Two macrocyclic pentaaza compounds containing pyridine evaluated as novel chelating agents in...

Journal of Inorganic Biochemistry 105 (2011) 292ndash301

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Journal of Inorganic Biochemistry

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Two macrocyclic pentaaza compounds containing pyridine evaluated as novelchelating agents in copper(II) and nickel(II) overload

Ana S Fernandes a M Faacutetima Cabral a Judite Costa a Matilde Castro a Rita Delgado bcMichael GB Drew d Vitor Feacutelix e

a iMedUL Faculdade de Farmaacutecia Universidade de Lisboa Av Prof Gama Pinto 1649-003 Lisboa Portugalb Instituto de Tecnologia Quiacutemica e Bioloacutegica Universidade Nova de Lisboa Av da Repuacuteblica 2780-157 Oeiras Portugalc Instituto Superior Teacutecnico Av Rovisco Pais 1049-001 Lisboa Portugald School of Chemistry University of Reading Whiteknights Reading RG6 6AD UKe Departamento de Quiacutemica CICECO and Secccedilatildeo Autoacutenoma de Ciecircncias da Sauacutede Universidade de Aveiro 3810-193 Aveiro Portugal

Corresponding author Fax +351 217 946 470E-mail address jcostaffulpt (J Costa)

0162-0134$ ndash see front matter copy 2010 Elsevier Inc Aldoi101016jjinorgbio201011014

a b s t r a c t

a r t i c l e i n f o

Article historyReceived 1 July 2010Received in revised form 17 November 2010Accepted 19 November 2010Available online xxxx

KeywordsMacrocyclic compoundsStability constantsChelation therapyCopper(II) complexNickel(II) complex

Two pentaaza macrocycles containing pyridine in the backbone namely 3691218-pentaazabicyclo[1231]octadeca-1(18)1416-triene ([15]pyN5) and 36101319-pentaazabicyclo[1331]nonadeca-1(19)1517-triene ([16]pyN5) were synthesized in good yields The acidndashbase behaviour of these compounds wasstudied by potentiometry at 2982 K in aqueous solution and ionic strength 010 M in KNO3 The protonationsequence of [15]pyN5 was investigated by 1H NMR titration that also allowed the determination ofprotonation constants in D2O Binding studies of the two ligands with Ca2+ Ni2+ Cu2+ Zn2+ Cd2+ and Pb2+

metal ions were performed under the same experimental conditions The results showed that all thecomplexes formed with the 15-membered ligand particularly those of Cu2+ and especially Ni2+ arethermodynamically more stable than with the larger macrocycle Cyclic voltammetric data showed that thecopper(II) complexes of the two macrocycles exhibited analogous behaviour with a single quasi-reversibleone-electron transfer reduction process assigned to the Cu(II)Cu(I) couple The UVndashvisible-near IRspectroscopic and magnetic moment data of the nickel(II) complexes in solution indicated a tetragonaldistorted coordination geometry for the metal centre X-band EPR spectra of the copper(II) complexes areconsistent with distorted square pyramidal geometries The crystal structure of [Cu([15]pyN5)]

2+ determinedby X-ray diffraction showed the copper(II) centre coordinated to all five macrocyclic nitrogen donors in adistorted square pyramidal environment

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1 Introduction

The therapy for metal overload pathologies usually involves theadministration of suitable chelators to selectively remove the metal fromthe body Regarding copper(II) and nickel(II) metal ions there is still aneed for safe and efficient chelating agents as the existing ones havea number of drawbacks such as toxic side effects and controversialefficiency [1]

Copper as an essential element is a component of many metallopro-teins and enzymes and plays a vital role in electron transfer reactionsof many cellular processes However excessive copper can be verytoxic resulting in severe diseases [2] Certain chelating agents have beenshown to bind copper with high affinity Previous work on copper(II)chelation agents has focused on Wilsons disease which is an inheritedmetabolic disease of copper toxicity that is fatal if left untreated [3]D-Penicillamine has been one of the most commonly used chelatingagents for treatment of this disease When the patient cannot tolerate

treatment with D-penicillamine trien [NNprime-bis(2-aminoethyl)ethane-12-diamine] and ammonium tetrathiomolybdate are consideredsafer alternatives Trien is a lesser active agent for copper(II) removalin biological media than D-penicillamine and although both chelatorshave similar toxicity side effects are less frequent and generallymilder with D-penicillamine Ammonium tetrathiomolybdate actingdifferently from both D-penicillamine and trien has been used due toits lower toxic profile but it is still an experimental drug and its long-term efficacy is unknown [4]

Copper(II) chelation therapy attracts also attention in recentinvestigations and treatment of neurodegenerative disorders such asAlzheimer Parkinson and CreutzfeldtndashJakob [5] Furthermore anexcess of copper appears to be an essential co-factor for angiogenesisMoreover high levels of copper were found in many human cancersincluding prostate breast colon lung and brain Consequently thetherapeutic value of copper(II) chelators as anti-angiogenic moleculesin the treatment of these cancers has been reported [6] More recentlymixtures of copper(II) chelators and copper salts were found to act asefficient proteasome inhibitors and apoptosis inducers specifically incancer cells [7]

293AS Fernandes et al Journal of Inorganic Biochemistry 105 (2011) 292ndash301

On the other hand human exposure to nickel occurs primarily viainhalation and ingestion through occupational exposure anddiet leadingto adverse effects on human health Nickel allergy in the form of contactdermatitis is the most common and well-known reaction Although theaccumulation of nickel in the body through chronic exposure can causelung fibrosis kidney and cardiovascular diseases the most seriousconcerns relate to nickels carcinogenic activity Epidemiological studieshave clearly implicated nickel compounds as human carcinogens [89]All nickel compounds except for metallic nickel were classified ascarcinogenic to humans in 1990 by the International Agency for Researchon Cancer (IARC) [10]

Over the years various chelators have been investigated for theirability to bind nickel The most effective ones are EDTA DTPA(diethylenetriaminepentaacetic acid) diethyldithiocarbamate tet-raethylthiuram disulfide and clioquinol (5-chloro-8-hydroxy-7-iodoquinoline) all of them presenting considerable side effects[81112]

Therefore the development of novel chelators selective for nickel(II)and for copper(II) and exhibiting minor side effects is an imperativeresearch This led us to investigate the possible use of macrocycliccompounds In factmetal chelates ofmacrocycles often showpropertiesthat are particularly different from those of analogous open chainchelators Macrocycles having more rigid structures can impose specificcoordination geometry to the metal ion whereas open chain chelatorsadapt more easily to the geometric requirements of the metal centre[13] In the present work the synthesis and characterization of twopentaaza macrocyclic compounds containing pyridine in the backbone[15]pyN5 (3691218-pentaazabicyclo[1231]octadeca-1(18)1416-tri-ene) and [16]pyN5 (36101319-pentaazabicyclo[1331]nonadeca-1(19)1517-triene) cf Scheme 1 as well as the study of their copper(II)and nickel(II) complexes are reported in order to evaluate their possibleuseas chelatingagents Toaccomplish this aim theacidndashbasebehaviourofthese two macrocycles was studied and their ability to coordinate Cu2+

and Ni2+ and other divalent metal ions (Ca2+ and Zn2+ are included dueto their essential role in living organisms) was evaluated The adoptedstructures of the Cu(II) and Ni(II) complexes were also studied byspectroscopicmethods in solution and the single crystal X-ray diffractionof [Cu[15]pyN5](PF6)2 was determined Finally due to the important roleof the redox behaviour of the copper(II) complexes in biology somevoltammetric studies were carried out

2 Experimental section

21 General procedures

Elemental analysis was performed on a VarioEL CHNS analyserfrom vacuum-dried powder samples Melting points were determinedwith a Koumlpffer Melting Point apparatus

211 Reagents26-Pyridinedimethanol NNprime-bis(2-aminoethyl)ethane-12-diamine

and NNprime-bis(2-aminoethyl)13-propanediamine were purchased fromAldrich 26-Pyridinedicarbaldehyde was prepared by publishedmethods[14] All the commercially available chemicals were of reagent grade and

Scheme

used as supplied without further purification Organic solvents werepurified or dried by standard methods [15]

Caution Althoughnoproblemswere found in thiswork perchloratesin the presence of organic matter are potentially explosive and should beprepared in small quantities

