Aspirin-inspired organometallic compounds: Structural characterization and cytotoxicity

Article abstract of DOI:10.1016/j.jorganchem.2017.01.016

We report here the preparation of 2-hydroxy-4-(picolinamido)benzoic acid and its aspirin analogue 2-acetoxy-4-(picolinamido)benzoic acid. Both ligands were used to synthesize organoruthenium and -osmium complexes. The compounds were characterized by NMR spectroscopy, electrospray ionization mass spectrometry and elemental analysis. The molecular structures of Ru and Os complexes of 2-hydroxy-4-(picolinamido)benzoic acid were determined by single crystal X-ray diffraction analysis. They showed a typical piano-stool configuration and the ligand coordinated as an N,N-bidentate chelator to the metal center. The cytotoxic potential of the selected compounds was evaluated in human colon (HCT116, SW480), non-small cell lung (NCI-H460) and cervical (SiHa) carcinoma cells. Surprisingly none of the compounds exhibited antiproliferative activity given the fact that other metal complexes of aspirin derivatives have shown promising anticancer activity.

Full text of DOI:10.1016/j.jorganchem.2017.01.016

Accepted Manuscript
Aspirin-inspired organometallic compounds: Structural characterization and
cytotoxicity
Adnan Ashraf, Muhammad Hanif, Mario Kubanik, Tilo Söhnel, Stephen M.F.
Jamieson, Arindam Bhattacharyya, Christian G. Hartinger
PII:
S0022-328X(17)30043-8
10.1016/j.jorganchem.2017.01.016
JOM 19773
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 24 October 2016
Revised Date: 23 January 2017
Accepted Date: 24 January 2017
Please cite this article as: A. Ashraf, M. Hanif, M. Kubanik, T. Söhnel, S.M.F. Jamieson, A.
Bhattacharyya, C.G. Hartinger, Aspirin-inspired organometallic compounds: Structural characterization
and cytotoxicity, Journal of Organometallic Chemistry (2017), doi: 10.1016/j.jorganchem.2017.01.016.
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ACCEPTED MANUSCRIPT
Synopsis
Inspired by the promising anticancer activity of aspirin–metal conjugates, aspirin-derived ligands
were prepared and coordinated to organoruthenium and -osmium moieties. Most interestingly,
crystallographic studies revealed a network of hydrogen bonding and π-stacking. Surprisingly,
none of the prepared compounds showed antiproliferative activity.

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Aspirin-inspired Organometallic Compounds: Structural Characterization
and Cytotoxicity
Adnan Ashraf,a Muhammad Hanif,a,* Mario Kubanik,a Tilo Söhnel,a Stephen M. F.
Jamieson,b Arindam Bhattacharyya,c Christian G. Hartingera,*
a School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand.
b Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019,
Auckland 1142, New Zealand
c Department of Zoology, University of Calcutta, Kolkata-700019, India.
* School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand.
E-mail: c.hartinger@auckland.ac.nz; m.hanif@auckland.ac.nz; Fax: (+64)9 373 7599 ext
87422
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Abstract
We report here the preparation of 2-hydroxy-4-(picolinamido)benzoic acid and its aspirin
analogue 2-acetoxy-4-(picolinamido)benzoic acid. Both ligands were used to synthesize
organoruthenium and -osmium complexes. The compounds were characterized by NMR
spectroscopy, electrospray ionization mass spectrometry and elemental analysis. The
molecular structures of Ru and Os complexes of 2-hydroxy-4-(picolinamido)benzoic acid
were determined by single crystal X-ray diffraction analysis. They showed a typical piano-
stool configuration and the ligand coordinated as an N,N-bidentate chelator to the metal
center. The cytotoxic potential of the selected compounds was evaluated in human colon
(HCT116, SW480), non-small cell lung (NCI-H460) and cervical (SiHa) carcinoma cells.
Surprisingly none of the compounds exhibited antiproliferative activity given the fact that
other metal complexes of aspirin derivatives have shown promising anticancer activity.
Keywords: anticancer activity, aspirin, osmium complexes, ruthenium complexes, NSAIDs.
