Synthesis of N-Aryl- and N-alkyl-Substituted Imidazolium Silver Complexes: Cytotoxic Screening by Using Human Cell Lines Modelling Acute Myeloid Leukaemia

Article abstract of DOI:10.1002/cmdc.202000138

A series of N-aryl- and N-alkyl substituted imidazoles has been synthesised and complexed with Ag⁺ to obtain silver-NHC complexes of the form [Ag(NHC)₂]X. These silver-NHC complexes were tested in vitro against the human cell lines HL-60 and MOLM-13, which both model acute myeloid leukaemia (AML). A substantial difference in cytotoxicity was revealed varying in the range 13–4 μM and 22–9 μM for HL-60 and MOLM-13, respectively. Furthermore, this study revealed that when an alkyl group is installed on the imidazole scaffold, its position substantially influences the cytotoxicity of the corresponding silver NHC complex.

Full text of DOI:10.1002/cmdc.202000138

Accepted Article
Title: Synthesis of N-aryl and N-alkyl substituted imidazolium silver
complexes. Cytotoxic screening using human cell lines modelling
acute myeloid leukemia
Authors: Eirin Alme, Karl Wilhelm Törnroos, Bjørn Tore Gjertsen, and
Hans-René Bjørsvik
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemMedChem 10.1002/cmdc.202000138
Link to VoR: https://doi.org/10.1002/cmdc.202000138
01/2020

10.1002/cmdc.202000138
ChemMedChem
COMMUNICATION
Synthesis of N-aryl and N-alkyl substituted imidazolium silver
complexes. Cytotoxic screening using human cell lines modelling
acute myeloid leukemia
Eirin Alme,[a] Karl Wilhelm Törnroos,[a] Bjørn Tore Gjertsen,[b, c] and Hans-René Bjørsvik*[a]
[a]
Ms Eirin Alme, Prof. Dr. Karl Wilhem Törnroos, Prof. Dr. Hans-René Bjørsvik
Department of Chemistry
University of Bergen
Allégaten 41, 5007 Bergen (Norway)
E-mail: hans.bjorsvik@kj.uib.no
[b]
[b]
Prof. Dr. B. T. Gjertsen
Center for Cancer Biomarkers CCBIO,
Department of Clinical Science, University of Bergen,
5020 Bergen (Norway)
Prof. Dr. B. T. Gjertsen
Department of Internal Medicine
Hematology Section, Haukeland University Hospital
P.B. 1400, 5021 Bergen (Norway)
Supporting information for this article is available on the WWW under https://doi.org/10.1002/cmdc.2020xxxxxx
Abstract: A series of N-aryl and N-alkyl substituted imidazoles were
synthesized and complexed with Ag+ to obtain silver-NHC complexes
of the form [Ag(NHC)2]X. These silver-NHC complexes were tested
in-vitro versus the human cell lines HL-60 and MOLM-13 that both
model acute myeloid leukemia (AML). A substantial difference in
cytotoxicity was revealed varying in the range 13-4 µM (for HL-60)
and 22-9 µM (for MOLM-13), respectively. Furthermore, this study
revealed that when an alkyl group was installed on the imidazole
scaffold, its position influenced substantially the cytotoxicity of the
corresponding silver NHC complex.
potent than the corresponding Ag-NHC complex with a methyl
group installed on the same position (NHC-I). Previous
investigations in our laboratories suggest that backbone alkylation
might contribute to the ligand-to-metal donation (NHC®Ag),[21]
which was supported by the examinations of the complexes NHC-
I and NHC-II. Although, the effect of an overall more lipophilic
compound may also be the reason for the potent activity observed
for NHC-II silver complex.
In a previous study,[20] we were not able to conclude whether the
variation of cytotoxicity actually was due to the altered electronic
properties of the imidazole ring owing to the alkylated backbone[21]
or if the observed effect was only due to the overall augmented
lipophilicity of the actual Ag-NHC complex, backbone heptylated
NHC-II and backbone methylated NHC-I.
In our attempt to address this issue, in this study we have
designed and produced a small library of target molecules
(Figure 1) that was synthesized and examined against the two
human cell lines HL-60 and MOLM-13. Moreover, we wanted also
to further optimize the cytotoxicity towards a potent compound
where only a very small dose is required and thus fit into an
appropriate therapeutic window.
