Organoruthenium and Organoosmium Complexes of 2-Pyridinecarbothioamides Functionalized with a Sulfonamide Motif: Synthesis, Cytotoxicity and Biomolecule Interactions

Article abstract of DOI:10.1002/cplu.201800194

Anticancer-active Ru^II–η⁶-p-cymene complexes of bioactive 2-pyridinecarbothioamide ligands have been shown to have high selectivity for plectin and can be administered orally. Reported herein is the functionalization of a 2-pyridinecarbothioamide with a sulfonamide group and its conversion into M–η⁶-p-cymene complexes (M = Ru, Os). The presence of a sulfonamide moiety in many organic drugs and metal complexes endows these agents with interesting biological properties and can transform the latter into multi-targeted agents. The compounds were characterized with standard methods and the in vitro anticancer activity data was compared with studies on the hydrolytic stability of the complexes and their reactivity to small biomolecules. A molecular modeling study revealed plausible modes of binding of the complexes in the catalytic pocket of carbonic anhydrase II.

Full text of DOI:10.1002/cplu.201800194

Accepted Article
Title: Organoruthenium and -osmium Complexes of 2-
Pyridinecarbothioamides Functionalized with a Sulfonamide
motif: Synthesis, Cytotoxicity and Biomolecule Interaction
Authors: Jahanzaib Arshad, Muhammad Hanif, Ayesha Zafar, Sanam
Movassaghi, Kelvin Tong, Jóhannes Reynisson, Mario
Kubanik, Amir Waseem, Tilo Söhnel, Stephen Jamieson, and
Christian Hartinger
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10.1002/cplu.201800194
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Organoruthenium and -osmium Complexes of 2-
Pyridinecarbothioamides Functionalized with a Sulfonamide motif:
Synthesis, Cytotoxicity and Biomolecule Interaction
Jahanzaib Arshad,a,b Muhammad Hanif,a,* Ayesha Zafar,a Sanam Movassaghi,a
Kelvin K. H. Tong,a Jóhannes Reynisson,a Mario Kubanik,a Amir Waseem,b Tilo
Söhnel,a Stephen M. F. Jamieson,c Christian G. Hartingera,*
a School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland
1142, New Zealand.
b Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan.
c Auckland Cancer Society Research Centre, University of Auckland, Private Bag
92019, Auckland 1142, New Zealand
* School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland
1142, New Zealand. http://hartinger.auckland.ac.nz/
E-mail: c.hartinger@auckland.ac.nz; m.hanif@auckland.ac.nz; Fax: (+64) 9 373
7599 ext 87422
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Abstract
Anticancer active RuII(η6-p-cymene) complexes of bioactive 2-pyridinecarbothioamide
ligands (PCAs) were shown to have high selectivity for plectin and can be administered
orally (Chem. Sci., 2013, 4, 1837–1846 and Angew. Chem. Int. Ed., 2017, 56, 8267 –
8271). Herein, we report the functionalization of the PCA ligand with a sulfonamide
group and its conversion into M(η6-p-cymene) complexes (M = Ru, Os). The presence
of the sulfonamide motif in many organic drugs and metal complexes endowed these
agents with interesting biological properties and may result in the latter case in
multitargeted agents. The compounds were characterized with standard methods and
the in vitro anticancer activity data was compared with studies on the hydrolytic
stability of the complexes and their reactivity to small biomolecules. A molecular
modelling study against carbonic anhydrase II revealed plausible binding modes of the
complexes in the catalytic pocket.
Keywords
Anticancer Activity; Sulfonamide; Organoruthenium Compounds; Bioorganometallics;
2-Pyridinecarbothiamide Ligands.
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Introduction
Sulfonamides constitute an important class of pharmacologically active agents. Drugs
featuring this pharmacophore have been used for the treatment of a variety of
conditions, from infectious diseases to antiepileptic or antiobesity drugs.[1-4] The
sulfonamide group is known to form adducts with Zn2+ ions present in active sites of
metalloenzymes, particular in those that are overexpressed in diseased conditions.
For example, carbonic anhydrases (CAs)[5] and histone deacetylases (HDACs) are
Zn-containing metalloenzymes overexpressed in many tumors. These enzymes are
considered important targets in anticancer drug discovery.[6] Sulfonamides have been
extensively investigated to inhibit the activity of these enzymes, in particular CAs.
Under the basic conditions used in the enzyme inhibition assay, the deprotonated
nitrogen atom of the sulfamoyl moiety of these compounds binds to the Zn ion in the
active site of the enzyme and disrupts its catalytic process.[5-7] The remaining
components of the drugs’ structures are involved in various hydrophilic and/or
hydrophobic interactions with amino acid residues of the active site and/or water
molecules. This was demonstrated by X-ray crystallographic analysis of the adduct
formation between various CAs and many representatives of sulfonamide-based
inhibitors.[5,8-13] The sulfonamide acetazolamide and its derivatives were evaluated
both in in vitro and in vivo assays as human carbonic anhydrase IX (h-CA IX) targeted
anticancer agents,[14] whereas several examples including indisulam (E7070) and
SLC-0111 entered clinical trials for the treatment of various advanced solid
[15,16]
tumors.
