Synthesis and biological evaluation of thiolate gold(i) complexes as thioredoxin reductase (TrxR) and glutathione reductase (GR) inhibitors

Article abstract of DOI:10.1039/c9nj02502b

New gold(i) thiolate complexes [(PPh₂py)Au(SR)] (PPh₂py = 2-(diphenylphosphino)pyridine and SR = the deprotonated form of pyridine-2-thiol (HSpy, 1a), pyrimidine-2 thiol (HSpyN, 1b), benzothiazole-2-thiol (HSbt, 1c) and 2-thiazoline-2-thiol (HSt, 1d)) were prepared by the reaction of chloro gold complex [(PPh₂py)AuCl], A, with the corresponding in situ generated thiolate salt (through a nucleophilic substitution reaction) under inert conditions. All complexes are characterized by NMR spectroscopy and the structures of 1b and 1d were further identified by single crystal X-ray determination. An in vitro cytotoxicity evaluation against five human cancer cell lines including A549 (nonsmall cell lung cancer), SKOV3 (human ovarian cancer), MCF-7 (human breast cancer), SW1116 (colon cancer) and HeLa (cervical cancer) demonstrated the promising antitumor effects of 1b in comparison with standard auranofin and cisplatin. The effects of these compounds on the proliferation of non-tumoral breast cell line MCF-12A showed good selectivity among tumorigenic and non-tumorigenic cell lines. The results illustrated that 1b effectively produces cell death by inducing apoptosis in the MCF-7 human breast cancer cell line. These complexes inhibit both cytosolic (TrxR1) and mitochondrial (TrxR2) thioredoxin reductases as well as glutathione reductase (GR).

Full text of DOI:10.1039/c9nj02502b

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Synthesis and biological evaluation of thiolate gold(I) complexes as thioredoxin
reductases (TrxRs) and glutathione reductase (GR) inhibitors
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Masood Fereidoonnezhad,a,d Hasti Ahmadi Mirsadeghi,b Sedigheh Abedanzadeh,c Alireza
Yazdani,a,d Arsalan Alamdarlou,a,d Mojgan Babaghasabha,b Zainab Almansaf,e Zeinab Faghih,f
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‖
Zach McConnell,e Hamid R. Shahsavari,*b,e and M. Hassan Beyzavi*e
aToxicology Research Center; Department of Medicinal Chemistry, School of Pharmacy, Ahvaz
Jundishapur University of Medical Sciences, Ahvaz, Iran.
bDepartment of Chemistry, Institute for Advanced Studies in Basic Sciences (IASBS), Zanjan
45137-66731, Iran.
cInstitute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran.
dStudent Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
eDepartment of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas
72701, United States.
fPharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
‖On sabbatical leave from IASBS, Zanjan, Iran.
*Email: shahsavari@iasbs.ac.ir, hrshahsa@uark.edu (H.R.S.), beyzavi@uark.edu. (M.H.B.).
Abstract
New
gold(I)
thiolate
complexes
[(PPh2py)Au(SR)],
(PPh2py
=
2-
(diphenylphosphino)pyridine and SR = deprotonated form of pyridine-2-thiol (HSpy, 1a),
pyrimidine-2 thiol (HSpyN, 1b), benzothiazole-2-thiol (HSbt, 1c) and 2-thiazoline-2-thiol (HSt,
1d)) were prepared by the reaction of chloro gold complex [(PPh2py)AuCl], A, with the
corresponding in situ generated thiolate salt (through a nucleophilic substitution reaction) under
inert conditions. All complexes are characterized by NMR spectroscopy and the structures of 1b
and 1d were further identified by single crystal X-ray determination. An in vitro cytotoxicity
evaluation against five human cancer cell lines including A549 (nonsmall cell lung cancer),
SKOV3 (human ovarian cancer), MCF-7 (human breast cancer), SW1116 (colon cancer) and Hela
(cervical cancer) demonstrated the promising antitumor effects of 1b in comparison with standard
auranofin and cisplatin. The effects of these compounds on the proliferation of non-tumoral breast
1

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cell line (MCF-12A) showed good selectivity among tumorigenic and non-tumorigenic cell lines.
The results illustrated that 1b effectively produces cell death by inducing apoptosis in MCF-7
human breast cancer cell line. These complexes inhibit both cytosolic (TrxR1) and mitochondrial
(TrxR2) thioredoxin reductases as well as glutathione reductase (GR).
