- Email: [email protected]
Influence of the dissolution solvent on the cytotoxicity of octahedral cationic Ir(III) hydride complexes
Accepted Manuscript Influence of the dissolution solvent on the cytotoxicity of octahedral cationic Ir(III) hydride complexes Huaiyi Huang, Nicolas Humbert, Vincent Bizet, Malay Patra, Hui Chao, Clément Mazet, Gilles Gasser PII:
S0022-328X(16)30561-7
DOI:
10.1016/j.jorganchem.2016.12.010
Reference:
JOM 19733
To appear in:
Journal of Organometallic Chemistry
Received Date: 20 September 2016 Revised Date:
7 December 2016
Accepted Date: 8 December 2016
Please cite this article as: H. Huang, N. Humbert, V. Bizet, M. Patra, H. Chao, C. Mazet, G. Gasser, Influence of the dissolution solvent on the cytotoxicity of octahedral cationic Ir(III) hydride complexes, Journal of Organometallic Chemistry (2017), doi: 10.1016/j.jorganchem.2016.12.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
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Influence of the Dissolution Solvent on the Cytotoxicity of Octahedral Cationic Ir(III) Hydride Complexes Huaiyi Huang,a,b,# Nicolas Humbert,c,# Vincent Bizet,c Malay Patra,a Hui Chao,b,*
a
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Clément Mazet,c,*and Gilles Gasserd,* Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. b
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School, of
c
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Chemistry, Sun Yat-Sen University, Guangzhou, 510275, P. R. China.
Organic Chemistry Department, University of Geneva, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland.
Chimie ParisTech, PSL Research University, Laboratory for Inorganic Chemical Biology, F-75005 Paris, France.
*
Corresponding
authors:
M AN U
d
Email:
[email protected];
WWW:
http://www.unige.ch/sciences/chiorg/mazet/; Tel: +41 22 379 62 88; Email: [email protected]; Tel: +86 20 8411 0613; Email: gilles.gasser@chimie-
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paristech.fr; WWW: www.gassergroup.com; Tel.: +33 1 44 27 56 02.
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# these authors have contributed equally to the work.
Keywords: Bioorganometallic Chemistry, Dimethyl Formamide, Dimethyl Sulfoxide,
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Iridium, Ligand Exchange, Medicinal Organometallic Chemistry, Stability.
Highlights:
1) The stability in DMF and DMSO of Ir(III) complexes is presented. 2) The cytotoxicity of Ir(III) complexes is described. 3) The role of the solvents used in biological studies is discussed.
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ACCEPTED MANUSCRIPT Abstract The stability of a compound in the solvent in which it is dissolved is a fundamental parameter in medicinal chemistry. In this article, we report on the results of our investigations on the stability of five Ir(III) complexes in DMF and DMSO. Importantly, we demonstrate that the rate of ligand exchange/decomposition of these
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compounds has an influence on their in vitro anticancer properties. The compounds were generally found to be less toxic to cancer cells after having been dissolved for longer time (24 h) in DMSO compared to short incubation time (1 h) in the same solvent. On the contrary, only minor differences in cytotoxicity were observed when
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the compounds were dissolved in DMF, emphasizing that this solvent should be employed instead of DMSO when unstable compounds are investigated, provided that
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the concentration of DMF is kept at a low concentration level.
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1. Introduction The use of metal complexes to catalyze reactions in living cells is currently a hot topic in inorganic chemical biology.[1-3] Scientists have employed such a concept, for example, to kill cancer cells,[4-7] to cleave protecting groups in living cells,[8-10] to modify functional groups[11] or to form new C-C bonds (i.e. Suzuki-Miyaura cross
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coupling, Sonogashira coupling) in living cells with the view of cellular labelling, inhibition of cell functions or of modulators.[12] In order to achieve such goals, the stability of the metal complex in a biological environment (i.e. in the presence of air, water, thiols, etc.) to avoid poisoning the catalyst is a fundamental pre-requisite.[2, 9]
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However, it has not to be forgotten that this catalyst should also be stable in the solvent in which it is dissolved before being applied to living cells/organisms. Usually, the metal complex, which is very often not completely soluble in water, is
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first dissolved in DMSO or DMF. However, such solvents have the potential to act as ligands and coordinate to metal ion through their coordinating atoms (i.e. usually O for DMF and O and S for DMSO), thereby poisoning the catalyst. Some of us recently demonstrated that care needed to be taken with the biological assessment of Nheterocyclic-[Ru(η6-arene)Cl2] drug candidates since we observed a DMSO-mediated
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ligand dissociation.[13] Gottesmann and co-workers warned the community about the use of DMSO with platinum anticancer compounds in their paper entitled “Say not to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes”.[14] Another extremely important point to consider when using such an
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organic solvent to dissolve a compound is obviously the toxicity of the solvent itself. The toxicity of DMSO and DMF on cancer cells has been investigated by Arnaud,
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Ghafoori and co-workers.[15, 16] They could show that DMSO was generally less toxic than DMF but also that the toxicity was dependent on the type of cancer cell line studied.