The imidazole ring constitute an embedded moiety of an assorted
1
]
selection of biologically active compounds,[
including
and anticancer
2
]
3
]
4 ]
analgesic,[
antibacterial,[
cytotoxic,[
compounds.[5] Imidazole derivatives also constitute an essential
moiety in a variety of alkaloids,[6] as a precursor for N-heterocyclic
carbene (NHC) ligands[7 ] used in transition metal catalysis,[8 ]
furthermore NHCs have also been proven to be versatile
organocatalysts.[9]
Elemental silver has been known for its bactericidal properties
since ancient time.[ 10 ] Silver has reemerged as a workable
alternative for treatment of infections,[11 ] exemplified by silver
sulfadiazine introduced for clinical use in the early 1960s,[12,13]
which has been used as a topical antibacterial for second- and
third-degree burn treatment. NHC complexes of late transition
metals have materialized as promising compounds with potent
activity as antibacterial, antiviral, and anticancer agents.[14]
A long standing project in our laboratory has been dedicated to
the design and development of synthetic methods for late stage
functionalization of the imidazole ring,[15,16,17,18,19] whose results
have been exploited to realize total syntheses of Ag-NHC
complexes, see NHC-I and NHC-II[20] of Figure 1. The NHC-I and
NHC-II complexes were explored by in vitro experiments involving
the human cell lines HL-60 and MOLM-13 that both model acute
myeloid leukemia. These studies revealed NHC-II, which contain
a heptylated imidazole backbone, to be four- to six-fold more
Chemical Synthesis and Structure Determination
The syntheses of the defined target silver-NHC complexes,
whose structures are outlined in Figure 1, were accomplished
through two different pathways: (1) via a three-step linear
synthetic pathway that was used for the preparation of the
complexes NHC-IX, NHC-X, NHC-XII, and NHC-XIII, Scheme 1,
and (2) a second pathway that comprises a multicomponent
reaction (MCR) that produce the ligand with all functionality
embedded. The only additional reaction step was the silver
complexation to approach complex NHC-XI, see Scheme 2.
1
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10.1002/cmdc.202000138
ChemMedChem
COMMUNICATION
telescoped. The pre-cursor NHC ligand 14, was obtained by
reacting 2,4,6-trimethylaniline
2
and oxalaldehyde 3,
formaldehyde 4, and butan-1-amine 15 as described
elsewhere.[24]
From our previous work with complexes NHC-I and NHC-II, any
clear evidences existed on whether these complexes comprised
one or more NHC ligands. In this work, however, we were able to
produce crystals complex NHC-IX. X-ray crystallographic analysis
of the obtained crystals revealed the complex to hold two ligands,
see Figure 2.
By means of crystallography, we revealed that the silver-NHC
complex NHC-IX contained two ligands coordinated to a silver
atom, see Figure 2. From the NMR data alone, it was difficult to
determine whether one or two ligands were present in the Ag-
NHC complex.
For the silver-NHC complexes NHC-X, NHC-XI, NHC-XII, and
NHC-XIII, we were not able, even after several attempts to
produce crystals suitable for crystallography analysis. The NMR
spectra of these compounds revealed however a similar pattern
as for complex NHC-IX, but this did not deliver conclusive
evidence for whether the complexes consist of one silver atom
coordinated to two NHC-ligands. HRMS analyses of the
complexes revealed expected molecular formula for the bis-
ligand-Ag complexes.
O
O
1
NH4
H3C
CH3COOH H2O
2 NH2
Added over
30 min.
O
O
4
+
H
CH3COOH (aq)
70 oC, 18h
H
O
3
a
N
N
N
R Br
Br
Ag
Br
THF
Reflux, 18h
N
c
N
N
5
2
R
L
R
b
NHC-Ag
NHC-Ag
NHC-IX
c
R Br
L
b
Method
1
Figure 1. Designed and synthesized Ag-NHC complexes (NHC-IX – NHC-XIII)
and measurements of IC50 versus the two different human cell lines HL-60 and
MOLM-13. The silver-NHC complexes NHC-I and NHC-II were previously
published in a work from our laboratory.20 The numerical values provided on top
of bar diagram indicate highest tested concentration (>78 µM, >123 µM, etc.).
Br
HO
10
39%
45%
6
7
8
Br
Br
NHC-X
39%
35%
2
1
93%
67%
11
12
HO
NHC-XII
HO
The three-step NHC-ligand synthesis of Scheme 1 incorporated
the following procedures. Step (a): 2,4,6-Trimethylaniline 2,
oxalaldehyde 3, formaldehyde 4, and ammonium acetate 1 were
mixed and reacted by means of a MCR protocol[22] to obtain a the
intermediate 1-mesityl-1H-imidazole 5. In step (b) involved the
installation of the functionalized alkyl chains using the W-
bromoalkyl derivatives 6-9. The W-bromoalkyl derivatives were
installed on N3 of the imidazole ring to obtain the various NHC
ligand precursors 10-13. Ultimately, each of these NHC
precursors were complexed with silver[23] following one of two
distinct methods that involve AgNO3 or Ag2O as silver sources,
respectively.
