Fine tuning through valence bond tautomerization of ancillary ligands in ruthenium(ii) arene complexes for better anticancer activity and enzyme inhibition properties

Article abstract of DOI:10.1039/C6DT02969H

Four new ruthenium arene PTA type complexes have been synthesized using substituted picolinamide derivatives as ancillary ligands and characterized by spectroscopic methods. In one of the complexes, the ancillary ligand has shown an unprecedented valence-bond tautomerization in the presence of an ammonium salt to act as a polar neutral donor ligand making the ligand more prone towards substitution. The same compound has shown remarkable antiproliferative activity against three cancer cell lines with GI₅₀ values comparable to Adriamycin, a known therapeutic drug. Along with this it also strongly inhibits the action of thioredoxin reductase, which might be a probable reason for the enhanced proliferative action of the valence-bond tautomerized compound.

Full text of DOI:10.1039/C6DT02969H

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P. Mandal, N. Malviya, M. F. C. Guedes da Silva, S. S. Dhankhar, C. M. Nagaraja and M. M. Shaikh, Dalton
Trans., 2016, DOI: 10.1039/C6DT02969H.
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ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
rsc.li/dalton

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DOI: 10.1039/C6DT02969H
Synopsis:
A series of water soluble ruthenium arene RAPTA like compounds have been prepared One
of the four compounds with a novel valence tautomerized ancillary ligand shows marked
antiproliferarive and thiredoxin reductase inhibition property .

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Fine tuning through valence bond tautomerization of ancillary ligand in rutDhOeI:n10i.u10m39/(CI6ID)T02969H
arene complex for better anticancer activity and enzyme inhibition property
Poulami Mandal,a Novina Malviya,a M. Fátima C. Guedes da Silva,b Sandeep Singh
Dhankhar,c C. M. Nagaraja,c Shaikh M Mobin, a,d Suman Mukhopadhyay*, a,d
a Department of Chemistry, School of Basic Sciences, Indian Institute of Technology Indore,
Indore 453552, India.
b Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Technical University
of Lisbon, Avenida Rovisco Pais,1049-001, Lisbon, Portugal
c Department of Chemistry, Indian Institute of Technology Ropar, Rupnagar 140001, Punjab,
India
d Centre for Bioscience and Biomedical Engineering (BSBE), Indian Institute of Technology
Indore, Indore 453552, India.
Tel : +91 731 2438 735 Fax: +91 731 2361 482
E-mail: suman@iiti.ac.in
Introduction:
Arene ruthenium (II) complexes with pseudo-octahedral coordination environment also
named as piano-stool compounds have been studied in recent past widely for its probable
application in catalysis, crystal engineering and metallo-pharmaceuticals.1,2,3,4,5 However
because of the their lower toxicity, amphiphilic properties generated from hydrophobic arene
ring and hydrophilic metal center, improved selectivity and possibility to play around with
the ligands to improve the target (DNA, enzyme and proteins) specific chemotherapy make
them an important class of compounds with biological significance. Ruthenium arene
complexes have shown significant antimetastatic and anticancer properties which can be
modulated by factors like hydrophobicity of arene ligand and hydrogen bond forming ability
and other properties of the ancillary ligands.6,7,8 One of the biggest advantage of ruthenium
complexes is that they are transported inside cancer cells by transferrin enzyme, an iron
transporter in human body which promotes selective accumulation of ruthenium compound in
cancer cells which overexpress the transferring receptor.9 The problem of water solubility of

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the ruthenium-arene complexes has been largely tackled by introduction of DaOmI: 1p0h.10ip39h/Cil6iDcT02969H
ligand called PTA (PTA = 1,3,5-triaza-7-phospha-tricyclo-[3.3.1.1]decane) to generate a
ruthenium-arene-PTA combination known as RAPTA compounds.10,11,12,13 With fixation of
arene ring and PTA ligand coordinated to ruthenium center, scientists around the world have
tried different ancillary ligands with various characteristics to evaluate possible effect on
cytotoxicity and target specificity.14,15,16,17 Recently some reports have been published where
picolinamide or carboxamide complexes have shown interesting anticancer properties due to
18,19,20,21
their dynamic coordinating nature to the metal center.
To explore the reactivity of
picolinamide based ligands we have utilized nitro substituted aniline and 3-pyridyl and 4-
pyridyl amine to generate picolinamide derivatives to monitor the effect of ancillary ligands
on anticancer activity as to the best of our knowledge there is no report where N-substituted
picolinamide part as ancillary ligand is modulated in RAPTA scaffold to explore the
anticancer properties of the synthesized complexes. Interestingly one of the synthesized
compound has shown marked antiproliferative activity against different cancer cell lines. As
it is evident that RAPTA compounds work upon molecular targets other than DNA,22 we
have explored the interaction of the synthesized complexes with thioredoxin reductase (TrxR)
which is one of the key target identified23 previously because of the presence of selenolate
group which after reduction prefers to bind with softer metal ion making it suitable as a
pharmacological target suitable for metallodrugs.
Experimental section
Materials and Methods
All the chemical reagents required were purchased from Sigma and used without further
purification. NMR spectra were recorded on an AVANCE III 400 Ascend Bruker BioSpin
machine at ambient temperature. Mass spectrometric analyses were done on Bruker-
Daltonics, microTOF-Q II mass spectrometer and elemental analyses were carried out with a
ThermoFlash 2000 elemental analyzer. Spectrophotometric measurements were performed on
a Varian UV-Vis spectrophotometer (Model: Cary 100) (for absorption) and a Fluoromax-4p
spectrofluorometer from Horiba JobinYvon (Model: FM-100) (for emission) using a quartz
cuvette with path length of 1 cm. Far-ultraviolet (UV) (190 to 260 nm) spectra were recorded
in 0.1 cm path length cell (Hellma, Muellheim/Baden, Germany) using a step size of 0.5 nm,
bandwidth of 1 nm and a scan rate of 20 nm min−1. Conductivity measurements were done
using a Digital Conductivity Meter Model 611 instrument. Conductivities of complexes were

