WO2018028453A1 - Palladium (ii) complexes containing n-heterocyclic carbene ligand, synthesis, and applications in cancer treatment - Google Patents

Abstract

Palladium (II) N-heterocyclic carbene (NHC) complexes which are stable in the presence of biological thiols. A representative complex, [Pd (C^N^N) (N, N'-nBu ₂NHC) ] (CF ₃SO ₃) (Pd1d, HC^N^N ＝ 6-phenyl-2, 2'-bipyridine, N, N'-nBu ₂NHC＝N, N'-di-n-butylimidazolium), displays potent killing activity toward cancer cell lines but is less cytotoxic toward normal human fibroblast cell lines.

Description

PALLADIUM(II) COMPLEXES CONTAINING N-HETEROCYCLIC CARBENE LIGAND, SYNTHESIS, AND APPLICATIONS IN CANCER TREATMENT

Technical Field

Described herein are palladium(ll) N-heterocyclic carbene (NHC) complexes which are stable in the presence of biological thiols that display potent killing activity toward cancer cell lines.

Background

There has been a growing interest in expanding the horizon of applications of palladium complexes such as in functional molecular materials and organic electronics besides catalysis. In this regard, palladium(ll) complexes have recently been examined as potential chemotherapeutic agents against cancer. The anti-proliferative properties of palladium complexes have been reported since 1980s. Most of the reported palladium(ll) complexes showed some DNA binding properties. Yet, only a few palladium(ll) complexes demonstrated good in vivo anti-cancer activity. The main challenge for the development of anti-cancer palladium(ll) complexes is their undesirably high reactivity toward sulfur-containing bio-molecules such as glutathione (GSH) and ease of substitution reactions under physiological conditions, leading to low in vivo stability and multiple speciation that complicates the potential therapeutic applications.

Summary

Metal complexes which inhibit activities of enzymes such as histone deacetylase, telomerase, topoisomerase, thioredoxin reductase and protein kinases can be effective for treatment of cancers, including those with drug-resistance. Described herein are physiologically stable anti-cancer metal complexes advantageous in minimizing multiple speciation that affects biological activities and pharmacokinetic properties. The advantages are achieved, in part, by introducing strong metal-carbon bonds and multidentate ligands serving as effective ways to suppress demetalation of the metal complexes. N-Heterocyclic carbene (NHC) is strong σ-donor ligand that can form stable metal-carbene bond with most transition metal ions. In addition, NHC itself is relatively non-toxic and versatile structures of NHC can be easily prepared.

Together with the improved stability provided by the chelating effect of tridentate pincer ligands, described herein are a series of anti-cancer cyclometalated palladium(ll) complexes containing NHC ligands, [Pd(C^AN^AN)(NHC)]⁺, which are stable in the presence of biological thiols. The palladium(ll) NHC complexes display in vitro cytotoxicities, and in vivo anti-cancer activities. Proteomics and subsequent biochemical assays revealed that the complexes induce mitochondrial dysfunction and inhibit the cancer promoting epidermal growth factor receptor pathway. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a series of chemical structures of cyclometalated palladium(ll) complexes containing N-heterocyclic carbene (NHC) ligands as anti-cancer drugs can be exemplified as follows.

Figure 2 shows cellular uptake of palladium as quantified by ICP-MS. HeLa, NCI-H1650 and normal CCD-19Lu cells treated with the palladium complexes (5 μΜ) for 3 h.

Figure 3 shows a) Keynode network analysis of proteomic data obtained from HeLa cells treated with Pdld indicated that Pdld acts on EGFR/Grb2 pathway, b-d) Inhibitory effect of Pdld on EGFR signaling. Cancer cells were treated with indicated concentrations of Pdld for 2 h. After EGFR stimulation with EGF (50 ng/mL) for 15 min, cells were harvested and lysed, and the activation/phosphorylation of EGFR (pEGFR) and its downstream protein kinase (pERK) were analyzed by Western blotting, b) HeLa cervical cancer cells, c) NCI-H460 lung cancer cells, d) HCC-827 lung cancer cells.

Figure 4 shows a) Average tumor volumes of NCI-H460 xenografts bearing mice treated with solvent or Pdld through intraperitoneal injection of 8 repeated doses for 17 days. Error bars represent standard deviations, n = 5, * denote p<0.05 compared to solvent control, b) Body weight of mice in different groups

Figure 5 shows proposed anti-cancer mechanisms of action of [Pd(C^AN^AN)(NHC)]⁺.

Figure SI shows 500 MHz COSY NMR spectrum of Pdlb in CD₃CN at 298 K: a) COSY NMR spectrum, b) ^H NOESY NMR spectrum, c) ^XH NMR spectrum.

Figure S2 shows Perspective views of the X-ray crystal structures of the cation of Pdla (left) and Pdlc (right). Hydrogen atoms are not shown.

Figure S3 shows a) Pdld (50 μΜ) in CH₃CN (solid line), CH₂CI₂ (dashed line) and CH₃OH (dotted line) at 298 K. b) UV-vis absorption spectrum of Pdla-Pdld (50 μΜ) in DMSO:PBS (l:19).

Figure S4 shows UV-vis absorption spectrum of a) Pdla, C^AN^AN ligand, [Pd(C^AN^AN)CI] and (CH₃)₂NHC ligand (50 μΜ) in CH₃CN at 298 K and b) Pdla and Pd2a (50 μΜ) in CH₃CN.

Figure S5 shows a) ^XH NMR spectra (400 MHz, 298 or 310 K) of Pdlb (2 mM) and GSH (20 mM) in D₂0:DMSO (9:1 v/v) solution mixture after mixing for 1 h and 24 h. No significant changes of the signals of Pdlb were found, b) ESI-MS-TOF spectrum of a reaction mixture of Pdla with GSH at 1:10 ratio (reaction time: 1 h and 24 h).

Figure S6 shows a) ^xH NMR spectra (400 MHz, 298 K) of Pdiso (2 mM) and GSH (20 mM) in D₂0 (150 mM ammonium bicarbonate solution):DMSO (9:1, v/v) solution mixture just after mixing, b) ³¹P NMR spectra (400 MHz, 298 K) of PdPPh₃ (4 mM) and GSH (16 mM) in D₂0 (150 mM ammonium bicarbonate solution):DMSO (9:1, v/v) solution mixture just after mixing.

Figure S7 shows a) ESI-MS-TOF spectrum of fra/is-[Pd(N HC)₂CI₂] in CH₃OH/ammonium bicarbonate buffer (50 mM) (1:9, v/v). b) ESI-MS-TOF spectrum of a reaction mixture of of tra/is-[Pd(NHC)₂CI₂] with GSH at 1:100 ratio in CH₃OH/ammonium bicarbonate buffer (50 mM) (1:9, v/v). (reaction time: 1 h).

Figure S8 shows a) Representative LC-MS chromatograms and b) mass spectra of (top) cell extract of HeLa cells treated with Pdla (10 μΜ), (middle) Pdla (10 μΜ) in cell culture medium for 48 h, and (bottom) Pdla (10 μΜ) in CH₃CN.

Figure S9 shows Unbound palladium content after incubation of different palladium complexes with human serum albumin (HSA) for 2 h based on ICP-MS analysis.

Figure S10 shows Emission spectral changes of HSA (3 μΜ, in Tris-HCI buffer (0.05 M Tris, 0.1 M NaCI, pH 7.40)) with increasing concentration of Pdla, Pdld and Pd2a. The concentration of the compounds was varied from 0.0 to 56.61 μΜ. λ_ΘΧ = 280 nm.

Figure Sll shows Caspase-3 and caspase-9 activities in HeLa cells treated with or without Pdla and Pdld (0.5 μΜ) at different time points.

Figure S12 shows The expression and cleavage of PARP in HeLa cells after treatment with Pdla or Pdld (0.5 μΜ) for 0, 12, 24, or 48 h.

Figure S13 shows UV-vis spectral change of Pdld in PBS:DMSO (9:1, v/v) with increasing concentration of ctDNA. Inset: [DNA]/e_ap vs [DNA]. Absorbance was monitored at 323 nm.

Figure S14 shows (Left) Gel electrophoresis of 123 bp DNA ladder (50 μΜ base pairs) in 1.5 % agarose gel showing the mobility of the DNA in the absence (first lane), or the presence of Pdla-Pdld (forth to seventh lane) and ethidium bromide (EB, second and third lane) in a 1:1 or 1:10 molar ratio. (Right) Gel mobility shift assay showing the mobility of the DNA in the absence (first and last lane), or the presence of PdPPh₃ and Pdiso in a 1:1 or 1:10 molar ratio (third to sixth lane) and ethidium bromide (EB, second) in a 1:1 molar ratio

Figure S15 shows Assay on DNA double strand break after incubation of HeLa cells with [Pd(C^AN^AN)(N HC)]⁺ (1 μΜ). Foci formation indicated by arrows in the image of treated sample showing that cisplatin can induce DNA DSBs. The HeLa cells were also stained with DAPI, which displayed blue fluorescence for visualization of nucleus. Figure. S16 shows Effect of [Pd(CNN)(NHC)]⁺ complexes (2.5 μΜ) or CCCP (2.5 μΜ) or vehicle control on mitochondrial potential of HeLa cells after incubation for 2 h. a) Untreated cells showing orange fluorescence owing to the strong J-aggregate of JC-1. b) CCCP treated cells showing green fluorescence due to low ΔΨιη, resulting in cytosolic accumulation of monomeric JC-1. c and d) Cells treated with Pdla and Pdld (2.5 μΜ) respectively.

Figure S17 shows The cytotoxicity profile of a) Pdla or b) Pdld in HeLa cells pre-treated with or without the thiol anti-oxidant /V-acetylcysteine (NAC).

Figure S18 shows Positive MS/MS spectrum of [C₂₇H₃iN₄Pd]⁺ ion at m/z 517.2 with a collision energy at 37 V in a) tumor b) blood with Pdld (2 mg/kg) through intraperitoneal injection of 8 repeated doses for 17 days.

Figure S19 shows a) Average tumor volumes of HeLa xenografts bearing mice treated with solvent or Pdld through intraperitoneal injection of 7 repeated doses for 13 days. Error bars represent standard deviations, n = 5, * denote p<0.05 compared to solvent control, b) Body weight of mice in different groups, c) Photos showing mice bearing HeLa xenografts after treatment with solvent or Pdld through intraperitoneal injection of 7 repeated doses for day 13.

Figure S20 shows In vitro anti-angiogenic property of [Pd(C^AN^AN)(NHC)]⁺ complexes on MSI cells, a) The anti-angiogenic activity of Pdla and Pdld (0.25 or 0.5 μΜ) as revealed by the tube-formation assay after 1 h treatment, b) Wound healing assay on MSI cells, (i) After 48 h, cells migrated to the wounded area, (ii) No significant inhibitory effect incubation with Pdla at 0.5 μΜ. (iii) Migration was inhibited by incubation with Pdla at 0.5 μΜ. Pictures taken at 100x magnification.

Figure S21 to S28 shows characterization of the palladium(ll)-carbene complexes.

Detailed Description

Terms not specifically defined herein should be given the meanings that would be given to them by one of skill in the art in light of the disclosure and the context. As used in the specification, however, unless specified to the contra ry, the following terms have the meaning indicated and the following conventions are adhered to.

It is to be understood that unless otherwise indicated this invention is not limited to specific reactants, reaction conditions, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a catalyst" or "a complex" encompasses a combination or mixture of different catalysts or complexes as will as a single catalyst or complex, reference to "a substituent" includes a single substituent as well as two or more substituents that may or may not be the same, and the like.

