Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities

Accepted Manuscript Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities Ping-Yong Liao, Xin-Rong Wang, Xiang-Hua Zhang, Tai-Shan Hu, Mei-Zhen Zheng, Ying-Hua Gao, Yi-Jia Yan, Zhi-Long Chen PII: DOI: Reference:

S0045-2068(16)30410-2 http://dx.doi.org/10.1016/j.bioorg.2017.02.015 YBIOO 2025

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

14 December 2016 26 January 2017 26 February 2017

Please cite this article as: P-Y. Liao, X-R. Wang, X-H. Zhang, T-S. Hu, M-Z. Zheng, Y-H. Gao, Y-J. Yan, Z-L. Chen, Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities, Bioorganic Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bioorg.2017.02.015

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Synthesis of 2-morpholinetetraphenylporphyrins and their photodynamic activities Ping-Yong Liaoa, Xin-Rong Wanga, Xiang-Hua Zhangb, Tai-Shan Huc, Mei-Zhen Zhengc, Ying-Hua Gaoa, Yi-Jia Yanc, and Zhi-Long Chena* a

Department of pharmaceutical Science & Technology, College of Chemistry and Biology, Donghua University,

Shanghai 201620, P. R. China b

Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200433, China

c

Shanghai Xianhui Pharmaceutical Co. Ltd., Shanghai 200433, China

*

Corresponding author: Zhi-Long Chen, Tel/Fax: 86-21-67792654.E-mail: [email protected]

Abstract: A series of 2-morpholinetetraphenylporphyrins functionalized with various substituents (Cl, Me, MeO group) at 4-phenyl position were prepared via nucleophilic substitution of 2-nitroporphyrin copper derivatives with morpholine by refluxing under a nitrogen atmosphere and then demetalization. Their basic photophysical properties, intracellular localization, cytotoxicities in vitro and in vivo were also investigated. All synthesized photosensitizers exihibited longer maxima absorption wavelengths than Hematoporphyrin monomethyl ether (HMME). They showed low dark cytotoxicity compared with that of HMME and were more phototoxic than HMME against Eca-109 cells in vitro. M3 also exhibited better photodynamic antitumor efficacy on BALB/c nude mice at a lower concentration. Therefore, M3 is a promising antitumor photosensitizer in photodynamic therapy application. Keywords: photosensitizer, photodynamic therapy, porphyrin, antitumor

1. Introduction Photodynamic therapy (PDT) is a clinically approved, minimally invasive protocol for treatment and is an attractive therapeutic procedure utilizing a photosensitizer (PS) activated by light of appropriate wavelength (phototherapeutic window 600 − 850 nm) to generate highly reactive oxygen species (ROS)[1-4]. ROS are chemically reactive radicals or non-radical molecules derived from molecular oxygen and include singlet oxygen, peroxide, superoxide and the hydroxyl radical[5, 6]. The ROS mediated mechanism is the major cause underlying the efficacy of PDT[7]. ROS mainly initiates three biological mechanisms that make PDT an effective anticancer procedure: (1) direct tumor killing induced by the ROS; (2) tumor-associated vascular shutdown and massive ischemic death; (3) activation of antitumor immune memory and systemic response[8]. Compared with traditional cancer therapies such as surgery and chemotherapy, PDT has the advantage of dual selectivity in that the PS can be targeted to its destination cell or tissue without destructing the surrounding normal tissues[9]. Over the past decade, a substantial effort has been put into the development of various classes of PS, the synthesis of PS with desired physical, chemical and biological properties is considered to be an

important bottleneck in PDT[10]. Hematoporphyrin derivative (HPD, Photofrin), the first generation of porphyrin-based PS, was discovered by Lipson and Baldes in 1960s and has received regulatory approval for the treatment of various tumors in more than 40 countries throughout the world. It suffered several drawbacks such as chemical heterogeneity, insufficient selectivity and photocytotoxicity for tumor therapy[11]. Compared with HPD, Hematoporphyrin monomethyl ether (HMME) has a unique and defined molecular structure, which was a marketed drug in China. Experimental studies and clinical trials demonstrated that HMME has a strong photodynamic effect with low toxicity[12, 13]. The essential element in the development of PDT is the PS that absorbs light of an appropriate wavelength and gives desired therapeutic outcome. Another way to improve the efficacy of PDT is by reducing the dosage of PS and photo irradiation by increasing photocytotoxicity and selective accumulation in the tumor. As we all know, the limited solubility of porphyrin in commonly used solvents affects their optical properties[14], many methods have been proposed to overcome these drawbacks. One method relies on inducing steric isolation of the porphyrins’ core through their substitution with bulky groups[15]. Another method relies on the introduction to appropriate functional groups, such as morpholine, carboxylic or sulfonic groups[16]. In the present study, 2-morpholinetetraphenylporphyrins were synthesized by nucleophilic substitution of 2-nitroporphyrin copper derivatives with morpholine and demetalization. Their chemical characterizations, photophysical properties, subcellular localization and photodynamic activities in vitro and in vivo are investigated.

