# Synthesis, Spectroscopic, and Anticancerous Properties

Authors: Sahar I. Mostafa; Farid A. Badria
Journal: Metal-Based Drugs
 (2008)

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## Abstract

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, H1 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.


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## Body

## 1. 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 [4–7], 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 2-mercapto-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].As a continuation of our research in
4,6-diamino-5-hydroxy-2-mercaptopyrimidine complexes and their biological
activities [12], in this research, we report the complexes obtained from the reaction of
4,6-diamino-5-hydroxy-2-mercaptopyrimidine (Hdahmp) with [Pd(bpy)Cl2]
and [Ag(bpy)(H2O)2]NO3. They have been
investigated using IR, 1HNMR,
and mass spectra, conductivity and   thermal measurements. In addition, the
anticancer activity of these complexes against Ehrlich  ascites tumor cells (EACs) has been reported.


## 2. Experimental

### 2.1. Material and Methods

All manipulations were performed
under aerobic conditions using 4,6-diamino-5-hydroxy-2-mercaptopyrimidine and
all other reagents (Merck) as received. [Pd(bpy)Cl2] was synthesized
by the literature method [13].The cells
ofEhrlich 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].


### 2.2. Instrumentation

Microanalyses were determined by the
Micro Analytical Unit (Cairo University, Cairo, Egypt). Electronic spectra were recorded using a Unicam UV2–100 U.V.-vis. Spectrometer. IR spectra were measured as KBr discs on a Matson 5000
FT-IR spectrometer (Cairo University). 1H 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-Al2O3 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).


### 2.3. Synthesis of Complexes

#### 2.3.1. [Pd(bpy)(dahmp)]Cl·H2O

To a stirred suspension of
[Pd(bpy)Cl2] (0.17 g, 0.5 mmol) in methanol-benzene (3:2, V/V) (15 cm3), was added a methanolic solution of KOH (0.055 g, 1 mmol)
containing Hdahmp (0.08 g, 0.5 mmol). The resulting suspension was stirred for
two days and a brown complex was obtained. It was filtered off, washed with
water and methanol, and then air-dried. Conductivity data (10-3 M in
DMF): ΛM =
97.0 ohm-1 cm2 mol-1. Elemental Anal. Calc. for C14ClH15N6O2SPd:
C, 35.53; H, 3.17; N,17.76; S, 6.77; Cl, 7.51. Found C, 35.72; H, 3.11; N,
17.71; S, 6.85; Cl, 7.63.


#### 2.3.2. [Ag(bpy)(Hdahmp)]NO3

Silver nitrate (0.087 g, 0.5 mmol)
in water (2 cm3) was added to bpy (0.078 g, 0.5 mmol) in methanol
(35 cm3) 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 (10-3 M in DMF):  ΛM = 60.0 ohm-1 cm2 mol-1. Elemental Anal. Calc. for C14H14N7O4SAg:
C, 34.72; H, 2.89; N, 20.25; S, 6.61. Found C, 34.54; H, 2.78; N, 20.00; S,
6.42.


### 2.4. Conductivity Measurements

The conductivity values for [Pd(bpy)(dahmp)]Cl and
[Ag(bpy)(Hdahmp)]NO3 were determined. The compounds were dissolved in water and the
measurements were done at concentrations; 1, 0.8, 0.6, 0.4, and 0.2 mM. The
conductance values (ΛM) were calculated and plotted
against concentration [15].


### 2.5. 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 usingEhrlich 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 [16] (5-fu; 20 mg/kg/day of mice) for
comparison. Group (2) received Hdahmp, [Pd(bpy)(dahmp)]Cl, and
[Ag(bpy)(Hdahmp)]NO3 complexes (0.01 mg/mice/day). Group (3) is the
control one received physiological saline (0.9% sodium chloride).


## 2.1. Material and Methods

All manipulations were performed
under aerobic conditions using 4,6-diamino-5-hydroxy-2-mercaptopyrimidine and
all other reagents (Merck) as received. [Pd(bpy)Cl2] was synthesized
by the literature method [13].The cells
ofEhrlich 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].


