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Article Cite This: Organometallics XXXX, XXX, XXX−XXX

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Triazolyl RuII, RhIII, OsII, and IrIII Complexes as Potential Anticancer Agents: Synthesis, Structure Elucidation, Cytotoxicity, and DNA Model Interaction Studies Charles K. Rono,† William K. Chu,† James Darkwa,† Debra Meyer,‡ and Banothile C. E. Makhubela*,† †

Department of Chemistry, University of Johannesburg, Kingsway Campus, 2006 Auckland Park, South Africa Department of Biochemistry, University of Johannesburg, Kingsway Campus, 2006 Auckland Park, South Africa

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ABSTRACT: Novel conjugated ruthenium(II), rhodium(III), and iridium(III) organometallic complexes of triazoles 1 and 2 synthesized and evaluated for anticancer activity against cervical (HeLa), kidney (HEK293), nonsmall lung cancer (A549), and leukemia (MT4) cancer cell lines are reported herein. The complexes are κ2-N,C coordinated and have the formula [ML(Ar)Cl] (where L is 1-benzyl-4-phenyl1H-1,2,3-triazole for 1 and 1-benzyl-4-hydroxymethyl-1H1,2,3-triazole for 2, Ar is p-cymene for RuII and OsII and Cp* for RhIII and IrIII, and M is metal). NMR studies, including HMBC and NOESY, were employed to unambiguously elucidate their structures and provide their conformational information in solution. Single-crystal X-ray diffraction data have been used to establish the solid-state structures of selected complexes, which further confirm the structural elucidation by NMR. Dynamic NMR studies, such as differential transferred NOE, have been employed to distinguish between isomers 1a_I and 1a_II of ruthenium(II) complexes of triazole 1. The rhodium(III) (1b) and iridium(III) (1c) complexes exhibited good cytotoxic activities (CC50 = 4−6 μM) comparable to that of the drug auranofin against lung cancer A549 cell lines (CC50 = 4.69 μM). While triazole 1 based ruthenium(II) (1a) and osmium(II) (1d) complexes displayed modest anticancer activities against HeLa and HEK293 cell lines, the analogous rhodium(III) and iridium(III) complexes exhibited good potential (CC50 = 9−54 μM versus auranofin (3−9 μM)) against these cancer cell lines. Insightful NMR studies on the interaction between the DNA model guanosine 5′-GMP and the complexes 1b,c reveal a possible mode of action of the aquated complexes involving carbenylation with DNA bases or purines through the triazolyl proton H-5. From the findings, these complexes could possibly confer their cytotoxic activities through intercalation with the DNA of pathological cells. Therefore, carbenylation of the triazolylrhodium(III) and iridium(III) complexes by DNA guanosine 5′-GMP is proposed as a novel mode of DNA intercalation of these complexes in cancer cells.

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INTRODUCTION

cells with no or minimal toxicity to the host in comparison to platinum-based drugs.2,3 Transition metals other than platinum such as ruthenium,1,4 palladium,5 and gold6 have also been studied for their anticancer activities. These non-platinum-based drugs have shown remarkable cytotoxic results on several cancer cell lines (such as HeLa-cervical, C6-glioma, and Chinese Hamster Ovarian−Ovarian cancer cell lines) with minimal side effects in comparison to platinum-based drugs.7,8 Compounds of ruthenium are highly promising drugs and have been identified to be less toxic in comparison to platinum drugs and capable of overcoming resistance induced by platinum drugs on cancer cells.3,9 These observed activities in arene ruthenium compounds have been attributed to transportation by transferrin to tumor cells, although at binding sites different

Cancer is a group of diseases with enormous socioeconomic burden in addition to being the second and third leading causes of death in developed and developing nations, respectively. The discovery of cisplatin as a cancer regimen in combination with pre-cisplatin-era therapeutic organic compounds greatly improved the cure rate for cancer from 10% to an average of at least 90% in most types of cancers and a 100% cure rate in particular for testicular cancer. However, the clinical use of cisplatin as an antitumor drug is limited by dose-limiting side effects (such as neurotoxicity, hepatotoxicity, and nephrotoxicity) and inherent or acquired resistance following repeated treatment. This is often associated with platinum-based drugs.1 There is continuous effort being made in the design and synthesis of novel anticancer agents with the desired efficacy, selectivity, and broad spectrum activity.2 This includes metallodrugs capable of inducing apoptosis to tumor © XXXX American Chemical Society

Received: July 5, 2019

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DOI: 10.1021/acs.organomet.9b00440 Organometallics XXXX, XXX, XXX−XXX

Article

Organometallics

Scheme 1. One-Pot Synthesis of Ligands 1 and 2 through 1,3-Dipolar Cycloaddition and Their Complexation Protocol To Give Complexes 1a−d and 2a,ba

Reagents and conditions: (i) NaN3 (1.05 equiv), ACO/H2O (3/1 v/v, 0.5 M), 18 h, 40 °C; (ii) (a) phenylacetylene (1.0 equiv), CuSO4·5H2O (2 mol %), ascorbic acid (10 mol %), H2O, 6 h, 80 °C, 94%, (b) propargyl alcohol (1.02 equiv), CuSO4·5H2O (2 mol %), ascorbic acid (10 mol %), H2O, 4 h, 50 °C, 88%; (iii) (a) [Ru(p-cym)Cl2]2 (0.5 equiv), 24 h, 82%, (b) [Rh(Cp*)Cl2]2 (0.5 equiv), 72 h, 52%, (c) [Os(p-cym)Cl2]2 (0.5 equiv), 24 h, (d) [Ir(Cp*)Cl2]2 (0.5 equiv), 72 h. a