Sulfonamides are versatile chelating ligands and, depending on their structure, they
can act as mono-, bi- or tridentate donor systems to transition metals ions.[1,6,17,18] The
coordination complexes of different clinically-used sulfonamides with AgI, CoII, NiII, CuII
and ZnII have been evaluated for their biological properties.[19-21] In many examples,
the enzyme inhibitory activity of these metal complexes was better than of their ligands
alone, possibly due to synergistic effects between the metal ion and the sulfonamide
by interacting with different areas of the active site of the enzyme.[6,17] Similarly, Re
and 99mTc complexes of sulfonamides were developed for molecular imaging of h-CA
IX-expressing tumors (Figure 1).[22,23] Some of these compounds demonstrated
nanomolar affinities for the pharmaceutically-relevant isozymes h-CA IX and h-CA XII,
which was much higher than that of acetazolamide, a benchmark organic inhibitor for
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CAs. A co-crystal structure of a Re complex with h-CA II showed that the deprotonated
nitrogen of the sulfonamide group bound to the catalytically-active Zn center and the
[CpRe(CO)3] moiety showed hydrophobic interactions with Phe131, Leu198, and
Pro202.[24] Biological activity and structure-activity relationships (SAR) for
metallocenes functionalized with the sulfonamide pharmacophore through triazole,
triazole-ester, triazole-amide, amide and urea linkers were reported. These
compounds showed moderate to good inhibitory activity in vitro and some examples
demonstrated high selectivity for cancer-associated CA IX and CA XII compared to
off-target CA I and II.[25]
Figure 1. The structures of lead bioactive metal complexes bearing a sulfonamide
pharmacophore.
Ruthenium half-sandwich complexes of the general structure [(η6-arene)Ru(bipy)Cl]+
displayed very high affinity towards h-CA II.[26] The co-crystal structure of h-CA II with
the Ru(arene) complex revealed that the complex bound to the catalytic zinc site
through the sulfonamide moiety. The aryl spacer formed close contacts with the
hydrophobic residues of the enzymes and the Ru(arene) scaffold was positioned at
the entrance of the cavity. Interestingly, there was no direct interaction between the
ruthenium center and the protein, despite the presence of a labile chlorido ligand.[26]
We have recently developed organometallic anticancer complexes of 2-
pyridinecarbothioamide ligands (PCAs).[27-31] The Ru complex termed plecstatin-1
demonstrated target selectivity for plectin in an invasive B16 melanoma tumor model.
[30]
Herein, we report the functionalization of the PCA scaffold with the sulfonamide
pharmacophore and its coordination to RuII/OsII(cym) (cym = η6-p-cymene)
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organometallics. The compounds were evaluated for their tumor-inhibition potential
against a panel of human cancer cell lines and their stability in solution as well as their
reactivity toward small biomolecules. Their interaction with CA was studied by
molecular modelling.
Results and Discussion
Bioactive PCAs can act as S,N-bidentate ligands to metal ions to access a library of
organometallic and coordination compounds.[27,32,33] We functionalized a PCA ligand
with a sulfonamide, a motif found in many drugs and involved in interactions with the
active sites of CAs. The sulfonamide-substituted PCA 1 was prepared in a one-pot
synthesis by refluxing p-phenylenediamine sulfanilamide and elemental sulfur in 2-
picoline for 18 h with a catalytic amount of sodium sulfide (Scheme 1). After work up
and recrystallization from acetonitrile, 1 was obtained in a good yield of 67%. The
ligand was characterized by NMR spectroscopy, ESI-MS, elemental analysis and
single crystal X-ray diffraction. In the 1H NMR spectrum of 1, the thioamide proton was
detected at 12.48 ppm. This accounts for a downfield shift of ca. 2 ppm as compared
to the amide proton of picolinamide ligands.[34] The protons of the pyridine ring were
observed in the range of 7.6–8.7 ppm, while the signals assigned to the aromatic
phenyl protons were detected in the range of 7.8–8.2 ppm. In the 13C{H} NMR
spectrum the pyridine ring carbon atoms were detected in the range of 124–153 ppm
while the carbons of the aromatic ring resonated between 124.3 and 141.5 ppm. The
ESI-mass spectrum of the ligand featured the pseudomolecular ion [1 + Na]+ at m/z
316.0157 which is in close agreement with the calculated value.
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Scheme 1. Synthetic route to N-(4-sulfamoylphenyl)pyridine-2-carbothioamide 1 and its
organometallic RuII and OsII complexes 1a–1d with the numbering scheme used to assign the
signals in the NMR spectra.
The molecular structure of N-(4-sulfamoylphenyl)pyridine-2-carbothioamide 1 was
determined by single crystal X-ray diffraction analysis (Figure 2). Crystals were grown
by slow evaporation from a methanol-dichloromethane mixture at room temperature.
