JOURNAL OF

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Journal of Inorganic Biochemistry 99 (2005) 2217–2225

InorganicBiochemistry

Synthesis and characterization of dual function vanadyl, galliumand indium curcumin complexes for medicinal applications

Khosro Mohammadi a,1, Katherine H. Thompson a,*, Brian O. Patrick a, Tim Storr a,Candice Martins a, Elena Polishchuk a, Violet G. Yuen b, John H. McNeill b,

Chris Orvig a,*

a Medicinal Inorganic Chemistry Group, Chemistry Department, University of British Columbia, Vancouver, BC, Canada V6T 1Z1b Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

Received 6 June 2005; received in revised form 19 July 2005; accepted 8 August 2005Available online 19 September 2005

Abstract

Novel bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione (curcumin) complexes with the formula, ML3, where M isGa(III) or In(III), or of the formula, ML2 where M is [VO]2+, have been synthesized and characterized by mass spectrometry, infra-red and absorption spectroscopies, and elemental analysis. A new ligand, bis[4-acetyl-3-hydroxyphenyl]-1,6-heptadiene-3,5-dione(diacetylbisdemethoxycurcumin, DABC) was similarly characterized; an X-ray structure analysis was performed. Vanadyl com-plexes tested in an acute i.p. testing protocol in STZ-diabetic rats showed a lack of insulin enhancing potential. Vanadyl complexeswere, however, more cytotoxic than were the ligands alone in standard MTT (3-[4,5-dimethylthiazole-2-yl]ate, -2,5-diphenyl-tetra-zolium bromide) cytotoxicity testing, using mouse lymphoma cells. With the exception of DABC, that was not different fromVO(DABC)2, the complexes were not significantly different from one another, with IC50 values in the 5–10 lM range. Galliumand indium curcumin complexes had IC50 values in the same 5–10 lM range; whereas Ga(DAC)3 and In(DAC)3 (whereDAC = diacetylcurcumin) were much less cytotoxic (IC50 = 20–30 lM). Antioxidant capacity was decreased in VO(DAC)2,Ga(DAC)3, and In(DAC)3, compared to vanadyl, gallium and indium curcumin, corroborating the importance of curcumin�s freephenolic OH groups for scavenging oxidants, and correlated with reduced cytotoxic potential.� 2005 Elsevier Inc. All rights reserved.

Keywords: Vanadium; Gallium; Indium; Curcumin; Diacetylcurcumin; Cytotoxicity; Antioxidant capacity

1. Introduction

Curcumin, a naturally occurring extract of the spice,turmeric (Curcuma longa L.) has a long history of ther-apeutic use. It has been used as an antioxidant, an anti-tumour agent, and an anti-inflammatant [1–4], and hascompleted a Phase I trial in 15 colorectal cancer patients

0162-0134/$ - see front matter � 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2005.08.001

* Corresponding authors. Tel.: +1 604 822 4449/1776; fax: +1 604822 2847.

E-mail addresses: kthompso@chem.ubc.ca (K.H. Thompson),orvig@chem.ubc.ca (C. Orvig).1 Current address: Chemistry Department, University of Shiraz,

Shiraz 71454, Iran.

in the United Kingdom [5]. Evaluation of curcumin andstructural derivatives in cancer chemoprevention modelsystems has demonstrated a range of potencies depen-dent upon particular substituents on the aromatic moi-ety [6]. Vanadyl curcumin (VO(cur)2) was recentlysynthesized and characterized in our laboratory [7]. Itwas more effective as an anti-cancer agent and as aninhibitor of synoviocyte proliferation, compared touncomplexed curcumin or vanadyl ion alone; it alsoproved to be exceptionally non-toxic in vivo, with noevidence of negative symptomatology during a month-long treatment period, at doses up to and including2.0 mmol kg�1 d�1 [7].

V

O

O

O

O

R

O

O

O

R

Fig. 2. The structure of bis(maltolato)oxovanadium(IV), BMOV,R = CH3, and bis(ethylmaltolato)oxovanadium(IV), BEOV,R = C2H5, used for comparison with vanadyl curcumin and analogsin bioactivity testing for insulin enhancing potential.

2218 K. Mohammadi et al. / Journal of Inorganic Biochemistry 99 (2005) 2217–2225

Compared to curcumin, diacetylcurcumin (DAC) haspreviously been shown to have greater efficacy as an NOscavenger [8]. A manganese diacetylcurcumin complexwas significantly more effective as an antioxidant scav-enger [than the comparable manganese curcumin com-plex], and displayed neuroprotective effects as well [9].We expected that new analogs of the curcumin ligand,including DAC, complexed to oxovanadium(IV), mightbe more effective than is vanadyl curcumin, as medicinalinorganic therapeutic agents. Thus, we prepared severalcurcumin derivatives by modifying slightly the ligand,first by separating out individual components of a typi-cal commercial curcumin, containing small amounts ofboth demethoxycurcumin (DMC) and bisdemethoxy-curcumin (BDC) [10], then by replacing the hydroxylsubstituents on the aromatic rings with acetyl groupsin curcumin [8] and in BDC (Fig. 1).

Oxovanadium(IV) complexes with maltol or ethyl-maltol, both approved in many countries as food addi-tives, have shown considerable promise as anti-diabeticagents [11–13]. Compared to other vanadium-contain-ing drug candidates, bis(maltolato)oxovanadium(IV)(BMOV) and bis(ethylmaltolato)oxovanadium(IV)(BEOV) (Fig. 2) are unsurpassed as orally available glu-cose- and lipid-lowering insulin mimetics, whetheradministered acutely or chronically. Complexation ofvanadyl with a known antioxidant, curcumin, has thepotential to improve synergistically the potency of avanadyl-based hypoglycemic agent [14,15].

