Tuning the coordination properties of phenothiazine by regioselective introduction of diphenylphosphanyl groups

Article abstract of DOI:10.1039/c4dt02665a

The palladium and platinum complexes of the newly synthesized 1-(diphenylphosphino)-10-methyl-10H-phenothiazine (1) and the previously reported 3-(diphenylphosphino)-10-alkyl-10H-phenothiazine [alkyl = Me (2), Et (3)] and 4-(diphenylphosphino)-10-ethyl-10H-phenothiazine (4) were prepared. Density functional calculations were carried out to explain the electronic properties of compounds 1, 3 and 4. Compounds 1, 3 and 4 can interact with DNA, as was observed in agarose gel electrophoresis experiments. In addition, the cytotoxicity of the platinum complexes of ligands 2 and 4 towards breast, colorectal and hepatocarcinoma cell lines was studied. This journal is

Full text of DOI:10.1039/c4dt02665a

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Tuning the coordination properties of
phenothiazine by regioselective introduction
Cite this: DOI: 10.1039/c4dt02665a
of diphenylphosphanyl groups†
a,d
a
b
c
d
a,e
I. H. Filip, E. Gál, I. Lupan, M. Perde-Schrepler, P. Lönnecke, M. Surducan,
a
d
a
L. I. Găină, E. Hey-Hawkins and L. Silaghi-Dumitrescu*
The palladium and platinum complexes of the newly synthesized 1-(diphenylphosphino)-10-methyl-10H-
phenothiazine (1) and the previously reported 3-(diphenylphosphino)-10-alkyl-10H-phenothiazine [alkyl
=
Me (2), Et (3)] and 4-(diphenylphosphino)-10-ethyl-10H-phenothiazine (4) were prepared. Density
Received 1st September 2014,
Accepted 27th October 2014
functional calculations were carried out to explain the electronic properties of compounds 1, 3 and 4.
Compounds 1, 3 and 4 can interact with DNA, as was observed in agarose gel electrophoresis experi-
ments. In addition, the cytotoxicity of the platinum complexes of ligands 2 and 4 towards breast, colo-
rectal and hepatocarcinoma cell lines was studied.
DOI: 10.1039/c4dt02665a
www.rsc.org/dalton
properties of phenothiazine with the coordination ability of
the phosphanyl group towards transition metals already used
Introduction
Research in the field of bis-phosphine ligands based on in cancer therapy as a continuation of our search for new palla-
9
–11
heterocyclic aromatic backbones has evolved rapidly in the last dium and platinum compounds with antitumor activity.
few decades, mainly because of their applications in a great The first examples of phenothiazine-based ligands containing
1
variety of catalytic processes. Since the synthesis of Xantphos
phosphorus are the reaction products obtained by phosphoryl-
many similar ligands have been developed and successfully ation of 10-methyl-phenothiazine with phosphorus tribromide
2
,3
12
used in catalysis (e.g., hydroformylation
or asymmetric used for the preparation of phosphorus(V) ligands. We have
4
allylic alkylation ) due to their large bite angles. The electronic previously reported the preparation of some mono- and bis-
and steric properties of these ligands were tuned to study the (diphenylphosphino)phenothiazines: 4-(diphenylphosphino)-
effects on the geometry of the palladium(II) complexes and 10-alkyl-10H-phenothiazine (alkyl = methyl, ethyl, isopropyl),
5
their catalytic activity. There are only a few examples of mono- 2- and 3-(diphenylphosphino)-10-methyl-10H-phenothiazine,
6
–8
phosphines with a xanthene-type backbone,
found applications in telomerisation of buta-1,3-diene and 10H-phenothiazine.
methanol. Herein we report the coordination behaviour of a new
Phenothiazine ligands with diphenylphosphanyl groups are phenothiazinyl-phosphine regioisomer, 1-(diphenylphos-
good candidates for the preparation of transition metal com- phino)-10-methyl-10H-phenothiazine (1), and the previously
plexes not only with catalytic properties but also with biologi- reported 3-(diphenylphosphino)-10-alkyl-10H-phenothiazine
cal activity. Therefore, we decided to combine the well-known [alkyl = Me (2), Et (3)] and 4-(diphenylphosphino)-10-ethyl-
and they have bis-phosphines 3,7- and 4,6-bis(diphenylphosphino)-10-methyl-
1
3,14
1
3,14
10H-phenothiazine (4)
towards transition metals (Pd, Pt)
and the biological activity of 1, 3 and 4 and of transition metal
complexes 11, 14 and 15.
a
Babeş-Bolyai University, Faculty of Chemistry and Chemical Engineering,
RO-400028 Cluj-Napoca, Romania. E-mail: lusi@chem.ubbcluj.ro
b
Institute for Interdisciplinary Research in Bio-Nano Sciences, Babeş-Bolyai
University, RO-400271 Cluj-Napoca, Romania
c
Ion Chiricuţã Oncology Institute Cluj-Napoca, Radiobiology and Tumour Biology
Department, RO-400010 Cluj-Napoca, Romania
Results and discussion
d
Universität Leipzig, Institut für Anorgansiche Chemie, D-04103 Leipzig, Germany
e
National Institute for Research and Development of Isotopic and Molecular
The diphenylphosphino-phenothiazines 1–3 were prepared by
Technologies, RO-400293 Cluj-Napoca, Romania
lithiation of the corresponding bromo-10-alkyl-phenothiazine
†
Electronic supplementary information (ESI) available: DFT calculations, cyclic
n
with BuLi in diethyl ether at −78 °C (according to a slightly
voltammograms and interactions with DNA. CCDC 1019584–1019589. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c4dt02665a
1
5
modified procedure by Katritzky et al. ), followed by electro-
philic substitution with diphenylchlorophosphine. The lithia-
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Scheme 1 Synthesis of diphenylphosphino-phenothiazines 1–4.
t
tion of 10-ethylphenothiazine with BuLi in THF at 0 °C in the
Table 1 Absorption and emission data of compounds 1, 3–7, and
presence of TMEDA (TMEDA = N,N,N′,N′-tetramethylethylene- 10–15
diamine) and the reaction with Ph PCl afforded 4 (Scheme 1).
2
1
13
31
Absorption
Quantum Stokes
Compounds 1–4 were identified by H, C and P NMR
λmax.abs (nm)
Emission
yield
Φ (%)
shift
3
1
(cm−1)
spectroscopy and elemental analysis. The signals in the
NMR spectra are in the range of −6.9 to −13.2 ppm [−8.9 (1),
6.9 (2), −7.0 (3) and −13.2 ppm (4)]. The C–P coupling
P
Compound exptl./calcd
λmax.em (nm)
a
1
3
4
5
6
7
1
1
1
1
1
1
253/242; 313/292
262/255; 311/282, 289 458
464
0.5
4.6
0.5
5.2
4.1
0.9
4.8
3.5
10 397
10 320
10 857
12 048
11 514
8921
11 356
10 359
10 849
10 131
7816
−
a
1
2
3
a
constants ( JPC = 5.6–10.9 Hz;
JPC = 17.9–30.8 Hz;
J
PC
=
252/242; 305/296
254, 309, 565
256, 310, 571
228, 281, 356
227, 263, 335, 400
234, 268, 318
229, 263, 336, 404
227, 267, 318
228, 287, 417
229, 327
456
366
363
375
375
371
368
366
b
5
.7–8.9 Hz) have values similar to those of other phosphorus-
b
1
6
b
aryl containing compounds.
b
0
1
2
3
4
5
The electronic properties of phenothiazinyl-phosphines 1, 3
and 4 were investigated by absorption/emission UV/Vis spec-
troscopy and cyclic voltammetry. UV absorption and emission
data of dilute solutions are listed in Table 1.
