Microwave assisted synthesis of rhodium(Ⅰ) N-heterocyclic carbene complexes and their cytotoxicity against tumor cell lines

{"full_text": " Journal of Organometallic Chemistry 964 (2022) 122300\n\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Organometallic Chemistry\n journal homepage: www.elsevier.com/locate/jorganchem\n\n\n\n\nMicrowave assisted synthesis of rhodium(\u2160) N-heterocyclic carbene\ncomplexes and their cytotoxicity against tumor cell lines\nJanina Schmidt, Jessica W\u00f6lker, Petra Lippmann, Ingo Ott\u2217\nInstitute of Medicinal and Pharmaceutical Chemistry, Technische Universit\u00e4t Braunschweig, Beethovenstr. 55, 38106 Braunschweig, Germany\n\n\n\n\na r t i c l e i n f o a b s t r a c t\n\nArticle history: Organometallic rhodium(\u2160) complexes of the general type [(COD)(NHC)RhCl] (where COD = 1,5-\nReceived 14 January 2022 cyclooctadiene, NHC = N-heterocyclic carbene) were prepared according to a two-step transmetalation\nRevised 18 February 2022\n method via a silver carbene intermediate. The procedure was supported by microwave irradiation allow-\nAccepted 18 February 2022\n ing reduced reaction periods. The complexes were evaluated for cytotoxic effects in selected cancer cell\nAvailable online 22 February 2022\n lines, namely HT-29 colon adenocarcinoma, MDA-MB-231 breast carcinoma and MCF-7 breast adenocar-\nKeywords: cinoma, and generally triggered strong cytotoxic effects with IC50 values in the low micromolar range.\nBioorganometallic chemistry Evaluation of structure-activity relationships clearly indicated a preference for branched isopropyl side\nCarbene chains at the nitrogen atoms of the NHC ligands versus non-branched ethyl side chains. Further studies\nCytotoxicity on selected examples in HT-29 cells indicated that this could be the result of a higher cellular uptake.\nRhodium \u00a9 2022 Elsevier B.V. All rights reserved.\n\n\n\n\n1. Introduction plexes with COD ligands had been described soon after the discov-\n ery of the antitumor activity of cisplatin [26,27]. Rh(\u2160)-based poly-\n Cisplatin and other square-planar platinum species have been meric nanomicelles have also demonstrated in-vivo anticancer ac-\nplaying a major role in clinical cancer chemotherapy over many tivity [28]. In particular very few studies on [(COD)(NHC)RhX] an-\ndecades and have shaped the \ufb01eld of inorganic medicinal chem- ticancer complexes are available, however, with very promising re-\nistry. Motivated by the tremendous success of the platinum anti- sults [2\u201314].\ncancer drugs, a considerable number of different metal-based po- Many of the investigated complexes show appreciable cyto-\ntential anticancer agents has been designed and studied for appli- toxicity against cultured tumor cell lines and the overcoming of\ncation in tumor therapy, however, without reaching the success of drug resistance in leukemia cells was reported for a selected com-\nthe lead compound cisplatin [1]. Interestingly, among the numer- plex [2]. Studies on the effects on the cellular signalling in can-\nous investigated metallodrug candidates there are comparably few cer cell lines revealed strong phosphorylation (activation) of sev-\nnon-platinum examples that exhibit a cisplatin-like square-planar eral relevant proteins, including the heat show protein 27 (HSP27)\ngeometry at the metal center. and mitogen-activated protein kinases (MAPKs), such as p38 and\n We and others have recently reported on the antitumor po- ERK1/2, indicative of a general cellular stress response [3]. Impor-\ntential of square-planar organometallic rhodium(\u2160) complexes of tantly, such biological response pattern was different from that ob-\nthe type [(COD)(NHC)RhX] (where COD = 1,5-cyclooctadiene, served with cisplatin. An other example showed good antitumor\nNHC = N-heterocyclic carbene and X = halide, see Fig. 1 for some activity in-vivo [12].\nselected examples) [2\u201314]. Based on the square-planar geometry and the fact that Rh(\u2160) is\n Rhodium complexes in general have been less frequently stud- isoelectronic with Pt(\u2161), the DNA has been considered as a rele-\nied as anticancer drugs and the majority of the recently investi- vant molecular target for [(COD)(NHC)RhX] complexes. In fact, one\ngated types are Rh(\u2162) complexes [15\u201325]. complex was shown to halt DNA replication and alter cell migra-\n If compared with other metal complexes, the number of reports tion [4]. An other example showed a higher amount of binding to\non rhodium(\u2160) anticancer agents is still relatively low, although the DNA than cisplatin in precipitation experiments with isolated\nthe antitumor potential of square-planar organometallic Rh(\u2160) com- DNA, however, cellular uptake experiments indicated that only a\n very small percentage of the complex reached the nuclei of cancer\n cells [2]. For complexes that contained a DNA intercalating naph-\n \u2217\n Corresponding author.\n thalimide structure in the NHC ligand, both intercalative interac-\n E-mail address: ingo.ott@tu-bs.de (I. Ott). tion with the DNA (related to the naphthalimide moiety) and co-\n\n\nhttps://doi.org/10.1016/j.jorganchem.2022.122300\n0022-328X/\u00a9 2022 Elsevier B.V. All rights reserved.\n\fJ. Schmidt, J. W\u00f6lker, P. Lippmann et al. Journal of Organometallic Chemistry 964 (2022) 122300\n\n\n\n\n Fig. 1. Examples of previously studied [(COD)(NHC)RhCl] complexes [2,9,10].\n\n\n\nordinative binding (under loss of the chlorido ligand) to the model 2. Experimental\nnucleoside base 9-ethylguanine were con\ufb01rmed [7,8].\n Besides DNA, also protein targets have been considered for 2.1. General\n[(COD)(NHC)RhX] complexes [29]. Protein crystallography studies\nof selected complexes with hen-egg-white lysozyme as a model Chemicals and reagents were obtained from Sigma\u2013Aldrich, TCI,\nprotein showed major adducts at a histidine residue. Interestingly, Alfa Aesar and ACROS unless otherwise noted. NMR spectra were\noxidation of Rh(\u2160) to Rh(\u2162) was noted changing the geometry of recorded on a Bruker DRX-400 AS or an AV III HD 500 NMR spec-\nthe rhodium center from square-planar to octahedral [10]. Binding trometer. Positive-ion ESI (electrospray ionization) mass spectra\nto histidine was also noted for a Rh(\u2162) complex with a NHC and were recorded on a Finnigan MAT95 XL. Elemental analyses were\na cyclopentadienyl ligand [30]. In this case the NHC ligand was re- conducted in a Flash EA1112 apparatus. A VictorTM X4 Perkin\u2013\nplaced upon binding to the protein. For similar iridium complexes Elmer 2030 multilabel reader was used for the biological assays.