Five-membered [C,N] and [N,O] metallocyclic complexes of palladium(II) with monoalkyl [α-(4-benzeneazoanilino)-N-benzyl] phosphonates: Synthesis, characterization and antitumour activity

Article abstract of DOI:10.1016/S0277-5387(00)00339-9

The synthesis, spectroscopic and biological properties of the novel palladium(II) complexes with monoethyl (HL1) and monobutyl (HL2) esters of [α-(4-benzeneazoanilino)-N-benzyl]phosphonic acid, have been prepared and studied. These potential polydentate ligands form two types of metallocyclic compounds, those with [C,N] and [N,O] five-membered chelate rings. The former are cyclopalladated chloro-bridged binuclear complexes, [PdL(μ-Cl)]₂, in which the deprotonated ligand undergoes palladation at the azo nitrogen and the ortho-carbon, while the latter mononuclear complexes, PdL₂, contain the organophosphorus ligand bonded through the aniline nitrogen and the deprotonated phosphonic acid oxygen. The complexes were identified and characterized by elemental analysis, magnetic and conductance measurements as well as by IR, ¹H, ¹³C and ³¹P nuclear magnetic resonance and ESI-mass spectroscopic studies. The in vitro antitumour activity of all the complexes was evaluated against the human KB cell line as a preliminary screening for their biological activity. The coordination behaviour of monoalkyl benzeneazophosphonates as well as the spectral and biological properties of their complexes were compared with the results reported for the corresponding dialkyl phosphonates and their palladium(II) complexes. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0277-5387(00)00339-9

www.elsevier.nl/locate/poly
Polyhedron 19 (2000) 937–948
Five-membered [C,N] and [N,O] metallocyclic complexes of
palladium(II) with monoalkyl [a-(4-benzeneazoanilino)-N-
benzyl]phosphonates: synthesis, characterization and antitumour activity
a
a
b
Ljerka Tusˇek-Bozˇic´ a, , Marijana Komac , Manda Curic´ , Antonin Lycˇka ,
´
*
Martina D’Alpaos c, Vito Scarcia d, Ariella Furlani d
a
ˇ
´
Department of Physical Chemistry, RuLer Boskovic Institute, Bijenicˇka c. 54, 10000 Zagreb, Croatia
b Research Institute for Organic Syntheses, 532 18 Pardubice-Rybitvi, Czech Republic
c Servizio di Spettrometria di Massa, Area di Ricerca del CNR, Corso Stati Uniti 4, 35100 Padua, Italy
d
´
Dipartimento di Scienze Biomediche, Universita degli Studi di Trieste, Via L. Giorgieri 7, 34127 Trieste, Italy
Received 29 November 1999; accepted 21 February 2000
Abstract
The synthesis, spectroscopic and biological properties of the novel palladium(II) complexes with monoethyl (HL1) and monobutyl (HL2)
esters of [a-(4-benzeneazoanilino)-N-benzyl]phosphonic acid, have been prepared and studied. These potential polydentate ligands form
two types of metallocyclic compounds, those with [C,N] and [N,O] five-membered chelate rings. The former are cyclopalladated chloro-
bridged binuclear complexes, [PdL(m-Cl)]2, in which the deprotonated ligand undergoes palladation at the azo nitrogen and the ortho-
carbon, while the latter mononuclear complexes, PdL2, contain the organophosphorus ligand bonded through the aniline nitrogen and the
deprotonated phosphonic acid oxygen. The complexes were identified and characterized by elemental analysis, magnetic and conductance
1
measurements as well as by IR, H, 13C and 31P nuclear magnetic resonance and ESI-mass spectroscopic studies. The in vitro antitumour
activity of all the complexes was evaluated against the human KB cell line as a preliminary screening for their biological activity. The
coordination behaviour of monoalkyl benzeneazophosphonates as well as the spectral and biological properties of their complexes were
compared with the results reported for the corresponding dialkyl phosphonates and their palladium(II) complexes.
q2000 Elsevier
Science Ltd All rights reserved.
Keywords: Palladium(II) complexes; Metallocycles; Aminophosphate complexes; Spectroscopy; Antitumour activity
1. Introduction
metal has a strong preference for N-donor ligands. Monoden-
tate ligands of this type readily undergo substitutionreactions
[14–16], however, the chelate effect is important in favour-
ing the final stability of a complex and there is a well devel-
oped chemistry for chelating ligands that combine a soft and
a hard donor centre. There are known cyclometallated com-
plexes in which the ligand is bonded through the carbon,
nitrogen and oxygen atoms [17,18]. Derivatives of amino-
carboxylic and aminophosphonic acids as [N,O] chelate
ligands are of particular interest owing to their relevance to
the natural systems and promising biological activity [19–
22].
Our systematic investigation has been directed to the syn-
thesis and characterization of new biological active palla-
dium(II) complexes with dialkyl and monoalkyl esters of
phosphonic acids derived from quinoline[14–16,21–23]and
aniline [13,24,25]. It was found that most of thesecomplexes
Organopalladium chemistry is one of the most extensive
and varied areas of transition metal chemistry. Special atten-
tion has been paid to metallocyclic complexes with nitrogen
donor ligands, such as various alkyl and aryl substituted
amines and imines, azo, hydrazo and heterocyclic com-
pounds. The chelate ring generally possesses three to seven
members, with the five-membered ring being most favoured.
