DOI: 10.1002/chem.201403214

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& Protoporphyrin IX

Synthesis of New Chlorin e6 Trimethyl and Protoporphyrin IX Dimethyl Ester Derivatives and Their Photophysical and Electrochemical Characterizations Jos C. J. M. D. S. Menezes,[a] M. Amparo F. Faustino,[a] Kleber T. de Oliveira,[b] Marciana P. Uliana,[b] Vitor F. Ferreira,[c] Steffen Hackbarth,[d] Beate Rçder,[d] Thiago Teixeira Tasso,[e] Taniyuki Furuyama,[e] Nagao Kobayashi,*[e] Artur M. S. Silva,[a] M. GraÅa P. M. S. Neves,*[a] and Jos A. S. Cavaleiro*[a]

Abstract: In view of increasing demands for efficient photosensitizers for photodynamic therapy (PDT), we herein report the synthesis and photophysical characterizations of new chlorin e6 trimethyl ester and protoporphyrin IX dimethyl ester dyads as free bases and ZnII complexes. The synthesis of these molecules linked at the b-pyrrolic positions to pyrano[3,2-c]coumarin, pyrano[3,2-c]quinolinone, and pyrano[3,2-c]naphthoquinone moieties was performed by using the domino Knoevenagel hetero Diels–Alder reaction. The a-methylenechromanes, a-methylenequinoline, and ortho-quinone methides were generated in situ from a Knoevenagel reaction of 4-hydroxycoumarin, 4-hydroxy-6-methylcoumarin, 4-hydroxy-N-methylquinolinone, and 2-hydroxy1,4-naphthoquinone, respectively, with paraformaldehyde in dioxane. All the dyads as free bases and as ZnII complexes were obtained in high yields. All new compounds were fully

characterized by 1D and 2D NMR techniques, UV/Vis spectroscopy, and HRMS. Their photophysical properties were evaluated by measuring the fluorescence quantum yield, the singlet oxygen quantum yield by luminescence detection, and also the triplet lifetimes were correlated by flash photolysis and intersystem crossing (ISC) rates. The fluorescence lifetimes were measured by a time-correlated single photon count (TCSPC) method, fluorescence decay associated spectra (FDAS), and anisotropy measurements. Magnetic circular dichroism (MCD) and circular dichroism (CD) spectra were recorded for one ZnII complex in order to obtain information, respectively, on the electronic and conformational states, and interpretation of these spectra was enhanced by molecular orbital (MO) calculations. Electrochemical studies of the ZnII complexes were also carried out to gain insights into their behavior for such applications.

Introduction Tetrapyrrolic derivatives like the heme group (FeII complex of protoporphyrin IX) and chlorophylls are well known for their role in vital processes like respiration and photosynthesis, respectively.[1, 2] However, interdisciplinary studies involving these types of macrocycles have pointed out the significant uses of their derivatives in catalysis, as sensors or biocides, new electronic materials, and in medicine.[3, 4] In particular, their use in medicine is mainly concerned with the detection and photodynamic therapy of cancer cells and other diseases, like wet age-related macular degeneration (AMD), acne, and also in microbial photoinactivation processes.[3–5] Detection application exploits the fluorescent properties of these macrocycles and the photodynamic approach is based on their selective accumulation in cancer cells and on the properties of their triplet states to participate in electron transfer, hydrogen abstraction, or interaction with oxygen in the formation of singlet oxygen (1O2) as well as other cytotoxic oxygen species (radicals). This form of phototherapy, wherein the exposure of the photosensitizer to light results in the production of reactive oxygen species, which becomes toxic to

[a] Dr. J. C. J. M. D. S. Menezes, Prof. M. A. F. Faustino, Prof. A. M. S. Silva, Prof. M. G. P. M. S. Neves, Prof. J. A. S. Cavaleiro Department of Chemistry and QOPNA, University of Aveiro 3810-193 Aveiro (Portugal) Fax: (+ 351)234-401-470 E-mail: [email protected] [email protected] [b] Prof. K. T. d. . Oliveira, Dr. M. P. Uliana Departamento de Qumica, Universidade Federal de S¼o Carlos 13565-905 S¼o Carlos - SP (Brazil) [c] Prof. V. F. Ferreira Departamento de Qumica Orgnica, Universidade Federal Fluminense 24020-141 Niteri, Rio de Janeiro (Brazil) [d] Dr. S. Hackbarth, Prof. B. Rçder Institut fr Physik, Humboldt-Universitt zu Berlin Newtonstrasse 15, 12489 Berlin (Germany) [e] Dr. T. Teixeira Tasso, Dr. T. Furuyama, Prof. N. Kobayashi Department of Chemistry, Graduate School of Science Tohoku University, Sendai 980-8578 (Japan) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201403214. Chem. Eur. J. 2014, 20, 1 – 13

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Full Paper targeted malignant and other diseased cells, is known as photodynamic therapy (PDT).[6] This modality of therapy is being used clinically in different countries and it is recognized as a treatment strategy minimally invasive and with less side effects.[7] Most drug formulations in use are based on derivatives obtained from functionalized natural protoporphyrin IX or chlorophylls or others obtained by synthesis.[3, 4] For instance, a recent work using trispolyethylene glycol ester derivative of chlorin e6 (a derivative of chlorophyll a) showed increased uptake, stability to hydrolysis, and effective photokilling in human ovarian cancer cells.[8] Chlorin e6 also showed effective photodynamic inactivation against human corneal endothelial cells in vitro by decreasing the viability and the proliferation, and also triggered apoptosis.[9] In a comparative study the use of exogenous protoporphyrin IX (PP-IX) and its dimethyl ester (PME) with the endogenous PP-IX produced through 5-aminolevulinic acid (5-ALA) and ALA methyl ester (AME) in nasopharyngeal carcinoma cells clearly showed higher intake/selectivity of PME for photodynamic diagnosis and therapy in vivo.[10] It is known that the presence of pyrano[3,2-c]coumarin,[11, 12] pyrano[3,2-c]quinoline,[13, 14] and pyrano[3,2-c]naphthoquinone[15, 16] motifs in many natural products like the isoethuliacoumarins (A, B, and C), the ethuliacoumarins (A and B), flindersine, simulenoline, and a- and b-lapachone are responsible for a wide range of interesting biological properties. These derivatives are known to have antihelminthic, antimolluscicidal, anticoagulant, antibacterial, antifungal, antitumor, and antiChagas disease activities.[11–16] The incorporation of such units in the natural-based chlorin e6 and protoporphyrin IX cores can be a good strategy in the search for new drugs for PDT due to a synergic effect involving the two structural moieties. These hybrid molecules should have the advantage of being less toxic to normal tissue, because the basic core structure is derived from natural chlorin e6 or protoporphyrin IX. In addition the complimentary absorption features around l = 650 nm of the new chlorin e6 derivatives is highly interesting in PDT and relatively to porphyrins will allow deeper treatments of tumors.[17] Recently, we reported the functionalization of b-vinyl-mesotetraphenylporphyrin[18] and b-vinyl-corrole[19] through the hetero Diels–Alder reaction with o-quinone methides (o-QMs) and a-methylenechromane generated in situ from a Knoevenagel reaction of 2-hydroxy-1,4-naphthoquinone,[18] 4-hydroxycoumarins,[18, 19] with paraformaldehyde or o-hydroxybenzyl alcohol[18] by heating. The efficiency of the approach prompted us to extend the studies to macrocycles like the ZnII chlorin e6 trimethyl ester 1 and the ZnII protoporphyrin IX dimethyl ester 7. For these studies we selected 4-hydroxycoumarin, 4-hydroxy-6-methylcoumarin, 4-hydroxy-N-methylquinolin-2-one, and 2-hydroxy-1,4-naphthoquinone and paraformaldehyde to generate, respectively, the a-methylenechromanes 6 a and 6 b, the a-methylenequinoline 6 c, and the o-quinone methide 6 d. The novel dyads, ZnII complexes and free bases, of the chlorin e6 derivatives 2–5 (Scheme 1) and the protoporphyrin IX derivatives 8–11 (Scheme 2) were obtained in good to excellent yields and were fully characterized. A detailed evaluation of their photophysical and electrochemical properties were also &

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Scheme 1. Synthesis of the chlorin e6 trimethyl ester dyads 2–5.

Scheme 2. Synthesis of the protoporphyrin IX dimethyl ester dyads 8–11.

carried out bearing in mind their potentialities for future applications in medicine.

Results and Discussion Chemistry The synthetic strategy to prepare the new dyads 2–5 from ZnII chlorin e6 trimethyl ester 1 is shown in Scheme 1 and was based on the previously developed o-methylene-carbonyl hetero Diels–Alder approach.[18] All the reactions were performed by heating compound 1 to reflux in 1,4-dioxane with the intermediates 6 a–6 d (Scheme 1), which were generated in situ by using the adequate 4-hydroxycoumarin, 4-hydroxy-62

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Full Paper methylcoumarin, 4-hydroxy-N-methylquinolin-2-one, and 2-hydroxy-1,4-naphthoquinone, and eight equivalents of paraformaldehyde. All reactions were completed when TLC controls showed the total consumption of the starting chlorin 1; in all cases this disappearance was accompanied by the formation of a new product which, after workup and chromatography, was identified by a detailed spectroscopic analysis (see the Supporting Information for NMR discussion) as being the expected regioisomeric adduct/dyad 2 a–5 a, constituted by an inseparable diastereomeric mixture (ratio determined by NMR). The reactions were carried out initially with one equivalent of each precursor of intermediates 6 a–6 c; the TLC control showed, after 1 h; a partial consumption of chlorin 1 and so another equivalent was necessary to be added in each case for the reaction to go on to completion. For the adducts 2 a–4 a the yields obtained are in the range from 75 to 91 % (Table 1,

Table 2. Yields of dyads 8 a–11 a obtained from the reaction of the protoporphyrin IX dimethyl ester 7 with the intermediates 6 a–6 d in 1,4-dioxane, which was heated to reflux.

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8

6 a–6 d [equiv]

t [h]

Product

Yield [%]

d.r.

6a 6b 6c 6d 6a 6b 6c 6d

1+1 1+1 1+1 2+4 4 4 4+4 4+4

2 2 2 5 1 1 2 2

2a 3a 4a 5a 2a 3a 4a 5a

88 91 75 75 97 96 89 86

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t [h]

Product

Yield [%]

d.r.

