Synthesis, characterization, in vitro cytotoxicity and anti-inflammatory activity of palladium(II) complexes with tertiary phosphines and heterocyclic thiolates: Crystal structure of [PdC28H19N8PS2]

Article abstract of DOI:10.1016/j.jorganchem.2008.01.005

The new four-coordinated mononuclear palladium(II) complexes 1-9 with chelating heterocyclic thiolates and tertiary phosphines with general formula [Pd(L)_nCl(R′R₂P)] (L = Pym2SH (pyrimidine-2-thiolate), Pur6SH (purine-6-thiolate), Py2SH (pyridine-2-thiolate), R₃P = PPh₃, P(o-tolyl)₃, PPh₂Cl), n = 1, 2) have been synthesized by the direct reaction of [PdCl₂(R′R₂P)₂] with polyfunctional heterocyclic thiolates which display a wide variety of coordinations. These compounds were characterized by elemental analysis, FT-IR and multinuclear (¹H, ¹³C and ³¹P) NMR. The X-ray diffraction study of non-ionic compound 5 showed that the thiolate acts as unidentate and that the chelating (-N,S) ligand adopts a slightly distorted square planar geometry around the palladium atom. In vitro the anti-inflammatory inhibition of compounds 1-9 was 10-15% greater than that of the standard drug Declofenac. Compounds 1 and 4 showed mostly a moderate to low cytotoxicity against seven human tumor cell lines whereas compound 3 was somewhat more active.

Full text of DOI:10.1016/j.jorganchem.2008.01.005

Available online at www.sciencedirect.com
Journal of Organometallic Chemistry 693 (2008) 1117–1126
www.elsevier.com/locate/jorganchem
Synthesis, characterization, in vitro cytotoxicity and
anti-inflammatory activity of palladium(II) complexes
with tertiary phosphines and heterocyclic thiolates:
Crystal structure of [PdC28H19N8PS2]
a,
a
b,
c
*
Farkhanda Shaheen , Amin Badshah , Marcel Gielen *, Christine Gieck ,
Mariyam Jamil d, Dick de Vos e
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Vrije Universiteit Brussel, Faculty of Engineering, HNMR Unit, B-1050 Brussels, Belgium
c
`
Universita del Piemonte Orientale, 15100 Alessandria, Italy
d Department of Biology, Faculty of Biological Science, Quaid-I-Azam University, Islamabad 45320, Pakistan
e Pharmachemie BV, PO Box 552, 2003 RN Haarlem, The Netherlands
Received 12 August 2007; received in revised form 6 January 2008; accepted 6 January 2008
Available online 11 January 2008
Abstract
The new four-coordinated mononuclear palladium(II) complexes 1–9 with chelating heterocyclic thiolates and tertiary phosphines
with general formula [Pd(L)nCl(R0R2P)] (L = Pym2SH (pyrimidine-2-thiolate), Pur6SH (purine-6-thiolate), Py2SH (pyridine-2-thio-
late), R3P = PPh3, P(o-tolyl)3, PPh2Cl), n = 1, 2) have been synthesized by the direct reaction of [PdCl2(R0R2P)2] with polyfunctional
heterocyclic thiolates which display a wide variety of coordinations. These compounds were characterized by elemental analysis,
FT-IR and multinuclear (1H, 13C and 31P) NMR. The X-ray diffraction study of non-ionic compound 5 showed that the thiolate acts
as unidentate and that the chelating (–N,S) ligand adopts a slightly distorted square planar geometry around the palladium atom. In
vitro the anti-inflammatory inhibition of compounds 1–9 was 10–15% greater than that of the standard drug Declofenac. Compounds
1 and 4 showed mostly a moderate to low cytotoxicity against seven human tumor cell lines whereas compound 3 was somewhat more
active.
Ó 2008 Elsevier B.V. All rights reserved.
Keywords: Thiolate; Palladium; Anti-inflammatory activity; Cytotoxity
1. Introduction
catalytic applications, the co-ordination chemistry of
heterocyclic thiolates and their corresponding anions as
ligands is very rich, combining S and N ends [5] and mim-
icking the electronic and structural properties of the active
sites of metallo-enzymes [6]. Palladium thiolates have sig-
nificant applications in biological systems due to the palla-
dium–sulfur interaction [7]. In some cases, the metal
complexes of these bases, especially those of Pt(II) and
Pd(II), show a higher anticancer activity than the free
ligands [8]. On the other hand, phosphines and phosphine
metal containing complexes have been actively studied due
to their extensive uses as antitumor and anti-inflammatory
In the last several decades, the chemistry of palla-
dium(II) complexes containing heterocyclic thiolate ligands
has received considerable attention due to their wide-range
of applications such as fungicide [1,2], in the pharmaceuti-
cal industry [3] and as electrical conductors [4]. For
*
Corresponding authors. Tel.: +32 2 629 32 79; fax: +32 2 629 32 81
(M. Gielen).
E-mail addresses: fshaheenpk@yahoo.com (F. Shaheen), mgielen@
vub.ac.be (M. Gielen).
0022-328X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2008.01.005

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F. Shaheen et al. / Journal of Organometallic Chemistry 693 (2008) 1117–1126
drugs [9]. Heterocyclic thiolates ligands contain at least one
deprotonated heterocyclic thioamid group
2.3. Synthesis (see Scheme 1)
(N–C–S)ꢀ and can act not only as monodentate, involving
either the exocyclic sulfur (g1-S) [10] or the endocyclic
nitrogen (g1-N) [11] but also as chelating and bridging
ligands [12]. Purine-6-thiolate (Pur6SH) has attracted some
attention because of its multiple and versatile binding sites
such as N1, N3, N7, N9, S6, while from the N,S-chelating
site, two nitrogen atoms (N1 and N7) can compete to be
involved in co-ordination to the central palladium atom.
