Synthesis and Investigation of Photophysical and ... - ACS Publications

Article pubs.acs.org/jmc

Cite This: J. Med. Chem. 2017, 60, 10220−10230

Synthesis and Investigation of Photophysical and Biological Properties of Novel S‑Containing Bacteriopurpurinimides Andrey F. Mironov,† Mikhail A. Grin,† Ivan V. Pantushenko,† Petr V. Ostroverkhov,† Yan A. Ivanenkov,∇,∼ Gleb I. Filkov,◆ Ekaterina A. Plotnikova,‡ Tatyana A. Karmakova,‡ Anna V. Starovoitova,§ Nelli.V. Burmistrova,§ Vadim V. Yuzhakov,§ Yuri S. Romanko,§ Maxim A. Abakumov,∥,○ Anastasiya A. Ignatova,⊥,¶ Alexey V. Feofanov,⊥,¶ Mikhail A. Kaplan,§ Raisa I. Yakubovskaya,‡ Anatoliy A. Tsigankov,# and Alexander G. Majouga*,∇,○ †

Moscow Technological University, 86 Vernadsky Avenue, Moscow 119571, Russia Branch, National Medical Radiology Research Center, P. A. Herzen Moscow Oncology Research Institute, 3 2nd Botkinskiy Proezd, Moscow 125284, Russia § A. Tsyb Medical Radiological Research Centre (A. Tsyb MRRC), 10 Zhukov Street, Obninsk, Kaluga Region, 249031, Russia ∥ Pirogov Russian National Research Medical University (RNRMU), 1 Ostrovitianov Street, Moscow 117997, Russia ⊥ Shemyakin−Ovchinnikov Institute of Bioorganic Chemistry, RAS, GSP-7, Ulitsa Miklukho-Maklaya 16/10, Moscow 117997, Russia # Institute of Fundamental Problems of Biology, Russian Academy of Sciences Pushino, Moscow Region 142290, Russia ∇ Chemistry Department, Moscow State University, Leninskie Gory, Building 1/3, GSP-1, Moscow 119991, Russia ○ National University of Science and Technology MISiS, Leninsky Prospect 4, Moscow 119049, Russia ◆ Moscow Institute of Physics and Technology (MIPT), 9 Institutskiy Per., Dolgoprudny, Moscow Region 141700, Russia ¶ Biology Faculty, Lomonosov Moscow State University, Leninskie Gory 1/70, Moscow 119992, Russia ∼ Institute of Biochemistry and Genetics Ufa Science Centre Russian Academy of Sciences (IBG RAS), Ufa, Bashkortostan, Russian Federation ‡

S Supporting Information *

ABSTRACT: Novel hybrid molecule containing 2-mercaptoethylamine was synthesized starting from O-propyloxime-Npropoxy bacteriopurpurinimide (dipropoxy-BPI), which was readily oxidized in oxygen atmosphere yielding the corresponding disulfide analogue (disulfide-BPI). Spectral, photophysical, photodynamic, and biological properties of compound were properly evaluated. Compounds bearing disulfide moiety can directly interact with glutathione (GSH), thereby reducing its intracellular concentration. Indeed, mice sarcoma S37 cell line was treated in vitro with disulfide-BPI, yielding a CC50 value of 0.05 ± 0.005 μM. A relatively high level of singlet oxygen was detected. It was demonstrated (by fluorescence) that the PS was rapidly accumulated in a cancer nest (S37) at a relatively high level after 2 h upon intravenous administration. After 24 h, no traces of the molecule were detected in the tumor mass. Moreover, high photodynamic efficiency was demonstrated at doses of 150−300 J/ cm2 against two different in vivo tumor models, achieving 100% regression of cancer growth.

■

INTRODUCTION Photodynamic therapy (PDT) is a novel noninvasive method for cancer diagnostics and therapy.1−3 The properties of photosensitizers (PSs) have a great impact on their efficacy, pharmacodynamics, and pharmacokinetics.4−6 The derivatives of naturally occurred bacteriochlorophyll-a can be reasonably regarded as promising PSs with a broad range of medical applications.7−11 Particularly, they have attracted a considerable © 2017 American Chemical Society

attention due to a wide spectrum of valuable features, especially in the field of cancer diagnostics and treatment. Bacteriochlorophylls (BChl) can be accumulated specifically in cancer cells and then upon laser irradiation produce photodynamic effect or fluorescence. The unique spectral characteristics of Received: October 12, 2017 Published: December 4, 2017 10220

DOI: 10.1021/acs.jmedchem.7b00577 J. Med. Chem. 2017, 60, 10220−10230

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of O-Propyloxime-N-propoxybacteriopurpurinimide 4

Scheme 2. Synthesis of S-Containing Bacteriopurpurinimides 5 and 6

a considerable attention during the past years.18 However, although the synthetic approach to S-containing photosensitizers has been reported previously,18,19 no pathways have been published for BChl. Herein, we report the synthesis and characterization as well as stability of novel bacteriochlorin PS containing disulfide group. Photodynamic efficiency was estimated in vitro and in vivo, particularly using S37 mice sarcoma and M1 rats sarcoma. The important influence of S−S moiety on tumor progression was clearly demonstrated. The detailed histological study and a functional-morphological study of the tumor after PDT with disulfide-BPI showed high photodynamic efficiency of the developed Bchl.

BChl, including the absorption at a wavelength of 800 nm, significantly broaden the scope of PDT of cancer imaging, especially in the case of deep-seated and pigmented tumors.12 Considering a relatively low or moderate chemical stability of natural pigments, an unmet demand for novel stable and effective analogues has been dramatically increased in recent years. Therefore, versatile and convenient synthetic approaches are relevant for the construction of such molecules with enhanced spectral and photophysical properties, activity, and selectivity as well as for the achievement of an appropriate lipophilicity/hydrophilicity ratio. Nanostructural PSs can be used to solve this issue, resulting in an enhanced penetration and loading into the tumor mass due to their effective extravasation from defect neovasculature.13−16 The mechanism of action of some thiol-containing compounds is directly associated with their tendency to be reversibly oxidized in the presence of GSH. Considering the photodynamic effect as the main contributing factor to initiate the oxidation of crucial cellular components by reactive oxygen spices (ROS) changes in redox intracellular state via GSH interaction with the molecules containing disulfide groups significantly contribute to the light induced phototoxicity to cancer cells.17 Therefore, the synthesis of novel PSs containing disulfide moiety has attracted

