Thiourea and Guanidine Compounds and Their Iridium Complexes in Drug-Resistant Cancer Cell Lines: Structure-Activity Relationships and Direct Luminescent Imaging.

Kent Academic Repository Full text document (pdf) Citation for published version Thomas, Samuel and Balónová, Barbora and Cinatl, Jindrich and Wass, Mark N. and Serpell, Christopher J. and Blight, Barry A. and Michaelis, Martin (2019) Thiourea and Guanidine Compounds and their Iridium Complexes in Drug-Resistant Cancer Cell Lines: Structure-Activity Relationships and Direct Luminescent Imaging. Working paper. ChemRxiv 10.26434/chemrxiv.8856146.v1 DOI https://doi.org/10.26434/chemrxiv.8856146.v1 Link to record in KAR https://kar.kent.ac.uk/78375/ Document Version Pre-print Copyright & reuse Content in the Kent Academic Repository is made available for research purposes. Unless otherwise stated all content is protected by copyright and in the absence of an open licence (eg Creative Commons), permissions for further reuse of content should be sought from the publisher, author or other copyright holder. Versions of research The version in the Kent Academic Repository may differ from the final published version. Users are advised to check http://kar.kent.ac.uk for the status of the paper. Users should always cite the published version of record. Enquiries For any further enquiries regarding the licence status of this document, please contact: researchsupport@kent.ac.uk If you believe this document infringes copyright then please contact the KAR admin team with the take-down information provided at http://kar.kent.ac.uk/contact.html Thiourea and Guanidine Compounds and their Iridium Complexes in Drug-Resistant Cancer Cell Lines: Structure-Activity Relationships and Direct Luminescent Imaging Samuel J. Thomas,a Barbora Balónová,b Jindrich Cinatl jr.,c Mark N. Wass,a Christopher J. Serpell,d* Barry A. Blight,b* Martin Michaelisa* School of Biosciences, Stacey Building, University of Kent, CT2 7NJ, UK University of New Brunswick, Department of Chemistry, Fredericton, New Brunswick, E3B 5A3, Canada c Institute of Medical Virology, Goethe University Frankfurt, Paul-Ehrlich-Strasse 40, 60596 Frankfurt am Main, Germany d School of Physical Sciences, Ingram Building, University of Kent, Canterbury, CT2 7NH, UK a b Abstract Thiourea and guanidine units are found in nature, medicine, and materials. Their continued exploration in applications as diverse as cancer therapy, sensors, and electronics means that their toxicity is an important consideration. We have systematically synthesised a set of thiourea compounds and their guanidine analogues, and elucidated structure-activity relationships in terms of cellular toxicity in three ovarian cancer cell lines and their cisplatin-resistant sub-lines. We have been able to use the intrinsic luminescence of iridium complexes to visualise the effect of both structure alteration and cellular resistance mechanisms. These findings provide starting points for the development of new drugs and consideration of safety issues for novel thiourea- and guanidine-based materials. Introduction Thiourea and guanidine derivatives are versatile compounds that have previously been synthesised for use in a variety of industries from materials manufacturing to medical research. The two functional groups represent modifications of urea, with the oxygen being replaced by a sulfur, or by an NH unit, respectively. Compared to urea, thioureas are more acidic and poorer hydrogen bond acceptors. In contrast, guanidines are basic and protonated under most aqueous conditions (pKa = 13.6 for unsubstituted guanidine). While urea is of low toxicity (oral LD50 in rats of 8471 mg/kg1), showing only irritant properties at high concentrations, guanidine is listed as harmful, with an LD50 of 1120 mg/kg for the hydrochloride.2 Thiourea (LD50 of 1750 mg/kg) is a carcinogen and teratogen and has harmful effects on the thyroid.3 Guanidines occur in biology in the form of the nucleobase guanine and the amino acid arginine, and are also found in urine.4,5 These compounds have also been employed in the catalytic synthesis in green chemistry,6 as potential antivirals for the treatment of poliovirus,7 and as adhesive promotors in the materials industry.8 We are currently investigating guanidine-based molecules as components in advanced organic LED materials.9 A focal point of guanidine research has been in medicinal chemistry. For example, metformin (N,N-dimethylbiguanide) is widely used in the treatment of type 2 diabetes.10 Further research has been conducted on their antimicrobial and anticancer properties.11– 13 In these cases, the compounds studied included chalcones14 and also platinum centred derivatives.15 Findings like these support advances in synthesising further derivatives of guanidine compounds to target new areas of cancer therapeutics, and further illustrate the biological importance of these simple and easily manipulatable structures. Compared to guanidines, thioureas are found more rarely, for example in 2-thiouridine in tRNAs.16 Within chemistry, interest has centred on their hydrogen bonding capability which has enabled sensing of anions,17 self-assembly,18 and organocatalysis,19 