction was quenched with water (5 mL), the layers separated and the aqueous layer extracted with DCM (35 mL). The combined organic extracts were washed with saturated aq. sodium bicarbonate (10 mL) and brine (10 mL), passed through a phase separator cartridge (Biotage) and concentrated in vacuo to afford the title compound as a yellow oil (308 mg, 1.44 mmol, 94% over 2 steps).
[1227] (c) Thioacetic acid 4,4-difluoro-cyclohexylester I-23
[1228] To a solution of methanesulfonic acid 4,4-difluoro-cyclohexyl ester I-22 (82 mg, 0.38 mmol) in DMA (2 mL) was added potassium thioacetate (131 mg, 1.1 mmol). The reaction was heated at 80 C. for 18 h. The reaction was cooled to rt, Et.sub.2O (10 mL) and water (10 mL) were added. The layers were separated and the aqueous layer extracted with Et.sub.2O (310 mL). The combined organic extracts were washed with water (10 mL) and brine (10 mL) before passing through a phase separator cartridge (Biotage). The crude residue was purified by column chromatography (Biotage SP1, 25 g KP-Sil, eluting with a gradient of isohexane to EtOAc) to afford the title compound as a pale yellow oil (42 mg, 0.21 mmol, 57%).
[1229] Several of the requisite chloro(trialkyl phosphine) gold(I) complexes VII, necessary for coupling with precursors III, IV, V, VI and IX required synthesis from commercial starting materials:
[1230] Dimethylethylphosphine gold(I) chloride I-27
##STR00064##
[1231] (a) Dimethylphosphine borane I-25
[1232] Cerium(III) chloride (25 g, 101.4 mmol) was suspended in THF (100 mL) and stirred at rt for 1 h. Sodium borohydride (3.8 g, 101.4 mmol) was then added and the suspension stirred at rt for a further 1 h. The reaction was cooled to 0 C. at which point dimethylphosphine oxide I-24 (2.6 g, 33.8 mmol) was added dropwise followed by lithium aluminium hydride (1M in THF, 40.7 mL, 40.7 mmol) also dropwise. The reaction was stirred at rt O/N before diluting with toluene (50 mL) then quenching with water (25 mL) and aqueous HCl (6N, 25 mL). The suspension was filtered through celite and the layers separated. The aqueous phase was extracted with DCM (340 mL) and the combined organic extracts washed with brine (140 mL) and passed through a phase separator cartridge (Biotage). Concentration in vacuo gave the crude product as a yellow oil which was purified by column chromatography (Biotage Isolera Four, 25 g KP-Sil column eluting with a gradient of isohexane to 20% EtOAc/isohexane) to provide the title compound as a colourless oil (1.49 g, 19.6 mmol, 58%).
[1233] (b) Dimethylethylphosphine borane I-26
[1234] Dimethylphosphine borane I-25 (100 mg, 1.3 mmol) was dissolved in THF (3 mL) and the colourless solution cooled to 0 C. NaH (60% dispersion in mineral oil, 53 mg, 1.3 mmol) was added in one portion, whereupon effervescence was observed. The opaque reaction was stirred at rt for 10 min then cooled to 0 C. whereupon iodoethane (0.12 mL, 1.4 mmol) was added in one portion. When TLC had indicated completion of the reaction, water (10 mL) and Et.sub.2O (10 mL) were added and the phases separated. The aqueous phase was extracted with Et.sub.2O (215 mL) and the combined organic extracts washed with brine (120 mL) before passing through a phase separator cartridge (Biotage). Concentration in vacuo gave the crude product as a colourless gum. Purification by column chromatography (Biotage Isolera Four, 10 g KP-Sil column, eluting with a gradient of isohexane to 20% EtOAc/isohexane) provided the title compound as a white solid (122 mg, 1.1 mmol, 90%).
[1235] (c) Dimethylethylphosphine gold(I) chloride I-27
[1236] Dimethylethylphosphine borane I-26 (225 mg, 2.0 mmol) was dissolved in THF (5 mL) and the colourless solution degassed with nitrogen for 5 min. DABCO (640 mg, 6.0 mmol) was added and the reaction sealed with a Teflon screw cap. The reaction was heated to 100 C. and stirred at this temperature for 4 h before cooling in an ice bath and adding a solution of chloro(tetrahydrothiophene)gold(I) (640 mg, 2.0 mmol) in 5 mL dry DCM. After stirring at rt O/N the reaction was diluted with DCM (10 mL) and water (10 mL) and the phases separated. The aqueous phase was extracted with DCM (220 mL) and the combined organic extracts washed with brine (20 mL) before passing through a phase separator cartridge (Biotage). Concentration in vacuo gave the crude product as a brown oil which was purified by column chromatography (Biotage SP1, 25 g KP-Sil eluting with 25% EtOAc/isohexane to 60% EtOAc/isohexane) to provide the title compound as a white solid (265 mg, 0.82 mmol, 41%). .sup.1H-NMR (400 MHz, CDCl.sub.13): ppm 1.85 (2H, dq, J=10.9, 7.6 Hz), 1.57 (6H, d, J=11.1 Hz), 1.26 (3H, dt, J=20.5, 7.6 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm 4.07 (s).
[1237] 1-Methylphospholane gold(I) chloride I-30 and 1-methylphosphinane gold(I) chloride I-31
##STR00065##
[1238] (a) 1-Methylphospholaneborane I-28
[1239] The bis-Grignard reagent was prepared by treating magnesium (1.0 g, 0.04 mol) with 1,4-dibromobutane (4.3 g, 20 mmol) in dry THF (50 mL) at 65 C. for 3 h. The reaction mixture was cooled to 0 C. before adding a cooled (10 C.) solution of dichloromethyl phosphine (2.3 g, 20 mmol) in dry THF (25 mL) dropwise maintaining a temperature of 10 C. The mixture was stirred O/N at rt. Borane-THF complex (1.0 M, 20 mL, 20 mmol) was added dropwise and the reaction mixture stirred for additional 4 h. The reaction mixture was poured onto a mixture of ice (200 g) and aqueous HCl (2M, 100 mL) with vigorous stirring. The aqueous phase was extracted with DCM (3100 mL) and the combined organic extracts dried over MgSO.sub.4. Concentration in vacuo gave the crude product as a yellow oil which was purified by column chromatography (Biotage SP1, 50 g KP-Sil column, eluting with isohexane to DCM) to provide the title compound as a colourless oil (700 mg, 6.0 mmol, 30%).
[1240] (b) 1-Methylphosphinaneborane I-29
[1241] Procedure similar to that described for 1-methylphospholaneborane I-28 starting from 1,5-dibromopentane (4.6 g, 20 mmol) to provide the title compound as a colourless oil (546 mg, 4.2 mmol, 21%).
[1242] (c) 1-Methylphospholane gold(I) chloride I-30
[1243] Procedure similar to that described for dimethylethylphosphine gold(I) chloride I-27 starting from 1-methylphospholaneborane 1-28 (116 mg, 1.0 mmol) to provide the title compound as an off-white solid (200 mg, 0.6 mmol, 60%). .sup.1H-NMR (400MHz, CDCl.sub.3): ppm 2.35-2.19 (2H, m), 2.03-1.85 (6H, m), 1.55 (3H, d, J=10.6 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm 11.82 (s).
[1244] (d) 1-Methylphosphinane gold(I) chloride I-31
[1245] Procedure similar to that described for dimethylethylphosphine gold(I) chloride I-27 starting from 1-methylphosphinaneborane I-29 (130 mg, 1.0 mmol) to provide the title compound as an off-white solid (120 mg, 0.35 mmol, 35%). .sup.1H-NMR (400MHz, CDCl.sub.3): ppm 2.16-2.05 (2H, m), 1.95-1.64 (7H, m), 1.55 (3H, d, J=10.9 Hz) 1.39 (1H, m). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm -1.38 (s).
[1246] 4-Methyl-[1,4]oxaphosphinane gold(I) chloride I-34
##STR00066##
[1247] (a) 4-Methyl-[1,4]oxaphosphinaneborane I-33
[1248] To a solution of diethyl methylphosphonate (1.5 g, 10.0 mmol) in dry THF (30 mL) was added lithium aluminium hydride (1M in THF, 15 mL, 15.0 mmol) at 0 C., and the mixture allowed to warm to rt and stirred for 4 h. The reaction mixture was cooled to 0 C. whereupon BuLi (1.6 M in hexanes, 12.5 mL, 20 mmol) was added over 5 min and stirring continued at 0 C. for 45 min. 1-Bromo-2-(2-bromoethoxy)ethane (2.3 g, 10 mmol) was then added in one portion and the reaction mixture stirred for further 4 h. Borane-THF complex (1M in THF, 20 mL, 20 mmol) was added and the reaction mixture stirred at rt for an additional 72 h before being diluted with water (60 mL) and 2M HCl (aq., 160 mL) with vigorous stirring. The aqueous phase was extracted with DCM and the combined organic extracts dried over MgSO.sub.4. Concentration in vacuo gave the crude product which was purified by flash column chromatography (Biotage SP1, 25 g KP-Sil column eluting with isohexane to EtOAc) to provide the title compound as a colourless oil (220 mg, 1.7 mmol, 17%).
[1249] (b) 4-Methyl-[1,4]oxaphosphinane gold(I) chloride I-34
[1250] Procedure similar to that described for dimethylethylphosphine gold(I) chloride I-27 starting from 4-methyl-[1,4]oxaphosphinaneborane I-33 (220 mg, 1.7 mmol) to provide the title compound as an off-white solid (186 mg, 0.5 mmol, 32%). .sup.1H NMR (400 MHz, CDCl.sub.3): ppm 4.19-3.95 (4H, m), 2.24-2.13 (2H, m), 2.09-2.01 (2H, m), 1.75 (3H, d, J=11.1 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm 7.26 (s),
[1251] Diethylmethylphosphine gold(I) chloride I-36
##STR00067##
[1252] (a) Diethylmethylphosphine borane I-35
[1253] To a cold (0) solution of diethylchlorophosphine (1.0 g, 8.0 mmol) in THF (20 mL) under inert atmosphere was slowly added methylmagnesium chloride (3M in THF, 2.7 mL, 8.0 mmol). After warming to rt and being stirred for 4 h, the reaction was cooled to 0 C. prior to the addition of borane-THF complex (1 M in THF, 8 mL, 8.0 mmol). The reaction mixture was allowed to warm up to rt O/N, then was diluted with Et.sub.2O (30 mL) and water (20 mL). The phases were separated and the organic layer was washed with water (210 mL) and brine (10 mL) before being dried over MgSO.sub.4 and concentrated in vacuo to provide the title compound as a colourless oil (388 mg, 3.2 mmol, 40%).
