Organometallic Gold(III) Complexes Similar to Tetrahydroisoquinoline

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Organometallic Gold(III) Complexes Similar to Tetrahydroisoquinoline (THIQ) Induce ER-stress-mediated Apoptosis and Pro-death Autophagy in A549 Cancer Cells Ke-Bin Huang, Feng-Yang Wang, Xiao-Ming Tang, Hai-Wen Feng, Zhen-Feng Chen, Yan-Cheng Liu, You-Nian Liu, and Hong Liang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01694 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

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Journal of Medicinal Chemistry

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Organometallic Gold(III) Complexes Similar to Tetrahydroisoquinoline (THIQ) Induce ER-stress-mediated Apoptosis and Pro-death Autophagy in A549 Cancer Cells

Ke-Bin Huang+ a, Feng-Yang Wang+ b, Xiao-Ming Tanga, Hai-Wen Fenga, Zhen-Feng Chen*a, Yan-Cheng Liua, You-Nian Liub and Hong Liang*a,b a

State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry &

Pharmacy, Guangxi Normal University Guilin, Guangxi, 541004, P. R. China. b

College of Chemistry and Chemical Engineering, Central South University Changsha, Hunan 410083, P. R. China.

+

These authors contributed equally to this work.

TITLE RUNNING HEAD: Organometallic Gold(III) Complexes Similar to Tetrahydroisoquinoline as Chemotherapy Agent with Multiple Modes of Tumor Cell Death Keywords: Organometallic Gold(III) Complexes; Mitochondria-targeted; ER-stress; Apoptosis; Pro-death Autophagy

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Abstract: Agents induced both apoptosis and autophagic death can be effective chemotherapeutic drugs. In our present work, we synthesized two organometallic gold(III) complexes harbouring C^N ligands that structurally resemble THIQ, Cyc-Au-1 (AuL1Cl2, L1 = 3,4-dimethoxyphenethylamine) and Cyc-Au-2 (AuL2Cl2, L2 = methylenedioxyphenethylamine). In screening their in vitro activity, we found both gold complexes to exhibit lower toxicity, lower resistance factors and better anticancer activity than cisplatin. The organometallic gold(III) complexes accumulate in mitochondria and induce elevated ROS and an ER stress response through mitochondrial dysfunction. These effects ultimately result in simultaneous apoptosis and autophagy. Importantly, compared to cisplatin, Cyc-Au-2 exhibits lower toxicity and better anticancer activity in a murine tumor model. To the best of our knowledge, Cyc-Au-2 is the first organometallic Au(III) compound that induces apoptosis and autophagic death. Based on our results, we believe Cyc-Au-2 to be a promising anticancer agent or lead compound for further anticancer drug development.

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1. Introduction Cisplatin and its analogues, carboplatin and oxaliplatin, have been successfully used chemotherapeutically against various solid tumours.1,2 However, the utilization of platinum anticancer drugs in the clinic has been hampered by drug resistance and serious side effects. 3,4 Therefore, extensive studies aimed at identifying new metal-based complexes with reduced side effects and enhanced activity have been conducted.5 Some non-platinum complexes exhibit properties suitable for drug development, such as alterable oxidation states, variable coordination geometry, improved solubility and flexible substitution kinetic pathways.6-8 In this regard, non-platinum metal medicines, including NAMI-A, KP1019 and auranofin, have been reported to display potent antitumor activities, with some of these compounds now in clinical trials or even clinical use.9,10 In particular, there are reports indicating that Au coordination compounds have the potential to conquer platinum drug resistance through their distinctly different anticancer mechanisms.11-13 However, the low physiological stability of both gold(I) and gold(III) compounds hampers their general therapeutic utility in vivo.14,15 Even if some phosphine-Au(I) compounds have been confirmed to be stable in vivo, in many cases the stability of Au(I) and Au(III) compounds is uncertain.16 “X,L-pincer (C^N) ligands” dramatically increase the stability of the corresponding cyclometalated complex. For example, by taking advantage of the strong σ-donor strength of C-Au(I/III), as well as the ease of ligand modification of this moiety, 17-21 organometallic gold complexes with pincer (C^N) ligand have been shown to exhibit both enhanced stabilization of the Au cation in physiological environments and promising anticancer potential.22-27 Furthermore, organometallic gold complexes have also been reported to trigger antiproliferative effects via direct interaction of the Au atom with sulphur or selenium donor atoms of enzymes such as thioredoxin reductase (TrxR), cathepsins, and poly(adenosine diphosphate (ADP)-ribose) polymerase1 (PARP-1), as well as aquaporins, indicating that several mechanisms may contribute to the overall pharmacological activity of organometallic Au complexes.12,28,29 Over the years, organometallic Au coordination compounds stabilized with (C^N), (C^N^C) or (C^N^N) pincer ACS Paragon Plus Environment

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ligands have attracted the attention of many chemists.30,31 1,2,3,4-Tetrahydroisoquinoline (THIQ), a privileged heterocyclic scaffold, is commonly found in naturally occurring alkaloids and possesses a diverse range of therapeutic characteristics, including antitumor, antibacterial, antiviral, anticoagulant, anti-inflammatory, anti-alzheimer and anticonvulsant activities.32 In the last decades, several fused THIQ alkaloids have been discovered as potent anticancer antibiotics. For example, trabectedin (ecteinascidin 743, Et-743) was approved by the European Commission in 2007 and the USFDA in 201533 for the treatment of soft tissue sarcomas. Since THIQ has been shown to elicit certain biological effects, a series of metal-based complexes which are directly coordinated by the N atom of THIQ has been developed and assessed for anticancer activity.34-36 As far as we know, organometallic compounds with a skeletal structure similar to THIQ, in which the metal atom is inserted into the N-heterocyclic scaffolds of THIQ, have been reported,37 but not yet considered as bioactive agents. Inspired by the anticancer properties of both organometallic Au complexes and THIQ, we designed two organometallic Au complexes with a similar skeletal structure to THIQ, with C^N ligands based on a simple and economical phenethylamine scaffold. Two novel complexes, Cyc-Au-1 (AuL1Cl2, L1 = 3,4-dimethoxyphenethylamine) and Cyc-Au-2 (AuL2Cl2, L2 = methylenedioxyphenethylamine), can be generated by a simple and economical method. In these complexes, the gold(III) atom substitutes a hydrogen atom from that carbon by cyclometalation, which is similar to 6,7dimethoxy-1,2,3,4-tetrahydroisoquinoline

