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In vitro anticancer and antibacterial activities of octahedral ruthenium(III) complexes with hydroxamic acids. Synthesis and spectroscopic characterization
ISSN 1070-3632, Russian Journal of General Chemistry, 2016, Vol. 86, No. 2, pp. 360–367. © Pleiades Publishing, Ltd., 2016.
In Vitro Anticancer and Antibacterial Activities of Octahedral
Ruthenium(III) Complexes with Hydroxamic Acids.
1
Synthesis and Spectroscopic Characterization
Raj Kaushal and Sheetal
Department of Chemistry, National Institute of Technology, Hamirpur, Himachal Pradesh-177005, India
e-mail: kaushalraj7823@rediffmail.com
Received July 14, 2015
Abstract—Five new ruthenium(III) complexes of the general formulas [RuCl(HO)L] (1–4) and [RuCl(HO)(HL)]
2 2 3 2 2
(5), where L = benzohydroximato (1), salicylhydroximato (2), acetohydroximato (3), hydroxyureato (4), LH =
N-hydroxy-N-phenylbenzamide (5), were synthesized by reaction of RuCl ·3H O with the corresponding
3 2
hydroxamic acids at a molar ratio of 1:2 molar. The complexes were characterized by elemental analyses and
FT-IR, UV-Vis, 1H and 13C NMR, and mass spectra. The complexes showed higher antibacterial activity
against ten pathogenic bacterial strains than the corresponding ligands. The anticancer activity of the complexes
against IMR-32 (neuroblastoma) cancer and CHO (Chinese hamster ovary) normal cell lines was evaluated
using MTT assay with respect to camptothecin as control. Complex 5 was found to exhibit an appreciable
cytotoxicity against IMR-32 cell line with an IC value of 102.27 μM.
50
Keywords: Ruthenium complexes, hydroxamic acids, antibacterial and anticancer activity
DOI: 10.1134/S1070363216020274
Diverse biological activities of metal ions and nium possesses antitumor properties [12]. Remarkable
naturally occurring inorganic or organic metal com- antitumor properties are mainly attributed to organic
plexes in health and pathological conditions have ruthenium(III) complexes trans-linked to N-hetero-
stimulated the development of metal complexes with cyclic ligands and inorganic ruthenium(III) complexes
potential medical applications [1–3]. Relevant pub- with cis- or trans-linked ammine ligands. Imidazolium
lished data show anticancer and antibacterial activities trans-[tetrachloro(dimethyl sulfoxide)(imidazole)ruthe-
of organic and inorganic complexes derived from nate(III)] (NAMI-A) is the first ruthenium-based drug
transition metals such as platinum, ruthenium, tita- with effective antineoplastic activity on solid tumors,
nium, iridium, and rhodium [4–8]. Chemotherapy with whereas KP1019 {indazolium trans-[tetrachlorobis-
platinum complexes is now one of the pillars in the (indazole)ruthenate(III)]} exhibited activity against
treatment of cancer [9]. However, severe side effects colon carcinomas and a variety of primary explanted
and efficiency against a restricted spectrum of tumors, human tumors [13]. These complexes recently com-
as well as acquired or intrinsic resistance, restrain their pleted Phase I clinical trial without any unexpected
successful therapeutic use. toxicity and showed a maximum-tolerated dose, profile
of adverse events, and dose limiting toxicity similar to
Therefore, alternative metal complexes are being
those observed in preclinical models where active
presently evaluated in clinical trials. Ruthenium metal
effects on metastases were observed [14] (Scheme 1).
complexes are used in different fields of chemistry,
In medicinal applications, hydroxamic acid moieties
particularly in bioinorganic and medicinal chemistry;
are used in the design of therapeutics targeting car-
some examples are shown below [10, 11]. The first
diovascular diseases, insecticides, and antimicrobial
systematic investigation of ruthenium complexes and
agents [15–17]. Hydroxamic acids are usually used as
their antitumor properties was done in the early 1980s
supporting ligands in chemistry and biology because of
with fac-[Ru(NH ) Cl ] and cis-[RuCl (NH ) Cl] pre-
3 3 3 2 3 4
their tautomerism and potential chelating properties
ceded by the discovery made in the 1970s that ruthe-
[18, 19]. Hydroxamic acid tautomers can exist as
different conformers [20], but they preferentially
1 The text was submitted by the authors in English. assume Z-hydroxamic structure as shown in Scheme 1.
360
In Vitro ANTICANCER AND ANTIBACTERIAL ACTIVITIES 361
Scheme 1.
