Copper (II) complexes possessing alkyl-substituted polypyridyl ligands: Structural characterization and in vitro antitumor activity

    Copper (II) complexes possessing alkyl-substituted polypyridyl ligands: Structural characterization and in vitro antitumor activity Noah R. Angel, Raneen M. Khatib, Julia Jenkins, Michelle Smith, Justin M. Rubalcava, Brian Khoa Le, Daniel Lussier, Zhuo (Georgia) Chen, Fook S. Tham, Emma H. Wilson, Jack F. Eichler PII: DOI: Reference:

S0162-0134(16)30275-6 doi:10.1016/j.jinorgbio.2016.09.012 JIB 10084

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

20 November 2015 16 September 2016 29 September 2016

Please cite this article as: Noah R. Angel, Raneen M. Khatib, Julia Jenkins, Michelle Smith, Justin M. Rubalcava, Brian Khoa Le, Daniel Lussier, Zhuo (Georgia) Chen, Fook S. Tham, Emma H. Wilson, Jack F. Eichler, Copper (II) complexes possessing alkyl-substituted polypyridyl ligands: Structural characterization and in vitro antitumor activity, Journal of Inorganic Biochemistry (2016), doi:10.1016/j.jinorgbio.2016.09.012

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ACCEPTED MANUSCRIPT Copper (II) complexes possessing alkyl-substituted polypyridyl ligands: Structural characterization and in vitro antitumor activity

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Noah R. Angel1‡, Raneen M. Khatib1‡, Julia Jenkins1, Michelle Smith1, Justin M. Rubalcava,1 Brian Khoa Le1, Daniel Lussier1, Zhuo (Georgia) Chen2, Fook S. Tham1, Emma H. Wilson3,

University of California, Riverside Department of Chemistry, 501 Big Springs Rd., Riverside,

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1

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Jack F. Eichler1*

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CA, 92521 Emory University Winship Cancer Institute

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University of California, Riverside School of Medicine, Division of Biomedical Sciences

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2

*

Corresponding Author: [email protected]

These authors contributed equally to this work

Abstract

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‡

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In an effort to find alternatives to the antitumor drug cisplatin, a series of copper (II) complexes possessing alkyl-substituted polypyridyl ligands have been synthesized. Eight new complexes are reported herein: µ-dichloro-bis{2,9-di-sec-butyl-1,10phenanthrolinechlorocopper(II)} {[(di-sec-butylphen)ClCu(µ-Cl)2CuCl(di-sec-butylphen)]}(1), 2-secbutyl-1,10-phenanthrolinedichlorocopper(II) {[mono-sec-butylphen) CuCl2} (2), 2,9-di-n-butyl-1,10phenanthrolinedichlorocopper(II) {[di-n-butylphen) CuCl2}(3), 2-n-butyl-1,10phenanthrolinedichlorocopper(II) {[mono-n-butylphen) CuCl2} (4), 2,9-di-methyl-1,10phenanthrolineaquadichlorocopper(II) {[di-methylphen) Cu(H2O)Cl2}(5), µ-dichloro-bis{6-secbutyl-2,2’-bipyridinedichlorocopper(II)} {(mono-sec-butylbipy) ClCu(µ-Cl)2CuCl(mono-sec-butylbipy)} (6), 6,6’-di-methyl-2,2’-bipyridinedichlorocopper(II) {6,6’-di-methylbipy) CuCl2} (7), and 4,4’dimethyl-2,2’-bipyridinedichlorocopper(II) {4,4’-di-methylbipy) CuCl2} (8). These complexes have

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ACCEPTED MANUSCRIPT been characterized via elemental analysis, UV-Vis spectroscopy, and mass spectrometry. Single crystal X-ray diffraction experiments revealed the complexes synthesized with the di-sec-butylphen

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ligand (1) and mono-sec-butylbipy ligand (6) crystallized as dimers in which two copper(II) centers

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are bridged by two chloride ligands. Conversely, complexes 2, 7, and 8 were isolated as

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monomeric species possessing distorted tetrahedral geometries, and the [(di-methylphen)Cu(H2O)Cl2] (5) complex was isolated as a distorted square pyramidal monomer

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possessing a coordinating aqua ligand. Compounds 1-8 were evaluated for their in vitro

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antitumor efficacy. Compounds 1, 5, and 7 in particular were found to exhibit remarkable activity against human derived lung cancer cells, yet this class of copper(II) compounds had

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minimal cytotoxic effect on non-cancerous cells. In vitro control experiments indicate the

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activity of the copper(II) complexes most likely does not arise from the formation of CuCl2 and free polypyridyl ligand, and preliminary solution state studies suggest these compounds are

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generally stable in biological buffer. The results presented herein suggest further development of this class of copper-based drugs as potential anti-cancer therapies should be pursued. Keywords: copper(II), polypyridyl, phenanthroline, bipyrydine, synthesis, anticancer

1. Introduction The use of transition metals in anticancer therapies has been a continuously growing field of study since the drug cisplatin (cis-diamminedichloroplatinum(II)) was approved for the clinical treatment of testicular and ovarian cancers in the late 1970’s [1]. One well-established area of research has focused on the evaluation of gold(III) complexes possessing alkylsubstituted 1,10-phenanthroline (alkylphen) and alkyl-substituted 2,2’-bipyridine (alkylbipy) ligands as potential alternatives to cisplatin [2-5]. Preliminary results in our lab found the free 2

ACCEPTED MANUSCRIPT polypyridyl ligands were more active antitumor agents than the corresponding gold(III) complexes, suggesting the gold complexes may simply release the ligand in a cellular

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environment [2]. A more recent report demonstrated that an expanded library of gold(III)

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complexes possessing alkyl-substituted polypyridyl ligands exhibited in vitro antitumor efficacy not correlated to the activity of the free ligands. These results suggest the gold(III) complexes

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initiate tumor cell death via a distinct mechanism compared to the polypyridyl ligands [3].

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Given these recent findings, it was desired to use the alkyl-substituted polypyridyl ligand scaffolds to make metallotherapeutic agents based on alternative metal centers. Evaluating the

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antitumor activity of alkylphen and alkylbipy complexes with different metals will provide an opportunity to further corroborate the finding that the metal-based drugs demonstrate therapeutic

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activity not dependent on the ligand activity, and potentially lead to the discovery of metallotherapies with more pronounced antitumor activity. Due to the increasing number of

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reports of copper(II) complexes displaying promising anticancer activity, the fact copper drugs potentially have fewer issues in regards to systemic toxicity than platinum or gold therapeutics, and the low cost and convenience in working with cupric salt starting materials, copper(II) analogs were the preliminary targets. Copper(II) is found to form numerous types of coordination compounds with varying geometries, and copper is a co-factor in enzymes that play critical roles in biological redox chemistry and mitochondrial respiration [6]. The proper regulation of copper ions in the body is critical from a physiological standpoint and abnormal levels of copper are associated with a variety of pathological states, including cancer [7]. In particular, analysis of ex vivo tumors has previously found higher levels of copper species present in cancer cells compared to normal tissues [8], and many cancer drugs have thus focused on targeting the regulation of copper in

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ACCEPTED MANUSCRIPT tumor cells as a mechanism of imparting therapeutic efficacy [9]. Despite the fact copper levels in biological systems need to be tightly regulated, there is a growing interest in developing

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copper-based coordination complexes as potential anticancer agents [10]. The rapid increase in

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publications devoted to investigating copper-based chemotherapies lies in part with the fact copper(II) has been shown to be a highly effective chemotherapeutic agent while also remaining

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selective for cancer cell lines, and has been found to initiate tumor cell death by activating a p53-

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dependent apoptosis pathway [11], by damaging DNA via the participation of reactive oxygen species [12], and/or by inhibiting intracellular proteins such as topoisomerases and proteasomes

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[7]. Finally, given the fact copper (II) is a naturally occurring in biological species and has a permitted daily exposure (PDE) that is 25 times higher than platinum [13], copper-based drugs

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are expected to have fewer limitations in regards to systemic toxicity compared to platinumbased drugs. A comprehensive review of potential copper(II) anticancer compounds by Santini,

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et al. summarizes the wide variety of ligand scaffolds that have been employed to make copper(II) complexes, and discusses the potential cytotoxic mechanisms initiated by copperbased drugs [7].

Studies on the structural characterization and biological activity of five- and sixcoordinate copper (II) complexes possessing polypyridyl-based ligands are commonly found in the literature [14]. However, surprisingly few reports can be found in which alkyl-substituted polypyridyl ligands have been used to synthesize four-coordinate distorted tetrahedral copper (II) complexes [15], and to our knowledge no [(alkylphen)CuCl2] or [(alkylbipy)CuCl2] complexes have been evaluated as potential anticancer therapies. Hence, it was desired to determine if fourcoordinate copper (II) complexes possessing alkylphen or alkylbipy ligands might prove to be more potent antitumor agents than the previously reported gold (III) analogues [3], and the synthesis

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ACCEPTED MANUSCRIPT and characterization of a library of cupric complexes possessing alkyl-substituted polypyridyl ligands and the in vitro efficacy of these complexes is described herein.

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2. Experimental section 2.1General procedures

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2,9-di-methyl-1,10-phenanthroline , 4-methyl-1,10-phenanthroline, 6,6’-di-methyl-2,2’-

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bipyridine, 4,4’-di-methyl-2,2’-bipyridine, CuCl2 starting materials, and all solvents were

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ordered from Sigma Aldrich and were used without any further purification. All other alkylpolypyridyl ligands were synthesized as previously described [3]. The mass spectrometry

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analyses were obtained using a Linden LIFDI probe, with an extraction voltage of 11 kV and an emitter current variable from 0 – 100 mA; the Linden equipment is coupled to a Waters GCT

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which was purchased with an FD option. UV-vis spectra were obtained in a 1.0 cm quartz cuvette using a Cary 50 UV-vis spectrophotometer. IR spectra were obtained from neat solids

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using a Bruker Alpha Attenuated Total Reflectance (ATR) FT-IR Spectrometer. The elemental analyses were carried out by Atlantic Microlab Inc. (Norcross, GA). All cell culturing was conducted under sterile conditions in a laminar flow hood, and all drugs were dissolved in DMSO and sterifiltered prior to being used in sulforhodamine B (SRB) tumor cell growth assays. Absorbance readings for the SRB assays were collected using a SpectraMax Series microplate reader. 2.2 Synthesis of [(di-sec-butylphen) ClCu(µ-Cl)2CuCl(di-sec-butylphen)] (1) Using a synthesis scheme adapted from Yang, et al. [15], 100.0 mg (0.342 mmol) of 2,9di-sec-butyl-1,10-phenanthroline were weighed out into an Erlenmeyer flask. To this was added 46.0 mg (0.342 mmol) of anhydrous CuCl2 dissolved in approximately 50 mL of DCM (dichloromethane, CH2Cl2). The solution changed from colorless to an orange color within an 5

ACCEPTED MANUSCRIPT hour of stirring, and the reaction mixture was subsequently stirred overnight at ambient conditions. The next day, the solution was filtered through a glass frit and the clear orange

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solution was transferred to a glass vial. The DCM was removed by rotary evaporation, the residual orange solid was dissolved in a minimum of hot acetonitrile and allowed to cool to room

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temperature, and the orange solution was placed in a -10oC freezer for recrystallization.

