Synthesis, Singlet Oxygen Photogeneration and DNA Photocleavage of Porphyrins with Nitrogen Heterocycle Tails

Eight novel compounds were prepared by reaction of 5-(bromo- propoxyphenyl)-10,15,20-triphenylporphyrin with oxazole thiols, 1,3,4-oxadiazole thiols and 1,3,4-thiadiazole thiols, and their structures confirmed by UV-vis, IR, 1H-NMR, MS and elemental analysis. The assessment of indirectly measured 1O2 production rates against 5,10,15,20-tetraphenyl porphyrin (H2TPP) were described and the relative singlet oxygen production yields were: porphyrin 5 > porphyrins 1, 3, 4, 6-8, H2TPP > porphyrin 2. Porphyrin 4 and porphyrin 7 showed substantial photocleavage activities toward DNA, with over 75% cleavage observed at 40 µM. It suggested that these those porphyrins with nitrogen heterocycle tails are potential photosensitive agents.


Introduction
The human vascular endothelial growth factor (VEGF) gene locates at chromosome 6P 21,3 , whose length is 28 Kb. The VEGF gene, of 14 Kb length, consists alternately of eight extrons and seven

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introns. The encoding product is a homogenous dimeric glycoprotein of 34-35 KD [1]. Against a target of VEGFR-2 tyrosine kinase VEGFR-2 plays a main receptor function in tumor angiogenesis mediated by VEGF, which prevents the activation of VEGFR-2 enzyme and limits VEGF signal transduction so as to inhibit tumor growth for cancer treatment. Compounds with oxazole, 1,3,4-oxadiazole and 1,3,4-thiadiazole structures display certain capability of acting in the VEGFR-2 tyrosine kinase domain and of binding VEGFR-2 in competition with ATP to prevent the growth of tumor vessels and cause tumor death [2][3][4]. So far, research on the use of angiogenesis inhibitors against the tumor metastasis has become a major topic.
Meanwhile, porphyrin-based compounds with unique structures have special affinity interactions with tumor cells and they can selectively remain in the tumor tissues [5,6]. Photodynamic therapy (PDT) is a promising therapy against cancer with intrinsic selectivity, which is used to eliminate deviant tissues, such as tumors. While gathering selectively around the tumors, the photosensitizers can absorb visible light under illumination and generate active oxygen (such as the singlet oxygen, 1 O 2 , generated when porphyrins are photoactivated by absorption at the Soret band [7]), which leads to tumor cell death [8,9]. The 1 O 2 is produced by energy transfer from the photoexcited sensitizer (in its triplet state) to the ground-state (triplet) oxygen. This is known as the Type II photosensitization mechanism, which is considered to be more important in PDT [10,11]. The DNA photocleavage activity of a photosensitizer depends on its 1 O 2 yield [12,13].
Porphyrins linked with some anticancer drugs can effectively localize in tumor cells and increase the cell cytotoxicity through the synergistic effect of porphyrins and anticancer drugs [14]. To enhance targeted therapy role of pophyrins against the VEGFR-2 tyrosine kinase target some porphyrins with nitrogen heterocycle tails were prepared using as starting materials oxazole thiols, 1,3,4-oxadiazole thiols and 1,3,4-thiadiazole thiols linked by -O(CH 2 ) 3 -S-spacer group (shown in Scheme 1). Their 1 O 2 productivity and DNA photocleavage activities were then investigated.

Photogeneration of 1 O 2
DNA photocleavage of the porphyrins was the result of generating the singlet oxygen. 1,3-diphenylisobenzofuran (DPBF) was able to capture the 1 O 2 photogenerated by the porphyrins, which reduced its own light activity [15,16]. The relationship between A/A 0 abosorbed by DPBF and illumination time indirectly reflected 1 O 2 yield of those porphyrins compared with H 2 TPP.
As shown in Figure 1 for H 2 TPP and porphyrins 1-8, the absorbance of DPBF at 418 nm decreased in the presence of each porphyrin with increasing illumination time. From the slope of the line, the relative rates of 1 O 2 photogeneration of the porphyrins could be compared. With the increasing of the line slopes, 1 O 2 yield was higher. It could be seen that the order was porphyrin 5 > porphyrins 1, 3, 4, 6-8, H 2 TPP > porphyrin 2. The ability of photogenerating 1 O 2 by the photosensitizers might be greatly affected through the interaction between the chromophoric groups [17]. The yield of 1 O 2 photogeneration by porphyrins 1, 3, 4, 6-8 was similar to that of H 2 TPP. The reason was that the conjugated system was not connected to the structure, but rather flexible chains were used, which made the porphyrins with oxazoles, oxadiazoles and thiadiazole rings show no significant change of 1 O 2 photogeneration compared to H 2 TPP. For copper (II) porphyrinate 2 no 1 O 2 photogenration was seen. This phenomenon has previously been observed in other paramagnetic metalloporphyrins with partially filled d orbitals, such as Ag II -TPP [18] and Co II -TCNPP [19], presumably due to a metal-facilitated relaxation of the porphyrin excited triplet state that precludes bimolecular quenching by oxygen. In contrast, Zn-porphyrin 5 showed a greater yield of 1 O 2 , as other Zn (II)-porphyrin derivatives, and this is attributed to their correspondingly high triplet quantum yield, ΦT [20].