In this work, we were interested in investigating the stability in DMF and DMSO of five Ir(III) complexes recently reported by some of us[17-19] in view of potential application in living cells (Figure 1). It has been previously shown that Ir(I) and Ir(III) complexes can indeed coordinate with DMSO[20] or DMF.[21-26] Specifically, in this contribution, we report on the in-depth examination of the stability of five Ir(III) complexes in DMSO and DMF at different time points. In addition, the toxicity profile of these compounds is evaluated on two different cancer cell lines, namely the
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ACCEPTED MANUSCRIPT human ovarian cancer A2780 and the cervical cancer HeLa cell lines, depending on the solvent used to prepare stock solutions. The latter assessment is highly relevant since the investigation of Ir(I)[27-34] and Ir(III)[35-43] complexes in medicinal
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chemistry is currently a topic of growing interest.
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Figure 1. Structures of the Ir(III) complexes studied in this work.
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2. Results and Discussion 2.1 NMR Stability Studies With the five Ir(III) complexes in hand,[17-19] the first experiment was to assess their stability using 1H and 31P{1H} NMR spectroscopy. For this purpose, an exact amount
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of the respective compounds was dissolved in d6-DMSO or d7-DMF (0.5 mL) and spectra were recorded after 2 and 24 h as well as after other time points when the compounds were found to decompose (all spectra can be found in the SI). A summary of our observations is presented in Table 1. Ir1 and the minor diastereoisomer of Ir4
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were found to be the only complexes of the series to completely disintegrate over 24 h. The other complexes were found to decompose in a much lower extend. Worthy of
complex Ir3.
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note, no major differences were observed between the two solvents except for
Table 1. Assessment of the stability of the Ir(III) complexes by NMR spectroscopy (see SI for the NMR spectra).
DMSO 2 h
Ir1a
ca. 15% decomposition
Ir2a
No change
Ir3b
No change
DMSO 24 h
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Compound
Ir4a
Faster decomposition of the minor diastereoisomer
Ir5c
Traces of decomposition
Complete decomposition Traces of decomposition Traces of decomposition of the minor diastereoisomer Minor diastereomer fully decomposed. Traces of decomposition of the major diastereoisomer ca. 15% decomposition
DMF 2 h
DMF 24 h
ca. 20% decomposition
Complete decomposition Traces of decomposition Minor diastereomer fully decomposed Minor diastereomer fully decomposed. Traces of decomposition of the major diastereoisomer ca. 20% decomposition
No change Traces of decomposition
Faster decomposition of the minor diastereoisomer
Traces of decomposition
a
20 mg of the complex was dissolved in the appropriate deuterated solvent (0.5 mL) and 1H and 31P{1H} NMR spectra were periodically recorded. b 6 mg of the complex was dissolved in the appropriate deuterated solvent (0.5 mL) and 1H and 31P{1H} NMR spectra were periodically recorded. c 14 mg of the complex was dissolved in the appropriate deuterated solvent (0.5 mL) and 1H and 31P{1H} NMR spectra were periodically recorded. 5
ACCEPTED MANUSCRIPT 2.2 Cytotoxicity Studies The cytotoxicity of Ir1-Ir5 was examined on the human ovarian epithelial A2780 and on cervical can cell lines. The well-known anticancer agent cisplatin was used as a reference in the cytotoxicity assay (Table 2). The stock solutions were prepared in two different solvents, namely DMSO and DMF. Of important note, the intrinsic
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toxicity of DMSO and DMF on these cancer cells lines was assessed by treating the cells with the culture medium containing 0.5% DMSO or DMF (the highest concentration used in the cellular studies). For DMSO, no cell toxicity could be detected among the concentration range. For DMF, about 10% inhibition on cell
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viability was observed when the cells were treated with 0.5% of DMF. However, when the IC50 values of the Ir(III) complexes were determined, we have used the values of cells treated with the same amount of solvent as a control. Hence, the
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potential intrinsic toxicity of DMF can be ignored. Two time points were investigated, namely 1 h and 24 h. Of note, for technical reasons, we relinquished to perform these experiments directly after dissolution since this would lead to some difference between the samples – it takes a bit of time to fill the holes of the 96-well tissue culture plate. As can be seen in Table 2, after 1 h incubation with the solvents (DMF
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or DMSO), Ir2-Ir5 were found to have a relatively high toxicity towards both cancer cell lines while Ir1 seemed to be less toxic. The IC50 values after 1 h in DMSO or DMF were nearly identical except for Ir1 which was found to be more cytotoxic in DMF than in DMSO, although the difference was not very significant if the
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experimental errors are taking into account. The distinctiveness of Ir1 compared to Ir2-Ir5 correlates well with its instability unveiled during our NMR studies.