22 Synthesis of the macrocycles

221 Synthesis of the macrocycle [15]pyN5

To a stirred solution of freshly prepared 26-pyridinedicarbalde-hyde (234 g 18 mmol) in methanol (40 mL) was added a solution ofPb(NO3)2 (61 g 18 mmol) in water (80 mL) To the resulting solutionwas added dropwise with rapid stirring a solution of NNprime-bis(2-aminoethyl)ethane-12-diamine (335 g 18 mmol) in methanol(40 mL) over a period of 3 h The solution was stirred while heatingunder reflux for 7 h during which time an intense deep red colourdeveloped After reflux the solution was cooled to 5 degC and sodiumborohydride (436 g 455 mmol) was added in small portions over60 min The yellow solution obtainedwas stirred at room temperaturefor 30 min and then heated on a hot water-bath at 60 degC for 30 minbefore being left overnight at room temperature Leadwas removed bytreating the mixture with Na2S9H2O (10 g 42 mmol) followed byheating on a hot water-bath for 30 min The solution was then cooledand lead(II) sulphidewas removed byfiltration through a bed of CeliteThe filtrate was extracted with dichloromethane (4times50 mL) thecombined extracts were dried with anhydrous MgSO4 and the dichlor-omethane was removed with a rotary evaporator to leave a light yellowoil This oil was dissolved in methanol and 37 hydrochloric acid wasadded until pHasymp2 During the addition an off-white solid precipitatedwhichwas identified as the pure desired compound Yield 85Mp 280ndash282 degC (decomp) 1H NMR (D2O pD=510) δ 321 (4H s (singlet) NndashCH2) 341 (4H t (triplet) NndashCH2ndashCH2ndashN) 351 (4H t NndashCH2ndashCH2ndashN)457 (4H s NndashCH2ndashpy) 753 (2H d (doublet) py) and 799 (1H t py)ppm 13C NMR (D2O pD = 510) δ 4392 (NndashCH2ndashCH2ndashN) 4564 (NndashCH2ndashCH2ndashN) 4633 (NndashCH2) 4992 (NndashCH2ndashpy) 12407 (py) 14033 (py)and 15094 (py) ppm Found C 3703 H 716 N 1656 Calc forC13H23N5middot4HClmiddot15H2O C 3698 H 716 N 1659


A procedure analogous to that described for [15]pyN5 was usedreplacing NNprime-bis(2-aminoethyl)ethane-12-diamine by NNprime-bis(2-aminoethyl)13-propanediamine The product was obtained as a thickyellow oil which was purified by passing through a neutral aluminacolumn (25times30 cm) and eluting with dichloromethanendashmethanol(1005 vv) The pure compound was dissolved in methanol and 37hydrochloric acidwas added until pHasymp2 An off-white salt precipitatedYield 46 Mp 266ndash268 degC (decomp) 1H NMR (D2O pD=255) δ 221(2 H q (quintuplet) CH2ndashCH2ndashN) 337 (4H t CH2ndashCH2ndashN) 365 (4H tNndashCH2ndashCH2ndashN) 371 (4H t NndashCH2ndashCH2ndashN) 463 (4H s NndashCH2ndashpy)756 (2H d py) and 800 (1H t py) ppm 13C NMR (D2O pD = 255) δ2130 (CH2ndashCH2ndashN) 4225 (NndashCH2ndashCH2ndashN) 4320 (NndashCH2ndashCH2ndashN)4414 (CH2ndashCH2ndashN) 5135 (NndashCH2ndashpy) 12450 (py) 14044 (py) and15068 (py) ppm Found C 3759 H 780 N 1537 Calc for C14H25-

N5middot4HClmiddot2H2O C 3780 H 750 N 1570

1

294 AS Fernandes et al Journal of Inorganic Biochemistry 105 (2011) 292ndash301

223 Synthesis of the copper(II) complex [Cu[15]pyN5](PF6)2An aqueous solution of Cu(ClO4)26H2O (0150mmol 0056 g) was

added to a stirred solution of [15]pyN5 (0150 mmol 00593 g) dissolvedin theminimumvolume of water (asymp 1 mL) Then 00489 g (0300 mmol)of NH4PF6was added and themixturewas stirred at 60 degC for 1 h The pHof the solutionwas increased to 62 by addition of a solution of KOH01 Mand the solventwas evaporatedunder vacuum The residuewas taken in aminimum amount of methanolndashacetonitrile (1025) Blue crystals wereformed in about 4 weeks by slow evaporation of the solvent mixture at4 degC Yieldasymp 80

23 Potentiometric measurements

231 Reagents and solutionsStock solutions of the ligands were prepared at ca 250times10minus3 M

Metal ion solutions were prepared at about 0025 to 0050 M from nitratesalts (analytical grade) in demineralized water (from a MilliporeMilli-Qsystem) and were standardized by titration with Na2H2EDTA [16]Carbonate-free solutions of the titrant KOH were prepared at ca010 M by dilution of a commercial ampoule of Titrisol (Merck) withdemineralized water under a stream of pure argon gas These solutionswere discarded every time carbonate concentrationwas about 05 of thetotal amountofbase For theback titrations a0100 Mstandard solutionofHNO3 prepared from a Merck ampoule was used The titrant solutionswere standardized (tested by Gran method) [17] For the competitiontitrations a standard K2H2EDTA aqueous solution was used

232 Equipment and work conditionsThe potentiometric setup for conventional titrations consisted of a

50 mL glass-jacketed titration cell sealed from the atmosphere andconnected to a separate glass-jacketed reference electrode cell by aWilhelm-type salt bridge containing 010 M KNO3 solution An Orion720A+measuring instrument fitted with a Metrohm 60150100 glasselectrode and a Metrohm 60733100 AgndashAgCl reference electrode wasused for themeasurements The ionic strength was kept at 010plusmn001 Mwith KNO3 temperature was controlled at 2982plusmn01 K by circulatingwater through the jacketed titration cell using a Huber Polystat cc1thermostat and atmospheric CO2 was excluded from the titration cellduring experiments by passing argon across the top of experimentalsolution Titrant solutionswere added through capillary tips at the surfaceof the experimental solution by a Metrohm Dosimat 765 automaticburette Titration procedure was automatically controlled by softwareafter selection of suitable parameters allowing for long unattendedexperimental runs

233 MeasurementsThe [H+] of the solutions was determined by the measurement of

the electromotive force of the cell E=Eo+Q log[H+]+Ej The termpH is defined as minuslog [H+] Eo and Q were obtained by titrating asolution of known hydrogen-ion concentration at the same ionicstrength using the acid pH range of the titration The liquid-junctionpotential Ej was found to be negligible under our experimentalconditions The value of Kw was determined from data obtained in thealkaline range of the titration considering Eo and Q valid for the entirepH range and found to be equal to 10minus1380 M2 The potentiometricequilibrium measurements were carried out using 2000 mL of ca250times10minus3 M ligand solutions diluted to a final volume of 3000 mLin the absence of metal ions and in the presence of each metal ion forwhich the CMCL ratio was 11 For the reactions of Cu2+ with bothligands competition titrations were performed K2H4EDTA was usedas the reference ligand for which values of protonation and stabilityconstants were determined before under the same experimentalconditions log K1

H=1022 log K2H=616 log K3

H=271 log K4H =20

log KCuEDTA=1923 log KCuHEDTA=306 log KCuEDTAOH=1133 [18]Ratios of 07511 and 111 (CLCLCCu) were used for L=[15]pyN5

and [16]pyN5 respectively and Lprime=EDTA The competition reactions

reached equilibrium upon 15 to 20 min at each point in the pH rangewhere the competition reaction took place The same values for thestability constants were obtained in both directions of the reactionthe direct curve titrating with KOH and the back titration with HNO3

234 Calculation of equilibrium constantsOverall equilibrium constants βi

H and βMmHhLl (being βMmHhLl=[MmHhLl][M]m[H]h[L]l) were calculated by fitting the potentiometricdata from protonation or complexation titrations with the HYPERQUADprogram [19] Species distribution diagrams were plotted from thecalculated constants with the HYSS program [20] Only mononuclearspecies ML MHL and MH-1L were found for the metal complexes of theligands (beingβMH-1L=βMLOHtimesKw)Differences in logunits between thevalues of βMHL (or βMH-1 L) and βML constants provide the stepwisereaction constants The species considered in a particular model werethose that could be justified by the principles of coordination chemistryThe errors quoted are the standard deviations of the overall stabilityconstants given directly by the program for the input data which includeall the experimental points of all titration curves and determined by thenormal propagation rules for the stepwise constants

Protonation constants were obtained from ca 180 experimentalpoints and stability constants for each metal ion were determinedfrom 120 to 180 experimental points (2 or 3 titration curves)

24 NMR measurements

241 Characterization of the macrocyclesThe 1H (40013 MHz) and 13C NMR (10062MHz) spectra were

recorded on a Bruker Avance-400 spectrometer at 294 K probetemperature Chemical shifts (δ) were given in ppm and couplingconstants (J) in Hz The NMR spectra were performed in CDCl3 (δ ppm1H 726 13C 7716) or in D2O The reference used for the 1H NMRmeasurements in D2O was 3-(trimethylsilyl)propionic acid-d4-sodiumsalt (DSS) and in CDCl3 the solvent itself (at 726 ppm) For 13C NMRspectra 14-dioxane (δ ppm 1H 375 13C 6720) was used as internalreference 2D NMR spectra correlation spectroscopy (COSY) hetero-nuclear multiple quantum coherence (HMQC) and heteronuclearmultiple bond correlation (HMBC) were acquired using gradient pulseprograms from Bruker library Phase-sensitive nuclear Overhauser effectspectroscopy (NOESY) was performed using a mixing time of 15 s Twoand monodimensional FIDs were processed using the TopSpin softwareversion13 fromBruker Peakassignmentswerebasedonpeak integrationand multiplicity for 1H spectra and on 2D experiments for 13C spectra

