Ruthenium tetrazene complexes bearing glucose moieties on their periphery: Synthesis, characterization, and in vitro cytotoxicity

Article abstract of DOI:10.1002/aoc.5896

Ruthenium tetrazene complexes with general formula [Cp*RuCl(1,4-R₂N₄)] (Cp* = η⁵-C₅Me₅), where R = benzyl (1), 2-fluorobenzyl (2), β-d-glucopyranosyl-unprotected (3a) and acyl-protected (3b–d), 2-acet

Full text of DOI:10.1002/aoc.5896

Received: 31 January 2020
DOI: 10.1002/aoc.5896
Revised: 6 April 2020
Accepted: 26 April 2020
F U L L P A P E R
Ruthenium tetrazene complexes bearing glucose moieties
on their periphery: Synthesis, characterization, and in vitro
cytotoxicity
Vojteˇch Hamala1,2 | Andrea Martišová3 | Lucie Červenková Šťastná1
|
Jindřich Karban1 | Andrej Dancˇo4 | Adam Šimarek4 | Martin Lamacˇ4
|
Michal Horácˇek4 | Tamara Kolářová3 | Roman Hrstka3 | Róbert Gyepes4,5
|
Jiří Pinkas4
1Academy of Sciences of the Czech
Ruthenium tetrazene complexes with general formula [Cp*RuCl(1,4-R2N4)]
(Cp* = η5-C5Me5), where R = benzyl (1), 2-fluorobenzyl (2), β-D-glucopyr
anosyl-unprotected (3a) and acyl-protected (3b–d), 2-acetamido-β-D-
glucopyranosyl-unprotected (4a) and acyl-protected (4b–d), propyl-β-D-glu-
copyranoside-unprotected (5a), and O-acetylated (5b), were synthesized and
characterized using nuclear magnetic resonance and electrospray ionization–
mass spectrometry. In addition, the molecular structure of 3b was determined
using X-ray crystallography. The cytotoxicity of complexes against ovarian
(A2780, SK-OV-3) and breast (MDA-MB-231) cancer cell lines and non-
cancerous cell line HEK-293 was evaluated and compared to cisplatin activity.
The carbohydrate-modified complexes bearing acyl-protecting groups
exhibited higher efficacy (in low micromolar range) than unprotected ones,
where the most active 4d was superior to cisplatin up to five times against all
investigated cancer cell lines; however, no significant selectivity was achieved.
The complex induced apoptotic cell death at low micromolar concentrations
(0.5 μM for A2780 and HEK293; 2 μM for SK-OV-3 and MDA-MB-231).
Republic, Institute of Chemical Process
Fundamentals, v.v.i., Rozvojová 135,
Prague 6, 165 02, Czech Republic
2Faculty of Chemical Technology,
University of Chemistry and Technology
Prague, Technická 5, Prague 6, 166 28,
Czech Republic
3Regional Centre for Applied and
Molecular Oncology, Masaryk Memorial
Cancer Institute, Žlutý kopec 7, Brno,
65653, Czech Republic
4Academy of Sciences of the Czech
Republic, J. Heyrovský Institute of
Physical Chemistry, v.v.i., Dolejškova
2155/3, Prague 8, 182 23, Czech Republic
5Department of Inorganic Chemistry,
Charles University, Hlavova 2030, Prague
2, 128 43, Czech Republic
Correspondence
Jiří Pinkas, J. Heyrovský Institute of
Physical Chemistry, Academy of Sciences
of the Czech Republic, v.v.i., Dolejškova
2155/3, 182 23 Prague 8, Czech Republic.
Email: jiri.pinkas@jh-inst.cas.cz
K E Y W O R D S
anticancer activity, glucose derivatives, ruthenium complexes, tetrazene ligands
Funding information
Grantová Agentura České Republiky,
Grant/Award Number: 17-05838S;
Ministerstvo Zdravotnictví Ceské
Republiky, Grant/Award Number: DRO
MMCI, 00209805; Ministry of Health of
the Czech Republic; the Czech
Science Foundation
V. Hamala and A. Martišová contributed equally to this study.
Appl Organomet Chem. 2020;e5896.
wileyonlinelibrary.com/journal/aoc
© 2020 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.5896

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HAMALA ET AL.
1 | INTRODUCTION
Cp* = η5-C5Me5, COD = 1,4-cyclooctadiene) reacted with
azides Ph(CH2)nN3 (n = 1, 2) to give the same products
[Cp*RuCl{1,4-((CH2)nPh)N4}].[15,16] Ruthenium(II) bisamine
complex [Cp˄RuCl(NH)3] (where Cp˄ = η5-(1-methoxy-
2,4-di-tert-butyl-3-neopentylcyclopentadienyl)) was success-
fully used for the synthesis of tetrazenes with pendant arene
groups [Cp˄RuCl(1,4-R2N4)] (where R = Ph,4-MeOC4H4,
4-CF3C6H4) by Park et al.[17] Also Ru(III) precursor
[Cp˄RuCl2]2 reacted with various azide derivatives and gen-
erated [Cp˄RuCl(1,4-R2N4)] complexes, where R = Bn[18]
and 1-phenylethyl.[19] Surprisingly, the aforementioned
reports do not contain any information about the cytotoxic-
ity of ruthenium tetrazene complexes.
Here, we publish the synthesis, characterization, and
anticancer properties of a family of ruthenium tetrazene
complexes. As our team is interested in the conjugation
of carbohydrates with transition metal complexes,[20,21]
the main aim of the work was focused on tetrazenes
containing glucose or glucosamine moieties on their
periphery. Two initial assumptions were used for the
introduction of carbohydrate moieties into the tetrazene
ligand. The first one was based on the well-known “War-
burg effect,” that is, high glucose uptake by cancer
cells.[22] Second, we proposed to use a glucose moiety as
a pattern tailoring lipophilic properties of the whole met-
allodrug. Therefore, the effect of carbohydrate-protecting
O-acyl groups on the lipophilicity and cytotoxicity of the
ruthenium complex was evaluated.
Cancer still remains one of the main health problems
worldwide, although successful early case detection com-
bined with new therapeutic options led to almost 30%
decrease in overall mortality within the past 25 years.[1]
The success in cancer treatment by platinum-based drugs
(mainly cisplatin cis-[PtCl2(NH3)2]) led to an enormous
interest in other metal-based drugs (so-called meta-
llodrugs). Among others, ruthenium complexes played a
dominant role, whereas (ImH)+[trans-RuCl4(κ-S-dmso)
(κ-S-Im)]− (NAMI A)[2] and [(η6-arene)RuCl2(pta)][3]
(RAPTA, pta = l,3,5-triaza-7-phosphatricyclo[3.3.1.1]dec-
ane) were the most prominent ones. Both complexes
exhibited antimetastatic effect against cancer cells,
whereas the former even entered phase I and phase II clin-
ical trials (albeit not continuing further due to efficiency
under expectations). In addition, several other types of
highly cytotoxic ruthenium complexes were developed by
several laboratories, where IndH+[trans-RuCl4(κ-2-N-
Ind)2]− (Ind = indazol, KP1039) by Keppler,[4] [(η6-arene)
Ru (en)Cl][PF6] (en = ethylenediamine) by Sadler,[5] and
thiolato-bridged diruthenium complexes by Süss-Fink[6]
are representative examples. The effect of organic ligand(s)
on the complex cytotoxicity and selectivity was found cru-
cial and widely reviewed.[7–9]
Tetrazene ligands represent a wide family of poten-
tially redox-active ligands, which could adopt three
mesomeric forms: neutral tetraazadiene, anionic radical,
and dianionic with a central double bond.[10,11] Gener-
ally, the ligand is built up directly on a metal center
either by insertion of an organic azide into metal–imido
bond or by coupling of two organic azides with the evolu-
tion of dinitrogen. In the case of ruthenium central atom,
the coupling proceeded at the Ru(II) center, that is,
Ru(III) is reduced in situ by an excess of organic azide,
whereas the formed complexes are exclusively diamag-
netic irrespective of their geometry. The first ruthenium
tetrazene complexes [RuCl2(PMe3)2{1,4-Mes2N4}] (where
Mes = mesitylene) and [(η6-p-cymene)Ru{1,4-(R)MesN4}]
(where R = 2,4,6-t-Bu3C4H2 or Mes) were synthesized by
the reaction of mesityl azide with ruthenium precursors
2 | RESULTS AND DISCUSSION
Starting azides L1–L3b, L4a, and L5a/b were purchased
from a commercial vendor or prepared by literature
methods (for details, see the “Experimental” section).