■

RESULTS AND DISCUSSION Chemistry. In the present work, dipropoxy-BPI (3, Scheme 1) was selected as a template compound. It was clearly demonstrated that ester (4) was quite stable and showed high photoinduced oxidative damage in cancer cells in vitro and in vivo. The maximum absorption wavelength was 800 nm. During a previous study,20 bacteriopurpurinimide 3 was obtained by the reaction of bacteriopurpurin 1 with propoxyamine in the presence of pyridine, in turn, compound 1 was 10221



Article

Figure 1. Intracellular distribution of compound 4 (row A) and compound 6 (row B) in S37 sarcoma cells. Column I: cell images in transmitted light (bars are 10 μm). Column II: confocal fluorescence images showing the intracellular distribution of 4 and 6. Cells were incubated with compounds 4 or 6 at 1 μM for 4 h. Column III: typical spectra of the intracellular fluorescence of compounds 4 and 6 are very similar to those observed for the same compounds in 1% Cremophor EL (CrEL).

were studied: (a) compound 3 was treated with cysteamine in the presence of EEDQ followed by a mild oxidation under oxygen atmosphere; (b) the desired compound was prepared by the reaction of 3 with cystamine disulfide. Thinlayer chromatography (TLC) monitoring also revealed that disulfide analogue 6 was readily formed in a solution of compound 5 in dichloromethane after 2 h. The detailed synthetic procedure for compounds 5 and 6 as well as the related analytical data (1H NMR, and UV−vis) are presented in the Experimental Section. MALDI-TOF spectra of compounds 4 and 6 (Figures S1, S2), as well as electronic absorption spectrum (Figure S3), are provided in Supporting Information. Purity of compounds 4 and 6 was proved by HPLC (Figure S5). The oxidation potential of disulfide-BPI (6) toward the intracellular GSH content was assessed by TLC monitoring. Thus, the S−S bond of compound 6 was rapidly reduced, yielding the oxidized form of glutathione (Supporting Information, Figures S4, S6, S7). As briefly mentioned above, the underlying mechanism of action of compound 6 is directly associated with its oxidative capacity, resulting in decreased stress resistance of cancer cells against ROS accumulation. Considering the obtained results, compound 6 can be fairly regarded as a pro-drug, which under the intracellular environment undergoes rapid reduction by GSH. In contrast to the data published recently by R. Pandey and co-workers,24 we clearly demonstrated that SH group could be easily oxidized in

readily synthesized via the allomerization of bacteriochlorophyll-a, which was produced by Rhodobacter capsulatus (strain B10). Bacteria were grown upon chemoheterotrophic conditions following the procedure described earlier.21−23 Using an optimized synthetic route (Scheme 1) we obtained compound 3 with good yield, up to 60%. At the first step, bacteriopurpurin 1 was easily converted into the corresponding intermediate compound 2 using hydroxylamine. Compound 2 was then treated with propyl iodide in the presence of K2CO3 to afford compound 3 in 55% yield. The resulting ester 4 was prepared by the reaction of 3 with diazomethane in diethyl ether. At each step, the efficiency of conversion was properly assessed using spectrophotometric analysis. Thus, the main absorption band of Q2 was shifted from 817 to 808 nm during the synthesis of intermediate compound 2 from bacteriopurpurin 1, while the formation of product 3 was accompanied by a subsequent shift to a wavelength of 800 nm. It should be especially noted that this approach greatly broadens the scope of diversity for this class of compounds possessing photodynamic activity. Dialkoxy derivatives of BPI were synthesized via the reaction of compound 1 with 2 equiv of alkoxyamines.20 However, considering a limited availability of alkoxyamines, alternative synthetic routes are strongly needed to obtain such molecules and their direct analogues. Dipropoxy-BPI (3) was further used to prepare dimeric compound 6 containing disulfide bond (Scheme 2). Two different synthetic routes 10222



Article

Figure 2. Photoinduced cytotoxicity of compounds 4 and 6 in sarcoma S37 cells (*: compound was not isolated from the medium).

in vitro26,30 and in vivo31) to photodynamic action initiated by PSs localized in lipid droplets. Microspectroscopy measurements revealed that the intracellular fluorescence spectra of compounds 4 and 6 were very similar in shape and peak maximum. In addition, they were similar to the spectra recorded from these compounds in solution with 1% CrEL (Figure 1, III). Previous studies showed that CrEL emulsion was well suited to model cellular lipid-like environment of hydrophobic chlorine and bacteriochlorine PSs.26,30,32 Thus, intracellular spectral characteristics of compounds 4 and 6 are consistent with their localization in lipid structures, e.g., lipid droplets and membrane-bound organelles. Photoinduced Toxicity of Compounds 4 and 6 in Sarcoma S37 Cell Line. It was demonstrated that compound 4 showed the maximal photoinduced cytotoxicity after 6 h incubation (CC50 = 0.11 ± 0.01 μM) of S37 cells with PS, whereas derivative 6 exhibited a 2-fold increase in activity (CC50 = 0.05 ± 0.01 μM) after a 4 h period (Figure 2). Two different assays were carried out to elucidate the photoinduced activity of the tested samples. During the first assay, the evaluated molecule was kept in the culture medium upon irradiation, whereas in the second one, the compound was isolated from the medium prior to the study (Figure 2). In the absence of light radiation compounds 4 and 6 were not toxic for S37 cells up to concentration of 15 μM during 24 h incubation period. Photoinduced cytotoxicity of the evaluated compounds was compared with that published for Bchl a derivatives33 as well as with PSs currently investigated in clinics (CC50 = 0.04−16.1 μM, dependent on cell type).34,35 For instance, the most structurally related compound, padeliporfin potassium, demonstrated CC50 values in the range of 1−4 μM upon PDT.33 The obtained in vitro results have revealed the tested molecules as possessing a significant photoinduced antitumor effect. Therefore, they were subsequently evaluated in vivo using S37 sarcoma mice (vide infra). Biodistribution of Compounds 4 and 6 in Animals. Biodistribution of the studied compounds was assessed using local fluorescence spectroscopy and optical imaging techniques in S37 mice sarcoma. It was revealed that the fluorescence maximum for the studied compounds was at a wavelength of 798 ± 2 nm. In animals, after iv injection of PSs at a dose of 2.5 mg/kg, they rapidly saturated tumor nest as well as neighboring

