Synthesis and evaluation of novel chlorophyll a derivatives as potent photosensitizers for photodynamic therapy

European Journal of Medicinal Chemistry 187 (2020) 111959

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis and evaluation of novel chlorophyll a derivatives as potent photosensitizers for photodynamic therapy Ying-Hua Gao a, Xue-Xue Zhu a, Wei Zhu a, Dan Wu b, Dan-Ye Chen a, Yi-Jia Yan c, Xiao-Feng Wu c, Donal F. O’Shea b, **, Zhi-Long Chen a, * a b c

Department of pharmaceutical Science & Technology, College of Chemistry and Biology, Donghua University, Shanghai, 201620, China Department of Chemistry, Royal College of Surgeons in Ireland, 123 St. Stephen’s Green, Dublin 2, Ireland Shanghai Xianhui Pharmaceutical Co., Ltd, Shanghai, 200433, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 October 2019 Received in revised form 20 November 2019 Accepted 8 December 2019 Available online xxx

Chlorophyll a exhibits excellent photosensitive activity in photosynthesis. The unstability limited its application as photoensitizer drug in photodynamic therapy. Here a series of novel chlorophyll a degradation products pyropheophorbide-a derivatives were synthesized and evaluated for lung cancer in PDT. These compounds have strong absorption in 660e670 nm with high molar extinction coefficient, and fluorescence emission in 660e675 nm upon excitation with 410e415 nm light. They all have much higher ROS yields than pyropheophorbide-a, and compound 10 was even higher than [3-(1hexyloxyethyl)]-pyrophoeophorbide a (HPPH). Distinctive phototoxicity was observed in vitro and the inhibition effect was in light dose-dependent and drug dose-dependent style. They can effectively inhibit the growth of lung tumor in vivo. Among them, compound 8 and 11 have outstanding photodynamic anti-tumor effects without obvious skin photo-toxicity, so they can act as new drug candidates for photodynamic therapy. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Chlorophyll derivatives Photosensitizer Photodynamic therapy Tumor

1. Introduction Photodynamic therapy (PDT) is a non-invasive or minimally invasive therapeutic technique with photo-imaging or photodiagnosis effects. With the optical fiber targeted laser irradiation, the light energy was absorbed by photosensitizer and then transferred to oxygen to produce high oxidative free radicals such as singlet oxygen which could react with electron-rich biomolecules to result in the necrosis and apoptosis of diseased tissue selectively [1]. So far, PDT has been widely used in the treatment of tumors, macular degeneration, pointed condyloma, actinic keratosis and other diseases in clinics [2e5]. Chlorophyll-a is an excellent photosynthesis pigment with high extinction coefficient at 660 nm and good singlet oxygen production [6]. Due to its unstability, chlorophyll-a degradation products were attracted attention in PDT. N-Aspartyl chlorin e6 (Npe6, Laserphylline) is the first chlorophyll-derived photosensitizer used

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (D.F. O’Shea), [email protected] (Z.-L. Chen). https://doi.org/10.1016/j.ejmech.2019.111959 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

in clinics, which was obtained from chlorin e6 by amidation of the acetic acid residue with L-aspartic acid and it had good hydrophilicity with strong absorption at 664 nm [7,8]. Nakashizuka et al. found that Laserphylline could seal new blood vessels without obvious damage to the surrounding tissues in PDT of choroidal neovascularization (CNV) [9]. Kevin M. Smith and Pandey, K. et al. synthesized series of chlorophyll derivatives and reported that compound 3-(1-hexyloxyethyl) ether of the pheophorbide a methyl ester had prominent photodynamic efficiency compared with the related chlorin e6 derivative in which the isocyclic ring E was cleaved. In addition, the longer-chain pyropheophorbide a ethers showed increased photodynamic activities due to slow clearance from plasma and tissues [10e12]. They also reported some chlorophyll-based photosensitizers for fluorescence imaging and photodynamic therapy with light in the near-infrared region [13]. Among these derivatives, HPPH was developed as a photodynamic anti-tumor drug which is in clinical evaluation [14e16]. Deyu Xu and Zhi-Long Chen et al. extracted and degraded chlorophyll-a from silkworm into series of chlorin e6 derivatives, pyropheophoride a derivatives and purpurin
18 derivatives with obvious photodynamic activities [17e24]. Pyropheophorbide-a, which is also called abalone toxin

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discovered by Hashimoto in 1962 from abalone pancreas [25], was derived from algal chlorophyll after the algae had been swallowed and metabolized. Under exposure to sunlight, it could cause serious skin photo-allergic reactions and result in some symptoms such as erythema, ulcers, partial necrosis after the abalone’s liver and other internal organs were eaten by human, cats or rats [26e29]. 5-aminolevulinic acid (5-ALA) can be used in PDT with safety as the prodrug to biosynthesize photosensitizer protoporphyrin IX with high selectivity in tumors and some other cells in rapid proliferation stage [30e32]. The derivatives of 5-ALA such as 5-ALA esters and N-acylated-5-ALA compounds are also effective prodrugs because they can be hydrolyzed into 5-ALA by related enzymes in vivo [33], which was also confirmed by our previous studies [34]. As an endogenous small molecular with amino group, carbonyl group, carboxylic group and only 5 carbons in its structure, 5-ALA is also a suitable compound used in the modification of photosensitizer to adjust the hydrophilicity and lipophilicity with the potential to produce protoporphyrin IX. Here, a series of pyropheophorbide-a derivatives were prepared after pyropheophorbide-a was linked with 5-ALA and then further modified, their biological activities in vitro and in vivo were also evaluated. 2. Results and discussion 2.1. Synthesis and characterization The synthesis of 173-N-(2-oxo-4-carboxybutyl) pyropheophorbide-a amide (3) was shown in Scheme 1. Pyropheophorbide-a (1) from silkworm excrements was reacted

with methyl 5-aminolevulinate hydrochloride in dichloromethane in the presence of EDCI-HOBt as condensation agent [35] and N, Ndiisopropylethylamine as base at rt for 6 h to give 173- N-(2-oxo-4methoxycarbonylbutyl) pyropheophorbide-a amide (2) with 96.1% yield. 2 was then hydrolyzed with lithium hydroxide solution in THF at rt for 1 h to give 173- N-(2-oxo-4-carboxybutyl) pyropheophorbide-a amide (3) with yield of 90.5%. Compound 2 was added into 30% hydrobromic acid/acetic acid solution at rt for 4 h to afford bromide 4, and the Br group of 4 was then replaced by alkyloxy groups after excess alcohols were added to the above solution. The resulted ether derivatives were hydrolyzed with 1 M lithium hydroxide solution in THF at rt for 1 - 3 h to give the desired alkyl ether derivatives (5e13) (Scheme 1). Compound 5 could also be synthesized with another five-step process (Scheme 2). The pyropheophorbide-a (1) was reacted with 30% HBr/AcOH at rt for 4 h and the Br group was selectively added to the secondary carbon of the peripheral C]C bond to give bromide (14) [10]. The Br group of 14 was then substituted by methoxy group at rt for 4 - 5 h to give ether derivative (15) in 62.4% yield [36]. After hydrolyzation of 15 with 2 M NaOH in THF at room temperature for 0.5 h, compound 16 was obtained with a yield of 90.6%. 16 was activated with HOBt-EDCI and coupled with methyl 5-aminolevulinate ester hydrochloride in N, N-diisopropylethylamine at room temperature for 6 h to form compound 17 in 59.7% yield. Compound 5 was obtained after hydrolyzation of 17 with lithium hydroxide solution in THF at room temperature for 1 h in 80.5% yield. The total isolated yield is 32.5% from compound 1 with Scheme 2 while dramatically increased to 54.5% from compound 1 with Scheme 1. So the method describes in Scheme 1 was used to synthesize other compounds 5e13.

Scheme 1. Reagents and reaction conditions: (a) EDCI, HOBt, DIPEA, CH2Cl2, r.t., 6 h, 96.1%; (b) 1 M LiOH, THF, r.t., 1 h, 90.5%; (c) 30% HBr/HOAc, rt, 4 h; (d) ROH, r.t., 4e5 h; 1 M LiOH, THF, r.t., 1e3 h.

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Scheme 2. Reagents and reaction conditions: (a) 30% HBr/AcOH, r.t., 4h. (b) ROH, r.t., 4e5 h, 62.4%. (c) 2M NaOH, THF, r.t., 0.5 h, 90.6%. (d) EDCI, HOBt, DIPEA, CH2Cl2, rt, 6 h, 59.7%. 1 M LiOH, THF, r.t., 1 h, 80.5%.

