Palladium complexes of bidentate pyridine N-heterocyclic carbenes: Optical resolution, antimicrobial and cytotoxicity studies

Article abstract of DOI:10.1002/aoc.4377

A series of bidentate pyridine-functionalized palladium N-heterocyclic carbene (Pd NHC) complexes with various wingtip substituents (R?=?methyl, phenyl and tert-butyl) have been synthesized and evaluated for their potential biomedical applications as anti

Full text of DOI:10.1002/aoc.4377

Received: 16 October 2017
DOI: 10.1002/aoc.4377
Revised: 28 February 2018
Accepted: 2 March 2018
F U L L P A P E R
Palladium complexes of bidentate pyridine N‐heterocyclic
carbenes: Optical resolution, antimicrobial and cytotoxicity
studies
Kar Bee Choo | Wee Li Mah | Sui Mae Lee | Wai Leng Lee | Yuen Lin Cheow
School of Science, Monash University
A series of bidentate pyridine‐functionalized palladium N‐heterocyclic carbene
Malaysia, Jalan Lagoon Selatan, 47500
Bandar Sunway, Selangor, Malaysia
(Pd NHC) complexes with various wingtip substituents (R = methyl, phenyl
and tert‐butyl) have been synthesized and evaluated for their potential biomed-
Correspondence
ical applications as antimicrobials and antiproliferative drug candidates. The
obtained Pd NHC complexes were applied in a standard broth microdilution
assay for determination of their antimicrobial activities against thirteen strains
of pathogenic microorganisms. In addition to that, cytotoxic activities of the Pd
NHC complexes were also evaluated against three human cancer cell lines,
namely breast (MCF‐7), colon (HCT116) and oral (H103) cancer cells, using
a standard MTT assay. Upon coordination to palladium, the Pd NHC com-
plexes show significant antimicrobial activities with minimum inhibitory con-
centrations in the micromolar range, and they are cytotoxic to the tested
carcinomas with IC50 ranging from 13 to 38 μM. Evidences for influence of
both wingtip substituents and optical isomerism on the biological activities of
the complexes have been found.
Yuen Lin Cheow, School of Science,
Monash University Malaysia, Jalan
Lagoon Selatan, 47500 Bandar Sunway,
Selangor, Malaysia.
Email: cheow.yuen.lin@monash.edu
Funding information
Ministry of Higher Education (MOHE),
Malaysia via Fundamental Research
Grant Scheme, Grant/Award Number:
(FRGS) (FRGS/1/2014/ST01/MUSM/03/1)
KEYWORDS
antimicrobial activity, cytotoxicity, N‐heterocyclic carbene, palladium NHC complexes
1 | INTRODUCTION
transition metal complexes containing improved organic
ligands, in particular the group 10 metals, using cisplatin
The clinical success of platinum‐based drugs like cisplatin
and its second‐generation analogues, carboplatin and
oxaliplatin, in the treatment of different types of cancer
has had an enormous impact on the discovery of novel
metallopharmaceutical agents.[1] However, platinum‐
based drugs have several substantial drawbacks including
poor aqueous solubility (1 mg ml−1), severe side effects
like nephrotoxicity, neurotoxicity, ototoxicity, nausea
and vomiting, effectiveness against a relatively narrow
range of tumour types, as well as drug resistance phe-
nomena, which have lowered the applicability and effi-
cacy of these agents.[2] These unresolved disadvantages
have stimulated research on the development of novel
as a prototype.
Metal N‐heterocyclic carbene (NHC) complexes
have received ample attention due to their highlighted
applications in catalysis, material science and biomedi-
cine.[3] Research on their therapeutic potential is one
of the most active areas within the emerging field of
bioorganometallic chemistry, with over 300 transition
metal NHC complexes being reported as potential antimi-
crobials and anti‐tumour drugs over the past three
years.[4] Their therapeutic potential is further accentuated
by a number of patents held by several universities and
‘big pharma’ in recent years.[5] NHCs are a class of
ligands similar to phosphine ligands, which are well
Appl Organometal Chem. 2018;e4377.
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Copyright © 2018 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4377

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CHOO ET AL.
known for their capability to form complexes with almost
all main group metals and transition metals. They are
generally derived from persistent carbenes which are sta-
ble compounds of divalent carbons. NHCs are generally
defined as heterocyclic species containing a carbene car-
bon and at least one nitrogen atom within the ring
structures.[6]
A number of group 10 metal NHC complexes have
been reported for their potential anticancer activities.
Recent studies have depicted the possible mode of action
of group 10 metal NHC complex‐based anticancer drug‐
like candidates as these metal centres appear promising
in altering cell migration, DNA replication and conden-
sation or DNA fragmentation.[7] Apart from the interfer-
ence with nucleic acids, a recent review has provided a
general insight into the mechanisms of anticancer action
of group 10 metal complexes, which include reactive
The impetus for the antimicrobial study came from
the knowledge that a number of elemental metals are
well known for their antimicrobial activities, for example
silver. Though there is no previous report on the antimi-
crobial properties of any Pd NHC complexes, a number
of transition metal NHC complexes have been shown to
exhibit significant antimicrobial activities against Gram‐
positive and/or Gram‐negative bacteria with minimum
inhibitory concentration (MIC) below 10 μg ml−1
,
including silver, gold, rhodium and ruthenium NHC
complexes among others.[3n,5c,9,12] Other organometallic
complexes containing palladium as the metal centre that
exhibit significant antimicrobial activities have also been
reported previously. For instance, a binuclear palladium
complex with N4O4 Schiff base ligand, a palladium
dicyclohexyldithiocarbamate complex and a palladium
N‐methylbenzylpiperidinedithiocarbamate complex have
been shown to be effective against a range of Gram‐
positive and Gram‐negative bacteria, as well as Candida
oxygen
species
production
associated
with
redox mechanisms, cell cycle arrest, endoplasmic reticu-
lum stress‐mediated apoptosis, mitochondria‐associated
apoptosis and binding to cytochrome, and binding to
essential amino acids causing protein dysfunctions to
name a few.
albicans at MIC below 50 μg ml−1 [13]
Thus, in order to
.
explore the potential biomedical applications of the syn-
thesized Pd NHC complexes, we decided to study their
antimicrobial properties.
Both belonging to group 10, palladium and platinum
display similar chemistry. Hence palladium has become
a target for chemotherapeutics development especially
as a component of anticancer drugs. There are a number
of novel mononuclear, binuclear and multinuclear palla-
dium NHC (Pd NHC) complexes with reduced cross‐
resistance to cisplatin, decreased toxicity and high
specificity that have been developed. Ray et al. reported a
study of two Pd NHC complexes: one bis(NHC) complex
and one mixed complex with NHC and pyridine ligands.