2. Results and Discussion 2.1. Synthesis and characterization The synthesis of 2-morpholinetetraphenylporphyrins with different substituents on 4-phenyl (M1 M3) is outlined in Scheme 1.

Scheme 1. Reagents and reaction conditions: (a) K2CO3, morpholine, reflux; (b) H2SO4, TFA, 30 min.

The compounds 1a-c were synthesized according to literature procedures[17-19]. Briefly, pyrrole and benzaldehyde derivatives were added to propinoic acid. After refluxing, the solution was cooled to room temperature and filtered to give purple tetraphenylporphyrin derivatives. Then, they were coordinated with copper atom, nitrated with cupric nitrate trihydrate to produce 2-nitro-5,10,15,20tetraphenylporphyrin copper (II) (1a-c). 2a-c were prepared by the nucleophilic substitution of 1a-c with dry morpholine under reflux at nitrogen atmosphere. Demetalization of 2a-c with H2SO4 and CF3COOH afforded target molecules (M1 - M3). The identities of M1 - M3 were elucidated by 1 HNMR,

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C-NMR and HR-MS. All spectra are provided in supplementary information (Figure S1 -

Figure S9). 2.2. Photophysical Properties The UV-vis spectra of M1 - M3 and HMME showed long wavelength absorption maxima in visible bands at 652, 653, 660, 612 nm respectively. M1 - M3 exihibited longer maxima absorption wavelength than HMME. Among them, M3 possessed the largest molar absorption coefficient at a long wavelength of 660 nm. The fluorescence excitation and emission spectra of M1 - M3 and HMME were investigated. As shown in Table 1, M1 - M3 displayed a fluorescence excitation band at approximately 420 nm and emission band at 662 - 672 nm, and the shapes of their excitation spectra were similar to those of the absorption spectra (Figure S10). Table 1 The absorption and emission properties of compounds M1 - M3 and HMME Compd

Absorption max(nm)(104 M-1cm-1) Soret

Q Band

emission max(nm) Excitation

emission

Stokes shift[a](nm)

M1

418 (10.5)

523 (1.21)

596 (0.42)

652 (0.23)

418

662

10

M2

418 (13.85)

524 (1.67)

600 (0.53)

653 (0.33)

418

666

13

M3

421 (16.55)

527 (1.36)

600 (0.50)

660 (0.41)

421

672

12

HMME

400 (16.05)

532 (0.88)

568 (0.68)

612 (0.46)

400

613

1

[a] Stokes shift at longest wavelength absorption.

We next experimentally investigated the reactive oxygen species (ROS) photogeneration of M1 - M3 and HMME. The efficiency of the ROS generation upon photo irradiation is an important measure of the cytotoxicity of PS. The quantum yield of singlet oxygen value (Φ Δ) was determined by using 1,3diphenylisobenzofuran (DPBF) as the scavenger and Rose Bengal as the reference (Φ Δ = 0.47)[20]. The data were plotted as ln[DPBF0]/[DPBFt] versus irradiation time t, straight lines were obtained for the sensitizers, and the slope for each compound was obtained after fitting with a linear function. The experimental results are shown in Figure 1. The ΦΔ values of M1 - M3 and HMME were calculated by the following equation[21]: ΦΔS =ΦΔR kS/kR. Where, k is the slope of the photodegradation rate of DPBF; the superscripts S and R stand for sample and reference, respectively. Table 2 summarized the quantum yield of singlet oxygen values. The ΦΔ value of M1 and HMME were compared, and ΦΔ value of M3 exhibited about 2.5 folds higher than that of HMME.

