Palladium(II) complexes of quinolinylaminophosphonates: Synthesis, structural characterization, antitumor and antimicrobial activity

Article abstract of DOI:10.1016/j.jinorgbio.2011.03.011

Three types of palladium(II) halide complexes of quinolinylaminophosphonates have been synthesized and studied. Diethyl and dibutyl [α-anilino-(quinolin-2-ylmethyl)]phosphonates (L1, L2) act as N,N-chelate ligands through the quinoline and aniline nitroge

Full text of DOI:10.1016/j.jinorgbio.2011.03.011

Journal of Inorganic Biochemistry 105 (2011) 867–879
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Palladium(II) complexes of quinolinylaminophosphonates: Synthesis, structural
characterization, antitumor and antimicrobial activity
Marina Juribašić a, Krešimir Molčanov a, Biserka Kojić-Prodić a, Lisa Bellotto b, Marijeta Kralj c,
Franca Zani d, Ljerka Tušek-Božić a,
⁎
a
Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička 54, HR-10002 Zagreb, Croatia
CNR, Istituto di Scienze e Tecnologie Molecolari, Sezione di Padova, Corso Stati Uniti 4, 35100 Padova, Italy
Division of Molecular Medicine, Ruđer Bošković Institute, Bijenička 54, HR-10002 Zagreb, Croatia
Dipartimento Farmaceutico, Universitá degli Studi di Parma, 43124 Parma, Italy
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Three types of palladium(II) halide complexes of quinolinylaminophosphonates have been synthesized and
studied. Diethyl and dibutyl [α-anilino-(quinolin-2-ylmethyl)]phosphonates (L1, L2) act as N,N-chelate
ligands through the quinoline and aniline nitrogens giving complexes cis-[Pd(L1/L2)X2] (X Cl, Br) (1–4). Their
3-substituted analogues [α-anilino-(quinolin-3-ylmethyl)]phosphonates (L3, L4) form dihalidopalladium
complexes trans-[Pd(L3/L4)2X2] (5–8), with trans N-bonded ligand molecules only through the quinoline
nitrogen. Dialkyl [α-(quinolin-3-ylamino)-N-benzyl]phosphonates (L5, L6) give tetrahalidodipalladium
complexes [Pd2(L5/L6)3X4] (9–12), containing one bridging and two terminal ligand molecules. The bridging
molecule is bonded to the both palladium atoms, one through the quinoline and the other through the
aminoquinoline nitrogen, whereas terminal ligand molecules are coordinated each only to one palladium via
the quinoline nitrogen. Each palladium ion is also bonded to two halide ions in a trans square-planar fashion.
The new complexes were identified and characterized by elemental analyses and by IR, UV–visible, 1H, 13C and
31P nuclear magnetic resonance and ESI-mass spectroscopic studies. The crystal structures of complexes 1–4
and 6 were determined by X-ray structure analysis. The antitumor activity of complexes in vitro was
investigated on several human tumor cell lines and the highest activity with cell growth inhibitory effects in
the low micromolar range was observed for dipalladium complexes 11 and 12 derived from dibutyl ester L6.
The antimicrobial properties in vitro of ligands and their complexes were studied using a wide spectrum of
bacterial and fungal strains. No specific activity was noted. Only ligands L3 and L4 and tetrahalidodipalladium
complexes 9 and 11 show poor activities against some Gram positive bacteria.
Received 30 September 2010
Received in revised form 16 March 2011
Accepted 16 March 2011
Available online 24 March 2011
Keywords:
Palladium(II) complex
Quinolinylaminophosphonate complex
Spectroscopy
Crystal structure
Antitumor activity
Antimicrobial activity
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
[10]. It is generally considered that the antitumor properties of
complexes strongly depend on the nature of the ligands and the metal
Considerable research efforts have been made to develop novel
metal-based antitumor complexes with the aim of improving effective-
ness and reducing the severe side effects of the current clinical platinum
chemotherapeutic agents such as cisplatin and its analogues [1,2]. The
special attention has been directed to platinum complexes with
different structural features than those of already used drugs [3–7]
and complexes of the other platinum-group metals like ruthenium,
rhodium, palladium and iridium [8–11]. Additionally, few complexes of
other transition metals, like titanium, gallium and gold have also been
found to be active and have entered clinical trials [10–12]. Recent
studies ondesignof lesstoxic and more selective metal-based antitumor
drugs are focused on complexes with biologically interesting ligands
coordination pattern. With this regard, very interesting ligand candi-
dates are derivatives of aminophosphonic acids owing to their broad-
spectrum of biological activities and potential applications in the
pharmacological and agrochemical fields [13–15]. With high affinity
towards bone and other calcified tissues, these compounds may be
utilized for drug design against bone diseases [16]. Our specific interest
in the area of phosphonate chemistry has been directed towards dialkyl
and monoalkyl esters of aromatic aminophosphonic acids and their
complexes. Different types of palladium(II) and platinum(II) complexes
with alkyl quinolinylmethylphosphonates and anilinobenzylphospho-
nates, such as molecular dihalide adducts with trans and cis configu-
rations, mononuclear and binuclear metallocyclic and ion-pair halide
salt complexes, have been synthesized, structurally characterized and
screened on their antitumor and antiviral activity [17–24]. Some of
these complexes demonstrated notable antitumor activity in vitro
against some animal and human tumor cell lines, but showed no or only
⁎
Corresponding author. Tel.: +385 1 4571217; fax: +385 1 4680245.
E-mail address: tusek@irb.hr (L. Tušek-Božić).
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.03.011

868
M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
marginal antiviral effects based on inhibition of virus-induced cyto-
pathogenicity in various cell cultures.
sealed MW process vials (5 mL) with magnetic stirring using CEM
Discover® LabmateTH/ExplorerPLS® monomode reactor. Melting
points were determined on a hot stage microscope and are
uncorrected. Infrared spectra were recorded on an ABB Bomem
MB102 spectrometer using KBr (4000–400 cm−1) and polyethylene
(400–200 cm−1) pellets. The one- and two-dimensional 1H, 13C and
31P NMR spectra were recorded with Bruker XWIN-600 Fourier-
transform spectrometer operating at 600.13 (and 300.13) MHz,
150.90 MHz and 242.92 MHz for 1H, 13C and 31P nucleus, respectively.
Spectra were recorded in CDCl3 at 302 K (unless stated otherwise, at
253 and 323 K) containing SiMe4 as an internal standard for 1H and
13C spectra or 85% H3PO4 as an external standard for 31P spectra. The
The coordination chemistry of palladium(II) is very similar to that
of platinum(II), but the higher liability in ligand exchange at Pd-
centre (105-fold vs Pt) may cause rapid hydrolysis processes leading
to dissociation of complex and formation of very reactive species
unable to reach their biological targets. These problems could be
overcome by using the bulky heterocyclic and chelating ligands. A
number of palladium complexes with aromatic N- and N,N-containing
ligands such as derivatives of pyridine, quinoline, pyrazole and 1,10-
phenanthroline [9,25–27], as well as those with N,S-chelating ligands
such as derivatives of thiosemicarbazones and dithiocarbamates, have
shown very promising antitumor characteristics [28–30]. Some
palladium(II) complexes exhibit significant antimicrobial activity
[30]. In addition, quinoline moiety has pharmaceutical significance
as important constituent in the structure of antimicrobial agents
[31,32] and, recently, several quinoline phosphonates have been
reported to be efficacious against bacteria and fungi [33].
Continuing our research program on preparation of metal complexes
with dialkyl esters of various aromatic aminophosphonic acids with
potential biological activity, in the present work we describe the
synthesis, spectroscopic and biological properties of palladium(II)
complexes with recently reported dialkyl quinolinylaminophosphonate
(Fig. 1) [34,35]. Crystal structures of some palladium complexes were
determined. Antimicrobial properties of free ligands and their complexes
were studied in vitro on a wide spectrum of bacterial and fungal strains.
The antiproliferative activity in vitro of complexes was investigated on a
panel of human cell lines (T-lymphoblast leukemia cells and breast, colon
and lung carcinoma). The results obtained were compared with those
previously reported for palladium(II) halide complexes of quinolinyl-
methylphosphonates [19,24], and discussed with respect to their metal-
binding behaviour and structure–activity relationships.
following techniques were used: 1H, 13C, APT, 31P, 1H-1H COSY, 1H-13
C
HMQC, 1H-13C HMBC. All two-dimensional experiments were per-
formed by standard pulse sequences, using Bruker XWINNMR
software Version 3.5. The ESI mass spectrometric measurements
were performed on a LCQDeca ion trap instrument (Thermo, San Jose,
CA USA) operating in the positive ion mode. Compounds were
dissolved in 1:1000 MeCN (9–12). The UV–vis spectra were obtained
in chloroform solution with a HP Agilent 8453 diode array
spectrophotometer in the 250–600 nm range. Conductance measure-
ments were carried out at room temperature using a CD 7A Tacussel
conductance bridge for 10−3 M solutions in DMF and methanol.
Elemental analyses (C, H and N) were performed on a Perkin-Elmer
Analyser PE 2400 Series 2 in the Ruđer Bošković Institute.
2.2. Synthesis of ligands
Diethyl and dibutyl [α-anilino-(quinolin-2-ylmethyl)]phospho-
nates (L1, L2), diethyl [α-anilino-(quinolin-3-ylmethyl)]phosphonate
(L3) and diethyl and dibutyl [α-(quinolin-3-ylamino)-N-benzyl]
phosphonates (L5, L6), prepared according to previously described
methods [34,35], were purified by column chromatography per-
formed on silica gel (130–270 mesh, 60 Å, Aldrich). New dibutyl [α-
anilino-(quinolin-3-ylmethyl)]phosphonate (L4) was prepared by the
same method under microwave irradiation.
2. Experimental
2.1. General methods
Equimolar amounts of quinoline-3-carboxaldehyde (1.65 mmol) and
aniline (1.65 mmol) were mixed and dibutyl phosphite in c.a. 10% molar
excess (1.82 mmol) was added. Reaction mixture was quickly stirred,
irradiated at max. 50 W for 10 min on 115 °C and chromatographed on
All reagents and solvents were high purity products purchased
from commercial sources and used without additional purification.
The microwave (MW) irradiation experiments were carried out in
Fig. 1. Structures and numbering scheme of palladium(II) complexes with diethyl and dibutyl [α-anilino-(quinolin-2-ylmethyl)]phosphonate (1–4), diethyl and dibutyl [α-anilino-
(quinolin-3-ylmethyl)]phosphonate (5–8) and diethyl and dibutyl [α-(quinolin-3-ylamino)-N-benzyl]phosphonate (9–12).