PCA 1 crystallized in the monoclinic space group Cc (compare Table 3 for the
crystallographic parameters). The hydrogen and oxygen atoms of the sulfonamide
group were involved in intermolecular H bonds with other molecules of 1. The pyridine
and benzene rings were found to be disordered indicating a strong displacement along
the S2-C10-C7-N2 and C6-C5-C2 axes in the molecule.
Figure 2. Molecular structure of N-(4-sulfamoylphenyl)pyridine-2-carbothioamide 1 drawn at
50% probability level.
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Compound 1 was converted into the corresponding RuII(cym) and OsII(cym)
complexes 1a–1d in good yields (53–88%). The reactions were performed under
nitrogen atmosphere by reacting 1 (2 eq.) with [Ru/Os(cym)X2]2 (1 eq.) in a mixture of
tetrahydrofuran and dichloromethane at 40 °C for 4 h (Scheme 1). The red to dark
red/black products were obtained after filtration[27] and were characterized by 1D and
1
2D NMR spectroscopy, ESI-MS and elemental analysis. The H NMR spectra of all
complexes were recorded in d4-MeOD (Figures S1–S4). Due to the fast H/D exchange
in protic deuterated solvents, the thioamide proton was not detected while the spectra
recorded for 1a and 1d in DMSO-d6 featured peaks at around 7.3 ppm absent in the
former (Figure S5). The H4 and H1 protons of the pyridine ring were deshielded due
to coordination of the pyridine nitrogen atom causing a shift by ca. 1 ppm. The nature
of the metal ion had only a slight effect on the 1H and 13C{1H} NMR chemical shifts of
the PCA ligand. The 13C{1H} spectra (Figures S6–S9) contained most of the expected
peaks but some of the quaternary carbon atoms were not detected, presumably
because of too low concentration of the samples. Importantly, the spectra showed
significant differences for the aromatic p-cymene C–H atoms for the Ru complex 1a
as compared to its Os counterpart 1b. These carbon atoms resonated about 10 ppm
downfield in case of 1a as compared 1b. Similar shifts have been observed for related
compounds while in other cases the shifts were less pronounced.[35-37]
The molecular structure of a single crystal formed from slow diffusion of diethyl ether
into methanol solution of 1d was determined by single crystal X-ray diffraction analysis
(Figure 3; compare Table 3 for the crystallographic parameters). The Os center
adopted a pseudooctahedral coordination geometry and 1 coordinated to the metal
ion as an anionic N,S-bidentate ligand after deprotonation of the amide group.
Therefore, we label this compound as 1dneutral. This is in contrast to all other molecular
structures of related Ru and Os complexes where the PCA ligand was neutral and a
complex cation was formed.[27-29] The Os–cymcentroid and Os–Cl distances were
1.671 Å and 2.442(4) Å and therefore similar to those reported for related
complexes.[27-29] The Os–S1 and Os–N1 bond lengths were 2.355(4) and 2.133(1) Å.
The C6–S1 bond (1.754(15) Å in 1dneutral) was elongated as compared to 1.655(5) Å
for 1, indicating a higher single bond character. The C6–N2 distance of 1.251(19) Å in
1dneutral was slightly shorter compared to a bond length of 1.345(6) Å in 1,
demonstrating increased double bond character upon coordination of the Os center to
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the S atom and deprotonation of the amide group. The latter bond is hardly modified
when PCA coordinates as a neutral ligand to a metal center.[31]
Figure 3. Molecular structure of 1dneutral drawn at 50% probability level.
To confirm the ionic nature of the complexes, conductivity measurements were
performed for 1 and its complexes 1a–d in acetonitrile. All the complexes showed
higher conductivity than the neutral ligand (Table S1), indicating their ionic nature.
However, it should be noted that the conversion of the cationic form into the neutral
form may be accompanied by the release of HCl.
The formation of the complexes was also confirmed by ESI-MS. In light of the
molecular structure of 1dneutral, which features the PCA ligand in its deprotonated form
coordinated to Os, it is interesting to note that the mass spectrum of 1d recorded in
positive ion mode featured a peak at an m/z value assigned to [M – Cl]+ ions but the
most abundant peak was from a [M– 2Cl – H]+ species, which was the only peak found
for the Ru complexes. The elemental analysis data of the complexes were in close
agreement with the theoretical values for the protonated complexes with a chlorido
counterion.
Stability in aqueous solution and reactivity toward amino acids
The aqueous stability of complexes 1a–1d was determined by NMR spectroscopy and
ESI-MS. The compounds were dissolved in D2O and 1H NMR spectra were recorded
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after 0.25, 1, 3, 24, 48, 72, 96 and 120 h. The compounds underwent chlorido/aqua
ligand exchange reactions within 15 min of incubation in D2O. There was no change
in the spectrum over a period of 120 h, indicating the high stability of the formed aqua
species.
