Heteroatom ChemistryVolume 19, Number 7, 2008

Synthesis, Characterization, and In VitroAntimicrobial and Antitumor Activities of SomeTetraphenylstibonium(V) CarboxylatesKiran Singhal, Rahul Mishra, and Prem RajDepartment of Chemistry, University of Lucknow, Lucknow 226007, India

Received 11 September 2007; revised 12 March 2008

ABSTRACT: Several hitherto unreported pentaco-ordinated tetraphenylstibonium(V) carboxylates ofgeneral formula (C6H5)4SbL, where L = o-OHC6H5

COO , (C6H5)2C(OH)COO , 2-(6-OCH3C10H6)CH(CH3)COO , ArCH(OH)COO (Ar = C6H5, p-CF3-

C6H4, and p OCH3C6H4),N

N COO–and N N

COO–

CH2CH3

H3C

O

have been prepared and characterized by elementalanalysis, solid state IR, 1H NMR, and 13C NMRspectra, conductivity, and molecular weight mea-surements. Spectroscopic data together with solutionphase studies conform to the requirement of triagonal-bipyramidal configuration for these compounds. Theywere tested for in vitro antifungal (against Aspergillusflavus and Aspergillus niger) and antibacterial(against Staphylococcus aureus and Klebsiellapneumoniae) activities. The in vitro antitumoractivity of all stibonium carboxylates was examinedagainst MCF-7 cell line. A few of them were foundto exhibit moderate to significant biological activ-ity. C© 2008 Wiley Periodicals, Inc. Heteroatom Chem19:688–693, 2008; Published online in Wiley InterScience(www.interscience.wiley.com). DOI 10.1002/hc.20498

Correspondence to: Kiran Singhal; e-mail: singhal.kiran@gmail.com.

Contract grant sponsor: CSIR, New Delhi, India.Contract grant numbers: 01/(1923)04/EMR-II, (09/107(0242)/

2002-EMR-I).c© 2008 Wiley Periodicals, Inc.

INTRODUCTION

In sharp contrast to the well-documented tetraaryl-stibonium -halides, -pseudohalides, -carboxylates,-sulfates, -chromates, -permagnates, -molydates,and so on [1–14], published data on synthesis,structure, antimicrobial, and antitumor activities oftetraarylstibonium derivatives isolated in combina-tion of biologically active carboxylic acids are quitelimited [6–8,14–17]. As a part of our research pro-gram on biologically active organoantimony com-pounds, we have now isolated a series of tetraphenyl-stibonium derivatives of α- and β-hydroxy carboxylicacids, naproxen (antiinflammatory), nalidixic acid(a broad spectrum antibiotic), and 2-pyrazine car-boxylic acid. Our interest was focused on inves-tigating in vitro antimicrobial and antitumor ac-tivities and examining whether these stiboniumderivatives will behave as covalent compounds withmonomeric constitution or charged tetraphenylsti-bonium, Ph4Sb+moiety with the presence of ionicspecies in the solution. Solid state IR, multinuclearNMR, and some solution phase studies have beenmade to ascertain the mode of bonding together within vitro antifungal, antibacterial, and antitumor ac-tivities to establish their biological efficacy.

RESULTS AND DISCUSSION

Tetraphenylstibonium carboxylates 1–8 can be ob-tained conveniently by the reaction of tetraphenyl-stibonium(V) bromide with the corresponding silversalts of carboxylic acid (Eq. (1)).

