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Research ArticleEfficient Electrochemical N-Alkylation of N-Boc-Protected4-Aminopyridines Towards New Biologically Active Compounds
Marta Feroci1 Isabella Chiarotto1 Gianpiero Forte1 Giovanna Simonetti2
Felicia Diodata DrsquoAuria2 Louis Maes3 Daniela De Vita4 Luigi Scipione4 Laura Friggeri4
Roberto Di Santo4 and Silvano Tortorella4
1 Department of Basic and Applied Sciences for Engineering Sapienza University of Rome Via Castro Laurenziano 7 00161 Rome Italy2 Department of Public Health and Infectious Diseases Sapienza University of Rome Piazzale Aldo Moro 5 00185 Rome Italy3 Laboratory for Microbiology Parasitology and Hygiene (LMPH) Faculty of Pharmaceutical Biomedical and Veterinary SciencesAntwerp University 2610 Antwerp Belgium
4 ldquoIstituto Pasteur-Fondazione Cenci Bolognettirdquo Department of ldquoChimica e Tecnologie del Farmacordquo Sapienza University of RomePiazzale Aldo Moro 5 00185 Rome Italy
Correspondence should be addressed to Marta Feroci martaferociuniroma1it
Received 22 November 2013 Accepted 21 January 2014 Published 5 March 2014
Academic Editors L Palombi and R Pohl
Copyright copy 2014 Marta Feroci et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
The use of electrogenerated acetonitrile anion allows the alkylation of N-Boc-4-aminopyridine in very high yields under mildconditions andwithout by-productsThehigh reactivity of this base is due to its large tetraethylammoniumcounterion which leavesthe acetonitrile anion ldquonakedrdquo The deprotection of the obtained compounds led to high yields in N-alkylated 4-aminopyridinesNonsymmetrically dialkylated 4-aminopyridines were obtained by subsequent reaction of monoalkylated ones with t-BuOK andalkyl halides while symmetrically dialkylated 4-aminopyridines were obtained by direct reaction of 4-aminopyridinewith an excessof t-BuOK and alkyl halides Some mono- and dialkyl-4-aminopyridines were selected to evaluate antifungal and antiprotozoalactivity the dialkylated 4-aminopyridines 3ac 3ae and 3ff showed antifungal towards Cryptococcus neoformans whereas 3cc 3eeand 3ff showed antiprotozoal activity towards Leishmania infantum and Plasmodium falciparum
1 Introduction
N-Alkylated 4-aminopyridine is a commonmoiety in biolog-ically active molecules It is present in fact in compoundswith different activities such as inhibitors of p38120572 MAPkinase [1] inhibitors of HIV-EP1 cellular transcription factor[2] inhibitors of coagulation Factor Xa [3] and 120573-chemokinereceptor CCR5 antagonists in anti-HIV therapy [4] inparticular we have focused our work on the developmentof new CYP51 inhibitors active both on fungal strains [5]and Trypanosoma Cruzi [6] Many literature data evidencedthat the pyridine group can efficaciously replace the heme-iron chelating azole moiety present in classical azole CYP51inhibitors and therefore the alkylation of 4-aminopyridine(4AP) represents an important goal in organic synthesis todevelop novel classes of antifungal and antiparasitic drugs[7 8]
Due to the wide presence of these products the alkylationof 4-aminopyridine (4AP) is therefore an important goal inorganic synthesis
Different approaches to obtainN-alkylated 4-aminopyri-dines have been reported in the literature Some examplesare the efficient condensation of 4AP with alcohols catalyzedby benzaldehyde [9] or copper [10 11] or magnetite [12] thereaction of 4AP with an acyl chloride and the followingreduction of the amide with LiAlH
4[13]
The most straightforward method however is the directalkylation of 4AP with alkyl halides although it suffersfrom some drawbacks The two different nitrogen atomscompete in the alkylation reaction and usually the morenucleophilic pyridine nitrogen atom reacts faster leading tothe corresponding pyridinium salt (Scheme 1) [14 15]
In these case the use of a very strong base is therefore nec-essary n-BuLi was successfully used by Singh and coworkers
Scheme 1 Reaction of 4-aminopyridine with alkyl halides
[16] obtaining N-methyl- and N-ethyl-4-aminopyridines in74ndash80 yields
A viable alternative is the enhancement of the nucle-ophilicity of the amine nitrogen atom (versus the pyridineone) allowing the use of weaker bases An example is theactivation of 2-aminopyridine as formyl or Boc derivativeat the amine nitrogen atom [17] with subsequent deproto-nation using sodium hydride alkylation and deprotectionwith trifluoroacetic acid The deprotonation of N-Boc-2-aminopyridine with NaH needs a careful control of thetemperature (0ndash5∘C) and is carried out in anhydrous DMFwith a vigorous stirring required to keep the suspension fluid
In this context we envisaged the possibility to alkylateN-Boc-4-aminopyridine (N-Boc-4AP) usingmilder reactionconditions that is using electrogenerated tetraethylammo-nium cyanomethanide (Et4N+minusCH
2CN) [18] This base the
acetonitrile anion can be easily obtained by cathodic gal-vanostatic reduction of a solution of acetonitrile contain-ing tetraethylammonium hexafluorophosphate as supportingelectrolyte (Scheme 2) without by-products (the reagent isthe electron) and it was successfully used by us in a goodvariety of reactions [19] such as the selective N-alkylationof bifunctional compounds [20] the Gewald reaction [21]the synthesis of 120573-lactams [22] and the synthesis of car-bamates [23] The actual mechanism for the formation ofacetonitrile anion is not known but a hypothesis based onthe direct reduction of the tetraethylammonium cation hasbeen reported (Scheme 2) [24]
The high reactivity of this base is ascribable to thelarge tetraethylammonium counterion which renders theacetonitrile anion extremely reactive Moreover its reactionas a base gives no by-products as the protonation restores themolecule of solvent
2 Results and Discussion
The reaction of electrogenerated acetonitrile anion with4AP followed by an alkyl halide leads to poor yields indesired compound with the pyridinium salt being the majorproduct This prevents the direct use of minusCH
2CN with 4AP
On the other hand if the amine nitrogen is activated asBoc derivative (N-Boc-4AP) the deprotonationalkylationreaction using acetonitrile anion leads to products 1 in veryhigh yields (Scheme 3 and Table 1 entries 1ndash6) The classicdeprotection with trifluoroacetic acid allows obtaining thedesired products 2 (Scheme 3 and Table 1 entries 1ndash6)
The data in Table 1 highlight that the reaction of depro-tonation of N-Boc-4AP using electrogenerated acetonitrileanion alkylation with both alkyl and benzyl halides anddeprotection with trifluoroacetic acid is very efficient withoverall yields of 78ndash86 However when the alkylating agentis a bromoacetophenone the yields in alkylated product are
lower and inmost cases the deprotection reaction leads to thedealkylation of the starting material (Table 1 entries 7ndash10)
As many biologically active compounds contain thedialkylated 4-aminopyridine moiety we tried to carry out asecond alkylation on products 2andashj using acetonitrile anionbut as expected the high nucleophilicity of the pyridinenitrogen led to the synthesis of the corresponding pyridiniumsalt
We thus carried out this second alkylation using strongbases the most efficient being t-BuOK in DMSO (Scheme 4)although the yields in dialkylated 4AP were not very highThe results of this reaction are reported in Table 2
In order to obtain symmetrically dialkylated 4AP 4APwas subjected to deprotonation with t-BuOK in DMSOadding an excess of alkylating agent The reaction led toa mixture of mono- and dialkylated 4-aminopyridines inmoderate to acceptable yields The results are reported inTable 3
3 Biological Activity
A selection of synthesized compounds was in vitro testedto evaluate antifungal activity against different strains of Calbicans C parapsilosis and Cryptococcus neoformans dataare reported in Table 4 As can be evidenced the nonsym-metrical dialkylated 4APs 3ac and 3ae showed a moderateantifungal activity towards C albicans and C parapsilosiswith MIC
50values of 32 120583gmL and showed an interesting
activity against Cryptococcus neoformans with MIC50values
of 04 and 4 120583gmL respectively Otherwise the symmetricaldialkylated 4APs 3cc 3ee and the Boc-protected monoalky-lated 4APs 1b 1e 1f showed poor antifungal activity withMIC50and MIC
100ge 64 120583gmL
Furthermore the symmetrical dialkylated 4APs 3cc 3eeand 3ff were in vitro tested to evaluate the activity againstTry-panosoma cruzi Trypanosoma brucei Leishmania infantumand Plasmodium falciparum the results are summarized inTable 5
As can be evidenced all tested compounds showed amoderate activity versus P falciparum and an interestingactivity towards L infantum with IC
50values lower than the
reference drug miltefosine otherwise they resulted scarcelyactive against T cruzi and T brucei Moreover these com-pounds also showed low toxic activity versus growingMRC-5cells
4 Conclusion
In conclusion we demonstrated the usefulness of electro-generated acetonitrile anion in the alkylation of N-Boc 4-aminopyridines both from the point of viewof the high yieldsand of the cleanliness of the reaction (no by-products) Thedeprotection of N-Boc 4-aminopyridines allowed obtainingmonoalkylated 4-aminopyridine in very high yields Thefollowing alkylation by means of t-BuOK and alkyl halidesled to nonsymmetrically dialkylated 4-aminopyridine whilesymmetrically dialkylated products were obtained directlyfrom 4-aminopyridine by reaction with an excess of t-BuOKand alkyl halide
ISRN Organic Chemistry 3
Table 1 Alkylation reaction of 119873-Boc-4AP using electrogenerated acetonitrile anion in MeCN-01M TEAHFP followed by deprotectionwith trifluoroacetic acida
Entry RndashX 1 yield 2 yield
1 Br6N
NBoc
6
1a gt95
N
HN 6
2a 91
2Br
N
NBoc
Ph
1b 85
N
HN Ph
2b 93
3Cl
N
NBoc
1c 85
N
HN
2c gt95
4 Br
Cl
Cl N
NBoc
Cl
Cl
1d 95
N
HN
Cl
Cl
2d 91
5Br
FN
NBoc
F
1e 93
N
HN
F
2e 90
6Br
F3C N
NBoc
CF3
1f 90
N
HN
CF3
2f 87
7
OBr
N
NBoc
O
1g 34N
HNO
2g trb
4 ISRN Organic Chemistry
Table 1 Continued
Entry RndashX 1 yield 2 yield
8
OBr
FN
NBoc
O
F
1h 38
N
HNO
F
2h trb
9
OBr
ClN
NBoc
O
Cl
1i 56
N
HNO
Cl
2i trb
10
OBr
ON
NBoc
O
O
1j 48
N
HNO
O
2j 73aThe reduction was conducted under galvanostatic conditions (20mA cmminus2) on Pt electrodes in a divided cell at rt on 20mLMeCN-01M TEAHFP solutioncontaining 1mmol of 4AP At the end of the electrolysis 1mmol of alkylating agent was added After 2 h at rt usual workup afforded the products Deprotectionwas carried out as described in the experimental part All the yields are in isolated products bWhen compounds 2gndashi were subjected to deprotection withtrifluoroacetic acid a large amount of 4AP was obtained
Overall reaction Possible mechanism
CH3CNEt4N-PF6 Et4N+minusCH2CN Et4N++ eminus
Et4N∙
Et4N∙
Et∙ + eminus
Etminus + CH3CNEtminus
minusCH2CN
Et3N + Et∙+eminus
Scheme 2 Electrogeneration of acetonitrile anion
N N
NHBoc
N
NBoc R
N
HNR
4AP N-Boc-4AP
NH2
(Boc)2OCH3CN rt
(1)
(2)
minusCH2CN CF3CO2HCH2Cl2RndashX
1andashj 2andashj
Scheme 3 Synthesis of N-alkyl-4-aminopyridine
Furthermore it can also be concluded that themonoalky-lation of the 4AP leads to inactive products and otherwiseinteresting activity against fungi and some protozoa can beobtained by dual symmetrical or nonsymmetrical dialkyla-