The UV/Vis spectra of compounds 1, 3 and 4 exhibit two
absorption maxima around 253 and 310 nm, in accordance
b
b
2.8
2.9
0.3
b
b
370
No fluorescence
observed
a
b
Relative to perylene. Relative to naphthalene. The excitation wavelength
with the TD-DFT calculated UV/Vis spectra. A strong contribu- is italicized. The concentrations of the solutions used for fluorescence
−7
−7
−7
measurements were 5.1 × 10 M (1), 2.1 × 10 M (3), 9.2 × 10 M (4),
tor to the absorption band around 250 nm is a formal exci-
tation of the lone pair of electrons at the phosphorus atom
into the π* orbitals of the phenothiazine with some delocaliza-
tion onto the phenylphosphine substituents (ESI, Table S1 and
−10
−8
−5
−12
1
0
M (5–7, 10–13), 10 M (14) and 10 to 10
M (15). Quantum yields
were calculated according to ref. 17.
Fig. S1†). The absorption around 310 nm is mainly related to while in the S states of 1 and 4 this planarity appears to be
1
excitations of phenothiazine orbitals with little participation of hampered by steric repulsion between the nitrogen and phos-
the diphenylphosphanyl group and with important contri- phorus atoms in 1 and the sulfur and phosphorus atoms in 4;
butions from the heteroatoms into LUMOs dominated by the the N⋯P distance of 3.3 Å and the S⋯P distance of 3.3 Å are
rest of the π* system.
shorter than the sums of the van der Waals radii (3.4 and
1
8
The similarity of the UV/Vis spectra of 1, 3 and 4 is in con- 3.6 Å, respectively). These distortions from planarity are also
trast to the dramatic difference in fluorescence quantum coupled with a deviation from planarity of the substituted
yields. To provide more details on this issue, DFT calculations benzene unit in the phenothiazine, both of which thus offer
[
B3LYP/6-311G(2d,2p)] were performed on the ground states reasons for a clear difference in excited-state properties
and excited states of 1, 3 and 4. The resulting geometries are between isomer 3 on the one hand and isomers 1 and 4 on the
shown in Fig. 1. In the ground state, 1, 3 and 4 display typical other hand, including fluorescence: in the first excited singlet
phenothiazine geometries, folded along the N–S axis. The first states of 1 and 4, the aromatic π system is disrupted and the
excited singlet (S
1
) state of 3 features a planar phenothiazine, system is destabilised by the steric repulsion, so that these
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Table 2 Cyclic voltammetric data E (V) for 1, 3 and 4
Compound
E
1/2(1)
E
1/2(2)
E
1/2(3)
1
3
4
0.976
0.827
0.825
—
1.109
1.112
—
—
1.446
Fig. 1 Optimised geometries for 1, 3, and 4 and their first excited
singlet states (1*, 3*, and 4*), viewed along the N–S axis (N: blue,
S: yellow, P: orange).
excited states are expected to have a shorter lifetime as well as
supplementary mechanisms for dissipating energy, e.g. due to
the additional conformational space provided by the distortion
(hence the reduced availability for fluorescence).
Cyclic voltammetry (CV) was carried out in dichloro-
methane, with ferrocene/ferrocenium (Fc/Fc ) as the internal
standard, scanning in the anodic (up to 1.8 V) and cathodic
+
Scheme 2 Synthesis of transition metal complexes 7–15 (cod = 1,5-
cyclooctadiene; Ptz = phenothiazine).
(
up to −0.2 V) region. In the case of compounds 3 and 4, the
1
9
first oxidation potential is due to the phenothiazine moiety
to investigate the coordination chemistry of N-substituted phe-
nothiazines, palladium(II) complexes were prepared by reaction
of N-alkyl-Ptz (alkyl = methyl and ethyl; Ptz = phenothiazine)
and is quasireversible for both compounds, while the second
oxidation potential for compound 3 (1.109 V) and the second
and third oxidation potentials (1.112 V and 1.446 V, respectively)
for compound 4 are generated by phosphorus oxidation and
the processes are irreversible (ESI, Fig. S2†). Compound 1
shows only one oxidation potential (Table 2).
with [PdCl (cod)] (cod = 1,5-cyclooctadiene) in 2 : 1 molar ratio
2
(
Scheme 2). Complexes 5 and 6 were obtained as dark purple
solids. The formation of the two complexes was demonstrated
1
13
by X-ray diffraction measurements; the H and C NMR
spectra are similar to those of the starting materials. Complex
Transition metal complexes
26
6
has already been reported in the literature but its solid-
Phenothiazine, its N-alkyl derivatives and metal phenothiazine state structure was not confirmed.
complexes are known to be biologically active compounds.
Suitable crystals of 5 and 6 for X-ray diffraction measure-
Although many transition metal phenothiazine complexes ments were obtained from dichloromethane at room tempera-
have been synthesised and characterised, there are only a few ture in one week. The solid-state structures are presented in
2
0–25
reports of their crystal and molecular structures.
In order Fig. 2 (Table 3).
Fig. 2 Molecular structures of complexes 5 and 6. Thermal ellipsoids are drawn at 50% probability. All hydrogen atoms are omitted for clarity.
Selected bond lengths (Å) and angles (°) for 5: Pd(1)–Cl(1) 2.3083(6), Pd(1)–S(1) 2.3267(6), S(1)–C(7) 1.770(2), S(1)–C(1) 1.756(2), N(1)–C(2) 1.396(3),
N(1)–C(8) 1.395(3), N(1)–C(13) 1.452(3), Cl(1)–Pd(1)–S(1) 84.35(2), Cl(1’)–Pd(1)–S(1) 95.65(2), C(1)–S(1)–C(7) 99.00(1), Pd(1)–S(1)–C(7) 103.06 (8),
Pd(1)–S(1)–C(1) 111.26(8), C(2)–N(1)–C(8) 119.3(2), C(8)–N(1)–C(13) 119.2(2); for 6: Pd(1)–Cl(1) 2.2921(5), Pd(1)–S(1) 2.3338(5), S(1)–C(7) 1.749(2),
S(1)–C(1) 1.761(2), N(1)–C(2) 1.400(2), N(1)–C(8) 1.401(2), N(1)–C(13) 1.472(2), C(13)–C(14) 1.528(3), Cl(1)–Pd(1)–S(1) 95.36(2), Cl(1’)–Pd(1)–S(1)
84.64(2), C(1)–S(1)–C(7) 98.32(9), Pd(1)–S(1)–C(7) 110.19(7), Pd(1)–S(1)–C(1) 106.76(7), C(2)–N(1)–C(8) 120.4(2), C(8)–N(1)–C(13) 118.1(2).
This journal is © The Royal Society of Chemistry 2014
Dalton Trans.

