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MICROREVIEW

DOI: 10.1002/ejoc.201301695

1,2,3-Triazole in Heterocyclic Compounds, Endowed with Biological Activity,through 1,3-Dipolar Cycloadditions

Antonino Lauria,*[a] Riccardo Delisi,[a] Francesco Mingoia,[b] Alessio Terenzi,[a]

Annamaria Martorana,[a] Giampaolo Barone,[a] and Anna Maria Almerico[a]

Keywords: Cycloaddition / Nitrogen heterocycles / Domino reactions / Homogeneous catalysis / Enzyme catalysis /Biological activity / Medicinal chemistry

1,3-Dipolar cycloaddition reactions can be considered apowerful synthetic tool in the building of heterocyclic rings,with applications in different fields. In this review we focuson the synthesis of biologically active compounds possessingthe 1,2,3-triazole core through 1,3-dipolar cycloaddition reac-tions. The 1,2,3-triazole skeleton can be present as a singledisubstituted ring, as a linker between two molecules, or em-

Introduction

The ever increasing demand for new effective drugs andthe requisite process of lead discovery and optimisationhave resulted in a continuous search for simple and efficientmethods for the generation of virtual screening and bio-logical libraries. For these reasons, medicinal chemists areparticularly attracted to synthetic methodologies that alloweasy access to large databases of compounds.

The identification of these rapid synthetic strategiesshould allow the medicinal chemist to assemble a largenumber of potentially active compounds in a very shortperiod of time, speeding up the process of discovery andlead optimisation. The key features of the ideal synthesis,alongside rapidity, are efficiency, versatility and selectiv-ity.[1,2] One series of synthetic methodologies that fulfillthese criteria is that of the 1,3-dipolar cycloaddition reac-tions (1,3-DCRs). Currently they constitute a highly versa-tile and powerful tool for the construction of pharmacolog-ically active compounds with one or more five-memberedheterocyclic rings.

In the landscape of 1,3-DCRs, copper or ruthenium havebeen the catalysts of choice for perhaps the best-known ex-ample: azide–alkyne cycloaddition (CuAAC or RuAAC)between the azide moiety and a terminal alkyne group to

[a] Università di Palermo, Dipartimento di Scienze e TecnologieBiologiche, Chimiche e Farmaceutiche (STEBICEF),Via Archirafi 32, 90123 Palermo, ItalyE-mail: [email protected]://portale.unipa.it/persone/docenti/l/antonino.lauria

[b] Istituto per lo Studio dei Materiali Nanostrutturati (ISMN) –Consiglio Nazionale delle Ricerche (CNR),Via Ugo La Malfa 153, 90146 Palermo, Italy

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bedded in a polyheterocycle. The cycloaddition reactions areusually catalysed by copper or ruthenium. Domino reactionscan be achieved through dipolarophile anion formation, gen-erally followed by cyclisation. The variety of attainable het-erocyclic structures gives an illustration of the importance ofthe 1,2,3-triazole core in medicinal chemistry.

afford the 1,2,3-triazole ring, also known as Huisgen cyclo-addition. Although this reaction was discovered at the be-ginning of the 20th century, its real potential and mecha-nism of reaction were uncovered only in the 1960s, by Hu-isgen et al.[3] Huisgen’s work led to the postulation of a syn-thetic route that could provide a huge variety of chemicalstructures in a very short time with different and, at thesame time, outstanding biological properties, giving enor-mous energy to modern drug discovery.

This review focuses on the medicinal chemistry applica-tions of heterocyclic compounds containing the 1,2,3-tri-azole moiety, either as a component fused in a polycyclicstructure or as a functionalised moiety, and on the syntheticprocedures based on exploiting 1,3-DCRs to yield such het-erocyclic derivatives.

The 1,2,3-Triazole Ring in Medicinal Chemistry

Among the 1,2,3-triazole heterocyclic compounds, 1,4-disubstituted derivatives are the most common triazolessynthesisable by means of 1,3-DCRs. Many heterocyclescontaining embedded triazole cores have been shown topossess a wide range of biological activity.

The 1,2,3-triazole core is stable against acidic and basichydrolysis as well as against oxidative and reductive condi-tions, reflecting a high aromatic stabilisation and relativeresistance to metabolic degradation.[4] At the same time, ithas a high dipole moment, of about 5 D,[5] and participatesactively in hydrogen bond formation as well as in dipole–dipole interactions.[6]

A. Lauria et al.MICROREVIEWThese features make the variously substituted triazole

moiety structurally similar to the amide bond, mimicking aZ or an E amide bond, depending on its substitutionpattern (Figure 1, parts a and b, respectively). In particular,

Antonino Lauria was born in 1969 in Palermo (Italy). He graduated in chemistry with full marks and honours at theUniversity of Palermo (1994). In 1996 he became Assistant Professor and in 2003–2004 he joined the research group ofProf. Alan R. Katritzky at the University of Florida in Gainesville (USA), working on studies on solubility throughcomputational chemistry. In 2006 he became Associate Professor in Medicinal Chemistry. His scientific activity is mainlydevoted to the design, the synthesis, the chemical behaviour and the structure–activity relationships (SARs) of newheterocyclic compounds of biological interest. In particular, his most recent studies are devoted to the development of newclasses of compounds structurally related to well-known anticancer drugs.

Riccardo Delisi was born in Palermo, Italy in 1988. He graduated in chemistry and pharmaceutical technology with fullmarks and honours at the University of Palermo (2011). He is currently a PhD student focusing his research on optimi-sation of heterocycle lead compounds as anticancer drugs under the supervision of Prof. A. Lauria and Dott. F. Mingoia.

Francesco Mingoia graduated in pharmacy at Palermo University (Italy) with full marks and honours. In 1990, he wasselected by the Italian National Research Council (CNR) for a fellowship training. Subsequently, before the completionof his PhD on pharmaceutical science, under the supervision of Prof A. M. Almerico, he was hired at the CNR. In 2002–2004, he joined Prof. G. Poli’s research group (Pierre et Marie Curie University/CNRS, Paris VI, France) collaboratingon Pd-catalysed organic synthesis. Currently he is a researcher at the Institute for Nanostructured Materials (ISMN),Palermo division. His current research interests focus on synthetic methodologies for new heterocyclic compounds andtheir use in medicinal chemistry.

Alessio Terenzi was born in Palermo (1981), Italy. He obtained his master’s degree in chemistry at the University ofPalermo (2007), where he also completed his PhD (2011) on metal complexes able to interact with DNA, under thesupervision of Dr. G. Barone. During his PhD, he worked as a Marie Curie Early Stage Researcher in Prof. Hannon’sgroup at the University of Birmingham (2010). He is currently a postdoctoral fellow at the University of Palermo. Hisresearch interests focus on interaction studies between synthetic compounds and non-canonical DNA structures.

Annamaria Martorana was born in Turin (Italy). She received her master’s degree in chemistry and pharmaceuticaltechnology with full marks and honours at the University of Palermo (2001), where she also obtained her PhD degreeon “isoindole chemistry” in medicinal chemistry (2005). During her postdoctoral work she was visiting researcher at theCentre for Biomolecular Sciences, Cancer Research, University of Nottingham (UK), under the supervision of EmeritusProfessor Malcolm F. G. Stevens (2005–2006). Currently, she is Assistant Professor at the University of Palermo. Herresearch interests are focused on design and synthesis of new heterocyclic molecules of biological interest.

Giampaolo Barone obtained his PhD in chemistry in 1998 at the University of Palermo, where he is currently AssistantProfessor of General and Inorganic Chemistry. He has been a postdoc at the University of Bath (UK) in 1999 and atthe EMBL-Hamburg outstation (Germany) in 2001. His research interests are mainly focused on the synthesis of newDNA-binding metal complexes and on the experimental and computational study of chemical and spectroscopic propertiesof metal compounds.

