it is present , in fact , in compounds with different activities such as inhibitors of p38 map kinase , inhibitors of hiv - ep1 cellular transcription factor , inhibitors of coagulation factor xa , and -chemokine receptor ccr5 antagonists in anti - hiv therapy ; in particular we have focused our work on the development of new cyp51 inhibitors , active both on fungal strains   and trypanosoma cruzi .
many literature data evidenced that the pyridine group can efficaciously replace the heme - iron chelating azole moiety present in classical azole cyp51 inhibitors and , therefore , the alkylation of 4-aminopyridine ( 4ap ) represents an important goal in organic synthesis to develop novel classes of antifungal and antiparasitic drugs [ 7 , 8 ] . due to the wide presence of these products ,
the alkylation of 4-aminopyridine ( 4ap ) is therefore an important goal in organic synthesis .
some examples are the efficient condensation of 4ap with alcohols catalyzed by benzaldehyde   or copper [ 10 , 11 ] or magnetite , the reaction of 4ap with an acyl chloride , and the following reduction of the amide with lialh4 . the most straightforward method , however , is the direct alkylation of 4ap with alkyl halides , although it suffers from some drawbacks .
the two different nitrogen atoms compete in the alkylation reaction and usually the more nucleophilic pyridine nitrogen atom reacts faster , leading to the corresponding pyridinium salt ( scheme 1 ) [ 14 , 15 ] . in these case , the use of a very strong base is therefore necessary : n - buli was successfully used by singh and coworkers , obtaining n - methyl- and n - ethyl-4-aminopyridines in 7480% yields .
a viable alternative is the enhancement of the nucleophilicity of the amine nitrogen atom ( versus the pyridine one ) , allowing the use of weaker bases .
an example is the activation of 2-aminopyridine as formyl or boc derivative at the amine nitrogen atom , with subsequent deprotonation using sodium hydride , alkylation , and deprotection with trifluoroacetic acid .
the deprotonation of n - boc-2-aminopyridine with nah needs a careful control of the temperature ( 05c ) and is carried out in anhydrous dmf , with a vigorous stirring required to keep the suspension fluid . in this context , we envisaged the possibility to alkylate n - boc-4-aminopyridine ( n - boc-4ap ) using milder reaction conditions , that is , using electrogenerated tetraethylammonium cyanomethanide ( et4nch2cn ) .
this base , the acetonitrile anion , can be easily obtained by cathodic galvanostatic reduction of a solution of acetonitrile containing tetraethylammonium hexafluorophosphate as supporting electrolyte ( scheme 2 ) , without by - products ( the reagent is the electron ) , and it was successfully used by us in a good variety of reactions , such as the selective n - alkylation of bifunctional compounds , the gewald reaction , the synthesis of -lactams , and the synthesis of carbamates . the actual mechanism for the formation of acetonitrile anion is not known , but a hypothesis based on the direct reduction of the tetraethylammonium cation has been reported ( scheme 2 ) .
the high reactivity of this base is ascribable to the large tetraethylammonium counterion , which renders the acetonitrile anion extremely reactive .
moreover , its reaction as a base gives no by - products , as the protonation restores the molecule of solvent .
the reaction of electrogenerated acetonitrile anion with 4ap , followed by an alkyl halide , leads to poor yields in desired compound with the pyridinium salt being the major product .
this prevents the direct use of ch2cn with 4ap . on the other hand , if the amine nitrogen is activated as boc derivative ( n - boc-4ap ) , the deprotonation / alkylation reaction using acetonitrile anion leads to products1 in very high yields ( scheme 3 and table 1 , entries 16 ) .
the classic deprotection with trifluoroacetic acid allows obtaining the desired products2 ( scheme 3 and table 1 , entries 16 ) .
the data in table 1 highlight that the reaction of deprotonation of n - boc-4ap using electrogenerated acetonitrile anion , alkylation with both alkyl and benzyl halides , and deprotection with trifluoroacetic acid is very efficient , with overall yields of 7886% .
however , when the alkylating agent is a bromoacetophenone , the yields in alkylated product are lower and in most cases the deprotection reaction leads to the dealkylation of the starting material ( table 1 , entries 710 ) .
as many biologically active compounds contain the dialkylated 4-aminopyridine moiety , we tried to carry out a second alkylation on products 2a  j using acetonitrile anion but , as expected , the high nucleophilicity of the pyridine nitrogen led to the synthesis of the corresponding pyridinium salt .