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Introduction
Although the application of platinum-based drugs has improved the overall outcome of
cancer therapy, undesired side effects, a limited spectrum of treatable tumors and resistance
are key limitations in their clinical use [1,2]. This led to the search for non-platinum
metallotherapeutics [3-11], among which ruthenium complexes occupy a prominent place
due to their relatively low general toxicity [4,5,9]. The ruthenium complexes KP1019
(indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)])/KP1339 {sodium trans-
[tetrachloridobis(1H-indazole)ruthenate(III)]}
and
NAMI-A
{imidazolium
trans-
[tetrachlorido(dimethylsulfoxide)(imidazole)ruthenium(III)]} have been tested in clinical
trials [5,12,13]. The discovery of the anticancer activity of RAPTA-C [Ru(cym)(PTA)Cl2]
(PTA = 1,3,5-triaza-7-phosphatricyclodecane; cym = η6-p-cymene) and RAED-C
[Ru(cym)(en)Cl]PF6 (en = 1,2-ethylenediamine) fueled research into half-sandwich RuII(η6-
arene) complexes [8,14-17]. The presence of the arene moiety not only enhances the
hydrophobic character of the complexes but also assists in stabilizing the ruthenium in 2+
oxidation state [14]. The osmium analogues of many recently reported organoruthenium
compounds were also investigated on their suitability as anticancer agents. Os complexes are
however still relatively less well explored compared to their Ru congeners. Interestingly, it is
hard to predict the biological effect when replacing a Ru center in a complex with Os. In
some cases, the Ru complexes are more potent than the Os analogues, while in others the
substitution does not impact the antiproliferative activity or the osmium complexes are found
to be more cytotoxic than their ruthenium counterparts [10].
Picolinic acid and its derivatives have been used as ligands for the preparation of anticancer
active organometallic complexes with Ru, Os, Rh and Ir centers [18-22]. Picolinato Os
complexes were found to be active against ovarian human carcinoma, while both Ru and Os
picolinamide compounds inhibited cell proliferation of colon, ovarian, and cisplatin-resistant
ovarian human carcinoma cells [18,20]. Picolinamide derivatives can act as N,O- or N,N-
coordinating bidentate ligands depending on the pH value of the solution (Figure 1)
[18,22,23]. The coordination mode of the picolinamide ligands impacted aquation and
cytotoxicity [18]. Furthermore, while Ru(cym) complexes of picolinamide in N,N-
coordination mode selectively target the DNA base guanine [18], the analogous
thiocarbamide derivatives, coordinating to a Ru(arene) moiety through an S,N-donor system,
were shown to interact preferentially with proteins [24].
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Figure 1. pH-dependent interconversion between N,N- and N,O-coordination modes in picolinamide
Ru(cym) complexes.
Recent studies have shown that aspirin and other non-steroidal anti-inflammatory drugs
(NSAIDs) are effective for chemoprevention of cancer [25-27], while several metal
complexes of NSAIDs have also showed promising anticancer activity [28-30]. Combining
an organometallic moiety with bioactive ligands has opened up new avenues in the
development of multi-targeted drugs [29,31-40]. With the aim of introducing aspririn-
inspired properties into organometallic anticancer compounds, we synthesized salicylic acid-
functionalized picolinamide and acetylated it. By using 4-aminosalicylic acid as the starting
material, the key functional groups of aspirin remained unmodified. The prepared
picolinamide derivatives were then coordinated to organo-Ru and –Os fragments with
different halides and studied for the anticancer properties.
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Results and Discussion
Aspirin is a well-known NSAID and recent reports highlighted its chemotherapeutic potential
against cancer [25]. Prolonged and dose-dependent use of aspirin was found to reduce the
risk of colon, gastrointestinal, lung and breast cancer [26,41,42]. In an attempt to exploit the
advantageous biological properties of aspirin, 2-picolinamide was functionalized with 4-
aminosalicylic acid to yield 1 (Scheme 1). The hydroxyl functional group of compound 1 was
acetylated with acetic anhydride/acetic acid to obtain the aspirin analogue of 2-picolinamide
2. Both 1 and 2 were converted into the respective half-sandwich RuII and OsII
organometallics by refluxing them with different dimeric [M(cym)X2]2 precursors. 2-
Picolinamide ligands may behave as N,N- and N,O-bidentate chelators depending on the pH
of the solution. When 1 was coordinated to the metal centers, only N,N-coordination was
observed upon reflux in a dichloromethane and acetone mixture (9:1). In contrast, for 2 both
N,N- and N,O-coordination modes were observed, the latter however to a minor degree and
only when iodido complexes were prepared (data not shown). Attempts to push the
equilibrium to either side showed that at pH ˂ 2 the compounds start to decompose, as
demonstrated by 1H NMR spectroscopy.
Scheme 1. Preparation of the organoruthenium and -osmium compounds based on aspirin-inspired
ligands derived from 4-picolinamide.