In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

The term "alkyl" as used herein refers to a linear, branched, or cyclic saturated hydrocarbon group typically although not necessarily containing 1 to about 20 carbon atoms, preferably 1 to about 12 carbon atoms, 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally, although again not necessarily, term "cycloalkyl" intends a cyclic alkyl group, typically having 4 to 8, preferably 5 to 7, carbon atoms. The term "substituted alkyl" refers to alkyl substituted with one or more substituent groups, and the terms

"heteroatom-containing alkyl" and "heteroalkyl" refer to alkyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the term "alkyl" includes linear, branched, cyclic, unsubstituted, substituted, and/or

heteroatom-containing alkyl.

The term "alkane" as used herein refers to a linear, branched, or cyclic saturated hydrocarbon typically although not necessarily containing 1 to about 20 carbon atoms, preferably 1 to about 12 carbon atoms, 1 to 6 carbon atoms, such as methane, ethane, propane, butane, octane, decane, and the like.

The term "alkoxy" as used herein intends an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group maybe represented as -O-alkyl where alkyl is as defined above, such as methoxy, ethoxy, propoxy, butoxy, octoxy, decoxy, and the like.

The term "aromatic ring" as used herein and unless otherwise specified, refers to a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Preferred aromatic ring contain 5 to 20 carbon atoms, and particularly preferred aromatic ring contain 5 to 14 carbon atoms. Exemplary aromatic ring contains one aromatic ring or two fused or linked aromatic rings, e.g., benzene, naphthalene, thiophene, benzothiophene, anthracene, pyrene, furan, pyrimidine, pyrrole, pyridine, fluorene, carbozole, carborane, isoquinoline, 1-isoquinoline, 2-quinoline, and the like. "Substituted aromatic ring" refers to an aromatic ring moiety substituted with one or more substituent groups.

By "substituted" , as alluded to in some of the aforementioned definitions, is meant that, at least one hydrogen atom bound to a carbon (or other) atom is replaced with one or more non-hydrogen substituents. Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, sulfhydryl, C1-C20 alkoxy, C2-C20 alkenyloxy, C2-C20 alkynyloxy, C5-C20 aryloxy, C6-C20aralkyloxy,

C6-C20 alkaryloxy, acyl (including C2-C20 alkylcarbonyl (-CO-alkyl) and

C6-C20 arylcarbonyl

(-CO-aryl)), acyloxy (-O-acyl, including C2-C20 alkylcarbonyloxy (-O-CO-alkyl) and

C6-C20 arylcarbonyloxy (-O-CO-aryl)), C2-C20 alkoxycarbonyl (-(CO)-O-alkyl),

C6-C20aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo),

C2-C20 alkylcarbonato (-O-(CO)-O-alkyl), C6-C20 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylato (-COO-), carbamoyl (-(CO)-NH2),

mono-(Cl-C20 alkyl)-substituted carbamoyl

(-(CO)-NH(C1-C20 alkyl)), di-(Cl-C20 alkyl)-substituted carbamoyl

(-(CO)-N(C1-C20 alkyl)2), mono-(C5-C20 aryl)-substituted carbamoyl (-(CO)-NH-aryl), di-(C5-C20 aryl)-substituted carbamoyl (-(CO)-N(C5-C20 aryl)2),

di-N-(Cl-C20 alkyl),N-(C5-C20 aryl)-substituted carbamoyl, thiocarbamoyi (-(CS)-NH2), mono-(Cl-C20 alkyl)-substituted thiocarbamoyi (-(CO)-NH(C1-C20 alkyl)),

di-(Cl-C20 alkyl)-substituted thiocarbamoyi (-(CO)-N(C1-C20 alkyl)2),

mono-(C5-C20 aryl)-substituted thiocarbamoyi (-(CO)-NH-aryl),

di-(C5-C20 aryl)-substituted thiocarbamoyi

(-(CO)-N(C5-C20 aryl)2), di-N-(Cl-C20 alkyl),N-(C5-C20 aryl)-substituted

thiocarbamoyi, carbamido (-NH-(CO)-NH2), cyano(-C=N), cyanato (-0-C≡N), thiocyanato (-S-C=N), isocyano (-N+≡C-), formyl (-(CO)-H), thioformyl (-(CS)-H), amino (-NH2), mono-(Cl-C20 alkyl)-substituted amino, di-(Cl-C20 alkyl)-substituted amino, mono-(C5-C20 aryl)-substituted amino, di-(C5-C20 aryl)-substituted amino,

C2-C20 alkylamido (-NH-(CO)-alkyl), C6-C20 arylamido (-NH-(CO)-aryl), imino

(-CR=NH where R = hydrogen, C1-C20 alkyl, C5-C20 aryl, C6-C20 alkaryl,

C6-C20 aralkyl, etc.), C2-C20 alkylimino (-CR=N(alkyl), where R = hydrogen,

C1-C20 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), arylimino (-CR=N(aryl), where R = hydrogen, C1-C20 alkyl, C5-C20 aryl, C6-C20 alkaryl, C6-C20 aralkyl, etc.), nitro (-N02), nitroso (-NO), sulfo (-S02-OH), sulfonato (-S02-0-), C1-C20 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), C5-C20 arylsulfanyl (-S-aryl; also termed "arylthio"), C1-C20 alkyldithio

(-S-S-alkyl), C5-C20 aryldithio (-S-S-aryl), C1-C20 alkylsulfinyl (-(SO)-alkyl),

C5-C20 arylsulfinyl (-(SO)-aryl), C1-C20 alkylsulfonyl (-S02-alkyl), C5-C20 arylsulfonyl

(-S02-aryl), boryl (-BH2), borono (-B(OH)2), boronato (-B(OR)2 where R is alkyl or other hydrocarbyl), phosphono (-P(0)(OH)2), phosphonato (-P(0)(0-)2), phosphinato

(-P(0)(0-)), phospho (-P02) phosphino (-PH2), silyl (-SiR3 wherein R is hydrogen or hydrocarbyl), and silyloxy (-O-silyl); and the hydrocarbyl moieties C1-C20 alkyl

(preferably C1-C12 alkyl, more preferably C1-C6 alkyl), C2-C20 alkenyl (preferably

C2-C12 alkenyl, more preferably C2-C6 alkenyl), C2-C20 alkynyl (preferably

C2-C12 alkynyl, more preferably C2-C6 alkynyl), C5-C20 aryl (preferably C5-C14 aryl),

C6-C20 alkaryl (preferably C6-C16 alkaryl), and C6-C20 aralkyl (preferably

C6-C16 aralkyl).

In addition, the aforementioned functional groups may, if a particular group permits, be further substituted with one or more additional functional groups or with one or more hydrocarbyl moieties such as those specifically enumerated above. Analogously, the above-mentioned hydrocarbyl moieties may be further substituted with one or more functional groups or additional hydrocarbyl moieties such as those specifically enumerated.

The "pharmaceutically acceptable anion" as used herein and unless otherwise specified, may be a chloro, bromo, iodo, tartrate, hydrogen tartrate, succinate, maleate, fumarate, sulfate, hydrogen sulfate or methylsulfate anion.

A series of chemical structures of cyclometalated palladium(ll) complexes containing N-heterocyclic carbene (NHC) ligands as anti-cancer drugs can be exemplified as follows.

PdPPh₃ [Pd(C^AN^AN)CI] frans-[Pd(NHC)₂CI₂]

N-Heterocyclic carbene (NHC) ligand is a strong σ-donor ligand that can form stable metal-C bond and can be prepared easily and is non-toxic. Complexes can contain tridendate CN N ligand to improve the stability. Pdiso, PdPPh₃, [Pd(C^AN^AN)CI] and tra/is-[Pd(NHC)₂CI₂] were synthesized to compare with [Pd(C^AN^AN)(NHC)]⁺ in terms of the physiological stability and cytoxicities.

Cyclometalated pa lladium(l l) complexes containing N HC ligands with different alkyl chain length (Pdla-Pdld Figure 1) were prepared. I n general, a mixture of [Pd(C^AN^AN)CI] and the corresponding N HC ligand in the presence of KOtBu was heated under reflux for 24 h to give pure palladium(l l) N HC complexes after purification by column chromatography and re-crystallization. The complexes were characterized by FAB-MS, ^XH NMR, ¹⁹F NMR and ³¹P NMR spectroscopies, and elemental analyses (Supporting I nformation). Other palladium(l l) complexes, Pd2a, PdPPh₃, Pdiso, tra/is-[Pd(NHC)₂CI₂] and [Pd(C^AN^AN)CI], were also prepared for comparative study. Crystals of Pdla and Pdlc suitable for X-ray crystallography were obtained by diffusion of pentane or diethyl ether into dichloromethane solutions. Perspective views of the crystal structures are shown in Figure S2. The Pd-C_carbene distances of Pdla and Pdlc are 1.995 A. Crystallographic and structural refinement data of Pdla and Pdlc and selected bond angles/distances are summarized in Table S1-S6 in the Supporting Information.

Table 1 below shows In vitro cytotoxic IC50 values (μΜ, 72 h) of the palladium(ll) complexes and cisplatin toward human cell lines of lung cancer (NCI-H1650 and NCI-H460), breast cancera (MDA-MB-231), cervical cancer (HeLa), ovarian cancer (A2780) and its cisplatin resistant clone (A2780cis), and normal lung fibroblast (CCD-19L.il). NCI-H1650 NCI-H460 MDA-MB-231 HeLa A2780cis A2780 CCD-19LU

Complex [M

Pd1a 2.5±0.2 2.1±0.2 0.9±0.05 1.8±0.1 2.2±0.2 1.7±0.2 32.1±1.1

Pd1b 1.2±0.09 1.1+0.1 0.9±0.06 1.4±0.2 1.8±0.2 1.5±0.2 22.1±1.9

Pd1c 0.5±0.03 0.4±0.05 0.8±0.05 0.5±0.05 1.2±0.1 1.6±0.1 15.4±1.1

Pd1d 0.09±0.01 0.08±0.01 0.5±0.06 0.1 ±0.01 0.5±0.05 0.2±0.03 11.8±1.2

Pd2a 0-9±0.1 1.1±0.09 0.4±0.04 0.4±0.03 2.5±0.2 1.3±0.2 8-9±0.7 ilra 7S-[Pd(NHC >100 >100 >100 >100 >100 >100 >100

[Pd(C^AN^AN)CI] >100 >100 >100 >100 >100 >100 >100

PdCb >100 >100 >100 >100 >100 >100 >100

Pdiso >50 >50 >50 >50 >50 >50 >50

PdPPha 14.5±1.2 15.2±1.4 18.2±1.7 11.2±1.0 2.2±0.1 3.2±0.3 25.5±2.1 cisplatin 15.5±0.9 9.5±0.8 20.5±1.5 12.1±1.1 30.5±3.1 1.5±0.1 95.5±8.1

The UV-visible absorption data and spectra of the [Pd(C^AN^AN)(N HC)]⁺ complexes are depicted in Table S7 and Figure S3 and S4. Complexes Pdla-Pdld show strong absorption bands in the region of 233-330 nm and a weaker absorption at ca. 383 nm, which are attributable to the intraligand (I L) charge transfer transitions.