Depending on the nature of metal ion and co-ligands, metal complexes are prone to
undergo ligand exchange when encountered with biomolecules such as proteins. In
order to understand the nature of such interactions, reactions of 1a and 1d with the
amino acids L-cysteine (Cys), L-methionine (Met), and L-histidine (His) were monitored
1
by H NMR spectroscopy in D2O. Despite that both 1a and 1d, undergo immediate
hydrolysis, they did not react with amino acids within 24 h of incubation at 1 : 1 and 1
: 2 (complex : amino acid) molar ratio (Figure 4 for His), after which another equivalent
of amino acid was added and the reaction was followed for another 96 h. The 1H NMR
spectra however remained largely unchanged with only a minor amount of another
species (< 5%) forming, possibly due to adduct formation with the amino acids. This
low reactivity was further confirmed by ESI-MS, where no adduct formation was
observed with amino acids. The relative high stability of the aqua species of these
complexes is unique compared to that of analogous Ru PCA complexes.
Figure 4. 1H NMR spectroscopic study of the reaction between 1a and His in D2O, monitored
for 72 h. The peaks of His are highlighted in grey boxes.
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In vitro anticancer activity
The antiproliferative activity of ligand 1 and its respective complexes 1a–1d was
determined in human HCT116 colorectal, H460 non-small cell lung, SiHa cervical, and
SW480 colon carcinoma cells (Table 1). The sulfonamide-substituted PCA ligand 1
was moderately active only in the HCT116 cancer cell line with an IC50 value of 105
μM. The Ru(cym) and Os(cym) complexes were inactive in all tested cancer cell lines.
This is surprising given the fact that plecstatin-1 and other related derivatives were
highly cytotoxic (Table 1).[27,29,30] The low potency may be related to the comparatively
low lipophilicity of ligand 1 (clogP = -0.148) as compared to N-(4-fluorophenyl)pyridine-
2-carbothioamide in F-SN (clogP = 1.832),[29] possibly interfering with efficient
accumulation in cancer cells. Another explanation may be that the sulfonamide
substituent hinders the interaction of the complex with plectin, which was identified as
the target for plecstatin-1.[30]
Table 1. In vitro anticancer activity (IC50 values) of ligands 1, its respective Ru/Os(cym)
complexes 1a, 1b, 1c and 1d, and related compounds F-SN and plecstatin-1 in human
colorectal (HCT116), non-small cell lung (NCI-H460) cervical (SiHa) and colon carcinoma
(SW480) cells (exposure time 72 h). The clogP values for the PCAs 1 and F-SN are also given.
Compound
IC50 value (µM)
clogP
HCT116
105 ± 3
>211
NCI-H460
>300
SiHA
SW480
>300
1
>300
>300
- 0.148
1a
>300
>300
-
1b
>300
>300
>300
>300
-
1c
>300
>300
>300
>300
-
1d
>300
>300
>300
>300
-
1.832
-
F-SN [29]
plecstatin-1 [29]
5.7 ± 0.7
6.5 ± 0.3
7.8 ± 1.8
10 ± 2
16 ± 6
8.3 ± 0.7
33 ± 2
9.9 ± 0.7
Molecular Modelling
As crystal structure of h-CA II with a co-crystallized Ru complex (SRX) featuring a
sulfonamide functional group has been reported (PDB ID: 3PYK),[38] we modelled
ligand 1 and both possible enantiomers of its chiral Ru and Os complexes 1a (1aE1
and 1aE2) and 1d (1dE1 and 1dE2), respectively, into the catalytic pocket using a
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molecular dynamics approach. The results were compared to that of a co-crystallized
Ru complex (SRX) with a sulfonamide functional group. All the compounds were found
to interact through H bonds with Thr residues in close proximity to the Zn ion in the
active site, to which the sulfonamide moieties bound (Table 2). In addition, they formed
lipophilic interactions with Val121, Leu60, and Leu198, as did SRX (in addition to
Pro202). The ligand and its complexes practically adopted the same conformation,
independent of the chirality at the metal center. The predicted pose of 1aE2 is shown
in Figure 5a with its hydrogen bonds with Thr199 and Thr200 via the oxygen atom of
the sulfonamide group. Complex 1aE2 is residing deep in the catalytic site of the
enzyme showing an excellent fit (Figure 5b), as did all the other complexes, and blocks
access to the Zn ion coordinated to His94, His96, and His119. This demonstrates that
the enzyme is a viable target, which however would have to be verified experimentally.
Figure 5. The modelled configuration of 1aE2 in the catalytic site of carbonic anhydrase II (PDB
ID 3PYK). a) Hydrogen bonds are depicted as green dotted lines between the metal complex
and the amino acids Thr199, and Thr200. Lipophilic interactions are represented as purple
dotted lines with Val121, Leu60 and Leu198. b) The enantiomer 1aE2 is shown in the binding
pocket with the protein surface rendered. Red depicts a negative partial charge on the surface,
blue depicts a positive partial charge and grey shows neutral/lipophilic areas.
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Table 2. The H bonds and lipophilic interactions of the modelled compounds with amino acid
residues of carbonic anhydrase II.