we thus carried out this second alkylation using strong bases , the most efficient being t - buok in dmso ( scheme 4 ) , although the yields in dialkylated 4ap were not very high .
symmetrically dialkylated 4ap , 4ap was subjected to deprotonation with t - buok in dmso , adding an excess of alkylating agent .
the reaction led to a mixture of mono- and dialkylated 4-aminopyridines , in moderate to acceptable yields .
a selection of synthesized compounds was in vitro tested to evaluate antifungal activity against different strains of c. albicans , c. parapsilosis , and cryptococcus neoformans ; data are reported in table 4 . as can be evidenced the nonsymmetrical dialkylated 4aps 3ac and 3ae showed a moderate antifungal activity towards c. albicans and c. parapsilosis with mic50 values of 32  g / ml and showed an interesting activity against cryptococcus neoformans , with mic50 values of 0.4 and 4  g / ml , respectively .
otherwise , the symmetrical dialkylated 4aps 3cc , 3ee and the boc - protected monoalkylated 4aps 1b , 1e , 1f showed poor antifungal activity with mic50 and mic100  64  g / ml . furthermore , the symmetrical dialkylated 4aps 3cc , 3ee , and 3ff were in vitro tested to evaluate the activity against trypanosoma cruzi , trypanosoma brucei , leishmania infantum , and plasmodium falciparum ; the results are summarized in table 5 .
as can be evidenced , all tested compounds showed a moderate activity versus p. falciparum and an interesting activity towards l. infantum with ic50 values lower than the reference drug miltefosine ; otherwise , they resulted scarcely active against t. cruzi and t. brucei .
in conclusion , we demonstrated the usefulness of electrogenerated acetonitrile anion in the alkylation of n - boc 4-aminopyridines , both from the point of view of the high yields and of the cleanliness of the reaction ( no by - products ) .
the deprotection of n - boc 4-aminopyridines allowed obtaining monoalkylated 4-aminopyridine in very high yields .
the following alkylation , by means of t - buok and alkyl halides , led to nonsymmetrically dialkylated 4-aminopyridine , while symmetrically dialkylated products were obtained directly from 4-aminopyridine by reaction with an excess of t - buok and alkyl halide .
furthermore , it can also be concluded that the monoalkylation of the 4ap leads to inactive products and otherwise interesting activity against fungi and some protozoa can be obtained by dual , symmetrical , or nonsymmetrical dialkylation of the amino group of 4ap ; these active molecules can be considered as lead compound to develop new antifungal and antiprotozoal compounds .
to a solution of di - tert - butyl dicarbonate ( 3  mmol ) in acetonitrile ( 3  cm ) at room temperature 4-aminopyridine ( 3  mmol ) was slowly added .
the solvent was evaporated and the crude 4-[n-(tert - butoxycarbonyl)amino]pyridine ( > 95% ) was used in the electrolyses without further purification .
rf ( 30% ethyl acetate in light petroleum ether ) 0.20 ; h nmr ( 200  mhz , cdcl3 )  1.53 ( s,9h ) , 6.9 ( bs , 1h ) , 7.32 ( dd ,  j = 4.8 , 1.6  hz , 2h ) , 8.45 ( dd ,  j = 4.8 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.2 , 81.7 , 112.3 , 145.6 , 150.3 , 151.9 ; eims , m / z : 194 ( m , 1% ) , 137 ( 2% ) , 121 ( 5% ) , 120 ( 8% ) , 94 ( 50% ) , 78 ( 4% ) , 57 ( 100% ) . constant current electrolyses ( i = 25  ma  cm ) were performed under a nitrogen atmosphere , at 20c , using an amel model 552 potentiostat equipped with an amel model 731 integrator .
all the experiments were carried out in a divided glass cell separated through a porous glass plug filled up with a layer of gel ( i.e. , methyl cellulose 0.5% volume dissolved in dmf - et4npf6 1.0  mol  dm ) ; pt spirals ( apparent areas 0.8  cm ) were used both as cathode and anode .
mecn - et4npf6 0.1  mol  dm was used as solvent - supporting electrolyte system ( catholyte : 20  cm ; anolyte : 5  cm )
after 145  c were passed , the current was switched off and 1  mmol of alkylating agent was added to the catholyte .
the solution was kept under stirring at room temperature for 2 hours ; then the solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography , using a mixture of ethyl acetate / light petroleum ether 2/8 in volume , obtaining the pure products .
flash column chromatography was carried out using merck 60 kieselgel ( 230400 mesh ) under pressure .
gc - ms measurements were carried out on se 54 capillary column using a fisons 8000 gas chromatograph coupled with a fisons md 800 quadrupole mass selective detector .
h and c nmr spectra were recorded at room temperature using a bruker ac 200 spectrometer using cdcl3 as internal standard . 

