COMPOUNDS

Abstract

Compounds of general formula (I) wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and X are as defined herein are inhibitors of the epithelial sodium channel (ENaC) and are useful for the treatment or prevention respiratory diseases and conditions, skin conditions and ocular conditions.

##STR00001##

Claims

1. A compound of general formula (I) including all tautomeric forms, all enantiomers and isotopic variants and salts thereof: ##STR00316## wherein X.sup.− is an anion; R.sup.1 is: i. H or halo; or ii. -L.sup.1R.sup.10, wherein L.sup.1 is: —Z.sup.1—, -Q.sup.1-, —Z.sup.1Q.sup.1-, -Q.sup.1Z.sup.1—, —Z.sup.1Q.sup.1Z.sup.2—, -Q.sup.1Q.sup.2-, -Q.sup.1Q.sup.2Z.sup.1—, -Q.sup.1Q.sup.2Z.sup.1Q.sup.3Z.sup.2—, —Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3-; —OZ.sup.1—, -OQ.sup.1-, —OZ.sup.1Q.sup.1-, —OQ.sup.1Z.sup.1—, —OZ.sup.1Q.sup.1Z.sup.2—, —OQ.sup.1Q.sup.2-, —OQ.sup.1Q.sup.2Z.sup.1—, —OQ.sup.1Q.sup.2Z.sup.1Q.sup.3Z.sup.2—, —OZ.sup.1Q.sup.1OQ.sup.2OQ.sup.3-, —Z.sup.1N(R.sup.7)Z.sup.2—, -Q.sup.1N(R.sup.7)Z.sup.2—, —Z.sup.1N(R.sup.7)Z.sup.2Q.sup.1-, -Q.sup.1N(R.sup.7)Z.sup.2Q.sup.2Z.sup.3—; —Z.sup.1O(CH.sub.2CH.sub.2C).sub.nZ.sup.2—, —Z.sup.1O(CH.sub.2CH.sub.2O).sub.nQ.sup.1-, —Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2Q.sup.1, —Z.sup.1O(CH.sub.2CH.sub.2O).sub.nQ.sup.1Z.sup.2—, -Q.sup.1Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2—, -Q.sup.1Z.sup.1O(CH.sub.2CH.sub.2O).sub.nQ.sup.1-, -Q.sup.1Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2Q.sup.1, —Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2Q.sup.1Z.sup.3—; —C(O)Z.sup.1—, —C(O)Q.sup.1-, —C(O)Z.sup.1Q.sup.1-, —C(O)Z.sup.1Q.sup.1Z.sup.2—, —C(O)Q.sup.1Z.sup.1—, —C(O)Q.sup.1Q.sup.2-, —C(O)Q.sup.1Q.sup.2Z.sup.1—, —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1—, —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1Q.sup.2-, —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1Q.sup.2Q.sup.3-, —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1Q.sup.2Z.sup.2—, —C(O)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3-; —C(O)N(R.sup.7)Z.sup.1—, —C(O)N(R.sup.7)Q.sup.1-, —C(O)N(R.sup.7)Z.sup.1Q.sup.1-, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2—, —C(O)N(R.sup.7)Q.sup.1Z.sup.1—, —C(O)N(R.sup.7)Q.sup.1Q.sup.2-, —C(O)N(R.sup.7)Q.sup.1Q.sup.2Z.sup.1—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Q.sup.2Z.sup.2—, —C(O)N(R.sup.7)Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2—, —C(O)N(R.sup.7)Z.sup.1O(CH.sub.2O).sub.nZ.sup.2—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2N(R.sup.8)Z.sup.3—, —C(O)N(R.sup.7)Z.sup.1N(R.sup.8)Z.sup.2—, —C(O)N(R.sup.7)Q.sup.1Z.sup.1N(R.sup.8)Z.sup.2—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3-, —C(O)N(R.sup.7)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3Z.sup.2—; —Z.sup.1C(O)N(R.sup.7)Z.sup.2—, —Z.sup.1C(O)N(R.sup.7)Q.sup.1-, —Z.sup.1C(O)N(R.sup.7)Z.sup.2Q.sup.1-, —Z.sup.1C(O)N(R.sup.7)Q.sup.1Z.sup.2—, —Z.sup.1C(O)N(R.sup.7)Q.sup.1Q.sup.2-, —Z.sup.1C(O)Q.sup.1-, —Z.sup.1C(O)Q.sup.1Z.sup.2—, —Z.sup.1C(O)Q.sup.1Q.sup.2-, —Z.sup.1C(O)N(R.sup.7)Q.sup.1Q.sup.2Z.sup.2—; —C(O)OZ.sup.1—, —C(O)OQ.sup.1-, —C(O)OZ.sup.1Q.sup.1-, —C(O)OZ.sup.1Q.sup.1Z.sup.2—, —C(O)OQ.sup.1Z.sup.1—, —C(O)OQ.sup.1Q.sup.2-, —C(O)OQ.sup.1Q.sup.2Z.sup.1—; -Q.sup.1C(O)Q.sup.2-, Q.sup.1C(O)Z.sup.1—, -Q.sup.1C(O)Q.sup.2Z.sup.1—, Q.sup.1C(O)Q.sup.2Q.sup.3-, Q.sup.1C(O)Z.sup.1Q.sup.2-, or Q.sup.1C(O)Q.sup.2Q.sup.3Z.sup.1—; —C(═NR.sup.9)N(R.sup.7)Z.sup.1—, —C(═NR.sup.9)N(R.sup.7)Q.sup.1-, —C(═NR.sup.9)N(R.sup.7)Z.sup.1Q.sup.1-, —C(═NR.sup.9)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2—, —C(═NR.sup.9)N(R.sup.7)Q.sup.1Z.sup.1—, —C(═NR.sup.9)N(R.sup.7)Q.sup.1Q.sup.2- or C(═NR.sup.9)N(R.sup.7)Q.sup.1Q.sup.2Z.sup.1—; wherein each of Z.sup.1, Z.sup.2 and Z.sup.3 is independently C.sub.1-12 alkylene, C.sub.2-12 alkenylene, C.sub.2-12 alkynylene any of which is optionally substituted by one or more substituents selected from halo, OH, C(O)NR.sup.15R.sup.16, C(O)OR.sup.15 and NR.sup.15R.sup.16; each R.sup.15 and R.sup.16 is independently H or C.sub.1-6 alkyl or R.sup.15 and R.sup.16 together with the nitrogen atom to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing one or more further heteroatoms selected from N, O and S; each of Q.sup.1, Q.sup.2 and Q.sup.3 is independently carbocyclyl, heterocyclyl, aryl or heteroaryl any of which is optionally substituted with one or more substituents selected from halo, OH, C14 alkyl, C.sub.1-4 haloalkyl, C(O)NR.sup.15R.sup.16, C(O)OR.sup.15 and NR.sup.15R.sup.16, and, for cycloalkyl and heterocyclyl groups, oxo, wherein R.sup.15 and R.sup.16 are as defined above; n is 1 to 6; each R.sup.7 and R.sup.8 is independently selected from H or C.sub.1-12 alkyl optionally substituted with one or more halo or OH groups, or when an R.sup.7 and an R.sup.8 or two R.sup.8 groups are attached to a nitrogen atom they may, together with the nitrogen atom combine to form a 5- or 6-membered heterocyclic ring optionally comprising one or more further heteroatoms selected from N, O and S; R.sup.9 is H or C.sub.1-6 alkyl; R.sup.10 is H, —N(R.sup.7)R.sup.8, —N(R.sup.7)C(═NR.sup.9)N(R.sup.8).sub.2, —N(R.sup.7)—C(O)OR.sup.8, OR.sup.7 or —C(O)OR.sup.7; or a cationic group selected from —N(R.sup.7)—C(O)—(C.sub.1-3 alkylene)-N.sup.++(R.sup.8).sub.3 and -N.sup.++(R.sup.8).sub.3, in which case, an additional anion X.sup.− will be required; and R.sup.7, R.sup.8 and R.sup.9 are as defined above; or iii. —R.sup.12, —OR.sup.12 —SO.sub.2R.sup.12, —C(O)OR.sup.12, —C(O)NR.sup.12R.sup.13, —C(═NR.sup.9)NR.sup.12R.sup.13, -Q.sup.1R.sup.12—, -Q.sup.1OR.sup.12-Q.sup.1SO.sub.2R.sup.12, -Q.sup.1C(O)OR.sup.12, -Q.sup.1C(O)NR.sup.12R.sup.13, -Q.sup.1C(═NR.sup.7)NR.sup.12R.sup.13, -Q.sup.1Q.sup.2OR.sup.12, -Q.sup.1SO.sub.2R.sup.12, -Q.sup.1Q.sup.2C(O)OR.sup.12, -Q.sup.1Q.sup.2C(O)NR.sup.12R.sup.13 or -Q.sup.1Q.sup.2C(═NR.sup.9)NR.sup.12R.sup.13; wherein Q.sup.1 and Q.sup.2 are defined as above; and each R.sup.12 and R.sup.13 is independently H, C.sub.1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl or C3-8 heterocyclyl, any of which is optionally substituted by one or more substituents selected from halo, OR.sup.7, C(O)OR.sup.7, —N(R.sup.7)R.sup.8 and C(O)N(R.sup.7)R.sup.8 and, in the case of cycloalkyl or heterocyclyl groups, oxo; wherein R.sup.7, R.sup.8 and R.sup.9 are as defined above; each of R.sup.2 and R.sup.3 is independently C.sub.1-10 alkyl, wherein one or more —CH.sub.2— groups is optionally replaced by —O—, —S— or —NR.sup.7— provided that adjacent —CH.sub.2— groups are not so replaced and which is optionally substituted with one or more substituents selected from halo, OH, SH, N(R.sup.7)R.sup.8, aryl, heteroaryl, cycloalkyl, heterocyclyl, —C(O)OR.sup.7, —C(O)N(R.sup.7)R.sup.8, OR.sup.7 and N(R.sup.7)R.sup.8, wherein R.sup.7 and R.sup.8 are as defined above; R.sup.4 is H, halo, cyano, C.sub.1-6 alkyl, C(O)OR.sup.16 or C(O)N(R.sup.16)R.sup.17; wherein alkyl groups are optionally substituted with one or more substituents selected from halo, —OW and N(R.sup.7)R.sup.8, wherein R.sup.7 and R.sup.8 are as defined above; each R.sup.16 and R.sup.17 is independently H or C.sub.1-6 alkyl or R.sup.16 and R.sup.17 together with the nitrogen atom to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing one or more further heteroatoms selected from O, N and S.

2. The compound according to claim 1, wherein the compound of general formula (IA): ##STR00317## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and X.sup.− are as defined for general formula (I); or the compound is of general formula (IB): ##STR00318## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and X.sup.− are as defined for general formula (I).

3. The compound according to claim 1, wherein R.sup.1 is H, halo, —R.sup.12, —C(O)OR.sup.12, or —OR.sup.12.

4. The compound according to claim 3, wherein R.sup.1 is H, chloro, C(O)OH, methyl, trifluoromethyl, methoxy or trifluoromethoxy.

5. The compound according to claim 1, wherein R.sup.1 is -L.sup.1R.sup.10.

6. The compound according to claim 5, wherein L.sup.1 is: —Z.sup.1—, Q.sup.1-, Z.sup.1Q.sup.1-, -Q.sup.1Z.sup.1—, —Z.sup.1Q.sup.1Z.sup.2—, -Q.sup.1Q.sup.2-, -Q.sup.1Q.sup.2Z.sup.1—, or -Q.sup.1Q.sup.2Z.sup.1Q.sup.3Z.sup.2—; —OZ.sup.1Q.sup.1-, or —OZ.sup.1Q.sup.1Z.sup.2—; —Z.sup.1N(R.sup.7)Z.sup.2—, or -Q.sup.1Z.sup.1N(R.sup.7)Z.sup.2—; —C(O)Q.sup.1-, —C(O)Q.sup.1Z.sup.1—, —C(O)Q.sup.1Q.sup.2-, —C(O)Q.sup.1Q.sup.2Z.sup.1—, —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1—, or —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1Q.sup.2-; —C(O)N(R.sup.7)Z.sup.1—, —C(O)N(R.sup.7)Q.sup.1-, —C(O)N(R.sup.7)Z.sup.1Q.sup.1-, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2—, —C(O)N(R.sup.7)Q.sup.1Z.sup.1—, —C(O)N(R.sup.7)Q.sup.1Q.sup.2-, —C(O)N(R.sup.7)Q.sup.1Q.sup.2Z.sup.1—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Q.sup.2Z.sup.2—, —C(O)N(R.sup.7)Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2—, —C(O)N(R.sup.7)Z.sup.1O(CH.sub.2O).sub.nZ.sup.2—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2N(R.sup.8)Z.sup.3—, —C(O)N(R.sup.7)Z.sup.1N(R.sup.8)Z.sup.2—, —C(O)N(R.sup.7)Q.sup.1Z.sup.1N(R.sup.8)Z.sup.2—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3-, or —C(O)N(R.sup.7)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3Z.sup.2—; —C(O)OZ.sup.1—, —C(O)OZ.sup.1Q.sup.1-, or C(O)OZ.sup.1Q.sup.1Z.sup.2—; or -Q.sup.1C(O)O.sup.2—, Q.sup.1C(O)Z.sup.1—, -Q.sup.1C(O)Q.sup.2Z.sup.1—, Q.sup.1C(O)Q.sup.2Q.sup.3-, Q.sup.1C(O)Z.sup.1Q.sup.2- or Q.sup.1C(O)Q.sup.2Q.sup.3Z.sup.1—.

7. The compound according to claim 6, wherein L.sup.1 is: —Z.sup.1—, -Q.sup.1, -Q.sup.1Z.sup.1—, -Q.sup.1Q.sup.2-, or -Q.sup.1Q.sup.2Z.sup.1—; —OZ.sup.1—; —C(O)O.sup.1—, or —C(O)Q.sup.1Z.sup.1—; or —C(O)N(R.sup.7)Z.sup.1—, C(O)N(R.sup.7)Q.sup.1-, —C(O)N(R.sup.7)Z.sup.1Q.sup.1-, —C(O)N(R.sup.7)Q.sup.1Z.sup.1—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Q.sup.2Z.sup.2—, —C(O)N(R.sup.7)Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2— or —C(O)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2N(R.sup.8)Z.sup.3—.

8. The compound according to claim 5, wherein R.sup.10 is H, —N(R.sup.7)R.sup.8, —N(R.sup.7)C(═NR.sup.9)N(R.sup.8).sub.2, —N(R.sup.7)C(O)OR.sup.8, —N(R.sup.7)—C(O)—(C.sub.1-3 alkylene)-N.sup.+(R.sup.8).sub.3, —N.sup.+(R.sup.8).sub.3, OR.sup.7, or —C(O)OR.sup.7.

9. The compound according to claim 8, wherein R.sup.10 is H, —N(R.sup.7)R.sup.8, —N(R.sup.7)C(═NR.sup.9)N(R.sup.8).sub.2, —N(R.sup.7)C(O)OR.sup.8— or —C(O)OR.sup.7.

10. The compound according to claim 9, wherein R.sup.10 is H and: a) L.sup.1 is —OZ.sup.1—, wherein Z.sup.1 is C.sub.1-4 alkylene; or L.sup.1 is -Q.sup.1-, -Q.sup.1Q.sup.2- or —C(O)N(R.sup.7)Q.sup.1-, where the Q.sup.1 group or, for -Q.sup.1Q.sup.2-, the Q.sup.2 group, is a nitrogen-containing heterocyclyl group which is linked to the R.sup.10 group via a ring nitrogen atom; or c) L.sup.1 is: —Z.sup.1—, Q.sup.1, -Q.sup.1Q.sup.2, or -Q.sup.1Q.sup.2Z.sup.1—, —OZ.sup.1—, —OQ.sup.1Z.sup.1—, or —OQ.sup.1Q.sup.2Z.sup.1—; —C(O)Z.sup.1—, —C(O)Q.sup.1Z.sup.1—, or —C(O)Q.sup.1Q.sup.2Z.sup.1—; —C(O)N(R.sup.7)Z.sup.1—, —C(O)N(R.sup.7)Q.sup.1Z.sup.1—, or —C(O)N(R.sup.7)Q.sup.1Q.sup.2Z.sup.1—; —C(O)OZ.sup.1—, —C(O)OQ.sup.1Z.sup.1—, —C(O)OQ.sup.1Q.sup.2-, or C(O)OQ.sup.1Q.sup.2Z.sup.1—; or —C(═NR.sup.9)N(R.sup.7)Z.sup.1—, —C(═NR.sup.9)N(R.sup.7)Q.sup.1Z.sup.1— or C(═NR.sup.9)N(R.sup.7)Q.sup.1Q.sup.2Z.sup.1—; or d) L.sup.1 is: -Q.sup.1Z.sup.1—, —Z.sup.1Q.sup.1Z.sup.2—, or -Q.sup.1Q.sup.2Z.sup.1—; —OQ.sup.1Z.sup.1—, —OZ.sup.1Q.sup.1Z.sup.2—, or —OQ.sup.1Q.sup.2Z.sup.1—; —Z.sup.1O(CH.sub.2CH.sub.2O).sub.nQ.sup.1Z.sup.2—, -Q.sup.1Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2—, or —Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2Q.sup.1Z.sup.3—; —C(O)Z.sup.1Q.sup.1Z.sup.2—, —C(O)Q.sup.1Z.sup.1—, —C(O)Q.sup.1Q.sup.2Z.sup.1—, —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1Q.sup.2Z.sup.2—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2—, or —C(O)N(R.sup.7)Q.sup.1Z.sup.1—; —C(O)N(R.sup.7)Q.sup.1Q.sup.2Z.sup.1—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Q.sup.2Z.sup.2—, or —C(O)N(R.sup.7)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3Z.sup.2—; Z.sup.1C(O)N(R.sup.7)Q.sup.1Z.sup.2—, —Z.sup.1C(O)Q.sup.1Z.sup.2—, or Z.sup.1C(O)N(R.sup.7).sup.7Q.sup.1Q.sup.2Z.sup.2—; —C(O)OZ.sup.1Q.sup.1Z.sup.2—, —C(O)OQ.sup.1Z.sup.1—, or —C(O)OQ.sup.1Q.sup.2Z.sup.1—; Q.sup.1C(O)Q.sup.2Z.sup.1—, or Q.sup.1C(O)Q.sup.2Q.sup.3Z.sup.1—; or —C(═NR.sup.9)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2—, —C(═NR.sup.9)N(R.sup.7)Q.sup.1Z.sup.1— or —C(═NR.sup.9)N(R.sup.7)Q.sup.1Q.sup.2Z.sup.1—; and the cyclic group Q.sup.1, Q.sup.2 or Q.sup.3 is a nitrogen containing heterocyclyl group linked to Z.sup.1 or Z.sup.2 or Z.sup.3 via a ring nitrogen atom; or e) L.sup.1 contains a moiety Z.sup.1, Z.sup.2 or Z.sup.3 which is linked directly to R.sup.10 and is a C.sub.1-12 alkylene group substituted by 2 to 11 OH groups.

11. The compound according to claim 9, wherein R.sup.10 is H and L.sup.1 contains a moiety Z.sup.1, Z.sup.2, or Z.sup.3; wherein Z.sup.1, Z.sup.2, or Z.sup.3 is linked directly to R.sup.10 and is —CH.sub.2[CH(OH)].sub.4—CH(OH)— such that the group Z.sup.1R.sup.10, Z.sup.2R.sup.10, or Z.sup.3R.sup.10 is a moiety —CH.sub.2[CH(OH)].sub.4—CH.sub.2OH.

12. The compound according to claim 8, wherein: a) R.sup.10 is —C(O)OR.sup.7; and L.sup.1 is: -Q.sup.1- or -Q.sup.1Q.sup.2- where Q.sup.1 or, for -Q.sup.1Q.sup.2 is a carbocyclyl or heterocyclyl group and is linked to R.sup.10 via a ring carbon atom; or C(O)N(R.sup.7)Q.sup.1, wherein Q.sup.1 is a is a carbocyclyl or heterocyclyl group and is linked to R.sup.10 via a ring nitrogen atom; or b) R.sup.10 is —N(R.sup.7)R.sup.8, —N(R.sup.7)C(═NR.sup.9)N(R.sup.8).sub.2 or —N(R.sup.7)C(O)OR.sup.8—; and L.sup.1 is: —Z.sup.1—, —OZ.sup.1—; —C(O)N(R.sup.7)Z.sup.1—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Q.sup.2Z.sup.2—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2N(R.sup.8)Z.sup.3, or —C(O)N(R.sup.7)Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2; or —C(O)N(R.sup.7)Q.sup.1-, —C(O)N(R.sup.7)Z.sup.1Q.sup.1- or —C(O)Q.sup.1-, wherein Q.sup.1 is a carbocyclyl or heterocyclyl group and is linked to R.sup.10 via a ring carbon atom; or C(O)Q.sup.1Z.sup.1—.

13. The compound according to claim 12, wherein R.sup.10 is —N(R.sup.7)R.sup.8; and each of R.sup.7 and R.sup.8 is independently either H or C.sub.1-8 alkyl optionally substituted with one or more OH groups.

14. The compound according to claim 13, wherein R.sup.7 and/or R.sup.8 is a moiety —CH.sub.2[CH(OH)].sub.4—CH.sub.2OH.

15. The compound according to claim 13, wherein R.sup.10 is —N{CH.sub.2[CH(OH)].sub.4—CH.sub.2OH}.sub.2.

16. The compound according to claim 12, wherein R.sup.10 is —N(R.sup.7)C(═NR.sup.9)N(R.sup.8).sub.2; and each of R.sup.7 and R.sup.9 is H or C.sub.1-4 alkyl; and one or both R.sup.8 groups are —CH.sub.2[CH(OH)].sub.4—CH.sub.2OH.

17. The compound according to claim 16, wherein each of R.sup.2 and R.sup.3 is independently C.sub.1-10 alkyl in which one or more —CH.sub.2— groups is optionally replaced by —O— or —S— and which is optionally substituted.

18. The compound according to claim 17, wherein R.sup.2 and R.sup.3 are the same or different and are both unsubstituted C.sub.1-4 alkyl.

19. The compound according to claim 1, wherein R.sup.4 is H or methyl.

20. The compound according to claim 1, having a cation selected from: 2-[({6-amino-3-methyl-1H-pyrazolo[3,4-b]pyrazin-5-yl}formamido)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium; 2-[({6-amino-3-methyl-1H-pyrazolo[3,4-b]pyrazin-5-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium; 2-[({6-amino-1H-pyrazolo[3,4-b]pyrazin-5-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium; or 2-[({6-amino-1H-pyrazolo[3,4-b]pyrazin-5-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetic acid; and the anion X.sup.−.

21. A process for the preparation of a compound of general formula (I) according to claim 1, comprising: A. reacting a compound of general formula (II) or a salt or activated derivative thereof: ##STR00319## with a salt of general formula (III): ##STR00320## wherein X.sup.− is the same or different from the X.sup.− of the product of general formula (I); or B. reacting a compound of general formula (XVIII): ##STR00321## with a compound of general formula (IX):
R.sup.2—X.sup.1  (IX) wherein R.sup.2 is as defined for general formula (I) and X.sup.1 is a leaving group such as halo; or with a compound of general formula (IXa):
R.sup.2a—X.sup.1  (IXa) wherein X.sup.1 is as defined above for general formula (IX) and R.sup.2a is a protected R.sup.2 group; or C. converting a compound of general formula (I) in which R.sup.3 comprises a —C(O)OR.sup.7 group wherein R.sup.7 is other than H or a —C(O)N(R.sup.7)R.sup.8 group; to a compound of general formula (I) in which R.sup.3 comprises a —C(O)OH or C(O)O.sup.− group; by hydrolysis; or D. converting a compound of general formula (I) wherein R.sup.1 is L.sup.1R.sup.10 where R.sup.10 is —N(R.sup.7)—C(O)OR.sup.8; to a compound of general formula (I) wherein R.sup.10 is —NHR.sup.7; by hydrolysis; or E. converting a compound of general formula (I) wherein R.sup.1 is L.sup.1R.sup.10, and L.sup.1 comprises a moiety Q.sup.1, Q.sup.2 or Q.sup.3 which is linked to R.sup.10 via a ring nitrogen atom; and R.sup.10 is C(O)OR.sup.7; to a compound of general formula (I) wherein R.sup.1 is L.sup.1R.sup.10 and R.sup.10 is H; by hydrolysis; or F. converting a compound of general formula (I) wherein R.sup.1 is L.sup.1R.sup.10 and R.sup.10 is —NH.sub.2; to a compound of general formula (I) wherein R.sup.1 is L.sup.1R.sup.10 and R.sup.10 is N(R.sup.7)R.sup.8 where R.sup.7 is CH.sub.2—R.sup.7a and R.sup.8 is CH.sub.2—R.sup.8a and one of R.sup.7a and R.sup.8a is C.sub.1-11 alkyl optionally substituted with one or more halo or OH groups; and the other of R.sup.7a and R.sup.8a is H or C.sub.1-11 alkyl optionally substituted with one or more halo or OH groups; by reductive amination with an aldehyde, acetal or cyclic hemiacetal equivalent compound; or G. converting a compound of general formula (I) wherein R.sup.1 is L.sup.1R.sup.10; and L.sup.1 comprises a moiety Q.sup.1, Q.sup.2 or Q.sup.3 linked to R.sup.10 via a ring nitrogen atom; and R.sup.10 is H; to a compound of general formula (I) wherein R.sup.1 is L.sup.1R.sup.10; and L.sup.1 comprises a moiety Q.sup.1, Q.sup.2 or Q.sup.3 linked to a Z.sup.1, Z.sup.2 or Z.sup.3 moiety via a ring nitrogen atom, wherein Z.sup.1, Z.sup.2 or Z.sup.3 is CH.sub.2—C.sub.1-11 alkyl optionally substituted with one or more halo or OH groups; and R.sup.10 is H; by reductive amination with an aldehyde, acetal or cyclic hemiacetal equivalent compound; or H. converting a compound of general formula (I) in which R.sup.1 is L.sup.1R.sup.10 and R.sup.10 is NH.sub.2; to a compound of general formula (I) in which R.sup.1 is L.sup.1R.sup.10 and R.sup.10 is —NHC(═NR.sup.9)N(R.sup.8).sub.2; by reaction with a carboximidamide or a salt thereof; or I. converting a compound of general formula (I) in which R.sup.1 is C(O)OH to: a compound of general formula (I) in which R.sup.1 is —C(O)NR.sup.12R.sup.13; or a compound of general formula (I) in which R.sup.1 is L.sup.1R.sup.10 and L.sup.1 is —C(O)N(R.sup.7)Z.sup.1—, —C(O)N(R.sup.7)Q.sup.1-, —C(O)N(R.sup.7)Z.sup.1Q.sup.1-, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2—, —C(O)N(R.sup.7)Q.sup.1Z.sup.1—, —C(O)N(R.sup.7)Q.sup.1Q.sup.2-, —C(O)N(R.sup.7)Q.sup.1Q.sup.2Z.sup.1—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Q.sup.2Z.sup.2—, —C(O)N(R.sup.7)Z.sup.1O(CH.sub.2CH.sub.2O).sub.nZ.sup.2— —C(O)N(R.sup.7)Z.sup.1O(CH.sub.2O).sub.nZ.sup.2—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1Z.sup.2N(R.sup.8)Z.sup.3—, —C(O)N(R.sup.7)Z.sup.1N(R.sup.8)Z.sup.2—, —C(O)N(R.sup.7)Q.sup.1Z.sup.1N(R.sup.8)Z.sup.2—, —C(O)N(R.sup.7)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3-, or —C(O)N(R.sup.7)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3Z.sup.2—; or L.sup.1 is —C(O)Q.sup.1-, —C(O)Q.sup.1Z.sup.1—, —C(O)Q.sup.1Q.sup.2-, —C(O)Q.sup.1Q.sup.2Z.sup.1—, —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1—, —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1Q.sup.2-, —C(O)Q.sup.1N(R.sup.7)C(O)Z.sup.1Q.sup.2Q.sup.3- or —C(O)Q.sup.1NR(R.sup.7)C(O)Z.sup.1Q.sup.2Z.sup.2—, wherein Q.sup.1 is a heterocyclyl ring linked to the —C(O) moiety via a ring nitrogen atom; by reaction with an appropriate amine or ammonium salt.

22-24. (canceled)

25. A method for the treatment or prophylaxis of respiratory diseases and conditions, skin conditions or ocular conditions, the method comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 1.

26. The method according to claim 25, wherein: the respiratory diseases and conditions are selected from cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis, including non-cystic fibrosis bronchiectasis, asthma and primary ciliary dyskinesia; the skin conditions are selected from psoriasis, atopic dermatitis and ichthyosis; and the ocular condition is dry eye disease.

27. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable excipient.

28. The pharmaceutical composition according to claim 27, wherein the pharmaceutical composition is formulated for nasal, bronchial (inhaled) or buccal administration as a dry powder for inhalation, an aerosol or a spray.

29. The pharmaceutical composition according to claim 27, further including an additional active agent selected from: β2 adrenoreceptor agonists such as metaproterenol, isoproterenol, isoprenaline, albuterol, salbutamol, formoterol, salmeterol, indacaterol, terbutaline, orciprenaline, bitolterol mesylate and pirbuterol; antihistamines, for example histamine H.sub.1 receptor antagonists such as loratadine, cetirizine, desloratadine, levocetirizine, fexofenadine, astemizole, azelastine and chlorpheniramine or H.sub.4 receptor antagonists; dornase alpha; corticosteroids such as prednisone, prednisolone, flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate mometasone furoate and fluticasone furoate; Leukotriene antagonists such as montelukast and zafirlukast; CFTR repair therapies e.g. CFTR potentiators such as Ivacaftor and CFTR correctors such as Lumacaftor and Tezacaftor; TMEM16A modulators, particularly TMEM16A potentiators; and Antibiotics.

30. A product comprising a compound according to claim 1 and an additional agent useful in the treatment or prevention of respiratory conditions as a combined preparation for simultaneous, sequential or separate use in the treatment of a respiratory disease or condition, wherein the additional active agent is selected from: β2 adrenoreceptor agonists such as metaproterenol, isoproterenol, isoprenaline, albuterol, salbutamol, formoterol, salmeterol, indacaterol, terbutaline, orciprenaline, bitolterol mesylate and pirbuterol; antihistamines, for example histamine H.sub.1 receptor antagonists such as loratadine, cetirizine, desloratadine, levocetirizine, fexofenadine, astemizole, azelastine and chlorpheniramine or H.sub.4 receptor antagonists; dornase alpha; corticosteroids such as prednisone, prednisolone, flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate mometasone furoate and fluticasone furoate; Leukotriene antagonists such as montelukast and zafirlukast; and Antibiotics.

31. (canceled)

Description

EXAMPLES

[0420] All reactions involving moisture-sensitive reagents were carried out under a nitrogen atmosphere using standard vacuum line techniques and oven-dried glassware. Commercial anhydrous solvents were used in reactions and HPLC grade solvents were employed for work-up and chromatography. Water was purified using an Elix UV-5 system. All other reagents were used as supplied without prior purification. Reported yields are corrected for LC/MS purity (determined by UV (215 nm) or ELS detection) unless otherwise stated. Sealed tube reactions were carried out in heavy wall Ace pressure tubes. Microwave experiments were carried out using a Biotage Initiator+. Flash column chromatography was carried out using a Biotage Isolera 4 using Biotage SNAP columns. NMR spectra were recorded on a Bruker Avance III HD 500 MHz or a Bruker Avance III HD 250 MHz using the solvent as internal deuterium lock. Spectra were recorded at room temperature unless otherwise stated.

[0421] Analytical LC/MS were carried out on the following systems:

[0422] System A: stationary phase: Kinetex Core-Shell C18 2.1×50 mm, 5 μm, 40° C.; detection UV 215 nm-ELS-MS; mobile phase: A, water+0.1% formic acid; B, MeCN.sup.+0.1% formic acid; gradient (A:B ratio, time): 95:5-0:100, 1.20 min; 100:0, 0.10 min; 100:0-5:95, 0.01 min; flowrate: 1.2 ml/min;

[0423] System B: stationary phase: Phenomenex Gemini-NX C18 2.0×100 mm, 3 μm, 40° C.; detection UV 215 nm-ELS-MS; mobile phase: A, 2 mM ammonium bicarbonate pH 10; B, MeCN; gradient (A:B ratio, time): 95:5-0:100, 5.5 min; 0:100, 0.4 min; 0:100-95:5, 0.02 min; flowrate: 0.6 ml/min;

[0424] System C: stationary phase: Phenomenex Kinetex-XB C18 2.1×100 mm, 1.7 μm, 40° C.; detection UV 215 nm-ELS-MS; mobile phase: A, water+0.1% formic acid; B, MeCN.sup.+0.1% formic acid; gradient (A:B ratio, time): 95:5-0:100, 5.30 min; 100:0, 0.50 min; 100:0-5:95, 0.02 min; 5:95, 1.18 min; flowrate: 0.6 ml/min;

[0425] System D: stationary phase: Waters CSH C18 2.1×100 mm, 1.7 μm, 40° C.; detection UV 215 nm-ELS-MS; mobile phase: A, 5 mM ammonium acetate pH 7; B, MeCN; gradient (A:B ratio, time): 95:5-0:100, 5.30 min; 100:0, 0.50 min; 100:0-5:95, 0.02 min; 5:95, 1.18 min; flowrate: 0.6 ml/min;

[0426] System E: stationary phase: Waters CSH C18 2.1×100 mm, 1.7 μm, 40° C.; detection UV 215 nm-ELS-MS; mobile phase: A, water+0.1% formic acid; B, MeCN.sup.+0.1% formic acid; gradient; gradient (A:B ratio, time): 95:5-0:100, 1.10 min; 100:0, 0.25 min; 100:0-5:95, 0.05 min; 5:95, 0.1 min; flowrate: 0.9 ml/min;

[0427] System F: stationary phase: Phenomenex Gemini-NX C18 2×50 mm 3 μm, 40° C.; detection UV 215 nm-ELS-MS; mobile phase: A, 2 mM ammonium bicarbonate pH 10; B, MeCN; gradient (A:B ratio, time): 99:1-0:100, 1.80 min; 100:0, 0.20 min; 0:100-99:1, 0.01 min; flowrate 1 mL/min

[0428] The following abbreviations and terms have the indicated meanings throughout:

AcOH glacial acetic acid
CDI 1,1′-carbonyldiimidazole
CV column volumes
dd doublet of doublets

DIPEA N,N-diisopropylethylamine

[0429] DMAP 4-dimethylaminopyridine

DMF N,N-dimethylformamide

[0430] DMSO dimethyl sulfoxide
ELS evaporative light scattering
ESI electrospray ionisation
EtOH ethanol
EtOAc ethyl acetate
HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide
hexafluorophosphate
HPLC high-performance liquid chromatography
LC/MS liquid chromatographymass spectrometry
m multiplet
mCPBA meta-chloroperoxybenzoic acid
MeCN acetonitrile
MeOH methanol
NMR nuclear magnetic resonance
q quartet
RT room temperature
R.sub.t retention time
s singlet
t triplet
tBME methyl tert-butyl ether
TFA trifluoroacetic acid
THF tetrahydrofuran

A. Synthesis of Intermediates

Intermediate 1 Synthesis of 2-amino-5-bromo-3-(methoxycarbonyl)pyrazin-1-ium-1-olate

[0431] ##STR00047##

[0432] mCPBA (70%, 15.9 g, 64.7 mmol) was added to a mixture of methyl 3-amino-6-bromopyrazine-2-carboxylate (10.0 g, 43.1 mmol) in CHCl.sub.3 (100 ml). The reaction mixture was heated at 60° C. for 2 h then allowed to cool to RT. The resultant mixture was cooled (0° C.) then filtered. The collected solid was washed with cold (0° C.) CHCl.sub.3 (50 ml) then recrystallized from EtOH and dried in vacuo to afford the product as an orange solid (2.19 g, 20%).

[0433] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.85 (s, 1H), 7.79 (s, 2H), 3.89 (s, 3H).

[0434] LC/MS (System A): m/z (ESI.sup.+)=248 [M(.sup.79Br)H.sup.+], 250 [M(.sup.81Br)H.sup.+], R.sub.t=0.64 min, UV purity=99%.

