Synthesis, Characterization, and Biological Profiling of Ruthenium(II)-Based 4-Nitro- And 4-Amino-1,8-naphthalimide Conjugates

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Synthesis, Characterization, and Biological Proﬁling of

Ruthenium(II)-Based 4‑Nitro- and 4‑Amino-1,8-naphthalimide

Conjugates

Robert B. P. Elmes,* Gary J. Ryan, Maria Luisa Erby, Daniel O. Frimannsson, Jonathan A. Kitchen,

Mark Lawler, D. Clive Williams, Susan J. Quinn,* and Thor ﬁ nnur Gunnlaugsson *

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ABSTRACT: We report the synthesis, photophysical character-

ization, and biological evaluation of four DNA-binding ruthenium-

(II) polypyridyl 4-nitro- and 4-amino-1,8-naphthalimide con-

jugates. A meta arrangement around the ring connecting the 1,8-

naphthalimide to a bipyridine ligand creates a cleft, the result of

which renders the shape of the complex complementary to that of

DNA. We have demonstrated that each complex exhibits water

solubility and a distinctive set of photophysical properties that has

allowed the nature of their interaction with DNA to be probed by

various ground- and excited-state titrations. Furthermore, by

varying the ancillary ligands, we also demonstrate their ability to

act as DNA photocleavers, where all compounds have been found

to cleave supercoiled DNA with high eﬃciency. Detailed cellular

uptake experiments revealed that the conjugates accumulate in the cytoplasm and nucleus of HeLa cells, showing characteristic red

metal-to-ligand charge-transfer emission, and also exhibit photoactivated cytotoxicity within the cells upon irradiation at 450 nm. A

comparison between the meta and para arrangements of the 1,8-naphthalimide moiety relative to the Ru(II) center suggests

increased DNA binding in the case of the meta arrangement; however, bipyridine−4-amino-1,8-naphthalimide conjugates appear to

show superior phototoxicity in comparison to their 4-nitro derivatives.

showing water solubility, photostability, reduced dark toxicity,

useful spectroscopic properties, and signiﬁcant DNA binding

aﬃnity.³¹⁻³⁶However, unlike classical PDT agents, ruthenium-

(II) polypyridyl complexes can also initiate apoptosis in cells

by other means than through singlet oxygen activation, as

recently outlined by us and others in the ﬁeld.³⁷⁻⁴²In fact,

often more than one activation pathway is available to such

potential therapeutics, which makes ruthenium(II) polypyridyl

complexes highly versatile and exciting therapeutic agents for

cancer and other diseases. The study of such agents in vitro

and in vivo has been extensively featured in the review by

Poynton and co-workers among others.^7,43−46

INTRODUCTION

■

Ruthenium(II) polypyridyl complexes have been extensively

studied in recent years for the most part because of their rich

photophysical characteristics, which enable their application in

diverse scientiﬁc disciplines such as solar energy conversion,^1,2

photocatalysis,^3,4molecular machines,^5,6and biological imag-

ing,⁷⁻⁹to name just a few key areas. With their cationic nature

and well-deﬁned spatial geometry, ruthenium(II) polypyridyls

are well-known to bind to nucleic acids and, through

modulation of their photophysical properties, are capable of

reporting on the binding event.¹⁰⁻¹⁵Several research groups

have contributed to a detailed understanding of the binding of

such complexes to oligonucleotides through combinations of

various spectroscopic methods and biochemical investiga-

tions.¹⁶⁻²²This work has also provided detailed mechanistic

insight into their photochemistry with oligonucleotides that

often result in photocleavage of DNA through oxidation and/

or photoadduct formation.²³⁻²⁵Indeed, the inherent excited-

state reactivity of π-deﬁcient ruthenium(II) polypyridyls has

led to increasing interest in ruthenium(II) polypyridyl

complexes as potential photodynamic therapy (PDT)

agents.²⁶⁻³⁰Such ruthenium(II) complexes display many

properties that make them a promising class of PDT agents,

Our interest in ruthenium(II) polypyridyls has focused on

novel ruthenium(II) conjugates, where tethering the metal

center to various other functional subunits such as gold

Received: May 12, 2020

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Figure 1. Novel ruthenium(II) 1,8-naphthalimide conjugates 1−4 and previously reported complexes 5 and 6.⁵³

49,50

nanoparticles,^47,48Troger’s bases,

and appended organic

sensors.⁵⁹⁻⁶¹However, their biological proﬁling has demon-

strated that minor structural changes can have a signiﬁcant

impact on their biological properties. Hence, with this is mind,

we set about developing another family of ruthenium(II) 1,8-

naphthalimide conjugates by (a) modifying the structure from

a para-substituted “linear” arrangement to a meta-substituted

“wedged” orientation and (b) replacing the bipyridine ligand

with the 1,4,5,8-tetraazaphenanthrene (TAP) ligand (Figure 1,

1 and 2), which become highly oxidizing upon visible

excitation.^62,63These modiﬁcations were expected to eﬀect

increased DNA binding aﬃnity and DNA photocleavage

eﬃciency by placing the metal center and activated TAP

ligands in the optimal positions to ensure a tight ﬁt in the DNA

helix.

Herein we report our ﬁndings where we have synthesized

four novel ruthenium(II) 1,8-naphthalimide conjugates 1−4

(Figure 1). As eluded to above, it was expected that the 1,8-

naphthalimide would contribute to high-aﬃnity DNA binding

through intercalation, while the Ru(II) center should

contribute to the interaction through electrostatic association

with the double helix or insertion into the grooves of DNA.

Our design would make use of a rigid aromatic group as the

linking moiety, allowing for control of the orientation of the

components of the complex with respect to each other, where a

meta arrangement around the connecting ring with respect to

the Ru(II) center was chosen to compare the binding aﬃnities

with those of previously reported structures 5 and 6 and to

investigate the eﬀect of substitution on their DNA binding

̈

chromophores⁵¹⁻⁵³has yielded various modiﬁed designs that

display high-aﬃnity DNA targeting, enhanced photophysical

properties, and eﬀective DNA photocleavage that we have

shown can aﬀect numerous biological pathways and initiate

apoptosis. We have also investigated the various binding

modes of metal-ion complexes with oligonucleotides using

ultrafast spectroscopy such as transient IR and transient

absorption.^19,23Recent reports point to the importance of the

binding mode of the metal center to DNA to enable the most

eﬃcient photoreaction and thus DNA cleavage.^19,37Indeed,

the number of attached chromophores and the nature of the

linker between the metal center and appended organic groups

appear to strongly aﬀect the subsequent photoreactivity.⁵²An

example of this was previously reported in the case of rigid

linear ruthenium polypyridyl complexes with a 4-nitro- or 4-

amino-1,8-naphthalimide appended group; see Figure 1 (5 and

6).⁵³These complexes were found to cleave plasmid DNA

upon visible-light excitation, where the substituent was found

to determine the extent of DNA cleavage with the nitro

derivative, resulting in the nicking of DNA, while the amino

derivative resulted in the formation of nicked and linear DNA.

The 4-nitro- or 4-amino-1,8-naphthalimide compounds are

in their own right important therapeutics, as we and others

have shown in the past, being able to induce apoptosis and

other biological processes.⁵⁴⁻⁵⁸The internal charge-transfer

excited state of the amino version has also been extensively

exploited in the development of “oﬀ−on” switches and

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a

Scheme 1. Complexation of Ligand 7 with Ru(TAP)₂Cl₂and Reduction of the Resulting Complex

a

Reagents and conditions: (i) Ru(TAP)₂Cl₂, DMF/H₂O, argon; (ii) MeOH, Pd/C, 1 atm of H₂.

−

Figure 2. (a) View of the molecular structure of the cation of 6. Interstitial CH₃CN molecules and PF₆are removed for clarity and ellipsoids

shown at 50% probability. (b) Ball-and-stick representation of the cation of 3 as determined by crystallography.

aﬃnity. In addition, the attached 1,8-naphthalimide structures

should serve to further increase the excited-state reactivity,

while simultaneously providing a known set of photophysical

properties that could be used to monitor the binding process.

For compounds 1 and 2, it was expected that the inclusion of

two TAP ligands in their design would serve to increase the

DNA cleavage eﬃciency of these systems and, moreover, to

provide a direct comparison between the Ru(bpy)₂(L) and

Ru(TAP)₂(L) systems (L = bipyridine−1,8-naphthalimide

conjugate).

95% and 98% yield, respectively, using Pd/C under a H₂

atmosphere (exempliﬁed in Scheme 1 for 1 and 2).

Synthesized as their chloride salts by stirring a solution of

the complexes in methanol (MeOH) with an Amberlite ion-

exchange resin, 1−4 were fully water-soluble. The successful

1

synthesis of compounds 1−4 was conﬁrmed by H and ¹³C

NMR, high-resolution mass spectrometry (HRMS), and

elemental analysis (see below and the Supporting Information

1

for further details). The H and ¹³C NMR spectra showed

well-resolved resonances for the polypyridyl as well as

naphthalimide components. HRMS showed the expected

isotopic distribution patterns, and elemental analysis conﬁrmed

that the complexes were obtained as pure materials. Moreover,

as stated above, complexes 5 and 6 had previously been

developed in our laboratory and were remade for comparison

purposes here. Their characterization matched that of our

previously published work.⁵³

In the case of 3 and 6, crystals that were found to be suitable

for X-ray diﬀraction analysis were grown upon slow

evaporation. Unfortunately, the data set collected for 3 was

of poor quality and only allowed for connectivity to be

established. Small red platelike crystals of 6 were obtained by

evaporation from acetonitrile (MeCN), and the low-temper-

ature (117 K) structure was determined. The structure of 6 is

shown in Figure 2a, having crystallized in the triclinic space

RESULTS AND DISCUSSION

■

Synthesis of Napthalimide−Polypyridine Ligands

and Ruthenium(II) Complexes. The formation of the

complexes 1−4 involved the synthesis of the conformationally

restricted bipyridine-1,8-naphthalimide ligand 7, which was

synthesized according to a modiﬁed procedure originally

reported by Johansson et al.⁶⁴(see the Supporting

Information), before reaction with Ru(bpy)₂Cl₂or Ru-

(TAP)₂Cl₂using a N,N-dimethylformamide (DMF)/water

(H₂O) mixture was conducted under microwave-assisted

conditions. This yielded 1 in 52% yield and 3 in 75% yield

after puriﬁcation using (ﬂash) column chromatography,

followed by counterion exchange and crystallization (see the

Experimental Section and further details in the Supporting

Information). Reduction to the corresponding 4-amino-1,8-

naphthalimide analogues 2 and 4 was successfully achieved in

̅

group P1 and containing one molecule of 6, one full

occupancy, and a half-occupancy interstitial CH₃CN molecule

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in the asymmetric unit. The Ru(II) center in 6 is coordinated

by two unsubstituted 2,2′-bipyridine molecules and one

molecule of 7, giving an overall N6 coordination sphere that

adopts a distorted octahedral geometry (S = 71°). Bond

lengths [2.068(5)−2.085(5) Å] and angles [cis bond angles =

78.9(2)−97.4(2)°; trans bond angles = 171.1(2)−174.1(2)°]

to the Ru(II) center are typical for RuN6 complexes. The

solvent mask routine in OLEX2 was required to mask the

−

electron density from one severely disordered PF₆counter-

anion. The amino group attached to the 4 position of 1,8-

naphthalimide is disordered over two sites with relative

occupancies of 0.65 and 0.35 (a common feature for 4-

substituted 1,8-naphthalimide compounds). The ability of

naphthalimide groups to be involved in π-based interactions is

well-known, and within this complex, the naphthalimide

moiety is involved in a strong anion···π interaction with the

−

PF₆counterion (centroid···F2 = 3.322 Å). Interestingly,

unlike other naphthalimide-containing complex structures,^65,66

no immediately obvious structure extension inﬂuences (e.g.,

π−π stacking between neighboring complexes, formation of π-

stacked layers, etc.) are arising from the naphthalimide moiety.