CH 3 + CF SO–
O 3 3
CH
3
S
Cl
H+
N+ N
Cl H+ NH N
Ru
Ru N N NH
Cl Cl
Cl Cl Ru Cl N+ N
N NH Cl Cl
N
NH
NH
NAMI-A
KP1019 O OH
2+ HN
+
O
H 2 N NH 2 N+ N
Ru X Ru 2CF 3 SO 3 – NH
Cl H 2 N S N+ N N Ru
C
NH
2
O
RM175 (X = PF )
6 DW1/2
ONCO4417 (X = Cl–)
Scheme 2.
OH OH H
O N HO N O N HO N
H OH OH
R R R R
Hydroxamic acid Hydroximic acid Hydroxamic acid Hydroximic acid
Z conformer Z isomer E conformer E isomer
H R HO R
H
R N
Mn+
N or
N
O OH –H– O Mn O + Mn+ O
The great majority of the investigated systems dis- carbonyl group and nitrogen atom. Ruthenium(III)
played coordination of the metal ion to the carbonyl complexes of general formulas [RuCl(H O)(L) ] (1–4)
2 2
oxygen atom and NHOH group. The O,O-coordination and [RuCl (H O)(HL) ] (5) were synthesized in
3 2 2
mode confirmed by X-ray crystallography involves quantitative yields by the reaction of RuCl · 3H O with
3 2
deprotonation of the OH group and chelation by the 2 equiv of the corresponding hydroxamic acid in
carbonyl oxygen atom [21]. On the other hand, N,O- ethanol under reflux (Scheme 3). The hydroxamic
coordination via deprotonation of the NH group was acids used as ligands were benzohydroxamic acid (L1),
observed for amino hydroxamic acids like RR′NC(O)· salicylhydroxamic acid (L2), acetohydroxamic acid
NHOH [22, 23] (Scheme 2). (L3), N-hydroxyurea (L4), and N-hydroxy-N-phenyl-
benzamide (L5). The proposed structures of 1–5 were
Herein, we report the synthesis, characterization, consistent with their elemental analyses (Table 1) and
and biological evaluation (anticancer and antibacterial) spectroscopic data (Table 2; see Experimental). The
of five ruthenium(III) complexes with hydroxamic ligands and complexes 1–5 are air-stable but somewhat
acids differing by the substituents attached to the hygroscopic when exposed to air for a long time.
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 86 No. 2 2016
362 RAJ KAUSHAL, SHEETAL
Scheme 3.
HO
R HO
R' EtOH, reflux O N R Ph OH 2 N Ph
O N OH + RuCl 3 · 3 H 2 O HO N Ru O or O Ru O Ph
N
R Cl Ph Cl Cl
OH 2 OH Cl
L1–L5 1–4 5
1, R = Ph; 2, R = 2-HOC H ; 3, R = Me; 4, R = H N.