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obtained with 1 mM solutions in acetonitrile. [Ru(L1)(η6-p-cymene)Cl] 1 andD[ORI: 1u0(.1L031V9)i/(eCwη6A6Dr-tTic0le2O96nl9inHe
benzene)Cl] 3 [HL1 = pyridine-2-carboxylic acid (4-nitro-phenyl)amide] were prepared by
literature method.24 Ligand HL2 [HL2 = 4-(N-(2-Pyridyl)carbamoyl pyridine] and HL3 [HL3 =
3-(N-(2-Pyridyl)carbamoyl) pyridine], were also prepared by method reported earlier.25 The
sulforhodamine B (SRB) growth inhibition (GI50) assays were carried out by Advanced
Center for Treatment, Research and Education in Cancer (ACTREC), Mumbai by following
the literature procedure.26
[RuL1(η6-p-cymene)PTA][BF4] 2 . 2H2O
0.05g (0.09 mmol) of compound [RuL1(η6-p-cymene)Cl] 1 was dissolved in 20 mL of
methanol. A methanolic solution (10 mL) of 1,3,5-triaza-7-phosphoadamantane (PTA)
(0.01g, 0.09 mmol) was added dropwise to the solution of compound 1. The reaction mixture
was stirred for 2 h at room temperature and then filtered. The filtrate was reduced in vacuo to
a volume of ca. 5 mL. An excess of diethyl ether was added to accomplish precipitation. The
precipitate was filtered, washed with diethyl ether and dried in vacuo to afford yellow
coloured crystalline solid as product. Yield: 76%. The yellow coloured compound was
dissolved in methanol. A methanolic soln of NaBF4 (0.1 g, 0.08 mmol) was added to it and
stirred for 2 h. The resulting solution was then filtered and filtrate was then concentrated in
vacuo to a volume of ca.5 mL. The filtrate was kept in refrigerator for overnight, from which
1
crystals suitable for X-ray analysis were obtained. Yield: 62%. H NMR (400.13MHz, 298K
1
,d6-DMSO): δ 8.98 [d, 1H , 3J (1H - H) = 4.0 Hz, CH of C5H4N], 8.29 [m, 3H, 1XCH of
3
1
C5H4N and 2XCH of C6H4NO2], 8.10 [d, 1H, J (1H - H) = 8.0 Hz, CH of C5H4N], 7.84 [t,
1
1
1H , 3J (1H - H) = 12.0 Hz, CH of C5H4N], 7.57 [d, 2H , 3J (1H - H) = 12.0 Hz, CH of
1
1
C6H4NO2], 6.26 [d,1H, 3J (1H - H) = 4.0 Hz, CH of C6H4], 5.92 [d,1H, 3J (1H - H)=8.0Hz,
3
CH of C6H4], 5.75 [d, 1H , 3J (1H - 1H)=8.0Hz CH of C6H4], 5.43[d, 1H, J (1H - 1H)=8.0Hz
CH of C6H4] , 4.37 [m.6H,3xNCH2N], 4.00 [m ,6H, 3xPCH2N], 2.84 [m, 1H, CH(CH3)2],
2.28 [s, 3H, C6H4CH3], 0.98 [d, 3H, CH(CH3)2], 0.74 [d, 3H, CH(CH3)2] (Fig. S1). 13C NMR
( 100.61MHz, d6-DMSO): δ 167.0 [C of CON], 157.7 [C of C6H4NO2], 156.3 [C of C5H4N],
154.1 [CH of C5H4N], 142.3 [CH of C5H4N], 140.7 [C of C6H4NO2], 128.9 [CH of C5H4N],
127.1 [2xCH of C6H4NO2], 126.7 [CH of C5H4N], 124.1 [2xCH of C6H4NO2], 119.1 [C of
C6H4], 101.1 [C of C6H4], 91.4 [CH of C6H4], 90.1 [CH of C6H4], 88.0 [CH of C6H4], 87.4
[CH of C6H4], 71.4 [3xNCH2N of PTA], 50.0-48.5 [3xPCH2N of PTA], 30.4 [C6H4CH3],
21.9 [CH(CH3)2], 20.9 [CH(CH3)2], 18.1 [CH of CH(CH3)2] (Fig. S2). 31P NMR (d6-DMSO,
126 MHz) : δ - 34.4 (Fig. S3). ESI-MS (+ve mode) : [RuL1(η6-p-cymene)PTA]+: 635.16

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(m/z) (Fig. S4). Anal. Calcd for C28H38BF4N6O5PRu: C, 44.40; H, 5.06; N, 11.09. Found:
DOI: 10.1039/C6DT02969H
C, 44.53; H, 5.11; N, 10.89.
[RuL1(η6-benzene)PTA][PF6] 4 .CH3OH
Compound [RuL1(η6-benzene)Cl] 3 (0.08g, 0.1 mmol) was dissolved in a mixture of
methanol (15 mL) and dichloromethane (15 mL). A methanolic solution (10 mL) of 1,3,5-
triaza-7-phosphoadamantane (PTA) (0.02g, 0.1 mmol) was added dropwise to the solution of
compound 3. The reaction mixture was stirred for 2 h at room temperature and then filtered.
The filtrate was reduced in vacuo to a volume of ca. 5 mL. An excess of diethyl ether was
added to accomplish precipitation. The precipitate was filtered, washed with diethyl ether and
dried in vacuo, affording a yellow solid. Yield: 62%. The yellow coloured compound was
dissolved in methanol. A methanolic solution of NH4PF6 (0.12 g, 0.1 mmol) was added to it
and stirred for 2 h. The resulting solution was then filtered and it was concentrated in vacuo
to a volume of ca.5 mL.The filtrate was kept for vapour diffusion with diethyl ether, from
1
which crystals suitable for X-ray analysis were obtained. Yield: 52%. H NMR(400.13MHz,
1
298K, d6-DMSO): δ 8.92 [d, 1H , 3J (1H - H) = 4.0 Hz CH of C5H4N], 8.27-8.24 [m, 2H
,2xCH of C6H4NO2], 8.10 [d, 1H , 3J (1H - 1H) = 8.0 Hz, CH of C5H4N], 7.93 [br.s,1H,CH of
C5H4N], 7.76 [t.1H, 3J (1H - 1H) = 12.0 Hz C5H4N], 7.54 [d, 2H, 3J (1H - 1H) = 12.0 Hz, 2xCH
of C6H4NO2], 6.00 [s, 6H ,6xCH of C6H6], 4.37 [m, 6H ,3xNCH2N of PTA], 3.98 [s, 6H
13
,3xPCH2N of PTA] (Fig. S5). C NMR ( 100.61MHz, d6-DMSO): δ 169.9 [C of CON],
158.8 [C of C5H4N], 155.9 [CH of C5H4N], 154.6 [C of C6H4NO2], 143.9 [C of C6H4NO2],
139.2 [CH of C5H4N], 129.4 [CH of C5H4N], 127.8 [2xCH of C6H4NO2], 127.2 [CH of
C5H4N], 124.2 [2xCH of C6H4NO2], 85.9 [6xCH of C6H6], 72.5 [3xNCH2N of PTA], 52.2
[3xPCH2N of PTA] (Fig. S6). 31P NMR (126 MHz, d6-DMSO): δ -39.16[s, PTA], -
144.1[sept, PF6] (Fig. S7). ESI-MS (+ve mode) : [RuL1(η6-benzene)PTA]+: 579.1(m/z) (Fig.
S8). Anal. Calcd for C25H30F6N6O4P2Ru: C, 39.74; H, 4.00; N, 11.12. Found: C, 39.56; H,
4.32; N, 10.89.
[RuL2(η6-p-cymene)Cl] 5
0.05g of [(η6-p-cymene)RuCl(μ-Cl)]2 (0.08 mmol) was dissolved in 20 mL of
dichloromethane and the ligand (HL2) [4-(N-(2-Pyridyl)carbamoyl pyridine] (0.32g,
0.16mmol), dissolved in methanol was added dropwise. The mixture was stirred at room
temperature for 4 h . The resulting solution was then filtered and evaporated in vacuo to ca.5

