Photostable Platinated Bacteriochlorins as Potent Photodynamic Agents

Ngoc An Le, Vipin Babu, Martina Kalt, Lukas Schneider, Frank Schumer, and Bernhard Spingler*

Article

Photostable Platinated Bacteriochlorins as Potent Photodynamic

Agents

Cite This: J. Med. Chem. 2021, 64, 6792 −6801

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sı Supporting Information

ABSTRACT: Photodynamic therapy (PDT) is used to treat various

cancerous diseases. Recently, we have demonstrated that platinated

pyridyl-substituted porphyrins are potent agents for PDT with very high

phototoxicity (IC₅₀down to 17 nM) and excellent phototoxic indices of

higher than 5800 (p.i. = IC (dark)/IC (light)) [Rubbiani, R. et al.,

Chem. Commun. 2020, 56, 14373]. However, the absorption of

porphyrins is not ideal for the treatment of larger tumors because they

essentially do not absorb light between 650 and 850 nm. Herein, we

report stable conjugates of a novel bacteriochlorin with cisplatin and

transplatin. They exhibit extremely high phototoxicity (IC values down

to 6 nM, irradiated with a 750 nm LED at a ﬂuence of 5 J/cm ), very low

dark toxicity, and thereby extremely high phototoxic indices up to 8300. Based on these exciting results, we believe that platinated

bacteriochlorins are promising candidates for further investigation as novel PDT anticancer agents.

INTRODUCTION

to induce excellent phototoxicity (IC₅₀down to 17 nM), only

minor dark toxicities, and therefore also brilliant phototoxicity

indices up to 5800.

■

Photodynamic therapy (PDT) is an emerging method for the

treatment of diﬀerent types of cancer including skin cancer,

lung cancer, oesophageal cancer, gastric carcinoma, breast

cancer, cervical cancer, brain tumors, head and neck tumors,

pancreatic cancer, and bladder cancer. It is considered less

invasive than surgery and has no long-term side eﬀects, which

are often seen in chemotherapy and radiotherapy. This

method is based on the activation of a photosensitizer (PS) by

light. The activated photosensitizer produces diﬀerent reactive

oxygen species (ROS), which oxidize biomolecules and cellular

structures, leading to cell death via necrosis or apoptosis. In

addition, PDT has demonstrated the ability to eliminate

tumors by damaging the microvasculature and by triggering an

13,15

However, the absorption of porphyrins is not ideal for the

treatment of deep-seated or larger tumors because they only

weakly absorb in the phototherapeutic window (650−850

nm), which allows deeper penetration into tissues. Ultra-

violet−visible (UV−vis) absorption spectra of porphyrins

present an intensive Soret band at about 420 nm and weak

Q bands between 500 and 650 nm. Therefore, shifting the

absorption of porphyrins into the red region is highly desirable.

For this purpose, bacteriochlorins (BChls), the analogues of

porphyrins with two reduced pyrrole rings, are possibly good

alternatives due to the presence of intense Q bands at 720−

17−20

immune response, which expands the application of PDT.

850 nm.

During the past 30 years, an increasing number of new PSs for

PDT emerged. Some of them are undergoing diﬀerent stages

of clinical trials, and a few have been already approved for

clinical use in many countries, for example, Photofrin,

BChl derivatives have received considerable attention as

second-generation photosensitizers for photodynamic therapy

due to absorption of wavelengths in the far-red region as well

as extraordinary phototoxicities. However, they are generally

susceptible to oxidation, being easily oxidized to corresponding

chlorins and porphyrins, which limits their application in PDT.

Herein, we report the development of a stable pyridyl-

substituted BChl and its platinated conjugates with either

Verteporﬁn, and Foscan.

The majority of PSs are cyclic tetrapyrroles and their

analogues, like (extended) porphyrins, chlorins, bacteriochlor-

ins, and phthalocyanines. They are the most extensively

studied PSs for PDT due to their strong absorption, high ROS

,10

production eﬃciency, and good biocompatibility.

Among

Received: January 12, 2021

Published: May 14, 2021

them, pyridyl-substituted porphyrins have attracted consid-

erable attention due to the wide range of modiﬁcation via

alkylation and complexation with diﬀerent transition metals,

yielding products that have important roles in supramolecular

11−14

chemistry and biomedical application including PDT.