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Very interestingly, after 24 h incubation with DMSO, the cytotoxicity of Ir1-Ir5 decreased significantly with IC50 values increasing by 2-3 times. On the contrary, the IC50 of Ir1-Ir5 are nearly identical after 1 h or 24 h incubation in DMF (within the error experimental). A similar trend in the change of IC50 values for the control cisplatin was also observed when DMSO was used to prepare the stock solution. This result is consistent with those reported earlier on the effect of DMSO in cisplatin’s cytotoxicity.[14] These outcomes are, in a first sight, counter-intuitive. Indeed, since similar results were obtained during the NMR stability studies for both solvents on all compounds tested in this study, it could have been anticipated that similar biological results should be obtained. Overall, these results clearly demonstrate that the solvent used to prepare stock solution has a direct influence on the cytotoxicity of the 6
ACCEPTED MANUSCRIPT complexes and that the use of a less coordinating solvent such as DMF should be preferred over a relatively strong coordinating solvent such as DMSO, since more consistent results are usually obtained – the IC50 values are not dramatically changing.
Table 2. IC50 values (µM) for Ir1-Ir5 after dissolution in DMSO or DMF.
DMSO
DMF
DMF
DMSO
(1 h)a
(24 h)b
(1 h)a
(24 h)b
(1 h)a
Ir1
16.5 ± 3.1
>20
9.4 ± 1.6
15.3 ± 5.1
>20
Ir2
9.2 ± 1.4
16.2 ± 3.4
4.4 ± 0.8
6.2 ± 0.9
11.7 ± 2.2
Ir3
6.1 ± 2.4
14.6 ± 2.5
5.3 ± 1.1
5.9 ± 1.0
Ir4
5.8 ± 0.9
>20
4.8 ± 1.4
4.1 ± 0.7
Ir5
5.4 ± 1.7
18.7 ± 3.1
4.6 ± 2.1
5.3 ± 1.2
Cisplatin
4.5 ± 0.1
32.7 ± 6.1
3.7 ± 0.3
4.7 ± 0.7
DMF
DMF
(24 h)b
(1 h)a
(24 h)b
>20
14.3 ± 4.5
16.2 ± 5.1
>20
9.1 ± 1.4
9.3 ± 2.0
7.1 ± 0.6
>20
6.5 ± 0.8
5.6 ± 0.7
6.6 ± 1.3
>20
7.5 ± 1.5
8.1 ± 1.3
7.3 ± 0.8
>20
4.3 ± 0.5
5.5 ± 0.9
8.7 ± 0.1
>100
5.3 ± 0.9
6.7 ± 0.2
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2.4 ± 0.7
Cisplatinc
DMSO
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DMSO
Compound
a
HeLa cell line
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A2780 cell line
9.3 ± 1.5
The stock solutions were prepared 1 h before adding into the culture medium. b The stock solutions were prepared 24 h before adding
into the culture medium. The final percentage of DMSO and DMF were kept below 0.5%. c The stock solution of cisplatin was freshly
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prepared in sterile saline (2 mM).
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3. Conclusion In this work, the stability of five Ir(III) complexes in DMF and DMSO was analyzed by 1H and
31
P{1H} NMR spectroscopy. This parameter is an important factor in
medicinal chemistry when the activity of a drug candidate is investigated. Specifically, we could show that the solvent used to dissolve the organometallic
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compounds studied in this work had a significant influence on the cytotoxic profiles. The Ir(III) complexes as well as cisplatin were, in general, found to be less toxic to HeLa and A2780 cancer cells after having been dissolved for long incubation times (24 h) in DMSO compared to short incubation times in the same solvent (1 h). In
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contrast, only minor differences in cytotoxicity were observed when the compounds were dissolved in DMF. Overall, this study emphasizes that DMF should be employed in in vitro cytotoxic assay on A2780 and HeLa cancer cell lines instead of
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DMSO when unstable inorganic compounds are investigated since more consistent
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results are usually obtained - the IC50 values are not dramatically changing.
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4. Experimental Section 4.1 Materials Commercial reagents were purchased from Sigma-Aldrich, Acros Organics or Strem Chemicals and used without purification unless otherwise noted. Air sensitive compounds such as phosphines or metal complexes were stored under inert
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atmosphere inside a MBraun glovebox. Solvents were dried over activated alumina columns and further degassed by three successive “freeze-pump-thaw” cycles if necessary. 4.2 Instrumentation and Methods
H, 31P{1H}, 19F{1H} and 13C{1H} spectra were recorded on ARX-300, ARX-400 and
ARX-500 Bruker Avance spectrometers. 1H and
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1
C NMR chemical shifts are given
in ppm relative to SiMe4, with the solvent resonance used as internal reference.