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1. Introduction
The chemistry of gold is one of the most remarkable areas in the basic and applied
chemistry research. Gold complexes have recently grown increasing attention in the design of new
metal-based anticancer drugs in order to overcome the drawbacks of platinum-based
chemotherapeutic agents.1-5 There have been extensive studies on the therapeutic applications of
gold complexes particularly antiproliferative properties against wide range of cancerous cells.6-9
Although, DNA damage is believed to be the dominant mechanism for platinum anticancer agents,
later studies have shown that thiol-containing proteins/enzymes, can play important roles in the
mechanisms of action of anticancer gold complexes based on the strong binding affinity of gold
ions with thiols.10-13 Phosphine gold(I) thiolate complexes are promising anticancer agents for in
vitro and in vivo antiproliferative activities.14-21 The well-known orally active phosphine Au(I)
thiolate complex, auranofin, has exhibited in vitro anticancer activities toward human cancer cell
lines.22-24 Therefore, a wide investigation has been developed to introduce new phosphine gold(I)
thiolate complexes for treatment of various diseases, including several types of leukemia,
carcinomas, and parasitic, bacterial, and viral infections.6, 25 The nature of ligands in the
coordination sphere around the metallic center has essential role to induce specific properties such
as stability / reactivity and lipophilicity/hydrophilicity to the metal-based drug. The coordination
of thiolate moieties to the gold center has been shown to improve promising in vitro and in vivo
cytotoxic properties of gold complex. In this regard, we designed to prepare a series of structural
analogues
of
2-(diphenylphosphino)pyridine
Au(I)
thiolate
complexes.
2-
(Diphenylphosphino)pyridine, bearing two different hard and soft coordination donor sites, is
undoubtedly one of the most attractive family of phosphine compounds which plays an important
role in organometallic and coordination chemistry.26, 27 It is an unsymmetrical hemilabile ligand
that exhibits different physical or chemical properties including luminescence,28-30 various
coordination modes,31, 32 catalytic,33-35 and biological activity.36
2

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Here we present the synthesis, characterization and chemotherapeutic properties of 2-
(diphenylphosphino)pyridinegold(I) thiolate complexes of general formula [(PPh2py)Au(SR)], 1a-
d, SR = deprotonated form of pyridine-2-thiol (HSpy, 1a), pyrimidine-2 thiol (HSpyN, 1b),
benzothiazole-2-thiol (HSbt, 1c) and 2-thiazoline-2-thiol (HSt, 1d), bearing PPh2py as a P
monodentate ligand. The cytotoxic activities of gold(I) complexes have been evaluated against
five cancer cell lines, A549, SKOV3, MCF-7, SW1116 and Hela, by means of the MTT assay
(MTT= 3-(4,5-dimethylthiazol-yl)-2,5-diphenyl-tetrazolium bromide). The effects of these
compounds on the proliferation of non-tumoral cell line (MCF-12A; normal human epithelial
breast cell line) was also investigated. Moreover, the ability of 1b was evaluated to inhibit the
proliferation of human breast cancer cells, MCF7, by measuring cell death via induction of
apoptosis. In vitro inhibition of the complexes on thioredoxin reductase (TrxR) and glutathione
reductase (GR) were also acquired.
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2. Result and Discussion
2.1. Synthesis and characterization of complexes
The known precursor complex [(PPh2py)AuCl], A,37 was synthesized according to the
reaction of the PPh2py ligand (2-(diphenylphosphino)pyridine) with [AuCl(SMe2)]38 (SMe2 =
dimethylsulfide). Addition of A to the corresponding thiolate salt prepared in situ by its
deprotonation in basic media, lead to produce new gold(I)thiolate complexes[(PPh2py)Au(SR)],
1a–d, SR = deprotonated form ofpyridine-2-thiol (HSpy, 1a), pyrimidine-2 thiol (HSpyN, 1b),
benzothiazole-2-thiol (HSbt, 1c) and 2-thiazoline-2-thiol (HSt, 1d). General synthetic route is
clearly shown in Scheme 1. Eisenberg has also reported 1a, however, utilizing a different synthetic
route.29 All procedures were performed under an argon atmosphere at ambient temperature. In
addition, the complexes are well stable at room temperature under the exclusion of light.
1
All complexes were characterized by NMR spectroscopy. The H and 31P{1H} NMR
spectra of each complex show the characteristic resonances of the functional groups present in the
molecules while the numerical NMR labeling are shown in Scheme 2. The 1H NMR and 31P{1H}
NMR spectra of 1d are shown in Figure 1, for clarity. The 31P{1H} NMR spectrum of A displays
only one singlet resonance at δ = 32.2 ppm corresponding to the P coordinated PPh2Py moiety
which has been shifted to the downfield (36–37 ppm) in new synthesized complexes (1a–d). In
the 1H NMR spectra of all five complexes (A and 1a–d), the adjacent proton to the pyridyl moiety
3

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of PPh2py (H6) has been distinguished as a doublet signal located at higher fields (ca. 8.80 ppm)
with 3JHH values of 4.6 Hz due to the electronic character of nitrogen atom. Aromatic protons were
assigned at ca. 7–8 ppm, however, protons placed near nitrogen atoms were evidently presented
at the higher chemical shifts. In the 1H NMR spectrum of 1d, aliphatic protons of 2-thiazoline-2-
thiolate ligand (H4' andH5') appeared with triplet pattern at 3.41 and 4.30 ppm, respectively (Figure
1).