## 2.2. Instrumentation

Microanalyses were determined by the
Micro Analytical Unit (Cairo University, Cairo, Egypt). Electronic spectra were recorded using a Unicam UV2–100 U.V.-vis. Spectrometer. IR spectra were measured as KBr discs on a Matson 5000
FT-IR spectrometer (Cairo University). 1H 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-Al2O3 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).


## 2.3. Synthesis of Complexes

### 2.3.1. [Pd(bpy)(dahmp)]Cl·H2O

To a stirred suspension of
[Pd(bpy)Cl2] (0.17 g, 0.5 mmol) in methanol-benzene (3:2, V/V) (15 cm3), was added a methanolic solution of KOH (0.055 g, 1 mmol)
containing Hdahmp (0.08 g, 0.5 mmol). The resulting suspension was stirred for
two days and a brown complex was obtained. It was filtered off, washed with
water and methanol, and then air-dried. Conductivity data (10-3 M in
DMF): ΛM =
97.0 ohm-1 cm2 mol-1. Elemental Anal. Calc. for C14ClH15N6O2SPd:
C, 35.53; H, 3.17; N,17.76; S, 6.77; Cl, 7.51. Found C, 35.72; H, 3.11; N,
17.71; S, 6.85; Cl, 7.63.


### 2.3.2. [Ag(bpy)(Hdahmp)]NO3

Silver nitrate (0.087 g, 0.5 mmol)
in water (2 cm3) was added to bpy (0.078 g, 0.5 mmol) in methanol
(35 cm3) 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 (10-3 M in DMF):  ΛM = 60.0 ohm-1 cm2 mol-1. Elemental Anal. Calc. for C14H14N7O4SAg:
C, 34.72; H, 2.89; N, 20.25; S, 6.61. Found C, 34.54; H, 2.78; N, 20.00; S,
6.42.


## 2.3.1. [Pd(bpy)(dahmp)]Cl·H2O

To a stirred suspension of
[Pd(bpy)Cl2] (0.17 g, 0.5 mmol) in methanol-benzene (3:2, V/V) (15 cm3), was added a methanolic solution of KOH (0.055 g, 1 mmol)
containing Hdahmp (0.08 g, 0.5 mmol). The resulting suspension was stirred for
two days and a brown complex was obtained. It was filtered off, washed with
water and methanol, and then air-dried. Conductivity data (10-3 M in
DMF): ΛM =
97.0 ohm-1 cm2 mol-1. Elemental Anal. Calc. for C14ClH15N6O2SPd:
C, 35.53; H, 3.17; N,17.76; S, 6.77; Cl, 7.51. Found C, 35.72; H, 3.11; N,
17.71; S, 6.85; Cl, 7.63.


## 2.3.2. [Ag(bpy)(Hdahmp)]NO3

Silver nitrate (0.087 g, 0.5 mmol)
in water (2 cm3) was added to bpy (0.078 g, 0.5 mmol) in methanol
(35 cm3) 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 (10-3 M in DMF):  ΛM = 60.0 ohm-1 cm2 mol-1. Elemental Anal. Calc. for C14H14N7O4SAg:
C, 34.72; H, 2.89; N, 20.25; S, 6.61. Found C, 34.54; H, 2.78; N, 20.00; S,
6.42.


## 2.4. Conductivity Measurements

The conductivity values for [Pd(bpy)(dahmp)]Cl and
[Ag(bpy)(Hdahmp)]NO3 were determined. The compounds were dissolved in water and the
measurements were done at concentrations; 1, 0.8, 0.6, 0.4, and 0.2 mM. The
conductance values (ΛM) were calculated and plotted
against concentration [15].