M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
869
silica gel column using the mixture ethyl acetate:hexane=2:1. Red-
brown microcrystalline product L4 was obtained by evaporation of the
solvent in vacuo. Yield: 86%. M.p. 52–54 °C. Anal. Calc. for C24H31N2O3P: C,
67.59; H, 7.33; N 6.57%. Found: C, 67.42; H, 7.05; N, 6.66%.
methanol (2 mL). After vigorous stirring for 3 h, the fine powder
precipitate that formed was separated, washed with ice-cold
methanol:water=1:1 and dried in vacuo over P2O5.
trans-[Pd(L3)2Cl2] (5). Pale yellow solid. Yield: 95%. M.p. 139–142 °C.
Anal. Calc. for C40H46N4O6P2Cl2Pd: C, 52.33; H, 5.05; N, 6.10%. Found:
C, 51.56; H, 5.03; N, 6.34%. IR (ν, cm−1): 3302 m (ν(N–H)); 1602 s,
1504 s (ν(C=N), ν(C=C), δ(NH)); 1240 s (ν(P=O)); 1047 vs, 1020 vs
(ν(PO–C)); 971 m-s (ν(P–O–C), νaliph(C–C)); 350 w-m (ν(Pd–Cl)). UV–
vis (λmax, nm (log(ε, M−1cm−1))): 302 (4.16), 313 (4.15), 324 (4.11).
trans-[Pd(L3)2Br2] (6). Yellow solid. Yield: 90%. M.p. 158–160 °C.
Anal. Calc. for C40H46N4O6P2Br2Pd: C, 47.71; H, 4.60; N, 5.56%. Found: C,
47.08; H, 4.64; N, 5.76%. IR (ν, cm−1, sh-shoulder): 3293 m (ν(N–H));
1602 s, 1503 s (ν(C=N), ν(C=C), δ(NH)); 1273 sh, 1265 sh,
1231 s (ν(P=O)); 1048 s, 1015 vs (ν(PO–C)); 966 m-s (ν(P–O–C),
2.3. Synthesis of complexes
2.3.1. General procedure for complexes cis-[PdLX2] (1–4)
A solution of Na2[PdX4] (X=Cl, Br 0.22 mmol) in methanol (2 mL)
was added dropwise via micro syringe to a stirred solution of the ligand
(L=L1 or L2; 0.20 mmol) in methanol (2 mL). Microcrystalline red
product precipitated immediately, and after stirring for 5 min was
filtered off, washed with ice-cold methanol and dried in vacuo over P2O5.
By prolonged stirring the initial red reaction mixture was slowly turned
to ochre colour giving after a few days a mixture of the complexes, yellow
aniline palladium dihalide adduct trans-[Pd(PhNH2)2X2] (X=Cl, Br)
[36] and grey N,O-chelate bis(quinolin-2-ylcarboxylato)palladium(II)
complex [37].
ν
aliph(C–C)); 299 w (ν(Pd–Br)). UV–vis (λmax, nm (log(ε, M−1cm−1))):
301 (4.29), 313 (4.27), 325 (4.21). Crystals suitable for X-ray diffraction
were grown by slow evaporation from an ethyl acetate (EtOAc) solution
at room temperature.
cis-[Pd(L1)Cl2] (1). Red solid. Yield: 77%. M.p. 180–183 °C. Anal. Calc.
for C20H23N2O3PCl2Pd: C, 43.86; H, 4.23; N, 5.11%. Found: C, 42.89; H, 4.22;
N, 5.26%. IR (ν, cm−1, vs—very strong, s—strong, m—medium, w—weak):
3206 w (ν(N–H)); 1594 m, 1512 m-s, 1493 m-s (ν(C=N), ν(C=C),
δ(NH)); 1256 s, 1231 m (ν(P=O)); 1048 s, 1037 s, 1026 vs, 1010 vs
(ν(PO–C)); 983 m-s, 973 m-s, 949 m, 930 w (ν(P–O–C), νaliph(C–C));
340 m, 325 m (ν(Pd–Cl)). UV–vis (λmax, nm (log(ε, M−1cm−1))): 316
(4.01), 328 (3.99), 405 (2.88). Crystals suitable for X-ray diffraction were
grown by slow evaporation from the acetone solution at room
temperature.
cis-[Pd(L1)Br2] (2). Dark red solid. Yield: 82%. M.p. 166–168 °C.
Anal. Calc. for C20H23N2O3PBr2Pd: C, 37.73; H, 3.17; N, 4.40%. Found: C,
37.33; H, 3.72; N, 4.46%. IR (ν, cm−1): 3211 w (ν(N–H)); 1594 m,
1511 m, 1493 m (ν(C=N), ν(C=C), δ(NH)); 1258 s, 1231 m
(ν(P=O)); 1048 s, 1034 s, 1020 vs, 1009 s (ν(PO–C)); 973 m, 947
w-m, 928 w (ν(P–O–C), νaliph(C–C)); 288 w, 261 w (ν(Pd–Br)). UV–
vis (λmax, nm (log(ε, M−1cm−1))): 315 (4.08), 328 (4.07), 398 (3.08).
Crystals suitable for X-ray diffraction were grown by slow evaporation
from the acetone solution at room temperature. Single crystals of the
acetonitrile monosolvate (2·MeCN) were obtained by slow evapora-
tion from an acetonitrile solution in the refrigerator kept at +4 °C.
cis-[Pd(L2)Cl2] (3). Orange-red solid. Yield: 71%. M.p. 166–168 °C.
Anal. Calc. for C24H31N2O3PCl2Pd: C, 47.74; H, 5.17; N, 4.64%. Found:
C, 47.49; H, 5.11; N, 4.83%. IR (ν, cm− 1): 3206 w (ν(N–H)); 1596 m,
1513 m-s, 1493 m (ν(C=N), ν(C=C), δ(NH)); 1258 vs, 1232 m
(ν(P=O)); 1061 m-s, 1026 vs, 1004 vs (ν(PO–C)); 977 s, 955 m, 928
trans-[Pd(L4)2Cl2] (7). Pale yellow solid. Yield: 95%. M.p. 193–194 °C.
Anal. Calc. for C48H62N4O6P2Cl2Pd: C, 55.96; H, 6.07; N, 5.44%. Found: C,
55.90; H, 6.21; N, 5.53%. IR (ν, cm−1): 3304 m (ν(N–H)); 1602 s, 1501 s
(ν(C=N), ν(C=C), δ(NH)); 1268 sh, 1235 s (ν(P=O)); 1061 m-s,
1021 vs (ν(PO–C)); 990 vs, 958 sh (ν(P–O–C), νaliph(C–C)); 355 m
(ν(Pd–Cl)). UV–vis (λmax, nm (log(ε, M−1cm−1))): 302 (4.16), 313
(4.15), 324 (4.11).
trans-[Pd(L4)2Br2] (8). Yellow solid. Yield: 86%. M.p. 196–197 °C.
Anal. Calc. for C48H62N4O6P2Br2Pd: C, 51.51; H, 5.58; N, 5.01%. Found:
C, 51.34; H, 5.63; N, 5.06%. IR (ν, cm−1): 3304 m (ν(N–H)); 1602 s,
1499 s (ν(C=N), ν(C=C), δ(NH)); 1267 w, 1235 s (ν(P=O));
1061 m, 1021 s (ν(PO–C)); 989 s, 958 sh (ν(P–O–C), νaliph(C–C));
299 w (ν(Pd–Br)). UV–vis (λmax, nm (log(ε, M−1cm−1))): 301 (4.26),
313 (4.23), 325 (4.18).
2.3.3. General procedure for complexes [Pd2L3X4] (9–12)
A solution of Na2[PdX4] (X=Cl, Br; 0.15 mmol) in methanol (2 mL)
was added dropwise to a stirred solution of the ligand (L=L5 or L6;
0.20 mmol) in methanol (2 mL). Fine powder product precipitated
immediately, and after stirring for 3 h was filtered off, washed with
ice-cold methanol and dried in vacuo over P2O5.
[Pd2(L5)3Cl4] (9). Pale yellow solid. Yield: 86%. M.p. N200 °C
decomp. Anal. Calc. for C60H69N6O9P3Cl4Pd2: C, 50.33; H, 4.96; N,
5.87%. Found: C, 50.09; H, 5.11; N, 5.89%. IR (ν, cm−1, br—broad):
3267 m-s br (ν(N–H)); 1619 s, 1609 s, 1541 m, 1493 m (ν(C=N),
ν(C=C), δ(NH)); 1239 s, 1223 vs (ν(P=O)); 1048 vs, 1018 vs (ν(PO–
C)); 975 s (ν(P–O–C), νaliph(C–C)); 348 w-m (ν(Pd–Cl)). UV–vis
(λmax, nm (log(ε, M−1cm−1))): 260 (4.92), 308 (3.92), 368 (4.24).
ESI MS: m/z 1489 (8%), [M+Na]+; 1118 (19%), [(M+Na)-L]+; 941
(95%), [(M+Na)-PdLCl2]+; 571 (11%), [(M+Na)-PdL2Cl2]+; 535
w (ν(P–O–C), νaliph(C–C)); 340 m, 325 m (ν(Pd–Cl)). UV–vis (λmax
,
nm (log(ε, M−1cm− 1))): 316 (4.00), 328 (3.97), 405 (2.88). Crystals
suitable for X-ray diffraction were grown by slow evaporation from
an acetone or chloroform solution at room temperature.
cis-[Pd(L2)Br2] (4). Red solid. Yield: 67%. M.p. 155–158 °C. Anal.
Calc. for C24H31N2O3PBr2Pd: C, 41.61; H, 4.51; N, 4.04%. Found: C,
41.45; H, 4.32; N, 3.93%. IR (ν, cm−1): 3216 w (ν(N–H)); 1596 m,
1513 m, 1493 m (ν(C=N), ν(C=C), δ(NH)); 1258 vs, 1232 w-m
(ν(P=O)); 1062 m-s, 1022 vs, 1003 s (ν(PO–C)); 975 s, 957 w-m, 926
(23%), [(M+Na)-PdL2Cl2-HCl]+; 393 (9%) [(M+Na)-Pd2L2Cl4]+
=
[L+Na]+
.
[Pd2(L5)3Br4] (10). Ochre solid. Yield: 82%. M.p. N200 °C decomp.
Anal. Calc. for C60H69N6O9P3Br4Pd2: C, 44.16; H, 4.35; N, 5.15%. Found:
C, 44.38; H, 4.41; N, 5.72%. IR (ν, cm−1): 3266 m br (ν(N–H)); 1617 s,
1609 s, 1543 w-m, 1493 m (ν(C=N), ν(C=C), δ(NH)); 1239 s, 1223 vs
(ν(P=O)); 1048 vs, 1022 vs (ν(PO–C)); 975 m-s (ν(P–O–C), νaliph(C–
C)); 292 w (ν(Pd–Br)). UV–vis (λmax, nm (log(ε, M−1cm−1))): 257
(5.05), 311 (4.13), 368 (4.26). ESI MS: m/z 1667 (b1%), [M+Na]+;
1296 (9%), [(M+Na)-L]+; 1030 (100%), [(M+Na)-PdLBr2]+; 660
(23%), [(M+Na)-PdL2Br2]+; 579 (32%), [(M+Na)-PdL2Br2-HBr]+; 393
(19%) [(M+Na)-Pd2L2Br4]+ = [L+Na]+.
[Pd2(L6)3Cl4] (11). Pale yellow solid. Yield: 77%. M.p. N200 °C
decomp. Anal. Calc. for C72H93N6O9P3Cl4Pd2: C, 52.92; H, 5.74; N,
5.14%. Found: C, 54.20; H, 6.12; N, 5.36%. IR (ν, cm−1): 3273 m br (ν(N–
H)); 1618 s, 1609 s, 1542 m, 1493 m (ν(C=N), ν(C=C), δ(NH)); 1240 s,
1222 s (ν(P=O)); 1063 s, 1022 vs (ν(PO–C)); 994 vs (ν(P–O–C), νaliph
(C–C)); 348 w-m (ν(Pd–Cl)). UV–vis (λmax, nm (log(ε, M−1cm−1))):
w (ν(P–O–C), νaliph(C–C)); 284 w, 261 w (ν(Pd–Br)). UV–vis (λmax
,
nm (log(ε, M−1cm−1))): 315 (4.07), 328 (4.05), 404 (3.08). Crystals
suitable for X-ray diffraction were grown by slow evaporation of a
solvent from the acetone or dichloromethane solution at room
temperature or an acetonitrile solution in the refrigerator at +4 °C.
The structure determination has been of limited accuracy, and the
ORTEP view of the asymmetric unit is given in the Supplementary
data.
2.3.2. General procedure for complexes trans-[PdL2X2] (5–8)
A solution of Na2[PdX4] (X=Cl, Br; 0.11 mmol) in methanol (2 mL)
and water (0.5 mL) was added dropwise avoiding any insolubilization
to a stirred solution of the ligand (L=L3 or L4; 0.20 mmol) in