Platinated tetrapyridyl-porphyrins were reported by our group

2021 American Chemical Society

792

J. Med. Chem. 2021, 64, 6792−6801

Article

Figure 1. Synthesis of BChls 6, 6-cPt, and 6-tPt.

cisplatin or transplatin and the biological evaluation of these

compounds for PDT.

analyzed with a Vanquish Horizon UHPLC System (Thermo Fisher

Scientiﬁc, Waltham) connected to a Vanquish eλ detector and a

timsTOF Pro TIMS-QTOF high-resolution mass spectrometer

The group of Lindsey has developed a number of

approaches for de novo multistep synthesis of BChls bearing

geminal dialkyl groups, preventing them from oxidation to

(Bruker Daltonics, Bremen, Germany). Elemental microanalyses

were performed on a LecoCHNS-932 elemental analyzer. The

26,27

22−24

synthesis of the known (2,6-di ﬂ uorophenyl)dipyrromethane

(2)

chlorins and porphyrins.

Arnaut et al. reported another

is described in the Supporting Information . UV−vis spectra were

recorded on a Cary 50 Scan Varian spectrophotometer. Fluorescence

spectra were recorded on an LS50B luminescence spectrometer.

Fluorescence lifetime, phosphorescence, and phosphorescence life-

time were recorded on an Edinburgh FLS920-stm spectrophotometer.

Transient absorption spectra were recorded on an Edinburgh LP920

spectrometer. Slopes and areas under the curves were calculated using

the data analysis and graphing software Origin Pro 2019. Crystallo-

graphic data were collected at 160.0(1) K on a Rigaku-Oxford

Diﬀraction XtaLAB Synergy-S dual-source diﬀractometer. This is a

kappa-axis four-circle goniometer with a Dectris Pilatus3 R 200K

HPC (Hybrid Photon Counting) detector and Cu and Mo PhotonJet

microfocus X-ray sources. Suitable crystals were covered with oil

method to stabilize meso-tetraphenyl-substituted bacteriochlor-

ins by introducing electron-withdrawing groups to the phenyl

rings to increase the oxidation potential of BChls, and

substituents at ortho-positions of the phenyl rings provide

steric hindrance for the pyrroline rings. Both eﬀects contribute

to the stabilization of BChls against oxidation. Compared to

Lindsey’s challenging and low-yielding multistep de novo

syntheses, Arnaut’s method is much simpler, but the scope

of functionalities modiﬁcation is narrower.

In this paper, we report new stable platinated BChls, which

show extremely high phototoxicity together with excellent

phototoxic indices upon irradiation at 750 nm.

(Inﬁneum V8512, formerly known as Paratone N), placed on a nylon

loop that is mounted on a CrystalCap Magnetic pin (Hampton

EXPERIMENTAL SECTION

Research), and immediately transferred to the diﬀractometer. The

■

Pro

program suite CrysAlis was used for data collection, numerical and

Instrumentation and Methods. All chemicals were of reagent-

grade quality or better, obtained from commercial suppliers and used

without further puriﬁcation. All solvents were of analytical grade.

Synthesis of BChl 6 and its platinum conjugates was performed in the

multiscan absorption correction, as well as data reduction. The

structures were solved with the dual-space algorithm using SHELXT

and were reﬁned by full-matrix least-squares methods on F with

SHELXL-2018 using the Olex2 GUI. The graphical output was

195

dark. H-, Pt-, and F-NMR spectra were recorded in deuterated

solvents on Bruker AV-400 (400 MHz) and AV-500 (500 MHz)

spectrometers at room temperature. The chemical shifts are reported

in ppm (parts per million). The residual deuterated solvent peaks and

K [PtCl ] measured in D O have been used as internal and external

produced with the help of the program Mercury. CCDC 2024670-

2024671 contain the supplementary crystallographic data for this

paper. These data are provided free of charge by The Cambridge

Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures .

Synthesis and Characterization. 5,15-Bis(3,5-dichloropyridin-

4-yl)-10,20-bis(2,6-diﬂuorophenyl) Porphyrin (4). (2,6-

195

references for H-NMR and Pt-NMR spectra, respectively. The

abbreviations for the peak multiplicities are as follows: s (singlet), d

26,27

(

doublet), dd (doublet of doublets), t (triplet), q (quartet), m

multiplet), and br (broad). Coupling constants J are given in hertz

Hz). ESI mass spectra were recorded on a Waters AQUITY-Bruker

Di ﬂ uorophenyl)dipyrromethane (2)

