Photocytotoxicity of Thiophene- And Bithiophene-Dipicolinato Luminescent Lanthanide Complexes

Luminescent Lanthanide Complexes

Article

Photocytotoxicity of Thiophene- and Bithiophene-Dipicolinato

⊥

Carime V. Rodrigues, Katherine R. Johnson, Vincent C. Lombardi, Marcelo O. Rodrigues,

Josiane A. Sobrinho, and Ana de Bettencourt-Dias*

Cite This: J. Med. Chem. 2021, 64, 7724 −7734

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sı Supporting Information

ABSTRACT: New thiophene-dipicolinato-based compounds, K nTdpa (n = 1, 2), were

III

isolated. Their anions are sensitizers of lanthanide ion (Ln ) luminescence and singlet

oxygen generation ( O ). Emission in the visible and near-infrared regions was observed

III

2−

for the Ln complexes with eﬃciencies (ϕ ) ϕ = 33% and ϕ = 0.31% for 1Tdpa

2−

III

and ϕ = 0.07% for 2Tdpa . The latter does not sensitize Eu emission. Fluorescence

imaging of HeLa live cells incubated with K [Eu(1Tdpa) ] indicates that the complex

permeates the cell membrane and localizes in the mitochondria. All complexes generate

III

O in solution with eﬃciencies (ϕ ) as high as 13 and 23% for the Gd complexes of

1_O

−

2−

3−

Tdpa and 2Tdpa , respectively. [Ln(nTdpa) ] (n = 1, 2) are phototoxic to HeLa

3−

cells when irradiated with UV light with IC values as low as 4.2 μM for [Gd(2Tdpa) ]

3−

and 91.8 μM for [Eu(1Tdpa) ] . Flow cytometric analyses indicate both apoptotic and

necrotic cell death pathways.

INTRODUCTION

a distinct advantage. The additional ability to luminesce is

one such example as it provides imaging and tracking ability in

solution or within biological tissues. This allows researchers to

■

Singlet oxygen ( O ) is a reactive oxygen species directly

linked to tissue damage and cellular death. O -generating

photosensitizers are useful in photodynamic therapy (PDT), a

cancer treatment option that uses light to excite photo-

sensitizers to generate O within diseased cells, resulting in

cell death. PDT is minimally invasive with reduced patient

pain, short recovery times, and a lower likelihood of infection.

Porﬁmer sodium, or Photofrin, is a porphyrin-based

photosensitizer that was approved by the U.S. Food and

Drug Administration in 1993 for the treatment of bladder

cancer and has since been approved for several other cancers,

including endobronchial and esophageal cancers.

in-based compounds are among the most studied photo-

sensitizers for PDT.

compounds include a toxic heavy metal to promote

intersystem crossing (ISC) for more eﬃcient O generation.

In addition, many show poor solubility, aggregation, which

shortens the lifetime of the excited state resulting in lower O

generation quantum yields,

study the compound’s mechanism of action. Bioimaging can

be accomplished with lanthanide ion (Ln ) complexes, as the

metal ions, which are less costly than other metals used as

photosensitizers, have unique luminescence properties. They

emit light due to parity-forbidden 4f−4f transitions. Because

the 4f orbitals are shielded by the 5s and 5p, the f−f transitions

are not drastically aﬀected by the coordination environment.

Thus, these ions are attractive for bioimaging applications, as

they display long luminescence decay lifetimes, narrow, metal-

speciﬁc emission bands, and large Stokes shifts of sensitized

emission (vide infra), which enable time-gated luminescence

imaging with resulting increased signal-to-noise ratio, making it

easier to discriminate the metal-centered emission from

III

−5

Porphyr-

While eﬃcient, several of the

22,23

bioﬂuorescence.

Since the transitions are forbidden, the promotion of metal-

centered emission is more easily achieved through coordinated

sensitizers, or antennae, in a process referred to as the antenna

eﬀect. The sensitizer (ligand or antenna) absorbs a photon,

and through a sequence of energy transfer (ET) steps, the

0,11

and high dark toxicity, which

More recent work highlights

2,13

limits dosage concentration.

the progress made in the development of improved photo-

III

emissive excited state of the Ln is populated, as illustrated in

sensitizers, with complexes of Ru , Pd , and Pt with triplet

excited states appropriately positioned to promote the

14−18

Received: March 10, 2021

Published: May 21, 2021

formation of O .

However, while these compounds are

promising, ruthenium, palladium, and platinum are costly rare

metals. Thus, there is a fundamental interest in the

development of new types of eﬃcient O generators.

Ideal photosensitizers for PDT need to be eﬀective at low

doses, and those with built-in multifunctional capabilities show

2021 American Chemical Society

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Journal of Medicinal Chemistry

Article

Figure 1. In addition, the triplet ( T) state of the sensitizer,

which is directly involved in the population of the emissive

thus not viable for imaging and therapy applications in

biological systems. More recently, we described a family of

24,25

III

oligothiophene-based sensitizers of Ln emission, which show

state, can transfer energy to O to generate cytotoxic O .

33,34

wavelength-dependent O generation

but are also not

water-soluble. Another series of oligothiophene-based ligands

that were water-soluble were studied for their cytotoxicity

toward HeLa cells. However, the latter displayed only weak

III

Eu -centered luminescence due to two water molecules

directly coordinated to the metal ion and were thus not useful

for bioimaging.

Oligothiophenes are naturally occurring plant pigments

known for their ability to generate O with high ϕ

1_O

Because of this property, oligothiophenes and thienyl-

derivatized compounds are reported to have applications as

6−38

antiviral, anticancer, and antifungal agents.

Thus,

thiophene-based functional groups are promising because of

9,40

their ability to enhance ISC

and of their interesting photophysical properties.

Dipicolinato-based compounds have been studied exten-

and eﬃciently generate O

3,34,42−46

47−51

sively for cell imaging.