The remaining acetic (L4b), propionic (L3c, L4c), and
butyric (L3d, L4d) acid esters were prepared by esterifi-
cation of the corresponding azide derivative, as described
in Supporting Information.
Ruthenium tetrazene complexes were synthesized
according to Scheme 1 under argon atmosphere. A
tetrameric structure of precursor [Cp*RuCl]4 was
activated by refluxing it in anhydrous acetonitrile.
Thus, in situ-generated [Cp*Ru(MeCN)3]Cl was reacted
[RuCl2(PMe3)3] and [(η6-p-cymene)Ru
=
N(2,4,6-t-
Bu3C4H2)] by Danopoulos et al.[12,13] At the same
time, dithiacarbamato ruthenium complex [Ru(Et2dtc)2
(1,4-Ts2N4)] (where Et2dtc = N,N-diethyldithiocarbamate
and Ts = tosyl) was synthesized, which possessed a trigo-
nal prismatic geometry around ruthenium atom.[14]
Other tetrazene complexes were identified as by-products
generated within azide-alkyne “click” cycloaddition reac-
tions catalyzed by coordinatively unsaturated cyclo-
pentadienyl ruthenium species. Either [Cp*RuCl(COD)]
or [Cp*RuCl(i-Pr)3] ruthenium precursors (where
with
a slightly substoichiometric amount (usually
1.95–1.98 eq.) of the corresponding azide in acetonitrile.
The color of the reaction mixture changed almost
instantly from orange to green, and gas evolution could
be observed in most cases. After evaporation of vola-
tiles, the crude products could be treated/manipulated
without inert atmosphere. The products were usually
purified using column chromatography on silica gel to
obtain the desired products as green solids or waxes.

HAMALA ET AL.
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2.1 | Molecular structure of 3b
Dark-green crystals suitable for X-ray diffraction analysis
were grown by diffusion of the hexane layer into a solu-
tion of 3b in CH2Cl2. The species 3b crystallized in a
monoclinic space group P21 (no. 4) and possessed four
symmetrically independent molecules in the crystal
structure (Figure S35). Selected geometric parameters are
provided in the Supporting Information (Table S1); the
molecular structure of one independent molecule and
selected bond distances and angles are shown in
Figure 1.
Ruthenium possessed a distorted tetrahedral environ-
ment, where the chlorine ligand, the centroid of the
cyclopentadienyl ring (denoted as Cg), and both terminal
nitrogen atoms of the tetrazene ligand were coordinated
to the metal center. Ruthenium and four nitrogen atoms
of tetrazene constituted almost the ideal plane with a
deviation up to 0.064 and 0.055 Å for nitrogen and
ruthenium atoms, respectively. The bonding situation
in the tetrazene ligand showed comparable values
for bonds between nitrogen atoms situated on the
lateral (N1–N2 1.318(6)−1.322(6) Å; N3–N4 1.300(6)
−1.323(5) Å) and central positions (N2–N3 1.311(5)
−1.318(6) Å). This is quite a different situation from pre-
viously published tetrazene ruthenium complexes
[RuCl2(PMe3)2{1,4-Mes2N4}][12] and [Cp˄RuCl(1,4-R2N4)]
(where R = Ph, Bn),[18] where the former exhibited neu-
tral tetraazadiene property (the difference between the
lateral and central bonds was 0.025 and 0.031 Å) and the
latter rather asymmetric tetrazene ligand. However, in
the case of 3b, this situation could be affected by the
SCHEME 1 Preparation of ruthenium tetrazene complexes
1–5b
Tetrazenes were obtained in moderate to good yields
in the range 22–83%. It is noteworthy that the reaction
is tolerant to all functional groups present in the
starting azides (i.e., hydroxyl, ester, amide), and we did
not observe any transformation/deprotection during
the reaction.
In addition to the azide derivatives described earlier,
adamantyl azide was subjected to reaction. However,
the reaction of the bulky azide with [Cp*Ru(MeCN)3]Cl
did not lead to a characteristic change in color (from
orange to green), and only limited gas evolution was
observed (for details, see Supporting Information).
After heating of the reaction mixture to reflux, 1H
NMR (nuclear magnetic resonance) spectroscopy
exhibited a rather complicated mixture of products. We
propose that the bulky adamantyl group precluded the
formation of tetrazene framework, as was described
previously for the reaction of [Cp*RuCl(NH3)2] with
2,6-diisopropylphenyl azide.[17]
All prepared complexes were characterized by 1H
NMR and 13C NMR, and all the obtained data corrobo-
rated the proposed structures. Electrospray ionization–
mass spectrometry (ESI–MS) always showed a molecular
ion as
a [M +
Na]+ ion. Coordination of two
glucopyranosyl moieties to ruthenium can be easily dem-
1
onstrated using H NMR spectroscopy. The product for-
mation can be traced according to the characteristic,
ꢀ1.2–1.5 ppm, large down-field shift of anomeric protons
(in comparison to the starting azide). Moreover, the coor-
dination to ruthenium causes the formation of two sets of
signals, each belonging to one of the two nonequivalent
glucopyranosyl moieties. The complexes exhibited good
stability toward air and water. The stability of 3b in water
solution was investigated in detail. The solution of 3b in
aqueous dmso-d6 was repeatedly monitored using 1H
NMR spectroscopy for 5 weeks and did not show any
deterioration (Figure S1).
FIGURE 1 Selected molecular structure of 3b at the 30%
probability level with partial labeling. Hydrogen atoms are omitted
for clarity. Selected range of bond distances (Å) and angles (ꢁ): Ru–
Cl 2.343(1)−2.355(1); Ru–Cg 1.875(2)−1.880(2); Ru–N1 1.949(3)
−1.967(4); Ru–N4 1.983(4)−1.993(4); N1–N2 1.318(6)−1.322(6); N2–
N3 1.311(5)−1.318(6); N3–N4 1.300(6)−1.323(5); Cl–Ru–N1
88.94(13)−96.69(13); Cl–Ru–N4 86.39(12)−92.42(13); N1–Ru–N4
73.22(14)−73.47(17)

4 of 12
HAMALA ET AL.
presence of either four independent molecules in the
structure or bulky glycopyranosyl substituents.
However, the situation was less clear for complexes bear-
ing O-acyl-protected carbohydrate derivatives, although all
of them possessed log P values within the region. The com-
plexes exhibited increasing cytotoxicity in the order
3a ꢀ 3d < 3c < 3b for β-D-glucopyranosyl-substituted,
4a ꢀ 4b < 4c < 4d for 2-acetamido-β-D-glucopyranosyl-
substituted, and 5a < 5b for propyl β-D-glucopyranosides.
In this respect, a dramatic decrease in the activity for 3d
should be mentioned, which we tentatively attribute to its
decreased aqueous solubility. This result suggests that
lipophilicity/aqueous solubility should be well balanced in
metallodrug design. The resolution of this problem for 3d
(by the introduction of noncoordinating anion instead of
chloride ligand) is currently under investigation.