## 2.5. 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 usingEhrlich 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 [16] (5-fu; 20 mg/kg/day of mice) for
comparison. Group (2) received Hdahmp, [Pd(bpy)(dahmp)]Cl, and
[Ag(bpy)(Hdahmp)]NO3 complexes (0.01 mg/mice/day). Group (3) is the
control one received physiological saline (0.9% sodium chloride).


## 3. Results and Discussion

### 3.1. Synthesis of Complexes

Table1 lists two new complexes of
4,6-diamino-5-hydroxy-2-mercaptopyrimidine (Hdahmp). The elemental analyses of
the isolated complexes agree with the assigned formula. The conductivities (ΛM) in DMF  at room
temperature showed the electrolytic character of these complexes [4, 16].Table 1
Spectral data of Hdahmp and its complexes.
IR spectral dataComplexesν(OH)νs(NH2)νas(NH2)δ(NH)ν(C=C) ν(C=N)ν(NCS)ν(CN)ν(M–O)ν(M–N)ν(NCS)ν(CS)[Pd(bpy)(dahmp)]Cl—3397 33603165164015561450136252440912551176[Ag(bpy)(Hdahmp)]NO33308339631711650154314791396—412 372*12771207*ν(Ag–S).The complex [Pd(bpy)(dahmp)]Cl was prepared
from [Pd(bpy)Cl2] and Hdahmp in methanol-benzene in presence of
aqueous base, while [Ag(bpy)(Hdahmp)]+ was made from aqueous AgNO3 with bpy and Hdahmp in methanol.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.


### 3.2. Vibration Spectra

The characteristic IR bands observed
and vibration assignments of 4,6-diamino-5-hydroxy-2-mercaptopyrimidine
(Hdahmp) complexes are reported in Table1. The spectrum of Hdahmp supports the
existence of the thione form in the solid phase (Scheme 1). This can be
attributed to the presence of ν(NH) stretch at
2970 cm-1 [17], the absence of ν(SH) near 2600 cm-1, and the
production of the characteristic thioamide bands due to extensive coupling of δ(NH), ν(C=N), ν(NCS), and ν(C=S) at 1652, 1562, 1455, and (1375, 1268,
1177) cm-1, respectively [18–20].Scheme 1
4,6-diamino-5-hydroxy-2-mercaptopyrimidine (Hdahmp).In the spectrum of
[Pd(bpy)(dahmp)]Cl, the stretching vibration ν(OH) at 3305 cm3 in the free ligand
is missing in the complex [21]. The bands at 3390 and 3185 cm-1,
arising from νs(NH2)
and νas(NH2),
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 acts as a mononegative
bidentate ligand, coordinating the metal ion through the deprotonated hydroxyl
and amino N(6)H2 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)]NO3 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
νs(NH2), νas(NH2),
and ν(OH) stretches
more or less in the same position as in the free ligand [18, 22, 24].Scheme 2
Structure of [Pd(bpy)(dahmp)]Cl.Scheme 3
Structure of [Ag(bpy)(Hdahmp)2]NO3.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].The spectrum of [Ag(bpy)(Hdahmp)]NO3 shows new strong
band near 1370 cm-1 assigned to the ionic uncoordinated NO3- [16, 26, 27].


### 3.3. Electronic Spectra

The electronic spectrum of [Pd(bpy)(dahmp)]+ complex shows bands at 475 and 326 nm due to 1A1g→1B1g and 1A1g→1E1g, transitions in a square-planar configuration [4, 23, 27]. Also, the spectrum of [Ag(bpy)(Hdahmp)]+ shows bands at 467, 382, and 277 nm; the latter two may arise from charge
transfer of the type ligand (π)→b1g (Ag+) and ligand (σ)→b1g (Ag+), respectively, in a typically
distorted square planar environment around the metal ion [4, 28, 29].


