Rapid Synthesis and Antiproliferative Properties of Polyazamacrocycle-Based Bi- and Tetra-Gold(I) Phosphine Dithiocarbamate Complexes

Article abstract of DOI:10.1002/cbic.201900227

A family of bi- and tetrametallic gold(I) phosphine dithiocarbamate complexes were synthesized, starting from cyclam and dimethylcyclam polyazamacrocycles, respectively, along with their monometallic gold(I) chloridophosphine precursors. Their antiproliferative properties were evaluated on two cancer cell lines (A549 and NSCLC-N6-L16). Most of the mono- and bimetallic complexes displayed strong activities and, in particular, one bimetallic derivative showed antiproliferative properties in the low micromolar range. Insights into the structure–activity relationships are given, along with determination of the thioredoxin reductase inhibition potential, two-photon imaging of the fluorescent derivatives, and evaluation of gold uptake.

Full text of DOI:10.1002/cbic.201900227

Accepted Article
Title: Rapid synthesis and anti-proliferative properties of
polyazamacrocycle-based bi- and tetra-gold(I) phosphine
dithiocarbamate complexes
Authors: Océane Flores, denis velic, Nesrine Mabrouk, Ali Bettaieb,
Christophe Tomasoni, jean-Michel Robert, Catherine Paul,
Christine Goze, Christos Roussakis, and Ewen Bodio
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10.1002/cbic.201900227
ChemBioChem
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Rapid synthesis and anti-proliferative properties of
polyazamacrocycle-based bi- and tetra-gold(I) phosphine
dithiocarbamate complexes
Océane Florès,[a] Denis Velic,[b] Nesrine Mabrouk,[c] Ali Bettaïeb,[c] Christophe Tomasoni,[b] Jean-Michel
Robert,[b] Catherine Paul,[c] Christine Goze,*[a] Christos Roussakis,[b] and Ewen Bodio*[a]
Abstract: A family of bimetallic and of tetrametallic phosphine-gold(I)-
dithiocarbamate complexes were synthesized starting from cyclam
and dimethylcyclam polyazamacrocycles respectively, along with
their monometallic chlorido-phosphine gold(I) precursors. Their
antiproliferative properties were evaluated on two cancer cell lines
(A549 and NSCLC-N6-L16). Most of the mono and bimetallic
complexes displayed strong activities, and especially one bimetallic
derivatives showed anti-proliferative properties in the low micromolar
range. Insights in the structure activity relationships were given, along
with determination of thioredoxin reductase inhibition potential, two-
photon imaging of the fluorescent derivatives, and evaluation of gold
uptake.
potential of gold complexes as anticancer drug. The interest was
even recently boosted by the repurposing of auranofin – a drug
usually prescribed for fighting rheumatoid arthritis – to treat
different types of cancer (see its entrance in clinical trials for the
treatment of Recurrent Epithelial Ovarian, Primary Peritoneal, or
Fallopian Tube Cancer [NCT01747798, NCT03456700]; of
several types of leukemia [NCT01419691], lung cancer
[NCT01737502]…).
Based on this compound, numerous studies highlight the interest
to stabilize gold(I) ion by a phosphine and a thiol ligand.[5–11] As a
matter of fact, some of us recently proved the stability of P-Au(I)
in vitro and in vivo thanks to an optical smart probe.[12,13] More
recently, the research for stabilizing gold(III) ion revealed the
potential of another sulfur-containing ligand: dithiocarbamate,
resulting in very promising complexes.[14,15] This type of ligand is
also interesting from a synthetic point of view, because they can
be easily synthesized in one step, starting from a primary or
secondary amine.
Introduction
The use of gold in therapy – a.k.a. Chrysotherapy – dates back to
Chinese antiquity (2500 B.C.). Gold was employed to treat
different disorders such as inflammation, infection and
tuberculosis.[1–3] In modern medicine, gold complexes are mainly
known for its anti-arthritic properties.[1,4] The study of its
anticancer properties is more recent. Indeed, following the
success of cisplatin and especially the discovery of its limitations,
more and more researchers have decided to investigate the
Dithiocarbamate ligands are not only used for stabilizing gold(III),
several studies reported on the design of promising phosphine
gold(I) dithiocarbamate complexes. In their review on gold
complexes, Tiekink and coll. resume the recent advances in this
domain.[16] In particular, they indicated that, if the phosphine
ligand bears phenyl rings, the complexes induced preferentially
apoptosis,[17] that the development of bimetallic complexes lead
to outstanding activities,[18] and that, most of the time gold
dithiocarbamate complexes display better activities than
cisplatin.[19]
[a]
Ms. O. Florès, Dr C. Goze, and Dr E. Bodio*
CNRS, Université Bourgogne Franche-Comté
ICMUB UMR6302 - CNRS
F-21000 Dijon, France.