These compounds are used successfully in organic synthesis
[1–3], homogeneous catalysis [4], asymmetric synthesis
[5], photochemistry [6] and optical resolution [7,8], and
are rather promising as liquid crystals [9,10] and potential
biologically active materials [11–13]. Complexes with O-
bonding ligands are less abundant because palladium as a soft
* Corresponding author. Tel.: q385-1-4680097; fax: q385-1-4680245;
e-mail: tusek@rudjer.irb.hr
0277-5387/00/$ - see front matter q2000 Elsevier Science Ltd All rights reserved.
PII S0277-5387(00)00339-9
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Lj. Tusˇek-Bozˇic´ et al. / Polyhedron 19 (2000) 937–948
displayed a certain in vitro antitumour activity againsthuman
and animal tumour cell lines. The greatest activity was found
forcomplexesofdialkylestersof[a-(4-benzeneazoanilino)-
N-benzyl]phosphonic acid [13]. These ligands with several
donor atoms serving as potential sites for metal coordination,
i.e. the anilino and azo nitrogen as well as the phosphoryl
oxygen, form two types of palladium(II) halide complexes:
adducts and cyclometallated derivatives. The former are
square planar with trans-bonded ligands through the azo
nitrogen, while in the latter the deprotonated ligand under-
goes palladation at the azo nitrogen and the ortho-carbon
forming the binuclear organopalladium complexes. The ani-
lino and phosphoryl groups are not involved in metal coor-
dination and are free to be involved in hydrogen bonding,
which is known to have some importance in metal–DNA
approaching and binding interactions [26]. In the present
paper we describe the synthesis as well as spectroscopicanal-
yses and biological properties of new palladium(II) com-
plexes of monoethyl (HL1) and monobutyl (HL2) esters of
[a-(4-benzeneazoanilino)-N-benzyl]phosphonic acid. In
the course of these investigations, the results obtained are
compared with those obtained for palladium complexes of
the corresponding dialkyl benzeneazophosphonates [13],
and are discussed with respect to their structure–stabilityand
structure–activity relationships. Differences in their com-
plex-forming behaviour mainly arise from the presence of
the phosphonic acid group in monoester derivatives, which
enables these ligands to coordinate to the palladium ion in
either their molecular or anionic form.
Fig. 1. Proposed chemical structures and numbering scheme of palladium
complexes with monoalkyl [a-(4-benzeneazoanilino)-N-benzyl]phos-
phonates (HL1 and HL2).
ing group [27,28]. Our results presented here, as well as
those obtained for the cyclopalladated complexes of the cor-
responding dialkyl phosphonates [13], are in accordance
with this assumption. In both cases the palladation occurred
in the anilinobenzyl-substituted aromatic ring. It may be pre-
sumed that the simplified mechanism of cyclopalladation
includes the initial coordination of azo nitrogen to metal ion
followed by electrophilic substitution at the ortho-carbon
atom. It is worth noting that in the case of dialkyl phosphon-
ates, the complexes with monodentate azo N-bonded ligands
could easily be isolated, while all efforts to prepare dihalide
adducts of the monoester ligands were unsuccessful.
2. Results and discussion
The prepared complexes are red powder like or microcrys-
talline compounds, stable in the solid state under normal
laboratory conditions. Their diamagnetic behaviour suggests
square-planar stereochemistry around the palladium(II) ion.
The molar conductance values (below 15 S cm2 moly1 in
DMF) correspond to those of non-electrolytes. The com-
plexes are insoluble in water, slightly soluble or soluble in
common organic solvents, and very soluble in DMF and
DMSO. In general, the binuclear complexes are more soluble
than the mononuclear complexes, as are complexes of butyl
ester with respect to complexes of its ethyl analogue. Because
of the insolubility of the prepared complexes in suitable sol-
vents, or because of their instability, we could not grow crys-
tals for X-ray analysis. The structure of the complexes, as
well as their stability and mode of decomposition, were
deduced mainly from their IR, multinuclear magnetic reso-
nance and mass spectroscopic studies. A detailed spectro-
scopic analysis of the free organophosphorus ligands has also
been described.