[1254] (b) Diethylmethylphosphine gold(I) chloride I-36
[1255] Procedure similar to that described for dimethylethylphosphine gold(I) chloride I-27 starting from diethylmethylphosphine borane I-35 (385 mg, 3.2 mmol) to provide the title compound as a white solid (475 mg, 1.41 mmol, 44%). .sup.1H NMR (400 MHz, CDCl.sub.3): ppm 1.95-1.75 (4H, m), 1.52 (3H, d, J=10.6 Hz), 1.21 (6H, dt, J=19.7, 7.6 Hz), 1.75 (3H, d, J=11.1 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm 18.13 (s).
[1256] 1,4-Dimethyl-[1,4]azaphosphinane gold(I) chloride I-39
##STR00068##
[1257] (a) 1,4-Dimethyl-[1,4]azaphosphinane 4-oxide I-37
[1258] A solution of methylphosphonic dichloride (2.0 g, 15 mmol) in THF (30 mL) was cooled to 78 C. A solution of vinylmagnesium bromide (1M in THF, 30 mL, 30.0 mmol) was added dropwise, and the resulting mixture was allowed to slowly warm up to rt O/N. The reaction mixture was then transferred to a sealed tube, into which methylamine (2M in THF, 9 mL,18 mmol) was added, followed by MeOH (30 mL). The tube was sealed and heated at 70 C. O/N, after which time the reaction was cooled to rt, and the solvent removed under reduced pressure. The residue was dissolved in a minimum amount of water/MeOH, loaded onto a SCX-2 cartridge (Biotage), washed with water, MeOH and finally NH.sub.3/MeOH solution (2M). Evaporation of the solvent under reduced pressure afforded the title compound as a pale yellow crystalline solid (620 mg, 4.2 mmol, 28%).
[1259] (b) 1,4-Dimethyl-[1,4]azaphosphinane borane I-38
[1260] Procedure similar to that described for dimethylphosphine borane I-25 starting from 1,4-dimethyl-[1,4]azaphosphinane 4-oxide I-37 (300 mg, 2.0 mmol) to provide the title compound as a 1:1 mixture (.sup.1H NMR) with the bis-borane complex I-123 as a white solid (130 mg).
[1261] (c) 1,4-Dimethyl-[1,4]azaphosphinane gold(I) chloride I-39
[1262] Procedure similar to that described for dimethylethylphosphine gold chloride I-27 starting from the 1:1 mixture of 1,4-dimethyl-[1,4]azaphosphinane borane I-38 and, 1,4-dimethyl-[1,4]azaphosphinane diborane I-123 (130 mg) to provide the title compound as a brown solid (170 mg, 0.46 mmol, 23% over 2 steps. .sup.1H-NMR (400MHz, CDCl.sub.3): ppm 2.90-2.68 (4H, m), 2.35 (3H, s), 2.22-2.15 (2H, m), 2.10-1.98 (2H, m), 1.65 (3H, d, J=11.1 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm 7.30 (s).
[1263] [Hydroxymethyl(methyl)phosphanyl]methanol I-40
##STR00069##
[1264] [Hydroxymethyl(methyl)phosphanyl]methanol I-40
[1265] A solution of tetrakis(hydroxymethyl)phosphonium chloride (aq., 80%, 7.5 mL, 50 mmol) was concentrated in vacuo to remove the water before adding TEA (30 mL). The resulting mixture was stirred at rt for 18 h. After filtration to remove salts, the supernatant was dissolved in THF (60 mL) and cooled to 40 C. before adding Mel (1.8 mL, 29 mmol). The solution was allowed to warm up to rt O/N and concentrated in vacuo before adding TEA (20 mL). concentration in vacuo provided the title compound as a yellow oil (1.77 g, 16.38 mmol, 33%) which was used straight away in the next step.
[1266] 4-Methyl-[1,4]sulfonylphosphinane gold(I) chloride I-44
##STR00070##
[1267] (a) 4-Methyl-[1,4] sulfonylphosphine oxide I-42
[1268] To a solution of [hydroxymethyl(methyl)phosphanyl]methanol I-40 (1.7 g, 15.6 mmol) in pyridine (40 mL) was added divinylsulfone (1.7 mL, 16 mmol). The resulting mixture was heated at 130 C. for 5 h. After cooling to rt, MeOH was added to quench the excess of divinylsulfone and the resulting mixture stirred at rt O/N. After concentration in vacuo, acetone (20 mL) was added and the resulting suspension was stirred at rt O/N. The resulting precipitate was collected by filtration and washed with acetone (210 mL) to provide the title compound as a beige solid (1.3 g, 7.1 mmol, 46%).
[1269] (b) 4-Methyl-[1,4]sulfonylphosphinaneborane I-43
[1270] Cerium (Ill) chloride (1.63 g, 6.6 mmol) was suspended in THF (30 mL) and stirred at rt for 30 min. Sodium borohydride (250 mg, 6.6 mmol) was then added and the suspension stirred at rt for a further 30 min. The reaction was cooled to 0 C. at which point 4-methyl-[1,4]sulfonylphosphine oxide I-42 (400 mg, 2.2 mmol) in THF (30 mL) was added dropwise followed by lithium aluminium hydride (1 M in THF, 2.65 mL, 2.65 mmol) also dropwise. The reaction was allowed to warm to rt O/N before being cooled to 0 C. and quenched with a 10% aq. Rochelle salt solution (20 mL). The aqueous layer was extracted with EtOAc (310 mL)and the organics combined, washed with brine and dried with Na.sub.2SO.sub.4. Concentration of the filtrate in vacuo provided the title compound as a white solid (140 mg, 0.76 mmol, 35%).
[1271] (c) 4-Methyl-[1,4]sulfonylphosphinane gold(I) chloride I-44
[1272] Prepared according to the procedure described for dimethylethylphosphine gold(I) chloride I-27 starting from 4-methyl-[1,4]sulfonylphosphinaneborane I-43 (135 mg, 0.7 mmol) to provide, after column chromatography (Biotage Isolera 4, 10 g KP-Sil) eluting with DCM to 5% MeOH in DCM, the title compound as a white solid (31 mg, 0.1 mmol, 11%). .sup.1H-NMR (400MHz, DMSO-d6): ppm 3.60-3.25 (4H, m), 2.63-2.55 (4H, m), 1.82 (3H, d, J=11.9 Hz). .sup.31P-NMR (162 MHz, DMSO-d6): ppm 4.35 (s).
[1273] Dimethyl[2-(methyl)thiazole]phosphine gold(I) chloride I-49 and dimethyl[2-(methyl) oxazole]phosphine gold(I) chloride I-50
##STR00071##
[1274] (a) 2-(Chloromethyl)thiazole I-45
[1275] To a solution of 2-(hydroxymethyl)thiazole (500 mg, 4.3 mmol) in DCM (25 mL) at 0 C. was added dropwise thionyl chloride (4.4 mL, 60.8 mmol). After stirring for 5 h, the solution was concentrated in vacuo. The resulting solid was triturated with Et.sub.2O (20 mL2) to provide the title compound as a yellow solid (550 mg, 4.1 mmol, 95%).
[1276] (b) 2-(Chloromethyl)oxazole I-46
[1277] To a solution of 2-(hydroxymethyl)oxazole (450 mg, 4.5 mmol) in DCM (25 mL) at 0 C. was added dropwise thionyl chloride (3.25 mL, 45 mmol). After stirring for 1 h, water (50 mL) and EtOAc (60 mL) were added. The phases were separated and the organic extracts Concentrated in vacuo to provide the title compound as a white solid (200 mg, 1.7 mmol, 37%).
[1278] (c) Dimethyl[2-(methyl)thiazole]phosphine borane I-47
[1279] To a solution of dimethylphosphine borane I-25 (200 mg, 2.6 mmol) in THF (30 mL) at 0 C. was added NaH (60% dispersion in mineral oil, 112 mg, 2.8 mmol) in one portion whereupon effervescence was observed. The opaque reaction was stirred at rt for 10 min then cooled back to 0 C. whereupon 2-(chloromethyl)thiazole I-45 (341 mg, 2.6 mmol) and Nal (383 mg, 2.6 mmol) were added. The mixture was allowed to warm to rt O/N, then water (10 mL) and DCM (10 mL) were added and the phases separated. The aqueous phase was extracted with DCM (215 mL) and the combined organic extracts washed with brine (20 mL) before passing through a phase separator cartridge (Biotage). Concentration in vacuo gave the crude product which was purified by column chromatography (Biotage Isolera 4, KP-Sil 25 g) eluting with DCM to 3% MeOH in DCM to provide the title compound as a yellow oil (73 mg, 0.4 mmol, 16%).
[1280] (d) Dimethyl[2-(methyl)oxazole]phosphine borane I-48
[1281] Procedure similar to that described for dimethyl[2-(methyl)thiazole]phosphine borane I-47 starting from 2-(chloromethyl)oxazole I-46 (200 mg, 1.8 mmol). Purification by column chromatography (Biotage Isolera 4, 10 g KP-Sil) eluting with isohexane to 50% EtOAc/isohexane provided the title compound as a colourless oil (183 mg, 1.1 mmol, 64%).
[1282] (e) Dimethyl[2-(methyl)thiazole]phosphine gold(I) chloride I-49
[1283] Procedure similar to that described for dimethylethylphosphine gold(I) chloride I-27 starting from dimethyl[2-(methyl)thiazole]phosphine borane I-47 (73 mg, 0.41 mmol) to provide the title compound as an off-white solid (17 mg, 0.04 mmol, 11%). .sup.1H-NMR (400MHz, CDCl.sub.3): ppm 7.76 (1H, d, J=3.3 Hz), 7.37 (1H, dd, J=3.3, 1.2 Hz), 3.73 (2H, d, J=11.4 Hz), 1.70 (6H, d, J=10.6 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm 4.52 (s).