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Red/orange crystals were isolated (59.9 mg, 41% yield) after 2 days. Elemental analysis

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(C40H48N4Cl4Cu2): Calculated: C = 56.27%, H = 5.75%; Experimental: C = 56.38%, H = 5.75%. IR: νmax/cm-1 3045 (CH), 2960 (CH), 2926 (CH), 2872 (CH), 1622, 1500, 863, 607. UV-vis:

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λmax (DMSO, 20°C)/nm (ε, M-1cm-1) 228 (43,779), 279 (34,376), 298 (Sh, 14, 905), 334 (Sh, 3011), 456 (Sh, 1331), 765 (65). Mass spectrometry: Molecular ion (m/z): [Cu(di-secphen)(Cl)]+ = 390.0909 amu (theoretical = 390.0919 amu); [Cu2(di-sec-butylphen)2(Cl)3]+ =

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815.1566 amu (theoretical = 815.1531 amu).

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2.3 Synthesis of [(mono-sec-butylphen)CuCl2] (2) This compound was synthesized in an analogous fashion to compound 1 by reacting 53.2 mg (0.225 mmol) of 2-sec-butyl-1,10-phenanthroline and 20.36 mg (0.225 mmol) of anhydrous CuCl2. This complex was isolated as yellow/green crystals after recrystallization from acetonitrile at -10°C over a two day period (22 mg, 26% yield). Elemental analysis (C16H16N2Cl2Cu): Calculated: C = 51.83%, H = 4.35%; Experimental: C = 51.98%, H = 4.41%. IR: νmax/cm-1 3053 (CH), 2955 (CH), 2926 (CH), 2855 (CH), 1587, 1496, 1395, 1151, 856, 732; UV-vis: λmax (DMSO, 20°C)/nm (ε, M-1cm-1) 228 (33,604), 275 (30,189), 296 (Sh, 12,561), 300(10863), 440 (380), 775 (125). Mass spectrometry: Molecular ion (m/z): [Cu(mono-secbutyl

phen)(Cl)]+ = 334.0303 amu (theoretical = 334.0293 amu); [Cu2(mono-sec-butylphen)2(Cl)3]+ =

703.0289 amu (theoretical = 703.0279 amu). 6

ACCEPTED MANUSCRIPT 2.4 Synthesis of [(di-n-butylphen)CuCl2] (3) This compound was synthesized in an analogous fashion to compound 1 by reacting 51.0

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mg (0.174 mmol) of 2,9-di-n-butyl-1,10-phenanthroline and 23.0 mg (0.174 mmol) of anhydrous

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CuCl2. This complex was isolated as red/orange crystals after recrystallization from acetonitrile

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at -10°C over a two day period (43 mg, 58% yield). Elemental analysis indicated the complex was isolated as an acetonitrile adduct. Elemental analysis (C20H24N2Cl2CuCH3CN): Calculated:

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C = 56.47%, H = 5.82%; Experimental: C = 56.90%, H = 5.61%. IR: νmax/cm-1 3341 (w), 3039

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(CH), 2953 (CH), 2925 (CH), 2856 (CH), 1442, 850, 672. UV-vis: λmax (DMSO, 20°C)/nm (ε, M-1cm-1) 243 (22,543), 279 (14,701), 296 (Sh, 8,434), 463 (Sh, 987), 781 (64). Mass

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spectrometry: Molecular ion (m/z): [Cu(di-n-butylphen)(Cl)]+ = 390.0908 amu (theoretical =

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390.0919 amu); [Cu2(di-n-butylphen)2(Cl)3]+ = 815.1561 amu (theoretical = 815.1531 amu).

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2.5 Synthesis of [(mono-n-butylphen)CuCl2] (4) This compound was synthesized in an analogous fashion to compound 1 by reacting 50.0 mg (0.212 mmol) of 2-n-butyl-1,10-phenanthroline and 28.45 mg (0.212 mmol) of anhydrous CuCl2. This complex was isolated as yellow/green crystals after recrystallization from acetonitrile at -10°C over a two day period (22 mg, 28% yield). Elemental analysis (C16H16N2Cl2Cu): Calculated: C = 51.83%, H = 4.35%; Experimental: C = 51.81%, H = 4.34%. IR: νmax/cm-1 3058 (CH), 2959 (CH), 2925 (CH), 2866 (CH), 1613, 1497, 1396, 856, 732, 648. UV-vis: λmax (DMSO, 20°C)/nm (ε, M-1cm-1) 229 (23,907), 277 (21, 443), 298 (Sh, 8,845), 334 (Sh, 1440), 345 (Sh, 967), 433 (Sh, 309), 753 (112). Mass spectrometry: Molecular ion (m/z): [Cu(mono-n-butylphen)(Cl)]+ = 334.0277 amu (theoretical = 334.0293 amu); [Cu2(mono-nbutyl

phen)2(Cl)3]+ = 703.0272 amu (theoretical = 703.0279 amu).

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ACCEPTED MANUSCRIPT 2.6 Synthesis of [(di-methylphen)Cu(H2O)Cl2] (5) This compound was synthesized in an analogous fashion to compound 1 by reacting

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500.0 mg (2.30 mmol) of 2,9-di-methyl-1,10-phenanthroline and 392.3 mg (2.30 mmol) of

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CuCl22H2O. This complex was isolated as light green crystals after recrystallization from

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acetonitrile at -10°C over a two day period (678 mg, 76% yield). Elemental analysis (C14H12N2Cl2CuH2O): Calculated: C = 46.62%, H = 3.91%; Experimental: C = 46.32%, H =

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3.88%. IR: νmax/cm-1 3218 (CH), 3194 (CH), 3183 (CH), 3169 (CH), 1503, 860, 730, 680, 529.

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UV-vis: λmax (DMSO, 20°C)/nm (ε, M-1cm-1) 286 (25,474), 337 (Sh, 2,897), (463 (Sh, 503), 806 (135). Mass spectrometry: Molecular ion (m/z): [Cu(di-methylphen)(Cl)2]+ = 305.9983 amu

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(theoretical = 305.9980 amu); [Cu2(di-methylphen)2(Cl)3]+ = 646.9623 amu (theoretical = 646.9653

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amu).

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2.7 Synthesis of [(mono-sec-butylbipy) ClCu(µ-Cl)2CuCl(mono-sec-butylbipy)] (6) This compound was synthesized in an analogous fashion to compound 1 by reacting 314.0 mg (1.48 mmol) of 6-sec-butyl-2,2’-bipyridine and 252.0 mg (1.48 mmol) of anhydrous CuCl2. The DCM solvent was removed by rotary evaporation and the green solid was redissolved in acetonitrile. Attempts to obtain X-ray quality crystals from acetonitrile were unsuccessful, therefore the solvent was removed by rotary evaporation and the resulting solid was washed multiple times with ice cold deionized water. The dark green powder was subsequently washed multiple times with cold diethyl ether, and the final product was dried in vacuo at 50°C overnight (49 mg, 9% yield). Elemental analysis indicated the complex was isolated as an acetonitrile adduct. Elemental analysis (C14H16N2Cl2CuCH3CN): Calculated: C = 49.56%, H = 4.94%; Experimental: C = 49.56%, H = 4.88%. IR: νmax/cm-1 3071 (CH), 3037

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ACCEPTED MANUSCRIPT (CH), 2964 (CH), 2928 (CH), 2873 (CH), 1560, 1445, 1159, 1023, 776, 538. UV-vis: λmax (DMSO, 20°C)/nm (ε, M-1cm-1) 293 (15,881), 316 (Sh, 8,318), 807 (90). Mass spectrometry:

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Molecular ion (m/z): [Cu(mono-sec-butylbipy(Cl)]+ = 291.9825 amu (theoretical = 291.9823 amu);

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[Cu2(mono-sec-butylbipy)2(Cl)3]+ = 618.9361 amu (theoretical = 618.9340 amu). The isolated dark green powder was re-dissolved in DCM, and light green X-ray quality crystals were obtained by

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2.8 Synthesis of [(6,6’-di-methylbipy)CuCl2] (7)

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slow evaporation over a two day period.

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This compound was synthesized in an analogous fashion to compound 1 by reacting 250.0 mg (1.36 mmol) of 6,6’ di-methyl-2,2’-bipyridine and 231.1 mg (1.36 mmol) of anhydrous

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CuCl2 . This complex was isolated as orange crystals after recrystallization from acetonitrile at -

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10°C over a two day period (72 mg, 15% yield). Elemental analysis (C12H12N2Cl2Cu):

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Calculated: C = 45.23%, H = 3.80%; Experimental: C = 44.73%, H = 3.74%. IR: νmax/cm-1 3618 (w), 3506 (w), 3090 (CH), 3058 (CH), 2923 (CH), 1599, 1481, 1332, 1015, 794, 650. UV-vis: λmax (DMSO, 20°C)/nm (ε, M-1cm-1) 297 (21,442), 305 (Sh, 16,601), 321 (Sh, 5,316), 806 (100). Mass spectrometry: Molecular ion (m/z): [Cu(6,6’-di-methylbipy)(Cl)]+ = 305.9983 amu (theoretical = 305.9980 amu); [Cu2(6,6’-di-methylbipy)2(Cl)3]+ = 646.9623 amu (theoretical = 646.9653 amu). 2.9 Synthesis of [(4,4’-di-methylbipy)CuCl2] (8) This compound was synthesized in an analogous fashion to compound 1 by reacting 250.0 mg (1.36 mmol) of 4,4’ di-methyl-2,2’-bipyridine and 231.0 mg (1.36 mmol) of anhydrous CuCl2 . This complex was isolated as blue/green crystals after recrystallization from methanol at -10°C over a two day period (236 mg, 49% yield). Elemental analysis (C12H12N2Cl2Cu): Calculated: C = 45.23%, H = 3.80%; Experimental: C = 45.05%, H = 3.75%. IR: νmax/cm-1 3064 9

ACCEPTED MANUSCRIPT (CH), 3055 (CH), 2973 (CH), 2918 (CH), 2186 (w), 2162 (w), 1613, 758, 491, 423. UV-vis: λmax (DMSO, 20°C)/nm (ε, M-1cm-1) 216 (18,957), 255 (Sh, 7,495), 300 (10,330), 309 (9,973), 731

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(109) Mass spectrometry: Molecular ion (m/z): [Cu(4,4’-di-methylbipy)(Cl)]+ = 281.9973 amu

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(theoretical 281.9980 amu).

