Synthesis, characterization and cytotoxicity of new gold(III) complexes with 1,2-diaminocyclohexane: Influence of stereochemistry on antitumor activity

Polyhedron 50 (2013) 434–442

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Synthesis, characterization and cytotoxicity of new gold(III) complexes with 1,2-diaminocyclohexane: Influence of stereochemistry on antitumor activity Said S. Al-Jaroudi a, Mohammed Fettouhi a, Mohammed I.M. Wazeer a, Anvarhusein A. Isab a,⇑, Saleh Altuwaijri b a b

Department of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran 31261, Saudi Arabia Clinical Research Laboratory, SAAD Research Development Center, SAAD Specialist Hospital, Al-Khobar 31952, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 27 July 2012 Accepted 13 November 2012 Available online 1 December 2012 Keywords: Gold(III) complex 1,2-Diaminocyclohexane Anticancer Gastric carcinogenesis (SGC-7901) Prostate (PC-3)

a b s t r a c t Gold(III) complexes of the type [(DACH)AuCl2]Cl, derived from sodium tetrachloroaurate(III) dihydrate NaAuCl42H2O, where DACH is diaminocyclohexane, have been synthesized. These potential metallodrug compounds were characterized using various spectroscopic and analytical techniques, including elemental analysis, UV–Vis, infrared spectroscopy, solution as well as solid NMR spectroscopy and X-ray crystallography. The potential of the synthesized gold(III) complexes as anti-cancer agents was investigated by measuring some relevant physicochemical and biochemical properties, such as the stability of the Au–N bonds by vibrational stretching from far-IR as well as cytotoxicity and the stomach cancer cell inhibiting effect. The solid-state 13C NMR chemical shift shows that the ligand is strongly bound to the gold(III) center via N atoms. An X-ray crystallography study of the complexes shows that the cyclohexyl ring adopts a chair conformation and the gold coordination sphere adopts a distorted square planar geometry. The cis isomer in solution showed higher activity towards the inhibitory effect of human cancer cell lines such as prostate cancer (PC-3) and gastric carcinoma (SGC-7901) than that of the trans isomer. The cytotoxicity of the cis isomer complex has also been estimated in PC-3 and SGC-7901 cells. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction The serendipitous discovery of cisplatin by Rosenberg in 1965 heralded a new area of anticancer drug research based on metallopharmaceuticals [1]. Cisplatin has been one of the most successful chemotherapies in the last 30 years and has been used to treat numerous types of cancers, including testicular, ovarian, head– neck and bladder tumors. Despite having great utility as a chemotherapeutic agent, cisplatin does have drawbacks; tumors often develop resistance to the drug and patients routinely experience severe side effects throughout the course of the treatment [2]. Subsequently, researchers are continually looking for therapeutic alternatives that might alleviate these limitations. Unfortunately, they have several major drawbacks. Common problems include cumulative toxicities of nephrotoxicity and cytotoxicity [3–6]. In addition to the serious side effects, the therapeutic efficacy is also limited by inherent or treatment-induced resistant tumor cells. These drawbacks have provided the motivation for alternative chemotherapeutic strategies. To circumvent the problem of drug-resistance in cisplatin-resistant cells, gold(III)-based com-

⇑ Corresponding author. E-mail address: [email protected] (A.A. Isab). 0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.poly.2012.11.034

plexes have been designed as potential alternatives to cisplatin [7–11]. Gold(III) compounds have greatly attracted researchers’ attention in the last decade for their outstanding cytotoxic actions. It is a metal ion which typically adopts a four-coordinate, square-planar geometry and is therefore expected to mimic the structural and electronic properties of platinum(II). Recent studies have shown that several gold(III) complexes are highly cytotoxic against different tumor cells [12–14], including some which are active even against the cisplatin-resistant cell lines [8,15–17]. Several lines of evidence suggest that gold(III) compounds produce their antiproliferative effects through innovative and non-conventional modes of action. For instance, the hypothesis that their biological effects are mediated by an antimitochondrial mechanism rather than by direct DNA damage, as it is the case for cisplatin and its analogs, has gained much credit during the last few years [10]. The strict relationship to platinum(II) compounds makes gold (III) complexes good candidates for development and testing as anticancer drugs, although the relatively high kinetic liability and the usually high redox potentials have largely hindered such investigations. These problems can possibly be circumvented by forming gold(III) compounds with one or more multidentate nitrogen-donor ligands to enhance the stability of the gold(III) complexes [18–20]. Some recent studies reporting that novel