242 NMR titration measurementsThe titration of [15]pyN5 (0010 M in D2O) was carried out in the

NMR tube The pD values were adjusted by adding DCl or CO2-freeKOD solutions Theminuslog [H] was measured directly in the NMR tubewith a combined glass AgndashAgCl microelectrode (Mettler-ToledoU402-M3-S7200) coupled with an Orion 3 Star pH meter Theelectrode was previously standardized with commercial aqueousbuffer solutions and the pD values were calculated according to theequation pD=pH+(040plusmn002) where pH is the direct pHreading [21] The dissociation constants in D2O (pKD) were calculatedfrom the NMR titration data using a non-linear least-squares curve-fitting procedure that minimizes the sum of the squares of thedeviations of the observed and calculated values of the chemicalshifts These pKD values were converted to pKH values obtained inwater by the equation pKD=011+110timespKH [21]

243 Magnetic momentsMagnetic moments were measured at 294 K using solutions of Ni

[15]pyN52+ (238times10minus2 M pH 645) and Ni[16]pyN5

2+ (223times10minus2 M632) in D2O The 1HNMR spectra of the solutionswith DSS as internalreference were acquired in a tube containing an internal capillary


filled with D2O and DSS and the corresponding magnetic momentscalculated from the shift (Δδ) between both reference signals [22]

25 Spectroscopic studies

Electronic spectra were recorded with a UNICAM model UV-4(UVndashvisible) or a Shimadzu model UV-3100 (UVndashvisible-near IR)spectrophotometers using aqueous solutions of Ni2+ and Cu2+

complexes of both macrocycles (10times10minus2 to 10times10minus3 M) at pHs663 to 705

EPR spectroscopy measurements of copper(II) complexes of [15]pyN5 and [16]pyN5 were recorded at 99 K with a Bruker EMX 300spectrometer equipped with continuous-flow cryostats for liquidnitrogen operating at X-band The complexes were prepared at about10times10minus3 M and pH values of 496 723 and 979 for Cu[15]pyN5

2+ and504 723 and 992 for Cu[16]pyN5

2+ in 1 M NaClO4 aqueous solution

26 Electrochemical studies

A BAS CV-50W Voltammetric Analyzer connected to BASWin-dows data acquisition software was used Cyclic voltammetricexperiments were performed in a glass cell MF-1082 from BAS in aC-2 cell enclosed in a Faraday cage at room temperature under argonThe reference electrode was AgndashAgCl (MF-2052 from BAS) filled withNaCl3 MinwaterstandardizedfortheredoxcoupleFe(CN)63minusFe(CN)64minus The auxiliary electrode was a 75-cm platinum wire (MW-1032 from BAS) with a gold-plated connector The working electrodewas a glassy carbon (MF-2012 from BAS)

Copper(II) complexes of [15]pyN5 and [16]pyN5 (163times10minus3 MpH=705 and 146times10minus3 M pH=709 respectively) were preparedin 01 M KNO3 in water The solutions were deaerated by an argonstream prior to all measurements and were kept under argon duringthe measurements Between each scan the working electrode waselectrocleaned by multi-cycle scanning in the supporting electrolytesolution polished on diamond 1 μm and on alumina 03 μm cleanedwith water and sonicated before use according to standardprocedures

Cyclic voltammograms with sweep rate ranging from 25 to1000 mV sminus1 were recorded in the region from +12 to minus12 V Atthis potential range the ligands were found to be redox inactive Thehalf-wave potentials E12 were obtained by averaging the anodic andcathodic peak potentials All potential values are reported relative tothe AgndashAgCl reference electrode and the E12 andΔEp of the Fe(CN)63minusFe(CN)64minus couple under our experimental conditions were 196 mVand 73 mV respectively

27 X-ray crystallography

Blue crystals of [Cu[15]pyN5](PF6)2 with suitable quality for singlecrystal X-ray diffraction determination were grown up from metha-nolndashacetonitrile solution

Crystal data C13H21CuF12N5 Mr=60083 monoclinic spacegroup P21c Z=4 a=88619(9) b =149388(14) c=166689(16)Aring β=103674(9)deg U=21442(4) Aring3 ρ(calc)=1861 Mg mminus3 μ(Mo-Kα) = 1283 mmminus1

X-ray datawere collected at 150(3) K on a CCDX-calibur plate systemusing graphite monocromatized Mo-Kα radiation (λ=071073 Aring) atReading University The selected crystal was positioned at 50 mm fromthe CCD and the frames were taken using a counting time of 2 s Theprocessing of the data was carried out with the Crysalis program [23]Intensities were corrected for empirical absorption effects with theABSPACK program [24] The structure was solved by direct methods andbysubsequentdifferenceFourier synthesesand refinedby fullmatrix leastsquares on F2 using the SHELX-97 suite [25] Anisotropic thermalparameters were used for the non-hydrogen atoms The hydrogenatoms bonded to carbon and nitrogen atomswere included in refinement

in calculated positions with isotropic parameters equivalent to 12 timesthose of the atom to which they were attached The final refinement of298 parameters converged to final R and Rw indices R1=00467 andwR2=01028 for 2861 reflections with IN2σ(I) and R1=00992 andwR2=01078 for all 6262 hkl data Molecular diagrams presented aredrawn with graphical package software PLATON [26]

3 Results and discussion

31 Synthesis and characterization of the macrocycles

Compounds [15]pyN5 and [16]pyN5 were prepared in good yieldby [11] condensation of 26-pyridinedicarboxaldehyde and NNprime-bis(2-aminoethyl)ethane-12-diamine (trien) and NNprime-bis(2-ami-noethyl)13-propanediamine respectively using Pb2+ as the tem-plate ion followed by reduction of the resulting tetraimines withsodium borohydride The pure products were obtained as tetrahy-drochloride salts in 85 and 46 yields respectively The lower yieldof the later compound results from the unfavourable adoptedgeometry of the lead(II) complex during the cyclization reaction [27]However Ca2+ or Ba2+ did not lead to better yields

Both macrocycles were synthesized by different and more timeconsuming procedures [28ndash30] Stetter et al [28] prepared [15]pyN5

following amodified Richman and Atkinsmethod [31] in 78 yield andRiley et al [29] followed the same procedure with minor changesKimura et al [30] prepared [16]pyN5 in unspecified yield by refluxingthe bisdiethyl esters of pyridine-26-dicarboxylic acid and NNprime-bis(2-aminoethyl)13-propanediamine in ethanol and high dilution followedby reduction of the resulted diamide with diborane in tetrahydrofuran

1D and 2D NMR spectroscopy were used for characterization of [15]pyN5 and [16]pyN5 The chemical shifts and the corresponding assign-ments were accomplished by 1H 13C COSY HMQC HMBC and NOESY atpD 510 and 255 respectively as described in Appendix A of theSupplementary material (cf Table S1 and Figs S1ndashS5)

32 Acidndashbase behaviour of the ligands

The acidndashbase behaviour of [15]pyN5 and [16]pyN5 was studied bypotentiometry in water at 2982 K and ionic strength 010 M in KNO3The former compound was also studied by 1H NMR spectroscopy Thedetermined protonation constants are collected in Table 1 togetherwith the values of the related [15]aneN5 and [16]aneN5 compounds(cf Scheme 1) for comparison Both compounds have five basiccentres however only three constants for [15]pyN5 and four for [16]pyN5 could be accurately determined by potentiometry and one morefor [15]pyN5 was obtained by 1H NMR The two compounds exhibithigh and fairly high values respectively for the first two protonationconstants corresponding to the protonation of nitrogen atoms inopposite positions minimizing the electrostatic repulsion betweenpositive charges of the ammonium groups formed The third andfourth constants are much lower due to the stronger electrostaticrepulsions as they correspond to protonation of nitrogen atoms atshort distances from already protonated ones and to the limitedmotion allowed in the ring backbone The increase in basicity of thesetwo last centres in [16]pyN5 is correlated with the increase of thelength of the chain between contiguous nitrogen atoms The valuesreported before (in NaClO4 medium) [29ndash33] shown in Table 1 differslightly from ours however for the first time we were able toaccurately determine the fourth protonation constant

The overall basicity and all the stepwise protonation constants of [15]pyN5and [16]pyN5 (see Scheme1andTable1) are smaller than that of thecorresponding macrocycles without pyridine as expected taking intoaccount the electron withdrawing effect of the pyridine ring

1H NMR spectroscopic titration of [15]pyN5 was carried out inorder to understand its protonation sequence and to determine thelower protonation constants In Fig 1 is shown the spectrum of the

Table 1Stepwise protonation constants (log Ki

H) of [15]pyN5 [16]pyN5 and other similar compounds for comparisona T=2982 K I=010 M in KNO3

