In vitro cytotoxicity and catalytic evaluation of dioxidovanadium(v) complexes in an azohydrazone ligand environment

Article abstract of DOI:10.1039/c9nj01815h

Three new anionic dioxidovanadium(v) complexes (HNEt₃)[VO₂(L)^1-3] (1-3) of tridentate binegative aroylhydrazone ligands containing the azobenzene moiety were synthesized and structurally characterized. The aroylhydrazone ligands (H₂L^1-3) were derived from the condensation of 5-(arylazo) salicylaldehyde derivatives with the corresponding aroyl hydrazides. All the synthesized ligands and metal complexes were successfully characterized by several physicochemical techniques, namely, elemental analysis, electrospray ionization mass spectrometry, spectroscopic methods (IR, UV-vis and NMR), and cyclic voltammetry. Single-crystal X-ray diffraction crystallography of 1-3 revealed five-coordinate geometry, where the ligand coordinates to the metal centre in a binegative tridentate O, N, O coordinating anion and two oxido-O atoms, resulting in distortion towards the square pyramidal structure. The complexes were further evaluated for their in vitro cytotoxicity against HeLa and HT-29 cancer cell lines. All the complexes manifested a cytotoxic potential that was found to be comparable with that of clinically referred drugs, while complex 3 proved to be the most cytotoxic among the three complexes for both cell lines, which may be due to the synergistic effect of the naphthyl substituent in the azohydrazone ligand environment coordinated to the vanadium metal. The synthesized complexes 1-3 were probed as catalysts for the oxidative bromination of thymol and styrene as a functional mimic of vanadium haloperoxidases (VHPOs). All the reactions provided high percentages of conversion (>90%) with a high turnover frequency (TOF) in the presence of the catalysts 1-3. In particular, for the oxidative bromination of thymol, the percentage of conversion and TOF were in the ranges of 98-99% and 5380-7173 (h^-1), respectively. Besides, 3 bearing the naphthyl substituent showed the highest TOF among all the complexes for the oxidative bromination of both thymol and styrene.

Full text of DOI:10.1039/c9nj01815h

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In vitro cytotoxicity and catalytic evaluation of dioxidovanadium(V)
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complexes in azohydrazone ligand environment
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Monalisa Mohanty,a Shailendra K. Maurya,b Atanu Banerjee,a Sushree Aradhana Patra,a Mannar R. Maurya,b
Aurélien Crochet,c Krzysztof Brzezinski,d and Rupam Dinda*,a
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aDepartment of Chemistry, National Institute of Technology, Rourkela, 769008 Odisha, India. E-mail: rupamdinda.nitrkl@gmail.com.
bDepartment of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247667, India
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cDepartment of Chemistry, Fribourg Center for Nanomaterials, University of Fribourg, CH-1700 Fribourg, Switzerland
dInstitute of Chemistry, University of Bialystok, Ciolkowskiego 1K, 15-245, Bialystok, Poland.
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Abstract
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Three new anionic dioxidovanadium(V) complexes (HNEt3)[VO2(L)1-3] (1–3) of tridentate binegative aroylhydrazone ligands
containing azobenzene moiety have been synthesized and structurally characterized. The aroylhydrazone ligands (H2L1-3) were
derived from the condensation of 5-(arylazo) salicylaldehyde derivatives with corresponding aroyl hydrazides. All the
synthesized ligands and metal complexes were successfully characterized by several physicochemical techniques namely,
elemental analysis, electrospray ionization mass spectrometry, spectroscopic methods (IR, UV–Vis and NMR), and cyclic
voltammetry. Single crystal X-ray diffraction crystallography of 1–3 revealed five-coordinate geometry, where the ligand
coordinates to the metal centre in a binegative tridentate O,N,O coordinating anion and two oxido–O atoms, resulting in a
distorted towards square pyramidal structure. The complexes were further evaluated for their in vitro cytotoxicity against HeLa
and HT-29 cancer cell lines. All the complexes manifested cytotoxic potential that was found to be comparable with clinical
referred drugs. While, complex 3 proved to be the most cytotoxic among the three complexes for both the cell lines, which may
be due to the synergistic effect of the napthyl substituent in the azohydrazone ligand environment coordinated to the
vanadium metal. The synthesized complexes 1–3 were probed as catalysts for the oxidative bromination of thymol and styrene
as a functional mimic of vanadium haloperoxidases (VHPOs). All the reactions gave a high percentage of conversion (>90%) with
a high turnover frequency (TOF), in the presence of the catalysts 1–3. In particular, for the oxidative bromination of thymol the
percentage of conversion and TOF lies in the range of 98–99% and 5380–7173 (h−1) respectively. Besides, 3 bearing the napthyl
substituent showed the highest TOF among all the complexes for both oxidative bromination of thymol and styrene.
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Keywords: Dioxidovanadium(V) / Azohydrazones / X-ray crystallography / In vitro cytotoxicity / HeLa / HT-29 /oxidative
bromination of thymol / oxidative bromination of styrene
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Introduction
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Paramount interest is being accredited to the coordination chemistry of vanadium complexes due to its significance in various
biochemical, pharmacological, and catalytic activities.1 As vanadium metal possess the ability to assume various oxidation states
of +3, +4 and +5, it shows diverse biological roles and interacts with different biomolecules.2 For instance, various marine as
well as terrestrial organisms assemble vanadium for their life processes,3 namely vanadium haloperoxidase (V-HPOs) that is
found in marine algae can facilitate the oxidative halogenation of the aromatic organic complexes in the presence of organic
hydroperoxides, hydrogen peroxide or molecular oxygen.4 It also plays a vital role in enzymes, such as vanadium-dependent
nitrogenases, nitrate reductases and amavadin.5,6 Further, the pharmacological potential of vanadium complexes has been
successfully utilized in the treatment of type I and type II diabetes, while in the past decade it has also been studied as an anti-
tumor agent.7
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In this context, vanadium complexes have exhibited promising cytotoxicity against a wide spectrum of human cancer cells
lines.7 These metal complexes have shown reduced side effects, more potent action, better selectivity and higher cytotoxicity
both in vitro and in vivo models.1b,8 Besides, an vanadium complex i.e. Metvan has induced apoptosis in testicular and ovarian
tumors.9 Vanadium complexes demonstrates apoptosis inducing ability along with higher cytotoxicity in comparison to those of
free ligand.10 And as the pharmacological properties of metal complexes are a resultant of both the metal centre and the ligand
backbone, it is highly beneficial to employ bioactive ligands in the design of metal based drug. One of such ligand is
aroylhydrazones, –NH–N=CRR’ (R and R’ = H, alkyl, aryl) they are known to manifest a wide variety of properties like
antimicrobial and antitumor and therefore represent an important class of compounds with profound applications in chemistry
and medicine.11 In addition, the use of azobenzene moiety in the ligand backbone is of considerable interest because they
exhibit interesting biological properties. As, DNA is the primary intracellular target of any anti-cancer drugs, these azobenzene
derivatives which usually exists in their stable trans-isomer form are known to interact with DNA through base stacking
method.12 Further, higher substitution and planarity of ligands like benzyl and napthyl groups can also lead to an enhanced DNA
interaction.13
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On the other hand, the capability of vanadium to easily interconvert between various oxidation states, high affinity for oxygen
and also its Lewis acidic nature makes vanadium appropriate and relevant for applications in redox and Lewis acid catalyzed
reactions.14 Consequently, vanadium has been widely used in catalytic oxidation reactions of various organic substrates
including sulphoxidation,15 bromination,16 oxidation of alcohols,17 epoxidation of olefins,18 hydroxylation of aromatic
hydrocarbons.19 Moreover, the combination of vanadium metal with aroylhydrazone ligands is a good choice for catalytic
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oxidation because of the latter’s ability to resist oxidation.20 Therefore, the use of vanadium aroylhydrazone complexes which
are of low cost, environmental friendly complexes and which can also alleviate the oxidizing nature of the oxidants21 and in a
way generates new generation catalysts with a high turnover number (TON), selectivity and sustainability is the need of the
hour.