The stability of the palladium(ll) complexes in the presence of biologically important thiols was examined. Pdlb (2 mM) in aqueous buffer solution (150 mM ammonium bicarbonate)/DMSO (4:1, v/v) mixture was stable in the presence of 10-fold excess of GSH (20 mM) at 40 °C and revealed no significant changes of the signals of Pdlb in the ¹H NMR spectrum for 24 h. (Figure S5). I n contrast, upon addition of excess GSH (20 mM) to Pdiso (2 mM) or PdPPh₃ (4 mM), the ^XH NMR signals in aromatic region (6-8.5 ppm) of Pdiso were different from those obtained without GSH (Figure S6a), while the ³¹P NMR signal of PdPPh₃ revealed a significant change in chemical shift from 40 ppm to -8 ppm (Figure S6b). These findings suggest that, unlike the [Pd(C^AN^AN)(N HC)]⁺ complexes, palladium(l l) phosphine and isocyanide complexes undergo reactions readily with GSH. The solution stability of [Pd(C^AN^AN)(NHC)]⁺ in the presence of GSH was further examined by electrospray ionization mass spectrometry (ESI-MS). A mixture of Pdla (0.2 mM) and 100-fold excess of GSH (20 mM) in aqueous buffer solution (50 mM ammonium bicarbonate, pH 7.6) was freshly prepared and the mass spectrum was recorded (Figure S5b). The peak at m/z = 433.3, corresponding to [Pdla-PF₆]⁺, was still observed after 24 h of incubation with GSH . In contrast, fra/is-[Pd(N HC)₂CI₂] (0.2 mM) was found to react readily with GSH (20 mM) in pH 7.6 buffer solution, as revealed by the vanishing of the ion peak of tra/is-[Pd(NHC)₂CI₂] (m/z = 503.4) and the emergence of [Pd(NHC)₂(GSH)]⁺ adduct peak (m/z = 772.3) upon incubation with GSH for 1 h (Figure S7). I n addition, the in vitro stability of Pdla was also studied by LC-MS. No new peaks were found in the LC-MS chromatograms obtained from incubation of cell culture medium or HeLa cell extract with Pdla (10 μΜ) for 48 h (Figure S8). Furthermore, [Pd(C^AN^AN)(N HC)]⁺ complexes were at least 5-fold less inhibitory toward thio-dependent enzyme thioredoxin reductase compared to the thiol reactive Pdiso and PdPPh₃ (Table S8). We also investigated the reaction of Pdld with large excess of non-thiol biological reductants, ascorbic acid. The absorption spectra of Pdld did not have significant changes and ascorbic acid oxidation, as determined by monitoring the absorbance at 265 nm, was not obvious up to 24 h.

As serum albumin is known to strongly bind with and hence lower the bioavailability of drug molecules, the level of free palladium(ll) complexes in the presence of human serum albumin (HSA) was examined by measuring the amount of unbound palladium in the solution mixture of the complexes (2 μΜ) and HSA (60 μΜ) by inductively coupled plasma mass spectrometry (ICP-MS) (Figure S9). It was found that more than 60 % of Pdla and 55 % of Pdld were left unbound after a 2-h incubation, while less than 16 % of unbound palladium was found for other palladium(ll) complexes. This suggests that [Pd(C^AN^AN)(NHC)]⁺ complexes have weaker interaction with HSA compared to other palladium(ll) complexes. By protein fluorescence titration experiments, the binding constants of Pdla, Pdld and Pd2a with HSA were determined to be (2.5±0.1)xl0⁵, (4.7±0.3)xl0⁴ and (1.8±0.2)xl0⁶ M^_1, respectively (Figure S10).

The in vitro cytotoxicities of the palladium(ll) complexes toward different human cancer cell lines including a cervical epithelial cancer (HeLa), lung cancer (NCI-H1650 and NCI-H460), an aggressive triple-negative breast cancer (MDA-MB-231), and an ovarian cancer (A2780) and its cisplatin resistant clone (A2780cis) were investigated (Table 1). All the [Pd(C^AN^AN)(NHC)]⁺ complexes were found to display promising anti-proliferative activity toward the cancer cell lines with IC₅₀ values of 0.09-2.5 μΜ, which were up to 172-fold more cytotoxic than cisplatin. The [Pd(C^AN^AN)(NHC)]⁺ complexes displayed similar cytotoxicities in both A2780 and A2780cis while cisplatin is relatively less cytotoxic (~20-fold) towards the cisplatin-resistant A2780cis than the A2780. Notably, [Pd(C^AN^AN)(NHC)]⁺ complexes were less cytotoxic toward a human normal lung fibroblast cell line (CCD-19Lu), e.g. Pdld showed an IC₅₀ value of 11.8 μΜ toward CCD-19L.U, which was about 140-fold higher than that toward NCI-H1650 cells. On the other hand, other palladium(ll) complexes were found to show much lower cytotoxicity toward the cancer cells. This may be attributable to the aforementioned poor stability of these complexes in cellular conditions (Figure S6 and S7) and their more significant binding with serum proteins (Figure S9).

The involvement of apoptosis in the cytotoxic action of the [Pd(C^AN^AN)(NHC)]⁺ complexes on cancer cells was examined. HeLa cells treated with Pdla or Pdld (0.5 μΜ) showed dramatic increase in the cell population with reduced DNA content (sub-Gl phase in flow cytometry analysis) (Table S9), increases in the enzymatic activities of caspase-3 and caspase-9 (Figure Sll) and cleavage of PARP-1 (Figure S12), signifying induction of apoptosis.

The cellular uptake of [Pd(C^AN^AN)(NHC)]⁺ and other palladium(ll) complexes was investigated by measuring palladium metal content in cell lysates using ICP-MS (Figure 2). Cancer cells (HeLa and NCI-H1650) were found to show 2-fold higher cellular upake of [Pd(C^AN^AN)(NHC)]⁺complexes as compared to the normal CCD-19Lu cells. On the other hand, PdPPh₃, Pdiso, irans-[Pd(NHC)₂CI₂] and [Pd(C^AN^AN)CI] all displayed much lower uptake into cancer and normal cells compared to that of [Pd(C^AN^AN)(NHC)]⁺ complexes (with up to 50-fold difference) (Table 1) in the design of palladium medicines for cancer treatment.

The binding constants, K_b, for Pdla, Pdld and Pd2a with calf-thymus DNA (ct DNA) determined from UV-visible absorption data and Scatchard plots were found to be (14.2±0.8)xl0³, (8.5±0.6)xl0³ and (9.5±0.7)xl0³ M^"1, respectively (Figure S13). The K_b values of Pdla, Pdld and Pd2a are markedly lower than those of other palladium(ll) complexes in the literature (K_b = 10^4"5 M¹) ⁴⁰' ^9] and also the typical DNA intercalator ethidium bromide (K_b = 10⁷) by up to several orders of magnitude. ^[4] In a gel mobility shift assay, DNA ladder (123 bp) mixed with [Pd(C^AN^AN)(NHC)]⁺ at molar concentration equivalent to that of base pairs did not show retarded mobility (Figure S14). To examine whether [Pd(C^AN^AN)(NHC)]⁺ complexes trigger DNA damage such as double strand break (DSB) inside the cells, fluorescence microscopic examination of phosphorylated Histone 2AX (γΗ2ΑΧ) was performed. γΗ2ΑΧ is an established marker for DSB, forming foci at the site of DNA breaks and appearing as punctate staining in the nucleus in immunofluorescence staining experiments.'¹⁰¹ It was found that HeLa cells displayed γΗ2ΑΧ foci after treatment with cisplatin for 24 h but not with the [Pd(C^AN^AN)(NHC)]⁺ complexes (Figure S15). These results suggest that DNA is unlikely to be a major molecular target of [Pd(C^AN^AN)(NHC)]⁺.

As mitochondria is known to play key roles in activating cell apoptosis, the effect of Pdla and Pdld on mitochondria was investigated by a fluorescent probe of mitochondrial membrane potential, JC-1. HeLa cells treated with Pdla or Pdld (2.5 μΜ) for 2 h followed by staining with JC-1 displayed green fluorescence, while orange fluorescence was observed in HeLa cells treated with solvent control (Figure S16). The green fluorescence from cells treated with [Pd(C^AN^AN)(NHC)]⁺ indicates disruption of the mitochondrial membrane potential by the complexes that leads to cytosolic accumulation of green-emissive monomeric JC-1. To assess whether production of reactive oxygen species (ROS), which may be augmented owing to mitochondrial dysfunction, is involved in the [Pd(C^AN^AN)(NHC)]⁺-induced cytotoxicity, HeLa cells were treated with Pdla or Pdld in the presence of excess cell permeable anti-oxidant N-acetylcysteine (NAC) and no significant change in cell viability was found (Figure S17). These data are also consistent with the in v/^'tro stability of the [Pd(C^AN^AN)(NHC)]⁺ in the presence of thiols.

A proteomic analysis of changes in protein expression mediated by Pdld was performed using HPLC-LTQ-Orbitrap MS followed by computational pathway analysis. The lists of proteins (Table S10) whose expressions were altered by Pdld were uploaded to ExPlain™ which is connected to the BIOBASE database for upstream searching of keynodes for pathway and keynode analysis. Through the pathway analysis, it was found that 24 and 56 regulated pathways were identified from the HeLa cells treated with Pdld for 2 and 10 h, respectively (Table Sll). Many of these pathways were associated with apoptotic cell death and angiogenesis. Furthermore, the Keynode network analysis^{1 J} deduced that Pdld acts on an upstream signaling pathway involving the growth factor receptor-bound protein 2 (Grb2) that is a key adaptor protein of diversified cancer-promoting growth factor receptors such as epidermal growth factor receptor (EGFR) (Figure 3a). To verify the effect of Pdld on EGFR signaling pathway as implied by proteomic analysis, the EGF stimulated phosphorylation of EGFR and its downstream protein kinase ERK1/2 in HeLa cells were studied by Western blotting experiment (Figure 3b). Incubation of HeLa cells with Pdld for 2 h led to significant decreases in phosphorylation of EGFR and ERK1/2 in a dose-dependent manner, suggesting that Pdld inhibits EGFR signaling. Similar EGFR inhibition was also demonstrated in two lung cancer cell lines (NCI-H460 and HCC-827) under the same experimental conditions (Figure 3c & 3d).

The in vivo anti-cancer activity of Pdld was examined. Treatment of nude mice bearing xenografts of NCI-H460 cancer cells with Pdld at 1 mg/kg and 2 mg/kg through intraperitoneal injection of 8 repeated doses for 17 days was found to inhibit tumor growth by 32 % (p<0.05) and 61 % (p<0.05), respectively, as compared to those treated with solvent control (Figure 4a). Importantly, no mouse death or significant body weight loss was found after the treatment with Pdld at these dosages (Figure 4b). At the end of the experiments (two days after the last compound administration), mice were sacrificed and the in vivo stability of Pdld was studied by LC-MS/MS. Intact cation of Pdld was found in tumor (0.048±0.015 g /g tissue) and blood plasma (9±3 nM) (Figure S18). The in vivo anti-cancer activity of Pdld was also examined in nude mice bearing xenografts of HeLa cancer cells with Pdld at 1 mg/kg and 3 mg/kg through intraperitoneal injection of 7 repeated doses for 13 days. It was found to inhibit tumor growth by 40 % (p<0.05) and 55 % (p<0.05), respectively, as compared to those treated with solvent control (Figure S19). Furthermore, the anti-cancer properties of [Pd(C^AN^AN)(NHC)]⁺ complexes in terms of their anti-angiogenic activities were investigated. Incubation of mouse endothelial cells (MSI) with Pdla and Pdld at sub-cytotoxic concentrations (0.25 and 0.5 M) resulted in distortion of the endothelial tube formation in Matrigel (Figure S20) and the cell migration in wound healing assay (Figure S21).