Anna Maria Almerico graduated in chemistry with full marks and honours in 1977 at the University of Palermo. Duringher academic career, she has been visiting Professor at the School of Chemical Sciences, University of East Anglia (UK,1983–1984), at the College of Pharmacy – Ohio State University (USA, 1986–1987) and at Masaryk University – Brno(Czech Republic, 1993). Currently she is Full Professor of Medicinal Chemistry in the Faculty of Pharmacy – Universityof Palermo. Her scientific interests are mainly devoted to the design and synthesis of new nitrogen heterocycles of bio-logical interest. In particular, her most recent studies are devoted to the preparation and evaluation of the biologicalproperties of compounds related to well-known anticancer/antiviral/antiparasitic drugs.

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many features underline the fact that the 1,4-disubstitutedtriazole moiety shows similarity with a Z amide bond: i) therole of the carbonyl oxygen lone pair of the amide bond isplayed by the 3-nitrogen atom lone pair, ii) the polarised

1,2,3-Triazole Units in Biologically Active Heterocycles

Figure 1. Structural similarity between the amide and 1,2,3-triazole moieties: a) Z amide bond and 1,4-disubstituted 1,2,3-triazole; b) Eamide and 1,5-disubstituted 1,2,3-triazole.

C–H bond (in the 5-position) acts as a hydrogen bondingdonor, in the same way as the amide N–H bond, and iii) theelectrophilic and polarised C4 is electronically similar to thecarbonyl carbon.

This notwithstanding, the overall dipolar moment of thetriazole system is larger than that of the amide bond. Asa consequence, its hydrogen-bonding donor and acceptorproperties are more marked than those of an amide.Furthermore, there is an overall increase of about 1.1 Å inthe distance between the substituents, which are linkedthrough two atoms in the amide but through three atomsin the triazole ring. Finally, the major difference lies in atompolarisation: in fact, the electrophilic carbonyl carbon is re-placed by a negatively polarised nitrogen atom.[7]

In addition to the potential of triazoles in mimicking theamide moiety and consequently the peptide bond (see be-low), their capability to act as bioisosteres of the acyl phos-phate and trans-olefinic moieties has also been predicted.[8]

Pagliai et al. described the replacement of the trans-olefinicmoiety of resveratrol (1, an anticancer, anti-inflammatoryand blood-sugar-lowering drug) with a triazole nucleus incompounds 2 and 3 (Figure 2).[8] The idea was supportedby molecular modelling calculations (MM2) that suggestedthat this modification did not alter the spatial positioningand the gap between the phenolic hydroxy groups, crucialfor the biological activity of the phytoalexin.

Thus, a parallel combinatorial approach was used tosynthesise several resveratrol analogues incorporating thetriazole moiety, with the aim of finding derivatives thatmaintained some, but not all, properties of the parent com-pound. Seventy-two compounds were submitted for bio-logical screening, and five new lead compounds were iden-tified.[8]

The effective replacement of the trans-olefin moiety witha 1,4-disubstituted triazole unit is not limited to resveratrol.

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Figure 2. trans-Olefinic moiety of resveratrol (1) replaced by a tri-azole ring.

The same replacement in diethylstilbestrol led to analoguemolecules with estrogenic activity.[9]

By the Grimm bioisosteric rule, other good candidatesfor bioisosteric substitution with triazole units should befive-membered rings containing two nitrogen atoms in vari-ous positions. Surprisingly, the literature does not presentas many data as would be expected. Despite this, a goodexample of the success of this substitution is represented bythe work on fipronil (4, Figure 3), an insecticide that actsboth as a non-competitive antagonist on insect GABA re-ceptors and as a blocker of insect glutamate-gated chlorinechannels.[10]

A library of 30 1-phenyl-1H-1,2,3-triazole derivatives 5was synthesised, with replacement of the pyrazole ring witha triazole. Surprisingly, in contrast with their 1,5 counter-parts, several 1,4 regioisomers were more potent competi-tive inhibitors than fipronil. This evidence strengthens theidea that triazole is a Grimm bioisostere of pyrazole.[10]

Another interesting and convenient use of triazoles wasin the generation of non-classical bioisosteres of potentiallylabile functional groups (e.g., ester, amide, phosphate) with

A. Lauria et al.MICROREVIEW

Figure 3. Structures of fipronil (4), 1-phenyl-1H-1,2,3-triazole de-rivatives (5) and arecoline (6), as well as of the triazoles 7 and 8,non-classical bioisosteres of the arecoline ester group.

heterocyclic rings capable of mimicking the same electro-static potential maps.[11] Analogously, the triazole moiety(derivatives 7 and 8) has been shown to act as a non-classi-cal bioisostere of the ester group in arecoline (6, Figure 3),a muscarinic agonist,[12,13] although in this case other het-erocycles have proved to be better mimetics.

Predicted bioisosterism failed in the development of anew series of enzyme inhibitors involved in the synthesis ofsiderophores (iron-chelators required for growth and viru-lence in Mycobacterium tuberculosis).[14]

In this case, the authors attempted to substitute the acylphosphate group in compound 9, fundamental as a spacerand multiple H-bonding site, but with the inconvenient ofbeing a good leaving group, with the more stable 1,2,3-tri-azole ring of compound 10 (Figure 4). Unfortunately, the1,4-disubstituted triazole analogues were ineffective as in-hibitors. Molecular modelling suggested that this lack ofactivity was due to the planar geometry of the triazole unit,which prevented it from acting as a hydrogen-bond ac-ceptor.[14]

Figure 4. The failure of replacement of an acyl phosphate unit witha 1,2,3-triazole moiety.

Among the best-known examples of triazole-containingdrugs, an eminent role is played by tazobactam (11, Fig-ure 5), a commercially available β-lactamase inhibitor, incombination with broad-spectrum antibiotics. Indeed, bio-logical studies performed on tazobactam and related tri-azole-containing compounds 12 (Figure 5) showed that


compounds of this class are potent β-lactamase inhibitorswith higher potency than sulbactam and clavulanic acid,underlining a pivotal role played by the triazole ring interms of potency.[15,16]

Figure 5. Example of β-lactamase inhibitors incorporating the1,2,3-triazole moiety.

Still in the antibiotics field, triazoles have been also usedto improve pharmacokinetic properties of desired drugs.For example, cephalosporins with good oral availabilitywere obtained by linking the triazole moiety to the cephalo-sporin core as in 13 (Figure 5).[17]

Pyrimidine nucleoside analogues have been extensivelystudied as antiviral agents, and the triazole moiety has beenused as surrogate of the pyrimidine ring to give compound14 (Figure 6), which was reported to have both antiviraland cytotoxic properties.[18]

Figure 6. Analogues of the pyrimidine nucleosides incorporatingthe triazole moiety.

A similar analogue, compound 15 (Figure 6), bearing agood leaving group, was synthesised to be used as an alkyl-ating agent.[19]

Synthesis of 1,2,3-Triazoles through 1,3-DCRs

The 1,2,3-triazole core, often found in heterocyclic struc-tures with biological activities, plays different roles: as a di-substituted bioisostere (generally in the 1,4-positions), asa linker of two biologically active molecules, or as a coreembedded in a polycyclic skeleton. The synthesis of the tri-azole core, through 1,3-DCRs, can be performed by cata-lytic or non-catalytic synthetic pathways. Interestingly,structural and energetic details of the reaction mechanismof a 1,3-DCR can be obtained by DFT calculations, as re-


cently reported.[20] Such an approach could be promisinglyextended to the study of metal-catalysed 1,3-DCRs.

1,4-Disubstituted 1,2,3-Triazoles

Cycloaddition of azides 16 and terminal alkynes 17, tobuild 1,2,3-triazoles 18 and 19, represents an importantclass of 1,3-DCRs with wide applicability in the synthesisof heterocyclic biologically active compounds (Figure 7).

Figure 7. 1,3-DCRs of azides and terminal alkynes.

The potential of this reaction type is very high, becausealkyne and azide moieties can be incorporated into a widerange of compounds. However, for more than 40 years thisreaction suffered from a lack of selectivity, yielding mix-tures of the 1,4- and the 1,5-regioisomers.[4] Furthermore,this transformation requires heating and long reaction timesto reach completion and the two regioisomers are some-times laborious to purify by classical chromatographic pro-cedures.

