Bis(pentahydroxyhexyl)amino substituted 2-{[(3-amino-pyrazin-2-yl)formamido]methyl}-1H-1,3-benzodiazol-3-ium derivatives as ENaC inhibitors for treating respiratory diseases

Abstract

The present invention discloses bis(pentahydroxyhexyl)amino substituted 2-{[(3-amino-pyrazin-2-yl)formamido]methyl}-1H-1,3-benzodiazol-3-ium derivatives of formula (I) as inhibitors of ENaC and are of use in the treatment of respiratory diseases and conditions, skin conditions or ocular conditions, wherein the respiratory disease or condition is e.g. cystic fibrosis, chronic obstructive pulmonary disease (COPD), chronic bronchitis, emphysema, bronchiectasis, including non-cystic fibrosis bronchiectasis, and asthma; the skin condition is e.g. psoriasis, atopic dermatitis and ichthyosis; and the ocular condition is e.g. dry eye disease. ##STR00001##

Claims

1. A compound of general formula (I) including all tautomeric forms, all enantiomers and isotopic variants and salts thereof: ##STR00104## wherein X.sup.− is an anion; R.sup.1 is halo, cyano, C.sub.1-3 alkyl, C.sub.1-3 haloalkyl, or —S(C.sub.1-3 alkyl); R.sup.2 is H or NH.sub.2; each of R.sup.3 and R.sup.4 is independently C.sub.1-10 alkyl, wherein one or more —CH.sub.2— groups is optionally replaced by —O—, —S— or provided that adjacent —CH.sub.2— groups are not so replaced, and which is optionally substituted with one or more substituents selected from halo, —OR.sup.6, —SR.sup.6, —NR.sup.6R.sup.7, aryl, heteroaryl, cycloalkyl, heterocyclyl, —C(O)OR.sup.6, or —C(O)NR.sup.6R.sup.7; wherein each R.sup.5, R.sup.6 and R.sup.7 is independently selected from H or Ci-4 alkyl; 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—, or -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—, or —OZ.sup.1Q.sup.1OQ.sup.2OQ.sup.3-; —NR.sup.8Z.sup.1—, —NR.sup.8Q.sup.1-, —NR.sup.8Z.sup.1Q.sup.1-, —NR.sup.8Q.sup.1Z.sup.1—, —NR.sup.8Z.sup.1Q.sup.1Z.sup.2—, —NR.sup.8Q.sup.1Q.sup.2-, —NR.sup.8Q.sup.1Q.sup.2Z.sup.1—, —NR.sup.8Q.sup.1Q.sup.2Z.sup.1Q.sup.3Z.sup.2—, or —NR.sup.8Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3; —Z.sup.1NR.sup.8Z.sup.2—, -Q.sup.1-Z.sup.1NR.sup.8Z.sup.2—, —Z.sup.1NR.sup.8Z.sup.2Q.sup.1-, or -Q.sup.1Z.sup.1NR.sup.8Z.sup.2Q.sup.2Z.sup.3—; —Z.sup.1O(CH.sub.2CH.sub.2O).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.1NR.sup.8C(O)Z.sup.1—, —C(O)Q.sup.1NR.sup.8C(O)Z.sup.1Q.sup.2-, —C(O)Q.sup.1NR.sup.8C(O)Z.sup.1Q.sup.2Q.sup.3-—C(O)Q.sup.1NR.sup.8C(O)Z.sup.1Q.sup.2Z.sup.2—, or —C(O)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3-; —C(O)NR.sup.8Z′—, —C(O)NR.sup.8Q.sup.1-, —C(O)NR.sup.8Z.sup.1Q.sup.1-, —C(O)NR.sup.8Z.sup.1Q.sup.1Z.sup.2—, —C(O)NR.sup.8Q.sup.1Z.sup.1—, —C(O)NR.sup.8Q.sup.1Q.sup.2-, —C(O)NR.sup.8Q.sup.1Q.sup.2Z.sup.1—, —C(O)NR.sup.8Z.sup.1Q.sup.1Q.sup.2Z.sup.2, —C(O)NR.sup.8(CH.sub.2CH.sub.2O).sub.nZ.sup.1—C(O)NR.sup.8Z.sup.1O(CH.sub.2O).sub.nZ.sup.2—, —C(O)NR.sup.8Z.sup.1Q.sup.1Z.sup.2NR.sup.9Z.sup.3—, —C(O)NR.sup.8Z.sup.1NR.sup.9Z.sup.2—, —C(O)NR.sup.8Q.sup.1Z.sup.1NR.sup.9Z.sup.2—, —C(O)NR.sup.8Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3, or —C(O)NR.sup.8Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3Z.sup.2; —Z.sup.1C(O)NR.sup.8Z.sup.2—, —Z.sup.1C(O)NR.sup.8Q.sup.1-, —Z.sup.1C(O)NR.sup.8Z.sup.2Q.sup.1-, —Z.sup.1C(O)NR.sup.8Q.sup.1Z.sup.2—, —Z.sup.1C(O)NR.sup.8Q.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-, or —Z.sup.1C(O)NR.sup.8Q.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-, or —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—; or C(═NR.sup.10)NR.sup.8Z.sup.1—, C(═NR.sup.10)NR.sup.8Q.sup.1-, C(═NR.sup.10)NR.sup.8Z.sup.1Q.sup.1-, C(═NR.sup.10)NR.sup.8Z.sup.1Q.sup.1Z.sup.2—, C(—NR.sup.10)NR.sup.8Q.sup.1Z.sup.1—, —C(═NR.sup.10)NR.sup.8Q.sup.1Q.sup.2, or C(═NR.sup.10)NR.sup.8Q.sup.1Q.sup.2Z.sup.1; each of Z.sup.1, Z.sup.2 and Z.sup.3 is independently C.sub.1-12 alkylene, C.sub.2-12 alkenylene, or C.sub.2-12 alkynylene any of which is optionally substituted by one or more halo, OH, C(O)NR.sup.11R.sup.12, C(O)OR.sup.11 or NR.sup.11R.sup.12; each R.sup.11 and R.sup.12 is independently H or C.sub.1-6 alkyl; 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, C(O)NR.sup.11R.sup.12, C(O)OR.sup.11 or NR.sup.11R.sup.12, or, for carbocyclyl and heterocyclyl groups, oxo, wherein R.sup.11 and R.sup.12 are as defined above; n is 1 to 6; each R.sup.8 and R.sup.9 is independently selected from H or C.sub.1-12 alkyl optionally substituted with one or more halo or OH groups; and R.sup.10 is H or C.sub.1-6 alkyl.

2. The compound according to claim 1, wherein the compound is of general formula (IA): ##STR00105## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, L.sup.1 and X.sup.− are as defined for general formula (I); or the compound is of general formula (TB): ##STR00106## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, L.sup.1 and X.sup.− are as defined for general formula (I).