2.1. Synthesis and properties
Investigations of the chelating behaviour of monoalkyl[a-
(4-benzeneazoanilino)-N-benzyl]phosphonates (HL1 and
HL2) towards the palladium(II) ion have shown that these
ligands act in a bidentate manner forming two types of metal-
locyclic complexes, those with [C,N] and [N,O] five-mem-
bered chelate rings (Fig. 1). The reaction of HL1 and HL2
2y
with PdCl4 (molar ratio M:Ls1:1) in methanol affords
cyclopalladated binuclear complexes with metal–metal
chloro bridge, [PdL(m-Cl)]2 (1 and 3), in which the depro-
tonated ligand undergoes palladation at the azo nitrogen and
the ortho-carbon. In basic media the phosphonate acid group
is deprotonated and, thus, the reaction of sodium salts of
monoalkyl phosphonates (NaL1 and NaL2) with PdCl42y in
water (M:Ls1:2) gives [N,O] chelate complexes, PdL2 (2
and 4), with coordinated ligand through the aniline nitrogen
and the phosphonic acid oxygen. It should be noted that in
an unsymmetricallysubstitutedazobenzenederivative,ortho-
palladation can occur in either benzene ring with the possi-
bility of obtaining an isomeric mixture, but in general it has
been noticed that the palladium–carbon s-bond is formed
preferentially with thebenzeneringhavinganelectron-donat-
2.2. Infrared spectra
There are great differences in the IR spectra of the two
types of complexes, supporting different metal–ligand inter-
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939
actions in these compounds. Significant differences may be
noticed between 1600 and 1530 cmy1 where the NH defor-
mation modes along with the benzene ring stretching vibra-
tions are found. A very strong band at 1576 cmy1 with a
shoulder at 1555 cmy1 is characteristic for the cyclopalla-
dated complexes 1 and 3 with an aryl carbon–metal s bond,
while the chelate complexes 2 and 4 in this frequency region
are characterized by two very intense bands near 1600 and
1585 cmy1. There are marked differences also between 1250
and 950 cmy1 where absorption bands appearassociatedwith
the acidic monoalkyl phosphonate group PO(OR)(OH)
[29,30]. In the cyclopalladated complexes two bands at 1237
and 1198 cmy1 could be ascribed to the P_O stretching
absorption, while the PO–H stretching vibration is superim-
posed on the PO–C stretching, giving broad and complex
and the most abundant fragment ion is formed by cleavage
of the C–P bond and complete loss of the phosphonate ester
group. The abundance of the protonated molecular ion isonly
about 5% as is shown in Fig. 2 for HL2, as an example of the
common decomposition pattern for both alkyl monoesters in
positive/negative ESImassspectra. Allpalladiumcomplexes
have been investigated by spectrometric measurements car-
ried out under positive/negative-ion ESI conditions with dif-
ferent cone voltages, using methanol as the solvent and the
mobile phase. The negative-ion mass spectra give more rel-
evant structural information than the spectra obtained in the
positive-ion mode. The molecular ion as well as the fragment
ions containing palladium are identified by the presence of
the characteristic clusters of isotopic peaks covering about
10 m/z units, due to the presence of the numerous palladium
isotopes. The fragmentation pathway depends mainly on the
type of complex and the major structurally informative frag-
ments in negative-ion mass spectra are summarized in Table
1. The first point to be noted is that all complexes exhibit the
deprotonated molecular ion and its relative abundance is
about 3% for the cyclopalladated binuclear complexes 1 and
3 and about 1% for the mononuclear palladium complexes 2
and 4. There are great differences in mass spectroscopic
behaviour between these two types of complexes.
bands between 1030 and 950 cmy1
.
In the spectra of the mononuclear complexes 2 and 4 there
isno evidenceof thebandsassociatedwithP–O–Hvibrations,
since in these complexes the phosphonate ligands act in the
anionic form. This is confirmed by the presence of the
n(PO2y) absorptions as medium or strong bands between
1230 and 1200 cmy1 for the antisymmetricandbetween1070
and 1025 cmy1 for the symmetric mode of this vibration. In
the latter frequency range the n(PO–C) absorption is super-
imposed upon that of the symmetric PO2y vibration[31,32].
The chloro-bridged cyclopalladated complexes 1 and 3 in
the far-IR region show two Pd–Cl stretching bands. The
higher-frequency band at 307 cmy1 is attributed to the vibra-
tion of the Pd–Cl bond trans to the nitrogen atom, while the
lower-frequency band at 255 cmy1 isascribedtothevibration
of the Pd–Cl bond trans to the s-bonded carbon. This is a
consequence of a greater trans influence of a s-bonded car-
bon compared to that of a nitrogen atom [33].
The fragmentation pathways of the cyclopalladated com-
plexes, including possible structures of Pd-containing ions,
proposed on the basis of the accurate mass measurementsand
isotopic cluster analysis are reported in Scheme 1 and Fig.
3(a). The loss of Clx produces the fragment ion which cor-
responds to binuclear monobridged species. In this respect it
2.3. Mass spectra
Electrospray ionization mass spectrometry (ESI-MS) is a
soft ionization technique which hasrecentlybeenwidelyused
in the structural analysis of non-volatile and thermally labile
species such as large biomolecules [34,35] and various coor-
dination metalcompounds, especiallypolynuclearcomplexes
[36,37]. This method allows pre-existing ions in solution to
be very gently transferred to the gas phase with minimal
fragmentation, followed by conventional mass spectrometry.