All complexes were characterized with 1D and 2D NMR spectroscopy, high resolution mass
spectroscopy, microanalysis and X-ray crystallography. The acetylation of 1 resulted in a
characteristic shift of the signals assigned to H-10 and H-14 (see Scheme 1 for the NMR
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numbering scheme used) in the downfield region of the 1H NMR spectra from 7.59 and 7.26
ppm to 7.81 and 7.74 ppm, respectively. An additional signal was observed at 2.31 ppm in 2
which was assigned to the methyl group. After complex formation the H-6 signal in all
complexes was considerably moved downfield to chemical shifts > 9.2 ppm, compared to
8.69 ppm for the uncoordinated ligands 1 and 2. The aromatic protons of cymene gave four
doublets in case of N,N coordination mode while in case of N,O coordination only two
doublets were observed.
Figure 2. Comparison of the 1H NMR spectra of ligand 2 and its Ru(cym) complex.
In the 13C{1H} NMR spectra of both 1 and 2, the carbonyl carbon atoms C-15 were present in
the most deshielded region at approximately δ = 171 ppm. After complexation to Ru or Os,
the most significant shifts were observed for the amide carbonyl carbon atoms C-7 which
appeared in the range 171–168 ppm in 1a–1c and 2a, as compared to 162 and 165 ppm for 1
and 2, respectively. The signals assigned to C-2 and C-9, adjacent to the nitrogen atoms
coordinated to the metal ions, were also shifted downfield. For the Ru complexes the
aromatic carbon atoms of p-cymene appeared in the range 86–84 ppm while in case of the Os
complex, these signals appeared around 76 ppm. In case of 2 and 2a, an additional signal was
present at δ = 21 ppm, which was assigned to the methyl carbon of the acetyl group.
The formation of both the ligands and the complexes was also confirmed by ESI-MS analysis
in positive ion mode. The mass spectra featured the pseudomolecular [M + Na]+ ions for the
ligands and [M – X]+ ions for the metal complexes. The m/z values obtained were very
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similar to the theoretical values and the recorded isotopic pattern matched well the modeled
counterparts.
Single crystals suitable for X-ray diffraction analysis of compounds 1a–1c were obtained in
methanol and diethyl ether by using slow diffusion method. The molecular structures of 1a–
1c showed pseudo-octahedral geometry around the metal center. Selected bond lengths are
given in Table 1. In all cases, ligand 1 formed a five-membered ring with the metal centers
showing N,N coordination through the nitrogen atoms of the pyridyl and carboxamide
moieties. Compounds 1a (Figure 3) and 1c (Figure 4) crystallized in the chiral space group
P21 and therefore only one of the two enantiomers with the Ru acting as the chiral center
were identified. Furthermore, the molecular structures contained co-crystallized methanol
which formed H bonds O5–H···O2 to the phenolic hydroxyl group. In contrast, 1b
crystallized in the non-chiral space group P21/c and both enantiomers were detected.
Figure 3. Molecular structure of 1a drawn at 50% probability level, with the inter- (orange) and
intramolecular (black) H bonds indicated as dashed lines. The H bond from the O1 atom to the next
molecule of 1a was omitted for clarity, as was one of the positions of the disordered MeOH found in
the structure.
In all structures, both intramolecular and intermolecular hydrogen bonding was observed.
Intramolecular H bonds O2–H···O3 involved the hydroxyl and carboxylic acid functional
groups, while intermolecular hydrogen bonds were formed between the carboxamide oxygen
O1 and the carboxylic acid hydroxyl group. These intermolecular H bonds extended to a one-
dimensional network between molecules of the Ru complexes 1a and 1c and the p-cymene
ligands interacted with the substituted phenyl groups by π stacking (3.312 and 3.274 Å for 1a
and 1c, respectively).
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Figure 4. Molecular structure of 1c forming a zigzag chain through intermolecular hydrogen bonding
between the carboxylic acid group of one molecule and the carboxamide of another. The inter- and
intramolecular H bonds are shown as orange and black dashed lines, respectively.
In case of the Os compound 1b (Figure 5), hydrogen bonds were only detected between
molecules of the same enantiomers and the two chains of enantiomers were connected
through π stacking between the p-cymene ligands and the substituted phenyl groups of the
same enantiomers (3.283 Å), while the two different enantiomers were also found to interact
with each other through π stacking (3.380 Å).
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Figure 5. Molecular structure of the Os complex 1b drawn at 50% probability level. The π-stacking
interaction between the same enantiomers of 1b are indicated by a black dashed line, while the π-
stacking between the two enantiomers is shown as an orange dashed line between the pyridine
moieties of the ligands.
As expected, the Ru–I bond distance in 1c (2.7149(6) Å) was longer than the Ru–Cl bond in
1a (2.4143(15) Å) which was similar to the Os–Cl bond length in 1b (Table 1). The M–N
distances were similar in all cases indicating similar electronic conditions around the metal
center. This implies that in all cases the ligands were coordinated as carboxamidates to the
metal centers after deprotonation of the amide group.
