Synthesis, Spectroscopic, and Anticancerous Properties of Mixed Ligand Palladium(II) and Silver(I) Complexes with 4,6-Diamino-5-hydroxy-2-mercaptopyrimidine and 2,2′-Bipyridyl

Synthesis of two new water-soluble mixed ligand [Pd(bpy)(dahmp)]Cl and [Ag(bpy)(Hdahmp)]NO3 complexes (dahmp and Hdahmp are the deprotonated monoanion and the protonated neutral 4,6-diamino-5-hydroxy-2-mercaptopyrimidine, resp.) is reported. The composition of the reported complexes was discussed on the bases of IR, 1H NMR, and mass spectra, as well as conductivity and thermal measurements. The reported complexes display a significant anticancer activity against Ehrlich ascites tumor cells (EACs). The higher activity of these complexes with their higher conductivity values corresponds to their complete ionization in aqueous solution.


INTRODUCTION
Currently, cisplatin is being used as an anticancer agent in several human cancers, particularly, testicular and ovarian cancers [1,2]. Side effects, especially nephrotoxicity, of this drug limit its widespread use in high dose [3]. The need to develop new complexes with reduced nephrotoxicity and higher activity has stimulated the synthesis of many new complexes. Over the past years, a renewed interest in Pd(II) complexes as potential anticancer agents has developed. Though a number of interesting Pd(II) targets have been investigated [4][5][6][7], the biological utility of such agents continues to be questioned, this may be due to the poor solubility of common Pd(II) complexes under physiologic conditions. Many studies, including nucleic and amino acid derivatives, showed that 2-mercaptopyrimidine and 2mercapto-4-aminopyrimidine are able to inhibit the synthesis of t-RNA [8]. Thus, they may act as valuable substrates in the synthesis of antitumor chemotherapeutic agents [9]. Also, the effect of 2-mercaptopyrimidine-5-carboxylic acid and S-analogy of pyrimidinic bases on oral epidermoid human carcinoma (KB) have been reported [10,11].

Metal-Based Drugs
The cells of Ehrlich ascites (EACs) tumor were obtained from National Cancer Institute (Cairo, Egypt). After harvesting and preparation of the cells, their total number and viability were determined by counting using Trypan blue [14].

Instrumentation
Microanalyses were determined by the Micro Analytical Unit (Cairo University, Cairo, Egypt). Electronic spectra were recorded using a Unicam UV 2-100 U.V.-vis. Spectrometer. IR spectra were measured as KBr discs on a Matson 5000 FT-IR spectrometer (Cairo University). 1 H NMR spectra were measured on a Varian Gemini WM-200 spectrometer (Laser Centre, Cairo University). Thermal analysis measurements were made in the 20-800 • C range at the heating rate of 10 • C min −1 , using α-Al 2 O 3 as a reference, on a Shimadzu Thermogravimetric Analyzer TGA-50. Conductimetric measurements were carried out at room temperature on a YSI Model 32 conductivity bridge. Mass spectra were recorded on a Matson MS 5988 spectrometer (Micro Analytical Unit, Cairo University).

[Ag(bpy)(Hdahmp)]NO 3
Silver nitrate (0.087 g, 0.5 mmol) in water (2 cm 3 ) was added to bpy (0.078 g, 0.5 mmol) in methanol (35 cm 3 ) to produce a colorless solution, to which Hdahmp (0.08 g, 0.5 mmol) was added. The reaction mixture was stirred in dark for 3 hours to produce a pale brown solid. It was filtered off, washed with little water, methanol, and diethyl ether, then dried in vacuo. Conductivity data (