O
Br
NHC-XIII
32%
1
61%
13
H3C
O
9
Scheme 1. Synthesis of the Ag-NHC complexes NHC-IX, NHC-X, NHC-XII, and
NHC-XIII. Reagents and conditions: (a) ammonium acetate 1, 2,4,6-
trimethylaniline 2, oxalaldehyde 3, formaldehyde 4 in CH3COOH and H2O, 70
oC, 18 h. b) THF, reflux, 18 h, R—Br. (c) Method 1: AgNO3, K2CO3, DCM, 20 oC,
24 h. Method 2: Ag2O, DCM, 20 oC, 24h.
The silver NHC complex NHC-XI was obtained via a different
three-step synthesis, whereof the two first steps (a) and (b) were
2
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10.1002/cmdc.202000138
ChemMedChem
COMMUNICATION
myeloid leukemia with complex karyotype and deletions of both
alleles of tumor suppressor TP53 (see atcc.org for details about
cell lines). HL-60 reflects thereby the most therapy refractory
leukemia. The monocytic derived MOLM-13 model a more
therapy responsive leukemia, but likely dependent on its mutated
receptor tyrosine kinase FLT3 gene, and with severe chromatin
remodulations due to the MLL gene fusion. MOLM-13 is in general
highly sensitive for ATP antagonists that inhibits FLT3.
Surprisingly, we find that six of the synthesized Ag-NHC
complexes were more effective in HL-60 than in MOLM-13 (Figure
1). We may speculate that the mechanism of action of the Ag-
NHC is reflected in the biology of the two cell lines. Particularly
NHC-X and NHC-XII would be interesting to test in normal bone
marrow cells and other tumor cells to determine if cytotoxicity is
beneficial for cancer cell eradication. NHC-XII may be a complex
that has lost its discriminative power between HL-60 and MOLM-
13, but represents a cytotoxic compound with a wider application.
Overall, the structure–activity optimization accomplished in
present study afforded a substantial augmentation of the cytotoxic
activity. Comparison of the most active silver complex NHC-XII
synthesized in this study with the Ag-NHC complexes prepared
in our previous works revealed a high potency improvements:
NHC-I : NHC-XII = 20 for the HL-60 cell line and NHC-I : NHC-XII
= 18 for the MOLM-13 cell line, and NHC-II : NHC-XII = 3.5 for the
HL-60 cell line and NHC-II : NHC-XII = 3.3 for the MOLM-13 cell
line.
Figure 2. X-ray crystal structure of the cationic moiety of silver-NHC complex
NHC-IX. The distance C1-Ag is 2.068(3) Å, the angle C1-Ag-C1’ is 180.00(0)°
and the angle between NHC-planes is 0.0(2) °. The symmetry operator for C1
to C1’ is (-x+1, -y+1, -z+1). The atomic displacement factors are given at a 50 %
probability. Hydrogens are omitted for clarity. Further details are provided in the
supporting information file.
O
CH3COOH
ZnCl2 , MgSO4
60 oC, 25 min.
H
H
3M HCl
KPF6
N
N
2
4
NH2
PF6
14
a
b
O
Conclusions
NH2 CH3
15
Five
different
silver-3-alkyl-1-mesityl-1H-imidazol-3-ium
O
3
H3C
complexes (NHC-IX–NHC-XIII) were synthesized, characterized,
and biologically evaluated for cytotoxicity. The complexes were
found to be cytotoxic in the micromolar concentration range
towards the two human cell lines HL-60 and MOLM-13. Space
demanding substituents installed on the N1 of the imidazole ring
concurrently with an alkyl chain on N3 augmented the cytotoxicity
of the silver NHC complex, revealing that the ligand molecular
structure is of paramount importance for the cytotoxicity of the
final silver-NHC complex. X-ray crystallography shown that a bis-
ligand silver complex (NHC-IX) was formed, which apparently act
in opposition to a mechanism of action that comprises “slow
release” of Ag+ ions. For now, we suggest that the mechanism of
action involves the entire Ag-NHC complex participating in an
outer sphere SET process[26] intercepting a redox process such
as the thioredoxin (Trx) system[27] that recently was observed as
a modulator of tumor expansion.[28] The Trx system plays an
important role for DNA synthesis and as a means to cope with
oxidative stress.[29] Moreover, the Trx system serves as a large
disulfide reductase, which catalyzes proteindisulfid-ditiol
AgNO3, K2CO3,
N
DCM, 20 oC, 24 h
PF
Ag
6
c
2
N
NHC-XI
H3C
Scheme 2. Synthesis of (1-butyl-3-mesityl-2,3-dihydro-1H-imidazol-2-yl)
silver(I) hexafluorophosphate NHC-XI. Reagents and conditions: (a) 2,4,6-
trimethylaniline 2, oxalaldehyde 3, butan-1-amine 15, Reflux, 25 min. (b) 3M
HCl, KPF6. (c) AgNO3, K2CO3, DCM, 20 oC, 24 h.