### 3.4.H1 NMR Spectra

The1H NMR spectrum of
Hdahmp in d6-DMSO shows two singlets at δ 6.07 and
6.18 ppm arising from N(4)H2 and N(6)H2, respectively
(Scheme 1). The proton of the hydroxyl group O(5)H appears as a broad singlet
at δ 9.13 ppm and the N(1)H proton gives a singlet
at δ 7.43 ppm. In the 1H NMR spectrum of
[Pd(bpy)(dahmp)]+, the proton of the hydroxyl group is not observed
while the resonance arising from N(6)H2 is shifted to lower field
[24, 30]. Also, the resonances arising from N(4)H2 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]. The 1H NMR
spectrum of [Ag(bpy)(Hdahm)]+ confirms the neutral bidentate behavior of  Hdahmp through the thione sulfur and the
cyclic N(3) center, as there is a slight shift from the free ligand spectrum
(Table 2). Also, the bpy ligand, in the complexes, shows upfield shifts as
compared with [Pd(bpy)Cl2] or [Ag(bpy)(H2O)2]+.
This is interpreted in terms of strong binding of dahmp- to Pd(II)
or Ag(I) in comparison to binding of chloride and aquo species, respectively
[4–6].Table 2
1H NMR spectral data of Hdahmp and its complexes.
CompoundsN(1)HN(4)H2O(5)HN(6)HHdahmp7.436.079.136.18[Pd(bpy)(dahmp)]+—*6.08—6.46[Ag(bpy)(Hdahmp)]+—*6.169.206.24*Signal
interefered with Ph or bpy protons.


### 3.5. Mass Spectra

The mass spectrum of
[Pd(bpy)(dahmp)]Cl·H2O shows a signal at m/e 474 (Cacld.
472.9) with 2.60% abundance. The spectrum shows signals at 355, 215, and 129
corresponding the loss of (H2O, C2H3N3S),
(Cl, Pd) [4], and (N2, C2H2NO) fragments,
respectively.


### 3.6. Thermal Measurements

The thermal decomposition of
[Pd(bpy)(dahmp)]Cl·H2O and [Ag(bpy)(Hdahmp)]NO3 was
studied by using the thermogravimetry (TG) technique. The thermogram of
[Pd(bpy)(dahmp)]Cl·H2O shows four TG inflections in the ranges 32–148, 150–360, 361–475, and 476–593°C. The first weight loss may arise from the
elimination of crystal lattice water (Calcd. 3.81, Found 3.73%) [4]. The second
step may arise from the release of half Cl2 and C3H5N3 fragments (Calcd. 25.06, Found 24.48%), the third step is due to the removal of
CNS and half bpy (C5H4N) fragments (Calcd. 28.76, Found
29.22%) [27], while the fourth step is attributed to the removal of the other
half of bpy species (Calcd. 16.49, Found 16.55%) followed by the formation of
PdO at 665°C (Calcd. 25.88, Found 26.36%) [4, 27]. The thermogram of
[Ag(bpy)(Hdahmp)]NO3 shows the first-step weight loss of 9.29% between 198 and 252°C, which
corresponds to the release of NO2 species (Calcd. 9.51%). The second
decomposition step occurs between 253 and 352°C, this weight loss is
attributed the loss of C3H6N3 fragment (Calcd.
17.36, Found 17.89%). The third TG inflection between 432–520°C,
may  arise from the elimination of bpy, CS,
three quarter species (Calcd. 49.18,
Found 51.09 %), leaving Ag2O representing (Found 23.00%) [5, 27].


### 3.7. Conductivity Measurements

Figure1 shows the plots of the conductivities
of [Pd(bpy)(dahmp)]Cl and [Ag(bpy)(Hdahmp)]NO3 against
concentrations. It is clear that as the concentration increases, the
conductivity increases, indicating the complete ionization of the complexes
species [31]. Since the conductivity for Cl- and NO3- is 76 and 71 ohm cm2, respectively [31, 32], this suggests that the
complexes ionized completely in aqueous media [33].Figure 1
Conductance-concentration relationship of the complexes.