6 4 2
Comparison of the FT-IR spectra of newly syn- 3020 cm–1), indicating tautomerization of the ligand
thesized ruthenium(III) complexes 1–5 with those of upon complex formation [24]. The weak overtones at
the free ligands (Table 2) confirmed the assumed coor- 2000–1700 cm–1 in the spectra of 1, 2, and 5 were
dination modes. Complexes 1–5 lacked N–H stretching assigned to the aromatic groups in the ligands. Strong
vibration band typical of free ligands L1–L4 (3150– carbonyl stretching bands at 1665–1600 cm–1 in the
Table 1. Yields, decomposition points, and elemental analyses of ruthenium(III) complexes 1–5
Found, % Calculated, %
Comp. Yield,
Color Formula
no. % C H Cl N Ru C H Cl N Ru
1 Dark green 68.3 237–240 39.40 3.27 8.30 3.25 23.85 C H N O ClRu 39.41 3.28 8.32 3.28 23.86
14 14 2 5
2 Reddish brown 73.6 295–298 36.58 3.01 7.62 6.06 22.01 C H N O ClRu 36.61 3.05 7.73 6.10 22.02
14 14 2 7
3 Light green 80.3 225–228 15.85 3.28 11.71 9.24 33.60 C H N O ClRu 15.87 3.30 11.73 9.26 33.64
4 10 2 5
4 Dark black 84.0 270–276 7.67 2.59 11.59 18.29 31.15 C H N O ClRu 7.88 2.62 11.65 18.38 30.18
2 8 4 5
5 Dark green 75.0 237–241 47.68 3.65 16.30 4.25 15.58 C H N O Cl Ru 47.88 3.68 16.34 4.29 15.61
26 24 2 5 3
Table 2. FT-IR and UV-Vis spectra of ruthenium(III) complexes 1–5
ν, cm–1 λ , nm
Complex max
or ligand O–H C–H N–H C=O C=N C–O N–O δ(C–H)a Ru–N Ru–Cl n–π* π–π*
L1 3372 3040, 2980 3294 1640 – – 995 795 – – 290 210
1 3431 3010, 2971 – 1633 1271 921 798 560 467 283 205
L2 3289 3945, 2930 3282 1625 – – 950 756 – – 271 215
2 3444 3038, 2925 – 1603 1265 932 759 562 460 265 210
L3 3394 2980, 2899 3230 1625 – – 963 – – 280 210
3 3424 2972, 2894 – 1630 1289 951 538 446 272 208
L4 3420 – 3314 1656 – – 993 – – 290 215
4 3442 – 3342 1643 1267 945 509 439 283 209
L5 3430 2930, 2850 1625 – – 995 775 – – 345 225
5 3418 2925, 2845 – 1619 1287 994 778 568b 486 330 218
a Out-of-plane aromatic C–H bending vibration mode.
b ν(Ru–O).
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 86 No. 2 2016
In Vitro ANTICANCER AND ANTIBACTERIAL ACTIVITIES 363
spectra of free ligands shifted to lower frequencies after the complexation, presumably because the N–OH
(1645–1602 cm–1) and decreased in intensity in going moiety was not involved in the coordination [1, 25].
to the corresponding complexes and were assigned to Protons in the coordinated water molecule resonated at
ν(C=N) [25]. The sharp band at 995–950 cm–1 ascribed δ = 3.22–3.45 ppm. The spectra of 1, 2, and 5
to N–O bending mode moves to lower wave numbers contained aromatic proton signals in the region δ =
at 995–921 cm–1 in the spectra of the complexes 6.73–7.99 ppm, complex 3 showed a singlet at δ =
suggesting tautomerization of the ligand during 2.14 ppm due to methyl protons, and the singlet at δ =
coordination [26]. The bands at 798–756 cm–1 in the 5.44 ppm in the spectrum of 4 was assigned to the NH
2
spectra of 1, 2, and 5 correspond to out-of-plane group.
bending vibrations of aromatic C–H bonds. The O–H
The 13C NMR spectra of newly synthesized ruthe-
stretching band present in the spectrum of L5 at
nium(III) complexes 1–5 showed all expected signals
3430 cm–1 remained apparent after complexation
and were generally consistent with the 1H NMR data.
(3418 cm–1), indicating that this group is not involved
The carbonyl carbon signal appeared at δ = 158.05–
in the coordination to ruthenium ion. The carbonyl C
159.18 ppm in the spectra of the free ligands
peak at 1653 cm–1 in the spectrum of L5 shifted to
disappeared after coordination to ruthenium ion in
1617 cm–1 due to coordination of the carbonyl oxygen
support to the ligand tautomerization, and a new peak
atom to ruthenium. Thus, the IR spectrum of 5
appeared at δ = 150.83–165.98 ppm due to C=N
supports coordination of the ligand to ruthenium ion C
group. The C=N signal of 4 was observed at δ =
through the carbonyl oxygen atom. The absorption in C
152.78 ppm.
the ranges 509–568 and 439–486 cm–1 was assigned to
ν(Ru–N)[or ν(Ru–O)] and ν(Ru–Cl) stretching The mass spectra of 1–5 contained the molecular
vibrations, respectively [27]. A broad band centered at ion peaks and those resulting from successive elimina-
3424–3344 cm–1 was observed in the spectra of tion of coordinated water molecule, chlorine atom, and
complexes 1–4 due to O–H vibrations of the hydroximate ligand from the molecular ion. These data
coordinated water molecule in the respective clearly showed that mononuclear complex was formed
complexes. in each case.
