New bis-(3-hydroxy-4-pyridinone)-NTA-derivative: Synthesis, binding ability towards Ca2+, Cu2+, Zn2+, Al3+, Fe3+ and biological assays

Accepted Manuscript New bis-(3-hydroxy-4-pyridinone)-NTA-derivative: Synthesis, binding ability towards Ca2+, Cu2+, Zn2+, Al3+, Fe3+ and biological assays

Anna Irto, Paola Cardiano, Karam Chand, Rosalia Maria Cigala, Francesco Crea, Concetta De Stefano, Lurdes Gano, Giuseppe Gattuso, Silvio Sammartano, Maria Amélia Santos PII: DOI: Reference:

S0167-7322(18)33944-8 doi:10.1016/j.molliq.2018.09.107 MOLLIQ 9713

To appear in:

Journal of Molecular Liquids

Received date: Revised date: Accepted date:

31 July 2018 15 September 2018 22 September 2018

Please cite this article as: Anna Irto, Paola Cardiano, Karam Chand, Rosalia Maria Cigala, Francesco Crea, Concetta De Stefano, Lurdes Gano, Giuseppe Gattuso, Silvio Sammartano, Maria Amélia Santos , New bis-(3-hydroxy-4-pyridinone)-NTA-derivative: Synthesis, binding ability towards Ca2+, Cu2+, Zn2+, Al3+, Fe3+ and biological assays. Molliq (2018), doi:10.1016/j.molliq.2018.09.107

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

New bis-(3-hydroxy-4-pyridinone)-NTA-derivative: synthesis, binding ability towards Ca2+, Cu2+, Zn2+, Al3+, Fe3+ and biological assays

PT

Anna Irtoa, Paola Cardianoa, Karam Chandb, Rosalia Maria Cigalaa, Francesco Creaa, Concetta De

RI

Stefanoa, Lurdes Ganoc, Giuseppe Gattusoa, Silvio Sammartanoa,*, Maria Amélia Santosb,*1

a

Viale F. Stagno d'Alcontres, 31 – 98166 Messina, Italy b

SC

Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali, Università di Messina,

Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovísco Pais 1,

c

NU

1049-001 Lisboa, Portugal

Centro de Ciências e Tecnologias Nucleares (C2TN), Instituto Superior Técnico, Universidade de

MA

Lisboa, Estrada Nacional 10, 2695-066 Bobadela LRS, Portugal

Abstract

D

A new bis-(3-hydroxy-4-pyridinone) ligand (NTA(PrHP)2), derived from nitrilotriacetic acid

PT E

(NTA), was synthesized and evaluated for its selective solution metal complexation capacity as well as its in vivo sequestering power for hard metal cations. After the study of its acid-base properties and determination of the protonation constants, the binding ability of NTA(PrHP)2

CE

towards divalent (Ca2+, Cu2+, Zn2+) and trivalent (Al3+, Fe3+) metal cations was investigated by means of UV-Vis spectrophotometric, potentiometric and

1

H NMR measurements. The

AC

determined speciation models consist of MpLqHr species with different stoichiometry. The obtained stability constants for the ML species follow the trend: Fe3+ > Al3+ > Cu2+ > Zn2+ > Ca2+. Furthermore, the sequestering ability of the ligand towards those metal cations was investigated by the determination of the pL0.5 and pM parameters calculated at different pHs and pH = 7.4, respectively. High pL0.5 values were obtained with significant variation of the sequestering ability with the investigated pH range. Analysis of the pM values at pH = 7.4 showed that the metal-ligand affinity follows the trend: Fe3+ > Al3+ > Cu2+ > Zn2+ > Ca2+. Finally, in vivo assays were performed to verify the efficacy of NTA(PrHP)2 as sequestering agent towards trivalent metal cations, when administered to mice preloaded with the radiotracer 67Ga–citrate. * Corresponding authors: email [email protected] (M. A. Santos).

address:

[email protected]

(S.

Sammartano);

email

address:

1

ACCEPTED MANUSCRIPT

Keywords: bis-(3-hydroxy-4-pyridinone); speciation; acid-base properties; metal chelation; bioassays.

1. Introduction

PT

The onset of diseases due to the accumulation of hard metal cations into the human body led the scientific community to look for new chelating agents, which should fulfil some

RI

important criteria, such as: absence of toxicity, economic availability, drug-likeness properties, affinity towards biological membranes, and higher metal chelating capacity and specificity than

SC

the commercial molecules.

In 1966, the first clinically approved iron chelator was the Deferoxamine (DFO or

NU

Desferal®), a tris-hydroxamate ligand isolated from Streptomyces pilosum[1]. Despite the high thermodynamic stability of its complex with Fe3+ featuring 1:1 stoichiometry[2], DFO was

MA

expensive and, on the other hand, its hydrophilicity, high molecular weight and peptidic nature, hindered its gastrointestinal (GI) absorption and oral activity, with the concomitant need of daily subcutaneous administration and serious problems of patient noncompliance[3-5]. This

D

hexadentate ligand was also used for the treatment of diseases linked to aluminium overload like

PT E

the osteomalacia[6]. These limitations led to a continuing research activity which, two decades later, allowed the discovery of a bidentate ligand, the 1,2-dimethyl-3-hydroxy-4-pyridinone, marketed as deferiprone (DFP or Ferriprox®)[7]. This iron-chelator is inexpensive and orally active, being approved in 1999 as second-line therapy for patients affected by thalassemia[8].

CE

However, the stability of the Fe3+/DFP complexes[9, 10] is lower than the corresponding Fe3+/DFO species, so that higher dosages of deferiprone are required for the effective removal of

AC

the metal cation from the body. To overcome these drawbacks, we have assisted to an intensive research on 3-hydroxy-4-pyridinones (3,4-HPs) derivatives, aimed to improve their effectiveness at physiological pH range as well as in all the biological conditions, low costs, oral activity and the absence of side effects[5, 11-15]. In particular, quite an amount of mono-3,4HP derivatives have been developed mostly aimed either to increase their bioavailability and affinity towards the biological sites and/or to improve the chelating efficiency[16-18]. This last challenge has been mostly focused on the development of bis-3,4-HP and tris-3,4-HP derivatives, by attaching two or three 3-hydroxy-4-pyridinone arms to a backbone[16, 19-25]. In particular, we have mainly used backbone supports based on iminopolyacidic cores aimed to compromise the lipohydrophilic balance with the chelating efficacy, such as IDA (iminodiacetic acid)[24], EDTA 2

ACCEPTED MANUSCRIPT (ethylenediaminotetracetic

acid)[21,

25],

NTA

(nitrilotriacetic

acid)[19]

or

NTP

(nitrilotriproprionic acid)[20] supporting skeletons. This paper deals with the synthesis and the study of metal complexation behaviour of a new bis-(3-hydroxy-4-pyridinone) ligand (NTA(PrHP)2, Fig. 1), containing two 3,4-HP binding units linked to NTA. Therefore, this ligand appears as an homologue of the tris-(3,4-HP), NTA(PrHP)3[19], and the bis-(3,4-HP) ((IDA(PrHP)2)[24] previously reported (see Fig. 1). After the study of its acid-base properties, the metal complexation and speciation model in aqueous

PT

solution, containing divalent (Ca2+, Cu2+, Zn2+) and trivalent (Al3+, Fe3+) metal cations, was performed in NaCl ionic medium (I = 0.15 mol L-1), since it is the major inorganic constituent of

RI

most of natural[26, 27] and biological fluids, namely blood plasma (I ~ 0.16 mol L-1)[28]. This

SC

investigation involved a series of analytical techniques, namely UV-Vis spectrophotometry, spectrofluorimetry, potentiometry (ISE-H+) and 1H NMR spectroscopy. The acid-base properties

NU

of the bis-(3-hydroxy-4-pyridinone) were studied at T = 298.15 and also at 310.15 K (physiological conditions). Moreover, the investigation of the sequestering ability of the ligand towards the metal cations of interest, involved the determination of the pL0.5[29] and the pM[30]

MA

parameters calculated at different pHs and pH = 7.4, respectively. Finally, biodistribution studies in mice previously injected with the radiotracer 67Ga–citrate were performed to verify the in vivo

PT E

2. Materials and methods

D

chelating ability of NTA(PrHP)2 towards trivalent metal cations.

2.1. Chemicals for solution studies

CE

The reagents were of the highest available purity and all the solutions were prepared with analytical grade water (R = 18 MΩ cm-1) and grade A glassware. The strong acid (HCl) and

AC

carbonate free strong base (NaOH) solutions were prepared from dilution of concentrated ampoules (Riedel-deHäen) and standardized against primary standard substances like sodium carbonate and potassium hydrogen phthalate, respectively. Sodium hydroxide solutions were preserved from atmospheric CO2 by means of soda lime traps. The solutions of Ca2+, Cu2+, Zn2+, Al3+ and Fe3+ were prepared by weighing the corresponding Fluka salts, such as CaCl2 dihydrate, CuCl2 dihydrate, ZnCl2, AlCl3 hexahydrate and FeCl3 hexahydrate, without further purification. Their purity, checked performing titrations with EDTA standard solutions, was in all the cases ≥ 98%.[31] Furthermore, known concentrations of inorganic acid (HCl) were added to the salts solutions to prevent hydrolysis of the metal cations. The ionic medium (NaCl) aqueous solutions were prepared by weighing the pure salt (Fluka), previously dried in an oven for at least 2 hours, at T = 383.15 K. 3

ACCEPTED MANUSCRIPT 2.2. Analytical equipments and procedures 2.2.1. UV-Vis Spectrophotometric tools and procedure An UV–Vis spectrophotometer (Varian Cary 50 model) equipped with an optic fiber probe with a fixed path length of 1 cm was used for the spectrophotometric experiments. The instrument was connected to a computer and a Varian Cary WinUV software was used for the acquisition of the signal of absorbance (A) vs. wavelength (λ/nm). Meanwhile potentiometric

PT

data were collected by means of a combined glass electrode (Ross type 8102, from ThermoOrion), connected to a potentiometer. The titrant was delivered in the measurement cell using a

RI

Metrohm 665 automatic burette and a magnetic stirrer ensured the homogeneity of the solutions during the titrations. Before starting the experiments, nitrogen was bubbled in the solutions for at

SC

least 5 minutes, to avoid the possible presence of atmospheric oxygen and carbon dioxide. To investigate the acid-base properties of the ligand, 25 mL of solution containing

NU

NTA(PrHP)2 (1.0·10-5 ≤ cL/ mol L-1 ≤ 2.1·10-5), hydrochloric acid (2.0·10-3 ≤ cHCl/mol L-1 ≤ 4.0·10-3) and the ionic medium NaCl at I = 0.15 mol L-1, was analyzed in the pH range 2.0–10.7.

MA

The experiments were carried out in the wavelength range 200  λ/nm  800 and T = 298.15 K and 310.15 K (physiological conditions). For the investigation of the metal-ligand interactions, the titrations were performed at the same ionic strength and range of λ/nm used for the acid-base

D

measurements and at T = 298.15 K. Different concentrations Ca2+, Cu2+, Zn2+, Al3+ and Fe3+

PT E

(5.0·10-6 ≤ cMn+/mol L-1 ≤ 1.5·10-4) and ligand (1.0·10-5 ≤ cL/mol L-1 ≤ 2.1·10-4) were selected for the measurements carried out in the pH range 2.0–11.0.

CE

2.2.2. Spectrofluorimetric equipment and procedure A FluoroMax-4 spectrofluorometer purchased by Horiba Jobin-Yvon, equipped with F3006 Autotitration Injector with two Hamilton Syringes (Gastight 1725 and 1001 TLLX models)

AC

with 250 μL and 1 mL of capacity, respectively, was used for the spectrofluorimetric experiments. The resolutions of the selectors of wavelength and titrant additions were 0.3 nm and 0.25 μL, respectively. The instrument was equipped with a Peltier Sample Cooler (F-3004 model) controlled by a Peltier Thermoelectric Temperature Controller model LFI-3751. The system was set by the FluorEssence 2.1 software by Horiba Jobin-Yvon. The measurements were accomplished by means of a 1 cm light path Hellma type 101-OS precision cell, with a combined glass microelectrode (model biotrode 6.0224.100, Metrohm) to collect e.m.f. values, a magnetic stirrer to homogenize the experiment solutions and an anti-diffusion burette tip. The burette tip and the electrode were both positioned to avoid interferences with the light beam. The automatic acquisition of data (emission intensity vs. titrant volume) was performed using the FluorEssence 4

ACCEPTED MANUSCRIPT 2.1 software. The best experimental conditions were determined through preliminary evaluations in which parameters such as equilibration time, scan rate, scan range and integration time, excitation and emission wavelengths, were systematically changed to select the values providing the best signal/noise ratio. A detailed description on the spectrofluorimetric equipment and procedure was already reported in a previous work [16]. Spectrofluorimetric experiments were carried out at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K and 310.15 K. The measurements were carried out by titrating with NaOH standard, 2

PT

mL of a solution containing the ligand (1.0·10-5 ≤ cL/mol L-1 ≤ 2.1·10-5), the strong acid (2.0·10-3 ≤ cHCl/mol L-1 ≤ 4.0·10-3) and the supporting electrolyte at the desired ionic strength value. For

RI

each addition (mL) of base the emission intensity of signal (CPS) was recorded in the

value, in the pH range 2.0–11.0.