Small, poor-quality crystals of 3 were obtained as red blocks;

however, the diﬀraction data were not of suﬃcient quality to

allow anything other than atom connectivity to be reported.

The connectivity is similar to that of 6, where the Ru(II)

center is coordinated by two unsubstituted 2,2′-bipyridine

molecules and one molecule of 3, giving an overall N6

coordination sphere with octahedral geometry (Figure 2b).

Photophysical Characterization. Having successfully

synthesized the Ru(TAP)₂complexes 1 and 2 and the

Ru(bpy)₂complexes 3 and 4, their spectroscopic properties

were investigated in 10 mM phosphate-buﬀered aqueous

solutions at pH 7.4. Examination of the UV/vis absorption

spectrum of 1 (Figure 3a) revealed absorption bands

characteristic of both the ruthenium(II) polypyridyl and 4-

nitro-1,8-naphthalimide chromophores, where the intense

band centered around 275 nm was attributed to π−π*

intraligand transitions, the band at 358 nm to the 1,8-

naphthalimide π−π* transitions, and the band centered at 415

nm to the metal-to-ligand charge-transfer (MLCT) transitions

of the ruthenium(II) polypyridyl center. Similar bands were

observed for 3 at 287, 351, and 457 nm, respectively. Each

complex displayed two MLCT transitions that are similar to

their parent complexes Ru(TAP)₂(bpy)²⁺(412 and 465 nm)

Figure 3. UV/vis absorption and ﬂuorescence excitation and emission

spectra of (a) 1 and (b) 2 (10 μM) in 10 mM phosphate buﬀer at pH

7.4.

In the case of the 4-nitro-1,8-naphthalimide-containing

complexes 1 and 3, excitation of the naphthalimide absorbance

bands did not yield naphthalimide-based emission but resulted

in MLCT-based emission at 645 and 625 nm, respectively,

which is similar to that observed for the parent complexes,

Ru(TAP)₂(bpy)²⁺(649 nm) and Ru(bpy)₃²⁺(617 nm).⁶⁷The

absence of 1,8-naphthalimide-associated emission suggested

that sensitization of the MLCT excited state occurred through

an energy-transfer process, in a manner similar to that of

complexes that we have previously described.⁶⁸The MLCT

origin of emission for 1 was conﬁrmed by the excitation

spectrum recorded for the emission at 645 nm (Figure 3a, red

trace), where, in contrast to the absorption spectrum, there is

an absence of the naphthalimide band. Excitation of 2 and 4 at

425 or 450 nm resulted in similar behavior, in that no emission

was observed from 1,8-naphthalimide, with emission bands

centered at 645 and 625 nm for the MLCT emission. The

MLCT origin of emission was also conﬁrmed for 2 by the

excitation spectra recorded for the emission at 625 nm, which

closely mirrored the absorption spectrum (Figure 3b, red

trace). This again demonstrates a very eﬃcient energy transfer

from the naphthalimide excited state to the Ru(II)-based

MLCT state. The Φ_Fvalue for 1 was found to be 0.022

( 10%), which is approximately half that reported for

[Ru(TAP)₂(bpy)]²⁺but higher than that reported for the

Ru(bpy)₂analogue 3, which exhibited a value of 0.001.^67,68

Conversely, the Φ_Fvalue for 2 was found to be 0.004 ( 10%),

a value lower than that described for its Ru(bpy)₂analogue 4,

which was reported as 0.018. The reduction in the quantum

yields, and the resulting weak emission displayed by these

complexes, is attributed to an interaction between 1,8-

2+

and Ru(bpy)₃(424 and 452 nm) (see the Supporting

Information).⁶⁷Notably, bands characteristic of 1,8-naphtha-

limide and Ru(II)-based MLCT were resolved. This is

important because it allows for direct addressing of both

parts of the complex in a semiselective manner, and as such,

this is a useful property to examine the interaction and

contribution of both components upon binding of the

complexes to DNA. The absorption properties of the amino

derivative Ru(TAP)₂complex 2 were shown to be somewhat

diﬀerent from those of 1 (Figure 3b). The band at 275 nm was

still present, although its molar absorptivity was slightly

increased; a broad band was observed at 425 nm

corresponding to both the 4-amino-1,8-naphthalimide and

Ru(II)-based MLCT transitions.

Complex 4 showed behavior similar to that observed for 2,

with slightly shifted maxima at 287 and 451 nm (see the

Supporting Information). A summary of the absorption

properties of 1−4 is given in Table 1.

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Table 1. Absorption and Emission Properties of 1−4 in Aerated 10 mM Phosphate Buﬀer, at pH 7.4, at 298 K

λ

(nm) [ε (M⁻¹cm⁻¹)] in phosphate buﬀer ( 10%)

max

complex

π−π* IL

π−π* Naph

MLCT

λ

(nm)

Φ_f( 10%)

τ_em(ns) (air)

emission

1

2

3

4

275 [61250]

275 [65700]

287 [78159]

287 [53055]

358 [15300]

415 [15000]

425 [22900]

457 [14765]

451 [17502]

645

0.022

0.004

0.001

0.018

437

166

645

625

351 [18787]

naphthalimide and the MLCT triplet state. Similar phenomena

have been observed for Ru(TAP)₂−aminoquinoline conjugates

and were attributed to electron-transfer processes between the

Ru(TAP)₂center (chromophore) and organic moiety

(quencher).⁶⁹

We anticipate that a similar process is likely to be occurring

here, where the excited state of the metal complex is eﬃciently

quenched by electron transfer to the 1,8-naphthalimide moiety.

Interestingly, the quantum yield of amino-substituted 2 is

greatly reduced in comparison to the nitro derivative 1,

suggesting that aminonaphthalimide is a more eﬃcient

quencher than its nitro counterpart in the case of the

Ru(TAP)₂(bpy) systems. Conversely, for the Ru(bpy)₃

complexes, the quantum yield of amino-substituted 4 is greater

than the nitro derivative 3, suggesting that 4-nitro-1,8-

naphthalimide is a more eﬃcient quencher than its amino

analogue in this instance. Excited-state lifetime measurements

(τ_em) followed the same trend, where 1 and 2 showed τ_em

values of 437 and 166 ns, respectively, with the lower τ_emvalue

being exhibited by 2 because of its ability to eﬃciently quench

the MLCT-based emission. The main diﬀerence in the

emission properties between the bpy- and TAP-based

complexes originates from the diﬀerence in the level of bpy

and TAP π* orbitals. The TAP π* orbital is lower in energy

than that of the bpy ligand.⁶⁷This leads to the TAP complexes

being highly oxidizing in their excited state, inducing an

eﬃcient electron transfer from aminonaphthalimide. The

variation in the reduction potential of 4-amino-1,8-naphthali-

mide (more easily oxidized) compared with the 4-nitro species

results in more eﬃcient quenching of the excited state by 4-

aminonapthalimide by electron transfer, thus resulting in an

increased ﬂuorescence quantum yield being displayed by 1.

The λ_max, τ_em, and Φ_Fvalues for 1−4 are summarized in Table

1.

Figure 4. (a) Changes in the UV/vis spectrum of 1 (8 μM) with

increasing additions of stDNA (0−210 μM). Inset: Plot of (ε_a− ε_f)/

(ε_b− ε_f) versus [DNA] (M⁻¹, P) using data with P/D values between

0 and 12 and the best ﬁt of the data (red ---) using the Bard equation.

(b) Changes in the UV/vis spectrum of 3 (6.9 μM) upon the addition

of stDNA (0−21.39 μM base pairs). Inset: Plot of (ε_a− ε_f)/(ε_b− ε_f)

versus equivalents of DNA and the corresponding nonlinear ﬁt. All in

10 mM phosphate buﬀer at pH 7.4.

DNA Binding Studies: Absorption Titrations. On the

basis of our previous work on Ru(bpy)₃−4-nitro- and −4-

amino-1,8-naphthalimide conjugates, it was expected that 1−4

would interact strongly with DNA, resulting in signiﬁcant

modulations of their photophysical properties.⁵³Due to their

bifunctional nature, it was also expected that the complexes

would interact with DNA through a combination of electro-

static and π-stacking interactions, where the metal complex

should bind externally to the phosphate backbone, while the

1,8-naphthalimide moieties would bind through intercalative

or groove binding interactions. To determine the nature of

their binding aﬃnity for DNA, UV/vis absorption spectrosco-

py was ﬁrst utilized to probe the interaction of 1−4 with DNA.

Titrations were carried out by the addition of small aliquots of

stDNA to a 10 mM phosphate buﬀer solution of each complex

at pH 7.4, until a plateau in the absorbance was reached.

examples, the changes in the ground state of 1 and 3 are shown

in Figure 4 (the results for 2 and 4 are shown in the

Supporting Information). The titration of 1−4 with stDNA

resulted in signiﬁcant changes to their absorption spectra.

Complex 1 exhibited a 37% decrease in absorbance at 358 nm

with a concomitant decrease of 6% in the MLCT band at 415

nm. Similarly, 3 exhibited a 45% decrease in absorbance at 351

nm with a concomitant decrease of 21% in the MLCT band at

457 nm. The absorbance changes displayed by 2 culminated in

a 22% decrease in the band centered at 425 nm, while 4

exhibited a 29% hypochromism of the MLCT band. The

deﬁned hypochromicities observed in the ground-state spectra

of all complexes immediately indicate strong interactions

occurring with DNA. In the cases of 1 and 3, the large degree

of hypochromicity seen in the π−π* bands suggests

intercalation of the 1,8-naphthalimide moiety between the

stacked bases, as expected with such planar structures.