In 2002, two research groups independently reported thatCuAAC proceeds as much as 107 times more rapidly thanthe uncatalysed version.[21,22] More importantly, at roomtemperature or with only moderate heating, use of the cop-per catalyst affords the exclusive formation of the 1,4-re-gioisomer 18 (Figure 7). To date, this near-perfect type ofcycloaddition is the most popular reaction in click chemis-try, characterised by high reliability and total specificity.[23]

This aspect is interesting because, as shown below, thecatalytic properties of copper metal or salts can be attrib-uted to the reversible CuII–CuI reaction and to the coordi-nation chemistry properties of CuI,II complexes with the re-action intermediates of 1,3-DCRs. Moreover, it is knownthat many CuII complexes are strong DNA-binders, and forthis reason endowed with cytotoxic properties.[24] Variousmethods are highly efficient in generating the active catalystCuI for the CuAAC reaction. One of the most commontechniques is in situ reduction of CuII salts, such asCuSO4·5H2O[22] or copper acetate,[25] forming CuI salts. So-dium ascorbate is typically used as reducing agent, in anexcess from three- to tenfold,[22] although other reducingagents, including hydrazine, have also been used with rea-sonable success.[26] This method constantly reduces CuII toCuI, maintaining significantly high levels of the catalyticspecies. This strategy has many advantages: firstly it ischeap, and it can also be performed in water and does notrequire a deoxygenated atmosphere. The main disadvantageis that the reducing agent can reduce CuII to Cu0. This cangenerally be prevented by using a suitable ratio of reducingagent to catalyst and/or by adding a copper-stabilisingagent such as tris-3-(hydroxypropyltriazolylmethyl)amine(THPTA).[27]


A second way to obtain the catalyst is to add a CuI saltsuch as CuBr, CuI or Cu(CH3CN)4PF6 in situ. Althoughthis method does not require a reducing agent, it is less usedthan the first one, because it has to be carried out in adeoxygenated environment and in organic solvent.[22] More-over, CuI salt catalysis is not as reliable as the CuII reductiveprocedure.

The use of oxidising copper metal with amine salts alsogenerates active catalysts. In this case there are a few disad-vantages: it is more expensive, requires longer reactiontimes (as well as larger amounts of copper metal) and,above all, needs a slightly acid environment to dissolve themetal, which might harm any acid-sensitive functionalgroups present in the reactants.[28]

In general, cycloadditions proceed through concertedmechanisms. However, experimentally measured kineticdata[29] and molecular modelling[28] performed on theCuAAC reaction indicate the preference for a stepwise reac-tion pathway (a stepwise catalysed CuAAC reaction has anactivation barrier 11 kcalmol–1 lower than a concerted cata-lysed reaction).[30] On the basis of experimental evidenceand the knowledge that CuI can readily insert itself intoterminal alkynes, giving Sonogashira coupling,[31] it was hy-pothesised that the first step of the reaction involves π com-plexation of a CuI dimer to the alkyne (Figure 8). There-after, removal of the terminal hydrogen from the alkyne oc-curs to form Cu-acetylide 20. There are several differentways in which the CuI-acetylide complexes can be formed,depending on the reaction conditions utilised: in aqueoussolvent the π complexation of CuI lowers the pKa of theterminal alkyne by as much as 9.8 pH units, allowing thedeprotonation and formation of Cu-acetylide 20 withoutthe addition of a base. If an aprotic solvent such as aceto-nitrile, dichloromethane or toluene is used, in the presenceof stoichiometric amount of CuI salts [e.g., CuI,[32]

Figure 8. Proposed reaction mechanism of CuI-catalysed 1,3-DCRs.

A. Lauria et al.MICROREVIEWCu(CH3CN)4PF6,[32] CuBr(PPh3)4 or CuIP(OEt)3][33,34]

then a base, usually a tertiary amine (e.g., TEA, DIEA),should be added to induce the deprotonation step crucialfor the complexation.[35] In the next step, azide nitrogen dis-places one of the ligands from a second Cu in the CuI-acetylide complex to form 21. This process activates the az-ide to nucleophilic attack from the acetylene carbon, dueto proximity and electronic factors, allowing easy attack ofanother nitrogen on the second carbon of the alkyne, lead-ing to a metallocycle (not shown for simplicity). The me-tallocycle then contracts, forming the corresponding tri-azole 22. Final protonation releases the CuI catalyst fromthe 1,2,3-triazole to give product 23 and to restart anothercatalytic cycle (Figure 8).

The search for better copper-based catalysts is still ongo-ing. For example, very recently, copper on charcoal hasbeen developed as a simple, inexpensive and efficientheterogeneous catalyst for triazole formation.[36] The poten-tial advantages of this “new type” of catalyst are numerous.It can be deployed in a large variety of solvents, it is easilyremovable by filtration and it can be used in at least twoadditional cycloadditions without loss of activity.[36] It hasalso been demonstrated that its use reduces or eradicatesthe contamination of the product by copper. Finally, theuse of a base is not strictly necessary for effective catalysisof the cycloaddition.

An evolution of this kind of catalysis consists of the useof CuI-modified zeolites (a family of aluminosilicate min-erals, highly porous and therefore with large surface areas)that exhibit, relative to charcoal, high concentrations ofactive sites, high thermal/hydrothermal stability and highsize selectivity.[37]

In 2012, Duan et al. reported an interesting example ofCuI catalysis.[38] In that work the author underlines the roleplayed by Cu2O nanoparticles (Cu2O-NPs) as catalysts ofintramolecular 1,3-DCRs, to afford the anticancer activetriazole analogues of epothilones. Under the classical catal-ysis conditions, with CuI/DIEA (N,N-diisopropylethyl-amine) or Cu(CN)4PF6, the cyclisation product 25 was ob-tained only in trace amounts or no reaction was observed.However, cyclisation compound 25 was successfully ob-tained in good yield when the reaction was catalysed byCu2O-NPs in acetonitrile as solvent (Figure 9).

Figure 9. Possible role of Cu2O nanoparticles in catalysing intra-molecular 1,3-DCRs.

The efficiency of Cu2O-NPs has also been evaluated inthe cyclisation of a variety of ring systems. The higheryields observed in all these kinds of reactions demonstrate


that Cu2O-NPs are exploitable catalysts for promoting in-tramolecular macrocyclic ring formation.[38] Moreover, theCu2O-NPs catalyst demonstrates less cytotoxicity andhigher efficiency than traditional CuI-generating systems.[39]

To obtain 1,4-disubstituted 1,2,3-triazoles in higheryields with shorter reaction times and under mild reactionconditions, Krim and co-workers decided to induce 1,3-DCRs by applying microwave (MW) irradiation.[40] Indeed,the use of MW-assisted organic synthesis has attracted con-siderable interest over the last two decades, often leading toremarkable decreases in reaction times, significant enhance-ments of yields, easier workups and better regioselectiv-ity.[41] In the above article the cycloaddition of a terminalalkyne and an azide was carried out under MW conditionswith CuI as catalyst and under solvent-free conditions, lead-ing to the desired product in a quantitative yield and witha reaction time of one minute. In contrast, when the samereaction was carried out under conventional heating condi-tions the reaction time was increased to 3 h and the yielddecreased to 83%. The formation of 1,4-disubstituted tri-azoles was unequivocally established. This innovative ap-proach gave a new, straightforward and convenient method-ology for the synthesis of new 1,4-disubstituted 1,2,3-tri-azoles in higher yields and shorter times than under theclassical CuI-catalysed cycloaddition.[40]

With the aim of developing an efficient, high yieldingand less time-consuming library of compounds containingboth spiro-oxindole and pyrrolidine/pyrrolizidine ringslinked to the well-known bioactive natural core androgra-pholide, Hazra et al. decided to explore the applicability ofan azomethine ylide cycloaddition to a conjugated doublebond under the MW irradiation conditions describedabove.[42] Also in this case the MW-irradiated reaction gavethe best yield (80%) in a shorter time (15–20 min) than un-der non-irradiated conditions. Moreover, control of the rel-ative stereochemistry at the spiro centre was observed.Interestingly, reactions of this kind proved chemo-, stereo-and regioselective.