6a 6a 6a 6b 6b 6c 6c 6d 6d 6d

2+2 2+2 2+6 2+2 6 6+6+3 8+4 6+2+2 6 4+4

5 1 2 1 2 4 3 24 16 3

8a 8a 8a 9a 9a 10 a 10 a 11 a 11 a 11 a

86 77 82 77 81 78 67 85 64 63

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Structural characterization of the new chlorin e6 and protoporphyrin IX dyads

entries 1–3) after 2 h of reaction. For the less reactive intermediate, the o-QM 6 d, the assay was started with two equivalents of its precursor but another four equivalents were required for a full conversion of the starting chlorin. The adduct was isolated in 75 % yield after 5 h of reaction (Table 1, entry 4). From additional experiments it was observed that the reaction time and the adduct yields for 2 a–5 a can be improved if the reactions are performed in the presence of a slight excess of each intermediate (Table 1, entries 5–8). Adducts 2 a and 3 a were isolated in yields of approximately 96 % in reactions with four equivalents of the corresponding intermediates, after 1 h of reaction and no addition of extra portions of the a-methylenechromanes. However, for adducts 4 a and 5 a, although the yields suffer a significant improvement (89 and 86 %, respectively, vs. 75 %) the addition of other four equivalents of the precursors of 6 c and 6 d (Table 1, entries 7 and 8) was required for a total consumption of the starting chlorin. When this approach was extended to the protoporphyrin IX dimethyl ester 7 (Scheme 2) we had to take into account the presence of the two vinyl groups and the difficulty to separate the two monosubstituted adducts from each other and from the disubstituted one (detected from the beginning of the reaction from preliminary experiments) by chromatography. In Chem. Eur. J. 2014, 20, 1 – 13

6a–6 d [equiv]

reactions under conditions leading only to the bis-adducts 8 a– 11 a, these have been isolated as diastereomeric mixtures in yields ranging from 77 to 86 % (Table 2). The reaction times and the amounts of the precursors of the intermediates 6 a– 6 d added are summarized in Table 2. As observed in the chlorin series, compounds 6 c and 6 d (see Scheme 1) were the less reactive species. Knowing that the presence or absence of a metal ion in the inner core of the macrocycle can influence the photophysical and the electrochemical features of this series of derivatives, the free base derivatives 2b–5b and 8b–11b were also obtained in good to excellent yields (67–95 %), after demetallation of the corresponding ZnII complexes by using a solution of trifluoroacetic acid (TFA) in chloroform (see Table S1 in the Supporting Information).

Table 1. Reaction optimization studies and yields of dyads 2 a–5 a obtained from the reaction of chlorin e6 trimethyl ester 1 with intermediates (6 a–6 d) in 1,4-dioxane, which was heated to reflux. Intermediate

Intermediate

The structural elucidation of all compounds involved the use of 1D (1H and 13C) and 2D [(1H,1H)-COSY, NOESY, (1H,13C)-HSQC, and (1H,13C)-HMBC] NMR techniques, HRMS, and UV/Vis spectroscopy (see the Supporting Information for a detailed discussion and the NMR spectra). Photophysical characterization Considering the potential application of the two series of derivatives as photosensitizers for PDT, the fluorescence quantum yields (Ffl) of the chlorins 2–5 and the protoporphyrins 8–11 and their ability to generate singlet oxygen were studied. For a better comparison of the results and to derive conclusions free of solvent effects, the photophysical studies were performed in dimethylformamide (DMF) in which all compounds and reference compounds are soluble. The methods selected—steady state absorption and fluorescence, time-resolved single photon counting (TCSPC), flash-photolysis and time-resolved singlet oxygen luminescence detection—have been described.[20–22] The steady state absorption spectra of all the new compounds show the expected profile related with the oxidation 3

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Full Paper state of the parent macrocycles. In the protoporphyrin series the ZnII complexes show the typical two Q-bands in the red region, whereas the free bases present four Q-bands; in this series the Soret bands appear at l = 411 nm for the ZnII complexes and at approximately l = 401 nm for the corresponding free bases. In the ZnII complex and the free-base chlorin e6 series, the typical and prominent Q-band associated to the reduced ring appears at approximately l = 633 and 657 nm, respectively, and the Soret band at l = 411 and 396 nm, respectively, (see Figure 1 for the representative compounds 2 a and 2 b).

Table 3. Quantum yields of fluorescence (Ffl), photosensitized generated singlet oxygen (FD), singlet oxygen decay time (tD), first excited tripletstate lifetimes (tT) obtained by laser flash photolysis, and intersystem crossing quantum yield (FISC) obtained with ps-TAS of compounds 1–5 and 7–11 and TPP in DMF.

1 2a 3a 4a 5a 2b 3b 4b 5b 7 8a 9a 10 a 11 a 8b 9b 10 b 11 b TPP

The excitation wavelength for both steady state fluorescence and flash photolysis experiments was l = 532 nm. Pulsed excitations for TCSPC and singlet oxygen luminescence detection were done with the second harmonic of the Nd-YAG laser systems at l = 532 nm. The new ZnII–chlorin e6 dyads exhibit a major fluorescence band centered between l = 640–646 nm and a shoulder type band at l = 675–690 nm (see Figure 1 for hybrid 2 a), which indicates a slight blue shift (around 4 nm) when compared with the starting ZnII–chlorin e6 trimethyl ester 1 with emission bands centered at l = 650 and 705 nm. In case of the free-base chlorin e6 series, the major fluorescence band appears centered between l = 662–664 nm, whereas the second band appears at l = 705 nm in case of compounds 2 b and 3 b and at l = 694 nm for compounds 4 b and 5 b (see Figure 1 for 2 b). In this chlorin e6 series (free-base and ZnII complexes) the fluorescence quantum yields, calculated by internal reference method by using meso-tetraphenylporphyrin (TPP) as standard (Ffl = 0.11 in DMF), vary between 0.13 and 0.20 for derivatives 2–4 (Table 3). A different situation was observed for compounds 5 a and 5 b, which show exceptionally low values (< 0.01). Presumably this behavior can be explained by probable light-induced electron transfer from the porphyrin to the naphthoquinone, which occurs in competition with fluorescence resulting in an observable decrease of the fluorescence values. With the electrochemical data obtained for compounds 5 a and 11 a (see below), we were able to calculate the driving force for the electron-transfer process, which was &

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FD[b]  0.05

tD [ms]  0.3 ms

tT [ms][c]  0.05 ms

tT [ms][d]  0.1 ms

FISC[e]  0.03

0.14 0.15 0.17 0.16 0.01 0.13 0.16 0.20 0.01 0.03 0.04 0.03 0.04 0.00 0.07 0.08 0.08 0.00 0.11

0.53 0.36 0.32 0.52 0.01 0.61 0.62 0.60 0.04 0.67 0.72 0.65 0.68 0.00 0.72 0.69 0.71 0.02 0.65

13.7 13.6 13.0 14.3 11.9 15.2 15.2 15.4 12.6 15.2 13.8 13.9 13.8 12.8 13.8 14.2 14.1 12.9 15.3

0.44 0.49 0.47 0.48 0.45 0.39 0.39 0.38 0.38 0.40 0.48 0.49 0.47 0.61 0.52 0.52 0.5 0.50 0.50

0.45 0.47 0.46 0.54 0.66 0.41 0.40 0.38 0.49 0.47 0.50 0.47 0.48 0.83 0.51 0.53 0.50 0.63 0.51

– 0.57 0.62 0.53 – 0.60 0.59 0.59 – 0.75 0.89 0.75 – 0.91 0.87 0.87 – –

[a] Optical density (O.D.) for all samples at l = 532 nm was 0.02. [b] O.D. for all samples at l = 532 nm was 0.2. [c] Data obtained from 1O2 kinetics and error of  0.1 for compounds 5 and 11. [d] Data obtained from laser flash photolysis with excitation at the longest Q-band; probed with LED/ interference filters (full width at half maximum (FWHM) = 10 nm) at l = 488 nm for compounds 2 a–5 a and l = 461 nm for the other compounds. [e] O.D. for samples 2 a, 3 a, and 4 a at l = 633 nm and compounds 2 b, 3 b, and 4 b at l = 657 nm was 1.0. O.D. for samples 8 a, 9 a, and 10 a at l = 540 nm and for compounds 8 b, 9 b, and 10 b at l = 496 nm was 1.0. ISC not determined for compounds 5 a, 5 b, 11 a, 11 b, and TPP.

Figure 1. Representative normalized absorption (solid lines) and fluorescence (dashed lines) spectra of the ZnII–chlorin e6 2 a (red lines) and of the corresponding free-base compound 2 b (green lines) in DMF.

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Ffl[a]  0.05

found to be highly exothermic (see an estimation for compound 5 a in the Supporting Information). The detailed study of the photophysical properties of these compounds (5 a and 5 b) to understand the quenching effects are being currently evaluated by advanced photophysical methods (femtosecond transient absorption spectra) and fall beyond the scope of discussion in the present work. For the protoporphyrin IX dyads 8 a–10 a the major emission band centered at l = 578 nm and the minor one at l = 633 nm (see Figure 2 for the representative compound 8 a). These emission bands are also slightly blue shifted when compared with the ones of the starting material 7 (l = 588 and 643 nm). In the free-base dyads 8 b–10 b the bands shift towards the red region and appear centered at l = 622 and 689 nm, respectively. A minor band along with a shoulder-type band were also consistently observed at l = 652 and between 627– 674 nm for these protoporphyrin IX free-base dyads (see Figure 2 for 8 b). A cursory glance at the tabulated values for the fluorescence quantum yields (Ffl) reveals that the chlorin e6 dyads 2–4 (a and b) have higher Ffl (0.13–0.20) when compared with the ones of the corresponding protoporphyrin dyads (8 a/b–10 a/ b; 0.03–0.08). Again, in the protoporphyrin IX series the adducts 11 a and 11 b, obtained from the o-QM 6 d, do not show 4

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Full Paper gregation.[23] Analogously, these arguments can also be extended to the present cases of dyads as a possible explanation for their self-aggregation, because in this case there is a pyran ring at the 3-position of the chlorin macrocycle, the ZnII metal ion, and the ester carbonyl group at the 13-position, all aligned on the required axis. To obtain more information about the recovery of the ground-state population after photoexcitation, the picosecond transient absorption spectra (ps-TAS) of compounds 2 a–4 a, 2 b–4 b, 8 a–10 a, and 8 b–10 b were recorded. As expected, the recovery of the ground-state population was found to be monoexponential. By using the compensation method,[24] the intersystem crossing (ISC) S1!T1 quantum yields, FISC, were estimated (Table 3). From the values in Table 3, it can be seen that the chlorin e6 dyads 2 a–4 a and 2 b–4 b have efficient intersystem crossing (ISC), whereas the protoporphyrin IX dyads have the highest ISC rates, which translates into higher singlet oxygen quantum yields. The UV/Vis spectra of all compounds were recorded before and after the ps-TAS measurements (data not shown) and indicated good photostability under the used conditions. Because various types of related effects, like auto fluorescence of tissues, makes it difficult to quantify the intensity in detection images during fluorescence diagnosis, it is important to measure the characteristic fluorescence lifetimes of such fluorophores to consider their potential use as diagnostic agents. If the lifetimes of such molecules will be relatively longer compared to the auto fluorescence lifetime, it will be helpful during the measurements inside cells.[25] The fluorescence lifetime measurements (TCSPC data; Table 4) have a general pattern in the exponential decay. The ZnII complexes (2 a–4 a and 2 b–4 b; 8 a–10 a, and 8 b–10 b) of the chlorin e6 and protoporphyrin IX series or the corresponding free bases follow a similar pattern of decay. A good fit is observed (i.e., lower reduced chi2 values) for all compounds only when two components are used (double exponential fit).

