Metalo components exhibiting significant anticancer and antibacterial properties: a novel sandwich-type like polymeric structure

Four new dicyanoargentate(I)-based complexes 1–4 were synthesized from certain metal ions with a tetradentate ligand [N, N-bis (2-hydroxyethyl) -ethylenediamine; N-bishydeten] and determined by diverse procedures (elemental, thermal, FT-IR, ESI–MS for 1–3 and, magnetic susceptibility and EPR for 1, and 2) including crystal analysis of 4. The crystal method revealed that complex 4 has a sandwich-type like polymeric chemical structure with layers formed by [Cd(N-bishydeten)2]2+ cations and [Ag(CN)2]− anions. The complexes were further characterized by fluorescence and UV spectroscopy to determine their physicochemical features. The complexes displayed a DNA binding activity within the same range as found for cisplatin, in addition to their strong stability in the presence of the physiological buffer system. The complexes were also investigated for pharmacological properties like interaction with DNA/Bovine serum albumin, anticancer and antibacterial activities. Physicochemical studies of DNA with the complexes suggested that the interaction mode between them are possibly both intercalative and groove binding types. These spectroscopic measurements also show that there may be a binding tendency between BSA and the complexes via hydrogen or Van der Waals bonds. The viability tests demonstrated that all the complexes exhibited antibacterial (1–4) and anticancer effects (2–4) toward ten diverse bacterial strains and three tumor cells (HT-29 colon adenocarcinoma, HeLa cervical cancer, and C6 glioma), respectively.


Experimental section
Synthesis. The synthesis acts were performed in the room temperature. The KCN (153 mg, 1.175 mmol) was added into a magnetically stirred solution of AgNO 3 (200 mg, 1.177 mmol) in ethyl alcohol (20 mL)/ water (10 mL). Firstly, the Ni(II), Cu(II), Zn(II) and Cd(II) salts (1 mmol) were added to the clear solution of K[Ag(CN) 2 ] (1 mmol, 0.199 g) prepared in the water-ethyl alcohol (volume ratio of 2:1) mixture. Afterward, the obtained metal salt solution was added to the auxiliary ligand N-bishydeten (2 mmol, 0.296 g) solution prepared in the alcohol, and it was stirred for about one hour. The resulting product was filtered, and also the clear filtrate was left to crystallize under room conditions. Complexes 1-3 were obtained in low yields as powder crystals, while complex 4 also formed in low yields, but as single crystals (Table S1), (Fig. 1). The reason that complexes are obtained in low yields may be a consequence of the very high tendency of N-bishydeten to produce stable complexes in the solution media or due to the steric hindrance around the coordination centre 30,33,46-51 . [Ni (N-bishydeten)Ag 3 (CN) 5 ] (1). Pink precipitates were recorded with a yield of 43% for 1. Anal. Calc. for C 11  [Cu(N-bisydeten)Ag 3 (CN) 5 ] (2). Light green precipitates were recorded with 40% yield for 2. Anal. Calc. for C 11    Characterization of 1-4. The structures of complexes 1-4 were determined by elemental analysis, IR, EPR (for 1 and 2), ESI-MS (for 1-3) and X-ray crystallography (for 4) techniques, and the proposed molecular formulas were estimated by thermal analyses (DTA and TG/DTG) and magnetization measurement (for 1 and 2) techniques. Thermal analysis is like fingerprinting of materials, such that, each obtained thermal analysis curve is specific to the tested specimen, provided that a correct structure is proposed. For instance, the mass loss indicated by the TG curve of a synthesized compound can be interpreted correctly only if a correct molecular formula is introduced. Besides, the experimental effective magnetic moment (μ effE ) of a complex is consistent with the theoretical effective magnetic moment (μ effT ) to the extent that a correct molecular formula makes the calculation. On the other hand, the typical peaks appearing in the ESI-MS spectra of 1-3 are attributed to [M + 2H] + . According to the characterization results, complexes 1-3 may have a molecular structure as given in Figure S1. The images in Figs. 2 and 3, s5, s6, s7, and s8 were generated by using K. Brandenburg, Diamond-Crystal and Molecular Structure Visualization, Crystal Impact GbR, Vers. 4.5.2, Bonn, Germany, 2018.