Intermediate 2 Synthesis of methyl 3-amino-6-bromo-5-chloropyrazine-2-carboxylate

[0435] ##STR00048##

[0436] Phosphorus oxychloride (1.61 ml, 17.3 mmol) was added dropwise over 5 min to a cooled (0° C.) mixture of 2-amino-5-bromo-3-(methoxycarbonyl)pyrazin-1-ium-1-olate, Intermediate 1 (2.15 g, 8.66 mmol) in DMF (20 ml). The resultant mixture was allowed to warm to RT then stirred at RT for 1 h. Additional phosphorus oxychloride (0.80 ml, 8.6 mmol) was added then the reaction was left to stir at RT for 20 min. The resultant mixture was added dropwise to water (150 ml) over 10 min. The resultant mixture was left to stand at RT for 16 h then cooled (0° C.) and filtered. The collected solid was washed with cool (5° C.) water then dried in vacuo to afford the product as a pale orange solid (1.99 g, 86%).

[0437] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.84 (s, 2H), 3.85 (s, 3H).

[0438] LC/MS (System A): m/z (ESI.sup.+)=266 [M(.sup.35Cl.sup.79Br)H.sub.1], 268 [M(.sup.35Cl.sup.81Br/.sup.37Cl.sup.79Br)H.sub.1], 270 [M(.sup.37Cl.sup.81Br)H.sub.1], R.sub.t=1.03 min, UV purity=100%.

Intermediate 3 Synthesis of methyl 3-{bis[(tert-butoxy)carbonyl]amino}-6-bromo-5-chloropyrazine-2-carboxylate

[0439] ##STR00049##

[0440] Di-tert-butyl dicarbonate (1.74 g, 7.99 mmol) was added portionwise over 3 min to a solution of methyl 3-amino-6-bromo-5-chloropyrazine-2-carboxylate, Intermediate 2 (968 mg, 3.63 mmol) and DMAP (22 mg, 0.18 mmol) in CH.sub.2Cl.sub.2 (10 ml). The reaction mixture was stirred at RT for 1 h. Additional di-tert-butyl dicarbonate (500 mg, 2.29 mmol) and DMAP (6 mg, 0.05 mmol) were added then the reaction was left to stir at RT for 45 min. The reaction mixture was concentrated in vacuo then the crude material was purified by flash column chromatography on a silica column (100 g). The column was eluted with EtOAc:heptane, increasing the gradient linearly from 0:100 to 34:76 over 8.5 CV. The desired fractions were combined then evaporated to afford the product as a pale yellow solid (1.55 g, 89%).

[0441] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 3.90 (s, 3H), 1.37 (s, 18H).

[0442] LC/MS (System F): m/z (ESI.sup.+)=466 [M(.sup.35Cl.sup.79Br)H.sub.1], 468 [M(.sup.35Cl.sup.81Br/.sup.37Cl.sup.79Br)H.sub.1], 470 [M(.sup.37Cl.sup.81Br)H.sub.1], R.sub.t=2.06 min, UV purity=98%.

Intermediate 4 Synthesis of methyl 6-acetyl-3-amino-5-chloropyrazine-2-carboxylate

[0443] ##STR00050##

Degassed MeCN (20 ml) was added to a flask containing methyl 3-{bis[(tert-butoxy)carbonyl]amino}-6-bromo-5-chloropyrazine-2-carboxylate, Intermediate 3 (98%, 1.55 g, 3.25 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (228 mg, 0.325 mmol) and CuI (124 mg, 0.649 mmol). Tributyl(1-ethoxyvinyl)stannane (2.19 ml, 6.49 mmol) was added then the resulting mixture was stirred at 40° C. for 50 min. Aqueous HCl solution (4.0 M, 20 ml, 80 mmol) was added then the reaction mixture was stirred at 60° C. for 1.5 h. The reaction mixture was allowed to cool to RT then added dropwise over 5 min to saturated aqueous NaHCO.sub.3 solution (120 ml). The resultant mixture was extracted with EtOAc (150 ml). The phases were separated then aqueous KF solution (1 M, 100 ml, 100 mmol) was added to the organic layer. The mixture was stirred vigorously for 0.5 h then filtered through a Celite® pad (filter material).

[0444] The pad was rinsed with EtOAc (30 ml) then the combined filtrates were transferred to a separating funnel. The phases were separated then the organic phase was washed with water (2×120 ml) and brine (120 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude material was purified by flash column chromatography on a silica column (100 g). The column was eluted with EtOAc:heptane, increasing the gradient linearly from 0:100 to 54:46 over 9 CV. The desired fractions were combined then evaporated to afford the product as a pale orange solid (265 mg, 36%).

[0445] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.62 (s, 1H), 7.96 (s, 1H), 3.89 (s, 3H), 2.51 (s, 3H+solvent).

[0446] LC/MS (System A): m/z (ESI.sup.+)=230 [M(.sup.35Cl)H.sub.1], 232 [M(.sup.37Cl)H.sub.1], R.sub.t=0.94 min, UV purity=100%.

Intermediate 5 Synthesis of methyl 6-amino-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxylate

[0447] ##STR00051##

[0448] Hydrazine hydrate (161 μL, 3.31 mmol) was added to a mixture of methyl 6-acetyl-3-amino-5-chloropyrazine-2-carboxylate, Intermediate 4 (380 mg, 1.65 mmol) in EtOH (20 ml). The resulting mixture was heated to 50° C. for 1 h. Additional hydrazine hydrate (50 μL, 1.0 mmol) was added then the reaction was left to stir at 50° C. for a further 16 h. The reaction was allowed to cool to RT then concentrated in vacuo. The residue was suspended in refluxing EtOAc (25 ml). The mixture was allowed to cool to RT then filtered. The collected solid was washed with EtOAc (5 ml) then dried in vacuo to afford the product as a grey solid (366 mg, 85%).

[0449] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 12.90 (s, 1H), 7.54 (s, 2H), 3.89 (s, 3H), 2.42 (s, 3H). NMR estimated purity=80%.

[0450] LC/MS (System A): m/z (ESI.sup.+)=208 [MH.sup.+], R.sub.t=0.75 min, UV purity=95%.

Intermediate 6 Synthesis of 1,5-bis(1H-imidazole-1-carbonyl)-3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-amine

[0451] ##STR00052##

[0452] Aqueous LiOH solution (1.0 M, 360 μl, 0.36 mmol) was added to a mixture of methyl 6-amino-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxylate, Intermediate 5 (80%, 56 mg, 0.22 mmol) in MeOH (1 ml). The resulting mixture was stirred at 50° C. for 6 h then stirred at RT for 16 h. Additional LiOH solution (1.0 M, 30 μl, 0.030 mmol) was added then the reaction was heated at 50° C. for 4 h. Additional LiOH solution (1.0 M, 50 μl, 0.050 mmol) was added then the reaction was heated at 50° C. for 2 h. The reaction mixture was concentrated in vacuo to afford an orange solid (56 mg). The solid thus obtained was suspended in DMF (1 ml) then CDI (91 mg, 0.56 mmol) and imidazole hydrochloride (35 mg, 0.34 mmol) were added. The resultant mixture was stirred at RT for 0.5 h. Additional CDI (65 mg, 0.40) was added then the reaction was stirred at RT for 45 min.

[0453] Additional imidazole hydrochloride (50 mg, 0.48 mmol) and CDI (70 mg, 0.43 mmol) were added then the reaction was left to stir at RT for 15 min. The reaction mixture was cooled (0° C.) then cold (5° C.) water (2 ml) was added. The resultant mixture was stirred at RT for 5 min then left to stand at RT for 10 min. The mixture was filtered then the collected solid was washed with water and dried in vacuo to afford the product as an orange solid (21 mg, 26%).

[0454] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 8.66-8.55 (m, 2H), 8.24 (s, 2H), 7.94-7.88 (m, 2H), 7.18-7.13 (m, 2H), 2.53 (s, 3H). Residual DMF (4.0 wt %) and imidazole (4.4 wt %). Overall NMR purity=92%.

[0455] LC/MS (System A): R.sub.t=0.72 min, UV purity=100%.

Intermediate 7 Synthesis of 6-amino-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxylic acid; trifluoroacetic acid

[0456] ##STR00053##

[0457] Aqueous LiOH solution (1.0 M, 2.7 ml, 2.7 mmol) was added to a mixture of methyl 6-amino-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxylate, Intermediate 5 (80%, 310 mg, 1.20 mmol) in MeOH (2.5 ml). The resultant mixture was stirred at 50° C. for 1.5 h then concentrated in vacuo to afford a brown/orange solid (335 mg). A sample (165 mg) of the residue thus obtained was treated with CH.sub.2Cl.sub.2 (4 ml) and TFA (0.5 ml). The resultant mixture was stirred at RT for 20 min then concentrated in vacuo. The residue thus obtained was suspended in water (4 ml) with sonication. The solid was collected by filtration and washed with water. The solid was then treated with CH.sub.2Cl.sub.2 (4 ml) and TFA (0.5 ml). The resultant mixture was stirred at RT for 10 min then concentrated in vacuo to afford the product as a brown solid (149 mg, 37%).

[0458] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 12.96 (s, 1H), 7.57 (s, 2H), 2.42 (s, 3H).LC/MS (System A): m/z (ESI.sup.+)=194 [MH.sup.+], R.sub.t=0.60 min, UV purity (254 nm)=91%.

Intermediate 8—Synthesis of 5-(I H-imidazole-1-carbonyl)-3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-amine

[0459] ##STR00054##

[0460] CDI (95 mg, 0.58 mmol) was added to a solution of 6-amino-3-methyl-1H-pyrazolo[3,4-b]pyrazine-5-carboxylic acid; trifluoroacetic acid, Intermediate 7 (91%, 149 mg, 0.441 mmol) in DMF (2 ml). The resulting mixture was stirred at RT for 0.5 h. CH.sub.2Cl.sub.2 (4 ml) was added then the reaction was left to stand for 20 min. The solid was collected by filtration, rinsed with CH.sub.2Cl.sub.2, then dried in vacuo to afford the product as a yellow solid (74 mg, 63%).

[0461] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.05 (s, 1H), 8.68-8.64 (m, 1H), 7.94-7.90 (m, 1H), 7.68 (s, 2H), 7.14-7.10 (m, 1H), 2.44 (s, 3H). 5 wt % residual DMF.

[0462] Overall purity estimate=91% LC/MS (System A): m/z (ESI.sup.+)=244 [MH.sup.+], R.sub.t=0.72 min, UV purity=96%.

Intermediate 9—Synthesis of 1-[(4-methoxyphenyl)methyl]-4-nitroso-1H-pyrazol-5-amine hydrochloride

[0463] ##STR00055##

[0464] HCl solution in dioxane (4.0 M, 20 ml, 80 mmol) was added dropwise over 5 min to a cooled (0° C.) solution of 1-[(4-methoxyphenyl)methyl]-1H-pyrazol-5-amine (4.00 g, 19.7 mmol) in EtOH (40 ml). Amyl nitrite (2.64 ml, 19.7 mmol) was added dropwise over 1 min then the resultant solution was left to stir at 0° C. for 3 h. The resultant suspension was filtered, then the collected solid was rinsed with cold (0° C.) EtOH then dried in vacuo to afford the product as a pale peach solid (2.05 g, 38%).

[0465] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.20-7.47 (m, 3H), 7.32-7.16 (m, 2H), 6.96-6.84 (m, 2H), 5.18-5.01 (m, 2H), 3.75-3.70 (m, 3H).

[0466] LC/MS (System A): m/z (ESI.sup.+)=233 [MH.sup.+], R.sub.t=0.88 min, UV purity=99%.

Intermediate 10—Synthesis of 6-amino-1-[(4-methoxyphenyl)methyl]-1H-pyrazolo[3,4-b]pyrazine-5-carboxylic acid

[0467] ##STR00056##

[0468] Potassium tert-butoxide (3.93 g, 35.0 mmol) was added portionwise over 5 min to a cooled (0° C.) suspension of 1-[(4-methoxyphenyl)methyl]-4-nitroso-1H-pyrazol-5-amine hydrochloride, Intermediate 9 (0.950 g, 3.50 mmol) and 2-cyanoacetic acid (1.19 g, 14.0 mmol) in EtOH (19 ml). The reaction was allowed to warm to RT then heated under microwave irradiation at 140° C. for 1 h 30 min. The resultant suspension was filtered then the collected solid was rinsed with EtOH and dried in vacuo. The solid thus obtained was suspended in water (15 ml) then the resultant suspension was acidified to pH=3 by drop-wise addition of aqueous HCl solution (2 M). The resultant suspension was filtered then the collected solid was rinsed with water (3 ml) and dried in vacuo to afford the product as a brown solid (214 mg, 20%).

[0469] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.26 (s, 1H), 8.22 (s, 1H), 7.81 (s, 2H), 7.21-7.15 (m, 2H), 6.91-6.84 (m, 2H), 5.37 (s, 2H), 3.71 (s, 3H).

[0470] LC/MS (System A): m/z (ESI.sup.+)=300 [MH.sup.+], R.sub.t=0.96 min, UV purity=100%.

Intermediate 11—Synthesis of 6-amino-1H-pyrazolo[3,4-b]pyrazine-5-carboxylic acid; trifluoroacetic acid

[0471] ##STR00057##

[0472] A solution of 6-amino-1-[(4-methoxyphenyl)methyl]-1H-pyrazolo[3,4-b]pyrazine-5-carboxylic acid, Intermediate 10 (190 mg, 0.635 mmol) and anisole (1.0 ml) in TFA (4 ml) was heated under microwave irradiation for 15 min at 100° C. The reaction mixture was concentrated in vacuo then the residue was suspended in toluene (5 ml). The mixture was sonicated then the solid was collected by filtration, rinsed with toluene, then dried under suction to afford the product as a dark brown solid (131 mg, 70%).

[0473] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.70-12.47 (m, 2H), 8.19 (s, 1H), 7.64 (s, 2H).

[0474] LC/MS (System A): m/z (ESI.sup.+)=180 [MH.sup.+], R.sub.t=0.15 min, ELS purity=100%.

Intermediate 12—Synthesis of 5-(1H-imidazole-1-carbonyl)-1H-pyrazolo[3,4-b]pyrazin-6-amine

[0475] ##STR00058##

[0476] CDI (104 mg, 0.640 mmol) was added to a solution of 6-amino-1H-pyrazolo[3,4-b]pyrazine-5-carboxylic acid; trifluoroacetic acid, Intermediate 11 (125 mg, 0.426 mmol) in DMF (2 ml). The reaction mixture was left to stir at RT for 3 h. Additional CDI (69 mg, 0.43 mmol) was added then the reaction was stirred at RT for a further 1 h. The reaction mixture was concentrated to ˜1 ml under a stream of nitrogen then diluted with CH.sub.2Cl.sub.2 (2 ml). The resultant suspension was filtered then the collected solid was rinsed with CH.sub.2Cl.sub.2 (2×1 ml) and dried under suction to afford the product as a dark yellow/green solid (70 mg, 72%).

[0477] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.43 (s, 1H), 8.63-8.59 (m, 1H), 8.30 (s, 1H), 7.91-7.87 (m, 1H), 7.66 (s, 2H), 7.11 (dd, J=1.6, 0.8 Hz, 1H).

[0478] LC/MS (System E): m/z (ESI.sup.+)=230 [MH.sup.+], R.sub.t=0.32 min, UV purity=100%.

Intermediate 13—Synthesis of 2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione

[0479] ##STR00059##

[0480] Intermediate 13 was synthesised according to literature procedures (US 2015/0018314 A1).

Intermediate 14—Synthesis of 2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium iodide

[0481] ##STR00060##

[0482] Iodoethane (715 μl, 8.95 mmol) was added to a suspension of 2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 13 (3.00 g, 8.95 mmol) in MeCN (20 ml). The mixture was heated at 80° C. for 4 h. Iodoethane (715 μl, 8.95 mmol) was added and mixture was stirred at 80° C. for 16 h. Iodoethane (715 μl, 8.95 mmol) was added and mixture was stirred at 80° C. for a further 24 h the allowed to cool to RT. The mixture was concentrated in vacuo to approximately one third of the original volume. The precipitate was collected by filtration then washed with MeCN to afford a grey solid (2.6 g). The filtrate was concentrated to afford a dark grey solid. The two batches of solid thus obtained were combined and suspended in MeCN (20 ml). Iodoethane (715 μl, 8.95 mmol) was added then the reaction mixture was stirred at 80° C. for 18 h then at 100° C. for 4 h. The reaction mixture was split into two equal portions in pressure tubes. Iodoethane (300 μl, 3.75 mmol) was added to both reaction mixtures then the pressure tubes were sealed and left to heat at 100° C. for 16 h. The reaction mixtures were allowed to cool to RT then combined. The resultant mixture was concentrated in vacuo to ˜5 ml then filtered. The collected solid was washed with the minimum of MeCN (0.5 ml) to yield the product as a grey solid (2.37 g). The filtrate was concentrated under reduced pressure to afford a dark brown solid, which was triturated with EtOAc (˜10 ml) and filtered. The filtrate was left to stand for 16 h then it was filtered again. The solids obtained from the EtOAc filtrations were combined and dried to yield an additional batch of the product as a grey solid (1.24 g). The two batches of product obtained were combined as an EtOAc suspension then evaporated and dried under vacuum to afford the product as a grey solid (3.61 g, 81%).

[0483] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 8.02 (d, J=9.2 Hz, 1H), 7.98-7.86 (m, 4H), 7.63 (d, J=2.2 Hz, 1H), 7.31 (dd, J=9.1, 2.3 Hz, 1H), 5.40 (s, 2H), 4.74-4.60 (m, 4H), 3.92 (s, 3H), 1.49-1.33 (m, 6H).

[0484] LC/MS (System A): m/z (ESI.sup.+)=364 [M.sup.+], R.sub.t=0.93 min, UV purity=99%.

Intermediate 15—Synthesis of 2-(aminomethyl)-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium iodide

[0485] ##STR00061##

[0486] Hydrazine hydrate (1.18 ml, 24.1 mmol) was added to a suspension of 2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium iodide, Intermediate 14 (2.37 g, 4.82 mmol) in MeOH (25 ml). The mixture was then heated at 75° C. for 2.5 h then left to cool to RT over 16 h. The reaction mixture was concentrated in vacuo and the resulting solid was suspended in CH.sub.2Cl.sub.2:MeOH (10:1) then filtered. The collected solid was washed with CH.sub.2Cl.sub.2. The filtrate was concentrated in vacuo to afford the product as a grey solid (1.89 g, >99%).

[0487] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.95 (d, J=9.1 Hz, 1H), 7.58 (d, J=2.3 Hz, 1H), 7.25 (dd, J=9.1, 2.3 Hz, 1H), 4.65-4.50 (m, 4H), 4.25 (s, 2H), 3.91 (s, 3H), 1.43 (m, 1.50-1.39 6H).

[0488] LC/MS (System A): m/z (ESI.sup.+)=234 [M.sup.+], R.sub.t=0.16 min, ELS purity=92%.

Intermediate 16 Synthesis of methyl 2-({[tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate

[0489] ##STR00062##

[0490] A mixture of 2-{[(tert-butoxy)carbonyl]amino}acetic acid (8.57 g, 48.9 mmol), HATU (20.5 g, 53.8 mmol) and DIPEA (17.0 ml, 97.8 mmol) in DMF (200 ml) was stirred at RT for 1 h. Methyl 4-amino-3-(ethylamino)benzoate (9.59 g, 48.9 mmol) was added portionwise then rinsed into the reaction with THF (20 ml). The reaction mixture was stirred at RT for 18 h. A solution of 2-{[(tert-butoxy)carbonyl]amino}acetic acid (0.857 g, 4.89 mmol), HATU (1.86 g, 4.89 mmol) and DIPEA (1.70 ml, 9.78 mmol) in DMF (3 ml) was stirred at RT for 15 min then added to the main reaction. The resulting solution was stirred at RT for 3 h. A solution of 2-{[(tert-butoxy)carbonyl]amino}acetic acid (0.857 g, 4.89 mmol), HATU (1.86 g, 4.89 mmol) and DIPEA (1.70 ml, 9.78 mmol) in DMF (3 ml) was stirred at RT for 15 min then added to the main reaction. The resulting solution was stirred at RT for 64 h. The reaction mixture was added to saturated aqueous NaHCO.sub.3 solution (200 ml). EtOAc (150 ml) and water (100 ml) were added then the phases were separated. The aqueous phase was extracted with EtOAc (2×150 ml), then the combined organic phases were washed with water (4×100 ml) and brine (50 ml) then dried over Na.sub.2SO.sub.4, filtered and evaporated to afford the crude intermediate as a black oil (18 g). The oil thus obtained was dissolved in acetic acid (80 ml) and stirred at 70° C. for 1 h. The reaction was allowed to cool to RT then evaporated to afford a brown solid. The solid was suspended in EtOAc (200 ml) then filtered and was washed with EtOAc, then dried under vacuum to afford a pale pink solid (6.5 g). The solid thus obtained was suspended in EtOAc (200 ml). The resulting suspension was heated at 50° C. for 15 min then allowed to cool to RT. The solid was collected by filtration to afford the product as a white solid (2.43 g). The filtrate was again filtered and the solid was collected by filtration, washed with EtOAc:heptane then dried under vacuum to afford a second batch of the product as a white solid (1.34 g). The filtrate was transferred to a separating funnel then extracted with saturated aqueous NaHCO.sub.3 solution (3×100 ml), water (100 ml) and brine (50 ml) then dried over Na.sub.2SO.sub.4, filtered and evaporated to a yellow solid which was suspended in the minimum volume of EtOAc:heptane (1:4) and filtered then dried under vacuum to afford a third batch of the product as a white solid (1.77 g). The filtrate from the first filtration was transferred to a separating funnel then extracted with saturated aqueous NaHCO.sub.3 solution (3×100 ml), water (100 ml) and brine (50 ml) then dried over Na.sub.2SO.sub.4, filtered and evaporated to a dark brown solid. The solid was suspended in EtOAc (50 ml) then filtered. The solid was dried under vacuum to afford a fourth batch of the product as a white solid (3.4 g). The filtrate was evaporated to afford a dark solid (8 g). The solid thus obtained was dissolved in CH.sub.2Cl.sub.2 then evaporated onto silica (16 g). The crude material was purified by flash column chromatography on a silica column (100 g). The column was eluted with EtOAc:heptane, increasing the gradient linearly from 0:100 to 100:0 over 10 CV. The desired fractions were combined and evaporated to afford a brown solid. The solid thus obtained was suspended in EtOAc:heptane (1:4, 20 ml) then filtered. The solid was washed with EtOAc:heptane then dried under vacuum to afford a fifth batch of the product as a white solid (1.45 g). The filtrate was concentrated in vacuo then the residue was suspended in EtOAc, filtered and dried under vacuum to afford a sixth batch of the product as an off-white solid (0.32 g).

[0491] The 6 batches of solid were combined as an EtOAc suspension then evaporated and dried under vacuum to yield the product as an off-white solid (10.7 g, 66%).

[0492] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.16 (d, J=1.1 Hz, 1H), 7.82 (dd, J=8.4, 1.6 Hz, 1H), 7.66 (d, J=8.4 Hz, 1H), 7.52 (t, J=4.9 Hz, 1H), 4.47 (d, J=5.8 Hz, 2H), 4.35 (q, J=7.2 Hz, 2H), 3.88 (s, 3H), 1.46-1.22 (m, 12H).

[0493] LC/MS (System A): m/z (ESI.sup.+)=334 [MH.sup.+], R.sub.t=0.98 min, UV purity=100%.

Intermediate 17—2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylic acid

[0494] ##STR00063##

[0495] Aqueous LiOH solution (2.0 M, 16 ml, 32 mmol) was added to a suspension of methyl 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate, Intermediate 16 (6.91 g, 20.7 mmol) in THF (100 ml). The reaction mixture was stirred at 50° C. for 16 h then allowed to cool to RT. The reaction mixture was concentrated in vacuo then the resulting solid was suspended in water (50 ml). Aqueous HCl solution (2 M) was added dropwise until pH 4 was reached. The resultant suspension was filtered then the solid was washed with the minimum of water and MeCN then dried under vacuum to afford the product as a white solid (6.05 g, 90%).

[0496] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 12.77 (s, 1H), 8.18-8.07 (m, 1H), 7.80 (dd, J=8.4, 1.5 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.52 (t, J=5.4 Hz, 1H), 4.47 (d, J=5.8 Hz, 2H), 4.34 (q, J=7.2 Hz, 2H), 1.46-1.21 (m, 12H).

[0497] LC/MS (System A): m/z (ESI.sup.+)=320 [MH.sup.+], R.sub.t=0.84 min, UV purity=99%.

Intermediate 18—Synthesis of 2-(aminomethyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylic acid dihydrochloride

[0498] ##STR00064##

[0499] HCl solution in dioxane (4.0 M, 14 ml, 56 mmol) was added to a suspension of 2-({[tert-butoxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylic acid, Intermediate 17 (3.55 g, 11.1 mmol) in MeCN (60 ml). The reaction mixture was stirred at RT for 4 h then filtered. The solid was dried under vacuum to afford the product as a white solid (3.39 g, 98%).

[0500] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.78 (s, 3H), 8.25 (s, 1H), 7.88 (dd, J=8.5, 1.2 Hz, 1H), 7.74 (d, J=8.4 Hz, 1H), 4.54-4.47 (m, 2H), 4.38 (q, J=7.2 Hz, 2H), 1.34 (t, J=7.2 Hz, 3H).

[0501] LC/MS (System A): m/z (ESI.sup.+)=220 [MH.sup.+], R.sub.t=0.16 min, ELS purity=94%.

Intermediate 19—Synthesis of 2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylic acid

[0502] ##STR00065##

[0503] NaHCO.sub.3 (4.83 g, 57.5 mmol) was added portionwise to a cooled (0° C.) suspension of 2-(aminomethyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylic acid dihydrochloride, Intermediate 18 (4.20 g, 14.4 mmol) in water (40 ml). The reaction mixture was allowed to warm to RT then a solution of benzyl 2,5-dioxopyrrolidin-1-yl carbonate (3.94 g, 15.8 mmol) in THF (40 ml) was added dropwise over 15 min. The reaction mixture was left to stir at RT for 16 h. The resultant mixture was extracted with EtOAc (50 ml). The phases were separated then the organic phase was washed with water (3×10 ml). The combined aqueous phases were acidified to pH 5 by addition of aqueous HCl solution (2 M), resulting in precipitation of a solid. The resultant suspension was filtered then the solid was dried under vacuum to afford the product as a white solid (3.5 g, 69%).

[0504] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.12 (s, 1H), 7.97 (t, J=5.8 Hz, 1H), 7.80 (dd, J=8.4, 1.4 Hz, 1H), 7.62 (d, J=8.4 Hz, 1H), 7.42-7.09 (m, 5H), 5.06 (s, 2H), 4.54 (d, J=5.9 Hz, 2H), 4.33 (q, J=7.0 Hz, 2H), 1.28 (t, J=7.1 Hz, 3H).

[0505] LC/MS (System A): m/z (ESI.sup.+)=354 [MH.sup.+], R.sub.t=0.89 min, UV purity=100%.

Intermediate 20—tert-butyl 2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate

[0506] ##STR00066##

[0507] 1,1-Di-tert-butoxy-N,N-dimethylmethanamine (6.77 ml, 28.3 mmol) was added to a suspension of 2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylic acid, Intermediate 19 (2.50 g, 7.08 mmol) in α,α,α-trifluorotoluene (50 ml). The reaction mixture was heated at 100° C. for 1 h. The reaction mixture was allowed to cool to RT then 1,1-di-tert-butoxy-N,N-dimethylmethanamine (6.77 ml, 28.3 mmol) was added dropwise over 15 min. The resultant mixture was heated at 100° C. for 45 min. The reaction mixture was cooled to 50° C. then 1,1-di-tert-butoxy-N,N-dimethylmethanamine (3.38 ml, 14.2 mmol) was added dropwise over 5 min. The resultant mixture was heated at 100° C. for 0.5 h then allowed to cool to RT. The reaction mixture was partitioned between EtOAc (50 ml) and water (50 ml). The phases were separated then the organic phase was washed with water (2×30 ml), saturated aqueous NaHCO.sub.3 solution (20 ml) and brine (10 ml) then dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford a beige solid (2.5 g). The solid thus obtained was suspended in MeCN (10 ml). The solid was collected by filtration then dried under vacuum to afford the product as an off-white solid (2.30 g, 79%).

[0508] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.07 (s, 1H), 7.97 (m, 1H), 7.76 (dd, J=8.4, 1.5 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 7.34 (m, 5H), 5.07 (s, 2H), 4.55 (d, J=6.0 Hz, 2H), 4.38-4.25 (m, 2H), 1.57 (s, 9H), 1.29 (m, 3H).

[0509] LC/MS (System A): m/z (ESI.sup.+)=410 [MH.sup.+], R.sub.t=1.17 min, UV purity=99%.

Intermediate 21—Synthesis of 2-({[(benzyloxy)carbonyl]amino}methyl)-6-[(tert-butoxy)carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0510] ##STR00067##

[0511] Three reactions were run independently as follows then combined for work-up. Reaction 1: a suspension of tert-butyl 2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate, Intermediate 20 (800 mg, 1.95 mmol) and iodoethane (629 μl, 7.82 mmol) in MeCN (10 ml) was heated under microwave irradiation for 2 h at 120° C. The reaction was retreated with iodoethane (629 μl, 7.82 mmol) then the reaction mixture was heated under microwave irradiation for a further 2 h at 120° C. Reaction 2: a suspension of tert-butyl 2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate, Intermediate 20 (800 mg, 1.95 mmol) and iodoethane (629 μl, 7.82 mmol) in MeCN (10 ml) was heated under microwave irradiation for 1 h 45 min at 120° C. The reaction was retreated with iodoethane (629 μl, 7.82 mmol) then the reaction mixture was heated under microwave irradiation for a further 1.5 h at 120° C. Reaction 3: a suspension of tert-butyl 2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate, Intermediate 20 (700 mg, 1.71 mmol) and iodoethane (591 μl, 6.84 mmol) in MeCN (10 ml) was heated under microwave irradiation for 1.5 h at 120° C. The reaction was retreated with iodoethane (629 μl, 7.82 mmol) then the reaction mixture was heated under microwave irradiation for a further 1.5 h at 120° C. The three reactions were combined and concentrated in vacuo. The crude material was purified by flash column chromatography on C18 (60 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 5%, 2 CV; 5-31%, 5 CV; 31%, 4 CV; 31-59%, 6 CV; 59-100%, 3 CV; 100% 1 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a white foam (2.13 g, 67%).

[0512] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.54 (s, 1H), 8.43 (t, J=5.1 Hz, 1H), 8.22-8.12 (m, 2H), 7.42-7.27 (m, 5H), 5.06 (s, 2H), 4.90 (d, J=5.3 Hz, 2H), 4.79-4.59 (m, 4H), 1.61 (s, 9H), 1.47-1.36 (m, 6H).

[0513] LC/MS (System A): m/z (ESI.sup.+)=438 [M.sup.+], R.sub.t=1.07 min, UV purity=100%.

Intermediate 22—Synthesis of 2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium hydrobromide bromide

[0514] ##STR00068##

[0515] HBr solution in AcOH (33 wt %, 4.28 ml, 18.8 mmol) was added to a solution of 2-({[(benzyloxy)carbonyl]amino}methyl)-6-[(tert-butoxy)carbonyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide, Intermediate 21 (2.13 g, 3.77 mmol) in AcOH (10 ml). The reaction mixture was stirred at RT for 0.5 h. The resultant suspension was concentrated in vacuo then azeotroped with MeCN. The solid thus obtained was suspended in the minimum volume of MeCN then filtered and dried under vacuum to afford the product as a white solid (1.52 g, 99%).

[0516] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.69 (s, 1H), 8.31-8.20 (m, 2H), 4.85-4.63 (m, 6H), 1.53-1.40 (m, 6H).

[0517] LC/MS (System A): m/z (ESI.sup.+)=248 [M.sup.+], R.sub.t=0.15 min, ELS purity=100%. Intermediate 23 Synthesis of (9H-fluoren-9-yl)methyl 4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}piperidine-1-carboxylate; formic acid

##STR00069##

[0518] A mixture of 9H-fluoren-9-ylmethyl 4-aminopiperidine-1-carboxylate hydrochloride (commercially available)(7.80 g, 21.7 mmol) and 4,6-O-benzylidene-D-glucopyranose (23.3 g, 86.9 mmol) in MeOH (110 ml) was stirred at RT for 0.5 h. NaCNBH.sub.3 (5.46 g, 86.9 mmol) was added then the reaction was heated at 60° C. The reaction was stirred at 60° C. for 18 h. The reaction was recharged with 4,6-O-benzylidene-D-glucopyranose (23.3 g, 86.9 mmol) then left to stir at 60° C. for a further 6 h. The reaction was allowed to cool to RT then added to saturated aqueous NaHCO.sub.3 solution (200 ml) and EtOAc (200 ml). The resultant mixture was filtered through a Celite® pad then the filtrate was transferred to a separating funnel. The phases were separated then the organic phase was washed with brine:water (1:1, 2×200 ml), brine (100 ml), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue was suspended in MeCN (200 ml) and tBME (250 ml) the filtered. The solid obtained was suspended in MeOH then filtered. The combined filtrates were concentrated in vacuo. The crude material thus obtained was purified by flash column chromatography on C18 (400 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 20%, 1 CV; 20-50%, 10 CV; 50-100%, 2 CV; 100%, 2 CV. The desired fractions were combined and concentrated in vacuo to remove most of the MeCN and some of the water then the residual aqueous solution was lyophilised to afford the product as an off-white solid (12.6 g, 66%).

[0519] .sup.1H NMR (500 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.90 (d, J=7.4 Hz, 2H), 7.64-7.54 (m, 2H), 7.45-7.27 (m, 14H), 5.46 (s, 2H), 5.23-5.07 (m, 2H), 4.88-4.21 (m, 6H), 4.13 (dd, J=10.5, 5.3 Hz, 2H), 4.02-3.67 (m, 9H), 3.61 (d, J=9.2 Hz, 2H), 3.50 (t, J=10.5 Hz, 2H), 2.64-2.56 (m, 3H), 2.42-2.31 (m, 2H), 1.69-1.51 (m, 2H), 1.31-0.90 (m, 2H). LC/MS (System A): m/z (ESI.sup.+)=827 [MH.sup.+], R.sub.t=1.08 min, UV purity=100%.

Intermediate 24 Synthesis of (1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](piperidin-4-yl)amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol

[0520] ##STR00070##

[0521] Piperidine (9.01 ml, 91.2 mmol) was added to a solution of (9H-fluoren-9-yl)methyl 4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}piperidine-1-carboxylate; formic acid, Intermediate 23 (12.6 g, 14.4 mmol) in THF (150 ml). The reaction was left to stir at RT for 16 h then concentrated in vacuo. The crude solid material was suspended in MeOH (100 ml) then heated to dissolve. The solution was allowed to cool then concentrated in vacuo until solid was observed. The resultant suspension was stirred at RT for 15 min then filtered. The filtrate was concentrated in vacuo until solid was observed. The resultant suspension was stirred at RT for 15 min then filtered. The filtrate was purified by flash column chromatography on C18 (400 g). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 1 CVs; 10-25%, 6 CVs; 25%, 2 CVs; 25-50%, 1 CV; 50-100%, 1 CVs; 100%, 2 CVs. The desired fractions were combined and concentrated in vacuo to remove the majority of the solvent. The residual solution thus obtained was lyophilised to afford a pale-yellow solid (6.35 g). The solid thus obtained was partitioned between EtOAc (100 ml) and saturated aqueous NaHCO.sub.3 solution (100 ml). The phases were separated then the aqueous phase was extracted with CHCl.sub.3:1 PA (2:1, 100 ml) and n-BuOH (2×100 ml). The combined organic phases were dried over Na.sub.2SO.sub.4 then concentrated in vacuo. The residue was dissolved in 1:2 MeCN:water then lyophilised to afford the product as a white solid (5.81 g, 67%).