The ESI mass spectrometric investigation of monoalkyl ani-
linobenzylphosphonates and their palladium(II) complexes
has shown that their mass fragmentation pattern is much less
extensive than that obtained for the corresponding dialkyl
phosphonates and their palladium complexes studied under
fast atom bombardment (FAB) conditions [24,25]. In the
ESI negative-ion mass spectra of the free non-coordinated
organophosphorus ligands, the deprotonated molecular ion
was observed as the base and the only intense peak, since the
phosphonic acid group could easily be deprotonated to give
an anion. In the corresponding spectra obtained in the posi-
tive-ion mode, the ligand fragmentation is more pronounced
Fig. 2. Positive-ion and negative-ion ESI mass spectra of HL2 in a mixture
of acetonitrile and water (50:50) with 0.1% acetic acid at a cone voltage of
50 V.
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Table 1
Major ion fragments in negative-ion mass spectra of Pd(II) complexes a
Ionic species b
m/z (relative intensity %) c
1
2
3
4
[(M–2H)qNa]y
[M–H]y
1091 (7)
1069 (3)
1056 (3.5)
1034 (9)
998 (11)
1147 (4)
1125 (3)
1112 (3.5)
1090 (11)
1054 (5)
893 (1)
949 (0.9)
[(M–2H)–ClqNa]yx
[(M–H)–Cl]yx
[(M–H)–Cl–HCl]yx
[(HL)2–2HqNa]y
[(HL)2–H]y
811 (1)
789 (0.4)
867 (0.9)
845 (0.5)
[PdLCl2qMe]y
585 (5)
570 (12)
556 (15)
549 (8)
534 (100)
531 d
499 (14)
426 (6)
409 (29)
394 (63)
613 (5)
598 (17)
584 (15)
577 (10)
562 (100)
559 d
527 (10)
454 (3)
437 (11)
422 (79)
[PdLCl2]y
[(PdLCl)–2HqNa]y
[(PdLCl)–HqMe]y
[(PdLCl)–H]y
[(PdL)–HqMeOH]yx
[(PdL)–H]yx
531 (0.8)
499 (0.7)
426 (0.6)
409 (0.7)
394 (100)
559 (1)
527 (0.3)
454 (0.6)
[LqMeOH]y
[LqMe]y
[L]y
422 (100)
a In methanol, cone voltage 50 V.
b LsL1, L2.
c Ion masses referenced to isotopes 106Pd and 35Cl.
d Partly overlapped with [(PdLCl)–H]y ion.
could be pointed out that some monohalide-bridgedbinuclear
palladium complexes have been isolated and characterized
[38,39]. Thefurtherlossofhydrogenchlorideinvolvesstruc-
tural rearrangement of the species giving a dinuclearcomplex
in which are two cyclopalladated moieties bonded through
the deprotonated phosphonic acid groups. The loss of one
palladium and one ligand molecule from the parent complex
ion leads to a deprotonated dichloromonopalladatedcomplex
[PdLCl2]y, which by the sequential losses of HCl and Clx
leads finally to the orthometallated species [(PdLCl)–H]y
and [(PdL)–H]yx, respectively. The former mono-chloro-
palladium fragment ion is the base peak in the spectra ofthese
complexes. The high stability of such metallated systems is
in agreement with the reported high stabilityofpalladium(II)
five-membered cyclometallated systems having nitrogen
donor ligands [24,40,41]. Of the organic fragments, the most
intense peak (60–80%) corresponds to the deprotonated
ligand molecule.
Investigations of decomposition properties of the mono-
nuclear complexes 2 and 4 indicate much less stability of
these compounds with respect to the binuclear cyclopalla-
dated complexes. The predominant pathway for their frag-
mentation is complete dissociation of the phosphonate ligand
molecules (Fig. 3(b)). The deprotonated ligand ion is the
major and the only intense fragment ion in the spectraofthese
complexes. The relative abundance of all other fragment ions
is very small amounting up to 1%. The most abundant pal-
ladium containing fragment ions are the parent molecular ion
[(PdL2)–H]y, and the ion containing only one [N,O]
bonded organophosphorus ligand. Dimerization of the free
organophosphorus ligand gives rise to [(HL)2–H]y ions at
m/z 798 and 845, respectively. The sodium adducts are also
present.
It is worth noting that the spectra of complexes show some
ions based on addition of methanol as solvent molecules as
well as addition of the sodium ion either totheparentcomplex
or to the ligand molecules or their fragments; this is in agree-
ment with the ESI mass results obtained for various complex
compounds [37,42]. Some of the most abundant adduct ions
are given in Table 1.
2.4. NMR spectra
1H, 13C and 31P NMR data of free ligands HL1 and HL2,
sodium salt NaL2 and both types of palladium complexes
derived from the butyl ester (complexes 3 and 4), obtained
in CDCl3 solution, are summarized in Tables 2 and 3, and
presented in Figs. 4–7. The numbering scheme is shown in
Fig. 1. Complexes of ethyl ester are not sufficiently soluble
for spectral analysis. The results obtained are compared and
discussed with those reported for dialkyl [a-(4-benzene-
azoanilino)-N-benzyl]phosphonates and their palladium
complexes [13,43]. The broadening of H/C aromatic
absorptions in the free monoesters with respect to those
observed in the corresponding diesters could be connected
with their dimeric structure in chloroform solution [30]. The
most pronounced broadening could be observed for protons
H-9,11 and H-15,17, as shown in Figs. 4 and 5(a). The slight
upfield shift (up to 0.25 ppm) of most of the aromaticprotons
with respect to those in diesters could be attributed to mutual
interligand shielding between two hydrogen bonded mono-
ester molecules lying parallel to one another. The ligand
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941
Scheme 1. ESI negative-ion fragmentation pathway for complexes 1 and 3.
association is ascribed to the strong intermolecular hydrogen
bonding between the phosphonic acid groups, which is con-
firmed by the presence of a very broad absorptionoftheacidic
OH resonance around 10 ppm. In the case of sodium salt
(Fig. 5(c)) the signal broadening of the aromatic protons is
not visible, as well as in the spectra of monoesters in DMSO
solution (Fig. 5(b)), in which there is no ligand association.