rf ( 80% ethyl acetate in dichloromethane ) 0.60 ; h nmr ( 200  mhz , cdcl3 )  0.88 ( t ,  j = 6.5  hz , 3h ) , 1.201.30 ( m , 10h ) , 1.491.86 ( m , 3h ) , 1.50 ( s , 9h ) , 3.69 ( app  t , j = 7.6  hz , 2h ) , 7.24 ( dd ,  j = 6.2 , 1.6  hz , 2h ) , 8.51 ( dd ,  j = 6.2 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  14.0 , 22.6 , 26.7 , 28.2 , 28.4 , 29.1 , 31.7 , 48.7 , 81.4 , 118.8 , 150.0 , 150.1 , 153.4 . 

rf ( 50% ethyl acetate in light petroleum ether ) 0.46 ; h nmr ( 200  mhz , cdcl3 )  1.48 ( s , 9h ) , 1.862.02 ( m , 2h ) , 2.64 ( t , j = 7.6  hz , 2h ) , 3.74 ( app  t ,  j = 7.6  hz , 2h ) , 7.127.33 ( m , 7h ) , 8.49 ( dd , j = 4.8 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.2 , 30.0 , 33.0 , 48.3 , 81.6 , 118.9 , 126.1 , 128.3 , 128.5 , 141.0 , 149.7 , 150.3 , 153.4 ; eims , m / z : m absent , 212 ( 5% ) , 107 ( 100% ) , 105 ( 5% ) , 91 ( 25% ) , 78 ( 27% ) , 77 ( 12% ) . 

rf ( 60% ethyl acetate in light petroleum ether ) 0.58 ; h nmr ( 200  mhz , cdcl3 )  1.45 ( s , 9h ) , 4.94 ( s , 2h ) , 7.207.37 ( m , 7h ) , 8.46 ( dd ,  j = 4.8 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.1 , 52.5 , 82.1 , 118.2 , 126.3 , 127.3 , 128.7 , 137.7 , 150.2 , 150.1 , 153.5 ; eims , m / z : m absent , 227 ( 4% ) , 183 ( 14% ) , 91 ( 100% ) , 78 ( 7% ) , 57 ( 51% ) . 
 tert - butyl ( 2,6-dichlorobenzyl)pyridin-4-ylcarbamate 
  1d .
rf ( 50% ethyl acetate in light petroleum ether ) 0.60 ; h nmr ( 200  mhz , cdcl3 )  1.48 ( s , 9h ) , 5.29 ( s , 2h ) , 7.027.21 ( m , 5h ) , 8.41 ( dd ,  j = 4.8 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.2 , 46.7 , 81.7 , 121.5 , 128.6 , 129.5 , 131.7 , 136.1 , 148.1 , 149.9 , 153.3 ; eims , m / z : 352 ( m , 1% ) , 252 ( 3% ) , 163 ( 6% ) , 161 ( 30% ) , 159 ( 42% ) , 78 ( 76% ) , 51 ( 100% ) . 
 tert - butyl ( 4-fluorobenzyl)pyridin-4-ylcarbamate 
  1e .
rf ( 50% ethyl acetate in light petroleum ether ) 0.49 ; h nmr ( 200  mhz , cdcl3 )  1.45 ( s , 9h ) , 4.89 ( s , 2h ) , 6.977.22 ( m , 6h ) , 8.47 ( app d , j = 6.0  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.1 , 51.8 , 82.2 , 116.6 ( d ,  j = 21.5  hz ) , 118.4 , 128.1 ( d , j = 8.0  hz ) , 133.4 ( d ,  j = 3.2  hz ) , 149.9 , 150.3 , 153.4 , 162.2 ( d , j = 245.4  hz ) ; eims , m / z : 302 ( m , 1% ) , 245 ( 4% ) , 201 ( 53% ) , 108 ( 100% ) , 78 ( 42% ) , 57 ( 100% ) . 
 tert - butyl ( 4-trifluoromethylbenzyl)pyridin-4-ylcarbamate 
  1f .
rf ( 50% ethyl acetate in light petroleum ether ) 0.41 ; h nmr ( 200  mhz , cdcl3 )  1.46 ( s , 9h ) , 4.99 ( s , 2h ) , 7.207.36 ( m , 4h ) , 7.61 ( app d ,  j = 8.4  hz , 2h ) , 8.49 ( app d , j = 6.2  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.1 , 52.2 , 82.5 , 118.1 , 124.0 ( q ,  j = 271.9  hz ) , 125.7 ( q ,  j = 3.7  hz ) , 126.6 , 129.7 ( q ,  j = 32.3  hz ) , 141.9 , 149.8 , 150.4 , 153.3 ; eims , m / z : m absent , 251 ( 9% ) , 158 ( 34% ) , 145 ( 2% ) , 78 ( 25% ) , 69 ( 9% ) , 57 ( 100% ) . 

