Monomeric and Dimeric Oxidomolybdenum(V and VI) Complexes, Cytotoxicity, and DNA Interaction Studies: Molybdenum Assisted C=N Bond Cleavage of Salophen Ligands

Article

pubs.acs.org/IC

Monomeric and Dimeric Oxidomolybdenum(V and VI) Complexes,

Cytotoxicity, and DNA Interaction Studies: Molybdenum Assisted

CN Bond Cleavage of Salophen Ligands

Sudarshana Majumder,^†Sagarika Pasayat,^†Alok K. Panda,^‡Subhashree P. Dash,^†,§Satabdi Roy,^†

Ashis Biswas,^‡Mokshada E. Varma,^∥Bimba N. Joshi,^∥Eugenio Garribba,^⊥Chahat Kausar,^#

Samir Kumar Patra,^#Werner Kaminsky,^¶Aurelien Crochet,^¥and Rupam Dinda*^,†

́

^†Department of Chemistry, National Institute of Technology, Rourkela 769008, Odisha, India

^‡School of Basic Sciences, Indian Institute of Technology Bhubaneswar, Bhubaneswar 751013, Odisha, India

^§Department of Basic Sciences, Paralamaharaja Engineering College, Sitalapalli, Brahmapur, Odisha 761003, India

^∥Bioprospecting Group, Agharkar Research Institute, G.G. Agharkar Road, Pune 411004, India

^⊥Dipartimento di Chimica e Farmacia, Universita

̀

di Sassari, Via Vienna 2, I-07100 Sassari, Italy

^#Department of Life Science, National Institute of Technology, Rourkela 769008, Odisha, India

^¶Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195, United States

^¥Department of Chemistry, Fribourg Center for Nanomaterials, University of Fribourg, CH-1700 Fribourg, Switzerland

S

* Supporting Information

ABSTRACT: Four novel dimeric bis-μ-imido bridged metal−metal

bonded oxidomolybdenum(V) complexes [Mo^VO₂L′₂¹⁻⁴] (1−4)

2

(where L′¹⁻⁴are rearranged ligands formed in situ from H₂L¹⁻⁴) and

a new mononuclear dioxidomolybdenum(VI) complex [Mo^VIO₂L⁵] (5)

synthesized from salen type N₂O₂ligands are reported. This rare series

of imido-bridged complexes (1−4) have been furnished from

rearranged H₃L′¹⁻⁴ligands, containing an aromatic diimine (o-

phenylenediamine) “linker”, where Mo assisted hydrolysis followed by

−CN bond cleavage of one of the arms of the ligand H₂L¹⁻⁴took

place. A monomeric molybdenum(V) intermediate species [Mo^VO-

(HL′¹⁻⁴)(OEt)] (I_d¹⁻⁴) was generated in situ. The concomitant

deprotonation and dimerization of two molybdenum(V) intermediate species (I_d¹⁻⁴) ultimately resulted in the formation of a

bis-μ-imido bridge between the two molybdenum centers of [Mo^VO₂L′₂¹⁻⁴] (1−4). The mechanism of formation of 1−4 has

2

been discussed, and one of the rare intermediate monomeric molybdenum(V) species I_d⁴has been isolated in the solid state and

characterized. The monomeric dioxidomolybdenum(VI) complex [Mo^VIO₂L⁵] (5) was prepared from the ligand H₂L⁵where the

aromatic “linker” was replaced by an aliphatic diimine (1,2-diaminopropane). All the ligands and complexes have been

characterized by elemental analysis, IR, UV−vis spectroscopy, NMR, ESI-MS, and cyclic voltammetry, and the structural features

of 1, 2, 4, and 5 have been solved by X-ray crystallography. The DNA binding and cleavage activity of 1−5 have been explored.

The complexes interact with CT-DNA by the groove binding mode, and the binding constants range between 10³and 10⁴M⁻¹.

Fairly good photoinduced cleavage of pUC19 supercoiled plasmid DNA was exhibited by all the complexes, with 4 showing the

most promising photoinduced DNA cleavage activity of ∼93%. Moreover, in vitro cytotoxic activity of all the complexes was

evaluated by MTT assay, which reveals that the complexes induce cell death in MCF-7 (human breast adenocarcinoma) and

HCT-15 (colon cancer) cell lines.

localized into Mo^V−Mo^Vsingle bonds.⁸However, examples of

INTRODUCTION

■

bis-μ-imido bridged metal−metal bonded oxidomolybdenum-

(V) complexes are few in the literature⁹although molybdenum-

(V) complexes with terminal imide groups¹⁰have been long

known.

The interesting coordination behavior and structural integrity

of metal−metal bonded molybdenum(V) complexes make their

chemistry a subject of enthusiastic research.^1,2In the past two

decades, there have been extensive studies on binuclear and

polynuclear molybdenum(V) complexes containing metal−

metal bonds, where molybdenum is found to be bridged

through oxido,³sulﬁdo,⁴telluro,⁵chloride,⁶or methoxido⁷

functionalities and the d-electrons of the Mo^Vcenter are

Received: June 20, 2017

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DOI: 10.1021/acs.inorgchem.7b01578

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Inorganic Chemistry

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On the other hand, despite the tremendous success of

platinum anticancer drugs,^11,12there is an acute need for the

development of alternative chemotherapeutic agents in cancer

therapy, due to the severe side eﬀects and resistance

phenomena of the established drugs.¹³⁻¹⁶Generally anticancer

agents which damage or block DNA synthesis are approved for

clinical use.¹⁷In this regard, the oxidative nature of metal

complexes of salens have encouraged biochemists to develop

novel DNA/RNA modiﬁers as well as biomolecular

probes.¹⁸⁻²¹It has been established that the DNA interaction

and antiproliferative potential of the metallo−salen complexes

depend on the type of the central metal and the position and

nature of the substituents present on the salen moiety.^18,21,22In

addition, the bridging aliphatic or aromatic diimine moiety of

salen type ligands has also been reported to signiﬁcantly

inﬂuence their cytotoxic eﬃciencies.^22a,d,23

relevant to mention that, to the best of our knowledge, this is

the ﬁrst report of some bis-μ-imido bridged dimeric

V

oxidomolybdenum(V) complexes [Mo₂O₂L′₂¹⁻⁴] (1−4)

synthesized from partially reduced salophen ligands, which

have been structurally characterized. 1−5 were tested for their

potential of exhibiting DNA binding and cleavage activity, and

their in vitro antiproliferative activity was assayed against the

MCF-7 and HCT-15 cell lines.

EXPERIMENTAL SECTION

■

Materials and Methods. All chemicals were procured commer-

cially and used as received. [MoO₂(acac)₂] was synthesized according

to a reported procedure.⁴²A Vario EL cube CHNS elemental analyzer

was used for elemental analysis measurement. A PerkinElmer

Spectrum RX I spectrophotometer was employed for recording IR

spectra. A Bruker Ultrashield 400 MHz spectrometer was used for

1

measuring H and ¹³C NMR spectra where SiMe₄was used as the

In recent times, the studies on DNA interactions and

antiproliferative activities of molybdenum complexes have

gained substantial interest.²⁴⁻³⁴However, structural reports

on oxidomolybdenum complexes with salen type ligands are

few³⁵⁻³⁷and their pharmacological properties remain practi-

cally unexplored.

internal standard. A PerkinElmer Lambda 25 spectrophotometer was

employed to measure the electronic spectra. A Sherwood Scientiﬁc

AUTOMSB sample magnetometer was used for measuring magnetic

susceptibility. Conductivity was measured with a Eutech CON 700

conductivity meter. ESI-MS were recorded on a SQ-300 MS

instrument operating in ESI mode. Electrochemical data were

recorded using a CH-Instruments (model no. CHI6003E) electro-

chemical analyzer. The cyclic voltammetry experiments were

performed using Pt working and auxiliary electrodes, SCE as reference

electrode, and TBAP as supporting electrolyte. TBAP (tetrabutylam-

monium perchlorate) was purchased from commercial sources, dried

properly, and used as a supporting electrolyte for cyclic voltammetry

experiments. EPR spectra were recorded from 0 to 8000 G at 298 K

(room temperature) with an X-band (9.4 GHz) Bruker EMX

spectrometer equipped with an HP 53150A microwave frequency

counter; the microwave frequency used was in the range 9.40−9.41

GHz. The supercoiled (SC) pUC19 DNA from Escherichia coli DH5α

cells was puriﬁed by using a GeneJET Plasmid Isolation Kit (Thermo

Scientiﬁc, USA). Calf thymus (CT) DNA (biochemistry grade) was

procured from SRL (India). Agarose (molecular biology grade) was

procured from Sigma-Aldrich (USA). MCF-7 (human breast

adenocarcinoma cell line), HCT-15 (colon cancer cell line), and

HaCaT (immortalized human keratinocytes) were purchased from

NCCS, Pune. Penicillin, streptomycin, DMEM, and fetal bovine serum

were procured from Invitrogen, and MTT was procured from

Spectrochem. Propidium iodide was purchased from Sigma-Aldrich

(USA). Caution! Although no difficulties were encountered during the

course of this work, attention is drawn to the potentially hazardous nature

of perchlorates.

Our research group has been involved in the synthetic,

structural, and biological studies of transition metal complexes

in the recent few years.³⁸In continuation of our research,

herein we have reported four novel dimeric bis-μ-imido bridged

metal−metal bonded oxidomolybdenum(V) complexes

[Mo₂O₂L′₂¹⁻⁴] (1−4) (where L′¹⁻⁴are rearranged ligands;

V

discussed later) and a new monomeric dioxidomolybdenum-

(VI) complex [Mo^VIO₂L⁵] (5) synthesized from salen type

N₂O₂ligands. As the site speciﬁc imine reduction of salen type

ligands is quite unusual in the literature³⁹and it has been

reported that molybdenum assisted reduction of aromatic

imines is more feasible than that of aliphatic imines,⁴⁰in this

work an aromatic and an aliphatic diamine were judiciously

chosen for preparing ligands to study their inﬂuence on the

control of molecular structure of oxidomolybdenum complexes,

as well as to investigate their impact on DNA interactions and

cytotoxicity studies. It was observed that, during metalation

with MoO₂(acac)₂, the salophen ligands H₂L¹⁻⁴containing the

aromatic diimine bridges transformed to new rearranged

ligands H₃L′¹⁻⁴through molybdenum assisted hydrolysis

followed by −CN bond cleavage of one of the arms of the

salophen ligands. The corresponding molybdenum(V) inter-

mediate [Mo^VO(HL′¹⁻⁴)(OEt)] (I_d¹⁻⁴) was generated from an

initially formed dioxidomolybdenum(VI) species

[Mo^VIO₂(HL¹⁻⁴)(EtOH)(acac)] (I¹⁻⁴) during the course of

the reaction (one of the molybdenum(V) intermediate species

Synthesis of Ligands H₂L¹⁻⁵. The salophen ligands H₂L¹⁻⁴were

prepared in a fair yield by the condensation of o-phenylenediamine

with the corresponding aldehydes (salicylaldehyde (H₂L¹), o-vanillin

(H₂L²), 5-bromosalicylaldehyde (H₂L³), and 2-hydroxy-1-naphthalde-

hyde (H₂L⁴)) in a 1:2 ratio in ethanol by a adapting a reported

procedure.⁴³The resulting dark orange compounds were ﬁltered and

then washed with ethanol and ﬁnally dried over fused CaCl₂. H₂L⁵was

synthesized by condensing 1,2-diaminopropane with o-vanillin as

reported in the literature.⁴⁴Elemental analysis results and NMR (¹H

and ¹³C) and IR data for all of these conﬁrmed their preparation. The

¹H NMR data of H₂L¹⁻⁵have been included in Table S1.