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gold(III) compounds show favorable antitumor properties both in vitro and in vivo have raised new interest in this research area [21–23]. Although, there are a multitude of structural analogs of the antitumor agent cis-diamine dichloroplatinum(II) (cisplatin) [24], only a few are presently used in clinical practice [25], including trans1,2-diaminocyclohexane (DACH) dichloroplatinum(II) [26]. Since the molecule is chiral, the relevance of stereochemical issues has been addressed by a number of investigators [27].The ligand DACH has three isomeric forms: the enantiomers (IR,2R-DACH) (trans-lDACH), (lS,2S-DACH) (trans-DACH) and the diastereoisomer Pt(1R,2S-DACH) (cis-DACH). In spite of conflicting views [28–32], the consensus is that the (R,R) isomer is generally more active than the (S,S) isomer [33,34], although activity has also been demonstrated with the (R,S) isomer [35]. With regard to the stereochemistry of the complexes, the DACH platinum compounds, Pt(IR,2RDACH) and Pt(lS,2S-DACH), have a higher antitumor activity than the Pt(IR,2S-DACH) complex [36–38]. In the early 1990s, a few gold compounds were prepared and characterized for their antitumor activity with positive results [39,40] Recently, the use of various Au(III) complexes with novel functionality has elicited more interest due to their distinct physical and chemical properties, stability under physiological conditions and outstanding cytotoxic effects [41,42]. Cis-diaminedichloroplatinum(II) (cisplatin) is one of the most widely used anticancer drugs today. However, platinum compounds possessing the 1,2-diaminocyclohexane (DACH) carrier ligand offer advantages over cisplatin with regard to bioavailability, activity and decreased renal toxicity [43]. Furthermore, the success of oxaliplatin, which incorporates the 1R,2R-DACH carrier ligand as a Pt(II) complex, raised considerable research interest over the past three decades in platinum–DACH complexes. Over the past several years, significant effort has been devoted to the study of the antitumor activity of platinum–DACH complexes, whereas gold–DACH complexes [44] have received relatively little attention, although, Au(III) has a fairly rich biological chemistry. For instance, it is redox active, can be coordinated by amino acids and proteins, is able to deprotonate and bind to the amide N of peptides and it is capable of cross-linking histidine imidazole rings [45]. As in the case of the parent cisplatin, the antitumor activity of platinum–DACH is accompanied by some toxicity. The emergence of resistance and low water solubility, that can affect the pharmacokinetics, are additional features that must be improved in the quest for a more effective analog [46]. As a continuation of our intrinsic interest in the synthesis of gold(III) complexes and to better understand the chemical and physical behavior of biologically relevant mono-(DACH) gold(III) complexes, the chiral isomers [cis-(±)-1,2-(DACH)AuCl2]Cl (1), [trans(±)-1,2-(DACH)AuCl2]Cl (2) and [(1S,2S)-(+)-1,2-(DACH)AuCl2]Cl (3) have been synthesized and fully characterized by IR, NMR, elemental analysis and UV–Vis. Scheme 1 illustrates the structures of the ligands and Scheme 2 shows the structures of the complexes. Their cytotoxicity has been tested in vitro in human gastric carcinoma cell line SGC-7901 and prostate cancer cell line PC-3. In this study, the influence of the relative stereochemistry of (DACH) gold(III) complexes on their antitumor activities was addressed. These compounds are sparingly water soluble.

