Journal of Porphyrins and Phthalocyanines J. Porphyrins ......KEYWORD INDEX (cumulative) Journal of...

CONTENTS

Journal of Porphyrins and PhthalocyaninesJ. Porphyrins Phthalocyanines 2017; 21: 1–76

See Paolo Ascenzi*, Chiara Ciaccio, Giovanna De Simone, Roberto Santucci and Massimo Coletta pp. 1–9

Horse heart carboxymethylated-cytc (CM-cytc) displays myoglobin-like properties due to the cleavage of the heme-Fe-Met80 axial bond. Under anaerobic conditions, the addition of NO to CM-cytc(III) leads to the transient formation of CM-cytc(III)-NO in equilibrium with CM-cytc(II)-NO+. In turn, CM-cytc(II)-NO+ is converted to CM-cytc(II) by OH−-based catalysis. Then, CM-cytc(II) binds NO very rapidly leading to CM-cytc(II)-NO.

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Articles

pp. 1–9Reductive nitrosylation of ferric carboxy-methylated-cytochrome cPaolo Ascenzi*, Chiara Ciaccio, Giovanna De Simone, Roberto Santucci and Massimo Coletta

Horse heart carboxymethylated-cytochrome c displays myoglo-bin-like properties due to the cleavage of the heme-Fe-Met80 axial bond. Carboxymethylation facilitates the reductive nitro-sylation of ferric cytochrome c and NO binding to the ferrous derivative.

pp. 10–15Stabilization of meso-tetraferrocenyl-porphyrin

blueKalil Cristhian Figueiredo Toledo, Bruno Morandi Pires, Juliano Alves Bonacin* and Bernardo Almeida Iglesias*

In this manuscript, we have studied a strategy to stabilize films of meso-te traferrocenyl-porphyrin (TFcP) with Prussian blue (PB) on electrodes and we have used the chemically modified electrode by the composite in sensing of dopamine.

CONTENTS

J. Porphyrins Phthalocyanines 2017; 21: 1–76

pp. 16–23Lyotropic liquid crystalline phthalocyanines con taining 4-((S)-3,7-dimethyloctyloxy)phenoxy moieties

The novel metal free phthalocyanine and its copper complex which are octa-substituted at the peripheral positions with 4-((S)-3,7-dimethyl-o ctyloxy)phenoxy moieties were synthesized and characte rized. The mesomorphic behavior and aggregation properties of these new materials are described.

pp. 24–30

carrier free 90yttrium labelled porphyrin as a possible agent for targeted therapy of tumorMahvash Abedi*, Mohammad Reza Nabid,

Nasim Vahidfar

The radiolabeling of 5,10,15,20-tetrakis(phenyl)porphyrin (H2TPP) was performed using the carrier free Y-90 which was prepared by the use of a home-made yttrium imprinted sorbent with a suitable radiochemical purity (95 ± 2% ITLC, 99 ± 0.5% HPLC) and specific activity (1.0 ± 0.1 GBq/mmol). Furthermore, the biodistribution study demonstrated that the kidneys could mostly remove the radio-complexes from the blood circulation and in lesser extent from the liver. As a result it is expected that due to its lipophilicity the higher mitochondrial content and thus, tumor cell uptake of this radiolabeled porphyrin happens and therefore 90Y-TPP could act as an efficient potential agent for targeted therapy of tumor.

pp. 31–36

tophysical properties of thiophene-substituted rare-earth bisphthalocyanines

A series of bis[octakis-(2-thienyl)phthalocyaninato] rare-earth metal(III) bis - phthalocyanine complexes of Pr, Sm and Gd were synthesized for the first time. The new compounds were characterized by UV-vis, NMR, FT-IR, mass spectros-copies as well as elemental analysis and cyclic voltammetry. Production of singlet oxygen was also estimated by 9,10-dimethylanthracene method.

pp. 37–47Symmetrical and difunctional substituted co balt

Synthesis and catalytic activity

Oscar Koifman

Series of phthalonitriles bearing with benzoic acid fragments was synthesized. Symmetrically and bifunctionally substituted phthalo-cyanines were obtained using these phthalonitriles. Their spectral properties and catalytic activity for aerobic oxidation of thiols were studied.

J. Porphyrins Phthalocyanines 2017; 21: 1–76

CONTENTS

pp. 48–58

mesomorphism of novel octakis(m-chloro pyri-dyloxy)phthalocyanato copper(II) com plexesKazuchika Ohta*, Kaori Adachi and Mikio Yasutake

Three novel octakis(m-chloropyridyloxy)phthalocyaninato copper(II) comple xes, [x-PyO(m-Cl)]8PcCu (x = 2, 3, 4: 2a–2c), have been synthesized to investigate their mesomorphism. Mesomorophism appears for the [2-PyO(m-Cl)]8PcCu (2a) and [4-PyO(m-Cl)]8PcCu (2c) derivatives, but not for the [3-PyO(m-Cl)]8PcCu (2b) derivative. The mesomorphism of 2b may be suppressed by the intramolecular N…Cl halogen bond.

pp. 59–66

explanation of its high photo-activity

Ye Tian, Jianting Yao, Yangdong Zheng*, Zhiguo Zhang* and Wenwu Cao*

Extinction coefficients of DVDMS at 405 and 630 nm are 4.36 × 105 and 1.84 × 104 M-1.cm-1. ΦF of DVDMS is 0.026 and its ΦΔ is 0.92. Although ΦΔ of DVDMS is only 10% higher than that of Photofrin® (0.83), its 10-fold greater extinction coefficient leads to an amazing reduction of dosage (about 1/10 of Photofrin®). Fluorescence diagnosis ability of DVDMS at 0.2 mg/kg is comparable to that of 2 mg/kg Photofrin® under the illumination of 405 nm laser because of the large difference of extinction coefficients.

pp. 67–76Synthesis of a novel CB2 cannabinoid-porphyrin conjugate based on an antitumor chromeno pyra-zo ledione

Nadine Jagerovic*

The synthesis of a cannabinoid-porphyrin conjugate based on an anti-tumor chromenopyrazoledione is reported. The novel conjugate binds weakly but selectively to CB2R.

AUTHOR INDEX (cumulative)

AAbedi, Mahvash 24Adachi, Kaori 48Ascenzi, Paolo 1

BBahrami-Samani, Ali 24Bilgin-Eran, Belkıs 16Bonacin, Juliano Alves 10Bureš, Filip 31

CCerný, Jirí 31Cao, Wenwu 59Chen, Tong 59Ciaccio, Chiara 1Coletta, Massimo 1

DDe Simone, Giovanna 1Dokládalová, Lenka 31

FFan, Ming 59Fang, Qicheng 59Fernández-Ruiz, Javier 67

GGüzeller, Dilek 16

IIglesias, Bernardo Almeida 10

JJagerovic, Nadine 67

KKoifman, Oscar 37Korkut, Sibel Eken 16Kuzmin, Ilya 37

LLycka, Antonín 31

MMaizlish, Vladimir 37Mikysek, Tomáš 31Morales, Paula 67Moreno, Laura 67Morozova, Anastasiya 37

NNabid, Mohammad Reza 24

OOcak, Hale 16Ohta, Kazuchika 48

PPires, Bruno Morandi 10

RRazumov, Mikhail 37

SSener, M. Kasım 16Santucci, Roberto 1Shirvani-Arani, Simindokht

24

TTian, Ye 59Toledo, Kalil Cristhian Figueiredo

10

VVahidfar, Nasim 24Vashurin, Artur 37

WYao, Jianting 59Yasutake, Mikio 48

ZZang, Lixin 59Zhang, Zhiguo 59Zhao, Huimin 59Zheng, Yangdong 59Znoyko, Serafima 37


JPP Volume 21 - Numbers 1 - Pages 1–76

KEYWORD INDEX (cumulative)


JPP Volume 21 - Numbers 1 - Pages 1–76

Aantitumor 67

Bbenzoic acids 37bioconjugate 674-bromo-5-nitro-phthalonitrite 37

Ccancer 67cannabinoid 67catalysis 37chromenopyrazole 67cobalt phthalocyanines 37columnar mesophase 48copper 16cyclic voltammetry 31

Ddopamine 10

Eelectroanalysis 10extinction coefficient 59

Fferric carboxymethylated-

cytochrome c 1

ferrocenyl-porphyrins 10fluorescence quantum yield 59flying-seed-like liquid crystals 48

Hheme-Fe atom coordination 1

Kkinetics 1

Lliquid crystalline 16lyotropic mesophase 16

Mmetallomesogen 48

Nnitric oxide binding 1

Ooxidation 37

Pphotodynamic therapy 59photophysical properties 59phthalocyanine 16, 48

porphyrin 10, 24Prussian blue 10

Rradiolabeling 24rare-earth phthalocyanine 31reductive nitrosylation 1

Ssinglet oxygen 31singlet oxygen quantum yield

59sinoporphyrin sodium 59synthesis 37

Ttargeted therapy 24tetraphenylporphyrin 67tumor 24

Uunsymmetrical phthalocyanine

UV-vis spectroscopy 31

Yyttrium-90 24


DOI: 10.1142/S1088424616501273

Published at http://www.worldscinet.com/jpp/

Copyright © 2017 World Scientific Publishing Company

INTRODUCTION

Eukaryotic cytochromes c (cytc) display a pivotal role in mitochondrial respiration and apoptosis [1, 2]. Cytc, which is located between the inner and the outer membrane of mitochondria, conveys electrons from complex III (HQH2-cytc reductase) to complex IV (cytc oxidase) of the respiratory chain. The hexa-coordination of the heme-Fe atom is maintained during the redox reaction avoiding the energy barrier that would slow-down electron transfer due to a site reorganization [3, 4]. On the other hand, at least 15% of mitochondrial cytc is bound to cardiolipin (CL), a lipid largely located in the

inner mitochondrial membrane [1, 5–13]. The interaction of cytc with CL weakens or cleaves the heme-Fe-Met80 distal bond [5, 9–18], inducing the appearance of myoglobin-(Mb-)like properties in the CL-cytc complex [6, 19–31]. This leads then to switch cytc functions from mitochondrial respiration to apoptosis [2, 32]. Furthermore, CL binding to cytc induces the drastic decrease of the protein formal standard potential, which is, at neutral pH, -150 mV (vs. NHE) [9], in place of +250 mV (vs. NHE), which is the typical value of the unmodified protein [33]. As a whole, CL-cytc could play either pro-apoptotic effects, catalyzing CL peroxidation and the subsequent cytc translocation into the cytoplasm, or anti-apoptotic actions, facilitating the binding and detoxification of reactive nitrogen and oxygen species and, in turn, the inhibition of CL peroxidation [1, 2].

Reductive nitrosylation of ferric carboxymethylated-

cytochrome c

Paolo Ascenzi*a, Chiara Ciacciob,c, Giovanna De Simoned, Roberto Santuccib

and Massimo Colettab,c

a Interdepartmental Laboratory of Electron Microscopy, Roma Tre University, Via della Vasca Navale 79, I-00146 Roma, Italy b Department of Clinical Sciences and Translational Medicine, University of Roma “Tor Vergata”, Via Montpellier 1, I-00133 Roma, Italy c Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Via Celso Ulpiani 27, I-70126 Bari, Italy d Department of Science, Roma Tre University, Viale Guglielmo Marconi 446, I-00146 Roma, Italy

ABSTRACT: Horse heart carboxymethylated-cytc (CM-cytc) displays myoglobin-like properties due to the cleavage of the heme-Fe-Met80 axial bond. Here, reductive nitrosylation of CM-cytc(III) between pH 8.5 and 9.5, at T = 20.0 °C, is reported. Under anaerobic conditions, the addition of NO to CM-cytc(III) leads to the transient formation of CM-cytc(III)-NO in equilibrium with CM-cytc(II)-NO+. In turn, CM-cytc(II)-NO+ is converted to CM-cytc(II) by OH−-based catalysis. Then, CM-cytc(II) binds NO very rapidly leading to CM-cytc(II)-NO. Kinetics of NO binding to CM-cytc(III) is independent of the ligand concentration, k values ranging between 3.6 ± 0.4 s-1 and 7.1 ± 0.7 s-1. This indicates that the formation of the CM-cytc(III)-NO complex is rate-limited by the cleavage of the weak heme-Fe(III) distal bond (likely Lys79). The conversion of CM-cytc(III)-NO to CM-cytc(II)-NO is rate-limited by the OH--mediated reduction of CM-cytc(II)-NO+ (hOH- = (1.2 ± 0.1) × 103 M-1.s-1). Lastly, the very fast nitrosylation of CM-cytc(II) takes place, values of lon ranging between 5.3 × 106 M-1.s-1 and 1.4 × 107 M-1.s-1. These results indicate that CM-cytc behaves as the cardiolipin-cytc complex highlighting the role of the sixth axial ligand of the heme-Fe atom in the modulation of the metal-based reactivity.

KEYWORDS: ferric carboxymethylated-cytochrome c, reductive nitrosylation, nitric oxide binding, heme-Fe atom coordination, kinetics.

*Correspondence to: Paolo Ascenzi, tel: +39 06-5733-3621, fax: +39 06-5733-6321, email: [email protected]

Copyright © 2017 World Scientific Publishing Company J. Porphyrins Phthalocyanines 2017; 21: 2–9

2 P. ASCENZI ET AL.

An alternative way to switch cytc from a hexa-coordinated electron transfer to a penta-coordinated globin-like protein is represented by the non-physiological carboxymethylation of Met65 and Met80 residues; carboxymethylated cytc (CM-cytc) indeed represents an invaluable physico-chemical model to investigate the role of the heme-Fe axial coordination on the metal reactivity [34]. Of note, ferrous horse heart CM-cytc (CM-cytc(II)) binds CO, NO, and O2 [35–38], whereas the ferric derivative (CM-cytc(III)) binds anionic ligands [39] and catalyzes peroxynitrite detoxification [28]. Furthermore, carboxymethylation affects the redox properties of cytc, whose redox potential, Eo, drops down by about 500 mV with respect to the value of the native protein (-218 mV instead of +250 mV vs. NHE) [33].

To highlight the Mb-like properties of CM-cytc, the reductive nitrosylation of CM-cytc(III) has been investigated between pH 8.5 and 9.5, at T = 20.0 °C. The study has been limited to this range, since at lower pH values no OH--dependent reductive nitrosylation occurs and at higher pH values a stable CM-cytc(III)-OH complex is present. Over the pH range explored, NO binding to CM-cytc(III) is limited by the cleavage of the weak heme-Fe distal bond (likely Lys79). Then, CM-cytc(II)-NO+, which is in equilibrium with CM-cytc(III)-NO, is converted to CM-cytc(II) by OH--based catalysis. Lastly, CM-cytc(II) binds very rapidly NO, in the excess of the gaseous ligand. Interestingly, also native cytc(III) and CL-cytc(III) have been reported to undergo reductive nitrosylation at alkaline pH [29, 40–42]. Of note, the reductive nitrosylation of heme-proteins may represent a biologically-relevant event since it leads to the oxidation and trapping of two NO molecules [29].

2. MATERIALS AND METHODS

Ferric horse heart cytc (cytc(III)) was obtained from Sigma-Aldrich (St. Louis, MO, USA). CM-cytc(III), carboxymethylated at positions 65 and 80, was prepared from the native form as detailed elsewhere [35]. The chemical modification of CM-cytc(III) was confirmed by amino acid analysis of CM-cytc(III) hydrolyzed with 6.0 M HCI prior to and after performic acid oxidation, according to [35]. As expected [35], the amino acid analysis indicated that 1.8 ± 0.1 Met residues were modified. The CM-cytc(III) concentration was determined spectrophotometrically at 408 nm (ε408 nm = 1.06 × 105 M-1 cm-1), at neutral pH [34]. CM-cytc(II) was obtained by adding sodium dithionite (final concentration, 2.0 × 10-3 M) to the heme-protein solution. Absorbance spectra of CM-cytc derivatives obtained here correspond to those previously reported, confirming the presence of a sixth axial ligand in CM-cytc(III) (likely Lys79), which replaces Met80, which cannot coordinate the heme’s iron because of carboxymethylation [34, 38].

Gaseous NO (from Linde Caracciolossigeno S.r.l., Roma, Italy) was purified by flowing through a NaOH column in order to remove acidic nitrogen oxides. The stock NO solution was prepared anaerobically by keeping in a closed vessel the degassed 5.0 × 10-3 M bis-tris propane buffer solution (pH 7.0) under NO at P = 760.0 mm Hg (T = 20.0 °C). The solubility of NO in the aqueous buffered solution is 2.05 × 10-3 M, at P = 760.0 mm Hg and T = 20.0 °C [43]. The concentration of NO in solution was determined, under anaerobic conditions and in the absence of the gaseous phase, by titration of ferrous horse hearth myoglobin (from Sigma-Aldrich, St. Louis, MO, USA), which was monitored by visible absorption spectroscopy [44].

Sodium dithionite concentration lower than 5.0 × 10-3 M does not react significantly with NO [45]. Moreover, the rate of NO reduction by dithionite (1.4 × 103 M-1.s-1, at pH 7.0 and 20.0 °C) [46] is small compared with the rate of CM-cytc(II) nitrosylation (ranging between 5.3 × 106 M-1.s-1 and 1.4 × 107 M-1.s-1 over the pH range explored; Table 1).

All the other chemicals were obtained from Merck AG (Darmstadt, Germany). All products were of analytical grade and used without purification unless stated.

Kinetics and thermodynamics of the reductive nitrosylation of CM-cytc(III) were analyzed in the frame-work of the minimum reaction mechanism (Scheme 1), accounting for the weakly-bound sixth axial ligand (likely Lys79), which is in equilibrium with the penta-coordinated CM-cytc(III) species (i.e. CM-cytc(III)*) [2, 29, 40–42, 47–55]:

Scheme 1. Mechanism of reductive nitrosylation of CM-cytc(III)

CM-cytc(III) + NO CM-cytc(III)* + NO

k-L

CM-cytc(III)-NO

kon

koff

(a)

CM-cytc(III)-NO CM-cytc (II)-NO+fast

(b)

CM-cytc(II)-NO+ + OH- CM-cytc(II) + HNO2

hOH-or hH2O

(c)

CM-cytc(II) + NO CM-cytc(II)-NO

lon

loff

(d)

where k-L is the apparent first-order rate constant for the dissociation of the weakly bound axial sixth ligand (possibly Lys79) of CM-cytc(III), kon and koff are the second-order associa tion and the first-order dissociation rate constants for the nitrosylation and denitrosylation of CM-cytc(III), respectively, hOH- and hH2O are the second-order and the first-order rate constants for the OH-- and H2O-catalyzed reduction of CM-cytc(III)-NO+ to produce CM-cytc(II) and HNO2, respectively, and lon


REDUCTIVE NITROSYLATION OF FERRIC CARBOXYMETHYLATED-CYTOCHROME c 3

and loff are the second-order association and the first-order dissociation rate constants for the nitrosylation and denitrosylation of CM-cytc(II), respectively.

Values of the apparent first-order rate constants for the reductive nitrosylation of CM-cytc(III) (i.e. k and h, respectively; see reactions (a) and (c), in Scheme 1) were obtained by rapid-mixing the CM-cytc(III) solution (final concentration, 2.6 × 10-6 M) with the NO solution (final concentration, 5.0 × 10-5 M to 5.0 × 10-4 M) under anaerobic conditions. No gaseous phase was present. The irreversible reductive nitrosylation of CM-cytc(III) was monitored by single-wavelength stopped-flow spectro scopy between 380 and 460 nm, the wavelength interval was 5 nm.

Values of k and h were obtained according to Equations 1–3 [2, 29, 47–56]:

[CM-cytc(III)]t = [CM-cytc(III)]i × e-k × t (1)

[CM-cytc(III)-NO]t = [CM-cytc(III)]i × k

× ((e-k × t/(h – k)) + (e-h × t/(k – h))) (2)

[CM-cytc(II)-NO]t = [CM-cytc(III)]i

– [CM-cytc(III)]t + [CM-cytc(III)-NO]t (3)

Values of the apparent second-order rate constant and of the apparent first-order rate constant for the OH-- and H2O-catalyzed conversion of CM-cytc(II)-NO+ to CM-cytc(II) (i.e. hH2O and hOH-, respectively; see reaction (c) in Scheme 1) were determined from the dependence of h on [OH-] according to Equation 4 [2, 29, 47–55]:

h = hOH- × [OH-] + hH2O (4)

Values of the apparent pseudo-first-order rate constant for NO binding to CM-cytc(II) (i.e. l; see reaction (d) in Scheme 1) were obtained by rapid-mixing the CM-cytc(II) solution (final concentration, 1.4 × 10-6 M) with the NO solution (final concentration, 5.0 × 10-6 M to 2.0 × 10-5), under anaerobic conditions. No gaseous phase was present. The nitrosylation of CM-cytc(II) was monitored by single-wavelength stopped-flow spectroscopy between 380 nm and 460 nm, the wavelength interval was 5 nm. Values of l were obtained according to Equation 5 [29]:

[CM-cytc(II)]t = [CM-cytc(II)]i × e-l × t (5)

Values of the apparent second-order rate constant for NO binding to CM-cytc(II) (i.e. lon; see reaction (d) in Scheme 1) were determined from the dependence of l on the NO concentration (i.e. [NO]) according to Equation 6 [29]:

l = lon × [NO] (6)

The reductive nitrosylation of CM-cytc(III) was investigated between pH 8.5 and 9.5 (5.0 × 10-2 M bis-tris propane buffer), at T = 20.0 °C.

All kinetic experiments have been carried out with the BioLogicSFM 2000 rapid-mixing stopped-flow apparatus (Claix, France); the dead-time of the stopped-flow apparatus was 1.4 ms and the observation chamber was 1 cm.

The results are given as mean values of at least four experiments plus or minus the corresponding standard deviation. All data were analyzed using the MATLAB program (The Math Works Inc., Natick, MA, USA).

RESULTS AND DISCUSSION

NO converts irreversibly CM-cytc(III) to CM-cytc(II)- NO between pH 8.5 and 9.5, at T = 20.0 °C. In fact, pumping off NO or bubbling helium through the CM-cytc(II)-NO solution leads to CM-cytc(II) not to CM-cytc(III). Of note, the denitrosylation process of CM-cytc(II)-NO (i.e. the formation of CM-cytc(II)) needs about 8 to 10 h to be completed (i.e. loff ≈ 1 × 10-4 s-1; see Scheme 1). The pH range over which this reaction is observed is limited because at pH < 8.5 only the reversible nitrosylation of CM-cytc(III) takes place and at pH > 9.5 a stable CM-cytc(III)-OH complex impairs the NO binding.

Mixing CM-cytc(III) and NO solutions induces the appearance of the difference absorbance spectrum of CM-cytc(III) minus CM-cytc(III)-NO with maximum at ~400 nm and ~430 nm, and minimum at ~415 nm (see Fig. 1, panel A). Then, the maximum and minimum of the difference absorbance spectrum of CM-cytc(III) minus CM-cytc(III)-NO shift to ~390 nm and ~415 nm corresponding to the minimum and maximum of the difference absorbance spectrum of CM-cytc(III)-NO minus CM- cytc(II)-NO (see Fig. 1, panel A). The difference absor-bance spectra obtained by mixing CM-cytc(III) and NO solutions correspond to those obtained by adding gaseous NO to the CM-cytc(III) solution. Also the irreversible conversion of cytc(III) to cytc(II)-NO and of CL-cytc(III) to CL-cytc(II)-NO by NO is charac terized by the formation of the cytc(II)-NO+ and CL-cytc(II)-NO+ transient species, respectively, which can be detected spectro - photometrically [26, 29, 40–42].

The time course for the reductive nitrosylation of CM-cytc(III) corresponds to a biphasic process (Fig. 1, panel B).

The first step of kinetics for CM-cytc(III) reductive nitrosylation (indicated by reaction (a) in Scheme 1) shows rates (i.e. k) independent of the NO concentration over the pH range, with values of k ranging between 3.6 s-1 and 7.1 s-1 (Fig. 1, panel C, and Table 1). Therefore, the observed rate constant k should correspond to k-L in Scheme 1, representing the rate limiting step of CM- cytc(III) nitrosylation, i.e. the hexa- to penta-coordination transition of the heme-Fe(III) atom preceding the formation of the CM-cytc(III)-NO adduct. Values of k−L for CM-cytc(III) reductive nitrosylation are similar to those reported for the reductive nitrosylation of CL-cytc(III) (ranging between 7.4 s-1 and 1.2 × 101 s-1) [29].

According to saturation kinetics [57, 58], values of kon for CM-cytc(III) nitrosylation (ranging between 7.2 × 104 M-1.s-1 and 1.4 × 105 M-1.s-1) have been estimated.


4 P. ASCENZI ET AL.

Moreover, the saturation of CM-cytc(III) by NO at the lowest ligand concentration explored (= 5 × 10-5 M) indicates that K values are ≤ 5 × 10-6 M (Table 2), as reported for CL-cytc(III) nitrosylation [29]. Then, values

of koff (= kon × K) for NO dissociation from CM- cytc(III)-NO are ≤ 7.0 × 10-1 s-1 (Table 2).

The second step of the CM-cytc(III) reductive nitrosylation (indicated by reactions (b)–(d) in Scheme 1) displays rates (i.e. h) independent of the NO concentration, at fixed pH (see Fig. 1, panel D). In contrast with values of k, values of h for CM-cytc(III) reductive nitrosylation increase from 4.1 × 10-3 s-1, at pH 8.5, to 3.9 × 10-2 s-1, at pH 9.5, and they are similar to those reported for the reductive nitrosylation of CL-cytc(III) over the same pH range (increasing from 8.8 × 10-3 s-1, at pH 8.5, to 9.5 × 10-3 s-1, at pH 9.5) [29].

According to Scheme 1, the value of h increases linearly on [OH-] (Fig. 2 and Table 1). The slope and the y intercept of the plot of h vs. [OH-] correspond to hOH- (= 1.2 × 103 M-1.s-1) and hH2O (= 8.7 × 10-4 s-1), respectively. Values of hOH- and hH2O for CM-cytc(III) reductive nitrosylation are similar to those reported for the reductive nitrosylation of CL-cytc(III) (corresponding to 3.0 × 103 M-1.s-1 and 1.5 × 10-3 s-1, respectively) [29].

Mixing CM-cytc(II) and NO solutions induces the appearance of the difference absorbance spectrum of CM-cytc(II) minus CM-cytc(II)-NO with maximum

Fig. 1. Kinetics of CM-cytc(III) reductive nitrosylation at pH 9.1 and T = 20.0 °C. (a) Difference kinetic absorbance spectra of CM-cytc(III) minus CM-cytc(III)-NO (open circles) and of CM-cytc(III)-NO minus CM-cytc(II)-NO (open squares). (B) Normalized averaged time courses of CM-cytc(III) reductive nitrosylation. The NO concentration was 1.0 × 10-4 M (trace a), and 4.0 × 10-4 M (trace b). The time course analysis according to Equations 1–3 allowed the determination of the following values of k = 7.5 ± 0.8 s-1 (trace a) and 6.8 ± 0.7 s-1 (trace b) and h = (17.1 ± 1.6) × 10-3 s-1 (trace a) and (15.4 ± 01.5) × 10-3 s-1 (trace b). For clarity, trace b has been upshifted of 0.3 units. (c) Dependence of pseudo-first-order rate-constant k for CM-cytc(III) reductive nitrosylation on the NO concentration. The average value of k is 7.1 ± 0.7 s-1. (d) Dependence of the first-order rate-constant h for CM-cytc(III) reductive nitrosylation on the NO concentration. The average value of h is (1.6 ± 0.2) × 10-2 s-1. Where not shown, standard deviation is smaller than the symbol. For details, see text

Table 1. Values of kinetic parameters for reductive nitrosylation of CM-cytc(III)a

pH k-L (s-1) h × 103 (s-1) lon (M-1.s-1)

8.5 6.4 ± 0.6 4.1 ± 0.41 (7.9 ± 1.0) × 106

8.8 3.6 ± 0.4 7.9 ± 0.79 —

9.1 7.1 ± 0.7 15.0 ± 1.5 (1.4 ± 0.3) × 107

9.4 6.9 ± 0.7 31.0 ± 3.1 —

9.5 4.8 ± 0.5 39.0 ± 3.9 (5.3 ± 0.9) × 106

a According to Scheme 1, k-L represents the apparent first- order rate for the dissociation of the weakly bound axial sixth ligand (possibly Lys79) of CM-cytc(III), h indicates the first- order rate constant for the conversion of CM-cytc(III)-NO+ to CM-cytc(II), and lon is the second-order association rate constant for the nitrosylation of CM-cytc(II). T = 20.0 °C.



Tabl

e 2.

Val

ues

of k

inet

ic a

nd th

erm

odyn

amic

par

amet

ers

for

irre

vers

ible

red

uctiv

e ni

tros

ylat

ion

of f

erri

c he

me-

prot

eins

Hem

e-pr

otei

nk -

L (

s-1)

k on

(M-1.s

-1)

k off (

s-1)

K (

M)

k off / k

on (

M)

h OH

- (M

-1.s

-1)

h H2O

(s-1

)l o

n (M

-1.s

-1)

l off (

s-1)

Myc

obac

teri

um t

uber

culo

sis

trH

b-N

—1.

4 ×

105a

1.6a

1.6

× 10

-5a

1.1

× 10

-5 a

1.7

× 10

2b6.

4 ×

10-4

b1.

6 ×

107a

—

Myc

obac

teri

um t

uber

culo

sis

trH

b-O

—9.

2 ×

103a

2.1a

1.9

× 10

-4a

2.3

× 10

-4 a

2.4

× 10

2b2.

9 ×

10-4

b2.

3 ×

105a

—

Cam

pylo

bact

er j

ejun

i trH

b-P

—1.

7 ×

107a

8.1a

6.5

× 10

-5a

7.4

× 10

-5 a

9.1

× 10

2b4.

8 ×

10-4

b1.

7 ×

107a

—

Met

hano

sarc

ina

acet

ivor

ans

Pgb

d—

4.8

× 10

4c2.

6c6.

1 ×

10-5

c5.

4 ×

10-5

c2.

9 ×

103d

4.1

× 10

-4d

2.7

× 10

7c—

Gly

cine

max

Lb

—1.

4 ×

105e

3.0e

2.1

× 10

-5e

2.1

× 10

-5 e

3.3

× 10

3f3.

0 ×

10-4

f2.

5 ×

108g

—

Scap

harc

a in

aequ

ival

vis

HbI

—3.

2 ×

101h

<1

× 10

-3h

—3.

1 ×

10-5

h>

2 ×

106i

—1.

6 ×

107j

—

Hor

se h

eart

Mb

—6.

8 ×

104k

5.2k

1.2

× 10

-4 k

7.6

× 10

-5 k

3.9

× 10

2l—

1.6

× 10

7k4.

3 ×

10-4

k

Sper

m w

hale

Mb

—1.

9 ×

105m

1.4

× 10

n7.

7 ×

10-5

m—

3.2

× 10

2o—

1.7

× 10

7p1.

2 ×

10-4

p

Hum

an N

gb f

ast p

hase

7.0q

2.1

× 10

1r2.

5 ×

10-3

r—

1.2

× 10

-4 r

≥2 ×

106s

——

—

Hum

an N

gb s

low

pha

se0.

6q2.

9r2.

0 ×

10-3 r

—1.

9 ×

10-4

r≥5

× 1

05s—

——

Hum

an H

b —

——

8.3

× 10

-5t

—3.

2 ×

103u

1.1

× 10

-3u

2.4

× 10

7p~3

.4 ×

10-5

p

HSA

-hem

e-Fe

—2.

1 ×

104v

3.1

× 10

-1v

1.8

× 10

-5v

1.5

× 10

-5 v

4.4

× 10

3w3.

5 ×

10-4

w6.

3 ×

106x

—

Rab

bit H

PX-h

eme-

Fe—

1.3

× 10

1y≤1

0-4 y

—≤8

× 1

0-6 y

≥7 ×

105z

—6.

3 ×

103a

a9.

1 ×

10-4

aa

CL

-hor

se h

eart

cyt

c7.

5bb5.

5 ×

105c

c≤3

dd≤5

× 1

0-6bb

—3.

0 ×

103e

e1.

5 ×

10-3

ee1.

4 ×

107b

b<1

× 1

0-4bb

—1.

8 ×

106b

b≤9

cc—

——

——

—

CM

-hor

se h

eart

cyt

c≥3

.6ff

≥1.4

× 1

05ff

≤7.1

× 1

0-1ff

≤5 ×

10-6

ff

—1.

2 ×

103

gg8.

7 ×

10-4

gg9.

1 ×

106f

f≈1

× 1

0-4ff

a pH

9.0

and

T =

20.

0 °C

. F

rom

[55

]. b T

= 2

0.0

°C.

Fro

m [

55].

c pH 7

.2 a

nd T

= 2

2.0

°C.

Fro

m [

54].

d T =

22.

0 °C

. F

rom

[54

]. e p

H 7

.3 a

nd T

= 2

0.0

°C.

Fro

m [

50].

f T =

20.

0 °C

. F

rom

[50

]. g p

H 7

.0 a

nd

T =

20.

0 °C

. F

rom

[74

]. h pH

7.5

and

T =

20.