4

I_dhas been isolated and characterized). Finally, the

molybdenum(V) intermediate (I_d¹⁻⁴) underwent deprotona-

tion and concomitant dimerization to form bis-μ-imido bridged

metal−metal bonded oxidomolybdenum(V) complexes

[Mo₂O₂L′₂¹⁻⁴] (1−4). In contrast, the ligand H₂L⁵with an

V

aliphatic imine component produced

a simple

H₂L¹. Yield: 65%. Anal. Calcd for C₂₀H₁₆N₂O₂: C, 75.93; H, 5.10;

N, 8.86. Found: C, 75.92; H, 5.17; N, 8.81. IR (KBr pellet, cm⁻¹):

3320 ν(O−H), 1623 ν(CN). ¹³C NMR (100 MHz, DMSO-d₆): δ

(ppm) = 164.51, 160.84, 142.72, 133.91, 132.91, 128.28, 120.21,

119.94, 119.55, 117.13.

dioxidomolybdenum(VI) complex [Mo^VIO₂L⁵] (5) where no

such ligand rearrangement took place. The key role of

molybdenum in the ligand transformation can be elucidated

by taking into account the reports of several other transition

metal complexes⁴¹using similar ligands, where no such −CN

bond cleavage takes place.

H₂L². Yield: 67%. Anal. Calcd for C₂₂H₂₀N₂O₄: C, 70.20; H, 5.36;

N, 7.44. Found: C, 70.23; H, 5.34; N, 7.41. IR (KBr pellet, cm⁻¹):

3484 ν(O−H), 1611 ν(CN). ¹³C NMR (100 MHz, DMSO-d₆): δ

(ppm) = 161.33, 157.54, 151.59, 146.37, 129.37, 127.65, 122.48,

121.67, 120.86, 118.19, 79.57.

All the ligands and complexes have been characterized by

various physicochemical techniques, and the structural features

of 1, 2, 4, and 5 have been solved by X-ray crystallography. It is

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DOI: 10.1021/acs.inorgchem.7b01578

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Table 1. Crystal Data and Reﬁnement Data of Complexes 1, 2, 4, and 5

1

2

4

5

formula

M

C₂₆H₁₈Mo₂N₄O₄

642.32

C₂₈H₂₂Mo₂N₄O₆

702.38

C₃₄H₂₂Mo₂N₄O₄

742.44

C₁₉H₂₀MoN₂O₆

468.31

cryst sym

space group

a (Å)

triclinic

monoclinic

P2₁/n

triclinic

P1

̅

P1

̅

P1

̅

8.5389(13)

10.4521(14)

13.565(2)

88.732(9)

80.213(9)

70.999(9)

1127.3(3)

2

9.4562(5)

9.6471(5)

14.4626(7)

79.619(2)

79.536(3)

88.060(2)

1276.17(11)

2

8.9152(3)

17.7572(6)

17.7893(6)

90

11.9576(11)

12.8243(13)

13.8984(14)

76.348(8)

77.430(8)

78.140(8)

1994.6 (3)

4

b (Å)

c (Å)

α (deg)

β (deg)

γ (deg)

V (Å³)

Z

D_calc(g cm⁻³

μ(Mo Kα) (mm⁻¹

104.473(3)

90

2726.84(16)

4

)

1.892

1.828

1.808

1.556

)

1.156

1.036

7.948

0.695

F(000)

636

700

1480

952

max/min transm

θ (max) (deg)

temp (K)

0.988/0.893

26.77

0.9597/0.9125

28.36

0.933/0.793

73.97

0.933/0.793

25.00

110

100

200

reﬂns collected

10634

54801

8414

9605

a

R

[I > 2σ(I)]

b

0.0538

0.0250

0.0487

0.0513

R_w[all data]

0.1103

0.0629

0.1336

0.1035

S [goodness of ﬁt]

max/min res (e Å⁻³

∑ || F | − |F ||

0.974

1.062

1.040

0.826

)

0.914/−0.895

0.658/−0.670

1.549/−0.662

0.483/−0.692

a

b

o

c

R =

. R_w=

∑[w(F ²− F ²)²]/∑[w(F ²)²] .

o

c

o

∑(|F |)

o

V

2

H₂L³. Yield: 62%. Anal. Calcd for C₂₀H₁₄Br₂N₂O₂: C, 50.66; H,

[Mo₂O₂L′₂] (2). Yield: 32%. Anal. Calcd for C₂₈H₂₂Mo₂N₄O₆: C,

47.88; H, 3.16; N, 7.98. Found: C, 47.84; H, 3.12; N, 7.94. IR (KBr

pellet, cm⁻¹): 1598 ν(CN), 946, 912 ν(MoO). UV−vis

(DMSO): λ_max, nm (ε, dm³mol⁻¹cm⁻¹): 483 (11914), 395

(20699), 328 (46037), 275 (52678). ¹³C NMR (100 MHz, DMSO-

d₆): δ (ppm) = 155.28, 153.70, 151.87, 148.94, 134.32, 129.02, 126.17,

122.34, 118.89, 118.21, 116.53, 115.70, 115.57, 55.74. ESI-MS: m/z

702.16 [M]⁺.

2.98; N, 5.91. Found: C, 50.62; H, 2.95; N, 5.90. IR (KBr pellet,

cm⁻¹): 3480 ν(O−H), 1611 ν(CN). ¹³C NMR (100 MHz, DMSO-

d₆): δ (ppm) = 162.67, 159.92, 142.45, 136.15, 134.23, 128.64, 121.88,

120.11, 119.63, 110.36.

H₂L⁴. Yield: 68%. Anal. Calcd for C₂₈H₂₀N₂O₂: C, 80.75; H, 4.84;

N, 6.73. Found: C, 80.71; H, 4.82; N, 6.70. IR (KBr pellet, cm⁻¹):

3488 ν(O−H), 1619 ν(CN). ¹³C NMR (100 MHz, DMSO-d₆): δ

(ppm) = 169.06, 157.82, 138.93, 137.27, 133.46, 129.46, 128.66,

127.89, 127.34, 124.11, 121.95, 121.06, 120.17, 109.70.

V

3

[Mo₂O₂L′₂] (3). Yield: 35%. Anal. Calcd for C₂₆H₁₆Br₂Mo₂N₄O₄:

C, 39.03; H, 2.02; N, 7.00. Found: C, 39.01; H, 2.00; N, 7.02. IR (KBr

pellet, cm⁻¹): 1597 ν(CN), 911, 883 ν(MoO). UV−vis

(DMSO): λ_max, nm (ε, dm³mol⁻¹cm⁻¹): 484 (9759), 383 (17993),

276 (57553). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) = 162.90,

154.61, 152.45, 137.33, 136.86, 134.63, 129.94, 124.63, 121.76, 119.02,

H₂L⁵. Yield: 64%. Anal. Calcd for C₁₉H₂₂N₂O₄: C, 66.65; H, 6.48;

N, 8.18. Found: C, 66.63; H, 6.45; N, 8.12. IR (KBr pellet, cm⁻¹):

3692 ν(O−H), 1627 ν(CN). ¹³C NMR (100 MHz, DMSO-d₆): δ

(ppm) = 167.54, 165.68, 152.01, 151.66, 148.43, 148.37, 123.62,

123.56, 118.70, 118.61, 118.33, 118.20, 115.17, 115.04, 64.44, 63.84,

56.14, 56.02, 20.33.

+

116.30, 116.22, 110.88. ESI-MS: m/z 800.64 [(M − H) ].

V

4

[Mo₂O₂L′₂] (4). Yield: 26%. Anal. Calcd for C₃₄H₂₂Mo₂N₄O₄: C,

55.00; H, 2.99; N, 7.55. Found: C, 55.02; H, 2.94; N, 7.52. IR (KBr

pellet, cm⁻¹): 1565 ν(CN), 937, 912 ν(MoO). UV−vis

(DMSO): λ_max, nm (ε, dm³mol⁻¹cm⁻¹): 486 (15710), 397

(17833), 349 (30945), 279 (55015). ¹³C NMR (100 MHz, DMSO-

d₆): δ (ppm) = 193.30, 169.13, 157.79, 138.91, 138.86, 137.30, 133.46,

129.47, 129.30, 128.66, 127.89, 127.32, 124.72, 124.11, 121.98, 121.07,

109.68. ESI-MS: m/z 742.28 [M]⁺.

V

Synthesis of Complexes [Mo₂O₂L′₂¹⁻⁴] (1−4). [MoO₂(acac)₂]

(1 mmol) was added to a reﬂuxing solution of the appropriate ligand

H₂L¹⁻⁴(1 mmol) in 20 mL of ethanol; the color initially changed to

pale blue and then to dark red. The change of color from pale blue to

red was very fast for 1−3, while for 4 the color remained blue until 15

min. The reﬂux was continued for 3 h and ﬁltered. A dark red residue

was obtained. Red crystals of 1, 2, and 4 were obtained by

recrystallizing the residue in DMSO. The X-ray quality crystals

obtained were ﬁltered and washed with ethanol. Due to the poor

quality of the crystals of 3, its X-ray structure could not be solved.

Elemental analysis results and IR, NMR (¹H, ¹³C), UV−vis, and ESI-

Synthesis of the Intermediate Species [Mo^VO(HL′⁴)(OEt)]

4

(I_d). [MoO₂(acac)₂] (1 mmol) was added to a reﬂuxing solution of

H₂L⁴(1 mmol) in ethanol. The solution turned pale blue, the reaction

was stopped after 15 min when the solution was still blue, and the

4

mixture was ﬁltered. Blue residue of I_dwas obtained. Elemental

1

MS data for all of the complexes veriﬁed their preparation. The H

analysis results and IR, UV−vis, EPR, ESI-MS, and cyclic voltammetry

data for the complex veriﬁed its preparation. The corresponding

intermediates [Mo^VO(HL′¹⁻³)(OEt)] (I_d¹⁻³) could not be isolated

because of their very fast conversion into the dimeric species

V

NMR data of [Mo₂O₂L′₂¹⁻⁴] (1−4) and [Mo^VIO₂L⁵] (5) have been

included in Table S1.