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As a part of our ongoing research on oxidovanadium(V) metal complexes of aroylazines in relation to their biological10a-c and
catalytic applications22a and some non-oxido vanadium(IV) complexes, which are very stable in solid as well as in solution
state,22b the present work reports the synthesis of three new dioxidovanadium(V) complexes (HNEt3)[VO2(L)1-3] (1–3) of
tridentate binegative aroylhydrazone ligands containing azobenzene moiety H2L1-3. Also it would be relevant to mention here
that, hydrogen bonding interactions for the design and synthesis of extended frameworks via supramolecular interactions has
been on the frontline of research, especially in the fields of molecular recognition and engineering of molecular solids.23 In this
study the use of triethylamine as a source of counter ion for the formation of polymeric aggregates has been made. This
counter ion plays a pivotal role because it increases the stability of the components of the supramolecular interactions through
hydrogen bonding networks by charge neutralization. Furthermore, these complexes also have enhanced water solubility
making them important subjects for biological and catalytic studies. All the synthesized ligands and metal complexes were
successfully characterized by elemental analysis, as well as various spectroscopic techniques (IR, UV–Vis and NMR). Considering
the above discussed biological and catalytic relevance of vanadium complexes containing O, N donor ligands, we have studied
the complexes for their antiproliferative efficacy against human cervical cancer (HeLa) and human colorectal adenocarcinoma
(HT-29) cell lines and also for their catalytic potential for the oxidative bromination of thymol and styrene as a functional mimic
of vanadium haloperoxidases (VHPOs).
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Experimental Section
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General Materials and methods
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All the chemicals were procured commercially and were used in their original form. VO(acac)2 was prepared as described in the
literature.24 Reagent grade solvents were dried and distilled prior to use. HeLa and HT-29 cell lines were obtained from National
Centre of Cell Science (NCCS), Pune, India. Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate buffered saline
(DPBS), trypsin EDTA solution, fetal bovine serum (FBS), antibiotic−antimitotic solution were procured from Himedia, Mumbai
India. While MTT (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium) and DAPI (4′,6-diamidino-2-phenylindole
dihydrochloride) were purchased from Sigma-Aldrich, India. Elemental analyses were performed on a Vario ELcube CHNS
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Elemental analyzer. IR spectra were recorded on a Perkin–Elmer Spectrum RXI spectrometer. H and 13C NMR spectra were
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studied with a Bruker Ultrashield 400 MHz spectrometer using SiMe4 as internal standard and 51V was studied using VOCl3 as an
internal standard. Electronic spectra were examined on a Lamda25, PerkinElmer spectrophotometer. Electrochemical data
were collected using PAR electrochemical analyser and a PC–controlled potentiostat/galvanostat (PAR 273A) at 298 K in a dry
nitrogen atmosphere. Cyclic voltammetry experiments were carried out with platinum working electrode, platinum auxiliary
electrode and SCE reference electrode. Commercially available TBAP (tetrabutyl ammonium perchlorate) was dried and used as
a supporting electrolyte for recording cyclic voltammograms of the complexes. ESI-MS of the complexes were recorded on a
Bruker MAT SSQ 710 spectrometer, employing complex concentration of 100 pmol/μL. A Shimadzu 2010 plus gas-
chromatograph fitted with an Rtx-1 capillary column (30 m × 0.25 mm × 0.25 μm) and a flame ionization detector (FID) were
utilized to analyse the reaction products. The quantification of the products was made on the basis of the relative peak area of
the respective product. Identity of these products was established using a GC-MS model Perkin-Elmer, Clarus 500 and
comparing the fragments of each product with the available library. The percent conversion of substrate and selectivity of the
products was calculated from GC data using the formulae:
Peak area of a substrate
Total area of substrate + products
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% Conversion of substrate
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100
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Peak area of a product
Total area of products
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% Selectivity of a product =
Caution: Although no problems were encountered during the course of this work, attention is drawn to the potentially
explosive nature of perchlorates.
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Synthesis of Ligands [H2L1-3
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The 5-(arylazo) salicyladehyde containing the azobenzene moiety was prepared as reported in the literature.25 The ligands [H2L1-
3] were then synthesized by condensing 5-(arylazo) salicyladehyde with their corresponding aroyl hydrazides (benzoic hydrazide
(H2L1), salicyclic hydrazide (H2L1) and 3-hydroxy-2-naphthoic hydrazide (H2L1) in a 1:1 molar ratio in an ethanolic medium. The
resulting light orange product was filtered, washed and dried over fused CaCl2.
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H2L1: Yield: 0.24 g (72%). Anal. Calcd for C20H16N4O2: C, 69.76; H, 4.68; N, 16.27. Found: C, 69.54; H, 4.59; N, 16.10 %. FTIR (KBr,
νmax/cm–1): ν(O–H) 3412, ν(N–H) 3056, ν(C=O) 1675, ν(C=N) 1612, ν(N=N) 1494. 1H NMR (400 MHz, DMSO-d6): δ (ppm) = 12.23
(s, 1H, OH), 11.83 (s, 1H, NH), 8.78 (s, 1H, CH), 8.25 7.13 (m, 13H, aromatic). 13C NMR (100 MHz, DMSO-d6): δ (ppm) =
168.49, 164.67, 152.48, 146.82 145.60, 133.26, 132.50, 131.35, 129.87, 129.03, 128.16, 126.30, 123.84, 122.75, 120.26, 117.78.
H2L2: Yield: 0.28 g (71%). Anal. Calcd for C20H16N4O3: C, 66.66; H, 4.48; N, 15.55. Found: C, 66.88; H, 4.89; N, 15.05 %. FTIR (KBr,
νmax/cm–1): ν(O–H) 3202, ν(N–H) 3059, ν(C=O) 1644, ν(C=N) 1607, ν(N=N) 1493. 1H NMR (400 MHz, DMSO-d6): δ (ppm) = 12.10
(s, 1H, OH), 11.88(s, 1H, phenolic OH) 11.82 (s, 1H, NH), 8.8 (s, 1H, CH), 8.26.9 (m, 12H, aromatic). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 163.55, 160.58, 153.64, 150.89, 148.71, 146.99, 146.49, 145.36, 141.78, 130.37, 128.78, 126.45, 123.91,
122.57, 121.33, 118.72, 114.81, 112.87.
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H2L3: Yield: 0.26 g (69%). Anal. Calcd for C24H18N4O3: C, 70.23; H, 4.42; N, 13.65. Found: C, 70.68; H, 4.47; N, 13.75 %. FTIR (KBr,
νmax/cm–1): ν(O–H) 3161, ν(N–H) 3056, ν(C=O) 1633, ν(C=N) 1625, ν(N=N) 1522. 1H NMR (400 MHz, DMSO-d6): δ (ppm) = 12.13
(s, 1H, OH), 12.01 (s, 1H, napthoic OH), 11.82 (s, 1H, NH), 8.81 (s, 1H, CH), 8.487.15 (m, 14H, aromatic). 13C NMR (100 MHz,
DMSO-d6): δ (ppm) = 164.23, 160.71, 154.47, 152.47, 147.27, 145.59, 136.37, 131.40, 130.79, 129.90, 129.15, 128.78, 127.24,
126.34, 124.31, 123.53, 122.76, 122.64, 120.65, 120.23, 117.80, 111.03.
Synthesis of dioxidovanadium (V) complexes (HNEt3)[VO2(L)1-3] (1–3)
The (HNEt3)[VO2(L)1-3] (1–3) complexes were synthesized by refluxing the azohydrazone ligands [H2L1-3] and [VO(acac)2] in an
alcohol medium using triethylamine as a base, for 3 h. Dark brown crystals, suitable for X-ray analysis were obtained from the
filtrate after 2−4 days.