from those of iron(III),10 and the selective activation to a more reactive species by the reducing environment of the solid tumor cells in comparison to healthy cells.3 In addition, arene ruthenium compounds possess similar ligand exchange kinetics in comparison to platinum(II) compounds and are hence designed to mimic platinum drugs, particularly targeting DNA.3,1112 The observed improved performance of NAMI-A, an imidazole ruthenium(III) complex, warrants further studies on related azaarene ruthenium and complexes of isoelectronic metals such as rhodium and iridium. The copper(I)-catalyzed Huisgen cycloaddition, which has been popularized as a “click” reaction, has seen 1,2,3-triazoles come into the limelight of organic synthesis and medicinal and materials applications.13 The heterocycle of 1,2,3-triazole has a great potential in coordination chemistry as an N-donor ligand due to its ease of synthesis and the wide functional group tolerance of the click reaction.14 In our effort to develop compounds with potential anticancer properties, we have synthesized a library of triazole-based hybrids, aryltriazoles, and their conjugated PGM-based complexes, including aryltriazole 1. Recently, Keppler and co-workers reported on the anticancer properties of conjugated ruthenium(II) and osmium(II) complexes of 1-benzyl-4-phenyl-1H-1,2,3-triazole (1).15 Intrigued by the activities of these compounds, herein we report on the marked bioactivity of the seemingly less active 1-benzyl-4-phenyl-1H-1,2,3-triazole (1) (about 10-fold weaker than cisplatin in lung cancer A549). As a consequence of complexation to form five-membered rhodium(III) and iridium(III) complexes, these metallacyles proved to be highly

active against a panel of four cancer cell lines, including non small cell lung cancer (A549), human kidney adenocarcinoma (HEK293), cervical cancer (HeLa), and leukemia (MT4). Furthermore, an effort has been made to elucidate the geometry of the complexes in solution through dynamic NMR studies such as transferred NOE of the protons in close conformational proximity and differential 1H−13C HMBC correlations and hence their stabilities in both solution and the solid state. Notably, a concentration-dependent self-association in solution was assumed to cause the spectral “roofing” effect of two vicinal p-cymene protons as observed by Keppler and co-workers15b in their recent study on the potent anticancer triazolyl ruthenium(II) complexes of N- and S-donor complexes of triazole 1. In this study, we report the isolation of two isomers of triazolyl ruthenium(II) complexes of 1benzyl-4-phenyl-1H-1,2,3-triazole (1), namely 1a_I (previously isolated) and 1a_II, and their solution state supported NMR structural elucidation, with the p-cymene region of 1a_II exhibiting the “roofing” characteristics in solution, invariant of the concentration and temperature effects.

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RESULTS AND DISCUSSION

Synthesis and Structure Elucidation of Aryl-1H-1,2,3triazole-Based Cyclometalated Ruthenium(II), Osmium(II), Rhodium(III), and Iridium(III) Complexes. The synthesis of ligands 1 and 2 was achieved following established literature protocols as described in the Experimental Section.16 The reaction of 1-benzyl-4-phenyl-1H-1,2,3-triazole (1) with 0.5 equiv of (p-cymene)ruthenium(II) dichloride dimer, in the presence of a slight excess of sodium acetate as a C−H B


Article

Organometallics activating agent, afforded the cyclometalated ruthenium(II) complex 1a (Scheme 1) in good yield as a yellow solid. A similar synthetic route was used in the syntheses of the other complexes, including 1b, which was obtained as a yelloworange solid in moderate yield using (Cp*)rhodium(III) dichloride dimer as the precursor. The (Cp*)iridium(III) dichloride dimer precursor gave complex 1c as a yellow solid in good yield, and (p-cymene)osmium(II) dichloride dimer afforded complex 1d as a yellow solid in good yield as well (Scheme 1) The synthesis of complex 2b was carried out using a procedure similar to that described for complex 1b with 1benzyl-4-hydroxymethyl-1H-1,2,3-triazole (2) as the ligand. The reaction went to completion in 12 h to give an orange solid in good yield (82%). Notably, an oxidation side product of hydroxymethyltriazole 2 to the triazolyl carboxaldehyde 3 (Scheme 1) was observed in low yield (8% yield) as a white solid. The triazole carboxaldehyde 3 was confirmed by the disappearance of the methylene protons at δH 4.67 ppm in 2 and the emergence of a new peak at δH 10.09 ppm corresponding to the carboxaldehyde (Figures S1−S3).16a,17 Complexation of 2 to form 2b was achieved through acetateassisted deprotonation of the triazolyl methanol to form an active and coordinating triazolylmethoxide species, as evidenced by the disappearance of the hydroxyl peak at δH 3.15 ppm in 2. In addition, a downfield shift of the triazolyl proton at δH 7.50 ppm (from δH 7.42 ppm in the ligand) and an upfield shift of the methylene protons of the triazolylmethoxide at δH 4.67 ppm (from δH 4.71 ppm in the ligand) implied successful coordination of the ligand to the rhodium(III) center, through the γ-nitrogen (Nγ) of the triazole moiety and the oxygen of the methyloxy group, in a bidentate manner. Unlike the case for complex 1b, there were slight downfield shifts in the triazole proton and benzyl methylene protons in complex 2b, probably due to the electron-withdrawing effect of the rhodium center upon coordination. In addition, the highresolution mass spectrometry fragmentation pattern of 2b, in the positive mode, revealed corroborative peaks at m/z 190.0976 corresponding to [(M − Rh(Cp*)Cl) + H]+, m/z 272.9912 corresponding to [Rh(Cp*)Cl]+, and 426.1052 as the base peak corresponding to [M − Cl]+ (Figure S11). For complex 2a, a complex mixture of side products was obtained and the yield was significantly low. Molecular Structures for Complexes 1a_I, 1b and 1c in the Solid State. Complex 1a_I is a yellow solid which crystallized in the triclinic P1̅ space group with four molecules in a unit cell volume upon slow diffusion of diethyl ether into a saturated chloroform solution of the complex, as reported previously.15a The molecular structure of 1a_I adopted a pseudo-octahedral “piano stool” geometry (Rup-cymene plane centroid distance (dpi) 1.691 Å) with the chloride and the triazole moiety as the legs of the stool while the p-cymene moiety is the “seat”, as shown in the plot of displacement thermal ellipsoids for complex 1a_I (Figure 1). It is notable that complex 1a_I is asymmetric at the metal center. Consequently, 1a_I exists as a dimeric parallel pair of these unit molecules in a mirror image fashion of R and S isomers with the Ru−Cl bonds facing each other in a pair while the isopropyl group of the p-cymene is oriented to the back of the molecule in close proximity to the benzyl moiety. Characteristic intermolecular hydrogen bonds between R isomers and S isomers (including H9AACl1 (2.777 Å) and H2ACl1 (2.721 Å)) as well as strong intramolecular hydrogen bonds in

Figure 1. Molecular structure of 1a_I. Hydrogen atoms with probable intramolecular interactions (at 30% of the adduct formed

