Gold(i) compounds with lansoprazole-type ligands: Synthesis, characterization and anticancer properties in vitro

Article abstract of DOI:10.1039/c4md00241e

A number of gold(i) complexes containing the proton pump inhibitor (PPI) lansoprazole and its reduced precursor 2-((3-methyl-4-(2,2,2-trifluoroethoxy) pyridin-2-yl)methylthio)-1H-benzo[d]imidazole have been synthesized and their biological effects have been evaluated in human cancer and non-tumorigenic cells in vitro. The lansoprazole-based compounds appear to act through a V-H ⁺-ATPase-mediated mechanism.

Full text of DOI:10.1039/c4md00241e

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Gold(I) compounds with lansoprazole-type ligands:
synthesis, characterization and anticancer
properties in vitro†
Cite this: Med. Chem. Commun., 2014,
5, 1418
M. Serratice,‡a B. Bertrand,‡bc E. F. J. Janssen,b E. Hemelt,b A. Zucca,a F. Cocco,a
a
b
*
*
M. A. Cinellu and A. Casini
A number of gold(I) complexes containing the proton pump inhibitor (PPI) lansoprazole and its reduced
precursor 2-((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl)methylthio)-1H-benzo[d]imidazole have
been synthesized and their biological effects have been evaluated in human cancer and non-
tumorigenic cells in vitro. The lansoprazole-based compounds appear to act through a V-H+-ATPase-
mediated mechanism.
Received 6th June 2014
Accepted 21st July 2014
DOI: 10.1039/c4md00241e
www.rsc.org/medchemcomm
Gold compounds have a long and important tradition in the alteration of the tumour microenvironment via changes in
medicine, the so-called Chrysotherapy that derives its name the pH gradient between the extracellular environment and the
from Chryses, a golden-haired heroine of Greek Mythology. cell cytoplasm.9 In fact, the extracellular pH of solid tumours is
Such a tradition dates back to the Chinese (2500 B.C.) and signicantly more acidic than that of normal tissues,10 thus
especially ourished in Europe during the late Middle Age and impairing the uptake of weakly basic chemotherapeutic drugs
the Renaissance. In early times of modern pharmacology, gold and reducing their anticancer effect. Therefore, a strategy to
compounds were widely used for the treatment of several revert drug resistance is the use of agents that disrupt the pH
diseases, especially as anti-infective and anti-tubercular agents. gradient in tumours by inhibiting the function of pumps
Nowadays, gold-based drugs are only used as a treatment for generating the pH gradient itself, such as vacuolar H+-ATPases
severe rheumatoid arthritis, because of their systemic toxicity (V-H+-ATPases).11,12
and poor stability. Moreover, in recent years, gold compounds
A class of V-H+-ATPase inhibitors, called proton pump
hold great promise as potential anticancer drugs.1,2 In fact, a inhibitors (PPIs), have emerged as the drug class of choice for
conspicuous number of coordination and organometallic gold treating patients with peptic diseases.13,14 These drugs inhibit
complexes, with highly different chemical structures, were gastric acid secretion by targeting the gastric acid pump, but
reported to manifest outstanding antiproliferative properties in they also directly inhibit V-H+-ATPases. PPIs are in general
cancer cells in vitro, and sometimes in vivo.3–5
protonable weak bases selectively accumulating in acidic
With the aim of developing new gold-based cytotoxic agents, spaces. Currently, there are ve PPIs available for treating
our groups have been particularly focusing on the study of gastric and duodenal ulcer disease, namely omeprazole, pan-
gold(I) and gold(III) complexes with nitrogen donor ligands and toprazole, esomeprazole, rabeprazole and lansoprazole. They
on the investigation of their biological mechanisms of action.6–8 are all substituted benzimidazoles that behave as prodrugs that
It is worth mentioning that resistance to cytotoxic agents is a need to be activated in vivo.13
major problem in treating cancer. The mechanisms underlying
Lansoprazole (HL2, Fig. 1) has a structure that combines a