[0522] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.48 (dd, J=7.5, 2.0 Hz, 4H), 7.38-7.28 (m, 6H), 5.50 (s, 2H), 4.23 (dd, J=10.6, 5.4 Hz, 2H), 4.00-3.88 (m, 4H), 3.85 (dd, J=5.5, 2.4 Hz, 2H), 3.70 (dd, J=9.3, 2.4 Hz, 2H), 3.60 (t, J=10.5 Hz, 2H), 3.09-3.02 (m, 1H), 3.00-2.91 (m, 1H), 2.78 (dd, J=13.4, 3.7 Hz, 2H), 2.75-2.65 (m, 1H), 2.59 (dd, J=13.4, 8.8 Hz, 2H), 2.54-2.47 (m, 1H), 2.37-2.28 (m, 1H), 1.81-1.70 (m, 2H), 1.55-1.49 (m, 1H), 1.42-1.35 (m, 1H).

[0523] LC/MS (System A): m/z (ESI.sup.+)=605 [MH.sup.+], R.sub.t=0.77 min, UV purity=100%.

Intermediate 25 Synthesis of (2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl](piperidin-4-yl)amino}hexane-1,2,3,4,5-pentol dihydrochloride

[0524] ##STR00071##

[0525] A mixture of (1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl](piperidin-4-yl)amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol, Intermediate 24 (5.81 g, 8.93 mmol) and aqueous HCl solution (2 M, 90 ml, 180 mmol) was stirred at RT for 18 h. The reaction mixture was concentrated in vacuo then re-dissolved in water and lyophilised to afford the product as a white solid (4.14 g, 92%).

[0526] .sup.1H NMR (500 MHz, D.sub.2O) δ 4.34-4.21 (m, 2H), 4.10-3.97 (m, 1H), 3.95-3.76 (m, 6H), 3.75-3.34 (m, 10H), 3.25-3.12 (m, 2H), 2.51-2.35 (m, 2H), 2.27-1.94 (m, 2H).

[0527] LC/MS (System A): m/z (ESI.sup.+)=429 [MH.sup.+], R.sub.t=0.12 min, ELS purity=100%.

Intermediate 26 Synthesis of tert-butyl N-[(1-ethyl-6-fluoro-1H-1,3-benzodiazol-2-yl)methyl]carbamate

[0528] ##STR00072##

[0529] A solution of 2-{[(tert-butoxy)carbonyl]amino}acetic acid (1.70 g, 9.73 mmol), HATU (4.07 g, 10.7 mmol) and DIPEA (3.39 ml, 19.5 mmol) in DMF (20 ml) was stirred at RT for 20 min. A solution of 1-N-ethyl-5-fluorobenzene-1,2-diamine (1.05 g, 9.73 mmol) in THF (10 ml) was added and the resulting mixture was stirred at RT for 16 h. The reaction mixture was poured onto saturated aqueous NaHCO.sub.3 solution (80 ml). EtOAc (100 ml) and water (50 ml) were added then the phases were separated. The aqueous phase was extracted with EtOAc (2×50 ml) then the combined organic phases were washed with water (4×50 ml) and brine (50 ml), then dried over Na.sub.2SO.sub.4, filtered and evaporated to afford the intermediate as a black oil (4 g). The intermediate was dissolved in acetic acid (30 ml) then heated at 60° C. for 4 h. The reaction mixture was allowed to cool to RT then stirred at RT for 16 h. The resulting mixture was evaporated then the resulting residue was partitioned between EtOAc (150 ml) and water (100 ml). The aqueous phase was extracted with EtOAc (50 ml) then the combined organic phases were washed with water (4×50 ml) and brine (50 ml), then dried over Na.sub.2SO.sub.4, filtered and evaporated to a black solid (4 g). The solid was dissolved in the minimum of CH.sub.2Cl.sub.2/MeOH then evaporated onto silica (9 g).

[0530] The crude material was purified by flash column chromatography on a silica column (120 g). The column was eluted with CH.sub.2Cl.sub.2:MeOH, increasing the gradient linearly from 100:0 to 95:5 over 10 column volumes. The desired fractions were combined and evaporated to a black solid (2.9 g). The solid thus obtained was dissolved in EtOAc (100 ml) and extracted with saturated aqueous sodium bicarbonate solution (3×50 ml) and water (50 ml) then dried over Na.sub.2SO.sub.4, filtered and evaporated to a black solid (2.5 g). The solid was dissolved in the minimum of CH.sub.2Cl.sub.2/MeOH then evaporated onto silica (10 g). The material was further purified by flash column chromatography on a silica column (120 g). The column was eluted with EtOAc:heptane, increasing the gradient linearly from 0:100 to 100:0 over 10 column volumes. The desired fractions were combined and evaporated to afford the product as a pink solid (1.78 g, 62%).

[0531] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.56 (dd, J=8.8, 4.9 Hz, 1H), 7.50-7.41 (m, 2H), 7.01 (ddd, J=9.9, 8.9, 2.5 Hz, 1H), 4.42 (d, J=5.9 Hz, 2H), 4.24 (q, J=7.2 Hz, 2H), 1.45-1.20 (m, 12H).

[0532] LC/MS (System A): m/z (ESI.sup.+)=294 [MH.sup.+], R.sub.t=0.92 min, UV purity=100%.

Intermediate 27—Synthesis of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-ium iodide

[0533] ##STR00073##

[0534] Iodomethane (497 μl, 7.98 mmol) was added to a suspension of tert-butyl N-[(1-ethyl-6-fluoro-1H-1,3-benzodiazol-2-yl)methyl]carbamate, Intermediate 26 (780 mg, 2.66 mmol) in MeCN (12 ml) in a pressure tube. The tube was sealed then heated at 75° C. for 4 h. The reaction mixture was allowed to cool to RT then concentrated in vacuo to afford the product as a pale yellow solid (1.16 g, 99%).

[0535] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.14-8.04 (m, 2H), 7.94 (t, J=5.2 Hz, 1H), 7.68-7.56 (m, 1H), 4.73 (d, J=5.4 Hz, 2H), 4.58 (q, J=7.2 Hz, 2H), 4.08 (s, 3H), 1.38 (d, J=11.2 Hz, 12H).

[0536] LC/MS (System A): m/z (ESI.sup.+)=308 [M.sup.+], R.sub.t=0.87 min, UV purity=99%.

Intermediate 28—Synthesis of 2-(aminomethyl)-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-ium hydrochloride iodide

[0537] ##STR00074##

[0538] HCl solution in dioxane (4.0 M, 3.3 ml, 13.2 mmol) was added to a solution of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-fluoro-3-methyl-1H-1,3-benzodiazol-3-ium iodide, Intermediate 27 (1.16 g, 2.66 mmol) in MeCN (5 ml). The reaction was stirred at RT for 0.5 h then concentrated in vacuo. The solid was azeotroped with MeCN (10 ml) then dried under vacuum to yield the product as a yellow/green solid (870 mg, 88%).

[0539] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.21 (s, 3H), 8.25-8.14 (m, 2H), 7.69 (td, J=9.3, 2.4 Hz, 1H), 4.76 (s, 2H), 4.70 (q, J=7.2 Hz, 2H), 4.19 (s, 3H), 1.42 (t, J=7.2 Hz, 3H).

[0540] LC/MS (System A): m/z (ESI.sup.+)=208 [M.sup.+], R.sub.t=0.14 min, ELS purity=100%.

Intermediate 29—Synthesis of 2-[(6-chloro-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione

[0541] ##STR00075##

[0542] A mixture of N-phthaloylglycine (2.15 g, 10.5 mmol), TBTU (3.52 g, 11.0 mmol) and triethylamine (2.31 ml, 13.0 mmol) in DMF (30 ml) was stirred at RT for 45 min. A solution of 5-chloro-1-N-ethylbenzene-1,2-diamine (1.70 g, 9.96 mmol) in THF (20 ml) was added and the resulting mixture was stirred at RT for 18 h. The reaction mixture was added to saturated aqueous NaHCO3 solution (100 ml) which caused a pale brown solid to precipitate from solution. The solid was filtered, washed with water and dried under vacuum. The solid thus obtained was triturated in MeCN then filtered and dried under vacuum to afford the intermediate as a pale pink solid (5.4 g). The solid thus obtained was added portion-wise to acetic acid (30 ml). The resulting suspension was heated at 100° C. for 45 min then allowed to cool to RT over 16 h. The resulting suspension was filtered and washed with EtOAc then dried under vacuum to afford the product as a pale pink solid (585 mg). The solid was suspended in MeCN (5 ml) then MeCN:water (1:1, 1 ml) was added. The resulting suspension was filtered then the solid was dried under vacuum to afford the product as a pale pink solid (430 mg, 13%). The filtrate was again filtered, then the solid was washed with EtOAc and dried under vacuum to afford a second batch of product as a pale pink solid (2.00 g). The solid was suspended in MeCN (20 ml) then MeCN:water (1:1, 5 ml) was added. The resulting suspension was filtered then the solid was dried under vacuum to afford second batch of the product as a pale pink solid (1.33 g, 38%). The two batches of product were as a suspension in MeCN then concentrated in vacuo and dried under vacuum to afford the product as a pale pink solid (1.76 g, 51%).

[0543] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 8.02-7.85 (m, 4H), 7.76 (d, J=1.9 Hz, 1H), 7.49 (d, J=8.6 Hz, 1H), 7.15 (dd, J=8.6, 2.0 Hz, 1H), 5.13 (s, 2H), 4.39 (q, J=7.1 Hz, 2H), 1.37 (t, J=7.2 Hz, 3H).

[0544] LC/MS (System A): m/z (ESI.sup.+)=340 [M(.sup.35Cl)H.sub.1], 342 [M(.sup.37Cl)H.sub.1], R.sub.t=1.12 min, UV purity=99%.

Intermediate 30 Synthesis of 6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-ium iodide

[0545] ##STR00076##

[0546] A mixture of 2-[(6-chloro-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 29 (600 mg, 1.77 mmol) and iodomethane (330 μl, 5.30 mmol) in MeCN (6 ml) was heated at 75° C. in a sealed tube for 1.5 h then allowed to cool to RT. Iodomethane (165 μl, 2.65 mmol) was added then the reaction was heated at 80° C. for 5 h. The reaction was allowed to cool to RT then filtered to afford a solid which was washed with MeCN and dried under vacuum to afford the product as a yellow solid (644 mg, 73%).

[0547] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.40 (d, J=1.8 Hz, 1H), 8.13 (d, J=8.9 Hz, 1H), 7.91 (ddt, J=18.3, 5.8, 3.1 Hz, 4H), 7.79 (dd, J=8.9, 1.9 Hz, 1H), 5.40 (s, 2H), 4.74 (q, J=7.2 Hz, 2H), 4.14 (s, 3H), 1.43 (t, J=7.2 Hz, 3H).

[0548] LC/MS (System A): m/z (ESI.sup.+)=354 [M(.sup.35Cl).sup.+], 356 [M(.sup.37Cl).sup.+], R.sub.t=0.90 min, UV purity=97%.

Intermediate 31 Synthesis of 2-(aminomethyl)-6-chloro-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-ium iodide

[0549] ##STR00077##

[0550] Hydrazine hydrate (446 μl, 9.17 mmol) was added to a suspension of 6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-3-methyl-1H-1,3-benzodiazol-3-ium iodide, Intermediate 30 (640 mg, 1.33 mmol) in MeOH (8 ml) in a pressure tube. The tube was sealed and heated at 75° C. for 3 h then allowed to cool to RT. The resulting suspension was filtered and the solid was washed with MeOH (10 ml). The filtrate was concentrated in vacuo to afford an orange solid, which was suspended in CH.sub.2Cl.sub.2 (10 ml) then filtered and washed through with CH.sub.2Cl.sub.2. The solid thus obtained was suspended in CH.sub.2Cl.sub.2 (10 ml). A few drops of MeOH were added and the suspension was sonicated. The resulting suspension was filtered then the solid was dried under vacuum to afford the product as an off-white solid (360 mg, 77%).

[0551] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.31 (d, J=1.9 Hz, 1H), 8.06 (d, J=8.9 Hz, 1H), 7.73 (dd, J=8.8, 1.9 Hz, 1H), 4.60 (q, J=7.2 Hz, 2H), 4.25 (s, 2H), 4.06 (s, 3H), 2.52-2.10 (s, 2H+DMSO), 1.41 (t, J=7.2 Hz, 3H).

[0552] LC/MS (System A): m/z (ESI.sup.+)=224 [M(.sup.35Cl).sup.+], 226 [M(.sup.37Cl).sup.+], R.sub.t=0.16 min, ELS purity=100%.

Intermediate 32—Synthesis of 2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-ium iodide

[0553] ##STR00078##

[0554] Iodomethane (590 μl, 9.47 mmol) was added to a suspension of 2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 13 (1.59 g, 4.74 mmol) in MeCN (13 ml) in a pressure tube. The mixture was heated at 80° C. for 4 h then allowed to cool to RT. The resulting suspension was reduced to approximately half of the original volume under a stream of nitrogen. The solid was collected by filtration then washed with further MeCN (3 ml) to yield the product as a white solid (1.99 g, 87%).

[0555] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.01-7.84 (m, 5H), 7.62 (d, J=2.2 Hz, 1H), 7.32 (dd, J=9.2, 2.3 Hz, 1H), 5.37 (s, 2H), 4.71 (q, J=7.1 Hz, 2H), 4.11 (s, 3H), 3.92 (s, 3H), 1.42 (t, J=7.2 Hz, 3H).

[0556] LC/MS (System A): m/z (ESI.sup.+)=350 [M.sup.+], R.sub.t=0.87 min, UV purity=99%.

Intermediate 33—Synthesis of 2-(aminomethyl)-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-ium iodide

[0557] ##STR00079##

[0558] Hydrazine hydrate (1.17 ml, 24.0 mmol) was added to a suspension of 2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-3-methyl-1H-1,3-benzodiazol-3-ium iodide, Intermediate 32 (2.29 g, 4.80 mmol) in MeOH (25 ml). The mixture was heated at 75° C. for 1 h. The reaction was concentrated in vacuo and the resulting solid was suspended in CH.sub.2Cl.sub.2:MeOH (10:1). The solid was collected by filtration and washed with CH.sub.2Cl.sub.2. The filtrate was concentrated in vacuo to afford the product as a pale brown solid (1.60 g, 96%).

[0559] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.91 (d, J=9.1 Hz, 1H), 7.57 (d, J=2.3 Hz, 1H), 7.26 (dd, J=9.1, 2.3 Hz, 1H), 4.58 (q, J=7.2 Hz, 2H), 4.23 (s, 2H), 4.03 (s, 3H), 3.91 (s, 3H), 1.42 (t, J=7.2 Hz, 3H).

[0560] LC/MS (System A): m/z (ESI.sup.+)=220 [M.sup.+], R.sub.t=0.14 min, ELS purity=100%.

Intermediate 34—Synthesis of tert-butyl N-{[1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-2-yl]methyl}carbamate

[0561] ##STR00080##

[0562] Intermediate 34 was synthesised according to literature procedures (WO 2009019506 A1).

Intermediate 35—Synthesis of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium iodide

[0563] ##STR00081##

[0564] Iodomethane (381 μl, 6.12 mmol) was added to a suspension of tert-butyl N-{[1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-2-yl]methyl}carbamate, Intermediate 34 (700 mg, 2.04 mmol) in MeCN (10 ml) in a pressure tube. The tube was sealed and heated at 75° C. for 8 h then allowed to cool to RT. The reaction mixture was concentrated in vacuo to afford the product as a pale yellow solid (1.01 g, >99%).

[0565] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.64 (s, 1H), 8.29 (d, J=8.7 Hz, 1H), 8.08 (d, J=8.7 Hz, 1H), 7.98 (t, J=5.2 Hz, 1H), 4.80 (d, J=5.6 Hz, 2H), 4.73 (q, J=7.2 Hz, 2H), 4.14 (s, 3H), 1.40 (m, 12H).

[0566] LC/MS (System A): m/z (ESI.sup.+)=358 [M.sup.+], R.sub.t=0.91 min, UV purity=98%.

Intermediate 36—Synthesis of 2-(aminomethyl)-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium hydrochloride iodide

[0567] ##STR00082##

[0568] HCl solution in dioxane (4.0 M, 2.8 ml, 11 mmol) was added to a solution of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-methyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium iodide, Intermediate 35 (1.07 g, 2.21 mmol) in MeCN (5 ml). The reaction was stirred at RT for 16 h then concentrated in vacuo to yield the product as an off-white solid (875 mg, 94%).

[0569] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.14 (s, 3H), 8.74 (s, 1H), 8.38 (d, J=8.8 Hz, 1H), 8.14 (dd, J=8.8, 1.3 Hz, 1H), 4.84 (d, J=5.5 Hz, 4H), 4.24 (s, 3H), 1.45 (t, J=7.2 Hz, 3H).

[0570] LC/MS (System A): m/z (ESI.sup.+)=258 [M.sup.+], R.sub.t=0.17 min, ELS purity=100%.

Intermediate 37—Synthesis of N-ethyl-2-nitro-5-(trifluoromethoxy)aniline

[0571] ##STR00083##

[0572] Two identical reactions were carried out in parallel as follows then combined for work-up. Ethylamine solution in THF (2.0 M, 4.1 ml, 8.2 mmol) was added to a suspension of 2-chloro-1-nitro-4-(trifluoromethoxy)benzene (1.00 g, 4.14 mmol) and K.sub.2CO.sub.3 (1.71 g, 6.21 mmol) in THF (12 ml) in a pressure tube. The tube was sealed then heated at 50° C. for 16 h then allowed to cool to RT. Additional ethylamine solution in THF (2.0 M, 2.1 ml, 4.2 mmol) was added then the reaction was heated at 50° C. for 24 h. The combined reactions were filtered then the solid thus obtained was rinsed with EtOAc (100 ml). The combined filtrates were extracted with saturated aqueous NaHCO.sub.3 solution (2×100 ml), water (50 ml) and brine (50 ml) then dried over Na.sub.2SO.sub.4, then filtered and evaporated to an orange oil. The crude material was purified by flash column chromatography on C18 (120 g). The column was eluted with MeCN:water using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-100%, 20 CVs; 100%, 2 CV. The desired fractions were combined and concentrated in vacuo to remove most of the MeCN. The residual aqueous mixture was extracted with EtOAc (100 ml). The organic phase was separated, then dried over Na.sub.2SO.sub.4 and evaporated to afford the product as a bright orange oil (1.42 g, 69%).

[0573] .sup.1H NMR (500 MHz, CDCl.sub.3) δ 8.23 (d, J=9.4 Hz, 1H), 8.04 (s, 1H), 6.61 (d, J=2.0 Hz, 1H), 6.48 (ddd, J=9.4, 2.4, 1.2 Hz, 1H), 3.33 (qd, J=7.2, 5.1 Hz, 2H), 1.39 (t, J=7.2 Hz, 3H).

[0574] LC/MS (System A): m/z (ESI.sup.+)=251 [MH.sup.+], R.sub.t=1.32 min, UV purity=100%.

Intermediate 38 Synthesis of tert-butyl N-([1-ethyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-2-yl]methyl) carbamate

[0575] ##STR00084##

[0576] A suspension of palladium on carbon (10 wt %, 0.3 g) and N-ethyl-2-nitro-5-(trifluoromethoxy)aniline, Intermediate 37 (1.42 g, 5.68 mmol) solution in EtOH (40 ml) was stirred under a hydrogen atmosphere at RT for 16 h. The reaction mixture was filtered through glass fibre filter paper and washed through with EtOAc. The filtrate was concentrated in vacuo and then diluted with THF (10 ml). The resulting solution was added to a pre-mixed solution of 2-{[(tert-butoxy)carbonyl]amino}acetic acid (994 mg, 5.68 mmol), HATU (2.37 g, 6.24 mmol) and DI PEA (1.98 ml, 11.4 mmol) in DMF (20 ml). The reaction mixture was stirred at RT for 68 h then poured onto saturated aqueous NaHCO.sub.3 (80 ml). EtOAc (50 ml) and water (50 ml) were added and the phases were separated. The aqueous phase was extracted with EtOAc (2×50 ml) then the combined organic phases were washed with water (4×50 ml) and brine (50 ml) then dried over Na.sub.2SO.sub.4, filtered and evaporated to a black oil. The oil thus obtained was dissolved in acetic acid (10 ml) and the resulting solution was heated at 70° C. for 1.5 h. The reaction solution was concentrated in vacuo then the residue was partitioned between EtOAc (50 ml) and saturated aqueous NaHCO.sub.3 solution (50 ml). The organic phase was washed with saturated aqueous NaHCO.sub.3 solution (4×50 ml), water (50 ml) and brine (10 ml) then dried over Na.sub.2SO.sub.4, filtered and evaporated to a brown solid (1.95 g). The crude material was dissolved in CH.sub.2Cl.sub.2/MeOH then evaporated onto silica (10 g) and purified by flash column chromatography on a silica column (120 g). The column was eluted with EtOAc:heptane, increasing the gradient linearly from 0:100 to 100:0 over 10 column volumes. The desired fractions were combined and evaporated to yield the product as a pink solid (1.04 g, 47%).

[0577] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.70-7.61 (m, 2H), 7.49 (d, J=5.1 Hz, 1H), 7.19-7.10 (m, 1H), 4.44 (d, J=5.9 Hz, 2H), 4.29 (q, J=7.1 Hz, 2H), 1.45-1.23 (m, 12H).

[0578] LC/MS (System A): m/z (ESI.sup.+)=360 [MH.sup.+], R.sub.t=1.11 min, UV purity=92%.

Intermediate 39 Synthesis of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-ium iodide

[0579] ##STR00085##

[0580] Iodomethane (208 μl, 3.34 mmol) was added to a suspension of tert-butyl N-{[1-ethyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-2-yl]methyl} carbamate, Intermediate 38 (92%, 400 mg, 1.02 mmol) in MeCN (5 ml) in a pressure tube. The tube was sealed then heated at 75° C. for 5 h then allowed to cool to RT. The reaction mixture was concentrated in vacuo to yield the product as a dark green solid (552 mg, >99%).

[0581] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.30 (s, 1H), 8.18 (d, J=9.1 Hz, 1H), 7.95 (t, J=5.2 Hz, 1H), 7.76 (d, J=9.1 Hz, 1H), 4.75 (d, J=5.4 Hz, 2H), 4.64 (q, J=7.1 Hz, 2H), 4.10 (s, 3H), 1.44-1.25 (m 12H).

[0582] LC/MS (System A): m/z (ESI.sup.+)=374 [M.sup.+], R.sub.t=0.96 min, UV purity=93%.

Intermediate 40—Synthesis of 2-(aminomethyl)-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-ium hydrochloride iodide

[0583] ##STR00086##

[0584] HCl solution in dioxane (4.0 M, 1.4 ml, 5.6 mmol) was added to a solution of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-methyl-6-(trifluoromethoxy)-1H-1,3-benzodiazol-3-ium iodide, Intermediate 39 (93%, 550 mg, 1.02 mmol) in MeCN (5 ml). The reaction was left to stir at RT for 16 h then concentrated in vacuo. The solid was azeotroped with MeCN (10 ml) then dried under vacuum to yield the product as a brown solid (480 mg, 94%).

[0585] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.11 (s, 3H), 8.40 (d, J=1.6 Hz, 1H), 8.28 (d, J=9.1 Hz, 1H), 7.83 (dd, J=9.1, 1.2 Hz, 1H), 4.80 (s, 2H), 4.75 (q, J=7.2 Hz, 2H), 4.21 (s, 3H), 1.42 (t, J=7.2 Hz, 3H).

[0586] LC/MS (System A): m/z (ESI.sup.+)=274 [M.sup.+], R.sub.t=0.37 min, ELS purity=87%.

Intermediate 41—Synthesis of 6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0587] ##STR00087##

[0588] A mixture of 2-[(6-chloro-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 29 (850 mg, 2.50 mmol) and iodomethane (2.0 ml, 25 mmol) in MeCN (12 ml) was heated under microwave irradiation for 3 h at 120° C. The resulting solution was left to stand at RT for 64 h, resulting in precipitation of a solid. The solid was collected by filtration then washed with MeCN and dried under vacuum to afford the product as a pale yellow solid (850 mg, 67%).

[0589] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.41 (d, J=1.8 Hz, 1H), 8.18 (d, J=8.9 Hz, 1H), 7.98-7.94 (m, 2H), 7.93-7.89 (m, 2H), 7.79 (dd, J=8.9, 1.9 Hz, 1H), 5.43 (s, 2H), 4.74-4.66 (m, 4H), 1.46-1.38 (m, 6H).

[0590] LC/MS (System A): m/z (ESI.sup.+)=368 [M(.sup.35Cl).sup.+], 370 [M(.sup.37Cl).sup.+], R.sub.t=0.93 min, UV purity=98%.

Intermediate 42—Synthesis of 2-(aminomethyl)-6-chloro-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0591] ##STR00088##

[0592] Hydrazine hydrate (787 μl, 16.2 mmol) was added to a suspension of 6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide, Intermediate 41 (845 mg, 1.62 mmol) in MeOH (6 ml) in a pressure tube. The tube was sealed and heated at 80° C. for 4 h then allowed to cool to RT. The reaction mixture was concentrated in vacuo. The resultant residue was suspended in CH.sub.2Cl.sub.2:MeOH (9:1, 20 ml) then filtered. The filtrate was concentrated in vacuo to afford the product as a yellow solid (301 mg, 49%).

[0593] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.32 (d, J=1.8 Hz, 1H), 8.11 (d, J=8.9 Hz, 1H), 7.73 (dd, J=8.8, 1.9 Hz, 1H), 4.65-4.56 (m, 4H), 4.29 (s, 2H), 1.46-1.40 (m, 6H).

[0594] LC/MS (System F): m/z (ESI.sup.+)=238 [M(.sup.35Cl).sup.+], 240 [M(.sup.37Cl).sup.+], R.sub.t=2.00 min, UV purity=97%.

Intermediate 43—Synthesis of 3-benzyl-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium bromide

[0595] ##STR00089##

[0596] A mixture of 2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 13 (500 mg, 1.49 mmol) and (bromomethyl)benzene (531 μl, 4.47 mmol) in MeCN (5 ml) was heated at 80° C. in a sealed tube for 16 h. The reaction mixture allowed to cool to RT then filtered. The solid was washed with MeCN then dried under vacuum to afford the product as a white solid (665 mg, 87%).

[0597] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.84-7.79 (m, 3H), 7.78-7.74 (m, 2H), 7.71 (d, J=2.3 Hz, 1H), 7.29 (dd, J=9.2, 2.3 Hz, 1H), 7.09 (t, J=7.7 Hz, 2H), 7.00-6.94 (m, 3H), 5.85 (s, 2H), 5.50 (s, 2H), 4.79 (q, J=7.2 Hz, 2H), 3.94 (s, 3H), 1.51 (t, J=7.2 Hz, 3H).

[0598] LC/MS (System A): m/z (ESI.sup.+)=426 [M.sup.+], R.sub.t=1.02 min, UV purity=99%.

Intermediate 44—Synthesis of 2-(aminomethyl)-3-benzyl-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium bromide

[0599] ##STR00090##

[0600] A mixture of 3-benzyl-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium bromide, Intermediate 43 (665 mg, 1.31 mmol) and hydrazine hydrate (638 μl, 13.1 mmol) in MeOH (5 ml) was stirred at 80° C. for 2 h in a sealed tube. The reaction mixture was allowed to cool then concentrated in vacuo to a yellow solid. The residue was suspended in CH.sub.2Cl.sub.2 (20 ml) with sonication. The resultant suspension was filtered then the solid was re-suspended in CH.sub.2Cl.sub.2:MeOH (1:1, 30 ml) with sonication. The resultant suspension was filtered then the combined filtrates were concentrated in vacuo to afford the product as a yellow solid (526 mg, 60%).

[0601] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.71 (d, J=9.1 Hz, 1H), 7.62 (d, J=2.3 Hz, 1H), 7.41-7.31 (m, 5H), 7.20 (dd, J=9.1, 2.3 Hz, 1H), 5.86 (s, 2H), 4.63 (q, J=7.2 Hz, 2H), 4.34 (s, 2H), 3.90 (s, 3H), 1.48 (t, J=7.2 Hz, 3H).

[0602] LC/MS (System A): m/z (ESI.sup.+)=296 [M.sup.+], R.sub.t=0.76 min, UV purity=56%.

Intermediate 45—Synthesis of tert-butyl N-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]carbamate

[0603] ##STR00091##

[0604] HATU (8.53 g, 22.3 mmol) and DIPEA (7.1 ml, 41 mmol) were added to a solution of N-(tert-butoxycarbonyl)glycine (3.93 g, 22.4 mmol) in DMF (40 ml). The resulting solution was stirred at RT for 0.5 h then a solution of 1-N-ethyl-5-methoxybenzene-1,2-diamine (3.39 g, 20.4 mmol) in THF (20 ml) was added. The reaction was left to stir at RT for 80 min then the reaction mixture was diluted with EtOAc (200 ml) and water (150 ml). The phases were separated then the organic phase was washed with water (3×150 ml) and brine (150 ml). The combined organic phases were dried over Na.sub.2SO.sub.4 then concentrated in vacuo to afford the crude intermediate as a dark red viscous oil. The intermediate was taken up in AcOH (40 ml) and the resulting solution was stirred at 60° C. for 18 h. The reaction mixture was allowed to cool then concentrated in vacuo. The residue was then dissolved in EtOAc (200 ml) then the pH was adjusted to 9 by the addition of saturated aqueous NaHCO.sub.3 solution. The phases were separated and the organic phase was washed with water (2×150 ml) and brine (150 ml), then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the crude product as a dark red oil. The crude material was purified by flash column chromatography on a silica column (340 g). The column was eluted with CH.sub.2Cl.sub.2:MeOH, increasing the gradient linearly from 100:0 to 92:8 over 10 column volumes. The desired fractions were combined and evaporated to a viscous dark red oil which solidified on standing to yield the product as a dark red solid (5.02 g, 77%).

[0605] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.47-7.39 (m, 2H), 7.06 (d, J=2.4 Hz, 1H), 6.78 (dd, J=8.7, 2.4 Hz, 1H), 4.39 (d, J=5.9 Hz, 2H), 4.22 (q, J=7.1 Hz, 2H), 3.81 (s, 3H), 1.39 (s, 9H), 1.28 (t, J=7.2 Hz, 3H).

[0606] LC/MS (System A): m/z (ESI.sup.+)=306 [MH.sup.+], R.sub.t=0.88 min, UV purity=96%.

Intermediate 46 Synthesis of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-ium bromide

[0607] ##STR00092##

[0608] Methyl bromoacetate (395 μl, 4.18 mmol) was added to a suspension of tert-butyl N-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]carbamate, Intermediate 45 (96%, 500 mg, 1.39 mmol) in MeCN (4 ml) in a pressure tube. The tube was sealed and the resulting mixture was stirred at 75° C. for 4 h then allowed to cool to RT. The resultant suspension was filtered then the solid was washed with cold MeCN and dried under vacuum to afford the product as a white solid (454 mg, 71%).

[0609] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.90 (d, J=9.2 Hz, 1H), 7.86 (t, J=5.3 Hz, 1H), 7.62 (d, J=2.2 Hz, 1H), 7.29 (dd, J=9.1, 2.3 Hz, 1H), 5.58 (s, 2H), 4.75 (d, J=5.4 Hz, 2H), 4.65 (q, J=7.2 Hz, 2H), 3.92 (s, 3H), 3.75 (s, 3H), 1.43 (t, J=7.2 Hz, 3H), 1.35 (s, 9H).

[0610] LC/MS (System A): m/z (ESI.sup.+)=378 [M.sup.+], R.sub.t=0.88 min, UV purity=100%.

Intermediate 47 Synthesis of 2-(aminomethyl)-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-ium hydrochloride bromide

[0611] ##STR00093##

[0612] A mixture of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-methoxy-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-ium bromide, Intermediate 46 (454 mg, 0.990 mmol) and HCl solution in dioxane (4.0 M, 2.5 ml, 10 mmol) was stirred at RT for 0.5 h. The reaction mixture was concentrated in vacuo to afford the product as a pale orange foam (580 mg, 96% yield corrected for 35 wt % residual dioxane observed in NMR).

[0613] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.86 (s, 3H), 7.97 (d, J=9.2 Hz, 1H), 7.68 (d, J=2.3 Hz, 1H), 7.35 (dd, J=9.2, 2.3 Hz, 1H), 5.77 (s, 2H), 4.79 (s, 2H), 4.72 (q, J=7.2 Hz, 2H), 3.94 (s, 3H), 3.77 (s, 3H), 1.47 (t, J=7.2 Hz, 3H).

[0614] LC/MS (System A): m/z (ESI.sup.+)=278 [M.sup.+], R.sub.t=0.14 min, ELS purity=95%.

Intermediate 48 Synthesis of 2-({[(tert-butoxy)carbonyl]amino}methyl)-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium bromide

[0615] ##STR00094##

[0616] 2-Bromoacetamide (691 mg, 5.01 mmol) was added to a suspension of tert-butyl N-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]carbamate, Intermediate 45 (85%, 600 mg, 1.67 mmol) in MeCN (6 ml) in a pressure tube. The tube was sealed then the reaction mixture was stirred at 80° C. for 5 h. The reaction was allowed to cool to RT then stirred at RT for 64 h. The reaction mixture was heated to 80° C. for a further 2 h then allowed to cool to RT. The resultant mixture was concentrated in vacuo to a viscous red oil. The crude material was purified by flash column chromatography on a silica column (50 g). The column was eluted with CH.sub.2Cl.sub.2:MeOH, using the following gradient (% MeOH, column volumes): 0%, 1 CV; 0-9%, 8 CV; 9%, 3 CV; 9-14%, 2 CV, 14%, 1 CV; 14-20%, 2 CV. The desired fractions were combined and evaporated to afford the product as a pale magenta foam (638 mg, 84%).

[0617] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.90 (s, 1H), 7.86-7.77 (m, 2H), 7.63 (s, 1H), 7.60 (d, J=2.2 Hz, 1H), 7.29 (dd, J=9.1, 2.3 Hz, 1H), 5.30 (s, 2H), 4.70 (d, J=5.4 Hz, 2H), 4.62 (q, J=7.2 Hz, 2H), 3.92 (s, 3H), 1.42 (t, J=7.2 Hz, 3H), 1.36 (s, 9H).

[0618] LC/MS (System A): m/z (ESI.sup.+)=363 [M.sup.+], R.sub.t=0.83 min, UV purity=98%.

Intermediate 49—Synthesis of 2-(aminomethyl)-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium hydrochloride bromide

[0619] ##STR00095##

[0620] A mixture of 2-({[(tert-butoxy)carbonyl]amino}methyl)-3-(carbamoylmethyl)-1-ethyl-6-methoxy-1H-1,3-benzodiazol-3-ium bromide, Intermediate 48 (98%, 638 mg, 1.41 mmol) and HCl solution in dioxane (4.0 M, 3.5 ml, 14 mmol) was stirred at RT for 0.5 h. The reaction mixture was concentrated in vacuo to afford the product as a purple solid (719 mg, 97%-yield corrected for 28 wt % residual dioxane observed in NMR).

[0621] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.91 (s, 3H), 8.35 (s, 1H), 7.98-7.89 (m, 2H), 7.68 (d, J=2.3 Hz, 1H), 7.38 (dd, J=9.2, 2.3 Hz, 1H), 5.45 (s, 2H), 4.77 (s, 2H), 4.71 (q, J=7.2 Hz, 2H), 3.94 (s, 3H), 1.46 (t, J=7.2 Hz, 3H).

[0622] LC/MS (System A): m/z (ESI.sup.+)=263 [M.sup.+], R.sub.t=0.15 min, ELS purity=100%.

Intermediate 50—Synthesis of 2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-ium iodide

[0623] ##STR00096##

[0624] A mixture of 2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 13 (500 mg, 1.49 mmol), 1-chloro-2-(methylsulfanyl)ethane (730 μl, 7.45 mmol) and NaI (1.12 g, 7.45 mmol) in MeCN (8 ml) was heated to 100° C. in a sealed tube for 5 h then allowed to cool to RT. The reaction mixture was concentrated in vacuo to a brown solid. The solid thus obtained was suspended in MeOH (8 ml) with sonication. The resultant suspension was filtered then the solid was washed with MeOH and dried under vacuum to afford the product as a brown solid (1.13 g, 50%) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.04 (d, J=9.2 Hz, 1H), 7.99-7.88 (m, 4H), 7.64 (t, J=2.6 Hz, 1H), 7.32 (dd, J=9.2, 2.3 Hz, 1H), 5.47-5.37 (m, 2H), 4.90-4.68 (m, 4H), 4.11 (s, 1H), 3.92 (d, J=1.4 Hz, 3H), 3.03 (t, J=7.1 Hz, 2H), 2.15 (s, 2H), 1.42 (q, J=6.9 Hz, 3H).