0 °C

. F

rom

[48

]. i T

= 2

0.0

°C.

Der

ived

fro

m [

48].

j pH

7.0

and

T =

20.

0 °C

. F

rom

[72

]. k p

H 9

.2 a

nd T

= 2

0.0

°C.

Fro

m [

52].

l T =

20.

0 °C

. D

eriv

ed f

rom

[52

].

m p

H 8

.79

and

room

tem

pera

ture

. Fro

m [

47].

n pH

8.7

9 an

d ro

om t

empe

ratu

re. D

eriv

ed f

rom

[47

]. o R

oom

tem

pera

ture

. Fro

m [

47].

p pH

7.0

and

T =

20.

0 °C

. Fro

m [

46].

q pH

7.0

and

T =

25.

0 °C

. Fro

m [

76].

r p

H 7

.0 a

nd r

oom

tem

pera

ture

. F

rom

[49

]. s R

oom

tem

pera

ture

. D

eriv

ed f

rom

[49

]. t p

H 7

.1 a

nd r

oom

tem

pera

ture

. F

rom

[47

]. u R

oom

tem

pera

ture

. F

rom

[47

]. v p

H 7

.5 a

nd T

= 2

0.0

°C.

Fro

m [

53].

w T

=

20.0

°C.

Fro

m [

53].

x pH

7.0

and

T =

20.

0 °C

. F

rom

[73

]. y p

H 7

.0 a

nd T

= 1

0.0

°C.

Fro

m [

51].

z T =

10.

0 °C

. F

rom

[51

]. aa

pH

7.0

and

T =

10.

0 °C

. F

rom

[75

]. bb

pH

7.1

to

9.5

and

T =

20.

0 °C

. F

rom

[29

].

cc p

H 7

.4 a

nd T

= 2

2 °C

. Aft

er n

anos

econ

d fl

ash

phot

olys

is, t

he ti

me

cour

se o

f N

O r

e-bi

ndin

g to

the

hem

e-F

e(II

I) a

tom

is b

ipha

sic;

the

appa

rent

rat

e co

nsta

nt o

f ea

ch e

xpon

enti

al d

epen

ds li

near

ly o

n th

e N

O

conc

entr

atio

n, y

ield

ing

valu

es o

f th

e se

cond

-ord

er r

ate

cons

tant

for

NO

bin

ding

to

CL

-cyt

c(II

I) (

i.e.

kon

). F

rom

[26

]. dd

kof

f = k

on ×

K.

Fro

m [

29].

ee T

= 2

0.0

°C.

Fro

m [

29].

ff p

H 8

.5 t

o 9.

5 an

d T

= 2

0.0

°C.

Pre

sent

stu

dy. gg

T =

20.

0 °C

.


6 P. ASCENZI ET AL.

and minimum at ~408 nm and ~430 nm, respectively (see Fig. 3, panel A). The absorbance spectrum obtained by mixing CM-cytc(II) and NO solutions corresponds to those obtained by adding gaseous NO to either cytc(II) or cytc(III) or CL-cytc(II) or CL-cytc(III) or CM-cytc(II) or CM-cytc(III) solutions or given in the literature [38–40, 42].

The time course of CM-cytc(II) nitrosylation corresponds to a mono-exponential process for more than 82% of its course (Fig. 3, panel B), over the pH range explored. Values of the pseudo-first-order rate constant for NO binding to CM-cytc(II) (i.e. l) depend linearly on the NO concentration (Fig. 3, panel C). The analysis of data according to Equation 6 allowed to determine values of lon ranging between 5.3 × 106 M-1.s-1 and 1.4 × 107 M-1.s-1 over the pH range explored (see Table 1). Values of lon for CM-cytc(II) nitrosylation are similar to those reported for CL-cytc(II) nitrosylation (ranging between 9.6 × 106 M-1.s-1 and 2.1 × 107 M-1.s-1) [29].

In order to better understand the mechanism of regulation of the reductive nitrosylation process a comparison with other hemoproteins indeed is important. In this respect, comparative data, reported in Tables 1 and 2 allow the following considerations of general significance.

(i) The high reactivity of NO towards ferric Mycobacterium tuberculosis truncated hemoglobin-N (trHb-N) [55], Mycobacterium tuberculosis trunc ated hemoglobin-O (trHb-O) [55], Campylobacter jejuni truncated hemoglobin-P (trHb-P) [55], Methano-sarcina aceti vorans protoglobin (Pgb) [54], Glycine max leghe moglobin (Lb) [50], horse myoglobin (Mb) [52], sperm whale Mb [47], human hemoglobin (Hb) [47], and human serum heme-albumin (HSA-heme-Fe) [53] reflects the penta-coordination of the heme-Fe atom.

(ii) NO binding to human Ngb [49], rabbit HPX-heme-Fe [51], CL-cytc(III) [29], and CM-cytc(III) (present study) is limited by the cleavage of the labile sixth axial ligand of the heme-Fe(III)

preceding the formation of the ferric nitrosylated heme-protein.

(iii) The very low reactivity of ferric penta-coordinated Scapharca inaequivalvis hemoglobin I (HbI) has been postulated to reflect either the non-occurrence

Fig. 2. Dependence of first-order rate-constant h for CM-cytc(III) reductive nitrosylation on the OH- concentration. The continuous line was generated from Equation 4 with hOH- = (1.2 ± 0.1) × 103 M-1.s-1 and hH2O = (8.7 ± 1.1) × 10-4 s-1. The CM-cytc(II) concentration was 2.6 × 10-6 M. Where not shown, standard deviation is smaller than the symbol. For details, see text

Fig. 3. Kinetics of NO binding to CM-cytc(II), at T = 20.0 °C. (a) Difference kinetic absorbance spectrum of CM-cytc(II) minus CM-cytc(II)-NO. (b) Normalized averaged time courses of CM-cytc(II) nitrosylation, at pH 9.1. The NO concentration was 5.0 × 10-6 M (trace a) and 2.0 × 10-5 M (trace b). The time course analysis according to Equation 5 allowed the determination of the following values of l = 6.4 × 101 s-1 (trace a) and 2.8 × 102 s-1 (trace b). (C) Dependence of the pseudo-first-order rate-constant l for CM-cytc(II) nitrosylation on the NO concentration, at pH 8.5 (circles), 9.1 (triangles), and 9.5 (squares). The continuous lines were generated from Equation 6 with lon = (7.9 ± 1.0) × 106 M-1.s-1 (pH 8.5), (1.4 ± 0.3) × 107 M-1.s-1 (pH 9.1), and (5.3 ± 0.9) × 106 M-1.s-1 (pH 9.5). The CM-cytc(II) concentration was 1.4 × 10-6 M. Where not shown, standard deviation is smaller than the symbol. For details, see text



of the heme-Fe(III)-NO intermediate or the con-comitant S-nitrosation of Cys92 or the modulation of heme-Fe(III) nitrosylation by the Cys92 redox state [48].

(iv) Values of koff for NO dissociation from ferric nitrosylated heme-proteins are lower than 9 s-1, reflecting the different hydrogen bonding networks stabilizing the heme-bound ligand (see [29, 47–55] and the present study).

(v) Although values of kon and koff for NO binding to ferric heme-proteins (see [26, 29, 47, 50, 52–55] and the present study) are very different, values of K (= koff/kon) match each other, indicating the occurrence of kinetic compensation phenomena.

(vi) With the exception of Scapharca inaequivalvis HbI [48], human Ngb [49], and rabbit hemopexin [51], values of hOH- for ferric heme-protein nitrosylation (see [29, 47, 50, 52–55] and the present study) span over two orders of magnitude, possibly reflecting the different anion accessibility to the heme pocket and the heme-Fe(III) protein reduction potentials [43, 47, 50, 59–70]. Moreover, the linear dependence of h on [OH-] indicates that no additional elements appear to be involved in the irreversible reductive nitrosylation of ferric heme-proteins. Furthermore, the OH- anions catalyzes the conversion of heme-Fe(II)-NO+ to heme-Fe(II) + NO3

– more efficiently than H2O. This may reflect either the higher affinity of negatively-charged ligands for ferric heme-proteins with respect to uncharged compounds and/or the deprotonation rate of the incoming ligand (see [43, 59, 63]).

(vii) Values of kon for NO binding to ferrous heme-proteins considered are larger than 2.3 × 105 M-1.s-1 (see [29, 46, 52, 54, 55, 71–74] and the present study) with the exception of rabbit HPX-heme-Fe [75] showing a hexa-coordinated bis-histidyl heme-Fe atom [66].

(viii) For hexa-coordinated ferrous Scapharca inaequi-valvis HbI [72], rabbit HPX-heme-Fe [75], CL-cytc, [29], and CM-cytc (present study), the transient penta-coordinated ferrous species was never detected; accordingly, the rate of NO binding to the heme-Fe(II) atom is faster than that of the OH–-dependent conversion of the heme-Fe(III)-NO+ species to the heme-Fe(II) form.

(ix) The very low values of loff for NO dissociation from ferrous nitrosylated heme-proteins (≤ 9.1 × 10-4 s-1) reflect the different hydrogen bonding networks stabilizing the heme-bound ligand (see [29, 46–53] and the present study).

CONCLUSION

In conclusion, present results highlight the role of the axial coordination of the heme-Fe atom in modulating the metal center reactivity. Thus, different rate-limiting steps

affect the reductive nitrosylation of ferric heme-proteins according to whether the starting species is penta- or hexa-coordinated. As a matter of fact, the reactivity of penta-coordinated ferric heme-proteins with NO is much faster than the overall reductive nitrosylation process, which is rate-limited by the OH--mediated reduction of the heme-Fe(II)-NO+ complex to heme-Fe(II) + NO2

- [47, 50, 52–55]. On the other hand, in hexa-coordinated ferric heme-proteins, NO binding represents the rate-limiting step of the whole process [29, 40–42, 49]. As a consequence, the reductive nitrosylation shows different features in heme-proteins, reflecting the different accessibility of NO and OH- to the metal center. An interesting point concerns the higher rates observed for the reductive nitrosylation of CL-cytc(III) with respect to CM-cytc(III), envisaging the possibility that CL induces a larger conformational change, exceeding the simple cleavage of the Fe-Met80 bond, which also occurs in CM-cytc(III).

Acknowledgements

This work was partly supported by Roma Tre University (CLA 2015 to P.A).

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‡ Abbreviations: CL, bovine cardiolipin; CL-cytc, cardiolipin-cytochrome c complex; CL-cytc(III), ferric CL-cytc; CL-cytc(II), ferrous CL-cytc; CM-cytc, carboxymethylated-cytc; CM-cytc(III), ferric CM-cytc; CM-cytc(II), ferrous CM-cytc; CM-cytc(III)-NO, nitrosylated CM-cytc(III); CM-cytc(II)-NO, nitrosylated CM-cytc(II); cytc, horse heart cytochrome c; Hb, hemoglobin; HPX, hemo-pexin; HSA, human serum albumin; Lb, leghemo-globin; Mb, myoglobin; Ngb, neuroglobin; Pgb, protoglobin; trHb, truncated Hb.

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DOI: 10.1142/S1088424616501224



INTRODUCTION

Supramolecular systems based on porphyrin-ferrocene covalently linked have been broadly studied due to its electronic structure [1], photophysical properties [2] and electrochemical behavior [3–5]. Ferrocene (Fc) can be considered as strong acceptor of electrons and supramolecular porphyrins can have their electron acceptor–donor character modulated according to peripheral group attached [6]. The combination of their chemistry can generate new properties and produces new possibilities in the research field of photoinduced electron transfer processes, mimetism of photosynthetic active sites, development of molecular-based electronic devices and electrochemical sensors [3, 4, 7–9].

One of the strategies to take advantage of porphyrin-ferrocene system is its use in chemically modified electrodes as electroactive layer, because the chemical characteristics can be transferred to electrode’s surface and it can be used in sensing of molecular targets [10]. On

the other hand, one challenge to modify electrodes with the meso-tetraferrocenyl-porphyrin molecule (TFcP), which structure is presented at Fig. 1, is the fact that TFcP is oxidized on electrode during anodic sweep. Because of this, the positive charge is produced in the ferrocene group and the molecular film of TFcP is not stable on the electrode’s surface. Consequently the film is solubilized in polar solvents, such as water.

In order to improve the stability of the film of TFcP in glassy carbon electrode, we used a composite formed by TFcP and Prussian blue (PB), which is a well-known compound derived from the reaction between hexacyanidoferrate(II) and iron(III). PB is a metal-cyanide framework [11] with remarkable electro-chemistry behavior and has been extensively used as artificial peroxidase enzyme due to its ability to catalyze hydrogen peroxide reduction [12] or can be applied in the electro-oxidation of biological relevant molecules such as ascorbic acid [13] and cysteine [14]. Furthermore, the synergic effect between PB and porphyrins can produce new properties or alter original characteristics and therefore these new features need to be researched.

The new composite formed by TFcP/Prussian blue has shown promising results in sensing of dopamine. This neurotransmitter is involved in several metabolic processes, playing a crucial role in central nervous, renal

Stabilization of meso-tetraferrocenyl-porphyrin films

by formation of composite with Prussian blue

Kalil Cristhian Figueiredo Toledo#a, Bruno Morandi Pires#a,

Juliano Alves Bonacin#*a and Bernardo Almeida Iglesias#*b◊

a Institute of Chemistry, P.O. Box 6154, University of Campinas — UNICAMP, 13083-970, Campinas — SP, Brazil b Departament of Chemistry, Universidade Federal de Santa Maria, 97105-900, Santa Maria — RS, Brazil

Received 15 June 2016Accepted 1 September 2016

ABSTRACT: Supramolecular systems based on porphyrin-ferrocene have attracted the attention to inorganic electrochemistry due to unique electronic properties and synergic behavior between porphyrin ring and peripheral ferrocene group. In order to improve the stability of the films of ferrocenyl-porphyrin on electrode’s surface, we used a combination of tetraferrocenylporphyrin and Prussian blue. The new structure formed was very stable and could be used in dopamine sensing, showing satisfactory analytical response comparable to other chemically modified electrodes the described in the literature.

KEYWORDS: porphyrins, ferrocenyl-porphyrins, Prussian blue, electroanalysis, dopamine.

◊ SPP full member in good standing

*Correspondence to: Juliano A. Bonacin, email: [email protected]; Bernardo A. Iglesias, email: [email protected]#These authors have equally contributed as first authors.


STABILIZATION OF meso-TETRAFERROCENYL-PORPHYRIN FILMS 11

and hormonal systems. In addition, it is also related to cognitive functions and some neurological diseases, such as Parkinson’s disease and schizophrenia which have been associated to low levels of dopamine [15]. Therefore, monitoring the levels of this biomolecule is of great interest, and many analytical methods have been created with this purpose. In this scenario, electroanalytical methods for determination dopamine appear as sensitive, rapid and cheap for this purpose [16].

In this manuscript, we have studied a strategy to stabilize films of meso-tetraferrocenyl-porphyrin (TFcP) with Prussian blue (PB) on electrodes and we have used the chemically modified electrode by the composite in sensing of dopamine.


TFcP was prepared by classical condensation between ferrocene carbaldehyde and pyrrole, using Lindsey’s conditions, as described previously [17] (Scheme 1).

The 1H NMR spectrum of TFcP is similar to that reported by Nemykin and co-workers [17] and consists of singlets corresponding to the β-pyrrolic protons (9.60 ppm; 8H), unsubstituted cyclopentadienyl ring (3.97 ppm; 20H) and

inner NH protons (-0.49 ppm; 2H), along with two multiplets corresponding to the substituted Cp rings (5.33 ppm; 8H for α-Cp-H and 4.76 ppm; 8H for β-Cp-H). The 1H and 13C NMR spectrum is presented in the Supplementary material (see Supporting information section, Figs S1 and S2).

HRMS-ESI mass spectra of TFcP are presented in the Supplementary material (Fig. S3) and almost exclusively consist of protonated molecular ions ([M + H]+). In the fully HRMS-ESI spectrum of protonated TFcP (see Fig. S3), the protonated molecular ion first eliminates an FeCp group, with the formation of ion [M – FeCp]+ (Fig. S3).

UV-vis spectrum of TFcP in DMSO solution consists of an intense Soret band (B band) at 433 nm with a prominent shoulder at 483 nm and, characteristic for metal-free porphyrins, two Q-bands observed at 662 and 733 nm, Q1 and Q2 respectively (Fig. 2). When the electronic spectrum is compared to the well-known metal-free meso-tetraphenyl-porphyrin, all bands in the UV-vis spectrum of TFcP are shifted to low energy positions, reflecting a greater electron-donating ability of ferrocenyl substituents (FeCp) in comparison to the phenylones [1, 17].

The electrochemical behavior of TFcP was investi-gated by cyclic voltammetry (CV) method using dry dichloromethane as solvent. The results are graphically

Fig. 1. Structural comparison of meso-tetraphenyl-porphyrin TPP and meso-tetraferrocenyl-porphyrin — TFcP

Scheme 1. Scheme of synthetic pathway of the tetraferrocenyl-porphyrin (TFcP)


12 K. C. F. TOLEDO ET AL.

presented in Fig. 3, and the redox potentials are indicated in Table 1. TFcP undergo two quasi-reversible one-electron reduction processes with potentials close to meso-tetraarylsubstituted metal-free porphyrins indicating the formation of respective porphyrin anions and dianions [9]. In anodic region, it is possible to observe three oxidation processes in CV. The first process at E1/2 = +0.674 V vs. SHE its can be assigned to the Fc ring oxidation, probably the process Fe2+/Fe3+. The last two anodic and irreversible processes can be attributed to the porphyrin ring oxidation by the formation of π-cation radical and dication species [18].

Porphyrins are known to produce stable films with interesting the electrochemical behavior [19, 20]. In the case of ferrocenyl-porphyrin as TFcP, the film is stable but when a high anodic potential is applied, the ferrocene (peripheral group) and porphyrin are oxidized and the film becomes water soluble, consequently the film is leached after three electrochemical cycles, as can be observed in Figs 4(a) and 4(d). Using a Britton-Robinson

buffer solution pH = 7.0 + KCl 0.10 M the same behavior was observed.

According to Figs 4(b) and 4(e), PB films are more stable than TFcP, but after 100 cycles the film is leached too, even in acid pH. The oxidation of PB film can be seen at potential of 0.20 V vs. Ag/AgCl and a continuous decrease in the intensity of anodic and cathodic current is observed after each cycle. An alternative to obtain stable films is to use a matrix of PB to stabilize the porphyrin macrocycle. The Fe3+ ions cannot reach all cyanides sites during Prussian blue formation and hence could have negative regions along the structure of the PB. The negative portion distributed along the PB film can attract electrostatically TFcP after its oxidation. The observed effect is a stabilization of the hybrid film on the GCE, as can be observed in Figs 4(c) and 4(f). Based on the small variation of the current after successive voltammetric cycles presented in Fig. 4(f), it is possible to suggest that PB can stabilize the porphyrin on electrode’s surface. So, the hybrid film could be applied in electrochemical sensing of molecules.

The micrographs of the TFcP, Prussian blue, and TFcP/Prussian blue films can be found in the Supplementary material. According to micrographs of the films of TFcP, it was observed a good coverage of the film over the electrode (Figs S5–S7) characteristic of porphyrin films. On the other hand, it is possible to see a kind of needle-shaped structure for Prussian blue on the surface of the electrode (Figs S8–S10). When the electrode is modified by both is possible to observe a mixture of TFcP spread with Prussian blue structure, which shows the high coverage of the surface (Figs S11–S13).

In the presence of dopamine (DA), the modified electrode has an electrocatalytic response in 0.60 V vs. Ag/AgCl. In bare electrode this process occurs in 0.75 V vs. Ag/AgCl and can cause poisoning of the electrode after additions of DA from the stock solution. The current of peak obtained by electrochemical measurement on the modified electrode showed a linear dependence with the concentration of DA (Fig. 5). A linear increase of current was observed with the increment of DA concentration in the range of 5.0 μM to 87.0 μM (inset of Fig. 5). In this range, the detection

Fig. 3. Cyclic voltammogram of TFcP in dry dichloromethane solution, using 0.1 M TBAPF6, at scan rate of 100 mV.s-1

Fig. 2. UV-vis electronic spectrum of TFcP in DMSO solution

Table 1. Redox potentials value of TFcP

TFcP Potential (in V; vs. SHE)

Ered1 -1.274a

Ered2 -1.805a

Eox1 +0.674c

Eox2 +1.340b

Eox3 +1.784b

a Epc = cathodic peak. b Epa = anodic peak; c E1/2 = Epa + Epc/2.



Fig. 4. (a) Cyclic voltammograms of GCE modified by TFcP in HCl 0.10 M + KCl 0.10 M after 3 cycles; (b) Cyclic voltammograms of GCE modified by PB in HCl 0.10 M + KCl 0.10 M after 100 cycles; (c) Cyclic voltammograms of GCE modified by hybrid film PB + TFcP in HCl 0.10 M + KCl 0.10 M after 100 cycles; (d) Decrease of the current caused by successive cycles of voltammetry for GCE modified by TFcP; (e) Decrease of the current caused by successive cycles of voltammetry for GCE modified by PB; (f) Comparison of decrease of the current caused by successive cycles of voltammetry for GCE modified by TFcP, PB and hybrid film PB/TFcP


14 K. C. F. TOLEDO ET AL.

limit found was 2.44 μM, which is in the same in the same order of magnitude of other works in the literature [15, 16, 21].

EXPERIMENTAL

General synthesis and methods

All reagents and solvents used in this work were in analytical or high purity grade, and were employed with no further purification.

The free-base 5,10,15,20-tetrakisferrocenyl-porphyrin derivative was synthesized according to previously described procedures by Nemykin and co-workers [17]. 1H NMR, 13C NMR and HRMS-ESI spectra can be found in the Supplementary material (Figs S1 and S2).

Preparation of meso-tetra(ferrocenyl)porphyrin (TFcP). A mixture of ferrocenecarbaldehyde (500 mg; 2.33 mmol), pyrrole (167 mg; 2.5 mmol) and BF3·Et2O (31.6 μL; 0.25 mmol) in dry CH2Cl2 (80 mL) was kept for 24 h at room temperature in argon atmosphere. After this period, p-chloranil (808 mg; 3.3 mmol) was added and the resulting mixture was refluxed for 3 h. After solvent evaporation, the residue was purified by column chromatography using silica gel in a toluene-triethylamine mixture (100:1; v/v) as the eluent. Finally, the product was recrystallized from toluene-hexane mixture (1:1; v/v) to obtain a dark-greenish solid.

5,10,15,20-Tetrakis(ferrocenyl)porphyrin. Yield 750 mg (0.717 mmol; 30%). UV-vis (DMSO): λmax, nm (ε × 104) 433 (12.3), 483 (sh), 662 (1.59) and 733 (1.38). 1H NMR (600 MHz, CDCl3, TMS): δ, ppm 9.60 (8H, s, β-pyrrole), 5.33 (8H, m, α-Cp), 4.76 (8H, m, β-Cp), 3.97 (20H, s, CpH), -0.49 (2H, s, inner NH). 13C NMR

(150 MHz, CDCl3, TMS): δ, ppm 144.9 (α-pyrrole), 124.5 (β-pyrrole), 117.2 (meso-C), 89.1 (Cp), 76.8 (α-Cp), 71.1 (Cp-H), 69.0 (β-Cp). HRMS-ESI: m/z 1047.1188 (calcd. for [M + H]+ 1047.4214).

Formation of PB films

Initially the glassy carbon electrode (GCE) was cleaned with 1.0, 0.5 and 0.3 μm alumina slurry on a felt cloth, rinsed with distilled water and then dried at room temperature. Afterwards, the GCE was firstly modified with Prussian Blue. PB was synthesized by mixing 10.0 mL of a 1.0 mM potassium ferrocyanide solution with 10.0 mL of 2.0 mM iron(III) chloride. A dark blue solid was obtained and separated by centrifugation. Modification is performed by drop casting of 10.0 μL of the PB (0.5 mg.mL-1) on the electrode surface. Thereafter, the modified electrode was dried for approximately 1 h with a hairdryer. A second layer was composed of TFcP in DCM (1.0 mM–1.05 mg.mL-1) and was dropped on the electrode (10.0 μL) and it was dried for approximately 1 h with a hairdryer. After this, more layers were deposited in the follow order: PB-TFcP-PB. Measurements have been performed in a three-system cell, with Ag/AgCl as a reference electrode, Pt wire as auxiliary and the GCE as working electrode in HCl-KCl 0.10 M. After that, aliquots of 50.0 μL of 1.0 mM solution of dopamine (DA) were added to the cell. The solution was analyzed by square wave voltammetry, in the range of 0.0 to 0.8 V vs. Ag/AgCl using a potenciostat/galvanostat AutoLab EcoChimie PGSTAT32N system. The amplitude used for SWV was 0.05 V and the frequency 25.00 Hz. Micrographs of PB, TPcP and PB/TPcP/PB film on electrode’s surface were obtained using a scanning electron microscopy JEOL model JMS6360-Lv.

Fig. 5. SWV of modified electrode in HCl 0.1 M and KCl 0.1 M in different concentrations of dopamine. Inset: response curve obtained from the SWV measurements



CONCLUSION

Hybrid films formed by TFcP and Prussian blue on GCE had its electrochemical stability improved when compared to TFcP and Prussian singly. Prussian blue probably compensates the positive charge formed during the oxidation of TFcP by formation of negative-positive ionic pair. The observed behaviors in the modified electrodes open new possibilities to use this kind of system in other electrocatalytic studies. In addition, the sensing of dopamine by hybrid film (TFcP/Prussian blue) presented linearity in the range of 5.0 μM to 87.0 μM and detection limit of 2.44 μM which can be considered satisfactory according to the literature.

Acknowledgements

The authors acknowledge the financial support by FAPESP (grant no. 2013/22127-2), FAEPEX (grant no. 2163/15) and CNPq (grant nos. 459923/2014-5 and 443625/2014-0), respectively.

Supporting information

NMR spectra, HR-mass spectroscopy spectra and all micrograph of TFcP, Prussian blue, and TFcP/Prussian blue hybrid films (Figs S1–S13) are given in the supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

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DOI: 10.1142/S1088424616501261



INTRODUCTION

Phthalocyanines (Pcs) are remarkable macrocyclic compounds with conjugated 18 π-electrons and four N-fused isoindole units in the inner core. Their bright colors, conductivity, chemical and thermal stability have made them very desirable for a various potential application in many fields of science and technology. They have been well-studied as semiconductor devices [1], chemical sensors [2], catalysts [3], non-linear optics [2, 4], optical data storage [5], electrochromic materials [6–8] and photodynamic therapy [9]. Pcs also show a strong tendency to exhibit a stable columnar thermotropic and lyotropic liquid crystal behavior [10–12].

Columnar mesophases are classified according to the nature of intra column order and the two-dimensional lattice symmetry of the columns such as nematic, hexagonal columnar and rectangular columnar [12, 13]. Phthalocyanine-based columnar LCs are important class

of materials for possible applications due to their high charge-carrier mobility, photoconductivity along the columnar stacks, strong absorption in the visible and near IR regions and broad mesophase range [10–13]. The strong intermolecular π–π orbital overlap of Pc rings is significant for molecular stacking in the columns [14]. The first thermotropic liquid crystalline phthalocyanine compound was reported by Simon and co-workers [15]. In addition to, the first studies on lyotropic mesomorphism exhibited by phthalocyanine derivatives were reported by Usoltseva and co-workers [16–19]. Since then, a wide variety of Pcs have been investigated to produce more easily controllable macroscopic structures [20]. Thermotropic behavior of phthalocyanine-based LCs can be varied by various central metal atom [21–23], the different position of substitution, the nature [24, 25] or length of side chains [15, 23, 26–29]. When the peripheral aliphatic side chains of various disk-like cores are branched, the mesomorphic range is widened [30, 31]. The introduction of branched chains in liquid crystals often reduces melting and clearing temperatures [30-33]. Additionally, the size of the branches has a strong effect on the phase stability [33]. The use of branched chains has been successfully applied in the case of several discotic

Lyotropic liquid crystalline phthalocyanines containing

4-((S)-3,7-dimethyloctyloxy)phenoxy moieties

Sibel Eken Korkut*, Hale Ocak, Belkıs Bilgin-Eran, Dilek Güzeller

and M. Kasım Sener*

Department of Chemistry, Yildiz Technical University, Davutpasa Campus, Esenler, Istanbul TR-34220, Turkey

Received 5 August 2016Accepted 9 October 2016

ABSTRACT: The novel metal free phthalocyanine and its copper complex which are octa-substituted at the peripheral positions with 4-((S)-3,7-dimethlyoctyloxy)phenoxy moieties were synthesized and characterized by FT-IR, 1H NMR and mass spectroscopy. Their mesomorphic properties were studied by polarizing optical microscopy. The spectroscopic properties and aggregation behaviors of the novel phthalocyanines were also investigated by UV-vis spectroscopy in different solvents with same concentration as well as in a wide range of concentrations of chloroform. Both compounds with chloroform and n-dodecane clearly show the lyotropic columnar mesophase in a wide temperature range whereas thermotropic liquid crystalline behavior for both compounds is not observed. Both of these novel compounds show no aggregation in toluene, tetrahydrofuran, dichloromethane and chloroform.

KEYWORDS: phthalocyanine, liquid crystalline, lyotropic mesophase, copper.

*Correspondence to: Sibel Eken Korkut, email: [email protected], tel: +90 212-383-4172, fax: +90 212-383-4134; M. Kasım Sener, tel: +90 212-383-4176, fax: +90 212-383-4134, email: [email protected]


LYOTROPIC LIQUID CRYSTALLINE PHTHALOCYANINES CONTAINING 4-((S)-3,7-DIMETHYLOCTYLOXY)PHENOXY MOIETIES 17

liquid crystals forming columnar mesophases [34-39]. To date, some optically active phthalocyanine derivatives containing peripheral chiral moieties such as (S)-3,7-dimethyloctoxy [12, 34, 40, 41] or (S)-2-methylbutoxy [42] on the α- or β-position of the phthalocyanine ring had been synthesized and investigated. The studies on phthalocyanines with branched aliphatic tails derived from (S)-citronellol showed that the introduction of branched chains onto the peripheral positions reduces the melting transition and give rise to a chiral columnar mesophase Colh* at room temperature and an achiral rectangular columnar mesophase Colr at elevated temperature [41]. One of striking result of the branching effect has been reported for phthalocyanine derivatives containing peripheral chiral (S)-2-methylbutanol moieties on the α- or β-position of the phthalocyanine. The position of chiral units on phthalocyanine ring influence the handedness and mesophase textures of liquid crystals [42].

In this work, the novel metal free (2) and copper (3) phthalocyanines containing eight 4-((S)-3,7-dimethlyoctyloxy)phenoxy groups at the β-position of phthalocyanine ring were synthesized and characterized to study the influence of branching and position of branched tails on liquid crystal behavior and aggregation properties. Thermotropic liquid crystalline behavior for the novel phthalocyanines were not observed whereas the lyotropic columnar mesophase with a wide mesomorphic range appeared by using solvents such as chloroform and n-dodecane. The mesomorphic behavior of the phthalocyanines were investigated by optical polarizing microscopy. The spectroscopic and aggregation pro perties of the novel phthalocyanines were investigated in different solvents (toluene, THF, dichloromethane) at a concentration of 1 × 10-5 M and in chloroform at different concentrations.

EXPERIMENTAL

IR spectra were recorded on a Perkin Elmer Spectrum One FT-IR (ATR sampling accessory) spectrophotometer, and electronic spectra on an Agilent 8453 UV-vis spectrophotometer. 1H NMR spectra were recorded on a Bruker Ultra Shield Plus 400 MHz spectrometer, in CDCl3

solutions with tetramethylsilane as an internal standard. Mass spectra were measured on a Bruker Microflex LT MALDI-TOF MS and Micro TOF ESI-MS. Melting point was determined on an Electrothermal Gallenkamp apparatus. Transition temperatures were measured and optical investigations were performed on samples between ordinary glass slides by using a Mettler FP-82HT hot stage and a control unit in conjunction with a Leica Leitz DMR polarizing microscope and Leica DFC295 camera.

4-((S)-3,7-dimethlyoctyloxy)phenol was prepared according to the reported procedures [43]. All other reagents and solvents were reagent grade quality and were obtained from commercial suppliers. The homogeneity of the products was tested in each step by TLC (SiO2).

Synthesis

4,5-Bis[4-((S)-3,7-dimethlyoctyloxy)-phenoxy]phthalonitrile (1). 4,5-Dichlorophthalonitrile (0.100 g, 0.50 mmol) was dissolved in 10 mL of dry DMF at 50 °C under Ar atmosphere and 4-((S)-3,7-dimethlyoctyloxy)phenol (0.250 g, 1.00 mmol) was added. After stirring for 30 min, finely ground anhydrous potassium carbonate (0.345 g, 2.49 mmol) was added in portions during 2 h with efficient stirring. The reaction mixture was stirred under Ar atmosphere at 50 °C for 48 h. The reaction mixture was cooled to room temperature and then poured into 150 mL of ice-water and stirred for 1 h. The white solution was extracted with chloroform (3 × 15 mL). After evaporation of solvent, the precipitate was filtered off and washed with cold ethanol. The compound was soluble in chloroform, THF and dichloromethane. Yield 0.300 g (47%), mp 69–70 °C. FT-IR: νmax, cm-1 (ATR) 3075–3046 (CH, aromatic), 2953–2869 (CH, aliphatic), 2228 (C≡N). 1H NMR (400 MHz; CDCl3): δ, ppm 7.2–6.9 (m, aromatic H, 10H), 4.0–3.9 (m, OCH2, 4H), 1.9–0.7 (m, CH, CH2, CH3, 38H). MS (LC): m/z calcd. 624; found 642 [M + H2O]+.