In summary, [Pd(C^AN^AN)(NHC)]⁺ complexes demonstrate high stability in vitro and in the presence of physiological thiols. Eventually the complexes show potent in vitro cytotoxicities against cancer cells as well as effective in vivo anti-cancer activities toward tumor xenograft in nude mice with no observable toxicity. The mechanism of anti-cancer action of the complexes, as investigated by proteomics analysis and biochemical assays, involved induction of mitochondrial dysfunction and inhibition of EGFR signaling pathway in association with apoptotic cell death of the cancer cells (Figure 5). This highlights the promising prospect of using NHC and pincer type cyclometalated ligands in the development of palladium(ll) complexes for anti-cancer treatment.

The examples, as shown in the Supporting Information, illustrate embodiments for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

With respect to any figure or numerical range for a given characteristic, a figure or a parameter from one range may be combined with another figure or a parameter from a different range for the same characteristic to generate a numerical range.

Other than in the operating examples, or where otherwise indicated, all numbers, values and/or expressions referring to quantities of ingredients, reaction conditions, etc., used in the specification and claims are to be understood as modified in all instances by the term "about."

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

The following Supporting Information as referred to above is incorporated herein by reference.

Supporting Information: Anti-cancer Cyclometalated Palladium(ll) Complexes with N-Heterocyclic Carbene Ligands Exhibit High Stability and Cytotoxicity In Vitro and Suppress Tumor Growth In Vivo (Tommy Tsz-Him Fong, Chun-Nam Lok, Clive Yik-Sham Chung, Yi-Man Eva Fung, Pui-Keong Chow, Pui-Ki Wan, and Chi-Ming Che*)

Materials and methods

Analytical grade organic solvents were used in all experiments unless otherwise stated. Pd(C^AN^AN)Cl [HC^AN^AN = 6-phenyl-2,2'-bipyridine],^[1] l,3-dimethyl-lH-imidazol-3-ium bromide, l,3-diethyl-lH-imidazol-3-ium bromide, l,3-dipropyl-lH-imidazol-3-ium bromide, l,3-dibutyl-lH-imidazol-3-ium bromide were synthesized according to the literature procedure. ^[2] Pd(R-C^AN^AN-R')Cl (R— C^AN^AN— R' = 3-(6'-aryl-2'-pyridinyl)isoquinoline)^[3] and

[Pd(C^AN^AN)(PPh₃)](CF₃S0₃) (PdPPh₃)^[1] have been reported previously. UV/vis spectra were recorded on a Perkin-Elmer Lambda 19 UV/vis spectrophotometer. For MTT and protein assays, the absorbance was quantified using Perkin-Elmer Fusion Reader (Packard Bioscience Company). Fast Atom Bombardment (FAB) mass spectra were obtained on a Finnigan Mat 95 mass spectrometer. 1H NMR and ¹³C NMR spectra were obtained on DPX 300, 400 M Bruker FT-NMR spectrometers relative to the signal of tetramethylsilane. Elemental analysis was performed by the Institute of Chemistry at the Chinese Academy of Science, Beijing. Other chemicals unless otherwise stated were purchased from Sigma-Aldrich Co. Single crystals X-ray diffraction

Single crystals of complexes Pdla and Pdlc that were suitable for X-ray diffraction studies were grown by diffusing pentane or Et₂0 into a concentrated solution of the complexes in dichloromethane. The X-ray diffraction data were collected on a Bruker X8 Proteum diffractometer. The crystal was kept at 100 K during data collection. The diffraction images were interpreted and the diffraction intensities were integrated by using the program SAINT. Multi-scan SADABS was applied for absorption correction. By using 01ex2,^[4] the structure was solved with the ShelXS^[5] structure solution program using direct methods and refined with the XL^[5] refinement package using least squares minimization. The positions of the H atoms were calculated on the basis of the riding mode with thermal parameters equal to 1.2 times that of the associated C atoms and these positions participated in the calculation of the final R indices. In the final stage of least-squares refinement, all non-hydrogen atoms were refined anisotropically. Crystallographic parameters are summarized in Table SI . CCDC 1448613-1448614 contain the supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Center via www.ccdc.cam.ac.uk/data_request/cif.

Synthesis and characterization of the palladium(II)-carbene complexes

Complex Pdla. A mixture of [Pd(C^AN^AN)Cl] (80.0 mg, 0.215 mmol), l,3-dimethyl-lH-imidazol-3-ium bromide (25.1 mg, 0.258 mmol) and KOtBu (29.0 mg, 0.258 mmol) in 30 mL dimethylformamide were heated to reflux for 24 h. Excess ammonium hexafluorophosphate was added and the reaction mixture was further refluxed for 1 h. After cooling to room temperature, DMF was removed by vacuum distillation and the residue was washed with water and hence diethyl ether. Light yellow product was obtained after silica-gel chromatography separation with CH₂C1₂ and CH₃CN (13 : 1, v/v) as eluent. Yield: 50 %. ¹H NMR (400 MHz, CD₃CN, 25 °C): 8.00 (d, J= 8.0 Hz, 1 H), 7.83-7.92 (m, 3 H), 7.74 (m, 1 H), 7.63 (d, J= 8.0 Hz, 1 H), 7.40 (d, J = 8.1 Hz, 1 H), 7.27 (m, 1 H), 7.06 (s, 2 H), 6.88 (t, J= 7.6 Hz, 1 H), 6.75 (t, J = 7.5 Hz, 1 H), 6.07 (d, J = 7.5 Hz, 1 H), 3.65 (s, 6 H); ³¹P NMR (161 MHz, CD₃CN): -144.53; ¹⁹F NMR (376 MHz, CD₃CN): -73.86; positive FAB-MS: m/z 433 [M - PF₆]⁺; Elemental Analysis: calcd for C₂iHi₉F₆N₄PPd: C, 43.58; H, 3.31; N, 9.68; found: C, 43.72; H, 3.32; N, 9.66. See Figure S21.

Complex Pdlb. The procedure is similar to that for Pdla except l,3-diethyl-lH-imidazol-3-ium bromide was used. Yield: 45 %. 1H NMR (300 MHz, CDC1₃, 25 °C): 8.26 (d, J = 8.1 Hz, 1 H), 8.10-8.14 (m, 1 H), 8.06-8.09 (m, 2 H), 7.96 (d, J = 8.0 Hz, 1 H), 7.71 (d, J = 8.1 Hz, 1 H), 7.59-7.62 (m, 1 H), 7.53 (d, J = 7.7 Hz, 1 H), 7.26 (s, 2 H), 7.12 (t, J = 7.6 Hz, 1 H), 6.99 (t, J = 7.5 Hz, 1 H), 6.29 (d, J = 7.5 Hz, 1 H), 4.34-4.46 (m, 4 H, -CH₂-), 1.45 (t, J= 7.4 Hz, 6 H, CH₃); ³¹P NMR (400 MHz, CD₃CN): -144.24; ¹⁹F NMR (376 MHz, CD₃CN): -73.25; Positive FAB-MS: m/z 461 [M - PF₆]⁺; Elemental Analysis, calcd for C₂₃H₂₃F₆N₄PPd: C, 45.45; H, 3.98; N, 9.22; found: 45.10, H 3.88, N 9.00. See Figure S22.

Complex Pdlc. The procedure is similar to that for Pdla except l,3-propyl-lH-imidazol-3-ium bromide was used. Yield: 48 %. 1H NMR (400 MHz, CDC1₃, 25 °C): 8.29 (d, J= 8.3 Hz, 1 H), 7.98-8.17 (m, 4 H), 7.73 (d, J= 7.9 Hz, 1 H), 7.58-7.62 (m, 1 H), 7.53 (d, J = 7.6 Hz, 1 H), 7.27 (s, 2 H), 7.12 (t, J = 7.4 Hz, 1 H), 6.99 (t, J = 7.5 Hz, 1 H), 6.31 (d, J = 7.5 Hz, 1 H), 4.20^1.39 (m, -N-CH2- on -«Pr, 4 H), 1.89 (sextet, J = 7.3 Hz, 4 H, -CH2- on - «Pr), 0.87 (t, J = 7.3 Hz, 6 H, -CHs on - «Pr); ³¹P NMR (161 MHz, CDsCN): -144.76; ¹⁹F NMR (376 MHz, CDsCN): -73.23; Positive FAB-MS: m/z 489 [M - PF₆]⁺; Elemental Analysis, calcd for C₂5H₂₇F₆N₄PPd · (H₂0)o_.5: C, 46.63; H, 4.38; N, 8.70; found: 46.50, H 4.26, N 8.49. See Figure S23.

Complex Pdld. The procedure is similar to that for Pdla except l,3-dibutyl-lH-imidazol-3-ium bromide was used. Yield: 52 %. 1H NMR (400 MHz, CDCI₃, 25 °C): 8.46 (d, J= 8.0 Hz, 1 H), 8.02-8.23 (m, 4 H), 7.71 (d, J= 7.9 Hz, 1 H), 7.63-7.64 (m, 1 H), 7.53 (d, J = 7.6 Hz, 1 H), 7.27 (s, 2 H), 7.14 (t, J = 7.4 Hz, 1 H), 6.99 (t, J = 7.5 Hz, 1 H), 6.30 (d, J = 7.0 Hz, 1 H), 4.25-4.42 (m, 4 H, -N-CH₂- on -«Bu), 1.84 (sextet, -CH₂- on -«Bu, J = 7.1 Hz, 4 H), 1.28 (sextet, 4H, J = 7.6 Hz, -CH₂- on -«Bu), 0.80 (t, J = 7.3 Hz, 6 H, -CHs on -«Bu); ³¹P NMR (161 MHz, CDsCN): -144.14; ¹⁹F NMR (376 MHz, CDsCN): -73.66; Positive FAB-MS: m/z 517 [M - PF₆]⁺; Elemental Analysis, calcd for C₂₇H₃iF₆N₄PPd: C, 48.92; H, 4.71; N, 8.45; found: 48.76, H 4.86, N 8.38. See Figure S24.

Complex Pd2a. The procedure is similar to that for Pdla except π-extended Pd(R-C^AN^AN-R')Cl was used. Yield: 45 %. 1H NMR (300 MHz, CDC1₃, 25 °C): 8.93 (s, 1 H), 8.52 (s, 1 H), 8.41 (d, J = 7.8 Hz, 1 H), 8.0-8.06 (m, 2 H), 7.87-7.92 (m, 1 H), 7.74-7.83 (m, 2 H), 7.65 (d, J = 7.8 Hz, 2 H), 7.54 (s, 2 H), 7.27 (s, 2 H), 7.18 (t, J = 7.4 Hz, 1 H), 7.05 (t, J = 7.4 Hz, 1 H), 6.36 (d, J = 7.1 Hz, 1 H), 4.04 (s, 6 H), 1.24-1.67 (s, 18 H); positive FAB-MS: m/z 671 [M - CF₃S0₃]⁺; Elemental Analysis, calcd for C₄₀H₄iF₃N₄O₃PdS · 2H₂0: C, 56.04; H, 5.29; N, 6.54; found: 55.95, H 5.07, N 6.78. See Figure S25.