2.2. Lipid
water partition coefficient Lipid - water partition coefficient (log P) is an important factor to influence photodynamic anti-tumor effect [37]. To adjust the hydrophilicity and lipophilicity of photosensitizers, pyropheophorbide-a was integrated with 5-ALA to increase the efficiency of compound to enter tumor cells. The equilibrium concentration of the compounds in octanol and water was determined by shake-flask method in test. Log P was defined by the logarithm of the ratio of a drug’s equilibrium concentration in octanol vs that in water. The Log P values of compounds 3, 5e13 were shown in Table 1. All log P values of compounds were in the range of 1.5 - 2.7. Compared with pyropheophorbide-a, compound 3 had better amphiphilicity. The C chain extension of ether compounds could increase its lipid solubility. The presence of hydroxyl and ether linkages in the glycol ether could influence the solubility of the compounds. 2.3. Photophysical properties The UVevis spectra of new compounds 3, 5e13 were measured

Table 1 ClogP and LogP values for compounds. compound

ClogP

logP

pyropheophorbide-a 3 5 6 7 8 9 10 11 12 13

5.83 4.20 3.67 4.01 4.34 4.48 4.89 5.06 3.15 3.52 4.13

3.77 2.28 1.91 2.16 2.34 2.36 2.61 2.67 1.59 1.82 2.22

± ± ± ± ± ± ± ± ± ± ±

0.24 0.17 0.11 0.13 0.15 0.19 0.12 0.21 0.14 0.16 0.12

in DMSO. These compounds all have strong absorption in 660e670 nm with high molar extinction coefficient (Fig. 1 and Table S1). The Q band absorption was observed at 669 nm for compound 3, which was accompanied by a redshift. The spectra of synthesized compounds at different concentration (1e32 mM) were showed in (Fig. 1c, 1d, and Fig. S13). The absorption intensity of these compounds was correlated positively with their concentration (Fig. S13). The strong absorption in the near-infrared region of new compounds could benefit the effects in PDT of tumors. Upon excitation with light 410e415 nm all compounds exhibited fluorescence emission within 661e669 nm (Fig. 2a and 2b). The 3D fluorescence matrix spectra clearly showed the high absorption intensity varied from 660 nm to 675 nm (Fig. 2c, 2d and Fig. S14). The strong emission at 661e669 nm of new PSs could benefit the photo-diagnosis or photo-imaging effects of tumors. 2.4. ROS generation efficiency It was reported that after absorbed by cancer cells, the photosensitizer was promoted from the ground state (S0) to the excited state (S1) under a certain wavelength of laser irradiation, and further transited to the triplet excited state (T1) through the intersystem crossing. Energy transfer between the excited triplet state of photosensitizer and ground state of oxygen is important to generate large amounts of ROS, essential for PDT [38]. ROS generation was determined with 1,3-diphenylisobenzofuran (DPBF) as a ROS scavenger [39]. DPBF could capture and react with ROS and reduce its absorbance around 410 nm (Fig. S15). After the data were plotted as ln[DPBF]0/[DPBF]t 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, as shown in Fig. 3a and 3b. All new compounds (3, 5e13) had much higher yields of ROS than pyropheophorbide-a with the Rose bengal as standard [40,41] (Table 2). Singlet oxygen is a very important ROS which could produce a series of physiological changes and cytotoxicity in clinical PDT [42]. In order to further confirm the generation of singlet oxygen, 2, 2, 6,

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Fig. 1. The absorption spectra of the compounds (3, 5e13) in DMSO at concentration of 1  10
3 mM (a, b); the absorption of compounds 8, 11 at different concentration (1e32 mM) in DMSO (c, d).

6-tetramethylpiperidine (TEMP) as a spin trap was employed by ESR measurement [43]. As a stable substance, TEMPO was produced from the reaction of TEMP with 1O2 and could be detected. There was no obvious TEMPO signal detected without irradiation in DMF. However, the intensities of signals enhanced progressively with photo-irradiation time increased. So compound 8 and 11 generated plenty of singlet oxygen after laser irradiation at 650 nm (Fig. 3c and 3d). The quantum yields of ROS could be reduced by the aggregation of photosensitizer which could be influenced by the periphery groups of tetrapyrrole ring [44]. It was showed that the introduction of 5-ALA to pyropheophoebide-a, and the modification of 3ethylene group by long liner alkyl ether chain in pyropheophoebide-a-ALA conjugation compound 3, could raise the ROS yield which may be caused by the inhibition of selfaggregation. 2.5. Cellular uptake and subcellular localization Time-dependent uptake of compounds on Human non-small cell lung cancer cell line A549 was shown in Fig. 4. The intracellular fluorescence intensity of PSs increased quickly within 4 h, to a plateau at about 12 h and maintained within 24 h. The uptake of compounds 8e11 was significantly higher than other compounds. Many researchers found that the intracellular location of the photosensitizer coincides with the primary site of photodamage [45e47]. Two representative compounds 8, 11 were selected to

study the photodynamic anti-tumor mechanism in vitro and in vivo. Fluorescence probes Mito-Tracker Green, Lyso-Tracker Blue, and DiOC6(3) were used to investigate the subcellular localization of compound 8 and 11 in mitochondria, lysosomes, and endoplasmic reticula. The A549 cells were cultured with compounds 8, 11 for 4 h and then incubated with these probes separately. As shown in Fig. 5 (a, compound 8; b, compound 11), the red fluorescence of compounds 8, 11 overlapped with Mito-Tracker Green, Lyso-Tracker Blue, and DiOC6(3), which indicated that the photosensitizers were accumulated and localized in mitochondria, lysosome, and endoplasmic reticula. 2.6. PDT with A549 induced necrotic cell death The generation of intracellular ROS was detected by DCFH-DA staining with high efficiency and practicability [48e50]. DCFH-DA could enter the cells and react with ROS to result in the emission of 525 nm green fluorescence under 488 nm laser irradiation monitored by fluorescence microscopy (  200). A549 cells were incubated 4 h with photosensitizer 8 and 11, followed by irradiation with 2 J/cm2 650 nm laser and then incubated for 20 min with DCFH-DA reagent. Green fluorescence could be observed, while no fluorescence could be traced in the photosensitizer control group without light irradiation and in the laser control group without photosensitizer, which indicated that the photosensitizer 8 and 11 could generate ROS under laser irradiation. (Fig. S16). The cytotoxic effects of the synthesized compounds were tested

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Fig. 2. The fluorescence emission (3, 5e10) in DMSO (a); the fluorescence emission (11e13) in DMSO (b); the 3D fluorescence matrix spectra of compound 8 (c); the 3D fluorescence matrix spectra of compound 11 (d).

on A549 cells with or without 650 nm laser treatment. HPPH was set as a control group. A549 cells were incubated with PSs at different concentrations (0.25, 0.5, 1, 2, 4, 6, 8, 10 mM) for 24 h without light, and MTT assays were then performed 48 h later. The TC50 values of compounds 3, 5e8, 11e13 were significantly higher than HPPH (Table S2, Fig. 6a and 6b). To evaluate the photodynamic antitumor activities, A549 cells were incubated with compounds 3, 5e13 (0.25, 0.5, 1, 2, 4, 6, 8, 10 mM) for 24 h, followed by exposure to 1 J/cm2 of 650 nm light and then MTT assays were performed. All new PSs can inhibit tumor cells proliferation. The therapeutic ratio can be obtained by comparing TC50 and IC50 values [51]. The results indicated that the values of therapeutic ratio with new PSs 8e12 were lager than HPPH (Table S2). Meanwhile, the increasing phototoxicities to A549 cells corresponded with the increasing concentrations of new compounds (Fig. 6c and 6d). In order to visualize cell viability caused by PDT against A549 cells, the microscopic analysis was performed. Calcein stain was measured after treating with laser alone, compounds 8, 11 followed by laser irradiation for 0 or 1 J/cm2. The cells were stained with DAPI (blue color, first column) and Calcein (green color, second column) and no apoptotic lesions cells were observed in groups treated with laser alone and PSs without laser (Fig. S17a). However, the viability of cells in PS - PDT groups was 30.69% and 34.15%, which was consistent with the results of phototoxicity in vitro (Fig. S17b). Photosensitizers localized in mitochondria are most likely to induce cell death in an apoptotic way, and ROS caused by PDT could trigger cell apoptosis [52]. To verify the apoptotic and necrotic cell death induced by compounds 8, 11-mediated PDT, A549 cells were stained with Annexin V as the marker for apoptosis and PI for