Table 1. Selected bond lengths [Å] observed in the molecular structures of 1a, 1b and 1c.
Bond
Bond length / Å
1b
1a
2.4143(15)
2.092(5)
2.077(4)
1.356(7)
1.321(7)
1c
2.7149(6)
2.088(5)
2.083(4)
1.345(8)
1.320(8)
M–X
2.413(2)
2.098(7)
2.087(7)
1.353(11)
1.325(11)
M–N1
M–N2
C6–N1
C1–N2
The Ru and Os complexes as well as the ligands 1 and 2 were tested on their cytotoxicity
against human colon HCT116 and SW480, non-small cell lung NCI-H460, and cervical SiHa
carcinoma cells after 72 h incubation (Table 2). All tested compounds were inactive against
these cell lines, as was aspirin in NCI-H460 and HCT116. In fact it was only possible to
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determine IC50 values in all four cell lines for the closest aspirin derivative, i.e., 2, while for
most of the other compounds the IC50 could not be reached due to limited solubility. This is
surprising given the fact that coordination of an aspirin derivative functionalized with an
alkyne to Co carbonyl complexes resulted in IC50 values in the low µM range in MCF-7
breast cancer cells [43] and the Pt derivative asplatin gave IC50 values around 1 µM in HeLa
cervical and HepG2 liver cancer cells [30]. On the other hand aspirin was found inactive in
these cell lines, as reported also by other groups for a variety of cell lines [44-46]. This may
be related to aspirin exhibiting its anticancer activity by acting on the tumor
microenvironment, e.g. through preventing prostaglandin E2-mediated evasion of T-cell
dependent tumor control, rather than on individual cancer cells [47].
Table 2. IC50 values determined for the aspirin-inspired ligands and their Ru and Os complexes
against human colon HCT116 and SW480, non-small cell lung NCI-H460, and cervical SiHa
carcinoma cells after 72 h incubation.
Compound
IC50 values / µM
SiHa HCT116
NCI-H460
1569 ± 239
˃ 426
SW480
-
aspirin
-
949 ± 108
˃ 426
325 ± 56
˃ 1054
˃ 280
˃ 426
˃ 1054
˃ 280
474 ± 47
˃ 280
1
˃ 1054
˃ 280
1a
1b
2
˃ 280
˃ 280
399 ± 15
˃ 280
290 ± 24
˃ 280
323 ± 34
˃ 280
2a
Conclusions
Picolinamides are an extensively studied class of ligands used for a wide variety of
applications. By preparing 2-picolinamides functionalized with 4-aminosalicylic acid and
subsequent acetylation we gained access to derivatives related to aspirin. The coordination of
aspirin derivatives to metal centers has been reported to lead to metal complexes with
anticancer activity. Therefore we used these ligands to prepare organo-Ru and -Os
complexes. 2-Picolinamide can coordinate to metal centers either as an N,N- or N,O-bidentate
ligand. While, N,N-coordination is thermodynamically favorable, the coordination mode is
pH-dependent. In this case, the amide functional group underwent deprotonation and the
ligands coordinated to the RuII and OsII centers through their pyridyl and carboxamide
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nitrogen atoms to obtain stable N,N-type of complexes. This was confirmed by single crystal
1
X-ray diffraction analysis and H NMR spectroscopy. In the molecular structures of the
complexes a multitude of H bonds were detected leading to a 1D network that was cross-
linked by π stacking interactions mostly between the phenolic ligands and the p-cymene
moieties coordinated to the metal centers. The obtained metal complexes and their ligands
were assayed on their antiproliferative activity against a series of different cancer cell lines
but none of the compounds were significantly potent cell growth inhibitors.
Acknowledgments
We thank the University of Auckland, the Austrian Science Fund (Schrödinger fellowship to
M.H.), the Higher Education Commission of Pakistan (IRSIP Scholarship to A. A.), the
India-New Zealand Education Council, Education New Zealand, the India-New Zealand
Research Institute, the Royal Society of New Zealand and COST CM1105 for funding.
Experimental
Material and methods
All the reactions were carried out under inert conditions. All the chemicals were of analytical
grade and used without further purification. Picolinic acid, α-terpinene, triethyl amine, 4-
aminosalicylic acid, thionyl chloride, acetic acid, acetic anhydride and hydrazine
hydrochloride were obtained from Sigma Aldrich. RuCl3·3H2O (40.4%) and OsO4 were
purchased from Precious Metals Online. Methanol, dichloromethane and dimethyl formamide
were dried according to standard procedures. The dimers bis[dichlorido(η6-p-
cymene)ruthenium(II)] [48], bis[diiodido(η6-p-cymene)ruthenium(II)] [49], bis[dichlorido(η6-
p-cymene)osmium(II)] [50] were synthesized by adapting reported procedures.