3

Changes in the ruthenium(II) complex MLCT band and in

the 1,8-naphthalimide π−π* band were monitored for 1 and 3,

while those changes at the broad band centered at 425 or 451

nm were monitored in the cases of 2 and 4. As representative

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Table 2. DNA Binding Parameters from Fits to Absorbance Data

complex

λ(Naph) hypochromism (%)

λ(MLCT) hypochromism (%)

binding constant K_b(M⁻¹

)

binding site size n (base pairs)

R²

1

2

3

4

5

6

37

6

22

21

29

15

30

1.3 × 10⁶( 0.1)

5.8 × 10⁶( 1.2)

1.9 × 10⁷( 0.6)

1.1 × 10⁷( 0.5)

4.5 × 10⁶( 0.7)

1.3 ( 0.03)

1.5 ( 0.03)

1.36 ( 0.02)

1.50 ( 0.06)

0.98 ( 0.02)

0.99

0.98

0.99

45

34

Similarly, the changes in the MLCT region conﬁrms that the

metal center of 1 and 3 is tightly bound to DNA. Because of

the overlap of the MLCT and naphthalimide bands of 2 and 4,

it is more diﬃcult to draw ﬁrm conclusions regarding the

separate portions of this system. However, the large

absorbance decrease observed was also coupled with a slight

red shift of the absorbance maxima in both cases, behavior

often associated with classical intercalation.⁷⁰

Analysis of the ground-state titration data using the Bard

equation⁷¹conﬁrmed that 1−4 show a strong aﬃnity for DNA

(summarized in Table 2). Complex 1 showed binding with K_b

= 1.3 × 10⁶( 0.1) and n = 1.3 ( 0.03), with 2 exhibiting

values of K_b= 5.8 × 10⁶( 1.2) and n = 1.5 ( 0.03). 3 gave K_b

= 1.9 × 10⁷( 0.1) and n = 1.36 ( 0.02), while 4 showed K_b=

1.1 × 10⁷( 0.7) and n = 1.5 ( 0.06). From UV/vis

absorption studies, it is clear that 1−4 bind DNA with high

aﬃnity, having binding constants on the order of 10⁶−10⁷M⁻¹.

Furthermore, the nature of the interaction with the binding

aﬃnity for DNA appears to be sensitive to the arrangement of

the ruthenium(II) and 1,8-naphthalimide components around

the connecting ring, where the meta arrangement results in a

more intimate association of the Ru(bpy)₂complexes 3 and 4

in comparison to the para derivatives 5 and 6 previously

reported [K_b= 4.5 × 10⁶( 0.7) and 3.0 × 10⁶( 1.0),

respectively].⁵³

The interaction of 1 and 2 with DNA at varying ionic

strengths was also examined using UV/vis absorption spec-

troscopy, where, at both 50 mM and 100 mM NaCl

concentrations, signiﬁcant spectroscopic changes were also

observed. Absorption decreases were observed in all cases (see

the Supporting Information) and conﬁrmed retention of a high

aﬃnity for DNA, even at very high NaCl concentrations. For

example, at 50 mM NaCl, 1 exhibited K_b= 7.4 × 10⁵( 0.9),

while at 100 mM NaCl, 1 had K_b= 4.3 × 10⁵( 1.8). The

overall study revealed an expected trend in the binding

aﬃnities of these complexes for DNA with K_bat 10 mM

phosphate buﬀer ≥ 50 mM NaCl ≥ 100 mM NaCl showing an

obvious eﬀect of the NaCl concentration on the binding

process (a summary of the results obtained are presented in

Table S1).

It is well-known that electrostatic DNA binding of

ruthenium(II) complexes is dependent on the ionic strength

of the medium.⁷²Under high salt conditions, we have observed

a decrease in the binding strength relative to that in 10 mM

phosphate buﬀer alone; nevertheless, because 1 and 2 retain a

high aﬃnity for DNA, even in 100 mM NaCl solution, it can

be concluded that, although electrostatic interactions have a

role to play in the binding process, it is the bifunctional nature

of these systems comprising an intercalating 1,8-naphthlimide

and a cationic Ru(II) center that governs the overall

association of these complexes with DNA. Previously reported

ruthenium(II) complexes that also display both electrostatic

and intercalative interactions with DNA exhibit binding

constants of a similar order of magnitude.^37,51,52

DNA Binding Studies: Emission Dependence Titra-

tions at Low Ionic Strength. In order to further conﬁrm this

interaction and monitor any excited-state redox processes, the

excited-state properties of 1−4 were also monitored as a

function of added DNA. As expected, the emission spectra of

all complexes studied were found to change dramatically in the

presence of increasing concentrations of DNA; however, the

results observed diﬀered signiﬁcantly in each case. The MLCT-

based emission of 1 in aqueous solution was shown to undergo

excited-state quenching, with a ca. 30% decrease being

observed upon the addition of stDNA (P/D of 0 → 30),

with the main changes occurring at P/D of 0 → 3. Similarly, 2

was also shown to have its MLCT-based emission quenched

but to a larger degree showing quenching of ca. 43% upon the

addition of stDNA (P/D of 0 → 30), with the main changes

occurring between a shorter P/D range of 0 → 2 (see the

Supporting Information).

The observed quenching of the excited state is most likely

due to photoelectron transfer between guanine residues and the

³MLCT state of the complexes, as previously described for

systems containing two or more TAP ligands.⁷³A biphasic

binding proﬁle with an immediate ﬂuorescence decrease

followed by a more gradual increase to a plateau over a P/D

range of 3 → 30 was observed for both complexes, which

suggests that these complexes distribute themselves diﬀerently

on the polynucleotide depending on the availability of binding

sites. The extra enhancement of luminescence quenching

might originate from DNA-induced stacking of the com-

plexes.⁷⁴For comparison, the emission proﬁles for each of the

systems, 1 and 2, are shown in the Supporting Information,

demonstrating the greater emission modulation experienced by

2 upon titration with DNA. This observation correlates well

with the results seen with the UV/vis absorption studies and

further reﬂects the high DNA binding aﬃnity of this system.

In a manner similar to that of the Ru(TAP)₂(bpy)

complexes, both Ru(bpy)₃complexes 3 and 4 underwent

two distinct phases of change in their emission proﬁles. 3

underwent an initial rapid decrease in the emission intensity

over a P/D range of 0 → 2 followed by a more gradual increase

to a plateau (P/D of 2 → 45), while the amino-functionalized

complex 4 also experienced two distinct phases of change. The

ﬁrst region in a P/D range of 0 → 2 is steep and represents

most of the intensity change before becoming more gradual,

reaching a plateau at approximately P/D = 30 with an overall

2.6-fold emission enhancement. This enhancement is likely

due to tight binding of 1,8-naphthalimide, which holds the

metal center in close proximity to the DNA, with subsequent

loss of quenching. Interestingly, the emission changes

displayed by complex 4 were signiﬁcantly greater than those

for its previously reported analogue 6.

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The increased enhancement that occurs for this system is

exempliﬁed in Figure 5, where the emission proﬁles of both

as described above, where a range of values were obtained for

K_bfrom ﬁts to the absorbance data (Table S2). The results

highlighted that 1 and 2 exhibited strong binding aﬃnity for

both [poly(dAdT)]₂and [poly(dGdC)]₂, as observed for

stDNA, but no major preference for either was observed (see

the Supporting Information).

The excited-state interactions of 1 and 2 with the

homopolymers were also monitored and saw substantial

diﬀerences between [poly(dAdT)]₂and [poly(dGdC)]₂,

3

where the characteristic MLCT emission bands centered at

645 nm were seen to experience an overall increase upon the

addition of [poly(dAdT)]₂, while, conversely, the emission was

seen to be eﬀectively quenched in the presence of [poly-

(dGdC)]₂. For comparison, the emission proﬁles for 1 in the

presence of stDNA, [poly(dAdT)]₂, and [poly(dGdC)]₂are

shown in Figure 6.

Figure 5. Comparison of the emission intensity changes for

2+

■

▲

●

Ru(bpy)₃(blue ), 4 (red ), and 6 (black ) upon the addition

of DNA in 10 mM phosphate buﬀer at pH 7.4.

complexes are compared with that of Ru(bpy)₃²⁺. This greater

enhancement may be related to the arrangement of the

constituents of the complex around the connecting ring. A

meta arrangement creates a cleft, the result of which renders

the shape of the complex more complementary to that of

DNA. Upon insertion of 1,8-naphthalimide into the helix, the

Ru(II) center in 4 is held more tightly bound to the DNA

backbone than that in 6 and, as a result, is more eﬀectively

protected from quenching. This result suggests that the meta

arrangement of the connecting ring in the bpy−1,8-

naphthalimide ligand is optimal to ensure a tight binding of

the Ru(II) metal center to the DNA backbone.

Figure 6. Relative changes in the integrated emission intensity of 1 (8

■

μM; λ_ex415 nm) with increasing concentrations of stDNA (red ),

◆

▲

[poly(dAdT)]₂(green ), and [poly(dGdC)]₂(blue ) in 10 mM

phosphate buﬀer at pH 7.4.

DNA Binding Studies: Emission Dependence Titra-

tions at High Ionic Strength. The susceptibility of the DNA

binding aﬃnity of 1 and 2 to the ionic strength was also

investigated by ﬂuorescence titrations at varying concen-

trations of NaCl (50 and 100 mM), and as seen above from

UV/vis absorption titrations, the overall emission modulations

upon the addition of DNA decrease as a function of added

NaCl. Salt back-titrations were carried out to exemplify the

eﬀect of increasing NaCl concentration (see the Supporting

Information). The ﬂuorescence emission was seen to be

aﬀected by increasing NaCl concentration, gradually increasing

toward that of the free compound but reaching a plateau at ca.

140 mM NaCl, with about a 40% increase toward that of the

free species. No further changes occurred with the addition of

up to 250 mM concentration of NaCl, indicating that the

majority of the compound remains bound even at high ionic

strength. These results give further conﬁrmation that the DNA

binding strength is dependent on the ionic strength of the

medium for complexes of this type, but even at very high NaCl

concentrations, the complexes were not fully displaced; this is

most likely due to 1,8-naphthalimide providing high aﬃnity

binding through intercalation into DNA.