In our previous study [7], cytotoxicity of vanadyl cur-cumin was shown in two assays used in anticancer drugscreening, smooth muscle cell proliferation and mouselymphoma cell culture. In mouse lymphoma cells, IC50

values for VO(cur)2 and curcumin were 10 and 33 lM,respectively. An additive effect, in which the observed ef-fect is at least equal to the sum of the effects of the indi-vidual components, is implied (but not proven) by theVO(cur)2 complex (composed of two ligands and onevanadyl ion), that resulted in 50% inhibition at less thanhalf the concentration required for the ligand alone (cur-cumin) [7]. Herein we attempt to further improve on thispotential therapeutic efficacy by using several curcumin

OH O

R2R3

R1OOR1

122'

33'

4' 45

5'

66'

77'

88'

99'1010'

Curcumin (Cur) R1= H, R2 = R3= OCH3

Demethoxycurcumin (DMC) R1 = R3= H, R2= OCH3

Bisdemethoxycurcumin (BDC) R1= R2= R3= HDiacetylcurcumin (DAC) R1= Ac, R2= R3= OCH3

Diacetylbisdemethoxycurcumin (DABC) R1= Ac, R2= R3= H

Fig. 1. The structure of curcumin and curcumin derivatives used asligands in this study.

derivatives as ligands, complexed to vanadyl ion or toone of two group 13 metal ions, gallium and indium.

Gallium and indium are also of major current interestas components of medicinal inorganic therapeutic anddiagnostic agents. Ga was the second metal, after Pt,to be used in cancer treatment – its anticancer propertieswere described for the first time in 1971 [16,17]. Galliumnitrate is an approved treatment for malignancy-associ-ated hypercalcemia [18]. The anticancer activities oftris(8-quinolinolato)gallium(III) and the tris(malto-lato)gallium(III) are also known [19–21]. A number ofprevious studies from our laboratories focused on thetris(bidentate ligand) ML3 Ga(III) and In(III) com-plexes of pyrones and pyridinones [22–24].

In this investigation, we have extended our studies ofmetal complexation by the b-diketones, curcumin andderivatives, to include one novel ligand, diacetylbisde-methoxycurcumin (DABC), and eight new metal coordi-nation complexes of curcumin and derivatives, includingvanadyl, gallium and indium-based compounds. A prin-cipal objective in this investigation was to study the glu-cose-lowering capabilities and anticancer potential ofthe individual components of commercial curcumin,along with acetylated derivatives (Fig. 1) and their asso-ciated vanadyl complexes. A secondary objective of thisinvestigation was to evaluate the therapeutic potential ofseveral gallium and indium complexes with curcuminand DAC. A corollary objective, relevant to both theprimary and secondary objectives, was to test thehypothesis that free radical scavenging activity wouldbe impaired, at least partially, in the new acetylatedcomplexes and that this might affect cytotoxicity as wellas antioxidant potential, in which the phenolic OHgroups, known to be essential for antioxidant activity[25–29], were blocked by acetyl moieties.

2. Experimental

2.1. Chemicals

All solvents (Sigma/Aldrich) and chemicals were re-agent grade and used without further purification unlessotherwise specified: vanadyl sulfate trihydrate, vanadylacetylacetonate, indium nitrate hydrate, triethylamine,

K. Mohammadi et al. / Journal of Inorganic Biochemistry 99 (2005) 2217–2225 2219

potassium persulfate, Trolox ((s)-(�)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) (Aldrich Chem-ical, Milwaukee, WI); carboxymethylcellulose, MTT(3-[4,5-dimethylthiazole-2-yl]ate, -2,5-diphenyl-tetrazo-lium bromide), ABTS (2,2 0-azino-bis(3-ethylbenzthiazo-line-6-sulfonic acid) diammonium salt) (Sigma, St.Louis, MO); curcumin, silica gel (Sigma Chemical, Mis-sassauga, ON); gallium nitrate hydrate (Alfa AESAR);dimethylsulfoxide-d6 (DMSO-d6, Cambridge IsotopeLaboratories, Inc.). Water was deionized (BarnsteadD8902 and D8904 cartridges) and distilled (CorningMP-1 Megapure Still) before use. The yields are for ana-lytically pure compounds with calculations based on themetal ion, where present.

2.2. Analytical instruments

IR spectra were recorded in the solid state (KBr pel-lets) in the range 500-4000 cm�1 using an ATI MattsonGalaxy Series FTIR 5000 spectrometer and were refer-enced to polystyrene. Elemental analyses were carriedout on a Carlo Erba analytical instrument by M. Lakha,Chemistry Department, UBC. Mass spectra (+ ion)were obtained on methanol solutions with a MicromassLCT MS50 Electrospray Ionization Mass Spectrometer.UV–vis spectra were recorded on a Hewlett Packard8453 UV–vis spectrophotometer, with temperature con-trolled by a Fisher ISOTEMP 1016D circulating bath(30.0 ± 0.1 �C). A Molecular Devices MicroplateReadere was used to read the absorbance at 570 nmin the 96-well plates. 1H NMR spectra were recordedon a Bruker AM-300 instrument at 300.13 MHz.

2.3. Separation of curcumin derivatives and preparation of

complexes

Curcumin was separated into individual componentsby modification of a previous method [30]. Curcuminwas dissolved in acetone, and separated by silica gelchromatography (98:5:2 CHCl3:MeOH:AcOH eluent).Curcumin eluted first, followed by a mixture of curcu-min and DMC, then pure BDC. Chromatographicseparation of the second phase mixture (95:5CH2Cl2:MeOH eluent) yielded pure DMC. Purity ofthe three separated components was verified by TLC(thin layer chromatography), NMR, IR, ESI-MS (elec-trospray ionization mass spectrometry), and EA (ele-mental analysis), and agreed with literature values[10,30].

2.3.1. Diacetylcurcumin, DAC, and

diacetylbisdemethoxycurcumin, DABC

DAC was prepared according to the literature method[5], and DABC was prepared by adapting that method.BDC and acetic anhydride were combined in dry pyri-dine, and recrystallized with EtOAc/hexane (60/40) to give

DABC in 93% yield. 1H NMR (CDCl3): 2.30 (6H, s,2 · COCH3), 5.82 (s, 1H, H1), 6.56 (d, 2H, J = 16.0 Hz,H3, H

03), 7.12 (d, 4H, J = 8.7 Hz, H7, H

07, H9, H

09), 7.55

(d, 4H, J = 8.7 Hz, H6, H06, H10, H0

10), 7.63 (d, 2H,J = 16.0 Hz, H4, H0

4). IR (KBr, cm�1): �3000, 2890(m=C–H,C–H), 1754 (mC@O,ester), 1641 (mC@O), 1515 (mC@C),1381 (mC–H), 1227 (mC–O,C–C–C), 1026 (mC–H), 973 (mH–C@C–

H,trans), 844(mC–H, aromatic). UV/Vis: kmax (nm, MeOH):313, 400. MS (+ES-MS): m/z = 415 [M + Na]+, 393[M + H]+. Anal. Calc. (Found): C23H20O6; C, 70.40(70.10); H, 5.14 (5.22%). X-ray crystal structure analysisconfirmed the compound identity (vide infra).