The final structure depends on the stability of N1/S6 or
N7/S6 end of the formed square planar structure. With
pyridine-2-thiolate (Py2SH) and pyrimidine-2-thiolate
(Pym2SH), coordination can occur through the S and
N,S-chelating sites. In our previous work, we have studied
the coordination behavior of heterocyclic thiolates with
palladium (II) [13]. As a continuation of our studies on ver-
satile coordination modes, effective biological activities and
in connection with the current interest in the coordination
chemistry of palladium(II) compounds with heterocyclic
thiolates, we selected three interesting ligands: derivatives
of pyrimidine, purine and pyridine: pyrimidine-2-thiolate
(Pym2SH), purine-6-thiolate (Pur6SH) and pyridine-2-
thiolate (Py2SH) of which a series of palladium(II) com-
plexes were synthesized.
2.3.1. Synthesis of the precursor [Pd(PR2R0)Cl2]
The precursor [Pd(PR2R0)2Cl2)], where R = phenyl,
o-tolyl and R0 = phenyl, o-tolyl and chloro, respectively,
were synthesized according to literature methods [14,15,
20] by the reaction of palladium(II) chloride in 25 cm3 of
ethanol with a few drops of 1 N hydrochloric acid and
PR2R0 in 25 cm3 acetone (PdCl2:PR2R0 = 1:2). The reac-
tion mixture was refluxed for 2 h to yield a pale-yellow pre-
cipitate that was washed with an excess of ethanol, dried in
air and recrystallized from THF, to yield light yellow crys-
tals after 48 h.
2.3.2. Synthesis of chloro-(pyrimidine-2-thiolato)(triphen-
ylphosphine)palladium(II) (1)
The ligand Pym2SH (0.039 g, 0.34 mmol) was mixed
with a suspension of PdCl2(PPh3)2 (0.15 g, 0.34 mmol)
(1:1 molar ratio) in 25 cm3 of CH2Cl2. The reaction mix-
ture was refluxed for an hour to yield a clear solution.
The solvent was evaporated under reduced pressure. The
solid product was recrystallized from a mixture of CH2Cl2
and n-hexane (3:1 molar ratio). (Yield: 85%). Anal. Calc.
for C22H18ClN2PPdS calc (obs): C, 51.28 (51.23); H, 3.52
(3.46); N, 5.44 (5.45); S, 6.22 (6.24). FT-IR: 1595,
m(C@N), 340, m(Pd–S), 443, m(Pd N), 289, m(Pd–Cl)
3
2. Experimental
cmꢀ1
;
1H NMR (DMSO-d6): 6.98 (d, JHH = 6.5 Hz,
3
H(4)) ppm, 8.23 (d, JHH = 7.3 Hz, H(6)) ppm, 6.46 (m,
1H(5)) ppm, 7.2-7.6 (m, 15H, Ph-H) ppm. 13C NMR
(DMSO-d6): 187.25(C2), 158.91(C4), 109.2(C5), 162.34
(C6); 31P{1H} NMR (CDCl3): d 31.5 (s, PPh3).
2.1. Reagents
Pyrimidine-2-thione, purine-6-thione, pyridine-2-thione,
triphenylphosphine, tris-o-tolylphosphine, chlorodiphenyl-
phosphine and palladium(II) chloride were of analytical
grade and were purchased from Aldrich (USA). All the
organic solvents were dried before use according to stan-
dard procedures [14].
2.3.3. Synthesis of chloro-(pyrimidine-2-thiolato)-
(tris-o-tolylphosphino)palladium(II) (2)
(Yield: 85%). Anal. Calc. for C25H24ClN2PPdS calc
(obs): C, 53.87 (53.83); H, 4.34 (4.32); N, 5.02 (5.01); S,
5.75 (5.72). FT-IR: 1598, m(C@N), 329, m(Pd–S), 440,
2.2. Instrumentation
m(Pd N), 301, m(Pd–S) cmꢀ1
;
1H NMR (DMSO-d6):
3
8.29 (d, JHH = 6.9 Hz, 2H(4 or 6)) ppm, 8.34 (d,
3JHH = 7.3 Hz, 2H(4 or 6)) ppm, 6.95 (m, 1H(5)) ppm,
7.2–7.5 (m, 12H, tolyl-H) ppm; 1.9–2.3 (m, 9H, –CH3)
ppm; 13C NMR (DMSO-d6): 177.35 (C2), 156.23 (C4),
106.99 (C5), 161.24 (C6), 31P{1H} NMR (CDCl3): d
32.85, (s, P(o-Tol)3).
Elemental analyses were carried out on a Fisons EA1108
CHNS-O microanalyser. FT-IR spectra were recorded on a
Bio-Rad Excalibur apparatus FT-IR, model FTS 3000 MX
using KBr discs from 4000 to 400 cmꢀ1 and CsI discs from
500 to 200 cmꢀ1 with a Perkin–Elmer FT-IR Nexus
spectrometer.
The 1H and 13C NMR spectra were recorded in
DMSO-d6 solutions on a Bruker 300 MHz spectrometer
operating at 300 and 75.5 MHz, respectively. The 31P
NMR spectra were recorded in CDCl3 solutions on a
Bruker AMX-400 MHz spectrometer operating at 160
MHz.
Single crystal X-ray diffraction data of 5 was collected at
293 K using an Oxford Diffraction Xcalibur 2 diffractome-
ter with Mo Ka radiation. Data reduction and analytical
absorption correction were performed using the CRYSALIS
RED software suite [15]. The structure was solved using
SIR2004 [16] and refined using SHELXL97 [17].