2,3,9,10,16,17,23,24-Octakis-[4-((S)-3,7-dimethlyo-ctyloxy)-phenoxy]phthalocyanine (2). A mixture of compound 1 (0.100 g, 0.16 mmol) and a catalytic amount of DBU in 1-pentanol (2 mL) was fused in a glass tube. The mixture was heated to 140 °C and stirring for 24 h in an oil bath. The reaction mixture was cooled to room temperature. After evaporation of solvent, 2 was chromatographed on silica gel and eluted with hexane:ethylacetate (4:1). Yield 0.030 g (27%). FT-IR: νmax, cm-1 (ATR) 3292 (NH), 3046 (CH, aromatic), 2953–2868 (CH, aliphatic). 1H NMR (400 MHz; CDCl3): δ, ppm 7.4–6.7 (br, aromatic H, 40H), 4.0–3.9 (br, OCH2, 16H), 2.0–0.7 (m, CH, CH2, CH3, 152H). UV-vis (CHCl3): λmax, nm 703, 669, 343.

2,3,9,10,16,17,23,24-Octakis-[4-((S)-3,7-dimethlyoc - tyloxy)-phenoxy]copper phthalocyanine (3). A mixture of compound 1 (0.100 g, 0.16 mmol) and anhydrous metal salt (0.002 g, 0.016 mmol CuCl2) and a catalytic amount of DBU in 1-pentanol (2 mL) was fused in a glass tube. The mixture was heated to 140 °C and stirred for 24 h in an oil bath. The reaction mixture was cooled to room temperature. After evaporation of solvent, the precipitate was filtered off and washed with hot methanol, respectively. 3 was chromatographed on silica gel and eluted with DCM:hexane (2:1). Yield 0.060 g (48%). FT-IR: νmax, cm-1 (ATR) 3044 (CH, aromatic), 2952–2869 (CH, aliphatic). UV-vis (CHCl3): λmax, nm 684, 340. MS (MALDI-TOF, DBH): m/z calcd. 2559.9; found 2561.9 [M + 2H]+.


Synthesis and characterization

The synthetic route for phthalonitrile 1 and novel phthalocyanines 2 and 3 are summarized in Scheme 1.


18 S. E. KORKUT ET AL.

A novel compound 4,5-[4-((S)-3,7-dimethlyoctyloxy)-phenoxy]-phthalonitrile 1 was prepared and used as a starting material for the preparation of peripherally octa-substituted phthalocyanines. Cyclotetramerization of the phthalonitrile derivative 1 to the metal free phthalocyanine (2) and its metal complex (3) were accomplished in 1-pentanol in the presence of a catalytic amount of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) as a strong base at reflux temperature in a glass tube. Novel phthalocyanines (2, 3) were purified by column chromatography on silica gel and gave yields 27% for 2 and 48% for 3.

Characterization of the new phthalocyanines involves combination of methods including FT-IR, 1H NMR and electronic spectroscopy, mass spectroscopy techniques. The data are consistent with the assigned structures. The results are given in the experimental section. The IR spectra of the phthalocyanines confirmed the formation of the macrocycles (see Figs S1, S4, S6). After cyclotetramerization of 1 into the metal-free phthalocyanine (2) and its metal complex (3), the sharp peak for the CN vibration at 2228 cm-1 disappeared, respectively. The IR spectra of the phthalocyanines 2

and 3 are very similar, except the metal free (2), showing an NH strecthing band peak at 3292 cm-1 in the inner core. The synthesized phthalocyanine derivatives 2 and 3 showed the characteristic vibrations belonging to aromatic CH stretching at 3046 and 3044 cm-1. The novel phthalocyanines clearly prove the presence of the long alkyl chain by the intense absorption peak for aliphatic group at 2953–2868 (for 2) and 2952–2869 (for 3) cm-1.

The 1H NMR spectra of 1 in CDCl3 was recorded. The 1H NMR spectrum of 1 showed signals ranging from 7.2 to 6.9 ppm, belonging to aromatic protons, integrating for 10 protons for 1 as expected (see Fig. S2). Also, the –OCH2 protons were observed at 4.0–3.9 ppm. The rest of the aliphatic CH, CH2, CH3 protons were observed between 1.9–0.7 ppm. In the ESI-MS spectrum of 1, we observed the [M + H2O]+ peak at values of m/z 642 amu (see Fig. S3).

1H NMR spectrum of metal free phthalocyanine (2) exhibited the aromatic protons in the range of 7.4–6.7 ppm, integrating for 40 protons for 2 as expected (see Fig. S5). Also, the –OCH2 protons were observed between 4.0–3.9 ppm. The rest of the aliphatic CH, CH2,

N N

NN

N N

N

N

ORRO

RO

RO

OR

OR

ORRO

M= 2H (2), Cu(II) (3)

OR =

M

*

CN

CN

Cl

Cl

OHO

K2CO3, DMF

RO

RO CN

CN

1

*

1-pentanol,140 °C, 24 h

.

Scheme 1. Synthesis of phthalocyanines 2 and 3



CH3 protons were observed between 2.0–0.7 ppm. In the MALDI-TOF mass spectrum of 3, we observed the [M + 2H]+ peak at values of m/z 2561.9 amu (see Fig. S7).

The UV-vis spectra of the new compounds (2 and 3) exhibited typical spectrum of phthalocyanines, which includes two distinct bands, one of them is in the visible region at 600–700 nm (Q-band) and the other one is in the UV region at about 300–500 nm (B-band). This spectrum of metal-free phthalocyanine (2) in chloroform shows a doublet in the Q-band region at 669 and 704 nm, and the B-band is recorded at 344 nm; while CuPc (3) give intense single Q-bands at 684 nm, and B-bands at 341 nm, respectively.

For phthalocyanines, the aggregation is usually detected from optical absorption studies. It is dependent on the concentration, nature of the solvent, nature and number of the substituents [10], complexed metal ions and temperature [44]. It has been well-established that non-aggregated Pcs are extremely important for their various applications [45–48]. In this study, the aggregation behavior of the phthalocyanine complexes (2 and 3) were investigated in different solvents such as chloroform, THF, dichloromethane and toluene (see Fig. 1 as an example for complex 2 (a), 3 (b)). The Beer–Lambert law was obeyed for all of these compounds at concentrations ranging from 1.4 × 10-6 to 4 × 10-6 M (see Fig. 2 as examples for

Fig. 1. UV-vis absorption spectra of compound 2 (a) and 3 (b) in different solvents. Concentration 1 × 10-5 M



complexes 2 (a) and 3 (b)). While the concentration was increased, the intensity of absorption of the Q-band also increased but a blue shift of the Q-bands was not observed at about 640 nm because of the non-aggregated species. Non-aggregation of both compounds in any of the studied solvents and under a wide range of concentrations in chloroform may be explained by the effect of a larger steric hindrance of 4-((S)-3,7-dimethlyoctyloxy)phenoxy groups at the β-position of phthalocyanine ring.

Compounds 2 and 3 had the high solubility in a number of organic solvents, such as toluene, tetrahydrofuran (THF), dichloromethane (DCM) and chloroform (CHCl3) whereas they were insoluble in dimethylformamide (DMF) and dimethylsulfoxide (DMSO).

The mesogenic properties of the phthalocyanines

Liquid crystal phase transition temperatures of phthalocyanines 2 and 3 were investigated by polarizing optical microscopy in the range of 20–280 °C. Thermo-tropic mesomorphism was not observed for both compounds 2 and 3 up to 280 °C. Heating was not applied to the samples at temperatures higher than 280 °C under polarizing optical microscopy due to the fact that decomposition started for both compounds. However, lyotropic mesomorphism in the phthalocyanines 2 and 3 was appeared when both compounds were dissolved in chloroform and n-dodecane solvents. In order to test the lyotropic behavior, the samples of compounds 2 and

Fig. 2. UV-vis absorption spectra changes of compound 2 (a) and 3 (b) in chloroform at different concentrations



3 were prepared by using one or two drops of solvents and then they were placed between two ordinary glass slides. A partial evaporation of the solvent occurred before investigation so the concentration can not exactly be determined. On cooling of the compound 2 in chloroform from 108 to 36 °C, the columnar (Col) mesophase appeared and this was characterized by a dendritic texture growing in size (see Fig. 3). On cooling of the compound 2 in n-dodecane, a fan-like texture of Col mesophase started to appear at a higher temperature than that of compound 2 in chloroform. Additionally, the fan-like texture of Col mesophase of compound 2 in n-dodecane distinctly exists in broad temperature ranges (280–20 °C) (see Fig. 4). This stability can be explained by the strong π-stacking interactions arising from the phthalocyanine cores. The effect of nature of the solvent was clearly observed on mesomorphic temperatures.

When compound 3 in chloroform was slowly cooled down from the isotropic phase at 173 °C, the mesophase appeared with a spherulitic texture, which is characteristic for a columnar mesophase. There are some straight linear defects which are characteristic

for ordered columnar mesophases [12] (see Fig. 5). Mesophase remained unchanged until 52 °C and then crystallization was occured. As expected, the transition temperatures of the copper complex of phthalocyanine 3

Fig. 3. The dendritic texture of Col mesophase of compound 2 in chloroform at 87 °C on cooling

Fig. 4. The fan-like textures of Col mesophase of compound 2 in n-dodecane at 273 °C and 119 °C on cooling

Fig. 5. A spherulitic texture of a Col mesophase of 3 in chloroform at 119 °C and 73 °C on cooling



in chloroform is higher than that of metal free precursor 2. When the solution of copper complex 3 with n-dodecane was investigated under the polarization microscope on gradually cooling, a dendritic texture appeared in the range of 240–38 °C (see Fig. 6). In comparison with the metal free phthalocyanine 2 dissolved in n-dodecane, the solvent nature results in increasing transition temperatures and a broadening of the mesophase range. As a result, a Col phase exists in broad temperature ranges in n-dodecane concentrated solutions.

CONCLUSION

In this study, new metal free (2) and copper (3) phthalocyanines which are octa-substitued with 4-((S)-3,7-dimethlyoctyloxy)phenoxy moieties at the β-position of phthalocyanine ring have been synthesized and characterized by classical spectroscopic methods (1H NMR, FT-IR, UV-vis and mass spectroscopy). The mesomorphic behavior and aggregation properties were determined by polarizing optical microscopy and UV-vis spectroscopy, respectively. None of the new phthalocyanines showed aggregation in any of the studied solvents with same concentration (toluene, THF, DCM and CHCl3) as well as in a wide range of concentrations of chloroform. The mesomorphic investigations showed that the novel metal free phthalocyanine and its copper complex exhibit the lyotropic mesomorphism by using chloroform and n-dodecane. It is well-known that the position such as being closer to phthalocyanine ring (α or β-position) [42] and the nature of substituent (branching, the presence of molecular chirality or chain length) [41, 49, 50] in phthalocyanine ring strongly influences the mesophase type which is exhibited. The (S)-3,7-dimethlyoctyloxy group is linked to phthalocyanine ring via a phenyl ring. As a result of this, the effect of molecular chirality on mesomorphism for compounds presented here is not as strong as that in phthalocyanines carrying the (S)-3,7-dimethlyoctyloxy group which was

directly linked on β-position phthalocyanine ring [41]. Also, the extension of discotic core by the introduction of phenyl ring peripherically disturbed the π-stacking interactions and gives rise to thermotropically non-mesogenic materials on the contrary of reported analogs [12, 34, 40, 41]. For the concentrated solution of both compound 2 and 3 with n-dodecane, a columnar mesophase (Col) was observed in broader temperature ranges than that of observed in chloroform. It clearly reveals that the using of a suitable solvent encourages the formation of stable columnar phases.


Figures S1–S7 are given in the supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

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DOI: 10.1142/S1088424617300038



INTRODUCTION

Radionuclide therapy (RNT) has an important role in nuclear medicine, with progressive efficacy on clinical phase. Development in radiopharmaceutical manufacture and the administration of a multidisciplinary procedure in clinical medicine have propelled radionuclide therapy processes and usage towards personalized treatment, therapeutic result, patient comfort and radiation security. The radionuclide therapy concept has been applied even before the 1960s [1, 2]. The unsealed sources radionuclides were extensively applied for the diagnosis and treatment of various diseases e.g. cancers or other species of pathological conditions, such as hyperthyroidism using Na131I (radioiodine therapy) and rheumatoid arthritis [2–6].

Developments in the realizing of tumor biology, recombinant antibody approach, synthetic chemistry and other related technologies have lately conducted to multiple advances in the progression and clinical utilizations of many endo-radiotherapeutic agents. During the past decade, RNT using the targeting potential of radiolabeled compounds, selectively deliver cytotoxicity of radiation to the treating location [7, 8]. However, due to RNT efficacy in treating the malignancies and the advance of a wide range of new products, the requirement for therapeutic nuclear medicine is expected to represent express growth.

Yttrium-90, is a pure beta particle emitter (half-life = 64.053 h) that decays into stable zirconium-90, due to being capable of producing a great radiation dose it can be used for killing the tumor cells. The average and maximum penetration of the irradiation is 2.5 and 10 mm, respectively, with an energy up to 2.27 MeV (100%) and a linear energy transfer (LET) of almost 0.2 keV/μm [9, 10].

Preparation and biological evaluation of a carrier free 90yttrium labelled porphyrin as a possible agent for targeted

therapy of tumor

Mahvash Abedi*a,b, Mohammad Reza Nabida, Simindokht Shirvani-Aranib,

Ali Bahrami-Samanib and Nasim Vahidfarb

a Department of polymer, Faculty of Chemistry, Shahid Beheshti University, G. C., 1983963113, Tehran, Iran b Nuclear Science and Technology Research Institute (NSTRI), P.O. Box: 11365-8486, Tehran, Iran

Received 8 June 2016Accepted 25 July 2016

ABSTRACT: In this research article, 5,10,15,20-tetrakis(phenyl)porphyrin (H2TPP) was produced and characterized. Then, radiolabeling of H2TPP was performed using the carrier free Y-90 which was prepared by the use of a home-made yttrium imprinted sorbent. The radiolabeling procedure was accomplished at 60 °C during 12 h with a suitable radiochemical purity (95 ± 2% ITLC, 99 ± 0.5% HPLC) and specific activity (1.0 ± 0.1 GBq/mmol). The obtained radio-labeled H2TPP in final formulation was kept for a week in order to investigate the complex stability. Accordingly, the partition coefficient was calculated as log P = 2.05. Furthermore, the biodistribution of the 90Y–TPP was determined in vital organs of normal wild-type rats using scarification studies. The kidneys could mostly remove the radio-complexes from the blood circulation and in lesser extent from the liver. As a result it is expected that due to its lipophilicity the higher mitochondrial content and thus, tumor cell uptake of this radiolabeled porphyrin happens and therefore 90Y–TPP could act as an efficient potential agent for targeted therapy of tumor.

KEYWORDS: porphyrin, yttrium-90, radiolabeling, tumor, targeted therapy.

*Correspondence to: Mahvash Abedi, email: [email protected], fax: +98 2188-221-116, tel: +98 2188-221-117


PREPARATION AND BIOLOGICAL EVALUATION OF A CARRIER FREE 90YTTRIUM LABELLED PORPHYRIN 25

This remarkable radioisotope owing to contribution in non-invasive radio targeted therapy and direct intra- tumoral injection has received great attention [11–18]. Hence numerous studies have represented the safety and also therapeutic efficacy of yttrium-90 [19–24]. According to the long range of penetration as well as ability of delivering high energy simultaneously Y-90 emmits β-radiations to the target cell. More than 90% of emitted radiation is enough to affect a range of 100 to 200 diameters of cells [25, 26].

The cell integrity is affected by the beta particles incident trough both the primary radiation effect that causes irreparable structural damage of double-stranded nuclear DNA and secondary radiation effect that as a result of cytosol water radiolysis increases the amount of toxic free radicals at this point [27, 28].

Not only the cytocidal properties but also the possibility of coordination with numerous tissue targeting-chelating agent results in considering 90Y as a great therapeutic tool which has been applied for treatment of different disorders such as hepatocellular carcinoma, neuroendocrine tumours, colorectal cancer with metastatic liver and non-Hodgkin’s lymphomas [28].

The name of porphyrin is derived from the greek word for purple because of the most porphyrins and metalloporphyrins owing to extensive π-conjugation are intensely colored, usually dark red or purple. Porphyrins are synthesized from repeated condensations of an aldehyde with pyrrole via electrophilic substitution reaction, Ring closure and finally spontaneous oxidizing in the air.

Porphyrins are naturally occurring macrocyclic compounds which diverse synthetic routes [29, 30] toward them has expanded because of their numerous potential applications [31–34] namely such as anti-inflammatory agents [35], antioxidants [36, 37], molecular magnetic resonance imaging contrast agents [38], anti-protozoal [39], antibacterial [40], anticancer [41, 42] and as a photodynamic therapy for molecular degeneration [43].

Since porphyrin rings has shown a great ability in complexation of metals, many radiolabeled porphyrins have been synthesized for the theranostic (both therapeutic and diagnostic) applications applying radionuclides including 99mTc [44, 45], 111In [46], 109Pd [47, 48], 188Re [49] and 166Ho [50, 51]. Moreover few radiolabeled conjugated porphyrin molecules with higher stability such as 177Lu–DOTA porphyrins [52] and 90Y–DOTA porphyrins [53] successfully have been prepared. Aforementioned potential therapeutic tumor targeting agents are under developing owing to the favorable pharmacological behaviors of porphyrins such as serum solubility, ease of (II) and (III) valent metallic radionuclide complexation, rapid blood washout and last but not the least the tumor avidity properties [54].

In this study, after the production of medical grade 90Y by using a home-made yttrium imprinted sorbent, 5,10,15,20-tetrakis(phenyl)porphyrin (H2TPP) was synthesized. The radiolabeling of H2TPP with 90Y

was then performed to produce 90Y–TPP. The final formulation, 90Y–TPP, was quality controlled before and after passing through Sep-pak® C18 cartridge using ITLC and HPLC, it’s the stability was checked up to one week and the partition coefficient was then determined. Biodistribution studies of the labeled compound in normal wild-type rats have been investigated and have been compared with those of free yttrium-90 that has been formerly performed.

EXPERIMENTAL

Materials and methods

Carrier free Y-90 was produced and purified at the Nuclear Science and Technology Research Institute (NSTRI), using a homemade ionic imprinted polymeric sorbent with a purity of >99.99%, as described earlier [56]. All other chemicals were purchased from Sigma-Aldrich Chemical Co. UK (Dorset, UK). Radio-chromatography was performed using Whatman® paper (on a thin-layer chromatography scanner (Bioscan AR2000, Paris, France). An Alliance high performance liquid chromatography (HPLC) instrument from Waters Corporation (Milford, MA, USA) was used to conduct

quality control studies. Animal studies were performed in accordance with the United Kingdom Biological Council’s Guidelines on the Use of Living Animals in Scientific Investigations, second edition. Sep-pak® C18 cartridges were obtained from Waters Corporation.

Laboratory coats, gloves and enclosed footwear were worn at all times in the lab when working with radioactive compounds. Safety glasses were also worn. Operations were carried out in a glove box. The hands and the body were kept at the maximum practicable distance from high-specific-activity radionuclides by the use of tongs or other handling devices. We used suitable shielding materials (such as perspex for beta radiations) wherever possible.

Synthesis of tetraphenyl porphyrin (H2TPP)

H2TPP (Fig. 1) was synthesized according to the previous literature, using freshly distilled benzaldehyde, pyrrole, and propionic acid, followed by oxidation [30]. The chlorin content of the crude reaction mixtures averaged around 10%. Yield 20%, mp 248–250 °C. 1H NMR (CDCl3): δ, ppm -2.8 (2 H, NH), 7.71–7.82 (12 H), 8.14–8.27 (8 H), 8.85 (8 H). 13C NMR (CDCl3): δ, ppm 120.20 (C), 126.74 (CH), 127.76 (CH), 131.16 (CH), 134.62 (CH), 142.22 (C), 145.6 (C). UV-vis (toluene): λmax, nm (ε) 418 (413200), 514 (19060), 549 (8080), 594 (5380), 648 (3870). IR (KBr): ν, cm-1 3320, 3055, 3025, 1595.

Radiolabeling of porphyrin with Y-90

The carrier-free 90Y was prepared using an acryl amide-based sorbent, as reported elsewhere[55]. The


26 M. ABEDI ET AL.

beta spectrum of the prepared carrier-free Y-90 is shown in Fig. 2. The acidic solution (2 mL) of [90Y] Y (NO3)3 (100–300 MBq) was transferred to a vial and evaporated via a flow of N2gas at 60–70 °C. A solution of 5 mg/mL H2TPP in absolute ethanol was prepared. Then 50 μL of the prepared solution was added to a vial containing an acetate buffer with a pH of 5.5 (450 μL), and the reaction was allowed to continue at 60 °C for 12 h. The active solution was then further purified using a Sep-pak® C18 cartridge. The radiochemical purity of the final solution was investigated using two systems for instant thin-layer chromatography (ITLC) (A: 10 mM DTPA pH 4; and B: Whatman No. 2 paper and 10% NH4OAc and methanol 1:1 as the mobile phase mixture) and high performance liquid chromatography (HPLC) analysis (before and after purification using Sep-pak®) with a C-18 column (25 cm × 5 mm) at a flow rate of 1 mL/min. Water and acetonitrile containing 0.1% trifluoroacetic acid (TFA) were used as solvents for gradient elution.

Purification of 90Y-TPP–complex

Purification of the 90Y–TPP complex conjugate was carried out using a Sep-pak C18 cartridge, following

the procedure described below. The cartridge was preconditioned with 4 mL of ethanol, followed by 2 mL of distilled water. The crude complex mixture was loaded in the cartridge and subsequently washed with 4 mL of distilled water, whereby uncomplexed 90Y was eluted from the column. Finally, eluting the cartridge with 1 mL of ethanol yielded the radiochemically pure 90Y-TPP complex. The ethanol was removed by gentle heating, and the radiolabeled complex was reconstituted in normal saline for further studies.

Quality control of 90Y-TPP complex

The radiolabeling yield was determined using radio-phase thin-layer chromatography (RTLC) and confirmed by HPLC studies. As mentioned above, the RTLC studies were performed using two systems (A: Whatman No. 2 paper as the stationary phase and 10 mM DTPA pH 4 as the mobile phase; and B: Whatman No. 2 paper and 10% NH4OAc and methanol 1:1 as the mobile phase mixture). The HPLC analysis was carried out with a C-18 column (25 cm × 5 mm) at a flow rate of 1 mL/min. Water (a) and acetonitrile containing 0.1% TFA (b) were used as the mobile phase. A gradient elution, as reported by Pillai [53], was adopted to determine the complexation, using solvents as follows: 0–28 min: 90% a–10% b; 28–30 min: 10% a; 30–32 min, 10% a–90%b. It should be mentioned that HPLC analysis was performed after one week’s incubation in a phosphate buffer solution, using a HPLC system with a gamma-RAM™ detector.

Determination of partition coefficients

In order to determine the octanol-water partition coefficients (log P), a mixture of 1 mL of buffered aqueous solution containing 5 MBq of the [90Y]-TPP and 1 mL of the octanol at 37 °C and pH ~7 was vortexed (2 min) and then left for almost 5 min. The resultant two phases were separated by 5 min centrifugation (at > 12000 × g). The activity containment of each phase was then counted in an automatic well-type counter. An aliquot of 500 μL was taken from the organic phase (octanol), and the same experiment was repeated three or four times with freshly buffered aqueous solutions. Three or four independent experiments were performed, and the average of the second and the third extractions was obtained and reported as log P.

Stability measurements

The stability of the complex was investigated using the paper chromatography method. For this purpose, a radiolabeled complex was sampled at time intervals for one week at room temperature (r.t) and investigated via the paper chromatography system described above.

In order to investigate serum stability, freshly prepared healthy human serum (300 μL) was introduced into a vial containing a radiolabeled complex solution

Fig. 1. Structure of H2TPP

Fig. 2. Beta spectrum for carrier free Y-90 prepared at NSTRI



(7.4 MBq, 200 μCi, 100 μL) and kept at 37 °C for one week. Trichloroacetic acid (10%, 100 μL) was added to a sample of the above mixture (50 μL), followed by centrifuging (3000 rpm, 5 min). Finally, the supernatant was investigated via the paper chromatography system.

In another set of experiments, the same samples obtained after one week’s incubation were studied using HPLC analysis according to the solvent/stationary phase system mentioned above. The results obtained from HPLC also confirmed the high stability of the prepared formula, even after one week.

Biodistribution in wild-type rats

In order to investigate the biodistribution of the radiolabeled product, pre-counted aliquots (3.7 MBq per 10 μL) of the final formula were injected into wild-type rats. The prepared formula was injected through the tail vein of each animal by syringe, and animals were sacrificed using the animal care protocols at selected times post-anesthesia at 2 and 4 h and 1, 2, and 6 days post-injection, after which blood samples were taken from the rodent aortas. All tissue and organs (blood, heart, lung, stomach, intestine, liver, spleen, kidneys, muscle, and bone) were excised, washed with saline and then weighed. The radioactivity associated with each one was measured with a high-purity germanium (HPGe) detector. The percentage of injected dose per gram of tissues was calculated.


Production, purification, and quality control of 90Y-TPP

In our previous investigation of medical-grade 90-yttrium, an efficient acryl amide-based sorbent was synthesized using the atom-transfer radical-polymerization (ATRP) approach [56], followed by radiolabeling H2TPP to develop a tumor targeting agent. [90Y]Y(NO3)3 was added to TPP in the presence of an acetate buffer with a pH of 5.5 to produce the 90Y–TPP complex. HPLC analysis in this step showed that a small portion of Y-90 remained in an un-complexed form (Fig. 3a). Therefore, the active solution was further purified using a Sep-pak® C18 cartridge (Fig. 3b). The radiochemical purity of the resultant 90Y–TPP solution, as well as [90Y]Y(NO3)3, was investigated using two

systems for ITLC (A: 10 mM DTPA pH 4, Table 1; and B: Whatman No. 2 paper and 10% NH4OAc and methanol 1:1 as the mobile phase mixture). The free yttrium cation in 90Y3+ form remained at the origin of the tlc-paper (Rf 0.22), while the 90Y–TPP complex migrated to a higher Rf (Rf 0.95). ITLC studies confirmed the successful production of a single radiolabeled compound.

The active complex due to the engagement of -NH polar functional groups in its structure not only demonstrates different chromatographic properties but also is more lipophilic. The water solubility of the radio-complex leads to fewer unnecessary uptakes in tissues, including the liver and fat, and faster kidney wash-out, demonstrating the relative lipophilicity.

Partition coefficient of 90Y–TPP

As expected from the chemical formula in Fig. 4, the lipophilicity of the 90Y–TPP compound is not high, owing

Fig. 3. High-performance liquid chromatography profile of (a) unpurified 90Y–TPP (b) purified 90Y–TPP (c) purified 90Y–TPP after one week incubation in human serum at 37 °C

Table 1. ITLC results for 90YCl3 solution and final [90Y]–TPP solution using system A: 10 mM DTPA pH 4

Rf Radiochemical purity Rf Radiochemical purity

Y-90 0.8 100% 0.2 100%

[90Y]–TPP 0.2 >95% 0.8 >95%


28 M. ABEDI ET AL.

to the ionic nature of the radio-complex. The measured octanol/water partition coefficient, P, for the complex was found to depend on the pH of the solution. At pH = 7, the log P was 2.05.

Stability measurements

The chemical stability of the 90Y–TPP complex was high enough to warrant further investigations. No loss of 90Y from the complex was observed by incubation of [90Y]–TPP in freshly prepared human serum for 24 h at 37 °C by ITLC. No peak related to free Y-90 (non-complexed 90Y) was observed. The obtained result illustrates that the radiochemical purity of the complex

remained at 98% for 24 h under physiological conditions. Performing HPLC analysis after one week’s incubation in the phosphate buffer solution, using the abovementioned system, also confirmed the stability (Fig. 3c).

Biodistribution studies

As was reported in [50, 57], in the case of free radio-lanthanides, represented by Y-90 (which is generally categorized as radio-lanthanide) in the present study, the radioactivity is mainly located in the kidneys, liver, and bone. Since [90Y] YCl3 is water soluble, the excretion occurs via the urinary tract. It is transferred in plasma in a protein-bounded form and finally accumulates in the liver [57]. The uptake of 90Y–TPP pre-formulated with the normal saline (pH 6.5–7) in different organs and tissue of wild-type rats at different post-injection times is shown in Table 2. As mentioned earlier, after the sacrifice of the rats by CO2 asphyxiation, dissection began by drawing blood from the aorta. Then blood, heart, lung, stomach, intestine, liver, spleen, kidneys, muscle, and bone samples were collected, followed by calculation of the tissue uptakes per g of tissue (%ID/g). The radiolabeled porphyrin has an ionic nature, which results in a high uptake in organs and tissues such as kidney, blood, lung, and spleen; therefore, the excretion takes place through the urinary tract after 24 h. Low intestinal activity demonstrates a low hepatobiliary excretion route. As can be seen from the data (Table 1), the intestine uptake was low, which similarly confirms the low hepatobiliary excretion rate. As mentioned earlier, 90Y–TPP is washed out from the blood circulation after 24 h, although the blood wash-out mechanisms are different from those of free cations, but the free Y-90 also shows a fast wash-out. Unlike free radio lanthanides, represented

Fig. 4. The structure of Y-TPP

Table 2. Biodistribution pattern of 90Y–TPP complex in wild type normal rat organ/tissues

Organ/tissue % injected dose

2 h 4 h 24 h 48 h 144 h

Blood 2.91 (0.15) 1.62 (0.12) 0.21 (0.09) 0.13 (0.05) 0.00 (0.00)

Heart 0.52 (0.22) 0.45 (0.23) 0.35 (0.23) 0.05 (0.01) 0.00 (0.00)

Lung 1.20 (0.28) 0.95 (0.15) 0.57 (0.13) 0.17 (0.05) 0.00 (0.00)

Stomach 0.62 (0.23) 0.51 (0.05) 0.37 (0.22) 0.23 (0.03) 0.12 (0.01)

Intestine 0.50 (0.27) 0.49 (0.14) 0.24 (0.11) 0.18 (0.09) 0.02 (0.01)

Liver 0.92 (0.03) 0.81 (0.21) 0.41 (0.10) 0.22 (0.01) 0.11 (0.02)

Spleen 0.99 (0.02) 1.68 (0.01) 0.41 (0.03) 0.15 (0.01) 0.00 (0.00)

Kidney 4.90 (0.14) 2.51 (0.23) 1.90 (0.21) 0.98 (0.02) 0.84 (0.32)

Muscle 0.15 (0.01) 0.09 (0.14) 0.03 (0.01) 0.02 (0.01) 0.00 (0.00)

Bone 0.81 (0.02) 0.49 (0.05) 0.24 (0.28) 0.12 (0.09) 0.00 (0.00)

The figure in the parentheses indicates standard deviations. Three animals were used for each time point.



here by Y-90, which have significant spleen radioactivity uptake 2 h post-injection, the highest accumulation value for the prepared formula was 1.68% ± 0.02, which happened 4 h post-injection. Although both free Y-90 and 90Y–TPP bone uptakes decrease with the passing of time, in all time intervals, bone uptake related to the free Y-90 is much higher.

CONCLUSION

5,10,15,20-Tetrakis(phenyl)porphyrin (H2TPP) was synthesized and successfully radiolabeled with 90Y, obtained from a homemade yttrium-imprinted sorbent. Total labeling and formulation of [90Y]–TPP took about 12 h at 60 °C (radiochemical purity: 95 ± 2% ITLC, 99 ± 0.5% HPLC, and specific activity: 1.0 ± 0.1 GBq/mmol). The complex was stable in its final formulation and human serum for one week. The partition coefficient was calculated for the compound (log P = 2.05). Biodistribution studies in wild-type rats showed that the complex is mostly washed out from the blood circulation through the kidneys and, to a lesser extent, from the liver. The kidney/liver, kidney/muscle, and kidney/blood ratios 4 h post-injection were 3.09, 27.9, and 1.54, respectively. As it was observed in biodistribution studies, the synthesized radiolabeled-porphyrin (90Y–TPP) shows acceptable stability and also retention in targeting tumoral/cancerous tissues and it could be a potential candidate for nuclear therapy.

Acknowledgements

The authors are grateful for support of this study by Nuclear Science and Technology Research Institute and Shahid Beheshti University Research Council.

