Indian Journal of Che mistry Vol. 40A, December 200 I , pp. 1288-1294

Synthesis, spectroscopic, luminescence, electrochemical and antibacterial studies of ruthenium(II) polypyridyl complexes containing 3-hydroxyflavones as

co-ligand

Lall an Mishra· & Ashok K. Singh .

Fac ulty of Sc ience, Che mistry Department , Banaras Hindu Uni versity, Varanasi 221 005 , India

Received 27 October 2000; revised 30 January 2001

Some mononuclear Ru(ll ) polypyridy l (2,2'-bipyridy l/1, 10-phenanthro line) compl exes contammg 3-hydroxy-4'-2 4

substituted Oavones (L H and L H) as co-ligands have been sy nthesised and characteri sed on the bas is o f the ir elemental I I I

analyses, FAB mass and spectral (IR , UV/visible, H NMR, and H- H COS Y NMR) data . Luminescence, e lectroche mi cal and anti bacteri al properties of the ligands and their Ru(ll ) compl exes have also been d iscussed.

3-Hydroxy fl avone (fl avonol) deri vat ives have been used as important yell ow dyes since ancient time. For example, weld; a yellow dye deri ved from the seeds and leaves of Raseda lutelo L, commonly known as dye's rocket, is used in the production of Lincoln green' . Another fl avone deri vati ve, quercetin has also been reported to have been used as dye but it degrades in li ght. Both type of dyes have been reported2 to show wide sp·ectrum antiviral and antitumor activities also. It has been observed that these fl avones interfe re with the initi al steps of the sy nthesis of viral RNA. Although the molecul ar mechani sm of such activities is not understood completely, yet there is a probability th at they inhibit the fo rmati on of minus strand RN A of poliov irus by interacting with one of the proteins involved in the binding of the virus-repli cation complex to ves icu lar membrane where virus rep lication takes place. Thus, the li mited attention paid to chelates of fl avanols and their bioacti viti es, prompted us to attach some flava nol deri vati ves with ruthenium(H) polypyridy ls, as polypyridyls of Ru(JI ) are objects of interest in view of their long-li ved charge-transfer excited state properties bes ides their signi fica nt redox behaviour3.

Materials and Methods Analytical grade solvents, 4-benzyloxybenzaldehyde,

4-chlorobenzaldehyde, 2'-hydroxyacetophenone and RuC1 3.3H20 were purchased fro m Aldrich and used as such, whereas cis-[Ru(bpy)2 CI 2].2H20 (bpy = 2,2'-bipy ridine) and [Ru(phenh CI2] (phen = I, I 0-phen-anthroline) were prepared using reported method

4•

Progress of the reactions was monitored by thin layer

chromatographic technique. All the complexes were prepared under nitrogen atmosphere. UV /Vi s spectra of the complexes were recorded at room temperature using a Varian Cary 2390 spectrophotometer. However, their 1 H NMR spectra were recorded on Jeol FX 90Q FT NMR spectrometer. FAB-mass and elemental analyses data were obtained from CDRI Lucknow, India. Electrochemi cal studi es of the complexes were carri ed out on a Electrochemi cal Interface SI 1287 potentiostat. The lumi nescence data were obtained from the University of Sevill a, Spain.

Synthesis of ligands The li gands were synthes ised usin g a reported2

method (Scheme-l ). 2'-Hydroxy-acetophenone (0.02 mol, 2.72g) and 4-benzy loxybenzaldehyde (0.02 mol, 4.24 g) were di ssolved together in ethanol (200 cm3) under stirring to whi ch aqueous NaOH (50%, 12cm3) was added dropwise . After stirri ng the reaction mi xture fo r 96 h at roo m temperature, it was diluted with water and then acidified with HCI (1 0%). The precipitate thus obtained was filtered off and crystalli zed from eth anol and dried in vacuo. 2'-Hydroxychalcone (L 1 H) thus obtained (330 mg) was di ssolved in MeOH (l 5 cm3) under sti rri ng and to thi s warm H20 2 (30%; I 0 cm

