Journal of Inorganic Biochemistry 130 (2014) 69–73

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Synthesis, crystallographic characterization and electrochemical property of a copper(II) complex of the anticancer agent elesclomol Nha Huu Vo a, Zhiqiang Xia a, Jason Hanko b, Tong Yun a, Steve Bloom a, Jianhua Shen a, Keizo Koya a, Lijun Sun a, Shoujun Chen a,⁎ a b

Synta Pharmaceuticals Corp., 45 Hartwell Avenue, Lexington, MA 02421, USA SSCI Inc., 3065 Kent Avenue, West Lafayette, IN 47906, USA

a r t i c l e

i n f o

Article history: Received 23 August 2013 Received in revised form 7 October 2013 Accepted 7 October 2013 Available online 12 October 2013 Keywords: Elesclomol Copper(II) complex Crystallographic characterization Electrochemical property Anticancer activity ROS

a b s t r a c t Elesclomol is a novel anticancer agent that has been evaluated in a number of late stage clinical trials. A new and convenient synthesis of elesclomol and its copper complex is described. X-ray crystallographic characterization and the electrochemical properties of the elesclomol copper(II) complex are discussed. The copper(II) cation is coordinated in a highly distorted square-planar geometry to each of the sulphur and amide nitrogen atoms of elesclomol. Electrochemical measurements demonstrate that the complex undergoes a reversible one-electron reduction at biologically accessible potentials. In contrast the free elesclomol is found electrochemically inactive. This evidence is in strong support of the mechanism of action we proposed for the anticancer activity of elesclomol. © 2013 Elsevier Inc. All rights reserved.

Elesclomol (N′1,N′3-dimethyl-N′1,N′3-di(phenylcarbonothio-yl) malonohydrazide, 1) is a novel small molecule anticancer drug candidate that is discovered and originated from our lab [1–4]. It exhibits strong antitumor activities against a broad range of cancer cell lines including MDR (multi-drug resistance) cell lines [4]. It is believed that elesclomol exerts its anticancer activity via the induction of reactive oxygen species (ROS) in cancer cells, which results in apoptosis [5]. Recent biological data support the hypothesis that elesclomol generates ROS via its chelation with copper(II) and redox cycling of copper(II) [6]. The data suggest that elesclomol obtains copper(II) outside the cell – from serum as well as from purified ceruloplasmin the primary copper-binding protein in blood – and requires copper(II) for its cellular entry and cytotoxicity [7]. On the other hand, copper(II) complexes are of continuing interest for their potential applications as molecular imaging agents [8–10]. They were investigated as anticancer agents for their capability to induce the formation of ROS and to inhibit proteasome activities in cancer cells [11–15]. Recent publications on elesclomol [6,16] prompt us to communicate our earlier results in the synthesis, crystallographic characterization, and the electrochemical property measurements of the elesclomol copper(II) complex (2). The syntheses of elesclomol (1) and its copper(II) complex (2) are illustrated in Scheme 1. Direct acylation of two equivalents of Nmethylbenzothiohydrazide [17] with one equivalent of malonyl chloride ⁎ Corresponding author. Tel.: +1 7815417255; fax: +1 7812748228. E-mail address: [email protected] (S. Chen). 0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2013.10.005