Table 3 Summary of data collection, structure solution and refinement details for 5–7, 10, 14 and 15
Compound
5
6
7
10
14
15
Formula
Formula weight
Temperature [K]
Crystal system
Space group
Unit cell
a [Å]
b [Å]
c [Å]
α [°]
β [°]
C
26
H
22Cl
2
2
N PdS
2
C
28
H
26Cl
2
N
2
PdS
2
C
25
H
20Cl
2
NPPdS·CH Cl
2 2
C
50
H
40Cl
2 2
N P
2
PdS
2
C
26
H
22Cl
2
NPPdS
26 2
C H22Cl NPPtS
677.47
293(2)
603.87
130(2)
Monoclinic
P2 /c
631.93
130(2)
Monoclinic
P2 /c
659.67
180(2)
Triclinic
Pˉ1
972.20
293(2)
Triclinic
Pˉ1
588.78
293(2)
Monoclinic
P2 /n
Monoclinic
P2 /n
1
1
1
1
10.1581(3)
8.0485(2)
15.1612(4)
90
109.362(3)
90
1169.44(6)
2
1.715
9.9826(3)
15.5847(4)
8.2241(2)
90
105.269(3)
90
1234.31(6)
2
1.700
10.4334(2)
11.1888(3)
13.1883(3)
78.881(2)
87.795(2)
62.256(2)
1334.66(6)
2
8.453(1)
10.8921(7)
19.511(1)
11.2482(7)
90
97.910(1)
90
2367.7(3)
4
1.652
10.8975(7)
19.615(1)
11.2702(8)
90
97.995(1)
90
2385.6(3)
4
1.886
10.438(1)
12.960(2)
97.823(2)
97.519(2)
97.248(2)
1110.9(3)
1
1.453
0.742
496
γ [°]
V [Å ]
3
Z
−3
ρcalcd. [g cm
]
1.641
1.251
660
−1
μ [mm
F(000)
]
1.220
608
1.160
640
1.181
1184.0
6.276
1312.0
Crystal size [mm]
0.15 × 0.15 × 0.02
2.848–30.507
8742
3575 (0.0406)
100.0%
0.3 × 0.15 × 0.02
2.881–30.508
14 390
3759 (0.0364)
99.9%
0.3 × 0.25 × 0.2
2.863–33.728
36 807
10 662 (0.0269)
99.8%
0.32 × 0.25 × 0.20
1.60–25.00
10 581
3890 (0.0332)
99.5%
0.32 × 0.20 × 0.18
2.09–25.02
22 472
4176 (0.0374)
100%
0.30 × 0.26 × 0.23
2.08–25.02
22 638
4203 (0.0442)
99.9%
ΘMin–ΘMax [°]
Collected reflections
Indep. reflections (Rint
Completeness to ΘMax
)
T
max./Tmin
1/0.99342
0/152
1.032
1/0.97637
0/161
1.069
1/0.98005
7/313
1.042
0.866/0.797
0/268
1.075
0.816/0.704
0/290
1.141
0.326/0.255
0/290
1.162
Restraints/parameters
GooF (F )
2
R
R
1
, wR
, wR
2
(I > 2σ(I))
(all data)
0.0353, 0.0689
0.0519, 0.0743
0.476/−0.748
0.0301, 0.0640
0.0376, 0.0669
0.626/−0.598
0.0344, 0.0830
0.0414, 0.0868
2.188/−1.471
0.0401, 0.0876
0.0475, 0.0906
0.498/−0.262
0.0364, 0.0783
0.0414, 0.0804
0.490/−0.384
0.0358, 0.0720
0.0409, 0.0739
1.263/−1.037
1
2
−
3
Residual electron density [e·Å
]

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The bond lengths and angles have similar values to those
2
0
observed in dichlorobis(phenothiazine-κS)palladium(II) (I),
that is they are not affected by alkylation of the nitrogen atom
[cf. Pd–S 2.3267(6) Å (in 5), 2.3338(5) Å (in 6), 2.3378(5) Å (in I);
Cl–Pd–S 84.35(2)° (in 5), 84.64(2)° (in 6), 84.05(2)° (in I)]. The
palladium atom has a slightly distorted square-planar geome-
try with the sulfur atoms in a trans arrangement. The dihedral
angle between the phenylene planes of the phenothiazine
moiety is 141.8° in 5 and 145.9° in 6.
The reaction of N-alkyl-phenothiazines (alkyl = methyl and
Fig. 3 Molecular structure of complex 7. Thermal ellipsoids are drawn
at 50% probability. Hydrogen atoms are omitted for clarity. Selected
bond lengths (Å) and angles (°): Pd(1)–N(1) 2.161(1), Pd(1)–P(1) 2.1829(5),
ethyl) with [PtCl (cod)] was attempted, but formation of the
2
corresponding platinum complexes could not be observed,
even upon increasing the temperature or the reaction time, Pd(1)–Cl(2) 2.2936(4), Pd(1)–Cl(1) 2.3925(5), N(1)–C(1) 1.473(2), N(1)–C(7)
.479(2), N(1)–C(13) 1.519(2), N(1)–Pd(1)–P(1) 86.18(4), P(1)–Pd(1)–Cl(2)
1
although the synthesis of a similar platinum(II) complex,
namely [PtCl (SPh ) ], has already been reported.
2 2 2
2
7
86.99(2), N(1)–Pd(1)–Cl(1) 94.68(4), P(1)–Pd(1)–Cl(1) 176.74(2), Cl(2)–
Pd(1)–Cl(1) 92.44(2), C(12)–P(1)–C(14) 105.52(9), C(14)–P(1)–C(20)
In order to study the coordination mode (monodentate,
bidentate, and preferred donor atom(s)) of the phosphanyl-
108.01(9), C(12)–P(1)–Pd(1) 102.51(7), C(14)–P(1)–Pd(1) 119.60(6), C(20)–
P(1)–Pd(1) 113.02(7), C(1)–N(1)–Pd(1) 117.70(1), C(7)–N(1)–Pd(1)
substituted phenothiazine ligands 1–4, reactions with 112.50(1), C(13)–N(1)–Pd(1) 98.90(1).
MCl (cod)] (M = Pd, Pt) in dichloromethane were performed
1 : 1 for 1 and 4, 2 : 1 (L : M) for 2 and 3, Scheme 2). In the
resulting metal(II) complexes 7–15, the signal of the phos-
[
2
(
28
complexes). The Pd–P and Pd–Cl bonds are shorter in the
3
1
1
phorus atom in the P{ H} NMR spectra is shifted to low field
chelate complex 7 (2.1829(5) Å and 2.2936(4) Å, respectively)
compared to the free ligand [1: −8.9 ppm, 7: 42.6 ppm,
: 14.8 ppm ( JPtP = 3808 Hz), 9: 14.9 ppm ( JPtP = 2714 Hz);
: −6.9 ppm, 10: 22.3 ppm, 11: 13.2 ppm ( JPtP = 3699 Hz);
: −7.0 ppm, 12: 22.1 ppm, 13: 13.0 ppm ( J
: −13.2 ppm, 14: 59.5 ppm, 15: 36.6 ppm ( JPtP = 3490 Hz)].