Their usefulness can be attributed

Figure 1. Simpliﬁed energy-level diagram illustrating the ET processes

to the well-documented facile formation of the stable 3:1

III

52−57

for Ln ion sensitization and O generation in this work. The ligand

ligand/metal complex,

the Ln from vibrational quenching in aqueous solution.

which provides better shielding of

III

absorbs energy hν to populate a singlet excited state ( S). A triplet

excited state ( T) is formed after ISC. T can transfer energy to the

Therefore, in pursuit of complexes with the dual properties of

emissive f* excited state which decays by luminescence (L) to the

light emission and O generation, we coupled two moieties, a

ground state f or can lead to O generation. The decay of O to

III

chelator capable of sensitizing Ln -based emission based on

triplet oxygen O₂is seen through the emission at 1270 nm.

Nonradiative (NR) pathways (dotted lines) can lead to the quenching

of excited states. Other possible radiative processes are ﬂuorescence

pyridine-bis(carboxylate) as well as a functional group capable

of generating O based on thiophene or 2,2′-bithiophene, to

generate new ligands that promote both properties when

(F) and phosphorescence (P). Energy levels are not drawn to scale.

III

bound to Ln ions. The resulting 3:1 ligand-to-metal Ln ion

complexes, shown in Scheme 1, are trianionic and water-

soluble. We report here the synthesis and photophysical

III

Ln -based systems work well as multifunctional platforms

6−28

with imaging capabilities, as recently demonstrated.

For

−

III

properties of the two new ligands, nTdpa , and their

respective luminescent Ln ion complexes, [Ln(nTdpa) ]

example, a Tb DOTA-based complex that displays green

Tb -centered emission with an eﬃciency of 24% and O

III

3−

III

(

n = 1 or 2, Ln = Eu , Gd , or Yb ). We show that

generation eﬃciency (ϕ ) of 12% was used to image NIH-

1_O

3−

[Eu(1Tdpa)₃] is useful for cell ﬂuorescence imaging and

III

T3 cells. While this complex displayed eﬃcient Tb

report the results of its colocalization studies, as well as the O

luminescence, ϕ

fell well below the eﬃciencies of organic

generation ability, photocytotoxicity, and dark cytotoxicity of

photosensitizers. Patra and coworkers described Eu^I^I^Iand

3−

[Eu(1Tdpa)

cells.

]

and the [Ln(2Tdpa)

]

complexes in HeLa

III

Tb complexes which penetrate the cell membrane of H460

and MCF-7 cells. The IC₅₀value of the more phototoxic Tb^I^I^I

complex is 7.94 ± 0.65 μM in H460 cells. The authors

mention that the cells remained viable in the presence of the

RESULTS AND DISCUSSION

■

H 1Tdpa and H 2Tdpa were synthesized by Suzuki coupling

complexes in the dark.

thienyl- or bithienyl-functionalized borolane with brominated

Our group has described dually functional complexes with

naphthalimide-containing antennae that sensitize visible-

dipicolinic ester following a modi ﬁ ed literature procedure

(

Scheme 2 and Supporting Information). Subsequent

III

(

Eu ) and near-infrared (NIR)-emitting (Yb , Nd , and

saponiﬁcation of diethyl 4-(thiophen-2-yl)pyridine-2,6-dicar-

boxylate (n = 1) or diethyl 4-(2,2′-bithiophen-5-yl)pyridine-

III

Er ) ions in addition to generating O with ϕ in the range

¹O

1−64%. These complexes are not water-soluble and are

2,6-dicarboxylate (n = 2) led to the formation of H 1Tdpa and

Scheme 1. [Ln(1Tdpa) ]³⁻(Left) and [Ln(2Tdpa)₃]³⁻(Right) Described Here

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Article

Scheme 2. Synthesis of H nTdpa (n = 1 or 2)

III

Table 1. Quantum Yields of Fluorescence (ϕ ), Quantum Yields of Ln Luminescence (ϕ ), and Ln Luminescence_a

−

Lifetimes (τ ) for [Ln(nTdpa) ] (n = 1 or 2) in Water (pH = 7.3) and Ethanol (Italicized Rows) at 25.0 ± 0.1 °C

III

ϕ^F(%) Gd^I^I^I

ϕ^Y^b(%)

ϕ^E^u(%)

τ^Y^b(μs)

τ^E^u(ms)

compound

ϕ (%) no Ln

7 ± 1

−

[

Ln(1Tdpa) ]³

3 ± 0

0.003 ± 0.000

33.3 ± 0.2

1.15 ± 0.00

.03 ± 0.00

1.57 ± 0.00

± 1

6 ± 1

0.31 ± 0.00

0.0007 ± 0.0001

0.07 ± 0.00

41.0 ± 0.0

8.59 ± 0.01

[

Ln(2Tdpa) ]³

28 ± 8

57 ± 4

55 ± 2

1 ± 1

9.76 ± 0.10

Ln^I^I^I= Gd , Yb , or Eu . Measured in 95% ethanol. Measured in D O. Yb emission is too low to reliably determine the lifetime.

III

H 2Tdpa in 23% overall yield for both compounds. H 1Tdpa

nm for the former and 364 nm for the latter (Figures S12 −

S16 ). The emission spectra display maxima at 380 nm (λ

and H 2Tdpa were characterized using NMR spectroscopy and

exc

mass spectrometry (Figures S1 −S4). To study the photo-

physical properties of these new compounds in aqueous

solution, H 1Tdpa and H 2Tdpa were deprotonated with

300 nm) with an associated quantum yield of 7% and 464 nm

−

(λ_e_x_c= 360 nm) with a quantum yield of 28% for [1Tdpa]

2−

and [2Tdpa] , respectively (Table 1), and are similar to other

9,60

K CO .

thienyl-based compounds in water.