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2.10 X-ray Crystallography

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Single crystals were coated with paratone oil and mounted on a cryoloop glass fiber. Xray intensity data were collected at 100(2) K on a Bruker APEX2 [14] platform-CCD X-ray

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diffractometer system (fine focus Mo-radiation, λ = 0.71073 Å, 50 kV/35 mA power). The CCD detector was placed at a distance of 5.0800 cm from the crystal. For complex 1, a total of 3600

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frames were collected for a sphere of reflections (with scan width of 0.3° in ω, starting ω and 2θ

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angles at −30°, and ϕ angles of 0°, 90°, 120°, 180°, 240°, and 270° for every 360 frames, 20

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s/frame exposure time). For complex 2, a total of 4800 frames were collected for a sphere of reflections (with scan width of 0.3o in ω and φ, starting ω and 2θ angles of –30o, and φ angles of 0o, 90o, 120o, 180o, 240o, and 270o for every 600 frames, 1200 frames with φ-scan from 0-360o, 20 sec/frame exposure time). For complexes 5-7, a total of 2880 frames were collected for a sphere of reflections (with scan width of 0.5o in ω and φ, starting ω and 2θ angles of –30o, and φ angles of 0o, 90o, 120o, 180o, 240o, and 270o for every 360 frames, and 720 frameswith φ-scan from 0o-360o, 10 sec/frame exposure time). For complex 8, a total of 6000 frames were collected for a sphere of reflections (with scan width of 0.3o in ω and φ, starting ω and 2θ angles of –30o, and φ angles of 0o, 45o, 90o, 120o, 180o, 225o, 240o, and 270o for every 600 frames, and 1200 frames with φ-scan from 0-360o, 10 sec/frame exposure time). The frames were integrated using the Bruker SAINT software package [17] and using a narrow-frame integration algorithm. Absorption corrections were applied to the raw intensity data using the Siemens Area-Detector 10

ACCEPTED MANUSCRIPT ABSorption (SADABS) correction program [18]. The Bruker SHELXTL software package [19] was used for phase determination and structure refinement. Direct methods of phase

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determination followed by two Fourier cycles of refinement led to an electron density map from

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which most of the non-hydrogen atoms were identified in the asymmetric unit of the unit cell. With subsequent isotropic refinement, all of the non-hydrogen atoms were identified. Atomic

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coordinates, isotropic and anisotropic displacement parameters of all the non-hydrogen atoms

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were refined by means of a full-matrix least-squares procedure on F2. The H-atoms were included in the refinement in calculated positions riding on the atoms to which they were

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attached.

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2.11 SRB Colorimetric Assays

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To test the effects of compounds 1–8 on the growth of A549 lung cancer cells,

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sulforhodamine B (SRB) cytotoxicity assays were done as described by Skehan, et al. [20] Cells were cultured in Roswell Park Memorial Institute (RPMI) media supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% L-glutamine. The cells were collected, and diluted in RPMI media such that the cells could be seeded in 96-well plates at a density of 4000 cells/well. The 96-well plate was incubated overnight at 37 °C under a 5% CO2 atmosphere. Sterifiltered DMSO stock solutions of the drugs were added to the wells in various concentrations (0–25 µM; drug concentrations were calculated assuming monomeric species were present in solution) and the 96-well plate was incubated at 37 °C under a 5% CO2 for an additional 72 hours. The supernatant cell culture medium was then removed and the cells were fixed for 1 hour with 10% cold trichloroacetic acid (100 μL per well). The trichloroacetic acid was discarded and the plates were washed 5 times with de-ionized water and air dried. After being stained with 0.4% SRB (50 μL per well) and incubated at room temperature for 10 11

ACCEPTED MANUSCRIPT minutes, the cells were washed 5 times with 1% acetic acid and air dried. The bound SRB was dissolved in 10 mM unbuffered Tris, pH 10.5 (100 μL per well) for 10 minutes at room

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temperature, and the absorbance at 492 nm was measured using a microplate reader. The percent

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cell growth was then calculated based upon the absorbance values relative to control cells not containing any drug. Each drug concentration was plated in triplicate to yield a percent growth

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vs. drug concentration curve, and these growth curves were subsequently repeated two additional

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times. The growth curves from the three experiments were then plotted in GraphPad Prism and best-fit curves were used to generate the IC50 values for each experiment. SRB assays were

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conducted in an analogous fashion with non-cancerous human foreskin fibroblast (HFF) cells. Each compound was plated in triplicate at one concentration and the percentage growth of the

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HFF cells was compared to percentage growth of the A549 at the same concentration.

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2.12 UV-vis Absorption Spectroscopy – Buffer Stability and Temperature Dependent Spectra For buffer stability testing, a 3.0 x 10-3 M stock solution in DMSO was made for compounds 1-8. Aliquots of the DMSO stock solutions were added to 0.10 M phosphate buffer (pH 7.4) in order to make final working solutions of 3.0 x10-5 M, and these solutions were placed in a 1.0 cm quartz cuvette. In order to determine if the complexes undergo decomposition in a biological medium, a Cary 50 UV-vis spectrophotometer was used to obtain spectra once every hour for a 20-24 hour period. For the temperature-dependent absorption measurements, a 3.0 x 10-3 M solution in DMSO was made for compounds 6 and 7. These solutions were placed in a 1.0 cm quartz cuvette, and the UV-vis spectra were obtained on a Varian Cary 500 Scan UV-Vis-NIR Spectrophotometer

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ACCEPTED MANUSCRIPT equipped with a Peltier Temperature Controller. The spectra were obtained between a temperature range of 25oC and 85oC in 10oC intervals.

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3. Results and Discussion:

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3.1 Synthesis and characterization

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Compounds 1-8 were synthesized following a procedure adapted from Yang, et al. [15],

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in which anhydrous CuCl2 or CuCl22H2O was reacted with one equivalent of the appropriate polypyridyl ligand in dichloromethane solvent (see Scheme 1). After stirring overnight, the

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compounds were recrystallized from acetonitrile (1-5, 7) or methanol (8) to yield analytically pure solids that were green/yellow or orange in color. An analytically pure sample of compound

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6 was obtained by removing the acetonitrile solvent by rotary evaporation, washing the resulting

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solid multiple times with cold water and then diethyl ether, and drying the solid in vacuo. The

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ratio of polypyridyl ligand to copper for all of the complexes was confirmed by elemental analysis and mass spectrometry, though it is noted both monomeric and dimeric species were observed in the mass spectra for compounds 1-7. It is likely the monomeric and dimeric species both exist in equilibrium in solution, as [Cu(polypyridyl)Cl]+ and [Cu2(polypyridyl)2Cl3]+ fragments were detected in the mass spectra of the complexes. Though the elemental analysis percent composition data cannot differentiate between the monomeric and dimeric species, the general formulae [(Rpolypridyl)CuCl2]x (x = 1 or 2) were confirmed (see experimental section). The UV-Vis absorption spectra for compounds 1-8 can be generalized by intense absorption maxima at 290-300 nm, intermediate absorption maxima at 300-450 nm, and weak absorption maxima at 750-850 nm. These are tentatively assigned as intraligand transitions, metal-to-ligand charge transfer bands, and d-d transitions, respectively, and these absorption

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ACCEPTED MANUSCRIPT maxima correspond to the previously reported UV-Vis spectra of copper(II) complexes possessing N,N-diisopropylpicolinamide ligands [21]. Given the fact the mass spectrometry data

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indicated an equilibrium mixture of monomeric and dimeric species exists in solution, and

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because thermochromic copper(II) complexes have been previously reported and exhibit interesting spectroscopic properties [22], preliminary variable temperature UV-Vis experiments

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were carried out to confirm if dynamic exchange between monomer and dimer is observed in

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solution. Indeed, the temperature-dependent UV-vis experiments revealed compounds (6) and (7) possess thermochromic isosbestic points in which the absorption maxima at 800-850 nm

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decreased with increasing temperature, while the absorption maxima at 450-500 nm increased with increased temperature (see Appendix 4). It is proposed the changes in these absorption

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maxima correspond to a temperature-dependent change in the concentrations of the monomeric and dimeric species, though it is possible the temperature dependent isosbestic points could arise

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due to solvent coordination effects. 3.2 X-ray Crystallography

Single crystals suitable for X-ray diffraction were obtained for compounds 1, 2, and 5-8. Compounds 2 and 8 were isolated as yellow/green crystals and compound 7 was isolated as orange crystals, and all three were found to possess distorted tetrahedral geometries in which the copper(II) centers are coordinated by one polypyridyl ligand and two chloro ligands (see Figure 1C, 1D, and 1E for compounds 2, 7, and 8, respectively). The bond angles range from 82.58º137.12º for compounds 2 and 7, whereas they range from 80.56º-160.70º for compound 8. The geometry for compound 8 is distorted such that it might be more appropriate to classify it as a distorted see-saw shape. Regardless of the semantics involved in naming the geometries/shapes of compounds 2, 7, and 8, Jahn-Teller distortion of tetrahedral copper(II) d9 complexes has been 14

ACCEPTED MANUSCRIPT previously observed in the literature [23], and a copper(II) complex possessing a substituted phenanthroline ligand reported by Yang, et al. {[(dpp)CuCl2]); dpp = 2,9-diphenyl-1,10-

PT

phenanthroline} was found to have angles about the copper(II) center ranging from 80.56º-

RI

104.90º [15]. Similar distorted tetrahedral geometries were also observed with the copper(II) complexes possessing tetraalkoxyaluminate ligands, in which the angles around the copper(II)

SC

center ranged from 78.0º - 139.0º [23]. Those previous findings generally align with the distorted

NU

tetrahedral geometry reported here, though it appears compound 8 undergoes an unusually high degree of distortion. To better compare the distortion in geometry, the four coordinate geometry

MA

index (τ4) [24] has been calculated for compunds 2, 7, and 8 and compared to previously reported (polypyridyl)CuCl2 complexes (see Table 2). The τ4 indices confirm the notion that

D

compound 8 (τ4 = 0.60) is the most severely distorted from ideal tetrahedral and approaches the

TE

see saw geometry (τ4 for the see saw geometry = 0.43). More interestingly, the steric bulk of the

diphenyl)

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polypyridyl substituents does not appear to correlate to increased geometric distortion, as the (2,9phenCuCl2 (τ4 = 0.68) and (2,9-di-naphthyl)phenCuCl2 (τ4 = 0.69) complexes [15] are less

distorted from ideal tetrahedral geometry than (mono-sec-butyl)phenCuCl2 (τ4 = 0.65) complex (2). Additionally, the unsubstituted (bipy)CuCl2 (τ4 = 0.57) complex reported by Wang [25] is more distorted from ideal tetrahedral geometry than compounds 7 and 8, and the [(di-methylphen)CuCl2] complex reported by Wang and Zhong [26], and the (6,6’-di-methylbipy)CuCl2 (τ4 = 0.65) complex (7) is less distorted than the less sterically demanding (4,4’-di-methylbipy)CuCl2 complex (8). It appears from these data the electronic properties of the polypyridyl ligands more strongly impacts the geometric parameters for these four-coordinate complexes. For compound 2, the Cu-N interatomic distances ranged from 1.9919(18)-2.0253(18) Å, while the Cu-Cl interatomic distances ranged between 2.2037(6)-2.2168(6) Å. The interatomic

15

ACCEPTED MANUSCRIPT distances for Cu-N in compound 7 were between 1.9779(13)-2.0196(13) Å, and the Cu-Cl distances ranged from 2.2067(4)-2.2309(4) Å. In compound 8, the Cu-N interatomic distances

PT

were noted to be between 1.9955(10)-2.0198(11) Å, while for Cu-Cl interatomic distances varied

RI

between 2.2336(3)-2.2336(3) Å. The Cu-N interatomic distances for compounds 2, 7, and 8 generally fall within the same range, though it is noted the Cu-Cl distances in compound 8 are

SC

slightly elongated compared to compounds 2 and 7. The ligand scaffold does not seem to be the

NU

primary factor in regards to the level of geometrical distortion observed, as compound 2 possesses a phenanthroline-based ligand whereas compound 7 possesses a bipyridine-based

MA

ligand. However, it is apparent subtle changes to the alkyl substituents on the bipyridine ligand can significantly impact the geometry around the copper(II) center, given the fact compound 8

TE

D

(4,4’-dimethylbipyridine ligand) has significantly more pronounced Jahn-Teller distortion and slightly longer Cu-Cl interatomic distances compared to its structural analogue compound 7

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(6,6’-dimethyl-bipyridine ligand). In comparing the interatomic distances of the [(dpp)CuCl2] complex reported by Yang, et al. [Cu-N = 2.012(2)-2.023(2) Å; Cu-Cl = 2.200(1)-2.217(1) Å] [15] with the distorted tetrahedral copper(II) complexes reported here, it is observed that compounds 2, 7, and 8 have similar Cu-N distances, though compound 8 appears to have slightly elongated Cu-Cl distances.