For 1 in the presence of [poly(dAdT)]₂, an initial slight

decrease in emission was followed by a sharp 56% increase

culminating in a plateau at P/D = 10, while upon titration with

[poly(dGdC)]₂, the emission was eﬃciently quenched by 62%

at P/D = 10. Similarly, in the case of 2 upon titration with

[poly(dAdT)]₂, the observed ﬂuorescence at 645 nm was seen

to experience an initial slight decrease followed by a

subsequent 5% overall increase, with the main changes

occurring at P/D of 0 → 4. In a manner similar to that of 1,

upon the addition of [poly(dGdC)]₂, 2 was shown to have its

excited state eﬀectively quenched by 54%, with the main

changes occurring at P/D of 0 → 2. As previously mentioned,

because of the π-deﬁcient nature of the Ru(II) cores of 1 and

3

2, the observed quenching of the MLCT states upon the

addition of stDNA is expected to be as a result of

photoinduced electron transfer reactions with guanine

nucleobases, a well-documented behavior of complexes

containing at least two TAP ligands.^73,75,76The observed

luminescence enhancement in the presence of [poly(dAdT)]₂

for both 1 and 2 most likely results from shelter from

nonradiative deactivation processes of the ³MLCT excited

state aﬀorded by the double-helix microenvironment.⁷⁷

DNA Binding Studies: Thermal Denaturation Studies.

Given the above spectroscopic results, thermal denaturation

studies were also carried out on 1−4 in order to further

elucidate the interaction of these complexes with DNA. In the

DNA Binding Studies: Polynucleotide Binding. Studies

were also performed with synthetic polynucleotides, [poly-

(dAdT)]₂and [poly(dGdC)]₂, in order to investigate whether

any sequence-speciﬁc binding could be observed. The

interaction of 1 and 2 with both [poly(dAdT)]₂and

[poly(dGdC)]₂was measured ﬁrst using UV/vis absorption

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Figure 7. CD curves of (a) ct-DNA (150 μM) in 10 mM phosphate buﬀer at pH 7, in the absence and presence of 3 at varying ratios and (b) the

diﬀerence spectra obtained. (c) ct-DNA (150 μM) in 10 mM phosphate buﬀer at pH 7, in the absence and presence of 5 at varying ratios and (b)

the diﬀerence spectra obtained.

absence of a ruthenium(II) polypyridyl complex, the T_mvalue

for DNA was determined to be 68 °C, and in all cases, the

melting transition for DNA in the presence of the complexes

showed large perturbations at all P/D ratios evaluated (see the

Supporting Information). The changes reﬂected stabilization of

the helix structure as a function of the complex concentration.

At P/D = 25, 10, and 5, the melting transition had not gone to

completion at 90 °C, and, consequently, it was not possible to

ﬁt the data to a sigmoidal function and, as such, exact melting

values could not be reported but may be regarded as being

greater than 75 °C. It is noteworthy that the amino complex 2

resulted in a slightly greater stabilization of DNA compared

with complex 1. This result is complementary to those results

from the UV/vis absorption and emission studies and

emphasizes the higher aﬃnity of 2 in comparison to the

nitro analogue 1.

The diﬀerent extents of DNA stabilization between the

linear and wedged systems were also demonstrated with these

experiments. The nitro complex 3 resulted in a signiﬁcantly

greater shift to higher temperature in the melting proﬁle

compared with its linear analogue 5, while the shifts of the

amino derivatives 4 and 6 to DNA were less obvious. However,

in both cases, the wedged systems result in greater DNA

stabilization than the linear analogues. This result is

complementary to those from the UV/vis absorption and

emission studies and emphasizes the importance of this minor

structural modiﬁcation, from a linear to a wedged orientation,

where the arrangement of the ruthenium(II) and 1,8-

naphthalimide components around the connecting ring has a

profound eﬀect on the DNA binding aﬃnity.

DNA Binding Studies: Circular (CD) and Linear

Dichroism (LD). CD titrations were carried out on Ru(bpy)₂

complexes 3−6, in order to further investigate the inﬂuence

that the arrangement of the ruthenium(II) and 1,8-

naphthalimide components around the connecting ring has

on DNA binding. The spectra obtained for the nitro complexes

3 and 5 are depicted in Figure 7.

For complex 3, a small induced circular dichroism (ICD)

signal is observed at long wavelength corresponding to the

MLCT absorption, in addition to large changes in the DNA

region, attributable to both changes in the CD signal of the

DNA itself and ICD of the metal complex in this region. These

results are indicative of association of the metal center in the

grooves, which results in an ICD signal.⁷⁸In contrast, no

changes were seen for the 1,8-naphthalimide moieties in these

complexes. Signiﬁcantly diﬀerent CD behavior was exhibited

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Figure 8. LD spectra of stDNA (150 μM) in 10 mM phosphate buﬀer, at pH 7.4, in the absence and presence of (a) 1 and (b) 2 at varying ratios.

by the linear nitro complex 5, where changes at the MLCT

band were similar to those observed for 3. However, these

were concomitant with changes in the region of absorption of

1,8-naphthalimide. From examination of the 300−400 nm

region, two bands became apparent: a positive signal at 378 nm

and a negative signal at 340 nm with a crossover at 355 nm.

This type of behavior suggests the formation of dimers or

higher-order complexes by stacking in the grooves or externally

to the double helix. Such results have previously been observed

for species such as methylene blue.⁷⁹It is likely that the

absence of CD signals in this region for complex 3 is due to the

diﬀerent orientations of the 1,8-naphthalimides when stacked

within the DNA helix. The two amino derivatives 4 and 6

displayed CD behavior similar to each other, where the most

signiﬁcant feature observed was growth of a band in the region

of absorption of the metal center. Large changes were also

observed in the region of absorption of DNA. The overall

results point to a binding mode in which the metal center is

tightly associated with the helix, thus experiencing its chirality.

LD studies can also provide strong evidence for the binding

mode of metal complexes to DNA, and, hence, LD titrations

with complexes 1 and 2 were carried out in order to gain better

insight into their speciﬁc interaction with the DNA helix.

Again, the behavior of 1 was of particular interest because it

showed distinct absorption bands for the 1,8-naphthalimide

and ruthenium(II) polypyridyl portions of the structure.

Consequently, it was expected that its LD behavior would

give valuable information on the binding geometry of each of

the separate functional moieties. The LD spectra resulting

from the addition of 1 to stDNA and a comparison with its

absorption spectrum are shown in Figure 8a, with those of 2

shown in Figure 8b. Changes are immediately obvious outside

of the DNA absorption region in the case of both complexes,

further conﬁrming their strong interaction with DNA in

addition to giving information on the speciﬁc mode of

interaction.

band is seen to be split in two, with a positive band centered at

ca. 415 nm and a negative band with a maximum at ca. 475

nm. This behavior would suggest that the positive band at 415

nm is a result of MLCT from the Ru(II) center to the ancillary

TAP ligands lying in the grooves of the DNA helix. Conversely,

the negative MLCT band at 475 nm may be associated with an

MLCT from the Ru(II) center to the bipyridine fragment of

the naphthalimide ligand, further conﬁrming the proposed

intercalative geometry of the 1,8-naphthalimide portion.

Similar behavior was seen for 2, whereby a negative signal is

observed at 460 nm at P/D = 25, and this negative band

exhibits a further decrease at P/D = 2.5. While it is more

diﬃcult to draw ﬁrm conclusions for 2 because of the overlap

of both the 1,8-naphthalimide and MLCT absorption bands,

the negative band centered at ca. 460 nm suggests that the 1,8-

naphthalimide portion of this conjugate is, again, most likely

intercalated between the nucleobases, as was seen for 1.

Considered with the other spectroscopic studies presented so

far, it is likely that, in all cases, the DNA binding involves the

insertion of 1,8-naphthalimide into the helix, with tight

association of the metal center, through external binding or

partial insertion into the grooves. The lower aﬃnity observed

for the −NO₂-substituted complexes is likely to be due to

steric repulsion caused by the nitro group, which can be

twisted slightly out of plane because of steric repulsion from

the neighboring H atom in the 5 position of the ring system.

The resultant diminished planarity makes intercalation or

insertion into the DNA helix less favorable for the nitro-

substituted species, while the amino-substituted complexes do

not experience this type of twisting, and as such its presence

may allow for more complete insertion into the helix, resulting

in stronger binding aﬃnity.⁸⁰

DNA Photocleavage Experiments of 1−4. Having

clearly identiﬁed that 1−4 bind avidly to DNA while displaying

varying spectroscopic behavior, it was anticipated that each

complex, in addition to binding to DNA, may show

photoreactivity with the DNA bases, as has previously been

observed for 5 and 6 and various other ruthenium(II)

polypyridyl complexes.^52,53In order to probe this behavior,

DNA photocleavage experiments were carried out by treating

pBR322 plasmid DNA (1 mg/mL) with each of the complexes

1−4 at varying ratios in a 10 mM phosphate buﬀer solution

before exposure to light irradiation. As shown in Figure 9, the

In the case of 1, when stDNA was treated with a low loading

of the complex (P/D = 25), a small negative signal is evident at

ca. 360 nm, suggesting that the 1,8-naphthalimide portion of

the complex is lying perpendicular to the DNA helix axis, i.e., is

intercalated. This observation is further emphasized upon

higher loading of 1 (P/D = 2.5), whereby a more strongly

negative signal is observed at ca. 365 nm, with the maximum

being slightly red-shifted by 5 nm. More complex behavior is

seen in the MLCT absorption region of 1, where the broad

2+

presence of Ru(bpy)₃(P/D = 20) resulted in an increase to

40% open form after irradiation, conﬁrming its eﬀectiveness as

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1

presence of the O₂scavenger sodium azide (NaN₃) showed

2+

that the photocleavage eﬃciency of Ru(bpy)₃is almost

1

completely inhibited by the presence of the O₂scavenger

while the eﬃciency of the nitro-substituted complex 3 or 5

appeared insensitive to the presence of NaN₃, suggesting that

damage mediated by singlet oxygen was not important for

these derivatives.

The amino-substituted conjugates 4 and 6 were shown to be

moderately sensitive to the presence of the singlet oxygen

scavenger, with both inducing approximately the same amount

of DNA damage in the absence of NaN₃, although it was

reduced. Nevertheless, in the presence of NaN₃, both 4 and 6

still displayed substantially improved cleavage eﬃciency over

the reference Ru(bpy)₃²⁺. To further investigate the involve-

Figure 9. Agarose gel electrophoresis of pBR322 DNA (1 mg/mL)

after 30 min of irradiation (2 J/cm²) in 10 mM phosphate buﬀer, at

pH 7. 4. Lane 1: plasmid DNA control. Lane 2: Ru(bpy)₃(P/D =

20). Lanes 3 and 4: 1 and 2 in the dark (P/D = 20). Lanes 5 and 6: 1

and 2 (P/D = 20). Lanes 7 and 8: 3 and 4 (P/D = 20). Lanes 9 and

10: 1 and 2 + NaN₃(P/D 20).

2+

1

ment of O₂in the DNA photocleavage process, studies were

a DNA photocleaver. Importantly, upon incubation in the

dark, none of 1−4 showed any dark-state DNA cleavage.