Figure 11. Sections of 1H NMR spectra showing the interconversion of solvated complex 1c through rearrangement of κS-DMSO complex 1c_II (lower spectrum) below 273 K to κO-DMSO complex 1c_I (upper spectrum) favored in solution at or above 298 K.

extraction using chloroform as a yellow-brown solid upon solvent removal. 1c_I displayed an observed characteristic m/z 640.1983 corresponding to a cationic DMSO-coordinated fragment of the complex (Figure S17). Interestingly, complex 1c_I was found to interconvert from complex 1c_I in solution, at a temperature of 298 K and/or above, to complex 1c_II at low temperatures below 298 K through rearrangement of κO-coordinated 1c-DMSO complex 1c_I to κS-coordinated complex 1c_II, as revealed by the 1H NMR of the complex collected within 5 min upon dissolution in chloroform (Figure 11, bottom). When the solution of complex 1c_II was left standing for 15 min, it led to the establishment of an equilibrium of complexes 1c_I and 1c_II, as shown in Figure 11 (top). It is notable that when the solution was left for a long while (greater than 4 h) at room temperature of 298 K, complex 1c_I was the predominant isomer in solution (Scheme 3). Interestingly, the κO-DMSO complex 1c_I apparently is the I


Article

Organometallics Table 2. Crystal and Structure Refinement Data for Complexes 1a_I and 1b,c empirical formula formula wt temp/K cryst syst space group a/Å b/Å c/Å α/deg β/deg γ/deg V/Å3 Z ρcalc/g cm−3 μ/mm−1 F(000) cryst size/mm3 radiation 2θ range for data collection/ deg index ranges no. of rflns collected no. of indep rflns no. of data/restraints/params goodness of fit on F2 final R indexes (I ≥ 2σ(I)) final R indexes (all data) largest diff peak, hole/e Å−3

1a_I

1b

C25H26ClN3Ru 505.01 99.99 triclinic P1̅ 10.7397(12) 11.7225(13) 18.898(2) 91.300 97.124 106.103(2) 2264.1(4) 4 1.482 0.827 1032.0 0.54 × 0.12 × 0.11 Mo Kα (λ = 0.71073) 2.176−56.63

C26H27.5Cl4N3Rh 626.72 100.0 triclinic P1̅ 10.8444(13) 15.2391(18) 16.967(2) 80.370(2) 81.202(2) 72.252(2) 2617.4(5) 4 1.590 1.082 1270.0 1.706 × 0.333 × 0.25 Mo Kα (λ = 0.71073) 2.448−46.67

C25H27ClN3Ir 597.14 150.0 triclinic P1 10.5971(11) 10.7081(13) 10.9685(12) 84.925(6) 72.798(6) 70.832(6) 1123.0(2) 2 1.766 6.080 584.0 0.369 × 0.044 × 0.025 Mo Kα (λ = 0.71073) 4.244−53.8

1c

−14 ≤ h ≤ 13, −13 ≤ k ≤ 15, −23 ≤ l ≤ 25 35589 11237 (Rint = 0.0438, Rσ = 0.0506) 11237/0/547 1.015 R1 = 0.0364, wR2 = 0.0794 R1 = 0.0548, wR2 = 0.0896 1.07, −0.59



(II), rhodium(III), osmium(II), and iridium(III) complexes display characteristic η interactions upon coordination to 1benzyl-4-phenyl-1H-1,2,3-triazole (1). Furthermore, we observed novel carbenylation of the DNA model guanosine-5′monophosphate upon incubation of these complexes at 310 K, which provides a fundamental insight into the possible mode of action of the complexes upon interaction with the molecular target DNA. This results from the deprotonation of the triazole proton (H-5) of the representative Nγ∩C complex 1b or 1c by the amine of a guanosine DNA model. Consequently, the generated carbene is capable of inducing further oxidative stress to the cancer cells, which are already under redox stress, due to the high metabolic demand of the proliferating cancer cells. Therefore, carbenylation of the triazolyl rhodium(III) and iridium(III) complexes by a DNA model nucleoside is proposed as a novel mode of interaction. Taking into consideration the fact that mismatched DNA bases (overexpressed in cancer cells) extrude into the groove of the DNA, we believe this mode of action represents a dual diagnostic as well as design strategy in the development of multifaceted anticancer agents.

(Figure S27). In some cases, for example, serum albumin interaction with conjugated metal complexes have been shown to lower their bioactivities due to the stable protein−metal complexes which limit the release as well as alter the activity of the bioactive agents in a pathological environment.21b Recently, Teo et al.20b provided further evidence on the marked variations in the activity of organogold complexes leading to a general decrease in their bioactivities for those complexes which exhibited interactions with human bovine serum albumin. From these experiments, the observed negligible affinities of 1b,c with protein biomolecules imply that they are relatively stable in the presence of proteins and possibly exert their activities through alternative modes of action. While the environment of the cell is complex and rich in biomolecules, a scrambling experiment (“one pot”) involving amino acids and 5′-GMP revealed the selectivity of 1c to DNA model guanosine over amino acids within an experimental time of 24 h (Figure S28).

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CONCLUSIONS In this study novel rhodium(III) and iridium(III) complexes were synthesized and have been shown to confer remarkable cytotoxic activity to the somewhat less active 1-benzyl-4phenyl-1H-1,2,3-triazole (CC50 > 100 μM) against a panel of four representative cancer cell lines, including the MT4 (leukemia, CC50 = 25−32 μM), HeLa (cervical, CC50 = 29− 54 μM), HEK293 (kidney adenocarcinoma, CC50 = 9−15 μM), and A549 (lung cancer, CC50 = 4−6 μM) cell lines. The activities of the two new rhodium(III) and iridium(III) complexes are comparable to that of auranofin (CC50 = 4.69 μM) in lung cancer A549 cell lines. The triazolyl ruthenium-

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EXPERIMENTAL SECTION

Materials, Physical Measurements, and General Procedure. Chemical reagents, including phenylacetylene, propargyl alcohol, sodium azide, 1,2,3,4,5-pentamethylcyclopentadiene, ruthenium trichloride hydrate, rhodium trichloride hydrate, osmium trichloride hydrate, and iridium trichloride hydrate, were purchased from SigmaAldrich Chemical Co. Inc. Analytical grade solvents were obtained from Rochelle Chemical Industries. All of the reagents and solvents were used as received without further purifications unless otherwise J