R NH2

S NH2

R NH2

R NH2

S NH2

S NH2

H2N

6 5

H2N

6

Cl

1

5

Au

Au 4

2

4

Cl

H2N

3

Cl

1 2

Cl

H2N

3

1, Cis-( ± )-1,2-(DACH)AuCl 3 H2N

6 5

H2N

6

Cl

1

5

Au

Au 4

2 3

Cl

1

4

Cl

H2N

2 3

H2N

Cl

2, Trans -( ± )-1,2-(DACH)AuCl 3

H2N

6 5

1

4

2

Cl Au

3

H2N

Cl

3, (S,S) -(+)-1,2-(DACH)AuCl 3 Scheme 2. Chemical structures of the synthesized gold(III) complexes.

2. Experimental 2.1. General procedures All commercial reagents were purchased from Aldrich and used as received unless otherwise stated. The 1H and 13C NMR experiments were performed on a Bruker Advance 400 or Jeol JNM-LA 500 spectrometer. 1H and 13C NMR chemical shifts were given as values with reference to tetramethylsilane (TMS) as an internal standard.

2.2. Synthesis of the Au(III) complexes Gold complexes of cis-(±)-1,2-diaminocyclohexane (1), trans(±)-1,2-diaminocyclohexane (2) and the purely optical active isomer of (S,S)-(+)-1,2-diaminocyclohexane (3) were synthesized by a general method described in literature for similar compounds [47], by dissolving of 199 mg (0.50 mmol) sodium tetrachloroaurate(III) dihydrate (NaAuCl42H2O) in a minimum amount of absolute ethanol at ambient temperature. In a separate beaker, a solution of 57 mg (0.50 mmol) of the diaminocyclohexane in the least amount of absolute ethanol was prepared, both solutions were mixed (total of 40 ml) and stirred for around 30 min until a clear solution was obtained, which was filtered and concentrated to 10 ml solvent then left for crystallization in the refrigerator. The produced solid was dried under vacuum. The product was obtained in a yield of 91–98%. The complexes prepared in the present study were characterized by their physical properties, NMR, IR, elemental analysis and X-ray crystallography. All the data support the formation of the desired DACH complexes. Melting points and elemental analyses for the complexes are presented in Table 1 (See Supplementary data for Tables 1–7).

S NH2

2.3. Electronic spectra

Trans

R NH2 Cis

Scheme 1. Isomerization structures of diaminocyclohexane (DACH).

Electronic spectra were obtained for the diaminocyclohexane gold(III) complexes using a Lambda 200, Perkin-Elmer UV–Vis spectrometer. The resulting absorption data are shown in Table 2.

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2.4. IR and far-IR studies The solid-state IR spectra of the ligands and their gold(III) complexes were recorded on a Perkin-Elmer FTIR 180 spectrophotometer using KBr pellets over the range 4000–400 cm1. The selected IR frequencies are given in Table 3. Far-infrared spectra were recorded for complexes (1)–(3) at 4 cm1 resolution at room temperature in cesium chloride disks on a Nicolet 6700 FT-IR with a far-IR beam splitter. Far-IR data for the complexes studied are depicted in Table 4. 2.5. Solution NMR measurements All NMR measurements were carried out on a Jeol JNM-LA 500 NMR spectrophotometer at 297 K. The 1H NMR spectra were recorded at a frequency of 500.00 MHz. The 13C NMR spectra were obtained at a frequency of 125.65 MHz with 1H broadband decoupling and they were referenced relative to TMS. The spectral conditions were: 32 k data points, 0.967 s acquisition time, 1.00 s pulse delay and 45° pulse angle. The 1H and 13C NMR chemical shifts are given in Tables 5 and 6, respectively, according to Scheme 2. 2.6. Solid state NMR studies 13