870
M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
260 (4.91), 308 (3.97), 369 (4.23). ESI MS: m/z 1657 (7%), [M+Na]+;
1231 (15%), [(M+Na)-L]+; 1053 (100%), [(M+Na)-PdLCl2]+; 627
(10%), [(M+Na)-PdL2Cl2]+; 591 (15%), [(M+Na)-PdL2Cl2-HCl]+; 449
(27%) [(M+Na)-Pd2L2Cl4]+ = [L+Na]+.
Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK;
fax: +44 1223 336033; or deposit@ccdc.cam.ac.uk.
2.5. Antitumor evaluation assays
[Pd2(L6)3Br4] (12). Yellow solid. Yield: 82%. M.p. N200 °C decomp.
Anal. Calc. for C72H93N6O9P3Br4Pd2: C, 48.04; H, 5.29; N, 4.67%. Found:
C, 48.33; H, 5.57; N, 4.83%. IR (ν, cm−1): 3268 m br (ν(N–H)); 1618 s,
1608 s, 1543 w-m, 1493 m (ν(C=N), ν(C=C), δ(NH)); 1240 s, 1222 vs
(ν(P=O)); 1062 m, 1023 vs (ν(PO–C)); 993 s (ν(P–O–C), νaliph(C–C));
289 w (ν(Pd–Br)). UV–vis (λmax, nm (log(ε, M−1cm−1))): 257 (5.05),
311 (4.11), 369 (4.25). ESI MS: m/z 1835 (5%), [M+Na]+; 1408 (5%),
[(M+Na)-L]+; 1142 (100%), [(M+Na)-PdLBr2]+; 716 (17%), [(M+
Na)-PdL2Br2]+; 635 (33%), [(M+Na)-PdL2Br2-HBr]+; 449 (15%) [(M+
Na)-Pd2L2Br4]+ = [L+Na]+.
The experiments were carried out on five human cell lines, which
are derived from four cancer types. The following cell lines were used:
SW 620 and HCT 116 (colon carcinoma), H 460 (lung carcinoma),
MCF-7 (breast carcinoma) and MOLT-4 (T-lymphoblast leukemia).
The cells were cultured as monolayers and maintained in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicilin and
100 mg/mL streptomycin in a humidified atmosphere with 5% CO2
at 37 °C.
The growth inhibition activity was assessed according to the
slightly modified procedure performed at the National Cancer
Institute, Developmental Therapeutic Program [43]. The panel cell
lines were inoculated onto a series of standard 96-well microtiter
plates on day 0, at 1×104 to 3×104 cells/mL, depending on the
doubling times of specific cell line. Test agents were then added in
five- or ten-fold dilutions (10−8 to 10−4 M) and incubated for further
72 h. Working dilutions were freshly prepared on the day of testing.
The solvent (DMSO) was also tested for eventual inhibitory activity by
adjusting its concentration to be the same as in working concentra-
tions. After 72 h of incubation the cell growth rate was evaluated by
performing the MTT assay [44], which detects dehydrogenase activity
in viable cells. The absorbance (OD, optical density) was measured on
a microplate reader at 570 nm. The absorbance is directly propor-
tional to the number of living, metabolically active cells [44]. The
percentage of growth (PG) of the cell lines was calculated according to
one or the other of the following two expressions.
2.4. X-ray structure analysis
Single crystals of 1–4 and 6 were measured on an Oxford Diffraction
Xcalibur Nova R diffractometer (microfocus Cu tube, CCD detector).
Program package CrysAlis PRO [38] was used for data reduction. The
structures were solved using SHELXS97 and refined with SHELXL97
[39]. The models were refined using the full-matrix least squares
refinement; all non-hydrogen atoms were refined anisotropically.
Hydrogen atoms (except NH) were treated as constrained entities,
using the command AFIX in SHELXL97 [39]. Molecular geometry
calculations were performed by PLATON [40] and the molecular
graphics were prepared using ORTEP-3 [41] and CCDC-Mercury [42].
Crystallographic and refinement data for the structures reported in
this paper are shown in Table 1.
Crystallographic data for the structural analysis have been
deposited with the Cambridge Crystallographic Data Centre, CCDC
No. 794995-795000. The data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
If (mean ODtest −mean ODtzero)≥0, then PG=100×(mean ODtest
mean ODtzero)/(mean ODctrl−meanODtzero). If (mean ODtest −mean
ODtzero)b0, then: PG=100×(mean ODtest−mean ODtzero)/ODtzero
−
,
Table 1
Crystallographic data, collection and structure refinement data of complexes 1–4 and 6.
Complex
1
2
2·MeCN
3
4
6·2EtOAc
Empirical formula
Formula weight
C
20H23Cl2N2O3PPd
C20H23Br2N2O3PPd
636.59
C22H26Br2N3O3PPd
677.63
C24H31Cl2N2O3PPd
603.78
C24H31Br2N2O3PPd
692.68
C48H62Br2N4O10P2Pd
1183.16
547.67
Crystal dimensions/mm
Colour
Solvent used for crystallization
Space group
a/Å
b/Å
c/Å
α/°
0.23×0.13×0.06
Light red
Acetone
P 212121
15.4282(1)
16.0030(1)
18.4946(1)
90
0.20×0.15×0.10
Red
Acetone
P 21/c
10.6650(1)
15.8511(2)
14.6300(2)
90
110.284(1)
90
0.15×0.12×0.10
Red
MeCN
0.15×0.09×0.07
Orange
Acetone, CHCl3
P 21/c
10.1639(1)
12.6778(1)
20.3398(2)
90
95.803(1)
90
0.15×0.12×0.10
Red
Acetone, CH2Cl2, MeCN
P 21/c
27.0301(9)
18.0654(5)
22.8664(7)
90
105.803(3)
90
0.16×0.14×0.11
Yellow
EtOAc
À
P 21/n
P 1
9.44584(6)
15.47859(10)
18.10265(12)
90
104.7280(7)
90
9.3411(4)
9.7802(4)
14.9894(5)
97.122(3)
94.837(3)
93.570(9)
1
1350.33(9)
1.455
5.528
Multi-scans
0.405, 0.544
2.98–75.00
11≥h≥−11
12≥k≥−10
18≥l≥−15
11,764
β/°
γ/°
90
90
8
Z
4
4
4
16
V/Å3
4566.27(5)
1.593
9.571
Multi-scans
0.462, 1.000
3.65–76.21
18≥h≥−18
20≥k≥−18
22≥l≥−23
15,786
2319.86(5)
1.823
11.337
Multi-scans
0.124, 0.322
4.26–76.55
13≥h≥−13
19≥k≥−19
18≥l≥−18
22,070
2559.80(3)
1.758
10.330
Multi-scans
0.489, 1.000
3.81–76.15
11≥h≥−11
18≥k≥−19
21≥l≥−22
13,211
2607.47(4)
1.538
8.437
Multi-scans
0.352, 0.550
4.11–77.42
12≥h≥−12
15≥k≥−15
23≥l≥−25
26,496
10743.9(6)
1.713
9.846
Multi-scans
0.295, 1.000
2.44–76.57
30≥h≥−33
21≥k≥−21
24≥l≥−28
49,100
Dcalc/g cm−3
μ/mm−1
Absorption correction
Tmin, Tmax
Θ range/°
Range of h, k, l
Reflections collected
Independent reflections
Observed reflections (I≥2σ)
Rint
7748
8163
0.0197
4338
4812
0.0327
4836
5301
0.0191
4844
5402
0.0375
14,282
21,488
0.0546
4864
5483
0.0239
R (F)
0.0296
0.0804
0.0286
0.0819
0.0259
0.0713
0.0341
0.0973
0.0925
0.3150
0.0429
0.1405
Rw (F2)
Goodness of fit
No. of parameters
F(000)
1.052
613
2208.0
0.520, –0.740
1.051
294
1248.0
0.978, –0.913
1.053
331
1336.0
0.431, –0.564
1.082
317
1232.0
0.787, –0.603
1.046
1413
5504.0
1.385, –1.471
1.220
382
604.0
1.082, –0.840
Δρmax, Δρmin (eÅ−3
)