The assignment of all aromatic H/C resonances was con-
firmed unambiguously by two-dimensional homonuclearand
heteronuclear NMR experiments. As examples, Fig. 4 shows
a 1H–1H COSY spectrum of HL1 and Fig. 6 shows a part of
the long-range 1H–13C HMBC spectrum ofsodiumsaltNaL2.
In addition, the signal assignment of the carbons C(1)–C(6)
was supported with the long-range phosphorus–carbon
couplings.
The spectra of both types of complexes are rathercomplex,
caused either by the removal of the degeneracy of resonances
due to metal coordination (complex 3) or by the presence of
more isomeric forms (complex 4). The most pronounced
chemical shift changes with respect to the uncomplexed
ligand could be noticed for the atoms involved in metal bind-
ing and those in the vicinity of the ligation site. In the binu-
clear complex 3, with palladation at the azo nitrogen and the
ortho-carbon, there are significantdifferencesintheB-phenyl
and C-phenyl rings of the ligand molecule. The proton NMR
spectrum shows a great broadening of all signals and an
extensive overlappingofthe aromaticresonances(Fig.5(c))
due to the dimeric nature of this complex. The non-equiva-
lence of the aromatic protons H(8) and H(12) and their
upfield shifts of 0.41 and 0.19 ppm, respectively, supports
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Fig. 3. Negative-ion ESI mass spectra of complexes 1 (a) and 4 (b) in methanol at a cone voltage of 50 V.
the metallation at C-11 of the B-phenyl ring. The shielding
of these protons is caused by the flow of electron densityfrom
the d orbital of the palladium atom into this aromatic ring.
Resonances of the other aromatic protons could not be clearly
resolved owing to complex overlap of signals between 7.20
and 7.80 ppm. The 31P NMR spectrum of this complex shows
a broad signal at 21.88 ppm (Fig. 7(a)). The 13C NMR
spectrum of this complex gives much more spectral infor-
mation. Comparing with the spectrum of the free ligand, the
most important changes appear in resonances related to car-
bons C-7-C-12, as an identification of changes in the electron
density of this aromatic ring due to ortho-metallation. For
this reason none of these carbons are still equivalent. The
palladium-bound C(11) is greatly shifteddownfieldby31.66
ppm, C(9) and C(12) are shifted to a lesser extent by 5.87
and 2.93 ppm, respectively, while an upfield shift of 7.07
ppm for the C(8) atom, para to the Pd atom, clearly indicates
the existence of some metal-to-ligand p-back-bonding. As
expected, the overall spectral pattern of complex 3 is very
similar to that of the cyclopalladated complex of dibutyl ben-
zeneazophosphonate owing to the same palladium–ligand
bonding [43].
The spectra of mononuclear complex 4 show a great com-
plexity arising from the existence of more isomeric species
in dynamic equilibrium with differentmagneticenvironment.
This can be interpreted by the presence of a new chiral centre
on the nitrogen atom due to its coordination to palladium, in
addition to that at the adjacent PCH asymmetric carbon and
phosphorus atoms, as well as by the restricted rotationaround
the C–N linkage. As a consequence, the proton NMR spec-
trum of this complex shows a great broadening of all proton
resonances (Fig. 5(d)), while its 13C NMR spectrum is char-
acterized by extensive signal splitting and multiple overlap
of signals, which prevent signal assignments for almost all
aromatic proton and carbon atoms. The existence of more
isomeric species is visible in the 31P NMR spectrum of this
complex. There are few signals in the range 5–25 ppm as was
shown in Fig. 7(b). The two most intense signals are at 14.46
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Table 2
1H NMR data a
HL1
HL2 b
NaL2
3
4
c
c
H-2,6
H-3,5
H-4
7.54 d
J(HH)s7.5
7.35 t
J(HH)s7.3
7.29 t
7.54 d (7.56)
J(HH)s7.6 (7.0)
7.35 t (7.36)
7.37 d
J(HH)s7.0
7.21 t
J(HH)s7.0
7.17 t
c
c
c
c
J(HH)s7.3 (7.2)
7.29 t d (7.32)
J(HH)s7.4
6.75 d
J(HH)s8.8
7.73 br s
J(HH)s7.4 (7.1)
6.72 d (6.76)
J(HH)s8.2 (8.8)
7.72 br s (7.81 d)
J(HH)s(8.7)
7.63 d (7.85)
J(HH)s6.5 (7.6)
7.22 br m f (7.47 d)
J(HH)s(7.3)
7.31 t g (7.39)
J(HH)s7.6
6.66 d
J(HH)s8.4
7.69 d
J(HH)s8.6
7.78 d
J(HH)s7.5
7.43 t
H-8,12
H-9,11
H-14,18
H-15,17
H-16
6.53, 6.31 br s e
6.62 br s
c
c
c
c
c
c
c
c
7.61 d
J(HH)s7.1
7.21 br m f
J(HH)s7.5
7.31 t g
7.36 t h
J(HH)s7.3
4.97 d
J(HH)s7.4
J(HH)s7.2
4.46 dd
PCH
4.95 d (4.92)
4.91 br s
4.80 br d
2J(PC)s22.8
2J(PC)s22.9 (23.9)
2J(PC)s23.2
J(NH)s6.4
6.22 br s
2J(PC)s23.8
j
NH
OH
OCH2
7.18 i
7.20 i (5.83)
6.09 br s
9.80 k
10.40 k
3.28 br s l
3.58, 3.53 m n
j
3.99, 3.82 m m
3.94, 3.76 m m
3.90, 3.65 br s n
3.70 br m n
(3.64; 3.91; 4.12) o
1.52 m p (1.42; 1.63) o
1.31 m r (1.23; 1.38) o
0.85 t (0.84; 0.90) o
J(HH)s7.3 (7.3)
OCH2CH2CH2CH3
OCH2CH2CH2CH3
CH3
1.36 m p
1.12 m r
0.72 t
1.46 br s
1.25 br s
0.83 br s
1.40 br s
1.15 br s
0.75 br s
1.21 t
J(HH)s7.1
J(HH)s7.2
a Spectra recorded in CDCl3 (d in ppm, coupling constants in Hz). Multiplicities: s, singlet; d, doublet; dd, doublet of doublet; t, triplet; m, multiplet; br, broad
signal.