(THIQ-1)

and

3-dioxolo[4,5-g]-5,6,7,8-tetrahydroisoquinoline

(THIQ-2), respectively. In the present study, we characterize these complexes and investigate their in vitro/vivo anti-tumour activity, including their mechanism of action. 2. Results and discussion 2.1 Synthesis and characterization General procedure for synthesis of gold(III) complexes: 30 mL anhydrous methanol and dichloromethane (1:1) were used to dissolve the ligands (0.1 mmol). KAuCl4 (0.1 mmol) was then added to the solution. The resultant mixture was ACS Paragon Plus Environment 4

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refluxed for 1 day protected from light. The yellowish solution formed was filtered. The filtrate was then crystallized by slow evaporation and the crystals of Cyc-Au-1 and Cyc-Au-2 were harvested, respectively. They were characterized by elemental analysis, ESI-MS and 1H NMR. In the 1H-NMR spectrum of Cyc-Au-2 in d6-DMSO, an aromatic proton appears at 7.0−6.5 ppm and two alkyl group protons appear at ~3.70 ppm. Two −OCH3 protons of Cyc-Au-1 appear at ~3.74 ppm, while the -OCH2O- protons of Cyc-Au-2 appear at 5.96 ppm. Almost all peaks of the 1H NMR spectra for the two Au complexes have been split; the chemical splitting of the protons is strong evidence that the ligands remain coordinated to the Au(III) ions.

[Scheme 1]

2.2 Crystal structure We determined the crystal structures of gold complexes Cyc-Au-1 and Cyc-Au-2 by single crystal x-ray diffraction. The crystallographic data and refinement details are listed in Table S1. The molecular structures of Cyc-Au-1 and Cyc-Au-2 are depicted in Figure 1. The Au(III) centers are coordinated by two chlorine groups and a C, N-bidentate chelating (η2) ligand. One six-membered chelating ring similar to the N-heterocyclic scaffolds of THIQ is formed. The geometry of the Au atom is a distorted square plane. The bite angle C−Au−N deviates from 90°. The carbon atom can be viewed as strongly coordinated to the metal, e.g., in Cyc-Au-2, Au1−C9 = 2.060 (3) Å, while the chloride ion forms a weaker bond to Au (III) (Au1−Cl1 = 2.2966 (8) Å, Au1−Cl2 = 2.2824 (9) Å), which are stronger than general Au−Cl (2.3707(9) distances reported in the literature.38 [Figure 1] 2.3 Stability of Cyc-Au-1 and Cyc-Au-2 in solution The stabilities of gold complexes were confirmed by HPLC and UV-vis absorption spectroscopy in TBS solution (Tris-KCl-HCl buffer, pH 7.35). The time-dependent (0, 24 and 48 h) HPLC chromatograms demonstrate that Plus Environment the solutions exhibit no apparent changes ACS overParagon the time course, demonstrating that the two gold compounds are 5


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stable in TBS for 48 h at room temperature (Figure S1). To further confirm the HPLC results, we also acquired time-dependent UV-vis absorption spectra of the two gold complexes (Figure S2). As expected, no apparent redor blueshift appears after incubating the complexes for up to 48 h under physiological conditions. Therefore the Au(III) complexes are stabile in solution. 2.4 Determination of Log P o/w Based on Lipinski’s rule, hydrophilicity and lipophilicity are crucial to drug development.39 A high hydrophilicity interferes with a drug’s ability to penetrate biological membranes. On the contrary, a high lipophilicity can hinder biomedical applications.40 The lipophilicity (log P o/w values) of the gold complexes was investigated using the shake-flask technique.41 The log P o/w values of Cyc-Au-1 and Cyc-Au-2 are 4.21 and 2.42, respectively (Fig. S3). These values indicate that both Cyc-Au-1 and Cyc-Au-2 are sufficiently lipophilic to satisfy the general lipophilicity requirement of drugs. Cyc-Au-1, with two methoxyl groups, possesses a higher log P o/w value, which translates into a higher membrane permeability than Cyc-Au-2, as corroborated by the results of cellular uptake experiments. 2.5 Interaction of DNA and TrxR Even though additional biological targets, including enzymes, RNA and other proteins, are present in tumor cells, DNA is generally believed to be a primary molecular target of metallodrugs.42 There are several covalent and/or non-covalent binding modes by which transition metal complexes interact with DNA, including intercalative, groove binding and electrostatic. Conformational changes in DNA can be detected with considerable sensitivity by examining melting temperature (Tm). An interaction (typically covalent) that destabilizes the double helix causes a decrease in the Tm, while intercalation or other stabilizing interactions lead to an increase in the Tm.43 To assess whether the compounds interact directly with DNA, thermal denaturation experiments of DNA treated with Cyc-Au-1, Cyc-Au-2 or THIQ derivatives were carried out. The melting curves of the DNA−compound systems are shown in Fig S4. All Tm of the DNA in ACS Paragon Plus Environment 6

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the presence of any of the four compounds show an increase, suggesting that all the tested compounds may intercalate into DNA. The interaction of the two gold complexes with DNA were weaker than the corresponding THIQ derivatives (Cyc-Au-1 to THIQ-1 and Cyc-Au-2 to THIQ-2), while Cyc-Au-2 and THIQ-2 have a more potent effect over their respective sister compound (Cyc-Au-2 to Cyc-Au-1 and THIQ-2 to THIQ-1). This is not surprising since the five-membered methoxy ring is a classical group present in DNA intercalating molecules. Based on the above results, it appears that the first site of the N-heterocyclic scaffold of THIQ and the five-membered methoxy ring affect the interaction of the compounds with DNA. Furthermore, the difference in DNA intercalation tendency may be one of the factors affecting the cytotoxicity of the compounds. As discussed in the introduction, TrxR has generally been considered one of the main targets of gold compounds. Therefore, we studied the TrxR inhibitory potential of Cyc-Au-1 and Cyc-Au-2 on isolated enzyme using an established procedure (DTNB reduction assay). The corresponding THIQ derivatives, THIQ-1 and THIQ-2, were used as the respective controls. The two gold complexes displayed marked TrxR inhibitory activity, the IC50 values of which were 0.253 and 0.107 µM, respectively. In contrast, the two THIQ derivatives were virtually inactive (IC50 > 30 µM). Although the complexes show structural analogy to THIQ, their inhibitory activity against TrxR may mainly comes from their gold centers. However, the TrxR inhibitory potential of Cyc-Au-1 and Cyc-Au-2 are much lower than that of auranofin (IC50 = 4 nM),44 the obvious difference of TrxR inhibitory potential between the new synthetic gold complexes and auranofin maybe also related to their different valence state of gold metal center, different coordination number, the stability of released group besides their completely different modes of structure. Gold(I) compounds generally display a higher inhibitory potency than gold(III) compounds. Gold(I/III) agents with less coordination number and one labile ligand generally show a better inhibitory potency because which may easily allow formation of a direct gold-semenium bond. In addition, gold(III) compounds may act through a different mechanism than metal coordination to active site selenol, causing for instance oxidative damage to the enzyme.45 ACS Paragon Plus Environment 7