688

Synthesis, Characterization and Antimicrobial and -Antitumor Activities of Tetraphenylstibonium(V) Carboxylates 689

Ph4SbBr + AgL → Ph4SbL + AgBr (1)

Where

(8)N

NO–

C

O

i3

2 1

(7);

NNH3C

CH2 CH3

CO–

O

O

12

34

4"5

6

7

88"

(6);

8"8

7

6

54"

43

21

CH CO–

OCH3

CH3O

α

[R = –H (3), –CF3 (4), and –OCH3(5)];

12

3i CH C

OH

O–

OR

α

(2);

αC C

OH

Ph O–

Oi3'

2 1

(1);12

3

i

OH

O–C

O

L =

′

′′

′′

′′

′

′ ′′

′′

′

′

′′′′

′′′

′

′

′

′ ′

′′

′′

The newly synthesized carboxylates are whitecrystalline solids except compounds 2 and 7, whichare off-white and yellowish in color, respectively.These compounds are soluble in common organicsolvents except in few nonpolar solvents, that is,petroleum ether and n-hexane. The molecular weightdata in freezing benzene indicate that these carboxy-lates have molecular monomeric constitution. Themolar conductance values for 10−3 M solutions inacetonitrile at room temperature ranges between 10and 18 ohm−1 cm2 Mol−1, which show the absenceof ionic species in solution [18]. The physical andchemical data are listed in Table 1.

IR Spectra

The infrared spectra of compounds 1–8 wererecorded in solid state, using KBr pellets in the rangeof 4000–400 cm−1. IR absorption bands due to phenylgroups bonded to antimony are identical to thosereported earlier [2]. Absorption frequencies of diag-

TABLE 1 Some Physical and Analytical Data of Tetraphenylstibonium(V) Carboxylates

Elemental Analysis (%): Found (Calculated)

Compound No. Formula Molecular Weight mp (◦C) Yield (%) C H N

1 C31H25O3Sb 567.09 166 71.0 65.02 (65.65) 4.32 (4.40) –2 C38H31O3Sb 657.16 160 64.6 70.10 (69.45) 4.69 (4.71) –3 C32H27O3Sb 581.10 142 68.4 66.05 (66.14) 4.68 (4.64) –4 C33H26O3F3Sb 649.11 162 67.0 60.93 (61.06) 4.09 (4.00) –5 C33H29O4Sb 611.11 88 66.7 64.72 (64.85) 4.80 (4.74) –6 C38H33O3Sb 659.16 58 63.2 69.38 (69.24) 4.92 (5.00) –7 C36H31O3N2Sb 661.14 186 61.5 65.31 (65.40) 4.79 (4.68) 4.12 (4.23)8 C29H23O2N2Sb 553.06 180 68.0 63.08 (62.97) 4.02 (4.15) 4.94 (5.06)

TABLE 2 Characteristic IR Absorption Bands (cm−1) ofTetraphenylstibonium(V) Derivatives

ν (OCO)CompoundNo. νasym νsym �ν ν (O H) ν (Sb C)

1 1643 (m) 1356 (s) 287 3340 (w) 454 (m)2 1626 (vs) 1325 (vs) 301 3363 (s) 456 (m)3 1620 (vs) 1321 (vs) 299 3405 (s) 454 (s)4 1598 (s) 1301 (vs) 297 3426 (w) 455 (s)5 1609 (s) 1302 (s) 307 3445 (w) 455 (m)6 1635 (s) 1265 (m) 370 – 458 (s)7 1718 (s) 1360 (m) 358 – 456 (m)8 1656 (vs) 1349 (m) 307 – 455 (s)

vs: very strong, s: strong, m: medium, w: weak.

nostic value and their possible assignments for rep-resentative compounds are given in Table 2.

The broad band due to ν(OH) present in COOHgroup of corresponding carboxylic acid ligand is ab-sent in all carboxylates. Whereas ν(OH) for com-pounds 1–5, that is, α-hydroxy and β-hydroxy deriva-tives shows absorption in the range of 3445–3340cm−1 as weak or strong band. IR spectroscopyprovides a method of assessing carboxylate coor-dination modes from the position and separation(�ν) between the antisymmetric and symmetricCO2-stretching modes. In the IR spectra of thesecompounds, the carboxylate bands are observedin the characteristic region: νasym(OCO) between1718 and 1598 cm−1 and νsym (OCO) between 1360and 1265 cm−1. The magnitude of difference �ν

[�ν = νasym (OCO) − νsym (OCO)] suggests the pres-ence of a monodentate “ester like” COO group witha pentacoordinate antimony atom consistent withtriagonal-bipyramidal geometry [7,18,19]. The ν(Sb-C)-stretching mode corresponding to y-mode ap-peared between 458 and 451 cm−1 and correspondswell with those reported earlier [20,21].

