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Synthesis, characterization, anticancer and antimicrobial study of arene ruthenium(II) complexes with 1,2,4-triazole ligands containing an α-diimine moiety
Z. Naturforsch. 2018; aop
Joel M. Gichumbi, Holger B. Friedrich*, Bernard Omondi, Geraldine G. Lazarus,
Moganavelli Singh and Hafizah Y. Chenia
Synthesis, characterization, anticancer and antimicrobial study
of arene ruthenium(II) complexes with 1,2,4-triazole ligands
containing an α-diimine moiety
https://doi.org/10.1515/znb-2017-0145 further investigated for its antimicrobial activity against
Received September 15, 2017; accepted October 15, 2017 six Gram-positive and four Gram-negative bacteria.
Abstract: The reaction of the ruthenium arene dimers [(η6- Keywords: anticancer; antimicrobial susceptibility; arene
arene)Ru(μ-Cl)Cl] (where arene = benzene or p-cymene) ruthenium; crystal structure; MTT assay; triazole ligands.
2
with the ligands 4-benzylidene-3,5-di(2′-pyridyl)-
4-amino-1,2,4-triazole (L1), 2-methoxybenzylidene-
3,5-di(2′-pyridyl)-4-amino-1,2,4-triazole (L2),
4-methylbenzyli dene-3,5-di(2′-pyridyl)-4-amino-1,2,4- 1 Introduction
triazole (L3) and indole-3-carbaldehyde-3,5-di(2′-pyridyl)-
4-amino-1,2,4-triazole (L4) in a 1:2 ratio gives the new Medicinal inorganic chemistry is a field which is experi-
complexes [(η6-arene)RuCl(L)]+ [arene = CH (with L = L1(1), encing fast growth, increasing prominence and fascinat-
6 6
L2(3), L4(7), with PF− as a counter ion, and L4 (6), with Cl− ing opportunities enabled by the design and tuning of
6
as a counter ion) or p-cymene with L = L1(2), L2(4), L3(5), the metal-based compounds as therapeutic agents [1].
L4(8) with PF− as a counter ion]. All complexes were fully Application of inorganic compounds in cancer treatment
6
characterized using 1H and 13C NMR, elemental analyses, has particularly received wide attention [2] due to the
UV/Vis and IR spectroscopy. The single crystal X-ray struc- increasing global burden of cancer, largely because of the
tures of ligand L2 and complex 1 have been determined. aging and growth of the world population, alongside an
The structure of 1 has the Ru atom coordinated with the increasing adoption of cancer-causing behavior, particu-
arene group and to the N,N′-bidentate ligand and to the Cl larly smoking [3]. Cisplatin is the most widely used drug
atom. The arene group occupies the apex, while the ligand for cancer treatment [4], but in spite of its wide applicabil-
and the Cl atom are at the base of a pseudo-octahedral ity, it is associated with high toxicity which leads to bad
three-legged piano stool. The cytotoxicity of these mono- side effects, and the cancer can develop resistance to it [2].
nuclear complexes was established in the human epithe- Currently, efforts are targeted at developing alternatives
lial colorectal adenocarcinoma cell line (Caco-2) and for to platinum-based drugs, with improved aspects which
selectivity in the non-cancerous human embryonic kidney include oral administration, lower side effects and lower
cell line (HEK293), using 5-fluorouracil (5-FU) as the refer- costs [5].
ence anticancer drug. Compounds 1 and 7 were relatively Within this context, compounds based on a number
inactive toward the Caco-2 tumor cells (IC > 200), while of transition and non-transition metals are being investi-
50
complexes 2–5 showed moderate anti-proliferative prop- gated [6]. Amongst these, ruthenium appears to be very
erties (IC > 100–200). Compound 6, however, displayed promising because it offers low toxicity, variable oxida-
50
better anti-proliferative properties with an IC value lower tion states and selectivity toward cancer cells and mimics
50
than that of the reference drug, 5-FU, and was therefore the binding of iron to biomolecules [7]. A number of ruthe-
nium complexes have been shown to have high in vivo and
in vitro activity, with KP1019 and NAMI-A perhaps being
the most promising potential next-generation anticancer
*Corresponding author: Holger B. Friedrich, School of Chemistry
and Physics, University of KwaZulu-Natal, Private Bag X54001, drugs based on clinical trials [2]. In addition, preliminary
Durban 4000, South Africa, e-mail: friedric@ukzn.ac.za clinical studies suggest that KP1019 has potential in the
Joel M. Gichumbi and Bernard Omondi: School of Chemistry treatment of drug-resistant tumors, since it remains effec-
and Physics, University of KwaZulu-Natal, Private Bag X54001,
tive against cancer cell lines that are very resistant to the
Durban 4000, South Africa
anticancer drugs doxorubicin and adriamycin [8]. NAMI-A
Geraldine G. Lazarus, Moganavelli Singh and Hafizah Y. Chenia:
has been shown to specifically target tumor metasta-
School of Life Sciences, University of KwaZulu-Natal, Private Bag
X54001, Durban 4000, South Africa ses, preventing both development and growth, and has
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2 J.M. Gichumbi et al.: Synthesis, characterization, anticancer and antimicrobial study of arene ruthenium(II)
significantly greater activity than cisplatin on these cell dinucleating ligands that should be able to bridge two
types [9]. metal ions by means of the N unit of the central tria-
2
Ruthenium half-sandwich complexes have also zole ring [23]. This ligand is potentially an ideal starting
been shown to exhibit excellent anticancer proper- material for the synthesis of more complex ligands by
ties, and research involving these is ongoing [2, 10]. Dif- derivatization of the 4-amino group. This strategy was ini-
ferent researchers have shown that in half-sandwich tially explored by Lagrenee et al. by reacting the triazole
ruthenium(II) arene compounds, the type of arene and of with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone [24]. The
the ancillary ligands plays a very important role in deter- ligand has also been derivatized to give various substitu-
mining the anticancer activity of the compounds [11]. ents on the 4-amino group [25].
Thus, a possibility exists for fine-tuning their pharmaco- The therapeutic importance of 1,2,4-triazole and the
logical properties by systematic variation of the substitu- continuing interest in half-sandwich ruthenium(II) arene
ents [5]. In this work we have varied the arene ring and compounds in anticancer and antimicrobial studies
used some triazole ligands. led us to investigate some reactions of [(η6-arene)Ru(μ-
There are many studies on the application of ruthe- Cl)Cl] (where the arene is benzene or p-cymene) with
2
nium in anticancer applications; however, there have ligands formed from derivatization of the 4-amino group.