tert - butyl ( 2-oxo-2-phenylethyl)pyridin-4-ylcarbamate 
  1  g. rf ( 60% ethyl acetate in light petroleum ether ) 0.50 ; h nmr ( 200  mhz , cdcl3 )  1.46 ( s ,  9h ) , 5.09 ( s , 2h ) , 7.26 ( d ,  j = 6.4  hz , 2h ) , 7.487.68 ( m , 3h ) , 7.99 ( d ,  j = 8.2  hz , 2h ) , 8.478.55 ( m , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.1 , 55.5 , 82.5 , 118.7 , 127.9 , 128.9 , 133.9 , 134.7 , 150.2 , 153.2 , 193.7 . 

tert - butyl ( 2-(4-fluoropheny)-2oxoethyl)pyridin-4-ylcarbamate 
  1  h. rf ( 50% ethyl acetate in light petroleum ether ) 0.50 ; h nmr ( 200  mhz , cdcl3 )  1.46 ( s , 9h ) , 5.05 ( s , 2h ) , 7.157.27 ( m , 4h ) , 7.998.06 ( m , 2h ) , 8.52 ( dd , j = 5.0 , 1.4  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.1 , 55.4 , 82.6 , 116.2 ( d ,  j = 22.0  hz ) , 118.8 , 130.6 ( d ,  j = 9.4  hz ) , 131.1 ( d ,  j = 3.2  hz ) , 150.1 , 150.2 , 153.2 , 166.2 ( d ,  j = 256.1  hz ) , 192.2 . 
 tert - butyl ( 2-(4-chloropheny)-2oxoethyl)pyridin-4-ylcarbamate 
  1i .
rf ( 20% ethyl acetate in dichloromethane ) 0.30 ; h nmr ( 200  mhz , cdcl3 )  1.45 ( s , 9h ) , 5.04 ( s , 2h ) , 7.21 ( dd ,  j = 4.6 , 1.6  hz , 2h ) , 7.50 ( d ,  j = 8.8  hz , 2h ) , 7.93 ( d ,  j = 8.0  hz , 2h ) , 8.51 ( dd ,  j = 4.6 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.1 , 55.4 , 82.6 , 118.8 , 129.3 , 133.0 , 140.5 , 149.9 , 150.3 , 153.1 , 192.7 . 
 tert - butyl ( 2-(4-methoxypheny)-2oxoethyl)pyridin-4-ylcarbamate 
  1j .
rf ( 40% ethyl acetate in dichloromethane ) 0.50 ; h nmr ( 200  mhz , cdcl3 )  1.46 ( s , 9h ) , 3.90 ( s , 3h ) , 5.04 ( s , 2h ) , 6.99 ( d ,  j = 9.0  hz , 2h ) , 7.24 ( dd ,  j = 4.8 , 1.6  hz , 2h ) , 7.97 ( d , 
j = 9.0  hz , 2h ) , 8.50 ( dd ,  j = 4.8 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.1 , 55.1 , 55.5 , 82.3 , 114.1 , 118.7 , 127.8 , 130.2 , 150.2 , 153.3 , 164.1 , 192.0 . to a solution of 1 ( 1  mmol ) in ch2cl2 ( 5  cm ) , kept at 0c , 1  cm of cf3cooh was added .
the solution was then mixed with aqueous sodium carbonate till ph 8 and extracted with ethyl acetate .
the solvent was removed under reduced pressure and the mixture was purified by flash chromatography , yielding pure compound 2 . 