Email: christine.goze@u-bourgogne.fr, ewen.bodio@u-bourgogne.fr
Dr D. Velic, Mr C. Tomasoni, Prof. J.-M. Robert, Prof. C. Roussakis
Université de Nantes - UFR Sciences Pharmaceutiques;
Laboratoire IIciMed EA1155 Département de cancérologie;
9 rue bias 44035 Nantes France
Ms. N. Mabrouk, Prof. A. Bettaïeb, Dr C. Paul
EPHE, PSL Research University
Laboratoire d'Immunologie et Immunothérapie des Cancers
75000, Paris, France
[b]
[c]
[c]
The previous studies highlight therefore the interest of being able
to design gold(I)-phosphine complexes, where the phosphine
bears three aryl substituents to favor cell death, and the gold ion
is stabilized by a dithiocarbamate ligand. Moreover, in parallel, it
could also be really attracting to find a strategy enabling a rapid
access to the polymetallic version of this kind of complexes to
maximize their activity. Therefore, we decided to develop
polymetallic dithiocarbamate complexes bearing various
triarylphosphine ligands. Usually, bi/polygold(I) complexes are
based on a polyphosphine ligand.[20–23] In our case, we decided to
draw our inspiration from Wilton-Ely and coll.,[24] and to use a
polyazamacrocycle – cyclam and dimethylcyclam – as a template
for the design of bi and tetragold(I) complexes.
Ms. N. Mabrouk, Prof. A. Bettaïeb, Dr C. Paul
Univ. de Bourgogne Franche Comté
LIIC, EA7269
F-21000 Dijon, France
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Results and Discussion
P-2 was synthesized following a reported three-step synthesis
(Scheme 2).[12,25] P-2 was introduced on C-0 and D-0 as for P-1
and led to the polymetallic complexes C-2 and D-2 (Figure 1).
Synthesis of the complexes:
Since the introduction of the gold moieties proceeded smoothly
onto C-2 and D-2, we decided to further introduce gold(I)
phosphine complexes that can be easily functionalized. In
particular, we chose a triarylphosphine bearing a carboxylic acid
function. This phosphine was easily coordinated to gold(I) to give
the complex P-3. P-3 was then activated using usual peptide
coupling conditions and reacted with an amine (benzylamine and
a phosphonium derivative, respectively) (Scheme 3). It is worth
noting that the amine reacts selectively on the activated carboxylic
acid position and the corresponding complexes P-4 and P-5 can
be easily purified by precipitation in a mixture DCM/pentane
(Scheme 3).
The synthesis of C-1 was performed according to Wilton-Ely’s
procedure:[24] Cyclam macrocycle
C was deprotonated in
presence of NaH and reacted with CS2 to lead to the
tetrakisdithiocarbamate salt C-0, which was directly reacted with
four equivalents of the gold-phosphine P-1 ([Au(PPh3)Cl]) to give
the desired tetrametallic complex C-1 (Scheme 1). Starting from
the dimethylcyclam D, the same methodology was applied for
synthesizing the bimetallic complex D-1. The reactions can be
monitored by 31P-NMR, thanks to the shift in the resonance
observed going from P-1 (singlet at 33.2 ppm) to the
dithiocarbamate complexes C-1 and D-1 (singlets at 36.1 and
36.2 ppm respectively). Interestingly, the 1H,13C,31P-NMR spectra
of C-1 and D-1 are very simple and well defined, highlighting the
high degree of symmetry of these complexes.
Scheme 3: syntheses of P-3, P-4, and P-5 complexes.
Since C-1 and C-2 were much less soluble either in organic or in
aqueous solvent, we decided to introduce our functionalized
phosphines P-3, P-4, and P-5 only on the dimethylcyclam
derivative D-0. The resulting bimetallic complexes D-3, D-4, and
D-5 were obtained and characterized (Figure 2).