Ghdhayed, Haque, and collaborators[25] claimed that it is possible
to produce either mono-ligand-silver or bis-ligand-silver
complexes from the same NHC ligand. The key factor that
induced the formation of the mono- or bis-NHC Ag complex was
the reaction temperature used during the silver complexation. The
bis-NHC-Ag complex was produced when the complexation was
conducted at 20 oC, while the mono-NHC-Ag complex was
produced when the reaction mixture was refluxed (that is at 82 oC)
in CH3CN as the reaction medium. We conducted a similar
experiment to see if this observation was of general validity using
our ligand (that was used for NHC-IX). Our trials resulted in the
formation of the bis-NHC-Ag complex both at low and high
reaction temperature.
transformation with
a preserving CGPC-active site motif.
Reductase catalyzes TrxR selenium-sulfide-selenium-thiol
transformation and supplies electrons to a wide range of
enzymes.[29] Such a biological interaction involving Au-NHC
complexes towards TrxR have been revealed,[30] which make it
reasonable to believe that an analogous mechanism of action
might occurs with Ag-NHCs.
The brief structural-activity studies carried through in this study,
afforded an Ag-NHC complex (NHC-XII) comprising substantial
enhanced cytotoxicity, NHC-I versus NHC-XII » 20 ´. Further
structure-activity optimization studies involving novel silver-NHC
complexes are underway and will be reported in near future.
Mechanism of action studies are currently in course of planning.
Biology
The screening method used in this study was based on WST-1
and reflects cellular metabolism and mitochondrial activity, and
has been described in detail previously.[20] We used the cell line
HL-60 in this screen to test the new silver-NHC complexes NHC-
IX, NHC-X, NHC-XII, and NHC-XIII against a model for acute
3
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10.1002/cmdc.202000138
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COMMUNICATION
Experimental part
Chemistry
General Methods
phases were combined and washed with MTBE (3 ´ 10 mL). The
water was then removed under reduced pressure using a rotary
evaporator. The resulting target product was isolated as a brown
waxy compound in an isolated yield of 39% (0.71 g). 1H NMR (500
MHz, DMSO-d6) δ 9.48 (s, 1H), 8.12 (s, 1H), 7.95 (s, 1H), 7.16 (s,
2H), 4.29 (t, J = 6.9 Hz, 2H), 2.34 (s, 3H), 2.03 (s, 6H), 1.90 (p, J
= 7.4 Hz, 2H), 1.44 – 1.40 (m, 2H), 1.36 – 1.32 (m, 2H), 1.28 –
1.24 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 140.19, 137.30,
134.26, 131.14, 129.23, 123.88, 123.17, 60.42, 49.22, 32.22,
29.10, 25.30, 24.78, 20.57, 16.88.
1H and 13C NMR spectra were recorded at ambient temperature
at frequencies of 500 MHz and 125 MHz, respectively. The
chemical shifts were reported in ppm relative to residual CHCl3 for
proton (d = 7.26 ppm) and CDCl3 for carbon (d = 77.16 ppm) and
with DMSO-d6 for proton (d = 2.50 ppm) and for carbon (d = 39.0
ppm) with an external reference (TMS). Multiplicities are
abbreviated as follows: singlet (s), doublet (d), triplet (t), multiplet
(m), broad (br) and overlapped (o).
3-(8-hydroxyoctyl)-1-mesityl-1H-imidazolium bromide[31] 12
[NEW]. 1-(2,4,6-trimethylphenyl)-1H-imidazole 5 (5.00 mmol,
0.92 g), 8-bromo-1-octanol 8 (5 mmol, 0.65 mL) and THF (9.20
mL) were transferred to a round bottle flask (25 mL) equipped with
a condenser and a magnetic stirring bar. The mixture was heated
at reflux and stirred for 18 h. Then, the mixture was diluted with
MTBE (15 mL) whereupon the product precipitated. The mixture
was extracted with distilled water (2 ´ 15 mL) (give the product
some time to dissolve). The aqueous phases were combined and
washed with MTBE (3 ´ 10 mL). The water was then removed
under reduced pressure using a rotary evaporator. Target product
was obtained as a brown waxy compound in an isolated yield of
35% (0.69 g). 1H NMR (500 MHz, DMSO-d6) δ 9.49 (s, 1H), 8.12
(s, 1H), 7.95 (s, 1H), 7.15 (s, 2H), 4.29 (t, J = 7.0 Hz, 2H), 2.33 (s,
3H), 2.02 (s, 6H), 1.90 (p, J = 14.3 2H), 1.40 (p, J = 6.7 Hz, 2H),
1.26 (m, 10H). 13C NMR (126 MHz, DMSO-d6) δ 140.24, 137.24,
134.26, 131.14, 129.23, 123.93, 123.16, 60.63, 49.29, 32.43,
28.99, 28.71, 28.26, 25.36, 25.30, 20.56, 16.82.