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2.6 In vitro cytotoxic activity and resistance of Cyc-Au-1 and Cyc-Au-2

The in vitro cytotoxicity of ligands L-1 and L-2, THIQ-1 and THIQ-2 and the two gold complexes Cyc-Au-1 and Cyc-Au-2 were examined in BEL-7404, SK-OV-3, HCT116, HepG-2 and normal HL-7702, and WI-38 cells, with auranofin and cisplatin as the positive controls. The IC50 values of Cyc-Au-1 and Cyc-Au-2 (IC50 values: 1.58~18.74 µM) are significantly lower than those of the two ligands L-1 and L-2 (IC50 values > 28.25 µM) and the two tetrahydroisoquinolines THIQ-1 and THIQ-2 (IC50 values > 17.5 µM), suggesting that organometallic complexes in which an Au replaces the first CH2 group of the N-heterocyclic scaffolds of THIQ possess enhanced cytotoxic activities (Figure 3A and Table S2). The gold(III) complex Cyc-Au-2 (IC50 values: 1.58~7.35 µM) displays a higher cytotoxicity than cisplatin (IC50 values: 8.45~21.58 µM) and Cyc-Au-1 (IC50 values: 9.26~18.74 µM), all of which are less potent than auranofin (0.52~1.24 µM). Compared to their toxicity in cancer cells, Cyc-Au-1 and Cyc-Au-2 exhibited lower in vitro cytotoxic activity in non-tumoral lung fibroblast WI-38 and human liver HL-7702 cells (IC50 values: 24.65~44.24 µM), demonstrating a selectivity of cytotoxicity. It should be noted that the two gold compounds also showed lower cytotoxicity than the two positive controls in the normal human liver HL-7702 cells (IC50 values for cisplatin: 17.86 µM, auranofin: 3.56 µM) and lung fibroblast WI-38 cells (IC50 values for cisplatin: 18.65 µM, auranofin: 9.67 µM). Furthermore, Cyc-Au-2 also exhibits a similar selectivity of cytotoxicity compared to reported cyclometalated gold(III) complexes which possess higher druggablility.46,47 Based on their structures and results of interaction of Au compounds with DNA and TrxR, it can be proposed that the cytotoxicity of Cyc-Au-1 and Cyc-Au-2 originates from interactions with DNA and/or proteins via the planar ligands and gold metal centre .28,48

Acquired resistance is known to be a major obstacle to drug therapy.49 Based on the success of the initial cell-based experiments described above, the cytotoxicity of Cyc-Au-1 and Cyc-Au-2 was further evaluated in cis-platin-sensitive (A549) and cis-platin-resistant (A549/CDDP) tumor cells using the MTT method. Cisplatin and auranofin were used as ACS Paragon Plus Environment positive controls. Cyc-Au-2 exhibited a more potent cytotoxic effect in both A549 and A549/CDDP cancer cells than 8

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Cyc-Au-1 or cisplatin (Figure 2B). Importantly, as in the case of auranofin (RF, 1.16), Cyc-Au-1 and Cyc-Au-2 both have considerably lower resistance factors (RF, 1.42 and 1.15, respectively), compared to cisplatin (7.12). Such a RF difference between the gold complexes and cisplatin may be a function of their spatial configurations, resulting in different targets and mechanisms of action in cells. Since Cyc-Au-2 displays a higher cytotoxicity in A549 cells, it was used in subsequent experiments examining the mechanism of cytotoxicity in the A549 cancer cell line.

[Figure 2] 2.7 Cellular uptake and membrane transport pathway The ability of metal-based anticancer drugs to be taken up by cells is an important factor that influences the compounds’ anti-proliferative performance.50 To investigate the cellular uptake properties of Cyc-Au-1 and Cyc-Au-2, we used ICP-MS to investigate the levels of the two Au compounds in A549 cells. After the cells were treated with a gold complex (10 µM) for 24 h, the amount of gold in whole-cell lysates followed the lipophilicity results: n (Cyc-Au-1) > n (Cyc-Au-2) (Figure 3A). However, considering the results of the cytotoxicity experiments, the anti-proliferative ability of Cyc-Au-1 and Cyc-Au-2 does not rely solely on the cellular uptake. Similar results using gold complexes were also reported by Antonello Merlino and coworkers.51,52 Because the nucleus and mitochondria are thought to be two main targets of anticancer drugs, we also investigated the distribution of the Au complexes in the subcellular compartments. The accumulation of the two Au complexes in mitochondria was considerably higher than in nuclei. Meanwhile, the accumulation of Cyc-Au-2 in mitochondria was higher than that of Cyc-Au-1, which may, at least in part, be responsible for the elevated cytotoxicity of Cyc-Au-2. Based on these results, the mitochondria are considered the main target of the two Au compounds in A549 cancer cells, a finding similar to the mechanism of action of cyclometalated gold(III) compounds reported by Contel’s group.53 The routes that small molecules can take when entering cells include energy-independent (passive diffusion and facilitated diffusion) or energy-dependent (active transport and endocytosis) pathways. We assessed the cellular uptake ACS Paragon Plus Environment 9