The UV-Vis spectra of hydroxamic acid ligands Complexes 1–5, as well as free ligands L1–L5,
L1–L5 and newly synthesized ruthenium(III) com- were tested by the agar well diffusion method for
plexes 1–5 were recorded in ethanol. The spectra antibacterial activity against ten pathogenic bacterial
displayed two bands with their maxima in the ranges strains, namely Enterobacter aerogenes MTCC 7325,
λ = 265–345 (n→π*) and 208–225 nm (π→π*) (Table 2). Micrococcus luteus MTCC 1809, Staphylococcus
Blue shift and hyperchromic effect were observed due aureus MTCC 3160, Staphylococcus epidermidis
to coordination with ruthenium ion. The shift was due MTCC 3086, Aeromonas hydrophila MTCC 1739,
to donation of lone pair of the nitrogen and oxygen Aclaligenes faecalis MTCC 126, Shigella sonnei
atoms of the ligand to ruthenium ion, i.e., L→M MTCC 2957, Klebsiella pneumoniae MTCC 3384,
charge transfer. It indicates coordination of the ligands Pseudomonas aeruginosa MTCC 1035, and Salmonella
to ruthenium through nitrogen and oxygen atoms. typhimurium MTCC 1253. All complexes were generally
Since the metal ion has d5 configuration, it has the more active than the corresponding ligands, and their
same multiplicity as the ground state term; therefore, activity was either comparable or even higher than the
all electronic transitions in the spectra are spin activity of ampicillin taken as control (Table 3). All
forbidden [1]. complexes 1–5 turned out to be superior to ampicillin
in the activity against P. aeruginosa, complexes 1 and
Complexes 1–5 displayed in the 1H NMR spectra
2 showed a high activity against A. faecalis, complex 3
singlets due to OH proton in the region δ = 10.20–
was potent against K. pneumoniae, and complex 5
12.50 ppm, while no NH signal typical of free ligands
displayed the largest inhibition zone on A. hydrophila.
L1–L4 was present. Furthermore, the OH signal of
complexes 1–4 appeared in a weaker field relative to The minimum inhibitory concentrations (MICs) of
the corresponding signal of the free ligand. These ruthenium(III) complexes 1–5 toward bacterial strains
findings suggest deprotonation of L1–L4 and their are presented in Table 4. It was found that complexes 1
coordination to ruthenium through the nitrogen atom. and 5 are efficient against E. aerogenes, A. faecalis, S.