M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
871
where the mean ODtzero is the average of optical density measurements
before exposure of cells to the test compound, the mean ODtest is the
average of optical density measurements after the desired period of time
and the mean ODctrl is the average of optical density measurements after
the desired period of time with no exposure of cells to the test
compound.
Each test was performed in quadruplicate in three individual
experiments. The results are expressed as IC50, which is the concentra-
tion necessary for 50% of inhibition. The IC50 values for each compound
are calculated from concentration–response curves using linear regres-
sion analysis by fitting the test concentrations that give PG values above
and below the reference value (i.e. 50%). If however, for a given cell line
all of the tested concentrations produce PGs exceeding the respective
reference level of effect (e.g. PG value of 50), then the highest tested
concentration is assigned as the default value, which is preceded by a “N”
sign. Each result is a mean value from three separate experiments.
act as bidentate ligands through the quinoline and aniline nitrogens
giving complexes cis-[Pd(L1/L2)X2] (1–4) with the five-membered N,N-
chelate ring and which was supported by their X-ray analysis. These
complexes were isolated immediately after their formation, i.e. after
about 5 min of stirring the reactants, while prolonged stirring caused
their slow decomposition and the formation of a mixture of complexes,
which were detected as an aniline palladium dihalide adduct trans-[Pd
(PhNH2)2X2] (X=Cl, Br) [36] and N,O-chelate bis(quinolin-2-ylcarbox-
ylato)palladium(II) complex [37]¸ confirmed by the spectroscopic and
X-ray crystallographic studies. It is interesting to note that in the
microwave-assisted synthesis of L1and L2by one-pot three-component
reaction of quinoline-2-carboxaldehyde, aniline and dialkyl phosphite,
as the main product was isolated quinoline-2-carboxanilide and not
dialkyl phosphonates as in the conventional thermal reaction [34].
Favoured formation of amide, and not of required imine, by reaction of
quinoline-2-carboxaldehyde and aniline may arise from proximity of
the acidic aldehyde hydrogen with respect to the quinoline nitrogen.
The 3-substituted quinoline analogues, dialkyl [α-anilino-(quinolin-
3-ylmethyl)]phosphonates (L3, L4) form dihalidopalladium complexes
with 1:2 metal-to-ligand ratio, trans-[Pd(L3/L4)2X2] (5–8), containing
trans N-bonded ligand molecules only through the quinoline nitrogen. It
is unlikely that such bulky two phosphonate ligand molecules could be
placed in cis position with respect to the metal ion as well as it may be
presumed that the steric effects inhibit the aniline nitrogen coordination
and 1:1 metal-to-ligand chelate formation as was obtained in the
complexes 1–4. The coordination behaviour of these ligands was
supported by the X-ray analysis of complex 6. The situation is more
complicated in the case of dialkyl [α-(quinolin-3-ylamino)-N-benzyl]
phosphonates (L5, L6), which give tetrahalidodipalladium complexes,
[Pd2(L5/L6)3X4] (9–12) containing three ligand molecules. A bridging
molecule is bonded to the both palladium atoms, one through the
quinoline nitrogen and the other through the aminoquinoline nitrogen.
The two other terminal ligand molecules are coordinated each only to
one palladium via the quinoline nitrogen and each palladium ion is also
bonded to two halide ions in a trans square-planar fashion. It could be
concluded that the geometric and steric requirements of the ligand
containing two or more donor atoms are very important in determining
the number of donor atoms that will coordinate and the final geometry
of the metallic species.
The novel complexes prepared are microcrystalline or powder-like
diamagnetic compounds, coloured yellow to dark-red, and they are
stable in the solid state under normal laboratory conditions. From the
very low molar conductance in DMF and methanol solutions (b10 S cm2
mol−1) it may be concluded that these complexes are true molecular
additioncompounds. In general, all the complexes are insoluble in water
and soluble or slightly soluble in a number of common organic solvents.
The complexes 1–4 are the most soluble whereas complexes 9–12 are
the least soluble compounds. Elemental and spectroscopic (IR, NMR,
MS) analyses of complexes clearly confirm the1:1, 1:2 and 2:3 metal-to-
ligand ratio in complexes 1–4, 5–8 and 9–12, respectively. It is worth
mentioningthat the great similarity in the spectroscopic patterns within
complexes of one type of quinolinylaminophosphonate ligands in-
dicates also the similar metal coordination patterns in their complexes,
as described below. In all the complexes the less electronegative and
coordinative poorphosphoryloxygen towards palladiumis not involved
in metal bonding and is free to be involved in hydrogen bonding.
Hydrogen bondingmight beof interest for thebiological activity of these
complex compounds. It is presumed that the possibility of hydrogen
bond formation might be important in the approaching and binding of
metal ions to nucleic acid fragments.
2.6. Antimicrobial activity
The antimicrobial activity of the synthesized compounds was
examined in vitro against ten Gram positive bacteria (Bacillus
megaterium BGSC 7A2, Bacillus subtilis ATCC 6633, Bacillus thuringien-
sis var. kurstaki BGSC 4D1, Sarcina lutea ATCC 9341, Staphylococcus
aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228 and clinical
isolates of Staphylococcus haemolyticus, Streptococcus agalactiae,
Streptococcus faecalis, Streptococcus faecium), eight Gram negative
bacteria (Enterobacter cloacae ATCC 23355, Escherichia coli ATCC 8739,
Haemophilus influenzae ATCC 19418, Proteus vulgaris ATCC 13315,
Pseudomonas aeruginosa ATCC 9027, Salmonella typhimurium ATCC
14028, Serratia marcescens ATCC 8100, a clinical isolate of Klebsiella
pneumoniae), two yeasts (Candida tropicalis ATCC 1369, Saccharomy-
ces cerevisiae ATCC 9763) and a mould (Aspergillus niger ATCC 6275).
The two-fold broth dilution technique [45] was used. The minimal
inhibitory concentrations (MIC, μg/mL) were defined as the lowest
concentrations of compound required to give complete inhibition of
the growth of each strain. All compounds were dissolved in DMSO,
then added to nutrient media (Haemophilus Test Medium for H.
influenzae, Mueller Hinton Broth for other bacteria and Sabouraud
Liquid Medium for fungi) to obtain final concentrations ranging from
1.5 μg/mL to 200 μg/mL. The amount of DMSO never exceeded 1% v/v.
Inoculum size was 5×105 bacteria/mL and 1×103 fungi/mL. The MICs
were read after incubation at 37 °C for 24 h (bacteria) and at 30 °C for
48 h (fungi). Media and media with 1% v/v DMSO were employed as
growth controls. Ciprofloxacin and miconazole were used as standard
antibacterial and antifungal drugs, respectively.
To detect the minimum bactericidal concentrations (MBC, μg/mL),
100 μL of liquid from each suspension remaining clear were
subcultured in fresh medium and the samples were incubated at
37 °C for 24 h. MBC values represent the lowest concentration of drug
needed for the reduction of the initial inoculum of 99.9%.
All experiments were performed in triplicate and repeated at least
three times.
3. Results and discussion
3.1. Synthesis and properties
All three types of quinoline-based aminophosphonate ligands
contain three potential donor atoms, quinoline nitrogen, aniline or
aminoquinoline nitrogen and phosphoryl oxygen, but their reaction
with Na2[PdCl4] (X=Cl, Br) in methanol afforded formation of
complexes with different metal–ligand interactions (Fig. 1). Their
structure was defined by elemental analysis, conductance measure-
ments, X-ray single-crystal diffraction and various spectroscopic
methods (IR, multinuclear NMR, UV–visible, ESI MS). It was found
that dialkyl [α-anilino-(quinolin-2-ylmethyl)]phosphonates (L1, L2)
3.2. X-ray studies
Molecular structures of complexes 1–4 and 6 are depicted in
Figs. 2–5 and Figs. S1 and S3 in Supplementary data. Some interesting
bond lengths and angles are listed in Table 2.