2.3.4. Synthesis of chloro-(pyrimidine-2-thiolato)(chloro-
diphenylphosphine)palladium(II) (3)
Pyrimidine-2-thione (0.039 g, 0.34 mmol) was mixed
with a suspension of PdCl2(ClPh2P)2 (0.15 g, 0.34 mmol)
in 20 cm3 of CH2Cl2 (1:1 molar ratio). The reaction mix-
ture was refluxed for half an hour to obtain a clear solu-
tion. The obtained product was recrystallized from
CH2Cl2/n-hexane by a slow evaporation at room tempera-
ture. (Yield: 83%). Anal. Calc. for C16H13Cl2N2PPdS calc
(obs): C, 40.57 (40.63); H, 2.77 (2.73); N, 5.91 (5.91); S,
6.77(6.76). FT-IR: 1592, m(C@N), 339, m(Pd–S), 423,
m(Pd N), 296, m(Pd–Cl) cmꢀ1
;
1H NMR (DMSO-d6):

F. Shaheen et al. / Journal of Organometallic Chemistry 693 (2008) 1117–1126
1119
Cl
N
Pd
N
S
PR2R'
CH2Cl2
(1-3)
1.hr reflux
N
N
PR2R'
Cl
S
SH
Pd
N
N
N
SH
N
H
N
(4, 6, 7)
N
+
CH2Cl2
[Pd(PR2 R')Cl2]
+
PR2R'
N
S
N
1.hr reflux
+
N
Pd
N
N
H
N
S
N
N
N
H
(5)
0.45CH2Cl2
SH
N
N
H
CH2Cl2
1. hr reflux
Cl
N
S
Pd
PR2R'
(8, 9)
R= phenyl, o-tolyl, R' = phenyl,o-tolyl for(1, 2, 4, 5, 6, 8, 9), Chloro for (3, 7)
Scheme 1. Synthetic scheme for compounds 1–9.
3
8.27 (d, JHH = 8.2 Hz 2H(4 or 6)) ppm, 8.25 (d,
and n-hexane (3:1). A slow evaporation at room tempera-
ture for several days resulted in small orange crystals
(Yield: 85%). Anal. Calc. for C28H21N8PPdS2 [0.45
CH2Cl2] calcd (obs): C, 43.11 (43.09); H, 2.71 (2.71); N,
14.39 (14.40); S, 8.22 (8.25). FT-IR: 1598–1640, m(C@N),
3JHH = 8.29 Hz, 2H(4 or 6)) ppm, 6.82 (m, 1H(5)) ppm,
7.4–7.8 (m, 10H, Ph-H) ppm; 13C NMR (DMSO-d6):
179.3 (C2), 151.2 (C4), 106.5 (C5), 168.54 (C6), 31P{1H}
NMR (CDCl3): d 30.6 (s, ClPh2P).
1
415, m(Pd–S), 397, m(Pd N) cmꢀ1; H NMR (DMSO-
3
3
2.3.5. Synthesis of chloro-(purine-6-thiolato)(trip-
henylphosphino)palladium(1I) (4)
d6): 6.88 (d, JHH = 7.0 Hz, 1H(8)) ppm, 7.23 (d, JHH =
7.0 Hz, 1H, NH(9)) ppm, 9.39 (s, 2H(2)) ppm, 7.4–7.6
(m, 15H, Ph-H) ppm; 13C NMR (DMSO-d6): 191.5 (C2),
180.8 (C4), 183.9 (C5), 188.5(C6), 185.9(C8), 31P{1H}
NMR (CDCl3): d 29.7 (s, PPh3).
(Yield: 87%). Anal. Calcd. for C23H18ClN4PPdS calcd
(obs): C, 49.74 (49.79); H, 3.26 (3.22); N, 10.08 (10.04);
S, 5.75 (5.77). FT-IR: 1590–1640, m(C@N), 305, m(Pd–Cl),
405, m(Pd–S), 399, m(Pd N) cmꢀ1; H NMR (DMSO-
1
3
d6): 6.92 (d, JHH = 7.9 Hz, 1H(8)) ppm, 7.85 (d,
2.3.7. Synthesis of chloro-(purine-6-thiolato)-
(tris-o-tolylphosphino)palladium(II) (6)
3JHH = 8.0 Hz, 1H(9)) ppm, 8.93 (s, 1H(2)) ppm, 7.4–7.6
(m, 15H, Ph-H) ppm; 13C NMR (DMSO-d6): 192.3 (C2),
182.5 (C4), 181.9 (C5), 187.5 (C6), 172.5 (C8), 31P{1H}
NMR (CDCl3): d 31.7 (s, PPh3).
(Yield: 86%). Anal. Calc. for C26H24ClN4PPdS2; calcd
(obs): C, 46.61 (46.64); H, 3.84 (3.85); N, 8.90 (8.90); S,
10.85 (10.89). FT-IR: 1590–1637, m(C@N), 373, m(Pd–S),
1
408, m(Pd N), 298, m(Pd–Cl) cmꢀ1; H NMR (DMSO-
3
2.3.6. Synthesis of bis-(purine-6-thiolato)(triphenyl-
phosphine)palladium(1I)dichloride (5)
A suitable crystal for X-ray diffraction was obtained by
dissolving 0.5 g of the sample in a small quantity of CH2Cl2
d6): 6.87 (d, JHH = 7.2 Hz, 1H(8)) ppm, 7.73 (d,
3JHH = 7.2 Hz, 1H, NH(9)) ppm, 8.45 (s, 1H(2)) ppm,
7.4–7.6 (m, 12H, tolyl-H) ppm, 1.95–2.44 (m, 9H, –CH3);
13C NMR (DMSO-d6): 189.5 (C2), 176.8 (C4), 179.9

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F. Shaheen et al. / Journal of Organometallic Chemistry 693 (2008) 1117–1126
(C5), 181.7 (C6), 178.1 (C8), 31P{1H} NMR (CDCl3): d
30.7 (s, (o-tolyl)3P).
refined by using the SHELX-97 program package [17].