Figure 2. Representative fluorescence spectra of the PP-IX derivatives 8 a (solid line) and 8 b (dashed line) in DMF with the optical density of 0.02 (lexc = 532 nm).

any fluorescence, due to the same reason mentioned for compounds 5 a and 5 b. Their quenching effects are being currently evaluated by advanced photophysical methods as mentioned earlier. Considering the singlet molecular-oxygen quantum yields (FD) with exception of compounds 5 and 11 all the prepared adducts (2–4 and 8–10) are able to generate singlet oxygen. A closer look warranted to conclude that the free-base chlorin e6 dyads show an unexpectedly higher ability to generate singlet oxygen than the ZnII counterparts; in the protoporphyrin IX series the difference between the free base and the ZnII adducts is not significant. One might suppose that Zn complexation increases the tendency especially of the chlorintype dyads to form aggregates, otherwise the reduction in FD would be difficult to explain. To evaluate this, the fluorescence (Ffl) and the singlet oxygen (FD) quantum yields of samples 2 a–4 a and meso-tetrakis(m-hydroxyphenyl)chlorin (m-THPC) as a reference were investigated in ethanol (see Table S2 in the Supporting Information). For adducts 2 a–4 a the fluorescence quantum yield decreased drastically in ethanol (Ffl  0.03) when compared with the ones determined in DMF (Ffl  0.16). This supports the assumption of a higher tendency to form aggregates after Zn complexation. Interestingly, this has no influence on the triplet behavior of the PSs. The triplet decay times are nearly unchanged taking into account the viscosity and oxygen solubility in the solvents. Also the ability of these adducts (2 a–4 a) to generate singlet oxygen is maintained in ethanol and the values of FD are similar to the ones obtained in DMF. A closer observation showed that on the removal of ZnII the observed effect for FD was neglected and all free-base dyads showed almost similar values (see Table 3). A similar behavior for ZnII–meso-tetraphenylporphyrin derivatives substituted at the beta position in comparison to their free bases was also observed.[22] Pigments related to chlorophylls having the three moieties: 1) a hydroxyl group on the 31 position, 2) a coordinated metal ion (Mg, Zn, or Cd), and 3) a carbonyl group at position 13 in a straight line on the N21–N23 axis, show self-agChem. Eur. J. 2014, 20, 1 – 13

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Table 4. Fluorescence lifetimes of the chlorin dyads 2 a–4 a and 2 b–4 b as well as of the protoporphyrin IX dyads 8 a–10 a and 8 b–10 b, from chlorin 1 and protoporphyrin 7, and comparison with TPP by the timecorrelated single photon counting (TCSPC) method.

1 2a 3a 4a 2b 3b 4b 7 8a 9a 10 a 8b 9b 10 b TPP

5

Monochromator l [nm]

tfl [ns]  0.2 ns

Amplitude [%]

tfl [ns]  0.05 ns

Amplitude [%]

Red. c2 value

660 660 660 660 660 660 660 588 578 578 578 622 622 622 660

3.1 3.5 3.5 3.4 5.4 5.4 5.3 2.1 2.2 2.2 2.2 15.1 14.8 15.0 10.3

89 85 85 87 82 82 79 89 91 87 88 92 92 92 100

0.54 0.79 0.76 0.77 0.37 0.28 0.27 0.32 0.56 0.45 0.50 0.79 0.69 0.63 –

11 15 15 13 17 18 21 11 9 13 12 7 8 8 –

1.11 1.20 1.16 1.12 0.99 1.07 1.03 1.19 1.18 1.09 1.15 1.00 0.99 1.04 1.12

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The Zn–chlorin e6 dyads have two fluorescence lifetimes approximately 3.5 and 0.8 ns for compounds 2 a–4 a, whereas the free bases have approximately 5.3 and 0.3 ns. Although in the Zn–protoporphyrin IX dyads 8 a–10 a the fluorescence lifetimes are of the order of about 2.2 and 0.5 ns, removal of the ZnII ion increased the lifetimes to approximately 15.0 and 0.7 ns, respectively (Table 4). To understand the two fluorescence lifetimes and the aggregation assumption we carried out fluorescence decay-associated spectra (FDAS) and anisotropy measurements for a representative sample from each series (i.e., compounds 2 a and 8 a). The fluorescence decay of compound 2 a in the wavelength range between l = 660 and 750 nm comprises two components with decay times of 3.3–3.5 ns, which is within the error margin, and 0.5–0.7 ns, which is just slightly above the error margin. The relative amplitude contribution of each of the two components changed with increments of wavelength (10 nm). The amplitude for the longer decay times (3.3–3.5 ns) decreased from 92 % at l = 660 nm to 74 % at l = 750 nm, whereas the amplitude for the shorter decay times (0.5–0.7 ns) increased from 8 % at l = 660 nm to 26 % at l = 750 nm accordingly (see Table S3 in the Supporting Information). This clearly indicates that the shorter decay time may belong to the dimers or other aggregates. Such aggregation should result in longer rotational decay times, as the aggregates are much bigger than the monomers, which results in slower rotation at the same temperature. Similar fluorescence decay of compound 8 a in the wavelength range between l = 570 and 660 nm comprised of two components with decay times of 2.2 and 0.5–1.0 ns with contributions of amplitude ranging between 91 and 9 % at l = 570 nm up to 95 and 5 % at l = 620 nm, respectively, for the two decay times mentioned (see Table S4 in the Supporting Information). A glance at the actual fluorescence lifetimes recorded at l = 578 nm and the FDAS analysis (between l = 570– 620 nm) indicates a close match between the lifetimes. We analyzed the time-resolved anisotropy decay at l = 660 and 720 nm for compound 2 a. Due to the substituent at the b position the aspect ratio of the geometric dimensions of the molecule is nearly two and hence we expected to identify up to two rotational decay times.[26] Doing so we found 240 and 380 ps at l = 660 nm (amplitude ratio 1:5), which are realistic values for derivatives of TPP of this size in DMF.[22] The same fit for the fluorescence anisotropy at l = 720 nm resulted in 320 and 780 ps with an amplitude ratio of 10:1. Although quite small, the slow component is found in each of the two-exponential fit of the fluorescence anisotropy at l = 720 nm, indicating the presence of some bigger compounds. If these compounds are dimers of the investigated dye it is not surprising that the amplitude of the long time is low, as aggregation usually quenches the fluorescence of the involved molecules. The shorter time found at l = 720 nm then probably is an overlay of the two rotational decay times found at l = 660 nm. However, the signal to noise ratio (SNR) of the fluorescence anisotropy measurements does not allow for a third decay time to be fitted. A similar anisotropy analysis in case of compound 8 a gave an anisotropy decay of (243  20) ps. &

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Although both free base and Zn complexes have been synthesized in this study, we have collected CD and MCD spectra of compound 2 a as a representative case of the zinc compounds (Figure 3). MCD spectra give information on the electronic structure so that it can enhance the interpretation of the electronic absorption spectra, whereas CD spectra generally afford implication on the conformation of a chromophoric compound.[27] As can be seen in Figure 3, the MCD of compound 2 a showed an intense peak at l = 635 nm corresponding to

Figure 3. MCD, CD, and electronic absorption spectra of compound 2 a in CHCl3 and its calculated CD and absorption spectra.

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Full Paper the absorption peak at l = 633 nm and two troughs at l = 594 and 559 nm associated with the absorption peaks at l = 593 and 558 nm, respectively. These characteristics strongly suggest that the absorption peaks at l = 633, 593, and 558 nm are assigned to the Qy00-, the Qx00-, and the Qx01-band, respectively. In addition, the Q MCD sign change of plus-to-minus in ascending energy experimentally indicate that the DLUMO is larger than the DHOMO.[27–30] In the Soret-band region, a dispersion type MCD appeared corresponding to the absorption peak at l = 412 nm, which is a pseudo Faraday A term observed for low-symmetry compounds. The CD spectra of compound 2 a showed negative and positive envelopes corresponding to the Q absorption peak at l = 633 nm and the Soret-band peak at l = 412 nm, respectively, which are similar to those reported previously for (proto)chlorophyll a, although a small positive and negative CD peak and trough at l = 557 and 443 nm, respectively, are not recognized in old literatures.[31, 32] When the first CD paper on (proto)chlorophyll a appeared in 1970,[31] the band at l = 558 nm in the absorption spectrum and at l = 557 nm in the CD spectrum was assigned to the Qx00-band. However, considering the MCD property found later than 1978 that the interacting bands give opposite MCD signs, the absorption band at l = 593 nm is most likely to be assigned as Qx00-band (plus a weak Qy01band). Calculated absorption and CD spectra are added in the lower region of Figure 3. Although the energy is slightly overestimated, the experimental absorption and CD spectra are roughly reproduced. Thus, the Qy00- and Qx00-band were calculated at l = 559 and 513 nm, and several weak absorption bands are seen at the longer wavelength side of the Soret band, plausibly corresponding to the CD trough at l = 443 nm, although they are not clearly recognized in the absorption spectrum. In the CD spectrum, a negative CD sign in the Qband region and a minus-plus-minus pattern observed in the l  500–350 nm region in the experiments are reproduced between l  400–320 nm in the calculations.

Table 5. Values of E [V] versus ferrocenium/ferrocene (Fc + /Fc) observed in the cyclic voltammograms of the compounds.