Equilibrium quotient [15]pyN5 [16]pyN5 [15]aneN5b [16]aneN5

b

[HL+][H+]times[L] 9616(8) 943c 911d 971(1) 948e 1085 1064[H2L2+][HL+]times[H+] 867(1) 880c 882d 832(3) 856e 965 949[H3L3+][H2L2+]times[H+] 533(2) 528c 527d 556(5) 583e 600 728[H4L4+][H3L3+]times[H+] 14(2)f ndash 237(8) b2e 174 171[H5L5+][H4L4+]times[H+] ndash ndash ndash ndash 116 145

[H4L4+][L]times[H+]4 2502 2596 ndash 2824 2912

a Values in parentheses are standard deviations on the last significant figureb T=2982 K I=02 M in NaClO4 ref [33]c T=2982 K I=01 M in NaClO4 ref [29]d T=2982 K I=01 M NaClO4 ref [32]e T=2982 K I=02 M in NaClO4 ref [30]f Determined in this work by 1H NMR spectroscopy using the calculated value of pKD4 and the equation pKD=011+110timespKH [21]


ligand at pD 510 and the titration curves for all resonances The 1HNMR spectrum exhibits six resonances in the 124ndash704 pD region butfor higher pD values Hd and He resonances overlap The resonances at

Fig 1 (a) 1H NMR titration curves for [15]pyN5 chemical shift δH (ppm)

799 and 753 ppm were assigned to Ha and Hd protons the twosinglets at 457 and 321 ppm to Hc and Hf protons and the triplets at351 and 341 ppm to Hd and He protons respectively

in function of pD (b) 1H NMR spectrum of [15]pyN5 (D2O pD 510)

Table 4pM values for [15]pyN5 H4EDTA and H5DTPA with some divalent metal ions

Ion [15]pyN5a H4EDTAb H5DTPAb

Ca2+ 500 789 655Ni2+ 1819 1568 1601Zn2+ 1424 1384 1444

a Calculated from the constants in Tables 1 and 2b Calculated from the values of the protonation constants and of the stability

constants reported in refs [1838] All the values calculated for 100 excess of freeligand at physiological conditions pH=740 CM=10times10minus5 M CL=20times10minus5 Musing the Hyss program [20]

Table 2Stepwise stability constants (log units) of the complexes of [15]pyN5 [16]pyN5 andother related ligands with several metal ionsa T=2982 K I=010 M

Equilibrium quotient [15]pyN5 [16]pyN5 [15]aneN5 [16]aneN5

[CaL2+][Ca2+]times[L] 321(2) ndash ndash ndash

[NiL2+][Ni2+]times[L] 2171(1) 1681(1) 181b 181c

[NiHL3+][NiL2+]times[H+] ndash 288(2) ndash ndash

[CuL2+][Cu2+]times[L] 2331(3) 2086(4) 283d 271d

[CuHL3+][CuL2+]times[H+] 231(4) 359(6) 43d ndash

[CuL2+][CuLOH+]times[H+] 1006(6) 1183(7) ndash ndash

[ZnL2+][Zn2+]times[L] 1776(2) 1530(1) 191e 179 e

[ZnHL3+][ZnL2+]times[H+] 268(3) 328(3) 31e 37 e

[CdL2+][Cd2+]times[L] 16853(7) 15532(4) 192e 181e

[CdHL3+][CdL2+]times[H+] 264(4) 305(2) 34e 39e

[PbL2+][Pb2+]times[L] 1544(2) 12932(6) 173e 143e

[PbHL3+][PbL2+]times[H+] 34(3) 361(5) 38e 50e

[PbL2+][PbLOH+]times[H+] ndash 1066(2) ndash ndash

a Values in parentheses are standard deviations on the last significant figureb T=3082 K ref [35]c Ref [36]d T=2982 K I=02 M polarographic method ref [37]e T=2982 K I=02 M ref [37]


The 1HNMR titration curves show the effect of successive protonationof basic centres of the molecule The first equivalent protonates mainlyN3 since the downfield shift of Hf resonance is larger followed by the shiftof He and Hd protons The Hc resonance has a very small shift in this pDrangemeaning a small percentageof protonationofN2 atoms The secondacid equivalent added protonates mainly the N2 centre as Hc Hb andHa resonancesmovedownfield SimultaneouslyN3 centrewasprotonatedto a low degree as the Hf He and Hd resonances show a slight downfieldshift The third acid equivalent (pD 721ndash312) continues protonatingthe N2 centre since c and d resonances shift downfield A slight shiftof He and Hf resonances reveals a small percentage of protonation on N3

centres The addition of one more equivalent of acid (pD 312ndash124) onlyprotonates N3 atoms as Hf He and Hd resonances move downfield Theabsence of any change on Ha Hb and Hc resonances suggests that N1 isnot protonated even at very low pD values

The 1H NMR titration also allowed the determination of the pro-tonation constants in D2O for [15]pyN5 pKD1=1061(7) pKD2=96(l)pKD3=629(7) and pKD4=16(2) These values are in agreement withthe equation for the correlation between the protonation constantsdetermined in H2O and in D2O for polyaza and polyoxandashpolyazamacrocyclic compounds pKD=011+110timespKH [21]

33 Thermodynamic stability of metal complexes

The stability constants of [15]pyN5 and [16]pyN5 with Ca2+ Ni2+Cu2+ Zn2+ Cd2+ and Pb2+ determined by potentiometric titrationsat the experimental conditions already indicated for the protonationconstants are collected in Table 2 together with those of the relatedmacrocycles [15]aneN5 and [16]aneN5 taken from the literature forcomparison Only mononuclear species (11 metal-to-ligand ratio)were found for the complexes of bothmacrocycles In most cases only

Table 3pM valuesa calculated for [15]pyN5 [16]pyN5 and other similar ligands with severaldivalent metal ions

Ion [15]pyN5 [16]pyN5 [15]aneN5 [16]aneN5

Ca2+ 500 ndash ndash ndash

Ni2+ 1819 1352 1238 1252Cu2+ 1979 1757 2278 2152Zn2+ 1424 1201 1338 1232Cd2+ 1334 1225 1348 1252Pb2+ 1192 965 1158 872

a Calculated from the constants in Tables 1 and 2 for 100 excess of free ligand atphysiological conditions pH=740 CM=10times10minus5M CL=20times10minus5M using theHyss program [20]

ML2+ and MHL3+ are formed but MLOH+ species were also found forCu2+ with [15]pyN5 and [16]pyN5 and Pb2+ with [16]pyN5 In allcases the proposed model was accepted by the HYPERQUAD program[19] using all data points from all titration curves with good statisticalparameters The complexes of Co2+ with both ligands wereimpossible to study due to their fast oxidation which occurred evenunder argon atmosphere owing to small oxygen impurities in thepurge gas The very low value for the Ca[16]pyN5

2+ was alsoimpossible to obtain by the method used Direct determinations ofthe stability constants of Cu[15]pyN5

2+ and Cu[16]pyN52+ were not

possible as ML2+ was completely formed in the beginning of thetitration (pHasymp22) and consequently reliable values for theconstants were obtained through a competition with a second ligandfor which the protonation and stability constants are accuratelyknown [34] Among the various ligands tried H4EDTA was chosen asthe best second ligand In spite of the higher overall basicity of [16]pyN5 this ligand forms ML2+ complexes less stable than those of [15]pyN5 (variations of 132 to 49 log units) being the largest decrease forthe nickel(II) complexes followed by the copper(II) ones Howevercontrary to [15]pyN5 [16]pyN5 forms ML2+ complexes with Cd2+

slightly more stable than with Zn2+ Differences in the cavity size ofboth ligands are responsible for this different behaviour

The comparisonof stability constant values of themetal complexes ofthe ligandswith andwithout pyridine (cf Scheme1 andTable 2) [35ndash38]reveals that the former complexes present lower values except for Ni[15]pyN5

2+ However stability constants do not provide directlycomparable basis for the measuring total ion sequestering abilities ofthe ligands at physiological conditions (pH 74) and therefore they wereused to calculate the pM values defined asminuslog [M2+ ] (cf Table 3) Theadvantage of comparing pM values rather than stability constants is thatthe pM values reflect the influence of ligand basicity and metal chelate

Fig 2 Species distribution curves for aqueous solutions containing Ni2+ Zn2+ Cd2+Pb2+ and [15]pyN5 (L) at 11111 molar ratio Percentages are given relative to thetotal amount of [15]pyN5 at an initial value of 167times10minus3 M

Table 5Spectroscopic UVndashvisible-near IR data and magnetic moments (μ) for the Ni(II) complexes of [15]pyN5 and [16]pyN5

Complex color pH UVndashvisible-near IRa λmaxnm (ε Mminus1 cmminus1) μ (MB)

Ni[15]pyN52+ (yellow) 669 1150 (47) 930 (108) 800 (sh 90) 600 (100) 530 (112) 306 (1726) 262 (248times103) 324

Ni[16]pyN52+ (blue) 686 1148 (187) 1060 (sh 293) 940 (418) 880 (sh 373) 810 (sh 271) 625 (178) 403 (182) 345 (820) 309 (1767) 262 (216times103) 337

a sh=shoulder


protonation The pM values for the nickel(II) complexes of [15]pyN5 and[16]pyN5 are significantly higher than those of [15]aneN5 and [16]aneN5

