Functionalized azobenzene platinum(II) complexes as putative anticancer compounds

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

https://doi.org/10.1007/s00775-021-01865-9

ORIGINAL PAPER

Functionalized azobenzene platinum(II) complexes as putative

anticancer compounds

Katia G. Samper · Julia Lorenzo · Mercè Capdevila · Òscar Palacios · Pau Bayón¹

1

2

1

Received: 4 February 2021 / Accepted: 20 March 2021 / Published online: 1 May 2021

©

Society for Biological Inorganic Chemistry (SBIC) 2021

Abstract

The synthesis and characterization of four platinum(II) complexes using azobenzenes conveniently functionalized as ligands

has been carried out. The characteristic photochemical behavior of the complexes due to the presence of azobenzene-type

ligands and the role of the ligands in the activation of the complexes has been studied. Their promising cytotoxicity observed

in HeLa cells prompted us to study the mechanism of action of these complexes as cytostatic agents. The interaction of the

compounds with DNA, studied by circular dichroism, revealed a diꢀerential activity of the Pt(II) complexes upon irradia-

tion. The intercalation abilities of the complexes as well as their reactivity with common proteins present in the blood stream

allows to conꢁrm some of the compounds obtained as good anticancer candidates.

Keywords Platinum complex · Azobenzene · Cisplatin resistance · Antitumor drug

Introduction

parts, metal ions and ligands, provides further possibilities

in drug design. The versatility of metal ions (several oxida-

tion states, diꢀerent coordination environments, etc.) as well

as the many possibilities in the design and synthesis of new

ligands open up an enormous number of possibilities in the

pursuit of new candidates with therapeutic properties. This is

especially necessary since most metal drug approaches pre-

sent a whole series of drawbacks. A broad set of unwanted

side eꢀects and low selectivity remain the most important

unresolved challenges [5]. Hence, new compounds and strat-

egies are required. A number of studies on new complexes

carried out in recent years have helped to progress in the

understanding of the mechanisms of action of these plati-

num-based drugs. Thus, apart from the accepted DNA inter-

action for cisplatin, other mechanisms have been revealed as

responsible for the action of other cytostatic agents based

equally on platinum complexes [3, 6–8].

According to World Health Organization (WHO), cancer is,

in the twenty-ꢁrst century, the ꢁrst or second leading cause

of death before age of 70 in 91 out of 172 countries [1]. It

also ranks it as the third or fourth in 22 additional countries.

Unfortunately, cancer incidence and mortality are rapidly

increasing in the world, due to aging and population growth

factors. Diagnosed cancer cases usually require treatments

based on radiotherapy, chemotherapy, surgery and others,

prevailing chemotherapy as one of the main strategies to

treat cancer.

Cisplatin became, decades ago, one of the most outstand-

ing advances in chemotherapy and in the ꢁght against can-

cer [2]. Since then, many eꢀorts have been devoted to the

development of new metal drugs [3, 4]. Combining both

With the aim of improving selectivity and eꢀectiveness,

one of the most promising ꢁelds in this concern is the so-

called phototherapy and more speciꢁcally the photoacti-

vated chemotherapy (PACT). This approach consists of the

induction of electronic and/or conformational changes in

the metal complex as a response to light. In this way, the

goal is to move from an inactive complex to another active

species. Thus, when and where the active drug is generated

can be controlled, and therefore selectivity can be improved,

consequently reducing side-effects, in comparison with

*

Òscar Palacios

oscar.palacios@uab.cat

Pau Bayón

pau.bayon@uab.cat

1

2

Departament de Química, Facultat de Ciències, Universitat

Autònoma de Barcelona, 08193-Cerdanyola del Vallès,

Barcelona, Spain

Institut de Biotecnologia i Biomedicina, Departments

Bioquímica i Biologia Molecular, Universitat Autònoma de

Barcelona, 08193-Cerdanyola del Vallès, Barcelona, Spain

Vol.:(0

1

123456789)

3

436

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

conventional chemotherapies [9, 10]. Speciꢁcally, PACT has

emerged as a good alternative to the conventional Pt(II)-

based therapies [11–19] and many eꢀorts have been made

in the design of pro-drugs based on Pt(IV) that lead to ꢁnal

Pt(II) active species [20].

a JASCO spectropolarimeter (model J-715, JASCO, Groß-

Umstadt, Germany) controlled with the J700 software,

(JASCO, Groß-Umstadt, Germany). Samples were ana-

lyzed in 1-cm capped quartz cuvettes, and the temperature

was maintained at 25 °C with a Peltier PTC-351S holder

(TE Technology, Traverse City, MI, USA). Spectra were

processed using the GRAMS 32 software (Thermo Fisher

Scientiꢁc, Waltham, MA, USA). Elemental analyses were

carried out with a Flash EA 2000 CHNS, Thermo Fisher

Scientiꢁc equipment with a TCD and a MAS 200 R autosa-

mpler for solid samples by the Servei d’Anàlisi Química

(SAQ) at the UAB. Fluorescence measurements were car-

ried out with a Perkin Elmer LS 55 50 Hz Fluorescence

Spectrometer using a 1-cm quartz cell. The data obtained

were corrected for the dilution eꢀects by means of the Orig-

inPro8 SR7 software (OriginLab Corporation, Northamp-

ton, MA, USA). Photoisomerization studies were carried

out with continuous irradiation using a 6-W UVP UVGL-55

handheld UV lamp at 254 nm and 365 nm. High-resolution

mass spectra (HRMS) were obtained with a Micro Tof-Q

Instrument (Brucker Daltonics GmbH, Bremen, Germany)

mass spectrometer working in high-resolution mode at SAQ

at the UAB.

Platinum-based PACT anticancer drugs can be activated

through several mechanisms [12]. Among them, photo-

switching seems to be a promising choice [21–24]. The

eꢀectiveness of these procedures depends on the eꢂciency

in light absorption, as well as on the reactivity of the corre-

sponding photoproducts formed. Some of the advantages of

photoswitches are the rapidity of response and that unwanted

by-products are not usually generated. Azobenzenes are

molecular motifs with good photostability and with well-

known properties as molecular switches. Azobenzene units

can easily absorb light, provoking its isomerization from

trans to cis conꢁguration. They have been used in various

applications, as therapeutic agents [25–27], in polymer

chemistry, materials and nanodevices [28–30].

In this work, azobenzene-based ligands are used for the

synthesis of Pt(II) complexes. In the literature, the number

of examples of platinum(II) complexes bearing azobenzene

ligands is scarce, even if some anticancer activity has been

suggested, mainly without taking into consideration the

isomerization of the ligands [31, 32]. In this work, we have

synthesized and characterized a new set of Pt(II)-azoben-

zene complexes with the aim of determining if the photoi-

somerization of azobenzenes can be useful and necessary

to improve the antiproliferative activity of the respective

complexes. Accordingly, their photochemical activity and

reactivity in front of several biomolecules have been studied.

Synthesis

All commercially available reagents and solvents were

used as received. Reactions were carried out under normal

atmospheric conditions with no eꢀorts to exclude moisture

or oxygen, unless it is indicated. The compounds tert-butyl

(3-bromopropyl)carbamate (2), tert-butyl (3-(4-nitrophe-

noxy)propyl)carbamate (3), tert-butyl (3-(4-aminophenoxy)

propyl)carbamate (4), (E)-4,4′-(diazene-1,2-diyl)diphenol

(10), (E)-4,4′-di(bromopropanoxy)azobenzene (11) were

synthetized as previously described from commercially

available reagents [33–36]. Complex [Pt(dmba)(dmso)Cl],

17 was synthesized according to earlier reported [37].

Experimental

Characterization of the ligands and complexes

synthesized

The characterizations of the ligands synthesized and the

corresponding Pt(II) complexes were carried out by several

techniques and methodologies.

Synthesis of tert‑butyl (E)‑(3‑(4‑((4‑hydroxyphenyl)

diazenyl)phenoxy)propyl) carbamate, 5

NMR spectra were registered at the Servei de Ressonàn-

cia Magnètica at the Universitat Autònoma de Barcelona

An adaption of the synthesis described [38] was followed.

To a solution of 4 (113 mg, 0.42 mmol) in 20 mL of dis-

tilled water, 42 µL of 32% hydrochloric acid (0.42 mmol,

1 equiv) were added and the resulting solution was cooled

below 5 ºC. An also cooled below 5 ºC solution of sodium

nitrite (30 mg, 0.42 mmol) in 20 mL of distilled water was

added dropwise to the previous cooled solution and stirred

for 90 min at a temperature lower than 5 ºC. The resulting

mixture was added dropwise to a cooled solution of phenol

(40 mg, 0.42 mmol), sodium hydroxide (17 mg, 0.42 mmol)

and sodium carbonate (47 mg, 0.44 mmol) in 50 mL of dis-

tilled water. After allowing stirring for 2 h at a temperature

(

UAB) using several Bruker spectrometers: DPX250

250 MHz), DPX360 (360 MHz), and ARX400 (400 MHz).

(

1

13

1

H and C{ H} NMR spectra were referenced using residual

solvent peaks, relative to tetramethylsilane (TMS) at 0 ppm.