Depending on the position of the phosphanyl substituent
1-, 3-, or 4-position), different bonding and coordination
modes are observed. 1-PPh - (1) and 4-PPh -substituted phe-
29
compared to the same bonds in trans-[PdCl (PPh ) ]
2
3 2
1
1
8
2
3
4
(
[
2.346(2) Å and 2.379(4) Å, respectively) or in di-μ-chlorobis-
bis(triphenylphosphine)palladium(II)]bis(tetrafluoroborate)
1
30
1
= 3672 Hz);
PtP
(2.290(2) Å and 2.386(3) Å, respectively). The dihedral angle
1
between the phenylene rings is smaller in complex 7 (132.1°)
31
compared to 10-methylphenothiazine (143.7°), due to the
(
rigid structure obtained after chelation. Two platinum com-
plexes were obtained (8 and 9) by the 1 : 1 reaction between 1
and [PtCl (cod)] as a 1 : 1.4 mixture (8 : 9) in solution. As 8
2
2
2
nothiazine (4) prefer a bidentate P,N (1) or P,S (4) bonding
mode resulting in five-membered chelate rings, while 3-PPh2-
and 9 could not be separated, their structures were elucidated
on the basis of the NMR spectra of the mixture. Two signals
with the appropriate platinum satellites were observed in the
substituted phenothiazines (2, 3) act only as monodentate
phosphine ligands.
An example of the preferred bidentate P,N bonding mode of 31
1
1
P{ H} NMR spectrum at 14.8 ppm ( J
= 3808 Hz) and
PtP
1
-PPh
2
-substituted phenothiazine (1) is the dark orange
1
1
4.9 ppm ( J
= 2714 Hz). Based on the trans influence on
PtP
palladium(II) dichloride complex 7. The molecular structure of
compound 7 was determined by X-ray diffraction measure-
ments. A summary of data collection, structure solution and
32
platinum complexes and taking into account the linear corre-
lation between the Pt–P coupling constant and the Pt–P bond
33
length, the first signal was assigned to complex 8 (isostruc-
refinement details is presented in Table 3. Compound 7 crys-
tallises in the triclinic space group P ˉ1 with one molecule in
the asymmetric unit. The asymmetric unit also contains an
additional dichloromethane molecule.
tural to palladium complex 7) with a bidentate P,N ligand, and
the second signal was assigned to complex 9, in which the
ligand is coordinated only through the phosphorus atom
(
Fig. 4).
Compound 1 acts as a bidentate P,N ligand towards Pd in
complex 7 (Fig. 3). The palladium atom is situated in a slightly
distorted square-planar geometry (P–Pd–Cl(1) 176.74(2)° and
N–Pd–Cl(2) 171.27(4)°). The angles around the P atom indicate
a tetrahedral environment and are in the range of observed
2
8
bond angles for similar compounds. The Pd–N (2.161(1) Å)
and Pd–P (2.1829(5) Å) bond lengths are similar to the values
reported for other palladium complexes with bidentate P,N
ligands (i.e., Pd–N is 2.100(5) Å in diiodo-{2-[bis(2-methyl-
phenyl)phosphinomethyl]pyridine}palladium(II), 2.131(9) Å in
diiodo-{N-[(Z)-2-(diphenylphosphine)-1-phenylethylidene]-2,6-
diisopropylaniline}palladium(II); the Pd–P bond lengths are
2
.230(2) Å and 2.223(3) Å, respectively, in the aforementioned Fig. 4 Proposed structure for complex 9.
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trans arrangement, and exhibits a slightly distorted square-
planar geometry (P(1)–Pd(1)–Cl(1) 91.55(3)°, P(1′)–Pd(1)–Cl(1)
88.45(3)°). The P–Pd bond is shorter in complex 7 (2.1829(5) Å),
in which a five-membered ring is formed by bidentate coordi-
nation of 1 to the palladium atom, compared to 2.3233(8) Å
in 10, which is similar to 2.347(1) Å in bis[1,1′-bis(diphenyl-
3
5
phosphino)ferrocenyl]palladium dichloride.
The dihedral
angle between the phenylene planes in 10 (143.0°) is similar to
the dihedral angles observed in non-chelate complexes 5 and
6
, but larger than that in the chelate complex 7 (132.1°). The
phosphorus atoms are coordinated in a distorted tetrahedral
fashion in 10 (i.e., C(1)–P(1)–C(14) 105.6(1)°, C(1)–P(1)–Pd(1)
Fig. 5 ORTEP drawing of 10. Thermal ellipsoids are drawn at 50% prob-
114.0(1)°,
C(14)–P(1)–Pd(1)
110.3(1)°,
C(14)–P(1)–C(20)
ability. Hydrogen atoms are omitted for clarity. Selected bond lengths
(
2
Å) and angles (°): Pd(1)–P(1) 2.3233(8), Pd(1)–P(1’) 2.3233(8), Pd(1)–Cl(1) 103.6(2)°).
.2878(9), Pd(1)–Cl(1’) 2.2878(9), P(1)–C(1) 1.811(3), P(1)–C(14) 1.813(3),
Ligand 4 reacts with [PdCl
: 1 molar ratio to give complexes which could be isolated
2 2
(cod)] and [PtCl (cod)] in
P(1)–C(20) 1.825(3), S(1)–C(3) 1.759(3), S(1)–C(7) 1.759(3), N(1)–C(13)
1
1.464(4), N(1)–C(4) 1.404(4), N(1)–C(12) 1.414(4), P(1)–Pd(1)–P(1’)
80.0(3), P(1)–Pd(1)–Cl(1) 91.55(3), Cl(1)–Pd(1)–Cl(1’) 180.0, C(1)–P(1)–
from the dichloromethane solution.
1
Crystals of palladium(II) complex 14 (orange) and platinum(II)
C(14) 105.6(1), C(1)–P(1)–Pd(1) 114.0(1), C(14)–P(1)–Pd(1) 110.3(1), C(14)–
P(1)–C(20) 103.6(2), C(3)–S(1)–C(7) 98.9(2), C(4)–N(1)–C(12) 119.6(3), complex 15 (yellow) with the 4-PPh
2
-substituted phenothiazine
C(4)–N(1)–C(13) 117.0(3), P(1)–C(1)–C(2) 122.1(2).
4
suitable for X-ray diffraction measurements were obtained
from dichloromethane/diethyl ether at room temperature in
one week (Fig. 6).
Similar coupling constants were observed in the case of
4-PPh -substituted phenothiazine 4 acts as a bidentate
2
1
[
2 3 2
PtCl (PPh ) ] ( JPt–P = 3679 Hz for the cis isomer and 2630 for ligand coordinating via the phosphorus(III) and sulfur atoms to
3
4
the trans isomer). The ESI mass spectrum of compound 9 Pd or Pt forming a five-membered chelate ring. A distorted
+
shows a peak corresponding to the [M − Cl] fragment at m/z = square-planar environment is observed (P–M–Cl(2) 173.69(4)°
1
025.
for 14 and 174.69(6)° for 15, and S–M–Cl(1) 175.28(3)° for 14
and 177.55(6)° for 15). Complexes 14 and 15 are isostructural.