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DOI: 10.1142/S1088424617500018



INTRODUCTION

The rare-earth phthalocyanines forming sandwich structures were first reported in 1965 [1]. Since then, they have been studied for various applications such as field-effect transistors [2], molecular semiconductors in electronic devices [3] and sensitizers for oxidation with TiO2 [4], etc. The compounds are liable to oxidation and reduction reactions. The redox reactions are mostly phthalocyanine ligand-based. During this reaction, change of color also takes place and this property may be used for electrochromic displays [5]. Widely studied are also their magnetic properties for use in single molecular magnets [6].

Thiophene moieties as strong donors are very often adopted for tailoring electronic properties of many classes of compounds studied for applications in organic electronics [7]. Surprisingly, there are not many symmetrical phthalocyanines (Pcs) bearing 2- or 3-thienyl substituents attached directly to the Pc ring documented in the literature [8–16]. They are either metal-free compounds or have zinc, copper, nickel or cobalt as the

central metal in the molecule. To our knowledge, there are no symmetrically thiophene-substituted rare-earth Pcs reported.

The aims of this work is to study the effect of thiophene-substituted rare-earth Pcs on their spectral and photo-physical properties. For this purpose, a set of three rare-earth compounds (Pc2Gd, Pc2Sm, Pc2Pr) was prepared and characterized by several methods.


Synthesis and purification

The octakis-thiophene-substituted rare-earth phthalo-cy anines (Pc2Gd, Pc2Sm and Pc2Pr) were prepared in a two-step, one-pot reaction in moderate yields of 30–45% according to the Scheme 1. In the first step, dilithium phthalocyanine is formed and in the subsequent step (without isolation of the intermediate) a metal exchange between Li and rare-earth metal takes place. The yields are higher than those for octa-alkoxy Pcs [17], but similar to octa-alkylthio Pcs [18]. Very often adopted one-step reaction — refluxing of dinitrile derivative with lanthanide acetate gave insoluble compounds, possibly resulting

Preparation, characterization and investigation of photo-

physical properties of thiophene-substituted rare-earth

bisphthalocyanines

Jirí Cerný*a, Lenka Dokládalováa, Antonín Lyckaa, Tomáš Mikysekb and Filip Burešc

a Center for Organic Chemistry, Rybitví c.p. 296, Rybitví 53354, Czech Republic b Department of Analytical Chemistry, University of Pardubice, Faculty of Chemical Technology, Studentská 573, Pardubice 53210, Czech Republic c Institute of Organic Chemistry and Technology, University of Pardubice, Faculty of Chemical Technology, Studentská 573, Pardubice 53210, Czech Republic

Received 15 August 2016Accepted 14 October 2016

ABSTRACT: Three bis[octakis-(2-thienyl)phthalocyaninato] rare-earth metal(III) phthalocyanine complexes (Pc2Pr, Pc2Sm, Pc2Gd) were synthesized for the first time. The new compounds were characterized by UV-vis, NMR, FT-IR, mass spectroscopies as well as elemental analysis and electrochemistry. Production of singlet oxygen was also estimated by 9,10-dimethylanthracene method.

KEYWORDS: rare-earth phthalocyanine, UV-vis spectroscopy, singlet oxygen, cyclic voltammetry.

*Correspondence to: Jirí Cerný, email: [email protected], tel: +420 466-825-661, fax: +420 466-823-900


32 J. CERNÝ ET AL.

from polymerization of thiophene during the reaction. The compounds are soluble in common organic solvents such as DMF, chloroform, dichloromethane, acetone, THF. Due to a high affinity to the standard adsorbents for chromatography (silica gel, alumina, cellulose), we used Bu4NBr for decreasing these interactions. The final purification was finished by washing of the isolated compounds with water and methanol to remove any residual Bu4NBr. A typical FT-IR spectrum of Pc2Pr is shown in supplementary data (Supplementary material, Fig. S1, see Supporting information) as well as mass spectra using MALDI-TOF for Pc2Gd, Pc2Sm and Pc2Pr (Figs S2–S4).

UV-vis spectra

Phthalocyanines exhibits in the visible region two main absorption bands. For unsubstituted phthalocyanines, first band is located around 350 nm and it is known as the B-band or Soret band. Usually, much higher intensity has a band at about 670 nm which is called the Q-band. However, the substitution has an effect mostly on the position of the Q-band. The rare-earth phthalocyanines have several redox states, the most important are a neutral form, a reduced (also called an anionic) form and an oxidized state. The isolated compounds were Bu4N salts and are thusly present in reduced forms.

Figure 1 shows spectra of Pc2Gd, Pc2Sm and Pc2Pr in DMF. Due to the presence of 16 thiophene substituents in the molecule, the Q-band is blue-shifted to 700 nm. This shift is very similar to octakis(hexylthio)-substituted rare-earth Pcs [18]. Very similar spectra were found in chloroform solutions if a small amount of triethylamine as a base was added to CHCl3 to neutralize present acids (Fig. S5).

Figure 2 shows a typical change of the spectra upon oxidation of Pc2Pr using Br2 in CHCl3. Two experiments were performed. For the first experiment, CHCl3 with triethylamine content (10-3 M) was used (Fig. 2). Increased concentration of Br2 added to Pc2Pr resulted in decrease of the intensity of the Q-band at 700 nm. A new band upon oxidation appeared in the 500–550 nm

region and it corresponds to the formation of π-radical cation. Also the original shoulder peak at about 730 nm is more broad. After addition of 20 μL of triethylamine to oxidized Pc, the spectrum reverts back to more than 95% of the initial values.

In the second experiment, as-received CHCl3 (with content of about 10–20 ppm HCl) was used (Fig. S6) and certain differences were found. First, a shoulder Q-band located at 730 nm is more intensive. Also the reaction of oxidized Pc2Pr with triethylamine is not recovering the original spectrum completely. Pc2Gd and Pc2Sm have the same spectral changes during oxidation with Br2.

From these two experiments it is apparent that the oxidation of reduced phthalocyanine is fully reversible if no acid is present. The formed neutral compound has also a second Q-band located at 730 nm of low intensity. The same absorption maximum has also metal-free phthalocyanine (H2Pc). H2Pc is formed during oxidation in the presence of a small amount of acid. It is also formed by reaction of Pc2Pr with acids. This reaction

Scheme 1. Synthetic route of bis[(octakis-(2-thienyl)phthalo-cyaninato] rare-earth metal(III) complexes Fig. 1. UV-vis spectra of Pc2Gd (––), Pc2Sm (----) and Pc2Pr

(….) in DMF (concentration: 10 mg/L)

Fig. 2. UV-vis spectra of Pc2Pr (––) and its oxidized forms in CHCl3 at concentration 10 mg/L with 0.001M triethylamine. Addition of 20 μL of 0.01 M Br2 (----), 35 μL of 0.01M Br2 (–·–) and 20 μL of triethylamine to oxidized species (····) of Pc2Pr (2 mL, 5 × 10-6 M)


PREPARATION, CHARACTERIZATION AND INVESTIGATION OF PHOTO-PHYSICAL PROPERTIES 33

is fast and irreversible. Therefore, any acidic conditions should be avoided.

NMR spectroscopy

The 1H NMR spectra of the samples were measured in CDCl3. Pc2Gd is paramagnetic and, thus, only Pc2Sm and Pc2Pr showed analyzable signals. Two sets of signals were present in the 1H NMR spectra of Pc2Sm and Pc2Pr. The ratios of two components were ca. 2.42:1 for Pc2Sm and ca. 3.4:1 for Pc2Pr after dissolving at laboratory temperature. The measurement of 1H NMR spectrum of Pc2Sm was repeated after heating of the sample. The ratio of the two components increased then to ca. 6.3:1. The changes of 1H NMR spectra patterns are known for analogous compounds and it is expected that the reason for such a behavior consists in a different aggregation of the molecules [13]. The 1H chemical shifts are reported in Experimental.

Singlet oxygen production

Phthalocyanines belong to the group of photosensi-tizers. Photosensitizers are compounds which are capable of generation of singlet oxygen upon their interaction with every-present diatomic oxygen and light of appropriate wavelength. Briefly, a phthalocyanine is excited by light from the state S0 to the state S1 (Scheme 2). Then, there are several possible reaction pathways. The most important is an intersystem crossing leading to the triplet state T1. Other two processes are either irradiative quenching or radiation in the form of fluorescence (hνf). The triplet state T1 then reacts with diatomic oxygen (3O2) to produce singlet oxygen (1O2). The other possibilities for quenching of the T1 are irradiative process or radiation as phosphorescence (hνF). So far, the quantum yield of singlet oxygen (Φ) for bisphthalocyanines was reported only for a few derivatives.

The measurement of singlet oxygen production was according to reported procedure using 9,10-dimethyl-anthracene (DMA) [19]. Briefly, the tested compounds were dissolved in DMF (1 mg.L-1). A sufficient amount (0.02 mL) of the concentrated freshly prepared solution

of DMA in DMF was added to the solution of 2 mL of phthalocyanine in a cuvette, to get the initial absorbance of DMA solution at 381 nm equal to about 1. The samples were then irradiated with laser light (Maestro CCM, λmax = 661 nm) to decrease the absorbance of DMA solution to ca. 0.2–0.3. The measurements were triplicated. The obtained half-times were corrected to the unit absorbance of the sample and related to the zinc phthalocyanine (Φ = 0.56) [20].

Surprisingly, the highest estimated singlet oxygen quantum yield Φ was to be 0.37 ± 0.02 for Pc2Gd. Only one Φ in DMF of 0.33 was found for lutetium bisphthalocyanine substituted with carboxyphenoxy groups [21]. Much lower values of Φ were found for Pc2Sm (Φ = 0.13 ± 0.01) and Pc2Pr (Φ = 0.21 ± 0.01). However, these Φ values are larger than those reported for unsubstituted rare-earth bisphthalocyanines (Φ less than 0.01) [22].

Electrochemistry

Electrochemical measurements were carried out in 1,2-dichloroethane containing 0.1 M Bu4NPF6. Cyclic voltammetry (CV) and rotating disk voltammetry (RDV) were used in a three electrode arrangement. The working electrode was platinum disk (2 mm in diameter) for CV and RDV experiments. As the reference and auxiliary electrodes were used saturated calomel electrode (SCE) separated by a bridge filled with supporting electrolyte and a Pt wire, respectively. All potentials are given vs. SCE. Voltammetric measurements were performed using a potentiostat PGSTAT 128N operated via NOVA 1.11 software.

The fundamental electrochemical characterization of studied phthalocyanines was performed in order to reach first oxidation (reduction) potentials (see Table 1) which are reflecting the effect of metal center as well as substitution moiety. Concerning the oxidation there are not big changes in oxidation potentials within the series (from +0.15 V to +0.19 V vs. ref.), the only difference is in the case of Pc2Pr which exhibits easiest oxidation. This is probably caused by structural effect of the Pr atom which has largest size in comparison with other two metals. In addition to this, the first oxidation of all three compounds is one-electron reversible redox process. Similar situation is when comparing first reduction potentials; they range from -0.70 V to -0.77 V vs. ref. The easiest reduction was observed for Pc2Gd. Again, there are just small differences between the first reduction potentials within the series, but it was also observed that the other reduction processes are close to the first reduction potential and almost merge into one. Nevertheless, the values of second potentials are displayed in Table 1 and they do not exhibit a change with the metal center. Moreover, the first reduction process was found to be irreversible for Pc2Pr and Pc2Sm, and one-electron reversible for Pc2Gd. Concerning the second oxidation process, it was found to be one electron reversible for Pc2Gd, and in case

Scheme 2. Jablonski diagram illustrating the electronic states of phthalocyanines. S 0 — base state, S1 — excited state, T1 — triplet state, ic — internal conversion, isc — intersystem crossing, hνf — fluorescence, hνF — fosforescence, 3O2 — triplet oxygen, 1O2 — singlet oxygen. States S0, S1 and T1 are related to a photosensitizer


34 J. CERNÝ ET AL.

of other two compounds only peak potentials from CV were registered, because more positive potentials, during the CV and RDV measurements, led to the formation of species which caused blocking of the electrode surface (see Supplementary material).

EXPERIMENTAL

General

Commercially available hydrates of Gd(OAc)3, Sm(OAc)3 and Pr(OAc)3 (Aldrich, Alfa Aesar) were dried under a high vacuum (10-4 mbar) at 120 °C overnight. 4,5-Diiodophthalodinitrile was prepared according to the literature procedure [23]. The starting 4,5-bis(2-thienyl)phthalodinitrile (1) was prepared by a modification [10] of the reported procedure [16]. All other reagents were supplied from commercial sources.

UV-vis spectra were measured within the range of 300–900 nm on a UNICAM UV/visible Spectrophotometer, Helios Beta. 1H NMR spectra were recorded on a Bruker Avance 400 II spectrometer operating at 400.13 MHz, compounds being dissolved in CDCl3. The 1H shifts were referenced to the signal of the internal tetramethylsilane (Me4Si, TMS) (δ = 0). IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer. Elemental analyses were obtained using a FISONS EA 1108 automatic analyzer. Matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) were measured on a MALDI mass spectrometer LTQ Orbitrap XL equipped with nitrogen laser. Positive-ion and linear mode of the compounds were obtained in trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile matrix using nitrogen laser accumulating 15 laser shots.

Synthesis

Preparation of 4,5-bis(2-thienyl)phthalonitrile (1). 4,5-Diiodophthalonitrile (20 g, 52.63 mmol) and 2-(tri-n-butylstannyl)thiophene (49.10 g, 131.58 mmol) were dissolved in 200 mL DMF. Argon was bubbled through the solution for 15 min whereupon PdCl2(PPh3)2 (4.06 g,

5.79 mmol) and the reaction mixture was stirred at 65 °C for 48 h. The solvents were evaporated in vacuo and the crude product was purified by flash chromatography (SiO2; CH2Cl2/hexane 1:1). Yield 12.46 g (81%) of yellow-white solid. 1H NMR (400 MHz, CDCl3): δH, ppm 7.90 (2H, s), 7.43 (2H, dd), 7.04 (4H, m). 13C NMR (100 MHz, CDCl3): δC, ppm 138.95, 138.19, 135.57, 129.10, 128.86, 127.69, 115.04, 114.25. MS (MALDI-TOF (DHB)): m/z 292.0132 [M]+.

Preparation of tetrabutylammonium salt of bis[4,5,4′,5′,4′′,5′′,4′′′,5′′′-octakis-(2-thienyl)phthalo-cyaninato]-gadolinium(III) (Pc2Gd). A round-bottom flask (250 mL) fitted with a thermometer, condenser and inert inlet was charged with the dinitrile 1 (1.4 g, 4.79 mmol), lithium (18.3 mg, 2.63 mmol) and dried pentan-1-ol (50 mL). The mixture was refluxed for 2 h under nitrogen. Then, the mixture was cooled to 50 °C and solution of anhydrous Gd(OAc)3 (200.6 mg, 0.6 mmol) was added in dried DMF (50 mL). The mixture was refluxed for 7 h and the solvent was evaporated under reduced pressure. The crude green-black compound was washed with water (2 × 50 mL), methanol (2 × 50 mL) and acetonitrile (2 × 50 mL). The product was purified by flash chromatography (cellulose with 2% loading of tetrabutylammonium bromide (Bu4NBr); eluents: CH2Cl2, hexane) and finally washed with water and methanol to remove any residual Bu4NBr. The pure product was obtained as a dark green powder. Yield 476 mg (29%), mp > 200 °C. Anal. calcd. for C144H100N17S16Gd (2736.31): C 63.15; H 3.68; N 8.69; S 18.73%. Found C 63.35; H 4.05; N 8.78; S 18.57. FT-IR: ν, cm-1 3100, 3068, 2951, 2924, 2853, 1594, 1530, 1483, 1443, 1408, 1389, 1349, 1291, 1236, 1208, 1090, 1037, 893, 849, 834, 763, 753, 697. UV-vis (DMF): λmax, nm (log ε) 376 (5.17), 633 (4.74), 663 (4.79), 700 (5.41). MS (MALDI-TOF(+)): m/z 2495.02 [MH+ – Bu4N].

Preparation of tetrabutylammonium salt of bis[4,5,4′,5′,4′′,5′′,4′′′,5′′′-octakis-(2-thienyl)phthalo-cyaninato]-samarium(III) (Pc2Sm). Compound Pc2Sm was prepared by the same procedure as Pc2Gd, starting with 1 (2 g, 6.84 mmol), lithium (26 mg, 3.75 mmol), pentan-1-ol (50 mL), anhydrous Sm(OAc)3 (280 mg, 0.86 mmol) in dried DMF (50 mL). The pure product was

Table 1. Electrochemical data of Pc2Gd, Pc2Sm and Pc2Pr

Compound E1/2 (ox1), Va E1/2 (ox2), Va E1/2 (red1), Va E1/2 (red2), Va EHOMO, eVb ELUMO, eVb ΔE, Vc

Pc2Gd 0.19 0.51 -0.70 -0.82 -4.59 -3.70 0.89

Pc2Sm 0.19 0.76 -0.77 -0.82 -4.59 -3.63 0.96

Pc2Pr 0.15 1.00 -0.73 -0.83 -4.55 -3.67 0.88

a E1/2 (ox1,2), E1/2(red1,2) are half-wave potentials of the first and second oxidation (reduction) measured by RDV. b EHOMO/

LUMO = -[E1/2 (ox1/red1) + 4.4] eV. All potentials are given vs. SCE. c ΔE = E1/2 (ox1) - E1/2 (red1), electrochemical gap.


PREPARATION, CHARACTERIZATION AND INVESTIGATION OF PHOTO-PHYSICAL PROPERTIES 35

obtained as a dark green powder. Yield 1.02 g (43%), mp > 200 °C. Anal. calcd. for C144H100N17S16Sm (2730.31): C 63.31; H 3.69; N 8.72; S 18.78%. Found C 63.06; H 4.08; N 8.52; S 18.33. FT-IR: ν, cm-1 3101, 3068, 2959, 2930, 2872, 1609, 1527, 1484, 1445, 1408, 1386, 1349, 1292, 1234, 1201, 1094, 1036, 894, 849, 833, 765, 752, 696. UV-vis (DMF): λmax, nm (log ε) 379 (5.30), 627 (4.96), 667 (4.98), 695 (5.69). 1H NMR (400 MHz; CDCl3; Me4Si): δH, ppm 0.15–0.19 (20H, m, -CH2CH3), 1.32 (8H, q, -CH2), 1.70 (8H, t, -NCH2), 7.12–7.15 (16H, m), 7.21–7.24 (16H, m), 7.41–7.42 (16H, m, 3 × thienyl), 9.16 (16H, s, Ar-H). MS (MALDI-TOF(+)): m/z 2490.00 [MH+ – Bu4N].

Preparation of tetrabutylammonium salt of bis[4,5,4′,5′,4′′,5′′,4′′′,5′′′-octakis-(2-thienyl)phthalo-cyaninato]-praseodymium(III) (Pc2Pr). Compound Pc2Pr was prepared by the same procedure as Pc2Gd, starting with 1 (1.68 g, 5.75 mmol), lithium (21.8 mg, 3.14 mmol), pentan-1-ol (50 mL), anhydrous Pr(OAc)3 (228.6 mg, 0.72 mmol) in dried DMF (50 mL). The pure product was obtained as a dark green powder. Yield 859 mg (44%), mp > 200 °C. Anal. calcd. for C144H100N17S16Pr (2719.30): C 63.53; H 3.70; N 8.75; S 18.85%. Found C 63.87; H 4.01; N 8.65; S 18.33. FT-IR: ν, cm-1 3100, 3068, 2960, 2930, 2872, 1610, 1527, 1484, 1445, 1407, 1386, 1349, 1292, 1234, 1200, 1094, 1036, 894, 849, 832, 765, 752, 696. UV-vis (DMF): λmax, nm (log ε) 380 (5.37), 627 (5.02), 667 (5.03), 694 (5.73). 1H NMR (400 MHz; CDCl3; Me4Si): δH, ppm -0.34 (8H, m, -CH2), -0.20–0.22 (8H, m, -CH2), -0.04 (12H, t, -CH3), 0.80 (8H, m, -NCH2), 7.12–7.14 (16H, m), 7.24 (16H, m), 7.41–7.42 (16H, m, 3 × thienyl), 9.20 (16H, s, Ar-H). MS (MALDI-TOF(+)): m/z 2478.98 [MH+ – Bu4N].

CONCLUSION

A series of three rare-earth metal(III) bisphthalocyanines (Pc2Gd, Pc2Sm, Pc2Pr) bearing 2-thienyl substituents in the form of tetrabutylammonium salts was synthesized and purified by flash chromatography. The compounds exhibit good solubility in many organic solvents such as DMF, THF, chloroform, dichloromethane and acetone.

The spectra in DMF and chloroform show significant shifts to longer wavelengths compared to unsubstituted compounds and confirmed the anionic form of the prepared compounds. It was found that these compounds are very sensitive to any acid present in solutions. If the acid is neutralized, a reversible oxidation with Br2 was found. It is necessary for future work to avoid even mild acidic conditions.

Surprisingly, a low to moderate production of singlet oxygen generated with a red laser light was found. Apparently, the presence of thiophene substituents increases the ability of the molecules to produce singlet oxygen.

The electrochemical investigation of the title compounds has shown that the variation of central metal

in the studied phthalocyanines does not bring big changes in the first oxidation (reduction) and HOMO (LUMO) band, respectively. Anyway, in comparison to previously published electrochemical data [24], the introduced compounds proceed in easier oxidation (reduction), hence they exhibit smaller gap between first oxidation and reduction which is possible to correlate with the HOMO–LUMO gap.

Acknowledgements

This work was supported by the Czech Science Foundation, Grant no. 14-10279S.


A full list of IR spectrum of Pc2Pr, MALDI-TOF, UV-vis and electrochemical data for Pc2Pr, Pc2Gd, Pc2Sm (Figs S1–S14) is given in the supplementary material. This material is available free of charge via the Internet at http://www.worldscinet.com/jpp/jpp.shtml.

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3. Bouvet M, Gaudillat P and Suisse J-M. J. Porphy-rins Phthalocyanines 2013; 17: 628–635.

4. Marci G, Garcia-Lopez E, Mele G, Palmisano L, Dyrda G and Slota R. Catalysis Today 2009; 143: 203–210.

5. Nicholson MM. Phthalocyanines 1993; 3: 71–117. 6. Ishikawa N, Sugita M, Ishikawa T, Koshihara

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7. Lind SJ, Gordon KC, Gambhir S and Officer DL. Phys. Chem. Chem. Phys. 2009; 11: 5598–5607.

8. Wang R, Zhao Y, Zhu Ch and Huang X. J. Heter. Chem. 2015; 52: 1230–1233.

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DOI: 10.1142/S108842461750002X



INTRODUCTION

Carbon acids of metal complexes tetrapyrrolic macroheterocyclic compounds have solubility in water and buffer media. This fact combined with ability to coordinatively interact with biomolecules is used in novel methods of PDT, for example sonodynamic therapy [1] where joint action of sensitizer and supersonic waves is used. Non-covalent interaction between biopolymers and phthalocyanines containing hydrophobic and hydrophilic groups is promising for creation of novel sensitizers for PDT [2, 3]. Besides, catalytic activity in redox processes of mercaptans and olefins of these compounds is also promising [4–7].

Catalysts obtained by immobilization of metallo-phthalocyanines on solid-phase carriers, carbon nano-tubes, organic polymers are also perspective [8–10]. Carbon residues of phthalocyanine molecule may be used as anchoring groups for linking with biological

molecules containing fragments of primary amines [11], immobilization on polymer surface, for example silica [12] or nanotubes [13] that may be used to create novel materials for purposes mentioned above. Thus, investigations of phthalocyanine macrocycle structure expansion are actively continuing at the moment [14, 15].

The possibility to synthesize non-symmetrical difunc - tional phthalocyanines with specific location of substi-tuents on the periphery allows to control their physical and chemical properties and consequently significantly expand the applications range.

There is data about carboxyl-substituted metallo-phthalocyanines, in which the carboxyl-group is linked directly with isoindole fragment and via phenoxy-, phenylamino- or phenylsulfanyl groups in literature [16–19]. However, they are primarily concerned the tetrasubstituted and octasubstituted phthalocyanines containing symmetrical substituents.

Meanwhile, the study of influence of the substituent located in ortho-position towards benzoic acid fragment electronic and structural effects on physical and chemical properties of these phthalocyanine derivatives is beneficial.

Symmetrical and difunctional substituted cobalt

phthalocyanines with benzoic acids fragments: Synthesis

and catalytic activity

Artur Vashurin*a,c◊, Vladimir Maizlishb, Ilya Kuzmina, Serafima Znoykob,

Anastasiya Morozovab, Mikhail Razumova,c and Oscar Koifmana

a Research Institute of Macroheterocycles of Ivanovo State University of Chemistry and Technology, Ivanovo 153000, Russia b Department of Technology of Fine Organic Synthesis, Ivanovo State University of Chemistry and Technology, Ivanovo 153000, Russia c Kazan Federal University, Kazan, 420008, Russia

Received 4 July 2016Accepted 22 October 2016

ABSTRACT: Difunctional and symmetric phthalonitriles were synthesized by nucleophilic substitution of brome and nitro-group in 4-bromo-5-nitro-phthalonitrile for residues 4-amino-, 4-hydroxyl- and 4-sulfanyl benzoic acid. Symmetrical and difunctional substituted cobalt phthalocyanines were obtained by template synthesis based on mentioned phthalonitriles. Their spectral properties and catalytic activity in aerobic oxidation of sodium N,N-carbomoditiolate were investigated.

KEYWORDS: 4-bromo-5-nitro-phthalonitrile, benzoic acids, synthesis, cobalt phthalocyanines, catalysis, oxidation.


*Correspondence to: Artur Vashurin, email: [email protected]


38 A. VASHURIN ET AL.

Introduction of the substituent may lead to cardinal changes in the catalytic activity of the compounds.

Current work is devoted to the synthesis and study of catalytic properties of symmetric and difunctionally substituted derivatives of cobalt phthalocyanine contai-ning four to eight fragments of carbon acids included in peripheral substituents.


Synthesis of asymmetrical nitriles

Nucleophilic substitution of bromine atom in 4-bromo-5-nitrophthalonitrile (1) on benzoic acid fragment was used to synthesize compounds (2–4) (Scheme 1).

The synthesis was carried out in DMF in presence of anhydrous potassium carbonate under the temperature of 25–35 °C for 2 h. Reaction for 4-((4,5-dicyano-2-nitrophenyl)amino)benzoic acid (4) was performed in presence of triethylamine. Extraction of compounds 2 and 3 was carried out by acidification of reaction mixture with 5% solution of hydrochloric acid. The formed precipitate was filtered and washed with the solution of water and HCl (0.7%). During the synthesis the precipitate of dinitrile 4 was formed and then filtered off. Additional precipitation of the dinitrile 4 from the reaction mixture was done by transfer of the filtrate to bidistilled water, wherein the nitrile precipitate was formed and then filtered. Next the desired product was washed with acidificated water. The yields of 2–4 compounds were 34–63%. Composition and structure of nitriles (2–4) were confirmed by elemental analysis, 1H and 13C NMR and IR spectroscopies, mass-spectrometry.

IR spectra of all phthalonitriles have the band of cyano-groups valent vibrations in range of 2227–2237 cm-1 [20]. IR spectra of all synthesized compounds have vibration bands of –OH and –C=O bonds of benzoic acids carboxyl fragments in range of 3413–3418 cm-1 and 1708–1725 cm-1 respectively. There are bands of symmetrical 1346–1383 cm-1 and asymmetrical 1523–1558 cm-1 vibrations of nitro-groups N=O bonds for compounds (2–4). There is band of valent vibrations of the Ar–S–Ar bond on 1117 cm-1 in IR spectrum of phthalonitrile 3. For the phthalonitrile 4 containing para-aminobenzoic acid fragment the bands of valent

3195 cm-1 and deformation 1613 cm-1 vibrations of secondary amino N–H bond are observed. Valent vibration of Ar–O–Ar bond in range of 1245–1254 cm-1 is registered in IR spectrum of compound 2 that is in agreement with known data [20].

13C NMR spectra of all studied compounds have signals of carbon atoms on 167–168 ppm, which, in accordance with theoretically calculated spectra (Table 1) and known literature [21], have to be attributed to the carbon atoms of the carboxyl groups. Besides, there are signals of cyano carbon atoms on 109–115 ppm and on 107–115 and 120–126 ppm is cyano-substituted carbon atoms.

The influence of the bridge-group nature on position of carbon atom signal located in ortho-position towards the benzoic acid fragment is detected. Thus, the signals of nitro-substituted carbon atoms are situated on 120 ppm for the compounds 2 with hydroxyl-benzoic acid fragment, on 131 ppm for the compound 4 with amino-benzoic acid fragment and on 134 ppm in the NMR spectrum of the compound 3 with sulfanylphenyl-benzoic acid fragment.

The nature of bridge-group also has an effect on the position of C9 carbon atom signal (Table 1). Thus, the C9 atom has a shift from 145 to 159 ppm under the transformation from the phthalonitrile contain-ing residue of para-sulfanylbenzoic acid (3) to the compound (2) having 4-carboxyphenoxy-group. para-Carboxy phenylaminosubstituted phthalonitrile has an intermediate position (150 ppm).

1H NMR spectra of the compounds (2–4) have signals of the protons of phthalonitrile fragment benzyl rings and residue of corresponding benzoic acid in the range of weak field (Table 1). There is no proton signal of carboxyl-group in 1H NMR spectrum for the compounds (3, 4) due to exchange of the proton on deuterium atom of the solvent.

Synthesis of symmetric nitriles

The 4-bromo-5-nitrophthalonitrile (1) was used as init-ial compound to obtain 4,4′-((4,5-dicyano-1,2-phenylene)bis(oxy))dibenzoic acid (5), 4,4′-((4,5-dicyano-1,2-phenylene)bis(sulfanediyl))dibenzoic acid (6) and 4,4′-((4,5- dicyano-1,2-phenylene)bis(azanediyl))dibenzoic acid (7). Reaction was performed by nucleo philic substitution of the brome atom and nitro-group according to the Scheme 2. Synthesis was carried out in aqueous DMF 3:5 in presence of anhydrous potassium carbonate. Reaction was done under 100 °C during 24 h for simultaneous substitution of brome atom and nitro-group contained in 1 on benzoic acid fragment. Triethylamine was used in case of the compound 7 con taining para-aminobenzoic acid fragment as substituent.

Extraction of the compounds 5 and 6 was performed by pouring of the reaction mixture into 5% aqueous solution of hydrochloric acid. The formed precipitate was filtered off and washed with distilled water. The yields of the compounds 5 and 6 were 72 and 88% respectively.

CN

CN

Br

O2N

HX COOH CN

CNO2N

X

COOH

X = O (2); S (3); NH (4)1

50 °C, 2 h

Scheme 1. General scheme of bifunctional nitriles synthesis


SYMMETRICAL AND DIFUNCTIONAL SUBSTITUTED COBALT PHTHALOCYANINES WITH BENZOIC ACIDS FRAGMENTS 39

In case of phthalonitrile 7 the reaction mixture was poured in water, the desired product was extracted by chloroform and then the organic layer was exposed to liquid column chromatography on silicagel using chloroform as eluent. The phthalonitrile 7 was dried in vacuum under 70 °C after removing of the solvent. The yield of the desired product was 40%.

It should be noted that the compounds 5 and 6 may be obtained by the introduction of the second fragment of the corresponding derivative of the benzoic acid in the molecules 2 and 3 respectively instead of nitro-groups. Such synthesizes were done and characterized by lower yields of the final product. Herewith, implementation of the synthesis in two steps needs isolation of the

intermediate that increases duration of the synthesis and leads to additional losses during purification. Synthesis of the compound 7 from 4-((4,5-dicyano-2-nitrophenyl)amono)benzoic acid (4) was failed.

IR spectra of all phthalonitriles have the bond of valent vibrations in range of 2229–2237 cm-1 indicating cyano-groups. There is dependence of the position of this band from the nature of the bridge-group consisting of its shift in the field of increase in wave numbers according to the following order: 6 < 7 < 5. For bifunctional phthalonitriles the position of the valent vibrations band of cyano-group also depends on nature of the bridge-group and represents in a different sequence: 2 < 3 < 4.

Besides, spectra of all synthesized compounds have bands of valent vibrations of O–H and C=O bands of carboxy-groups in range of 3417–3457 cm-1 and 1706–1710 cm-1 respectively. There are no bands of N=O vibrations in IR spectra of symmetrically substituted phthalonitriles that indicates completed nucleophilic substitution of nitro-group in the compound 1. IR spectrum of the compound 6 shows band of valent vibrations of Ar–S–Ar bond on 1104 cm-1. For the compound 7 containing para-aminobenzoic acid fragment bands of valent 3195 cm-1 and deformation 1604 cm-1 vibrations of N–H bond of the secondary amino-group are registered. IR spectrum for the compound 5 has band of valent vibrations of Ar–O–Ar bond on 1220 cm-1.

Synthesis of phthalocyanine cobalt complexes

Cobalt complexes of substituted phthalocyanines were obtained by template method under heating of corresponding phthalonitrile (2–7) with cobalt chloride in presence of urea under the temperature of 190 °C for solidification of the reaction mixture according to Scheme 3.

