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Metal-based antitumor cytotoxic and antimicrobial activity pharmacological evaluation of Knoevenagel condensate -diketone Schiff base thiosemicarbazone Cu(II) and Zn(II) complexes.

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Full Paper
Received: 23 February 2009
Revised: 16 April 2009
Accepted: 16 April 2009
Published online in Wiley Interscience: 28 May 2009
(www.interscience.com) DOI 10.1002/aoc.1512
Metal-based antitumor, cytotoxic
and antimicrobial activity: pharmacological
evaluation of Knoevenagel condensate
β-diketone Schiff base thiosemicarbazone
Cu(II) and Zn(II) complexes
N. Ramana∗ , R. Jeyamurugana , B. Rajkapoorb and V. Mageshc
Knoevenagel condensate Schiff base ligands [L = 3-cinnamalideneacetylacetone-thiosemicarbazone (CAT)/3-cinnamalideneacetylacetoneethylthiosemicarbazone (CAET)/3-cinnamalideneacetylacetonephenylthiosemicarbazone (CAPT)] and
their copper/zinc complexes were synthesized. They were characterized by analytical and spectral techniques. From these data
it was found that the ligands adopt square-planar geometry on metalation with Cu2+ and Zn2+ . To evaluate the antitumor and
cytotoxic activity of the synthesized complexes in mice and human cancer cell lines, the antitumor activity of the complexes was
evaluated against an Ehrlich ascites carcinoma (EAC) tumor model. The activity was assessed using survival time and short-term
in vitro cytotoxic activity. Oral administration of complexes (100 mg/kg) increased the survival time. The cytotoxic activity
of complexes was evaluated using human breast cancer (MDA-MB-231), colon cancer (HCT-116) and nonsmall lung cancer
(NCI-H-23) cell lines. Both the complexes possessed significant antitumor and cytotoxic activity on EAC and human cancer cell
lines. The in vitro antimicrobial screening effect of the investigated compounds was also tested against the various organisms
c 2009 John Wiley & Sons, Ltd.
by well diffusion method. Copyright Keywords: Schiff base complexes; antitumor; cytotoxic; antimicrobial
Introduction
Appl. Organometal. Chem. 2009 , 23, 283–290
∗
Correspondence to: N. Raman, VHNSN College, Chemistry, Virudhunagar,
Tamilnadu, 626001, India. E-mail: drn raman@yahoo.co.in
a Research Department of Chemistry, VHNSN College, Virudhunagar- 626 001,
India
b Department of Pharmacology and Toxicology, St.John’s Pharmacy College,
Bangalore- 560 040, India
c Department of Biotechnology, Dr. MGR University, E. V. R. Road, Maduravoyal,
Chennai- 600 095, India
c 2009 John Wiley & Sons, Ltd.
Copyright 283
Cancer is a leading cause of mortality worldwide, and the failure
of conventional chemotherapy to effect a major reduction in
mortality indicates that new approaches are critically needed. The
recent approach of chemotherapy serves as an attractive method
to control malignancy.[1] In experimental cancer chemotherapy
studies, attempts are made to identify agents which can
exhibit any or a combination of the following characteristics:
(i) prevention of the initiation of tumors; (ii) delay or arrest of
the development of tumors; (iii) extension of cancer latency
periods; (iv) reduction of cancer metastasis and mortality; and
(v) prevention of recurrence of secondary tumors. The major focus
of research in chemotherapy for cancer in recent times includes
the identification, characterization and development of new and
safe cancer chemopreventive agents.[2]
Cisplatin is one of the most potent antitumor drugs available
for the therapeutic management of solid tumors, overian, lung,
head, neck and bladder cancers. Despite wide application as a
chemotherapeutic agent, cisplatin exhibits severe side effects,
such as nephrotoxicity, neurotoxicity, ototoxicity, nausea and
emetogenicity, which limit the possibilities for gaining therapeutic
benefits from dose intensification.[3 – 6] Thus, scientists are now
engaged in exploring transition metal-based complexes and other
metal-based complexes.
One of the major applications of the transition metal complexes
is for medical testing as antibacterial and antitumor agents aiming
toward the discovery of an effective and safe therapeutic regimen
for the treatment of bacterial infections and cancers.[7 – 10] In addition, many Schiff base complexes with metals have also provoked
wide interest because they possess a diverse spectrum of biological
and pharmaceutical activities, including antitumor and antioxidative activities.[11] Moreover, thiosemicarbazones, semicarbazones
and their metal complexes represent an interesting class of compounds with a wide range of pharmacological applications.[12]
Bearing all these in mind, we have synthesized and characterized
the novel Knoevenagel condensate β-diketone Schiff base of
thiosemicarbazone copper(II) and zinc(II) complexes. Usually such
complexes have pharmacological applications. Hence, we decided
to evaluate the antitumor, cytotoxic, antibacterial and antifungal
activities of the synthesized copper(II) and zinc(II) complexes as
compared with the standard drugs.