In contrast to compounds 2, 7, and 8, compound 5 was isolated as green crystals, and was found to possess a five-coordinate, distorted square pyramidal geometry in the solid state (see Figure 1F). The copper center is coordinated by the dimethylphen ligand, two chloro ligands, and a coordinating water molecule. The distorted square pyramidal geometry in compound 5 is characterized by the square planar base, which is comprised by one nitrogen donor from the methyl

phen ligand, the two chloro ligands, and the aqua ligand. The angles in the square planar

16

ACCEPTED MANUSCRIPT base range from 86.59º-94.82º. The axial position in the distorted square pyramidal geometry is occupied by the second nitrogen donor from the methyphen ligand. This axial ligand is distorted

PT

from the ideal 90º angle with the square planar base, evidenced by the O-Cu-N angle of 107.31º and the N-Cu-N angle of 80.11º. This distortion was previously observed in a copper(II) complex

RI

ion reported by Singh, et al. {[Cu2(Pyz)2(μ-Cl)2][ClO4]2; Pyz = N-{(Pyrazol-1-yl)methyl}-N-

SC

benzyl-2-(pyridin-2-yl)ethanamine}, in which the angles of the axial ligand were found to be

NU

79.3º and 162.7º [27]. The five coordinate geometric index (τ5) [28] was calculated for compound 5, and confirms this complex adopts what is best described as distorted square

MA

pyramidal (τ5 = 0.27; see Table 2).

D

(The Cu-N interatomic distances in compound 5 are also notable, as the axial Cu-N

TE

distance is longer than the Cu-N distance in the equatorial plane (Cu-Nax = 2.2326(15) Å, Cu-Neq = 1.9901(12) Å). Despite the fact the Cu-Nax interatomic distance is significantly longer than the

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Cu-N distance generally observed for polypyridyl nitrogen donor ligands bonded to copper(II), the Cu-Nax interatomic distance is less than the sum of the van der Waal’s radii (~ 2.95 Å). Though this type of asymmetry in the Cu-N interatomic distances was observed in the [Cu2(L)2(μ-Cl)2][ClO4]2 complex ion reported by Singh, et al., the asymmetry was not as pronounced (Cu-Neq = 1.977(4) Å and Cu-Nax = 2.085(4) Å) [27]. It is also noted the alkylsubstituted polypyridyl ligands have been previously observed to yield distortion of the axial ligand bond angles and asymmetric metal-nitrogen interatomic distances in pseudo-five coordinate gold(III) complexes [2,3]. More generally, the Cu-N interatomic distances for compound 5 are within the normal range reported for copper(II) complexes in a distorted square pyramidal geometry, though the Cu-Cl interatomic distances appear to be slightly elongated compared to the [Cu(Py3A)(Cl)(CH3OH)] (Py3A = N-(bis(2-pyridyl)methyl)-2-

17

ACCEPTED MANUSCRIPT pyridinecarboxamide) complex previously reported by Rowland, et al. {Compound 5: Cu-N = 1.9901(12)-2.2326(15) Å; Cu-Cl = 2.3030(4)-2.3048(4) Å; [Cu(Py3A)(Cl)(CH3OH)]: Cu-N =

PT

1.9223(15)-2.0316 (14) Å; Cu-Cl = 2.2253(5) Å} [29].

RI

Compounds 1 (orange crystals) and 6 (light green crystals) were unexpectedly found to

SC

possess a dimeric structure in the solid state (see Figure 1A and 1B). Generally, in compounds 1 and 6 the two copper(II) centers are bridged by two chloro ligands, and each metal center

NU

possesses a polypyridyl ligand and one terminal chloro ligand. For compound 1, the two copper centers adopt what can be described as a distorted square pyramidal geometry, in which the

MA

square planar base exhibits angles ranging from 92.78º-93.32º, and the axial N donor from the disec-butyl

D

phen ligand is elongated (Cu-Nax = 2.2561(14) Å; Cu-Neq = 1.9982(14) Å) and distorted

TE

slightly out of the ideal 90º axial plane (N-Cu-Cl = 79.51º; N-Cu-Cl = 110.70º). The distorted square pyramidal geometry for the copper centers in compound 1 is generally analogous to that

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observed in compound 5, though it is noted the Cu-Nax interatomic distance is slightly longer in compound 1. This is presumably due to the increased steric bulk of the di-sec-butylphen ligand compared to the di-methylphen ligand. The τ5 geometric index for compound 1 (τ5 = 0.13) indicates there is less distortion from ideal square pyramidal geometry around the copper(II) centers in compound 1 compared to compound 5 (see Table 2). Though an analogous dimeric copper(II) complex possessing polypyridyl-based ligands was not found in the literature, the [Cu2(Pyz)2(μCl)2][ClO4]2 complex ion reported by Singh, et al. does provide an example of a copper(II) dimer possessing a polydentate nitrogen donor ligand and bridging chloro ligands [27]. This complex ion also possesses a distorted square pyramidal geometry about the two copper centers, though the asymmetry in the Cu-N distances is not as distinct as that observed in compound 1 {[Cu2(Pyz)2(μ-Cl)2][ClO4]2; Cu-N = 1.977(4)-2.085(4) Å} [27]. The distortion of the axial

18

ACCEPTED MANUSCRIPT nitrogen donor from the ideal 90º angle with the square planar base is similar to that observed in compound 1 and compound 5 {[Cu2(Pyz)2(μ-Cl)2][ClO4]2; N-Cu-N = 79.3º; N-Cu-Cl = 102.1º}

PT

[27]. It is noted copper(II) dimeric structures with five-coordinate geometries have been previously reported with other ligand systems. For example a copper(II) dimer possessing

RI

terminal dimethylphen and chloro ligands, with bridging methoxy and phenoxy ligands was

SC

described by Boldron, et al. [30], and a copper(II) dimer possessing axial oxygen donors from

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the N,N-diisopropylpicolinamide ligand and bridging chloro ligands was reported by Lumb, et al. [21].

MA

Though compound 6 also possesses a dimeric structure in the solid state, the coordination environment of the two copper(II) centers in this complex differs from that observed in

TE

D

compound 1 and the strength of the interaction of the monomer subunits appears to be weaker in compound 6 (see Figure 1B). Compound 6 might be alternately viewed as two identical weakly

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interacting monomeric units, evidenced by the asymmetry in the interatomic distances of the bridging chloride ligands. The terminal chloro ligands have Cu-Cl distances of 2.2684(8) Å, while the bridging chloro ligands have Cu-Cl distances of 2.2923(7) Å and 2.5094(7) Å. The latter is significantly longer than the other two Cu-Cl interatomic distances but still shorter than the sum of the Van der Waals radii for copper and chlorine (~ 3.15 Å). The notion the monomeric units interact more weakly in compound 6 compared to compound 1 is further illustrated by comparing the asymmetry in the Cu-N and Cu-Cl distances; the Cu-N distances in 6 are more symmetrical {6: Cu-N = 2.033(2) Å and 2.040(2) Å; 1: Cu-N = 1.9982(14) Å and 2.2561(14) Å } whereas the Cu-Cl distances are less symmetrical {6: Cu-Cl = 2.268(7)(2) Å, 2.2923(8) Å, and 2.5094(7) Å; 1: Cu-Cl = 2.2741(4) Å, 2.2880(4) Å, and 2.3750(4) Å }. From the dimeric perspective, the two copper(II) centers in compound 6 adopt an approximately

19

ACCEPTED MANUSCRIPT trigonal bipyramidal geometry. The N(2)-Cu(1)-Cl(2) axes possess a bond angle of 172.43(6)º, while the three equatorial bond angles are 120.78(3)º, 131.76(6)º, and 107.41(6)º. The angles

PT

between the equatorial and axial atoms range from 80.58(9)-95.24(6)º. The τ5 geometric index for the copper(II) centers in compound 6 confirms the assignment of the distorted trigonal

RI

bipyramidal geometry (τ5 = 0.86; see Table 2). Analogous to compound 8, the monomeric

SC

subunits in compound 6 are more accurately described as having a see-saw geometry, with the

NU

monomers in 6 being even more distorted from the ideal tetrahedral geometry [the axial N(2)Cu(1)-Cl(2) angle is 172.43º and the angle of the N(1)-Cu(1)-Cl(1) base of the see saw is

MA

131.76(6)º]. The τ4 geometric index for the copper(II) center in the monomeric unit of compound 6 (τ4 = 0.57; see Table 2) indicates this approximates the see saw geometry. It is noted the light

TE

D

green color of compound 6 contrasts to the orange color of compound 1, and is likely due to the distinctive difference of the distorted square pyramidal coordination geometry in 1 that arises

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from the stronger interaction of the monomer subunits. However, no discernable trend can be found in terms of correlating the color of complexes 1-8 to their general structural properties (the dimeric complex 1 and monomeric complex 7 were isolated as orange crystals, whereas the dimeric complex 6 and monomeric complexes 2, 5, and 8 were isolated as green crystals). 3.3 In vitro Antitumor Activity

In order to quantify the anticancer efficacy of the library of copper complexes reported herein, sulforhodamine B (SRB) assays were used to obtain the 50% inhibitory concentration (IC50) values of each drug candidate. The antitumor activity of the copper(II) complexes was then compared to the free polypyridyl ligand activity, the activity of a previous gold(III) complex possessing the di-sec-butylphen ligand {[(di-sec-butylphen)AuCl3]} [2,3], and the clinically used drug cisplatin (see Table 3). It is noted the samples for the copper complexes 1-8 used for the SRB 20

ACCEPTED MANUSCRIPT experiments were prepared under the assumption monomeric species were present in solution. Even though previous SRB analyses found the free di-sec-butylphen ligand had more pronounced in-

PT

vitro antitumor activity than the corresponding gold(III) complex [(di-sec-butylphen)AuCl3] [1],

RI

more recent SRB analysis of a broader library of gold(III) complexes possessing alky-substituted phen and bipy ligands indicated the antitumor activity of the gold complexes was not dependent

SC

on the activity of the free ligand [3]. These data led to the desire to test analogous complexes

NU

with different metal centers, with the aim being to further test the notion the activity of metal-

search for more potent drug candidates.

MA

based drugs is not dependent on the activity of the free polypyridyl ligands, and to expand the

D

In general, the cupric complexes demonstrate much higher activity (i.e., lower IC50

TE

values) than the corresponding free ligands (see Table 3), demonstrating the metal-ligand complex in these metallotherapies likely plays a significant role in how these compounds inhibit

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cancer growth. It is also highlighted that most of the copper(II) complexes reported here have significantly more pronounced antitumor activity than the previously reported gold(III) complex, [(di-sec-butylphen)AuCl3], and the clinically used drug cisplatin (see Table 3). Most noteworthy are complexes 1, 5, and 7, all of which had an IC50 value below 0.2 µM. This activity is at least 23 times greater than that observed with cisplatin, and at least 1.7 times greater than that of the most potent gold(III) complex previously reported in our lab (see Table 3). Compounds 1-8 also appear to have more pronounced antitumor activity than previously reported copper(II) complexes possessing different ligand frameworks. Imidazophenanthroline copper(II) complexes previously reported by Nagababu, et al. were found to have IC50 values with A549 tumor cells ranging from 0.6-38.2 µM [31], and dinuclear copper(II) complexes bearing bis(triazacyclonane)

21

ACCEPTED MANUSCRIPT ligands reported by Montagner, et al. had IC50 values ranging from 11-90 µM against a panel of six different tumor cell lines [10].