Reaction mixture was cooled after the finish of the process, then stirred and transferred on filter, where it was washed first with acidified water to remove the products of interaction of urea and excess of cobalt chloride, then with acetone. Next the precipitate was

Table 1. Chemical shift in 13C 1H NMR spectra of bifunctional-substituted phthalonitriles, DMSO (d6), standart TMS

Number δ, ppm

X = O (2) X = S (3) X = NH (4)

Calcd. Exp. Calcd. Exp. Calcd. Exp.

C1 112 115 114 115 115 115

C2 110 109 110 113 110 111

C3 121 120 121 126 120 120

C4 109 109 113 115 107 107

C5 128 131 129 131 112 111

C6 143 144 129 131 150 150

C7 160 160 147 146 137 138

C8 123 120 133 134 130 131

C9 157 159 145 145 150 150

C10 118 116 129 131 124 120

C11 131 131 131 129 124 120

C12 124 128 131 128 122 120

C13 168 168 169 167 168 168

H1 8.85 8.83 9.10 9.00 9.13 8.85

H2 7.78 7.61 7.70 7.74 8.05 8.07

H3 7.87 7.85 7.83 7.95 7.34 7.61

H4 8.08 8.07 8.15 8.07 7.77 7.85

H5 11.72 9.10 11.72 — 10.20 —

–NH — — — — 10.20 8.83

CN

CN

Br

O2N

HX COOH CN

CNX

X

COOH

X = O (5); S (6); NH (7)

1

100 °C, 24 h

COOH

Scheme 2. General scheme of symmetrical nitriles synthesis



dried. Desired compounds were extracted by DMF, the solvent was removed. Obtained products are powders of dark-green color, insoluble in acetone, chloroform, partially soluble in water and well-soluble in aqueous-alkaline solutions. Solubility in DMF of Co(X)Pc4-6 is higher than Co(X)Pc1-3 caused by presence of additional centers of solvation presented by four carboxyl groups.

The band of valent vibrations of cyano-groups in range of 2225–2240 cm-1 [20] in IR spectra of synthesized cobalt phthalocyanines is not registered. This fact indicates absence of impurity of the starting compounds in investigated samples. There are bands of valent vibrations of functional groups bonds in all spectra of the synthesized macroheterocycles, which were registered under analysis of initial phthalonitiles (2–7) IR spectra. It suggests that they are still contained in molecules of obtained cobalt phthalocyanines. The shift of the C=O bonds position under transformation from phthalonitriles to phthalo-cyanines towards lower wave numbers is observed.

Electronic absorption spectra of Co(X)Pc in aqueous mediums (Fig. 1) have diffuse character that is character-istic for associated macrocycle forms. Previously [22] we have studied tetraderivative analogues of investigated compounds, which have no peripheral substituent in fifth position of annulated benzene ring. It was established that the nature of hetero-bridge affects aggregation processes in water-alkali media. Thus, there was no association for tetracarboxyl derivative containing amino-bridge observed, whereas it is predominant for phthalocyanine containing thio-bridge [22].

The nature of bridge heteroatom (X) has almost no influence on the position of long-wavelength absorption bands Co(X)Pc (Table 2).

Transition from aqueous and organic solutions to concentrated sulfuric acid is accompanied by bathochromic

shift of long-wavelength absorption bands. Comparison of UV-vis spectra of phthalocyanines combining fragments of benzoic acids and nitro-groups have showed that the value of bathochromic shift of Q-band under transition from DMF to concentrated sulfuric acid depends on the nature of bridge hetero-atom binding benzoic acid fragment and phthalocyanine molecule. The value of bathochromic shift of Q-band decreases in the following order Δλ Co(S)Pc2 > Co(NH)Pc3 > Co(O)Pc1.

An apparent aggregation of Co(NH)Pc3 leads to the conclusion about redistribution of electronic effects of substituents towards conjugated macrocycle π-system in case of octa-substituted phthalocyanines in contradistinction to tetra-derivatives. Intensification of dimerization of macrocycles containing nitro-groups as peripheral substituents is caused by electron acceptor properties of nitro-group introduced in ortho-position

CN

CN

X

COOH

RN

N

N

N

N

N

N

N

X

R X

R

X

RX

R

Co

2-7

HOOC

COOH

COOH

HOOCCoCl2x6H2O, 190 °C

O

H2N NH2

R = NO2 X = O (Co(O)Pc1); S (Co(S)Pc2); NH (Co(NH)Pc3)

X

COOH

or R = X = O (Co(O)Pc4); S (Co(S)Pc5); NH (Co(NH)Pc6)

Co(X)Pc

Scheme 3. General scheme of phthalocyanines synthesis

Figure 1. UV-vis spectra of Co(X)Pc (c 4.5 × 10-6 M) in DMF: (1) Co(O)Pc4, (2) Co(S)Pc5, (3) Co(NH)Pc6 at 298.15 K



relatively to the second substituent namely, the constri-ction of the electron density from the macrocyclic system and amplification of π–σ contraction and π–π repulsion processes. Obviously, this effect is not observed without strong electron acceptor group in macrocycle.

Based on these considerations we can conclude that these effects have to be aligned in phthalocyanines containing symmetrical peripheral substituents.

The law of Lambert–Bouguer–Beer is not observed in UV-vis spectra (Fig. 1) of Co(X)Pc4-6 solutions that also indicates aggregation of these compounds in solutions. The nature of bridge-heteroatom in that case significantly affects the position of absorption long-wavelength of the compounds (Table 2). Thus it was found that on condition of equal concentrations of the macrocycles in DMF solution the Q-band undergoes a bathochromic shift in the following order: Co(S)Pc5 > Co(O)Pc4 > Co(NH)Pc6. Aggregation degree of Co(O)Pc4 is higher compared to other studied compounds that is in agreement with our previous results [22] for their tetra-derivative analogues.

UV-vis spectra of ammonia water solution (5%) for Co(S)Pc5 (Fig. 2) has splitting of Q-band and two maximums registered, which have to be attributed to H-aggregates (660 nm) and monomer forms of phthalocyanine (700 nm). The monomer-associate equilibrium for Co(NH)Pc6 is shifted towards molecular forms of macrocycle evidenced by not broadened Q-band at 700 nm.

The position of maximums of Q-band for investigated macrocycles in sulfuric acid is bathochromic shifted. It is found that replacement of CoP(S)c2 nitro-groups by one more fragment of mercaptobenzoic acid under transformation to Co(S)Pc5 causes bathochromic shift for 180 nm. The shift is not so big in case of compounds having hydroxyl- and aminobenzoic acids fragments and it is 40 and 22 nm, respectively.

Catalytic activity in oxidation of N,N-carbomodithiolate

In our previous works [22, 23] it was established that tetra-derivative analogues of investigated compounds (Fig. 3) containing no substituents in ortho-position

Table 2. Position of long-wavelength absorption bands (λmax, nm) in UV-vis spectra of Co(X)Pc solutions at 298.15 K

Macrocycle Solvent

DMF NH4OH (5%) H2SO4

Co(O)Pc1 675 651, 701 772

Co(S)Pc2 608, 680 705 813

Co(NH)Pc3 609, 676 700 807

Co(O)Pc4 680 671 813

Co(S)Pc5 673 660; 700 993

Co(NH)Pc6 687 651; 701 829

Figure 2. UV-vis spectra of Co(X)Pc (c 5.5 × 106 M) in NH4OH 5% aqueous solution: (1) Co(O)Pc4, (2) Co(S)Pc5 at 298.15 K

N

N

N

N

N

N

N

N

X

X

X

X

Co

HOOC

COOH

COOH

HOOC

X = O (CoT(O)Pc); S (CoT(S)Pc); NH (CoT(NH)Pc)

Figure 3. Cobalt complexes with tetrasubstituted phthalocyanines



towards benzoic acid fragments are catalytically active in oxidation of sulfur compounds.

The mechanism of the process was suggested and approved. The limiting step is formation of RS· radicals according to the scheme RS ⋅ CoIIPc ⋅ O-

2 + H2O → RS + H2O ⋅ CoIIIPc ⋅ O2

2-. It was established that increase of electron acceptor ability of spacer bridge in the substituent on periphery of phthalocyanine leads to increase in catalytic activity [22, 23].

As it is seen from the data of Fig. 4 the kinetic curves of sodium N,N-carbomodithiolate (DTC) oxidation in presence of symmetrical octa-substituted phthalocyanines are described by formal kinetic equation of first order in substrate. Obtained kinetic data allowed us to calculate activation parameters for this process (Table 3). It should be noted that the reaction rate increases up to 80 times compared to non-catalytic oxidation [24].

Analysis of obtained results and literature data [25–27] suggests that for Co(X)Pc4-6 the mechanism including triple complex formation with followed elimination of RS· radical from it is realized. The mechanism is presented by Equations 1–5.

⎯⎯→+ ←⎯⎯ i1K- II I II2

.RS (Co Pc) RS Co Pc + Co Pc (1)

−⎯⎯→+ ←⎯⎯i i2

2

KI II2

. . .RS Co Pc O RS Co Pc O (2)

+ ⎯⎯⎯⎯→ +i i3 , slowlyII - III 2-2 2 2 2

. . . .RS Co Pc O H O RS H O Co Pc Ok

(3)

⎯⎯→4kIII 2- II -2 2 2 2

. .2H O Co Pc O 2Co Pc + H O + 2OH (4)

⎯⎯⎯⎯⎯→i Instantaneous2RS RSSR (5)

Based on this mechanism the influence of the peripheral substituent nature on catalytic activity of the macrocycle is definitely depends on heteroatom effects affecting metal cation of phthalocyanine molecule, which in turn determines the stability of triple complex and aggregation degree of macrocycles in solution.

Kinetic equation obtained using Michaelis-Menten formal kinetic describes these systems well and it is linearized in Lineweaver–Burke coordinates giving Equation 6.

=+

� �2 2

-

-

3 1 2 (CoPc) O

RS1 RS

K K.

1 K

k c cr c

c (6)

It should be noted that introduction of four additional benzoic acid fragments leads to intensification of specific solvation of the macrocycle on periphery. Besides, ionization of the macromolecule is strengthened. It leads to decrease of process rate because of competing interaction of the substrate and solvent with active centers of the catalyst. DTC oxidation rate constant decreases in the folloing order of catalysts Co(NH)Pc6 > Co(S)Pc5 ≥ Co(O)Pc4. The order is different from tetra-substituted one CoT(S)Pc > CoT(NH)Pc > CoT(O)Pc that is probably caused by difference in macrocycle association degree. Thus, CoT(NH)Pc in solution is monomeric and all its reaction centers are permanently occupied by solvent [22]. Whereas, there is possibility to compete for active center in associate Co(NH)Pc6.

The catalyst forms intermediate in solutions of high ionization degree of the macrocycle and high containing of OH-. The intermediate has oxidized thiocarbamic acid

Figure 4. Kinetic dependences of DTC oxidation (c 2.6 × 10-3 M) in presence of homogeneous phthalocyanine catalysts (c 5.6 × 10-5 M) (1) Co(NH)Pc6, (2) Co(O)Pc4, (3) Co(S)Pc5 in water-alkali solution (pH 11) at 298.15 K. The dotted line: the result of the formal processing of the kinetic equation

Table 3. Kinetic dependences of DTC oxidation (c 2.6 × 10-3 M) in presence of Co(X)Pc phthalocyanine catalysts (c 5.6 × 10-5 M) in water-alkali solution (pH 11)

Catalyst kw298 × 102, L.(mol.s)-1 E≠, kJ.mol-1 ΔS≠ J.(mol.K)-1 -lnA χ

Co(O)Pc1 7.01 -22.09 -378 8.7 69.05

Co(S)Pc2 26.91 -12.82 -339 7.3 89.42

Co(NH)Pc3 9.57 -22.95 -375 8.5 67.43

Co(O)Pc4 3.82 10.1 -281 9.1 57.71

Co(S)Pc5 4.58 22.4 -236 9.2 68.02

Co(NH)Pc6 17.56 12.1 -245 7.7 53.86



in outer coordination sphere forming hydrogen bond with coordinated oxygen molecule. Then there is transfer of the charge from the metal to oxygen. Increase of pH leads to competing of DTC coordination processes and dimerization due to intensification of last one. This complicates the electron transfer. That is why the authors [28] suggested that electron transfer is implemented in reaction of CoPcII … OH- complex with other hydroxyl-ion (CoPcII … OH-)2 + OH- → CoPcI … CoPcII … OH- + O- + H2O. CoPcI + O2 → CoPcII + O2

-, CoPcII + O2 → CoPcIII + O2-.

There is permanent formation of O2– and CoPcIII oxidized

forms under excess of oxygen in water-alkali system that complicates the triple complex formation on condition of high content of hydroxyl ions in the solution and consequently reduces the activity of the phthalocyanine as a catalyst. It is also a characteristic of investigated by us systems.

Absolutely unexpected results are obtained for Co(X)Pc1-3. There is inhibition of the oxidation under increase of the temperature while maintaining the formal first-order kinetic reaction on the substrate. The calculation of the activation parameters of the system showed negative energies of activation process (Table 3).These data characterizes the process as complex. Obviously, the rate constant of the process (3), which is limiting stage, is increased under increase of temperature. But the presence of strong acceptor groups (NO2) contained in the macrocycle in para-position towards benzoic acid fragments leads to contraction of π-electronic density from central metal cation and decrease of partially positive charge compensation [29]. Macrocycle-solvent coordination interaction compensates this charge.

Increase of activation entropy change indicates the desolvation of activated complex (Fig. 5) and introducing of substrate or oxygen into coordination sphere, which characterizes the transfer towards association-dissociation mechanism of the process.

Obviously, there is an exchange between DTC and coordinated by phthalocyanine ligand (Fig. 5) which can be presented by solvent or molecular oxygen [30]. The exchange depends essentially on electron-donor power of second ligand, for ions — on their basicity.

Wherein, the concentration of associated macrocycle forms participated in reaction (1) decreases under temperature increase. Decrease of dimer concentration is faster than increase of reaction (3) rate constant that leads to decrease of the total rate of the process. The total process rate changes in order CoT(S)Pc > CoT(NH)Pc > CoT(O)Pc, which correlates to the series of tetra-substituted CoT(X)Pc.

EXPERIMENTAL

Equipment

Elemental analysis has been carried out by means of chromatographic analyzer Flash HCNS-OEA 1112 (Germany). The flowrates of helium and oxygen were 140 mL/min and 250 mL/min, respectively; the temperature of the reactor was 1173 K, oxygen was supplied into the reactor for 250 mL/min with 12 s time delay.

FT-IR spectra were recorded using IR-Fourier spectrophotometer Avatar 360 (USA) in 400–4000 cm-1 frequency range.

NMR spectra of the solutions were recorded by means of NMR spectrometer Bruker AVANCE-500 (Germany) at operating frequency 500 MHz (1H) and 100 MHz (13C). Measurements were performed under the Fourier transformation conditions in 5 mm cells at various temperatures. Chemical shifts were measured with reference to the internal standard — tetramethylsilane (TMS). The accuracy of measurements was ±0.005 ppm.

Electron absorption spectra (UV-vis) were registered by means of Unico 2800 (USA) spectrophotometer in a spectral range of 200–1000 nm. Quarts optical cell were used for the measurements. UV-vis spectra were recorded at 298.15 ± 0.03 K.

Mass spectra were measured on an Axima MALDI-TOF mass-spectrometer (Shimadzu, Japan).

pH values of the solutions were controlled with pH meter S220 Seven Compact (Mettler Toledo, Switzerland). Relative error of pH determining was ±0.002.

Synthesis of bifunctional nitriles

4-bromo-5-nitrophthalonitrile (1) was synthesized according to the method recommended in [31]. Physical and chemical properties of the obtained compound are in good agreement with literature data. mp 140–142 °C (%). Elemental analysis found C, 38.10; N, 16.50; H, 0.68%. Anal. calcd. for (C8H2BrN3O2) C, 38.16; N, 16.67; H, 0.80. IR (KBr): ν, cm-1 2241 (C≡N), 1560 (NO2), 1341 (NO2), 813 (C–Br).

General method. 2.52 g (0.01 mol) 4-bromo-5-nitrophthalonitrile (1) and 0.01 mol of corresponding benzoic acid were dissolved in 50 mL of DMF and placed into two-necked flask equipped with a reflux. The

Co O O

X

C SHS

NR

R

where X = solvent or molecular oxygen

Figure 5. Exchange of ligands



solution of 1.38 g (0.01 mol) of anhydrous potassium carbonate in 7 mL of water was added to the mixture. The reaction mixture was stirred at 25 °C for 1 h. Obtained precipitate was filtered off and washed with 5% aqueous solution of hydrochloric acid until the colorless filtrate. Then it was dried on air under 70–80 °C. Extraction was performed by acidification of the reaction mixture by 5% solution of hydrochloric acid. The precipitate was filtered and washed with acidified water.

4-(4,5-Dicyano-2-nitrophenoxy)benzoic acid (2). It was synthesized from 1.38 g of para-hydroxybenzoic acid. The yield is 1.76 g (57%). Elemental analysis found C, 59.02; H, 2.32; N, 13.30%. Anal. calcd. for (C15H7N3O5) C, 58.26; H, 2.28; N, 13.59. IR (KBr): ν, cm-1 3014 (OH), 2231 (C≡N), 1719 (C=O), 1558 (NO2), 1380 (NO2), 1254 (Ar–O–Ar). MS (MALDI-TOF): m/z 309.29 [M], calcd. [M] 309.24.

4-((4,5-Dicyano-2-nitrophenyl)thio)benzoic acid (3). It was synthesized from 1.54 g of para-phenylsulfanylbenzoic acid. Yield 1.12 g (34%). Elemental analysis found C, 55.25; H, 2.02; N, 13.00%. Anal. calcd. for (C15H7N3O4S) C, 55.38; H, 2.17; N, 12.92. IR (KBr): ν, cm-1 3417 (OH), 2232 (C≡N), 1725 (C=O), 1550 (NO2), 1383 (NO2), 1117 (Ar–S–Ar). MS (MALDI-TOF): m/z 324.71 [M–H]-, calcd. [M] 325.65.

4-((4,5-Dicyano-2-nitrophenyl)amino)benzoic acid (4). It was synthesized from 1.37 r para-aminobenzoic acid in presence of 2 mL of triethylamine. Yield 1.96 g (63%). Elemental analysis found C, 58.20; H, 3.00; N, 18.10%. Anal. calcd. for (C15H7N4O4) C, 58.45; H, 2.62; N, 18.18. IR (KBr): ν, cm-1 3418 (OH), 3195 (NH), 2237 (C≡N), 1708 (C=O), 1613 (NH), 1523 (NO2), 1406 (NH), 1346 (NO2). MS (MALDI-TOF): m/z 332.01 [M + Na]+, calcd. [M] 308.25.

Synthesis of symmetrical nitriles

General methods. I variant. 2.52 g (0.01 mol) of 4-bromo-5-nitrophthalonitrile (1) and 0.02 mol of corresponding benzoic acid were dissolved in 50 mL of DMF and placed into two-necked flask equipped with reflux. The solution of 5.52 g (0.04 mol) of anhydrous potassium carbonate in 7 mL of water was added to the mixture. The reaction mixture was stirred under 100 °C for 24 h, then poured onto acidic water and desired product was collected from the filter. The precipitate obtained was filtered of and washed with 5% aqueous solution of hydrochloric acid until colorless filtrate.

4,4 ′-((4,5-Dicyano-1,2-phenylene)bis(oxy))dibenzoic acid (5). It was synthesized from 2.76 g of para-hydroxybenzoic acid. Yield 2.88 g (72%). Elemental analysis found C, 64.59; H, 3.12; N, 6.83%. Anal. calcd. for (C22H12N2O6) C, 66.00; H, 3.02; N, 7.00. 1H NMR (DMSO-d6): δ, ppm 10.58 s (COOH, 2H), 7.66 s (H1, 2H), 6.96–7.06 d (H2, 4H, J 2.01 Hz), 8.03–8.12 d (H3, 4H, J 2.05 Hz). IR (KBr): ν, cm-1 3457 (OH); 2237

(C≡N); 1708 (C=O); 1220 (Ar–O–Ar). MS (MALDI-TOF): m/z 399.30 [M–H]-; calcd. [M] 400.07.

4,4′-((4,5-Dicyano-1,2-phenylene)bis(sulfanediyl))dibenzoic acid (6). It was synthesized from 3.08 g of para-mercaptobenzoic acid. Yield 3.80 g (88%). Elemental analysis found C, 60.95; H, 2.93; N, 6.15; S, 14.35%. Anal. calcd. for (C22H12N2O4S2) C, 61.10; H, 2.80; N, 6.48; S, 14.83. 1H NMR (DMSO-d6): δ, ppm 10.55 s (COOH, 2H), 7.88 s (H1, 2H), 7.57–7.60 d (H2, 4H; J 2.05 Hz), 7.96–8.02 d (H3, 4H; J 2.04 Hz). IR (KBr): ν, cm-1 3417 (OH); 2229 (C≡N); 1710 (C=O); 1104 (Ar–S–Ar). MS (MALDI-TOF): m/z 431.28 [M–H]-; calcd. [M] 432.02.

4,4′-((4,5-Dicyano-1,2-phenylene)bis(azanediyl))dibenzoic acid (7). It was synthesized from 2.74 g of para-aminobenzoic in presence of 2 mL of triethylamine. After finish of the synthesis the desired compound was extracted by pouring of the reaction mixture onto water and then adding chloroform. Next the chloroform was separated from water part on separating funnel, after this the solvent was removed. The compound was column chromatographed on silica using chloroform as eluent. Yield 1.59 g (40%). Elemental analysis found C, 65.20; H, 3.33; N, 14.10%. Anal. calcd. for (C22H14N4O4) C, 66.33; H, 3.54; N, 14.06. 1H NMR (DMSO-d6): δ, ppm 8.58 s (COOH, 2H), 8.51 s (NH, 2H), 8.01 s (H1, 2H), 7.52–7.53 d (H2, 1H; J 2.05 Hz), 7.62–7.63 d (H3, 2H; J 2.01 Hz). IR (KBr): ν, cm-1 3418 (OH), 3195 (NH), 2234 (C≡N), 1706 (C=O), 1604 (NH), 1434 (NH). MS (MALDI-TOF): m/z 399.34 [M + H]+; calcd. [M] 398.38.

General methods. II variant. 0.1 mmol of nitrile (2–3) and 0.1 mmol of corresponding benzoic acid were dissolved in 5 mL of DMF and placed into two-necked flask equipped with reflux. The solution of 0.14 g (0.1 mmol) of anhydrous potassium carbonate in 0.7 mL of water was added to the mixture. It was stirred under 80–90 °C for 24 h. The precipitate obtained was filtered off and washed with aqueous solution of hydrochloric acid (5%) until colorless filtrate. Then it was dried on air under 70–80 °C.

The compound (7) was not obtained by this method.4,4 ′-((4,5-Dicyano-1,2-phenylene)bis(oxy))

dibenzoic acid (5). It was synthesized from 0.31 g of 4-(4,5-dicyano-2-nitrophenoxy)benzoic acid (2) and 0.14 g of para-hydroxybenzoic acid. Yield 0.21 g (52%). Elemental analysis found C, 64.63; H, 3.14; N, 6.67%. Anal. calcd. for (C22H12N2O6) C, 65.70; H, 3.02; N, 7.00. IR (KBr): ν, cm-1 3454 (OH); 2237 (C≡N); 1708 (C=O); 1220 (Ar–O–Ar).

4,4′-((4,5-Dicyano-1,2-phenylene)bis(sulfanediyl))dibenzoic acid (6). It was synthesized from 0.33 g 4-((4,5-dicyano-2-nitrophenyl)thio)benzoic acid (3) and 0.15 g para-mercaptobenzoic acid. Yield 0.27 g (62%). Elemental analysis found C, 60.90; H, 2.90; N, 6.24; S, 14.52%. Anal. calcd. for (C22H12N2O4S2) C, 61.10; H, 2.80; N, 6.48; S 14.83. IR (KBr): ν, cm-1 3417 (OH); 2229 (C≡N); 1710 (C=O); 1104 (Ar–S–Ar).



Synthesis of cobalt complex of phthalocyanines

General method. Carefully stirred mixture of 0.33 mmol of corresponding substituted phthalonitrile, 54 mg (0.20 mmol) of cobalt chloride hexahydrate and 60 mg (1 mmol) of urea was held under the temperature of 190–195 °C until solidifying of the reaction mixture. Next, it was stirred, washed with acidic water and acetone, then dried on air under 70–80 °C.

Cobalt tetra-4-nitro-tetra-5-(4-carboxyphenoxy)phthalocyaninate (Co(O)Pc1). It was synthesized from 101 mg of phthalonitrile 2. Yield 62 mg (58%). Elemental analysis found C, 55.10; H, 2.54; N, 13.15%. Anal. calcd. for (C60H28N12O20Co) C, 55.61; H, 2.18; N, 12.97. IR (KBr): ν, cm-1 3463 (OH), 1722 (C=O), 1509 (NO2), 1384 (NO2), 1263 (Ar–O–Ar). MS (MALDI-TOF): m/z 1294.90 [M]-, calcd. [M] 1295.09.

Cobalt tetra-4-nitro-tetra-5-(4-carboxyphenoxyul-phanyl)phthalocyaninate (Co(S)Pc2). It was synthe-sized from 108 mg of phthalonitrile 3. Yield 67 mg (60%). Elemental analysis found C, 53.17; H, 2.20; N, 12.00%. Anal. calcd. for (C60H28N12O16S4Co) C, 52.98; H, 2.07; N, 12.36. IR (KBr): ν, cm-1 3440 (OH), 1719 (C=O), 1589 (NO2), 1404 (NO2) 1194 (Ar–S–Ar), 1050 (N=N), 744 (C–N). MS (MALDI-TOF): m/z 1382.18 [M + Na], calcd. M 1359.01.

Cobalt tetra-4-nitro-tetra-5-(4-carboxyphenyla mino)phthalocyaninate (Co(NH)Pc3). It was synthe-sized from 101 mg of phthalonitrile 4. Yield 51 mg (48%). Elemental analysis found C, 56.00; H, 2.38; N, 18.00%. Anal. calcd. for (C60H32N16O16Co) C, 55.78; H, 2.50; N, 17.35. IR (KBr): ν, cm-1 3432 (OH), 3192 (NH), 1703 (C=O), 1379 (NO2). MS (MALDI-TOF): m/z 1326.36 [M + 2H2O – H]-, calcd. [M] 1291.15.

Cobalt octa-4,5-(4′-carboxyphenoxy)phthalocyani-nate (Co(O)Pc4). It was synthesized from 120 mg of phthalonitrile 5. Yield 82 mg (60%). Elemental analysis found C, 63.10; H, 3.04; N, 6.55%. Anal. calcd. for (C88H48N8O24Co) C, 63.66; H, 2.91; N. 6.75. IR (KBr): ν, cm-1 3443 (OH); 1712 (C=O); 1224 (Ar–O–Ar). MS (MALDI-TOF): m/z 1676.17 [M + H2O – H]+; calcd. [M] 1659.31.

Cobalt octa-4,5-(4′-carboxyphenylsulfanyl)phtha-lo cyaninate (Co(O)Pc5). It was synthesized from 130 mg of phthalonitrile 6. Yield 95 mg (64%). Elemental analysis found C, 59.17; H, 2.82; N, 6.10%. Anal. calcd. for (C88H48N8O16S8Co) C, 59.09; H, 2.70; N, 6.26. IR (KBr): ν, cm-1 3447 (OH), 1711 (C=O), 1104 (Ar–S–Ar). MS (MALDI-TOF): m/z 1789.78 [M + 2H]+; calcd. [M] 1787.03.

Cobalt octa-4,5-(4′-carboxyphenylamino)phthalo-cy aninate (Co(O)Pc6). It was synthesized from 120 mg of phthalonitrile 7. Yield 50 mg (37%). Elemental analysis found C, 63.70; H, 3.55; N, 13.18%. Anal. calcd. for (C88H56N16O16Co)C, 63.96; H, 3.42; N, 13.56. IR (KBr): ν, cm-1 3413 (OH); 3172 (NH); 1703 (C=O); 1602 (NH); 1434 (NH). MS (MALDI-TOF): m/z 1652.36 [M + H]+; calcd. [M] 1651.34.

Studies of catalytic activity

The catalytic activity of metallophthalocyanines was tested with a known [32, 33] reaction of sodium N,N-diethylcarbamodithioate (DTC) oxidation. It proceeds according to general scheme:

Advantages of this reaction are low toxicity of initial materials and possibility of monitoring of concentration

of initial and desired materials and identification of reaction products using electron absorption (UV-vis spectra) and FT-IR spectroscopy methods. Experiments to study the kinetics of DTC oxidation were carried out in thermostatic cell in which the solution of DTC of 650 mL was loaded.

The air needed for oxidation was fed via micro compressor with constant rate of 2 L.min-1. The reaction takes place in kinetic region under these parameters [23]. After establishing a constant temperature of reaction mixture, it was stirred and sample of 2 mL was taken to determine initial concentration of DTC, then compressor was turned on. This moment was taken as the beginning of the reaction. Samples of 2 mL were taken periodically during the experiment to determine current concentration

of DTC. The concentration of DTC was monitored by spectrophotometric method.

Under conditions of constant concentrations of oxygen and catalyst, constant pH of solution the rate of DTC oxidation is described by first order kinetic equation:

∂=

∂ obs DTC-c

k ct

(7)

where kobs — observed constant of the rate, s-1.It is confirmed by straightness of graphics in

coordinates lnc — t and constancy of rate constants calculated according to the equation:

=0

obs

ln c

ckt

(8)

N C

SNa

SC2H5

C2H5

2 + 1/2 O2 + H2O N C

S

SC2H5

C2H5

NC

S

S C2H5

C2H5

+ 2 NaOHcat

Scheme 4. Oxidation of DTC



where C0 — initial concentration of DTC, c — current (t) concentration of DTC.

The rate constants of (n + 1)-order were calculated using Equation 9.

=298 obs

DTCw i

kk

c (9)

The activation energy (E≠) for the studied temperature range was calculated by the Arrhenius equation in the integrated form:

≠ ⎛ ⎞= ⎜ ⎟⎝ ⎠

1 2 2

2 1 1

19.1 lg-

T T kE

T T k (10)

where T1 and T2 — temperatures and k2 and k1 — observed rate constants at current temperatures.

Entropy change for the formation of transition state ΔS≠ was calculated with Equation 11:

≠≠Δ = + −298S 19.1ln 253

298w

Ek (11)

Degree of transformation was calculated according to Equation 12:

= (c0–cτ)/c0 (12)

where C0 — initial concentration of DTC, cτ — current concentration of DTC.

Disulfide formation was monitored with FT-IR spectra, and 1H NMR, and 13C NMR. 1H NMR of DTC oxidation (500 MHz, D2O): δ, ppm 4.32 (m, J = 15 Hz, 4H, CH2); 1.39 (t, J = 5 Hz, 6H, CH3).

13C NMR of DTC oxidation (100 MHz): δ, ppm 11.65, 27.50, 49.11, 205.02. IR of DTC oxidation IR (KBr): ν, cm-1 2979 (–CH3 υas), 2847 (–CH2–υas), 1476 (–CH2–δ), 1378 (–C–N st), 1269 (–C = S st), 1075, (d, –C–S).

During oxidation of DTC the formation of diethylcarbamothioylsulfanyl-N,N-diethylcarbamodi-thioate (Thiuram E) is observed. 1H NMR of Thiuram E obtained by DTC oxidation (500 MHz, CDCl3): δ, ppm 3.71–3.77 (m, 8H, CH2); 1.29–1.23 (m, 12H, CH3).

13C NMR of Thiuram E obtained by DTC oxidation (100 MHz): δ, ppm 10.49, 25.72, 52.04, 51.26, 190.05. IR of Thiuram E obtained by DTC oxidation IR (KBr): ν, cm-1 2975 (–CH3 υas), 2861 (–CH2–υas), 1505 (–CH2–δ), 1380 (–C–N st), 1273 (–C=S st), 1143, 995 (–S–S–).

CONCLUSION

Bifunctional- and symmetrical substituted phthalo-nitriles and cobalt phthalocyanines based on it were synthesized in the work by nucleophilic substitution of brome in 4-bromo-5-nitrophthalonitrile on residues of benzoic acids. All synthesized macrocycles have catalytic activity in oxidation of N,N-carbomodithiolate. The nature

of peripheral substituents affects rate and mechanism of the oxidation significantly. Introduction of electron acceptor groups in periphery of the phthalocyanine molecule leads to increase of catalytic activity.

Acknowledgements

The work was supported by Russian Science Foundation project 14-23-00204 and partially supported by Russian President’s grant for young scientists — PhD (MK-2776.2015.3).