necrosis. After 4 h post-PDT, the proportion of apoptotic cells increased to 83.88% and 67.73% in compounds 8, 11 treated cells, respectively (Fig. 7). These results suggested that the apoptosis induced by compound 8 - PDT and compound 11 - PDT might be caused by mitochondria-dependent caspase pathway. The activity of caspase-3 was determined with the Caspase-3 Activity Kit. After PDT for compounds 8, 11, caspase-3 activity was significantly restrained compared to the control group (Fig. S18). 2.7. Photodynamic activity in vivo The in vivo PDT efficacy of new compounds was evaluated by monitoring the tumor growth in the subcutaneous A549 tumorbearing BALB/c nude mice. In the preliminary experiments, images of A549 tumor at 14 days after PDT and tumor weight were carried out on the new compounds (Fig. S19 and Table S3). Subsequently, A549 subcutaneous xenografts of 6e7 mm in diameter were randomly separated into 10 groups: negative control group without drug and laser irradiation, positive control group with HPPH and PDT, group with 120 J/cm2 laser irradiation and without drug, pyropheophorbide-a without laser irradiation, pyropheophorbide-a with laser irradiation, compound 8 with or without laser irradiation, compound 11 with or without laser irradiation. The mice were delivered after i. v. injection of the compound at a dose of 0.3 mg/kg body weight. After 14 days postPDT, there were no obvious differences observed between the negative control group, group with 120 J/cm2 laser irradiation and without drug, group with HPPH and without laser irradiation, groups with new compounds and without laser irradiation. By

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Fig. 3. Photo-oxidation of DPBF (6  10
5 M) with pyropheophorbide-a and 3, 5e13 (2  10
5 M) in DMF. The plot for the generation rate of ROS (3, 5e10) in DMF(a). The plot for the generation rate of ROS (11e13) in DMF(b). ESR spectra as a function of time of compound 8 (c). ESR spectra as a function of time of compound 11 (d).

Table 2 Spectroscopic properties of compounds 3, 5 - 13. Compounds

k [S
1]

FD

3 5 6 7 8 9 10 11 12 13 HPPH pyropheophorbide-a

0.0251 0.0195 0.0161 0.0180 0.0313 0.0353 0.0363 0.0270 0.0162 0.0329 0.0357 0.0081

1.255 0.975 0.805 0.900 1.565 1.765 1.815 1.350 0.810 1.645 1.785 0.405

k: the slope of the photodegradation rate of DPBF; FD: ROS relative quantum yields.

contrast, in compound - PDT groups, the tumors were appeared edema at 1e2 day(s) post- PDT, then transferred to black-brown scabs from day 3 to day 5, followed by scar thickening which was gradually resolved in the following 2 weeks and new tissue were generated (Fig. 8). From the tumor growth curves of different groups, significant tumor inhibition was observed in HPPH-PDT, 8 PDT and 11 - PDT groups compared to other control groups (Fig. 9a). Kaplan-Meier analysis showed that the survival time of the mice in 8 - PDT and 11 - PDT groups were significantly prolonged compared to other groups. Compound 8 and 11 were found to be as effective as HPPH group (Fig. 9b). After 1d post PDT, tumors were dissected and subjected to

Fig. 4. Time-dependent uptake of PSs (3, 5e13) on Human non-small cell lung cancer cell line A549. The error bars denote standard deviation of three replicates.

histopathological examination (H&E) for those 3 sacrificed mice. No obvious necrosis cells were observed in the negative control group, the group with laser irradiation and without drug, group with HPPH and without laser irradiation, groups with new compounds and without laser irradiation. However, necrosis leukocyte infiltration (yellow cut head instructions) and vascular rupture (red cut head instructions) obviously appeared in the compound - PDT

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Fig. 5. Identification of subcellular localization in A549 cells co-stained with Mito-Tracker, Lyso-Tracker, and ER-Tracker. Scale bar: 20 mm.

Fig. 6. The cell viability of A549 cells treated by compounds (HPPH, 3, 5e13) at the concentrations of 0.25e10 mM under dark in MTT assay (a, b). The compounds (3, 5e13, HPPH) at different concentrations (0.25, 0.5, 1, 2, 4, 6, 8, 10 mM) with light dose (1 J/cm2) in MTT assay, respectively (c, d).

groups (Fig. 9c). In vivo studies indicated that compound 8e11 could effectively inhibit the growth of the tumor. Meanwhile, we found that compound 8e11 were still effective when the dose was reduced to 0.15 mg/kg.

2.8. In vivo imaging of A549 tumor A549 tumor-bearing mice were intravenously injected with 0.3 mg/kg compounds and the fluorescence images were shown in

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Fig. 7. Cell apoptosis necrotic by PDT in A549 cells. The cells were loaded with compounds 8, 11, irradiated for 1 J/cm2, and incubated for 4 h, respectively. Cells were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) and analyzed by flow cytometric analysis (FACS).

Fig. 8. PDT efficacy of compounds in A549 tumor-bearing BALB/c nude mice.

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Fig. 9. Tumor volume at different time points after treatment (a); Kaplan-Meier survival curve of A549 tumor-bearing mice. *P < 0.05, **P < 0.01 for 8 - PDT group and 11 - PDT group, compared with untreated by log-rank test, respectively (n ¼ 5) (b); Representative histopathological images of PDT treated tumors (c).

Fig. 10. It was showed that the compounds (compound 8, left; compound 11, right) could be distributed in various tissues of the mice after the administration. The tumor signal could be distinguished from surrounding tissues signal from 3 h up to 24 h postinjection and the tumor uptake of compound reached a maximum at 24 h, then slightly decreased over time. The results showed that the compounds exhibited rapid tumor targeting characteristics and selective retention in tumor tissue in vivo. The A549 tumor-bearing mice were killed after intravenous injection of compounds 8, 11 at 6 h, 12 h, respectively (Fig. 10b). It was shown that the fluorescence intensity in tumor tissue was considerably higher than in the normal tissues including heart, spleen, lung, brain, and kidney at 12 h. High levels of compounds were also observed in the liver. The fluorescence intensity of the dissected organs and tissues suggested that compounds were selected and retained in tumor tissue (Fig. 10c). 2.9. Skin phototoxic effect analysis Effective evaluation of the skin phototoxicity of the compound is essential on account that skin-phototoxicity is the main side effect caused by photodynamic therapy in clinics [53]. Several models, such as KM mice model, SD rats model [54], ICR mice model [55] and Guinea pig model [56,57], were usually used to evaluate the skin phototoxicity. Here ICR mice model with therapeutic dose, Guinea pig model with therapeutic dose and relatively high dose were selected and performed. ICR mice were exposed to irradiation with simulated sunlight (10 mW/cm2) at 1, 3, 5, 7 days after intravenous injection of 0.45 mg/kg compounds of 8, 11, pyropheophorbide-a and HPPH. There was no erythema and

swelling in the ears and skin with a visual inspection, and no significant difference in the ear weight after irradiation compared with control (P > 0.05, Table 3). In addition, no effects on the body weight, heart rate, respiration rate and food consumption were observed compared to the control experiments. A representative H & E stained micrographs of the auricles and the exposed skin on the backs of mice after light irradiation were shown in Fig. 11 and S20. No pathological changes were found in the auricles of four groups for 1, 3, 5, 7 days compared to the control experiments. None dermal inflammation of all treatment groups was observed as well. There was no direct effect of skin phototoxicity caused by the compounds tested at the dose level of 0.45 mg/kg. Guinea pigs were exposed to irradiation with simulated sunlight (10 mW/cm2) at 1d after injection of therapeutic dose 0.45 mg/kg and high dose 4.5 mg/kg compounds of 8, 11, pyropheophorbide-a and HPPH. As in the ICR mice model, no significant skin phototoxicity was observed at the 0.45 mg/kg injection in all treatment groups. However, obvious skin phototoxic effects could be detected at a dose of 4.5 mg/kg. The weight of the left ear, right ear, and back skin were examined (Table 4). It was shown that the ear and back skin significantly thickened in pyropheophorbide-a group. The reaction of vasodilatation, edema, hyperemia were observed in pyropheophorbide-a group after exposure to sunlight. The nucleus pyknosis of the cuticle was shown and no clear margin could be seen. There was no significant difference of the ear, back skin and the cuticle nucleus in compounds 8, 11 and HPPH groups compared to the negative control group (Fig. 12). These results indicated that skin phototoxicity of compounds 8, 11 was lower than pyropheophorbide-a.