3) was added over a period of

15 min. The solution was diluted wi th H20 fo llowed by acidification by HCI ( I 0%). The CH2Cl2 layer, after drying on Na2S04, was evaporated and the residue obtai ned (eH) was crystallised from methanol; m.p. 168°C. 2'-Hydroxyacetophenone and 4-chlorobenzaldehyde were also condensed similarl y. The corresponding chalcone (L3H) isolated was then

MISHRA eta/.: Ru(ll ) POLYPYRIDYL COMPLEXES CONTAINING 3-H YDROXYFLAYONES 1289

+ 0 . EtOH OH POCH,$ CH, H NaOH (50%)

(~ = p~nyl)

Synthesis of ligands

Scheme I

H

LH

l NaOH(lO%) H,o, (30%)

OCH2~

rAIOH }-10;-cl ~L3H-

l NaOH(lO%) H,0,(30%) rf)(0)-(0)-cl ~0;-

cyclised into 3-hydroxyflavone (L 4H) (Scheme I). These products were characterised by comparing their IR, 1H NMR and 13C NMR with earlier reported5 data.

Synthesis of complexes Ruthenium (II) complexes (M 1 and M5) were

prepared by the following general procedure. An ethanolic solution (5 cm3) of cis-[Ru(bpyh Ch].2H20 (1 mmol, 520 mg) containing ethanolic solution (5 cm3) of respective ligands (L2H and L 4H) , (1 mmol, 344 mg and 272 mg respectively) and a few drops of Et3N were refluxed together for 14 h. After cooling at room temperature, respective solutions were concentrated in vacuo. To the concentrated solutions, saturated aqueous solution of NH4PF6 was added and the corresponding solids thus obtained were washed successively with H20 , EtOH and then Et20 , dried in vacuo and purified by column chromatographic technique using neutral alumina as column support and CH3CN aqueous KN03-water (7 : 1:0.5 v/v) as eluents6. Eluates thus obtained were concentrated and corresponding residues were then dissolved in acetone and reprecipitated by add iti on of saturated aqueous sol uti on of NH4PF6. The crystalline solids thus obtained were fi ltered and washed successively with H20 , EtOH and Et20 and then dried in vacuo. The complexes of the ligands L

2H and L

4H with

[Ru(phenh C1 2l (M2 and M6) were also prepared and

purified using simjlar procedures as above. The analytical data along with other physical

properties of the ligands and their complexes are given in Table I whereas a representative FAB-rriass spectrum of complex M

6 is shown in Fig. 1. The

complex M4

was prepared by direct debenzoylation2

of the complex M2

(1 mmol) in CH3COOH (10 cm3

)

and cone. HCI (5 cm3) for 6 h on a water-bath. After cooling at room temperature, solution was diluted with water (25 cm3) when a precipitate was obtained, which was filtered and washed successively with excess H20, EtOH and Et20 and was crystallized from EtOH. However, the complex M

3 was isolated

and purified using the method reported for M 1

condensing the debenzoylated2 ligand eH (L2,H) and cis-[Ru(bpyh CI2] .2H20.

Results and Discussion The complexes were found to be thermally stab le

and soluble in acetone, acetonitrile, DMF and DMSO. The molar conductances of the complexes recorded in CH3CN solution (10.

3 M) were found to be consistent with the reported values7 .

IR Spectra of chalcone (L1H and L

3H) gave

characteristic peaks at 3600, 1640 and 980 em· ' which are assigned to v( -OH), v(C=O) and v(CH=CH) respectively in view of earlier report5 . IR spectra of eH and L 4H also showed similar peaks as above except that the v(CH=CH) was absent and a new peak was observed at 3450 em· ' ass igned as v(OH). These evidences support the cyclisation of L 1H, L3H into corresponding L 2H and L 4H products. However, in the IR spectra of the complexes, a peak observed at -1620 cm- 1 in the spectra of the free ligands.

