Patent Abstract:
the present invention relates to macrocyclic amidinourea derivatives of formula 8 , methods of preparation and uses thereof , pharmaceutical compositions in particular to be used as chitinase inhibitors in the treatment of a fungal infection .

Detailed Description:
the compounds having formula 8 described in the invention can be synthesized as described in scheme 1 below : ( i ) allylnh 2 , edc , hobt , dipea , dmf ( ii ) dibal - h , dcm , r . t . ( iii ) thf , reflux , 12 h ( iv ) grubbs &# 39 ; cat . 2nd gen ., toluene or dcm 2 - 10 mm , 40 - 80 ° c . ( v ) h 2 , pd / c , etoh ( vi ) r 1 nboc ( c = nboc ) sme , thf , reflux , 12 h . wherein n 1 , n 2 , r 1 , r 2 , x , y and z are as defined herein above . unless otherwise indicated , commercially available reagents and solvents were used without further purification . specifically , the following abbreviations may have been used in the descriptions of the experimental methods : min : minutes ; h : hour ( s ); r . t . : room temperature , nmr : nuclear magnetic resonance ; mhz : megahertz ; 1 h : proton ; 13 c : carbon 13 ; mg : milligrams ; mmol : millimoles ; ml : milliliters ; μl : microliters ; n : normal ; nmr nuclear magnetic resonance ; lc - ms liquid chromatography mass spectrum ; tms : tetramethylsilane ; thf : tetrahydrofuran ; tf : trifluoromethylsulfonyl ; boc : tert - butyloxycarbonyl ; dcm : dichloromethane ; etoac : ethyl acetate ; edc : n -( 3 - dimethylaminopropyl )- n ′- ethylcarbodiimide hydrochloride ; hobt : 1 - hydroxybenzotriazole ; dmf : n , n - dimethylformamide ; dipea : n , n ′- diisopropylethylamine ; dibal - h : diisobutylaluminium hydride ; meoh : methyl alcohol ; meod : deuterated methyl alcohol ; etoh : ethyl alcohol ; et 2 o : diethyl ether ; et 3 n : triethylamine ; dmso : dimethyl sulfoxide ; cbzcl : benzyl chloroformate ; ep : petroleum ether ; dcc : n , n ′- dicyclohexyl methanediimine ; tmsci : trimethylsilyl chloride ; py : pyridine . except where indicated otherwise , all temperatures are expressed in ° c . ( degrees centigrade ) or k ( kelvin ). the structure of the intermediates and of the final compounds of the synthesis were confirmed by nmr and / or lc - ms analysis . the 1 h - nmr spectra were acquired with a varian gemini 200 ( 200 mhz ) or with a brucker avance dpx400 ( 400 mhz ). the 13 c - nmr spectra were acquired with a brucker avance dpx400 ( 400 mhz ). the chemical shifts are expressed in parts per million ( ppm , δ units ). the coupling constants are expressed in hertz ( hz ) and the splitting patterns are described as s ( singlet ), bs ( broad signal ), d ( doublet ), t ( triplet ), q ( quartet ), quint ( quintet ), m ( multiplet ). the lc - ms analyses were carried out on a system consisted of a varian apparatus ( varian inc ) including a vacuum solvent degassing unit , two pumps ( 212 - lc ), a triple quadrupole msd ( mod . 320 - lc ) mass spectrometer with es interface and varian ms workstation system control vers . 6 . 9 software . chromatographic separation was obtained using a pursuit c18 column ( 50 × 2 . 0 mm ) ( varian ) with 3 μm particle size and gradient elution : eluent a being ch 3 cn and eluent b consisting of an aqueous solution of formic acid ( 0 . 1 %). the analysis started with 0 % of eluent a , which was linearly increased up to 50 % in 10 min , then slowly increased up to 60 % up to 15 min . the flow rate was 0 . 3 ml / min and injection volume was 5 μl . the instrument operated in positive mode and parameters were : detector 1850 v , drying gas pressure 25 . 0 psi , desolvation temperature 300 . 0 ° c ., nebulizing gas 45 . 0 psi , needle 5000 v and shield 600 v . nitrogen was used as nebulizer and drying gas . collision induced dissociation was performed using argon as the collision gas at a pressure of 1 . 8 mtorr in the collision cell , the collision energy was set to 149 ev . three transitions were recorded at 279 . 8 ( m / z ), 321 . 9 ( m / z ) and 368 . 0 ( m / z ) setting capillary voltage to − 27 . 5 v , − 23 . 5 v and − 19 . 5 v respectively . for reasons of clarity , the detailed synthesis of example 16 has been reported in scheme 2 below . the yields were calculated assuming that products were 100 % pure if not stated otherwise . ( i ) brch 2 ch ═ ch 2 , k 2 co 3 , dcm , reflux ( ii ) nh 2 oh , py , etoh , reflux ( iii ) zn , hcl , thf , reflux ( iv ) ( bocnh ) 2 c = ntf , et 3 n , dcm ( v ) allylnh 2 , edc , hobt , dipea , dmf ( vi ) dibal - h , dcm , r . t . ( vii ) thf , reflux , 12 h ( viii ) grubbs &# 39 ; cat . 2nd gen ., toluene or dcm 2 - 10 mm , 40 - 80 ° c . ( ix ) h 2 , pd / c , etoh ( x ) crotylnboc ( c = nboc ) sme , thf , reflux , 12 h 2 - hydroxybenzaldehyde 9 ( 0 . 5 ml , 4 . 69 mmol , sigma - aldrich catalogue id : s356 ) was dissolved in ch 3 cn ( 10 ml ) and k 2 co 3 ( 0 . 97 ml , 7 . 03 mmol ) and 3 - bromoprop - 1 - ene ( 0 . 45 ml , 5 . 