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Fig. 10. In vivo distributions of compounds 8 and 11 in the tumor-bearing mice after intravenous injection by dosage 0.3 mg/kg. The distribution in mice at the different postinjection time (0 h, 2 h, 6 h, 12 h, 24 h) (a). In vivo fluorescence images of organs and tumor in A549 tumor-bearing mice after 6 h and 12 h post-injection of compound 8, 11, respectively (1, heart; 2, liver; 3, spleen; 4, Lung; 5, kidney; 6, brain; 7, tumor). (b). The fluorescence intensity of various organs and tissues at 6 h, 12 h, respectively (c).

Table 3 Ear weight of ICR mice after exposure to light irradiation at 1, 3, 5 and 7 days after intravenous injection of 0.45 mg/kg compounds. Group

Ear weight(mg)

control 0.45 mg/kg 0.45 mg/kg 0.45 mg/kg 0.45 mg/kg

12.6 13.1 12.8 12.9 13.3

1 day

a

pyropheophorbide-a HPPH 8 11

P > 0.05 compared with the control.

± ± ± ± ±

3 days 1.2 1.4a 1.5 a 1.6 a 1.1 a

12.8 12.7 13.3 13.1 13.2

± ± ± ± ±

5 days 1.5 2.1 1.7 1.6 1.5

a a a a

12.9 13.2 13.1 13.5 12.9

± ± ± ± ±

7 days 1.4 0.9 1.4 1.5 1.3

a a a a

13.2 13.3 13.2 12.9 13.4

± ± ± ± ±

1.6 1.5 1.1 1.2 1.7

a a a a

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Fig. 11. Representative HE micrographs of the auricles and the exposed skin on the backs of mice in compuounds 8, 11, HPPH and pyropheophorbide-a. The auricles of the mice (left ear) (a); The exposed neck skin(b); The skin of the backs of mice (c). Scale bar: 100 mm.

2.10. Conclusion Table 4 The weight of ears and back skin in Guinea pig model after exposure to irradiation with simulated sunlight at 1 day after intravenous injection of 4.5 mg/kg compounds. Group

weight(mg)

control 4.5 mg/kg 4.5 mg/kg 4.5 mg/kg 4.5 mg/kg

39.2 42.3 41.6 57.2 42.6

Left ear 8 11 pyropheophorbide-a HPPH

**P < 0.01 compared with the control.

± ± ± ± ±

1.8 2.7 2.6 2.5** 2.9

Right ear 40.4 43.6 41.9 59.2 42.2

± ± ± ± ±

2.1 3.3 2.5 1.9** 2.7

Back skin 72.4 ± 3.1 75.2 ± 2.6 74.3 ± 2.8 100.3 ± 2.4** 74.1 ± 3.2

A series of pyropheophorbide-a derivatives have been synthesized from pyropheophorbide-a. They all have strong absorption in 660e670 nm with molar extinction coefficient, fluorescence emission in 660e675 nm upon excitation with 410e415 nm light, and high ROS yields. These compounds exhibited higher phototoxicity and lower dark toxicity than pyropheophorbide-a to tumor cells in vitro. In vivo experiment showed that all compounds had obvious anti-tumor effect while efficacy of compounds 8e10 were more distinctive than HPPH. The skin photo-toxicity of 8 and 11 were weaker than pyropheophorbide-a. Compound 8 and 11 were found to localize in subcellular organelles, including lysosomes, mitochondria and endoplasmic reticulum. They were potent inducers of apoptosis and necrosis to cell destruction. So compounds 8e11 had outstanding photodynamic anti-tumor effects

Fig. 12. Representative HE micrographs of the auricles and the exposed skin on the backs of Guinea pig. The auricles of the Guinea pig (left ear) (a); the back skin of Guinea pig (b). Scale bar: 100 mm.

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without obvious skin photo-toxicity and could act as new drug candidates for photodynamic therapy. 3. Materials and methods 3.1. Materials All chemicals and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd and used without further purification. All solutions were freshly prepared. All reactions were carried out under inert an atmosphere of N2 with magnetic stirring and away from sunlight. Pyropheophorbide-a (1) and HPPH were donated by Shanghai Xianhui Pharmaceutical Co. Ltd. Chromatographic separation was carried out under pressure on silica gel using flashcolumn techniques. Reactions were monitored by thin-layer chromatography (TLC) using UV light (254 nm). (Yantai Jiangyou Chemical, China). Melting points were measured in a microscopic hot stage apparatus and were uncorrected. ESR measurements were performed with a Micro-ESR spectrometer (Bruker). 1 H NMR and 13C NMR spectra were recorded on a Bruker AMX400 and chemical shifts d (ppm) were referenced to TMS and referenced to the solvent indicated. ESI-mass spectra were carried out on a Micromass triple quadrupole mass spectrometer. MADLITOF mass spectra were recorded on an AB SCIEX 4800 Plus MALDI TOF/TOF™. HRMS spectra were recorded on a Brucker Daltonics APEXIII 7.0 T FT mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Elemental analysis was obtained from Midwest Microlab, LLC, Indianapolis, IN. Column chromatography was performed with silica gel H (300e400 mesh). UVevis absorption spectra were recorded on an ultravioletevisible spectrophotometer (Model V-530, Japan). Fluorescence spectra were measured on a fluorescence spectrophotometer (FluoroMax-4, France). 3.1.1. Synthesis of methyl 173-(4-oxo-carboxybutyl) pyropheophorbide-amide (2) Pyropheophorbide-a (100 mg, 0.19 mmol) was dissolved in dry CH2Cl2. HOBt (75.31 mg, 0.23 mmol) and EDCI (48 mg, 0.25 mmol) were added and stirred until completely dissolved to from a homogeneous solution. After 2 h, mixture of methyl 5-ALA$HCl (41.4 mg, 0.23 mmol) and DIPEA (0.15 ml, 0.76 mmol) in CH2Cl2 was added to the reaction mixture. The mixture was then stirred for 6 h at room temperature under N2. After the reaction completed, the solvent was evaporated. The residue was purified by flash chromatography (CH2Cl2: MeOH ¼ 100: 1) to give a dark green solid 2 (119.0 mg, 96.1%). 1H NMR (400 MHz, CDCl3): d ppm 9.45 (s, 1H), 9.37 (s, 1H), 8.59 (s, 1H), 7.98 (dd, J ¼ 18.2, 11.7 Hz, 1H), 6.28 (d, J ¼ 17.8 Hz, 1H), 6.17 (d, J ¼ 11.6 Hz, 1H), 5.30 (d, J ¼ 2.3 Hz, 1H), 5.10 (d, J ¼ 19.3 Hz, 1H), 4.50 (d, J ¼ 8.4 Hz, 1H), 4.35 (s, 1H), 3.82 (d, J ¼ 26.9 Hz, 1H), 3.72 - 3.62 (m, 3H), 3.61 (s, 3H), 3.57 (d, J ¼ 2.1 Hz, 3H), 3.41 (s, 3H), 3.22 (s, 3H), 2.65 (s, 1H), 2.44 (s, 3H), 2.34 (t, J ¼ 6.6 Hz, 1H), 2.21 (d, J ¼ 18.4 Hz, 3H), 1.81 (d, J ¼ 7.4 Hz, 4H),
1.80 (s, 1H). 13C NMR (100 MHz, CDCl3): d ppm 203.29, 196.21, 172.70, 172.18, 144.77, 136.14, 135.83, 130.46, 129.10, 122.56, 103.80, 97.03, 77.28, 51.85, 51.62, 49.99, 48.75, 48.04, 34.17, 32.28, 29.96, 27.25, 23.16, 19.36, 17.40, 12.13, 11.95, 11.17. LC-MS (ESI): 662.3 [Mþ1]þ. 3.1.2. Synthesis of 173-(4-oxo-carboxybutyl) pyropheophorbideamide (3) Compound 2 (100 mg, 0.15 mmol) was dissolved in THF (10 mL) and stirred under N2. The resulted solution was treated with 1M NaOH (0.75 mL, 0.75 mmol), and the resulting mixture was stirred at room temperature for 1 h. To the mixture 1M HCl was added to the mixture until pH 3 was attained. Then the suspension was extracted with DCM (3 ✕ 50 mL). The combined organic layers were