Results and discussion
IR spectra. The characteristic bands of the functional groups of all the complexes are presented in the "Experimental Section" and the IR spectra of the complexes and N-bishydeten ligand are depicted in Figure S2 (Supplementary Material). The most significant vibration frequency for cyanido complexes is known to be the peaks of the CN group. The frequency of the free CN group is different from that of the CN group, which is coordinated with the metal (M-C≡N) or bridged between metal centers (M-C≡N-M′). When the cyano group forms a bridge between the metal centers, the stretching vibration band of cyano usually splits as well, while the vibration frequency value shifts to a higher wavelength 54 . As clearly indicated by the IR spectra of 1-4 given in Figure S2, CN stretching vibration peaks have both shifted to higher frequencies, and the peaks are split. The splitting of cyano-stretching vibration peaks in 1-3 into two or three peaks is the most critical evidence that they are in the polymeric structures. Meanwhile, the polymeric complex 4 formed a single stretching vibration band ( Figure S2). In this part, the terminal cyanido ligand involved in hydrogen bonding (HBs) interactions (Table 1) 55,56 .
The sharp shift of υ(O-H) over 3000 cm −1 to a frequency higher than the O-H vibration of the ligand results from the free presence of the O-H group in the complexes. The υ(NH 2 ) stretching vibration of the N-bishydeten ligand, which is expected to emerge as a splitting peak from NH 2 -groups, also appeared at 3300-3100 cm −1 . Excessive splitting of the υ(C-H) stretching vibrations at 2980-2840 cm −1 may result from the different environment of the CH 2 groups as a result of the binding of the N-bishydeten ligand to the metal ions.
In addition, the absorption bands observed in the complexes in the range of 1600-993 cm −1 are stretching and bending vibrations caused by bonds between the atoms of C-, N-, O-and H-. These absorption bands are also present in the free ligand, N-bishydeten, but minor variations in the absorption bands of these functional groups have been observed in the complexes. For example, while the C-N stretching vibration was observed at 1151 cm −1 in the neutral ligand, this stretching vibration band in the complexes 1-4 was observed at 1197, 1141, 1122 and 1114 cm −1 , respectively. Also, as a result of the coordination of Ag I and M II ions with C-, O-and N-atoms, peaks which are thought to belong to Ag I -C, M II -N and M II -O stretching vibrations in the range of 600-400 cm −1 were found. Thermal analyses. Thermogravimetric/thermogravimetric derivative-differential thermal analysis (TG/ DTG-DTA) measurements of 1-4 also support the crystal composition (4) and the proposed structures (1-3) as shown in Figures S3 and S4. The TG/DTG curves of 1-4 are followed by a process in which a multi-step weight loss is observed from 35 to 1050 °C. The sharp peak at 300-400 °C in the thermal decomposition graph of complex 1 corresponds to an N-bishydeten ligand and two cyanide groups, while complex 2 corresponds only to the degradation of the N-bishydeten ligand at 500 °C ( Figure S4). The neutral N-bishydeten ligand is degraded in the initial steps of the thermal decomposition which is followed by the thermal degradation of the cyanido ligand.    Figure S5; Supplementary Material). In the polymer chains like the structure of complex 4, the dicyano silver moieties were adopted slightly bent like conformer using intramolecular argentophilic interaction (Ag1…Ag2…A3 and Ag4…Ag5…Ag4) ( Fig. 2 and Figure S6; Supplementary Material). In the structure, argentophilic interactions cooperatively act with HBs interaction (Table 1) Figure S5). On the other hand, All the N-Cd-N, C-N-Cd and C-Ag-C which deviates remarkably from the 90° and 180°, which were likely the outcome of the steric limitations arising from the form of the ligands (Scheme 1). The N2A-Cd1-N1, O2A-Cd1-O1A, N9A-Cd2-N8 and O4B-Cd2-O3B angles formed by Cd1 and Cd2 centers-N-bishydeten ligands are 76.5(6)°, 90.9(6)°, 71.9(4)° and 91.9(8)°, respectively. Additionally, the Cd2-N7-C8, C8-Ag1-C7 and C7-N4-Cd1 bond angles are 170.0(4)°, 158.4(2)° and 165.6(4)°, respectively, while the Cd2-N11-C18, C18-Ag4-C20 and C20-N13-Cd1 bond angles are 171.4(4)°, 171.1(2)° and 167.2(5)°, respectively (Table S2). As a result, the significant deviation from linearity of the Cd-N-C and C-Ag-C angles leads to the formation of arc-shaped chains at the Ag1 and Ag4-centered respectively as seen from the Fig. 2 and Figure S5.