\nof the type [Ir(NHC)(COD)Cl] a ligand exchange mechanism was Cell culture: MCF-7, MDA-MB-231, HT-29, Caco-2 and Calu-3 cells\nproposed, which consisted of an initial loss of the COD and chlo- were obtained from DSMZ (Braunschweig) and were maintained in\nride ligands and, depending on the conditions, further reaction of Dulbecco\u2019s modi\ufb01ed Eagle\u2019s medium (DMEM; 4.5 g/L \u1d05-glucose, l-\nthe remaining fragment with biomolecules or decomposition [31]. glutamine, pyruvate), supplemented with fetal bovine serum su-\nNotably, oxidation of the metal upon interaction with proteins has perior, standardized (Bio&Sell, 10% v/v) and gentamycin (50 mg/L)\nalso been con\ufb01rmed for such iridium complexes and they generally with a weekly passage.\ndisplayed very promising in-vitro anticancer activity [32\u201334].\n Moderate inhibition of the selenoenzyme thioredoxin reductase\n 2.2. General synthesis of the phenylimidazolium halides (2a\u20132h)\n(TrxR) was reported for some examples of [(COD)(NHC)RhX] com-\nplexes [2,9]. Such TrxR inhibition might indirectly contribute to\n An amount of 0.20 g of the respective phenylimidazole 1a - 1d\nthe anticancer mechanism as the resulting upregulation of reactive\n and 1.0 equivalent of K2 CO3 were added to 2.0 mL of acetonitrile, 3\noxygene species formation contributes to DNA damage [9].\n equivalents of ethyl iodiode (for 2a \u2013 2d) or 2-bromopropane (for\n [(COD)(NHC)RhX] complexes also showed good activity against\n 2e \u2013 2h), respectively, were added, and the mixture was reacted\nthe SARS-CoV-2 papain-like protease PLpro , however, due to their\n for 8-54 h in a microwave at 82\u00b0C, 50 W and 10 psi. The progress\ntoo strong cytotoxicity against host cells, they did not proceed to\n of the reaction was monitored by thin layer chromatography. The\nfurther antiviral studies [35].\n liquid phase was removed, the solid phase washed with methanol,\n Regarding the cellular localisation, besides the expected mito-\n and the volume of the combined liquid phases was reduced us-\nchondrial uptake, accumulation in the endoplasmic reticulum (ER)\n ing a rotary evaporator. Further work-up for 2a \u2013 2d: The residue\nwas reported by Metzler-Nolte et al. [11] calling for further inves-\n was dissolved in dichloromethane and \ufb01ltered using a syringe \ufb01lter\ntigation of possible induction of ER stress by the complexes.\n (0.2 \u03bcM) to remove the remaining K2 CO3 . Dichloromethane was re-\n Finally, the potential of [(COD)(NHC)RhX] complexes as\n moved by evaporation under reduced pressure resulting in an oily\nbioorganometallic catalysts, e.g. for hydrosilylation, should be\n residue with some precipitate. A small amount of acetonitrile was\nmentioned [36\u201338].\n added for dissolution and the product was precipitated with 10 mL\n Taken together, the ongoing studies on the mechanisms of\n of 1/1 ethylacetate/n-hexane (V/V) and repeatedly cooled for 24 h\n[(COD)(NHC)RhX] complexes indicate that such complexes have\n at -25\u00b0C until no further precipitate was obtained. The combined\nmultitarget anticancer properties that can be \ufb01ne-tuned by care-\n precipitates were dried using a rotary evaporator followed by dry-\nful optimization of the coordinated ligands, in particular the\n ing under reduced pressure at 40\u00b0C for a period of 3 days. Further\nNHC partial structures. Further studies involving different NHC\n work-up for 2e \u2013 2h: The products were puri\ufb01ed by column chro-\ncore structures and elucidation of structure-activity-relationships\n matography over silica with ethylacetate/n-hexane/methanol mix-\nshould therefore have a high priority in the further development\n tures (8/2/0 \u2192 8/2/1 \u2192 8/2/10) as eluent. The volume of the com-\nof the complexes.\n bined eluates was reduced with a rotary evaporator, \ufb01ltered using\n In the here reported work we introduced phenylimidazole de-\n a syringe \ufb01lter (0.2 \u03bcm), and dried under reduced pressure at 40\u00b0C\nrived NHC ligand structures, for which we had reported very\n for 3 days.\npromising results as ligands of bioactive gold complexes in recent\n 1,3-Diethyl-(4-phenyl)-1H-imidazol-3-iumiodid (2a) was al-\nstudies [39], into complexes of the general type [(COD)(NHC)RhCl]\n ready described in the literature [39].\naiming at establishing more structure-activity-relationships regard-\n 1,3-Diethyl-4-(4\u2019-chlorophenyl)-1H-imidazol-3-iumiodid (2b)\ning the NHC ligands. Thus, the in\ufb02uence of different halide sub-\n was already described in the literature [39].\nstituents on the para-position of the phenyl ring as well as the side\n 1,3-Diethyl-4-(4\u2019-chlorophenyl)-1H-imidazol-3-iumiodide (2c)\nchains (non-branched: ethyl, branched: isopropyl) on the NHC ni-\n general method with 1c, reaction period: 28 h, precipitate with\ntrogen atoms were evaluated. The resulting eight target complexes\n EtOAc, brown solid, yield: 35%\nwere investigated for cytotoxic effects in a panel of cancer lines. In 1 H NMR (400 MHz, CDCl ) \u03b4 10.33 (d, 4 J\n 3 H,H = 1.8 Hz, 1H, CH,\naddition to that, we have evaluated the application of microwave\n C2), 7.51 (m, 2H, 2CH, C6\u2019 + C2\u2019), 7.42 (m, 2H, 2CH, C3\u2019 + C5\u2019), 7.37\ntechnology in the synthesis of the target complexes.\n (d, 3 JH,H = 1.8 Hz, 1H, CH, C5), 4.50 (q, 3 JH,H = 7.4 Hz, 2H, CH2 ,\n\n 2\n\fJ. Schmidt, J. W\u00f6lker, P. Lippmann et al. Journal of Organometallic Chemistry 964 (2022) 122300\n\n\nC8), 4.29 (q, 3 JH,H = 7.4 Hz, 2H, CH2 , C6), 1.66 (t, 3 JH,H = 7.4 Hz, C9), 4.49 (hept, 3 JH,H = 6.7 Hz, 1H, CH, C6), 1.67 (m, 12H, 4CH3 ,\n3H, CH3 , C9), 1.51 (t, 3 JH,H = 7.3 Hz, 3H, CH3 , C7) ppm; 13 C NMR C7 + C8 + C10 + C11) ppm; 13 C NMR (121 MHz, CDCl3 ) \u03b4 135.65\n(101 MHz, CDCl3 ) \u03b4 137.34 (s, CH, C2), 136.77 (s, Cq , C4\u2019), 134.09 (s, CH, C2), 134.01 (d, 1 JBr,C = 3.22 Hz, Cq , C4\u2019), 133.88 (s, Cq , C4)\n(s, Cq , C4) 130.98, (s, Cq , C1\u2019), 129.87 (s, 2CH, C5\u2019 + C3\u2019), 123.28 132.86 (s, 2CH, C3\u2019 + C5\u2019), 132.63 (s, 2CH, C2\u2019 + C6\u2019), 124.06 (s, Cq ,\n(s, 2CH, C2\u2019 + C6\u2019), 119.37 (s, CH, C5), 45.72 (s, CH2 , C6), 43.49 (s, C1\u2019), 116.82 (s, CH, C5), 53.75 (s, CH, C6), 51.56 (s, CH, C9), 23.60 (s,\nCH2 , C8), 15.76 (d, 3 JC,C = 21.97 Hz, 2CH3 , C7 + C9) ppm; elemental 2CH3 , C7 + C8), 23.34 (s, 2CH3 , C10 + C11) ppm; elemental analy-\nanalysis: C13 H16 ClIN2 (calc./found [%]): C (43.06 / 43.02), H (4.45 / sis: C15 H20 Br2 N2 (calc./found [%]): C (46.42 / 39.99), H (5.19 / 4.28),\n4.60), N (7.73 / 7.56); MS (EI): m/z =235.10 [M-Br]+ N (7.22 / 7.54); MS (EI): m/z = 307.08 [M-Br]+\n 1,3-Diethyl-4-(4\u2019-bromophenyl)-1H-imidazol-3-iumiodide (2d)\nwas already described in the literature [39] 2.3. General synthesis of (COD)(NHC)RhCl complexes (3a\u20133h)\n 1,3-Diisopropyl-(4-phenyl)-1H-imidazol-3-iumbromid (2e)\ngeneral method with 1a, reaction period: 54 h, eluent for column An amount of 0.10 g (2 equivalents) of the respective\nchromatography: EtOAc:n-hexane:MeOH (8/2/1 \u2192 8/2/100), yellow phenylimidazolium precursor (2a \u2013 2h) was dissolved in 2.0 mL\npowder, yield: 11% dichloromethane, 1 equivalent of silver(I)oxide were added and the\n 1 H NMR (400 MHz, CDCl ): \u03b4 11.05 (d, 4 J\n 3 H,H = 1.7 Hz, 1H, C2), mixture was stirred for 15 min at room temperature. For trans-\n7.61 \u2013 7.52 (m, 3H, C4\u2019 + C3\u2019 + C5\u2019), 7.41 \u2013 7.38 (m, 2H, C2\u2019 + C6\u2019), metalation 1 equivalent of [Rh(COD)Cl]2 was added and reacted\n7.23 (d, 4 JH,H = 1.4 Hz, 1H, C5), 5.24 (hept, 3 JH,H =6.7 Hz, 1H, C9), for 3-5 h (see below) at 25\u00b0C, 10 psi and 50 W in a microwave\n4.52 (hept, 3 J H,H = 6.7 Hz, 1H, C6), 1.69 (d, 3 J H,H = 6.7 Hz, 12H, apparatus (CEM). The formed silver halide was removed by \ufb01ltra-\nC11 + C10) ppm; 13 C NMR (101 MHz, CDCl3 ): \u03b4 136.10 (s, CH, C2), tion and the product was puri\ufb01ed chromatography over silica with\n134.89 (s, Cq , C4), 130.86 (s, CH, C4\u2019), 129.64 (d, 3 Jc,c = 23,55 Hz, dichloromethane / methanol mixtures as eluent (1/0 \u2192 1/0.5). The\n4 CH, C6\u2019 + C2\u2019 + C3\u2019 + C5\u2019), 125.24 (s, Cq , C1\u2019), 116.12 (s, CH, C5), volume of the combined eluted pure fractions was reduced using\n53.47 (s, CH, C6), 51.44 (s, CH, C9), 23.63 (d, 2 Jc,c = 23,45 Hz, 4 a rotary evaporator, \ufb01ltered using a 0.2 \u03bcm syringe \ufb01lter, treated\nCH3 , C7 + C8 + C10 + C11) ppm; elemental analysis: C15 H21 BrN2 with n-hexane, and dried for 3 days at 40\u00b0C under reduced pres-\n(calc./found [%]): C (58.26 / 58.59), H (6.94 / 6.84), N (8.73 / 9.06); sure to yield the crystalline product.\nMS (EI): m/z =219.13 [M-Br+H]+ Chlorido[1,3-diethyl-(4-phenyl)-imidazol-2-ylidene(cycloocta-\n 1,3-Diisopropyl-4-(4\u2019-\ufb02uorophenyl)-1H-imidazol-3-iumbromid 1,5-diene)]rhodium(\u2160) (3a)\n(2f) general method with 2a; reaction period: 3 h, yellow solid, yield:\ngeneral method with 1b, reaction period: 38 h, white powder, 87%. Complex 3a was already reported in the literature [35].\nyield: 34% Chlorido[1,3-diethyl-4-(4\u2019-\ufb02uorophenyl)-imidazol-2-\n 1 H NMR (500 MHz, DMSO-d ) \u03b4 9.55 (d, 4 J ylidene(cycloocta-1,5-diene)]rhodium(\u2160) (3b)\n 6 H,H = 1.8 Hz, 1H,\nC2), 8.09 (d, 4 JH,H = 1.8 Hz, 1H, C5), 7.64 (m, 2H, C2\u2019 + C6\u2019), 7.45 general method with 2b, reaction period: 4 h, yellow powder,\n(m, 2H, C3\u2019 + C5\u2019), 4.69 (hept, 3 JH,H = 6.8 Hz, 1H, C9), 4.48 (hept, yield: 77%. Complex 3b was already reported in the literature [35].\n3J 3\n H,H = 6.8 Hz, 1H, C6), 1.55 (d, JH,H = 6.7 Hz, 6H, C10 + C11), Chlorido[1,3-diethyl-4-(4\u2019-chlorophenyl)-imidazol-2-\n1.45 (d, 3 JH,H = 6.7 Hz, 6H, C7 + C8) ppm; 13 C NMR (126 MHz, ylidene(cycloocta-1,5-diene)]rhodium(\u2160) (3c)\nDMSO-d6 ) \u03b4 163.01 (d, 1 JC,F = 248.1 Hz, Cq , C4\u2019), 133,43 (s, CH, C2), general method with 2c, reaction period: 5 h; yellow powder;\n132.75 (s, Cq , C4), 132.27 (d, 3 JC,F = 8.8 Hz, 2CH, C2\u2019 + C6\u2019), 122.13 yield: 46%\n 1 H NMR (400 MHz, CDCl ) \u03b4 7.43 \u2013 7.39 (m, 2H, 2CH, C2\u2019 + C6\u2019),\n(d, 4 JC,F = 3.2 Hz, Cq , C1\u2019), 118.70 (s, CH, C5), 116.26 (d, 3 JC,F = 22.0 3\nHz, 2CH, C3\u2019 + C5\u2019), 52.67 (s, CH, C6), 50.08 (s, CH, C9), 39.42 (dp, 7.28 \u2013 7.24 (m, 2H, 2 CH, C3\u2019 + C5\u2019), 6.81 (s, 1H, CH, C5), 5.09\n2J 3\n C,C = 42.0, JC,C = 21.0 Hz, 4CH3 , C7 + C8 + C10 + C11) ppm; \u2013 4.99 (m, 2H, 2 CH, 2 trans-CH-COD), 4.85 (dq, 2 JH,H = 13.8 Hz,\n19 F NMR (471 MHz, DMSO-d ) \u03b4 -110.06 (tt, 3 J 3J 2 3\n H,H = 7.3 Hz, 1H, CH2 , C8), 4.72 (dq, JH,H = 13.4 Hz, JH,H = 7.4\n 4\n 6 F,H = 8.9 Hz, JF,H\n= 5.4 Hz) ppm; elemental analysis: C15 H20 BrFN2 (calc./found [%]): Hz, 1H, CH2 , C6), 4.58 (dq, 2 JH,H = 14.67 Hz, 3 JH,H = 7.29 Hz, 1H,\nC (55.06 / 55.69), H (6.16 / 6.23), N (8.56 / 8.64); MS (EI): m/z CH2 , C6), 4.50 (dq, 2 JH,H = 13.8 Hz, 3 JH,H = 7.1 Hz, 1H, CH2 , C8),\n=246.18 [M-Br+H]+ 3.36 (m, 2H, 2 CH, 2 cis-CH-COD), 2.52 \u2013 2.33 (m, 4H, 2 CH2 axial,\n 1,3-Diisopropyl-4-(4\u2019-chlorophenyl)-1H-imidazol-3-iumiodide COD), 1.96 (m, 4H, 2 CH2 equatorial, COD), 1.56 (t, 3 JH,H = 7.3 Hz,\n(2g) 3H, CH3 , C9), 1.27 (t, 3 JH,H = 7.2 Hz, 3H, CH3 , C7) ppm; 13 C NMR\ngeneral method with 1c, reaction period: 25 h, white powder, (101 MHz, CDCl3 ) \u03b4 183.42 (d, 1 JRh,C = 51.02 Hz, Cq , C2), 134.97 (s,\nyield: 39% Cq , C4\u2019), 133.54 (s, Cq , C1\u2019), 130.02 (s, 2 CH, C2\u2019 + C6\u2019), 129.12 (s,\n 1 H NMR (500 MHz, CDCl ) \u03b4 11.02 (s, 1H, CH, C2), 7.54 (d, 2 CH, C3\u2019 + C5\u2019), 127.57 (s, Cq , C4\u2019), 118.45 (s, CH, C5), 98.45 (dd,\n 3\n 1J 2\n Rh,C = 6.84 Hz, JRh,C = 4.13 Hz, 2 CH, 2 trans-CH-COD), 68.33\n4J 4\n H,H = 7.9 Hz, 2H, 2CH, C2\u2019 + C6\u2019), 7.37 (d, JH,H = 8.1 Hz, 2H, 2CH,\nC3\u2019 + C5\u2019), 7.32 (s, 1H, CH, C5), 5.23 (hept, 3 JH,H = 6.7 Hz, 1H, CH, (d, JRh,C = 14.94 Hz, CH, cis-CH-COD), 67.99 (d, 1 JRh,C = 14.86 Hz,\n 1\n\nC9), 4.49 (hept, 3 JH,H = 6.7 Hz, 1H, CH, C6), 1.68 (dd, 3 JH,H = 6.3 CH, cis-CH-COD), 45.90 (s, CH2 , COD), 43.92 (s, CH2 , COD), 33.13 (s,\nHz, 12H, 4CH3 , C7 + C8 + C10 + C11) ppm; 13 C NMR (126 MHz, CH2 , COD), 32.77 (s, CH2 , COD), 29.04 (s, CH2 , C6), 28.72 (s, CH2 ,\nCDCl3 ) \u03b4 137.34 (s, CH, C2), 136.19 (s, Cq , C4\u2019), 133.64 (s, Cq , C4) C8), 16.33 (s, CH3 , C7), 16.19 (s, CH3 , C9) ppm; elemental analysis:\n131.12 (s, 2CH, C3\u2019 + C5\u2019), 129.88 (s, 2CH, C2\u2019 + C6\u2019), 123.66 (s, Cq , C21 H27 Cl2 N2 Rh (calc. / found [%]): C (52.41 / 52.90), H (5.66 / 5.74),\nC1\u2019), 116.69 (s, CH, C5), 53.60 (s, CH, C6), 51.53 (s, CH, C9), 23.68 N (5.82 / 5.54); MS (EI): m/z = 479.9 [M]+ , 444.0 [M-Cl]+\n(s, 2CH3 , C7 + C8), 23.42 (s, 2CH3 , C10 + C11) ppm; elemental Chlorido[1,3-diethyl-4-(4\u2019-bromophenyl)-imidazol-2-\nanalysis: C15 H20 BrClN2 (calc./found [%]): C (52.42 / 51.64), H (5.87 ylidene(cycloocta-1,5-diene)]rhodium(\u2160) (3d)\n/ 5.81), N (8.15 / 7.83); MS (EI): m/z =164.65 [M-Br]+ general method with 2d, reaction period: 5 h; yellow powder,\n 1,3-Diisopropyl-4-(4\u2019-bromophenyl)-1H-imidazol-3-iumbromide yield: 65%\n 1 H NMR (500 MHz, CDCl ) \u03b4 = 7.58 \u2013 7.55 (m, 2H, 2 CH,\n(2h) 3\ngeneral method with 1d, reaction period: 39 h, white powder, C2\u2019 + C6\u2019), 7.22 \u2013 7.19 (m, 2H, 2 CH, C3\u2019 + C5\u2019), 6.81 (s, 1H, CH, C5),\nyield: 66% 5.08 \u2013 5.00 (m, 2H, 2 CH, 