N
HNR
2N
NR
(1) t-BuOK DMSO rt
R998400
3
(2) R998400ndashX
Scheme 4 Alkylation of 4-alkylaminopyridine
tion of the amino group of 4AP these activemolecules can beconsidered as lead compound to develop new antifungal andantiprotozoal compounds
ISRN Organic Chemistry 5
Table 2 Alkylation reaction of 4-alkylaminopyridines with 119905-BuOK in DMSOa
Entry Starting 2 R1015840ndashX 3 yieldb
1N
HN
2a
6
Cl
N
N 6
3ac 29
2N
HN
2c
Br6N
N 6
3ac 34
3N
HN
2e
F Br6N
N 6
F
3ae 24
4N
HN
2e
F Br
N
N
F
3be 31a1mmol of 2 in 2mL of anhydrous DMSO at rt under N2 Then 15mmol of 119905-BuOK were added followed by 1mmol of halide after 20min The reaction waskept under stirring for 4 h bAll the yields are in isolated products
5 Materials and Methods
51 General Acetonitrile was distilled twice from P2O5and
CaH2 Commercially available reagents were used without
further purification The Boc protection of 4-aminopyridinewas carried out following the literature [25]
4-[N-(tert-Butoxycarbonyl)amino]pyridine N-Boc-4AP To asolution of di-tert-butyl dicarbonate (3mmol) in acetonitrile(3 cm3) at room temperature 4-aminopyridine (3mmol) wasslowly added This mixture was then allowed to stir for 3 h atroom temperatureThe solvent was evaporated and the crude4-[N-(tert-butoxycarbonyl)amino]pyridine (gt95)was usedin the electrolyses without further purification Rf (30 ethylacetate in light petroleum ether) 020 1H NMR (200MHzCDCl
52 Electrochemical ALkylation of N-Boc-4AP Constant cur-rent electrolyses (I = 25mA cmminus2) were performed undera nitrogen atmosphere at 20∘C using an Amel Model 552
potentiostat equipped with an Amel Model 731 integratorAll the experiments were carried out in a divided glasscell separated through a porous glass plug filled up witha layer of gel (ie methyl cellulose 05 volume dissolvedin DMF-Et
4NPF610mol dmminus3) Pt spirals (apparent areas
08 cm2) were used both as cathode and anode MeCN-Et4NPF601mol dmminus3 was used as solvent-supporting elec-
trolyte system (catholyte 20 cm3 anolyte 5 cm3) 1mmol ofN-Boc-4-aminopyridine was present in the catholyte After145 C were passed the current was switched off and 1mmolof alkylating agent was added to the catholyte The solutionwas kept under stirring at room temperature for 2 hours thenthe solvent was evaporated under reduced pressure and theresiduewas purified by flash column chromatography using amixture of ethyl acetatelight petroleum ether 28 in volumeobtaining the pure products
Flash column chromatography was carried out usingMerck 60 kieselgel (230ndash400 mesh) under pressure GC-MSmeasurements were carried out on SE 54 capillary columnusing a Fisons 8000 gas chromatograph coupled with aFisons MD 800 quadrupole mass selective detector 1H and13C NMR spectra were recorded at room temperature usinga Bruker AC 200 spectrometer using CDCl
3as internal
standard
6 ISRN Organic Chemistry
Table 3 Dialkylation reaction of 4-aminopyridine with 119905-BuOK in DMSOa
Entry RndashX 3 yieldb
1Br
N
N
3bb 36
2Cl
N
N
3cc 42
3Br
F N
N
F F
3ee 39
4Br
F3C N
N
CF3
3ff 65
F3C
a1mmol of 4AP in 2mL of anhydrous DMSO at rt under N2 Then 2mmol of 119905-BuOK were added followed by 2mmol of halide after 20min The reactionwas kept under stirring for 4 h bAll the yields are in isolated products
53 Deprotection of Compounds 1andashj To a solution of 1(1mmol) in CH
2Cl2(5 cm3) kept at 0∘C 1 cm3 of CF
3COOH
was addedThismixture was allowed to stir for 3 h at 0∘CThesolution was then mixed with aqueous sodium carbonatetill pH 8 and extracted with ethyl acetate The solvent
8 ISRN Organic Chemistry
was removed under reduced pressure and the mixture waspurified by flash chromatography yielding pure compound 2
54 Alkylation of Compounds 2ace To a solution of 2(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
15mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 1mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
55 Dialkylation of 4-Aminopyridine To a solution of 4AP(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
2mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 2mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
Organisms For the antifungal evaluation strains obtainedfrom the American Type Culture Collection (ATCC Rock-ville MD USA) the German Collection of Microorganisms(DSMZ Braunschweig Germany) and the PharmaceuticalMicrobiology Culture Collection (PMC Department ofPublic Health and Infectious Diseases ldquoSapienzardquo UniversityRome Italy) were tested The strains were Candida albicans(ATCC 10231 ATCC 10261 ATCC 24433 ATCC 900283153 PMC 1002 PMC 1011 and PMC 1030) C parapsilosisATCC22019 C parapsilosis DSM 11224 C tropicalis DSM11953 C tropicalis PMC 0908 C tropicalis PMC 0910 Cglabrata PMC 0805 C krusei DSM 6128 and C kruseiPMC 0613 Cryptococcus neoformans (DSM 11959 PMC2102 PMC 2107 PMC 2111 and PMC 2136) dermatophytes(Trichophyton mentagrophytes DSM 4870 T mentagrophytesPMC6509 Microsporum gypseum DSM 7303 and Mgypseum PMC 7331) All of the strains were stored andgrown in accordance with the procedures of the Clinical andLaboratory Standards Institute (CLSI) [26 27]
Antifungal Susceptibility Assays In vitro antifungal suscep-tibility was evaluated using the CLSI broth microdilutionmethods [26 27] Fluconazole and Amphotericin B wereused as reference drugs The final concentration rangedfrom 0125 to 64 120583gmL The compounds were dissolvedpreviously in DMSO at concentrations 100 times higherthan the highest desired test concentration and successivelydiluted in test medium in accordance with the proceduresof the CLSI [28] Microdilution trays containing 100 120583L ofserial twofold dilutions of compounds in RPMI 1640medium(Sigma-Aldrich St Louis MO USA) were inoculated withan organism suspension adjusted to attain a final inoculumconcentration of 10 times 103ndash15 times 103 cellsmL for yeasts and04 times 10
4ndash5 times 104 CFUmL for dermatophytes The panelswere incubated at 35∘C and observed for the presence of
growth at 48 h (Candida spp) and 72 h (C neoformans anddermatophytes)
The minimal inhibitory concentration (MIC) was foryeasts the lowest concentration that showed ge 50 growthinhibition compared with the growth control and for der-matophytes the lowest concentration that showed ge 80growth inhibition compared with the growth control TheMIC100
was the lowest drug concentration that prevented100 of growth with respect to the untreated controlAccording to CSI protocols the fluconazole MIC
50and the
amphotericin B MIC100
were calculated (2223) The resultswere expressed as the geometric mean (GM) of the MICvalues
562 Antiprotozoal Assay For the evaluation of antiproto-zoal and cytotoxic activity an integrated panel of microbialscreens and standard screening methodologies were adoptedas previously described [29] on the following organismschloroquine-resistant P falciparum K 1-strain L infantumMHOMMA (BE)67 amastigote stage suramin-sensitiveTrypanosoma brucei Squib-427 strain Trypanosoma cruziTulahuen CL2 (benznidazole-sensitive) strain human fetallung fibroblast cells (MRC-5 SV2)
All assays were performed in triplicate Compounds weretested at 5 concentrations (64 16 4 1 and 025120583gmL)to establish a full dose titration and determine the IC
50
(inhibitory concentration 50) The final in-test concentra-tion of DMSO did not exceed 05 which is known not tointerfere with the different assays [29]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was financially supported by Miur Italy Theauthors thank Mr Marco Di Pilato for his support in theexperimental part of the work
References
[1] G R Luedtke K Schinzel X Tan et al ldquoAmide-basedinhibitors of p38120572 MAP kinase Part 1 Discovery of novelN-pyridyl amide lead moleculesrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 8 pp 2556ndash2559 2010
[2] M Otsuka M Fujita Y Sugiura et al ldquoSynthetic inhibitors ofregulatory proteins involved in the signaling pathway of thereplication of human immunodeficiency virus 1rdquo Bioorganicand Medicinal Chemistry vol 5 no 1 pp 205ndash215 1997
[3] H Nishida Y Miyazaki T Mukaihira et al ldquoSynthesis andevaluation of 1-arylsulfonyl-3-piperazinone derivatives as afactor Xa inhibitor II Substituent effect on biological activitiesrdquoChemical and Pharmaceutical Bulletin vol 50 no 9 pp 1187ndash1194 2002
[4] C A Willoughby K G Rosauer J J Hale et al ldquo134Trisubstituted pyrrolidine CCR5 receptor antagonists bearing
10 ISRN Organic Chemistry
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006
Scheme 1 Reaction of 4-aminopyridine with alkyl halides
[16] obtaining N-methyl- and N-ethyl-4-aminopyridines in74ndash80 yields
A viable alternative is the enhancement of the nucle-ophilicity of the amine nitrogen atom (versus the pyridineone) allowing the use of weaker bases An example is theactivation of 2-aminopyridine as formyl or Boc derivativeat the amine nitrogen atom [17] with subsequent deproto-nation using sodium hydride alkylation and deprotectionwith trifluoroacetic acid The deprotonation of N-Boc-2-aminopyridine with NaH needs a careful control of thetemperature (0ndash5∘C) and is carried out in anhydrous DMFwith a vigorous stirring required to keep the suspension fluid
In this context we envisaged the possibility to alkylateN-Boc-4-aminopyridine (N-Boc-4AP) usingmilder reactionconditions that is using electrogenerated tetraethylammo-nium cyanomethanide (Et4N+minusCH
2CN) [18] This base the
acetonitrile anion can be easily obtained by cathodic gal-vanostatic reduction of a solution of acetonitrile contain-ing tetraethylammonium hexafluorophosphate as supportingelectrolyte (Scheme 2) without by-products (the reagent isthe electron) and it was successfully used by us in a goodvariety of reactions [19] such as the selective N-alkylationof bifunctional compounds [20] the Gewald reaction [21]the synthesis of 120573-lactams [22] and the synthesis of car-bamates [23] The actual mechanism for the formation ofacetonitrile anion is not known but a hypothesis based onthe direct reduction of the tetraethylammonium cation hasbeen reported (Scheme 2) [24]
The high reactivity of this base is ascribable to thelarge tetraethylammonium counterion which renders theacetonitrile anion extremely reactive Moreover its reactionas a base gives no by-products as the protonation restores themolecule of solvent
2 Results and Discussion
The reaction of electrogenerated acetonitrile anion with4AP followed by an alkyl halide leads to poor yields indesired compound with the pyridinium salt being the majorproduct This prevents the direct use of minusCH
2CN with 4AP
On the other hand if the amine nitrogen is activated asBoc derivative (N-Boc-4AP) the deprotonationalkylationreaction using acetonitrile anion leads to products 1 in veryhigh yields (Scheme 3 and Table 1 entries 1ndash6) The classicdeprotection with trifluoroacetic acid allows obtaining thedesired products 2 (Scheme 3 and Table 1 entries 1ndash6)