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pathways by incubating cells with the complexes at low temperature, or incubating cell lines with an endocytosis inhibitor CQ (chloroquine) or with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), a potent mitochondrial oxidative phosphorylation uncoupler.54 A549 cells incubated at 4 °C or pre-treated with CCCP (10 µM) exhibited a blockage of cellular uptake of Cyc-Au-1 and Cyc-Au-2, while pre-incubating A549 cells with CQ (50 µM) did not elicit an effect (Figure 3B). These results collectively indicate that Cyc-Au-1 and Cyc-Au-2 complexes are taken up by non-endocytic, energy-dependent active transport. [Figure 3] 2.8 Induction of mitochondrial dysfunction. Mitochondria are the central processing site for death signals originating from intrinsic and extrinsic apoptosis. The triggering of various death-pathways begins with the dysfunction of mitochondria and the release of death factors. Some cyclometalated gold(III) complexes have been shown to elicit measurable mitochondrial dysfunction and lead to tumor cells death.53,55 Our ICP-MS results also suggest that the mitochondria may be the main target of Cyc-Au-2. To assess the contribution of mitochondria in Cyc-Au-2-induced cancer cell death, we examined mitochondrial membrane potential,

which

is

measurably

affected

under

conditions

of

mitochondrial

dysfunction,

using

5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (a.k.a., JC-1). In those cells with unaffected mitochondrial function, JC-1 remains in the monomeric (green) form in the cytosol but the aggregated form (red) is also present in mitochondria, as the result of the mitochondrial membrane depolarization. In contrast, when the mitochondria are depolarized in cells with mitochondrial dysfunction, JC-1 remains in the monomeric form.43 When we treated A549 cells with Cyc-Au-2, we found a time-dependent enhancement in the green (monomeric) fluorescence versus the control (Figure 4A), further suggesting that mitochondrial dysfunction may be central to the process of death induced by Cyc-Au-2. [Figure 4]


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Mitochondria, the power plants of the cell, are vital to ATP production.56 A hallmark feature of cancer cells is the alteration of energy production pathways. This unique characteristic flags mitochondrial metabolism as a potential therapeutic target in malignancy.57 The cell Titer-Glo® luminescent cell viability test was used to observe the impact of Cyc-Au-2 on ATP production. As shown in Figure 4B, a dose-dependent decrease in ATP concentration was observed in cells incubated with Cyc-Au-2, after treatment with 12 µM Cyc-Au-2 for 12 h, the ATP level decreases to 24.1 ± 2.1% the level of untreated cells. These results indicate that Cyc-Au-2 can effectively impair mitochondrial energy metabolism in A549 cells, further confirming that mitochondrial dysfunction is involved in Cyc-Au-2 cytotoxicity. 2.9 ROS production and ER stress Mitochondria generate a large proportion of the reactive oxygen species (ROS) produced in cells. However, these organelles are also vulnerable to ROS attack.58 Furthermore, compromised mitochondria cause an increase in ROS levels, which can, in turn, lead to cellular dysfunction.59 Importantly, the death-inducing capacity of anticancer drugs has been associate with the production of ROS. In addition, chemotherapeutic agents’ ability to affect endoplasmic reticulum (ER) stress is a key element in drug efficacy. The simultaneous presence of elevated ROS and ER stress can increase the presentation of damage-associated molecular patterns, which were ultimately crucial for the death of the cancer cells.59 Some reported cyclometalated gold(III) complexes can induce a marked release of ROS and result in ER stress.60 Since we also observed mitochondrial accumulation of Cyc-Au-2, and concomitant mitochondrial membrane depolarization and impaired energy metabolism, we investigated the impact of drug treatment on intracellular ROS levels using 2′,7-dichlorodihydrouorescein diacetate (H2DCFDA), which is oxidized to fluorescent 2′,7′-dichlorouorescein (DCF) by cellular ROS.61 We simultaneously examined the effects on the ER using ER-Tracker Red by binding to the sulfonylurea receptors on the ER. As shown in Figure 5A, there was a clear colocalization of ROS with the ER after incubation for 6 h with Cyc-Au-2. Upon extending the incubation time to 18 h, the ROS became more diffuse and less colocalized, possibly ACS Paragon Plus Environment 11


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due to incremental ER membrane permeability relevant to ER stress-induced death. We next investigated whether the enrichment of ROS in the ER indeed induces ER stress. After incubation of A549 cells with 3 µM Cyc-Au-2 for 0-36 h, the expression of phosphorylated RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) and C/EBP homologous protein (CHOP) and phosphorylated eukaryotic initiation factor 2a (eIF2a), were all increased (Figure 6), indicating ER stress. To further confirm that Cyc-Au-2-induced ROS accumulation is due to mitochondrial dysfunction and a key factor leading to cell death, we co-treated A549 cells with a mitochondria-targeted superoxide dismutase mimetic (Mito-TEMPO, Mito) and Cyc-Au-2. The Mito-TEMPO dose-dependently increased the viability of Cyc-Au-2-treated cells from about 50% to approximately 90% of untreated controls (Figure 5B). Together these results indicate that mitochondrial dysfunction mediated by Cyc-Au-2 may result in the release of ROS and cause an ER stress response, ultimately leading to the death of A549 cells. [Figure 5] [Figure 6] 2.10 Cyc-Au-2-induced apoptotic pathways Various death-pathways are triggered when mitochondrial dysfunction and ER stress reach critical levels. In particular, apoptosis is one of the key mechanism resulting from mitochondrial dysfunction and ER stress. Thus, we investigated whether apoptosis was induced by Cyc-Au-2 in the A549 cells. The process of apoptosis is generally accompanied by distinct morphological changes, including membrane dysfunction and loss of microvilli, cytoplasmic and nuclear condensation and DNA fragmentation. In apoptosis, the exposure of phosphatidylserine (PS) usually occurs prior to the loss of plasma membrane integrity. However, healthy cells are un-reactive to annexin V-FITC and propidium iodide (PI), while those cells undergoing early-apoptosis are reactive to annexin-V FITC but remain un-reactive to PI and cells in the later stages of apoptosis or necrosis react with both annexin V-FITC and PI.62 As shown in Figure 7A, after treatment with 2.5 or 5.0 µM Cyc-Au-2 for 24 h, percentage of annexin V-positive and PI-negative cells (Q4) was enhanced from ACS Paragon Plus Environment 12

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28.6% to 56.9%. This finding was further confirmed by a membrane/DNA-specific (DAPI/DiD) co-staining assay, in which the nucleus is stained with a blue fluorescent dye while the cell membrane is stained with a red fluorescent dye. In the presence of 3.0 µM Cyc-Au-2 (24 h), cell membrane damage, nuclear condensation, DNA breakage and apoptotic bodies were markedly observed (Figure 7B). Basing on these results, we can conclude that apoptosis was the mode of A549 cell death caused by Cyc-Au-2.