oxygen atmosphere, furnishing the corresponding disulfide derivative. The same question is raised on a similar product obtained by the hydrolysis of structurally related tert-butyl esters upon the treatment with terephthalic acid. We therefore speculate that in both cases disulfide analogues have actually been formed under the applied conditions instead of thiols. ROS Generation. Using a method of chemical traps, compounds 4 and 6 were found to be able to generate singlet oxygen under irradiation with light (Supporting Information, Figure S8). In agreement with this result, the addition of singlet oxygen quencher, NaN3, suppressed the photoinduced fading of 4-nitroso-N,N-dimethylaniline mediated by compounds 4 or 6. It was shown that compounds 4 or 6 did not generate •OH radicals upon laser irradiation. Quantum yields of singlet oxygen (Φ(1O2)) were similar for compounds 4 and 6 and equal to 0.59 and 0.62, respectively. Therefore, neither S−S moiety nor close proximity of two chromophores in compound 6 affects the generation of singlet oxygen. The Φ(1O2) values of compounds 4 and 6 were better than Φ(1O2) of bacteriochlorin a ((Φ(1O2) = 0.3325), comparable with highly active PSs on the basis of cycloimide derivatives of bacteriochlorin p26 as well as with clinically tested PSs meso-tetra(3-hydroxyphenyl)chlorine (m-THPC) and mono-L-aspartyl chlorine e6 (NPe6) (Φ(1O2) about 0.6−0.727,28). Accumulation and Distribution of Compounds 4 and 6 in Sarcoma S37 Cells. The ability of bacteriochlorin to fluoresce in compounds 4 and 6 allowed us to use fluorescence microscopy and microspectroscopy to study the interactions 4 and 6 with cancer cells. It was found that the compounds readily penetrated through the cell membrane and had a similar intracellular distribution (Figure 1). PSs 4 and 6 did not penetrate through the nuclear membrane and were not deposited in cell membrane. They stained weakly cytoplasm and predominantly accumulated in cytoplasmic vesicles of submicron size (Figure 1, I and II). These light-contrast vesicles, which are clearly recognized in transmitted-light images of cells, are lipid droplets (also known as lipid bodies or lipid particles), consisting mainly of neutral lipids. Lipid droplets are lipid storage and metabolic organelles.29 Intracellular accumulation in lipid droplets is a property of chlorine and bacteriochlorine PSs with increased hydrophobicity.26,30 As was shown previously, cancers cells were highly sensitive (both 10223



Article

Figure 3. Accumulation of compounds 4 (A) and 6 (B) in mice inoculated with sarcoma S37 cells (fluorescent ru, or FRU: the measure of amplitude displayed along the Y-axis of the background plot).

Figure 4. Biodistribution of compounds 4 (A) and 6 (B) in visceral organs (mice).

Figure 5. Changes in tumor volume observed through the time in S37 sarcoma mice after the administration of compound 4 or 6 upon irradiation in a dose of 2.5 mg/kg (iv).

In skin, the maximum values of fluorescence for both compounds were similar (2.0 ± 0.2 ru). After 24 h, only a weak signal of 4 and 6 was observed in skin, and 88−92% signal reduction was reached compared to the initial value. Therefore, no acute skin toxicity was predicted for these compounds. The maximal values of fluorescence contrast (2.2 ± 0.3 ru) were registered in 15 min after injection for compound 4 and in 30 min for compound 6. Photosensitizers demonstrated a relatively high volume of distribution. They were readily

tissue. The maximum of normalized PS fluorescence in tumor was reached in 15 min after injection for compound 4 (4.4 ± 0.3 ru) and in 30 min for compound 6 (4.0 ± 0.3 ru). This indicated that the compounds were predominantly accumulated in tumor mass. Concentration of PSs in tumor was retained at a considerably high level during 2 h, then it was gradually reduced, and only background signal was registered after 24 h (Figure 3 and Supporting Information, Figure S9). 10224



Article

As shown in Figure 6, a significant decrease in tumor volume was registered on day 3 after PDT was applied; this effect was preserved up to day 12 (tumor volume in the untreated group was almost 30 times higher in contrast to the treated group, see Table 2). During the period of an exponential growth (days 11−23), tumor M1 parenchyma was found to have a solid-type structure with oxyphilous areas of spontaneous necrotic events observed in the central zones of tumor nodes (Figure 8a). Cells had a spindle-shaped morphology and were tightly packed. As clearly shown in Figure 8b,d, a dramatic reduction in the gradient of immunoimaging intensity from periphery to the central zone was registered in response to PCNA antibody. Multitudinous necrotic and apoptotic events were also observed (Figure 8c). An angiogenic state was visualized with CD31 antibody and depicted in Figure 8e,f. A dramatically different histological picture was obtained upon PDT with compound 6 (Figure 9a−f). Thus, on day 2 after PDT was applied, extensive tissue zones of dystrophic changes accompanied by extravasation were revealed. In several cases, the depth of damage reached the tumor nodes (Figure 9a). Minor fragments of the proliferated tumor parenchyma were deposited as a conglomerate of thin strips located close to hypodermis or as small branched islands positioned in tumor nest above tight-fitting muscles (Figure 9b). A great amount of tumor tissue was in a state of coagulative necrosis with eosinophilic periphery areas of lost vessels (Figure 9c). CD31dependent immunoimaging study showed that the destruction of tumor tissue was mainly associated with the disruption of tumor vasculature (Figure 9e,f). In turn, PCNA-imaging analysis demonstrated that the cells survived after PDT (Figure 9d) and located in peripheral parenchymal areas preserved the population potential.