PT

Even though the antiproliferative effects of the copper(II) complexes reported here are

RI

generally more promising than the free polypyridyl ligands and previously reported gold(III)

SC

complexes, the activity of the copper-based drugs is not consistent across the general class of polypyridyl ligands. There is a 25-fold difference between the most active and least active drugs,

NU

and in fact the most potent compounds reported here are not restricted to a single ligand class. Of

MA

the three complexes with IC50 values less than 0.2 µM, two of these possess phenanthrolinebased ligands, while the third possesses a bipyridine-based ligand. As previously reported with

D

alkyl-substituted polypyridyl gold(III) complexes, subtle modifications to the ligand backbone

TE

impart significant changes to the antitumor efficacy of the drugs. These results provide further evidence that the antitumor efficacy of metallotherapeutic drugs possessing alkyl-substituted

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phenanthroline and bipyridine ligands is not dependent on the inherent activity of the free ligands, and suggest finding the right combination of metal center and polypyridyl ligand can lead to enhanced drug activity.

Given the fact the copper complexes reported herein had such pronounced cytotoxic activity, it was prudent to determine if this class of compounds might also demonstrate antiproliferative or toxic effects against normal cells. Therefore, non-cancerous human foreskin fibroblast (HFF) cells were tested against the panel of copper drugs. The percent growth of HFF cells in the presence of a specific concentration of copper complex was compared to the percent growth of A549 cells treated with the same concentration of drug (see Figure 2 and Appendix 1). Even at concentrations greater than the IC50 value for each of the copper drugs, HFF cell growth was inhibited at a significantly lower rate than the A549 tumor cells. The HFF growth was 3-10 22

ACCEPTED MANUSCRIPT times higher compared to the growth of the A549 cells, and a Student’s T-test was carried out to confirm these differences in growth were statistically significant (see Figure 2 and Appendix 1).

PT

These results suggest this class of copper compounds has a suitable toxicity window in which

RI

normal cells are likely to be less affected by drug treatment. Given that previous reports indicate copper(II) complexes likely kill tumor cells by cleaving DNA via oxidation by reactive oxygen

SC

species or through intercalation into DNA base pairs, [32] the observation that fast-replicating

NU

tumor cells are more sensitive to the drug treatment is not completely unexpected. 3.4 Stability of copper(II) complexes

MA

The observation that compounds 1-7 exhibited antitumor efficacy greater than the corresponding free ligands suggests there is limited dissociation of the alkylphen or alkylbipy ligand

TE

D

from the copper(II) center in the cellular environment. However, it was desired to carry out additional stability studies and in vitro control experiments in order to further corroborate the

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notion the antiproliferative effects of the alkylpolypyridyl copper(II) complexes does not arise due to the release of free ligand and/or CuCl2 species. In a previous study of potential copper(II) drugs it was found CuCl2 had an IC50 of approximately 100 µM with A549 lung cancer cells [33], well above the IC50 levels observed in compounds 1-8. However, under the experimental conditions used here, CuCl2 inhibited A549 cell growth to a greater extent than reported by Primik, et al. [33]. Therefore, the growth of A549 tumor cells at concentrations near the IC50 values for compounds 1-8 was compared to the growth of these cells in the presence of CuCl2 at the same concentrations (see Figure 3 and Appendix 2). A qualitative comparison clearly indicates compounds 1-7 yield greater inhibition of tumor cell growth compared to CuCl2, and a Student’s t-test confirms the lower cell growth

23

ACCEPTED MANUSCRIPT for compounds 1-7 is statistically significant. It is noted compound 8 was observed to possess an IC50 value higher than that of CuCl2 (IC50 = 5.0 μM and 1.8 μM, respectively).

PT

Even though the fact CuCl2 has a significantly decreased effect on tumor cell growth

RI

compared to compounds 1-7, suggesting the efficacy of the copper(II) compounds does not arise

SC

from the release of a CuCl2 species, this does not preclude the possibility the copper polypyridyl complexes decompose in the intracellular environment and lead to an antitumor mechanism

NU

involving the synergistic action of CuCl2 and the free polypyryidyl ligand. Thus, a control

MA

experiment was carried out in which the fraction of cell growth for compound 7 at 0.2 μM was compared to the cell growth in the presence of 0.2 μM CuCl2, 0.2 μM 6,6’-dimethylbipy ligand,

D

and a physical mixture of 0.2 μM CuCl2 and 0.2 μM 6,6’-dimethylbipy. Even though binary

TE

copper(II) complexes with phen [(phen)CuX2] and bipy [(bipy)CuX2] ligands have been observed to possess extremely large binding constants (Kb ≈ 109 and 108, respectively) [34], the

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formation of [(alkylpolypyridyl)CuCl2] species from the physical mixture of ligand and CuCl2 in the biological milieu used for the cell growth was considered unlikely. The data from this control experiment clearly indicates the individual CuCl2 and free bipy ligand, as well as the physical mixture of the two species inhibit tumor cell growth to a much smaller extent than the [(6,6’dimethyl

bipy)CuCl2] complex, and a Student’s t-test confirms the decreased cell growth for the

copper(II) complex 7 is statistically significantly (see Figure 4). This control experiment indeed provides additional evidence the antitumor activity of the polypyridyl copper(II) complexes likely results from the intact complexes, and not due to the resulting ligand and/or copper species that would be present after complex dissociation. Finally, in order to provide further evidence the alkylphen and

alkyl

bipy copper(II)

complexes do not dissociate into free ligand and CuCl2 species in the cellular environment, the 24

ACCEPTED MANUSCRIPT stability of compounds 1-8 were monitored in the presence of phosphate buffer over a 20-24 hour period (see Figure 5 and Appendix 3). Though these experiments do not directly mimic the

PT

in vitro cellular solution conditions and the concentrations of the copper(II) complexes are

RI

significantly higher in these experimental conditions, these stability experiments at least provide some preliminary insight about the likelihood of complex dissociation in a physiological buffer

SC

environment. None of the compounds appeared to undergo any decomposition pathways

NU

involving dissociation of the polypyridyl ligand, as no significant changes in the absorption maxima for the intraligand (270-290 nm) or LMCT (400-500 nm) bands were observed.

MA

Compounds 1, 3, and 5 did exhibit an isosbestic point that resulted from a small decrease in the intraligand absorption intensity and concomitant increase in the LMCT absorption intensity (see

TE

D

Figure 5 and Appendix 3), however this is likely attributed to a change in the coordination environment in the aqueous solvent and not a release of the polypyridyl ligand. The remaining

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complexes undergo slow and subtle changes in concentration over a 20-24 hour time period, and no dissociation of the polypyridyl ligand is observed (see Figure 5 and Appendix 3). These results are in agreement with the previous report in which it was found the binary polypyridyl copper(II) complexes, [(phen)CuX2] and [(bipy)CuX2] have binding constants on the order of 108 and 109, respectively [34]. The changes in concentration observed in phosophate buffer are attributed to slight changes in the solubility of the copper(II) complexes in aqueous solvent, and changes in the absorption intensity arise due to either decreased solubility and/or changes in optical density (e.g., compound 7 undergoes an immediate increase in absorption intensity at all wavelengths due to an increase in optical density, followed by a slow decrease in absorption intensity over all wavelengths as the insoluble species settle out of solution; see Appendix 3). In summary, there is no evidence indicating the polypyridyl ligand undergoes complete dissociation

25

ACCEPTED MANUSCRIPT in biological buffer for any of the reported complexes, providing further evidence this class of metallotherapies likely initiates tumor cell death via a mechanism that involves the polypyridyl-

PT

copper(II) complex.

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4. Conclusion

SC

We have reported herein a facile synthetic approach that provides an opportunity to

NU

access copper(II) complexes possessing an array of alkyl-substituted phen and alkyl-substituted bipy ligands. Although this class of coordination complexes has been relatively unexplored, they

MA

do adopt distorted tetrahedral, distorted square pyramidal, or trigonal bipyramidal geometries often found with copper(II). Most notably, the copper(II) complexes reported herein possess

D

quite remarkable in vitro antitumor activity and selectivity for cancerous cells, and appear to

TE

initiate tumor cell death via a mechanism in which the copper(II) center remains coordinated to

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the polypyridyl ligand. However there is evidence these complexes undergo dynamic structural changes in solution that involve monomer/dimer exchange and/or changes in solvent coordination. Hence, future studies need to focus on learning more about the structural properties of these copper(II) complexes in solution, determining if there are structure-function relationships that can predict the antitumor efficacy of specific complexes, and elucidating the mechanism by which these metallotherapies initiate tumor cell death. Given the advantages copper(II) compounds potentially possess over platinum-based drugs, continuing to explore this new class of anticancer metallotherapies is warranted. 5. Acknowledgements We would like to acknowledge the National Institute of Health as well as the Maximizing Access to Research Careers program for funding this research. This project was supported by 26

ACCEPTED MANUSCRIPT Award Number T34GM062756 from the National Institute of General Medical Sciences. Additionally, we would like to acknowledge the University of California, Riverside Analytical

PT

Chemistry Instrumentation Facility and Ron New for performing the mass spectrometry

RI

analyses. Finally, we would like to thank the anonymous reviewers for their suggested additions

SC

and edits, which vastly improved the quality of the final manuscript.

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6. List of Abbreviations phen = 1,10-phenanthroline

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bipy = 2,2’-bipyridine

D

PDE = permitted daily exposure

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FD = field desorption

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LIFDI = Liquid Introduction Field Desorption Ionization

Waters GCT = time-of-flight based mass spectrometer UV-vis = ultraviolet-visible IR = infrared SRB = sulforhodamine B DCM = dichloromethane CCD = charge-coupled device A549 = human derived non-small cell lung cancer, adenocarcinoma

27

ACCEPTED MANUSCRIPT RPMI = Roswell Park Memorial Institute cell culture media

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DMSO = dimethyl sulfoxide

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HFF = human foreskin fibroblast cells

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IC50 = inhibitory concentration 50%

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LMCT = ligand-to-metal charge transfer 7. Works Cited

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[1] M. G. Apps, E. H. Y. Choi, N.J. Wheate, End. Can., 2015, 22(4), R219-R233.

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[2] C. D. Sanghvi, P. M. Olsen, C. Elix, S. (Bruce) Peng, D. Wang, Z. (Georgia) Chen, D. M.

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Shin, K. I. Hardcastle, C. E. MacBeth, J. F. Eichler J.Inorg. Biochem., 2013, 128, 68-76.