Heteroatom Chemistry DOI 10.1002/hc

690 Singhal, Mishra, and Raj

TABLE 3 1H NMR Data of Tetraphenylstibonium(V) Carboxylates in δ (ppm)

Compound No. α-H OCH3/CH3/CH2 Naphthyl/Naphthyridine/Pyrazine Ph/Ph′

1 – – – 6.54–7.74 (m)2 – – – 6.92–7.13 (m)3 4.68 (s) – – 7.25–7.48 (m)4 4.67 (s) – – 7.18–7.45 (m)5 4.69 (s) 3.77 (s) – 7.12–7.46 (m)6 3.46 (m) 3.90 (s) (OCH3), 1.49 (d) CH3 7.10–7.69 (m) 6.90–7.15 (m)7 – 2.61 (s) (CH3), 1.37 (t) 7.60–8.96 (m) 7.11–7.38 (m)

(CH3 of ethyl), 4.58 (m) (CH2)8 – – 8.82–9.20 (m) 6.71–6.98 (m)

s: single, d: doublet, t: triplet, m: multiplet.

1H NMR Spectra

The 1H NMR spectral data of compounds 1–8 arelisted in Table 3. All the spectral data of newlysynthesized carboxylates were compared with thoseof tetraphenylstibonium(V) bromide and the cor-responding carboxylic acids. The peak due to thephenyl group bonded to antimony and of the car-boxylate group appeared as a multiplet and is over-lapped. The β-hydrogen in the case of the naproxenderivative 6 was observed as a doublet because ofthe presence of one hydrogen at the α-position. Forcompound 7, the methyl protons ( CH3) attachedwith a heterocyclic ring and of the ethyl groupappeared at δ = 2.61 (s) and δ = 1.37 (t) ppm, re-spectively, whereas the CH2 proton appeared atδ = 4.58 ppm. In addition, the signals for three mag-netically nonequivalent protons of naphthyridine

TABLE 4 13C NMR Data of Tetraphenylstibonium(V) Carboxylates in δ (ppm)

Compound No.

13C NMR 1 2 3 4 5 6 7 8

o-C 138.02 135.73 135.91 135.72 135.71 152.46 149.28 140.22m-C 130.41 130.28 129.13 129.08 132.44 130.92 128.40 126.17p-C 135.70 135.97 136.61 136.83 136.86 149.31 145.05 137.141′-C 130.52, 160.08 128.71 127.68 126.35 130.32 129.27 – 143.812′-C 117.74, 119.03 128.03 126.72 126.35 128.34 120.14 148.21 144.523′-C 135.17 128.03 126.80 128.27 129.26 129.49 136.11 147.634′-C – 81.80 – – – 106.05 179.03 –5′-C – – – – – 126.81 118.13 –6′-C – – – – – 157.83 108.52 –7′-C – – – – – 126.81 164.46 –8′-C – – – – – 129.41 – –4′′-C 136.06 122.548′′-C – – – – – 135.18 149.51 –i-C 136.59 143.64 135.13 134.71 134.92 134.14 134.08 133.58i ′-C 116.09 137.58 141.68 142.66 142.62 – – 146.10α-C – – 73.85 73.93 76.80 46.24 – –CH2/CH3/ – – – 125.04 53.21 18.86 (CH3), 24.90(CH3), –CF3/OCH3 55.33 (OCH3) 46.65(CH2),

14.89 (CH3 of ethyl)>C O 173.62 174.99 174.63 173.73 174.91 182.33 166.52 166.35

ring were recorded in the range δ = 7.60–8.96 (m)ppm. The integration of the peaks corresponds wellwith the proposed structure of the compounds. Allthe protons in the carboxylates have been identified,and the total number of protons calculated from theintegration curve tallies with the expected molecularstructure. The PMR spectral data are similar to thosereported earlier for such compounds [1–3,22,23].