accumulated in visceral organs and tissues, especially in liver, kidney, and spleen. Then, the normalized fluorescence was reduced and after 24 h compound 4 was localized predominantly in spleen, while compound 6 was in kidney (Figure 4). As a result, it was clearly demonstrated that the studied molecules had a similar biodistribution and accumulated in tumor S37 cells, achieving the maximal contrast coefficient in 0.25 h after injection for compound 4 and in 0.5 h for compound 6. This time interval was selected for performing the study on photoinduced anticancer therapy (see below). Photoinduced Anticancer Activity of Compounds 4 and 6 in S37 Sarcoma Mice. The studied compounds were then evaluated in photoinduced anticancer therapy. Edema was observed for the following 4 days. No lethal outcomes among the animal population were registered after the course applied. As clearly seen in Figure 5, both photosensitizers demonstrated a promising anticancer activity. Thus, compound 4 administered 30 min prior to the irradiation at a dose of 2.5 mg/kg (iv) significantly attenuated tumor growth and progression as compared to the control group. If the compound was administered 15 min prior to the irradiation, higher efficiency was observed with ∼100% decrease in tumor volume (Table 1). Table 1. Anticancer Activity of Compounds 4 and 6 upon Photodynamic Therapy in S37 Mice Sarcoma (Light Dose was 150 J/cm3) time after irradiation (d) TGIa (%) therapy schedule 2.5 mg/kg, 15 min 2.5 mg/kg, 30 min 5.0 mg/kg, 15 min 2.5 mg/kg, 30 min 5.0 mg/kg, 30 min a

8

11

15

compound 4 100 100 88.6 81.5 100 100 compound 6 100 97.4 93.9 100 100 100 100 100 100

18

20

100 77.1 100

100 65.9 100

88.0 100

75.5 100

■

CONCLUSION In summary, we have synthesized and characterized novel derivatives of bacteriochlorophyll-a containing disulfide moiety. It was found that compound 6 undergoes rapid reduction in the presence of GSH. Therefore, we strongly speculate that the PS described herein can interact with a pool of intracellular GSH molecules leading to its depletion results in weaker resistance of tumor cells against ROS. Disulfide 6 and its derivative 4 demonstrated pronounced photoinduced cytotoxicity comparable with that reported for PS evaluated in clinics. The maximum TGI (up to 100%) was determined for compound 6. The achieved effect was dose-dependent, i.e., at a lower dose of the PS (2.5 mg/kg) tumor growth was maintained due to untreated peripheral areas. The results of the performed study clearly demonstrate that the PS is, actually, unevenly distributed within the solid tumor mass results in incomplete tumor growth inhibition and elimination of the nest. The time required for the accumulation of compounds 6 and 4 within the tumor nest was different. Thus, compound 4 reached the tumor site more rapidly in contrast to analogue 6 that is more profitable for clinical application.

TGI: tumor growth inhibition.

The obtained results demonstrated that compound 4 (2.5 mg/kg, iv, 30 min) produced the lowest therapeutic effect as the sustained tumor growth was observed in all the tested animals of this group resulted in no mice were cured. The most relevant photodynamic anticancer efficacy (TGI ∼ 100%) was reached at day 4 with compound 4 (the same dose, iv, black curve). The achieved effect was then observed for the subsequent period up to day 15 of the study. For both compounds, at a dose of 5 mg/kg, anticancer activity was significantly enhanced up to 100% (Table 1). Photoinduced Anticancer Activity of Compound 6 in M1 Rats Sarcoma. Because the time interval between the administration of compound 6 and irradiation (30 min) is preferable compared to 15 min for compound 4 from the point of view of practical use of the photodynamic therapy procedure, a disulfide derivative 6 has been chosen for further tests in M1 rats sarcoma. Compound 6 showed about 60% decrease in tumor volume at higher light dose of 300 J/cm3 on day 12 after PDT was applied (Table 2, Figures 6 and 7).

■

EXPERIMENTAL SECTION

Chemical Synthesis. Synthesis of compound (4) was carried out according to the previously described procedure.20 O-Propyloxime-N-propoxybacteriopurpurinimide sulfide (5) and disulfide (6) derivative EEDQ (21 mg, 0.0864 mmol) and cysteamine hydrochloride (13 mg, 0.1152 mmol) were added to a solution of 20 mg (0.0288 mmol) of compound 4 dissolved in 3 mL of methylene chloride. The reaction proceeded for 48 h under vigorous stirring. The 10225



Article

Table 2. Anticancer Activity of Compound 6 upon PDT in M1 Rats Sarcoma vs Untreated Control Group days after inoculation/days after PDT impact parameter

group

14/3

23/12

1.6 ± 0.3 0.59 ± 0.09*a

12 ± 3 0.44 ± 0.18**b

tumor volume (mm )

control PDT

1.22 ± 0.18 1.13 ± 0.18

volume of tumor parenchyma estimated by staining with hematoxylin and eosin (%)

control PDT

69 ± 6 39 ± 11*a

70 ± 4 23 ± 6**b

56 ± 3 30 ± 10

volume of tumor necrotic zones (%)

control PDT

32 ± 6 60 ± 11*a

30 ± 4 77 ± 6**b

44 ± 3 71 ± 10*a

volume of PCNA-positive cells in tumor parenchyma (FPCNA) (%)

control PDT

71 ± 3 26 ± 11**b

81 ± 3 38 ± 12**b

72 ± 5 39 ± 12*a

mitotic index (MI) (%)

control PDT

1.9 ± 0.2 1.2 ± 0.2*a

apoptotic index (AI) (%)

control PDT

3

a

12/1

0.29 ± 0.02 0.39 ± 0.04

1.84 ± 0.04 1.84 ± 0.11

1.9 ± 0.2 1.78 ± 0.19

0.31 ± 0.02 0.43 ± 0.01

0.37 ± 0.06 0.42 ± 0.09

*p < 0.05. b**p < 0.01 compared with the corresponding control value.

Figure 6. Anticancer effect of PDT-assisted treatment of M1 rats sarcoma with compound 6. progress of the reaction was monitored by TLC. After the reaction was completed, the formed products were extracted with methylene chloride to remove the excess of cysteamine and EEDQ. The solvent was then evaporated in vacuo. The dry residue was dissolved again in methylene chloride and recrystallized from hexane. Compound 5 was purified by TLC in methanol:methylene (1:40). The yield of compound 5 was 35%. MALDI, m/z: 756.416 (M+). Under oxygen atmosphere, compound 5 furnished the dimeric product 6. Compound 6 was also obtained by analogy to the synthetic procedure described above for compound 5 (dimeric cysteamine hydrochloride was used). The desired product was isolated in good yield (56%). MALDI, m/z: 1510,102 (M+). Found (%): C, 65.19; H, 6.91; N, 12.92. Calcd for C82H104N14O10S2 (%): C, 65.23; H, 6.94; N, 12.99. 1H NMR (300 MHz, CDCl3, δ, ppm): 8.61 (H, s, 5-H), 8.54 (H, s, 10-H), 8.38 (H, s, 20-H), 7,12 (m, 174-NH), 5.80 (H, m, 17-H), 4.46 (4H, m, −OCH2CH2CH3), 4.29 (2H, m, 7-H, 18-H), 4.1 (H, m, 8-H), 3.63 (3H, s, 12-CH3), 3.28 (3H, s, 2-CH3), 2,93 (2H, t, 176-CH2), 2,83 (2H, t, 177-CH2-S) 2.74 (3H, s, 32-CH3), 2.73 (H, m, 172-CH2), 2.35 (3H, m, 81-CH2, 171-CH2, 172-CH2), 2.11 (2H, m, 81-CH2, 171-