2.3.2. Complex syntheses and characterizationVanadyl complexes were prepared by addition of

�10 mL degassed MeOH or acetone to 0.50 mmol cur-cumin or derivative dissolved in MeOH with gentle heat-ing and stirring. Dropwise addition of vanadylacetylacetonate [VO(acac)2], 0.25 mmol, in �10 mL de-gassed methanol, followed by reflux for �2 h underAr, and then cooling to room temperature (RT), affor-ded a solid product that was then isolated by vacuumfiltration, washed with cold methanol and dried over-night in vacuo at RT. Gallium and indium complexeswere prepared similarly, using 0.60 mmol curcumin orderivative in 15 mL MeOH and dropwise addition ofGa(NO3)3 Æ 6H2O, or In(NO3)3 Æ �3H2O) (0.20 mmol),dissolved in 2 mL MeOH, followed by dropwise addi-tion of triethylamine, 0.60 mmol, reflux for 2 h, coolingto RT, vacuum filtration of the solid product, MeOHwash and drying overnight in vacuo at RT.

2.3.2.1. Bis(1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-hept-

adiene-3,5-dionato)oxovanadium(IV), vanadyl curcumin

[VO(cur)2]. 89% yield. IR (KBr, cm�1): �3495 (mO–H),�3010, 2935 (mC–H), 1626 (mC@O), 1591, 1489 (mC@C),1391 (mC–H), 1261–1151 (mC–O,C–C–C), 966 (mV@O), 847(mC–H, aromatic). UV/Vis: kmax (nm, MeOH): 273, 383.MS (+ES-MS, positive electrospray MS): m/z = 802[M + H]+. Anal. Calc. (Found): C42H38O13V; C, 62.92(62.85); H, 4.78 (5.02%).

2.3.2.2. Bis(1,7-[4-hydroxy-3-methoxyphenyl]-10,70-[3,4-

hydroxyphenyl-1,6-heptadiene-3,5-dionato)oxovanadium-

(IV), vanadyl demethoxycurcumin [VO(DMC)2]. 78%yield. IR (KBr, cm�1): �3395 (mO–H), �3180–2800 (mC–H),1600 (mC@O), 1561(mC@C), 1385 (mC–H), 1276–1170 (mC–O,

C–C–C), 982 (mV@O), 832 (mC–H, aromatic). UV/Vis: kmax

(nm, MeOH): 257, 423. MS (+ES-MS): m/z = 764 [M +Na]+, 742 [M + H]+. Anal. Calc. (Found): C40H34O11V Æ2H2O; C, 61.78 (62.17); H, 4.92 (4.93%).

2.3.2.3. Bis(1,7-bis[4-hydroxy-3-hydroxyphenyl]-1,6-hep-

tadiene-3,5- dionato)oxovanadium(IV) vanadylbisdeme-thoxycurcumin [VO(BDC)2]. 86% yield. IR (KBr,cm�1): �3375 (mO–H), �3124–3013 (mC–H), 1601 (mC@O),

2220 K. Mohammadi et al. / Journal of Inorganic Biochemistry 99 (2005) 2217–2225

1512, 1398 (mC@C), 1161 (mC–O), 972 (mV@O), 831 (mC–H,aromatic). UV/Vis: kmax (nm, MeOH): 249, 414. MS(+ES-MS): m/z = 682 [M + H]+. Anal. Calc. (Found):C38H30O9V Æ 3H2O; C, 62.04 (62.38); H, 4.93 (4.84).

2.3.2.4. Bis(1,7-bis[4-acetyl-3-methoxyphenyl]-1,6-hept-adiene-3,5-dionato)oxovanadium(IV) vanadyl diacetyl-

curcumin [VO(DAC)2]. 96% yield. IR (KBr, cm�1):�3418 (mO–H, H2O), �3123–2935 (mC–H), 1761 (mC@O, es-ter), 1624 (mC@O), 1508, 1396 (mC@C), 1296–1155 (mC–O,

C–C–C), 995 (mV@O), 855 (mC–H, aromatic). UV/Vis: kmax

(nm, MeOH): 281, 376. MS (+ES-MS): m/z = 992[M + Na]+, 969 [M]+. Anal. Calc. (Found): C50H50O17-

V Æ H2O; C, 60.79 (60.51); H, 4.90 (4.93%).

2.3.2.5. Bis(1,7-bis[4-acetyl-3-hydroxyphenyl]-1,6-hept-

adiene-3,5-dionato)oxovanadium(IV), vanadyl diac-

etylbisdemethoxycurcumin [VO(DABC)2]. 82% yield.IR (KBr, cm�1): �3250 (mO–H, H2O), 1763 (mC@O,ester),1622 (mC@O), 1516 (mC@C), 1370 (mC–H), 1206–1160(mC–O,C–C–C), 994 (mV@O), 840 (mC–H, aromatic). UV/Vis: kmax (nm, MeOH): 285, 400. MS (+ES-MS): m/z = 850 [M + H]+. Anal. Calc. (Found): C46H38O13V Æ2H2O; C, 62.38 (62.68); H, 4.78 (4.60%).