Fig. 1. First-order plots for the photodecomposition of DPBF photosensitized by compounds M1 - M3, HMME and the irradiation time. Table 2 Ratios of singlet oxygen yields and singlet oxygen values (Φ Δ) Compd M1 M2 M3 HMME

103×k [s-1] 1.58 3.19 4.31 1.56

2.3. Cytotoxicity on Eca-109 cells

ΦΔ 0.093 0.187 0.253 0.092

An effective PS needs to possess high phototoxicity and low dark toxicity. The dark toxicity and phototoxicity of M1 - M3 and HMME were evaluated in Eca-109 cells exposed to various concentrations of each compound using MTT assay. The dark and light cytotoxicity of DMEM medium containing different concentration of DMSO were investigated firstly. The data demonstrated the DMEM medium containing DMSO had no impact on the percentage cell survival (data not shown). The dark cytotoxicity of these PSs was evaluated at concentrations ranging from 0.001 to 100 µM (Figure S11). Compared to HMME, all PSs exhibited approximate dark cytotoxicity under 20 µM. After exposure to light, M1 - M3 displayed a prominent dose-dependent in the survival rate (Figure S12). At the same light dose, with an increase in the concentration of all the PSs from 1 µM to 16 µM, the cell viability revealed decrease results. At the same concentration of PSs, with an increase in the light dose from 1 J/cm2 to 16 J/cm2, the cell viability also revealed sharp decreasing results. The IC50 values of M1 - M3 and HMME were shown in Table 3. These data demonstrated that M1 - M3 were more toxic than HMME under light irradiation, at concentrations where their dark toxicity was negligible. These results suggest that M1 - M3 are promising photosensitizers for application in vitro. Table 3 Phototoxicity for M1 – M3 and HMME toward Eca-109 Cells Compound

IC50 (µM) [b]

M1

6.41*** 6.27***

M2 M3

5.81***

HMME

9.46 [b] Visible light  = 650 nm, light dose = 16 J/cm2. ***P<0.001 represents a significant difference relative to the control group.

2.4. Intracellular localization The subcellular localization of the PS is of special significance, since it determines the site of primary photodamage and the type of cellular response to the therapy. Its intracellular targets are close to the sites where the PS is located. Therefore, cellular structures containing PS would be preferentially damaged upon illumination[22]. The preferential sites of subcellular localization of M3 was investigated by confocal microscopy through exposure of Eca-109 cells to M3 for 12 h and staining them with fluorescent dye (Hoechst 33342) for cell nucleus. As shown in Figure 2, M3 was mainly found in cytoplasm and nuclear membranes corresponding to the red fluorescent and nucleus colocalized with the blue fluorescence of the nucleus probe.

Fig. 2. Intracellular location of M3 in Eca-109 cells. Eca-109 cells were incubated with 5 μM M3 for 4 h in dark, and then stained with Hoechst 33342. Red fluorescence corresponds to M3; blue fluorescence represents the signal for Hoechst 33342. 2.5. In vivo PDT efficacy PDT antitumor efficacy of M1 - M3 was evaluated on BABL/c nude mice bearing Eca-109 tumor. By comparing the tumor weight in different groups and defining group without any treatment as a control, the inhibition rates of tumor growth could be calculated. As illustrated in Figure 3a, the growth of implanted tumors among control group, light irradiation group and PS group was of no significant difference, M3 - PDT group has the best inhibition rate compared to other groups. Because of M3 best comprehensive performance, M3 was selected as a typical representative. In Figure 3b, the volume growth curves of tumors were provided. The tumor volume increased about 10 - folds for 14 days in control group. M3 - PDT group decreased the tumor volume at the 5th day post treatment, and the tumor growth was much slower than that in control group. Overall, these results demonstrated that compound M3 would be effective to tumor treament in vivo.

Fig. 3. (a) Inhibition rates of compounds M1 - M3 against Eca-109 cells. (b) Tumor volume of M3 at different time points post - PDT. The data shown are the means ± SD of three independent experiments.

3. Conclusions In conclusion, a series of 2-morpholinetetraphenylporphyrins were prepared via nucleophilic substitution of 2-nitroporphyrin copper derivatives with morpholine and demetalization. Their basic photophysical properties, intracellular localization, cytotoxicities in vitro and in vivo were also investigated. All synthesized PSs exihibited longer maxima absorption wavelengths than HMME. They also showed low cytotoxicity in the dark and compared to that of HMME. And they were more phototoxic than HMME against Eca-109 cells. In vivo experiments showed M3 exhibited better photodynamic antitumor efficacy on BALB/c nude mice at lower concentration. Therefore, M3 is a promising antitumor PS in PDT application.