For further support of the cyclisation, NMR ( 1 H and 13C) spectra of L 1H and L3H recorded in DMSO-d6 were compared with the corresponding NMR spectra for eH and L 4H. From the peak positions shown in Table 2, it is evident that HC=CH protons exhibit a doublet at 8 7.01-7.06 ppm in the spectrum of L 1H and L3H. Major peaks observed at 8 193.61, 163.55, 148.24 and 136.08 ppm in the 13C NMR spectrum of L 1 H are ass igned as C=O, C-OH (C-~) and (C-a) carbons, respectively which are shifted to 8 173.3, 155 .3 1, 145 .29 and 137.76 ppm, respectively in the 13C NMR spectrum of L

2H. Similary, peaks

observed at 8 193.85, 164.03, 144.29 and 136.08 ppm in the 13C NMR spectrum of L3H ass igned as above also shifted to 8173.81,155.76,144.17 and 138.94 respectively in the spectrum of L

4H. Thus, the

1290 INDIAN J CHEM, SEC A, DECEMBER 2001

Table I-Physical and analytical data of Ru (II) complexes

Complexes FAB-mass data Yield % Found (Calc.) (colour)

I 2 [M- PF; ] 757 (756) 48 .3 M [RuL (bpy)2]PF6;

(Dark brown) 2 2

[M-2 PF6- ] 805 (804) 57. 1 M [RuL (phen)2](PF6h .2H20

3 2' (Light brown)

M [RuL H(bpyh](PF6) 2.7H20 [M] 957 (957) 50.0 (Yelowish)

M4

[RuL2 'H(phenh]2PF6.EtOH [M] I 005 (I 005) 48.0 (Reddish yellow)

5 4 + 50.5 M [RuL (bpy)2] PF6.4EtOH [M- PF6- ] 685 (684) (Yellowish) 6 4 [M] 878 (878) 49.8 M [RuL (phen)2] PF6.EtOH

(B lack)

100 \S4

90 l\S

80

70 1!6

60

50 120

'0 640 28! 7ll

30 791

20 467

10 171

0 100 200 300 ,00 500 600 700 800 900

Fig. 1-FAB-mass spectrum of the complex [RuL \Phen)2](PF6)

cyclisation of L 1 and L3 into corresponding compounds L2H and L 4H is supported by their 1H and 13C NMR spectra.

Calc. (Found), % .c H

56.0 (55 .9) 3.4(3.4)

48.4 (48.8) 3 . .1 (2.7)

38.4 (38.3) 3.6 (3.6)

53.9 (54.3) 3.7 (3 .5)

50:8 (50.9) 4.7 (4.8)

53.3 (53.2) 3.5 (3.3)

.Qlll N (ohm·1 cm2 rno1" 1)

6.2 (6."2 ) 260

5.3 (4.9) 270

5.5 (5.1 ) 230

5.8(6.1 ) 260

5.5 (5.5 ) 150

5.3 (6.0) 147

/-

In the complexes M 1, M

5 and M

6, the deprotonation

of the -OH proton of 3-hydroxy group in eH-L 4H is supported by the number of counter-anions present in the molecular formula obtained on the basis of elemental analyses. This is further supported by the disappearance of -OH proton in their 1H NMR spectra. However, in the spectra of the complexes M

2,M3,M

4, OH proton did not deprotonate upon

coordination . Furthermore, 1 H NMR spectra of the complexes M

3 and M

4 did not show any peak at -8

5.2 ppm as assigned for> OCH2 protons in the spectra of the complexes M

1 and M

2• However, additional

peak due to -OH proton at 8 9.6 ppm and 8 9.5 ppm appeared in the spectra of M3 and M4 complexes respectively. In the spectra of the complexes peaks observed at 8 7 .1 2. - 7.42 ppm were assigned to four protons of aromatic group attached to -OCH2(Ph) (M

1

and M2

) along wi th four protons of aromatic group attached to chromone pati (Pht) at 8 8.0-8.6 ppm (Fig. 1 ).