C solid-state NMR spectra were recorded on a Bruker 400 MHz spectrometer at an ambient temperature of 25 °C. Samples were packed into 6 mm zirconium oxide rotors. Cross polarization and high power decoupling were employed. A pulse delay of 7.0 s and a contact time of 5.0 ms were used in the CPMAS experiments. The magic angle spinning rates were 4 and 8 kHz. Carbon chemical shifts were referenced to TMS by setting the high frequency isotropic peak of solid adamantane to 38.56 ppm. The solid NMR data are given in Table 7. 2.7. X-ray crystallography For each of (1) and (2), an X-ray quality single crystal, which was obtained from EtOH solution, was mounted in a thin-walled glass capillary on a Bruker-Axs Smart Apex diffractometer equipped with graphite monochromatized Mo Ka radiation (k = 0.71073 Å). The data were collected using SMART [48]. The data integration was performed using SAINT [49]. An empirical absorption correction was carried out using SADABS [50]. The structure was solved with direct methods and refined by full matrix least square methods based on F2, using the structure determination package SHELXTL [51] based on SHELX 97 [52]. Graphics were generated using ORTEP-3 [53] and MERCURY [54]. For compound (1), some of the hydrogen atoms of the disordered ethanol molecule could not be placed. For compound (2), one hydrogen atom of the water molecule was located on a Fourier Difference map and refined isotropically, the second one could not be placed reliably. All other H atoms were placed a calculated positions using a riding model. Crystal and structure refinement data are given in Table 8. Selected bond lengths and bond angles are given in Table 9.

Table 8 Crystal and structure refinement data for compounds (1) and (2). Compound

(1)

(2)

CCDC deposit No. Empirical formula Formula weight T (K) k (Å) Crystal system Space group Unit cell dimensions a (Å) b (Å) c (Å) b (°) V (Å3) Z q (g cm3) l (mm1) F(0 0 0) Crystal size (mm) h range (o) Limiting indices

831613 C14 H34 Au2 Cl6 N4 O 881.09 297(2) 0.71073 orthorhombic Pbcn

850216 C12 H30 Au2 Cl6 N4 O 853.03 295(2) 0.71073 monoclinic P21

19.792(1) 12.4662(7) 10.3212(6)

9.5898(7) 8.6106(6) 14.477(1) 95.307(1) 1190.3(2) 2 2.38 12.994 796 0.52  0.49  0.16 1.41–28.28 12 6 h 6 12 11 6 k 6 11 19 6 l 6 19 Tmin = 0.0564 and Tmax = 0.2244 5835/2/230 1.017 R1 = 0.0308 wR2 = 0.0739 R1 = 0.0329 wR2 = 0.0747 0.796 and 1.555

Max and min transmission Data/restraints/parameters Goodness-of-fit (GOF) on F2 Final R indices [I > 2r(I)] R indices (all data) Largest difference in Peak and hole (e Å3)

2546.5(2) 4 2.298 12.152 1656 0.40  0.37  0.26 1.93–28.29 26 6 h 6 26 16 6 k 6 16 13 6 l 6 13 Tmin = 0.0850 and Tmax = 0.1443 3162/0/128 1.051 R1 = 0.0246 wR2 = 0.0631 R1 = 0.0288 wR2 = 0.0654 1.766 and 1.544

Table 9 Selected bond lengths (Å) and bond angles (o) for compounds (1) and (2). (1)

(2)

Au1–N1 2.029(4) Au1–N2 2.030(3) Au1–Cl1 2.261(1) Au1–Cl2 2.268(1)

Au1–N1 2.029(6) Au1–N2 2.031(5) Au1–Cl1 2.274(2) Au1–Cl2 2.276(2)

Au2–N3 2.029(6) Au2–N4 2.054(7) Au2–Cl3 2.259(3) Au2–Cl4 2.266(2)

N1–Au1–N2 83.9(2) N1–Au1–Cl1 91.7(1) N2–Au1–Cl1 175.4(1) N1–Au1–Cl2 176.0(1) N2–Au1–Cl2 92.6(1) Cl1–Au1–Cl2 91.83(5)