High-resolution mass spectra were recorded on the Bruker micro TOF-Q II electrospray
ionization (ESI) mass spectrometer in positive ion mode. 1H and 13C{1H} NMR spectra were
recorded on Bruker DRX 400 MHz NMR spectrometers at ambient temperature at 400.13
(1H) and 100.61 MHz (13C{1H}), and 2D NMR data were collected in a gradient-enhanced
1
mode. H and 13C{1H} chemical shifts are reported vs SiMe4 and were determined by
1
reference to the residual H and 13C{1H} solvent peaks. All compounds were analyzed via
multinuclear 2D (1H-1H COSY, 1H-13C HSQC, and HMBC) NMR spectroscopic
experiments, allowing unambiguous assignments of the resonances.
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The X-ray diffraction data of crystals of 1a−1c were collected on a Bruker Smart APEX II
diffractometer with graphite-monochromatized Mo Kα radiation (λ = 0.71073 Å) at 100 K.
Data reduction was carried out using the SAINT program [51]. Semi-empirical absorption
corrections were applied based on equivalent reflections using SADABS [52]. The structure
solution and refinements were performed with the SHELXS-97 and SHELXL-2013 program
packages [53].
Table 3. XRD data for the compound 1a, 1b and 1c
Compound
CCDC
1a
1b
1c
1507689
1507690
1507691
Chemical
C23H23ClN2O4Ru ꢀ CH3OH
C23H23ClN2O4Os
C23H23IN2O4Ru ꢀ CH3OH
Formula
M (g mol-1)
559.99
100(2)
617.08
651.44
temperature (K)
100(2)
100(2)
crystal size (mm) 0.34 × 0.10 × 0.08
0.24 × 0.10 × 0.05
Monoclinic
P21/c
0.38 × 0.12 × 0.08
Monoclinic
P21
crystal system
space group
a (Å)
Monoclinic
P21
7.9960(2)
16.6547(5)
8.8148(2)
98.0820(10)
1162.22(5)
2
8.7053(3)
16.7894(5)
15.5887(5)
100.1845(15)
2242.49(13)
4
8.4582(5)
16.6622(9)
8.7067(5)
99.370(3)
1210.68(12)
2
b (Å)
c (Å)
β (º)
V (A3)
Z
Dc (mg m-3)
µ (mm-1)
F (000)
Θ range (deg)
h range
k range
1.623
1.828
1.787
0.828
5.838
1.959
572
1200
644
2.63 to 25.44
-9 to 9
1.80 to 27.68
-10 to 10
-21 to 20
-20 to 17
17438
2.37 to 27.95
-10 to 11
-19 to 21
-11 to 11
14746
-19 to 20
-10 to 10
11819
l range
no. refls.
no. parameters
Rint
313
285
302
0.0834
0.0570
0.0432
R1 (obs.)
wR2 (all data)
S
0.0289
0.0545
0.0324
0.0644
0.1126
0.0752
1.008
1.154
1.049
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Syntheses
2-Hydroxy-4-(picolinamido)benzoic acid 1
A mixture of picolinic acid (0.96 g, 7.84 mmol) and thionyl chloride (1.15 mL, 15.60 mmol)
was stirred in dry dichloromethane for 18 h at 40 °C under inert conditions. The solvent was
removed under vaccuum, the residue was dissolved in dry DMF (10 mL) and triethyl amine
(4.34 mL, 31.2 mmol) was added at 0 °C. After stirring for 5 min, 4-aminosalicylic acid (0.80
g, 5.22 mmol) was added and the reaction was stirred for 18 h at room temperature. The
solvent was removed under vacuum and the residue was treated with water (200 mL) and
HCl (35%; 3.3 mL), which resulted in the formation of off-white precipitates. The product
was filtered, washed with water and dried.
Yield: 89% (1.20 g, off-white), MS (ESI+): m/z = 281.0525 [M + Na]+ (calcd. 281.0538).