[0625] LC/MS (System A): m/z (ESI.sup.+)=410 [M.sup.+], R.sub.t=0.99 min, UV purity=66%.

Intermediate 51—Synthesis of 2-(aminomethyl)-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-ium iodide

[0626] ##STR00097##

[0627] A mixture of 2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-6-methoxy-3-[2-(methylsulfanyl)ethyl]-1H-1,3-benzodiazol-3-ium iodide, Intermediate 50 (66%, 1.00 g, 1.62 mmol) and hydrazine hydrate (226 μl, 4.65 mmol) in MeOH (5 ml) was heated to 80° C. in a sealed tube for 80 min. The reaction mixture was concentrated in vacuo to an orange solid. The solid thus obtained was suspended in CH.sub.2Cl.sub.2 (50 ml) with sonication. The suspension was filtered then the filtrate was concentrated in vacuo to afford the product as a pale orange solid (395 mg, 48%).

[0628] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.96 (d, J=9.1 Hz, 1H), 7.60-7.56 (m, 1H), 7.27 (dt, J=9.1, 2.1 Hz, 1H), 4.76 (t, J=6.9 Hz, 2H), 4.61 (q, J=6.1, 4.9 Hz, 2H), 4.30 (s, 2H), 3.92 (d, J=1.4 Hz, 3H), 3.01 (t, J=6.9 Hz, 2H), 2.41-2.30 (m, 2H), 2.13 (s, 3H), 1.45 (t, J=7.3 Hz, 3H).

[0629] LC/MS (System A): m/z (ESI.sup.+)=280 [M.sup.+], R.sub.t=0.65 min, UV purity=80%.

Intermediate 52—Synthesis of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-ium bromide

[0630] ##STR00098##

[0631] (2-Bromoethoxy)(tert-butyl)dimethylsilane (1.66 g, 6.96 mmol) was added to a suspension of tert-butyl N-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]carbamate, Intermediate 45 (85%, 500 mg, 1.39 mmol) in MeCN (5 ml) in a pressure tube. The tube was sealed then the reaction mixture was stirred at 80° C. for 0.5 h then at 100° C. for 16 h. The reaction was allowed to cool to RT then additional (2-bromoethoxy)(tert-butyl)dimethylsilane (1.00 g, 4.18 mmol) was added. The tube was sealed then the reaction was left to stir at 100° C. for a further 24 h. The reaction mixture was allowed to cool then concentrated in vacuo to a red oil. The crude material was purified by flash column chromatography on a silica column (25 g). The column was eluted with CH.sub.2Cl.sub.2:MeOH, increasing the gradient linearly from 100:0 to 90:10 over 10 column volumes. The desired fractions were combined and evaporated to yield a viscous dark red oil (615 mg). The material was further purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-36%; 36%, 2 CV; 36-50%, 4 CV; 50-100%, 3 CV; 100%, 3 CV. The desired fractions were combined and evaporated to yield the product as a viscous dark red oil (278 mg, 43%).

[0632] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.28 (s, 1H), 7.99-7.84 (m, 2H), 7.57 (d, J=2.2 Hz, 1H), 7.28 (dd, J=9.1, 2.3 Hz, 1H), 4.76 (d, J=5.5 Hz, 2H), 4.71-4.65 (m, 2H), 4.59 (q, J=7.1 Hz, 2H), 3.92 (s, 3H), 3.77 (t, J=4.7 Hz, 2H), 1.41 (t, J=7.2 Hz, 3H), 1.38 (d, J=2.8 Hz, 9H).

[0633] LC/MS (System A): m/z (ESI.sup.+)=350 [MH.sup.+], R.sub.t=0.88 min, UV purity=92%.

Intermediate 53 Synthesis of 2-(aminomethyl)-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-ium hydrochloride bromide

[0634] ##STR00099##

[0635] Hydrogen chloride solution in dioxane (4.0 M, 1.2 ml, 4.8 mmol) was added to a solution of 2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-3-(2-hydroxyethyl)-6-methoxy-1H-1,3-benzodiazol-3-ium bromide, Intermediate 52 (92%, 227 mg, 0.49 mmol) in MeCN (3 ml). The resulting mixture was stirred at RT for 1 h. The reaction mixture was concentrated in vacuo to afford the product as a dark purple solid (174 mg, 97%).

[0636] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.89 (s, 3H), 8.03 (d, J=9.2 Hz, 1H), 7.66 (d, J=2.3 Hz, 1H), 7.35 (dd, J=9.2, 2.3 Hz, 1H), 4.82-4.73 (m, 4H), 4.69 (q, J=7.2 Hz, 2H), 3.94 (s, 3H), 3.80 (t, J=4.6 Hz, 2H), 1.45 (t, J=7.2 Hz, 3H).

[0637] LC/MS (System F): m/z (ESI.sup.+)=250 [MH.sup.+], R.sub.t=1.58 min, UV purity=99%.

Intermediate 54 Synthesis of 2-{[(2-carboxyphenyl)formamido]methyl}-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-ium iodide

[0638] ##STR00100##

[0639] A mixture of 2-[(1-ethyl-6-methoxy-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 13 (2.00 g, 5.96 mmol) and 2-[2-(2-iodoethoxy)ethoxy]ethan-1-ol (90%, 8.62 g, 29.8 mmol) in MeCN (15 ml) was heated at 130° C. in a sealed tube for 16 h then allowed to cool to RT. The reaction mixture was concentrated in vacuo then the crude material was purified by flash column chromatography on C18 (12 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-14%, 2 CV; 14-18%, 1 CV; 18-27%, 2 CV; 27-31%, 0.5 CV; 31-60%, 0.5 CV; 60-100%, 1 CV; 100%, 1 CV. The desired fractions were combined and evaporated to yield the product as a yellow amorphous solid (1.72 g, 47%).

[0640] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 12.97-12.91 (m, 1H), 7.96 (d, J=9.2 Hz, 1H), 7.72 (dd, J=7.8, 1.2 Hz, 1H), 7.60 (dd, J=7.7, 1.3 Hz, 1H), 7.57 (d, J=2.3 Hz, 1H), 7.37 (td, J=7.5, 1.4 Hz, 1H), 7.30-7.24 (m, 2H), 5.04 (d, J=5.0 Hz, 2H), 4.91 (t, J=4.8 Hz, 2H), 4.77-4.62 (m, 3H), 3.91 (s, 3H), 3.83 (t, J=4.9 Hz, 2H), 3.51-3.46 (m, 2H), 3.43-3.39 (m, 4H), 3.36-3.28 (m, 2H+HDO), 1.41 (t, J=7.2 Hz, 3H).

[0641] LC/MS (System A): m/z (ESI.sup.+)=486 [M.sup.+], R.sub.t=0.80 min, UV purity=100%.

Intermediate 55 Synthesis of 2-(aminomethyl)-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-ium iodide

[0642] ##STR00101##

[0643] Hydrazine hydrate (639 μl, 13.2 mmol) was added to a solution of 2-{[(2-carboxyphenyl)formamido]methyl}-1-ethyl-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}-6-methoxy-1H-1,3-benzodiazol-3-ium iodide, Intermediate 54 (1.52 g, 2.48 mmol) in MeOH (12 ml) in a pressure tube. The tube was sealed and the reaction solution was heated at 75° C. for 3.5 h. The reaction was allowed to cool to RT then stirred at RT for 64 h. Additional hydrazine hydrate (639 μl, 13.2 mmol) was added and the reaction was heated at 75° C. for a further 18 h, then at 80° C. for a further 24 h. The reaction mixture was allowed to cool to RT then concentrated in vacuo to an orange solid. The solid thus obtained was suspended in CH.sub.2Cl.sub.2:MeOH (9:1, 30 ml) then filtered. The filtrate was concentrated in vacuo to afford the product as a viscous orange oil (1.21 g, 88%).

[0644] LC/MS (System F): m/z (ESI.sup.+)=338 [M.sup.+], Rt=1.41 min, UV purity=84%.

Intermediate 56—Synthesis of 3-benzyl-6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-3-ium bromide

[0645] ##STR00102##

[0646] Benzyl bromide (88 μl, 0.74 mmol) was added to a suspension of 2-[(6-chloro-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 20 (0.25 g, 0.74 mmol) in MeCN (5 ml) in a pressure tube. The tube was sealed then the reaction was stirred at 80° C. for 16 h then allowed to cool to RT. Benzyl bromide (88 μl, 0.74 mmol) was added then the reaction was stirred at 80° C. for a further 6 h. The reaction was allowed to cool to RT then left to stand at RT for 64 h. Benzyl bromide (88 μl, 0.74 mmol) was added then the reaction was stirred at 80° C. for a further 24 h. The reaction was allowed to cool to RT then filtered. The solid was washed with MeCN then dried under vacuum to afford the product as a white solid (370 mg, 97%).

[0647] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.50 (d, J=1.8 Hz, 1H), 7.98 (d, J=8.9 Hz, 1H), 7.85-7.68 (m, 5H), 7.14-7.05 (m, 2H), 6.99 (t, J=6.5 Hz, 3H), 5.89 (s, 2H), 5.52 (s, 2H), 4.82 (q, J=7.2 Hz, 2H), 1.51 (t, J=7.2 Hz, 3H).

[0648] LC/MS (System A): m/z (ESI.sup.+)=430 [M.sup.+], Rt=0.98 min, UV purity=99%.

Intermediate 57—Synthesis of 2-(aminomethyl)-3-benzyl-6-chloro-1-ethyl-1H-1,3-benzodiazol-3-ium bromide

[0649] ##STR00103##

[0650] Hydrazine hydrate (0.18 ml, 3.62 mmol) was added to a suspension of 3-benzyl-6-chloro-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-3-ium bromide, Intermediate 56 (370 mg, 0.724 mmol) in MeOH (4 ml) in a pressure tube. The tube was sealed then the reaction was stirred at 75° C. for 3 h. The reaction mixture was allowed to cool to RT then filtered. The filtrate was concentrated in vacuo to afford an orange solid. The solid thus obtained was suspended in CH.sub.2Cl.sub.2:MeOH (9:1, 10 ml) then filtered. The solid was dried under vacuum then suspended in MeOH. The suspension was filtered then the filtrate was concentrated in vacuo. The resultant residue was suspended in CH.sub.2Cl.sub.2:MeOH (9:1, 10 ml) with sonication. The resultant suspension was filtered then the filtrate was concentrated in vacuo to afford the product as a pale yellow solid (107 mg, 33%).

[0651] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.36 (d, J=1.8 Hz, 1H), 7.83 (d, J=8.9 Hz, 1H), 7.66 (dd, J=8.9, 1.9 Hz, 1H), 7.43-7.30 (m, 5H), 5.89 (s, 2H), 4.63 (q, J=7.2 Hz, 2H), 4.35 (s, 2H), 1.46 (t, J=7.2 Hz, 3H).

[0652] LC/MS (System A): m/z (ESI.sup.+)=300 [M.sup.+], Rt=0.81 min, UV purity=85%.

Intermediate 58—Synthesis of 3-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium bromide

[0653] ##STR00104##

[0654] Benzyl bromide (173 μl, 1.46 mmol) was added to a suspension of tert-butyl N-{[1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-2-yl]methyl}carbamate, Intermediate 34 (250 mg, 0.73 mmol) in MeCN (3 ml) in a pressure tube. The tube was sealed and heated at 80° C. for 16 h then the reaction mixture was concentrated in vacuo to afford an orange oil which solidified on standing. The resulting solid was suspended in MeCN (2 ml). The solid was collected by filtration then dried under vacuum to afford the product as a white solid (160 mg). The filtrate was concentrated in vacuo. The residue was suspended in the minimum volume of MeCN then filtered. The solid thus obtained was dried under vacuum to yield a second batch of product as an off-white solid (90 mg). The 2 batches of product were combined in MeCN then evaporated to afford the product as an off-white solid (250 mg, 64%).

[0655] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.69 (s, 1H), 8.03-7.87 (m, 3H), 7.43-7.36 (m, 3H), 7.31-7.23 (m, 2H), 5.92 (s, 2H), 4.97-4.85 (m, 2H), 4.78 (q, J=7.1 Hz, 2H), 1.49 (t, J=7.1 Hz, 3H), 1.30 (s, 9H).

[0656] LC/MS (System A): m/z (ESI.sup.+)=435 [M.sup.+], R.sub.t=1.05 min, UV purity=96%.

Intermediate 59—Synthesis of 2-(aminomethyl)-3-benzyl-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium hydrochloride bromide

[0657] ##STR00105##

[0658] HCl solution in dioxane (4.0 M, 0.61 ml, 2.4 mmol) was added to a solution of 3-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-1-ethyl-6-(trifluoromethyl)-1H-1,3-benzodiazol-3-ium bromide, Intermediate 58 (250 mg, 0.49 mmol) in MeCN (2.5 ml). The reaction was stirred at RT for 18 h then concentrated in vacuo. The residue was azeotroped with MeCN then dried under vacuum to afford the product as a pale yellow solid (209 mg, 95%).

[0659] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.18 (s, 3H), 8.77 (s, 1H), 8.10-7.99 (m, 2H), 7.51-7.46 (m, 2H), 7.44-7.34 (m, 3H), 6.05 (s, 2H), 4.94-4.79 (m, 4H), 1.53 (t, J=7.2 Hz, 3H).

[0660] LC/MS (System A): m/z (ESI.sup.+)=334 [M.sup.+], R.sub.t=0.86 min, UV purity=100%.

Intermediate 60—Synthesis of tert-butyl N-[(1-benzyl-1H-1,3-benzodiazol-2-yl)methyl]carbamate

[0661] ##STR00106##

[0662] Benzyl bromide (1.29 ml, 10.8 mmol) was added to a mixture of tert-butyl N-(1H-1,3-benzodiazol-2-ylmethyl)carbamate (85%, 3.15 g, 10.8 mmol) and K.sub.2CO.sub.3 (2.25 g, 16.3 mmol) in DMF (25 ml). The resulting mixture was stirred at RT for 16 h. The reaction mixture was partitioned between water (150 ml) and EtOAc (150 ml). The phases were separated then the aqueous phase was extracted with EtOAc (5×150 ml). The combined organics were dried over Na.sub.2SO.sub.4 then concentrated in vacuo to approximately 50 ml. The resultant slurry was recrystallized from the minimum volume of refluxing EtOAc to afford the product as white solid (2.22 g, 61%).

[0663] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.64-7.58 (m, 1H), 7.51-7.45 (m, 1H), 7.40-7.34 (m, 1H), 7.34-7.24 (m, 3H), 7.20-7.11 (m, 4H), 5.51 (s, 2H), 4.44 (d, J=5.8 Hz, 2H), 1.32 (s, 9H).

[0664] LC/MS (System A): m/z (ESI.sup.+)=338 [MH.sup.+], R.sub.t=1.02 min, UV purity=100%.

Intermediate 61—Synthesis of 1-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-ium bromide

[0665] ##STR00107##

[0666] A mixture of tert-butyl N-[(1-benzyl-1H-1,3-benzodiazol-2-yl)methyl]carbamate, Intermediate 60 (500 mg, 1.48 mmol) and methyl bromoacetate (421 μl, 4.45 mmol) in MeCN (5 ml) was stirred at 70° C. in a sealed tube for 16 h. The reaction mixture was allowed to cool then concentrated in vacuo to afford the product as a white solid (717 mg, 96%).

[0667] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.06 (d, J=8.3 Hz, 1H), 7.89 (t, J=5.4 Hz, 1H), 7.81 (d, J=8.2 Hz, 1H), 7.73-7.60 (m, 2H), 7.42-7.32 (m, 3H), 7.28 (d, J=6.8 Hz, 2H), 5.97 (s, 2H), 5.69 (s, 2H), 4.89 (d, J=5.4 Hz, 2H), 3.77 (s, 3H), 1.32 (s, 9H).

[0668] LC/MS (System A): m/z (ESI.sup.+)=410 [M.sup.+], R.sub.t=0.94 min, UV purity=97%.

Intermediate 62—Synthesis of 1-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-3-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-ium bromide

[0669] ##STR00108##

[0670] A mixture of 1-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-3-(2-methoxy-2-oxoethyl)-1H-1,3-benzodiazol-3-ium bromide, Intermediate 61 (350 mg, 0.71 mmol) in aqueous LiOH solution (1.0 M, 1.4 ml, 1.4 mmol) and MeOH (1.5 ml) was stirred at RT for 16 h. The reaction mixture was acidified to pH 3 by dropwise addition of aqueous HCl solution (1 M) then concentrated in vacuo to afford the crude intermediate as a beige solid (469 mg). The crude intermediate thus obtained was dissolved in DMF (4 ml) then HATU (340 mg, 0.89 mmol) and DIPEA (206 μl, 1.18 mmol) were added. Piperidine (88 μl, 0.89 mmol) was added to the reaction solution then the resulting mixture was stirred at RT for 45 min. Additional piperidine (150 μl, 1.52 mmol) and HATU (300 mg, 0.89 mmol) were added and the reaction was left to stir at RT for an additional 64 h. The reaction mixture was concentrated in vacuo then the crude material was purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 10-45%, 12 CV; 45%, 2 CV; 45-53%, 3 CV; 53-100%, 3 CV; 100%, 1 CV. The desired fractions were combined and evaporated to afford the product as a pale orange foam (209 mg, 53%).

[0671] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.98 (d, J=8.3 Hz, 1H), 7.83-7.76 (m, 2H), 7.70-7.58 (m, 2H), 7.43-7.32 (m, 3H), 7.25 (d, J=6.8 Hz, 2H), 5.97 (s, 2H), 5.76 (s, 2H), 4.78 (d, J=5.4 Hz, 2H), 3.58-3.51 (m, 2H), 3.47-3.42 (m, 2H), 1.76-1.62 (m, 4H), 1.51 (s (br), 2H), 1.32 (s, 9H).

[0672] LC/MS (System A): m/z (ESI.sup.+)=463 [M.sup.+], R.sub.t=1.00 min, UV purity=99%.

Intermediate 63 Synthesis of 2-(aminomethyl)-1-benzyl-3-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-ium hydrochloride bromide

[0673] ##STR00109##

[0674] A mixture of 1-benzyl-2-({[(tert-butoxy)carbonyl]amino}methyl)-3-[2-oxo-2-(piperidin-1-yl)ethyl]-1H-1,3-benzodiazol-3-ium bromide, Intermediate 62 (205 mg, 0.377 mmol) and

[0675] HCl solution in dioxane (4.0 M, 1.1 ml, 4.4 mmol) was stirred at RT for 0.5 h. The reaction mixture was concentrated in vacuo to afford the product as a viscous orange oil (219 mg, 92% yield corrected for 24 wt % residual dioxane observed in NMR).

[0676] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.15-8.09 (m, 1H), 7.91 (d, J=8.3 Hz, 1H), 7.74 (t, J=7.7 Hz, 1H), 7.68 (t, J=7.8 Hz, 1H), 7.44-7.33 (m, 3H), 7.32 (d, J=7.0 Hz, 2H), 6.05 (s, 2H), 5.97 (s, 2H), 4.74 (s, 2H), 3.63-3.58 (m, 2H), 3.48-3.45 (m, 2H), 1.79-1.71 (m, 2H), 1.71-1.63 (m, 2H), 1.57-1.46 (m, 2H).

[0677] LC/MS (System A): m/z (ESI.sup.+)=363 [M.sup.+], R.sub.t=0.82 min, UV purity=93%.

Intermediate 64 Synthesis of 6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-ium iodide

[0678] ##STR00110##

[0679] Intermediate 64 was synthesised by according to literature procedures (US 2015/0018313 A1).

Intermediate 65—Synthesis of 2-(aminomethyl)-6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0680] ##STR00111##

[0681] Morpholine (3.46 ml, 40.0 mmol) was added to a solution of 6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-ium iodide, Intermediate 64 (95%, 3.00 g, 4.00 mmol) in THF (50 ml). The reaction mixture was stirred at RT for 25 min then diluted with diethyl ether (150 ml). The resulting mixture was agitated then the supernatant was decanted off. The residual gum was washed further with ether (2×60 ml) then dried under vacuum. The residue was dissolved in THF then concentrated in vacuo afford the product as a pale orange solid (1.99 g, 83%).

[0682] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.95 (d, J=9.1 Hz, 1H), 7.65-7.61 (m, 1H), 7.25 (dd, J=9.1, 2.3 Hz, 1H), 7.11-7.05 (m, 1H), 4.61-4.53 (m, 4H), 4.25 (s, 2H), 4.11 (t, J=5.9 Hz, 2H), 3.36 (q, J=5.9 Hz, 2H), 1.45-1.41 (m, 6H), 1.39 (s, 9H).

[0683] LC/MS (System A): m/z (ESI.sup.+)=363 [M.sup.+], R.sub.t=0.76 min, UV purity=82%.

Intermediate 66—Synthesis tert-butyl N-[3-(3-fluoro-4-nitrophenoxy)propyl]carbamate

[0684] ##STR00112##

[0685] A suspension of 3-fluoro-4-nitrophenol (2.50 g, 15.9 mmol), tert-butyl (3-bromopropyl)carbamate (3.98 g, 16.7 mmol) and K.sub.2CO.sub.3 (2.64 g, 19.1 mmol) in acetone (15 ml) was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo then the residue was partitioned between EtOAc (50 ml) and water (50 ml). The phases were separated then the organic phase was extracted with water (2×50 ml) and brine (50 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the product as a viscous orange oil (4.65 g, 84%).

[0686] .sup.1H NMR (500 MHz, Acetone-d6) δ 8.14 (t, J=9.2 Hz, 1H), 7.16 (dd, J=13.7, 2.5 Hz, 1H), 6.96 (dd, J=9.3, 2.6 Hz, 1H), 6.92 (t, J=5.5 Hz, 1H), 4.13 (t, J=6.2 Hz, 2H), 3.07 (q, J=6.6 Hz, 2H), 1.84 (p, J=6.5 Hz, 2H), 1.37 (s, 9H).

[0687] LC/MS (System A): R.sub.t=1.22 min, UV purity=90%.

Intermediate 67—Synthesis of tert-butyl N-{3-[3-(ethylamino)-4-nitrophenoxy]propyl}carbamate

[0688] ##STR00113##

[0689] Ethylamine solution in THF (2.0 M, 10 ml, 20 mmol) was added to a mixture of tert-butyl N-[3-(3-fluoro-4-nitrophenoxy)propyl]carbamate, Intermediate 66 (90%, 4.65 g, 13.3 mmol) and K.sub.2CO.sub.3 (2.20 g, 16.0 mmol) in THF (30 ml). The reaction mixture was stirred at RT for 16 h then additional ethylamine solution in THF (2.0 M, 3.0 ml, 6.0 mmol) was added. The reaction mixture was left to stir at RT for a further 70 h then filtered. The filter pad was rinsed with EtOAc then the combined filtrate was extracted with water (2×150 ml) and brine (150 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the product as a viscous yellow/orange oil (4.69 g, 93%).

[0690] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.26 (t, J=5.0 Hz, 1H), 8.02 (d, J=10.0 Hz, 1H), 6.92-6.86 (m, 1H), 6.30-6.27 (m, 2H), 4.09 (t, J=6.3 Hz, 2H), 3.40-3.35 (m, 2H), 3.08 (q, J=6.7 Hz, 2H), 1.84 (p, J=6.5 Hz, 2H), 1.37 (s, 9H), 1.24 (t, J=7.1 Hz, 3H).

[0691] LC/MS (System A): m/z (ESI.sup.+)=340 [MH.sup.+], R.sub.t=1.30 min, UV purity=90%.

Intermediate 68—Synthesis of (9H-fluoren-9-yl)methyl N-{[6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1-ethyl-1H-1,3-benzodiazol-2-yl]methyl}carbamate

[0692] ##STR00114##

[0693] Palladium on carbon (10 wt %, 662 mg) was added to a solution of tert-butyl N-{3-[3-(ethylamino)-4-nitrophenoxy]propyl}carbamate, Intermediate 67 (90%, 4.69 g, 12.4 mmol) in EtOH (60 ml). The resulting mixture was stirred under a hydrogen atmosphere for 20 h. The reaction mixture was filtered through a Celite pad then the filtrate was concentrated in vacuo. The residue was dissolved in DMF (10 ml) to give a solution of the phenylenediamine intermediate. A solution of FMOC-glycine (3.88 g, 13.1 mmol), HATU (5.20 g, 13.7 mmol) and DIPEA (4.3 ml, 25 mmol) in DMF (20 ml) was stirred at RT for 0.5 h. The phenylenediamine DMF solution was then added and the resulting solution was stirred at RT for 1 h. Additional FMOC-glycine (2.00 g, 6.73 mmol and HATU (2.50 g, 6.57 mmol) were added then the reaction was left to stir at RT for a further 45 min. The reaction mixture was partitioned between EtOAc (100 ml) and water (100 ml). The phases were separated then the organic phase was washed with water (2×100 ml) and brine (100 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford a red solid. The solid thus obtained was dissolved in AcOH (20 ml) then heated at 60° C. for 16 h. The reaction was allowed to cool to RT then concentrated in vacuo. The residue thus obtained was treated with saturated aqueous NaHCO.sub.3 solution until pH 9 then partitioned between EtOAc (250 ml) and water (250 ml). The phases were separated then the organic phase was dried over Na.sub.2SO.sub.4 and concentrated in vacuo.

[0694] The crude material was purified by flash column chromatography on a silica column (340 g). The column was eluted with CH.sub.2Cl.sub.2:MeOH, using the following gradient (% MeOH, column volumes): 0%, 1 CV; 0-3.3%, 7 CV; 3.3%, 1 CV; 3.3-4.5%, 2 CV. The desired fractions were combined and evaporated to afford the product as a pale orange solid (4.73 g, 53%).

[0695] LC/MS (System A): m/z (ESI.sup.+)=571 [MH.sup.+], R.sub.t=1.18 min, UV purity=80%.

Intermediate 69—Synthesis of 6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium iodide

[0696] ##STR00115##

[0697] A mixture of 9H-fluoren-9-ylmethyl N-{[6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1-ethyl-1H-1,3-benzodiazol-2-yl]methyl}carbamate, Intermediate 68 (80%, 1.50 g, 2.10 mmol) and iodoethane (1.69 ml, 21.0 mmol) in THF (15 ml) was heated under microwave irradiation for 1.5 h at 120° C. The reaction mixture was concentrated in vacuo then the crude material was purified by flash column chromatography on a silica column (100 g).

[0698] The column was eluted with CH.sub.2Cl.sub.2:MeOH, using the following gradient (% MeOH, column volumes): 0%, 1 CV; 0-3.4%, 7 CV; 3.4-4.3%, 2 CV, 4.3-6.0%, 2 CV. The desired fractions were combined and evaporated to afford the product as a pale orange foam (1.03 g, 61%).

[0699] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.24 (t, J=5.1 Hz, 1H), 7.95 (d, J=9.1 Hz, 1H), 7.86 (d, J=7.5 Hz, 2H), 7.62 (d, J=7.5 Hz, 2H), 7.56 (s, 1H), 7.36 (t, J=7.4 Hz, 2H), 7.30-7.22 (m, 3H), 6.93-6.88 (m, 1H), 4.74 (d, J=5.1 Hz, 2H), 4.55-4.47 (m, 6H), 4.22 (t, J=5.8 Hz, 1H), 4.13 (t, J=6.0 Hz, 2H), 3.12 (q, J=6.4 Hz, 2H), 1.94-1.85 (m, 2H), 1.37-1.31 (m, 15H).

[0700] LC/MS (System A): m/z (ESI.sup.+)=599 [M.sup.+], R.sub.t=1.15 min, UV purity=90%.

Intermediate 70—Synthesis of 2-(aminomethyl)-6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0701] ##STR00116##

[0702] Morpholine (1.10 ml, 12.7 mmol) was added to a solution of 6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-ium iodide, Intermediate 69 (90%, 1.03 g, 1.27 mmol) in THF (10 ml). The reaction mixture was stirred at RT for 2 h then diluted with diethyl ether (40 ml). The mixture was agitated then the supernatant was decanted off. The procedure was repeated with further diethyl ether washes (2×20 ml). The residue thus obtained was dried under vacuum to afford the product as a pale orange foam (700 mg, 99%).

[0703] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.94 (d, J=9.1 Hz, 1H), 7.56 (d, J=2.1 Hz, 1H), 7.25 (dd, J=9.1, 2.2 Hz, 1H), 6.99-6.83 (m, 1H), 4.56 (q, J=7.2 Hz, 4H), 4.24 (s, 2H), 4.12 (t, J=6.2 Hz, 2H), 3.12 (q, J=6.6 Hz, 2H), 1.94-1.84 (m, 2H), 1.42 (t, J=7.2 Hz, 6H), 1.37 (s, 9H).

[0704] LC/MS (System A): m/z (ESI.sup.+)=377 [M.sup.+], R.sub.t=0.82 min, UV purity=91%.

Intermediate 71—Synthesis of 2-[(6-bromo-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione

[0705] ##STR00117##

[0706] A mixture of N-phthaloylglycine (13.1 g, 63.8 mmol), TBTU (21.5 g, 67.0 mmol) and triethylamine (14.1 ml, 79.1 mmol) in DMF (150 ml) was stirred at RT for 45 min. A solution of 5-bromo-1-N-ethylbenzene-1,2-diamine (13.1 g, 60.9 mmol) in THF (50 ml) was added and the resulting mixture was stirred at RT for 18 h. The reaction mixture was added onto saturated aqueous NaHCO3 solution (400 ml). The resulting precipitate was collected by filtration then washed with water and dried under vacuum to afford the intermediate as a light grey solid. The solid thus obtained was added portionwise to acetic acid (150 ml). The resulting suspension was heated at 100° C. for 2.5 h then allowed to cool to RT. The reaction mixture was concentrated in vacuo then the residue was partitioned between EtOAc (300 ml) and water (300 ml). The resulting precipitate was collected by filtration and washed with EtOAc (200 ml) and water (200 ml) then dried under vacuum to afford the product as a pink solid (17.9 g, 76%).

[0707] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.96 (dd, J=5.6, 3.0 Hz, 2H), 7.93-7.88 (m, 3H), 7.44 (d, J=8.5 Hz, 1H), 7.27 (dd, J=8.5, 1.9 Hz, 1H), 5.12 (s, 2H), 4.39 (q, J=7.2 Hz, 2H), 1.37 (t, J=7.2 Hz, 3H).

[0708] LC/MS (System A): m/z (ESI.sup.+)=384 [M(.sup.79Br)H.sub.1], 386 [M(.sup.81Br)H.sub.1], R.sub.t=1.12 min, UV purity=100%.

Intermediate 72—Synthesis of tert-butyl N-(3-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}prop-2-yn-1-yl)carbamate

[0709] ##STR00118##

[0710] CuI (0.25 g, 1.29 mmol) was added to a solution of 2-[(6-bromo-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 71 (5.00 g, 13.0 mmol) and tert-butyl N-(prop-2-yn-1-yl)carbamate (2.40 g, 15.5 mmol) in DMF (60 ml). Nitrogen was bubbled through the reaction mixture for 5 min then Pd(PPh.sub.3).sub.4 (0.74 g, 0.64 mmol) was added, followed by triethylamine (2.92 ml, 19.3 mmol). The reaction mixture was heated at 65° C. for 24 h then concentrated in vacuo. The crude material was purified by flash column chromatography on a silica column (100 g). The column was eluted with EtOAc:heptane, increasing the gradient linearly from 0:100 to 50:50 over 10 column volumes. The desired fractions were combined and evaporated to afford the product as a yellow foam (2.45 g, 39%).

[0711] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.99-7.88 (m, 4H), 7.71 (s, 1H), 7.46 (d, J=8.3 Hz, 1H), 7.35 (s, 1H), 7.16 (dd, J=8.3, 1.3 Hz, 1H), 5.13 (s, 2H), 4.40 (q, J=7.1 Hz, 2H), 4.02-3.97 (m, 2H), 1.41 (s, 9H), 1.36 (t, J=7.1 Hz, 3H).

[0712] LC/MS (System A): m/z (ESI.sup.+)=459 [MH.sup.+], R.sub.t=1.17 min, UV purity=95%. Intermediate 73—Synthesis of tert-butyl N-(3-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}propyl)carbamate

##STR00119##

[0713] Palladium on carbon (10 wt %, 557 mg) was added to a solution of tert-butyl N-(3-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}prop-2-yn-1-yl)carbamate, Intermediate 72 (2.4 g, 5.23 mmol) in EtOH (120 ml). The reaction mixture was stirred at RT under a hydrogen atmosphere for 48 h. The reaction was recharged with palladium on carbon (10 wt %, 278 mg) and stirred at RT under a hydrogen atmosphere for a further 24 h. The reaction was re-charged with palladium on carbon (10 wt %, 278 mg) and stirred at RT under a hydrogen atmosphere for a further 24 h. The reaction mixture was filtered through a Celite pad. The Celite pad was rinsed with EtOH (100 ml), MeOH (100 ml), EtOAc (100 ml), and DMF (5 ml). The combined filtrate was concentrated in vacuo then the crude material was purified by flash column chromatography on a silica column (25 g). The column was eluted with EtOAc:heptane, increasing the gradient linearly from 0:100 to 75:25 over 10 column volumes. The desired fractions were combined and evaporated to afford the product as a light yellow solid (1.20 g, 43%).

[0714] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.98-7.93 (m, 2H), 7.92-7.88 (m, 2H), 7.37-7.34 (m, 2H), 6.97 (dd, J=8.2, 1.5 Hz, 1H), 6.84 (t, J=5.3 Hz, 1H), 5.09 (s, 2H), 4.35 (q, J=7.2 Hz, 2H), 2.96-2.90 (m, 2H), 2.69-2.63 (m, 2H), 1.71 (p, J=7.3 Hz, 2H), 1.40-1.35 (m, 12H).

[0715] LC/MS (System A): m/z (ESI.sup.+)=463 [MH.sup.+], R.sub.t=1.07 min, UV purity=86%.

Intermediate 74 Synthesis of 6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0716] ##STR00120##

Iodoethane (1.04 ml, 13.0 mmol) was added to a solution of tert-butyl N-(3-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}propyl)carbamate, Intermediate 73 (86%, 1.20 g, 2.23 mmol) in MeCN (18 ml) in a pressure tube. The tube was sealed and heated at 110° C. for 4 h. The reaction was allowed to cool to RT then iodoethane (1.04 ml, 13.0 mmol) was added then the reaction was heated at 110° C. for a further 4 h. The reaction mixture was allowed to cool to RT then concentrated in vacuo to afford the product as a brown solid (1.52 g, >99%).

[0717] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.02 (d, J=8.6 Hz, 1H), 7.97-7.93 (m, 3H), 7.92-7.88 (m, 2H), 7.58 (d, J=8.6 Hz, 1H), 6.89 (s, 1H), 5.42 (s, 2H), 4.70-4.66 (m, 4H), 2.93 (q, J=6.2 Hz, 2H), 2.79 (t, J=7.4 Hz, 2H), 1.81-1.72 (m, 2H), 1.44-1.40 (m, 6H), 1.37 (s, 9H).

[0718] LC/MS (System A): m/z (ESI.sup.+)=491 [M.sup.+], R.sub.t=1.08 min, UV purity=91%.

Intermediate 75 Synthesis of 2-(aminomethyl)-6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0719] ##STR00121##

[0720] Hydrazine hydrate (609 μl, 12.5 mmol) was added to a solution of 6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide, Intermediate 74 (91%, 1.52 g, 2.23 mmol) in MeOH (20 ml) in a pressure tube. The tube was sealed then heated at 75° C. for 3 h. The reaction mixture was allowed to cool to RT then concentrated in vacuo. The residue was suspended in CH.sub.2Cl.sub.2:MeOH (9:1, 20 ml) then filtered. The filtrate was concentrated in vacuo to afford the product as a yellow foam (1.21 g, 89%).

[0721] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.95 (d, J=8.5 Hz, 1H), 7.88 (s, 1H), 7.51 (dd, J=8.5, 1.3 Hz, 1H), 6.91 (t, J=5.5 Hz, 1H), 4.60-4.52 (m, 4H), 4.26 (s, 2H), 2.93 (app. q, J=6.6 Hz, 2H), 2.78 (t, J=7.5 Hz, 2H), 1.80-1.70 (m, 2H), 1.46-1.40 (m, 6H), 1.37 (s, 9H).

