Molecular Sciences Synthesis and Characterization of Novel Purpurinimides as Photosensitizers for Photodynamic Therapy

A series of novel purpurinimides with long wavelength absorption were designed and synthesized to develop novel and potential photosensitizers. These compounds were investigated through reduction, oxidation, rearrangement reaction and amidation reactions of methyl pheophorbide a. They demonstrated a considerable bathochromic shift of the major absorption band in the red region of the optical spectrum (695–704 nm). Newly synthesized purpurinimides were screened for their antitumor activities, and showed higher photodynamic efficiency against A549 cell lines as compared to purpurin-18 methyl ester. The results revealed the novel purpurinimides could be potential photosensitizers.


Introduction
Photodynamic therapy (PDT) is a relatively new cancer modality that uses light, photosensitizers (PSs), and oxygen for the treatment of various forms of cancer by photodynamic action [1][2][3]. The PDT treatment derives great promise from the dual selectivity that is produced by both a preferential uptake of the drug by the diseased tissue and the restriction of carefully regulated light absorption onto the specific sites [4]. Regulatory approvals for the clinical use of PSs and PDT now exist in many countries around the world for treating cancers of the head and neck, brain, lung, pancreas, intraperitoneal cavity, breast, prostate and skin [5][6][7].
Chlorophyll (a natural dye) derivatives and related systems have been used as PSs in cancer phototherapy. Chlorophylls exhibit photophysical properties similar to porphyrin systems; the Q y bands of chlorins is normally red-shifted to 20-30 nm and has a 10 times greater absorption intensity compared to porphyrins, which make chlorin-containing systems better candidates for PDT [8].
Purpurinimides, derived from chlorophyll-a, are tumor-avid PSs and show a strong absorption in the near IR region with a high singlet oxygen ( 1 O 2 ) producing efficiency, a key cytotoxic agent in PDT application [9]. PSs with long wavelength absorption should exhibit deeper tissue penetration, which is very useful to treat large and deeply seated tumors [10][11][12]. Therefore, the synthesis of novel purpurinimides has become the focus in PDT studies.
For chlorophyll derivatives, the Q y bands were strongly affected by introduced different substituent groups to Q y axis (N 21 -N 23 , see Scheme 1). In continuation with our earlier efforts on PS design [13], it was thought worthwhile to synthesize new purpurinimide derivatives by incorporating the essential structural features of the above-mentioned potential cytotoxic drugs in order to obtain synergistic effects, which we report herein.
In the present work, we report the synthesis, structural characterization and biological evaluation of a series of novel purpurinimides through reduction, oxidation and rearrangement reactions followed by amidation reaction of methyl pheophorbide a (MPa).