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HSR/KOH/EtOH
P
Au
Cl
P
Au
SR
-KCl
N
N
A
1
S
S
N
N
R =
N
N
N
1a (Spy)
1b (SpyN)
1c (Sbt)
1d (St)
Scheme 1. Synthetic route for the preparation of 1. All reactions were carried out under an inert
gas at room temperature.
Suitable single crystals of 1b and 1d were obtained at room temperature by slow diffusion
of n-hexane into dichloromethane solution of complexes. The ORTEP view, crystal data and
structural refinement parameters are determined in Figure 2 and Table 1, respectively. Complexes
1b and 1d have been crystallized in the monoclinic space group P21/n. As shown in Figure 2, each
Au(I) center is located in a slightly distorted planar environment, surrounded by phosphorous atom
of PPh2py ligand (P1) and sulphur atom of thiolate moiety (S1). The angles subtended by the
ligands at the Au(I) centers (P1-Au1-S1 = 175.53 (8), 1b and 177.58 (10), 1d), deviate from the
linearity indicating the distorted planar environments. The distance between Au(I) and the thiolate
moieties (Au1-S1 = 2.308 Å (2), 1b and 2.307 Å (2), 1d) are in accordance with other Au(I) thiolate
complexes.39, 40
4

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Figure 1. (a) 1H and (b) 31P{1H} NMR spectra of 1d in CDCl3 at room temperature. The signals
assignments are depicted. The NMR solvent residual signal is shown by *.
Figure 2. Representations of the X-ray crystal structures of 1b (left) and 1d (right) showing all
non-hydrogen atoms as 50% thermal ellipsoids. Selected geometrical parameters (Å, °) for 1b:
Au1-P1 2.255(2); Au1-S1 2.308(2); P1-C51.809(7); S1-C11.759(8); P1-Au1-S1 175.53(8); Au1-
P1-C5110.9(2); Au1-S1-C1100.6(3). Selected geometrical parameters (Å, °) for 1d: Au1-P1
2.258(3); Au1-S1 2.307(2); P1-C 71.828(10); S1-C19 1.741(13); P1-Au1-S1 177.58(10); Au1-P1-
C7111.7(4); Au1-S1-C19101.8(4).
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Table 1. Crystallographic and structure refinement data for 1b and 1d.
1b
1d
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7
8
Formula
Formula weight
T (K)
C21H17AuN3PS
571.38
C20H18AuN2PS2
578.42
293(2)
293(2)
9
λ (Å)
0.71073
0.71073
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Crystal system
Space group
Crystal size(mm)
a (Å)
Monoclinic
P21/n
Monoclinic
P21/n
0.12 × 0.22 × 0.22
9.2477(18)
17.742(3)
12.591(3)
90
93.63(3)
90
2061.7(7)
4
1.841
2.654 - 24.998
1096
0.14 × 0.26 × 0.32
12.258(3)
9.6261(19)
18.107(4)
90
105.71(3)
90
2056.8(8)
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1.868
1.803 - 24.999
1112
b (Å)
c (Å)
α (°)
β (°)
γ (°)
V (Å3)
Z
Dcalc (g cm-1)
θmin, θmax (°)
F000
µ (mm-1)
Index ranges
7.325
7.440
-10 ≤ h ≤ 10
-20 ≤ k ≤ 21
-13 ≤ l ≤ 14
14486
-14 ≤ h ≤ 14
-11 ≤ k ≤ 11
-21 ≤ l ≤ 21
14476
Data collected
Unique data
3611
3636
a
R1 , wR2b (I > 2ϭ (I))
0.0338, 0.0628
0.0688, 0.0684
0.882
0.0462, 0.1026
0.0820, 0.1147
0.803
a
b
R1 , wR2 (all data)
GOF on F2 (S)
CCDC No.
1574994
1574995
aR1= Σ||Fo|-|Fc||/Σ|Fo|, bwR2 = [Σ(w(Fo Fc2)2)/Σw(Fo ) ]
2-
2 2 1/2
2.2. Biological activity studies
The IC50 of compounds on each cell line is presented in Table 2. Firstly, all of these
compounds display good selectivity between tumorigenic and non-tumorigenic cell lines (MCF-
12A; normal human epithelial breast cell line). They showed less cytotoxicity than cisplatin and
auranofin on MCF-12A. However, one-way ANOVA statistical analysis showed that the
differences between 1b and 1d with auranofin on MCF-12A is not statistically significant (Table
S1-S6). As could be observed, the cytotoxic effect of compounds on cancer cell lines varies from
moderate to good effect. The results were compared to cisplatin and auranofin as the reference
drugs. All of the synthesized compounds (except A and 1d which is not statistically significant)
showed greater anti-proliferative activity than cisplatin, against MCF-7 cell line. In comparison to
auranofin, 1b showed greater and 1c showed comparable cytotoxic activity on MCF-7 cell line.