16 mmol , sigma - aldrich catalogue id : 337528 ) were added . mixture was stirred at reflux for 12 hours . solvent was then evaporated in vacuum and the residue dissolved in etoac ( 5 ml ). water ( 10 ml ) was added and the mixture stirred for 10 min at room temperature . organic phase was then separated and the aqueous layer was extracted with etoac ( 2 × 10 ml ). the combined organic phases were dried over na 2 so 4 , filtered and concentrated under reduced pressure to give title compound 10 . yield 99 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ = 10 . 25 ( 1h , s ), 7 . 54 - 7 . 52 ( 1h , m ), 7 . 23 - 7 . 19 ( 1h , m ), 6 . 72 - 6 . 66 ( 2h , m ), 5 . 82 - 5 . 75 ( 1h , m ), 5 . 20 - 5 . 15 ( 1h , d , j = 17 . 6 hz ), 5 . 05 - 5 . 03 ( 1h , d , j = 10 . 8 hz ), 4 . 33 - 4 . 31 ( 2h , m ) ppm . 13 c nmr ( cdcl 3 ) δ 188 . 9 , 160 . 6 , 135 . 6 , 132 . 2 , 127 . 8 , 124 . 7 , 120 . 5 , 117 . 5 , 112 . 7 , 68 . 7 ppm , liquid chromatography mass spectroscopy ( lcms ) m / z ( es +) m / z : 347 . 0 [ 2m + na ] + , 185 . 0 [ m + na ] + , 163 . 0 [ m + h ] + . elemental analysis for c 10 h 10 o 2 : calcd . c , 74 . 06 ; h , 6 . 21 . found c , 74 . 36 ; h , 6 . 52 . 2 - prop - 2 - enoxybenzaldehyde 10 ( 4 . 69 mmol ) was dissolved in etoh ( 10 ml ) and pyridine ( 0 . 45 ml , 5 . 63 mmol ) and nh 2 oh ( 490 mg , 7 . 03 mmol ) were added . the mixture was heated at reflux for 2 h . brine was added , the aqueous layer was extracted with etoac ( 2 × 10 ml ) and the combined organic phases were dried over na 2 so 4 and filtered . the solvent was evaporated under reduced pressure , and the residue was purified by flash chromatography ( sio 2 ) using hexane - et 2 o , 3 : 1 as the eluent affording a mixture of e / z isomers of oxime . n -[( 2 - prop - 2 - enoxyphenyl ) methylidene ] hydroxylamine ( 818 mg , 4 . 62 mmol ) was dissolved in thf ( 30 ml ) and zn ( 3 . 00 g , 46 . 23 mmol ) was added . hcl 2n ( 12 ml ) was added and mixture was stirred at reflux for 3 h . the reaction mixture was cooled to room temperature , filtered on a celite pad to remove the excess zinc and condenser under reduced pressure . ph was adjusted to & gt ; 10 by addition of ammonium hydroxide . the aqueous layer was extracted with etoac ( 2 × 10 ml ) and the combined organic phases were dried over na 2 so 4 and filtered . the solvent was evaporated under reduced pressure to give title compound 11 . yield 99 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ = 7 . 06 - 7 . 01 ( 2h , m ), 6 . 77 - 6 . 73 ( 1h , m ), 6 . 67 - 6 . 65 ( 1h , m ), 5 . 92 - 5 . 84 ( 1h , m ), 5 . 28 - 5 . 24 ( 1h , d , j = 17 . 2 hz ), 5 . 13 - 5 . 10 ( 1h , d , j = 10 . 4 hz ), 4 . 36 - 4 . 35 ( 2h , m ), 3 . 70 - 3 . 68 ( 2h , m ), 1 . 65 ( 2h , br s ) ppm . 13 c nmr ( cdcl 3 ) δ 156 . 1 , 133 . 2 , 131 . 9 , 128 . 2 , 127 . 7 , 120 . 5 , 116 . 8 , 111 . 3 , 68 . 3 , 42 . 4 ppm . lcms m / z ( es +) m / z = 349 . 0 [ 2m + na ] + , 202 . 0 [ m + na ] + , 163 . 9 [ m + h ] + . elemental analysis for c 10 h 13 no : calcd . c , 73 . 59 ; h , 8 . 03 ; n , 8 . 58 . found c , 73 . 86 ; h , 8 . 47 ; n , 9 . 01 . procedure for the preparation of tert - butyl n -({[( tert - butoxy ) carbonyl ] amino }( trifluoromethanesulfonylimino ) methyl ) carbamate ( also named as 1 , 3 - di - boc - 2 -( trifluoromethylsulfonyl ) guanidine ) tert - butyl n —[ n -[( 2 - methylpropan - 2 - yl ) oxycarbonyl ] carbamimidoyl ] carbamate ( 7 . 12 g , 27 . 45 mmol , sigma - aldrich catalogue id : 496871 ) was dissolved in dry dcm ( 136 ml ) and et 3 n ( 4 . 2 ml , 30 . 19 mmol ) was added . the solution was cooled at − 78 ° c . and trifluoromethylsulphonyl trifluoromethanesulphonate ( sigma - aldrich , catalogue id : 91737 ) solution 1 m in methylene chloride ( 35 . 68 ml ) was added in one portion . the mixture was then allowed to warm to r . t . and stirred for 4 h . water was added to the mixture and organic layers were washed two times with brine while aqueous phase was extracted twice with etoac . finally combined organic phases were dried over na 2 so 4 , filtered and evaporated in vacuum . the solid residue was recrystallized from hexane led to the title compound tert - butyl n -({[( tert - butoxy ) carbonyl ] amino }( trifluoromethanesulfonylimino ) methyl ) carbamate ( also named as 1 , 3 - di - boc - 2 -( trifluoromethylsulfonyl ) guanidine ). yield 70 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ = 8 . 00 ( 2h , s ), 1 . 4 ( 18h , m ) ppm . 13 c nmr ( cdcl 3 ) δ 151 . 0 , 148 . 0 , 117 . 5 , 85 . 9 , 27 . 7 ppm . lcms m / z ( es +) m / z = 805 . 0 [ 2m + na ] + , 414 . 0 [ m + na ] + , 391 . 9 [ m + h ] + . elemental analysis for c 12 h 20 f 3 n 3 o 6 s : calcd . c , 36 . 83 ; h , 5 . 15 ; n , 10 . 74 . found c , 37 . 13 ; h , 5 . 35 ; n , 10 . 94 . ( 2 - prop - 2 - enoxyphenyl ) methanamine 11 ( 345 mg , 2 . 1 mmol ), et 3 n ( 0 . 58 ml , 4 . 2 mmol ) and 1 , 3 - di - boc - 2 -( trifluoromethylsulfonyl ) guanidine were mixed in dcm ( 25 ml ) and mixture stirred at room temperature overnight . the aqueous layer was separated and extracted with etoac ( 2 × 10 ml ) and the combined organic phases were dried over na 2 so 4 and filtered . the solvent was evaporated under reduced pressure , and the residue was purified by flash chromatography ( hexane - et 2 o , 8 : 2 ) affording guanidine title compound 1a . yield 75 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ = 11 . 