Anticancer activity against Ehrlich ascites carcinoma in mice
All the compounds were screened for their anticancer activity by dissolving samples in minimum amount of DMSO (Hdahmp) or water (complexes) and diluting with phosphate-buffered saline (PBS; pH = 7.2). The anticancer studies using Ehrlich ascites tumor cells (EACs) were carried out by incubating 0.2 mL of cells IP. All the treatments started 24 hours after inoculation for 45 days. The tumor-bearing mice were divided into three groups. Group (1) is the standard one that received the 5-florouracil [ (3) is the control one received physiological saline (0.9% sodium chloride). The complexes are powder-like, stable in the normal laboratory atmosphere, and soluble in water, DMF, or DMSO. We had hoped to characterize the structure of one of the complexes by single X-ray crystallography, but were thwarted on numerous occasions by very small crystal dimensions. Thus, the characterization of these complexes was based on the physical and spectroscopic techniques.
In the spectrum of [Pd(bpy)(dahmp)]Cl, the stretching vibration ν(OH) at 3305 cm 3 in the free ligand is missing in the complex [21]. The bands at 3390 and 3185 cm −1 , arising from ν s (NH 2 ) and ν as (NH 2 ), respectively [21], in the free ligand are shifted to lower wave numbers upon complexation [21,22]. The bands arising from ν(C=N), ν(NCS), and ν(C=S) are not affected while the bands arising from ν(NH) and δ(NH) stretches are slightly shifted to lower wave number in the complexes. This suggests that Hdahmp Table 1: Spectral data of Hdahmp and its complexes.   acts as a mononegative bidentate ligand, coordinating the metal ion through the deprotonated hydroxyl and amino N(6)H 2 groups, without any participation of the thione sulfur or cyclic nitrogen atoms in coordination [23]. This feature is further supported by the observation that a band near 1178 cm −1 arises from ν(C=S) stretch that remains unchanged [18] (Scheme 2). The vibrational spectrum of [Ag(bpy)(Hdahmp)]NO 3 suggests the participation of the thione sulphur and the cyclic N(3) in coordination due to the shift observed in the ν(C=S) and ν(N-C=S) stretching vibrations (Scheme 3). This suggestion is supported by the slight shift of ν(NH) and δ(NH) to lower wave number with the existence of ν s (NH 2 ), ν as (NH 2 ), and ν(OH) stretches more or less in the same position as in the free ligand [18,22,24]. Also, the bands of the free bpy ligand near 740 cm −1 are shifted to higher frequencies in the complexes (773 cm −1 ) [4,25].

1 H NMR spectra
The 1 H NMR spectrum of Hdahmp in d 6 -DMSO shows two singlets at δ6.07 and 6.18 ppm arising from N(4)H 2 and N(6)H 2 , respectively (Scheme 1). The proton of the hydroxyl group O(5)H appears as a broad singlet at δ9.13 ppm and the N(1)H proton gives a singlet at δ7.43 ppm. In the 1 H NMR spectrum of [Pd(bpy)(dahmp)] + , the proton of the hydroxyl group is not observed while the resonance arising from N(6)H 2 is shifted to lower field [24,30]. Also, the resonances arising from N(4)H 2 and N(1)H are slightly shifted to lower field. This is probably due to the decrease in the electron density caused by the withdrawing of electrons by the metal ions from the pyrimidine ring coordination centers [13,23].

Mass spectra
The

Thermal measurements
The  the complexes species [31]. Since the conductivity for Cl − and NO − 3 is 76 and 71 ohm cm 2 , respectively [31,32], this suggests that the complexes ionized completely in aqueous media [33].