31
P
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NMR chemical shifts are reported in ppm relative to H3PO4. 19F NMR chemical shifts are reported in ppm relative to CFCl3. 4.3 Synthesis
All complexes studied in this work were prepared as previously reported by Humbert and Mazet.[17-19] The analytical data matched those previously reported.[17-19]
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4.4 Cytotoxicity studies[44]
Cytotoxicity activity tests were performed using freshly prepared stock solutions in DMSO or DMF of the Ir(III) complexes (5 mM), respectively. The solutions were kept in the dark for 1 h or 24 h before cell culture medium was added. The final
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concentration of DMSO or DMF was less than 0.5 %. The stock solution of cisplatin was freshly prepared in sterile saline (2 mM). A fluorometric cell viability assay using
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resazurin (Promocell GmbH) was employed to compare the cytotoxicity of the complexes in the dark for 48 h. A2780 and HeLa cell lines were plated in triplicates in 96-well plates at a density of 4000 cells per well in 100 µL 24 h prior to treatment. Cells were then treated with increasing concentrations of compounds for 48 h. The medium was then replaced by 100 mL fresh culture medium containing resazurin (final concentration 0.2 mg mL-1). After 4 h incubation at 37 °C, fluorescence of the highly red fluorescent resorufin product was quantified at 590 nm emission with 540 nm excitation wavelength in a SpectraMax M5 microplate reader.
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ACCEPTED MANUSCRIPT Acknowledgements This work was financially supported by the Swiss National Science Foundation (Professorships N° PP00P2_133568 and PP00P2_157545 to G.G. as well as PP00P2_133482 to C.M.), the University of Zurich (G.G), the University of Geneva
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(C.M.), the 973 program (No. 2015CB856301 to H.C.), the National Science Foundation of China (Nos. 21471164 and 21525105 to H.C.) and the China Scholarships Council (Grant No. 201506380026 to H.H.). This work has received support under the program «Investissements d’Avenir » launched by the French
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Government and implemented by the ANR with the reference ANR-10-IDEX-0001-
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02 PSL (G.G.). The authors thank Dr. Tanmaya Joshi for useful discussions.
Appendix A. Supplementary material
NMR spectra of complexes Ir1-Ir5 in d6-DMSO and d7-DMF at different time
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intervals.
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[1] G. Gasser Inorganic Chemical Biology: Principles, Techniques and Applications, John Wiley & Sons, Ltd, Chichester, West Sussex, UK, 2014. [2] M. Patra, G. Gasser, ChemBioChem, 13 (2012) 1232-1252. [3] P.K. Sasmal, C.N. Streu, E. Meggers, Chem. Commun., 49 (2013) 1581-1587. [4] J.J. Soldevila-Barreda, I. Romero-Canelón, A. Habtemariam, P.J. Sadler, Nature Commun., 6 (2015) DOI: 10.1038/ncomms7582. [5] J.J. Soldevila-Barreda, P.J. Sadler, Curr. Opin. Chem .Biol., 25 (2015) 172-183, and references therein. [6] J.T. Weiss, J.C. Dawson, K.G. Macleod, W. Rybski, C. Fraser, C. Torres-Sánchez, E.E. Patton, M. Bradley, N.O. Carragher, A. Unciti-Broceta, Nature Commun., 5 (2014) DOI: 10.1038/ncomms4277. [7] S.J. Dougan, A. Habtemariam, S.E. McHale, S. Parsons, P.J. Sadler, Proc. Natl. Acad. Sci. U.S.A, 105 (2008) 11628-11633. [8] T. Völker, E. Meggers, Curr. Opin. Chem .Biol., 25 (2015) 48-54, and publications therein. [9] C. Streu, E. Meggers, Angew. Chem. Int. Ed., 45 (2006) 5645-5648, and references therein. [10] J. Li, J. Yu, J. Zhao, J. Wang, S. Zheng, S. Lin, L. Chen, M. Yang, S. Jia, X. Zhang, P.R. Chen, Nature Chem., 6 (2014) 352-361. [11] P.K. Sasmal, S. Carregal-Romero, A.A. Han, C.N. Streu, Z. Lin, K. Namikawa, S.L. Elliott, R.W. Köster, W.J. Parak, E. Meggers, ChemBioChem, 13 (2012) 