The data in Table 1 highlight that the reaction of depro-tonation of N-Boc-4AP using electrogenerated acetonitrileanion alkylation with both alkyl and benzyl halides anddeprotection with trifluoroacetic acid is very efficient withoverall yields of 78ndash86 However when the alkylating agentis a bromoacetophenone the yields in alkylated product are
lower and inmost cases the deprotection reaction leads to thedealkylation of the starting material (Table 1 entries 7ndash10)
As many biologically active compounds contain thedialkylated 4-aminopyridine moiety we tried to carry out asecond alkylation on products 2andashj using acetonitrile anionbut as expected the high nucleophilicity of the pyridinenitrogen led to the synthesis of the corresponding pyridiniumsalt
We thus carried out this second alkylation using strongbases the most efficient being t-BuOK in DMSO (Scheme 4)although the yields in dialkylated 4AP were not very highThe results of this reaction are reported in Table 2
In order to obtain symmetrically dialkylated 4AP 4APwas subjected to deprotonation with t-BuOK in DMSOadding an excess of alkylating agent The reaction led toa mixture of mono- and dialkylated 4-aminopyridines inmoderate to acceptable yields The results are reported inTable 3
3 Biological Activity
A selection of synthesized compounds was in vitro testedto evaluate antifungal activity against different strains of Calbicans C parapsilosis and Cryptococcus neoformans dataare reported in Table 4 As can be evidenced the nonsym-metrical dialkylated 4APs 3ac and 3ae showed a moderateantifungal activity towards C albicans and C parapsilosiswith MIC
50values of 32 120583gmL and showed an interesting
activity against Cryptococcus neoformans with MIC50values
of 04 and 4 120583gmL respectively Otherwise the symmetricaldialkylated 4APs 3cc 3ee and the Boc-protected monoalky-lated 4APs 1b 1e 1f showed poor antifungal activity withMIC50and MIC
100ge 64 120583gmL
Furthermore the symmetrical dialkylated 4APs 3cc 3eeand 3ff were in vitro tested to evaluate the activity againstTry-panosoma cruzi Trypanosoma brucei Leishmania infantumand Plasmodium falciparum the results are summarized inTable 5
As can be evidenced all tested compounds showed amoderate activity versus P falciparum and an interestingactivity towards L infantum with IC
50values lower than the
reference drug miltefosine otherwise they resulted scarcelyactive against T cruzi and T brucei Moreover these com-pounds also showed low toxic activity versus growingMRC-5cells
4 Conclusion
In conclusion we demonstrated the usefulness of electro-generated acetonitrile anion in the alkylation of N-Boc 4-aminopyridines both from the point of viewof the high yieldsand of the cleanliness of the reaction (no by-products) Thedeprotection of N-Boc 4-aminopyridines allowed obtainingmonoalkylated 4-aminopyridine in very high yields Thefollowing alkylation by means of t-BuOK and alkyl halidesled to nonsymmetrically dialkylated 4-aminopyridine whilesymmetrically dialkylated products were obtained directlyfrom 4-aminopyridine by reaction with an excess of t-BuOKand alkyl halide
ISRN Organic Chemistry 3
Table 1 Alkylation reaction of 119873-Boc-4AP using electrogenerated acetonitrile anion in MeCN-01M TEAHFP followed by deprotectionwith trifluoroacetic acida
Entry RndashX 1 yield 2 yield
1 Br6N
NBoc
6
1a gt95
N
HN 6
2a 91
2Br
N
NBoc
Ph
1b 85
N
HN Ph
2b 93
3Cl
N
NBoc
1c 85
N
HN
2c gt95
4 Br
Cl
Cl N
NBoc
Cl
Cl
1d 95
N
HN
Cl
Cl
2d 91
5Br
FN
NBoc
F
1e 93
N
HN
F
2e 90
6Br
F3C N
NBoc
CF3
1f 90
N
HN
CF3
2f 87
7
OBr
N
NBoc
O
1g 34N
HNO
2g trb
4 ISRN Organic Chemistry
Table 1 Continued
Entry RndashX 1 yield 2 yield
8
OBr
FN
NBoc
O
F
1h 38
N
HNO
F
2h trb
9
OBr
ClN
NBoc
O
Cl
1i 56
N
HNO
Cl
2i trb
10
OBr
ON
NBoc
O
O
1j 48
N
HNO
O
2j 73aThe reduction was conducted under galvanostatic conditions (20mA cmminus2) on Pt electrodes in a divided cell at rt on 20mLMeCN-01M TEAHFP solutioncontaining 1mmol of 4AP At the end of the electrolysis 1mmol of alkylating agent was added After 2 h at rt usual workup afforded the products Deprotectionwas carried out as described in the experimental part All the yields are in isolated products bWhen compounds 2gndashi were subjected to deprotection withtrifluoroacetic acid a large amount of 4AP was obtained
Overall reaction Possible mechanism
CH3CNEt4N-PF6 Et4N+minusCH2CN Et4N++ eminus
Et4N∙
Et4N∙
Et∙ + eminus
Etminus + CH3CNEtminus
minusCH2CN
Et3N + Et∙+eminus
Scheme 2 Electrogeneration of acetonitrile anion
N N
NHBoc
N
NBoc R
N
HNR
4AP N-Boc-4AP
NH2
(Boc)2OCH3CN rt
(1)
(2)
minusCH2CN CF3CO2HCH2Cl2RndashX
1andashj 2andashj
Scheme 3 Synthesis of N-alkyl-4-aminopyridine
Furthermore it can also be concluded that themonoalky-lation of the 4AP leads to inactive products and otherwiseinteresting activity against fungi and some protozoa can beobtained by dual symmetrical or nonsymmetrical dialkyla-
N
HNR
2N
NR
(1) t-BuOK DMSO rt
R998400
3
(2) R998400ndashX
Scheme 4 Alkylation of 4-alkylaminopyridine
tion of the amino group of 4AP these activemolecules can beconsidered as lead compound to develop new antifungal andantiprotozoal compounds
ISRN Organic Chemistry 5
Table 2 Alkylation reaction of 4-alkylaminopyridines with 119905-BuOK in DMSOa
Entry Starting 2 R1015840ndashX 3 yieldb
1N
HN
2a
6
Cl
N
N 6
3ac 29
2N
HN
2c
Br6N
N 6
3ac 34
3N
HN
2e
F Br6N
N 6
F
3ae 24
4N
HN
2e
F Br
N
N
F
3be 31a1mmol of 2 in 2mL of anhydrous DMSO at rt under N2 Then 15mmol of 119905-BuOK were added followed by 1mmol of halide after 20min The reaction waskept under stirring for 4 h bAll the yields are in isolated products
5 Materials and Methods
51 General Acetonitrile was distilled twice from P2O5and
CaH2 Commercially available reagents were used without
further purification The Boc protection of 4-aminopyridinewas carried out following the literature [25]
4-[N-(tert-Butoxycarbonyl)amino]pyridine N-Boc-4AP To asolution of di-tert-butyl dicarbonate (3mmol) in acetonitrile(3 cm3) at room temperature 4-aminopyridine (3mmol) wasslowly added This mixture was then allowed to stir for 3 h atroom temperatureThe solvent was evaporated and the crude4-[N-(tert-butoxycarbonyl)amino]pyridine (gt95)was usedin the electrolyses without further purification Rf (30 ethylacetate in light petroleum ether) 020 1H NMR (200MHzCDCl
52 Electrochemical ALkylation of N-Boc-4AP Constant cur-rent electrolyses (I = 25mA cmminus2) were performed undera nitrogen atmosphere at 20∘C using an Amel Model 552
potentiostat equipped with an Amel Model 731 integratorAll the experiments were carried out in a divided glasscell separated through a porous glass plug filled up witha layer of gel (ie methyl cellulose 05 volume dissolvedin DMF-Et
4NPF610mol dmminus3) Pt spirals (apparent areas
08 cm2) were used both as cathode and anode MeCN-Et4NPF601mol dmminus3 was used as solvent-supporting elec-
trolyte system (catholyte 20 cm3 anolyte 5 cm3) 1mmol ofN-Boc-4-aminopyridine was present in the catholyte After145 C were passed the current was switched off and 1mmolof alkylating agent was added to the catholyte The solutionwas kept under stirring at room temperature for 2 hours thenthe solvent was evaporated under reduced pressure and theresiduewas purified by flash column chromatography using amixture of ethyl acetatelight petroleum ether 28 in volumeobtaining the pure products
Flash column chromatography was carried out usingMerck 60 kieselgel (230ndash400 mesh) under pressure GC-MSmeasurements were carried out on SE 54 capillary columnusing a Fisons 8000 gas chromatograph coupled with aFisons MD 800 quadrupole mass selective detector 1H and13C NMR spectra were recorded at room temperature usinga Bruker AC 200 spectrometer using CDCl
3as internal
standard
6 ISRN Organic Chemistry
Table 3 Dialkylation reaction of 4-aminopyridine with 119905-BuOK in DMSOa
Entry RndashX 3 yieldb
1Br
N
N
3bb 36
2Cl
N
N
3cc 42
3Br
F N
N
F F
3ee 39
4Br
F3C N
N
CF3
3ff 65
F3C
a1mmol of 4AP in 2mL of anhydrous DMSO at rt under N2 Then 2mmol of 119905-BuOK were added followed by 2mmol of halide after 20min The reactionwas kept under stirring for 4 h bAll the yields are in isolated products
53 Deprotection of Compounds 1andashj To a solution of 1(1mmol) in CH
2Cl2(5 cm3) kept at 0∘C 1 cm3 of CF
3COOH
was addedThismixture was allowed to stir for 3 h at 0∘CThesolution was then mixed with aqueous sodium carbonatetill pH 8 and extracted with ethyl acetate The solvent
8 ISRN Organic Chemistry
was removed under reduced pressure and the mixture waspurified by flash chromatography yielding pure compound 2
54 Alkylation of Compounds 2ace To a solution of 2(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
15mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 1mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
55 Dialkylation of 4-Aminopyridine To a solution of 4AP(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
2mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 2mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
Organisms For the antifungal evaluation strains obtainedfrom the American Type Culture Collection (ATCC Rock-ville MD USA) the German Collection of Microorganisms(DSMZ Braunschweig Germany) and the PharmaceuticalMicrobiology Culture Collection (PMC Department ofPublic Health and Infectious Diseases ldquoSapienzardquo UniversityRome Italy) were tested The strains were Candida albicans(ATCC 10231 ATCC 10261 ATCC 24433 ATCC 900283153 PMC 1002 PMC 1011 and PMC 1030) C parapsilosisATCC22019 C parapsilosis DSM 11224 C tropicalis DSM11953 C tropicalis PMC 0908 C tropicalis PMC 0910 Cglabrata PMC 0805 C krusei DSM 6128 and C kruseiPMC 0613 Cryptococcus neoformans (DSM 11959 PMC2102 PMC 2107 PMC 2111 and PMC 2136) dermatophytes(Trichophyton mentagrophytes DSM 4870 T mentagrophytesPMC6509 Microsporum gypseum DSM 7303 and Mgypseum PMC 7331) All of the strains were stored andgrown in accordance with the procedures of the Clinical andLaboratory Standards Institute (CLSI) [26 27]
Antifungal Susceptibility Assays In vitro antifungal suscep-tibility was evaluated using the CLSI broth microdilutionmethods [26 27] Fluconazole and Amphotericin B wereused as reference drugs The final concentration rangedfrom 0125 to 64 120583gmL The compounds were dissolvedpreviously in DMSO at concentrations 100 times higherthan the highest desired test concentration and successivelydiluted in test medium in accordance with the proceduresof the CLSI [28] Microdilution trays containing 100 120583L ofserial twofold dilutions of compounds in RPMI 1640medium(Sigma-Aldrich St Louis MO USA) were inoculated withan organism suspension adjusted to attain a final inoculumconcentration of 10 times 103ndash15 times 103 cellsmL for yeasts and04 times 10
4ndash5 times 104 CFUmL for dermatophytes The panelswere incubated at 35∘C and observed for the presence of
growth at 48 h (Candida spp) and 72 h (C neoformans anddermatophytes)
The minimal inhibitory concentration (MIC) was foryeasts the lowest concentration that showed ge 50 growthinhibition compared with the growth control and for der-matophytes the lowest concentration that showed ge 80growth inhibition compared with the growth control TheMIC100
was the lowest drug concentration that prevented100 of growth with respect to the untreated controlAccording to CSI protocols the fluconazole MIC