1 2a 3a 4a 5a 7 8a 9a 10 a 11 a

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E Iox[a]

E IIox[b]

E IIIox[b]

E IVox[b]

2.11 1.91 1.89 1.91 1.82 1.94 1.89[b] 1.90[b] 1.93[b] –

1.78 1.71[b] 1.71[b] 1.80[b] 1.23 – – – – 1.27

0.13 0.13 0.14 0.12 0.14 0.28 0.29 0.30 0.30 0.15

0.47 0.47 0.47 0.46 0.45 0.45 0.55 0.55 0.57 –

0.73 0.73 0.73 0.72 0.74 0.71[c] 0.66 0.65 0.67 –

– 0.79[c] 0.85[c] 0.85[c] 0.87[c] – 0.80 0.81 0.84 –

exert significant influence in the electronic properties of the macrocycles. The first reduction process of compounds 5 a and 11 a can be assigned to the two-electron reduction of the quinone moiety. In compound 11 a, it occurs in a single two-electron step characterized by the increased peak current, whereas for compound 5 a the quinone may undergo two successive one-electron reductions, which is indicated by the shoulder of the 1.23 V peak at more anodic potential.[36]

Conclusion The synthesis and photophysical characterizations of new chlorin e6 trimethyl ester and protoporphyrin IX dimethyl ester dyads linked to pyrano[3,2-c]coumarin, pyrano[3,2-c]quinolinone, and pyrano[3,2-c]naphthoquinone moieties at the b positions as their ZnII and free-base forms is reported. All the dyads as the Zn complexes and their free bases by demetallation were obtained in good to excellent yields. The evaluation of their photophysical properties by measuring the fluorescence quantum yield, the singlet oxygen quantum yield by luminescence detection, and the triplet lifetimes indicated that all these compounds can be optimized for PDT applications. The electronic and conformational structures of a representative Zn complex were examined by using MCD and CD spectroscopy, respectively. Redox data were collected for the Zn complexes and it was found that peripheral modification of the ring can exert significant influence in the electrochemical properties of the macrocycles.

The electrochemical properties of the ZnII dyads 2 a–5 a and 8 a–11 a as well as of the precursor macrocycles 1 and 7 were studied by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in distilled DMF as solvent. The potential for all redox processes relative to the ferrocenium/ferrocene couple are resumed in Table 5 and the plots are included in the Supporting Information (Figures SF76–SF81). The precursor chlorin 1 and the precursor porphyrin 7 showed two and one reversible reduction processes, respectively, which are in agreement with previous data.[33–35] Functionalization of compounds 1 and 7 to yield compounds 2 a–4 a and 8 a–10 a lead to the irreversibility of the first and second reduction, respectively, and also the anodic shift of both processes, with the most noticeable shifts observed for the second reduction of compound 1 (  200 mV). The comparison between precursor 1 and compounds 2 a–4 a and that between precursor 7 and compounds 8 a–10 a indicate that peripheral modification of the ring can www.chemeurj.org

E Ired[a]

[a] Reversible processes (E1/2 value). [b] Irreversible processes. [c] Values taken from the DPV plots.

Electrochemical studies

Chem. Eur. J. 2014, 20, 1 – 13

E IIred[a]

Experimental Section General Chemistry: 1H NMR spectra were recorded at 300 or 500 MHz and 13 C NMR spectra were recorded at 75 or 126 MHz with Bruker Avance 300 and 500 spectrometers. CDCl3 was used as solvent unless specified. Chemical shifts (d) are expressed in parts per million relative to tetramethylsilane. The coupling constants (J) are given in Hertz. Unequivocal 1H assignments were made by using 2D COSY and NOESY experiments (mixing time of 800 ms), whereas 13 C assignments were made on the basis of DEPT-135 and 2D

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Full Paper HSQC and HMBC experiments (delay for long-range J(C,H) couplings were optimized for 7 Hz). HRMS (ESI) was performed with a Bruker maXis impact by using acetone and a Bruker Apex-Qe spectrometer by using chloroform as solvent and 3-nitrobenzyl alcohol (NBA) as matrix. The UV/Vis spectra were recorded with a Shimadzu UV-2501 PC spectrophotometer by using dimethylformamide (DMF) as solvent (1 cm path length quartz cell). Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra were collected with a JASCO-725 spectrodichrometer equipped with a JASCO electromagnet that produces magnetic fields of up to one Tesla without and with magnetic field, respectively, by using DMF as solvent. Preparative thin layer chromatography was carried out on 20 cm  20 cm glass plates coated with silica gel (1 mm thick, Merck). Analytical TLC was carried out on precoated sheets with silica gel (0.2 mm thick, Merck). Dioxane was dried over molecular sieves. All other solvents and reagents were used as received without further purification.

3-(5-Oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]chromen-2-yl)chlorin e6 trimethyl ester zinc(II) (2 a): 1H NMR (CDCl3, 500 MHz): d = 1.69 (t, J = 7.5 Hz, 3 H; H-82), 1.75 (d, J = 7.1 Hz 3 H; H-181), 1.80 (dd, J = 16.8, 8.1 Hz, 1 H; H-171), 2.09–2.21 (m, 2 H; H-171, H-172), 2.47–2.57 (m, 1 H; H-172), 2.74–2.78 (m, 1 H; H-3’), 2.89–2.97 (m, 1 H; H-3’), 3.03– 3.06 (m, 2 H; H-4’), 3.11 and 3.13 (2 s, 3 H; H-71), 3.38 and 3.39 (2 s, 3 H; H-21), 3.45 (s, 3 H; H-121), 3.56 and 3.57 (2 s, 3 H; H-174), 3.75 (q, J = 7.5 Hz, 2 H; H-81), 3.84 (s, 3 H; H-153), 4.19 (s, 3 H; H-132), 4.31 (d, J = 8.1 Hz, 1 H; H-17), 4.37 (q, J = 7.1 Hz, 1 H; H-18), 5.10 (d, J = 19.2 Hz, 1 H; H-151), 5.25 (d, J = 19.2 Hz, 1 H; H-151), 6.62 (dt, J = 11.2, 2.4 Hz, 1 H; H-2’), 7.11 (t, J = 7.6 Hz, 1 H; H-9’), 7.18 (t, J = 7.6 Hz, 1 H; H-9’), 7.36 (d, J = 8.4 Hz, 1 H; H-7’), 7.38 (d, J = 8.4 Hz, 1 H; H-7’), 7.47–7.51 (m, 1 H; H-8’), 7.50–7.54 (m, 1 H; H-8’), 7.80 (d, J = 7.9 Hz, 1 H; H-10’), 7.89–7.92 (m, 1 H; H-10’), 8.61 (s, 1 H; H-20), 9.50 (s, 1 H; H-5), 9.56 and 9.57 ppm (2 s, 1 H; H-10); 13C NMR (CDCl3, 75 MHz): d = 11.1 (C-21), 11.7 (C-71), 12.3 (C-121), 17.6 (C-82), 19.4 (C-81), 20.9 (C-4’), 22.8 (C-181), 29.7 (C-3’), 30.1 (C-171, 172), 38.8 (C-151), 47.2 (C-18); 51.7 (C-174), 52.2 (C-153), 52.3 (C-17), 52.8 (C132), 76.6 (C-2’), 93.5 (C-20), 101.1, 101.2 (C-5), 101.5 (C-4’a), 102.1 (C-15), 103.9 (C-10), 115.8, 115.9 (C-10’a), 116.6 (C-7’), 122.3, 122.5 (C-10’), 123.87, 123.94 (C-9’), 127.9 (C-13), 131.48, 131.51 (C-8’), 133.9, 134.0 (C-7), 134.3, 134.4 (C-2), 137.47, 137.52 (C-3), 141.6 (C8), 142.16, 142.17, 142.25, 142.27 (C-11,12), 143.7, 143.8 (C-4), 144.3 (C-9), 147.5 (C-6), 148.2 (C-14), 152.3, 152.36, 152.43, 152.48 (2  C6’a, 2  C-1), 160.7, 160.8 (C-10’b), 162.2 (C-16), 163.27, 163.32 (C-5’), 165.7 (C-19), 170.7 (C-131), 173.5 (C-152), 173.8 ppm (C-173); UV/Vis (DMF): lmax (log e) = 411 (5.12), 517 (3.44), 592 (3.87), 633 nm (4.70); HRMS (ESI-TOF): m/z calcd for C47H46N4O9Zn: 874.2551 [M] + C ; found: 874.2556.

Photophysics: Steady-state fluorescence spectra of the investigated compounds were measured in 1 cm  1 cm quartz optical cells by using a combination of a cw-Xenon lamp (XBO 150) and a monochromator (Lot-Oriel, bandwidth 10 nm) for excitation and a polychromator with a cooled CCD matrix as a detector system (Lot– Oriel, Instaspec IV).[37] As reference for measurements of the fluorescence quantum yield, Ffl, meso-tetraphenylporphyrin (TPP) in DMF (Ffl = 0.11) was used. Photosensitized steady-state singlet oxygen luminescence was measured at 1270 nm. A cwYb:YAG laser (Versadisk, ELS) equipped with a frequency doubling unit was used to excite the samples at l = 420 nm. The setup for detection of the luminescence signal has been reported previously.[38] To calculate the singlet oxygen quantum yield, FD, TPP in DMF was used as reference (FD = 0.65).[39]