been significantly fewer studies on the antimicrobial All the compounds of the general formula [(η6-arene)
properties of ruthenium [12–15]. The development of RuCl(L)]+ [arene = CH with L = L1(1), L2(3), L4(6, 7 – the
6 6
antimicrobials has been one of the major advances in counter ions differ) or p-cymene with L = L1(2), L2(4),
medical science [13, 15]. However, there has been the L3(5), L4(8)] [where L is 4-benzylidene-3,5-di(2-pyridinyl)-
emergence of drug-resistant populations of microorgan- 4-amino-1,2,4-triazole (L1), 2-methoxybenzylidene-
isms [13]. Infection by such drug-resistant pathogens has 3,5-di(2′-pyridyl)-4-amino-1,2,4-triazole (L2),
become a considerable cause of morbidity and death 4-methylbenzylidene-3,5-di(2′-pyridyl)-4-amino-1,2,4-
worldwide [13]. There is, therefore, the need for the triazole (L3) and indolyl-3-carbaldehyde-3,5-di(2′-pyridyl)-
development of new classes of antimicrobial compounds 4-amino-1,2,4-triazole (L4)] are new and were fully
rather than drugs necessarily based on analogues of characterized. Furthermore, the structures of complex 1
known scaffolds [13]. and ligand L2 were confirmed by single crystal X-ray crys-
Triazoles are a class of heterocyclic compound con- tallography. These complexes were then used in antican-
taining an aromatic five-membered ring system contain- cer studies against Caco-2 (human epithelial colorectal
ing three nitrogen atoms, which can be arranged in two adenocarcinoma). To test their selectivity, the compounds
different ways to give either 1,2,3-triazole or 1,2,4-tria- were also tested against non-cancerous human embry-
zole [16]. 1,2,4-Triazoles and their heterocyclic deriva- onic kidney (HEK 293) cells. In addition, complex 6 was
tives represent an interesting class of compounds with a tested against four Gram-positive and six Gram-negative
range of biological activities. A large number of 1,2,4-tri- bacteria.
azoles containing ring systems exhibit a wide spectrum
of chemotherapeutic activities, including antibacterial,
antifungal, antitubercular, analgesic, anti-inflamma-
2 Results and discussion
tory, anticonvulsant, antiviral, insecticidal and anti-
depressant activities [17–20]. In anticancer studies, the
therapeutic importance of 1,2,4-triazoles is well docu- 2.1 Synthesis and characterization
mented in drugs such as vorozole, letrozole and anas- of ligands
trozole, which contain a substituted 1,2,4-triazole ring.
These are currently being used for breast cancer treat- Ligands L1–L4 were synthesized in excellent yields by
ment [18]. the reaction of the respective aldehyde with the 4-amino
1,2,4-Triazole and its derivatives have gained attention group of 3,5-di(2-pyridyl)-4H-1,2,4-triazole in refluxing
as ligands because they combine the coordination chem- methanol using concentrated sulfuric acid as the catalyst
istry of pyrazoles and imidazoles and also show a strong (Fig. 1). L3 and L4 are new and all ligands were character-
property of acting as bridging ligands between two metal ized using FTIR, 1H and 13C NMR spectroscopy, elemental
centers [21]. There are very few reports of DNA targeting analyses and high-resolution mass spectra. Single crystal
triazoles based on dinuclear transition metal complexes X-ray diffraction for ligand L2 was also carried out.
[22]. The 4-substituted 3,5-di(2-pyridyl)-4H-1,2,4-tria- The 1H NMR spectra display a singlet at δ = 9.00–
zoles are bis-bidentate; hence, they have potential as 8.86 ppm for the proton bonded to the azomethine carbon,
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J.M. Gichumbi et al.: Synthesis, characterization, anticancer and antimicrobial study of arene ruthenium(II) 3
2.2 The synthesis and characterization
of complexes 1–8
[(η6-arene)Ru(μ-Cl)Cl] (where the arene is benzene or
2
p-cymene) react with the N,N′ ligands L1–L4 in methanol at
room temperature to afford the mononuclear complexes
1–8 (Fig. 3). The complexes were isolated as their hex-
Fig. 1: Synthesis of L1 to L4.
afluorophosphate salts, except for 6, which was isolated
as the chloro salt. These new complexes were obtained in
good yields and were characterized with FTIR, 1H and 13C
and the 13C NMR spectra show the resonance of the azome-
NMR spectroscopy, high-resolution mass spectra and ele-
thine carbon between 165 and 166 ppm. In the IR spectra,
mental analyses. All complexes were mononuclear irre-
there are sharp bands at 1630–1673 cm−1 corresponding to
spective of the ligand-to-metal ratio and were obtained as
the asymmetric stretching of the azomethine C=N bond,
orange or brown non-hygroscopic solids soluble in polar
indicating the successful condensation between the alde-
solvents such as acetonitrile, acetone and dimethyl sulph-
hyde and the amine. The UV/Vis data of the ligands (Fig. 2)
oxide (DMSO).
obtained in acetonitrile showed absorption bands in the
The formation of complexes 1–8 was confirmed by 1H
region 195–291 nm attributed to the ligand-centered n–π*
and 13C NMR, where the proton and carbon peaks of the
and π–π* transitions, in agreement with similar reported
(CH=N) group of the ligand and the complex were moni-
ligands [26].
tored. The proton of the (CH=N) group moves downfield
to around δ = 9.01–9.20 ppm from the region of δ = 8.59–
8.64 ppm of the free ligand, due to deshielding of the
proton of the (CH=N) group when the nitrogen atoms
coordinate with the ruthenium. The 1H NMR spectra for
complexes 2, 4, 5 and 8 suggest a loss of the two-fold sym-
metry of the p-cymene arene ligand upon coordination of
the metal with the ligands as evidenced by the 1H NMR
resonances of the i-propyl group of the p-cymene ligand
with its diastereotopic methyl hydrogens and carbons.
This shows that the complexes are chiral. In the 13C NMR
spectra, the carbon peak of the (CH=N) group shifted
from the region δ = 149.8–158.3 ppm for the free ligands
to around 150.0–160.5 ppm. For the [(η6-CH)RuCl(L)]
6 6
Fig. 2: UV/Vis spectra of ligands L1–L4 in acetonitrile. PF complexes, the CH singlet is shifted downfield on
6 6 6
R
Ar +
Ru Cl
R Cl N MeOH N N N N
Ru 2
N
+ 2
Cl Cl N
Ru N N N N N
Cl R
Ar
R = Ar = phenyl (1); 2-methoxyphenyl (3); 3-indole (6); 3-indole (7)
R = Ar = phenyl (2); 2-methoxyphenyl (4); 4-methylphenyl (5); 3-indole (8)
NB = 3-indole (6) has Cl– as the counter ion, the others PF –
6
Fig. 3: Preparation of complexes 1–8.
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4 J.M. Gichumbi et al.: Synthesis, characterization, anticancer and antimicrobial study of arene ruthenium(II)
Fig. 4: UV/Vis spectra of 1–8 in acetonitrile.
coordination with the bidentate ligand, when compared These assignments support those by other workers study-
to the starting material dimer. ing complexes with N,N′-bidentate ligands [31].
Furthermore, in the IR spectra of all compounds 1–8,
a strong absorption band is seen within the region 1573–
1590 cm−1. This band is due to the symmetrical vibration 2.3 Molecular and crystal structure
of the pyridine μ(CH=N) group, which is shifted to lower determination
wavenumbers than those of the uncoordinated triazole
ligand (1585–1598 cm−1). This indicates that these ligands The molecular structures of L2 and 1 are given in Figs. 5
are bonded to the ruthenium center through the pyridine and 6, while selected bond distances and angles are
nitrogen atom. Also apparent is a strong peak, seen for all shown in Table 1. L2 crystallizes with one molecule in the
of the compounds (except 6, which has Cl− as a counter asymmetric unit, while complex 1 crystallizes with three
ion) between 828 and 841 cm−1, which can be assigned to components, the cation [(η6-CH)RuCl(N–N)]+ (where
6 6
the PF− counter ion [27–29]. N–N = ligand), the counter anion PF− and a molecule of
6 6
The high-resolution mass spectra of 1–8 also confirm
that the mononuclear complexes were formed. The com-
pounds show the molecular ion of the N,N′-bidentate
complex [(η6-arene)RuCl(L)]+, with the expected pattern
characteristic of the isotopes of ruthenium.