Figure 7. Results of photodynamic therapy with compound 6 in M1sarcoma rats: (1) prior to irradiation, (2) after the course. 10226



Article

Figure 8. Histological section and functional morphology of M1 rats sarcoma in the control group on day 14 after inoculation. (a,c) Staining with hematoxylin and eosin, (b,d) immunohistochemical reaction of nucleus toward PCNA antibodies, (e) immunoimaging of vascular endothelium with CD31antibodies in peritumoral areas and in a “hot-spot” parenchyma (f). CH2), 1.80(3H, d, 7-CH3), 1.72 (9H, m, 18-CH3, −OCH2CH2CH3), 1.25 (4H, m, −OCH2 CH2CH3), 1.24 (3H, m, 82-CH3), 0.18 (s, NH), 0.12 (s, NH). UV−vis, λmax, nm (ε × 10−3, M−1 cm−1): 365 (100), 417 (53), 539 (40), 799 (49). ROS Generation. The photoinduced generation of singlet oxygen by compound 4 and 6 was investigated using a method of chemical traps following the previously described protocol.32,36 Rose Bengal dye was used as a standard photosensitizer with known quantum yield of singlet oxygen (Φ(1O2) = 0.75).37 Compound 4 (6.1 μΜ) or 6 (3.4 μΜ) dissolved in 1% solution of Cremophor EL or rose Bengal (2.3 μΜ) was supplemented with 4-nitroso-N,N-dimethylaniline (30 μΜ) and histidine (10 μΜ) in phosphate buffered saline. Then the mixture was exposed to laser radiation (λ = 532 nm, 1.1 mW). Fading of 4nitroso-N,N-dimethylaniline was monitored using absorption spectroscopy every 5−10 min. Quantum yields of singlet oxygen Φ(1O2) for compounds 4 and 6 were registered in parallel and calculated following a routine procedure.25,32,36 Measurements were repeated in two independent experiments, and calculated Φ(1O2) values were averaged. Photoinduced generation of •OH was assessed without histidine using the same approach. Microscopy and Microspectroscopy Measurements. Sarcoma S37 cells were incubated with compound 4 or 6 at 1 μM for 4 h and studied by laser scanning confocal microscopy and microspectroscopy. Confocal microspectroscopy measurements were performed using an experimental installation described in ref 38. Laser scanning confocal microscopy measurements were performed with LSM710-Confocor3 microscope (Zeiss, Germany) equipped by αPlan-Apochromat 63×/ 1.46 oil immersion objective (at 0.3 μm lateral and 1.5 μm axial resolution). Fluorescence was excited with the 543 nm wavelength (laser power of 10 μW) and detected at wavelengths over 655 nm with an APD detector.

In Vitro Study. S37 sarcoma cells were selected as a model line to study the photoinduced cytotoxicity and intracellular distribution of the compounds. Cytotoxicity was assessed using a routine MTT test. Cells were cultured under the standard conditions at 37 °C in a humidified atmosphere with 5% CO2 in DMEM medium supplemented with L-glutamine (2 mM) and fetal bovine serum (10%, PanEco, Russia). The photoinduced efficiency was estimated as follows: S37 cells were plated in a flat-bottomed 96-well microtiter plate (Costar, USA). The evaluated compounds were added 24 h after inoculation. The concentration was varied in the range of 0.05−28 μM. Then, the cells were irradiated using a halogen lamp through a broadband KS-19 filter with a light transmission (720 nm) to assess the photoinduced cytotoxicity of the studied PSs through 0.25, 0.5, 2, 4, and 6 h after incubation. The power density was 21.0 ± 1.0 mW/ cm2, while the calculated light dose was 10 J/cm2. Irradiation was carried out with and without the removal of the PS from the medium. After irradiation, the cells were incubated under the standard conditions during 24 h. To analyze the cytotoxicity of the PSs, the cells were placed in darkened conditions for 24 h. The survival rate was assessed by visual inspection and colorimetrically using MTT test. The inhibition of cell growth by more than 50% was considered as biologically relevant. This value was calculated as the average of three independent tests. In Vivo Study. All manipulations have been carried out in accordance with the national and international rules for the humane treatment of animals (Annex A to European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (ETS no. 123) and Guidance on the Maintenance and Care of Laboratory Animals (Article No. 5 of the Convention), Strasbourg, 2006. 10227