42 ( 1h , s ), 8 . 62 ( 1h , s ), 7 . 09 - 7 . 07 ( 1h , d , j = 7 . 6 hz ), 7 . 03 - 6 . 99 ( 1h , m ), 6 . 71 - 6 . 63 ( 2h , m ), 5 . 92 - 5 . 83 ( 1h , m ), 5 . 22 - 5 . 18 ( 1h , d , j = 17 . 6 hz ), 5 . 06 - 5 . 04 ( 1h , d , j = 10 . 4 hz ), 4 . 46 - 4 . 45 ( 2h , m ), 4 . 37 - 4 . 35 ( 2h , m ), 1 . 31 ( 9h , s ), 1 . 26 ( 9h , s ) ppm . 13 c nmr ( cdcl 3 ) δ 163 . 4 , 156 . 4 , 155 . 8 , 152 . 9 , 132 . 9 , 129 . 4 , 128 . 7 , 125 . 5 , 120 . 4 , 117 . 1 , 111 . 3 , 82 . 3 , 78 . 5 , 68 . 5 , 40 . 6 , 28 . 0 , 27 . 7 ppm . lcms m / z ( es +) m / z = 833 . 0 [ 2m + na ] + , 428 . 0 [ m + na ] + , 406 . 2 [ m + h ] + . elemental analysis for c 21 h 13 n 3 o 5 : calcd . c , 62 . 20 ; h , 7 . 71 ; n , 10 . 36 . found c , 62 . 56 ; h , 7 . 92 ; n , 10 . 85 . 8 - aminooctanoic acid ( 500 mg , 3 . 14 mmol , sigma - aldrich catalogue id : 855294 ) and k 2 co 3 ( 867 mg , 6 . 28 mmol ) were dissolved in thf ( 15 ml ) ( suspension ). then cbzcl ( 0 . 67 ml , 4 . 71 mmol ) was added and the mixture was stirred at r . t . overnight . the reaction was quenched with etoac and h 2 o . aqueous phase was separated and ph of aqueous phase was adjusted to ph = 2 by addition of hcl 4n and extracted twice with etoac . combined organic phase were then dried over na 2 so 4 , filtered and evaporated in vacuum affording the title compound 2a . yield 99 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ 11 . 01 ( 1h , s ), 7 . 28 - 7 . 19 ( 5h , m ), 5 . 11 - 5 . 07 ( 1h , d , j = 16 hz ), 5 . 05 - 5 . 03 ( 1h , d , j = 8 hz ), 3 . 18 - 3 . 16 ( 2h , m ), 2 . 44 - 2 . 40 ( 2h m ), 1 . 64 - 1 . 62 ( 2h , m ), 1 . 48 - 1 . 43 ( 2h , m ), 1 . 32 ( 6h , m ) ppm . 13 c nmr ( cdcl 3 ) δ 178 . 7 , 156 . 4 , 136 . 5 , 128 . 4 , 128 . 0 , 128 . 0 , 66 . 5 , 41 . 4 , 33 . 8 , 29 . 7 , 28 . 6 , 26 . 4 , 24 . 7 ppm . lcms m / z ( es +) m / z = 609 . 0 [ 2m + na ] + , 316 . 0 [ m + na ] + , 294 . 1 [ m + h ] + . elemental analysis for c 16 h 23 no 4 : calcd . c , 65 . 51 ; h , 7 . 90 ; n , 4 . 77 . found c , 65 . 96 ; h , 8 . 35 ; n , 5 . 04 . the 8 -( benzyloxycarbonylamino ) octanoic acid 2a ( 14 mmol ), hobt ( 424 mg , 3 . 14 mmol ), edc ( 487 mg , 3 . 14 mmol ), dipea ( 0 . 66 ml , 3 . 77 mmol ), allylamine ( 0 . 23 ml , 3 . 14 mmol ) were mixed in dmf ( 5 ml ). mixture was stirred overnight at room temperature . the reaction was quenched with nahco 3 , and then aqueous phase was separated and extracted with etoac ( 3 × 5 ml ). the solvent was evaporated under reduced pressure , and the residue was purified by flash chromatography ( etoacep , 4 : 1 ) affording benzyl n -[ 8 - oxo - 8 -( prop - 2 - enylamino ) octyl ] carbamate yield 75 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ 7 . 28 - 7 . 19 ( 5h , m ), 5 . 79 - 5 . 70 ( 1h , m ), 5 . 74 ( 1h , br s ), 5 . 11 - 5 . 07 ( 1h , d , j = 17 . 2 hz ), 5 . 03 - 5 . 01 ( 1h , d , j = 10 . 4 hz ), 5 . 00 ( 2h , s ), 4 . 78 ( 1h , br s ), 3 . 81 - 3 . 78 ( 2h , t , j = 4 . 8 hz ), 3 . 10 - 3 . 08 ( 2h , m ), 2 . 12 - 2 . 08 ( 2h , t , j = 7 . 6 hz ), 1 . 55 ( 2h , m ), 1 , 40 ( 2h , m ), 1 . 23 - 1 . 18 ( 8h , m ) ppm . 13 c nmr ( cdcl 3 ) δ 172 . 8 , 156 . 3 , 136 . 5 , 134 . 2 , 128 . 4 , 127 . 9 , 127 . 6 , 116 . 9 , 66 . 4 , 41 . 7 , 40 . 8 , 36 . 4 , 29 . 7 , 28 . 9 , 28 . 7 , 26 . 3 , 25 . 4 ppm . lcms m / z ( es +) m / z = 687 . 0 [ 2m + na ] + , 355 . 0 [ m + na ] + , 333 . 1 [ m + h ] + . elemental analysis for c 19 h 28 n 2 o 3 : calcd . c , 68 . 65 ; h8 . 49 ; n8 . 43 . found c , 69 . 06 ; h , 8 . 95 ; n , 8 . 93 . benzyl n -[ 8 - oxo - 8 -( prop - 2 - enylamino ) octyl ] carbamate ( 747 mg , 2 . 25 mmol ) was dissolved in ch 2 cl 2 dry ( 5 ml ) and cooled at − 78 ° c . dibal ( 3 . 37 ml , 3 . 37 mmol ) was added and mixture was stirred 1 h at − 78 ° c . then mixture was warmed at room temperature dibal ( 4 . 5 ml , 4 . 5 mmol ) was added again and mixture was stirred for ⅔ h . the reaction was quenched with etoac and rochelle salt . the crude title compound 3a was used in the next step . yield 99 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ = 7 . 28 - 7 . 19 ( 5h , m ), 5 . 79 - 5 . 70 ( 1h , m ), 5 . 11 - 5 . 07 ( 1h , d , j = 17 . 2 hz ), 5 . 03 - 5 . 01 ( 1h , d , j = 10 . 4 hz ), 5 . 00 ( 2h , s ), 3 . 22 - 3 . 20 ( 2h , m ), 2 . 09 - 2 . 05 ( 4h , m ), 1 . 40 - 1 . 50 ( 4h , m ), 1 . 23 - 1 . 18 ( 8h , m ) ppm . 13 c nmr ( cdcl 3 ) δ 156 . 3 , 141 . 2 , 134 . 2 , 129 . 0 , 128 . 1 , 127 . 9 , 116 . 0 , 65 . 0 , 52 . 1 , 49 . 8 , 41 . 9 , 31 . 0 , 29 . 9 , 29 . 4 , 27 . 0 , 26 . 8 ppm . lcms m / z ( es +) m / z = 659 . 0 [ 2 m + na ] + , 341 . 0 [ m + na ] + , 319 . 0 [ m + h ] + . tert - butyl n -[[( 2 - methylpropan - 2 - yl ) oxycarbonylamino ]-[( 2 - propoxyphenyl ) methylamino ] methyl ] carbamatebenzyl 8 -( allylamino ) octylcarbamate 1a ( 2 . 25 mmol ) and compound 3a ( 1 . 