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mL. Addition of excess diethyl ether to the filtrate leads to the formation of orange coloured
DOI: 10.1039/C6DT02969H
precipitate. The precipitate was filtered, washed with hexane and dried in open air affording
the title compound as orange solid. Yield 54%. 1H NMR (400.13MHz, 298K, CDCl3): δ 9.03
[br s, 1H , CH of C5H4N of 4-amino pyridine], 8.37 [broad, 2H ,CH of C5H4N of 4-amino
pyridine], 8.05 [d, 1H , CH of C5H4N of picolinic acid], 7.97 [t, 1H, CH of C5H4N of
picolinic acid ], 7.58 [t, 1H, CH of C5H4N of picolinic acid ], 6.81 [d, 2H ,CH of C5H4N of 4-
amino pyridine], 5.49 [d, 1H, ,CH of C6H4], 5.32 [d, 2H, , CH of C6H4], 5.28 [d, 1H, , CH of
C6H4], 2.46 [sept, 1H, CH(CH3)2], 2.23 [s, 3H, C6H4CH3], 1.05 [d, 3H, CH(CH3)2], 0.99 [d,
3H, CH(CH3)2] (Fig. S9). ESI-MS (+ve mode) : [RuL2(η6-p-cymene)]+: 434.1 (m/z) (Fig.
S10).
[Ru(HL2)(η6-p-cymene)PTA][PF6]2 6 .CH3OH
A 10 mL methanolic solution of 1,3,5-triaza-7-phosphoadamantane (0.35g, 0.22 mmol) was
added dropwise to a solution of compound 5 (0.1g, 0.22mmol) dissolved in a mixture of
methanol and dichloromethane (10mL + 10mL). The resulting solution was then stirred
overnight at room temperature and filtered. The filtrate was then evaporated to dryness to get
the crude product as yellow solid. Yield: 78%. The obtained compound was then dissolved
in methanol (0.5 g, 0.08 mmol) and a methanolic solution of NH4PF6 (0.03 g, 0.18 mmol) was
then added to it .The resulting solution was allowed to stir for 2h at room temperature. The
mixture was then filtered and concentrated to 5 mL and kept at 0°C for overnight. Yellow
coloured crystals suitable for single crystal XRD was obtained from the reaction mixture.
1
Yield: 70%. H NMR (400.13MHz, 298K ,d6-DMSO) : δ 9.00 [d, 1H , 3J (1H - 1H) = 4.0 Hz,
CH of C5H4N], 8.64 [d, 2H, 2xCH of substituted 4-amino pyridine], 8.36 [t, 1H , 3J (1H - 1H)
= 12.0 Hz, CH of C5H4N], 8.21 [d, 1H , 3J (1H - 1H) = 4.0 Hz, CH of C5H4N], 7.92 [d,1H, 3J
1
1
(1H - H) = 8.0 Hz, CH of C5H4N], 7.87 [d,2H, 3J (1H - H)=8.0Hz, 2xCH of substituted 4-
1
amino pyridine], 6.29 [d, 1H , 3J (1H - H)=4.0Hz CH of C6H4], 6.06[d, 1H, CH of C6H4] ,
5.87[d, 1H, CH of C6H4] ,5.78[d, 1H, CH of C6H4] , 4.41 [m, 6H,3xNCH2N], 3.99 [m, 6H,
3xPCH2N], 2.43 [s,3H, C6H4CH3], 2.26 [sept, 1H, CH of CH(CH3)2],0.99 [d, 3H, CH(CH3)2],
0.73[d,3H,CH(CH3)2] (Fig. S11). 13C NMR ( 100.61MHz, d6-DMSO) : δ 168.6 [C of CON] ,
156.7 [C of substitued 4-amino pyridine], 153.0 [2xCH of substituted 4-amino pyridine] ,
142.5 [CH of C5H4N], 141.0 [CH of C5H4N], 129.8 [CH of C5H4N], 127.5 [CH of C5H4N],
122.4 [CH of substitued 4-amino pyridine], 121.0 [CH of substitued 4-amino pyridine],101.6
[C of C6H4], 90.9 [C of C6H4], 87.9 [2xCH of C6H4], 78.5 [2xCH of C6H4], 71.2 [3xNCH2N
of PTA], 49.9-48.5 [3xPCH2N of PTA], 30.6 [C6H4CH3], 21.9 [CH(CH3)2], 20.9 [CH(CH3)2],

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Dalton Transactions
18.4 [CH of CH(CH3)2] (Fig. S12). 31P NMR (d6-DMSO, 126 MHz) : δ -38.9, -144.2 [VsieewpAtr,ticle Online
DOI: 10.1039/C6DT02969H
PF6] (Fig. S13). ESI-MS (+ve mode) : 296.1(m/z) :[RuL2(η6-p-cymene)]2+/2 (Fig. S14).Anal.
Calcd for C28H39F12N6O2P3Ru: C, 36.81; H, 4.30; N, 9.20. Found: C, 36.90; H, 4.54; N,
9.45
[RuL3(η6-p-cymene)Cl] 7
0.1g of [(η6-p-cymene)RuCl(μ-Cl)]2 (0.16mmol) was dissolved in 50 mL of methanol and
methanolic solution of
ligand (HL3) [3-(N-(2-Pyridyl)carbamoyl) pyridine] (0.65g,
0.32mmol) was added dropwise to that. The mixture was stirred at room temperature for 4 h .
The resulting solution was then filtered. The filtrate was evaporated in vacuo of ca.5 mL. An
excess of diethyl ether was added to accomplish precipitation. The precipitate was filtered,
washed with hexane and dried in open air affording the title compound as orange solid. Yield
42%. 1H NMR(400.13MHz, 298K, CDCl3): δ 9.54 [s, 1H CH of substituted 3-amino
pyridine], 9.02 [s, 1H ,CH of C5H4N], 8.76 [d, 1H , CH of C5H4N], 8.36 [d, 1H, CH of
substituted 3-amino pyridine], 8.12 [d, 1H, CH of substituted 3-amino pyridine], 7.96 [t , 1H ,
CH of C5H4N], 7.54 [t, 1H ,CH of C5H4N], 7.22 [t, 1H ,CH of substituted 3-amino pyridine],
5.65 [d, 1H, 3J (1H - 1H) = 7.8Hz ,CH of C6H4], 5.57 [d, 1H, CH of C6H4], 5.49 [d, 1H, 3J (1H
1
- H) = 7.7Hz, CH of C6H4], 5.30 [d, 1H, CH of C6H4], 2.85 [sept, 1H, CH(CH3)2], 2.26 [s,
3H, C6H4CH3], 1.14 [d, 3H, CH(CH3)2], 1.05 [d, 3H, CH(CH3)2] (Fig. S15) . ESI-MS (+ve
mode) : [RuL3(η6-p-cymene)]+: 434.0(m/z) (Fig. S16).
[RuL3(η6-p-cymene)PTA][BF4] 8
To a solution of compound 7 (0.1 g, 0.22 mmol) dissolved in methanol (20mL), a 10 mL
methanolic solution of 1,3,5-triaza-7-phosphoadamantane (0.35g, 0.22mmol) was added
dropwise. The resulting mixture was stirred overnight at room temperature and filtered. The
filtrate was then evacuated in vacuo to get the crude product as orange coloured solid. Yield:
70%. The orange solid was then dissolved in a mixture of dichloromethane and methanol
(5mL+5mL) and a methanolic solution of NaBF4 (0.006g, 0.05mmol) was added to it. The
resulting solution was the stirred at room temperature for 2 h and the filtered. The filtrate was
then concentrated in vacuo to 2 mL and kept for vapour diffusion with diethyl ether to get
orange coloured crystal of compound 8 . Yield: 65%. 1H NMR (400.13MHz, 298K ,d6-
DMSO) :δ 8.98 [d, 1H , 3J (1H - 1H) = 4.0 Hz, CH of C5H4N], 8.68 [s, 1H, CH of substituted
3-amino pyridine], 8.40 [br.s,1H, CH of C5H4N], 8.29 [t, 1H, 3J (1H - 1H) = 12.0 Hz, CH of