(HNEt3)[VO2(L)1] (1): Yield: 0.28 g (52%). Anal. Calcd for C31H44N5O4V: C, 61.88; H, 7.37; N, 11.64. Found: C: 61.72; H: 7.90; N:
11.47 %. FTIR (KBr, νmax/cm–1): ν(C=N) 1614, ν(N=N) 1463, ν(V=O) 942, 954. UV-Vis (DMSO): λmax, nm (ε, mol-1 cm-1): 396(8933),
344(10,600), 261(6133).1H NMR (400 MHz, DMSO-d6): δ (ppm) = 9.20 (s, 1H, CH), 8.916.94 (m, 13H, aromatic), 3.08 (m, 6H,
CH2), 1.16 (t, 9H, CH3). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 170.55, 168.58, 152.46, 146.71, 146.62, 146.49, 145.56,
131.37, 129.88, 126.54, 123.29, 122.75, 120.41, 117.72, 115.81, 112.67, 46.27, 9.22. 51V NMR (105MHz, DMSO-d6, ppm): δ = –
529. ESI-MS: (CH3OH; negative ion mode): m/z 425.1 [M–(Et3NH)+]+.
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(HNEt3)[VO2(L)2] (2): Yield: 0.34 g (55%). Anal. Calcd for C31H44N5O4V: C, 61.88; H, 7.37; N, 11.64. Found: C: 61.01; H: 7.70; N:
11.10 %. FTIR (KBr, νmax/cm–1): ν(O–H) 3550, ν(C=N) 1607, ν(N=N) 1479, ν(V=O) 947, 960. UV-Vis (DMSO): λmax, nm (ε, mol-1 cm-
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1): 395(8405), 346(10,328), 265(6620). H NMR (400 MHz, DMSO-d6): δ (ppm) = 12.15 (s, 1H, phenolic OH), 9.31 (s, 1H, CH),
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9.056.98 (m, 12H, aromatic), 3.08 (m, 6H, CH2), 1.16 (t, 9H, CH3). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 170.99, 168.28,
159.09, 157.00, 152.58, 143.76, 133.40, 131.52, 130.94, 129.86, 129.57, 126.14, 122.59, 121.57, 119.89, 119.28, 117.06, 115.72,
46.22, 9.12. 51V NMR (105MHz, DMSO-d6, ppm): δ = –537. ESI-MS: (CH3OH; negative ion mode): m/z 441.2 [M–(Et3NH)+]+ (2).
(HNEt3)[VO2(L)3] (3): Yield: 0.31 g (57%). Anal. Calcd for C35H46N5O5V: C, 58.49; H, 6.45; N, 9.75. Found: C: 58.07; H: 6.69; N: 9.86
%. FTIR (KBr, νmax/cm–1): ν(O–H) 3442, ν(C=N) 1609, ν(N=N) 1469, ν(V=O) 947, 959. UV-Vis (DMSO): λmax, nm (ε, mol-1 cm-1):
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390(9027), 342(10,756), 263(6135). H NMR (400 MHz, DMSO-d6): δ (ppm) = 12.04 (s, 1H, napthoic OH), 9.37 (s, 1H, CH),
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8.526.98 (m, 14H, aromatic), 3.08 (m, 6H, CH2), 1.15 (t, 9H, CH3). 13C NMR (100 MHz, DMSO-d6): δ (ppm) = 170.45, 168.39,
157.72, 155.22, 152.57, 143.81, 136.53, 131.63, 130.97, 129.86, 129.26, 128.48, 127.36, 126.33, 126.28, 123.86, 122.59, 121.64,
119.97, 119.58, 118.18, 110.75, 46.26, 9.15. 51V NMR (105MHz, DMSO-d6, ppm): δ = –537. ESI-MS: (CH3OH; negative ion mode):
m/z 491.1 [M–(Et3NH)+]+.
Crystallography
Single crystals of the complexes (1−3) suitable for X-ray diffraction study were obtained at room temperature by slow
evaporation from their ethanol/methanol solution. Crystals of complex 1−2 were mounted on a loop in oil on a Stoe IPDS2
diffractometer equipped with an Oxford Cryosystem open flow cryostat and a Mo Kα radiator (λ) 0.71073 Å. The crystals were
kept at 298(2) K (1) and at (2) during data collection, while the X-ray diffraction data of complex (3) was collected at 100(2) K on
SuperNova diffractometer (Rigaku) with a CCD detector and Cu Kα radiation. Crystallographic data and details of refinement of
all the complexes are summarized in Table 1. The structures were solved through Olex2,26 with the help of ShelXT27 structure
solution program using intrinsic phasing and refined with the ShelXL28 refinement package using Least Squares minimisation. All
hydrogen atoms were initially located in electron-density difference maps. C-H hydrogen atoms were constrained to idealized
positions, with C-H = 0.95-1.00 Å and with Uiso(H) = 1.5Ueq(C) for methyl hydrogen atoms and Uiso(H) = 1.2Ueq(C) for others. The
ammonium and hydroxyl hydrogen atoms were freely refined without any constraints or restraints, while the non-hydrogen
atoms were refined anisotropically. PLATON software29 was used to validate the crystallographic data.
26
27
28
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Table 1. Crystal Data and Structure Refinement Details for 1−3.
5
6
7
8
Complex
Empirical formula
1
C26 H30 N5 O4 V
527.49
298(2) K
0.71073
2
C26 H30 N5 O5 V
543.49
200(2) K
0.71073
3
C30 H32 N5 O5 V
Formula weight
Temperature
Wavelength[Å]
Crystal system
Space group
Unit cell dimensions
a [Å]
593.55
100 K
1.54184
triclinic
P -1
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Monoclinic
P 21
Monoclinic
P 21
6.9776(6)
8.6271(7)
22.0397(18)
90
6.8790(3)
8.5804(3)
22.3944(11)
90
9.1706(2)
11.5525(2)
15.4622(3)
109.451(2)
b [Å]
c [Å]
 [°]
 [°]
 [°]
94.002(7)
91.071(4)
93.169(2)
112.197(2)
1399.04(5)
90
90
3
1323.48(19)
2
1321.59(10)
2
Volume [Å ]
Z
2
Density calculated
3
3
1.409 g/cm-3
1.324 mg/m
1.366 mg/m
-1
0.415
0.420
3.37
Absorption coefficient [mm
F(000)
]
552
568
620
3
0.13 x 0.1 x 0.05
0.26 × 0.13 × 0.07
0.24 x 0.18 x 0.07
Crystal size [mm ]
Theta range for data collection [°]
Reflections collected
Independent reflections, R(int)
Data completeness [%]
5.072 to 50.428
17318
4686, 0.1554
98.8 %
4684/66/294
0.873
3.638 to 50.362
16895
4671, 0.0744
98.9 %
4671/3/340
1.002
4.4 to 76.4
59124
5860
99.8
5860/0/436
1.09
Data / restraints / parameters
2
Goodness-of-fit on F
Final R indices [I>2sigma(I)]
R1 = 0.0499, wR2 = 0.0919 R1
=
0.0643, wR2
=
=
R1 = 0.039, wR2=0.113
0.1478
R indices (all data)
R1 = 0.1219, wR2 = 0.1165 R1
=
0.0880, wR2
R1 = 0.0394, wR =
0.1608
0.019(17)
n/a
0.1129
-
n/a
Absolute structure parameter
Extinction coefficient
0.01(5)
n/a
0.217 and -0.37
-3
1.06 and -0.32
0.71 and -0.58
Largest diff. peak and hole e.Å
CCDC number
1903090
1903091
1902693
2
3
4
Cytotoxic study
MTT assay
5
6
The in vitro cytotoxicity of 1−3 was studied against human cervical cancer (HeLa), colon cancer (HT-29) and non-cancerous
human epidermal keratinocyte cells (HaCaT) through MTT dye reduction assay. Prior to the experiment, the cells were
maintained in DMEM media supplemented with 10% FBS and penicillin–streptomycin solution in a humidified (95% humidity)
CO2 incubator (5% CO2) at 37 °C with uniform passages at 70–80% confluence. For the present study, the cells were harvested
by trypsinization and seeded at a concentration of 1 × 104 cells per well into a 96-well plate. After 12 h of initial seeding, HeLa
and HT-29 cells were subjected to treatment with six different concentrations (100, 50, 10, 7, 5, 3 μg/mL) of each complex,
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while HaCaT cells were subjected to treatment with four different concentrations (100, 50, 10, 5 μg/mL) of each complex. The
final working concentration of the complexes was prepared in complete DMEM media. Untreated cells cultured in media alone
were taken as control. Subsequently after 12, 24 and 48 h, the viability of the cells was tested by MTT assay and the optical
density of these cells was measured at 595 nm using a micro plate reader spectrophotometer.10a Cytotoxicity of the compounds
was checked by measuring the percentage cell viability of the cells with respect to control. The experiments were performed
four times (n=4), and the data were expressed as mean ± SD. Single variance ANOVA under 95% confidence interval was used to
evaluate the statistical significance of the data.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
30
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33
34
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45
46
47
48
49
50
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56
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59
60
8
9
Nuclear staining assay
The nuclear morphological change during cell death of the cells in response to treatment with the complexes (1−3) was studied
through DAPI staining method.31 HeLa and HT-29 cells were treated with test compounds at a concentration of 10 μg/ml for 24
h. After treatment, the cells were fixed with 4% formaldehyde for 15 min, followed by permeabilization with 0.1% Triton X-100.