However, as can be seen in Figure 9, lanes 5 and 6, irradiation

and subsequent electrophoresis of pBR322 DNA in the

presence 1 and 2 at a P/D ratio = 20 showed clear evidence

of eﬃcient DNA photocleavage, with 1 showing complete

conversion of the plasmid to open-form DNA and 2 exhibiting

an increase in the open form to 75%. Furthermore, compared

with the Ru(bpy)₂analogues 3 and 4, which showed 15% and

48% cleavage, respectively, both 1 and 2 were found to be

signiﬁcantly more eﬀective photocleavage agents. When

carried out in a D₂O solution where reactive oxygen species

(ROS) has a longer lifetime and would be expected to induce

more eﬃcient damage of the DNA. Indeed, as demonstrated in

2+

Figure 10, Ru(bpy)₃cleavage was observed to be more

1

irradiated in the presence of the known O₂scavenger, NaN₃

(lanes 9 and 10), it was observed that the photocleavage

eﬃciencies of 1 and 2 were not reduced. The relative amounts

of supercoiled versus open-form DNA are summarized in

Table S3.

From the above results, it is clear that the Ru(TAP)₂(bpy)-

1,8-naphthalimide derivatives 1 and 2 display DNA photo-

cleavage with eﬃciencies greater than those of [Ru(bpy)₃]²⁺

and the Ru(bpy)₃-1,8-naphthalimide derivatives 3 and 4. In

addition, it appears that the nitro derivative 1 is a more

eﬀective photocleaver than its amino-substituted analogue 2,

most likely because of its increased Φ_Fvalue as described

above and the opposite trend seen for the Ru(bpy)₃-1,8-

naphthalimide derivatives. Moreover, the photocleavage

eﬃciencies of 1 and 2 were not aﬀected to any great extent

Figure 10. Eﬀect of irradiation in a D₂O solution on the

2+

■

photocleavage eﬃciency of Ru(bpy)₃(red ), 3 (purple ), 4

■

(green ), 5 (blue ) and 6 (black ). Solid bars represent cleavage

in a H₂O solution and hatched bars cleavage in a D₂O solution.

1

when irradiated in the presence of NaN₃, suggesting that O₂

eﬃcient in a D₂O solution than in a H₂O solution. Similarly,

complex 5 displayed an enhancement in the photocleavage

formation may not be the primary mechanism by which these

complexes exert their activity. Detailed biological proﬁling of 1

and 2 is ongoing and will be presented in a subsequent study;

however, given the results presented above, it was of interest to

gain further insight into the eﬀect of the arrangement of the

ruthenium(II) and 1,8-naphthalimide components around the

connecting ring. Thus, below we present more detailed studies

carried out on Ru(bpy)₃-1,8-naphthalimide derivatives 3−6.

More Detailed DNA Photocleavage Experiments of

3−6. The orientation of the components within these systems

was shown to have interesting consequences on the DNA

binding behavior; thus, it was expected that this would also be

reﬂected in the cleavage studies. Irradiation of DNA in the

absence and presence of each of the complexes, in a 10 mM

phosphate buﬀer solution, showed the nitro-substituted

conjugates 3 and 5 displaying poorer photocleavage eﬃciency

than their amino-substituted analogues 4 and 6. Some minor

diﬀerences were also apparent between the corresponding

linear and wedged derivatives, where slightly greater eﬃciency

was observed in both cases for complexes comprising a linear

arrangement of the 1,8-naphthalimide and ruthenium(II)

components (e.g., 5 and 6). Studies carried out in the

1

eﬃciency in the presence of D₂O, implicating O₂in the

mechanism of damage induced by the complex. This was in

contrast to the results obtained with the addition of NaN₃to

1

this system, where no sensitivity to the added O₂scavenger

was observed. Complex 3 displayed, however, no enhancement

in the cleavage eﬃciency in a D₂O solution.

Both 4 and 6 displayed improved photocleavage eﬃciency of

DNA in a D₂O solution, giving a quantitative conversion. This,

in conjunction with the results in the presence of NaN₃,

implicates ROS as important reactive species in the damage

induced to DNA by these complexes. 4 and 6 displayed

signiﬁcantly greater cleavage eﬃciency compared with the

reference Ru(bpy)₃²⁺, emphasizing the importance of the 1,8-

naphthalimide unit in these systems. In summary, the above

results demonstrate that complexes 3−6 display useful

photocleavage properties, whose eﬃciency depends largely

on substitution of the 1,8-naphthalimide moiety; the nitro-

substituted complexes 3 and 5 display cleavage eﬃciencies of

the same order as Ru(bpy)₃²⁺. The linear derivative 5 seemed

to display some dependence on ROS formation to exert

damage, whereas its wedged analogue 3 did not. Both of the

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■

▲

▽

Figure 11. Cellular uptake of 3−6 by K562 cells. Cells were treated with 5 (1.0 μM, black ), 6 (1.0 μM, red ), 3 (1.0 μM, green ), and 4 (1.0

◆

μM, blue ) for various times at 37 °C in the presence of 5% CO₂. Each point at the graph represents a mean from three separate experiments.

amino-substituted complexes 4 and 6 showed improved

cleavage eﬃciency over the reference Ru(bpy)₃²⁺. It was

expected that this was due to the high binding aﬃnity of these

systems for DNA, which places the metal center in close

proximity to the helix, whereby it may eﬀectively induce

damage. The damage to DNA caused by 4 and 6 was also

shown to involve ROS.

Having investigated the DNA binding and photocleavage

properties of 3−6 in detail and discovering that the

arrangement of the ruthenium(II) and 1,8-naphthalimide

components around the connecting ring has a profound eﬀect

on the binding mode and aﬃnity, we decided to also undertake

an investigation into their biological properties and ascertain

whether this small structural modiﬁcation might also lead to

diﬀering behavior in cellulo.

Cellular Uptake Experiments. Cellular uptake is of

paramount importance from the point of view of both

diagnostics and therapeutics, and, furthermore, access to

cellular DNA is important to the viability of 3−6 as a new

class of PDT agents. Our previous work and the work of others

have uncovered the ability of the ruthenium(II) polypyridyl

complexes to localize within cancer cells of various types in

vitro.^{7,26,30,44,81,82}With particular relevance to this study is the

observation that ruthenium(II) polypyridyl 1,8-naphthalimide

structural diﬀerence between the linear and wedged arrange-

ments in cellular uptake.

As shown in Figure 11, the diﬀerence in the emitted

ﬂuorescence becomes more evident when the maximum

ﬂuorescent intensity is plotted against time, where all

complexes were internalized within 9 h. Furthermore, the

maximum ﬂuorescence intensity was observed after 24 and 48

h and may be a consequence of high concentrations within the

cell or that these compounds are bound to a cellular target.

First, the uptake of each of the complexes into HeLa cells

was also veriﬁed by ﬂow cytometry studies, where at all time

points the amino-substituted complexes 4 and 6 displayed

similar intensities, suggesting that the uptake properties of

these two derivatives are quite similar in nature. Second, the

nitro-substituted complexes 3 and 5 displayed contrasting

uptake properties to each other, where the linear derivative 5

was observed to enter cells much more eﬀectively. Complex 5

was found to be the most readily taken up by the HeLa cells

after 24 h of incubation. Complex 5 was weakly emissive in

solution, possessing a Φ_Fvalue of 0.004 in H₂O, in comparison

to the amino complexes 4 and 6, which possessed Φ_Fvalues of

0.019 and 0.018, respectively. Furthermore, 5 displayed a

smaller luminescent enhancement in the presence of DNA

than 4 and 6 did. Therefore, for complex 5 to give rise to such

a large increase in the ﬂuorescence intensity, it must have been

present in the cells at a much higher concentration. The

reduced intensity displayed by HeLa cells incubated with 3

may also be reﬂective of the emission properties of the system;

this complex possessed the lowest quantum yield at 0.001.

Therefore, the amount of this complex that accumulates in the

cells will be approximately the same as that for 5, with the

diﬀerence in the intensities resulting from the diﬀering

quantum yields of emission possessed by the complexes.

Overall, the nitro complexes 3 and 5 appear to enter both

K562 and HeLa cells more rapidly than their amino analogues

4 and 6, but, importantly, these studies showed that each of 3−

6 can be internalized by both cell types successfully.

̈

Troger’s bases (themselves synthesized from complexes 4 and

6) undergo rapid cellular uptake and display intense

luminescence inside cells within 2 h of administration.⁴⁹

Thus, the ability of 3−6 to enter cancer cells was also

investigated using a number of techniques.

Flow cytometry was initially used to investigate cellular

uptake where both adherent (HeLa) and nonadherent (K562)

cells were investigated. 3−6 were rapidly internalized by K562

cells, where their ﬂuorescence increased steadily as a function

of time (30 min → 48 h). The intracellular ﬂuorescence

intensity was shown to increase after 30 min in all cases, with

the exception of the wedged nitro derivative 3. The results also

indicated that the wedged derivatives 3 and 4 displayed slower

uptake at earlier time points (within 1 h), while cells treated

with the linear derivatives 5 and 6 showed a gradual linear

increase of ﬂuorescence that was time-dependent (see the

Supporting Information). This indicates the importance of the

Cellular Localization Experiments. Confocal ﬂuores-

cence microscopy was employed in order to provide visual

evidence of the localization of 3−6 in HeLa cells. The results

obtained are exempliﬁed in Figure 12, which shows the

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Figure 12. Bright-light and confocal laser scanning microscopy images of HeLa cells treated with 3−6 and stained with Hoechst 3342. Cells were

incubated with 3−6 at 10 μM concentration for 24 h before their nucleus was stained with Hoechst 3342 (0.5 μM) for 15 min. The left panel

shows the bright-light images of the investigated cells. The middle panel shows the Hoechst 3342 nuclear stain (Ex. 405 nm/Em. 470−500 nm).

The right panel shows compounds 3−6 (Ex. 405 nm/Em > 650 nm). Scale bars represent 10 μM.

ﬂuorescence confocal laser scanning microscopy images of

HeLa cells after incubation with 3−6. The observed images

show the majority of cells exhibiting intense red ﬂuorescence

associated around the nucleus. While a considerable amount of

ﬂuorescence arising from 3 was also detected in the cytoplasm,

the other derivatives were mainly shown to localize in the

nucleus, implied by colocalization staining with the nuclear

stain Hoechst 3342. Interestingly, the ﬂuorescence intensities

of 1 and 2 measured within the nucleus are somewhat lower in

intensity in comparison to that observed surrounding the

nuclear membrane. Such behavior may imply the occurrence of

photoredox processes between 1 and 2 and the nuclear DNA,

causing the emission intensity of these complexes to be

eﬀectively quenched, as was observed in vitro during the

excited-state DNA titrations (the corresponding images for 1

and 2 are shown in the Supporting Information). In summary,

these results indicate that the combination of a ruthenium(II)

polypyridyl center with a more lipophilic 1,8-naphthalimide

unit is eﬀectively internalized by cells, enabling them to

selectively localize inside the nucleus and bind to cellular DNA.