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One-way ANOVA statistical analysis showed that the differences between 1a, 1b and 1c with
auranofin on MCF-7 is not statistically significant. However in the case of SKOV3 cell line, 1a
and 1b displayed significantly higher in vitro cytotoxicity with IC50 of 6.85 and 6.42 μM,
respectively, in comparison to cisplatin. This differences is statistically significant. 1a and 1b was
also showed comparable cytotoxic activity on SKOV3 cell line with auranofin (IC50= 5.28 μM)
and these differences is not statistically significant. Regarding A549, 1b showed comparable
cytotoxicity with auranofin as it showed a good anti-proliferative activity with IC50 of 7.67 μM,
comparing with that measured for auranofin which was 6.49 μM. However, one-way ANOVA
statistical analysis showed that the differences between 1b and auranofin on A549 is not
statistically significant. The cytotoxic activity of the synthesized compounds against Hela cell line
was also evaluated. 1b showed higher anti-proliferative activity than cisplatin and auranofin,
however one-way ANOVA statistical analysis showed that the differences between 1b and
auranofin on Hela is not statistically significant. All of the synthesized compounds showed higher
cytotoxic activity than cisplatin, against SW1116 cell line. The difference between 1b with IC50 of
3.31 μM, as the most cytotoxic compound in SW1116 cell line, and auranofin (IC50= 4.20 μM) is
not statistically significant.
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Structure-activity relationship studies revealed that the presence of non-aromatic ligand
(thiolated dihydrothiazole ring in 1d) decreased cytotoxicity comparing to the other thiolated
ligands. Among the aromatic thiolated ligands, 1b with a thiolated pyrimidine ring showed the
highest potency. Complex A which encompasses chloro group instead of thiolated ligand showed
lowest anti-proliferative activity against all of the studied cell lines.
Table 2. In vitro cytotoxic activity against studied tumorigenic and non-tumorigenic cell lines.a
Complex
(IC50 ± SD) μM
MCF-7 Hela
22.08 ± 1.53 16.15 ± 1.74 12.57 ± 0.72 11.21 ± 0.43 15.19 ± 0.61 89.35 ± 1.17
A549
SKOV3
SW1116
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A
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1d
12.71 ± 1.86
7.67 ± 1.16
6.85 ± 0.26
6.42 ± 0.27
7.24 ± 0.46 5.19 ± 0.62 7.06 ± 0.33 58.69 ± 1.28
5.38 ± 0.13 2.29 ± 0.28 3.31 ± 0.52 45.21± 2.11
6.06 ± 0.84 5.17 ± 0.59 9.81 ± 0.25 63.57 ± 1.09
7.93 ± 1.44 8.51 ± 1.12 12.21 ± 1.60 52.41 ± 2.13
14.48 ± 1.70 14.21 ± 1.08
18.09 ± 1.99 14.37 ± 0.42
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Cisplatin
5.32 ± 0.39
13.29 ± 1.24 10.52 ± 1.39 32.47 ± 1.03 41.69 ± 1.32 32.65 ± 1.22
5.28 ± 0.51 6.21 ± 0.89 2.22 ± 0.72 4.20 ± 0.65 49.18± 2.32
Auranofin 6.49 ± 0.71
a Compounds were dissolved in 1% of DMSO (as described in the Experimental section) and diluted with
media before addition to cell culture medium for a 72 hour incubation period. Each experiment was
separately repeated three times. Data are presented as mean ± SD.
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2.3. Determining apoptotic effect of 1b on MCF-7 cell line
Using an Annexin V apoptosis detection kit, we evaluated the apoptotic effect of 1b on
MCF-7 cell line. As a normal process, apoptosis or programmed cell death plays a crucial role in
tissue homeostasis. Loss of plasma membrane asymmetry is one of the morphologic features
happened during the early steps of apoptosis. In apoptotic cells, phosphatidylserine (PS) which is
normally found on the inner layer of the plasma membrane translocates to the external leaflet. It
thereby can be specifically recognized by a fluorochrome-labeled Annexin V which is a sensitive
probe with high affinity for PS. 7-AAD (7-Aminoactinomycin D), a DNA binding reagent was
also used to discriminate live healthy cells from dead cells (necrotic or late-apoptotic cells) through
penetrating into the nucleus of damaged cells. To determine the apoptotic effect of 1b, MCF-7
cells were treated with 3 different concentrations of this compound (2.5, 5 and 10 µM). As could
be observed in Figure 3, the percentage of the Annexin V+ cells which entered in early apoptotic
phase (right side), remarkably increased from 10.8% in untreated cells to 33.3%, 72.6% and 76.5%
in the cells following treatment with 2.5, 5 and 10 µM of 1b for 48 h, respectively. These results
indicated that this compound could obviously induce apoptosis in cancerous cells in a dose
dependent manner. As shown in Figure 3, after a 48-hour treatment with 1b, most cells were
undergoing apoptosis (Annexin V positive and 7-AAD negative). A minor population of them
were observed to be positive for both Annexin V and 7-AAD, which indicates that that they were
in the end stage of apoptosis or already dead.