The OH signal of L5 almost did not change its position aureus, and A. hydrophila. Complexes 2 and 3 inhibited
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 86 No. 2 2016
364 RAJ KAUSHAL, SHEETAL
Table 3. Antibacterial activity of complexes 1–5 and ligands L1–L5 against pathogenic bacterial strains
Inhibition zone diameter, mm
Microbial species
1 (L1) 2 (L2) 3 (L3) 4 (L4) 5 (L5) Ampicillin
S. typhimurium 11.5 (9.5) 9 (9) 9.5 (10) 9.5 (11.5) 10 (9.5) 16.0
E. aerogenes 13 (9.5) 11 (8) 9 (11) 8.5 (12) 13.5 (10.5) 15.0
S. epidermidis 10.5 (8) 12 (8.5) 10.5 (11.5) 8.5 (12.5) 8.5 (9.5) 16.0
A. faecalis 13 (9) 13.5 (9.5) 10.5 (10.5) 8.5 (9.5) 10 (12.5) 10.0
S. aureus 14 (8.5) 11 (9.5) 10.5 (8.5) 11 (12.5) 12.5 (12.5) 12.0
M. luteus 11 (9.5) 12.5 (11) 11 (9.5) 10 (9.5) 8.5 (8) 10.0
A. hydrophila 12 (9.5) 11.5 (9) 10 (9.5) 12.5 (13.5) 14.5 (8.5) 13.0
K. pneumoniae 9.5 (9.5) 10.5 (12.5) 13.5 (8.5) 10.5 (10.5) 10.5 (11) 11.0
P. aeruginosa 12.5 (11) 12.5 (10.5) 12.5 (14.5) 11.5 (10.5) 11.5 (15.5) 8.0
S. sonnei 11.5 (10) 8.5 (11.5) 8.5 (7) 10.5 (7.5) 9.5 (12.5) 13.0
Table 4. Minimum inhibitory concentrations (MIC, μg/L) of complexes 1–5 against pathogenic bacterial strains
Microbial species 1 2 3 4 5 Ampicillin
S. typhimurium 500.0 500.0 500.0 500.0 250.0 31.2
E. aerogenes 125.0 250.0 250.0 500.0 62.5 250.0
S. epidermidis 125.0 125.0 125.0 1000.0 1000.0 125.0
A. faecalis 62.5 62.5 125.0 500.0 500.0 500.0
S. aureus 31.2 125.0 125.0 500.0 62.5 125.0
M. luteus 250.0 125.0 250.0 250.0 500.0 250.0
A. hydrophila 31.2 250.0 500.0 125.0 31.2 31.2
K. pneumoniae 500.0 500.0 31.5 250.0 250.0 250.0
P. aeruginosa 62.5 62.5 62.5 125.0 125.0 62.5
S. sonnei 250.0 500.0 1000.0 500.0 62.5 31.2
the growth of A. faecalis, as well as M. luteus and K. magnitude than those of camptothecin taken as
pneumoniae, respectively, while complex 4 was less reference drug. The cytotoxicity of the complexes was
active than ampicillin against all the bacterial strains generally similar to or slightly higher than the
tested. cytotoxicity of the corresponding ligands. Complex 5
turned out to be the most active among the examined
The IC values of complexes 1–5 and the cor-
50 compounds against IMR-32 cancer cell line (IC =
responding ligands were calculated by using the best- 50
102 μM); however, it also showed an appreciable
fit regression model (Table 5). The cytotoxicity was
cytotoxicity toward CHO p-40 normal cell line (IC =
studied by in vitro MTT assay on IMR-32 (neuro- 50
180 μM against IC 467 μM for the free ligand).
blastoma) cancer and CHO p-40 (Chinese hamster 50
ovary) normal cell lines. The IC values of the In summary, five novel ruthenium(III) complexes
50
complexes and ligands with respect to both cancer and with hydroxamic acid ligands have been synthesized
normal cell lines were higher by at least an order of and characterized by spectroscopic data and elemental
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 86 No. 2 2016
In Vitro ANTICANCER AND ANTIBACTERIAL ACTIVITIES 365
Table 5. Cytotoxicity of ruthenium(III) complexes 1–5 and [Aquachlorobis(benzohydroximato-κO,κN)ruthe-
ligands L1–L5 against IMR-32 cancer and CHO normal cell nium(III)] (1). A solution of 0.1 g (0.418 mmol) of
lines benzohydroxamic acid in 20 mL of ethanol was added
under continuous stirring over a period of 2 h to a
IC , μM
50
Complex/ligand solution of 0.0547 g (0.209 mmol) of ruthenium(III)
IMR-32 CHO p-40
chloride. The mixture was heated for 16 h under
1/L1 166/218 225/596 reflux, and the reaction was assumed to be complete
when HCl no longer evolved and the color changed.