The UV/Vis spectra of complexes 1–8 were acquired
in acetonitrile as depicted in Fig. 4. The spectra are char-
acterized by an intense ligand localized π–π* transition
in the ultraviolet region, and a metal-to-ligand charge
transfer (MLCT) (dπ–π*) band in the visible region. The
MLCT bands are due to the low spin d6 configuration of the
ruthenium(II) providing filled orbitals of proper symmetry.
These can interact with low lying π* orbitals of the biden-
tate ligands. The absorption bands in the visible region at
420–431 nm and 350–370 nm can be assigned to the MLCT
transitions [30]. These peaks are absent in the UV/Vis
spectra of the ligands. The intense bands at 257–275 nm
and 300–312 nm can be assigned to the ligand-centered
n–π* and π–π* transitions. There is a bathochromic shift Fig. 5: Ortep view of L2; displacement ellipsoids are drawn at a 50%
of the ligand absorption from 195–255 nm to 255–291 nm. probability level, with the hydrogen atoms omitted for clarity.
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J.M. Gichumbi et al.: Synthesis, characterization, anticancer and antimicrobial study of arene ruthenium(II) 5
In the complex, the ruthenium atom is coordinated by
the N,N′-ligand and a chlorine atom and also to the arene
ring such that the overall structure can be described as a
“three-legged piano stool” (arene as apex and bidentate
ligand and chlorine atom as the legs), while the geometry
around the Ru(II) center is classical pseudo-octahedral.
The Ru–N bond distances are 2.051(6) and 2.157(5) Å
pyr
and are similar to those reported for related compounds
[29–33]. The N –Ru–N bond angle is 77.9(2)°, while
pyr py
the N –Ru–Cl angles are 85.81(14)° and 84.65(15)°, also
py
similar to those of related cationic complexes [29–33].
2.4 Stability of the complexes in different
solvents
Fig. 6: Ortep view of complex 1; displacement ellipsoids are drawn
at the 50% probability level, with the hydrogen atoms omitted for The stability of the metal-based complexes in solution is
clarity. important for determining their mode of action [34–37].
This is because the accepted mode of action for mononu-
clear complexes that contain a chloride ligand involves
Table 1: Selected bond lengths (Å) and angles (deg) for L2 and the aquation of the M–Cl bond to an intermediate M-aqua
complex 1. species which goes on to interact with DNA base pairs or
with proteins to form adducts [34]. Thus, the time-depend-
L2 1
ent hydrolysis of complex 3 in a [D]DMSO-DO (20:80)
6 2
Bond lengths solvent system was monitored by 1H NMR spectroscopy
C(2)–N1 1.3469 (13) Ru(1)–N(1) 2.1123 (17) and was taken as representative (Fig. S1; Supplementary
N(4)–N(5) 1.4170 (11) Ru(1)–N(2) 2.0523 (17)
Information). The presence of DMSO ensured the solubil-
N(2)–N(3) 1.3843 (13) Ru(1)–Cl(1) 2.3959 (16)
ity of the complexes. The complex was found to undergo
C(16)–N(16) 1.343 (14)
rapid hydrolysis. Equilibrium was reached by the first
N(5)–C(8) 1.2801 (12)
Bond angles NMR spectrum acquired in 5 min.
C(6)–N(1)–C(1) 116.45 (9) N(1)–Ru(1)–N(2) 76.11 (7) To confirm the hydrolysis of the complexes, NaCl
C(1)–N(4)–C(7) 105.70 (8) N(1)–Ru(1)–Cl(1) 84.12 (5) was added to the equilibrium solutions of the chloride
C(1)–N(2)–N(3) 107.76 (8) N(2)–Ru(1)–Cl(1) 85.31 (5)
complex and its aqua adduct. 1H NMR spectra were then
C(16)–N(6)–C(20) 116.87 (10)
recorded within 10 min of the NaCl addition. Upon the
addition of the NaCl, the 1H NMR peaks corresponding to
the chloride complexes increased in intensity, while the
acetonitrile, in the asymmetric unit. In the structure of peaks for the aqua adducts decreased (Fig. S1). This con-
L2, the three six-membered rings are twisted away from firms the formation of the aqua adducts and the revers-
the plane of the five-membered triazole ring. While the ibility of the process.
two pyridyl rings are only twisted by angles of 30.55(4)
and 38.57(4)° for the triazole ring, the methoxy substi-
tuted phenyl ring is almost orthogonal to the triazole 2.5 MTT cytotoxic tests results
ring, the dihedral angle between the two being 88.25(3)°.
The conformation of the ligand in complex 1 changes on The cytotoxicity tests for complexes 1–8 and the ligand
complexation and sees the pyridyl ring that participates L1 were carried out using the 3-(4,5-dimethyl-2-thiazolyl)-
in coordination to the Ru(II) center being coplanar with 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay on
the triazole moiety. The dihedral angle of the non-coordi- human ovarian cancer cell lines Caco-2 (human epithe-
nating ring to the coordinating moiety (triazole plus one lial colorectal adenocarcinoma). In order to assess the
pyridyl ring) is now only 20.78(7)°, whereas that of the selectivity of the compounds toward cancer cells rather
coordinating ring to the methoxy-substituted phenyl ring than normal cell lines, the antiproliferative effects on the
is halved to about 44.96(6)°. human embryonic kidney (HEK-293T, a model for healthy
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6 J.M. Gichumbi et al.: Synthesis, characterization, anticancer and antimicrobial study of arene ruthenium(II)
cells) cell lines were determined. In addition, for compari- more so than the known anticancer drug 5-FU, which was
son purposes, the cytotoxicity of 5-fluorouracil (5-FU), a not selective between the normal cell lines and the tumor
commercial anticancer drug, was evaluated under the cell lines (Table 2).
same conditions (Table 2). Compounds 1 and 8 were essentially inactive toward
In comparison to the free ligand L1, the ruthenium the Caco-2 tumor cells (IC > 200), complexes 2–5 and 7
50
arene complexes demonstrated a higher antiproliferative had low antiproliferative properties (IC > 100), while
50
activity against Caco-2. In addition, the compounds’ cyto- compound 6 had moderate antiproliferative properties.
toxicity was lower for non-tumor cell lines when compared Furthermore, compound 6 has a lower IC value than the
50
to that toward the cancer cell lines (Caco-2). This indicates reference drug 5-FU.
that the compounds were selective to the tumor cells,
2.6 Antimicrobial susceptibility tests
Table 2: In vitro cytotoxic effect of ruthenium compounds 1–8.
Complex 6, which showed promising antiproliferative
Compound no IC 50 (μm)a properties, and the ligand (Ligand L1) were investigated
HEK293 Caco-2 for their antimicrobial activity against six Gram-positive
and four Gram-negative bacteria (Table 3). Also, the activi-
L1 206 ± 16 300 ± 2
1 444 ± 4 250 ± 7 ties of these compounds in the inhibition of these bacte-
2 252 ± 3 134 ± 25 ria were compared to the established antibacterial drugs,
3 185 ± 17 106 ± 35 ampicillin (AMP10) and tetracycline (TE30). The ligand
4 151 ± 32 135 ± 15
did not demonstrate any antimicrobial activity against the
5 414 ± 12 146 ± 5
panel of bacterial strains tested. The complex showed an
6 245 ± 9 50 ± 12
7 256 ± 12 194 ± 15 intermediate activity at 40 μL for the Gram-positive bac-
8 607 ± 10 203 ±3 teria tested, but no activity against Mycobacterium smeg-
5-Fub 47 ± 1 74 ± 5 matis mc2155. In addition, for the Gram-negative bacteria,
the complex showed an intermediate activity against
aIC value corresponds to the concentration of the respective com-
50
pound required to affect 50% mortality in net cells. b5-Fluorouracil Escherichia coli ATCC 35218 and Klebsiella pneumoniae
(5-Fu) was used as a reference drug. ATCC 700603, but no activity against E. coli ATCC 25922.