NU

2.2.3. Potentiometric equipment and procedure

SC

wavelengths range 300  λ/nm  520 (λexc = 278 nm), together with the corresponding e.m.f.

Potentiometric titrations were performed with a Metrohm model 809 Titrando coupled to a

MA

combined glass electrode (Ross type 8102, from Thermo-Orion). The apparatus was linked to a computer and acquisition of e.m.f. values was performed by means of the Metrohm TiAMO 1.2 software. Estimated accuracy was ±0.15 mV and ±0.003 mL for e.m.f. and titrant volume

D

readings, respectively. The titrations were performed in thermostatted cells under magnetic

PT E

stirring and bubbling purified pre-saturated N2(g) into the solution to exclude the presence of O2(g) and CO2(g) inside. For each experiment, independent titrations of hydrochloric acid, with standard NaOH solutions were performed, at the same experimental conditions (ionic medium,

CE

ionic strength, temperature) than those used to study the systems, to refine the electrode potential value (E0), the acidic junction potential (Ej = ja[H+]), and the ionic product of water (Kw). In this

AC

order, the pH scale used was the free concentration scale and pH ≡ -log[H+], where [H+] is the free proton concentration. For the titrations, 80 – 100 data points were collected and the equilibrium state during the experiments was checked, by using the Metrohm TiAMO 1.2 software, by adopting precautions like checking the necessary time to reach equilibrium and performing back titrations. Further details on the experimental procedure are reported in the literature[16]. The binding ability of NTA(PrHP)2 towards Al3+ was investigated also by performing potentiometric titrations at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K and different ligand (5.0·10-4  cL/mol L-1  1.0·10-3) and metal cation (5.0·10-4  cAl3+/mol L-1  7.0·10-4) concentrations. 5

ACCEPTED MANUSCRIPT 2.2.4. 1H NMR apparatus and procedure 1

H NMR spectra at T = 298.15 K in 9:1 H2O/D2O mixture were recorded on a Bruker

AVANCE 300 operating at 300 MHz. The chemical shifts were measured with respect to 1,4dioxane and converted relative to tetramethylsilane (TMS), employing δ(dioxane) = 3.70 ppm. Prior to investigate the metals containing systems, the acid-base properties of NTA(PrHP)2 was checked by 1H NMR titrations on 5.0·10-3 mol L-1 ligand solutions, in the pH range between approx. 2.0 and 11.0 and I = 0.15 mol L-1 in NaCl(aq). The spectra of the systems containing

PT

NTA(PrHP)2 and Zn2+ or Al3+ were carried out by adding known amounts of a NaOH solution to mixtures of the ligand (5.0·10-3 mol L-1) and the metal cations (2.5·10-3 mol L-1), in the same

SC

2.3. Computer programs and equilibrium constants

RI

ionic medium and pH range already cited.

NU

The determination of all the parameters of the acid–base titrations (E0, pKw, liquid junction potential coefficient ja, analytical concentration of reagents) was performed using of the nonlinear least squares ESAB2M[32] computer program. The UV-Vis spectrophotometric and

MA

spectrofluorimetric data were elaborated using the HYPERQUAD 2008[33] computer program, whereas the potentiometric ones were analyzed by means of STACO[34] and HYPERQUAD

D

2008[33] computer programs. For NMR data treatment, HypNMR software was employed to

PT E

calculate the protonation constants and the individual chemical shifts belonging to each species present at equilibria for NTA(PrHP)2. All proton exchange reactions were found to be fast on the NMR time-scale. Since in the specific case of Al3+/L system two sets of signals were found in the spectra, that accounted for free and bound ligand, the corresponding average chemical shifts

CE

were both considered in the calculations[35]. The speciation diagrams were drawn using HySS[36] program which allowed also to calculate the species formation percentages.

AC

The stepwise (logKrH, eq. (1)) and overall (logβrH, eq. (2)) protonation constants of NTA(PrHP)2 (Lz-) are expressed by the following equilibria: H+ + H(r-1)L-(z-(r-1)) = HrL-(z-r)

logKrH

(1)

logβrH

(2)

and rH+ +Lz- = HrL-(z-r)

where r indicates the r-th protonation step and z is the charge of the completely deprotonated ligand. The hydrolysis reactions of the metal cations are given as follows: pMn+ + rH2O = Mp(OH)r(np-r) + rH+

logβrOH

(3) 6

ACCEPTED MANUSCRIPT The stability or formation constants of the complexes with the metal cations refer to the following equilibrium: pMn+ + qLz- + rH+ = MpLqHr(pn+r-qz)

logβpqr

(4)

The protonation and stability constants, concentrations and ionic strengths are expressed in the molar concentration (c, mol L-1) scale. Along the text, the charges of the species determined in all the cases are omitted for simplicity.

PT

2.4. General synthetic information

RI

All the reactions were TLC controlled, using as mobile phases S1 (DCM-MeOH 9.5:0.5), S2 (DCM-MeOH-NH4OH 8.5:1:0.5) and S3 (DCM-MeOH-NH4OH 8.5:2:0.5) solvents mixture

SC

(see Abbreviations). Ferric chloride, ninhydrine and Dragensdorf tests were used to check the possible presence of –OH, amino groups and quaternary nitrogen groups, respectively, into the

NU

reaction mixtures. The solvents, if necessary, were dried using the standard methods.[37] 1H and 13

C NMR investigations were performed, using Bruker AVANCE III 300 MHz and Bruker

MA

AVANCE III 400 MHz spectrometers, in deuterated solvents (D2O, Methanol-d4, DMSO-d6), whose peaks in the spectra were assigned taking into account the literature data.[38] Melting points determination was carried out by means of a Leica Galen III hot stage apparatus. Mass

D

spectra (ESI-MS) were obtained using a 500 MS LC Ion Trap (Varian Inc., Palo Alto, CA, USA)

PT E

mass spectrometer equipped with an ESI ion source, operating in the positive or negative ion mode. For the target final compound, the elemental analyses were performed using a Fisons EA1108 CHNS/O instrument at LAIST and were within the limit of ± 0.4%.

CE

2.5. Synthetic procedures

2.5.1. Synthesis of 2-(bis(2-((3-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)propyl)amino)-2-

AC

oxoethyl)amino)acetic acid (NTA(PrHP)2) 3-Benzyloxy-2-methyl-4-pyrone (1). 3-Hydroxy-2-methyl-4-pyrone (20.07 g, 159.15 mmol) was dissolved in 200 mL of MeOH and a solution of NaOH (6.98 g, 174.50 mmol) in 22 mL of water was added to this mixture, followed by dropwise addition of benzyl chloride (16.50 mL, 142.75 mol). The reaction mixture was heated under reflux (T = 348 K) for 20 h. The solvent was evaporated under reduced pressure and the remaining orange oil was dissolved in DCM (80 mL) and washed with 5% (w/v) of sodium hydroxide aqueous solution (3 × 30 mL) and water (2 × 30 mL). The organic phase was dried over anhydrous sodium sulfate and filtered. The solvent was rotoevaporated and the residue was dried in vacuum to give the title product as a pale yellow oil (32.93 g, η = 95%). TLCs were performed in S1 mixture. 1H NMR (400 MHz, MeOD-d4), 7

ACCEPTED MANUSCRIPT δ(ppm): 7.82 (1H, d, 6-HPy), 7.33 (5H, s, Ph), 6.35 (1H, d, 5-HPy), 5.03 (2H, s, CH2Ph), 2.05 (3H, s, CH3). 13C NMR (100 MHz, MeOD-d4), δ(ppm): 175.81, 160.98, 155.05, 143.49, 136.81, 128.76, 128.19, 128.18, 116.22, 73.37, 13.67; m/z (ESI-MS) = 217 (M+1). 1-(3-Aminopropyl)-3-(benzyloxy)-2-methylpyridin-4(1H)-one

(2).

A

solution

of

3-

benzyloxy-2-methyl-4-pyrone (10.41 g, 48.16 mol) in a EtOH/H2O mixture (6/4 mL) was added dropwise to a solution of 1,3-diaminopropane (4.50 mL, 52.97 mmol) in a 17 mL of EtOH, 13

PT

mL of H2O and NaOH aqueous solution (cNaOH = 2 mol L-1). The reaction mixture was left stirring under reflux at T = 348 K for 20 h. After cooling, HCl aqueous solution (cHCl = 2 mol L) was added until pH ~ 1 and EtOH was evaporated. To the remaining oily solution, water was

RI

1

added (50 mL), followed by extraction with Et2O (4 × 50 mL). The aqueous phase was

SC

alkalinized with a NaOH aqueous solution (cNaOH = 10 mol L-1) until pH ~ 12 and extracted with DCM (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate and filtered; then

NU

the solvent was rotoevaporated to dryness. To the dried residue, 3 mL of EtOH were added; this solution was acidified until pH ~ 2 with HCl-saturated EtOH to give a white precipitate that was

MA

recrystallized from EtOH-ACN, affording the pure product as the corresponding hydrochloride salt (2.66 g, η = 18%). TLC control of the reaction was carried out using S2 mixture, Rf = 0.45. m.p. 463–466 K. 1H NMR (400 MHz, D2O), δ (ppm): 8.13 (1H, d, 6-HPy), 7.38 (5H, s, Ph), 7.11

D

(1H, d, 5-HPy), 5.08 (2H, s, CH2Ph), 4.28 (2H, t, CH2NPy), 2.99 (2H, t, CH2NH2), 2.34 (3H, s,

PT E

CH3), 2.09 (2H, m, CH2CH2NH2). 13C NMR (100 MHz, D2O), δ (ppm): 164.96, 150.10, 142.97, 141.92, 135.19, 129.69, 129.27, 128.82, 113.56, 75.43, 53.21, 36.21, 27.28, 12.90; m/z (ESI-MS)

CE

= 273 (M+1).

2-(Bis(2-((3-(3-(benzyloxy)-2-methyl-4-oxopyridin-1(4H)-yl)propyl)amino)-2oxoethyl)amino)acetic acid (3). To a mixture of nitrilotriacetic acid (NTA, 0.35 g, 1.83 mmol)

AC

dissolved in 25 mL of dry and distilled DCM, N-methylmorpholine (NMM, 0.80 mL, 7.32 mmol) was added under nitrogen atmosphere. Few minutes later, when the reaction mixture became a clear solution, n-propylphosphonic anhydride (T3P, 1.20 mL, 4.02 mmol) was added dropwise under nitrogen and left stirring for 30 min. Meanwhile a solution of 1-(3-aminopropyl)3-(benzyloxy)-2-methylpyridin-4(1H)-one (1,12 g, 3.66 mmol) as hydrochloride salt, which had been neutralized with KOH (0.24 g, 4.40 mmol) in 20 mL of dry methanol, by stirring under nitrogen for 1.5 h, was filtered to remove the precipitate of KCl. The solvent was evaporated under vacuum, the mixture dissolved in dry DCM and added to the clear solution of above NTA mixture, under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 5 h and the course of the synthesis was monitored by TLC using the S3 mixture. DCM layer was 8

ACCEPTED MANUSCRIPT washed with brine solution and the reaction mixture was purified through column chromatography over silica in 9-10% MeOH-DCM system to give the desired compound (0.88 g, η = 69%). m. p. 351–353 K. 1H NMR (400 MHz, MeOD-d4), δ (ppm): 7.73 (2H, d, 6-HPy), 7.30-7.38 (10H, m, Ph), 6.46 (2H, d, 5-HPy), 5.05 (4H, s, CH2Ph), 3.99 (4H, t, CH2N), 3.43 (4H, s, NCH2CO), 3.29 (2H, s, CH2COOH), 3.23 (4H, t, CH2NH), 2.16 (6H, s, CH3), 1.86 (4H, t, CH2CH2CH2).13C NMR (100 MHz, MeOD-d4), δ (ppm): 177.56, 173.41, 172.71, 145.70, 143.59, 139.95, 137.04, 128.88, 128.02, 116.04, 73.07, 59.68, 51.61, 35.60, 30.00, 11.40. m/z (ESI-MS)

PT

= 699 (M)+, 722 (M+Na)+.

RI

2-Bis(2-((3-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)propyl)amino)-2-

SC

oxoethyl)amino)acetic acid (4, NTA(PrHP)2). In a hydrogenation flask, the intermediate 3 (0.70 g, 1 mmol) was dissolved in methanol and 10% Pd/C was added (0.21 g, 2 mmol) and the

NU

suspension mixture was stirred under a H2 atmosphere (4.5 atm) for 3 h at room temperature. The reaction mixture was filtered, the methanol was evaporated under reduced pressure and the solid product was dried under vacuum affording the pure final product (0.49 g, η = 95%); m. p.

MA

368–370 K. 1H NMR (400 MHz, MeOD-d4), δ (ppm): 7.57 (2H, d, 6-HPy), 6.30 (2H, d, 5-HPy), 3.99 (4H, t, CH2N), 3.30 (4H, s, NCH2CO), 3.21 (2H, s, CH2COOH), 3.18 (4H, t, CH2NH), 2.31 (6H, s, CH3), 1.86 (4H, t, CH2CH2CH2).

13

C NMR (100 MHz, DMSO-d6), δ (ppm): 173.70,

D

171.22, 169.29, 145.99, 138.07, 129.05, 111.09, 58.70, 56.40, 51.10, 35.88, 30.70, 11.70. m/z

PT E

(ESI-MS) = 520 (M +1). Elemental analysis calc. for (C24H33N5O8. 0.18 H2O): C 55.14, H 6.43, N 13.40%; found: C 55.46, H 6.39, N 13.45%.