[1284] (t) Dimethyl[2-(methyl)oxazole]phosphine gold(I) chloride I-50
[1285] Procedure similar to that described for dimethylethylphosphine gold(I) chloride I-27 starting from dimethyl[2-(methyl)oxazole]phosphine borane I-48 (180 mg, 1.1 mmol) to provide the title compound as a white solid (165 mg, 0.4 mmol, 39%). .sup.1H-NMR ppm (400MHz, CDCl.sub.3): 7.67 (1H, s), 7.11 (1H, s), 3.45 (2H, d, J=10.6 Hz), 1.69 (6H, d, J=10.6 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm 0.29 (s).
[1286] Dimethylcyclopentylphosphine gold(I) chloride I-52
##STR00072##
[1287] (a) Dimethylcyclopentylphosphine borane I-51
[1288] Procedure similar to that described for dimethylethylphosphine borane I-26 starting from dimethylphosphine borane I-25 (200 mg, 2.6 mmol) and bromocyclopentane (0.36 mL, 2.9 mmol) to provide the title compound as a colourless oil (208 mg, 1.4 mmol, 56%).
[1289] (b) Dimethylcyclopentylphosphine gold(I) chloride I-52
[1290] Procedure similar to that described for dimethylethylphosphine gold(I) chloride I-27 starting from dimethylcyclopentylphosphine borane I-53 (104 mg, 0.72 mmol) to provide the title compound as a colourless oil (58 mg, 0.16 mmol, 22%). .sup.1H-NMR (400 MHz, CDCl.sub.3): ppm 2.11-1.91 (3H, m), 1.85-1.74 (2H, m), 1.72-1.58 (4H, m), 1.56 (6H, d, J=10.6 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm 14.10 (s).
[1291] Tert-butyldimethylphosphine gold(I) chloride I-54
##STR00073##
[1292] Procedure similar to that described for dimethylethylphosphine gold(I) chloride I-27, starting from tert-butyldimethylphosphine borane I-53 (100 mg, 0.76 mmol) to provide the title compound as a white solid (73 mg, 0.21 mmol, 27%). .sup.1H-NMR (400 MHz, CDCl.sub.3): ppm 1.51 (6H, d, J=10.1 Hz), 1.21 (9H, d, J=16.7 Hz). .sup.31P-NMR (162 MHz, CDCl.sub.3): ppm 24.61 (s).
[1293] Mercapto-N,N-dialkyl-benzamides I-71 to I-81
##STR00074##
[1294] (a) 2,2-Disulfanediylbis(N,N-dimethylbenzamide) I-58
[1295] 2,2-Dithiobenzoic acid I-55 (500 mg, 1.6 mmol) was suspended in anhydrous toluene (5 mL) and DMF (31 L). Thionyl chloride (310 L, 4.3 mmol) was added and the reaction mixture stirred at 90 C. for 16 h. Dimethylamine hydrochloride (1.3 g, 16.3 mmol), DIPEA (5.7 mL, 32.6 mmol) and THF (10 mL) were then added and stirred at rt O/N. The reaction mixture was evaporated to dryness, suspended in DCM and washed sequentially with water, 10% aqueous K.sub.2CO.sub.3 and saturated aqueous citric acid. The organic layer was passed through a phase separator cartridge (Biotage) and concentrated in vacuo. The residue was purified by column chromatography (Biotage, Isolera 4, 25 g KP-Sil, eluting with EtOAc) to afford the title compound as a yellow solid (360 mg, 1.0 mmol, 62%).
[1296] The following dithiobenzamides I-59 to I-70 were prepared according to the procedure described for 2,2-disulfanediylbis(N,N-dimethylbenzamide) I-58. All reactions performed using 10 equivalents of the appropriate amine unless otherwise stated.
[1297] (b) 2,2-DisulfanediyIbis(N,N-diethylbenzamide) I-59
[1298] Using diethylamine (1.7 mL, 16.3 mmol) the title compound was provided as a yellow oil (164 mg, 0.4 mmol, 48%).
[1299] (c) 2,2 -Disulfanediylbis(N-methoxy-N-methylbenzamide) I-60
[1300] Using N,O-dimethylhydroxylamine hydrochloride (796 mg, 8.2 mmol) the title compound was provided as a colourless gum (106 mg, 0.3 mmol, 33%).
[1301] (d) (Disulfanediylbis(4,1-phenylene))bis(morpholinomethanone) I-61
[1302] Using morpholine (0.71 mL, 8.2 mmol) the title compound was provided as a yellow gum (203 mg, 0.5 mmol, 56%).
[1303] (e) (DisulfanediyIbis(4,1-phenylene))bis(thiomorpholinomethanone) I-62
[1304] Using thiomorpholine (0.82 mL, 8.2 mmol) the title compound was provided as an off-white solid (140 mg, 0.3 mmol, 36%).
[1305] (t) 2,2-DisulfanediyIbis(N-methyl-N-(2-(methylthio)ethyObenzamide) I-63
[1306] Procedure similar to that described for 2,2-disulfanediylbis(N,N-dimethylbenzamide I-58 except 2,2-dithiobenzoic acid I-55 (100 mg, 0.33 mmol) and N-methyl-2-(methylthio)ethanamine (100 mg, 0.95 mmol) were used. The title compound was provided as a yellow gum (63 mg, 0.13 mmol, 40%).
[1307] (g) 2,2-DisulfanediyIbis(N-isopropyl-N-methylbenzamide) I-64
[1308] Procedure similar to that described for 2,2-disulfanediylbis(N,N-dimethylbenzamide I-58 except 2,2-dithiobenzoic acid I-55 (250 mg, 0.82 mmol) and N-isopropylmethylamine (0.51 mL, 4.9 mmol) were used. The title compound was provided as a yellow gum (175 mg, 0.42 mmol, 51%).
[1309] (h) 2,2-DisulfanediyIbis(N,N-diisopropylbenzamide) I-65
[1310] Procedure similar to that described for 2,2-disulfanediylbis(N,N-dimethylbenzamide I-58 except 2,2-dithiobenzoic acid I-55 (250 mg, 0.8 mmol) and diisopropylamine (0.69 mL, 4.9 mmol) were used. The title compound was provided as a pale yellow solid (144 mg, 0.3 mmol, 37%).
[1311] (i) 4,4-DisulfanediyIbis(N,N-dimethylbenzamide) I-66
[1312] Procedure similar to that described for 2,2-disulfanediylbis(N,N-dimethylbenzamide I-58 except 4,4-dithiobenzoic acid I-57 (250 mg, 0.82 mmol) and dimethylamine hydrochloride (665 mg, 8.2 mmol) were used. The title compound was provided as a white solid (132 mg, 0.37 mmol, 45%).
[1313] W 2,2-DisulfanediyIbis(N-ethyl-N-isopropylbenzamide) I-67
[1314] Procedure similar to that described for 2,2-disulfanediylbis(N,N-dimethylbenzamide I-58 except 2,2-dithiobenzoic acid I-55 (250 mg, 0.82 mmol) and N-ethylisopropylamine (711 mg, 8.2 mmol) were used. The title compound was provided as a yellow gum (124 mg, 0.28 mmol, 34%).
[1315] (k) 3,3-DisulfanediyIbis(N,N-dimethylbenzamide) I-68
[1316] Procedure similar to that described for 2,2-disulfanediylbis(N,N-dimethylbenzamide I-58 except 3,3-dithiobenzoic acid I-56 (250 mg, 0.82 mmol) and dimethylamine hydrochloride (665 mg, 8.2 mmol) were used. The title compound was provided as a colourless gum (125 mg, 0.35 mmol, 42%).
[1317] (I) 4,4-DisulfanediyIbis(N-methoxy-N-methylbenzamide) I-69
[1318] Procedure similar to that described for 2,2-disulfanediylbis(N,N-dimethylbenzamide I-58 except 4,4-dithiobenzoic acid I-57 (250 mg, 0.82 mmol) and N,O-dimethylhydroxylamine hydrochloride (796 mg, 8.2 mmol) were used. The title compound was provided as a white solid (108 mg, 0.28 mmol, 34%).
[1319] (m)3,3-DisulfanediyIbis(N-methoxy-N-methylbenzamide) I-70
[1320] Procedure similar to that described for 2,2-disulfanediylbis(N,N-dimethylbenzamide I-58 except 3,3-dithiobenzoic acid I-56 (250 mg, 0.82 mmol) and N,O-dimethylhydroxylamine hydrochloride (796 mg, 8.2 mmol) were used. The title compound was provided as a pale yellow gum (165 mg, 0.42 mmol, 51%).
[1321] (n) 2-Mercapto-N,N-dimethyl-benzamide I-71
[1322] 2,2-Disulfanediylbis(N,N-dimethylbenzamide) I-58 (50 mg, 0.14 mmol) was dissolved in MeOH (4 mL) and water (3 mL) before adding TCEP.HCl (200 mg, 0.7 mmol) in one portion. The reaction mixture was stirred at rt O/N and the solvent removed under reduced pressure. The residue was dissolved in water and extracted with DCM. The combined organic extracts were passed through a phase separator cartridge (Biotage) and concentrated in vacuo to provide the title compound as a yellow oil (45 mg, 0.25 mmol, 89%).
[1323] (o) 2-Mercapto-N,N-diethyl-benzamide I-72
[1324] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 2,2-disulfanediylbis(N,N-diethylbenzamide) I-59 (40 mg, 0.09 mmol) and TCEP.HCl (138 mg, 0.48 mmol) were used. The title compound was provided as a colourless gum (28 mg, 0.13 mmol, 70%).
[1325] (p) 2-Mercapto-N-methoxy-N-methyl-benzamide I-73
[1326] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 2,2-disulfanediylbis(N-methoxy-N-methylbenzamide) I-60 (50 mg, 0.13 mmol) and TCEP.HCl (183 mg, 0.64 mmol) were used. The title compound was provided as a colourless gum (46 mg, 0.23 mmol, 92%).
[1327] (q) (2-Mercapto-phenyl)-morpholin-4-yl-methanone I-74
[1328] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except (disulfanediyIbis(4,1-phenylene))bis(morpholinomethanone) I-61 (42 mg, 0.09 mmol) and TCEP.HCl (135 mg, 0.47 mmol) were used. The title compound was provided as a white solid (39 mg, 0.17 mmol, 93%).