3. The compound according to claim 1 wherein R.sup.1 is halo or methyl.

4. The compound according to claim 1 wherein R.sup.2 is NH.sub.2.

5. The compound according to claim 1 wherein: one or both of R.sup.3 and R.sup.4 are methyl, ethyl, benzyl, pyridylmethyl, —CH.sub.2OH, —CH.sub.2NH.sub.2, —CH.sub.2CH.sub.2OH or CH.sub.2CH.sub.2NH.sub.2; or both R.sup.3 and R.sup.4 are C.sub.1-10 alkyl; or at least one of R.sup.3 and R.sup.4 is —CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OH or —CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OH.

6. The compound according to claim 1 wherein L.sup.1 is: —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.1NR.sup.8C(O)Z.sup.1—, —C(O)Q.sup.1NR.sup.8C(O)Z.sup.1Q.sup.2-, —C(O)Q.sup.1NR.sup.8C(O)Z.sup.1Q.sup.2Q.sup.3-—C(O)Q.sup.1NR.sup.8C(O)Z.sup.1Q.sup.2Z.sup.2—, or —C(O)Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3-; or —C(O)NR.sup.8Z.sup.1—, —C(O)NR.sup.8Q.sup.1-, —C(O)NR.sup.8Z.sup.1Q.sup.1, C(O)NR.sup.8Z.sup.1Q.sup.1Z.sup.2—, —C(O)NR.sup.8Q.sup.1Z.sup.1—, —C(O)NR.sup.8Q.sup.1Q.sup.2-, —C(O)NR.sup.8Q.sup.1Q.sup.2Z.sup.1, C(O)NR.sup.8Z.sup.1Q.sup.1Q.sup.2Z.sup.2, —C(O)NR.sup.8(CH.sub.2CH.sub.2O).sub.nZ.sup.1—C(O)NR.sup.8Z.sup.1O(CH.sub.2O).sub.nZ.sup.2—, —C(O)NR.sup.8Z.sup.1Q.sup.1Z.sup.2NR.sup.9Z.sup.3—, —C(O)NR.sup.8Z.sup.1NR.sup.9Z.sup.2—, —C(O)NR.sup.8Q.sup.1Z.sup.1NR.sup.9Z.sup.2—, —C(O)NR.sup.8Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3-, or —C(O)NR.sup.8Z.sup.1Q.sup.1OQ.sup.2OQ.sup.3Z.sup.2—.

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

8. The compound according to claim 1 wherein 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.1NR.sup.8C(O)Z.sup.1—, —C(O)Q.sup.1NR.sup.8C(O)Z.sup.1Q.sup.2-, —C(O)Q.sup.1NR.sup.8C(O)Z.sup.1Q.sup.2Q.sup.3-, or —C(O)Q.sup.1NR.sup.8C(O)Z.sup.1Q.sup.2Z.sup.2—, and Q.sup.1 is pyrrolidin-1-yl, piperidin-1-yl, or piperazin-1-yl; or L.sup.1 is -Q.sup.1C(O)Q.sup.2-, -Q.sup.1C(O)Q.sup.2Z.sup.1—, Q.sup.1C(O)Q.sup.2Q.sup.3-, or Q.sup.1C(O)Q.sup.2Q.sup.3Z.sup.1—, and Q.sup.2 is pyrrolidin-1-yl, piperidin-1-yl, or piperazin-1-yl.

9. The compound according to claim 1 selected from the following salts: 6-(4-{bis[(2S,3R,4R,5R)-2,3,4,5, 6-pentahydroxyhexyl]amino}piperidine-1-carbonyl)-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium; 2-{[(3-amino-6-bromopyrazin-2-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; 6-[4-({bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}methyl)piperidine-1-carbonyl]-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium; 6-[(3R)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl] amino}pyrrolidine-1-carbonyl]-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium; 6-[(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethyl)carbamoyl]-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium; 2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-6-{-1H-1,3-benzodiazol-3-ium; or 6-({2-[4′-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl] amino}ethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamoyl)-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium; wherein the counter ion is an anion X.sup.− as defined in claim 1.

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

Description

EXAMPLES

(1) 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. Analytical LC/MS were carried out on the following systems: 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+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; 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; 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+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; 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.

(2) The following abbreviations and terms have the indicated meanings throughout:

(3) AcOH glacial acetic acid

(4) CDI 1,1′-carbonyldiimidazole

(5) CV column volumes

(6) dd doublet of doublets

(7) DIPEA N,N-diisopropylethylamine

(8) DMF N,N-dimethylformamide

(9) DMSO dimethyl sulfoxide

(10) dppf 1,1′-bis(diphenylphosphino)ferrocene

(11) ELS evaporative light scattering

(12) ESI electrospray ionisation

(13) EtOAc ethyl acetate

(14) HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

(15) HBTU 3-[bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide hexafluorophosphate

(16) HPLC high-performance liquid chromatography

(17) LC/MS liquid chromatography-mass spectrometry

(18) m multiplet

(19) MeCN acetonitrile

(20) MeOH methanol

(21) NMR nuclear magnetic resonance

(22) q quartet

(23) RT room temperature

(24) Rt retention time

(25) s singlet

(26) t triplet

(27) TFA trifluoroacetic acid

(28) THF tetrahydrofuran

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

(29) ##STR00033##

(30) 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 washed 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 washed 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 column volumes. 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). 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%).

(31) .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).

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

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

(33) ##STR00034##

(34) 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 1 (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 volume of water and MeCN then dried under vacuum to afford the product as a white solid (6.05 g, 90%).

(35) .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).

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

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

(37) ##STR00035##

(38) 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 2 (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%).

(39) .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).

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

Intermediate 4—Synthesis of 2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylic acid

(41) ##STR00036##

(42) 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 3 (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%).

(43) .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).

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

Intermediate 5—tert-butyl 2-({[(benzyloxy)carbonyl]amino}methyl)-1-ethyl-1H-1,3-benzodiazole-6-carboxylate

(45) ##STR00037##

(46) 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 4 (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%).

(47) .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).

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

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

(49) ##STR00038##

(50) 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 5 (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 81 (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 81 (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%).

(51) .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).

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

Intermediate 7—Synthesis of 2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium hydrobromide bromide

(53) ##STR00039##

(54) 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 6 (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%).

(55) .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).

(56) LC/MS (System A): m/z (ESI.sup.+)=248 [M.sup.+], R.sub.t=0.15 min, ELS purity=100%.

Intermediate 8—Synthesis of 3-chloro-5-(1H-imidazole-1-carbonyl)pyrazine-2,6-diamine

(57) ##STR00040##

(58) CDI (705 mg, 4.35 mmol) was added to a suspension of 3,5-diamino-6-chloro-pyrazine-2-carboxylic acid (546 mg, 2.90 mmol) in DMF (6 ml). The resulting suspension was stirred at RT for 10 min. More DMF (4 ml) was added then the reaction was left to stir at RT for a further 17 h. The reaction mixture was cooled (0° C.) then water (20 ml) was added in portions (2 ml) over 0.5 h. The mixture was stirred at 0° C. for 1 h. The resulting suspension was filtered then the solid was rinsed with ice cold water (10 ml) and then dried in vacuo to afford an off-white solid (674 mg, 95%).