N. Raman et al.
Experimental
Materials and Methods
Elemental analyses (C, H, N and S) were carried out with a Calrlo
Erba 1108 analyzer. IR spectra of the samples were recorded
using KBr pellets and a Perkin–Elmer 783 spectrophotometer.
1 H-NMR spectra (300 MHz) of the samples were recorded in
CDCl3 and DMSO-d6 by employing TMS as internal standard on
a Bruker Avance DRX 300 FT-NMR spectrometer. Fast atomic
bombardment mass spectra (FAB-MS) were obtained using
a VGZAB-HS spectrometer in a 3-nitrobenzylalcohol matrix.
Electronic absorption spectra were recorded using a Shimadzu UV1601 spectrophotometer. Magnetic susceptibility measurements
of the complexes were carried out by Gouy balance using copper
sulfate as the calibrant. The purity of ligands and their complexes
was evaluated by thin-layer chromatography. The ligands (CAT,
CAET and CAPT) and their copper and zinc complexes[13] were
prepared according to literature procedure. Synthetic routes
of ligands and these copper and zinc complexes are shown in
Scheme 1.
Synthesis of Ligand CAT
Knoevenagel condensate β-diketone (3-cinnamalideneacetylacetone) (10 mM, 2.14 g) prepared as per the method adopted
by Raman et al.,[14] was refluxed with an ethanolic solution (30 ml)
of thiosemicarbazide (20 mM, 1.82 g) and 1 g of anhydrous K2 CO3
for about 12 h. The solvent was reduced to one-third and the
pasty mass so obtained was treated with hot water and set aside
in refrigerator for 10 h. The solid material formed was removed
by filtration and recrystallized from ethanol. Yield: 65%. IR (KBr):
3427 (NH2 ), 3237 (N(2) H), 1624 (C N), 789 (C S) cm−1 . 1 H-NMR
(δ): (phenyl multiplet) 6.4–6.9 (m); (CH3 ), 2.6 (s); (NH2 ), 7.5 (s),
(N(2) H), 10.2 (s). 13 C NMR (δ): (phenyl ring, C1 –C4 ), 126.6, 127.7,
125.2, 132.3; (C5 , C6 , C7 , C8 and C10 ), 128.1, 125.6, 138.2, 129.0,
20.5; (C9 and C11 ), 156.4, 184.5. MS m/z (%): 360 [M]+ (8.4); 238
[C14 H14 N4 ]+ (64.0); 210 [C14 H14 N2 ]+ (94.0); 182 [C14 H14 ]+ (76); 88
[CH3 N3 ]+ (21.2); 77 [C6 H5 ]+ (28.5); 66 [C5 H6 ]+ (26.5); 60 [CSNH2 ]+
(26.3); 44 [CS]+ (23.7). Anal. calcd for C16 H20 N6 S2 : C, 53.3; H, 5.6; N,
23.3; S,17.8; found: C, 53.1; H, 5.5; N, 23.0; S, 17. 5 (%). λmax in EtOH,
360.5 nm.
Synthesis of Ligand CAET and CAPT
Scheme 1. The outline of the syntheses of metal complexes.
284
Ligands CAET and CAPT were synthesized according to the above
described procedure by the replacement of thiosemicarbazide
by ethylthiosemicarbazide (2.38 g) and phenylthiosemicarbazide
(3.34 g) respectively. Ligand CAET: yield, 68%. IR (KBr): 3329 (N(4) H),
3224 (N(2) H), 1618 (C N), 812 (C S) cm−1 . 1 H-NMR (δ): (phenyl
multiplet), 6.3–6.8 (m); (CH3 ), 2.1 (s); (N(4) H), 7.4(d), (N(2) H), 10.3
(s), (CH2 ), 3.8 (m); (CH3 ) 2.6. 13 C NMR (δ): (phenyl ring, C1 –C4 ),
127.3, 128.5, 126.5, 134.4; (C5 , C6 , C7 , C8 and C10 ), 129.2, 125.2,
138.6, 129.7, 21.3; (C9 and C11 ), 158.3, 186.0; (R CH2 –CH3 ), 42.8,
23.2. MS m/z (%): 417 [M]+ (9.7); 387 [C18 H22 N6 S2 ]+ (6.5); 238
[C14 H14 N4 ]+ (61.6); 210 [C14 H14 N2 ]+ (91.0); 182 [C14 H14 ]+ (72.8);
77 [C6 H5 ]+ (28.5); 66 [C5 H6 ]+ (25.1); 44 [CS]+ (23.7). Anal. calcd for
C20 H28 N6 S2 : C, 57.7; H, 6.8; N, 20.2; S, 15.4; found: C, 57.5; H, 6.6; N,
20.0; S, 15.1 (%). λmax in EtOH, 364 nm. Ligand CAPT: yield, 64%.
IR (KBr): 3316 (N(4) H), 3218 (N(2) H), 1626 (C N), 798 (C S) cm−1 .