[0722] LC/MS (System A): m/z (ESI.sup.+)=361 [M.sup.+], R.sub.t=0.81 min, UV purity=80%.

Intermediate 76 Synthesis of tert-butyl 4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}-1,2,3,6-tetrahydropyridine-1-carboxylate

[0723] ##STR00122##

[0724] tert-Butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (966 mg, 3.12 mmol) was added to a solution of 2-[(6-bromo-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 71 (1.00 g, 2.60 mmol) in dioxane (10 ml) and water (2 ml). K.sub.2CO.sub.3 (1.08 g, 7.81 mmol) was added then nitrogen was bubbled through the resulting suspension for 10 min. Pd(dppf).sub.2Cl.sub.2 (190 mg, 0.26 mmol) was added then the reaction mixture was heated under microwave irradiation for 2 h at 85° C. The reaction mixture was partitioned between EtOAc (100 ml) and water (50 ml). The phases were separated then the organic phase was washed with brine (50 ml) then dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude material was purified by flash column chromatography on a silica column (50 g). The column was eluted with EtOAc:heptane, using the following gradient (% EtOAc, column volumes): 0%, 1 CV; 0-50%, 7 CV; 50-79%, 3 CV; 79%, 2 CV; 79-92%, 2 CV, 100%, 2 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a white solid (670 mg, 50%).

[0725] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.99-7.87 (m, 4H), 7.60 (s, 1H), 7.42 (d, J=8.5 Hz, 1H), 7.25 (dd, J=8.5, 1.5 Hz, 1H), 6.16 (s, 1H), 5.11 (s, 2H), 4.49-4.31 (m, 2H), 4.05-3.97 (m, 2H), 3.56 (t, J=5.6 Hz, 2H), 2.54 (s, 2H), 1.43 (s, 9H), 1.37 (t, J=7.1 Hz, 3H).

[0726] LC/MS (System A): m/z (ESI.sup.+)=487 [MH.sup.+], R.sub.t=1.18 min, UV purity=95%.

Intermediate 77 Synthesis of tert-butyl 4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylate

[0727] ##STR00123##

[0728] Palladium on carbon (10 wt %, 142 mg) was added to a solution of tert-butyl 4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}-1,2,3,6-tetrahydropyridine-1-carboxylate, Intermediate 76 (95%, 660 mg, 1.29 mmol) in EtOH (40 ml). The reaction mixture was stirred at RT under a hydrogen atmosphere for 16 h. The reaction was recharged with palladium on carbon (10 wt %, 140 mg) and stirred at RT under a hydrogen atmosphere for a further 48 h. The reaction mixture was filtered through Celite pad then concentrated in vacuo to afford the product as a colourless oil (635 mg, 94%).

[0729] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.96 (dd, J=5.6, 2.9 Hz, 2H), 7.91 (dd, J=5.6, 3.0 Hz, 2H), 7.45 (s, 1H), 7.38 (d, J=8.3 Hz, 1H), 7.02 (dd, J=8.4, 1.4 Hz, 1H), 5.09 (s, 2H), 4.37 (q, J=7.2 Hz, 2H), 4.33 (t, J=5.1 Hz, 1H), 4.10 (d, J=10.4 Hz, 2H), 2.84-2.75 (m, 2H), 1.78 (d, J=12.8 Hz, 2H), 1.58 (qd, J=12.7, 4.3 Hz, 2H), 1.42 (s, 9H), 1.37 (t, J=7.2 Hz, 3H).

[0730] LC/MS (System A): m/z (ESI.sup.+)=489 [MH.sup.+], R.sub.t=1.13 min, UV purity=93%.

Intermediate 78 Synthesis of 6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0731] ##STR00124##

Iodoethane (486 μl, 6.04 mmol) was added to a solution of tert-butyl 4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylate, Intermediate 77 (93%, 635 mg, 1.21 mmol) in MeCN (10 ml). The reaction mixture was heated under microwave irradiation for 3 h at 120° C. The reaction mixture was concentrated in vacuo to afford the product as a brown solid (765 mg, 84%).

[0732] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 8.07-8.01 (m, 2H), 7.98-7.88 (m, 4H), 7.64 (d, J=9.3 Hz, 1H), 5.43 (s, 2H), 4.69 (q, J=7.0 Hz, 4H), 4.20-4.04 (m, 2H), 3.05-2.71 (m, 3H), 1.85-1.75 (m, 2H), 1.67 (td, J=12.3, 3.5 Hz, 2H), 1.46-1.37 (m, 15H).

[0733] LC/MS (System A): m/z (ESI.sup.+)=517 [M.sup.+], R.sub.t=1.06 min, UV purity=86%.

Intermediate 79 Synthesis of 2-(aminomethyl)-6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0734] ##STR00125##

[0735] Hydrazine hydrate (284 μl, 5.82 mmol) was added to a solution of 6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide, Intermediate 78 (86%, 750 mg, 1.00 mmol) in MeOH (10 ml) in a pressure tube. The tube was sealed then the reaction mixture was heated at 75° C. for 5 h. The reaction mixture was allowed to cool to RT then concentrated in vacuo. The residue was suspended in CH.sub.2Cl.sub.2:MeOH (9:1, 20 ml) then filtered. The filtrate was concentrated in vacuo to afford the product as a yellow foam (605 mg, 85%).

[0736] .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 7.96 (d, J=8.7 Hz, 1H), 7.95 (s, 1H), 7.58 (d, J=8.7 Hz, 1H), 4.65-4.51 (m, 4H), 4.27 (s, 2H), 4.13 (d, J=13.3 Hz, 2H), 3.02-2.77 (m, 3H), 1.89-1.76 (m, 2H), 1.72-1.58 (m, 2H), 1.47-1.39 (m, 15H).

[0737] LC/MS (System A): m/z (ESI.sup.+)=387 [M.sup.+], R.sub.t=0.89 min, UV purity=72%.

Intermediate 80 Synthesis of tert-butyl 4-(4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}-1H-pyrazol-1-yl)piperidine-1-carboxylate

[0738] ##STR00126##

[0739] tert-Butyl 4-[4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (540 mg, 1.43 mmol) was added to a solution of 2-[(6-bromo-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione, Intermediate 71 (500 mg, 1.30 mmol) in dioxane (10 ml) and water (0.5 ml) in a pressure tube. Cs.sub.2CO.sub.3 (848 mg, 2.60 mmol) was added then the resulting suspension was de-gassed by bubbling a stream of nitrogen through the reaction mixture for 10 min. XPhos-Pd-G2 (61 mg, 0.078 mmol) was added then the nitrogen bubbling was continued for a further 5 min. The tube was sealed then heated at 100° C. for 16 h. The reaction mixture was concentrated in vacuo then partitioned between EtOAc (100 ml) and water (100 ml). The phases were separated then the organic phase was washed with water (50 ml) and brine (2×50 ml), then dried over MgSO.sub.4, filtered and evaporated. The crude material was purified by flash column chromatography on a silica column (50 g). The column was eluted with EtOAc:heptane, increasing the gradient linearly from 0:100 to 100:0 over 10 column volumes. The desired fractions were combined and evaporated to yield the product as a light yellow foam (226 mg, 31%).

[0740] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.28 (s, 1H), 7.98-7.94 (m, 2H), 7.94-7.89 (m, 3H), 7.81-7.76 (m, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.38 (dd, J=8.4, 1.5 Hz, 1H), 5.11 (s, 2H), 4.43-4.34 (m, 3H), 4.04 (dd, J=15.8, 8.8 Hz, 2H), 2.93 (s, 2H), 2.09-2.02 (m, 2H), 1.81 (qd, J=12.4, 4.3 Hz, 2H), 1.43 (s, 9H), 1.42-1.39 (m, 3H).

[0741] LC/MS (System A): m/z (ESI.sup.+)=555 [MH.sup.+], R.sub.t=1.13 min, UV purity=100%.

Intermediate 81—Synthesis of 6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0742] ##STR00127##

[0743] Iodoethane (159 μl, 1.98 mmol) was added to a solution of tert-butyl 4-(4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}-1H-pyrazol-1-yl)piperidine-1-carboxylate, Intermediate 80 (220 mg, 0.397 mmol) in MeCN (5 ml). The reaction mixture was heated under microwave irradiation for 2 h at 120° C. Iodoethane (130 μl, 1.62 mmol) was added then the reaction mixture was heated under microwave irradiation for 1 h at 120° C. The reaction mixture was concentrated in vacuo then azeotroped with diethyl ether. The residue was dried under vacuum to afford the product as a light yellow solid (280 mg, 80%).

[0744] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.50 (s, 1H), 8.29 (s, 1H), 8.12 (s, 1H), 8.10 (d, J=8.8 Hz, 1H), 7.98-7.94 (m, 3H), 7.94-7.89 (m, 2H), 5.42 (s, 2H), 4.74-4.65 (m, 4H), 4.48-4.35 (m, 1H), 4.06 (d, 2H), 2.95 (br. s, 2H), 2.08 (d, J=6.7 Hz, 2H), 1.81 (qd, J=12.7, 4.7 Hz, 2H), 1.49-1.40 (m, 15H).

[0745] LC/MS (System A): m/z (ESI.sup.+)=583 [M.sup.+], R.sub.t=1.13 min, UV purity=81%.

Intermediate 82—Synthesis of 2-(aminomethyl)-6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0746] ##STR00128##

[0747] Hydrazine hydrate (96 μl, 2.0 mmol) was added to a solution of 6-(1-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1H-pyrazol-4-yl)-2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide, Intermediate 81 (81%, 280 mg, 0.32 mmol) in MeOH (5 ml) in a pressure tube. The tube was sealed then heated at 75° C. for 1 h. The reaction mixture was allowed to cool to RT then concentrated in vacuo. The residue was suspended in CH.sub.2Cl.sub.2 (9:1, 20 ml) then filtered. The solid thus obtained was washed further CH.sub.2Cl.sub.2 (20 ml). The filtrates were combined and evaporated to dryness to afford the product as a light yellow solid (224 mg, >99%).

[0748] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.48 (s, 1H), 8.23 (s, 1H), 8.10 (s, 1H), 8.03 (d, J=8.7 Hz, 1H), 7.90 (dd, J=8.7, 1.3 Hz, 1H), 4.62-4.56 (m, 4H), 4.44-4.36 (m, 1H), 4.28 (s, 2H), 4.07 (d, J=10.4 Hz, 2H), 2.94 (br. s, 2H), 2.08 (d, J=10.4 Hz, 2H), 1.82 (tt, J=12.2, 6.2 Hz, 2H), 1.46-1.40 (m, 15H).

[0749] LC/MS (System A): m/z (ESI.sup.+)=453 [M.sup.+], R.sub.t=0.91 min, UV purity=83%.

Intermediate 83—Synthesis of 5-(2-aminoethoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium hydrochloride chloride

[0750] ##STR00129##

[0751] HCl solution in dioxane (4.0 M, 14 ml, 56 mmol) was added to a mixture of 6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-ium iodide, Intermediate 64 (4.28 g, 6.01 mmol) in MeCN (50 ml). The resulting mixture was stirred at RT for 20 min then concentrated in vacuo to afford the product as a brown/orange foam (3.87 g, 98%).

[0752] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.30 (t, J=5.3 Hz, 1H), 8.15 (s (br), 3H), 7.99 (d, J=9.1 Hz, 1H), 7.86 (d, J=7.5 Hz, 2H), 7.69-7.60 (m, 3H), 7.38-7.30 (m, 3H), 7.25 (t, J=7.3 Hz, 2H), 4.75 (d, J=5.2 Hz, 2H), 4.58-4.45 (m, 6H), 4.34 (t, J=4.9 Hz, 2H), 4.22 (t, J=6.0 Hz, 1H), 1.37-1.30 (m, 6H).

[0753] LC/MS (System A): m/z (ESI.sup.+)=485 [M.sup.+], R.sub.t=0.84 min, UV purity=85%.

Intermediate 84 Synthesis of 5-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium chloride

[0754] ##STR00130##

[0755] A mixture of 4,6-O-benzylidene-D-glucopyranose (95%, 6.67 g, 23.6 mmol), 5-(2-aminoethoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium hydrochloride chloride, Intermediate 83 (85%, 3.87 g, 5.91 mmol) and AcOH (1.35 ml, 23.6 mmol) in MeOH (100 ml) was stirred at RT for 0.5 h. NaCNBH.sub.3 (1.48 g, 23.6 mmol) was added then the resulting mixture was stirred at RT for 20 h. More MeOH (40 ml) was added then the reaction was left to stir at RT for a further 24 h. More MeOH (80 ml) was added, then the reaction was retreated with 4,6-O-benzylidene-D-glucopyranose (95%, 1.60 g, 5.67 mmol), AcOH (0.34 ml, 5.94 mmol) and NaCNBH.sub.3 (0.38 g, 6.05 mmol). The reaction was left to stir at RT for a further 92 h then added to saturated aqueous NaHCO.sub.3 solution (250 ml). The resultant suspension was stirred at RT for 20 min. The solid was collected by filtration then washed with water and dried under vacuum to afford the product as a pale pink solid (6.43 g, 89%).

[0756] LC/MS (System A): m/z (ESI.sup.+)=990 [M.sup.+], 496 [(M.sup.+)+H.sup.+], R.sub.t=0.93 min, UV purity=84%.

Intermediate 85—2-(aminomethyl)-5-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride

[0757] ##STR00131##

[0758] Morpholine (4.77 ml, 55.1 mmol) was added to a stirred mixture of 5-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium chloride, Intermediate 84 (84% 6.42 g, 5.26 mmol) in THF (60 ml). The resulting mixture was stirred at RT for 4 h. The reaction mixture was diluted with diethyl ether (150 ml). The resulting suspension was agitated then the suspension was decanted off, leaving behind a viscous oil. More diethyl ether (80 ml) was added to the oil residue then the mixture was sonicated. The resulting suspension was again decanted off to leave behind a viscous oil. The process was repeated once more with diethyl ether (80 ml) then the resulting viscous oil was dried under vacuum to afford the product as a pale purple foam (4.39 g, 85%).

[0759] LC/MS (System A): m/z (ESI.sup.+)=767 [M.sup.+], 384 [(M.sup.+)+H.sup.+], R.sub.t=0.75 min, UV purity=82%.

Intermediate 86 Synthesis of 2-(aminomethyl)-5-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium dihydrochloride chloride

[0760] ##STR00132##

[0761] A mixture of 2-(aminomethyl)-5-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium chloride, Intermediate 85 (82%, 1.50 g, 1.53 mmol) and aqueous HCl solution (2.0 M, 25 ml, 50 mmol) was stirred at RT for 1 h. The reaction mixture was concentrated in vacuo then the residue was dissolved in water (10 ml) and lyophilised to afford the product as a pale purple foam (1.53 g, >99%).

[0762] LC/MS (System A): m/z (ESI.sup.+)=591 [M.sup.+], 296 [(M.sup.+)+H.sup.+], R.sub.t=0.13 min, UV purity=70%.

Intermediate 87 Synthesis of 5-(3-aminopropoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium hydrochloride chloride

[0763] ##STR00133##

[0764] HCl solution in dioxane (4.0 M, 3.3 ml, 13 mmol) was added to a mixture of 6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-ium iodide, Intermediate 69 (95%, 1.00 g, 1.31 mmol) in MeCN (15 ml). The reaction mixture was stirred at RT for 0.5 h then concentrated in vacuo to afford the product as a viscous yellow oil (875 mg, >99% —yield corrected for 15 wt % residual dioxane observed in NMR).

[0765] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.30 (t, J=5.3 Hz, 1H), 8.00-7.85 (m, 6H), 7.65-7.59 (m, 3H), 7.37 (t, J=7.4 Hz, 2H), 7.31 (dd, J=9.1, 2.1 Hz, 1H), 7.27 (t, J=7.4 Hz, 2H), 4.76 (d, J=5.4 Hz, 2H), 4.58-4.46 (m, 6H), 4.27-4.19 (m, 3H), 3.05-2.95 (m, 2H), 2.12-2.06 (m, 2H), 1.34 (t, J=7.1 Hz, 6H).

[0766] LC/MS (System A): m/z (ESI.sup.+)=499 [M.sup.+], R.sub.t=0.89 min, UV purity=98%.

Intermediate 88 Synthesis of 5-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium hydrochloride chloride

[0767] ##STR00134##

[0768] A mixture of 4,6-O-benzylidene-D-glucopyranose (1.43 g, 5.32 mmol), 5-(3-aminopropoxy)-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium hydrochloride chloride, Intermediate 87 (85%, 875 mg, 1.30 mmol) and

[0769] AcOH (305 μl, 5.32 mmol) in MeOH (25 ml) was stirred at RT for 0.5 h. NaCNBH.sub.3 (334 mg, 5.32 mmol) was added then the resulting mixture was stirred at RT for 64 h. Additional 4,6-O-benzylidene-D-glucopyranose (500 mg, 1.86 mmol) and AcOH (110 μl, 1.92 mmol) was added. The mixture was stirred for 0.5 h then NaCNBH.sub.3 (115 mg, 1.83 mmol) was added. The resulting mixture was stirred at RT for a further 16 h. Saturated aqueous NaHCO.sub.3 solution (40 ml) was added over 5 min whereupon a white precipitate formed. The resultant suspension was filtered and the collected solid was washed with water then dried under vacuum to afford the product as a white solid (1.39 g, 60%).

[0770] LC/MS (System A): m/z (ESI.sup.+)=503 [(M.sup.+)+H.sup.+], R.sub.t=1.00 min, UV purity=60%. Intermediate 89 Synthesis of 2-(aminomethyl)-5-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium hydrochloride chloride

##STR00135##

[0771] Morpholine (659 μl, 7.62 mmol) was added to a mixture of 5-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propoxy)-1,3-diethyl-2-({[(9H-fluoren-9-ylmethoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-ium chloride hydrochloride, Intermediate 88 (60%, 1.32 g, 0.762 mmol) in THF (10 ml). The mixture was stirred at RT for 2.5 h. The reaction mixture was diluted with diethyl ether (20 ml). The resulting suspension was sonicated then the suspension was decanted off, leaving behind a viscous oil. More diethyl ether (20 ml) was added to the oil residue then the mixture was sonicated. The resulting suspension was again decanted off to leave behind a viscous oil. The process was repeated once more with diethyl ether (20 ml) then the resulting viscous oil was dried under vacuum to afford the product as a pale orange solid (639 mg, 79%).

[0772] LC/MS (System A): m/z (ESI.sup.+)=781 [M.sup.+], 391 [(M.sup.+)+H.sup.+], R.sub.t=0.78 min, UV purity=80%.

Intermediate 90 Synthesis of 2-(aminomethyl)-5-(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium hydrochloride chloride

[0773] ##STR00136##

[0774] A mixture of 2-(aminomethyl)-5-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propoxy)-1,3-diethyl-1H-1,3-benzodiazol-3-ium hydrochloride chloride, Intermediate 89 (80%, 375 mg, 0.367 mmol) and aqueous HCl solution (2.0 M, 8.0 ml, 16 mmol) was stirred at RT for 40 min. The reaction mixture was concentrated in vacuo then diluted with water and lyophilised to afford the product as a pale orange solid (305 mg, 98%).

[0775] LC/MS (System A): m/z (ESI.sup.+)=605 [M.sup.+], 303 [(M.sup.+)+H.sup.+], R.sub.t=0.13 min, UV purity=80%.

Intermediate 91—Synthesis of tert-butyl 4-{1-ethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylate

[0776] ##STR00137##

[0777] Step 1: A suspension of hydrazine hydrate (1.27 ml, 1.31 mmol) and tert-butyl 4-{2-[(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)methyl]-1-ethyl-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylate Intermediate 77 (2.56 g, 5.24 mmol) in MeOH (30 ml) was heated under reflux for 2.5 h then allowed to cool to RT. The resultant suspension was left to stand at RT for 16 h then filtered. The collected solid was washed solid with CH.sub.2Cl.sub.2 then the filtrate was concentrated in vacuo and azeotroped with MeCN to afford the intermediate as a white solid (1.24 g). Step 2: A solution of (2,5-dioxopyrrolidin-1-yl) 9H-fluoren-9-ylmethyl carbonate (1.16 g, 3.43 mmol) in MeCN (20 ml) was added dropwise over 10 min to a mixture of the intermediate from Step 1 and NaHCO.sub.3 (576 mg, 6.86 mmol) in MeCN (40 ml) and water (30 ml). The reaction mixture was stirred at RT for 21 h then partitioned between EtOAc (100 ml) and water (100 ml). The phases were separated then the organic phase was washed with water (2×50 ml), brine (50 ml), then dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The solid thus obtained was suspended in MeCN (150 ml) under reflux, with sonication at various intervals. The resultant suspension was allowed to cool to RT then filtered. The collected solid was dried under vacuum to afford the product as a white solid (1.52 g, 50% over 2 steps).

[0778] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.99 (s, 1H), 7.88 (d, J=7.5 Hz, 2H), 7.72 (d, J=7.4 Hz, 2H), 7.48 (d, J=8.2 Hz, 1H), 7.44-7.37 (m, 3H), 7.31 (t, J=7.4 Hz, 2H), 7.07 (d, J=8.3 Hz, 1H), 4.48 (d, J=5.7 Hz, 2H), 4.32 (d, J=7.1 Hz, 2H), 4.23 (t, J=6.9 Hz, 3H), 4.15-4.05 (m, 2H), 2.95-2.75 (m, 3H), 1.80 (d, J=12.0 Hz, 2H), 1.65-1.52 (m, 2H), 1.42 (s, 9H), 1.26 (t, J=7.1 Hz, 3H).

[0779] LC/MS (System A): m/z (ESI.sup.+)=581 [MH.sup.+], R.sub.t=1.12 min, UV purity=100%.

Intermediate 92—Synthesis of 6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium iodide

[0780] ##STR00138##

[0781] A suspension of tert-butyl 4-{1-ethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-6-yl}piperidine-1-carboxylate, Intermediate 91 (1.55 g, 2.67 mmol) and iodoethane (1.07 ml, 13.4 mmol) in MeCN (20 ml) was heated under microwave irradiation for 2.5 h at 120° C. The reaction mixture was concentrated in vacuo then the crude material was purified by flash column chromatography on a silica column (25 g). The column was eluted with CH.sub.2Cl.sub.2:MeOH, using the following gradient (% MeOH, column volumes): 0%, 1 CV; 0-2.3%, 5 CV; 2.3%, 2 CV; 2.3-10%, 7 CV, 10%, 7 CV. The desired fractions were combined and evaporated to afford the product as a light pink foam (1.60 g, 75%).

[0782] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.26 (t, J=5.2 Hz, 1H), 7.98 (d, J=9.1 Hz, 2H), 7.86 (d, J=7.5 Hz, 2H), 7.62 (d, J=7.7 Hz, 3H), 7.35 (t, J=7.4 Hz, 2H), 7.25 (t, J=7.3 Hz, 2H), 4.76 (d, J=5.2 Hz, 2H), 4.59-4.44 (m, 6H), 4.22 (t, J=5.6 Hz, 1H), 4.16-4.10 (m, 2H), 3.00-2.80 (m, 3H), 1.83 (d, J=12.1 Hz, 2H), 1.66 (qd, J=12.5, 4.0 Hz, 2H), 1.44 (s, 9H), 1.34 (t, J=7.0 Hz, 6H).

[0783] LC/MS (System A): m/z (ESI.sup.+)=609 [M.sup.+], R.sub.t=1.11 min, UV purity=92%.

Intermediate 93—Synthesis of 1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-6-(piperidin-4-yl)-1H-1,3-benzodiazol-3-ium hydrochloride iodide

[0784] ##STR00139##

[0785] HCl solution in dioxane (4.0 M, 2.0 ml, 8.0 mmol) was added to a solution of 6-{1-[(tert-butoxy)carbonyl]piperidin-4-yl}-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium iodide, Intermediate 92 (1.59 g, 1.99 mmol) in MeCN (50 ml). The reaction mixture was stirred at RT for 2 h then concentrated in vacuo and azeotroped with MeCN (3×25 ml) to afford the product as a yellow solid (1.34 g, >99%).

[0786] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 9.00 (br.s, 1H), 8.87 (br.s, 1H), 8.35 (t, J=5.1 Hz, 1H), 8.04 (d, J=8.6 Hz, 1H), 7.88 (s, 1H), 7.85 (d, J=7.6 Hz, 2H), 7.62 (d, J=7.5 Hz, 2H), 7.58 (d, J=8.5 Hz, 1H), 7.34 (t, J=7.4 Hz, 2H), 7.24 (t, J=7.4 Hz, 2H), 4.77 (d, J=5.1 Hz, 2H), 4.61-4.50 (m, 4H), 4.48 (d, J=5.9 Hz, 2H), 4.21 (t, J=5.8 Hz, 1H), 3.42 (d, 1H), 3.15-2.96 (m, 4H), 2.04-1.91 (m, 4H), 1.34 (t, J=7.1 Hz, 6H).

[0787] LC/MS (System A): m/z (ESI.sup.+)=509 [M.sup.+], R.sub.t=0.85 min, UV purity=100%.

Intermediate 94—Synthesis of 6-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium iodide

[0788] ##STR00140##

[0789] A solution of 1,3-diethyl-2-[({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)methyl]-6-(piperidin-4-yl)-1H-1,3-benzodiazol-3-ium hydrochloride iodide, Intermediate 93 (1.33 g, 1.98 mmol), 4,6-O-benzylidene-D-glucopyranose (1.06 g, 3.95 mmol) and AcOH (226 μl, 3.95 mmol) in MeOH (50 ml) was stirred at RT for 0.5 h. NaCNBH.sub.3 (248 mg, 3.95 mmol) was added then the resultant solution was stirred at RT for 24 h. The reaction mixture was recharged with 4,6-O-benzylidene-D-glucopyranose (1.06 g, 3.95 mmol) and AcOH (226 μl, 3.95 mmol) then left to stir at RT for 0.5 h. NaCNBH.sub.3 (150 mg, 2.37 mmol) was added then the resultant solution was stirred at RT for a further 16 h. Saturated aqueous NaHCO.sub.3 solution (50 ml) was added over 5 min then the resultant suspension was allowed to stand at RT for 0.5 h then filtered. The collected solid was washed with water (100 ml) then dried under vacuum for afford the product as a beige solid (1.24 g, 57%).

[0790] LC/MS (System A): m/z (ESI.sup.+)=381 [(M.sup.+)+H.sup.+], R.sub.t=0.94 min, UV purity=81%.

Intermediate 95 Synthesis of 2-(aminomethyl)-6-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

[0791] ##STR00141##

[0792] Morpholine (370 μl, 11.3 mmol) was added to a suspension of 6-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}-1,3-diethyl-2-[({[(9H-fluoren-9-yl)methodoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium iodide, Intermediate 94 (81% 1.00 g, 0.90 mmol) in THF (10 ml). The resulting suspension was sonicated for 10 min then left to stir at RT for 2.5 h. The reaction mixture was diluted with diethyl ether (25 ml). The resulting white precipitate was filtered and washed with diethyl ether (25 ml) then dried under vacuum to afford the product as a white solid (615 mg, 92%).

[0793] LC/MS (System A): m/z (ESI.sup.+)=539 [M.sup.+], R.sub.t=0.73 min, UV purity=90%.

Intermediate 96 Synthesis of tert-butyl N-[3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propyl]carbamate

[0794] ##STR00142##

[0795] A solution of 2,5-dioxopyrrolidin-1-yl 9H-fluoren-9-ylmethyl carbonate (3.87 g, 11.5 mmol) in MeCN (30 ml) was added dropwise over 20 min to a mixture of NaHCO.sub.3 (1.93 g, 23.0 mmol) and tert-butyl N-(3-aminopropyl)carbamate (2.00 g, 11.5 mmol) in MeCN (40 ml) and water (40 ml). The resultant mixture was stirred at RT for 1 h then filtered. The collected solid was washed with water (2×20 ml) then MeCN (2×20 ml), then dried under vacuum to afford a white solid (1.28 g). The solid thus obtained was suspended in EtOAc (10 ml) then filtered. The solid collected was dried under vacuum to afford a first batch of the product as a white solid (1.24 g). The MeCN/water filtrate was concentrated in vacuo then the resulting residue was partitioned between EtOAc (100 ml) and water (50 ml). The phases were separated then the organic phase was washed with water (2×50 ml), brine (20 ml), then dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford a white solid (2.30 g). The solid thus obtained was combined with the filtrate from the EtOAc trituration then the combined material was purified by flash column chromatography on a silica column (25 g). The column was eluted with heptane:EtOAc:MeOH using the following gradient: 100:0:0, 3 CV; 100:0:0-81:19:0, 3 CV; 81:19:0%, 2 CV; 81:19:0-61:39:0, 3 CV; 61:39:0, 5 CV; 61:39:0-12:88:0, 8 CV; 12:88:0-0:100:0, 2 CV; 0:100:0, 1 CV; 0:100:0-0:93:7, 4 CV; 0:93:7, 3 CV; 0:93:7-0:91:9, 1 CV. The desired fractions were combined and evaporated to afford a second batch of the product as a white solid (2.60 g), which was analytically identical to the first batch. Overall yield=3.84 g (84%).

[0796] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.89 (d, J=7.5 Hz, 2H), 7.73-7.59 (m, 2H), 7.41 (t, J=7.4 Hz, 2H), 7.36-7.29 (m, 2H), 7.22 (t, J=5.6 Hz, 1H), 6.74 (s, 1H), 4.29 (d, J=6.9 Hz, 2H), 4.21 (t, J=6.8 Hz, 1H), 3.04-2.78 (m, 4H), 1.56-1.43 (m, 2H), 1.37 (s, 9H).

[0797] LC/MS (System A): m/z (ESI.sup.+)=419 [(M.sup.+Na).sup.+], R.sub.t=1.25 min, UV purity=99%.

Intermediate 97 Synthesis of (9H-fluoren-9-yl)methyl N-(3-aminopropyl)carbamate hydrochloride

[0798] ##STR00143##

[0799] HCl solution in dioxane (4.0 M, 8.0 ml, 32 mmol) was added to a suspension of tert-butyl N-(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propyl)carbamate, Intermediate 96 (2.60 g, 6.56 mmol) in MeCN (40 ml). The reaction mixture was stirred at RT for 1 h then filtered.

[0800] The collected solid was rinsed with MeCN then dried under vacuum to afford the product as a white solid (1.89 g, 87%).

[0801] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.95-7.77 (m, 5H), 7.68 (d, J=7.4 Hz, 2H), 7.48-7.38 (m, 3H), 7.37-7.29 (m, 2H), 4.33 (d, J=6.8 Hz, 2H), 4.26-4.17 (m, 1H), 3.10-2.99 (m, 2H), 2.81-2.71 (m, 2H), 1.76-1.64 (m, 2H).

[0802] LC/MS (System A): m/z (ESI.sup.+)=297 [MH.sup.+], R.sub.t=0.91 min, UV purity=100%.

Intermediate 98 Synthesis of (9H-fluoren-9-yl)methyl N-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)carbamate; formic acid

[0803] ##STR00144##

[0804] A mixture of 9H-fluoren-9-ylmethyl N-(3-aminopropyl)carbamate hydrochloride, Intermediate 97 (900 mg, 2.70 mmol) and 4,6-O-benzylidene-D-glucopyranose (1.45 g, 5.41 mmol) in MeOH (40 ml) was stirred at RT for 1.5 h. AcOH (0.31 ml, 5.4 mmol) and NaCNBH.sub.3 (340 mg, 5.41 mmol) were added then the reaction was stirred at RT for 18 h. The reaction was recharged with 4,6-O-benzylidene-D-glucopyranose (1.45 g, 5.41 mmol) then the reaction was stirred at RT for 1 h. NaCNBH.sub.3 (340 mg, 5.41 mmol) was added then the reaction was left to stir at RT for a further 114 h. Saturated aqueous NaHCO.sub.3 solution (50 ml) was added dropwise over 10 min then the resultant mixture was partitioned between EtOAc (50 ml) and water (50 ml). The phases were separated then the organic phase was washed with saturated aqueous NaHCO.sub.3 solution (2×50 ml), water (50 ml) and brine (20 ml), then dried using Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford a white solid (2.25 g). The crude material thus obtained was purified by flash column chromatography on C18 (120 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-29%, 4 CV;

[0805] 29-39%, 2 CV; 39%, 2 CV; 39-47%, 1 CV; 47-73%, 1 CV; 73-100%, 1 CV; 100% 1 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a white solid (1.12 g, 49%).

[0806] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.16 (m, 1H), 7.88 (d, J=7.5 Hz, 2H), 7.67 (d, J=7.4 Hz, 2H), 7.45-7.37 (m, 6H), 7.36-7.25 (m, 8H), 7.24-7.16 (m, 1H), 5.49-5.39 (m, 2H), 5.27-5.01 (m, 2H), 4.32-4.25 (m, 2H), 4.23-4.16 (m, 1H), 4.16-4.09 (m, 2H), 3.84-3.74 (m, 4H), 3.73-3.66 (m, 2H), 3.64-3.56 (partially obscured m, 2H), 3.53-3.45 (obscured m, 2H), 2.99-2.89 (obscured m, 2H), 2.68-2.54 (obscured m, 6H), 1.60-1.48 (m, 2H).

[0807] LC/MS (System A): m/z (ESI.sup.+)=801 [MH.sup.+], R.sub.t=1.01 min, UV purity=100%.

Intermediate 99 Synthesis of (1R,2S)-3-[(3-aminopropyl)[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino]-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol; bis(formic acid)

[0808] ##STR00145##

[0809] Diethylamine (1.44 ml, 14.0 mmol) was added to a solution of 9H-fluoren-9-ylmethyl N-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)carbamate; formic acid, Intermediate 98 (1.32 g, 1.40 mmol) in THF (20 ml). The reaction mixture was stirred at RT for 16 h then concentrated in vacuo. The crude material thus obtained was purified by flash column chromatography on C18 (60 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 5%, 2 CV; 5-18%, 3 CV; 18%, 2 CV; 18-26%, 2 CV; 26-100%, 2 CV; 100% 1 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a white solid (710 mg, 80%).

[0810] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.36-8.24 (m, 2H), 7.44-7.38 (m, 4H), 7.38-7.29 (m, 6H), 5.44 (s, 2H), 4.18-4.08 (obscured m, 2H), 3.87-3.75 (obscured m, 4H), 3.73-3.65 (obscured m, 2H), 3.63-3.56 (obscured m, 2H), 3.53-3.44 (obscured m, 2H), 2.90-2.78 (m, 2H), 2.75-2.55 (obscured m, 6H), 1.78-1.65 (m, 1H), 1.55 (m, 1H).

[0811] LC/MS (System A): m/z (ESI.sup.+)=579 [MH.sup.+], R.sub.t=0.74 min, UV purity=100%.

Intermediate 100 Synthesis of (2R,3R,4R,5S)-6-[(3-aminopropyl)[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]hexane-1,2,3,4,5-pentol dihydrochloride

[0812] ##STR00146##

[0813] A mixture of (1R,2S)-3-[(3-aminopropyl)[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino]-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol; bis(formic acid), Intermediate 99 (335 mg, 0.50 mmol) and aqueous HCl solution (2 M, 5 ml, 10 mmol) was stirred at RT for 3 h. The reaction mixture was concentrated in vacuo then the residue was azeotroped with MeCN (3×10 ml) to afford the product as a colourless viscous oil (235 mg, 99%).

[0814] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 4.25-4.18 (m, 2H), 3.91-3.83 (m, 2H), 3.81-3.76 (m, 2H), 3.74-3.64 (m, 6H), 3.62-3.51 (m, 2H), 3.50-3.39 (m, 4H), 3.10-3.03 (m, 2H), 2.22-2.13 (m, 2H).

[0815] LC/MS (System A): m/z (ESI.sup.+)=403 [MH.sup.+], R.sub.t=0.12 min, ELS purity=100%.

Intermediate 101—Synthesis of tert-butyl 4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)piperidine-1-carboxylate

[0816] ##STR00147##

[0817] A solution of 2,5-dioxopyrrolidin-1-yl 9H-fluoren-9-ylmethyl carbonate (5.05 g, 15.0 mmol) in MeCN (50 ml) was added dropwise over 20 min to a mixture of tert-butyl 4-aminopiperidine-1-carboxylate (3.00 g, 15.0 mmol) and NaHCO.sub.3 (2.52 g, 30.0 mmol) in MeCN (50 ml) and water (50 ml). The resulting mixture was left to stir at RT for 16 h then partitioned between EtOAc (100 ml) and water (100 ml). The phases were separated then the organic phase was washed with water (100 ml) and brine (100 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the product as a white foam (6.20 g, 95%).