Compound
SRX
1
H bonds
Lipophilic interactions
Val121, Leu198, Pro202
Val121, Leu198
Thr199
Thr200
1aE1
Thr199, Thr200
Thr199, Thr200
Thr199, Thr200
Thr200
Val121, Leu198
1aE2
Val121, Leu198, Leu60
Val121, Leu198
1dE1
1dE2
Val121, Leu198, Leu60
Conclusions
We describe in this paper an approach where we borrowed the PCA pharmacophore
for functionalization with a sulfonamide and the preparation of its half sandwich
complexes to target the enzyme carbonic anhydrase. The Ru(cym) and Os(cym)
complexes were synthesized and thoroughly characterized. Interestingly, the
molecular structure of 1d suggests deprotonation of the carbothioamide moiety, while
similar structures crystallized in the protonated form, as did ligand 1. We evaluated the
compounds for their stability in aqueous solution and reactivity with biomolecules. The
compounds undergo a quick chlorido/aqua ligand exchange but are surprisingly
unreactive to amino acids. The antiproliferative activity was assayed in a small panel
of human cancer cell lines and an IC50 value could only be determined for ligand 1 in
HCT116 cells. While binding to CA II, as determined by molecular modelling studies,
may not result in anticancer activity, this shows that the compounds are still capable
of interacting with the Zn ion in the catalytic site of CA II.
Acknowledgments
We thank the University of Auckland (University of Auckland Doctoral Scholarship to
K. T.), the Higher Education Commission of Pakistan (IRSIP Scholarship to J. A.), and
the Royal Society of New Zealand for funding. We are grateful to Tanya Groutso and
Tony Chen for collecting the X-ray diffraction and MS data, respectively.
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Experimental
Materials and methods
All reactions were carried out under nitrogen atmosphere using standard Schlenk
techniques. Chemicals obtained from commercial suppliers were used as received
and were of analytical grade. Tetrahydrofuran (THF) and dichloromethane (DCM)
were first dried through a solvent purification system (LC Technology Solutions Inc.,
SP-1 solvent purifier), degassed under a N2 flow, and stored in a Schlenk flask.
Methanol was dried using standard procedures and stored over activated molecular
sieves (3 Å).
α-Terpinene, 2-picoline, and Na2S·9H2O were purchased from Merck, 4-
aminobenzenesulfonamide, sulfur, and OsO4 from Sigma-Aldrich, L-histidine, L-
methionine and L-cysteine from AK Scientific, and RuCl3·3H2O (99%) from Precious
Metals Online.
The
dimers
bis[dichlorido(η6-p-cymene)ruthenium(II)],[39]
bis[dibromido(η6-p-
cymene)ruthenium(II)],[40]
bis[diiodido(η6-p-cymene)ruthenium(II)],[40]
and
bis[dichlorido(η6-p-cymene)osmium(II)][41,42] were synthesized by adapting reported
procedures.
1H and 13C{1H} and 2D (COSY, HSQC, HMBC) NMR spectra were recorded on a
Bruker Avance AVIII 400 MHz NMR spectrometer at ambient temperature at 400.13
MHz (1H) or 100.61 MHz (13C{1H}). Chemical shifts are reported versus SiMe4 and
were determined by reference to the residual solvent peaks.
High resolution mass spectra were recorded on a Bruker micrOTOF-QII mass
spectrometer in positive electrospray ionization (ESI) mode. Elemental analyses were
carried out on an Exeter Analytical Inc-CE-440 Elemental Analyser and were
performed at the Campbell Microanalytical Laboratory, The University of Otago. X-ray
diffraction measurements of single crystals were carried out on a Bruker SMART
APEX2 diffractometer with a CCD area detector using graphite monochromated Mo-
Kα radiation (λ = 0.71073 Å). The molecular structures were solved and refined with
[43]
the SHELXL-2016
and Olex2[44,45] program packages. The molecular structures
were visualized using Mercury 3.9.
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Table 3. X-ray diffraction measurement parameters for 1 and 1dneutral
.
1
1dneutral
1829883
CCDC
1829882
C12H11O2N2S2
293.36
Formula
C22H24ClN3O2OsS2
652.21
Molecular weight (g mol-1)
Crystal size (mm)
0.32 × 0.10 × 0.08 0.26 × 0.10 × 0.08
Wavelength (Å)
0.71073
100(2)
monoclinic
Cc
0.71073
100(2)
Temperature (K)
Crystal system
monoclinic
P-1
Space group
a (Å)
4.8844(6)
28.476(3)
8.8935(9)
90
6.9829(7)
12.2144(10)
13.5379(12)
79.167(5)
83.956(6)
82.303(6)
1120.06(18)
2
b (Å)
c (Å)
α (°)
β (°)
94.869(7)
90
γ (°)
Volume (Å3)
1232.5(2)
4
Z
Calculated Density (mg/mm3)
Absorption coefficient (mm-1)
F(000)
1.581
1.934
0.433
6.024
608
636
Theta range (°)
25.233
24.403
Number of Parameters / Reflections (all) 204 / 2214
289 / 3613
Final R indices [I > 2σ(I)]
R1 = 0.0412
R1= 0.0844
wR2 = 0.0741
wR2 = 0.1594
R indices (all data)
R1 = 0.0514
R1 = 0.1071
wR2 = 0.0774
wR2 = 0.1668
Goodness-of-fit on F2
1.050
1.116
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General procedure for the synthesis of organo-Ru and -Os complexes
A solution of [M(cym)Cl2]2 (M = Ru, Os) in dry DCM was added to a stirred solution of
1 in dry THF. The reaction mixture was stirred for 4 h at 40 °C under nitrogen
atmosphere. A change in color from brown to deep red was observed immediately
after the addition of the dimeric precursor. The solvent was evaporated and the residue
was dissolved in a minimal volume of DCM, followed by addition of n-hexane that
resulted in immediate precipitation. After placing it in the fridge overnight, the
precipitate was filtered, and dried under reduced pressure.