2.3.2.6. Tris(curcuminato)gallium(III), Ga(cur)3. 70%yield. 1H NMR (DMSO-d6): 3.77 (18H, s, 6 · OCH3),5.95 (3H, s, 1-H), 6.72 (6H, d, J = 15.8 Hz, 3,3 0-H2),6.76 (6H, d, J = 8.3 Hz, 9,9 0-H2), 7.03 (6H, d,J = 8.3 Hz, 10,10 0-H2), 7.23 (6H, s, 6,6 0-H2), 7.41 (6H,d, J = 15.8 Hz, 4,4 0-H2), 9.53 (6H, s, 6 · OH). IR(KBr, cm�1): 3500–3200 (mO–H), 3150–2700 (mC–H),1624 (mC@O), 1600, 1507 (mC@C), 1394 (mC–H), 1286–1125 (mC–O,C–C–C), 972 (mH–C@C–H,trans), 847 (mC–H, aro-matic), 469 (mGa–O). UV-vis: kmax nm (10% DMSO/MeOH log(e)): 264 (4.9), 432 (5.4). MS (+ESI-MS):m/z = 1172 [ML3 + H]+, 803 [ML2]

+. Anal. Calc.(Found): C63H57GaO18 Æ H2O; C, 63.59 (63.97); H,5.00 (5.08%).

2.3.2.7. Tris(diacetylcurcuminato)gallium(III), Ga-

(DAC)3. 80% yield. 1H NMR (DMSO-d6): 2.24 (18H,s, 6 · O–CO–CH3), 3.77 (18H, s, 6 · OCH3), 6.13 (3H,s, 1-H), 6.98 (6H, d, J = 16.0 Hz, 3,3 0-H2), 7.10 (6H, d,J = 7.7 Hz, 9,9 0-H2), 7.23 (6H, d, J = 7.7 Hz, 10,10 0-H2), 7.43 (6H, s, 6,6 0-H2), 7.53 (6H, d, J = 16.0 Hz, 4,40-H2). IR (KBr, cm�1): 3200–2700 (mC–H), 1765 (mC@O,ester),1632 (mC@O), 1527, 1397 (mC@C), 1302–1125 (mC–O,C–C–C),992 (mH–C@C–H,trans), 839 (mC–H, aromatic), 457 (mGa–O).UV–vis: kmax nm (10% DMSO/MeOH log(e)): 258 (4.8),424 (5.4). MS (+ESI-MS): m/z = 1447 [ML3 + Na]+,971 [ML2]

+. Anal. Calc. (Found): C75H69GaO24 Æ 3H2O;C, 60.94 (60.82); H, 5.11 (4.96%).

2.3.2.8. Tris(curcuminato)indium(III), In(cur)3. 70%yield. 1H NMR (DMSO-d6): 3.76 (18H, s, 6 · OCH3),

5.89 (3H, s, 1-H), 6.75 (6H, d, J = 16.0 Hz, 3,3 0-H2),6.77 (6H, d, J = 7.7 Hz, 9,9 0-H2), 7.05 (6H, d,J = 7.7 Hz, 10,10 0-H2), 7.24 (6H, s, 6,6 0-H2), 7.43 (6H,d, J = 16.0 Hz, 4,4 0-H2), 9.54 (6H, s, 6 · OH). IR(KBr, cm�1): 3500–3200 (mO–H), 3200–2700 (mC–H),1624 (mC@O), 1596, 1511 (mC@C), 1391 (mC–H), 1286–1125 (mC–O,C–C–C), 984 (mH–C@C–H,trans), 815 (mC–H, aro-matic), 465 (mIn–O). UV–vis: kmax nm (10% DMSO/MeOH log(e)): 270 (5.1), 428 (5.5). MS (+ESI-MS):m/z = 1217 [ML3]

+, 849 [ML2]+. Anal. Calc. (Found):

C63H57InO18.H2O; C, 61.27 (61.60); H, 4.82 (4.91%).

2.3.2.9. Tris(diacetylcurcuminato)indium(III), In(DAC)3.

80% yield. 1H NMR (DMSO-d6): 2.25 (18H, s, 6 · O–CO–CH3), 3.77 (18H, s, 6 · OCH3), 6.05 (3H, s, 1-H),7.00 (6H, d, J = 16.0 Hz, 3,3 0-H2), 7.10 (6H, d,J = 8.1 Hz, 9,9 0-H2), 7.25 (6H, d, J = 8.1 Hz, 10,10 0-H2), 7.44 (6H, s, 6,6 0-H2), 7.55 (6H, d, J = 16.0 Hz,4,4 0-H2). IR (KBr, cm�1): 3250–2700 (mC–H), 1765(mC@O,ester), 1628 (mC@O), 1511, 1386 (mC@C), 1298–1116(mC–O,C–C–C), 988 (mH–C@C–H,trans), 839 (mC-H, aromatic),469 (mIn–O). UV–vis: kmax nm (10% DMSO/MeOHlog(e)): 256 (4.9), 406 (5.4). MS (+ESI-MS):m/z = 1491 [ML3 + Na]+, 1468 [ML3]

+. Anal. Calc.(Found): C75H69InO24; C, 61.31 (61.17); H, 4.73(4.81%).

2.4. X-ray crystallography

An X-ray crystal structure and atom labeling schemeof the novel acetylated curcuminoid, DABC, is pre-sented in Fig. 3, and crystal and refinement data are pre-sented in Table 1. A yellow needle crystal of C23H20O6

having approximate dimensions of 0.30 · 0.10 ·0.05 mm was mounted on a glass fiber. All measure-ments were made on a Bruker X8 APEX diffractometerwith graphite monochromated Mo Ka radiation. Thedata were collected at T = �100.0 ± 0.1 �C to a maxi-mum 2h value of 55.8�. Data were collected in a seriesof / and x scans in 0.50� oscillations with 15.0 s expo-sures at a crystal-to-detector distance of 38.03 mm.

Of the 20093 reflections that were collected, 2262were unique (Rint = 0.043); equivalent reflections weremerged. Data were collected and integrated using theBruker SAINT [31] software package. The linearabsorption coefficient, l, for Mo Ka radiation is0.97 cm�1. Data were corrected for absorption effectsusing the multi-scan technique (SADABS, an absorp-tion correction software package) [32], with minimumand maximum transmission coefficients of 0.737 and0.995, respectively. The data were corrected for Lorentzand polarization effects.

The structure was solved by direct methods [33]. Thematerial resides on a twofold rotation axis. As such, theC11–O3 bond (1.31 A) has only partial double bondcharacter, and the hydroxyl hydrogen (located in a

Fig. 3. An ORTEP drawing of diacetylbisdemethoxycurcumin (DABC) showing 50% probability ellipsoids.