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DOI: 10.1142/S1088424617500043



INTRODUCTION

In our laboratory, we have developed a new type of “flying-seed-like liquid crystals” since 2006 [1–6]. Very recently, we have synthesized novel phthalocyanine derivatives, [(x-C1)PhO]8PcCu (x = p, o, m: 1a–1c in Fig. 1), substituted by eight phenoxy groups having methoxy groups on their para, ortho, or meta positions

[4]. As a result, [(p-C1)PhO]8PcCu (1a) shows no mesophase, whereas [(o-C1)PhO]8PcCu (1b) shows a monotropic Colro(P2m) mesophase; [(m-C1)PhO]8PcCu (1c) shows two enantiotropic Colro(P21/a) columnar mesophases in a wide temperature region. Thus, we have revealed that mesomorphism could be induced by these novel bulky substituents instead of using long alkyl chains, and that appearance of the mesomorphism was greatly affected by the position of methoxy groups. Especially, it is very interesting that the derivative 1c having a methoxy group at the meta position tends to show enantiotropic mesophases, whereas the derivative 1a having a methoxy group at the para position tends to show no mesophase.

Therefore, in this study, we have synthesized novel octakis-(m-chloropyridyloxy)phthalocyaninato copper(II) complexes, [x-PyO(m-Cl)]8PcCu(x = 2, 3, 4: 2a–2c

Flying-seed-like liquid crystals 7†: Synthesis and

mesomorphism of novel octakis(m-chloropyridyloxy)

phthalocyanato copper(II) complexes

Kazuchika Ohta*a◊, Kaori Adachia and Mikio Yasutakeb

a Smart Material Science and Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 1-15-1 Tokida, Ueda, 386-8567, Japan b Comprehensive Analysis Center for Science, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan

Received 3 October 2016Accepted 29 October 2016

ABSTRACT: We have synthesized three novel octakis(m-chloropyridyloxy)phthalocyaninato copper(II) complexes, [x-PyO(m-Cl)]8PcCu(x = 2, 3, 4: 2a–2c), keeping a chlorine atom at the meta position on the 2-, 3- and 4-pyridyloxy group, in which the nitrogen atom is located at the 2-, 3- and 4-positions, respectively. Their phase transition behavior and the mesophase structure have been established by using a polarizing optical microscope, a differential scanning calorimeter, and a temperature-dependent small angle X-ray diffractometer. Very interestingly, the mesomorphism appears with strong dependence of the position of nitrogen in m-chloropyridyloxy group. The derivative [3-PyO(m-Cl)]8PcCu (2b) introduced a nitrogen atom at the 3-position is not mesogenic but crystalline. On the other hand, the derivative [2-PyO(m-Cl)]8PcCu (2a) introduced a nitrogen atom at the 2-position shows columnar mesomorphism only at very high temperatures over 325 °C. The derivative [4-PyO(m-Cl)]8PcCu (2c) introduced a nitrogen atom at the 4-position shows columnar mesomorphism in a very wide temperature region from rt to the decomposition temperature at 306 °C. From the viewpoint of N…Cl halogen bond, we have discussed about relationship between their mesomorphism and the position of nitrogen atom in m-chloropyridyloxy group.

KEYWORDS: flying-seed-like liquid crystals, metallomesogen, phthalocyanine, columnar mesophase.


*Correspondence to: Smart Material Science and Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 1-15-1 Tokida, Ueda, 386-8567, Japan. E-mail: [email protected]; Tel & FAX: +81-268-21-5492.† Part 6: Ref. 6 in this paper.


FLYING-SEED-LIKE LIQUID CRYSTALS 7 49

Fig. 1. Appearance of mesomorphism depending on the position of methoxy groups for [(x-C1)PhO]8PcCu (x = p, o, m: 1a–1c). ×: no mesophase appears; : monotropic mesophase appears; : enantiotropic mesophase appears


50 K. OHTA ET AL.

in Scheme 1), keeping a chlorine atom at the meta position on the 2-, 3- and 4-pyridyloxy group, in which the nitrogen atom is located at the 2-, 3- and 4-positions, respectively, and investigated the influence of the position of nitrogen atom in the m-chloropyridyloxy group on their mesomorphism.

EXPERIMENTAL

Synthesis

Scheme 1 shows synthetic route for novel flying-seed-like liquid crystals based on [x-PyO(m-Cl)]8PcCu(2a–2c: x = 2, 3, 4). They were prepared according to our previ-ously reported methods [12]. Phthalonitrile derivatives, [x-PyO(m-Cl)]2FN (3a–3c: x = 2, 3, 4), were synthesized from 4,5-dichlorophthalonitrile and the corresponding m-halogenohydroxy pyridine derivatives, x-PyOH(m-Cl) (x = 2, 3, 4), purchased from Tokyo Kasei. The target phthalocyanine derivatives, [x-PyO(m-Cl)]8PcCu (2a–2c: x = 2, 3, 4), could be synthesized from the corresponding phthalonitrile derivatives, [x-PyO(m-Cl)]2FN (3a–3c: x = 2, 3, 4). The detailed synthetic procedures were described below.

[2-PyO(m-Cl)]2FN (3a). A mixture of 4, 5-dichloro-phthalonitrile (104 mg, 0.525 mmol), 6-chloro-2-pyridine (175 mg, 1.35 mmol) and dry-DMSO (5 mL) was

stirred at 60 °C under a nitrogen atmosphere for 10 min. To the reaction mixture, one sixth of K2CO3 (484 mg, 3.50 mmol) was separately added every 5 min six times. It was heated up to 100 °C and kept the temperature. To promote the reaction, 427 mg (3.09 mmol) and 284 mg (2.05 mmol) of K2CO3 were added after 3.5 h and 6.0 h, respectively. After more than 2.0 h, the reaction mixture was cooled down to rt and extracted with chloroform. The organic layer was washed with water, dried over Na2SO4 and evaporated in vacuum. The residue was purified by column chromatography (silica gel, dichloromethane, Rf = 0.55) to obtain 57.4 mg of pale yellow solid. Yield 28.5%, mp 197 °C. IR (Kbr): ν, cm-1 2237 (–CN). 1H NMR (CDCl3, TMS): δ, ppm 7.79 (s, 2H, Ar), 7.58 (t, J = 9.10 Hz, 2H, Ar), 7.02 (d, J = 8.09 Hz, 2H, Ar), 6.69 (d, J = 8.08 Hz, 2H, Ar).

[3-PyO(m-Cl)]2FN (3b). A mixture of 4,5-dichloro-phthalonitrile (103 mg, 0.523 mmol), 3-chloro-5-pyridine (3-PyOH(m-Cl): 275 mg, 2.12 mmol), dry-DMSO (5 mL) was stirred at 60 °C under a nitrogen atmosphere for 10 min. To the reaction mixture, one sixth of K2CO3 (484 mg, 3.50 mmol) was separately added every 5 min six times. It was heated up to 100 °C and kept the temp-erature. To promote the reaction, 108 mg (0.776 mmol) of K2CO3 was added after 45 min. After more than 45 min, the reaction mixture was extracted with chloroform. The organic layer was washed with water, dried over Na2SO4 and evaporated in vacuum. The

ClCl

NC CN

AO

OA

CN

CN

N

O O

N

NN

N

N M N

N

O

O

O

O

O

O

A A

A

A

A

A

AA

K2CO3

A-OH

N

Cl

N

Cl

N

Cl

A

DMSO

2a-c: [x-PyO(m-Cl)]8PcCu

(x=2, 3, 4)

3a-c: [x-PyO(m-Cl)]2FN

CuCl2, DBU

1-hexanol

: c

: a

: b

2-PyO(m-Cl)

3-PyO(m-Cl)

4-PyO(m-Cl)

x-PyOH(m-Cl)

M = Cu

+

Scheme 1. Synthetic route for [x-PyO(m-Cl)]8PcCu(2a–2c). DMSO = dimethyl sulfoxide and DBU = 1,8-diazabicyclo[5, 4, 0] undec-7-ene



residue was purified by column chromatography (silica gel, dichloromethane: ethyl acetate = 8:2 (v/v), Rf = 0.47) to obtain 177.3 mg of pale yellow solid. Yield 88.7%, mp 179 °C. IR (KBr): ν, cm-1 2237 (–CN). 1H NMR (CDCl3, TMS): δ, ppm 8.45 (d, J = 5.81 Hz, 2H, Ar), 8.22 (d, J = 9.3 Hz, 2H, Ar), 7.32 (s, 2H, Ar), 7.31–7.29 (m, 2H, Ar).

[4-PyO(m-Cl)]2FN (3c). A mixture of 4, 5-dichloro-phthalonitrile (128 mg, 0.661 mmol), 2-chloro-4-hydroxypyridine (4-PyOH(m-Cl): 339 mg, 1.95 mmol), dry- DMSO (5 mL) was stirred at 60 °C under a nitrogen atmosphere for 10 min. To the reaction mixture, a sixth part of K2CO3 (1926 mg, 13.9 mmol) was separately added every 5 min six times. It was heated up to 100 °C and kept the temperature for 9 h. The reaction mixture was cooled down to rt, extracted with chloroform and washed with water. The organic layer was dried over Na2SO4 and evaporated in vacuum. The residue was purified by column chromatography (silica gel, dichloromethane:ethyl acetate = 9:1 (v/v), Rf = 0.67) to obtain 83.9 mg of pale yellow solid. Yield 33.1%, mp 154 °C. IR (KBr): ν, cm-1 2235(–CN). 1H NMR (CDCl3, TMS): δ, ppm 8.40 (d, J = 7.83 Hz, 2H, Ar), 7.63 (s, 2H, Ar), 6.84 (d, J = 2.27 Hz, 2H, Ar), 6.78 (dd, J1 = 6.31, J2 = 2.15, 2H, Ar).

[2-PyO(m-Cl)]8PcCu (2a). A mixture of [2-PyO(m-Cl)]2FN (3a: 91.0 mg, 0.237 mmol), CuCl2 (27.1 mg, 0.201 mmol), 1-hexanol (5 mL), DBU (3 drops) was refluxed under a nitrogen atmosphere for 4 h. After cooling to rt, methanol was poured into the reaction mixture to precipitate the target compound. The precipi tates were collected by filtration and the filtrate

was washed with methanol, ethanol and acetone, successively. The residue was resolved in a small amount of mixed solvents, chloroform:THF = 1:1 (v/v). The solution was poured into a column for the column chromatography (silica gel). The impurities were removed by using the first eluent of chloroform and then the target Pc complex 2a (Rf = 0.0 for chloroform) was collected by using the second eluent of THF. After evaporation of the solvent, the residue was purified by Soxhlet extraction using dry THF. The filtrate was evaporated and dried in vacuo to obtain 18.5 mg of green powder. Yield and MALDI-TOF Mass data: see Table 1. UV-vis spectral data: see Table 2. Phase transition behavior: see Table 3.

[3-PyO(m-Cl)]8PcCu (2b). A mixture of [3-PyO(m-Cl)]2FN (3b: 70.9 mg, 0.185 mmol), CuCl2 (40.3 mg, 0.230 mmol), 1-hexanol (5 mL), DBU (3 drops) was refluxed under a nitrogen atmosphere for 6 h. After cooling to rt, methanol was poured into the reaction mixture to precipitate the target compound. The precipitates were collected by filtration and the filtrate was washed with methanol, ethanol and acetone, successively. The residue was resolved in a small amount of mixed solvents, chloroform:THF = 1:1 (v/v). The solution was poured into a column for the column chromatography (silica gel). The impurities were removed by using the eluent of chloroform. Differently from 2a, the target Pc complex 2b (Rf = 0.0 for chloroform) was so tightly adhered to the silica gel that we could not remove it even by using THF. Therefore, the silica gel absorbed the Pc complex

Table 1. Yields and MALDI-TOF mass spectral data of [x-PyO(m-Cl)]8PcCu: 2a–2c

Compound Yield (%) Mol. formula (average mass) exact mass

Observed exact mass

2a: [2-PyO(m-Cl)]8PcCu 19.5 C72H32Cl8CuN16O8 (1596.30)1590.94

1591.87 (M+1)

2b: [3-PyO(m-Cl)]8PcCu 19.1 C72H32Cl8CuN16O8

(1596.30) 1590.94

1591.03

2c: [4-PyO(m-Cl)]8PcCu 9.12 C72H32Cl8CuN16O8

(1596.30) 1590.94

1591.01

Table 2. Electronic spectral data of [x-PyO(m-Cl)]8PcCu: 2a–2c

Compound Concentration# (X10-6 mol/l)

λmax (nm) (logε)

Soret-band Q-band

2a: [2-PyO(m-Cl)]8PcCu 4.96 274.4 (4.90) 348.1 (4.88) 609.4 (4.52) 634.3* (4.81) 672.6 (5.09)

2b: [3-PyO(m-Cl)]8PcCu$ — — — — — —

2c: [4-PyO(m-Cl)]8PcCu 6.29 252.9 (4.28) 352.4 (4.38) 606.7 (4.04) 639.8* (4.13) 670.8 (4.44)

#: In THF. *: Aggregation band of Q0–0. $: This derivative is insoluble in any solvents.


52 K. OHTA ET AL.

was poured into a big beaker and THF warmed at 60 °C was into the beaker with stirring. The resulted hot THF solution was collected by decantation. It was repeated 8 times. The combined THF solutions were evaporated to dryness. The residue was purified by Soxhlet extraction using dry THF. The filtrate was evaporated and dried in vacuo to obtain 14.1 mg of green powder. Yield and MALDI-TOF Mass data: see Table 1. UV-vis spectral data: see Table 2. Phase transition behavior: see Table 3.

[4-PyO(m-Cl)]8PcCu (2c). A mixture of [4-PyO(m-Cl)]2FN (3c: 81.5 mg, 0.213 mmol), CuCl2(27.6 mg, 0.205 mmol), 1-hexanol (5 mL), DBU(3drops) was refluxed under a nitrogen atmosphere for 3 h. After cooling to rt, methanol was poured into the reaction mixture to precipitate the target compound. The precipitates were collected by filtration and the filtrate was washed with methanol, ethanol and acetone, successively. The residue was resolved in a small amount of mixed solvents, chloroform:THF = 1:1 (v/v). The solution was poured into a column for the column chromatography (silica gel). The impurities were removed by using the first eluent of chloroform and the target Pc complex 2c (Rf = 0.0 for chloroform) was collected by using the second eluent of THF. After evaporation of the solvent, the residue was purified by Soxhlet extraction using dry THF. The filtrate was evaporated and dried in vacuo to obtain 12.3 mg of green powder. Yield and MALDI-TOF Mass data: see Table 1. UV-vis spectral data: see Table 2. Phase transition behavior: see Table 3.

Measurements

The compounds [x-PyO(m-Cl)]2FN (3a–3c) synthe-sized here were identified with an FT-IR spectro meter (Nicolet NEXUS 670) and 1H NMR spectrometer (BRUKER Ultrashield 400 M Hz). The MALDI-TOF mass spectral measurements of phthalocyanine derivatives [x-PyO(m-Cl)]8PcCu(2a–2c) were carried

out by using a Bruker Daltonics Autoflex III spectro-meter (matrix: dithranol). Electronic absorption (UV-vis) spectra of 2a and 2c were recorded by using a Hitachi U-4100 spectrophotometer. Phase transition behavior of 2a–2c was observed with a polarizing optical microscope (Nikon ECLIPSE E600 POL) equipped with a Mettler FP82HT hot stage and a Mettler FP-90 Central Processor, and a Shimadzu DSC-50 differential scanning calorimeter. The decomposition temperatures were measured by a Rigaku Thermo plus TG 820 thermogravity analyzer. The mesophases were identified by using a small angle X-ray diffractometer (Bruker Mac SAXS System) equipped with a temperature-variable sample holder with a Mettler FP82HT hot stage. The measurable range is from 3.0 Å to 110 Å and the temperature range is from rt to 375 °C.


Synthesis

Scheme 1 shows the synthetic route. The Pc compounds synthesized here were characterized by using MALDI-TOF mass spectra (Table 1) and UV-vis spectra (Table 2). However, one of these three derivatives, [3-PyO(m-Cl)]8PcCu (2b), was hardly soluble in any solvents that the UV-vis spectrum could not measured. Also these three Pc derivatives, 2a–2c, are much less flammable so that the elemental analyses were not carried out. However, it could be confirmed from their MALDI-TOF mass spectra and UV-vis spectra that the target derivatives, 2a–2c, were surely synthesized.

Phase transition behavior

Phase transitions of the present derivatives [x-PyO(m-Cl)]8PcCu (2a–2c) are summarized in Table 3. The

K

Colro1

K

Colro2

Colro1 Colro2

PhaseCompound Phase

dc.

(dc.)306[16.3]

325[25.6]

ca. 248

Tg = ca.172

(dc.)

T (°C) [ΔH (kJ.mol-1)]

2b: [3-PyO(m-Cl)]8PcCu

2c: [4-PyO(m-Cl)]8PcCu

2a: [2-PyO(m-Cl)]8PcCu

371[56.1]

(P2m) (P2m)

Glassy Coltet.o

(P2m) (P2m)

Relaxation

suppercooledColtet.o

218

Table 3. Phase transition temperatures of [x-PyO(m-Cl)]8PcCu: 2a–2c

Phase nomenclature: K = crystal, Coltet.o = tetragonal ordered columnar mesophase, Colro = rectangular ordered columnar mesophase and dc. = decomposition.



mesomorphism was established by polarizing microscopic observations, DSC, TG-DTA and small angle X-ray diffraction measurements.

As can be seen from this table, the freshly prepared sample of [2-PyO(m-Cl)]8PcCu (2a) at rt shows a mixture of glassy tetragonal ordered columnar (Coltet.o) mesophase and a small amount of crystalline (K) phase. When the mixture of glassy Coltet.o and K was heated from rt, a glass transition was observed at ca. 172 °C and then the resulted supercooled Coltet.o mesophase relaxed into the crystalline phase K at 218 °C. On further heating, the crystalline phase K melted into a rectangular ordered columnar (Colro1(P2m)) mesophase at 325 °C. The Colro1(P2m) mesophase transformed into another rectangular ordered columnar (Colro2(P2m)) mesophase at 371 °C accompanied with gradual decomposition. On the other hand, the derivative [3-PyO(m-Cl)]8PcCu (2b) showed a crystalline K phase from rt to the decomposition temperature of 248 °C without showing any mesophase. On the contrary, the derivative [4-PyO(m-Cl)]8PcCu (2c) showed a rectangular ordered columnar (Colro1(P2m)) mesophase even at rt. When it was heated from rt, the Colro1(P2m) mesophase transformed into another rectangular ordered columnar (Colro2(P2m)) mesophase at 306 °C accompanied with gradual decomposition.

Thus, the mesomorphism appeared with strong depen-dence of the position of nitrogen atom in m-chloro-pyridyloxy group. The derivative [2-PyO(m-Cl)]8PcCu (2a) introduced a nitrogen atom at the 2-position shows mesomorphism only at very high temperatures over 325 °C. The derivative [4-PyO(m-Cl)]8PcCu (2c) introduced a nitrogen atom at the 4-position shows mesomorphism in a very wide temperature region from rt to the decomposition temperature at 306 °C. On the other hand, the derivative [3-PyO(m-Cl)]8PcCu (2b) introduced a nitrogen atom at the 3-position is not mesogenic but crystalline.

Polarizing microscopic observations

Figure 2 shows photomicrographs of the Pc derivatives, [x-PyO(m-Cl)]8PcCu (2a–2c). The photomicrographs (a) and (b) show the Colro1(P2m) mesophase at 350 °C and the Colro2(P2m) mesophase at 375 °C for the derivative [2-PyO(m-Cl)]8PcCu (2a), respectively. As can be seen from these photomicrographs, when the cover glass was pressed at both 350 °C and 375 °C, the derivative 2a was spread to show stickiness together with birefringence between crossed Nicols. This means that these phases of Colro1 and Colro2 are mesomorphic.

The photomicrograph (c) shows the rigid crystals K of [3-PyO(m-Cl)]8PcCu (2b) at rt. When the cover glass was pressed, this derivative showed rigidity in a whole temperature region from rt to 248 °C.

The photomicrographs (d) and (e) show the Colro1(P2m) mesophase at rt and the Colro2(P2m) mesophase at 340 °C for the derivative [4-PyO(m-Cl)]8PcCu (2c), respectively. As can be seen from these photomicrographs, when

the cover glass was pressed at both rt and 340 °C, the derivative [4-PyO(m-Cl)]8PcCu (2c) was spread to show stickiness together with birefringence between crossed Nicols. This implies that these phases of Colro1 and Colro2 are mesomorphic. The derivatives 2a and 2c decompose without clearing into isotropic liquid (I.L.), so that their natural textures could not be obtained from the I.L. However, these mesophases could be confirmed from the stickiness with birefringence, as mentioned above.

Temperature-dependent X-ray diffraction measurements

In order to reveal the detailed mesophase structures, temperature-dependent small angle X-ray diffraction measurements were carried out for the mesogenic derivatives, [2-PyO(m-Cl)]8PcCu (2a) and [4-PyO(m-Cl)]8PcCu (2c). These X-ray diffraction (XRD) patterns and their X-ray data are summarized in Figs 3, 4 and Table 4, respectively.

As can be seen from Fig. 3(a), the mesophase at rt in the derivative [2-PyO(m-Cl)]8PcCu (2a) gave 11 sharp peaks. As can be seen from Table 4, all the peaks except for no. 3 and no. 7 could be well assigned to the reflections from a 2D tetragonal lattice having the lattice constant, a = 21.4 Å. Peak no. 7 could be assigned to the reflection corresponding to the stacking distance (h = 5.54 Å) in the intracolumnar disks. Therefore, this phase could be tentatively identified as a tetragonal ordered columnar (Coltet.o) mesophase, but this phase was rigid when it was pressed on the cover glass at rt. Moreover, Peak no. 3 could not be assigned to any reflections from the 2D tetragonal lattice. It may be a peak from a 3D crystalline lattice. Hence, this state at rt was identified as a mixture states of a glassy Coltet.o mesophase and small amount of a crystalline K phase.

As can be seen from Fig. 3(b), this derivative 2a at 350 °C gave eleven sharp peaks. All the peaks except for no. 7 could be well assigned to the reflections from a 2D rectangular (P2m) lattice having the lattice constants, a = 20.7 Å and b = 14.2 Å, as can be seen from Table 4. Peak no. 7 could be assigned to the reflection corresponding to the stacking distance (h = 5.15 Å) of intracolumnar disks. Therefore, this mesophase could be identified as a Colro1(P2m) mesophase. As can be seen from Fig. 3(c), this derivative 2a at 375 °C gave eleven sharp peaks. All the peaks except for no. 7 could be well assigned to the reflections from a 2D rectangular (P2m) lattice having the lattice constants, a = 20.0 Å and b = 14.2 Å. Peak no. 7 was assigned to the reflection corresponding to the stacking distance (h = 5.26 Å) of intracolumnar disks. Therefore, this mesophase was also identified as another Colro2(P2m) mesophase.

Figures 4(a) and 4(b) show the X-ray diffraction patterns of the derivative [4-PyO(m-Cl)]8PcCu(2c) at 150 °C and 340 °C, respectively. In the same manner above-mentioned for 2a, the mesophases in 2c at 150 °C and 340 °C could be also identified as a rectangular


54 K. OHTA ET AL.

ordered columnar mesophase Colro1(P2m) having the lattice constants, a = 19.6 Å, b = 16.0 Å and h = 4.42 Å, and another rectangular ordered columnar mesophase Colro2(P2m) having the lattice constants, a = 19.5 Å, b = 16.0 Å and h = 4.51 Å, respectively. These X-ray data are listed up also in Table 4.

Relationship between mesomorphism and position of the nitrogen atom in pyridyloxy group

As mentioned above, [2-PyO(m-Cl)]8PcCu (2a) and [4-PyO(m-Cl)]8PcCu (2c) show mesomorphism but [3-PyO(m-Cl)]8PcCu (2b) does not show it.

Figure 5 illustrates molecular structures of all the octakis(m-chloropyridyloxyphthalocyanato) copper(II) complexes, [x-PyO(m-Cl)]8PcCu (2a–2c). Only the nitro gen atom in the [3-PyO(m-Cl)]8PcCu (2b) deriva - tive can be close to the neighboring chlorine atom by rotation, so that an additional intramolecular N…Cl halogen bond may be formed in the nearest m-chloro-pyridyloxy groups [13]. Hence, the m-chloropyridyloxy groups fixed by the halogen bond may neither rotate nor flip-flop to form the soft parts essential for mesomorphism [1–6]. Therefore, the derivative 2b cannot show mesomorphism. Moreover, it is very interesting that the derivative [2-PyO(m-Cl)]8PcCu (2a) shows

Fig. 2. Photomicrographs. [2-PyO(m-Cl)]8PcCu (2a); (a) Colro1(P2m) at 350 °C; (b) Colro2(P2m) at 375 °C. [3-PyO(m-Cl)]8PcCu (2b); (c) K at rt. [4-PyO(m-Cl)]8PcCu (2c); (d) Colro1(P2m) at rt; (e) Colro2(P2m) at 340 °C



mesomorphism only at very high temperatures over 325 °C, whereas the derivative [4-PyO(m-Cl)]8PcCu (2c) shows meso morphism in a very wide temperature region from rt to the decomposition temperature at 306 °C. It may be attributed to the intermolecular N…Cl halogen bond that 2a can form but 2c cannot. In the very high temperature region, the intermolecular N…Cl halogen bond of 2a may be destroyed to start the rotation or flip-flop of the m-chloropyridiloxy groups. Therefore, the derivative 2a shows mesomorphism only at very high temperatures over 325 °C. On the other hand, the derivative 2c may form neither intramolecular nor intermolecular halogen bonds, so that derivative 2c shows mesomorphism in a very wide temperature region from rt to the decomposition temperature at 306 °C.

Thus, both intramolecular and intermolecular halogen bonds may more or less prevent inducing mesomorphism for the present octakis(m-chloropyridyloxy)phthalocyaninato copper(II) complexes. On the contrary, halogen bonds in all the liquid crystals reported to date promote to induce mesomorphism [14–28].

CONCLUSION

In this study, we have synthesized novel octakis(m-chloropyridyloxy)phthalocyaninato copper(II) complexes, [x-PyO(m-Cl)]8PcCu (x = 2, 3, 4: 2a–2c), keeping a chlorine atom at the meta position on the 2-, 3- and 4-pyridyloxy group, in which the nitrogen atom is located at the 2-, 3- and 4-positions, respectively, and investigated the influence of the position of nitrogen atom in the m-chloropyridyloxy group on their mesomorphism.

As a result, the derivative [2-PyO(m-Cl)]8PcCu (2a) introduced a nitrogen atom at the 2-position shows columnar mesomorphism only at very high temperatures over 325 °C. The derivative [3-PyO(m-Cl)]8PcCu (2b) introduced a nitrogen atom at the 3-position is not mesogenic but crystalline. On the other hand,

30252015105

30252015105

7

2

3 4

5

68

9

10

11

0.8

Glassy Coltet.o

+ K at r.t.

1

2θ

Inte

nsity (

a.u

.)

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3

4

56

7 89

1011

Colro1(P2m) at 350oC

0.8

Inte

nsity (

a.u

.)

1

2

2θ

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3 4

5 6

7 89

1011


0.8

Inte

nsity (

a.u

.)

1

2

2θ

(c)

(b)

(a)

Fig. 3. X-ray diffraction patterns of [2-PyO(m-Cl)]8PcCu (2a) at rt, 350 °C and 375 °C

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Inte

nsity (

a.u

.)


0.8

1

2

45

6

7

8

2θ

3

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0.8


7

1

2

3

4

5

6

8

2θ

Inte

nsity (

a.u

.)

910

11

12

13

14

(b)

(a)

Fig. 4. X-ray diffraction patterns of [4-PyO(m-Cl)]8PcCu (2c): (a) at 150 °C and (b) at 340 °C


56 K. OHTA ET AL.

Table 4. X-ray data of 2a and 2c

Compound mesophase lattice constants (Å)

Peak no. Spacing (Å) Miller indices (h k l)

Observed Calculated

2a: [2-PyO(m-Cl)]8PcCu

tentatively assigned glassy Coltet.o + K at r.t.a = 21.4h = 5.54Z = 1.0 for ρ = 1.8

1 2 3 4 5 6 7 8 91011

21.115.611.4

7.49 6.81 5.97 5.54 4.87 4.62 3.61 3.45

21.115.6—

7.60 6.73 5.99 5.54 4.79 4.56 3.58 3.45

(1 0 0)(1 1 0)

X(2 2 0)(3 1 0)(2 3 0)

h(4 2 0)(2 4 0)(0 6 0)(1 6 0)

Colro1 (P2m) at 350 °Ca = 20.7 b = 14.2h = 5.15Z = 1.0 for ρ = 1.8

1 2 3 4 5 6 7 8 91011

20.714.212.0

8.38 6.88 6.18 5.14 4.70 4.19 4.03 3.65

20.714.211.7

8.35 6.90 6.20 5.14 4.71 4.17 3.89 3.53

(1 0 0)(0 1 0)(1 1 0)(2 1 0)(3 0 0)(3 1 0)

h(0 3 0)(4 2 0)(3 3 0)(0 4 0)


1 2 3 4 5 6 7 8 91011

20.014.211.9

8.06 6.92 6.16 5.26 4.71 4.19 4.03 3.66

20.014.211.6

8.18 7.09 6.04 5.26 4.72 4.27 4.00 3.54

(1 0 0)(0 1 0)(1 1 0)(2 1 0)(0 2 0)(3 1 0)

h(4 1 0)(2 3 0)(5 0 0)(0 4 0)

2c: [4-PyO(m-Cl)]8PcCu


1 2 3 4 5 6 7 8 91011121314

19.616.012.0

7.89 6.69 6.16 5.38 4.96 4.58 4.42 4.04 3.80 3.64 3.41

19.616.012.4

8.00 6.54 6.20 5.33 4.91 4.68 4.42 4.00 3.81 3.61 3.41

(1 0 0)(0 1 0)(1 1 0)(0 2 0)(3 0 0)(2 2 0)(0 3 0)(4 0 0)(2 3 0)

h(0 4 0)(5 1 0)(4 3 0)(3 4 0)


1 2 3 4 5 6 7 8

19.516.07.976.194.514.183.713.08

19.516.0

8.01 6.19 4.51 4.17 3.70 3.10

(1 0 0)(0 1 0)(0 2 0)(2 2 0)

h(4 2 0)(2 4 0)(4 4 0)

h: Stacking distance = (0 0 1), ρ: Assumed density (g/cm3), X = a reflection from 3D crystalline lattice.



O

N

O ON

N

NN

N

N Cu N

N

N

O

N

ON

ON

O

O

N

N

Cl

Cl

Cl Cl

Cl

Cl

Cl

N

Cl

N

O O

N

N

NN

N

N Cu N

NO

N

ON

O

N

O

N

O

O

N

N

N

Cl Cl

Cl

Cl

ClCl

Cl

Cl

N

O O

N

N

NN

N

N Cu N

NO

N

O

N

O

N

O

N

O

O

N

N

N

Cl Cl

Cl

Cl

ClCl

Cl

Cl

O

O

N

N

Cl

Cl

O

O

N

N

Cl

Cl

O

O

N

N

Cl

Cl

(b) 2b: [3-PyO(m-Cl)]8PcCu

(c) 2c: [4-PyO(m-Cl)]8PcCu

(a) 2a: [2-PyO(m-Cl)]8PcCu

Appearance ofmesophase

Flip-flop

Fixed by N---Clhalogen bond

Flip-flop

Fig. 5. Appearance of the mesomorphism depending on both flip-flop of the bulky m-chloropyridyloxy groups and the N…Cl halogen bond. ×: no mesophase appears; : enantiotropic mesophase appears


58 K. OHTA ET AL.

the derivative [4-PyO(m-Cl)]8PcCu (2c) introduced a nitrogen atom at the 4-position shows columnar mesomorphism in a very wide temperature region from rt to the decomposition temperature at 306 °C. Thus, the mesomorphism appears with strong dependence of the position of nitrogen in m-chloropyridyloxy group.

We have discussed about relationship between their mesomorphism and the position of nitrogen atom in m-chloropyridyloxy group. The [3-PyO(m-Cl)]8PcCu (2b) derivative may form an additional intramolecular N…Cl halogen bond in the nearest m-chloropyridyloxy groups. Hence, the m-chloropyridyloxy groups fixed by the halogen bond may neither rotate nor flip-flop to prevent showing mesomorphism. The derivative [2-PyO(m-Cl)]8PcCu (2a) can form the intermolecular N…Cl halogen bond at lower temperatures, but the halogen bond may be destroyed at very high temperatures over 325 °C to show mesomorphism. On the other hand, the derivative [4-PyO(m-Cl)]8PcCu (2c) may form neither intramolecular nor intermolecular N…Cl halogen bond, so that it shows mesomorphism in a very wide temperature region from rt to 306 °C. Thus, appearance of the mesomorphism in the present [x-PyO(m-Cl)]8PcCu (2a–2c) derivatives strongly depends on the position of nitrogen atom in m-chloropyridyloxy group and it can be explained from the viewpoint of halogen bond.

REFERENCES

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2. Takagi Y, Ohta K, Shimosugi S, Fujii T and Itoh E. J. Mater. Chem. 2012; 22: 14418–14425.

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4. Yoshioka M, Ohta K and Yasutake M. RSC Advances 2015; 5: 13828–13839.