Anal. Found: C, 59.48; H, 3.96; N, 10.58. Calcd for C13H10N2O4·0.25H2O: C, 59.43; H, 4.03;
1
3
N, 10.66. H NMR (400.13 MHz, [d4]MeOH, 25 °C): δ = 8.49 (d, J = 8 Hz, 1H, H-6), 8.00
(d, 3J = 8 Hz, 1H, H-3), 7.98 (t, 3J = 8 Hz, 1H, H-4), 7.64 (d, 3J = 9 Hz, 1H, H-13), 7.41-7.38
3
4
13
(m, 2H, H-5, H-10), 7.05 (dd, J = 9 Hz, J = 1 Hz, 1H, H-14) ppm. C{1H} NMR (100.61
MHz, [d4]MeOH, 25 °C): δ = 171.7 (C-15), 163.7 (C-11), 162.0 (C-7), 149.6 (C-2), 148.5 (C-
6), 144.8 (C-9), 138.3 (C-4), 130.9 (C-13), 127.3 (C-5), 122.7 (C-3), 111.4 (C-10), 108.4 (C-
12), 107.2 (C-14) ppm.
2-Acetoxy-4-(picolinamido)benzoic acid 2
2-Hydroxy-4-(picolinamido)benzoic acid (0.40 g, 1.55 mmol) was placed in a freshly
prepared mixture of acetic acid (3 mL) and acetic anhydride (3 mL) in a 25 mL round bottom
flask. The reaction mixture was heated to reflux for 0.5 h. After cooling down the reaction
mixture was directly poured into 50 mL cold water and stirred vigorously to obtain the
desired product. The precipitated product was filtered and washed with water to give rise of
2.
Yield: 86% (0.40 g, off-white), MS (ESI+): m/z = [M + Na]+ Calcd. 323.0644, obs: 323.0636.
Anal. Found: C, 58.25; H, 4.27; N, 8.25. Calcd for C15H12N2O5·0.5H2O: C, 58.25; H, 4.24; N,
1
9.06. H NMR (400.13 MHz, [d4]MeOD, 25 °C): δ = 8.71-8.69 (m, 1H, H-6), 8.23-8.20 (m,
1H, H-3), 8.04 (d, 3J = 9 Hz, 1H, H-13), 7.98 (ddd, 3J = 8 Hz, 4J = 1 Hz, 1H, H-4), 7.81 (d, 4J
= 2 Hz, 1H, H-10), 7.73 (dd, 3J = 9 Hz, 4J = 2 Hz, 1H, H-14), 7.62-7.59 (m, 1H, H-5), 2.31 (s,
13
3H, H-17) ppm. C{1H} NMR (100.61 MHz, [d4]MeOH, 25 °C): δ = 171.4 (C-15), 167.2
(C-11), 164.8 (C-7), 153.2 (C-16), 150.7 (C-2), 149.7 (C-6), 144.5 (C-9), 139.1 (C-4), 133.8
(C-13), 128.3 (C-5), 123.6 (C-3), 120.1 (C-10), 118.0 (C-12), 115.7 (C-14), 21.1 (C-17) ppm.
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General procedure for the synthesis of the Ru and Os complexes
A suspension of ligand (0.40 mmol) and [M(cym)Cl2]2 (0.20 mmol) in dichloromethane
(20 mL) was subjected to reflux for 12 h to get an orange suspension. Then acetone was
added (3 mL) to the reaction mixture and continued to reflux for a further 2 h. The solvent
was evaporated completely under reduced pressure and the residue was washed with DCM
and filtered to collect the orange precipitates.
Chlorido(η6-p-cymene)[2-hydroxy-4-(picolinamido)benzoic acid]ruthenium(II) 1a
Complex 1a was synthesized by following the general procedure using ligand 1 (103 mg,
0.40 mmol) and [Ru(cym)Cl2]2 (122 mg, 0.20 mmol).
Yield: 66% (141 mg). MS (ESI+): m/z = [M – Cl]+ Calcd. 493.0701, obs: 493.0739. Anal.