The anionic slices {[Ag(μ-CN-) 2 ] 8 [Ag(CN)] 3 } 8− of the sandwich-type like structure are composed of the bridged Ag 8 (CN) 16 8− anions and the Ag 3 (CN) 3 groups involved in argentophilic interactions. The argentophilic interactions observed between Ag(I) centers (d 10 -d 10 ) are ligand-unbacked forces and have an essential role in complex stability and molecular clustering 57,58 . The Ag…Ag bond distances of the triple and quintet fragments of complex 4 have various values ranging from 3.12 to 3.23 Å which are below the sum of the vander Walls of two Ag atoms (3.44 Å) 59  EPR and magnetic properties. The powder EPR spectra of complex 1 containing Ag + and Ni 2+ ions at room temperature could not be observed. This situation may be because Ag + ion is diamagnetic and Ni 2+ ion does not signal because it has short relaxation times at room temperature. EPR spectrum analyzes of Ni 2+ ioncontaining complexes reveal that EPR signals cannot be obtained at room temperature, but very low-intensity peaks can be seen at very low temperatures 60 .
The powder EPR spectra of complex 2 are seen in Figure S9 in the Supplementary Material. The EPR spectra of 2 have been observed in the parallel and perpendicular components. The parallel peak is because the dc field is equal to the symmetry axis of the paramagnetic center. The values of g ⊥ and g // extracted from the powder www.nature.com/scientificreports/ spectrum of complex 2 are g // = 2.210, g ⊥ = 2.095, respectively. This spectrum belongs to Cu 2+ ion (S = 1/2, I = 3/2). It can be inferred from the order of g // > g ⊥ > g e (g e = 2.0023, free electron g value) that, Cu 2+ is located in distorted items (D 4h ) elongated along the ground state of the paramagnetic electron is d x 2 −y 2 ( 2 B 1g state) and z-axis [61][62][63][64] . When the Lande g values of Cu 2+ complexes containing tetracyanidometallate having neutral ligands are compared with those of complex 2, it is noticed that g amounts are g // > g ⊥ > g e 33,34,48,49,65-68 . The magnetic susceptibilities of 1 and 2 were recorded in the temperature of 10-300 K. The temperature dependence of magnetic (χ m ) and χ m T are seen in Figures S10 and S11 (Supplementary Material) for both complexes. The variable temperature dependence of χ m for both complexes were coordinated by the relation α + C/(T − θ) , which α is the temperature independent susceptibility (TIP) 69 . For 2, the determined results are: C = 0.588 ± 0.0003 emuK/mol Oe , α = 0.00027 ± 0.000003 emu/mol Oe and = −4.9 ± 0.009 K . As for 1, the determined fitting results:C = 2.56 ± 0.0005 emu/mol Oe , α = 0.00077 ± 0.000004 emu/mol Oe and = − 0.6 ± 0.002 K . The good magnetic moment for 1, µ eff , was determined as 4.52 in Bohr magneton ( µ B ) 70 .