4 mmol ) were mixed in thf ( 10 ml ), et 3 n ( 1 . 4 mmol ) was added and mixture was refluxed overnight . solvent was removed and the residue was purified by flash chromatography ( hexane - et 2 o from 9 : 1 to 1 : 1 ) affording the title compound 12 . yield 80 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ 12 . 27 ( 1h , s ), 8 . 47 ( 1h , s ), 7 . 26 - 7 . 21 ( 5h , m ), 7 . 18 - 7 . 12 ( 2h , m ), 6 . 83 - 6 . 76 ( 2h , m ), 6 . 05 - 5 . 97 ( 1h , m ), 5 . 75 - 5 . 67 ( 1h , m ), 5 . 36 - 5 . 32 ( 1h , d , j = 17 . 2 hz ), 5 . 21 - 5 . 18 ( 1h , d , j = 10 . 4 hz ), 5 . 07 - 5 . 01 ( 1h , m ), 5 . 00 - 4 . 99 ( 2h , m ), 4 . 98 - 4 . 96 ( 1h , m ), 4 . 52 - 4 . 47 ( 4h , m ), 3 . 86 - 3 . 84 ( 2h , m ), 3 . 37 - 3 . 33 ( 1h , m ), 3 . 18 - 3 . 14 ( 1h , m ), 3 . 09 - 3 . 06 ( 2h , m ), 1 . 43 ( 4h , m ), 1 . 37 ( 9h , m ), 1 . 21 - 1 . 16 ( 8h , m ) ppm . 13 c nmr ( cdcl 3 ) δ 163 . 6 , 156 . 3 , 153 . 9 , 153 . 1 , 136 . 7 , 135 . 3 , 134 . 6 , 129 . 2 , 128 . 8 , 128 . 2 , 128 . 0 , 127 . 8 , 127 . 0 , 120 . 4 , 117 . 1 , 115 . 4 , 111 . 4 , 81 . 7 , 68 . 6 , 66 . 1 , 50 . 3 , 48 . 3 , 40 . 9 , 40 . 2 , 29 . 8 , 29 . 2 , 28 . 5 , 27 . 9 , 27 . 8 , 26 . 8 , 26 . 5 ppm . lcms m / z ( es +) m / z = 672 . 3 . 0 [ m + na ] + , 688 . 2 [ m + k ] + , 650 . 3 [ m + h ] + . elemental analysis for c 36 h 51 n 5 o 6 : calcd . c , 66 . 54 ; h , 7 . 91 ; n , 10 . 78 . found c , 66 . 93 ; h , 8 . 11 ; n , 10 . 98 . the next step is a ring closing metathesis and was made by two different procedures : a . the starting material compound 12 ( 0 . 43 mmol ) was dissolved in dcm ( solution 2 mm ); second generation grubbs catalyst ( 0 . 043 mmol , sigma - aldrich catalogue id : 569747 ) was dissolved in 2 ml of dcm and added to previous solution via syringe pump over 4 h at 40 / 50 ° c . the solvent was evaporated under reduced pressure , and the residue was purified by flash chromatography ( hexane - et 2 o , 9 : 1 ) affording the title compound 13 . b . the starting material compound 12 ( 0 . 76 mmol ) was dissolved in toluene ( solution 10 mm ); grubbs catalyst ( 0 . 15 mmol ) was dissolved in 2 ml of toluene and added to previous solution via syringe pump over 4 h at 100 ° c . the solvent was evaporated under reduced pressure , and the residue was purified by flash chromatography ( hexane - et 2 o , 9 : 1 ) affording the title compound 13 . procedure b ; yield 30 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ 7 . 33 - 7 . 16 ( 5h , m ), 7 . 02 - 7 . 89 ( 4h , m ), 5 . 38 - 5 . 32 ( 2h , m ), 5 . 07 - 5 . 05 ( 2h , m ), 4 . 65 - 4 . 45 ( 4h , m ), 3 . 80 - 3 . 70 ( 2h , m ), 3 . 17 - 3 . 14 ( 4h , m ), 1 . 43 ( 9h , m ), 1 . 26 ( 12h , m ) ppm . 13 c nmr ( cdcl 3 ) δ 163 . 8 , 156 . 3 , 155 . 3 , 153 . 4 , 152 . 9 , 136 . 6 , 129 . 8 , 129 . 3 , 128 . 7 , 128 . 4 , 121 . 6 , 113 . 4 , 82 . 0 , 75 . 0 , 66 . 4 , 48 . 1 , 46 . 3 , 41 . 0 , 29 . 8 , 29 . 3 , 29 . 1 , 28 . 6 , 28 . 0 , 26 . 9 , 26 . 5 , 24 . 6 ppm . lcms m / z ( es +) m / z = 660 . 3 [ m + na ] + , 688 . 2 [ m + k ] + , 622 . 3 [ m + h ] + . elemental analysis for c 34 h 47 n 5 o 6 : calcd . c , 65 . 68 ; h , 7 . 62 ; n , 11 . 26 ; o , 15 . 44 . found c , 65 . 98 ; h , 7 . 91 ; n , 11 . 72 ; o , 15 . 83 . compound 13 ( 0 . 097 mmol ) was diluted in etoh ( 9 ml ) and pd / c , 10 % wt . ( 45 mg ) was added . h 2 was insufflated and the mixture was stirred for 5 h . the reaction mixture was filtered through celite and then solvent was evaporated in vacuum . crude title compound 14 was used in the next step without any further purification . yield 99 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ = 7 . 27 - 7 . 08 ( 2h , m ), 6 . 88 - 6 . 77 ( 2h , m ), 4 . 61 - 4 . 60 ( 2h , m ), 4 . 02 - 3 . 98 ( 2h , m ), 3 . 30 - 3 . 26 ( 4h , m ), 3 . 14 - 3 . 10 ( 2h , m ), 1 . 76 - 1 . 75 ( 2h , m ), 1 . 59 - 1 . 54 ( 6h , m ), 1 . 43 ( 9h , m ), 1 . 20 - 1 . 14 ( 8h , m ) ppm lcms m / z ( es +) m / z = 512 . 3 [ m + na ] + , 528 . 2 [ m + k ] + , 490 . 3 [ m + h ] + . to a stirred suspension of koh ( 2 . 8 mmol ) in a solution of ch 2 cl 2 / ch 3 cn ( 19 : 1 , 3 . 5 ml ), tetrabutylammonium bromide ( 0 . 2 mmol ) and tert - butyl n —[ n -[( 2 - methylpropan - 2 - yl ) oxycarbonyl ] carbamimidoyl ] carbamate ( 1 mmol , sigma - aldrich catalogue id : 496871 ) were added . after few minutes , a solution of ( e )- 1 - bromobut - 2 - ene ( 2 . 4 mmol , sigma - aldrich catalogue id : c86405 ) in ch 2 cl 2 / ch 3 cn ( 19 : 1 , 3 . 5 ml ) was added dropwise and the resulting solution was stirred at r . t . for 16 - 18 h . the reaction mixture was then poured on ice and the aqueous layer was extracted with ch 2 cl 2 ( 2 × 5 ml ). the combined organic phases were washed with brine , dried over na 2 so 4 , filtered and concentrated under reduced pressure . the crude products were purified by flash column chromatography ( sio 2 ) using 1 : 9 meoh / ch 2 cl 2 as the eluent to yield the title compound tert - butyl n - but - 2 - enyl - n —[ n ′-[( 2 - methylpropan - 2 - yl ) oxycarbonyl ]- n - methylsulfanylcarbamimidoyl ] carbamate . time 16 h , yield 70 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ 5 . 