[Figure 7]

Generally, there are two major pathways of apoptosis, namely the intrinsic and extrinsic pathways. The extrinsic pathway is death receptor-dependent. The two pathways involve a sequential activation of caspases, which ultimately hydrolyze cellular substrates, leading to biochemical and morphological alterations.63 In order to investigate the apoptotic pathway induced by Cyc-Au-2, the proteolytic activities of executioner caspase-3, Fas/TNF-mediated initiator caspase-8 and mitochondrial-mediated caspase-9, were evaluated.63 As indicated in Figure 8, a time-dependent enhancement in caspase-9/3 proteolytic activity was observed upon treatment with 2.5 µΜ Cyc-Au-2. However, there was no obvious change in the level of caspase-8 activity. This finding indicates that the intrinsic pathway likely plays a major role in A549 cell apoptosis induced by Cyc-Au-2. The intrinsic pathway is a cascade of processes in response to various stimuli. This apoptotic pathway begins with an accumulation of p53, succeeded by the regulation of Bcl-2 family proteins. To further confirm the above finding of intrinsic pathway induction, we examined the expression levels of several important proteins associated with the intrinsic pathway by means of Western blot. Cyc-Au-2 enhanced the expression levels of the pro-apoptosis proteins, Bad, Bax and p53, but suppressed the levels of pro-survival proteins, Bcl-xl and Bcl-2 (Figure 8). Such observations are consistent with the apoptosis inducted by chemotherapeutics of intrinsic pathways.63 [Figure 8] ACS Paragon Plus Environment 13


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2.11 Induction of pro-death autophagy The cell death pathways generally include apoptosis (Type I), autophagy (Type II), and necrosis (Type III).64 In response to stress signals such as hypoxia, increased reactive oxygen species and mitochondrial dysfunction, autophagy usually ensues.65 Since we observed increased ROS and mitochondrial dysfunction in response to Cyc-Au-2-treatment of A549 cancer cells, we explored whether the cytotoxic effect of Cyc-Au-2 was mediated via autophagy. The LC3-II, a general biomarker of autophagosomal and a cleaved conjugate of LC3-phosphatidyl-ethanolamine, was firstly determined through Western blot.66 LC3-II-conversion in A549 cells markedly increased in a time dependent manner (Figure 9). More importantly, the LC3 blots also show that Cyc-Au-2 treatment resulted in a general increase in total LC3 protein expression (the levels of LC3-I was enhanced with longer interaction times), which is in accordance with previous findings of autophagy-induced by chemotherapeutics. In autophagy, the adaptor protein SQSTM1 (sequestosome 1, p62) connects LC3 to ubiquitin in misfolded proteins. Thus, the autophagic process leads to a clearance of ubiquitylated proteins together with SQSTM1.67 We observed that SQSTM1 levels in A549 cells treated with Cyc-Au-2 was down-regulated (Figure 9), which further confirms that autophagy is induced by Cyc-Au-2. We also examined autophagy using green fluorescent protein (GFP)-fused LC3 (a specific label for autophagosome formation). As indicated in Figure 10A, the generation of GFP-LC3-labeled vacuoles in A549 cell lines was enhanced after incubation with 3.0 µM Cyc-Au-2 for 24 h. Our findings confirm that autophagy is associated with cell death induced by Cyc-Au-2. [Figure 9]

Due to its complexity, autophagy can have a pro-death or pro-survival role in the treatment of cancer.68 Herein, we used 3-methyladenine (3-MA) (a specific class III PI3K inhibitor to block autophagosome formation) to detect the relationship between autophagy and the anti-proliferative activity induced by Cyc-Au-2.69 The anti-proliferative effects in A549 cells were weakened by pre-incubation with 3-MA (1 and 2 mM) for 1 h (Figure 10B). We found, through MTT assay, that pre-incubation using the autophagic in A549 cells markedly blocked Cyc-Au-2-induced cell ACSinhibitor Paragon3-MA Plus Environment 14

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death in a dose dependent manner; in the presence of 2 mM 3-MA, cell death cut down by approximately 30%, compared to cells treated with only Cyc-Au-2 (8 µM, 48 h), indicating that Cyc-Au-2 can induce pro-death autophagy. [Figure 10] 2.12 The relationship between apoptosis and autophagy The apoptosis and autophagy signaling pathways are interconnected in a complex network of feedback loops and crosstalk. In order to examine the possible relationship between autophagic cell death and apoptosis induced by Cyc-Au-2 in A549 cell lines, we investigated the activity of Cyc-Au-2-induced caspase-3 in the presence of 3-MA. Interestingly, using caspase-3 cleavage as a proxy for apoptosis (Fig. 11A), the level of apoptosis induced by a 24 h treatment of A549 cells did not significantly change after using 3-MA to inhibit autophagy. We also assessed the level of autophagy induced by Cyc-Au-2 in the presence of the pan-caspase inhibitor, Z-VAD-FMK. The amount of LC3B-II in A549 cells was significantly increased after pharmacological inhibition of Cyc-Au-2-induced apoptosis by Z-VAD-FMK. Based on above results, we conclude that the apoptosis induced by Cyc-Au-2 does not rely on autophagy induced by Cyc-Au-2. However, Cyc-Au-2-induced autophagy can compensate for the inhibition of apoptosis by Z-VAD-FMK. [Figure 11] 2.13 Acute toxicity and in vivo anticancer activity After establishing the mechanism by which Cyc-Au-2 kills cancer cells in vitro, Cyc-Au-2's acute toxicity and in vivo anticancer activity were further studied in a mouse model. Acute toxicity was examined in KM mice with five mice in each group. Cyc-Au-2 was intraperitoneally administered at the doses provided in Table S3. In the mice, 20 µmol/kg cisplatin killed one out of the five, while all mice were killed by doses higher than 30 µmol/kg. In contrast, all mice administered Cyc-Au-2 survived at the 20 µmol/kg dose, and only one mouse was dead at 30 µmol/kg, demonstrating that the acute toxicity of Cyc-Au-2 is significantly lower than cisplatin. Considering the change of behavior and body



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weight of the mice, we preliminarily identified 20 µmol/kg as maximum tolerated dose and 30 µmol/kg as the lethal dose for Cyc-Au-2.