N1–Au1–N2 84.3(2) N1–Au1–Cl1 92.0(2) N2–Au1–Cl1 176.2(2) N1–Au1–Cl2 174.2(2) N2–Au1–Cl2 89.9(2) Cl1–Au1–Cl2 93.81(8)

N3–Au2–N4 84.1(2) N3–Au2–Cl3 92.2(2) N4–Au2–Cl3 176.2(2) N3–Au2–Cl4 176.4(2) N4–Au2–Cl4 92.3(2) Cl3–Au2–Cl4 91.4(1)

Similarly, in another set, compounds (1)–(3) and PC3 cells were kept for an entire day (24 h) and for 72 h (3 days). In the remaining set, Fig 7, compounds (1) and (2), with fixed concentrations, were employed to determine the growth inhibitory effect for both PC-3 and SGC-7901 cells. After being treated with (1) and (2), the cell viability was examined by an MTT assay. 2.9. Assessment of cell proliferation

2.8. Cell lines and reagents Human gastric cancer SGC-7901 and prostate cancer PC-3 cells were incubated. Trypan blue dye exclusion analysis and MTT assay were used to detect cell proliferation and to assess the inhibitory effect of the compounds (1)–(3) on the proliferation of the SGC7901 and PC-3 cells. In one culture plate, human gastric cancer SGC-7901 and PC-3 cells were treated with various concentrations of compounds (1)–(3), and the control (water), Figs. 1–3. In one set of plates, Figs. 4–6, compounds (1)–(3) and SGC-7901 cells were kept for an entire day (24 h) and for 72 h (3 days).

An MTT assay was used to obtain the number of living cells in the sample. SGC-7901 and PC-3 cells were seeded on 96-well plates at a predetermined optimal cell density to ensure exponential growth for the duration of the assay. After 24 h pre incubation, the growth medium was replaced with an experimental medium containing the appropriate drug or control. Six duplicate wells were set up for each sample, and cells untreated with drug served as a control. Treatment was conducted for 24 and 72 h. After incubation, 10 lL MTT (6 g/L, Sigma) was added to each well and the incubation was continued for 4 h at 37 °C. After removal of the

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Fig. 3. Effect of the (S,S)-(+)-1,2-(DACH)–gold(III) complex on cell growth in (A) PC3 and (B) SGC-7901 cells. The cells were treated with 10 lM of compound (3) for 1 day and 3 days. The anti-proliferative effect was measured by an MTT assay. The results are expressed as the mean, SD. ⁄P < 0.05. Fig. 1. Effect of the cis-(±)1,2-(DACH)–gold(III) complex on cell growth in (A) PC-3 and (B) SGC-7901 cells. The cells were treated with 10 lM of compound (1) for 1 day and 3 days. The anti-proliferative effect was measured by an MTT assay. The results are expressed as the mean, SD. ⁄P < 0.05.

Fig. 2. Effect of the trans-(±)1,2-(DACH)–gold(III) complex on cell growth in (A) PC3 and (B) SGC-7901 cells. The cells were treated with 10 lM of compound (2) for 1 day and 3 days. The anti-proliferative effect was measured by an MTT assay. The results are expressed as the mean, SD. ⁄P < 0.05.

medium, MTT stabilization solution (dimethylsulfoxide:ethanol = 1:1) was added to each well, and shaken for 10 min until all crystals were dissolved. Then, the optical density was detected in a micro plate reader at 550 nm wavelength using an ELISA reader. Each assay was performed in triplicate. The cell number and viability were determined by trypan blue dye exclusion analysis. 3. Results and discussion

Fig. 4. Effect of the cis-(±)-1,2-(DACH)–gold complex on cell growth in (A) PC-3 and (B) SGC-7901 cells. The cells were treated with various concentrations of compound (1) for 24 h. The anti-proliferative effect was measured by an MTT assay. The results are expressed as the mean, SD. ⁄P < 0.05.