50and the
amphotericin B MIC100
were calculated (2223) The resultswere expressed as the geometric mean (GM) of the MICvalues
562 Antiprotozoal Assay For the evaluation of antiproto-zoal and cytotoxic activity an integrated panel of microbialscreens and standard screening methodologies were adoptedas previously described [29] on the following organismschloroquine-resistant P falciparum K 1-strain L infantumMHOMMA (BE)67 amastigote stage suramin-sensitiveTrypanosoma brucei Squib-427 strain Trypanosoma cruziTulahuen CL2 (benznidazole-sensitive) strain human fetallung fibroblast cells (MRC-5 SV2)
All assays were performed in triplicate Compounds weretested at 5 concentrations (64 16 4 1 and 025120583gmL)to establish a full dose titration and determine the IC
50
(inhibitory concentration 50) The final in-test concentra-tion of DMSO did not exceed 05 which is known not tointerfere with the different assays [29]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was financially supported by Miur Italy Theauthors thank Mr Marco Di Pilato for his support in theexperimental part of the work
References
[1] G R Luedtke K Schinzel X Tan et al ldquoAmide-basedinhibitors of p38120572 MAP kinase Part 1 Discovery of novelN-pyridyl amide lead moleculesrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 8 pp 2556ndash2559 2010
[2] M Otsuka M Fujita Y Sugiura et al ldquoSynthetic inhibitors ofregulatory proteins involved in the signaling pathway of thereplication of human immunodeficiency virus 1rdquo Bioorganicand Medicinal Chemistry vol 5 no 1 pp 205ndash215 1997
[3] H Nishida Y Miyazaki T Mukaihira et al ldquoSynthesis andevaluation of 1-arylsulfonyl-3-piperazinone derivatives as afactor Xa inhibitor II Substituent effect on biological activitiesrdquoChemical and Pharmaceutical Bulletin vol 50 no 9 pp 1187ndash1194 2002
[4] C A Willoughby K G Rosauer J J Hale et al ldquo134Trisubstituted pyrrolidine CCR5 receptor antagonists bearing
10 ISRN Organic Chemistry
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006
Table 1 Alkylation reaction of 119873-Boc-4AP using electrogenerated acetonitrile anion in MeCN-01M TEAHFP followed by deprotectionwith trifluoroacetic acida
Entry RndashX 1 yield 2 yield
1 Br6N
NBoc
6
1a gt95
N
HN 6
2a 91
2Br
N
NBoc
Ph
1b 85
N
HN Ph
2b 93
3Cl
N
NBoc
1c 85
N
HN
2c gt95
4 Br
Cl
Cl N
NBoc
Cl
Cl
1d 95
N
HN
Cl
Cl
2d 91
5Br
FN
NBoc
F
1e 93
N
HN
F
2e 90
6Br
F3C N
NBoc
CF3
1f 90
N
HN
CF3
2f 87
7
OBr
N
NBoc
O
1g 34N
HNO
2g trb
4 ISRN Organic Chemistry
Table 1 Continued
Entry RndashX 1 yield 2 yield
8
OBr
FN
NBoc
O
F
1h 38
N
HNO
F
2h trb
9
OBr
ClN
NBoc
O
Cl
1i 56
N
HNO
Cl
2i trb
10
OBr
ON
NBoc
O
O
1j 48
N
HNO
O
2j 73aThe reduction was conducted under galvanostatic conditions (20mA cmminus2) on Pt electrodes in a divided cell at rt on 20mLMeCN-01M TEAHFP solutioncontaining 1mmol of 4AP At the end of the electrolysis 1mmol of alkylating agent was added After 2 h at rt usual workup afforded the products Deprotectionwas carried out as described in the experimental part All the yields are in isolated products bWhen compounds 2gndashi were subjected to deprotection withtrifluoroacetic acid a large amount of 4AP was obtained
Overall reaction Possible mechanism
CH3CNEt4N-PF6 Et4N+minusCH2CN Et4N++ eminus
Et4N∙
Et4N∙
Et∙ + eminus
Etminus + CH3CNEtminus
minusCH2CN
Et3N + Et∙+eminus
Scheme 2 Electrogeneration of acetonitrile anion
N N
NHBoc
N
NBoc R
N
HNR
4AP N-Boc-4AP
NH2
(Boc)2OCH3CN rt
(1)
(2)
minusCH2CN CF3CO2HCH2Cl2RndashX
1andashj 2andashj
Scheme 3 Synthesis of N-alkyl-4-aminopyridine
Furthermore it can also be concluded that themonoalky-lation of the 4AP leads to inactive products and otherwiseinteresting activity against fungi and some protozoa can beobtained by dual symmetrical or nonsymmetrical dialkyla-
N
HNR
2N
NR
(1) t-BuOK DMSO rt
R998400
3
(2) R998400ndashX
Scheme 4 Alkylation of 4-alkylaminopyridine
tion of the amino group of 4AP these activemolecules can beconsidered as lead compound to develop new antifungal andantiprotozoal compounds
ISRN Organic Chemistry 5
Table 2 Alkylation reaction of 4-alkylaminopyridines with 119905-BuOK in DMSOa
Entry Starting 2 R1015840ndashX 3 yieldb
1N
HN
2a
6
Cl
N
N 6
3ac 29
2N
HN
2c
Br6N
N 6
3ac 34
3N
HN
2e
F Br6N
N 6
F
3ae 24
4N
HN
2e
F Br
N
N
F
3be 31a1mmol of 2 in 2mL of anhydrous DMSO at rt under N2 Then 15mmol of 119905-BuOK were added followed by 1mmol of halide after 20min The reaction waskept under stirring for 4 h bAll the yields are in isolated products
5 Materials and Methods
51 General Acetonitrile was distilled twice from P2O5and
CaH2 Commercially available reagents were used without
further purification The Boc protection of 4-aminopyridinewas carried out following the literature [25]
4-[N-(tert-Butoxycarbonyl)amino]pyridine N-Boc-4AP To asolution of di-tert-butyl dicarbonate (3mmol) in acetonitrile(3 cm3) at room temperature 4-aminopyridine (3mmol) wasslowly added This mixture was then allowed to stir for 3 h atroom temperatureThe solvent was evaporated and the crude4-[N-(tert-butoxycarbonyl)amino]pyridine (gt95)was usedin the electrolyses without further purification Rf (30 ethylacetate in light petroleum ether) 020 1H NMR (200MHzCDCl
52 Electrochemical ALkylation of N-Boc-4AP Constant cur-rent electrolyses (I = 25mA cmminus2) were performed undera nitrogen atmosphere at 20∘C using an Amel Model 552
potentiostat equipped with an Amel Model 731 integratorAll the experiments were carried out in a divided glasscell separated through a porous glass plug filled up witha layer of gel (ie methyl cellulose 05 volume dissolvedin DMF-Et
4NPF610mol dmminus3) Pt spirals (apparent areas
08 cm2) were used both as cathode and anode MeCN-Et4NPF601mol dmminus3 was used as solvent-supporting elec-
trolyte system (catholyte 20 cm3 anolyte 5 cm3) 1mmol ofN-Boc-4-aminopyridine was present in the catholyte After145 C were passed the current was switched off and 1mmolof alkylating agent was added to the catholyte The solutionwas kept under stirring at room temperature for 2 hours thenthe solvent was evaporated under reduced pressure and theresiduewas purified by flash column chromatography using amixture of ethyl acetatelight petroleum ether 28 in volumeobtaining the pure products
Flash column chromatography was carried out usingMerck 60 kieselgel (230ndash400 mesh) under pressure GC-MSmeasurements were carried out on SE 54 capillary columnusing a Fisons 8000 gas chromatograph coupled with aFisons MD 800 quadrupole mass selective detector 1H and13C NMR spectra were recorded at room temperature usinga Bruker AC 200 spectrometer using CDCl
3as internal
standard
6 ISRN Organic Chemistry
Table 3 Dialkylation reaction of 4-aminopyridine with 119905-BuOK in DMSOa
Entry RndashX 3 yieldb
1Br
N
N
3bb 36
2Cl
N
N
3cc 42
3Br
F N
N
F F
3ee 39
4Br
F3C N
N
CF3
3ff 65
F3C
a1mmol of 4AP in 2mL of anhydrous DMSO at rt under N2 Then 2mmol of 119905-BuOK were added followed by 2mmol of halide after 20min The reactionwas kept under stirring for 4 h bAll the yields are in isolated products
53 Deprotection of Compounds 1andashj To a solution of 1(1mmol) in CH
2Cl2(5 cm3) kept at 0∘C 1 cm3 of CF
3COOH
was addedThismixture was allowed to stir for 3 h at 0∘CThesolution was then mixed with aqueous sodium carbonatetill pH 8 and extracted with ethyl acetate The solvent
8 ISRN Organic Chemistry
was removed under reduced pressure and the mixture waspurified by flash chromatography yielding pure compound 2
54 Alkylation of Compounds 2ace To a solution of 2(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
15mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 1mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
55 Dialkylation of 4-Aminopyridine To a solution of 4AP(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
2mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 2mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
Organisms For the antifungal evaluation strains obtainedfrom the American Type Culture Collection (ATCC Rock-ville MD USA) the German Collection of Microorganisms(DSMZ Braunschweig Germany) and the PharmaceuticalMicrobiology Culture Collection (PMC Department ofPublic Health and Infectious Diseases ldquoSapienzardquo UniversityRome Italy) were tested The strains were Candida albicans(ATCC 10231 ATCC 10261 ATCC 24433 ATCC 900283153 PMC 1002 PMC 1011 and PMC 1030) C parapsilosisATCC22019 C parapsilosis DSM 11224 C tropicalis DSM11953 C tropicalis PMC 0908 C tropicalis PMC 0910 Cglabrata PMC 0805 C krusei DSM 6128 and C kruseiPMC 0613 Cryptococcus neoformans (DSM 11959 PMC2102 PMC 2107 PMC 2111 and PMC 2136) dermatophytes(Trichophyton mentagrophytes DSM 4870 T mentagrophytesPMC6509 Microsporum gypseum DSM 7303 and Mgypseum PMC 7331) All of the strains were stored andgrown in accordance with the procedures of the Clinical andLaboratory Standards Institute (CLSI) [26 27]
Antifungal Susceptibility Assays In vitro antifungal suscep-tibility was evaluated using the CLSI broth microdilutionmethods [26 27] Fluconazole and Amphotericin B wereused as reference drugs The final concentration rangedfrom 0125 to 64 120583gmL The compounds were dissolvedpreviously in DMSO at concentrations 100 times higherthan the highest desired test concentration and successivelydiluted in test medium in accordance with the proceduresof the CLSI [28] Microdilution trays containing 100 120583L ofserial twofold dilutions of compounds in RPMI 1640medium(Sigma-Aldrich St Louis MO USA) were inoculated withan organism suspension adjusted to attain a final inoculumconcentration of 10 times 103ndash15 times 103 cellsmL for yeasts and04 times 10
4ndash5 times 104 CFUmL for dermatophytes The panelswere incubated at 35∘C and observed for the presence of
growth at 48 h (Candida spp) and 72 h (C neoformans anddermatophytes)
The minimal inhibitory concentration (MIC) was foryeasts the lowest concentration that showed ge 50 growthinhibition compared with the growth control and for der-matophytes the lowest concentration that showed ge 80growth inhibition compared with the growth control TheMIC100
was the lowest drug concentration that prevented100 of growth with respect to the untreated controlAccording to CSI protocols the fluconazole MIC
50and the
amphotericin B MIC100
were calculated (2223) The resultswere expressed as the geometric mean (GM) of the MICvalues
562 Antiprotozoal Assay For the evaluation of antiproto-zoal and cytotoxic activity an integrated panel of microbialscreens and standard screening methodologies were adoptedas previously described [29] on the following organismschloroquine-resistant P falciparum K 1-strain L infantumMHOMMA (BE)67 amastigote stage suramin-sensitiveTrypanosoma brucei Squib-427 strain Trypanosoma cruziTulahuen CL2 (benznidazole-sensitive) strain human fetallung fibroblast cells (MRC-5 SV2)
All assays were performed in triplicate Compounds weretested at 5 concentrations (64 16 4 1 and 025120583gmL)to establish a full dose titration and determine the IC
50
(inhibitory concentration 50) The final in-test concentra-tion of DMSO did not exceed 05 which is known not tointerfere with the different assays [29]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was financially supported by Miur Italy Theauthors thank Mr Marco Di Pilato for his support in theexperimental part of the work