34 - 5 . 21 ( 2h , m ), 3 . 69 - 3 . 68 ( 2h , d , j = 8 hz ), 2 . 02 - 2 . 01 ( 3h , s ), 1 . 15 ( 9h , s ), 1 . 12 ( 9h , m ) ppm . 13 c nmr ( cdcl 3 ) δ 162 . 8 , 157 . 7 , 151 . 5 , 129 . 6 , 126 . 5 , 82 . 0 , 82 . 4 , 45 . 4 , 27 . 9 , 17 . 5 , 15 . 3 ppm . lcms m / z ( es +) m / z = 710 . 9 [ 2m + na ] + , 367 . 0 [ m + na ] + , 345 . 1 [ m + h ] + elemental analysis for c 16 h 28 n 2 o 4 s : calcd . c , 55 . 79 ; h , 8 . 19 ; n , 8 . 13 . found c , 55 . 83 ; h , 8 . 21 ; n , 8 . 34 . to a stirred solution of the compound 14 ( 0 . 097 mmol ) in dry thf ( 5 ml ) a solution of tert - butyl n - but - 2 - enyl - n —[ n ′-[( 2 - methylpropan - 2 - yl ) oxycarbonyl ]- n - methylsulfanyl carbamimidoyl ] carbamate ( 0 . 10 mmol ) in dry thf ( 4 ml ) was added dropwise . then et 3 n ( 0 . 14 mmol ) was added . the mixture was then stirred at reflux overnight . the solvent was evaporated under reduced pressure , and the residue was purified by flash chromatography ( sio 2 ) using 1 : 1 hexane - et 2 o as the eluent affording title compound 15 . yield 50 %. 1 h nmr ( 400 mhz , cdcl 3 ) δ 11 . 97 ( 1h , s ), 8 . 44 ( 1h , s ), 7 . 28 - 6 . 74 ( 4h , m ), 5 . 57 - 5 . 55 ( 1h , m ), 5 . 44 - 5 . 41 ( 1h , m ), 4 . 09 - 4 . 07 ( 2h , d , j = 8 hz ), 4 . 02 - 4 . 00 ( 2h , d , j = 6 . 4 hz ), 3 . 31 - 3 . 30 ( 2h , m ), 3 . 17 - 3 . 10 ( 6h , m ), 1 . 60 - 1 . 58 ( 3h , d , j = 6 . 4 hz ), 1 . 42 - 1 . 36 ( 27h , m ), 1 . 22 - 1 . 17 ( 16h , m ) ppm . 13 c nmr ( cdcl 3 ) δ 163 . 9 , 158 . 0 , 155 . 5 , 153 . 4 , 153 . 0 , 130 . 3 , 128 . 3 , 128 . 2 , 127 . 9 , 126 . 1 , 120 . 4 , 111 . 7 , 81 . 9 , 67 . 1 , 47 . 4 , 46 . 3 , 43 . 7 , 37 . 1 , 39 . 5 , 29 . 3 , 28 . 5 , 28 . 0 , 26 . 9 , 26 . 7 , 24 . 5 , 23 . 8 , 20 . 9 , 17 . 6 ppm . elemental analysis for c 41 h 67 n 7 o 8 : calcd . c , 62 . 65 ; h , 8 . 59 ; n , 12 . 47 . found c , 63 . 08 ; h , 8 . 98 ; n , 12 . 84 . guanylated compound 15 ( 0 . 025 mmol ) was dissolved in dry dcm ( 30 ml for 1 mmol ) and treated with a 10 % solution of freshly distilled tfa . the resulting solution was stirred at room temperature under argon . after 24 h the reaction mixtures were concentrated under reduced pressure affording the crude title compound 16 ( brown oil ) in quantitative yield . yield quantitative . 1 h nmr ( 400 mhz , meod ) δ 7 . 33 - 6 . 94 ( 4h , m ), 5 . 68 - 5 . 64 ( 1h , m ), 5 . 44 - 5 . 41 ( 1h , m ), 4 . 44 - 4 . 38 ( 2h , m ), 4 . 21 - 4 . 20 ( 2h , m ), 3 . 68 - 3 . 67 ( 2h , d , j = 4 . 8 hz ), 3 . 54 ( 2h , m ), 3 . 30 - 3 . 28 ( 2h , m ), 3 . 12 - 3 . 09 ( 2h , t , j = 6 . 8 hz ), 1 . 65 - 1 . 64 ( 3h , m ), 1 . 55 - 1 . 45 ( 4h , m ), 1 . 32 - 1 . 21 ( 12h , m ) ppm . 13 c nmr ( meod ) δ 163 . 5 , 157 . 6 , 156 . 2 , 155 . 8 , 131 . 0 , 129 . 9 , 128 . 6 , 127 . 9 , 127 . 1 , 124 . 8 , 121 . 3 , 111 . 4 , 69 . 0 , 47 . 2 , 43 . 0 , 41 . 0 , 31 . 5 , 29 . 2 , 28 . 7 , 28 . 4 , 27 . 2 , 26 . 1 , 24 . 9 , 22 . 0 , 16 . 2 ppm . elemental analysis for c 26 h 43 n 7 o 2 : calcd . c , 64 . 30 ; h , 8 . 92 ; n , 20 . 19 . found c , 64 . 68 ; h , 9 . 08 ; n , 20 . 84 . compound 17 was synthesized starting from the appropriate intermediate 1f , according to the procedure as for example 16 . 1 h nmr ( 400 mhz meod ) δ 7 . 23 - 6 . 88 ( 4h , m ), 5 . 62 - 5 . 64 ( 1h , m ), 5 . 40 - 5 . 35 ( 1h , m ), 4 . 43 - 4 . 42 ( 2h , m ), 4 . 06 ( 2h , m ), 3 . 65 ( 2h , m ), 3 . 45 - 3 . 41 ( 2h , m ), 3 . 23 ( 2h , m ), 3 . 08 - 3 . 00 ( 2h , m ), 1 . 63 - 1 . 62 ( 3h , m ), 1 . 49 - 1 . 48 ( 6h , m ), 1 . 28 - 1 . 17 ( 12h , m ) ppm . 13 c nmr ( 100 mhz meod ) δ 163 . 5 , 156 . 7 , 156 . 2 , 154 . 7 , 130 . 0 , 128 . 5 , 127 . 9 , 127 . 1 , 124 . 8 , 120 . 0 , 111 . 1 , 64 . 6 , 49 . 2 , 46 . 6 , 42 . 4 , 41 . 0 , 39 . 6 , 28 . 7 , 28 . 3 , 27 . 0 , 26 . 0 ppm . lc - ms m / z ( es +) m / z = 522 . 3 [ m + na ] + , 538 . 0 [ m + k ] + , 500 . 2 [ m + h ] + compound 18 was synthesized starting from the appropriate intermediate 1g , according to the procedure as for example 16 . 1 h nmr ( 400 mhz meod ) δ 12 . 31 ( 1h , s ), 8 . 05 ( 1h , s ), 7 . 26 - 7 . 22 ( 2h , m ), 6 . 92 - 6 . 84 ( 2h , m ), 5 . 66 - 5 . 65 ( 1h , m ), 5 . 49 - 5 . 42 ( 1h , m ), 4 . 37 ( 2h , m ), 3 . 97 ( 2h , m ), 3 . 68 ( 2h , m ), 3 . 51 ( 2h , m ), 3 . 30 ( 2h , m ), 3 . 15 - 3 . 11 ( 4h , m ), 1 . 63 - 1 . 62 ( 3h , m ), 1 . 50 - 1 . 48 ( 6h , m ), 1 . 28 - 1 . 23 ( 12h , m ) ppm . 13 c nmr ( 100 mhz meod ) δ 163 . 7 , 157 . 5 , 156 . 1 , 155 . 8 , 130 . 3 , 128 . 5 , 124 . 9 , 119 . 8 , 110 . 9 , 67 . 2 , 53 . 3 , 46 . 42 . 5 , 41 . 0 , 29 . 0 , 28 . 7 , 28 . 4 , 27 . 1 , 26 . 7 , 26 . 4 , 26 . 4 , 26 . 3 , 26 . 2 , 26 . 1 , 26 . 0 ppm . compound 19 was synthesized according to scheme 1 by reacting intermediate 1b with intermediate 3a , and applying a similar procedure as for example 16 . the synthesis of intermediate 1 b is described in scheme 3 . reagents and conditions : ( i ) ( bocnh ) 2 c = ntf , et 3 n , dcm , ( ii ) tmsci , et 3 n , dcm , reflux ; ( iii ) dmap , dcc , but - 3 - en - 1 - ol ( sigma - aldrich , catalogue id : 496839 ), dcm , rt 24 h ; 3 - aminopropanoic acid ( 600 mg 6 . 