in EtOAc leads to the formation of elesclomol (1) as a yellow crystalline solid in 60–70% isolated yield. Compared to the synthetic methods we reported previously [4], this new procedure avoids the use of either explosive perchloric acid or a coupling agent such as N, N′dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The employment of a class 3 solvent (EtOAc) and the silica gel column-free isolation also makes it very suitable for the cGMP manufacturing of elesclomol. The copper(II) complex 2, a stable red solid, is formed by treating 1 with copper sulphate pentahydrate in aqueous acetone at rt (other copper(II) salts such as: copper(II) sulphate hexahydrate, copper(II) chloride, copper(II) bromide also yield similar results). Proton NMR spectrum of 2 consists of very broad signals, consistent with a typical paramagnetic nature of a copper(II) complex. X-ray diffraction quality single crystal of 2 (an orange plate of C19H18CuN4O2S2 having approximate dimensions of 0.38 × 0.38 × 0.18 mm3) was obtained by recrystallization from acetonitrile. Single crystal X-ray diffraction data collection was performed with Mo Kα radiation (λ = 0.71073 Å) on a Nonius KappaCCD diffractometer. Cell constants and an orientation matrix for data collection were obtained from least-squares refinement using the setting angles of 10,784 reflections in the range 3°bθb27°. The data were collected to a maximum 2θ value of 55.0%, at a temperature of 150 ± 1 K. A total of 10,784 reflections were collected, of which 4336 were unique. Lorentz and polarisation corrections were applied to the data. The linear absorption coefficient is 1.37 mm−1 for Mo Kα radiation. The structure was solved by direct methods using SIR2004. A total of 3548 reflections were used in the calculation. Of the 4336 reflections used in the refinements, only

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Scheme 1. The synthesis of elesclomol copper(II) complex 2.

the reflections with Fo2 N 2σ(Fo2) were used in calculating R. The goodness-of-fit parameter was 1.04. The highest peak in the final difference Fourier had a height of 0.41 e/Å3. The minimum negative peak had a height of −0.84 e/Å3. There is no residual electron density. The triclinic cell parameters and calculated volume are as follows: a =

6.7952(7) Å, b = 11.4847(11) Å, c = 12.7588(15) Å, α = 74.864(6)°, β = 89.909(7)°, γ = 88.297(7)°, and V = 960.72(18) Å3. The formula molecular weight of the asymmetric unit in the crystal structure of 2 form 1 is 462.05 g mol−1 with Z = 2, resulting in a calculated density of 1.597 g cm−3.

Fig. 1. ORTEP drawing of elesclomol copper(II) complex 2. Atoms are represented by 50% probability anisotropic thermal ellipsoids.

Fig. 2. ORTEP drawing of 2, highlighting the distorted elesclomol molecule and the copper(II) coordination sphere. Atoms are represented by 50% probability anisotropic thermal ellipsoids.

N.H. Vo et al. / Journal of Inorganic Biochemistry 130 (2014) 69–73

Fig. 3. Packing diagram of 2 viewed down the crystallographic a axis.

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An ORTEP drawing of the complex 2 is shown in Fig. 1. The quality of the structure obtained is high, as indicated by the R-value of 0.040 (4%). The asymmetric unit shown in Fig. 1 contains a single C19H18CuN4O2S2 molecule, and there are two molecules in the unit cell. The copper(II) ion is coordinated in a highly distorted square-planar geometry coordinated to each of the sulphur and amide nitrogen atoms of elesclomol. Whereas the non-hydrogen atoms for the copper(II) complex of diacetyl-bis(N4-methylthiosemicarbazone) (Cu(II)[ATSM]) are approximately coplanar [18], closer examination of 2 reveals that the elesclomol molecule is highly distorted. An ORTEP drawing highlighting the highly distorted copper(II) coordination sphere in 2 is provided in Fig. 2. The highly distorted square-planar geometry of complex 2 results in the out-of-planar trans-S\Cu\N (N7\Cu\S1 and N3\Cu\S9) bond angle of 161.69(6)o and 158.38(6)o, respectively. This distorted square-planar geometry is different from the wellstudied copper(II) complex of acetylacetonate bis(thiosemicarbazone) (Cu[AATS]) or diacetyl-bis(N4-methylthiosemicarbazone) (Cu[ATSM]) which adopts an essentially square-planar coordination [18–20]. The bond length for the two S\Cu bonds in complex 2 is 2.2346(7) and 2.2375(7) Å, respectively, and is very similar to that in Cu[AATS]. However the N\Cu bond distances of 1.944(2) and 1.948(2) Å are notably shorter than that in Cu[AATS] (1.9737(16) and 1.9893(16) Å. As a consequence, the S\Cu\S angle is wider in complex 2 (97.10(3)o) than in Cu[AATS] [91.728(19)o]. Similarly, the bridge CH2 group is under stress to accommodate the coordination geometry, as demonstrated by a C4\C5\C6 bond angle of 124.5(2)o that is considerably larger than what would be expected for a tetrahedral C5 carbon atom. Packing diagrams viewed along the a crystallographic axis is shown in Fig. 3. Since the amide nitrogen atoms are deprotonated in 2, there are no hydrogen bonds observed in the crystal lattice, suggesting that the lattice is held together largely by van der Waals' interactions. Intermolecular π–π interactions between the phenyl groups are observed. The low aqueous solubility of elesclomol copper(II) complex 2 prevents the measurement of its electrochemical properties in water or an aqueous based buffer system. Fig. 4 depicts cyclic voltammograms (CV) of free elesclomol 1 and complex 2 in DMSO with 0.1 M tetraethylammonium perchlorate (THP) as the electrolyte. Complex 2 exhibits a well-defined cyclic voltammogram with a reduction and oxidation potential of −378mV and −287mV, respectively. These redox potentials are well within the biologically accessible range. The currents from reduction and oxidation are 2.83 μA and 2.78 μA, with a ratio of 1.02. This process can be classified as a reversible process as a current ratio of one is considered as a true reversible reduction and oxidation process [21]. In contrast, the free elesclomol is not electrochemically active. Taken together with the findings that elesclomol can complex with copper(II) in cell culture media, this electrochemical property corroborates the