The final refinements were carried out on Fo . Atomic
2
scattering factors for spherical neutral free atoms were
taken from standard sources, and anomalous dispersion
corrections were applied [18]. Difference Fourier analysis,
which was performed after locating the atoms of the
[Pd(Pur6S)2(PPh3)] molecule, showed a strong disorder
for the solvent molecules. Calculations performed at an
intermediate stage, in which the relative positional occu-
pancies of the Cl positions were refined, revealed a full
occupancy for Cl1 and Cl2 and partial occupancies for
the remaining Cl atoms. Disorder of the solvent (0.45%)
molecules was modelled using the SQUEEZE routine of the
2.3.8. Synthesis of chloro-(purine-6-thiolato)(chloro-
diphenylphosphino)palladium(II) (7)
(Yield: 80%). Anal. Calc. for C17H13Cl2N4PPdS calcd
(obs): C, 39.75 (39.75); H, 2.55 (2.50); N, 10.91 (10.91);
S, 6.24 (6.25). FT-IR: 1595–1628, m(C@N), 295, m(Pd–Cl),
1
320, m(Pd–S), 473, m(Pd N) cmꢀ1; H NMR (DMSO-
3
d6): 6.72 [d, JHH = 7.0 Hz, 1H(8)] ppm, 7.96 (d,
3JHH = 7.0 Hz, 1H, NH(9)) ppm, 8.23 (s, 1H(2)) ppm,
7.1–7.4 (m, 10H, Ph-H) ppm. 13C NMR (DMSO-d6):
192.3 (C2), 166.3 (C4), 160.2 (C5), 182.2 (C6), 168.24
(C8), 31P{1H} NMR (CDCl3): d 27.0 (s, PClPh2).
PLATON software suite [19]. Final
R indices were
R1 = 0.0482, wR2 = 0.1315 for [I > 2r(I)] and R1 =
0.0590, wR2 = 0.1431 for all data.
2.3.9. Synthesis of chloro-(pyridine-2-thiolato)(trip-
henylphosphino)palladium(1I) (8)
(Yield: 87%). Anal. Calc. for C23H19ClNPPdS calcd
(obs): C, 53.71 (53.75); H, 3.72 (3.70); N, 2.72 (2.71); S,
6.23 (6.23). FT-IR: 1639, m(C@N), 295, m(Pd–Cl), 315,
2.5. Antifungal assay
The agar tube dilution method was used for antifungal
activity assay of plant extracts with some modifications
[21]. The compounds were solubilized in dimethylsulfox-
ide and a dilution was performed in Sabouraud dextrose
agar (SDA) (Merck) medium, and acidic Media (pH 5.5–
5.6) containing a relatively high concentration of glucose
(40%) were prepared by mixing (SDA) and 6.5 gm/mL
distilled water. The contents were dissolved and dispensed
as 4 mL volumes into screw capped tubes and autoclaved
at 121 °C for 20 min. Tubes were allowed to cool to
50 °C and non-solidified SDA is loaded with 67 lL of
compound pipetted out from the stock solution. This
gave the final concentration of 200 lg/mL of the com-
pound in media. Tubes were then allowed to solidify in
slanting position at room temperature. Tubes were pre-
pared in triplicate for each fungus species. Other media
supplemented with dimethylsulfoxide and reference anti-
fungal drugs were used as negative and positive control
respectively. Each tube was inoculated with 4 mm diame-
ter piece of inoculums, removed from a 7 days old cul-
ture of fungus. The tubes were incubated at 28 °C for 7
days. Cultures were examined twice weekly during the
incubation. The growth in the media was determined by
measuring the linear growth (mm) and the growth inhibi-
tion was calculated with reference to a negative control.
A growth control of the test strains and a susceptibility
standard test using Terbinafine 200 lg/mL as the refer-
ence system were performed by applying the same
technique.
m(Pd–S), 428, m(Pd N) cmꢀ1
;
1H NMR (DMSO-d6):
3
3
6.72 (d, JHH = 7.0 Hz, 1H(8)) ppm, 7.96 (d, JHH
=
7.0 Hz, 1H, NH(9)) ppm, 7.1–7.4 (m, 15H, Ph-H) ppm.
13C NMR (DMSO-d6): 192.3 (C2), 168.24 (C3), 106.3
(C4), 108.5 (C5), 182.2 (C6), 31P{1H} NMR (CDCl3): d
27.0 (s, PClPh2).
2.3.10. Synthesis of chloro-(pyridine-2-thiolato)-
(tris(o-tolyl)phosphino)-palladium(1I) (9)
(Yield: 75%). Anal. Calc. for C26H25ClNPPdS calcd
(obs): C, 56.12 (56.15); H, 4.53 (4.52); N, 2.52 (2.55);
S, 5.76 (5.76). FT-IR: 1628, m(C@N), 319, m(Pd–Cl),
1
354, m(Pd–S), 473, m(Pd N) cmꢀ1, H NMR (DMSO-
3
d6): 6.78 (d, JHH = 6.9 Hz, 1H(8)) ppm, 7.86 (d,
3JHH = 6.85 Hz, 1H, NH(9)) ppm, 7.1–7.4 (m, 10H,
Ph-H) ppm, 2.94 (m, 9H, –CH3) ppm. 13C NMR
(DMSO-d6): 191.3 (C2), 168.42 (C3), 105.7 (C4), 109.3
(C5), 182.9 (C6), 31P{1H} NMR (CDCl3), d 29.0 (s,
PClPh2).
2.4. Crystal structure determination
The structure of non-ionic complex 5 was determined
by single-crystal X-ray diffraction. An orange crystal
was selected from the reaction product and mounted with
epoxy glue on a glass capillary. The measurement was
carried out at 293 K using an Oxford Diffraction Xcalibur
2 diffractometer with Mo Ka radiation. The exposure
time was 10 s per frame, the crystal-detector distance,
60 mm. Data reduction and analytical absorption correc-
tion were performed using the CRYSALIS RED software suite
2.6. Anti-inflammatory activity
˚
[15]. The final lattice parameters (a = 31.6198(10) A; c =
Adult male Sprague–Dawley rats (180–230 g) were used
in all the experiments. The animals were housed under
optimum conditions of light and temperature (12 h light
and dark cycle and 22–38 °C), with food and water
provided ad libitum. The animals were divided into 11
groups (one control, one standard and nine test).