Since the first reports, hundreds of articles exploring thesynthetic possibilities of CuAAC have been published. Nev-ertheless, relatively few examples in which this reliablemethod is applied to steroid azides exist. In one of them,the authors attempted to develop an effective route for theproduction of various 2-triazolyl derivatives of choles-tanone 28 (Figure 10) with the aim of obtaining compoundsendowed with antiproliferative activity,[43] as well as othertriazolyl-steroids.[44] Several A-ring-substituted 1,2,3-tri-azolylcholestan-3-ones 28 were synthesised in very goodyields by treatment of 2α-azido-5α-cholestan-3-one (26)with various terminal alkynes 27 (Figure 10).

In these cases, the reactions were catalysed by CuI gener-ated in situ by the reduction of CuSO4 with sodium ascorb-ate. Furthermore, the particular feature of this work was anunusual two-phase solvent system (CH2Cl2 as co-solventwith water), applied to increase the solubility of the steroidin water and to eliminate the use of any ligands, thus simpli-fying the reaction protocol. These particular conditions al-lowed the desired compounds to be obtained in very good


Figure 10. Example of the synthesis of triazolyl-steroids throughCuAAC.

yields.[44] Probably for the same reasons, an unusual two-phase solvent system (tBuOH/H2O 1:1) was employed forthe CuAAC synthesis of 3-propargyloxy betulinic acid ana-logues 31 (Figure 11).[45] The goal was to obtain a moreefficacious and less toxic analogue 31 of betulinic acid (29),a cytotoxic agent, active against several human cancer celllines, with impressive IC50 values in the μm range.

Figure 11. Synthesis of betulinic acid analogues 31 through 1,3-DCRs.

In this case the chemical modification involved the incor-poration of the 1,2,3-triazole moiety at the C-3 position ofring A, with the aim of studying the antiproliferative ac-tivity of triazolyl-steroids. With this unusual solvent systema library of 18 triazole derivatives, offering structural diver-sity, was prepared in very high yields.[45] These compoundswere screened against a panel of nine human cancer celllines, and many produced dose-dependent growth inhibi-tion effects on several cancer cell lines.[45]

The same unusual solvent mix (tBuOH/H2O 1:1) wasalso crucial in another interesting synthesis route to obtain,through CuAAC reactions, a series of carbamate derivativesof 4β-(1,2,3-triazol-1-yl)podophyllotoxin 35 (Figure 12).[46]

Podophyllotoxin (32) is a strong antimicrotubule agent asan inhibitor of the colchicine-binding site on tubulin; how-ever, because of its high toxicity, it could not be used in


therapy.[47] All of the synthesised carbamates 35 showedmore potent anticancer activity, higher solubility and lesstoxicity than natural podophyllotoxin against a panel offour human cancer cell lines.[46]

Figure 12. Synthesis of carbamate derivatives 35.

The multi-solvent approach was also reported by Wuet al. in 2013.[48] The synthesis of 4�-(1,2,3-triazol-1-yl)-2�-deoxy-2�-fluoro-β-d-arabinofuranosyl-cytosines 37 throughCuAAC reactions between 4�-azido-arabinofuranosyl-cytos-ines 36 and appropriate alkynes was described (Figure 13).

Figure 13. The synthesis of β-d-arabinofuranosyl-cytosines 37.

Also in this case the noteworthy reaction feature lies inthe solvent system: a ternary water/tBuOH/acetonitrile mix-ture (1:1:1) allowed the targeted compounds to be obtainedin good yields (66–96%) without contamination with the1,5-regioisomers and with no trace amounts of alkynehomocoupling side reaction products (see below). The syn-thesised compounds 37 exhibited potent anti-HIV-1 activitywithout significant host cytotoxicity at the highest testedconcentration of up to 25 μm.[48]

An interesting overview of reaction conditions (solventsystem, additive, temperature), yields and regioisomerictrends of CuAAC was offered by Chen and co-workers in2011. The authors synthesised a series of podophyllotoxin

A. Lauria et al.MICROREVIEWTable 1. Reaction conditions for the 1,3-CDR between 4β-azido-podophyllotoxin (38) and methyl propiolate.[49]

Entry Catalyst Additive Solvent t [h] T [°C] Yield 1,4-triazole [%] Yield 1,5-triazole [%]

1 CuSO4·5H2O l-asc. acid tBuOH /H2O (1:1) 2 25 70 02 CuSO4·5H2O l-asc. acid tBuOH/H2O (1:1) 2 50 95 03 CuSO4·5H2O l-asc. acid tBuOH/H2O (1:1) 2 75 65 04 CuSO4·5H2O l-asc. acid THF/H2O (1:1) 2 25 0 05 CuSO4·5H2O l-asc. acid DMF/H2O (1:1) 2 25 80 06 CuSO4·5H2O l-asc. acid DMSO/H2O (1:1) 2 25 83 07 CuI 2,6-lutidine CHCl3 12 0 90 08 CuI pyridine CHCl3 12 0 20 179 CuI l-asc. acid tBuOH/H2O (1:1) 12 25 0 010 CuSO4·5H2O 2,6-lutidine tBuOH/H2O (1:1) 2 25 15 1011 CuI none PEG400/H2O (1:1) 2 25 95 012 none none CH2Cl2 6 25 15 12

Figure 14. Reaction between 4β-azido-podophyllotoxin (38) and methyl propiolate.[49]

analogues 39 and 40.[49] As can be clearly seen in Table 1,the CuAAC reactions resulted, as previously demonstrated,in 1,4-regioselective products (Figure 14).

In this work, it is important to underline that 2,6-lutidinewas used beneficially as an additive for CuI, relative toother alkaline additives, whereas l-ascorbic acid was neces-sary when CuSO4 was used to generate the active CuI.[49]

The solvent PEG400 was found to enhance the reaction ratein the Cu-catalysed reaction greatly without any additive(Entry 11).[49,50] In contrast, the condition used in Entries 4and 9 did not yield any product. Therefore, the traditionalthermal conditions without any catalyst were applied, giv-ing a mixture of the 1,4- and 1,5-disubstituted triazolyl-po-dophyllotoxins 39 and 40, but in very low yield (Entry 12).In most cases, the ratio of the two regioisomers was approx-imately 1:1. To confirm the structures of the series of 1,4-disubstituted or 1,5-disubstituted triazolyl-podophyllotox-ins, X-ray crystallography was used.[50]

Recently, researchers have turned their attention to thepossible benefits of innovative and new synthetic ap-proaches such as CuAAC for the synthesis of base- orsugar-modified nucleosides, nucleoside bioconjugates andmodified oligonucleotides.[51] Among them a relevant classof heterocyclic compounds with embedded 1,2,3-triazolemoieties, achieved through 1,3-DCRs, are the triazolyl-nu-cleosides.[51,52] For example, Benhida et al. isolated a seriesof nucleoside-substituted triazoles 43 with in vitro cyto-static activity. The synthesis was based on straightforward1,3-DCRs between 1-azido-ribose derivative 41 and ter-minal alkynes 42 with a cooperative effect of MW acti-vation and CuI catalysis (Figure 15).[53]


Figure 15. The synthesis of β-triazolylnucleosides 43.

Another unusual and fine case of catalysed reactions isthat of enzymatic 1,3-DCRs. By means of these reactions,it might be possible to overcome one of the main problemsof medicinal chemistry: target selectivity. The conventionalapproach used by medicinal chemists in drug discovery con-sists of the synthesis of a collection of compounds and sub-sequent biological screening. The procedure would beshortened and optimised if the biological target could ac-tively “choose” its best ligand. This futuristic approach wascarried out by Lewis and co-workers.[54]

The ingenious intuition of these researchers was to usean engineered enzyme that, at room temperature, couldbring azide and alkyne close together by “using itself” astheir reaction vessel.[54] By this strategy, only compoundsthat fit correctly into the active site of the enzyme shouldreact to give new potential ligands of the enzyme (Fig-ure 16).