V

1

[Mo₂O₂L′₂] (1). Yield: 34%. Anal. Calcd for C₂₆H₁₈Mo₂N₄O₄: C,

48.62; H, 2.82; N, 8.72. Found: C, 48.60; H, 2.81; N, 8.73. Main IR

peaks (KBr pellet, cm⁻¹): 1601 ν(CN), 951, 855 ν(MoO). UV−

vis (DMSO): λ_max, nm (ε, dm³mol⁻¹cm⁻¹): 465 (2824), 368 (6422),

320 (13313), 290 (18595). ¹³C NMR (100 MHz, DMSO-d₆): δ

(ppm) = 177.30, 172.51, 161.61, 147.74, 142.03, 137.44, 136.87,

133.79, 122.48, 121.97, 116.76, 112.23, 111.97. ESI-MS: m/z 642.15

[M]⁺.

V

[Mo₂O₂L′₂¹⁻³] (1−3).

In an alternative strategy, when [MoO₂(acac)₂] (1 mmol) was

added to a reﬂuxing solution of H₂L⁴(1 mmol) in ethanol in the

presence of few drops of water, the solution remained blue for 30 min

4

and blue crystalline residue of I_dwas now obtained in a higher yield.

[Mo^VO(HL′⁴)(OEt)] (I_d⁴). Yield: 15%. Anal. Calcd for

C₁₉H₁₇MoN₂O₃: C, 54.69; H, 4.11; N, 6.71. Found: C, 54.64; H,

C

DOI: 10.1021/acs.inorgchem.7b01578

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Inorganic Chemistry

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4.10; N, 6.73. IR (KBr pellet, cm⁻¹): 3062 ν(−NH), 1568 ν(−CN),

949 ν(MoO). UV−vis (DMSO): λ_max, nm (ε, dm³mol⁻¹cm⁻¹):

607 (4692), 562 (2917), 519 (1466), 323 (14222), 274(46436). ESI

ε_f), respectively; K_bis determined from the ratio of the slope to the

intercept.

Using Thermal Denaturation Technique. Thermal denaturation

studies of CT-DNA (100 μM) in the absence and presence of

molybdenum complexes (25 μM) were carried out by monitoring the

absorbance at 260 nm in the temperature range of 30−90 °C in 10

mM Tris−HCl buﬀer (pH 8.0) containing 1% DMF. The ramp rate

for each of the experiments was 0.5 °C/min. All the experiments were

carried out in absorbance mode using a Chirascan CD spectropo-

larimeter (Applied Photophysics, U.K.) equipped with a Peltier

temperature controller. The melting temperature (T_m) was

determined from the derivative plot (dA₂₆₀/dT vs T) of the melting

proﬁle.^47a

Using Circular Dichroism Spectroscopy. Circular dichroism (CD)

spectroscopy was studied using a Chirascan CD spectropolarimeter

(Applied Photophysics, U.K.) at 25 °C. CD spectra of CT-DNA (100

μM) in the absence and presence of molybdenum complexes (25 μM)

were obtained in the wavelength range of 240−400 nm in 10 mM

Tris−HCl buﬀer (pH 8.0) containing 1% DMF using quartz cell with

10 mm path length.^38c−g

Fluorescence Experiments. The ﬂuorescence spectra of the

complexes 1−5 (100 μM) as well as ligands (100 μM) in 10 mM

Tris−HCl buﬀer (pH 8.0) containing 1% DMF at 25 °C were

recorded using a spectroﬂuorimeter using Fluoromax 4P (Horiba

Jobin Mayer, USA).

DNA Cleavage Experiments. For all experiments, 300 ng of

supercoiled (SC) pUC19 DNA was used, in the presence of 50 mM

Tris−HCl buﬀer (pH 8.0) containing 1% DMF.

Chemically Induced DNA Cleavage. For chemical nuclease studies,

the cleavage experiments were executed in the dark employing

hydrogen peroxide (0.5 mM) as the oxidizing agent in the absence and

presence of the molybdenum complexes (1−100 μM). The incubation

temperature was ﬁxed at 37 °C for 3 h and analyzed for DNA cleaved

products by agarose gel electrophoresis.

Photoinduced DNA Cleavage. The photoinduced DNA cleavage

studies were carried out according to literature procedures.^38c−g

Brieﬂy, the photolytic DNA cleavage experiments were performed on

supercoiled (SC) pUC19 DNA (300 ng) with 1−5 (1−100 μM) in 50

mM Tris HCl buﬀer (pH 8.0) containing 1% DMF. The extent of

DNA cleavage was calculated from the intensities of the bands using

the UVP Gel Documentation System (Gel Doc It²). The error

observed in determining the band intensities was between 3−6%. Four

diﬀerent additives (two singlet oxygen quenchers (sodium azide and ^L-

histidine) and two hydroxyl radical quenchers (KI and ^D-mannitol))

were used to discern the mechanistic aspect behind the DNA cleavage.

These additives were added to the reaction mixture before the addition

of the complex. The concentration of each of the additives was 0.5

mM.

Cytotoxicity Studies. MTT Assay. MCF-7 cells were cultured in

DMEM having 10% FBS and penicillin (100 U/mL) and streptomycin

(100 μg/mL) in a humidiﬁed 5% CO₂incubator at a temperature of

37 °C. HCT 15 cells were cultured in RPMI 1640 containing 10% FBS

and penicillin (100 U/mL) and streptomycin (100 μg/mL) in a

humidiﬁed 5% CO₂incubator at 37 °C. HaCaT cells were cultured in

DMEM having 10% FBS and penicillin (100 U/mL) and streptomycin

(100 μg/mL) in a humidiﬁed 5% CO₂incubator at a temperature of

37 °C. The cytotoxicity was assayed by determining the viability of

MCF-7, HCT-15, and HaCaT cells after treatment by 1−5 through

MTT assay. Brieﬂy, the cells (10⁵cells/well) were seeded in 96-well

plates in DMEM containing 10% FBS. After cells reached conﬂuency,

treatment was given with varying concentrations (5 μM, 10 μM, 25

μM, and 50 μM) of the molybdenum complexes. The complexes were

dissolved in DMSO, and working solutions were prepared by diluting

the stock of a 5 mM concentration of each compound with plain

DMEM. Final working concentration of DMSO in assay was less than

2%. Untreated cells were taken as 100% survival. After the treatment,

medium was removed and cells were incubated with MTT (5 mg/mL)

for 4 h. The crystals formed were dissolved in DMSO, and absorbance

was read at 570 nm. IC₅₀was calculated for each compound by

treating the cells for 48 h.

+

MS: m/z 421.64 [(M + 2H) ].

Synthesis of [Mo^VIO₂L⁵] (5). [MoO₂(acac)₂] (1 mmol) was added

to a reﬂuxing solution of H₂L⁵(1 mmol) in 20 mL of ethanol; the

color changed to orange. The solution was reﬂuxed for 3 h. Dark

orange crystalline residue of 5 was obtained on ﬁltering the solution.

Some of the crystals were of good quality and were used directly for X-

ray analysis. Elemental analysis results and IR, NMR (¹H, ¹³C), UV−

vis, and ESI-MS data for the complex veriﬁed its preparation.

[Mo^VIO₂L⁵] (5). Yield: 42%. Anal. Calcd for C₁₉H₂₀MoN₂O₆: C,

48.73; H, 4.30; N, 5.98. Found: C, 48.71; H, 4.29; N, 5.94. IR (KBr

pellet, cm⁻¹): 1607 ν(CN), 907, 874 ν(MoO). UV−vis

(DMSO): λ_max, nm (ε, dm³mol⁻¹cm⁻¹): 438 (1871), 323 (7254),

278 (12155). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) = 164.21,

159.45, 150.85, 150.71, 149.61, 148.93, 132.08, 129.14, 126.65, 126.29,

124.98, 124.47, 123.62, 123.55, 122.39, 121.74, 121.67, 121.60, 121.29,

118.72, 118.64, 118.34, 118.20, 117.68, 117.24, 116.91, 116.78, 116.54,

115.18, 115.05, 67.85, 64.51, 63.90, 62.64, 38.51, 30.24, 28.82, 22.87.

ESI-MS: m/z 468.12 [M]⁺.

Crystallography. Clear inclusion free single crystals of 1, 2, 4, and

5 suitable for X-ray diﬀraction studies were mounted on loops with oil.

Crystallographic data and details of reﬁnement are collected in Table

1. The compounds 1, 2, and 5 crystallize in the triclinic space group

P1, and 4 crystallizes in monoclinic space group P2 /n. The unit cell

̅

1

parameters and the intensity data were recorded for complexes 1 and 2

on a Bruker APEX II employing graphite monochromated Mo Kα

radiation (λ = 0.71073 Å) at ∼110 K and ∼100 K respectively. The

data for complex 4 were collected on SuperNova AS2 Data Collections

using high intensity of SuperNova microfocus Cu Kα source (λ =

1.54184 Å) and a highly sensitive Atlas CCD detector at ∼100 K.

Single crystals of complex 5 were mounted on a Stoe IPDS 2

diﬀractometer equipped with an Oxford Cryosystem open ﬂow

cryostat employing a graphite monochromated Mo Kα radiation (λ =

0.71073 Å) at ∼200 K. The intensity data were corrected for Lorentz,

polarization, and absorption eﬀects. The data was integrated and scaled

using SAINT, SADABS within the APEX2 software package by

Bruker.^45aThe structures were solved using the SHELXS97^45band

reﬁned using SHELXL97⁴⁶computer programs. Hydrogen atoms were

set in geometrically idealized positions and constrained to ride on their

parent atoms with C−H distances in the range 0.95−1.00 Å. Isotropic

thermal parameters U_eqwere deﬁned such that they were 1.2U_eqof

their parent atom U_eqfor CH’s and 1.5U_eqof their parent atom U_eqin

the case of methyl groups. All the non-hydrogen atoms were reﬁned

anisotropically by full-matrix least-squares.

DNA Binding Experiments. Using Absorption Spectroscopy.