1

95

Pt NMR chemical shift scale is referenced relative to

K PtCl in D O set at—1616 ppm. All spectra have been

2

4

2

registered at 298 K except when speciꢁed.

UV–vis spectra were acquired using an Agilent 8453

Spectrophotometer with Diode-array detector in 1-cm thick

quartz cuvettes. CD measurements were performed using

1

3

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

437

1

+

1

13

lower than 10 ºC, the solution was neutralized using 32%

of hydrochloric acid. The brown light solid was ꢁltered

and recrystallized in a mixed solvent of ethanol and water

[M] : 496.2227, found: 496.2240. H/ H and H/ C cor-

relation were recorded.

(

1:1, v/v) to give 5 as a yellow/light-brown solid. (80 mg,

Synthesis of (E)‑1‑(4‑(3‑(chloro‑l5‑azanyl)propoxy)

phenyl)‑2‑(4‑(3‑(ethylthio)propoxy)phenyl)‑diazene, 8

1

5

6

3

1%). H NMR (250 MHz, CDCl ): δ 7.97–7.79 (m, 4H),

3

.99–6.92 (m, 4H), 4.77 (br, 1H), 4.07 (t, 2H, J =5.9 Hz),

.36 (q, 2H, J = 6.7 Hz, J = 12.8 Hz), 2.02 (quin, 2H,

The deprotection of the amine group was performed follow-

ing a previously described procedure [39]. 1 mL of concen-

trated HCl was added dropwise to a ꢃask with 7 (306 mg,

0.65 mmol) in ethyl acetate (5 mL), and the solution was

stirred at RT for 1 h. The reaction product was precipitated

with hexane. The solid was collected and washed several

times with 5 wt % sodium bicarbonate solution and dried

1

3

J = 6.0 Hz), 1.45 (s, 9H). C NMR (100.6 MHz, CDCl :

3

δ 160.8, 158.5, 156.4, 147.2, 147.1, 124.7, 124.5, 115.9,

1

14.8, 79.5, 66.1, 38.1, 29.7, 28.6 ppm. HRMS Calcd. for

1

+

C H N O [M] : 372.1923, found: 372.1916.

2

0

25

3

4

Synthesis of tert‑butyl (E)‑(3‑(4‑((4‑(3‑bromopropoxy)

phenyl)diazenyl)phenoxy)propyl)‑carbamate, 6

under vacuum to obtain 8 as a yellow solid. (203 mg, 84%).

1

H NMR (250 MHz, CD OD): δ 7.85 (m, 4H), 7.07 (m,

3

A solution of 5 (100 mg, 0.27 mmol) and potassium car-

bonate (186 mg, 1.35 mmol) in dmf (10 mL) was stirred

for 5 min at room temperature (RT). Commercially avail-

able 1,3-dibromopropane (81 mg, 41 µL, 0.4 mmol) was

added and after 90 min of stirring, the reaction mixture was

quenched with 20 mL of water. The product was extracted

with ethyl acetate (3×10 mL) and the organic fraction was

washed with brine (2 × 10 mL). The organic portion was

4H,), 4.19 (m, 4H), 3.07 (t, 2H, J= 7.17 Hz), 2.73 (t, 2H,

J = 7.03 Hz), 2.56 (q, 2H, J = 7.3 Hz), 2.11 (m, 4H), 1.26

1

3

(t, 3H, J=7.3 Hz). C NMR (100.6 MHz, CD OD): 162.7,

3

162.2, 148.4, 148.1, 125.3, 115.8, 67.8, 66.8, 38.9, 30.3,

30.1, 28.7, 26.6, 15.2. HRMS Calcd. for C H N O S

2

0

28

3

2

1

+

[M] : 374.1902, found: 374.1901.

Synthesis of (E)‑4,4′‑di(ethanethiolpropanoxy)azobenzene,

dried over MgSO , ꢁltrated and the solvent removed under

12

4

vacuum to aꢀord 6 as an orange solid. (127 mg, 95%).

1

H NMR (400 MHz, CDCl ): δ 7.91 (d, J = 7.6 Hz, 4H)

Ethanethiol (120 mg, 1.9 mmol) was added to a ꢃask with

11 (402 mg, 0.9 mmol) and potassium carbonate (609 mg,

4.4 mmol) in dmf (20 mL) and stirred at RT overnight.

Then, 30 mL of water were added, and the precipitate

was collected and washed with water (6×5 mL). The iso-

3

7

4

.06–6.96 (m, 4H), 4.74 (br, 1H), 4.20 (t, 2H, J=5.8 Hz),

.10 (t, 2H, J=6.0 Hz), 3.63 (t, 2H, J=6.4 Hz), 3.36 (q, 2H,

J=5.6 Hz, J=11.57 Hz), 2.36 (quin, 2H, J2=6.1 Hz), 2.02

1

3

(

quin, 2H, J=6.2 Hz), 1.45 (s, 9H) ppm. C NMR (CDCl ,

3

1

00.6 MHz): 161.0, 160.9, 156.2, 147.2, 147.1, 124.6, 114.9,

14.8, 79.5, 66.2, 65.8, 38.1, 32.4, 30.0, 29.7, 28.6 ppm.

lated solid was dried under vacuum to aꢀord 12 as a yel-

1

low solid (205 mg, 56%). H NMR (250 MHz, CDCl ): δ

3

1

+

HRMS Calcd. for C H BrN O [M] : 492.1498, found:

7.87 (d, 4H, J = 9.23 Hz), 7.00 (d, 4H, J = 9.13 Hz), 4.15

(t, 4H, J=6.35 Hz), 2.74 (t, 4H, J=6.95 Hz), 2.57 (q, 4H,

J = 7.32 Hz), 4.24 (quin, 4H, J = 6.65 Hz), 2.74 (t, 4H,

2

3

30

3

4

1

13

4

92.1485. H/ H and H/ C correlation were recorded.

1

+

Synthesis of tert‑butyl (E)‑(3‑(4‑((4′‑(3′‑(ethylthio)propoxy)

phenyl)diazenyl)phenoxy)propyl)‑carbamate, 7

J=6.95 Hz) ppm. HRMS Calcd. for C H N O S [M] :

22 30 2 2 2

419.1821 found: 419.1814.

A solution of 6 (724 mg, 1.95 mmol), ethanethiol (173 µL,

Synthesis of di‑µ‑chloro‑bis(N,N‑dimethylbenzylamine‑2‑C,N)

2

.35 mmol) and potassium carbonate (1.347 g, 9.75 mmol)

diplatinum(II) complex, [Pt(dmba)Cl] , 13

2

in dmf (25 mL) was stirred 5 h at RT. Then 30 mL of water

were added, the resulting solution was cooled and a yellow

precipitate appeared. The solid was ꢁltered, washed with

The synthesis was carried out adapting a previously

described synthesis [40]. To a ꢃask with K PtCl (908 mg,

2

4

water (3×5 mL) and dried under vacuum to obtain 7 as a

2.19 mmol) in distilled water (20 mL) a solution of N,N-

dimethylbenzylamine in methanol (18 mL) was added,

and the resulting mixture was stirred at RT for 2 days. The

black/brown solid formed was collected by ꢁltration and was

1

yellow solid. (635 mg, 69%). H NMR (250 MHz, CDCl ):

3

δ 7.86 (d, 4H, J = 8.2 Hz), 7.03–6.95 (m, 4H), 4.76 (br,

1

H), 4.15 (t, 2H, J=6.2 Hz), 4.10 (t, 2H, J=5.9 Hz), 3.35

(

q, 2H, J = 6.3 Hz, J = 12.3 Hz), 2.74 (t, 2H, J = 7.2 Hz),

.58 (q, 2H, J=7.2 Hz), 2.10 (quin, 2H, J= 7.0 Hz), 2.02

quin, 2H, J=6.2 Hz), 1.45 (s, 9H), 1.28 (t, 3H, J=5.9 Hz)

extracted with hot CHCl (150 mL). The solvent was evapo-

3

2

rated to dryness and the crude was puriꢁed by column (SiO ,

2

(

CHCl : AcOEt (9:1)) to obtain 13 as a white solid. (200 mg,

3

1

3

1

ppm. C NMR (100.6 MHz, CDCl ): 161.3, 161.1, 156.2,

28%). H NMR (250 MHz, CDCl ): δ 7.24–7.20 (m, 1H),

3

1

47.0, 146.9, 124.7, 114.9, 79.8, 69.2, 66.8, 38.1, 29.8, 29.3,

7.02–6.81 (m, 3H), 3.89 (s, 2H, J = 27 Hz), 3.01 (s, 6H,

1

95

2

8.6, 28.2, 26.2, 14.9 ppm. HRMS Calcd. for C H N O S

J=23 Hz). Pt NMR (128 MHz, CDCl ): -3657.9 ppm.