˚
18.0729(6) A) were calculated from all reflections observed
in the actual data collection. Systematic absences
indicated the rhombohedral centrosymmetric space group
R-3. The structure was solved by direct methods using
SIR2004 [16], which revealed the atomic positions, and

F. Shaheen et al. / Journal of Organometallic Chemistry 693 (2008) 1117–1126
1
1121
2.6.1. Preparation of test samples for bioassay
3.1. H, 13C and 31P NMR data
Test samples were suspended in 0.75% sodium carboxy-
methylcellulose (CMC) and were given orally to the test
animals. The animals of the control group received the
same experimental handling except that the drug treatment
was replaced with appropriate volume of the dosing vehi-
cle. Nine groups of three animals each received one com-
pound i.e. 1–9 at 25 mg/kg. The standard drug
Declofenac potassium 10 mg/5 mL was used as reference
drug.
The proton NMR spectra for complexes 1–9 shows the
resonance line pattern observed for the heterocyclicthiols
that has been assigned on the basis of literature data [25]
(see Scheme 2).
1
The H NMR spectra were consistent with structures
with two ligands, a heterocyclic thiolate (Pym2Sꢀ, Pur6Sꢀ
and Py2Sꢀ) and a tertiary phosphine (PPh3, (o-tolyl)3P,
ClPh2P). In compound 1, a doublet at 8.23 ppm (H(6),
3JHH = 7.3 Hz) and
a doublet at 6.98 ppm (H(4),
2.6.2. Carrageenan-induced hind paw oedema test
3JHH = 6.5 Hz) are observed at room temperature. The
The rat paw oedema was induced by subcutaneous injec-
tion of 0.1% carrageenan (50 lL) into the sub plantar
region of each animal left hind paw [22]. The measurement
of rat paw volumes was carried out by the plythesmograph-
ic method [23]. It was done by recording the rat paw vol-
ume before the carrageenan injection at 0 h and then at
1 h intervals for 4 h. Nine groups of three animals were
given 1–9 compounds (25 mg/kg), each group given one
compound. The standard drug Declofenac potassium
10 mg/5 mL was used as a positive control. The negative
control group received 0.75% CMC (carboxymethylcellu-
lose) sodium. One hour later, a volume of 50 lL of carra-
geenan at 0.1% in distilled water was injected into the
rat’s peritoneal cavity. The percentage oedema inhibition
was calculated for each animal group in comparison with
its control treated group.
H(6) doublet shows a downfield chemical shift as compared
to the free ligand at 6.46 (d, H(3), JHH = 7.0 Hz) which
3
indicates that Pym2Sꢀ coordinates to central metal
through N(1). The ring-proton H(5) of pyrimidine-2-thio-
late is observed at 6.46 ppm. The considerable chemical
shift differences of the PymSꢀ ligand in these complexes
with respect to the uncoordinated thiolate suggest a pymSꢀ
coordination to palladium. Almost the same chemical
shifts are observed in the complexes 2 and 3. The NH pro-
ton in all complexes shows downfield shifts whereas the
other protons show an upfield shift. The resonance of the
thiolate protons are downfield to TMS; these downfield
shifts are in accordance with a dominant r-delocalization
pattern of spin density and are consistent with the ground
state of the Pd(II) which has two unpaired electrons in
r-symmetry orbitals (dx2ꢀy2, dz2). However, the net spin
density in the dp orbitals could be polarized by the
2
2.7. Cytotoxicity of compounds 1, 3 and 4 against seven
human tumor cell lines
unpaired electrons in the dx2ꢀy and dz2 orbitals via spin–
orbital coupling and this mechanism can distribute spin
density into the thiolate rings.
The cytotoxicity of compounds 1, 3 and 4 was tested
in vitro by applying seven well characterized human tumor
cell lines (MCF7 and EVSA-T (breast cancers), WIDR
(colon cancer), IGROV (ovarian cancer), M19 MEL (mel-
anoma), A498 (renal cancer), H226 (non-small cell lung
cancer) and the microculture sulforhodamine B (SRB) test
of which the protocol was described elsewhere [13,24].
Cell lines WIDR, M19 MEL, A498, IGROV and H226
belong to the currently used anticancer screening panel of
the National Cancer Institute, USA.
In compound 5, the two Pur6Sꢀ ligands are in equiva-
lent in the solid state structure. However, as described ear-
lier, the room temperature 1H NMR spectrum of 5 showed
only one set of Pur6Sꢀ resonances. The apparent equiva-
lence of the two Pur6Sꢀ ligands in the spectrum at room
temperature can be attributed to some fluxionality in solu-
tion [26]. One set of Pur6Sꢀ acts as unidentate (S-bonded)
and the other one as bidentate (N,S-bonded) and consider-
able downfield chemical shifts at 6.99 ppm (d, 1H(8),
3
3JHH = 8 Hz), 7.23 ppm (d, 1H(9), JHH = 8 Hz), 9.39
The MCF7 cell line is estrogen receptor (ER)+/proges-
terone receptor (PgR)+ and the cell line EVSA-T is
(ER)ꢀ/(PgR)ꢀ.
ppm (s, 1H(2)) are observed in the purine ring protons
[10,27] after complexation to palladium(II). These results
are also confirmed by the single crystal X-ray diffraction
result. The same trend of chemical shifts in the single set
3. Results and discussion
The four-coordinate thiolate complexes of Pd(II) were
isolated from the reaction mixture of [Pd(PR2R0)2Cl2)
and the corresponding heterocyclic thiolates (1:1 and 1:2
molar ratio) in dichloromethane. The isolated complexes
are sketched in Scheme 1. During the synthesis the acidic
proton of the ligand is abstracted by the precursor provid-
ing the anion which is trapped by the metal substrate
(PdCl2) moiety to form the new complex with concomitant
release of HCl.