this phenomenon appear to prot of functions involved in the benzimidazole ring and a pyridine ring connected through a
control of cell homeostasis. A mechanism of resistance may be sulnyl linkage. The compound's activation occurs by addition
of two protons to the nitrogens on either side of the sulnyl
group. The second protonation on the benzimidazole ring
a
causes rearrangement of the sulnyl into a sulfenic acid or a
sulfenamide. The latter can react with the cysteine molecules of
the H+-ATPase to form one or two disulde bonds.15
PPI may revert chemoresistance and increase chemo-
sensitivity of different human tumour cells. Recent ndings
from Fais et al. have shown that PPI pretreatment sensitized
tumour cell lines to the effect of cisplatin, 5-uorouracil and
vinblastine.16 PPI pretreatment was associated with the
`
Universita degli Studi di Sassari, Dipartimento di Chimica e Farmacia, Via Vienna, 2,
Sassari, I-07100, Italy. E-mail: cinellu@uniss.it; Fax: +39 079 229559
bDept. of Pharmacokinetics, Toxicology and Targeting, Research Institute of Pharmacy,
University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands. E-mail: a.casini@rug.nl; Fax: +31 50 363 3274
c
´
´
Institut de Chimie Moleculaire de l'Universite de Bourgogne, UMR 6302 CNRS
´
Universite de Bourgogne, Dijon, France
† Electronic supplementary information (ESI) available: Experimental section and
analysis of compounds, NMR spectra. See DOI: 10.1039/c4md00241e
‡ These two authors contributed equally to the work.
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Fig. 1 Ligands HL1 and lansoprazole (HL2) and their mononuclear gold(I) complexes.
inhibition of V-H+-ATPase activity and an increase of both assess compounds' selectivity. Moreover, preliminary results on
extracellular pH and the pH of lysosomal organelles, consistent the possible contribution of a V-H+-ATPase-mediated mecha-
with a cytoplasmic retention of the cytotoxic drugs and targeting nism in the compounds' biological mode of action were
to the nucleus in the case of doxorubicin.16 In vivo experiments obtained.
showed that oral pretreatment with omeprazole induced a
Initially, lansoprazole (2-((3-methyl-4-(2,2,2-triuoroethoxy)-
sensitivity of the human solid tumors to anticancer drugs.16 pyridin-2-yl)methylsulnyl)-1H-benzo[d]imidazole) (HL2) was
Moreover, PPI pre-treatment of melanoma cells increased prepared by oxidation of 2-((3-methyl-4-(2,2,2-triuoroethoxy)
cellular uptake of cisplatin, as compared to untreated cells, in an pyridin-2-yl)methylthio)-1H-benzo[d]imidazole (HL1, Fig. 1)
acidic-depend manner.17
according to a recently reported method.21
Interestingly, other studies described the advantages of
Aerward, a variety of gold(I) complexes, both mono-
combining lansoprazole with metal ions, such as Ni(II), Co(II), (Fig. 1) and dinuclear (Fig. 2), have been prepared by reaction
Mn(II), Cu(II) and Hg(II), as well as inner transition metals of the two ligands with the appropriate gold(I) precursor. The
1
(Th(IV), Ce(IV), Nd(III) and Gd(III)), for therapeutic purposes as compounds have been characterized by H and 31P NMR, as
antibacterial or antifungal agents.18,19 Recently, the pump well as IR spectroscopy and elemental analysis (see ESI† for
inhibitor pantoprazole has also been linked to a Gd-based details).
compound to enhance the accumulation of the resulting MRI
contrast agent in the stomach and colon.20
Within this frame, we decided to explore the possibility to
couple gold ions to lansoprazole-type ligands in order to achieve
“bifunctional” metal compounds able to selectively target
cancer cells. Thus, we report here on the synthesis and chemical
characterization of a series of mono- and binuclear gold(I)
compounds bearing lansoprazole-type ligands. The anti-
proliferative properties of the compounds have been evaluated
in cancer cell lines, including the human ovarian cancer A2780
cells and their cisplatin resistant variant A2780cisR, as well as in
non-cancerous human embryonic kidney HEK-293T cells to
Fig. 2 Dinuclear complexes based on lansoprazole-type ligands.