[0818] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.89 (d, J=7.5 Hz, 2H), 7.69 (d, J=7.4 Hz, 2H), 7.41 (t, J=7.4 Hz, 2H), 7.33 (td, J=7.4, 0.9 Hz, 2H), 7.27 (d, J=7.7 Hz, 1H), 4.31 (d, J=6.7 Hz, 2H), 4.21 (t, J=6.6 Hz, 1H), 3.84 (br. d, J=11.7 Hz, 2H), 3.52-3.40 (m, 1H), 2.80 (br. s, 2H), 1.70 (d, J=10.8 Hz, 2H), 1.39 (s, 9H), 1.20-1.29 (m, 2H).

[0819] LC/MS (System A): m/z (ESI.sup.+)=445 [(M.sup.+Na).sup.+], R.sub.t=1.38 min, UV purity=97%.

Intermediate 102—Synthesis of (9H-fluoren-9-yl)methyl N-(piperidin-4-yl)carbamate hydrochloride

[0820] ##STR00148##

[0821] HCl solution in dioxane (4.0 M, 11 ml, 44 mmol) was added to a solution of tert-butyl 4-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)piperidine-1-carboxylate, Intermediate 101 (6.20 g, 14.7 mmol) in MeCN (100 ml). The resulting solution was stirred at RT for 2 h. The reaction was re-dosed with HCl solution in dioxane (4.0 M, 2.0 ml, 8.0 mmol) then the reaction mixture was left to stir at RT for a further 1 h. The reaction mixture was filtered then the collected solid was washed with MeCN then dried under vacuum to afford the product as a white solid (4.60 g, 87%).

[0822] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.85 (s, 1H), 8.69 (s, 1H), 7.89 (d, J=7.5 Hz, 2H), 7.69 (d, J=7.4 Hz, 2H), 7.50 (d, J=7.2 Hz, 1H), 7.42 (t, J=7.4 Hz, 2H), 7.36-7.31 (m, 2H), 4.33 (d, J=6.6 Hz, 2H), 4.22 (t, J=6.4 Hz, 1H), 3.62-3.54 (m, 1H), 3.21 (d, J=12.0 Hz, 2H), 2.96-2.88 (m, 2H), 1.88 (d, J=12.1 Hz, 2H), 1.67-1.50 (m, 2H).

[0823] LC/MS (System A): m/z (ESI.sup.+)=323 [MH.sup.+], R.sub.t=0.89 min, UV purity=100%.

Intermediate 103—Synthesis of (9H-fluoren-9-yl)methyl N-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}carbamate

[0824] ##STR00149##

[0825] 4,6-O-benzylidene-D-glucopyranose (2.99 g, 11.2 mmol) was added to a solution of (9H-fluoren-9-yl)methyl N-(piperidin-4-yl)carbamate hydrochloride, Intermediate 102 (2.00 g, 5.57 mmol) in MeOH (75 ml). The reaction mixture was stirred at RT for 20 min then AcOH (670 μl, 11.7 mmol) and NaCNBH.sub.3 (700 mg, 11.2 mmol) were added. The reaction mixture was stirred at RT for 22 h. Saturated aqueous NaHCO.sub.3 solution (50 ml) was added dropwise over 10 min. The resulting mixture was partitioned between EtOAc (200 ml) and water (100 ml). The phases were separated then the organic phase was washed with saturated aqueous NaHCO.sub.3 solution (100 ml), water (2×100 ml) and brine (2×100 ml). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the product as a white solid (3.13 g, 92%).

[0826] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.78 (d, J=7.4 Hz, 2H), 7.63 (d, J=7.1 Hz, 2H), 7.52-7.45 (m, 2H), 7.39-7.27 (m, 7H), 5.55 (s, 1H), 4.34 (d, J=6.4 Hz, 2H), 4.25 (dd, J=10.7, 5.4 Hz, 1H), 4.21-4.17 (m, 1H), 4.07-4.00 (m, 1H), 3.97-3.92 (m, 1H), 3.90-3.87 (d, J=6.4 Hz, 1H), 3.82 (d, J=9.3 Hz, 1H), 3.61 (t, J=10.5 Hz, 1H), 3.39-3.32 (m, 1H), 2.97-2.94 (m, 1H), 2.77-2.64 (m, 2H), 2.47 (dd, J=11.8, 6.7 Hz, 1H), 2.14 (t, J=10.5 Hz, 1H), 2.06-2.02 (m, 1H), 1.75 (dd, J=59.2, 11.6 Hz, 2H), 1.53-1.36 (m, 2H).

[0827] LC/MS (System A): m/z (ESI.sup.+)=575 [MH.sup.+], R.sub.t=0.96 min, UV purity=94%.

Intermediate 104—Synthesis of (1R,2S)-3-(4-aminopiperidin-1-yl)-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol

[0828] ##STR00150##

[0829] Diethylamine (2.64 ml, 25.6 mmol) was added to a solution of (9H-fluoren-9-yl)methyl N-{1-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]piperidin-4-yl}carbamate, Intermediate 103 (94%, 3.13 g, 5.12 mmol) in THF (40 ml). The reaction mixture was left to stir at RT for 1 h. The reaction mixture was re-dosed with diethylamine (2.64 ml, 25.6 mmol) then the reaction was left to stir at RT for a further 20 h. The reaction mixture was concentrated in vacuo then the resultant residue was suspended in EtOAc (10 ml) and water (10 ml). Diethyl ether (50 ml) was added then the mixture was sonicated. The resulting suspension was filtered then the collected solid was rinsed with diethyl ether (20 ml) then dried under vacuum to afford the product as a white solid (1.98 g, >99%).

[0830] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.41 (dd, J=6.5, 3.1 Hz, 2H), 7.38-7.31 (m, 3H), 5.49 (s, 1H), 4.11 (dd, J=10.5, 4.9 Hz, 1H), 3.81 (q, J=6.2 Hz, 1H), 3.79-3.69 (m, 3H), 3.52-3.48 (m, 1H), 2.83 (d, J=11.3 Hz, 1H), 2.58-2.52 (m, 1H), 2.45-2.45 (m, 2H+DMSO), 2.24 (dd, J=12.4, 6.1 Hz, 1H), 1.93-1.73 (m, 2H), 1.61 (d, J=12.5 Hz, 1H), 1.49 (d, J=12.1 Hz, 1H), 1.17-1.14 (m, 2H).

[0831] LC/MS (System A): m/z (ESI.sup.+)=353 [MH.sup.+], R.sub.t=0.13 min, ELS purity=100%.

Intermediate 105—Synthesis of (2R,3R,4R,5S)-6-(4-aminopiperidin-1-yl)hexane-1,2,3,4,5-pentol dihydrochloride

[0832] ##STR00151##

[0833] Aqueous HCl solution (2.0 M, 11 ml, 22 mmol) was added to a suspension of (1R,2S)-3-(4-aminopiperidin-1-yl)-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol, Intermediate 104 (400 mg, 1.13 mmol) in water (5 ml). The reaction mixture was left to stir at RT for 2 h then concentrated in vacuo. The resulting residue was dissolved in water (20 ml) then extracted with EtOAc (20 ml). The aqueous phase was concentrated in vacuo to afford the product as an off-white foam (279 mg, 73%).

[0834] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 4.26-4.18 (m, 1H), 3.87-3.74 (m, 4H), 3.74-3.62 (m, 4H), 3.61-3.42 (m, 2H), 3.29-3.12 (m, 2H), 2.28 (t, J=13.2 Hz, 2H), 2.15-1.92 (m, 2H).

[0835] LC/MS (System A): m/z (ESI.sup.+)=265 [MH.sup.+], R.sub.t=0.13 min, ELS purity=100%.

Intermediate 107 Synthesis of benzyl N-[1-(2-{[(tert-butoxy)carbonyl]amino}ethyl)piperidin-4-yl]carbamate

[0836] ##STR00152##

[0837] Triethylamine (515 μl, 3.69 mmol) was added to a solution of benzyl N-(4-piperidyl)carbamate hydrochloride (500 mg, 1.85 mmol) and tert-butyl N-(2-bromoethyl)carbamate (500 mg, 2.22 mmol) in MeCN (4 ml) in a pressure tube. The tube was sealed then the reaction mixture was heated at 85° C. for 16 h. Additional tert-butyl N-(2-bromoethyl)carbamate (150 mg, 0.67 mmol) was added then the reaction was left to heat at 85° C. for a further 1 h. The reaction mixture was concentrated in vacuo then the solid thus obtained was dissolved in the minimum of refluxing MeCN then allowed to cool to RT. The resultant suspension was filtered then the filtrate was concentrated in vacuo. The crude material was purified by flash column chromatography on a silica column (25 g). The column was eluted with CH.sub.2Cl.sub.2:MeOH, increasing the gradient linearly from 0-15% MeOH over 10 column volumes. The desired fractions were combined and evaporated to afford a viscous red oil (254 mg). The material thus obtained was partitioned between EtOAc (15 ml) and saturated aqueous NaHCO.sub.3 solution (15 ml). The phases were separated then the organic phase was washed with water (2×15 ml) and brine (15 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the product as a pale red solid (140 mg, 20%).

[0838] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.39-7.28 (m, 5H), 7.21 (d, J=7.6 Hz, 1H), 6.59 (t, J=5.3 Hz, 1H), 5.00 (s, 2H), 3.29-3.22 (m, 1H), 3.00 (q, J=6.4 Hz, 2H), 2.76 (d, J=11.5 Hz, 2H), 2.28 (t, J=6.9 Hz, 2H), 1.95 (t, J=11.0 Hz, 2H), 1.69 (d, J=10.5 Hz, 2H), 1.43-1.32 (m, 11H).

[0839] LC/MS (System A): m/z (ESI.sup.+)=378 [MH.sup.+], R.sub.t=0.85 min, UV purity=100%.

Intermediate 108 Synthesis of tert-butyl N-[2-(4-aminopiperidin-1-yl)ethyl]carbamate

[0840] ##STR00153##

[0841] A mixture of benzyl N-[1-(2-{[(tert-butoxy)carbonyl]amino}ethyl)piperidin-4-yl]carbamate, Intermediate 107 (140 mg, 0.370 mmol) and palladium on carbon (10 wt %, 20 mg) in EtOH (5 ml) was stirred under a hydrogen atmosphere at RT for 2 h. The reaction mixture was filtered through a Celite pad then the filtrate was concentrated in vacuo to afford the product as a colourless oil (124 mg, 96% yield corrected for 70% purity determined by NMR).

[0842] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 6.61-6.53 (m, 1H), 4.37 (s, 2H), 3.00 (q, J=6.5 Hz, 2H), 2.76-2.71 (m, 2H), 2.49-2.45 (m, 1H), 2.27 (t, J=7.0 Hz, 2H), 1.91 (t, J=10.6 Hz, 2H), 1.67-1.60 (m, 2H), 1.37 (s, 9H), 1.22-1.15 (m, 2H).

[0843] LC/MS (System A): m/z (ESI.sup.+)=244 [MH.sup.+], R.sub.t=0.14 min, ELS purity=100%.

Intermediate 109—Synthesis of bis(formic acid); tert-butyl N-[2-(4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}piperidin-1-yl)ethyl]carbamate

[0844] ##STR00154##

[0845] A mixture of tert-butyl N-[2-(4-aminopiperidin-1-yl)ethyl]carbamate, Intermediate 108 (70%, 725 mg, 2.08 mmol), 4,6-O-benzylidene-D-glucopyranose (3.57 g, 12.7 mmol) and AcOH (725 uL, 12.7 mmol) in MeOH (20 ml) was stirred at RT for 0.5 h. NaCNBH.sub.3 (795 mg, 12.7 mmol) was added then the resulting mixture was stirred at RT for 5 days. The reaction was retreated with 4,6-O-benzylidene-D-glucopyranose (1.80 g, 6.29 mmol) and AcOH (362 uL, 6.32 mmol) then the reaction was left to stir at RT for 0.5 h. NaCNBH.sub.3 (396 mg, 6.30 mmol) was added then the reaction was left to stir at RT for a further 18 h. The reaction was retreated with 4,6-O-benzylidene-D-glucopyranose (1.80 g, 6.29 mmol) and AcOH (362 uL, 6.32 mmol) then the reaction was left to stir at RT for 0.5 h. NaCNBH.sub.3 (396 mg, 6.30 mmol) was added then the reaction was left to stir at RT for a further 18 h. The reaction mixture was concentrated under a stream of nitrogen then saturated aqueous NaHCO.sub.3 solution was added dropwise until effervescence ceased. The resulting mixture was partitioned between saturated aqueous NaHCO.sub.3 solution (150 ml) and EtOAc (150 ml). The phases were separated then the organic phase was washed with NaHCO.sub.3 (150 ml), water (2×150 ml) and brine (150 ml), then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford a pale yellow solid. The crude material was purified by flash column chromatography on C18 (120 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-16%, 11 CV; 16%-100%, 4 CV; 100%, 1 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a colourless oil (383 mg, 18%).

[0846] LC/MS (System A): m/z (ESI.sup.+)=375 [(M.sup.+)+H.sup.+], 748 [MH.sup.+], R.sub.t=0.82 min, UV purity=83%.

Intermediate 110—Synthesis of (2R,3R,4R,5S)-6-{[1-(2-aminoethyl)piperidin-4-yl][(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}hexane-1,2,3,4,5-pentol trihydrochloride

[0847] ##STR00155##

[0848] A mixture of tert-butyl N-[2-[4-[bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino]-1-piperidyl]ethyl]carbamate, Intermediate 109 (83%, 360 mg, 0.356 mmol) and aqueous HCl solution (4.0 M, 3.6 ml, 14.4 mmol) was stirred at RT for 0.5 h then concentrated in vacuo. The residue was dissolved in water:MeCN (9:1, 12 ml) then lyophilised to afford the product as a white foam (220 mg, 94%).

[0849] .sup.1H NMR (500 MHz, Deuterium Oxide) δ 4.34-4.26 (m, 2H), 4.16-4.05 (m, 1H), 3.95-3.88 (m, 4H), 3.87 (d, J=2.9 Hz, 1H), 3.84 (d, J=3.0 Hz, 1H), 3.83-3.78 (m, 2H), 3.73-3.67 (m, 4H), 3.64-3.46 (m, 8H), 3.40-3.31 (m, 2H), 2.58 (d, J=14.0 Hz, 1H), 2.51 (d, J=13.7 Hz, 1H), 2.35-2.24 (m, 1H), 2.23-2.12 (m, 1H).

[0850] LC/MS (System F): m/z (ESI.sup.+)=472 [MH.sup.+], R.sub.t=0.29 min, ELS purity=88%.

Intermediate 111—Synthesis of (9H-fluoren-9-yl)methyl 4-({[(tert-butoxy)carbonyl]amino}methyl)piperidine-1-carboxylate

[0851] ##STR00156##

[0852] NaHCO.sub.3 (2.35 g, 28.0 mmol) was added portionwise over 1 min to a stirred solution of tert-butyl N-(4-piperidylmethyl)carbamate (3.00 g, 14.0 mmol) in MeCN (50 ml) and water (50 ml). A solution of (2,5-dioxopyrrolidin-1-yl) 9H-fluoren-9-ylmethyl carbonate (4.72 g, 14.0 mmol) in MeCN (50 ml) was added dropwise over 1 h then the reaction was left to stir at RT for 18 h. The reaction mixture was partitioned between EtOAc (100 ml) and water (100 ml). Brine was added to aid separation of phases. The phases were separated then the aqueous phase was extracted with EtOAc (50 ml). The combined organic extracts were washed with brine (70 ml), dried over MgSO.sub.4, filtered and concentrated in vacuo to afford the product as an off white solid (7.02 g, 96%).

[0853] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.89 (d, J=7.5 Hz, 2H), 7.62 (d, J=7.4 Hz, 2H), 7.41 (t, J=7.3 Hz, 2H), 7.33 (td, J=7.4, 0.9 Hz, 2H), 6.85 (t, J=5.8 Hz, 1H), 4.53-4.29 (m, 2H), 4.26 (t, J=6.3 Hz, 1H), 3.97-3.67 (m, 2H), 2.78 (t, J=6.1 Hz, 2H), 2.75-2.61 (m, 2H), 1.60-1.44 (m, 3H), 1.38 (s, 9H), 0.93-0.73 (m, 2H). 5 wt % residual solvent.

[0854] LC/MS (System A): m/z (ESI.sup.+)=459 [M.sup.+Na.sup.+], R.sub.t=1.40 min, UV purity=89%. Overall purity estimate=84%.

Intermediate 112—Synthesis of (9H-fluoren-9-yl)methyl 4-(aminomethyl)piperidine-1-carboxylate hydrochloride

[0855] ##STR00157##

[0856] HCl solution in dioxane (4.0 M, 12 ml, 48 mmol) was added drop-wise over 8 min to a stirred solution of (9H-fluoren-9-yl)methyl 4-({[(tert-butoxy)carbonyl]amino}methyl)piperidine-1-carboxylate, Intermediate 111 (7.00 g, 16.0 mmol) in MeCN (100 ml). The resulting solution was stirred at RT for 17 h then concentrated in vacuo to afford the product as a white solid (5.56 g, 82%).

[0857] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.04-7.82 (m, 5H), 7.62 (d, J=7.4 Hz, 2H), 7.42 (t, J=7.4 Hz, 2H), 7.34 (td, J=7.4, 0.9 Hz, 2H), 4.42-4.32 (m, 2H), 4.27 (t, J=6.3 Hz, 1H), 4.04-3.71 (m, 2H), 2.84-2.69 (m, 2H), 2.67 (d, J=6.8 Hz, 2H), 1.80-1.70 (m, 1H), 1.70-1.58 (m, 2H), 1.04-0.87 (m, 2H). 7 wt % residual dioxane.

[0858] LC/MS (System A): m/z (ESI.sup.+)=337 [MH.sup.+], R.sub.t=0.86 min, UV purity=95%. Overall purity estimate=88%.

Intermediate 113—Synthesis of (9H-fluoren-9-yl)methyl 4-({bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}methyl)piperidine-1-carboxylate

[0859] ##STR00158##

[0860] AcOH (3.11 ml, 54.3 mmol) was added to a solution of (9H-fluoren-9-yl)methyl 4-(aminomethyl)piperidine-1-carboxylate hydrochloride, Intermediate 112 (5.56 g, 14.9 mmol) and 4,6-O-benzylidene-D-glucopyranose (14.6 g, 54.3 mmol) in MeOH (100 ml).

[0861] The reaction was stirred at RT for 40 min then NaCNBH.sub.3 (3.41 g, 54.3 mmol) was added in portions over 50 min. The resulting solution was stirred at RT for 17 h. The reaction mixture was re-treated with 4,6-O-benzylidene-D-glucopyranose (7.29 g, 27.2 mmol) and AcOH (1.56 ml, 27.2 mmol) then stirred at RT for 30 min. NaCNBH.sub.3 (1.71 g, 27.2 mmol) was added in portions over 1 h. The resulting solution was stirred at RT for a further 70 h then added onto saturated aqueous NaHCO.sub.3 solution (200 ml) in portions over 30 min. The resultant suspension was stirred at RT for 1 h then filtered. The solid was washed with water (100 ml) then dried in vacuo to afford a white solid (13.8 g). A portion (5.55 g) of the crude material thus obtained was purified by flash column chromatography on C18 (400 g). The column was eluted with MeCN:H.sub.2O+0.1% NH.sub.4OH using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-57%, 16 CV; 57%, 9 CV; 59-63%, 2 CV; 100%, 3 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a beige solid (2.99 g, 23%).

[0862] .sup.1H NMR (500 MHz, CD.sub.3OD-d4) δ 7.86-7.75 (m, 2H), 7.65-7.55 (m, 2H), 7.53-7.44 (m, 4H), 7.43-7.37 (m, 2H), 7.36-7.27 (m, 8H), 5.51 (m, 2H), 4.64-4.36 (m, 5H), 4.27-4.20 (m, 3H), 4.04-3.92 (m, 4H), 3.86 (m, 2H), 3.71 (m, 2H), 3.64-3.57 (m, 2H), 2.73-2.58 (m, 3H), 2.54-2.44 (m, 2H), 2.36-2.23 (m, 2H), 1.81-1.42 (m, 3H), 0.89-0.58 (m, 2H).

[0863] LC/MS (System B): m/z (ESI.sup.+)=841 [MH.sup.+], R.sub.t=4.78 min, UV purity=95%.

Intermediate 114 Synthesis of (1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(piperidin-4-yl)methyl]amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol; bis(formic acid)

[0864] ##STR00159##

[0865] Piperidine (3.30 ml, 33.4 mmol) was added to a stirred solution of (9H-fluoren-9-yl)methyl 4-({bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}methyl)piperidine-1-carboxylate, Intermediate 113 (2.81 g, 3.34 mmol) in THF (40 ml). The reaction mixture was stirred at RT for 18 h then concentrated in vacuo. The residue was suspended in Et.sub.2O (30 ml) with sonication then the resultant suspension was filtered. The solid collected was rinsed with Et.sub.2O (20 ml) then dried under vacuum to afford a white solid (3.07 g). A sample (1.78 g) of the crude solid was purified by flash column chromatography on C18 (120 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-16%, 1.5 CV; 16%, 2.5 CV; 16-39%, 6 CV; 39-100%, 1.5 CV; 100% 2 CV. The remaining crude solid material was purified by flash column chromatography on C18 (120 g). The column was eluted with MeCN:water+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-14%, 2 CV; 14%, 2 CV; 14-17%, 1 CV; 17-55%, 7 CV; 55-100%, 1 CV; 100% 4 CV. The desired fractions from both columns were combined and concentrated in vacuo to afford the product as an off-white solid (1.58 g, 67%).

[0866] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.33 (s, 2H), 7.53-7.45 (m, 4H), 7.41-7.31 (m, 6H), 5.53 (m, 2H), 4.25 (m, 2H), 4.02 (m, 2H), 3.96 (m, 2H), 3.89 (m, 2H), 3.74 (m, 2H), 3.62 (m, 2H), 3.30-3.20 (m, 2H), 2.96-2.80 (m, 4H), 2.78-2.68 (m, 2H), 2.65-2.58 (m, 2H), 2.09-2.00 (m, 1H), 1.92-1.68 (m, 2H), 1.33-1.18 (m, 2H).

[0867] LC/MS (System A): m/z (ESI.sup.+)=619 [MH.sup.+], R.sub.t=0.73 min, UV purity=100%.

Intermediate 115 Synthesis of (2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(piperidin-4-yl)methyl]amino}hexane-1,2,3,4,5-pentol dihydrochloride

[0868] ##STR00160##

[0869] A solution of (1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(piperidin-4-yl)methyl]amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol; bis(formic acid), Intermediate 114 (1.52 g, 2.14 mmol) in aqueous HCl solution (2 M, 23 ml, 46 mmol) was stirred at RT for 4.5 h. The reaction was concentrated in vacuo to afford a viscous yellow gum (1.18 g, quantitative based on 93% estimated purity).

[0870] .sup.1H NMR (500 MHz, D.sub.2O) δ 4.32-4.21 (m, 2H), 3.89-3.81 (m, 4H), 3.81-3.75 (m, 2H), 3.72-3.59 (m, 4H), 3.56-3.48 (m, 6H), 3.39 (m, 2H), 3.09 (m, 2H), 2.40-2.27 (m, 1H), 2.21-2.13 (m, 1H), 2.06-1.99 (m, 1H), 1.66-1.53 (m, 2H).

[0871] LC/MS (System C): m/z (ESI.sup.+)=443 [MH.sup.+], R.sub.t=0.32 min, ELS purity=100%.

Intermediate 116 Synthesis of formic acid; tert-butyl (3R)-3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}pyrrolidine-1-carboxylate

[0872] ##STR00161##

[0873] 2-Picoline borane complex (0.86 g, 8.05 mmol) was added to a suspension of tert-butyl (3R)-3-aminopyrrolidine-1-carboxylate (500 mg, 2.68 mmol) and 4,6-O-benzylidene-D-glucopyranose (2.88 g, 10.7 mmol) in MeOH (5 ml). The mixture was heated at 60° C. for 17 h. The reaction mixture was allowed to cool to RT then concentrated in vacuo. The residue was partitioned between EtOAc (15 ml) and water (15 ml). The phases were separated then the organic phase was washed with water (15 ml) and brine (15 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude material was purified by flash column chromatography on C18 (60 g, Ultra). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-40%, 10 CV; 40-100%, 2 CV; 100%, 2 CV. The desired fractions were combined and concentrated in vacuo then the residual aqueous solution was lyophilised to afford the product as a white solid (1.39 g, 70%).

[0874] .sup.1H NMR (500 MHz, CD.sub.3OD-d4) δ 8.27 (s, 1H), 7.47 (dd, J=7.2, 2.3 Hz, 4H), 7.34 (qd, J=4.7, 1.8 Hz, 6H), 5.51 (s, 2H), 4.24 (dd, J=10.6, 5.4 Hz, 2H), 4.04 (s, 2H), 3.95 (td, J=9.9, 5.4 Hz, 2H), 3.89 (dd, J=5.3, 2.2 Hz, 2H), 3.75 (dd, J=9.4, 2.2 Hz, 2H), 3.69 (d, J=6.2 Hz, 1H), 3.59 (q, J=11.4, 11.0 Hz, 3H), 3.34-3.29 (m, 1H+CD.sub.3OD), 3.24-3.15 (m, 1H), 2.92 (d, J=48.5 Hz, 5H), 1.96 (d, J=31.5 Hz, 1H), 1.75 (s, 1H), 1.45 (s, 9H).LC/MS (System A): m/z (ESI.sup.+)=691 [MH.sup.+], R.sub.t=0.93 min, UV purity=100%.

Intermediate 117 Synthesis of (2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(3R)-pyrrolidin-3-yl]amino}hexane-1,2,3,4,5-pentol dihydrochloride

[0875] ##STR00162##

[0876] A suspension of formic acid; tert-butyl (3R)-3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}pyrrolidine-1-carboxylate, Intermediate 116 (1.39 g, 1.89 mmol) in aqueous HCl solution (2 M, 30 ml, 60 mmol) was stirred at RT for 18 h. The reaction mixture was concentrated in vacuo then re-dissolved in water (20 ml) and lyophilised to afford the product as a cream foam (1.11 g, quantitative based on 83% estimated purity).

[0877] .sup.1H NMR (500 MHz, D.sub.2O) δ 4.67 (p, J=8.4 Hz, 1H), 4.33-4.28 (m, 2H), 3.99-3.92 (m, 1H), 3.88 (dd, J=5.0, 2.3 Hz, 2H), 3.83 (dd, J=11.8, 3.0 Hz, 2H), 3.81-3.76 (m, 2H), 3.75-3.64 (m, 6H), 3.63-3.58 (m, 2H), 3.52-3.46 (m, 2H), 3.47-3.39 (m, 1H), 2.75-2.67 (m, 1H), 2.37-2.28 (m, 1H).LC/MS (System A): m/z (ESI.sup.+)=415 [MH.sup.+], R.sub.t=0.13 min, ELS purity=100%.

Intermediate 118 Synthesis of formic acid; tert-butyl (3S)-3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}pyrrolidine-1-carboxylate

[0878] ##STR00163##

[0879] 2-Picoline borane complex (861 mg, 8.05 mmol) was added to a suspension of tert-butyl (3S)-3-aminopyrrolidine-1-carboxylate (500 mg, 2.68 mmol) and 4,6-O-benzylidene-D-glucopyranose (2.88 g, 10.7 mmol) in MeOH (5 ml). The resultant mixture was heated at 60° C. for 16 h. The reaction mixture was allowed to cool to RT partitioned between EtOAc (20 ml) and water (20 ml). The phases were separated then the aqueous phase was extracted with EtOAc (20 ml). The combined organic phases were washed with water (20 ml) and 1:1 water:brine (20 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude material was purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-40%, 10 CV; 40-100%, 2 CV; 100%, 2 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a pale yellow foam (1.73 g, 87%).

[0880] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.25 (s, 1H), 7.54-7.46 (m, 4H), 7.41-7.30 (m, 6H), 5.54 (s, 2H), 4.30-4.22 (m, 2H), 4.14-4.07 (m, 2H), 4.01-3.92 (m, 2H), 3.91 (dd, J=5.3, 2.2 Hz, 2H), 3.82-3.72 (m, 3H), 3.68-3.53 (m, 3H), 3.41-3.33 (m, 1H), 3.25-3.04 (m, 4H), 3.04-2.89 (m, 2H), 2.17-2.07 (m, 1H), 1.99-1.83 (m, 1H), 1.56-1.36 (m, 9H).

[0881] LC/MS (System A): m/z (ESI.sup.+)=691 [MH.sup.+], R.sub.t=0.93 min, ELS purity=100%. Intermediate 119 Synthesis of (2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(3S)-pyrrolidin-3-yl]amino}hexane-1,2,3,4,5-pentol dihydrochloride

##STR00164##

[0882] A suspension of formic acid; tert-butyl (3S)-3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}pyrrolidine-1-carboxylate, Intermediate 118 (1.72 g, 2.33 mmol) in aqueous HCl solution (2 M, 30 ml, 60 mmol) and MeOH (1 ml) was stirred at RT for 4 h. The reaction mixture was concentrated in vacuo then re-dissolved in water and lyophilised to afford the product as a cream foam (1.05 g, 92%).

[0883] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 4.72 (p, J=8.2 Hz, 1H), 4.30 (s, 2H), 3.98-3.36 (m, 18H), 2.76-2.66 (m, 1H), 2.48-2.36 (m, 1H).

[0884] LC/MS (System A): m/z (ESI.sup.+)=415 [MH.sup.+], R.sub.t=0.13 min, ELS purity=100%.

Intermediate 120 Synthesis of formic acid; tert-butyl N-[(1r,4r)-4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}cyclohexyl]carbamate

[0885] ##STR00165##

[0886] 2-Picoline borane complex (939 mg, 8.78 mmol) was added to a suspension of tert-butyl N-(4-aminocyclohexyl)carbamate (627 mg, 2.93 mmol) and 4,6-O-benzylidene-D-glucopyranose (3.14 g, 11.7 mmol) in MeOH (6 ml). The mixture as heated at 60° C. for 16 h then concentrated in vacuo. The residue was partitioned between EtOAc (15 ml) and water (15 ml). The phases were separated then the organic phase was washed with water (10 ml) and brine (10 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude material was purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-40%, 10 CV; 40-100%, 2 CV; 100%, 2 CV. The desired fractions were combined and concentrated in vacuo then the residual aqueous solution was lyophilised to afford the product as a white solid (914 mg, 41% yield)).

[0887] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.52 (s, 1H), 7.54-7.48 (m, 4H), 7.44-7.32 (m, 6H), 5.56 (s, 2H), 4.31-4.23 (m, 2H), 4.18-4.06 (m, 2H), 4.02-3.92 (m, 4H), 3.82-3.75 (m, 2H), 3.64 (t, J=10.5 Hz, 2H), 3.30-3.01 (m, 6H), 2.00-1.76 (m, 4H), 1.56-1.39 (m, 10H), 1.36-1.15 (m, 2H), 1.09-0.97 (m, 1H).

[0888] LC/MS (System C): m/z (ESI.sup.+)=719 [MH.sup.+], R.sub.t=2.29 min, ELS purity=100%.

Intermediate 121—Synthesis of (2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(1r,4r)-4-aminocyclohexyl]amino}hexane-1,2,3,4,5-pentol dihydrochloride

[0889] ##STR00166##

[0890] A suspension of formic acid; tert-butyl N-[(1r,4r)-4-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}cyclohexyl]carbamate, Intermediate 120 (910 mg, 1.19 mmol) in aqueous HCl solution (2 M, 20 ml, 40 mmol) was stirred at RT for 18 h.

[0891] The reaction mixture was concentrated in vacuo then the residue was dissolved in water (20 ml) and lyophilised to afford the product as a cream foam (718 mg, quantitative based on 85% estimated purity).

[0892] .sup.1H NMR (500 MHz, D.sub.2O) δ 4.19-4.09 (m, 2H), 3.81-3.66 (m, 6H), 3.63-3.51 (m, 6H), 3.39-3.14 (m, 4H), 2.24-2.13 (m, 3H), 2.11-2.04 (m, 1H), 1.87-1.76 (m, 1H), 1.69-1.45 (m, 3H).

[0893] LC/MS (System A): m/z (ESI.sup.+)=443 [MH.sup.+], R.sub.t=0.13 min, ELS purity=100%.

Intermediate 122 Synthesis of (9H-fluoren-9-yl)methyl N-[(1s,4s)-4-{[(tert-butoxy)carbonyl]amino}cyclohexyl]carbamate

[0894] ##STR00167##

[0895] A solution of 9H-fluoren-9-ylmethyl carbonochloridate (4.04 g, 15.6 mmol) in THF (30 ml) was added dropwise over 5 min to a cooled (0° C.) mixture of tert-butyl N-[(1s,4s)-4-aminocyclohexyl]carbamate (3.35 g, 15.6 mmol) and aqueous sodium carbonate solution (1 M, 30 ml, 30 mmol) in THF (60 ml). The reaction was allowed to warm to RT then left to stir at RT for 16 h. The reaction mixture was diluted with water (100 ml) then extracted with EtOAc (100 ml). The organic phase was washed with water (100 ml) and brine (50 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the product as a pale beige foam (6.71 g, 91%).

[0896] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.92-7.83 (m, 2H), 7.74-7.56 (m, 2H), 7.45-7.36 (m, 2H), 7.36-7.28 (m, 2H), 7.17 (d, J=5.4 Hz, 1H), 6.70-6.58 (m, 1H), 4.44-4.19 (m, 3H), 3.43-3.33 (m, 2H), 1.65-1.21 (m, 17H).

[0897] LC/MS (System A): m/z (ESI.sup.+)=459 [M.sup.+Na.sup.+], R.sub.t=1.35 min, UV purity=93%.

Intermediate 123—Synthesis of (9H-fluoren-9-yl)methyl N-[(1s,4s)-4-aminocyclohexyl]carbamate hydrochloride

[0898] ##STR00168##

[0899] HCl solution in dioxane (4.0 M, 77 ml, 310 mmol) was added to a suspension of (9H-fluoren-9-yl)methyl N-[(1s,4s)-4-{[(tert-butoxy)carbonyl]amino}cyclohexyl]carbamate, Intermediate 122 (6.71 g, 15.4 mmol) in dioxane (80 ml). The reaction was left to stir at RT for 20 h.

[0900] The reaction mixture was concentrated in vacuo to afford the product as a white solid (6.04 g, 92%).

[0901] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.95-7.81 (m, 5H), 7.78-7.62 (m, 2H), 7.46-7.40 (m, 2H), 7.36-7.32 (m, 2H), 7.28-7.20 (m, 1H), 4.36-4.16 (m, 3H), 3.54-3.44 (m, 1H), 3.12-2.99 (m, 1H), 1.86-1.41 (m, 8H). Residual solvent estimate: 6.8 wt % LC/MS (System A): m/z (ESI.sup.+)=337 [MH.sup.+], R.sub.t=0.90 min, UV purity=94%.

Intermediate 124—Synthesis of (9H-fluoren-9-yl)methyl N-[(1s,4s)-4-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(2S,3R)-2,3-dihydroxy-3-[(5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}cyclohexyl] carbamate

[0902] ##STR00169##

[0903] A solution of (9H-fluoren-9-yl)methyl N-[(1s,4s)-4-aminocyclohexyl]carbamate hydrochloride, Intermediate 123 (4.70 g, 12.6 mmol) and 4,6-O-benzylidene-D-glucopyranose (10.1 g, 37.8 mmol) in MeOH (100 ml) was stirred at RT for 0.5 h. NaCNBH3 (3.17 g, 50.4 mmol) was added then the reaction mixture was left to heat at 60° C. for 4.5 h. More 4,6-O-benzylidene-D-glucopyranose (10.1 g, 37.8 mmol) was added then the reaction was left to heat at 60° C. for 20 h. More 4,6-O-benzylidene-D-glucopyranose (10.1 g, 37.8 mmol) was added then the reaction was left to heat at 60° C. for 19 h. The reaction was allowed to cool to RT then added to saturated aqueous NaHCO.sub.3 solution (100 ml) and EtOAc (100 ml). The resulting suspension was left to stir at RT for 2 h then filtered through a Celite pad. The pad was rinsed through with EtOAc (50 ml) then the filtrate was transferred to a separating funnel. Saturated aqueous NaHCO.sub.3 solution (50 ml) was added then the phases were separated. Water (150 ml) was added to the organic phase then the resultant mixture was left to stir at RT for a further 1 h. The resultant mixture was transferred to a separating funnel then the phases were separated. The organic phase was washed with water (150 ml) and brine (100 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford a golden viscous oil (19.1 g). The crude material was dissolved in refluxing isopropanol (200 ml). The mixture was stirred under reflux for 0.5 h then allowed to cool to RT. The solid was collected by filtration, then rinsed with IPA and dried under vacuum to afford the product as a cream solid (7.68 g). A sample of the solid thus obtained (3.0 g) was purified by flash column chromatography on C18 (120 g). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 20%, 2 CV; 20-50%, 10 CV; 50-100%, 2 CV; 100%, 2 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a white solid (1.46 g, 13%)).