SH
N
SH
N
SH
N
6
5
6
5
1N
7
9
N
3
3
N
N3
4
4
H
Pym2SH
Py2SH
Pur6SH
(I)
(II)
(III)
Scheme 2. Numbering scheme for the heterocyclic thiols.

1122
F. Shaheen et al. / Journal of Organometallic Chemistry 693 (2008) 1117–1126
of purine ring protons is observed in the compounds 4, 6
and 7. The absence of –SH signal is indicating a deprotona-
tion of Pur6SH and its coordination to palladium(II) as the
anionic Pur6Sꢀ moiety.
The 31P{1H} NMR spectra of the compounds run in
CH2Cl2, relative to the external reference 85% H3PO4, with
downfield shifts defined as positive. In complexes 1–9, a
single 31P resonance is observed at dP, 32.85–27.00 ppm
with a coordination shift (dcomplex–dligand).
The 1H NMR spectra of compounds 8 and 9 show
a downfield chemical shift for the ring-hydrogen H(6)
3
at 8.85 ppm (d, H(6), JHH = 7.4 Hz), indicative of an
3.2. Crystal structure determination
N,S– mode of co-ordination of the ligand Py2Sꢀ with
palladium; the H3 and H4 protons appear as a complex
In the structure of the non-ionic complex 5 (see Fig. 1),
the Pd atom is four-coordinate and exhibits a slightly dis-
torted square-planar geometry. One of the 6-mercaptopur-
ine molecules acts as monodentate and the other one as
bidentate, the fourth coordination site of the metal cation
being occupied by the triphenylphosphine. It is to be noted
that, although the NMR spectra show only one set of sig-
nals for the 6-mercaptopurine ligand, corresponding to a
quick fluctuation between monodentate and bidentate coor-
dination for the molecule in solution, no disorder of the
ligands was found. Both 6-mercaptopurine molecules are
approximately planar, with an angle of 78.4° between the
least square planes. The two chlorine atoms (Cl1 and Cl2)
are coordinated via hydrogen bonds to the 6-mercaptopur-
ine ligands, Cl2 is also connected to the PPh3 unit of the
adjacent molecule. The resulting crystal structure contains
a regular arrangement of one-dimensional channels running
along the c-axis. The walls of these channels are composed
of interlocking 1D helical chains consisting of the
Pd(Pur6S)2PPh3 system. The two chlorines of the 0.45
CH2Cl2 molecules are located in large pores (diameter:
pattern around 7.89 ppm. The downfield shift of the proton
3
H5 appearing at 6.43 ppm (t, 1H, pyS-H(5), JHH
=
6.8 Hz) suggests that the ligands are attached to the central
metal via N(1) and S in these complexes.
The 13C NMR spectra of complexes exhibit low-field
shifts. The N,S-chelating Pym2Sꢀ and Pur6Sꢀ moiety in
the metal complexes shows low-field shifts for C(2) and
C(6) carbons of the pyrimidine and purine rings [28], while
the S-bonded unidentate Pym2Sꢀ exhibits a downfield shift
for C(6). This is attributed to the delocalization of electron
density on the pyrimidine ring [10], increasing its aromatic-
ity relative to the free 2HPymS or N,S-chelating Pym2Sꢀ
ligand. The 13C NMR data of these compounds show the
presence of N,S-chelating Pym2Sꢀ, Pur6Sꢀ, PySꢀ anions,
1
in agreement with the solid state structure results. The H
and 13C NMR spectral data provide evidence for extensive
delocalization of the charge density along the R–P–Pd–
N,SR axis and the absence of any back bonding property
of S atoms makes it further necessary to disperse the excess
charge density received by the Pd metal centre onto the
ligands. Coordination by P and N atoms lowers the energy
of the p-orbitals of phenyl and thiolate moieties, thus
enhancing their Lewis acidity.
˚
6.8 A), thus stabilizing the porous framework and no disor-
der of ligand is found in complex 5. Although the electron
density of the Cl atoms of the dichloromethane unit was
Fig. 1. ORTEP diagram of [PdC28H21N8PS2] with atomic numbering scheme.

F. Shaheen et al. / Journal of Organometallic Chemistry 693 (2008) 1117–1126
1123
clearly visible during the refinement of the crystal structure,
the central carbon atoms could not be detected. However,
the distances between the chlorine atoms were in accordance
with values expected for the 1,3 Clꢁ ꢁ ꢁCl distance in the
dichloromethane molecule. The results indicate a rotational
disorder of the central CH2 unit around the Clꢁ ꢁ ꢁCl axis, the
C/H atoms of the solvent molecules were therefore mod-
elled using the SQUEEZE routine of the PLATON software suite
[19]. The total amount of solvent was determined by refining
the occupancy of the Cl atoms using fixed isotropic thermal
parameters. The results suggest 8.1 dichloromethane mole-
cules per unit cell (see Table 1).
indicative of a S,N-coordination [33,34] and of the forma-
tion of a four-membered chelate ring around the central
metal through
a donor atom set comprising the
imine-nitrogen and thiolate sulfur, respectively, while a
new absorption band due to m(Pd–Cl) appears at
295–315 cmꢀ1. The observed shifts in the ligand vibrations
upon coordination with respect to the free ligand are sim-
ilar in all complexes. Therefore the predicted S,N-coordi-
nation of the ligand is supported by the single-crystal
X-ray diffraction data. The coordination of PR3 to the cen-
tral metal atom exhibited expected results and the effect of
PR3 is also observed in some dithiocarbamate complexes
[35]. The appearance of a (P–C) vibration, which occurs
at 1474 cmꢀ1 in PPh3, is shifted toward 1460 cmꢀ1 upon
coordination. Pd–P vibration and low energy band of
Pd–Cl should appear below the detection limit due to high
trans influence of sulfur donor.