872
M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
Fig. 2. Ball and stick view of both molecules in the asymmetric unit of the complex 1.
Alkyl chains reveal two orientations in the crystal. More populated orientation is
shown. All hydrogen atoms are omitted for clarity. ORTEP view is given in
Supplementary data (Fig. S1).
Fig. 4. Molecular structure of the complex 3. Displacement ellipsoids are drawn at 50%
probability level and all hydrogen atoms are omitted for clarity.
described in this work. The interaction is blocked by positioning of the
phosphoryl group in the hydrophobic pocket of the aryl and alkyl groups.
Molecules of complex 2 in its acetonitrile solvate differ from those in the
solvent free structure in the conformation of the phenyl ring and the ester
groups. The difference can be assigned to the presence of the MeCN
molecules in the structure of the solvate.
In crystal structures, strong N–H···O=P hydrogen bonds are the
dominant intermolecular interaction (Table 3). Aminophosphonate
groups of two molecules of opposite chiralities form centrosymmetric
dimers, and in most of the described crystal structures, phosphoryl
oxygen also forms the hydrogen bond with the neighbouring aromatic
C–H donor functionality. Similar structural pattern was observed in
other aminophosphonates, like diethyl (α-anilino-N-phenyl)phos-
phonate [46] and its recently published substituted derivatives [47].
Hydrogen bonds C–H···X (X=Cl, Br) also contribute to the crystal
packing of complexes.
All chelate complexes (1–4) are square-planar and their characteristic
structural feature is the five-membered chelate ring Pd–N(quinoline)–C–
C–N(amino) that can be described either as twisted around the Pd–N
(amino) bond (1, 2, 2·MeCN) or as the envelope on N(amino) atom (3)
(Figs. 2–4 and Fig. S3). The chelate ring geometries observed in the
similar chelate dichloride palladium(II) complexes with α-amino-
methylpyridin-2-yl derivatives were analogous to ones described in
this work [48–51]. Two symmetrically independent molecules of
complex 1 are connected by the π–π interactions of quinoline rings
(Table S1, Fig. S9). Complex 2 crystallizes from acetone (Fig. 3a) and as a
monosolvate from MeCN (Fig. 3b). π–π interactions of quinoline rings are
observed in both structures (Table S1, Fig. S10). The unusual feature of
the crystal packing of the solvate is the absence of the characteristic
N–H···O=P hydrogen bonding observed in all other structures
Complex 6 is square-planar with trans conformation and the
palladium atom is situated in the crystallographic inversion centre
(Fig. 5). Crystal packing motif involves chains of ten-membered rings
C(13)[R22(10)] [52] formed by N–H···O=P hydrogen bonding of
aminophosphonate moieties along the c axis (Fig. 6). Two aminopho-
sphonate ligands are bonded to the palladium atom monodentately
through the quinoline nitrogen atom similar to the complexes trans-
[PdL2X2] (X=Cl, Br; L=diethyl quinolin-2- (2-dqmp) or quinolin-8-
ylmethylphosphonate (8-dqmp) [17,18], diethyl pyridin-3-
ylmethylphosphonate [53], and pyridin-2- (2-pmOpe) and pyr-
idin-4-ylmethylphosphate (4-pmOpe) [54]). Bond length Pd1–N1
is 2.033(3)Å and Pd1–Br1 is 2.4308(5)Å, similar to 2.064 Å and
2.435 Å, respectively, in trans-[Pd(2-dqmp)2Br2] [17]. The coordi-
nation sphere is almost an ideal square with the N1–Pd1–Br1 angle
of 89.90(10)°. Twisting angle between the coordination plane and
the plane of the bound quinoline ring is 74.88°. The similar values
80.63 and 79.00 were observed for trans-[Pd(2-dqmp)2Cl2] [17]
and trans-[Pd(2-pmOpe)2Cl2] [54], respectively. Observed value
can be rationalized by the need to accommodate present ethyl
Fig. 3. Molecular structure of a) the complex 2 and b) its solvate 2·MeCN. Displacement ellipsoids are drawn at 50% probability level and all hydrogen atoms are omitted for clarity.