Article

Figure 9. Results of histological and functional morphology studies performed on day 1 after PDT with compound 6. (a,c) Staining with hematoxylin and eosin, (b,d) immunohistochemical reaction of nucleus toward PCNA antibodies, (e) immunoimaging of vascular endothelium with CD31antibodies in peritumoral areas and in a “hot-spot” parenchyma (f). maximum fluorescence of bacteriochlorins. Fluorescent contrast (PC) was calculated as follows: FN(tumor)/FN(skin). Biodistribution of the studied PSs was assessed by not only using local fluorescence spectroscopy but also by optical imaging (IVIS Spectrum-CT, PerkinElmer). This method allows estimating fluorescent radiation through the skin of a living organism in dynamics. Fluorescence was excited at wavelengths of 710 and 745 nm. Light emission was detected at 780, 800, 820, and 840 nm. Spectral analysis was performed using Living Image 4.4 software (PerkinElmer) to distinguish the fluorescence signals from the tissue and the fluorophore. The intensity of fluorescence was measured in photon/ s/cm2. Inhalation anesthesia with 2% isoflurane (Abbott, USA) was used in the study. PS was administered in S37sarcomaxenograft mice at a dose of 2.5 mg/kg (iv). Then, the fluorescence of the sample was measured in the tumor, skin, and internal organs in vivo and ex vivo. Photoinduced Anticancer Activity of Compounds 4 and 6 in Sarcoma S37 Mice. Photodynamic therapy was performed remotely with the PSs on day 7 of tumor growth in sarcoma S37 mice at a dose of 2.5 and 5.0 mg/kg. Control animals received an intravenous isotonic 0.9% solution of sodium chloride. Droperidol was used as an anesthetic (injection solution, 2.5 mg/mL, FSUE Moscow Endocrine Plant, Russia) at a dose of 0.25 mg per animal (ip). Prior to PDT, the tumor volume was in the range of 140−160 mm3. LED source was used for the irradiation (wavelength of 800 ± 21 nm, FGUP SSC NIOPIK, Russia). The power density was 100 mW/cm2, while the energy density was 150 J/cm2. The time interval between the administration of PS and subsequent irradiation was 15 and 30 min. Animals without the tumor growth after the treatment were monitored during 90 d. The antitumor effect was assessed as the mean valueof the tumor volume in the control and experimental group, tumor growth inhibition (TGI), and the cure index (CI): V = d1 × d2 × d3 × 0.52, where d1, d2, and d3 are the three mutually perpendicular diameters of the tumor; TGI = [(Vt − Vc)/Vt] × 100%, where Vt and Vc are the

S37sarcoma mice (100 animals, 18−22 g) were obtained from the Andreyevka Laboratory Animal Farm of the FGBUN “NCBMT” FMBA of Russia. They were used to study the biodistribution of the evaluated compounds. The cell culture was implanted in immunocompetent F1 mice, and their metastatic events were spread predominantly via lymphogenic route. Adult wild-type female rats (60 animals, 190−330 g) inoculated with nonmetastatic sarcoma M1 were used to investigate the anticancer effect of the compounds as well. Tumor cells were implanted subcutaneously in left thigh area. Parameters of the therapy were different depending on the type of animals and tumor weight as well as main PK features. Inoculation as well as tumor volume/mass monitoring were performed by following the standard protocol. The animals were kept in standard housing conditions (humidity 50−60%, temperature 21 ± 1 °C, a 12 h light/ dark cycle with lights on at 8:00 am, individually ventilated cages) and given ad libitum access to standard rodent chow and water. All the experiments were carried out in full accordance with the approved guidance. In Vivo Biodistribution. The biodistribution study was performed ex vivo by using local fluorescence spectroscopy (LFS) immediately after the laboratory animal was sacrificed. Fluorescence was registered by a contact method using a laser spectral analyzer for the fluorescent diagnostics “LESA-06” (LLP “BioSpec”, Russia). Fluorescence was excited by He−Ne laser radiation (wavelength was 632.8 nm, spectral range was 400−900 nm). The samples of skin, muscle, liver, kidney, and spleen tumor were obtained from three animals (ex tempore, 0.25, 0.5, 2, 4, and 24 h). When the fluorescence was excited in the red region of the spectrum, the integrated fluorescence intensity in the spectral range of 640−900 nm was normalized to the integrated intensity of the backscattered diffuse scattering signal of the exciting laser radiation, thereby determining the normalized fluorescence (FN) of the studied tissues. The accumulation of the PSs in tissues was assessed by the max. FN values at a wavelength corresponding to the 10228



Article

average tumor volume in the test and control groups, respectively; CI = [Nc/Nt] × 100%, where Nc and Nt are the number of cured animals and the total number of animals in the group, respectively. Statistical analysis was performed using Microsoft Excel Software, OriginPro, STATISTICA and LivingImage 4.4. Linear values were compared by the Student’s test (p < 0.05). Photoinduced Anticancer Activity of Compound 6 in Sarcoma M1 Rats. Ten days after inoculation, rats were randomly distributed between two separate groups: the first group of 25 animals was used as a control population, while the second group was subjected to PDT with compound 6. The efficiency of PDT was readily assessed using the following equation: (Vi − V0)/V0, where V0 is the initial tumor volume mm3 (day 11 after inoculation), while Vi corresponds to tumor volume registered on day i. Compound 6 was administered intraperitoneally at a single dose of 2.5 mg/kg on day 11 after inoculation. After 2.5−3 h, the treatment with light irradiation (wavelength, 810 nm; power density, 0.51 W/cm2; radiant energy density, 300 J/cm2; diameter of the field of laser impact, 1.5 cm) was carried out for 10 min using laser apparatus “Latus”. Histological samples were isolated on day 12, 14, and 23 as well as on days 1, 3, and 12 for the control and treatment groups, respectively, after PDT. Five animals were analyzed at each time checkpoint (of total: 15 rats in each group), while the remaining 10 rats had been examined up to 32 days since inoculation. Immunohistochemical study and imaging were performed using biotin−extravidin−peroxidase complex with a mice antibody (clone PC10, “Thermo Fisher Scientific”, 1:200) targeted against PCNA (proliferating cell nuclear antigen) and a rabbit antibody (M-20-R, “Santa Crua”, 1:20) targeted against CD31 (cluster of differentiation 31).

■

propoxybacteriopurpurinimide; ROS, reactive oxygen spices; GSH, glutathione; PDT, photodynamic therapy; PS, photosensitizer; BChl, bacteriochlorophyll; m-THPC, meso-tetra(3hydroxyphenyl)chlorine; CrEL, Cremophor EL; EEDQ, N(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00577. Analytical and biological data (PDF) Molecular formula strings (XLSX)