Found: C, 51.89; H, 4.44; N, 5.23. Calcd for C23H23ClN2O4Ru·0.25H2O: C, 51.88; H, 4.45;
1
3
N, 5.26. H NMR (400.13 MHz, [d4]MeOH, 25 °C): δ = 9.27 (d, J = 5 Hz, 1H, H-6), 8.10
(ddd, 3J = 8 Hz, 4J = 1 Hz, 1H, H-4), 7.96 (dd, 3J = 8 Hz, 4J = 1 Hz, 1H, H-3), 7.90 (d, 3J = 9
3
4
Hz, 1H, H-13), 7.67 (ddd, J = 7 Hz, J = 1 Hz, 1H, H-5), 7.20-7.16 (m, 2H, H-10, H-14),
3
3
3
5.64 (d, J = 6 Hz, 1H, H-20/H-22), 5.43 (d, J = 6 Hz, 1H, H-20/H-22), 5.32 (d, J = 6 Hz,
3
1H, H-21/H-23), 4.98 (d, J = 6 Hz, 1H, H-21/H-23), 2.63-2.56 (m, 1H, H-26), 2.19 (s, 3H,
H-24), 1.08 (d, 3J = 7 Hz, 3H, H-25/H-27), 1.06 (d, 3J = 7 Hz, 3H, H-25/H-27) ppm. 13C{1H}
NMR (100.61 MHz, [d4]MeOH, 25 °C): δ = 173.2 (C-15), 168.6 (C-7), 163.5 (C-11), 159.7
(C-2) , 156.0 (C-9), 155.6 (C-6), 140.3 (C-4), 131.6 (C-13), 128.5 (C-5), 126.6 (C-3), 118.8
(C-10), 115.4 (C-12), 110.6 (C-14), 103.4 (C-21), 102.4 (C-18), 86.2 (C-20/C-22), 86.1 (C-
20/C-22), 85.1 (C-19/C-23), 85.0 (C-19/C-23), 32.1 (C-26), 22.5 (C-25/C-27), 22.2 (C-25/C-
27), 19.0 (C-24) ppm.
Chlorido(η6-p-cymene)[2-hydroxy-4-(picolinamido)benzoic acid]osmium(II) 1b
Complex 1b was synthesized by following the general procedure using ligand 1 (103 mg,
0.40 mmol) and [Os(cym)Cl2]2 (158 mg, 0.20 mmol).
Yield: 84% (214 mg). MS (ESI+): m/z = [M – Cl]+ Calcd. 583.1273, obs: 583.1270. Anal.
Found: C, 43.41; H, 3.81; N, 4.42. Calcd for C23H23ClN2O2Os·H2O: C, 43.49; H, 3.97; N,
4.41. 1H NMR (400.13 MHz, [d4]MeOH, 25 °C): δ = 9.20 (d, 3J = 6 Hz, 1H, H-6), 8.13 (ddd,
4
3J = 8 Hz, J = 6 Hz, 1H, H-4), 7.89 (dd, 3J = 8 Hz, 4J = 1 Hz, 1H, H-3), 7.79 (d, 3J = 9 Hz,
1H, H-13), 7.67 (ddd, 3J = 7 Hz, 4J = 1 Hz, 1H, H-5), 7.12-7.10 (m, 2H, H-10, H-14), 5.96 (d,
3
3
3J = 6 Hz, 1H, H-20/H-22), 5.68 (d, J = 6 Hz, 1H, H-20/H-22), 5.58 (d, J = 6 Hz, 1H, H-
14

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3
21/H-23), 5.17 (d, J = 6 Hz, 1H, H-21/H-23), 2.54-2.47 (m, 1H, H-26), 2.31 (s, 3H, H-24),
13
1.10-1.05 (m, 6H, H-25, H-27) ppm. C{1H} NMR (100.61 MHz, [d4]MeOH, 25 °C): δ =
173.2 (C-15), 171.2 (C-7), 163.5 (C-11), 159.5 (C-2), 156.0 (C-6), 155.5 (C-9), 140.5 (C-4),
131.6 (C-13), 129.3 (C-5), 127.0 (C-3), 119.2 (C-10), 115.7 (C-12), 110.8 (C-14), 95.4 (C-
21), 94.1 (C-18), 77.5 (C-20/C-22), 77.3 (C-20/C-22), 74.8 (C-19/C-23), 74.7 (C-19/C-23),
32.6 (C-26), 22.8 (C-25/C-27), 22.7 (C-25/C-27), 19.0 (C-24) ppm.
Iodido(η6-p-cymene)[2-hydroxy-4-(picolinamido)benzoic acid]ruthenium(II) 1c
Complex 1c was synthesized by following the general procedure using ligand 1 (77 mg,
0.30 mmol) and [Ru(cym)I2]2 (147 mg, 0.15 mmol).
Yield: 76% (141 mg). MS (ESI+): m/z = [M – I]+ Calcd. 493.0701, obs: 493.0712. Anal.