74 mmol , sigma - aldrich catalogue id : 146064 ) was dissolved in ch 2 cl 2 ( 10 ml ) and trimethylsilyl chloride ( 0 . 86 ml , 6 . 74 mmol ) was added dropwise at room temperature over 5 min . the reaction mixture was then refluxed for 1 h and then cooled to 0 ° c . triethylamine ( 1 . 22 ml , 8 . 76 mmol ) was then added followed by n , n ′- bis ( tert - butoxycarbonyl )- s - methylisothiourea ( 977 mg , 3 . 37 mmol ). the resulting mixture was refluxed for 1 h , then cooled down to room temperature and added with meoh ( 10 ml ) and stirred for 10 min . solvent was then evaporated and the residue diluted with water and then acidified with hcl 1n to ph 2 . the mixture was extracted with acoet ( 2 × 10 ml ). the organic layers were collected and washed with brine , dried over anhydrous na 2 so 4 and evaporated under reduce pressure . the residue was used in the next step without any further purification . to a solution of 3 -[[ n , n ′- bis [( 2 - methylpropan - 2 - yl ) oxycarbonyl ] carbamimidoyl ] amino ] propanoic acid ( 8 . 98 mmol ) in dry dcm ( 20 ml ), the but - 3 - en - 1 - ol ( 17 . 96 mmol , sigma - aldrich catalogue id : 496839 ) and dmap ( 0 . 89 mmol ) were added . the mixture was cooled down to 0 ° c . and dcc ( 13 . 47 mmol ) was then added . the mixture was allowed to warm to r . t . overnight while stirring under argon . the white precipitated formed during the reaction was filtered off . the solution was then washed with brine , dried over na 2 so 4 and concentrated in vacuum . the residue was purified by flash chromatography using hexane et 2 o 4 : 6 as eluent affording desired compound 1b , but - 3 - enyl 3 -[[ n , n - bis [( 2 - methylpropan - 2 - yl ) oxycarbonyl ] carbamimidoyl ] amino ] propanoate . 1 h nmr ( 400 mhz meod ) δ 12 . 31 ( 1h , s ), 8 . 05 ( 1h , s ), 5 . 64 ( 1h , m ), 5 . 42 ( 1h , m ), 4 . 13 ( 2h , m ), 3 . 78 ( 2h , m ), 3 . 64 ( 2h , m ), 3 . 43 ( 2h , m ), 3 . 08 ( 2h , m ), 2 . 51 ( 2h , m ), 1 . 63 ( 3h , m ), 1 . 55 ( 4h , m ), 1 . 49 ( 4h , m ), 1 . 27 ( 12h , m ) ppm . 13 c nmr ( 100 mhz meod ) δ 171 . 3 , 171 . 1 , 155 . 1 , 154 . 0 , 137 . 5 , 128 . 6 , 63 . 9 , 42 . 5 , 41 . 0 , 37 . 6 , 34 . 1 , 29 . 2 , 28 . 7 , 28 . 4 , 27 . 6 , 26 . 1 , 21 . 9 , 18 . 7 ppm . compound 20 was synthesized according to scheme 1 by reacting intermediate 1h with intermediate 3a , and applying a similar procedure as described for example 16 . reagents and conditions : ( i ) ( bocnh ) 2 c = ntf , et 3 n , dcm ( ii ) tmsci , et 3 n , dcm , reflux ; ( iii ) dmap , dcc , pent - 4 - en - 1 - ol ( sigma - aldrich , catalogue id : 111279 ), dcm , rt 24 h ; 3 - aminopropanoic acid ( 600 mg 6 . 74 mmol , sigma - aldrich catalogue id : 146064 ) was dissolved in ch 2 cl 2 ( 10 ml ) and trimethylsilyl chloride ( 0 . 86 ml , 6 . 74 mmol ) was added dropwise at room temperature over 5 min . the reaction mixture was then refluxed for 1 h and then cooled to 0 ° c . triethylamine ( 1 . 22 ml , 8 . 76 mmol ) was then added followed by n , n ′- bis ( tert - butoxycarbonyl )- s - methylisothiourea ( 977 mg , 3 . 37 mmol ). the resulting mixture was refluxed for 1 h , then cooled down to room temperature and added with meoh ( 10 ml ) and stirred for 10 min . solvent was then evaporated and the residue diluted with water and then acidified with hcl 1n to ph 2 . the mixture was extracted with acoet ( 2 × 10 ml ). the organic layers were collected and washed with brine , dried over anhydrous na 2 so 4 and evaporated under reduce pressure . the residue was used in the next step without any further purification . to a solution of 3 -[[ n , n ′- bis [( 2 - methylpropan - 2 - yl ) oxycarbonyl ] carbamimidoyl ] amino ] propanoic produced in the previous step ( 8 . 98 mmol ) in dry dcm ( 20 ml ), the pent - 4 - en - 1 - ol ( 17 . 96 mmol , sigma - aldrich catalogue id : 111279 ) and dmap ( 0 . 89 mmol ) were added . the mixture was cooled down to 0 ° c . and dcc ( 13 . 47 mmol ) was then added . the mixture was allowed to warm to r . t . overnight while stirring under argon . the white precipitated formed during the reaction was filtered off . the solution was then washed with brine , dried over na 2 so 4 and concentrated in vacuum . the residue was purified by flash chromatography using hexane - et 2 o 4 : 6 as eluent affording desired compound 1b , but - 3 - enyl 3 -[[ n , n ′- bis [( 2 - methylpropan - 2 - yl ) oxycarbonyl ] carbamimidoyl ] amino ] propanoate . 1 h nmr ( 300 mhz meod ) δ 12 . 31 ( 1h , s ), 8 . 05 ( 1h , s ), 5 . 65 ( 1h , m ), 5 . 43 ( 1h , m ), 4 . 06 ( 2h , m ), 3 . 68 ( 2h , m ), 3 . 58 ( 2h , m ), 3 . 44 ( 2h , m ), 3 . 30 ( 2h , m ), 3 . 11 - 3 . 07 ( 2h , m ), 2 . 62 - 2 . 59 ( 2h , m ), 1 . 63 ( 3h , d , j = 6 . 0 hz ), 1 . 51 ( 8h , m ), 1 . 27 ( 12h , m ) ppm . 13 c nmr ( 100 mhz meod ) δ 171 . 3 , 171 . 1 , 155 . 1 , 154 . 0 , 136 . 1 , 129 . 2 , 76 . 