rf ( 20% dichloromethane in ethyl acetate ) 0.16 ; h nmr ( 200  mhz , cdcl3 )  0.89 ( t ,  j = 7.2  hz , 3h ) , 1.251.34 ( 6h ) , 1.611.70 ( m , 2h ) , 2.893.12 ( m , 2h ) , 3.153.24 ( m , 2h ) , 5.435.48 ( m , 2h ) , 6.57 ( d ,  j = 5.2  hz , 2h ) , 8.11 ( d ,  j = 5.2  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  14.1 , 22.6 , 27.0 , 28.8 , 29.2 , 29.2 , 31.8 , 42.9 , 107.4 , 155.2 . 

rf ( ethyl acetate ) 0.46 ; h nmr ( 200  mhz , cd3cn )  1.912.06 ( m , 2h ) , 2.71 ( t ,  j = 7.4  hz , 2h ) , 3.173.27 ( m , 2h ) , 4.9 ( bs , 1h ) , 6.626.66 ( m , 2h ) , 7.147.46 ( m , 5h ) , 7.457.97 ( m , 2h ) ; c nmr ( 50  mhz , cd3cn )  29.8 , 32.5 , 42.0 , 107.3 , 125.9 , 128.3 , 128.4 , 141.0 , 141.5 , 157.8 ; eims , m / z : 212 ( m , 1% ) , 107 ( 100% ) , 91 ( 43% ) , 78 ( 13% ) . 

rf ( ethyl acetate ) 0.57 ; h nmr ( 200  mhz , cdcl3 )  4.46 ( d ,  j = 6.0  hz , 2h ) , 6.7 ( bs , 2h ) , 7.1 ( bs , 1h ) , 7.217.41 ( m , 5h ) , 8.0 ( bs , 2h ) ; c nmr ( 50  mhz , cdcl3 )  46.8 , 107.4 , 127.2 , 127.6 , 128.8 , 137.7 , 148.6 , 154.0 ; eims , m / z : 184 ( m , 15% ) , 183 ( 15% ) , 107 ( 5% ) , 91 ( 100% ) , 78 ( 16% ) . 

rf ( ethyl acetate ) 0.38 ; h nmr ( 200  mhz , cd3cn ) 4.59 ( s , 2h ) , 6.0 ( bs , 1h ) , 6.68 ( dd ,  j = 5.2 , 1.4  hz , 2h ) , 7.277.47 ( m , 3h ) , 8.41 ( app d ,  j = 5.4   hz , 2h ) ; c nmr ( 50  mhz , cd3cn )  42.2 , 107.4 , 128.7 , 130.4 , 132.8 , 136.0 , 147.3 , 154.6 ; eims , m / z : 256 ( m + 4 , 1% ) , 254 ( m + 2 , 7% ) , 252 ( m , 14% ) , 162 ( 10% ) , 160 ( 67% ) , 158 ( 100% ) , 78 ( 37% ) . 

rf ( ethyl acetate ) 0.27 ; h nmr ( 200  mhz , cdcl3 )  4.38 ( d , j = 5.4  hz , 2h ) , 5.1 ( bs , 1h ) , 6.5 ( bs , 2h ) , 7.027.10 ( m , 2h ) , 7.307.34 ( m , 2h ) , 8.2 ( bs , 2h ) ; c nmr ( 50  mhz , cdcl3 )  46.3 , 107.8 , 115.8 ( d ,  j = 21.5  hz ) , 129.0 ( d ,  j = 8.1  hz ) , 133.1 , 148.5 , 153.8 , 166.8 ( d ,  j = 205.9  hz ) , ; eims , m / z : 202 ( m , 5% ) , 107 ( 16% ) , 109 ( 100% ) , 78 ( 16% ) . 