Scheme 1: syntheses of C-0, C-1, D-0, and D-1 compounds.
Once these two model compounds validated, we decided to
introduce more sophisticated gold(I)-complexes. First, we
tethered a coumarin-based phosphine gold(I) complex P-2. We
previously designed the corresponding chlorido and glucose
derivatives complexes for investigating the mechanism of action
of phosphine-gold(I) complexes.[12,13]
.
Scheme 2: synthesis of P-2 complexes
Figure 2: Structures of P-3, P-4, P-5, D-3, D-4 and D-5 complexes.
The two reactions of the synthesis, i.e. formation of the
dithiocarbamate
+ complexation appeared to be almost
quantitative. However, significant amounts of the products were
lost during the purification process, more precisely during the
precipitation in cold methanol, in which the complexes are partially
soluble.
Figure 1: Structures of C-2 and D-2 complexes.
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Biological evaluation:
the fact that, for the second series of complexes P-2, C-2, and
D-2, which displayed limited solubility in cell medium at high
concentrations, any significant anti-proliferative properties was
found on the two tested cell lines.
From a biological point of view, it is interesting to note that at
physiological pH (7.4), complexes of series 1, 2, and 4 will be
neutral, the ones of series 3 will be anionic, and the ones of series
5 will be cationic. Moreover, the complexes P-2, C-2, and D-2
contain one or several coumarin moieties, which is known to be
biologically relevant. Indeed, several coumarin derivatives have
been reported to possess anti-cancer properties[26–29] and to
Concerning the monometallic complexes P-1, P-3, and P-5, they
display moderate to good anti-proliferative properties. The higher
activity of P-5 may be due to the combination of both the gold(I)
complex moiety and the phosphonium.
enable sometimes tracking by optical imaging[13,30,31]
. The
introduction of phosphonium moieties (complexes P-5 and D-5)
was also done on purpose: phosphonium derivatives often display
strong anti-proliferative properties and they favor mitochondria
uptake,[32–34] which are believed to be the organelles targeted by
gold(I) complexes.[7] Consequently, we may have a synergy
between the different parts of the molecules.
Interestingly, the bimetallic complexes D-1, D-3, and D-5 display
very promising IC50. Antiproliferative properties of D-1 and D-5 are
in the range of auranofin but the one of D-3 is more than three
time higher than this benchmark complex. Moreover, they all
display slightly higher to strongly higher activity than their mono-
metallic parents (1 to 1.5 for series 5, around 3 for series 1, and
more than 10 for series 3). Thus, the increased activities of D-1
and D-3 are not only due to the presence of two gold atoms in the
molecules.
The anti-proliferative properties of the resulting complexes were
evaluated on two cell lines: A549 and NSCLC-N6-L16 (an
adenocarcinoma and an epidermoid lung cancer, respectively).
NSCLC-N6-L16 is a cell line derived from a NSCLC of a
previously untreated patient (moderately differentiated classified
as T2N0M0)[35]. The A549 line was obtained from the ATCC
It is worth noting that P-3 and D-3 are twice more active on A549
than on NSCLC-N6-L16 cell line. This observation suggests that
these two complexes are cytostatic because A549 cells have a
(reference CCL-185)[36] and is known to have a wild type p53 gene, doubling time twice higher than NSCLC-N6-L16 cells (24 and 48h
while NSCLC-N6-L16 has a mutant p53 gene, similar to tumors in
situ. The IC50 were measured after incubation of the twelve
complexes for 48 h thanks to MTT tests. The tests were
performed using an increasing concentration of the products up
to 30 µg/mL. The results were converted in molar concentration
in order to facilitate the comparison between the different systems
(the cyclam and the dimethylcyclam have also been evaluated but
we did not observe any sign of toxicity) (Table 1).
respectively). On the contrary, P-1, D-1, P-4, P-5, and D-5 display
the same IC50 for both A549 and NSCLC-N6-L16 cell lines, which
suggests a cytotoxic action. Moreover, it seems that the
mechanism of action of the complexes may not be sensitive to the
status of p53 gene – A549 has a wild type p53 gene, while
NSCLC-N6-L16 has a mutant one.[37–42] This is interesting
because p53 gene would be implied in some resistance
phenomena. Therefore, compounds, which are not sensitive to its
status may induce less resistance phenomena and should not
display cross-resistance with platinum drugs.