(differences are in log units 581 for the 15-memberedmacrocycles and10 for the 16-membered ones) The zinc(II) cadmium(II) and lead(II)complexes with both series of macrocycles have pM values of the sameorder of magnitude while for copper(II) complexes the values aremarkedly higher for ligands without pyridine (differences in log unitsare 299 for the 15-membered and 395 for the 16-memberedmacrocycles) The last pM differences can in part be related to theconformation adopted by the ligands upon complexation Neverthelessa polarographic technique was used for stability constants determina-tions of Cu[15]aneN5

2+ and Cu[16]aneN52+ and additionally no

competition reaction with a second ligand was employed We areconvinced that those values should be confirmed for definitiveconclusions to be drawn

The very high stability constant value of Ni[15]pyN52+ led us to

evaluate the potential role of [15]pyN5 as chelator for removal ofexcess of nickel(II) from the body In Table 4 are collected the pMvalues for nickel(II) and other two important biological metal ionscalcium(II) and zinc(II) for our chelator together with clinically usedones namely H4EDTA and H5DTPA The [15]pyN5 presents not onlythe highest pNi value but also the largest selectivity (differencebetween pM values) towards zinc(II) and calcium(II)

Fig 3 X-band EPR spectra of Cu[15]pyN52+ (a) and Cu[16]pyN5

2+ complexes (b) in anaqueous solution of 10times10minus3 M and in 10 M of NaClO4 both recorded at 99 K andat pH 723 Microwave power of 20 mW modulation amplitude of 10 mT and thefrequency (ν) was of 941 GHz The simulated spectra are shown in gray theexperimental ones are in black

Additionally [15]pyN5 can be used in the quantitative determina-tion of Ni2+ in solutions containing also zinc(II) cadmium(II) andlead(II) (in similar amounts) as can be observed by the speciesdistribution diagram in Fig 2


341 Nickel(II) complexesThe UVndashvisible-near IR spectra for Ni[15]pyN5

2+ and Ni[16]pyN52+

complexes were recorded in water solutions at pH values 669 and 686respectively (cf Table 5) The electronic spectrum of the yellow Ni[15]pyN5

2+ exhibits two absorption bands of low intensities at 530 and930 nm and the charge transfer band at 262 nm The Ni[16]pyN5

2+

complex is blue and the spectrum also exhibits one intense peak that isascribed to a charge-transfer absorption band and bands at 625 and940 nm The 175 and 150 ratios between the near IR (ν1) and the visible(ν2) bands and the corresponding magnetic moments of 324 BM and of337 BM calculated for the two complexes respectively are characteristicof high-spin six coordinate nickel(II) centre in distorted tetragonalsymmetry [39] where the five positions are occupied by nitrogen atomsof the backbone and the last position by the solvent (water or metalcounter-ion nitrate) Therefore in solution both complexes exhibitstructures that are not quite different even though the Ni[15]pyN5

2+

presents stronger equatorial field and Ni[16]pyN52+ a more distorted

geometryFollowing considerations of Busch and co-workers [40] we

assigned the visible-near IR bands to 3B1grarr3B2g directly related to10Dqxy and 3B1g rarr 3Ega equal to the difference between 10Dqxy and354Dt transitions The values of the equatorial and axial ligand fieldwere calculated based on these assignments Dqxy=1887 cmminus1 andDqz=260 cmminus1 for Ni[15]pyN5

2+ and Dqxy=1600 cmminus1 andDqz=525 cmminus1 for Ni[16]pyN5

2+ Therefore Dqz is strongly influencedby the in-plane ligand field and decreases as Dqxy increases as found inother cases [41] Similar geometry was described for Ni(Me2[15]pyN5)2+

(Me2[15]pyN5=213-dimethyl-3691218-pentaazabicyclo[1231]-octadeca-1(18)1416-triene) based on spectroscopic studies in solutionand supported by molecular models [42]

342 Copper(II) complexesThe Cu[15]pyN5

2+ and Cu[16]pyN52+ exhibit broad bands in the

visible region due to the copper dndashd transitions with λmax at 610 and646 nm respectively The corresponding X-band EPR spectra exhibitthe four expected lines at low field due to the interaction of theunpaired electron spin with the copper nucleus and a strongunresolved band at high field see Fig 3 Bands in the visible region(λmax) and the hyperfine coupling constants Ai (i=x y and z) and gvalues obtained by the simulation of the spectra [43] are shown in

Table 6Spectroscopic X-band EPR data for the Cu(II) complexes of [15]pyN5 and [16]pyN5

Complex Visible band λmaxnm(ε Mminus1 cmminus1)

EPR parametersAitimes104 cmminus1

gx gy gz Ax Ay Az

Cu[15]pyN52+ 610 (150) 2035 2070 2210 269 400 1706

Cu[16]pyN52+ 646 (143) 2038 2077 2222 232 335 1638

Table 8Selected bond distances (Aring) and angles (deg) in the copper(II) coordination sphere

CundashN(4) 2060(3)CundashN(7) 1921(3) CundashN(10) 2011(2)CundashN(1) 2229(3) CundashN(13) 2034(3)N(7)ndashCundashN(10) 825(1) N(1)ndashCundash-N(4) 817(1)N(7)ndashCundashN(13) 1561(1) N(10)ndashCundashN(13) 854(1)N(7)ndashCundashN(4) 824(1) N(10)ndashCundashN(4) 1645(1)N(13)ndashCundashN(4) 1073(1) N(7)ndashCundashN(1) 1209(1)N(10)ndashCundashN(1) 1093(1) N(13)ndashCundashN(1) 826(1)

Table 7Cyclic voltammetric data for [15]pyN5 and [16]pyN5 copper(II) complexesa

Complex EpcmV EpamV ΔEpmV E12mV

Cu[15]pyN52+ minus749 minus673 76 minus711


a Scan rate=100 mV sminus1 E12 values (vs AgndashAgCl) were taken as the average of theanodic (Epa) and the cathodic peak potentials (Epc) ΔEp=|EpaminusEpc|


Table 6 These values are characteristic of mononuclear copper(II)complexes in rhombic symmetry with elongation of the axial bondsand a dx2minusy2 ground state Elongated octahedral or distorted squarepyramidal stereochemistries are consistent with these data buttrigonalndashbipyramidal or tetragonal geometries involving compressionof axial bonds can be excluded [44ndash46]

The g and Ai parameters are related to the electronic transitions bythe factors derived from ligand field theory [47ndash50] the g valuesincrease and the Az value decreases as the planar ligand field becomesweaker or as the axial ligand field becomes stronger and this occurswith the simultaneous red-shift of the dndashd absorption bands in theelectronic spectra This sequence in principle parallels the degree ofdistortion from square-planar to square pyramidal and then tooctahedral or tetragonal geometries [51ndash53] In agreement with thisthe Cu[15]pyN5

2+ complex exhibits the lowest gz value and the largestAz and simultaneously its absorption band is blue shifted in relation tothat of Cu[16]pyN5

2+ pointing to a stronger equatorial ligand fieldindicating similar structures for the complexes of both macrocyclesconsistent with distorted square pyramidal geometry as alsoobserved in the crystal [Cu[15]pyN5](PF6)2 (vide infra)

35 Cyclic voltammetry studies

The redox behaviour of Cu[15]pyN52+ and Cu[16]pyN5

2+ wasinvestigated by cyclic voltammetry in water In Table 7 are depictedtheir electrochemical data where Epa and Epc are the anodic and thecathodic peak potentials respectively and ΔEp=|EpaminusEpc| As can beseen the two complexes exhibit analogous electrochemical behaviourshowing a single quasi-reversible one-electron transfer reductionprocess at half-wave potential values E12 (vs AgndashAgCl) of minus711 mV(EpaminusEpc=76 mV) and minus609 mV (EpaminusEpc=80mV) respectively

Fig 4 Molecular structure of [Cu[15]pyN5]2+ complex with atomic labelling schemeadopted

that can be assigned to the Cu(II)Cu(I) couple Upon repetitive cyclingthe voltammetric response remained essentially unchanged This featureindicates that the initial copper complexes are regenerated during thepotential scan For both copper(II) complexes the E12 values wereindependent when the scan rate (ν) was varied between 25 and1000 mV sminus1 theΔEp values increased and thepeak current ratio (IpaIpc)was slightlydifferent but close tounity Furthermore a linear relationshipbetween the peak currents and the square root of the ν (ν12) wasobserved This fact implies that these electrochemical processes aremainly diffusion-controlled

The Cu[16]pyN52+ yields a E12 value that is shifted to less negative

indicating a easier reduction to Cu(I) than the corresponding valueobserved for Cu[15]pyN5

2+ This difference which is in agreementwith the stability constants discussed before can be rationalized interms of flexibility and size of the macrocyclic cavities in bothcomplexes the geometric requirements and the size of the metal ionin different oxidation states The reduction of Cu(II) (d9) to Cu(I) (d10)involves a drastic increase in the metal radius and a geometric changefrom pyramidal to tetrahedral Obviously the larger and more flexiblecavity of [16]pyN5 compared to that of [15]pyN5 tends to stabilizebetter the copper(I) complex