Table 1Crystal data for DABC

Empirical formula C23H20O6

Formula weight 392.39Crystal color, habit Yellow, needleCrystal system MonoclinicSpace group C2/c (No. 15)

Unit cell

a (A) 45.58(1)b (A) 5.676(1)c (A) 7.576(1)a (�) 90.0b (�) 94.765(9)c (�) 90.0V (A3) 1953.4(7)

Z 4T (�C) �100Dcalc. (g cm

�3) 1.334l (Mo Ka) (cm�1) 0.97Number of unique data 2262Number of observed data 2262Number of variables 137R 0.072Rw 0.112Goodness-of-fit 1.06

K. Mohammadi et al. / Journal of Inorganic Biochemistry 99 (2005) 2217–2225 2221

difference map and refined isotropically) is disorderedbetween two equivalent positions (one-half hydrogenon each). All non-hydrogen atoms were refined aniso-tropically. All other hydrogen atoms were included incalculated positions but not refined. The maximumand minimum peaks on the final difference Fouriermap corresponded to 0.22 and �0.19 e� A3, respec-tively. All refinements were performed using the SHEL-XTL crystallographic software package [34].

2.5. Biological studies

2.5.1. Animals

Male Wistar rats weighing 190–220 g (Animal CareUnit, University of British Columbia) were housed twoper cage (polycarbonate), and were maintained on a12:12 h light–dark schedule.Animalswere allowedunlim-ited access to rat chow (LabDiet Rodent Diet 5001,Ralston-Purina) and water. All experiments were per-formed in accordance with the principles and guidelinesof theCanadianCouncil onAnimalCare.Ratswere accli-matized for 7 days prior to induction of diabetes.

2.5.2. Diabetes induction

Animals were divided into eight groups: untreateddiabetic (control), diabetic treated with BMOV, andsix diabetic test groups (three pairs of vanadyl complexand ligand alone). Diabetes was induced by a singleintravenous injection of streptozotocin (STZ, 60 mgkg�1 in 0.9% NaCl, 1 mL kg�1) under light halothaneanesthesia. Diabetes was confirmed three days afterSTZ injection from tail-vein blood collection. Bloodsamples were spun at 10,000g for 25 min in a BeckmanAllegra 21R centrifuge and plasma glucose levels subse-quently determined (Beckman Glucose Analyzer 2,Beckman Instruments, La Brea, CA). Blood glucose lev-els >13 mM were accepted as diabetic. Acute (intraperi-toneal, i.p.) testing took place one week following STZinjections, and consisted of carboxymethylcellulose(CMC) suspensions of test compounds at a dose of0.10 mmol kg�1, or CMC alone, as outlined previously[7]. Response to compounds was measured as percentdecline in plasma glucose (compared to diabetic-un-treated) at 8, 12, 24, and 48 h after i.p. administrationof ligand or complex. In vivo anti-hyperglycemic activityutilized a normalized parameter, % glucose lowering (%GL) [7], calculated as follows:

%GL ¼ ð½glucose�control � ½glucose�testÞ=½glucose�controlð1Þ

Statistical analysis was performed with a one-wayANOVA (analysis of variance) test, p < 0.05. All dataare presented as means ± SEM.

2.5.3. Cell viability testing

Mouse lymphoma L1210 cells (from UBC ChemistryBiological Services Laboratory stock) were grown insuspension at 37 �C and 5% CO2 in RPMI 1640 medium(a medium developed at Roswell Park Memorial Insti-tute) supplemented with 10% heat-inactivated horse ser-um, 50 IU/mL penicillin, 50 lg/mL streptomycin,0.5 mM sodium pyruvate, 0.05% (w/v) pluronic acidF68 and 0.01 M HEPES, without phenol red. (Allreagents were purchased from GIBCO BRL, GrandIsland, NY). Cells were plated onto 96-well plates byaddition of 100 lL of the cell suspension (1 · 105 cells/mL) to each well. To prevent evaporation, 200 lL sterilewater was added to the perimeter wells. Cells were then

O OR2

R2

R1O OR1

M

n

[VO(cur) 2], VO(DAC)2] n = 2; M=VO, R1 = H, Ac; R2 = OCH3

[VO(BDC)2], VO(DABC)2] n = 2; M=VO, R1 = H, Ac; R2 = H Ga(cur)3], [Ga(DAC)3] n = 3; M = Ga, R1 = H, Ac; R2 = OCH3

[In(cur) 3], [In(DAC)3] n = 3; M = In, R1 = H, Ac; R2 = OCH3

Fig. 4. The structures of the metal complexes prepared in this study.

2222 K. Mohammadi et al. / Journal of Inorganic Biochemistry 99 (2005) 2217–2225

incubated at 37 �C for 24 h. Stock solutions of test com-pounds were made up in DMSO, to ensure complete sol-ubilization. Test compounds were added as serialdilutions in RPMI 1640 medium (200–3.13 lg/mL),100 lL/well, and the plate was incubated at 37 �C for3 days. MTT dye reduction, measured by A570 nm, wasused to determine cell viability, as previously described[35]. In brief, MTT, 50 lL of a 2.5 mg/mL solution insterile, distilled water, was added to each well; back-ground A570 nm absorbance was recorded; plates wereincubated for 3 h at 37 �C (in 5% CO2), then spun downfor 5 min at low speed, followed by removal of 230 lL ofunreacted MTT solution, dissolution of formazan crys-tals in 150 lL DMSO and reading of A570 nm. Dataare presented as ranges of values.

2.5.4. Trolox equivalent antioxidant assay

All complexes were tested for their antioxidant activ-ity using the Trolox (6-hydroxy-2,5,7,8-tetramethylchro-man-2-carboxylic acid) equivalent antioxidant capacity(TEAC) assay [36]. This assay is based on ABTS�+

(2,2 0-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)diammoniuim salt) radical cation decolorization, asmeasured spectrophotometrically by the change inabsorbance at 745 nm. Ligands, or their complexes,were initially dissolved in DMSO and then diluted inMeOH, so that addition of 20 lL of the resulting solu-tions to 2 mL of ABTS�+ stock solution caused a reduc-tion of 20–80% in the absorbance as a result of ABTS�+

radical reduction. To obtain this range, final concentra-tions for the compounds ranged 2.5–15 lM. A745 wasmeasured at 30 �C, immediately, and at 1, 3, and6 min, after initial mixing (performed in triplicate).The TEAC value of the sample was calculated basedon the inhibition exerted by the standard Trolox solu-tion at 3 min, by which time point all samples exhibiteda linear response to addition of ABTS�+ solution.