Article

Organometallics

7.43 (s, 1H (triazolic ArCHf), 1H), 7.32 (m, 3H, benzylArH(a,b)), 7.24 (d, 2Hc, benzylArH), 5.46 (s, 2H, benzylCH2(e)), 4.71 (s, 2H, triazolylCH2(h)), 3.47 (s, 1H, triazolylOHi). 13C{1H} NMR (CDCl3, 125 MHz): δ 148, 134.4, 129.0, 128.7, 128.0, 121.6, 56.2 (Ce), 54.1 (Ch). Synthesis of (1-Benzyl-1H-1,2,3-triazol-4-yl)carboxaldehyde (3). Compound 3 was obtained as a side product in the synthesis of compound 2 in low yield (∼10%) and isolated by flash silica gel column chromatography in 40% ethyl acetate in hexane as a white solid. 1H NMR (400 MHz, CDCl3): δ 10.10 (s, 1H), 7.97 (s, 1H), 7.40−7.25 (m, 6H), 5.57 (s, 2H). 13C{1H} NMR (100 MHz, CDCl3): δ 185.0 (CO), 148.0, 133.3, 129.3, 129.3, 128.5, 128.3, 125.0, 54.5. General Procedure for Synthesis of Triazolyl Cyclometalated 1a−d. To a stirred solution of 1-benzyl-4-phenyl-1H-1,2,3triazole (119 mg, 0.50 mmol, 2.0 equiv) in 4 mL of dry methanol was added anhydrous sodium acetate (46.2 mg, 1.1 equiv) at room temperature (295 K) ,and the solution was degassed with nitrogen and stirred under a nitrogen atmosphere.15a After 1 h, a degassed solution of the metal precursor (0.25 mmol, 1.0 equiv) in dry methanol (5 mL) was added in one portion and stirred until completion of the reaction in 24 h (TLC monitoring of the consumption of 1-benzyl-4-phenyl-1H-1,2,3-triazole). The mixture was filtered, and the solvent was removed by rotary evaporation. Dichloromethane (5 mL) was added to dissolve the complex and the mixture filtered to remove the insoluble salt impurities. Removal of dichloromethane from the filtrate by rotary evaporation afforded the target complex as yellow to yellow-orange solids. The complexes are insoluble in methanol but readily soluble in chlorinated solvents (DCM and CHCl3). Synthesis of 1-Benzyl-4-phenyl-1H-1,2,3-triazolyl(p-cymene)ruthenium(II) Chloride (1a). The conjugated 1-benzyl-4-phenyl-1H1,2,3-triazolyl(p-cymene)ruthenium(II) chloride was obtained by recrystallization of the isomeric mixture of ruthenium complexes in a dichloromethane/diethyl ether solvent mixture as a pure yellow solid in good yield (200 mg, 76% yield): λmax = 413.5 nm. Anal. Calcd for C25H26ClN3Ru: C, 59.46; H, 5.19; N, 8.32; Cl, 7.02; Ru, 20.01. Found: C, 59.43; H, 5.03; N, 8.31. 1H NMR (400 MHz in CDCl3): δ 8.13 (d, 2H, ArH2′′, J = 8.2 Hz), 7.48 (s, 1H, ArH5‑trz), 7.33 (m, 3H, ArH2′′,4′), 7.22 (m, 3H, ArH3′, 5′′), 7.08 (t, 1H, ArH3′′, J = 7.6 Hz), 6.94 (t, 1H, ArH4′′, J = 7.6 Hz), 5.54 (d, 1H, ArHh‑p‑ cym, J = 6.3 Hz), 5.47 (d, 1H, ArHi‑p‑ cym, J = 6.3 Hz), 5.45 (d, 1H, ArHc‑p‑ cym J = 6.3 Hz), 5.42 (d, 1H, CH2, Ha‑1, J = 15 Hz), 5.25 (d, 1H, CH2, Ha‑2, J = 15 Hz), 5.21 (d, 1H, ArHd‑p‑ cym,J = 6.3 Hz), 2.38 (sep, 1H, CHmethine), 2.01 (s, 3H, CH3), 0.87 (dd, 6H, CH3‑isoproyl,J = 28 Hz, 6.8 Hz). 13 C{1H} NMR (100 MHz, CDCl3): δ 176.3, 155.8, 139.5, 135.1, 134.1, 129.0, 128.8, 128.0, 127.6, 122.6, 122.2, 117.1, 99.6, 98.6, 89.1, °C = −250 (c = 1.0 mg/10 mL, 87.4, 85.3, 55.0, 30.9, 22.0, 18.7. [α]30.6 589nm CH2Cl2). Cyclometalated 1a_I and 1a_II were obtained in a one-pot reaction and separated from the isomeric mixture by slow recrystallization from a acetone/methanol solvent mixture (1/1 v/ v). Isomer 1a_I precipitated out easily from this mixture, while 1a_II precipitated out readily in acetone. Data for isomer 1a_I are as follows. 1H NMR (500 MHz, CDCl3): δ 8.17 (d, 1H, ArH, J = 7.5 Hz), 7.53 (s, 1H, ArH5‑trz), 7.41−7.34 (m, 3H, ArH), 7.28−7.22 (m, 3H, ArH), 7.13 (t, 1H, ArH, J = 7.2 Hz), 6.99 (t, 1H, ArH, J = 7.3 Hz), 5.51 (ddd, 4H, ArHp‑cymn, CHAB, J = 31.3 Hz, 28.7 Hz, 10.4 Hz), 5.26 (dd, 2H, ArH, CHAB, J = 17.3 Hz, 10.3 Hz), 2.42 (sep, 1H, CHmethine), 2.05 (s, 3H, CH3(me)), 0.91 (dd, 6H, CH3(i‑pr), J = 35.3 Hz, 6.9 Hz) (Figure S3). 13C{1H} NMR (125 MHz, CDCl3): δ 176.1, 155.4, 139.5, 135.0, 134.2, 129.0, 128.8, 128.0, 127.6, 122.5, 122.2, 117.0, 99.5, 98.7, 88.9, 87.2, 85.3, 83.2, 54.9, 30.7, 22.1, 22.0, 18.6 (Figure S4). Data for isomer 1a_II are as follows. 1H NMR (500 MHz, CDCl3): δ 8.17 (d, 1H, ArH, 3JHH = 7.5 Hz), 7.52 (s, 1H, ArH5‑trz), 7.41−7.36 (m, 3H, ArH), 7.31−7.23 (m, 4H, ArH), 7.14−7.07 (m, 1H, ArH), 6.99 (td, 1H, ArH, 3JHH = 7.4 Hz, 4JHH = 1.0 Hz), 5.58 (d, 1H, ArHp‑ cym, 3JHH = 6.0 Hz), 5.50 (dd, 3H, ArHp‑ cym, CHAB, 2JAB = 15.0 Hz, 3JHH = 7.1 Hz), 5.33 (d, 1H, CHAB, 2JAB = 15.0 Hz), 5.25 (d, 1H, ArHp‑ cym, 3JHH = 5.7 Hz), 2.43 (sep, 1H, CHmethine), 2.06 (s, 3H,