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[3] P. M. Olsen, C. Ruiz, D. Lussier, B. Khoa Le, N. Angel, M. Smith, C. (Brian) Hwang, R. Khatib, J. Jenkins, K. Adams, J. Getcher, F. Tham, Z. (Georgia) Chen, E. H. Wilson, J. F. Eichler, J.Inorg. Biochem., 2014, 141, 121-131. [4] L. Messori, F. Abbate, G. Marcon, P. Orioli, M. Fontani, E. Mini, T. Mazzei, S. Carotti, T. O'Connell, P. Zanello, J. Med. Chem., 2000, 43, 3541–3548. [5] M.A. Cinellu, L. Maiore, M. Manassero, A. Casini, M. Arca, H.H. Fiebig, G. Kelter, E. Michelucci, G. Pieraccini, C. Gabbiani, L. Messori, ACS Med. Chem. Lett., 2010, 1, 336–339. [6] a) C. Marzano, M. Pellei, F. Tisato, C. Santini, Medicinal Chemistry, 2009, 9, 185-211; b) S. Ebrahimipour, I. Sheikhshoaie, J. Castro, W. Hasse, M. Mohamadi, S. Foro, M. Shekhshoaie, S. Esmaeili-Mahini, Inorg. Chim. Acta, 2015, 430, 245-252.

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ACCEPTED MANUSCRIPT [7] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chem. Rev.., 2014, 114, 815-862.

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[8] S.B., Nayak, V.R. Bhat, D. Upadhyay, S.L. Udupa, Indian J. Physiol. Pharmacol. 2003, 47, 108. [9] X. Peng, V. Gandhi, Ther. Delivery, 2012, 3, 823.

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[10] a) D. Montagner, V. Gandin, C. Marzano, A. Erxleben, J. Inorg. Biochem., 2015, 145, 101-

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107; b) M.F. Primik, G. Mühlgassner, M.A. Jakupec, O. Zava, P.J. Dyson, V.B. Arion, B.K.

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Keppler, Inorg. Chem., 2010, 49 (1), 302–311; c) S.S. Bhat, A.A. Kumbhar, H. Heptullah, A.A. Khan, V.V. Gobre, S.P. Gejji, V.G. Puranik, Inorg. Chem., 2011, 50 (2), 545–558; d) R.

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Loganathan, S. Ramakrishnan, M. Ganeshpandian, N.S.P. Bhuvanesh, M. Palaniandavar, A. Riyasdeen, M.A. Akbarsha, Dalton Trans., 2015, 44, 10210-10227.

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[11] J. Liang, Y. Wang, K. Du, G. Li, R. Guan, L. Ji, H. Chao, J. Inorg. Biochem., 2014, 141, 17-

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[12]. A. Krause-Heuer, P. Leverett, A. Bolhuis, J. Aldrich-Wright, Aust. J. Chem, 2012, 65, 860-

[13] Guideline on the Specification Limits for Residues of Metal Catalysts, Committee for Human Medicinal Products, European Medicines Agency, 2007, 7 Westferry Circus, Canary Wharf, London, E14 4HB, UK. (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500 003586.pdf) [14] a) B. Wu, P. Yang, X. Huang, Y. Liu, X. Liu, C. Xia, Zeit. Anorg. Allg. Chem., 2006, 632, 684-688; b) S. Ramakrishnan, V. Rajendiran, M. Palaniandavar, V.S. Periasamy, B.S. Srinag, H. Krishnamurthy, M.A. Akbarsha, Inorg. Chem., 2009, 48 (4), 1309–1322; c) J.D. Ranford, P.J.

29

ACCEPTED MANUSCRIPT Sadler, D.A. Tocher, J. Chem. Soc., Dalton Trans, 1993, 3393-3399; d) M. Yadoshi, M. Odoko, N. Okabe, Chem. Pharm. Bull., 2007, 55, 853-860; e) O. Horvath, Coord. Chem. Rev., 1994,

PT

135-136, 303-324; e) M.T. Miller , P.K. Gantzel, T.B. Karpishin, Inorg. Chem., 1998, 37 (9),

RI

2285–2290.

SC

[15] P. Yang, X. Yang, B. Wu, Eur. J. Inorg. Chem., 2009, 2009(20), 2951.

NU

[16] APEX 2, version 2012.2.2, Bruker, Bruker AXS Inc., Madison, Wisconsin, USA, 2012. [17] SAINT, version V8.18C, Bruker, Bruker AXS Inc., Madison, Wisconsin, USA, 2011.

MA

[18] SADABS, version 2008/1, Bruker, Bruker AXS Inc., Madison, Wisconsin, USA, 2008. [19] SHELXTL, version 2008/4, Bruker, Bruker AXS Inc., Madison, Wisconsin, USA, 2008.

D

[20] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren, H.

TE

Bokesch, S. Kenney, M.R. Boyd, J. Nat. Can. Inst., 1990, 82, 107–12.

AC CE P

[21] I. Lumb, M. S. Hundal, M. Corbella, V. Gómez, G. Hundal, Eur.J. Inorg.Chem., 2013, 2013(27), 4799-4811.

[22] a) S. Choi, J. A. Larrabee, J. Chem. Educ., 1989, 66(9), 774-776; b) M. J. M. Van Oort, J. Chem. Educ., 1988, 65(1), 84.

[23] M. Veith, K. Valtchev, V. Huch, Inorg. Chem., 2008, 47(3), 1204–1217. [24] L. Yang, D.R. Powella, R. Houser, Dalton Trans., 2007, 955-964. [25] Y.Q. Wang, W.H. Bi, X. Li, R. Cao, Acta Cryst., Sec. E, 2004, E60, m876-m877. [26] B.S. Wang, H. Zhong, Acta Cryst., Sec. E, E65, m1156. [27] R. Singh, F. Lloret, R. Mukherjee, Zeit. Anorg. Allg. Chem, 2014, 640, 1086-1094.

30

ACCEPTED MANUSCRIPT [28] A.W. Addison, T.N. Rao, J. Reedijk, J. van Rijn, G.C. Verschoor, J. Chem. Soc., Dalton Trans., 1984, 1349-1356.

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[29] J. M. Rowland, M. M. Olmstead, P. K. Mascharak, Inorg. Chem., 2000, 39, 5326-5332.

SC

RI

[30] C. Boldron, D. Tooke, A. Spek, J. Reedijk, Ang. Chem., 2005, 44, 3585-3587. [31] P. Nagababu, A. Barui, B. Thulasiram, C. Devi, S. Satyanarayana, C. Patra, B. Sreedhar, J.

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Med. Chem., 2015, 58, 5226-5241.

MA

[32] a) F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Med. Res. Rev., 2010, 30(4), 708-749; b) Warren, H. Bokesch, S. Kenney, M.R. Boyd, J. Natl. Cancer Inst., (1990), 82,

D

1107–1112.

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[33] M.F. Primik, G. Muhlgassner, M.A. Jakupec, O. Zava, P.J. Dyson, V.B. Arim, B.K.

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Keppler, Inorg. Chem., 2010, 49(1), 302-311. [34] N. Turkel, C. Sahin, Chem. Pharm. Bull., 2009, 57(7), 694-699.

31

ACCEPTED MANUSCRIPT Schemes, Data Tables, and Figures

PT

Scheme 1: Synthesis of compounds 1-8.

R1 = H, R2 = sec-butyl (2) R1 = R2 = n-butyl (3) R1 = H, R2 = n-butyl (4)

NU

SC

RI

CuCl2

MA

CuCl22H2O

H2

TE

D

(5)

R1 = R2 = 6,6’-dimethyl (7) R1 = R2 = 4,4’-dimethyl (8)

AC CE P

CuCl2

CuCl2

R

R

1/2

R

(1)

CuCl2 1/2

(6)

32

R

ACCEPTED MANUSCRIPT

(1) Empirical

(2)

(5)

(6)

PT

Table 1: X-ray crystallographic data for compounds 1, 2, 5-8.

C40H48Cl4Cu2N4

C16H16Cl2CuN2

C14H14Cl2CuN2O

C28 H32 Cl4 Cu2 N4

Formula weight

853.70

370.75

360.71

Temperature

100(2) K

100(2) K

100(2) K

Wavelength

0.71073 Å

0.71073 Å

0.71073 Å

Crystal system

Monoclinic

Triclinic

Monoclinic

Space group

C 2/c (#15)

P -1 (#2)

P 21

a = 18.9314(5) Å

a = 7.7072(9) Å

a = 9.4396(3) Å

b = 12.4842(3) Å

b = 8.8724(10) Å

Unit cell

c = 16.9588(4) Å

dimensions

(7)

(8)

C12H12Cl2CuN2

C12H12Cl2CuN2

693.45

318.68

318.68

110(2) K

100(2) K

100(2) K

0.71073 Å

0.71073 Å

0.71073 Å

Triclinic

Monoclinic

Triclinic

P -1

P 21/c

P -1

a = 8.2703(4) Å

a = 7.0710(5) Å

a = 7.0026(3) Å

b = 8.0733(2) Å

b = 8.5937(4) Å

b = 9.5809(7) Å

b = 9.0676(4) Å

c = 11.8443(13) Å

c = 9.8120(3) Å

c = 11.4993(5) Å

c = 18.5036(14)Å

c = 11.1850(5)Å

 = 90°

 = 98.5854(16)°

 = 90°

 = 104.9232(7)°

 = 90°

 =108.0213(6)°

 = 99.7377(4)°

 = 97.6972(16)°

 =105.2850(5)°

 = 92.3516(7)°

 = 93.2257(12)°

 = 97.4234(6)°

 = 90°

 = 04.6855(15)°

 = 90°

 = 112.6589(6)°

 = 90°

 = 10.7754(6)°

Volume

3950.35(17) Å3

762.09(15) Å3

721.31(4) Å3

719.69(6) Å3

1251.57(16) Å3

608.23(5) Å3

Z

4

2

2

1

4

2

Density

1.435 Mg/m3

1.616 Mg/m3

1.661 Mg/m3

1.600 Mg/m3

1.691 Mg/m3

1.740 Mg/m3

1.381 mm-1

1.776 mm-1

=1.879 mm-1

1.875 mm-1

2.148 mm-1

2.210 mm-1

1768

378

366

354

644

322

0.435 x 0.121 x

0.226 x 0.156 x

0.334 x 0.236 x

0.372 x 0.211 x

0.365 x 0.141 x

0.267 x 0.152 x

0.106 mm3

0.056 mm3

0.079 mm3

0.109 mm3

0.122 mm3

0.089 mm3

1.963 to 29.129°

1.768 to 30.507°

2.152 to 30.508°

2.205 to 29.571°

1.988 to 30.508°

-25<=h<=25,

-11<=h<=10,

-13<=h<=13,

-11<=h<=11,

-9<=h<=9,

-9<=h<=9,

-17<=k<=17,

-12<=k<=12,

-11<=k<=11,

-11<=k<=11,

0<=k<=13,

-12<=k<=12,

-23<=l<=23

0<=l<=16

-14<=l<=14

-15<=l<=15

0<=l<=25

0<=l<=15

42884

7510

21203

21167

8370

4790

Absorption coefficient F(000)

SC

NU

MA

D

TE

AC CE P

(calculated)

RI

formula

Crystal size

Theta range for

1.856 to 29.130°

data collection

Index ranges

Reflections collected

33

ACCEPTED MANUSCRIPT Independent

5314 [R(int) =

4611 [R(int) =

4402 [R(int) =

3870 [R(int) =

3596 [R(int) =

3682 [R(int) =

reflections

0.0300]

0.0313]

0.0202]

0.0236]

0.0274]

0.0247]