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Figure 3. Flow cytometric analysis of apoptotic effect of 1b. MCF-7 cells (Human breast
carcinoma) were left untreated (A) or treated for 48 h with 2.5, 5 and 10 μM of 1b (B, C and D).
Untreated cells (A) were mostly negative for both Annexin V and 7-AAD and therefore are viable.
2.4. In vitro inhibition of thioredoxin reductase (TrxR) and glutathione reductase (GR)
Compounds A and 1a-d were in vitro tested for their ability to inhibit both cytosolic
(TrxR1) and mitochondrial (TrxR2) thioredoxin reductases as well as glutathione reductase (GR).
The results are compared with the reference drug, auranofin, as the potent inhibitor of TrxR
enzymes that it has been shown in vitro and in vivo to have anticancer potential.
As shown in Figure 4, the gold(I) complexes (A and 1a-d) at 20 µM are potent inhibitors
of TrxR1, like cisplatin, and auranofin (at 1µM), but testing of enzyme inhibition assay at lower
concentrations showed that the synthesized compounds slightly less evident inhibitors of TrxR2
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and, interestingly, also inhibit GR while cisplatin or auranofin (at 1 µM) is not inhibiting GR. The
dual TrxR1 + GR inhibition is interesting from a mechanistic stand point. GR is usually quite
sturdy against inhibitors of TrxR1, although we have seen similar effects with e.g. ”TRi-55” in
Arnér et al. work.41
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Figure 4. The inhibitor capacity of complexes A, 1a-d on TrxR1, TrxR2 and Gr. The gold(I)
complexes (A and 1a-d) at 20 µM are potent inhibitors of TrxR1 and TrxR2, like cisplatin, and
auranofin (at 1µM). They also inhibit GR while cisplatin or auranofin (at 1 µM) is not inhibiting
GR.
Using additional concentrations (0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM and 4 μM), the
second order rate constants in inhibition of compounds on TrxR1 and GR were determined. As it
was clear in Figure 5, a time delay in enzyme activity initiation after incubation with some of the
compounds, which it can be suggested is due to a slow non-specific binding of the compounds to
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bovine serum albumin (BSA) that eventually ”relieves” the reductase from non-irreversible
inhibition, while the irreversible inhibition, of course, cannot be altered (as reflected by the slope
in activity one it has started).
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Figure 5. %Inhibition of complexes A, 1a-d and auranofin against time of incubation with TrxR1.
The percentage of inactivation compared to DMSO control was plotted against time using
GraphPad Prism 5.0a (GraphPad software, Inc.). The resulting slopes (kobs), were plotted against
the inhibitor concentrations resulting in Kinact (the rate of enzyme inactivation). The Kinact for
complexes A, 1c and auranofin with TrxR1 were calculated in Figure 6. The Kinact of A and 1b is
greater than what it was measured for 1c and auranofin. It seemed, A is a good inhibitor of TrxR1
and more potent than auranofin. However one-way ANOVA statistical analysis showed that, this
differences between Kinact of 1b and 1c with auranofin is not statistically significant.
In addition, the inhibitory activity of complexes towards disulfide reductase GR has been
acquired. Figure 7 shows that, 1c revealed a significant ability of inhibiting GR similar to that
observed for auranofin. The rate of enzyme inactivation of 1c is greater than what it was calculated
for auranofin. Hence, 1c is a good inhibitor of GR.
3. Conclusion
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The synthesis of new 2-(diphenylphosphino)pyridine gold(I) complexes with various
heterocyclic thiolate ligands with good yields is reported. All of the new complexes were
characterized by NMR spectroscopy. The molecular and crystal structures of new compounds 1b
and 1d were determined by single crystal X-ray crystallography. Treatment of new synthesized
gold(I) thiolate complexes on five cancer cell lines, A549, SKOV3, MCF-7, SW1116 and Hela
demonstrated considerable cytotoxic activities. The effects of these compounds on the
proliferation of non-tumoral cell line (MCF-12A) showed good selectivity between tumorigenic
and non-tumorigenic cell lines. According to the results, 1b showed the highest potential cytotoxic
activity among the complexes and significantly exhibited higher in vitro cytotoxicity than cisplatin
against MCF-7 cells lines. Moreover, 1b, as the most effective compound, has indicated apoptosis-
inducing activities to MCF-7 cancer cell line in a dose dependent manner. These complexes were
a potent inhibitor of thioredoxin reductases (TrxR) and glutathione reductase (GR). Thiolate
ligands confer higher stability under physiological conditions, which leads to an enhancement of
their effectiveness as cytotoxic derivatives. Based on this study, 1b can be introduced as an
efficient anticancer agent for further in vitro and in vivo biological studies in order to develop the
non-platinum chemotherapeutic metallopharmaceuticals.
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Figure 6. Estimation of the rate of enzyme inactivation (Kinact) for A, 1c and auranofin with TrxR1.