3-(9-Methyl-5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]chromen-2-yl)chlorin e6 trimethyl ester zinc(II) (3 a): 1H NMR (CDCl3, 300 MHz): d = 1.69 (t, J = 7.6 Hz, 3 H; H-82), 1.74 (d, J = 7.1 Hz, 3 H; H-181), 1.80– 1.83 (m, 1 H; H-171), 2.12–2.21 (m, 2 H; H-171, H-172), 2.23 and 2.26 (2 s, 3 H; Ar-CH3), 2.45–2.60 (m, 1 H; H-172), 2.69–2.81 (m, 1 H; H-3’), 2.90–3.00 (m, 1 H; H-3’), 3.01–3.08 (m, 2 H; H-4’), 3.15 (s, 3 H; H-71), 3.39 and 3.40 (2 s, 3 H; H-21), 3.45 (s, 3 H; H-121), 3.57 and 3.58 (2 s, 3 H; H-174), 3.76 (q, J = 7.6 Hz, 2 H; H-81), 3.85 (s, 3 H; H-153), 4.20 (s, 3 H; H-132), 4.29–4.31 (m, 1 H; H-17), 4.38 (q, J = 7.1 Hz, 1 H; H-18), 5.12 (d, J = 19.2 Hz, 1 H; H-151), 5.26 (d, J = 19.2 Hz, 1 H; H-151), 6.61 (dt, J = 10.9, 2.6 Hz, 1 H; H-2’), 7.28–7.34 (m, 2 H; H-7’, H-8’), 7.65 (d, J = 1.9 Hz, 1 H; H-10’), 7.69 (d, J = 1.9 Hz, 1 H; H-10’), 8.61 and 8.62 (2 s, 1 H; H-20), 9.51 (s, 1 H; H-5), 9.57 and 9.58 ppm (2 s, 1 H; H-10); 13 C NMR (CDCl3, 75 MHz): d = 11.0 (C-21), 11.7 (C-71), 12.3 (C-121), 17.6 (C-82), 19.4 (C-81), 20.79, 20.81 (Ar-CH3), 20.9 (C-4’), 22.7 (C-181), 29.7 (C-3’), 30.0 (C-171), 30.7 (C-172), 38.8 (C-151), 47.2, 42.3 (C-18); 51.7 (C-174), 52.1 (C-153), 52.3 (C-17), 52.8 (C-132), 76.2, 76.3 (C-2’), 93.5, 93.6 (C-20), 101.2 (C-5), 101.4 (C-4’a), 102.2 (C-15), 103.9 (C10), 115.5 (C-10’a), 116.4 (C-7’), 122.1 (C-10’), 128.0 (C-13), 132.5 (C8’), 133.7 (C-7, C-9’), 134.0 (C-2), 137.5, 137.6 (C-3), 141.6 (C-8), 142.20, 142.23, 142.3 (C-11, C-12), 143.7, 143.8 (C-4), 144.3 (C-9), 147.6 (C-6), 148.2 (C-14), 150.6 (C-6’a), 152.37, 152.40 (C-1), 160.8 (C-10’b), 162.27, 162.30 (C-16), 163.47, 163.49 (C-5’), 165.7, 165.8 (C19), 170.6 (C-131), 173.5 (C-152), 173.6 (C-173) ppm; UV/Vis (DMF): lmax (log e): 411 (5.04), 515 (3.54), 592 (3.87), 633 nm (4.64); HRMS 8ESI-TOF): m/z calcd for C48H48N4O9Zn: 888.2707 [M] + C ; found: 888.2709.

Electrochemistry: Cyclic voltammetry and differential pulse voltammetry measurements were performed by using a Hokuto Denko HZ5000 potentiostat, in a cell under a nitrogen atmosphere containing a glassy carbon electrode (area = 0.07 cm2) as working electrode, a Pt wire as counter electrode, and Ag/AgCl as reference electrode. The sample solutions were prepared in a 1.0 mm concentration in DMF freshly distilled over CaH2, and tetrabutylammonium perchlorate (TBAP) was used as supporting electrolyte. The cyclic voltammograms were measured at a scan rate of 100 mV s1 and the potential values were corrected by using the ferrocenium/ ferrocene couple (Fc + /Fc) as internal standard. Computational methods: In order to help the interpretation of the assignment of the electronic absorption and CD spectra of the chlorin compound 2 a, the Gaussian 09[40] software package was used to carry out DFT and time-dependent (TD) DFT calculations by using the B3LYP functional with the 6-31G(d) basis set. General procedure for preparing chlorin e6 derivatives: In a round-bottom flask or sealed tube, equipped with a magnetic stirring bar, a solution of compound 6 a [formed in situ from 4-hydroxycoumarin (2.3 mg, 14.2 mmol) and paraformaldehyde (3.4 mg, 116 mmol)] [for 6 b: 4-hydroxy-6-methylcoumarin (2.5 mg), for 6 c: 4-hydroxy-N-methylquinolin-2-one (2.5 mg), for 6 d: 2-hydroxy-1,4naphthoquinone (2.5 mg)] in 1,4-dioxane (2.5 mL) and chlorin e6 (1) (10 mg, 14.2 mmol) was heated to reflux until consumption of the starting chlorin 1 (1–5 H; see Table 1). The quinone methide precursors were added at regular intervals. Dioxane was then removed under reduced pressure, chloroform (50 mL) was added to the residue and the mixture was washed with a saturated aqueous solution of sodium bicarbonate (2  20 mL). The organic phase was concentrated under vacuum and the residual crude product was purified by preparative TLC by using a mixture of toluene/ethyl acetate (8:2) as the eluent.

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Chem. Eur. J. 2014, 20, 1 – 13

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3-(6-Methyl-5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]quinolin-2-yl)chlorin e6 trimethyl ester zinc(II) (4 a): 1H NMR (300 MHz, CDCl3): d = 1.62–1.70 (m, 3 H; H-82), 1.74 (d, J = 7.1 Hz, 3 H; H-181), 1.81 (dd, J = 18.7, 10.0 Hz, 1 H; H-171), 2.08–2.28 (m, 2 H; H-171, H-172), 2.45–2.74 (m, 5 H; H-172, H-3’, H-4’), 3.07 and 3.08 (2 s, 3 H; H-71), 3.31 and 3.36 (2 s, 3 H; H-21), 3.32 (s, 3 H; N-CH3), 3.43 and 3.45 (2 s, 3 H; H121), 3.58 and 3.59 (2 s, 3 H; H-174), 3.65–3.76 (m, 2 H; H-81), 3.71–

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Full Paper 3.76 (m, 2 H; H-81), 3.85 and 3.86 (2 s, 3 H; H-153), 4.20 and 4.21 (2 s, 3 H; H-132), 4.31 (d, J = 10.0 Hz, 1 H; H-17), 4.37 (q, J = 7.1 Hz, 1 H; H-18), 5.12 (d, J = 19.0 Hz, 1 H; H-151), 5.27 (d, J = 19.0 Hz, 1 H; H151), 6.44 (t, J = 8.8 Hz, 1 H; H-2’), 6.92 (t, J = 7.6 Hz, 1 H; H-9’), 7.04 (dd, J = 16.0, 7.6 Hz, 1 H; H-9’), 7.27–7.37 (m, 1 H; H-7’), 7.46 (t, J = 7.6 Hz, 1 H; H-8’), 7.88 (d, J = 8.1 Hz, 1 H; H-10’), 7.98 (d, J = 8.1 Hz, 1 H; H-10’), 8.57 and 8.59 (2 s, 1 H; H-20), 9.50–9.55 ppm (m, 2 H; H5,10); 13C NMR (CDCl3, 75 MHz): d = 10.97, 11.00 (C-71), 11.6, 11.7 (C21), 12.29, 12.31 (C-121), 17.6 (C-82), 19.4 (C-81, C-4’), 22.7 (C-181), 29.01 (C-3’), 29.04 (C-171, N-CH3), 30.4 (C-172), 38.9 (C-151), 47.3, 47.4 (C-18), 51.6 (C-174), 52.1 (C-153), 52.3 (C-17), 52.7 (C-132), 75.5, 75.6 (C-2’), 93.3, 93.4 (C-20), 101.2, 101.4 (C-5), 101.9, 102.0 (C-15), 103.8 (C-10), 106.7, 106.9 (C-4’a), 113.9 (C-7’), 115.9, 116.2 (C-10’a), 121.5, 121.7 (C-9’), 122.4, 122.7 (C-10’), 127.8 (C-13), 130.0, 130.2 (C8’), 133.7, 133.8 (C-7), 134.0 (C-2), 138.0, 138.3 (C-6’a), 138.6, 138.7 (C-3), 141.4 (C-8), 142.0, 142.1 (C-11, C-12), 144.1 (C-4,9), 147.59, 147.64 (C-6), 148.1, 148.2 (C-14), 152.5, 152.6 (C-1), 156.9, 157.1 (C10’b), 162.01 (C-16), 162.11 (C-5’), 165.6, 165.7 (C-19), 170.7, 170.8 (C-131), 173.5, 173.56 (C-152), 173.64 ppm (C-173); UV/Vis (DMF): lmax (log e): 411 (4.99), 514 (3.63), 592 (3.87), 632 nm (4.58); HRMS (ESI-TOF): m/z calcd for C48H49N5O8Zn: 887.2867 [M] + C ; found: 887.2873.

3 H; H-153), 4.27 (s, 3 H; H-132), 4.43 (d, J = 9.6 Hz, 1 H; H-17), 4.45 (d, J = 9.6 Hz, 1 H; H-17), 4.49 (q, J = 7.0 Hz, 1 H;H-18), 5.26 (d, J = 18.7 Hz, 1 H; H-151), 5.39 (d, J = 18.7 Hz, 1 H; H-151), 6.70 (dd, J = 5.0, 2.4 Hz, 1 H; H-2’), 6.74 (dd, J = 5.0, 2.4 Hz, 1 H; H-2’), 7.18–7.30 (m, 2 H; H-9’), 7.46 (dd, J = 8.4, 0.8 Hz, 1 H; H-7’), 7.47 (dd, J = 8.4, 0.8 Hz, 1 H; H-7’), 7.54–7.62 (m, 2 H; H-8’), 7.91 (dd, J = 8.0, 1.5 Hz, 1 H; H-10’), 7.98 (dd, J = 8.0, 1.5 Hz, 1 H; H-10’), 8.84 (s, 1 H; H-20), 9.59 and 9.60 (2 s, 1 H; H-5), 9.74 ppm (s, 1 H; H-10); UV/Vis (DMF): lmax (log e): 396 (5.12), 497 (4.06), 524 (3.57), 601 (3.66), 657 nm (4.67); HRMS (ESI-TOF): m/z calcd for C47H49N4O9 : 813.3494 [M+H] + ; found: 813.3493. 3-(9-Methyl-5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]chromen-2-yl)chlorin e6 trimethyl ester (3 b): 1H NMR (300 MHz, CDCl3): d = 1.65 (s, 2 H; NH), 1.70 (t, J = 7.5 Hz, 3 H; H-82), 1.75 (d, J = 7.1 Hz, 3 H; H181), 2.17–2.22 (m, 3 H; 2  H-171, H-172), 2.27 and 2.31 (2 s, 3 H; ArCH3), 2.56–2.60 (m, 1 H; H-172), 2.76–3.08 (m, 2 H; H-3’), 3.11–3.14 (m, 2 H; H-4’), 3.14 (s, 3 H; H-71), 3.52 (s, 3 H; H-21), 3.60 (s, 3 H; H121), 3.64 (s, 3 H; H-174), 3.76–3.81 (m, 2 H; H-81), 3.79 (s, 3 H; H-153), 4.27 (s, 3 H; H-132), 4.42 (d, J = 7.9 Hz, 1 H; H-17), 4.47 (q, J = 7.1 Hz, 1 H; H-18), 5.25 (d, J = 18.7 Hz, 1 H; H-151), 5.38 (d, J = 18.7 Hz, 1 H; H-151), 6.69 (dt, J = 5.1, 2.6 Hz, 1 H; H-2’), 7.34–7.38 (m, 2 H; H-7’, H8’), 7.69 (s, 1 H; H-10’), 7.74 (d, J = 0.7 Hz, 1 H; H-10’), 8.84 (s, 1 H; H20), 9.60 and 9.61 (2 s, 1 H; H-5), 9.74 ppm (s, 1 H; H-10); UV/Vis (DMF): lmax (log e): 396 (5.10), 497 (4.03), 524 (3.49), 603 (3.59), 657 nm (4.65); HRMS (ESI-TOF): m/z calcd for C48H51N4O9 : 827.3651 [M+H] + ; found: 827.3667.