1 H-NMR (δ): (phenyl multiplet), 6.2–6.9 (m); (CH ), 2.6 (s); (N(4) H),
3
7.3 (s), (N(2) H), 10.2 (s); 2.1. 13 C NMR (δ): (phenyl ring, C1 –C4 ), 127.7,
128.1, 126.5, 135.1; (C5 , C6 , C7 , C8 and C10 ), 128.4, 125.3, 138.2,
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129.0, 20.3; (C9 , C11 ), 155.4, 187.3; (phenyl ring, -R), 139.6, 125.6,
128.4, 124.4. MS m/z (%): 513 [M]+ (9.2); 238 [C14 H14 N4 ]+ (64.0);
210 [C14 H14 N2 ]+ (94.0); 182 [C14 H14 ]+ (76); 66 [C5 H6 ]+ (24.3); 77
[C6 H5 ]+ (32.6); 44 [CS]+ (23.7). Anal. Calcd for C28 H28 N6 S2: C, 65.6;
H, 5.5; N, 16.4; S, 12.5; Found: C, 65.5; H, 5.0; N, 16.0; S, 12.1 (%).
λmax in EtOH, 372 nm.
Synthesis of Copper(II) Complexes
An ethanolic solution of the above ligand CAT (10 mM, 3.61 g)
was added to a solution of CuCl2 .2H2 O (10 mM, 1.7 g) in ethanol
(20 ml) and the mixture was refluxed for 1 h and concentrated
to one-third volume and kept at 0 ◦ C for 2 h. The solid product
formed was filtered, washed several times with small amounts of
ethanol and diethyl ether and dried in vacuo. [Cu(CAT)]: yield, 64%.
IR (KBr): 3422 (NH2 ), 1592 (C N), 612 (C–S), 980 (N–N), 432 (M–N),
c 2009 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2009, 23, 283–290
Metal-based antitumor, cytotoxic and antimicrobial activity
374 (M-S) cm−1 . MS m/z (%): 422 [M]+ (9.3); 358 [C16 H18 N6 S2 ]+
(6.3); 238 [C14 H14 N4 ]+ (54.0); 210 [C14 H14 N2 ]+ (92.0); 182 [C14 H14 ]+
(84.0); 77 [C6 H5 ]+ (28.3); 66 [C5 H6 ]+ (28.2); 60 [CSNH2 ]+ (26.3); 44
[CS]+ (23.7). Anal. Calcd for [CuC16 H18 N6 S2 ]: Cu, 15.1; C, 45.5; H,
4.3; N, 19.9; S,15.2; found: Cu, 15.0; C, 45.0; H, 4.1; N, 19.4; S, 15.2
(%). λM 10−3 (−1 cm2 mol−1 ), 2.1; µeff (BM), 1.86. λmax in DMF,
386, 520 and 582 nm.
Similarly, complexes [Cu(CAET)] and [Cu(CAPT)] were synthesized according to the above-described procedure. [Cu(CAET)]:
yield, 61%. IR (KBr): 3326 (N(4) H), 1585 (C N), 619 (C–S), 982 (N–N),
426 (M–N), 386 (M–S) cm−1 . MS m/z (%): 479 [M]+ (10.5); 415
[CuC20 H26 N6 S2 ]+ (8.7); 238 [C14 H14 N4 ]+ (60.4); 210 [C14 H14 N2 ]+
(85.0); 182 [C14 H14 ]+ (70.3); 66 [C5 H6 ]+ (26.5); 77 [C6 H5 ]+ (28.5);
44 [CS]+ (23.7). Anal. calcd for [CuC20 H26 N6 S2 ]: Cu, 13.3; C, 50.2; H,
5.5; N, 17.6; S,13.4; found: Cu, 13.0; C, 49.9; H, 5.2; N, 17.2; S, 13.1
(%). λM 10−3 (−1 cm2 mol−1 ), 2.3; µeff (BM), 1.84. λmax in DMF,
387, 526 and 587 nm. [Cu(CAPT)]: yield, 58%. IR (KBr): 3326 (N(4) H),
1585 (C N), 619 (C–S), 986 (N–N), 426 (M–N), 386 (M-S) cm−1 . MS
m/z (%): 574 [M]+ (10.4); 238 [C14 H14 N4 ]+ (59.5); 210 [C14 H14 N2 ]+
(89.0); 182 [C14 H14 ]+ (69.3); 66 [C5 H6 ]+ (22.9); 77 [C6 H5 ]+ (28.6); 44
[CS]+ (23.7). Anal. calcd for [CuC28 H26 N6 S2 ]: Cu, 11.1; C,58.6; H, 4.6;
N, 14.6; S, 11.2; found: Cu, 10.8; C, 58.1; H, 4.3; N, 14.1; S, 10.9 (%).
λM 10−3 (−1 cm2 mol−1 ), 2.7; µeff (BM), 1.87. λmax DMF, 389 nm,
528 nm and 607 nm.
plet), 6.8 (m); (CH3 ), 2.7 (s); (N(4) H), 7.4 (s) (CH2 ), 3.7 (m); (CH3 ), 2.2.