[1329] (r) (2-Mercapto-phenyl)-thiomorpholin-4-yl-methanone I-75
[1330] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except (disulfanediyIbis(4,1-phenylene))bis(thiomorpholinomethanone) I-62 (50 mg, 0.11 mmol) and TCEP.HCl (150 mg, 0.53 mmol) were used. The title compound was provided as a yellow solid (46 mg, 0.19 mmol, 91%)
[1331] (s) 2-Mercapto-N-(2-methylsulfanyl-ethyl)-benzamide I-76
[1332] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 2,2-disulfanediylbis(N-methyl-N-(2-(methylthio)ethyl)benzamide) I-63 (29 mg, 0.06 mmol) and TCEP.HCl (86 mg, 0.3 mmol) were used. The title compound was provided as a yellow gum (29 mg, 0.12 mmol, quantitative).
[1333] (t) N-Isopropyl-2-mercapto-N-methyl-benzamide I-77
[1334] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 2,2-disulfanediylbis(N-isopropyl-N-methylbenzamide) I-64 (43 mg, 0.10 mmol) and TCEP.HCl (148 mg, 0.52 mmol) were used. The title compound was provided as a yellow oil (42 mg, 0.2 mmol, 97%).
[1335] (u) N,N-Diisopropyl-2-mercapto-benzamide I-78
[1336] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 2,2-disulfanediyIbis(N,N-diisopropylbenzamide) I-65 (44 mg, 0.09 mmol) and TCEP.HCl (135 mg, 0.47 mmol) were used. The title compound was provided as a white solid (42 mg, 0.18 mmol, 94%).
[1337] (v) 4-Mercapto-N,N-dimethyl-benzamide I-79
[1338] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 4,4-disulfanediylbis(N,N-dimethylbenzamide) I-66 (47 mg, 0.13 mmol) and TCEP.HCl (188 mg, 0.66 mmol) were used. The title compound was provided as a colourless oil (50 mg, 0.28 mmol, quantitative).
[1339] (w) N-Ethyl-N-isopropyl-2-mercapto-benzamide I-80
[1340] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 2,2-disulfanediylbis(N-ethyl-N-isopropylbenzamide) I-67 (42 mg, 0.09 mmol) and TCEP.HCl (135 mg, 0.47 mmol) were used. The title compound was provided as a pale yellow oil (38 mg, 0.17 mmol, 90%).
[1341] (x) 3-Mercapto-N,N-dimethyl-benzamide I-81
[1342] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 3,3-disulfanediylbis(N,N-dimethylbenzamide) I-68 (42 mg, 0.12 mmol) and TCEP.HCl (168 mg, 0.59 mmol) were used. The title compound was provided as a pale yellow oil (43 mg, 0.24 mmol, quantitative).
[1343] (y) 4-Mercapto-N-methoxy-N-methyl-benzamide I-82
[1344] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 4,4-disulfanediylbis(N-methoxy-N-methylbenzamide) I-69 (63 mg, 0.16 mmol) and TCEP.HCl (230 mg, 0.8 mmol) were used. The title compound was provided as a colourless oil (56 mg, 0.28 mmol, 88%).
[1345] (z) 3-Mercapto-N-methoxy-N-methyl-benzamide I-83
[1346] Procedure similar to that described for 2-mercapto-N,N-dimethyl-benzamide I-71 except 3,3-disulfanediylbis(N-methoxy-N-methylbenzamide) I-70 (46 mg, 0.12 mmol) and TCEP.HCl (168 mg, 0.59 mmol) were used. The title compound was provided as a colourless oil (53 mg, 0.27 mmol, quantitative).
[1347] 5-(2-Methoxycarbonyl-ethylsulfanyl)-pyrimidine-4-carboxylic acid methyl ester I-86
##STR00075##
[1348] (a) 5-Bromo-pyrimidine-4-carboxylic acid methyl ester I-85
[1349] 5-Bromo-4-pyrimidine carboxylic acid I-84 (858 mg, 4.23 mmol) was dissolved in MeOH (15 mL) and thionyl chloride (77 L, 1.06 mmol) added dropwise at rt. The reaction mixture was heated to 70 C. and stirred at this temperature for 3 h. The reaction mixture was then cooled to rt and evaporated to dryness. The residue was re-dissolved in a mixture of water (25 mL) and saturated aq. NaHCO.sub.3 (25 mL) before extracting with EtOAc (350 mL). The combined organic extracts were then washed with saturated aqueous NaHCO.sub.3 (40 mL) and brine (40 mL) before drying over MgSO.sub.4. Concentration in vacuo provided the title compound as a brown solid (502 mg, 2.31 mmol, 55%).
[1350] (b) 5-(2-Methoxycarbonyl-ethylsulfanyl)-pyrimidine-4-carboxylic acid methyl ester I-86
[1351] A mixture of 5-bromo-pyrimidine-4-carboxylic acid methyl ester I-85, (500 mg, 2.3 mmol), methyl-3-mercaptopropionate (280 uL, 2.3 mmol), Pd.sub.2(dba).sub.3 (84 mg, 0.092 mmol), Xantphos (106 mg, 0.18 mmol), DIPEA (801 uL, 4.6 mmol) and dioxane (15 mL) was degassed with nitrogen and the mixture heated at 110 C. until LC-MS (AnalpH2_MeOH_4min) indicated completion of the reaction. The reaction mixture was concentrated in vacuo and the residue diluted with EtOAc (100 mL) before being washed with saturated aqueous NH.sub.4Cl (30 mL), saturated aqueous NaHCO.sub.3 (30 mL) and brine (30 mL). The organic phase was dried over MgSO.sub.4 before being concentrated in vacuo. The residue was purified by column chromatography (Biotage, Isolera 4, 100 g KP-Sil, eluting with 20% EtOAc/isohexane to EtOAc) to afford the title compound as an off-white solid (378 mg, 1.5 mmol, 64%).
[1352] Sulfanyl-propionic acid methyl esters I-94 to I-98
##STR00076##
[1353] (a) 5-Bromo-pyrimidine-4-carboxylic acid dimethylamide I-89
[1354] 5-Bromo-4-pyrimidine carboxylic acid I-84 (410 mg, 2.0 mmol) and dimethylamine hydrochloride (329 mg, 4.0 mmol) were combined and suspended in DCM (13 mL). DIPEA (1.1 mL, 6.1 mmol) was added followed by HATU (1.1 g, 2.9 mmol) and the reaction stirred at rt O/N. The reaction was diluted with DCM and washed with water and the layers separated. The aqueous fraction was extracted with DCM (2) and the combined organic extracts passed through a phase separator cartridge (Biotage) and concentrated in vacuo. The residue was purified by column chromatography (Biotage, Isolera 4, 50 g KP-Sil, eluting with 50% EtOAc/isohexane to EtOAc) to afford the title compound as a pale yellow oil (353 mg, 1.5 mmol, 76%).
[1355] (b) (5-Bromo-pyrimidin-4-yl)-(4-methyl-piperazin-1-yl)-methanone I-90
[1356] Procedure similar to that described for 5-bromo-pyrimidine-4-carboxylic acid dimethylamide I-89 except 1-methylpiperazine (546 82 L, 4.9 mmol) was used. No final purification was performed. The crude title compound was provided as a yellow oil (1.9 g, 6.7 mmol, >100%).
[1357] (c) 3-Bromo-N,N-dimethyl-isonicotinamide I-91
[1358] Procedure similar to that described for 5-bromo-pyrimidine-4-carboxylic acid dimethylamide I-89 except 3-bromoisonicotinic acid I-87 (350 mg, 1.7 mmol) and dimethylamine hydrochloride (141 mg, 1.7 mmol) were used. The title compound was provided as an orange oil (1.1 g, 6.7 mmol, >100%).
[1359] (d) 3-Bromo-pyridine-2-carboxylic acid dimethylamide I-92
[1360] Procedure similar to that described for 5-bromo-pyrimidine-4-carboxylic acid dimethylamide I-89 except 3-bromopyridine-2-carboxylic acid I-88 (350 mg, 1.7 mmol) and dimethylamine hydrochloride (141 mg, 1.7 mmol) were used. The crude title compound was provided as an orange oil (1.2 g, 6.7 mmol, >100%).
[1361] (e) (3-Bromo-pyridin-2-yl)-(4-methyl-piperazin-1-yl)-methanone I-93
[1362] Procedure similar to that described for 5-bromo-pyrimidine-4-carboxylic acid dimethylamide I-89 except 3-bromopyridine-2-carboxylic acid I-88 (400 mg, 2.0 mmol) and 1-methylpiperazine (0.27 mL, 2.4 mmol) were used. The title compound was provided as a colourless oil (520 mg, 1.8 mmol, 92%).
[1363] (t) 3-(4-Dimethylcarbamoyl-pyrimidin-5-ylsulfanyl)-propionic acid methyl ester I-94
[1364] Procedure similar to that described for 5-(2-methoxycarbonyl-ethylsulfanyl)-pyrimidine-4-carboxylic acid methyl ester I-86 except 5-bromo-pyrimidine-4-carboxylic acid dimethylamide I-89 (353 mg, 1.5 mmol) was used. The title compound was provided as a yellow oil (130 mg, 0.48 mmol, 31%).
[1365] (g) 3-[4-(4-Methyl-piperazine-1-carbonyl)-pyrimidin-5-ylsulfanyl]-propionic acid methyl ester I-95
[1366] Procedure similar to that described for 5-(2-methoxycarbonyl-ethylsulfanyl)-pyrimidine-4-carboxylic acid methyl ester I-86 except (5-bromo-pyrimidin-4-yl)-(4-methyl-piperazin-1-yl)-methanone I-90 (700 mg, 2.5 mmol) was used. Purification was carried out by preparative HPLC (basic conditions) to provide the title compound as a colourless oil (85 mg, 0.3 mmol, 11%).
[1367] (h) 3-(4-Dimethylcarbamoyl-pyridin-3-ylsulfanyl)-propionic acid methyl ester I-96
[1368] Procedure similar to that described for 5-(2-methoxycarbonyl-ethylsulfanyl)-pyrimidine-4-carboxylic acid methyl ester I-86 except 3-bromo-N,N-dimethyl-isonicotinamide I-91 (396 mg, 1.7 mmol) was used. Purification by reverse phase column chromatography (Biotage, Isolera 4, 120 g KP-C18-HS, eluting with water to MeOH) afforded the title compound as a yellow oil (185 mg, 079 mmol, 41%).