rf ( ethyl acetate ) 0.25 ; h nmr ( 200  mhz , cdcl3 )  4.51 ( d ,  j = 6.0  hz , 2h ) , 6.2 ( bs , 1h ) , 6.6 ( bs , 2h ) , 7.54 ( d ,  j = 8.0  hz , 2h ) , 7.67 ( d ,  j = 8.0  hz , 2h ) , 8.1 ( bs , 2h ) ; c nmr ( 50  mhz , cdcl3 )  45.4 , 107.8 , 124.4 ( q ,  j = 271.0  hz ) , 125.4 ( q ,  j = 3.9  hz ) , 128.4 ( q , j = 31.9  hz ) , 127.7 , 143.2 , 146.7 , 155.0 ; eims , m / z : 252 ( m , 80% ) , 183 ( 11% ) , 159 ( 100% ) , 107 ( 52% ) , 78 ( 31% ) . 
 1-(4-methoxyphenyl)-2-(pyridin-4-ylamine)ethan-1-one 
  2  g. rf ( 50% ethyl acetate in ethanol ) 0.15 ; h nmr ( 200  mhz , cdcl3 )  3.90 ( s , 3h ) , 4.57 ( d ,  j = 3.8  hz , 2h ) , 4.6 ( bs , 1h ) , 6.58 ( d , j = 6.2  hz , 2h ) , 6.98 ( d ,  j = 9.0  hz , 2h ) , 7.98 ( d ,  j = 9.0  hz , 2h ) , 8.19 ( d ,  j = 6.2  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  48.2 , 55.6 , 108.1 , 114.1 , 127.2 , 130.2 , 150.8 , 153.6 , 164.5 , 191.6 . to a solution of 2 ( 1  mmol ) in anhydrous dmso ( 2  cm ) , kept at rt under n2 , 1.5  mmol of t - buok was added .
this mixture was allowed to stir for 20  min at rt ; then 1  mmol of alkyl halide was added and the solution was kept under stirring at rt for 4  h. the solution was then mixed with water and extracted with dichloromethane .
the solvent was removed under reduced pressure and the mixture was purified by flash chromatography , yielding pure compound 3 . 

rf ( 80% ethyl acetate in ethanol ) 0.38 ; h nmr ( 200  mhz , cdcl3 )  0.860.92 ( m , 3h ) , 1.201.40 ( m , 10h ) , 1.631.70 ( m , 2h ) , 3.42 ( app t ,  j = 7.6  hz , 2h ) , 4.59 ( s , 2h ) , 6.51 ( d ,  j = 5.2  hz , 2h ) , 7.147.38 ( m , 5h ) , 8.18 ( d ,  j = 5.2  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  14.1 , 22.6 , 26.9 , 27.0 , 29.2 , 29.4 , 29.7 , 31.8 , 50.7 , 53.4 , 106.9 , 126.2 , 127.3 , 128.8 , 136.8 , 148.9 , 153.6 . 

rf ( ethyl acetate ) 0.40 ; h nmr ( 200  mhz , cdcl3 )  0.860.92 ( m , 3h ) , 1.231.35 ( m , 10h ) , 1.621.72 ( m , 2h ) , 3.42 ( app t ,  j = 7.8  hz , 2h ) , 4.58 ( s , 2h ) , 6.56 ( app d ,  j = 6.4  hz , 2h ) , 6.997.16 ( m , 4h ) , 8.17 ( app d ,  j = 6.4  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  14.1 , 19.2 , 22.6 , 26.9 , 29.2 , 29.3 , 31.7 , 51.3 , 53.3 , 107.4 , 116.1 ( d , j = 21.7  hz ) , 127.9 ( d ,  j = 8.1  hz ) , 130.9 ( d ,  j = 3.5  hz ) , 144.9 , 155.3 , 162.3 ( d ,  j = 246.5  hz ) . 