In palladium(II) and platinum(II) complexes, the two 3-PPh
2
-
substituted phenothiazines 2 and 3 act as monodentate The M–S (2.2575(8) Å for 14 and 2.239(1) Å for 15) and M–P
ligands and have a trans arrangement due to steric reasons. bonds (2.2236(9) Å for 14 and 2.215(1) Å for 15) and S–M–P
Dark orange crystals of palladium complex 10 were obtained bond angles (88.21(3)° for 14 and 88.71(5)° for 15) have
from dichloromethane/diethyl ether, and the molecular struc- comparable values to other five-membered P,S chelates like
2
16
ture was determined by X-ray crystallography (Fig. 5).
cis-[Pd{(1-P(Biph)-2-S-C H )-κ S,P} ]
(Biph = 1,1′-biphenyl-
6
4
2
2
16
In 10, the palladium atom is located on a crystallographic 2,2′-diyl), cis-[Pt{(1-P(Biph)-2-S-C H )-κ S,P} ] and [PtCl {(1-
inversion centre, is coordinated by two phosphine ligands in a SPh-8-PPh -Nap)-κ S,P}] (Nap = naphthalene-1,8-diyl) [M–S:
6
4
2
2
2
36
2
Fig. 6 Molecular structures of complexes 14 and 15. Thermal ellipsoids are drawn at 50% probability. Hydrogen atoms are omitted for clarity.
Selected bond lengths (Å) and angles (°) for 14: Pd(1)–Cl(1) 2.310(1), Pd(1)–P(1) 2.2236(9), Pd(1)–S(1) 2.2575(8), Pd(1)–Cl(2) 2.3552(9), S(1)–C(6)
1
1
.764(3), S(1)–C(7) 1.783(3), P(1)–Pd(1)–S(1) 88.21(3), P(1)–Pd(1)–Cl(1) 88.47(3), P(1)–Pd(1)–Cl(2) 173.69(4), Cl(1)–Pd(1)–Cl(2) 94.62(4), C(1)–P(1)–C(15)
07.4(2), C(1)–P(1)–Pd(1) 107.1(1), C(21)–P(1)–Pd(1) 114.9(1), C(15)–P(1)–C(21) 106.7(2), C(1)–P(1)–C(21) 105.0(2), C(6)–S(1)–Pd(1) 105.4(1), C(7)–S(1)–
Pd(1) 114.3(1), C(6)–S(1)–C(7) 96.15(1), C(1)–C(6)–S(1) 121.0(2); for 15: Pt(1)–Cl(1) 2.313(2), Pt(1)–P(1) 2.215(1), Pt(1)–S(1) 2.239(1), Pt(1)–Cl(2) 2.357(2),
S(1)–C(6) 1.767(5), S(1)–C(7) 1.791(5), P(1)–Pt(1)–S(1) 88.71(5), P(1)–Pt(1)–Cl(1) 90.10(5), P(1)–Pt(1)–Cl(2) 174.69(6), Cl(1)–Pt(1)–Cl(2) 91.94(6), C(1)–
P(1)–C(15) 107.4(3), C(1)–P(1)–Pt(1) 107.1(2), C(21)–P(1)–Pt(1) 115.4(2), C(15)–P(1)–C(21) 105.7(3), C(1)–P(1)–C(21) 105.5(2), C(6)–S(1)–Pt(1) 105.4(2),
C(7)–S(1)–Pt(1) 114.3(2), C(6)–S(1)–C(7) 95.7(2), C(1)–C(6)–S(1) 120.7(4).
Dalton Trans.
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2
.3110(5) Å, 2.3098(8) Å, 2.261(4) Å; M–P: 2.2567(4) Å, 2.2365(7) could be observed at 343 nm for complex 15. In the case of
Å, 2.221(4) Å; S–M–P: 87.37(2)°, 87.63(3)°, 90.5(2)°, respecti- complex 14, a clear distinction between the absorption
vely]. The Pd–P (2.2236(9) Å) bond length in complex 14 is maxima from 289 nm of complex 14 and 260 nm of DNA
similar to that observed in complex 7 (2.1829(5) Å), but cannot be made. The ability of complex 15 to displace
the deviation from square-planar geometry is smaller in the CT-DNA-bound ethidium bromide was investigated by fluore-
case of 7 [P(1)–Pd(1)–Cl(1) 176.74(2)° in 7, P(1)–Pd(1)–Cl(2) scence measurements (ESI, Fig. S5†). Only a small increase in
1
73.69(4)° in 14]. The phenothiazine unit is folded along the the ethidium bromide emission could be observed upon
N–S axis in the complexes in which five-membered rings are addition of the complexes to DNA-bound ethidium bromide so
formed through coordination. For example, in complex 10, in an intercalative binding of the studied complexes to DNA is
which two molecules of the ligand coordinate to the metal unlikely.
centre and there are no structural restraints, the N(1)–C(4)–
DNA melting experiments were carried out based on the
C(5) and S(1)–C(3)–C(2) bond angles are 122.7(3) and 118.4(2)°, intensity of the absorption maximum CT-DNA at 260 nm at
respectively, while in complex 7, in which the nitrogen atom different temperatures, in the absence and presence of com-
participates in chelation, the N(1)–C(7)–C(12) bond angle is plexes 14 and 15 (ESI, Fig. S6†). According to this, the Tm of
smaller (120.4(2)°) and the S(1)–C(8)–C(9) bond angle is larger free CT-DNA was 70.6 °C, while for DNA treated with com-
m
(119.4(2)°). However, in complex 14, in which P,S coordination plexes 14 and 15 (at a DNA–complex ratio of 1.5 : 1) T values
is observed, both the N(1)–C(5)–C(4) (124.1(3)°) and the S(1)– of 66.8 °C and 68.6 °C, respectively, were observed. The Tm of
C(6)–C(1) bond angles (121.0(2)°) are larger than in 10. The DNA treated with complexes 14 and 15 should increase com-
dihedral angle between the phenylene rings of the phenothi- pared to free DNA if intercalation would occur. The observed
azine moiety is 133.3° in 14 and 132.2° in 15, indicating a Tm values were not significantly different for the free DNA
similar deviation from planarity compared to complex 7 and the two tested complexes and none of the performed
(132.1°) and a smaller deviation compared to non-chelate experiments show intercalation of the tested complexes in
complex 10 (143.0°).
DNA.
Interactions with DNA
Chemotherapeutic anticancer drugs are known to interact with
DNA by intercalation or groove binding. Intercalation of
3
7
Cytotoxicity of complexes 11 and 15
molecules with DNA may lead to frameshift mutagenesis and Due to the proven antiproliferative effects of platinum(II) com-
3
8
chromosomal breakage.