References
[1] G R Luedtke K Schinzel X Tan et al ldquoAmide-basedinhibitors of p38120572 MAP kinase Part 1 Discovery of novelN-pyridyl amide lead moleculesrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 8 pp 2556ndash2559 2010
[2] M Otsuka M Fujita Y Sugiura et al ldquoSynthetic inhibitors ofregulatory proteins involved in the signaling pathway of thereplication of human immunodeficiency virus 1rdquo Bioorganicand Medicinal Chemistry vol 5 no 1 pp 205ndash215 1997
[3] H Nishida Y Miyazaki T Mukaihira et al ldquoSynthesis andevaluation of 1-arylsulfonyl-3-piperazinone derivatives as afactor Xa inhibitor II Substituent effect on biological activitiesrdquoChemical and Pharmaceutical Bulletin vol 50 no 9 pp 1187ndash1194 2002
[4] C A Willoughby K G Rosauer J J Hale et al ldquo134Trisubstituted pyrrolidine CCR5 receptor antagonists bearing
10 ISRN Organic Chemistry
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006
2j 73aThe reduction was conducted under galvanostatic conditions (20mA cmminus2) on Pt electrodes in a divided cell at rt on 20mLMeCN-01M TEAHFP solutioncontaining 1mmol of 4AP At the end of the electrolysis 1mmol of alkylating agent was added After 2 h at rt usual workup afforded the products Deprotectionwas carried out as described in the experimental part All the yields are in isolated products bWhen compounds 2gndashi were subjected to deprotection withtrifluoroacetic acid a large amount of 4AP was obtained
Overall reaction Possible mechanism
CH3CNEt4N-PF6 Et4N+minusCH2CN Et4N++ eminus
Et4N∙
Et4N∙
Et∙ + eminus
Etminus + CH3CNEtminus
minusCH2CN
Et3N + Et∙+eminus
Scheme 2 Electrogeneration of acetonitrile anion
N N
NHBoc
N
NBoc R
N
HNR
4AP N-Boc-4AP
NH2
(Boc)2OCH3CN rt
(1)
(2)
minusCH2CN CF3CO2HCH2Cl2RndashX
1andashj 2andashj
Scheme 3 Synthesis of N-alkyl-4-aminopyridine
Furthermore it can also be concluded that themonoalky-lation of the 4AP leads to inactive products and otherwiseinteresting activity against fungi and some protozoa can beobtained by dual symmetrical or nonsymmetrical dialkyla-
N
HNR
2N
NR
(1) t-BuOK DMSO rt
R998400
3
(2) R998400ndashX
Scheme 4 Alkylation of 4-alkylaminopyridine
tion of the amino group of 4AP these activemolecules can beconsidered as lead compound to develop new antifungal andantiprotozoal compounds
ISRN Organic Chemistry 5
Table 2 Alkylation reaction of 4-alkylaminopyridines with 119905-BuOK in DMSOa
Entry Starting 2 R1015840ndashX 3 yieldb
1N
HN
2a
6
Cl
N
N 6
3ac 29
2N
HN
2c
Br6N
N 6
3ac 34
3N
HN
2e
F Br6N
N 6
F
3ae 24
4N
HN
2e
F Br
N
N
F
3be 31a1mmol of 2 in 2mL of anhydrous DMSO at rt under N2 Then 15mmol of 119905-BuOK were added followed by 1mmol of halide after 20min The reaction waskept under stirring for 4 h bAll the yields are in isolated products
5 Materials and Methods
51 General Acetonitrile was distilled twice from P2O5and
CaH2 Commercially available reagents were used without
further purification The Boc protection of 4-aminopyridinewas carried out following the literature [25]
4-[N-(tert-Butoxycarbonyl)amino]pyridine N-Boc-4AP To asolution of di-tert-butyl dicarbonate (3mmol) in acetonitrile(3 cm3) at room temperature 4-aminopyridine (3mmol) wasslowly added This mixture was then allowed to stir for 3 h atroom temperatureThe solvent was evaporated and the crude4-[N-(tert-butoxycarbonyl)amino]pyridine (gt95)was usedin the electrolyses without further purification Rf (30 ethylacetate in light petroleum ether) 020 1H NMR (200MHzCDCl
52 Electrochemical ALkylation of N-Boc-4AP Constant cur-rent electrolyses (I = 25mA cmminus2) were performed undera nitrogen atmosphere at 20∘C using an Amel Model 552
potentiostat equipped with an Amel Model 731 integratorAll the experiments were carried out in a divided glasscell separated through a porous glass plug filled up witha layer of gel (ie methyl cellulose 05 volume dissolvedin DMF-Et
4NPF610mol dmminus3) Pt spirals (apparent areas
08 cm2) were used both as cathode and anode MeCN-Et4NPF601mol dmminus3 was used as solvent-supporting elec-
trolyte system (catholyte 20 cm3 anolyte 5 cm3) 1mmol ofN-Boc-4-aminopyridine was present in the catholyte After145 C were passed the current was switched off and 1mmolof alkylating agent was added to the catholyte The solutionwas kept under stirring at room temperature for 2 hours thenthe solvent was evaporated under reduced pressure and theresiduewas purified by flash column chromatography using amixture of ethyl acetatelight petroleum ether 28 in volumeobtaining the pure products
Flash column chromatography was carried out usingMerck 60 kieselgel (230ndash400 mesh) under pressure GC-MSmeasurements were carried out on SE 54 capillary columnusing a Fisons 8000 gas chromatograph coupled with aFisons MD 800 quadrupole mass selective detector 1H and13C NMR spectra were recorded at room temperature usinga Bruker AC 200 spectrometer using CDCl
3as internal
standard
6 ISRN Organic Chemistry
Table 3 Dialkylation reaction of 4-aminopyridine with 119905-BuOK in DMSOa
Entry RndashX 3 yieldb
1Br
N
N
3bb 36
2Cl
N
N
3cc 42
3Br
F N
N
F F
3ee 39
4Br
F3C N
N
CF3
3ff 65
F3C
a1mmol of 4AP in 2mL of anhydrous DMSO at rt under N2 Then 2mmol of 119905-BuOK were added followed by 2mmol of halide after 20min The reactionwas kept under stirring for 4 h bAll the yields are in isolated products
53 Deprotection of Compounds 1andashj To a solution of 1(1mmol) in CH
2Cl2(5 cm3) kept at 0∘C 1 cm3 of CF
3COOH
was addedThismixture was allowed to stir for 3 h at 0∘CThesolution was then mixed with aqueous sodium carbonatetill pH 8 and extracted with ethyl acetate The solvent
8 ISRN Organic Chemistry
was removed under reduced pressure and the mixture waspurified by flash chromatography yielding pure compound 2
54 Alkylation of Compounds 2ace To a solution of 2(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
15mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 1mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
55 Dialkylation of 4-Aminopyridine To a solution of 4AP(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
2mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 2mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
Organisms For the antifungal evaluation strains obtainedfrom the American Type Culture Collection (ATCC Rock-ville MD USA) the German Collection of Microorganisms(DSMZ Braunschweig Germany) and the PharmaceuticalMicrobiology Culture Collection (PMC Department ofPublic Health and Infectious Diseases ldquoSapienzardquo UniversityRome Italy) were tested The strains were Candida albicans(ATCC 10231 ATCC 10261 ATCC 24433 ATCC 900283153 PMC 1002 PMC 1011 and PMC 1030) C parapsilosisATCC22019 C parapsilosis DSM 11224 C tropicalis DSM11953 C tropicalis PMC 0908 C tropicalis PMC 0910 Cglabrata PMC 0805 C krusei DSM 6128 and C kruseiPMC 0613 Cryptococcus neoformans (DSM 11959 PMC2102 PMC 2107 PMC 2111 and PMC 2136) dermatophytes(Trichophyton mentagrophytes DSM 4870 T mentagrophytesPMC6509 Microsporum gypseum DSM 7303 and Mgypseum PMC 7331) All of the strains were stored andgrown in accordance with the procedures of the Clinical andLaboratory Standards Institute (CLSI) [26 27]
Antifungal Susceptibility Assays In vitro antifungal suscep-tibility was evaluated using the CLSI broth microdilutionmethods [26 27] Fluconazole and Amphotericin B wereused as reference drugs The final concentration rangedfrom 0125 to 64 120583gmL The compounds were dissolvedpreviously in DMSO at concentrations 100 times higherthan the highest desired test concentration and successivelydiluted in test medium in accordance with the proceduresof the CLSI [28] Microdilution trays containing 100 120583L ofserial twofold dilutions of compounds in RPMI 1640medium(Sigma-Aldrich St Louis MO USA) were inoculated withan organism suspension adjusted to attain a final inoculumconcentration of 10 times 103ndash15 times 103 cellsmL for yeasts and04 times 10
4ndash5 times 104 CFUmL for dermatophytes The panelswere incubated at 35∘C and observed for the presence of
growth at 48 h (Candida spp) and 72 h (C neoformans anddermatophytes)
The minimal inhibitory concentration (MIC) was foryeasts the lowest concentration that showed ge 50 growthinhibition compared with the growth control and for der-matophytes the lowest concentration that showed ge 80growth inhibition compared with the growth control TheMIC100
was the lowest drug concentration that prevented100 of growth with respect to the untreated controlAccording to CSI protocols the fluconazole MIC
50and the
amphotericin B MIC100
were calculated (2223) The resultswere expressed as the geometric mean (GM) of the MICvalues
562 Antiprotozoal Assay For the evaluation of antiproto-zoal and cytotoxic activity an integrated panel of microbialscreens and standard screening methodologies were adoptedas previously described [29] on the following organismschloroquine-resistant P falciparum K 1-strain L infantumMHOMMA (BE)67 amastigote stage suramin-sensitiveTrypanosoma brucei Squib-427 strain Trypanosoma cruziTulahuen CL2 (benznidazole-sensitive) strain human fetallung fibroblast cells (MRC-5 SV2)
All assays were performed in triplicate Compounds weretested at 5 concentrations (64 16 4 1 and 025120583gmL)to establish a full dose titration and determine the IC
50
(inhibitory concentration 50) The final in-test concentra-tion of DMSO did not exceed 05 which is known not tointerfere with the different assays [29]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was financially supported by Miur Italy Theauthors thank Mr Marco Di Pilato for his support in theexperimental part of the work
References
[1] G R Luedtke K Schinzel X Tan et al ldquoAmide-basedinhibitors of p38120572 MAP kinase Part 1 Discovery of novelN-pyridyl amide lead moleculesrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 8 pp 2556ndash2559 2010
[2] M Otsuka M Fujita Y Sugiura et al ldquoSynthetic inhibitors ofregulatory proteins involved in the signaling pathway of thereplication of human immunodeficiency virus 1rdquo Bioorganicand Medicinal Chemistry vol 5 no 1 pp 205ndash215 1997
[3] H Nishida Y Miyazaki T Mukaihira et al ldquoSynthesis andevaluation of 1-arylsulfonyl-3-piperazinone derivatives as afactor Xa inhibitor II Substituent effect on biological activitiesrdquoChemical and Pharmaceutical Bulletin vol 50 no 9 pp 1187ndash1194 2002
[4] C A Willoughby K G Rosauer J J Hale et al ldquo134Trisubstituted pyrrolidine CCR5 receptor antagonists bearing
10 ISRN Organic Chemistry
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006
Table 2 Alkylation reaction of 4-alkylaminopyridines with 119905-BuOK in DMSOa
Entry Starting 2 R1015840ndashX 3 yieldb
1N
HN
2a
6
Cl
N
N 6
3ac 29
2N
HN
2c
Br6N
N 6
3ac 34
3N
HN
2e
F Br6N
N 6
F
3ae 24
4N
HN
2e
F Br
N
N
F
3be 31a1mmol of 2 in 2mL of anhydrous DMSO at rt under N2 Then 15mmol of 119905-BuOK were added followed by 1mmol of halide after 20min The reaction waskept under stirring for 4 h bAll the yields are in isolated products
5 Materials and Methods