Both complexes demonstrated potent cytotoxic activity
against HeLa (cervical cancer), MCF‐7 (breast cancer)
and HCT116 (colon adenocarcinoma) cell lines in the
micromolar range.[9] Interestingly, the pyridine‐function-
alized Pd NHC complex was 2‐ to 20‐fold more cytotoxic
than cisplatin. Yet, high cytotoxicity against cancer cells
is not limited to Pd–bis(NHC) complexes. Wang et al. have
explored a heteroleptic Pd NHC‐π‐allyl complex contain-
ing a pendant amine group that exhibited up to 10‐fold
increased cytotoxicity compared to cisplatin when tested
against breast cancer cell lines (MCF‐7 and MDA‐MB‐
231) and glioblastoma cells (U‐87 MG).[10] Recently, a
new Pd NHC complex derived from a binuclear silver
NHC complex was shown to exhibit significant antiprolif-
erative activity against MCF‐7 cells with IC50 of 2.5 μM, a
value comparable with that of the standard drug used in
the study, tamoxifen (IC50 = 2.4 μM).[11] The promising
in vitro antiproliferative activities of Pd NHC complexes
have prompted us to explore their potential as anticancer
agents.
Previously, Leung's group reported the synthesis and
characterization of a series of novel cyclopalladated pyri-
dine‐functionalized NHC dichloride complexes showing
interesting conformational behaviour and exhibiting
remarkable activities in catalytic allylic alkylation reac-
tions.[14] In this paper, we report the influence of optical
isomerism and various wingtip substituents in the struc-
ture of pyridine‐functionalized Pd NHC complexes on
the antimicrobial activity and cytotoxicity against cancer
cells. As the literature implies, the metal centre plays an
important role in interacting with biological targets while
lipophilicity is maintained at an optimum level by
adjusting the side chain in the metal NHC complex.[15]
2 | RESULTS AND DISCUSSION
2.1 | Synthesis of Pd NHC Complexes
The synthesis of the targeted complexes followed
established procedures with some minor modifications
(Scheme 1).[14b,c] Initially, the commercially available
2‐benzoylpyridine (1) was reduced to phenyl(pyridine‐2‐
yl)methanol (2), followed by halogenation to give 2‐
(chloro(phenyl)methyl)pyridine (3). Then, the targeted
pyridine‐functionalized imidazolium salts 4a, 4b and 4c
and benzimidazolium salt 6 were obtained by reacting
with their respective imidazoles (1‐methylimidazole,
1‐phenylimidazole and 1‐tert‐butylimidazole) and 1‐
methylbenzimidazole under reflux for 48 h.[14b,16] The
pyridine‐functionalized Pd NHC complexes ( )‐5 and

CHOO ET AL.
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was observed at 10.39, 11.30 and 11.19 ppm for com-
pounds 4a, 4b and 4c, respectively; whereas the charac-
teristic benzimidazolium proton peak was found at
11.33 ppm for compound 6. Apart from that, their identi-
ties were also confirmed by the resonance of the various
wingtip groups on the heterocyclic ring, such as a singlet
1
in the H NMR spectra at 4.03 and 4.30 ppm for the
methyl group in compounds 4a and 6, respectively, as
well as a singlet at 1.70 ppm for the tert‐butyl group in
compound 4c. For all the salts, the proton at the chiral
centre has a downfield shift appearing in the aromatic
proton region (6.25 to 7.90 ppm) and was indistinguish-
able from other aromatic protons.
In this study, we synthesized the transition metal com-
plexes with NHC ligands via transmetalation through the
corresponding Ag(I)–NHC complexes which act as a trans-
fer agent. This method is very well established and very
attractive compared to other procedures, which often
require inert or harsher conditions.[17] In this case, silver
oxide acts as a base as well as a metalation agent. The sim-
plicity of the Ag–carbene bond formation attracts interest
because of the great affinity of the electron exchange taking
place between carbene C and Ag centre. The NHC species
are good electronic donors whereby Ag acts as an excellent
accepter of electron density. The overall electronic exchange
is the best example of a σ d donation from carbene C to the
Ag centre and a π back‐donation from Ag to carbene C.[15]
As expected, 1H NMR spectra of Pd NHC complexes in
DMSO‐d6 confirm the successful deprotonation of
imidazolium moieties indicating coordination of the ligand
to the metal centre. The characteristic change was the dis-
appearance of the characteristic NHCN proton signal of
imidazolium salts 4a–c and benzimidazolium salt 6 in
SCHEME 1 Synthesis pathway of racemic Pd NHC complexes.
Reagents and conditions: (i) NaBH4, EtOH, 1 h, 90 °C, 15 min, 1:1
EtOAc–H2O; (ii) methanesulfonyl chloride, Et3N, DCM, overnight, r.t.,
1:4 EtOAc–hexane; (iii) 1‐methylbenzimidazole, CH3CN, reflux, 48 h;
(iv) 1‐methylimidazole (a)/1‐phenylimidazole (b)/1‐tert‐butylimidazole
(c), CH3CN, reflux, 48 h; (v) Ag2O, CH2Cl2, r.t., overnight, in dark;
(vi) Pd(CH3CN)2Cl2, CH3CN, r.t., overnight, in dark
1
the H NMR spectra upon palladium coordination in ( )‐
5a–c and ( )‐7. Similarly, the proton at the chiral centre
shifted downfield and fell within the aromatic region, thus
unidentifiable from other aromatic protons. In addition,
there was a general downfield shift in the 1H NMR spectra
upon metal coordination. The obtained values lie within
the range reported in the literature.[14] In most cases, no
NCN resonance was observable the 13C NMR spectra of
the synthesized Pd NHC complexes in DMSO‐d6. This is
not unusual since metal NHC complexes with similar
behaviour have been reported. The reason for the absence
of carbene signals is still unclear; nevertheless a fast
dynamic behaviour combined with the poor relaxation of
quaternary NCN could account for it.[18]
( )‐7 could be obtained by reaction of the imidazolium
salts and benzimidazolium with silver(I) oxide, followed
by a transmetalation reaction using bis(acetonitrile)palla-
dium (II) chloride. Four pyridine‐functionalized Pd NHC
complexes were synthesized for the purpose of biomedi-
cal studies. All synthesized compounds and complexes
1
were characterized using H NMR and 13C NMR spec-
troscopy. The obtained spectroscopic data were consistent
with previous literature.[14]
2.2 | Optical Resolution of Pd NHC
Complexes
1
In the H NMR spectra, the acidic proton (NHCN)
signal of compounds 4 and 6 confirmed the formation
of the salts, resonating in a low field for all salts. For
example, the characteristic imidazolium proton peak
Following a literature method, the optical resolution of
the racemic Pd NHC complex ( )‐5a was initiated by

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CHOO ET AL.
SCHEME 2 Optical resolution of racemic complex ( )‐5a.
Reagents and conditions: (i) AgNO3, MeOH, r.t., 3 h, in dark; (ii)
sodium (Sc)‐phenylalanate, MeOH, r.t., 2 h; (iii) 1 M HCl, MeOH, r.
t., 1 h
SCHEME 3 Optical resolution of racemic complex ( )‐5b.