CE

2.6. Computational studies

The conformational analysis of the ZnLH32H2O, Al2L3H3 and Al2L2(OH)2 complexes was

AC

carried out with the classical molecular mechanics force field (MMFF) by using the Monte Carlo method to randomly sample the conformational space. The equilibrium geometries were then refined by semi-empirical methods (PM6), and finally optimized at the density functional level of theory (DFT, B3LYP functional) using the 6-31G(d) basis set. All quantum mechanical calculations were performed using Spartan’10[39] (Wavefunction, Inc.). 2.7. Animal studies Ethics - Animal studies were carried out under the supervision of well-experienced researcher in laboratory animal facilities licensed by the National Authority in compliance with the principles of laboratory animal science on animal care, protection and welfare. The research 9

ACCEPTED MANUSCRIPT project was also approved by the National Authority according to the National and European Legislation. Biodistribution studies -

67

Ga-citrate injection solution was prepared by dilution of

67

Ga

citrate from Mallinckrodt Medical B.V. with saline to obtain a final radioactive concentration of approximately 7.0-8.0 MBq/100 L. Biodistibution studies were carried out in groups of 3 female Balb-C mice (randomly bred, Charles River, from CRIFFA, France) weighing ca. 22-25

PT

g. Mice were intravenously (i.v.) injected with 100 L (7.0 – 8.0 MBq) of 67Ga citrate via the tail vein immediately followed by intraperitoneal (i.p.) injection of 0.5 mol of the ligand in 100 L

RI

saline. Animals were maintained on normal diet ad libitum and were sacrificed by cervical

SC

dislocation at 1 h and 24 h post-administration. The administered radioactive dose and the radioactivity in sacrificed animals were measured in a dose calibrator (Capintec CRC25R). The difference between the radioactivity in the injected and sacrificed animal was assumed to be due

NU

to whole body excretion. Tissue samples of main organs were then removed for counting in a gamma counter (Berthold LB2111, Berthold Technologies, Germany). Biodistribution results

MA

were expressed as percent of injected activity per gram of organ (% I.A./g) and presented as mean values ±SD.

PT E

3.1. Synthesis of the ligand

D

3. Results and discussion

The synthesis of NTA(PrHP)2 was carried through the 4 reaction steps summarized in

CE

Scheme 1. The first 2 steps lead to the preparation of the mono-hydroxypyridinone “arm”, following standard reactions. Starting from the naturally origin maltol, its hydroxyl was protected with a benzyl group, by reaction with benzyl chloride under basic conditions; the

AC

benzyloxypyrone was further transformed in the corresponding N-heterocycle (pyridinone), Nfunctionalized with the aminopropyl group, by reacting with diaminopropylamine, following a Michael addition. The condensation of two equivalents of 1-(3-aminopropyl)-3-(benzyloxy)-2methylpyridin-4(1H)-one (2) with the molecular scaffold nitrilotriacetic acid (NTA), was performed aided by the coupling agent the n-propylphosphonic anhydride (T3P or 2,4,6tripropyl-1,3,5,2,4,6-trioxatriphosphinane-2,4,6-trioxide)[40-42] in the presence of a base, leading to the formation of new amidic bonds. The synthetic procedure ended with a standard catalytic hydrogenolysis (H2, 10% Pd/C) to remove the benzyl protecting groups[43]. 3.2. Acid – base properties of the ligand 10

ACCEPTED MANUSCRIPT The structure of NTA(PrHP)2 (see Fig. 1) is featured by six different protonable sites: 1. two hydroxyl groups of the N-aromatoid rings; 2. an amino and a carboxylic groups on the NTA side of the molecule; 3. two pyridinone nitrogen atoms (proton supplied by excess of inorganic acid) [44]. For all the possible protonable groups the protonation constants were successfully determined, and the experimental results obtained are reported in Table 1. The analysis of recorded absorption spectra (Fig. S1) showed an absorption band with a

PT

λmax = 278 nm. An increase of its intensity until pH ~ 5 occurs, followed by a decrease of the signal and a batochromic shift. Furthermore, some isosbestic points at different wavelenghts can

RI

be observed, due to the sequential formation of the different protonated species.

SC

The temperature effect on the speciation was also studied by performing UV–Vis spectrophotometric measurements at I = 0.15 mol L-1 in NaCl(aq) and T = 310.15 K

NU

(physiological conditions). The protonation constants for all the species determined at T = 310.15 K, as listed in Table 1, show a common trend; in fact, the values of the logK1H and logK2H, probably related to the –OH groups, slightly increase upon heating, while an inverse behaviour

MA

involves the other protonation constants. The effect of this variable on the acid-base properties of NTA(PrHP)2 can be better estimated by the analysis of the distribution diagram in Fig. 2, built at

D

I = 0.15 mol L-1 in NaCl(aq) at T = 298.15 K and T = 310.15 K. The percentages of all the species exceed the 35% and their formation at higher temperature is shifted towards more acidic pHs

PT E

than at T = 298.15 K; at physiological pH values the main species is, in both cases, H2L. Furthermore, also spectrofluorimetric measurements were performed at I = 0.15 mol L-1 in NaCl(aq) at T = 298.15 K and T = 310.15 K, with the aim of confirming the speciation models and

CE

the results obtained by UV–Vis spectrophotometric data. λmax = 278 nm was selected as maximum excitation wavelength. In addition, some checks were carried out selecting as

AC

excitation wavelength the ones of isosbestic points, to verify whether the behaviour of the species was the same observed at λmax. In Fig. 3 an example of tridimensional titration curve of NTA(PrHP)2 is reported at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K, in the pH range 2.0– 11.0: a lowering of the signal increasing the pH was observed during the experiments. Owing to the complexity of the system, only some protonation constants were successfully refined. For example, at T = 298.15 K three protonation constants were determined, namely logK3H = 7.33±0.01, logK5H = 3.78±0.02 and logK6H = 2.60±0.03, comparable to the corresponding ones coming from the UV–Vis investigations, namely logK3H = 7.15, logK5H = 3.51, logK6H = 2.87. 3.3. Hydrolysis of the metal cations 11

ACCEPTED MANUSCRIPT The hydrolytic constants of the metal cations of interest and, in some case, the solubility product related to the formation of sparingly soluble species (Al(OH)3(s) and Fe(OH)3(s)), were already studied and are reported in literature (see Table S1) [10, 45-48]. 3.4. Stability of Mn+/Lz - complexes The study of the binding ability of NTA(PrHP)2 towards Ca2+, Cu2+, Zn2+, Al3+ and Fe3+ at

PT

I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K, allowed the determination of MpLqHr complexes with different stoichiometry, depending on the different charges and/or acid-base properties of

RI

the metal cations. 3.4.1. M2+/bis-(3-hydroxy-4-pyridinone) systems

SC

The elaboration of the UV-Vis spectrophotometric data related to the Ca2+, Cu2+, Zn2+/bisligand interactions in aqueous solution led to the determination of speciation models including

NU

protonated, simple metal-ligand and polynuclear species, namely the MLH3, MLH2, MLH and ML, in the pH range 2.0–11.0. The experimental formation constants of the M2+/NTA(PrHP)2

MA

complexes, obtained at the already mentioned experimental conditions, are reported in Table 2. The analysis of the data showed that the stability constant value of CuL species is higher than those of CaL and ZnL, probably due to the higher affinity of copper towards the amino groups.

D

As an example, in Fig. 4 the distribution diagram of Zn2+/NTA(PrHP)2 complexes at I = 0.15

PT E

mol L-1 in NaCl(aq) and T = 298.15 K, is reported. For all the species the formation percentages exceeds the 25%. Free Zn2+ is present in solution until pH ~ 2.8, while the ZnLH3 and ZnLH2 species reach formation percentages of 78 and 87% at pH ~ 2.3 and 4.0, respectively. ZnLH

CE

starts to form at pH ~ 3.0, this also being the main complex at physiological pH value. Finally, the formation of the 1:1 metal-ligand species (ZnL) begins at pH ~ 6.9 and achieves the 93% at

AC

pH ~ 11.0. The analysis of the titration curves obtained from UV-Vis spectrophotometric measurements at different pH values, showed a batochromic shift as already observed for the protonation reactions.

3.4.2. Al3+/bis-(3-hydroxy-4-pyridinone) interactions The investigation on the complexing ability of the ligand towards Al3+ at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K, showed the formation of five species, namely AlLH, AlL, Al2L, Al2L3H3 and Al2L2(OH)2, in the pH ranges 2.0–8.0 for potentiometric titrations, 2.0–4.0 for 1H NMR measurements and 2.0–11.0 for UV-Vis spectrophotometric checks, carried out at low components concentrations. The analysis of the experimental data led to the refinement of the

12

ACCEPTED MANUSCRIPT stability constant values of the complexes, which are in good accordance among the analytic techniques, confirming the speciation scheme determined (see Table 3). In Fig. S2 the distribution of the species determined by potentiometry at the cited conditions is reported. Except for AlL, all the other Al3+/NTA(PrHP)2 complexes exceed the 40% of formation. The free metal cation is present in solution from pH ~ 2.0, whilst the Al2L and AlLH species reach the 52% and 83% of formation at pH ~ 2.2 and 2.8, respectively. Al2L3H3 starts to form at pH ~ 2.6; this polynuclear complex covers almost all the pH range investigated

PT

and it is also the main species at physiological pH values. The formation of Al2L2(OH)2 species occurs at pH ~ 7.2 being the 43% of formation achieved at pH ~ 8.0.

RI

The absorption spectra recorded for this system are characterized by a similar trend than

SC

those obtained for the protonation reactions; they vary in a significant way along the pH scale, as the metal-ligand species absorb at different wavelengths. The band at 278 nm (λmax) presents an

NU

increasing of its intensity in the pH range 2.5–6.0, followed by a decrease and then by a batochromic shift. Moreover, some isosbestic points at different wavelengths occur.

The

interactions

between

MA

3.4.3. Fe3+/bis-(3-hydroxy-4-pyridinone) study Fe3+

and

NTA(PrHP)2

were

studied

by

UV-Vis

D

spectrophotometry at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K in the pH range 2.0–11.0, without the formation of sparingly soluble species. The elaboration of the experimental data led

PT E

to the determination of complexes with different stoichiometry, such as protonated (FeLH2, FeLH), simple metal-ligand (FeL), hydrolytic mixed (FeL(OH)) and polynuclear (Fe2L3, Fe2L3H) species. The experimental stability constants of the Fe3+/bis-(3-hydroxy-4-pyridinone) species

CE

are reported in Table 3.

The analysis of the absorption spectra (Fig. 5) recorded at different metal-to-ligand

AC

concentration ratios (e.g. cM : cL = 1 : 1, Fig. 5 a); cM : cL = 1 : 2, Fig. 5 b)), suggests the existence of different species for each stoichiometric condition. In the case of measurements performed at metal-to-ligand equimolar ratio (Fig. 5 a)), at pH ~ 2.2 a charge transfer (CT) band (λmax = 510 nm), between the oxygen atoms of the bis-(3-hydroxy-4-pyridinone) and the iron(III) can be observed, attributable to the formation of a bis-chelated[21, 24, 44] species (FeLH). Along the pH scale, the intensity of this band increases, and a hypsochromic shift and some isosbestic points at different wavelengths occur. In the pH range 4.0-5.2, the presence of a band at λmax = 475 nm indicates the formation of FeL complex[18]. Moreover the formation of mixed hydroxo–ligand species is evidenced by the presence of an absorption band at λmax = 465 nm at alkaline pH values. Concerning the experiments carried out at 1:2 stoichiometry (Fig. 5 b)), the 13

ACCEPTED MANUSCRIPT absorption spectra are featured also a band at λmax = 460 nm, typical of tris-chelated species[21, 24, 44], involving three 3-hydroxy-4-pyridinone moieties coordinated to the metal cation. Since it is not possible to provide all of the six coordination sites by one ligand with hydroxypyridinonate type chelates, in conditions of ligand excess, it can be assumed that one molecule of ligand can bridge two bis-chelated Fe3+ centres to complete the coordination sphere, corresponding to the formation of binuclear Fe2L3Hx species, where x = 0, 1.