Completeness to

100.0 %

100.0 %

99.8 %

100.0 %

100.0 %

100.0 %

Absorption

Semi-empirical

Semi-empirical

Semi-empirical

Semi-empirical

Semi-empirical

Semi-empirical

correction

from equivalents

from equivalents

from equivalents

from equivalents

from equivalents

from equivalents

Full-matrix least-

Full-matrix least-

Full-matrix

Full-matrix least-

Full-matrix

squares on F2

least-squares on

RI

Full-matrix least-

Refinement squares on F2

least-squares on F2

5314 / 216 / 282

4611 / 7 / 214

4402 / 1 / 189

squares on F2

SC

squares on F2 method

Data / restraints /

PT

theta = 25.242°

F2 3596 / 0 / 157

3682 / 0 / 157

1.048

1.047

1.068

parameters Goodness-of-fit

1.062

NU

3870 / 107 / 201

1.054

1.085

Final R indices

R1 = 0.0284, wR2

R1 = 0.0322, wR2

R1 = 0.0153,

R1 = 0.0398,

R1 = 0.0233,

R1 = 0.0199,

[I>2sigma(I)]

= 0.0676

= 0.0864

wR2 = 0.0398

wR2 = 0.0932

wR2 = 0.0558

wR2 = 0.0521

R indices (all

R1 = 0.0364, wR2

R1 = 0.0404, wR2

R1 = 0.0157,

R1 = 0.0453,

R1 = 0.0283,

R1 = 0.0211,

data)

= 0.0714

= 0.0901

wR2 = 0.0400

wR2 = 0.0968

wR2 = 0.0579

wR2 = 0.0527

Extinction

n/a

n/a

n/a

n/a

n/a

n/a

0.474 and -0.367

0.536 and -0.394

e.Å-3

e.Å-3

0.463 and -0.362 Largest diff. peak and hole

e.Å-3

D

TE

AC CE P

Coefficient

MA

on F2

0.680 and -0.514 e.Å-3

0.393 and -0.247 0.732 and -0.801 e.Å-3

e.Å-3

34

ACCEPTED MANUSCRIPT Table 2: Summary of selected τ4 and τ5 geometric indices. For τ4, a value of 1 corresponds to ideal tetrahedral geometry and a value of 0 corresponds to ideal square planar geometry [22. For τ5, a value of 1 corresponds to ideal trigonal bipyramidal geometry and a value of 0 corresponds to ideal square pyramidal [26].

NU

SC

RI

PT

τ index

AC CE P

TE

D

MA

Compound τ4 [(mono-sec-butylphen)CuCl2] (2) [(di-methylphen)CuCl2] [23] [(di-phenylphen)CuCl2] [13] [(di-naphthylphen)CuCl2] [13] [(bipy)CuCl2] [24] [(4,4’-dimethylbipy)CuCl2] (8) [(6,6’-dimethylbipy)CuCl2] (7) [mono-sec-butylbipy)CuCl3] monomer unit (6) τ5 [(di-sec-butylphen) ClCu(µ-Cl)2CuCl(di-sec-butylphen)] (1) [(mono-sec-butylbipy)ClCu(µ-Cl)2CuCl(mono-sec-butylbipy)] (6) [(2,9-di-methylphen) Cu(H2O)Cl2] (5)

35

0.65 0.69 0.68 0.69 0.57 0.60 0.65 0.57 0.13 0.86 0.27

ACCEPTED MANUSCRIPT

PT

Table 3: IC50 values for compounds 1-8 against A549 tumor cells (A549 = human derived nonsmall cell lung adenocarcinoma). For compounds in which greater than 50% cell growth was not observed, the percent growth at 0.2 µM is reported (i.e., these compounds have an IC50 value less than 0.2 µM). IC50 (μM)

RI

Complex Name

0.42±0.17

—

0.58±0.15

—

—

31.6 %

2.92±0.47

—

1.62±.022

—

1.08±0.28

—

0.211±.0098

—

0.62±0.16

—

0.342±.022

—

1.35±0.09

—

—

17.2 %

100% cell growth at 12

—

phen

mono-sec-butyl

phen

MA

[(mono-sec-butylphen)CuCl2] (2) di-n-butyl

phen

D

[(di-n-butylphen)CuCl2] (3)

di-methyl

phen

AC CE P

[(mono-n-butylphen)CuCl2] (4)

TE

mono-n-butyl

phen

NU

[(di-sec-butylphen)AuCl3] [(di-sec-butylphen) ClCu(µ-Cl)2CuCl(di-sec-butylphen)] (1)

[(2,9-di-methylphen) Cu(H2O)Cl2] (5) mono-sec-butyl

bipy

(relative to negative control)

SC

di-sec-butyl

% Growth at 0.2 μM

μM

[(mono-sec-butylbipy)ClCu(µ-Cl)2CuCl(mono-sec-butylbipy)] (6)

1.76±0.197

—

[(6,6’-dimethylbipy)CuCl2] (7)

—

15.3%

[(4,4’-dimethylbipy)CuCl2] (8)

5.00±0.144

—

Cisplatin

4.67±0.21

—

36

ACCEPTED MANUSCRIPT

PT

B

RI

A

N(1)

N(10)

Cl(2) Cu (2)

Cu(1)

NU

Cl(1)

SC

N(10)

Cl(1) Cl(2)

Cl(1A)

Cl(2B) N(1A)

N(1B)

Cu(1A) Cu(1B)

Cl(2A)

MA

N(1)

N(2A)

N(2B)

C

AC CE P

TE

D

Cl(1B)

D

N(1)

N(10)

N(10)

N(1)

Cu(1)

Cu(1)

Cl(1)

Cl(2)

Cl(2) Cl(1)

37

ACCEPTED MANUSCRIPT

E

NU

SC

RI

PT

F

N(1) N(2)

MA

Cu(1)

Cl(2)

AC CE P

TE

D

Cl(1)

38

N (10)

N (1)

Cl (2)

O (1) Cl (1)

ACCEPTED MANUSCRIPT

Figure 1: Ellipsoid representations for compounds 1, 2, 5-8, with selected interatomic distances (Å) and bond angles (o). Hydrogen atoms are omitted in Figure 1A for clarity. A (1): Cu(1)-N(1) 1.9982(14); Cu(1)-N(10) 2.2561(14); Cu(1)-Cl(1) 2.2741(4); Cu(1)-Cl(2) 2.2880(4); Cu(1)-Cl(2B) 2.375(4); N(1)-Cu(1)-N(10) 79.51(6); N(1)-Cu(1)-Cl(1)92.78(4); N(10)-Cu(1)-Cl(1) 101.35(3); N(1)-Cu(1)-Cl(2) 166.77(4); N(10)-Cu(1)-Cl(2) 110.70(4); Cl(1)-Cu(1)-Cl(2) 93.327(15); C(2)-N(1)-Cu(1)123.86(13); C(10B)-N(1)-Cu(1) 115.69(11); C(9)-N(10)-Cu(1) 133.20(12); C(10A)-N(10)-Cu(1) 107.85(11)

PT

B (6): Cu(1A)-N(2A) 2.033(2); Cu(1A)-N(1A) 2.040(2); Cu(1A)-Cl(1A) 2.2684(7); Cu(1A)-Cl(2A) 2.2923(8); Cu(1A)-Cl(2B) 2.5094(7); Cl(2A)-Cu(1B) 2.5094(7); Cu(1B)-N(1B) 2.040(2); Cu(1B)-N(2B) 2.033(2); N(2A)-Cu(1A)-N(1A) 80.58(9); N(2A)-Cu(1A)-Cl(1A) 95.29(6); N(1A)-Cu(1A)-Cl(1A) 131.76(6); N(2A)-Cu(1A)-Cl(2A) 172.43(6); N(1A)-Cu(1A)-Cl(2A) 93.56(7); Cl(1A)-Cu(1A)-Cl(2A) 92.19(3); N(2A)-Cu(1A)-Cl(2B) 91.59(7) N(1A)Cu(1A)-Cl(2B) 107.41(6); Cl(1A)-Cu(1A)-Cl(2B) 120.78(3); Cl(2A)-Cu(1A)-Cl(2B) 85.57(2);

SC

RI

C (2): Cu(1)-N(10) 1.9919(18); Cu(1)-N(1) 2.0253(18); Cu(1)-Cl(2) 2.2037(6); Cu(1)-Cl(1) 2.2168(6); N(10)-Cu(1)-N(1) 82.58(7); N(10)-Cu(1)-Cl(2) 99.26(5); N(1)-Cu(1)-Cl(2) 139.16(6); N(10)-Cu(1)-Cl(1) 137.69(5); N(1)-Cu(1)-Cl(1) 103.22(6); Cl(2)-Cu(1)-Cl(1) 102.16(2); C(2)-N(1)-Cu(1) 129.71(15); C(10B)-N(1)-Cu(1) 111.19(13)

D (7): Cu(1)-N(1) 1.9779(13); Cu(1)-N(2) 2.0196(13); Cu(1)-Cl(2) 2.2067(4); Cu(1)-Cl(1) 2.2309(4); N(1)-Cu(1)-N(2) 82.26(5); N(1)-Cu(1)-Cl(2) 137.12(4); N(2)-Cu(1)-Cl(2) 109.79(4); N(1)-Cu(1)-Cl(1) 101.35(4); N(2)-Cu(1)-Cl(1) 127.57(4); Cl(2)-Cu(1)-Cl(1) 102.240(17); C(2)-N(1)-Cu(1)

NU

125.09(11); C(6)-N(1)-Cu(1) 114.45(10); C(11)-N(2)-Cu(1) 127.13(11); C(7)-N(2)-Cu(1) 112.84(10)

E (8): Cu(1)-N(2) 1.9955(10); Cu(1)-N(1) 2.0198(11); Cu(1)-Cl(1) 2.2336(3); Cu(1)-Cl(2) 2.2336(3); N(2)-Cu(1)-N(1) 80.56(4); N(2)-Cu(1)-Cl(1)

MA

95.26(3); N(1)-Cu(1)-Cl(1) 151.49(3); N(2)-Cu(1)-Cl(2) 160.72(3); N(1)-Cu(1)-Cl(2) 94.00(3); Cl(1)-Cu(1)-Cl(2) 98.049(12); C(2)-N(1)-Cu(1) 126.88(9); C(6)-N(1)-Cu(1) 114.62(8); C(11)-N(2)-Cu(1) 125.18(9); C(7)-N(2)-Cu(1) 115.72(8); F (5): Cu(1)-O(1) 1.9579(13); Cu(1)-N(1) 1.9901(12); Cu(1)-N(10) 2.2326(15); Cu(1)-Cl(2) 2.3030(4); Cu(1)-Cl(1) 2.3048(4); O(1)-H(1W) 0.77(3);

D

O(1)-H(2W) 0.71(3); O(1)-Cu(1)-N(1) 171.27(6); O(1)-Cu(1)-N(10) 107.31(6); N(1)-Cu(1)-N(10) 80.11(5); O(1)-Cu(1)-Cl(2) 87.49(4); N(1)-Cu(1)-Cl(2)

TE

86.59(5); N(10)-Cu(1)-Cl(2) 100.59(4); O(1)-Cu(1)-Cl(1) 87.89(4); N(1)-Cu(1)-Cl(1) 94.82(5); N(10)-Cu(1)-Cl(1) 104.04(4); Cl(2)-Cu(1)-Cl(1) 155.204(17); Cu(1)-O(1)-H(1W) 120(2); Cu(1)-O(1)-H(2W) 123(2); H(1W)-O(1)-H(2W) 104(3); C(2)-N(1)-Cu(1) 124.36(10); C(10B)-N(1)-Cu(1.