for example. The thiourea functional group has been used widely in medicine, with 2-thiouracil itself used to treat thyroid disorders for some time.20 Thioureas are investigated as antiviral agents,21 including non-nucleoside HIV-1 reverse transcriptase inhibitors,22 insecticidal growth regulators,23 anti-inflammatory,23 and anticancer drugs.21 Thioureas show promise as anticancer drug candidates due to their sulfur atoms,24 with sulfur itself being a versatile and biologically important element to all living organisms.25 Thiourea derivatives have been synthesised with an assortment of partnering organic structures and demonstrated to exert anti-cancer effects in human cancer cell lines from different entities including breast and lung cancer as well as leukaemia26–28 29 Given the broad application of guanidines and thioureas in upcoming drugs and materials, their toxicity is of interest both from the standpoint of potentially beneficial or detrimental effects. In this light, we decided to perform a systematic screen of a series of thiourea and guanidine compounds for toxicity in ovarian cancer cell lines, including those which display resistance to cisplatin, the mainstay of ovarian cancer therapies and one of the most commonly used anticancer drugs.30,31 These studies identify trends which could be used as first pointers for initial toxicity predictions of similar compounds. We have also been able to make use of the intrinsic luminescence of iridium complexes to observe extent and distribution of uptake, and its relationship to guanidine or thiourea function. Results and Discussion N N H S NH2 N N MeCN 59% N N N N H S N H N N RNH2 R = n-Pr, 44% 1N R = n-Bu, 65% 2N R = n-C 3N R = Bn, 620% H13, 45% 4N NH N N H N H (Ir(ppy)2Cl)2 K2CO3 Toluene N H R N H N H Ir-2N N H N H R N R = n-Pr, 63% 1S R = n-Bu, 56% 2S R = n-C 3S R = Bn, 671% H13, 81% 4S R = n-Bu 44% N R N H 2 Ir NH N S N 2 Ir N N (Ir(ppy)2Cl)2 K2CO3 Toluene R = n-Bu 53% N HgO, NH3/MeOH CHCl3 S N H N H R Ir-2S Scheme 1. Synthesis of guanidine and thiourea compounds and their iridium complexes. A series of guanidine and thiourea compounds was synthesised based upon a 2-aminobenzimidazole unit; this was selected since it is a ‘drug-like’ unit which also has capacity to form the type of metal complexes which could be used in OLED materials. Monosubstitution of 1,1’-thiocarbonyldiimidazole was achieved by reaction with substoichiometric 2-aminobenzimidazole in acetonitrile. The second imidzoyl unit was displaced by a selection of amines (n-propylamine, n-butylamine,32 n-hexylamine, and benzylamine) in the presence of dimethylaminopyridine (DMAP) in dimethylformamide (DMF) to give compounds 1S to 4S in 63 - 81 % yield after purification.22 Portions of these compounds were converted to the corresponding guanidines by reaction with mercury(II) oxide and methanolic ammonia in chloroform to give 1N to 4N33,34 in 40 – 65% yield.35 Two iridium complexes9,36 based on these systems were prepared by reacting 2S and 2N separately with [Ir(ppy)2Cl]2 in toluene in the presence of potassium carbonate, giving bright yellow powders Ir-2S and Ir-2N in 53 % and 44 % yield respectively. All compounds were characterised by 1H and 13C NMR, EI-MS, and elemental analysis. To examine the biological activity of the compounds synthesised, they were tested for effects on the viability of the human ovarian cancer cell lines EFO-21, EFO-27 and COLO-704 and their cisplatinresistant sublines EFO-21rCDDP2000, EFO-27rCDDP2000 (both adapted to cisplatin 2µg/mL), and COLO704rCDDP1000 (adapted to cisplatin 1µg/mL) using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphennyltetrazolium bromide) (MTT) assay modified after Mosmann37 as previously described.38 Cisplatin is one of the most commonly used anticancer drugs, and resistance formation to cisplatin represents a major obstacle to the development of improved anticancer therapies.30,39,40. The assay principle is based on metabolization of the yellow MTT reagent into a purple insoluble formazan compound within the mitochondria of viable cells.37 This colour change from yellow to purple enables for the collection of rapid and coherent cell proliferation data.41 Concentrations that reduce cell viability by 50% relative to an untreated control (IC50) were calculated by treating the cell lines with sequential dilutions of the compounds. 40 35 IC50 (μM) 30 25 20 15 10 5 0 EFO-21 EFO-27 EFO-21rCDDP2000 1S 2S 3S 4S COLOEFO-27rCDDP2000 1N 2N 3N 4N 704 COLO704rCDDP1000 Figure 1. IC50 values for compounds 1S-4S and 1N-4N against six cancer cell lines. Error bars represent one standard deviation, over three repeats. All of the unmetallated compounds examined showed toxicity in all cell lines, with IC50 values in the low micromolar range (Fig. 1). While thioureas 1S, 2S, and 4S (IC50s across all cell lines averaging 1.29, 1.26, and 2.96 μM, respectively) were notably more toxic than their guanidine counterparts 1N, 2N, and 4N (18.7, 10.1, 6.8 μM respectively), the hexyl-appended 3S (15.8 μM) was less effective at inhibiting cell proliferation than 3N (9.0 μM). We propose that these differences relate to the impact that the longer chain makes on the hydrophobicity