2 trans-CH-COD), 4.84 (dq, 2 JH,H = 14.8\n 1 H NMR (500 MHz, CDCl ) \u03b4 10.70 (s, 1H, CH, C2), 7.81 (m, Hz, 3 JH,H = 6.78 Hz, 1H, CH2 , C8), 4.71 (dq, 2 JH,H = 13.4 Hz, 3 JH,H\n 3\n2H, 2CH, C2\u2019 + C6\u2019), 7.69 (d, 4 JH,H = 8.2 Hz, 1H, CH, C5), 7.58 = 7.4 Hz, 1H, CH2 , C6), 4.58 (dq, 2 JH,H = 14.8 Hz, 3 JH,H = 6.78 Hz,\n(m, 2H, 2CH, C3\u2019 + C5\u2019), 4.95 (hept, 3 JH,H = 6.7 Hz, 1H, CH, 1H, CH2 , C6), 4.51 (dq, 2 JH,H = 14.11 Hz, 3 JH,H = 7.23 Hz, 1H, CH2 ,\n C8), 3.41 \u2013 3.30 (m, 2H, 2 CH, 2 cis-CH-COD), 2.50 \u2013 2.33 (m, 4H,\n\n 3\n\fJ. Schmidt, J. W\u00f6lker, P. Lippmann et al. Journal of Organometallic Chemistry 964 (2022) 122300\n\n\n2 CH2 axial, COD), 2.04 \u2013 1.89 (m, 4H, 2 CH2 equatorial, COD), 1.55 C5), 5.96 (hept, 3 JH,H = 7.0 Hz, 1H, CH, C9), 5.88 (hept, 3 JH,H = 7.1\n(t, 3 JH,H = 7.3 Hz, 3H, CH3 , C9), 1.27 (t, 3 JH,H = 7.2 Hz, 3H, CH3 , Hz, 1H, CH, C6), 5.04 \u2013 4.98 (m, 2H, 2 CH, 2 trans-CH-COD), 3.45\nC7) ppm; 13 C NMR (126 MHz, CDCl3 ) \u03b4 = 183.46 (d, 1 JRh,C = 51.09 \u2013 3.38 (m, 2H, 2 CH, 2 cis-CH-COD), 2.49 \u2013 2.33 (m, 4H, 2 CH2\nHz, Cq , C2), 133.54 (s, Cq , C4), 132.08 (s, 2 CH, C2\u2019 + C6\u2019), 130.24 axial, COD), 2.01 \u2013 1.90 (m, 4H, 2 CH2 equatorial, COD), 1.52 (dd,\n(s, 2 CH, C3\u2019 + C5\u2019), 128.02 (s, Cq , C1\u2019), 123.11 (s, Cq , C4\u2019), 118.42 3J 4\n H,H = 6.8 Hz, JH,H = 0.8 Hz, 6H, 2 CH3 , C10 + C11), 1.42 (dd,\n(s, CH, C5), 98.45 (dd, 1 JRh,C = 6.9 Hz, 2 JRh,C = 5.0 Hz, 2 CH, 2 3J 4\n H,H = 7.1 Hz, JH,H = 4.3 Hz, 6H, 2CH3 , C7 + C8) ppm;\n 13 C NMR\n\ntrans-CH-COD), 68.33 (d, 1 JRh,C = 14.6 Hz, CH, cis-CH-COD), 68.01 (126 MHz, CDCl3 ) \u03b4 = 181.45 (d, 1 JRh,C = 50.64 Hz, Cq , C2), 135.34\n(d, 1 JRh,C = 14.6 Hz, CH, cis-CH-COD), 45.89 (s, CH2 , COD), 43.93 (s, (s, Cq , C4), 132.94 (s, Cq , C4\u2019), 132.68 (s, 2 CH, C2\u2019 + C6\u2019), 128.76\nCH2 , COD), 33.12 (s, CH2 , COD), 32.76 (s, CH2 , COD), 29.03 (s, CH2 , (s, Cq , C1\u2019), 128.32 (s, 2 CH, C3\u2019 + C5\u2019), 116.78 (s, CH, C5), 97.73\nC6), 28.72 (s, CH2 , C8), 16.33 (s, CH3 , C7), 16.19 (s, CH3 , C9) ppm; (dd, 1 JRh,C = 14.97 Hz, 2 JRh,C = 7.46 Hz, 2 trans-CH-COD), 64.64 (dd,\n 1J 2\nelemental analysis: C21 H27 BrClN2 Rh (calc. / found [%]): C (47.98 / Rh,C = 15.21 Hz, JRh,C = 7.53 Hz, 2 cis-CH-COD) 54.64 (s, CH, C6),\n49.02), H (5.18 / 5.43), N (5.33 / 5.14); MS (EI): m/z = 524.0 [M]+ , 52.78 (s, CH, C9), 33.02 (s, CH2 , COD), 32.76 (s, CH2 , COD), 28.94\n487.9 [M-Cl]+ . (s, CH2 , COD), 28.76 (s, CH2 , COD), 24.21 (s, CH3 , C7), 23.65 (s, CH3 ,\n Chlorido[1,3-diisopropyl-(4-phenyl)-imidazol-2- C8), 23.38 (s, CH3 , C10), 23.22 (s, CH3 ,C11) ppm; elemental analy-\nylidene(cycloocta-1,5-diene)]rhodium(\u2160) (3e) sis: C23 H31 Cl2 N2 Rh (calc. / found [%]): C (54.24 / 53.87), H (6.14 /\n general method with 2e; reaction period: 5 h; yellow powder, 6.35), N (5.50 / 5.10); MS (EI): m/z = 508.1 [M]+ , 472.7 [M-Cl]+\nyield: 57% Chlorido[1,3-diisopropyl-4-(4\u2019-chlorophenyl)-imidazol-2-\n 1 H NMR (500 MHz, CDCl ) \u03b4 = 7.44 \u2013 7.36 (m, 3H, 3 CH,\n 3 ylidene(cycloocta-1,5-diene)]rhodium(\u2160) (3h)\nC3\u2019 + C4\u2019 + C5\u2019), 7.34 \u2013 7.31 (m, 2H, 2 CH, C2\u2019 + C6\u2019), 6.71 (s, 1H, general method with 2h, reaction period: 5 h, eluent for column\nC5), 5.95 (h, 3 JH,H = 7.1 Hz, 1H, CH, C9), 5.88 (h, 3 JH,H = 6.8 Hz, 1H, chromatography: EtOAc:n- hexane:MeOH (8/ 2/0 \u2192 8/2/10), yellow\nCH, C6), 5.04 \u2013 4.97 (m, 2H, 2 CH, 2 trans-CH-COD), 3.46 \u2013 3.40 (m, powder, yield: 42%\n2H, 2 CH, 2 cis-CH-COD), 2.53 \u2013 2.33 (m, 4H, 2 CH2 axial, COD), 1 H NMR (600 MHz, CDCl ) \u03b4 = 7.55 \u2013 7.51 (m, 2H, 2 CH,\n 3\n2.01 \u2013 1.89 (m, 4H, 2 CH2 equatorial, COD), 1.52 (d, 3 JH,H = 6.8 Hz, C3\u2019 + C5\u2019), 7.21 \u2013 7.18 (m, 2H, 2 CH, C2\u2019 + C6\u2019), 6.71 (s, 1H, CH, C5),\n6H, 2 CH3 , C10 + C11), 1.43 (dd, 3 JH,H = 8.1 Hz, 4 JH,H = 7.0 Hz, 5.96 (hept, 3 JH,H = 7.0 Hz, 1H, CH, C9), 5.88 (hept, 3 JH,H = 6.8 Hz,\n6H, 2 CH3 , C7 + C8) ppm; 13 C NMR (126 MHz, CDCl3 ) \u03b4 = 180.54 1H, CH, C6), 5.05 \u2013 4.96 (m, 2H, 2 CH, 2 trans-CH-COD), 3.44 \u2013 3.37\n(d, 1 JRh,C = 50.6 Hz, Cq , C2), 134.28 (s, Cq , C4), 131.40 (s, CH, C4\u2019), (m, 2H, 2 CH, 2 cis-CH-COD), 2.54 \u2013 2.32 (m, 4H, 2 CH2 axial, COD),\n130.34 (s, CH, phenyl- CH), 129.04 (s, CH, phenyl- CH), 127.98 (s, 2.02 \u2013 1.88 (m, 4H, 2 CH2 equatorial, COD), 1.52 (dd, 3 JH,H = 6.84\nCH, phenyl-CH), 116.47 (s, CH, phenyl- CH), 97.55 (dd, 1 JRh,C = 14.82 Hz, 4 JH,H = 1.1 Hz, 6H, 2 CH3 , C10 + C11), 1.42 (dd, 3 JH,H = 7.0\nHz, 2 JRh,C = 7.03 Hz, 2 trans-CH-COD), 67.59 (dd, 1 JRh,C = 14.84 Hz, Hz, 3 JH,H = 4.2 Hz, 6H, 2 CH3 , C7 + C8) ppm; 13 C NMR (151 MHz,\n2J\n Rh,C = 7.07 Hz, 2 cis-CH-COD), 54.58 (s, CH, C6), 52.70 (s, CH, C9), CDCl3 ) \u03b4 = 181.29 (d, 1 JRh,C = 51.46 Hz, Cq , C2), 132.96 (s, Cq , C4)\n33.06 (s, CH2 , COD), 32.74 (s, CH2 , COD), 28.98 (s, CH2 , COD), 28.75 132.93 (s, 2 CH, C2\u2019 + C6\u2019), 131.29 (s, C3\u2019 + C5\u2019), 129.24 (s, Cq , C4\u2019),\n(s, CH2 , COD), 24.23 (s, CH3 , C7), 23.61 (s, CH3 , C8), 23.35 (s, CH3 , 123.54 (s, Cq , C1\u2019) 116.73 (s, CH, C5), 97.74 (dd, 1 JRh,C = 14.85 Hz,\n 2J 1\nC10), 23.22 (s, CH3 , C11) ppm; elemental analysis: C23 H32 ClN2 Rh Rh,C = 6,94 Hz, 2 CH, 2 trans-CH-COD), 67.64 (dd, JRh,C = 15,10\n(calc./found [%]): C (58.17 / 58.08), H (6.79 / 6.88), N (5.90 / 5.1); Hz, 2 CH, 2 cis-CH-COD), 54.65 (s, CH, C6), 52.79 (s, CH, C9), 33.02\nMS (EI): m/z = 474.13 [M]+ , 438.1 [M-Cl]+ . (s, CH2 , COD), 32.75 (s, CH2 , COD), 28.94 (s, CH2 , COD), 28.76 (s,\n Chlorido[1,3-diisopropyl-4-(4\u2019-\ufb02uorophenyl)-imidazol-2- CH2 , COD), 24,21 (s, CH3 , C7), 23.66 (s, CH3 , C8), 23.39 (s, CH3 ,\nylidene(cycloocta-1,5-diene)]rhodium(\u2160) (3f) C10), 23.22 (s, CH3 , C11) ppm; elemental analysis: C23 H31 BrClN2 Rh\ngeneral method with 2f, reaction period: 5 h, eluent for column (calc. / found [%]): C (49.89 / 50.38), H (5.64 / 5.88), N (5.06 / 5.1);\nchromatography: dichloromethane; yellow powder, yield: 89% MS (EI): m/z = 553.9 [M]+ , 518.9 [M-Cl]+\n 1 H NMR (500 MHz, CDCl ) \u03b4 = 7.34 \u2013 7.27 (m, 2H, 2 CH,\n 3\nC3\u2019 + C5\u2019), 7.12 \u2013 7.05 (m, 2H, 2 CH, C2\u2019 + C6\u2019), 6.71 (s, 1H, CH, C5),\n 2.4. Antiproliferative effects in tumor cells\n5.97 (hept, 3 JH,H = 7.0 Hz, 1H, CH, C9), 5.88 (hept, 3 JH,H = 6.8 Hz,\n1H, CH, C6), 5.01 (m, 2H, 2 CH, 2 trans-CH-COD), 3.50 \u2013 3.38 (m,\n The antiproliferative effects were determined according to stan-\n2H, 2 CH, 2 cis-CH-COD), 2.50 \u2013 2.33 (m, 4H, 2 CH2 axial, COD),\n dard protocols. In short: A volume of 100 \u03bcL of HT-29 cells\n2.02 \u2013 1.87 (m, 4H, 2 CH2 equatorial, COD), 1.52 (dd, 3 JH,H = 