The ﬂuorescence

III

The Ln complexes were obtained by reacting solutions of

spectra of these compounds in 95% ethanol are virtually

indistinguishable from those in water (Figures S15 and S16).

III

K nTdpa with LnCl (Ln = Gd , Eu , or Yb ) in water and

3−

stirring at 50 °C for 24 h before spectroscopic characterization.

The expected 3:1 ligand-to-metal stoichiometry of the

complexes in solution, analogous to known dipicolinato

[Gd(1Tdpa)₃] and [Gd(2Tdpa)₃] also ﬂuoresce in the

UV−vis region in water. When compared to the uncoordinated

ligands, there are no considerable changes to the emission

proﬁles (Figures S17 and S18). Fluorescence quantum yields

2−57

complexes,

was conﬁrmed through emission titration of

K nTdpa with the YbCl solution (Figures S5 and S6). The

(ϕ ), however, were 3 and 57%, respectively (Table 1). The

complexes were also characterized using mass spectrometry

Figures S7 −S11 ) and elemental analysis showing results

latter is higher than the ϕ of the free ligand likely due to

III

planarization of the ligand upon coordination to Gd and

consistent with that stoichiometry. Emission lifetimes (vide

infra) also conﬁrm the saturation of the coordination sphere

with the successful exclusion of water molecules, consistent

with the 3:1 stoichiometry. For our studies, we isolated the

some phosphorescence contribution due to improved ISC.

The ﬂuorescence spectra in 95% ethanol show no appreciable

change with respect to the potassium salts of the ligands

(Table 1 and Figures S19 and S20).

III

complexes K [Ln(nTdpa) ] (n = 1 or 2 and Ln = Eu , Yb ,

In water, both ligands sensitize Yb , a NIR emitter, and the

III

3−

or Gd ) after solvent evaporation and redissolved them in the

emission spectrum of [Yb(1Tdpa) ] with the characteristic

appropriate solvents.

metal-centered F → F transition (λ_m_a_x= 978 nm) is

7/2

The absorbance spectra of [1Tdpa]²and [2Tdpa]²⁻in

water are broad and structureless with maxima at 300 and 380

nm, respectively. The excitation spectra for both species match

closely the respective absorbance spectra with maxima at 300

−

seen when the complex is excited at 320 nm (Figure 2). A

−

similar emission pro ﬁ le is seen when [Yb(2Tdpa)₃] is excited

at 370 nm (Figure S21 ). Quantum yields of luminescence

(ϕ ) for both complexes were 0.003 and 0.0007% for

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All compounds generate O , which was conﬁrmed by the

appearance of a phosphorescence peak at 1270 nm in the

−

emission spectra (Figure S33). The ϕ

1_O

for 2Tdpa ,

3−

[

Gd(1Tdpa) ] , and [Gd(2Tdpa) ] are 45, 13, and 23%

in 95% ethanol, respectively (Table 2). It was not possible to

Table 2. Quantum Yields of Singlet Oxygen Generation (

) for [Ln(nTdpa)₃]³(n = 1 or 2) in 95% Ethanol at

−

1_O

5.0 ± 0.1 °C

compound

no Ln^I^I^I

Gd^I^I^I

Eu^I^I^I

Yb^I^I^I

−

[

Ln(1Tdpa) ]³

23 ± 0

13 ± 0

45 ± 4

Ln(2Tdpa) ]³

LE = O emission is observed but is too weak to reliably determine

1_O

determine ϕ _O

intensity (Figure S32 ). Determination of ϕ _O

in some cases due to low phosphorescence

−

Figure 2. Normalized emission spectra of the [Ln(1Tdpa) ]

III

was attempted in

complex [Ln = Eu (red) or Yb (teal)] in water at 25.0 ± 0.1

−

C (pH = 7.3); [complex] = 5 × 10 M; λ_e_x_c= 320 nm.

water, but due to the low dissolved O content (χ in water =

2 O2

−

−471

.2 × 10 , ethanol = 5.71 × 10

), the phosphorescence

−

3−

[

Yb(1Tdpa)₃]³and [Yb(2Tdpa) ] , respectively, and are

comparable to values for other luminescent Yb complexes in

water. Measurement of luminescence lifetimes were not

reliable due to low signal intensity.

When exciting [Eu(1Tdpa) ] at 320 nm, the spectrum

displays the characteristic Eu -centered transitions D → F

J = 0−4) (Figure 2). The ﬁne structure of the D → F

transition is consistent with a relatively low symmetry

The eﬃciency

of sensitized Eu emission (ϕ ) was 33.3% in water (Table

), which is more eﬃcient than what is reported for the

analogous dipicolinato complex (ϕ = 24% ). Luminescence

emission lifetimes (τ ) were obtained in water and deuterated

water and are 1.15 and 2.03 ms, respectively, and are similar to

(Figures S27 and

S28 and Table S1 ). In both cases, these data ﬁt a single

signal was too weak to allow for reliable results (Figure S33).

Nonetheless, the toxicity of these compounds was tested by

incubating human cervical cancer carcinoma (HeLa) cells with

III

2−

the 2Tdpa -based compounds.

3−

HeLa cells were selected for this study as they are an ideal

candidate due to their robust characteristics. Cell viability

was assessed using an MTT metabolic activity assay in the dark

and under UV light irradiation (λ_e_x_c= 365 nm). As shown in

Figure 3a, the viability of HeLa cells decreases with increasing

concentration of the photosensitizers when irradiated for 5

min. At most of the concentrations used, both Ln complexes,

[Gd(2Tdpa)₃]

cytotoxic than 2Tdpa . Control experiments show no

appreciable change to cell viability within 5 min of irradiation,

indicating that cell death is not observed in the absence of the

photosensitizers.

III

(

III 46,47,63

environment, distorted D , around Eu .