rf ( ethyl acetate : n - hexane : methanol 50  :  33  :  17 ) 0.48 ; h nmr ( 200  mhz , cdcl3 )  1.912.03 ( m , 2h ) , 2.68 ( t ,  j = 7.4  hz , 2h ) , 3.43 ( app t , j = 7.8  hz , 2h ) , 4.54 ( s , 2h ) , 6.44 ( app d ,  j = 5.4  hz , 2h ) , 6.967.36 ( m , 9h ) , 8.15 ( bs , 2h ) ; c nmr ( 50  mhz , cdcl3 )  28.1 , 33.0 , 49.9 , 52.9 , 106.8 , 115.8 ( d ,  j = 21.6  hz ) , 126.3 , 127.9 ( d ,  j = 8.0  hz ) , 128.3 , 128.6 , 132.1 ( d ,  j = 3.2  hz ) , 140.7 , 147.9 , 153.7 , 162.1 ( d , j = 245.8  hz ) . to a solution of 4ap ( 1  mmol ) in anhydrous dmso ( 2  cm ) , kept at rt under n2 , 2  mmol of t - buok was added .
this mixture was allowed to stir for 20  min at rt ; then 2  mmol of alkyl halide was added and the solution was kept under stirring at rt for 4  h. the solution was then mixed with water and extracted with dichloromethane .
the solvent was removed under reduced pressure and the mixture was purified by flash chromatography , yielding pure compound 3 . 

rf ( 80% ethyl acetate in dichloromethane ) 0.20 ; h nmr ( 200  mhz , cd3cn )  1.841.99 ( m , 4h ) , 2.65 ( t ,  j = 7.5  hz , 4h ) , 3.29 ( t ,  j = 7.5  hz , 4h ) , 6.30 ( d ,  j = 5.6  hz , 2h ) , 7.177.31 ( m , 10h ) , 8.13 ( d ,  j = 5.6  hz , 2h ) ; c nmr ( 50  mhz , cd3cn )  28.3 , 33.1 , 49.5 , 106.4 , 126.2 , 128.3 , 128.5 , 141.1 , 140.7 , 152.4 . 

rf ( 80% ethyl acetate in ethanol ) 0.32 ; h nmr ( 200  mhz , cdcl3 )  4.67 ( s , 4h ) , 6.58 ( dd ,  j = 4.8 , 1.6  hz , 2h ) , 7.197.40 ( m , 10h ) , 8.20 ( dd ,  j = 4.8 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  53.2 , 107.1 , 126.4 , 127.4 , 128.9 , 136.8 , 150.2 , 153.9 . 

di(4-fluorobenzyl)pyridin-4-amine 
 3ee 
 . rf ( 80% ethyl acetate in dichloromethane ) 0.40 ; h nmr ( 200  mhz , cdcl3 )  4.61 ( s , 4h ) , 6.56 ( dd ,  j = 5.0 , 1.6  hz , 2h ) , 6.997.19 ( m , 8h ) , 8.22 ( dd , j = 5.0 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  52.5 , 107.1 , 115.8 ( d ,  j = 21.6  hz ) , 128.1 ( d ,  j = 8.0  hz ) , 132.3 ( d ,  j = 3.2  hz ) , 150.3 , 153.6 , 162.2 ( d ,  j = 245.8  hz ) . 