M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
873
C=N bonds could be ascribed to π electronic redistribution in the
heterocyclic ligand caused by metallation of the quinoline nitrogen. In
this region occur also the NH deformation bands. The absorption of
the NH stretching vibrations in the free ligands is found at about
3300 cm−1 as a strong (L1–L4) or medium band (L5, L6), indicating
the existence of hydrogen bonding between the NH and P=O groups.
In the complexes 1–4 this band is reduced in intensity and is shifted to
lower frequencies by up to 100 cm−1 due to the bonding of the aniline
group to the metal ion, while in the complexes 5–8 with only the
quinoline nitrogen bonding it shows only a small higher frequency
shift and an intensity decrease. In dipalladium complexes 9-12, with
both nitrogen donors bonding, in the ν(NH) region appears a broad
absorption with maximum at lower frequency up to 40 cm−1 with
respect to the free ligands and is broadened on its higher frequency
side.
There are no great changes in the position of absorption bands of
the phosphonic ester group as it is not involved in the metal
coordination. The small variation in position of the ν(P=O) absorp-
tions displayed in the 1260–1230 cm—1 region is arising from their
sensitivity to the hydrogen bonding which may change the polarity of
the phosphoryl oxygen atom and brings about the appearance of these
bands at lower frequencies [55]. The spectra of all compounds show a
great complexity within 1075-900 cm—1 where occur the stretching
P-O-C and aliphatic C-C vibrations along with deformation modes of
the alkyl ester groups and =CH vibrations of the quinoline and
benzene rings [56].
Fig. 5. Molecular structure of the complex 6. Displacement ellipsoids are drawn at 30%
probability level and all hydrogen atoms and EtOAc molecules are omitted for clarity.
Palladium atom is positioned in the crystallographic inversion centre [0.5, 0.5, 0.5].
Unlabeled atoms are related by the symmetry operator i [1−x, 1−y, 1−z].
acetate molecules and the steric hindrance of two large aminopho-
sphonate ligands.
The far-IR spectra (400–200 cm—1) of complexes give insight into
the metal–ligand vibrations which are very useful for determining the
modes of ligand bonding. The position and the multiplicity of the
palladium–halogen stretching bands could undoubtedly confirm
either a cis or trans orientation of the terminal halogens in the
complexes [57]. The two ν(Pd-X) bands are visible for the complexes
1–4 as expected for the cis-square-planar complexes in C2v stereo-
chemistry. In the complexes (5–12) one band characteristic for the
trans-square-planar complexes in D2h stereochemistry is found. It is
worth noting that the presented spectroscopic data are in good
agreement with those obtained for the corresponding types of the
palladium complexes of quinolinylmethylphosphonates and anilino-
benzylphosphonates [17,18,21,22,58].
3.3. IR spectra
The IR frequencies associated with the main functional groups of
the complexes are listed in the experimental section. When
comparing the spectroscopic data of the complexes with those of
the free ligands [34,35], marked changes may be noticed in the ligand
bands arising from various modes of donor groups which are involved
in bonding to palladium. The results obtained are in accordance with
those obtained by X-ray structure analysis. The variation in position
and intensity of absorptions in the region of 1615–1500 cm−1
associated with the stretching modes of the quinoline C=C and
Table 2
Selected bonds (Å) and angles (°) for complexes 1–3 and 6. X=Cl or Br.
a
a
Bond
1_1
1_2
2
2·MeCN
3
6·2EtOAc
Pd1–X1
Pd1–X2
Pd1–N1
Pd1–N2
2.2778(12)
2.3002(10)
2.059(3)
2.2663(15)
2.3039(14)
2.056(3)
2.4079(4)
2.4297(4)
2.067(2)
2.076(2)
2.4137(3)
2.4305(3)
2.0758(18)
2.0703(19)
2.2704(8)
2.2937(9)
2.100(2)
2.062(2)
2.4308(5)
–
2.034(3)
–
2.069(3)
2.072(3)
Angle
X1–Pd1–N1
X1–Pd1–N2
X1–Pd1–X2
X2–Pd1–N1
N1–Pd1–N2
–
–
–
–
–
89.90(11)
92.02(8)
91.61(4)
96.74(9)
79.25(11)
92.39(9)
90.23(5)
97.77(9)
79.34(12)
92.61(7)
92.03(1)
96.22(7)
78.54(9)
90.54(5)
91.09(1)
98.69(5)
79.15(7)
89.78(7)
89.05(3)
100.26(7)
80.96(9)
–
–
–
–
a
Complex 1 comprises two molecules in an asymmetric unit. Data for both molecules are given. Molecule 1_1 contains Pd1 and molecule 1_2 contains Pd2. Accurate numbering of
atoms in the molecule 1_2 is: Pd(n+1), Cl(n+2), N(n+2), P(n+1), O(n+3), C(n+20), where n is the numbering of analogous atom in the molecule 1_1.
Table 3
Parameters for N–H···O=P hydrogen-bonding in the crystal structures of complexes 1–3 and 6.
Complex
d(D–H)/Å
d(H···A)/Å
d(D–H···A)/Å
α(D–H···A)/°
Sym. op. i
1
N2–HN1···O4i
N4–HN2···O1i
N2–HN···O1i
N2–HN···O1i
N2–HN···O1i
0.91
0.91
0.88(5)
0.91
0.92(5)
2.15
2.11
2.09(4)
2.13
2.12(5)
2.965(4)
2.916(4)
2.855(3)
2.954(3)
3.029(6)
149
147
145(5)
150
169(5)
−1+x, y, z
1+x, y, z
−x, −y, 1−z
1−x, −y, −z
1−x, 1−y, 2−z
2
3
6

874
M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
Fig. 6. Motif of the crystal structure of the complex 6 showing the C(13)[R22(10)] chain of rings parallel to the [001] direction. Alkyl chains reveal two orientations in the crystal. More
populated orientation is shown. Hydrogen atoms not participating in the N–H···O=P hydrogen-bonding are omitted for clarity.
3.4. NMR spectra
downfield shift (up to 0.06 ppm). In the carbon resonances the greatest
downfield shift show C-2 (4.23–4.68 ppm) and C-4 (2.56–2.86), the
smaller one exhibit C-3 (1.47–1.58 ppm), C-6 (1.39–1.43 ppm), C-7
(1.61–1.95 ppm)and C-8 (0.37–1.03 ppm), while for C-9 anupfield shift
(1.17–1.32 ppm) was observed. It is important to note that in the
spectra of these complexes some H/C resonances show either
broadening or multiplicity arising from the existence of more isomeric
species in dynamic equilibrium in solution negligibly differing magnet-
ically due to the restricted rotation around the Pd–N(quinoline)
coordination bond. The similar spectroscopic feature was found for
some palladium(II) and platinum(II) halide complexes of this type
The 1H, 13C and 31P NMR spectral studies of the complexes provide
important structural information of these compounds. The complete
1H and 13C data obtained on the basis of one- and two-dimensional
homo- and hetero-nuclear experiments are summarized in Supple-
mentary data according to the numbering scheme shown in Fig. 1. The
first point to be noted is that complexes retain their integrity in
chloroform solution and that the results obtained are in agreement
with those obtained by X-ray and IR analyses of complexes in the solid
state. The spectral data for the free ligands provided a basis for
interpreting the spectra of their complexes [34,35,56]. In all
compounds two sets of separate H/C resonances have been observed
for the diastereotopic alkyl ester groups of the P(O)(OR)2 moiety. The
spectral features are characterized by increased complexity arisen
also from the H/C-phosphorus splitting. Proton of the PCH group
appears either as a doublet with PH coupling or as a doublet of
doublets due to simultaneous coupling with phosphorus (21.6–
25.1 Hz) and NH proton (4.5–7.2 Hz). The NH proton appears as a
broad singlet or a broad doublet, while in the L4–L6 is visible a doublet
of doublets, due to couplings with phosphorus and PCH proton.
Carbon of the PCH group appears as a doublet with PC coupling of
140.7–151.7 Hz. The assignments of the aromatic H/C resonances
were unambiguously ascertained by using a combination of the 1D
and 2D multinuclear NMR analyses.
In the spectra of complexes, most of proton and carbon resonances
show a certain high-frequency shift with respect to the free ligands.
The decrease in the electron density in the heterocyclic aromatic ring
caused by the metal coordination of the quinoline nitrogen to
palladium atom and the proximity of the palladium atom which is
known to be magnetically anisotropic [59], bring about deshielding of
the atoms adjacent to the ligation sites. Thus, in the N,N-chelate
complexes 1–4 the greatest downfield shift in the 1H NMR spectra was
observed for the aromatic H-8 (1.22–1.26 ppm) and H-4 (0.39–
0.41 ppm) of the quinoline and H-4′ (0.52–0.53 ppm) and H-2′,6′
(0.27–0.28 ppm) of the aniline protons. Other aromatic protons
resonances exhibit a smaller frequency shift which varies from 0.06
to 0.19 ppm. For the PCH proton a slight upfield shift of 0.19–
0.21 ppm was observed, while the POCH2 protons exhibit a downfield
shift of 0.40–0.92 ppm. The other methylene protons as well methyl
protons are shifted downfield by up to 0.25 ppm. In the 13C NMR
spectra, the pronounced high-frequency shifts were found for the C-2,
C-4, C-2′,6′, C-4′, PCH and POCH2 carbons of 3.57–4.31, 4.35–4.80,
5.86–6.12, 8.89–9.07, 12.79–13.21 and 3.79–4.19 ppm, respectively.
The smaller downfield shifts exhibit C-5, C-8, C-10 and C-3′,5′ (up to
2.7 ppm), while for C-3, C-9 and C-1′ a slight upfield shielding was
observed (up to 2.9 ppm).
As expected, in the spectra of complexes 5–8 the significant changes
caused by complexation were observed only for the quinoline H/C
resonances. The greatest downfield shift exhibit protons H-8 (1.79–
1.88 ppm) and H-2 (0.54–0.56 ppm), the smaller one was found for H-7
(0.32–0.39 ppm) and H-4 (0.13–0.16 ppm), while all the other aromatic
as well the non-aromatic PCH and alkyl protons exhibit very small
Fig. 7. Parts of 1H NMR spectra of complexes a) 6 and b) 7 at various temperatures.