washed with water (3 ✕ 50 mL) and dried over anhydrous Na2SO4. The organic solvent was removed under reduced pressure and the residue was purified by column chromatography over silica gel (CH2Cl2: MeOH ¼ 50 : 1) to give compound 3 (88.6 mg, 90.5%) as a black solid. 1H NMR (400 MHz, DMSO‑d6): d ppm 9.57 (s, 1H), 9.30 (s, 1H), 8.88 (s, 1H), 8.25 (t, J ¼ 5.5 Hz, 1H), 8.15 (dd, J ¼ 18.1, 11.7 Hz, 1H), 6.34 (d, J ¼ 17.8 Hz, 1H), 6.17 (d, J ¼ 11.6 Hz, 1H), 5.12 (s, 1H), 4.58 (d, J ¼ 7.6 Hz, 1H), 4.30 (d, J ¼ 9.1 Hz, 1H), 3.96 (q, J ¼ 10.9, 7.4 Hz, 2H), 3.57 (d, J ¼ 12.0 Hz, 6H), 3.50 (s, 1H), 3.41 (s, 4H), 3.12 (s, 3H), 1.81 (d, J ¼ 7.2 Hz, 3H), 1.57 (t, J ¼ 7.5 Hz, 3H), 0.95 (t, J ¼ 7.2 Hz, 2H), 0.10 (s, 1H),
2.10 (s, 1H). 13C NMR (101 MHz, CDCl3): d ppm 203.29, 196.21, 172.70, 172.18, 144.77, 136.14, 135.83, 130.46, 129.10, 122.56, 103.80, 97.03, 77.28, 51.85, 51.62, 49.99, 48.75, 48.04, 34.17, 32.28, 29.96, 27.25, 23.16, 19.36, 17.40, 12.13, 11.95, 11.17. HRMS (MADLI-TOF): calcd for C38H42N5O5 [M þ H]þ 648.3176, found 648.3180. 3.1.2.1. General procedures for the preparation of the pyropheophorbide-a derivatives 5e13. Compound 2 (100 mg, 0.15 mmol) was treated with 30% HBr/acetic acid (5 ml), and the resulted solution was stirred at room temperature for 2 h. Several types of alcohol (50 ml) were added to the solution, and the mixture was stirred at 30e80  C for 4 h under N2 protection. Then 50 mL of DCM was added and separated, the combined organic layers were washed with water (3  50 mL). The solvent was evaporated and the residue was dissolved in THF (5 mL). The resulted solution was treated with 1M LiOH (0.75 mL, 0.75 mmol), and the resulting mixture was stirred at room temperature for 1e2 h. 1 M HCl solution was added to the mixture until pH 3 was attained. Then the suspension was extracted with DCM (3  50 mL). The combined organic layers were washed with water (3  50 mL) and dried over anhydrous Na2SO4. The organic solvent was removed under reduced pressure and the residue was purified by column chromatography over silica gel to afford black solid 5e13. 3.1.3. 3-(1-methyloxyethyl)-3-devinyl-173-(4-oxo-carboxybutyl)pyropheophorbide-amide (5) This product was prepared according to the general procedure. The crude porphyrin was purified by silica gel chromatography (CH2Cl2: MeOH ¼ 20 : 1) to give the dark green product 5. (58.3 mg, 56.8%) Mp > 300  C; 1H NMR (400 MHz, DMSO‑d6): d ppm 9.77 (s, 1H), 9.73 (s, 1H), 8.87 (s, 1H), 5.98 (s, 1H), 4.59 (d, J ¼ 8.0 Hz, 1H), 4.33 (s, 1H), 3.94 (d, J ¼ 7.3 Hz, 2H), 3.74 (s, 3H), 3.64 (s, 3H), 3.49 (s, 4H), 3.46 (s, 3H), 3.24 (s, 3H), 2.61 (d, J ¼ 7.8 Hz, 3H), 2.38 (d, J ¼ 6.8 Hz, 2H), 2.04 (d, J ¼ 6.3 Hz, 3H), 1.79 (d, J ¼ 7.5 Hz, 3H), 1.64 (t, J ¼ 7.0 Hz, 3H), 1.24 (d, J ¼ 12.5 Hz, 4H), 0.29 (s, 1H),
1.96 (s, 1H). 13C NMR (100 MHz, DMSO‑d6): d ppm. 206.03, 195.74, 173.96, 172.97, 172.70, 150.49, 148.47, 145.33, 141.17, 137.51, 136.37, 130.52, 128.33, 106.48, 104.68, 93.90, 74.11, 57.78, 57.07, 51.66, 49.92, 48.87, 47.97, 39.51, 34.49, 32.54, 30.68, 27.91, 24.54, 23.31, 19.12, 17.85, 12.07, 11.43, 11.38, 11.19. HRMS (MADLI-TOF): calcd for C39H46N5O6 [M þ H]þ 680.3443, found 680.3441. 3.1.4. 3-(1-ethyloxyethyl)-3-devinyl-173-(4-oxo-carboxybutyl)pyropheophorbide-amide (6) The crude porphyrin was purified by silica gel chromatography (CH2Cl2: MeOH ¼ 20 : 1) to give the dark green product 6 (58.8 mg, 56.1%). 1H NMR (400 MHz, DMSO‑d6): d ppm 12.10 (s, 1H), 9.83 (s, 1H), 8.86 (d, J ¼ 2.3 Hz, 1H), 6.05 (s, 1H), 5.24 (d, J ¼ 19.7 Hz, 2H), 4.33 (s, 1H), 3.93 (s, 2H), 3.75 (s, 4H), 3.40 (s, 5H), 3.24 (s, 3H), 2.62 (s, 6H), 2.38 (s, 2H), 2.16 (s, 2H), 2.03 (s, 3H), 1.79 (d, J ¼ 7.1 Hz, 3H), 1.65 (s, 3H), 1.30 (s, 6H), 0.32 (s, 1H),
1.96 (s, 1H). 13C NMR (100 MHz, DMSO‑d6): d ppm 195.76, 175.14, 172.98, 172.57, 162.01, 154.58, 150.46, 148.42, 145.30, 140.01, 137.45, 133.12, 130.47, 128.29, 106.43, 104.67, 97.60, 93.89, 72.32, 64.40, 51.61, 49.89, 49.08, 47.95,

Y.-H. Gao et al. / European Journal of Medicinal Chemistry 187 (2020) 111959

39.47, 35.91, 32.54, 30.78, 30.70, 24.91, 23.31, 19.11, 17.90, 16.00, 15.98, 12.07, 11.38, 11.12. HRMS (MADLI-TOF): calcd for C40H48N5O6 [M þ H]þ 694.3599, found 694.3599.

3.1.5. 3-(1-n-propyloxyethyl)-3-devinyl-173-(4-oxo-carboxybutyl)pyropheophorbide-amide (7) The crude porphyrin was purified by silica gel chromatography (CH2Cl2: MeOH ¼ 20 : 1) to give the dark green product 7 (51.3 mg, 48.0%). 1H NMR (400 MHz, DMSO‑d6): d ppm 12.09 (s, 1H), 9.80 (d, J ¼ 3.5 Hz, 1H), 9.67 (d, J ¼ 2.5 Hz, 1H), 8.85 (s, 1H), 8.19 (dt, J ¼ 6.5, 3.1 Hz, 1H), 6.01 (q, J ¼ 6.6 Hz, 1H), 5.22 (d, J ¼ 20.0 Hz, 2H), 4.58 (dt, J ¼ 7.4, 4.0 Hz, 1H), 4.32 (d, J ¼ 8.8 Hz, 1H), 4.00 - 3.85 (m, 2H), 3.76 3.63 (m, 3H), 3.58 (d, J ¼ 2.4 Hz, 3H), 3.40 (d, J ¼ 1.4 Hz, 3H), 3.22 (s, 3H), 2.62 (t, J ¼ 6.5 Hz, 3H), 2.48 (s, 5H), 2.39 (t, J ¼ 6.5 Hz, 2H), 2.04 (dd, J ¼ 6.6, 3.7 Hz, 3H), 1.79 (d, J ¼ 7.2 Hz, 3H), 1.62 (t, J ¼ 7.5 Hz, 4H), 1.31 - 1.11 (m, 9H), 0.92 (td, J ¼ 7.4, 3.0 Hz, 4H), 0.26 (d, J ¼ 4.3 Hz, 1H),
1.97 (d, J ¼ 3.0 Hz, 1H). 13C NMR (100 MHz, DMSO‑d6): d ppm 205.99, 195.72, 173.97, 172.96, 161.99, 154.59, 150.49, 148.48, 145.34, 141.15, 137.51, 136.33, 135.52, 130.53, 128.32, 106.46, 104.70, 97.76, 93.89, 72.51, 71.65, 70.85, 51.66, 49.92, 48.87, 47.9, 34.48, 32.53, 31.71, 30.68, 29.41, 27.89, 24.86, 23.41, 19.13, 17.88, 12.09, 11.63, 11.37, 11.22, 11.17. HRMS (MADLI-TOF): calcd for C41H50N5O6 [M þ H]þ 708.3756, found 708.3755.