Table 2Selected bond lengths [A] and angles [deg] in DABC

C(9)–C(10) 1.340(2)C(10)–C(11) 1.467(2)C(11)–O(3) 1.305(2)C(11)–C(12) 1.404(2)O(3)–H(1) 0.87(3)C(9)–C(10)–C(11) 123.4(1)C(9)–C(10)–H(10) 118.3O(3)–C(11)–C(12) 121.0(1)O(3)–C(11)–C(10) 119.1(1)C(11)–C(12)–H(12) 119.0

3. Results and discussion

Commercial curcumin was separated into its compo-nent parts by column chromatography, followed byrecrystallization. Curcumin, and its more �strippeddown� derivative, BDC, were used for synthesis of acet-ylated analogs, DAC and DABC. A series of noveloxovanadium(IV) curcuminoid complexes (VO(DMC)2,VO(BDC)2, VO(DAC)2) and VO(DABC)2 were synthe-sized and characterized, as well as two new Ga(III) com-plexes, Ga(cur)3 and Ga(DAC)3, and their In(III)analogs, In(cur)3 and In(DAC)3 (Fig. 4).

Spectroscopic characterization of the ligands andtheir complexes confirmed the proposed structures(Figs. 1 and 4). IR spectra of all ligands showed mC@O

in the typical 1600–1630 cm�1 range, which shifted tolower energy in the vanadyl complexes of the same

ligands. Vanadyl complexes also had no broad band inthe 2600–3400 cm�1 range, related to the stretching ofintramolecular H in the enol function, as noted for apreviously synthesized vanadyl 1,7-diaryl-1,6-heptadi-ene-3,5-dione, with a different pattern of hydroxylationon the aromatic rings [37]. Vanadyl complexes did showa mV@O medium intensity band at �975–995 cm�1. Aprominent band at 960–975 cm�1, due to the trans–CH@CH– group, also remained unchanged in the vana-dyl complexes. The curcumin ligands showed twoabsorption bands in the UV/Vis region: an n ! p* tran-sition at �360–430 nm and a p ! p* transition at �240–290 nm, which shifted to slightly higher energy in thevanadyl complexes, indicative of involvement of the car-bonyl group in metal complexation. Further evidence ofcomplexation comes from the mass spectra of all com-plexes, which show intense peaks for [VOL2]

+,[VOL2 + H]+ and [VOL2 + Na]+, and confirm a stoichi-ometry of 2:1 curcuminoid to oxovanadium(IV).

For the ligands DAC and DABC, protection of theOH hydrogens by acetyl groups was confirmed by ab-sence of a d � 9.5–10 ppm signal, typical of phenol ringOH hydrogens in curcuminoids, in the 1H NMR spec-tra. Lack of free aromatic OH groups could be expectedto compromise the antioxidant capacity of DAC andDABC compared to the other ligands tested, and thiswas borne out experimentally (vide infra).

Selected bond lengths and angles ofDABC fromX-raycrystallographic analysis are summarized in Table 2. In arelated structure of DAC, also solved by us, but recentlypublished by others [38], the presence of an intramolecu-

Table 3Mouse lymphoma cell cytotoxicity, and antioxidant assay results forligands and their vanadyl, gallium and indium complexes

Compounds Cytotoxicity in mouselymphoma IC50 (lM)a

TEACb

Curcumin 25–35 1.10 ± 0.15DMC 35–40 1.12 ± 0.32BDC 15–20 –c

DAC 10–15 –c

DABC 9–10 –c

VO(cur)2 5–6 2.67 ± 0.57VO(DMC)2 8–9 2.17 ± 0.46VO(BDC)2 5–6 0.95 ± 0.24VO(DAC)2 7–10 0.57 ± 0.08VO(DABC)2 5–7 0.62 ± 0.10Ga(cur)3 5–10 3.83 ± 1.12In(cur)3 5–10 3.81 ± 0.88Ga(DAC)3 25–30 1.27 ± 0.20In(DAC)3 20–25 0.82 ± 0.11

a Values are ranges of values from at least two separate plates.b TEAC = Trolox Equivalent Antioxidant Capacity.c Inhibition of absorbance reduction <25% compared to controls.

K. Mohammadi et al. / Journal of Inorganic Biochemistry 99 (2005) 2217–2225 2223

lar O3–H1 � � � O3* hydrogen bond is apparent, clearlysupporting a preferred keto-enol form for the b-diketonemoiety, in the solid state. The crystal structure of DABC(complete bond lengths and angles provided in Table S1)also reveals the formation of an intramolecular O3–H1 � � � O3* bond with an O3 � � � O3* distance of2.548(1) A, within the range of intramolecularly H-bonded b-diketone enols (2.37 6 O � � � O P 2.59 A).The angle ofO3–H1 � � � O3* is 151(3)�, in good agreementwith data of DAC and other b-diketones [38,39]. Of thepossible tautomeric forms, it appears that, in the crystalphase, b-diketones prefer the cis-enol arrangement stabi-lized by a strong intramolecular hydrogen bond. The b-diketone moiety exists exclusively as the keto-enol formin the solid-state structure, correlating well with the solu-tion NMR data.

Evidence for the formulation in the keto-enol formcomes from the observed O3–H1 of 0.87(3) A andO3* . . . H1 of 1.75 A, indicating a single bond betweenO3 and H1 and a hydrogen bond between O3* � � � H1(Table S2). Data are consistent with only one H-atomattached to the central C atom. It thus seems clear that,in the solid state, DABC exists wholly in the keto-enolform, in agreement with previous data for curcuminderivatives. All other bond lengths and angles are withintypical values [38,40].