Synthetic procedures
1-(2,4,6-trimethylphenyl)-1H-imidazole[22]
5
[25364-44-7].
Acetic acid (10 mL), aqueous formaldehyde 4 (40 mmol, 3.00 mL)
and aqueous glyoxal 3 (40 mmol, 4.60 mL) was transferred to a
three necked round bottom flask (50 mL) equipped with a
condenser and a magnetic stirring bar. The mixture was heated
at 70 oC, whereupon an mixture composed of acetic acid (10 mL),
ammonium acetate 1 (40 mmol, 3.08 g) in water (2 mL) and
mesitylamine 2 (39 mmol, 5.60 mL) were added drop-wise to the
solution over a period of 20 min. by means of a dropping-funnel.
The mixture was stirred at 70oC for another 18 h. The mixture was
then cooled at room temperature and drop-wise added to a stirred
aqueous solution of sodium bicarbonate (29.40 g / 300 mL). A
brown solid was formed, which was filtrated off by using a
Büchner funnel with filter paper and washed with water (3 ´ 20
mL). The product was left to dry, and finally recrystallized using
ethyl acetate to obtain target product in an isolated yield of 31%
(2.27 g). 1H NMR (500 MHz, DMSO-d6): δ = 7.62 (s, 1H), 7.17 (s,
1H), 7.11 (s, 1H), 7.03 (s, 2H), 2.29 (s, 3H), 1.92 (s, 6H) ppm.
13C NMR (126 MHz, DMSO-d6) δ = 137.97, 137.55, 134.66,
133.40, 128.74, 128.66, 120.34, 20.48, 16.91 ppm.
3-(6-ethoxy-6-oxohexyl)-1-mesityl-1H-imidazolium
bromide[31] 13 [NEW]. 1-(2,4,6-trimethyl-phenyl)-1H-imidazole
(10.0 mmol, 1.87 g) 5, ethyl-6-bromohexanoate 9 (10.0 mmol,
1.79 mL), and THF (19 mL) were transferred to a round bottle
flask (100 mL) equipped with a condenser and a magnetic stirrer
bar. The solution was heated at reflux and stirred for 18 h. The
mixture was diluted with MTBE (30 mL) and the product
precipitated. The mixture was extracted with distilled water (2 ´
15 mL) (give the product some time to dissolve). The aqueous
phases were combined and washed with MTBE (3 ´ 10 mL). The
water was then removed under reduced pressure using a rotary
evaporator. Target product was obtained as a brown waxy
compound in an isolated yield of 32% (1.30 g). 1H NMR (500 MHz,
DMSO-d6) δ 9.52 (s, 1H), 8.14 (s, 1H), 7.96 (s, 1H), 7.16 (s, 2H),
4.31 (t, J = 7.1 Hz, 2H), 4.04 (q, J = 7.1 Hz, 2H), 2.33 (s, 3H), 2.31
(t, J = 7.3 Hz, 2H) 2.02 (s, 6H), 1.92 (p, J = 7.2 Hz, 2H), 1.58 (p,
J = 7.4 Hz, 2H), 1.27 (p, J = 7.7 Hz, 2H), 1.17 (t, J = 7.1 Hz,
3H).13C NMR (126 MHz, DMSO-d6) δ 172.67, 140.22, 137.28,
134.27, 131.14, 129.23, 123.90, 123.15, 59.68, 49.07, 33.16,
28.70, 24.82, 23.63, 20.56, 16.85, 14.11.