Cytotoxicity and Phototoxicity Studies. In order to

investigate the cytotoxic and phototoxic properties of 3−6,

MTT cytotoxicity assays were carried out on K562 cells at

various concentrations in both the absence and presence of a

varied light dose (2, 4, and 8 J/cm²). The results of the toxicity

studies are shown in Figure 13 and summarized in Table 3.

The wedged 4-nitro derivative 3 showed the highest

cytotoxicity in the dark, suggesting that its high DNA binding

aﬃnity may give rise to some dark toxicity. 4−6, however,

showed minimal dark toxicity, exhibiting EC₅₀values that were

higher than 100 μM. Conversely, as shown in Table 4, the

wedged amino derivative 4 displayed the highest phototoxicity

with an EC₅₀value of 66.5 μM upon 2 J/cm²irradiation. The

linear 4-amino derivative also showed a cytotoxicity increase

upon irradiation with 2 J/cm²(>100 μM → 86.5 μM);

however, no apparent changes were exhibited upon irradiation

with the 4-nitro derivatives 3 and 5. These results correlated

well with the results observed from the DNA photocleavage

experiments, where the 4-amino derivatives 4 and 6 showed

considerable photocleavage activity.

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Figure 13. In vitro cyto- and phototoxicity of 3−6 against K562 cells after 24 h of incubation postirradiation: (A) dark controls; (B) 2 J/cm²; (C) 4

2

◆

■

▲

▼

J/cm ; (D) 8 J/cm . Cells were treated with 3 (green ), 4 (blue ), 5 (black ), and 6 (red ).

a

Table 3. Summary of EC₅₀Values (μM) for 3−6 after Irradiation with Diﬀerent Light Doses after 24 h Postirradiation

3

4

5

6

0 J/cm²

2 J/cm²

4 J/cm²

8 J/cm²

71.2 ( 6.9)

74.0 ( 7.5)

63.6 ( 9.5)

69.6 ( 8.4)

>100

66.5 ( 9.2)

41.7 ( 2.3)

24.4 ( 3.0)

86.5 ( 8.3)

69.2 ( 3.3)

53.4 ( 4.0)

73.1 ( 4.4)

a

Each value represents a mean calculated from a single experiment in triplicate ( SEM).

Table 4. Cell Cycle Analysis of K562 Cells Untreated or Treated with 3−6 and Irradiated with 4 J/cm²

NT

NT + Irr

3

4

5

6

sub-G₁

G₁

S

5.01 ( 0.3)

52.3 ( 0.6)

24.7 ( 0.3)

15.6 ( 0.5)

5.71 ( 0.4)

48.9 ( 0.6)

26.6 ( 0.5)

17.1 ( 0.4)

5.74 ( 0.1)

46.1 ( 1.0)

27.1 ( 0.6)

20.9 ( 0.6)

10.1 ( 0.1)

40.7 ( 0.4)

25.0 ( 0.8)

22.4 ( 0.3)

8.63 ( 0.4)

40.6 ( 0.4)

29.9 ( 0.1)

21.2 ( 0.1)

8.64 ( 0.3)

32.1 ( 0.4)

26.0 ( 0.4)

32.0 ( 0.1)

G₂/M

Similar results were obtained with increased light dose,

where the phototoxicity was the highest for 4 and 6, while the

4-nitro derivatives showed a minimal increase even at 8 J/cm².

These results conﬁrm the light-activated therapeutic potential

of 4 and 6, where the 4-amino derivatives were highly eﬀective

against the growth of K562 cells upon photoactivation,

particularly with a 8 J/cm²light dose.

In order to further probe the mechanism of action of these

compounds and to determine whether ROS played a role in

their photoactivity, further ﬂow cytometry experiments were

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■

Figure 14. Summary of ROS production in K562 cells after treatment with 3−5. Cells were treated with 5 (10 μM, green ), 6 (10 μM, purple ),

■

3 (10 μM, blue ), and 4 (10 μM, red ) for 24 h before they were irradiated (open bars) or left in the dark (solid bars). Vehicle-treated cells

■

.

(NT) are indicated with purple , and cells treated with 2 mM H₂O₂are represented by a gray

2

■

Figure 15. Cell cycle analysis of K562 cells irradiated with (a) 4 J/cm and (b) 8 J/cm . Cells were either left untreated (blue ) or treated with 5

■

(10 μM, green ), 6 (10 μM, pink ), 3 (10 μM, light blue ), and 4 (10 μM, red ) for 24 h before irradiation. Cells were analyzed 48 h after

irradiation. Untreated, irradiated cells are indicated with a solid gray plot.

conducted to measure the production of ROS arising from

cells treated with 3−6 both in the dark and after photo-

activation. Dihydroethidium (DHEM) was used to investigate

the involvement of ROS, where it was expected that the

production of ROS would convert DHEM to ethidium, giving

rise to increased ethidium ﬂuorescence upon its intercalation

into DNA. A summary of the results is shown in Figure 14,

where all compounds exhibited a small increase in ROS

formation in the dark with the exception of compound 3

despite this being the most cytotoxic compound in the dark, as

was discussed above, and indicating that 3 might exert its

biological activity through a diﬀerent mechanism. A minimal

increase in ROS formation was observed in untreated cells

after 8 J/cm²light dose, but, most interestingly, the linear 4-

amino derivative 6 showed the highest increase, while the

corresponding wedged 4-amino derivative 4 showed the

second-highest ROS formation. These results correlate well

with the phototoxicity analyses discussed above and may

suggest that the phototoxicity exerted by these compounds

involves the production of ROS. The 4-nitro derivatives 3 and

5 showed trends similar to those obtained from the

phototoxicity studies, where the linear derivative 5 showed a

minimal increase in the cytotoxicity upon irradiation and the

wedged 4-nitro derivative 3 showed small or no changes after

irradiation. It is clear from the above results that the 4-amino

substituents on the 1,8-naphthalimide chromophore are

important for ROS production, where they have been shown

to have higher ﬂuorescence quantum yields and also higher

DNA photocleavage eﬃciencies.

To further examine the eﬀects of these compounds on

cellular DNA, cellular growth, and initiation of apoptosis, a

ﬂow-cytometry-based cell cycle analysis assay was also

conducted. As shown in Figure 15 and Table 4, irradiation

with 4 J/cm²light doses leads to a minimal increase in cells

that are situated in the G₂/M phase of the cell cycle, indicating

that light irradiation promoted DNA damage, resulting in the

G₂/M block and activation of the DNA repair mechanism.

Furthermore, this light dose was shown to induce apoptosis to

a small population of cells that were incubated in the absence

of 3−6. However, in the presence of 3−6, cells were arrested in

the G₂/M phase to a larger extent than the untreated cells

upon irradiation with a 4 J/cm²light dose. Furthermore, the

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Figure 16. Summary of the results obtained from the ﬂow cytometry analysis of the MMP after treatment with 3−6 in the presence and absence of

a 8 J/cm²light. UL represents the upper left grid and corresponds to healthy cells. The upper right (UR) grid correlates to cells with partially

depolarized mitochondria, and the lower grids (LL and LR) represent cells that exhibited complete loss of the MMP. Each stacked bar corresponds

to a mean from two individual experiments.

linear 4-amino derivative 6 induced the highest amount of G₂/

M blocked cells, correlating well with the previous results.

Treatment with 3−6 also showed considerable induction of

apoptosis, especially by the wedged 4-amino derivative 4,

which showed 10% apoptosis. An overall trend similar to that

for the phototoxicity investigation was obtained, where the 4-

amino derivatives 4 and 6 were shown to be more phototoxic

than the 4-nitro derivatives 3 and 5. These results indicate that

treatment with 3−6 may cause apoptosis, induced by arresting

cells in the G₂/M phase of the cell cycle. The G₂/M

checkpoint has been shown to be activated upon DNA

damage, and in light of these results, it is possible that these

compounds are responsible for DNA photocleavage within

cells. The eﬀects were more pronounced with a 8 J/cm²light

dose, again indicating that increased light doses give rise to

increased toxicity (see the Supporting Information).

percentage of ROS. Furthermore, these results also indicated

rapid apoptosis induction by 3 compared with the other

complexes. Treatment with 5 upon irradiation resulted in an

increase in cells with partially depolarized mitochondria, while

the wedged derivatives 3 and 4 showed results similar to those

with the irradiated control.

CONCLUSIONS

■

In conclusion, we have reported the synthesis and photo-

physical characterization of four new DNA binding ruthenium

complexes 1−4 appended with either 4-amino- or 4-nitro-1,8-

naphthalimide. A meta arrangement around the ring

connecting 1,8-naphthalimide to a bipyridine ligand creates a

cleft, the result of which renders the shape of the complex

complementary to that of DNA. We have demonstrated that

1−4 show water solubility and a distinctive set of photo-

physical properties that has allowed the nature of their

interaction with DNA to be probed by various ground- and

excited-state titrations. From these titrations, DNA binding

aﬃnities with stDNA, [poly(dA-dT)]₂, and [poly(dG-dC)]₂

have been evaluated as being on the order of 10⁶M⁻¹.

Furthermore, given the π-deﬁcient character of 1 and 2, they

have been shown to exhibit highly oxidizing excited states that

are eﬀectively quenched by redox interactions with guanine

sites on DNA. Similarly, the tight binding, typical of

intercalative interaction, was also apparent from thermal

denaturation studies, where large shifts were observed in the

T_mvalues of stDNA in the presence of 1−4 at various binding

ratios. From CD and LD studies, an overall binding mode for

conjugates 1−4 with DNA was proposed, in which insertion of

1,8-naphthalimide into the helix occurs with concomitant

external association of the Ru(II) center. Because of the

excellent DNA binding ability exhibited by 1−4, their ability to

act as DNA photocleavers was also examined, where both 1

and 2 were found to cleave supercoiled DNA with much

increased eﬃciencies compared with the Ru(bpy)₃-1,8-

naphthalimide analogues 3 and 4. This is attributed to the

The eﬀects of 3−6 on the mitochondrial membrane

potential (MMP) was also evaluated using the ﬂow cytometry

JC-1 assay, whereby an increase in green ﬂuorescence is an

indicator of a loss of MMP.

A summary of the results is shown in Figure 16 with

corresponding plots found in the Supporting Information. The

results showed that the positive control, etoposide, displayed

an induction of cells exhibiting a complete loss of MMP of

30%. The cells that were treated with 3−6 in the dark did not

induce any major changes to the mitochondrial potential,

although treatment with compound 3 showed an increase in

the number of cells with partially depolarized mitochondria

compared with the untreated control. These results again agree

with the previously discussed cytotoxicity studies, where 3 was

found to be the most cytotoxic compound in the dark.