[Chlorido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-
carbothioamide)ruthenium(II)] chloride 1a
The synthesis of 1a was performed following the general complexation procedure,
using N-(4-sulfamoylphenyl)pyridine-2-carbothioamide (120 mg, 0.41 mmol) and
[Ru(cym)Cl2]2 (124 mg, 0.20 mmol). After completion of the reaction, the solvent was
concentrated in vacuum up to 5 mL and n-hexane was added for further precipitation
in the fridge. The solid product was filtered, followed by washing with dichloromethane
(2 × 10 mL) and drying in vacuum. Yield: 130 mg (53%), red solid. Elemental analysis
found:
C,
39.84;
H,
3.89;
N,
5.81,
calculated
for
C22H25Cl2N3O2RuS2·0.7CH2Cl2·1.25H2O: C, 40.00; H, 4.27; N, 6.17. MS (ESI+): m/zcalc
528.0353 [1a – 2Cl – H]+ (m/z 528.0356). 1H NMR (400.13 MHz, d4-MeOD, 25 °C): δ
3
3
= 9.66 (d, J(H1,H2) = 6 Hz, 1H, H-1), 8.43 (d, J(H4,H3) = 8 Hz, 1H, H-4), 8.29 (td,
3J(H3,H4)/(H3,H2) = 8 Hz, 4J(H3,H1) = 2 Hz, 1H, H-3), 8.09 (d, 3J(H9,H8)/(H11,H12) = 9 Hz, 2H, H-
9/H-11), 7.85 (t, 3J(H2,H3)/(H2,H1) = 8 Hz, 1H, H-2), 7.76 (d, 3J(H8,H9)/(H12,H11)= 9 Hz, 2H, H-
8/H-12), 6.05 (d, 3J(H15,H14) = 6 Hz, 1H, H-15), 5.94 (d, 3J(H17,H18) = 6 Hz, 1H, H-17), 5.91
3
3
(d, J(H18,H17) = 6 Hz, 1H, H-18), 5.65 (d, J(H14,H15) = 6 Hz, 1H, H-14), 2.74 (sept,
3J(H21,H20)/ (H21,H22) = 7 Hz, 1H, H-21), 2.21 (s, 3H, H-19), 1.21 (d, 3J(H20,H21) = 7 Hz, 3H,
3
13
H-20), 1.13 (d, J(H22,H21) = 7 Hz, 3H, H-22) ppm. C{1H} NMR (100.61 MHz, CDCl3
[0.3 mL] / d4-MeOD [0.1 mL], 25 °C): δ = 159.0 (C-5), 153.4 (C-1), 140.3 (C-10),
139.81 (C-3), 129.3 (C-9/C-11), 127.4 (C-2), 125.3 (C-4), 125.1 (C-8/C-12) 106.3 (C-
16), 103.6 (C-13), 88.1 (C-15), 87.8 (C-17), 85.4 (C-18), 84.3 (C-14), 31.4 (C-21), 22.8
(C-20), 21.7 (C-22), 18.3 (C-19) ppm.
15
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[Bromido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-
carbothioamide)ruthenium(II)] bromide 1b
The synthesis of 1b was performed following the general complexation procedure,
using N-(4-sulfamoylphenyl)pyridine-2-carbothioamide (100 mg, 0.34 mmol) and
[Ru(cym)Br2]2 (125 mg, 0.17 mmol). After completion of the reaction, the solvent was
concentrated in vacuum up to 5 mL and n-hexane was added for further precipitation
in the fridge. The solid product was filtered, followed by washing with dichloromethane
(2 × 10 mL) and drying in vacuum. Yield: 145 mg (62%), red solid. Elemental analysis
found: C, 39.31; H, 3.71; N, 5.75, calculated for C22H25Br2N3O2RuS2·0.2C4H8O: C,
38.96; H, 3.81; N, 5.98. MS (ESI+): m/zcalc 528.0353 [1b – 2Br – H]+ (m/z 528.0340).