Anticancerous activity
The reliable criteria for judging the efficacy of any anticancer drug are prolongation of life span, improving the clinical, hematological, and biochemical profile, as well as reduction in viable tumor cell count in the host [34,35]. We have reported that [PdL(pa)] + , [PdL'(pa)Cl], and [Pd(bpy)(cdhp)] (L = 2, 2 -bipyridyl; L = 9,10-phenanthroline, 2-(2pyridyl)quinoxaline); pa = anion of 2-pepiridine carboxylic acid; cdhp = dianion of 5-chloro-2,3-dihydroxypyridine) in water or DMSO exhibit potent cytotoxic activity against Ehrlich ascites tumor cells [4,5]. It is known that the anticancer available drugs inhibit the hematological and biochemical parameters (hemoglobin (Hb), red blood cells count (RBCs), and white blood cells count (WBCs); blood picture). The ultimate goal of this project is to develop mixed ligand complexes containing nitrogen bases effective against  cancer without side effects on the hematological and biochemical parameters.
In order to detect the influence of Hdahmp, [Pd(bpy)-(dahmp)]Cl, and [Ag(bpy)(Hdahmp)]NO 3 on the hematological status of EAC-bearing mice, a comparison study was made among three groups of mice (each group contains seven mice) from the second day after inoculation. Group (1) tumor-bearing mice treated with 5-fu (standard [36,37] 3 shows remarkable efficacy manifested by survival and activity, as well as reduction in the tumor size. The hematological parameters including hemoglobin (Hb), red blood cells count (RBCs), and white blood cells count (WBCs) data are reported in Table 3. It is clear that the hematological parameters of tumorbearing mice treated with Hdahmp, [Pd(bpy)(dahmp)]Cl, and [Ag(bpy)(Hdahmp)]NO 3 exhibits much better significant figures with the use of small doses of (0.01 mg/mice/day) compared with the standard (5-fu), the market drug (∼0.4 mg/mice/day).
There are reports that complexes containing pyridine ring (cyclic nitrogen) display significant anticancer activity [4,38]. Thus, the presence of the pyrimidine ring increases the anticancer activity and activates the binding of metal ion to the tumor DNA as it contains two cyclic nitrogen atoms [5].
The hematological parameters show that [Pd(bpy)-(dahmp)]Cl and [Ag(bpy)(Hdahmp)]NO 3 are more effective than Hdahmp itself, as the presence of both bpy and dahmp in the complexes possess a multiring planar area with nitrogen bases and hence higher hydrophobicity, which would lead the intercalation more deeply into the tumor DNA [5].
In order to investigate the action of Pd(II) and Ag(I) complexes in the tumor DNA, the intercalated complexes affecting the structure of the DNA prevent polymerase and other DNA binding proteins from functioning properly. As the complexes covantely bind to DNA with preferential binding to the N-7 position of guanine and adenine, they are able to bind two different sites on DNA, producing cross-links that cause increase in the viscosity in comparison to the nor-mal unbound DNA. The results are prevention of DNA synthesis, inhibition of transcription, and induction of mutations [5,39].
Regarding the tumor size and EAC count, in the control group was (220 × 10 6 cells/cm 3 ), reduced in using 5-fu to (80 × 10 6 cells/cm 3 ) while the strong reduction to 44.8 × 10 6 , 31.8×10 6 , and 30.4×10 6 cells per cm 3 was observed in using Hdahmp, [Pd(bpy)(dahmp)] + , and [Ag(bpy)(Hdahmp)] + , respectively. The strong reduction in EAC count and tumor size may be due to the reductive nature of many tumors that contain significant regions at low oxygen tension. Thus, they initiate a catalytic auto-oxidation process involving generation of reactive oxygen species. The coordinated dahmp and bpy may reduce toxic effects caused by xenobiotic core of the complexes, contribute to their anticancer action, and facilitate their transport through cell membrane [40]. Our investigations have shown that glutathione content, in liver and kidney, and glutathione-S-transferase activity were decreased, suggesting that the partial reduction products of oxygen, in the presence of the complexes, yield very reactive species, which could start catalytic oxidation of substrates and show antitumor action [41].

Effect of survival time
The mean survival time (MST) of groups 1 and 2 was compared with that of the control group using the following calculations [42]. Percentage (%) increase in lifespan over control = [(MST of treated group × 100/MST of control group) −100]; MST = days of each mice in the group/total number of mice. Percentage (%) increase in lifespan over control showed to be high in mice treated with Hdahmp, [Pd(bpy)(dahmp)]Cl, and [Ag(bpy)(Hdahmp)]NO 3 (Table 3).

The side effects and toxicity
The side effects and toxicity of Hdahmp, [Pd(bpy)-(dahmp)] + , and [Ag(bpy)(Hdahmp)] + have been detected. After the first week of the treatment, the mice show flu-like 6 Metal-Based Drugs attack and in the third week show spot dropping on the hair (alopecia). Fortunately, the solid organs have not been affected.
The study of detailed mechanism and in vivo anticancer screens (phase II & III) using the studied complexes are under way.

CONCLUSION
There are reports that complexes containing pyridine ring (cyclic nitrogen) display significant anticancer activity. The anticancer activity of the new water-soluble complexes, [Pd(bpy)(dahmp)]Cl and [Ag(bpy)(Hdahmp)]NO 3 , shows remarkable efficacy against Ehrlich ascites tumor cells (EACs) manifested by survival and activity, as well as reduction in the tumor size.