However, the MMP was aﬀected upon irradiation with a 8 J/

cm²light dose. The linear 4-amino derivative 6 showed the

highest percentage of cells with a complete loss of the MMP.

These results are in agreement with the cytotoxicity and ROS

production studies discussed above, where this compound was

shown to be the most cytotoxic and induce the highest

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8.88 (m, 2H, Ar−H), 8.74 (m, 2H, Ar−H), 8.69 (d, 1H, J = 7.92 Hz,

Ar−H), 8.66 (m, 4H, TAP-H), 8.51 (d, 1H, J = 7.92 Hz, Ar−H), 8.41

(d, 1H, J = 2.70 Hz, TAP-H), 8.33 (d, 1H, J = 2.46 Hz, TAP-H), 8.15

(m, 4H, 2Ar−H and 2TAP-H), 8.07 (d, 1H, J = 7.98 Hz, Ar−H), 7.94

(s, 1H, Ar−H), 7.84 (t, 1H, J = 7.84 Hz, Ar−H), 7.80 (d, 2H, J = 5.94

Hz, Ar−H), 7.64 (m, 2H, Ar−H), 7.41 (t, 1H, J = 6.12 Hz, Ar−H).

¹³C NMR (150 MHz, CD₃CN): δ_C164.7, 163.9, 158.1, 157.6, 154.2,

154.1, 151.1, 150.9, 150.5, 150.5, 150.5, 149.8, 149.7, 149.4, 146.6,

146.6, 146.5, 143.3, 143.3, 142.98, 142.95, 140.2, 138.0, 137.6, 133.9,

133.80, 133.79, 133.1, 132.2, 131.6, 131.1, 130.9, 130.6, 130.3, 129.0,

128.9, 128.8, 128.2, 126.0, 125.7, 125.2, 124.6, 124.3, 123.2.

ν_max(ﬁlm)/cm⁻¹: 1711 (CO), 1665 (CO), 1535 (NO₂stretch),

1355 (NO₂stretch). HRMS(-ES). Calcd for C₄₈H₂₈F₆N₁₂O₄PRu (M

nature of the photoactive TAP MLCT state that yields a

strongly oxidizing center TAP^•−, which because of the aﬃnity

of the naphthalimide is located in close proximity to the

guanine base.⁸³More detailed cellular uptake experiments were

carried out on complexes 3−6, with each complex being found

to accumulate in the cytoplasm and nucleus of HeLa cells,

showing characteristic red MLCT emission. 3−6 were also

chosen to proceed to more detailed biological investigation,

where they were shown to induce limited cytotoxicity in the

dark but to signiﬁcantly reduce cell numbers in the culture

upon irradiation at 450 nm, suggesting photoactivated

cytotoxicity within the cells. Interestingly, the trend obtained

for the phototoxicity was in correlation with DNA photo-

cleavage studies, where the 4-amino derivatives 4 and 6 were

shown to be the most eﬃcient photocleavers. 6 was shown to

induce the highest production of ROS after 8 J/cm²irradiation,

and cell cycle analysis showed that the complexes induced a

G₂/M block after irradiation at 8 J/cm². A similar trend was

observed for the cytotoxicity studies, where the 4-amino

derivatives 4 and 6 were shown to be superior to the 4-nitro

derivatives 3 and 5. Finally, the MMP was aﬀected by the

photoactivation of compounds 3−6, where the linear 4-amino

derivative 6 again showed the greatest increase in cells that had

completely lost their MMP. These investigations have shown

the potential of ruthenium(II) 1,8-naphthalimides as PDT

agents, and further studies are continuing to investigate the

biological proﬁles of complexes 1 and 2 and to identify suitable

candidates to progress to in vivo models.

+

+ PF₆): m/z 1083.1042. Found: m/z 1083.1088.

Ru(4-[N-(m-phenyl)-4-amino-1,8-naphthalimide]-2,2′-

bipyridine)(1,4,5,8-tetraazaphenanthrene)₂(PF₆)₂(2). Ru(4-[N-

(m-phenyl)-4-nitro-1,8-naphthalimide]-2,2′-bipyridine)(1,4,5,8-tet-

raazaphenanthrene)₂Cl₂(0.1 g, 0.081 mmol, 1 equiv) was dissolved in

HPLC-grade MeOH (20 mL) and 10% Pd/C added. The reaction

mixture was subjected to 3 atm of H₂for 24 h. The reaction mixture

was ﬁltered through a Celite plug and the solvent removed under

reduced pressure to give the product as a red/brown solid (0.092 g,

95%). The crystalline solid was converted to the chloride form of the

complex by stirring a solution of the PF₆salt in MeOH with

Amberlite anion exchange resin (Cl⁻form) for 1 h. Calcd for

C₄₈H₃₀F₁₂N₁₂O₂P₂Ru·2H₂O: C, 46.72; H, 2.78; N, 13.62. Found: C,

46.65; H, 2.30; N, 13.15. Mp: >250 °C. ¹H NMR (600 MHz,

CD₃CN): δ_H9.15 (d, 1H, J = 2.76 Hz, TAP-H), 9.14 (d, 1H, J = 2.70

Hz, TAP-H), 8.93 (d, 1H, J = 2.94 Hz, TAP-H), 8.92 (d, 1H, J = 2.76

Hz, TAP-H), 8.86 (d, 1H, J = 1.74 Hz, Ar−H), 8.74 (d, 1H, J = 8.16

Hz, Ar−H), 8.64 (m, 4H, TAP-H), 8.55 (d, 1H, J = 6.90 Hz, Ar−H),

8.43 (d, 1H, J = 8.88 Hz, Ar−H), 8.40 (d, 1H, J = 2.76 Hz, TAP-H),

8.34 (d, 1H, J = 2.70 Hz, TAP-H), 8.32 (d, 1H, J = 8.32 Hz, Ar−H),

8.15 (m, 3H, 2Ar−H and 2TAP-H), 8.00 (d, 1H, J = 8.70 Hz, Ar−H),

7.94 (s, 1H, Ar−H), 7.76 (m, 4H, Ar−H), 7.66 (dd, 1H, J = 1.98 and

6.12 Hz, Ar−H), 7.56 (d, 1H, J = 7.92 Hz, Ar−H), 7.40 (dt, 1H, J =

1.08 and 5.70 Hz, Ar−H), 6.97 (d, 1H, J = 8.28 Hz, Ar−H), 6.06 (s,

2H, NH₂). ¹³C NMR (150 MHz, CD₃CN): δ_C165.6, 164.8, 158.0,

157.6, 154.1, 154.0, 152.9, 151.0, 150.49, 150.48, 150.46, 149.74,

149.69, 149.4, 149.3, 146.57, 146.55, 146.5, 146.4, 143.31, 143.30,

143.0, 142.9, 140.2, 139.3, 137.1, 134.9, 133.8, 133.8, 133.8, 132.6,

131.4, 131.2, 129.7, 129.3, 128.9, 128.0, 125.8, 125.7, 125.6, 123.8,

123.0, 120.9, 110.8, 109.7. ν_max(ﬁlm)/cm⁻¹: 3356 (aromatic C−H

stretch), 1675 (CO), 1628 (CO), 1584 (NH bend), 1371 (C−

EXPERIMENTAL SECTION

■

4-[N-(m-Phenyl)-4-nitro-1,8-napthalimide]-2,2′-bipyridine

(7). 3-([2,2′-Bipyridin]-4-yl)aniline (0.54 g, 2.17 mmol, 1 equiv) and

4-nitro-1,8-naphthalic anhydride (0.53 g, 2.17 mmol, 1 equiv) were

suspended in HPLC-grade ethanol (EtOH; 30 mL), and the mixture

reﬂuxed in a pressure tube for 48 h. The reaction mixture was cooled

to room temperature before the product was collected by suction

ﬁltration and washed with EtOH (30 mL), giving the product as a

1

yellow/brown solid (0.86 mg, 84%). H NMR (400 MHz, CDCl₃):

δ_H8.92 (d, 1H, J = 8.7 Hz, Ar−H), 8.81 (d, 1H, J = 7.4 Hz, Ar−H),

8.75 (m, 2H, Ar−H), 8.7 (s, 1H, Ar−H), 8.68 (d, 1H, J = 5.0 Hz, Ar−

H), 8.46 (m, 2H, Ar−H), 8.05 (t, 1H, J = 7.8 Hz, Ar−H), 7.93 (d, 1H,

J = 8.0, Ar−H), 7.84 (t, 1H, J = 8.0 Hz, Ar−H), 7.72 (m, 2H, Ar−H),

7.59 (d, 1H, J = 5.0 Hz, Ar−H), 7.42 (d, 1H, J = 7.5 Hz, Ar−H), 7.32

(t, 1H, J = 6.0 Hz, Ar−H). ¹³C NMR (100 MHz, CDCl₃): δ_C162.2,

156.3, 155.5, 149.5, 149.3, 148.7, 147.7, 139.5, 136.5, 135.0, 132.5,

129.9, 129.8, 129.7, 129.4, 129.0, 128.6, 127.4, 126.9, 126.5, 123.6,

123.5, 123.4, 122.6, 121.2, 120.9, 118.6. ν_max(ﬁlm)/cm⁻¹: 1717

(−CO−N−CO−), 1524 (C−NO₂), 1350 (C−NO₂). HRMS(-ES).

Calcd for C₂₈H₁₇N₄O₄(M + H): m/z 473.1250. Found: m/z

473.1233.

+

N stretch). HRMS(-ES). Calcd for C₄₈H₃₀F₆N₁₂O₂PRu (M + PF₆):

m/z 1053.1300. Found: m/z 1053.1266.