2

5

36

3

4

3

1

3

438

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

Synthesis of Pt(dmba)(8)Cl, 14

(250 MHz, CDCl ): δ 7.83 (d, 4H, J = 9.10 Hz), 7.41 (d,

3

2

H, J = 25 Hz), 7.10–6.85 (m, 10H), 4.32–4.09 (m, 4H),

A previously sonicated suspension of 13 (20 mg,

3.94 (s, 4H, J = 20 Hz,), 3.75–3.42 (m, 2H), 3.53–3.16 (m,

2H), 2.99 (s, 12H, J = 17 Hz), 2.52–2.23 (m, 2H), 1.43 (t,

0

.028 mmol) in methanol (10 mL) was added to a solution

1

3

of 8 (23 mg, 0.056 mmol) and sodium methoxide (25%)

13 µL, 0.056 mmol) in methanol (5 mL) and the result-

6H, J = 7.71 Hz). C NMR (100.6 MHz, CDCl ): 160.8,

3

(

147.6, 147.2, 134.4, 131.8, 125.6, 124.5, 124.1, 121.8,

ing mixture was stirred overnight at 50 ºC. Afterwards, the

solution was evaporated until the appearance of a solid,

which was removed by ꢁltration. The ꢁltrate was evapo-

rated to dryness to obtain 14 as an orange crystalline solid,

which was washed with water, ethanol and diethyl ether.

114.9, 66.3, 52.6, 52.5, 33.9, 32.7, 28.0, 13.4. HRMS for

2

+

C H N O Pt S [M] : 536.1494 found: 538.1494. Anal.

4

0

54

4

2

2 2

Calcd. for C H Cl N O Pt S ·C H O (Mr 1220.31): C,

4

0

54

2

4

2

4

10

43.24; H, 5.28; N, 4.58. Found: C, 43.68; H, 5.08; N, 4.64.

1

95

(

10.8 mg, 25%). Pt NMR (128 MHz, CDCl ): − 3706

3

1

+

and−3303 ppm. HRMS Calcd. for C H N O PtS [M] :

Interaction of metal‑complexes with biomolecules

by mass spectrometry

2

9

39

4

2

7

02.2438 found: 702.2447.

For the study of the interaction of the metal-complexes

with several biomolecules, molecular mass determination

were performed by electrospray ionization mass spectrom-

etry at the SAQ in the UAB, equipped with a time-of-ꢃight

analyzer (ESI-TOF MS) using a Micro Tof-Q Instrument

(Brucker Daltonics GmbH, Bremen, Germany) calibrated

with ESI-L Low Concentration Tuning Mix (Agilent Tech-

nologies), interfaced with a Series 1100 HPLC pump (Agi-

lent Technologies) equipped with an autosampler, both

controlled by the Compass Software.

Synthesis of Pt(dmba)(7)Cl, 15

A previous sonicated suspension of 13 (44 mg, 0.060 mmol)

in methanol (13 mL) was added to a suspension of 7 (56 mg,

0

.118 mmol) in methanol (5 mL) and was stirred overnight

at 50 ºC. At that point, the solution was evaporated until pre-

cipitation starts. The solid was removed by ꢁltration, and the

ꢁ

ltrate was evaporated to dryness to obtain a brown crystal-

1

line solid identiꢁed as 15. (31 mg, 30%) H NMR (360 MHz,

CDCl ): δ 7.91–7.79 (m, 4H), 7.41 (d, 1H, J = 21.6 Hz),

The interaction of metal-complexes with proteins was

analyzed in positive mode. For each experiment, 20 μL of

3

7

.09–6.88 (m, 7H), 4.75 (br, 1H), 4.27–4.14 (m, 2H), 4.10

−

1

(

t, 2H, J=6.1 Hz), 3.94 (s, 2H, J=18 Hz), 3.72–3.43 (br m,

H), 3.41–3.27 (m, 2H), 2.99 (s, 6H, J=17 Hz), 2.95–2.70

sample was injected at 40 μL·min ; the capillary counter

electrode voltage was 4.5 kV; the desolvation temperature

2

−

1

(

m, 2H), 2.48–2.21 (m, 2H), 2.07–1.92 (m, 2H), 1.45 (s,

was 100 ºC; dry gas at 6 L·min . Spectra were collected

throughout an m/z range from 800 to 2500. The liquid car-

rier was an 85:15 mixture of 15 mM ammonium acetate

and acetonitrile, pH 7.0.

1

3

9

H), 1.45–1.37 (m, 3H) ppm. C NMR (100.6 MHz,

CDCl ): 160.9, 160.8, 156.2, 147.6, 147.2, 134.4, 131.8,

3

1

6

25.6, 124.5, 124.1, 121.8, 114.9, 114.8, 79.5, 75.3, 66.3,

1

95

6.2, 52.6, 38.1, 33.9, 32.7, 29.7, 28.6, 28.0, 13.4. Pt

The proteins used in this work were purchased from

Sigma-Aldrich: human serum albumin (A8763), trans-

ferrin (T3309), myoglobin (M6036) and cytochrome C

(C3484). The complementary oligonucleotides used in

this work, OP1 (5′-CACTTCCGCT-3′) and OP2 (5′-AGC

GGAAGTG-3′) were purchased from Euroꢁns MWG Syn-

thesis GmbH (Ebersberg, Germany). The corresponding

double-stranded oligonucleotide (DS) was obtained incu-

bating 50 mM solutions of each strand in MiliQ-water at

NMR (128 MHz, CDCl ): − 3706 ppm. HRMS Calcd. for

3

1

+

1

C H N O PtS [M] : 802.2960, found: 802.2952. H/ H

3

4

47

4

1

13

and H/ C correlation were recorded. Anal. Calcd. for

C H ClN O PtS·CH OH (Mr 869.29): C, 48.30; H, 5.91;

3

4

47

4

3

N, 6.44; S, 3.68. Found: C, 47.38; H, 5.42; N, 6.37, S, 2.81.

Synthesis of Pt (dmba) (12)Cl , 16

2

7

0 ºC for 2 h and further allowed to cool slowly overnight

A solution of 13 (61 mg, 0.08 mmol) in chloroform

17 mL) was added to a solution of 12 (35 mg, 0.08 mmol)

at room temperature.

(

Stock solutions of each protein and DS in water were

freshly prepared for each experiment. Stock solutions of

each complex were prepared in dmso or dmf.

All samples were prepared by incubating the corre-

sponding complex with the biomolecule at the designed

ratio (protein:complex 1:1, 1:5 or 1:10) for 24 h at 37 ºC.

Then the samples were injected without any further treat-

ment. All samples were injected at least in duplicate to

ensure reproducibility.

in chloroform (10 mL) and the resulting mixture reꢃuxed

overnight. Then, the solvent was evaporated to dryness

and the obtained oil was dissolved in the minimum quan-

tity of dichloromethane. Subsequently, methanol was

added until an orange precipitate appeared which was

collected and washed with methanol (2 × 5 mL) and die-

thyl ether (2 × 5 mL) and dried under vacuum to obtain

1

an orange solid identiꢁed as 16 (28 mg, 29%). H NMR

1

3

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

DNA interaction

439

complex during 24 h and 72 h and then 10 µL of Presto-

Blue® was added following the standard protocol. After

3 h incubation, ꢃuorescence (at 590 nm) was measured by a

microplate reader (Victor3). The relative cell viability (%)

for each sample related to the control well was calculated.

The concentration of the compound versus % viability was

plotted to produce the dose–response curves, which were

analyzed using a logistic sigmoid function ꢁt with GraphPad

Prism 6 software. The reported IC values are the average

CD measurements

Stock solutions of calf thymus DNA (3.5 mM, Sigma

Aldrich), TrisHCl (400 mM), and platinum complexes

(

15 mM) were used to prepare the diꢀerent samples. The

DNA concentration was determined spectrophotometri-

cally at 260 nm by using the molar absorptivity value of

5

0

–

1

–1

6

600 M ·cm per nucleotide. The ellipticity values are

of three independent experiments with six replicates per

concentration level.

given in millidegrees (mdeg).

Uptake studies of each complex in HeLa cells were car-

5

ried out in 6 well plates at a density of 2 × 10 cells/well

Competitive DNA‑binding experiments with ethidinium

in 1000 µl of culture medium and were allowed to grow

overnight. Then, cells were treated with 10 µM of each com-

plex during 3 h. After this time, the cell culture was col-

lected, and the cells were washed twice with PBS. Then, the

cells were trypsinized and collected. All the samples were

digested with ultra-pure nitric acid overnight and diluted to

the suitable dilution to be detected by ICP-MS (Agilent 7500

instrument, at SAQ in the UAB). All the experiments were

repeated twice to obtain the percentage of platinum inside

de cell from the total.

bromide

A solution of calf-thymus DNA (2.5 µM) in TrisHCl (5 mM)

was saturated with a solution of Ethidium Bromide (EB)

(

12.5 µM). Stock solutions of cisplatin and 14 and 15 (5 µM)

were used to titrate the DNA solution adding from 0 to a

total amount of 100 µM. After each addition, the solution

was stirred during 5 min before measurement The ꢃuores-

cence spectra of each equivalent added to the DNA-EB solu-

tion were measured exciting at 520 nm and recording the

emission between 550 and 700 nm. The maximum intensity

observed was at 608 nm. The spectra were analyzed accord-

ing to the classical Stern-Voltmer eq:

Results and discussion

ꢀ

I

Synthesis of azobenzene ligands

0

= 1 + K [Q]

SV

I

where I is the initial intensity of DNA-EB, I is the inten-

The diꢀerent ligands used contain an azobenzene unit con-

veniently functionalized to favor metal binding. Ligands 7

and 8 were synthesized following the strategy summarized

in Scheme 1.