Med. Chem. Commun., 2014, 5, 1418–1422 | 1419
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Three of the mononuclear complexes, namely [Au(HL1)Cl] and 1171 cmꢀ1, respectively. The proton spectrum in CD2Cl2
(1a) and [Au(HLn)(PPh3)](BF4) (n ¼ 1, 2a-BF4; n ¼ 2, 2b-BF4), shows well resolved signals for both the L1 and the saccharinate
contain the ligand – HL1 or HL2 – in its neutral form. The ligand in 1 : 1 molar ratio; most of the L1 signals are slightly
0
neutral complex 1a was prepared by reaction of HL1 with downeld shied with respect to the free ligand and the H3 and
0
[Au(THT)Cl] (THT ¼ tetrahydrothiophene). The two cationic H6 protons give rise to two multiplets, one partially overlapping
complexes 2a-BF4 and 2b-BF4, both featuring triphenyl- with the H4” and H5” resonances of the saccharinate ligand at
phosphine as ancillary ligand, were obtained by reaction of 7.76 ppm, the other at 7.67 ppm, thus suggesting coordination
the appropriate ligand with the solvento complex of the Au(sac) moiety to the deprotonated nitrogen atom of the
[Au(PPh3)(solv)](BF4), this was prepared in situ by reaction of imidazole ring. The 31P{1H} NMR spectra of 4a, 5a and 5b show
[Au(PPh3)Cl] with AgBF4 in dichloromethane.
Of the three possible coordination sites, i.e. the sulfur atom, consistent with linear gold(I) PPh3 (4a) and PTA (5a and 5b)
the pyridine and the imidazole nitrogen atom, the IR and H derivatives having a phosphorus trans to a nitrogen atom.8
singlet resonances at, respectively, d 32.2, ꢀ58.8 and ꢀ58.6 ppm
1
NMR data suggest that the latter is likely coordinated to the Au-
The binuclear complexes 7a-BF4 and 7b-BF4 (Fig. 2) were
Cl fragment in 1a. Indeed, its far IR spectrum shows the Au-Cl synthesized by reaction of the respective deprotonated ligand
stretching vibration at 341 cmꢀ1, consistent with a Au-Cl trans with 2 eq. of the solvento complex [(PPh3)Au(solv)](BF4) in
1
to a nitrogen atom, and the 1H NMR spectrum in CD2Cl2 shows dichloromethane. Their H NMR spectra are characterized by
two multiplets at d 7.71 and 7.63 ppm, each integrating for 1 well resolved signals with the correct L : PPh3 integral ratio. In
0
0
proton, attributed to H6 and H3 (numbering scheme in both cases, two signals are found in their 31P{1H} NMR spec-
Experimental section†), respectively, which in the free ligand trum at 31.2 and 33.1 (7a-BF4) and 31.0 and 33.2 (7b-BF4),
give rise to only one multiplet at 7.55 ppm, thus suggesting attributable to the Au(PPh3) coordinated respectively to the
involvement of the iminic nitrogen of the imidazole ring in the iminic and amidic nitrogen atom of the imidazole ring.
coordination to AuCl.
The new compounds were screened for their antiproliferative
In the case of 2a-BF4 and 2b-BF4, coordination of the AuPPh3 properties on human ovarian cancer cell lines sensitive (A2780)
fragment to a nitrogen atom is inferred by their 31P{1H} NMR and cisplatin resistant (A2780cisR) as well as in human embry-
spectrum, where a singlet resonance at d 31.6 and 31.2 ppm, onic kidney HEK-293T cells using the classical MTT assay (see
respectively, is consistent for linear gold(I) PPh3 derivatives Experimental for details†) in comparison to the “free” ligands
having a phosphorus trans to a nitrogen atom. The 1H NMR and cisplatin used as reference. A dose-dependent inhibition of
spectra of the two cationic complexes are characterized by broad cell growth was observed in all cell lines with IC50 values
signals, which suggest a uxionality process involving posi- ranging from ca. 0.7 to >70 mM aer 72 hours incubation as
tional exchange of the (PPh3)Au+ group between the two iminic depicted in Table 1.
nitrogen coordination sites. The site-exchange mode is repre-
Several conclusions can be drawn from the obtained results:
sented in Scheme 1. A similar behaviour was previously all compounds elicit good antiproliferative properties, while the
observed for analogous gold(I) complexes of 2-(20-pyridyl)benz- ligands are moderately toxic in the selected cell lines (the least
imidazole.8 All signals are shied upeld, as usually observed
for PPh3 complexes.