Figure 16. 1,3-DCRs catalysed by an Ach-E engineered enzyme.

This strategy was used to identify extremely potent inhib-itors of acetylcholinesterase (Ach-E), the key enzyme thathydrolyses acetylcholine at the synaptic cleft and constitutesa pharmacological target for many clinically relevant condi-tions, including Alzheimer’s disease and muscular dys-trophy. Reactions between phenanthridinium species 44 andtacrine derivatives 45 and other analogues functionalisedeither with azides or with terminal alkynes were carried outin the absence of catalyst. As expected, under these condi-tions, after 6 d, negligible amounts of products 46 wereformed. In contrast, when the reactions were carried out inthe presence of the engineered enzyme, any productsformed were the results of a rate-accelerating enzyme-tem-plating effect. Indeed, as hypothesised, out of all the pos-sible sets of reactions (�75 combinations) only six combi-nations took place. The result of this study was the in situdiscovery of the most potent non-covalent inhibitors ofAch-E ever discovered, with dissociation constants in thefemtomolar range (Kd 33–100 fm).[55,56]

The application of 1,3-DCR enzyme catalysis was alsodemonstrated for carbonic anhydrase inhibitors,[57] underli-ning the versatile application of this strategy with other en-zymes. This powerful technique could also be applicable inthe synthesis of other non-enzyme drug targets. Indeed, al-though only enzyme inhibitors have been synthesised todate, there is no reason to assume that other new receptorligands could not also be identified by this strategy.

1,5-Disubstituted 1,2,3-Triazoles

As previously seen, the “canonical” 1,3-DCR betweenazides and alkynes catalysed by CuI yields only the 1,4-di-substituted triazole system. In a few cases, however, tworegioisomers have been formed in ratios of approximately1:1. For this reason many research groups have tried to de-velop an orthogonal cycloaddition procedure to give the1,5-disubstituted regioisomers. The first approach was re-ported in 1994.[58] That work described how the substitutedtrimethylsilyl-acetylene (48) could direct the cycloadditionroute selectively to the 1,5-regioisomer 49. The trimethyl-


silyl group probably controls the regioselectivity by a com-bination of two factors: firstly, its steric hindrance precludesthe orientation of the transition state that leads to the 1,4-regioisomer, and secondly, the trimethylsilyl group is ableto stabilise a positive charge on acetylene at the β-carbonposition in the transition state (Figure 17).[58]

Figure 17. Steric influence on the regioselectivity of azide–alkyne1,3-DCRs.

In 2004, Krasinski et al. tried to synthesise the 1,5-disub-stituted triazole ring by exploiting reactions betweenbromomagnesium acetylides and organic azides.[59] The in-termediates 50 could be trapped with different electrophilesto form 1,4,5-trisubstituted 1,2,3-triazoles 51 regioselec-tively (Figure 18).

Figure 18. Direct synthesis of 1,4,5-trisubstituted 1,2,3-triazoles 51.

Furthermore, in following papers this strategy was suc-cessfully utilised to prepare a set of triazole-based mono-phosphine and sulfonyl ligands.[60,61] The main limitation ofreactions of this kind consists of the transformation of thealkyne group into the bromomagnesium acetylene with theGrignard reagent.[60] This step is particularly troublesomewhen carried out on the functionalised substrates typical ofmedicinal chemistry (e.g., esters and enolisable or hydrolys-able substrates). Thus, these reaction conditions are notusable in the synthesis of pharmaceutical compounds.

In 2005, pentamethylcyclopentadienyl ruthenium(II)complexes (Cp*Ru), such as Cp*RuCl(PPh3)2, were discov-ered to act as catalysts for 1,3-DCRs between alkynes andazides. Unlike all of the previously mentioned catalysts,Cp*Ru complexes afforded only 1,5-substituted 1,2,3-tri-azoles 54 (Figure 19).[62]

Figure 19. 1,3-DCRs of azides and terminal alkynes in the presenceof Cp*RuCl(PPh3)2 as catalyst.

It is also notable that catalysts of this kind, unlike theircopper counterparts, allow reactions between internal alk-ynes and azides.[62] This trend was confirmed by a system-atic study that explored the reaction products of several

A. Lauria et al.MICROREVIEWasymmetric internal alkynes with variously substituted az-ides.[63] Unfortunately, limited information detailing the roleof ruthenium(II) complexes in the catalytic mechanism isavailable.

Multicomponent Reactions and CuAAC

The potential of CuAAC reactions can be further ampli-fied by combining them with multicomponent reactions(MCRs). MCRs are a class of reactions in which three ormore substrates and/or adaptable molecules, are combinedin one-pot fashion to give a product that contains essentialparts of all of them, yielding complex structures in few syn-thetic steps.[64] Moreover, this procedure offers significantadvantages over conventional linear-type syntheses. Barbasand co-workers first developed the use of MCRs followedby CuAAC reactions.[65] They applied multicomponent re-actions between phosphoranes, aryl aldehydes and spiro-lactones, in the presence of l-proline as catalyst, to obtainvarious dispirolactones through a domino Wittig/Knoeven-agel/Diels–Alder reaction sequence. Dispirolactones ob-tained in this way showed antioxidant and free radical scav-enger activities.[65] These compounds were then treated withbenzyl azide in the presence of sulfate/metallic copper togenerate new chemical libraries 55, for high-throughputscreening, in a relatively short period of time (Figure 20).[65]

Figure 20. Libraries of spirotrione-triazoles 55 and triazolyl-di-hydropyrimidinone analogues 56.

Recently, a research group started investigations into theapplication of multicomponent, domino and cycloadditionreactions to the construction of new heterocycles, whichhave unearthed several compounds with marked ac-tivity.[66,67]

In another example, a library of triazolyl-dihydropyrim-idinone analogues 56 was synthesised under microwave irra-diation conditions, combining a Biginelli reaction withCuAAC, with the aim of improving the biological activityof the lead compound.[68]

Also remarkable is a recent report on the use of CuAACto generate 1,4,5-trisubstituted 1,2,3-triazoles throughmulticomponent reactions.[69] All the cases of multicompo-nent reactions and subsequent CuAAC cited above high-


light how reactions of this kind might play an eminent rolein lead identification and lead optimisation procedures andin the generation of chemical libraries useful in medicinalchemistry.

Uncatalysed Reactions

Although copper – and in some cases ruthenium – ap-pear to be the perfect catalysts and the synthetic protocolsare well tested, the applications that have been postulatedfor 1,3-DCRs between azides and alkynes still allow for fur-ther investigation into conditions that might favour particu-lar applications. Despite the great roles played by the vari-ous catalysts reported above, the use of copper salts hassome drawbacks. The copper used as the catalyst is cyto-toxic in nature. Moreover, excessive intake can lead to con-ditions such as hepatitis, Alzheimer’s disease and neurologi-cal disorders.[70] Furthermore, the catalysis can lead to alk-yne homocoupling. This drawback occurs when a terminalalkyne reacts with another alkyne but not with the expectedazide (Figure 21).

Figure 21. CuI-catalysed oxidative homocoupling of terminal alk-ynes.

These homocoupling side reactions require a CuI or CuII

catalyst and the presence of oxygen.[30] The stabilities ofsome azides might also be a limiting factor. In fact, if theratio of nitrogen atoms to carbon atoms is higher than one,the molecule could decompose before reacting with the Cu-acetylide complex. Fortunately, this is generally not a majorissue for pharmaceutical research, which tends to focus onlarger molecules with high numbers of carbon atoms.[30]

Efforts have been made to develop reactions that do notrequire metal catalyst.[71] New reactions of this kind in-clude: reactions between azides and activated alkynes, reac-tions between azides and electron-deficient alkynes, and re-actions between azides and arynes.[72] Other studies haveshown that 1,3-DCRs can also occur rapidly if the alkyneis first incorporated into an eight-membered ring, forming acyclooctyne of type 57.[72–74] The unusually high ring strainassociated with cyclooctyne (18 kcal mol–1) allows it to re-act selectively and rapidly with azide groups, giving the1,2,3-triazoles without the formation of side products or theaid of a catalyst (Figure 22).