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3
1
1
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DOI: 10.1039/C6DT02969H
substituted 3-amino pyridine], 8.07 [d, 1H , J ( H - H) = 12.0 Hz, CH of substituted 3-amino
pyridine], 7.84 [t, 1H , 3J (1H - 1H) = 4.0 Hz, CH of C5H4N], 7.76 [d,1H, 3J (1H - 1H) = 8.0 Hz,
CH of C5H4N], 7.54 [br.t,1H, 3J (1H - 1H)=8.0 Hz, CH of substituted 3-amino pyridine], 6.29
1
[d, 1H , 3J (1H - H)=4.0Hz CH of C6H4], 6.06 [d, 1H, CH of C6H4] , 5.87 [d, 1H, CH of
C6H4] ,5.78 [d, 1H, CH of C6H4] , 4.41 [m.6H, 3xNCH2N], 3.99 [m ,6H, 3xPCH2N], 2.43 [s,
3H, C6H4CH3], 2.26 [sept, 1H, CH of CH(CH3)2],0.93 [d, 3H, CH(CH3)2],
0.75[d,3H,CH(CH3)2] (Fig. S17). 13C NMR ( 100.61MHz, d6-DMSO) : δ 167.0 [C of CON] ,
157.2 [CH of C5H4N], 156.3 [CH of C5H4N] , 154.2 [C of substituted 3-amino pyridine],
147.5 [C of C5H4N substituted 3-amino pyridine], 144.5 [CH of C5H4N], 140.6 [CH of
substituted 3-amino pyridine], 133.5 [CH of C5H4N], 129.3 [CH of C5H4N],118.7 [CH of
substituted 3-amino pyridine], 115.2 [CH of substituted 3-amino pyridine], 101.8 [C of
C6H4], 93.5 [C of C6H4], 91.3 [CH of C6H4], 88.8[CH of C6H4], 87.5 [C of C6H4], 79.4-70.6
[3xPCH2N of PTA], 53.7- 46.2 [3xNCH2N of PTA], 30.2 [C6H4CH3], 21.9 [CH(CH3)2], 21.1
[CH(CH3)2], 17.9 [CH of CH(CH3)2] (Fig. S18). 31P NMR (d6-DMSO, 126 MHz) : δ -39.1
(Fig. S19). ESI-MS (+ve mode) : [RuL3(η6-p-cymene)PTA]+: 591.15 (m/z) (Fig. S20). Anal.
Calcd for C27H34BF4N6OPRu: C, 47.87; H, 5.06; N, 12.41. Found: C, 47.53; H, 5.31; N,
12.47
X-ray crystallography
Compound 2
X-ray single crystal of complex 2 was immersed in cryo-oil, mounted in
Nylon loops and measured at
a
temperature of 150
K
in
a
Bruker
AXS-KAPPA APEX II with graphite monochromated Mo-Kα (λ=0.71073)
radiation. Data were collected using omega scans of 0.5º per frame and
full
using Bruker SMART27 software and refined using Bruker SAINT28 on
all the observed reflections. Absorption corrections were applied
using SADABS.29 Structures were solved by direct methods by using
the SHELXS–97 and refined with
package30 SHELXL–2014/7.31
Calculations were performed using the WinGX System–Version 2014.1.32
Least square refinements with anisotropic thermal motion parameters
for all the non-hydrogen atoms and isotropic for the remaining atoms
sphere
of
data
were
obtained.
Cell
parameters
were
retrieved

Page 9 of 32
Dalton Transactions
were employed. All hydrogen atoms bonded to carbon were includedView Article Online
in
DOI: 10.1039/C6DT02969H
the model at geometrically calculated positions and refined using
a
riding model. Uiso(H) were defined as 1.2Ueq of the parent carbon
atoms for phenyl and methylene residues and 1.5Ueq of the parent
carbon atoms for the methyl groups. There was some void in the
structure which, despite the negligible volume and number of electrons
(63
Å3
and
3
electrons
per
unit
cell),
was
corrected
with
PLATON/SQUEEZE.33
Compound 4
X-ray single crystal structural data of compound 4 was collected on a Bruker D8 Venture
PHOTON 100 CMOS diffractometer equipped with a INCOATEC microfocus source and
graphite monochromated Mo Kα radiation (λ = 0.71073 Å) operating at 50 kV and 30 mA.
The program SAINT85 was used for integration of diffraction profiles and absorption
correction was done with SADABS86 program. The structure was solved by SIR 9287 and
refined by full matrix least-square method using SHELXL-201388 and WinGX system, v
2013.3.89. All the nonhydrogen atoms were located from the difference Fourier map and
refined anisotropically. All the hydrogen atoms were fixed by HFIX and placed in ideal
positions and included in the refinement process using riding model with isotropic thermal
parameters. Potential solvent accessible area or void space was calculated using the PLATON
multipurpose crystallographic software.
Compound 6 and Compound 8:
Single crystal X-ray structural studies of 6 and 8 were carried out on a CCD Agilent
Technologies (Oxford Diffraction) SUPER NOVA diffractometer. Data for the crystal 6 and
8 were collected at 293(2) K and 150(2) K respectively using graphite-monochromated
MoKα radiation (λ= 0.71073 Å). The strategy for the data collection was evaluated by using
the CrysAlisPro CCD software. The data were collected by the standard ‘phi-omega scan
techniques and were scaled and reduced using CrysAlis- Pro RED software. The structures
were elucidated by direct methods using SHELXS-97 and refined by full matrix least squares
with SHELXL-97, refining on F2.31 The positions of all the atoms were obtained by direct
methods. All non-hydrogen atoms were refined anisotropically. The remaining hydrogen