5. Ishikawa A, Ohta K, and Yasutake M. J. Porphyrins Phthalocyanines 2015; 19: 639–650.

6. Watarai A, Ohta K and Yasutake M. J. Porphyrins Phthalocyanines 2016; 20: 822–832.

7. Sanusi K, Antunes E and Nyokong T. J. Chem. Dalton Transactions, 2014; 43: 999–1010.

8. Sekhosana K, Amuhaya E and Nyokong T. Polyhe-dron 2016; 105: 159–169.

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12. Sato H, Igarashi K, Yama Y, Ichihara M, Itoh E and Ohta K. J. Porphyrins Phthalocyanines 2012; 16: 1148–1181.

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14. Metrangolo P, Praesang C, Resnati G, Liantonio R, Whitwood AC and Bruce DW. Chem. Commun., 2006; 3290–3292.

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19. Fourmigue M. Curr. Opin. Solid State Mat. Sci. 2009; 13: 36–45.

20. Bruce DW, Metrangolo P, Meyer F, Pilati T, Prae-sang C, Resnati G, Terraneo G, Wainwright SG and Whitwood AC. Chem.–Eur. J. 2010; 16: 9511–9524, S9511/1-S9511/19.

21. Priimagi A, Saccone M, Cavallo G, Shishido A, Pilati T, Metrangolo P and Resnati G. Adv. Mater. 2012; 24: OP345–OP352.

22. Wong JP-W, Whitwood AC and Bruce DW. Chem. —. Eur. J. 2012; 18: 16073–16089.

23. Vijayakumar VN and Madhu Mohan MLN. Phase Transitions 2012; 85: 113–130.

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DOI: 10.1142/S1088424617500055



INTRODUCTION

Photodynamic therapy (PDT) has successfully been used for the treatment of many kinds of cancers [1]. In PDT, three elements are involved: a photosensitizer (PS), light of appropriate frequency and molecular oxygen.

PDT is based on the uptake and retention of PSs in tumor cells or tissues, and activation with light, resulting in death of tumor cells by highly cytotoxic reactive oxygen species (ROS) [2–4]. The destruction of cancerous tissues occurs only if light and PSs reach the interior of cancer cells. Photosensitizer has preferential accumulation in cancer cells [5] while singlet oxygen (1O2) is locally generated and has a short effective radius [3], therefore, the cytotoxic of PDT effects is confined to cancer tissues with minimal side effects to normal tissues. Overall, PDT is one of the most selective treatments for oncological diseases with excellent targeting capability and few side effects. The

Photophysical properties of sinoporphyrin sodium

and explanation of its high photo-activity

Lixin Zanga, Huimin Zhao*b, Qicheng Fangc◊, Ming Fand, Tong Chend, Ye Tiane◊,

Jianting Yaoe, Yangdong Zheng*f, Zhiguo Zhang*a and Wenwu Cao*g

a Condensed Matter Science and Technology Institute, Harbin Institute of Technology, Harbin, 150080, China b School of Physics and Electronics, Shandong Normal University, Ji’nan, 250014, China c Institute of Materia Medica, Chinese Academy of Medical Science, Beijing, 100050, China d Shenzhen Micromed Tech. Co., Ltd., Shenzhen, 518109, China e Division of Cardiology, the First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, Harbin, 150001, China f Department of Physics, Harbin Institute of Technology, Harbin, 150001, China g Department of Mathematics and Materials Research Institute, The Pennsylvania State University, Pennsylvania, 16802, USA

Received 2 September 2016Accepted 7 November 2016

ABSTRACT: Sinoporphyrin sodium (DVDMS) is a novel photosensitizer with high photodynamic therapy (PDT) effect. Reasons for its high photo-activity were investigated according to the study of photophysical characteristics of DVDMS. Extinction coefficients (ε) of DVDMS at 405 nm and 630 nm are 4.36 × 105 and 1.84 × 104 M-1.cm-1; fluorescence quantum yield (ΦF) is 0.026; quantum yield of lowest triplet state formation is 0.94 and singlet oxygen quantum yield (ΦΔ) is 0.92. Although ΦΔ of DVDMS is only 10% higher than that of Photofrin® (0.83), the extinction coefficient of DVDMS at 630 nm is 10-fold greater than that of Photofrin®. This leads to its higher singlet oxygen generation efficiency (εΦΔ). The higher εΦΔ of DVDMS can result in an effective reduction of dosage (1/10 of Photofrin®) reaching the same cytotoxic effect as Photofrin®. Even though ΦF is approximately equal to that of Photofrin®, brightness (εΦF) of DVDMS is 10-fold greater than that of Photofrin® because of the 10-fold greater extinction coefficient. Thus, fluorescence diagnosis ability of 0.2 mg/kg DVDMS is comparable to that of 2 mg/kg Photofrin® used in PDT. Overall, the 10-fold greater extinction coefficients are responsible for the high brightness and singlet oxygen generation efficiency of DVDMS.

KEYWORDS: photodynamic therapy, sinoporphyrin sodium, photophysical properties, extinction coefficient, fluorescence quantum yield, singlet oxygen quantum yield.

SPP full member in good standing

*Correspondence to: Huimin Zhao, email: [email protected]; Yangdong Zheng, email: [email protected]; Zhiguo Zhang, email: [email protected]; Wenwu Cao, email: [email protected]


60 L. ZANG ET AL.

development of PDT is largely dependent on the progress of more efficient and practical PSs [6]. There are thousands of natural and synthetic photoactive compounds having photosensitizing potential [7–10], in which the Tookad is under the phase III clinical trials [11] and the redaporfin is under the phase III clinical trials [12].

Until now, only several PSs, Photofrin™, Visudyne™, Foscan™, Levulan™ and Metvix™, have been approved for clinical use by the drug regulation administrations of different countries. These available PSs are not satisfactory in several aspects, such as less efficient in ROS production, hydrophobic and long time residue in the body. These deficiencies of available PSs limit the development of PDT in clinical applications. In particular, the most commonly used PS in clinic is still Photofrin®

[3], which is a mixture of dimers and oligomers of hematoporphyrin. The first limitation of Photofrin® is that the esters or ethers linked with the porphyrin units were found to be unstable, and hydrolysis was also detected in the injection solution [13]. Composed of multiple components, Photofrin® has no controllable quality standards [14]. Besides, a relatively big dosage of Photofrin® is needed because it has a low extinction coefficient at its treatment wavelength (630 nm) [15]. Moreover, due to its strong toxicity to skin and eyes, patients are warned to avoid direct sunlight or bright light indoors for 4–6 weeks following the drug injection [16, 17]. To overcome these limitations of Photofrin®, Fang et al. from the Chinese Academy of Medical Sciences isolated an active compound from Photofrin® and named it sinoporphyrin sodium (DVDMS) [18]. DVDMS is patented in the People’s Republic of China. DVDMS, a newly developed PS with a single and well-defined chemical structure, has shown much higher photo-activity in preclinical studies than Photofrin® from the study of Fang et al. Hu et al. have reported that DVDMS-PDT showed a cell proliferation inhibition effect on a human esophageal cancer cell line (Eca-109 cells) [19]. DVDMS was observed to have higher brightness and singlet oxygen generation efficiency compared to other known PSs: hematoporphyrin, protoporphyrin IX (PpIX) and Photofrin® [20]. A safety evaluation of DVDMS in Beagle dogs has been conducted and DVDMS-PDT was demonstrated to be a safe and promising anti-tumor therapy in clinic [21]. In addition, it was proved that DVDMS has greater water-solubility, chemical stability and good targeting ability for tumor cells or tissues [22, 23]. In animal tests, DVDMS can be totally washed out of bodies after three days from the study of Fang et al. DVDMS has shown many excellent preclinical characteristics, but values of its photophysical parameters, such as extinction coefficients, fluorescence quantum yield, quantum yield of triplet state formation and singlet oxygen quantum yield (ΦΔ) of DVDMS are still lacking. Moreover, without these parameters, it is difficult to understand its excellent characteristics as well as the differences between DVDMS and other porphyrins.

In this work, reason for the high PDT effect of the new photosensitizer DVDMS was revealed. Photophysical characteristics of DVDMS were studied quantitatively for the use in PDT. UV-visible absorption spectrum and extinction coefficients of DVDMS were measured. Fluorescence quantum yield of DVDMS was determined based on a relative method using Rhodamine 6G as the reference. Quantum yield of the first triplet state formation at room temperature was calculated from low-temperature measurements. Singlet oxygen quantum yield of DVDMS was determined based on a relative method using Rose Bengal as the reference. Comparison of the preclinical properties between DVDMS and the most commonly used photosensitizer, Photofrin®, was performed. The better properties of DVDMS were explained based on the obtained quantitative results.

EXPERIMENTAL

Materials

Sinoporphyrin sodium (DVDMS) was kindly provided by Professor Qicheng Fang from the Chinese Academy of Medical Sciences. Protoporphyrin IX (PpIX) was purchased from Dalian Meilun Biological Tech. Co., Ltd.; Rhodamine 6G (R6G) was obtained from Sigma–Aldrich Company; 1,3-diphenylisobenzofuran (DPBF) and 4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein disodium salt (Rose Bengal) were bought from J&K Scientific Ltd.

Measurement

UV-visible absorption spectra were measured using a miniature fiber optic spectrometer (Ocean Optics QE65000) equipped with a deuterium lamp. Extinction coefficients (ε(λ)) of DVDMS in methanol were measured based on Beer-Lambert law, -log(I(λ)/I0(λ)) = ε(λ) . C . L, where I(λ) is the transmission spectrum of DVDMS, I0(λ) is the transmission spectrum of the blank (methanol), C is the concentration of DVDMS and L is the optical pathlength, the product ε(λ) . C . L is the absorbance. From Beer–Lambert law, the extinction coefficient at the specific wavelength can be obtained from the relationship between -log(I(λ)/I0(λ)) and C. To get this relationship, -log(I(λ)/I0(λ)) of DVDMS at various concentrations (six different concentrations) were measured. All spectra were calibrated using a mercury lamp.

In order to characterize the fluorescence emission ability of DVDMS, fluorescence quantum yield (ΦF) in air-saturated methanol was measured using a relative method with R6G as the reference (ΦF = 0.93) [24]. A laser centered at 532 nm (CLO Laser DPGL-500L) was chosen as the excitation light. The relationship between ΦF of DVDMS and that of R6G can be described by Equation 1 [25],

⎛ ⎞⎛ ⎞Φ = Φ ⎜ ⎟ ⎜ ⎟⎝ ⎠ ⎝ ⎠

rr

F F r

1,

A

A I (1)


PHOTOPHYSICAL PROPERTIES OF SINOPORPHYRIN SODIUM AND EXPLANATION OF ITS HIGH PHOTO-ACTIVITY 61

where the superscript “r” stands for the reference reagent. Ir and I are the integrated emission intensities of R6G and DVDMS. Ar and A are the absorption of the excitation light by R6G and DVDMS at 532 nm, which can be described by Equation 2.

e( )532 ( ) (1 e )− λ= λ × − λ,∫ CLA I d (2)

where I532 (λ) is the normalized emission spectrum of the 532 nm laser.

The determination of quantum yield of the first triplet state formation (Φt) at room temperature was performed using the method described in a previous work [8, 26]. Gadolinium ion was coordinated into the center of DVDMS to study the characteristic of the triplet state of DVDMS. The details were provided in the Supporting information.

In the measurement of singlet oxygen quantum yield (ΦΔ) of DVDMS in methanol, a relative spectrophoto-metric method was used with Rose Bengal (ΦΔ = 0.79) [27, 28] as the reference and DPBF as singlet oxygen trapping reagent [29–34]. The relative spectrophotometric method [5, 35] was based on Equation 3,

rr

r,Δ Δ

⎛ ⎞⎛ ⎞Φ = Φ ⎜ ⎟ ⎜ ⎟⎝ ⎠ ⎝ ⎠k A

A k (3)

where k is the photodegradation rate of DPBF, A is the absorption of excitation light by photosensitizers [36] described in Equation 2. The consumption of DPBF (15 μM) irradiated with a 532 nm laser (1 mW.cm-2) was monitored with UV-visible absorption spectra at different irradiation time. The degradation rate of DPBF (k) was determined according to the decrease of the absorption peak at 410 nm with time.


Extinction coefficients

Figure 1(a) shows the UV-visible absorption spectra of DVDMS (black) and Photofrin® (red) at the same concentration (6 mg/L) in methanol. Inset of Fig. 1(a) shows the chemical structure of DVDMS. Unlike normal porphyrin derivatives, DVDMS is a double ring porphyrin-related photosensitizer.

The absorption spectrum of DVDMS was found to be similar to other porphyrin-related photosensitizers, comprising the following bands (nomenclature given by Platt [37]): four Q bands, located at 508 nm, 541 nm, 578 nm and 630 nm, a B band (Soret band) centered at 388 nm. Table 1 shows the detailed comparison of DVDMS and Photofrin® with regard to the absorbance spectra shown in Fig. 1(a). It can be predicted that the mass extinction coefficient of DVDMS is about 7.5 times

that of Photofrin®. Thus, the molar extinction coefficient of DVDMS will be about 15 times that of Photofrin® because the molecular weight of DVDMS is about twice that of Photofrin®.

To verify the assumption above, the molar extinction coefficients (ε) of the five absorption peaks were determined using the absorption spectra of DVDMS at various concentrations (0–5 μM) as shown in Fig. 1(b). The relationship between the absorbance at a specific

Fig. 1. (Color online) (a) The UV-visible absorption spectra of DVDMS (black) and Photofrin® (red) at the same concentration (6 mg/L) in methanol as well as the chemical structure of DVDMS. (b) Absorption spectra of DVDMS at 0–5 μM, inset: relationship between the absorbance at a specific wavelength and the concentrations of DVDMS

Table 1. A comparison of DVDMS and Photofrin® with regard to the absorbance spectra shown in Fig. 1(a)

Samples Solvent Concentration, mg/L A (630 nm)

DVDMS methanol 6 0.090

Photofrin® methanol 6 0.012


62 L. ZANG ET AL.

wavelength and the concentrations were obtained to be linear and results are shown in the inset of Fig. 1(b). The ε at the specific wavelength is identical to the slope. ε of the five absorption peaks are: 5.28 × 105 M-1.cm-1 (388 nm), 6.23 × 104 M-1.cm-1 (508 nm), 4.00 × 104 M-1.cm-1 (541 nm), 3.22 × 104 M-1.cm-1 (578 nm) and 1.84 × 104 M-1.cm-1 (630 nm). From published works [38], the ε of Photofrin® at its treatment wavelength is 1.17 × 103 M-1.cm-1 (3 × 103 in Ref. 15). We can see that the ε of DVDMS at 630 nm is about one order of magnitude greater than that of Photofrin®. This result is identical to that shown in Fig. 1(a) and Table 1.

UV-blue lights (lamps, 405 nm lasers or 337 nm nitrogen lasers) were usually used in fluorescence diagnosis [39]. From published works, the ε of Photofrin® at 405 nm is about 24-fold greater than that at 630 nm [40, 41]. Thus, the ε of Photofrin® at 405 nm is about 2.81 × 104 M-1.cm-1. From calculations, the ε of DVDMS at 405 nm is 4.36 × 105 M-1.cm-1. It can be seen that the ε of DVDMS at 405 nm is one order of magnitude greater than that of Photofrin®. This means that DVDMS has better absorption ability of UV-blue lights.

Fluorescence quantum yield

Figure 2 presents the emission spectra of DVDMS (black) and Photofrin® (red) at the same concentration (6 mg/L) excited by a 405 nm diode laser. It can be seen that DVDMS also has two red photoluminescence peaks, centered at 626 nm and 690 nm. Generally, the luminescence spectrum reflects the structure of the ground state. This two peak fluorescence indicates that there are two vibrational levels in the ground state of DVDMS. From Fig. 2, one can see that the fluorescence intensity of DVDMS is much higher than that of Photofrin® at the same concentration. Table 2 presents the detailed comparison of DVDMS and Photofrin® with regard to the fluorescence spectra shown in Fig. 2. One can see that the fluorescence intensity of DVDMS is about one order of

magnitude higher than that of Photofrin®. This may be caused by its greater absorption ability of the excitation light.

To further verify the reason for the brighter fluorescence of DVDMS in cells compared with other photosensitizers described in a published work [20], fluorescence quantum yield (ΦF) of DVDMS was measured. In the measurement of ΦF of DVDMS, A and I for R6G and DVDMS were obtained based on Equations 1 and 2. From A, I and ΦF of R6G (0.93), ΦF of DVDMS was determined to be 0.026 with uncertainty of 0.002 in five-times repeated experiments. The experi-mental conditions and calculated ΦF of DVDMS in air saturated methanol are shown in Table 3. Results indicate that ΦF of DVDMS is approximately equal to that of Photofrin® (0.027) according to a previous work [42]. Nevertheless, brightness of fluorescence is related to not merely ΦF but also extinction coefficient (ε) at the excitation wavelength. The product of ε and ΦF was

Fig. 2. (Color online) The emission spectra of DVDMS (black) and Photofrin® at the same concentration (6 mg/L) excited by a 405 nm diode laser

Table 2. A comparison of DVDMS and Photofrin® with regard to the fluorescence spectra shown in Fig. 2

Samples Solvent Concentration, mg/L λexc, nm Intensity, a.u. at 630 nm

DVDMS methanol 6 405 3.24

Photofrin® methanol 6 405 0.34

Table 3. Experimental conditions and calculated fluorescence quantum yield of DVDMS in methanol

Samples Solvent Concentration, μM λexc, nm A I, a.u. ΦF

R6G methanol 2.5 532 3.40 3149820 0.9324

DVDMS methanol 6 532 1.92 50130 0.026



defined as brightness (εΦF) [43]. If 405 nm lasers are used as the excitation light in fluorescence diagnosis, brightness of DVDMS in air-saturated solution was obtained to be 11336 M-1.cm-1 based on the experimental data of extinction coefficient at 405 nm and fluorescence quantum yield, while brightness of Photofrin® was 760 M-1.cm-1 from Refs. 38 and 40–42. The brightness of DVDMS is over one order of magnitude greater than that of Photofrin®. Therefore, the reported brighter fluorescence emission of DVDMS compared to other photosensitizers in cells [20] is mainly caused by the higher absorption of excitation light by DVDMS.

Quantum yield of the first triplet state formation

To obtain the quantum yield of the first triplet state (T1) formation of DVDMS, the method reported in previous works [8, 26] was used. The detailed measurements and results are provided in Supporting information. Φt was calculated to be 0.94. From these data, we can conclude that 2.6% of the absorbed photons were converted to fluorescence, approximately 3.4% involved in non-radiative transitions from S1 to S0, and 94% transferred from S1 to T1 by intersystem crossing.

Singlet oxygen quantum yield

DVDMS was reported to have higher singlet oxygen generation efficiency compared to other photosensitizers [20]. To determine the singlet oxygen generation efficiency of DVDMS, firstly, the quantum yield of singlet oxygen for DVDMS was measured. In the measurement, a relative method based on Equation 3 was utilized with DPBF as singlet oxygen trap.

It has been well-demonstrated that the absorption spectra of DPBF remain unchanged after irradiated by 532 nm laser [10]. However, the absorption of DPBF in the mixture with RB or DVDMS decreased with irradiation time as shown in Figs 3(a) and 3(b), which demonstrates the production of singlet oxygen. Here, the absorption spectra of DPBF were obtained by using the transmission spectra of RB or DVDMS as the blank. The absorption spectra of the mixtures using the transmission spectrum of methanol as blank were provided as Fig. S2 in Supporting information. The value of absorption peak at 410 nm in Figs 3(a) and 3(b) was used to represent the concentration of DPBF ([DPBF]). The concentrations of DPBF and the irradiation time follow the first-order kinetic equation [29] as follows,

ln([DPBF]0/[DPBF]) = kt (4)

Here [DPBF0] is the initial concentration of DPBF and t is the irradiation time. Figure 3(c) shows the time dependence of ln([DPBF]0 / [DPBF]) in the mixture with RB or DVDMS under the irradiation of 532 nm light. The degradation rate k was determined by linear fitting of the experimental data.

To obtain the absorption (A) of excitation light by photosensitizers in Equation 3, Equation 2 was used. Based on the degradation rates k of DPBF, the absorption A and ΦΔ

r, ΦΔ of DVDMS was calculated to be 0.92 ± 0.01

Fig. 3. (Color online) The absorption spectra for DPBF in the mixture with RB (a) or DVDMS (b) at different irradiation time. (c) Time dependence of ln([DPBF0]/[DPBF]) in the mixture with RB or DVDMS under the irradiation of 532 nm light


64 L. ZANG ET AL.

based on Equation 3, which is 10% higher than that of Photofrin® (0.83) [44] in the same solvent. Experimental conditions and calculated ΦΔ of DVDMS in methanol are given in Table 4.

The 10% higher singlet oxygen quantum is not enough for the much higher singlet oxygen generation efficiency. The singlet oxygen generation efficiency is not only related to singlet oxygen quantum yield but also the extinction coefficient at the treatment wavelength. Similar to the definition of brightness, singlet oxygen generation efficiency (εΦΔ) was defined as the product of singlet oxygen quantum yield (ΔΔ) and extinction coefficient (ε) at treatment wavelength (λ = 630 nm). Based on the experimental data of ε and ΦΔ, singlet oxygen generation efficiency of DVDMS in air-saturated methanol was obtained to be 16928 M-1.cm-1, while singlet oxygen generation efficiency of Photofrin® is 971 M-1.cm-1 from Refs. 38 and 44. The singlet oxygen generation efficiency of DVDMS is over one order of magnitude greater than that of Photofrin®.

For comparison, the photophysical parameters of DVDMS and Photofrin® including the extinction coefficients, fluorescence quantum yield, singlet oxygen quantum yield, brightness, singlet oxygen generation efficiency as well as the dosage reaching the same fluorescence diagnosis and cytotoxic effect are listed in Table 5. All values of photophysical parameters (ε, ΦF, ΦΔ) of Photofrin® were from previous published works [38, 42, 44]. The higher brightness and singlet oxygen generation efficiency of DVDMS are in agreement with the biological results observed in preclinical studies [19, 20] and can be used to reveal the reasons for its higher PDT effect.

Based on obtained values of fluorescence quantum yield, quantum yield of the first triplet state formation and singlet oxygen, the energy transfer process of DVDMS is described in Fig. 4. It can be seen that 2.6% of the absorbed photons were converted to fluorescence,

approximately 3.4% involved in non-radiative transitions from S1 to S0, and 94% transferred from S1 to T1 via intersystem crossing. Then, 92% of the absorbed photons return to S0 by the interaction with surrounding oxygen molecules.

From the results above, ΦΔ of DVDMS is just a little higher than that of Photofrin® in the same solvent. Hence, the much higher extinction coefficient DVDMS at 630 nm is the main reason for its higher singlet oxygen generation efficiency compared to Photofrin®. If the dosage of DVDMS is set as 2 mg/kg, which is equal to the dosage of Photofrin®, the power density of irradiated light can be significantly decreased or the treatment time can be greatly shortened. On the other hand, the greater is the extinction coefficient, the smaller drug dosage is required to induce a cytotoxic response [45]. In other words, much smaller dosage of DVDMS (at least 1/10, 0.2 mg/kg) can reach the same cytotoxic effect as Photofrin® (2 mg/kg). Compared with the dosage of Photofrin® (2 mg/kg), the dosage of DVDMS for PDT can be set as 0.2 mg/kg in temporary. This may decrease the cost of PDT and greatly reduce the risk of provoking systemic toxic reactions in DVDMS-mediated PDT. From another point of view, the extinction coefficient at 405 nm of DVDMS is also one order of magnitude greater than that

Table 4. Experimental conditions and calculated singlet oxygen quantum yield of DVDMS in methanol

Samples Solvent Concentration, μM λexc, nm A k ΦΔ

RB methanol 5 532 3.50 0.190 0.7927

DVDMS methanol 2.5 532 0.71 0.045 0.92

Table 5. Comparison of the photophysical parameters between DVDMS and Photofrin®

PSs Solvent ε405, M-1.cm-1 ε630, M

-1.cm-1 ΦF ΦΔ Brightness, M-1.cm-1

Singlet oxygen generation efficiency, M-1.cm-1

DVDMS methanol 436000 18400 0.026 0.92 11336 16928

Photofrin® methanol 28100 117038 0.02742 0.8344 760 971

Fig. 4. The energy transfer processes for DVDMS



of Photofrin®. Brightness of DVDMS is about 10-fold greater than that of Photofrin®. Consequently, under the illumination of lights centered at 405 nm, the ability of fluorescence diagnosis based on 0.2 mg/kg DVDMS is comparable to that of 2 mg/kg Photofrin®. If the same dosage level is used in PDT, the DVDMS can produce 10-fold brighter fluorescence, which can greatly increase the diagnostic sensitivity.

CONCLUSION

In summary, reason for the advantages of DVDMS was investigated by determining its photophysical characteristics. Although the ΦΔ of DVDMS is just a little higher than that of Photofrin® (0.83), 1/10th dosage of DVDMS can reach the same cytotoxic effect as that of Photofrin®. The main reason was the 10-fold higher extinction coefficient of DVDMS than that of Photofrin® at 630 nm. Even though ΦF is approximately equal to that of Photofrin®, brightness (εΦF) of DVDMS is 10-fold greater than that of Photofrin® leading to the potential greater fluorescence diagnosis ability. Thus, the brightness of fluorescence for 0.2 mg/kg DVDMS is comparable to that for 2 mg/kg Photofrin® under the illumination of lights at about 405 nm. Our results provide a reasonable explanation for the high photo-activity of DVDMS. Overall, DVDMS is a much superior photosensitizer with brighter fluorescence and higher singlet oxygen generation efficiency resulted from the extremely high extinction coefficients.


Measurement of triplet state formation quantum yield; the full spectra of Fig. 3(a, b). This material is available free of charge via the Internet.

Acknowledgements

This research was supported in part by National Key Technology R&D Program of China (Grant no. 2013BAI03B06) and the National Natural Science Foundation of China (Grant no. 61308065).

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DOI: 10.1142/S1088424617500092



INTRODUCTION

Cancer is a multifactorial disease that involves numerous pathological processes. Therefore, the combination of different therapies represents a promising strategy in the treatment of malignant neoplasms. G-protein-coupled cannabinoid receptor type-1 (CB1R) and type-2 (CB2R) have emerged as promising therapeutic targets for cancer treatment [1]. We recently described chromeno-pyrazolediones with in vivo antitumor activity [2, 3]. The para-chromenopyrazoles were efficient for prostate cancer cell lines [3], while the ortho-chromenopyrazoles revealed to be potent for triple negative breast cancer [2]. Moreover, these later have shown selectivity for CB2R which is an advantage due to the lack of unwanted psychoactive effects generated by activation of CB1R in the brain. They

exert antitumor effect by inducing cell apoptosis through activation of CB2R and through oxidative stress. It is worthy to mention that these chromenopyrazolediones did not show cytotoxicity on organs such as liver, spleen, lungs, and heart in vivo [2].

Another effective and minimally invasive therapy for cancer treatment is the photodynamic therapy (PDT) [4, 5]. PDT has already been clinically approved for the treatment of various types of malignant disorders such as bladder, lung or esophageal cancer [6]. This technique involves the administration of a photosensitizer (PS) followed by its activation in the solid tumor by light irradiation at a specific wavelength. In the presence of tissue oxygen, the photoactive sensitizer triggers a series of photochemical processes that lead to direct cancer cell death and tumor microvascular damage [7, 8]. Different cell death pathways may be evoked by PDT: apoptosis, necrosis and autophagy [9]. The presence of high amount of collagen and lipid contributes to a preferential accumulation of the photosensitizer by malignant cell types. Therefore, this therapeutic procedure exerts a certain cytotoxic selectivity for cancer cells. In this

Synthesis of a novel CB2 cannabinoid-porphyrin conjugate

based on an antitumor chromenopyrazoledione

Paula Moralesa†, Laura Morenoa, Javier Fernández-Ruizb and Nadine Jagerovic*a

a Instituto de Química Médica (IQM), Consejo Superior de Investigaciones Científicas (CSIS), Unidad Asociada l+D+i IQM/Universidad Rey Juan Carlos (URJC), Calle Juan de la Cierva 3, 28006 Madrid, Spain b Instituto Universitario de Investigación en Neuroquímica, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), 28040 Madrid, Spain

Received 7 November 2016Accepted 12 December 2016

ABSTRACT: With the objective of developing an antitumor agent, the synthesis of a chromeno-pyrazoledione conjugated to a tetraphenylporphyrin is described. A complete conformational analysis of the novel porphyrin conjugate was performed using ab initio Hartree–Fock calculations at the 6-31G* level. The novel conjugate (14) shows stronger absorption intensity for both Soret and Q-bands than the free meso-tetraphenylporphyrin. It binds weakly but selectively to the cannabinoid receptor type-2. During the synthetic approach, a new tetraphenylporphyrin, 5-[4-(3,5-dioxomorpholino)phenyl]-10,15,20-triphenylporphyrin (10), has been characterized.

KEYWORDS: tetraphenylporphyrin, chromenopyrazole, cannabinoid, bioconjugate, cancer, antitumor.

*Correspondence to: Nadine Jagerovic, email: [email protected], tel: +34 915-622-900, fax: +34 915-644-853

†Current address: Department of Chemistry and Biochemistry, University of North Carolina Greensboro, Greensboro, North Carolina, USA


68 P. MORALES ET AL.

context, the strategy proposed here consists in combining PDT with cannabinoid antitumor agents.

These last years, strategies have been explored in which porphyrins are conjugated to molecules showing preferential accumulation for tumor tissues or having affinity for receptors expressed in tumors [10]. Most intensive efforts have been generated for the use of carriers such as nanoparticles [11–13], liposomes [14], polymers [15], translocator protein [16], glycoprotein [17], antibodies [18], or cyclodextrins [19] to enhance the efficiency of the photosensitizers. Another strategy is the conjugation of a therapeutic drug to a porphyrin with two different approaches: combining a photosensitizer with a therapeutic agent or using porphyrins as carriers due to their ability to accumulate in cancer tissues as compared to normal tissues [20]. For instance, the photosensitizer temoporfin has been conjugated to non-steroidal anti-inflammatory compounds to improve the post-PDT treatment tumor regrowth [21]. The cytotoxic agent trilobolide, a sesquiterpene lactone inductor of nitroxic oxide, has been lately conjugated to porphyrin to increase its taking up by cancer cells [22]. The use of porphyrins as translocation vectors has also been examined with the anticancer agent doxorubicin that has been conjugated to porphyrazine through an acid-labile oxime linker [23].

The aim of the current study is the synthesis of a cannabinoid-porphyrin conjugate based on our anti tumor chromenopyrazoledione (Fig. 1) [3]. During the course of this research, Bai et al. [24] reported the first CB2R-targeted photosensitizer (IR700DX-mbc94). Phototherapy treatment using IR700DX-mbc94 greatly inhibited the growth of expressed CB2R tumors but not tumors that were not expressing CB2R [25].


Synthesis

Firstly, 7-(1,1-dimethylheptyl)-1,4-dihydro-4,4-dime-thylchromeno[4,3-c]pyrazol-6,9-dione (5) was synthe - sized according to the route previously described by us (Scheme 1) [3]. Preparation of the porphyrin moiety started from the commercially available meso-tetraphenylporphyrin (TPP, 6) which was regioselectively

para-nitrated to 5-(p-nitrophenyl)-10,15,20-triphenyl-porphyrin (7). The mononitro functionality was introduced using 1.8 equiv of sodium nitrite in the presence of TFA. This regiospecific mild procedure for electrophilic nitration at the para position of the phenyl groups in TPP was previously reported by Luguya et al. [26]. This approach provides selective control in the number of nitrated phenyl groups by varying the amount of sodium nitrite and the duration of the reaction. Nitroporphyrin 7 was then easily reduced with tin (II) chloride to obtain 5-(p-aminophenyl)-10,15,20-triphenylporphyrin 8 (Scheme 2).

The conversion of the amino group of porphyrin 8 to the carboxylic acid 9, was achieved by reaction with diglycolic anhydride in DMF [27]. Unfortunately, the coupling of porphyrin 9 with chromenopyrazoledione 5 that was attempted through the following procedures was not achieved in our hands. We first proposed the conversion of the carboxylic acid 9 to the corresponding acid chloride by thionyl chloride followed by reaction with 5. This procedure failed to give the desired amide. Then, different coupling reagents such as carbodiimides [carbonyldiimidazole (CDI)] or more potent coupling reagents such as phosphonium [(benzotriazol-1-yloxy)-tris[pyrrolidino] phosphonium hexafluorophosphate (PyBOP)] or uronium salts [hexafluorophosphate salt of the O-(7-azabenzotriazolyl)tetramethyl uranium (HATU)] in the presence of a base and dry DMF as solvent were also unsuccessful to give the desired cannabinoid-porphyrin conjugate. In most of these attempts, intramolecular cycli-zation of the 2-(2-amino-2-oxoethoxy)acetic acid group of compound 9 underwent the formation of a morpholine-3,5-dione affording porphyrin 10. To our knowledge, 5-[4-(3 ,5-dioxomorphol ino)phenyl]-10,15,20-triphenylporphyrin 10 has never been described in the literature. This intramolecular cyclization has scarcely

O

NHN O

O

Fig. 1. para-Chromenopyrazoledione: an antitumoral agent

R1

OH

HO O

OH O OH

R1O

O OH

R1 O

OH

R1

HN N

R1 = 1,1-dimethylheptyl

(i) (iii)(ii)

O

O

R1

HN N

(iv)

O31 2 4 5

Scheme 1. Synthesis of 7-(1′,1′-dimethylheptyl)-dihydro-4,4-dimethylchromeno[4,3-c]pyrazol-6,9-dione. Reaction conditions. (i) 3,3-dimethylacrylic acid, CH3SO3H, P2O5, 70 °C, M.W., 10 min (81%). (ii) NaH, THF, M.W., 46 °C, 20 min then ethyl formate, THF, M.W., 46 °C, 20 min (76%). (iii) H2N–NH2, EtOH, 16 h, room temperature, (81%). (iv) [bis(trifluoro-acetoxy)iodo]benzene, ACN/H2O (6:1), 15 min, room temperature (21%)


SYNTHESIS OF A NOVEL CB2 CANNABINOID-PORPHYRIN CONJUGATE BASED 69

been studied in the literature in which the morpholine-3,5-dione was described as a by-product [28–30].