Reagents and conditions: (i) AgNO3, MeOH, r.t., 3 h, in dark; (ii)
sodium (Sc)‐prolinate, MeOH, r.t., 2 h; (iii) 1 M HCl, MeOH, r.t., 1 h
prolinate without changing the counter anion present in
the diastereomic complexes.[14a] In our case, the two
resulting diastereomers could not be separated resulting
in several failed attempts of crystallization. The problem
was finally overcome by changing the counter anion.
After switching the counter anion from chloride to
nitrate, successful optical resolution of racemic complex
( )‐5b was achieved using sodium‐(Sc)‐prolinate as the
auxiliary ligand (Scheme 3). Fractional crystallization
from methanol and diethyl ether yielded the less soluble
diastereomers (Sc,Sc)‐10b. The identity of (Sc,Sc)‐10b
was confirmed through NMR and in agreement with
the literature.[14a] Slow diffusion of diethyl ether into a
methanol solution of the (Rc,Sc)‐10b‐enriched mother
liquor is performed to allow the remaining (Sc,Sc)‐10b
to crystallize out. Similarly, the optically active (S)‐5b
was obtained by stirring a methanol solution of diastereo-
mers (Sc,Sc)‐10b with 1 M HCl. The optically active (S)‐
5b was isolated as a yellow powder. Unfortunately, the
optical resolution of ( )‐5c was unsuccessful despite
changing the ancillary chloride ligand and use of various
sodium–amino acid salts. Meanwhile, the optical resolu-
tion of ( )‐7 is on‐going.
switching the counter ions present in the diastereomic
complexes from chloride to nitrate, and sodium‐(Sc)‐
phenylalanate was used as the resolving reagent
(Scheme 2).[14b] Fractional crystallization method was
employed to yield the less soluble diastereomers (Rc,Sc)‐
9a from methanol and diethyl ether. Whereas the opti-
cally active (R)‐5a was obtained by stirring an equimolar
methanol solution of diastereomers (Rc,Sc)‐9a with 1 M
1
HCl. Their identities were confirmed with H NMR and
13C NMR, also in agreement with a previous report.[14b]
Slow diffusion of diethyl ether into the methanol solution
of the (Sc,Sc)‐9a‐enriched mother liquor is performed to
allow the remaining (Rc,Sc)‐9a to crystallize out. The dia-
stereomers (Rc,Sc)‐9a and (Sc,Sc)‐9a were distinguished
with the presence of two distinct sets of proton reso-
nances for each of the diastereomers. For example, NH2
in (Rc,Sc)‐9a was characterized as a doublet and a
multiplet at 5.00 and 5.98–6.01 ppm, respectively, while
in (Sc,Sc)‐9a, the NH2 protons were present at around
7.00 ppm.
The literature has reported the optical resolution of
the racemic Pd NHC complex ( )‐5b using sodium‐(Sc)‐

CHOO ET AL.
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TABLE 1 Cell viability (half inhibitory concentration) IC50
values of screened imidazolium salts, Pd NHC complexes and cis-
platin against human breast MCF‐7, human colon HCT116 and
human oral H103 tumour cells incubated for 48 h
cell lines. This structure–activity relationship may be
attributed to the central metal atom as explained by
Tweedy's chelation theory and overall lipophilicity of
the compounds.[19] Besides that, it is known that the ste-
ric factors (bulk, size and shape) of drug can affect how
easily it can approach and interact with a binding site.
Bulky substituents may act like a steric shield and hin-
der the ideal interaction between a compound and its
binding site; alternatively, they may help to orientate
the compound appropriately for maximum binding and
increased activity.[20] The literature has suggested that
the mode of action of Pd NHC complexes is due to cova-
lent bonding at the minor groove of DNA causing cross‐
linking of the bases (especially with guanine), and the
square planar configuration enabled intercalation of the
compounds with the DNA involving a π–π stacking.[21]
A mode of action involving a block of the cell cycle at
the G2/M phase also has been proposed for Pd NHC
complexes.[10]
Half inhibitory concentration, IC50 (μM)a
Complex
MCF‐7
HCT116
H103
4a
>40
>40
>40
>40
36.70
>40
>40
25.84
>40
>40
5.83
>40
>40
>40
>40
>40
13.93
22.69
>40
34.53
>40
>40
5.44
4b
>40
4c
>40
( )‐5a
( )‐5b
( )‐5c
(Rc,Sc)‐9a
(R)‐5a
(Sc,Sc)‐10b
(S)‐5b
Cisplatin
>40
18.64
38.60
>40
36.57
>40
23.11
8.37
Interestingly, the optically active enantiomer (R)‐5a
was shown to be cytotoxic with IC50 around 25–36 μM
against the three cell lines tested as compared to the race-
mic Pd NHC complex ( )‐5a (Table 1). Conversely, as
opposed to the enantiomer (R)‐5a which has enhanced
cell cytotoxicity against the carcinomas after optical reso-
lution, the IC50 values (S)‐5b were higher than that of
racemic Pd NHC complex ( )‐5b with reduced cytotoxic-
ity. Similar to their antimicrobial properties, it is specu-
lated that the cell toxicity of (R)‐enantiomers is more
evident compared to (S)‐enantiomers. The cell cytotoxic-
ity effects of these active complexes were comparable to
those of other reported organic anticancer agents, includ-
ing noscapine (IC50 of 20–35 μM) and estramustine (IC50
of 0.5–17 μM).[22] Yet, a trans‐Pd NHC complex with
impressive cell cytotoxicity against HeLa, HCT116 and
MCF‐7 carcinoma cells with IC50 of 0.8 to 4 μM, and
twice as effective as cisplatin, has been reported.[10] On
the other hand, in a recent study where Kumar et al.
investigated five enantiomeric pairs of Pd NHC com-
plexes derived from 1,2,4‐triazole, no influence on the
cytotoxicity was observed in regards to the chirality as
both enantiomers of all five enantiometic pairs exhibited
near‐equal activities, whereby one of the enantiomeric
pair was shown to be 3‐ to 27‐fold more active than the
benchmark cisplatin against human carcinoma cell
lines.[23] Mechanistic studies of the potent chiral Pd
NHC complex revealed that it interfered with cancer cell
proliferation by arresting the cells at the G2 phase; caused
production of reactive oxygen species in the mitochondria
which led to cell death; and was involved in inducing
p53‐dependent programmed cell death in MCF‐7 cells.[23]
Nevertheless, it is known that for a racemic complex, in
this case Pd NHC complexes ( )‐5, each enantiomer
aCells were exposed to various concentrations of complexes for 48 h and cell
viability was assessed using MTT assay. The IC50 values were calculated from
a graph of mean SEM, which are experiments performed in quadruplicate,
and tabulated.