Table 3: Antimicrobial susceptibility test results of complex 6 and ligand L1 with zones of inhibition given to the nearest mm.
Compound Gram-positive bacteria
(20 mg mL−1)
B. subtilis E. faecalis S. aureus S. aureus S. saprophyticus M. smegmatis
ATCC 6653 ATCC 51299 ATCC29213 ATCC 43300 ATCC 35552 mc2155
10 20 40 10 20 40 10 20 40 10 20 40 10 20 40 10 20 40
Ligand 1 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R)
Compound 6 0 (R) 0(R) 12(I) 0 (R) 0 (R) 10(I) 0 (R) 0 (R) 12 (I) 0 (R) 0 (R) 10 (I) 0 (R) 0 (R) 12 (I) 0 (R) 0 (R) 0 (R)
DMSO 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R)
Tetracycline 36 (S) 0 (R) 0 (R) 0 (R) 28 (S) 36 (S) 26 (S) na
(TE30) – 30 μg
Ampicillin 40 (S) 25 (S) 25 (S) 20 (S) 11 (R) na
(AMP10) – 10 μg
Compound Gram-negative bacteria
E. coli ATCC 25922 E. coli ATCC 35218 K. pneumoniae P. aeruginosa
ATCC 700603 ATCC 27853
10 20 40 10 20 40 10 20 40 10 20 40
Ligand 1 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R)
Compound 6 0 (R) 0 (R) 0 (R) 0(R) 0(R) 12(I) 0(R) 0 (R) 10 (I) 0(R) 0 (R) 7 (R)
DMSO 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R) 0 (R)
Tetracycline (TE30) – 30 μg 27 (S) 23 (S) 12 (R) 15 (I)
Ampicillin (AMP10) – 10 μg 20 (S) 0 (R) 0 (R) 0 (R)
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Of interest is the activity shown by the complex against ionization (ESI) was in positive mode with acetonitrile
the β-lactam resistant E. coli ATCC 35218 (TEM-containing as the mobile phase. The sample (10 μL) was injected at
strain) and sulfhydryl variable-containing K. pneumoniae a flow rate 0.3 mL min−1. Electronic spectra were deter-
ATCC 700603, which are Gram-negative and show resist- mined in acetonitrile using a Perkin-Elmer LAMBDA 35
ance to antimicrobials. UV/Vis spectrophotometer. 1H and 13C NMR spectra were
obtained using a Brucker Top Spin 400 MHz spectrometer
in deuterated [D]DMSO from Sigma-Aldrich. An Ernst-
6
3 Conclusions
Leitz-Wetzlar hot stage microscope was used to obtain
the melting points which are uncorrected. The dimers
To summarize, a new series of cationic mononuclear com- [(η6-arene)Ru(μ-Cl)Cl], where the arene is benzene or
2
plexes were prepared from a dimeric ruthenium arene p-cymene, were synthesized by published methods [38].
precursor and a series of dipyridyl-triazole ligands. The The ligands 3,5-di(2′-pyridyl)-4-amino-1,2,4-triazole and
complexes were characterized using a number of spec- 4-benzylidene-3,5-di(2′-pyridyl)-4-amino-1,2,4-triazole
troscopic and analytical techniques. The structures of (L1) were synthesized according to the reported method
ligand L2 and complex 1 have been determined. Complex [26]. In addition, the ligand 4-methylbenzylidene-3,5-
1 displayed a pseudo-octahedral three-legged piano stool di(2′-pyridyl)-4-amino-1,2,4-triazole was also synthesized
geometry. Cytotoxicities of these mononuclear complexes according to literature procedures [25].
were established against the cancer cell line Caco-2 and
non-cancer cell line HEK293. Compounds 1 and 8 were
4.1 In vitro anticancer activity
least active against the Caco-2 tumor cells (IC > 200),
50
complexes 2–5 showed moderate anticancer activities
4.1.1 Chemicals
(IC > 100), while compound 6 produced the highest
50
anticancer activity, with an IC value lower than that of
50 Eagle’s minimum essential medium (EMEM) with
the control standard drug 5-Fu. Furthermore, compound
l-glutamine (4.5 g L−1), trypsin-versene mixture and anti-
6 displayed cell specificity showing lower activity in
biotic was purchased from Lonza BioWhittaker (Verviers,
the normal cell line compared to that of the cancer cell
Liège, Belgium). MTT reagent, phosphate buffered saline
line, suggesting its potential as a possible anticancer
(PBS) tablets and DMSO were purchased from Merck.
compound. Complex 6, which showed promising anti-
Fetal bovine serum (FBS) was purchased from Hyclone
proliferative properties, and the ligand (Ligand L1) were
GE Healthcare (UT, USA). All tissue culture consumables
investigated for their antimicrobial activity against six
were obtained from Corning Incorporated (New York City,
Gram-positive and four Gram-negative bacteria. The activ-
USA). All other chemicals and reagents were of analytical
ity shown by the complex against the β-lactam resistant
grade. Ultrapure deionized 18 MΩ water (Milli-Q50) was
E. coli ATCC 35218 and extended-spectrum β-lactam resist-
used throughout.
ant K. pneumoniae ATCC 700603 was noteworthy.
4.1.2 Cell culture
4 Experimental section
Caco-2 (human epithelial colorectal adenocarcinoma) cell
All manipulations were done under nitrogen with stand- lines were maintained at 37°C under 5% CO, in culture
2
ard Schlenk techniques. Acetonitrile (Merck, Darm- flasks containing 5 mL of complete medium [EMEM sup-
stadt, Germany) was dried over PO , diethyl ether was plemented with 10% (v/v) gamma-irradiated FCS and
4 10
distilled over sodium/benzophenone, while Mg/I was antibiotic (100 μg mL−1 penicillin, 100 μg mL−1 streptomy-
2
used to dry ethanol. The chemicals 1,4-cyclohexadiene, cin, 0.25 μg mL−1 amphotericin B)].