13 C NMR (δ): (phenyl ring, C –C ), 127.6, 128.6, 126.7, 134.8; (C ,
1
4
5
C6 , C7 , C8 and C10 ), 129.7, 125.6, 138.8, 129.9, 21.6; (C9 and C11 ),
163.0, 178.9; (R CH2 –CH3 ), 42.9, 23.4. MS m/z (%): 480 [M]+ (8.9).
Anal. calcd for [ZnC20 H26 N6 S2 ]: Zn, 13.6; C, 50.1; H, 5.5; N, 17.5; S,
13.4; found: Zn, 13.2; C, 49.8; H, 5.5; N, 17.3; S, 13.1 (%). λM 10−3
(−1 cm2 mol−1 ), 2.3. λmax in DMF, 387.4 nm. [Zn(CAPT)]: yield,
66%. IR (KBr): 3314 (N(4) H), 1590 (C N), 632 (C–S), 1006 (N–N),
423 (M–N), 387 (M-S) cm−1 . 1 H-NMR: δ (phenyl multiplet), 6.8 (m);
δ (CH3 ), 2.2 (s); δ (N(4) H), 7.4 (d). 13 C NMR (δ): (phenyl ring, C1 –C4 ),
127.9, 128.5, 126.8, 135.4; (C5 , C6 , C7 , C8 and C10 ), 128.7, 125.6,
138.5, 129.3, 20.5; (C9 , C11 ), 164.8, 179.5; (phenyl ring, -R), 139.8,
125.9, 128.6, 124.5. MS m/z (%): 576 [M]+ (9.6). Anal. calcd for
[ZnC28 H26 N6 S2 ]: Zn, 11.3; C, 58.4; H, 4.6; N, 14.6; S,11.1; found: Zn,
11.0; C, 58.0; H, 4.3; N, 14.0; S, 10.7 (%). λM 10−3 (−1 cm2 mol−1 ),
1.3. λmax in DMF, 390 nm.
Results and Discussion
The ligands and their complexes were found to be stable in air.
The ligands were soluble in common organic solvents but their
complexes were soluble only in DMF and DMSO.
Elemental Analysis and Molar Conductance
Synthesis of Zinc(II) Complexes
A solution of ligand CAT (10 mM, 3.61 g) in ethanol (20 ml) was
added to a solution of ZnCl2 (10 mM, 1.36 g) in ethanol (10 ml) and
the mixture was refluxed for 1 h and concentrated to one-third
volume and kept at 0 ◦ C for 2 h. The solid product formed was
filtered, washed several times with small amounts of ethanol and
diethyl ether and dried in vacuo. [Zn(CAT)]: yield, 67%. IR (KBr):
3424 (NH2 ), 1589 (C N), 616 (C–S), 997 (N–N), 437 (M–N), 368
(M–S) cm−1 . 1 H-NMR (δ): (phenyl multiplet), 6.2–6.8 (m); (CH3 ),
2.3 (s); (NH2 ), 7.4 (s). 13 C NMR (δ): (phenyl ring, C1 –C4 ), 126.9,
128.0, 125.5, 132.4; (C5 , C6 , C7 , C8 and C10 ), 128.3, 125.8, 138.4,
129.4, 20.8; (C9 and C11 ), 162.4, 178.6. MS m/z (%): 424 [M]+ (10.2).
Anal. calcd for [ZnC16 H18 N6 S2 ]: Zn, 15.4; C, 45.3; H, 4.3; N, 19.8; S,
15.1; found: Zn, 15.0; C, 45.0; H, 4.0; N, 19.7; S, 15.0 (%). λM 10−3
(−1 cm2 mol−1 ), 1.8. λmax in DMF, 383 nm.
Similarly, complexes [Zn(CAET)] and [Zn(CAPT)] were synthesized according to the above-described procedure. [Zn(CAET)]:
yield, 62%. IR (KBr): 3328 (N(4) H), 1584 (C N), 627 (C–S), 1002
(N–N), 442 (M–N), 378 (M–S) cm−1 . 1 H-NMR (δ): (phenyl multi-
The elemental analysis results for the metal complexes agree
with the calculated values showing that the complexes have 1 : 1
metal–ligand ratio. The observed low molar conductances of the
complexes in DMF for 10−3 M solutions at room temperature are
consistent with the non-electrolytic nature of the complexes due
to there being no counter (chloride) ions in the structures of the
Schiff base metal complexes.
Mass Spectra
The FAB mass spectra of synthesized ligands and their complexes
were recorded and the obtained molecular ion peaks confirmed
the proposed formulae. The mass spectrum of CAT ligand showed
peak at m/z 360 (8.4%) corresponding to [C16 H20 N6 S2 ]+ ion. Also,
the spectrum exhibited the fragments at m/z 44, 60, 77 and
88 corresponding to [CS]+ , [CSNH2 ]+ , [C6 H5 ]+ and [CH3 N3 ]+ ,
respectively. The mass spectrum of [CuC16 H18 N6 S2 ] showed
peaks at 422, 423 and 424 with 9.3, 5.7, and 3.2% abundances,
respectively. The one at 422 may represent the molecular ion peak
of the complex and the other peaks are isotopic species (Fig. 1).