M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
875
(trans-[ML2X2], M=Pd, Pt) with various quinoline and aniline de-
rivatives [59–61]. In complex 5 two closely spaced signals are visible for
the H-8, PCH, NH and C-2 resonances, while in complex 7 signals for the
PCH, NH, C-2 and C-9 resonances are separated. The most of other H/C
resonances exhibit only slight broadening. The situation is more
complicated in the bromide complexes 6 and 8, where for almost all
H/C resonances the separated signals are visible. In the proton spectra
resonances are doubled, but in the 13C spectra of complex 6 even three
sets of carbon signals are shown for the C-2, C-3, C-8 and PCH
resonances, while in complex 8 only C-2 and PCH exhibit three
separated signals. The existence of rotational isomers in these
complexes, which distribution depends on both, the phosphonate
(ethyl/butyl) and halogen (Cl/Br) ligand, was confirmed by variable
temperature 1H NMR measurements. By comparing the spectra at room
temperature with those at 253 and 323 K, more pronounced changes
were observed in the spectra recorded at lower temperature, where
more signals of different intensities may be seen for almost all aromatic
protons (Fig. 7). The connectivity from 2D NMR experiments enabled
the assignments of H/C absorptions of all isomers and the most
populated isomer could be ascribed to the less sterically hindered one.
The spectra of complexes 9–12 confirm their structure of having
two terminal and one bridging ligand molecules (see Figs. 8 and 9).
Either the broadening, presumably in the poorly soluble chloride
complexes 9 and 11, or the presence of two sets of signals in the ratio
2:1 were found for almost all H/C resonances in the corresponding
bromide complexes 10 and 12. The same signal ligands ratio was
observed in the spectra of these complexes recorded at lower
temperature (253 K, see Fig. 9). Similarly as in the complexes 5–8,
the greatest downfield shift is observed for the protons H-8 (1.62–
1.82 ppm), H-2 (0.52–0.57 ppm) and H-7 (0.28–0.33 ppm) as well as
the carbons C-2 (3.84–4.96 ppm) and C-4 (2.59–3.10 ppm). In
general, the most of H/C resonances of the bridging ligand exhibit
negligibly greater high-frequency shift with respect to that of the
Fig. 9. Part of the 1H NMR spectra of the complex 12 recorded at 302 and 253 K.
terminal ligand resonances. The exceptions are the NH and PCH
proton resonances.
The valuable structural information about the complexes could be
obtained by their 31P NMR spectra, which data are given in Table 4 and
partly presented in Fig. 10. All diester ligands L1–L6 show a sharp
absorption between 20.6 and 21.5 ppm. Due to the vicinity of the
palladium atom in the chelate complexes 1–4 this absorption is
shifted upfield by c.a. 6–7 ppm. As expected, more complicated
spectral patterns are in the complexes with the 1:2 metal-to-ligand
ratios, due to the presence of rotational isomers and their temperature
redistribution. In spectra of the chloride complexes 5 and 7 at 302 K
Fig. 8. Parts of a) 1H and b) APT NMR spectra of the complex 10. Signals of the bridging ligand are denoted with an asterisk (*).

876
M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
Table 4
31P NMR data (δ, ppm) for ligands L1–L6 and their complexes 1–12.a
δ/ppm
δ/ppm
δ/ppm
L1
L2
L3
20.56
20.46
21.16
1
3
5
13.56
14.24
2
4
6
13.58
14.19
302 K: 20.44, 20.40 br (1:1.7)
253 K: 20.62, 20.51, 20.50, 20.41 (1:2.3:1.3)
302 K: 20.82 br
253 K: 20.56, 20.50, 20.46 (1.6:1:1)
20.63,e 20.55 br (2:1)
21.33,e 21.27 br (2:1)
302 K: 20.84, 20.78, 20.75, 20.73 (1:1:1:1)
253 K: 20.57, 20.47, 20.43, 20.39 (2:1:3:1.3)
302 K: 20.88, 20.85, 20.80, 20.79 (1:1.3)
b
c
L4
21.18
7
8
d
253 K: 20.63, 20.58, 20.51, 20.48 (1:1.6)
L5
L6
21.45
21.39
9
11
10
12
20.66,e 20.62 br (2:1)
21.37, 21.33 br (2:1)
a
Spectra recorded at 302 K unless stated otherwise. Ratio of signal intensities given in parentheses. br—broad.
Partly overlapped signals at 20.51 and 20.50 ppm. Their joint intensity is 2.3.
Partly overlapped signals at 20.88 and 20.85 ppm and those at 20.80 and 20.79 ppm. Ratio of joint intensities of pairs is 1:1.3.
Partly overlapped signals at 20.63 and 20.58 ppm and those at 20.51 and 20.48 ppm. Ratio of joint intensities of pairs is 1:1.6.
Signal of terminal ligands is broadened towards higher field due to the signal of the bridging ligand.
b
c
d
e
appear either two closely spaced absorptions at about 20.4 ppm (5) or
a bit broader absorption at 20.8 ppm (7). At lower temperature
(253 K) in these complexes are present four and three signals,
respectively. In the corresponding bromide complexes 6 and 8, both in
the spectra at room and lower temperature, there are clearly visible
four singlets between 20.7 and 20.9 ppm, but a different intensity
ratio of signals and small chemical shift differences could be observed
at various temperatures (Fig. 10b). In the spectra of complexes 9–12
there are two broadened singlets in the ratio 2:1 between 20.6 and
21.3 ppm arisen from two equivalent terminal and one bridging
ligand molecules (Fig. 10c). In complexes 9–11 these absorptions are
partly overlapped.
ligand charge-transfer transitions. The relatively dark colour of
complexes 1–4 indicates strong charge-transfer absorptions originat-
ing in the ultraviolet and trailing off into the visible region, while the
relatively pale colour of the other complexes indicates that the
quinoline ligand has a low ligand field strength in forming these
complexes. In addition, the intensity of absorption bands is slightly
higher for the bromide complexes than their chloride analogues,
which could be ascribed to the increased ligand participation in the
occupied MO-levels, derived mainly from the metal d-levels.
3.6. Mass spectra
The structure of the complexes was also supported by the mass
spectra carried out under positive-ion ESI conditions, but here are
given only the major structurally informative data for the complexes
9–12, as for this type of palladium complexes we could not grow
crystals for X-ray analysis. The mass spectrometric behaviour and
fragmentation pathways of the free ligands and their complexes
studied by collisional experiments on the ESI-generated species (ESI
MSn) will be published in detail elsewhere. In the ESI spectra (positive
ions) all the complexes show the presence of the molecular ion as
sodium adduct [M+Na]+ supporting the 1:1, 1:2 and 2:3 metal-to-
ligand ratio in complexes 1–4, 5–8 and 9–12, respectively. Formation
of sodiated molecular ions, very often present instead of the
corresponding protonated molecular ions, is the normal feature in
the ESI MS spectra of compounds investigated under positive ion
electrospray ionization conditions. In the case of complexes 9–12 the
important diagnostic fragment ions are those obtained by losses of
one ligand molecule as well as PdLX2, PdL2X2, PdL2X3 and Pd2L2X4
from the sodiated molecular ion finally leading to the sodium adduct
3.5. UV–vis spectra
The electronic absorption spectra of complexes obtained in
chloroform solution in the 250–600 nm region show several over-
lapping bands, which complicate the assignation of the transitions
involved. Quinolinyaminophosphonates alone show few intense band
maxima between 270 and 350 nm due to π–π* transitions, which is in
accordance with the spectra of the substituted quinoline and aniline
derivatives [19,62]. All palladium(II) complexes are diamagnetic with
a square-planar geometry, for which three spin-allowed d–d transi-
tions are expected, corresponding to transitions from the three lower
lying d-levels to the empty dx2
orbitals [63]. The ground state is
− y2
1A2g and the excited states related to these transitions are A2g 1B1g
,
1
and 1Eg in order of increased energy. Overlapping of the strong
absorptions from the charge-transfer or intraligand π–π* transitions
prevents the observation and detection of the expected bands [19,21].
The most prominent absorption is around 400 (1–4), 324 (5–8) and
368 (9–12)nm, respectively, which could be ascribed to the metal-to-
of the free ligand [L+Na]+
.
Fig. 10. 31P{1H} NMR spectra of complexes a) 4, b) 6 and c) 12.