α-phellandrene, 2-pyridinecarbonitrile, 2-methoxy ben-
zaldehyde, 3-indole carboxaldehyde and 4-methyl ben-
zaldehyde were bought from Sigma-Aldrich and used as 4.2 Cytotoxicity tests
supplied. The elemental analyses were obtained using a
Thermal-Scientific Flash 2000 CHNS/O instrument. The 4.2.1 MTT assay
IR spectra were obtained in the solid state with a Perkin
Elmer Spectrum 100 ATR spectrophotometer. A Waters Originally, the human embryonic kidney (HEK293) cells
Micromass LCT Premier TOF-MS was used to obtain were obtained from the Anti-viral Gene Therapy Unit, at
the mass spectra by direct infusion. The electrospray the Medical School of the University of the Witwatersrand,
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SA. The human colorectal adenocarcinoma cells ( Caco-2) Pseudomonas aeruginosa ATCC 27853) and six Gram posi-
were obtained from Highveld Biologicals (Pty) Ltd, SA. tive (Bacillus subtilis ATCC 6633, Enterococcus faecalis
These cells were grown to semi-confluency in tissue ATCC 51299, M. smegmatis mc2155, Staphylococcus aureus
culture flasks (25 cm2) in EMEM (Lonza BioWhittaker, ATCC 29213, S. aureus ATCC 43300 and Staphylococcus
Belgium) which was supplemented with antibiot ics saprophyticus ATCC 35552) bacterial strains, which were
(100 μg mL−1 penicillin, 100 μg mL−1 streptomycin) and grown on tryptic soy agar plates overnight, were resus-
10% FBS. The cells were seeded at a density of 1.8 × 103 pended in sterile distilled water. Subsequently, the turbid-
cells per well in a 96-well plate which contained 100 μL ity of the cell suspensions was adjusted to be equivalent
of medium. These cells were subsequently incubated for to that of a 0.5 McFarland standard. Mueller-Hinton agar
24 h at 37°C in 5% CO. The medium was then removed plates were then inoculated with these suspensions by
2
and 100 μL of fresh medium added. Compounds 1–8 at streaking swabs over the entire agar surface. Thereafter,
concentrations of 50, 100, 150 and 200 μg mL−1 were then the respective compounds were applied to the disks [40].
added to the cells in triplicate and incubated for 48 h at The plates were subsequently incubated at 37°C for 18 h,
37°C. Cells given no test compounds were used as positive except M. smegmatis mc2155 plates which were incubated
controls (100% viability). at 37°C for 48 h. The testing was carried out in duplicate
The MTT assay methodology was based on the method and the standard antimicrobial agent controls were tet-
reported by Mosmann [39]. Thus, the metabolic activity of racycline (TE30) and ampicillin (AMP10) disks (Oxiod,
the cells was measured by reduction of MTT to formazan UK), while DMSO-impregnated disks were evaluated and
using the succinate-tetrazolium reductase system. Once averaged. The zone diameter criteria used for assigning
incubated for 48 h, the used medium was replaced with resistance or susceptibility to the complexes investigated
100 μL fresh medium and 100 μL of MTT (5 mg mL−1 in were as follows: susceptible (S) ≥ 15 mm, intermediate
PBS). The cells were then further incubated at 37°C for 4 h, (I) = 11–14 mm and resistant (R) ≤ 10 mm. The following
after which the MTT and the medium were removed. DMSO criteria were used for assigning resistance or susceptibil-
(200 μL) was then added to each well which dissolved the ity to AMP10: (S) ≥ 17 mm, (I) = 14–16 mm and (R) ≤ 13 mm.
formazan salt, giving a purple solution. The absorbance The criteria used for TE30 were as follows: (S) ≥ 19 mm,
of this solution was determined using a Mindray 96A (I) = 15–18 mm and (R) ≤ 14 mm [30].
microplate reader (Vacutec, Germany) at wavelengths of
570 nm (detection λ) and 630 nm (reference λ for nonspe-
cific signals). The cell viability (%) was directly correlated
4.4 General procedure for the synthesis
with absorbance and was calculated in comparison to the
of the ligands, N-benzylidene-3,5-di(2′-
untreated control as shown:
pyridyl)-4-amino-1,2,4-triazole
[(OD Treated–OD Treated)/
570 630
(OD Control–OD Control)]×100. 3,5-di(2′-Pyridyl)-4-amino-1,2,4-triazole (2 g, 8.4 mmol),
570 630
the respective aldehyde (8.6 mmol) and one drop of HSO
2 4
All the tests were carried out in triplicate and Micro- were dissolved in methanol (20 mL). The mixture was
soft Excel 2010™ was used for the calculations of the con- heated to reflux for 6 h under nitrogen. The solution was
centration at which 50% cell death occurred (IC ). then cooled and the solvent concentrated under reduced
50
pressure to about 2 mL. The product was precipitated by
the addition of diethyl ether. The precipitate was filtered,
washed with ether, recrystallized from ethanol and dried
4.3 E valuation of antimicrobial activity
under vacuum.
by disk diffusion assay
The disk diffusion method was used to determine the
antimicrobial susceptibility to the compounds [40]. Thus, 4.4.1 2-Methoxybenzylidene-3,5-di(2′-pyridyl)-4-amino-
stock solutions of the complexes (20 mg) were made up 1,2,4-triazole (L2)
in DMSO (1 mL). Sterile blank disks (6 mm; MAST, UK)
were impregnated with, respectively, 10 μL (0.2 mg), M.p. 150.2°C – UV/Vis (MeCN): λ = 292 nm. – IR (KBr,
max
20 μL (0.4 mg) and 40 μL (0.8 mg) of these stock solutions cm−1): ν = 1673.5 (CH=N). – 1H NMR (400 MHZ, [D]DMSO,
6
and left to dry for 1 h. Four Gram-negative (E. coli ATCC 25°C, TMS): δ = 9.00 (s, 1H, CH=N), 8.61–8.59 (m, 2H, Py),
25922, E. coli ATCC 35218, K. pneumoniae ATCC 700603, 8.09–8.06 (m, 2H, Py), 8.02–8.00 (m, 2H, Py), 7.98–7.83
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(m, 1H, Py), 7.59–7.55 (m, 1H, Py), 7.51–7.48 (m, 2H, Ar), 7.15 1H, Ar), 7.40 (s, 3H, Ar), 6.21 (s, 6H, CH). – 13C NMR ([D]
6 6 6
(dd, 2J = 8.2 Hz, 1H, Ar), 7.06 (t, J = 7.48 Hz, 1H, Ar), 3.75 DMSO): δ = 173.11 (CH=N), 157.16 (Py), 149.44 (Py), 149.44
HHH
(s, 3H, OCH). – 13C NMR ([D]DMSO): δ = 165.17 (CH=N), (Py), 144.2 (Py), 143.49 (Py), 140.67 (Py), 138.24 (Py),
3 6
159.21 (Py), 149.84 (Py), 149.38 (Py), δ 146.33 (Py), 137.22 134.11 (Py), 130.65 (Pyra), 129.7 (Pyra), 129.46 (Ar), 128.28
(Py), 134.61 (Py), 127.32 (Pyra), 124.45 (Pyra), 120.89 (Ar), (Ar), 127.52 (Ar), 126.21 (Ar), 124.97 (Ar), δ 124.54 (Ar),
119.89 (Ar), 112.30 (Ar), 55.89 (–OCH). – HRMS ((+)ESI): 86.06 (CH). – HRMS ((+)ESI): m/z = 541.05 (calcd. 541.05
3 6 6
m/z = 379.1295 (calcd. 379.1278 [C H NONa]+, [M + Na]+). – [C H ClNRu]+, [M–PF]+). – Anal. calcd. for [C H ClNRu]
20 16 6 25 20 6 6 25 20 6
Anal. calcd. for C H NO: C 67.40, H 4.53, N 23.58; found C PF: C 43.77, H 2.94, N 12.25; found C 44.25, H 2.67, N 12.35.
20 16 6 6
66.90, H 4.49, 23.87. White powder; yield 70%. Brown solid; yield 76%.