(5.16 g, 20 mmol; see the

Supporting Information for a detailed synthetic description) and 3,5-

dichloro-4-pyridinecarboxaldehyde (3) (3.5 g, 20 mmol) were

dissolved in 1.5 L of DCM. A mixture of TFA (25 mL) and DCM

UPLC-MS system. High-resolution MS samples (1 μL injection) were

793

J. Med. Chem. 2021, 64, 6792−6801

Journal of Medicinal Chemistry

condition for the DCM to evaporate giving crystals (see Figure 2 ),

Article

(

500 mL) was added to this solution (Figure 1). The reaction mixture

was stirred at room temperature for 30 min. Afterward, DDQ (10 g,

4 mmol) and methanol (100 mL) were added and the reaction was

also not be done with more than one gram of para-toluenesulfonyl

hydrazide. The reaction mixture was cooled down to room

temperature, and DCM (100 mL) was added; the formed insoluble

solid was ﬁltered oﬀ, and the ﬁltrate was washed three times with

NaOH (0.5 M, 100 mL) then one time with water (100 mL). The

continued for 1 h. The reaction mixture was washed three times with

a 10% solution of potassium carbonate (1 L), dried over anhydrous

magnesium sulfate, and ﬁltered through a pad of silica. Ethanol (200

mL) was added, and the solution was left under atmospheric

organic phase was dried over MgSO . Then, the solvent was

evaporated to give an oily residue, which was diluted with 5 mL of

DCM before hexane was added to the solution. The precipitate was

ﬁltered oﬀ and puriﬁed by column chromatography (silica gel,

MeOH/DCM 3:100). The red-green fraction was collected, and the

solvent was evaporated to yield the product BChl 6 as a dark green

solid (68 mg, 68%). UV−vis (DCM/MeOH 10:1): λ (nm, log ε) =

max

350 (5.1), 377 (5.09), 510 (4.77), 750 (5.03). H-NMR (400 MHz,

DMSO-d ): δ = 9.11 (s, 4H), 8.33−8.19 (m, 4H), 8.16 (d, J = 4.9,

H), 7.85 (q, J = 4.9, 2H), 7.82−7.71 (m, 2H), 4.15−3.99 (m, 3H),

.95−3.87 (m, 2H), 2.63 (d, J = 4.8, 6H), −1.23 (s, 2H). F-NMR

(376 MHz, DMF-d ): δ = 103.72 (d, J = 10.1, 2F), −106.68 (dd, J =

0.4, 2.8, 2F). (+)-MS (ESI): m/z 1017.15 [M + H] , 509.17 [M +

H] . HR-MS-ESI [M + H] : calcd 1017.05649, found: 1017.05268.

Elemental analysis calcd (%) for C H Cl F N O S ·1.95H O: C

0.24, H 3.25, N 10.65; found: C 50.59, H 3.42, N 10.3.

[

cis-PtCl(NH ) ] -5,15-bis(3,5-dichloropyridin-4-yl)-10,20-bis(2,6-

2 2

diﬂuoro-3-N-methylsulfamoylphenyl) Bacteriochlorin Nitrate (6-

cPt). Cisplatin (0.074 mmol, 22.2 mg) and silver nitrate (0.074 mmol,

12.6 mg) were dissolved in 2 mL of DMF and stirred at room

temperature in the dark for 24 h. The resulted turbid solution was

centrifuged to remove white silver chloride. The supernatant was

added to a solution of BChl 6 (0.0295 mmol, 30 mg) in 1 mL of DMF

and stirred at 60 °C for 48 h. After that, the mixture was cooled down

to room temperature and the DMF was evaporated. MeOH (10 mL)

was added before the mixture was sonicated for 5 min and centrifuged

to remove the excess of cisplatin. The supernatant was precipitated

with diethyl ether. The solid was ﬁltered and further dried in vacuum

to yield the product 6-cPt as a dark green solid (30 mg, 61%). UV−

Figure 2. Displacement ellipsoid representation of the crystal

structure of 4 at 50% probability.

vis (DCM/MeOH 10:1): λ (nm, log ε) = 350 (4.90), 377 (4.97),

max

511 (4.63), 753 (4.89). H-NMR (400 MHz, DMF-d ): δ = 9.52 (s,

H), 8.55−8.43 (m, 2H), 8.43−8.25 (m, 4H), 7.92−7.66 (m, 4H),

.30 (s, 6H), 4.94 (s, 6H), 4.31−4.13 (m, 4H), 4.13−3.96 (m, 4H),

which were then ﬁltered and washed with methanol (100 mL) and

acetone (100 mL) to yield porphyrin (4) as a purple solid (650 mg,

.80 (s, 6H), −1.24 (s, 2H). F-NMR (376 MHz, DMF-d ): δ =

195

−

103.77 (d, J = 10.2), −106.66 (d, J = 10.4). Pt-NMR (107.27

%). NMR spectra could not be obtained due to low solubility.

MHz, DMF-d ): δ = −2292. HR-MS-ESI [M] : calcd 771.01228,

(+)-MS (ESI): m/z 827.19 [M + H] , 414.14 [M + 2H] . Elemental

found: 771.01090. Elemental analysis calcd (%) for

C H Cl F N O Pt S : C 31.65, H 2.53, N 11.74; found: C

analysis calcd (%) for C H Cl F N ·0.2CH OH·2H O: C 58.33, H

14 10

2 2

.88, N 9.67; found: C 58.09, H 2.62, N 9.63.