Ru(4-[N-(m-phenyl)-4-nitro-1,8-naphthalimide]-2,2′-

bipyridine)(bipyridine)₂(PF₆)₂(3). 7 (0.29 g, 0.62 mmol, 1 equiv)

was dissolved in DMF and H₂O added until it began to precipitate. A

few drops of DMF was added to fully dissolve the ligand, and

Ru(bpy)₂Cl₂·2H₂O (0.32 g, 0.62 mmol, 1 equiv) was added. The

solution was saturated with argon by bubbling for 10 min. The

reaction mixture was heated at reﬂux under an Ar atmosphere for 24

h. The solvent was removed under reduced pressure, and the resulting

residue was redissolved in H₂O and ﬁltered. The ﬁltrate was reduced

in volume, and to it was added a concentrated aqueous solution of

NH₄PF₆. The resulting precipitate was extracted with CH₂Cl₂and

dried over MgSO₄, and the solvent was removed under reduced

pressure. The product was puriﬁed by silica ﬂash column

chromatography, eluting with 40:4:1 CH₃CN/H₂O/aqueous

NaNO₃(sat). The chloride form of the complex was reformed by

stirring a solution of the PF₆salt in MeOH with Amberlite ion-

exchange resin (Cl⁻form) for 1 h, giving the product as a red/brown

solid (0.45 g, 75%). Calcd for C₄₈H₃₂F₁₂N₈O₄P₂Ru: C, 49.03; H,

2.74; N, 9.53. Found: C, 48.87; H, 2.90; N, 9.25. Accurate MS. Calcd

Ru(4-[N-(m-phenyl)-4-nitro-1,8-naphthalimide]-2,2′-

bipyridine)(1,4,5,8-tetraazaphenanthrene)₂(PF₆)₂(1). 7 (0.104

g, 0.22 mmol, 1 equiv) and Ru(TAP)₂Cl₂(0.118 g, 0.22 mmol, 1

equiv) were suspended in DMF/H₂O (1:1), and the suspension was

degassed by bubbling with argon for 15 min. The reaction mixture

was heated at 150 °C for 40 min using microwave irradiation before

being allowed to cool and ﬁltered. The solvent from the resulting

solution was removed under reduced pressure before redissolution in

H₂O (5 mL). The PF₆salt of the complex was formed by the addition

of a concentrated ethanolic solution of NH₄PF₆, with the resulting

precipitate being collected by centrifugation. The dried solid was

redissolved in MeCN before puriﬁcation by the slow diﬀusion of

diethyl ether into MeCN, giving the product as a red/brown solid

(0.140 g, 52%). The crystalline solid was converted to the chloride

form of the complex by stirring in MeOH with Amberlite anion

1

for C₄₈H₃₂N₈O₄Ru (M²⁺): m/z 886.1590. Found: m/z 886.1614. H

NMR (CD₃CN, 600 MHz): δ_H8.87 (1H, dd, J = 1.0 and 8.8 Hz, Ar−

H), 8.83 (1H, d, J = 1.9 Hz, Ar−H), 8.73 (1H, dd, J = 0.9 and 7.3 Hz,

Ar−H), 8.72 (1H, d, J = 8.2 Hz, Ar−H), 8.70 (1H, d, J = 8.0 Hz, Ar−

H), 8.57 (1H, d, J = 3.4 Hz, Ar−H), 8.56 (3H, m, 3 × Ar−H), 8.51

1

exchange resin (Cl⁻form) for 1 h. Mp: >250 °C. H NMR (600

MHz, CD₃CN): δ_H9.15 (m, 2H, TAP-H), 8.93 (m, 2H, TAP-H),

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(1H, d, J = 7.9 Hz, Ar−H), 8.13−8.05 (7H, m, 7 × Ar−H), 7.95 (1H,

t, J = 1.9 Hz, Ar−H), 7.84−7.77 (7H, m, 7 × Ar−H), 7.69 (1H, dd, J

= 2.0 and 6.1 Hz, Ar−H), 7.60 (1H, m, Ar−H), 7.46−7.42 (5H, m, 7

× Ar−H). ¹³C NMR (CD₃CN, 150 MHz): δ_C164.7, 163.9, 158.6,

158.0, 152.8, 152.70, 152.67, 152.6, 151.0, 149.3, 138.8, 138.7, 138.0,

137.9, 133.1, 131.8, 131.5, 131.0, 130.9, 130.5, 130.3, 128.9, 128.7,

128.6, 128.5, 128.2, 125.7, 125.6, 125.3, 125.2, 124.6, 124.3, 122.9. IR

ν_max(cm⁻¹): 1715 (w, −CO−N−CO), 1528 (m, C−NO₂), 1349 (m,

C−NO₂).

Ru(4-[N-(m-phenyl)-4-amino-1,8-naphthalimide]-2,2′-

bipyridine)(bipyridine)₂(PF₆)₂(4). Ru(4-[N-(m-phenyl)-4-nitro-

1,8-naphthalimide]-2,2′-bipyridine)(bipyridine)₂Cl₂(0.14 g, 0.12

mmol, 1 equiv) was dissolved in HPLC-grade MeOH and 10% Pd/

C added. The reaction mixture was subjected to 3 atm of H₂for 24 h

before being ﬁltered through a Celite plug and the solvent removed

under reduced pressure, giving the product as an orange solid (0.13 g,

98%). Calcd for C₄₈H₃₄F₁₂N₈O₂P₂Ru·2.5CH₃CN: C, 50.99; H, 3.35;

N, 11.78. Found: C, 51.14; H, 3.65; N, 11.54. Accurate MS. Calcd for

C₄₈H₄₃N₈O₂Ru (M²⁺): m/z 856.1848. Found: m/z 856.1818. ¹H

NMR (CD₃CN, 600 MHz): δ_H8.71 (1H, s, Ar−H), 8.54 (5H, m, 5 ×

Ar−H), 8.35 (1H, d, J = 7.1 Hz, Ar−H), 8.23 (1H, d, J = 8.3 Hz, Ar−

H), 8.09 (5H, m, 5 × Ar−H), 7.97 (1H, t, J = 7.9 Hz, Ar−H), 7.80

(1H, d, J = 4.9 Hz, Ar−H), 7.75 (5H, m, 5 × Ar−H), 7.66 (1H, d, J =

6.0 Hz, Ar−H), 7.63 (1H, d, J = 7.6 Hz, Ar−H), 7.54 (1H, t, J = 7.6

Hz, Ar−H), 7.50−7.38 (5H, m, 7 × Ar−H), 6.70 (1H, br d, J = 6.8

Hz, NH₂). ¹³C NMR (CH₃CN, 150 MHz): δ_C164.6, 163.9, 157.4,

156.9, 156.8, 156.78, 156.77, 151.8, 151.6, 151.5, 151.4, 151.3, 147.4,

138.2, 137.8, 137.7, 137.6, 135.8, 133.6, 131.3, 131.1, 129.9, 128.6,

128.2, 127.5, 127.49, 127.48, 126.1, 124.5, 124.3, 124.2, 124.1, 123.9,

122.0, 121.1, 119.4, 109.1, 108.5. IR ν_max(cm⁻¹): 3354 (w, −NH₂),

1684 (m, −CO−N−CO−), 1634 (s, −NH₂).

Thorﬁnnur Gunnlaugsson − School of Chemistry, Trinity

Biomedical Sciences Institute, Trinity College Dublin (TCD),

The University of Dublin, Dublin 2, Ireland; Synthesis and Solid

State Pharmaceutical Centre (SSPC), Limerick, County

Limerick, Ireland; orcid.org/0000-0003-4814-6853;

Phone: +353 896 3459; Email: gunnlaut@tcd.ie

Authors

Gary J. Ryan − School of Chemistry, Trinity Biomedical Sciences

Institute, Trinity College Dublin (TCD), The University of

Dublin, Dublin 2, Ireland

Maria Luisa Erby − School of Biochemistry and Immunology,

Trinity Biomedical Sciences Institute, Trinity College Dublin,

The University of Dublin, Dublin 2, Ireland

Daniel O. Frimannsson − School of Chemistry, Trinity

Biomedical Sciences Institute, Trinity College Dublin (TCD),

The University of Dublin, Dublin 2, Ireland; School of Medicine,

Institute of Molecular Medicine, St. James’s Hospital, Trinity

College Dublin, Dublin 8, Ireland

Jonathan A. Kitchen − School of Chemistry, Trinity Biomedical

Sciences Institute, Trinity College Dublin (TCD), The

University of Dublin, Dublin 2, Ireland; Chemistry, School of

Natural and Computational Sciences, Massey University,

Auckland 0745, New Zealand; orcid.org/0000-0002-7139-

5666

Mark Lawler − Institute for Health Sciences, Centre for Cancer

Research and Cell Biology, School of Medicine, Dentistry and

Biomedical Sciences, Queen’s University of Belfast, Belfast BT9

7BL, Northern Ireland

D. Clive Williams − School of Biochemistry and Immunology,

Trinity Biomedical Sciences Institute, Trinity College Dublin,

The University of Dublin, Dublin 2, Ireland

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01395.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.inorgchem.0c01395

Details of all general experimental techniques (pages 2−

5), synthesis details of 7, 9, 12, and 13 and

characterization of the spectra of compounds 1−4, 7,

9, 12, and 13 (pages 6−16), photophysical inves-

tigations for each system as well as tables containing

binding constants (pages 17−32), and additional

biological data (pages 33−38) (PDF)

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This publication has emanated from research supported, in

part, by a research grant from Science Foundation Ireland

(RFP and PI Awards 10/45 IN.1/B2999 and 13/IA/1865 to

T.G.), Enterprise Ireland (funding to T.G. and M.L.), and

TCD. R.B.P.E., T.G., and S.J.Q. thank the SFI-funded SSPC

Pharmaceutical Centre (SFI Research Centres Phase 2: 12/

RC/2275_P2) cofunded under the European Regional

Development Fund. R.B.P.E. also acknowledges the Irish

Research Council for PhD funding and SFI for opportunistic

funding received through the SFI Infrastructure Award SFI 16/

RI/3399. We particularly thank Professor John M. Kelly

(TCD) for his advice, inspiration, support, and continuous

mentoring during the development of this and other

ruthenium(II) polypyridine-based projects. We thank Dr.

Tom McCabe (TCD) for helping with the collections of

crystallographic data sets and Dr. John O’Brien (TCD) for his

endless help with collating NMR data.

Accession Codes

CCDC 2002583 contains the supplementary crystallographic

data for this paper. These data can be obtained free of charge

via www.ccdc.cam.ac.uk/data_request/cif, or by emailing

data_request@ccdc.cam.ac.uk, or by contacting The Cam-

bridge Crystallographic Data Centre, 12 Union Road,

Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

AUTHOR INFORMATION

■

Corresponding Authors

Robert B. P. Elmes − Department of Chemistry, Maynooth

University, National University of Ireland, Maynooth W23

F2K8, County Kildare, Ireland; Synthesis and Solid State

Pharmaceutical Centre (SSPC), Limerick, County Limerick,

Ireland; orcid.org/0000-0001-7898-903X; Phone: +353

1708 4615; Email: robert.elmes@mu.ie

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Pharmaceutical Centre (SSPC), Limerick, County Limerick,

Ireland; orcid.org/0000-0002-7773-8842; Phone: +353

7162407; Email: susan.quinn@ucd.ie

■

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T

https://dx.doi.org/10.1021/acs.inorgchem.0c01395

Inorg. Chem. XXXX, XXX, XXX−XXX

Article Doi

Synthesis, Characterization, and Biological Profiling of Ruthenium(II)-Based 4-Nitro- And 4-Amino-1,8-naphthalimide Conjugates

DOI: 10.1021/acs.inorgchem.0c01395

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