The neutral complexes 4a, 5a and 5b and the anionic Table 1 Cell viability (IC50 values) of gold(I) compounds and ligands
HL1 and lansoprazole (HL2) in human ovarian carcinoma cell lines
sensitive (A2780) or resistant to cisplatin (A2780cisR) and in HEK-293T
complex Na-3a, contain the ligand in its deprotonated form.
The saccharinate complex Na-3a was obtained from reaction of
cells after 72 h incubation
HL1 with Na[Au(sac)2]22 (sacH ¼ saccharine) in acetonitrile
IC50a (mM)
solution. The triphenylphosphine complex 4a was synthesized
by reaction of the solvento complex [Au(PPh3)(solv)](BF4) with
HL1 and KOH in acetone/MeCN/H2O, while the PTA (PTA ¼
1,3,5-triaza-7-phosphaadamantane) complexes 5a and 5b have
been obtained by reaction of [Au(PTA)Cl] with HL1 and HL2,
respectively, and KOH in H2O/MeCN solution.
Compound
A2780
A2780cisR
HEK-293T
HL1
26.3 ꢁ 8.5
45.6 ꢁ 2.6
20.2 ꢁ 4.0
>100
66.8 ꢁ 10.4
59.0 ꢁ 15.2
16.0 ꢁ 4.9
>100
>70
>70
Lansoprazole (HL2)
PPh3
PTA
18.6 ꢁ 2.6
>100
The IR spectrum of Na-3a shows the C]O and the SO2
stretching vibrations of the saccharinate ligand at 1689, 1290
1a
10.9 ꢁ 0.5
3.5 ꢁ 0.3
1.1 ꢁ 0.3
8.3 ꢁ 2.1
3.0 ꢁ 0.5
9.0 ꢁ 0.8
16.2 ꢁ 1.1
1.0 ꢁ 0.1
1.5 ꢁ 0.3
2.4 ꢁ 0.6
28.2 ꢁ 2.4
4.2 ꢁ 0.8
0.7 ꢁ 0.1
13.4 ꢁ 3.8
2.8 ꢁ 0.7
10.3 ꢁ 2.7
13.2 ꢁ 4.6
1.2 ꢁ 0.3
0.9 ꢁ 0.4
35.0 ꢁ 7.0
19.9 ꢁ 0.5
6.8 ꢁ 1.0
5.3 ꢁ 0.4
12.4 ꢁ 0.5
3.9 ꢁ 0.5
10.5 ꢁ 1.2
12.1 ꢁ 0.5
2.7 ꢁ 0.8
5.0 ꢁ 0.9
12.0 ꢁ 1.9
2a-BF4
2b-BF4
Na-3a
4a
5a
5b
7a-BF4
7b-BF4
Cisplatin
Scheme
1
Proposed site-exchange mode of the (PPh3)Au+
a
The reported values are the mean
determinations.
ꢁ
SD of at least three
group between the two iminic nitrogen coordination sites in 2a-BF4
and 2b-BF4.