Dalton Transactions
Page 10 of 32
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atoms were placed in geometrically constrained positions and refined wiDthOI: 1i0s.o10t3r9o/Cp6iDcT02969H
temperature factors, generally 1.2Ueq of their parent atoms.
Protein binding experiments:
Protein binding studies of the synthesised compounds were carried out by tryptophan
fluorescence quenching experiments using human serum albumin (HSA).The excitation
wavelength for HSA was at 280 nm and the quenching of the emission intensity of the
tryptophan residues of HSA at 345 nm was monitored using the complexes as quencher with
increased concentration. The excitation and emission slit widths and scan rates were kept
constant throughout the experiment. A 10 μM stock solution of HSA was prepared using 50
mM tris buffer solution and stored at 4°C for further use. Stock solutions of 5 mM strength
were made using synthesized compounds. Fluorometric titration was carried out taking 2 mL
of the protein solution and fluorescence intensity was measured as blank. For titration, each
time, 10 μL of the stock solution was added to the protein solution and fluorescence intensity
was measured. For all the four complexes, up to 100μL of the solution was added to measure
fluorescence quenching. The fluorescence quenching data was further analyzed by the Stern–
Volmer equation, which again can be expressed in terms of bimolecular quenching rate
constant and average life time of the fluorophore as shown in following equation.
퐹0
[ ]
= 1 + 퐾푞휏0 푄 = 1 + 퐾푠푣[푄]
퐹
where F0 and F are the fluorescence intensities in the absence and the presence of a quencher,
kq is the bimolecular quenching rate constant, τ0 is the average lifetime of fluorophore in the
absence of a quencher and [Q] is the concentration of a quencher (metal complexes). KSV is
the Stern–Volmer quenching constant in M−1.
Inhibition of Trx-R enzyme:
The inhibition of Trx-R enzyme was carried out using DTNB as substrate according to the
protocol given by sigma Aldrich with subsequent modification. Colorimetric assays were

Page 11 of 32
Dalton Transactions
performed with a microplate reader to determine the inhibition of Trx-R. The compoundViwewaAsrticle Online
DOI: 10.1039/C6DT02969H
dissolved to make stock solution in DMSO (5 mM). Mammalian thioredoxin reductase,
supplied in 50 mM TrisHCl (pH = 7.4), 10 mM EDTA, 300 mM NaCl and 10% glycerol was
diluted 20 times. The reaction mixture in each well consists of working buffer [180 μL; 100
mM potassium phosphate with 10 mM EDTA and 0.24 mM nicotinamide adenine
dinucleotide phosphate (NaDPH)], 1X assay buffer (8 μL), TrxR enzymes (2 μL), and
positive control (cisplatin) or the ruthenium complex. The reaction mixture were incubated
for 15 min at 298 K and then a 100 mM DMSO solution (6 μL) of DTNB was added.
Formation of 5-thio-2 nitrobenzoic acid (5-TNB) was monitored with a microplate reader at
412 nm after 15 and 30 min. Non-interference with the assay component was ensured by a
negative control experiment with an enzyme free solution. Following equation was used to
determine the TrxR enzyme activity.
Unit/mL = [ΔA412/min(TrxR) X dil X vol]/enzvol
ΔA412/min = change in absorbance at 412 nm per minute, ΔA412/min(TrxR) = [ΔA412/min
(sample) - ΔA412/min (blank, without enzyme)], dil = sample dilution factor, vol = volume of
the reaction and enzvol = volume of the enzyme.
Results and discussion:
Syntheses of complexes
Room temperature substitution of chloride ligand in ruthenium (II) complexes of general
formula [Ru(L)(η6-arene)Cl] (HL1 = pyridine -2-carboxylic acid(4-nitro-phenyl)-amide, η6-
arene = p-cymene 1 and HL1 = pyridine -2-carboxylic acid(4-nitro-phenyl)-amide, η6-arene
= benzene 3, HL2 = 4-(N-(2-Pyridyl)carbamoyl pyridine , η6-arene = p-cymene, 5 HL3 = 3-
(N-(2-Pyridyl)carbamoyl) pyridine,
η6-arene = p-cymene 7 ) with 1,3,5-triaza-7-
phosphoadamantane (PTA) yielded water soluble complexes [Ru(L)(η6-arene)(PTA)]+ [2, 4,
8] and [Ru(HL)(η6-arene)(PTA)]++ [6] (Scheme 1). Compound 2 and compound 8 were
recrystallized in presence of tetrafluoroborate ion in dichloromethane and methanol mixture
to generate single crystals of [RuL1(η6-p-cymene)PTA]BF4 2 and single crystals of
[RuL3(η6-p-cymene)PTA]BF4 8 for solid state characterization whereas single crystals for
compound 4 and compound 6 were obtained in presence of NH4PF6 as [RuL1(η6-
benzene)PTA]PF6 and [Ru(HL2)(η6-p-cymene)PTA](PF6)2, respectively. All the PTA
compounds were as yellow/yellow-orange powders in moderate to good yields (52% - 78%).

Dalton Transactions
Page 12 of 32
2
6
3
6
View Article Online
DOI: 10.1039/C6DT02969H
Compound [RuL (η -p-cymene)Cl] 5 and [RuL (η -p-cymene)Cl] 7 were synthesized by the
reaction of N-substituted picolinamide with [Ru(η6-arene)Cl2] in an identical method adopted
for compound 1 and 3.
The 1H NMR spectra for complex 2 and 4 display peaks at 8.98 to 7.57 ppm typical for the
aromatic ring for ligand L1. Lack of an NH signal indicates the deprotonation of the ligand.
The signals of the arene ring are observed 6.26 to 5.43 ppm and 2.84, 2.28, 0.98 and 0.72
ppm for p-cymene and 6.0 ppm for coordinated benzene ring. The resonance due to the PTA
is shifted towards lower field with respect to uncoordinated PTA confirming the
1
coordination. Although usually free PTA shows only two singlets in H NMR, however we
have observed multiplets when coordinated to metal center as reported earlier.34 Pyridinyl
signals for the ligand HL2 in compound 5 and 6 are observed in the range 9.03 to 6.81 ppm.
Similar peaks in the range of 9.54 to 7.22 were observed for ligand L3 in compound 7 and 8.
13
The C spectra for compounds 2, 4, 6 and 8 also corroborates with the proposed structure.
The 31P NMR spectra of compounds 2, 4 , 6 and 8 show peaks in the range of -30 to -39 ppm
as reported earlier.10 The ESI mass spectra of 2, 4 , 6, 8 revealed two main peak envelopes.
The higher one corresponds to
[RuL(arene)(PTA)]+ and the other being due to
[RuL(arene)]+. Compound 5 and 7 produce ESI-MS peak for [RuL(p-cym)]+ moiety.
The solid state structure of the four compounds (2, 4, 6 and 8)
were investigated by X-ray crystallography. All the compounds possess the characteristic
psudo-octahedral geometry exhibited by all RAPTA complexes. The η6 π-bonded arene ring
occupying three coordination positions of octahedron, along with two coordination sites
occupied by nitrogen donor of N-substituted picolinamide moiety and the other coordination
site is occupied by PTA ligand through the phosphorus donor. The asymmetric unit of 2
comprises one complex cation, one BF4 anion and two water molecules (Fig 1). The (L1)
ligand binds the metal in a N,N-chelating fashion, thus giving rise to a five-membered
RuNCCN metallacycle, the ruthenium coordination environment being then fulfilled by the
P-atom of PTA and by the -bonded aromatic ring of p-cymene. Bond distances and angles
are in the range of those found in other ruthenium compounds and PTA or arene
derivatives.14 As a result of steric restrictions, the anionic organic ligand is twisted, as
measured by the angle (25.03 º) between the least-square planes of its aromatic and pyridinyl
ring; in addition, the least-square planes of the cymene and of the metallacycle rings are