36 X-ray structure of the copper(II) complex

The single crystal structure of [Cu[15]pyN5](PF6)2 was determinedby X-ray diffraction The molecular structure of [Cu[15]pyN5]2+

presented in Fig 4 shows the metal centre coordinated by the fivenitrogen donor atoms from [15]pyN5 Selected bond distances andangles given in Table 8 indicate that the copper(II) centre hasdistorted square pyramidal geometry The basal plane is formed bythe nitrogen atoms N(3) N(4) N(7) and N(10) with the trans anglesN(7)ndashCundashN(13) and N(10)ndashCundashN(4) of 1561(1) and 1645(1)degrespectively The apical position is occupied by the remainingnitrogen donor N(1) which is 2263(3) Aring from the least squaresplane defined by the basal nitrogen donors The copper centre is 0238(1) Aring from this plane towards the apical site leading to a CundashN(1)distance of 2229(3) Aring On the other hand the N(7)ndashCundashN(1) angle of1209(1)deg seems to indicate a tendency of the metal coordination

Table 9Dimensions of the NndashHmiddotmiddotmiddotF hydrogen bonds for [Cu[15]pyN5](PF6)2

d(HmiddotmiddotmiddotF)Aring d(NmiddotmiddotmiddotF)Aring bNndashHmiddotmiddotmiddotFdeg

N(1)ndashH(1) 244 3319(4) 163 F(21)N(4)ndashH(4) 230 3034(3) 138 F(12) [minusx+1 minusy+1 minusz]N(4)ndashH(4) 249 3186(3) 133 F(26) [x minusy+12 zminus12]N(10)ndashH(10) 228 3129(3) 156 F(13)

[minusx+1 yminus12minusz+12]N(10)ndashH(10) 243 3061(3) 127 F(11)

[minusx+1 yminus12minusz+12]N(10)ndashH(10) 243 2975(4) 119 F(22)N13ndashH(13) 237 3084(3) 136 F(12) [minusx+1 minusy+1 minusz]N(13)ndashH(13) 253 3296(3) 142 F(16) [minusx+1 minusy+1 minusz]

Fig 5 Crystal packing diagram showing the 1D chain formed by the interaction between the PF6minus counter-ions and [Cu[15]pyN5]2+ complexes via NndashHmiddotmiddotmiddotF hydrogen bonds (dashedred lines) (For interpretation of the references to color in this figure legend the reader is referred to the web version of this article)


environment for a trigonal bipyramidal geometry However thetrigonal index defined as t=(βminusα)60 (where β and α are the largestangles in the metal coordination sphere with βNα) [54] is only 013indicating undoubtedly the existence of a copper square pyramidalmetal coordination sphere taking into account that this angularparameter has an ideal value of 0 for the square pyramidal geometrywith C2v symmetry and 1 for the trigonal bipyramidal geometry withD3h symmetry Furthermore the basal nitrogen donors display anaverage tetrahedral distortion ofplusmn0123(1) Aring which is consistentwith the spectroscopic data of the complex reported above

To achieve the geometric arrangement described the macrocycleis folded through the axis defined by the nitrogen atoms contiguous tothe pyridine ring (N(4) and N(10)) leading to a dihedral anglebetween the equatorial plane and the plane defined by the nitrogenatoms N(1) N(4) and N(10) of 6480(9)deg

The CundashN(sp2) distance is shorter than the remaining four CundashN(sp3)distances following the usual structural trend found for copper complexesof tetraazamacrocyles incorporating the ndashNCH2(pyridine)CH2Nndash frag-ment [55] Furthermore the CundashN distances of [Cu[15]pyN5]2+ comparewellwith those found for the related 15-memberedmacrocyclic complex[Cu(Me2[15]pyN5)]2+whichexhibits a similar coordinationenvironment[56]

InTable 9 are gathered theNndashHmiddotmiddotmiddotFhydrogenbonds found in the solidstate for [Cu[15]pyN5](PF6)2 The [Cu[15]pyN5]2+ cations and PF6minus anionsare assembled into 1D infinite chains by multiple NndashHmiddotmiddotmiddotF hydrogenbonds along the [001] crystallographic direction Furthermore one ofthese chains presented in Fig 5 shows that there are eight independenthydrogen bonds derived from the interaction of all NndashHbinding groups ofeach [Cu[15]pyN5]2+ complex with two independent counter-ions Inaddition two of these intermolecular bonding interactions are bifurcatedone trifurcated andone almost linearwith anNmiddotmiddotmiddotFdistance of 3318(4)Aringand an NndashHmiddotmiddotmiddotF angle of 163deg

4 Conclusions

Two macrocyclic ligands having five donor nitrogen atoms one ofthem being a pyridine [15]pyN5 and [16]pyN5 have been synthe-sized In spite of being known for several years scarce quantitativeevaluation of the binding ability of these macrocycles to chelate metalions has been carried out until the present work Here the acidndashbasereactions of both macrocycles have been studied and their stabilityconstants with several metal ions of biological relevance or ability toact as toxic agents were determined by accurate techniques Theincrease of the cavity size of the macrocycles from 15 to 16 membersled to a decrease of all the stability constants without any specialincrease of selectivity Therefore from both chelators the [15]pyN5 isthe more promising for the aimed medical applications However acomparison of this ligand with [15]aneN5 revealed that the lattermacrocycle is a better chelator for copper(II) although a definitiveconclusion implies the redetermination of the stability constant of Cu[15]aneN5

2+ using accurate methods Nevertheless the pCu valuecalculated for [15]pyN5 of 1979 (cf Table 3) is much higher than the

1636 value determined under the same conditions for the clinicallyavailable copper(II) chelator trien [57]

Concerning nickel(II) [15]pyN5 is a very strong chelator and selectivetowards zinc(II) and calcium(II) essential metal ions and therefore itsevaluation for chelation therapy is pertinent These encouraging chemicalresults warrant further studies

Abbreviations[15]pyN5 3691218-pentaazabicyclo[1231]octadeca-1

(18)1416-triene[16]pyN5 36101319-pentaazabicyclo[1331]nonadeca-1

(19)1517-triene[15]aneN5 1471013-pentaazacyclopentadecane[16]aneN5 1581114-pentaazacyclohexadecaneDTPA diethylenetriaminepentaacetic acidDSS 3-(trimethylsilyl)propionic acid-d4-sodium saltpM concentration of free metal ion in solutiontrien NNprime-bis(2-aminoethyl)ethane-12-diamine

Acknowledgments

The authors acknowledge the financial support from Fundaccedilatildeo para aCiecircncia e a Tecnologia (FCT) with co-participation of the EuropeanCommunity fund FEDER (project no PTDCQUI671752006) The authorswish to thank the Elemental Analysis Service Unit of ITQB-UNL forproviding analytical data ASF acknowledges Fundaccedilatildeo para a Ciecircncia e aTecnologia Portugal for the financial support (PhD grant SFRHBD287732006) We also thank the EPSRC (UK) and the University ofReading for funds for the diffractometer

Appendix A Supplementary data

Crystallographic data for the structure [Cu[15]pyN5](PF6)2 in thispaper have been deposited with the Cambridge Crystallographic DataCentre as supplementary publication number CCDC782478 Copy of thedata can be obtained free of charge on application to CCDC 12 UnionRoad Cambridge CB2 1EZ UK [fax +44(0) 1223 336033 or e-maildepositccdccamacuk] Supplementary data to this article can befound online at doi101016jjinorgbio201011014

References

[1] M Blanuša VM Varnai M Piasek K Kostial Curr Med Chem 12 (2005) 2771ndash2794[2] T Wang Z Guo Curr Med Chem 13 (2006) 525ndash537[3] K Camphausen M Sproull S Tantama S Sankineni T Scott C Meacutenard CN

Coleman MW Brechbiel Bioorg Med Chem 11 (2003) 4287ndash4293[4] S Bolognin D Drago L Messori P Zatta Med Res Rev 29 (2009) 547ndash570[5] E Gaggeli H Kozlowsi D Valensin G Valensin Chem Rev 106 (2006) 1995ndash2044[6] F Tisato CMarzanoM PorchiaM Pellei C SantiniMed Res Rev 30 (2010) 708ndash749[7] KG Daniel P Gupta RH Harbach WC Guida QP Dou Biochem Pharmacol 67

(2004) 1139ndash1151[8] O Andersen Chem Rev 99 (1999) 2683ndash2710[9] E Denkhaus K Salnikow Crit Rev Oncol Hematol 42 (2002) 35ndash56


[10] International Agency for Research on Cancer IARC monographs on the evaluationof carcinogenic risks to humans IARC Lyon 1990

[11] J Saary R Qureshi V Palda J DeKoven M Pratt S Skotnicki-Grant L Holness J AmAcad Dermatol 53 (2005) 845ndash855

[12] JP Thyssen T Menneacute Chem Res Toxicol 23 (2010) 309ndash318[13] RD Hancock AE Martell Chem Rev 89 (1989) 1875ndash1914[14] J Costa R Delgado Inorg Chem 32 (1993) 5257ndash5265[15] DD Perrin WLF Armarego Purification of Laboratory Chemicals 3rd ed