Fig. 2- 1H- 1H COSY NMR spccti"U m of complex [RuL \bpy)2]PF6 in DMSO-c/6 at room t emp~ra t re.

However, five protons of aromatic group attached to -OCH2(Ph

MISHRA et al.: Ru(ll) POL YPYRIDYL COMPLEXES CONTAINING 3-HYDROXYFLA VONES 1291

Table 2- 1H NMR data of ligands and their Ru(ll) complexes

Compound 8 ppm

L1H 5.19 (2H, s, -OCH2), 7.0 (2H, d, Ph) 7.06 (2H, d, CH=CH), 7.42 (5H, m, Ph Ph-OCH2 > PhOH.

Thus, based on the elemental analysis and spectroscopic data, structures for the complexes are proposed as shown in Structure I.

1292 IND IAN J CHEM, SEC A, DECEMBER 200 1

Table 4 - Electrochemical data* of li gands and their Ru (II ) complexes

Compound Oxidation peaks, £ 112/V Reduction peaks, £ 112/V

0.4, 0.95 , 1.0, 1.36

0.18, 1.0

0.07 ,0.5, 1.08, 1.5 0.6, 1.05, 1.05, 1.3 1 0.36,0.54, 1.03, 1.5 0.32,0.64,0.8 1' 1.32 0.1 8,0.54,0.86, 1.12 0.18,0.54,0.9, 1.46

- 0.54,- 1.06,- 1.65 - 1.5,- 1.0

- 1.95,- 1.45,- 1.62 - 0.93,- 1.30, - 1.45, - 1.8 1 - 1.70, - 1.54, -0.45 -0.7 1, -1.45,- 1.92 - 0.78,- 1.33,- 1.9 -0.76, -1.25, -1.54, -0.9

*Obtain in acetonitrile solution (I o·3 M) containing 0.1 M tetrabutylammonium perchlorate (TBAP) at room temperature using Ag/AgCI as a reference electrode. Scan rate was 100 mY/s.

Electrochemistry The electrochemical behaviour of the free ligands

and their metal complexes were studied by cyclic voltammetry in CH3CN (10.

3M) containing TBAP as supporting electrolyte (warning :perchlorate salts are explosive , use of small amounts is recommended) graphite disc as working electrode, platinum wire as an auxiliary electrode and Ag/ Ag + as reference electrode. The cyclic voltammogram of free fl avone L2H showed three oxidation peaks at 0.4, 1.0, 1.36 V with very weak conesponding reductions. However, in the cyclic voltammogram of its metal complexes (Mt & M2) three oxidation peaks were also observed at 0.5, (1.08-1.05) and 1.34 V with concomitant increase in the current intensity of the peaks at 1.08-1.05 V indicating that the ox idation Ru(II)- Ru(III) also occurs in the same potential range. In the complexes M

5 and M

6 stronger oxidati on peaks were

observed as compared to those obtained for I ?

complexes M and M- e,xcept that the peaks are diminished in the case of M

5 and M

6 (Fig. 3)

com plexes with concomitant en hancement in peak intensity at 0.54 V. This indicates that in these complexes Ru (II) -7 Ru(HI) ox idation occurs at 0.54 V. Additionally , it was quite interesti ng to note that in the cyclicvoltammogram of the complexes M3 and M

4

Ru(U) -7 Ru(III) oxidati on occurred at (1.03-0.80) V. Thus the oxidation of Ru (II) -7 Ru(III) is found to be tuned with the functiona lity present at the para position of terminal aromati c group and oxidation potential is found to decrease with the decreasing electron donor power of the groups attached, viz. , PhOCH2 ~ PhOH > PhCI. Reduction of ligands L

2H and L 4H was found to occur at 0.6, 0.3, 1.06 to 1.0 V (broad) which in complexes containing polypyridy l