Synthesis and Characterization
In our quest to compare the effect of imide analogs for quantitative structure-activity relationship (QSAR) studies [14], several attempts were made to convert the 3-vinyl group into the ethyl group and dimethyloxyethyl group by reduction and oxidation reaction.
The synthetic strategies adopted for the target compounds are shown in Scheme 1. MPa 1, as an important starting material, was isolated from chlorophyll paste (Excrementum bombycis). The vinyl group at 3-position of MPa 1 was selectively hydrogenated by using Pd/C as a catalyst to yield meso-pheophorbide a 2, which was converted to mesopurpurin-18 methyl ester 3 via air oxidation. Reaction of MPa 1 with Ti(NO 3 ) 3 at 0 °C produced the intermediate adduct 5 in high yield. Following the same procedure, 3-(2,2-dimethoxyethyl)-3-devinyl-purpurin-18 methyl ester 6 was obtained in 40% yield. From the reaction between two key intermediates 3 and 6, and the corresponding amines, (a) N,N-dimethyl ethylamine; (b) N,N-diethyl ethylamine; (c) N-isopropyl ethylamine; (d) N,N-dimethylpropyl ethylamine; and (e) imidazolyl propylamine, successfully afforded the final purpurinimides 4a-4e and 7a-7e in excellent yield. All the purpurinimide compounds were successfully characterized by a combination analysis of 1 H NMR and UV-vis spectroscopies, and elemental analysis. The structures of these novel purpurinimides were confirmed by 1 H NMR spectroscopy ( Figure 1). Compared to the mesopurpurin-18 methyl ester 3 the 1 H NMR spectra of 4a-4e showed each triplet at  The spectroscopic properties of the purpurinimides in dichloromethane are shown in Figure 2 and summarized in Table 1. In the electronic absorption spectra, the long wavelength bands (Q y band) of mesopurpurinimides 4a-4e were observed in the range of 694-698 nm, compared with mesopurpurin-18 methyl ester 3 (686.7 nm), these mesopurpurinimides show a bathochromic shift of the Q y band. The electronic spectrum of 3-(2,2-dimethoxyethyl)-3-devinyl-purpurinimides shows a bathochromic shift of the Q y bands from 690.3 (compound 6) to 700.1 (compound 7a), 699.6 (compound 7b), 703.6 (compound 7c), 698.9 (compound 7d) and 698.8 nm (compound 7e). These purpurinimides had the "ideal" photochemical properties required for an effective PDT agent.  Fluorescence (emission) spectra of purpurinimides 4a-4c and 7a-7c in dimethylsulfoxide (DMSO) are shown in Figure 3. For all measurements the excitation wavelength was 530 nm. Three broad emission bands were observed (for 4a, 550-644, 645-692, and 693-800 nm) with their emission maxima at 718 (4a), 717 (4b), 604 (4c), 720 (7a), 719 (7b), and 657 (7c) nm. In addition, elemental analysis data for the purpurinimides reveal a good match between calculated and experimental values to the compounds, respectively.

Singlet Oxygen Study
Figure 5 reveals relative difference of 1 O 2 photogeneration between the purpurinimides 4a-4c and 7a-7c using 1,3-diphenylisobenzofuran (DPBF) as a selective 1 O 2 acceptor [15]. Among the purpurinimides, 4c and 7a showed relatively higher 1 O 2 photogeneration, results which are slightly different from the in vitro results. Consequently, this result proves that the increased photodynamic activity attributed to the purpurinimides induced 1 O 2 photogeneration through cellular penetration and localization of the purpurinimides into the cells.

The Hydrophobicity Property Study
It is generally believed that the hydrophobicity parameter (logarithm of the partition coefficient between n-octanol and water; log P) is closely related to the cellular uptake. We applied a program module of the ACD/Labs software (version 12.01, Advanced Chemistry Development, Inc., Toronto, ON, Canada) to calculate the lipophilicity of the synthesized purpurinimides. Results are summarized in Table 3. All the purpurinimides have log P values up to log 5, which theoretically should be expected to give good PDT tumor response.

General Methods
The 1 H NMR spectra were recorded on a Varian-500 MHz spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts are given as δ values using TMS as the internal standard and J values in Hz. The UV-visible spectra were recorded on S-3100 spectrophotometer (Scinco, Seoul, Korea) using dichloromethane as solvent. Elemental analysis was performed on Flash 2000 series of automatic elemental analyzer (Thermo Fisher Scientific, Milano, Italy) at Biohealth Products Research Center (BPRC), Inje University, Korea. Fluorescence spectra were obtained using a LS-50B Perkin Elmer luminescence spectrometer (Perkin Elmer, Waltham, MA, USA) at the Center for Research Facilities, Gyeongsang National University, Korea. The hydrophobicity parameter (logarithm of the partition coefficient between n-octanol and water; log P) was calculated on the basis of the purpurinimide structure using ACD/Labs software (version 12.01). Melting points (uncorrected) were measured on an Electrothermal IA9000s Series digital melting point apparatus. Thin-layer chromatography (TLC) was done on Merck silica gel 60 glass sheets (Cat. HX948839, layer thickness 0.25 mm) (Merck, Darmstadt, Germany). Column chromatography was performed over silica gel 60 (230-400 mesh) (Merck). In some cases, preparative TLC plates were also used for the purification (Analtech precoated silica gel GF glass plate, Cat. 01012, layer thickness 0.5 mm) (Merck). Materials obtained from commercial suppliers were used without further purification. MPa 1 [16], mesopurpurin-18 methyl ester 3 [9,16] and 3-(2,2-dimethoxyethyl)-3-devinyl-purpurin-18 methyl ester 6 [9,17] were prepared according to the literature procedures.