Finally, we investigated the therapeutic potential of Cyc-Au-2 in an in vivo model of metastatic cancer. Cisplatin or Cyc-Au-2 was intraperitoneally administered to A549 xenograft tumor bearing mice after the tumor reached approximately 1.2 cm in diameter (day 0). Cisplatin (3 mg/kg) or Cyc-Au-2 (10 mg/kg) were intraperitoneally administered on days 0, 3, 6, 9 and 12. These doses and the adminstration frequency were adopted based on the acute toxicity of the drugs (Table S2) and the state of the mice. Under above conditions, although the cisplatin and Cyc-Au-2 both elicited a statistically significant inhibition of growth with respect to the vehicle, the antitumor effect of Cyc-Au-2 in vivo was greater than that of cisplatin (Figure 12a). The body weights of animals administered the metallocomplexes proceeded to increase (Figure 12b), but an obvious body weight difference was found between the Cyc-Au-2 group and the cisplatin group with the body weight of mice in the cisplatin-treated group lower than that in the Cyc-Au-2-treated group. These results further show the limited side effects of treatment with Cyc-Au-2. At the end of the experiment, we collected and weighed the tumors, and calculated the inhibition rate of tumor growth (IRT). Cyc-Au-2 showed an IRT as high as 62.5%, which is significantly higher than the 44.3% achieved by cisplatin (Figure 12c). This difference can also be seen in the tumor images shown in Figure 12d. Although the xenograft models are different than those used by Che and co-workers,70 when compared with the effects of their binucear organometallic gold(III) complex Au360 and gold(III) porphyrin complex gold-1a,71,72 Cyc-Au-2 displays a simillar inhibition of tumour growth but better in vivo safety. Taken together, these results show that Cyc-Au-2 has considerably improved in vivo therapeutic effects, and lower side effects, compared to cisplatin. [Figure 13]


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3. Conclusion In this work, we describe two novel organometallic gold(III) complexes similar to the structure of tetrahydroisoquinolines, with C^N ligands based on the simple and economical phenethylamine scaffold. We have evaluated the stability, lipophilicity, ability to be taken up by cells and the anticancer activity of these gold complexes in detail. Cyc-Au-2 exhibits a greater anti-proliferative activity against the tested cancer cells, including cisplatin-resistant cells. Importantly, this complex also shows low toxicity against the non-tumoral HL-7702 cell line. Cyc-Au-2 mainly affects mitochondria, where it initiates a cascade of events indicative of mitochondrial dysfunction, including ATP depletion, mitochondrial membrane depolarization, increased ROS levels and ER stress. Additional experiments with Cyc-Au-2 implicate both pro-death autophagy and the intrinsic apoptotic pathway in the complex’ cytotoxicity. Furthermore, the level of autophagy inversely correlated with the amount of apoptosis. In a mouse model of cancer, Cyc-Au-2 displayed lower toxicity and better anticancer activity than cisplatin. Although the complexes show structural analogy to THIQ, how this fact is related to the in vitro and in vivo effects of the complexes requires future investigation. Our study indicates that Cyc-Au-2 is a promising candidate anticancer drug or model compound for anticancer drug development.

4. Experimental section The relative purity of all target compounds used in the biophysical and biological studies was ≥95%, which were routinely checked by HPLC (waters e2695 ). 4.1 Materials and reagents All chemicals were provided by Alfa Aesar or Sigma unless otherwise indicated. Unless specifically noted, all materials were applied as received without further purification. The TBS buffer solution (pH 7.35, 5 mM Tris, 50 mM NaCl) was generated using double distilled water. DNA loading buffer (6×) and TBE buffer (1×) were purchased from commercial sources. The test compounds were dissolved in DMSO to make 2.0 mM stock solutions for in vitro cytotoxicity ACS Paragon Environment assays. However, cisplatin was directly dissolved in 0.9%Plus NaCl saline to obtain a 2.0 mM stock solution. The final ex17


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perimental solutions of the test compounds were diluted in TBS with a maximum DMSO concentration of 0.1%.

4.2 Synthesis and characterization The general procedure for Cyc-Au-1 and Cyc-Au-2 synthesis are as follows: Equimolar amounts (0.2 mmol) of 3,4-methylenedioxyphenethylamine or 3,4-dimethoxyphenethylamine (Alfa Aesar) and KAuCl4 (0.2 mmol, Alfa Aesar) were added to a solution of methanol/dichloromethane (1:1) and the solutions were refluxed for 24 h in the dark. The resulting yellowish solution were filtered and the filtrate was volatilized slowly. After 5 days, light yellow crystals were obtained. Cyc-Au-1: Yield: 55 %. 1H NMR (400 MHz, DMSO-d6) δ 6.84 (d, J = 8.1 Hz, 1H), 6.70 (dd, J = 8.1, 1.6 Hz, 1H), 3.74 (s, 3H), 3.72 (s, 3H), 2.81 – 2.67 (t, J = 5.4 Hz, 2H), 2.57 (t, J = 7.2 Hz, 2H). Anal. Found (%): C, 26.86; H, 2.93; N, 3.13. Calcd

for C10H14NO2Cl2Au: C, 26.54; H, 2.89; N, 3.10. ESI-MS: m/z 490.4 [M – Cl + DMSO]+ Cyc-Au-2: Yield: 43 %.1H NMR (400 MHz, DMSO-d6) δ 6.81 (dd, J = 7.7, 3.6 Hz, 1H), 6.65 (dd, J = 7.9, 1.7 Hz, 1H), 5.96 (s, 2H), 2.73 (t, J = 7.2 Hz, 1H), 2.56 (t, J = 7.2 Hz, 1H). Anal. Found (%): C, 25.08; H, 2.10; N, 3.25. Calcd for C9H10NO2Cl2Au: C, 25.54; H, 2.19; N, 3.19. ESI-MS: m/z 474.9 [M – Cl + DMSO]+ 4.3 In vitro cytotoxicity Human cancer cells A549, SK-OV-3, BEL-7404, A549/DDP, HepG-2 and HCT-116 were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences. Cancer cells were cultivated in RPMI-1640 medium (RPMI-1640 medium, gibco) supplemented with 2 mM glutamine (Sigma), 10% (v/v) fetal bovine serum (FBS, gibco), 100 U/mL streptomycin (Sigma) and 100 U/mL penicillin (Sigma) at 37 °C, under a humidified atmosphere of 95% air /5% CO2. Cyc-Au-1 and Cyc-Au-2 were dissolved to stock concentrations of 2 mM with DMSO (Sigma), however cisplatin (Sigma) used as positive control was directly dissolved in 0.9% NaCl. The cytotoxicity of all complexes against the six human tumour cells and two normal cancer cell lines, HL-7702 and WI-38, was detected by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT, Sigma) assay according to reported ACS Paragon Plus Environment 18