3
1H NMR (400.13 MHz, d4-MeOD, 25 °C): δ = 9.66 (d, J(H1,H2) = 6 Hz, 1H, H-1), 8.45
(d, 3J(H4,H3) = 8Hz, 1H, H-4), 8.30 (td, 3J(H3,H4)/(H3,H2) = 8 Hz, 4J(H3,H1) = 2 Hz, 1H, H-3),
3
8.11 (d, J(H9,H8)/(H11,H12) = 9 Hz, 2H, H-9/H-11), 7.83 (m, 3H, H-2/H-8/H-12), 6.05 (d,
3J(H15,H14) = 6 Hz, 1H, H-15), 5.94 (d, 3J(H17,H18) = 7 Hz, 1H, H-17), 5.90 (d, 3J(H18,H17)
=
6 Hz, 1H, H-18), 5.69 (d, 3J(H14,H15) = 6 Hz, 1H, H-14), 2.81 (sept, 3J(H21,H20)/ (H21,H22) = 7
3
Hz, 1H, H-21), 2.28 (s, 3H, H-19), 1.21 (d, J(H20,H21) = 7 Hz, 3H, H-20), 1.15 (d,
3J(H22,H21) = 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, CDCl3 [0.3 mL] / d4-
MeOD [0.1 mL], 25 °C): δ = 158.8 (C-1), 153.3 (C-7), 142.9 (C-10), 140.0 (C-3), 129.5
(C-9/C-11), 127.5 (C-2), 125.9 (C-4), 125.7 (C-8/C-12) 107.6 (C-16), 103.4 (C-13),
87.8 (C-15), 87.4 (C-17/C-18), 85.1 (C-14), 31.3 (C-21), 22.5 (C-20), 21.6 (C-22), 18.9
(C-19) ppm.
[Iodido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-carbothioamide)ruthenium(II)]
iodide 1c
The synthesis of 1c was performed following the general complexation procedure,
using N-(4-sulfamoylphenyl)pyridine-2-carbothioamide (80 mg, 0.27 mmol) and
[Ru(cym)I2]2 (133 mg, 0.14 mmol). After completion of the reaction, the solid product
was filtered, followed by washing with dichloromethane (2 × 10 mL) and
tetrahydrofuran (1 × mL) and drying in vacuum. Yield: 187 mg (88%), Red solid.
Elemental analysis found: C, 35.99; H, 3.72; N, 4.72, calculated for
C22H25I2N3O2RuS2·0.75 C4H8O: C, 35.89; H, 3.74; N, 5.02. MS (ESI+): m/zcalc 528.0353
[1c – 2I – H]+ (m/z 528.0340). 1H NMR (400.13 MHz, d4-MeOD, 25 °C): δ = 9.63 (d,
3J(H1,H2) = 6 Hz, 1H, H-1), 8.42 (d, 3J(H4,H3) = 8Hz, 1H, H-4), 8.25 (td, 3J(H3,H4)/(H3,H2) = 8
Hz, 4J(H3,H1) = 2 Hz, 1H, H-3), 8.09 (d, 3J(H9,H8)/(H11,H12) = 9 Hz, 2H, H-9/H-11), 7.77 (m,
16
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3H, H-2/H-8/H-12), 6.03 (d, 3J(H15,H14) = 6 Hz, 1H, H-15), 5.88 (d, 3J(H17,H18) = 7 Hz, 1H,
H-17), 5.85 (d, 3J(H18,H17) = 7 Hz, 1H, H-18), 5.70 (d, 3J(H14,H15) = 6 Hz, 1H, H-14), 2.89
(sept, 3J(H21,H20)/(H21,H22) = 7 Hz, 1H, H-21), 2.37 (s, 3H, H-19), 1.21 (d, 3J(H20,H21) = 7 Hz,
3H, H-20), 1.17 (d, 3J(H22,H21) = 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, CDCl3
[0.3 mL] / d4-MeOD [0.1 mL], 25 °C): δ = 159.7 (C-1), 139.2 (C-3), 128.7 (C-9/C-11),
128.2 (C-2), 127.5 (C-4), 124.9 (C-8), 124.6 (C-12), 87.7 (C-15), 87.4 (C-17), 85.7 (C-
18), 85.2 (C-14), 31.5 (C-21), 22.4 (C-20), 21.6 (C-22), 19.4 (C-19) ppm.
[Chlorido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-carbothioamide)osmium(II)]
chloride 1d
The synthesis of 1d was performed following the general complexation procedure,
using N-(4-sulfamoylphenyl)pyridine-2-carbothioamide (90 mg, 0.31 mmol) and
[Os(cym)Cl2]2 (121 mg, 0.15 mmol). After work up the solid product was washed with
dichloromethane (2 × 10 mL) and the solvent was removed on a rotary evaporator.