3.1.6. 3-(1-n-butyloxyethyl)-3-devinyl-173-(4-oxo-carboxybutyl)pyropheophorbide-amide (8) The crude porphyrin was purified by silica gel chromatography (CH2Cl2: MeOH ¼ 20 : 1) to give the dark green product 8 (46.7 mg, 42.8%). 1H NMR (400 MHz, DMSO‑d6): d ppm 12.08 (s, 1H), 9.80 (d, J ¼ 3.7 Hz, 1H), 9.63 (d, J ¼ 3.7 Hz, 1H), 8.84 (s, 1H), 8.26 - 8.12 (m, 1H), 5.99 (q, J ¼ 6.6 Hz, 1H), 5.34 - 4.96 (m, 3H), 4.58 (q, J ¼ 7.4 Hz, 1H), 4.31 (d, J ¼ 8.9 Hz, 1H), 3.99 - 3.86 (m, 2H), 3.68 (dd, J ¼ 16.7, 10.7, 5.4 Hz, 4H), 3.63 - 3.48 (m, 6H), 3.40 (s, 11H), 3.21 (s, 4H), 2.62 (q, J ¼ 10.0, 8.2 Hz, 4H), 2.48 (s, 5H), 2.39 (t, J ¼ 6.5 Hz, 3H), 2.05 1.95 (m, 4H), 1.79 (d, J ¼ 7.2 Hz, 4H), 1.63 (dt, J ¼ 15.2, 8.3 Hz, 8H), 1.13 (s, 5H), 0.81 (td, J ¼ 7.3, 3.1 Hz, 5H), 0.23 (d, J ¼ 4.9 Hz, 1H),
1.98 (d, J ¼ 3.0 Hz, 1H). 13C NMR (100 MHz, DMSO‑d6): d ppm 206.02, 195.72, 174.00, 172.94, 172.65, 150.45, 145.31, 137.46, 136.29, 130.49, 128.27, 106.43, 104.65, 97.76, 93.88, 68.84, 51.63, 49.91, 48.87, 47.96, 34.48, 32.52, 32.27, 30.68, 29.39, 27.89, 24.86, 23.31, 19.51, 19.10, 17.87, 14.21, 12.05, 11.32, 11.13. HRMS (MADLI-TOF): calcd for C42H52N5O6 [M þ H]þ 722.3912, found 722.3910.

3.1.7. 3-(1-n-pentyloxyethyl)-3-devinyl-173-(4-oxo-carboxybutyl)pyropheophorbide-amide (9) The crude porphyrin was purified by silica gel chromatography (CH2Cl2: MeOH ¼ 20 : 1) to give the dark green product 9 (32 mg, 28.8%).1H NMR (400 MHz, DMSO‑d6): d ppm 12.21 -11.84 (m, 1H), 9.81 (d, J ¼ 2.3 Hz, 1H), 9.74 (s, 1H), 8.85 (s, 1H), 8.19 (t, J ¼ 5.5 Hz, 1H), 5.98 (q, J ¼ 6.5 Hz, 1H), 5.17 (dd, J ¼ 47.9, 20.1 Hz, 3H), 4.58 (d, J ¼ 7.6 Hz, 1H), 4.32 (d, J ¼ 8.3 Hz, 1H), 4.10 (s, 1H), 3.95 - 3.89 (m, 2H), 3.70 (d, J ¼ 8.2 Hz, 2H), 3.62 (s, 2H), 3.39 (s, 2H), 3.22 (s, 2H), 3.17 (s, 3H), 2.61 (t, J ¼ 6.3 Hz, 3H), 2.36 (dd, J ¼ 14.0, 7.7 Hz, 3H), 2.16 (dd, J ¼ 20.5, 10.1 Hz, 3H), 2.04 (dd, J ¼ 6.3, 3.4 Hz, 3H), 1.78 (d, J ¼ 7.2 Hz, 3H), 1.63 (t, J ¼ 7.5 Hz, 5H), 1.14 (d, J ¼ 7.2 Hz, 5H), 0.65 (d, J ¼ 6.8 Hz, 4H), 0.30 (s, 1H),
1.96 (s, 1H).13C NMR (151 MHz, DMSO‑d6): d ppm 206.47, 195.77, 172.95, 172.73, 161.95, 154.55, 150.41, 148.39, 145.29, 141.09, 140.04, 137.40, 136.25, 135.23, 133.19, 130.40, 128.21, 106.37, 104.59, 97.81, 93.89, 72.52, 69.08, 51.58, 49.89, 48.96, 47.93, 34.92, 34.72, 29.37, 28.55, 23.31, 22.39, 19.05, 17.87, 14.23, 11.99, 11.34, 11.11. HRMS (MADLI-TOF): calcd for C43H53N5O6 [M þ H]þ 735.4033, found 736.4033.