0

sity recorded after adding each equivalent of complex, K

sv

is the Stern-Voltmer constant and [Q] is the complex con-

centration. The data obtained were corrected for the dilution

eꢀects by means of the OriginPro8 SR7 software (OriginLab

Corporation, Northampton, MA, USA).

3-Bromopropylamine Bromhydrate, 1, was protected in

excellent yield as its Boc-carbamate, following a standard

procedure [25]. Then, a nucleophilic substitution from a

p-nitrophenol and the product 2 in basic conditions [38] was

carried out, to ꢁnally obtain 3 in 54% yield. The product

was apparently an oil, which crystallized after 2 days at RT

as a white solid. Diꢀerent strategies were tried in order to

achieve the reduction of the nitro group to the corresponding

aniline, 4. Neither Fe in acetic acid and ethanol [41, 42] nor

zinc dust attempts worked. In both cases, a mixture of diꢀer-

ent products together with starting material were obtained.

Finally, a hydrogenation succeeded using 10% of Pd/C as

the catalyst in the presence of a catalytic amount of acetic

Cell culture and cytotoxicity assays

Human cancer cells (HeLa) were obtained from American

Type Culture Collection (ATCC, Manassas, VA, USA) and

were routinely cultured in MEM (modiꢁed Eagle’s medium)

alpha (Invitrogen) containing 10% heat-inactivated fetal

bovine serum (FBS) at 37 ºC in a humidiꢁed CO atmos-

2

phere. The cytotoxicity of each complex was evaluated using

®

PrestoBlue Cell Reagent (Life Technologies) assay. Stock

solutions of each complex were prepared in dmso and dmf.

All working concentrations were prepared in MEM alpha

medium (at maximum dmso concentration of 0.2%). Cells

acid in H at 2-bar pressure in a Fisher-Porter vessel. Aniline

2

4

was obtained as brown oil in 85% yield, which was used

3

without further puriꢁcation in the next step.

were seeded in 96 well plates at a density of 5×10 cells/

An adaption of the synthesis described by J. Kim and

co-workers [38] was followed for azobenzene 5 by means of

an azo coupling reaction between the corresponding diazo-

nium salt from 4 and an activated phenol. The activation of

well in 100 µl of culture medium and were allowed to grow

overnight. After this time, cells were treated with diꢀerent

concentrations (0, 2.5, 5, 10, 25, 50, 100, or 200 µM) of each

1

3

440

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

Scheme 1. Synthetic strategy

for ligands 7 and 8

HO

NO₂

(

Boc) O CH Cl

2 2 2

NH₃⁺Br^-

Br

NHBoc

Br

DMF K CO

2

3

Et N 8h RT

3

97%

54%

1

2

O

N

NHBoc

Br

DMF K2CO3

95%

O

NHBoc

O

NHBoc

Pd/C H₂

EtOH

85%

1) HCl, NaNO2

N

Br

2

) Phenol, NaOH,

NaCO

3

NO₂

NH₂

3

4

51%

OH

5

O

N

O

NHBoc

EtSH, K CO

NHBoc

NH₂

1) HCl, AcOEt

N

2

3

RT, 1h

N

O

N

O

DMF, RT 5h

9 %

2) NaHCO₃

84 %

S

6

Br

S

O

6

7

8

the phenol from its ionized form was required because the

neutral molecule is not suꢂciently nucleophilic [41, 42]. For

that reason, the pH was controlled in order to get mild basic

medium to provide the reaction. The reaction took place

selectively in the para position of the phenol, which attacks

the electrophile diazonium salt to generate 5, as a yellow/

light-brown solid, in a 51% yield after recrystallization.

Azobenzene 6 was synthetized in 95% yield by means

of a monosubstitution reaction of 1,3-dibromopropane by

the corresponding phenolate of 5 using K CO as base.

potassium ethanethiolate at room temperature. A yellow

solid was obtained in 69% yield which was identiꢁed as

diazobenzene, 7. The amine group deprotection in 7 was

easily carried out with concentrated HCl at RT, followed by

precipitation of the corresponding ammonium salt in hexane.

After washing several times with sodium bicarbonate solu-

tion, amine 8 was obtained in 84% yield.

Ligand 12 was synthetized starting from 4-aminophe-

nol, 9 (Scheme 2) [35]. Coupling reaction of commercially

available 9 in a neat reaction with potassium hydroxide at

high temperature yielded 10 as a brown solid in 36%. The

2

3

Afterwards, the resulting bromide in 6 was substituted by

OH

O

Br

SEt

OH

EtSH

K CO DMF,

Br

KOH

00ºC

6 %

N

O

N

O

2

3

K CO DMF,

RT, overnight

5 %

2

3

2

3

RT, overnight

NH₂

Br

56 %

SEt

5

9

OH

1

0

11

12

Scheme 2. Synthetic strategy for ligand 12

1

3

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

441

1

95

1

195

synthesis was continued in a similar manner as for 8, but

introducing, in this case, two bromopropyl chains. This dou-

ble substitution required longer reaction times; up to 19 h

instead of 90 min from 5 to 6. In this way, 11 was obtained

as a yellow solid in 55% yield. Finally, the double substitu-

tion of both bromide groups by the corresponding thioether

in basic conditions gave azobenzene 12 as a yellow solid in

Pt-NMR chemical shifts by means of an H- Pt-HMBC

experiment revealed two peaks corresponding to diꢀerent

platinum(II)-complexes,−3706 and -3303 ppm (SI, Figure

S20), which are the expected for platinum in the+2 oxida-

tion state [46].

Two hypotheses regarding possible structures for the syn-

thetized 14 complex were postulated: (a) the complex in

solution was completely in a dynamic equilibrium between

two or more compounds, which does not allow to obtain

5

6% yield.

1

Synthesis of Pt‑compounds

a clear H-NMR spectrum; (b) two or more isomers were

formed in the reaction, which could be also according to the

obtained HR-MS data.

The new Pt(II) compounds were synthesized using the pre-

viously described ligands. The synthesis and puriꢁcation of

each compound required a speciꢁc strategy.

In the bibliography, diꢀerent examples of hemilabile mol-

ecules of bidentate ligands are reported, and especially for

those ligands with two donor centers with diꢀerent strengths

[45]. In these studies, the hemilabile ligands experience

dynamic equilibrium with the Pt(II) center, and the recorded

Di-µ-chloro-bis(N,N-dimethylbenzylamine-2-C,N)

diplatinum(II) complex, [Pt(dmba)Cl] , 13 (Scheme 3) was

2

prepared as a common platinum(II)-complex precursor. The

synthesis was carried out from an adaption of the work by

Ryabov and co-workers [40]. After a 2-days reaction of the

commercially available starting material K PtCl and two

1

H-NMR spectra also show broad signals. In our case, 8

presents two donor centers which could act as hemilabile

ligands. In order to prove the ꢁrst hypothesis (a), diꢀerent

2

4

1

equivalents of dmba in a mixture of water/methanol, and col-

umn puriꢁcation a stable dimer 13 was obtained as a white

solid. This complex bears N,N-dimethylbenzylamine (dmba)

as bidentate ligand by means of a chelate bond between the

nitrogen atom of the amine and a carbon atom of the benzyl

group with the platinum(II) center. This chelate ring confers

higher stability to the platinum(II)-complex than most other

bidentate ligands due to its kinetic inertness [43, 44].

H-NMR spectra were recorded at diꢀerent temperatures. If

the complex undergoes a dynamic equilibrium, the change

of the temperature will modify its equilibrium constant [45]

1

and, therefore, the H-NMR signal distribution. However,

no changes were observed in the diꢀerent spectra recorded

neither when the temperature was increased or decreased.