They were subsequently stained with DAPI for 5 min at 37 °C and were washed with PBS. Untreated cells were used as control
experiments. The cells were then examined by confocal microscopy (Leica, SP8).
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22
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Catalytic reactions of (HNEt3)[VO2(L)1-3] (1–3)
Oxidative bromination of thymol
Oxidative bromination of thymol was carried out taking complexes 1–3 as catalyst precursors. As a representative, thymol (1.5
g, 10 mmol) was added to an aqueous solution (20 mL) of KBr (1.19 g, 10 mmol), 30% aqueous H2O2 (1.13 g, 10 mmol) in a 50
mL reaction flask. The catalyst (0.0005 g) and 70% HClO4 (1.43 g, 10 mmol) was added, and the reaction mixture was stirred at
room temperature. In all batches, the experimental conditions (e.g., stirring speed, the size of the magnetic bar and reaction
flask) were similarly maintained. After 1 h, the products were extracted in the hexane layer and were injected into the GC. The
identity of all the products was confirmed by GC-MS.
Oxidative bromination of styrene
Styrene (1.04 g, 10 mmol) was added to an aqueous solution (20 mL) of KBr (1.19 g, 10 mmol) and 30% aqueous H2O2 (1.13 g,
10 mmol) in a 50 mL reaction flask. The catalyst (0.0005 g) and 70% HClO4 (1.43 g, 10 mmol) was added, and the reaction
mixture was stirred at room temperature. In all batches, the experimental conditions (e.g., stirring speed, the size of the
magnetic bar and reaction flask) were similarly maintained. The effect of various parameters such as the amount of catalyst,
oxidant, NaHCO3, and solvent were checked to optimize the conditions for the best performance of the catalyst. After 1 h, the
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products were extracted in the hexane layer and were injected into the GC. The identity of all the products was confirmed by
GC-MS.
7
8
3
Results and discussion
9
4
5
Synthesis
10
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13
14
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18
19
20
21
22
23
24
25
26
27
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29
30
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32
33
34
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45
46
47
48
49
50
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52
53
54
55
56
57
58
59
60
In the present study, three dioxidovanadium(V) complexes (HNEt3)[VO2(L)1-3] (1–3) have been synthesized from azohydrazone
ligands [H2L1-3]. The synthesis of the complexes is summarized in Scheme 1. The reaction of the azohydrazone with VO(acac)2
proceeded smoothly in the molar ratio of 1:1 in alcohol medium in the presence of triethylamine as a base. Single crystals of
complexes 1−3 were obtained directly from the slow evaporation of the filtrate of the reaction mixture. Further, the purity of
these complexes was confirmed by elemental analyses. NMR spectra and ESI-MS were consistent with the X-ray structures. The
complexes were soluble in CH3CN, ethanol, methanol, DMF, DMSO and partially soluble in water. Magnetic susceptibility and
molar conductivity data revealed that all the complexes (1−3) are diamagnetic and electrically non-conducting in solution. The
detailed characterization of all the complexes has been discussed in the respective section (IR, UV-Vis, NMR, ESI-MS and X-Ray
crystallography).
6
7
8
9
10
11
12
13
R
N
O
N
O
VO(acac)2
EtOH/MeOH, Et3N
V
H
Et
Et
R
N
N
H
N
O
N
N
O
N
N
Reflux
O
Et
H2L1-3
OH
(HNEt3)[VO2(L)1-3
]
R
Complex
(HNEt3)[VO2(L)1](1)
(HNEt3)[VO2(L)2](2)
HO
HO
(HNEt3)[VO2(L)3](3)
14
15
Scheme 1. Schematic Representation for the Synthesis of (HNEt3)[VO2(L)1-3] (1–3).
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2
Spectral Characteristics
The spectral characteristics of all the ligands (H2L1–3) and complexes (1–3) are given in the Experimental Section. The FTIR
spectra of the ligands show a broad band in the range of 3202−3412 cm-1 due to the stretching vibration of a −OH group. Due to
the presence of a –NH group in the ligand a broad band is observed between 3056−3059 cm-1 while the −C=O stretching
frequency is obtained in the range of 1644−1675 cm-1. The stretching frequency for –C=N and –N=N is obtained in the range of
16121625 cm-1 and 1494−1522 cm-1 respectively. The disappearance of −NH, −C=O, −OH stretching bands in the IR spectra of
the complexes (1−3) indicate the formation of metal complexes. However, there is a peak for the pending phenolic OH (2) and
napthoic OH (3) group present in the ligand backbone in the range 3442−3550 cm-1. The sharp peak at 1609−1614 cm-1 is
probably due to the −C=N− moiety in the complex formed.32 The presence of two strong peaks in the range 942−960 cm-1 is due
to the V=O stretching which indicates the presence of dioxido group in the complexes.10a,c,33
3
7
8
9
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
6
7
8
9
10
11
12
13
14
The electronic spectra of all the complexes were recorded in DMSO and were of similar pattern. They exhibited medium
intensity bands in the range 396−390 nm which can be assignable to ligand to metal charge transfer (LMCT) and also displayed
strong intensity bands within 261−342 nm range, due to ligand centered transitions.32a,b A representative absorbance spectrum
of 1 is given in Figure 1.
2.0
344
1.5
1.0
0.5
0.0
396
261
300
400
500
600
700
Wavelength (nm)
15
16
Figure 1. UV–vis spectra of 1 (1.5 × 10−4 M) in DMSO.
17
18
The 1H and 13C NMR of all the ligands and their corresponding compounds were recorded in DMSO–d6 and the data are given in
1
the experimental section. The H NMR spectrum of the free ligands exhibits singlet resonance in the range δ = 12.23–11.88
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2
ppm due to −OH group. However, the ligand H2L2 and H2L3 exhibit one extra singlet peak in the region δ = 12.01–11.88 ppm for
phenolic OH (H2L2) and napthoic OH (H2L3). The singlets in the range δ = 11.83–11.82 ppm and δ = 8.80–8.78 ppm are due to
−NH group and azomethine –CH proton respectively. The aromatic protons of the ligands are clearly observed in the expected
region δ = 8.48–6.9 ppm as multiplets.32a,b In the NMR spectra of complexes 1−3, the peak for the aromatic −OH and −NH
proton disappeared due to the deprotonation at the time of complexation, while the peak in the region δ = 12.15–12.04 ppm is
retained for that of pending phenolic OH (2) and napthoic OH (3) group present in the ligand backbone. The signal for
azomethine –CH proton in the complexes appear in the range δ = 9.37–9.20. The aromatic signals for the complexes appear in
the range of δ = 9.0–6.9 ppm as multiplet. There is two additional peak observed at the aliphatic region δ = 3.08 and at δ = 1.15
ppm that is attributed to the presence of a triethylamine moiety. In the 13C NMR spectra of the ligands H2L1–3, spectral signals
for the aromatic carbons are found in the downfield region in the range δ = 168.49−111.03 ppm. While in the 13C NMR spectra
of complexes 1−3, signals for the aromatic carbons are found in the downfield region in the range δ = 170.99−110.75 ppm. Also,
two additional peaks are found in the up field region (δ = 46.27−9.12) due to the presence of two aliphatic carbon atoms
(−CH2CH3) of triethylamine group. The 51V NMR spectra of 1–3 shows a singlet in the range δ ꓿ −537 to −529 ppm. Chemical
shifts obtained in this range are typical for dioxidovanadium(V) species.10a,c
3
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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39
40
41
42
43
44
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49
50
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53
54
55
56
57
58
59
60
5
6
7
8
9
10
11
12
13
14
15
16
17
Mass spectra of all the complexes were recorded in methanol in the ESI mode, by applying a complex concentration of 100
pmol/μL. The characteristic ion peaks for 1−3 appear at m/z 425.1 [M–(Et3NH)+]+, 441.2 [M–(Et3NH)+]+ and 491.1 [M–(Et3NH)+]+
respectively, in the negative ion mode. Figure S1 depicts the representative ESI-MS of 1.