Complex PdlSO. To a yellow suspension of [Pd(C^AN^AN)Cl] (0.10 g, 0.22 mmol) in CH₃CN:CHC1₃ (1 : 1, v/v; 20 mL) at room temperature was added excess tert-butylisocyanide (0.5 mL, 4.4 mmol). Upon stirring, the color of the mixture changed to pale yellow after 30 min. Addition of excess LiC10₄ in acetonitrile followed by diethyl ether yielded a white precipitate, which was collected and dried. Recrystallization by slow diffusion of diethyl ether into an acetonitrile solution of the crude product afforded white crystals: yield 0.12 g, 91 %. 1H NMR (400 MHz, DMSO-i¾, 25 °C): 8.66 (s, 1 H), 8.57 (d, J = 7.8 Hz, 1 H), 8.29-8.36 (m, 3 H), 8.14 (d, J = 6.8 Hz, 1 H), 7.82 (d, J = 7.6 Hz, 2 H), 7.21-7.27 (m, 3 H), 1.70 (s, 9 H); positive FAB-MS: m/z 378 [M⁺ - C≡NtBu + CH₃CN]; Elemental Analysis, calcd for C₂iH₂₀ClN₃O₄Pd: C, 48.48; H, 3.87; N, 8.08; found: C, 48.62; H, 3.69; N, 8.18. See Figure S26. w?S-[Pd(NHC)₂(Cl)₂]. It was synthesized according to a modification of a procedure reported in the literature. ^[6] l,3-Dibutyl-lH-imidazol-2-ium bromide (0.274 g, 1.26 mmol) and Ag₂0 (0.146 g, 0.63 mmol) in dichloromethane (30 mL) were stirred at room temperature for 4 h. The solution was then filtered by celite, and the filtrate was evaporated under reduced pressure.

[l,3-Dibutyl-lH-imidazol-2-ylidene]AgCl was crystallized from the acetonitrile solution of the crude product by slow-evaporation technique. Yield = 0.117 g (29 %); 1H MR (400 MHz, CDC1₃): 7.23 (s, 2 H), 4.17 (t, J = 7.1 Hz, 4 H), 1.84 (pentet, J = 7.2 Hz, 4 H), 1.36 (sextet, J = 7.3 Hz, 4 H), 0.95 (t, J = 7.3 Hz, 6 H). The trans- [Pd( HC)₂(Cl)₂] was then synthesized by heating the acetonitrile solution (15 mL) of [l,3-dibutyl-lH-imidazol-2-ylidene]AgCl (0.117 g, 0.36 mmol) and Pd(COD)Cl₂ (47.4 mg, 0.17 mmol) under reflux for 6 h. The volume of solution mixture was reduced to ~1 mL, and the light yellow crystal of trans- [Pd( HC)₂(Cl)₂] was obtained by slow evaporation of the acetonitrile solution. Yield = 30 mg (32.8 %); 1H NMR (400 MHz, CDC1₃): 6.82 (t, J = 3.9 Hz, 4 H), 4.49 (m, 8 H), 2.08 (m, 8 H), 1.47 (m, 8 H), 1.01 (t, J = 6.3 Hz, 12 H); ¹³C{1H} NMR (150 MHz, CDC1₃): 169.1, 120.5, 50.7, 33.0, 20.1, 13.8; Positive ESI-MS: m/z = 503.4 [M - Cl]⁺; Elemental Analysis, calcd for C₂₂H₄oCl₂N₄Pd: C, 49.12; H, 7.50; N, 10.42; found: C, 48.72; H, 7.89; N, 10.28. See Figure S27 and S28.

Stability analysis

Solution stabilities of the palladium(II) complexes were investigated by ESI-MS, ³¹P NMR, and 1H NMR. The aqueous solution with 10 % DMSO containing the complexes (2 mM) and GSH (20 mM) was analyzed by ESI-MS, ³¹P NMR, and 1H NMR to examine the formation of complex-GSH adduct.

The stability of Pdla was also examined in HeLa cells using LC/MS. Pdla (1 μΜ) was added to the HeLa cells for 2 h, 8 h, 24 h, and 48 h. HeLa cells were washed with PBS twice and 500 μΕ of miliQ water was added and incubated for 10 min at room temperature. It was then diluted by 20-fold with acetonitrile. Solutions were then centrifuged at 15000 rpm for 15 min at 4°C. The supernatant was kept and dried. Acetonitrile (200 μΕ) was added and centrifuged again and the supernatant was then transferred to UPLC vials for LC/MS analysis.

Interaction with human serum albumin

The binding with human serum albumin (HSA) was determined by a modified procedure. ^[7] In general, a 280 mg amount of HSA was dissolved in 7 mL of minimal essential medium. Pd(C^AN^AN)Cl and tra«s-[Pd(NHC)₂(Cl)₂] was added to the HSA-containing medium with final concentration of 2 μΜ and the mixture was shaken at 50 rpm. After 2 h, 100 μL of the solution was taken, and treated with 500 μΐ, of cold (-20 °C) acetone, and stored at -20 °C for 30 min to allow precipitation of the protein fraction. Afterwards, the solution was centrifuged at 13000 rpm for 1 min at 4 °C. The supernatant was taken and the protein precipitates were treated with 500 μί, of 60 % HN0₃ (*) at 60-70 °C for 2 h and at room temperature for 12 h. The final volume was adjusted to 10 mL for inductively coupled plasma-mass spectrometry (ICP-MS) analysis. The total palladium content was measured by digesting the solutions of palladium and HSA mixture without precipitation. The percentage of bound palladium was determined as the palladium contents in precipitated proteins divided by the total palladium contents.

(*) Caution: Concentrated HN0₃ is highly reactive with acetone, causing explosion. The acetone should be evaporated before adding HN0₃, and the digestion needs to be handled carefully!

Cell culture

All cell lines were obtained from American Type Culture Collection (ATCC). Bronchioalveolar carcinoma (NCI-H1650), breast adenocarcinoma (MDA-MB-231), cervical epithelial carcinoma (HeLa), and normal lung fibroblast (CCD-19Lu) were maintained in Eagle's minimum essential medium. All cell culture media were supplemented with 10 % (v/v) fetal bovine serum, L-glutamine (2 mM) and penicillin/streptomycin (100 U/mL). Cells were incubated in 5 % C0₂ humidified air atmosphere at 37 °C and subcultured when 80 % confluence was reached.

Cytotoxicity assay

The cytotoxic properties of the complexes were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ^[8] Cells were seeded in 96-well plates and incubated overnight prior to be examined. All of the complexes were dissolved in DMSO. The concentration of the complexes was calculated according to the elemental composition of the complexes determined by the elemental analyses. Media in the presence of the tested complexes were added and serially diluted to various concentrations (i.e. from 50 μΜ to 0.2 μΜ), and media containing cisplatin were used as the positive control. The maximum concentration of DMSO in media did not exceed 0.1 % (v/v). The cells were incubated for 72 h and followed by the addition of MTT solution. The cells were further incubated for 3 h and solubilization buffer (100 μΐ,, 10 % SDS in 0.01 M HC1) was subsequently added. The absorption intensities at 580 nm in each well were measured by a Perkin-Elmer FusionTM a-FP plate reader. The IC₅₀ values of the complexes (concentrations at which could inhibit cellular growth by 50 % compared to the negative control) were determined from the plots of the cell viability percentage versus the complex concentration. For each set of data, at least three independent experiments have been done.

Cellular uptake measurement

Cellular uptake experiments were conducted according to the literature method with some modifications. The cells were seeded at a density of 1 x 10⁶ cells/dish in 6-cm culture dishes. After 24 h incubation, cells were left untreated or treated with the metal complexes. Cells were harvested after 24 and 48 h, trypsinized and washed four times with cold PBS. Milli-Q water (500 μί) was added and monolayer cells were scraped off from the culture dishes. All samples (300 μΐ,) were digested in 70 % HN0₃ (500 uL) at 70 °C for 2 h, which were then diluted 1 : 100 in water for ICP-MS analysis.

In vivo tumor growth inhibition experiment

Female BALB/cAnN-nu (nude) mice, 4-7 week old, were purchased from Charles River Laboratories (Wilmington, MA). Mice were maintained under specific pathogen-free conditions. All animal experiments were conducted under the guidelines approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong. To establish tumors, 5 ^χ 10⁶ HeLa cells suspended in PBS (100 μΐ,) were injected into the right and left back flanks of each mouse respectively by subcutaneous injection. The mice were divided into different groups with a solvent control and drug treated groups after the tumor volume had reached about 30 mm³ (2-3 days after tumor inoculation). Mice were treated with the compounds or solvent control according to the body weight once every 2-3 days by intraperitoneal injection until the mice were sacrificed. Tumor sizes were measured once every 2-3 days using a digital caliper and tumor volumes were calculated by the formula V = ab^{2 χ} 0.52, where a and b were the longest and the shortest diameters of the tumor. The volume of PET diluent injected was kept / L per injection in all mice, and mice in all group received the same amount of PET diluent.

Western blotting analysis

HeLa cells (5 10⁵) were cultured on 60 mm dish and incubated overnight before experiments. HeLa cells were treated with Pdld for various concentrations (0.5, 2 and

4 μΜ). After 2 h incubation, cells were harvested and lysed using the lysis buffer (150 mM NaCl, 100 mM Tris-HCl pH 7.4, 10 % glycerol, 1 % Triton X-100, 10 mM NaF,

5 mM sodium pyrophosphate, 5 mM sodium orthovanadate, 0.1 % SDS) with protease inhibitor cocktail. The cellular protein content was quantified by the Protein Assay (Bio-Rad). Total protein extracts (40 μg) were loaded onto 12.5 % SDS-polyacrylamide gel, separated by electrophoresis followed by transfer of proteins from the gel to polyvinylidene fluoride (PVDF) membranes. After the transfer of protein, the membrane was then blocked with 3 % BSA in TBST buffer and incubated with corresponding primary antibodies at 4 °C overnight. Primary antibodies of PARP (1 : 1500), cleaved PARP (1 : 1500), EGFR (1 : 1000), pEGFR (1 : 1000), ERK 1/2 (1 : 1500), pERK 1/2 (1 : 1500), GAPDH (1 :2000) and Actin (1 :2000) were obtained from Cell Signaling Technology. After washing, the membrane was incubated with secondary antibody conjugated with horseradish peroxidase (1 :5000) for 90 min. The immunoreactive signals were detected using enhanced chemiluminenscene kit (GE healthcare) following the procedures given in the user manual. Equal loading of each lane was confirmed by the intensity of β-actin.

Immunofluorescence staining

Cells (2 10⁴) were seeded on glass coverslips and treated with freshly prepared palladium(II) complexes (1 μΜ) or cisplatin (10 μΜ) following overnight culture at 37 °C, 5 % C0₂. Briefly, for γΗ2ΑΧ staining, cells were fixed in 4 % paraformaldehyde/PBS (15 min, RT), permeabilized with 0.3 % Triton X-100/PBS (15 min, on ice), blocked in 0.2 % BSA/PBS (1 h, RT), stained for γΗ2ΑΧ (Cell Signaling, #2577L, 1 : 100 dilution, at 4 °C, 2 h), and labeled with secondary antibodies (Invitrogen, Alexa Fluor® 568 dye, 1 :200 dilution, 2 h, RT). For DNA staining, cells were stained with DAPI (1 :400 dilution, at 4 °C, 15 min). Coverslips were then visualized using a fluorescence microscope (ZEISS).

Purified TrxR enzyme assay Recombinant rat TrxRl (ICMO Corp, Sweden; 1 nM) was reduced with NADPH (0.2 mM) and then incubated with the metal complexes (1 nM to 1 μΜ) for 30 min in a 100 mM potassium phosphate buffer, pH 7.4 and 1 mM EDTA. The enzyme activities (initial rates of increases in O.D.₄i₂„) were measured using 3 mM DT B.