3.3. FT-IR results
Deprotonation of the ligand is accompanied by a sub-
stantial modification of their FT-IR spectra with m(S–H)
(2600–2500 cmꢀ1) absent from the spectra of the metal
derivatives 1–9 and a significant perturbation of the
‘‘thioamide” bands [29]. In all cases, shifts to shorter wave-
lengths are observed for m(C@N) and m(C–S) relative to the
free thione, the 650 cmꢀ1 band m(C@S) is absent. Addi-
tional weak or medium intensity bands for m(Pd–S)
(ꢂ385 cmꢀ1) as well as m(Pd–N) (ꢂ399–443 cmꢀ1) are
observed [30,31]. Such vibrational activities and replace-
ment of the acidic hydrogen(s) by palladium [32] are clearly
3.4. Antifungal activity
The results of antifungal assays showed (Table 2) that
the compounds 1–9 possess antifungal activity against the
following five fungi: Fusarium moniliformes, Fusarium sao-
lani, Mucor sp., Aspergillus niger, Aspergillus fumigatus.
The rank-order of the % of inhibition effect for the fungi
is Fusarium moniliformes > Fusarium saolani > Aspergillus
fumigatus ꢂ Aspergillus niger > Mucor sp. and histogram,
which clearly indicated that palladium(II) complexes are
significantly active against these fungi (detailed data in
Table 2).
Table 1
Crystal data and structure refinement for complex 5
Compounds 1, 2, 4, 5 and 7 at concentration of 200 lg/
mL inhibited the growth of F. moniliformes by 78–67%.
Compounds 3, 6 and 8 showed a moderate growth inhibi-
tion of 65–52%. Comparatively compound 9 showed a
lower (50%) inhibition than the other ones. The com-
pounds 8 and 9 showed the highest activity against the
A. fumigatus i.e. 65% and 62%, respectively. The growth
inhibition of all compounds against F. moniliformes is
Empirical formula
[C28H21N8PS2Pd] 0.45 CH2Cl2
Formula weight
Temperature (K)
778.16
293(2)
0.7107
Rhombohedral
R-3
˚
Wavelength (A)
Crystal system
Space group
Unit cell dimensions
˚
a (A)
31.6198(10)
18.0729(6)
15648.6(9)
18
1.478
0.876
˚
c (A)
Volume (A )
3
˚
Z
Dcalc (Mg/m3)
Absorption coefficient (mmꢀ1
F(000)
)
Table 2
Anti-fungal activity of palladium(II) complexes
6953
Crystal size (mm)
Theta range for data collection (°)
Index ranges
0.19 ꢃ 0.11 ꢃ 0.06
4.10–20.81
ꢀ31 6 h 6 31, ꢀ31 6 k6 31,
ꢀ18 6 l 6 18
29242
3609 [0.0396]
99.2%
Analytical
0.903 and 0.791
Compounds
Growth effect %Inhibition (Fungus)
F. F. Mucor A.
moniliformes solani sp.
A.
niger fumigatus
1
2
3
4
5
6
7
8
9
78.28
67.11
65.26
68.89
75.66
52.68
67.56
52.89
49.98
58
30
22
45
51
50
56
45
54
25
0
10
0
33.33 30.33
40.44 35.38
39.44 45.64
58.69 53.08
52.32 50.25
51.11 58.00
47.25 48.55
54.76 65.34
32.62 61.87
Reflections collected
Independent reflections [Rint
Completeness to theta = 20.81°
Absorption correction
Maximum and minimum
transmission
]
0
25
10
18
28
22
100
Refinement method
Full-matrix least-squares on F2
3609/0/397
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2r(I)]
R indices (all data)
1.077
Linear length in ꢀve 45
90
39
R1 = 0.0477, wR2 = 0.1282
R1 = 0.0586, wR2 = 0.1393
control
Largest difference in peak and hole 0.935 and ꢀ0.610
Terbinafine, used as control inhibited all fungi at 200 lg/mL.
ꢀve control group with 0.75% CMC (carboxymethylcellulose) sodium.
ꢀ3
˚
(e A
)

1124
F. Shaheen et al. / Journal of Organometallic Chemistry 693 (2008) 1117–1126
Table 3
3.5. Anti-inflammatory activity
Anti-inflammatory activity of palladium(II) compounds of heterocyclic
thiolates on carrageenan induced rat paw oedema
During the initial pharmacological evaluation of com-
pounds 1–9, it was suggested that the in vitro drugs’ potency
relative to other NSAIDs (nonsterodial anti-inflammatory
drugs) produce their therapeutic activities through the inhi-
bition of cyclooxygenase (COX), the enzyme that makes
prostagladins (PGs). COX has at least two isoenzymes i.e.
COX-1 which constitutes the PGs that protect the stomach
and the kidney and COX-2 which induces the inflammation
stimuli, such as cytokines and produces PGs that contribute
to the pain and swelling of inflammation. In the present
work, the anti-inflammatory action results of the synthe-
sized compounds 1–9 show that the injection of carrageenan
into the rat paw induces the liberation of bradykinin, which
later induces the biosynthesis of prostaglandin and other
autacoids (i.e. hormones that produce their physiological
effect by transportation of blood) that are responsible for
the formation of the inflammatory exudates [36]. From
the carrageenan-induced oedema tests involving both
COX-1 and COX-2 activities [37], it is not as clear whether
Compounds % oedema inhibition at time (h)
First hour
Second
hour
Third hour
Fourth
hour
Standard
2.32 4.52
3.49 4.32 68.70 3.05
74.14 2.72
drug
1
2
3
4
5
6
7
8
9
26.74 2.34 35.11 4.06 45.19 20.78 70.41 6.35
18.17 2.12 20.17 7.72 24.42 3.43
29.84 3.51 36.78 6.13 69.57 2.84
24.32 1.02
93.87 2.35
83.46 3.53
91.46 2.25
92.87 2.34
85.46 2.25
87.54 2.14
88.68 1.08
22.51 1.51
32.7 6.13 55.33 3.12
23.84 3.23 30.91 5.32 68.66 2.72
18.93 4.21 29.32 3.49 73.12 2.64
25.73 4.23 35.38 5.32 69.47 2.52
19.54 1.78 36.98 2.35 76.46 2.36
18.45 3.78 28.45 3.65 72.65 2.99
Values are mean SEM; n = 3 in each group.