■

REFERENCES

(1) Bonnett, R. Chemical Aspects of Photodynamic Therapy; CRC Press, Boca Raton, FL, 2000. (2) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Photodynamic Therapy for Cancer. Nat. Rev. Cancer 2003, 3, 380−387. (3) Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.; Piette, J.; Wilson, B. C.; Golab, J. Photodynamic Therapy of Cancer: An Update. Ca-Cancer J. Clin. 2011, 61 (4), 250−281. (4) Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R. K. The Role of Porphyrin Chemistry in Tumor Imaging and Photodynamic Therapy. Chem. Soc. Rev. 2011, 40, 340−362. (5) Manivannan, E.; Nayan, J. P.; Pandey, R. R. In Porphyrin-Based Multifunctional Agents for TumorImaging and Photodynamic Therapy (PDT); Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press: Boston, 2011; pp 249−323. (6) Allison, R. R.; Downie, G. H.; Cuenca, R.; Hu, X. H.; Childs, C. J. H.; Sibata, C. H. Photosensitizers in Clinical PDT. Photodiagn. Photodyn. Ther. 2004, 1 (1), 27−42. (7) Moser, J. G. Photodynamic Tumor Therapy, 2nd and 3rd Generation Photosensitizers; Harwood Academic Publishers: London, 1998; pp 3−8. (8) Koudinova, N. V.; Pinthus, J. H.; Brandis, A.; Brenner, O.; Bendel, P.; Ramon, J.; Eshhar, Z.; Scherz, A.; Salomon, Y. Photodynamic Therapy with Pd-Bacteriopheophorbide (TOOKAD): Successful in Vivo Treatment of Human Prostatic Small Cell Carcinoma Xenografts. Int. J. Cancer 2003, 104, 782−789. (9) Brandis, A.; Mazor, O.; Neumark, E.; Rosenbach-Belkin, V.; Salomon, Y.; Scherz, A. Novel Water-Soluble Bacteriochlorophyll Derivatives for Vascular-Targeted Photodynamic Therapy: Synthesis, Solubility, Phototoxicity and the Effect of Serum Proteins. Photochem. Photobiol. 2005, 81 (4), 983−992. (10) Grin, M. A.; Mironov, A. F.; Shtil, A. A. Bacteriochlorophyll A, and Its Derivatives: Chemistry and Perspectives for Cancer Therapy. Anti-Cancer Agents Med. Chem. 2008, 8 (6), 683−697. (11) Staron, J.; Boron, B.; Karcz, D.; Szczygieł, M.; Fiedor, L. Recent Progress in Chemical Modifications of Chlorophylls and Bacteriochlorophylls for the Applications in Photodynamic Therapy. Curr. Med. Chem. 2015, 22 (26), 3054−3074. (12) Pandey, R. K.; Goswami, L. N.; Chen, Y.; Gryshuk, A.; Missert, J. R.; Oseroff, A.; Dougherty, T. J. Indium as a central metal enhances the photosensitizing efficacy of benzoporphyrin derivatives. Lasers Surg. Med. 2006, 38 (5), 445−467. (13) Cheng, Y.; Meyers, J. D.; Broome, A.-M.; Kenney, M. E.; Basilion, J. P.; Burda, C. Deep Penetration of a PDT Drug into Tumors by Noncovalent Drug-Gold Nanoparticle Conjugates. J. Am. Chem. Soc. 2011, 133 (8), 2583−2591. (14) Srivatsan, A.; Jenkins, S. V.; Jeon, M.; Wu, Z.; Kim, C.; Chen, J.; Pandey, R. K. Gold Nanocage-Photosensitizer Conjugates for DualModal Image-Guided Enhanced Photodynamic Therapy. Theranostics 2014, 4 (2), 163−174. (15) Qiao, W.; Wang, B.; Wang, Y.; Yang, L.; Zhang, Y.; Shao, P. Cancer Therapy Based on Nanomaterials and Nanocarrier Systems. J. Nanomater. 2010, 2010, 796303. (16) Suvorov, N. V.; Grin, M. A.; Popkov, A. M.; Garanina, A. S.; Mironov, A. F.; Majouga, A. G. Novel Photosensitizer Based on Bacteriopurpurinimide and Magnetite Nanoparticles. Makrogeterotsikly 2016, 9 (2), 175−179. (17) Wang, C.; Liu, L.; Cao, H.; Zhang, W. Intracellular GSHActivated Galactoside Photosensitizers for Targeted Photodynamic Therapy and Chemotherapy. Biomater. Sci. 2017, 5 (2), 274−284.

AUTHOR INFORMATION

Corresponding Author

*Phone: +74959394020. E-mail: [email protected] or [email protected]. ORCID

Alexander G. Majouga: 0000-0002-5184-5551 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was supported by Russian Science Foundation (RSF grant: no. 16-13-10092, synthesis and chemical modification of bacteriopurpurinimides, 17-74-30012, in vitro study, IBG RAS Ufa) as well as by Russian Foundation for Basic Research (RFBR grants: OFI no. 15-29-01156, 16-03-00519a, 16-3360180\15). The authors also acknowledge financial support obtained from Ministry of Education and Science of Russia (MISiS, K2-2017-069).

■

ABBREVIATIONS USED dipropoxy-BPI, O-propyloxime-N-propoxybacteriopurpurinimide; disulfide-BPI, disulfide analogue of O-propyloxime-N10229