Fluorescence quenching experiment

Fluorescence quenching experiment was followed with a reported procedure. ^[5] In brief, a solution of HSA (3 μΜ, in Tris-HCl buffer (0.05 M Tris, 0.1 M NaCl, pH 7.40)) was excited at 280 nm and the emission spectrum was recorded. Then the solution was added with the aliquots of stock solution of the complex (5 mM/10 mM) and the emission spectra were recorded at the same excitation wavelength after equilibration for 1 min per aliquot until saturation point was almost reached. The binding constant was determined by applying the following equation

log[(Io-I)/I] = logX + nlog[Q]

where I₀ and I are the fluorescence intensity of HSA without and with complex, respectively; [Q] is complex concentration. Plot of log[(Io-I)/I] versus log[Q] gave the y-intercept equaling to logK, and the binding constant can be obtained accordingly.

Absorption-titration experiment

Absorption titration experiment was performed according to a reported procedure. ^J The absorption spectra of a solution of the complex in PBS (with 5 % DMSO) was recorded and the solution was added with the aliquots of stock solution of ctDNA (2.63 mM) and the absorption spectra were recorded after equilibration for 1 min per aliquot until saturation point was almost reached. The binding constant was determined by applying the Scatchard equation:

[DNA]/Asap = [ϋΝΑ]/Δε+ 1/(Δε^χ¾), where Aeap = |eA-eF| where εΑ = Abs/[complex], and Δε = |eB-6F| where εΒ and εΡ correspond to the extinction coefficients of the DNA-bound and -unbound complex, respectively. Plot of [ϋΝΑ]/Δε3ρ versus [DNA] gave a slop equaling to l/Δε and a y intercept equaling to 1/(Δε^χ¾), and K was obtained from the ratio of the slop to the y-intercept.

Measurement of mitochondrial membrane potential

HeLa cells (2 10⁴ cells/mL) were seeded in glass-bottomed dishes with supplemented culture medium and incubated for 24 h and then were treated with 2.5 μΜ or 5 μΜ of the complexes or vehicle control for 2 h in a C0₂ incubator at 37 °C. JC-1 (5 μΜ) staining solution was then added to the cells and incubation was continued for 30 min. After washing with serum free culture medium twice, the cells were visualized using a fluorescence microscopy. For positive control, carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 50 μΜ) and JC-1 (5 μΜ) were co-incubated with HeLa cells for 30 min and examined with fluorescence microscope. The fluorescence intensity for both J-aggregates and monomeric forms of JC-1 were measured with a 96-well plate reader (J-aggregates: excitation/emission = 535/580 nm; JC-1 monomers: excitation/emission = 485/530 nm). Proteomic analysis

Sample preparation

HeLa cells were treated with Pdld at 0.5 μΜ concentration for 2 and 10 h in a C0₂ incubator at 37 °C. Equal amount of DMSO were added to HeLa cells as a positive control. The cells were then washed with cold PBS twice and the cells were lysed with a urea lysis buffer (20 mM Tris-HCl, 8 M urea, protein phosphatase inhibitor cocktail, pH 8.0). The cells were then scraped in 1.5 mL Eppendorf tubes and spin down at 10,000 rpm for 15 min at 4 °C. The supernatant was kept and protein concentration was then measured. 50 μg of protein were then precipitated by adding 5x volume of ice-cold acetone (storage at -20 °C). The samples were mixed and kept at -20 °C for 30 min followed by centrifugation at 13,000 rpm for 20 min at 4 °C. The solvents were discarded and the pellets were dried by SpeedVac (Thermo Fisher Scientific). The dried samples were re-suspended with 25 μΐ, of Suspension Buffer (100 mM Tris, 8 M urea, pH 8.5) and DTT was added to make a final concentration of 5 mM to denature the samples at 60 °C. After denaturing for 20 min, iodoacetamide was added to make a final concentration of 25 mM. Then the samples were kept in the dark for 30 min at 25 °C, 100 mM Tris (pH 8.5) was added to dilute the concentration of urea to 1 M, and 0.5 trypsin was added to each reaction mixture, and followed by digestion for overnight at 37 °C. The reaction mixtures were then acidified by addition of 10 μΐ, formic acid to stop the proteolysis. After centrifugation at 14,000 rpm for 15 min, the supernatants were transferred to new tubes (to be frozen at -80 °C for long term storage). The resulting peptides were desalted and enriched by Stage Tips. For each sample, three biological replicates were prepared. The samples were re-dissolved with H₂0 (containing 0.1 % formic acid, v/v) for subsequent HPLC-MS/MS analysis.

HPLC-MS/MS analysis

MS analysis was performed with a LTQ Orbitrap Velos Orbitrap MS (Thermo) connected online with a HPLC. The analytical column was a self-packed PicoTip® column (360 μπι outer diameter, 75 μπι inner diameter, 15 μπι tip, New Objective) packed with 10 cm length of C18 material (ODS-A CI 8 5-μιη beads, YMC) with a high-pressure injection pump (Next Advance). The mobile phases of HPLC are A (0.1 % formic acid in HPLC grade H₂0, volume percentage) and B (0.1 % formic acid in HPLC grade acetonitrile, volume percentage). Three micrograms of sample was loaded onto the analytical column by the auto-sampler and rinsed with 2 % B for 6 min and subsequently eluted with a linear gradient B from 2 % to 40 % for 120 min. For the MS analysis, LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) was operated in a data-dependent mode cycling through a high-resolution (6000 at 400 mlz) full scan MSI (300-2000 mlz) in Orbitrap followed by CID MS2 scans in LTQ on the 20 most abundant ions from the immediate preceding full scan. The selected ions were isolated with a 2-Da mass window and put into an exclusion list for 60 seconds after they were first selected for CID.

Proteins identification and quantification

The raw data were directly used for protein identification and quantification using MaxQuant (Version 1.5.0.25). The data were searched against IPI human database (Version 3.87), in which trypsin specificity was used with up to two missed cleavages allowed. Methionine oxidation was set as a variable modification, and iodoacetamide derivative of cysteine was set as a fixed modification. Default settings were used for mass tolerance for MSI and MS2. The false discovery rate (FDR) was determined by searching against a reverse database and kept FDR at 1 %.

Signaling pathway analysis

Lists of quantified proteins (shown as their Protein IDs) were uploaded to the ExPlain™ tool (version 3.1, BIOBASE) and converted to genes (shown as their Gene Symbols) for further signaling pathway analysis. Details of procedure for pathway analysis have been described previously. ^{[9 10]}

Intracellular reactive oxygen species (ROS) measurement

The production of intracellular ROS was measured in the HeLa cell line using the oxidation-sensitive fluorescent dye dichlorodihydrofluorescein acetate (DCF-DA). HeLa cells were exposed to 0.5 μΜ of complexes for 1-2 h prior to staining with DCF. An increase in green fluorescence intensity was used to quantify the generation of intracellular ROS. After adding DCF at a final concentration of 5 μΜ to the culture medium, the cells were incubated at 37 °C for an additional 1 h, harvested, washed with PBS, and the fluorescent signal was measured immediately with a plate reader (excitation/emission = 440/530 nm).

Caspase activities

The activities of caspase-3 and caspase-9 were determined by caspase substrates Ac-DEVD-AMC and Ac-LHED-AMC, respectively. Briefly, cells were collected by scrapping and pelleted by centrifugation. Cell lysis buffer was then added (0.5 ml/10 cm plate) to cell pellet and homogenized. The cell lysates were clarified by centrifugation. 200 μΐ of fluorescent substrate solution (50 μΜ) and 25 μΐ lysate solution were mixed in a black microplate. The plate was incubated at 37°C. RFU was read on a fluorescence plate reader with excitation at 380 nm and emission at 460 nm. The protein concentrations of the lysates were measured by Biorad protein assays.

Cell cycle arrest

HeLa cells (2 ^χ 10⁵) were treated with complexes (0.5 μΜ) for 12, 24 and 48 h. Cells were collected by trypsinization and fixed in 70 % ethanol at -20 °C for 30 min. After fixation, cells were washed twice with PBS and treated with RNase A (0.1 mg/mL) for 1 h at 37 °C. Cells were then stained with propidium iodide (10 μg/mL). The fluorescence signals were manipulated with FACSCalibur flow cytometry (BD Biosciences). 10,000 ungated events were acquired for each sample. Populations of cells at different phases of cell cycle were analyzed with CellQuest Pro software.

Tube-formation assay

By using the In Vitro Angiogenesis Kit, 10x diluent buffer and ECMatrix™ solution were mixed in a ratio of 1 :9. The mixture (50 μL) was transferred to each well of a 96-well plate. The matrix solution was incubated at 37 °C to allow polymerization. After 1 h, MSI cells (50,000) premixed with different concentrations of Pdla Pdld (0.25 or 0.5μΜ) in 100 μΐ, of DMEM medium were added to the top of the polymerized matrix. After incubation at 37 °C for 8 h, tube formation was examined under an inverted light microscope at lOOx magnification and was quantified using the Sigma Scan Pro. software. The percentage of inhibition was calculated based on the distance measured relative to the untreated control.

Wound-healing assay

MSI cells were cultured on 60-mm culture dishes at a density of 2 ^χ 10⁶ cells/dish. After 24 h, a single wound was created in the middle of the cell monolayer by gently removing the attached cells with a sterile plastic pipette tip. The cells were washed twice with PBS and solutions of different concentrations of Pdla Pdld (0.25 or 0.5 μΜ) in 5 mL of DMEM were added. After incubation at 37 °C for 8 h, migration of the cells into the wound was observed under an inverted microscope.

Quantification of Pdld in mouse tumors and plasma using LC-MS MS

Nude mice bearing xenografts of NCI-H460 cancer cells were treated with Pdld at 2 mg/kg through intraperitoneal injection of 8 repeated doses for 17 days. The mice were subjected to cardiac puncture. Blood (about 600-800 μΐ.) collected was added with Na₂EDTA (20 mg/mL) in a ratio of 10: 1 to prevent coagulation, centrifuged (3500 rpm for 15 min at 4 °C) and plasma were transferred to a centrifuge tube. The mouse tumors were harvested and dissected into pieces and weighed. Each of weighed tissues was homogenized in lxPBS (1 :3, w/v) by tissue homogenizer. Both plasma and homogenized tissue samples were stored at -80 °C until they were analyzed.

Chromatographic and mass spectrometric conditions

The analysis was performed on a Waters AC QUIT Y™ UPLC system and a Waters Q-TOF Premier ™ mass spectrometer with an electrospray ionization source (Micromass MS Technologies). The data acquisition and analysis were performed using Waters MassLynx version 4.1. Separation was achieved by a Waters ACQUITY ™ BEH Ci8 column (100 2.1 mm i.d., 1.7 μΜ) connected to a Waters ACQUITY™ BEH Ci8 guard column (5 x 2.1 mm i.d., 1.7 μΜ) with gradient elution system consisted of 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). Separation was achieved by using: 5-55 % B from 0 to 3 min; 55-90 % B from 3 to 15 min; 90-100 % B from 15 to 25 min; 100 % B from 25 to 30 min and returned to initial conditions and equilibrated for 3 minutes. The flow rate was 0.4 mL/min and the injection volume was 2 μυ

The mass spectrometer was operated in positive mode and the conditions were optimized as follows: source temperature, 120 °C; desolvation temperature, 500 °C; nebulization gas flow rate, 800 L/h; cone gas flow rate, 20 L/h; capillary voltage, 3 kV; sampling cone voltage, 25 V. The data were collected into two separate data channels with the instrument spending 0.5 s on data acquisition for each channel and a 0.1 s inter-channel delay. The TOF scan range was from 100 to 1000 Da. The optimized fragmentation transition for quantification of Pdld was mlz 517.2→337.0 with a collision energy of 37 eV.