Testing for biological activity of the ligands and com-plexes included both in vivo administration of compounds(i.p.) toassess antidiabetic efficacyand invitro cell studiesofcytotoxic potential. In the acute testing protocol for insulinenhancing potential, no compounds in this series showedany significant response. DMC and VO(DAC)2 appearedminimally responsive (10.8 ± 0.7%GL and 12.0 ±0.7%GL, respectively); DAC and VO(DMC)2 also didnot significantly lower plasma glucose (�5.1 ± 2.2%GLand2.2 ± 0.2%GL, respectively)within the 72 h time frameof this study. Curcumin and VO(cur)2 also had very slightplasma glucose-lowering (17.2 ± 1.6%GL and 13.7 ±0.8%GL, respectively), not significantly different from un-treated diabetic rats, and corroborating previous results[7], in which VO(cur)2 had a maximal %GL of 13.9 ± 0.9at 12 h. By comparison, the BMOV test group used as abenchmark in this series significantly lowered plasma glu-cose (47 ± 13.4%GL, 24 h, after the same ED50 dose =0.10 mmol kg�1) in STZ-diabetic rats.

Cytotoxicity testing of ligands and compounds (Table3) in mouse lymphoma cells corroborated earlier resultsfor vanadyl curcumin and curcumin alone [7], andshowed roughly similar cytotoxicity for all new vanadylcomplexes. The same trend was apparent for Ga(cur)3and In(cur)3; however, Ga(DAC)3 and In(DAC)3 wereboth substantially less cytotoxic. For ligands alone,cytotoxicity in this assay followed the general trend:DABC > DAC > BDC > cur > DMC.

The greater cytotoxicity of the acetylated analogs ofcurcumin (DAC and DABC) compared to curcumin

and DMC contrasts with the greater NO scavenging[30] and Phase 2 detoxification enzyme induction [8],seen previously for curcumin compared to DAC andDMC. In both of the latter studies, curcumin was morepotent than were acetylated or demethoxylated deriva-tives of curcumin. For induction of quinone reductase(a Phase 2 detoxification enzyme, used as a measure ofchemopreventive potential [41]) the key features of acurcumin analog that determined potency were presenceof hydroxyl groups at the ortho position on the aromaticrings, and a b-diketone functionality. In our series, dia-cetyl curcumin analogs obviously lacked hydroxylgroups on the aromatic rings; however, cytotoxicity inmouse lymphoma cells was not impaired for ligandsalone or for vanadyl complexes; if anything, it wasenhanced. For Ga(DAC)3 and In(DAC)3, comparedto Ga(cur)3 and In(cur)3, this situation was reversed(Table 3).

Comparison of antioxidant potential among thecomplexes indicated that the predominant determinantof antioxidant capacity was the ligand, with VO(cur)2and VO(DMC)2 roughly twice as effective as were cur-cumin and DMC alone, and Ga(cur)3 and In(cur)3roughly three times as effective, in line with the formu-lations ML2 for vanadyl complexes and ML3, with Mbeing the metal ion and L, the ligand. The exceptionswere for acetylated ligands, in which ligands alonehad too low a response to be quantified by the assayconditions we used; VO(DAC)2 and VO(DABC)2 hadvery low TEAC values compared to the other vanadylcomplexes, and both Ga(DAC)3 and In(DAC)3 showedabout one-third the antioxidant capacity compared toGa(cur)3 and In(cur)3. Our corollary hypothesis thatfree aromatic ring OH groups would be important to

2224 K. Mohammadi et al. / Journal of Inorganic Biochemistry 99 (2005) 2217–2225

antioxidant capacity of these complexes was thusconfirmed.

4. Conclusions

A series of curcumin derivatives and their VO2+,Ga(III), and In(III) complexes have been synthesizedand characterized, both chemically and biologically.Acetylation of curcumin and BDC significantly de-creased the antioxidant potential of compounds contain-ing these ligands, but did not affect cytotoxicity inmouse lymphoma cells. The new vanadyl complexesdid not display any significant glucose-lowering poten-tial, nor were they more effective than previously synthe-sized vanadyl curcumin. The novel ligand, DABC,displayed moderate cytotoxicity (IC50 � 10 lM), andreadily formed a vanadyl bisligand complex, which alsoshowed modest anticancer potential. Gallium and in-dium tris complexes of curcumin had much lower IC50

values than did their diacetylcurcumin analogs in mouselymphoma cells; further ligand refinements may yieldgallium and indium curcuminoids with potential as diag-nostic or therapeutic agents.

5. Abbreviations

DMC

demethoxycurcumin BDC bisdemethoxycurcumin DAC diacetylcurcumin DABC diacetylbisdemethoxycurcumin BMOV bis(maltolato)oxovanadium(IV) BEOV bis(ethylmaltolato)oxovanadium(IV) STZ streptozotocin CMC carboxymethylcellulose DMSO dimethylsulfoxide ESI-MS electrospray ionization mass spectrometry +ES-MS positive electrospray mass spectrometry SADABS an X-ray absorption correction software

package

EA elemental analysis RT room temperature TLC thin layer chromatography TEAC trolox equivalent antioxidant capacity,

i.p., intraperitoneal

IC50 concentration at which 50% inhibition

of cell growth occurs

ABTS�+ 2,2 0-azinobis-(3-ethylbenzothiazoline-6-

sulfonic acid) diammoniuim salt

MTT 3-[4,5-dimethylthiazole-2-yl]ate, -2,5-

diphenyl-tetrazolium bromide

RPMI 1640 a medium developed at Roswell

Memorial Institute

ANOVA analysis of variance

Acknowledgements

Acknowledgement is made to Cheri Barta for X-raycrystallographic expertise, the Canadian Institutes ofHealth Research (CIHR) for operating funds, theNatural Sciences and Engineering Research Council(NSERC) for a Discovery Grant and a PostgraduateScholarship (T.S.) and the Ministry of Science, Researchand Technology of I.R. Iran for salary support (K.M.).

Appendix A. Supplementary data

Table S1 giving bond lengths and angles in DABCwas deposited. Supplementary data associated with thisarticle can be found, in the online version, atdoi:10.1016/j.jinorgbio.2005.08.001.

References

[1] B. Joe, M. Vijaykumar, B.R. Lokesh, Crit. Rev. Food Sci. Nutr.44 (2004) 97–111.

[2] R.N. Chopra, J.C. Chopra, K.L. Handa, L.D. Kapur, IndigenousDrugs of India, second ed., Dhur, Calcutta, 1958.

[3] S.D. Deodhar, R. Sethi, R.C. Srimal, Indian J. Med. Res. 71(1980) 632–634.