AC CE P

114.75(10); C(9)-N(10) Cu(1) 133.48(12); C(10A)-N(10)-Cu(1) 107.14(11).

39

ACCEPTED MANUSCRIPT

***

***

0.8

***

RI

0.6

*

PT

***

***

*

***

1

SC

0.4 0.2

NU

Fraction of Cell Growth

1.2

A549

AC CE P

TE

D

MA

0

HFF

Figure 2: SRB data for compounds 1-8. Comparison of the percent growth of A549 human lung cancer cells to non-cancerous HFF cells in the presence of specified drug concentration. A Student’s T test was used to determine if the average growth rates were statistically different (p values < 0.05 indicate statistically different values; *** p < 0.001; ** p < 0.01; * p < 0.05).

40

*

**

*

**

**

*

*

2

4 3

PT

1

6 7

5

a

RI

1.2 1 0.8 0.6 0.4 0.2 0

NU

SC

Fraction of Cell Growth

ACCEPTED MANUSCRIPT

copper polypyridyl complexes

CuCl2

MA

Figure 3: SRB fractional growth of A549 lung cancer cells for compounds 1-8 with comparison to A549 cell growth in the presence of CuCl2. A Student’s T test was used to determine if the average growth rates were statistically different (p values < 0.05 indicate statistically different values; *** p < 0.001; ** p < 0.01; * p < 0.05).

TE

D

The IC50 of CuCl2 (1.83 μM) was lower than the IC50 of compound 8.

AC CE P

a

41

ACCEPTED MANUSCRIPT 1.2 **

1

PT RI

0.8

***

SC

0.6 0.4

NU

Fraction of Growth

***

MA

0.2 0

CuCl2 (0.2 µM)

TE

D

Compound 7 (0.2 µM)

6,6'-di-methyl-bipy (0.2 µM)

CuCl2 + 6,6'-dimethyl-bipy (0.2 µM)

AC CE P

Figure 4: Fraction of growth of A549 tumor cells for compound 7 at 0.2 μM in comparison to CuCl2 (0.2 μM), 6,6’-dimethylbipy (0.2 μM), and a control in which cells were incubated with 0.2 μM CuCl2 and 0.2 μM 6,6’-dimethylbipy. A Student’s T test was used to determine if the average growth rates of the CuCl2, free ligand, and CuCl2/ligand combination were statistically different from compound 7 (p values < 0.05 indicate statistically different values; *** p < 0.001; ** p < 0.01; * p < 0.05).

42

ACCEPTED MANUSCRIPT

Compound 1

0.8 0.7

0 hours 1 hour

PT

0.6

2 hours

0.5

3 hours

RI

0.4

4 hours

Absorbance

5 hours

SC

0.3 0.2

NU

0.1 0 275

325

375 425 475 Wavelength (nm)

525

575

TE

D

MA

225

24 hours

0.4

Compound 5 0 hours

AC CE P

0.35

1 hour

0.3

2 hours

0.25

3 hours

Absorbance 0.2

4 hours 22 hours

0.15 0.1 0.05

0 240

290

340

390 440 490 Wavelength (nm)

43

540

590

640

625

ACCEPTED MANUSCRIPT 0.35

Compound 6

PT

0.3 0 hours

0.25

RI

1 hour

0.2

SC

Absorbance

NU

0.15

0.1

3 hours 4 hours 5 hours 24 hours

MA

0.05

2 hours

0 325

375 425 475 Wavelength (nm)

D

275

TE

225

525

575

625

AC CE P

Figure 5: UV-vis spectra of compounds 1, 5, and 6 in 0.10 M phosphate buffer, pH 7.4. Spectra were obtained for 3.0 x10-5 M solutions of each complex every hour for a 24 hour period.

44

ACCEPTED MANUSCRIPT Appendices

Fraction of Growth (A549)

[(di-sec-butylphen) ClCu(µ-Cl)2CuCl(di-sec-

NU

0.0937 ± 0.0006

butyl

phen)] (1) (1.6μM)

MA

[(mono-sec-butylphen)CuCl2] (2) (3.2μM)

Student’s T test

Growth (HFF)

(p value, two-tailed)

0.830 ± 0.003 0.0002

D

TE

0.0001

0.553 ± 0.015

1.368 ± 0.004

[(mono-n-butylphen)CuCl2] (4) (0.2μM)

AC CE P

0.0001

0.857 ± 0.001

0.362 ± 0.000

[(di-n-butylphen)CuCl2] (3) (0.2μM)

Fraction of

SC

Compound and Concentration

RI

PT

Appendix 1: SRB data for compounds 1-8. Comparison of the percent growth of A549 human lung cancer cells to non-cancerous HFF in the presence of specified drug concentration. A Student’s T test was used to determine if the average growth rates were statistically different (p values < 0.05 indicate statistically different values).

0.0141 0.389± 0.006

1.764 ± 0.008

[(dimethylphen)CuCl2] (5) (0.2μM)

0.0002 0.174 ± 0.001

0.841 ± 0.001

[(mono-sec-butylbipy)CuCl2] (6) (1.6μM)

< 0.0001 0.481 ± 0.008

1.879 ± 0.004

[(6,6’-dimethylbipy)CuCl2] (7) (0.2μM)

0.0011 0.150 ± 0.002

1.538 ± 0.005

[(4,4’-dimethylbipy)CuCl2] (8) (6μM)

0.0005 0.192 ± 0.006

45

1.82 ± 0.01

ACCEPTED MANUSCRIPT Appendix 2: SRB fractional growth of A549 lung cancer cells for compounds 1-8 with comparison to A549 cell growth in the presence of CuCl2. A Student’s T test was used to determine if the average growth rates were statistically different (p values < 0.05 indicate statistically different values). Fraction of Growth of

Student’s T test

Specified

CuCl2 at Same

(p Value, two-tailed)

0.1731 +/- 0.0060

butyl

0.9796 +/- 0.0592

0.0097

0.4130+/- 0.0044

0.6122 +/- 0.0588

0.0305

[(di-n-butylphen)CuCl2] (3) (0.4 μM)

0.2274 +/- 0.0036

1.065 +/- 0.021

0.0123

[(mono-n-butylphen)CuCl2] (4) (0.4 μM)

0.3413 +/- 0.0006

1.065 +/- 0.021

0.0208

[(dimethylphen)CuCl2] (5) (0.2 μM)

0.1740 +/- 0.0006

0.9796 +/- 0.0592

0.0097

0.6122 +/- 0.0588

0.0445

D

MA

[(mono-sec-butylphen)CuCl2] (2) (1.6 μM)

0.481 ± 0.008

TE

[(mono-sec-butylbipy)CuCl2] (6) (1.6 μM) [(6,6’-dimethylbipy)CuCl2] (7) (0.2 μM)

0.150 ± 0.002

0.9796 +/- 0.0592

0.0084

[(4,4’-dimethylbipy)CuCl2] (8)a

-

-

-

AC CE P

a

NU

phen)] (1) (0.2 μM)

Concentration

SC

Concentrations [(di-sec-butylphen) ClCu(µ-Cl)2CuCl(di-sec-

PT

Fraction of Growth at

RI

Compound and Concentration

The IC50 of CuCl2 (1.83 μM) was lower than the IC50 of compound 8.

46

ACCEPTED MANUSCRIPT

Compound 2

2

0 hours

PT

1 hour 1.5

2 hours

RI

3 hours Absorbance

4 hours

SC

1

5 hours

22 hours

NU

0.5

340

440 540 Wavelength (nm)

640

AC CE P

TE

D

240

MA

0

Compound 3

1.4 1.2

0 hours

1

1 hour 2 hours

0.8

3 hours

Absorbance

4 hours

0.6

24 hours 0.4 0.2 0 240

340

440

540

Wavelength (nm)

47

640

ACCEPTED MANUSCRIPT 3

Compound 4

2.5

0 hours

2

PT

1 hour

3 hours 4 hours

SC

Absorbance 1.5

RI

2 hours

5 hours

24 hours

NU

1

MA

0.5

0

340 440 Wavelength (nm)

540

AC CE P

TE

D

240

Compound 7

0.3

0.25

0 hours 1 hour 2 hours 3 hours 4 hours 5 hours 24 hours

0.2

Absorbance 0.15 0.1 0.05 0 200

400 600 Wavelength (nm)

48

800

640

ACCEPTED MANUSCRIPT

0.35 0.3

0 hours

0.25

PT

Compound 8 1 hour

RI

2 hours

0.2

3 hours

Absorbance

SC

0.15

NU

0.1 0.05

300

400

500 600 Wavelength (nm)

D

200

MA

0

4 hours 5 hours 24 hours

700

800

AC CE P

TE

Appendix 3: UV-vis spectra of compounds 2-4, and 7-8 in 0.10 M phosphate buffer, pH 7.4. Spectra were obtained for 3.0 x10-5 M solutions of each complex every hour for a 20-24 hour period.

49

ACCEPTED MANUSCRIPT A

PT

0.8

RI

0.7

0.6

SC

0.5

NU

Absorbance 0.4

0.3

0

45C 55C

65C 75C 85C

400

500

600 Wavelength (nm)

700

800

900

TE

300

D

0.1

0.8

AC CE P

B

35C

MA

0.2

25C

0.7

0.6

0.5

25C 35C

Absorbance 0.4

45C 55C 65C 75C

0.3

85C

0.2

0.1

0 350

450

550

650 Wavelength (nm)

750

850

Appendix 4: Temperature-Dependent UV-vis spectra for compounds 6 and 7. A: [(6,6’-dimethyl bipy) CuCl2] (7) in DMSO; 25-85oC; B: [(mono-sec-butylbipy) CuCl2] (6); each complex was prepared as a 3.0 x 10-3 M solution in DMSO; 25-85oC. 50

ACCEPTED MANUSCRIPT

SC

RI

PT

Graphical abstract

in vitro tumor cell assay

NU

N(10) N(1)

CELL DEATH

MA

Cu(1)

Cl(2)

AC CE P

TE

D

Cl(1)

Treatment of lung cancer cell line A549 with of alkyl-substituted polypyridyl copper(II) complexes shows promising antiproliferative activity as well as a high degree of selectivity for cancerous cells over benign somatic cells.

51

ACCEPTED MANUSCRIPT Copper (II) complexes possessing alkyl-substituted polypyridyl ligands: Structural characterization and in vitro antitumor activity

PT

Noah R. Angel1‡, Raneen M. Khatib1‡, Julia Jenkins1, Michelle Smith1, Brian Khoa Le1, Daniel

RI

Lussier1, Zhuo (Georgia) Chen2, Fook S. Tham1, Emma H. Wilson3, Jack F. Eichler1*

SC

Highlights:

NU

-Eight new copper(II) coordination complexes synthesized and characterized. -Copper(II) complexes possess alky-substituted 1,10-phenanthroline and 2,2’-bipyridine ligands.

MA

-Five copper(II) complexes characterized by single crystal X-ray diffraction. -Three distorted tetrahedral, one distorted square pyramidal, and one dimeric complex described.

AC CE P

TE

D

-Copper(II) complexes exhibit general in-vitro antitumor activity against lung tumor cells.

52