Table 1: Determination of the IC50 values (μM) of auranofin and complexes P-1
to P-5, C-1 and C-2, and D-1 to D-5 by MTT assay at 48 h on A549 and NSCLC-
N6-L16 (values are presented as the mean ± SEM of at least three independent
experiments with three replicates) (N.T.: not tested).
To try to go deeper in the understanding of the in vitro behavior of
the complexes, we evaluated gold uptake in cells by ICP-MS
measurements (see Figure S 38 in ESI). For this study, the
complexes have been incubated with A549 cells at 15 µM for 1 h.
Then, the cells have been washed, lysed, and prepared for ICP-
MS dosage. The highest gold uptakes were observed for the
polymetallic complexes (excepted for the first series). These
results seem obvious but are inconsistent with anti-proliferative
assays (no correlation between IC50 and gold uptake). This may
be because low soluble complexes, especially C-1 and C-2, form
aggregates in cells, and it is not possible to differentiate by ICP-
MS available gold from aggregates.
Complexes IC50-A549 (µM)
IC50 - NSCLC-N6-L16 (µM)
Auranofin
P-1
10 ± 3
29 ± 11
-
N.T.
28 ± 10
C-1
-
D-1
8.8 ± 0.2
> 40
12.7 ± 0.2
P-2
-
C-2
-
-
D-2
-
-
P-3
35.1 ± 0.1
3.2 ± 0.9
13.5 ± 1.1
-
18.8 ± 0.9
< 2.4
D-3
P-4
11.3 ± 0.3
-
Most of the previous reported studies dealing with gold complexes,
especially gold(I) derivatives, hypothesize that their main targets
are thioredoxin reductases (TrxRs).[7,43,44] These selenoenzyme,
overexpressed in numerous cancer cells, are localized in
cytoplasm and especially in mitochondria and are implied in
oxidative stress regulation.[45–47] The inhibition mechanism seems
consisting in the exchange of the X ligand borne by gold atom
(here chlorido or dithiocarbamate ligand) by the selenium of the
active site selenol of TrxRs.[48–51] Their inhibition lead to death of
D-4
P-5
D-5
8.7 ± 0.1
8.4 ± 0.5
12.0 ± 0.7
8.3 ± 1.7
The first conclusion that can be drawn from these data is the fact
that the tetrametallic complexes C1 and C2 did not display any
significant anti-proliferative properties. This may be explained by
their lower solubility in cell media. This hypothesis is confirmed by
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the cells. Thus, we investigated the ability of some of our
compounds (P-1, P-3, D-1, and D-3) to inhibit TrxRs and compare
them to auranofin. We used A549 cell line and a commercially
available kit, which was already used for evaluating gold(I)-based
complexes inhibition of TrxRs.[43] The tests revealed that at 5 µM
all the tested complexes significantly inhibit TrxRs (Figure 3 and
Figure S 36 and Figure S 37 in ESI). After auranofin, P-1 displays
the best inhibition properties, and D-1, P-3, and D-3 are around
three time less active than auranofin. These results highlight the
ability of our complex to interact with TrxRs and proteins bearing
thiol or selenol residues. However, there is no clear relationship
between antiproliferative properties (IC50 values) and the ability of
the complex to inhibit TrxRs. This observation was previously
reported in different studies and puts forward the question if TrxR
is really the main cellular target of gold(I) complexes.[43,52]
NT
P-2 (2D)
P-2 (3D)
Figure 4: Two-photon fluorescence microscopy experiments in the A549 cell
line. Cells were treated (P-2) or not (NT) with 50 μM P-2 compounds for 1h at
37 °C, excitation was at 720 nm and detection was around 400-492 nm. A 3-D
image was obtained with P-2 treatment.
Concerning C-2 and D-2, despite their high absorption coefficient
(123,000 and 63,000 mol.L-1.cm-1 respectively, at 347 nm in
DMSO), they are more difficult to observe. Indeed, the presence
of numerous heteroatoms such as sulfurs or nitrogens must
quench their fluorescence via photo-induced electron transfer
(PET) (we already experiment this phenomenon when a coumarin
moiety was tethered to imidazole derivatives[31]). However, we
succeeded in obtaining an image of D-2, when we increased the
power of the laser from 0.8% to 4%. On this image, it seems that
D-2 is present in cells as aggregates, which confirms our
hypothesis (Figure 5).