4 , 41 . 1 , 41 . 0 , 37 . 3 , 36 . 8 , 29 . 4 , 29 . 2 , 29 . 3 , 29 . 0 , 28 . 8 , 28 . 4 , 28 . 5 , 26 . 1 , 21 . 8 , 19 . 0 ppm . compound 21 was synthesized according to scheme 1 by reacting intermediate 1c with intermediate 3a and applying a similar procedure as described for example 16 . ( i ) br ( ch 2 ) 3 ch ═ ch 2 ( 1 . 2 eq ), k 2 co 3 ( 1 . 2 eq ), ch 3 cn , reflux , 12 h ( ii ) ethinylmagnesium bromide ( 1 . 5 eq ), thf , − 15 ° c ., 4 h ( iii ) n 3 ph ( 2 . 5 eq ), naascorbate ( 0 . 1 eq ), cuso 4 ( 0 . 01 eq ), h 2 o / t - buoh , mw , 125 ° c ., 30 ′ ( iv ) mno 2 ( 10 eq ), dcm , r . t . 18 h ( v ) nh 2 oh ( 2 . 5 eq ), py ( 1 . 2 ), etoh , reflux , 4 h ( vi ) zn ( 10 . 0 eq ), hcl ( 2n , 10 . 0 eq ), thf , reflux , 2 h ( vii ) ( bocnh 2 ) 2 c = tf ( 1 . 1 eq ), et 3 n ( 2 . 0 ), dcm , r . t . 18 h 1 h nmr ( 400 mhz , meod ) δ 8 . 56 ( 1h bs ), 7 . 87 - 7 . 85 ( 2h , d , j = 7 . 2 hz ), 7 . 59 - 7 . 55 ( 2h , t , j = 8 . 0 hz ), 7 . 50 - 7 . 47 ( 1h , t , j = 7 . 2 hz ), 7 . 39 - 7 . 35 ( 1h , t , j = 7 . 2 hz ), 7 . 30 - 7 . 29 ( 1h , d , j = 4 . 0 hz ), 7 . 07 - 7 . 05 ( 1h , t , j = 8 . 4 hz ), 6 . 90 ( 2h , bs ), 6 . 35 ( 1h , s ), 5 . 73 - 5 . 67 ( 1h , m ), 5 . 47 - 5 . 43 ( 1h , m ), 4 . 13 ( 2h , s ), 3 . 71 - 3 . 70 ( 2h , d , j = 5 . 6 hz ), 3 . 47 ( 2h , s ), 3 . 33 ( 2h , s ), 3 . 07 - 3 . 04 ( 2h , t , j = 6 . 8 ), 1 . 69 - 1 . 67 ( 3h , d , j = 6 . 0 hz ), 1 . 59 - 1 . 52 ( 8h , m ), 1 . 28 - 1 . 23 ( 12h , m ) 13 c nmr ( 400 mhz , meod ) δ 163 . 6 , 162 . 4 , 156 . 3 , 156 . 1 , 153 . 1 , 152 . 1 , 149 . 0 , 137 . 5 , 137 . 0 , 131 . 5 , 131 . 2 , 130 . 4 , 129 . 5 , 128 . 8 , 127 . 6 , 124 . 9 , 120 . 5 , 120 . 15 , 119 . 4 , 119 . 2 , 115 . 3 , 97 . 01 , 67 . 6 , 66 . 4 , 50 . 4 , 48 . 9 , 48 . 3 , 47 . 5 , 45 . 5 , 42 . 5 , 41 . 0 , 30 . 7 , 30 . 1 , 29 . 2 , 28 . 8 , 28 . 4 , 26 . 1 , 17 . 5 lcms m / z ( es +) m / z = 329 . 1 [ m + 2h ] 2 + , 657 . 3 [ m + h ] + , 695 . 3 [ m + na ] + compound 22 was synthesized according to according to scheme 1 by reacting intermediate 1d with intermediate 3a and applying a similar procedure as for example 16 . ( i ) pph 3 ( 1 . 1 eq ), thf , reflux , 12 h ( ii ) ch 2 o ( 1 . 8 eq ), koh ( 2 . 0 eq ), dcm , r . t ., 1 h ( iii ) salicylaldehyde ( 1 . 0 eq ), k 2 co 3 ( 1 . 5 eq ), ch 3 cn , reflux , 12 h ( iv ) nh 2 oh ( 2 . 5 eq ), py ( 1 . 2 eq ), etoh , reflux , 4 h ( v ) zn ( 10 . 0 eq ), hcl ( 2n , 10 eq ), thf , reflux , 2 h ( iv ) ( bocnh 2 ) 2 c = tf ( 1 . 1 eq ), et 3 n ( 2 . 0 eq ), dcm , r . t . 18 h . 1 h nmr ( 400 mhz , meod ) δ 7 . 46 - 7 . 44 ( 1h , d , j = 6 . 0 hz ), 7 . 35 ( 2h , m ), 7 . 23 ( 3h , m ) 6 . 98 ( 1h , m ), 6 . 87 - 6 . 88 ( 1h , t , j = 4 . 5 ), 6 . 84 ( 1h , m ), 5 . 75 - 5 . 69 ( 1h , m ), 5 . 51 - 5 . 48 ( 1h , m ), 5 . 07 ( 2h , s ), 4 . 46 - 4 . 40 ( 2h , d , j = 24 hz ), 3 . 74 - 3 . 72 ( 2h , d , j = 5 . 6 hz ), 3 . 40 - 3 . 30 ( 4h , m ), 3 . 17 - 3 . 13 ( 2h , t , j = 8 . 8 ), 2 . 63 - 2 . 59 ( 2h , t , j = 7 . 4 ), 1 . 72 - 1 . 69 ( 3h , d , j = 6 . 0 hz ), 1 . 57 - 1 . 54 ( 4h , m ), 1 . 32 - 1 . 28 ( 10h , m ) 13 c nmr ( 400 mhz , meod ) δ 163 . 7 , 156 . 4 , 155 . 9 , 153 . 2 , 136 . 8 , 136 . 6 , 135 . 3 , 134 . 7 , 133 . 7 , 129 . 7 , 129 . 6 , 129 . 4 , 128 . 6 , 128 . 1 , 125 . 7 , 125 . 6 , 124 . 9 , 121 . 1 , 119 . 5 , 114 . 8 , 111 . 9 , 76 . 2 , 48 . 4 , 47 . 5 , 42 . 5 , 41 . 0 , 40 . 4 , 37 . 4 , 29 . 2 , 28 . 9 , 28 . 8 , 28 . 7 , 28 . 5 , 28 . 4 , 28 . 1 , 26 . 1 , 16 . 3 lcms m / z ( es +) m / z = 281 . 7 [ m + 2h ] 2 + , 562 . 3 [ m + h ] + . compound 23 was synthesized according to according to scheme 1 by reacting intermediate 1e with intermediate 3a and applying a similar procedure as for example 16 . ( i ) pph 3 ( 1 . 1 eq ), thf , reflux , 12 h ( ii ) ch 2 o ( 1 . 8 eq ), koh ( 2 . 0 eq ), dcm , r . t ., 1 h ( iii ) salicylaldehyde ( 1 . 0 eq ), k 2 co 3 ( 1 . 5 eq ), ch 3 cn , reflux , 12 h ( iv ) nh 2 oh ( 2 . 5 eq ), py ( 1 . 2 eq ), etoh , reflux , 4 h ( v ) zn ( 10 . 0 eq ), hcl ( 2n , 10 eq ), thf , reflux , 2 h ( iv ) ( bocnh 2 ) 2 c = tf ( 1 . 1 eq ), et 3 n ( 2 . 0 eq ), dcm , r . t . 18 h . 1 h nmr ( 400 mhz , meod ) δ 7 . 31 - 7 . 19 ( 7h , m ), 7 . 07 ( 1h , s ), 6 . 97 - 6 . 93 ( 1h , t , j = 7 . 2 ), 5 . 75 - 5 . 70 ( 1h , m ), 5 . 59 - 5 . 47 ( 1h , m ), 5 . 13 ( 2h , s ), 4 . 53 ( 2h , s ), 3 . 74 - 3 . 72 ( 2h , d , j = 4 . 8 hz ), 3 . 35 ( 2h , s ), 3 . 17 - 3 . 13 ( 4h , t , j = 7 . 2 ), 2 . 66 - 2 . 65 ( 2h , d , j = 5 . 2 ), 2 . 09 ( 2h , s ), 1 . 71 - 1 . 69 ( 3h , d , j = 6 . 4 hz ), 1 . 56 - 1 . 48 ( 4h , m ), 1 . 27 - 1 . 19 ( 10h , m ) 13 c nmr ( 400 mhz , meod ) δ 161 . 7 , 157 . 2 , 155 . 9 , 154 . 1 , 153 . 2 , 147 . 8 , 141 . 1 , 136 . 6 , 130 . 1 , 130 . 0 , 128 . 8 , 128 . 2 , 127 . 9 , 127 . 4 , 125 . 9 , 125 . 4 , 124 . 8 , 122 . 7 , 120 . 2 , 111 . 4 , 70 . 2 , 69 . 2 , 44 . 8 , 44 . 4 , 42 . 6 , 42 . 5 , 41 . 0 , 37 . 4 , 31 . 5 , 29 . 2 , 28 . 8 , 28 . 7 , 28 . 5 , 27 . 5 , 27 . 1 , 26 . 1 , 25 . 8 , 16 . 