2.3 | Cytotoxicity Studies of Pd NHC
Complexes
It is well known that metal complexes generally display
cytotoxic activity. In this study, the cytotoxic effects of
the synthesized ligands and Pd NHC complexes were
evaluated using in vitro 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐
diphenyltetrazolium bromide (MTT) assays against
human breast (MCF‐7), human colon (HCT116) and
human oral (H103) cancer cell lines. The obtained IC50
values are summarized in Table 1. Cisplatin was utilized
as a positive control as it is an anti‐tumour drug. These
cell lines were exposed to various concentrations of com-
plexes, from 0 to 40 μM, for 48 h. The MTT assay results
show that the synthesized ligands and complexes have an
inhibitory effect on these cancer cells in a dose‐dependent
manner.
Notably, the corresponding precursor ligands,
imidazolium salts 4, shows no cytotoxicity up to 40 μM
suggesting that the growth inhibition of cancer cells is
likely the consequence of incorporation of metal cations
(Table 1). Instead, the cytotoxic effects observed in the
racemic Pd NHC complexes ( )‐5 is speculated to be
mainly due to the metal–ligand interaction with cellular
components. In general, the bulkier Pd NHC complexes
( )‐5b and ( )‐5c have shown only moderate cytotoxic
effect compared to their analogue ( )‐5a, with IC50 rang-
ing from 13.93 to 38.60 μM against the tested carcinoma

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CHOO ET AL.
possesses its own pharmacological activities that can be
null, similar, different or opposite.[24] Even though the
Pd NHC complexes showed lower cytotoxic effects com-
pared to cisplatin, the racemic Pd NHC complexes ( )‐
5b and ( )‐5c have exhibited substantial cell cytotoxic-
ities against the carcinoma cells tested. Therefore, it is
important to separate the enantiomers via optical resolu-
tion and evaluate their respective bioactivities. Also, the
IC50 values of metal NHC complexes have been previ-
ously reported to vary within different cell lines; hence
it is conceivable that these Pd NHC complexes could
show greater potency in other cell types.
showed antimicrobial activities at 5 mM or below against
some pathogens, this is not comparable to other
imidazolium salts which were reported to have MIC
values in the micromolar range.[26] Nevertheless, the anti-
microbial activities increased markedly upon coordina-
tion to palladium (( )‐5a–c). For example, Pd NHC
complex ( )‐5c displayed antimicrobial activities against
all the microorganism strains tested, with the lowest
MIC value of 16 μM (or 7 μg ml−1) against B. subtilis
(Table 2). The lack of antimicrobial activity of Pd NHC
complex ( )‐5a compared to ( )‐5b and ( )‐5c was not
surprising. This is because antimicrobial activity is often
dependent on the chain length of the N‐substituted
imidazolium salts, in which the lowest MIC values were
achieved by compounds with long alkyl chains (C8 to
C16).[26b,d] Longer alkyl chains are thought to be associ-
ated with disruption of bacterial membranes, probably
due to the integration of the long hydrocarbon chain with
the lipid bilayer of the cell membrane, leading to leakage
of cell contents.[27] Also, the extended conjugated ring of
the benzimidazolium salt 6 and the racemic Pd NHC
complex ( )‐7 have been shown to exhibit no improved
antimicrobial activities against the microorganisms tested
compared to their imidazolium‐based counterparts,
imidazolium salt 4a and Pd NHC complex ( )‐5a. These
results agree with reports in the literature; the side groups
on the benzimidazole ring influence the antimicrobial
activities, where the combined steric and electronic con-
straints imposed by the methyl group and pyridine moi-
ety may be responsible for the decreased activities.[28]
Remarkably, while the racemic complex ( )‐5a did
not show any antimicrobial activity at the highest con-
centration tested (0.5 mM), its corresponding purified
enantiomer (R)‐5a was shown to inhibit the microorgan-
isms tested. In contrast, this is not the same for Pd NHC
complex 5b, where both the racemic ( )‐5b and the opti-
cally pure (S)‐5b did not show any antimicrobial activity
at the highest concentration tested (0.5 mM). This may
be attributed to the antimicrobial property of Pd NHC
complexes being more prominent in the (R)‐enantiomers
instead of (S)‐enantiomers. It is not uncommon that there
is only one major bioactive enantiomer out of two in
racemic drugs.[29] The most famous example would be
thalidomide, where (R)‐thalidomide is effective against
morning sickness while (S)‐thalidomide is teratogenic.[30]
Therefore, optical resolution is important so that each of
the enantiomers can be evaluated for their respective bio-
active potentials.
2.4 | Antimicrobial Studies of Pd NHC
Complexes
The antimicrobial effects of metal NHC complexes have
recently attracted increased attention. In this study, we
screened
the
synthesized
pyridine‐functionalized
imidazolium salts 4a–c, benzimidazolium salts 6, racemic
Pd NHC complexes ( )‐5a–c and ( )‐7, the diastereomers
(Rc,Sc)‐9a and (Sc,Sc)‐10b as well as the optically active
enantiomers (R)‐5a and (S)‐5b against a series of patho-
genic microorganisms including Gram‐negative and
Gram‐positive bacteria as well as Candida albicans by
using broth microdilution assay. An antibiotic, chloram-
phenicol, was used as the reference drug in the antimicro-
bial assay. Chloramphenicol is a type of broad‐spectrum
antibiotic that exerts antimicrobial effect through protein
synthesis inhibition by binding to the 50S ribosomal sub-
unit. The MIC values of the synthesized complexes for the
tested microorganism strains are presented in Table 2.
Overall, the Gram‐positive bacterial strains were
inhibited by low micromolar concentrations of the syn-
thesized complexes, whereas substantially lower effica-
cies against Gram‐negative bacterial strains were
observed. The different susceptibility of Gram‐positive
and Gram‐negative bacteria can be attributed to their
morphological differences. The outer peptidoglycan layer
of Gram‐positive bacteria is not an effective permeability
layer. On the other hand, the outer membrane of Gram‐
negative bacteria contains structural lipopolysaccharide
components, thereby making the cell wall impermeable
to lipophilic solutes while porins, membrane transport
proteins, constitute a selective barrier to the hydrophilic
solutes. The compounds synthesized were not soluble in
a wide array of solvents but DMSO, and they were made
up of several conjugated rings, which give rise their
hydrophobicity (i.e. lipophilicity). Therefore, the lack of
efficacy against Gram‐negative bacteria was not
surprising.[25]
The findings suggest that the antimicrobial activity
occurs due to a synergy between the metal and the ligand,
as evidenced by the improved antimicrobial activity of the
synthesized Pd NHC complexes despite the poorer anti-
microbial activities as observed in the imidazolium salts
It was found that all microorganisms were consider-
ably resistant to the ligands 4a–c and 6. Although 4a–c

CHOO ET AL.