3-(5,10-Dioxo-3,4,5,10-tetrahydro-2 H-benzo[g]chromen-2-yl)chlorin e6 trimethyl ester zinc(II) (5 a): 1H NMR (300 MHz, CDCl3): d = 1.67 (t, J = 7.6 Hz, 3 H; H-82), 1.73 J = 7.2 Hz, (d, 3 H; H-181), 1.80–1.83 (m, 1 H; H-171), 1.97–2.24 (m, 2 H; H-171, H-172), 2.48–2.54 (m, 1 H; H-172), 2.65–2.85 (m, 2 H; H-3’), 2.85–3.06 (m, 2 H; H-4’),3.20 and 3.21 (2 s, 3 H; H-71), 3.35 (s, 3 H; H-21), 3.42 (s, 3 H; H-121), 3.57 (s, 3 H; H-174), 3.73 (dd, J = 16.3, 8.3 Hz, 2 H; H-81), 3.83 (s, 3 H; H-153), 4.17 and 4.18 (2 s, 3 H; H-132), 4.28 (d, J = 9.1 Hz, 1 H;H-17), 4.35 (q, J = 7.2 Hz, 1 H; H-18), 5.10 (d, J = 19.1 Hz, 1 H; H-151), 5.23 (d, J = 19.1 Hz, 1 H; H-151), 6.53 (d, J = 9.5 Hz, 1 H; H-2’), 7.60–7.75 (m, 2 H; H-7’, H-8’), 7.96–8.06 (m, 2 H; H-6’, H-9’), 8.56 (s, 1 H; H-20), 9.40 and 9.42 (2 s, 1 H; H-5), 9.49 and 9.52 ppm (2 s, 1 H; H-10); 13C NMR (75 MHz, CDCl3): d = 11.1 (C-71), 11.7 (C-21), 12.3 (C-121), 17.5 (C-82), 19.4 (C81), 20.1 (C-4’), 22.8 (C-181), 29.0 (C-171), 29.3 (C-3’), 30.7 (C-172), 38.9 (C-151), 47.2 (C-18), 51.6 (C-174), 52.1 (C-153), 52.3 (C-17), 52.7 (C-132), 76.3, 76.4 (C-2’), 93.4 (C-20), 100.8, 100.9 (C-5), 101.9 (C-15), 103.8 (C-10), 121.78, 121.83 (C-4’a), 126.0, 126.1, 126.2, 126.3 (C-6’, C-9’), 127.7 (C-13), 130.9, 131.0 (C-9’a), 131.9, 132.0 (C-5’a), 133.05, 133.09 (C-8’), 133.76, 133.82 (C-7), 133.87, 133.94 (C-7’), 134.6, 134.7 (C-2), 137.3 (C-3), 141.36, 141.39 (C-8); 141.9, 142.08, 142.11 (C-11, C-12), 143.9, 143.97, 144.03, 144.1 (C-4, C-9), 147.6, 147.7 (C-6), 148.0 (C-14), 152.6 (C-1), 156.1, 156.2 (C-10’a), 162.0 (C-16), 165.6 (C-19), 170.8 (C-131), 173.6 (C-152), 173.9 (C-173), 179.37 and 179.45 (C-5’), 184.3, 184.4 ppm (C-10’); UV/Vis (DMF): lmax (log e): 411 (5.07), 517 (3.59), 592 (3.90), 633 nm (4.66); HRMS (ESI-TOF): m/z calcd for C48H46N4O9Zn: 886.2551 [M] + C ; found: 886.2542.

3-(6-Methyl-5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]quinolin-2-yl)chlorin e6 trimethyl ester (4 b): 1H NMR (300 MHz, CDCl3): d = 1.61 (s, 2 H; NH), 1.67 (t, J = 7.4 Hz, 3 H; H-82), 1.76 and 1.77 (2 d, J = 7.1 Hz, 3 H; H-181), 2.17–2.28 (m, 3 H; 2  H-171, H-172), 2.51–2.61 (m, 1 H; H-172), 2.75–3.01 (m, 2 H; H-3’), 3.09 and 3.10 (2 s, 3 H; H71), 3.14–3.42 (m, 2 H; H-4’), 3.51 and 3.52 (2 s, 3 H; H-21), 3.59 (s, 3 H; H-121), 3.64 (s, 3 H; H-174), 3.72–3.78 (m, 2 H; H-81), 3.79 (s, 3 H; H-153), 3.88 (s, 3 H; N-CH3), 4.27 (s, 3 H; H-132), 4.43 (d, J = 8.1 Hz, 1 H; H-17), 4.48 (q, J = 7.1 Hz, 1 H; H-18), 5.25 (d, J = 18.6 Hz, 1 H; H151), 5.38 (d, J = 18.6 Hz, 1 H; H-151), 6.63–6.67 (m, 2 H; H-2’), 7.16– 7.23 (m, 2 H; H-9’), 7.48 (d, J = 8.1 Hz, 1 H; H-7’), 7.49 (d, J = 8.1 Hz, 1 H; H-7’), 7.58–7.63 (m, 2 H; H-8’), 8.13 and 8.19 (2 dd, J = 8.0, 1.4 Hz, 1 H; H-10’), 8.82 (s, 1 H; H-20), 9.67 and 9.69 (2 s, 1 H; H-5), 9.72 ppm (s, 1 H; H-10); UV/Vis (DMF): lmax (log e): 396 (5.10), 497 (4.03), 524 (3.55), 603 (3.61), 657 nm (4.62); HRMS (ESI-TOF): m/z calcd for C48H52N5O8 : 826.3810 [M+H] + ; found: 826.3805.

Decomplexation procedure: The ZnII chlorins 2 a–5 a were treated with a solution of 5 % TFA in chloroform (1 mL). The reaction mixture was stirred at 0 8C for 10 min and then neutralized with a saturated aqueous solution of NaHCO3. The aqueous phase was extracted with chloroform, the organic phase was dried over Na2SO4 and evaporated under vacuum to dryness. The resulting residue was purified by preparative TLC by using mixtures of ethyl acetate and toluene as eluent.

3-(5,10-Dioxo-3,4,5,10-tetrahydro-2 H-benzo[g]chromen-2-yl)chlorin e6 trimethyl ester (5 b): 1H NMR (300 MHz, CDCl3): d = 1.66 (s, 2 H; NH), 1.70 (t, J = 7.6 Hz, 3 H; H-82), 1.75 (d, J = 7.1 Hz, 3 H; H-181), 2.13–2.37 (m, 3 H; 2  H-171, H-172), 2.54–2.62 (m, 1 H; H-172), 2.70– 2.95 (m, 2 H; H-3’), 2.97–3.21 (m, 2 H; H-4’), 3.24 (s, 3 H; H-71), 3.49 and 3.50 (2 s, 3 H; H-21), 3.59 (s, 3 H; H-121), 3.63 (s, 3 H; H-174), 3.73–3.81 (m, 2 H; H-81), 3.77 (s, 3 H; H-153), 4.26 (s, 3 H; H-132), 4.42 (d, J = 8.3 Hz, 1 H; H-17), 4.43–4.53 (m, 1 H; H-18), 5.25 (d, J = 18.3 Hz, 1 H; H-151), 5.37 (d, J = 18.3 Hz, 1 H; H-151), 6.66 (dt, J = 5.3, 3.2 Hz, 2 H; H-2’), 7.74–7.82 (m, 2 H; H-7’, H-8’), 8.17–8.23 (m, 2 H; H-6’, H-9’), 8.79 and 8.80 (2 s, 1 H; H-20), 9.56 and 9.57 (2 s, 1 H; H5), 9.71 ppm (s, 1 H; H-10); UV/Vis (DMF): lmax (log e): 396 (5.09), 497 (4.02), 524 (3.48), 603 (3.58), 657 nm (4.64); HRMS (ESI-TOF): m/ z calcd for C48H49N4O9 : 825.3494 [M+H] + ; found: 825.3494.

3-(5-Oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]chromen-2-yl)chlorin e6 trimethyl ester (2 b): 1H NMR (300 MHz, CDCl3): d = 1.67 (s, 2 H; NH), 1.70 (t, J = 7.2 Hz, 3 H; H-82), 1.76 and 1.77 (2 d, J = 7.0 Hz, 3 H; H-181), 2.08–2.28 (m, 3 H; 2  H-171, H-172), 2.53–2.61 (m, 1 H; H172), 2.77–2.85 (m, 1 H; H-3’), 2.91–3.08 (m, 1 H; H-3’), 3.12–3.16 (m, 2 H; H-4’), 3.12 and 3.13 (2 s, 3 H; H-71), 3.52 (s, 3 H; H-21), 3.59 (s, 3 H; H-121), 3.64 (s, 3 H; H-174), 3.77 (q, J = 7.2 Hz, 2 H; H-81), 3.79 (s,

General procedure for preparing the protoporphyrin IX derivatives: In a round-bottom flask or sealed tube, equipped with a magnetic stirring bar, a solution of compound 6 a [2 equiv, formed in situ from 4-hydroxycoumarin (9.9 mg, 61 mmol) and paraformaldehyde (14.7 mg, 489 mmol)]) [for 6 b: 4-hyroxy-6-methylcoumarin (10.8 mg), for 6 c: 4-hydroxy-N-methylquinolin-2-one (10.7 mg), for 6 d: 2-hydroxy-1,4-naphthoquinone (10.7 mg)] in 1,4-