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procedures.73 The cells were incubated with complexes Cyc-Au-1 or Cyc-Au-2 for 48 h in five different concentrations. The concentration range of each complex was selected based on the cytotoxicity. The growth inhibitory rates of the complexes were calculated as (ODcontrol − ODtest)/ODcontrol × 100%. The IC50 values of the complexes were calculated using the Bliss method (n = 5). All data were independently tested at least in triplicate. 4.4 Analysis of apoptosis by confocal microscopy and flow cytometry The apoptosis-inducing ability of Cyc-Au-2 was first examined in A549 cells using flow cytometry. A549 cell lines in the exponential growth phase were culture for 12 h in 6-well plates and then treated with Cyc-Au-2 (2.5 or 5.0 µM). After 24 h treatment, the cells were collected, washed thrice in PBS and re-suspended in 120 µL binding buffer (130 mM NaCl, 2.5 mM CaCl2, and 10 mM Hepes/NaOH, pH 7.4) at a final concentration of 1×106 cells/mL. The resuspended cells were further treated using 10 µL (5 µg/mL) annexin V-FITC (R&D, prepared in 10 mM NaCl, 0.02% NaN3 (R&D) and 50 mM Tris, 1% bovine serum albumin(Abcam), pH 7.4) and 10 µL propium iodide (PI, R&D; 20 µg/mL) for 20 min at 25 °C protected from light. A Becton-Dickinson FACSC alibur platform was used to evaluate apoptosis by flow cytometry. 74 In the confocal microscopy experiments, A549 cells were grown to 80% confluence on chamber slides. Cyc-Au-2 (3.0 µM) was added to the culture medium for 24 h at 37 °C. The A549 cells were then washed thrice using PBS buffer, stained using a solution including 100 mg/mL DiD (Sigma) and 100 mg/mL DAPI (Sigma) for 40 min and imaged under a Zeiss LSM7 microscope.75 4.5 Analysis of GFP-LC3 Lipofectamine®3000 (Thermo Fisher Scientific, USA) was used to transfect A549 cells with an expression vector encoding GFP-LC3. After 24 h, Cyc-Au-2 was added to the culture medium for an additional 24 h. After washing, confocal microscopy was used to visualize the GFP-LC3 (488 nm excitation/500-540 nm emission).



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4.6 Inhibition of autophagy A549 cells grown to confluence in 96-well plates were pre-incubated without or with 3-MA (1 or 2 mM, Sigma) for 1 h. Cyc-Au-2 was then added into the culture medium at the corresponding concentrations for 48 h. The MTT method was applied to assess cell viability. 4.7 In vivo anticancer activity The acute toxicity was examined using KM mice (male, approximately 25 g, 5 weeks old). The Peking Union Medical College of China provided all mice. The mice were raised in the experimental animal facilities at Guangxi Medical University in a humidity- and temperature-controlled environment with a 12 h dark /12 h light cycle. All mice were fed commercial chow and water ad libitum. All experiments were carried out following the Guide for the Care and Use of Laboratory Animals of Guangxi Medical University. In addition, equimolar amphiphilic supramolecular PEG5000-PLA3000 (Sigma, nPEG : nAu=1:1) was used to enhance the solubility of Cyc-Au-2 in 0.9% NaCl solution as previously described,76 since Cyc-Au-2 precipitates as soon as the DMSO solution of Cyc-Au-2 is diluted into to 20 µM/kg (0.5 ml) in 0.9% NaCl. For acute toxicity experiments, the dose of cisplatin and Cyc-Au-2 were administered as shown in table S3, with each experimental group containing five mice. Nude mice bearing A549 tumors were randomly divided into three groups (five mice per group). The vehicle was a solution containing the equivalent amount of PEG5000-PLA3000. Cisplatin or Cyc-Au-2 were administered by intraperitoneal injection after the tumors reached 1.2 cm in diameter (day 0). Cisplatin (3 mg/kg) or Cyc-Au-2 (10 mg/kg) was injected on days 0, 3, 6, 9, and 12. The volume (mm3) of tumor was calculated according to the formula: tumor volume = (longest diameter) × (shortest diameter)2 × 0.5. The mice were euthanized after a treatment of 18 days at which time the tumors were excised from the mice and weighed. The inhibition rates of tumor growth (IRT) were calculated as follows: IRT = 100% × (average weight of the tumor control group – average weight of the experimental tumor group)/average weight of the tumor control group. ACS Paragon Plus Environment 20

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4.8 Statistics The data processing included the Student’s t-test, with p ≤ 0.05 considered significant, using SPSS 13.0.

ASSOCIATION CONTENT

Supplementary Information:

The methods of experiments; the stability of HPLC and UV-vis absorption spectra for gold complexes, Lipophilicity (log Po/w) of the Au(III) complexes and the melting curves of ct-DNA, ESI-MS spectrum of Cyc-Au-1 and Cyc-Au-2 complexes, 1H-NMR spectrum of Cyc-Au-1 and Cyc-Au-2 complexes, Crystal data and details of structure refinement for Cyc-Au-1 and Cyc-Au-2, Table S2. IC50 (µM) values for complexes against six human tumor cell lines and two normal cell lines, Table S3. the results of acute toxicity for cisplatin and Cyc-Au-2 (alive/total), reference (pdf).

Molecular Formula Strings(csv).

AUTHOR INFORMATION

Corresponding Authors: Zhen-Feng Chen, PhD Telephone: (086) 773-2120998; Fax:(086) 773-21209958. E-mail: [email protected]; Hong Liang, PhD Telephone: (086) 773-2120998; Fax: (086) 773-21209958; and E-mail: [email protected]. Conflict of interest: The authors declared no competing financial interest.