b Data for dibutyl [a-(4-benzeneazoanilino]-N-benzyl]phosphonate from Ref. [43] are given in parentheses for comparison.
c Resonance not resolved owing to extensive broadening and overlapping of signals between 6.8–8.0 ppm.
d Partly overlapped with H16.
e No equivalent protons. The first value is related to H-8 and the second to H-12.
f Overlapped with NH. Value obtained from a 1H–1H COSY experiment.
g Partly overlapped with H-4. Value obtained from a 1H–1H COSY experiment.
h Value obtained from a 1H–13C HMBC experiment. Signal partly overlapped with H-2,6.
i Overlapped with H-15,17. Tentative value obtained from a 1H–13C HMBC experiment.
j Resonance overlapped with aromatic protons.
k Very broad signal due to strong hydrogen bonding.
l OH resonance from H2O.
m Centres of two multiplets. Separate resonances for two geminal protons.
n Centres of two multiplets. Resonances for two geminal protons are partly overlapped.
o Values in parentheses related to protons of two diastereotopic butyl groups of dibutyl ester.
p Appears as quintet with J(HH);7.2 Hz.
r Appears as sextet with J(HH);7.4 Hz.
and 18.90 ppm. The fact that the 31P NMR chemical shifts of
the complex do not differ much from that observed for the
free ligand and itssodium salt at 18.59and19.19ppm, respec-
tively, supports the conclusion that coordination of phos-
phonate to metal ion is predominantly ionic and scarcely
affects the electron density of the phosphonate phosphorus
[44].
The NMR spectral studies have shown that complexes
retain their integrity in chloroform solution, but decompose
in DMSO. This solvent with strong complexing ability initi-
ates dissociation of the complex moiety by displacing the
organophosphorus ligand. As could be expected, the decom-
position process is much more pronounced in the [N,O]
chelate complexes.
In conclusion, the results obtained by NMR studies are in
agreement with those of mass spectrometry, indicating that
the five-membered [C,N] chelate ring in the binuclear
chloro-bridged complexes is much more stable than the five-
membered [N,O] ring in the mononuclear chelate com-
plexes. This behaviour is in accordance with the assumption
that the chelate ring where the cyclopalladation is assisted by
the coordination of a nitrogen atom hasa remarkablestability.
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Table 3
13C NMR data a
HL1
HL2 b
NaL2
3
C-1
136.30
136.42 (135.50)
138.52
135.15
127.82
128.69
128.15
147.90
2J(PC)s3.5
127.47
C-2,6
C-3,5
C-4
127.89
128.00 (127.79)
3J(PC)s5.0 (5.5)
128.57 (128.54)
4J(PC)s2.3 (2.4)
127.76 (127.95)
5J(PC)s2.7 (3.1)
148.43 (149.08)
3J(PC)s4.9
128.49
3J(PC)s4.1
128.44
4J(PC)s2.3
127.69
4J(PC)s2.3
127.27
5J(PC)s2.9
147.60
5J(PC)s3.4
150.12
C-7
3J(PC)s14.0
113.86
C-8
C-12
C-9
C-11
C-10
C-13
C-14,18
C-15,17
C-16
PCH
114.55
114.55
124.20
124.20
141.42
154.28
120.48
128.86
114.57 (113.31)
114.57 (113.31)
124.04 (124.79)
124.04 (124.79)
141.59 (145.19)
153.74 (152.88)
120.68 (122.16)
128.96 (128.77)
129.55 (129.52)
56.92 (55.99)
107.50
117.50
129.91
155.70
144.50
150.28
123.62
128.05
129.01
56.10
113.86
124.90
124.90
145.21
152.97
122.30
128.90
129.66
129.45
56.86
56.82
1J(PC)s146.2
62.87
1J(PC)s146.2
1J(PC)s139.3
66.47
1J(PC)s147.1
67.46
OCH2
66.64 (66.99; 67.10) c
2J(POC)s7.3 (7.1)
13.60 (13.39; 13.43) c
2J(POC)s7.1
16.42
2J(POC)s6.0
13.77
CH3
13.56
32.31
18.50
3J(POCC)s6.2
OCH2CH2CH2CH3
OCH2CH2CH2CH3
32.61 (32.19; 32.40) c
3J(POCC)s6.2 (5.5)
18.69 (18.37; 18.52) c
32.73
3J(POCC)s6.9
18.74
a Spectra recorded in CDCl3 (d in ppm, coupling constants in Hz).
b Data for dibutyl [a-(4-benzeneazoanilino]-N-benzyl]phosphonate from Ref. [43] are given in parentheses for comparison.
c Values of dibutyl ester are related to two diastereotopic butyl groups.