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Figure 7. %Inhibition of complex 1c and Auranofin on GR as well as their Kinact estimation. 1c is
more potent than auranofin in inhibition of GR.
4. Experimental
4.1. General procedures and materials
All reactions were carried out under an argon atmosphere using standard Schlenk
31
techniques. NMR spectra (1H and P{1H}) were recorded on a Bruker Avance DPX 400 MHz
instrument at 298 K. All chemical shifts are reported in ppm (part per million) relative to their
corresponding external standards (SiMe4 for 1H (400 MHz) and 85% H3PO4 for 31P (162 MHz))
and all the coupling constants (J values) are given in Hz. The microanalyses were performed using
a vario EL CHNS elemental analyzer. 2-(Diphenylphosphino)pyridine (PPh2py), pyridine-2-thiol
(HSpy), pyrimidine-2-thiol (HSpyN), benzothiazole-2-thiol (HSbt) and 2-thiazoline-2-thiol (HSt)
were purchased from Aldrich or Acros. Also, all solvents were purchased from Aldrich and used
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without further purification. Complex [(PPh2py)AuCl],A, was prepared as reported in literature.37
The chemical shift assignments are based on the following NMR labeling for the ligands as are
shown in Scheme 2. Additional data for A: NMR data in CDCl3: δ (1H)8.79 (d, 3JHH = 4.7 Hz, 1H,
H6), 7.98 (t, 3JHH = 7.9 Hz, 1H, H3),7.80 (tdd, 3JHH = 7.9, 4JHH = 1.8 Hz, 4JPH = 3.7 Hz, 1H, H4),
7.69 (ddd, 3JHH = 7.2,4JHH = 1.4 Hz, 3JPH = 13.4 Hz, 4H, Ho), 7.43-7.54 (m, 6H, Hm and Hp), 7.40
(td, 3JHH = 7.8, 4JHH = 1.6 Hz, 1H, H5) ppm. δ (31P{1H}) 32.2 (s, 1P) ppm.
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Hm
H4
H5
H6
H3
Ho
Hp
Hm
Ph
P
N
Ho
Au
PPh2py
H4'
H4'
H4'
H4'
H5'
H6'
H3'
H5'
H4'
H5'
S
S
N
S
S
H4'
N
S
N
N
N
S
H6'
Au
Au
H5'
H5'
H7'
Au
Au
St
Spy
SpyN
Sbt
Scheme 2. Numerical Scheme for 1H NMR assignment.
4.2. Synthesis of complexes
4.2.1. [(PPh2py)Au(κ1-S-Spy)], 1a.
Complex A (100 mg, 0.202mmol) was added to an ethanolic solution of potassium
pyridine-2-thiolate ligand (KC5H4NS) under inert atmosphere condition [KC5H4NS prepared by
dissolving of KOH (15 mg, 0.262mmol, 1.3eq) in 10 mL of absolute ethanol and was treated with
pyridine-2-thiol (23 mg, 0.202mmol) and stirred for 15 min]. Yellow solution was formed and
after stirring for 2 h at room temperature and the solvent was removed under reduced pressure and
the residue was extracted with CH2Cl2 (10 mL). The obtained colorless solution was filtered
through celite and the filtrate was concentrated to a small volume (~ 1 mL)under vacuum, and
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diethyl ether (5 mL) was added to give 1a as a pale yellow solid, which was filtered and washed
with diethyl ether(3  3 mL) and dried. Yield: 94 mg, 82%. Elem. Anal. Calcd. forC22H18AuN2PS
(570.40); C, 46.32; H, 3.18; N, 4.91. Found: C, 46.39; H, 3.21; N, 4.97.NMR data in CDCl3: δ
(1H) 8.79 (d, 3JHH = 4.6 Hz, 1H, H6), 8.28 (d, 3JHH = 5.4 Hz, 1H, H6'), 8.15 (t, 3JHH = 7.7 Hz, 1H,
H3),7.76-7.82 (m, 5H, H4 and Ho), 7.44-7.52 (m, 7H, H4', Hm and Hp), 7.37 (td, 3JHH = 7.7, 4JHH
1.4 Hz, 1H, H5), 7.31(td, 3JHH = 7.6, 4JHH = 1.8 Hz, 1H, H3'),6.87(td, 3JHH = 7.2, 4JHH = 1.1 Hz, 1H,
H5') ppm. δ (31P{1H}) 36.1 (s, 1P) ppm.
=
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The other new complexes were made similarly using A and the appropriate thiol ligands.
4.2.2. [(PPh2py)Au(κ1-S-SpyN)], 1b.