Figure 22. Strain-promoted azide–alkyne [3+2] cycloaddition(SPAAC) reaction.

Because of the high efficiency of these reactions, this ap-proach was used by a research team in the conjugation (seebelow) of methotrexate (MTX) to a dendrimer platform.[75]

MTX is a well-known and extensively characterised anti-neoplastic agent that displays increased activity when con-jugated to a dendrimer.[76] By use of a copper-free click con-jugation protocol, a modified MTX (in which an azide moi-ety had been added) was joined to the newly synthesisedcyclooctyne dendrimer platform in a stoichiometric ratioand with very good yields.[75] These results indicate the fea-sibility of this non-copper-based strategy for reproduciblysynthesising dendrimer conjugates with varied functionali-ties.

Metal-free click reactions, however, have their ownlimitations. Most of these reactions involve large compli-cated pathways, such as the incorporation of the alkyne ina cyclooctyne. Moreover, the uncatalytic method producesmixtures of regioisomers 58 and 59. All of these inconve-niencies make reactions of this type poorly applicable topharmaceutical applications, so the major proportion ofthem prove to be of limited utility.[72]

The Triazole Ring as Spacer or Linker

The triazole moiety is an ideal linker: it provides greatwater solubility and is very similar to amide bonds (seeabove) and relatively resistant to hydrolysis reactions,[27] soit is stable enough under typical biological conditions.Moreover, this ring is extremely rigid, and for this reasonthe two linked substances cannot interact between them-selves.[27] These features allow the triazole moiety to be seenas an inactive linker or spacer, although it is not possibleto exclude the possibility that, under particular conditions,it may act as a biological entity on its own.

Conjugation with Biomacromolecules

CuAAC has been successfully adopted as a powerful toolfor joining lipids to proteins. Indeed, although many cyto-solic and membrane cellular proteins are subject to naturalsingle or double lipidation, synthetic conjugation of pro-teins with lipids still represents a formidable challenge. Thefirst example of a reaction of this kind was reported in2005.[77] In this pioneering work, the authors convertedphosphatidylethanolamine into the corresponding azide de-rivative, which reacted successfully with terminal alkyneproteins to give the lipoprotein analogues 60 (Figure 23).


Figure 23. Lipoprotein analogues 60 and sugar-coupled ferrocene61, both obtained by CuAAC.

Using this strategy, the authors developed an easy anduseful method exploitable in the synthesis of lipoproteinand lipid analogues that could be employed as models inthe study of lipids behaviour in biological environments orin biological membranes (e.g., lipid rafts or compartmen-talisation).

This trend has also had a major impact in carbohydratechemistry. Linking of a sugar molecule to a poorly solublecentral core (e.g., peptide, drug, etc.) could enhance solubil-ity and accordingly improve the pharmacokinetic profile.This aspect is well treated in a work published in 2004, inwhich the authors, in order to increase solubility, tried cou-pling of the antimalarial drug ferrocene to sugars throughCuAAC to provide 61 (Figure 20).[78] Although no bio-logical data are available, it was demonstrated that thesecompounds maintain electrochemical behaviour similar tothat of ferrocene.[78]

Another example was applied in the conjugation of dif-ferent sugar moieties to the decapeptide tyrocidine. This li-pophilic peptide, targeting the lipid bilayer of bacteria, pres-ents pronounced antibiotic properties, but possesses a verylow solubility as well as a low therapeutic index. In orderto improve its safety profile and solubility, this drug hasbeen coupled to mono-, di- and trisaccharides to generatedifferent kinds of cyclic peptides by CuAAC. The triazole-grafted glycopeptides synthesised in this way showed ahigher therapeutic index than tyrocidine, as well as in-creased solubility.[79]

Another interesting example is to be found in the synthe-sis of multivalent neoglycoconjugates 62, in which, in orderto synthesise compounds with improved tumour growth in-hibition properties, CuAAC reactions were used to linkmultiple sugars on a central aromatic scaffold (Fig-ure 24).[80]

A similar topic was subsequently investigated by Liebertet al., who used CuAAC to modify the surface of cellulosewith the aim of increasing the overall cellulose affinitytowards its desired target.[81] The first step involved the in-


Figure 24. Multivalent neoglycoconjugates 62 efficiently achievedby regiospecific catalytic 1,3-CDR.

troduction of the azide moiety into cellulose through twosimple actions: tosylation of the secondary alcohol groups,followed by treatment with sodium azide. After this, threedifferent terminal alkynes, all of them low-molecular-weightcompounds (methyl propiolate, 2-ethynylaniline and 3-eth-ynylthiophene) were added, together with a CuII salt cata-lyst and sodium ascorbate.

In the same year, Hafren et al. reported the preparationof modified cellulose by a slightly different approach.[82] Inthat work, terminal alkynes were introduced onto the sec-ondary alcohol groups of cellulose in one step by use ofhex-5-ynoic acid. The product was then allowed to reactwith 3-azidocoumarinin in the presence of a copper catalystto give a coumarin cellulose compound.

Another interesting use of the triazole system as a linkerbetween two chemical entities is the application of CuAACin the immobilisation of small molecules (e.g., chromo-phores) on macromolecular structures. The possible appli-cations of these reactions are endless. In fact, by usingCuAAC it is possible to generate fluorogenic compoundsthat allow tracking of biomolecules in the cell.[83] A clearexample of this kind is the in situ generation of fluorescentDNA probes 65, an indispensable tool in molecular biology(Figure 25).[84]

Figure 25. CuAAC (click reaction) for the multiple postsyntheticlabelling of alkyne-modified DNA.

Another example can be found in the bioconjugation ofmany organic molecules (e.g., biotin and maltose) to goldnanoparticles that are used in cancer cell diagnostics. In thisreaction, gold nanoparticles were functionalised with azidemoieties and the molecule to be attached was functionalisedso as to contain a terminal alkyne moiety for subsequentcycloaddition to form the 1,2,3-triazole ring.[85] This tech-nique has been successfully used, for example, to comparethe activities of various enzymes in breast cancer cells[86,87]

or to identify the targeting site of an unknown class of mol-ecules.[88]


Site targeting through the use of liposomes represents an-other interesting use of conjugation.[89] Liposomes arewidely used as drug carriers in drug delivery systems, andthe possibility of coupling a specific and functionalised li-gand (an α-d-mannosyl derivative or an azido modified ly-sine) to their surfaces endows them with cell selectivity. Toachieve this goal, mannosyl or lysine residues functionalisedwith azide groups were conjugated to the surfaces of lipo-somes bearing alkyne moieties. Triazole linkers were thusgenerated by use of a catalytic amount of ascorbate/CuSO4

and a stabilising agent that protects the CuI oxidation statefrom degradation pathways. The importance of the stabiliz-ing agent lies in protection of the bilayers, to avoid thechange in the particle size of the liposome. These mannosyl-ated/lysinated liposomes would then be used for specificallydirecting the contents of liposomes to cells expressing thereceptor affinity for the “joined” ligand, such as humandendritic cells.[89]

In a similar way, Cavalli et al. investigated the in situ sur-face modification of liposomes, bearing terminal alkynegroups at their surfaces, with the aid of azide-functionalisednitro-benzoxadiazole and CuBr as catalyst.[90]

The Triazole Ring as a Linker in Multitarget Drugs

Although 1,3-DCRs were initially developed as a drugdiscovery tool, nowadays the second most common phar-maceutical application of reactions of this kind is the gener-ation of dimers, chimeras and multivalent drugs in whichpolymer chains or biomolecules are linked through theirterminal functionalities with other functional groups (Fig-ure 26).

Figure 26. Linkage of two different molecules through a 1,2,3-tri-azole ring.

Clear examples in which a triazole is an inactive linkerare compound 66, a multivalent antibiotic obtained by link-ing linezolid with a macrolide, or compound 67, in whichvancomycn is linked with a cephalosporin (Figure 27).[91,92]

The triazole linker has also been used to generate biden-tate inhibitors, in which the molecules interact with two dif-ferent binding sites of the same target (Figure 28).