13

3.1.8. 3-(1-n-hexyloxyethyl)-3-devinyl-173-(4-oxo-carboxybutyl)pyropheophorbide-amide (10) The crude porphyrin was purified by silica gel chromatography (CH2Cl2: MeOH ¼ 20 : 1) to give the dark green product 10 (40 mg, 35.3%). 1H NMR (400 MHz, DMSO‑d6): d ppm 12.22 - 11.93 (m, 1H), 9.81 (d, J ¼ 2.3 Hz, 1H), 9.74 (s, 1H), 8.85 (s, 1H), 8.19 (t, J ¼ 5.5 Hz, 1H), 5.98 (q, J ¼ 6.5 Hz, 1H), 5.17 (dd, J ¼ 47.9, 20.1 Hz, 3H), 4.58 (d, J ¼ 7.6 Hz, 1H), 4.32 (d, J ¼ 8.3 Hz, 1H), 4.10 (s, 1H), 3.95 - 3.89 (m, 2H), 3.70 (d, J ¼ 8.2 Hz, 2H), 3.62 (s, 2H), 3.39 (s, 2H), 3.22 (s, 2H), 3.17 (s, 3H), 2.61 (t, J ¼ 6.3 Hz, 3H), 2.36 (dd, J ¼ 14.0, 7.7 Hz, 3H), 2.16 (dd, J ¼ 20.5, 10.1 Hz, 3H), 2.04 (dd, J ¼ 6.3, 3.4 Hz, 3H), 1.78 (d, J ¼ 7.2 Hz, 3H), 1.63 (t, J ¼ 7.5 Hz, 5H), 1.14 (d, J ¼ 7.2 Hz, 5H), 0.65 (d, J ¼ 6.8 Hz, 4H), 0.30 (s, 1H),
1.96 (s, 1H).13C NMR (151 MHz, DMSO‑d6): d ppm 206.14, 195.70, 174.31, 172.86, 172.68, 161.82, 154.50, 150.36, 148.34, 145.19, 141.04, 139.97, 137.34, 136.16, 135.15, 133.10, 130.36, 128.11, 106.35, 104.46, 97.80, 93.84, 72.46, 69.04, 51.59, 49.89, 48.88, 47.92, 34.57, 32.52, 31.47, 30.66, 30.08, 28.13, 25.99, 24.86, 23.30, 22.41, 19.02, 17.76, 14.13, 11.94, 11.30, 11.10. HRMS (MADLI-TOF): calcd for C44H55N5O6 [M þ H]þ 749.4240, found 750.4240. 3.1.9. 3-(1-ethoxyloxyethyl)-3-devinyl-173-(4-oxo-carboxybutyl)pyropheophorbide-amide (11) The crude porphyrin was purified by silica gel chromatography (CH2Cl2: MeOH ¼ 20 : 1) to give the dark green product 11 (39.7 mg, 37.0%). 1H NMR (400 MHz, DMSO‑d6): d ppm 9.81 (s, 1H), 9.65 (s, 1H), 8.85 (s, 1H), 7.94 (t, J ¼ 6.2 Hz, 1H), 6.10 (q, J ¼ 6.8 Hz, 1H), 5.22 (d, J ¼ 20.1 Hz, 1H), 5.11 (s, 1H), 4.71 (s, 2H), 4.57 (q, J ¼ 7.7, 7.2 Hz, 1H), 4.29 (dd, J ¼ 18.4, 8.5 Hz, 1H), 4.01 - 3.80 (m, 6H), 3.70 (tt, J ¼ 15.5, 6.1 Hz, 6H), 3.58 (d, J ¼ 15.9 Hz, 5H), 3.42 (s, 4H), 3.23 (s, 5H), 3.13 (dd, J ¼ 14.0, 6.0 Hz, 1H), 2.64 (dt, J ¼ 26.6, 7.7 Hz, 2H), 2.30 (t, J ¼ 7.7 Hz, 2H), 1.90 - 1.73 (m, 5H), 1.62 (t, J ¼ 7.5 Hz, 4H), 1.16 (s, 2H), 0.81 (dd, J ¼ 11.0, 6.1 Hz, 1H), 0.25 (s, 1H),
1.97 (s, 1H). 13C NMR (100 MHz, DMSO‑d6): d ppm 195.76, 173.24, 172.92, 172.54, 162.00, 154.69, 150.49, 148.43, 145.24, 141.13, 137.46, 136.51, 130.48, 128.26, 109.33, 106.42, 104.64, 97.84, 93.84, 72.66, 71.08, 66.03, 65.18, 61.07, 59.35, 51.71, 49.91, 47.97, 43.16, 32.73, 30.52, 28.37, 24.82, 23.29, 19.10, 17.90, 12.06, 11.40, 11.18. HRMS (MADLI-TOF): calcd for C40H48N5O7 [M þ H]þ 710.3548, found 710.3549. 3.1.10. 3-(1-methoxyethyloxyethyl)-3-devinyl-173-(4-oxocarboxybutyl)-pyropheophorbide-amide (12) The crude porphyrin was purified by silica gel chromatography (CH2Cl2: MeOH ¼ 20 : 1) to give the dark green product 12 (31.4 mg, 28.7%).. 1H NMR (400 MHz, DMSO‑d6): d ppm 9.76 (d, J ¼ 15.4 Hz, 1H), 9.30 (s, 1H), 8.54 (s, 1H), 6.03 - 5.88 (m, 1H), 5.02 (d, J ¼ 19.9 Hz, 1H), 4.47 (s, 1H), 4.28 (s, 1H), 3.88 - 3.70 (m, 4H), 3.71 - 3.55 (m, 6H), 3.25 (d, J ¼ 3.0 Hz, 4H), 2.45 - 2.26 (m, 7H), 2.17 - 2.04 (m, 5H), 1.77 (d, J ¼ 6.8 Hz, 4H), 1.63 (s, 7H), 1.33 (s, 5H), 1.28 (s, 8H),
1.94(s, 1H). 13 C NMR (100 MHz, DMSO‑d6): d ppm 144.92, 137.61, 123.99, 119.11, 103.96, 97.89, 77.24, 73.32, 73.21, 72.32, 68.50, 59.12, 51.31, 50.08, 47.81, 34.23, 33.81, 31.95, 31.46, 30.21, 29.72, 29.68, 29.46, 29.39, 29.08, 23.07, 22.72, 19.41, 17.37, 14.15, 11.93, 11.27, 11.06. HRMS (MADLI-TOF): calcd for C41H50N5O7 [M þ H]þ 724.3705, found 724.5060. 3.1.11. 3-(1-n-propoxyoxyethyloxyethyl)-3-devinyl-173-(4-oxocarboxybutyl)-pyropheophorbide-amide (13) The crude porphyrin was purified by silica gel chromatography (CH2Cl2: MeOH ¼ 20 : 1) to give the dark green product 13 (23.5 mg, 20.7%). 1H NMR (400 MHz, DMSO‑d6): d ppm 9.78 (d, J ¼ 3.9 Hz, 1H), 9.62 (s, 1H), 8.86 (s, 1H), 8.22 (dq, J ¼ 12.6, 6.4 Hz, 2H), 6.09 (q, J ¼ 6.6 Hz, 1H), 5.24 - 5.15 (m, 1H), 5.11 (s, 2H), 4.58 (d, J ¼ 7.5 Hz, 1H), 4.31 (d, J ¼ 9.0 Hz, 2H), 4.03 - 3.88 (m, 4H), 3.73 - 3.61 (m, 7H), 3.55 (d, J ¼ 3.0 Hz, 9H), 3.23 (s, 5H), 2.62 (p, J ¼ 8.6, 7.3 Hz, 5H), 2.38

14

Y.-H. Gao et al. / European Journal of Medicinal Chemistry 187 (2020) 111959

(d, J ¼ 6.3 Hz, 4H), 2.04 (dd, J ¼ 6.6, 3.8 Hz, 4H), 1.80 (t, J ¼ 7.4 Hz, 6H), 1.58 (dt, J ¼ 13.4, 7.5 Hz, 5H), 1.44 (ddt, J ¼ 11.1, 7.1, 3.7 Hz, 3H), 0.79 (dtd, J ¼ 26.1, 7.8, 5.2 Hz, 11H), 0.20 (s, 1H),
2.01 (s, 1H). 13C NMR (100 MHz, DMSO‑d6): d ppm 206.04, 195.74, 174.01, 172.94, 172.79, 172.68, 161.98, 148.44, 145.24, 141.18, 137.47, 136.41, 130.49, 128.28, 106.45, 104.63, 97.82, 93.90, 72.45, 70.32, 68.66, 51.64, 49.91, 48.88, 47.97, 34.48, 32.53, 30.68, 27.89, 24.79, 23.32, 22.87, 19.09, 17.87, 12.42, 12.05, 11.36, 11.16, 10.86. HRMS (LC-MS-TOF): calcd for C43H54N5O7 [M þ H]þ 752.4018, found 752.4027. 3.1.12. Methyl 3-(1-methyloxyethyl)-3-devinyl-173-(4-oxocarboxybutyl)-pyropheophorbide-amide (17) Compound 16 (125.1 mg, 0.18 mmol) was dissolved in dry CH2Cl2. HOBt (75.2 mg, 0.22 mmol) and EDCI (46.3 mg, 0.22 mmol) were added and stirred. After 2 h, a pre-mixed mixture of methyl 5ALA$HCl (41.4 mg, 0.23 mmol) and DIPEA (0.15 ml, 0.76 mmol) in CH2Cl2 were added to the reaction mixture. The mixture was stirred for 6 h at room temperature under N2 and then the solvent was evaporated. The residue was purified by flash chromatography (CH2Cl2: MeOH ¼ 100 : 1) to give a dark green solid 17 (91.4 mg, 59.7%). 1H NMR (400 MHz, CCl3D): d ppm 9.75 (s, 1H), 9.58 (s, 1H), 8.61 (s, 1H), 5.90 (d, J ¼ 7.1 Hz, 1H), 5.31 (d, J ¼ 20.0 Hz, 1H), 5.15 (d, J ¼ 19.8 Hz, 1H), 4.55 (s, 1H), 3.76 (s, 2H), 3.71 (s, 3H), 3.64 - 3.55 (m, 5H), 3.44 (s, 3H), 3.31 (s, 3H), 2.70 (s, 1H), 2.40 (s, 3H), 2.20 - 2.11 (m, 3H), 1.85 (d, J ¼ 7.1 Hz, 3H), 1.73 (t, J ¼ 7.7 Hz, 3H), 0.88 (s, 1H),
1.76 (s, 1H). LC-MS (ESI): 694.4 [Mþ1]þ. 3.2. Determination of Clog P and log P Theoretical values of log P (Clog P) were calculated using Chemdraw 14.0 (CambridgeSoft corporation). log P was estimated using an atom/fragment contribution method developed at SRC. The descriptor for the lipophilicity of a drug is the octanol/water partition coefficient (log P), experimental logPow determinations by the “shake-flask” method which is a classic measure [58]. The equation consisted of two types of descriptors (Cn-o and Cw) which was measured by UVevisible absorption spectra (Model V-530, Japan).

Pow ¼

Cn
o Cw

Cn-o: the concentration of drugs in octanol (mg/L); Cw: the concentration of drugs in water (mg/L).