DNA intercalators and groove- plexes against diverse tumour types, reports on new platinum
binding compounds have found clinical applications as anti- complexes and their cytotoxic activity are constantly
3
9
40–43
cancer and antibacterial agents. To evaluate the potential increasing.
activity of phenothiazines in this biological context, the inter- The cytotoxicity of platinum(II) complexes 11 and 15 was
action of phenothiazinyl-phosphines 1, 3 and 4 and metal assessed against breast, colorectal and hepatocarcinoma cell
complexes 14 and 15 with DNA was investigated by agarose gel lines. The platinum(II) complex 8 could not be obtained in
electrophoresis (ESI, Fig. S3†).
pure form, and complex 13 is similar to its methyl analogue
These experiments indicated that phenothiazinyl-phos- 11; therefore, these complexes were not tested. The viability of
phines 1 and 3 interact with DNA and change its physical pro- all three cell lines decreased proportionally with the increase
perties, as the modified DNA does not migrate during in the concentration of both compounds (Fig. 7). The concen-
electrophoresis and can be observed at the starting line, trations of compound 15 which reduced the viability by 50%
whereas there was no significant interaction for phenothiazi- (IC50) were 28.07 µM for the breast carcinoma (MCF7) cell line,
nyl-phosphine 4 at low concentrations. For the highest tested 94.22 µM for the hepatocarcinoma (HepG2) cell line, and 110
concentrations of all three compounds, the disappearance of µM for colorectal carcinoma (DLD1). For compound 11, the
some relaxed form of DNA and an agglomeration of DNA mole- toxicity against all three cell lines was similar, with no signifi-
cules that did not migrate and remained in the wells was cant differences: IC50 MCF7 = 46.75 µM; IC50 DLD1 = 51.58 µM;
observed, similar to other phenothiazine-containing com- IC50 HepG2 = 48.49 µM.
pounds, such as 2-amino-10-methyl-10H-phenothiazine and a
When compared with the two-way ANOVA test, the toxicity
of compound 15 was found to be significantly higher towards
1
9
phenothiazine analogue of Tröger’s base.
II
II
The Pd and Pt complexes of 4-PPh
2
-substituted phe- MCF7 cells compared to DLD1 and HepG2 (Table 4). Com-
nothiazine 4 are capable of binding DNA to generate molecular pound 11 had similar toxicities against all tumor cell lines
aggregates with different migration capacities in the electro- tested. When comparing the two compounds, the toxicity of
phoresis experiment compared to the free DNA.
compound 11 was significantly higher than that of compound
To evaluate the intercalation capacity of complexes 14 and 15 for the HepG2 and DLD1 (p < 0.001), while against the
5 in DNA, the electronic absorption spectral titration in the MCF7 compound 15 was more effective (p < 0.01). The IC50
1
presence of increasing amounts of CT-DNA was performed values for both compounds were comparable to those obtained
ESI, Fig. S4†). CT-DNA absorption increased during titration, for cisplatin and oxaliplatin for the MCF7 cell line (ESI,
(
but no other relevant hypochromic or hyperchromic effect Fig. S7†).
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Fig. 7 The cytotoxicity of platinum(II) complexes 11 and 15 against breast (MCF7), colorectal (DLD1) and hepatocarcinoma (HepG2) cell lines. The
IC50 values were calculated using a nonlinear regression and a four-parameter sigmoidal curve fit, each point representing the mean ± SEM from
three separate measurements.
Table 4 IC50 of complexes 11 and 15 against the tested cell lines
Experimental
IC50 (µM)
MCF7
HepG2
DLD1
General remarks
All manipulations were carried out by standard Schlenk and
110 ± 0.83 vacuum-line techniques under an atmosphere of dry argon
Complex 11
Complex 15
Cisplatin
46.75 ± 3.3
28.07 ± 0.24
25.18 ± 0.14
38.46 ± 0.12
48.49 ± 2.02
94.22 ± 1.2
13.09 ± 0.08
20.46 ± 0.21
51.58 ± 0.6
32.07 ± 0.54
51.41 ± 0.07
using anhydrous solvents. 1-Bromo-10-methyl-phenothia-
Oxaliplatin
44
45
45
zine,
3
10-methyl-phenothiazine,
10-ethyl-phenothiazine,
1
5
-bromo-10-methyl-phenothiazine,
nothiazine, [PdCl
3-bromo-10-ethyl-phe-
(cod)] were prepared
1
5
46
46
2
(cod)] and [PtCl
2
according to literature procedures. All other chemicals were
used as purchased. CT-DNA lyophilized powder was purchased
Conclusions
In the pursuit of new compounds with biological activity we from Sigma-Aldrich. THF was dried over sodium/benzophe-
combined the well-known properties of phenothiazine with none, distilled under an atmosphere of dry argon and stored
the coordination ability of the phosphorus towards transition over molecular sieves. CH
metals already used in cancer therapy. Reactions of phenothia- CaH , distilled and kept under argon. CDCl
zinyl-phosphine ligands, 1-(diphenylphosphino)-10-methyl- LiAlH , distilled and stored over molecular sieves. The infrared
0H-phenothiazine (1), 3-(diphenylphosphino)-10-alkyl-10H- spectra were recorded on a Bruker Vector 22 IR spectrometer
2
Cl
2
and TMEDA were refluxed over
2
3
was dried over
4
1
−
1
phenothiazine (alkyl = Me (2), Et (3)) and 4-(diphenylpho- scanning between 4000 and 400 cm using KBr pellets. The
sphino)-10-ethyl-10H-phenothiazine (4) with [MCl (cod)] (M =
Pd, Pt) afforded three coordination patterns for the three regio- 300 and Bruker Avance 600 spectrometers. H and C chemi-
isomers (1, 3 and 4), characterised by spectroscopy methods cal shifts are quoted in parts per million (ppm) at 600.13 MHz
and X-ray structure analysis: chelating with nitrogen and phos- or 300.11 MHz for H and at 150.90 MHz or 75.46 MHz for
1
13
31
H, C and P NMR spectra were recorded on Bruker Avance
2
1
13
1
13
C
3
1
phorus (the first example in phenothiazine chemistry), relative to tetramethylsilane (TMS). P NMR chemical shifts
2
[MCl
2
{(10-methyl-1-diphenylphosphino-phenothiazine)-κ N,P}] were determined relative to 85% H
3 4
PO as the external stan-
(
M: Pd, Pt), monodentate through phosphorus, [MCl {(10- dard (at 242.94 MHz or 121.49 MHz). J values are given in Hz.
2
alkyl-3-diphenylphosphino-phenothiazine)-κP}] (alkyl: Me, Et; EI-MS spectra were recorded on a GC-MS Shimadzu QP-2010
M: Pd, Pt) and chelating with sulfur and phosphorus, Plus instrument. ESI-MS spectra were recorded on an Orbitrap
2
[
(
MCl {(10-ethyl-4-diphenylphosphin-phenothiazinyl)-κ S,P}] Thermo mass spectrometer. A Perkin Elmer Lambda 35 UV/Vis
2
M: Pd, Pt). The biological activity of the ligands (1, 3 and 4) spectrometer with 1.0 cm quartz cells was used for absorbance
and the transition metal complexes 11 ([PtCl
2
(10-Me-3-PPh
2
-
studies. Fluorescence studies were conducted on a Perkin
Ptz) ]), 14 ([PdCl {(10-Et-4-PPh -Ptz)-κ S,P}]) and 15 ([PtCl {(10- Elmer LS 55 instrument. Thermal denaturation studies were
2
2
2
2
2
2
Et-4-PPh
2
-Ptz)-κ S,P}]) was also reported.