stated. All reactions involving the synthesis of ligands were carried out in air. Manipulations of metal complexes and air-sensitive reagents were performed using either standard Schlenk line techniques under a nitrogen or argon atmosphere or a nitrogen- or argon-filled MBraun glovebox. Dichloromethane for metal complex syntheses was dispensed from a PureSolv solvent purification system. Ethanol and methanol were redistilled and stored over molecular sieves for use in syntheses of metal complexes. Analytical thin-layer chromatography (TLC) was performed using aluminum TLC plates precoated with silica gel 60 F254. Flash column chromatography was performed using 70−230 mesh (63−200 μm) silica gel. 1 H NMR spectra were recorded at 400 MHz (and 500 MHz), 13C and DEPT-135 NMR spectra were recorded at 100 MHz (and 125 MHz based on a 500 MHz 1H NMR spectrometer), and 2D (COSY, 1 H−13C HSQC, 1H−13C HMBC, and 1H−15N HMBC) were recorded at room temperature with Bruker Avance III 400 and 500 MHz FT NMR spectrometers. All chemical shifts (δ) were reported in parts per million (ppm) relative to tetramethylsilane (δ = 0.00). Coupling constants (J values) are given in Hertz, and proton assignments were confirmed with COSY and/or 1H−15N HMBC. Multiplicity is indicated as follows: d = doublet, m = multiplet, and t = triplet. 13C NMR spectral assignments were confirmed using 1H−13C HSQC and 1H−13C HMBC. The spectra were generated in solutions of MeOD-d4 (δH 4.78 (s), 3.30 (s) and δC 49.0), CDCl3-d (δH 7.24 (s) and δC 77.0), or DMSO-d6 (δH 2.45 (s) and δC 39.5). NMR data were acquired using TopSpin pulse sequences supplied from the Bruker NMR spectrometer manufacturer and processed using MestRenova v6.0.2−5475. Specific rotation was recorded using MCP200 polarimeter (Anton Paar). UV-Vis spectra were recorded in DMSO using Shimadzu UV-1800 spectrophotometer. Single-Crystal X-ray Diffraction Studies. In a typical experiment, a suitable crystal was selected and mounted on a “Bruker APEX-II CCD” diffractometer. The crystal was kept at 99.99 K (1a_I), 100.0 K (1b), or 150.0 K (1c) during data collection. Using Olex2,34 the structure was solved with the olex2.solve35 structure solution program using Charge Flipping and refined with the ShelXL36 refinement package using least-squares minimization. The CCDC deposition numbers for 1b,c are 1892482 and 1892492, respectively. Table 2 gives the crystal and structure refinement data. Synthesis of 1-Benzyl-4-phenyl-1H-1,2,3-triazole (1). A mixture of benzyl azide (10 mmol, 1.33 g, 1.0 equiv), phenylacetylene (1.12 mL, 10 mmol, 1.0 equiv), CuSO4·5H2O (50.0 mg, 2 mol %), and ascorbic acid (176 mg, 10 mol %) in 10 mL of distilled water was refluxed at 80 °C. After 4 h, a white precipitate was formed and TLC (40% ethyl acetate in hexane) showed the disappearance of the benzyl azide spot. Precipitation was enhanced by cooling the reaction mixture to room temperature to afford the target product as a white solid. Recrystallization of the product in dichloromethane layered with hexane afforded the target product 1 as a pure white crystalline solid in 80% yield (1.86 g): λmax = 285.5 nm. 1H NMR (400 MHz in CDCl3-d): δ 7.77 (d, 2H, ArH2′, 3J = 7.2 Hz), 7.64 (s, 1H, ArH5‑trz), 7.37 (m, 5H, ArHBn), 7.29 (br.s, 3H, ArH3′/4′), 5.55 (s, 2H, CH2(Bn)). Synthesis of 1-Benzyl-4-hydroxymethyl-1H-1,2,3-triazole (2). Compound 2 was synthesized following a protocol already established in the literature using copper(I)-catalyzed azide−alkyne 1,3-dipolar cycloaddition. Propargyl alcohol (0.2 mL, 3.36 mmoL, 1.1 equiv) was added to a stirred solution of benzyl azide (399 mg, 3.00 mmol, 1.0 equiv) in 5 mL of deionized water/methanol (1/1 v/v) at 50 °C. Subsequently, a solution of copper(II) sulfate pentahydrate (15 mg, 2 mol %, 0.06 mmol) and ascorbic acid (52.8 mg, 10 mol %, 0.3 mmol) in 1 mL of water was added to the mixture dropwise. After 4 h, the reaction was complete, as evidenced by the disappearance of the spot corresponding to benzyl azide starting material. Methanol was removed under rotary evaporation. A 5 mL portion of saturated ammonium chloride was added to the mixture and extracted with 3 × 20 mL of dichloromethane. The combined organic layer was dried over anhydrous MgSO4, filtered off, and concentrated to dryness under a rotary evaporator to give the target 1-benzyl-4-hydroxymethyl-1H-1,2,3-triazole in good yield, 82% (466 mg), as a white crystalline solid: λmax = 268.0 nm. 1H NMR (CDCl3, 500 MHz): δ K