,15-Bis(3,5-dichloropyridin-4-yl)-10,20-bis(2,6-diﬂuoro-3-N-

methylsulfamoylphenyl) Porphyrin (5). A mixture of porphyrin 4

31.73, H 2.45, N 11.67.

[

trans-PtCl(NH ) ] -5,15-bis(3,5-dichloropyridin-4-yl)-10,20-bis-

2 2

(

2,6-diﬂuoro-3-N-methylsulfamoylphenyl) Bacteriochlorin Nitrate

(

500 mg, 0.6 mmol) and chlorosulfonic acid (8 mL) was heated at 80

(

6-tPt). Transplatin (0.074 mmol, 22.2 mg) and silver nitrate (0.074

°C for 3 h. Afterward, the reaction mixture was cooled down and 500

mmol, 12.58 mg) were dissolved in 2 mL of DMF and stirred at room

temperature in the dark for 24 h. The resulted turbid solution was

centrifuged to remove white silver chloride. The supernatant was

added to a solution of BChl 6 (0.0295 mmol, 30 mg) in 1 mL of DMF

and stirred at 60 °C for 48 h. After that, the mixture was cooled down

to room temperature and the DMF was evaporated. MeOH (10 mL)

was added before the mixture was sonicated for 5 min and centrifuged

to remove the excess of transplatin. The supernatant was precipitated

with diethyl ether. The solid was ﬁltered and further dried in vacuum

to yield the product 6-tPt as a dark green solid (40 mg, 82%). UV−vis

mL of DCM were added and the excess of acid was extracted with a

% aqueous potassium carbonate solution (500 mL). The solution

was dried over anhydrous magnesium sulfate, and a solution of

methylamine in THF (2 M, 2 mL) was added. After 30 min, the

solution was ﬁltered through a pad of silica gel and the product was

eluted with a mixture of MeOH/DCM (3:100). The solvent mixture

was then evaporated, yielding product (5) as a red solid (375 mg,

1%). H-NMR (400 MHz, DMSO-d ): δ = 9.25 (s, 4H), 9.08 (d, J =

0.4, 8H), 8.50−8.39 (m, 2H), 7.97−7.89 (m, 4H), 2.73 (d, J = 4.8,

H), −2.98 (s, 2H). F-NMR (376 MHz, DMSO-d ): δ = −101.71

(DCM/MeOH 10:1): λ (nm, log ε) = 351 (4.93), 378 (4.96), 512

max

(

dd, J = 28.1, 9.4, 2F), −104.97 (dd, J = 38.5, 9.4, 2F). (+)-MS (ESI):

+ 2+

(4.63), 752 (4.88). H-NMR (500 MHz, DMF-d ): δ = 9.72 (s, 4H),

m/z 1013.17 [M + H] , 507.07 [M + 2H] .

,15-Bis(3,5-dichloropyridin-4-yl)-10,20-bis(2,6-diﬂuoro-3-N-

methylsulfamoylphenyl) Bacteriochlorin (6). A mixture of porphyrin

.52−8.45 (m, 2H), 8.40−8.34 (m, 2H), 8.31−8.24 (m, 2H), 7.89−

.80 (m, 4H), 4.79 (s, 12H), 4.29−4.18 (m, 4H), 4.10−3.97 (m, 4H),

.82 (d, J = 5.0, 6H), −1.24 (s, 2H). F-NMR (376 MHz, DMF-d ):

(100 mg, 0.099 mmol) and para-toluenesulfonyl hydrazide (1.0 g,

δ = 103.79 (dd, J = 10.3, 4.1), −106.64 (d, J = 10.1). Pt-NMR

bottom ﬂask. The mixture was degassed by three cycles of evacuating

and reﬁlling with nitrogen, then left under high vacuum for 1 h, and

purged again with nitrogen. Afterward, the mixture was carefully

heated to 120 °C under a nitrogen atmosphere for 3 h. There is the

danger of deﬂagration of para-toluenesulfonyl hydrazide under a

(107.27 MHz, DMF-d

771.01228, found: 771.00606. Elemental analysis calcd (%) for

·0.4 DMF·3H O: C 30.97, H 2.92, N 11.50;

found: C 30.78, H 3.23, N 11.23.

): δ = −2301. HR-MS-ESI [M] : calcd

42Cl

N O

10Pt

The photophysical and cell biology studies are described in the

nitrogen atmosphere above 127 °C. Therefore, this reaction should

Supporting Information.