(40 000–28 570 cm1), which correspond to LMCT transitions, a signal at 300 nm that could be assigned to a Cl ? Au charge transfer by analogy to the absorption spectrum of auric acid, which gives a band at 320 nm [55], where this transition has a high extinction coefficient and cannot be assigned to the symmetry forbidden d–d transition. According to crystal field theory for d8 complexes, the LUMO orbital is dx2 —y2 , so the ligand to metal charge transfer could be due to a pr ? dx2 —y2 transition [56]. It is evident that the electronic spectra of these compounds are stable and consistent, which means that the gold centers remain in the +3 oxidation states.

3.1. Electronic spectra

3.2. IR and far-IR spectroscopic studies

The kmax values for the complexes studied are shown in Table 2. The Au(III) complexes show absorptions in the region 250–350 nm

Table 3 lists the significant IR bands of the free DACH ligands and the gold(III) complexes. The N–H stretching band, which

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Au–N bond [59]. The red shift of the DACH complexes with respect to auric acid shows a weakening of the Au–Cl bond. 3.3. Solution NMR characterization

Fig. 5. Effect of the trans-(±)-1,2-(DACH)–gold complex on cell growth in (A) PC-3 and (B) SGC-7901 cells. The cells were treated with various concentrations of compound (2) for 24 h. The anti-proliferative effect was measured by an MTT assay. The results are expressed as the mean, SD. ⁄P < 0.05.

The 1H and 13C NMR chemical shifts of compounds (1)–(3), along with the free ligands, are listed in Tables 5 and 6, respectively. Also, the 1H and 13C NMR spectra of complexes (1)–(3) depicted one half of the total expected signals because of the C2 symmetry axis. The 1,2-diaminocyclohexane ring is considered as a rigid conformer that allows the equatorial H3 and H4 protons to be distinguished from the axial H3 and H4 protons at room temperature. A 1H NMR downfield shift was observed for the complexes with respect to the free diamine ligands. A significant downfield shift was observed at 3.59 ppm for complex (1) with respect to the free DACH ligand at 2.23 ppm. This can be attributed to donation of nitrogen lone pairs to the gold center that causes deshielding of the proton(s) next to the bonding nitrogen. On the other hand, the 13C NMR downfield shift was observed only for the carbon next to the bonding nitrogen and the other carbons in the complex showed an upfield shift. For instance, the chemical shifts of C3 and C4 for complex 1 were observed at 26.78 and 21.43 ppm, respectively, whereas for the free diamine ligand they occur at 35.26 and 26.36 ppm. It is also worth mentioning that complexes (1)–(3), even though they have same DACH skeleton, their NMR chemical shifts are not the same due to differences in their stereochemistry upon complexation. 3.4. Solid-state NMR At the spinning rate of 8 kHz, only the isotropic signals were observed for the carbons, indicating a small anisotropy due to the sp3 hybridization of these atoms. Compared to the solution chemical shifts, substantial deshielding in the solid state is observed, with a similarity in the chemical shifts amongst all the synthesized complexes (Table 7), which is a clear indication of the stability of the prepared complexes. The solid state NMR spectrum of complex (1) showed two sets of peaks with equal intensity, which supports the idea of the inequivalency of all six carbon atoms of DACH. This indicates that complex (1) lacks C2 symmetry in the solid state. 3.5. X-ray crystal structure

Fig. 6. Effect of the (S,S)-(+)-1,2-(DACH)–gold(III) complex on cell growth in (A) PC3 and (B) SGC-7901 cells. The cells were treated with various concentrations of compound (3) for 24 h. The anti-proliferative effect was measured by an MTT assay. The results are expressed as the mean, SD. ⁄P < 0.05.