11161120. [12] R.M. Yusop, A. Unciti-Broceta, E.M.V. Johansson, R.M. Sanchez-Martin, M. Bradley, Nature Chem., 3 (2011) 239-243. [13] M. Patra, T. Joshi, V. Pierroz, K. Ingram, M. Kaiser, S. Ferrari, B. Spingler, J. Keiser, G. Gasser, Chem. Eur. J., 19 (2013) 14768-14722. [14] M.D. Hall, K.A. Telma, K.E. Chang, T.D. Lee, J.P. Madigan, J.R. Lloyd, I.S. Goldlust, J.D. Hoeschele, M.M. Gottesman, Cancer Res., 74 (2014) 3913-3922. [15] L. Jamalzadeh, H. Ghafoori, R. Sariri, H. Rabuti, J. Nasirzade, H. Hasani, M.R. Aghamaali, Avicenna J. Med. Biochem., 4 (2016) e33453. [16] G. Da Violante, N. Zerrouk, I. Richard, G. Provot, J.C. Chaumeil, P. Arnaud, Biol. Pharm. Bull., 25 (2002) 1600-1603. [17] N. Humbert, C. Mazet, Chem. Commun., (2016) 10629-10631. [18] N. Humbert, D.J. Vyas, C. Besnard, C. Mazet, Chem. Commun., 50 (2014) 10592-10595. [19] H. Li, C. Mazet, Org. Lett., 15 (2013) 6170-6173. [20] R. Dorta, H. Rozenberg, L.J.W. Shimon, D. Milstein, Chem. Eur. J., 9 (2003) 5237-5249. [21] A.R. Siedle, R.A. Newmark, J. Am. Chem. Soc., 111 (1989) 2058-2062. [22] A.R. Siedle, R.A. Newmark, Organometallics, 8 (1989) 1442-1450. [23] A.R. Siedle, R.A. Newmark, K.A. Brown-Wensley, R.P. Skarjune, L.C. Haddad, K.O. Hodgson, A.L. Roe, Organometallics, 7 (1988) 2078-2079. [24] H. Chen, X. Li, R. Bai, Y. Wu, Y. Fan, J. Chao, Organometallics, 32 (2013) 6226-6231. [25] J.A. Broomhead, W. Grumley, Inorg. Chem., 10 (1971) 2002-2009. [26] N. Curtis, G. Lawrance, A. Sargeson, Aust. J. Chem., 36 (1983) 1327-1339. [27] Y. Gothe, T. Marzo, L. Messori, N. Metzler-Nolte, Chem. Commun., 51 (2015) 3151-3153. 11
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ACCEPTED MANUSCRIPT Highlights: 1) The stability in DMF and DMSO of Ir(III) complexes is presented. 2) The cytotoxicity of Ir(III) complexes is described.
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3) The role of the solvents used in biological studies is discussed.
S0022-328X(16)30561-7
DOI:
10.1016/j.jorganchem.2016.12.010
Reference:
JOM 19733
To appear in:
Journal of Organometallic Chemistry
Received Date: 20 September 2016 Revised Date:
7 December 2016
Accepted Date: 8 December 2016
Please cite this article as: H. Huang, N. Humbert, V. Bizet, M. Patra, H. Chao, C. Mazet, G. Gasser, Influence of the dissolution solvent on the cytotoxicity of octahedral cationic Ir(III) hydride complexes, Journal of Organometallic Chemistry (2017), doi: 10.1016/j.jorganchem.2016.12.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
Influence of the Dissolution Solvent on the Cytotoxicity of Octahedral Cationic Ir(III) Hydride Complexes Huaiyi Huang,a,b,# Nicolas Humbert,c,# Vincent Bizet,c Malay Patra,a Hui Chao,b,*
a
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Clément Mazet,c,*and Gilles Gasserd,* Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. b
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School, of
c
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Chemistry, Sun Yat-Sen University, Guangzhou, 510275, P. R. China.
Organic Chemistry Department, University of Geneva, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland.
Chimie ParisTech, PSL Research University, Laboratory for Inorganic Chemical Biology, F-75005 Paris, France.
*
Corresponding
authors:
M AN U
d
Email:
[email protected];
WWW:
http://www.unige.ch/sciences/chiorg/mazet/; Tel: +41 22 379 62 88; Email: [email protected]; Tel: +86 20 8411 0613; Email: gilles.gasser@chimie-
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paristech.fr; WWW: www.gassergroup.com; Tel.: +33 1 44 27 56 02.
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# these authors have contributed equally to the work.
Keywords: Bioorganometallic Chemistry, Dimethyl Formamide, Dimethyl Sulfoxide,
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Iridium, Ligand Exchange, Medicinal Organometallic Chemistry, Stability.
Highlights:
1) The stability in DMF and DMSO of Ir(III) complexes is presented. 2) The cytotoxicity of Ir(III) complexes is described. 3) The role of the solvents used in biological studies is discussed.