[1369] (i) 3-(2-Dimethylcarbamoyl-pyridin-3-ylsulfanyl)-propionic acid methyl ester I-97
[1370] Procedure similar to that described for 5-(2-methoxycarbonyl-ethylsulfanyl)-pyrimidine-4-carboxylic acid methyl ester I-86 except 3-bromo-pyridine-2-carboxylic acid dimethylamide I-92 (396 mg, 1.7 mmol) was used. Purification by reverse phase column chromatography (Biotage, Isolera 4, 120 g KP-C18-HS, eluting with water to MeOH) afforded the title compound as a yellow oil (145 mg, 0.5 mmol, 32%).
[1371] (j) 3-[2-(4-Methyl-piperazine-1-carbonyl)-pyridin-3-ylsulfanyl]-propionic acid methyl ester I-98
[1372] Procedure similar to that described for 5-(2-methoxycarbonyl-ethylsulfanyl)-pyrimidine-4-carboxylic acid methyl ester I-86 except (3-bromo-pyridin-2-yl)-(4-methyl-piperazin-1-yl)-methanone I-93 (520 mg, 1.8 mmol) was used. Purification was carried out by preparative HPLC (basic conditions) to provide the title compound as a white solid (157 mg, 0.5 mmol, 27%).
[1373] 3-(Pyrimidin-5-ylsulfanyl)-propionic acid methyl ester I-101 and 3-(2-methyl-pyrimidin-5-ylsulfanyl)-propionic acid methyl ester I-102
##STR00077##
[1374] 3-(Pyrimidin-5-ylsulfanyl)-propionic acid methyl ester I-101
[1375] Procedure similar to that described for 5-(2-methoxycarbonyl-ethylsulfanyl)-pyrimidine-4-carboxylic acid methyl ester I-86 except 5-bromopyrimidine I-99 (300 mg, 1.9 mmol) was used. The title compound was provided as a yellow oil (231 mg, 1.2 mmol, 62%).
[1376] In a slight modification to the above procedure, purification by preparative HPLC (acidic conditions) also provided the title compound as a colourless oil (1.2 g, 5.8 mmol, 92%).
[1377] 3-(2-Methyl-pyrimidin-5-ylsulfanyl)-propionic acid methyl ester 1-102
[1378] Procedure similar to that described for 5-(2-methoxycarbonyl-ethylsulfanyl)-pyrimidine-4-carboxylic acid methyl ester I-86 except 5-bromo-2-methylpyrimidine I-100 (300 mg, 1.7 mmol) was used. The title compound was provided as a colourless oil (101 mg, 0.48 mmol, 27%).
[1379] Thioacetic acid 2, 6-dimethyl-tetrahydro-pyran-4-yl ester I-106
##STR00078##
[1380] (a) 2, 6-Dimethyl-tetrahydro-pyran-4-ol I-104
[1381] 2,6-Dimethyltetrahydro-4H-pyran-4-one (as a mixture of diastereoisomers) I-103 (360 mg, 2.77 mmol) was dissolved in anhydrous MeOH (10 mL) and sodium borohydride (116 mg, 2.77 mmol) added portion-wise at 0 C. The reaction mixture was allowed to warm to rt over the course of 18 h whereupon the reaction was quenched with saturated aq. ammonium chloride. The aqueous phase was extracted with Et.sub.2O (2) and the combined organic extracts washed with brine before passing through a phase separator cartridge (Biotage). Concentration in vacuo provided the title compound as a mixture of diastereoisomers (242 mg) which was used without further purification.
[1382] (b) Methanesulfonic acid 2, 6-dimethyl-tetrahydro-pyran-4-yl ester I-105
[1383] To a cooled (0 C.) solution of 2,6-dimethyl-tetrahydro-pyran-4-ol I-104 (242 mg, 1.9 mmol) in anhydrous DCM (10 mL) was added mesyl chloride (0.17 mL, 2.2 mmol) followed by TEA (0.51 mL, 3.7 mmol). The reaction mixture was stirred at 0 C. for 4 h whereupon water was added and the aqueous layer extracted with DCM (2). The combined organic extracts were washed with saturated sodium bicarbonate and brine before passing through a phase separator (Biotage). Concentration in vacuo provided the crude title compound as a mixture of diastereoisomers as a yellow oil (490 mg).
[1384] (c) Thioacetic acid 2, 6-dimethyl-tetrahydro-pyran-4-yl ester I-106
[1385] Methanesulfonic acid 2,6-dimethyl-tetrahydro-pyran-4-yl ester (490 mg, crude) I-105 was dissolved in DMA (7 mL) and potassium thioacetate (640 mg, 5.5 mmol) added in one portion. The reaction mixture was heated at 80 C. for 24 h. The reaction mixture was diluted with water and the aqueous residue extracted with Et.sub.2O (3). The combined organic extracts were concentrated in vacuo. The residue was purified by column chromatography (Biotage, SP1, 10 g KP-Sil, eluting with isohexane to 20% EtOAc/isohexane) to afford the title compound as a mixture of diastereoisomers (159 mg, 0.85 mmol, 46% over 3 steps).
[1386] 4-Methyl-tetrahydro-pyran-4-thiol I-110
##STR00079##
[1387] (a) 1,6-Dioxa-spiro[2.5]octane I-108
[1388] Trimethylsulfoxonium iodide (286 mg, 13 mmol) was dissolved in DMSO (20 mL) under an atmosphere of nitrogen. NaH (60% dispersion in mineral oil, 520 mg, 13 mmol) was then added portion-wise (NB. vigorous effervescence observed). The resultant suspension was stirred at rt for 1 h whereupon tetrahydro-4H-pyran-4-one I-107 (0.93 mL, 10 mmol) was added dropwise. The reaction mixture was stirred at rt for an additional 1 h where it was then poured into a water/ice slurry. The aqueous phase was extracted with Et.sub.2O (3) and the combined organic extracts washed with water and brine before drying over MgSO.sub.4. Concentration in vacuo provided the title compound as a pale yellow oil (725 mg, 6.3 mmol, 63%).
[1389] (b) 6-Oxa-1-thia-spiro[2.5]octane I-109
[1390] 1,6-Dioxa-spiro[2.5]octane I-108 (725 mg, 6.3 mmol) was dissolved in anhydrous MeOH (20 mL) and thiourea (480 mg, 6.3 mmol) added. The reaction mixture was heated at 80 C. for 4.5 h at which point water was added. The aqueous phase was extracted with Et.sub.2O (3) and the combined organic extracts washed with brine before drying over MgSO.sub.4. The residue was purified by column chromatography (Biotage, SP1, 10 g KP-Sil, eluting with isohexane to 20% EtOAc/isohexane) to afford the title compound (130 mg, 1 mmol, 16%).
[1391] (c) 4-Methyl-tetrahydro-pyran-4-thiol I-110
[1392] 6-Oxa-1-thia-spiro[2.5]octane I-109 (130 mg, 1 mmol) was dissolved in THF (3.5 mL) and heated to 70 C. under an atmosphere of nitrogen. Lithium aluminium hydride (1 M in THF, 0.5 mL, 0.5 mmol) was then added and the reaction mixture stirred for 1 h. The reaction mixture was cooled to 0 C. and HCl (1 N, 3.5 mL) added dropwise. The aqueous phase was then extracted with Et.sub.2O (210 mL) and the combined organic extracts concentrated in vacuo. Purification by column chromatography (Biotage, SP1, 10 g KP-Sil, eluting with pentane to 10% Et.sub.2O/pentane) afforded the title compound (56 mg, 0.42 mmol, 42%).
[1393] ()Thioacetic acid S-((3S, 4S)-3-methyl-tetrahydro-pyran-4-yl)ester I-113
##STR00080##
[1394] (a) 3-Methyl-tetrahydro-pyran-4-one I-124
[1395] Diisopropylamine (1.1 mL, 6.0 mmol) in THF (10 mL) was cooled to 78 C. and n-butyllithium (1.6 M hexanes, 3.8 mL, 6.0 mmol) added dropwise. The reaction mixture was stirred at 78 C. and allowed to gradually warm to rt over the course of 2 h before cooling to 78 C. once again. Tetrahydro-4H-pyran-4-one I-107 (500 mg, 5.0 mmol) as a solution in THF (20 mL) and HMPA (0.88 mL) was then added dropwise and the reaction was subsequently stirred at 78 C. and allowed to gradually warm to rt over the course of 2 h. The reaction mixture was cooled to 0 C. whereupon saturated aqueous ammonium chloride was added and the aqueous phase extracted with Et.sub.2O (2). Concentration under reduced pressure provided the crude residue which was purified by column chromatography (Biotage, SP1, 25 g KP-Sil, eluting with isohexane to 20% EtOAc/isohexane) to afford the title compound (400 mg, 2.8 mmol, 56%).
[1396] (b) 3-Methyl-tetrahydro-pyran-4-ol I-111
[1397] Procedure similar to that described for 2,6-dimethyl-tetrahydro-pyran-4-ol I-104 except 3-methyl-tetrahydro-pyran-4-one I-124 (350 mg, 2.5 mmol) was used. The crude title compound was provided as a mixture of diasteroisomers (440 mg).
[1398] (a) () (3S, 4R)-3-Methyl-tetrahydro-pyran-4-ol I-112a
[1399] Procedure similar to that described for methanesulfonic acid 2,6-dimethyl-tetrahydro-pyran-4-yl ester I-105 except 3-methyl-tetrahydro-pyran-4-ol I-111 (140 mg, 1.2 mmol) was used. The title compound was provided (127 mg, 0.65 mmol, 54%). The trans-isomer I-112b was also isolated (30 mg, 0.15 mmol, 13%).
[1400] (b) () Thioacetic acid S-((3S, 4S)-3-methyl-tetrahydro-pyran-4-yl)ester I-113
[1401] Procedure similar to that described for thioacetic acid 2,6-dimethyl-tetrahydro-pyran-4-yl ester I-106 except () (3S, 4R)-3-methyl-tetrahydro-pyran-4-ol I-112a (111 mg, 0.57 mmol) was used. The title compound was provided (44 mg, 0.25 mmol, 44%).
[1402] 4-Acetylsulfanyl-piperidine-1-carboxylic acid methyl ester I-121 and 4-acetylsulfanyl-piperidine-1-carboxylic acid ethyl ester I-122
##STR00081##
[1403] (a) 4-Oxo-piperidine-1-carboxylic acid methyl ester I-115
[1404] To a cooled (0 C.) solution of 4-piperidone I-114 (300 mg, 3.0 mmol) in water (2 mL) was added a solution of potassium carbonate (1.05 g, 7.6 mmol) in water (5 mL) followed by methyl chloroformate (350 l, 4.5 mmol). The reaction mixture was stirred at 0 C. for 3 h. The mixture was diluted with DCM, the layers separated and the aqueous phase extracted with DCM (3). The combined organic extracts were passed through a phase separator cartridge (Biotage) and concentrated in vacuo. Purification by flash column chromatography (Biotage SP1, 25 g KP-Sil, eluting with isohexane to EtOAc) provided the title compound as a colourless oil (320 mg, 2.0 mmol, 68%).