4. Experimental Section 4.1. Materials and instrumentations All solvents and reagents were obtained from commercial suppliers and used without further purification unless otherwise stated. Melting points were obtained on a “stuart” Bibby apparatus and are uncorrected. 1H-NMR and

13

C-NMR spectra were recorded on a Bruker 400 MHz spectrometer.

Chemical shifts were reported as δ values relative to the internal standard tetramethylsilane. MALDITOF mass spectra were recorded on an AB SCIEX 4800 Plus MALDI TOF/TOFTM. HR-MS spectra were recorded on a Thermo Fisher Scientific LTQ FT Ultra Mass Spectrometer. Column chromatography was performed using silica gel H (300 - 400 mesh). UV-vis absorption spectra were recorded on an ultraviolet visible spectrophotometer (Model V-530, Japan). Fluorescence spectra were measured on a Fluorescence Spectrophotometer (FluoroMax-4, France). 4.2. General procedure for the synthesis of 2a-c A solution of 1 (0.23 mmol) and K2CO3 (360 mg, 2.56 mmol) in dry morpholine (12 mL) was stirred at reflux for about 10 h under nitrogen, at which time no more starting material was detected by TLC. The mixture was cooled, diluted with dichloromethane (150 mL), washed with water (3  100 mL) and saturated brine (3  100 mL), dried over anhydrous Na2SO4 and evaporated to dryness. The resulting crude porphyrin was purified by silica gel chromatography eluted with dichloromethane/petroleum ether to give the desired product 2.

4.2.1. 2-morpholine-5,10,15,20-tetra(4-chlorinephenyl)porphyrin copper (II) (2a) The crude porphyrin was purified by silica gel chromatography eluted with dichloromethane/ petroleum ether (1:4) to give the dark green product 2a (124 mg, 60%). Mp > 300 oC. MS (MALDITOF): m/z 897.1 [M + H]+. 4.2.2. 2-morpholine-5,10,15,20-tetra(4-methylphenyl)porphyrin copper (II) (2b) The crude porphyrin was purified by silica gel chromatography eluted with dichloromethane/ petroleum ether (1: 2) to give the dark green product 2b (71.5 mg, 38%). Mp > 300 oC. MS (MALDITOF): m/z 817.3 [M + H]+. 4.2.3. 2-morpholine-5,10,15,20-tetra(4-methoxylphenyl)porphyrin copper (II) (2c) The crude porphyrin was purified by silica gel chromatography eluted with dichloromethane/ petroleum ether (1:1) to give the desired product 2c (109.1 mg, 53.9%). Mp > 300 oC. MS (MALDITOF): m/z 881.3 [M + H]+. 4.3. General procedure for the synthesis of M1 - M3 To compound 2 (1 mmol) in a round-bottomed flask, CF3COOH (8 mL) was added. Concentrated H2SO4 (2 mL) was then added dropwise to this stirred mixture until the porphyrin was dissolved. The reaction continued with stirring at room temperature for 30 min, after which the mixture was poured into ice water (100 mL), then the pH was adjusted to 7 by adding aqueous NaHCO3. The water phase was extracted with dichloromethane (3  50 mL). The combined organic layer was washed with water (3  100 mL) and saturated brine (3  100 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to give purple solid of compound M. 4.3.1. 2-morpholine-5,10,15,20-tetra(4-chlorinephenyl)porphyrin (M1) M1 was prepared by following the above general procedure (yield: 718.3 mg, 86%). Mp > 300 oC. 1H NMR (400 MHz, CDCl3): δ = 8.80 - 8.68 (m, 5H), 8.63 (d, J = 4.9 Hz, 1H), 8.19 - 8.07 (m, 9H), 7.78 7.68 (m, 8H), 3.42 (d, 4H), 3.08 (d, J = 17.9 Hz, 4H), -2.68 (s, 2H) ppm. 13C NMR (100 MHz, CDCl3): δ = 140.84, 140.47, 140.03, 139.72, 136.53, 135.65, 135.56, 135.33, 134.42, 134.38, 134.30, 134.11, 127.16, 127.05, 119.71, 118.86, 118.12, 117.40, 117.25, 66.41, 54.23 ppm. HRMS (DART Positive) m/z [M + H]+ calcd for C48H34N5Cl4O: 836.1512; found: 836.1496. 4.3.2. 2-morpholine-5,10,15,20-tetra(4-methylphenyl)porphyrin (M2)