Page 13 of 32
Dalton Transactions
View Article Online
49.84 º apart. The Ru-P bond is nearly orthogonal to the metallacycle (88.95 º), but markedly
DOI: 10.1039/C6DT02969H
away from the plane of the arene (130.40 º). The isolated ruthenium complexes form a 1D
hydrogen bonded chain along b-axis through C32-H32A...O3 and C34-H34B...O3. The
-
counter BF4 anion and two water molecules as solvent of crystallization cemented these
parallel chains through further hydrogen bondings [C22-H22...F1, C27-H27B...F1, C12-
H12...F1, C30-H30C...F2, C31-H31A...F3, O1S-H1A...F3, C25-H25...F4, C33-H33A...O1S,
O2S-H2A...N31] for a 2D hydrogen bonded polymer in bc-plane.
Compound 4 also have a similar kind of structure like compound 2 (Fig 2). However, the
twisting in the anionic ligand is much greater here which is found to be 56.3º between the
least-square planes of its aromatic and pyridinyl rings. In compound 4 every complex
molecule is found to be interacting to three other surrounding molecules through C13-
H13...O1, C21-H21...O1, C6-H6...O2, C11-H11...N4, C9-H9...N5. It gives a complicated 3D
-
packing which is further reinforced by octahedral PF6 counteranion [C2-H2...F3, H19-
H19...F3, C17-H17A...F2 and C22-H22...F1] and one methanol as solvent molecules.
Compound 6 has shown to be crystallizing in monoclinic space group P21/n. Two pyridyl ring
in the ligand was found to be twisted at an angle of 37.43° (Fig 3). A methanol molecule is
also observed as solvent of crystallization. However the most interesting observation is that
the presence of two hexafluorophosphate ion per ruhtenium center indicating the neutral
nature of the substituted ligand. A closer observation of the crystal structure reveals
comparative shorter C-N bond distance of amino pyridyl fraction of the ligand [C7-N2
1.396(5) Å] along with C8-C9 and C10-C11 bond distance in pyridyl ring [1.365(6) and
1.366(6) Å, respectively) indicating the protonation of the 4-pyridyl nitrogen in presence of
ammonium salt during the complexation which leads to essentially change of valence-bond
representation of the ligand as per scheme 2. Protonation at 4-pyridyl nitrogen atom gives a
partial double bond character to the C7-N2 bond making it a comparatively labile in nature.
A similar kind of observation is reported earlier for an alkyl pyridine palladium complex.35
This subtle change in the system makes the ligand relatively weaker with respect to complex
8 which is evident through comparatively longer bond length between donating nitrogen atom
and ruthenium center [N(1)-Ru(1) and N(2)-Ru(2)]. There are two crystallographically
independent PF6- ions present. One of them connect three ruthenium cationic complex
-
through C25-H25A...F1, C23-H23A...F2, C2-H2...F6 and C15-H15...F4. The other PF6
connects four such complexes through C27-H27A...F8, C18-H18...F7, C25-H25B...F9, C24-
H24A...F12 and C11-H11...F12. The N-pyridyl hydrogen has shown a strong hydrogen bond

Dalton Transactions
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with methanol molecule which act as solvent of crystallization through N3-DHO3I:N10..1.0.O39/1C16D1T02969H
[1.896 Å]. All these hydrogen bonds leads to formation of 3D-hydrogen bonded network .
Compound 8 crystallized in triclinic space group C2/c. Two pyridyl ring in the ligand was
-
found to be twisted at an angle of 45.91° (Fig 4). One BF4 ion per ruthenium center is
observed indicating that the ligand as an uninegative charge unlike compound 6. This is due
to the inability to stabilize the alternative valence bond sturcture as per previous case by the
pyridyl N-atom at 3-position. Each BF4- ion is connected to six complex units via hydrogen
bond, viz. C18-H18...F3, C24-H24B...F3, C26-H26B...F2, C27-H27B...F3, C25-H25B...F1,
C19-H19...F1, C1-H1...F1 and C3-H3...F1. Furthermore each molecule is connected to two
different molecules through amide O atom via hydrogen bonding [C9-H9...O1, C8-H8...O1,
C23-H23B...O1,] and PTA N-atom [C24-H24A...N6]. Altogether it forms a hydrogen
bonded 3D network stabilized by counteranion.
Conductivity measurements
In order to investigate the probable structures of compound 6 and 8 in solution, the
conductivity of their solution was measured. Conductivity values for compound 6 in
acetonitrile and water were found to be 270 Ω-1 cm2 mol-1 and 280 Ω-1 cm2 mol-1 attesting
the 1:2 composition of the complex with retention of the dicationic nature of the ruthenium
complex moiety.36 On the other hand compound 8 is showing molar conductance around 106
Ω-1 cm2 mol-1 indicative of 1:1 composition as per solid state crystal structure.36 The
conductance measurement of compound 5 and 7 provide the value corresponding to non-
electrolyte which is in agreement with the solid state structure.
Protein Binding Studies:
As a probable candidate of tumour growth inhibition agent we were interested to study the
interaction of the synthesized complexes with different biomolecules. However, we did not
find any interaction like binding or cleavage to CT-DNA by any of the synthesized
complexes. On the other side all the complexes have shown interesting interaction with serum
albumin which is important because of its ability to carry active molecules in the blood
through their non-covalent conjugation. These are the group of proteins which can bind metal

Page 15 of 32
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ions and complexes and transport through blood streams. Fluorescence quenching experiment
DOI: 10.1039/C6DT02969H
was carried out to understand the mechanism of action between synthesized complexes and
HSA (human serum albumin). The high sensitivity of the tryptophan moiety and its
surrounding local environment present in the albumin towards intrinsic fluorescent spectra of
proteins can provide certain useful information of structure and dynamics which are often
utilized in the study of protein folding and association reaction.
Separate solutions of HSA were titrated against addition of different compounds. In case of
HSA binding, addition of reported compounds to the respective protein solution causes
significant decrease of initial fluorescence intensity for the compounds number 2, 4, 6, 8
respectively. In case of compound no. 2 and 4, plots [F0/F vs [Q] ] with upward curvature
towards y-axis. were obtained, signifying concurrent quenching by collision as well as by
complex formation with the same quencher.37To get further insight in the quenching process,
the fluorescence quenching data were analyzed with Stern-Volmer equation as described in
experimental section. The value of KSV obtained by linear plot of F0/F vs [Q] follows the
order 2 > 4 > 8 > 6. The nitro substituted ligand based complexes (2 and 4) are capable of
quenching the tryptophan fluorescence more strongly with respect to pyridyl ligand based
complexes (6 and 8). This may be due to the presence of substituted phenyl ring instead of
pyridyl group which may lead to stronger hydrophobic interaction with the protein. Similarly
p-cymene complex 2 has been found to be more interactive with protein due to enhanced
hydrophobic interaction of methyl and isopropyl groups of p-cymene with tryptophan site.38
The kq value obtained for all the complexes are found to be in the range of 8.04 X 1011 - 6.41
X 1013 L mol-1 S-1, indicating the involvement of static quenching mechanism.39 To
determine the binding constant and number of binding sites, the Scatchard equation was
employed, which is given by
푙표푔 [퐹0−퐹] = log 퐾a + 푛 log[푄]
퐹
Where Ka and n are the binding constant and number of binding sites respectively, and F0 and
F are the fluorescence intensities in the absence and presence of the quencher respectively.
Thus, a plot of log [(F0 − F)/F] versus log[Q] can be used to determine the value of binding
constant (from the intercept) and number of binding sites (from the slope). Using Scatchard
plot, binding constants and n values were also calculated for all the compounds which are