[4] M.C.Y. Heng, US Patent Application 20010051184, 2001.[5] R.A. Sharma, S.A. Euden, S.L. Platton, D.N. Cooke, A.

Shafayat, H.R. Hewitt, T.H. Marczylo, B. Morgan, D. Heming-way, S.M. Plummer, M. Pirmohamed, A.J. Gescher, W.P.Steward, Clin. Cancer Res. 10 (2004) 2854–6847.

[6] S. Gafner, S.-K. Lee, M. Cuendet, S. Barthelemy, L. Vergnes, S.Labidalle, R.G. Mehta, C.W. Boone, J.M. Pezzuto, Phytochem-istry 65 (2004) 2849–2859.

[7] K.H. Thompson, K. Bohmerle, E. Polishchuk, C. Martins, P.Toleikis, J. Tse, V. Yuen, J.H. McNeill, C. Orvig, J. Inorg.Biochem. 98 (2004) 2063–2070.

[8] Y. Sumanont, Y. Murakami, M. Tohda, O. Vajragupta, K.Matsumoto, H. Watanabe, Biol. Pharm. Bull. 27 (2004) 170–173.

[9] O. Vajragupta, P. Boonchoong, H. Watanabe, M. Tohda, N.Kammasud, Y. Sumanont, Free Rad. Biol. Med. 35 (2003) 1632–1644.

[10] H. Ahsan, N. Parveen, N.U. Khan, S.M. Hadi, Chem.–Biol.Interact. 121 (1999) 161–175.

[11] K.H. Thompson, J.H. McNeill, C. Orvig, Chem. Rev. 99 (1999)2561–2571.

[12] J.H. McNeill, V.G. Yuen, H.R. Hoveyda, C. Orvig, J. Med.Chem. 35 (1992) 1489–1491.

[13] K.H. Thompson, B.D. Liboiron, Y. Sun, K.D.D. Bellman, I.A.Setyawati, B.O. Patrick, V. Karunaratne, G. Rawji, J. Wheeler, K.Sutton, S. Bhanot, C. Cassidy, J.H. McNeill, V.G. Yuen, C.Orvig, J. Biol. Inorg. Chem. 8 (2003) 66–74.

[14] L.C.Y. Woo, V.G. Yuen, K.H. Thompson, J.H. McNeill, C.Orvig, J. Inorg. Biochem. 76 (1999) 251–257.

[15] T. Storr, D. Mitchell, P. Buglyo, K.H. Thompson, V.G. Yuen,J.H. McNeill, C. Orvig, Bioconjugate Chem. 14 (2003) 212–221.

[16] M.M. Hart, R.H. Adamson, Proc. Natl. Acad. Sci. USA 68 (1971)1623–1626.

[17] M.M. Hart, C.F. Smith, S.T. Yancey, R.H. Adamson, J. Natl.Cancer Inst. 47 (1971) 1121–1127.

K. Mohammadi et al. / Journal of Inorganic Biochemistry 99 (2005) 2217–2225 2225

[18] M.J. Abrams, B.A. Murrer, Science 261 (1993) 725–730.[19] P. Collery, J.L. Domingo, B.K. Keppler, Anticancer Res. 16

(1996) 687–691.[20] L.R. Bernstein, Pharmacol. Rev. 50 (1998) 665–682.[21] V.B. Arion, M.A. Jakupec, M. Galanski, P. Unfried, B.K.

Keppler, J. Inorg. Biochem. 91 (2002) 298–305.[22] Z. Zhang, D.M. Lyster, G.A. Webb, C. Orvig, Nucl. Med. Biol. 19

(1992) 327–335.[23] H.R. Hoveyda, S.J. Rettig, C. Orvig, Inorg. Chem. 32 (1993)

4909–4913.[24] H.R. Hoveyda, V. Karunaratne, S.J. Rettig, C. Orvig, Inorg.

Chem. 31 (1992) 5408–5416.[25] P. Vankatesan, M.N.A. Rao, J. Pharm. Pharmacol. 52 (2000)

1123–1128.[26] Y.-M. Sun, H.-Y. Zhang, D.-Z. Chen, C.-B. Liu, Org. Lett. 4

(2002) 2909–2911.[27] L.R.C. Barclay, M.R. Vinqvist, Org. Lett. 2 (2000) 2841–2843.[28] S.V. Jovanovic, S. Steenken, C.W. Boone, M.G. Simic, J. Am.

Chem. Soc. 121 (1999) 9677–9681.[29] S.V. Jovanovic, C.W. Boone, S. Steenken, M. Trinoga, R.B.

Kaskey, J. Am. Chem. Soc. 123 (2001) 3064–3068.[30] O. Vajragupta, P. Boonchoong, L.J. Berliner, Free Rad. Res. 38

(2004) 303–314.

[31] SAINT. Version 6.02. Bruker AXS Inc., Madison, Wisconsin,USA, 1999.

[32] SADABS. Bruker Nonius area detector scaling and absorp-tion correction – V2.05, Bruker AXS Inc., Madison, Wisconsin,USA.

[33] A. Altomare, M. Cascarano, C. Giacovazzo, A. Guagliardi, J.Appl. Cryst. 26 (1993) 343–350, SIR92.

[34] SHELXTL Version 5.1. Bruker AXS Inc., Madison, Wisconsin,USA, 1997.

[35] T.J. Mosmann, Immunol. Meth. 65 (1983) 55–63.[36] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M.

Yang, C. Rice-Evans, Free Rad. Biol. Med. 26 (1999)1231–1237.

[37] K. Krishnankutty, V.D. John, Synth. React. Inorg. Metal-Org.Chem. 33 (2003) 343–358.

[38] J.T. Mague, W.L. Alworth, F.L. Payton, Acta Cryst. C 60 (2004)o608–o610.

[39] A.P. Gilli, V. Bertolasi, L. Pretto, V. Frretti, G. Gilli, J. Am.Chem. Soc. 126 (2004) 3845–3855.

[40] C.R. Grija, N.S. Begum, A.A. Syed, V. Thiruvenkatam, ActaCryst. C 60 (2004) o611–o613.

[41] A.T. Dinkova-Kostova, P. Talalay, Carcinogenesis 20 (1999) 911–914.