PT

3.5. 1H NMR titrations and computational studies

RI

In order to obtain further information on the acid-base behavior of NTA(PrHP)2 as well as its coordination capability towards Zn2+ and Al3+, several 1H NMR spectra in a H2O/D2O

SC

mixture were collected, in the pH range between approx. 2.0 and 11.0, under the conditions already described in the Materials and methods section. The spectra recorded on the metal-free

NU

system showed a single set of average resonances, so that fast mutual exchange among all the species was assumed. The observed average chemical shifts of the 1HNMR spectra of

MA

NTA(PrHP)2, showed pH-dependence, namely a shielding effect upon pH increasing. In particular, as already detected for L2 ((S)-2-amino-4-((2-(3-hydroxy-2-methyl-4-oxopyridin1(4H)-yl)ethyl)amino)-4-oxobutanoic acid))[16] system, the doublets found for the aromatoid

D

protons, a and b in Fig. 1, shifted towards lower chemical shifts at acidic pHs, suggesting that the

PT E

first deprotonations occurred on the quaternarized pyridinone nitrogens. The cited resonances remained virtually unchanged up to pH ~ 9.0, then the peaks were involved in a shift, wider for a protons, that can be explained in terms of further deprotonation processes occurring in the same

CE

region of the molecule, at high pH values, involving the hydroxylic groups. A similar behavior with respect to the one discussed was found also for c methylic and d methylene groups, whilst

AC

the resonances due to e and f protons, as expected, were not involved in significant variations upon pH increasing. A deshielding comparable to the one occurring for a and b signals was also observed, up to pH = 6.0, for g and h methylenes peaks, suggesting that the nearby –COOH and nitrogen amino moieties were involved in deprotonations, in the acidic range. Since the extent of the g and h shifts towards lower ppm was quite similar, from the direct observation of the spectra a clear deprotonation sequence could not be assessed. As stated, all the recorded spectra on NTA(PrHP)2 solutions showed a single set of resonances, deriving from the rapidly exchanging species on the NMR time scale, so that each observed chemical shift is actually the mol-fraction weighted average of the δ values of all the species present at equilibrium, at each investigated pH. These average chemical shifts were employed as input data in HypNMR software to calculate the protonation constants and the individual chemical shifts. As can be inferred from 14

ACCEPTED MANUSCRIPT Table 1, the as such refined protonation constants for NTA(PrHP)2 are in an excellent agreement with the ones obtained from UV-Vis spectrophotometry data elaboration. In addition, in Table S2 are collected the calculated individual chemical shifts for each species present at equilibria; unfortunately, these refined values did not provide other additional information on the deprotonation sequence. However, to further highlight the reliability of the calculation, Fig. 6 shows the complete overlapping of the experimental and calculated chemical shifts for selected nuclei.

PT

The speciation models proposed for the complexing ability of NTA(PrHP)2 towards Zn2+ and Al3+ were further ascertained by 1H NMR titrations carried out on metals containing

RI

systems, in the conditions pointed out in the Materials and methods section. As found for

SC

NTA(PrHP)2 titrations, also in the case of Zn2+/L solutions, the collected spectra showed a single set of resonances. The effect of the presence of Zn2+ on the ligand average chemical shifts

NU

involved mainly the aromatoid core, since the resonances due to a, b, c and d protons, at pH > 4.0, underwent a shielding with respect to the metal-free solutions, thus suggesting that at the cited pHs, the pyridinone moieties are responsible for the interaction with the metal. An opposite

MA

behavior was observed for h methylenes, adjacent to the carboxylic group, whose signal shifted towards higher ppm starting from pH ~ 4.2 on. In the pH range between neutrality and 11.0, the f

D

methylenes resonance was upfield shifted, suggesting that also the amidic moieties may be involved in the coordination. The observed chemical shifts were once again employed to

PT E

calculate the stability constants of the metal-containing species as well as the individual chemical shifts of each nucleus in the complexes formed. More in detail, due to the low number of available data at higher pHs, ZnL stability constant was not refined, whereas the other three logβ,

CE

namely logβ113, logβ112 and logβ111 were successfully calculated, their being values absolutely comparable to the ones gained by means of UV-Vis spectrophotometry findings elaboration, as

AC

reported in Table 2. In addition, the individual chemical shifts for the most abundant species present at equilibria, i.e. ZnLH3, ZnLH2 and ZnLH were obtained as well (Table S3). This allowed to compare them to the free ligand ones; more in detail, for both ZnLH2 and ZnLH, the protons belonging or close to the aromatoid moiety, namely a, b, c and d as well as f, g and h appeared shifted with respect the corresponding metal-free species calculated signals, indicating the involving of the whole ligand in the metal-ligand interaction, whereas for ZnLH3, the peaks involved in a considerable shifting were g and h only, thus suggesting that, for this complex, the preferential site for the interaction with the ligand was the NTA core rather than hydroxo-oxo moiety. Computational studies at the density functional level (B3LYP functional, 6-31G(d) basis set)[49-51] provided further insight into the structural features of the ZnLH3 complex. Geometry 15

ACCEPTED MANUSCRIPT optimization of the equilibrium conformer of ZnLH32H2O revealed that the Zn2+ cation is indeed nestled within the bowl-shaped cavity generated by the ligand (Fig. 7), rather than being chelated by the terminal pyridinone rings, thus confirming the evidence gathered by NMR spectroscopy. The metal is held there by three ZnO close contacts (2.05–2.24 Å) with the carbonyl oxygen atoms from the two amide groups and the carboxylic acid moiety. A weaker interaction with the tertiary amine nitrogen atom (3.07 Å), two water molecules and the oxygen atom of a folded-in pyridinone group complete the coordination sphere.

PT

In the case of the spectra recorded on NTA(PrHP)2 and Al3+ mixtures, in the whole pH

RI

range investigated, two different sets of signals were detected for a, b, c and d protons, due to bound and free ligand, as found for similar systems[16, 21, 24]. As a common feature, the

SC

presence of Al3+ is responsible for a general broadening of the spectra[11], this effect being more pronounced for “bound” signals. As an example, the spectra of metal free and metal containing

NU

solutions, recorded at a pH of approx. 2.2, are compared in Fig. 8, where this evidence is clearly shown. From the observation of the experimental data, since the “bound” peaks were observed

MA

only for some nuclei, it can be suggested that the coordination towards Al3+ occurred via the aromatoid part of the ligand, probably accounting for the most abundant species formed on the whole pH range studied. From speciation studies obtained from UV-Vis spectrophotometry and

D

potentiometry investigations, it appeared that, together with the free ligand (due to its excess in

PT E

solution, in the selected conditions) other complexes should be found, whose stoichiometry depended on the pH. As no other information can be gained from the simple observation of NMR spectra, the data were employed to calculate the stability constants by HypNMR computer

CE

software, using, as known values, the data refined from the protonation investigations (i.e. protonation constants and individual species chemical shifts) obtained previously. As can be inferred from Tables 3 and S3, only the stability constants due to AlLH, AlL and Al2L were

AC

refined and, consequently, the individual chemical shifts of the corresponding complexes. The so calculated logβ were in good agreement with the ones refined starting from potentiometric and spectrophotometric findings. The comparison between the complex species calculated individual chemical shifts with the ones refined for the metal-free ligand system, indicated that for AlLH and Al2L no significant variations occurred, whereas for AlL all the signals, except e and f, were shifted, suggesting, once again, that all the coordination sites of the ligand were involved in the metal-ligand interaction. Unfortunately, since the most abundant species expected to form in the whole pH range investigated, namely Al2L3H3 and Al2L2(OH)2, are polynuclear and HypNMR did not allow to gain reliable data on polynuclear species, their logβ as well as the individual chemical shifts could not be refined from the NMR experimental data. The density functional 16

ACCEPTED MANUSCRIPT (B3LYP/6-31G(d)) optimized geometry of these two complexes showed that, even though in both cases the aluminium cations are bound to the pyridinone arms, their geometry appears quite different. In the Al2L3H3 case, an approximately D3-symmetric structure is evidenced, with the three ligand molecules arranged in a C-shaped conformation that allows only one pyridinone moiety of each ligand to interact with one of the Al3+ cations (Fig. 9 a): top, b): side). Both metal atoms display a slightly distorted octahedral coordination sphere, with the six pyridinone oxygen atoms from three different ligand strands (AlO 2.30–2.44 Å for the carbonyl oxygens, 1.76–

PT

1.82 Å for the C–O–). The AlAl distance is 8.73 Å. Regarding the Al2L2(OH)2 complex, two

RI

hydroxide ions are placed in a bridging position between the metals (Fig. 9 c)). The Al3+ ions and the oxygen atoms lie on the same plane in a diamond-like arrangement (AlO 1.88–1.89 Å,

SC

AlAl 2.94 Å, OO 2.35 Å). Interestingly, in this case the two ligand molecules are arranged in a more pinched C-conformation, in such a way that each one uses both of its hydroxypyridinone

NU

chelating moieties to coordinate to a different aluminium cation.

MA

3.6. Literature data comparison 3.6.1. Acid-base properties of the ligands

The acid-base behaviour of the bis-(3-hydroxy-4-pyridinone) studied at I = 0.15 mol L-1 in

D

NaCl(aq) and T = 298.15 K, could be compared with a quite good agreement with the deferiprone

PT E

literature data, reported in Table 4 and obtained by Bretti and colleagues [52], Clevette et al.[9] at the same experimental conditions, and by Crisponi et al.[3, 4] and Clarke et al.[53] at I = 0.10 mol L-1 in KCl(aq) and T = 298.15 K. Martell et al.[10] reported data for the protonation of

CE

nitrilotriacetic acid at I = 0.10 mol L-1 in Na+ supporting electrolyte. Some differences are evidenced, as it can be observed from the data in Table 4, probably because of electronic effects

AC

and for the substitution of two carboxylic groups of the NTA with two 3-hydroxy-4-pyridinone chains. Santos et al.[25] published the protonation constants of the ligand EDTA(PrHP)2 at I = 0.20 mol L-1 in KCl(aq) and T = 298.15 K and the results [24] of a study performed at the same temperature and I = 0.10 mol L-1 in KNO3(aq) on the product IDA(PrHP)2. Furthermore, Cappai et al.[19] reported the acid-base behaviour of the ligand NTA(PrHP)3 at I = 0.10 mol L-1 in NaCl(aq) and T = 298.15 K. The values determined by all these authors, listed in Table 4, are quite in good agreement with those obtained in this work at the already mentioned experimental conditions. 3.6.2. Stability of Mn+/bis-(3-hydroxy-4-pyridinone) systems Comparisons with the data reported in the literature about the complexation of the metal cations either with EDTA(PrHP)2[21], IDA(PrHP)2[24], NTA(PrHP)3[19] or with DFP and 17

ACCEPTED MANUSCRIPT NTA[9, 10, 53, 54], from which NTA(PrHP)2 is derivative, can be made. The literature data listed for the Mn+/Lz- systems concerning the common species to those determined in this paper, are reported in Table 5. Martell et al.[10] and Clarke et al.[53] reported the results of some studies focused on Ca2+, Cu2+ and Zn2+ interactions with NTA and those of Cu2+ and Zn2+ with deferiprone, respectively, at I = 0.10 mol L-1 in Na+ background electrolyte and T = 298.15 K. The stability of the M2+/NTA(PrHP)2 species resulted to be higher than those of the systems just mentioned (see

PT

Table 5); therefore, from the thermodynamic point of view, the synthesized compound may be a good alternative to the use of NTA and DFP for the treatment of metal cations overload from the

RI

human body. Furthermore, Gama et al.[21] obtained for the Zn2+/EDTAPr(3,4-HP)2 system, at I

SC

= 0.20 mol L-1 in KCl(aq) and T = 298.15 K, a same speciation model of that reported in this paper. The author considered also the formation of other species, here not refined, such as ZnLH4

NU

and the polynuclear Zn2LH complex. Santos et al.[24] published the results of a study performed on the Zn2+/IDAPr(3,4-HP)2 complexation at I = 0.10 mol L-1 in KNO3(aq) and T = 298.15 K, obtaining a simple speciation model constituted by ZnLH3, ZnLH and ZnL. From the comparison

MA

between the literature data related to the ZnL species (Table 5) and the corresponding ones reported in Table 3 at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K, the following trend of

D

stability was observed bis-hydroxypyridinone ligands: NTA(PrHP)2 > IDA(PrHP)2 > EDTA(PrHP)2.

PT E

Concerning the Al3+/bis-ligand systems, Gama et al.[21], Santos and co-workers[24] and Cappai et al.[19] obtained similar speciation scheme with EDTA(PrHP)2, IDA(PrHP)2 and NTA(PrHP)3. The models presented in the literature consider also other complexes, here not

CE

refined, such as the protonated AlLH4, AlLH3, AlLH2, mixed hydrolytic (AlLOH) and polynuclear Al2L3, Al2L3H and Al2L3H2 species. Clevette et al.[9] and Martell et al.[10] reported, at T =

AC

298.15 K and I = 0.15 mol L-1 in NaCl(aq) and I = 0.10 mol L-1 in Na+ background electrolyte, respectively, the formation of a Al(DFP) and Al(NTA) species with logβ110 = 11.91 and 13.30, respectively, against a value of logβ110 = 26.19±0.08 determined here for Al3+/NTA(PrHP)2 system, at the already mentioned experimental conditions. Also the results of studies on complexation of iron(III) with EDTA(PrHP)2, IDA(PrHP)2 and NTA(PrHP)3 were published by Gama and coworkers[21], Santos and colleagues[24] and Cappai et al.[19] respectively, with speciation schemes, listed in Table 5, that are in agreement with those reported in this paper. For the FeL species the following trend of stability was observed: NTA(PrHP)3 > EDTA(PrHP)2 > NTA(PrHP)2 > IDA(PrHP)2. Except for NTA(PrHP)3, since the other compounds have the same alkyl-HP arm the difference can be attributed to the 18

ACCEPTED MANUSCRIPT presence of extra-carboxylic groups (two, one or zero, respectively) or to the higher flexibility associated to the EDTA skeleton. In the papers, also the formation of other protonated and polynuclear species (e.g. FeLH4, FeLH3, Fe2L3H2, Fe2L3H3) was determined. Furthermore, from the analysis of the literature data reported by Motekaitis et al.[54] and Martell et al.[10] (Table 5), the stability of the FeL species of the Fe3+/deferiprone and Fe3+/NTA systems, respectively, resulted lower than those occurring for Fe3+/NTA(PrHP)2 complexes. Consequently, also in this case, synthesized bis-ligand can be an efficient tool with respect to DFP and NTA for the

PT

treatment of human body with problems of hard trivalent metal cations overload.