In this part, 10 K, for 1 and 2 could be very tiny antiferromagnetic interplay in the chemical structure, as observed in the insertion of Figures S10 and S11. The magnetic of 1 and 2 recorded a resemblance to tetracyanidometallate containing similar neutral ligands 33,34,48,49,[65][66][67][68] . They even exhibited antiferromagnetic properties at low temperatures as in complexes 1 and 2 33,48,49,65-68,71,72 . DNA topoisomerase I, DNA restriction endonucleases, and DNA binding studies. Determination of DNA topoisomerase I enzyme inhibitory activities. DNA topoisomerase that is an important target for anticancer agents are nuclear enzymes and alter the topological state of DNA molecule during the cell division and another cellular process such as replication and transcription 73,74 . Today, some topoisomerase inhibitor compounds like Camptosar, topotecan, and irinotecan have been utilized in clinical practice. Hence, to figure out the antiproliferative activities of these molecules includes inhibition of DNA topoisomerase I, we investigated the effects of these molecules on the recombinant act of topoisomerase I enzyme. The results showed that IC50 concentration of these compounds, for instance Camptothecin (Fig. 4), inhibited the DNA relaxation activity of topoisomerase I that they can be used as a new topoisomerase I inhibitor which acts through binding to topoisomerase I. The results of other studies also revealed that metal complexes bind to topoisomerase I and inhibited it 70,75,76 Determination of DNA restriction endonucleases activity. In the presence of the 3 and 4, DNA digestion was complete, and two bands were observed near the well at the top of the lanes (Lanes 1 and 2). Treatment of KpnI and BamHI with 3 and 4 failed to inhibit the restriction endonucleases activity of these enzymes. These results indicated that 3 and 4 did not bind to pTOLT plasmid DNA. However, the 2 caused the formation of two DNA  In addition to UV-Visible absorption spectroscopy technique, the ethidium bromide exchange studies were also conducted to determine the binding affinity between 2, 3, 4 and DNA. The emission spectrum data EB bound to DNA in the presence and absence of 2, 3, and 4 are depicted in Figure S13 (Supplementary Material). The decreases in the fluorescence intensity of EB-DNA in the presence of 2, 3, and 4 implied that they might intercalate into a pair of the DNA. The quenching of EB to CT-DNA by the 2 is in harmony with the Stern-Volmer equation, which provides more evidence about the interaction between 2 and DNA, and is shown in Figure S13  Stability study. The results of these molecules were conducted by utilizing a simple spectrophotometric assay. The molecules in physiological buffer (Phosphate buffered saline, 0.1 M, pH 7.4) were performed at regular intervals for 24 h. There were no changes in absorbance up to 24 h in complexes. Thus, the silver compounds proved to have the ability of high solution stability in a buffer ( Table 2).
This study was assessed using the absorbance values of eight diverse concentrations of the molecules within the same day and between various days. The repeatability, inner-and intra-day precision of the work displayed at since % RSD < 2% for the molecules. These molecules remained fairly stable (Figure S14 Table 2). The plots in measuring of the compounds were found to be linear in the scanning concentration range, and the linearity values of 1, 2, 3 and 4 were 0.95-0.98 for all ( Table 2). The lowest amounts that could be detected ( (Fig. 6). To determine whether selectively killed the cancer agents in the absence of being detrimental to the standard cells, we determined the antiproliferative actions of our molecules towards colon, cervical, a normal cell line (Vero), and brain cancer cell lines. BCPA test effects implied that 2 (0.87-3.64 µM), 3 (2.37-3.34 µM), 4 (0.48-0.63 µM) and [Ag(CN) 2 ] − (5.08-5.63 µM) ligand disclosed very high antiproliferative effects on these cells, while ligand, N-bishydeten, seen no antiproliferative effects towards cancer with the same administrative dose (data not shown). Antiproliferative activities of complex 2 were higher on HT29 (0.87 ± 0.09 µM) and C6 (0.95 ± 0.09 µM) cells in comparison to Vero cells (Fig. 6 and, Table 3). This text means that complex 2 has an interesting selectivity towards cancer cells. Tumor specificity index (TSI) and IC 50 amounts to be used in consequent studies were recorded by performing the BrdU ELISA method, and these are given in Table 3.
The find tumor specificity index results divided by the sum of the IC 50 amounts from normal cells (Vero) to the sum of the IC 50 values of the cancer cells (C6, HeLa, HT29) ( Table 3). Molecule 2 recorded the best selectivity for the HT29 (3.23 TSI) and C6 (2.96 TSI) cells over the Vero cells while compounds 3 (1.29 TSI) and 4 (1.31 TSI) displayed poor selectivity for the HT29 cells. The cell proliferation results disclosed that the Ag(I) molecules were remarkably more antiproliferative than cisplatin and 5FU (Fig. 6) 30-34,58 . Cytotoxic profile of the Ag(I) compounds. LDH test results revealed that 2, 3, and 4 exhibited the identical cytotoxic effects as the 5FU, Indeed, bridging ligand [Ag(CN) 2 ] − , caused greater cytotoxicity than positive control on some cell lines ( Figure S15; Supplementary Material). It is observed that [Ag(CN) 2 ] − is a highly toxic molecule towards both standard and tumorigenic cells. However, it was found to have a limited effect while examining the contribution of [Ag(CN) 2 ] − to the antiproliferative and cytotoxic activities of our compounds. As seen in Fig. 6 and Figure S15, the antiproliferative and cytotoxic activity of 2, 3, and 4 were lower than bridging ligand, recording that the cytotoxicity of [Ag(CN) 2 ] − reduced to safe levels in Ag(I) compounds. All compounds and 5FU or cisplatin (9-11%) tested and also were found to be moderately cytotoxic against HT29 cells. How-  (Table 3). However, it is necessary to conduct in vivo studies in order to determine the real cytotoxic effect of these compounds. An ideal anticancer drug would exterminate cancer cells without disturbing normal cells and has cytostatic profiles that can activate apoptosis 78 .