(59) .sup.1H NMR (500 MHz, DMSO-d6) δ 8.54-8.52 (m, 1H), 8.27-7.26 (m, 5H), 7.08-6.99 (m, 1H). 3 wt % residual DMF.

(60) LC/MS (System A): m/z (ESI.sup.+)=239 [M(.sup.35Cl)H.sup.+], 241 [M(.sup.37Cl)H.sup.+], R.sub.t=0.15 and 0.52 min, ELS purity=82+18%.

Intermediate 9—Synthesis of 6-carboxy-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide

(61) ##STR00041##

(62) A suspension of 3-chloro-5-(1H-imidazole-1-carbonyl)pyrazine-2,6-diamine, Intermediate 8 (352 mg, 1.47 mmol) and 2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium hydrobromide bromide, Intermediate 7 (603 mg, 1.47 mmol) in DMF (15 ml) was stirred at RT. After a 3 min, DMSO (0.1 ml) was added then the reaction mixture was left to stir at RT for 23 h. The reaction mixture was concentrated in vacuo then azeotroped with MeCN (20 ml). The residue thus obtained was suspended in MeCN (20 ml) with sonication. The resulting suspension was cooled (0° C.). The solid was collected by filtration, rinsed with ice-chilled MeCN (10 ml), then dried in vacuo to afford the product as an orange solid (701 mg, 91%).

(63) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.55 (s, 1H), 8.99 (t, J=5.3 Hz, 1H), 8.64-8.57 (m, 1H), 8.22 (dd, J=8.7, 1.3 Hz, 1H), 8.17 (d, J=8.7 Hz, 1H), 7.78-6.44 (m, 4H), 5.01 (d, J=5.3 Hz, 2H), 4.76 (q, J=7.1 Hz, 2H), 4.69 (q, J=7.1 Hz, 2H), 1.48-1.34 (m, 6H). 5 wt % residual DMF.

(64) LC/MS (System A): m/z (ESI.sup.−)=418 [M(.sup.35Cl).sup.+], 420 [M(.sup.37Cl).sup.+], R.sub.t=0.79 min, UV purity=99%.

Intermediate 10—Synthesis of 5-bromo-3-(1H-imidazole-1-carbonyl)pyrazin-2-amine

(65) ##STR00042##

(66) CDI (2.23 g, 13.8 mmol) was added to a suspension of 3-amino-6-bromopyrazine-2-carboxylic acid (2.00 g, 9.17 mmol) in DMF (20 ml). The reaction was stirred at RT for 16 h. The reaction mixture was cooled (0° C.) then diluted with water (20 ml). The solid was collected by filtration then washed with the minimum volume of water and cooled (0° C.) MeCN then dried under vacuum to afford the product as a yellow solid (2.23 g, 86%).

(67) .sup.1H NMR (250 MHz, DMSO-d.sub.6) δ 8.58-8.52 (m, 2H), 7.96-7.82 (m, 3H), 7.15-7.07 (m, 1H).

(68) LC/MS (System A, MeOH quench): m/z (ESI.sup.+)=232 [Methyl ester M(.sup.79Br)H.sup.+], 234 [Methyl ester M(.sup.81Br)H.sup.+]), R.sub.t=0.87 min, UV purity=95%.

Intermediate 11—Synthesis of 2-{[(3-amino-6-bromopyrazin-2-yl)formamido]methyl}-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide

(69) ##STR00043##

(70) A solution of 5-bromo-3-(1H-imidazole-1-carbonyl)pyrazin-2-amine, Intermediate 10 (40 mg, 0.15 mmol) and 2-(aminomethyl)-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium hydrobromide bromide, Intermediate 7 (61 mg, 0.15 mmol) in DMF (1 ml) was stirred at RT for 17 h. The reaction was filtered then the solid collected was washed with MeCN and dried under vacuum to afford the product as an off-white solid (31 mg, 37%).

(71) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 13.58 (s, 1H), 9.60 (t, J=5.3 Hz, 1H), 8.62 (s, 1H), 8.42 (s, 1H), 8.26-8.16 (m, 2H), 7.67 (s, 2H), 5.10 (d, J=5.3 Hz, 2H), 4.80-4.65 (m, 4H), 1.44-1.39 (m, 6H).

(72) LC/MS (System C): m/z (ESI.sup.+)=447 [M(.sup.79Br).sup.+], 449 [M(.sup.81Br).sup.+], R.sub.t=1.29 min, UV purity=94%.

Intermediate 12—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

(73) ##STR00044##

(74) A mixture of 9H-fluoren-9-ylmethyl 4-aminopiperidine-1-carboxylate hydrochloride (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%).

(75) .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).

(76) LC/MS (System A): m/z (ESI.sup.+)=827 [MH.sup.+], R.sub.t=1.08 min, UV purity=100%.

Intermediate 13—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

(77) ##STR00045##

(78) Piperidine (9.01 ml, 91.2 mmol) was added to a solution of (9H-fluoren-9-yl)methyl 4-{bis[(2S,3R)-2,3-didroxy-3-dihydroxy-3-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]propyl]amino}piperidine-1-carboxylate; formic acid, Intermediate 12 (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:IPA (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%).

(79) .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).

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

Intermediate 14—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

(81) ##STR00046##

(82) 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 13 (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%).

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

Intermediate 15—Synthesis of (9H-fluoren-9-yl)methyl 4-({[(tert-butoxy)carbonyl]amino}methyl)piperidine-1-carboxylate

(84) ##STR00047##

(85) 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%).

(86) .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.

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

(88) Combined estimate purity=84%.

Intermediate 16—Synthesis of (9H-fluoren-9-yl)methyl 4-(aminomethyl)piperidine-1-carboxylate hydrochloride

(89) ##STR00048##

(90) 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 15 (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%).

(91) .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.

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

(93) Combined estimated purity=88%.

Intermediate 17—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

(94) ##STR00049##

(95) 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 16 (5.56 g, 14.9 mmol) and 4,6-O-benzylidene-D-glucopyranose (14.6 g, 54.3 mmol) in MeOH (100 ml). 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 CVs; 10-57%, 16 CVs; 57%, 9 CV; 59-63%, 2 CVs; 100%, 3 CVs. The desired fractions were combined and concentrated in vacuo to afford the product as a beige solid (2.99 g, 23%).

(96) .sup.1H NMR (500 MHz, CD.sub.3OD-d.sub.4) 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 (s, 2H), 4.64-4.36 (m, 5H), 4.27-4.20 (m, 3H), 4.04-3.92 (m, 4H), 3.86 (dd, J=5.7, 2.3 Hz, 2H), 3.71 (dd, J=9.3, 2.4 Hz, 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). LC/MS (System B): m/z (ESI.sup.+)=841 [MH.sup.+], R.sub.t=4.78 min, UV purity=95%.