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CHOO ET AL.
and PdCl2. Based on Tweedy's chelation theory, the ion
polarity of Pd(II) is reduced by overlapping of the ligand
orbitals and the exchange of the partial positive charge
of the metal ion to the donor atoms of the ligands;
thereby the delocalization of π‐electrons on the chelate
ring increases and improves the lipophilicity of the com-
plexes.[31] The increased lipophilicity allows the complex
to penetrate the lipid membrane and block the metal
binding sites on enzymes, which might result in the dis-
turbance of cellular respiration and protein synthesis,
thus inhibiting cell growth.[32] In general, DNA is consid-
ered to be the main target of group 10 metallodrugs
through the formation of intra‐ and inter‐strand adducts,
while metal bonding to sulfur‐containing groups in pro-
teins was also reported.[33]
Rudolph AUTOPOL VI automatic polarimeter at
1
436 nm. H NMR and 13C NMR spectroscopy was per-
formed with a Bruker Advance 300 NMR spectrometer.
The number of protons (n) for a given resonance is indi-
cated by n H. Coupling constants are reported as a J value
1
in Hz. H NMR signals are reported as δ in units of parts
per million (ppm) downfield from SiMe4 (δ = 0.00 ppm).
13C NMR signals are reported as δ in units of parts per
million (ppm) relative to the signal of chloroform‐d
(δ = 77.16 ppm, triplet) and DMSO‐d6 (δ = 39.52 ppm,
septet). All chemical shifts reported are referenced to
the chemical shifts of their respective residual solvent res-
onances. Unless stated otherwise, all NMR experiments
were carried out at 300 K. See the supporting information
for the NMR spectra of all synthesized compounds,
imidazolium salts and Pd NHC complexes.
3 | CONCLUSIONS
4.2 | Synthesis of Phenyl(pyridin‐2‐yl)
methanol (2)
A series of pyridine‐functionalized imidazolium salts and
Pd NHC complexes were synthesized and characterized
by spectroscopic techniques. Antimicrobial activities and
cytotoxic effects of these ligands and Pd NHC complexes
against three human cancer cell lines were reported.
Although the mechanism of action is not known at pres-
ent, it is clear from this work that the nature of various
wingtip substituents of the NHC ligand has a profound
effect on the biological activities of the resulting Pd
NHC complexes. Besides, optical isomerism might play
a role in the biological activity, as illustrated by the race-
mic Pd NHC complexes ( )‐5a and ( )‐5b, and their
respective enantiomers. Therefore, it is important to
resolve the racemic metal NHC complexes into their
respective enantiomers. The optical resolution of the
racemic Pd NHC complexes ( )‐5c and ( )‐7 is currently
in progress.
The method for reduction of 2‐benzoylpyridine to 2 was
modified from the literature.[16] Sodium borohydride
(2.04 g, 54 mmol) in 50 ml of 95% ethanol was added
dropwise into a solution of 2‐benzoylpyridine (5 g,
27 mmol) in 50 ml of 95% ethanol on an ice bath. The
reaction mixture was stirred continuously for 1 h until
the reaction was completed. Then, 100 ml of water was
added to the reaction mixture and the solution was
heated at 90 °C for 15 min. Then, the solution was
extracted with ethyl acetate thrice and the solvent was
evaporated under reduced pressure to afford a pale green
oil. Slow evaporation of a solution in ethanol at room
temperature yielded the clear crystal solid, 5.01 g,
96.43%. 1H NMR (300 MHz, CDCl3, δ, ppm): 5.29 (d, 1 H,
JH,H = 4.5 Hz, OH), 5.75 (d, 1 H, JH,H = 4.5 Hz, H1),
7.13–7.39 (m, 7 H, arom.), 7.60 (td, 1 H, JH,H = 7.8 Hz,
JH,H = 1.8 Hz, arom.) and 8.55 (dq, 1 H, JH,H = 5.1 Hz,
JH,H = 1.2 Hz, arom.). 13C NMR (75 MHz, CDCl3, δ,
ppm): 75.00 (C1), 121.35, 122.42, 127.07, 127.82, 128.56,
136.83, 143.23, 147.83 and 160.91 (arom.). MS m/z:
[M + H]+ calcd for C12H12NO 186.2298, found 186.0912.
4 | EXPERIMENTAL
4.1 | General Procedures
All the commercially available chemicals and solvents
were used without prior drying or purification. Reactions
involving air‐sensitive compounds were performed under
a positive pressure of nitrogen. Phenyl(pyridine‐2‐yl)
4.3 | Synthesis of 2‐(Chloro(phenyl)
methyl)pyridine (3)
methanol
(PdCl2(CH3CN)2),
(2), bis(acetonitrile)palladium
chloride
1‐tert‐
Compound 2 (1.85 g, 10.5 mmol) and triethylamine
(3.4 ml, 24.4 mmol) in 32 ml of CH2Cl2 was stirred in
an ice bath. Methanesulfonyl chloride (1.2 ml, 15.8 mmol)
was added dropwise into the stirring solution. The reac-
tion mixture was left to warm up slowly to room temper-
ature. The reaction mixture was allowed to stir overnight
and then poured into a saturated aqueous NaHCO3
solution. The aqueous layer was extracted with
1‐phenylimidazole and
butylimidazole were prepared according to literature
methods with some modifications. Mass spectra were
recorded with an Agilent 1290 Infinity LC system coupled
to an Agilent 6520 Accurate‐Mass Q‐TOF mass spectrom-
eter with dual ESI source. Optical activity of the diaste-
reomers and enantiomers was determined using a

CHOO ET AL.
9 of 13
chloroform thrice. The combined organic layers were
washed with water, dried over anhydrous MgSO4 and
evaporated in vacuo to give a red liquid. The resultant
red liquid was subjected to column chromatography
(ethyl acetate–hexane 1:4 v/v) to give a yellow oil,
1.52 g, 74.32%. The spectral data for this compound are
consistent with those reported in the literature.[14b] 1H
NMR (300 MHz, CDCl3, δ, ppm): 6.16 (s, 1 H, H1), 7.20
(ddd, 1 H, JH,H = 7.5 Hz, JH,H = 4.8 Hz, JH,H = 1.2 Hz,
arom.), 7.25–7.37 (m, 3 H, arom.), 7.45–7.49 (m, 2 H),
7.55 (dt, 1 H, JH,H = 7.8 Hz, JH,H = 0.9 Hz, arom.), 7.70
(td, 1 H, JH,H = 7.8 Hz, JH,H = 1.8 Hz, arom.) and 8.57
(dq, 1 H, JH,H = 4.8, JH,H = 0.9 Hz, H9). 13C NMR
(75 MHz, CDCl3, δ, ppm): 64.54 (C12), 122.09 and
122.81 (arom.), 127.78 (C10), 128.28 (C12), 128.64 and
137.05 (arom.), 139.96 (C12), 149.19 (C8) and 159.75
(C9). MS m/z: [M + H]+ calcd for C12H11NCl 204.6754,
found 204.0574.