RI

3.7. Sequestering ability

SC

The sequestering ability a ligand towards one or more metal cations can be studied at various pH, ionic strength, ionic medium and temperature values, by the determination of an

NU

empirical parameter, the pL0.5, which represents the total concentration of ligand required to sequester the 50% of a metal cation present in trace (10-12 mol L-1) in solution. This parameter,

MA

already proposed by this research group[29], is expressed by a sigmoidal Boltzmann type equation (eq. (5)): 1 1 10(pL pL0.5 )

(5)

D

M=

log cL, at xM = 0.5.

PT E

where xM is the mole fraction of metal cation complexed by the ligand, pL = -log cL and pL0.5 = The study of the sequestering ability of NTA(PrHP)2 towards Ca2+, Zn2+, Cu2+, Al3+ and

CE

Fe3+ was carried out at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K and different pH values. From the analysis of the pL0.5 values reported in Table 6, it was observed that the sequestration of the metal cations by the ligand is quite strong, which can be mostly due to the presence of two

AC

3-hydroxy-4-pyridinone chelating units, although the NTA moiety may also be involved. The sequestering ability of NTA(PrHP)2 varies along the investigated pH ranges and this behaviour can be explained taking into account the acid-base properties of the 3,4-HP and of the metal cations. Indeed, the tendency of the ligand to sequester the metal cations increases upon pH increasing due to its deprotonation, but at the same time the hydrolysis of Ca2+, Zn2+, Cu2+, Al3+ and Fe3+ occurs and the competition between NTA(PrHP)2 with OH- to bind the metal cations decreases its sequestering ability. Consequently, to achieve an optimal sequestration, it is mandatory to reach a good balance between the ligands protonation and the metal cations hydrolysis; obviously also the presence or absence of the free metal cation in solution and the formation of both metal-ligand complexes with different stoichiometry and of sparingly soluble 19

ACCEPTED MANUSCRIPT species influence the sequestering ability. An example of sequestration diagram of NTA(PrHP)2 towards Al3+ is reported in Fig. 10 a), drawn at I = 0.15 mol L-1 in NaCl(aq), T = 298.15 K and pH range investigated by UV-Vis spectrophotometry (2.0 ≤ pH ≤ 11.0). It can be noted that the sequestering ability of the ligand increases with the pH values up to pH = 5.0. Furthermore, it assumes similar values in the range 5.0 ≤ pH ≤ 8.1 because, according to the distribution diagram in Fig. S2, in this pH range there is only one major complex, the Al2L3H3 polynuclear species, that reaches formation percentages higher than 90%. Finally, at 9.0 ≤ pH ≤ 11.0, the pL0.5 values

PT

decrease, probably due to the formation in high amounts of ternary mixed hydrolytic Al2L2(OH)2 species. The variations of pL0.5 as a function of parameters like the pH, often follow linear or

RI

polynomial models, as can be seen in Fig. 10 b), for the dependence of pL0.5 on pH for the

SC

Al3+/NTA(PrHP)2 system.

The calculation of pL0.5 values resulted, for several metal-ligand systems,[29] a very

NU

efficient tool for the study of the sequestering ability of ligands towards different metal cations. Nevertheless, if like in our case some systems present polynuclear species (e.g. Al2L, Al2L3H3, Fe2L, Fe2L3, Fe2L3H), they should not be formed at the trace concentration in which the pL0.5 is

MA

calculated. Consequently, this calculation would not consider all the metal-ligand complexes refined in the speciation model, and making comparisons with not containing polynuclear

D

species systems would not be correct. Therefore, to compare objectively the efficiency of a ligand towards different metal cations, the determination of pM values[30] (see pM values

PT E

analysis section) was also considered, because it takes into account the concentration of free metal cation, without undergoing drawbacks related to the presence of different stoichiometry

CE

species. 3.8. pM values analysis

AC

The difficulties found by pL0.5 parameter to explain the different affinity towards various metal cations by NTA(PrHP)2, due to the presence of polynuclear species for some systems, may be overcome by the calculation of pM parameter[30] (pM = -log [M]free with cM = 1.0·10-6 mol L1

and cL = 1.0·10-5 mol L-1) at physiological pH (pH = 7.4). This tool can allow either the

investigation of the chelating affinity of a ligand towards different metal cations or the comparison between different ligands behaviour at the same experimental conditions. Thus, the pM values (Table 7) of all the Mn+/NTA(PrHP)2 systems investigated in this paper were calculated and compared to each other and with the data obtained for ligands with similar molecular structures (IDA(Pr(HP)2, EDTA(PrHP)2, NTA(PrHP)3)[19, 21, 24] and other

20

ACCEPTED MANUSCRIPT ones of clinical and biological interest (e.g. DFP, NTA, DTPA, DOTA, DFB, transferrin),[10, 53, 55, 56] taking into account literature stability constants values. The analysis of the pM values reported in Table 7 showed that the trend at pH = 7.4 is: Fe3+ > Al3+ > Cu2+ > Zn2+ > Ca2+, as illustrated in Fig. 11 a): it could be probably explained considering the “hard-soft acids and bases” theory (HSAB),[57-59] where a hard acid-hard base or soft acid-soft-base interactions are kinetically and thermodynamically favored if compared with hard-soft ones. On this basis, the affinity between hard-metal cations (acids: e.g. Fe3+ and

PT

Al3+) and NTA(PrHP)2 hard-base functional groups (bases: -OH, -COOH) is higher than those with borderline acids such as Cu2+ and Zn2+. Besides, the ligand presents a lower efficacy

RI

towards Ca2+, hard metal too, maybe due to its higher atomic radius than the other metal cations

SC

under study and lower charge than Fe3+ and Al3+, probably less promoting the M-L interaction. Furthermore, the analysis of the pM data calculated at pH = 7.4 showed that, at the already

NU

mentioned conditions, the chelating efficacy of NTA(PrHP)2 towards M2+ and Al3+ is higher than DFP, complexones and, where possible, also DFB and tranferrin. For example, at micromolar conditions the Ca2+ and Cu2+-affinity follows the trend NTA(PrHP)2 > DTPA > NTA, with ΔpCa

MA

= 10.1, 11.1 and ΔpCu = 6.7, 7.8 (Fig. 11 b)), respectively. Also in terms of Zn2+-chelating ability, as it can be observed from the data in Table 7, the synthesized compound could be a better chelator than either EDTA(PrHP)2 and IDA(PrHP)2 (ΔpZn = 6.7, 7.7, respectively), or

D

DFP, DTPA, NTA and DFB (ΔpZn = 1.3, 2.6, 5.8, 10.8, respectively), while the NTA(PrHP)2

PT E

comparison with DOTA showed similar pZn values (17.4 vs. 17.9). The pAl values calculated for the new ligand showed a stronger metal chelating affinity than all the other ligands of clinical and biological application (ΔpAl = 2.1, 5.3, 6.2, 6.9, 8.2, 8.6, for DFB, DFP, DTPA, transferrin,

CE

DOTA, NTA, respectively), as well as EDTA(PrHP)2, IDAPr(3,4-HP)2 and NTA(PrHP)3 (ΔpAl = 2.4, 2.6, 1.6 respectively). On the contrary, concerning the Fe3+-chelating efficiency, DFB,

AC

DTPA, DOTA, EDTA(PrHP)2, IDA(PrHP)2 and NTA(PrHP)3 present higher pM values than NTA(PrHP)2, while an opposite trend characterizes transferrin, DFP and NTA (ΔpFe = 3.3, 4.3, 5.1, respectively, Table 7). 3.9. Pharmacokinetic properties To evaluate the drug-likeness of the ligand in study, NTA(PrHP)2, in comparison with the series of 3-hydroxy-4-pyridinone analogues presented in Fig. 1, some descriptors of their pharmacokinetic profiles were calculated using QikProp program, v. 2.5[60]. Thus, parameters such as the calculated octanol–water partition coefficient (clogP), the ability to cross the BBB (log BB), the capacity to be absorbed through the gastro-intestinal gut, and binding with human 21

ACCEPTED MANUSCRIPT serum albumin, have been calculated and are summarized in Table S4. Analysis of the data in this table shows that although only DFP present no violations to the criteria of the Lipinski´s rule [61], NTA(PrHP)2 presented appropriate lipophilicity (clogP < 5), molecular weights (~ 500) only exceed the number of H-bond acceptors (< 10) or H-bond donors (< 5) which correspond to practically only one violation. Furthermore, in comparison with NTA(PrHP)2, the analogues with the better iron chelating capacity (EDTA(PrHP)2 and NTA(PrHP)3)[19, 21] present higher difficulty on membrane permeation, such as the brain-blood membrane, mostly due to the higher

PT

hydrophilicity and molecular weight.

RI

3.10. Biological assays

67

Ga-

SC

Biodistribution studies were carried out in Balb-C mice previously injected with

citrate to assess the ability of the new bis-3 hydroxy-4-pyridinone ligand NTA(PrHP)2 as in vivo the radiotracer

67

NU

chelating agent for the mobilization of trivalent metal cations. Since the biodistribution profile of Ga-citrate in mice is well-known, we evaluated the effect of the ligand on the

MA

biokinetics and elimination of the radiometal by intraperitoneal administration of 0.5 mole of the NTA(PrHP)2 solution immediately after intravenous administration of the radiotracer. Tissue distribution of

67

Ga in major organs up to 24 h is presented in Fig. 12. Data from this study

D

indicate that the radiometal is rapidly cleared from the bloodstream (0.7±0.3 and 0.06±0.03%

PT E

I.A./g of blood at 1 and 24 h, respectively) mainly by the kidneys. Clearance from the major organs is also very rapid and the radioactivity uptake in most organs, except the kidneys, is inferior to 0.5% I.A./g 24 h after injection. The overall rate of radioactivity excretion was high, 84.6±4.0 and 95.0±2.5 % I.A. at 1 h and 24 h, respectively (see Table 5S and Fig. 13).

CE

Altogether, these results clearly demonstrated that the co-administration of this ligand interferes in the usual biodistribution of the radiotracer leading to a rapid clearance from most organs and a

AC

high increase in the excretion rate from whole animal body through the urinary tract. Such enhancement in the in vivo elimination of the

67

Ga clearly indicate that the NTA(PrHP)2 has a

high in vivo chelating ability, therefore acting as decorporating agent of this radiometal. A comparative analysis of these biodistribution and excretion profiles of

67

Ga with those

obtained in the same animal model with the co-administration of the well-known iron chelating drug DFP at 1 h and 24 h, is presented in Fig. 13 to assess the potential usefulness of our ligand to complex in vivo and eliminate trivalent metal cations. Data from biodistribution profile of the radiotracer

67

Ga-citrate itself in the same animal model was also introduced in the graphic for

comparative purposes. These data clearly indicate an important increase in the elimination of the radiotracer when the NTA(PrHP)2 is administered. Indeed, 1 h after injection more than 80% of 22

ACCEPTED MANUSCRIPT the injected activity was already excreted while after DFP administration such excretion was not so pronounced. Hence, our data lead us to conclude that NTA(PrHP)2 efficiently chelates the radiometal in our animal model which suggests that it can be a very promising candidate as chelating agent of other trivalent metal cations. 4. Conclusions A new bis-(3-hydroxy-4-pyridinone) derivative of NTA, NTA(PrHP)2, was synthesized

PT

and then the acid-base properties and the complexing ability of the ligand towards divalent (Ca2+,

RI

Cu2+, Zn2+) and trivalent (Al3+, Fe3+) metal cations were investigated in NaCl(aq) medium. The speciation study of the bis-(3-hydroxy-4-pyridinone) in aqueous solution started from the

SC

analysis of the acid-base properties, by UV-Vis spectrophotometric and spectrofluorimetric titrations at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K and at T = 310.15 K (physiological

NU

conditions). To confirm the protonation profile also 1H NMR measurements were performed at I = 0.15 mol L-1 and T = 298.15 K. The protonation constants determined the different analytical techniques were in quite good agreement and they also agree with data reported in the literature

MA

for similar compounds.[10, 24, 25, 52] The binding ability of the ligand towards Ca2+, Cu2+, Zn2+, Al3+ and Fe3+ was investigated by potentiometric and UV-Vis spectrophotometric

D

experiments, carried out at T = 298.15 K. The speciation models obtained consisted of MpLqHr

PT E

species with different stoichiometry, including protonated, simple metal-ligand, hydrolytic mixed and polynuclear species, taking into account the different charges and structures of the ligands and the various hydrolytic behaviours of the metal cations. The stability constants refined 1

CE

for the ML species follow the trend: Fe3+ > Al3+ > Cu2+ > Zn2+ > Ca2+. H NMR and computational studies allowed to suggest, for ZnLH3 species, an interaction of the

metal cation with the carbonyl oxygen atoms from the two amide groups and the carboxylic acid

AC

from the NTA moiety. Concerning the Al3+/NTA(PrHP)2 interactions, computational studies indicated for the Al2L3H3 complex an approximately D3-symmetric structure, with the three ligand molecules arranged in a C-shaped conformation that allows each pyridinone moiety to interact with a different Al3+ cation. On the other hand, the Al2L2(OH)2 complex is featured with two hydroxide ions placed in a bridging position between the metals. Furthermore, the sequestering ability of the ligand towards the metal cations was investigated by the determination of pL0.5 and pM values at different experimental pHs and pH = 7.4, respectively. It was observed that NTA(PrHP)2 ligands assume high pL0.5 values and its sequestering ability towards the metal cations varies during the whole pH range of investigation. The analysis of the pM values calculated at pH = 7.4 showed that the metal-ligand affinity 23

ACCEPTED MANUSCRIPT follows the trend: Fe3+ > Al3+ > Cu2+ > Zn2+ > Ca2+. Biodistribution studies in mice indicated that the ligand has high in vivo chelating ability for trivalent metal cations promoting a rapid elimination of the radiometal from the animal body, mainly by urinary excretion. Acknowledgements The authors from the University of Messina thank MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca) for financial support (co-funded PRIN project with prot.