Intermediate 18—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)

(97) ##STR00050##

(98) Piperidine (3.3 ml, 33.41 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 17 (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%).

(99) .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 (s, 2H), 4.25 (dd, J=10.7, 5.4 Hz, 2H), 4.02 (dt, J=8.9, 4.5 Hz, 2H), 3.96 (td, J=10.1, 5.4 Hz, 2H), 3.89 (dd, J=5.2, 2.4 Hz, 2H), 3.74 (dd, J=9.4, 2.4 Hz, 2H), 3.62 (t, J=10.5 Hz, 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).

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

Intermediate 19—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 dihydrochioride

(101) ##STR00051##

(102) 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 18 (1.52 g, 2.14 mmol) in aqueous HCl (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).

(103) .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 (d, J=6.9 Hz, 2H), 3.09 (tt, J=13.0, 3.3 Hz, 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).

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

Intermediate 20—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

(105) ##STR00052##

(106) 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 Methanol (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 CVs; 10-40%, 10 CVs; 40-100%, 2 CVs; 100%, 2 CVs. 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%).

(107) 1H NMR (500 MHz, Methanol-d4) δ 8.29 (s, 1H), 7.53-7.44 (m, 4H), 7.42-7.30 (m, 6H), 5.53 (s, 2H), 4.26 (dd, J=10.6, 5.4 Hz, 2H), 4.10-4.01 (m, 2H), 4.01-3.92 (m, 2H), 3.91 (dd, J=5.3, 2.2 Hz, 2H), 3.77 (dd, J=9.4, 2.2 Hz, 2H), 3.74-3.67 (m, 1H), 3.66-3.54 (m, 3H), 3.26-3.17 (m, 1H), 3.09-2.80 (m, 5H), 2.04-1.86 (m, 1H), 1.86-1.68 (m, 1H), 1.47 (s, 9H). LC/MS (System A): m/z (ESI.sup.+)=691 [MH.sup.+], R.sub.t=0.93 min, UV purity=100%.

Intermediate 21—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

(108) ##STR00053##

(109) 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 20 (1.39 g, 1.89 mmol) in aqueous HCl (2 M, 30 ml, 60 mmol) was stirred at RT for 18 h.

(110) 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).

(111) .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).

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

Intermediate 22—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

(113) ##STR00054##

(114) 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 CVs; 10-49%, 8 CVs; 49-54%, 0.5 CVs; 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%).

(115) .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).

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

Intermediate 23—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

(117) ##STR00055##

(118) 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 22 (1.5 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).

(119) .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).

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

Intermediate 24—Synthesis of tert-butyl N-[2-(2-{2-[2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)ethoxy]ethoxy}ethoxy)ethyl]carbamate

(121) ##STR00056##

(122) NaHCO.sub.3 (0.57 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. 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).

(123) .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).

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

Intermediate 25—Synthesis of (9H-fluoren-9-yl)methyl N-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamate hydrochloride

(125) ##STR00057##

(126) 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 24 (83%, 1.61 g, 2.61 mmol) in MeCN (16 ml). The resulting solution was left to stir at RT for 22 h then concentrated in vacuo to afford the product as a yellow gum (1.57 g, quantitative based on 75% estimated purity).

(127) .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.

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

Intermediate 26—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-di hydroxy-15-[(4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl]-3,6,9-trioxa-12-azapentadecan-1-yl]carbamate; formic acid

(129) ##STR00058##

(130) 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 25 (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 CVs; 10-31%, 8 CVs; 31%, 4.5 CV; 31-35%, 1.5 CVs; 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 CVs; 10-27%, 4 CVs; 27%, 0.5 CV; 27-68%, 9 CV; 68%, 0.5 CVs; 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%).

(131) .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 27—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)

(132) ##STR00059##

(133) Piperidine (1.34 ml, 13.6 mmol) was added to a stirred solution of (9H-fluoren-9-yl)methyl N-[(14 S, 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 26 (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). The column was eluted with MeCN:H.sub.2O+0.1% formic acid using the following gradient (% MeCN, column volumes): 10%, 2 CVs; 10-13%, 1.5 CVs; 13%, 2.5 CV; 13-22%, 5.5 CVs; 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%).

(134) .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).

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

Intermediate 28—Synthesis of (14S,15R,16R,17R)-1-amino-12-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]-3,6,9-trioxa-2-azaoctadecane-14,15,16,17,18-pentol dihydrochloride

(136) ##STR00060##

(137) 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 27 (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).

(138) .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).

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

Intermediate 29—Synthesis of tert-butyl N-[2-(4-bromophenyl)ethyl]carbamate

(140) ##STR00061##

(141) 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%).

(142) .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).

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

Intermediate 30—Synthesis of tert-butyl N-{2-[4′-(cyanomethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate

(144) ##STR00062##

(145) A mixture of tert-butyl N-[2-(4-bromophenyl)ethyl]carbamate, Intermediate 29 (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)C.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%).

(146) .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).

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

Intermediate 31—Synthesis of tert-butyl N-{2-[4′-(2-aminoethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate

(148) ##STR00063##

(149) A mixture of tert-butyl N-{2-[4′-(cyanomethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamate, Intermediate 30 (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%).

(150) .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).

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

Intermediate 32—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

(152) ##STR00064##

(153) 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 31 (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.

(154) 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 CVs; 10-59%, 10 CVs; 59%, 2 CV; 59-100%, 8 CVs; 100%, 2 CVs. The desired fractions were combined and concentrated in vacuo to afford the product as a white solid (932 mg, 78%).

(155) .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).

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

Intermediate 33—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

(157) ##STR00065##

(158) 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 32 (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 the 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).

(159) .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).

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

Intermediate 58—Synthesis of 6-(2-{[(tert-butoxy)carbonyl]amino}ethoxy)-1,3-diethyl-2-({[(9H-fluoren-9-yl methoxy)carbonyl]amino}methyl)-1H-1,3-benzodiazol-3-ium iodide

(161) ##STR00066##

(162) Intermediate 58 was synthesised by according to literature procedures (US 2015/0018313 A1).

Intermediate 60—Synthesis tert-butyl N-[3-(3-fluoro-4-nitrophenoxy)propyl]carbamate

(163) ##STR00067##

(164) 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%).

(165) .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).

(166) LC/MS (System A): m/z (ESI.sup.+)=R.sub.t=1.22 min, UV purity=90%.

Intermediate 61—Synthesis of tert-butyl N-{3-[3-(ethylamino)-4-nitrophenoxy]propyl}carbamate

(167) ##STR00068##

(168) 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 60 (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 (150 ml). The organic phase was washed with water (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%).

(169) .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).