1
ACCEPTED MANUSCRIPT Abstract The stability of a compound in the solvent in which it is dissolved is a fundamental parameter in medicinal chemistry. In this article, we report on the results of our investigations on the stability of five Ir(III) complexes in DMF and DMSO. Importantly, we demonstrate that the rate of ligand exchange/decomposition of these
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compounds has an influence on their in vitro anticancer properties. The compounds were generally found to be less toxic to cancer cells after having been dissolved for longer time (24 h) in DMSO compared to short incubation time (1 h) in the same solvent. On the contrary, only minor differences in cytotoxicity were observed when
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the compounds were dissolved in DMF, emphasizing that this solvent should be employed instead of DMSO when unstable compounds are investigated, provided that
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the concentration of DMF is kept at a low concentration level.
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1. Introduction The use of metal complexes to catalyze reactions in living cells is currently a hot topic in inorganic chemical biology.[1-3] Scientists have employed such a concept, for example, to kill cancer cells,[4-7] to cleave protecting groups in living cells,[8-10] to modify functional groups[11] or to form new C-C bonds (i.e. Suzuki-Miyaura cross
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coupling, Sonogashira coupling) in living cells with the view of cellular labelling, inhibition of cell functions or of modulators.[12] In order to achieve such goals, the stability of the metal complex in a biological environment (i.e. in the presence of air, water, thiols, etc.) to avoid poisoning the catalyst is a fundamental pre-requisite.[2, 9]
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However, it has not to be forgotten that this catalyst should also be stable in the solvent in which it is dissolved before being applied to living cells/organisms. Usually, the metal complex, which is very often not completely soluble in water, is
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first dissolved in DMSO or DMF. However, such solvents have the potential to act as ligands and coordinate to metal ion through their coordinating atoms (i.e. usually O for DMF and O and S for DMSO), thereby poisoning the catalyst. Some of us recently demonstrated that care needed to be taken with the biological assessment of Nheterocyclic-[Ru(η6-arene)Cl2] drug candidates since we observed a DMSO-mediated
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ligand dissociation.[13] Gottesmann and co-workers warned the community about the use of DMSO with platinum anticancer compounds in their paper entitled “Say not to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes”.[14] Another extremely important point to consider when using such an
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organic solvent to dissolve a compound is obviously the toxicity of the solvent itself. The toxicity of DMSO and DMF on cancer cells has been investigated by Arnaud,
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Ghafoori and co-workers.[15, 16] They could show that DMSO was generally less toxic than DMF but also that the toxicity was dependent on the type of cancer cell line studied.
In this work, we were interested in investigating the stability in DMF and DMSO of five Ir(III) complexes recently reported by some of us[17-19] in view of potential application in living cells (Figure 1). It has been previously shown that Ir(I) and Ir(III) complexes can indeed coordinate with DMSO[20] or DMF.[21-26] Specifically, in this contribution, we report on the in-depth examination of the stability of five Ir(III) complexes in DMSO and DMF at different time points. In addition, the toxicity profile of these compounds is evaluated on two different cancer cell lines, namely the
3
ACCEPTED MANUSCRIPT human ovarian cancer A2780 and the cervical cancer HeLa cell lines, depending on the solvent used to prepare stock solutions. The latter assessment is highly relevant since the investigation of Ir(I)[27-34] and Ir(III)[35-43] complexes in medicinal
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chemistry is currently a topic of growing interest.
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Figure 1. Structures of the Ir(III) complexes studied in this work.
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2. Results and Discussion 2.1 NMR Stability Studies With the five Ir(III) complexes in hand,[17-19] the first experiment was to assess their stability using 1H and 31P{1H} NMR spectroscopy. For this purpose, an exact amount
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of the respective compounds was dissolved in d6-DMSO or d7-DMF (0.5 mL) and spectra were recorded after 2 and 24 h as well as after other time points when the compounds were found to decompose (all spectra can be found in the SI). A summary of our observations is presented in Table 1. Ir1 and the minor diastereoisomer of Ir4
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were found to be the only complexes of the series to completely disintegrate over 24 h. The other complexes were found to decompose in a much lower extend. Worthy of
complex Ir3.
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note, no major differences were observed between the two solvents except for
Table 1. Assessment of the stability of the Ir(III) complexes by NMR spectroscopy (see SI for the NMR spectra).