2.2 | Cytotoxicity studies
The cytotoxicity of new compounds against selected can-
cer cell lines A2780 and SK-OV-3 derived from ovarian
cancer, and MDA-MB-231 representing triple negative
breast cancer, was evaluated by MTT assay and is pres-
ented, together with compound lipophilicity, in Table 1
as IC50 values after 72 h incubation. Human embryonal
kidney cell line HEK-293 was used to evaluate the effect
of tested compounds on noncancerous cells.
Carbohydrate-free complexes 1 and 2 exhibited promis-
ing cytotoxicity at low micromolar (7.5 and 4.8 μM) con-
centrations against cisplatin-sensitive cells A2780 and only
about an order of magnitude worse (24 and 54 μM) against
intrinsically cisplatin-resistant cell line SK-OV-3.[23]
This itself is a reason for further investigation of various
tetrazene complexes. However, our main interest lies in
carbohydrate-substituted complexes 3–5 and particularly
in determining how increasing lipophilicity of complexes
from unprotected (3a, 4a, 5a) to butyryl-protected (3d and
4d) ones influences the cytotoxicity. Generally, complexes
3a, 4a, and 5a with unprotected carbohydrate hydroxyls
exhibited none or only negligible activity against all studied
lines. These results were consistent with the complexes’
poor lipophilicity (see Table 1) positioned outside of
Lipinski's druggable region (log P from −0.4 to 5.6).[24] This
indicated the importance of lipophilic functionality in
the design of (metalo)drugs, as noted previously.[25,26]
Among all complexes, three most active ones contained
lipophilic propionyl and butyryl groups and followed the
order 3c < 3b < 4d, where the most active one exhibited
activities substantially higher than cisplatin.
To determine the mechanism of cell death in
response to 4d, the induction of programmed cell death
was investigated using Annexin V/PI (prodidium iodide)
staining, which enables the detection of both early and
late apoptotic cells (Figure 2). Consistent with data from
MTT assay, the induction of apoptosis was detected in
A2780 and HEK293 cells exposed to lower doses from
0.5 μM of 4d. In contrast, the induction of apoptotic cell
death in SK-OV-3 and MDA-MB-231 cells required
higher doses of ꢀ2 μM of 4d.
To measure the proliferative capacity of cells exposed
to 4d, we used clonogenic assay (Figure 3). As the graph
shows, colony formation in cell culture medium was
increasingly inhibited with increasing concentration of
TABLE 1 Tested compounds’ lipophilicity (log P) and their cytotoxicity (IC50, μM) against cell lines as listed after 72 h treatment
Compound
Log P
0.6
A2780
SK‐OV‐3
24
MDA‐MB‐231
ND
HEK‐293
12
1
7.5 0.8
4.8 1.3
65 16
3
1
2
0.5
54 10
>100
ND
26 10
>100
3a
−2.3
0.9
ND
3b
0.31 0.25
0.68 0.21
>100
2.5 0.7
5.3 0.2
>100
ND
0.9 0.4
1.9 0.8
>100
3c
0.9
4.3 1.2
>100
3d
0.6
4a
−2.4
0.3
23
4
>100
ND
58
8
4b
50 10
>100
>100
>100
4c
1.6
1.49 0.05
0.36 0.06
>100
14.7 0.8
2.01 0.21
>100
6.6 2.3
1.02 0.25
>100
4.2 2.1
0.60 0.19
>100
4d
0.2
5a
−2.4
0.6
5b
29
7
43
8
>100
22
1
Cisplatin
ND
1.7 0.3
5.6 1.0
3.7 0.6
3.8 0.5
Note: ND, not determined.

HAMALA ET AL.
5 of 12
FIGURE 2 Determination of programmed cell death. The graphs were constructed from three independent biological experiments. *,
p < 0.05, NT represents untreated cells serving as control
and SK-OV-3 cells exposed to 4d was observed (data not
shown).
3 | CONCLUSIONS
We have shown that various organic azides react
smoothly with commercially accessible Ru(II) precursor
[Cp*RuCl]4 after its thermal activation in acetonitrile.
The reaction produced air- and water-stable ruthenium
tetrazene complexes that could be easily purified using
column chromatography on silica. It should be noted that
FIGURE 3 Clonogenic assay. Relative numbers of colonies in
organic azides are popular building blocks widely used in
percentage were calculated with respect to colonies developed from
“click chemistry”[27–29] and available from many com-
untreated cells. *, p < 0.05
mercial vendors. Moreover, they could be prepared from
primary amines by their reaction with fluorosulfuryl
4d, but at the same time the ability of particular cell lines
to develop colonies after being challenged with 4d
showed the same trend as MTT assay. More sensitive
A2780 and HEK-293 cells developed fewer colonies, espe-
cially in response to higher doses of 4d. On the contrary,
the most resistant SK-OV-3 cells formed most colonies
despite the presence of relatively high doses of 4d
(Figure 3).
The effect of 4d on cell cycle was also determined.
Although SK-OV-3 and MDA-MB-231 showed differences
in the fractions of cells in the individual cell-cycle phases
compared to A2780 and HEK-293 cells, no significant
effect of 4d on cell-cycle distribution was observed.
Similarly, no effect on cell migration of MDA-MB-231
azide, as was demonstrated by more than 1200 exam-
ples.[30] Therefore, a large family of ruthenium tetrazene
complexes could be quickly synthesized and tested.
The evaluation of in vitro cytotoxicity revealed a sub-
stantially stronger cytotoxicity for O-acyl (propionyl and
butyryl) complexes than for unprotected ones. The most
active complex 4d showed IC50 values up to five times
better compared to cisplatin against all cancer cell lines
under study; however, no significant selectivity against
cancer cell lines was achieved. The complex induced apo-
ptotic cell death at low micromolar concentrations
(0.5 μM for A2780 and HEK293; 2 μM for SK-OV-3; and
MDA-MB-231), whereas no significant cell-cycle pertur-
bation was observed.

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HAMALA ET AL.
4 | EXPERIMENTAL
evaporated to dryness, and purified using column
chromatography.
Air-sensitive compounds were manipulated under an
argon atmosphere using standard Schlenk techniques.
Anhydrous acetonitrile was obtained from Sigma-Aldrich
(St. Louis, MO, USA) and stored over MS 3 Å. [Cp*RuCl]4
was obtained from Strem Chemicals (Newburyport,
MA, USA). Benzyl azide (L1), 2-fluorobenzyl azide
(L2), adamantyl azide, and 2,3,4,6-tetra-O-acetyl-β-
D-glucopyranosyl azide (L3b) were obtained from
Sigma-Aldrich. β-D-glucopyranosyl azide (L3a),[31]
2-acetamido-2-deoxy-β-D-glucopyranosyl azide (L4a),[31]
4.1.2 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-dibenzyltetrazene)] (1)
[Cp*RuCl]4 (0.136 g, 0.125 mmol) dissolved in 8 ml of
acetonitrile was reacted with a solution of benzyl azide
(L1) in dichloromethane (2.0 ml 0.5 M solution, 1.00 mmol)
for 30 min according to the general procedure. Column
chromatography (silica gel, eluent tetrahydrofuran [THF]/
heptane, 2/5, v/v; Rf = 0.32) produced 0.240 g (79%) of 1 as
a green-brown amorphous solid.