rf ( 80% ethyl acetate in dichloromethane ) 0.25 ; h nmr ( 200  mhz , cdcl3 )  4.73 ( s , 4h ) , 6.58 ( dd , j = 5.0 , 1.6  hz , 2h ) , 7.367.59 ( m , 8h ) , 8.26 ( dd , j = 5.0 , 1.6  hz , 2h ) ; c nmr ( 50  mhz , cdcl3 )  53.1 , 107.1 , 123.3 ( q ,  j = 3.7  hz ) , 123.8 ( q ,  j = 272.3  hz ) , 124.5 ( q ,  j = 3.8  hz ) , 129.7 , 131.4 ( q ,  j = 32.4  hz ) , 137.7 , 150.4 , 153.4 . 
 organisms . for the antifungal evaluation , strains obtained from the american type culture collection ( atcc , rockville , md , usa ) , the german collection of microorganisms ( dsmz , braunschweig , germany ) , and the pharmaceutical microbiology culture collection ( pmc , department of public health and infectious diseases ,  sapienza  university , rome , italy ) were tested .
the strains were candida albicans ( atcc 10231 , atcc 10261 , atcc 24433 , atcc 90028 , 3153 , pmc 1002 , pmc 1011 , and pmc 1030 ) , c. parapsilosis atcc22019 , c. parapsilosis dsm 11224 , c. tropicalis dsm 11953 , c. tropicalis pmc 0908 , c. tropicalis pmc 0910 , c. glabrata pmc 0805 , c. krusei dsm 6128 , and c. krusei pmc 0613 , cryptococcus neoformans ( dsm 11959 , pmc 2102 , pmc 2107 , pmc 2111 , and pmc 2136 ) , dermatophytes ( trichophyton mentagrophytes dsm 4870 , t. mentagrophytes pmc6509 , microsporum gypseum dsm 7303 , and m. gypseum pmc 7331 ) .
all of the strains were stored and grown in accordance with the procedures of the clinical and laboratory standards institute ( clsi ) [ 26 , 27 ] . 

the final concentration ranged from 0.125 to 64  g / ml . the compounds were dissolved previously in dmso at concentrations 100 times higher than the highest desired test concentration and successively diluted in test medium in accordance with the procedures of the clsi .
microdilution trays containing 100  l of serial twofold dilutions of compounds in rpmi 1640 medium ( sigma - aldrich , st .
louis , mo , usa ) were inoculated with an organism suspension adjusted to attain a final inoculum concentration of 1.0  101.5  10 cells / ml for yeasts and 0.4  105  10 cfu / ml for dermatophytes .
the panels were incubated at 35c and observed for the presence of growth at 48  h ( candida spp . ) and 72  h ( c. neoformans and dermatophytes ) .
the minimal inhibitory concentration ( mic ) was , for yeasts , the lowest concentration that showed  50% growth inhibition compared with the growth control and , for dermatophytes , the lowest concentration that showed  80% growth inhibition compared with the growth control .
the mic100 was the lowest drug concentration that prevented 100% of growth with respect to the untreated control . according to csi protocols , the fluconazole mic50 and the amphotericin b mic100 were calculated ( 22,23 ) .
the results were expressed as the geometric mean ( g  m ) of the mic values .
for the evaluation of antiprotozoal and cytotoxic activity an integrated panel of microbial screens and standard screening methodologies were adopted as previously described   on the following organisms : chloroquine - resistant p. falciparum k 1-strain ; l. infantum mhom / ma ( be)/67 amastigote stage ; suramin - sensitive trypanosoma brucei squib-427 strain ; trypanosoma cruzi tulahuen cl2 ( benznidazole - sensitive ) strain ; human fetal lung fibroblast cells ( mrc-5 sv2 ) .
compounds were tested at 5 concentrations ( 64 , 16 , 4 , 1 , and 0.25  g / ml ) to establish a full dose titration and determine the ic50 ( inhibitory concentration 50% ) .
the final in - test concentration of dmso did not exceed 0.5% , which is known not to interfere with the different assays .
 the use of electrogenerated acetonitrile anion allows the alkylation of n - boc-4-aminopyridine in very high yields , under mild conditions and without by - products . 
 the high reactivity of this base is due to its large tetraethylammonium counterion , which leaves the acetonitrile anion  naked . 
  the deprotection of the obtained compounds led to high yields in n - alkylated 4-aminopyridines . 
 nonsymmetrically dialkylated 4-aminopyridines were obtained by subsequent reaction of monoalkylated ones with t - buok and alkyl halides , while symmetrically dialkylated 4-aminopyridines were obtained by direct reaction of 4-aminopyridine with an excess of t - buok and alkyl halides . some mono- and dialkyl-4-aminopyridines 
 were selected to evaluate antifungal and antiprotozoal activity ; the dialkylated 4-aminopyridines 3ac , 3ae and 3ff showed antifungal towards cryptococcus neoformans ; whereas 3cc , 3ee and 3ff showed antiprotozoal activity towards leishmania infantum and plasmodium falciparum . 