Chem. Eur. J. 2014, 20, 1 – 13

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Full Paper meso); 13C NMR (126 MHz, CDCl3): d = 11.6 (CH3), 11.7 (CH3), 12.12 (CH3), 12.14 (CH3), 21.4 (C-4’), 21.7, 21.8 (C-131, C-171), 29.3 (N-CH3), 30.8 (C-3’), 31.0 (C-3’), 36.87, 36.92 (C-132, C-172), 51.7 (2  OCH3), 75.9, 76.1 (C-2’), 96.5, 96.6 (C-meso), 97.79, 97.81 (C-meso), 98.4 (Cmeso), 99.4 (C-meso), 107.1, 107.2, 113.89, 113.95 (C-7’), 116.2, 116.3, 121.60, 121.65, 121.74 (C-9’), 122.5, 122.7 (C-10’), 130.2, 130.25 (C8’), 137.2, 137.3, 137.4, 137.5, 137.6, 138.1, 138.5, 139.0, 145.20, 145.24, 146.0, 146.9, 147.3, 147.56, 147.59, 147.9, 148.2, 157.2, 163.1, 173.5 (C-133), 173.6 ppm (C-173); UV/Vis (DMF) lmax (log e): 411 (5.56), 539 (4.21), 575 nm (4.08); HRMS (ESI-TOF): m/z calcd for C58H54N6O8Zn: 1026.3289 [M+H] + ; found: 1026.3275.

dioxane (4 mL), and porphyrin 7 (20 mg, 30.6 mmol) was heated to reflux until consumption of the starting porphyrin 7 (1–24 h). The quinone methide-type precursors were added at regular intervals. Dioxane was then removed under reduced pressure, chloroform (50 mL) was added to the residue, and the mixture was washed with a saturated aqueous solution of sodium bicarbonate (2  20 mL). The organic phase was concentrated under vacuum and the residual crude product was purified by column chromatography by using a solution of chloroform/methanol (95:5) followed by preparative TLC by using toluene/ethyl acetate (2:1) as the eluent. {3,8-Bis(5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]chromen-2-yl)-13,17bis[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethylporphyrinato}zinc(II) (8 a): 1H NMR (500 MHz, [D6]acetone): d 2.91–3.07 (m, 4 H; H-3’, H-4’), 3.16–3.22 (m, 4 H; H-3’, H-4’), 3.28 (t, J = 7.5 Hz, 2 H; H132),3.31 (t, J = 7.5 Hz, 2 H; H-172),3.44 (s, 3 H; CH3), 3.60 (s, 3 H; OCH3), 3.61 (s, 3 H; OCH3), 3.67 (s, 3 H; CH3), 3.68 (s, 3 H; CH3), 3.85 (s, 3 H; CH3), 4.38 (t, J = 7.5 Hz, CH2, 2 H; H-131), 4.42 (t, J = 7.5 Hz, 2 H; CH2, H-171), 7.15–7.37 (m, 4 H; 2  H-2’, 2  H-9’), 7.45–7.49 (m, 2 H; 2  H-7’),7.65–7.70 (m, 2 H; 2  H-8’), 7.97 (d, J = 8.1 Hz, 1 H; H10’), 8.02–8.06 (m, 1 H; H-10’), 10.18 (s, 1 H; H-meso), 10.28 (s, 1 H; H-meso), 10.36 and 10.37 (2 s, 1 H; H-meso), 10.53 ppm (s, 1 H; Hmeso); 13C NMR (75 MHz, [D6]acetone): d = 11.6 (CH3), 11.7 (CH3), 12.1 (CH3), 12.2 (CH3), 21.8 (2  C-4’), 22.4 (C-131, C-171), 31.08 (C-3’), 31.11 (C-3’), 37.6 (C-132, C-172), 51.7 (2  OCH3), 77.78 (C-2’), 77.81 (C-2’), 97.9 (C-meso), 98.7 (C-meso), 99.1 (C-meso), 99.9 (C-meso), 102.9, 117.3 (2  C-7’), 123.30 (C-10’), 123.34 (C-10’), 124.8 (C-9’), 124.9 (C-9’), 132.47 (C-8’), 132.50 (C-8’), 137.9, 138.6, 138.7, 140.5, 146.2, 147.2, 149.3, 149.7, 153.60, 153.61 (C-10’a), 161.1 (2  CO), 173.8 ppm (C-133, C-173); UV/Vis (DMF): lmax (log e): 411 (5.65), 540 (4.31), 576 nm (4.19); HRMS (ESI-TOF): m/z calcd for C56H48N4O10Zn: 1000.2656 [M] + ; found: 1000.2654.

{3,8-Bis(5,10-dioxo-3,4,5,10-tetrahydro-2 H-benzo[g]chromen-2-yl)13,17-bis[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethylporphyrinato}zinc(II) (11 a): 1H NMR (500 MHz, [D6]DMSO): d = 2.77–2.86 (m, 2 H; H-3’), 2.95–3.04 (m, 2 H; H-3’), 3.09–3.20 (m, 4 H; 4  H-4’), 3.28 (t, J = 7.4 Hz, 2 H; H-132), 3.31 (t, J = 7.4 Hz, 2 H; H-172), 3.52 (s, 3 H; CH3), 3.57 (s, 3 H; OCH3), 3.58 (s, 3 H; OCH3), 3.64 (s, 6 H; 2  CH3), 3.76 (s, 3 H; CH3), 4.35 (t, J = 7.4 Hz, 2 H; H-131), 4.39 (t, J = 7.4 Hz, 2 H; H-171), 7.05 (dd, J = 11.6, 2.4 Hz, 1 H; H-2’), 7.09 (dd, J = 11.4, 2.6 Hz, 1 H; H-2’), 7.87–7.92 (m, 2 H; H-7’, H-8’), 7.93–7.97 (m, 2 H; H-7’, H-8’), 8.11–8.14 (m, 2 H; H-6’, H-9’), 8.15–8.17 (m, 2 H; H-6’, H9’), 10.10 and 10.11 (2 s, 1 H; H-meso), 10.21 (s, 1 H; H-meso), 10.24 (s, 1 H; H-meso), 10.34 and 10.35 ppm (2 s, 1 H; H-meso); 13C NMR (126 MHz, [D6]DMSO): d = 11.4 (CH3), 11.5 (CH3), 11.9 (CH3), 11.95 (CH3), 20.2 (C-4’), 20.21 (C-4’), 21.5 (C-131), 21.6 (C-171), 29.1 (C-3’), 29.2 (C-3’), 36.9 (C-132), 37.0 (C-172), 51.5 (2  OCH3), 75.9 (C-2’), 75.91 (C-2’), 96.8 (C-meso), 97.8 (C-meso), 98.3 (C-meso), 99.1 (Cmeso), 122.18, 122.24, 125.9, 126.1 (2  C-6’, C-9’), 131.0, 131.92 (2  C-5’a, C-9’a); 133.7, 134.6 (2  C-7’, C-8’), 136.6, 136.9, 137.7, 137.9, 138.1, 138.7, 139.5, 143.9, 144.9, 145.7, 146.7, 147.2, 147.90, 147.94, 148.2, 156.20, 156.21, 173.20 (C-133), 173.21 (C-173), 179.49 (CO), 179.53 (CO), 184.09 (CO), 184.11 ppm (CO); UV/Vis (DMF): lmax (log e): 411 (5.54), 539 (4.16), 575 nm (4.03); HRMS (ESI-TOF): m/z calcd for C58H49N4O10Zn: 1025.2735 [M+H] + ; found: 1025.2718.

{3,8-Bis(6-methyl-5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]chromen2-yl)-13,17-bis[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethylporphyrinato}zinc(II) (9 a). 1H NMR (300 MHz, CDCl3): d = 2.25 (s, 3 H; ArCH3), 2.30 (s, 3 H; Ar-CH3), 2.84–2.96 (m, 2 H; H-3’, H-4’), 3.14–3.24 (m, 10 H; 3  H-3’, 3  H-4’, 2  H-132, 2  H-172), 3.42 (s, 3 H; CH3), 3.43 (s, 3 H; CH3), 3.59 (s, 3 H; CH3), 3.61 (s, 3 H; OCH3), 3.65 (s, 3 H; OCH3), 3.81 (s, 3 H; CH3), 4.23–4.39 (m, 4 H; 2  H-131, 2  H-171), 6.90–6.96 (m, 2 H; 2  H-2’), 7.32–7.36 (m, 4 H; 2  H-7’, 2  H-8’), 7.75 (d, J = 8.0 Hz, 1 H; H-10’), 7.83 (d, J = 8.0 Hz, 1 H; H-10’), 9.85 (s, 1 H; H-meso), 10.08 and 10.10 (2 s, 1 H; H-meso), 10.17 (s, 1 H; H-meso), 10.39 ppm (s, 1 H; H-meso); 13C NMR (75 MHz, CDCl3): d = 11.59 (CH3), 11.6 (CH3), 12.1 (CH3), 12.2 (CH3), 20.8 (Ar-CH3), 20.9 (Ar-CH3), 21.1 (C-4’), 21.2 (C-4’), 21.68 (C-131), 21.71 (C-171), 30.4 (C-3’), 30.7 (C-3’), 36.8 (C-132, C-172), 51.7 (2  OCH3), 77.2 (C-2’), 97.0 (C-meso), 98.2 (2  C-meso), 99.3 (C-meso), 101.6, 101.7, 115.6, 116.6 (2  C-7’), 122.0 (2  C-10’), 132.7 (2  C-8’), 133.7 (2  C-9’), 136.4, 136.6, 137.8, 138.5, 139.4, 145.0, 145.9, 146.9, 147.3, 148.1, 148.5, 150.8 (2  C6’a), 160.9 (2  C-10’b), 163.4 (2  CO), 173.4 (C-133), 173.5 ppm (C173); UV/Vis (DMF): lmax (log e): 411 (5.56), 539 (4.21), 576 nm (4.08). HRMS (ESI-TOF): m/z calcd for C58H53N4O10Zn: 1028.2969 [M] + ; found: 1028.2978.