DMSO 2 h
Ir1a
ca. 15% decomposition
Ir2a
No change
Ir3b
No change
DMSO 24 h
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Compound
Ir4a
Faster decomposition of the minor diastereoisomer
Ir5c
Traces of decomposition
Complete decomposition Traces of decomposition Traces of decomposition of the minor diastereoisomer Minor diastereomer fully decomposed. Traces of decomposition of the major diastereoisomer ca. 15% decomposition
DMF 2 h
DMF 24 h
ca. 20% decomposition
Complete decomposition Traces of decomposition Minor diastereomer fully decomposed Minor diastereomer fully decomposed. Traces of decomposition of the major diastereoisomer ca. 20% decomposition
No change Traces of decomposition
Faster decomposition of the minor diastereoisomer
Traces of decomposition
a
20 mg of the complex was dissolved in the appropriate deuterated solvent (0.5 mL) and 1H and 31P{1H} NMR spectra were periodically recorded. b 6 mg of the complex was dissolved in the appropriate deuterated solvent (0.5 mL) and 1H and 31P{1H} NMR spectra were periodically recorded. c 14 mg of the complex was dissolved in the appropriate deuterated solvent (0.5 mL) and 1H and 31P{1H} NMR spectra were periodically recorded. 5
ACCEPTED MANUSCRIPT 2.2 Cytotoxicity Studies The cytotoxicity of Ir1-Ir5 was examined on the human ovarian epithelial A2780 and on cervical can cell lines. The well-known anticancer agent cisplatin was used as a reference in the cytotoxicity assay (Table 2). The stock solutions were prepared in two different solvents, namely DMSO and DMF. Of important note, the intrinsic
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toxicity of DMSO and DMF on these cancer cells lines was assessed by treating the cells with the culture medium containing 0.5% DMSO or DMF (the highest concentration used in the cellular studies). For DMSO, no cell toxicity could be detected among the concentration range. For DMF, about 10% inhibition on cell
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viability was observed when the cells were treated with 0.5% of DMF. However, when the IC50 values of the Ir(III) complexes were determined, we have used the values of cells treated with the same amount of solvent as a control. Hence, the
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potential intrinsic toxicity of DMF can be ignored. Two time points were investigated, namely 1 h and 24 h. Of note, for technical reasons, we relinquished to perform these experiments directly after dissolution since this would lead to some difference between the samples – it takes a bit of time to fill the holes of the 96-well tissue culture plate. As can be seen in Table 2, after 1 h incubation with the solvents (DMF
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or DMSO), Ir2-Ir5 were found to have a relatively high toxicity towards both cancer cell lines while Ir1 seemed to be less toxic. The IC50 values after 1 h in DMSO or DMF were nearly identical except for Ir1 which was found to be more cytotoxic in DMF than in DMSO, although the difference was not very significant if the
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experimental errors are taking into account. The distinctiveness of Ir1 compared to Ir2-Ir5 correlates well with its instability unveiled during our NMR studies.
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Very interestingly, after 24 h incubation with DMSO, the cytotoxicity of Ir1-Ir5 decreased significantly with IC50 values increasing by 2-3 times. On the contrary, the IC50 of Ir1-Ir5 are nearly identical after 1 h or 24 h incubation in DMF (within the error experimental). A similar trend in the change of IC50 values for the control cisplatin was also observed when DMSO was used to prepare the stock solution. This result is consistent with those reported earlier on the effect of DMSO in cisplatin’s cytotoxicity.[14] These outcomes are, in a first sight, counter-intuitive. Indeed, since similar results were obtained during the NMR stability studies for both solvents on all compounds tested in this study, it could have been anticipated that similar biological results should be obtained. Overall, these results clearly demonstrate that the solvent used to prepare stock solution has a direct influence on the cytotoxicity of the 6
ACCEPTED MANUSCRIPT complexes and that the use of a less coordinating solvent such as DMF should be preferred over a relatively strong coordinating solvent such as DMSO, since more consistent results are usually obtained – the IC50 values are not dramatically changing.
Table 2. IC50 values (µM) for Ir1-Ir5 after dissolution in DMSO or DMF.
DMSO
DMF
DMF
DMSO
(1 h)a
(24 h)b
(1 h)a
(24 h)b
(1 h)a
Ir1
16.5 ± 3.1
>20
9.4 ± 1.6
15.3 ± 5.1
>20
Ir2
9.2 ± 1.4
16.2 ± 3.4
4.4 ± 0.8
6.2 ± 0.9
11.7 ± 2.2
Ir3
6.1 ± 2.4
14.6 ± 2.5
5.3 ± 1.1
5.9 ± 1.0
Ir4
5.8 ± 0.9
>20
4.8 ± 1.4
4.1 ± 0.7
Ir5
5.4 ± 1.7
18.7 ± 3.1
4.6 ± 2.1
5.3 ± 1.2
Cisplatin
4.5 ± 0.1
32.7 ± 6.1
3.7 ± 0.3
4.7 ± 0.7
DMF
DMF
(24 h)b
(1 h)a
(24 h)b
>20
14.3 ± 4.5
16.2 ± 5.1
>20
9.1 ± 1.4
9.3 ± 2.0
7.1 ± 0.6
>20
6.5 ± 0.8
5.6 ± 0.7
6.6 ± 1.3
>20
7.5 ± 1.5
8.1 ± 1.3
7.3 ± 0.8
>20
4.3 ± 0.5
5.5 ± 0.9
8.7 ± 0.1
>100
5.3 ± 0.9
6.7 ± 0.2
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2.4 ± 0.7
Cisplatinc
DMSO
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DMSO
Compound
a
HeLa cell line
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A2780 cell line
9.3 ± 1.5
The stock solutions were prepared 1 h before adding into the culture medium. b The stock solutions were prepared 24 h before adding
into the culture medium. The final percentage of DMSO and DMF were kept below 0.5%. c The stock solution of cisplatin was freshly
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prepared in sterile saline (2 mM).