18
19
20
21
22
23
24
25
26
27
28
Electrochemical properties
The redox properties of the complexes (1−3) were studied in DMSO solution in the presence of 0.1 M TBAP as electrolyte using
SCE as reference electrode, Pt as working electrode, while Pt wire was used as auxiliary electrode at a variable scan rate of 50,
100 and 150 mV s−1. The voltammograms of all the complexes shows a similar pattern and the redox potential data of all the
complexes are summarized in Table 2. As a representative, the voltammogram of 1 has been discussed herein and is depicted in
Figure 2 and the rest of the voltammograms of 2 and 3 are given in Figures S2–S3. There are irreversible single electron
cathodic responses of 1 at −0.33, −0.66 and −1.25 V and anodic response at 0.71 V which may be assigned to the ligand centred
reduction and oxidation respectively. Conversely, the complex displays quasi reversible single electron transfer redox at E1/2
value at −1.61 V which is designated to the V(V)/V(IV) reduction couple of 1 containing reduced ligand.34
As a representative, voltammogram of the ligand H2L1 is shown in Figure S4. The voltammogram at different scan rates were
compared to confirm the quasi-reversible nature of the redox processes. Also, identical experimental conditions were
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maintained to verify the single electron processes and its current height was compared with that of the standard
ferrocene−ferrocenium couple.
7
8
3
Table 2. Cyclic Voltammetric Resultsa of 1–3 at 298 K in DMSO.
9
Complex
Ec1/2 (V)
ΔEcP (mV)
249
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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52
53
54
55
56
57
58
59
60
1
2
3
-1.61
-1.58
248
-1.55
240
a
4
5
In DMSO at a variable scan rate 50, 100 and 150 mV s-1. Ec = (Ea + Ec )/2, where Ea and Ec are anodic and cathodic peak
1/2
P
P
P
P
potentials vs. SCE, respectively.  EcP = EaP – Ec .
P
V(V)
V(IV)
150Scan
100Scan
50Scan
10A
V(V)
V(IV)
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
Potential vsSCE
6
7
Figure 2. Cyclic Voltammogram of 1 (10−3 M) in DMSO.
8
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X-ray structure description
5
6
7
8
1
2
3
Even though the preliminary characterization data (elemental analysis, H and 13C NMR and IR) suggested the presence of a
ligand, two oxido groups and a counter ion in the complexes 1–3, they could not manifest any definite stereochemistry for the
complexes or predict the coordination mode of H2L1–3. In order to validate the coordination mode of the ligands and the
supramolecular geometries possible in these complexes, the structure of 1–3 has been determined by X-ray crystallography.
4
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
31
32
33
34
35
36
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39
40
41
42
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44
45
46
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48
49
50
51
52
53
54
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56
57
58
59
60
5
1
6
The X–ray structures of the complexes reveal that all the evidences provided by the H NMR spectra is correct. The relevant
7
bond parameters are mentioned in Table 3, while the molecular structure along with the atom numbering scheme for 1–3 is
depicted in Figure 3. The structure reveals that the vanadium atom which is present in a penta-coordinated fashion is bound by
a tridentate binegative anion coordinating via phenolate oxygen-O(2), imine nitrogen-N(2) and enolic oxygen-O(1) and two
oxido-O(3), O(4) atoms, the charge of this moiety being balanced by a triethylamine cation. The resulting O4N donor set in the
complexes 1–3 is distorted toward square pyramidal based on a 휏 value in the range 0.08–0.2429 which is evident from the ideal
values of 0.0 and 1.0 for square pyramidal and trigonal bipyramidal geometries, respectively.35
8
9
10
11
12
1
2
3
13
Figure 3. The molecular structure of 1–3 with atomic numbering scheme used.
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Table 3. Selected Geometric Parameters for 1–3.
5
6
7
8
Bond distances (Å)
V(1)–O(1)
1
2
3
1.956(5)
1.903(5)
1.610(5)
1.636(5)
2.146(6)
1.398(8)
1.226(8)
1.970(5)
1.910(5)
1.603(5)
1.639(5)
2.124(6)
1.416(8)
1.233(10)
1.982(1)
1.902(1)
1.611(1)
1.636(2)
2.150(1)
1.392(2)
1.258(2)
V(1)–O(2)
V(1)–O(3)
V(1)–O(4)
V(1)–N(2)
N(1)–N(2)
N(3)-N(4)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bond angles (°)
O(1)–V(1)–N(2)
O(2)–V(1)–N(2)
O(2)–V(1)–O(1)
O(3)–V(1)–N(2)
O(3)–V(1)–O(1)
O(3)–V(1)–O(2)
O(3)–V(1)–O(4)
O(4)–V(1)–N(2)
O(4)–V(1)–O(1)
O(4)–V(1)–O(2)
73.2(2)
82.1(2)
73.9(2)
81.2(2)
73.53(5)
81.79(6)
151.65(6)
112.22(7)
101.14(7)
100.96(7)
110.31(8)
136.99(7)
92.44(7)
96.31(7)
148.0(2)
106.8(2)
102.0(3)
104.4(3)
109.7(3)
143.1(3)
93.7(2)
149.1(2)
109.0(3)
101.4(3)
103.8(3)
109.1(3)
141.5(3)
93.3(2)
94.0(3)
95.1(3)
2
3
The azohydrazone ligands form a six membered and a five membered chelate ring at the V(V) metal center with O(2)–V(1)–N(2)
and O(1)–V(1)–N(2) bite angles of 82.1(2)°– 81.79(6)° and 73.9(2)°–73.2(2)° respectively. One of the two oxido groups O(4) is
located terminally at the apical position of the square pyramid and trans to the imine nitrogen N(2), while the other oxido
group O(3) is located in the axial plane. V–O(4) bond distances are slightly longer than that of V–O(3) due to the trans effect,
however such bond length are longer than that of the monoalkoxido vanadium(V) complexes containing only one terminal
oxygen atom.32b The angle between the two oxido atoms O(3) and O(4), i.e. O(3)–V(1)–O(4) lies in the range 110.31(8)°–
109.1(3). Both the V═O bond are shorter than their corresponding V–O(phenolate) and V–O (enolate) bonds suggesting
stronger binding of the oxido group compared to those of the phenolate and enolate oxygen atoms.36
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
The supramolecular architectures show that the complexes are bound to the triethylamine cation moiety by V═O(4)…H–N(5)
hydrogen bond (red dotted lines, Figure 4), whereas there is an additional intra molecular hydrogen bonding O(5)–H…N(1)
present for complex 2 and 3 (blue dotted lines, Figure 4(b) and (c)) involving the hydrogen of napthoic OH and imine nitrogen
N(1) atom. Detail parameter of the classical hydrogen bonding of the complexes (1−3) has been listed in Table 4.
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(a)
(b)
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(c)
1
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4
Figure 4. Supramolecular architectures of (a) 1, (b) 2 and (c) 3. Dashed lines represents intramolecular (blue) and intermolecular
(red) hydrogen bonds, and for clarity only hydrogen atoms involved in these interactions are shown. For (c) only more
populated location of the disordered protonated amine is shown.
5
Table 4. Hydrogen bond geometry for 1–3.
Complex
D-H…A
D-H [Å]
0.86(3)
0.90(3)
0.95(8)
0.83(3)
0.86(2)
H…A [Å]
1.81(3)
1.79(3)
1.77(5)
1.84(3)
1.86(2)
D…A [Å]
2.668(9)
2.681(9)
2.589(9)
2.605(2)
2.711(2)
D-H…A []
176(1)
169(1)
142(4)
151(2)
172(2)
1
2
N5-H5N-O4
N5-H5N-O4
O5-H5O-N1
O5-H5…N1
N5-H5B…O4i
3
6
7
i symmetry code: (i) –x+2, -y+1, -z+1
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1
2
Cytotoxic study
MTT assay
3
Following successful synthesis and characterization, in vitro cytotoxicity of the complexes (1−3) was evaluated by determining
the viability of cervical cancer (HeLa) and human colon cancer (HT-29) cell lines through MTT assay. These studies were
performed in order to probe and observe the influence of the vanadium metal centre in the aroylhydrazone ligand environment
appended with an azobenzene moiety, on the antiproliferative efficacy of the reported complexes. The analysis revealed that
all the complexes were quite effective in killing the cancer cells and were highly cytotoxic even at a low concentration of
3 μg/ml. It is also observed that, upon increase in time of exposure of the complexes (3 μg/ml) from 12 to 24 h there is a
gradual decrease in the HeLa and HT-29 cell viability (Figure 5A and 5B). Following that, after 48 h of exposure there is almost
60 and 70% cell death with respect to the control for HeLa and HT-29 cell lines respectively, at the highest concentration of the
complexes (100 μg/ml) tested (Figure 5C and 5D). These set of complexes showed similar cytotoxicity towards both the cancer
cells.