285
Figure 1. FAB Mass spectrum of [Cu(CAT)].
Appl. Organometal. Chem. 2009, 23, 283–290
c 2009 John Wiley & Sons, Ltd.
Copyright www.interscience.wiley.com/journal/aoc
N. Raman et al.
44 and 77 represent CS and C6 H5 moieties respectively. The m/z
of all the fragments of ligands and their complexes confirmed
the stoichiometry of the complexes to be of the type [ML]. This
was further supported by the mass spectra of all the complexes.
The observed peaks were in good agreement with their empirical
formulae, as indicated from microanalytical data. Thus, the mass
spectral data reinforced the conclusion drawn from the analytical
and conductance values.
Infrared Spectra
The IR spectra provide valuable information regarding the nature
of the functional group attached to the metal atom. In all the
complexes, it was found that the ν(N(2) H) band, originally present
in the ligand system at 3218–3237 cm−1 , disappeared and a
new band appeared at 1618–1624 cm−1 due to ν (C N). The
ν (C N) band present in the ligand shifted to lower frequency
by ca 20 cm−1 on complexation.[15] This was further supported
by the presence of a new band at ca 423–442 cm−1 , assigned
to ν (M–N).[16] The absence of thioamide band ν (HN–C S)
at ca 785–815 cm−1 and the appearance of new band at ca
612–632 cm−1 confirmed the conversion of ν (C S) into ν (C–S)
band[17] and a new band around 368–387 was assigned to ν (M–S)
band. The reduction of thioamide band ν (N C–SH) observed at
ca 980 cm−1 suggests that coordination occurs through S atoms.
These data reveal that the ligands behave as tetradentate and are
coordinated to the metal ions through the azomethine nitrogen
and thiolate sulfur.
Electronic Absorption Spectra and Magnetic Measurements
The electronic absorption spectra of the complexes were recorded
in DMF solution. The spectra of the Cu(II) complexes showed
two bands in the visible region, around 17 500–16 500 and
19 300–18 500 cm−1 , which were assigned to 2 B1g → 2 A1g and
2B
2
1g → Eg transitions, respectively. The electronic spectral data
suggested a square-planar geometry around the Cu(II) ion. The
observed magnetic moment of the Cu(II) complexes (1.84–1.87
BM) at room temperature were non-coupled mononuclear
complexes of magnetically diluted d9 system with S = 1/2
spin-state square-planar structure. The monomeric nature of the
complexes was further supported by the microanlytical and FAB
mass spectral data. The electronic absorption spectra of the Zn(II)
complexes showed bands (26 000–25 600 cm−1 ) assigned to intraligand charge transfer transitions.[18]
Scheme 2. Mass fragment pattern of [Cu(CAT)].
1 H NMR Spectra
286
The strongest peak (base beak) at m/z 358 represents the stable
species (C16 H18 N6 S2 ). Also, the spectrum exhibited the fragments
(Scheme 2) at m/z 44, 60, 77 and 88 corresponding to [CS]+ ,
[CSNH2 ]+ , [C6 H5 ]+ and [CH3 N3 ]+ , respectively.
The FAB mass spectrum of [Cu(CAET)] exhibited peak at 479
(10.5%), 480, (4.6%) and 481 (3.8%) representing the molecular
ion peak, [CuC20 H26 N6 S2 ] and other peaks are isotopic species.
The second peak at m/z 415 represents the [C20 H26 N6 S2 ]+ . Also,
the spectrum exhibited the fragments at m/z 385, 44 and 77
represent C18 H20 N6 S2 , CS and C6 H5 moieties respectively. The
mass spectrum of [CuC28 H26 N6 S2 ] showed peaks at m/z 574, 575,
and 576 with 10.4, 5.8 and 4.2% abundances, respectively. The
highest abundance peak 574 represents the molecular ion peak
of [Cu(CAPT)] complex. The strongest peak (base beak) at m/z
572 represents the stable species (C28 H26 N6 S2 ). The peaks at m/z
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The 1 H NMR spectra of the ligands and their zinc complexes
were recorded in CDCl3 and DMSO-d6 respectively. The free
ligands exhibited a singlet at δ 10.2–10.3 ppm due to the –N(2) H
proton. The absence of this signal in the spectra of the complexes
suggested that this proton had been lost via thioenolization of
C S group and coordination of the sulfur atom to the metal ion.