3 sterile plastic microtitration plates containing flat - bottomed wells were used . the plates contained serial dilution of the antifungal agents with a volume of assay medium of 100 μl / well . two drug - free medium wells were used as sterility and growth controls . the trays were inoculated with 100 μl / well of the final inoculum , with the exception of sterility control wells . the range of concentrations tested for each drug was 1 . 25 - 80 μm . the microtitration plates were incubated at 37 ° c . for 24 h . the minimal inhibitory concentrations ( mics ) were determined at 24 h both visually and spectrophotometrically measuring the turbidity at 595 nm with a varian model 1475 spectrophotometer . results of biological tests are shown in table 2 , biological data are reported as mic 90 that is the minimum concentration required to inhibit the growth of at least the 90 % of the strains tested for each species . compounds 16 , 17 and 18 possessing an aromatic ring fused to the macrocyclic core proved to be more active when compared to compounds 19 and 20 bearing an ester moiety . it is noteworthy that compounds 18 and 20 , with a macrocyclic core of 15 atoms showed one of the highest activity toward c . albicans , c . guillermondii and c . parapsilosis when compared with homologue compounds with a lover number of atoms in the macrocyclic core . in particular compounds 18 and 20 are more active than fluconazole on c . guillermondii and c . krusei . compounds 16 - 19 were thus assayed against mutant c . albicans and c . glabrata strains bearing mutation on erg11 , cdr1 , cdr2 , snq2 and mdr1 genes , these mutants were obtained from clinical isolates and have been reported in the scientific literature . the erg11 gene , which encodes the cytochrome p450 , confers resistance to drugs belonging to the azole class , such as voriconazole and fluconazole . cdr1 and cdr2 genes encodes for atp binding cassette ( abc ) transporters , their overexpression confers resistance to the azole class . mdr1 gene , which encodes a membrane transport protein of the major facilitator superfamily , confers resistance to a broad range of drugs and toxic compounds . the snq2 gene encodes for yet another abc transporter that confers resistance to azole and non - azole drugs . voriconazole , an azole active on fluconazole resistant strains , was chosen as reference compound . data are reported in table 3 as mic value ( minimum inhibitory concentration ( mic ) is the lowest concentration of an agent that will inhibit the visible growth of a microorganism after overnight incubation ) obtained as the mean of three measurements for each strain . all compounds 16 - 19 proved to be active against c . albicans and c . glabrata fluconazole resistant strains . compound 17 resulted more active than voriconazole itself against mutant strains dsy289 , dsy296 and dsy775 . compound 18 was more active than fluconazole but in general less active than compounds 16 and 17 and voriconazole , except for dsy289 strain . chitinase inhibition assays were conducted against an example of the gh18 family of chitinases , i . e the chitinase enzyme extracted from trichoderma viride . this enzyme has an high degree of identity compared to chitinase 4 of candida albicans , and is commercially available from sigma - aldrich ( catalogue id : c8241 ). the chitinolytic enzymes from t . viride are a mixture of extracellular chitinolytic enzymes , which exhibit exo - and endochitinase activities . the major activity was found to be that of n - acetyl - β - glucosaminidase . the substrate used was the np - glcnac ( sigma - aldrich , catalogue id : n9376 ) dissolved in mes 50 mm at ph6 . 0 ; the final concentration was 250 μm . the enzyme was resuspended at 0 . 11 mg / ml in mes 50 mm at ph 6 . 0 , with a final concentration for the assay of 73 nm diluting with mes 50 mm and bsa 20 μg / ml at ph 6 . 0 . a stock solution of the inhibitors was prepared at 50 mm in dmso 100 %. the mixture with the inhibitor at various concentrations and the enzyme was prepared directly into the reading plate and left to incubate at room temperature for 20 minutes or three hours . at the end of the incubation times , the substrate was added and the reading was conducted by recording the values up to a maximum time of 10 minutes . the substrate concentration was measured by reading uv absorption at 300 nm ( ε m 300 : 11100 ), while , the product of hydrolysis were measured at 400 nm ( δε m 400 : 2120 ). both ic 50 and k i can be calculated . the results for compound 17 , one of the most active compound against candida strains , are reported in table 4 . for comparison , the inventors have also tested compounds disclosed in wo2009 / 113033 ( reference compounds 34 - 39 ). compound 17 ( with the aromatic ring fused in the macrocyclic core ) is an active inhibitor of the chitinase enzyme . this compound is more active than compounds 34 - 39 . it is worth to note that the ic 50 values decrease with time , suggesting a slow binding of the inhibitor . 1 . groll , a . h . ; 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