Furthermore, it is known that palladium asa softmetalprefers
nitrogen bonding over that of oxygen and therefore the coor-
dination of oxygen is more easily displaced. On the other
hand, it is worth noting that our recently reported investiga-
tions of the coordination of monoethyl 2-quinolylmethyl-
phosphonate to the palladium(II) ion have shown that this
monophosphonate ligand forms a very stable PdL2 complex
with a six-membered [N,O] chelate ring via the quinoline
nitrogen and the phosphonic acid oxygen [14], in spite of
the general belief that the six-membered ring is lessfavoured.
The differences observed in stability between the six-mem-
bered [N,O] ring in the quinoline complex and a five-mem-
bered ring in the benzeneazoanilino phosphonate complex
could be ascribed on one side to the relatively low basicity
of the benzalaniline nitrogen compared with that of the quin-
oline nitrogen, caused by participation of its electron pair in
resonance with the adjacent azobenzene p-system, and, on
the other side, to the influence of steric factors. The benze-
neazoaniline ligands are more bulky than the quinoline deriv-
ative. 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,
the final geometry, and the stability of the metallic species.
2.5. In vitro antitumour activity
The antitumour activity of the complexes was determined
in vitro on the KB cell line derived from a human oral epi-
dermoid carcinoma as a preliminary screening for their bio-
logical activity. The results obtained have shown that the
concentration of the complex required to inhibit tumour cell
growth by 50% was higher than 10y5 mol ly1. There are no
significant differences in response between complexes of
ethyl and butyl esters, nor between the two types of com-
plexes in spite of their differential structural properties and
most probably different DNA binding and nicking ability.
When comparing results of the in vitro assays related to the
cyclopalladated complexes 1 and 3 with those obtained for
the corresponding palladated azobenzene complexes of
dialkyl phosphonates [13], it could be seen that diester com-
plexes show about ten times higher activity (-5=10y6 mol
ly1) than the monoester complexes which may be ascribed
to their greater lipophilicity and solubility. In this regard it is
worth noting that similar effects were observed for the anti-
tumour activity of palladium(II) complexes with diethyl and
monoethyl esters of quinolylmethylphosphonic acids
[15,16,21]. The lipophilicity of the complexesincreaseswith
increasing bulkiness from monoester to diester derivatives
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945
Fig. 4. 1H–1H COSY spectrum of HL1 in CDCl3.
and may facilitate transport across the cellular membrane
[45,46].
Fig. 5. Aromatic part of 1H NMR spectra: (a) HL2 in CDCL3, (b) HL2 in
DMSO-d6, (c) 3 in CDCl3 and (d) 4 in CDCl3.
3. Experimental
night under vacuum. Yield 0.156 g (56%). Anal. Found: C,
47.13; H, 4.17; N, 8.09; Pd, 19.11. Calc. for C42H42N6-
O6Pd2P2Cl2: C, 47.03; H, 3.95; N, 7.84; Pd, 19.84%. IR:
n(C_C), d(N–H), 1576vs, 1554sh; n(P_O), 1237w,
1198w-m; n(PO–C), n(P–OH), 1031s, 997m; n(Pd–Cl),
307w, 255vw.
3.1. Materials
Monoethyl and dibutyl [a-(4-benzeneazoanilino)-N-ben-
zyl]phosphonates (HL1 and HL2) and their sodium salts
(NaL1 and NaL2), prepared as previously described [47],
were purified by recrystallization from absolute ethanol prior
to use. All other reagents and solvents were analytical grade
products and were used without purification.
3.2.2. Pd(L1)2 (2)
A concentrated aqueous solution of K2[PdCl4] (0.081 g,
0.25 mmol) was added dropwise to a stirred solution ofNaL1
(0.203 g, 0.49 mmol) in water (10 ml). The reactionmixture
was stirred continuously for 3 h at room temperature. The red
precipitate formed was filtered off, washed with cold water
and dried under vacuum over P2O5 for 24 h. Yield 0.214 g
(52%). Anal. Found: C, 56.41; H, 4.97; N, 9.37; Pd, 12.41.
Calc. for C42H42N6O6PdP2: C, 56.35; H, 4.73; N, 9.40; Pd,
11.89%. IR:n(C_C), d(N–H), 1601vs, 1583vs;nas(PO2y),
1224m, 1207m; nsym(PO2y), n(PO–C), 1070m, 1046s.