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toxic being PTA ligand with IC50 higher than 100 mM). Secondly, lansoprazole also showed similar increase in ATP levels
each gold complex shows similar cell growth inhibition in both (following the order 2b-BF4 [ 7b-BF4 > 5b z lansoprazole, see
A2780 and A2780cisR cells. Notably, most of the new Fig. 3), although it must be noted that for compound 5b we had
compounds (made exception for 1a) are more active than to reach 5 mM concentration to obtain a signicant shi
cisplatin towards the resistant cell line A2780cisR, indicating compared to controls. Conversely, ligand HL1 and its derivatives
that different mechanisms of action take place with respect to did not show such an effect at any of the tested concentrations,
classical DNA alkylating agents as platinum drugs. This is not supporting the idea that other molecular mechanisms may be at
surprising since gold-based compounds have been shown to the basis of their antiproliferative properties. Specically,
mainly target proteins/enzymes in cancer cells.23
gold(I) complexes with phosphine ligands have been oen
The most effective derivatives of the series, even more potent described as potent inhibitors of intracellular seleno-enzymes
than cisplatin, are the mononuclear compound 2b-BF4 deriva- such as thioredoxin reductase.24 A possible reason for this
tive of lansoprazole, and the binuclear complexes 7a-BF4 and markedly different behaviour between the two classes of
7b-BF4 (IC50 in the 1 mM range), all of them containing PPh3 compounds is that the lack of the sulnyl group in HL1 does not
ligands and being positively charged in solution. The gold(I) allow activation of the ligand and its conversion into the sul-
derivatives bearing either the 1,3,5-triaza-7-phosphaada- fenamide responsible for V-H+-ATPase inhibition.
mantane (5a and 5b) or the saccharine (Na-3a) ligands are ca.
10-fold less effective. In general, the activities of the lansopra-
Conclusions
zole gold(I) complexes are very similar to those containing the
reduced HL1 ligand, but with the same secondary ligand.
In summary, we have synthesized and characterized nine new
gold(I) complexes bearing lansoprazole-type moieties. The
series includes mononuclear complexes containing the neutral
However, compound 2a-BF4 is less potent in cancer cells than
the lansoprazole derivative 2b-BF4. Moreover, compounds 2b-
BF4 and 7b-BF4 show certain selectivity towards the cancer cells,
ligand (1a, 2a-BF4, and 2b-BF4), mononuclear compounds
being ca. 4-5-fold less effective in the non-cancerous HEK-293T
bearing the ligand in its deprotonated form (Na-3a, 4a, 5a and
cell line.
5b), as well as the cationic binuclear complexes (7a-BF4 and
7b-BF4). The compounds have shown to possess enhanced
antiproliferative properties in human ovarian cancer cells
sensitive and resistant to cisplatin with respect to the “free”
In order to assess if the various gold complexes may exert
their biological effects through a V-H+-ATPase-mediated mech-
anism, we evaluated the levels of intracellular ATP in A2780 cells
treated with the compounds for 3 h as described in the Exper-
lansoprazole-type ligands.
imental section.† Interestingly, and in accordance with previ-
Preliminary data on the lansoprazole-based complexes
ously reported data for omeprazole and esomeprazole,16 cells
suggest that inhibition of V-H+-ATPase activity may represent an
treated with lansoprazole present higher levels of intracellular
important aspect of its mechanism of action compared to the
ATP than untreated cells (4.5 ꢁ 0.3 vs. 2.9 ꢁ 0.3 pmol per mg
case of gold(I) complexes containing the lansoprazole reduced
precursor. Certainly, several differences in the compounds'
structure may account for the observed diverse biological
protein, respectively), suggesting inhibition of the V-H+-ATPase
activity (Fig. 3). In addition, all the compounds based on
effects, including differences in transport mechanisms.
Although further studies are necessary to validate the
mechanisms of action of these new complexes, we believe that
our results hold promise to design novel cytotoxic metal
compounds that can be activated via the microenvironmental
acidication by cancerous tissues and whose biological targets
may differ from those of classical Pt(II) chemotherapic agents.
Acknowledgements
A. C. thanks the University of Groningen for funding (Rosalind
Franklin Fellowship) and the University of Sassari for a visiting
professorship. M. A. C. gratefully acknowledges the Regione
`
Autonoma della Sardegna (RAS) for the grant “Premialita
Regionale 2011”. B. B. thanks the ERASMUS program for a
mobility fellowship to the University of Sassari. Authors
acknowledge COST Action CM1105 for nancial support and
fruitful discussion.
Fig. 3 Intracellular ATP levels in A2780 cells untreated (Ctl) and
treated with fixed concentration of compounds (2 mM, and 5 mM for 5b)
for 3 hours at 37 ꢂC. The difference in cellular ATP levels for HL2 and Notes and references
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