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The complexes exhibited IC50 values in the range of 4.7 − 5.5 μM and 5.29 −7.48 μM against HeLa and HT-29 cells (Table 5)
respectively. Out of the three complexes, the maximum cytotoxicity against HeLa cells was shown by 3, while the other two
complexes gave near about same IC50 values. A similar fashion was also seen for HT-29 cells, where 3 was the maximum
cytotoxic, followed by 2 and 1. Also it seems that there is a tendency for a higher toxicity of the compounds being tested for
HeLa than for HT-29 cells. The complexes showed higher antiproliferative activity as compared to their corresponding ligand
and metal precursor towards both the cancer cells (IC50 values >100 μM). In order to determine the cytotoxic effect of the
present complexes on the viability of physiological cells, we have performed similar study on human skin keratinocyte cells
(HaCaT). It was noticed that there is subtle decrease in the HaCaT cell viability upon increase in the time of exposure from 12 to
24 h of the complexes (5 μg/ml) (Figure 5E). The results (Figure 5F, Table 5) signify that unlike for the cancers cells, the
complexes are less toxic towards HaCaT at lower concentrations and are cytotoxic only at concentration of 50 μg/ml and higher
at 48h of exposure. This can be an indication that the complexes may be selective in nature towards cancer cells. A feasible
rationalization would be the decrease in the polarity of the ligand and the central metal ion after charge equilibration which
aids in the permeation of the complexes through the lipid layer and possible higher uptake by the cell membrane.37
Consequently leading to synergistic effects and improving the cytotoxicity of the overall complex. Furthermore, the presence of
a napthyl substituent13 in the ligand backbone can be attributed to the lower IC50 value of 3 in comparison to other complexes.
28
29
Under similar experimental conditions, commonly used chemotherapeutic drugs like cisplatin and cyclophosphamide have
shown comparable effectiveness against HeLa with IC50 values of 12.2, and 1689 μM, and against HT-29 cells, IC50 values of 70
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and 21.5 μM respectively.38 The present series of vanadium complexes in an azohydrazone ligand environment have shown
improved in vitro cytotoxicity results against HT-29 cell lines in comparison to our earlier reports on arylazovanadium(IV)
complexes10e and also to other vanadium complexes (>47 μM against HT-29 for [VO(sal-L-tryp)(acetylethTSC)]·C2H5OH [VO(sal-
L-tryp)(Me-ATSC)], and [VO(salL-tryp)(N-ethhymethohcarbthio)]·H2O),39 whereas the antiproliferative potential of 1−3 was also
better than or comparable to our earlier reports on vanadium10 and molybdenum hydrazone40 complexes against HeLa cells.
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A)
B)
*
*
*
*
*
*
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C)
E)
D)
F)
*
*
45
46
47
1
Figure 5. Effect of 1−3 (3 μg/ml) on cell viability of HeLa and HT-29 cells at 12h (A) and 24h (B) exposure and at 48 h (C and D)
exposure taking 3, 5, 7, 10, 50, 100 μg/ml concentration of the complexes. Effect of 1−3 (5 μg/ml) on cell viability of HaCaT at
12h and 24h (E) of exposure and at 48 h (F) exposure taking 5, 10, 50, 100 μg/ml concentration of the complexes. The cell
viability was calculated by MTT assay. Data reported as the mean ± SD for n = 4. *P < 0.05 was considered statistically
significant.
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Table 5. IC50 value of 1‒3 against HeLa, HT-29 and HaCaT Cells after 48 h Exposure.
5
6
7
IC50 (µM)
Complex
HeLa cells HT-29 cells HaCaT cells
8
9
5.5 ± 0.75 7.48 ± 1.04 84.68 ± 0.6
5.46 ± 1.59 6.73 ± 0.91 75.15 ± 3.3
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4.7 ± 0.55
5.29 ± 1.1 68.71 ± 0.29
2
3
Nuclear staining assay
4
The mechanism of cell death in response to treatment with the complexes was studied through the staining of dying cells with
DAPI, which is a fluorescence dye specific for nucleus.10e These DAPI stained nuclei was used to check the chromatin
condensation or fragmentation during apoptosis (type I programmed cell death) which is a hallmark feature of nuclear
alteration.10 Many of the cytotoxic agents and clinically active compounds are known to disrupt the nuclear integrity and
cytoskeletal structure of the cell during their course of their action,41 like cisplatin (complexation of nucleic acids),42 doxorubicin
(intercalation of nucleic acids)43 and taxol (stabilization of microtubules).44 Figure 6 depict the DAPI staining images of the
complexes against HeLa and HT-29 cell lines. It can be seen from the images that the number of DAPI stained nuclei were
reduced in number in comparison to the control for both the cell lines. Along with that, treated HT-29 cells exhibited shrinking
morphology and condensed chromatin bodies for all the complexes. While in addition, treated HeLa cells also reveal nuclear
blebbings and fragmentation. This phenomenon is a clear indication of apoptosis taking place in the cells. Till date there are a
number of cytotoxic complexes that have reported such kind of phenomenon and on this basis we hypothesize that this set of
compounds may possess comparable properties.10
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HeLa
Control
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HT-29
Control
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3
4
Figure 6. Morphology of HeLa (above panel) and HT-29 (lower panel) cells treated with complexes 1−3 at a concentration of 10
μg/ml for 24 h. The cells were visualized under confocal microscope after staining with DAPI (blue color, nucleus). The scale bar
corresponds to 50 μm for control, while for the complexes the scale bar corresponds to 25 μm.
5
6
Catalytic activity studies of 1–3
7
Oxidative bromination of thymol
8
There are various reports in the literature of vanadium complexes behaving as structural and/or functional mimics of vanadium
haloperoxidases (VHPOs).45 These complexes can catalyze various oxidation reactions in the presence of peroxides46 and
organic substrates.47 So, in the first case, the studied complexes (1−3) have been used to evaluate their efficiency towards
catalytic oxidative bromination of thymol (a monoterpene) in the presence of KBr, 70% aqueous HClO4 and 30% H2O2 in
aqueous solution under appropriate reaction conditions. All reactions were carried out in a 50 mL reaction flask at room
temperature. The bromination reaction takes place on the most activated site as a result of electrophilic aromatic substitution
in the phenolic ring. Thus, the oxidative bromination of thymol led to the formation of 2-bromothymol and 4–bromothymol
while further bromination of these also gave some 2, 4– dibromothymol (Scheme 2).
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Br
Br
Br
Br
Catalyst
+
+
H2O2, KBr, HClO4
OH
OH
Thymol
OH
OH
2-Bromothymol
4-Bromothymol 2,4-dibromthymol
1
2
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Scheme 2. Products of the oxidative bromination of thymol.
3
4
In order to obtain best suited reaction conditions for the maximum oxidative bromination products, reaction conditions were
optimized considering 2 as a representative catalyst. Thus, for 10 mmol of thymol (1.50 g), three different amounts of catalyst
(0.0005, 0.001, 0.002 g), three different amounts of 30% aqueous H2O2 (10, 20 and 30 mmol), three different amounts of KBr
(10, 20 and 30 mmol) and three different amounts of 70% aqueous HClO4 (10, 20 and 30 mmol, were added in three equal
portions to the reaction mixture, first portion at t = 0 and other two portions after every 30 min intervals were taken in 20 mL
water and the reaction was carried out at room temperature for 2h. The amount of HClO4 and its addition to the reaction was
found to be crucial to bring out effective catalytic bromination and selectivity of products.