Indeed, in this case the binding of one molecular compo-nent to the target with high affinity should bring the secondcomponent, with lower affinity, closer to its target, in thisway increasing the potencies and affinities of both compo-nents (Figure 28).[93]

Yao and co-workers published an interesting article thatrefers to this topic in 2006.[94] That paper reports the firsthigh-throughput-amenable CuAAC assembly of a 66-mem-ber library consisting of triazole-linked bidentate com-pounds for in situ screening on a panel of protein tyrosinephosphatase inhibitors. The same researchers described asolid-phase strategy for the productive synthesis of azide


Figure 27. Compounds 66 (linezolid linked with a macrolide) and67 (vancomycin linked with a cephalosporin).

Figure 28. Schematic representation of a triazole linker used to gen-erate bidentate inhibitors, in which the molecules interact with twodifferent binding sites of the same target.

libraries suitable for building bioactive compound librar-ies.[95] Later, this approach allowed the construction ofnearly 3500 bidentate triazolyl isoxazole acid derivatives asnew protein tyrosine phosphatase inhibitors with promisingpharmacological profiles.[96]

Due to the presence of the indole nucleus in a lot ofnatural products with biological activities, Zhang and co-workers developed an indole-based bicyclic salicylic acidderivative for creation of a 212-member CuAAC library.[97]

A similar idea was exploited to identify selective matrixmetalloprotease (MMP) inhibitors. In this case, the activecompounds were eight different substituted succinyl hydro-xamates (well known to interact with a zinc ion present inthe active site of the enzyme), each bearing a terminal alk-yne. These were coupled with twelve different hydrophobicazides by a parallel combinatorial approach. Two of themwere found to be inhibitors of MMP 7, with activity in themicromolar range.[98]

The triazole moiety has also been used as a linker inthe synthesis of homodimer drugs, a successful strategy toincrease the potencies or solubilities of compounds. Thefirst attempt to use a triazole group as a spacer on the basisof these assumptions was performed with vitamin D3 dimerderivatives. Unfortunately, although the reaction was suc-cessful, the obtained dimers were biologically inactive.[99]


Another research team tried to use the same reaction toobtain a homodimer of daunorubicin. In this case the syn-thesised compound showed a higher DNA intercalatingcapability.[100] Indeed, it is well known that daunorubicinbinds DNA in a stoichiometric ratio of 2:1, but in this casethe length and the flexibility of the triazole moiety producethe best distance between the two monomers, giving a mo-lecule with a better DNA binding ability.

This topic was also addressed in another paper, in which1,3-DCRs between azides and alkynes were used to synthe-sise artemisinin homodimers 72 (Figure 29), new artemisi-nin derivatives obtained by joining two artemisinin mo-lecules without destroying the endoperoxide linkage.[101]

Molecules of this class have been reported to display muchhigher anticancer activities than many of their mono-mers.[102] The strategy adopted was simple but ingenious.Firstly, artemisinin lactol 69 was synthesised from artemisi-nin (68) by NaBH4 reduction in methanol at low tempera-ture (Figure 29). 10α-Azidoartemisinin 71 was synthesisedby treating artemisinin lactol 69 with NaN3 and bromotri-methylsilane in dichloromethane under inert atmosphere.Then, the alkyne precursors 70 were synthesised, againfrom artemisinin lactol 69, by treatment with alkynylalcohols. The subsequent 1,3-DCRs between the differentsynthesised derivatives of artemisinin were carried out inthe presence of CuSO4 and sodium ascorbate in dichloro-methane and water at room temperature to furnish the de-sired artemisinin homodimers 72.

Figure 29. Use of 1,3-DCRs to synthesise artemisinin homodimers72.

It is worth pointing out that the stereochemistry of theC-10 centres of azidoartemisinin and the alkyne derivativesof artemisinin remained identical for each dimer.


The 1,2,3-Triazole Ring Embedded inPolyheterocycles

In recent decades 1,3-DCRs have seen various applica-tions in the synthesis of new annelated heterocyclic com-pounds. In particular, they proved to be very efficacious asfirst steps in domino reactions leading to several flat po-lyheterocycle systems, potential DNA intercalators throughπ–π stacking interactions. A domino reaction is a processinvolving two or more bond-forming transformations (usu-ally C–C or C–N bonds), which take place under the samereaction conditions without additional reagents and cata-lysts, and in which the subsequent reactions take place asa consequence of the functionality formed in the previousstep.[76]

An example of application of such a sequential pro-cess[103] involves the synthesis of the pyrrolo[3,4-e][1,2,3]tri-azolo[1,5-a]pyrimidine core 76 by means of domino reac-tions. The key intermediate of this synthesis was the 3-azidopyrrole 73 (Figure 30).

Figure 30. Synthesis of the pyrrolo[3,4-e][1,2,3]triazolo[1,5-a]pyr-imidine core 76 by means of domino reactions.

The azido moiety acts as 1,3-dipolar component in 1,3-DCRs with anions 74, obtained by deprotonation of α-methylene groups in substituted acetonitrile dipolarophiles(Figure 30). The intermediates 75 each bear an aminogroup susceptible to immediate further cyclisation with theortho-carboxyethyl moiety to provide the pyrrolo[3,4-e][1,2,3]triazolo[1,5-a]pyrimidin-5-ones 76. Although thistype of anionic hetero-domino reaction is well known anddescribed in aromatic series, it has only in a few cases beenapplied to pentatomic heterocycles, mainly to thiophene de-rivatives.[104] This represented the first example in which the1,3 dipolar reaction was applied to azidopyrrole deriva-tives.[103]

To evaluate the potential ability of the polycyclic ringsystem to interact with DNA, the LUMO and HOMO en-ergies were calculated. The analysis of the data underlinedthat the HOMOs (–8.86 eV) and LUMOs (–0.80 eV) of thesynthesised pyrrolo-triazolo-pyrimidine derivatives were


comparable with those of well-known intercalating agentssuch as doxorubicin (HOMO –9.10 eV, LUMO–1.57 eV).[103]

In 2002, in connection with these studies, another paperreported the application of the same synthetic approach tothe synthesis of the members of the tricyclic pyrrolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine system 78 (Figure 31), iso-mers of 76.[105]

Figure 31. Synthesis of members of the tricyclic pyrrolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine system 78 by means of domino re-actions.

It is well known that the reactivity of pyrrole derivativescan change widely on switching the substituents or on mov-ing them from β- to α-position.[106] Thus, the authors de-cided to investigate the reactivity of 3-azidopyrrole-2-carb-oxylate compounds.

In this case, after 24 h at room temperature, the reactionshowed mainly unreacted starting material. Knowing thatin the final step of the domino reaction ethanol is both theproduct and the solvent, the authors decided to remove itunder reduced pressure. By this expedient, a compound 78was obtained. Therefore, it seems that in this case the bond-forming efficiency depends on the amount of ethanol pres-ent in the reaction environment, in contrast with what wasobserved in the case of ethyl 3-azidopyrrole-4-carboxylate.All the derivatives of the annelated 1,2,3-triazolo[1,5-a]pyr-imidine ring system were investigated for their biologicalproperties and showed moderate antiproliferative activity.

In order to improve the biological activity and to extendthe application of 1,3-DCRs to other heterocycles, ethyl 2-azidoindole-3-carboxylate 79 was used (Figure 32).[107]

Figure 32. Application of 1,3-DCRs to ethyl 2-azidoindole-3-carb-oxylate 79 to obtain other heterocycles with improved biologicalactivity.