biological findings demonstrating copper(II) as essential to the in vitro ROS induction activity of elesclomol [6]. In summary, the copper(II) complex (2) of the novel anticancer agent elesclomol was prepared under “click chemistry” conditions and its structure was determined by X-ray crystallography. Complex 2 exhibits a well-defined cyclic voltammogram with an anodic peak (Epa) and a cathodic peak (Epc) of −378 mV and −287 mV, respectively. In contrast to the coplanar geometry observed for the copper(II) complexes of other widely-studied thiosemicarbazones such as ATSM and AATS, the copper(II) ion is coordinated in a highly distorted square planar geometry to each of the sulphur and amide nitrogen atoms of elesclomol. While the cyclic voltammogram of 2 displays a welldefined reversible reduction–oxidation process, elesclomol itself is electrochemically inactive. This observation is in strong support of the mechanism of action (MOA) we proposed for elesclomol. Furthermore, the convenient preparation of elesclomol and its derivatives makes them readily available for biomarker studies. Further investigation on other metal complexes with elesclomol or its derivatives should point to a broader application in the field of molecular imaging for cancer therapy. Abbreviations MDR Multidrug resistance ROS Reactive oxygen species DCC N,N′-dicyclohexylcarbodiimide EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide cGMP Current good manufacturing practices ORTEP The oak ridge thermal ellipsoid plot ATSM Diacetyl-bis(N4-methylthiosemicarbazone) AATS Acetylacetonate bis(thiosemicarbazone) THP Tetra-ethylammonium perchlorate CV Cyclic voltammogram MOA Mechanism of actions Epa Anodic peak Epc Cathodic peak

Acknowledgement The authors are indebted to Tracy Olson, Nicholas Triano, Yumiko Wada, Patricia Rao, Weiwen Ying, Masa Nagai, Dinesh Chimmanamada, Jun Jiang, Noriaki Tatsuta and Andrew Sonderfan for helpful discussion and valuable suggestions. Appendix A. Supplementary data Synthetic procedures for 1 and 2, crystallographic data in CIF format, tables of data collection parameters, and procedures for CV measurement. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinorgbio.2013.10.005.

4 3

Current (µA)

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Potential (V) vs. Ag/AgCl Fig. 4. CV of elesclomol (1) and elesclomol copper(II) complex (2) at a scan rate of 50 mV/s. Solid line: 2, 1.2 mM in DMSO with 0.1 M THP; dot line: 1, 1.7 mM in DMSO with 0.1 M THP.

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