The complexes showed multiples in the region δ 6.2–6.9 ppm
attributed to the aromatic protons, which appeared at almost
the same position as in the respective ligands. The –NH2 group
gave singlet at δ 7.5 ppm in the free ligand (CAT) and remained
unchanged in the complex [Zn(CAT)]. This shows that the –NH2
group was not taking part in complexation. The –N(4) H group
proton gave a signal at δ 7.3–7.4 ppm in free ligands (CAET and
CAPT) as well as in their zinc complexes. It also confirmed that the
–N(4) H group was not involved in complexation.
c 2009 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2009, 23, 283–290
Metal-based antitumor, cytotoxic and antimicrobial activity
Figure 2. 13 C NMR spectrum of [Zn(CAPT)].
Figure 3. Structure of the ligands.
Figure 4. Structure of the complexes.
13 C NMR Spectra
The 13 C NMR spectra of the ligands and their zinc complexes were recorded in DMSO-d6 . The aromatic carbon peaks
were observed at δ 125.0–140.0 ppm. The ligands showed
thioamide carbon (HN–C S) signal at δ 184.5–187.5 ppm,
which was significantly shifted upfield (δ 178.5–179.5 ppm) in
the complexes (Fig. 2). This was due to the enolization of
the thione group and formation of a new azomethine linkage. The ligands showed a signal at δ 156.0–159.0 ppm due
to the presence of azomethine–carbon in the ligands. This
signal also underwent a downfield shift in the complexes (δ
162.0–165.0 ppm), indicating coordination of azomethine nitrogen to metal. The solvent DMSO-d6 carbon peak appeared at δ
39.7 ppm.
Based on the above analytical and spectral data, the structures
of the ligands and their complexes are shown in Figs 3 and 4,
respectively.
Pharmacological Methods
Antitumor activity
Appl. Organometal. Chem. 2009, 23, 283–290
Effect of Cu(II) and Zn(II) Complexes on Survival Time[20]
Animals were inoculated at 1 × 106 cells/mouse on day ‘0’ and
treatment with copper(II) and zinc(II) complexes started 24 h after
inoculation, at a dose of 100 mg/kg/d, p.o. The control group was
treated with the same volume of 0.9% sodium chloride solution.
All the treatments were given for 9 days. The median survival
c 2009 John Wiley & Sons, Ltd.
Copyright www.interscience.wiley.com/journal/aoc
287
Adult Swiss female albino mice (20–25 g) were procured from
the animal house, St John’s Pharmacy College, Bangalore, India
and used throughout the study. They were housed in microlon
boxes in controlled environment (temperature 25 ± 2 ◦ C and 12 h
dark/light cycle) with standard laboratory diet and water adlibitum.
The experiments were performed in accordance with guidelines
established by the European community for the care and use of
laboratory animals, and were approved by the Institutional Animal
Ethics Committee (IAEC) of St John’s Pharmacy College, Bangalore,
India.
EAC cells were obtained courtesy of Amala Cancer Research
Centre, Trissur, India. They were maintained by weekly intraperitoneal inoculation of 106 cells/mouse.[19] All values are expressed
as means ± SEM. The data were statistically analyzed by oneway ANOVA followed by Dunnett’s test; p-values <0.05 were
considered significant.
N. Raman et al.
time (MST) of each group, comprising six mice, was noted. The
antitumor efficiency of complexes was compared with that of
5-fluorouracil (Dabur Pharmaceutical Ltd, India; 5-FU,
20 mg/kg/day i.p. for 9 days). The MST of the treated groups
was compared with those of control groups by the following
calculation:
Increase in life span =
T −C
× 100
C
where T = number of days treated animals survived and C =
number of days control animals survived.
The effect of complexes on the survival of tumor-bearing mice
showed the MST for the control group to be 16 days, while it
was 35.75 (123.4%) and 32.25 days (101.56%), respectively, for
the group treated with 5-FU and [Zn(CAT)], respectively. These
results are almost comparable to that for 5-FU, the standard drug
(Table 1). The reliable criterion for evaluating an anticancer drug
is prolongation of lifespan of the animal.[21] Our results show
an increase in lifespan mice treated with the complexes. These
results clearly demonstrate the antitumor effect of complexes
against EAC.
Effect of Cu(II) and Zn(II) Complexes on Cytotoxicity in Vitro
Short-term cytotoxicity was assessed by incubating 1 × 106 EAC
cells in 1 ml phosphate buffer saline with varying concentrations
(100–400 µg/ml) of the Cu(II) and Zn(II) complexes at 37 ◦ C for
3 h in CO2 atmosphere ensured using a McIntosh field jar. The
viability of the cells was determined by the trypan blue exclusion
method.[22]
The short-term in vitro cytotoxicity study of EAC-bearing mice
showed the GI50 of complexes to be 100, 200, 300 and 400 µg/ml.
The GI50 values for all of the complexes are given in Table 2. Of all
of the complexes tested, [Zn(CAT)] had the lowest GI50 value of
137 µg/ml, which is similar to that of the standard 5-FU (132 µg/ml).
From this result [Zn(CAT)] complex had higher cytotoxicity and
anticancer effect on cancer cell lines than the other complexes.