6.8\n (2,565 cells/mL), MDA-MB-231 cells (4,120 cells/mL) and MCF-7\nHz, 4 JH,H = 1.1 Hz, 6H, 2 CH3 , C10 + C11), 1.41 (dd, 3 JH,H = 8.4\n cells (4,840 cells/mL) was transferred into the wells of a 96-well\nHz, 4 JH,H = 7.0 Hz, 6H, 2 CH3 , C7 + C8) ppm; 13 C NMR (126\n plates and incubated at 37\u00b0C under 5% CO2 for 72 h. Stock so-\nMHz, CDCl3 ) \u03b4 = 180.96 (d, 1 JRh,C = 50.89 Hz, Cq, C2), 163.12\n lutions of the compounds were freshly prepared in dimethylfor-\n(d, 1 JC,F = 248.89 Hz, Cq, C4\u2019) 133.30 (d, 4 JC,F = 8.35 Hz, 2 CH,\n mamide (DMF) and diluted with the cell culture medium to obtain\nC2\u2019 + C6\u2019), 133.02 (s, Cq, C4), 126.24 (d, 4 JC,F = 3.6 Hz, Cq, C1\u2019),\n various concentrations (\ufb01nal concentration of DMF: 0.1 % v/v). Af-\n116.81 (s, CH, C5), 115.11 (d, 3 JC,F = 23,79 Hz, 2 CH, C3\u2019 + C5\u2019),\n ter 72 h (HT-29) or 96 h (MDA-MB-231, MCF-7) of exposure, the\n97.69 (dd, 1 JRh,C = 16.25 Hz, 2 JRh,C = 7.47 Hz, 2 trans-CH-COD),\n biomass of the cells was determined via crystal violet staining and\n67.60 (dd, 1 JRh,C = 16.30 Hz, 2 JRh,C = 7.33 Hz, 2 cis-CH-COD), 54.62\n the IC50 value was determined as the concentration that caused\n(s, CH, C6), 52.73 (s, CH, C9), 33.01 (s, CH2 , COD), 32.78 (s, CH2 ,\n 50% inhibition of cell proliferation relative to an untreated control.\nCOD), 28.93 (s, CH2 , COD), 28.78 (s, CH2 , COD), 24.21 (s, CH3 , C8),\n The results were calculated as the mean values of three indepen-\n23.59 (s, CH3 , C7), 23.36 (s, CH3 , C10), 23.22 (s, CH3 , C11) ppm;\n19 F NMR (471 MHz, CDCl ) \u03b4 -112.19 (tt, 3 J 4 dent experiments.\n 3 F,H = 8.6 Hz, JF,H = 5.3\n 1\nHz, F) ppm; elemental analysis: C23 H31 ClFN2 Rh (calc. / found [%]):\nC (56.05 / 56.03), H (6.34 / 6.31), N (5.68 / 5.63); MS (EI): m/z = 2.5. Toxic effects against cell layers with high con\ufb02uency\n492.0 [M]+ , 456.0 [M-Cl]+\n Chlorido[1,3-diisopropyl-4-(4\u2019-chlorophenyl)-imidazol-2- Caco-2 and Calu-3 cells were grown as almost con\ufb02uent mono-\nylidene(cycloocta-1,5-diene)]rhodium(\u2160) (3g) layers in 96-well plates. Complex 3f was dissolved as stock solution\ngeneral method with 2g, reaction period: 5 h, yellow powder; in DMF and diluted with cell culture medium to graded concentra-\nyield: 42% tions. The cell layers were incubated with the drug containing me-\n 1 H NMR (500 MHz, CDCl ) \u03b4 = 7.39 \u2013 7.36 (m, 2H, 2 CH,\n 3 dia for 24 h at 37\u00b0C / 5% CO2 in an incubator. The cell viability was\nC3\u2019 + C5\u2019), 7.28 \u2013 7.24 (m, 2H, 2 CH, C2\u2019 + C6\u2019), 6.71 (s, 1H, CH, determined by crystal violet staining and cell viability was calcu-\n\n 4\n\fJ. Schmidt, J. W\u00f6lker, P. Lippmann et al. Journal of Organometallic Chemistry 964 (2022) 122300\n\n\n\n\n Scheme 1. Synthesis of [(COD)(NHC)RhCl] complexes a) ethyl iodide or 2-bromopropane, K2 CO3 (82\u00b0C, 10 psi, 50 W), b) Ag2 O (r.t.), [Rh(COD)Cl]2 (25\u00b0C, 10 psi, 50 W)\n\n\n\n\nlated as percentage of an untreated control. Results were obtained silver intermediate using the rhodium dimer bis[chlorido(1,5-\nin three independent experiments. cyclooctadiene)rhodium(I)] for up to 18 h [2,3,35]. In this work,\n the same reaction mechanism was supported by microwave irra-\n2.6. Cellular uptake diation (50 W) leading overall to convenient reaction periods (up\n to 5 h in total) for the target complexes 3a \u2013 3h. After removal of\n For cellular-uptake studies HT-29 cells were grown until at least the formed silver halide by \ufb01ltration, the pure products were ob-\n70% con\ufb02uency in 75 cm2 cell culture \ufb02asks. Stock solutions of tained by chromatography over silica and precipitation as yellow\ncomplex 3b or 3f in DMF were freshly prepared and diluted with solids in yields of 42 \u2013 89%. The high purity of all target complexes\ncell culture medium to the desired concentration of 2.0 \u03bcM (\ufb01nal was con\ufb01rmed by elemental analyses and mass spectrometry con-\nDMF concentration: 0.1% v/v). The cell culture medium of the cell \ufb01rmed the expected molecular ion [M]+ signals for 3a \u2013 3h.\nculture \ufb02asks was replaced with 10 mL of the cell culture medium In the 1 H-NMR spectra several diagnostic features can be ob-\ncontaining the test compound and the \ufb02asks were incubated for 1, served, which clearly con\ufb01rm coordination of the NHC ligand to\n3, 6 or 24 h at 37\u00b0C / 5% CO2 . Afterwards the medium was removed the metal. Thus, the hydrogen signal at the C2 carbon of the im-\nand the cells were washed with phosphate-buffered saline pH 7.4. idazole structure (at approx. 9-11 ppm in the spectra of 2a \u2013 2h)\nAfter trypsinization, the cell pellets were isolated by centrifugation, is absent and the hydrogen at C5 of the imidazole appears as sin-\nresuspended in 1\u20135 mL puri\ufb01ed water, lysed by ultrasonication and glet as a consequence of the missing long-range coupling to the\nappropriately diluted using puri\ufb01ed water. The rhodium content absent C2 hydrogen. In the complexes with ethyl side chains, 3a \u2013\nof the samples was determined by AAS and the protein content 3d, the CH2 hydrogens appear as individual non-equivalent signals\nwas determined by the Bradford method as described in our recent with dq splitting after metal coordination, which can be ascribed\npublication in more detail [2]. The results were obtained in two in- to rotations around the Rh-C bond [40]. In the 13 C-NMR spectra,\ndependent experiments and were expressed as nmol rhodium per the signals for the C2 carbon are signi\ufb01cantly down\ufb01eld-shifted\nmilligram of cellular protein. in comparison with the respective signals of the imidazolium NHC\n precursors (180.5 \u2013 183.5 ppm for 3a \u2013 3h vs 133.4 \u2013 137.4 ppm\n3. Results for 2a \u2013 2h) and show a characteristic 1 J rhodium-carbon coupling\n of 50.6 \u2013 51.5 Hz.