3-azidopropyl-β-D-glucopyranoside
(L5a),[32]
and
3-azidopropyl 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-
glucopyranoside (L5b)[33] were synthesized according to
the literature. The synthesis of 2,3,4,6-tetra-O-propionyl-
β-D-glucopyranosyl azide (L3c), 2,3,4,6-tetra-O-butyryl-β-
D-glucopyranosyl azide (L3d), 2-acetamido-2-deoxy-
3,4,6-tri-O-acetyl-β-D-glucopyranosyl azide (L4b), 2-ace-
tamido-2-deoxy-3,4,6-tri-O-propionyl-β-D-glucopyranosyl
azide (L4c), and 2-acetamido-2-deoxy-3,4,6-tri-O-butyryl-
β-D-glucopyranosyl azide (L4d) is described in the
Supporting Information.
4.1.3 | NMR spectra in line with
literature[16]
1H NMR (CD2Cl2, 300 MHz): 7.39–7.25 (m, 10H, Ph),
5.60, 5.36 (2 × d, 2 × 2H, J = 14.1 Hz, CH2), 1.65 (s, 15H,
CH3 Cp*). 13C{1H} NMR (CD2Cl2, 75 MHz): 136.4 (Cipso
,
Ph), 129.3, 128.6 (2 × 2CH, Ph), 127.9 (CH, Ph), 100.5 (Cq
Cp*), 70.4 (CH2), 10.4 (CH3 Cp*). HRMS–ESI (m/z):
[M + Na]+, 100%, calculated for C24H29ClN4RuNa
533.1019; found 533.1017.
1H (400.1/300 MHz), 13C (100.6/75 MHz), and 19F
(282 MHz) NMR spectra were measured using a Bruker
Avance 400 or Varian Mercury 300 spectrometer at
25 ꢁC. The 1H NMR and 13C NMR spectra were
referenced to the solvent signal (δ/ppm; δH/δC: CDCl3,
7.26/77.16, dmso-d6, 2.50/39.52; CD2Cl2, 5.32/53.84).
High-resolution mass spectrometry (HRMS) spectra
were measured with a Bruker MicrOTOF-QIII spectro-
meter using acetonitrile solutions of the samples. The
spectrometer was calibrated using sodium formate, and
electrospray ionization source in positive mode was used
for all analyses. Melting points were determined on a
Koffler block and were not corrected.
4.1.4 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
{1,4-bis(2-fluorobenzyl)tetrazene}] (2)
[Cp*RuCl]4 (0.189 g, 0.174 mmol) dissolved in 10 ml of
acetonitrile was reacted with a solution of 2-fluorobenzyl
azide (L2) in methyl tert-butyl ether (2.5 ml 0.5 M
solution, 1.25 mmol) for
3
days according to
the general procedure. Evaporation of the reaction
mixture produced a crude product, which contained
ꢀ5 mol% of 2-fluorobenzaldehyde (singlet at 10.38 ppm
characteristic of aldehydic CHO proton).[34] Purification
of the crude material using column chromatography (sil-
ica gel, eluent dichloromethane, Rf = 0.64) produced 2 as
a green solid. Yield 205 mg (64%).
4.1.1 | General procedure for the
preparation of ruthenium tetrazene
complexes
[Cp*RuCl]4 was mixed with dry acetonitrile
(c = 0.0125 mol/l) under argon atmosphere, and the
suspension was heated by a heat gun until the solid
dissolved completely and a transparent orange solution
was obtained. Dried ligand (7.8 eq.) was dissolved
(or suspended) in dry acetonitrile (c = 0.2 mol/l), and the
resulting solution (or suspension) was added dropwise to
the stirred solution of the ruthenium precursor. An
almost-immediate change in color (orange to green) and
gas evolution were observed. The reaction mixture was
stirred at room temperature for the desired time,
Mp 114 ꢁC. 1H NMR (CDCl3, 300 MHz): 7.31–7.23 (m,
4H, o-F-C6H4), 7.12–7.06 (m, 4H, o-F-C6H4), 5.62, 5.40
(2 × d, 2 × 2H, J = 15.2 Hz, CH2), 1.69 (s, 15H, CH3 Cp*).
13C{1H} NMR (CDCl3, 75 MHz): 160.4 (d, 1JCF = 246.0 Hz,
CF, o-F-C6H4), 131.7 (d, JCF = 3.8 Hz, CH, o-F-C6H4),
129.6 (d, JCF = 8.1 Hz, CH, o-F-C6H4), 124.2 (d,
2
JCF = 3.6 Hz, CH, o-F-C6H4), 122.9 (d, JCF = 14.7 Hz,
CCH2, o-F-C6H4), 115.2 (d, JCF = 21.6 Hz, CH, o-F-C6H4),
3
100.3 (Cq Cp*), 63.1 (d, JCF = 3.7 Hz, CH2), 10.1
(Cq Cp*). 19F NMR (CDCl3, 282 MHz): −117.7 (m, 2F,
o-F-C6H4).

HAMALA ET AL.
7 of 12
4.1.5 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
[M + Na]+, 100%, calculated for C38H53ClN4O18RuNa
1013.1987; found 1013.1982.
(1,4-bis(β-D-glucopyranosyl)tetrazene)] (3a)
[Cp*RuCl]4 (0.267 g, 0.246 mmol) dissolved in 10 ml of ace-
tonitrile reacted with solid L3a (0.393 g, 1.92 mmol) for
48 h. The product precipitated during the reaction from
the reaction mixture. Filtration and washing with dry ace-
tonitrile produced 9 as a green dust. Yield 0.479 g (83%).
4.1.7 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-bis(2,3,4,6–tetra-O-propionyl-β-D-
glucopyranosyl)tetrazene)] (3c)
1
ꢁ
Mp 130 C (decomp.). H NMR (dmso-d6, 400 MHz):
δ 5.49 (d, 1H, J = 8.4 Hz, H-10), 5.38 (d, 1H, J = 8.7 Hz,
H-1), 5.21 (d, 1H, J = 4.8 Hz, OH-3/4), 5.16–5.13 (m, 3H,
OH-3/4, OH-30, OH-40), 4.59 (t, 1H, J = 5.7 Hz, OH-60),
4.52 (t, 1H, J = 5.2 Hz, OH-6), 4.18 (ddd, 1H, J = 8.7, 8.4,
2.9 Hz, H-20), 3.73–3.59 (m, 5H, H-2, 2H-6, OH-20, H-60),
3.51–3.40 (m, 6H, H-3, H-4, H-5, H-30, H-50, H-60),
3.35–3.27 (m, 2H, OH-2, H-40), 1.75 (s, 15H, CH3 Cp*).
13C{1H} NMR (dmso-d6): δ 103.3 (Cq Cp*), 102.3 (C-1),
100.2 (C-10), 80.3 (C-50), 80.0 (C-5), 77.3 (C-30), 76.2 (C-3),
74.8 (C-20), 74.1 (C-2), 69.8 (C-40), 69.6 (C-4), 61.0 (C-60),
60.6 (C-6), 10.2 (CH3 Cp*). HRMS–ESI (m/z): [M + Na]+,
100%, calculated for C22H37ClN4O10RuNa 677.1137;
found 677.1132.
[Cp*RuCl]4 (0.068 g, 0.063 mmol) reacted with L3c
(0.201 g, 0.468 mmol) according to the general procedure.
Column chromatography (35 g silica gel, eluent PE:
EtOAc 2:1, Rf = 0.22) produced 3c as a green gel. Yield
0.195 g (70%).