The postulation of the second hypothesis (b) entailed

that the reaction of 8 with the platinum(II) precursor, 13,

could evolve into diꢀerent constitutional isomers. The sulfur

atom is softer than the nitrogen atom, as primary amines are

hard bases. Consequently, the coordination of the ꢁrst to the

Pt(II) center should be more favorable than the coordination

of the latter. However, to verify the existence of diꢀerent

isomers, a new Pt(II)-complex was synthetized. The previ-

ously synthetized 7 was used as a ligand, which diꢀers from

8 only in the amine-protection. The protecting Boc group

should block the coordination of the nitrogen atom to the

metal center, and apparently, only the sulfur atom should

coordinate to the Pt(II). The synthesis of this complex was

carried out in similar conditions than those used for the syn-

thesis of 14, but without using sodium methoxide. After an

The platinum(II)-complex, 14, bearing ligand 8 was syn-

thetized as follows. Azobenzene 8 was treated with a previ-

ously sonicated solution containing the precursor complex

1

3 in basic conditions, using sodium methoxide. In this way,

complex 14 was obtained in 25% yield, as an orange solid

(

Scheme 3). The mass spectrum (SI, Figure S21) conꢁrms

the formulated complex by means of the observation of a

+

peak that corresponds to [M-Cl] . Moreover, this peak is

also consistent with the expected isotopic pattern. However,

1

a complex H-NMR spectrum was obtained, with several

peaks at the aliphatic area. In addition, a super-broad signal

1

95

was acquired at around−3000 ppm for Pt-NMR experi-

ment (SI, Figure S19). Only an indirect acquirement of the

Scheme 3. Synthesis of

Pt(dmba)(8)Cl], 14

O

[

NH₂

Cl

N

MeOH, MeONa

+

1/2

Pt

[Pt(dmba)(8)Cl]

N

O

50 ºC overnight

N

Cl

N

25%

14

S

13

8

1

3

442

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

Scheme 4. Synthesis of

Pt(dmba)(7)Cl], 15

O

NHBoc

O

N

[

NHBoc

^C^H^C^l3^,

reflux overnight

N

+

[Pt (dmba) Cl ]

2

15

N

O

N

O

13

30%

S

Pt

Cl

N

7

the aliphatic signals near to the coordinated atom are shifted

and broader. Consequently, their multiplicity cannot be

N

Pt

195

1

Pt

observed, and neither the Pt- H couplings as satellites (SI,

NH

2

Cl

N

Cl

^N^H2

195

S

Figure S22). The Pt-NMR chemical shift was−3706 ppm,

S

O

which ꢁts with one of those two found for 14. Therefore, one

isomer of 14 corresponds to a complex bound to the sulfur

N

O

N

O

N

O

atom of the thioether group, 14 . Consequently, the other

S

1

4_S

14_N

isomer might be that one coordinated to the amino group,

1

4 (Fig. 1).

N

Fig. 1 Proposed structures for both isomers of 14. According to simi-

lar examples in literature, S,N-trans conꢁguration has been tentatively

A dinuclear complex, 16, bearing azobenzene 12 was syn-

thetized. This reaction was performed with an equimolar

amount of the precursor complex 13 in chloroform. After

precipitation with methanol, dinuclear complex 16 was iso-

lated by simple ꢁltration as an orange solid in 29% yield

(Scheme 5).

assigned for Pt complexes in this work [22]. By analogy, 14 has

N

been also proposed as N,N-trans

overnight-reaction [Pt(dmba)(7)Cl], 15 was obtained in 30%

yield as a brown solid (Scheme 4).

The formation of complex 16 was corroborated by dif-

1

13

1

The complex was fully characterized by H-, C{ H}-

ferent techniques. By comparison of H-NMR spectra of

1

95

1

and Pt{ H}-NMR spectroscopy, the corresponding two-

dimensional NMR spectroscopy, HR-MS and elemental

analysis (SI, Figures S22-S27).

free ligand 12 and complex 16, the signals corresponding to

the neighboring thioether protons, H-3 and H-4, are shifted

to the lower ꢁeld. This eꢀect can be also observed for pro-

tons in beta position though in a minor degree (SI, Figure

The peak observed in the mass spectrum (SI, Figure S27)

was consistent with the complex formulated, exhibiting the

1

95

S33). Pt NMR chemical shift at -3705 ppm was obtained

1

195

expected isotopic pattern. The H-NMR spectrum suggested

indirectly by H- Pt-HMBC recorded spectrum (SI, Fig-

ure S34). This chemical shift corroborates the coordination

the coordination of the platinum atom to the sulfur, because

Scheme 5. Synthesis of dinu-

clear complex 16 from azoben-

N

Pt

Cl

S

zene 12 and precursor 13

O

N

S

^C^H^C^l3^,

reflux overnight

N

+

[Pt (dmba) Cl ]

2

16

N

O

N

O

1

3

2

9%

S

Pt

N

Cl

12

1

3

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

443

sphere of the complex. In addition, the mass spectrum

Despite both bands are found in the ligands and in the com-

plexes, this demonstrates that these units are not electroni-

cally coupled in the ground state of complexes 14 and 15.

Azobenzene derivatives can undergo trans-to-cis isomeri-

zation upon irradiation at the wavelength where the π→π*

absorption band of the trans-isomer appears [21]. Upon irra-

diation for 1 min at 365 nm, diluted solutions of the trans-

isomers of 7, 8, 14 and 15, were analyzed by UV–vis in

ACN as solvent. In all the cases the photostationary states

(PSS), i.e., the maximum amount of the cis-isomer that can

be achieved, were obtained (Fig. 3). The cis-isomer spec-

tra for all compounds show the same noticeable diꢀerences

with the corresponding trans-isomer spectra: (a) intensity

decrease and hypsochromical shift of the π→π* electronic

transition band, which are ascribed to the loss of planarity

of the cis-azobenzene moiety; and b) a slight increase of the

n→π* transition absorption band, at 450 nm.

2

+

showed a peak that corresponds to [M-Cl-Cl] . Moreover,

its expected isotopic pattern was observed (SI, Figure S32).

All of these results allow us to conꢁrm the formulated-com-

plex, as long as its purity by elemental analysis.

Photochemistry

Azobenzene-derivative molecules, as explained before, are

well-known to have a characteristic reversible photoisomeri-

zation from its trans-to-cis isomeric form and vice versa.

For that reason, the photophysical properties of the 7, 8 and

1

2 synthetized azobenzene ligands and their corresponding

Pt(II)-complexes (14, 15 and 16) were investigated. The type

of ligands used in this work contain, in both rings of the

azobenzene moiety, an ether group substituted in para posi-

tion, and therefore both rings show equal electronic prop-

erties. However, the substituted group in each chain could

inꢃuence their kinetic photoisomerization by means of, for

example, steric impairments.

Both complexes, 14 and 15, present a characteristic band

around 280 nm, which seems to be related to the dmba

ligands of the complexes (Fig. 3a, b). Although this band

is not shifted in their cis-isomeric form regarding to that of

their trans-isomer, the intensity is slightly higher. Moreo-

ver, the absorption bands that correspond to the azobenzene

moieties of the cis-isomers of 14 and 15 are overlapped with

those of their respective ligands, 8 and 7. Therefore, these

results corroborate that the metal-complex and the azoben-

zene moiety are not electronically coupled in the cis-state.

As expected for azobenzene-derivative switches, cis-to-

trans back isomerization of ligands cis-7 and cis-8, were

observed as well as for complexes cis-15 and cis-14 both

The absorption spectra of the thermally stable trans-iso-

mer of ligands 7, 8 and complexes 14 and 15 were recorded

(

Fig. 2). As expected, all these compounds show the same

absorption bands, which are characteristic of azobenzene-

type compounds: a very intense π→π* transition band local-

ized at 358 nm, together with a much weaker n →π* band

around 450 nm, in the blue-green region of the visible spec-

trum. The spectra obtained for 14 and 15 suggest that the

azobenzene moiety is far away from the metallic center, as

they are very similar to those recorded for the free ligands.

Fig. 2 Absorption spectra of

compounds 7, 8, 14 and 15 in

ACN. Measurements were car-

ried out at low concentrations

–

5

(

~10 M) to minimize aggrega-

tion processes

1

3

444

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

(a)

(b)

0,8

1

0

,2

,0

,8

,6

,4

,2

,0

-

ttrraanns-sRX1153

ttrraanns-sR

-

X1145

PSS

S

PSS

0,6

0,4

0,2

0,0

250

2

50

300

350

400

450

500

550

600

300

350

400

450

500

550

600

Wavelength (nm)

(c)

(d)

1,0

0

,8

,6

,4

,2

,0

-

ttrraanns-sRX710

-

ttrraanns-sRX814

PSS

P

PSS

S

0

,8

,6

,4

,2

0,0

250

2

50

300

350

400

450

500

550

600

300

350

400

450

500

550

600

Wavelength (nm)

Fig. 3 Absorption spectra in ACN of the trans-isomer and the PSS for complexes: (a) 14, (b) 15, and their corresponding ligands: (c) 8 and (d) 7

by photoexposition and thermally. In all cases, quantitative

conversion to their trans-isomers was observed.

Table 1 Parameters of the thermal cis→trans back isomerization in

ACN of compounds 7, 8, 14 and 15

Due to the putative diꢀerent reactivity of both Pt(II)-

isomers, and to check the stability of the cis isomer, the

thermally cis-to-trans isomerization process was studied.