The binding of the dimeric oxidomolybdenum(V) and monomeric

dioxidomolybdenum(VI) complexes to calf thymus DNA (CT-DNA)

was examined as previously described.^38c−gBrieﬂy, the UV−vis

titration experiments were carried out with variable CT-DNA

concentrations (0−50 μM) against a ﬁxed concentration of

molybdenum complex (25 μM) in 10 mM Tris−HCl buﬀer (pH

8.0) containing 1% DMF using a Lambda 35 PerkinElmer (USA)

spectrophotometer. The binding of ligands to CT-DNA was also

studied. To do this the concentration of ligand was ﬁxed at 25 μM and

was titrated with variable DNA concentration (0−250 μM) in 10 mM

Tris−HCl buﬀer (pH 8.0) containing 1% DMF. The data obtained

from the titration experiments were then ﬁt to the following equation

to obtain binding constant K_b:^38c−g

[DNA]

ε_a− ε_f

[DNA]

ε_b− ε_f

1

=

+

K_b(ε_b− ε_f)

(1)

where [DNA] is the concentration of DNA base pairs, ε_a, ε_f, and ε_b

correspond to apparent extinction coeﬃcients for the complex, i.e.,

Abs/[complex] in the presence of DNA, in the absence of DNA, and

fully bound DNA, respectively. A plot of [DNA]/(ε_a− ε_f) vs [DNA]

resulted in a slope and intercept equal to 1/(ε_b− ε_f) and 1/K_b(ε_b−

D

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V

Scheme 1. Synthesis of Dimeric Oxidomolybdenum(V) Complexes [Mo₂O₂L′₂¹⁻⁴] (1−4) and Monomeric

Dioxidomolybdenum(VI) Complex [Mo^VIO₂L⁵] (5)

V

1−4

Scheme 2. Schematic Pathway for the Formation of [Mo₂O₂L′₂¹⁻⁴] (1−4) via the Intermediate [Mo^VO(HL′¹⁻⁴)(OEt)] (I_d

)

Mo(V) intermediate, [Mo^VO(HL′¹⁻⁴)(OEt)] (I_d¹⁻⁴), and

ﬁnally dimeric bis-μ-imido bridged oxidomolybdenum(V)

Propidium Iodide Staining. Propidium iodide (or PI) is an

intercalating agent which has the ﬂuorescence excitation maximum is

535 nm, and the emission maximum is 617 nm.^47bPI staining was

carried out to visualize the morphology of the nuclei after the

treatment with 1−5. The MCF-7 (human breast cancer cells), HCT-

15 (colon cancer cells), and HaCaT (immortalized human

keratinocytes) cells were grown in the 96-well plate. After reaching

approximately 90% conﬂuency, the cells were treated with varying

concentrations (5, 10, 25, 50 μM) of 1−5 and were incubated for 48 h.

Cells were observed under an inverted ﬂuorescence microscope after

treatment to check for morphological changes, during cell death. Then

the cells were washed twice with PBS (pH 7.4) and ﬁxed after

incubation for 15 min with 3.7% of formaldehyde. The cells were

further washed twice with PBS and treated with 0.2% Triton X-100 in

PBS for 30 s. Again, the washing was repeated with PBS and PI

solution (10 μg/mL) was added, and the sample was kept in the dark

for 15 min. At the end, the cells were further washed with PBS and

imaged under a ﬂuorescence microscope (FLoid, Life Technologies

and Olympus IX 70).

V

complexes [Mo₂O₂L′₂¹⁻⁴] (1−4) were formed, containing

rearranged ligands H₃L′¹⁻⁴furnished by Mo assisted hydrolysis

and −CN bond cleavage (Scheme 2). One of the

4

intermediates, I_d, has been isolated in the solid state and

characterized by UV−vis, IR, EPR, ESI-MS, and cyclic

voltammetry.

V

1−4

The detailed mechanism of the formation of [Mo₂O₂L′₂

]

(1−4) is shown in Scheme 3, taking H₂L²as a representative

among the four ligands, which illustrates that at ﬁrst a

dioxidomolybdenum(VI) species [Mo^VIO₂(HL²)(acac)-

(EtOH)] (I²) is formed, and then a molybdenum assisted

hydrolysis of one of the coordinated −CN bonds of the

2

salophen ligand leads to the formation of I_{b ,}via the formation

of I_a²and release of Hacac.^47cThe pendant phenolic OH then

coordinates to the metal center, and removal of the aldehyde

(o-vanillin) as an acid (o-vanillic acid)^47dtakes place in lieu of

the reduction of Mo(VI) species (I_b) to Mo(IV) species (I_c²).

2

RESULTS AND DISCUSSION

■

The Mo(IV) species, I_c², then undergoes a fast comproportio-

Synthesis. Scheme 1 portrays the synthesis of four dimeric

nation^47ereaction with a Mo(VI) species present in the

oxidomolybdenum(V) complexes [Mo₂O₂L′₂¹⁻⁴] (1−4),

V

2

where L′¹⁻⁴indicates the rearranged form of the ligands

H₂L¹⁻⁴, and a monomeric dioxidomolybdenum(VI) complex

(5), via two diﬀerent reaction pathways (path a and path b),

from salen type ligands (H₂L¹⁻⁵).

reaction medium to form a Mo(V) intermediate I_d. Finally

deprotonation and dimerization of I_doccurs with the loss of

2

ethanol, and μ-imido bridges between two molybdenum

V

2

fractions are furnished to yield [Mo₂O₂L′₂] (2).

In the above reaction, water plays a signiﬁcant role. In fact,

when the same reaction is carried out in the presence of few

drops of water, it has been found that the yield of 2 increases

When the ligands were subjected to reﬂux in the presence of

MoO₂(acac)₂, H₂L¹⁻⁴(which contained the aromatic diimine)

followed the reaction path a (Scheme 1) which involved a

E

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V

2

Scheme 3. Proposed Mechanistic Pathway of Formation of Dimeric Oxidomolybdenum(V) Complex [Mo₂O₂L′₂] (2) and the

Corresponding Oxidovanadium(IV) Complex, [V^IVOL²]^41a,b

signiﬁcantly. There are reports of vanadium complexes of

similar ligands where such hydrolysis and −CN bon cleavage

of salophens did not take place in the presence of VO(acac)₂

(Scheme 3).^41a,bIt has also been found in the literature that, in

the presence of several other transition metals (Ti,^41cCr,^41d

Mn,^41eFe,^41fCo,^41gNi,^41hCu,⁴¹ⁱZn,^41jW,^35cOs,^41kand Pt^41l),

−CN bond cleavage of salophens does not take place, which

proves that the hydrolysis and −CN bond cleavage of

salophens in complex 1−4 is molybdenum assisted. X-ray

crystallography (discussed later) revealed the presence of a

localized metal−metal single bond between the two

molybdenum(V) centers of all the oxidomolybdenum(V)

(5) took place via path b (Scheme 1), where no such

hydrolysis and −CN bond cleavage of H₂L⁵occurred in the

presence of MoO₂(acac)₂owing to the fact that, during the

−CN bond cleavage, the corresponding intermediate,

[Mo^VIO₂(HL⁵)(EtOH)(acac)(H₂O)] (I_a⁵), would not get any

resonance stabilization due to the absence of aromatic

backbone in the ligand environment, which is available in the

case of I_a¹⁻⁴. 1−5 were completely soluble in DMF and DMSO

and partly soluble in CH₃CN, CH₂Cl₂, and H₂O. The

complexes were stable in both solution and solid phases. The

solution phase stability of the complexes for the biological

assays was conﬁrmed by electronic absorption and NMR

studies (Figure S1A −C). From the magnetic susceptibility and

molar conductivity data, it could be concluded that all the

V

complexes [Mo₂O₂L′₂¹⁻⁴] (1−4). The formation of the

monomeric dioxidomolybdenum(VI) complex [Mo^VIO₂L⁵]

F

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4

Figure 1. ESI-MS spectrum of [Mo^VO(HL′⁴)(OEt)] (I_d).

complexes (1−5) are diamagnetic and electrically nonconduct-

ing in solution. The oxidomolybdenum(V) complexes (1−4)

are silent in electron paramagnetic resonance (EPR) spectros-

copy, which may be attributed to the antiferromagnetic

coupling^48aof electron spins of the two 4d¹Mo(V) ions and

the formation of an Mo−Mo bond.^1,3i,48b,c

UV−Vis Spectroscopy. The electronic absorption spectra

4

of the complexes 1−5 and I_dwere recorded in DMSO. The

spectral data are summarized in the Experimental Section. The

spectra of 1−5 display a shoulder in the 438−486 and two

strong absorptions in the 275−397 nm range which are

assignable to LMCT and intraligand transitions, respective-

ly.^49a−cThe absorption spectrum of [Mo^VO(HL′⁴)(OEt)] (I_d)

4

IR Spectroscopy. Selected IR data of all the ligands

(H₂L¹⁻⁵) and the complexes 1−5 are given in the Experimental

Section. The ligands showed a minimum intensity IR band in

the range of 3320−3692 cm⁻¹due to ν(OH) stretching.⁴³

Distinct bands within the range of 1611−1627 cm⁻¹due to

ν(CN) of azomethine groups of the salen type ligands^32,43

(H₂L¹⁻⁵) were noticed. In the complexes (1−5), the ν(CN)

stretching was shifted to lower frequencies (1565−1607 cm⁻¹),

which might be attributable to the lower CN bond order due

to the coordination to the metal atom.³³In addition, the

complexes 1−5 display a pair of sharp strong peaks in the range

855−951 cm⁻¹due to ν(MO) stretch.^49a−cThe intermediate

exhibits peaks at 607, 562, 519, 323, and 274 nm. The band at

323 and 274 nm is attributed to the intraligand charge transfer

band; the bands at 562 and 519 nm are assigned as LMCT

bands.^49a−cThe low energy transition band at 607 nm is

located in the d−d transition region as evidenced from previous

reports of oxidomolybdenum complexes.^49c−eHowever, the

unusually high intensity of this band (ε_max, 4692 dm³mol⁻¹

cm⁻¹), which is too high for a pure d−d transition,^38aand

analogy with other molybdenum(V) complexes^49fsuggest that

this transition may be due to the presence of a mixture of

LMCT and d−d transitions. The representative spectra of

4

[Mo^VO(HL′⁴)(OEt)] (I_d), which has been isolated, showed a

complex 4 and I_dare given in Figure S3.

strong oxido peak at 949 cm⁻¹due to a single ν(MO)

stretch.^49a−cAbsorption at 3062 cm⁻¹is observed which can be

attributed to the ν(NH) stretching;^38dthe peak at 1568 cm⁻¹is

due to the −CN stretching^38aof the imine bond of the

salophen ligand. Representative spectra of complex 4 and its

NMR Spectroscopy. ¹H and ¹³C NMR of all the

1

compounds were recorded in DMSO-d₆. The H NMR data

are collected in Table S1, and the ¹³C NMR data are given in

the Experimental Section. The coordinating modes of H₂L¹⁻⁵

1

were conﬁrmed by comparing their H NMR spectral patterns

4

corresponding intermediate I_dare given in Figure S2.

with those of the corresponding complexes. The representative

G

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Inorganic Chemistry

¹H NMR spectra of H₂L², [Mo₂^VO₂L′₂] (2), and

Article

2

consist of a strong central signal due to the coupling between

an unpaired electron with the nonmagnetic nuclei ⁹²Mo, ⁹⁴Mo,

⁹⁶Mo, ⁹⁸Mo, and ¹⁰⁰Mo (I = 0; total natural abundance 74.5%)

and six hyperﬁne lines due to the coupling with ⁹⁵Mo and ⁹⁷Mo

nuclei (I = 5/2; total abundance 25.5%), indicated by the

asterisks in Figure 2. No signals below 3250 G and above 3650

G were revealed. These features demonstrate that the oxidation

state of Mo is +V with only one electron in the 4d orbitals

(conﬁguration 4d¹).^51aThe examination of the EPR spectra

indicates the presence of only one species in the organic

V

3

[Mo₂O₂L′₂] (3) are given in Figures S4a, S4b, and S5,

respectively. The ¹H NMR spectrum of the free ligands exhibits

resonance in the range δ = 12.96−15.13 ppm due to phenolic

OH and at δ = 8.52−9.68 ppm due to the azomethine −CH

protons, respectively.^50aThe signals for the aromatic protons

from ligands are observed in the expected region δ = 6.75−8.54

ppm. Singlets for the −OCH₃protons of H₂L²and H₂L⁵are

observed at around 3.74 ppm. The aliphatic protons of H₂L⁵

appear as two doublets at 3.73 and 1.31 ppm for −CH₂and

−CH₃respectively, and a multiplet is observed at 3.77 ppm for

−CH proton.⁵⁰In the NMR spectra of complexes, the peak for

the aromatic −OH proton was not observed due to the

deprotonation of phenolic group. The NMR spectrum of

complex 5 exhibits double the number of protons due to the

presence of two entities in one unit.