40

20

0

MISHRA et al.: Ru(II) POLYPYRIDYL COMPLEXES CONTAINING 3-HYDROXYFLAVONES 1293

Table 5-Luminescence data* of Ru (II) complexes

Compound Amax (em) !Rei# (em) Rel (nm)

Mt 412,530 0.01, 0.15 0.004, 0.006 , 416,530 0.01, 0.25 0.0007,0.01 M-

MJ 413,473,529 0.06, 0.06,0.19 0.003,0.003, 0.008

M4 411,530 O.Q7, 0.20 0.003,0.008 Ms M6

*Recorded in acetonitrile at 25°C. Solutions were 10·6 M (Acx =440 nm), #Relative to standard [Ru(bpy)3f+ (-)No luminescence was observed.

.--,200 :J

.Q

£ Vl c

$>! c

0

450 500 550

Wavelength {nm)

600

Fi g. 4-Luminesccnce spectrum of the complex !RuL\ bpyh l PF6 in CH3CN ( 10-

6M) at room temperature.

region at -450 nm for ruthenium complexes 10 is quite weak and the emissions are mainly originating from the ligand part. Since the redox orbitals of both free ligands and Ru(Il) complexes are found to li e at similar energies as is ev ident from their oxidation potential data , it was found difficult to make clear distinction on the origin of the emissions. Excited state life time measurement of all the compounds in CH3CN (10'

3 M) was made using a single photon counting FL 900 CD Edinburgh Analytical Instrument at the Physical Chemistry Department, University of Sevilla, Spain and the experimental data were processed by the deconvolution method and was found to be < Ins in a ll compounds. Since measuring limit of the instrument was upto I ns level, so we could not measure below thi s region.

Thus redox and emission behaviour of the free ligands and their Ru(ll) complexes were found quite similar indicating that energy gaps between LUMO of

Table 6-Antibacterial activity of the ligands and their Ru (II) complexes agai nst E. Coli in DMSO*

Compound (%)inhibition

L2H 17.95

L4H 17.30

Mt 37.52 Mz 17.30 MJ 40.78 M4 20.00 Ms 37.52 M6 75.04

*Concentration of the solution was 10 J1M.

free ligands and HOMO of the metal in complexes are very low.

Antibacterial activity Antibacterial activity of the free ligands and their

complexes was evaluated in DMSO at 10 pM concentration and the data shown in Table 6 are expressed as the (%)inhibition in growth of bacteria against the control (free DMSO) in which growth was considered to be I 00%. The activity data were obtained by subtracting any inhibition shown by the solvent (DMSO). The data show that activity in all cases could be related to the presence of groups at 4-position of terminal phenyl ring (Fig. I) and is found to be most significant in case there is a ch loro group attached at position 4-of the ring. However, in other cases where OCH2Ph or OH groups are at 4-position activity remain s almost the same. Another feature for the variation of activity could be related with the te rminal ligand attached to ruthenium in the complexes. Activity is hi gher when it is I , I 0-phenanthroline ring whereas it is lower in case of 2,2'-bipyridine ring as terminal li gand. This findin g is consistent with the reported observation3· 11 • In order to explore the molecular mechanism of the antibacterial behaviour, we allowed the most act ive compound (M

6) to interact with plasmid DNA

PBR322 at different compound-nuc leotide binding ratios (D/N). Since, DNA is thought to be most common site for interaction in view of an earlier report 12. Similar electrophoretic mobility 13 of the DNA in the presence and absence of compound clearly indicates that the re is no binding of the compound with the tertiary structure of the D A. Thus antibacterial property of the complexes foll ows a different mechanism; probably it is in consonance with the theoretical density calculation made by Mishra et al. 14.

1294 INDIAN J CHEM, SEC A, DECEMBER 2001

Acknowledgement One of the authors (LM) acknowledges UGC, New

Delhi for financial assistance and Dr. A K Tripathi , Biotech. , BHU, Varanasi for his help in evaluating antibacterial activity.

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