Standard and sample preparation Stock solution (2 μΜ) of each analyte was prepared using methanol. They were subjected to serial dilution to give concentrations of Pdld at: 0.005, 0.01, 0.05, and 0.1 μΜ. A simple and rapid protein precipitation method was used for sample preparation. A volume of 100 μΙ_, from each concentration of each analyte was transferred to a centrifuge tube and evaporated to dryness by a concentrator. A blank plasma/tumor homogenate (100 μΐ.) was added and followed by acetonitrile (300 μΐ.) and the mixture was vortexed for 10 s and centrifuged (15000 rpm) for 15 min. The supernatant was collected and evaporated to dryness by a concentrator. Methanol (100 μΐ,) was added and then vortexed again for 10 s and centrifuged (15000 rpm) for 15 min. The supernatant (2 μ ) was subject to LC-MS analysis. Analyte-treated plasma and tumor were prepared in a similar manner.

Supporting Tables

Table SI. Crystal data of complex Pdla.

Complex Pdla

Empirical formula C₂iH₁₉F₆N₄PPd

Formula weight 578.28

Temperature/K 100

Crystal system Triclinic

Space group P-l

a/A 11.865(2)

b/A 13.505(3)

c/A 14.862(3)

a/° 84.748(4)

β/° 69.465(4)

γ 73.759(4)

Volume/A³ 2141.1(7)

Z 4

pcalcg/cm³ 1.794

μ/mm^"1 8.338

F(000) 1151.0

Crystal size/mm³ 0.4 0.3 0.2

Radiation ΟιΚα (λ = 1.54178)

2Θ range for data collection/⁰ 6.36 to 132.14

Index ranges -13 < h < 14, -15 < k < 15, -17 < 1 < 17

Reflections collected 25385

Independent reflections 7257 [R_int = 0.0588, R_sigma = 0.0503]

Data/restraints/parameters 7257/0/599

Goodness-of-fit on F² 1.044

Final R indexes [1>=2σ (I)] Ri = 0.0441, wR₂ = 0.1209

Final R indexes [all data] Ri = 0.0446, wR₂ = 0.1215

Largest diff peak/hole / e A^"3 1.42/-0.96

Table S2. Crystal Data of complex Pdlc. Complex Pdlc

Empirical formula C₂5H₂₇F₆N₄PPd

Formula weight 634.87

Temperature/K 100.0

Crystal system tetragonal

Space group 1-4

a/A 19.703(2)

b/A 19.703(2)

c/A 14.3173(16)

a/° 90

β/° 90

γ/° 90

Volume/A³ 5558.2(13)

o o

pcalcg/cm³ 1.517

μ/mm^"1 6.481

F(000) 2560.0

Crystal size/mm³ 0.2 0.2 0.2

Radiation CuKa ^ = 1.54178)

2Θ range for data collection/⁰ 6.344 to 133.708

Index ranges -19 ≤h ≤23, -21 ≤k ≤23, -17 ≤1 ≤12

Reflections collected 27804

Independent reflections 4840 [Rin_t = 0.0658, Rsigma = 0.0459]

Data/restraints/parameters 4840/54/358

Goodness-of-fit on F² 1.085

Final R indexes [1>=2σ (I)] Ri = 0.0387, wR₂ = 0.1107

Final R indexes [all data] Ri = 0.0387, wR₂ = 0.1108

Largest diff. peak/hole / e A^"3 1.32/-0.37

Flack parameter -0.010(6)

Table S3. Bond distances of Pdla.

Atom Atom Distance [A] Atom Atom Distance [A]

Pdl Nl 1.988(3) N7 C41 1.448(5)

Pdl N2 2.154(3) N7 C38 1.361(5)

Pdl C 17 1.995(4) N7 C39 1.383(5)

Pdl C I 1.982(4) N5 C28 1.348(5)

Nl C7 1.359(5) N5 C32 1.346(5)

Nl Cl l 1.341(5) N8 C40 1.380(5)

N2 C 12 1.358(5) N8 C38 1.354(5)

N2 C 16 1.346(5) N8 C42 1.466(5)

N4 C 17 1.355(5) C22 C27 1.424(6)

N4 C21 1.461(5) C22 C23 1.389(6)

N4 C 19 1.378(5) C33 C32 1.487(6)

N3 C17 1.351(5) C33 C34 1.374(6)

N3 C 18 1.384(5) C27 C28 1.479(6)

N3 C20 1.454(6) C27 C26 1.391(6)

C2 C I 1.404(6) C28 C29 1.394(6)

C2 C3 1.386(6) C31 C32 1.385(6)

C13 C 12 1.384(6) C31 C30 1.392(6)

C13 C14 1.378(6) C37 C36 1.384(6)

CI C6 1.424(6) C36 C35 1.382(7)

C12 C l l 1.488(6) C40 C39 1.337(7)

C14 C 15 1.381(6) C26 C25 1.383(6)

C7 C8 1.389(6) C29 C30 1.381(6)

C7 C6 1.465(6) C35 C34 1.383(6)

C4 C5 1.388(6) C23 C24 1.396(6)

C4 C3 1.394(6) C25 C24 1.396(7)

C9 C IO 1.388(6) PI F5 1.586(3)

C9 C8 1.387(6) PI F3 1.591(3)

C18 C 19 1.336(6) PI Fl 1.595(3)

CIO C l l 1.381(6) PI F6 1.599(3)

C5 C6 1.399(6) PI F4 1.574(3)

C16 C 15 1.392(6) PI F2 1.590(3)

Pd2 N6 2.129(3) P2 F12 1.590(3)

Pd2 N5 1.986(3) P2 F8 1.570(3)

Pd2 C22 2.015(4) P2 F9 1.617(4)

Pd2 C38 1.996(4) P2 Fl l 1.593(3)

N6 C33 1.366(5) P2 F10 1.572(4)

N6 C37 1.337(5) P2 F7 1.562(4) Table S4. Bond distances of Pdlc.

Atom Atom Distance [A] Atom Atom Distance [A]

Pdl C16 2.009(6) C13 C14 1.377(11)

Pdl Nl 1.990(5) C7 C6 1.389(8)

Pdl N2 2.142(5) C3 C4 1.388(9)

Pdl C17 1.995(6) C14 C15 1.401(10)

PI F3 1.607(6) C4 C5 1.392(9)

PI F4 1.602(6) C5 C6 1.481(8)

PI F5 1.611(6) F1A P1A 1.56(3)

PI F6 1.586(7) F2A P1A 1.56(2)

PI F2 1.592(7) N3 C17 1.347(8)

PI Fl 1.620(7) N3 C18 1.373(10)

C16 Cl l 1.422(9) N3 C20 1.469(9)

C16 C15 1.380(9) N4 C19 1.372(10)

Nl CIO 1.342(8) N4 C17 1.356(8)

Nl C6 1.337(8) N4 C23 1.461(8)

N2 C5 1.365(8) C19 C18 1.324(12)

N2 CI 1.340(9) C22 C21 1.514(10)

Cl l C12 1.400(9) C25 C24 1.518(10)

Cl l CIO 1.482(8) C23 C24 1.508(10)

C12 C13 1.388(10) C21 C20 1.515(10)

C2 C3 1.386(10) F6A P1A 1.55(2)

C2 CI 1.393(10) F5A P1A 1.56(2)

CIO C9 1.390(9) F4A P1A 1.56(2)

C8 C7 1.384(9) F3A P1A 1.56(2)

C8 C9 1.396(9)

Table S5. Bond angles of Pdla.

Atom Atom Atom Angle [·] Atom Atom Atom Angle [·]

Nl Pdl N2 77.86(13) C40 N8 C42 124.6(3)

Nl Pdl C17 174.32(14) C38 N8 C40 111.0(3)

C17 Pdl N2 107.01(14) C38 N8 C42 124.3(3)

CI Pdl Nl 82.02(15) C27 C22 Pd2 111.6(3)

CI Pdl N2 159.86(14) C23 C22 Pd2 131.2(3)

CI Pdl C17 93.04(15) C23 C22 C27 117.2(4)

C7 Nl Pdl 117.1(3) N6 C33 C32 115.4(3)

Cl l Nl Pdl 120.1(3) N6 C33 C34 121.7(4)

Cl l Nl C7 122.5(3) C34 C33 C32 122.9(4)

C12 N2 Pdl 113.0(3) C22 C27 C28 116.3(4)

C16 N2 Pdl 128.7(3) C26 C27 C22 121.0(4)

C16 N2 C12 118.3(4) C26 C27 C28 122.8(4)

C17 N4 C21 124.1(3) N5 C28 C27 112.4(3)

C17 N4 C19 111.3(3) N5 C28 C29 119.2(4)

C19 N4 C21 124.5(3) C29 C28 C27 128.4(4)

C17 N3 C18 111.2(3) C32 C31 C30 118.1(4)

C17 N3 C20 124.2(3) N5 C32 C33 113.4(4)

C18 N3 C20 124.5(3) N5 C32 C31 120.3(4)

N4 C17 Pdl 126.1(3) C31 C32 C33 126.3(4)

N3 C17 Pdl 129.6(3) N6 C37 C36 122.4(4)

N3 C17 N4 104.0(3) C35 C36 C37 118.8(4)

C3 C2 CI 122.3(4) C39 C40 N8 107.1(4)

C14 C13 C12 119.6(4) C25 C26 C27 120.6(4)

C2 CI Pdl 131.2(3) N7 C38 Pd2 128.1(3)

C2 CI C6 116.2(4) N8 C38 Pd2 127.5(3)

C6 CI Pdl 112.6(3) N8 C38 N7 104.2(3)

N2 C12 C13 121.7(4) C30 C29 C28 119.0(4)

N2 C12 Cl l 115.2(3) C36 C35 C34 119.3(4)

C13 C12 Cl l 123.1(4) C40 C39 N7 106.9(4)

C13 C14 C15 119.4(4) C22 C23 C24 121.7(4)

Nl C7 C8 119.1(4) C26 C25 C24 119.3(4)

Nl C7 C6 112.2(3) C23 C24 C25 120.2(4)

C8 C7 C6 128.6(4) C29 C30 C31 120.9(4)

C5 C4 C3 119.1(4) C33 C34 C35 119.3(4)

C8 C9 CIO 121.1(4) F5 PI F3 89.99(17)

C19 C18 N3 106.7(3) F5 PI Fl 89.81(16)

Cl l CIO C9 118.3(4) F5 PI F6 179.1(2)

C4 C5 C6 120.5(4) F5 PI F2 91.5(2)

Nl Cl l C12 113.7(3) F3 PI Fl 178.0(2)

Nl Cl l CIO 120.3(4) F3 PI F6 90.11(17)

CIO Cl l C12 126.0(4) Fl PI F6 90.12(16) C9 C8 C7 118.6(4) F4 PI F5 89.46(19)

CI C6 C7 116.0(4) F4 PI F3 90.3(2)

C5 C6 CI 121.4(4) F4 PI Fl 91.7(2)

C5 C6 C7 122.5(4) F4 PI F6 89.7(2)

N2 C16 C15 122.5(4) F4 PI F2 178.7(2)

C2 C3 C4 120.6(4) F2 PI F3 90.6(2)

C14 C15 C16 118.6(4) F2 PI Fl 87.4(2)

C18 C19 N4 106.8(3) F2 PI F6 89.4(2)

N5 Pd2 N6 78.61(13) F12 P2 F9 88.23(19)

N5 Pd2 C22 81.94(15) F12 P2 Fl l 178.8(3)

N5 Pd2 C38 177.13(14) F8 P2 F12 91.04(16)

C22 Pd2 N6 160.55(15) F8 P2 F9 86.2(2)

C38 Pd2 N6 103.67(14) F8 P2 Fl l 89.13(19)

C38 Pd2 C22 95.78(16) F8 P2 F10 175.0(3)

C33 N6 Pd2 112.8(3) Fl l P2 F9 90.6(3)

C37 N6 Pd2 128.5(3) F10 P2 F12 89.90(17)

C37 N6 C33 118.5(4) F10 P2 F9 88.9(3)

C38 N7 C41 124.5(3) F10 P2 Fl l 89.8(2)

C38 N7 C39 110.8(3) F7 P2 F12 91.2(2)

C39 N7 C41 124.7(3) F7 P2 F8 90.9(3)

C28 N5 Pd2 117.8(3) F7 P2 F9 177.0(3)

C32 N5 Pd2 119.7(3) F7 P2 Fl l 90.0(3)

C32 N5 C28 122.5(3) F7 P2 F10 94.0(3)

Table S6. Bond angles of Pdlc.