Pergamon Oxford 1988[16] G Schwarzenbach W Flaschka Complexiometric Titrations Methuen amp Co London

1969[17] FJ Rossotti HJ Rossotti J Chem Educ 42 (1965) 375ndash378[18] R Delgado MC Figueira S Quintino Talanta 45 (1997) 451ndash462[19] P Gans A Sabatini A Vacca Talanta 43 (1996) 1739ndash1753[20] L Alderighi P Gans A Ienco D Peters A Sabatini A Vacca Coord Chem Rev 184

(1999) 311ndash318[21] R Delgado JJR Frauacutesto da Silva MTS Amorim MF Cabral S Chaves J Costa

Anal Chim Acta 245 (1991) 271ndash282[22] DF Evans J Chem Soc (1959) 2003ndash2005[23] CRYSALIS Oxford Diffraction Ltd 2005[24] ABSPACK Oxford Diffraction Ltd 2005[25] GM Sheldrick Acta Cryst A64 (2008) 112ndash122[26] AL Spek PLATON A Multipurpose Crystallographic Tool Utrecht University

Utrecht The Netherlands 2010[27] NV Gerbeleu VB Arion J Burgess Template Synthesis of Macrocyclic

Compounds Wiley-VCH Weinheim 1999[28] H Stetter W Frank R Mertens Tetrahedron 37 (1981) 767ndash772[29] DP Riley SL Henke PJ Lennon RH Weiss WL Neumann WJ Rivers KW

Aston KR Sample H Rahman C Ling J Shieh DH Busch W Szulbinski InorgChem 35 (1996) 5213ndash5231

[30] E Kimura M Kodama R Machida K Ishizu Inorg Chem 21 (1982) 595ndash602[31] JE Richman TJJ Atkins Am Chem Soc 96 (1974) 2268ndash2270[32] A Dees A Zahl R Puchta NJR E-Hommes FW Heinemann I Ivanovic-

Burmazovic Inorg Chem 46 (2007) 2459ndash2470

[33] M Kodama E Kimura Dalton Trans (1978) 104ndash110[34] J Costa R Delgado MGB Drew V Feacutelix Dalton Trans (1998) 1063ndash1071[35] M Kodama E Kimura S Yamaguchi Dalton Trans (1980) 2536ndash2538[36] M Kodama T Koike N Hoshiga R Machida E Kimura Dalton Trans (1984) 673ndash678[37] M Kodama E Kimura Dalton Trans (1978) 1081ndash1085[38] LD PettitHKJ Powell IUPACStabilityConstantsDatabase AcademicSoftware Timble

2003[39] X Cui MJ Calhorda PJ Costa R Delgado MGB Drew V Feacutelix Helv Chim Acta

87 (2004) 2613ndash2628[40] LY Martin CR Sperati DH Busch J Am Chem Soc 99 (1977) 2968ndash2981[41] L Sacconi F Mani A Bencini in G Wilkinson RD Gillard JA McCleverty (Eds)

Comprehensive Coordination Chemistry Pergamon Press Oxford 1987[42] MC Rakowski M Rycheck DH Busch Inorg Chem 14 (1975) 1194ndash1200[43] F Neese Diploma Thesis University of Konstanz Germany June 1993[44] J Costa R Delgado MC Figueira RT Henriques M Teixeira Dalton Trans (1997)

65ndash73[45] MC Styka RC Smierciak EL Blinn RE DeSimone JV Passarielo Inorg Chem

17 (1978) 82ndash86[46] BJ Hathaway Coord Chem Rev 52 (1983) 87ndash169[47] HR Gersmann JD Swalen J Chem Phys 36 (1962) 3221ndash3233[48] H Yokoi M Sai T Isobe S Ohsawa Bull Chem Soc Jpn 45 (1972) 2189ndash2195[49] PW Lau WC Lin J Inorg Nucl Chem 37 (1975) 2389ndash2398[50] Y Li Bull Chem Soc Jpn 69 (1996) 2513ndash2523[51] AW Addison M Carpenter LK-M Lau M Wicholas Inorg Chem 17 (1978)

1545ndash1552[52] MJ Maroney NJ Rose Inorg Chem 23 (1984) 2252ndash2261[53] P Barbaro C Bianchini G Capannesi L Di Luca F Laschi D Petroni PA Salvadori

A Vacca F Vizza Dalton Trans (2000) 2393ndash2401[54] AW Addison TN Rao J Reedjik J van Rijn GC Verschoor Dalton Trans (1984)

1349ndash1356[55] FH Allen Acta Cryst B58 (2002) 380ndash388[56] MGB Drew S Hollis PC Yates Dalton Trans (1829ndash1834)[57] R Delgado S Quintino M Teixeira A Zhang Dalton Trans (1996) 55ndash63


On the other hand human exposure to nickel occurs primarily viainhalation and ingestion through occupational exposure anddiet leadingto adverse effects on human health Nickel allergy in the form of contactdermatitis is the most common and well-known reaction Although theaccumulation of nickel in the body through chronic exposure can causelung fibrosis kidney and cardiovascular diseases the most seriousconcerns relate to nickels carcinogenic activity Epidemiological studieshave clearly implicated nickel compounds as human carcinogens [89]All nickel compounds except for metallic nickel were classified ascarcinogenic to humans in 1990 by the International Agency for Researchon Cancer (IARC) [10]

Over the years various chelators have been investigated for theirability to bind nickel The most effective ones are EDTA DTPA(diethylenetriaminepentaacetic acid) diethyldithiocarbamate tet-raethylthiuram disulfide and clioquinol (5-chloro-8-hydroxy-7-iodoquinoline) all of them presenting considerable side effects[81112]

Therefore the development of novel chelators selective for nickel(II)and for copper(II) and exhibiting minor side effects is an imperativeresearch This led us to investigate the possible use of macrocycliccompounds In factmetal chelates ofmacrocycles often showpropertiesthat are particularly different from those of analogous open chainchelators Macrocycles having more rigid structures can impose specificcoordination geometry to the metal ion whereas open chain chelatorsadapt more easily to the geometric requirements of the metal centre[13] In the present work the synthesis and characterization of twopentaaza macrocyclic compounds containing pyridine in the backbone[15]pyN5 (3691218-pentaazabicyclo[1231]octadeca-1(18)1416-tri-ene) and [16]pyN5 (36101319-pentaazabicyclo[1331]nonadeca-1(19)1517-triene) cf Scheme 1 as well as the study of their copper(II)and nickel(II) complexes are reported in order to evaluate their possibleuseas chelatingagents Toaccomplish this aim theacidndashbasebehaviourofthese two macrocycles was studied and their ability to coordinate Cu2+

and Ni2+ and other divalent metal ions (Ca2+ and Zn2+ are included dueto their essential role in living organisms) was evaluated The adoptedstructures of the Cu(II) and Ni(II) complexes were also studied byspectroscopicmethods in solution and the single crystal X-ray diffractionof [Cu[15]pyN5](PF6)2 was determined Finally due to the important roleof the redox behaviour of the copper(II) complexes in biology somevoltammetric studies were carried out

2 Experimental section

21 General procedures

Elemental analysis was performed on a VarioEL CHNS analyserfrom vacuum-dried powder samples Melting points were determinedwith a Koumlpffer Melting Point apparatus

211 Reagents26-Pyridinedimethanol NNprime-bis(2-aminoethyl)ethane-12-diamine

and NNprime-bis(2-aminoethyl)13-propanediamine were purchased fromAldrich 26-Pyridinedicarbaldehyde was prepared by publishedmethods[14] All the commercially available chemicals were of reagent grade and

Scheme

used as supplied without further purification Organic solvents werepurified or dried by standard methods [15]

Caution Althoughnoproblemswere found in thiswork perchloratesin the presence of organic matter are potentially explosive and should beprepared in small quantities

22 Synthesis of the macrocycles


To a stirred solution of freshly prepared 26-pyridinedicarbalde-hyde (234 g 18 mmol) in methanol (40 mL) was added a solution ofPb(NO3)2 (61 g 18 mmol) in water (80 mL) To the resulting solutionwas added dropwise with rapid stirring a solution of NNprime-bis(2-aminoethyl)ethane-12-diamine (335 g 18 mmol) in methanol(40 mL) over a period of 3 h The solution was stirred while heatingunder reflux for 7 h during which time an intense deep red colourdeveloped After reflux the solution was cooled to 5 degC and sodiumborohydride (436 g 455 mmol) was added in small portions over60 min The yellow solution obtainedwas stirred at room temperaturefor 30 min and then heated on a hot water-bath at 60 degC for 30 minbefore being left overnight at room temperature Leadwas removed bytreating the mixture with Na2S9H2O (10 g 42 mmol) followed byheating on a hot water-bath for 30 min The solution was then cooledand lead(II) sulphidewas removed byfiltration through a bed of CeliteThe filtrate was extracted with dichloromethane (4times50 mL) thecombined extracts were dried with anhydrous MgSO4 and the dichlor-omethane was removed with a rotary evaporator to leave a light yellowoil This oil was dissolved in methanol and 37 hydrochloric acid wasadded until pHasymp2 During the addition an off-white solid precipitatedwhichwas identified as the pure desired compound Yield 85Mp 280ndash282 degC (decomp) 1H NMR (D2O pD=510) δ 321 (4H s (singlet) NndashCH2) 341 (4H t (triplet) NndashCH2ndashCH2ndashN) 351 (4H t NndashCH2ndashCH2ndashN)457 (4H s NndashCH2ndashpy) 753 (2H d (doublet) py) and 799 (1H t py)ppm 13C NMR (D2O pD = 510) δ 4392 (NndashCH2ndashCH2ndashN) 4564 (NndashCH2ndashCH2ndashN) 4633 (NndashCH2) 4992 (NndashCH2ndashpy) 12407 (py) 14033 (py)and 15094 (py) ppm Found C 3703 H 716 N 1656 Calc forC13H23N5middot4HClmiddot15H2O C 3698 H 716 N 1659