7.67–7.79 (m, 5 H, arom.), 7.96 (t, 1 H, JH,H = 1.8 Hz, arom.),
8.23 (s, 1H, H10), 8.60 (dq, 1 H, JH,H = 0.9 Hz, JH,H = 4.8 Hz,
H21) and 11.30 (s, 1 H, H7). 13C NMR (75 MHz, CDCl3, δ,
ppm): 65.82 (C10), 119.47 (C8), 121.71 (C20), 123.44
(C18), 123.80, 124.81, 129.10, 129.25, 129.41, 130.19,
130.56, 134.54, 136.28, 136.42, 137.71 and 149.34 (arom.),
155.02 (C17). MS (ESI) m/z: [M − Cl] + calcd for
C21H18N3 312.1501, found 312.1508.
4.4.3 | Synthesis of 1‐tert‐butyl‐3‐
[phenyl(pyridin‐2‐yl)methyl]‐1H‐
imidazolium chloride (4c)
Brown solid, 8.24 g, 54.00%. 1H NMR (300 MHz,
CDCl3, δ, ppm): 1.70 (s, 9 H, H17), 7.27–7.37 (m, 5
H), 7.50–7.55 (m, 2H), 7.72 (td, 1 H, JH,H = 1.8 Hz,
JH,H = 7.8 Hz), 7.81–7.83 (m, 1 H), 8.14 (s, 1 H), 8.59
(dq, 1 H, JH,H = 0.9 Hz, JH,H = 4.8 Hz, H1) and 11.19
(s, 1 H, H13). 13C NMR (75 MHz, CDCl3, δ, ppm):
30.05 (C17), 60.13 (C16), 65.26 (C6), 117.97 (C15),
122.50 (C14), 123.60 (C2), 124.87 (C4), 128.91, 129.07,
136.53 and 136.96 (arom.), 137.55 (C3), 149.30 (arom.)
and 155.44 (C1). MS (ESI) m/z: [M − Cl]+ calcd for
C19H22N3 292.1814, found 292.3259.
4.4 | Synthesis of Pyridine‐Functionalized
Imidazolium Salts 4
Liquid 1‐methylimidazole (a) or 1‐phenylimidazole (b) or
1‐tert‐butylimidazole (c) (1 mol equiv.) was added to a
stirring solution of compound 3 (1 mol equiv.) in 50 ml
of CH3CN. The reaction mixture was heated at refluxing
temperature for 48 h. Then, the reaction mixture was
reduced in vacuo and the resulting oil was stirred in
diethyl ether. The diethyl ether layer was decanted away
to give ligands 4a, 4b and 4c. The spectral data for these
compounds are consistent with those reported in the
literature.[14]
4.5 | Synthesis of Pyridine‐Functionalized
Benzimidazolium Salt 6
Liquid 1‐methylbenzimidazole (1 mol equiv.) was added
to a stirring solution of compound 3 (1 mol equiv.) in
50 ml of CH3CN. The reaction mixture was heated at
refluxing temperature for 48 h. Then, the reaction mix-
ture was reduced in vacuo and the resulting oil was
stirred in diethyl ether. The diethyl ether layer was
decanted away to give ligand 6. The spectral data are con-
sistent with those reported in the literature.[14c] Brown
4.4.1 | Synthesis of 1‐methyl‐3‐
[phenyl(pyridin‐2‐yl)methyl]‐1H‐
imidazolium chloride (4a)
1
oil, 2.55 g, 62.3%. H NMR (300 MHz, CDCl3, δ, ppm):
Hygroscopic off‐white solid, 2.97 g, 45.75%. 1H NMR
(300 MHz, CDCl3, δ, ppm): 4.03 (s, 3 H, CH3), 7.26–7.37
(m, 4 H, arom.) 7.41–7.46 (m, 3 H, arom.), 7.63–7.76 (m, 4
H, arom.), 8.57–8.60 (m, 1 H, H13) and 10.39 (s, 1 H, H4).
13C NMR (75 MHz, CDCl3, δ, ppm): 36.63 (C1), 65.98
(C5), 122.48 (C2), 122.58 (C3), 123.78 (C14), 124.36 (C16),
128.84, 129.24, 129.34, 136.43 and 137.61 (arom.), 137.85
(C15), 149.52 (arom.) and 155.09 (C13). MS (ESI) m/z:
[M − Cl]+ calcd for C16H16N3 250.1344, found 250.1952.
4.30 (s, 3 H, H1), 7.28–7.30 (m, 1 H, H18), 7.31 (m, 5 H,
arom.), 7.46–7.49 (m, 1 H, arom.), 7.55–7.64 (m, 2 H,
arom.), 7.64–7.70 (m, 2 H, arom.), 7.75–7.78 (m, 1 H,
arom.), 7.82 (td, 1 H, JH,H = 7.8, JH,H = 1.8 Hz, arom.),
8.56 (dd, 1 H, JH,H = 6, JH,H = 1.2 Hz, H17), 11.23 (s, 1
H, H8). 13C NMR (75 MHz, CDCl3, δ, ppm): 33.93 (C1),
66.83 (C9), 112.56, 115.80, 124.12, 124.59, 126.78, 126.91,
128.88, 129.44, 129.55, 131.40, 134.71, 138.11, 143.81,
149.71 and 154.55 (arom.), 160.20 (C17). MS (ESI) m/z:
[M − Cl]+ calcd for C20H18N3 300.14, found 299.14.
4.4.2 | Synthesis of 1‐phenyl‐3‐
[phenyl(pyridin‐2‐yl)methyl]‐1H‐
imidazolium chloride (4b)
4.6 | Synthesis of Pyridine‐Functionalized
Pd NHC Complexes
1
Brown oil, 2.71 g, 28.81%. H NMR (300 MHz, CDCl3, δ,
To a solution of pyridine‐functionalized NHC ligands 4a,
4b, 4c or 6 (1 mol equiv.) in 30 ml of CH2Cl, Ag2O
ppm): 7.34–7.39 (m, 3 H, arom.), 7.49–7.61 (m, 5 H, arom.),

10 of 13
CHOO ET AL.
(0.5 mol equiv.) was added in the dark. The reaction mix-
ture was stirred at room temperature for 12 h and was fil-
tered through a short plug of celite. A PdCl2(CH3CN)2
suspension (1 mol equiv. in 100 ml of CH3CN) was added
to the filtrate in the dark. The reaction mixture was then
allowed to stir overnight at room temperature and was fil-
tered through celite the next day. The filtrate was reduced
in vacuo to approximately 50 ml and then 200 ml of
diethyl ether to yield the Pd NHC complexes. The spectral
data for these compounds are consistent with those
reported in the literature.[14]
4.6.4 | Synthesis of racemic complex ( )‐7
1
Orange‐yellow solid 1.63 g. 61.10%. H NMR (300 MHz,
DMSO‐d6, δ, ppm): 4.19 (s, 3 H, H1), 7.34–7.38 (m, 3 H,
Ph), 7.48–7.57 (m, 2 H, Ph), 7.67 (d, 1 H, JH,H = 1.5 Hz,
arom.), 7.80–7.82 (m, 1 H, H19), 7.93–8.00 (m, 1 H, H9),
8.16–8.21 (m, 2 H, H17 and H4), 8.25–8.28 (m, 1 H,
H18), 9.03–9.25 (m, 1 H, H20). 13C NMR (75 MHz,
DMSO‐d6, δ, ppm): 35.77 (C1), 63.71 (C9), 111.33,
112.52, 124.57, 125.05, 127.28, 127.45, 128.96, 129.47,
132.42, 134.67, 136.26, and 138.51 (arom.), 143.20 (C18),
150.32 and 155.07 (arom.).