13C NMR Spectra

The 13C NMR data of compounds 1–8 have beenrecorded, using CDCl3 as solvent (reference δ = 77.0ppm). The chemical shift details are summarized inTable 4. The number of signals found correspondsto the presence of magnetically nonequivalent car-bon atoms. The position of the aromatic carbon sig-nals remains almost unchanged in the case of α- and

Heteroatom Chemistry DOI 10.1002/hc

Synthesis, Characterization and Antimicrobial and -Antitumor Activities of Tetraphenylstibonium(V) Carboxylates 691

TABLE 5 Biological Activity of Tetraphenylstibonium(V) Carboxylates

Antimicrobial Activity

Antifungal Antibacterial

Aspergillus Aspergillus Staphylococcus Klebsiellaflavus niger aureus pneumoniae

Colony Colony Antitumour ActivityDiameter Inhibition Diameter Inhibition

Compound no. (cm) (%) (cm) (%) Cell count × 104 Activity

1 1.7 15 1.4 30 – – 10.29 ± 1.08 –2 1.6 20 1.3 35 + ++ 10.02 ± 0.62 +3 1.4 30 1.7 15 ++ ++ 10.20 ± 1.12 +4 1.4 30 1.6 20 ++ ++ 09.98 ± 0.58 +5 No activity – 1.4 30 – ++ 11.27 ± 1.92 –6 1.4 30 1.5 25 ++ +++ 10.38 ± 1.14 –7 – – – – – – 08.23 ± 0.48 ++8 1.7 15 1.4 30 + ++ 11.12 ± 2.12 –Control 2.0 – 2.0 – – – 10.22 ± 1.25 –

β-hydroxy carboxylic acids complexes as comparedto that of ligand. The α-carbon in most of the com-pounds is deshielded and shifts to lower field in com-parison to the free acids. The ipso-carbon of the lig-ands in all the complexes also shifted downfield inthe complexes. The position of carboxylate carbon(>C O) in all the tetraphenylstibonium carboxylatesshifts to lower field as compared to the free acid, in-dicating the participation of the carboxylate groupin coordination to metal [24].

Thus, on the basis of monomeric and non-conducting nature of tetraphenylstibonium com-pounds and the absence of bridging CO group(s)confirmed from spectral analysis, tetraphenylstibo-nium carboxylates can be assigned pentacoordi-nated triagonal-bipyramidal structure with the car-boxylate group at apical position in the solid state.These results are in good agreement with those re-ported for similar stibonium salt [9,10,22,23].

Antifungal Activity

The in vitro antifungal activity of compounds 1–6and 8 was evaluated against Aspergillus flavus andAspergillus niger at 10 μg mL−1 of the test com-pound, and the percentage inhibition was calculatedby the colony diameter of control and test samples(Table 5). Most of the compounds show poor tomoderate activity against both the strains. The com-pounds 3, 4, and 6 show moderate activity, whereascompounds 1 and 2 are poorly active against A.flavus. Surprisingly, compound 5 was found inac-

tive against A. flavus. The activity against A. niger ofcompounds 1 and 2 was found moderate, whereascompounds 3, 4, and 6 were poorly active. It is in-teresting to note that the carboxylates, which arepoorly active against A. flavus, show enhanced activ-ity against A. niger. The maximum activity was ob-served for tetraphenylstibonium benzilate 2 againstA. niger.