III

3−

and [Yb(2Tdpa) ] , are more photo-

−

III

47,64,65

what is seen for other Eu complexes

exponential decay and are thus consistent with the presence of

HeLa cells were treated with the photosensitizers under the

same conditions in the dark to test the compound’s dark

toxicity. Cell viability does not decrease below 80% for 3.00,

6.25, and 12.5 μM (Figure 3b), and some dark cytotoxicity is

observed at higher concentrations of 25 and 50 μM for all

photosensitizers. IC₅₀values in the dark and with irradiation

were determined and are summarized in Table 3. Similar

III

only one Eu species in solution. From the lifetimes in H O

and D O, we calculated the number of water molecules

III

66,67

coordinated to Eu , q, in this complex;

the value of ∼0

coordinated water molecules is consistent with a coordination

environment composed of three nTdpa²ligands.

−

III

Unlike what is observed for the Gd complexes and the

ligands, the Ln luminescence properties are impacted by the

III

3−

potencies for [Gd(2Tdpa) ]

observed, and 2Tdpa is notably less potent (Figure S36),

which is likely due to the 3:1 ligand-to-metal stoichiometry of

and [Yb(2Tdpa)₃ ]

are

−

change in solvent, as one would expect from the nature of the

emitting species. The exchange of water for 95% ethanol

reduces O−H quenching (Figures S24 −S26), which results in

the complex. These IC₅₀values compare favorably to those of

III

74−76

an increase in ϕ (Table 1). Since the Yb luminescence

known photosensitizers.

The phototoxicity indices (PTIs)

increases in both complexes, we were able to measure τ as

of the complexes (Table 3), after a short irradiation period of 5

min, compare favorably with those of reported photo-

sensitizers, despite being lower than the values published for

−

3−

.59 and 9.76 μ s for [Yb(1Tdpa)₃] and [Yb(2Tdpa) ] ,

respectively (Figures S30 and S31 and Table S2 ), which are

68,69

15,17,18

3−

similar to known complexes.

Both lifetimes ﬁt a single

the most eﬃcient ones.

The [Eu(1Tdpa)₃] complex

exponential decay consistent with a unique coordination

environment around Yb , and the similar values for both

was also tested for cytotoxicity with HeLa cells when irradiated

and in the dark (Figures S37 and S38). IC₅₀determination

showed that it is signi ﬁ cantly less potent than the 2Tdpa -

III

2−

complexes indicate similar coordination geometries. The

eﬃciencies ϕ , ϕ , τ , and ϕ also increase signiﬁcantly

derivatives (Table 3, Figure S39 ). This decrease in cytotoxicity

3−

when water is exchanged for 95% ethanol for [Ln(1Tdpa)₃]

Tables 1 and S3) again due to reduced quenching through

is consistent with our observation of the low-intensity O

(

emission at 1270 nm (Figure S32).

O−H oscillators of the bulk solvent. It is important to note

Although we do not anticipate leaching of the metal ion

from these complexes, control experiments using the LnCl₃

salts were conducted and con ﬁ rmed that the free metal ions are

III

that the Eu emission proﬁle remains the same, indicating that

the symmetry around the metal center does not change as a

result of changing solvent.

not cytotoxic (Figures S34 −S38), as previously observed.

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Figure 3. Cell viability (%, MTT assay) as a function of incubation concentration of 2Tdpa (yellow) and [Gd(2Tdpa)₃]³⁻(blue), or

2−

Yb(2Tdpa)₃]³(green) (a) after 5 min of irradiation with 365 nm light and (b) in the dark. Control experiments were performed under the same

−

[

experimental conditions.

The combined data suggest that these photosensitizers are

potential photodynamic therapeutics with low dark toxicity.

The cell death mechanism was studied by ﬂow cytometry of

HeLa cells stained with Annexin V-ﬂuorescein isothiocyanate

26% necrotic and 16% apoptotic. In the dark, 91% of

combined cells remain viable (Figure 4a). After irradiating

−

with light, cells incubated with [Gd(2Tdpa)₃] are 44%

necrotic and 28% apoptotic, yet less than 25% of combined

cells show as necrotic or apoptotic when in the dark (Figure

(

FITC) and propidium iodide (PI) after incubating the cells

with 12.5 μM of the 2Tdpa -based compounds in the dark

2−

3−

4b,e). Likewise, cells incubated with [Yb(2Tdpa)₃] show

and after a short irradiation period of 5 min. Figure 4 shows

32% of cells as necrotic and 19% as late apoptotic in the light

(Figure 4c,f). Figure S40 shows control experiments in the

dark and under light irradiation, indicating that all cells remain

viable in the absence of a photosensitizer.

−

the cell population of viable cells (FITC /PI ), early-stage

−

apoptotic cells (FITC /PI ), late-stage apoptotic cells

−

(

FITC /PI ), and necrotic cells (FITC /PI ) after treat-

−

3−

2−

ment with 2Tdpa , [Gd(2Tdpa) ] , or [Yb(2Tdpa) ] .

These results indicate that 2Tdpa -mediated photolysis

Cells treated with these compounds show minimal cell death in

the dark (Figure 4a−c). However, when cells treated with

these compounds are exposed to UV light, an increased

percentage of cells indicate late apoptotic and necrotic

processes. Cells incubated with 2Tdpa²(Figure 4d) are

induces cell death by necrosis. PDT photosensitizers have been

observed to trigger cell death by both necrosis and apoptosis

78,79

and the latter is the most common pathway.

In fact, the

cell death mechanism for the FDA-approved drug, Photofrin,

when used in combination with carboplatin, shows signiﬁcant

−

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Journal of Medicinal Chemistry

characteristics of both pathways have been observed, several

Article

−

Table 3. IC₅₀Values and PTI for the 2Tdpa -Based

Compounds and K [Eu(1Tdpa) ] Compared to Known

Photosensitizers

others have suggested that the balance between apoptosis and

necrosis is dependent on multiple factors such as photo-

sensitizer dose, cell type, genetic and metabolic potential, and

83−86

the photosensitizers subcellular localization.