### 3.8. 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-(2′-pyridyl)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)]NO3 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]). Group (2) tumor-bearing
mice treated with Hdahmp, [Pd(bpy)(dahmp)]Cl, and [Ag(bpy)(Hdahmp)]NO3.
Group (3) is the control tumor-bearing mice. The anticancer activity of Hdahmp,
[Pd(bpy)(dahmp)]Cl, and [Ag(bpy)(Hdahmp)]NO3 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 tumor-bearing mice treated with Hdahmp, [Pd(bpy)(dahmp)]Cl, and
[Ag(bpy)(Hdahmp)]NO3 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).Table 3
Haematological and biochemical parameters of Hdahmp and its complexes.
ParametersCompoundHb(1) (12–16 g/dl)RBCs(2) (4.0–6.0×106 cell/cm3)HCT(3) (35.0–50.0%)WBCs(4) (4000–11000×106 cell/cm3)EAC Count (×106 cell/cm3)MSt/day(5)Hdahmp10.76.340.3880044.813.2[Pd(bpy)(dahmp)]Cl11.46.440.31200031.811.4[Ag(bpy)(Hdahmp)]NO311.06.341900030.411.25-fu10.26.037.776008013.6Control (0.9% NaCl)7.84.7222.224002209.0(1)Hb
= hemoglobin, (2)RBCs = red blood cells count, (3)HCT =
hemato crate value, (4)WBCs =
white blood cells count values in normal mice are in parentheses, (5)the
mean survival time.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)]NO3 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 normal 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 (220×106 cells/cm3),
reduced in using 5-fu to (80×106 cells/cm3) while the
strong reduction to 44.8×106, 31.8×106, and 30.4×106 cells per cm3 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].In order to detect the influence of
the solvent in the cytotoxicity of Hdahmp, [Pd(bpy)(dahmp)]+, and
[Ag(bpy)(Hdahmp)]+. As expected, the water-soluble [Pd(bpy)(dahmp)]+ and [Ag(bpy)(Hdahmp)]+ are less kidney toxic.


### 3.9. 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)]NO3 (Table 3).


### 3.10. 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 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.


## 3.1. Synthesis of Complexes

Table1 lists two new complexes of
4,6-diamino-5-hydroxy-2-mercaptopyrimidine (Hdahmp). The elemental analyses of
the isolated complexes agree with the assigned formula. The conductivities (ΛM) in DMF  at room
temperature showed the electrolytic character of these complexes [4, 16].Table 1
Spectral data of Hdahmp and its complexes.
IR spectral dataComplexesν(OH)νs(NH2)νas(NH2)δ(NH)ν(C=C) ν(C=N)ν(NCS)ν(CN)ν(M–O)ν(M–N)ν(NCS)ν(CS)[Pd(bpy)(dahmp)]Cl—3397 33603165164015561450136252440912551176[Ag(bpy)(Hdahmp)]NO33308339631711650154314791396—412 372*12771207*ν(Ag–S).The complex [Pd(bpy)(dahmp)]Cl was prepared
from [Pd(bpy)Cl2] and Hdahmp in methanol-benzene in presence of
aqueous base, while [Ag(bpy)(Hdahmp)]+ was made from aqueous AgNO3 with bpy and Hdahmp in methanol.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.