M2 was prepared by following the above general procedure (yield: 695 mg, 92%). Mp > 300 oC. 1H NMR (400 MHz, CDCl3): δ = 8.83 - 8.78 (m, 3H), 8.77 - 8.72 (m, 2H), 8.67 (d, J = 4.9 Hz, 1H), 8.20 8.16 (m, 3H), 8.11 (dd, J = 7.8, 3.6 Hz, 6H), 7.57 (t, J = 7.6 Hz, 8H), 3.28 (m, 8H), 2.76 - 2.69 (m, 12H), -2.59 (s, 2H) ppm. 13C NMR (100 MHz, CDCl3): δ = 139.79, 139.47, 139.02, 138.66, 137.33, 137.28, 137.22, 137.08, 135.52, 134.71, 134.60, 134.33, 127.58, 127.51, 127.48, 127.40, 120.80, 119.87, 118.55, 118.25, 66.50, 54.21, 21.59, 21.54 ppm. HRMS (DART Positive) m/z [M + H]+ calcd for C52H46N5O: 756.3697; found: 756.3685. 4.3.3. 2-morpholine-5,10,15,20-tetra(4-methoxylphenyl)porphyrin (M3) M3 was prepared by following the above general procedure (yield: 786.5 mg, 96%). Mp > 300 oC. 1H NMR (400 MHz, CDCl3): δ = 8.83 (d, J = 6.7 Hz, 3H), 8.80 - 8.74 (m, 2H), 8.66 (d, J = 4.8 Hz, 1H), 8.19 - 8.13 (m, 9H), 7.31 (dd, J = 8.5, 3.1 Hz, 6H), 7.24 (d, J = 8.1 Hz, 2H), 4.13 - 4.07 (m, 12H), 3.43 (m, 4H), 3.13 (m, 4H), -2.53 (s, 2H) ppm.

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C NMR (100 MHz, CDCl3): δ = 159.50, 159.42, 159.38,

159.28, 136.68, 135.87, 135.71, 135.42, 135.15, 134.85, 134.36, 134.07, 120.52, 119.54, 118.19, 117.88, 112.43, 112.3, 112.25, 112.21, 66.52, 55.65, 55.58, 54.13 ppm. HRMS (DART Positive) m/z [M + H]+ calcd for C52H46N5O5: 820.3493; found: 820.3478. 4.4. Photophysical and photochemical measurements 4.4.1. Absorption and emission spectra UV-visible absorption spectra were recorded on an ultraviolet visible spectrophotometer (Model V530, Japan). Fluorescence spectra were measured on a Fluorescence Spectrophotometer (FluoroMax-4, France). Slits were kept narrow to 1 nm in excitation and 1 or 2 nm in emission. Right angle detection was used. All the measurements were carried out at room temperature. Drugs were dissolved in DMSO to get 5µM solutions. 4.4.2. Singlet oxygen generation detection Luminescence spectra of singlet oxygen sensitized by each PS solution were recorded on a spectrometer. The singlet oxygen ability of PSs was monitored by chemical oxidation of DPBF. The PSs (0.5 μM) were treated with DPBF (20 μM) in DMSO. PS solution irradiated with 650 nm light at the laser intensity of 5 mW/cm2. The natural logarithm values of absorption of DPBF at 410 nm were plotted against the irradiation time and fit by a first - order linear least squares model to get the singlet oxygen generation rate of the photosensitized process. The rate constant was converted into 1O2 quantum yield by comparison with the rate constant for DPBF photo-oxidation sensitized by Rose Bengal.