2/L2 221/251 198/249
The solution was kept overnight at 0°C in a refri-
3/L3 191/191 176/176 gerator, and the light reddish brown crystalline
material was filtered off, repeatedly washed with
4/L4 149/137 349/413
petroleum ether, recrystallized from ethanol, and dried
5/L5 102/121 180/466 in a vacuum desiccator over P O . 1H NMR spectrum,
2 5
Camptothecin 7.9 9.2 δ, ppm: 10.25 s (NOH), 7.58 t (1H), 7.32 q (2H), 7.95
d (2H), 3.22 s (OH). 13C NMR spectrum, δ , ppm:
C
126.21 (Co), 127.22 (Cm), 132.21 (Cp), 134.88 (Ci),
164.53 (C=N). Mass spectrum, m/z: 426.511 [M]+,
analyses. On the basis of the spectral data, coor- 408.284 [M – H O]+, 372.754 [M – H O – Cl]+,
2 2
dination of the ligands to ruthenium ion through the 236.724 [M – H O – Cl – C H NO ]+, 154.213 [M –
2 7 6 2
hydroxyl oxygen and nitrogen atoms or carbonyl C H N O ]+, 136.723 [C H NO ]+.
17 12 2 4 7 6 2
carbon atom and distorted octahedral geometry of the
Complexes 2–4 were synthesized in a similar way.
complexes have been proposed. Antibacterial activity
The yields, melting points, elemental analyses, and IR
of the synthesized complexes against A. hydrophila, S.
and UV spectra of 1–5 are given in Tables 1 and 2.
aureus, and K. pneumoniae has been revealed, and
their cytotoxicity with respect to IMR-32 cancer and [Aquachlorobis(2-hydroxybenzohydroximato-
CHO p-40 normal cell lines has been evaluated by the κO,κN)ruthenium(III)] (2). 1H NMR spectrum, δ,
MTT assay. The complexes are generally more active ppm: 12.50 s (NOH), 11.53 s (C 6 H 4 OH), 6.75 d (1H),
than the corresponding ligands. 7.11 t (1H), 7.29 q (1H), 7.81 d (1H), 3.42 s (OH). 13C
NMR spectrum, δ , ppm: 113.91 (C3), 121.21 (C5),
C
EXPERMENTAL 128.55 (C6), 132.56 (C4), 120.11 (C1), 156.78 (C2),
162.98 (C=N). Mass spectrum, m/z: 458.540 [M]+,
Ruthenium(III) chloride, benzohydroxamic acid, 440.531 [M – H 2 O]+, 405.551 [M – H 2 O – Cl]+,
salicylhydroxamic acid, acetohydroxamic acid, N- 253.421 [M – H 2 O – Cl – C 7 H 6 O 3 N]+, 154.591 [M –
hydroxyurea, and N-hydroxy-N-phenylbenzamide were C H O N]+, 152.130 [C H O N]+.
14 12 6 7 6 3
commercial products purchased from Aldrich and [Aquachlorobis(acetohydroximato-κO,κN)ruthe-
Merck and were used as received after checking their nium(III)] (3). 1H NMR spectrum, δ, ppm: 2.14 s
melting or boiling points. All reagents and solvents (CH ), 3.28 s (OH), 10.57 s (NOH). 13C NMR spec-
3
were of analytical grade and were purified by standard trum, δ , ppm: 13.89 (CH ), 165.98 (C=N). Mass spec-
C 3
procedures [32]. The FT-IR spectra were recorded in trum, m/z): 302.342 [M]+ [C H N O ClRu]+, 284.391
4 10 2 5
KBr on a Perkin Elmer 1600 spectrometer. The [M – H O]+, 248.807 [M – H O – Cl+], 174.811 [M –
2 2
electronic absorption spectra were measured in the H O – Cl – C H O N]+, 154.351 [M – C H O N ]+.
2 2 4 2 4 8 4 2
range λ 200–600 nm on a Perkin Elmer Lambda750
[Aquachlorobis(N′-hydroxycarbamimidato-
UV-Vis spectrophotometer from solutions in methanol.
κO,κN)ruthenium(III)] (4). 1H NMR spectrum, δ,
The 1H and 13C NMR spectra were recorded on Bruker
ppm: 3.35 s (OH), 5.54 s (2H, NH ), 10.20 s (NOH).