\n3.1. Chemistry\n 3.2. Cell growth inhibitory effects\n The NHC ligands of the target [(COD)(NHC)RhCl] complexes\nconsist of a phenylimidazole core structure. In a \ufb01rst step of For evaluation of cytotoxic effects by complexes 3a \u2013 3h we\nthe synthesis procedure (see Scheme 1), the respective phenylim- selected three relevant adherent cancer cell lines, namely HT-29\nidazoles 1a \u2013 1d were N-alkylated with ethyl iodide or 2- colon adenocarcinoma, MDA-MB-231 breast carcinoma and MCF-\nbromopropane, respectively, under basic conditions supported by 7 breast adenocarcinoma. The cell growth inhibitory effects of 2a\n50 W microwave irradiation to yield the phenylimidazolium \u2013 2h and 3a \u2013 3h were determined and expressed as IC50 values\nhalides 2a \u2013 2h. The [(COD)(NHC)RhCl] complexes of our pre- (see Table 1). The metal free NHC ligand precursors 2a \u2013 2h did\nvious projects had been prepared by reaction of the respective not reach IC50 values up to a concentration of 100 \u03bcM and can\nphenylimidazolium cations with half equivalent of silver iodide therefore be considered as inactive, which is in strong agreement\nfor 4h, followed by a transmetalation reaction of the formed with our previous results obtained with some of the compounds\n\n 5\n\fJ. Schmidt, J. W\u00f6lker, P. Lippmann et al. Journal of Organometallic Chemistry 964 (2022) 122300\n\n\n Table 1 mined in comparison to those obtained with the ethyl analogue 3b\n Cell growth inhibition (IC50 values, \u03bcM) of 2a \u2013 2h and 3a \u2013 3h against\n (see Fig. 2). For this purpose, HT-29 cells were exposed to 2.0 \u03bcM\n selected cancer cell lines; the values between brackets represent the exper-\n imental errors. of 3b or 3f over periods of up to 24 h and the cellular rhodium lev-\n els were determined by high-resolution continuum source atomic\n R1 R2 HT-29 MCF-7 MDA-MB-231\n absorption spectroscopy (HRCS-AAS).\n 2a \u2013 2h >100 >100 >100 Both complexes showed a rapid uptake within the \ufb01rst hour\n 3a -H -CH2 CH3 6.4 (\u00b10.5) 4.5 (\u00b10.4) 2.0 (\u00b10.7) of incubation and decreased values after longer periods (24 h). In\n 3b -F -CH2 CH3 6.7 (\u00b11.9) 4.3 (\u00b10.6) 2.4 (\u00b10.3)\n all cases the levels obtained with 3f were signi\ufb01cantly higher than\n 3c -Cl -CH2 CH3 7.2 (\u00b12.0) 4.9 (\u00b12.0) 2.5 (\u00b10.7)\n 3d -Br -CH2 CH3 9.2 (\u00b10.3) 5.1 (\u00b10.5) 2.7 (\u00b10.6) those found with 3b, which indicates that the higher cytotoxicity\n 3e -H -CH(CH3 )2 3.1 (\u00b10.6) 1.8 (\u00b10.2) 1.2 (\u00b10.2) of the isopropyl derivatives could in fact be related to a higher ac-\n 3f -F -CH(CH3 )2 1.9 (\u00b10.2) 1.7 (\u00b10.1) 1.0 (\u00b10.1) cumulation in the cells.\n 3g -Cl -CH(CH3 )2 4.1 (\u00b10.9) 2.9 (\u00b10.6) 1.4 (\u00b10.2)\n 3h -Br -CH(CH3 )2 2.9 (\u00b11.0) 2.2 (\u00b11.0) 1.4 (\u00b10.6)\n 4. Discussion and conclusions\n\n A series of eight [(COD)(NHC)RhCl] complexes was prepared\n and studied as possible anticancer agents.\n [(COD)(NHC)RhCl] complexes can be prepared by different syn-\n thesis procedures [41]. A convenient frequently used method is\n the procedure reported by Chianese et al. that goes back to the\n transmetalation method of Wang and Lin [42,43]. In the \ufb01rst\n step of this method, the imidazolium NHC precursor is treated\n with silver oxide leading to the formation of silver carbene com-\n plexes that can be reacted in a second step with the rhodium(\u2160)\n dimer [Rh(COD)Cl]2 to obtain the neutral target species of the\n type [(COD)(NHC)RhCl]. In our previous studies we had applied\n a one-pot version of this method without isolation of the sil-\n ver NHC intermediates [2,3,35]. The time required for the trans-\nFig. 2. Cellular rhodium levels (nmol Rh per milligram cellular protein) of HT-29\n metalation reaction depended on the nature of the NHC precur-\ncells after exposure to 2.0 \u03bcM of 3b or 3f.\n sor. Extended reaction periods were necessary in particular in\n case of phenylimidazole-based NHC precursors [35]. Alternatively,\n[39]. In contrast, all [(COD)(NHC)RhCl] complexes displayed good [(COD)(NHC)RhCl] complexes can also be prepared by base catal-\ncell proliferation activity with IC50 values in the range of 0.9 to ysed one-pot methods [11,44].\n9.2 \u03bcM. A clear preference for the isopropyl side chain over the In this work we have used a modi\ufb01ed version of the previous\nethyl side chain was evident, as in all cases the IC50 values of the one-pot procedure by applying 50 W microwave irradiation. Com-\nisopropyl substituted complexes 3e \u2013 3h were lower than that of plexes 3a and 3b had already been reported and prepared accord-\nthe corresponding ethyl derivative (compare results for 3a vs 3e, ing to the non-microwave procedure with a 4 h reaction with silver\n3b vs 3f, 3c vs 3g, and 3d vs 3h). Regarding the para-substituents oxide followed by 18 h reaction with [Rh(COD)Cl]2 at room tem-\non the phenyl ring of the NHC ligands, the differences in the IC50 perature [35]. The microwave assisted procedure allowed to signif-\nvalues within 3a \u2013 3d and 3e \u2013 3h, respectively, were small. How- icantly shorten the reaction periods with 15 min for the treatment\never, among the more active isopropyl derivatives the para \ufb02uorine with silver oxide and 3 h for 3a and 4 h for 3b, respectively. In\ncompound 3f turned out to be the most active complex against all conclusion, the method might provide a suitable improvement for\nstudied cell lines and reached very promising IC50 values between the accelerated synthetic access to [(COD)(NHC)RhCl] complexes\n0.9 and 2.0 \u03bcM. from NHC precursors that otherwise require extended reaction pe-\n The most active derivative 3f was subjected to further studies riods. The microwave-assisted procedure does not yet represent an\nregarding toxic effects against cell layers with high con\ufb02uency, in optimized method and in particular the effect of heating versus\norder to distinguish between proliferation inhibition and \u201cdirect\u201d microwave irradiation would be worthwhile to evaluate.\ntoxicity against cells. For this purpose, we used an assay setup that The target compounds were studied for their antiproliferative\nwe had recently used to check for host cell toxicity before sub- effects in selected cancer cell lines and displayed strong cytotoxic\njecting compounds to antiviral studies in infected host cells [35]. effects with IC50 values in the micromolar range. There was a clear\nFor this purpose, Caco-2 human colon adenocarcinoma and Calu-3 preference for the branched (isopropyl) side chains at the NHC ni-\nlung adenocarcinoma cells were grown until almost con\ufb02uent lay- trogen atoms versus the non-branched (ethyl) side chains. In pre-\ners and then exposed for 24 h to different concentrations of 3f. vious studies, in which a benzimidazole based ligand was used in-\nUnder these conditions 3f triggered IC50 values of 2.50(\u00b10.12) \u03bcM in stead of the phenylimidazole moiety, shorter methyl side chains\nCaco-2 cells and 3.34(\u00b10.93) \u03bcM in Calu-3 cells. Although these val- were preferred over ethyl groups [2]. Within the more active iso-\nues are higher than the IC50 values of 3f for proliferation inhibi- propyl derivatives of this report, the compound with the \ufb02uorine\ntion, the margin between toxicity against extended cell layers and substituent on the phenyl ring of the NHC ligand, 3f, triggered\nproliferation inhibition is rather small and does not indicate a pref- the highest activity in all studied cell lines. Cellular uptake stud-\nerence for proliferation inhibition over direct toxic effects against ies indicated that the enhanced activity of the isopropyl derivatives\nthe cells. could primarily be a consequence of stronger accumulation in the\n cells. Taken together, this indicates that careful optimization of the\n3.3. Cellular uptake NHC core structures in relation to the side chains at the NHC ni-\n trogen atoms can be an useful strategy to \ufb01ne-tune and optimize\n In order to evaluate if the enhanced activity of the deriva- the antiproliferative effects of [(COD)(NHC)RhCl] complexes. With\ntives with the isopropyl side chain was a consequence of a higher IC50 values between 0.9 and 2.0 \u03bcM, 3f exceeded the activity of\nlipophilicity resulting in a higher uptake of the complexes, the cel- related monocarbene gold complexes of the type (NHC)AuCl [31].