1H NMR (dmso-d6, 400 MHz): δ 6.17 (d, 1H,
J = 9.4 Hz, H-10), 6.09 (dd, 1H, J = 9.5, 8.9 Hz, H-2), 5.85
(d, 1H, J = 8.9 Hz, H-1), 5.73 (t, 1H, J = 9.5 Hz, H-3),
5.56 (t, 1H, J = 9.4 Hz, H-30), 5.27 (t, 1H, J = 9.4 Hz, H-
20), 5.22 (dd, 1H, J = 10.2, 9.4 Hz, H-40), 5.10 (dd, 1H,
J = 10.0, 9.5 Hz, H-4), 5.53 (ddd, 1H, J = 10.0, 6.2, 2.2 Hz,
H-5), 4.45 (ddd, 1H, J = 10.2, 4.5, 2.2 Hz, H-50), 4.31 (dd,
1H, J = 12.7, 4.5 Hz, H-60), 4.11 (dd, 1H, J = 12.4, 6.2 Hz,
H-6), 4.03–3.94 (m, 2H, H-6, H-60), 2.38–2.15 (m, 12H,
CH2CH3), 2.08–1.84 (m, 4H, CH2CH3), 1.77 (s, 15H, CH3
Cp*), 1.08–0.93 (m, 18H, CH3), 0.75, 0.73 (2 × t, 2 × 3H,
J = 7.5 Hz, CH3). 13C{1H} NMR (dmso-d6, 101 MHz): δ
173.2 (2 × CO), 173.1, 173.0, 172.7, 172.6, 171.6, 171.5
(6 × CO), 103.9 (Cq Cp*), 98.4 (C-10), 95.8 (C-1), 73.7 (C-
30), 73.4 (C-50), 73.1 (C-3), 73.0 (C-5), 71.3 (C-20), 70.1 (C-
2), 68.0 (C-4), 66.9 (C-40), 62.0 (C-6), 61.2 (C-60), 27.0,
26.93, 26.85, 26.83, 26.81, 26.75, 26.64, 26.56
(8 × OCH2CH3), 10.0 (CH3 Cp*), 9.00, 8.97, 8.9, 8.81
(4 × CH2CH3), 8.8 (2 × CH2CH3), 8.5, 8.4 (2 × CH2CH3).
HRMS–ESI (m/z): [M + Na]+, 100%, calculated for
C46H69ClN4O18RuNa 1125.3241; found 1125.3240.
4.1.6 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-bis(2,3,4,6–tetra-O-acetyl-β-D-
glucopyranosyl)tetrazene)] (3b)
[Cp*RuCl]4 (0.256 g, 0.236 mmol) dissolved in 10 ml of
acetonitrile reacted with L3b (745 mg, 2.00 mmol)
dissolved in the same solvent (20 ml) according to the
general procedure. Column chromatography (silica gel,
eluent dichloromethane:THF 1:4, Rf = 0.89) produced 3b
as a green amorphous solid. Yield 0.774 g (82%).
1
ꢁ
Mp 124 C (decomp.). H NMR (dmso-d6, 400 MHz):
δ 6.16 (d, 1H, J = 9.3 Hz, H-10), 6.06 (dd, 1H, J = 9.3,
8.9 Hz, H-2), 5.85 (d, 1H, J = 8.9 Hz, H-1), 5.70 (dd, 1H,
J = 9.8, 9.3 Hz, H-3), 5.53 (dd, 1H, J = 9.4, 9.3 Hz, H-30),
5.27 (t, 1H, J = 9.3 Hz, H-20), 5.19 (dd, 1H, J = 10.3,
9.4 Hz, H-40), 5.08 (t, 1H, J = 9.8 Hz, H-4), 4.51 (ddd, 1H,
J = 9.8, 6.2, 2.3 Hz, H-5), 4.43 (ddd, 1H, J = 10.3, 4.6,
2.2 Hz, H-50), 4.28 (dd, 1H, J = 12.4, 4.6 Hz, H-60), 4.09
(dd, 1H, J = 12.4, 6.2 Hz, H-6), 4.03 (dd, 1H, J = 12.4,
2.3 Hz, H-6), 3.97 (dd, 1H, J = 12.4, 2.2 Hz, H-60), 2.04,
2.03, 2.00 (3 × s, 3 × 3H, CH3 OAc), 1.95 (2 × s, 2 × 3H,
CH3 OAc), 1.92 (s, 3H, CH3 OAc), 1.78 (s, 15H, CH3 Cp*),
1.69, 1.58 (2 × s, 2 × 3H, CH3 OAc). 13C{1H} NMR (dmso-
d6, 101 MHz): δ 169.9 (CO), 169.8 (2 × CO), 169.7, 169.4,
169.3, 168.28, 168.26 (5 × CO), 103.9 (Cq Cp*), 98.2 (C-10),
95.9 (C-1), 73.8 (C-30), 73.3 (C-50), 73.1 (C-3), 73.0 (C-5),
71.4 (C-20), 70.2 (C-2), 68.2 (C-4), 67.1 (C-40), 62.1 (C-60),
61.3 (C-6), 20.9, 20.8, 20.49, 20.48, 20.44, 20.41, 20.39,
20.3 (8 × CH3 OAc), 10.0 (CH3 Cp*). HRMS–ESI (m/z):
4.1.8 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-bis(2,3,4,6–tetra-O-butyryl-β-D-
glucopyranosyl)tetrazene)] (3d)
[Cp*RuCl]4 (0.080 g, 0.074 mmol) reacted with L3d (0.280 g,
0.577 mmol) according to the general procedure. Column
chromatography (45 g silica gel, eluent PE:EtOAc 5:2,
Rf = 0.34) produced 3d as green gel. Yield 0.276 g (77%).
1H NMR (dmso-d6, 400 MHz): δ 6.14 (d, 1H, J = 9.4 Hz,
H-10), 6.09 (dd, 1H, J = 9.5, 8.8 Hz, H-2), 5.83 (d, 1H,
J = 8.8 Hz, H-1), 5.74 (t, 1H, J = 9.5 Hz, H-3), 5.59 (dd, 1H,
J = 9.7, 9.1 Hz, H-30), 5.35 (dd, 1H, J = 9.4, 9.1 Hz, H-20),
5.21 (dd, 1H, J = 10.2, 9.7 Hz, H-40), 5.11 (dd, 1H, J = 10.0,
9.5 Hz, H-4), 5.51 (ddd, 1H, J = 10.0, 5.2, 2.8 Hz, H-5), 4.44
(ddd, 1H, J = 10.2, 4.6, 2.2 Hz, H-50), 4.26 (dd, 1H, J = 12.7,
4.6 Hz, H-60), 4.06–4.00 (m, 2H, H-6), 3.94 (dd, 1H, J = 12.7,
2.2 Hz, H-60), 2.35–2.12 (m, 12H, OCH2CH2), 2.03–1.81 (m,

8 of 12
HAMALA ET AL.
4H, OCH2CH2), 1.77 (s, 15H, CH3 Cp*), 1.57–1.42 (m, 12H,
OCH2CH2), 1.30–1.20 (m, 4H, OCH2CH2), 0.89–0.81 (m,
18H, CH3), 0.69 (t, 3H, J = 7.5 Hz, CH2CH3), 0.67 (t, 3H,
J = 7.4 Hz, CH2CH3). 13C{1H} NMR (dmso-d6, 101 MHz): δ
172.28, 172.27, 172.1, 172.0, 171.7, 171.56, 170.62, 170.59
(8 × CO), 103.9 (Cq Cp*), 98.2 (C-10), 95.8 (C-1), 73.6 (C-30),
73.5 (C-50), 73.0 (C-5), 72.9 (C-3), 71.0 (C-20), 70.0 (C-2), 68.0
(C-4), 67.1 (C-40), 61.8 (C-6), 61.2 (C-60), 35.5, 35.4, 35.3,
35.24, 35.17, 35.13, 35.10, 35.05 (8 × OCH2CH2), 17.8, 17.73
(2 × CH2CH3), 17.70 (2 × CH2CH3), 17.68, 17.5, 17.3, 17.2
(4 × CH2CH3), 13.30 (2 × CH2CH3), 13.31 (CH2CH3), 13.30
(2 × CH2CH3), 13.28, 13.25, 13.2 (3 × CH2CH3), 10.0 (CH3
Cp*). HRMS–ESI (m/z): [M + Na]+, 100%, calculated for
C54H85ClN4O18RuNa 1237.4495; found 1237.4489.