4.4.2 Indole-3-carbaldehyde-3,5-di(2′-pyridyl)-4-amino- 4.5.2 Complex 2
1,2,4-triazole (L4)
M.p. 241°C (dec.) – UV/Vis (MeCN): λ = 298 nm. – IR (KBr,
max
M.p. 148.6°C – UV/Vis (MeCN): λ = 288 nm. – IR (KBr, cm−1): ν = 1574.8 υ(CH=N) , 827.6 (P–F). – 1H NMR (400
max Pyridine
cm−1): ν = 1630.6 (CH=N). – 1H NMR (400 MHZ, [D]DMSO, MHZ, [D]DMSO, 25°C, TMS): δ = 9.64 (d, J = 5.68 Hz, 1H,
6 6 HH
25°C, TMS): δ = 12.07 (s, 1H, NH), 8.86 (s, 1H, CH=N), 8.64 Py), 9.11 (s, 1H, CH=N), 8.56 (d, J = 4.8 Hz, 1H, Py), 8.33 (d,
HH
(s, 2H, Py), 8.29–7.99 (m, 6H, Py), 7.60–7.50 (m, 3H, Indole), J = 4.4 Hz, 3H, Py), 8.18 (m, 1H, Py), 7.98 (d, J = 7.12 Hz,
HH HH
7.27–7.15 (m, 2H, Indole). – 13C NMR ([D]DMSO): δ = 184.93 2H, Py), 7.86 (d, 1H, J = 4.96 Hz, 1H, Ar), 7.73 (m, 1H, Ar),
6 HH
(C–N), 166.04 (CH=N), 149.30 (Py), 145.70 (Py), 138.42 7.64–7.60 (m, 3H, Ar), 6.24 (q, 2H, p-cymene), 5.99 (q, 2H,
(Py), 137.57 (Py), 137.26 (Py), 135.79 (Py), 124.88 (Pyra), p-cymene), 2.86 (sep, CH, p-cymene), 2.17 (s, 3H, CH),
3
124.52 (Pyra), 124.31 (Pyra), 123.31 (Ar), 122.07 (Ar), 120.77 1.19 (m, 6H, (CH)). – 13C NMR( [D]DMSO): δ = 173.23
3 2 6
(Ar), 118.13 (Ar), 113.39 (Ar), 109.85 (Ar). – HRMS ((+)ESI): (CH=N), 156.73 (Py), 149.63 (Py), 149.42 (Py), 144.22 (Py),
m/z = 387.1584 (calcd. 388.1281 [C H NNa]+, [M + Na]+). – 143.46 (Py), 140.55 (Py), 138.25 (Py), 134.11 (Pyra), 130.6
21 15 7
Anal. calcd. for C H N: C 69.03, H 4.14, N 26.83; found C (Pyra), 127.66 (Ar), 126.22 (Ar), 124.01 (Ar), 104.11 (Ar), 101.7
21 15 7
69.43, H 4.09, N 26.34. Red powder; yield 66%. (Ar), 86.2 (Ar, p-cymene), 84.69 (Ar, p-cymene), 83.72 (Ar,
p-cymene), 82.59 (Ar, p-cymene), 30.46 (CH, p-cymene),
22.08 (CH, p-cymene), 21.38 (CH, p-cymene),18.05 (CH,
3 3 3
p-cymene). – HRMS ((+)ESI): m/z = 597.1107 (calcd. 597.1107
4.5 General method: synthesis of complexes
[C H ClNRu]+, [M–PF]+). – Anal. calcd. for [C H ClNRu]
1–8 29 28 6 6 29 28 6
PF: C 46.94, H 3.80, N 11.33; found C 46.75, H 3.51, N 11.19.
6
Orange solid; yield 82%.
A mixture of [(η6-arene)Ru(μ-Cl)Cl] (arene = benzene
2
or p-cymene) (0.16 mmol) and the triazole ligand
(0.33 mmol) was stirred in dry methanol (30 mL) for 3 h at
4.5.3 Complex 3
ambient temperature. The solvent was reduced to 10 mL
and two equivalents of NH PF were added. The excep-
4 6 M.p. 225°C (dec.) – UV/Vis (MeCN): λ = 310 nm. – IR
tion was compound 6, which was obtained as a chloro max
(KBr, cm−1): ν = 1589.9 (CH=N) , 830.0 υ(P–F). – 1H NMR
salt. The compounds formed were filtered, recrystallized Pyridine
(400 MHZ, [D]DMSO, 25°C, TMS): δ = 9.74 (d, J = 5.48 Hz,
from acetonitrile, washed with EtO and then dried under 6 HH
2 1H, Py), 9.18 (s, 1H, CH=N), 8.61 (d, J = 4.48 Hz, 1H, Py),
vacuum. HH
8.36–8.27 (m, 3H, Py), 8.17–8.11 (m, 2H, Py), 7.87–7.86 (m,
1H, Py), 7.85–7.83 (m, 2H, Ar), 7.23–7.16 (m, 2H, Ar), 6.20 (s,
6H, CH), 3.77 (s, 3H, OCH). – 13C NMR: δ = 168.11 (CH=N),
6 6 3
4.5.1 Complex 1 160.01 (Py), 157.13 (Py), δ 149.74 (Py), 144.24 (Py), 143.6
(Py), 140.64 (Py), 138.21 (Py), 128.10 (Pyra), 127.45 (Pyra),
M.p. 225°C (dec.) – UV/Vis (MeCN): λ = 298 nm. – IR 126.05 (Ar), 125.10 (Ar), 124.39 (Ar), 121.21 (Ar), 118.64
max
(KBr, cm−1): ν = 1574.2 (CH=N) , 828.4 (P–F). – 1H NMR (Ar), 112.65 (Ar), 86.03 (CH), 55.99 (-OCH). – HRMS ((+)-
Pyridine 6 6 3
(400 MHZ, [D]DMSO, 25°C, TMS): δ = 9.75 (d, J = 5.14 Hz, ESI): m/z = 571.0596 (calcd. 571.0587 [C H ClONRu]+,
6 HH 26 22 6
1H, Py), 9.09 (s, 1H, CH=N), δ 8.56 (d, J = 4.36 Hz, 1H, Py), [M–PF]+). – Anal. calcd. for [C H ClONRu]PF: C 43.62,
HH 6 26 22 6 6
8.35 (t, J = 7.08 Hz, 3H, py), 8.18 (t, J = 7.96 Hz, 1H, Py), H 3.10, N 11.74; found C 43.19, H 2.70; N 11.13. Orange solid;
HH HHH
7.98 (m, 2H, Py), 7.87–7.83 (m, 1H, Ar), 7.73 (t, J = 7.52 Hz, yield 76%.