Atom Atom Atom Angle ['] Atom Atom Atom Angle [·]

C16 Pdl N2 160.7(2) C13 C14 C15 120.8(6)

Nl Pdl C16 82.2(2) C16 C15 C14 120.8(6)

Nl Pdl N2 78.6(2) C3 C4 C5 119.5(6)

Nl Pdl C17 175.6(2) N2 C5 C4 121.1(6)

C17 Pdl C16 93.9(2) N2 C5 C6 116.0(5)

C17 Pdl N2 105.2(2) C4 C5 C6 122.9(5)

F3 PI F5 178.0(5) Nl C6 C7 120.1(5)

F3 PI Fl 89.4(4) Nl C6 C5 113.8(5)

F4 PI F3 88.9(4) C7 C6 C5 126.1(5)

F4 PI F5 89.1(4) CIO C9 C8 118.1(5)

F4 PI Fl 89.9(4) C17 N3 C18 110.6(6)

F5 PI Fl 90.5(4) C17 N3 C20 125.4(5)

F6 PI F3 88.9(5) C18 N3 C20 124.0(6)

F6 PI F4 177.8(5) C19 N4 C23 125.4(6)

F6 PI F5 93.1(5) C17 N4 C19 109.9(6)

F6 PI F2 89.7(4) C17 N4 C23 124.5(6)

F6 PI Fl 90.2(4) C18 C19 N4 107.7(7)

F2 PI F3 90.9(4) N3 C17 Pdl 129.3(5)

F2 PI F4 90.2(4) N3 C17 N4 104.8(5)

F2 PI F5 89.2(4) N4 C17 Pdl 125.3(4)

F2 PI Fl 179.7(5) N4 C23 C24 112.8(6)

Cl l C16 Pdl 111.3(4) C22 C21 C20 111.8(6)

C15 C16 Pdl 130.7(5) C19 C18 N3 106.9(7)

C15 C16 Cl l 118.0(6) N3 C20 C21 112.9(5)

CIO Nl Pdl 117.6(4) C23 C24 C25 111.8(6)

C6 Nl Pdl 119.5(4) N2 CI C2 121.9(6)

C6 Nl CIO 122.8(5) F1A P1A F2A 179.3(15)

C5 N2 Pdl 112.0(4) F1A P1A F5A 88.4(15)

CI N2 Pdl 128.7(5) F6A P1A F1A 89.2(15)

CI N2 C5 119.3(6) F6A P1A F2A 91.1(15)

C16 Cl l CIO 116.6(5) F6A P1A F5A 92(2)

C12 Cl l C16 121.0(6) F6A P1A F4A 177(2)

C12 Cl l CIO 122.4(5) F6A P1A F3A 90(2)

C13 C12 Cl l 119.4(6) F5A P1A F2A 91.0(15)

C3 C2 CI 119.2(6) F4A P1A F1A 89.0(15)

Nl CIO Cl l 112.2(5) F4A P1A F2A 90.6(15)

Nl CIO C9 119.8(5) F4A P1A F5A 85.4(19)

C9 CIO Cl l 128.0(5) F4A P1A F3A 92(2)

C7 C8 C9 120.8(6) F3A P1A F1A 88.2(15)

C14 C13 C12 120.1(6) F3A P1A F2A 92.4(15)

C8 C7 C6 118.3(5) F3A P1A F5A 176(2)

C2 C3 C4 119.0(6) Table S7. UV-visible absorption data of the palladium(II) complexes.

Complex λ max/nm (ε/dm³ mol^cm^"1)

Pdla 251 (sh, 23100), 270 (sh, 18100), 312 (9300), 332 (sh, 6500), 383 (sh,

1600)

Pdlb 251 (sh, 25600), 270 (sh, 19800), 312 (10200), 332 (sh, 7000), 383 (sh,

1650)

Pdlc 251 (sh, 26700), 270 (sh, 21300), 312 (10700), 332 (sh, 7300), 383 (sh,

1600)

Pdld 251 (sh, 25100), 270 (sh, 19700), 312 (10300), 332 (sh, 7400), 383 (sh,

1700)

Pd2a 254 (sh, 35400), 292 (sh, 32200), 347 (10800), 365 (sh, 9200), 400 (sh,

3350)

Pdiso 233 (sh, 66000), 266 (sh, 16800), 320 (8650), 386 (2800)

Table S8. Inhibition of purified TrxR by palladium(II) Pdla-Pdld, Pdiso, PdPPh₃, tra¾s-[Pd( HC)₂Cl₂], [Pd(C^AN^AN)Cl] and auranofin.

Complex IC₅₀ (nM)

Pdla 350±20.2

Pdlb 382±21.1

Pdlc 351±25.8

Pdld 321±29.8

Pdiso 41.0±3.12

PdPPh₃ 21.2±1.43 trans- Pd(NRC)₂C\₂] 418±28.1

[Pd(C^AN^AN)Cl] 310±25.1

auranofin 14±0.53

Table S9. The percentages of cells in each phase of the cell cycle after Pdla and

Pdld (0.5 μΜ) treatment at different time points.

Complex Treatment time [h] Sub-Gl [%] G0/G1 [%] S [%] G2/M [%]

Control 12 1.8±0.1 65.7±5.8 11.2±1.1 21.0±1.7

Pdld 12 4.2±0.3 58.9±5.3 14.7±1.1 21.6±1.8

Control 24 1.7±0.2 59.9±4.9 17.0±1.5 21.4±1.8

Pdla 24 2.6±0.2 58.0±5.6 17.5±1.5 22.1±2.1

Pdld 24 22.7±2.1 38.5±3.2 14.2±1.2 23.7±1.9

Control 48 8.0±0.7 68.4±6.1 8.2±0.7 15.3±1.2

Pdla 48 46.0±3.8 29.3±1.2 4.4±0.3 19.5±2.0

References

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Claims

Claims What is claimed is:

1. A pharmaceutical composition for treatment of cancer comprising a cyclometalated N-heterocyclic carbene complex having the formula I:

or a pharmaceutically acceptable salt thereof, wherein

R¹, R², R³, R⁴, and R⁵ are each independently alkyl or -H;

R⁶ and R⁷ are independently an alkyl chain;

A is a OS0₂CF₃, CI, PF₆ or a pharmaceutically acceptable anion;

n is +1;

b is -1; and

y is 1.

2. A pharmaceutical composition of I of claim 1, wherein,

R¹, R², R³, R⁴, and R⁵ are each -H;

R⁶ and R⁷ is a C₂ alkyl chain;

A is a OSO2CF3, CI, PF₆ or a pharmaceutically acceptable anion;

n is +1;

b is -1; and

y is 1.

3. A pharmaceutical composition of I of claim 1, wherein,

R¹, R², R³, R⁴, and R⁵ are each -H;

R⁶ and R⁷ is a C₃ alkyl chain;

A is a OS0₂CF₃, CI, PF₆ or a pharmaceutically acceptable anion;

n is +1;

b is -1; and

y is 1.

4. A pharmaceutical composition of I of claim 1, wherein,

R¹, R², R³, R⁴, and R⁵ are each -H;

R⁶ and R⁷ is a C₄ alkyl chain;

A is a OS0₂CF₃, CI, PF₆ or a pharmaceutically acceptable anion; n is +1;

b is -1; and

y is 1.

5. A pharmaceutical composition of I of claim 1, wherein,

R¹, R², R³, R⁴, and R⁵ are each -H;

R⁶ and R⁷ is a combination of Ci to C₄ alkyl chain;

A is a OS0₂CF₃, CI, PF₆ or a pharmaceutically acceptable anion;

n is +1;

b is -1; and

y is 1.

6. A pharmaceutical composition for treatment of cancer comprising a cyclometalated complex having the formula I I :

tl

R¹, R², R³, R⁴, and R⁵ are each independently alkyl or -H;

R⁶ is

A is a OSO2CF3, CI, CI0₄ or a pharmaceutically acceptable anion;

n is +1;

b is -1; and

y is 1.

7. A pharmaceutical composition of I I of claim 5, wherein,

R¹, R², R³, R⁴, and R⁵ are each -H;

R⁶ is

A is a OS0₂CF₃, CI or a pharmaceutically acceptable anion;

n is +1;

b is -1; and

y is 1.

8. A pharmaceutical composition for treatment of cancer comprising a cyclometalated N-heterocyclic carbene complex having the formula I II :

IN

or a pharmaceutically acceptable salt thereof, wherein

R¹, R², R³, and R⁴ are each independently an alkyl chain.

9. A pharmaceutical composition of II I of claim 7, wherein

R¹, R², R³, and R⁴ are each C₂ alkyl chain, C₃ alkyl chain or C₄ alkyl chain.

10. A pharmaceutical composition for treatment of cancer comprising a cyclometalated N-heterocyclic carbene complex having the formula IV: IV

or a pharmaceutically acceptable salt thereof, wherein

R¹ is substituent containing any aromatic ring/substituted aromatic ring not limited to benzene, naphthalene, thiophene, benzothiophene, anthracene, pyrene, furan, pyrimidine, pyrrole, pyridine, fluorene, carbozole, carborane; or any alkoxy chain not limited to methoxy, ethoxy; or substituent containing any saturated hydrocarbon chain not limit to alkane, ethane, propane, butane;

R² and R³ are independently a C C₄ alkyl chain; A is a OS0₂CF₃, CI, PF₆ or a pharmaceutically accepted anion;

n is +1;

b is -1;

y is l;

Ring A is any aromatic ring/substituted aromatic ring providing an anionic carbon donor to the palladium(ll) ion; it is not limited to benzene, naphthalene, thiophene, benzothiophene, anthracene, pyrene, furan, pyrimidine, pyrrole, pyridine, fluorene, carbozole, carborane; and

Ring B is any aromatic ring/substituted aromatic ring with a nitrogen coordinating to palladium(ll) ion; it is not limited to pyrimidine, pyridine, isoquinoline, 1-isoquinoline, 2-quinoline.