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Organometallics

complex 2b as a pure orange solid in good yield (201 mg, 82%): λmax = 401 nm. Anal. Calcd for C20H25ClN3ORh: C, 52.02; H, 5.46; Cl, 7.68; N, 9.10; O, 3.46; Rh, 22.28. Found: C, 50.5; H, 4.57; N, 8.91. 1 H NMR (400 MHz, CDCl3): δ 7.50 (s, 1H, ArHtrz H‑5), 7.36 (m, 3H, ArH2′, 4′), 7.28 (t, 2H, ArH3′, J = 2.8 Hz, J = 4.4 Hz), 5.49 (s, 2H, CH2(Bn)), 4.67 (s, 2H, CH2(methyloxy), 1.56 (s, 15H, CH3(Cp*)). HRMSESI: calcd m/z for C24H20ClN5O [M −Cl] 426.1053, found [M − °C = 0 (c = 1.0 mg/10 mL, CH2Cl2). Cl]+ 426.1052. [α]27.5 589nm For complex 2a, a complex mixture of side products was obtained and the yield of this conjugated complex 2a, was very low upon preparative TLC isolation. Interaction of the Triazolyl Conjugated Metal Complexes with DNA Model Nucleoside Guanosine-5′-monophosphate (5′-GMP). In order to evaluate the probable interaction of DNA, as the primary molecular target of most organometallic anticancer agents, the reactivities of complexes 1b,c as representative complexes were evaluated by incubating them with an equimolar amount of the nonbuffered aqueous solution of DNA model nucleoside 5′-GMP (10% DMSO-d6 in D2O) at room temperature of 298 K for 72 h. The experiment was repeated at a temperature of 313 K and timedependent 1H NMR and 31P{1H} NMR spectra were recorded at intervals of 12 h for 72 h. NMR Studies on Interaction of the Triazolyl Conjugated Metal Complexes with Bovine Serum Albumin (BSA) as a Protein Model. An aqueous solution of BSA in D2O (pD 7.9) was incubated at 310 K with an aqueous solution of complex 1b or 1c in 10% DMSO-d6 in D2O and monitored at regular 12 h intervals for 72 h by 1H NMR spectroscopy and total correlation spectroscopy (TOCSY, at the beginning and at the end of the experiment). Inspection of the time-dependent 1H NMR spectra of complexes 1b,c and comparative analysis (stacking, using topspin software) of the generated spectra of the complex 1b (or 1c)/BSA mixture were employed in examining the presence or absence of interactions between the complexes and BSA. Hydrolysis of Complex 1c. Into a 0.3 mL DMSO-d6 solution of complex 1c (3.0 mg, 5.0 mmoL, 1.0 equiv) in a 5 mm diameter NMR tube was added 0.1 mL of D2O, and the mixture was incubated at 298 K. Consequently, the proton NMR was recorded after 5 min upon addition of D2O and subsequently at intervals of 2.5 min for a period of 1 h, at which point the only detectable species on the basis of 1H NMR was the aquated species. The relative conversion and/or yield was determined by quantitative monitoring of the relative amount given by the integral of triazole proton H*-5 (with an asterisk *, shown in red in Scheme 4) of the aquated complex 1c to that of