4.5. In vitro experiments 4.5.1. Cell lines and culture conditions Eca-109 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences. All cell culture related reagents were purchased from Shanghai Ming Rong Bio-Science Technology Co., Ltd. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS, BioChrom, Cambridge, UK) in 5% CO2 at 37 oC in a humidified incubator. Cells in the exponential phase of growth were used in each experiment. 4.5.2. MTT cell viability assay Cytotoxicity studies were done with synthesized PSs and HMME in Eca-109 cells. Cells were incubated with various concentrations in DMEM medium with 10% (v/v) FBS, collected with 0.25% (w/v) trypsin, and seeded in 96-well plates at 1 × 105 cells per well. The cells were allowed to attach to the bottom of the wells and uptake for 24 h in the dark. PS in DMEM medium and 2% DMSO (different concentrations range from 0.001 to 100 µM) was administered to cells and allowed to uptake for 24 hours in the dark. DMEM medium containing photosensitizer was removed and cells were washed twice with fresh PBS before irradiation with different light doses (0, 1, 2, 4, 6, 8, 16 J/cm2) using a MRL-III-650 nm laser. The cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide (MTT) colorimetric assay 24 h after treatment. In parallel, non-irradiated cells were used to investigate the dark cytotoxicity. The percentage cell survival was calculated by normalization with respect to the value for no drug treatment. Data were obtained by using three independent sets of experiments done in triplicate for each concentration. 4.5.3. Intracellular localization The cells were plated on poly-L-lysine coated coverslips in 12-well plates at the density of 1 × 10 5 cells/mL. After 24 h, the cells were incubated in the dark at 37 °C with 5 µM drug for 4 h, then rinsed in the DMEM medium and incubated with Hoechst 33342 (1 µg/mL) for 10 min at 37 °C. After rinsing three times with PBS, the coverslips were fixed with 4% (w/v) paraformaldehyde at 4 °C for 30 min. The cells were observed by a confocal fluorescence microscopy (Carl Zeiss LSM 700,Jena, Germany). The drug was excited at 417 nm and monitored through a 600 - 700 nm band-pass filter, and Hoechst 33342 was excited at 350 nm and blue fluorescence was detected through a 450 - 500 nm band-pass filter. 4.6. In vivo experiments

4.6.1. Tumor xenograft mice model Four-weeks-old female BALB/c nude mice were obtained from the Shanghai SLAC Laboratory Animal Company and housed in dedicated pathogen-free barrier facilities. And 5 × 106 Eca-109 cells were injected subcutaneously in 200 µL PBS into right forelimb. All animal protocols were approved by the Animal Care and Use Committee of Donghua University. 4.6.2. In vivo PDT efficacy When the tumor reached approximately 100 mm3 in size (about 14 days after inoculation), the tumorbearing mice were divided into 4 groups: control, light, PS and PS - PDT (each group contained 6 mice). PS was injected into the tail vein of mice in the PS and PS - PDT groups at a dose of 5 mg/kg (PS was dissolved in PBS containing 1% DMSO). Then the mice were restrained in plastic plexiglass holders without anesthesia and treated with laser light (650 nm, 100 J/cm2, 180 mW/cm2) except for the control and PS group. The power was monitored during the entire treatment. Post - PDT, the mice were observed daily for tumor regrowth or tumor cure. Visible tumors were measured every two days using two orthogonal measurements L and W (perpendicular to L), and the volumes were calculated using the formula V = LW2/2 and recorded. After PS - PDT at 14 days, animals were sacrificed and the tumor was removed and weighed, and tumor inhibition rates were calculated. 4.7. Statistical analysis All experiments were performed in triplicate and the data were expressed as mean plus and minus the standard error of the mean. Analysis of variance (ANOVA) and Student’s t-test were used to determine the statistically significant difference among different groups when appropriate. 4.8. Human & animal welfare The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Donghua University.

Acknowledgements This work was supported by National Natural Science Foundation of China (No. 21372042, 81301878, 21402236), Foundation of Shanghai Government (No. 15XD1523400, 14140903500, 15431904100, 15411960400, 14ZR1439900, 14ZR1439800, 15ZR1439900, 16ZR1400600) and Foundation of Songjiang Government (No. 14SJGGYY08, 15SJGG45). References

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Graphical abstract

compound M3 is an effective and a highly promising antitumor agent for photodynamic therapy.

Highlights

1. A series of 2-morpholinetetraphenylporphyrin derivatives were synthesized. 2. All target compounds exhibited low cytotoxicity and comparable with that of Hematoporphyrin monomethyl ether (HMME) in the dark. 3. They were more phototoxic than HMME in vitro experiment. 4. M3 exhibited best photodynamic antitumor efficacy on BALB/c nude mice bearing Eca-109 tumor. 5. M3 is a powerful and promising antitumor photosensitizer for photodynamic therapy.