[1405] (b) 4-Hydroxy-piperidine-1-carboxylic acid methyl ester I-117
[1406] To a solution of 4-oxo-piperidine-1-carboxylic acid methyl ester I-115 (315 mg, 2.0 mmol) in MeOH (5 mL) at 0 C. was added sodium borohydride (114 mg, 3.0 mmol). The reaction mixture was stirred at 0 C. for 2 h. The reaction was quenched with saturated aqueous ammonium chloride (5 mL), MeOH was removed in vacuo and the aqueous layer was extracted with DCM (3). The combined organic extracts were passed through a phase separator cartridge (Biotage) and concentrated in vacuo to afford the crude product which was purified by column chromatography (Biotage, SP1, 25 g KP-Sil, eluting with isohexane to EtOAc) to afford title compound as a colourless oil (140 mg, 0.88 mmol, 44%).
[1407] (c) 4-Methanesulfonyloxy-piperidine-1-carboxylic acid methyl ester I-119
[1408] To a solution of 4-hydroxy-piperidine-1-carboxylic acid methyl ester I-117 (140 mg, 0.88 mmol) in DCM (3 mL) was added mesyl chloride (82 L, 1.0 mmol) and TEA (245 L, 1.76 mmol). The reaction mixture was stirred at 0 C. for 1 h. The reaction was quenched with water (5 mL), the layers separated and the aqueous layer extracted with DCM (35 mL). The combined organic extract was washed with saturated aqueous sodium bicarbonate (10 mL) and brine (10 mL), passed through a phase separator cartridge (Biotage) and concentrated in vacuo. The crude product was purified by flash column chromatography (Biotage SP1, 10 g KP-Sil, eluting with isohexane to EtOAc) to afford the title compound as a colourless oil (168 mg, 0. 71 mmol, 80%).
[1409] (d) 4-Acetylsulfanyl-piperidine-1-carboxylic acid methyl ester I-121
[1410] To a solution of 4-methanesulfonyloxy-piperidine-1-carboxylic acid methyl ester I-119 (168 mg, 0.71 mmol) in DMA (4 mL) was added potassium thioacetate (243 mg, 2.1 mmol). The reaction was heated at 80 C. for 18 h. The reaction was cooled to rt and Et.sub.2O (10 mL) and water (10 mL) were added. The layers were separated and the aqueous layer extracted with Et.sub.2O (310 mL). The combined organic extract was washed with water (10 mL) and brine (10 mL) before passing through a phase separator cartridge (Biotage). The crude residue was purified by flash column chromatography (Biotage SP1, 25 g KP-Sil, eluting with isohexane to EtOAc) to afford the title compound as a pale orange oil (78 mg, 0.36 mmol, 51%).
[1411] (e) 4-Oxo-piperidine-1-carboxylic acid ethyl ester I-116
[1412] Procedure similar to that described for 4-oxo-piperidine-1-carboxylic acid methyl ester I-115 except ethyl chloroformate (0.43 mL, 4.5 mmol) was used. The title compound was provided as a colourless oil (332 mg, 1.9 mmol, 65%).
[1413] (t) 4-Hydroxy-piperidine-1-carboxylic acid ethyl ester I-118
[1414] Procedure similar to that described for 4-hydroxy-piperidine-1-carboxylic acid methyl ester I-117. The title compound was provided as a colourless oil (337 mg, 1.9 mmol, 100%).
[1415] (g) 4-Methanesulfonyloxy-piperidine-1-carboxylic acid ethyl ester I-120
[1416] Procedure similar to that described for 4-methanesulfonyloxy-piperidine-1-carboxylic acid methyl ester I-119. The title compound was provided as a colourless oil (423 mg, 1.7 mmol, 94%).
[1417] (h) 4-Acetylsulfanyl-piperidine-1-carboxylic acid ethyl ester I-122
[1418] Procedure similar to that described for 4-acetylsulfanyl-piperidine-1-carboxylic acid methyl ester I-121. The title compound was provided as a pale red oil (218 mg, 0.9 mmol, 59%).
[1419] Thioacetic acid S-(tetrahydro-pyran-2-yl) ester I-126
##STR00082##
[1420] Potassium thioacetate (460 mg, 4.0 mmol) was dissolved in conc. HCl (32%, 10.2M) and cooled to 0 C. Dihydropyran I-125 (0.37 mL, 4.0 mmol) was then added dropwise and the reaction mixture stirred at 0 C. for 2 h whereupon the reaction mixture was concentrated in vacuo. The residue was purified by column chromatography (Biotage, SP1, 10 g KP-Sil, eluting with isohexane to 10% EtOAc/isohexane) to afford the title compound (630 mg, 3.9 mmol, 98%).
EXAMPLE 1
[1421] Compounds of the formula I were synthesised via the coupling of chloro(trialkyl phosphine) gold(I) complexes of formula VII with thiol derivatives of general formula III:
##STR00083##
[1422] Method A: To a stirred suspension of chlorophosphine gold(I) compound VII (0.32 mmol) in EtOH (1 mL) at 0 C. was slowly added the appropriate thiol III (0.32 mmol) as a solution in aqueous K.sub.2CO.sub.3 (10% w/v, 1 mL). The reaction mixture was then stirred at 0 C. for 1 h before it was diluted with water (5 mL) and extracted with DCM (415 mL). The combined organic extracts were passed through a phase separator cartridge (Biotage) and the solvent evaporated to provide the title compound I.
[1423] Method B: As Method A, except the thiol III was pre-dissolved in a mixture of K.sub.2CO.sub.3 (aq., 1 mL) and EtOH (1 mL).
[1424] Method C: As Method A, except the reaction was heated at 50 C. for 18 h.
[1425] Method D: The appropriate thiol III (0.17 mmol) and chlorophosphine gold(I) compound VII (0.17 mmol) were combined and dissolved in DCM (5 mL) under an atmosphere of nitrogen. The solution was cooled to 0 C. before TEA (0.34 mmol) was added dropwise over 5 min. The reaction was stirred at 0 C. for 45 min whereupon the reaction was diluted with water (15 mL) and the layers separated. The aqueous residue was extracted with DCM (210 mL) and the combined organic extracts washed with brine (115 mL) before passing through a phase separator cartridge (Biotage). Concentration in vacuo afforded the title compound I.
[1426] Method E: To a stirred suspension of chlorophosphine gold(I) compound VII (0.32 mmol) in EtOH or MeOH (1 mL) at 0 C. was slowly added the appropriate thiol III (0.32 mmol) as a solution in 10% K.sub.2CO.sub.3 (aq., 1 mL). The reaction was stirred at 0 C. for 1 h before diluting with water (5 mL) and acidifying to pH3 with KHSO.sub.4 (aq.). The aqueous layer was extracted with DCM (415 mL) and the combined organic extracts passed through a phase separator cartridge (Biotage) before concentrating in vacuo to provide the title compound I.
[1427] Method F: As method A except MeOH was used as the reaction solvent and aqueous K.sub.2CO.sub.3 (10% w/v) was added to a stirring solution of chlorophosphine gold(I) compound VII and thiol III. The resultant precipitate that formed during the reaction was collected by filtration and was washed with a combination of MeOH, EtOH, water, Et.sub.2O or hexane to provide the title compound.
[1428] Method G: As method A except after stirring at 0 C. for 1 h, water was added and the resultant precipitate collected by filtration. The solid was washed with a combination of MeOH, EtOH, water, Et.sub.2O or hexane to provide the title compound.
[1429] Method H: As method A except after aqueous work up, the product was purified by trituration.
[1430] Method I: As method E except after aqueous work up, the product was purified by trituration.
[1431] Method J: As method F except EtOH was used as the reaction solvent.
[1432] Method K: Thiol III (0.1 mmol) was dissolved in THF (10 mL) and NaH (60% dispersion in mineral oil, 0.2 mmol) added. The reaction mixture was stirred at rt for 15 mins whereupon chlorophosphine gold(I) compound VII (0.1 mmol) was added. The reaction mixture was stirred at rt for 18 h before water (10 mL) was added followed by aqueous KHSO.sub.4 (2M) until pH 6 was reached. The aqueous layer was extracted using EtOAc (330 mL) and the combined organic extracts concentrated in vacuo to provide the crude product. Trituration with 1:1 Et.sub.2O/isohexane provided title compound I.
[1433] Method L: As method A except MeOH was used as the reaction solvent.
[1434] Method M: The appropriate thiol III (0.32 mmol) was dissolved in EtOH (2.0 mL) and aqueous NaOH (1M, 2 mL) added. The reaction was then cooled to 0 C. and chlorophosphine gold(I) complex VII (0.32 mmol) was added in one portion. The reaction was stirred at 0 C. for 1 h whereupon the reaction was poured into water and extracted with DCM (2). The combined organic extracts were washed with brine and passed through a phase separator cartridge (Biotage) and concentrated in vacuo to afford the product I.
[1435] The solvent (or combination of solvents) used for trituration and isolation of target compounds I can be selected from the following: MeOH, EtOH, water, Et.sub.2O, EtOAc, isohexane or DCM.
[1436] Some of the compounds were prepared using methods in which minor modifications to the general methods were made; specifically, these methods involved small changes to the stoichiometry of reagents (1-2 equivalents), duration of reaction (1-18 h) and volume of solvent (1-2 mL).
[1437] The following compounds were made using these methods:
TABLE-US-00002 TABLE 1 Compound Analytical Data Structure Number Method Physical appearance/Yield
[1438] Compounds of the formula I, synthesised from thiol precursors IV, V, VI and IX, were synthesised via a one-pot, two-step procedure comprising thiol deprotection and coupling in situ to chloro(trialkyl phosphine) gold(I) complex VII.