important, while, against Mucor sp., the activity of all com-
pounds is not significant. Compounds 1–7 showed moder-
ate activities against A. niger and A. fumigatus (see Fig. 2).
100
80
60
40
20
0
Antifungal Activity of Palladium Compounds
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
Compounds
3rd hour
2nd hour
Compounds
4th hour
Ist hour
Fusarium
moniliformes
Aspergillus
Niger
Fig. 3. Inhibition of prostaglandin biosynthesis by palladium compounds:
at the 4th hour, compounds 3, 7 and 9 showed maximum (89–93%)
oedema inhibition, compounds 1, 5, 6 and 8 show a moderate one,
compound 4 a low one and 2, a very low inhibition, while the result of the
standard drug is 74.1 2.7% at the 4th hour. Three or more estimates
were averaged, standard error of the mean SEM; n = 3 in each group is
shown in Table 3.
Aspergillus
Fumigatus
Fusarium solani
Mucor sp
Fig. 2. Comparison of Growth% inhibition of palladium compounds
against five fungi.
Table 4
Inhibition doses ID50 in vitro cytotoxicities of compounds 1,3 and 4 and of six reference compounds doxorubicin (DOX), cisplatin (CPT), 5-fluorouracil
(5-FU), methotrexate (MTX), etoposide (ETO) and taxol (TAX)
Compound
A498
EVSA-T
H226
IGROV
M19MEL
MCF-7
WiDr
1
2335
5428
12603
90
2253
143
37
1314
<3
1097
327
1561
8
422
475
5
5166
2756
11727
199
3269
340
2287
3934
<3
192
1396
7802
60
169
297
7
3382
3445
6630
16
558
442
23
2338
1705
3212
10
699
750
18
2890
7033
2686
11
967
225
<3
3
4
DOX
CPT
5FU
MTX
ETO
TAX
317
<3
580
<3
505
<3
2594
<3
150
<3

F. Shaheen et al. / Journal of Organometallic Chemistry 693 (2008) 1117–1126
1125
the complexes of palladium(II) with heterocyclic thiolates/
Acknowledgement
tertiary phosphine act by inhibiting COX-1 alone or by
inhibiting both COX-1 and COX-2. Therefore, it is sug-
gested that the mechanism of action of the compounds
may be related to a prostaglandin synthesis inhibition, as
described for the anti-inflammatory mechanism of declofe-
nac potassium in the inhibition of the inflammatory process
induced by carrageenan [38] (see Fig. 3).
The authors thank Professor Davide Viterbo for his
crystallographic support and higher Education Commo-
tion Islamabad, Pakistan for financial support through
Project No. 20-623/R&D/06/258.
Appendix A. Supplementary material
3.6. Cytotoxicity of compounds 1, 3 and 4 against seven
human tumor cell lines
CCDC 643263 contains the supplementary crystallo-
graphic data for compound 5. These data can be obtained
free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif. Supple-
mentary data associated with this article can be found,
in the online version, at doi:10.1016/j.jorganchem.
2008.01.005.
Table 4 shows the experimental results.
Compounds 1 and 4 showed mostly a moderate to low
cytotoxicity against the seven well characterized human
tumor cell lines whereas 3 was somewhat more active.
4. Conclusion
References
The synthesized complexes 1–9 were characterized by
FT-IR and multinuclear NMR spectroscopy, and single-
crystal X-ray diffraction. Each technique suggests that
structural changes occur in heterocyclic thiols upon depro-
tonation and coordination to palladium takes place
through S,N– and S atoms, the other coordination posi-
tions being occupied by chloride ions and phosphorus
atoms to achieve a square planar geometry around palla-
dium. The coordination position has also been confirmed
by the novel crystal structure of complex 5, [bis(purine-6-
thiolato)(triphenylphosphine)]palladium(II). The major
purpose of this work was to study the structural relation-
ship of compounds with biological systems, the study of
anti-inflammatory properties to determine the relationship
between N,S– and NCSS- compounds and the anti-inflam-
mation actions (i.e. carrageenan-induced paw oedema for-
mation). The tests were optimized by using NSAIDs
(nonsteriodal anti-inflammatory drugs) Declofenac (stan-
dard drug), response was produced which blocked the
oedema by 74% and the compounds shows 10–15% greater
inhibition than the standard drug. However, the
rank-order potency of these compounds 1–9 (accordingly
heterocyclic thiolates) as inhibitors of COX in vitro is
purine-6-thiolate > pyrimidine-2-thiolate > pyridine-2-thio-
late. Further studies will be needed to delineate the differen-
tial effects of heterocyclic thiolate palladium(II) complexes
in systems that involve both COX-1 and COX-2. At pres-
ent, inhibition of COX is clearly the most likely mechanism
underlying the actions of (N,S)-thiolates. This is based not
only on its ability to inhibit COX in vitro and block pros-
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COX-independent activities. Most notably, palladium–
thiolate complexes may not interact with a variety of key
receptors, channels, or enzymes known to be involved in
pain transmission mechanisms. Compounds 1 and 4
showed mostly a moderate to low cytotoxicity against the
seven human tumor cell lines whereas compound 3 was
somewhat more active.
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