The promising luminescence properties of the Eu complex

prompted us to evaluate its use as a luminescent dye for

compound

^I^C⁵0 (μM) hν

^I^C50 (μM) no hν

PTI

III

K 2Tdpa

10.5 ± 0.8

4.2 ± 0.5

6.3 ± 0.6

91.8 ± 5.1

7.1

47.5 ± 2.5

39.7 ± 4.8

43.8 ± 3.6

435.3 ± 5.3

>41

4.5

9.5

7.0

4.7

K [Gd(2Tdpa) ]

cellular imaging, including identifying its ability to permeate

K [Yb(2Tdpa) ]

79,84

the cell membrane

and its subcellular location. Therefore,

K [Eu(1Tdpa) ]

3−

^w^eⁱⁿ^c^u^b^a^t^e^d^H^e^L^a^c^e^l^l^s^wⁱ^t^h¹²^.⁵^μ^M^o^f^[^E^u⁽¹^T^d^p^a⁾3^] (pH

7.4), Hoechst 33342 (1 μg/mL), and MitoTracker Green

Photofrin

Re-NH₂

17.3 ± 2.9

8.1 ± 0.3

>100

>50

FM (50 nM) to determine cell permeability and subcellular

[

VO(cur)(phen)]Cl

localization. The live-cell images in Figure 5 show that Hoechst

Data taken from ref 76; IC₅₀for this compound was expressed as 4.3

μg/mL (light) and 25 μg/mL (dark) in HeLa cells (MW: 605.7 g/

mol) after 24 h incubation and irradiation with a broad-spectrum

halogen lamp. Data taken from ref 74; IC for this compound was

determined after 4 h incubation and irradiation with 350 nm light.

Data taken from ref 75; cur = curcumin; phen = 1,10-phenanthro-

III

3342 and Eu luminescence emission are observed when the

cells are irradiated with 405 nm light (top center and right),

and MitoTracker Green FM luminescence emission is

observed when the cells are irradiated with 488 nm light

III

(top left). The Eu compound appears to permeate the cell

line; IC₅₀for this compound was determined after 4 h incubation and

irradiation with a broad-spectrum source.

membrane of HeLa cells and co-localize in the same region as

MitoTracker Green FM dye, indicating some cell speciﬁcity

(

bottom center).

Other Eu complexes which penetrate the membrane of

III

necrosis in HeLa cells. Conventionally, cell necrosis has been

considered an unregulated process, which is independent of

HeLa cells were described by Yuan and coworkers who

III

apoptosis. However, recent studies have demonstrated

mechanisms with characteristics related to apoptosis and

necrosis pathways. Although it is unclear to us why

reported Eu complexes based on a derivative of 4′-(10-

methyl-9-anthryl)-2,2′:6′,2″-terpyridine and display lumines-

cence within HeLa cells, yet are weakly luminescent compared

87,88

to the complex reported here (ϕ = 0.88%).

These

2−

3−

Figure 4. Flow cytometry quantiﬁcation of HeLa cells stained with Annexin V-FITC and PI using 2Tdpa , [Yb(2Tdpa) ] , [Gd(2Tdpa) ] as

PDT sensitizers in the dark (a−c) and after light irradiation (d−f), respectively. Light/dark negative and light/dark positive controls were also

examined (Figure S40).

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Figure 5. Live cell images of HeLa cells incubated with (top left) MitoTracker Green FM (50 nM, λ = 488 nm), (top center) Hoechst 33342 (1

exc

3−

μg/mL, λ_e_x_c= 405 nm), (top right) [Eu(1Tdpa)₃] (12 μM, λ_e_x_c= 405 nm), and (bottom center) merged image.

authors also characterized the complexes’ photophysical

enhanced stability, in which the pyridine-based chelator shows

properties in solution at high pH, which is not near the

higher denticity, as well as biocompatibility for diagnostic and

therapeutic applications. Ultimately, our work shows that the

targeted strategy of combining known cytotoxic compounds

pH of HeLa cells (7.9 ± 0.1 ) or within the typical pH range

of animal cells (6.5−7.5 ). Chauvin, Bu

nzli, and coworkers

reported a Eu complex based on a hexadentate bis-

benzimidazole)pyridine ligand with a quantum yield of 11%

at pH = 7.4. Bioimaging of these complexes indicated

III

with chelators capable of sensitizing Ln -centered emission

(

was successfully pursued and, as a strategy, should be viable for

a variety of other systems as well.

cytoplasmic localization of HeLa cells. Patra and coworkers

synthesized Eu and Tb complexes with a dopamine-

functionalized diethylenetriaminepentaacetic acid ligand and

reported ϕ and ϕ of 3.8 and 2.2%, respectively, at pH =

.2. A high concentration (100 μM) of each Ln complex for

III

EXPERIMENTAL SECTION

■

General Information. All the reagents used were of analytical

grade, commercially obtained, and used as received. Solvents were

dried by standard methods. The syntheses were completed under the

III

N atmosphere unless otherwise speciﬁed. The purity of the organic

bioimaging, likely due to their poor luminescence eﬃciency,

compounds and metal complexes was found to be at least 95% and

indicated cytoplasmic localization of the complexes within

III

was assessed by H NMR spectroscopy, high-resolution mass

HeLa cells. In contrast, the luminescent Eu compound

spectrometry (spectra in ESI), and combustion microanalysis where

appropriate. Combustion microanalyses of the compounds used in the

cytotoxicity studies were performed by Galbraith Laboratories

studied here eﬃciently sensitizes luminescence and coupled

with its ability to generate O under irradiation (Figures S32,

S37, and S39) provides a dual functionality that is absent from

the above examples.