PT

2015MP34H3).

The authors from (IST) University of Lisbon thank the Portuguese Fundação para a

RI

Ciência e Tecnologia (FCT) for financial support for the projects UID/QUI/00100/2013 and

SC

PEst-C/SAU/LA0001/2011-2013, and the postdoctoral fellowship (KC). Acknowledgements are also due to the Portuguese NMR (IST-UL Center) and Mass Spectrometry Networks (Node IST-

NU

CTN) for providing access to their facilities. References

MA

[1] H. Bickel, G.E. Hall, W. Keller-Schierlein, V. Prelog, E. Vischer, A. Wettstein, Stoffwechselprodukte von Actinomyceten. 27. Mitteilung. Über die Konstitution von

D

Ferrioxamin B, Helv. Chim. Acta, 43 (1960) 2129-2138.

PT E

[2] J.B. Neilands, Microbial Iron Compounds, Annu. Rev. Biochem., 50 (1981) 715-731. [3] G. Crisponi, V.M. Nurchi, M.A. Zoroddu, Iron chelating agents for iron overload diseases, Thalassemia Reports, 4 (2014) 2046.

[4] G. Crisponi, M. Remelli, Iron chelating agents for the treatment of iron overload, Coordin.

CE

Chem. Rev., 252 (2008) 1225-1240.

[5] M.A. Santos, S. Chaves, 3-hydroxypyridinone derivatives as metal sequestering agents for

AC

therapeutic use, Future Med. Chem., 7 (2015) 383-410. [6] R.A. Yokel, P. Ackrill, E. Burgess, J.P. Day, J.L. Domingo, T.P. Flaten, J. Savory, Prevention and treatment of aluminum toxicity including chelation therapy: status and research needs, J.Toxicol. Environ. Health, 48 (1996) 667-684. [7] R.C. Hider, G. Kontoghiorghes, J. Silver, M.A. Stockham, UK Patent 2117766, DOI (1982). [8] H. Nick, Iron chelation, quo vadis?, Curr. Opin. Chem. Biol., 11 (2007) 419-423. [9] D.J. Clevette, W.O. Nelson, A. Nordin, C. Orvig, S. Sjoeberg, The complexation of aluminum with N-substituted 3-hydroxy-4-pyridinones, Inorg. Chem., 28 (1989) 2079-2081.

24

ACCEPTED MANUSCRIPT [10] A.E. Martell, R.M. Smith, R.J. Motekaitis, NIST Critically selected stability constants of metal complexes database, 8.0, National Institute of Standard and Technology, Garthersburg, MD, 2004. [11] P. Cardiano, C. Foti, F. Giacobello, O. Giuffrè, S. Sammartano, Study of Al 3+ interaction with AMP, ADP and ATP in aqueous solution, Biophys. Chem., 234 (2018) 42-50. [12] R. Cusnir, C. Imberti, R.C. Hider, P.J. Blower, M.T. Ma, Hydroxypyridinone Chelators: From Iron Scavenging to Radiopharmaceuticals for PET Imaging with Gallium-68, Int. J. Mol.

PT

Sci., 18 (2017) 116.

perspectives, Coordin. Chem. Rev., 228 (2002) 187-203.

RI

[13] M.A. Santos, Hydroxypyridinone complexes with aluminium. In vitro/vivo studies and

SC

[14] M.A. Santos, Recent developments on 3-hydroxy-4-pyridinones with respect to their clinical applications: Mono and combined ligand approaches, Coordin. Chem. Rev., 252 (2008)

NU

1213-1224.

[15] M.A. Santos, S.M. Marques, S. Chaves, Hydroxypyridinones as “privileged” chelating structures for the design of medicinal drugs, Coordin. Chem. Rev., 256 (2012) 240-259.

MA

[16] A. Irto, P. Cardiano, K. Chand, R.M. Cigala, F. Crea, C. De Stefano, L. Gano, S. Sammartano, M.A. Santos, Bifunctional 3-hydroxy-4-pyridinones as effective aluminium

D

chelators: synthesis, solution equilibrium studies and in vivo evaluation, J. Inorg. Biochem., 186 (2018) 116-129.

PT E

[17] J.I. Lachowicz, V.M. Nurchi, G. Crisponi, M.G. Jaraquemada-Pelaez, M. Arca, A. Pintus, M.A. Santos, C. Quintanova, L. Gano, Z. Szewczuk, M.A. Zoroddu, M. Peana, A. DomínguezMartín, D. Choquesillo-Lazarte, Hydroxypyridinones with enhanced iron chelating properties.

CE

Synthesis, characterization and in vivo tests of 5-hydroxy-2-(hydroxymethyl)pyridine-4(1H)one, Dalton Trans., 45 (2016) 6517-6528.

AC

[18] M.A. Santos, M. Gil, L. Gano, S. Chaves, Bifunctional 3-hydroxy-4-pyridinone derivatives as potential pharmaceuticals: synthesis, complexation with Fe(III), Al(III) and Ga(III) and in vivo evaluation with 67Ga, J. Biol. Inorg. Chem., 10 (2005) 564. [19] R. Cappai, K. Chand, J.I. Lachowicz, S. Chaves, L. Gano, G. Crisponi, V.M. Nurchi, M. Peana, M.A. Zoroddu, M.A. Santos, A new tripodal-3-hydroxy-4-pyridinone for iron and aluminium sequestration: synthesis, complexation and in vivo studies, New J. Chem., 42 (2018) 8050-8061. [20] S. Chaves, S.M. Marques, A.M.F. Matos, A. Nunes, L. Gano, T. Tuccinardi, A. Martinelli, M.A. Santos, New Tris(hydroxypyridinones) as Iron and Aluminium Sequestering Agents: Synthesis, Complexation and In Vivo Studies, Chem. Eur. J., 16 (2010) 10535-10545. 25

ACCEPTED MANUSCRIPT [21] S. Gama, P. Dron, S. Chaves, E. Farkas, M.A. Santos, A bis(3-hydroxy-4-pyridinone)EDTA derivative as a strong chelator for M3+ hard metal ions: complexation ability and selectivity, Dalton Trans., DOI (2009) 6141-6150. [22] A. Nunes, M. Podinovskaia, A. Leite, P. Gameiro, T. Zhou, Y. Ma, X. Kong, U.E. Schaible, R.C. Hider, M. Rangel, Fluorescent 3-hydroxy-4-pyridinone hexadentate iron chelators: intracellular distribution and the relevance to antimycobacterial properties, J. Biol. Inorg. Chem., 15 (2010) 861-877.

PT

[23] S. Piyamongkol, T. Zhou, Z.D. Liu, H.H. Khodr, R.C. Hider, Design and characterisation of novel hexadentate 3-hydroxypyridin-4-one ligands, Tetrahedron Lett., 46 (2005) 1333-1336.

RI

[24] M.A. Santos, S. Gama, L. Gano, G. Cantinho, E. Farkas, A new bis(3-hydroxy-4-

SC

pyridinone)-IDA derivative as a potential therapeutic chelating agent. Synthesis, metalcomplexation and biological assays, Dalton Trans., DOI 10.1039/B409357G(2004) 3772-3781.

NU

[25] M.A. Santos, S. Gama, L. Gano, E. Farkas, Bis(3-hydroxy-4-pyridinone)-EDTA derivative as a potential therapeutic Al-chelating agent. Synthesis, solution studies and biological assays, J. Inorg. Biochem., 99 (2005) 1845-1852.

MA

[26] J. Buffle, Complexation Reactions in Aquatic Systems: an Analytical Approach, Ellis Horwood, Chichester, 1988.

D

[27] F.J. Millero, Physical Chemistry of Natural Waters, John Wiley & Sons, Inc., New York, 2001.

PT E

[28] C. Lentner, Geigy Scientific Tables, 8th ed., CIBA-Geigy, Basilea, Switzerland, 1981. [29] F. Crea, C. De Stefano, C. Foti, D. Milea, S. Sammartano, Chelating agents for the sequestration of mercury(II) and monomethyl mercury(II), Curr. Med. Chem., 21 (2014) 3819-

CE

3836.

[30] K.N. Raymond, C.J. Carrano, Coordination chemistry and microbial iron transport, Acc.

AC

Chem. Res., 12 (1979) 183-190. [31] H.A. Flaschka, EDTA Titration, Pergamon Press, London, 1959. [32] C. De Stefano, P. Princi, C. Rigano, S. Sammartano, Computer Analysis of Equilibrium Data in Solution. ESAB2M: An Improved Version of the ESAB Program, Ann. Chim. (Rome), 7 (1987) 643-675. [33] P. Gans, A. Sabatini, A. Vacca, Investigation of equilibria in solution. Determination of equilibrium constants with the HYPERQUAD suite programs., Talanta, 43 (1996) 1739-1753. [34] C. De Stefano, C. Foti, O. Giuffrè, P. Mineo, C. Rigano, S. Sammartano, Binding of Tripolyphosphate by Aliphatic Amines: Formation, Stability and Calculation Problems, Ann. Chim. (Rome), 86 (1996) 257-280. 26

ACCEPTED MANUSCRIPT [35] C. Frassineti, S. Ghelli, P. Gans, A. Sabatini, M.S. Moruzzi, A. Vacca, Nuclear Magnetic Resonance as a Tool for Determining Protonation Constants of Natural Polyprotic Bases in Solution, Anal. Biochem., 231 (1995) 374-382. [36] L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini, A. Vacca, Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species, Coord. Chem. Rev., 184 (1999) 311-318. [37] W.L.F. Armarego, D.D. Perrin, Purification of Laboratory Chemicals, 4 th ed., Butterworth-

PT

Heinemann Press, Oxford, 1999.

[38] H.E. Gottlieb, V. Kotlyar, A. Nudelman, NMR Chemical Shifts of Common Laboratory

RI

Solvents as Trace Impurities, J. Org. Chem., 62 (1997) 7512-7515.

SC

[39] Y. Shao, L.F. Molnar, Y. Jung, J. Kussmann, C. Ochsenfeld, S.T. Brown, A.T.B. Gilbert, L.V. Slipchenko, S.V. Levchenko, D.P. O'Neill, R.A. DiStasio Jr, R.C. Lochan, T. Wang, G.J.O.

NU

Beran, N.A. Besley, J.M. Herbert, C. Yeh Lin, T. Van Voorhis, C.S. Hung, A. Sodt, R.P. Steele, V.A. Rassolov, P.E. Maslen, P.P. Korambath, R.D. Adamson, B. Austin, J. Baker, E.F.C. Byrd, H. Dachsel, R.J. Doerksen, A. Dreuw, B.D. Dunietz, A.D. Dutoi, T.R. Furlani, S.R. Gwaltney,

MA

A. Heyden, S. Hirata, C.-P. Hsu, G. Kedziora, R.Z. Khalliulin, P. Klunzinger, A.M. Lee, M.S. Lee, W. Liang, I. Lotan, N. Nair, B. Peters, E.I. Proynov, P.A. Pieniazek, R.Y. Min, J. Ritchie,

D

E. Rosta, D.C. Sherrill, A.C. Simmonett, J.E. Subotnik, H.L. Woodcock III, W. Zhang, A.T. Bell, A.K. Chakraborty, D.M. Chipman, F.J. Keil, A. Warshel, W.J. Hehre, H.F. Schaefer III, J.

PT E

Kong, A.I. Krylov, P.M.W. Gill, M. Head-Gordon, Advances in methods and algorithms in a modern quantum chemistry program package, Phys. Chem. Chem. Phys., 8 (2006) 3172-3191. [40] T.I. Al-Warhi, H.M.A. Al-Hazimi, A. El-Faham, Recent development in peptide coupling

CE

reagents, J. Saudi Chem. Soc., 16 (2012) 97-116. [41] J.R. Dunetz, Y. Xiang, A. Baldwin, J. Ringling, General and Scalable Amide Bond

AC

Formation with Epimerization-Prone Substrates Using T3P and Pyridine, Org. Lett., 13 (2011). [42] E. Valeur, M. Bradley, Amide bond formation: beyond the myth of coupling reagents, Chem. Soc. Rev., 38 (2009) 606-631. [43] L. Saghaie, M.M. Sadeghi, A. Nikazma, Synthesis, analysis and determination of partition coefficients of N-arylhydroxypyridinone derivatives as iron chelators, Res. Pharm. Sci., 1 (2006) 40-48. [44] M.A. Santos, M. Gil, S. Marques, L. Gano, G. Cantinho, S. Chaves, N-Carboxyalkyl derivatives of 3-hydroxy-4-pyridinones: synthesis, complexation with Fe(III), Al(III) and Ga(III) and in vivo evaluation, J. Inorg. Biochem., 92 (2002) 43-54. [45] C.F. Baes, R.E. Mesmer, The Hydrolysis of Cations, John Wyley & Sons, New York 1976. 27

ACCEPTED MANUSCRIPT [46] R.M. Cigala, C. De Stefano, A. Giacalone, A. Gianguzza, Speciation of Al3+ in fairly concentrated solutions (20 –200mmol L-1) at I = 1 mol L-1 (NaNO3), in the acidic pH range, at different temperatures, Chem. Spec. Bioavail., 23 (2011) 33-37. [47] F.J. Millero, R. Woosley, The Hydrolysis of Al(III) in NaCl solutions-A Model for Fe(III), Environ. Sci. Technol., 43 (2009) 1818-1823. [48] D. Pettit, K.K. Powell., Stability Constants Database, Academic software, Otley, UK, 1997. [49] S. Colombo, C. Coluccini, M. Caricato, C. Gargiulli, G. Gattuso, D. Pasini, Shape

PT

selectivity in the synthesis of chiral macrocyclic amides, Tetrahedron, 66 (2010) 4206-4211. [50] C. Gargiulli, G. Gattuso, A. Notti, S. Pappalardo, M.F. Parisi, A DFT study on a

RI

calix[5]crown-based heteroditopic receptor, Supramol. Chem., 22 (2010) 358-364.