Yield: 168 mg (80%), black solid. Elemental analysis found: C, 39.28; H, 3.94; N, 5.87;
S, 8.96, calculated for C22H25Cl2N3O2OsS2·0.1C6H14: C, 38.93; H, 3.82; N, 6.03; S,
9.20. MS (ESI+): m/zcalc 618.0925 [1d – 2Cl – H]+ (m/z 618.0918), m/zcalc 654.0692 [1d
–Cl]+ (m/z 654.0665). 1H NMR (400.13 MHz, d4-MeOD, 25 °C): δ = 9.50 (d, 3J(H1,H2)
6 Hz, 1H, H-1), 8.43 (d, 3J(H4,H3) = 9 Hz, 1H, H-4), 8.21 (t, 3J(H3,H4)/(H3,H2) = 8 Hz, 1H, H-
=
3
3
3), 8.04 (d, J(H9,H8)/(H11,H12) = 9 Hz, 2H, H-9/H-11), 7.73 (d, J(H2,H3)/(H2,H1) = 8 Hz, 1H,
H-2), 7.63 (d, 3J(H8,H9)/(H12,H11) = 9 Hz, 2H, H-8/H-12), 6.14 (d, 3J(H15,H14) = 6 Hz, 1H, H-
15), 6.06 (d, 3J(H17,H18) = 6 Hz, 1H, H-17), 6.02 (d, 3J(H18,H17) = 6 Hz, 1H, H-18), 5.75 (d,
3
3J(H14,H15) = 6 Hz, 1H, H-14), 2.64 (sept, J(H21,H20)/ (H21,H22) = 7 Hz, 1H, H-21), 2.27 (s,
3
3
3H, H-19), 1.19 (d, J(H20,H21)= 7 Hz, 3H, H-20), 1.08 (d, J(H22,H21) = 7 Hz, 3H, H-22)
ppm. 13C{1H} NMR (100.61 MHz, CDCl3 [0.3 mL] / d4-MeOD [0.1 mL], 25 °C): δ = 158.4
(C-1), 139.5 (C-3), 129.5 (C-9/C-11), 127.3 (C-2), 125.2 (C-4), 124.2 (C-8/C-12), 96.3
(C-13), 79.4 (C-15), 78.9 (C-17), 76.3 (C-18), 73.7 (C-14), 31.0 (C-21), 22.6 (C-20),
21.5 (C-22), 18.1 (C-19) ppm.
Stability in aqueous solution and reactivity with amino acids
The hydrolytic stability of 1a–1d was studied by dissolving the compounds (1–2
mg/mL) in D2O. 1H NMR spectra were recorded after 0.5, 2, 24, 48, 72, 96 and 120 h
and ESI-mass spectra after 0.5, 24, 96 h and 7 d. To determine the reactivity with the
17
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amino acids Cys, His and Met, 1a or 1d (1–2 mg/mL) was dissolved in D2O and 2
equivalents of the respective amino acids were added. The incubation mixture was
analyzed by 1H NMR spectroscopy and ESI-MS after 0.5, 2, 24, 48, 72, and 96 h.
Sulforhodamine B Cytotoxicity Assay
HCT116, SW480 and NCI-H460 cells were supplied by ATCC, while SiHa cells were
from Dr. David Cowan, Ontario Cancer Institute, Canada. The cells were grown in
αMEM (Life Technologies) supplemented with 5% fetal calf serum (Moregate Biotech)
at 37 °C in a humidified incubator with 5% CO2.
The cells were seeded at 750 (HCT116, NCI-H460), 4000 (SiHa) or 5000 (SW480)
cells/well in 96-well plates and left to settle for 24 h. The compounds were added to
the plates in a series of 3-fold dilutions, containing a maximum of 0.5% DMSO at the
highest concentration. The assay was terminated after 72 h by addition of 10%
trichloroacetic acid (Merck Millipore) at 4 °C for 1 h. The cells were stained with 0.4%
sulforhodamine B (Sigma-Aldrich) in 1% acetic acid for 30 min in the dark at room
temperature and then washed with 1% acetic acid to remove unbound dye. The stain
was dissolved in unbuffered Tris base (10 mM; Serva) for 30 min on a plate shaker in
the dark and quantified on a BioTek EL808 microplate reader at an absorbance
wavelength of 490 nm with 450 nm as the reference wavelength to determine the
percentage of cell growth inhibition by determining the absorbance of each sample
relative to a negative (no inhibitor) and a no-growth control (day 0). The IC50 values
were calculated with SigmaPlot 12.5 using a three-parameter logistic sigmoidal
dose−response curve between the calculated growth inhibition and the compound
concentration. The presented IC50 values are the mean of at least 3 independent
experiments, where 10 concentrations were tested in duplicate for each compound.
Conductivity measurements
The conductivity in acetonitrile was determined for ligand 1 and complexes 1a–d
(0.1 mM) on an Oakton CON 700 Conductivity/°C/°F Benchtop Meter at room
temperature.
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Calculated logarithmic octanol/water partition coefficient (clogP)
ChemBioDrawUltra 15.0 was used to determine the calculated logarithmic octanol-
water partition coefficient (clogP) of 1.
Molecular Modelling
Scigress Ultra version F.J 2.6[46] was used for the modelling of the ligands into the
crystal structure of human carbonic anhydrase II (PDB ID 3PYK).[38] Hydrogen atoms
were added to the structures and the ligands were built into the binding pocket based
on
co-crystallized
[chlorido{N-[di(pyridin-2-yl-κN)methyl]-4-
sulfamoylbenzamide}{(1,2,3,4,5,6-η)-(1R,2R,3R,4S,5S,6S)-1,2,3,4,5,6-
hexamethylcyclohexane-1,2,3,4,5,6-hexayl}ruthenium(II)]. The ligands were first
structurally optimized followed by short 1 ps molecular dynamics simulations using the
MM2 force field.[47]
19
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