It reacted with the sodium salts of acetonitrile derivativesto give polycyclic compounds of type 80 in good yields (80–90%). It is noteworthy that 79 was very prone to undergo1,3-DCR and that the further ring closure to the pyrimidine


system was easier than in the case of the pyrrole ring.[107]

Another interesting peculiarity was seen in the energeticstudy of HOMO and LUMO: the calculated values for theindole compounds (LUMO –1.055 eV and HOMO–9.034 eV) were in fact closer to those of the well-knownintercalating agent doxorubicin than the calculated valuesfor pyrrole compounds seen above. Moreover, preliminarydocking studies to test their DNA binding capability werecarried out, and the results predict good intercalating capa-bility of this type of polyheterocycles. However, in the anti-proliferative tests most of the annelated triazolopyrimidineswere inactive up to 10–4 m concentration. Nevertheless, it isworth considering that, in the case of the antitumor classof actinomycins, the presence of side chains plays a funda-mental role in the DNA interacting capability. A few com-pounds designed with suitable chains in the N4 position,selected by molecular docking, were prepared. They showeda remarkable improvement on the original lead compoundantitumor activity at micromolar level.[108]

To exploit domino 1,3-DCRs further for the synthesis ofbiologically active compounds, the synthetic approach toindolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidines 82 (Figure 33),isomers of the previously discussed derivatives, was investi-gated.[109]

Figure 33. 1,3-DCRs for the synthesis of indolo[2,3-e][1,2,3]tri-azolo[1,5-a]pyrimidines 82.

Thus, ethyl 3-azidoindole-2-carboxylate 81 led to com-pounds 82 containing the 4H-indolo[2,3-e][1,2,3]triazolo-[1,5-a]pyrimidine ring system. It is worth mentioning thatin some cases it was impossible to obtain the correspondingtriazolo-pyrimidines, probably because of lower reactivityof some acetonitriles and/or the instability of the 3-azido-indole. For a preliminary evaluation of the ability of theobtained derivatives to interact with DNA, molecular dock-ing studies were also performed.[110] The in silico approachshowed that the triazolo-pyrimidines 82 are good DNA-binders, suggesting that they could be potential antitumordrugs.

In a recent work, chemometric features correlating withincreasing antitumor activity of the indolo[3,2-e][1,2,3]tri-azolo[1,5-a]pyrimidine derivatives 82 were analysed. Thesefindings were applied in the lead optimisation of annelatedtriazolo[1,5-a]pyrimidines, having a different ring fusionand consequently different aromatic features, to obtain newannelated benzothieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimid-ines 83 and pyrido[3�,2�:4,5]thieno[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine derivatives 84, that represent thio and aza-thioisosteres of the previously synthesised indolo[2,3-e][1,2,3]-triazolo[1,5-a]pyrimidine core 82 (Figure 34).[111]


Figure 34. The thio and aza-thio isosteres of the indolo[2,3-e]-[1,2,3]triazolo[1,5-a]pyrimidine core 82.

The synthetic methodology involved a 1,3-DCR, carriedout in dry ethanol, in which the azido moieties of ethyl 3-azidobenzo[b]thiophene-2-carboxylate (85a) or of the azaanalogue ethyl 3-azidothieno[2,3-b]pyridine-2-carboxylate(85b) played the dipolar component role, while the di-polarophiles were the sodium salts of acetonitriles (Fig-ure 35). As previously observed for the pyrrolo[2,3-e]-[1,2,3]triazolo[1,5-a]pyrimidines, this process was stronglyinfluenced by the amount of the solvent used in the reac-tion. Use of smaller volume of dry ethanol allowed the iso-lation of the cyclised compounds in good yields.

Figure 35. Synthetic pathway involving 1,3-DCRs, carried out indry ethanol, to obtain the angular isomers 86.

Both series of synthesised compounds were tested againsta panel of 60 human tumour cell lines and, as previouslyobserved in the case of the indolo-triazolo-pyrimidine de-rivatives, they showed poor antiproliferative activity, with asimilar response in all panels.[111]

Therefore, on the basis of the previous findings in theseries of pyrrolo-pyrimidine derivatives,[112] with the aim ofenhancing the biological activity of the benzo-thieno-tri-azolo-pyrimidine and pyrido-thieno-triazolo-pyrimidinecompounds, the same authors decided to apply the chemo-metric protocol Virtual Lock and Key (VLAK). This is acomputational methodology applied in an attempt to im-prove the activity of heterocyclic derivatives with poor bio-logical profiles.[113,114] An in-house database of structurescontaining the annelated benzo-thieno[2,3-e][1,2,3]tri-azolo[1,5-a]pyrimidine and pyrido[3�,2�:4,5]thieno[2,3-e]-[1,2,3]triazolo[1,5-a] pyrimidine systems suitably function-alised with a large number of side chains was built and sub-

A. Lauria et al.MICROREVIEWmitted to VLAK in order to identify new potential antican-cer derivatives and to choose the right side chain in thisway.[111]

Also in this case, the VLAK protocol was applied withuse of the good biological results obtained for theindolo[3,2-e][1,2,3]triazolo[1,5-a]pyrimidine derivatives 82discussed above as a guideline. The synthesis of the com-pounds selected on the basis of the VLAK protocol wassuccessfully achieved, and variously substituted thieno-tri-azolo-pyrimidines showed antitumour activity up to sub-micromolar concentration.[111]

Following the synthesis of the already mentioned angularsystems 87, incorporating a triazolo-pyrimidine moiety, thesame authors observed and reported peculiar behaviour ofthese class of compounds, known in literature as theDimroth rearrangement. This rearrangement consists of aring-opening followed by ring-closure of the pyrimidinering in the presence of water, giving the corresponding lin-ear systems 88 (Figure 36).[103,108,111]

Figure 36. Dimroth rearrangement to give linear systems 88.

However an unexpected Dimroth rearrangement in thereaction leading to annelated thieno[2,3-e][1,2,3]triazolo-[1,5-a]pyrimidine core 86 was observed.[115] This allowedthe isolation of the linear thieno[3,2-d][1,2,3]triazolo[1,5-a]-pyrimidine isomer 89. By decorating the linear isomer with

Figure 37. Dimroth rearrangement leading to linear isomer 89;most active compound of the series 90.


the same chains that had improved the biological activityof the angular isomers, new annelated thieno[3,2-d][1,2,3]-triazolo[1,5-a]pyrimidines were designed and synthesised.The biological results showed that the new derivatives ex-hibited strong antiproliferative activity up to nanomolarconcentrations. In vivo screenings of the most active com-pound, N-[2-(1H-imidazol-4-yl)ethyl]-4-(3-phenyl-10-oxo-4,10-dihydrobenzothieno[3,2-d][1,2,3]triazolo[1,5-a]pyr-imidin-4-yl)butanamide (90), showed low toxicity and highpotency (Figure 37).

Conclusions

This review has surveyed the occurrence of the 1,2,3-tri-azole nucleus in biologically active heterocyclic compounds.The synthesis of such compounds is achievable through 1,3-dipolar cycloadditions through both catalytic and uncata-lytic routes. In the former case, copper-catalysed azide–alk-yne cycloaddition (CuAAC) is the most efficacious: a clickreaction characterised by high reliability and total specific-ity in producing the 1,4-disubstituted triazole regioisomers.Other innovative synthetic approaches to 1,2,3-triazoles,such as engineering enzymatic strategy, Cu2O nanoparticlecatalysis and application of MWs, are mentioned. The po-tential of CuAAC reactions has been further amplifiedthrough their inclusion in multicomponent reaction pro-cesses, such as a Wittig/Knoevenagel/Diels–Alder sequence.In few cases two regioisomers have been formed, with aratio of approximately 1:1. Orthogonal cycloadditions togive exclusively 1,5-disubstituted regioisomer are reviewed.The CuAAC reaction, with the insertion of a triazole ring,revealed itself to be a powerful tool for joining lipids toproteins. The biological importance of the 1,2,3-triazolesystem as a linker between two active molecules to improvetheir pharmacokinetic and/or pharmacodynamic profileshas also been discussed. Use of 1,3-CDRs in a series ofdomino reactions is also described, with dipolarophile anionformation and cyclisation steps involved in the syntheticpathways. Linear 1,2,3-triazole isomers were finallyachieved through an unexpected Dimroth rearrangement.

Acknowledgments

This work was in part supported by an FFR2012 – University ofPalermo grant (CUP:B78C12000380001).

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Received: November 11, 2013Published Online: March 31, 2014

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