Yield: 86 mg, 74%.Elem. Anal. Calcd. forC21H17AuN3PS (571.39): C, 44.14; H, 3.00; N,
7.35. Found: C, 44.23; H, 3.04; N, 7.42. NMR data in CDCl3: δ (1H) 8.83 (d, 3JHH = 4.8 Hz, 1H,
3
3
H6), 8.41 (d, JHH = 4.9 Hz, 2H, H4'), 8.24 (td, JHH = 8.1 Hz,4JHH = 1.0 Hz, 1H, H3), 7.87 (ddd,
3JHH = 7.9,4JHH = 1.1 Hz, 3JPH = 14.0 Hz, 4H, Ho), 7.83 (tdd, 3JHH = 7.7, 4JHH = 1.9 Hz, 4JPH = 3.6
Hz, 1H, H4), 7.47-7.56 (m, 6H, Hm and Hp), 7.41 (m, 1H, H5), 6.88 (t, 3JHH = 4.9 Hz, 1H, H5') ppm.
δ (31P{1H}) 37.1 (s, 1P) ppm.
4.2.3. [(PPh2py)Au(κ1-S-Sbt)], 1c.
Yield: 100 mg, 79%.Elem. Anal. Calcd. forC24H18AuN2PS2 (626.49): C, 46.01; H, 2.90;
3
N, 4.47. Found: C, 45.95; H, 2.98; N, 4.59. NMR data in CDCl3: δ (1H) 8.82 (d, JHH = 4.6 Hz,
1H, H6), 8.10 (t, 3JHH = 7.7 Hz, 1H, H3),7.78-7.85 (m, 5H, H4 and Ho),7.73 (d, 3JHH = 7.8 Hz, 1H,
H7'), 7.59 (d, 3JHH = 7.9 Hz, 1H, H4'), 7.47-7.55 (m, 6H, Hm and Hp), 7.42 (m, 1H, H5), 7.32 (t, 3JHH
= 7.8 Hz, 1H, H6'), 7.18 (t, 3JHH = 7.8 Hz, 1H, H5') ppm. δ (31P{1H}) 36.7 (s, 1P) ppm.
4.2.4. [(PPh2py)Au(κ1-S-St)], 1d.
Yield: 106 mg, 91%.Elem. Anal. Calcd. forC20H18AuN2PS2 (578.44): C, 41.53; H, 3.14;
3
N, 4.84. Found: C, 41.65; H, 3.20; N, 4.91. NMR data in CDCl3: δ (1H) 8.79 (d, JHH = 4.6 Hz,
1H, H6), 8.08 (t, 3JHH = 7.8 Hz, 1H, H3),7.74-7.83 (m, 5H, H4 and Ho), 7.44-7.53 (m, 6H, Hm and
3
3
Hp), 7.39 (m, 1H, H5), 4.30 (t, JHH = 8.0 Hz, 2H, H5'), 3.41 (t, JHH = 8.0 Hz, 2H, H4') ppm. δ
(31P{1H}) 36.6 (s, 1P) ppm.
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4.3. Crystal structure determination and refinement
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The X-ray diffraction measurement was carried out on STOE IPDS2T diffractometer with
graphite-monochromated Mo Kα radiation. The single crystals suitable for X-ray analysis were
obtained from CH2Cl2/n-hexane solution (at room temperature) and mounted on a glass fiber and
used for data collection. Cell constants and an orientation matrix for data collection were obtained
by least-square refinement of the diffraction data for 1b and 1d. Diffraction data were collected in
a series of ω scans in 1° oscillations and integrated using the Stoe X-AREA software package.42
Numerical absorption correction was applied using X-Red32 software. The structure was solved
by direct methods and subsequent difference Fourier maps and then refined on F2 by a full-matrix
least-squares procedure using anisotropic displacement parameters. Atomic factors are from the
International Tables for X-ray Crystallography. All non-hydrogen atoms were refined with
anisotropic displacement parameters. Hydrogen atoms were placed in ideal positions and refined
as riding atoms with relative isotropic displacement parameters. All refinements were performed
using the X-STEP32, SHELXL-2014 and WinGX-2013.3 programs.43-47 Crystallographic data for
the structural analysis has been deposited with the Cambridge Crystallographic Data Centre, No.
CCDC-1574994 (for 1b) and CCDC-1574995 (for 1d).
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4.4. Biological Assay
4.4.1. Cell Lines and Cell Culture
Human cancer cell lines, MCF-7 (breast cancer), SKOV3 (ovarian cancer), SW1116 (colon
cancer), Hela (cervical cancer) and A549 (non-small cell lung cancer) were purchased from
National Cell Bank of Iran (NCBI, Pasteur Institute, Tehran, Iran). All cancer cells were cultured
in RPMI 1640 (BioIdea), except MCF-7 which were cultured in DMEM medium supplemented
with 10% fetal bovine serum (FBS; Gibco) and 1% pencilin-streptomycin and were incubated at
37 °C in humidified CO2 incubator. MCF-12A was cultured in medium containing DMEM/ Ham’s
F12 in the presence of 2.5% horse serum and supplemented with epidermal growth factor,
hydrocortisone, bovine insulin, cholera toxin, and antibiotic/antimycotic. 3-(4,5-dimethylthiazol-
yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was applied for assessing the cytotoxic
activities of A and 1a-d as previously described.48-50 Briefly, the cells were trypsinized and plated
in 96-well microplates per well. The cells were then treated with different concentrations of the
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gold complexes (1 to 100 μM) in triplicate manner. Following 48 h incubation at 37 °C in
humidified CO2 incubator, in order to dissolve the formazan crystals, the media were completely
discarded and replaced with 150 μL of 0.5 mg/mL MTT solution and incubated for 3-4 h at 37 °C.