51 General Acetonitrile was distilled twice from P2O5and
CaH2 Commercially available reagents were used without
further purification The Boc protection of 4-aminopyridinewas carried out following the literature [25]
4-[N-(tert-Butoxycarbonyl)amino]pyridine N-Boc-4AP To asolution of di-tert-butyl dicarbonate (3mmol) in acetonitrile(3 cm3) at room temperature 4-aminopyridine (3mmol) wasslowly added This mixture was then allowed to stir for 3 h atroom temperatureThe solvent was evaporated and the crude4-[N-(tert-butoxycarbonyl)amino]pyridine (gt95)was usedin the electrolyses without further purification Rf (30 ethylacetate in light petroleum ether) 020 1H NMR (200MHzCDCl
52 Electrochemical ALkylation of N-Boc-4AP Constant cur-rent electrolyses (I = 25mA cmminus2) were performed undera nitrogen atmosphere at 20∘C using an Amel Model 552
potentiostat equipped with an Amel Model 731 integratorAll the experiments were carried out in a divided glasscell separated through a porous glass plug filled up witha layer of gel (ie methyl cellulose 05 volume dissolvedin DMF-Et
4NPF610mol dmminus3) Pt spirals (apparent areas
08 cm2) were used both as cathode and anode MeCN-Et4NPF601mol dmminus3 was used as solvent-supporting elec-
trolyte system (catholyte 20 cm3 anolyte 5 cm3) 1mmol ofN-Boc-4-aminopyridine was present in the catholyte After145 C were passed the current was switched off and 1mmolof alkylating agent was added to the catholyte The solutionwas kept under stirring at room temperature for 2 hours thenthe solvent was evaporated under reduced pressure and theresiduewas purified by flash column chromatography using amixture of ethyl acetatelight petroleum ether 28 in volumeobtaining the pure products
Flash column chromatography was carried out usingMerck 60 kieselgel (230ndash400 mesh) under pressure GC-MSmeasurements were carried out on SE 54 capillary columnusing a Fisons 8000 gas chromatograph coupled with aFisons MD 800 quadrupole mass selective detector 1H and13C NMR spectra were recorded at room temperature usinga Bruker AC 200 spectrometer using CDCl
3as internal
standard
6 ISRN Organic Chemistry
Table 3 Dialkylation reaction of 4-aminopyridine with 119905-BuOK in DMSOa
Entry RndashX 3 yieldb
1Br
N
N
3bb 36
2Cl
N
N
3cc 42
3Br
F N
N
F F
3ee 39
4Br
F3C N
N
CF3
3ff 65
F3C
a1mmol of 4AP in 2mL of anhydrous DMSO at rt under N2 Then 2mmol of 119905-BuOK were added followed by 2mmol of halide after 20min The reactionwas kept under stirring for 4 h bAll the yields are in isolated products
53 Deprotection of Compounds 1andashj To a solution of 1(1mmol) in CH
2Cl2(5 cm3) kept at 0∘C 1 cm3 of CF
3COOH
was addedThismixture was allowed to stir for 3 h at 0∘CThesolution was then mixed with aqueous sodium carbonatetill pH 8 and extracted with ethyl acetate The solvent
8 ISRN Organic Chemistry
was removed under reduced pressure and the mixture waspurified by flash chromatography yielding pure compound 2
54 Alkylation of Compounds 2ace To a solution of 2(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
15mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 1mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
55 Dialkylation of 4-Aminopyridine To a solution of 4AP(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
2mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 2mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
Organisms For the antifungal evaluation strains obtainedfrom the American Type Culture Collection (ATCC Rock-ville MD USA) the German Collection of Microorganisms(DSMZ Braunschweig Germany) and the PharmaceuticalMicrobiology Culture Collection (PMC Department ofPublic Health and Infectious Diseases ldquoSapienzardquo UniversityRome Italy) were tested The strains were Candida albicans(ATCC 10231 ATCC 10261 ATCC 24433 ATCC 900283153 PMC 1002 PMC 1011 and PMC 1030) C parapsilosisATCC22019 C parapsilosis DSM 11224 C tropicalis DSM11953 C tropicalis PMC 0908 C tropicalis PMC 0910 Cglabrata PMC 0805 C krusei DSM 6128 and C kruseiPMC 0613 Cryptococcus neoformans (DSM 11959 PMC2102 PMC 2107 PMC 2111 and PMC 2136) dermatophytes(Trichophyton mentagrophytes DSM 4870 T mentagrophytesPMC6509 Microsporum gypseum DSM 7303 and Mgypseum PMC 7331) All of the strains were stored andgrown in accordance with the procedures of the Clinical andLaboratory Standards Institute (CLSI) [26 27]
Antifungal Susceptibility Assays In vitro antifungal suscep-tibility was evaluated using the CLSI broth microdilutionmethods [26 27] Fluconazole and Amphotericin B wereused as reference drugs The final concentration rangedfrom 0125 to 64 120583gmL The compounds were dissolvedpreviously in DMSO at concentrations 100 times higherthan the highest desired test concentration and successivelydiluted in test medium in accordance with the proceduresof the CLSI [28] Microdilution trays containing 100 120583L ofserial twofold dilutions of compounds in RPMI 1640medium(Sigma-Aldrich St Louis MO USA) were inoculated withan organism suspension adjusted to attain a final inoculumconcentration of 10 times 103ndash15 times 103 cellsmL for yeasts and04 times 10
4ndash5 times 104 CFUmL for dermatophytes The panelswere incubated at 35∘C and observed for the presence of
growth at 48 h (Candida spp) and 72 h (C neoformans anddermatophytes)
The minimal inhibitory concentration (MIC) was foryeasts the lowest concentration that showed ge 50 growthinhibition compared with the growth control and for der-matophytes the lowest concentration that showed ge 80growth inhibition compared with the growth control TheMIC100
was the lowest drug concentration that prevented100 of growth with respect to the untreated controlAccording to CSI protocols the fluconazole MIC
50and the
amphotericin B MIC100
were calculated (2223) The resultswere expressed as the geometric mean (GM) of the MICvalues
562 Antiprotozoal Assay For the evaluation of antiproto-zoal and cytotoxic activity an integrated panel of microbialscreens and standard screening methodologies were adoptedas previously described [29] on the following organismschloroquine-resistant P falciparum K 1-strain L infantumMHOMMA (BE)67 amastigote stage suramin-sensitiveTrypanosoma brucei Squib-427 strain Trypanosoma cruziTulahuen CL2 (benznidazole-sensitive) strain human fetallung fibroblast cells (MRC-5 SV2)
All assays were performed in triplicate Compounds weretested at 5 concentrations (64 16 4 1 and 025120583gmL)to establish a full dose titration and determine the IC
50
(inhibitory concentration 50) The final in-test concentra-tion of DMSO did not exceed 05 which is known not tointerfere with the different assays [29]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was financially supported by Miur Italy Theauthors thank Mr Marco Di Pilato for his support in theexperimental part of the work
References
[1] G R Luedtke K Schinzel X Tan et al ldquoAmide-basedinhibitors of p38120572 MAP kinase Part 1 Discovery of novelN-pyridyl amide lead moleculesrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 8 pp 2556ndash2559 2010
[2] M Otsuka M Fujita Y Sugiura et al ldquoSynthetic inhibitors ofregulatory proteins involved in the signaling pathway of thereplication of human immunodeficiency virus 1rdquo Bioorganicand Medicinal Chemistry vol 5 no 1 pp 205ndash215 1997
[3] H Nishida Y Miyazaki T Mukaihira et al ldquoSynthesis andevaluation of 1-arylsulfonyl-3-piperazinone derivatives as afactor Xa inhibitor II Substituent effect on biological activitiesrdquoChemical and Pharmaceutical Bulletin vol 50 no 9 pp 1187ndash1194 2002
[4] C A Willoughby K G Rosauer J J Hale et al ldquo134Trisubstituted pyrrolidine CCR5 receptor antagonists bearing
10 ISRN Organic Chemistry
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006
Table 3 Dialkylation reaction of 4-aminopyridine with 119905-BuOK in DMSOa
Entry RndashX 3 yieldb
1Br
N
N
3bb 36
2Cl
N
N
3cc 42
3Br
F N
N
F F
3ee 39
4Br
F3C N
N
CF3
3ff 65
F3C
a1mmol of 4AP in 2mL of anhydrous DMSO at rt under N2 Then 2mmol of 119905-BuOK were added followed by 2mmol of halide after 20min The reactionwas kept under stirring for 4 h bAll the yields are in isolated products
53 Deprotection of Compounds 1andashj To a solution of 1(1mmol) in CH
2Cl2(5 cm3) kept at 0∘C 1 cm3 of CF
3COOH
was addedThismixture was allowed to stir for 3 h at 0∘CThesolution was then mixed with aqueous sodium carbonatetill pH 8 and extracted with ethyl acetate The solvent
8 ISRN Organic Chemistry
was removed under reduced pressure and the mixture waspurified by flash chromatography yielding pure compound 2
54 Alkylation of Compounds 2ace To a solution of 2(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
15mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 1mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
55 Dialkylation of 4-Aminopyridine To a solution of 4AP(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
2mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 2mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
Organisms For the antifungal evaluation strains obtainedfrom the American Type Culture Collection (ATCC Rock-ville MD USA) the German Collection of Microorganisms(DSMZ Braunschweig Germany) and the PharmaceuticalMicrobiology Culture Collection (PMC Department ofPublic Health and Infectious Diseases ldquoSapienzardquo UniversityRome Italy) were tested The strains were Candida albicans(ATCC 10231 ATCC 10261 ATCC 24433 ATCC 900283153 PMC 1002 PMC 1011 and PMC 1030) C parapsilosisATCC22019 C parapsilosis DSM 11224 C tropicalis DSM11953 C tropicalis PMC 0908 C tropicalis PMC 0910 Cglabrata PMC 0805 C krusei DSM 6128 and C kruseiPMC 0613 Cryptococcus neoformans (DSM 11959 PMC2102 PMC 2107 PMC 2111 and PMC 2136) dermatophytes(Trichophyton mentagrophytes DSM 4870 T mentagrophytesPMC6509 Microsporum gypseum DSM 7303 and Mgypseum PMC 7331) All of the strains were stored andgrown in accordance with the procedures of the Clinical andLaboratory Standards Institute (CLSI) [26 27]
Antifungal Susceptibility Assays In vitro antifungal suscep-tibility was evaluated using the CLSI broth microdilutionmethods [26 27] Fluconazole and Amphotericin B wereused as reference drugs The final concentration rangedfrom 0125 to 64 120583gmL The compounds were dissolvedpreviously in DMSO at concentrations 100 times higherthan the highest desired test concentration and successivelydiluted in test medium in accordance with the proceduresof the CLSI [28] Microdilution trays containing 100 120583L ofserial twofold dilutions of compounds in RPMI 1640medium(Sigma-Aldrich St Louis MO USA) were inoculated withan organism suspension adjusted to attain a final inoculumconcentration of 10 times 103ndash15 times 103 cellsmL for yeasts and04 times 10
4ndash5 times 104 CFUmL for dermatophytes The panelswere incubated at 35∘C and observed for the presence of
growth at 48 h (Candida spp) and 72 h (C neoformans anddermatophytes)
The minimal inhibitory concentration (MIC) was foryeasts the lowest concentration that showed ge 50 growthinhibition compared with the growth control and for der-matophytes the lowest concentration that showed ge 80growth inhibition compared with the growth control TheMIC100