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (Nos. 21401031, 21431001, 81473102, 21761005), IRT_16R15, and Natural Science Foundation of Guangxi Province (Nos. 2015GXNSFAA139043, 2016GXNSFGA380005) and the Program for Key Scientific Research of Guangxi Normal University (2014ZD005) as

well as “BAGUI Scholar” program of Guangxi Province of China. ACS Paragon Plus Environment 21


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ABBREVIATIONS USED

THIQ: Tetrahydroisoquinoline (THIQ); Cyc-Au-1: (AuL1Cl2, L1 = 3,4-dimethoxyphenethylamine); Cyc-Au-2: (AuL2Cl2, L2 = methylenedioxyphenethylamine); NAMI-A, KP1019; PARP-1:diphosphate (ADP)-ribose) polymerase1; TrxR: thioredoxin reductase; THIQ-1: 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline; THIQ-2: 3-Dioxolo[4,5-g]-5,6,7,8tetrahydroisoquinoline;

CCCP:

carbonyl

cyanide

2′,7-dichlorodihydrouorescein

diacetate;

PERK:

phosphorylated

initiation

factor

eukaryotic

mitochondria-targeted

superoxide

dismutase

3-chlorophenylhydrazone;

protein 2a;

kinase-like

CHOP:

mimetic;

C/EBP

CQ:

endoplasmic homologous

SQSTM1:sequestosome

1;

chloroquine; reticulum protein; 3-MA:

H2DCFDA:

kinase;

eIF2a:

Mito-TEMPO: 3-methyladenine;

IRT :inhibition rate of tumour growth.

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Figure Legends Fig. 1 ORTEP drawing of Cyc-Au-1/Cyc-Au-2. The hydrogen atoms have been omitted for clarity. Selected bond lengths (Å) and angles (°) for Cyc-Au-1: Au1–Cl1 2.394 (2), Au1–N1 2.037 (9), Au1–Cl2 2.273 (3), Au1–C10, 2.027 (7); Cl2–Au1–Cl1 91.12(11), N1–Au1–Cl1 87.4(3), N1–Au1–Cl2 178.2(3), C10–Au1–Cl1 175.7(2), C10–Au1–Cl2 91.2(2), C10–Au1–N1 90.3(3); for Cyc-Au-2: Au1–Cl1 2.2966 (8), Au1–N1 2.050 (3), Au1–Cl2 2.3924 (9), Au1–C9 2.060 (3); Cl2–Au1–Cl1 91.32 (3), N1–Au1–Cl1 175.18 (8), N1–Au1–Cl2 83.92 (8), C9–Au1–Cl1 91.47 (9), C10–Au1–Cl2

177.20 (9), C9–Au1–N1 93.29 (11).

Fig. 2 (A) IC50 (µM) values for gold complexes Cyc-Au-1 and -2 in six human tumor cell lines and one human liver cell line; (B) IC50 (µM) and resistance factors of Cyc-Au-2 and cisplatin in A549 and A549/CDDP cells. Resistance factor (RF) is defined as IC50 in A549 CDDP cells / IC50 in A549 cells.

Fig. 3 (A) Cellular uptake of complexes and accumulation of complexes in the nuclei and mitochondria, as determined by ICP-MS, in A549 cells after 48 h of treatment with 10 µM of complexes; (B) Uptake of complexes by A549 cells pre-incubated with chloroquine (50 µM) or CCCP (10 µM) for 1 h. **p < 0.02.


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Fig. 4 (A) Loss of ∆Ψm by Cyc-Au-2 treatment. Cells were incubated with Cyc-Au-2 for 0.5 h, treated with JC-1 and examined by fluorescence microscopy; (B) Decrease of cellular ATP levels in A549 cells incubated with Cyc-Au-2. Cells were treated with the indicated concentrations of Cyc-Au-2 for 12 h. The luminescence intensity was measured in a microplate reader. **p < 0.02.

Fig. 5 (A) Confocal micrographs of cells after incubation with the indicated formulation and treatment with DCFH (ROS indicator, green) and ER-Tracker red (Red); (B) Cyc-Au-2-induced cell death is prevented by a mitochondria-targeted superoxide scavenger.

Fig. 6 WB analysis of ER-stress markers after incubation of A549 cells with Cyc-Au-2 (at the IC50 concentration) for the indicated incubation times.

Fig. 7 (A) Propidium iodide/annexin V staining of A549 cells incubated with Cyc-Au-2 (at the IC50 and 2x the IC50 concentration) examined by flow cytometry; (B) DiD/DAPI assay of A549 cells incubated with Cyc-Au-2 (at IC50 concentration) measured by confocal microscope.

Fig. 8 WB analysis of mitochondria-mediated apoptosis markers after incubation with Cyc-Au-2 at the indicated concentrations.

Fig. 9 WB analysis of key proteins of autophagy in Cyc-Au-2-treated A549 cells. The cells were incubated with Cyc-Au-2 for the durations shown at the IC50 concentration. The data represent the mean±SEM of three different experiments. **p < 0.05, compared to the untreated group.

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Fig. 10 (A) Representative fluorescence micrographs showing GFP-LC3 in A549 cells incubated with Cyc-Au-2 (3.0 µM) for 24 h; (B) The inhibition of the anti-tumor activity of Cyc-Au-2 by 3-MA. Cells were pre-incubated with 3-MA (1 or 2 mM) for 1 h and then treated with Cyc-Au-2 at the concentrations shown for 48 h. **p < 0.02.

Fig. 11 Relationship between autophagic cell death and apoptosis induced by Cyc-Au-2 in A549 cells. (A) Effect of inhibition of autophagic cell death on Cyc-Au-2-induced caspse-3 cleavage in A549 cells; (B) Effect of apoptosis inhibition on Cyc-Au-2-induced autophagy in A549 cells.

Fig. 12 (a) Tumor growth profiles in mice (A549 xenograft mouse model). Squares, vehicle; circles, cisplatin; triangles, Cyc-Au-2. (b) Effect of drug treatment on body weight. Squares, vehicle; circles, cisplatin; triangles, Cyc-Au-2. (c) Tumor weights and inhibition of tumor growth in mice treated with the indicated drugs or vehicle control at the experimental endpoints. (d) Images of representative tumors from the experiment described in d. IRT = 100% × (mean tumor weight of the control group – mean tumor weight of the experimental group)/mean tumor weight of the control group.


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Scheme and Figures Scheme 1. The synthetic route of Cyc-Au-1 and Cyc-Au-2

Scheme 1

Figure 1

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Figure 2

Figure 3

Figure 4


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Figure 5

Figure 6

Figure 7

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Figure 8

Figure 9


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Figure 10

Figure 11

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Figure 12


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Table of Contents graphic

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44x25mm (300 x 300 DPI)


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Organometallic Gold(III) Complexes Similar to Tetrahydroisoquinoline

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