4

solution (I_d), characterized by g_iso= 1.955 and A_iso= 40.4 ×

10⁻⁴cm⁻¹. These spin Hamiltonian parameters are consistent

with a Mo(N₂^O₂) equatorial coordination mode^51band fall in

the range expected by the correlation between g_isoand A_iso

proposed some years ago by Enemark and co-workers.^51c

As it can be observed, the spectral signals decrease

signiﬁcantly in the time range 0−26 min (at t = 26 min, only

the central absorption is detected and the signal-to-noise ratio

is very low) and disappear completely for time longer than 30

min, suggesting a probable antiferromagnetic^48acoupling

between the unpaired electrons of the Mo(V) centers in the

bis-μ-imido bridged oxidomolybdenum(V) complex (4)

In the ¹³C NMR spectra of the ligands H₂L¹⁻⁵, spectral

signals for the aromatic carbons are found in the range of δ =

109.70−169.06 ppm.^50bThe signals for the aliphatic carbons of

H₂L⁵are found in the range δ = 20.33−64.44 ppm, and the

signal for −OCH₃carbon of H₂L²appears at 79.57 ppm, while

in the ¹³C NMR spectra of complexes 1−5, signals for the

aromatic carbons are found in the downﬁeld region in the range

δ = 109.68−193.30 ppm.⁵⁰The signals for −OCH₃carbon and

aliphatic carbons of the complexes 2 and 5 appear in the range

δ = 22.87−67.85 ppm. Figure S6 shows the ¹³C NMR spectrum

of 3.

4

formed upon the dimerization of two intermediate I_dspecies

(Scheme 3).

Electrochemical Properties. Electrochemical properties of

the complexes 1−5 have been studied by cyclic voltammetry in

DMF solution, and that of I_d⁴has been studied in DMSO (0.1

M TBAP). The CV traces of all the binuclear

ESI-MS. ESI-MS spectra of 1−5 and I_d⁴have been recorded

in DMSO. The characteristic molecular ion peaks for 1−5

appear at 642.15 [M]⁺, 702.16 [M]⁺, 800.64 [(M − H)⁺],

742.28 [M]⁺, and 468.12 [M]⁺, respectively, while the

oxidomolybdenum(V) complexes [Mo₂O₂L′₂¹⁻⁴] (1−4) are

V

of similar pattern, and a representative voltammogram of

V

4

[Mo₂O₂L′₂] (4) is discussed herein. The redox potentials of

4

+

all the complexes are summarized in Table 2. It is observed that

molecular ion peak for I_dappears at 421.64 [(M + 2H) ].

4

The representative ESI-MS spectra of I_dand 3 are given in

4

Table 2. Cyclic Voltammetric Results for 1−5 and (I_d) at

Figure 1 and Figure S7, respectively.

a

298 K

EPR Spectroscopy of the Intermediate Monomeric

Mo(V) Compound [Mo^VO(HL′⁴)(OEt)] (I_d). EPR spectra

4

E^c_1/2(V),

E_pc(V)

ΔE_p

complex

(mV)

E^a_1/2(V)

(mV)

were recorded at room temperature (298 K) as a function of

4

the time on I_d(Schemes 2 and 3), dissolved in a degassed

[Mo₂^VO₂L′₂] (1)

−0.61, −1.36

−0.63, −1.33

−0.62, −1.35

−0.63, −1.37

−0.89, −1.69

−0.91

372

340

365

370

270

286

0.76, 1.14 300, 130

0.75, 1.11 300, 120

0.76, 1.13 310, 140

0.78, 1.14 310, 110

1

DMSO solution. These are shown in Figure 2. The spectra

[Mo₂^VO₂L′₂] (2)

2

[Mo₂^VO₂L′₂] (3)

3

[Mo₂^VO₂L′₂] (4)

4

[Mo^VIO₂L⁵] (5)

[Mo^VO(HL′⁴)(OEt)]

4

(I_d)

a

4

Solvent: DMF for 1−5 and DMSO for I_d. Pt working and auxiliary

electrodes and SCE as reference electrode and TBAP as supporting

electrolyte. Scan rate: 50 mV/s. E_1/2= (E_pa+ E_pc)/2, where E_paand E_pc

are anodic and cathodic peak potentials vs SCE, respectively. ΔE_p=

E_pa− E_pc.

4 exhibits a single electron quasi-reversible reductive response

at E_1/2value of −0.63 V and an irreversible reductive response

at −1.37 V (Figure 3a) which can be assigned to the reduction

of Mo(V) → Mo(IV) of the two corresponding molybdenum

centers.⁵²Further, two single electron quasi-reversible oxidative

responses at E_1/2value of 0.78 and 1.14 V can be attributed to

the oxidation of Mo(V) → Mo(VI) in the binuclear species.

In the CV trace of the monomeric dioxidomolybdenum(VI)

complex [Mo^VIO₂L⁵] (5), a single electron quasi-reversible

response at E_1/2value of −0.89 V and an irreversible reductive

response at −1.69 V are observed (Figure 3b). The response at

E_1/2= −0.89 V can be attributed to the Mo(VI) → Mo(V)

Figure 2. EPR spectra recorded at 298 K as a function of the time on

I_d⁴dissolved in a degassed DMSO solution: (a) t = 7 min; (b) t = 14

min; (c) t = 20 min; (d) t = 26 min. With the asterisks the six

hyperﬁne lines due to the coupling between the unpaired electron with

⁹⁵Mo and ⁹⁷Mo nuclei are indicated.

H

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4

Figure 3. Cyclic voltammograms of (a) [Mo₂O₂L′₂] (4) and (b) [Mo^VIO₂L⁵] (5).

reduction, while the peak at −1.69 V is due to the reduction of

the salen type ligand.^53a

(1−4) is given in Scheme 2). Disregarding the metal−metal

bond, each molybdenum atom exists in a distorted square-

pyramidal geometry where the MoN₃O₂coordination environ-

ment comprises two imido-bridged nitrogen atoms, one imino

nitrogen atom, and one phenoxido oxygen atom describing the

equatorial plane, and one oxido oxygen as an axial group.

Distortion of the square plane is observed from the diﬀerence

in the Mo−O(1)/O(2), Mo−N(1)/N(3), Mo−N(2), and

Mo−N(4) bond lengths as well as the diﬀerence in the bond

angles at the Mo atom [74.6(7) to 109.4(2)°]. The lengths of

the MoO, Mo−O, Mo−N_imino, and Mo−N_imidobonds are

usual.^{1,9e,49a−c,54}Both Mo acceptor centers are displaced from

the average equatorial plane described by O(1)/O(2)−N(1)/

N(3)−N(2)−N(4) toward the axial oxido oxygen atom O(3)/

O(4) by about 0.63(1) Å, the distance of O(3)/O(4) from the

same plane being about 2.3 Å. In the aldehyde oxidoreductase

of Desulfovibrio gigas, similar displacement of the Mo atom from

the equatorial plane toward the apical oxido oxygen atom is also

found.⁵⁵Due to this displacement, the Mo^Vacceptor center

becomes somewhat inaccessible to the donor approaching from

the opposite direction. Therfore, it is evident that

The CV trace of I_d⁴(Figure 4) shows a single electron quasi-

reversible reductive response at E_1/2value of −0.91 V due to

4

Figure 4. Cyclic voltammogram of [Mo^VO(HL′⁴)(OEt)] (I_d).

V

[Mo₂O₂L′₂¹⁻⁴] (1−4) has a square-pyramidal structure with

the sixth position, trans to the oxido oxygen atom O(3)/O(4),

vacant. The shorter length of the Mo−N_imidobond compared to

the Mo−N_iminebond indicates that the deprotonated bridging

imido-N coordinates more strongly to the metal center than the

imino group. The main reason for the structural distortion of

complexes 1, 2, and 4 from the ideal geometry is their observed

average bite angle of the tridentate ligands (79.15°), which are

signiﬁcantly deviated from 90°. The Mo(1)−Mo(2) bond

distance in 1, 2, and 4 ranging between 2.64 and 2.66 Å

indicates the formation of a localized metal−metal single

bond.^{1,3i,j,48b,c,56}Here, it is worth mentioning that this is the

ﬁrst structural evidence of metal−metal bonded

oxidomolybdenum(V) complexes synthesized from salen type

ligands containing an aromatic imine group. Earlier there have

been reports on molybdenum complexes of similar ligands

where molybdenum(VI) oxido^35a−cor peroxido^35dstructures

were proposed on the basis of assumptions from spectral data.

However, the structures of 1, 2, and 4 clearly indicate that in

situ hydrolysis and −CN bond cleavage of one of the arms of

the salophen ligands, containing an aromatic imine moiety,

takes place in the presence of molybdenum acetylacetonate

leading to the formation of dimeric oxidomolybdenum(V)

complexes.

the reduction of Mo(V) → Mo(IV).^49f,53b,cNo metal centered

oxidation peaks were observed during the anodic scans,

4

indicating that I_dis not electrochemically oxidizable to

Mo(VI) species under these conditions.^49f,53bAn irreversible

peak at 1.16 V is observed (Figure S8) which may be attributed

to the oxidation of the ligand.^53dUnder identical experimental

conditions, the single electron processes were veriﬁed by

comparing its current height with that of the standard

ferrocene−ferrocenium couple.

Description of X-ray Structure of Dinuclear Mo(V),

V

[Mo₂O₂L′₂^1,2,4] (1, 2, and 4), and Mononuclear Mo(VI),

[Mo^VIO₂L⁵] (5), Complexes. The molecular structure of the

V

dinuclear [Mo₂O₂L′₂^1,2,4] (1, 2, and 4) and mononuclear

[Mo^VIO₂L⁵] (5) complexes was determined by the single

crystal X-ray diﬀraction technique, and ORTEP representations

for the complexes are shown in Figure 5. The relevant bond

distances and angles are collected in Tables 3 and 4.

The structures of 1, 2, and 4 contain dinuclear [Mo₂O₂]⁶⁺

V

cores with Mo−Mo bonds. The rearranged ligands (H₃L′^1,2,4

)

coordinate each molybdenum atom through one phenoxido

oxygen, one imino nitrogen, and one bridging imido nitrogen in

V

1−4

a tridentate chelating manner (formation of [Mo₂O₂L′₂

]

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V

1

V

2

V

4

Figure 5. ORTEP representations of compounds (a) [Mo₂O₂L′₂] (1), (b) [Mo₂O₂L′₂] (2), (c) [Mo₂O₂L′₂] (4), and (d) [Mo^VIO₂L⁵] (5) with

displacement parameters of 1, 2, and 4 at 50% and 5 at 30% probability.