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Article

Table 1. Photophysical Data for Bacteriochlorins

triplet-state

lifetime (MeOH)

absorption (DMF)

ﬂuorescence (MeOH) (ex. 355 nm)

singlet oxygen quantum yield

λ_m_a_x(nm) log (ε_m_a_x

)

λ_m_a_x(nm) τ_F(ns)

Φ_F

N (μs) Air (ns) Φ_Δ₍_d₎(CD OD) Φ_Δ₍_i_n_d₎(MeOH) Φ (DMF) ×10

750

753

752

650

5.03

4.89

4.88

4.47

751

754

653

1.75

1.67

1.63

0.0095 ± 0.0013

0.0093 ± 0.0008

0.0034 ± 0.0008

0.089

10.3

10.1

13.3

237

221

197

0.93 ± 0.06

1.02 ± 0.06

0.68 ± 0.05

0.81 ± 0.04

0.90 ± 0.05

0.64 ± 0.03

0.43

0.99 ± 0.05

-cPt

-tPt

2.60 ± 0.14

8.43 ± 0.23

42,43

Foscan

Φ : Photodecomposition quantum yield.

Table 2. (Photo)cytotoxicity (IC₅₀[μM]) of the BChls in HeLa (Cervical Cancer), A2780 (Ovarian Cancer), and CP70

(

Cisplatin-Resistant Ovarian Cancer) Cell Lines in the Dark and Upon Irradiation

HeLa

dark

A2780

dark

CP70

dark

light

p.i.

light

p.i.

903

472

light

p.i.

0.210 ± 0.004

0.046 ± 0.018

0.011 ± 0.004

124 ± 19

40 ± 19

44 ± 14

590

870

4000

0.124 ± 0.07

0.053 ± 0.008

0.006 ± 0.002

112 ± 3

25 ± 5

>50

0.132 ± 0.082

0.050 ± 0.002

0.008 ± 0.003

>100

>50

>758

>1000

>6250

-cPt

-tPt

>8333

Cells were incubated with the compounds for 4 h, then the media was replaced, and ﬁnally the cells were irradiated with light at 750 nm (5 J/

cm ). Each of the independent experiments is an average value of triplicates (p.i.: phototoxic index).

Table 3. Comparison of Phototoxic Indexes (p.i.) of Pyridyl-Containing Photosensitizers after Transplatination (in the HeLa

Cell Line)

compound

λ (irradiation) [nm] p.i. of PS-(py)_np.i. of PS-(py-[Pt(NH ) Cl] ) improvement of p.i. by transplatination

refs

tetra(4′-pyridyl)-porphyrin

tetrapyridino-phthalocyanine

pyridyldistyryl-BODIPY

420

>600

657

17.3

0.8

1210

7.5

543

9.4

6.8

dipyridyl-bacteriochlorin 6-tPt

750

590

4000

this work

RESULTS AND DISCUSSION

successfully applied. The reagent p-toluenesulfonyl hydrazide

is used as a precursor of diimide, which acts as a reducing agent

in this reaction. Upon careful heating to 120 °C, p-

toluenesulfonyl hydrazide melts, acting as a solvent for the

reaction, and then decomposes to liberate diimide, the

reduction agent. The reduction of porphyrin 5 to BChl 6

was successful.

■

The design of BChl 6 (Figure 1) was inspired by Arnaut’s

5,34

13,15

work,

our previous work,

and the diﬀerent availability

of 3,5-dihalogen-4-pyridinecarbaldehydes. Pyridyl groups in

meso-positions provide the possibility for platination with

either activated cis- or transplatin. Fluoro- and chloro-

substituents provide steric hindrance for the pyrroline rings

Platination of the pyridyl moieties in BChl 6 was carried out

with either cisplatin or transplatin. These reagents were treated

with one equivalent of silver nitrate in DMF and then reacted

in a 1:1 ratio with the pyridyl moieties of BChl 6 at elevated

temperatures to form the ﬁnal products, which were

characterized by NMR spectroscopy ( H, F,

+)-ESI-MS and elemental analysis. Table 1 presents the most

important photophysical properties of BChl 6 and its

platinated products 6-cPt and 6-tPt. All BChls have similar

UV−vis spectra with a strong sharp Q band at around 750 nm

and two Soret bands in the range 340−380 nm. Platination and

^m^e^t^h^y^l^a^tⁱ^oⁿ^l^e^a^d^t^o^a^s^lⁱ^g^h^t^r^e^d^s^hⁱ^f^t^o^f^t^h^e^Qy band.

Fluorescence spectra show very small Stokes shifts, indicating

similarity between the molecular geometries of ground state

and excited states (Figure S2).

However, the ﬂuorescence quantum yields are very low

(<1%). This is mainly explained by the heavy atom eﬀect of

the four chlorine atoms and was also observed by Arnaut and

against oxidation. Sulfonamide groups on phenyl rings

together with pyridyl rings provide strong electron-with-

drawing eﬀects on the system; thus, they increase the oxidation

potential of the BChls and thereby stabilize them against

oxidation.