Dalton Transactions
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represented in table 2. The value of n for all the complexes are in the range ~1, indicating
DOI: 10.1039/C6DT02969H
comparable binding property of the complexes with HSA.
Growth Inhibition of ruthenium-arene complexes:
The antiproliferative activities of compound 2, 4, 6, and 8 were studied against three cell lines
viz. breast cancer cell line MCF 7, ovarian carcinoma cell line A2780 and lungs cancer cell
line A549. SRB assay is utilized to obtain the results in terms of GI50 (concentration of drug
that produces 50% inhibition of the cells), TGI (concentration of the drug that produces total
inhibition of the cells) and LC50 (concentration of the drug that kills 50% of the cells). All
cancer cells were exposed to 24h to increasing concentrations of the compounds, and their
proliferation were determined. Adriamycin, a chemotherapeutic drug was used as a positive
control. Though compound 2, 4 and 8 do not show much cell growth inhibition, however
quite interestingly compound 6 have shown remarkable cell growth inhibition properties
against all cell lines almost to the tune of adriamycin in terms of molar concentration. All the
data are summarized in table 3 and in fig 5.
Stability study of compound 6 and 8:
To understand what might leading compound 6 to be so active against cancer cell lines
whereas similar compound 8 is non active, we have performed the solution stability study of
complex 6 and 8. Many a times cleavage of M-L bond is found to be the first step for the
activation of Ru-complexes in solution.40 Thus it is of considerable interest to know the
nature of species generated in solution which is responsible for its cytotoxic behaviour. Thus,
the hydrolytic behaviour of the compound 6 was studied to check its stability under
pseudopharmacological conditions. The decomposition nature of the compound was studied
in 5 mM NaCl solution (corresponding to the low intracellular NaCl concentration in cells)
and in 100 mM NaCl solution (assuming to the higher NaCl levels in blood plasma).
Compound 6 was dissolved in aqueous NaCl (c= 5 or 100 mM in D2O containing 10% D6-
DMSO) and maintained at 37°C for 7 days. Decomposition of the compound was monitored
31
using P NMR spectroscopy. Compound 6 underwent immediate transformation in both the
solutions (5 mM and 100 mM NaCl) with the substitution of picolinamide ligand with two
chloride ions to form [Ru(cym)(PTA)Cl2] which is evident by the typical 31P singlet peak at -
35.5 ppm (Fig 6 and Fig 7),41 whereas no peak, corresponding to the starting complex 6 at -

Page 17 of 32
Dalton Transactions
31
View Article Online
DOI: 10.1039/C6DT02969H
38.9 ppm was observed any more. Analysing the P NMR data, it can be said that compound
6 is unstable in intracellular NaCl concentration in cells as well as in extracellular blood
plasma. It undergoes instant transformation to form RAPTA-C predominantly. However, a
similar study with complex 8 has shown the compound is quite stable at both 5 mM and 100
31
mM concentration of NaCl without showing any other P peak even after 7 days except that
characteristic one for complex 8 itself at -37.8 ppm (Fig 8 and Fig 9). This results are in
agreement with the observation of solid state structure determination where a valence bond
tautomer of ligand HL2 gets stabilized making the substituted picolinamide ligand weaker one
with respect to ligand HL3 in complex 8. Perhaps this dissociation of the ligand is playing
some important role in the observed antiproliferative property of complex 6.
As complex 6 has shown tendency to form [Ru(cym)(PTA)Cl2] in NaCl solution therefore to
check the possibility that if [Ru(cym)(PTA)Cl2] is the actual active species for
antiproliferative properties of complex 6, we have performed cytotoxicity study of
[Ru(cym)(PTA)Cl2] against all three cancer cell lines. It has been observed that that GI50
values for [Ru(cym)(PTA)Cl2] is quite high against all the three cell lines (table 5 ). Thus, it
can be concluded that [Ru(cym)(PTA)Cl2] is not the responsible entity for the
antiproliferative activity of compound 6. In physiological medium, after the ligand gets
liberated from the coordination sphere owing to its labile nature, some other biomolecule like
thioredoxin reductase; which is already present in the physiological medium might get
attached to ruthenium centre which activates the compound to act as an antiproliferative
agent.
Mammalian Trx-R Inhibition:
To further investigate the possible pathway by which compound 6 is showing growth
inhibition properties we have carried out mammalian thioredoxin reductase inhibition
properties of the synthesized complexes. Mammalian thioredoxin reductases (TrxR) are large
homodimeric proteins that play an important role in the intracellular redox metabolism, along
with a few other biochemical systems.23 Thioredoxin reductases are characterized by broad
substrate specificity and by easily accessible redox centers. In mammalian TrxR, the redox
center consists of a cysteine selenocysteine redox pair that approaches the N-terminal active
site of the other subunit for electron transfer.42 The active site selenolate group, after
reduction, manifests a large propensity to react with “soft” metal ions, making TrxR a likely
pharmacological target for a vast array of metallodrugs. This is probably the reason why
various gold(I) and platinum(II) compounds were earlier reported to be potent inhibitors of

Dalton Transactions
Page 18 of 32
mammalian thioredoxin reductase.43,44 Recent studies suggest that someDOrI:u10th.10e3n9i/Cu6mDT02969H
complexes also show Trx-R inhibition owing to its ‘soft’ nature.42,45 Complexes 2, 4, 6 and 8
have been tested for TrxR inhibition at various concentration. Whereas compound 2, 4 and 8
have shown very little activity towards Trx-R, compound 6 was found highly active against
the enzyme. The IC50 value was found to be 11.07μM which might be due to the weakly
bound substituted picolinamide ligand which dissociates easily in presence of thioredoxin
reductase with selenocysteine redox center. The selenocysteine group might get coordinated
to ruthenium center inducing inhibition of the enzyme which might be a plausible reason of
inherent tumor growth inhibition property of compound 6.
View Article Online
Conclusion:
Four new ruthenium (II) complexes containing N-substituted picolinamide ligand with
amphiphilic PTA ligands have been synthesized and characterised. All the complexes are
water soluble and their interaction with HSA protein molecules have been explored.
Cytotoxicities for the prepared compounds have been evaluated for different cell lines.
Compound 2, 4 and 8 are having general molecular formula [RuL(6-arene)(PTA)]X where
the ligands (L) behave as monoanionic ligand. Whereas in compound 6 the ligand takes a
valence bond tautomerised protonated form which behaves as neutral polar ligand making the
complex dicationic in nature. Interestingly among the four compounds, compound 6 show
very promising antiproliferative activity against all the three cell lines with GI50 value
comparative to adrimycin, a cytotoxic drug in μM scale. To understand the mechanistic
pathway, protein binding studies, hydrolysis and ligand substitution and mammalian Trx-R
inhibition studies were carried out for all the complexes. Though all the compounds show
significant binding with HSA protein, whereas, only compound 6 strongly inhibits Trx-R
activity which is probably having some correlation with its strong antiproliferative property.
Supporting Information :
CCDC 1036656, 1449639, 1477542 and 1444481 contain the supplementary
crystallographic data for 2, 4, 6 and 8 respectively. These data can be obtained free of charge
via
http://www.ccdc.cam.ac.uk/conts/retrieving.html,
or
from
the
Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-
336-033; or e-mail: deposit@ccdc.cam.ac.uk.