DNA photocleavage ability
The DNA photocleavage of porphyrins 1-8 and H 2 TPP was detected by monitoring the conversion of supercoiled form (form I) to the nicked circular form (form II). Only porphyrins 4, 7 with cationic groups showed certain photocleavage activies towards DNA, with over 75% cleavage activities observed at 40 µM (see Figure 2). The reason might be that porphrins 4, 7 with single cationic groups can bind the anionic area of DNA via electrostatic interactions. Although porphyrins 4, 7 have relatively weaker electrostatic interaction with DNA compared with other cationic porphyrins, such as meso-tetrakis(N-methylpyridinium-4-yl)porpyrin, H 2 TMPyP, (98% cleavage activity at 2.5 µM [19]), porphyrins 4, 7 also showed a good DNA photocleavage activity at higher concentrations. As for the remaining porphyrins and H 2 TPP, their insignificant DNA photocleavage activities (data not shown) were likely the result of a lack of a binding interaction with DNA and a more effectively oxidative attack from close range despite the high 1 O 2 yields. This suggests the importance of close-range interactions with the target for the effect of the drugs and the positive charge of porphyrin structures to affect the interaction with DNA [21,22].  In PDT, a good cellular uptake effect depends more on lipophilicity of a photosensitizer than on its 1 O 2 yield [23]. For example, H 2 TMPyP showed strong DNA binding and photocleavage activity, but its PDT efficacy was poor due to its poor cellular uptake. Hence, more porphyrins with both hydrophilic and lipophilic groups, such as porphyrins 4 and 7, can be an effective PDT agent.

Preparation of porphyrin 4
Porphyrin 3 (50 mg, 0.059 mmol) was added to CH 3 I (10 mL). After heating at reflux for 4 h under nitrogen, the solution was concentrated to dryness in vacuum. The precipitate was purified on a silica gel column and eluted with chloroform and methanol (v/v 20:1). The second fraction was the blue-violet title product. Yield: 53 mg, 90%. 1

Preparation of porphyrin 7
Porphyrin 6 (50 mg, 0.058 mmol) was added to CH 3 I (10 mL). After heating at reflux for 4 h under nitrogen, the solution was concentrated to dryness in vacuum. The precipitate was purified on a silica gel column and eluted with chloroform and methanol (v/v 20:1). The second fraction was the blue-violet title product. Yield: 54 mg, 92%. 1  14 mmol) and 5-(2-hydroxyphenyl)-2-thioxo-1,3,4-oxadiazoline (13 mg, 0.14 mmol) were dissolved in dry DMF (25 mL) and anhydrous potassium carbonate (1 g) was added to the solution. Under nitrogen protection, the mixture was heated to 65 °C for 5 h. After cooling to room temperature, the reaction mixture was poured into water saturated with sodium chloride (30 mL) and filtered. The precipitate was purified on a silica gel column and eluted with chloroform. The second fraction was the blue-violet title product. Yield: 130 mg, 61%. 1

Measurement of singlet oxygen production rate
1,3-Diphenylisobenzofuran (DPBF) was used as a selective singlet oxygen ( 1 O 2 ) acceptor, which was bleached upon reaction with 1 O 2 . Eight sample solutions of DPBF in DMSO (50 μM) containing, respectively no porphyrin (control sample), H 2 TPP (1 μM) and porphyrins 1-8 (1 μM) were prepared in the dark. Each sample container was covered with aluminum foil with a yellow filter (with cutoff wavelength <500 nm) on one side. The samples were then exposed to light (50 watt) through the filter. After irradiation, visible spectra of the sample solutions were measured spectrophotometrically. The normalized absorbances of DPBF at 418 nm in these samples were reported as a function of the photo-irradiation time. From this plot, the rates of 1 O 2 production of porphyrins 1-8 relative to those of H 2 TPP were determined.

DNA photocleavage assay
The DNA photocleavage activities of porphyrins 1-8 and H 2 TPP were measured using the plasmid DNA relaxation assay. Briefly, the plasmid DNA (pBluescript, 0.5 μg), enriched with the covalently-closed circular or supercoiled conformer (Form I), and the one-phor-all plus buffer (10 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, pH 7.5) was vortexed. Aliquots of the DNA were pipetted into different Eppendorf tubes. Various amounts of autoclaved water (control sample) or prophyrins (test sample) were added into the Eppendorf tubes to give a final volume of 20 μL in each sample tube. The sample mixtures were then photo-irradiated at 400-450 nm for 60 min using a transilluminator (Vilber Lourmat) containing 4 × 15 W light tubes (Aqua Lux) with maximum emission at 435 nm. After photo-irradiation, 2 μL of the 6x sample dye solution (which contained 20% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol FF) was added to each Eppendorf tube and mixed well by centrifugation. The sample mixtures were loaded onto a 0.8% (v/v) agarose gel (Gel dimension: 13 cm × 10 cm), with 1× TBE buffer (89 mM Tris-borate, 1 mM EDTA, pH 8) used as supporting electrolyte, and electrophoresized at 1.3 Vcm −1 for 3 h using a mini gel set (CBS Scientific Co., Model No. MGU-502T). After electrophoresis, the gel was stained with 0.5 μg/mL ethidium bromide solution for 30 min and then destained using deionized water for 10 min. The resulting gel image was viewed under 365 nm and captured digitally using a gel documentation system (BioRad).

Conclusions
Eight porphyrins with nitrogen heterocycle tails were synthesized and characterized. Compared with H 2 TPP, the 1 O 2 yields of porphyrins 1-8 were measured indirectly and their DNA photocleavage activities were tested. Two porphyrins with cationic groups, 4 and 7, which showed certain singlet oxygen yields and DNA photocleavage activities could be potenial photosensitizers. The in vitro PDT activities against VEGFR-2 receptors and tumor angiogenesis are currently under investigation.