4.6.1 | Synthesis of racemic complex ( )‐5a
4.7 | Optical Resolution of Racemic Pd
NHC Complex ( )‐5a
1
Orange‐yellow solid 2.10 g, 37.55%. H NMR (300 MHz,
DMSO‐d6, δ, ppm): 3.96 (s, 3 H, H2), 7.26 (s, 1 H, H5),
7.35–7.50 (m, 5 H, arom.), 7.60–7.65 (m, 1 H, H15), 7.81
(d, 1 H, JH,H = 1.8 Hz, H4), 8.00–8.02 (m, 1 H, H13),
8.20 (td, 1 H, JH,H = 1.5 Hz, JH,H = 7.8 Hz, H14) and
9.10–9.16 (m, 1 H, H16). 13C NMR (75 MHz, DMSO‐d6,
δ, ppm): 38.35 (C2), 67.07 (C5), 122.89 (C3), 124.62 (C4),
125.60 (C15), 126.81 (C13), 127.45, 128.81, 129.28 and
140.94 (arom.), 152.08 (C14), and 155.06 (C16).
To a suspension of the racemic complex ( )‐5a (2.08 g,
4.86 mmol) in 20 ml of MeOH, AgNO3 (1.74 g, 9.72 mmol)
was added. The reaction mixture was allowed to stir in
the dark at room temperature for 3 h and was filtered
through a short plug of celite. The orange filtrate was
treated with sodium (Sc)‐phenylalanate (0.11 g,
4.86 mmol) in 100 ml of MeOH. The reaction was then
stirred for 2 h and was concentrated to approximately
50 ml and was left to stand overnight. From the
diastereomic mixture, only complex (Rc,Sc)‐9a crystal-
lized out as off‐white crystals the next day and was iso-
lated and washed with MeOH. The mother liquor was
enriched with (Sc,Sc)‐9a. The spectral data for the diaste-
reomer are consistent with previous literature.[14b]
Diastereomer (Rc,Sc)‐9a. Off‐white solid. [α]436 + 78.6
(c 0.05, DMSO), lit.[14b] [α]436 + 83.3 (c 0.5, DMSO).
1H NMR (300 MHz, DMSO‐d6, δ, ppm): 2.63 (dd, 1 H,
JH,H = 6.0 Hz, JH,H = 14.4 Hz, H18), 3.17 (d, 1 H,
JH,H = 5.4 Hz, H19), 3.47–3.54 (m, 1 H, H19), 3.75 (s, 3
H, H2), 4.99 (d, 1 H, JH,H = 9.9 Hz, NH), 5.99–6.02 (m, 1
H, NH), 6.94–6.98 (d, 2 H, arom.), 7.17–7.26 (m, 4 H,
arom.), 7.34–7.42 (m, 3 H, arom.), 7.47–7.52 (m, 2 H,
arom.), 7.60 (d, 1 H, JH,H = 1.8 Hz, arom.), 7.75–7.80
(m, 1 H, arom.), 7.91 (d, 1 H, JH,H = 1.8 Hz, arom.),
8.15–8.18 (m, 1 H, arom.), 8.35 (td, 1 H, JH,H = 1.8 Hz,
JH,H = 7.8 Hz, arom.) and 8.72 (dd, 1 H, JH,H = 1.5 Hz,
JH,H = 5.7 Hz, H16). 13C NMR (75 MHz, DMSO‐d6, δ,
ppm): 36.66 (C2), 48.48 (C19), 62.13 (C5), 65.80 (C18),
123.02 (C3), 124.04 (C4), 126.06 (C15), 126.23 (C13),
126.50, 127.08, 127.98, 128.36, 128.71, 129.00, 136.73,
139.14, 141.54, 151.14 and 152.52 (Ph), 154.15 (C16)
and 178.64 (C17).
4.6.2 | Synthesis of racemic complex
(
)‐5b
1
Orange‐yellow solid 1.50 g, 39.88%. H NMR (300 MHz,
DMSO‐d6, δ, ppm): 7.36–7.55 (m, 10 H, arom.), 7.69
(t, 1 H, JH,H = 6.9 Hz, JH,H = 1.5 Hz, H20), 7.82–7.87
(m, 3 H, arom.), 8.03–8.10 (m, 2 H, H18 and H9), 8.23
(td, 1 H, JH,H = 8.1 Hz, JH,H = 1.5 Hz, H19) and
9.20–9.23 (m, 1 H, H21). 13C NMR (75 MHz, DMSO‐d6,
δ, ppm): 67.08 (C10), 123.07 (C9), 123.90 (C8), 124.83
(C20), 125.13 (C18), 127.05, 127.98, 128.36, 128.57,
128.75, 137.73 and 139.08 (arom.) 140.42 (C19), 154.06
(arom.) and 154.90 (C21).
4.6.3 | Synthesis of racemic complex ( )‐5c
1
Orange‐yellow solid 4.39 g. 60.00%. H NMR (300 MHz,
DMSO‐d6, δ, ppm): 1.88 (s, 9 H, H17), 7.23 (s, 1 H, H6),
7.36–7.46 (m, 1 H, arom.), 7.58–7.62 (m, 1 H, H2), 7.70
(s, 1 H, H15), 7.87 (s, 1 H, H14), 7.99–8.02 (m, 1 H, H4),
8.14–8.20 (m, 1 H, H3) and 8.96 (d, 1 H, JH,H = 5.1 Hz,
H1). 13C NMR (75 MHz, DMSO‐d6, δ, ppm): 29.99
(C17), 58.67 (C16), 66.61 (C6), 120.27 (C15), 121.61
(C14), 123.99 (C2), 124.98 (C4), 126.21, 126.95, 127.43,
136.67 and 139.06 (arom.), 149.64 (C3), 153.86 (arom.)
and 153.98 (C1).
The optically active (R)‐5a was obtained by stirring a
methanol solution of diastereomer (Rc,Sc)‐9a with 1 M
HCl. The optically active (R)‐5a was isolated as a yellow
powder, 59%. [α]436 + 87.4 (c 0.05, DMSO), lit.[14b]
[α]436–12.5 (c 0.5, DMSO).

CHOO ET AL.