SC

[51] G. Gattuso, A. Notti, M.F. Parisi, I. Pisagatti, M.E. Amato, A. Pappalardo, S. Pappalardo, Threading the Calix[5]arene Annulus, Chem. Eur. J., 16 (2010) 2381-2385.

NU

[52] C. Bretti, R.M. Cigala, F. Crea, G. Lando, S. Sammartano, Thermodynamics of proton binding and weak (Cl-, Na+ and K+) species formation, and activity coefficients of 1,2-dimethyl3-hydroxypyridin-4-one (deferiprone), J. Chem. Thermodyn., 77 (2014) 98-106.

MA

[53] E.T. Clarke, A.E. Martell, Stabilities of 1,2-dimethyl-3-hydroxy-4-pyridinone chelates of divalent and trivalent metal ions, Inorg. Chim. Acta, 191 (1992) 57-63.

D

[54] R.J. Motekaitis, A.E. Martell, Stabilities of the iron(III) chelates of 1,2-dimethyl-3-hydroxy4-pyridinone and related ligands, Inorg. Chim. Acta, 183 (1991) 71-80.

PT E

[55] W.R. Harris, V.L. Pecoraro, Thermodynamic binding constants for gallium transferrin, Biochemistry, 22 (1983) 292-299.

[56] W.R. Harris, J. Sheldon, Equilibrium constants for the binding of aluminum to human

CE

serum transferrin, Inorg. Chem., 29 (1990) 119-124. [57] R.G. Pearson, Hard and Soft Acids and Bases, J. Am. Chem. Soc., 85 (1963) 3533-3539.

AC

[58] R.G. Pearson, Hard and soft acids and bases, HSAB, part 1: Fundamental principles, J. Chem. Educ., 45 (1968) 581. [59] R.G. Pearson, Hard and soft acids and bases, HSAB, part II: Underlying theories, J. Chem. Educ., 45 (1968) 643. [60] L.L.C. Schrödinger, QikProp, version 2.5, New York, NY, 2005. [61] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Adv. Drug Deliver. Rev., 46 (2001) 3-26.

28

ACCEPTED MANUSCRIPT Abbreviations NTA(PrHP)2

=

2-(bis(2-((3-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)propyl)amino)-2-

oxoethyl)amino)acetic acid; 1

H NMR = Proton Nuclear Magnetic Resonance;

13

PT

C NMR = Carbon-13 Nuclear Magnetic Resonance;

TLC = Tin Layer Chromatography;

RI

BnCl = benzyl chloride;

SC

MeOH = methanol;

NU

EtOH = ethanol; DCM = dichloromethane;

MA

NMM = N-methylmorpholine;

T3P = n-propylphosphonic anhydride or 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane-2,4,6-

ACN = acetonitrile;

CE

Et2O = diethyl ether;

PT E

DMSO = dimethyl sulfoxide;

D

trioxide;

L2

=

AC

10% Pd/C = palladium on activated carbon, 10% (w/w); ((S)-2-amino-4-((2-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl)amino)-4-

oxobutanoic acid)).

29

ACCEPTED MANUSCRIPT Tables Table 1 Protonation constantsa,b of NTA(PrHP)2 determined by UV–Vis spectrophotometry and 1H NMR spectroscopy at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 and 310.15 K

T = 310.15 K

T = 298.15 K

logβ1H

10.84±0.03c

10.95±0.02c

10.85±0.002c

logβ2H (logK2H)

20.501±0.007 (9.661)d

20.99±0.03 (10.04)d

20.48±0.003 (9.64)d

logβ3H (logK3H)

27.65±0.14 (7.15)

27.13±0.03 (6.14)

28.07±0.014 (7.59)

logβ4H (logK4H)

32.21±0.07 (4.56)

31.25±0.01 (4.12)

32.28±0.014 (4.21)

logβ5H (logK5H)

35.72±0.09 (3.51)

34.54±0.02 (3.29)

35.79±0.010 (3.51)

logβ6H (logK6H)

38.59±0.07 (2.87)

36.95±0.04 (2.41)

38.59±0.007 (2.80)

NU

SC

PT

T = 298.15 K

RI

H NMR spectroscopy

logβrH equilibrium refers to eq. (2); b logKrH equilibrium refers to eq. (1); c ±Std. deviation; constant

values

calculated

by

means

of

eq.

(1).

PT E

D

MA

stability

CE

stepwise

d

AC

a

1

UV-Vis spectrophotometry

logβrH a (logKrH)b

30

ACCEPTED MANUSCRIPT Table 2 Experimentala,b and averagec stability constants of M2+/NTA(PrHP)2 species determined by UVVis spectrophotometric and 1H NMR spectroscopic titrations at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K 1

H NMR

Mean Zn2+/L

spectroscopy

valuesc

UV-Vis spectrophotometry logβpqra

Ca2+

Cu2+

Zn2+

(logK113)

(11.11)

(11.21)

(10.28)

logβ112

35.50±0.06

35.77±0.03

34.96±0.01

(logK112)

(15.00)

(15.27)

(14.46)

logβ111

29.508±0.007

31.73±0.02

29.72±0.03

(logK111)

(18.668)

(20.89)

logβ110

19.70±0.02

21.08±0.01

37.93±0.03d

37.93±0.27e

(10.28)

(10.28)

34.58±0.10

34.77±0.24 (14.27)

30.58±0.03

30.15±0.36

(18.88)

(19.74)

(19.31)

19.85±0.07

-

19.85±0.07

NU

(14.08)

logβpqr refer to eq. (4); b logKpqr refer to equilibria: M2+ + HrL(r-3) = MLHr(r-1); c values obtained

MA

a

38.86±0.01d 37.93±0.03d

RI

38.76a±0.03d

SC

logβ113

PT

(logKpqr)b

by average of UV–Vis spectrophotometric and e

H NMR spectroscopic data for

errors on weighed data.

AC

CE

PT E

D

Zn2+/NTA(PrHP)2 system; d ±Std. Deviation;

1

31

ACCEPTED MANUSCRIPT Table 3 Experimentala,

b-h

and averagei stability constants of M3+/NTA(PrHP)2 species obtained by

different analytical techniques at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K UV-Vis

Potentiometry

spectrophotometry

(ISE-H+)

logβpqra

Mean valuesi

1

H NMR

spectroscopy

UV-Vis spectrophotometry

Al3+

Fe3+

logβ112

-

-

PT

(logKpqr)b-h -

-

RI

(logK112)b

37.67±0.09m (17.17)

30.72±0.02l

30.42±0.02l

30.46±0.06l

30.49±0.10l

34.06±0.08

(logK111)b

(19.88)

(19.58)

(19.62)

(19.65)

(23.22)

logβ110

26.810±0.008

26.19±0.08

26.35±0.17

26.39±0.13

27.25±0.05

logβ11-1

-

-

-

-

17.34±0.05

(logK11-1)

32.49±0.01

31.95±0.04

(logK210)d

(5.68)

(5.76)

logβ22-2

43.25±0.06

42.82±0.19

(logK22-2)e

(10.76)

(10.87)

logβ230

-

(logK230) logβ231

-

D

CE

(logK231)g

-

PT E

f

-

90.88±0.03

90.53±0.06

(logK233)h

(9.61)

(9.26)

AC

32.06±0.15

42.01±0.18

(5.63)

(5.67)

(14.76)

-

43.03±0.18

-

(10.97) -

-

69.17±0.03 (14.67)

logβ233 a

(3.64)

31.98±0.03

MA

logβ210

NU

c

SC

logβ111

-

-

81.058±0.008 (15.72)

-

90.70±0.16

-

(9.43)

b

logβpqr refer to eq. (4); logK112 and logK111 refer to equilibrium: M3+ + HrL(r-3) = MLHr(r+); c

logK11-1 refers to equilibrium: Fe3+ + L3- + H2O = FeL(OH)- + H+; d logK210 refers to equilibrium: M3+ + ML0(aq) = M2L3+; e logK22-2 refers to equilibrium: Al2L3+ + L3- + 2H2O = Al2L2(OH)22- + 2H+;

f

logK230 refers to equilibrium: 2FeL0(aq) + L3- = Fe2L33-;

g

logK231 refers to equilibrium:

2FeL0(aq) + HL2- = Fe2L3H2-; h logK233 refers to equilibrium: 2AlL0 (aq) + H3L0(aq) = Al2L3H30(aq);

i

values obtained by average of UV–Vis spectrophotometric, potentiometric and 1H NMR spectroscopic data for Al3+/NTA(PrHP)2 system; l ±Std. Deviation; m errors on weighed data.

32

ACCEPTED MANUSCRIPT Table 4 Literature protonation constants of some metal chelators at T = 298.15 K, different ionic strenghts and ionic media logK1H

logK2H

logK3H

logK4H

logK5H

logK6H

DFPa

9.82

3.69

-

-

-

-

-

[52]

DFPa

9.86

3.70

-

-

-

-

-

[9]

DFPb

9.82

3.66

-

-

-

-

-

[3,

b

9.77

3.68

-

-

NTAd

9.46

2.52

1.81

1.00

EDTA(PrHP)2e

10.12

9.53

7.04

IDA(PrHP)2f

9.94

9.44

5.42

NTA(PrHP)3g

10.75

9.85

9.34

4] -

[53]

-

-

-

[10]

4.03

3.38

2.84

-

[25]

3.54

3.11

-

-

[24]

4.19

-

-

1.20

[19]

SC

RI

-

logK7H Ref.

-

NU

DFP

a

PT

Ligand

at I = 0.15 mol L-1 in NaCl(aq); b at I = 0.10 mol L-1 in KCl(aq); c at I = 0.16 mol L-1 in NaCl(aq); d

MA

at I = 0.10 mol L-1 in Na+ background electrolyte; e at I = 0.20 mol L-1 in KCl(aq); f at I = 0.10 mol

AC

CE

PT E

D

L-1 in KCl(aq); g at I = 0.10 mol L-1 in NaCl(aq).

33

ACCEPTED MANUSCRIPT Table 5 Literature Mn+/Lz- systems data at T = 298.15 K and different ionic strength and ionic media System

MLH3 MLH2 MLH

ML

MLOH

M2 L

M2L2(OH)2

M2L3

Ca2+/NTAa

-

-

-

6.30

-

-

-

-

Cu2+/DFPa

-

-

-

10.62

-

-

-

-

Cu2+/NTAb

-

-

14.30 12.70

-

-

-

33.44

29.45

21.18 11.34

-

21.63

27.28

-

18.92 13.30

-

-

Zn2+/DFPa

-

-

-

7.19

-

-

Zn2+/NTAb

-

-

-

10.05

-

Al3+/EDTA(PrHP)2c

-

-

27.58

-

Al3+/IDA(PrHP)2a

-

-

25.37 20.35

Al3+/NTA(PrHP)3d

-

-

30.40 26.58

-

-

Zn2+/EDTA(PrHP)2c 2+

Zn /IDA(PrHP)2

3+

Al /DFP 3+

a

e b

Al /NTA

-

Fe3+/EDTA(PrHP)2c

-

Fe3+/IDA(PrHP)2a

-

C A

-

Ref.

-

-

[10]

-

-

[53]

-

-

[10]

T P

-

I R -

-

-

[21]

-

-

-

-

[24]

-

-

-

[53]

-

-

-

-

[10]

-

N A

-

27.26

-

-

-

-

[21]

-

-

32.05

-

-

76.64

[24]

-

-

-

-

-

-

[19]

M

-

C S U

-

-

D E 11.91

-

-

-

-

-

-

[9]

-

13.30

-

-

-

-

-

-

[10]

PT

E C

M2L3H M2L3H3

37.15

34.19 29.62

22.87

37.21

-

73.60

82.40

-

[21]

-

31.16 26.16

-

-

-

74.26

79.90

-

[24]

Fe3+/NTA(PrHP)3d

-

38.04

-

33.11

-

-

-

-

-

-

[19]

Fe3+/DFPc

-

-

-

15.10

-

-

-

-

-

-

[54]

Fe3+/NTAf

-

-

-

16.00

11.64

-

-

-

-

-

[10]

a

at I = 0.10 mol L-1 in KCl(aq); b at I = 0.10 mol L-1 in Na+ background electrolyte; c at I = 0.20 mol L-1 in KCl(aq); d at I = 0.10 mol L-1 in NaCl(aq);

e

at I = 0.15 mol L-1 in NaCl(aq); f at I = 0.10 mol L-1 in K+ ionic medium. 34

ACCEPTED MANUSCRIPT Table 6 pL0.5a values of Mn+/bis-(3-hydroxy-4-pyridinone) systems at different pHs, I = 0.15 mol L-1 in

pH

pL0.5a

Ca2+/NTA(PrHP)2

4.0

11.4

7.4

12.7

8.1

12.7

4.0

11.8

7.4

12.7

8.1

12.7

4.0

10.8

7.4

12.7

8.1

12.7

Cu /NTA(PrHP)2

NU

Zn2+/NTA(PrHP)2

3+

Fe3+/NTA(PrHP)2

AC

CE

PT E

D

MA

Al /NTA(PrHP)2

a

SC

2+

RI

System

PT

NaCl(aq) and T = 298.15 K

2.5

4.4

3.5

8.6

4.0

10.4

5.0

10.6

6.0

12.7

7.4

12.6

8.1

12.4

9.0

11.1

10.0

8.7

11.0

5.8

2.5

8.7

4.0

11.9

6.0

12.4

7.4

12.5

8.1

12.0

11.0

7.0

calculated by using eq. (5).