Figure 3: Slopes of these curves were used to calculate the TrxR activity rate
(A.U.). Data are plotted as mean values +/- S.D. of three independent
experiments. All results were statistically analyzed using a t-test/two tailed
(**p<0,01, ***p<0,001).
We also investigated the possibility to image the coumarin-based
complexes P-2, C-2, and D-2 in order to obtain data that are more
reliable. Coumarins are a class of fluorophore that can be excited
around 300-370 nm and detected around 380-450 nm. The
problem is that an excitation at so low wavelengths induces strong
auto-fluorescence of the cells and hampers the detection of the
compounds. However, some of us previously reported that
coumarin derivatives can be imaged by two-photon
microscopy,[13,31] which allow an excitation around 700 to 750 nm
where auto-fluorescence is very limited. P-2 can be easily imaged
(Figure 4). As, we previously observed, P-2 accumulates in lipid
rafts (lipophilic micro domains of cell membrane). This localization
is particularly clear on the 3D image.
Figure 5: Two-photon fluorescence microscopy experiments in the A549 cell
line. Cells were treated (D-2) or not (NT) with 50 μM D-2 compounds for 1h at
37 °C, excitation was at 720 nm and detection was around 400-492 nm.
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Conclusions
MTT assay
A549 cells (2.5x103 cells/well) and NSCLC-N6-L16 cells (1x104 cells/well)
were seeded in 96-wells clear flat-bottom plates (Falcon). Cells were
incubated with different concentrations of each molecules to determine
IC50 for 48h (37 °C, 5% CO2). Cell growth was estimated by a colorimetric
assay based on the conversion of tetrazolium dye (MTT) to a blue
formazan product by living cells. Optical density at 570 nm corresponding
to solubilized formazan was read for each well on a Titertek Multiskan MKII.
Several new bi- and tetrametallic gold(I)-dithiocarbamate
complexes based on cyclam and dimethylcyclam scaffold were
synthesized and fully characterized by usual spectroscopic
technics. These sophisticated molecules can be easily
synthesized in one to three days at a global yield of 10 to 50%
starting from the free phosphine, [Au(tht)Cl], and the
polyazamacrocycle. Their C1 or C4 symmetry was highlighted by
the simplicity of their NMR spectra. The anti-proliferative
properties of the seven polymetallic complexes, as well as their
five monometallic chlorido-gold(I)-phosphine parent complexes,
were evaluated on A549 and NSCLC-N6-L16 cell lines. It appears
that highly lipophilic complexes did not display any significant
activity due to the formation of aggregates in cells. On the contrary,
less lipophilic complexes show moderate to high anti-proliferative
properties. One monometallic (P-5) and two bimetallic complexes
(D-1 and D-5) display activity in the range of auranofin, while the
bimetallic complex D-3 is three times more active. The results
suggest that anionic complexes are cytostatic, while neutral and
cationic complexes are cytotoxic. It also appears that anti-
proliferative properties of the complexes seems to be
independent from p53 gene status, which is interesting with
respect to resistance phenomena. Some preliminary mechanistic
studies showed that the complexes enter the cells and are able to
inhibit TrxRs. However, we did not find any direct correlation
either between gold uptake and antiproliferative activity or
between TrxR inhibition rate and antiproliferative activity.
TrxRs activity assay
The TrxRs activity was measured with a commercially available kit (Sigma
aldrich). The assay is based on the reduction of 5,5’-dithiobis(2-
nitrobenzoic)acid(DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB)
by TrxRs. The yellow color product is measurable at 405 nm by
spectrophotometry. The cells (A549) were previously incubated with or
without auranofin, P1, P3, D1, or D3 at 5 µM during 48h. The cells were
harvested, lysed, and TrxRs activity was measured according to the
manufacturer’s instructions. The linear increase in absorbance at 405 nm
was measured on a (Perkin Elmer Wallac 1420 Victor2 Microplate Reader
during 12 minutes). The TrxRs activity rate was calculated from the slope
of absorbance at 405 nm versus time and normalized by the total protein
in sample.