3-(3-hydroxypropyl)-1-(2,4,6-trimethylphenyl)
imidazolium
31
]
bromide[
10 [656832-57-4]. 1-(2,4,6-trimethylphenyl)-1H-
imidazole 5 (10.0 mmol, 1.86 g), 3-bromo-1-propanol 6 (10 mmol,
0.90 mL) and THF (18 mL) was added to a round bottle flask
equipped with a condenser and a stirring bar. The solution was
heated and stirred at reflux for 18 h. The mixture was then diluted
with methyl tert-butyl ether (MTBE) (30 mL) whereupon the
product precipitated. The mixture was extracted with distilled
water (2 ´ 30 mL) (give the product some time to dissolve). The
aqueous phases were combined and washed with MTBE (3 ´ 20
mL). The water was then removed under reduced pressure using
a rotary evaporator. Resulting in a light brown waxy compound
1
that was isolated in a yield of 39% (1.27 g). H NMR (500 MHz,
DMSO-d6) δ 9.49 (s, 1H), 8.13 (s, 1H), 7.94 (s, 1H), 7.15 (s, 2H),
4.38 (t, J = 6.9 Hz, 1H), 3.47 (t, J = 5.8 Hz, 1H), 2.34 (s, 3H), 2.05
(m, 2H), 2.03 (s, 6H). 13C NMR (126 MHz, DMSO-d6) δ 140.14,
137.42, 134.28, 131.13, 129.17, 123.74, 123.25, 57.21, 46.94,
31.96, 20.53, 16.84 ppm.
3-butyl-1-mesityl-1H-imidazol-3-ium hexafluorophosphate[32]
14 [1176199-40-8]. 2,4,6-trimethylaniline 2 (0.14 mL, 1.0 mmol),
butylamine 15 (0.1 mL, 1.0 mmol), and acetic acid (0.26 mL, 4.5
mmol) were transferred to a round bottom flask (250 mL)
equipped with a magnetic stirrer bar. The mixture was heated at
60 oC for 5 min. Then, MgSO4 (0.241 g, 2.0 mmol) was added to
the mixture (denoted as mixture A). Another mixture B was
prepared by transferring to a round bottle flask equipped with a
magnetic stirrer bar: glyoxal 3 (0.12 mL, 1.0 mmol, 40 % weight
in aqueous solution), formaldehyde 4 (0.1 mL, 1.0 mmol, 37% in
3-(6-hydroxyhexyl)-1-(2,4,6-trimethylphenyl)
imidazolium
bromide[31] 11 [656832-48-3]. 1-(2,4,6-trimethylphenyl)-1H-
imidazole 5 (5.0 mmol, 0.93 g), 6-bromo-1-hexanol 7 (5.0 mmol,
0.90 mL), and THF (9.20 mL) were transferred to a round bottle
flask (25 mL) equipped with a condenser and a magnetic stirrer
bar. The mixture was heated at reflux and stirred for 18 h. The
mixture was diluted with MTBE (15 mL) whereupon the product
precipitated. The mixture was extracted with distilled water (2 ´
15 mL) (give the product some time to dissolve). The aqueous
4
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10.1002/cmdc.202000138
ChemMedChem
COMMUNICATION
aqueous solution), and acetic acid (0.26 mL, 4.5 mmol) that was
heated at 60 oC for 5 min. whereupon ZnCl2 (0.164, 1.2 mmol) and
Et2O (1.2 mL) were added. Mixture B was then added to mixture
(s, 4H), 6.93 (t, J = 1.6 Hz, 2H), 3.95 (t, J = 6.9 Hz, 4H), 2.37 (s,
6H), 1.84 (s, 12H), 1.68 – 1.73 (m, 4H), 1.20 – 1.11 (m, 4H), 0.89
(t, J = 7.3 Hz, 6H).13C NMR (126 MHz, CDCl3) δ 139.32, 135.49,
134.83, 129.16, 122.68, 121.99, 51.40, 33.23, 30.90, 21.07, 19.45,
17.42, 13.59. HRMS (ESI) m/z: calcd for C32H44N4107Ag
591.26169; found 591.26193 and calcd for C32H44N4109Ag
593.26135 found 593.26604.
o
A and the combined mixtures was stirred at 60 C for 25 min.
o
before cooling at 20 C. DCM (25 mL) and HCl(aq) (3M, 50 mL)
was added and the resulting mixture was stirred at 20 oC for 1 h.
The post-reaction mixture was then transferred to a separatory
funnel where the organic phase was separated, and water (50
mL) and potassium hexafluorphosphate (0.184 g, 1.0 mmol) was
Bis(1-(8-hydroxyoctyl)-3-mesityl-2,3-dihydro-1H-imidazol-2-
yl) silver(I) bromide NHC-XII was isolated in a yield of 67%
(using 0.6 mmol of substrate 12 and 0.6 mmol of AgNO3 with a
reaction time of 24 h).1H NMR (500 MHz, CDCl3) δ 6.87 (s, 8H),
4.10 (s, 4H), 3.54 (t, J = 6.6 Hz, 4H), 2.27 (s, 6H), 1.85 (s, 12H),
1.78 (s, 4H), 1.49 (m, 4H), 1.25 (s, 16H).13C NMR (126 MHz,
CDCl3) δ 135.41, 134.71, 129.36, 128.98, 122.76, 99.99, 62.53,
51.91, 32.61, 31.30, 29.16, 28.95, 26.17, 25.59, 21.10, 17.62.