\nlular rhodium levels of the most active complex, 3f, were deter- Additional studies evaluating the toxicity of 3f against non prolif-\n\n 6\n\fJ. Schmidt, J. W\u00f6lker, P. Lippmann et al. Journal of Organometallic Chemistry 964 (2022) 122300\n\n\nerating cells indicated that the complex causes both proliferation [18] C. Yang, W. Wang, J.-X. Liang, G. Li, K. Vellaisamy, C.-Y. Wong, D.-L. Ma, C.-\ninhibition and direct toxic effects against the cells. H. Leung, A Rhodium(III)-Based Inhibitor of Lysine-Speci\ufb01c Histone Demethy-\n lase 1 as an Epigenetic Modulator in Prostate Cancer Cells, J. Med. Chem. 60\n (2017) 2597\u20132603, doi:10.1021/acs.jmedchem.7b00133.\nDeclaration of Competing Interest [19] K.K.H. Tong, M. Hanif, S. Movassaghi, M.P. Sullivan, J.H. Lovett, K. Hummitzsch,\n T. S\u00f6hnel, S.M.F. Jamieson, S.K. Bhargava, H.H. Harris, C.G. Hartinger, Triazolyl-\n Functionalized N-Heterocyclic Carbene Half-Sandwich Compounds: Coordina-\n The authors declare that they have no known competing \ufb01nan-\n tion Mode, Reactivity and in vitro Anticancer Activity, ChemMedChem 16\ncial interests or personal relationships that could have appeared to (2021) 3017\u20133026, doi:10.1002/cmdc.202100311.\nin\ufb02uence the work reported in this paper. [20] T.-M. Khan, N.S. Gul, X. Lu, R. Kumar, M.I. Choudhary, H. Liang, Z.-F. Chen,\n Rhodium(iii) complexes with isoquinoline derivatives as potential anticancer\n agents: in vitro and in vivo activity studies, Dalton Trans 48 (2019) 11469\u2013\nSupplementary materials 11479, doi:10.1039/c9dt01951k.\n [21] D.-L. Ma, M. Wang, Z. Mao, C. Yang, C.-T. Ng, C.-H. Leung, Rhodium com-\n Supplementary material associated with this article can be plexes as therapeutic agents, Dalton Trans. 45 (2016) 2762\u20132771, doi:10.1039/\n c5dt04338g.\nfound, in the online version, at doi:10.1016/j.jorganchem.2022. [22] C.-H. Leung, H.-J. Zhong, D.S.-H. Chan, D.-L. Ma, Bioactive iridium and rhodium\n122300. complexes as therapeutic agents, Coord. Chem. Rev. 257 (2013) 1764\u20131776,\n doi:10.1016/j.ccr.2013.01.034.\nReferences [23] Y. Geldmacher, M. Oleszak, W.S. Sheldrick, Rhodium(III) and iridium(III) com-\n plexes as anticancer agents, Inorg. Chim. Acta 393 (2012) 84\u2013102, doi:10.1016/\n [1] K.D. Mjos, C. Orvig, Metallodrugs in medicinal inorganic chemistry, Chem. Rev. j.ica.2012.06.046.\n 114 (2014) 4540\u20134563, doi:10.1021/cr400460s. [24] M. Sohrabi, M. Saeedi, B. Larijani, M. Mahdavi, Recent advances in biological\n [2] L. Oehninger, L.N. K\u00fcster, C. Schmidt, A. Mu\u00f1oz-Castro, A. Prokop, I. Ott, activities of rhodium complexes: Their applications in drug discovery research,\n A chemical-biological evaluation of rhodium(I) N-heterocyclic carbene com- Eur. J. Med. Chem. 216 (2021) 113308, doi:10.1016/j.ejmech.2021.113308.\n plexes as prospective anticancer drugs, Chem. Eur. J. 19 (2013) 17871\u201317880, [25] S. Medici, M. Peana, V.M. Nurchi, J.I. Lachowicz, G. Crisponi, M.A. Zoroddu, No-\n doi:10.1002/chem.201302819. ble metals in medicine, Coord. Chem. Rev. 284 (2015) 329\u2013350, doi:10.1016/j.\n [3] L. Oehninger, S. Spreckelmeyer, P. Holenya, S.M. Meier, S. Can, H. Alborzinia, ccr.2014.08.002.\n J. Schur, B.K. Keppler, S. W\u00f6l\ufb02, I. Ott, Rhodium(I) N-Heterocyclic Carbene [26] T. Giraldi, G. Zassinovich, G. Mestroni, Antitumour action of planar,\n Bioorganometallics as in Vitro Antiproliferative Agents with Distinct Effects organometallic rhodium(I) complexes, Chem.-Biol. Interact. 9 (1974) 389\u2013394\n on Cellular Signaling, J. Med. Chem. 58 (2015) 9591\u20139600, doi:10.1021/acs. 10.1016/0 0 09-2797%2874%2990 020-9.\n jmedchem.5b01159. [27] T. Giraldi, G. Sava, G. Bertoli, G. Mestroni, G. Zassinovich, Antitumor Ac-\n [4] J.R. McConnell, D.P. Rananaware, D.M. Ramsey, K.N. Buys, M.L. Cole, tion of Two Rhodium and Ruthenium Complexes in Comparison with cis-Di-\n S.R. McAlpine, A potential rhodium cancer therapy, Bioorg. Med. Chem. Lett. amminedichloroplatinum(II), Cancer Res 37 (1977) 2662\u20132666.\n 23 (2013) 2527\u20132531, doi:10.1016/j.bmcl.2013.03.016. [28] J. Wang, J.-J. Nie, P. Guo, Z. Yan, B. Yu, W. Bu, Rhodium(I) Complex-Based Poly-\n [5] P.V. Simpson, C. Schmidt, I. Ott, H. Bruhn, U. Schatzschneider, Synthesis, Cellu- meric Nanomicelles in Water Exhibiting Coexistent Near-Infrared Phosphores-\n lar Uptake and Biological Activity Against Pathogenic Microorganisms and Can- cence Imaging and Anticancer Activity in Vivo, J. Am. Chem. Soc. 142 (2020)\n cer Cells of Rhodium and Iridium N-Heterocyclic Carbene Complexes Bearing 2709\u20132714, doi:10.1021/jacs.9b11013.\n Charged Substituents, Eur. J. Inorg. Chem. (2013) 5547\u20135554 2013, doi:10.1002/ [29] D. Loreto, A. Merlino, The interaction of rhodium compounds with proteins:\n ejic.201300820. A structural overview, Coord. Chem. Rev. 442 (2021) 213999, doi:10.1016/j.ccr.\n [6] W. Streciwilk, A. Terenzi, X. Cheng, L. Hager, Y. Dabiri, P. Prochnow, J.E. Bandow, 2021.213999.\n S. W\u00f6l\ufb02, B.K. Keppler, I. Ott, Fluorescent organometallic rhodium(I) and ruthe- [30] M.P. Sullivan, M. Cziferszky, I. Tolbatov, D. Truong, D. Mercadante, N. Re,\n nium(II) metallodrugs with 4-ethylthio-1,8-naphthalimide ligands, Eur. J. Med. R. Gust, D.C. Goldstone, C.G. Hartinger, Probing the Paradigm of Promiscu-\n Chem. 156 (2018) 148\u2013161, doi:10.1016/j.ejmech.2018.06.056. ity for N-Heterocyclic Carbene Complexes and their Protein Adduct Formation,\n [7] W. Streciwilk, A. Terenzi, F. Lo Nardo, P. Prochnow, J.E. Bandow, B.K. Keppler, Angew. Chem. Int. Ed. 60 (2021) 19928\u201319932, doi:10.1002/anie.202106906.