## 3.2. Vibration Spectra

The characteristic IR bands observed
and vibration assignments of 4,6-diamino-5-hydroxy-2-mercaptopyrimidine
(Hdahmp) complexes are reported in Table1. The spectrum of Hdahmp supports the
existence of the thione form in the solid phase (Scheme 1). This can be
attributed to the presence of ν(NH) stretch at
2970 cm-1 [17], the absence of ν(SH) near 2600 cm-1, and the
production of the characteristic thioamide bands due to extensive coupling of δ(NH), ν(C=N), ν(NCS), and ν(C=S) at 1652, 1562, 1455, and (1375, 1268,
1177) cm-1, respectively [18–20].Scheme 1
4,6-diamino-5-hydroxy-2-mercaptopyrimidine (Hdahmp).In the spectrum of
[Pd(bpy)(dahmp)]Cl, the stretching vibration ν(OH) at 3305 cm3 in the free ligand
is missing in the complex [21]. The bands at 3390 and 3185 cm-1,
arising from νs(NH2)
and νas(NH2),
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 acts as a mononegative
bidentate ligand, coordinating the metal ion through the deprotonated hydroxyl
and amino N(6)H2 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)]NO3 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
νs(NH2), νas(NH2),
and ν(OH) stretches
more or less in the same position as in the free ligand [18, 22, 24].Scheme 2
Structure of [Pd(bpy)(dahmp)]Cl.Scheme 3
Structure of [Ag(bpy)(Hdahmp)2]NO3.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].The spectrum of [Ag(bpy)(Hdahmp)]NO3 shows new strong
band near 1370 cm-1 assigned to the ionic uncoordinated NO3- [16, 26, 27].


## 3.3. Electronic Spectra

The electronic spectrum of [Pd(bpy)(dahmp)]+ complex shows bands at 475 and 326 nm due to 1A1g→1B1g and 1A1g→1E1g, transitions in a square-planar configuration [4, 23, 27]. Also, the spectrum of [Ag(bpy)(Hdahmp)]+ shows bands at 467, 382, and 277 nm; the latter two may arise from charge
transfer of the type ligand (π)→b1g (Ag+) and ligand (σ)→b1g (Ag+), respectively, in a typically
distorted square planar environment around the metal ion [4, 28, 29].


## 3.4.H1 NMR Spectra

The1H NMR spectrum of
Hdahmp in d6-DMSO shows two singlets at δ 6.07 and
6.18 ppm arising from N(4)H2 and N(6)H2, respectively
(Scheme 1). The proton of the hydroxyl group O(5)H appears as a broad singlet
at δ 9.13 ppm and the N(1)H proton gives a singlet
at δ 7.43 ppm. In the 1H NMR spectrum of
[Pd(bpy)(dahmp)]+, the proton of the hydroxyl group is not observed
while the resonance arising from N(6)H2 is shifted to lower field
[24, 30]. Also, the resonances arising from N(4)H2 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]. The 1H NMR
spectrum of [Ag(bpy)(Hdahm)]+ confirms the neutral bidentate behavior of  Hdahmp through the thione sulfur and the
cyclic N(3) center, as there is a slight shift from the free ligand spectrum
(Table 2). Also, the bpy ligand, in the complexes, shows upfield shifts as
compared with [Pd(bpy)Cl2] or [Ag(bpy)(H2O)2]+.
This is interpreted in terms of strong binding of dahmp- to Pd(II)
or Ag(I) in comparison to binding of chloride and aquo species, respectively
[4–6].Table 2
1H NMR spectral data of Hdahmp and its complexes.
CompoundsN(1)HN(4)H2O(5)HN(6)HHdahmp7.436.079.136.18[Pd(bpy)(dahmp)]+—*6.08—6.46[Ag(bpy)(Hdahmp)]+—*6.169.206.24*Signal
interefered with Ph or bpy protons.


## 3.5. Mass Spectra

The mass spectrum of
[Pd(bpy)(dahmp)]Cl·H2O shows a signal at m/e 474 (Cacld.
472.9) with 2.60% abundance. The spectrum shows signals at 355, 215, and 129
corresponding the loss of (H2O, C2H3N3S),
(Cl, Pd) [4], and (N2, C2H2NO) fragments,
respectively.