HRMS (ESI) m/z: calcd for C40H60N4O2107Ag 735.37672; found
735.37700 and calcd for C40H60N4O2109Ag 737.37638 found
737.38167.
o
added. The mixture was stirred at 20 C 1 h, before the organic
phase was separated and dried over MgSO4. The organic phase
was filtered, and the solvent removed under reduced pressure
using a rotary evaporator. The resulting product was a yellow
waxy compound in an isolated yield of 73% (0.28 g). 1H NMR (500
MHz, DMSO-d6) δ 9.43 (s, 1H), 8.11 (s, 1H), 7.95 (s, 1H), 7.16 (s,
2H), 4.29 (t, J = 7.1 Hz, 2H), 2.34 (s, 3H), 2.02 (s, 6H), 1.89 (p, J
= 7.3 Hz, 2H), 1.29 (h, J = 7.4 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H).
13C NMR (126 MHz, DMSO-d6) δ 140.26, 137.20, 134.27, 131.14,
129.23, 123.96, 123.14, 49.06, 31.00, 20.54, 18.73, 16.79, 13.22.
Silver(I) NHC complexes[33] NHC-IX, NHC-X, NHC-XI, NHC-XII,
NHC-XIII. The Imidazolium salt (10, 11, 12, 13, 14) (0.60 mmol),
AgNO3 (0.102-0.154 g, 0.60-0.90 mmol), and DCM (30 mL) were
transferred to a round bottle flask (50 mL) equipped with a
magnetic stirrer bar. The reaction mixture was stirred for 2 min.
whereupon K2CO3 (10 mmol) was added, and then stirred for
another 3.5-24 h. The reaction mixture was then filtered through
a pad of Celite and the solvent volume was reduced until a volume
of »2 mL using a rotary evaporator. Target silver complex
precipitated from the concentrate when hexane (15 mL) was
added.
Bis(1-(6-ethoxy-6-oxohexyl)-3-mesityl-2,3-dihydro-1H-
imidazol-2-yl) silver(I) bromide NHC-XIII was isolated in a yield
of 61% (using 0.6 mmol of substrate 13 and 0.6 mmol of AgNO3
with a reaction time of 24 h). 1H NMR (500 MHz, CDCl3) δ 7.47 (d,
J = 1.7 Hz, 2H), 6.98 (d, J = 1.7 Hz, 2H), 6.95 (s, 4H), 4.11 (q, J
= 7.1 Hz, 8H), 2.35 (s, 6H), 2.29 (t, J = 7.4 Hz, 4H), 1.90 (s, 12H),
1.87 – 1.80 (m, 4H), 1.64 (p, J = 7.6 Hz, 4H), 1.25 (t, J = 7.1 Hz,
10H).13C NMR (126 MHz, CDCl3) δ 173.09, 139.16, 135.37,
134.62, 129.13, 122.68, 121.81, 60.15, 51.50, 33.81, 31.00, 30.85,
25.72, 24.23, 20.98, 17.47, 14.16. HRMS (ESI) m/z: calcd for
C40H56N4O4107Ag 763.33525; found 763.33556 and calcd for
C40H56N4O4109Ag 765.33491; found 765.33260.
Bis(1-(3-hydroxypropyl)-3-mesityl-2,3-dihydro-1H-imidazol-
2-yl) silver(I) bromide NHC-IX was isolated in a yield of 45%
(using 0.6 mmol of substrate 10 and 0.9 mmol of AgNO3 with a
reaction time of 3.5 h), or a yield of 52% when using 0.6 mmol of
substrate 10 and 0.6 mmol of AgNO3 and a reaction time of 24 h).
1H NMR (500 MHz, CDCl3) δ 7.20 (s, 2H), 6.85 (s, 6H), 4.42 (t, J
= 6.9 Hz, 4H), 3.63 (t, J = 5.4 Hz, 4H), 2.40 (s, 6H), 2.06 (m, 4H),
1.74 (s, 12H). 13C NMR (126 MHz, CDCl3) δ 138.73, 135.49,
Crystallography
A colourless crystal specimen with needle morphology was
mounted inside a MiTiGen MicroLoop containing Parabar 10312
oil (Hampton Research) and diffraction data were collected at 100
K using an Oxford Cryosystems, Cryostream 700 Plus N2 open
flow blower, on the PILATUS@SNBL diffractometerat the BM01A
station of the Swiss–Norwegian Beamlines at the ESRF
134.91, 129.05, 122.59, 120.99, 57.25, 48.59, 33.91, 21.19, 17.41. (Grenoble, France). The energy (wavelength) of the synchrotron
HRMS (ESI) m/z: calcd for C30H40N4O2107Ag 595.22022; found
595.22061 and calcd for C30H40N4O2109Ag 597.21988; found
597.21736.
radiation was set to 17.1774 keV (0.72179 Å) using a 16-bunch
electron storage ring mode. The crystals scatter weakly and the
Bragg profiles are split in two components. The data were
collected by way of one φ scans of 1.456 images for a total of 24
min., with an angular step of 0.5° in a shutter-free mode with the
PILATUS2M detector.