Antibacterial Activity

The compounds 1–6 and 8 were assayed for in vitroantibacterial activity against two human pathogenicstrain, namely, Staphylococcus aureus and Klebsiellapneumoniae (Table 5). It was surprising to note thatsalicylic acid derivative 1 does not show any activ-ity against both the strains, in contrast compounds2–5 were found moderately active against K. pneu-moniae; the similar pattern was observed for com-pounds 3, 4, and 6 against S. aureus. The naproxenderivative 6 exhibited maximum activity among allthe tetraphenylstibonium carboxylates against K.pneumoniae.

Antitumor Activity

The in vitro antitumor activity of compounds 1–8was carried out against human breast adenocarci-noma cancer cell line (MCF-7) by the MTT method(Table 5). Compounds 1, 5–6, and 8 were found inac-tive, whereas compounds 2–4 were found to exhibit

Heteroatom Chemistry DOI 10.1002/hc

692 Singhal, Mishra, and Raj

low activity. The nalidixic acid derivative 7 showsmaximum activity.

EXPERIMENTAL

Tetraphenylstibonium(V) bromide, Ph4SbBr, wasprepared by using the reported method [25]. All thecarboxylic acids (all from Aldrich) were used in theform of their silver salts. Naproxen was procuredin the form of tablets (500 mg/tablet), extracted withmethanol and ethyl acetate and subsequently crystal-lized from ethyl acetate and chloroform; mp 156◦C.All the reactions were carried out under dark inan atmosphere of nitrogen under anhydrous condi-tions. IR spectra were recorded in solid state, usingKBr pellets on FT-IR spectrophotometer (Shimadzu8201 PC) over the spectral range 4000–400 cm−1.1H and 13C NMR spectra were recorded on BruckerDRX-300 instrument using TMS and CDCl3 as refer-ence, respectively. The stoichiometry of compoundswas established by elemental analysis on a semimi-croscale, using elemental analyzer, Elemental VarioEL III, Carbo Erba 1108.

Synthesis of 1–8

A heterogeneous solution of tetraphenylstibo-nium(V) bromide (1.0 mmol) and silver salt of cor-responding acid (1.0 mmol) in THF (20 mL) wasstirred at room temperature for 24 h. The yellowishprecipitate of AgBr, thus formed was filtered off. Onthe removal of solvent from filtrate under vacuum,the crystalline solid was obtained. The obtained solidwas recrystallized from a mixture of THF and hexane(1:3).

Biological Activity

The antifungal activity of compounds was tested bypotato dextrose agar plate diffusion method [26], us-ing 10 μg mL−1 of test compound against A. niger andA. flavus. The percentage inhibition of compoundswas calculated by the reported method [27].

Percentage inhibition = C − TC

× 100

where C is diameter of fungus in control and .T isdiameter of fungus in test compounds.

Antibacterial activity of compounds was deter-mined by the disk-diffusion method [28]. The steriledisk of 5 mm diameter of filter paper (Whatman No.1) impregnated with the test compound (10 μg mL−1

in ethanol) was placed on the nutrient agar plateat 37◦C for 72 h. The inhibition zone around thedried impregnated disks was measured after 72 h.

The activity was classified as “highly active (+++)”(diameter = 10–15 mm), “moderately active (++)”(diameter = 5–10 mm), “slightly active (+)” (diame-ter ∼5 mm), and “inactive (-)” (diameter <5 mm).

The antitumor activity of all the compoundswas performed by incubating the human breastcancer cell line (MCF-7) with test compoundsat 1.0 μg mL−1 doses for 96 h, and the cellgrowth count was measured by the MTT as-say [29]. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was dissolved in aphosphate-buffered saline at a concentration of 5 μgmL−1. The 50 μL of the MTT solution added to eachwell and mixed thoroughly to dissolve the crystals ofthe purple-colored zone. The plates were then readon a microplate reader at a wavelength of 670 nm.The readings were presented as optical density.

ACKNOWLEDGMENTS

We are grateful to National Botanical Research Insti-tute (NBRI), Lucknow, India, for biological activityof compounds and Regional Sophisticated Instru-mentation Centre (RSIC), Lucknow, India, for mi-croanalytical and spectral data.

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