THF 4:1, Rf = 0.42) produced 4b as a green amorphous
solid. Yield 0.270 g (56%).
1H NMR (dmso-d6, 400 MHz): δ 7.26 (d, 1H,
J = 8.2 Hz, NH), 6.89 (d, 1H, J = 6.4 Hz, NH0), 6.21 (d,
1H, J = 9.5 Hz, H-10), 6.12 (d, 1H, J = 9.3 Hz, H-1), 5.93
(t, 1H, J = 9.7 Hz, H-30), 5.70 (t, 1H, J = 9.8 Hz, H-3),
5.06 (dd, 1H, J = 9.7, 9.4 Hz, H-40), 5.02 (dd, 1H, J = 9.8,
9.4 Hz, H-4), 4.80 (ddd, 1H, J = 9.8, 9.3, 8.2 Hz, H-2), 4.33
(dd, 1H, J = 12.6, 4.7 Hz, H-60), 4.23–4.11 (m, 3H, H-5, H-
6, H-50), 4.03–3.95 (m, 3H, H-6, H-20, H-60), 2.03, 2.01,
2.00 (3 × s, 3 × 3H, CH3 OAc), 1.96 (s, 2 × 3H, CH3 OAc),
1.93 (s, 3H, CH3 OAc), 1.76 (s, 15H, CH3 Cp*), 1.51, 1.48
(2 × s, 2 × 3H, CH3 NHAc). 13C{1H} NMR (dmso-d6,
101 MHz): δ 170.0, 169.94, 169.91, 169.6, 169.5, 169.42,
169.39, 169.1 (8 × CO), 103.4 (Cq Cp*), 97.3 (C-10), 96.3
(C-1), 73.7 (C-50), 73.5 (C-5), 72.7 (C-3), 71.6 (C-30), 68.8
(C-4), 68.4 (C-40), 62.2 (C-6), 61.6 (C-60), 55.2 (C-20), 53.3
(C-2), 23.3, 22.9 (2 × CH3 NHAc), 20.54, 20.51, 20.48,
20.46, 20.44, 20.42 (6 × CH3 OAc), 10.2 (CH3 Cp*).
HRMS–ESI (m/z): [M + Na]+, 100%, calculated for
C38H55ClN6O16RuNa 1011.2306; found 1011.2311.
4.1.9 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-bis(2-acetamido-2-deoxy-β-D-
glucopyranosyl)tetrazene)] (4a)
[Cp*RuCl]4 (0.238 g, 0.270 mmol) reacted with L4a
(0.518 g, 2.10 mmol) according to the general procedure.
The product was precipitated from the reaction mixture,
filtered, and washed with dry THF producing 4a as a
green powder. Yield 0.563 g (71%).
4.1.11 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-bis(2-acetamido-2-deoxy-3,4,6–tri-O-
propionyl-β-D-glucopyranosyl)tetrazene)]
(4c)
1
ꢁ
Mp 124 C (decomp.). H NMR (dmso-d6, 400 MHz):
δ 7.18 (d, 1H, J = 8.2 Hz, NH), 6.59 (brs, 1H, NH0), 5.97
(d, 1H, J = 9.5 Hz, H-10), 5.87 (d, 1H, J = 9.3 Hz, H-1),
5.16 (d, 1H, J = 5.5 Hz, OH-30), 5.11 (d, 1H, J = 5.5 Hz,
OH-4), 5.09 (d, 1H, J = 5.7 Hz, OH-40), 5.03 (d, 1H,
J = 5.3 Hz, OH-3), 4.57 (t, 1H, J = 5.7 Hz, OH-6), 4.46
(ddd, 1H, J = 9.3, 9.1, 8.2 Hz, H-2), 4.41 (ddd, 1H,
J = 9.1, 8.9, 5.5 Hz, H-30), 4.26 (brs, 1H, OH-60), 3.86
(ddd, 1H, J = 9.4, 9.1, 5.3 Hz, H-3), 3.76–3.66 (m, 2H, H-
6, H-60), 3.62–3.41 (m, 5H, H-5, H-6, H-20, H-50, H-60),
3.32–3.25 (m, 2H, H-4, H-40), 1.72 (s, 15H, CH3 Cp*),
1.57, 1.51 (2 × s, 2 × 3H, CH3 NHAc). 13C{1H} NMR
(dmso-d6, 101 MHz): δ 170.1, 169.2 (2 × CO), 102.5
(Cq Cp*), 97.7 (C-10), 97.6 (C-1), 80.2 (C-5), 79.8 (C-50),
74.2 (C-3), 72.0 (C-30), 71.0 (C-40), 70.8 (C-4), 61.1 (C-60),
61.0 (C-6), 59.0 (C-20), 56.0 (C-2), 23.8, 23.4 (2 × CH3
NHAc), 10.3 (CH3 Cp*). HRMS–ESI (m/z): [M + Na]+,
100%, calculated for C26H43ClN6O10RuNa 759.1669;
found 759.1664.
[Cp*RuCl]4 (0.087 g, 0.080 mmol) reacted with L4c
(0.258 g, 0.623 mmol) according to the general procedure.
Column chromatography (40 g silica gel, eluent EtOAc,
Rf = 0.44) produced 4c as a green amorphous solid. Yield
0.125 g (36%).
1H NMR (dmso-d6, 400 MHz): δ 7.25 (d, 1H,
J = 8.4 Hz, NH), 6.92 (d, 1H, J = 7.4 Hz, NH0), 6.18 (d,
1H, J = 9.6 Hz, H-10), 6.11 (d, 1H, J = 9.3 Hz, H-1), 5.92
(t, 1H, J = 9.7 Hz, H-30), 5.71 (t, 1H, J = 9.8 Hz, H-3),
5.09 (t, 1H, J = 9.7 Hz, H-40), 5.04 (t, 1H, J = 9.8 Hz, H-
4), 4.81 (ddd, 1H, J = 9.8, 9.3, 8.4 Hz, H-2), 4.37 (dd, 1H,
J = 12.6, 4.7 Hz, H-60), 4.27–4.19 (m, 2H, H-5, 50), 4.17
(dd, 1H, J = 12.4, 6.0 Hz, H-6), 4.08 (ddd, 1H, J = 9.7, 9.6,
7.4 Hz, H-20), 4.02 (dd, 1H, J = 12.4, 1.8 Hz, H-6), 3.96
(dd, 1H, J = 12.6, 2.0 Hz, H-60), 2.33–2.16 (m, 12H,
CH2CH3), 1.76 (s, 15H, CH3 Cp*), 1.50, 1.47 (2 × s,
2 × 3H, CH3 NHAc), 1.03–0.96 (m, 18H, CH2CH3).
13C{1H} NMR (dmso-d6, 101 MHz): δ 173.26, 173.25,
172.9, 172.8, 172.7, 172.6, 169.9, 169.1 (8 × CO), 103.4 (Cq
Cp*), 97.4 (C-10), 96.3 (C-1), 73.8 (C-50), 73.6 (C-5), 72.6
(C-3), 71.7 (C-30), 68.6 (C-4), 68.3 (C-40), 62.1 (C-6), 61.5
(C-60), 55.0 (C-20), 53.3 (C-2), 26.91, 26.85, 26.84, 26.81,
26.7, 26.6 (6 × CH2CH3), 23.3, 22.9 (2 × CH3 NHAc), 10.2
(CH3 Cp*), 9.13, 9.07, 8.94, 8.91 (4 × CH2CH3), 8.8
(2 × CH2CH3). HRMS–ESI (m/z): [M + Na]+, 100%,
4.1.10 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-bis(2-acetamido-2-deoxy-3,4,6–tri-O-
acetyl-β-D-glucopyranosyl)tetrazene)] (4b)
[Cp*RuCl]4 (0.134 g, 0.123 mmol) reacted with L4b
(0.358 g, 0.96 mmol) according to the general procedure.