HHH
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4.5.4 Complex 4 (d, J = 5.44 Hz, 1H, Py), 9.01 (s, 1H, CH=N), 8.61 (d,
HH
J = 4.6 Hz, 1H, Py), 8.37 (m, 1H, Py), 8.31–8.25 (m, 3H,
HH
M.p. 220°C (dec.) – UV/Vis (MeCN): λ = 294 nm. – IR (KBr, Py), 8.22–8.11 (m, 2H, Py), 7.83–7.82 (m, 1H, Ar), 7.60–7.57
max
cm−1): ν = 1589.96 υ(CH=N) , 836.9 (P–F). – 1H NMR (400 (m, 2H, Ar), 7.36 (m, 2H, Ar), 6.22 (s, 6H, CH). – 13C NMR:
Pyridine 6 6
MHZ, [D]DMSO, 25°C, TMS): δ = 9.63 (d, J = 5.44 Hz, 1H, δ = 167.99 (CH=N), 150.35 (Py), 149.68 (Py), 149.38 (Py),
6 HH
Py), 9.20 (s, 1H, CH=N), 8.60–8.59 (m, 1H, Py), 8.36–8.33 (m, 144.32 (Py), 143.89 (Py), 140.48 (Py), 137.95 (Py), 137.56 (Py),
2H, Py), 8.28–8.26 (m, 1H, Py), 8.18–8.11 (m, 2H, Py), 7.87– 128.29 (Pyra), 127.29 (Pyra), 125.85 (Ar), 124.95 (Ar), 124.19
7.84 (m, 1H, Py), 7.72–7.62 (m, 2H, Ar), 7.24–7.07 (m, 2H, Ar), (Ar), 123.80 (Ar), 123.40 (Ar), 121.56 (Ar), 112.86 (Ar), 109.21
6.23 (d, J = 6.12 Hz, 1H, p-cymene), 6.17 (m, 1H, p-cymene), (Ar), 86.01 (CH). – HRMS ((+)-ESI): m/z = 580.0590 (calcd.
HH 6 6
5.99 (m, J = 6.06 Hz, 2H, Ar, p-cymene), 3.77 (s, 3H, OCH), 580.0590 [C H ClNRu]+). – Anal. calcd. for [C H ClNRu]
HH 3 27 21 7 27 21 7
2.82 (sep, 1H, CH(CH)), 2.17 (s, 3H, CH), 1.19 (m, 6H, (CH)). Cl: C 52.69, H 3.44, N 15.93; found C 52.20, H 3.74, N 15.43.
3 3 3 2
– 13C NMR: δ = 168.63 (CH=N), 160.54 (Py), 157.24 (Py), 150.36 Orange solid; yield 82%.
(Py), 144.81 (Py), 141.03 (Py), 138.72 (Py), 136.57 (Py), 128.64
(Py), 126.57 (Pyra), 125.62 (Pyra), 124.89 (Ar), 121.74 (Ar),
119.28 (Ar), 113.20 (Ar), 104.8 (Ar), 101.9 (Ar), 86.61 (Ar, 4.5.7 Complex 7
p-cymene), 85.20 (Ar, p-cymene), 84.30 (Ar, p-cymene),
83.43 (Ar, p-cymene), 56.53 (OCH), 30.96 (CH), 22.60 (CH), M.p. 124°C (dec.) – UV/Vis (MeCN): λ = 301 nm. – IR
3 3 max
21.96 (CH), 18.60 (CH). – HRMS ((+)-ESI): m/z = 627.1218 (KBr, cm−1): ν = 1617.62 (CH=N) , 830.96 (P–F). – 1H
3 3 Pyridine
(calcd. 627.1218 [C H ClONRu]+, [M–PF]+). – Anal. calcd. NMR (400 MHZ, [D]DMSO, 25°C, TMS): δ = 12.45 (s, 1H,
30 30 6 6 6
for [C H ClONRu]PF: C 46.67, H 3.92, N 10.88; found C N=H), 9.74 (d, J = 5.44 Hz, 1H, Py), 9.00 (s, 1H, CH=N),
30 30 6 6 HH
46.43, H 3.39, N 10.38. Orange solid; yield 82%. 8.61 (d, J = 4.44 Hz, 1H, Py), 8.39 (d, J = 7.84 Hz, 1H,
HH HH
Py), 8.30–8.27 (m, 3H, Py), 8.13–8.11 (m, 2H, Py), 7.83(t,
J = 6.20 Hz, 1H, Ar), 7.60 (d, J = 8.48 Hz, 2H, Ar),
HHH HH
4.5.5 Complex 5 7.36–7.28 (m, 2H, Ar), 6.21 (s, 6H, CH). 13C NMR: δ 167.96
6 6
(CH=N), 150.34 (Py), 149.68 (Py), 149.38 (Py), 144.32 (Py),
M.p. 231°C (dec.) – UV/Vis (MeCN): λ = 299 nm. – 143.89 (Py), 140.46(Py), 138.04 (Py), 137.94 (Py), 137.54 (Py),
max
IR (KBr, cm−1): ν = 1585.7 υ(CH=N) , 832.4 (P–F). 127.27 (Pyra), 125.85 (Ar), 124.95 (Ar), 124.19 (Ar), 123.83
Pyridine
– 1H NMR (400 MHZ, [D ]DMSO, 25°C, TMS): δ = 9.63 (Ar), 123.41(Ar), 122.53(Ar), 121.57 (Ar), 112.84 (Ar), 109.25
6
(d, J = 5.44 Hz, 1H, Py); 9.04 (s, 1H, CH=N), 8.55 (d, (Ar), 86.01 (CH). – HRMS ((+)-ESI): m/z = 580.0590 (calcd.
HH 6 6
J = 4.4 Hz, 1H, Py), 8.32 (m, 3H, Py), 8.17–8.13 (m, 1H, 580.0590 [C H ClNRu]+). – Anal. calcd. for [C H ClNRu]
HH 27 21 7 27 21 7
Py), 7.87–7.83 (m, 3H, 2Py, 1Ar), 7.62–7.59 (m, 1H, Ar), PF: C 44.73, H 2.93, N 13.52; found C 45.12, H 3.05, N 13.92.
6
7.44 (t, J = 4.4 Hz, 2H, Ar), 6.24 (q, 2H, p-cymene), 5.99 Orange solid; yield 82%.
HH
(q, 2H, p-cymene), 2.83 (sep, 1H, CH(CH)), 2.4 (s, 3H,
3 2
CH), 2.1 (s, 3H, CH), 1.19 (m, 6H, (Me), cymene). – 13C
3 3 2
NMR: δ = 173.03 (CH=N); 149.81 (Py), 149.72 (Py), 149.42 4.5.8 Complex 8
(Py), 144.91 (Py), 144.22 (Py), 143.49 (Py), 138.22 (Py),
130.06 (Py), 129.75 (Pyra), 128.00 (Pyra), 127.66 (Ar), M.p. 136°C (dec.) – UV/Vis (MeCN): λ = 287nm. – IR
max
126.17 (Ar), 124.93 (Ar), 104.09 (Ar), 101.42 (Ar), 86.23 (Ar, (KBr, cm−1): ν = 1573.0 (CH=N) , 841.1 (P–F). – 1H
Pyridine
p-cymene), 84.68 (Ar, p-cymene), 83.72 (Ar, p-cymene), NMR (400 MHZ, [D]DMSO, 25°C, TMS): δ = 12.40 (s, 1H,
6
82.90 (Ar, p-cymene), 30.45 (CH), 22.08 (CH), 21.38 (CH), N=H), 9.65 (d, J = 5.36 Hz,1H, Py), 9.02 (s, 1H, CH=N),
3 3 HH
18.05 (CH). – HRMS ((+)-ESI): m/z = 611.1279 (calcd. 8.60 (d, J = 4.52 Hz, 1H, Py), 8.40 (d, J = 7.08 Hz, 1H,
3 HH HH
611.1224 [C H ClN Ru]+, [M–PF ]+). – Anal. calcd. for Py), 8.30–8.22 (m, 3H, Py), 8.15–8.11 (m, 2H, Py), 7.83 (d,
30 30 6 6
[C H ClN Ru]PF: C 47.66, H 4.00, N 11.12; found C 47.50, J = 6.56 Hz, 1H, Ar), 7.60 (t, J = 7.56 Hz, 2H, Py ), 7.34
30 30 6 6 HH HHH
H 3.89, N 11.3. Orange solid; yield 85%. (t, J = 9.04 Hz, 2H, Ar), 6.24 (d, J = 6.42 Hz, 1H, Ar,
HHH HH
p-cymene), 6.20 (d, J = 6.04 Hz, 1H, Ar, p-cymene), 6.00
HH
(d, J = 6.08 Hz, 1H, Ar, p-cymene), 5.97 (d, J = 6.12 Hz,
HH HH
4.5.6 Complex 6 1H, Ar, p-cymene), 2.88 (sep, 1H, CH(CH)), 2.18 (s, 3H,
3 2
CH), 1.19 (m, 6H,(CH)). – 13C NMR: δ = 168.81 (Py), 150.58
3 3 2
M.p. 124°C (dec.) – UV/Vis (MeCN): λ = 310 nm. – IR (CH=N), 149.66 (Py), 144.34 (Py), 143.84 (Py), 140.34
max
(KBr, cm−1): ν = 1613.8 υ(CH=N) . – 1H NMR (400 (Py), 137.97 (Py), 137.55 (Py), 124.95 (Pyra), 124.20 (Pyra),
Pyridine
MHZ, [D]DMSO, 25°C, TMS): δ = 12.45 (s, 1H, N=H), 9.76 112.85 (Ar), 109.20 (Ar), 104.02 (Ar), 101.35 (Ar), 86.21 (Ar,
6
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p-cymene), 84.64 (Ar, p-cymene), 83.75 (Ar, p-cymene), the structure refinement and crystal data information for
82.88 (Ar, p-cymene), 30.47 (CH), 22.08 (CH), 21.43 (CH), ligand L2 and complex 1.