In vitro two-photon microscopy:
The adenocarcinoma lung cancer A549 cells (2x105 cells) were treated 1h
at 37°C with 50 µM of P-2, C-2 and D-2. After paraformaldehyde fixation
(2%) and cytospin (300 rpm, 5 min), two-photon fluorescence images were
collected on a Nikon A1-MP scanning microscope (Nikon, Japan). Imaging
was carried out with a ×60 Plan Apo IR objective (NA: 1.27, Water
Immersion, Nikon, Japan) at a scanning speed of 0.5 frame per second.
Excitation at 720 nm was provided by an IR laser (Chameleon, Coherent).
Fluorescence emission was collected using a 400-492 nm band-pass
emission filter (FF01-492/SP, Semrock). Images were treated with NIS
software (Nikon, Japan).
This study proves the interest of developing bimetallic complexes
for increasing the activity of compounds. Moreover, it enables the
discovery of three very promising compounds (D-1, D-3, and D-5),
D-3, which is active in the low micromolar range can be
considered as a lead compound and worth being studied in details
in the future to investigate deeply its biological behavior in vitro
and to test its potential in vivo.
Acknowledgements
The Ministère de l’Enseignement Supérieur et de la Recherche,
the Centre National de la Recherche Scientifique (CNRS), the
École Pratique des Hautes Études (EPHE), and the French
Research National Agency (ANR) via project JCJC ‘‘SPID” ANR-
16-CE07-0020 and project JCJC “WazaBY” ANR-18-CE18-0012
are gratefully acknowledged. Chematech® society is warmly
thanked for providing cyclam and dimethylcyclam. This work is
part of the projects ‘‘Pharmacoimagerie et agents théranostiques”
et ‘‘Chimie durable, environnement et agroalimentaire” supported
by the Université de Bourgogne and the Conseil Régional de
Bourgogne through the Plan d’Actions Régional pour l’Innovation
(PARI) and the European Union through the PO FEDER-FSE
Bourgogne 2014/2020 programs. Dr M. Laly is acknowledged for
ICP-MS analyses. Ms M.-J. Penouilh and Dr Q. Bonin are
gratefully acknowledged for HR-MS and NMR analyses.
Plateforme DimaCell, U. Bourgogne Franche-Comté, F21000
Dijon, France. E.B. and C.R. sincerely thanks Dr M. Malleter for
enabling this collaboration. FrenchBIC and GDR AIM are
acknowledged for fruitful discussion.
Experimental Section
Chemical synthesis
All synthetic details are provided in ESI part.
Biological part
Cell lines and cultures
Two cell lines, A549 and NSCLC-N6-L16, were used in this study
originating from an adenocarcinoma and an epidermoid lung cancer,
respectively. NSCLC-N6-L16 is a cell line derived from a NSCLC of a
previously untreated patient (moderately differentiated classified as
T2N0M0).[35] The A549 line was obtained from the ATCC (reference CCL-
185)[36] and is known to have a wild type p53 gene, while NSCLC-N6-L16
has a mutant p53 gene, similar to tumors in situ.
Keywords: gold complex • polymetallic • cancer •
dithiocarbamate • phosphine
The cell lines were cultured in RPMI-1640 enriched with 100 IU penicillin,
100 μg.mL-1 streptomycin, 2 mM glutamine and 5% fetal bovine serum.
Cell culture plates were maintained in humidified incubators at 37 °C in a
5% CO2 atmosphere. NSCLC-N6-L16 has a cell doubling time of 48h in
vitro, and A549 24h.
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10.1002/cbic.201900227
ChemBioChem
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Bi- and tetrametallic phosphine-
gold(I)-dithiocarbamate complexes
were synthesized starting from cyclam
and dimethylcyclam
O. Florès, D. Velic, N. Mabrouk, A.
Bettaïeb, C. Tomasoni, J.-M. Robert, C.
Paul, C. Goze, C. Roussakis,* and E.
Bodio*
polyazamacrocycles and their
antiproliferative properties were
evaluated on two cancer cell lines
(A5549 and NSCLC-N6-L16).
Page No. – Page No.
Rapid synthesis and anti-proliferative
properties of polyazamacrocycle-
based bi- and tetra-gold(I) phosphine
dithiocarbamate complexes
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.