Column chromatography (30 g silica gel, eluent EtOAc:

HAMALA ET AL.
9 of 12
calculated for C44H67ClN6O16RuNa 1095.3247; found
1095.3253.
H-5), 2.33–2.29, 2.16–2.08 (2 × m, 2 × 2H, CHH), 1.69 (s,
15H, CH3 Cp*). 13C{1H} NMR (dmso-d6, 101 MHz): δ
103.0, 102.8 (2 × C-1), 99.4 (Cq Cp*), 76.8 (2C-3, 2C-5),
73.6, 73.5 (2 × C-2), 70.12, 70.09 (2 × C-4), 66.61, 66.56
(2 × OCH2), 63.7, 63.5 (2 × NCH2), 61.13, 61.10 (2 × C-6),
29.2, 29.1 (2 × CH2), 9.6 (CH3 Cp*). HRMS–ESI (m/z):
[M + Na]+, 100%, calculated for C28H49ClN4O12RuNa
793.1976; found 793.1981.
4.1.12 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-bis(2-acetamido-2-deoxy-3,4,6–tri-O-
butyryl-β-D-glucopyranosyl)tetrazene)] (4d)
[Cp*RuCl]4 (0.054 g, 0.050 mmol) reacted with L4d
(0.178 g, 0.390 mmol) according to the general procedure.
Column chromatography (40 g silica gel, eluent PE:
EtOAc 3:2, Rf = 0.30) produced 4d as a green amorphous
solid. Yield 0.145 g (63%).
4.1.14 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-bis(3-(2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl)-propyl)tetrazene)] (5b)
1H NMR (dmso-d6, 400 MHz): δ 7.27 (d, 1H,
J = 8.4 Hz, NH), 6.89 (d, 1H, J = 7.3 Hz, NH0), 6.19 (d,
1H, J = 9.5 Hz, H-10), 6.09 (d, 1H, J = 9.3 Hz, H-1), 5.93
(t, 1H, J = 9.8 Hz, H-30), 5.70 (t, 1H, J = 9.8 Hz, H-3),
5.11 (t, 1H, J = 9.8 Hz, H-40), 5.06 (t, 1H, J = 9.8 Hz, H-
4), 4.82 (ddd, 1H, J = 9.8, 9.3, 8.4 Hz, H-2), 4.32 (dd, 1H,
J = 12.7, 4.6 Hz, H-60), 4.26–4.19 (m, 2H, H-5, 50), 4.14
(dd, 1H, J = 12.4, 5.6 Hz, H-6), 4.05–4.01 (m, 2H, H-6, H-
20), 3.96 (dd, 1H, J = 12.7, 1.8 Hz, H-60), 2.30–2.14 (m,
12H, OCH2CH2), 1.76 (s, 15H, CH3 Cp*), 1.55–1.44 (m,
18H, 6OCH2CH2, 2CH3 NHAc), 0.88–0.80 (m, 18H,
CH2CH3). 13C{1H} NMR (dmso-d6, 101 MHz): δ 172.37,
172.36, 171.9, 171.8, 171.7, 171.6, 169.9, 169.0 (8 × CO),
103.4 (Cq Cp*), 97.4 (C-10), 96.4 (C-1), 73.8 (C-50), 73.6 (C-
5), 72.5 (C-3), 71.5 (C-30), 68.5 (C-4), 68.2 (C-40), 61.9 (C-
6), 61.5 (C-60), 55.1 (C-20), 53.2 (C-2), 35.4, 35.3, 35.24,
35.18, 35.17, 35.1 (6 × OCH2CH2), 23.3, 22.9 (2 × CH3
NHAc), 17.9, 17.83, 17.75, 17.73 (4 × CH2CH3), 17.70
(2 × CH2CH3), 13.38, 13.37, 13.31 (3 × CH2CH3), 13.28
(2 × CH2CH3), 13.26 (CH2CH3), 10.2 (CH3 Cp*). HRMS–
[Cp*RuCl]4 (0.178 g, 0.164 mmol) reacted with L5b
(0.600 g, 1.39 mmol) overnight according to the general
procedure. Column chromatography (20 g silica gel, elu-
ent hexane:EtOAc 1:3, Rf = 0.70) produced 5b as a green
solid. Yield 0.451 g (62%).
1
ꢁ
Mp 64 C. H NMR (dmso-d6, 400 MHz): δ 5.28, 5.27
(2 × dd, 2 × 1H, J = 9.8, 9.4 Hz, H-3), 4.90 (t, 2H,
J = 9.8 Hz, H-4), 4.82–4.78 (m, 4H, H-1, H-2), 4.42–4.31
(m, 2H, NCHH), 4.23–4.14 (m, 4H, NCHH, H-6),
4.04–3.97 (m, 4H, H-6, H-5), 3.84–3.75 (m, 2H, OCHH),
3.63–3.54 (m, 1H, OCHH), 3.45–3.39 (m, 1H, OCHH),
2.32–2.13 (m, 4H, CHH), 2.02, 2.01, 1.98 (3 × s, 3 × 6H,
CH3 OAc), 1.95, 1.94 (2 × s, 2 × 3H, CH3 OAc), 1.67 (s,
15H, CH3 Cp*). 13C{1H} NMR (dmso-d6, 101 MHz): δ
170.0 (2 × CO-6), 169.6 (2 × CO-3), 169.3 (2 × CO-4),
169.2, 169.1 (2 × CO-2), 99.54 (C-1), 99.46 (Cq Cp*), 99.4
(C-1), 72.1, 72.0 (2 × C-3), 71.01, 70.97 (2 × C-2), 70.6,
70.5 (2 × C-5), 68.2 (2 × C-4), 67.4, 67.0 (2 × OCH2), 63.2,
62.8 (2 × NCH2), 61.8 (2 × C-6), 28.8, 28.7 (2 × CH2),
20.5, 20.4 (2 × 3 CH3 OAc), 20.3 (2 × CH3 OAc), 9.5 (CH3
Cp*). HRMS–ESI (m/z): [M + Na]+, 100%, calculated for
C44H65ClN4O20RuNa 1129.2826; found 1129.2826.
ESI (m/z): [M
+
Na]+, 100%, calculated for
C50H79ClN6O16RuNa 1179.4188; found 1179.4187.
4.1.13 | [η5-(C5Me5)RuCl-1,4-κ2-N,N0-
(1,4-bis(3-(β-D-glucopyranosyl)-propyl)
tetrazene)] (5a)
4.2 | X-ray crystallography
Diffraction data for 3b were collected on a Bruker
D8 VENTURE diffractometer (Mo-Kα radiation,
λ = 0.71073 Å) at 150 K using an Oxford Cryostream
cooler. The collected data were reduced using the diffrac-
tometer software. The phase problem was solved by
intrinsic phasing, and structure models were refined by
full-matrix least squares on F2 (SHELX-2018/3).[35] All
non-hydrogen atoms were refined anisotropically. Hydro-
gen atoms were placed in idealized positions and refined
isotropically using the riding model. Solvent molecules
present in the structure could not be refined with satis-
factory results, and their contribution to diffraction
data was subtracted using the SQUEEZE procedure
[Cp*RuCl]4 (0.360 g, 0.332 mmol) reacted with L5a
(0.684 g, 2.59 mmol) overnight according to the general
procedure. Column chromatography (20 g silica gel, elu-
ent acetone:EtOH 10:1, Rf = 0.80) produced 5a as a green
foam. Yield 0.154 g (22%).