M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
877
Table 5
breast carcinoma (MCF-7) and T-lymphoblast leukemia cells (MOLT-
4). The results, expressed as concentration of the complex required to
inhibit the tumor cell growth by 50% (IC50), are summarized in Table 5
and the concentration–response profiles for the chloride complexes
are given in Fig. 11.
Inhibitory effects of palladium complexes 1–12 on the growth of human tumor cell
lines.
IC50 (μM)
Complex
SW 620
H 460
MCF-7
HCT 116
61
MOLT-4
It was shown that complexes possess differential antiproliferative
activity against the examined cell lines at the tested concentration
range (0.01–100 μM). In general, it was shown that there is no
significant difference in sensitivity between the five tumor cell lines,
neither between the corresponding chloride and bromide complexes.
Only HCT 116 cell was slightly more sensitive than the other cell lines.
This is somewhat in contrast to the sensitivity of these five cell lines to
cisplatin; for example cell lines H 460 and MOLT-4 show more
pronounced sensitivity to this chemotherapeutic. Also, tetrachlorido
complex 9 was slightly more active with respect to its tetrabromido
analogue 10. Most importantly, complexes of dibutyl esters in all three
types of complexes are significantly more effective than those of
diethyl esters, which may be partly ascribed to the greater
lipophilicity and membrane permeability of the former complexes.
The lipophilicity of the complexes increases from diethyl to dibutyl
derivatives and may facilitate transport through the cellular mem-
branes [64]. Among complexes of butyl esters the most significant
activity at low micromolar range, which is comparable to the activity
of the reference compound cisplatin, show dipalladium complexes 11
1
2
3
4
5
6
7
8
9
10
11
12
N100
92
3
N100
85 12
18 0.1
7
74 26
N100
89
15
7
2
83 12
15 0.3
13 0.3
36 21
42 11
70 23
10
11
20
7
7
2
19
4
15 0.2
31 27
31 13
12 0.6
14
1
21 0.6
46 25
≥100
39 19
24 0.1
13
3
4
7
17
18
3
2
0.1
1
1
0.4
0.2
10
9
1
4
4
4
5
13
2
3
0.1
0.2
0.5
3
0.6
0.4
3
17 12
14
5
2
0.7
20
8
9
5
11
13
2
2
2
0.6
5
15 0.5
13 0.6
3
4
7
0.5
0.1
4
8
8
4
6
3
6
Cisplatin
0.3 0.02
10
5
7
0.2 0.04
3.7. Antitumor activity
The antiproliferative activity of the complexes was determined in
vitro on five human tumor cell lines derived from four cancer types:
colon carcinoma (SW 620 and HCT 116), lung carcinoma (H 460),
Fig. 11. Concentration–response profiles for the chloride complexes (1, 3, 5, 7, 9, 11) tested on various human tumor-cell lines in vitro. The cells were treated with the complexes at
different concentrations, and the percentage of growth (PG) was calculated. Each point represents a mean value of four parallel samples in three individual experiments.

878
M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
and 12 (IC50 2–8 μM), somewhat less active are mononuclear
complexes 7 and 8 (IC50 4–20 μM) and 3 and 4 (IC50 11–21 μM)
with trans- and cis-configuration, respectively. There are much
greater variations in response between complexes of diethyl esters.
Thus, while dipalladium complexes 9 and 10 show pronounced
activity (IC50 5–18 μM), mononuclear complexes display either
modest activity as in the case of complexes 5 and 6 (IC50 13–
46 μM), with one exception of complex 5 on MCF-7 cells with
IC50 N100, or have extremely modest antiproliferative activity as
complexes 1 and 2 (IC50 N60 μM). It is important to note that free
dibutyl esters were found to show extremely low, if any, antitumor
activity in MOLT-4 cells with IC50 value between N100 and N400 μM
[34], and therefore, it may be presumed that the free ligands do not
participate a lot in activity of the complexes. Furthermore, it is
interesting to compare the results of the in vitro assays on the same
tumor cell line related to the palladium dihalide complexes of
diethyl quinolin-2- and quinolin-8-ylmethylphosphonates [19,24].
Cis-complexes with 1:1 metal-to-ligand ratio of diethyl quinolin-2-
ylmethylphosphonate are more active than the cis-complexes 1 and
2, while those with 1:2 ratio and trans-configuration are less active
than complexes 5 and 6 [19]. On the other side, trans-complexes of
quinolin-8-ylmethylphosphonate show significant antitumor ac-
tivity with IC50 b10 μM. These findings support the general
assumption that the relationship between structure and activity
is extremely complex and depend on a lot of factors. Thus, the
differences in lability between the halide groups in different type of
complexes may alter the biochemical properties of the complexes,
as the breaking ability of the Pd–halogen bond is presumed to be
the crucial step in complex reaction with DNA strands. In addition,
the introduction of the second metal binding unit, which can also
interfere with nucleobases of DNA, may enhance the cytotoxic
effect [65]. This is in accordance with finding that dinuclear
complexes 9–12 show the higher activity with respect to mononu-
clear complexes 1–8.
contain three potential donor atoms, quinoline nitrogen, amino
nitrogen and phosphoryl oxygen, but their coordination behaviour
towards palladium(II) ion is different. In complexes either quinoline
or both quinoline and amino nitrogens are involved in metal(s)
bonding forming mononuclear dihalide adducts either with cis- or
trans-configuration as well as dinuclear tetrahalide complexes.
Phosphoryl oxygen is not coordinated and is free to be involved in
hydrogen bonding, which is the main feature of crystal structures of
complexes. The stereochemistry of the complexes, the nature of
metal–ligand binding and hydrogen bond interactions are investigat-
ed by spectroscopy and X-ray structure analysis. Biological properties
of complexes were examined by screening of their ability to inhibit
the cancer growth in vitro in a panel of human tumor cell lines and
their antimicrobial activity in a wide spectrum of bacterial and fungal
strains. While no specific antimicrobial effects of both the free
organophosphorus ligands and their palladium(II) halide complexes
were noted, the majority of complexes demonstrated cytostatic
activity, which was especially pronounced in the case of dipalladium
tetrahalide complexes with IC50 b10 μM. It may be concluded that
palladium complexes of investigated quinolinylaminophosphonates
represent an interesting class of new complex compounds from the
viewpoint of their physicochemical, structural and biological
properties.
Supplementary data associated with this article, including addi-
tional crystallographic and spectroscopic data along with the results
of antimicrobial screening, can be found in the online version at
doi:10.1016/j.jinorgbio.2011.03.011.
Abbreviations
M.p.
Melting point
Decomp. Decomposition
DMF
MTT
Dimethylformamide
Tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide]
Molecular Orbital
3.8. Antimicrobial activity
MO
MW
Microwave
The three different series of complexes 1–4, 5–8 and 9–12 were
tested for their in vitro antimicrobial activity against a large number of
standard microorganisms representative of Gram positive and Gram
negative bacteria, yeasts and moulds. The starting ligands L1–L6 and
palladium salts Na2[PdCl4] and Na2[PdBr4] were also included for a
comparison.
UV–vis Ultraviolet–visible
APT
COSY
HMQC
HMBC
Attached Proton Test
COrrelation SpectroscopY
Heteronuclear Multiple Quantum Coherence
Heteronuclear Multiple Bond Correlation
ESI MS ElectroSpray Ionization Mass Spectroscopy
Most of the tested compounds are devoid of antibacterial and
antifungal properties up the concentration of 200 μg/mL. Only a weak
inhibition could be detected towards some Gram positive bacteria
with MIC values of 200 μg/mL. In this regard, L3 and L6 ligands and
complex 11 inhibit the growth of B. thuringiensis var. kurstaki and a
similar behaviour is shown by ligand L6 and complex 9 against B.
megaterium and S. epidermidis. These compounds act as bacteriostatic
agents, exhibiting minimal bactericidal concentrations higher than
the corresponding MICs (MBCs N200 μg/mL).
Although the low degree of antibacterial activity prevents from
establishing extensive structure–activity relationships, it is interest-
ing to observe that both the active ligands L3 and L6 are 3-substituted
quinolines and that only dinuclear tetrachlorodipalladium complexes
(compounds 9 and 11) exhibited antibacterial properties. Further-
more, the different lipophilicity of the ethyl and butyl substituents at
the phosphonate moiety does not significantly affect the inhibition of
the growth of tested microorganisms.
ORTEP
CCD
PG
Oak Ridge Thermal Ellipsoid Plot
Charge-Coupled Device
Percentage of Growth
OD
Optical Density
IC50
MIC
MBC
Concentration necessary for 50% of inhibition of cell growth
Minimal inhibitory concentration
Minimum bactericidal concentration
SW 620 Human colorectal adenocarcinoma cell line
H 460
MCF-7
HCT 116 Human colon cancer cell line
MOLT-4 Human acute lymphoblastic leukemia cell line
Human large cell lung cancer cell line
Human breast adenocarcinoma cell line
Acknowledgements
4. Conclusion
The financial support of the Croatian Ministry of Science, Education
and Sports (grant Nos. 098-0982915-2950, 098-1191344-2943 and
098-0982464-2514) is gratefully acknowledged. The authors would
like to thank B. Sc. S. Roca and Mr. Sc. Ž. Marinić for recording the NMR
spectra and Dr. L. Šuman for screening the antitumor assays.
A series of novel palladium(II) chloride and bromide complexes
with three types of quinolinylaminophosphonates have been synthe-
sized and structurally characterized. All organophosphorus ligands

M. Juribašić et al. / Journal of Inorganic Biochemistry 105 (2011) 867–879
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