(

Knoxville, TN).

Synthesis of H nTdpa. H nTdpa (n = 1 or 2) was synthesized as

shown in Scheme 2.

Synthesis of Diethyl 4-Bromopyridine-2,6-dicarboxylate (1). This

compound was synthesized using a modiﬁed literature procedure.

The product was puriﬁed using a silica column and hexanes/ethyl

acetate (1:1) as the eluent. The pure compound was isolated as a

CONCLUSIONS

■

In summary, two thiophene-containing molecules were

synthesized and used as sensitizers for O generation and

III

Ln luminescence in the visible and NIR region. O₂

white powder. Yield: 2.0 g, 81%.

generation was observed for all complexes discussed here,

H NMR (CDCl , 400 MHz): 8.43 (s, py, 2H), 4.50 (q, CH , 4H),

and 1.46 (t, CH , 6H) ppm.

and the most eﬃcient O generation was observed for the

−

Synthesis of 4,4,5,5-Tetramethyl-2-(thiophen-2-yl)-1,3,2-dioxa-

borolane (2). Potassium acetate (1.1 mg, 11 mmol), bis(pinacolato)-

Tdpa -based compounds. These displayed photocytotoxicity

toward HeLa cells after only a short irradiation period of 5 min

and IC values were determined and are in the range 4.2−10.5

diboron (930 mg, 3.7 mmol), and Pd(dppf)Cl (130 mg, 0.02 mmol)

were added to a round-bottom ﬂask containing dry toluene (10 mL)

and heated to 80 °C for 24 h. The reaction was cooled and poured

into water, and the product was extracted with diethyl ether (3 × 10

mL). The combined organic phases were washed with brine and dried

3−

μM, while the IC value for the red emissive [Eu(1Tdpa) ]

is 91.8 μM. All compounds displayed low dark toxicity.

3−

[

Eu(1Tdpa)₃] was further used as a luminescent dye for live-

cell imaging, indicating that this compound penetrates the cell

membrane where it localizes in the mitochondria.

over Na SO . The solvent was removed under reduced pressure to

2 4

yield yellow crystals. Yield: 730 mg, 94%.

H NMR (CDCl , 400 MHz): 7.66 (d, Th, 1H), 7.64 (d, Th, 1H),

These improved luminescence characteristics, water sol-

ubility, and photocytotoxicity toward HeLa cells make these

compounds potentially interesting for PDT. As the pyridine-

based ligands can be easily functionalized, while not discussed

here, they serve as the basis for new compounds with improved

characteristics, such as a red-shifted excitation wavelength, and

.19 (dd, Th, 1H), and 1.35 (s, CH , 12H) ppm.

Synthesis of Diethyl 4-(Thiophen-2-yl)pyridine-2,6-dicarboxylate

(

3). This compound was synthesized using a modiﬁed literature

procedure. 2 (103 mg, 0.49 mmol), 1 (100 mg, 0.33 mmol), and

XPhosPd G2 (26 mg, 0.033 mmol) were dissolved in deoxygenated

tetrahydrofuran (THF) (2 mL). K PO in deoxygenated water (1.4

730

J. Med. Chem. 2021, 64, 7724−7734

Journal of Medicinal Chemistry

The Supporting Information is available free of charge at

Article

mL, 0.5 M) was added and stirred for 4 h at 50 °C. Distilled water (5

mL) was used to quench the reaction, and the product was extracted

with CH Cl (3 × 10 mL). The combined organic phases were dried

Elemental analysis calculated for K 1Tdpa: C, 40.60; H, 1.55; N,

4.30. Found: C, 40.40; H, 2.24; N, 3.65.

Elemental analysis calculated for K 2Tdpa·2H O: C, 40.62; H,

over Na SO and ﬁltered, and the solvent was removed under reduced

2.50; N, 3.16. Found: C, 40.15; H, 2.14; N, 2.64.

pressure. The product was puriﬁed using a silica column, with 1:1

hexanes/ethyl acetate as the eluent, and was isolated as light yellow

Synthesis of Lanthanide Complexes (K

[Ln(nTdpa)

], n = 1

or 2). All metal complexes were prepared by mixing 1 equiv of LnCl

III

(

Ln = Yb , Gd , or Eu ) with 3 equiv of K 1Tdpa or K 2Tdpa in

crystals. Yield: 50 mg, 50%.

water and stirred for 24 h at 50 °C.

H NMR (CDCl , 400 MHz): 8.44 (s, py, 2H), 7.69 (d, Th, 1H),

K [Gd(1Tdpa) ].

.69 (d, Th, 1H), 7.52 (d, Th, 1H), 7.19 (dd, Th, 1H), 4.52 (q, CH₂,

Yield: 95%.

[

H), and 1.48 (t, CH , 6H) ppm.

^G^d⁽¹^T^d^p^a⁾3^]+ 2K⁺.

−

Synthesis of 4-(Thiophen-2-yl)pyridine-2,6-dicarboxylic Acid

ESI-HRMS [C 33

976.8147 (exp) (Figure S8 ).

[Eu(1Tdpa) ].

Yield: 94%.

^E^u⁽¹^T^d^p^a⁾3^]+ 2K⁺.

H 215 GdK N O S

]⁻m/z: 976.8338 (calc),

2 3 12 3

(H 1Tdpa). NaOH (45 mg, 1.1 mmol) was dissolved in distilled

water (2 mL) and added dropwise to a solution of 3 (140 mg, 0.46

mmol) in 1:5 methanol/water (6 mL). The reaction was stirred at 60

C for 4 h and cooled in an ice bath. HCl (1 M) was added until pH

2, upon which an oﬀ-white precipitate formed. The precipitate was

ﬁltered, washed with cold water, and dried under low pressure. Yield:

−

[

∼

−

ESI-HRMS [C H EuK N O S ] m/z: 971.8331 (calc),

33 215

12 3

71.8295 (exp) (Figure S7 ).