A
higher DNA carried out with a CECIL CE7450 spectrophotometer equipped
binding capacity was observed for ligand 3 compared to com- with a Peltier temperature-controlling programmer. Cyclic
pounds 1 and 4. Complexes 14 and 15 also interact with DNA, voltammograms were recorded on a, Gamry Instruments,
generating molecular aggregates. Compound 15 showed Reference 600 instrument, using a solution of 0.1 M tetrabutyl-
2 2
better cytotoxic activity against breast carcinoma, but had ammonium hexafluorophosphate in CH Cl as the supporting
a lesser effect on hepatocarcinoma and colorectal carcinoma electrolyte; starting potential: −2.0 V vs. SCE, scan rate v =
cell lines than compound 11. Theoretical calculations 50 mV s− . The reference electrode consisted of a silver wire
1
(DFT and TD-DFT) explained the better fluorescence quantum (diameter ∼1 mm, length ∼10 cm) coated with AgCl and
yield observed for ligand (3) compared to the other dipped into a 1 M KCl solution. A glassy carbon electrode
regioisomers.
(3 mm) was used as the working electrode and a Pt wire as the
Dalton Trans.
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counter electrode. Potentials are reported vs. ferrocenium/ removed in vacuo. The resulting yellow oil was purified by flash
+
ferrocene (FcH /FcH). The concentration of 1, 3 and 4 was chromatography on silica.
.1 × 10− M. The elemental analyses (C, H, N, S) were per-
2
1-(Diphenylphosphino)-10-methyl-10H-phenothiazine
(1).
1
formed on a Vario EL – Heraeus. The melting points were 1.43 g 1-bromo-10-methyl-phenothiazine (4.9 mmol), 6.93 ml
determined using an Electrothermal 9100 instrument and are n-butyllithium (11.09 mmol), and 0.9 ml chlorodiphenylphos-
uncorrected.
phine (11.09 mmol) were added into 8 ml THF. The resulting
yellow oil was purified by flash chromatography on silica with
n-heptane and triethylamine (0.1%) as eluents to afford the
product as a white solid. 1 (0.51 g, 26%). mp = 123–125 °C.
Crystallographic data collection and structural refinement
For compounds 5–7, the data were collected on a Gemini diffr-
actometer (Agilent Technologies) using MoKα radiation (λ =
Found: C 75.8, H 5.15, N 3.4, S 7.7. Calc. for C25
H20NPS:
C 75.5, H 5.1, N 3.5, S 8.1%. UV-vis: λ (CH CN)/nm 253 and
max
3
0
.71073 Å), and ω-scan rotation. Data reduction was performed
3
−1
−1
4
7
313sh (ε/dm mol cm 19 738). Fluorescence: λexc = 313 nm,
with CrysAlis Pro including the program SCALE3 ABSPACK
for empirical absorption correction. The structures were solved
by direct methods and the refinement was performed with
SHELXL-2013. With the exception of one disordered solvent
molecule in 7, all non-hydrogen atoms were refined with aniso-
tropic thermal parameters and hydrogen atoms were calcu-
lated on idealised positions. For compounds 10, 14 and 15,
the data were collected on a Bruker SMART APEX diffract-
ometer, using graphite-monochromated MoKα radiation (λ =
7
λ
em = 464 nm, Φ = 0.5% in CH
perylene solution in cyclohexane). IR (KBr, ˜ν max/cm
3045 (w), 2962 (w), 2861 (w), 1582 (w), 1472 (s), 1432 (s),
3
CN, c = 5.1 × 10− M (standard:
−
1
)
4
8
1398 (m), 1328 (m), 1260 (s), 1093 (s), 11 020 (s), 869 (w),
7
99 (s), 747 (s), 693 (s). δH (600.13 MHz, CDCl ) 3.44 (3 H, d,
3
5
4
3
J
J
PH 1, 11-H), 6.26 (1 H, dd, JHH 1, JHH 8, 9-H), 6.65 (1 H, ddd,
4
3
3
3
HH 1, JHH 8, JPH 4, 2-H), 6.89 (1 H, t, JHH 8, 7-H), 6.93 (1 H,
4
3
4
3
dd, J 1, J 8, 6-H), 6.97 (1 H, td, J 2, J 8, 8-H), 7.12
HH
HH
HH
HH
4
3
(
(
1 H, dd, JHH 2, JHH 8, 4-H), 7.28–7.34 (10 H, m, Ph), 7.43
1 H, td,
0
.71073 Å). For this purpose the crystals were attached on cryo-
4
3
P 3
JPH 2, JHH 8, 3-H). δ (242.94 MHz, CDCl ) −8.9.
loops to a glass fibre and data were collected at room tempera-
ture (293 K). The structures were refined with anisotropic
thermal parameters. The hydrogen atoms were refined with
a riding model and a mutual isotropic thermal parameter.
For structure solving and refinement the software package
δ (150.90 MHz, CDCl ) 43.2 (d, J 17, 11-C), 123.9 (9-C), 126.4
C
3
PC
3
(
(
(
7-C), 127.2, 127.6 (6-C, 8-C), 127.26 (d, JPC 8.9, 4a-C), 127.6
5a-C), 128.5 (d, JPC 6.6, 3-C), 128.6 (d, JPC 7.4, 14-C), 128.9
15-C), 132.3 (d, J 5.6, 1-C), 132.9 (d, J 18.1, 2-C), 133.2
(d, JPC 1.2, 4-C), 134.2 (d, JPC 21, 13-C), 137.0 (d, JPC 10.9,
3
3
1
2
PC
PC
4
2
1
4
8
SHELX-97 was used.
CCDC 1019584 (5), 1019585 (6),
2
12-C), 147.6 (9a-C), 148.26 (d,
JPC 17.9, 10a-C). EI-MS: m/z
1
019586 (7), 1019587 (10), 1019588 (14) and 1019589 (15)
+
397 [M] .
contain the supplementary crystallographic data for this
paper.
DFT calculations
Geometries for the ground state (S ) and the first excited
0
1
singlet state (S ) of compounds 1, 3 and 4 were obtained using
DFT and TD-DFT, respectively, with the B3LYP functional
together with the 6-311G(2d,2p) basis set as implemented in
4
9
Gaussian 09. Frequency calculations were used to confirm
the S0 and S1 geometries as local minima on the potential-
energy surface. UV/Vis spectra were simulated using TD-DFT
with a total number of 50 states.
3
-(Diphenylphosphino)-10-methyl-10H-phenothiazine
(2).
1
.43 g 3-bromo-10-methyl-phenothiazine (4.9 mmol), 6.93 ml
n-butyllithium (11.09 mmol), and 0.9 ml chlorodiphenylphos-
phine (11.09 mmol) were added into 8 ml THF. The resulting
yellow oil was purified by flash chromatography on silica with
toluene–n-heptane = 2 : 3 and triethylamine (0.1%) as eluents
to afford the product as a pale yellow solid. 2 (0.60 g, 31%). mp
141–142 °C (lit., 112.5 °C). Found: C 75.7, H 5.2, N 3.4,
General procedure for the synthesis of 1-(diphenylphosphino)-
1
1
1
0-methyl-10H-phenothiazine (1), 3-(diphenylphosphino)-
0-methyl-10H-phenothiazine (2) and 3-(diphenylphosphino)-
0-ethyl-10H-phenothiazine (3)
18
=
S 8.2. Calc. for C25H20NPS: C 75.5, H 5.1, N 3.5, S 8.1%.