The media containing MTT was then replaced with 150 μL of DMSO. The plate were incubated
for more 30 min at 37 °C in the dark. The absorbance of each well was obtained at 490 nm using
a microplate ELISA reader. The data were analyzed using Excel 2013 and CurveExpert 1.4 and
the concentration with 50% inhibitory effect was reported as IC50 of each compound. Each
experiment was separately repeated three times. Data are presented as mean ± SD.
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4.4.2. Apoptosis detection
PE Annexin V Apoptosis Detection Kit I from BD Biosciences was used for detection of
apoptosis. To do this, 50000 cells was cultured in complete culture media in a 24-well culture
plate. The cells were then treated with different concentrations of complex 1b (2.5, 5 and 10 µM)
o
and incubated at 37 C with 5% CO2 for 48 hours. An untreated sample with equivalent DMSO
concentration was also used as the control. The cells were then harvested, washed with the cold
PBS 1X and re-suspended in 1X Binding Buffer. 1 x 105 of the cells in 50 µl Binding Buffer were
transferred to the tubes, stained with 2 µl of FITC-conjugated Annexin V as well as 2 µl of 7-AAD
(7-Aminoactinomycin D) solution and incubated for 15 min at room temperature in the dark. 300
µl of 1X Binding Buffer was added to each tube and analyzed immediately by four-color
FACSCalibur flow cytometer (BD Bioscience, USA). At least 50000 cells were acquired. The data
were analyzed by flowJo software packages.
4.4.3. Trx1/insulin assay
130 nM TrxR1 was reduced in 200 μM NADPH in Tris-EDTA (TE) buffer and incubated
at room temperature with increasing concentrations (0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM and
4 μM) of Auranofin and the gold(I) complexes (A and 1a-d) for different time points (5, 30, 60
and 90 minutes). Aliquots were removed and added to a mixture containing 25 μM Trx1, 160 μM
insulin and 200 μM NADPH in TE buffer. NADPH consumption at 30°C was measured at 340 nm
using the VersaMax microplate reader. The percentage of inactivation compared to DMSO control
was plotted against time using GraphPad Prism 5.0a (GraphPad software, Inc.). The resulting
slopes (kobs), were plotted against the inhibitor concentrations resulting in Kinact.
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4.4.4. Trx2/insulin assay
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130 nM TrxR2 was reduced in 200 μM NADPH in Tris-EDTA (TE) buffer and incubated
at room temperature with Auranofin in 1 μM and the gold(I) complexes (A and 1a-d) in 20 μM
for different time points (5, 30, 60 and 90 minutes). Aliquots were removed and added to a mixture
containing 25 μM Trx2, 160 μM insulin and 200 μM NADPH in TE buffer. NADPH consumption
at 30°C was measured at 340 nm using the VersaMax microplate reader.
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4.4.5. Glutathione reductase assay
160 nM GR was reduced by 250 μM NADPH in 0.1 M potassium phosphate buffer, pH
7.0 containing 1 mM EDTA. Auranofin and the gold(I) complexes (A and 1a-d) was added at
increasing concentrations (0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM and 4 μM) and incubated
with the enzyme at room temperature. At various time points (5, 30, 60 and 90 minutes), aliquots
were removed and GR activity was measured in the presence of 100 μM NADPH, 1 mM
glutathione disulfide (GSSG) and 1 mM EDTA in potassium phosphate buffer at 340 nm using the
VersaMax microplate reader.
Acknowledgements
M.H.B. gratefully acknowledges the financial support through the startup funds from the
University of Arkansas. The Institute for Advanced Studies in Basic Sciences (IASBS) Research
Council, the Department of Medicinal Chemistry, School of Pharmacy, Ahvaz Jundishapur
University of Medical Sciences (GP95120 and B-9550) and the Iran National Science Foundation
(Grant no 96007334) are also gratefully acknowledged. H.R.S. wish to acknowledge IASBS for
their sabbatical leave at University of Arkansas. Prof. Elias Arnér and Belen Espinosa are
gratefully acknowledged for performing the cytosolic (TrxR1), mitochondrial (TrxR2) thioredoxin
reductases and glutathione reductase (GR) tests.
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Synthesis and biological evaluation of thiolate gold(I) complexes as
thioredoxin reductases (TrxRs) and glutathione reductase (GR)
inhibitors
Gold(I) complexes with 2-(diphenylphosphino)pyridine and thiolate ligands are prepared and
studied for their antiproliferative effects in tumor and normal cells in vitro. The complexes are
also revealed to efficiently inhibit the thioredoxin reductases (TrxR) and glutathione reductase
(GR).