6 mg, 61%.

Elemental analysis calculated for K [Eu(1Tdpa) ]·6H O: C, 35.42;

H NMR (D O, 400 MHz): 8.32 (s, py, 2H), 8.01 (d, Th, 1H),

H, 2.43; N, 3.76. Found: C, 35.67; H, 1.93; N, 3.16.

.81 (d, Th, 1H), and 7.23 (dd, Th, 1H) ppm.

−

K [Yb(1Tdpa) ].

HR ESI-MS (C H NO S) : 248.0023 (calc), 248.0019 (exp).

Yield: 91%.

[Yb(1Tdpa)

Synthesis of 2-(2,2′-Bithiophen-5-yl)-4,4,5,5-tetramethyl-1,3,2-

−

]³+ 2H⁺.

dioxaborolane (4). This compound was synthesized using a modiﬁed

]⁻m/z: 917.9168 (calc),

literature procedure. N-Butyllithium (2.6 mL, 6.5 mmol, 2.5 M in

hexane) was added dropwise to a chilled mixture of THF (50 mL)

and 2,2′-bithiophene (1.0 g, 6.0 mmol) and stirred for 1 h at −78 °C.

ESI-HRMS [C 33

917.9208 (exp) (Figure S9 ).

[Gd(2Tdpa) ].

Yield: 98%.

215 YbH

-Isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.4 mL, 8.2

3−

[

^G^d⁽²^T^d^p^a⁾3^]+ H + K .

ESI-HRMS [C H GdKN O S ] m/z: 1184.8411 (calc),

mmol) was added dropwise at −78 °C. The reaction was allowed

to reach RT and stirred for an additional 20 h. Saturated NH Cl

−

45 224

12 6

184.8529 (exp) (Figure S10 ).

solution (20 mL) was used to quench the reaction, and the product

was extracted with diethyl ether (3 × 15 mL). The combined organic

phases were washed with water (3 × 10 mL) and brine (3 × 10 mL)

and dried over Na SO . Excess solvent was removed under reduced

Elemental analysis calculated for K [Gd(2Tdpa) ]: C, 42.81; H,

.68; N, 3.33. Found: C, 42.67; H, 2.02; N, 3.16.

K [Yb(2Tdpa) ].

Yield: 91%.

[

pressure, and the product was puriﬁed using a silica column and 4:1

petroleum ether/ethyl acetate as the eluent. The pure product was

^Y^b⁽²^T^d^p^a⁾3^]+ 2H⁺.

−

ESI-HRMS [C H YbK N O S ] m/z: 1162.9000 (calc),

isolated as a blue oil. Yield: 1.2 g, 69%.

45 224

12 6

1162.9177 (exp) (Figure S11 ).

H NMR (CDCl , 400 MHz): 7.53 (d, Th, 1H), 7.25 (d, Th, 1H),

Elemental analysis calculated for K [Yb(2Tdpa) ]·2H O: C, 41.12;

.23 (d, Th, 2H), 7.02 (t, Th, 1H), and 1.35 (s, CH , 12H) ppm.

Synthesis of Diethyl 4-(2,2′-Bithiophen-5-yl)pyridine-2,6-dicar-

H, 1.92; N, 3.20. Found: C, 41.02; H, 1.90; N, 3.06.

boxylate (5). This compound was synthesized using a modiﬁed

literature procedure. 4 (440 mg, 1.5 mmol), 1 (300 mg, 1 mmol),

and XPhosPd G2 (78 mg, 0.1 mmol) were dissolved in deoxygenated

THF (4 mL). K PO in deoxygenated water (4.2 mL, 0.5 M) was

ASSOCIATED CONTENT

sı Supporting Information

■

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01805.

added, and the reaction was stirred for 4 h at 50 °C. The reaction was

quenched with water (5 mL), and the product was extracted with

CH Cl (3 × 10 mL). The combined organic phases were dried over

Molecular formula strings, selected experimental details

and instrument information, NMR and high-resolution

mass spectra, speciation studies, absorption, excitation

and emission spectra, emission lifetimes, eﬃciencies of

light emission and singlet oxygen generation, cell

viability studies, and IC₅₀values and ﬂow cytometry

experiments (PDF )

Na SO and ﬁltered, and the solvent was removed under reduced

pressure. The product was puriﬁed using a silica column, and 1:1

hexanes/ethyl acetate was used as the eluent. The product was

isolated as green crystals. Yield: 210 mg, 54%.

H NMR (acetone-d , 400 MHz): 8.40 (s, py, 2H), 7.92 (d, Th,

H), 7.54 (d, Th, 1H), 7.46 (d, Th, 1H), 7.40 (d, Th, 1H), 7.15 (t,

Th, 1H), 4.45 (q, CH , 4H), and 1.42 (t, CH , 6H) ppm.

Synthesis of 4-(2,2′-Bithiophene)pyridine-2,6-dicarboxylic Acid

SMILES identiﬁers for developed compounds (CSV)

(

H 2Tdpa). NaOH (45 mg, 1.1 mmol) was dissolved in distilled water

(

2 mL) and then added dropwise to a solution of 5 (150 mg, 0.39

mmol) in 1:5 methanol/water (6 mL). The reaction was stirred at 60

C for 4 h and cooled in an ice bath. HCl (1 M) was added until pH

2, forming a yellow precipitate. The product was ﬁltered, washed

AUTHOR INFORMATION

∼

■

Corresponding Author

with cold water, and dried under reduced pressure, yielding a yellow−

Ana de Bettencourt-Dias − Department of Chemistry,

University of Nevada, Reno, Reno, Nevada 89557, United

States; orcid.org/0000-0001-5162-2393; Email: abd@

unr.edu

brown powder. Yield: 930 mg, 76%.