General Procedure
In a typical experiment, mesopurpurin-18 methyl ester 3 (200 mg) and excess of each corresponding amine (0.15 mL) were dissolved in toluene (20 mL), and the mixture was refluxed under nitrogen atmosphere. After TLC showed complete consumption of purpurin-18 methyl ester, the mixture was cooled to room temperature, and then the solvent and excess amines were removed. The crude product was purified using silica column chromatography or preparative TLC plates with 10% methanol in dichloromethane to give corresponding purpurinimide 4a, 4b, 4c, 4d and 4e as a purple solid, respectively.

General Procedure
In a typical experiment, 3-(2,2-dimethoxyethyl)-3-devinyl-purpurin-18 methyl ester 6 (200 mg) and excess of each corresponding amine (0.15 mL) were dissolved in toluene (20 mL), and the mixture was refluxed under nitrogen atmosphere. After TLC showed complete consumption of purpurin-18 methyl ester, the mixture was cooled to room temperature, and then the solvent and excess amines were removed. The crude product was purified using silica column chromatography or preparative TLC plates with 10% methanol in dichloromethane to give corresponding purpurinimide 7a, 7b, 7c, 7d and 7e as a purple solid, respectively.

In Vitro Photosensitizing Efficacy
A549 cell lines were cultured at 37 °C in a humidified 5% CO 2 incubator using RFMI 1640 growth medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. For phototoxicity studies, A549 cells were plated in 96-well plates at a density of 1 × 10 5 cells/well. After 24 h of incubation, 100 μL of 1, 2, 5, 10 and 15 μM purpurinimides were added in each well, respectively. Plates were returned to the incubator for 24 h. And then the cells were replaced with fresh media and exposed to light (640-710 nm, total dose 2.0 J/cm 2 for 15 min). Following illumination, the plates were incubated at 37 °C in the dark. After 3, 12 and 24 h incubations, MTT solution was added into each well and the absorbance was measured by fluorescence multi-detection reader (BioTek, Synergy HT, Winooski, VT, USA) at 450 nm. Each group consisted of 3 wells. The percentage cell survival was calculated by normalization with respect to the value for no PS treatment (control).

Conclusions
We described the synthesis of novel purpurinimides, mesopurpurin-18-N-aminoimides and 3-(2,2-dimethoxyethyl)-3-devinyl-purpurin-18-N-aminoimides with various amines (N,N-dimethyl ethylamine, N,N-diethyl ethylamine, N-isopropyl ethylamine, N,N-dimethylpropyl ethylamine and imidazolyl propylamine). The final desired purpurinimides were obtained in excellent yield. The purpurinimides were characterized by a combination analysis of 1 H NMR, UV-vis and photoluminescence spectroscopies, and elemental analysis. 1 H NMR spectroscopy confirms the structures of purpurinimides using ammonium formation after methylation, resulting in significant down field shift of the methyl proton signals. In the electronic absorption spectra, compared with starting materials, these purpurinimides show a bathochromic shift (8-13 nm) of the Q y band, resulting in a long wavelength absorption (695-704 nm) which should be helpful for promoting deep light penetration into tumor tissue because of minimal light scattering. Fluorescence spectra present three broad emission bands at 550-800 nm range. Preliminary in vitro studies demonstrate that the new purpurinimides revealed excellent photodynamic efficacy (IC 50 0.28-1.27 μM at 12 h incubation time after photoirradiation), which corresponds the excellent 1 O 2 photogeneration of the purpurinimides. Among the purpurinimides, mesopurpurin-18-N-(N,N-dimethyl)ethylimide 4a presents the best photodynamic activity result. The photodynamic activity is relatively higher in the order of 4a > 4b > 4c > 7c > 7a > 7b, results which are related to the hydrophobicity property (log P). For the purpurinimides to be potential candidates for PDT, further in vivo studies (e.g., pharmacokinetics and tissue distribution tests) are desirable, which is currently under investigation. This result could be useful for synthesis and development of new potential PSs as well as for understanding of QSAR study in PDT.