EXAMPLE 2
[1439] ##STR00149##
[1440] The appropriate protected thiol IV or V (0.33 mmol) was dissolved in MeOH (1 mL) and aqueous NaOH (10% w/v, 0.3 mL) added in one portion. The reaction was heated to 100 C. in a microwave reactor for 1 h, whereupon the reaction was cooled to 0 C. and the chlorophosphine gold(I) compound VII (0.33 mmol) added in one portion. The reaction was stirred at 0 C. for 1 h before it was diluted with water (5 mL) and extracted with DCM (415 mL). The combined organic extracts were passed through a phase separator cartridge (Biotage) and the solvent evaporated to provide the title compound I.
[1441] The following compounds were made using this method:
TABLE-US-00003 TABLE 2 Compound Analytical Data Structure Number Physical appearance/Yield
EXAMPLE 3
[1442] ##STR00153##
[1443] Under an atmosphere of nitrogen, the appropriate protected thiol VI (0.11 mmol) as a solution in degassed EtOH (1.0 mL) and aqueous NaOH (1 M, 1.0 mL) was added to chlorophosphine gold(I) compound VII (0.11 mmol) in one portion. The reaction was stirred at rt for 2 h whereupon water (10 mL) was added and the mixture extracted with DCM (310 mL). The combined organic extracts were passed through a phase separator cartridge (Biotage) and concentrated in vacuo to afford the crude product which was triturated with pentane/Et.sub.2O (2) to afford the title compound I.
[1444] Compound 78 was prepared and isolated as described in the general method except MeOH was used as the reaction solvent.
[1445] The following compounds were made using this method:
TABLE-US-00004 TABLE 3 Compound Analytical Data Structure Number Physical appearance/Yield
EXAMPLE 4
[1446] ##STR00160##
[1447] The appropriate protected thiol IX (0.18 mmol) was dissolved in MeOH (2.0 mL) and aqueous NaOH (10% w/v, 0.5 mL) added. The reaction mixture was heated to 100 C. in a microwave reactor for 1 h. The reaction was then cooled to 0 C. and chlorophosphine gold(I) complex VII (0.18 mmol) was added in one portion. The reaction was stirred at 0 C. for 1 h whereupon the reaction was poured into water (10 mL) and extracted with DCM (315 mL). The combined organic extracts were passed through a phase separator cartridge (Biotage) and concentrated in vacuo to afford the product I.
[1448] Compound 52 was prepared and isolated as described in the general method except after stirring at 0 C. for 1 h, water was added followed by acidification to pH 3 with aqueous KHSO.sub.4 (2M).
[1449] The methyl ester in I-86 is also hydrolysed to the carboxylic acid during the reaction to prepare compound 52.
[1450] Compound 65 was prepared as described in the general method except the title compound was isolated by trituration.
[1451] The solvent (or combination of solvents) used for trituration and isolation of target compounds I can be selected from the following: MeOH, EtOH, water, Et.sub.2O, EtOAc, isohexane or DCM.
[1452] The following compounds were made using this methods:
TABLE-US-00005 TABLE 4 Compound Analytical Data Structure Number Physical appearance/Yield
EXAMPLE 4
[1453] Growth Media
TABLE-US-00006 Tryptic Soy Broth Formula/Litre Pancreatic Digest of Casein 17.0 g Enzymatic Digest of Soybean 3.0 g Sodium Chloride 5.0 g Di-potassium hydrogen Phosphate 2.5 g Glucose 2.5 g
[1454] Directions for use: Dissolve 30 g of the medium in one litre of purified water, mix thoroughly, and then autoclave at 121 C. for 15 minutes.
TABLE-US-00007 Luria Broth Formula/Litre Tryptone 10.0 g Yeast Extract 5.0 g NaCl 5.0 g
[1455] Directions for use: Dissolve components in 1 litre of distilled or deionized water and sterilize by autoclaving at 121 C. for 15 minutes.
TABLE-US-00008 Mueller Hinton II Broth (Cation-Adjusted) Formula/Litre Beef Extract 3.0 g Acid Hydrolysate of Casein 17.5 g Starch 1.5 g *Adjusted and/or supplemented as required with appropriate salts to provide 20-25 mg/L of calcium and 10-12.5 mg/L of magnesium and as additionally required to meet performance criteria.
[1456] Directions for use: Dissolve components in 1 litre of distilled or deionized water and sterilize by autoclaving at 121 C. for 15 minutes.
TABLE-US-00009 Brain Heart Infusion Broth Formula/Litre Brain Heart Infusion solids 12.5 g Beef heart infusion solids 5 g Proteose peptone 10 g Glucose 2 g Sodium Chloride 5 g Di-sodium Phosphate 2.5 g
[1457] Directions for use: Dissolve components in 1 litre of purified water. Heat the mixture with frequent agitation to completely dissolve the medium, and sterilize by autoclaving at 121 C. for 15 minutes.
[1458] Growth assay for S.aureus.
[1459] Stock solution of the test compounds (20 mg/ml) in dimethyl sulfoxide (DMSO) were serially diluted in DMSO and each diluted compound added in duplicate to a 96-well plate to a final DMSO concentration of 2% (v/v). An overnight culture of S. aureus (Oxford strain) grown in tryptic soy broth (TSB) was diluted to approximately 510.sup.7 cfu/ml and 150 l of this sample was added to each well of the 96-well plates. Control wells included an untreated control with bacteria in TSB in the presence of 2% DMSO and a negative sample (containing 150p1 TSB growth media in the presence of 2% DMSO). Plates were incubated in a shaking incubator at 37 C. for 22-24 hours and bacterial growth assessed by absorbance at a wavelength of 595 nm. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of compound that inhibited growth compared to the no-treatment control.
[1460] Variation of growth assays for:
[1461] Klebsiella pneumoniae, Acinetobacter baumannnii or E.coli (ATCC 25922): use of 1/100 overnight dilution to set up assay, medium used: Luria broth (LB); incubation without shaking.
[1462] P.aeruginosa (ATCC 27853): use of 1/100 overnight dilution to set up assay, medium used: Cation adjusted Mueller Hinton broth (CaMHB); incubation without shaking.
TABLE-US-00010 K. P. S. aureus pneumoniae E. coli aeruginosa MIC MIC MIC MIC Compound (g/mL) (g/mL) (g/mL) (g/mL) 1 1.6 6.3 6.3 6.3 2 1.6-3.1 6.3 12.5 6.3 3 1.6 6.3 6.3 12.5 4 0.8 1.6 1.6 3.1 5 0.8-1.6 3.1 1.6 25 6 0.8 3.1-6.3 1.6-3.1 12.5-25 7 0.8 1.6-3.1 1.6 12.5 8 0.8 3.1 1.6 12.5 9 0.8-1.6 3.1 1.6 12.5 10 0.8-1.6 3.1 1.6-3.1 12.5 11 0.8 3.1-6.3 1.6-3.1 6.3-12.5 12 0.8 3.1-6.3 1.6-3.1 6.3-12.5 13 0.8-1.6 3.1 3.1-6.3 6.3-12.5 14 0.8-1.6 3.1-6.3 6.3-12.5 12.5 15 1.6 3.1 1.6-3.1 12.5 16 1.6 6.3 3.1 12.5 17 1.6 3.1 1.6-3.1 12.5 18 1.6 6.3 3.1 12.5 19 0.8 3.1-6.3 1.6-3.1 12.5 20 0.8 3.1-6.3 3.1-6.3 12.5 21 0.8 6.3 6.3 12.5 22 0.8 3.1-6.3 3.1 12.5 23 0.8-1.6 6.3 3.1-6.3 25 24 0.8 6.3 3.1-6.3 100 25 <0.8 3.1-6.3 1.6 26 <0.8 3.1-6.3 1.6-3.1 27 0.8-1.6 25 12.5 28 <0.8 6.3 12.5 29 0.8-1.6 12.5 3.1 30 <0.8 12.5 3.1-6.3 31 3.1 25-50 6.3 32 0.8 3.1-6.3 1.6 33 3.1 6.3 6.3 6.3 34 0.8 6.3 3.1-6.3 12.5-25 35 0.8-1.6 3.1-6.3 6.3 12.5 36 <0/8 6.3 3.1 37 >0.8 6.3 3.1 38 <0.8 3.1-6.3 1.6 39 0.8-1.6 3.1-6.3 1.6-6.3 25 40 0.8 1.6-3.1 0.8-1.6 12.5 41 0.8 1.6-3.1 0.8-1.6 6.3-12.5 42 0.8 3.1-6.3 0.8-1.6 12.5-25 43 1.6-3.1 12.5-25 6.3-12.5 50-100 44 0.8-3.1 50-100 1.6-12.5 25-50 45 0.8 3.1-6.3 1.6-3.1 6.3-12.5 46 0.8-1.6 3.1-6.3 0.8-1.6 12.5 47 0.8 6.3 1.6-25 6.3-25 48 0.8 6.3-12.5 1.6-3.1 12.5 49 0.8 6.3-12.5 1.6-3.1 12.5 50 0.8-1.6 6.3 0.8-3.1 6.3-12.5 51 1.6-6.3 6.3-25 6.3-12.5 6.1-12.5 52 0.8 6.1-12.5 0.8-3.1 6.3-100 (*25 n = 8) 53 0.8-3.1 3.1-12.5 1.6-3.1 6.3-25 54 0.8-3.1 6.1-12.5 3.1-12.5 12.5-25 55 0.8 6.3-12.5 0.8-1.6 6.3-12.5 56 0.8 12.5 1.6-3.1 6.3-12.5 57 0.8-1.6 6.3 1.6-3.1 6.3-12.5 58 1.6-3.1 3.1 1.6-3.1 3.1-6.3 59 0.8-3.1 6.3 1.6-3.1 6.3-25 60 0.8-1.6 6.3 1.6-3.2 6.3-12.5 61 1.6-3.1 25-50 12.5-25 25->100 62 0.8-3.1 12.5-25 3.1-12.5 50-100 63 0.8-1.6 3.1-6.3 1.6-6.3 6.3-12.5 64 0.8-1.6 3.1-6.3 1.6-3.2 6.3-12.5 65 0.8-1.6 12.5 3.1-6.3 6.3-25 66 3.1-6.3 3.1 3.1-6.3 6.3-12.5 67 0.8 12.5-25 1.6 3.1-12.5 68 0.8-12.5 6.3-25 1.6-12.5 12.5-25 69 3.1-6.3 >100 12.5-25 >100 70 1.6-3.1 25-50 6.3-12.5 25 71 0.8-1.6 1.6 1.6-3.1 6.3-25 73 0.8-1.6 6.3-12.5 3.1 25->100 74 0.8-1.6 12.5-25 3.1 6.3-12.5 75 1.6-3.1 1.6 6.3 6.3-25 76 0.8 50 25-50 >100 77 1.6 25 25 50 78 6.3 100 100 100 79 6.3 12.5-25 12.5-25 12.5-25 80 0.8 2.3 3.1 6.3 81 0.8 3.1 3.1-6.3 6.3 82 0.8-1.6 3.1 2.4 6.3 83 0.8 3.1-6.3 3.1-6.3 3.1-12.5 84 1.6 3.1 6.3-12.5 12.5-50 85 0.8-1.6 25-50 12.5-25 12.5->100 86 0.8-1.6 >100 50->100 50->100 87 1.2 6.3 3.1-6.3 12.5-25 88 0.8-1.6 1.6-6.3 90 1.6 25 12.5-50 >100 91 0.4-1.6 3.1 1.6-6.3 1.6-6.3 I-27 0.8 4.7 2.4 3.1 I-30 0.8-6.3 3.1-6.3 1.6-6.3 6.3-12.5 I-31 0.8 6.3 6.3-12.5 12.5-25 I-34 1.2-3.1 6.3 3.1 I-36 6.3 I-39 1.6-3.1 6.3 6.3 12.5 I-44 6.3-25 50-100 25-100 6.3-50 I-49 0.8-1.6 12.5-25 6.3-12.5 25 I-50 1.6-3.1 12.5-25 6.3-12.5 12.5-25 I-52 0.8 6.3 6.3 6.3 I-54 12.5 12.5 I-130 0.8 9.4 6.3 9.4 *= geometic mean
[1463] Inhibition of Neisseria gonorrhoeae (NCTC 8375) growth on solid media
[1464] N. gonorrhoeae was grown for 48 hours at 37 C. on Chocolate agar plates (BD Diagnostics). A culture loop-full of bacterial culture was picked from the plate and re-suspended in 50 l sterile phosphate buffered saline. The suspension was spread evenly onto the surface of a fresh chocolate agar plate and left to dry (approximately 5 minutes). Small discs of blotting paper were placed on the surface of the agar plate and 3 l of test compounds (at 20 mg/ml) were applied to the discs. The plates were incubated overnight at 37 C. and zones of clearance around the disc were measured.