11 of 13
(for Pd complexes) or 5 mM and 3.9 μM (for NHC
ligands) in 100 μl of broth. Then, 100 μl of 1:100 diluted
bacterial suspensions was added into each well prior to
incubation at 37 °C for 24 h. The well with the lowest
concentration of compounds that had no observed
growth was determined as the MIC. The content of clear
wells was streaked on Mueller–Hinton agar (MHA) and
incubated overnight. The minimum bactericidal concen-
tration was determined as the lowest concentration of
compound in which the inoculum did not give bacterial
growth on the MHA. The experiment was performed in
triplicate.
4.8 | Optical Resolution of Racemic Pd
NHC Complex ( )‐5b
To a suspension of the racemic complex ( )‐5b (0.26 g,
0.53 mmol) in 20 ml of MeOH, AgNO3 (0.18 g, 1.06 mmol)
was added. The reaction mixture was allowed to stir in
the dark at room temperature for 3 h and was filtered
through celite. The orange filtrate was treated with
sodium (Sc)‐prolinate (0.07 g, 0.53 mmol) in 100 ml of
MeOH. The reaction was then stirred for 2 h and was
concentrated to approximately 50 ml and was left to stand
overnight. From the diastereomic mixture, only complex
(Sc,Sc)‐10b crystallized out as off‐white crystals the next
day and was isolated and washed with MeOH. The
mother liquor was enriched with (Rc,Sc)‐10b. The spec-
tral data for the diastereomer are consistent with previous
literature.[14c]
4.10 | Evaluation of Cytotoxicity
Cytotoxicity studies of the synthesized compounds and
metal NHC complexes along with cisplatin were carried
out using HCT116, H103 and MCF‐7 cell lines. MTT cell
viability assay was performed to evaluate the cell cytotox-
icity effects of the synthesized compounds.[34] The three
human carcinoma cell lines were from American Type
Culture Collection (ATCC). The compounds tested were
prepared in 2% DMSO in PBS solution. Positive control
utilized in this assay was cisplatin (prepared in 0.9% NaCl
solution). The cells were seeded in a 96‐well microtiter
plate at a density of 5000 cells per well in 100 μl of cell
culture medium and incubated at 37 °C (5% CO2) for
24 h. After 18 h of seeding, the medium was removed
and then the cells were incubated at 37 °C for 48 h in
the CO2 incubator with absence and/or presence of vari-
ous concentrations of compounds of interest ranging
from 0 to 40 μM. After incubation, 10 μl of MTT solution
(5 mg ml−1 in PBS) was added into each well. These
plates were incubated again for 4 h in the CO2 incubator
at 37 °C. After that, the medium–MTT solutions are
removed and the purple formazan crystals formed were
dissolved in 200 μl of DMSO. The resulting MTT products
were determined by measuring the absorbance at 570 nm,
with reference wavelength at 620 nm. The cell viability
was determined by using the formula: cell viability
(%) = (optical density of sample/optical density of con-
trol) × 100 (solvent controls set to 100% viable cells).
IC50 values were defined as the concentrations that show
50% inhibition of proliferation on any tested cell line.
Diastereomer (Sc,Sc)‐10b. Off‐white solid. [α]436
–
153.2 (c 0.05, DMSO), lit.[14c] [α]436–167 (c 0.5, DMSO).
1H NMR (300 MHz, DMSO‐d6, δ, ppm): 1.07–1.12 (m, 1
H, H24), 1.20–1.26 (m, 1 H, H25), 1.46–1.50 (m, 1 H,
H25), 1.69–1.75 (m, 1 H, H26), 2.15–2.18 (m, 1 H, H26),
7.39–7.49 (m, 6 H, arom.), 7.62–7.64 (m, 1 H, arom.),
7.67–7.72 (m, 2 H, arom.), 7.75–7.79 (m, 1 H, arom.),
7.87–7.90 (m, 2 H, arom.), 7.93–7.94 (m, 1 H, arom.),
8.13 (d, 1 H, JH,H = 2.1 Hz, arom.), 8.22 (dd, 1 H,
JH,H = 7.8 Hz, JH,H = 1.5 Hz, arom.), 8.35 (td, 1 H,
JH,H = 7.8 Hz, JH,H = 1.5 Hz, H19) and 8.75 (dd, 1
H, JH,H = 6.0 Hz, JH,H = 1.8 Hz, H21). 13C NMR
(75 MHz, DMSO‐d6, δ, ppm): 22.90 (C24), 28.27
(C25), 49.36 (C26), 66.80 (C10), 67.07 (C23), 124.47
(C9), 124.82 (C8), 126.40 (C20), 126.62 (C18),
127.14, 127.32, 129.02, 129.47, 130.20, 130.35, 153.31
and 153.45 (Ph), 154.96 (C21) and 178.13 (C22).
The optically active (S)‐5b was obtained by stirring a
methanol solution of diastereomer (Sc,Sc)‐10b with 1 M
HCl. The optically active (S)‐5b was isolated as a yellow
powder in 50% yield. [α]436–45.7 (c 0.05, DMSO), lit.[14c]
[α]436–27.8 (c 0.2, MeOH).
4.9 | Evaluation of Antimicrobial Activity
The broth microdilution assay was conducted with 96‐
well microtiter plates according to a protocol stated by
the Clinical and Laboratory Standards Institute with
some amendments.[25] The compounds tested were pre-
pared in 10% DMSO in phosphate‐buffered saline (PBS)
solution. Overnight bacteria culture in Mueller–Hinton
broth (MHB) was adjusted to match 0.5 McFarland
standard and diluted 1:100 in MHB. Into each well of
the 96‐well microtiter plate, the compounds of interest
were serially diluted two‐fold with MHB to obtain final
working concentrations between 0.5 mM and 0.39 μM
ACKNOWLEDGEMENTS
We are grateful for financial support from the Ministry of
Higher Education (MOHE), Malaysia via Fundamental
Research Grant Scheme (FRGS) (FRGS/1/2014/ST01/
MUSM/03/1) and the School of Science, Monash Univer-
sity Malaysia.

12 of 13
CHOO ET AL.
C. Tessier, W. Kofron, R. Simons, J. Garrison, Carbene porphy-
rins and carbene porphyrinoids, methods of preparation and
uses thereof, Google Patents, 2004; f) W. J. Youngs, C. A.
Tessier, J. Garrison, C. Quezada, A. Melaiye, M. Panzner, S.
Durmas, Metal complexes of N‐heterocyclic carbenes as radio-
pharmaceuticals and antibiotics, Google Patents, 2005.
ORCID
Yuen Lin Cheow http://orcid.org/0000-0002-0336-2328
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Additional Supporting Information may be found online
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How to cite this article: Choo KB, Mah WL, Lee
SM, Lee WL, Cheow YL. Palladium complexes of
bidentate pyridine N‐heterocyclic carbenes: Optical
resolution, antimicrobial and cytotoxicity studies.
Appl Organometal Chem. 2018;e4377. https://doi.
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