For that, back isomerization from the PSS to the more

stable trans-isomer was followed by UV–vis absorption

spectroscopy. After irradiation, the spectra were recorded

periodically and furtherly, the values of the absorbance

at the π → π* band, i.e. at the maximum absorption of

the trans-isomer, were plotted over time (SI, Figures S36

and S37). The data was adjusted to a monoexponential

function, indicating that this process follows a ꢁrst-order

kinetic rate. The equation derived from this adjustment

allowed to obtain the rate constant of each isomerization

and also the half-lives of each cis-isomer (Table 1).

k_c_i_s_→_t_r_a_n_s₍_s−1

cis

t₁_∕₂(h)

Compound

)

–

5

6

7

8

1

6.0×10

3,8×10

2,0×10

1.5×10

3.2

50,0

9,6

4^a

5

–5

–

5

12.8

a) Values for 14 were listed just to give a notion of their

order of magnitude as they have been calculated from a

mixture of two species 14 and 14

S

N.

1

3

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

445

Differences between the rate constants of the free

azobenzene 7 or its corresponding complex 15 might be

ascribed to the bulkier complex introduced in one of the

phenyl groups of the azobenzene, which seems to provoke

a decrease of the cis-to-trans thermal back isomerization

rate [24].

All the half-live of the cis-isomers calculated are in the

timescale of hours at room temperature. However, some

diꢀerences can be observed among them. The half-live of

cis-7 is shorter than that of 8. This might be attributed to

the polarity of the molecule. On one hand, azobenzene-7

contains a Boc and a thioethane groups in the alkyl chains,

while 8 exhibits diꢀerent polarity, due to the amine group

Aiming to quantify the amount of trans- and cis-azoben-

1

zenes in the PSS, H-NMR was considered a good choice

(

Scheme1). Because the back-isomerization rate depends

because azobenzenes present diꢀerent electronic conꢁgu-

ration and polarity at each isomeric form, thus possess-

ing unique or speciꢁc NMR spectra. With the intention of

obtaining comparative results with the study by UV–vis

on the polarity of the solvent [50], the inꢃuence of the

substituent groups in azobenzene molecules seems to be

essential.

absorption spectroscopy, it was decided to use CD CN as

3

1

Fig. 4 a H-NMR spectrum of ligand trans-7 in CD CN, 298 K.

NMR spectrum in CD CN, 298 K of complex 15 after irradiation at

3

1

b) H-NMR spectrum after irradiation of trans-7 at 365 nm (PSS-

365 nm (PSS-15). (∎=Signals assigned to the cis-7 isomer.=⋏signals

7

), signals corresponding to cis-7 and trans-7 isomers are visible.

1 1

assigned to cis-15)

c) H-NMR spectrum of ligand trans-15 in CD CN, 298 K. d) H-

3

1

3

446

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

solvent. Though both, ligand 7 and complex 15, were totally

band around 450 nm being characteristic of azobenzene-type

bands.

soluble in CD CN, 8 and 14 showed low solubility in this

3

solvent, therefore we decided to focus our study on 7 and

Next, the trans to cis photoisomerization was carried

out, at 365 nm, for ligand 12 and the corresponding dinu-

clear complex 16. Only 1 min of irradiation was required

to achieve the PSS for both 12 and 16 (SI, Figure S40).

Although this experiment was performed in a diꢀerent sol-

vent, CH Cl instead of ACN, no eꢀect of the solvent was

1

5 (Fig. 4).

1

The H-NMR spectrum of PSS-15 in CD CN (Fig. 4d)

3

shows some broad signals that correspond to those protons

that are close to the metal-center, due to their long relaxation

time. Accordingly, the signals corresponding to the protons

distant to the metal center can be observed clearly. In addi-

tion, in the aromatic region, a unique signal about 6.8 ppm

related to the aromatic protons of the cis-7 (Fig. 4b) was

obtained, while two diꢀerent signals for these protons were

obtained for cis-15 (Fig. 4d), showing some loss of sym-

metry that indicates the preferred coordination of the metal

center to one of the two chains of the ligand. Moreover,

aromatic protons of the azobenzene moiety of the cis-15

were assigned by a 2D-NOESY experiment. Both signals

at 6.85 and 6.80 ppm are correlated by distance with those

at 3.99 and 4.18 ppm, related to the α-oxygen protons (SI,

Figure S35).

2

observed in the trans to cis photoisomerization. However,

the kinetic values of the photo-back isomerization show that

the half-life values for cis-12 and cis-16 are shorter than

the values reported above for ligand 7 and its correspond-

ing Pt(II)-complex 15 (Table 1). However, these values are

related to the photo-exposure of the solution to light, being

this a faster reaction than the thermal-back isomerization.

Also, the diꢀerences in values could be related to the sol-

vent, with diꢀerent polarity and viscosity than ACN [25].

Nevertheless, these values show again a proportionality

between the kinetic constants of cis-12 and cis-16. The cis to

trans back-isomerization for the cis-azobenzene coordinated

to Pt(II) center, 16, proceeds slower than for the free azoben-

zene, 12. Therefore, again the Pt(II) center might modify the

kinetics of the isomerization process.

Once the speciꢁc signals were identiꢁed, the cis:trans

ratios in the PSS were determined as 3:1 for complex 15

while 4:1 for ligand 7 (SI, Figures S38 and S39, respec-

tively). This similarity reveals that their direct trans-to-cis

photoisomerization is not noticeably aꢀected by the intro-

duction of the Pt(II) center.

Biological assessments

Due to their poor solubility in ACN, the photochemical

characterization of 12 and the dinuclear complex 16 was car-

ried out in CH Cl . Absorption spectra of both compounds

The in vitro antitumoral activity of complexes 14 and 15

were investigated in human cells from the epithelial cervix,

HeLa cell lines, which present adenocarcinoma disease.

These complexes are soluble enough in a<0.1% dmso dilu-

2

overlap, indicating that the ligand-to-metal coordination

does not modify the absorption of the azobenzene moiety

tion in H O required for these experiments. The in vitro anti-

2

(

Fig. 5). Both compounds show a very intense π→π* tran-

cancer activity was evaluated by means of two experiments:

the determination of their cytotoxicity, by calculation of the

IC value; and measurement of the uptake in HeLa cells of

sition band localized at 358 nm and a much weaker n→π*

5

0

each complex.

1

0

,2

,0

,8

,6

,4

,2

,0

trans-A o-C (25)

1

z

2

Cytotoxicity

trans-Pt-C6 (49)

16

The IC value is an indicative of the cytotoxicity of a com-

5

0

plex. Additionally, these values can be compared among

themselves and with that of cisplatin (or another reference

compound), which is used as a model in cytotoxicity studies.

Table 2 summarizes the IC values calculated for each

5

0

platinum(II)-complex and ligand reported in this work with

the aim of evaluating the eꢀect of the azobenzene ligands. In

addition, and because dmso is known to signiꢁcantly modify

the activity of some metal-based anticancer agents [54], the

IC values of complexes 14 and 15 were determined in

5

0

2

50

300

350

400

450

500

550

600

dmso and in dmf media. Besides, to cover the possibility

of dmso displacement of azobenzene ligands, a homolo-

gous complex [Pt(dmba)(dmso)Cl], 17 [54], was also tested

Wavelength (nm)

Fig. 5 Normalized absorption spectra of compounds 12 and 16 in

(

Table 2).

CH Cl . Measurements were carried out at low concentrations to

2

–

5

minimize aggregation processes (~10 M)

1

3

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

447

Table 2 IC₅₀values measured for ligands and complexes of this work

and cisplatin towards HeLa cells at 24 h and 72 h

Entry

Compound

solvent

IC₅₀(24 h)

IC₅₀(72 h)

1

2

3

4

5

6

7

8

dmso

dmf

dmso

water

>200

24± 1

11± 2

41± 6

5± 2

18± 4

20

>200

18± 2

4.9± 0.6

37± 4

4.0± 0.1

13± 2

4

a

14

15

17

a

cisplatin

a

IC values for 14 and 17 in dmf are very similar with those obtained

5

0

in dmso

Fig. 6 Percentage of Pt(II) inside HeLa cells after uptake studies with

1

0 µM of complex exposure for 3 h. Samples were analyzed by ICP-

MS after being digested with ultra-pure concentrated nitric acid. The

stock solution was also measured and set as 100% of the amount of

platinum used, together with the amount “external-platinum”. Com-

plexes 14 and 17 are containing dmso as a solvent

First, cytotoxicity for ligands 7 and 8 was checked. As

shown in Table 2, while 7 does not exhibit high cytotoxic

activity, 8 shows IC values in the micromolar range.

5 min and afterwards was incubated in the dark for 72 h;

(c) the third cell-plate was irradiated at 365 only after

72 h of incubation. Disappointingly, although slightly dif-

ferences on the cell viability for 1 µM dose of 14 can be

observed, they were not relevant enough (Figure S42).

5

0

Interestingly, the Pt(II)-complex, 14, bearing ligand, 8,

presents better values of IC . Therefore, it can be con-

5

0

cluded that the complexation of the azobenzene ligand

enhances its cytotoxic activity.