[1465] HepG2 Cell Inhibition Assay
[1466] Cell counting kit-8 (Sigma, CCK-8) assays were performed to assess the effect of compounds on cell viability. The assay is based on the reduction of a water-soluble tetrazolium salt (WST-8) by cellular dehydrogenases to a formazan dye which can be detected spectroscopically. 96-well plates were seeded with the human hepatocyte cell line (HepG2) at approximately 810.sup.3 cells per well in Eagle's Minimum Essential Medium (EMEM) with Earle's salts and sodium bicarbonate supplemented with 10% heat-inactivated foetal bovine serum 2 mM glutamine and 1% non-essential amino acids (NEAA). The following day serial dilutions of compounds (dissolved and diluted in DMSO) were added to the cells in duplicates. Control wells included an untreated control where cells were grown in the presence of 1% DMSO and a medium only control (plus 1% DMSO). After 24 hours CCK-8 reagent (10 l) was added to each well and cell viability was assessed by measuring the absorbance at a wavelength of 450 nm after 2-3h hours. Only living cells can reduce the tetrazolium salts into coloured formazan products. Results were expressed as 50% growth inhibition (TD.sub.50) values compared to untreated control.
TABLE-US-00011 HepG2 cell Compound TD.sub.50 (g/mL) 1 2 2 6 3 32 4 4 5 3 6 11.5 7 6 8 3 9 3 10 2 11 2 12 5 13 2.5 14 3 15 23 16 17 17 3.5 18 18 23 2 25 22 26 13 27 22 28 11 29 35 30 19 31 45 32 11 33 4 34 1 35 4
Efficacy Studies in the Galleria mellonella Model
[1467] G. mellonella larvae at 5.sup.th or 6.sup.th instar stage were purchased from a commercial supplier and used within 3 days. Prior to infection larvae were kept at room temperature. Larvae were infected with bacteria (various Gram positive and negative bacteria, including S.aureus, K.pneumoniae, E.coli and P.aeruginosa) using a sterile Hamilton syringe. Bacteria cultures were grown overnight, washed 3 in PBS and resuspended in PBS. Larvae were wiped with 70% ethanol and 10 l of bacteria solution (to cause 80%-100% death within 3-4 days) was injected into the bottom right proleg of the larvae. Larvae injected with 10 l of PBS were used as negative controls. Larvae were then placed in petri dishes (1 dish per condition) containing filter paper at the bottom of the dish at 37 C. After various time points post infection (1-6h), larvae were taken from the incubator wiped again with 70% ethanol and injected with 10 l of various concentrations of compound, dissolved in either 5% dimethyl sulfoxide, 5% ethanol or 5% 1-methyl-2-pyrrolidinone into a proleg on the left hand-side. Control larvae received 10 l of 5% solvent. Ten larvae were injected for each condition. To assess the toxicity of the compound, larvae were injected with various concentrations of compound alone. Larvae were returned to a 37 C. incubator and checked daily. Larvae were considered dead when no movement occurred when touched with a blunt pair of forceps. Black or discoloured larvae which still showed movement were considered to be alive. Numbers of dead larvae were recorded each day.
Primary Cells Viability Assay
[1468] Neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated from venous blood obtained from healthy volunteers as previously described (Nauseef, Methods in Molecular Biology, 412 (2007), pp. 15-20). In brief, heparinised blood was diluted 1:1 with 3% Dextran-500 PBS solution (Sigma) to allow for erythrocyte sedimentation. Buffy coat was centrifuged over Hypaque-Ficoll (GE Lifescience) and PBMCs were carefully collected from the interface of the Hypaque-Ficoll and the upper liquid layer. Pelleted neutrophils were collected after hypotonic lysis of residing erythrocytes. Isolated cells were washed and suspended in culture media (RPMI+10% FBS) at 2 106 cells/mL. Cell suspensions were transferred into 96-well plates containing compound serially diluted in DMSO (1% final volume). After 24 hours, the reaction was stopped and cells were stained with AnnexinV and 7-AAD. Results were determined by FACSCalibur and viability was defined for AnnexinV/7-AAD double negative cells population.
Biofilm Prevention Assay (S. aureus)
[1469] The effect of a test compound on the formation of a S. aureus biofilm was assessed using a biofilm prevention assay as described by Merritt et al. Current Protocols in Microbiology, 2011, 1B.1.1-1B.18 with slight modifications. Briefly, S. aureus was grown overnight in tryptic soy broth (TSB) and diluted to 1/100 before 150 L was added to the wells of a flat bottomed 96-well plate. Three microliters of compound at the appropriate dilution in DMSO was added to the wells in duplicate. Controls included a positive control with bacteria alone in TSB with 2% DMSO and a negative (no bacteria) control with 150 L TSB containing 2% DMSO. Plates were sealed with AeraSeal and incubated at 37 C. for 24 hours. Plates were then washed three times with PBS, dried at 60 C. for 1 hour and stained with crystal violet for 1 hour. The plates were again washed three times with water, then dried 33% acetic acid was added to re-solubilize the crystal violet stain bound to the adherent cells. Absorbance was then measured at 595 nm and expressed as a percentage of the bacteria only control. A biofilm inhibitory concentration (BIC.sub.90) was determined as the concentration at which biofilm mass (measured by crystal violet staining) was reduced by at least 90% compared to untreated controls.
[1470] The effect of a test compound on preformed S. aureus biofilms can also be assessed. Briefly S.aureus was plated in 96-well plates as described above and incubated at 37 C. for 24 hours. Biofilms were then washed 3 times with TSB and 150 L of fresh TSB and 3 L of compound at the appropriate dilution in DMSO was added to the wells in duplicate. Plates were again sealed with AeraSeal and reincubated at 37 C. for 24 hours. Biofilm was then detected as described above.
TABLE-US-00012 S. aureus BF dispersal Compound BIC.sub.90 (g/mL) 6 0.8 10 0.8 11 0.8 12 1.6 18 0.8 27 0.8 28 0.8 29 0.8 36 0.8
Biofilm Assay for A. baumannii
[1471] A.baumannii was grown overnight in LB broth and diluted 1/00- 1/500 before 200 L was added to the wells of a flat bottomed 96-well plate with TSP 96 pins lid inserted. Plates with pins were incubated at 37 C. for 24 hours. Pins were washed with sterile phosphate buffered saline three times and exposed to compounds at pre-determined concentration in LB broth for 24 hours. Pins were washed again and either stained with crystal violet as described in the S.aureus biofilm assay, or incubated with LB media for 24 hours and the minimum biofilm eradication concentration (MBEC) was measured as the lowest concentration of compounds preventing further planktonic growth.
TABLE-US-00013 Compound Minimum biofilm eradication conc. (MBEC) g/mL 6 10 18 2.5 28 20 29 20 31 20 32 10 36 10 37 20 38 10 39 10 40 5 41 5 62 10
Persister Cell Assay
[1472] To determine whether S. aureus persister cells were susceptible to treatment with a test compound, a persister cell (or SCV) isolate hemB mutant of NCTC 8325-4 was used (Von Eiff et al., (1997) J Bacteriol 179:4706-4712). This persister cell variant displays varying resistance to erythromycin and the aminoglycosides gentamicin and kanamycin. Growth assays were performed essentially as described above with the bacteria being grown in TSB. Disc assays were also performed by plating bacteria on TSB agar. Discs impregnated with an amount of test compound were placed on top of the agar. The plates were incubated overnight at 37 C. and any zone of bacterial inhibition was observed.
Sensitivity of Multidrug Resistant Clinical Isolates
[1473] The activity of test compounds against multi-drug resistant bacterial strains was assessed by the disk diffusion assay; a standardised method to assess for the antimicrobial susceptibility of microorganisms (adapted from EUCAST, Version 5, January 2015). In brief, bacterial cultures were suspended in phosphate buffer and spread evenly onto blood agar plates. Cellulose disks were placed onto the agar plates and 3 l test compound (60 g/disk) were pipetted to the centre. A panel of standard antibiotics disks (Sigma) were used to control for the antimicrobial resistance profile of the individual strains (quantity as indicated in the table). The plates were then placed into a thermo-incubator and were cultured at 37 C. over-night. Activity was recorded by measuring the zone of clearance (mm) around the disks.
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