Avance II and JNM-ECS400 spectrometers at 400 and 2
13C NMR spectrum: δ 152.78 ppm (C=N). Mass spec-
100 MHz, respectively, from solutions in DMSO-d C
6 trum, m/z: 304.031 [M]+, 286.071 [M – H O]+, 250.502
using tetramethylsilane as internal standard. Electro- 2
[M – H O – Cl+], 175.527 [M – H O – Cl – CH N O ]+,
spray ionization mass spectra (ESI-MS) were obtained 2 2 3 2 2
154.341 [M – C H N O ]+.
on a Bruker micrOTOF-Q I0356 mass spectrometer; 2 6 4 4
sample solutions were infused directly at a rate of 5– [Aquatrichlorobis(N-hydroxy-N-phenylbenzamide-
30 μL/min. κO)ruthenium(III) (5). 1H NMR spectrum, δ, ppm:
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 86 No. 2 2016
366 RAJ KAUSHAL, SHEETAL
3.42 s (OH), 7.55 d (2H), 7.14 q (2H), 6.90 t (1H) nella typhimurium MTCC 1253. Complexes 1–5 were
(C H CO); 7.95 d (2H), 7.35 q (2H), 7.79 t (1H) dissolved in DMSO to a final concentration of
6 5
(C H N). 13C NMR spectrum, δ , ppm: 115.92 (Co′), 1 mg/mL. A loop full of the given test strain was
6 5 C
126.22 (Cm′), 123.58 (Cp′), 133.78 (Ci′), 158.66 (C=N), inoculated in 25 mL of N-broth (nutrient broth) and
125.21 (Co), 128.11 (Cm), 130.18 (Cp), 150.97 (Ci), incubated for 24 h at 37°C. A Petri dish (100 mm in
160.53 (C=O). Mass spectrum, m/z: 631.365 [M]+, diameter) was charged with 28–30 mL of the nutrient
633.346 [M – H O]+, 607.843 [M –H O – Cl+], agar medium which was inoculated by the pour-plate
2 2
394.830 [M – H O – Cl – C H NO ]+, 225.321 [M – technique with 0.1 mL of the activated strain at 40–45°C.
2 13 11 2
C H N O ]+, 213.461 [C H NO ]+. The complete procedure of the plate preparation was
26 22 2 4 13 10 2
done in a laminar airflow to maintain strict sterile and
Cytotoxicity assay. Preliminary in vitro cytotoxic
aseptic condition. The medium was allowed to
studies were performed using MTT assay [28] on
solidify, a well was made in the plates with the use of a
IMR-32 (neuroblastoma) cancer and CHO p-40
cup-borer (0.85 cm), and the well was filled with a test
(Chinese hamster ovary) normal cell lines obtained
sample solution. Pure solvent was used as control. The
from the ATCC (American Tissue Cell Culture
plates were incubated for 24 h at 37°C, and the
Collection) and maintained in Dulbecco’s Modified
inhibition zone was measured. Mean values from two
Eagle Medium containing 10% (v/v) of fetal bovine
runs for each bacterial strain and each complex
serum (FBS), 1% (v/v) of penicillin or streptomycin,
(ligand) were calculated.
and 1% (v/v) of L-glutamine. The cell cultures were
seeded in 96-well plates containing 200-μL wells at a
ACKNOWLEDGMENTS
density of 5000 cells per 200 μL of medium and were
incubated for 24 h at 37°C to allow their exponential
The authors acknowledge the contribution of Prof
growth. Complexes were dissolved in a minimum
Saroj Arora, Department of Botanical and Enviro-
amount of DMSO to obtain stock solutions with a
mental Sciences, Guru Nanak Dev University,
concentration of 5 × 10–3 M. The cells were then
Amritsar, Punjab, India and Prof Kiran Nehra, Depart-
treated with varying concentrations of the complexes
ment of Biotechnology, Deenbandhu Chhotu Ram
and incubated for 48 h at 37°C. The solutions were
University of Science and Technology, Murthal,
then removed from the wells, the cells were washed
Sonipat (Haryana), India for providing necessary
with a phosphate buffer, and fresh medium was added
facilities to carry out in-vitro analyses.
to the wells. After incubation for 24 h at 37°C,
individual wells were treated with 200 μL of a solution
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