was the lowest drug concentration that prevented100 of growth with respect to the untreated controlAccording to CSI protocols the fluconazole MIC
50and the
amphotericin B MIC100
were calculated (2223) The resultswere expressed as the geometric mean (GM) of the MICvalues
562 Antiprotozoal Assay For the evaluation of antiproto-zoal and cytotoxic activity an integrated panel of microbialscreens and standard screening methodologies were adoptedas previously described [29] on the following organismschloroquine-resistant P falciparum K 1-strain L infantumMHOMMA (BE)67 amastigote stage suramin-sensitiveTrypanosoma brucei Squib-427 strain Trypanosoma cruziTulahuen CL2 (benznidazole-sensitive) strain human fetallung fibroblast cells (MRC-5 SV2)
All assays were performed in triplicate Compounds weretested at 5 concentrations (64 16 4 1 and 025120583gmL)to establish a full dose titration and determine the IC
50
(inhibitory concentration 50) The final in-test concentra-tion of DMSO did not exceed 05 which is known not tointerfere with the different assays [29]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was financially supported by Miur Italy Theauthors thank Mr Marco Di Pilato for his support in theexperimental part of the work
References
[1] G R Luedtke K Schinzel X Tan et al ldquoAmide-basedinhibitors of p38120572 MAP kinase Part 1 Discovery of novelN-pyridyl amide lead moleculesrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 8 pp 2556ndash2559 2010
[2] M Otsuka M Fujita Y Sugiura et al ldquoSynthetic inhibitors ofregulatory proteins involved in the signaling pathway of thereplication of human immunodeficiency virus 1rdquo Bioorganicand Medicinal Chemistry vol 5 no 1 pp 205ndash215 1997
[3] H Nishida Y Miyazaki T Mukaihira et al ldquoSynthesis andevaluation of 1-arylsulfonyl-3-piperazinone derivatives as afactor Xa inhibitor II Substituent effect on biological activitiesrdquoChemical and Pharmaceutical Bulletin vol 50 no 9 pp 1187ndash1194 2002
[4] C A Willoughby K G Rosauer J J Hale et al ldquo134Trisubstituted pyrrolidine CCR5 receptor antagonists bearing
10 ISRN Organic Chemistry
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006
53 Deprotection of Compounds 1andashj To a solution of 1(1mmol) in CH
2Cl2(5 cm3) kept at 0∘C 1 cm3 of CF
3COOH
was addedThismixture was allowed to stir for 3 h at 0∘CThesolution was then mixed with aqueous sodium carbonatetill pH 8 and extracted with ethyl acetate The solvent
8 ISRN Organic Chemistry
was removed under reduced pressure and the mixture waspurified by flash chromatography yielding pure compound 2
54 Alkylation of Compounds 2ace To a solution of 2(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
15mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 1mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
55 Dialkylation of 4-Aminopyridine To a solution of 4AP(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
2mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 2mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
Organisms For the antifungal evaluation strains obtainedfrom the American Type Culture Collection (ATCC Rock-ville MD USA) the German Collection of Microorganisms(DSMZ Braunschweig Germany) and the PharmaceuticalMicrobiology Culture Collection (PMC Department ofPublic Health and Infectious Diseases ldquoSapienzardquo UniversityRome Italy) were tested The strains were Candida albicans(ATCC 10231 ATCC 10261 ATCC 24433 ATCC 900283153 PMC 1002 PMC 1011 and PMC 1030) C parapsilosisATCC22019 C parapsilosis DSM 11224 C tropicalis DSM11953 C tropicalis PMC 0908 C tropicalis PMC 0910 Cglabrata PMC 0805 C krusei DSM 6128 and C kruseiPMC 0613 Cryptococcus neoformans (DSM 11959 PMC2102 PMC 2107 PMC 2111 and PMC 2136) dermatophytes(Trichophyton mentagrophytes DSM 4870 T mentagrophytesPMC6509 Microsporum gypseum DSM 7303 and Mgypseum PMC 7331) All of the strains were stored andgrown in accordance with the procedures of the Clinical andLaboratory Standards Institute (CLSI) [26 27]
Antifungal Susceptibility Assays In vitro antifungal suscep-tibility was evaluated using the CLSI broth microdilutionmethods [26 27] Fluconazole and Amphotericin B wereused as reference drugs The final concentration rangedfrom 0125 to 64 120583gmL The compounds were dissolvedpreviously in DMSO at concentrations 100 times higherthan the highest desired test concentration and successivelydiluted in test medium in accordance with the proceduresof the CLSI [28] Microdilution trays containing 100 120583L ofserial twofold dilutions of compounds in RPMI 1640medium(Sigma-Aldrich St Louis MO USA) were inoculated withan organism suspension adjusted to attain a final inoculumconcentration of 10 times 103ndash15 times 103 cellsmL for yeasts and04 times 10
4ndash5 times 104 CFUmL for dermatophytes The panelswere incubated at 35∘C and observed for the presence of
growth at 48 h (Candida spp) and 72 h (C neoformans anddermatophytes)
The minimal inhibitory concentration (MIC) was foryeasts the lowest concentration that showed ge 50 growthinhibition compared with the growth control and for der-matophytes the lowest concentration that showed ge 80growth inhibition compared with the growth control TheMIC100
was the lowest drug concentration that prevented100 of growth with respect to the untreated controlAccording to CSI protocols the fluconazole MIC
50and the
amphotericin B MIC100
were calculated (2223) The resultswere expressed as the geometric mean (GM) of the MICvalues
562 Antiprotozoal Assay For the evaluation of antiproto-zoal and cytotoxic activity an integrated panel of microbialscreens and standard screening methodologies were adoptedas previously described [29] on the following organismschloroquine-resistant P falciparum K 1-strain L infantumMHOMMA (BE)67 amastigote stage suramin-sensitiveTrypanosoma brucei Squib-427 strain Trypanosoma cruziTulahuen CL2 (benznidazole-sensitive) strain human fetallung fibroblast cells (MRC-5 SV2)
All assays were performed in triplicate Compounds weretested at 5 concentrations (64 16 4 1 and 025120583gmL)to establish a full dose titration and determine the IC
50
(inhibitory concentration 50) The final in-test concentra-tion of DMSO did not exceed 05 which is known not tointerfere with the different assays [29]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was financially supported by Miur Italy Theauthors thank Mr Marco Di Pilato for his support in theexperimental part of the work
References
[1] G R Luedtke K Schinzel X Tan et al ldquoAmide-basedinhibitors of p38120572 MAP kinase Part 1 Discovery of novelN-pyridyl amide lead moleculesrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 8 pp 2556ndash2559 2010
[2] M Otsuka M Fujita Y Sugiura et al ldquoSynthetic inhibitors ofregulatory proteins involved in the signaling pathway of thereplication of human immunodeficiency virus 1rdquo Bioorganicand Medicinal Chemistry vol 5 no 1 pp 205ndash215 1997
[3] H Nishida Y Miyazaki T Mukaihira et al ldquoSynthesis andevaluation of 1-arylsulfonyl-3-piperazinone derivatives as afactor Xa inhibitor II Substituent effect on biological activitiesrdquoChemical and Pharmaceutical Bulletin vol 50 no 9 pp 1187ndash1194 2002
[4] C A Willoughby K G Rosauer J J Hale et al ldquo134Trisubstituted pyrrolidine CCR5 receptor antagonists bearing
10 ISRN Organic Chemistry
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006
54 Alkylation of Compounds 2ace To a solution of 2(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
15mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 1mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
55 Dialkylation of 4-Aminopyridine To a solution of 4AP(1mmol) in anhydrous DMSO (2 cm3) kept at rt under N
2
2mmol of t-BuOK was added This mixture was allowedto stir for 20min at rt then 2mmol of alkyl halide wasadded and the solution was kept under stirring at rt for4 h The solution was then mixed with water and extractedwith dichloromethane The solvent was removed underreduced pressure and the mixture was purified by flashchromatography yielding pure compound 3
Organisms For the antifungal evaluation strains obtainedfrom the American Type Culture Collection (ATCC Rock-ville MD USA) the German Collection of Microorganisms(DSMZ Braunschweig Germany) and the PharmaceuticalMicrobiology Culture Collection (PMC Department ofPublic Health and Infectious Diseases ldquoSapienzardquo UniversityRome Italy) were tested The strains were Candida albicans(ATCC 10231 ATCC 10261 ATCC 24433 ATCC 900283153 PMC 1002 PMC 1011 and PMC 1030) C parapsilosisATCC22019 C parapsilosis DSM 11224 C tropicalis DSM11953 C tropicalis PMC 0908 C tropicalis PMC 0910 Cglabrata PMC 0805 C krusei DSM 6128 and C kruseiPMC 0613 Cryptococcus neoformans (DSM 11959 PMC2102 PMC 2107 PMC 2111 and PMC 2136) dermatophytes(Trichophyton mentagrophytes DSM 4870 T mentagrophytesPMC6509 Microsporum gypseum DSM 7303 and Mgypseum PMC 7331) All of the strains were stored andgrown in accordance with the procedures of the Clinical andLaboratory Standards Institute (CLSI) [26 27]
Antifungal Susceptibility Assays In vitro antifungal suscep-tibility was evaluated using the CLSI broth microdilutionmethods [26 27] Fluconazole and Amphotericin B wereused as reference drugs The final concentration rangedfrom 0125 to 64 120583gmL The compounds were dissolvedpreviously in DMSO at concentrations 100 times higherthan the highest desired test concentration and successivelydiluted in test medium in accordance with the proceduresof the CLSI [28] Microdilution trays containing 100 120583L ofserial twofold dilutions of compounds in RPMI 1640medium(Sigma-Aldrich St Louis MO USA) were inoculated withan organism suspension adjusted to attain a final inoculumconcentration of 10 times 103ndash15 times 103 cellsmL for yeasts and04 times 10
4ndash5 times 104 CFUmL for dermatophytes The panelswere incubated at 35∘C and observed for the presence of
growth at 48 h (Candida spp) and 72 h (C neoformans anddermatophytes)
The minimal inhibitory concentration (MIC) was foryeasts the lowest concentration that showed ge 50 growthinhibition compared with the growth control and for der-matophytes the lowest concentration that showed ge 80growth inhibition compared with the growth control TheMIC100
was the lowest drug concentration that prevented100 of growth with respect to the untreated controlAccording to CSI protocols the fluconazole MIC
50and the
amphotericin B MIC100
were calculated (2223) The resultswere expressed as the geometric mean (GM) of the MICvalues
562 Antiprotozoal Assay For the evaluation of antiproto-zoal and cytotoxic activity an integrated panel of microbialscreens and standard screening methodologies were adoptedas previously described [29] on the following organismschloroquine-resistant P falciparum K 1-strain L infantumMHOMMA (BE)67 amastigote stage suramin-sensitiveTrypanosoma brucei Squib-427 strain Trypanosoma cruziTulahuen CL2 (benznidazole-sensitive) strain human fetallung fibroblast cells (MRC-5 SV2)
All assays were performed in triplicate Compounds weretested at 5 concentrations (64 16 4 1 and 025120583gmL)to establish a full dose titration and determine the IC