[0904] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.35 (s, 1H), 7.85-7.77 (m, 2H), 7.67-7.58 (m, 2H), 7.53-7.43 (m, 4H), 7.41-7.26 (m, 10H), 5.52 (s, 2H), 4.58-4.35 (m, 2H), 4.31-4.23 (m, 2H), 4.23-4.13 (m, 3H), 4.00-3.89 (m, 4H), 3.75 (m, 2H), 3.71-3.65 (m, 1H), 3.62 (m, 2H), 3.44-3.32 (m, 4H), 1.89-1.26 (m, 8H).

[0905] LC/MS (System A): m/z (ESI.sup.+)=841 [MH.sup.+], R.sub.t=1.09 min, UV purity=99%.

Intermediate 125 Synthesis of (1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(1s,4s)-4-aminocyclohexyl]amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol; bis(formic acid)

[0906] ##STR00170##

[0907] Piperidine (615 uL, 6.23 mmol) was added to a solution of (9H-fluoren-9-yl)methyl N-[(1s,4s)-4-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(2S,3R)-2,3-dihydroxy-3-[(5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}cyclohexyl]carbamate, Intermediate 124 (1.46 g, 1.74 mmol) in THF (10 ml). The reaction was left to stir at RT for 21 h then concentrated in vacuo. The residue was suspended in MeOH (10 ml) then filtered. The filtrate was concentrated in vacuo then suspended in MeOH (5 ml) and filtered. The filtrate was purified by flash column chromatography on C18 (60 g). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-32%, 7 CV; 32-40%, 1 CV; 12 CV; 40-100%, 2 CV; 100%, 2 CV. The desired fractions were combined then concentrated in vacuo. The residual aqueous solution was lyophilised to afford the product as a white solid (388 mg, 31%).

[0908] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.40 (s, 2H), 7.51-7.43 (m, 4H), 7.40-7.28 (m, 6H), 5.51 (s, 2H), 4.29-4.20 (m, 2H), 4.08-4.02 (m, 2H), 4.00-3.92 (m, 2H), 3.88 (dd, J=5.0, 2.5 Hz, 2H), 3.72 (dd, J=9.3, 2.5 Hz, 2H), 3.61 (t, J=10.5 Hz, 2H), 3.39-3.34 (m, 1H), 3.07-2.89 (m, 5H), 1.97-1.62 (m, 8H).

[0909] LC/MS (System A): m/z (ESI.sup.+)=619 [MH.sup.+], R.sub.t=0.76 min, UV purity=100%.

Intermediate 126 Synthesis of (2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl][(1s,4s)-4-aminocyclohexyl]amino}hexane-1,2,3,4,5-pentol

[0910] ##STR00171##

[0911] A solution of (1R,2S)-3-{[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl][(1s,4s)-4-aminocyclohexyl]amino}-1-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propane-1,2-diol; bis(formic acid), Intermediate 125 (385 mg, 0.622 mmol) in aqueous HCl solution (2 M, 6 ml, 12 mmol) was stirred at RT for 4 h then concentrated in vacuo. The resulting oil was dissolved in water (10 ml) then lyophilised to afford a white foam (323 mg). The material thus obtained was dissolved in water/MeOH then loading on to a pre-wetted SCX cartridge (5 g). The cartridge was eluted with MeOH then the product was released by extensive elution with 7 M ammonia solution in MeOH. The ammonia eluent was concentrated in vacuo. The residue was dissolved in water/MeCN then lyophilised to afford the product as a white solid (212 mg, 77%).

[0912] .sup.1H NMR (500 MHz, D.sub.2O) δ 4.30-4.20 (m, 2H), 3.90-3.62 (m, 12H), 3.57-3.49 (m, 1H), 3.43-3.31 (m, 2H), 2.21-2.09 (m, 3H), 2.06-1.90 (m, 4H), 1.85-1.70 (m, 2H).

[0913] LC/MS (System A): m/z (ESI.sup.+)=443 [MH.sup.+], R.sub.t=0.14 min, ELS purity=100%.

Intermediate 127—Synthesis of formic acid; tert-butyl N-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)-N-methylcarbamate

[0914] ##STR00172##

[0915] alpha-picoline borane (0.861 g, 8.05 mmol) was added to a suspension of tert-butyl N-(3-aminopropyl)-N-methylcarbamate (0.505 g, 2.68 mmol) and 4,6-O-benzylidene-D-glucopyranose (2.88 g, 10.7 mmol) in MeOH (5 ml). The mixture was heated at 60° C. for 16 h. The reaction mixture was allowed to cool to RT then partitioned between EtOAc (20 ml) and water (20 ml). The phases were separated then the aqueous phase was extracted with EtOAc (20 ml). The combined organic phases were washed with water (20 ml) and 1:1 water:brine (20 ml) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude material was purified by flash column chromatography on C18 (60 g). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-40%, 10 CV; 40-100%, 2 CV; 100%, 2 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a pale yellow foam (1.19 g, 60%).

[0916] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.40 (s, 1H), 7.57-7.44 (m, 4H), 7.44-7.32 (m, 6H), 5.55 (s, 2H), 4.31-4.23 (m, 2H), 4.24-4.15 (m, 2H), 4.00-3.90 (m, 4H), 3.79 (dd, J=9.4, 2.2 Hz, 2H), 3.64 (t, J=10.5 Hz, 2H), 3.41-3.31 (m, 3H), 3.25-3.08 (m, 4H), 2.76-2.64 (m, 3H), 1.88-1.75 (m, 2H), 1.50-1.39 (m, 9H).

[0917] LC/MS (System A): m/z (ESI.sup.+)=693 [MH.sup.+], R.sub.t=0.94 min, ELS purity=100%.

Intermediate 128 Synthesis of (2R,3R,4R,5S)-6-{[3-(methylamino)propyl][(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}hexane-1,2,3,4,5-pentol dihydrochloride

[0918] ##STR00173##

[0919] A suspension of formic acid; tert-butyl N-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)-N-methylcarbamate, Intermediate 127 (1.18 g, 1.60 mmol) in aqueous HCl solution (2 M, 30 ml, 60 mmol) and MeOH (1 ml) was stirred at RT for 4 h then concentrated in vacuo. The residue was dissolved in water (30 ml) then lyophilised to afford the product as a cream foam (770 mg, 99%).

[0920] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 4.29-4.20 (m, 2H), 3.96-3.86 (m, 2H), 3.83-3.77 (m, 2H), 3.77-3.65 (m, 6H), 3.63-3.39 (m, 6H), 3.18-3.10 (m, 2H), 2.77 (s, 3H), 2.28-2.18 (m, 2H).

[0921] LC/MS (System A): m/z (ESI.sup.+)=417 [MH.sup.+], R.sub.t=0.13 min, ELS purity=100%.

Intermediate 129—Synthesis of formic acid; tert-butyl N-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethyl)carbamate

[0922] ##STR00174##

[0923] 4,6-O-Benzylidene-D-glucopyranose (10.05 g, 37.45 mmol) was added to a solution of tert-butyl N-(2-aminoethyl)carbamate (1.50 g, 9.36 mmol) in MeOH (50 ml). The reaction was stirred at RT for 15 min before then AcOH (2.14 ml, 37.5 mmol) was added. The reaction was stirred at RT for a further 15 min then NaCNBH.sub.3 (2.35 g, 37.5 mmol) was added portionwise over 5 min. The reaction was stirred at RT for 16 h then saturated aqueous NaHCO.sub.3 solution (50 ml) was added dropwise over 15 min. Further saturated aqueous NaHCO.sub.3 solution (50 ml) was added, followed by EtOAc (50 ml). The reaction was stirred at RT for 15 min then transferred to a separating funnel. More EtOAc (100 ml) was added then the phases were separated. The aqueous phase was extracted with EtOAc (150 ml), then the combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (4×200 ml) and brine (50 ml), then dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude material was purified by flash column chromatography on C18 (400 g, Ultra). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-49%, 8 CV; 49-54%, 0.5 CV; 54-100%, 1 CV. The desired fractions were combined and concentrated in vacuo then the residual aqueous solution was lyophilised to afford the product as a white solid (2.77 g, 42%).

[0924] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.34 (s, 1H), 7.53-7.43 (m, 4H), 7.40-7.26 (m, 6H), 5.52 (s, 2H), 4.25 (dd, J=10.7, 5.4 Hz, 2H), 4.14 (q, J=5.7 Hz, 2H), 3.94 (td, J=10.0, 5.4 Hz, 2H), 3.89 (dd, J=5.0, 2.3 Hz, 2H), 3.75 (dd, J=9.4, 2.2 Hz, 2H), 3.61 (t, J=10.5 Hz, 2H), 3.26-2.99 (m, 8H), 1.42 (s, 9H).

[0925] LC/MS (System A): m/z (ESI.sup.+)=665 [MH.sup.+], R.sub.t=0.94 min, UV purity=100%.

Intermediate 130 Synthesis of (2R,3R,4R,5S)-6-[(2-aminoethyl)[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]hexane-1,2,3,4,5-pentol dihydrochloride

[0926] ##STR00175##

[0927] A suspension of formic acid; tert-butyl N-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethyl)carbamate, Intermediate 129 (1.50 g, 2.11 mmol) in aqueous HCl (2 M, 21 ml, 42 mmol) was stirred at RT for 68 h. The reaction was concentrated in vacuo, then re-dissolved in MeCN/water and concentrated in vacuo. The residue was re-dissolved in MeCN/water then lyophilised to afford the product as a white solid (1.03 g, 99% based on 94% estimated purity).

[0928] .sup.1H NMR (500 MHz, D.sub.2O) δ 4.30 (dt, J=8.9, 4.7 Hz, 2H), 3.88 (dd, J=4.9, 2.2 Hz, 2H), 3.86-3.71 (m, 6H), 3.71-3.66 (m, 4H), 3.61-3.50 (m, 6H).

[0929] LC/MS (System A): m/z (ESI.sup.+)=389 [MH.sup.+], R.sub.t=0.14 min, ELS purity=100%.

Intermediate 131 Synthesis of tert-butyl N-[2-(2-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}ethoxy)ethyl]carbamate

[0930] ##STR00176##

[0931] NaHCO.sub.3 (0.574 g, 6.84 mmol) was added portionwise over 2 min to a stirred solution of tert-butyl N-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethyl]carbamate (1.00 g, 3.42 mmol) in MeCN (15 ml) and water (15 ml) RT. A solution of (2,5-dioxopyrrolidin-1-yl) 9H-fluoren-9-ylmethyl carbonate (1.15 g, 3.42 mmol) in MeCN (15 ml) was added dropwise over 30 min.

[0932] The resulting solution was stirred at RT for 18 h. EtOAc (30 ml) and brine (10 ml) were added then the phases were separated. The aqueous phase was extracted with EtOAc (20 ml). The combined organic extracts were washed with brine (45 ml), dried over MgSO4, then concentrated in vacuo to afford the product as a yellow gum (1.61 g, 76% yield).

[0933] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.89 (d, J=7.5 Hz, 2H), 7.72-7.61 (m, 2H), 7.41 (t, J=7.4 Hz, 2H), 7.37-7.27 (m, 3H), 6.74 (t, J=5.4 Hz, 1H), 4.29 (d, J=6.9 Hz, 2H), 4.21 (t, J=6.9 Hz, 1H), 3.55-3.44 (m, 8H), 3.40 (t, J=6.0 Hz, 2H), 3.36 (t, J=6.1 Hz, 2H), 3.13 (q, J=5.9 Hz, 2H), 3.05 (q, J=6.0 Hz, 2H), 1.36 (s, 9H).

[0934] LC/MS (System A): m/z (ESI.sup.+)=537 [M.sup.+Na.sup.+], R.sub.t=1.28 min, UV purity=83%.

Intermediate 132—Synthesis of (9H-fluoren-9-yl)methyl N-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamate hydrochloride

[0935] ##STR00177##

[0936] HCl solution in dioxane (4 M, 2 ml, 8 mmol) was added to a stirred solution of tert-butyl N-[2-(2-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}ethoxy)ethyl]carbamate, Intermediate 131 (83%, 1.61 g, 2.61 mmol) in MeCN (16 ml). The resulting solution was left to stir at RT for 22 then concentrated in vacuo to afford the product as a yellow gum (1.57 g, quantitative based on 75% estimated purity).

[0937] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.89 (d, J=7.5 Hz, 2H), 7.83 (s, 3H), 7.69 (d, J=7.4 Hz, 2H), 7.42 (t, J=7.4 Hz, 2H), 7.37-7.29 (m, 3H), 4.30 (d, J=6.9 Hz, 2H), 4.21 (t, J=6.8 Hz, 1H), 3.60-3.57 (m, 2H), 3.56-3.48 (m, 8H), 3.40 (t, J=6.0 Hz, 2H), 3.13 (q, J=5.9 Hz, 2H), 2.98-2.92 (m, 2H). 20 wt % residual solvent.

[0938] LC/MS (System A): m/z (ESI.sup.+)=415 [MH.sup.+], R.sub.t=0.89 min, UV purity=92%.

Intermediate 133—Synthesis of (9H-fluoren-9-yl)methyl N-[(14S,15R)-12-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]-14,15-dihydroxy-15-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]-3,6,9-trioxa-12-azapentadecan-1-yl]carbamate; formic acid

[0939] ##STR00178##

[0940] AcOH (737 μL, 12.9 mmol) was added to a solution of (9H-fluoren-9-yl)methyl N-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamate hydrochloride, Intermediate 132 (75%, 1.57 g, 3.22 mmol) and 4,6-O-benzylidene-D-glucopyranose (3.46 g, 12.9 mmol) in MeOH (30 ml). The reaction was stirred at RT for 45 min. NaCNBH.sub.3 (809 mg, 12.9 mmol) was added portionwise over 50 min. The resultant solution was stirred at RT for 40 h. The reaction mixture was treated with 4,6-O-benzylidene-D-glucopyranose (1.73 g, 6.45 mmol), AcOH (368 μL, 6.43 mmol) and MeOH (10 ml). The reaction was stirred at RT for 1 h then NaCNBH.sub.3 (403 mg, 6.41 mmol) was added portionwise over 20 min. MeOH (10 ml) was added then the reaction mixture was stirred at RT for 70 h. Saturated aqueous NaHCO.sub.3 solution (100 ml) was added portionwise over 5 min then EtOAc (100 ml) was added. The phases were separated then the aqueous phase was extracted with EtOAc (3×30 ml). The combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (2×50 ml), brine (50 ml), then dried over MgSO.sub.4, filtered and concentrated in vacuo to afford a brown gum (3.86 g). A portion (2 g) of the crude material was purified by flash column chromatography on C18 (120 g, Ultra). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-31%, 8 CV; 31%, 4.5 CV; 31-35%, 1.5 CV; 35-47%, 1 CV; 47-61%, 1.5 CV; 100% 2 CV. The remaining crude material was purified by flash column chromatography on C18 (120 g, Ultra). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-27%, 4 CV; 27%, 0.5 CV; 27-68%, 9 CV; 68%, 0.5 CV; 68-78%, 2 CV; 78-100%, 1.5 CV; 100% 1 CV. The desired fractions from both columns were combined and concentrated in vacuo to afford the product as a light brown resin (1.30 g, 48%).

[0941] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.44 (s, 1H), 7.81 (d, J=7.5 Hz, 2H), 7.68-7.59 (m, 2H), 7.52-7.44 (m, 4H), 7.44-7.37 (m, 2H), 7.37-7.30 (m, 8H), 5.52 (s, 2H), 4.36 (d, J=6.8 Hz, 2H), 4.28-4.15 (m, 5H), 3.98-3.88 (m, 4H), 3.75 (dd, J=9.4, 2.3 Hz, 2H), 3.67-3.44 (m, 14H), 3.40-3.33 (m, 4H), 3.31-3.21 (m, 4H).LC/MS (System A): m/z (ESI.sup.+)=919 [MH.sup.+], R.sub.t=1.02 min, UV purity=93%.

Intermediate 134 Synthesis of (14S,15R)-1-amino-12-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]-15-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]-3,6,9-trioxa-12-azapentadecane-14,15-diol: bis(formic acid)

[0942] ##STR00179##

[0943] Piperidine (1.34 ml, 13.6 mmol) was added to a stirred solution of (9H-fluoren-9-yl)methyl N-[(14S,15R)-12-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]-14,15-dihydroxy-15-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]-3,6,9-trioxa-12-azapentadecan-1-yl]carbamate; formic acid, Intermediate 133 (93%, 1.25 g, 1.20 mmol) in THF (15 ml). The resulting solution was stirred at RT for 6 h then concentrated in vacuo. The residue thus obtained was suspended in Et.sub.2O (10 ml) with sonication. The supernatant was decanted off then the process was repeated with more Et.sub.2O (10 ml). The residue thus obtained was purified by flash column chromatography on C18 (120 g, Ultra).

[0944] The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-13%, 1.5 CV; 13%, 2.5 CV; 13-22%, 5.5 CV; 22-60%, 5.5 CV; 60-83%, 2 CV; 100% 2 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a yellow solid (849 mg, 89%).

[0945] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.40 (s, 2H), 7.54-7.43 (m, 4H), 7.41-7.28 (m, 6H), 5.53 (s, 2H), 4.26 (dd, J=10.7, 5.4 Hz, 2H), 4.20-4.12 (m, 2H), 3.97-3.87 (m, 4H), 3.74 (dd, J=9.4, 2.6 Hz, 2H), 3.70-3.54 (m, 14H), 3.29-3.19 (m, 3H), 3.16-3.08 (m, 2H), 3.07-2.99 (m, 1H), 2.98-2.86 (m, 2H).

[0946] LC/MS (System A): m/z (ESI.sup.+)=697 [MH.sup.+], R.sub.t=0.74 min, UV purity=100%.

Intermediate 135 Synthesis of (14S,15R,16R,17R)-1-amino-12-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]-3,6,9-trioxa-12-azaoctadecane-14,15,16,17,18-pentol dihydrochloride

[0947] ##STR00180##

[0948] A solution of (14S,15R)-1-amino-12-[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]-15-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]-3,6,9-trioxa-12-azapentadecane-14,15-diol; bis(formic acid), Intermediate 134 (845 mg, 1.07 mmol) in aqueous HCl solution (2 M, 10 ml, 20 mmol) was stirred at RT for 5.5 h then concentrated in vacuo. The residue thus obtained was re-dissolved in water (15 ml) then lyophilised to afford a pale yellow gum (660 mg, quantitative based on 96% estimated purity).

[0949] .sup.1H NMR (500 MHz, D.sub.2O) δ 4.30-4.22 (m, 2H), 3.96-3.91 (m, 2H), 3.88-3.82 (m, 4H), 3.81-3.63 (m, 17H), 3.59-3.48 (m, 5H), 3.26-3.20 (m, 2H).

[0950] LC/MS (System C): m/z (ESI.sup.+)=521 [MH.sup.+], R.sub.t=0.33 min, ELS purity=100%.

Intermediate 136 Synthesis of tert-butyl N-[2-(4-bromophenyl)ethyl]carbamate

[0951] ##STR00181##

[0952] Di-tert-butyl dicarbonate (3.93 g, 18.0 mmol) was added to a cooled (0° C.) stirred solution of 2-(4-bromophenyl)ethanamine (3.00 g, 15.0 mmol) in THF (20 ml). The resulting solution was allowed to warm to RT then stirred at RT for 18 h. The reaction mixture was partitioned between EtOAc (30 ml) and saturated aqueous NaHCO.sub.3 solution (50 ml). The phases were separated then the aqueous phase was extracted with EtOAc (15 ml). The combined organic phases were washed with brine (50 ml), dried over MgSO.sub.4, then concentrated in vacuo. The crude material was dissolved in the minimum volume of CH.sub.2Cl.sub.2, pre-adsorbed onto silica, then purified by flash column chromatography on a silica column (25 g). The column was eluted with EtOAc:heptane, using the following gradient (% EtOAc, column volumes): 0%, 1 CV; 0-8%, 2.5 CV; 8-12%, 1 CV; 12%, 3.5 CV; 12-27%, 5.5 CV; 27-30%, 0.5 CV; 30%, 2 CV; 30-90%, 4 CV; 100% 1 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a white solid (3.99 g, 88%).

[0953] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.50-7.42 (m, 2H), 7.19-7.11 (m, 2H), 6.86 (t, J=5.3 Hz, 1H), 3.12 (q, J=6.6 Hz, 2H), 2.70-2.63 (m, 2H), 1.41-1.26 (m, 9H).

[0954] LC/MS (System A): R.sub.t=1.27 min, UV purity=99%.

Intermediate 137 Synthesis of tert-butyl N-{2-[4′-(cyanomethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate

[0955] ##STR00182##

[0956] A mixture of tert-butyl N-[2-(4-bromophenyl)ethyl]carbamate, Intermediate 136 (4.09 g, 13.6 mmol), [4-cyanomethyl)phenyl]boronic acid (2.63 g, 16.4 mmol) and K.sub.2CO.sub.3 (5.65 g, 40.9 mmol) in 1,4-dioxane (105 ml) was degassed by bubbling a stream nitrogen through the mixture for 5 min. Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (445 mg, 0.545 mmol) was added and degassing was continued for a further 5 min. The reaction mixture was heated at 80° C. for 15 h then at 100° C. for 7 h. The reaction was allowed to cool to RT then retreated with K.sub.2CO.sub.3 (3.76 g, 27.2 mmol) and degassed for 5 min. Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (445 mg, 0.545 mmol) was added then the mixture was degassed for a further 5 min. The resultant mixture was heated at 100° C. for 24 h then allowed to cool to RT. The reaction was retreated with K.sub.2CO.sub.3 (1.88 g, 13.6 mmol) and [4-cyanomethyl)phenyl]boronic acid (0.88 g, 5.5 mmol) then degassed for 10 min. Pd(dppf)Cl.sub.2.CH.sub.2Cl.sub.2 (445 mg, 0.545 mmol) was added then the mixture was degassed for a further 5 min. The reaction was heated at 100° C. for 18 h then allowed to cool to RT. The reaction mixture was filtered then the collected solids were washed with EtOAc (50 ml). The combined filtrate was concentrated in vacuo. The residue was re-dissolved in EtOAc:heptane (1:1) then filtered through a silica pad. The pad was rinsed with EtOAc:heptane (1:1, 200 ml). The filtrate was concentrated in vacuo to afford an off-white solid (3.94 g). The silica pad was rinsed through further with EtOAc (200 ml) to afford a brown solid (1.68 g). The brown solid from the EtOAc filtrate was pre-adsorbed onto silica, then purified by flash column chromatography on a silica column (50 g). The column was eluted with EtOAc:heptane, using the following gradient (% EtOAc, column volumes): 0%, 1 CV; 0-30%, 11 CV; 30%, 20 CV; 30-45%, 4.5 CV; 45%, 7.5 CV; 45-50%, 1 CV; 50%, 15 CV. The desired fractions were combined and concentrated in vacuo to afford an off-white solid (1.00 g, 21%).

[0957] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.67 (d, J=8.2 Hz, 2H), 7.59 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.2 Hz, 2H), 7.28 (d, J=8.1 Hz, 2H), 6.90 (t, J=5.5 Hz, 1H), 4.07 (s, 2H), 3.17 (q, J=6.5 Hz, 2H), 2.73 (t, J=7.4 Hz, 2H), 1.44-1.29 (m, 9H).

[0958] LC/MS (System A): R.sub.t=1.27 min, UV purity=97%.

Intermediate 138—Synthesis of tert-butyl N-{2-[4′-(2-aminoethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate

[0959] ##STR00183##

[0960] A mixture of tert-butyl N-{2-[4′-(cyanomethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate, Intermediate 137 (570 mg, 1.69 mmol), aqueous ammonia solution (35%, 0.5 ml) and aqueous Raney nickel slurry (50%, 2 ml) in EtOH (15 ml) and DMF (5 ml) was stirred under a hydrogen atmosphere for 18 h. The reaction mixture was filtered through a Celite pad. The pad was rinsed with EtOH (50 ml) and MeOH (100 ml) then the combined filtrate was concentrated in vacuo. The residue was azeotroped with heptane (3×100 ml) then dried in vacuo to afford the product as an off-white solid (515 mg, 84%).

[0961] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.60-7.50 (m, 4H), 7.31-7.22 (m, 4H), 6.89 (t, J=5.3 Hz, 1H), 3.19-3.13 (m, 2H), 2.78 (t, J=7.2 Hz, 2H), 2.72 (t, J=7.4 Hz, 2H), 2.66 (t, J=7.2 Hz, 2H), 1.43-1.29 (m, 9H).

[0962] LC/MS (System A): m/z (ESI.sup.+)=341 [MH.sup.+], R.sub.t=0.93 min, UV purity=94%.

Intermediate 139—Synthesis of tert-butyl N-{2-[4′-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate

[0963] ##STR00184##

[0964] AcOH (0.33 ml, 5.8 mmol) was added to a solution of tert-butyl N-{2-[4′-(2-aminoethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate, Intermediate 138 (94%, 515 mg, 1.42 mmol) and 4,6-O-benzylidene-D-glucopyranose (1.58 g, 5.89 mmol) in MeOH (50 ml). The reaction was left to stir at RT for 50 min then NaCNBH.sub.3 (370 mg, 5.89 mmol) was added portionwise over 25 min. The resulting solution was stirred at RT for 24 h. Further 4,6-O-benzylidene-D-glucopyranose (790 mg, 2.94 mmol), AcOH (0.17 ml, 3.0 mmol) and MeOH (50 ml) were added then the reaction was left to stir at RT for 40 min. NaCNBH.sub.3 (185 mg, 2.94 mmol) was added portionwise over 20 min then the reaction was left to stir at RT for 68 h. Further 4,6-O-benzylidene-D-glucopyranose (790 mg, 2.94 mmol), AcOH (0.17 ml, 3.0 mmol) and MeOH (50 ml) were added then the reaction was left to stir at RT for 30 min. NaCNBH.sub.3 (185 mg, 2.94 mmol) was added portionwise over 20 min then the reaction was left to stir at RT for 18 h. Further 4,6-O-benzylidene-D-glucopyranose (790 mg, 2.94 mmol) and MeOH (25 ml) were added then the reaction heated at 40° C. for 18 h. The reaction mixture was allowed to cool to RT then saturated aqueous NaHCO.sub.3 solution (40 ml) was added in portions over 15 min. The resultant mixture was stirred at RT for 30 min then the solid was collected by filtration, rinsed with water (10 ml), then dried in vacuo. The crude solid material thus obtained was purified by flash column chromatography on C18 (120 g). The column was eluted with MeCN:H.sub.2O+0.1% NH.sub.4OH using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-59%, 10 CV; 59%, 2 CV; 59-100%, 8 CV; 100%, 2 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a white solid (932 mg, 78%).

[0965] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.57-7.49 (m, 2H), 7.46-7.37 (m, 6H), 7.34-7.29 (m, 6H), 7.28-7.23 (m, 2H), 7.09-7.01 (m, 2H), 6.90 (t, J=5.6 Hz, 1H), 5.48 (s, 2H), 5.14 (d, J=5.8 Hz, 2H), 4.50-4.40 (m, 4H), 4.12 (dd, J=10.5, 5.3 Hz, 2H), 3.87-3.75 (m, 4H), 3.75-3.69 (m, 2H), 3.67-3.60 (m, 2H), 3.50 (t, J=10.4 Hz, 2H), 3.21-3.10 (m, 2H), 2.82-2.65 (m, 8H), 2.57 (dd, J=12.9, 8.9 Hz, 2H), 1.43-1.29 (m, 9H).

[0966] LC/MS (System B): m/z (ESI.sup.+)=845 [MH.sup.+], R.sub.t=4.80 min, UV purity=100%.

Intermediate 140—Synthesis of (2R,3R,4R,5S)-6-({2-[4′-(2-aminoethyl)-[1,1′-biphenyl]-4-yl]ethyl}[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino)hexane-1,2,3,4,5-pentol dihydrochloride

[0967] ##STR00185##

[0968] A suspension of tert-butyl N-{2-[4′-(2-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}ethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate, Intermediate 139 (932 mg, 1.10 mmol) in aqueous HCl solution (2 M, 8.5 ml, 17 mmol) was stirred at RT for 24 h then further aqueous HCl solution (2 M, 8.5 ml, 17 mmol) was added. The reaction was left to stir at RT for a further 24 h. The reaction mixture was heated at 40° C. for 4 h then concentrated in vacuo. The residue thus obtained was dissolved in water (15 ml) then lyophilised to afford the product as a white resin (753 mg, quantitative based on 94% estimated purity).

[0969] .sup.1H NMR (500 MHz, D.sub.2O) δ 7.77-7.70 (m, 4H), 7.53-7.48 (m, 2H), 7.48-7.43 (m, 2H), 4.30-4.19 (m, 2H), 3.87-3.61 (m, 12H), 3.61-3.45 (m, 4H), 3.33 (t, J=7.4 Hz, 2H), 3.29-3.16 (m, 2H), 3.07 (t, J=7.4 Hz, 2H).

[0970] LC/MS (System A): m/z (ESI.sup.+)=569 [MH.sup.+], R.sub.t=0.15 min, ELS purity=100%.

Intermediate 141—Synthesis of (2S)-2-{[(benzyloxy)carbonyl]amino}-4-{[(tert-butoxy)carbonyl]amino}butanoic acid

[0971] ##STR00186##

[0972] NaHCO.sub.3 (3.13 g, 37.3 mmol) was added to a solution of (2S)-4-amino-2-{[(benzyloxy)carbonyl]amino}butanoic acid (4.70 g, 18.6 mmol) in THF (50 ml) and water (50 ml). The reaction was stirred at RT for 5 min then a solution of di-tert-butyl dicarbonate (4.88 g, 22.4 mmol) in THF (50 ml) was added dropwise over 10 min. The reaction was stirred at RT for 16 h then concentrated in vacuo to remove the majority of the THF. The residual aqueous solution was acidified to pH 2 by dropwise addition of 2 M aqueous HCl solution then extracted with EtOAc (100 ml then 50 ml). The combined organic phases were washed with water (50 ml) and brine (50 ml), then dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford the product as a colourless oil (6.65 g, 98%).

[0973] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 12.60 (s, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.41-7.24 (m, 5H), 6.87-6.76 (m, 1H), 5.03 (s, 2H), 4.00-3.92 (m, 1H), 3.06-2.90 (m, 2H), 1.92-1.77 (m, 1H), 1.71-1.59 (m, 1H), 1.37 (s, 9H).

[0974] LC/MS (System A): m/z (ESI.sup.+)=375 [M.sup.+Na.sup.+], R.sub.t=1.06 min, UV purity=97%.

Intermediate 142—Synthesis of tert-butyl N-[(3S)-3-{[(benzyloxy)carbonyl]amino}-3-carbamoylpropyl]carbamate

[0975] ##STR00187##

[0976] Isobutyl carbonochloridate (2.94 ml, 22.7 mmol) was added dropwise to a cooled (0°) solution of (2S)-2-{[(benzyloxy)carbonyl]amino}-4-{[(tert-butoxy)carbonyl]amino}butanoic acid, Intermediate 141 (6.15 g, 17.5 mmol) and N-methylmorpholine (2.88 ml, 26.2 mmol) in THF (100 ml). The reaction was allowed to warm to RT then stirred at RT for 17 h. The solution was cooled to 0° C. then ammonia solution in MeOH (7 M, 12 ml, 84 mmol) was added dropwise over 5 min. The reaction mixture was allowed to warm to RT then stirred at RT for 3 h. The resultant suspension was filtered then the filtrate was concentrated in vacuo. The residue was partitioned between EtOAc (200 ml) and saturated aqueous NaHCO.sub.3 solution (100 ml). The phases were separated then the organic layer was washed with water (50 ml). The organic phase was concentrated in vacuo to afford an off white solid. The solid thus obtained was suspended in MeCN (50 ml) then filtered. The solid was dried in vacuo to afford the product as a white solid (2.56 g, 40%) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.42-7.24 (m, 7H), 7.08-6.97 (m, 1H), 6.80-6.68 (m, 1H), 5.09-4.92 (m, 2H), 3.98-3.86 (m, 1H), 3.03-2.88 (m, 2H), 1.82-1.68 (m, 1H), 1.67-1.54 (m, 1H), 1.44-1.30 (m, 9H).

[0977] LC/MS (System A): m/z (ESI.sup.+)=374 [M.sup.+Na.sup.+], R.sub.t=1.52 min, UV purity=97%.

Intermediate 143—Synthesis of tert-butyl N-[(3S)-3-amino-3-carbamoyl propyl]carbamate

[0978] ##STR00188##

[0979] A mixture of tert-butyl N-[(3S)-3-{[(benzyloxy)carbonyl]amino}-3-carbamoylpropyl]carbamate, Intermediate 142 (2.06 g, 5.85 mmol) and 10% Pd/C (50% wet, 0.31 g) (50% wet) in THF (12 ml) and EtOH (12 ml) was stirred under an atmosphere of hydrogen for 18 h. The reaction was filtered through glass fibre filter paper, then concentrated in vacuo to afford the product as a white solid (1.27 g, quantitative).

[0980] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.26 (s, 1H), 6.91 (s, 1H), 6.83-6.69 (m, 1H), 3.11-2.92 (m, 3H), 1.80-1.59 (m, 3H), 1.46-1.32 (m, 10H).

[0981] LC/MS (System A): m/z (ESI.sup.+)=218 [MH.sup.+].

Intermediate 144—Synthesis of tert-butyl N-[(3S)-3-[(3-{[(benzyloxy)carbonyl]amino}propyl)amino]-3-carbamoylpropyl]carbamate

[0982] ##STR00189##

[0983] Sodium triacetoxyborohydride (2.37 g, 11.2 mmol) was added portionwise over 5 min to a solution of tert-butyl N-[(3S)-3-amino-3-carbamoylpropyl]carbamate, Intermediate 143 (1.62 g, 7.46 mmol) and benzyl N-(3-oxopropyl)carbamate (1.55 g, 7.46 mmol) in THF (40 ml). The reaction was stirred at RT for 16 h then water (50 ml) was added. The mixture was extracted with EtOAc (4×50 ml). The combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (2×50 ml) and brine (10 ml), then dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford the product as a colourless oil (2.89 g, 85%).

[0984] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.39-7.22 (m, 5H), 5.06 (s, 2H), 3.25-3.06 (m, 5H), 2.69-2.45 (m, 2H), 1.85-1.61 (m, 4H), 1.42 (s, 9H).

[0985] LC/MS (System A): m/z (ESI.sup.+)=409 [MH.sup.+], R.sub.t=0.87 min, UV purity=90%.

Intermediate 145—Synthesis of tert-butyl N-(3-{[(benzyloxy)carbonyl]amino}propyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate

[0986] ##STR00190##

[0987] A solution of di-tert-butyl dicarbonate (1.85 g, 8.49 mmol) in THF (10 ml) was dropwise over 5 min to a solution of tert-butyl N-[(3S)-3-[(3-{[(benzyloxy)carbonyl]amino}propyl)amino]-3-carbamoylpropyl]carbamate, Intermediate 144 (2.89 g, 7.07 mmol)) and triethylamine (1.47 ml, 10.6 mmol) in THF (30 ml). The reaction mixture was left to stir at RT for 16 h then concentrated in vacuo. The resultant oil was partitioned between EtOAc (50 ml) and water (50 ml). The phases were separated then the organic phase was washed with saturated aqueous NaHCO.sub.3 solution (50 ml) and brine (20 ml), then dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude material thus obtained was purified by flash column chromatography on a silica column (50 g). The column was eluted with CH.sub.2Cl.sub.2:MeOH, increasing the gradient linearly from 100:0 to 92:8 over 15 CV. The desired fractions were combined and concentrated in vacuo. The material thus obtained was further purified by flash column chromatography on C18 (120 g). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 5%, 2 CV; 5-100%, 20 CV; 100%, 2 CV. The desired fractions were combined and concentrated in vacuo to afford the product as an orange oil (0.42 g, 12%).