Determination of the apoptotic effect of the Ag(I) complexes by DNA laddering method. DNA
laddering revealed the 2, 3, and 4 induced the organization of DNA fragmentation in cancer cells in comparison to the standard cells (Fig. 7). Here, appearances of apoptotic morphology and DNA fragmentation may be a result of the activation of the extrinsic apoptotic pathways, including Ca 2+ dependent endonucleases. Apoptosis assay is determined by controlling cell death which included cleavage of DNA molecule into regular fragments. In this part, we observed that our molecules could act through containing apoptosis on some cells. More studies were conducted to obtain the antiproliferative and apoptotic potentials of Ag molecules which are consistent with this work 70,75,75,79,80 .
The apoptotic effect of the Ag(I) molecules at the single cell level. Ag(I) compounds were found to exhibit a possess pharmacological effect and vigorous antiproliferative property. We have evaluated the apoptotic effect of the molecules on HT29 cells utilizing the TUNEL method as an immunohistological study to reveal their mechanism of action on cells. The apoptotic activity of the Ag(I) compounds was examined in human colon cancer cells, HT29. The TUNEL method stressed the formation of the apoptosis in the HT29 cells with concentrations of 0.87 ± 0.09 μM for 2 and 2.37 ± 0.32 μM for 3 and 0.48 ± 0.08 μM for 4 for 24 h. In Fig. 7, HT29 cells treated with Ag(I) compounds caused green fluorescence, indicating fragmented DNA in apoptotic cells. As illustrated in Fig. 8, TUNEL results depicted that 2, 3, and 4 significantly triggered apoptosis on HT29    Fig. 9, the increase in BSA concentrations leads to a change in the absorption of the complexes resulting in hypochromism for 1 and 3 and hyperchromism for 2 and 4. These results suggested that an interaction exists between these complexes and BSA similar to the DNA binding mode of the complexes. In addition, the molecules caused an upward trend in the BSA absorbance and exhibited a slight redshift, indicating the presence of van der Waals or hydrogen bonds between BSA and them.
The effect of the Ag(I) molecules on HeLa cell migration. The capacity of the cancer cell migration is an excellent target for anticancer agents because tumor cells may escape from the apoptosis mechanism by using its migration capability. Successful cancer treatment involves both inhibition of cancer proliferation and suppression of the migration effect as plenty of cancer cells exhibit potent cell growth and invasive behavior. Ag(I) compounds at 50% maximal inhibitory concentration (IC 50 ) decelerated HeLa cell migration and enhanced apoptotic stress ( Figure S16). The 2, 3 and 4 can also restrict the level of development of HeLa cells, indicating they could be entered to preclinical trials. Ag(I) compounds at 30% maximal inhibitory concentration (IC 30 ) managed to inhibit tumor cell migration with low cytotoxicity (data not shown). In addition, this cytotoxic ability of Ag(I) compounds at 20% maximal inhibitory concentration (IC 20 ) allowed the suppression of cell migration without damaging the cell membrane at non-toxic concentrations (data not shown). After 72 h of incubation, the migration rate of the treated cells failed to fill the gap ( Figure S16; Supplementary Material, Day 2), and untreated HeLa cells accomplished to form throughout the gap. While untreated HeLa cells filled 100% of the gap, compounds 2, 3, and 4 managed to spread to 9%, %11, 15% of the gap, respectively ( Figure S16; Supplementary Material, Day 2). In addition, the media with Ag(I) compounds were replaced with fresh media following the 72-h incubation, but the HeLa cells could not fill the gap (data not shown). That is, the Ag(I) compounds may exhibit cytostatic effect by inhibiting cell growth and multiplication. The result was consistent with that of the other studies on Ag(I) complexes containing different ligands 73,74,78,80,81,83-87 . The effect of the Ag(I) complexes in the morphology of the cells. As shown in Figure S17 Figure S17 have also displayed the normal structure of the most control cells. Most of the treated cells had an abnormal fibroblast-like appearance and were detached from the plate surface. Moreover, the cells began to separate from one another and to appear smaller. These situations were consistent with the outcomes of TUNEL methods, and this finding was similar to those of previous studies [83][84][85][86][87] . According to information found in literature [88][89][90] , the appearance of the cells treated with 2, 3 and 4 clearly indicated the the quality and the number of cells in the flask monolayer were reduced.