Table 3. Selected Bond Distances (Å) and Angles (deg) for

Complexes 1, 2, and 4

Table 4. Selected Bond Distances (Å) and Angles (deg) for

Complex 5

1

2

4

bond distances (Å)

bond angles (deg)

Bond Distances (Å)

Mo1−O1

2.101(4)

1.918(4)

1.689(5)

1.701(5)

2.133(7)

2.279(8)

O1−Mo1−N1

89.4(2)

167.0(2)

82.3(2)

Mo1−O1

Mo2−O2

Mo1−O3

Mo2−O4

Mo1−N1

Mo1−N2

Mo2−N2

Mo2−N3

Mo1−N4

Mo2−N4

Mo1−Mo2

2.028(4)

2.020(5)

1.683(4)

1.687(5)

2.170(7)

1.947(5)

1.938(5)

2.175(5)

1.935(6)

1.955(5)

2.650(1)

2.016(2)

2.020(2)

1.693(2)

1.696(2)

2.167(2)

1.961(2)

1.962(2)

2.187(2)

1.948(2)

1.960(2)

2.656(3)

2.034(3)

2.035(5)

1.698(4)

1.703(4)

2.163(4)

1.974(4)

1.979(4)

2.153(4)

1.963(4)

1.966(4)

2.640(6)

Mo1−O2

Mo1−O3

Mo1−O4

Mo1−N1

Mo1−N2

O2−Mo1−N2

O5−Mo1−N2

O4−Mo1−N2

N1−Mo1−N2

O1−Mo1−O2

167.0(2)

71.9(2)

102.9(2)

as the basal plane occupied by one tertiary nitrogen N(1) and

two phenolate oxygen atoms O(1) and O(2), all from the

tetradentate ligand (L⁵)²⁻, together with a terminal oxido ligand

O(3). Another terminal oxido oxygen atom O(4) and one

tertiary nitrogen N(2), from the ligand, are attached to the

metal atom to ﬁll its two axial sites and form a trans O(4)−

Mo−N(2) angle of 167.0(2)°. The Mo atom shifts 0.315(3) Å

out of this mean meridional plane toward the O(4) atom. The

rather long Mo−N distance 2.28(8) Å of the tertiary nitrogen

N(2), from the ligand, to the sixth coordination position lying

trans to the oxido-oxygen O(4) may be due to the displacement

of the Mo atom from the equatorial mean plane toward O(4).

Bond Angles (deg)

O1−Mo1−N1

O2−Mo2−N3

O3−Mo1−O1

O4−Mo2−O2

N2−Mo1−N1

N4−Mo2−N3

Mo1−N2−Mo2

Mo1−N4−Mo2

N4−Mo1−O1

N2−Mo2−O2

O3−Mo1−Mo2

O4−Mo2−Mo1

83.7(2)

84.1(2)

106.9(2)

106.8(2)

75.2(2)

75.4(2)

86.0(2)

85.9(2)

87.1(2)

87.6(2)

106.7 (2)

105.6(2)

82.7(7)

82.6(1)

109.0(2)

109.4(2)

75.0(2)

75.8(2)

83.8(2)

84.4(2)

87.7(2)

86.4(2)

105.4(1)

82.9(6)

108.5(7)

109.2(7)

75.7(7)

74.6(7)

85.2(7)

85.6(7)

85.7(7)

86.8(7)

108.9(6)

108.0(5)

2+

The Mo−O(3) and Mo−O(4) bond distances of the MoO₂

group are unexceptional.^38b,47e,57All these structural features as

well as bond length and angle values are in good accordance

with similar cis-MoO₂L complexes of related N₂O₂ligands

reported earlier.^58a,bThe evaluation of structure of 5 involves

application of SQUEEZE to remove the contribution of

disordered solvent from the reﬁnement, which was ﬁnished

before the new SHELX version was available (see also the

The mononuclear dioxidomolybdenum(VI) complex (5)

shows a distorted octahedral geometry involving NO₃donors

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related B-level CheckCIF alert). Also, recently a

dioxidouranium(VI) complex with a similar tetradentate salen

ligand as H₂L⁵has been reported in the literature where the

metal is bound to the N₂O₂donors in the same fashion.^58c

DNA Binding Studies. In order to understand the binding

of the molybdenum complexes 1−5 to CT-DNA, diﬀerent

spectroscopic techniques were used.

Absorption Spectroscopic Studies. The absorption

bands of the complexes 1−5 originating in the regions 430−

485 and 275−390 nm are attributed to L−Mo(dπ) LMCT and

intraligand transitions, respectively.^49aThe equilibrium binding

constant (K_b) of the complexes to CT-DNA was determined by

UV−vis titration experiments (Table 5 and Figure 6). On

usually gives a large positive ΔT_mvalue.⁶²Complex 4 showed a

slightly negative ΔT_mvalue of −2.00 °C (Table 5 and Figure 7)

possibly due to the destabilization of the DNA double helix.⁶³

Circular Dichroism Studies. In order to study the

conformational changes in CT-DNA upon the interaction

with complexes, circular dichroism (CD) spectroscopy was

used.⁶⁴Two conservative CD bands in the UV region are

observed for CT-DNA: a positive band at 275 nm due to base

stacking interaction and a negative band at 245 nm due to right-

handed helicity.^65aInteraction of small molecules by groove

binding mode shows less or no perturbation on the base

stacking and helicity bands, whereas, during intercalation mode,

intensity changes of both bands can be induced, therefore

modulating the right-handed B-conformation of DNA.⁶⁴The

CD spectra of CT-DNA (100 μM) upon the interaction with

1−5 showed very minute changes, for the positive band at 275

nm as well as for the negative band at 245 nm (Figure S10),

which suggests that the interaction of the oxidomolybdenum

complexes 1−5 were groove binding (possibly minor groove

binding mode) in nature. From the above DNA binding studies

it may be depicted that the naphthyl group interacts with the

DNA by groove binding mode, possibly by van der Waals

forces of interaction, apart from this the naphthyl group can

also interact with the DNA by hydrophobic interactions.^65b,c

Competitive DNA Binding Studies. Competitive DNA

binding studies by ﬂuorescence measurements using DAPI

could not be performed as the complexes 1−4 as well as the

ligands H₂L¹⁻⁴exhibited an intense ﬂuorescence in the same

region. In order to assess the major groove and intercalative

binding mode, competitive binding experiments were carried

out with methyl green (MG) and ethidium bromide (EB),

respectively.^38eBut, we did not observe any quenching in the

ﬂuorescence of MG and EB bound to CT-DNA (672 and 597

nm, respectively) upon the successive addition of the

complexes 1−5.^38eHence, the complexes do not interact

with CT-DNA either by major groove or intercalative mode of

binding. In another way, these two competitive binding results

hinted toward the possibility of interaction of these complexes

with the CT-DNA via minor groove binding mode. In fact,

thermal denaturation and circular dichroism ﬁndings also

indicated a similar possibility, i.e., the complexes 1−5 possibly

interact with the CT-DNA by minor groove binding mode.

DNA Cleavage Studies. Chemically Induced DNA

Cleavage. The chemically induced DNA cleavage activity of

1−5 (1−100 μM) was performed in the dark, using supercoiled

pUC19 DNA (300 ng) in 50 mM Tris−HCl buﬀer (pH 8.0) in

1% DMF, in the presence of hydrogen peroxide (500 μM) as

the oxidizing agent. The molybdenum complexes do not exhibit

any chemically induced DNA cleavage activity.

Table 5. DNA Binding Parameters for 1−5

a

b

complex

binding constant (K_b) (M⁻¹

)

ΔT_m(°C)

1

2

3

4

5

2.50 × 10⁴

9.36 × 10³

8.44 × 10³

3.20 × 10⁴

9.90 × 10⁴

+1.50

+1.00

+1.50

−2.00

+1.50

a

DNA binding constants were determined by the UV−vis spectral

b

method. Change in the melting temperature of CT-DNA.

addition of CT-DNA to the complex solution, the UV−vis

absorption bands in the region 275−390 nm showed a

hypochromic shift for 1−5 and an appreciable hyperchromic

shift in the LMCT band for complex 5 (Figure 6). Both the

hypochromism for 1−5 and hyperchromism for 5 indicate the

interaction between the oxidomolybdenum complexes and CT-

DNA. The hypochromic shift due to the intraligand transition

for the complexes 1−5 was taken to calculate the binding

aﬃnity of the complexes with CT-DNA. The resulting

hypochromicity of 1−5 in the intraligand transition band is

possibly due to the interaction between the electronic states of

the chromophores of the ligands and the DNA bases.^59−61a

The binding aﬃnity of the interaction between CT-DNA and

each of the molybdenum complexes 1−5 is indicated by the

binding constant, K_b, which was calculated using eq 1. The data

reported in Table 5 reveal that the DNA binding strengths of

the molybdenum complexes are in the order 5 > 4 > 1 > 2 > 3,

with the K_bvalues of 1−5 ranging from 9.90 × 10⁴to 8.44 ×

10³M⁻¹. The binding aﬃnity of the ligands to CT-DNA gave

values lower than their corresponding molybdenum complexes

(Figure S9 and Table S2). The order of the binding aﬃnity can

mainly be attributed to the small size of the complexes and

incorporation of the electron donating groups attached to the

aromatic rings in the ligands.^61b,cApart from this, both the

phenyl and the naphthyl groups in these ligands may also play

an important role in the interaction of the complexes with CT-

DNA.^61d

Thermal Denaturation Studies. In order to investigate

the stability of the CT-DNA in the presence and absence of the

molybdenum complexes 1−5, DNA melting experiments were

performed.^47aThe melting temperature (T_m) of CT-DNA in

the absence of any complex was ∼66 °C, which is in accordance

with our previous reports.^38c−gUpon interaction with 1−3 and

5, the T_mof CT-DNA increased very slightly (∼1.00−1.50 °C),

whereas, upon interaction with 4, the same was decreased

slightly (∼2.0 °C) in Figure 7 and Table 5. The low ΔT_mvalues

indicate that 1−3 and 5 may possibly bind to CT-DNA by

groove binding mode rather than an intercalative mode, which

Photoinduced DNA Cleavage. To evaluate whether 1−5

possess photonuclease activity, a photoinduced DNA cleavage

activity assay was carried out. The photoinduced DNA nuclease

activity of 1−5 on irradiation of UVA light of 350 nm for 3 h in

the absence and presence of the complexes was studied using

supercoiled (SC) pUC19 DNA (300 ng) in 50 mM Tris−HCl

buﬀer (pH 8.0) containing 1% DMF (Figure S11). The extent

of the DNA cleavage was conﬁrmed by the decrease in the

supercoiled pUC19 DNA (form I) and subsequent formation

of nicked circular DNA (form II) and linear DNA (form III).