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

Intermediate 62—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

(171) ##STR00069##

(172) Palladium on carbon (10 wt %, 662 mg) was added to a solution of tert-butyl N-{3-[3-(ethylamino)-4-nitrophenoxy]propyl}carbamate, Intermediate 61 (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.

(173) 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%).

(174) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 7.97 (d, J=5.8 Hz, 1H), 7.91-7.87 (m, 2H), 7.72 (d, J=7.7 Hz, 2H), 7.42 (dt, J=13.6, 8.0 Hz, 3H), 7.35-7.27 (m, 3H), 7.06 (s, 1H), 6.93-6.85 (m, 1H), 6.79 (d, J=8.6 Hz, 1H), 4.46 (d, J=5.6 Hz, 2H), 4.32 (t, J=6.8 Hz, 2H), 4.24-4.16 (m, 3H), 4.01 (t, J=6.1 Hz, 2H), 3.11 (q, J=6.6 Hz, 2H), 1.89-1.82 (m, 2H), 1.37 (s, 9H), 1.25 (t, J=7.0 Hz, 3H).

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

Intermediate 63—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

(176) ##STR00070##

(177) A mixture of (9H-fluoren-9-yl)methyl N-{[6-(3-{[(tert-butoxy)carbonyl]amino}propoxy)-1-ethyl-1H-1,3-benzodiazol-2-yl]methyl}carbamate, Intermediate 62 (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). 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%).

(178) .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).

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

Intermediate 65—Synthesis of 2-[(6-bromo-1-ethyl-1H-1,3-benzodiazol-2-yl)methyl]-2,3-dihydro-1H-isoindole-1,3-dione

(180) ##STR00071##

(181) 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 NaHCO.sub.3 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%).

(182) .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).

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

Intermediate 66—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

(184) ##STR00072##

(185) 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 65 (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%).

(186) .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).

(187) LC/MS (System A): m/z (ESI.sup.+)=459 [MH.sup.+], R.sub.t=1.17 min, UV purity=95%.

Intermediate 67—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

(188) ##STR00073##

(189) 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 66 (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%).

(190) .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).

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

Intermediate 68—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

(192) ##STR00074##
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 67 (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%).

(193) .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).

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

Intermediate 69—Synthesis of 2-(aminomethyl)-6-(3-{[(tert-butoxy)carbonyl]amino}propyl)-1,3-diethyl-1H-1,3-benzodiazol-3-ium iodide

(195) ##STR00075##

(196) 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 68 (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%).

(197) .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).

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

(199) Intermediate 69 may be reacted with a compound of general formula (IV) to give a compound of formula:

(200) ##STR00076##

(201) This compound may be treated with HCl solution in dioxane to remove the protecting group, yielding a compound of the formula:

(202) ##STR00077##

(203) This compound may then be stirred at room temperature with 4,6-O-benzylidene-D-glucopyranose and acetic acid in methanol, followed by the addition of NaCNBH.sub.3. After further stirring, a benzylidene protected intermediate will be obtained. This can then be treated with aqueous HCl to give a product of formula:

(204) ##STR00078##

(205) An analogous process is described in our co-pending application WO 2018/096325 (see Example 45).

Intermediate 86—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

(206) ##STR00079##

(207) 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 58 (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%).

(208) .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).

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

Intermediate 87—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

(210) ##STR00080##

(211) 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 86 (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%).

(212) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.76 (d, J=8.5 Hz, 3H), 7.59 (d, J=8.0 Hz, 2H), 7.53-7.49 (m, 2H), 7.47-7.43 (m, 4H), 7.37-7.34 (m, 3H), 7.33-7.25 (m, 10H), 7.24-7.20 (m, 2H), 5.50 (s, 2H), 4.63-4.48 (m, 7H), 4.30-4.16 (m, 6H), 4.08-4.03 (m, 2H), 3.99-3.93 (m, 5H), 3.91-3.86 (m, 1H), 3.78-3.73 (m, 3H), 3.67-3.56 (m, 4H), 3.16-3.06 (m, 2H), 3.00-2.93 (m, 2H), 2.85-2.77 (m, 2H), 1.49-1.37 (m, 6H).

(213) 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 88—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

(214) ##STR00081##

(215) 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 87 (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).

(216) 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%).

(217) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.81-7.66 (m, 1H), 7.51-7.14 (m, 14H), 5.52-5.43 (m, 2H), 4.65-4.48 (m, 5H), 4.35-4.30 (m, 2H), 4.27-4.11 (m, 4H), 4.08-3.85 (m, 7H), 3.81-3.72 (m, 4H), 3.68-3.63 (m, 6H), 3.63-3.53 (m, 3H), 3.16-3.01 (m, 2H), 2.98-2.90 (m, 1H), 2.84-2.80 (m, 6H), 1.57-1.45 (m, 6H).

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

Intermediate 89—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

(219) ##STR00082##

(220) 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 88 (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%).

(221) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.01 (d, J=9.2 Hz, 1H), 7.73 (d, J=2.2 Hz, 1H), 7.55-7.51 (m, 1H), 4.93 (s, 2H), 4.79-4.69 (m, 4H), 4.68-4.62 (m, 3H), 4.32-4.19 (m, 2H), 4.07-3.94 (m, 2H), 3.92-3.84 (m, 10H), 3.81-3.58 (m, 16H), 3.33-3.29 (m, 8H), 3.26-3.21 (m, 6H), 1.65-1.59 (m, 6H).

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

(223) Intermediate 89 may be reacted with a compound of general formula (IV) to give a compound of general formula (I). An analogous process is described in our co-pending application WO 2018/096325 (see Example 43).

Intermediate 90—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

(224) ##STR00083##

(225) 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 63 (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).

(226) .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).

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

Intermediate 91—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

(228) ##STR00084##

(229) 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 90 (85%, 875 mg, 1.30 mmol) and 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%).

(230) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 8.24 (s, 1H), 7.88-7.80 (m, 3H), 7.64-7.57 (m, 3H), 7.48-7.19 (m, 26H), 5.46 (dd, J=15.4, 7.6 Hz, 2H), 4.73 (s, 2H), 4.48 (s, 9H), 4.21 (d, J=5.4 Hz, 3H), 4.10 (s, 5H), 3.82-3.67 (m, 4H), 1.91 (s, 2H), 1.32 (s, 6H).

(231) LC/MS (System A): m/z (ESI.sup.+)=503 [(M.sup.+)+H.sup.+], R.sub.t=1.00 min, UV purity=60%.

Intermediate 92—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

(232) ##STR00085##

(233) 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-yl)methoxy]carbonyl}amino)methyl]-1H-1,3-benzodiazol-3-ium hydrochloride chloride, Intermediate 91 (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%).