The structure suffers from disorder of the anionic (Br-) site,
situated on an inversion centre. The positional disorder can in
addition possibly be chemical as a fraction of the site might be
occupied by a NO3- ion. As this disorder cannot be resolved and
because there is no independent proof of the chemical
composition, the structure is only described with respect to the
cationic metal complex moiety. The Bragg diffraction pattern also
shows twinning. Only the major part of the two components has
been integrated. Further details are provided in the supporting
information file.
Bis(1-(6-hydroxyhexyl)-3-mesityl-2,3-dihydro-1H-imidazol-2-
yl) silver(I) bromide NHC-X was isolated in a yield of 93% (using
0.6 mmol of substrate 11 and 0.6 mmol of AgNO3 with a reaction
time of 24 h).1H NMR (500 MHz, CDCl3) δ 7.38 (s, 2H), 6.94 (s,
6H), 4.13 (s, 4H), 3.61 (t, J = 6.4 Hz, 4H), 2.36 (s, 6H), 1.88 (s,
12H), 1.83 (s, 4H), 1.62 – 1.52 (m, 5H), 1.49 – 1.39 (m, 4H), 1.29
(s, 4H).13C NMR (126 MHz, CDCl3) δ 139.29, 135.43, 134.74,
129.26, 122.67, 121.69, 61.91, 51.72, 32.32, 31.29, 30.95, 26.00,
25.21, 21.11, 17.58. HRMS (ESI) m/z: calcd for C36H52N4O2107Ag
679.31412; found 679.31443 and calcd for C36H52N4O2109Ag
681.31378; found 681.30640.
Bis(1-butyl-3-mesityl-2,3-dihydro-1H-imidazol-2-yl) silver PF6
[33]
NHC-XI
was isolated in a yield of 83% (using 0.6 mmol of
Biological activity screening.
substrate 11 and 0.6 mmol of AgNO3 with a reaction time of
24 h).1H NMR (500 MHz, CDCl3) δ 7.31 (t, J = 1.7 Hz, 2H), 6.95
The human cell lines HL-60 and MOLM-13 (American Type
Culture Collection) were cultured in RPMI 1640, 2 mM L-
5
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10.1002/cmdc.202000138
ChemMedChem
COMMUNICATION
Glutamine, 50 UmL-1 penicillin/streptomycin (Sigma Aldrich) and
10% fetal bovine serum (Biowest) and incubated in humidified
atmosphere at a temperature of 37oC under 5% CO2. The cells (2
´ 105 cells ´ mL-1) were treated with various concentrations
(0.001 µM ® 100 µM) of the five different silver N-heterocyclic
carbene complexes (NHC-IX, NHC-X, NHC-XI, NHC-XII, and
NHC-XIII) for a period of 24 h. The metabolic cell activity, that is
the cell viability, was measured by using the WST-1 cell
proliferation agent (Roche) as described by the supplier protocol
and read on a luminescence plate reader (Infinite 200 Pro, Tecan).
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We are grateful to the Swiss-Norwegian beamlines at the ESRF,
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beamline BM01A. E.A. is grateful to the University of Bergen,
Department of Chemistry for research fellowship funding. BTG
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Conflict of Interest
[25] a) R.A. Haque, M.Z. Ghdhayeb, S. Budagumpi, A.W. Salman, M.B.K.
Ahamed, A.M.S.A. Majid. Inorg. Chim. Acta 2013, 394, 519–525; b) M.Z.
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The authors declare no conflict of interest.
Keywords: cytotoxicity • leukemia • metallodrugs • synthesis •
NHC-silver
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6
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10.1002/cmdc.202000138
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Entry for the Table of Contents
Ag-NHC complexes were designed, synthesised, and biological evaluated versus human cell lines modelling acute myeloid leukemia
to reveal high cytotoxicity.
Institute and/or researcher Twitter usernames:
ORCID
•
•
•
•
Hans-René Bjørsvik: 0000-0001-9593-6079
Karl Wilhelm Törnroos: 0000-0001-6140-5915
Bjørn Tore Gjertsen: 0000-0001-9358-9704
Eirin Alme: 0000-0003-0690-3465
7
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