The percentage of net DNA cleavage by the complexes was

estimated using the following equation:

K

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Figure 6. Electronic absorption spectra of 1 (a), 2 (b), 3 (c), 4 (d), and 5 (e) (25 μM) upon the titration of CT-DNA (0−50 μM). The experiments

were performed in 10 mM Tris−HCl buﬀer (pH 8.0) containing 1% DMF. Arrow indicates the changes in absorbance with respect to an increase in

the CT-DNA concentration. The linear ﬁt of [DNA]/(ε_a− ε_f) vs [DNA] is given in the inset. The binding constant (K_b) was calculated using eq 1.

The subscripts s and c refer to the sample and control,

respectively.⁶⁵Since we did not observe a DNA band

corresponding to form III in our photonuclease studies, the

concentration of form III is put as zero for both “control” and

“sample”. The DNA cleavage activity of the molybdenum

complexes 1−5 was carried out in a concentration dependent

manner from 1 to 100 μM. The net DNA cleavage percent by

1−5 was plotted with increasing concentration of the

molybdenum complexes (Figure 8). Approximately 1−5%

DNA cleavage was observed in the presence of 1 μM of 1−3

and 5 whereas at the same concentration 4 showed a DNA

cleavage of ∼37% (Figure 8). The DNA cleavage activity for

each of the complexes was saturated at a concentration of 50

μM. At this concentration, the photonuclease activity of 1, 2, 3,

4, and 5 was approximately, 17, 26, 36, 93, and 4%, respectively.

This indicated that 4 has the most promising photonuclease

activity among the complex series. Cleavage activity of 1−5

varies due to the diﬀerent functional groups present in the

ligand moieties.^38c−gComplex 4 exhibits the highest photo-

nuclease activity, which may be due to the presence of extended

aromatic ring on the naphthyl group^61dpresent in the ligand

moiety. Apart from this, the presence of the phenyl group and

the electron donating groups on the aromatic rings might also

inﬂuence the DNA cleavage activity of the complexes.^61cFrom

Figure 7. Derivative plot of thermal denaturation of CT-DNA (100

μM) in the absence and presence of 1−5 (25 μM) in 10 mM Tris−

HCl buﬀer (pH 8.0) containing 1% DMF. The ΔT_m(°C) of the

complexes as compared to CT-DNA is shown in the inset.

Net DNA cleavage %

form II_s+ 2 × form III_s

form I_s+ form II_s+ 2 × form III_s

=

form II_c+ 2 × form III_c

form I_c+ form II_c+ 2 × form III_c

−

(2)

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MCF-7 (human breast adenocarcinoma) and HCT-15 (colon

cancer) cells using the MTT assay as per our previous

protocol.^67i,jThe ligands H₂L¹⁻⁵and MoO₂(acac)₂gave high

IC₅₀values (>200 μM), whereas 1−5 gave values in the range

28.52−11.13 μM in MCF-7 cells and between 76.30 and 32.20

μM in HCT-15 cells (Table 6). In contrast, cisplatin, a widely

Table 6. Cytotoxicity of 1−5 as Measured by MTT Assay in

MCF-7, HCT-15, and HaCaT Cells

a

IC₅₀(μM)

complex

MCF-7

HCT-15

HaCaT

1

2

3

4

5

11.13 0.65

13.25 1.32

28.52 2.65

12.43 0.94

27.31 1.83

32.20 0.92

52.54 2.68

76.30 1.84

54.92 1.06

55.61 0.65

200.74 0.12

179.47 1.02

477.26 2.36

370.13 0.51

96.00 0.09

Figure 8. Concentration dependent DNA cleavage of 1−5; 300 ng of

SC pUC19 DNA at diﬀerent concentration of the complexes [1−100

μM in 50 mM Tris−HCl buﬀer (pH 8.0) containing 1% DMF] was

photoirradiated with UVA at 350 nm for 3 h. The net DNA cleavage

percent was determined using eq 2. Each of the data points measured

is mean standard deviation from triplicate experiments.

a

Values are mean SD, n = 3.

the control experiments it could be concluded that neither

DMF (1%) nor the ligand molecules H₂L¹⁻³and H₂L⁵, at a

concentration of 50 μM, showed any photoinduced DNA

cleavage activity, implying that DMF and the ligands H₂L¹⁻³

and H₂L⁵are cleavage inactive under similar experimental

conditions (Figure S12). However, the ligand molecule H₂L⁴, at

a concentration of 50 μM, showed a DNA cleavage activity of

∼20% (Figure S12), which increased signiﬁcantly upon

complexation (∼93% at a 50 μM complex concentration) to

molybdenum (Figure 8). This indicated that the better activity

of complex 4 was due to the presence of naphthyl moiety in the

ligand H₂L⁴.^66a

The photoinduced DNA cleavage activity of 1−5 was

examined in the presence of various additives, to discern the

mechanistic pathways of the photocleavage reactions. There can

be two possible mechanistic pathways by which the DNA

cleavage reaction proceeds, which involve molecular oxygen: (i)

a type II process where a singlet oxygen species (¹O₂) is

involved or (ii) a photoredox process where reactive hydroxyl

radicals (^•OH) are involved.^66bWhen NaN₃(a singlet oxygen

quencher) was added, the DNA cleavage activity of 4 was

inhibited by ∼24%, while in the presence of KI (a hydroxyl

radical quencher), it was inhibited by ∼54% (Figure S13 and

Figure S14). Further, in the presence of ^L-histidine (singlet

oxygen quencher) and ^D-mannitol (hydroxyl radical quencher),

the DNA cleavage activity of 4 was restrained by ∼14 and

∼19%, respectively (Figure S13 and Figure S14). However, no

inhibition of DNA cleavage was observed for 1−3 and 5 in the

presence of the additives (Figure S13). Therefore, it can be

concluded that 4 exhibits photoinduced DNA cleavage activity

probably via both singlet oxygen and hydroxyl radical pathways.

However, the mechanistic pathways associated with the

photoinduced DNA cleavage by 1−3 and 5 cannot be distinctly

ascertained.

used chemotherapeutic drug, is comparatively eﬀective in

MCF-7^68aand HCT-15^68bcells with an IC₅₀value of 7.8 and

12.20 μM respectively. The marked decrease in the inhibitory

ability of H₂L¹⁻⁴as well as their lower binding propensity to

CT-DNA compared to 1−5 indicates that incorporation of

molybdenum has a signiﬁcant eﬀect on cytotoxicity. This is

possibly because the polarity of the ligand and the central metal

ion is reduced by coordination through the charge equilibra-

tion. This favors the permeation of the complexes through the

lipid layer of the cell membrane.^68c,69The results of DNA

binding/cleavage ability for the ligands as well as the complexes

also indicate that the metal complexes exhibit greater biological

activities than the free ligands.⁷⁰

The cytotoxic activities of the complex series 1−5 can be

judged from their IC₅₀values (Table 6), with their dose

dependency illustrated in Figures 9 and 10.

Figure 9. Eﬀect of the complexes (1−5) on cell viability and growth of

MCF-7 cells. The cells were treated with diﬀerent concentrations of

the test compounds for 48 h, and then cell viability was calculated by

MTT assay. Data reported as the mean SD for n = 3.

The above results of the DNA interaction studies of the

synthesized molybdenum complexes 1−5 are in accordance

with previous reports of DNA binding/cleavage studies of

related oxidomolybdenum(VI)^67a−dand those of other

transition metal^67e−hsalen complexes. However, the DNA

cleavage activity of complex 4 (∼93% at a 50 μM complex

concentration) is exceptionally better than in the previous

reports.

IC₅₀values of the complexes 1, 4, and 2 ranges between

11.13 and 13.25 μM in the case of MCF-7, and in the case of

HCT-15 it ranges between 32.20 and 54.92 μM, while 3 and 5

have IC₅₀values in the range 27.31−28.52 μM in the case of

MCF-7 and between 55.61 and 76.30 in HCT-15 cells. Since

the variation in the IC₅₀values obtained for complexes 1, 4, and

2 is somewhat diﬀerent from that of 3 and 5, these complexes

can be combined into two groups, i.e., “group 1” corresponding

to the complexes with higher cytotoxicity (1, 4, and 2) and

Cytotoxicity Studies. MTT Assay. The antiproliferative

eﬃcacy of 1−5 was assayed by determining the viability of

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moiety.^66aAlso, the introduction of aromatic diimine bridges in

the salen complexes (1, 2, and 4) increases their cytotoxicity, as

evident from the higher IC₅₀value of complex 5 (less

cytotoxicity), which contains an aliphatic diimine bridge.^22c

The MTT assay for determining the cytotoxicity of metal

complexes on the HaCaT (immortalized human keratinocytes)

cell line was also performed. The very high IC₅₀values of 1−5

(96.00−477.26 μM, Table 6) and their dose dependency

(Figure S15) in HaCaT cells indicated that the compounds

exhibited no substantial cytotoxicity to normal keratinocytes.

In the recent past, some oxidomolybdenum(VI) complexes

have been reported to have signiﬁcant cytotoxic activities

against various human cancer cell lines including HL-60 and

K562 (leukemia), A-549 (lung cancer), HeLa (cervical

carcinoma), and MCF-7 (breast carcinoma).^24,34,67b,71Salen

complexes of some transition metals have also been reported to

show appreciable cytotoxic activities against human cancer cell

lines.^22a,c,72In few cases the results of the above studies are in

accordance with the synthesized molybdenum complexes 1−5,

while in some cases they exhibit better cytotoxicity than 1−5 in

terms of IC₅₀values.

Nuclear Morphological Studies. Investigation of apoptotic

potential of compounds is usually performed by DAPI staining

experiments.^38c−gHowever, it was found that the synthesized

molybdenum complexes 1−4 themselves exhibited ﬂuorescence

emission at the same wavelengths as DAPI (blue region).

Hence, the use of DAPI as staining agent was inappropriate in

the present situation, and therefore it was replaced by

propidium iodide, which exhibited red ﬂuorescence, to examine

the eﬀect of the test complexes on nuclear morphology of

MCF-7 and HCT-15 cells. To check the live cells in diﬀerent

treatment groups, the density of the PI stained nuclei was used.

Figure 11 and Figure 12 show images taken after PI staining in

Figure 10. Eﬀect of the complexes (1−5) on cell viability and growth

of HCT-15 cells. The cells were treated with diﬀerent concentrations

of the test compounds for 48 h and then cell viability was calculated by

MTT assay. Data reported as the mean SD for n = 3.