Although no influence of solvent was detected for 14

Uptake assessment

(

Figure S41), 15 showed notable differences depending

on the solvent (Table 2, entries 4 and 5). Complex 15

presents much better results in dmf than in dmso. This

fact could be attributed to the putative dmso influence

removing the azobenzene or maybe the chloride ligand,

resulting in a less active complex.

For the uptake studies, cells were treated with each complex

at 10 µM for 3 h. Then, the amount of platinum in the cell

culture and the cell pellets were quantiꢁed by ICP-MS.

The amount of platinum measured inside the cells was

diꢀerent for each complex (Fig. 6): while 14 in dmso and 15

in dmf show the major quantity inside the cell, the amount

of the other complexes (17, cisplatin and 15 in dmso) is

very low.

IC values were calculated at two different exposure

5

0

times: 24 h and 72 h. However, these values do not dif-

fer so much among them, indicating that the activity of

all the studied compounds occurs in the first 24 h. Only

in the case of 14 a slight improvement of the IC value

As previously observed, the activity of complex 15

showed a certain dependence on the medium used, dmso

or dmf. Here, the uptake of the complex in the cells was

practically avoided when dmso was used as a solvent. Taking

together these observations, it seems reasonable to postu-

late that the low uptake of the complex could be one of the

reasons of the decrease of the cytotoxic activity in dmso,

compared with that obtained when dmf was used as a sol-

vent (Table 2). However, the use of dmso as a solvent for

the uptake studies with 14 did not have the same eꢀect as

for 15. The percentage of Pt(II) inside the cells when they

were treated with 14 in dmso was about 53% of the total

dose (Fig. 6). This fact suggests that the presence of the

free amine in 14 may play a decisive role during the inter-

nalization of the complex. Moreover, this conclusion could

also explain the activity of ligand 8. Low quantities of Pt(II)

were detected inside the cells when they were treated with

5

0

was observed after 72 h of complex exposition (Table 2,

entry 3). Interestingly, 14 in dmso and 15 in dmf (Table 2,

entries 3 and 5) show better IC values than cisplatin for

5

0

those experiments at 24 h of complex exposition. How-

ever, the values are similar after 72 h, suggesting that the

activity of cisplatin is kinetically slower than that of our

synthetized-complexes, 14 and 15.

The molecular switch character of azobenzenes is well

known and the opportunity it represents in the field of

bioscience [55]. Therefore, it seemed reasonable to assess

whether the activity of the Pt-complexes depended on

irradiation. To this aim, 14 was taken as a model and it

was submitted to three different circumstances: (a) a cell-

plate was treated with 14 for 72 h avoiding any exposure

to irradiation; (b) a second cell-plate was treated with the

corresponding dose of 14, was irradiated at 365 nm for

1

3

448

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

complex 17. This result suggests that azobenzene ligands, 7

and 8, might be critical in the internalization process.

classical DNA intercalating agents, one promising feature

of some platinum(II)-complexes is their intercalation with

DNA [61]. For that reason, the potential intercalation abili-

ties of 14, 15 and 17 were studied by ꢃuorescence experi-

ments by means of a conventional competition experiment

with Ethidium Bromide (EB). As expected for cisplatin,

complex 17 did not show any changes in the ꢃuorescence

spectra (SI, Figure S43 c and d). This result indicates that

17, despite not intercalating with DNA, it could interact

through other mechanisms. Thus, a more extensive study

would be required. Although 14 and 15 exhibit a certain

degree of intercalation into DNA, their calculated Stern–

DNA‑binding studies

The anticancer activity of Pt(II)-complexes is attributed to

DNA-binding, being this one of the proposed mechanisms

of action of cisplatin [56]. For that reason, the DNA inter-

actions of 14, 15 and 17 were studied. These studies were

carried out by circular dichroism (CD) and ꢃuorescence

measurements using calf-thymus DNA.

3

−1

Circular dichroism measurements Calf-thymus DNA (ct-

DNA) presents a characteristic feature of the CD spectrum

with small amplitude bands: a positive band ca. 280 nm and

a negative one ca. 245 nm [29]. Modiꢁcations in the CD

spectrum are attributed to DNA conformational changes

because of drug interactions with the biomolecule [57].

Only minor changes in the CD spectrum are attributed to

monofunctional platinum(II) adducts, while more dramatic

changes are related to bifunctional adducts [60].

Volmer quenching constants (K ) are 1.76·10 M and

SV

3

−1

3.46·10 M , respectively. These values indicate that these

complexes are very weak intercalators [61]. The apparent

binding constant for these complexes was not calculated

because 50% of the initial ꢃuorescence was not reached [62].

Interaction of the complexes with proteins

To evaluate the interaction of the synthesized complexes

towards the main proteins in blood stream and other ones

related to metal detoxiꢁcation, complexes 14 and 17 were

solubilized in dmso and incubated at 37 ºC with myoglobin,

cytochrome C, transferrin and albumin (HSA) at diꢀerent

protein:Pt ratios and followed by ESI–MS (Fig. 8 and Tables

S2 and S3). Interestingly, not all the proteins showed the

same reactivity with the studied platinum(II) complexes.

Notice that since dmso was used as solubilizing solvent,

some peaks related to the interaction of 14 and 17 with some

of the studied protein showed a dmso molecule coordinated

to the metal moiety.

The interactions of both complexes, 14 and 15, with DNA

were studied for the trans- and cis-isomers. Cis-conꢁgura-

tions were obtained after irradiation of the stock solutions

of each complex before being added to the corresponding

DNA solution. Moreover, to ensure that the cis-isomer was

present, the solutions with DNA and the corresponding com-

plex were also irradiated during the experiment. A blank

sample of DNA was also irradiated to guarantee its stability

under UV light. The corresponding experiment with 15 was

achieved in acetonitrile/water solvent, while those with 14

and 17 were carried out in dmf/water.

Diꢀerent reactivity was measured for trans- and cis-14

The results obtained show poor interaction of 14 with

proteins. No peaks related to covalent binding of the com-

plex to the protein were observed for the incubations with

cytochrome C and HSA (Fig. 8b, c). Moreover, the peak

of the intact myoglobin was the major one recorded in the

mass spectrum when incubating 14 with the protein. At low

protein:Pt ratios, a variety of species with one Pt(II)-adduct

and diꢀerent types of ligands attached were observed. How-

ever, the main species observed are those peaks related to

the protein with one molecule of 14 attached without the

azobenzene and the chloride ligands, but with a coordinated

dmso, and with that without azobenzene ligand and with

a coordinated dmso (Fig. 8a). This variety of species can

be explained when considering that 14 is formed by two

isomers, 14 and 14 , thus the ꢁrst one should have more

(

Fig. 7, a and b). While modiꢁcations of the initial spectrum

of the DNA by the trans-isomer complex were independ-

ent in the [14]/[DNA] ratio, the experiment performed with

cis- 14 showed a decrease in the positive band dependent

on the [14]/[DNA] ratio. The results show minor changes

in the CD spectra when trans-15 was incubated with calf

thymus DNA. Slight changes were observed for the cis-15 at

high [15]/[DNA] ratios (Fig. 7c, d). However, when 17 was

incubated with DNA, no changes in the CD spectrum of the

ct-DNA were observed, suggesting that 17 does not induce

conformational changes in the helicity of DNA (Fig. 7e).

Interestingly, the comparison of the behavior of 17 with

some of the changes observed for both 15 and 14, can indi-

cate that azobenzene ligands should be involved in the inter-

action with DNA, because when the ligands are replaced by

a dmso molecule, i.e. for 17, no changes in the CD spectra

are observed.

S

N

aꢂnity for Cys residues, while the second for His. Addi-

tionally, 14 interacts with transferrin forming Pt(II)-adducts

with up to 3 platinum moieties attached to the protein. These

adducts correspond to the Pt(II) and the dmba ligand, once

released the azobenzene and chloride ligands. However, the

Competitive DNA‑binding experiments with ethidinium

bromide Although azobenzene ligands are not known as

1

3

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

449

DNA

(

a) 20

(b) 15

ri=0.1

ri=0.5

ri=1

DNA

ri=0.1

ri=0.5

ri=1

1

0

5

0

5

1

5

0

5

0

5

ri=2

1

ri=2

-

-10

-

-15

-

20

25

30

35

-

10

15

20

25

trans-14

cis-14

-40

2

50

275

300

325

350

250

275

300

325

350

Wavelength (nm)

DNA

(

c)

4

2

0

(d)

4

2

ri=0,3

ri=0,5

ri=1

DNA

ri=0,3

ri=0,5

ri=1

0

-

2

-2

trans-15

cis-15

2

25

250

275

300

225

250

275

300

Wavelength (nm)

(

e)

4

2

0

2

4

DNA

ri=0,3

ri=0,5

ri=1

-

1

7

-

2

40

260

280

300

320

Wavelength (nm)