(234) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.83-7.77 (m, 1H), 7.54-7.44 (m, 5H), 7.39-7.21 (m, 10H), 5.54-5.44 (m, 2H), 4.68-4.55 (m, 6H), 4.40-4.32 (m, 2H), 4.31-4.07 (m, 6H), 4.04-3.87 (m, 6H), 3.79-3.74 (m, 2H), 3.61-3.55 (m, 2H), 2.83-2.76 (m, 2H), 2.74-2.65 (m, 2H), 2.03-1.96 (m, 1H), 1.60-1.52 (m, 6H).

(235) 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 93—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

(236) ##STR00086##

(237) 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 92 (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 (note to afford the product as a pale orange solid (305 mg, 98%).

(238) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 7.98 (d, J=9.2 Hz, 1H), 7.62 (d, J=2.1 Hz, 1H), 7.49 (dd, J=9.2, 2.2 Hz, 1H), 4.92 (s, 2H), 4.76-4.69 (m, 4H), 4.37 (t, J=5.7 Hz, 2H), 4.25 (dd, J=9.0, 4.1 Hz, 2H), 3.89-3.82 (m, 7H), 3.80-3.73 (m, 4H), 3.72-3.53 (m, 13H), 3.50-3.43 (m, 2H), 3.24-3.21 (m, 4H), 2.49-2.33 (m, 2H), 1.61 (t, J=7.3 Hz, 6H).

(239) 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%.

(240) Intermediate 93 may be reacted with a compound of general formula (IV) to give a compound of general formula (I). An analogous process is described in our co-pending application WO 2018/096325 (see Example 44).

Intermediate 100—Synthesis of tert-butyl N-[3-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propyl]carbamate

(241) ##STR00087##

(242) 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%).

(243) .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).

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

Intermediate 101—Synthesis of (9H-fluoren-9-yl)methyl N-(3-aminopropyl)carbamate hydrochloride

(245) ##STR00088##

(246) 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-yl)methoxy]carbonyl}amino)propyl]carbamate, Intermediate 100 (2.60 g, 6.56 mmol) in MeCN (40 ml). The reaction mixture was stirred at RT for 1 h then filtered. The collected solid was rinsed with MeCN then dried under vacuum to afford the product as a white solid (1.89 g, 87%).

(247) .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).

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

Intermediate 102—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; bis(formic acid)

(249) ##STR00089##

(250) A mixture of (9H-fluoren-9-yl)methyl N-(3-aminopropyl)carbamate hydrochloride, Intermediate 101 (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 NaSO.sub.4, filtered and concentrated in vacuo dryness 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; 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, 46%).

(251) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ 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, 2tH), 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).

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

Intermediate 103—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)

(253) ##STR00090##

(254) Diethylamine (1.44 ml, 14.0 mmol) was added to a solution 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; bis(formic acid), Intermediate 102 (1.12 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, 76%).

(255) .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).

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

Intermediate 104—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

(257) ##STR00091##

(258) 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 103 (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%).

(259) .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).

(260) LC/MS (System A): m/z (ESI.sup.+)=404 [MH.sup.+], R.sub.t=0.12 min, ELS purity=100%.

(261) Intermediate 104 may be reacted with a compound of general formula (II) using a similar method to that described in Examples 1-7 below to obtain a compound of general formula (I).

Intermediate 113—Synthesis of benzyl N-[1-(2-{[(tert-butoxy)carbonyl]amino}ethyl)piperidin-4-yl]carbamate

(262) ##STR00092##

(263) 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%).

(264) .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).

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

Intermediate 114—Synthesis of tert-butyl N-[2-(4-aminopiperidin-1-yl)ethyl]carbamate

(266) ##STR00093##

(267) A mixture of benzyl N-[1-(2-{[(tert-butoxy)carbonyl]amino}ethyl)piperidin-4-yl]carbamate, Intermediate 113 (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).

(268) .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).

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

Intermediate 115—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

(270) ##STR00094##

(271) A mixture of tert-butyl N-[2-(4-aminopiperidin-1-yl)ethyl]carbamate, Intermediate 114 (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%).

(272) .sup.1H NMR (500 MHz, CD.sub.3OD) δ 8.38 (s, 2H), 7.52-7.48 (m, 4H), 7.39-7.35 (m, 6H), 5.54 (s, 2H), 4.26 (dd, J=10.7, 5.4 Hz, 2H), 4.07 (s (br), 2H), 4.02-3.94 (m, 2H), 3.93-3.90 (m, 2H), 3.79-3.73 (m, 2H), 3.63 (t, J=10.5 Hz, 2H), 3.27-3.20 (m, 3H), 3.17-2.92 (m, 6H), 2.69 (s (br), 1H), 2.38 (s (br), 1H), 2.23-2.08 (m, 1H), 1.94-1.85 (m, 2H), 1.83-1.72 (m, 1H), 1.67-1.57 (m, 1H), 1.46 (s, 9H).

(273) 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 116—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

(274) ##STR00095##

(275) A mixture 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, Intermediate 115 (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%).

(276) .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).

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

(278) Intermediate 116 may be reacted with a compound of general formula (II) using a similar method to that described in Examples 1-7 below to obtain a compound of general formula (I).

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

(279) ##STR00096##

(280) A suspension of 6-carboxy-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide, Intermediate 9 (322 mg, 0.646 mmol) and CDI (157 mg, 0.968 mmol) in DMF (3 ml) was stirred at RT for 4 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 14 (388 mg, 0.775 mmol) and rinsed in with further DMF (1 ml). The reaction was left to stir at RT for 16 h then 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% TFA using the following gradient (% MeCN, column volumes): 2%, 2 CVs; 2-20%, 15 CVs; 20%, 3 CV; 20-100%, 2 CVs; 100%, 2 CVs. The desired fractions were combined and concentrated in vacuo at RT. The residual material was diluted with water then lyophilised to afford the product as a white solid (300 mg, 44%).

(281) .sup.1H NMR (500 MHz, DMSO-d.sub.6+D.sub.2O) δ 8.13-8.04 (m, 2H), 7.69 (d, J=8.6 Hz, 1H), 4.97 (s, 2H), 4.74-4.55 (m, 5H), 4.09-3.92 (m, 2H), 3.85-3.75 (m, 1H), 3.64-3.56 (m, 4H), 3.52-3.07 (m, 13H), 2.94-2.77 (m, 1H), 2.24-1.56 (m, 3H), 1.42-1.34 (m, 6H). LC/MS (System D): m/z (ESI.sup.+)=828 [M(.sup.35Cl).sup.+], 830 [M(.sup.37Cl).sup.+], R.sub.t=1.34 min, UV purity=99%.