195

Pt), HR-

The synthesis of BChl 6 started with the preparation of

porphyrin 4 (Figure 1). The MacDonald [2+2] macro-

cyclization is the most common and versatile method for the

(

35,36

synthesis of trans-A B -porphyrins.

Despite many reported

methodologies, the synthesis of porphyrins still faces the

problem of low yields and diﬃcult puriﬁcation.

particular, the synthesis of pyridyl-substituted porphyrins is

6−39

known to be scarcely eﬀective with standard condensation

procedures; moreover, ortho-substituents provide extremely

hindered active sites, leading to unsuccessful synthesis and

6,41

high degree of scrambling.

Therefore, a laborious

optimization was done to ﬁnally achieve a satisfactory yield

of 8% for porphyrin 4 (Supporting Information Table S1 ). The

crystal structure of 4 could be obtained and is depicted in

Figure 2.

Sulfonamide groups were introduced to porphyrin 4 by

means of chlorosulfonation followed by reaction with methyl-

amine. The next step is the reduction of porphyrin 5 to BChl 6.

The solvent-free reduction reported by Pereira et al. was

co-workers for their octachloro-substituted bacteriochlorins.

It is interesting to note that diﬀerent peripheral substitutions of

both bacteriochlorin systems modulate the ﬂuorescence

quantum yields, albite without increasing these quantum yields

above 1%. Furthermore, the singlet oxygen yields of 6, 6-cPt,

and 6-tPt are inﬂuenced by the diﬀerent peripheral

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J. Med. Chem. 2021, 64, 6792−6801

Article

Figure 3. Cellular co-localization of compounds 6 (A), 6-cPt (B), and 6-tPt (C) (always at 15 μM), as well as the negative control with no added

BChl (D) in HeLa cells.

substituents, despite the fact that the triplet lifetimes of the

three BChls are pretty similar.

1270 nm, using MB as standard (Figure S3). The results

derived from the direct method are for most compounds

within a 3σ range compared to those obtained by the indirect

The transient absorption spectra of BChls 6 , 6-cPt, and 6-

tPt in methanol, presented in Figures S10 −S13, exhibit

positive peaks at 780 nm indicating the triplet absorption of

the compounds. Therefore, the triplet-state lifetimes of the

compounds were derived from the kinetic analysis of the decay

at 780 nm with monoexponential ﬁtting. The lifetimes were

measured for both nitrogen-saturated and air-saturated

solutions. A signiﬁcant decrease of the lifetime in the presence

of air was observed due to the triplet-state quenching of

molecular oxygen. The long triplet lifetimes in the range of 10

μs provide suﬃcient time to transfer energy to molecular

oxygen to form singlet oxygen.

method. All studied compounds exhibited excellent Φ values,

thus showing that they all have potential as PDT agents.

Additionally, the ability of the three bacteriochlorins 6, 6-cPt,

and 6-tPt to generate hydroxyl radicals was determined with

the APF hydroxyl radical sensor (Figures S7 and S8 ). The

•

results show that the rate of the OH radical generation for the

both platinated compounds 6-cPt and 6-tPt is 2- to 3-fold

higher compared to the non-platinated BChl 6.

The photodecomposition quantum yield (Φ ) is a parameter

used to characterize the photostability of a PS; it refers to the

number of decomposed molecules divided by the number of

photons absorbed by the system. The photodecomposition of

BChls follows a ﬁrst-order kinetic with respect to irradiation

time; therefore, it should be calculated as the following ratio:

initial rate of disappearance of photosensitizer molecules

The ability to produce ROSs eﬃciently is the key

characteristic of a good photodynamic agent. The majority of

PSs act via type II photosensitization mechanism, making the

singlet oxygen quantum yield one of the most important

parameters to characterize PSs for PDT applications. Addi-

tionally, hydroxyl radicals can be generated by both type I

divided by the initial rate of absorption of photons.

−

Generally, a PS with Φ lower than 10 is accepted as

photosensitization pathway and type II pathway via singlet

suﬃciently photostable for PDT. In this study, Φ of all BChls

oxygen. In this study, the singlet oxygen quantum yields Φ_Δ

were determined by two diﬀerent methods, an indirect method

and a direct method. The indirect method employs DPBF as a

singlet oxygen quencher and methylene blue (MB) as the

were determined indirectly relative to the standard zinc

−

phthalocyanine (ZnPc) in DMSO (Φ = 2.51 × 10 ).