CH3(me)), 0.92 (dd, 6H, CH3(i‑pr), 3JHH = 32.8 Hz, 3JHH = 6.9 Hz) (Figure S5). 13C{1H} NMR (125 MHz, CDCl3): δ 176.2, 155.5, 139.5, 135.0, 134.1, 129.1, 128.9, 128.1, 127.6, 122.6, 122.2, 116.9, 99.5, 98.8, 88.9, 87.3, 85.3, 83.2, 55.0, 30.7, 22.1, 22.0, 18.7 (Figure S6). Synthesis of 1-Benzyl-4-phenyl-1H-1,2,3-triazolyl(pentamethylcyclopentadienyl)rhodium(III) Chloride (1b). The conjugated 1-benzyl-4-phenyl-1H-1,2,3-triazolyl(pentamethylcyclopentaidenyl)rhodium(III) chloride was obtained by recrystallization in chloroform as a yellow solid in good yield (180 mg, 68% yield): λmax = 304.5 nm. Anal. Calcd for C25H27N3ClRh: C, 59.12; H, 5.36; Cl, 6.98; N, 8.27; Rh, 20.26. Found: C, 58.99; H, 5.35; N, 8.25. 1H NMR (400 MHz in CDCl3): δ 7.73 (d, 1H, ArH2‑Ph, J = 7.6 Hz), 7.50 (s, 1H, ArH5′‑trz), 7.3 (d, 2H, ArH2′‑Bn, J = 6.0 Hz), 7.25 (m, 3H, ArH3′,4′‑Bn), 7.21 (d, 1H, ArH5‑Ph, J = 8.4 Hz), 7.13 (t, 1H, ArH3‑Ph, J = 7.4 Hz), 6.96 (t, 1H, ArH4−Ph, J = 7.4 Hz), 5.37 (dd, 2H, CH2(Bn), J = 20 Hz, J = 15 Hz), 1.66 (s, 15H, CH3(cp*)). 13C{1H} NMR (100 MHz, CDCl3): δ 173.7 (d, 1JRhC(trz) = 60 Hz), 155.3, 136.7, 134.7, 134.2, 129.1, 128.8, 128.6, 128.1, 125.7, 122.7, 121.7, °C = +400 (c = 1.0 117.0, 95.7 (d, 1JRhC(Cp*) = 60 Hz), 54.9, 9.1.[α]30.7 589nm mg/10 mL, CH2Cl2). HRMS(+ve): base peak at m/z 472.1261 corresponding to C25H27RhN3 [M − Cl]+ (calcd m/z 472.1260). HRMS(−ve): peak at m/z 542.0665 corresponding to C25H27RhClN3 [M + Cl]− (calcd m/z 542.0637). Synthesis of 1-Benzyl-4-phenyl-1H-1,2,3-triazolyl(pentamethylcyclopentadienyl)iridium(III) Chloride (1c). The conjugated 1-benzyl-4-phenyl-1H-1,2,3-triazolyl(pentamethylcyclopentaidenyl)iridium(III) chloride was obtained by recrystallization in chloroform as a yellow solid in good yield (262 mg, 83% yield): λmax = 297 nm. Anal. Calcd for C25H27N3ClIr: C, 50.28; H, 4.56; N, 7.04, Cl, 5.94; Ir, 32.19. Found: C, 50.26; H, 4.55; N, 7.06. 1H NMR (400 MHz in CDCl3): δ 7.74 (d, 1H, ArH, J = 7.6 Hz), 7.50 (s, 1H, ArH), 7.34 (d, 2H, ArH, J = 1.2 Hz), 7.32 (d, 1H, ArH), 7.26 (d, 3H, ArH, J = 8.0 Hz), 7.08 (t, 1H, ArH, J = 6.4 Hz), 6.93 (t, 1H, ArH, J = 7.2 Hz), 5.37 (dd, 2H, CH2(Bn), J = 33.6 Hz, J = °C = +420 (c 1.0 mg/10 mL, 14.8 Hz), 1.73 (s, 15H, CH3(Cp*)). [α]26.3 589nm CH2Cl2). HRMS(+ve): base peak at m/z 562.1834 C25H27IrN3 [M − Cl]+ (calcd m/z 562.1834). Synthesis of 1-Benzyl-4-phenyl-1H-1,2,3-triazolyl(p-cymene)osmium(II) Chloride (1d). The conjugated 1-benzyl-4-phenyl-1H1,2,3-triazolyl(p-cymene)osmium(II) chloride was obtained by recrystallization in a dichloromethane/diethyl ether solvent mixture as a yellow solid in good yield (208 mg, 66% yield): λmax = 386.5 nm. Anal. Calcd for C25H26ClN3Os: C, 50.54; H, 4.41; Cl, 5.97; N, 7.07; Os, 32.02. Found: C, 50.48; H, 4.27; N, 7.21. 1H NMR (400 MHz, DMSO-d6): δ 8.47 (s, 1H, ArH), 7.91 (d, 1H, ArH, J = 7.2 Hz), 7.35 (m, 6H, ArHBn, H‑3′), 6.89 (t, 1H, ArH, J = 7.2 Hz), 6.86 (t, 1H, ArH, J = 7.2 Hz), 5.74 (s, 2H, CH2), 5.65 (d, 1H, ArH, J = 5.6 Hz), 5.50 (d, 2H, ArH, J = 6 Hz), 5.45 (d, 1H, ArH, J = 5.2 Hz), 2.17 (sep, 1H, °C = CHmethine), 2.00 (s, 3H, CH3), 0.75 (d, 6H, CH3(i‑pr)). [α]29.5 589nm −150 (c 1.0 mg/10 mL, CH2Cl2). HRMS(+ve): base peak at m/z 560.1737 for C25H28N3Os [M − Cl]+ (calcd m/z 560.1868). Synthesis of 1-Benzyl-4-methyloxy-1H-1,2,3-triazolyl Complexes 2a,b. To a stirred solution of 1-benzyl-4-hydroxymethyl-1H1,2,3-triazole (99 mg, 0.52 mmol, 2.0 equiv) in 5 mL of dry methanol was added anhydrous sodium acetate (46 mg, 1.1 equiv) at room temperature (295 K), and the solution was degassed with nitrogen and stirred under a nitrogen atmosphere. After 30 min, a degassed solution of [Ru(p-cymene)Cl2]2 for 2a or [Rh(Cp*)Cl2]2 (160 mg, 0.26 mmol, 1.0 equiv) for 2b in dry methanol (5 mL) was added in one portion and stirred further until completion of the reaction in 12 h (TLC monitoring of the disappearance of 1-benzyl-4-hydroxymethyl-1H-1,2,3-triazole spot). The mixture was filtered, and the solvent was removed from the filtrate under rotary evaporation. Dichloromethane (20 mL) was added to dissolve the conjugated complex in the product mixture and the solution filtered to remove the insoluble salt impurities of acetate as a white residue. Removal of dichloromethane from the filtrate by rotary evaporation afforded the crude complex as an orange solid. Recrystallization in acetone of the crude product gave the target conjugated rhodium(III) chloride

Scheme 4. Aquation of Complex 1c

triazole proton H-5 (shown in blue in Scheme 4) of the chlorinated complex 1c. 1H NMR (400 MHz, DMSO-d6): δ 8.78 (s, 1H), 8.47 (s, 3H), 7.67 (s, 1H), 7.59 (d, J = 7.2 Hz, 4H), 7.48−7.30 (m, 23H), 7.20 (d, J = 3.6 Hz, 2H), 6.99 (t, J = 6.7 Hz, 3H), 6.91 (t, J = 7.0 Hz, 3H), 5.78 (s, 2H), 5.75−5.65 (m, 7H), 1.63 (d, J = 23.2 Hz, 61H). Note: the 1H integral values reported at mean time t ≥ 5 min correspond to both the chloride and aquated complex 1c, showing their relative amounts in the solution. The proton signals corresponding to the aquated complex are downfield relative to those of the chloride complex 1c (e.g., for triazole H-5 proton at t = 5 min, δH (aquated) was 8.78 ppm (25%) while that of δH (chloride) was 8.47 ppm (75%)). Interaction with Amino Acids. In a typical experiment, complex 1c (3 mg, 5.0 × 10 −3 mmol, 1.0 equiv) in 0.4 mL of 50% DMSO-d6 in distilled H2O (2/2 v/v) was added to an aqueous solution of amino acid (1.0 equiv; 0.6 mg of L-cysteine or 0.6 mg of DL (or L)-proline or L