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DOI: 10.1039/C6DT02969H
Acknowledgement :
We are grateful for the financial support received from SERB, DST Govt of India through
research grant no SR/S1/IC-43/2012. We are also thankful to the Sophisticated Instrument
Center, IIT Indore for the structure elucidation (Compound 6 and 8).

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DOI: 10.1039/C6DT02969H
Schemes:
Scheme 1: Synthesis of compounds [Ru(L1)(η6-p-cymene)PTA][BF4] 2, [Ru(L1)(η6-
benzene)PTA][PF6] 4,
[Ru(HL2)(η6-p-cymene)PTA][PF6]2 6, and [Ru(L3)(η6-p-
cymene)PTA][BF4] 8

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DOI: 10.1039/C6DT02969H
Scheme 2: Valence bond tautomerism of compound 6
Figures:
Fig 1: The solid state crystal structure of compound 2 with partial atomic numbering scheme

Dalton Transactions
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DOI: 10.1039/C6DT02969H
Fig 2: The solid state crystal structure of compound 4 with partial atomic numbering scheme
Fig 3: The solid state crystal structure of compound 6 with partial atomic numbering scheme

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View Article Online
DOI: 10.1039/C6DT02969H
Fig 4: The solid state crystal structure of compound 8 with partial atomic numbering scheme
MCF7
A2780
A549
100
80
60
40
20
0
0.00
0.02
0.04
0.06
0.08
Concentration(M)
Fig 9: analysis of GI50 value of Compound 6 against different cell lines using
adriamycin as positive control

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DOI: 10.1039/C6DT02969H
Fig 6: 31P NMR of compound 6 in 5mM NaCl solution after 7 days. 31P NMR of compound 6
in 10% D6-DMSO [inset].

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DOI: 10.1039/C6DT02969H
Fig 7: 31P NMR of compound 6 in 100mM NaCl solution after 7 days. 31P NMR of
compound 6 in 10% D6-DMSO [inset].
31
Fig 8: 31P NMR of compound 8 in 5 mM NaCl solution after 7 days. P NMR of compound
8 in 10% D6-DMSO [inset].

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DOI: 10.1039/C6DT02969H
Fig 9: 31P NMR of compound 8 in 100mM NaCl solution after 7 days. 31P NMR of
compound 8 in 10% D6-DMSO [inset].

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DOI: 10.1039/C6DT02969H
Tables:
Table 1: Summary of the Crystallographic data for complexes 2, 4, 6, 8
2
4
6
8
formula
[C28H34N6O3PRu]
[BF4].2(H2O)
757.49
[C24H26N6O3PRu]
[PF6].(CH3OH)
[C27H35N6OPRu] [C27H34N6OPRu]
BF4
2[PF6].(CH4O)
Formula wt
Crys syst
space group
a(Å)
755.52
913.63
677.45
Monoclinic
C 2/c
Triclinic
P -1
Triclinic
P-1
Monoclinic
P 21/n
9.2108(7)
10.1314(8)
18.1559(12)
105.174(3)
98.350(3)
92.197(3)
1612.69
10.219(5)
10.410(5)
15.762(5)
76.497(5)
79.789(5)
63.638(5)
1455.6(11)
2
8.9084(2)
35.5854(8)
11.1155(3)
90
39.549(2)
12.8897(4)
12.5616(3)
90
b (Å)
c (Å)
α (deg)
β (deg)
γ (deg)
V (Å3)
96.018(2)
90
90.442(3)
90
3504.29(15)
4
6403.4(4)
8
Z,
2
molecules/cell
Density(g/cm3 1.560
)
1.724
0.731
1.732
0.687
1.405
0.593
Absorp coeff 0.606
(mm-1)
λ [Mo-Kα] (Å) 0.71073
0.71073
293
0.71073
293(2)
0.71073
150(2)
T(K)
150

Dalton Transactions
Page 28 of 32
View Article Online
DOI: 10.1039/C6DT02969H
no of reflns 43759
collected
68017
7243
52669
11685
25101
5967
No
of
5639
independent
reflns
5626
8373
4828
No of obsd
reflns
6673
0.0348
0.0854
1.052
0.0458
0.1222
1.133
R1
0.0419
0.1422
1.04
0.0607
0.1717
1.119
wR2
GOF
Table 2: Selected Bond Lengths (Å) for Compounds 2, 4, 6 and 8
Compound Ru(1)-P(1)
Ru(1)-N(1)
2.118(2)
2.092(3)
2.086(3)
2.079(3)
Ru(1)-N(2)
2.072(2)
2.096(3)
2.115(3)
2.100(3)
Ru(1)-Ccentroid
1.735
2.3112(7)
2.327(1)
2.319(1)
2.319(1)
2
4
6
8
1.735
1.736
1.736

Page 29 of 32
Dalton Transactions
View Article Online
DOI: 10.1039/C6DT02969H
Table 3: Selected Bond Angles (°) for Compounds 2, 4, 6 and 8
Compound P(1)-Ru(1)-N(1)
P(1)-Ru(1)-N(2)
86.90(7)
N(1)-Ru(1)-N(2)
77.80(9)
87.54(7)
87.37(8)
85.14(9)
83.70(9)
2
4
6
8
88.84(8)
76.9(1)
89.38(9)
77.9(1)
90.30(8)
77.2(1)
Table 4: Various parameters obtained from HSA interaction with the prepared compounds
HSA
Ksv(M-1)
3.21x104
2.87x104
Kq(M-1S-1)
5.19x1012
4.64x1012
Ka(M-1)
2.24x105
1.14x107
n
Compound 2
Compound 4
1.23
1.55
Compound 6
Compound 8
4.97x103
5.60x103
8.04x1011
8.28x1011
1.41x104
4.87x104
1.09
1.24
Table 5: GI50 value (μM) of the prepared compounds in human cancer cells
Compound
MCF7
A2780
A549
>80
>80
>80
>80
<0.1
>80
>80
<0.1
2
>80
>80
4
<0.1
>80
<0.1
>80
6
8
[Ru(cym)(PTA)Cl2]
Adriamycin
>80
>80
<0.1
<0.1