\n I. Ott, Synthesis and Biological Evaluation of Organometallic Complexes Bear- [31] Y. Gothe, T. Marzo, L. Messori, N. Metzler-Nolte, Cytotoxic activity and protein\n ing Bis-1,8-naphthalimide Ligands, Eur. J. Inorg. Chem. (2018) 3104\u20133112 2018, binding through an unusual oxidative mechanism by an iridium(I)-NHC com-\n doi:10.10 02/ejic.20180 0384. plex, Chem. Commun. 51 (2015) 3151\u20133153, doi:10.1039/c4cc10014j.\n [8] W. Streciwilk, A. Terenzi, R. Misgeld, C. Frias, P.G. Jones, A. Prokop, B.K. Keppler, [32] I.M. Daubit, N. Metzler-Nolte, On the interaction of N-heterocyclic carbene\n I. Ott, Metal NHC Complexes with Naphthalimide Ligands as DNA-Interacting Ir+I complexes with His and Cys containing peptides, Dalton Trans 48 (2019)\n Antiproliferative Agents, ChemMedChem 12 (2017) 214\u2013225, doi:10.1002/cmdc. 13662\u201313673, doi:10.1039/c9dt01338e.\n 201600557. [33] Y. Gothe, T. Marzo, L. Messori, N. Metzler-Nolte, Iridium(I) Compounds as\n [9] J.-J. Zhang, J.K. Muenzner, M.A. Abu El Maaty, B. Karge, R. Schobert, S. W\u00f6l\ufb02, Prospective Anticancer Agents, Chem. Eur. J. 22 (2016) 12487\u201312494, doi:10.\n I. Ott, A multi-target caffeine derived rhodium(i) N-heterocyclic carbene com- 1002/chem.201601542.\n plex, Dalton Trans. 45 (2016) 13161\u201313168, doi:10.1039/c6dt02025a. [34] Y. Gothe, I. Romero-Canel\u00f3n, T. Marzo, P.J. Sadler, L. Messori, N. Metzler-\n[10] I.M. Daubit, M.P. Sullivan, M. John, D.C. Goldstone, C.G. Hartinger, N. Metzler- Nolte, Synthesis and Mode of Action Studies on Iridium(I)-NHC Anticancer\n Nolte, A Combined Spectroscopic and Protein Crystallography Study Reveals Drug Candidates, Eur. J. Inorg. Chem. (2018) 2461\u20132470 2018, doi:10.1002/ejic.\n Protein Interactions of RhI(NHC) Complexes at the Molecular Level, Inorg. 201800225.\n Chem. 59 (2020) 17191\u201317199, doi:10.1021/acs.inorgchem.0c02438. [35] M. Gil-Moles, S. T\u00fcrck, U. Basu, A. Pettenuzzo, S. Bhattacharya, A. Rajan,\n[11] I.M. Daubit, S. Wortmann, D. Siegmund, S. Hahn, P. Nuernberger, N. Metzler- X. Ma, R. B\u00fcssing, J. W\u00f6lker, H. Burmeister, H. Hoffmeister, P. Schneeberg,\n Nolte, Unveiling Luminescent IrI and RhI N-Heterocyclic Carbene Complexes: A. Prause, P. Lippmann, J. Kusi-Nimarko, S. Hassell-Hart, A. McGown, D. Guest,\n Structure, Photophysical Speci\ufb01cs, and Cellular Localization in the Endoplasmic Y. Lin, A. Notaro, R. Vinck, J. Karges, K. Cariou, K. Peng, X. Qin, X. Wang,\n Reticulum, Chem. Eur. J. 27 (2021) 6783\u20136794, doi:10.1002/chem.202100375. J. Skiba, \u0141. Szczupak, K. Kowalski, U. Schatzschneider, C. Hemmert, H. Gor-\n[12] R. Fan, M. Bian, L. Hu, W. Liu, A new rhodium(I) NHC complex inhibits TrxR: nitzka, E.R. Milaeva, A.A. Nazarov, G. Gasser, J. Spencer, L. Ronconi, U. Kortz,\n In vitro cytotoxicity and in vivo hepatocellular carcinoma suppression, Eur. J. J. Cinatl, D. Bojkova, I. Ott, Metallodrug Pro\ufb01ling against SARS-CoV-2 Target\n Med. Chem. 183 (2019) 111721, doi:10.1016/j.ejmech.2019.111721. Proteins Identi\ufb01es Highly Potent Inhibitors of the S/ACE2 interaction and the\n[13] I. Slimani, S. S\u0327ahin-B\u00f6l\u00fckbas\u0327\u0131, M. Ulu, E. Evren, N. G\u00fcrb\u00fcz, I\u0307. \u00d6zdemir, Papain-like Protease PLpro, Chem. Eur. J. 27 (2021) 17928\u201317940, doi:10.1002/\n N. Hamdi, I\u0307. \u00d6zdemir, Rhodium(i ) N-heterocyclic carbene complexes: synthe- chem.202103258.\n sis and cytotoxic properties, New J. Chem. 45 (2021) 5176\u20135183, doi:10.1039/ [36] A. Monney, M. Albrecht, A chelating tetrapeptide rhodium complex comprised\n d1nj00144b. of a histidylidene residue: biochemical tailoring of an NHC-Rh hydrosilylation\n[14] I.M. Daubit, J. Wolf, N. Metzler-Nolte, Rhodium(I) and Iridium(I) N-Heterocyclic catalyst, Chem. Commun. 48 (2012) 10960\u201310962, doi:10.1039/c2cc35491h.\n carbene complexes of imidazolium functionalized amino acids and peptides, J. [37] A. Monney, E. Alberico, Y. Ortin, H. M\u00fcller-Bunz, S. Gladiali, M. Albrecht, Stere-\n Organomet. Chem. 909 (2020) 121096, doi:10.1016/j.jorganchem.2019.121096. ospeci\ufb01c synthesis and catalytic activity of L-histidylidene metal complexes,\n[15] Y.-B. Peng, W. He, Q. Niu, C. Tao, X.-L. Zhong, C.-P. Tan, P. Zhao, Mitochondria- Dalton Trans 41 (2012) 8813\u20138821, doi:10.1039/c2dt30799e.\n targeted cyclometalated rhodium(III) complexes: synthesis, characterization [38] A. Monney, F. Nastri, M. Albrecht, Peptide-tethered monodentate and chelating\n and anticancer research, Dalton Trans 50 (2021) 9068\u20139075, doi:10.1039/ histidylidene metal complexes: synthesis and application in catalytic hydrosi-\n D1DT01053K. lylation, Dalton Trans 42 (2013) 5655\u20135660, doi:10.1039/c3dt50424g.\n[16] J. Liang, A. Levina, J. Jia, P. Kappen, C. Glover, B. Johannessen, P.A. Lay, Reactiv- [39] C. Schmidt, B. Karge, R. Misgeld, A. Prokop, R. Franke, M. Br\u00f6nstrup, I. Ott,\n ity and Transformation of Antimetastatic and Cytotoxic Rhodium(III)-Dimethyl Gold(I) NHC Complexes, Chem. Eur. J. 23 (2017) 1869\u20131880, doi:10.1002/chem.\n Sulfoxide Complexes in Biological Fluids: An XAS Speciation Study, Inorg. 201604512.\n Chem. 58 (2019) 4880\u20134893, doi:10.1021/acs.inorgchem.8b03477. [40] M.J. Doyle, M.F. Lappert, Activation parameters for rotation about an M\u2013\n[17] B.Y.T. Lee, M.P. Sullivan, E. Yano, K.K.H. Tong, M. Hanif, T. Kawakubo-Yasukochi, C carb bond from temperature dependent 1 H NMR spectra of Rh I car-\n S.M.F. Jamieson, T. Soehnel, D.C. Goldstone, C.G. Hartinger, Anthracenyl Func- bene complexes, J. Chem. Soc., Chem. Commun. (1974) 679\u2013680, doi:10.1039/\n tionalization of Half-Sandwich Carbene Complexes: In Vitro Anticancer Activ- C39740 0 0 0679.\n ity and Reactions with Biomolecules, Inorg. Chem. 60 (2021) 14636\u201314644,\n doi:10.1021/acs.inorgchem.1c01675.\n\n\n 7\n\fJ. Schmidt, J. W\u00f6lker, P. Lippmann et al. Journal of Organometallic Chemistry 964 (2022) 122300\n\n\n[41] J.M. Praetorius, C.M. Crudden, N-Heterocyclic carbene complexes of rhodium: [43] A.R. Chianese, X. Li, M.C. Janzen, J.W. Faller, R.H. Crabtree, Rhodium and Irid-\n structure, stability and reactivity, Dalton Trans. (2008) 4079\u20134094, doi:10. ium Complexes of N-Heterocyclic Carbenes via Transmetalation: Structure and\n 1039/b802114g. Dynamics, Organometallics 22 (2003) 1663\u20131667, doi:10.1021/om021029.\n[42] H.M.J. Wang, I.J.B. Lin, Facile Synthesis of Silver(I)\u2212Carbene Complexes. Use- [44] C. K\u00f6cher, W.A. Herrmann, Heterocyclic carbenes. One-pot synthesis of\n ful Carbene Transfer Agents, Organometallics 17 (1998) 972\u2013975, doi:10.1021/ rhodium and iridium carbene complexes, J. Organomet. Chem. 532 (1997) 261\u2013\n om9709704. 265, doi:10.1016/S0022- 328X(96)06732- 0.\n\n\n\n\n 8\n\f", "pages_extracted": 8, "text_length": 71177}