## 3.6. Thermal Measurements

The thermal decomposition of
[Pd(bpy)(dahmp)]Cl·H2O and [Ag(bpy)(Hdahmp)]NO3 was
studied by using the thermogravimetry (TG) technique. The thermogram of
[Pd(bpy)(dahmp)]Cl·H2O shows four TG inflections in the ranges 32–148, 150–360, 361–475, and 476–593°C. The first weight loss may arise from the
elimination of crystal lattice water (Calcd. 3.81, Found 3.73%) [4]. The second
step may arise from the release of half Cl2 and C3H5N3 fragments (Calcd. 25.06, Found 24.48%), the third step is due to the removal of
CNS and half bpy (C5H4N) fragments (Calcd. 28.76, Found
29.22%) [27], while the fourth step is attributed to the removal of the other
half of bpy species (Calcd. 16.49, Found 16.55%) followed by the formation of
PdO at 665°C (Calcd. 25.88, Found 26.36%) [4, 27]. The thermogram of
[Ag(bpy)(Hdahmp)]NO3 shows the first-step weight loss of 9.29% between 198 and 252°C, which
corresponds to the release of NO2 species (Calcd. 9.51%). The second
decomposition step occurs between 253 and 352°C, this weight loss is
attributed the loss of C3H6N3 fragment (Calcd.
17.36, Found 17.89%). The third TG inflection between 432–520°C,
may  arise from the elimination of bpy, CS,
three quarter species (Calcd. 49.18,
Found 51.09 %), leaving Ag2O representing (Found 23.00%) [5, 27].


## 3.7. Conductivity Measurements

Figure1 shows the plots of the conductivities
of [Pd(bpy)(dahmp)]Cl and [Ag(bpy)(Hdahmp)]NO3 against
concentrations. It is clear that as the concentration increases, the
conductivity increases, indicating the complete ionization of the complexes
species [31]. Since the conductivity for Cl- and NO3- is 76 and 71 ohm cm2, respectively [31, 32], this suggests that the
complexes ionized completely in aqueous media [33].Figure 1
Conductance-concentration relationship of the complexes.


## 3.8. 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-(2′-pyridyl)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)]NO3 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]). Group (2) tumor-bearing
mice treated with Hdahmp, [Pd(bpy)(dahmp)]Cl, and [Ag(bpy)(Hdahmp)]NO3.
Group (3) is the control tumor-bearing mice. The anticancer activity of Hdahmp,
[Pd(bpy)(dahmp)]Cl, and [Ag(bpy)(Hdahmp)]NO3 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 tumor-bearing mice treated with Hdahmp, [Pd(bpy)(dahmp)]Cl, and
[Ag(bpy)(Hdahmp)]NO3 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).Table 3
Haematological and biochemical parameters of Hdahmp and its complexes.
ParametersCompoundHb(1) (12–16 g/dl)RBCs(2) (4.0–6.0×106 cell/cm3)HCT(3) (35.0–50.0%)WBCs(4) (4000–11000×106 cell/cm3)EAC Count (×106 cell/cm3)MSt/day(5)Hdahmp10.76.340.3880044.813.2[Pd(bpy)(dahmp)]Cl11.46.440.31200031.811.4[Ag(bpy)(Hdahmp)]NO311.06.341900030.411.25-fu10.26.037.776008013.6Control (0.9% NaCl)7.84.7222.224002209.0(1)Hb
= hemoglobin, (2)RBCs = red blood cells count, (3)HCT =
hemato crate value, (4)WBCs =
white blood cells count values in normal mice are in parentheses, (5)the
mean survival time.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)]NO3 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 normal 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 (220×106 cells/cm3),
reduced in using 5-fu to (80×106 cells/cm3) while the
strong reduction to 44.8×106, 31.8×106, and 30.4×106 cells per cm3 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].In order to detect the influence of
the solvent in the cytotoxicity of Hdahmp, [Pd(bpy)(dahmp)]+, and
[Ag(bpy)(Hdahmp)]+. As expected, the water-soluble [Pd(bpy)(dahmp)]+ and [Ag(bpy)(Hdahmp)]+ are less kidney toxic.


## 3.9. 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)]NO3 (Table 3).


## 3.10. 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 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.


## 4. 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)]NO3, shows
remarkable efficacy against Ehrlich ascites tumor cells (EACs) manifested by survival and activity,
as well as reduction in the tumor size.



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*Source: 723634-2008-04-08.xml*