Moreover, all the complexes were more selective cytotoxic against
EAC cell line.
Cytotoxic Studies
Human breast cancer (MAD-MB-231), colon cancer (HCT-116) and
nonsmall lung cancer (NCI-H 23) cells were obtained from National
Table 1. Effect of Cu(II) and Zn(II) complexes treatment on the survival
of tumor-bearing mice
Treatment
Tumor control
5-FU
[Cu(CAT)]
[Cu(CAET)]
[Cu(CAPT)]
[Zn(CAT)]
[Zn(CAET)]
[Zn(CAPT)]
MST
Increase in life span
16 ± 0.5774
35.75 ± 2.75∗
29.75 ± 3.735
27.25 ± 1.42∗∗
21.5 ± 1.190
32.25 ± 2.11∗
28.75 ± 2.543∗
25.5 ± 1.42
–
123.4
85.94
70.31
34.38
101.56
79.69
59.38
288
N = 6; days of drug treatment = 9.
∗ p < 0.01; ∗∗ p < 0.05 Vs tumor control.
Data were analyzed by one-way ANONA followed by Dunnett’s test.
www.interscience.wiley.com/journal/aoc
Table 2. In vitro cytotoxic activity of Cu(II) and Zn(II) complexes in
EAC cell line
GI50 (µg/ml)
Treatment compounds
[Cu(CAT)]
[Cu(CAET)]
[Cu(CAPT)]
[Zn(CAT)]
[Zn(CAET)]
[Zn(CAPT)]
5-FU
162
182
252
137
180
182
132
Average of three determinations, three replicates.
GI50 , drug concentration inhibiting 50% cellular growth following 3 h
of drug exposure.
Table 3. Cytotoxic effect of Cu(II) and Zn(II) in human cancer cell lines
MDA-MB-231 (µg/ml) NCI-H 23 (µg/ml) HCT-116 (µg/ml)
Complexes GI50
[Cu(CAT)]
[Cu(CAET)]
[Cu(CAPT)]
[Zn(CAT)]
[Zn(CAET)]
[Zn(CAPT)]
TGI
LC50
0.45
35
0.1 >100
0.1
4
0.05
53
0.4
5
0.02
9.2
>100
>100
>100
>100
>100
>100
GI50 TGI LC50 GI50 TGI LC50
24
1
21
31
22
9
52
42
50
55
50
39
78
75
78
82
76
84
2
4
6
1
0.3
1
30
34
38
12
9
13
70
79
83
79
56
69
Average of three determinations, three replicates.
GI50 , drug concentration inhibiting 50% cellular growth following 72 h
of drug exposure.
TGI, total cellular growth inhibition.
LC50 , concentration of drug resulting in a 50% reduction at the end of
drug treatment as compared with that at the beginning.
Centre for Cell Science (Pune, India). Stock cells of MAD-MB-231,
HCT-116 and NCI-H 23 cell lines were cultured in RPMI-1640 or
DMEM supplemented with 10% in activated newborn calf cerum,
pencillin (100 IU/ml), streptomycin (100 µg/ml) and amphotericinB (5 µg/ml) under a humidified atmosphere of 5% CO2 at 37 ◦ C until
confluent. The cells were dissociated in 0.2% trypsin and 0.02%
EDTA in phosphate buffer saline solution. The stock culture was
grown in 25 cm2 tissue-culture flasks, and cytotoxicity experiments
were carried out in 96-well microtiter plates (Tarsons India, Kolkata,
India).
Cell lines in the exponential growth phase were washed,
trypsinized and resuspended in complete culture media. Cells
were plated at 10 000 cells/well in 96-well microtiter plates
and incubated for 24 h, during which a partial monolayer
formed. They were then exposed to various concentration of
the complexes (0.1–100 µg/ml) and 5-FU. Control wells received
only maintenance medium. The plates were incubated at 37 ◦ C in
a humidified incubator with 5% CO2 for a period of 72 h. At the
end of 72 h, viability was determined by MTT assay.[23]
The cytotoxic activity of the complexes was evaluated on human
tumor cell lines MDA-MB-231 (human breast cancer), NCI-H-23
(nonsmall cell lung cancer) and HCT-116 (colon cancer) using
standard MTT-dye reduction assay. When the cells were treated
for 72 h with various concentrations of complexes (5–100 µg/ml),
relative cell survival progressively decreased in a dose-dependent
manner. The GI50 , TGI and LC50 of the complexes are shown in
c 2009 John Wiley & Sons, Ltd.
Copyright Appl. Organometal. Chem. 2009, 23, 283–290
Metal-based antitumor, cytotoxic and antimicrobial activity
Table 4. Antibacterial activity of the ligands and their complexes
Minimum inhibitory concentration (MIC) (×104 µM)
Sample no.
1.