3.3. Ultravioletevisible spectroscopy Absorption spectra were recorded using Ultravioletevisible Spectrophotometer (Model V-530, Japan). Spectra were collected from 300 nm to 800 nm in 1 nm steps. The compounds were dissolved in DMSO at a concentration of 1 mM.The samples were tested with the concentration ranges from 1 mM to 32 mM. All experiments were performed at room temperature using quartz cuvettes. 3.4. Excitation and emission spectra Excitation and emission spectra were obtained using Fluorescence Spectrometer (FluoroMax-4, France) from 350 nm to 640 nm and 600 nme750 nm in 1 nm steps, respectively. The compounds were dissolved in DMSO at a concentration of 1 mM. The samples were tested with the concentration at 5 mM. All experiments were

performed at room temperature using quartz cuvettes. 3.5. Detection of ROS 2 mM photosensitizers and 60 mM DPBF were mixed and irradiated, respectively. The reaction was monitored spectrophotometrically by measuring the decrease of optical density every 10 S at an absorbance of 410 nm of DPBF. Rose Bengal was also dissolved in DMF and used as a standard to measure ROS yields (FD ¼ 0.47) [59]. Rose Bengal, one of the permitted artificial food dyes in Japan, is an anionic water-soluble red additive which has a high quantum yield of ROS upon light irradiation [60]. The FD values of compounds were calculated by the following equation:

FSD ¼

KS  FRD KR

where, k is the slope of the photodegradation rate of DPBF; the superscripts/subscripts S and R stand for the sample and the reference, respectively. TEMP and PSs were dissolved in DMF, respectively. The 20 mL TEMP (0.2 M) and 20 mL compound were mixed in capillary tubes, which were irradiated every 20 S in the presence of oxygen using a 5 mW Nd: YAG laser (650 nm) as the light source. The irradiation area of the sample was 10 mm in diameter. ESR assays were all carried out at room temperature operating at 9.65 GHz of the microwave frequency, 15 mW of microwave power, 3400e3500 G of scanning field, 0.8 G of modulation amplitude, 15.5 S of the scan time, and 32 scan number. 3.6. Cell culture and uptake A549 cells, purchased from Institute of Biochemistry and Cell Biology, CAS, were maintained of F-12K medium supplemented with 10% FBS, 100 IU/ml penicillin, and 100 mg/mL streptomycin at 37  C in a humidified atmosphere of 5% CO2. To monitor the uptake of PSs, A549 cells were grown on 24 wells and incubated with PSs for different times (0.5, 1, 2, 4, 8, 12, 24 h). The medium was then removed, digested and the cells were collected in centrifuge tubes. The fluorescence intensity was measured with a fluorescence spectrophotometer. The A549 cells were grown on coverslips in 35-mm Petri dishes at 1  104 cells/mL and then incubated with the photosensitizers of 1 mM in complete medium for 4 h. The cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min and subsequently loaded with 0.2 mM Mitro-Tracker Green, 5 mM Lyso-Tracker Blue, 0.5 mM ER-Tracker Green for 30 min at 37  C in the complete medium, respectively. After washing with PBS three times, cells were observed using a fluorescence microscope (Carl Zeiss LSM700, Jena, Germany). Experiments were repeated three times in minimal ambient light. 3.7. Cytotoxicity The photosensitizers in DMSO (1 mmol/L) was filtered and sterilized. A semiconductor laser (650 nm) was employed as a light source in PDT. Cells were cultured in 96 - well plate at 5  104 cells/ 100 mL per well for 24 h. In dark toxicity assay, the A549 cells were treated with 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10 mM of PSs. In phototoxicity assay, the cells were treated with compounds in different concentrations of 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10 mM. Light exposure was regulated by irradiation time, with the intensity of 1 J/cm2, obtained with illumination times for 40 s at the density of 25 mW/ cm2. Afterward, 20 mL MTT was added per well for 4 h and the

Y.-H. Gao et al. / European Journal of Medicinal Chemistry 187 (2020) 111959

570 nm absorbance was measured with a microplate reader. Data were presented as mean ± standard deviation (SD). Then the cell viability was calculated according to the following formulation: cell viability (%) ¼ ODexpriment/ODcontrol  100%. All experiments were carried out in triplicate. 3.8. FACS analysis After the PDT treatment in vitro, the A549 cells were harvested and stained with annexin V and propidium iodide (PI) in Annexin V-FITC apoptosis detection kit I (KeyGen BioTech, China) according to the manufacturer’s instructions. Stained cells were analyzed on FACS scan flow cytometer with CELL Quest software (Becton Dickinson, San Jose, CA, USA). At least 10,000 events were collected for each sample. The analyses of compounds were performed at 4 h after PDT, respectively. 3.9. In vivo tumor growth and PDT-resistant cancer 6

The A549 cells (2  10 ) were suspended in 0.2 mL PBS, then injected subcutaneously into flanks of male BALB/c nude mice (5e6 weeks old, weight range 14e16 g). The tumor growth was monitored for about 14 days when the tumor grew to 6e7 mm and the tumor dimensions were determined by caliper measurements. The mice bearing tumors were randomized into ten groups: control group, laser radiation group, drug group receiving 0.3 mg/kg compound without laser application, and PDT group which was given 0.3 mg/kg drug via the tail vein followed by laser radiation after 24 h. The laser irradiation was performed with a diode laser (650 nm) and a light dose of 120 J/cm2 was delivered at a dose rate of 180 mW/cm2. The volume of each tumor was calculated as a  b2/2 (where a is the longitudinal diameter, b is the short diameter). 3.10. In vivo optical imaging To inquired the bio-distribution of the compound in A549 tumor-bearing mice model. The mice were injected with 1 mg/kg compound via the tail vein when tumors grew to approximately 150e170 mm3 in volume (n ¼ 3). The images were obtained using an IVIS spectrum small animal imaging system (PerkinElmer, USA) at a different time (0e24 h) and were analyzed using Living Image 4.5.1 software (PerkinElmer, USA). To investigate the organ distribution of different compound, the mice were anesthetized and killed after injection compound at 6 h, 12 h. Subsequently, the heart, liver, spleen, lung, kidney, brain, and tumor of the mice were then separated and placed in a Petri dish. The fluorescence intensity was measured which the excitation and emission were set at 420 nm and 650 nm, respectively. 3.11. Skin phototoxicity ICR mice (90 males and 90 females; 20e22 g body weight) were used as an animal model to investigate the skin phototoxicity. The mice were assigned randomly four groups: control (no treatment; n ¼ 36), 0.45 mg/kg compound 8 treatment (n ¼ 36), 0.45 mg/kg 11 treatment (n ¼ 36), 0.45 mg/kg pyropheophorbide-a treatment (n ¼ 36), and 0.45 mg/kg HPPH treatment (n ¼ 36). The fur of each mouse was removed from the back before dosing. After the injection of 0.45 mg/kg (i. v.), the mice were irradiated at 1 d, 3 d, 5 d, and 7 d, respectively. The simulated solar light source used in this project is Osram simulated solar light (wavelength range 250e800 nm), with the light intensity of 10 mW/cm2 and irradiation time of 10 min. 6 mice from each group were sacrificed 1 d after receipt of light irradiation. Both ears were made to a circle

15

(diameter 8 mm) and weight was calculated. A guinea pig model was used in skin phototoxicity experiments with therapeutic dose (0.45 mg/kg) and relatively high dose (4.5 mg/kg). The guinea pig was injected with compounds 8, 11, pyropheophorbide-a and HPPH, respectively. This experiment was consistent with ICR mouse model test method. The auricular and exposed skin samples were also obtained from the mice, fixed in 4% paraformaldehyde, embedded in paraffin wax, sectioned, and stained with HE for microscopic examination. 3.12. Histological examination The tumors were examined photographically at 1d post PDT. The excised tumors were fixed in 4% formaldehyde in PBS, embedded in paraffin, sectioned and stained with hematoxylin-eosin (H&E) reagent. 3.13. Statistical analysis Graphics were created by origin pro 8.0 (Graph Software, USA). All results are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed t-tests or two-way analysis of variance. A P value of <0.01 and < 0.05 was considered statistically significant. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21977016), Foundation of Shanghai Science and Technology Committee (No. 17431902600, 17430741800, 17430711900, 18430713000, 18430731600; 19410711000); The Fundamental of Research Fund for the Central Universities (No. 17D110513). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.111959. Abbreviations PDT DMSO DMF DPBF ROS DCFH-DA MTT

TC50 IC50

photodynamic therapy Dimethyl sulfoxide; N,N-Dimethyl formamide; 1,3-diphenylisobenzofuran reactive oxygen species 2, 7-dichlorodihydrofluorescein diacetate 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide, thiazolyl blue tetrazolium bromide. Half dark toxic concentration the concentration of a photosensitizer inhibits 50% of the cells under light

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