occurs around 3300 cm1 for the free ligands, shifts towards a higher frequency (blue shift) upon complexation by about 150 cm1. Another important vibrational band observed in IR spectra is the C–N stretching, which also showed a slight shift to higher wavenumber, indicating a shorter C–N bond in the complexes than in the free ligands. Far-IR spectra showed absorption bands at 353 and 367 cm1 for the symmetric and asymmetric stretching of the Au–Cl bond, which is consistent with an Au–Cl stretching mode trans to nitrogen [57,58]. Another group of bands at 395 and 437 cm1 could be assigned to the symmetric and asymmetric stretching of the

Fig. 8 shows the crystal structure of complex (1). The gold(III) ion is bonded to two nitrogen atoms of the cis-cyclohexane-1,2diamine ligand and two chloride ions in a distorted square planar geometry. The two Au–N bond distances are not significantly different (2.029(4) Å), while the Au–Cl bond distances are 2.261(1) and 2.268(1) Å (Tables 8 and 9). The Cl-Au-Cl and N-Au-N bond angles are 91.83(5) and 83.9(2)°, respectively. The later value reflects the chelation strain of the diamine ligand. These values are similar to those found in dichloro-(ethylenediamine-N,N0 )-gold(III) chloride dihydrate [47] and dichloro-(1,2-ethanediamine)-gold(III) nitrate [60]. The cyclohexyl ring adopts a chair conformation. The square planar geometry and the five-membered ring strain impose an N1–C1–C2–N2 torsion angle of 49.80°. All the amine hydrogen atoms are engaged in hydrogen bonding with the Cl counter ion. To the best of our knowledge, this is the first X-ray structure of a gold complex based on cyclohexane-1,2-diamine [61]. A molecule of ethanol is present in the lattice. It presents an orientation disorder on a twofold rotation axis. The metal complex molecules pack head to tail to generate molecular chains along the c axis, which in turn pack into layers parallel to the ac plane (Fig. 9). These are separated by sheets hosting columns of disordered ethanol molecules and Cl counter ions, having hydrogen bonding interactions with the NH2 groups of adjacent layers.

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Fig. 7. Effect of compounds 1 and 2 on cell growth in (A) PC-3 and (B) SGC-7901 cells. The cells were treated with 10 lM of compounds 1 and 2 for 1 day, 2 days and 3 days. The anti-proliferative effect was measured by an MTT assay. The results were expressed as the mean, SD. ⁄P < 0.05.

Fig. 8. X-ray structure of compound (1).

The crystal structure of complex (2) is depicted in Fig. 10. In this case, the asymmetric unit contains two cationic molecules of the gold complex, two chloride counter ions and one crystallization

water molecule. Similarly to (1), in both molecules the gold(III) ion is bonded to two nitrogen atoms of the trans-cyclohexane1,2-diamine ligand and two chloride ions (Fig. 10). The geometry

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Fig. 9. Molecular packing in compound (1).

Fig. 10. X-ray structure of compound (2).

is distorted square planar, with Au–N and Au–Cl bond distances in the ranges 2.029(6)–2.054(7) and 2.259(3)–2.276(2) Å, respectively, and with similar N–Au–N bond angles of 84.1(2) and 84.3(2)°, in addition to Cl–Au–Cl bond angles in the range 91.4(1)–93.81(8)°. The coordination sphere bond distances and bond angles are similar to those of compound (1). The two cyclohexyl rings adopt a chair conformation with N1–C1–C6–N2 and N3–C7–C12–N4 torsion angles of 55.78 and 52.33°, respectively. Hydrogen bonding interactions take place between the amino groups and the chloride counter ions. 3.6. Effect of compounds (1), (2) and (3) on cell proliferation The bioassay tests were completed for compounds (1)–(3) under various experimental conditions. The cytotoxicity assay was performed with various concentrations of the synthesized gold(III)