The results are presented in Table 1; all compounds have

standard in DMF (Φ = 0.52, Figure S6). The direct method

lower Φ values compared to those of ZnPc and Foscan.

measures the phosphorescence of singlet oxygen at around

Therefore, the photodecomposition quantum yields of the

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Figure 4. Collapse studies of MMP with compound 6-tPt in HeLa cells. Image series (A−G) show cells with (A) solvent vector, no irradiation as

negative control, (B) solvent vector and irradiation as negative control, (C) CCCP as positive control, (D) 15 μM 6-tPt, no irradiation, (E) 0.1 μM

-tPt, no irradiation, (F) 15 μM 6-tPt with irradiation, and (G) 0.1 μM 6-tPt with irradiation. Irradiation was executed at 750 nm for 30 min (2.5

J/cm ).

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Intracellular localization is an important characterization for

novel PDT agents, which shows the primary target of

photodamage induced by the photosensitizers. HeLa cells

were treated with BChls, Hoechst 33342, and Mito BacMam

GFP (Figure 3). The images show that the PSs are taken up by

the cells; however, platinated BChls 6-cPt and 6-tPt do not

localize in the nucleus. This is in contrast to a tetracationic

platinated porphyrazine, which localizes in the nuclei. BChls

and lipophilic mono- and dicationic PSs are reported to mainly

52,53

localize in the mitochondria.

Interestingly, when Mito-

Tracker Green was applied instead of Mito BacMam GFP, we

found that the ﬂuorescence intensity of the tracker signi ﬁ cantly

decreased compared to the negative control (Figure S15).

MitoTracker Green is a cationic dye, which is known to adhere

to mitochondria via interaction with the negatively charged

mitochondrial membrane potential (MMP). While 6-tPt is

also a cationic species, its application hindered the

accumulation of the dye to the mitochondria, which leads to

reduced adherence of MitoTracker Green to the mitochondria.

The most promising PS, 6-tPt was investigated further for its

intracellular localization with other organelles (ER, golgi, and

lysozyme; Figures S16 and S17 ). However, due to its low

ﬂuorescence quantum yield, more precise co-localization

quantiﬁcations were not obtainable as the ﬂuorescence signal

was too weak for the calculation of the Pearson correlation

Figure 5. Intracellular ROS levels in HeLa cells treated with 10 nM

compound 6-tPt for 4 h, irradiated with 750 nm light for 2.5 J/cm .

The ROS signal is indicated as DCF ﬂuorescence, measured using the

BL1 channel (excitation 488 nm, emission 530 nm) on an Attune

NxT Cytometer and is plotted against side scatter (SSC). Results are

expressed as a mean percentage of ROS-positive cells over total cell

population, obtained from three independent experiments with a

minimum of 100 000 cells analyzed per condition. The error bars

represent the standard deviation (S.D).

coeﬃcient.

The collapse of the MMP, a marker of apoptosis, was

conﬁrmed with the Mitochondrion Membrane Potential Kit

MAK147 (Sigma-Aldrich, Figure 4). The dye accumulates in

healthy cells with normal mitochondrial membrane potential

synthesized BChls 6, 6-cPt, and 6-tPt proved their suﬃcient

photostability for PDT.

(

MMP), resulting in a ﬂuorescence signal (λ = 540/λ = 590

ex em

In vitro cytotoxicity assays were performed to evaluate the

anticancer activity of 6, 6-cPt, and 6-tPt (Table 2). All BChls

have excellent phototoxicity (IC₅₀values in the nanomolar

range) and high phototoxic indices against human cervical

cancer HeLa cells, ovarian cancer cells (A2780), and cisplatin-

resistant ovarian cancer cells (CP70) (see Tables S2 and S3 for

a comparison with other bacteriochlorins and chlorins) upon

nm). Cells are incubated at the same concentration as in the

co-localization studies (15 μM) and at 0.1 μM. Carbonyl

cyanide m-chlorophenyl hydrazone (CCCP) was used as a

positive control to demonstrate the disruption of MMP

(CCCP, Figure 4C).

Negative controls were run with the solvent vehicle

(MeOH) and either with or without irradiation (Figure

4A,B). No diﬀerences can be detected after the irradiation.

irradiation at 750 nm with a lower light dose (5 J/cm )

13,25

compared with previous studies.

Platination signiﬁcantly

Upon irradiation (2.5 J/cm ) and treatment with either 15 or

increased phototoxicity. Especially, 6-tPt has an extremely low

0.1 μM 6-tPt, a total collapse of the MMP was detected,

similar to that in the positive control (Figure 4C,F,G). The

ﬂuorescence signal of the sample with 0.1 μM 6-tPt was too

low to be detected (Figure 4E,G). Interestingly, there seems to

be relocalization of the PS after irradiation (Figure 4D,F),

which unfortunately cannot be further investigated because of

the very low ﬂuorescence quantum yield of the PS. Such a

relocation of PS upon irradiation has been described before by

IC value of 6 nM in the A2780 ovarian cancer cell line, which

led to an extremely high phototoxic index of higher than 8300.