5
6
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8
9
10
11
12
13
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Details of all reaction conditions and the corresponding conversions of thymol are summarized in Figure 7 and Table 6. The
data presented in Table 6 are based on two sets of reaction for each experiment and obtained conversions are within ± 1%. It
may be concluded from the data presented in Table 6 that the optimized reaction conditions (entry no. 8) for the best
conversion of 10 mmol (1.50 g) of thymol with the catalyst 2 are those using 0.0005 g catalyst, 30 mmol 30% H2O2, 20 mmol
KBr, and 20 mmol 70% HClO4. Under these conditions the obtained conversion is 98% where the selectivity of different major
products follows the order: 4-bromothymol (70%) > 2,4-dibromothymol (19%) > 2-bromothymol (11%).
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(b)
(a)
60
50
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30
20
10
0
70
60
50
40
30
20
10
0
0.5mg
1mg
2mg
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10 mmol
20 mmol
30 mmol
0
20
40
60
80 100 120
0
20 40 60 80 100 120
Time/min
Time/min
(d)
(c)
80
70
60
50
40
30
20
10
0
100
80
60
40
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0
10 mmol
20 mmol
30 mmol
10 mmol
20 mmol
30mmol
0
20 40 60 80 100 120
Time/min
0
20 40 60 80 100 120
Time/min
1
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2
3
Figure 7. (a) Effect of variation of amount of catalyst on the oxidative bromination of thymol. Reaction conditions: thymol (1.5
g, 10 mmol), 30% aqueous H2O2 (1.13 g, 10 mmol), KBr (1.19 g, 10 mmol), and HClO4 (1.43 g, 10 mmol) at room temperature.
(b) Effect of variation of amount of oxidant on the oxidative bromination of thymol. Reaction conditions: thymol (1.5 g, 10
mmol), catalyst 2 (0.5 mg, 7.5 × 10-4 mmol), KBr (1.19 g, 10 mmol) and HClO4 (1.43 g, 10 mmol) at room temperature. (c) Effect
of varying amount of additive (KBr) on the oxidative bromination of thymol. Reaction conditions: thymol (1.5 g, 10 mmol),
catalyst 2 (0.5 mg, 7.5 × 10-4 mmol), 30% aqueous H2O2 (3.39 g, 30 mmol) and HClO4 (1.43 g, 10 mmol) at room temperature. (d)
Effect of varying amount of HClO4 on oxidative bromination of thymol. Reaction conditions: thymol (1.5 g, 10 mmol), catalyst 2
(0.5 mg, 7.5 × 10-4 mmol), 30% aqueous H2O2 (3.39 g, 30 mmol) and KBr (2.38 g, 20 mmol) at room temperature.
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1
2
Table 6. Conversion of thymol (1.5 g, 0.010 mol) using 2 as a catalyst, turn over frequency, and selectivity of different products
for 2h of reaction time under different reaction conditions.
6
7
8
9
Entry
KBr
H2O2
HClO4
Catalyst
Conv.
[%]
TOF
[h-1]
Selectivity [%]b
[g (mmol)]
[g (mmol)]
[g (mmol)]
[mg (mmol)]
2-Brth
4-Brth
2,4-diBrth
1
2
3
4
5
6
7
8a
9
1.19 (10)
1.19 (10)
1.19 (10)
1.19 (10)
1.19 (10)
2.38 (20)
3.57 (30)
2.38 (20)
3.57 (20)
1.13 (10)
1.13 (10)
1.13 (10)
2.26 (20)
3.39 (30)
3.39 (30)
3.39 (30)
3.39 (30)
3.39 (30)
1.43 (10)
1.43 (10)
1.43 (10)
1.43 (10)
1.43 (10)
1.43 (10)
1.43 (10)
2.86 (20)
4.29 (30)
0.5 (7.5 × 10-4)
1 (1.5 × 10-3)
52
44
47
53
61
71
68
98
99
3466
1466
783
13
13
9
82
84
82
67
81
73
77
70
46
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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28
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30
31
32
33
34
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37
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41
42
43
44
45
46
47
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49
50
51
52
53
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56
57
58
59
60
3
2 (3.0 × 10-3)
0.5 (7.5 × 10-4)
0.5 (7.5 × 10-4)
0.5 (7.5 × 10-4)
0.5 (7.5 × 10-4)
0.5 (7.5 × 10-4)
0.5 (7.5 × 10-4)
8
3533
4066
4733
4533
6533
6600
10
2
23
7
11
12
11
12
16
11
19
42
aThe optimized conditions mentioned here are the best among the different sets of reactions carried out. b2-Brth = 2-
bromothymol, 4-Brth = 4-bromothymol and 2,4-diBrth = 2,4-dibromothymol.
3
4
5
6
7
Other two catalysts i.e. 1 and 3 was also tested under the above optimized reaction conditions and the results are presented in
Table 7. It is clear from the data that both complexes show similar catalytic activity (ca. 99%) (also see Figure 8) along with high
turnover frequency. Again 4-bromothymol is highest in selectivity. Blank reaction under above reaction conditions gave only
38% conversion (Table 7).
8
9
Comparing the present sets of complexes with literature it was found that they have shown similar and even better conversion
with higher turnover frequency to that previously reported for [VVO(acac)(L)] [Hacac = acetyl acetone, H2L = 6,6'-(2-(pyridin-2-
yl)ethylazanediyl)bis(methylene)bis(2,4-di-tert-butylphenol)] (ca. 99%),48 Cs(H2O)[VVO2{3,5-bis(2-hydroxyphenyl)-1-phenyl-
1,2,4-triazole}] (99%),49 and [K(H2O)2][VO2L] (H2L = ligands derived from 4,6-diacetyl resorcinol and isonicotinoyl hydrazide,
nicotinoyl hydrazide benzoyl hydrazide or 2-furoyl hydrazide (90-98%)50 signifying them as an efficient catalyst for oxidative
bromination of thymol. Interestingly, 3 containing a naphthyl substituent in the ligand backbone showed the highest turnover
frequency among the series.
10
11
12
13
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2
Table 7. Conversion, turn over frequency and selectivity parameters for various catalysts for the oxidative bromination of
thymol.
Catalyst
TOF [h-1]a
Conv. [%]
Selectivity [%]
7
8
9
2- Brth
11
4-Brth
70
2,4-diBrth
19
1
2
3
6533
5380
7173
98
99
99
38
10
11
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6
8
3
76
77
84
18
15
13
Without catalyst
aTOF values calculated at 2 h reaction time.
3
100
80
60
40
20
0
1
2
3
Blank
0
20 40 60 80 100 120
Time/min
4
5
Figure 8. Plot representing conversion of thymol in the presence of 1−3.
6
7
Oxidative bromination of Styrene
In the second case, the complexes (1−3) have also been used to evaluate their efficiency towards catalytic oxidative
bromination of styrene. We have considered 2, as a representative catalyst again and the oxidative bromination of styrene was
carried out at room temperature in the presence of KBr, 70% aqueous HClO4 and 30% aqueous H2O2 in water which led to the
formation of (a) 2-bromo-1-phenylethanol and (b) 1-phenylethane-1,2-diol, though literature reports the formation of one
more product i.e. 1,2-dibromo-1-phenylethane (Scheme 3).22a,51,52
8
9
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HO
HO
Br
OH
Br
Br
Catalyst
+
+
H2O2, KBr, HClO4
7
8
9
(c)
(a)
(b)
1
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2
3
Scheme 3. Expected products upon oxidative bromination of styrene: (a) 1,2–dibromo-1-phenylethane, (b) 2-bromo-1-
phenylethane-1-ol and (c) 1-phenylethane-1,2-diol.
4
5
Consequently, the reaction was investigated by changing different parameters that may affect the rate of styrene bromination
and the selectivity of different products. Thus, for 10 mmol of styrene (1.04 g, 10 mmol), three different amounts of catalyst
(0.0005, 0.001, 0.002g), three different amounts of 30% aqueous H2O2 (10, 20 and 30 mmol), three different amounts of KBr
(10, 20 and 30 mmol) and three different amounts of 70% aqueous HClO4 (10, 20 and 30 mmol) was added in three equal
portions to the reaction mixture as mentioned above. Details of all reaction conditions and the corresponding conversion of
styrene are summarized in Table 8 (also see Figure S5). From the data presented in Table 8 it is clear that the optimized
reaction conditions (entry no. 8) for the maximum conversion of 10 mmol (1.04 g) of styrene are those using 0.001g (1.5 × 10-3
M) of catalyst, 3.39 g (30 mmol) of 30% H2O2, 2.38 g (20 mmol) of KBr, and 2.86 g (20 mmol) of 70% HClO4. Under these
conditions the obtained conversion is 99% where the selectivity of the two products follows the order: 1-phenylethane-1,2-diol
(71%) > 2-bromo-1-phenylethanol (29%).