1
Found: C, 43.45; H, 4.13; N, 4.32. Calcd for C23H24IN2O4Ru: C, 43.58; H, 4.05; N, 4.42. H
NMR (400.13 MHz, [d4]MeOH, 25 °C): δ = 9.27 (d, 3J = 5 Hz, 1H, H-6), 8.05 (td, 3J = 8 Hz,
4J = 1 Hz, 1H, H-4), 7.94 (dd, 3J = 8 Hz, 4J = 1 Hz, 1H, H-3), 7.89 (d, 3J = 8 Hz, 1H, H-13),
7.61 (ddd, 3J = 7 Hz, 4J = 2 Hz, 1H, H-5), 7.29 (d, 4J = 2 Hz, H-10), 7.25 (dd, 3J = 8 Hz, 4J =
3
3
2 Hz, 1H, H-14), 5.47 (d, J = 6 Hz, 2H, H-20/H-22), 5.36 (d, J = 6 Hz, 1H, H-21/H-23),
4.93 (d, 3J = 6 Hz, 1H, H-21/H-23), 2.74-2.68 (m, 1H, H-26), 2.39 (s, 3H, H-24), 1.08 (d, 3J =
3
7 Hz, 3H, H-25/H-27), 1.05 (d, J = 7 Hz, 3H, H-25/H-27) ppm. 13C{1H} NMR (100.61
MHz, [d4]MeOH, 25 °C): δ = 173.4 (C-15), 168.6 (C-7), 163.4 (C-11), 16.1 (C-2), 157.2 (C-
6), 156.4 (C-9), 140.1 (C-4), 131.8 (C-13), 128.4 (C-5), 127.0 (C-3), 119.5 (C-10), 116.0 (C-
12), 110.8 (C-14), 106.6 (C-21), 100.6 (C-18), 86.6 (C-20/C-22), 85.8 (C-20/C-22), 85.6 (C-
19/C-23), 85.5 (C-19/C-23), 32.7 (C-24), 23.1 (C-26), 22.5 (C-25/27), 21.4 (C-25/27) ppm.
Chlorido(η6-p-cymene)[2-acetoxy-4-(picolinamido)benzoic acid]ruthenium(II) 2a
Complex 2a was synthesized by following the general procedure using 2 (120 mg, 0.40
mmol) and [Ru(cym)Cl2]2 (122 mg, 0.20 mmol).
Yield: 63% (141 mg). MS (ESI+): m/z = [M – Cl]+ Calcd. 535.0807, obs: 535.0814; [M – Cl –
CH3CO]+ Calcd. 493.0701, obs: 493.0707. Anal. Found: C, 52.33; H, 4.61; N, 4.55. Calcd for
C25H25ClN2O5Ru: C, 52.68; H, 4.42; N, 4.91. 1H NMR (400.13 MHz, [d4]MeOH, 25 °C): δ =
9.29 (dd, 3J = 5 Hz, 4J = 1 Hz, 1H, H-6), 8.10 (ddd, 3J = 8 Hz, 4J = 1 Hz, 1H, H-4), 8.07 (d, 3J
= 8 Hz, 1H, H-13), 7.97 (dd, 3J = 8 Hz, 3J = 1 Hz, 1H, H-3), 7.67 (ddd, 3J = 6 Hz, 4J = 1 Hz,
1H, H-5), 7.60 (dd, 3J = 6 Hz, 4J = 1 Hz, 1H, H-14), 7.47 (d, 4J = 1 Hz, 1H, H-10), 5.67 (d, 3J
= 6 Hz, 1H, H-20/H-22), 5.43 (dd, 3J = 6 Hz, 4J = 1 Hz, 1H, H-20/H-22), 5.32 (dd, 3J = 6 Hz,
3
4J = 1 Hz, 1H, H-21/H-23), 5.04 (d, J = 6 Hz, 1H, H-21/H-23), 2.58-2.51 (m, 1H, H-26),
15

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3
13
2.34 (s, 3H, H-17), 2.18 (s, 3H, H-24), 1.06 (d, J = 7 Hz, 6H, H-25, H-27) ppm. C{1H}
NMR (100.61 MHz, [d4]MeOH, 25 °C): δ = 171.9 (C-15), 168.9 (C-7), 167.5 (C-11), 158.5
(C-16), 156.0 (C-2), 155.9 (C-6), 152.6 (C-9), 14.5 (C-4), 132.9 (C-13), 128.7 (C-5), 123.7
(C-3), 125.3 (C-10), 123.2 (C-14), 120.2 (C-12), 103.5 (C-21), 103.0 (C-18), 86.5 (C-20/C-
22), 86.3 (C-20/C-22), 85.1 (C-19/C-23), 84.9 (C-19/C-23), 32.2 (C-26), 22.5 (C-25/C-27),
22.2 (C-25/C-27), 21.1 (C-17), 18.9 (C-24) ppm.
Biological Studies
The antiproliferative activity of the compounds in HCT116, NCI-H460, SiHa and SW480
cells was determined using the sulforhodamine B assay as described previously [38,54].
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Highlights
•
•
•
•
Preparation of aspirin-derived organometallic compounds
Picolinamide-based ligands act as bidentate chelators to the metal center
Structural characterization shows a network of hydrogen bonding and
Complexes were non-cytotoxic in human cancer cells
π stacking interactions