35

ACCEPTED MANUSCRIPT Table 7 pM values calculated for different Mn+/L systems based on 3-hydroxy-4-pyridinone ligands and different marketed ligands at pH = 7.4 from literature stability constants at T = 298.15 K. pM

Ref.

System

pM

Ref.

Ca2+/NTA(PrHP)2

17.2

This work

Zn2+/NTAe

11.6

[10]

Cu2+/NTA(PrHP)2

19.4

This work

Al3+/NTAe

12.8

[10]

Zn2+/NTA(PrHP)2

17.4

This work

Fe3+/NTAe

18.5

[10]

Al3+/NTA(PrHP)2

21.4

This work

Ca2+/DTPAe

3+

2+

e

7.1

[10]

12.7

[10]

23.6

This work

Cu /DTPA

Al3+/NTA(PrHP)3a

19.8

[19]

Zn2+/DTPAe

14.8

[10]

Fe3+/NTA(PrHP)3a

26.6

[19]

Al3+/DTPAe

15.2

[10]

Zn2+/EDTA(PrHP)2b

10.7

[21]

Fe3+/DTPAe

24.6

[10]

Al3+/EDTA(PrHP)2b

19.0

[21]

Zn2+/DOTAe

17.9

[10]

Fe3+/EDTA(PrHP)2b

26.3

[21]

Al3+/DOTAe

13.2

[10]

24.3

[10]

Al3+/IDAPr(PrHP)2c

18.8

Fe3+/IDAPr(PrHP)2c

25.8

Zn2+/DFPd

16.1

Al3+/DFPd

16.1

2+

e

Ca /NTA

SC

Fe /DOTA

e

6.6

[55]

[24]

Al3+/DFBd

19.3

[55]

[53]

Fe3+/DFBd

26.5

[55]

[53]

Al3+/transferrinf

14.5

[56]

19.3

[53]

Fe3+/transferrine

20.3

[55]

6.1

[10]

11.6

[10]

D

Zn2+/DFBf

CE

Cu2+/NTAe

[24]

3+

[24]

PT E

Fe3+/DFPd

NU

9.70

Zn /IDAPr(PrHP)2

MA

c

RI

Fe /NTA(PrHP)2

2+

a

PT

System

at I = 0.10 mol L-1 in NaCl(aq); b at I = 0.20 mol L-1 in KCl(aq); c at I = 0.10 mol L-1 in KNO3(aq); d at

AC

I = 0.10 mol L-1 in KCl(aq); e in K+ background electrolyte; f at I = 0.027 mol L-1 in NaHCO3(aq).

36

ACCEPTED MANUSCRIPT

PT

Figs. and schemes

EDTA(PrHP)2

NU

SC

RI

IDA(PrHP)2

NTA(PrHP)3

DFP

MA

NTA(PrHP)2

Fig. 1. Structure of the synthesized bis-(3-hydroxy-4-pyridinone) NTA(PrHP)2, of previous reported analogues (two bis-(3-hydroxy-4-pyridinones), IDA(PrHP)2 and EDTA(PrHP)2, and structure

stand

for

the

1

H

NMR

titrations

peaks

assignment.

AC

CE

PT E

NTA(PrHP)2

D

one tris-(3-hydroxy-4-pyridinone), NTA(PrHP)3) and the drug DFP. The letters in the

37

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

PT E

Scheme 1. Synthetic procedure used for NTA(PrHP)2 ligand. Reagents and conditions: a) BnCl, NaOH, MeOH/H2O, reflux T = 348 K, 20 h; b) NaOH, EtOH/H2O, reflux T = 348 K, 20 h; 20 h;

AC

CE

c) dry DCM, NMM, T3P, 5 h; d) H2, 10% Pd/C, MeOH, p = 4.5 atm, 3-4 h.

38

ACCEPTED MANUSCRIPT

100 4

5

1

3 2 2

6

3

6

40

7 1

PT

% NTA(PrHP)2

80 60

5

4

20

4

6

8

10

SC

pH

RI

7

0

Fig. 2. Distribution diagram of NTA(PrHP)2 at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K

NU

(solid line) and T = 310.15 K (dashed line). cL = 1.0·10-5 mol L-1.

AC

CE

PT E

D

MA

Species: 1. H6L; 2. H5L; 3. H4L; 4. H3L; 5. H2L; 6. HL; 7. L.

39

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 3. Emission intensity of NTA(PrHP)2 (cL = 1.0·10-5 mol L-1) as a function of the wavelength

AC

CE

PT E

D

MA

NU

(nm) at λexc = 278 nm, different pH values, I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K.

40

ACCEPTED MANUSCRIPT

100

4

3 2

60

20

PT

40 1

0 4

6

pH

8

10

SC

2

RI

2+

80

Zn

5

NU

Fig. 4. Distribution diagram of Zn2+/NTA(PrHP)2 complexes at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K. cZn2+ = 8.0·10-6 mol L-1; cL = 2.0·10-5 mol L-1.

AC

CE

PT E

D

MA

Species: 1. free Zn2+; 2. ZnLH3; 3. ZnLH2; 4. ZnLH; 5. ZnL.

41

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

pH = 10.0, max = 465 nm pH = 5.2, max = 475 nm

400

500

600

 / nm

700

pH = 3.2, max = 465 nm pH = 2.2, max = 510 nm

800

PT

pH = 2.2, max = 510 nm

pH = 10.5, max = 460 nm

400

RI

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

A

A

ACCEPTED MANUSCRIPT

SC

a)

500

600

 / nm

700

800

b)

Fig. 5. UV-Vis spectrophotometric titration curves of NTA(PrHP)2 at I = 0.15 mol L-1 in

CE

PT E

D

MA

; b) cFe3+ = 3.0·10-4 mol L-1, cL = 6.6·10-4 mol L-1.

AC

1

NU

NaCl(aq), T = 298.15 K and at different pH values. a) cFe3+ = 2.0·10-4 mol L-1, cL = 2.0·10-4 mol L-

42

ACCEPTED MANUSCRIPT

3.9

8.0

3.8

7.9

3.7 CH g 2

3.6

7.7 7.6

3.5

7.5

3.4

7.4

3.3

7.3 4

6

pH

8

2

10

4

RI

2

PT

CH b

7.8

SC

1)

6

pH

8

10

2)

Fig. 6. Observed (□) and calculated (○) values of chemical shifts of b (1) and g (2) nuclei of

AC

CE

PT E

D

MA

NU

NTA(PrHP)2 vs. pH, at cL = 5.0·10-3 mol L-1, I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K.

43

RI

PT

ACCEPTED MANUSCRIPT

SC

Fig. 7. Ball-and-stick view of the DFT-optimized geometry (B3LYP/6-31G(d)) of the ZnLH32H2O complex. Oxygen: red; carbon: grey; hydrogen: white, nitrogen: blue; zinc: dark

AC

CE

PT E

D

MA

NU

blue. Hydrogen atoms linked to carbon atoms have been omitted for clarity.

44

SC

RI

PT

ACCEPTED MANUSCRIPT

NU

Fig. 8. 1H NMR spectra of: 1) NTA(PrHP)2 (cL = 5.0·10-3 mol L-1) and 2) Al3+/ NTA(PrHP)2 system (cAl3+ = 2.5·10-3 mol L-1, cL = 5.0·10-3 mol L-1) at pH ~ 2.2, I = 0.15 mol L-1 in NaCl(aq)

AC

CE

PT E

D

MA

and T = 298.15 K.

45

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

b)

AC

CE

PT E

D

MA

a)

c)

Fig. 9. Ball-and-stick view of the DFT-optimized geometry (B3LYP/6-31G(d)) of the Al2L3H3 (a, top, b, side) and Al2L2(OH)2 (c) complexes. Oxygen: red; carbon: grey; hydrogen: white, nitrogen: blue; aluminium: pink. Some hydrogens have been omitted for clarity.)

46

ACCEPTED MANUSCRIPT

1.0

12

0.8

pH = 7.4, 8.1

10

pH = 4.0

0.4

pH = 11.0

0.2

pH = 3.5, 10.0

pH = 2.5

pH = 9.0

8 6

pH = 5.0

4

0.0 2

4

2

6 8 10 12 14 16 -logcNTAPr(3,4-HP) 2

SC

a)

4

RI

0

PT

0.6

pL0.5

Al complexed

14

pH = 6.0

6 pH

8

10

12

b)

Fig. 10. Sequestration diagram of NTA(PrHP)2 towards Al3+ at different pH values as molar

NU

fraction of metal cation complexed vs. the total ligands concentration (as -logc) (a) and trend of

AC

CE

PT E

D

MA

pL0.5 vs. pH values (b) at I = 0.15 mol L-1 in NaCl(aq) and T = 298.15 K.

47

ACCEPTED MANUSCRIPT

3+

30 Al

pM

25

3+

25

2+

Cu

2+

Zn

20 Ca

2+

pM

Fe

30

15

20

2+

Cu -NTA(PrHP)2

15 2+

Cu -DTPA

10

5

5

4

6

7.4 8

2

10

pH

a)

6

7.4 8

10

pH

b)

Fig. 11. pM values calculated vs. pH for the different M /NTA(PrHP)2 (a, Ca2+ = ■, Cu2+ = Δ,

SC

n+

4

RI

2

2+

Cu -NTA

PT

10

Zn2+ = ◊, Al3+ = □, Fe3+ = ○) and Cu2+/NTA(PrHP)2 (□) , Cu2+/NTA (○) and Cu2+/DTPA (Δ) (b)

AC

CE

PT E

D

MA

NU

systems. pM= -log[M]free with cM = 1.0·10-6 mol L-1 and cL = 1.0·10-5 mol L-1.

48

ACCEPTED MANUSCRIPT 2.5

% I.A. / g

2 1.5 1

0 Liver

Intestine Heart

Kidney

Bone

Stomach

Bis24 NTAPr(3,4-HP)2 h

SC

Bis 1hNTAPr(3,4-HP)2

Lung

RI

Blood

PT

0.5

Fig. 12. Biodistribution of 67Ga after intraperitoneal injection of NTA(PrHP)2, expressed as % I.

AC

CE

PT E

D

MA

NU

A./g, at 1 and 24 h post-injection in female Balb-C mice (n = 3).

49

ACCEPTED MANUSCRIPT

100

60 40 20 0 1h

Citrate Liver

DFP Heart

Lung

1h

24 h

Bis NTAPr(3,4NTA(PrHP) 2 HP)2 Kidney

Bone

Excretion

NU

Blood

24 h

RI

24 h

SC

1h

PT

%I.A. / g

80

Fig. 13. Biodistribution data in the most relevant organs, expressed as % I.A./g for 67Ga-citrate and

67

Ga-citrate with co-administration of NTA(PrHP)2 or DFP 1 and 24 h after administration

AC

CE

PT E

D

MA

in female Balb-C mice (n = 3).

50

ACCEPTED MANUSCRIPT Graphical abstract

Fe

30

3+

Al

20 Ca

4

6

7.4 8 pH

10

NU

SC

2

RI

5

a)

2+

15 10

NTA(PrHP)2

Cu

2+

Zn

PT

pM

25

3+

2+

M3+ Excretion DFP

MA

24 h

D

24 h 1h

PT E

NTA(PrHP)2

1h

CE

0

20

40

60

80

100

b)

AC

A new bis-(3-hydroxy-4-pyridinone) ligand, NTA(PrHP)2, with illustration of the its metal chelation capacity and selectivity based on the pM values calculated vs. pH for the different Mn+/ligand systems (a, Ca2+ = ■, Cu2+ = Δ, Zn2+ = ◊, Al3+ = □, Fe3+ = ○) and its in vivo excretion capacity in animal model (mice overload with 67Ga) (b).

51

ACCEPTED MANUSCRIPT Highlights 

A new bis-(3-hydroxy-4-pyridinone) ligand derived from nitrilotriacetic acid was synthesized.



Its acid-base properties and binding ability towards M2+ and M3+ were investigated.



The sequestration of the ligand towards Mn+ was studied by the determination of pL0.5 values at different pHs. Analysis of pM values at pH = 7.4 showed that the metal-ligand affinity follows the trend Fe3+ > Al3+

PT



> Cu2+ > Zn2+ > Ca2+.

CE

PT E

D

MA

NU

SC

RI

Bioassays indicated that the ligand has high in vivo chelating ability towards trivalent metal cations.

AC



52