Article

(18) Guo, X.; Wang, L.; Wang, S.; Li, Y.; Zhang, F.; Song, B.; Zhao, W. Syntheses of New Chlorin Derivatives Containing Maleimide Functional Group and Their Photodynamic Activity Evaluation. Bioorg. Med. Chem. Lett. 2015, 25 (19), 4078−4081. (19) Pantiushenko, I. V.; Rudakovskaya, P. G.; Starovoytova, A. V.; Mikhaylovskaya, A. A.; Abakumov, M. A.; Kaplan, M. A.; Tsygankov, A. A.; Majouga, A. G.; Grin, M. A.; Mironov, A. F. Development of Nanostructured IR-Photo-Sensitizers Based on Bacteriochlorophyll-a Derivatives and Gold Nanoparticles for Photodynamic Therapy of Cancer. Biochemistry (Moscow) 2015, 80, 891−902. (20) Chissov, V. I.; Yakubovskaya, R. I.; Mironov, A. F.; Grin, M. A.; Plotnikova, E. A.; Morozova, N. B.; Tsygankov, A. A. Composition for photodynamic therapy of cancer. Patent RU no. 2521327, 2012. (21) Tsygankov, A. A.; Laurinavichene, T. V.; Gogotov, I. N. Laboratory Scale Photobioreactor. Biotechnol. Tech. 1994, 8 (8), 575− 578. (22) Tsygankov, A. A.; Laurinavichene, T. V.; Bukatin, V. E.; Gogotov, I. N.; Hall, D. O. Biomass Production by Continuous Cultures of Rhodobacter Capsulatus Grown in Various Bioreactors. Prikl. Biokhim. Mikrobiol. 1997, 33 (5), 549. (23) Patrusheva, E. V.; Fedorov, a. S.; Belera, V. V.; Minkevich, I. G.; Tsygankov, A. A. Synthesis of Bacteriochlorophyll-a by the Purple Nonsulfur Bacterium Rhodobacter Capsulatus. Appl. Biochem. Microbiol. 2007, 43 (2), 187−192. (24) Srivatsan, A.; Jeon, M.; Wang, Y. F.; Chen, Y. H.; Kim, C.; Pandey, R. K. A Novel Bacteriochlorin-Gold Nanoparticle Construct for Photoacoustic Imaging. J. Porphyrins Phthalocyanines 2016, 20 (1− 4), 490−496. (25) Hoebeke, M.; Damoiseau, X. Determination of the Singlet Oxygen Quantum Yield of Bacteriochlorin-a: A Comparative Study in Phosphate Buffer and Aqueous Dispersion of Dimiristoyl-L-AlphaPhosphatidylcholine Liposomes. Photochem. Photobiol. Sci. 2002, 1 (4), 283−287. (26) Sharonov, G. V.; Karmakova, T. A.; Kassies, R.; Pljutinskaya, A. D.; Grin, M. A.; Refregiers, M.; Yakubovskaya, R. I.; Mironov, A. F.; Maurizot, J. C.; Vigny, P.; Otto, C.; Feofanov, A. V. Cycloimide Bacteriochlorin P Derivatives: Photodynamic Properties and Cellular and Tissue Distribution. Free Radical Biol. Med. 2006, 40 (3), 407− 419. (27) Mojzisova, H.; Bonneau, S.; Maillard, P.; Berg, K.; Brault, D. Photosensitizing Properties of Chlorins in Solution and in MembraneMimicking Systems. Photochem. Photobiol. Sci. 2009, 8, 778−787. (28) Spikes, J. D.; Bommer, J. C. Photosensitizing Properties of Mono-L-Aspartyl Chlorin e6 (NPe6): A Candidate Sensitizer for the Photodynamic Therapy of Tumors. J. Photochem. Photobiol., B 1993, 17 (2), 135−143. (29) Zweytick, D.; Athenstaedt, K.; Daum, G. Intracellular Lipid Particles of Eukaryotic Cells. Biochim. Biophys. Acta, Rev. Biomembr. 2000, 1469 (2), 101−120. (30) Feofanov, A.; Sharonov, G.; Grichine, A.; Karmakova, T.; Pljutinskaya, A.; Lebedeva, V.; Ruziyev, R. R.; Yakubovskaya, R.; Mironov, A.; Refregier, M.; Maurizot, J.; Vigny, P.; Grichine, A.; Yakubovskaya, R. Comparative Study of Photodynamic Properties of 13,15-N-Cycloimide Derivatives of Chlorin p6. Photochem. Photobiol. 2004, 79 (2), 172−188. (31) Karmakova, T.; Feofanov, A.; Pankratov, A.; Kazachkina, N.; Nazarova, A.; Yakubovskaya, R.; Lebedeva, V.; Ruziyev, R.; Mironov, A.; Maurizot, J. C.; Vigny, P. Tissue Distribution and in Vivo Photosensitizing Activity of 13,15-[N-(3-Hydroxypropyl)]cycloimide Chlorin p6 and 13,15-(N-Methoxy)cycloimide Chlorin p6Methyl Ester. J. Photochem. Photobiol., B 2006, 82 (1), 28−36. (32) Feofanov, A. V.; Nazarova, A. I.; Karmakova, T. A.; Plyutinskaya, A. D.; Grishin, A. I.; Yakubovskaya, R. I.; Lebedeva, V. S.; Ruziev, R. D.; Mironov, A. F.; Maurizot, J. C.; Vigny, P. Photobiological Properties of 13,15-N-(Carboxymethyl)- and 13,15-N-(2Carboxyethyl)cycloimide Derivatives of Chlorin p6. Russ. J. Bioorg. Chem. 2004, 30 (4), 374−384. (33) Mazor, O.; Brandis, A.; Plaks, V.; Neumark, E.; RosenbachBelkin, V.; Salomon, Y.; Scherz, A. WST11, a Novel Water-Soluble

Bacteriochlorophyll Derivative; Cellular Uptake, Pharmacokinetics, Biodistribution and Vascular-Targeted Photodynamic Activity Using Melanoma Tumors as a Model. Photochem. Photobiol. 2005, 81 (2), 342−351. (34) Sehgal, I.; Sibrian-Vazquez, M.; Vicente, M. G. H. Photoinduced Cytotoxicity and Biodistribution of Prostate Cancer Cell-Targeted Porphyrins. J. Med. Chem. 2008, 51 (19), 6014−6020. (35) Králová, J.; Bříza, T.; Moserová, I.; Dolenský, B.; Vašek, P.; Poučková, P.; Kejík, Z.; Kaplánek, R.; Martásek, P.; Dvořaḱ , M.; Král, V. Glycol Porphyrin Derivatives as Potent Photodynamic Inducers of Apoptosis in Tumor Cells. J. Med. Chem. 2008, 51 (19), 5964−5973. (36) Grichine, A.; Feofanov, A.; Karmakova, T.; Kazachkina, N.; Pecherskih, E.; Yakubovskaya, R.; Mironov, A.; Egret-Charlier, M.; Vigny, P. Influence of the Substitution of 3-Vinyl by 3-Formyl Group on the Photodynamic Properties of Chlorin P6: Molecular, Cellular and in Vivo Studies. Photochem. Photobiol. 2001, 73 (3), 267−277. (37) Lee, P. C. C.; Rodgers, M. A. J. Laser Flash Photokinetic Studies of Rose Bengal Sensitized Photodynamic Interactions of Nucleotides and DNA. Photochem. Photobiol. 1987, 45, 79−86. (38) Efremenko, A. V.; Ignatova, A. A.; Borsheva, A. A.; Grin, M. A.; Bregadze, V. I.; SIvaev, I. B.; Mironov, A. F.; Feofanov, A. V. Cobalt Bis(dicarbollide) versus Closo-Dodecaborate in Boronated Chlorin e(6) Conjugates: Implications for Photodynamic and Boron-Neutron Capture Therapy. Photochem. Photobiol. Sci. 2012, 11 (4), 645−652.

10230


Synthesis and Investigation of Photophysical and ... - ACS Publications

Recommend Documents