To avoid this intramolecular cyclization, another synthetic approach using butane-1,3-dione unstead of 1,1′-oxybis(ethane-2-one) as linker between the porphyrin and the chromenopyrazole was attempted without success.

After these consecutive synthetic failures in obtaining the desired cannabinoid-porphyrin conjugate, we decided to use the piperazine derivative 12 as starting material for the coupling with chromenopyrazole 5. Interestingly, the piperazine moiety is an appropriate linker because of its low toxicity and biotransformation that involves several well-known metabolic reactions [31]. 5-(4α-piperazineacetylamidophenyl)-10,15,20-triphenyl-porphyrin (12) was previously described by Gaware et al.

[32] as intermediate in the preparation of a conjugate of tetraphenylporphyrin with glucosamines. Thus, amino-porphyrin 8 was firstly acylated using bromoacetyl bromide to give 5-(4α-bromoacetylamidophenyl)-10,15,20-triphe-nylporphyrin (11). Then, a nucleophilic substitution with piperazine afforded the nucleophilic porphyrin

intermediate 5-(4α-piperazineacetylamidophenyl)-10,15,- 20-triphenylporphyrin (12) (Scheme 3). Finally, the synthesis of the porphyrin-cannabinoid conjugate 14 was achieved as depicted in Scheme 4. Acylation of chromenopyrazoledione 5 using bromoacetyl bromide afforded the substituted chromenopyrazole 13 that was then allowed to alkylate the piperazine intermediate 9 affording the desired conjugate 14.

Attempts to directly link the aminoporphyrin 8 to compound 13 did not give the porphyrin-chromeno-pyrazoledione conjugate in our experiments. This fact may be due to the weak nucleophilicity of the aminoporphyrin [33].

Conformational analysis of the porphyrin-chromenopyrazoledione conjugate

A complete conformational analysis of the novel porphyrin conjugate 14 was performed using ab initio Hartree–Fock calculations at the 6-31G* level as encoded in Spartan ´08 (Wave function, Inc., Irvine CA). As displayed in Fig. 2, the global minimum energy conformer

NNH

N HN

NNH

N HN

NO2

NNH

N HN

NH2

(i)

7 8

(ii)

NNH

N HN

NH

9

(iii)

O

O

O

OH

NNH

N HN

NH

O

O

O

NN

O

O

O

6

NNH

N HN

ON

O

O

10

(iv)

2-(2-Oxo-2-((4-(10,15,20-triphenylporphyrin-5-yl)phenyl)amino)ethoxy)acetic acid

Scheme 2. Synthesis of porphyrin derivatives. Reaction conditions. (i) NaNO2 (1.8 equiv), TFA, 25 °C, 3 min (49%). (ii) SnCl2, conc. HCl, 65 °C, 1 h (96%). (iii) Diglycolic anhydride, DMF, rt, 24 h (85%). (iv) (a) SOCl2, toluene, 120 °C, 30 min, MW; (b) chromenopyrazole 5, NaH, CH2Cl2, rt, overnight



of conjugate 14 adopts an expanded spatial conformation whereas folded conformers (Fig. 2b) exert higher relative energy values. Nonetheless, these theoretical values are calculated under vacuum conditions. Physiological conditions may influence these conformations.

Photophysicochemical properties

The UV-vis spectra of the porphyrin-chromeno-pyrazoledione conjugate 14 and the free meso-tetraphenyl - porphyrin (TPP) were recorded at 0.1 mM. A Soret

band with absorption maxima near 420 nm and medium Q-bands at 500–700 nm were observed for both porphyrins (Fig. 3). As clearly depicted in Fig. 3, compound 14 shows stronger absorption intensity for both Soret and Q-bands than the free TPP (Table 1). This suggests aggregation processes for the conjugate 14 that are less intense than for TPP. Broadening of Soret band is characteristic of π–π stacking and hydrophobic interactions in porphyrin systems. The aggregate formation is clearly affected by the ionic strength of the solvent (Fig. 3) [32, 34].

NNH

N HN

NH2

NNH

N HN

NH

O Br

NNH

N HN

NH

O N

NH

8 11 12

(i) (ii)

Scheme 3. Synthesis of the porphyrin-piperazine intermediate 12. Reaction conditions. (i) Bromoacetyl bromide, Et3N, CH2Cl2, 25 °C, 1 h (31%). (ii) Piperazine, CH2Cl2, 25 °C, 45 min (96%)

O

NHN

O

NN

O

Br

NNH

N HN

NH

O N

N

O

NN

O

O

O

O

O

O

O

13 14

(i) (ii)

5

Scheme 4. Synthesis of the porphyrin-chromenopyrazoledione conjugate 14. Reaction conditions. (i) Bromoacetyl bromide, Et3N, CH2Cl2, 25 °C, 1 h (37%). (ii) Porphyrin 9, Et3N, CH2Cl2, 25 °C, overnight (12%)

Fig. 2. (a) Global minimum energy conformer of compound 14 (ΔE: -0.11 Kcal/mol). (b) Higher energy conformer of compound 14 showed for comparison (ΔE: 4.55 Kcal/mol)



The fluorescence intensity of the TPP and compound 14 in aqueous solution at 0.1 mM did not show detectable emission. This absence of fluorescence may be caused by porphyrin-solvent interactions promoting non-radiative decay or self-aggregation of porphyrin molecules. Excitation of compound 14 dissolved in DCM (0.1 mM) at 418 nm resulted in the fluorescence spectrum displayed in Fig. 4.

Cannabinoid receptor affinity

The cannabinoid binding affinity of the porphyrin-chromenopyrazoledione conjugate 14 was evaluated by radioligand competition experiments for both receptor types CB1R and CB2R. The porphyrin-piperazine intermediate 12 was also appraised in these assays. As depicted in Table 2, the new conjugate 14 displayed very low affinity for CB2R and did not bind to CB1R. Thus, compound 14 does not retain the affinity of its

Fig. 3. UV-vis absorption spectra of 14 and tetraphenylporphyrin (TPP) at constant concentration (0.1 mM) in different solvents at room temperature

Fig. 4. Fluorescence spectrum of compound 14 (0.1 mM) under excitation with light of 418 nm in dichloromethane (slit width: 15–15 nm, and 1 cm path length)

Table 1. Absorption maxima and molecular extinction coefficients of 14 and tetraphenylporphyrin (TPP) in different solvents at room temperature

TPP (water/DMSO) Compd 14 (water/DMSO) Compd 14 (dioxane) Compd 14 (DCM)

λmax, nm ε, M-1 cm-1 λmax, nm ε, M-1 cm-1 λmax, nm ε, M-1 cm-1 λmax, nm ε, M-1 cm-1

Soret band 418 17763 423 69244.8 418 201449 418 173102.4

517 1984 518 6985.6 514 10276 515 7721

Q-band 552 968 553 3979.2 549 5752 550 3782

591 688 592 2417.6 591 3564 590 2300

648 479 648 1731.2 647 2809 646 1795



chromenopyrazole precursor 5. The TPP intermediate 12 did not show binding affinity for both receptor types.

EXPERIMENTAL

Chemistry

General methods and materials. Reagents and solvents were purchased from Sigma-Aldrich Co., Fluorochem, Acros Organics, Manchester Organics and Lab-Scan and were used without further purification or drying. Silica gel 60 F254 (0.2 mm) thin layer plates were purchased from Merck GmbH. Products were purified using flash column chromatography (Merck Silica gel 60, 230–400 mesh). The compounds were characterized by a combination of NMR experiments, HPLC-MS, and high-resolution mass spectrometry (HRMS). HPLC-MS analysis was performed on a Waters 2695 HPLC system equipped with a photodiode array 2996 coupled to Micromass ZQ 2000 mass spectrometer (ESI-MS), using a reverse-phase column SunFireTM (C-18, 4.6 × 50 mm, 3.5 μm) in gradient A: CH3CN/0.1% formic acid, B: H2O/0.1% formic acid visualizing at λ = 254 nm. Flow rate was 1 mL/min. 1H NMR and 13C NMR spectra were recorded on a Bruker (300 and 75 MHz) at 25 °C. Samples were prepared as solutions in deuterated solvent and referenced to internal non-deuterated solvent peak. Chemical shifts were expressed in ppm. Coupling constants are given in hertz (Hz). The purity of the novel compounds was determined by LC coupled to HRMS. The experiment was performed in a LC-MS hybrid quadrupole/time of flight (QTOF) analyzer equipped with an Agilent 1200 LC coupled to an Agilent 6500 Accurate Mass (1–2 ppm mass accuracy) using electrospray ionization in the positive mode (ESI+). Elemental analyses of the compounds were performed using a LECO CHNS-932 apparatus. Deviations of the elemental analysis results from the calculated are within ± 0.4%.

UV-vis measurements were recorded on a Perkin-Elmer Lambda 25 UV-vis spectrometer. Fluorescence emission spectra for quantum yield were obtained using a SPEX FluoroMax spectrometer (Spectrocell Corporation, Oreland, PA, USA).

Synthesis

7-(1,1-Dimethylheptyl)-5-hydroxy-2,2-dimethyl-chroman-4-one (2) [3]. 5-(1,1-Dimethylheptyl)resorcinol (1) (2.50 g, 10.59 mmol) and 3,3-dimethylacrylic acid (1.59 g, 15.88 mmol) both dissolved in methanesulfonic acid (16 mL, 0.24 mmol) were added to P2O5 (1.20 g, 8.81 mmol) under nitrogen atmosphere. Then, the reaction mixture was stirred 8 h at 70 °C. Afterwards, water was added (50 mL) and the product was extracted with EtOAc (3 × 50 mL). The combined organic layers were dried over MgSO4. The organic solvent was evaporated under reduced pressure and the crude was purified by column chromatography on silica gel (hexane/EtOAc, 5:1), obtaining the desired compound as a pale yellow solid. Yield 2.77 g (81%), mp 50–52 °C. 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 11.53 (s, 1H), 6.45 (d, J = 1.6 Hz, 1H), 6.37 (d, J = 1.6 Hz, 1H), 2.71 (s, 2H), 1.60–1.49 (m, 2H), 1.47 (s, 6H), 1.22 (s, 6H), 1.23–1.17 (m, 6H), 1.11–0.94 (m, 2H) 0.84 (t, J = 6.6 Hz, 3H). 13C NMR (75 MHz; CDCl3): δC, ppm 197.4, 162.6, 161.3, 159.5, 106.6, 105.7, 105.3, 78.9, 48.1, 44.0, 38.7, 31.7, 29.9, 22.6, 28.4, 26.7, 24.6, 14.0. HPLC-MS: [A, 80→95%], tR: 4.94 min, (95%). MS (ES+): m/z 319 [M + H]+. Anal. calcd. for C20H30O3: C, 75.43; H, 9.50. Found C, 75.52; H 9.64.

7-(1,1-Dimethylheptyl)-5-hydroxy-3-hydroxy-methylene-2,2-dimethylchroman-4-one (3) [3]. A solu - tion of 2 (0.40 g, 1.25 mmol) in anhydrous THF (8 mL) was added to a vial containing dry sodium hydride (0.30 g, 12.57 mmol) under nitrogen atmosphere. The mixture was irradiated under microwave at 45 °C for 25 min. Subsequently, ethyl formate (2.88 mL, 37.70 mmol) was added to the sealed vial and it was irradiated under microwave at 45 °C for 25 min. Water was added and the product was extracted with EtOAc (3 × 5 mL). The combined organic layers were dried over MgSO4 and the solvent was evaporated under reduced pressure. The crude was purified by column chromatography on silica gel (hexane/EtOAc, 4:1), to afford compound 3 as a yellow oil. Yield 0.33 g (76%). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 13.49 (d, J = 11.7 Hz, 1H), 11.28 (s, 1H), 7.34 (d, J = 11.7 Hz, 1H), 6.47 (d, J = 1.6 Hz, 1H), 6.36 (d, J = 1.6 Hz), 1.58 (s, 6H), 1.56–1.46 (m, 2H), 1.22 (s, 6H), 1.14–1.28 (m, 6H), 1.10–1.04 (m, 2H), 0.84 (t, J = 6.5 Hz, 3H). 13C NMR (75 MHz, CDCl3): δC, ppm 189.4, 162.7, 161.6, 161.5, 158.7, 114.4, 107.4, 106.2, 104.9, 78.3, 44.4, 38.8, 31.7, 29.9, 22.6, 28.4, 28.2, 24.6, 14.1. HPLC-MS: [A, 80→95%], tR: 2.88 min, (97%). MS (ES+): m/z 347 [M + H]+. Anal. calcd. for C21H30O4: C, 72.80; H, 8.73. Found C, 73.07; H 8.64.

Table 2. Binding affinity of the chromenopyrazole 5, the porphyrin-chromenopyrazoledione conjugate 14, the porphyrin intermediate 12 and the reference cannabinoid WIN55,212-2 for hCB1R and hCB2R

Compound CB1R Ki, μMa CB2R Ki, μMa

5 0.32 ± 0.23 0.13 ± 0.02

12 > 40 > 40

14 > 40 13.79 ± 0.20

WIN55,212-2 0.04 ± 0.08 0.003 ± 0.002

aValues obtained from competition curves using [3H]CP55940 as radioligand for hCB1R and hCB2R and are expressed as the mean ± SEM of at least three experiments.



7-(1,1-Dimethylheptyl)-2,4-dihydro-4,4-dime-thylchromeno[4,3-c]pyrazol-9-ol (4) [3]. A solution of 3 (0.50 g, 1.44 mmol) and anhydrous hydrazine (0.11 mL, 3.61 mmol) in EtOH (9 mL) was stirred during 4 h at 40 °C. The solvent was evaporated under reduced pressure and the crude was purified by column chromatography on silica gel (hexane/EtOAc, 2:1) to furnish 4 as a yellow oil. Yield 0.40 g (81%). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 7.32–7.29 (br s, 1H), 6.58 (d, J = 1.5 Hz, 1H), 6.51 (d, J = 1.5 Hz), 6.48 (s, 1H), 1.63 (s, 6H), 1.58–1.52 (m, 2H), 1.25 (s, 6H), 1.18 (s, 6H), 1.12–1.05 (m, 2H), 0.83 (t, J = 6.7 Hz, 3H). 13C NMR (75 MHz; CDCl3): δC, ppm 153.7, 153.5, 153.4, 144.1, 129.1, 123.4, 106.8, 106.5, 101.7, 77.0, 44.9, 38.4, 32.2, 30.4, 30.0, 29.3, 25.0, 23.1, 14.5. HPLC-MS: [A, 80 95%], tR: 3.80 min, (98%). MS (ES+): m/z 343 [M + H]+. Anal. calcd. for C21H30N2O2: C, 73.65; H, 8.83. Found C, 74.01; H, 8.59.

7-(1,1-Dimethylheptyl)-1,4-dihydro-4,4-dimethyl-chromeno[4,3-c]pyrazol-6,9-dione (5) [3]. To a solution of 7-(1,1-dimethylheptyl)-1,4-dihydro-4,4-dimethyl - chromeno[4,3-c]pyrazol-9-ol (4) (130 mg, 0.38 mmol) in MeCN/H2O (6:1, 2.5 mL) a solution of BTIB (490 mg, 1.14 mmol) in MeCN/H2O (6:1, 2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 15 min, neutralized with aqueous NaHCO3 saturated solution, and extracted with diethyl ether. The organic layer was washed with H2O, dried over MgSO4 and concentrated. Column chromatography on silica gel (hexane/EtOAc, 1:2) afforded the title compound as a red solid. Yield 29 mg (21%). mp 85–86 °C. 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 8.41 (br s, 1H), 7.40 (s, 1H), 6.69 (s, 1H), 1.59–1.57 (br s, 6H), 1.55–1.48 (m, 2H), 1.30 (s, 6H), 1.27–1.23 (m, 6H), 1.19–1.12 (br s, 2H), 0.86–0.82 (m, 3H). 13C NMR (75 MHz; CDCl3): δC, ppm 184.1, 180.9, 160.2, 161.2, 137.8, 132.0, 130.4, 129.5, 113.8, 78.6, 43.3, 30.9, 29.6, 28.7, 27.4, 25.1, 23.2, 21.8, 14.0. HPLC-MS: [A, 70% 100%], tR: 3.37 min (98%). MS (ES+): m/z 357 [M + H]+. Anal. calcd. for C21H28N2O3: C, 70.76; H, 7.92. Found C, 71.03; H, 8.24.

5-(4-Nitrophenyl)-10,15,20-triphenylporphyrin (7) [26]. To a solution of meso-tetraphenylporphyrin (TPP, 6) (500 mg, 0.81 mmol) in TFA (25 mL) sodium nitrite (99 mg, 1.40 mmol) was added and the reaction mixture was stirred for 3 min at room temperature. After that, the crude was poured into water and extracted three times with CH2Cl2. The organic layers combined and washed with saturated aqueous NaHCO3 and water. The mixture was dried over anhydrous Na2SO4 and the solvent was removed under vacuum. Flash column chromatography (CH2Cl2) provided the title compound as a purple solid. Yield 262 mg (49%). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 9.03–8.99 (m, 2H), 8.86–8.79 (m, 6H), 8.59 (d, J = 8.1 Hz, 2H), 8.28 (d, J = 8.1 Hz, 2H), 8.19–8.12 (m, 6H), 7.81–7.64 (m, 9H), -2.77 (s, 2H). HPLC-MS: [iso 95%–5%], tR: 10.0 min

(94%). MS (ES+): m/z 660 [M + H]+. HRMS calcd. for C44H29N5O2: 659.2321. Found 659.2298.

5-(4-Aminophenyl)-10,15,20-triphenylporphyrin (8) [26]. 5-(4-Nitrophenyl)-10,15,20-triphenylporphyrin (7) (101 mg, 0.15 mmol) was dissolved in concentrated hydrochloric acid (10 mL) and, while stirring, tin(II) chloride (162 mg, 0.85 mmol) was carefully added. The mixture was heated to 65 °C for 1 h under nitrogen atmosphere. The crude was then poured into cold water and neutralized with ammonium hydroxide until pH 8. The aqueous solution was extracted with CH2Cl2 until colorless. The organic layers were combined, dried over Na2SO4, and the solvent was removed under reduced pressure. Flash column chromatography (CH2Cl2) afforded the title compound as a purple solid (92 mg, 96% yield). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 8.93–8.91 (m, 2H), 8.79–8.67 (m, 6H), 8.22–8.19 (m, 6H), 8.10 (d, J = 7.8 Hz, 2H), 7.84–7.75 (m, 9H), 7.02 (d, J = 7.8 Hz, 2H), 4.02 (s, 2H), -2.69 (s, 2H). HPLC-MS: [iso 95%–5%], tR: 6.13 min (99%). MS (ES+): m/z 630 [M + H]+. HRMS calcd. for C44H31N5: 629.2579. Found 629.2583.

2-{2-Oxo-2-[(4-(10,15,20-triphenylporphyrin-5- yl)phenyl)amino]ethoxy}acetic acid (9) [27]. To a solution of aminoporphyrin 8 (340 mg, 0.53 mmol) in DMF (3 mL) diglycolic anhydride (93 mg, 0.80 mmol) was added. The reaction was stirred at room temperature overnight. The crude was diluted with CHCl3 and hexane until precipitation occurred. The precipitate was filtered and washed with water to remove residual anhydride and then dried under vacuum to obtain the title compound as a purple solid. Yield 340.9 mg (85%). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 9.01–8.94 (m, 2H), 8.82–8.79 (m, 6H), 8.31–8.16 (m, 10H), 7.78–7.64 (m, 9H), 4.61 (s, 2H), 4.45 (s, 2H), -2.75 (br s, 2H). HRMS calcd. for C48H35N5O4: 745.2689. Found 745.2701.

5-[4-(3,5-Dioxomorpholino)phenyl]-10,15,20-triphenylporphyrin (10). A solution of compound 9 (40 mg, 0.05 mmol) in toluene (2 mL) and SOCl2 (6 μL, 0.08 mmol) was heated at 120 °C under microwave irradiation conditions for 30 min. The solvent was removed under vacuum and the corresponding acyl chloride was used for the next step without further purification. A solution of 5 (14 mg, 0.04 mmol) in anhydrous CH2Cl2 (1 mL) was added to a precooled suspension of NaH (3 mg, 0.12 mmol) in CH2Cl2, the mixture was stirred for 10 min under nitrogen atmosphere. After that, the acyl chloride (30 mg, 0.04 mmol), dissolved in anhydrous CH2Cl2 (1 mL), was rapidly added and the reaction was stirred for 30 min. The reaction mixture was then diluted with CH2Cl2 and washed with water and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. Column chromatography on silica gel (MeOH/CH2Cl2, 1:12) afforded the title undesired compound as a purple solid. Yield 15 mg (52%). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 8.99–8.87 (m, 2H), 8.80–8.72 (m, 6H), 8.23–8.15 (m, 8H), 7.97 (d,



J = 8.0 Hz, 2H), 7.82–7.74 (m, 9H), 4.39 (s, 4H), -2.88 (s, 2H). HPLC-MS: [iso 95%–5%], tR: 4.31 min (99%). MS (ES+): m/z 758 [M + H]+. HRMS calcd. for C48H33N5O3: 727.2583. Found 727.2602.

5-(4α-Bromoacetylamidophenyl)-10,15,20-tri-phenylporphyrin (11) [32]. A solution of amino-porphyrin 8 (600 mg, 0.95 mmol) in CH2Cl2 (10 mL) and Et3N (0.29 mL, 2.01 mmol) was stirred under N2 atmosphere. Bromoacetylbromide (0.11 mL, 1.33 mmol) was added dropwise at room temperature and the reaction mixture was stirred for 1 h. The crude was diluted in CH2Cl2, washed with water and brine and extracted three times. The combined organic layers were dried over Na2SO4 and concentrated under vacuum. Flash column chromatography on silica gel (CH2Cl2) afforded the title compound as a purple solid. Yield 220 mg (31%). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 8.84–8.79 (m, 2H), 8.74–8.68 (m, 6H), 8.37–8.34 (br s, 1H), 8.26–8.11 (m, 10H), 7.79–7.63 (m, 9H), 4.22 (s, 2H), -2.81 (br s, 2H). HPLC-MS: [iso 95%–5%], tR: 6.27 min (99%). MS (ES+): m/z 751 [M + H]+. HRMS calcd. for C46H32BrN5O: 749.1790. Found 749.1814.

5-(4α-Piperazineacetylamidophenyl)-10,15,20-triphenylporphyrin (12) [32]. A mixture of bromo acety-lated porphyrin 11 (50 mg, 0.06 mmol) and piperazine (34 mg, 0.39 mmol) in CH2Cl2 (3 mL) were stirred at room temperature for 1 h under N2 atmosphere. The reaction mixture was then diluted with CH2Cl2 and washed with water and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. Column chromatography on silica gel (MeOH/CH2Cl2, 1:12) afforded the title compound as a purple solid. Yield 29 mg (96%). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 9.07–8.99 (br s, 1H), 8.88–8.73 (m, 8H), 8.21–8.09 (m, 10H), 7.87–7.70 (m, 9H), 4.07 (s, 2H), 2.95–2.89 (br t, 4H), 2.73–2.68 (br t, 4H), 2.43 (br s, 1H), -2.82 (s, 2H). HPLC-MS: [A, 60%→95%], tR: 2.42 min (90%). MS (ES+): m/z) 756 [M + H]+. HRMS calcd. for C50H41N7O: 755.3373. Found 755.3351.

2-(2-Bromoacetyl)-7-(1,1-dimethylheptyl)-1,4-dihydro-4,4-dimethylchromeno[4,3-c]pyrazol-6,9-dione (13). Compound 5 (35 mg, 0.10 mmol) was dissolved in anhydrous CH2Cl2 (3 mL) and stirred under N2 atmosphere. Et3N (30 μL, 0.21 mmol) was added, followed by dropwise addition of bromoacetylbromide (13 μL, 0.15 mmol) at room temperature. Stirring was continued at room temperature for 1 h. The reaction mixture was diluted in CH2Cl2, then washed with water and brine and the product was extracted three times. The combined organic layers were then dried over Na2SO4 and the solvent was removed under vacuum. Column chromatography on silica gel (hexane/EtOAc, 1:2) afforded the title compound as an orange oil. Yield 17 mg (37%). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm7.74 (s, 1H), 6.85 (s, 1H), 4.52 (s, 2H), 1.64–1.59 (br s, 6H), 1.54–1.47 (m, 2H), 1.38 (s, 6H), 1.33–1.27 (m, 6H), 1.22–1.17 (br s, 2H), 0.99–0.87 (m, 3H). HPLC-MS: [A,

80% → 95%], tR: 4.16 min (93%). MS (ES+): m/z 477 [M + H]+. HRMS calcd. for C23H29BrN2O4: 476.1311. Found 476.1328.

Porphyrin-chromenopyrazoledione conjugate (14). Compound 12 (14 mg, 0.02 mmol) dissolved in anhydrous CH2Cl2 (2 mL) was stirred in Et3N (3 μL, 0.02 mmol) under N2 atmosphere for 5 min. Compound 13 (17 mg, 0.04 mmol), dissolved in anhydrous CH2Cl2 (2 mL), was rapidly added and the reaction was stirred overnight at room temperature. The reaction mixture was diluted in CH2Cl2, then washed with water and brine and the product was extracted three times. The combined organic layers were then dried over Na2SO4 and the solvent was removed under vacuum. Column chromatography on silica gel (MeOH/CH2Cl2, 1:12) afforded the title compound as a purple solid. Yield 2.50 mg (12%). 1H NMR (300 MHz; CDCl3; Me4Si): δH, ppm 9.19–9.14 (br s, 1H), 8.91–8.79 (m, 8H), 8.24–8.11 (m, 10H), 7.81 (s, 1H), 7.67–7.54 (m, 9H), 6.79 (s, 1H), 4.13 (s, 2H), 3.88 (s, 2H), 2.88–2.76 (br t, 4H), 2.63–2.60 (br t, 4H), 1.72–1.63 (m, 6H), 1.58–1.46 (m, 2H), 1.41 (s, 6H), 1.41–1.34 (m, 6H), 1.30–1.21 (br s, 2H), 0.85 (t, J = 7.0 Hz, 3H), -2.78 (s, 2H). 13C NMR (75 MHz; CDCl3): δC, ppm 182.7, 181.3, 170.1, 162.8, 161.5, 143.6, 139.4, 137.6, 136.1, 134.9, 134.0, 131.7, 130.9, 130.2, 128.8, 127.3, 126.5, 121.0, 119.7, 118.1, 114.3, 77.5, 62.6, 54.6, 54.2, 46.7, 44.0, 31.6, 29.9, 28.1, 26.9, 25.3, 22.8, 22.0, 14.1. HPLC-MS: [A, 60% 95%], tR: 11.28 min (91%). MS (ES+): m/z 1152 [M + H]+. HRMS calcd. for C73H69N9O5: 1151.5422. Found 1151.5456.

Cannabinoid binding experiments

Membranes from transfected cells with human canna-binoid receptors (RBHCB1M400UA and RBXC-B2M400UA) were supplied by Perkin-Elmer Life and Analytical Sciences (Boston, MA). The protein concen-tration for the CB1R membranes was 8.0 mg.mL-1, whereas for the CB2R membranes the protein concentration was 4.0 mg.mL-1 or 3.6 mg.mL-1 depending on the batch. The commercial membranes were diluted (approximatively 1:20) with the binding buffer (50 mM TrisCl, 5 mM MgCl2.H2O, 2.5 mM EDTA, 0.5 mg.mL-1 BSA and pH = 7.4 for CB1R binding; 50 mM TrisCl, 5 mM MgCl2.H2O, 2.5 mM EGTA, 1 mg.mL-1 BSA and pH = 7.5 for CB2R binding). The final membrane protein concentration was 0.4 mg.mL-1 of incubation volume and 0.2 mg/mL of incubation volume for the CB1R and the CB2R assays, respectively. The radioligand used was [3H]-CP55940 (PerkinElmer) at a concentration of membrane KD × 0.8 nm, and the final volume was 200 μL for CB1R binding and was 600 μL for CB2R binding. 96-Well plates and the tubes necessary for the experiment were previously siliconized with Sigmacote (Sigma).

Membranes were resuspended in the corresponding buffer and were incubated with the radioligand and each compound (10-4–10-11 M) for 90 min at 30 °C. Non-specific



binding was determined with 10 μM WIN55212-2 and 100% binding of the radioligand to the membrane was determined by its incubation with membrane without any compound. Filtration was performed by a Harvester®

filtermate (Perkin-Elmer) with Filtermat A GF/C filters pretreated with polyethylenimine 0.05%. After filtering, the filter was washed nine times with binding buffer, dried and a melt-on scintillation sheet (MeltilexTM A, Perkin Elmer) was melted onto it. Then, radioactivity was quantified by a liquid scintillation spectrophotometer (Wallac MicroBeta Trilux, Perkin-Elmer). Competition binding data were analyzed by using GraphPad Prism program and Ki values are expressed as mean ± SEM of at least three experiments performed in triplicate for each point.

CONCLUSIONS AND FUTURE PERSPECTIVES

With the purpose of developing an antitumor agent, chromenopyrazoledione 5 was conjugated to a tetraphenylporphyrin derivative. This macrocycle may confer to our cannabinoid a more specific tumor tissue delivery and may enable the development of target-selective phototherapy approaches. The novel conjugate 14 binds weakly but selectively to CB2R. Further studies involving 14 will consist of in vivo assays to study its metabolism processes. Additionally, the synthetic design in this study provided a methodology to prepare a new tetraphenylporphyrin, 5-[4-(3,5-dioxomorpholino)phenyl]-10,15,20-triphenylporphyrin (10).

Acknowledgements

N.J. and L.M. are indebted to Kevin M. Smith and Graça H. Vicente for their encouragement and assistance. Financial support by Spanish Grants from the Spanish Ministry MINECO/FEDER and SAF2015-68580-C2, from CAM S2010/BMD-2308.

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CONTENTS


Reductive nitrosylation of ferric carboxymethylated-cytochrome c 1Paolo Ascenzi*, Chiara Ciaccio, Giovanna De Simone, Roberto Santucci and Massimo Coletta

Stabilization of meso-tetraferrocenyl-porphyrin films by formation of composite with Prussian blue 10 Kalil Cristhian Figueiredo Toledo, Bruno Morandi Pires, Juliano Alves Bonacin* and Bernardo Almeida Iglesias*

Lyotropic liquid crystalline phthalocyanines containing 4-((S)-3,7-dimethyloctyloxy)phenoxy moieties 16Sibel Eken Korkut*, Hale Ocak, Belkıs Bilgin-Eran, Dilek Güzeller and M. Kasım S¸ener*

Preparation and biological evaluation of a carrier free 90yttrium labelled porphyrin as a possible agent for targeted therapy of tumor

Mahvash Abedi*, Mohammad Reza Nabid, Simindokht Shirvani-Arani, Ali Bahrami-Samani 24 and Nasim Vahidfar

Preparation, characterization and investigation of photo-physical properties of thiophene-substituted 31 rare-earth bisphthalocyanines

Jirí Cerný*, Lenka Dokládalová, Antonín Lycka, Tomáš Mikysek and Filip Bureš

Symmetrical and difunctional substituted cobalt phthalocyanines with benzoic acids fragments: Synthesis 37 and catalytic activity

Artur Vashurin*, Vladimir Maizlish, Ilya Kuzmin, Serafima Znoyko, Anastasiya Morozova, Mikhail Razumov and Oscar Koifman

Flying-seed-like liquid crystals 7†: Synthesis and mesomorphism of novel octakis(m-chloropyridyloxy)- 48 phthalocyanato copper(II) complexes

Kazuchika Ohta*, Kaori Adachi and Mikio Yasutake

Photophysical properties of sinoporphyrin sodium and explanation of its high photo-activity 59 Lixin Zang, Huimin Zhao*, Qicheng Fang, Ming Fan, Tong Chen, Ye Tian, Jianting Yao, Yangdong Zheng*, Zhiguo Zhang* and Wenwu Cao*

Synthesis of a novel CB2 cannabinoid-porphyrin conjugate based on an antitumor chromenopyrazoledione 67Paula Morales, Laura Moreno, Javier Fernández-Ruiz and Nadine Jagerovic*

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