Example 2—Synthesis of 2-{[(3-amino-6-bromopyrazin-2-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

(282) ##STR00097##

(283) A solution of 2-{[(3-amino-6-bromopyrazin-2-yl)formamido]methyl}-6-carboxy-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide, Intermediate 11 (200 mg, 0.379 mmol), HBTU (144 mg, 0.379 mmol) and 4-methylmorpholine (83 μL, 0.76 mmol) in DMF (4 ml) was stirred at RT for 2 h. (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 14 (223 mg, 0.454 mmol) was added then the reaction was left to stir at RT for 16 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:H.sub.2O+0.1% TFA using the following gradient (% MeCN, column volumes): 5%, 1.5 CVs; 5-13%, 4 CVs; 13%, 0.5 CV; 13-17%, 2.5 CVs; 17%, 11 CV; 17-25%, 2 CV; 25%, 2.5 CV; 25-30%, 0.5 CV; 30% 1 CV; 30-33%, 0.5 CV; 33%, 3 CV; 36-100%, 2 CV; 100%, 3 CVs. The desired fractions were combined and concentrated in vacuo at RT. The residual material was diluted with water/MeCN then lyophilised to afford the product as a yellow solid (67 mg, 16%).

(284) .sup.1H NMR (500 MHz, DMSO-d.sub.6+D.sub.2O) δ 9.64 (t, J=5.4 Hz, 1H), 8.39 (s, 1H), 8.17-8.04 (m, 2H), 7.76-7.65 (m, 1H), 5.06 (d, J=5.1 Hz, 2H), 4.76-4.53 (m, 5H), 4.12-3.89 (m, 2H), 3.72-3.06 (m, 17H), 2.95-2.79 (m, 1H), 2.28-1.51 (m, 4H), 1.50-1.28 (m, 6H).

(285) LC/MS (System D): m/z (ESI.sup.+)=429 [(M(.sup.79Br)+H).sup.2+], 430 [(M(.sup.81Br)+H).sup.2+], R.sub.t=1.58 min, UV purity=100%.

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

(286) ##STR00098##

(287) A mixture of 6-carboxy-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide, Intermediate 9 (150 mg, 0.301 mmol) and CDI (73 mg, 0.45 mmol) in DMF (1.5 ml) was stirred at RT for 3 h. Additional CDI (45 mg, 0.28) was added then the reaction was left to stir at RT for 16 h. (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, Intermediate 19 (93%, 186 mg, 0.336 mmol) was added, followed by DMF (0.5 ml). The reaction mixture was stirred at RT for 25 h then 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% TFA using the following gradient (% MeCN, column volumes): 2%, 2 CVs; 2-10%, 7 CVs; 10-11%, 1 CV; 11%, 5 CVs; 11-20%, 7 CV; 20-100%, 2 CV; 100%, 1.5 CVs. The desired fractions were combined and concentrated in vacuo at RT. The residual material was dissolved in MeCN/water then lyophilised to afford the product as a yellow solid (55 mg, 16%). .sup.1H NMR (500 MHz, DMSO-d.sub.6+D2O) δ 8.04 (d, J=8.6 Hz, 1H), 8.02 (s, 1H), 7.70-7.58 (m, 1H), 4.95 (s, 2H), 4.72-4.57 (m, 4H), 4.54-4.38 (m, 1H), 4.05-3.95 (m, 2H), 3.68-3.61 (m, 2H), 3.58 (dd, J=11.0, 3.1 Hz, 2H), 3.54-3.45 (m, 3H), 3.43-3.36 (m, 4H), 3.34-3.23 (m, 4H), 3.20-3.01 (m, 3H), 2.94-2.72 (m, 1H), 2.26-1.47 (m, 2H), 1.42-1.33 (m, 7H), 1.32-1.11 (m, 2H).

(288) LC/MS (System D): m/z (ESI.sup.+)=842 [M(.sup.35Cl).sup.+], 844 [M(.sup.37Cl).sup.+], R.sub.t=1.34 min, UV purity=96%.

Example 4—Synthesis of 6-[(3R)-3-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}pyrrolidine-1-carbonyl]-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetic acid trifluoroacetate

(289) ##STR00099##

(290) A mixture of 6-carboxy-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide, Intermediate 9 (128 mg, 0.256 mmol) and CDI (62 mg, 0.38 mmol) in DMF (1.5 ml) was stirred at RT for 6 h. An aliquot (0.75 ml) of the reaction mixture was added to (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, Intermediate 21 (83%, 106 mg, 0.180 mmol). The reaction was left to stir at RT for 22 h then diluted with water (1 ml). The crude material was purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:H.sub.2O+0.1% TFA using the following gradient (% MeCN, column volumes): 2%, 2 CVs; 2-3%, 1 CVs; 3%, 3 CV; 3-13%, 8 CVs; 13%, 3 CV; 13-20%, 6 CV; 20-100%, 2CV; 100%, 2 CVs. The desired fractions were combined and concentrated in vacuo at RT. The residual material was dissolved in MeCN/water then lyophilised to afford the product as a yellow solid (37 mg, 27%).

(291) .sup.1H NMR (500 MHz, DMSO-d.sub.6+D.sub.2O) δ 8.13 (s, 1H), 8.11-8.01 (m, 1H), 7.76 (d, J=8.8 Hz, 1H), 4.96 (s, 2H), 4.74-4.51 (m, 4H), 4.41-4.19 (m, 1H), 4.12-3.94 (m, 2H), 3.75-3.21 (m, 17H), 2.39-2.04 (m, 3H), 1.44-1.32 (m, 6H).

(292) LC/MS (System D): m/z (ESI.sup.+)=814 [M(.sup.35Cl).sup.+], 816 [M(.sup.37Cl).sup.+], R.sub.t=1.25 min, UV purity=97%.

Example 5—Synthesis of 6-[(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethyl)carbamoyl]-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetic acid trifluoroacetate

(293) ##STR00100##

(294) A mixture of 6-carboxy-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide, Intermediate 9 (150 mg, 0.301 mmol) and CDI (73 mg, 0.45 mmol) in DMF (1.5 ml) was stirred at RT for 4.5 h. (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, Intermediate 23 (94%, 166 mg, 0.338 mmol) was added then the reaction was left to stir at RT for 16 h then diluted with water (1 ml). The crude material was purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:H.sub.2O+0.1% TFA using the following gradient (% MeCN, column volumes): 2%, 2 CVs; 2-10%, 6.5 CVs; 10%, 7.5 CV; 10-20%, 7.5 CVs; 20-100%, 2 CV; 100%, 2 CVs.

(295) The desired fractions were combined and concentrated in vacuo at RT. The residual material was dissolved in MeCN/water then lyophilised to afford the product as a yellow solid (93 mg, 30%).

(296) .sup.1H NMR (500 MHz, DMSO-d.sub.6+D.sub.2O) δ 8.38 (s, 1H), 8.11 (d, J=8.8 Hz, 1H), 8.09-8.04 (m, 1H), 5.05-4.91 (m, 2H), 4.73-4.58 (m, 4H), 4.09-3.97 (m, 2H), 3.69-3.64 (m, 3H+HDO), 3.62-3.25 (m, 15H), 1.50-1.31 (m, 6H).