Decomplexation procedure: The ZnII protoporhyrins 8 a–11 a were treated with a mixture of 5 % TFA in chloroform (10 mL). The reaction mixture was stirred at RT for 20 min and then neutralized with a saturated aqueous solution of NaHCO3. The aqueous phase was extracted with chloroform, the organic phase was dried over Na2SO4 and evaporated under vacuum to dryness. The resulting residue was purified by preparative TLC by using mixtures of ethyl acetate and toluene or chloroform and methanol as eluents. 3,8-Bis(5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]chromen-2-yl)-13,17bis[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethylporphyrin (8 b): 1 H NMR (300 MHz, CDCl3): d = 3.60 (s, 2 H; NH), 2.84–3.23 (m, 8 H; H-3’, H-4’), 3.28 (t, J = 7.2 Hz, 2 H; H-132), 3.33 (t, J = 7.2 Hz, 2 H; H172), 3.41 and 3.42 (2 s, 3 H; CH3), 3.53 and 3.54 (2 s, 3 H; CH3), 3.67 (s, 6 H; 2  OCH3), 3.70 (s, 3 H; CH3), 3.81 (s, 3 H; CH3), 4.40 (t, J = 7.2 Hz, 2 H; H-131), 4.45 (t, J = 7.2 Hz, 2 H; H-171), 6.86–6.91 (m, 2 H; 2  H-2’), 7.21–7.29 (m, 2 H; 2  H-9’), 7.50 (d, J = 8.3 Hz, 2 H; 2  H7’), 7.57–7.63 (m, 2 H; 2  H-8’), 7.94–7.98 (m, 1 H; H-10’), 8.01–8.04 (m, 1 H; H-10’), 10.16 (s, 1 H; H-meso), 10.21 and 10.24 (2 s, 1 H; Hmeso), 10.24 (s, 1 H; H-meso), 10.34 and 10.35 ppm (2 s, 1 H; Hmeso); UV/Vis (DMF): lmax (log e): 401 (5.34), 496 (4.20), 532 (3.93), 568 (3.83), 621 nm (3.62); HRMS (ESI-TOF): m/z calcd for C56H51N4O10 : 939.3598 [M+H] + ; found: 939.3592.

{3,8-Bis(6-N-methyl-5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]quinolin2-yl)-13,17-bis[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethylporphyrinato}zinc(II) (10 a): 1H NMR (300 MHz, CDCl3): d = 2.86–3.05 (m, 8 H; 4  H-3’, 4  H-4’), 3.15–3.24 (m, 4 H; 2  H-132, 2  H-172), 3.35 (s, 3 H; CH3), 3.36 (s, 3 H; CH3), 3.52 (s, 3 H; CH3), 3.56 (s, 3 H; N-CH3), 3.58 (s, 3 H; N-CH3), 3.64 (s, 3 H; OCH3), 3.65 (s, 3 H; OCH3), 3.76 (s, 3 H; CH3), 4.23–4.32 (m, 4 H; 2  H-131, 2  H-171), 6.79–6.82 (m, 2 H; 2  H-2’), 7.08–7.19 (m, 2 H; 2  H-9’), 7.33–7.37 (m, 2 H; 2  H-7’), 7.50–7.61 (m, 2 H; 2  H-8’), 8.14–8.16 (m, 1 H; H-10’), 8.23 (d, J = 8.5 Hz, 1 H; H-10’), 9.72 (s, 1 H; H-meso), 9.97 and 9.99 (2 s, 1 H; Hmeso), 10.19 (s, 1 H; H-meso), 10.38 and 10.40 ppm (2 s, 1 H; H-

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3,8-Bis(6-methyl-5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]chromen-2yl)-13,17-bis[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethylporphyrin (9 b): 1H NMR (300 MHz, CDCl3): d = 3.58 (s, 2 H; NH), 2.25 (s, 3 H; Ar-CH3), 2.29 (s, 3 H; Ar-CH3), 2.80–3.00 (m, 4 H; H-3’, H-4’), 3.04– 3.24 (m, 4 H; H-3’, H-4’), 3.28 (t, J = 7.2 Hz, 2 H; 2  H-132), 3.33 (t, J = 7.2 Hz, 2 H; 2  H-172), 3.44 (s, 3 H; CH3), 3.57 (s, 3 H; CH3), 3.66 and 3.67 (2 s, 3 H; OCH3), 3.70 (s, 3 H; CH3), 3.81 (s, 3 H; CH3), 4.41 (t, J =

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Full Paper 7.2 Hz, 2 H; 2  H-131), 4.45 (t, J = 7.2 Hz, 2 H; 2  H-171), 6.84–6.93 (m, 2 H; 2  H-2’), 7.38–7.39 (m, 4 H; 2  H-7’, 2  H-8’), 7.72 (s, 1 H; H10’), 7.80 (s, 1 H; H-10’), 10.16 (s, 1 H; H-meso), 10.23 (s, 1 H; Hmeso), 10.24 (s, 1 H; H-meso), 10.36 ppm (s, 1 H; H-meso); UV/Vis (DMF): lmax (log e): 401 (5.54), 496 (4.22), 532 (3.98), 569 (3.89), 621 nm (3.67); HRMS (ESI-TOF): m/z calcd for C58H55N4O10 : 967.3913 [M+H] + ; found: 967.3904. 3,8-Bis(6-N-methyl-5-oxo-2,3,4,5-tetrahydro-2 H-pyrano[3,2-c]quinolin2-yl)-13,17-bis[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethylporphyrin (10 b): 1H NMR (300 MHz, CDCl3): d = 3.60 (s, 2 H; NH), 2.85–3.24 (m, 8 H; 4  H-3’, 4  H-4’), 3.27 (t, J = 7.5 Hz, 2 H; 2  H132), 3.32 (t, J = 7.5 Hz, 2 H; 2  H-172), 3.37 and 3.44 (2 s, 3 H; CH3), 3.48 and 3.50 (2 s, 3 H; CH3), 3.66 and 3.67 (2 s, 3 H; OCH3), 3.70 (s, 3 H; CH3), 3.79 and 3.80 (2 s, 3 H; N-CH3), 3.90 (s, 3 H; N-CH3), 3.91 (s, 3 H; CH3), 4.39 (t, J = 7.5 Hz, 2 H; 2  H-131), 4.45 (t, J = 7.5 Hz, 2 H; 2  H-171), 6.75–6.88 (m, 2 H; 2  H-2’), 7.14–7.21 (m, 2 H; 2  H-9’), 7.49–7.53 (m, 2 H; 2  H-7’), 7.59–7.67 (m, 2 H; 2  H-8’), 8.17 (dd, J = 7.9, 1.3 Hz, 1 H; H-10’), 8.24 (dd, J = 7.9, 1.3 Hz, 1 H; H-10’), 10.13 (s, 1 H; H-meso), 10.22 (s, 1 H; H-meso), 10.30 and 10.33 (2 s, 1 H; Hmeso), 10.45 and 10.46 ppm (2 s, 1 H; H-meso); UV/Vis (DMF): lmax (log e): 400 (5.31), 497 (4.21), 530 (3.96), 568 (3.84), 621 nm (3.63); HRMS (ESI-TOF): m/z calcd for C58H57N6O8 : 965.4232 [M+H] + ; found: 965.4224.

Keywords: Chlorin e6 · hetero Diels–Alder · Knoevenagel · photophysics · porphyrinoids

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3,8-Bis(5,10-dioxo-3,4,5,10-tetrahydro-2 H-benzo[g]chromen-2-yl)13,17-bis[2-(methoxycarbonyl)ethyl]-2,7,12,18-tetramethylporphyrin (11 b): 1H NMR (300 MHz, CDCl3 and one drop of TFA): d = 2.03–2.42 (m, 4 H; 4  H-3’), 2.95–3.09 (m, 4 H; 4  H-4’), 3.13–3.16 (m, 4 H; 2  H-132, 2  H-172), 3.63 (s, 3 H; CH3), 3.67 (s, 3 H; OCH3), 3.68 (s, 3 H; OCH3), 3.70 (s, 6 H; 2  CH3), 3.73 (s, 3 H; CH3), 4.44–4.49 (m, 4 H; 2  H-131, 2  H-171), 6.73–6.80 (m, 2 H; 2  H-2’), 7.87–7.92 (m, 4 H; 2  H-7’, 2  H-8’), 8.25–8.29 (m, 2 H; H-6’, H-9’), 8.33–8.42 (m, 2 H; H-6’, H-9’), 10.65 and 10.68 (2 s, 1 H; H-meso), 10.91 and 10.95 (2 s, 1 H; H-meso), 11.02 and 11.22 (2 s, 1 H; H-meso), 11.29 and 11.43 ppm (2 s, 1 H; H-meso); UV/Vis (DMF): lmax (log e): 400 (5.25), 497 (4.17), 530 (3.93), 567 (3.83), 621 nm (3.64); HRMS (ESI-TOF): m/z calcd for C58H51N4O10 : 963.3560 [M+H] + ; found: 963.3589.

Acknowledgements Thanks are due to the University of Aveiro, Fundażo para a CiÞncia e a Tecnologia (FCT, Portugal), the European Union, QREN, FEDER, and COMPETE for funding the Organic Chemistry Research Unit (QOPNA) (project PEst-C/QUI/UI0062/2013, FCOMP-01–0124-FEDER-037296) and the Portuguese National NMR Network (RNRMN) are gratefully acknowledged. J.C.J.M.D.S.M. thanks QOPNA for a research grant and Dr. Ronald Steffen, Tobias Bornhtter, and Dr. Eugeny Ermilov from the Institut fr Physik, Humboldt-Universitt zu Berlin for help during measurements and fruitful discussions. Thanks are also due to the FCT (Portugal)-CAPES (Brazil) collaborative programme, and to CNPq and FAPESP/S¼o Paulo Research Foundation, Brazil, (2013/06532-4). In Japan, this work was partly supported by Grant-in-Aids for Scientific Research on Innovative Areas (No. 25109502, “Stimuli-responsive Chemical Species”), Scientific Research (B) (No. 23350095), Challenging Exploratory Research (No. 25620019), and Young Scientist (B) (No. 24750031) from the MEXT. Chem. Eur. J. 2014, 20, 1 – 13

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FULL PAPER & Protoporphyrin IX J. C. J. M. D. S. Menezes, M. A. F. Faustino, K. T. d. . Oliveira, M. P. Uliana, V. F. Ferreira, S. Hackbarth, B. Rçder, T. Teixeira Tasso, T. Furuyama, N. Kobayashi,* A. M. S. Silva, M. G. P. M. S. Neves,* J. A. S. Cavaleiro* Natural hybrid photosensitizers: The synthesis of new chlorin e6 trimethyl ester and protoporphyrin IX dimethyl ester dyads as free bases and ZnII complexes in high yields is reported. All compounds were fully characterized by

Chem. Eur. J. 2014, 20, 1 – 13

1D and 2D NMR techniques, UV/Vis spectroscopy, and HRMS. Their photophysical and electrochemical properties were evaluated. Magnetic circular dichroism and circular dichroism spectra were also recorded.

Synthesis of New Chlorin e6 Trimethyl and Protoporphyrin IX Dimethyl Ester Derivatives and Their Photophysical and Electrochemical Characterizations

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