Article

Organometallics 0.78 mg of L-histidine in 0.2 mL of 50% D2O in distilled H2O. The 1H NMR of the reaction mixture was recorded in DMSO-d6 at 298 K (Figures S25−27). Subsequently, the temperature of the reaction mixture was raised to 313 K over 15 min and the 1H NMR recorded at this temperature. The reaction mixture was then incubated at 313 K for 48 h while the 1H NMR spectra were recorded at intervals of 6 h. In a similar fashion, complex 1a was incubated with these amino acids including histidine in order to draw comparison with regard to their respective behaviors in an aqueous environment in the presence of these biomolecules. Scrambling Experiment between Amino Acid and Nucleobases. A scrambling experiment to evaluate the competitive interaction of DNA model guanosine 5′-GMP in the presence of proline or histidine as an amino acid was conducted to evaluate if the observed carbenylation of 1c by 5′-GMP could be observed in the presence of biomolecules, mainly proteins. pKa Determination Experiment of 1c. Into a DMSO solution (0.2 mL) of aquated complex 1c (3.0 mg, 5 μmol, 1.0 equiv) was added 0.1 mL of a stock solution of triethylamine (TEA) in D2O (2.02 mg, 0.4 mL, 20 μmol). The pH of the solution was recorded as soon as the addition was made in a vial and the 1H NMR recorded within a mean time of 5 min upon recording of the pH value of the mixture (ca. 7 min). A scatter plot of the pH versus the resonance shift δ of TEA CH2 with curve fitting, done using Origin8 software, was used to estimate the pKa (inflection point of the curve) of the triazole H-5. From experimental observations, the pKa of the proton is between 8.0 and 10.5 (approximately 9, based on Figure S23), as these notably correspond to the lowest limit (after t = 12 h, when the proton has been completely deprotonated) and maximum limit (at t = 5 min, upon interacting with an equimolar amount of triethylamine). For purposes of comparison, the experiment involving TEA was conducted under the same conditions as that of interaction with the DNA model guanosine, 5′-GMP. Cell Culture and in Vitro Cytotoxicity Studies. The synthesized triazolyl conjugated cyclometalates were submitted to ADC Mintek, Johannesburg, South Africa, for cell culture and cytotoxicity assays against a panel of four cell lines: namely, HeLa for cervical cancer, MT4 for leukemia, HEK293 for kidney adenocarcinoma, and A549 for lung cancer. A colorimetric MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) in vitro cytotoxicity assay was conducted to determine the change in cell viability, by measuring the absorbance of the treated cells relative to the untreated cells. The MTS compound (yellow) is metabolized by viable cells to form a dark blue-purple compound, formazan, visible through UV−vis spectroscopy at 490 nm. The absorbance is directly proportional to the cell viability. Briefly, the HeLa, MT4, A549, and HEK293 cells were grown using standard tissue culture techniques. The cells were seeded at 1 × 105 cells/mL (for HEK293 and A549) and 4 × 105 cells/mL (for HeLa and MT4) and incubated in 96-well microtiter plates at 37 °C overnight, with the subsequent addition of the triazole derivatives at six 2-fold dilutions of 100, 50, 25, 12.5, 6.25, 3.125, and 0 μM. The cells were incubated for 4 days, whereupon MTS (5 μL) was added to each well. The absorbance values were measured at 490 nm for three independent measurements each in duplicate after 1, 2, and 4 h incubation periods, averaged and recorded as mean ± standard error of mean for n = 6. From the data, dose−response cell viability curves for the various cell lines were plotted and the concentrations that induced 50% decrease in cell viabilities (CC50) for the respective compounds were determined for each cell line. Cellular Uptake of Organometallic Drugs by ICP-MS. HEK 293 cell lines were seeded at a density of 1 × 105 cells/mL and incubated in a 96-well plate at 37 °C overnight for 24 h. Subsequently, the triazole-based organometallic drugs (1a−d and 2b) were added in triplicate each at 50 μM/well and the cells incubated further at 37 °C. After 48 h, the culture medium was removed and the cells were washed three times with cold phosphate buffered saline (PBS). Thereafter, the cells were harvested by trypsinization and cold PBS added to completely remove the cells from the wells. Then, the suspensions were centrifuged at 800g for 5 min at 4 °C and the

supernatant removed. Ice-cold cell lysis buffer was added to the pellets, which were resuspended to form homogeneous cell suspensions and then incubated in ice. After 30 min, the cells were centrifuged at 800g for 5 min at 4 °C and the supernatant was transferred and stored in clean centrifuge vials. For inductively coupled plasma mass spectrometry (ICP-MS) analysis, the cell lysates were transferred into clean 20 mL glass vials containing 2 mL of 65% HNO3 and heated at 80 °C for 2 h. When it was cooled to room temperature, the colorless mixture was filtered off through a 0.45 μm filter and the filtrate spiked with gallium-69 (50 μg/L) as an internal standard.37 103Rh, 102Ru, 193Ir, and 192Os metals in the cells were qualified and quantified by ICP-MS using an ICPMS-2030 instrument equipped with an As-10 autosampler and micromix nebulizer, with the instrument settings optimized for maximum sensitivity to each of these metals. The amount of each metal in the cell was estimated by a calibration-curve method, processed using LabSolutions ICP-MS version 1.02 software, and the results are reported as mean ± standard deviation (n = 3). Statistical Analysis. The results were recorded as mean ± SEM. A two-paired t test was used to analyze the mean difference between two groups while one-way ANOVA with Bonferroni posthoc t test correction was used in the analysis of multiple comparisons. The criterion for statistical significance was set at p < 0.05.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.9b00440. 1

H NMR, 13C{1H} NMR, HSQC, 1H−13C HMBC, and HRMS-ESI spectra (PDF) Accession Codes

CCDC 1892482, 1892492, and 1895247 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ data_request/cif, or by emailing [email protected]. uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*E-mail for B.C.E.M.: [email protected]. ORCID

Banothile C. E. Makhubela: 0000-0002-2292-7400 Author Contributions

The manuscript was written through contributions of all authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the TWAS in partnership with the South African National Research Foundation (Grant Nos. 99992, 95517, and 99269) for financial support. We greatly acknowledge the assistance offered by Ms. Hendriëtte van der Walt (Nanotechnology Innovation Centre (NIC), ADC DST/ Mintek) in conducting cytotoxicity assays. We are also grateful to Mr. Mutshinyalo Nwamadi (NMR facility-Spectrau, UJ) for NMR technical assistance, Dr. Maritjie Stander (CAF, Stellenbosch University) for HRMS analysis, and Dr. Edwin Madala (Department of Biochemistry, UJ) for generously M


Article

Organometallics

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providing bovine serum albumin for performing protein interaction studies.

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ABBREVIATIONS TLC, thin layer chromatography; HRMS-ESI, high-resolution mass spectrometry−electron spray ionization technique; NMR, nuclear magnetic resonance; HSQC, homonuclear single quantum correlation; HMBC, heteronuclear multiple bond correlation; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlational spectroscopy; DNA, DNA; CC50, cytotoxic concentration inducing 50% decrease in cell viability of the maximal response; 1,3-DC, 1,3-dipolar cycloaddition; 5′GMP, guanosine-5′-monophosphate; BSA, bovine serum albumin

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REFERENCES

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