[0988] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.42-7.28 (m, 5H), 7.27-6.96 (m, 3H), 6.92-6.61 (m, 1H), 5.08-4.93 (m, 2H), 4.43-3.93 (m, 1H), 3.21-2.78 (m, 6H), 2.03-1.52 (m, 4H), 1.46-1.30 (m, 18H).

[0989] LC/MS (System A): m/z (ESI.sup.+)=509 [MH.sup.+], R.sub.t=1.16 min, UV purity=95%.

Intermediate 146 Synthesis of tert-butyl N-(3-aminopropyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate

[0990] ##STR00191##

[0991] A mixture of tert-butyl N-(3-{[(benzyloxy)carbonyl]amino}propyl)-N-[(1 S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate, Intermediate 145 (780 mg, 1.47 mmol) and 10% Pd/C (50% wet, 80 mg) in EtOH (20 ml) was stirred under an atmosphere of hydrogen for 40 h at RT. The reaction mixture was filtered through glass fibre filter paper then concentrated in vacuo to afford the product as a white foam (580 mg, quantitative based on 95% estimated purity).

[0992] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.19 (s, 1H), 7.01 (s, 1H), 6.89-6.66 (m, 1H), 4.40-3.85 (m, 1H), 3.18-2.83 (m, 5H), 2.49-2.37 (m, 2H+solvent), 2.05-1.82 (m, 1H), 1.74-1.30 (m, 22H).

[0993] LC/MS (System A): m/z (ESI.sup.+)=375 [MH.sup.+], R.sub.t=0.80 min, UV purity=100%.

Intermediate 147 Synthesis of formic acid; tert-butyl N-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate

[0994] ##STR00192##

[0995] 4,6-O-Benzylidene-D-glucopyranose (1.60 g, 5.96 mmol) was added to a solution of tert-butyl N-(3-aminopropyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate, Intermediate 146 (0.558 g, 1.49 mmol) in MeOH (25 ml). The reaction mixture was stirred at RT for 15 min then AcOH (0.341 ml, 5.96 mmol) was added. The reaction mixture was stirred at RT for a further 15 min then NaCNBH.sub.3 (0.375 g, 5.96 mmol) was added portion-wise over 5 min. The reaction mixture was stirred at RT for 64 h. The reaction was re-treated with 4,6-O-benzylidene-D-glucopyranose (1.6 g, 5.96 mmol) and stirred for a further 24 h at RT. Saturated aqueous sodium bicarbonate solution (25 ml) was added dropwise over 5 min. EtOAc (20 ml) was added then the resultant mixture was left to stir at RT for 15 min. The phases were separated then the organic phase was washed with saturated aqueous sodium bicarbonate solution (4×50 ml) and brine (25 ml). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude material was purified by flash column chromatography on C18 (120 g). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CV; 10-46%, 8 CV; 46-52%, 1 CV; 52%, 2 CV; 52-97, 2 CV. The desired fractions were combined and concentrated in vacuo to afford the product as a white solid (690 mg, 50%).

[0996] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.39 (s, 1H), 7.54-7.42 (m, 4H), 7.41-7.26 (m, 6H), 5.59-5.47 (m, 2H), 4.33-4.13 (m, 4H), 4.02-3.87 (m, 5H), 3.81-3.70 (m, 2H), 3.67-3.55 (m, 2H), 3.42-3.34 (m, 6H+solvent), 3.20-2.99 (m, 4H), 1.98-1.78 (m, 4H), 1.52-1.33 (m, 18H).

[0997] LC/MS (System A): m/z (ESI.sup.+)=879 [MH.sup.+], R.sub.t=0.95 min, UV purity=100%.

Intermediate 148 Synthesis of (2S)-4-amino-2-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]butanamide trihydrochloride

[0998] ##STR00193##

[0999] A suspension of formic acid; tert-butyl N-(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)-N-[(1S)-3-{[(tert-butoxy)carbonyl]amino}-1-carbamoylpropyl]carbamate, Intermediate 147 (690 mg, 0.746 mmol) in aqueous HCl solution (2.0 M, 7.5 ml, 15 mmol) was stirred at RT for 16 h. The reaction mixture was concentrated in vacuo. The residue was dissolved in MeCN/water then lyophilised to afford the product as a white solid (500 mg, quantitative based on 91% estimated purity).

[1000] .sup.1H NMR (500 MHz, D.sub.2O) δ 4.31-4.21 (m, 2H), 4.15-4.08 (m, 1H), 3.91-3.74 (m, 6H), 3.73-3.61 (m, 4H), 3.58-3.42 (m, 6H), 3.29-3.10 (m, 4H), 2.43-2.20 (m, 4H).

[1001] LC/MS (System A): m/z (ESI.sup.+)=503 [MH.sup.+], R.sub.t=0.13 min, ELS purity=100%.

Intermediate 149 Synthesis of 4-[4-(4-aminobutyl)phenyl]-2-[(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)amino]butanamide; tetrakis(acetic acid)

[1002] ##STR00194##

[1003] Intermediate 149 was synthesised according to literature procedures (WO2014/099673 A1).

Intermediate 150 Synthesis of 4-[4-(4-aminobutyl)phenyl]-2-[(3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}propyl)amino]butanamide trihydrochloride

[1004] ##STR00195##

[1005] A solution of 4-[4-(4-aminobutyl)phenyl]-2-[(3-{bis[(2S,3R)-2,3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}propyl)amino]butanamide; tetrakis(acetic acid), Intermediate 149 (96%, 140 mg, 0.13 mmol) in aqueous HCl solution (2 M, 5 ml, 10 mmol) was stirred at RT for 2 h. The reaction mixture was concentrated in vacuo then lyophilised to afford the product as a beige solid (107 mg, quantitative based on 88% estimated purity).

[1006] .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.22-7.14 (m, 4H), 4.27-4.18 (m, 2H), 4.06-3.99 (m, 1H), 3.91-3.85 (m, 2H), 3.83-3.75 (m, 2H), 3.75-3.63 (m, 6H), 3.63-3.39 (m, 6H), 3.23-3.08 (m, 2H), 2.96-2.88 (m, 2H), 2.76-2.60 (m, 4H), 2.39-2.13 (m, 4H), 1.75-1.62 (m, 4H).

[1007] LC/MS (System A): m/z (ESI.sup.+)=635 [MH.sup.+], R.sub.t=0.16 min, ELS purity=100%.

B. SYNTHESIS OF EXAMPLE COMPOUNDS

Example 1 Synthesis of 2-[({6-amino-3-methyl-1H-pyrazolo[3,4-b]pyrazin-5-yl}formamido)methyl]-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium trifluoroacetate (Compound 1)

[1008] ##STR00196##

[1009] A mixture of 1,5-bis(1H-imidazole-1-carbonyl)-3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-amine, Intermediate 6 (92%, 21 mg, 0.057 mmol) and 2-(aminomethyl)-1,3-diethyl-6-methoxy-1H-1,3-benzodiazol-3-ium iodide, Intermediate 15 (50 mg, 0.14 mmol) in DMF (1 ml) was stirred at RT for 16 h. The reaction mixture was concentrated in vacuo then the crude material was purified by flash column chromatography on C18 (12 g). The column was eluted with MeCN:H.sub.2O+formic acid using the following gradient (% MeCN, column volumes): 5%, 2 CV; 5-31%, 14.5 CV; 31-98%, 4.5 CV. The desired fractions were combined then concentrated in vacuo. The material thus obtained was further purified by flash column chromatography on C18 (12 g). The column was eluted with MeCN:H.sub.2O+TFA using the following gradient (% MeCN, column volumes): 2%, 2 CV; 2-33%, 16 CV; 33-100%, 5 CV. The desired fractions were combined then lyophilised to afford the product as a yellow solid (2.5 mg, 8.3%).

[1010] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 12.90 (s, 1H), 9.65 (t, J=5.4 Hz, 1H), 7.97 (d, J=9.1 Hz, 1H), 7.88-7.49 (m, 3H), 7.28 (dd, J=9.1, 2.3 Hz, 1H), 5.08 (d, J=5.4 Hz, 2H), 4.70-4.61 (m, 4H), 3.91 (s, 3H), 2.46 (s, 3H), 1.43-1.39 (m, 6H).

[1011] LC/MS (System C): m/z (ESI.sup.+)=409 [M.sup.+], R.sub.t=1.73 min, UV purity=99%.

Example 2 Synthesis of 2-[({6-amino-3-methyl-1H-pyrazolo[3,4-b]pyrazin-5-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium formate (Compound 2)

[1012] ##STR00197##

[1013] A mixture of 5-(1H-imidazole-1-carbonyl)-3-methyl-1H-pyrazolo[3,4-b]pyrazin-6-amine, Intermediate 8 (91%, 75 mg, 0.28 mmol) and 2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium hydrobromide bromide, Intermediate 22 (152 mg, 0.370 mmol) in DMF (1.5 ml) was stirred at RT for 16h. The reaction mixture was concentrated in vacuo then the residue was suspended up in MeCN (4 ml) with sonication. The solid was collected by filtration and rinsed with MeCN. The solid thus obtained was dissolved in MeOH (50 ml) then concentrated in vacuo to afford a viscous magenta oil. The crude material was purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:H.sub.2O+formic acid using the following gradient (% MeCN, column volumes): 5%, 2 CV; 5-32%, 13 CV; 32-100%, 2.5 CV. The desired fractions were combined then concentrated in vacuo to afford the product as an orange solid (54 mg, 41%).

[1014] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 12.93 (s, 1H), 9.71 (t, J=5.4 Hz, 1H), 8.41 (s, 1H), 8.32 (5, 1H), 8.19 (dd, J=8.6, 1.0 Hz, 1H), 7.96 (d, J=8.6 Hz, 1H), 7.64 (s, 2H), 5.12 (d, J=5.4 Hz, 2H), 4.77-4.64 (m, 4H), 2.46 (s, 3H), 1.47-1.38 (m, 6H).

[1015] LC/MS (System C): m/z (ESI.sup.+)=423 [M.sup.+], R.sub.t=1.23 min, UV purity=99%. Example 3 Synthesis of 2-[({6-amino-1H-pyrazolo[3,4-b]pyrazin-5-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide (Compound 3)

##STR00198##

[1016] A suspension of 5-(1H-imidazole-1-carbonyl)-1H-pyrazolo[3,4-b]pyrazin-6-amine, Intermediate 12 (70 mg 0.29 mmol) and 2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium hydrobromide bromide, Intermediate 22 (120 mg, 0.293 mmol) in DMF (1.5 ml) was stirred at RT for 64 h. The reaction mixture was concentrated under a stream of nitrogen then the residue was suspended in MeCN (3 ml) with sonication. The solid was collected by filtration, rinsed with MeCN, then dried under suction to afford the product as a light brown solid (141 mg, 93%).

[1017] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.33 (s, 1H), 9.94 (t, J=5.3 Hz, 1H), 8.62-8.60 (m, 1H), 8.23 (dd, J=8.7, 1.3 Hz, 1H), 8.21-8.16 (m, 2H), 7.77 (s, 2H), 5.11 (d, J=5.3 Hz, 2H), 4.79 (q, J=7.1 Hz, 2H), 4.72 (q, J=7.1 Hz, 2H), 1.45-1.39 (m, 6H).

[1018] LC/MS (System C): m/z (ESI.sup.+)=409 [M.sup.+], R.sub.t=1.12 min, UV purity=99%.

Example 4 Synthesis of 2-[({6-amino-1H-pyrazolo[3,4-b]pyrazin-5-yl}formamido)methyl]-6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetic acid trifluoroacetate (Compound 4)

[1019] ##STR00199##

[1020] A solution of 2-[({6-amino-1H-pyrazolo[3,4-b]pyrazin-5-yl}formamido)methyl]-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide, Example 3 (135 mg, 0.276 mmol) and CDI (66 mg, 0.41 mmol) in DMF (1 ml) was stirred at RT for 3.5 h. The resultant solution was added to (2R,3R,4R,5S)-6-{[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl](piperidin-4-yl)amino}hexane -1,2,3,4,5-pentol dihydrochloride, Intermediate 25 (164 mg, 0.328 mmol) and rinsed in with DMF (0.5 ml). The reaction mixture was stirred at RT for 16 h then concentrated under a stream of nitrogen. The crude material was purified by flash column chromatography on C18 (12 g). The column was eluted with MeCN:H.sub.2O+TFA using the following gradient (% MeCN, column volumes): 2%, 2 CVs; 2-20%, 15 CVs; 20-100%, 2 CVs; 100%, 2 CVs.

[1021] The desired fractions were combined and concentrated in vacuo. The material thus obtained was further purified by flash column chromatography on C18 (12 g). The column was eluted with MeCN:H.sub.2O+TFA using the following gradient (% MeCN, column volumes): 2%, 2 CVs; 2-20%, 15 CVs; 20-100%, 2 CVs; 100%, 2 CVs. The desired fractions were combined and concentrated in vacuo then lyophilised to afford the product as a yellow solid (80 mg, 28%).

[1022] .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.34 (s, 1H), 9.97 (t, J=5.1 Hz, 1H), 8.33-8.14 (m, 4H), 8.15-7.40 (m, 3H), 5.74-5.40 (m, 2H), 5.10 (d, J=5.0 Hz, 2H), 5.00-3.59 (m, 25H), 3.29-2.78 (m, 8H), 2.26-1.31 (m, 10H).

[1023] LC/MS (System D): m/z (ESI.sup.+)=819 [M.sup.+], R.sub.t=1.16 min, UV purity=100%.

Example 5 Synthesis of Further Compounds of the Invention

[1024] Compounds of general formula (I) with cations of the formula:

##STR00200##

are prepared as shown in Table 1 below using methods similar to those set out in Examples 1-4 and the Examples of co-pending application PCT/GB2017/053499.

TABLE-US-00001 TABLE 1 Compd No A Method  5 [00201] Intermediate 6 + Intermediate 28 As Example 2 of PCT/GB2017/053499  6 [00202] Intermediate 6 + Intermediate 31 As Example 3 of PCT/GB2017/053499  7 [00203] Intermediate 6 + Intermediate 33 As Example 4 of PCT/GB2017/053499  8 [00204] Intermediate 6 + Intermediate 36 As Example 5 of PCT/GB2017/053499  9 [00205] Intermediate 6 + Intermediate 40 As Example 6 of PCT/GB2017/053499  10 [00206] Intermediate 6 + Intermediate 44 As Example 8 of PCT/GB2017/053499  11 [00207] Intermediate 6 + Intermediate 47 As Example 9 of PCT/GB2017/053499  12 [00208] Base hydrolysis of Compound 11 using aqueous LiOH As Example 10 of PCT/GB2017/053499  13 [00209] Intermediate 6 + Intermediate 49 As Example 11 of PCT/GB2017/053499  14 [00210] Intermediate 6 + Intermediate 51 As Example 12 of PCT/GB2017/053499  15 [00211] Intermediate 6 + Intermediate 53 As Example 13 of PCT/GB2017/053499  16 [00212] Intermediate 6 + Intermediate 55 As Example 14 of PCT/GB2017/053499  17 [00213] Intermediate 6 + Intermediate 57 As Example 16 of PCT/GB2017/053499  18 [00214] Intermediate 6 + Intermediate 59 As Example 17 of PCT/GB2017/053499  19 [00215] Intermediate 6 + Intermediate 63 As Example 18 of PCT/GB2017/053499  20 [00216] Intermediate 6 + Intermediate 42 As Examples 20 and 21 of PCT/GB2017/053499  21 [00217] Intermediate 6 + Intermediate 65 As Example 22 of PCT/GB2017/053499  22 [00218] Intermediate 6 + Intermediate 70 As Example 23 of PCT/GB2017/053499  23 [00219] Intermediate 6 + Intermediate 75 As Example 24 of PCT/GB2017/053499  24 [00220] Intermediate 6 + Intermediate 79 As Example 25 of PCT/GB2017/053499  25 [00221] Intermediate 6 + Intermediate 82 As Example 26 of PCT/GB2017/053499  26 [00222] Deprotection of Compound 21 using HCl solution in dioxane As Example 27 of PCT/GB2017/053499  27 [00223] Deprotection of Compound 22 using HCl solution in dioxane As Example 28 of PCT/GB2017/053499  28 [00224] Deprotection of Compound 23 using HCl solution in dioxane As Example 29 of PCT/GB2017/053499  29 [00225] Deprotection of Compound 24 using HCl solution in dioxane As Example 30 of PCT/GB2017/053499  30 [00226] Deprotection of Compound 25 using HCl solution in dioxane As Example 31 of PCT/GB2017/053499  31 [00227] Reaction of Compound 26 with 1H-1,2,4-triazole-1- carboximidamide hydrochloride As Example 32 of PCT/GB2017/053499  32 [00228] Reaction of Compound 27 with 1H-1,2,4-triazole-1- carboximidamide hydrochloride  33 [00229] Reaction of Compound 28 with 1H-1,2,4-triazole-1- carboximidamide hydrochloride As Example 33 of PCT/GB2017/053499  34 [00230] Reaction of Compound 2 with tert- Butyl N-(3-amino- propyl)carbamate As Example 35 of PCT/GB2017/053499  35 [00231] Reaction of Compound 2 with tert- Butyl 4- aminopiperidine-1- carboxylate As Example 36 of PCT/GB2017/053499  36 [00232] Reaction of Compound 2 with tert- Butyl N-(piperidin-4- yl)carbamate. As Example 37 of PCT/GB2017/053499  37 [00233] Reaction of Compound 2 with tert- butyl N-[1-(2- aminoethyl)-4- piperidyl]carbamate As Example 38 of PCT/GB2017/053499  38 [00234] Deprotection of Compound 34 using HCl in dioxane. As Example 39 of PCT/GB2017/053499  39 [00235] Deprotection of Compound 35 using HCl in dioxane As Example 40 of PCT/GB2017/053499  40 [00236] Deprotection of Compound 36 using HCl in dioxane As Example 41 of PCT/GB2017/053499  41 [00237] Deprotection of Compound 37 using HCl in dioxane As Example 42 of PCT/GB2017/053499  42 [00238] Intermediate 6 + Intermediate 86 As Example 43 of PCT/GB2017/053499  43 [00239] Intermediate 6 + Intermediate 90 As Example 44 of PCT/GB2017/053499  44 [00240] Reaction of Compound 28 with 4,6-O-benzylidene-D- glucopyranose followed by deprotection with HCl. As Example 45 of PCT/GB2017/053499  45 [00241] Reaction of Compound 30 with 4,6-O-benzylidene-D- glucopyranose followed by deprotection with HCl. As Example 47 of PCT/GB2017/053499  46 [00242] Reaction of Compound 2 with Intermediate 100. As Example 48 of PCT/GB2017/053499  47 [00243] Reaction of Compound 2 with Intermediate 105. As Example 49 of PCT/GB2017/053499  48 [00244] Reaction of Compound 2 with Intermediate 25 using a method similar to that of Example 4  49 [00245] Reaction of Compound 2 with Intermediate 110. As Example 51 of PCT/GB2017/053499  50 [00246] Reaction of Compound 2 with Intermediate 115 As Example 52 of PCT/GB2017/053499  51 [00247] Reaction of Compound 2 with Intermediate 117 As Example 53 of PCT/GB2017/053499  52 [00248] Reaction of Compound 2 with Intermediate 119 As Example 54 of PCT/GB2017/053499  53 [00249] Reaction of Compound 2 with Intermediate 121 As Example 55 of PCT/GB2017/053499  54 [00250] Reaction of Compound 2 with Intermediate 126 As Example 56 of PCT/GB2017/053499  55 [00251] Reaction of Compound 2 with Intermediate 128 As Example 57 of PCT/GB2017/053499  56 [00252] Reaction of Compound 2 with Intermediate 130 As Example 58 of PCT/GB2017/053499  57 [00253] Reaction of Compound 2 with Intermediate 135 As Example 59 of PCT/GB2017/053499  58 [00254] Reaction of Compound 2 with Intermediate 140 As Example 60 of PCT/GB2017/053499  59 [00255] Reaction of Compound 2 with Intermediate 148 As Example 61 of PCT/GB2017/053499  60 [00256] Reaction of Compound 2 with Intermediate 150 As Example 62 of PCT/GB2017/053499 118 [00257] Reaction of Compound 2 with Intermediate 95 followed by deprotection with aqueous HCl

[1025] Compounds of the formula:

##STR00258##

are prepared as shown in Table 2 below using methods similar to those set out in Examples 1-4 and the Examples of co-pending application PCT/GB2017/053499.

TABLE-US-00002 TABLE 2 Compd No A Method  61 [00259] Intermediate 12 + Intermediate 15 Method as for Example 1  62 [00260] Intermediate 12 + Intermediate 28 As Example 2 of PCT/GB2017/053499  63 [00261] Intermediate 12 + Intermediate 31 As Example 3 of PCT/GB2017/053499  64 [00262] Intermediate 12 + Intermediate 33 As Example 4 of PCT/GB2017/053499  65 [00263] Intermediate 12 + Intermediate 36 As Example 5 of PCT/GB2017/053499  66 [00264] Intermediate 12 + Intermediate 40 As Example 6 of PCT/GB2017/053499  67 [00265] Intermediate 12 + Intermediate 44 As Example 8 of PCT/GB2017/053499  68 [00266] Intermediate 12 + Intermediate 47 As Example 9 of PCT/GB2017/053499  69 [00267] Base hydrolysis of Compound 68 using aqueous LiOH As Example 10 of PCT/GB2017/053499  70 [00268] Intermediate 12 + Intermediate 49 As Example 11 of PCT/GB2017/053499  71 [00269] Intermediate 12 + Intermediate 51 As Example 12 of PCT/GB2017/053499  72 [00270] Intermediate 12 + Intermediate 53 As Example 13 of PCT/GB2017/053499  73 [00271] Intermediate 12 + Intermediate 55 As Example 14 of PCT/GB2017/053499  74 [00272] Intermediate 12 + Intermediate 57 As Example 16 of PCT/GB2017/053499  75 [00273] Intermediate 12 + Intermediate 59 As Example 17 of PCT/GB2017/053499  76 [00274] Intermediate 12 + Intermediate 63 As Example 18 of PCT/GB2017/053499  77 [00275] Intermediate 12 + Intermediate 42 As Examples 20 and 21 of PCT/GB2017/053499  78 [00276] Intermediate 12 + Intermediate 65 As Example 22 of PCT/GB2017/053499  79 [00277] Intermediate 12 + Intermediate 70 As Example 23 of PCT/GB2017/053499  80 [00278] Intermediate 12 + Intermediate 75 As Example 24 of PCT/GB2017/053499  81 [00279] Intermediate 12 + Intermediate 79 As Example 25 of PCT/GB2017/053499  82 [00280] Intermediate 12 + Intermediate 82 As Example 26 of PCT/GB2017/053499  83 [00281] Deprotection of Compound 78 As Example 27 of PCT/GB2017/053499  84 [00282] Deprotection of Compound 79 using HCl solution in dioxane As Example 28 of PCT/GB2017/053499  85 [00283] Deprotection of Compound 80 using HCl solution in dioxane As Example 29 of PCT/GB2017/053499  86 [00284] Deprotection of Compound 81 using HCl solution in dioxane As Example 30 of PCT/GB2017/053499  87 [00285] Deprotection of Compound 82 using HCl solution in dioxane As Example 31 of PCT/GB2017/053499  88 [00286] Reaction of Compound 83 with 1H-1,2,4-triazole-1- carboximidamide hydrochloride As Example 32 of PCT/GB2017/053499  89 [00287] Reaction of Compound 84 with 1H-1,2,4-triazole-1- carboximidamide hydrochloride  90 [00288] Reaction of Compound 85 with 1H-1,2,4-triazole-1- carboximidamide hydrochloride As Example 33 of PCT/GB2017/053499  91 [00289] Reaction of Example 3 with tert-Butyl N- (3-amino- propyl)carbamate As Example 35 of PCT/GB2017/053499  92 [00290] Reaction of Compound 3 with tert- Butyl 4- aminopiperidine-1- carboxylate As Example 36 of PCT/GB2017/053499  93 [00291] Reaction of Compound 3 with tert- Butyl N-(piperidin-4- yl)carbamate. As Example 37 of PCT/GB2017/053499  94 [00292] Reaction of Compound 3 with tert- butyl N-[1-(2- aminoethyl)-4- piperidyl]carbamate As Example 38 of PCT/GB2017/053499  95 [00293] Deprotection of Compound 91 using HCl in dioxane. As Example 39 of PCT/GB2017/053499  96 [00294] Deprotection of Compound 92 using HCl in dioxane As Example 40 of PCT/GB2017/053499  97 [00295] Deprotection of Compound 93 using HCl in dioxane As Example 41 of PCT/GB2017/053499  98 [00296] Deprotection of Compound 94 using HCl in dioxane As Example 42 of PCT/GB2017/053499  99 [00297] Intermediate 12 + Intermediate 86 As Example 43 of PCT/GB2017/053499 100 [00298] Intermediate 12 + Intermediate 90 As Example 44 of PCT/GB2017/053499 101 [00299] Reaction of Compound 38 with 4,6-O-benzylidene-D- glucopyranose followed by deprotection with HCl. As Example 45 of PCT/GB2017/053499 102 [00300] Reaction of Compound 30 with 4,6-O-benzylidene-D- glucopyranose followed by deprotection with HCl. As Example 47 of PCT/GB2017/053499 103 [00301] Reaction of Compound 3 with Intermediate 100. As Example 48 of PCT/GB2017/053499 104 [00302] Reaction of Compound 3 with Intermediate 105. As Example 49 of PCT/GB2017/053499 105 [00303] Reaction of Compound 3 with Intermediate 110. As Example 51 of PCT/GB2017/053499 106 [00304] Reaction of Compound 3 with Intermediate 115 As Example 52 of PCT/GB2017/053499 107 [00305] Reaction of Compound 3 with Intermediate 117 As Example 53 of PCT/GB2017/053499 108 [00306] Reaction of Compound 3 with Intermediate 119 As Example 54 of PCT/GB2017/053499 109 [00307] Reaction of Compound 3 with Intermediate 121 As Example 55 of PCT/GB2017/053499 110 [00308] Reaction of Compound 3 with Intermediate 126 As Example 56 of PCT/GB2017/053499 111 [00309] Reaction of Compound 3 with Intermediate 128 As Example 57 of PCT/GB2017/053499 112 [00310] Reaction of Compound 3 with Intermediate 130 As Example 58 of PCT/GB2017/053499 113 [00311] Reaction of Compound 3 with Intermediate 135 As Example 59 of PCT/GB2017/053499 114 [00312] Reaction of Compound 3 with Intermediate 140 As Example 60 of PCT/GB2017/053499 115 [00313] Reaction of Compound 3 with Intermediate 148 As Example 61 of PCT/GB2017/053499 116 [00314] Reaction of Compound 3 with Intermediate 150 As Example 62 of PCT/GB2017/053499 117 [00315] Reaction of Compound 12 with Intermediate 95 followed by deprotection with aqueous HCl

C. BIOLOGICAL EXAMPLES

Example 6 Short circuit current assay to determine ENaC blocker potency in human bronchial epithelial cells

[1026] Cell culture Human bronchial epithelial cells (HBECs) (Lonza, UK) were cultured using a modification of the method described by Coote et al, (2008). Cells were seeded into plastic T-75 flasks and grown in Bronchial Epithelial Cell Growth Medium (BEGM) (Lonza, UK) supplemented with bovine pituitary extract (52 ng/mL), hydrocortisone (0.5 μg/mL), human recombinant Epidermal Growth Factor (0.5 ng/mL), epinephrine (0.5 ng/mL), transferrin (10 ng/mL), insulin (5 ng/mL), retinoic acid (0.1 ng/mL), triiodothyronine (6.5 ng/mL), gentamycin (50 μg/mL) and amphotericin-B (50 ng/mL). Medium was changed every 48 hours until cells were 90% confluent. Cells were then passaged and seeded (8.25×10.sup.5 cells/insert) onto polycarbonate Snapwell™ inserts (Costar, UK) in differentiation media containing 50% DMEM in BGEM with the same supplements as above but without triiodothyronine and a final retinoic acid concentration of 50 nM (all-trans retinoic acid; Sigma-Aldrich, UK). Cells were maintained submerged for the first 7 days in culture after which time they were exposed to an apical air interface for the remainder of the culture period. From the first day of establishment of an ALI, HBEC were fed with a DMEM:HAMS F-12 (1:1) media containing 2% Ultroser G (Pall BioSepra, France) with gentamycin (50 μg/mL) and amphotericin B (50 ng/mL). Cells were used for short-circuit current assay between days 14-21 after the establishment of the ALI. At all stages of culture, cells were maintained at 37° C. in 5% CO.sub.2 in an air incubator.

Short-circuit current (ISC) measurements

[1027] Snapwell inserts were mounted in Costar Vertical Diffusion Chambers (Costar, UK) and were bathed with continuously gassed Ringer solution (5% CO.sub.2 in O.sub.2; pH 7.4) maintained at 37° C. containing (in mM): 120 NaCl, 25 NaHCO.sub.3, 3.3 KH.sub.2PO.sub.4, 0.8 K.sub.2HPO.sub.4, 1.2 CaCl.sub.2, 1.2 MgCl.sub.2 and 10 glucose. The solution osmolarity was always between 280-300 mOsm/kg H.sub.2O for all physiological salt solutions used. Cells were voltage clamped to 0 mV (model EVC4000, WPI). Transepithelial resistance (RT) was measured by applying a 2 mV pulse at 30 s intervals and calculating RT by Ohm's law. Data were recorded using a PowerLab workstation (ADInstruments, UK).

[1028] ENaC blocker compounds were added to the apical chamber from a 1000-fold stock solution (prepared in DMSO) to achieve a cumulative concentration response in terms of the inhibition of the basal ISC. At the completion of the concentration response, a supra-maximal concentration of amiloride (10 μM) was added. The concentration of test compound that induced a 50% inhibition of the total amiloride-sensitive ISC (IC.sub.50) was calculated using GraphPad Prism v6.05. The results are presented in Table 3, from which it can be seen that the compounds of the present invention have ENaC inhibiting activity.

TABLE-US-00003 TABLE 3 Example Averages .Math. ENaC No. IC.sub.50 (nM) Avg 1 2 2 91.5 3 106 4 47

REFERENCES

[1029] App E M, King M, Helfesrieder R, Köhler D and Matthys H. Acute and long-term amiloride inhalation in cystic fibrosis lung disease. A rational approach to cystic fibrosis therapy. Am Rev Respir Dis., 1990, 141(3):605-12. [1030] Botero-Velez M, Curtis J J and Warnock D G. Brief report: Liddle's syndrome revisited—a disorder of sodium reabsorption in the distal tubule. N Engl J Med., 1994, 330(3):178-81. [1031] Boucher R C. Evidence for airway surface dehydration as the initiating event in C F airway disease. J Intern Med., 2007, 261(1):5-16. [1032] Bowler I M, Kelman B, Worthington D, Littlewood J M, Watson A, Conway S P, Smye S W, James S L and Sheldon T A. Nebulised amiloride in respiratory exacerbations of cystic fibrosis: a randomised controlled trial. Arch Dis Child., 1995, 73(5):427-30. [1033] Chang S S, Grunder S, Hanukoglu A, Rösler A, Mathew P M, Hanukoglu I, Schild L, Lu Y, Shimkets R A, Nelson-Williams C, Rossier B C and Lifton R P. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet., 1996, 12(3):248-53. [1034] Coote K, Atherton-Watson H C, Sugar R, Young A, MacKenzie-Beevor A, Gosling M, Bhalay G, Bloomfield G, Dunstan A, Bridges R J, Sabater J R, Abraham W M, Tully D, Pacoma R, Schumacher A, Harris J, Danahay H. Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease. J Pharmacol Exp Ther. 2009 May; 329(2):764-74. [1035] Coote K J, Atherton H, Young A, Sugar R, Burrows R, Smith N J, Schlaeppi J M, Groot-Kormelink P J, Gosling M, Danahay H. The guinea-pig tracheal potential difference as an in vivo model for the study of epithelial sodium channel function in the airways. Br J Pharmacol. 2008 December; 155(7):1025-33. [1036] Fajac I, Hubert D, Guillemot D, Honore I, Bienvenu T, Volter F, Dall'Ava-Santucci J and Dusser D J. Nasal airway ion transport is linked to the cystic fibrosis phenotype in adult patients. Thorax, 2004, 59(11):971-6. [1037] Frateschi S, Charles R-P, Hummler E. The Epithelial Sodium Channel ENaC and its Regulators in the Epidermal Permeability Barrier Function. The Open Dermatology Journal, 2010, 4: 27-35. [1038] Graham A, Hasani A, Alton E W, Martin G P, Marriott C, Hodson M E, Clarke S W and Geddes D M. No added benefit from nebulized amiloride in patients with cystic fibrosis. Eur Respir J., 1993, 6(9):1243-8. [1039] Hirsh A J, Zhang J, Zamurs A, Fleegle J, Thelin W R, Caldwell R A, Sabater J R, Abraham W M, Donowitz M, Cha B, Johnson K B, St George J A, Johnson M R, Boucher R C. Pharmacological properties of N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02), a novel epithelial sodium channel blocker with potential clinical efficacy for cystic fibrosis lung disease. J Pharmacol Exp Ther. 2008 April; 325(1):77-88. [1040] Kellenberger S and Schild L. Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure. Physiol Rev., 2002 82(3):735-67. [1041] Kerem E, Bistritzer T, Hanukoglu A, Hofmann T, Zhou Z, Bennett W, MacLaughlin E, Barker P, Nash M, Quittell L, Boucher R and Knowles M R. Pulmonary epithelial sodium-channel dysfunction and excess airway liquid in pseudohypoaldosteronism. N Engl J Med., 1999, 341(3):156-62. [1042] Knowles M R, Stutts M J, Spock A, Fischer N, Gatzy J T and Boucher R C. Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science, 1983, 221(4615):1067-70. [1043] Knowles M R, Church N L, Waltner W E, Yankaskas J R, Gilligan P, King M, Edwards L J, Helms R W and Boucher R C. A pilot study of aerosolized amiloride for the treatment of lung disease in cystic fibrosis. N Engl J Med., 1990, 322(17):1189-94. [1044] Leal T, Fajac I, Wallace H L, Lebecque P, Lebacq J, Hubert D, Dall'Ava J, Dusser D, Ganesan A P, Knoop C, Cumps J, Wallemacq P and Southern K W. Airway ion transport impacts on disease presentation and severity in cystic fibrosis. Clin Biochem., 2008, 41(10-11):764-72. [1045] Matsui H, Grubb B R, Tarran R, Randell S H, Gatzy J T, Davis C W and Boucher R C. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell, 1998, 95(7):1005-15. [1046] Middleton P G, Geddes D M and Alton E W. Effect of amiloride and saline on nasal mucociliary clearance and potential difference in cystic fibrosis and normal subjects. Thorax, 1993, 48(8):812-6. [1047] Noone P G, Regnis J A, Liu X, Brouwer K L, Robinson M, Edwards L and Knowles M R. Airway deposition and clearance and systemic pharmacokinetics of amiloride following aerosolization with an ultrasonic nebulizer to normal airways. Chest, 1997, 112(5):1283-90. [1048] Perazella M A. Drug-induced hyperkalemia: old culprits and new offenders. Am J Med., 2000, 109(4):307-14. [1049] Pons G, Marchand M C, d'Athis P, Sauvage E, Foucard C, Chaumet-Riffaud P, Sautegeau A, Navarro J and Lenoir G. French multicenter randomized double-blind placebo-controlled trial on nebulized amiloride in cystic fibrosis patients. The Amiloride-AFLM Collaborative Study Group. Pediatr Pulmonol., 2000, 30(1):25-31. [1050] Thelin W R, Johnson M R, Hirsh A J, Kublin C L, Zoukhri D. Effect of Topically Applied Epithelial Sodium Channel Inhibitors on Tear Production in Normal Mice and in Mice with