2
3
4
5
6
7
8
9
10
Compound
Staphylococcus aureus
Bacillus subtilis
Escherichia coli
Klebsiella pneumonia
CAT
CAET
CAPT
[Cu(CAT)]
[Cu(CAET)]
[Cu(CAPT)]
[Zn(CAT)]
[Zn(CAET)]
[Zn(CAPT)]
Streptomycin
14.4
11.5
8.7
2.1
1.7
1.0
3.8
2.7
2.0
1.7
14.9
12.2
9.6
2.8
2.1
1.2
4.5
3.5
2.3
2.5
16.0
11.0
8.4
2.1
1.3
0.7
3.3
2.5
1.7
1.3
16.6
13.4
10.0
2.1
1.7
1.0
4.7
3.3
2.3
2.0
Table 5. Antifungal activity of the ligands and their complexes
Minimum inhibitory
concentration (MIC) (×104 µM)
Sample
Aspergillus Fusarium Curvularia Rhizoctonia
no.
Compound
niger
solani
lunata
bataicola
1.
2
3
4
5
6
7
8
9
10
CAT
CAET
CAPT
[Cu(CAT)]
[Cu(CAET)]
[Cu(CAPT)]
[Zn(CAT)]
[Zn(CAET)]
[Zn(CAPT)]
Nystatin
16.6
12.9
9.4
3.8
3.5
2.0
6.4
5.0
3.2
1.0
17.5
13.4
9.6
5.0
4.2
2.4
6.8
5.4
3.6
1.7
18.6
14.6
10.5
4.7
2.9
2.3
6.1
4.2
2.6
0.9
19.7
15.8
11.5
5.9
3.8
1.9
6.1
4.4
2.8
1.5
Table 3 for all three cell lines. Based on cytotoxicity results, the
[Zn(CAT)] complexes produced a more potent cytotoxic effect on
these three human cancer cell lines than other complexes.
Conclusion
Antimicrobial assays
The synthesized complexes were characterized by analytical and
spectral data. Based on the data, they adopt square-planar
geometry. The antitumor and in vitro cytotoxic activities of these
metal complexes were examined. The results suggest that all the
complexes produced a potent cytotoxic effect against human
tumor cell lines as well as EAC. The in vitro antibacterial and
antifungal activity of ligands and their complexes was also
evaluated by well diffusion method. The results obtained from
antifungal and antibacterial tests together showed that all the
complexes are more active towards bacteria than fungi. It has
been found that the activities of the complexes are higher than
those of the ligands.
Acknowledgments
The authors gratefully acknowledge the financial support of this
work by the Department of Science and Technology, New Delhi,
India. They express their heartfelt thanks to the VHNSN College
Managing Board for providing the research facilities.
c 2009 John Wiley & Sons, Ltd.
Copyright www.interscience.wiley.com/journal/aoc
289
The synthesized ligand and their complexes were tested for their
in vitro antimicrobial activity against the Gram-positive bacteria
Staphylococcus aureus and Bacillus subtilis and the Gram-negative
bacteria Escherichia coli and Klebsiella pneumoniae by the well
diffusion method, using agar nutrient as the medium, and fungi
Aspergillus niger, Fusarium solani, Curvularia lunata and Rhizoctonia
bataicola by the well diffusion method using potato dextrose agar
as medium. The stock solution (10−2 M) was prepared by dissolving
the compounds in DMSO and the solutions were serially diluted in
order to find the MIC values. In a typical procedure,[24] a well was
made on the agar medium inoculated with microorganisms. The
well was filled with the test solution using a micropipette and the
plate was incubated, 24 h for bacteria and 72 h for fungi at 35 ◦ C.
During this period, the test solution diffused and the growth of the
inoculated microorganisms was affected. The inhibition zone was
developed, at which the concentration was noted. Streptomysin
and nystatin were used as control drugs. The minimum inhibitory
concentration (MIC) values of the compounds against the growth
of microorganisms are summarized in Tables 4 and 5.
Appl. Organometal. Chem. 2009, 23, 283–290
A comparative study of MIC values of the Schiff base and its
complexes indicates that the metal complexes exhibit higher
antimicrobial activity than the free ligand and the control
(streptomycin). Such increased activity of the complexes can be
explained on the basis of the Overtones concept[25] and Tweedy’s
chelation theory.[26] According to the Overtones concept of cell
permeability, the lipid membrane that surrounds the cell favors
the passage of only the lipid-soluble materials due to which
liposolubility is an important factor that controls the antimicrobial
activity. On chelation, the polarity of the metal ion will be reduced
to a greater extent due to the overlap of the ligand orbital and
partial sharing of the positive charge of the metal ion with donor
groups. Further, it increases the delocalization of π -electrons over
the whole chelate ring and enhances the lipophilicity of the
complexes. This increased lipophilicity enhances the penetration
of the complexes into lipid membranes and blocking of the
metal binding sites in the enzymes of microorganisms. The results
obtained from antifungal and antibacterial tests together showed
that all complexes tested are more active towards bacteria than
fungi. Moreover, copper complexes are more active than zinc
complexes against microorganisms.
N. Raman et al.
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