The phototoxic index (p.i.) is calculated by dividing the

IC (dark) value by the IC (light) value. The p.i. should be as

high as possible, which indicates that the compound in non-

irradiated tissues does not cause any harm to the patient. On

the other hand, platination also slightly increases the dark

toxicity as minor dark toxicity was observed for BChls 6-cPt

and 6-tPt (40 and 44 μM, respectively). It is however diﬃcult

to explain the order of the observed IC (light) values (6 >

57−60

several research groups.

To demonstrate that the phototoxicity is attributed to the

increased level of intracellular ROS, the intracellular ROS

levels were measured using the nonﬂuorescent 2′,7′-dichlor-

‑cPt > 6-tPt) solely with the singlet oxygen quantum yields

(

6‑cPt > 6 > 6-tPt), the ability to generate hydroxyl radical (6

odihydroﬂuorescein diacetate (H DCF-DA) followed by

6-cPt ∼ 6-tPt), or a combination of both eﬀects. It seems

FACS analysis. After cell uptake, H DCF-DA is hydrolyzed

that other eﬀects are relevant as well. All three bacteriochlorins

exhibited light-dose-dependent phototoxicity when HeLa cells

treated with the compounds were irradiated with increasing

light doses at 750 nm (1.25−5 J/cm ) (see Table S4).

Additionally, we investigated the eﬀect of the transplatination

to H DCF, which then upon oxidation by diverse ROS species

(like H O in the presence of Fe(II), hydroxyl radical, and

singlet oxygen ) is converted to the ﬂuorescent 2′,7′-

dichloroﬂuorescein (DCF). The results show that, when

HeLa cells were treated with 6-tPt and then irradiated with

light at 750 nm, the percentage of cells with increased ROS

levels was signiﬁcantly higher compared to treatment in the

dark or the light irradiation alone. In cells treated with 6-tPt

followed by light irradiation, ROS levels increased 42-fold

(

Table 3). The reaction of pyridyl-containing photosensitizers

with monoactivated transplatin yields transplatinated photo-

sensitizers, which have improved phototoxic indexes between

.8- and 70-fold in all systems we have studied so far.

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J. Med. Chem. 2021, 64, 6792−6801

Journal of Medicinal Chemistry

The Supporting Information is available free of charge at

Article

compared with untreated cells, 21-fold compared with light-

treated cells, and 3.5-fold compared with cells treated with 6-

tPt in the dark (Figures 5 and S18).

ABBREVIATIONS USED

■

APF, 4-aminophenyl-ﬂuorescein; BChl, bacteriochlorin;

CCCP, carbonyl cyanide m-chlorophenyl hydrazone; DCM,

dichloromethane; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzo-

quinone; DMSO, dimethylsulfoxide; DMF, N,N-dimethylfor-

mamide; DPBF, 1,3-diphenylisobenzofuran; ESI, electrospray

CONCLUSIONS

In summary, the BChls 6, 6-cPt, and 6-tPt have an excellent

phototoxicity proﬁle under a low-intensity irradiation of 5 J/

■

ionization; H DCF-DA, 2′,7′-dichlorodihydroﬂuorescein diac-

cm at 750 nm, with high potency toward diﬀerent cancer cell

etate; IC , dye concentration required to kill 50% of the cells;

lines, including cisplatin-resistant cancer cells. Especially, the

compound 6-tPt exhibits extremely high phototoxicity (HeLa:

IC = 11 nM, A2780: IC = 6 nM, CP70: IC = 8 nM) and

LED, light-emitting diode; MB, methylene blue; MeOH,

methanol; MMP, mitochondrial membrane potential; p.i.,

phototoxic index: ratio of IC (dark) to IC (light); PDT,

very low dark toxicity to give unprecedented phototoxic

indexes higher than 8300. Cell localization experiments

showed that the compounds predominantly localize in

mitochondria. These favorable characteristics demonstrate

that the transplatinated BChls are promising candidates to be

investigated as novel PDT anticancer agents in vivo.

photodynamic therapy; PS, photosensitizer; ROS, reactive

oxygen species; τ , triplet-state lifetime; TFA, triﬂuoroacetic

acid; THF, tetrahydrofuran; UPLC, ultrahigh-performance

liquid chromatography; ZnPc, unsubstituted zinc(II) phthalo-

cyanine; Φ , photodecomposition quantum yield; Φ ,

ﬂuorescence quantum yield; Φ , singlet oxygen quantum yield

ASSOCIATED CONTENT

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00052.

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