3 3
18.08 (CH). – HRMS ((+)-ESI): m/z = 636.1219 (calcd. CCDC 1573551 and 1573552 contain the supplemen-
3
636.1319 [C H ClNRu]+). – Anal. calcd. for [C H ClNRu] tary crystallographic data for this paper. These data
31 29 7 31 29 7
PF: C 58.53, H 4.60, N 15.41; found C 58.65, H 4.69, N 15.45. can be obtained free of charge from The Cambridge
6
Orange solid; yield 82%. Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
4.6 X-ray crystallography
5 Supplementary information
The crystals of the ligand L2 and complex 1 were obtained
by solvent diffusion. A solution of L2 in dry dichlorometh-
Figure S1 (1H NMR of the hydrolysis of complex 3) is given
ane and a solution of compound 1 in dry acetone were,
as Supplementary Information available online (https://
respectively, layered with four equivalents of hexane.
doi.org/10.1515/znb-2017-0145).
Both were kept for 2 days at room temperature in the
dark. Suitable crystals of L2 and complex 1 were glued
Acknowledgments: We thank the NRF and UKZN for
onto the tip of a glass fiber and subsequently mounted
financial support. In addition, we acknowledge Prinsloo
under a stream of cold nitrogen at T = 100(1) K. Using a
Xolisa Phiri for her technical assistance during antimicro-
video camera, they were centered in the X-ray beam of
bial studies. Joel M. Gichumbi acknowledges Prof. E. N.
the Bruker Smart APEX II diffractometer. Direct methods
Njoka for his support.
using SHELXS [41] were used to solve the structures
which were refined using SHELXL [42, 43]. Table 4 shows
References
Table 4: Summary of the crystal data for the ligand L2 and complex 1.
[1] G. Ludwig, G. N. Kaluđerović, M. Bette, M. Block, R. Paschke,
Compound L2 1 D. Steinborn, J. Inorg. Biochem. 2012, 113, 77.
[2] M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger,
Formula C H NO C H ClNRuPF
20 16 6 27 23 7 6 B. K. Keppler, Dalton Trans. 2008, 14, 183.
Formula weight 356.39 727.01
[3] A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward, D. Forman,
Crystal size, mm3 0.37 × 0.34 × 0.34 0.32 × 0.28 × 0.25
Ca-Cancer J. Clin. 2011, 61, 69.
Crystal system Orthorhombic Triclinic
[4] G. Ludwig, G. N. Kaluđerović, M. Bette, M. Block, R. Paschke,
Space group Pbcn P1̅
D. Steinborn, J. Inorg. Biochem. 2012, 113, 77.
a, Å 11.4021(9) 8.7684(8)
[5] R. K. Gupta, R. Pandey, G. Sharma, R. Prasad, B. Koch, S.
b, Å 14.6609(11) 11.5083(10)
Srikrishna, P.-Z. Li, Q. Xu, D. S. Pandey, Inorg. Chem. 2013, 52,
c, Å 20.8271(16) 14.5497(13)
3687.
α, deg 90 88.407(4)
[6] H. M. Wallace, P. J. Hergenrother, P. J. Sadler, Chem. Soc. Rev.
β, deg 90 85.626(4)
2015, 44, 8771.
γ, deg 90 78.552(3)
[7] P. Govender, A. K. Renfrew, C. M. Clavel, P. J. Dyson,
V, Å3 3481.6(5) 1434.7(2)
B. Therrien, G. S. Smith, Dalton Trans. 2011, 40, 1158.
Z 8 2
[8] P. Heffeter, M. Pongratz, E. Steiner, P. Chiba, M. A. Jakupec,
D , Mg m−3 1.379 1.683
calcd L. Elbling, B. Marian, W. Korner, F. Sevelda, M. Micksche, B. K.
T, K 173(2) 173(2)
Keppler, W. Berger, J. Pharmacol. Exp. Ther. 2005, 312, 281.
μ, mm−1 0.090 0.770
[9] B. M. Blunden, A. Rawal, H. Lu, M. H. Stenzel, Macromolecules
λ, Å 0.71073
2014, 47, 1646.
F(000), e 1488 728
[10] S. Thangavel, R. Rajamanikandan, H. B. Friedrich, M. Ilanchelian,
θ /θ , deg 1.956/28.465 2.273/25.569
min max B. Omondi, Polyhedron 2016, 107, 124.
No. of reflns. Collected 62 379 17 875
[11] L. K. Filak, S. Göschl, P. Heffeter, K. Ghannadzadeh Samper,
No of indep. reflns./R 4376/0.0245 5239/0.0230
int A. E. Egger, M. A. Jakupec, B. K. Keppler, W. Berger, V. B. Arion,
Completeness to θ , % 100 97.8
max Organometallics 2013, 32, 903.
Absorption correction Semi-empirical from equivalents
[12] P. Appelt, F. D. Fagundes, G. Facchin, M. Gabriela Kramer,
Refinement Full-matrix least squares on F2
D. F. Back, M. A. A. Cunha, B. Sandrino, K. Wohnrath,
Goodness-of-fit on F2 1.037 1.074
M. P. de Araujo, Inorg. Chim. Acta 2015, 436, 152.
R1/wR2 (I > 2σ(I)) 0.0376/0.0943 0.0238/0.0595
[13] F. Li, J. G. Collins, F. R. Keene, Chem. Soc. Rev. 2015, 44, 2529.
R1/wR2 (all data) 0.0422/0.0996 0.0231/0.0591
[14] E. A. Nyawade, H. B. Friedrich, B. Omondi, H. Y. Chenia,
Largest diff. peak/hole, e Å−3 0.28/−0.30 0.67/−0.56
M. Singh, S. Gorle, J. Organomet. Chem. 2015, 799–800, 138.
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