“group 2” corresponding to the complexes with lower

cytotoxicity (3 and 5). The cytotoxicity order of the complexes

matches completely neither with the DNA binding order nor

with the photonuclease activity order. However, group 1

complexes, with higher cytotoxicity, do exhibit comparatively

higher photonuclease activity than group 2 complexes, which

have lower cytotoxicity. The variation in results of cytotoxicity

of the complexes may be aﬀected by the various functional

groups in the salen ligands. The presence of the electron

withdrawing Br group in complex 3 leads to an increase in the

IC₅₀(less cytotoxicity),¹⁸while complex 1, which has no

substituents on the ligand fragment, and complex 2, which

contains an electron releasing −OMe group, exhibit lower IC₅₀

(higher cytotoxicity).¹⁸The considerable eﬃcacy of 4 in MCF-

7 cells, as also inferenced from DNA binding/cleavage studies,

may be due to the presence of the naphthyl group in the ligand

Figure 11. Eﬀect of treatment of complexes on the nuclear morphology of MCF-7 cells. The morphological changes in nuclei of MCF-7 cells from

control and treated groups that were washed twice with PBS (pH 7.4) and ﬁxed after incubation for 15 min with 3.7% of formaldehyde. The cells

were further washed twice with PBS and treated with 0.2% Triton X-100 in PBS for 30 s. Again, the washing was repeated with PBS and PI solution

(10 μg/mL) was added, and the sample was kept in the dark for 15 min. At the end, the cells were further washed with PBS and imaged under a

ﬂuorescence microscope (FLoid, Life Technologies). Arrows indicate the morphological changes in nuclei of MCF-7 cells observed on applying

increasing concentrations (5, 10, 25, and 50 μM) of 2 in comparison to control. The scale bar corresponds to 100 μm.

N

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Article

Figure 12. Eﬀect of treatment of complexes on the nuclear morphology of HCT-15 cells. The morphological changes in nuclei of HCT-15 cells from

control and treated groups that were washed twice with PBS (pH 7.4) and ﬁxed after incubation for 15 min with 3.7% of formaldehyde. The cells

were further washed twice with PBS and treated with 0.2% Triton X-100 in PBS for 30 s. Again, the washing was repeated with PBS and PI solution

(10 μg/mL) was added, and the sample was kept in the dark for 15 min. At the end, the cells were further washed with PBS and imaged under a

ﬂuorescence microscope (Olympus IX 70). Arrows indicate the morphological changes in nuclei of HCT-15 cells observed on applying increasing

concentrations (5, 10, 25, and 50 μM) of 2 in comparison to control. The scale bar corresponds to 50 μm.

red ﬂuorescence at diﬀerent concentrations of 2 on MCF-7 and

HCT-15 cells, respectively. Figures S16 and S17 show images

taken after PI staining of 1 and 3−5 on MCF-7 and HCT-15

cells, respectively. It is observed from the ﬁgures that the

number of PI stained nuclei was reduced in treated wells with

increasing concentrations of 1−5, indicating dose dependent

cell death.^73,74The gray scale images at 48 h after the treatment

with increasing concentrations of 1−5 are shown in Figure 11b

and Figure 12b for MCF-7 and HCT-15 cells, respectively. The

distinct nuclear condensation and fragmentation (indicated by

arrows) were seen in treated cells (Figures 11 and 12)

compared to control cells (Figures S16A and S17A).

cleavage in the ligand took place and a monomeric

dioxidomolybdenum(VI) complex [Mo^VIO₂L⁵] (5) was

furnished. The structures of dimeric metal−metal bonded

oxidomolybdenum(V) complexes synthesized from salen type

ligands containing aromatic imines were established for the ﬁrst

time through X-ray crystallography, and the mechanistic

pathway of their formation was also explained. The

4

disappearance of EPR signals in the Mo(V) intermediate I_d

after 30 min also proved the formation of corresponding

diamagnetic dimeric bis-μ-imido bridged oxidomolybdenum(V)

complexes, where antiferromagnetic coupling^48aoccurs be-

tween the two Mo(V) centers. Comparison with several other

transition metal complexes of similar ligand systems,⁴¹where

no such ligand transformation takes place, proved that the

hydrolysis and −CN bond cleavage of salophen ligands was

Mo assisted.

In vitro biological studies reveal that 1−5 show appreciable

DNA binding propensity. The CD and thermal melting studies

revealed that the complexes possibly interact with CT-DNA in

a manner consistent with groove binding mode, with binding

constants ranging from 10³to 10⁴M⁻¹. 1−5 exhibited good

photoinduced cleavage of pUC19 supercoiled plasmid DNA,

with 4 giving the maximum photoinduced DNA cleavage

activity of ∼93%. It is concluded from the mechanistic study

that the photolytic DNA cleavage of 4 proceeded via both

singlet oxygen and hydroxyl radical pathways, whereas the

mechanistic pathways associated with 1−3 and 5 could not be

distinctly ascertained. Also, results of antiproliferative activities

of the synthesized complexes suggested that they are

signiﬁcantly cytotoxic toward MCF-7 and HCT-15 cell lines.

The complexes containing the aromatic diimine moiety (1, 2,

CONCLUSIONS

■

Five salen type ligands from an aromatic diamine, o-

phenylenediamine (H₂L¹⁻⁴), and an aliphatic diamine, 1,2-

diaminopropane (H₂L⁵), were synthesized. H₂L¹⁻⁴furnished

dimeric bis-μ-imido bridged oxidomolybdenum(V) complexes

[Mo₂O₂L′₂¹⁻⁴] (1−4), where L′¹⁻⁴indicates the rearranged

V

form of the ligands H₂L¹⁻⁴, via an interesting mechanistic

pathway which involves the formation of rare monomeric

molybdenum(V) intermediate [Mo^VO(HL′¹⁻⁴)(OEt)] (I_d

)

1−4

by the molybdenum assisted hydrolysis of the imine bond and

subsequent −CN bond cleavage of the salophens. The

deprotonation and dimerization of Mo(V) intermediates ﬁnally

resulted in the formation of two imido bridges between the

Mo(V) centers containing a metal−metal bond. One of the

molybdenum(V) intermediates, [Mo^VO(HL′⁴)(OEt)] (I_d),

4

was isolated in the solid state and characterized by spectral

(IR, UV−vis, EPR), ESI-MS, and cyclic voltammetry studies. In

the case of H₂L⁵, no such imine hydrolysis or −CN bond

O

DOI: 10.1021/acs.inorgchem.7b01578

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Giester, G. Oxomolybdenum Coordination Compounds with Pyridine.

Syntheses and Structures of [Mo₄O₈(OCH₃)2Cl₂L₄]·2L (L = Py, 4-

MePy), [Mo₄O₈(OCH₃)₄(4-MePy)₄], [Mo₆O₁₂(OCH₃)6Py₄] and

[Mo₁₀O₂6Py₈]·7Py. Inorg. Chim. Acta 2000, 307, 33−41. (d) Zhou,

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and 4) were found to be more cytotoxic than the complex

containing the aliphatic diimine (5). The presence of the

electron withdrawing Br group in 3 lowered its cytotoxicity.

The better activity of 4 may be attributed to the presence of the

naphthyl moiety in the ligand system. Also, 1−5 did not exhibit

signiﬁcant cytotoxicity to HaCaT cells (immortalized human

keratinocytes). The present results will encourage further work

on oxidomolybdenum complexes for the development of metal-

based agents for anticancer studies.

̌ ̌

1464. (e) Modec, B.; Brencic, J. V. Novel Hydrogenmaleato

Molybdenum(V) Complexes Based on a Dinuclear Metal−Metal

Bonded Unit: Syntheses and Structural Characterization of

( P y H ) ₃[ M o ₂O ₄C l ₄( O O C C H

=

C H C O O H ) ] a n d

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.inorg-

chem.7b01578.

(PyH)₃[Mo₂O₄Br₄(OOCCHCHCOOH)] CH₃CN. Inorg. Chem.

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■

S

¹H NMR, UV−vis, FTIR, ESI-MS, absorbance, and CD

spectra, binding constant values, absorption spectral

traces, cyclic voltammogram, electronic absorption

spectra, and gel diagrams (PDF)

Accession Codes

CCDC 1491689−1491692 contain the supplementary crystal-

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

charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-

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

Cambridge Crystallographic Data Centre, 12 Union Road,

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

Online 2005, 61, m1553−m1555. (j) Tasn

̌

er, M.; Prugoveck

̌

i, B.;

Calogovic, D.

̌

Soldin, Z.; Prugovecki, S.; Rukavina, L.; Matkovic-

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Synthesis and Characterization of Oxomolybdenum(V) Dinuclear

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Characterization of a Dinuclear Mo^Vand a Hexanuclear Mixed-

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AUTHOR INFORMATION

Corresponding Author

*E-mail: rupamdinda@nitrkl.ac.in.

ORCID

Alok K. Panda: 0000-0002-4452-993X

Eugenio Garribba: 0000-0002-7229-5966

Rupam Dinda: 0000-0001-9452-7791

■

́ ́

137. (l) Cindric, M.; Pavlovic, G.; Vrdoljak, V.; Kamenar, B.

Hexanuclear Complexes of Molybdenum(V) Containing

[Mo₆O₁₂(OCH₃)₄(acac)₃]⁻Anion. Polyhedron 2000, 19, 1471−1478.

(m) Majumdar, M.; Patra, S. K.; Bera, J. K. Oxidative Route to

Polyoxomolybdates from Quadruply Bonded [Mo^II≡Mo^II] Precursor:

Structural Characterization of

a Tetranuclear Cluster

[Mo₄Cl₅O₈(pyNP)₂] (pyNP = (2-(2-pyridyl)1,8-naphthyridine)).

Polyhedron 2007, 26, 1597−1602. (n) Sreehari, S.; Luck, R. L. J.

Cluster Sci. 2010, 21, 525−541.

Notes

The authors declare no competing ﬁnancial interest.

(4) (a) Hsieh, T.-C.; Gebreyes, K.; Zubieta, J. Studies of

Molybdenum Thiolato-Complexes. The Chemical Characterization

and the Crystal and Molecular Structures of Two Sulphido-Bridged

Mo^VDimers, [PhCH₂PPh₃]₂[Mo₂O₂S₂(SC₂H₄S)₂] and

[HNEt₃]₂[Mo₂S₄(SC₂H₄S)₂]. Transition Met. Chem. 1985, 10, 81−

84. (b) Martinez, M.; Ooi, B.-L.; Sykes, A. G. Reaction Paths in the

Formation of Triangular and Cuboidal Molybdenum/Sulfur Cluster

Complexes as Aqua Ions by Reduction of Molybdenum(V) Dimers. J.

Am. Chem. Soc. 1987, 109, 4615−4619. (c) Koffi-Sokpa, E. I.; Calfee,

D. T.; Allred, B. R. T.; Davis, J. L.; Haub, E. K.; Rich, A. K.; Porter, R.

A.; Mashuta, M. S.; Richardson, J. F.; Noble, M. E. Activation of

Ligand Reactivity: Thiolate C−S and Dithiophosphate Ester C−O

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ACKNOWLEDGMENTS

■

The authors thank the reviewers for their comments and

suggestions, and Prof. J. C. Pessoa and Dr. A. Buchholz for

fruitful discussions which were helpful in preparing the revised

version of the manuscript. Funding for this research was

provided by Council of Scientiﬁc and Industrial Research,

Government of India (Grant No. 01(2735)/13/EMR-II) and

DBT, Government of India [Grant No. 6241 P112/RGCB/

PMD/DBT/RPDA/2015].

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Monomeric and Dimeric Oxidomolybdenum(V and VI) Complexes, Cytotoxicity, and DNA Interaction Studies: Molybdenum Assisted C=N Bond Cleavage of Salophen Ligands

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