(297) LC/MS (System D): m/z (ESI.sup.+)=788 [M(.sup.35Cl).sup.+], 790 [M(.sup.37Cl).sup.+], R.sub.t=1.30 min, UV purity=97%.

Example 6—Synthesis of 2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-6-{[(14S,15R,16R,17R)-14,15,16,17,18-pentahydroxy-12-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]-3,6,9-trioxa-12-azaoctadecan-1-yl]carbamoyl}-1H-1,3-benzodiazol-3-ium trifluoroacetic acid trifluoroacetate

(298) ##STR00101##

(299) A mixture of 6-carboxy-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide, Intermediate 9 (150 mg, 0.301 mmol) and CDI (73 mg, 0.45 mmol) in DMF (1.5 ml) was stirred at RT for 4.5 h. Additional CDI (73 mg, 0.45) was added then the reaction was left to stir at RT for 18 h. The reaction mixture was added to (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, Intermediate 28 (96%, 214 mg, 0.346 mmol). The resultant mixture was left to stir at RT for 21 h then diluted with water (2 ml). The crude material was purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:H.sub.2O+0.1% TFA using the following gradient (% MeCN, column volumes): 2%, 2 CVs; 2-9%, 6 CVs; 9%, 1.5 CV; 9-14%, 4 CVs; 14%, 6 CV; 14-19%, 4 CV; 19-20%, 0.5 CV; 20-100%, 2 CVs; 100% 1.5 CV. The desired fractions were combined and concentrated in vacuo at RT. The residual material was dissolved in MeCN/water then lyophilised to afford a yellow solid (74 mg). The material thus obtained was further purified by flash column chromatography on C18 (12 g). The column was eluted with MeCN:H.sub.2O+0.1% TFA using the following gradient (% MeCN, column volumes): 2%, 2 CVs; 2-14%, 10 CVs; 14%, 5.5 CV; 14-20%, 5 CVs; 20-100%, 2 CVs; 100% 2 CV. The desired fractions were combined and concentrated in vacuo at RT. The residual material was dissolved in MeCN/water then lyophilised to afford the product as an off-white solid (9 mg, 3%).

(300) .sup.1H NMR (500 MHz, DMSO-d.sub.6+D.sub.2O) δ 8.41 (s, 1H), 8.10-8.07 (m, 2H), 5.05-4.92 (m, 2H), 4.71-4.57 (m, 4H), 4.04-3.93 (m, 2H), 3.67-3.62 (m, 2H), 3.61-3.17 (m, 28H), 1.48-1.30 (m, 6H).

(301) LC/MS (System D): m/z (ESI.sup.+)=461 [(M(.sup.35Cl)+H).sup.2+], 462 [(M(.sup.37Cl)+H).sup.2+], R.sub.t=1.48 min, UV purity=96%.

Example 7—Synthesis of 6-({2-[4′-(2-{bis[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino}ethyl)-[1,1′-biphenyl]-4-yl]ethyl}carbamoyl)-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium trifluoroacetic acid trifluoroacetate

(302) ##STR00102##

(303) A mixture of 6-carboxy-2-{[(3,5-diamino-6-chloropyrazin-2-yl)formamido]methyl}-1,3-diethyl-1H-1,3-benzodiazol-3-ium bromide, Intermediate 9 (128 mg, 0.256 mmol) and CDI (62 mg, 0.38 mmol) in DMF (1.5 ml) was stirred at RT for 6 h. An aliquot (0.75 ml) of the reaction mixture was added to (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, Intermediate 33 (94%, 154 mg, 0.226 mmol). Additional DMF (0.75 ml) was used to rinse the intermediate solution into the reaction vessel. The reaction mixture was stirred at RT for 16 h then concentrated under a stream of nitrogen. The residue thus obtained was diluted with water (1 ml) then purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:H.sub.2O+0.1% TFA using the following gradient (% MeCN, column volumes): 2%, 2 CVs; 2-6%, 3 CVs; 6%, 0.5 CV; 6-10%, 3.5 CVs; 10%, 4 CV; 10-20%, 8.5 CV; 20-100%, 2 CV; 100%, 2 CVs. The desired fractions were combined and concentrated in vacuo at RT. The residual material was dissolved in MeCN/water then lyophilised to afford a yellow oil. The material thus obtained was further purified by flash column chromatography on C18 (30 g). The column was eluted with MeCN:H.sub.2O+0.1% TFA using the following gradient (% MeCN, column volumes): 10%, 2 CVs; 10-21%, 2 CVs; 21%, 0.5 CV; 21-25%, 1 CVs; 25%, 4 CV; 25-35%, 2 CV; 35%, 1 CV; 35-84%, 9 CV; 100%, 3.5 CVs. The desired fractions were combined and concentrated in vacuo at RT. The residual material was dissolved in MeCN/water then lyophilised to afford the product as a yellow solid (35 mg, 23%).

(304) .sup.1H NMR (500 MHz, DMSO-d.sub.6+D.sub.2O) δ 8.29 (s, 1H), 8.05-7.90 (m, 2H), 7.61-7.50 (m, 4H), 7.38-7.27 (m, 4H), 4.94 (s, 2H), 4.68-4.52 (m, 4H), 4.09-4.06 (m, 2H+HDO), 3.73-3.21 (m, 18H), 3.08-2.97 (m, 2H), 2.95-2.84 (m, 2H), 1.41-1.33 (m, 6H). LC/MS (System D): m/z (ESI.sup.+)=485 [(M(.sup.35Cl)+H).sup.2+], 486 [(M(.sup.37Cl)+H).sup.2+], R.sub.t=2.03 min, UV purity=100%.

(305) Other Compounds of the Invention

(306) Intermediate 7 may be reacted with intermediate 8 to give a compound of the formula:

(307) ##STR00103##

(308) This compound may be reacted with Intermediate 104 or Intermediate 116 to yield further compounds of general formula (I).

BIOLOGICAL EXAMPLES

Example 8—Short Circuit Current Assay to Determine ENaC Blocker Potency in Human Bronchial Epithelial Cells

(309) Cell Culture

(310) 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 ALl, 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 ALl. At all stages of culture, cells were maintained at 37° C. in 5% CO.sub.2 in an air incubator.

Example 9—Bronchoalveolar Lavage

(311) A 0.1 mg/mL solution of ENaC inhibitor in 5% dextrose was administered intratracheally to a rat weighing 225-250 g. A volume of 1 mL/Kg was used. After 6 hours, lungs were lavaged with 3×4 mL of sterile saline. A 1 mL aliquot was subsequently snap frozen. Lungs were excised, weighed and snap frozen. Compound levels in the BAL and lung tissue were subsequently determined using LC/MS/MS bioanalysis.

(312) The results from Examples 8 and 9 are presented in Table 1.

(313) TABLE-US-00001 BAL @ 6 hours Example ENaC (ng/mL) 1 6 222 2 18 3 4 210 4 11 524 5 5 471 6 4 251 7 1 645