6
7
8
9
10
11
12
13
14
15
Table 8. Conversion of styrene (1.04 g, 10 mmol), using 2 as a catalyst, turn over frequency, and product selectivity for 2 h of
reaction time under different reaction conditions.
Entry
KBr
[g (mmol)]
H2O2
[g (mmol)]
HClO4
[g (mmol)]
Catalyst
[mg (mmol)]
Conv.
[%]
TOF [h-
Selectivity [%]a
1
]
a
b
1
2
1.19 (10)
1.19 (10)
1.19 (10)
1.19 (10)
2.38 (20)
3.57 (30)
2.38 (20)
2.38 (20)
2.38 (20)
1.13 (10)
1.13 (10)
1.13 (10)
2.26 (20)
3.39 (30)
3.39 (30)
3.39 (30)
3.39 (30)
1.43 (10)
1.43 (10)
1.43 (10)
1.43 (10)
1.43 (10)
1.43 (10)
2.86 (20)
4.29 (30)
4.29 (30)
0.5 (7.5 × 10-4)
1 (1.5 × 10-3)
2 (3.0 × 10-3)
1 (1.5 × 10-3)
1 (1.5 × 10-3)
1 (1.5 × 10-3)
1 (1.5 × 10-3)
1 (1.5 × 10-3)
blank
43
53
47
65
87
88
99
99
35
2866
1766
783
12
14
13
14
19
27
29
20
24
88
86
87
86
81
73
71
80
76
3
4
2166
2900
2933
3300
3300
-
6
7
8*
9
10
3.39 (30)
a
a = 2-bromo-1-phenylethanol and b = 1-phenylethane-1,2-diol.
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As observed in earlier reports cis-[VO2]-complexes51 the reaction of complexes with KBr in the presence of H2O2 and HClO4,
catalytically generate HOBr and/or Br+, Br2, Br3- which react with styrene to give bromonium ion as an intermediate. The
nucleophile Br– as well as H2O both may attack on the α-carbon of the intermediate to give 1,2-dibromo-1-phenylethane and 2-
bromo-1-phenylethane-1-ol, respectively. The nucleophile H2O may further attack on the α–carbon of 2-bromo-1-
phenylethane-1-ol to give 1-phenylethane-1,2-diol.52 All these justify the formation of 1-phenylethane-1,2-diol in highest yield.
2
3
4
10
11
5
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
7
Other two complexes i.e. 1 and 3 were also tested under the above optimized reaction conditions for the maximum conversion
of styrene. The results are summarized in Table S1 and Figure S6. It is clear from the data that other two complexes show
comparable catalytic activity (99%) along with high turnover frequency and within the two products, the selectivity of 1-
phenylethane-1,2-diol is always higher than that of 2-bromo-1-phenylethanol. Blank reaction gave only 35% conversion. Thus,
these complexes have potential catalytic activity.
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Catalytic activity of the complexes towards oxidative bromination of styrene also compares well with previous reports of
[VVO(OEt)(EtOH)(L)] (H2L = Schiff bases derived from 2-hydroxy-1-acetonaphthone or 2-hydroxy-1-napthaldehyde and
salicylhydrazide, benzoylhydrazide and anthranylhydrazide) (96-97% conversion with 63-69% selectivity towards diol)51,
[MoVIO2(X-Hsal-dahp)(H2O)] [(X-sal = salicylaldehyde and its derivatives, dahp = 1,3-diamino-2-hydroxypropane) (97-99%
conversion with 61% selectivity towards diol)52 and shows increased that is 99% conversion with 75% selectivity towards diol.
Further, catalytic activity of VO(acac)2 has also been carried out under the same reaction conditions where thymol gave 60±1%
conversion while styrene gave ca. 65% conversion, which is less than what we observed using complexes (1−3) reported here as
catalysts.
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20
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Reactivity of complexes with H2O2
It has been previously accounted that dioxidovanadium(V) complexes on reaction with H2O2 gives the corresponding
oxidoperoxidovanadium(V) species.49 Similar species has been generated for the present study in MeCN and the progress of
reaction has been monitored through UV–vis spectroscopy. For all the complexes similar observations were made and as a
representative, the result of 1 has been discussed here. The gradual addition of one drop portion of 30% H2O2 (0.108 g, 0.95
mmol) dissolved in 5 mL of MeCN to 25 mL of 3.82 × 10-5 M solution of 1 in MeCN lead to the decrease in the intensities of the
385, 341 and 291 nm bands (Figure 9). The decrease in the above bands happens due to the ligand to metal charge transfer and
intra ligand transitions. These changes clearly suggests the formation of oxidoperoxidovanadium(V) species in solution.48,49,50,53
The reactivity of 2 and 3 has been depicted in Figures S7 and S8 respectively.
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1
341
385
2
1
0
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
291
300
400
500
Wavelength (nm)
1
2
Figure 9. Plots representing the Spectral changes during titration of 1 with H2O2. Spectra were obtained after successive
3
4
addition of one drop portion 30% H2O2 (0.108 g, 0.95 mmol) dissolved in 5 mL of MeCN to 25 mL of 3.82 × 10-5 M solution of 1 in
MeCN.
5
Conclusions
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7
Concisely, this report presents the synthesis of three new dioxidovanadium(V) complexes of tridentate binegative
azohydrazone ligands (H2L1-3) and their characterization by various analytical techniques (IR, UV–vis, 1H and 13C NMR
spectroscopy, ESI-MS and cyclic voltammetry). The molecular structures of 1–3 have been established by single crystal X-ray
diffraction analysis, which revealed that vanadium is present in a distorted square pyramidal geometry with O4N coordination
spheres. These complexes have been evaluated for their in vitro cytotoxicity against HT-29 colon and HeLa cervical carcinoma
cells. Though all the complexes were found to be cytotoxic in nature, 3 proved to be the most potent among them. The
cytotoxicity of these complexes may be assigned to the azobenzene moiety in the ligand backbone synergizing with the
different substituents along with the vanadium metal. Further, the catalytic potential of 1–3 was also tested for oxidative
bromination of thymol and styrene. In coherence with the antiproliferative activity, for all the cases, the percentage of
conversion (>90%) was also found to be significantly high in the presence of the catalysts with a high turnover frequency.
Especially, for the oxidative bromination of thymol, the percentage of conversion and TOF lies in the range of 98–99% and
5380–7173 (h−1) respectively. Besides, for all the catalytic reactions 3 showed the highest TOF value may be due to the naphthyl
substituent. Also, the brominated organic compounds synthesized through oxidative bromination of the corresponding
precursor (thymol and styrene) using 1–3 as catalyst have been identified as useful starting material for several organic
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transformations along with biological and pharmaceutical importance.54 Again, oxidative bromination reactions using these
metal complexes as catalyst can be considered as a favorable tool to replace the toxic and corrosive brominating agents (i.e.
Br2).54 Therefore, the results of the present study stimulate further insights into the mechanistic pathway of vanadium
complexes in azohydrazone ligand environment in relation to their antiproliferative and catalytic activity and also to study the
correlation of the catalytic efficiency of these complexes to that of their biological potential.
2
3
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59
60
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7
Conflicts of interest
There are no conflicts to declare.
8
Acknowledgements
9
R.D thanks DBT, Govt. of India [Grant No. 6241 P112/RGCB/PMD/DBT/RPDA/2015] and CSIR, Govt. of India [Grant No.
01(2963)/18/EMR-II] for funding this research. R. D. thanks Dr Indranil Banerjee for discussion related to biological study.
10
11
Notes and references
12
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Corresponding Author
*E-mail: rupamdinda@nitrkl.ac.in (Rupam Dinda)
CCDC contains the supplementary crystallographic data for 13. . These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associated with this
article can be found, in the online version, at DOI: XXXXX
ORCID
Rupam Dinda: 0000-0001-9452-7791
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