complexes on PC-3 and SGC-7901 cells. The experimental PC-3 and SGC-7901 cells were treated with various concentrations of (1), (2) and (3) for 24–72 h, the cell viability was determined as described above by an MTT assay and the results are shown in Tables 10 and 11, as well as in Figs. 1–7. As depicted in Figs. 1–3, the cis-(±)-1,2(DACH)-gold complex exhibited potentially high activity against the gastric cancer cell SGC-7901 and human prostate cancer cells after 24 and 72 h of treatment with 10 lM, whereas, trans-(±)1,2-(DACH) and purely chiral trans-()-1,2-(DACH) gold complexes showed moderate inhibition against SGC-7901 and PC-3 cell lines under the same assay experimental conditions. From Figs. 4–6, it is also quite clear that the gold(III) complexes under study showed a concentration dependent cytotoxic effect on cancerous PC-3 and SGC-7901 cells. It can be concluded from Figs. 2, 3, 5 and 6 that there is no significant difference in the bioactivity between the trans-(1R,2R)-(DACH) isomer and the trans-(1S,2S)-(DACH) isomer.

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Table 10 Effect of compound (1) on cell/proliferation and the cell cycle of the PC3 and SGC-7901 cell lines (Mean, SD) after incubation for 24 and 72 h. PC3 Cell line Group Control (1)

Day 1 (24 h) 0.75016 ± 0.02511 0.70412 ± 0.29933

SGC-7901 Cell line Day 3 (72 h) 1.3910 ± 0.11711 0.81748 ± 0.17350

Table 11 Cytotoxicity of compound (1) towards different tumor cell lines. The data were collected after 72 h exposure to the compound. Compound

SGC-7901

PC3

(1)

8.5 ± 0.23

8.1 ± 0.17

To the best of our knowledge, these are the first bioassay tests that have been reported for gold(III) complexes based on cyclohexane1,2-diamine. In the time dependent activity studies, it is revealed that after 72 h of the experiment for (1) on the PC-3 cell, the cell proliferation is bit higher than that of the SGC-7901 cells at a fixed 10 lM concentration (Fig. 1). Furthermore, in Fig. 7, the cytotoxicity results demonstrate that compound (1) at 10 lM concentration has a higher cytotoxic effect in comparison with the same concentration of compound (2). 4. Conclusion Gold, along with its therapeutic and beneficial effect on human health, is amongst the most ancient of all metals used in medicine. The use of gold complexes in modern medicine has allowed information regarding toxicological and clinical administration to become available, along with valuable studies concerning its metabolism and molecular targets. Therefore, gold has become one of the most promising metals for drug development in medicine. Three mono gold(III) complexes based on DACH with different configurational structures were prepared. These gold(III) complexes were characterized using elemental analyses, solution and solid NMR, UV, IR, far-IR spectroscopy and X-ray crystallography. The analytical data strongly support the formation of the [(DACH)AuCl2]Cl type complex. Also, X-ray crystallography demonstrates that the gold coordination sphere of this complex has a distorted square planar geometry. According to our biological assays, complex (1), with a cis configuration, is a more promising candidate as an anti-cancer agent than the trans isomers, complexes (2) and (3). The exact mechanisms are not clearly known, but the inhibitory effect of [(cis-DACH)AuCl2]Cl on the proliferation of rapidly dividing cells may be attributed to the induction of cell cycle blockage, interruption of the cell mitotic cycle, programmed cell death (apoptosis) or premature cell death (necrosis). Therefore, [(cis-DACH)AuCl2]Cl might be a promising chemo preventative and chemotherapeutic agent against human gastric carcinogenesis. As the cytotoxic activity of the [(cis-DACH)AuCl2]Cl complex is high towards some cancer cell lines, further biological evaluation for this class of complex is worthy of effort, especially in order to evaluate activities in vivo. Acknowledgement The author(s) would like to acknowledge the support provided by King Abdulaziz City for Science and Technology (KACST) through the Science & Technology Unit at King Fahd University of Petroleum & Minerals (KFUPM) for funding this work through Project No. 10-BIO1368-04 as part of the National Science, Technology and Innovation Plan.

Day 1 (24 h) 0.54516 ± 0.02483 0.79107 ± 0.40634

Day 3 (72 h) 1.091 ± 0.068 0.85672 ± 0.23955

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