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3. Conclusion In this work, the stability of five Ir(III) complexes in DMF and DMSO was analyzed by 1H and
31
P{1H} NMR spectroscopy. This parameter is an important factor in
medicinal chemistry when the activity of a drug candidate is investigated. Specifically, we could show that the solvent used to dissolve the organometallic
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compounds studied in this work had a significant influence on the cytotoxic profiles. The Ir(III) complexes as well as cisplatin were, in general, found to be less toxic to HeLa and A2780 cancer cells after having been dissolved for long incubation times (24 h) in DMSO compared to short incubation times in the same solvent (1 h). In
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contrast, only minor differences in cytotoxicity were observed when the compounds were dissolved in DMF. Overall, this study emphasizes that DMF should be employed in in vitro cytotoxic assay on A2780 and HeLa cancer cell lines instead of
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DMSO when unstable inorganic compounds are investigated since more consistent
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results are usually obtained - the IC50 values are not dramatically changing.
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4. Experimental Section 4.1 Materials Commercial reagents were purchased from Sigma-Aldrich, Acros Organics or Strem Chemicals and used without purification unless otherwise noted. Air sensitive compounds such as phosphines or metal complexes were stored under inert
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atmosphere inside a MBraun glovebox. Solvents were dried over activated alumina columns and further degassed by three successive “freeze-pump-thaw” cycles if necessary. 4.2 Instrumentation and Methods
H, 31P{1H}, 19F{1H} and 13C{1H} spectra were recorded on ARX-300, ARX-400 and
ARX-500 Bruker Avance spectrometers. 1H and
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1
C NMR chemical shifts are given
in ppm relative to SiMe4, with the solvent resonance used as internal reference.
31
P
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NMR chemical shifts are reported in ppm relative to H3PO4. 19F NMR chemical shifts are reported in ppm relative to CFCl3. 4.3 Synthesis
All complexes studied in this work were prepared as previously reported by Humbert and Mazet.[17-19] The analytical data matched those previously reported.[17-19]
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4.4 Cytotoxicity studies[44]
Cytotoxicity activity tests were performed using freshly prepared stock solutions in DMSO or DMF of the Ir(III) complexes (5 mM), respectively. The solutions were kept in the dark for 1 h or 24 h before cell culture medium was added. The final
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concentration of DMSO or DMF was less than 0.5 %. The stock solution of cisplatin was freshly prepared in sterile saline (2 mM). A fluorometric cell viability assay using
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resazurin (Promocell GmbH) was employed to compare the cytotoxicity of the complexes in the dark for 48 h. A2780 and HeLa cell lines were plated in triplicates in 96-well plates at a density of 4000 cells per well in 100 µL 24 h prior to treatment. Cells were then treated with increasing concentrations of compounds for 48 h. The medium was then replaced by 100 mL fresh culture medium containing resazurin (final concentration 0.2 mg mL-1). After 4 h incubation at 37 °C, fluorescence of the highly red fluorescent resorufin product was quantified at 590 nm emission with 540 nm excitation wavelength in a SpectraMax M5 microplate reader.
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ACCEPTED MANUSCRIPT Acknowledgements This work was financially supported by the Swiss National Science Foundation (Professorships N° PP00P2_133568 and PP00P2_157545 to G.G. as well as PP00P2_133482 to C.M.), the University of Zurich (G.G), the University of Geneva
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(C.M.), the 973 program (No. 2015CB856301 to H.C.), the National Science Foundation of China (Nos. 21471164 and 21525105 to H.C.) and the China Scholarships Council (Grant No. 201506380026 to H.H.). This work has received support under the program «Investissements d’Avenir » launched by the French
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Government and implemented by the ANR with the reference ANR-10-IDEX-0001-
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02 PSL (G.G.). The authors thank Dr. Tanmaya Joshi for useful discussions.
Appendix A. Supplementary material
NMR spectra of complexes Ir1-Ir5 in d6-DMSO and d7-DMF at different time
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intervals.
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ACCEPTED MANUSCRIPT Highlights: 1) The stability in DMF and DMSO of Ir(III) complexes is presented. 2) The cytotoxicity of Ir(III) complexes is described.
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3) The role of the solvents used in biological studies is discussed.