3.2. Preparation of complexes
3.2.1. [Pd(L1)Cl]2 (1)
The reaction of HL1 (0.205 g, 0.52 mmol) and
Na2[PdCl4] (0.160 g, 0.54 mmol) in methanol (10 ml) at
room temperature gave after stirring for 5 days a precipitate,
which was separated, washed with cold methanol, and dried
under vacuum. The dried complex was dissolved in hot chlo-
roform (5 ml), and after filtering to remove some black
decomposition products, the solution was evaporated to dry-
ness yielding a dark red glassy solid which was stored over-
3.2.3. [Pd(L2)Cl]2 (3)
This complex was prepared in a similar manner ascomplex
1 by stirring the reaction mixture of HL2 (0.204 g, 0.48
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Lj. Tusˇek-Bozˇic´ et al. / Polyhedron 19 (2000) 937–948
Fig. 6. Aromatic part of the long-range 1H–13C HMBC spectrum of NaL2.
3.2.4. Pd(L2)2 (4)
This complex was prepared by almost the same procedure
as complex 2 by reaction of sodium salt of L2 (0.206 g, 0.46
mmol) and K2[PdCl4] (0.081 g, 0.25 mmol) in water (5
ml). Yield 0.175 g (80%). Anal. Found: C, 57.96; H, 5.39;
N, 8.96; Pd, 11.40. Calc. for C46H50N6O6PdP2: C, 58.08; H,
5.30; N, 8.84; Pd, 11.19%. IR: n(C_C), d(N–H), 1598vs,
1589vs; nas(PO2y), 1205s br; nsym(PO2y), n(PO–C),
1063s, 1025s.
3.3. Physical measurements and analyses
Melting points were determined on a hot stage microscope
and are uncorrected. Infrared spectra were recorded on a
Perkin Elmer 580 B spectrophotometer using KBr (4000–
250 cmy1) and polyethylene (400–200 cmy1) pellets.
The electrospray mass spectrometric measurements were
performed on a Navigator (Finnigan, San Jose, CA) instru-
ment equipped with ESI ion source. The ligands were
recorded in positive and negative ion modes using methanol
or a mixture of acetonitrile and water (50:50) with 0.1%
acetic acid as the solvent and the mobile phase. Complexes
were recorded in methanol in both modes. The sample solu-
tion (10 ml of 10y5 mol ly1) was introduced into the ion
source, at a flow rate of 0.3 ml miny1, using a Rheodyne
external loop injector coupled to a Thermo Separation (San
Jose, CA) spectra series HPLC pump. The measurements
were performed using nitrogen as nebulizing gas, and using
different cone voltages (30, 50 and 120 V). Expected natural
abundance isotope clusters patterns for various ion clusters
were calculated with the Isotopic Distribution Program
Fig. 7. 31P NMR spectra of complexes 3 (a) and 4 (b).
mmol) and Na2[PdCl4] (0.145 g, 0.49 mmol) in methanol
(5 ml) for 3 days. Yield 0.114 g (42%). Anal. Found: C,
48.65; H, 4.49; N, 7.17; Pd, 18.87. Calc. for C46H50N6-
O6Pd2P2Cl2: C, 48.95; H, 4.47; N, 7.45; Pd, 18.86%. IR:
n(C_C), d(N–H), 1576vs, 1555sh; n(P_O), 1235w,
1198m; n(PO–C), n(P–OH), 1023m-s, 997m-s; n(Pd–Cl),
306w, 255vw.
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Lj. Tusˇek-Bozˇic´ et al. / Polyhedron 19 (2000) 937–948
947
(IDS) contained in the FTMS 7.01 application program of
the NICOS operating system using polynomial expansion
based on natural abundances of the Pd and Cl isotopes.
our cell growth showed 50% inhibition (IC50), and was cal-
culated by linear regression analysis [52]. The activity
threshold was set at 10y5 mol ly1, since this appears to be a
fairly realistic cutoff point for most compounds [53].
1
The H and 13C one- and two-dimensional NMR spectra
were performed with Varian broadband Gemini 300 and Bru-
ker AMX 360 spectrometers, operating at 75.46 and 90.56
MHz for the 13C resonance, respectively. Spectra were
recorded in CDCl3 or DMSO-d6 containing SiMe4 asaninter-
nal standard. Digital resolution in 1H NMR spectra was 0.25
Hz, while in 13C NMR it was 0.60 Hz per point. The narrower
spectral regions of special interest were measured also with
a smaller spectral width and greater digital resolution (down
to 0.2 Hz). The following techniqueswereused:proton-noise
decoupling, attached proton test (APT), 1H–1H COSY,
NOESY and 1H–13C COSY, long-range 1H–1H COSY,
HMQC and HMBC. The 1H–1H COSY spectra wereobtained
in the magnitude mode, while NOESY spectra were obtained
in the phase-sensitive mode. All two-dimensional experi-
ments were performed by standard pulse sequences, using
Gemini Data System software Version 6.3 Revision A (for
VXR-4000) and Bruker microprograms of UXNMR soft-
ware Version 940501.3 [48]. For proton decoupling Waltz-
16 modulation was used. 31P NMR spectra were measured at
145.79 MHz on a Bruker AMX 360 spectrometer in CDCl3
with external 85% H3PO4 in a coaxial capillary.
Acknowledgements
Financial support granted by the Ministry of Science,
Technology and Informaticsof theRepublicofCroatia(grant
No. 009806-07) is gratefully acknowledged. The authors
ˇ
´
would like to thank Mr Z. Marinic and B. Metelko (RuKer
ˇ
´
Boskovic Institute) forsome oftheNMRspectra. A.L.thanks
the Grant Agency of the Czech Republic (grant No. 104-99-
0835) for financial support.
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