Fig. 7 Circular dichroism spectra of calf thymus DNA incubated

with: (a) trans-14 and (b) cis-14. In both cases, the concentration of

ct-DNA was 50 µM in 10 mM Tris–HCl and 50 mM NaCl, while the

complex was ranging from 0 to 100 µM. c trans-15 and (d) cis-15. In

both experiments, the concentration of ct-DNA was 30 µM in 10 mM

Tris–HCl and 50 mM NaCl, while the complex was ranging from 0 to

30 µM. All experiments were carried out at T=296 K and pH=7.4

main peak observed in the mass spectra corresponds to a one

Pt(II)-adduct (Fig. 8c and SI, Table S1).

and 1:10) (Table S2). This complex, in contrast with 14,

interacts with all the studied proteins. Only minor peaks

were detected with platinum(II)-adducts with cytochrome

C in the mass spectra recorded, being the intact protein the

The interaction of 17 with the selected proteins was

also studied, at diꢀerent protein:Pt molar ratios (1:1, 1:5

1

3

450

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

Fig. 8 Mass spectra of 14 recorded after incubation for 24 h at

7 °C with (a) myoglobin (Myo) and (b) cytochrome C (Cyt C)

at a 1:10 (protein:Pt) molar ratios, (c) transferrin (Tf) at a 1:5

protein:Pt) molar ratios and (d) human serum albumin (HSA) at a

and azobenzene (8) ligands (738.25–409.01 mass units at the corre-

sponding charge state); the notation “+n◊” indicates a mass increase

corresponding to the addition of “n” molecules of complex 14 after

elimination of azobenzene (8)) ligand (738.25–373.51 mass units at

the corresponding charge state); the notation “+n●” indicates a mass

increase corresponding to the addition of “n” molecules of complex

14 after elimination of azobenzene (8)) ligand, and the coordination

of one dmso (738.25–373.51+78.1 mass units at the corresponding

charge state); the notation “+n□” indicates a mass increase corre-

sponding to the addition of “n” molecules of complex 14 after elimi-

nation of chloride ligand (738.25–35.5 mass units at the correspond-

ing charge state)

3

(

1

:1 (protein:Pt) molar ratios. The numbers proceeded by the “+”

symbol in the boxes denote the charge state of the peaks. The nota-

tion “+n○”, where n is a number after the name of the protein,

denotes a mass increase corresponding to the addition of “n” mol-

ecules of complex 14 to the protein after the elimination of chlo-

ride and azobenzene (8) ligands, and the coordination of one dmso

(

738.25–409.01+78.1 mass units at the corresponding charge state);

the notation “+n■” indicates a mass increase corresponding to the

addition of “n” molecules of complex 14 after elimination of chloride

major peak even at a 1:10 protein:Pt molar ratio. When 17

interacts with myoglobin (that contains only His, without

Cys residues), major species with the complex after the

release of chloride ligands were detected. This behavior can

be attributed to the presence of only His residues in this

protein, facilitating the release of the weakest ligand in the

complex, while the S-donor ligand of the initial complex has

not been practically replaced. Regarding the interaction of

complex 17 with transferrin and HSA, only the mass spectra

of the samples incubated at 1:1 protein:Pt molar ratio were

obtained. Higher concentrations of complex in solution may

cause the destruction of the protein. In both cases, the main

peaks detected when both proteins interact with 17 showed

the total release of chlorine and dmso ligands, as expected

due to the presence of His in Cys residues in both proteins.

When comparing the behavior of both complexes against

proteins, it can be observed a higher integrity of 14 than of

bound to the platinum moiety. This means that the stability

of the complex in front of proteins is high enough and that

the only way for them to interact requires the practically total

destruction of the complex.

On the contrary, the interaction of 17 with the proteins

exhibits certain selectivity. The interaction of proteins con-

taining His required the release of chloride, while with those

containing also Cys, the release of dmso was also observed.

This particular behavior was also observed in other Pt(II)

complexes containing similar ligands, suggesting certain

lability of the ligands [63].

Conclusions

The synthesis and characterization of three new azobenzene

ligands and of their corresponding platinum(II) complexes

revealed that Pt(II) complexation can improve the half-life of

their less thermodynamically favored cis isomers in respect

to that of the free ligands. The cis–trans azobenzene ratio

1

7 in the presence of proteins. Interestingly, when 14 inter-

acts with the proteins studied, the release of chloride and of

the azo-ligand 8 is required, while only dmso is maintained

1

3

JBIC Journal of Biological Inorganic Chemistry (2021) 26:435–453

451

in the photostationary state (PSS) of ligand 7 and its corre-

sponding platinum(II) complex, 15, revealed the diꢀerenti-

ated inꢃuence of the platinum center in isomerization kinet-

ics. The cytotoxicity studies in HeLa cell lines allowed to

observe that complexes 14 and 15 present better IC values

platinum complexes. Eur J Med Chem 140:349–382. https://doi.

org/10.1016/j.ejmech.2017-09-034

4

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chemistry. Chem Rev 114:4540–4563. https://doi.org/10.1021/

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5

0

than that of cisplatin after 24 h of incubation (thus exhibit-

ing faster action than cisplatin), as well as that the inꢃuence

of the cis–trans ligand isomerization in terms of cytotoxic-

ity does not show signiꢁcant diꢀerences. Evaluation of cell

uptake revealed a crucial role of the ligands in the internali-

zation of complexes, resulting them to be the key point for

the improvement in contrast with cisplatin in some cases.

The interaction of the compounds with DNA, either by

direct binding or by intercalation mechanism, conꢁrmed that

the cis–trans isomerization of the ligands can aꢀect the way

in which the complexes interact with DNA but in any case,

the intercalating abilities of these complexes are very weak.

Finally, the interaction of complexes 14 and 17 with proteins

by ESI–MS allows to conclude that the azobenzene ligand

plays a crucial role in the survival of complex 14 against the

diꢀerent proteins to which it has been exposed.

6

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improving platinum anticancer drugs-phenanthriplatin. Anti-

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Dasari S, Tchounwou PB (2014) Cisplatin in cancer therapy:

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In summary, this work aims to contribute to the ꢁeld of

understudied platinum(II) complexes with azobenzene-type

ligands and thus provide knowledge about these metal com-

plexes that undoubtedly show great potential in the discov-

ery of new applications in diꢀerent ꢁelds.

particles for eꢂcient light-controlled gene delivery and syner-

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12. Shi H, Imberti C, Sadler PJ (2019) Diazido platinum(IV) com-

plexes for photoactivated anticancer chemotherapy. Inorg Chem

Front 6:1623–1638. https://doi.org/10.1039/C9QI00288J

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3. Morales K, Samper KG, Pena Q, Hernando J, Lorenzo J, Rod-

ríguez-Diéguez A, Capdevila M, Figueredo M, Palacios O, Bayón

P (2018) Squaramide-based Pt(II) complexes as potential oxy-

gen-regulated light-triggered photocages. Inorg Chem 57:15517–

15525. https://doi.org/10.1021/acs.inorgchem.8b02854

Supplementary Information The online version contains supplemen-

tary material available at https://doi.org/10.1007/s00775-021-01865-9.

Acknowledgements We acknowledge the Spanish Ministerio de

Ciencia e Innovación for ꢁnancial support (projects CTQ2010-15380,

CTQ2013-41161-R, and the MINECO-FEDER (BIO2015-67358-C2-

14. Srivastava P, Singh K, Verma M, Sivakumar S, Patra AK (2018)

Photoactive platinum(II) complexes of nonsteroidal anti-inꢃam-

matory drug naproxen: Interaction with biological targets, anti-

oxidant activity and cytotoxicity. Eur J Med Chem 144:243–254.

https://doi.org/10.1016/j.ejmech.2017.12.025

2

-P and RTI2018-098027-B-C22). We are also members of the Grup

de Recerca de la Generalitat de Catalunya, ref. 2017SGR-864. K. G.

S. acknowledges to the UAB the PIF grant. We also thank the Servei

d’Anàlisi Química (SAQ) at the Universitat Autònoma de Barcelona

for allocating instrument time.

15. Reessing F, Szymanski W (2017) Beyond photodynamic therapy:

light-activated cancer chemotherapy. Curr Med Chem 24:4905–

4

950. https://doi.org/10.2174/0929867323666160906103223

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6. Najjar A, Rajabi N, Karaman R (2017) Recent approaches to

platinum(IV) prodrugs: a variety of strategies for enhanced deliv-

ery and eꢂcacy. Curr Pharm Des 23:2366–2376. https://doi.org/

Declarations

Conflict of interest The authors declare that there is no known com-

peting ꢁnancial interests or personal relationships that could have ap-

peared to inꢃuence the work reported in this paper.

1

0.2174/1381612823666170201161037

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The cis-diammineplatinum(II) complex of curcumin: a dual action

DNA crosslinking and photochemotherapeutic. Angew Chem Int

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In vitro and in vivo optimization of an anti-glioma modality based

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Article Doi

Functionalized azobenzene platinum(II) complexes as putative anticancer compounds

DOI: 10.1007/s00775-021-01865-9

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