balance of the molecule. The guanidine compounds are expected to be protonated under these conditions, and addition of a long chain will produce a cationic surfactant-type molecule which could disrupt biological membranes.42 With other side chains, the charged guanidiniums may not be sufficiently lipophilic to enter the biological membranes when compared to the neutral thioureas. This is supported by ClogP value calculation (Table S1, supplementary information) – only 3N (ClogP = 2.75) is as lipophilic as any of the S series (lowest ClogP is for 1S, at 2.58). In the case of 3S, the lipophilicity may be so high that it prevents the compound leaving the membrane and entering the cells. Despite the variation in activity of the individual compounds, the average sensitivity of the cell lines was remarkably consistent, with the parental EFO21, EFO-27, and COLO-704 lines have average IC50s of 8.0, 8.7, and 8.1 μM, and their cisplatin-resistant sublines having average IC50s of 14.5, 14.1, and 12.1 μM, respectively. Hence overall, the cisplatin resistant sublines were also more resistant to our compounds, however this effect was larger with 1N4N than with 1S-4S (average ratio of resistant/parental IC50s = 1.81 versus 1.23). 18 16 IC50 (μM) 14 12 10 8 6 4 2 0 EFO-21 EFO21rCDDP2000 EFO-27 Ir-2S EFO27rCDDP2000 COLO704 COLO704rCDDP1000 Ir-2N Figure 2. IC50 values for compounds Ir-2S and Ir-2N against six cancer cell lines. Error bars represent one standard deviation over three repeats. The same studies were then conducted with the iridium complexes Ir-2S and Ir-2N (Fig. 2). Here, the guanidine compound (average IC50 = 1.4 μM) was approximately ten times more toxic across the board than Ir-2S (average IC50 = 13.5 μM). This is a reverse of what was seen for the unmetallated compounds 2S and 2N. For both EFO-21 and EFO-27 lines, the cisplatin-resistant sublines were slightly more tolerant of all compounds (IC50s on average 25% higher), but when COLO-704rCDDP1000 cells were treated with Ir-2S the IC50 was reduced to 45% of the value of the parental cell line, suggesting that a different mechanism of resistance is in place here. In this case, both complexes are uncharged at pH 7, and since the chemical difference is on the interior of the molecule, they could be expected to have similar cell penetration properties, with the difference in toxicity being due to processing within the cell. Figure 3. Confocal images of cells treated with Ir-2S and Ir-2N. Emission from metal complexes are green, and nuclei are shown in blue (stained with DAPI). All cells were treated with compounds at their IC50 concentrations. The brightness, gain and excitation of the confocal microscopes laser for compound expression was kept consistent throughout the assays, although slight adjustments were made to the brightness of the DAPI stain to achieve a clear nuclei mapping image. Taking advantage of the intrinsic luminescence of the iridium complexes, we were able to study whether the difference in toxicity related to cell uptake or internal processing using confocal microscopy (Fig. 3). Using EFO-21, EFO-21rCDDP2000, EFO-27, and EFO-27rCDDP2000 cells, which were amenable to this technique due to good adhesion properties, cell lines were dosed with Ir-2S and Ir2N at their IC50 concentrations. Ir-2S and Ir-2N have similar excitation and emission spectra in terms of peak position and shape, but the quantum yield of Ir-2N is roughly ten times that of Ir-2S (Fig. S1, supplementary information), since the IC50s of Ir-2S are approximately ten times higher than those of Ir-2N (Table S2, supplementary information), the total brightness for full uptake of either molecule should be similar, enabling comparison of uptake efficiency. Cell nuclei were stained with DAPI. In all cases, the compounds were observed to be primarily located in the cytoplasm, with little change in cell morphology compared to untreated cells. In EFO-21 and EFO-21rCDDP2000 cells, there was clearly greater uptake of the more toxic Ir-2N compared to Ir-2S. This is impressive given the tenfold difference in concentrations. In EFO-27 cells, the Ir-2N uptake was only slightly higher than that of Ir2S, while in EFO-27rCDDP2000 cells, the total level of uptake was approximately equal. However, in the latter case, distribution of Ir-2S was notably punctate compared to a more even distribution of Ir-2N. Whilst further analysis would be required to depict what may cause these discrepancies, this indicates cell line-specific differences in cellular uptake and intracellular distribution, which may cause the differences observed in toxicity. Conclusions All compounds tested showed notable toxicity. On the one hand, this shows that use of similar thiourea and guanidine structures in materials applications should be accompanied by caution about release. On the other hand, these structures could be useful scaffolds for cancer chemotherapeutics; in particular for drug resistance – the series 1S-4S showed minimal reduction in efficacy in drug resistant cell lines, and Ir-2S was in fact more effective in one resistant subline. Notably, cisplatin is a mainstay of ovarian cancer therapy. 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