1H NMR (dmso-d6, 400 MHz): δ 4.99, 4.98 (2 × d,
2 × 1H, J = 4.5 Hz, OH), 4.93 (d, 2H, J = 4.5 Hz, OH),
4.89 (d, 2H, J = 4.5 Hz, OH), 4.50–4.40 (m, 4H, 2OH,
2NCHH), 4.34–4.25 (m, 2H, NCHH), 4.18, 4.16 (2 × d,
2 × 1H, J = 7.7 Hz, H-1), 3.89–3.82 (m, 2H, OCHH),
3.69–3.65 (m, 2H, H-6), 3.61–3.49 (m, 2H, OCHH),
3.46–3.41 (m, 2H, H-6), 3.15–2.96 (m, 8H, H-2, H-3, H-4,

10 of 12
HAMALA ET AL.
implemented in PLATON.[36] Molecular graphics was
performed using the PLATON program.
CCDC 1979972 contains supplementary crystallo-
graphic data deposited for 3b. These data can be obtained
free of charge from The Cambridge Crystallographic Data
Centre at www.ccdc.cam.ac.uk/data_request/cif.
added to water and 1-octanol to a final concentration of
1 mM. The same volumes of water and octanol con-
taining the same complex were equally mixed and
shaken for 5 min. Then the tubes were shortly spun, and
samples from aqueous and organic layers were subjected
to UV–Vis. spectrophotometry (NanoDrop 2000, Thermo-
Fisher Scientific). Absolute amounts of individual com-
plexes were calculated by constructing respective
calibration curves (data not shown). Log P was defined as
the logarithm of the ratio of the concentrations of a sol-
ute between the two solvents.
4.3 | Cell lines and cytotoxicity testing
ꢁ
All human cells were maintained at 37 C in humidified
atmosphere with 5% CO2. A2780 cells were grown in high-
glucose RPMI-1640 and SK-OV-3 cells in McCoy's 5ª, and
HEK 293 and MDA-MB-231 cells were maintained in high-
glucose Dulbecco's modified Eagle's medium (DMEM, all
media were from Sigma-Aldrich (St. Louis, MO, USA)).
Each media was supplemented with 10% fetal bov
ine serum (Gibco, ThermoFisher Scientific, Waltham,
MA, USA), 300 μg/ml of L-glutamine (Sigma-Aldrich),
and 100 μg/ml of HyClone Penicillin–Streptomycin 100X
solution (BioSera, Nuaille, France). The culture medium
was changed during each cell passage every 2–3 days.
Cells were grown to 60–80% confluence before experi-
mental treatments with tested compounds at concentra-
tions from 1 to 100 μM. Cells were mycoplasma free
throughout the experiments.
Cells were seeded in a 96-well plate at a density of
3000 cells/well for cytotoxicity testing. The next day,
these cells were exposed to selected compounds for 72 h.
The cell viability was measured using colorimetric MTT
assay as described previously.[37] Data from MTT assay
were analyzed in GraphPad Prism software and
expressed as IC50 values (compound concentrations that
produce 50% of cell metabolic inhibition). All experi-
ments were performed independently at least thrice.
4.6 | Cell-cycle determination
Cells (2 × 105) were seeded in six-well plates and treated
with 0.1, 0.5, or 1 μM 4d for 48 h. The cells were then
washed twiꢁce with PBS and fixed in 70% ethanol over-
night at 4 C. After incubation, the cells were washed
with PBS and stained with 1 ml of PI staining solution
per sample (0.1% Triton X-100, 10 μg/ml propidium
iodide, 100 μg/ml DNAse-free RNAase A, Sigma-Aldrich)
for 30 min at room temperature in the dark. After incuba-
tion, the fluorescence was measured using a flow
cytometer (FACS Verse, BD Biosciences, Franklin Lakes,
NJ, USA) and evaluated using BD FACSuite v 1.0.6. In
total, 10,000 events per sample were recorded.
4.7 | Annexin V cell death analysis
Cells (105) were seeded in 12-well plates, and then A2780
and HEK-293 cells were treated with 0.1, 0.5, and 1 μM of
4d for 24 h, whereas SK-OV-3 and MDA-MB-231 cells
were exposed to 1, 2, and 4 μM of 4d for 24 h. Then, the
cells were collected with Accutase and washed twice with
PBS. Cell pellets were then resuspended in 50 μl of
staining solution prepared from 1× binding buffer (20×
Annexin V Binding Buffer, MACS Miltenyi Biotec), 1 μl
of FITC-Annexin V (Biolegends, San Diego, CA, USA),
and 0.5 μl of 1 mg/ml PI Sigma-Aldrich (St. Louis, MO,
USA) and incubated for 20 min in the dark at room tem-
perature. The fluorescence signal was detected using a
flow cytometer (FACS Verse, BD Biosciences). A total of
10,000 events were recorded for each sample. The per-
centage of apoptotic cells was quantified using FCS
Express 4 software (BD Biosciences).
4.4 | Clonogenic assay
A total of 200 cells per well were seeded in six-well plates,
and next day these cells were treated with 4d at 100-,
500- and 1000-nM concentration. Ten days later, the cells
were washed twice with phosphate-buffered saline (PBS)
and fixed for 30 min in methanol, and developed colonies
were then visualized using Giemsa–Romanowski staining
and counted.
4.5 | Determination of partition
coefficient (log P) by “shake flask” method
4.8 | Wound healing assay
The tested compounds were dissolved at 20-mM concen-
tration in dimethyl sulfoxide. Particular stocks were
Confluent cells grown in 12-well plates were scraped with
a sterile micropipette tip and then incubated in serum-

HAMALA ET AL.
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ACKNOWLEDGMENTS
This work was supported by the Czech Science Founda-
tion (Grantová Agentura České Republiky, project num-
ber 17-05838S) and Ministry of Health of the Czech
Republic (Ministerstvo Zdravotnictví České Republiky,
project DRO MMCI, 00209805). The authors thank
Dr. Lidmila Petrusová for melting point determinations.
Jiří Pinkas thanks Ms. Ivana Dobiášová for assistance in
the preparation of 5a and 5b.
CONFLICT OF INTEREST
[25] C. T. Campbell, U. Aich, C. A. Weier, J. J. Wang, S. S. Choi,
M. M. Wen, K. Maisel, S. G. Sampathkumar, K. J. Yarema,
J. Med. Chem. 2008, 51, 8135.
[26] S. M. Meier-Menches, C. Gerner, W. Berger, C. G. Hartinger,
B. K. Keppler, Chem. Soc. Rev. 2018, 47, 909.
The authors declare no competing financial interest.
FUNDING INFORMATION
This work was supported by the Czech Science Founda-
tion (project number 17-05838S) and Ministry of Health
of the Czech Republic (project DRO MMCI, 00209805).
[27] P. Thirumurugan, D. Matosiuk, K. Jozwiak, Chem. Rev. 2013,
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[29] X. Y. Jiang, X. Hao, L. L. Jing, G. C. Wu, D. W. Kang,
X. Y. Liu, P. Zhan, Expert Opin. Drug Discov. 2019, 14, 779.
[30] G. Meng, T. Guo, T. Ma, J. Zhang, Y. Shen, K. B. Sharpless,
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ORCID
Jiří Pinkas https://orcid.org/0000-0003-3478-0802
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How to cite this article: Hamala V, Martišová A,
Červenková Šťastná L, et al. Ruthenium tetrazene
complexes bearing glucose moieties on their
periphery: Synthesis, characterization, and in vitro
cytotoxicity. Appl Organomet Chem. 2020;e5896.
https://doi.org/10.1002/aoc.5896
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