H NMR (DMSO-d , 400 MHz): 9.87 (s, OH, 2 H), 8.16 (s, py,

H), 7.86 (d, Th, 1H), 7.60 (d, Th, 1H), 7.46 (d, Th, 1H), 7.41 145

(d, Th, 1H), and 7.15 (t, Th, 1H) ppm.

−

ESI-MS (C H KNO S ) : 367.9459 (calc), 367.9460 (exp).

4 2

Authors

Synthesis of K nTdpa (n = 1 or 2). K CO (0.5 mmol) and

Carime V. Rodrigues − Department of Chemistry, University

of Nevada, Reno, Reno, Nevada 89557, United States;

Laboratório de Inorganica e Materiais, Instituto de Química,

Universidade de Brasília, Brasilia 70910-900, Brazil

H nTdpa (n = 1 or 2) (0.5 mmol) were dissolved in distilled water

and stirred for 30 min, yielding colorless or yellow solutions for

K 1Tdpa and K 2Tdpa, respectively. KOH and HCl were used, as

appropriate, to adjust the pH to 7.2.

731

J. Med. Chem. 2021, 64, 7724−7734

Journal of Medicinal Chemistry

orcid.org/0000-0002-1323-0222

Article

Katherine R. Johnson − Department of Chemistry, University

of Nevada, Reno, Reno, Nevada 89557, United States;

Vincent C. Lombardi − Department of Microbiology and

Immunology, University of Nevada, Reno, Reno, Nevada

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Materiais, Instituto de Química, Universidade de Brasília,

Brasilia 70910-900, Brazil

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Josiane A. Sobrinho − Department of Chemistry, University of

Nevada, Reno, Reno, Nevada 89557, United States

imaging and theranostics. Theranostics 2012, 2, 916−966.

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Complete contact information is available at:

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pathways to water-soluble phthalocyanines and close analogs. Coord.

Chem. Rev. 2010, 254, 2792−2847.

(

10) Jiang, X.-J.; Lo, P.-C.; Tsang, Y.-M.; Yeung, S.-L.; Fong, W.-P.;

Author Contributions

C.V.M. and K.R.J. contributed equally to this manucript.

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⊥

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The authors declare no competing ﬁnancial interest.

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ACKNOWLEDGMENTS

■

The National Science Foundation (CHE-1800392 to AdBD) is

gratefully acknowledged for ﬁnancial support. Shanti Rawat

and Prof. Jorge H.S.K. Monteiro are acknowledged for their

support and guidance. Janina S. Ruprecht is acknowledged for

collecting mass spectra. This work was carried out with support

from the Coordination for the Improvement of Higher

Education Personnel, (CAPES-Brazil) Process

(12) Dummin, H.; Cernay, T.; Zimmermann, H. W. Selective

photosensitization of mitochondria in hela cells by cationic Zn(II)-

phthalocyanines with lipophilic side-chains. J. Photochem. Photobiol., B

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nonporphyrin photosensitizers in oncology: Preclinical and clinical

advances in photodynamic therapy. Photochem. Photobiol. 2009, 85,

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(

88881.186961/2018-01 to CVSR-Capes ScholarBrazil).

14) Potocny, A. M.; Pistner, A. J.; Yap, G. P. A.; Rosenthal, J.

Research reported in this publication used the Cellular and

Molecular Imaging (CMI) core facility supported by the

National Institute of General Medical Sciences of the National

Institutes of Health under grant number P20 GM103650.

Electrochemical, spectroscopic, and O sensitization characteristics of

synthetically accessible linear tetrapyrrole complexes of palladium and

platinum. Inorg. Chem. 2017, 56, 12703−12711.

(15) Potocny, A. M.; Riley, R. S.; O ’Sullivan, R. K.; Day, E. S.;

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phototoxicity index. Inorg. Chem. 2018, 57, 10608−10615.

ABBREVIATIONS

■

O singlet oxygen S, singlet; T, triplet; DOTA, dodecane

tetraacetic acid; dppf, diphenylphosphineferrocene; ET, energy

transfer; exp, experimental; FITC, ﬂuorescein isothiocyanate;

ISC, inter-system crossing; L, luminescence; Ln , lanthanide-

16) Wang, J.; Potocny, A. M.; Rosenthal, J.; Day, E. S. Gold

nanoshell-linear tetrapyrrole conjugates for near infrared-activated

dual photodynamic and photothermal therapies. ACS Omega 2020, 5,

III

(

26−940.

(

III); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-

17) Sainuddin, T.; McCain, J.; Pinto, M.; Yin, H.; Gibson, J.; Hetu,

lium bromide; NIR, near-infrared; nTdpa, oligo(n)thiophene-

functionalized dipicolinic acid; PDT, photodynamic therapy;

PI, propidium iodide; PTI, phototoxicity index (while PI is the

abbreviation commonly seen in the literature we use this one

here to distinguish from propidium iodide); THF, tetrahy-

drofuran; XPhosPd G2, Buchwald second-generation Pd(0)

catalyst.

M.; McFarland, S. A. Organometallic Ru(II) photosensitizers derived

from π -expansive cyclometalating ligands: Surprising theranostic PDT

effects. Inorg. Chem. 2016, 55, 83−95.

(18) Soliman, N.; McKenzie, L. K.; Karges, J.; Bertrand, E.; Tharaud,

M.; Jakubaszek, M.; Guérineau, V.; Goud, B.; Hollenstein, M.; Gasser,

G.; Thomas, C. M. Ruthenium-initiated polymerization of lactide: A

route to remarkable cellular uptake for photodynamic therapy of

cancer. Chem. Sci. 2020, 11, 2657−2663.

(19) Haxel, G. B.; Hedrick, J. B.; Orris, G. J. Rare Earth Elements-

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