A procedure analogous to that described for [15]pyN5 was usedreplacing NNprime-bis(2-aminoethyl)ethane-12-diamine by NNprime-bis(2-aminoethyl)13-propanediamine The product was obtained as a thickyellow oil which was purified by passing through a neutral aluminacolumn (25times30 cm) and eluting with dichloromethanendashmethanol(1005 vv) The pure compound was dissolved in methanol and 37hydrochloric acidwas added until pHasymp2 An off-white salt precipitatedYield 46 Mp 266ndash268 degC (decomp) 1H NMR (D2O pD=255) δ 221(2 H q (quintuplet) CH2ndashCH2ndashN) 337 (4H t CH2ndashCH2ndashN) 365 (4H tNndashCH2ndashCH2ndashN) 371 (4H t NndashCH2ndashCH2ndashN) 463 (4H s NndashCH2ndashpy)756 (2H d py) and 800 (1H t py) ppm 13C NMR (D2O pD = 255) δ2130 (CH2ndashCH2ndashN) 4225 (NndashCH2ndashCH2ndashN) 4320 (NndashCH2ndashCH2ndashN)4414 (CH2ndashCH2ndashN) 5135 (NndashCH2ndashpy) 12450 (py) 14044 (py) and15068 (py) ppm Found C 3759 H 780 N 1537 Calc for C14H25-

N5middot4HClmiddot2H2O C 3780 H 750 N 1570

1












H=271 log K4H =20







24 NMR measurements














2+ and504 723 and 992 for Cu[16]pyN5
























b


[H4L4+][L]times[H+]4 2502 2596 ndash 2824 2912









Ca2+ 500 789 655Ni2+ 1819 1568 1601Zn2+ 1424 1384 1444






[NiL2+][Ni2+]times[L] 2171(1) 1681(1) 181b 181c


[CuL2+][Cu2+]times[L] 2331(3) 2086(4) 283d 271d



[ZnL2+][Zn2+]times[L] 1776(2) 1530(1) 191e 179 e


[CdL2+][Cd2+]times[L] 16853(7) 15532(4) 192e 181e


[PbL2+][Pb2+]times[L] 1544(2) 12932(6) 173e 143e













Ni2+ 1819 1352 1238 1252Cu2+ 1979 1757 2278 2152Zn2+ 1424 1201 1338 1232Cd2+ 1334 1225 1348 1252Pb2+ 1192 965 1158 872














a sh=shoulder













2+ and Ni[16]pyN52+



2+


2+













gx gy gz Ax Ay Az

Cu[15]pyN52+ 610 (150) 2035 2070 2210 269 400 1706

Cu[16]pyN52+ 646 (143) 2038 2077 2222 232 335 1638



































4 Conclusions








Acknowledgments




References





































H=271 log K4H =20







24 NMR measurements














2+ and504 723 and 992 for Cu[16]pyN5
























b


[H4L4+][L]times[H+]4 2502 2596 ndash 2824 2912









Ca2+ 500 789 655Ni2+ 1819 1568 1601Zn2+ 1424 1384 1444






[NiL2+][Ni2+]times[L] 2171(1) 1681(1) 181b 181c


[CuL2+][Cu2+]times[L] 2331(3) 2086(4) 283d 271d



[ZnL2+][Zn2+]times[L] 1776(2) 1530(1) 191e 179 e


[CdL2+][Cd2+]times[L] 16853(7) 15532(4) 192e 181e


[PbL2+][Pb2+]times[L] 1544(2) 12932(6) 173e 143e













Ni2+ 1819 1352 1238 1252Cu2+ 1979 1757 2278 2152Zn2+ 1424 1201 1338 1232Cd2+ 1334 1225 1348 1252Pb2+ 1192 965 1158 872














a sh=shoulder













2+ and Ni[16]pyN52+



2+


2+













gx gy gz Ax Ay Az

Cu[15]pyN52+ 610 (150) 2035 2070 2210 269 400 1706

Cu[16]pyN52+ 646 (143) 2038 2077 2222 232 335 1638



































4 Conclusions








Acknowledgments




References
































2+ and504 723 and 992 for Cu[16]pyN5
























b


[H4L4+][L]times[H+]4 2502 2596 ndash 2824 2912









Ca2+ 500 789 655Ni2+ 1819 1568 1601Zn2+ 1424 1384 1444






[NiL2+][Ni2+]times[L] 2171(1) 1681(1) 181b 181c


[CuL2+][Cu2+]times[L] 2331(3) 2086(4) 283d 271d



[ZnL2+][Zn2+]times[L] 1776(2) 1530(1) 191e 179 e


[CdL2+][Cd2+]times[L] 16853(7) 15532(4) 192e 181e


[PbL2+][Pb2+]times[L] 1544(2) 12932(6) 173e 143e













Ni2+ 1819 1352 1238 1252Cu2+ 1979 1757 2278 2152Zn2+ 1424 1201 1338 1232Cd2+ 1334 1225 1348 1252Pb2+ 1192 965 1158 872














a sh=shoulder













2+ and Ni[16]pyN52+



2+


2+













gx gy gz Ax Ay Az

Cu[15]pyN52+ 610 (150) 2035 2070 2210 269 400 1706

Cu[16]pyN52+ 646 (143) 2038 2077 2222 232 335 1638



































4 Conclusions








Acknowledgments




References





























b


[H4L4+][L]times[H+]4 2502 2596 ndash 2824 2912









Ca2+ 500 789 655Ni2+ 1819 1568 1601Zn2+ 1424 1384 1444






[NiL2+][Ni2+]times[L] 2171(1) 1681(1) 181b 181c


[CuL2+][Cu2+]times[L] 2331(3) 2086(4) 283d 271d



[ZnL2+][Zn2+]times[L] 1776(2) 1530(1) 191e 179 e


[CdL2+][Cd2+]times[L] 16853(7) 15532(4) 192e 181e


[PbL2+][Pb2+]times[L] 1544(2) 12932(6) 173e 143e













Ni2+ 1819 1352 1238 1252Cu2+ 1979 1757 2278 2152Zn2+ 1424 1201 1338 1232Cd2+ 1334 1225 1348 1252Pb2+ 1192 965 1158 872














a sh=shoulder













2+ and Ni[16]pyN52+



2+


2+













gx gy gz Ax Ay Az

Cu[15]pyN52+ 610 (150) 2035 2070 2210 269 400 1706

Cu[16]pyN52+ 646 (143) 2038 2077 2222 232 335 1638



































4 Conclusions








Acknowledgments




References




























Ca2+ 500 789 655Ni2+ 1819 1568 1601Zn2+ 1424 1384 1444






[NiL2+][Ni2+]times[L] 2171(1) 1681(1) 181b 181c


[CuL2+][Cu2+]times[L] 2331(3) 2086(4) 283d 271d



[ZnL2+][Zn2+]times[L] 1776(2) 1530(1) 191e 179 e


[CdL2+][Cd2+]times[L] 16853(7) 15532(4) 192e 181e


[PbL2+][Pb2+]times[L] 1544(2) 12932(6) 173e 143e













Ni2+ 1819 1352 1238 1252Cu2+ 1979 1757 2278 2152Zn2+ 1424 1201 1338 1232Cd2+ 1334 1225 1348 1252Pb2+ 1192 965 1158 872














a sh=shoulder













2+ and Ni[16]pyN52+



2+


2+













gx gy gz Ax Ay Az

Cu[15]pyN52+ 610 (150) 2035 2070 2210 269 400 1706

Cu[16]pyN52+ 646 (143) 2038 2077 2222 232 335 1638



































4 Conclusions








Acknowledgments




References






























a sh=shoulder













2+ and Ni[16]pyN52+



2+


2+













gx gy gz Ax Ay Az

Cu[15]pyN52+ 610 (150) 2035 2070 2210 269 400 1706

Cu[16]pyN52+ 646 (143) 2038 2077 2222 232 335 1638



































4 Conclusions








Acknowledgments




References




























































4 Conclusions








Acknowledgments




References


























Two macrocyclic pentaaza compounds containing pyridine evaluated as novel chelating agents in...

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