IHC investigation of slides treated by Ag(I) molecules. Immunohistochemistry staining was found
to reduce the expression of Bcl-2 and increase the expression of P53 in Ag(I) complexes-treated the cells, which emphasizes the apoptotic effects of these molecules (Figures S18 and S19). These findings are agreeable with those of similar works 91 . The results also revealed that Ag(I) complexes treated cells significantly reduced the expression of cytokeratins (CK20 and CK7) releasing from proliferating or apoptotic cells. This condition can be associated with the reduced metastatic capability via an anti-migratory potential of these molecules due to the influenced intermediate filament (IF) proteins. there are some powerful links among the pathogen bacterial flora (i.e., septicemia) or the opportunistic agents (i.e., pneumonia infections) and certain cancer kinds like urogenital, cervical, stomach cancers, liver, and lymphoproliferative disturbances 70 . Therefore, the pathogen bacterial flora and the opportunistic agents may be taken into consideration both in the management of cancer patients and in individual susceptibility to cancer 80 . Indeed, dual acting factors with antimicrobial and antiproliferative potentials can result in improved therapeutic efficacy for cancer cell patients or reduced cancer predisposition. In light of this information, the antimicrobial activities of 1-4 were also tested against four gram-positive bacteria and five gram-negative bacteria. The experiments were conducted in triplicate to prevent possible errors, and SCF [Sulbactam (30 µg) + Cefoperazone (75 µg)] was used as a standard drug 53,92,93 . Results were 4 > 2 > 3 > SCF > 1 > KCN for antibacterial activities while for S. enteridis, and S. gallinarum were 4 > SCF > 1 > KCN sequence of antibacterial effects (Tables 4 and 5). The bacterial inhibition sites of complexes  In this study, molecules 4 and 1 were subjected to MIC, and the findings profiles are submitted in Table 5. Sulbactam (30 µg) + Cefoperazone (75 µg) (105 µg/disc), were used as the standard and investigated by the Serial microdilution method to obtain MIC values in Mueller-Hinton Broth for the antibacterial test. The inhibition zones and MIC amounts for strains for 4 and 1 were recorded in the range of 15-37 mm and 15.62-125 μg/mL, respectively (Tables 5 and 4). Four types of gram-positive bacterial strains (St. pyogenez, B. subtilis, B. cereus, S. aureus) and five types of the gram-negative (S. enteridis, E. aerogenes, P. aeruginosa, E. coli, and S. gallinarum) were sensitive to 4 and 1. For the 4, the MIC and inhibition zones values of the bacterial strains were found as 31.25-125 μg/mL and 25-37 mm, respectively (Tables 5 and 4). In Table 5

Conclusion
In this present study, four different complexes were synthesized using Ni 2+ (1), Cu 2+ (2), Zn 2+ (3), Cd 2+ (4), K [Ag(CN) 2 ], and N-bishydeten and characterized by some advanced analytical techniques. Complex 4 consisting of [Cd(N-bishydeten)]4[Ag(CN) 2 ]8[Ag(CN)] has a sandwich-type layered structure verified by the crystal method. In addition, the complexes were studied for their pharmacological properties, and they exhibited very strong anticancer (2-4) and antimicrobial activities (1-4). The compounds, especially 2, possessed more selective cytotoxic activity than the positive control against cancer cells, particularly HT29. The interaction of 1-4 with CT-DNA and BSA was shown with respect to the spectral changes in their absorbance, and their binding affinity was found to be very similar to the currently used anticancer agents such as cisplatin and 5FU. In future studies, we will try to improve the amount and functionality of our Ag(I) complexes using different ligands, metal salts, and new methods. Since the in vitro biological properties of these Ag(I) complexes can be used mainly against some cancer cell lines, in vivo anticancer study is very important to reveal the mechanism of action. In summary, our results show that these molecules are potentially valuable drug candidates and are suitable for further pharmacological testing.