50
(inhibitory concentration 50) The final in-test concentra-tion of DMSO did not exceed 05 which is known not tointerfere with the different assays [29]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was financially supported by Miur Italy Theauthors thank Mr Marco Di Pilato for his support in theexperimental part of the work
References
[1] G R Luedtke K Schinzel X Tan et al ldquoAmide-basedinhibitors of p38120572 MAP kinase Part 1 Discovery of novelN-pyridyl amide lead moleculesrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 8 pp 2556ndash2559 2010
[2] M Otsuka M Fujita Y Sugiura et al ldquoSynthetic inhibitors ofregulatory proteins involved in the signaling pathway of thereplication of human immunodeficiency virus 1rdquo Bioorganicand Medicinal Chemistry vol 5 no 1 pp 205ndash215 1997
[3] H Nishida Y Miyazaki T Mukaihira et al ldquoSynthesis andevaluation of 1-arylsulfonyl-3-piperazinone derivatives as afactor Xa inhibitor II Substituent effect on biological activitiesrdquoChemical and Pharmaceutical Bulletin vol 50 no 9 pp 1187ndash1194 2002
[4] C A Willoughby K G Rosauer J J Hale et al ldquo134Trisubstituted pyrrolidine CCR5 receptor antagonists bearing
10 ISRN Organic Chemistry
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006
Organisms For the antifungal evaluation strains obtainedfrom the American Type Culture Collection (ATCC Rock-ville MD USA) the German Collection of Microorganisms(DSMZ Braunschweig Germany) and the PharmaceuticalMicrobiology Culture Collection (PMC Department ofPublic Health and Infectious Diseases ldquoSapienzardquo UniversityRome Italy) were tested The strains were Candida albicans(ATCC 10231 ATCC 10261 ATCC 24433 ATCC 900283153 PMC 1002 PMC 1011 and PMC 1030) C parapsilosisATCC22019 C parapsilosis DSM 11224 C tropicalis DSM11953 C tropicalis PMC 0908 C tropicalis PMC 0910 Cglabrata PMC 0805 C krusei DSM 6128 and C kruseiPMC 0613 Cryptococcus neoformans (DSM 11959 PMC2102 PMC 2107 PMC 2111 and PMC 2136) dermatophytes(Trichophyton mentagrophytes DSM 4870 T mentagrophytesPMC6509 Microsporum gypseum DSM 7303 and Mgypseum PMC 7331) All of the strains were stored andgrown in accordance with the procedures of the Clinical andLaboratory Standards Institute (CLSI) [26 27]
Antifungal Susceptibility Assays In vitro antifungal suscep-tibility was evaluated using the CLSI broth microdilutionmethods [26 27] Fluconazole and Amphotericin B wereused as reference drugs The final concentration rangedfrom 0125 to 64 120583gmL The compounds were dissolvedpreviously in DMSO at concentrations 100 times higherthan the highest desired test concentration and successivelydiluted in test medium in accordance with the proceduresof the CLSI [28] Microdilution trays containing 100 120583L ofserial twofold dilutions of compounds in RPMI 1640medium(Sigma-Aldrich St Louis MO USA) were inoculated withan organism suspension adjusted to attain a final inoculumconcentration of 10 times 103ndash15 times 103 cellsmL for yeasts and04 times 10
4ndash5 times 104 CFUmL for dermatophytes The panelswere incubated at 35∘C and observed for the presence of
growth at 48 h (Candida spp) and 72 h (C neoformans anddermatophytes)
The minimal inhibitory concentration (MIC) was foryeasts the lowest concentration that showed ge 50 growthinhibition compared with the growth control and for der-matophytes the lowest concentration that showed ge 80growth inhibition compared with the growth control TheMIC100
was the lowest drug concentration that prevented100 of growth with respect to the untreated controlAccording to CSI protocols the fluconazole MIC
50and the
amphotericin B MIC100
were calculated (2223) The resultswere expressed as the geometric mean (GM) of the MICvalues
562 Antiprotozoal Assay For the evaluation of antiproto-zoal and cytotoxic activity an integrated panel of microbialscreens and standard screening methodologies were adoptedas previously described [29] on the following organismschloroquine-resistant P falciparum K 1-strain L infantumMHOMMA (BE)67 amastigote stage suramin-sensitiveTrypanosoma brucei Squib-427 strain Trypanosoma cruziTulahuen CL2 (benznidazole-sensitive) strain human fetallung fibroblast cells (MRC-5 SV2)
All assays were performed in triplicate Compounds weretested at 5 concentrations (64 16 4 1 and 025120583gmL)to establish a full dose titration and determine the IC
50
(inhibitory concentration 50) The final in-test concentra-tion of DMSO did not exceed 05 which is known not tointerfere with the different assays [29]
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
This work was financially supported by Miur Italy Theauthors thank Mr Marco Di Pilato for his support in theexperimental part of the work
References
[1] G R Luedtke K Schinzel X Tan et al ldquoAmide-basedinhibitors of p38120572 MAP kinase Part 1 Discovery of novelN-pyridyl amide lead moleculesrdquo Bioorganic and MedicinalChemistry Letters vol 20 no 8 pp 2556ndash2559 2010
[2] M Otsuka M Fujita Y Sugiura et al ldquoSynthetic inhibitors ofregulatory proteins involved in the signaling pathway of thereplication of human immunodeficiency virus 1rdquo Bioorganicand Medicinal Chemistry vol 5 no 1 pp 205ndash215 1997
[3] H Nishida Y Miyazaki T Mukaihira et al ldquoSynthesis andevaluation of 1-arylsulfonyl-3-piperazinone derivatives as afactor Xa inhibitor II Substituent effect on biological activitiesrdquoChemical and Pharmaceutical Bulletin vol 50 no 9 pp 1187ndash1194 2002
[4] C A Willoughby K G Rosauer J J Hale et al ldquo134Trisubstituted pyrrolidine CCR5 receptor antagonists bearing
10 ISRN Organic Chemistry
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006
4-aminoheterocycle substituted piperidine side chainsrdquo Bioor-ganic andMedicinal Chemistry Letters vol 13 no 3 pp 427ndash4312003
[5] D de Vita L Scipione S Tortorella et al ldquoSynthesis andantifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivativesrdquo European Journal of MedicinalChemistry vol 49 pp 334ndash342 2012
[6] L Friggeri L Scipione R Costi et al ldquoNew promising com-pounds with in vitro nanomolar activity against TrypanosomaCruzirdquoMedicinal Chemistry Letters vol 4 pp 538ndash541 2013
[7] C K Chen P S Doyle L V Yermalitskaya et al ldquoTrypanosomaCruzi CYP51 inhibitor derived from a Mycobacterium tubercu-losis screen hitrdquo PLoS Neglected Tropical Diseases vol 3 no 2article e372 2009
[8] T Y Hargrove Z Wawrzak P W Alexander et al ldquoCom-plexes of Trypanosoma Cruzi Sterol 14120572-Demethylase (CYP51)with two pyridine-based drug candidates for chagas diseasestructural basis for pathogen selectivityrdquo Journal of BiologicalChemistry vol 288 pp 31602ndash31615 2013
[9] Q Xu Q Li X Zhu and J Chen ldquoGreen and scalable aldehyde-catalyzed transition metal-freeDehydrative N-Alkylation ofamides and amines with alcoholsrdquo Advanced Synthesis andCatalysis vol 355 pp 73ndash80 2013
[10] Q Li S Fan Q Sun H Tian X Yu and Q Xu ldquoCopper-catalyzed N-alkylation of amides and amines with alcoholsemploying the aerobic relay race methodologyrdquo Organic andBiomolecular Chemistry vol 10 no 15 pp 2966ndash2972 2012
[11] A Martınez-Asencio D J Ramon and M Yus ldquoN-Alkylationof poor nucleophilic amines and derivatives with alcohols by ahydrogen autotransfer process catalyzed by copper(II) acetatescope andmechanistic considerationsrdquo Tetrahedron vol 67 no17 pp 3140ndash3149 2011
[12] R Martınez D J Ramon and M Yus ldquoSelective N-monoalkylation of aromatic amines with benzylic alcoholsby a hydrogen autotransfer process catalyzed by unmodifiedmagnetiterdquo Organic and Biomolecular Chemistry vol 7 no 10pp 2176ndash2181 2009
[13] E J Delaney L E Wood and I M Klotz ldquoPoly(ethylenimines)with alternative (alkylamino)pyridines as nucleophilic cata-lystsrdquo Journal of the American Chemical Society vol 104 no 3pp 799ndash807 1982
[14] T Zhao and G Sun ldquoSynthesis and characterization of antimi-crobial cationic surfactants aminopyridinium saltsrdquo Journal ofSurfactants and Detergents vol 9 no 4 pp 325ndash330 2006
[15] T Ito T Ikemoto Y Isogami et al ldquoPractical synthesis of low-density lipoprotein receptor upregulator N-[1-(3-phenylpro-pane-1-yl)piperidin-4-yl]-5-thia-18b-diazaacenaphthylene-4-carboxamiderdquo Organic Process Research and Development vol6 no 3 pp 238ndash241 2002
[16] O M Singh S J Singh N K Su and S-G Lee ldquoReaction oflithioamines with alkyl halides a convenient direct synthesisof N-alkylaminopyridinesrdquo Bulletin of the Korean ChemicalSociety vol 28 no 1 pp 115ndash117 2007
[17] D M Krein and T L Lowary ldquoA convenient synthesis of 2-(alkylamino)pyridinesrdquo Journal of Organic Chemistry vol 67no 14 pp 4965ndash4967 2002
[18] The direct cathodic reduction of N-Boc-4AP led to its depro-tection and to the formation of 4AP
[19] L Rossi M Feroci and A Inesi ldquoThe electrogeneratedcyanomethyl anion in organic synthesisrdquo Mini-Reviews inOrganic Chemistry vol 2 no 1 pp 79ndash90 2005
[20] M Feroci D de Vita L Scipione G Sotgiu and S Tor-torella ldquoElectrogenerated acetonitrile anion induced selectiveN-alkylation of bifunctional compoundsrdquo Tetrahedron Lettersvol 53 no 20 pp 2564ndash2567 2012
[21] M Feroci I Chiarotto L Rossi and A Inesi ldquoActivationof elemental sulfur by electrogenerated cyanomethyl anionsynthesis of substituted 2-aminothiophenes by the Gewaldreactionrdquo Advanced Synthesis and Catalysis vol 350 no 17 pp2740ndash2746 2008
[22] M Feroci ldquoSynthesis of 120573-lactams by 4-exo-tet cyclizationprocess induced by electrogenerated cyanomethyl anion part 2Stereochemical implicationsrdquoAdvanced Synthesis and Catalysisvol 349 no 13 pp 2177ndash2181 2007
[23] M Feroci M A Casadei M Orsini L Palombi and A InesildquoCyanomethyl anioncarbon dioxide system an electrogener-ated carboxylating reagent Synthesis of carbamates under mildand safe conditionsrdquo Journal of Organic Chemistry vol 68 no4 pp 1548ndash1551 2003
[24] M Feroci M Orsini G Sotgiu L Rossi and A Inesi ldquoElec-trochemically promoted C-N bond formation from acetylenicamines and CO
2 Synthesis of 5-methylene-13-oxazolidin-2-
onesrdquo Journal of Organic Chemistry vol 70 no 19 pp 7795ndash7798 2005
[25] Y Basel and A Hassner ldquoDi-tert-butyl dicarbonate and4-(dimethylamino)pyridine revisited Their reactions withamines and alcoholsrdquo Journal of Organic Chemistry vol 65 no20 pp 6368ndash6380 2000
[26] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts Approved Standard 3rd edition CLSIDocument M27A3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[27] CLSI Reference Method for Broth Dilution Antifungal Sus-ceptibility Testing of Filamentous Fungi Approved Standard2nd edition CLSI Document M38-A2 Clinical and LaboratoryStandards Institute Wayne Pa USA 2008
[28] CLSI Reference Method for Broth Dilution Antifungal Suscep-tibility Testing of Yeasts 3rd inFormational Supplement CLSIDocumentM27-S3 Clinical and Laboratory Standards InstituteWayne Pa USA 2008
[29] P Cos A J Vlietinck D V Berghe and LMaes ldquoAnti-infectivepotential of natural products how to develop a stronger in vitrorsquoproof-of-conceptrsquordquo Journal of Ethnopharmacology vol 106 no3 pp 290ndash302 2006