−
1
The starting material (1-bromo-10-methyl-10H-phenothiazine IR (KBr, ˜ν max/cm ) 3052 (w), 3006 (w), 2963 (w), 2881 (w),
for 1, 3-bromo-10-methyl-10H-phenothiazine for and 2817 (w), 1590 (w), 1563 (w), 1457 (vs), 1428 (w), 1370 (m),
-bromo-10-ethyl-10H-phenothiazine for 3) was dissolved in 1329 (m), 1289 (w), 1256 (m), 1138 (w), 1093 (w), 1020 (m),
dry THF and cooled to −78 °C, and then n-butyllithium was 997 (w), 814 (m), 745 (vs), 695 (s). δH (600.13 MHz, CDCl )
2
3
3
3
3
added dropwise. After 2 h of stirring at low temperature, a THF 3.37 (3 H, s, 11-H), 6.79 (1 H, d, JHH 8, 9-H), 6.82 (1 H, d, JHH
solution of chlorodiphenylphosphine was added dropwise and 8, 1-H), 6.94 (1 H, t, JHH 8, 7-H), 7.08 (1 H, dd, JHH 7, JHH 2,
the solution was stirred overnight. The reaction mixture was 6-H), 7.11 (1 H, dd, J 8, J 1, 8-H), 7.14–7.19 (2 H, m, 2-H,
3
3
4
3
4
HH
HH
diluted with 100 ml of dichloromethane and washed with 4-H), 7.28–7.31 (4 H, m, Ph), 7.32–7.34 (6 H, m, Ph). δ
water (3 × 100 ml). The organic phase was dried over an- (242.94 MHz, CDCl ) −6.9. δ (150.90 MHz, CDCl ) 35.5 (11-C),
hydrous sodium sulfate, filtered and then the solvent was 114.3 (d, J 8.4, 1-C), 114.3 (9-C), 122.9 (7-C), 123.3 (5a-C),
P
3
C
3
3
PC
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3
3
1
6
2
1
23.9 (d, JPC 7.7, 4a-C), 127.4, 127.7 (6-C, 8-C), 128.6 (d, JPC After stirring for 16 h at room temperature, the reaction
1
2
.8, 14-C), 128.8 (15-C), 130.1 (d, J 10.7, 3-C), 132.4 (d, J
mixture was diluted with 100 ml dichloromethane and washed
PC 22.9, 4-C), with water (3 × 100 ml). The organic phase was dried over
JPC 10.5, 12-C), 145.5 (10a-C), 146.7 (9a-C). EI-MS: anhydrous sodium sulfate, filtered and then the solvent was
PC
PC
2
2
0, 2-C), 133.6 (d,
JPC 19.2, 13-C), 133.8 (d, J
1
37.5 (d,
+
m/z 397 [M] .
removed in vacuo. The resulting yellow oil was purified by flash
chromatography on silica with toluene–n-heptane = 1 : 2 and
triethylamine (0.1%) as eluents to afford the product as a light
yellow crystalline powder. 4 (0.33 g, 18%). mp = 143–145 °C
1
9
(
lit., 151–152 °C). Found: C 75.7, H 5.5, N 3.4, S 7.6. Calc. for
22NPS: C 75.9, H 5.4, N 3.4, S 7.8%. UV-vis: λmax (CH CN)/
nm 252 and 305sh (ε/dm mol cm 19 935). Fluorescence:
exc = 305 nm, λem = 456 nm, Φ = 0.5% in CH CN, c = 9.2 ×
0− M (standard: perylene solution in cyclohexane). IR (KBr,
C
26
H
3
3
−1
−1
λ
3
7
1
−
1
3
-(Diphenylphosphino)-10-ethyl-10H-phenothiazine (3). 1.5 g ˜ν max/cm ) 3075 (vw), 3042 (vw), 2990 (w), 2937 (vw), 2793 (vw),
-bromo-10-ethyl-phenothiazine (4.9 mmol), 6.93 ml n-butyl- 1586 (w), 1554 (w), 1475 (m), 1442 (s), 1401 (w), 1384 (w),
lithium (11.09 mmol), and 0.9 ml chlorodiphenylphosphine 1322 (w), 1285 (w), 1245 (m), 1195 (vw), 1158 (vw), 1135 (w),
11.09 mmol) were added into 8 ml THF. The resulting yellow 1097 (w), 780 (w), 759 (m), 722 (w), 699 (m), 563 (vw). δ
3
(
H
3
3
oil was purified by flash chromatography on silica with (300.11 MHz, CDCl ) 1.43 (3 H, t, JHH 7, 12-H), 3.94 (2 H, q,
3
3
3
toluene–n-heptane = 2 : 3 and triethylamine (0.1%) as eluents
to afford the product as a yellow solid. 3 (0.78 g, 39%). mp = m, 1-H, 7-H, 9-H), 7.02–7.16 (3 H, m, 2-H, 6-H, 8-H), 7.32–7.35
68–169 °C. Found: C 75.5, H 5.5, N 3.7, S 7.6. Calc. for (10 H, m, Ph). δ (121.49 MHz, CDCl ) −13.2. δ (75.46 MHz,
22NPS: C 75.9, H 5.4, N 3.4, S 7.8%. UV-vis: λmax (CH
CN)/ CDCl ) 13.2 (12-C), 42.1 (11-C), 115.1, 115.5 (1-C, 9-C), 122.5
nm 262 and 311sh (ε/dm mol cm 33 570). Fluorescence: (7-C), 124.7 (d, JPC 6.6, 4-C), 126.8 (6-C, 8-C), 127.4 (3-C), 127.8
JHH 7, 11-H), 6.39 (1 H, dd, JHH 7, JPH 4, 3-H), 6.85–6.90 (3 H,
1
C
P
3
C
26
H
3
3
3
−1
−1
1
3
2
λexc = 311 nm, λem = 458 nm, Φ = 4.6% in CH CN, c = 2.1 × (2-C), 128.7 (d, JPC 7.2, 15-C), 128.9 (16-C), 130.3 (d, JPC 30.8,
3
0− M (standard: perylene solution in cyclohexane). IR (KBr, 4a-C), 134.1 (d,
7
2
JPC 19.9, 14-C), 136.0 (5a-C), 136.2 (d, J
1
1
˜
ν
PC
−
1
3
max/cm ) 3068 (w), 3050 (w), 2974 (w), 2930 (w), 2851 (w),
590 (w), 1565 (m), 1460 (vs), 1433 (w), 1384 (m), 1357 (w), 411 [M] .
10.2, 13-C), 145.2 (9a-C), 145.6 (d, J 5.7, 10a-C). EI-MS: m/z
PC
+
1
1
9
δ
326 (m), 1251 (w), 1234 (m), 1160 (w), 1134 (w), 1107 (m),
97 (w), 890 (w), 812 (m), 745 (vs), 695 (s), 637 (vw), 598 (vw).
(300.11 MHz, CDCl ) 1.42 (3 H, t, J 7, 12-H), 3.92 (2 H, q,
3
H
3
HH
3
J
HH 7, 11-H), 6.82–6.93 (3 H, m, 1-H, 7-H, 9-H), 7.04–7.20 (4 H,
m, 2-H, 4-H, 6-H, 8-H), 7.28–7.35 (10 H, m, Ph).
δ
P
(
121.49 MHz, CDCl ) −7.0. δ (75.46 MHz, CDCl ) 13.0 (12-C),
3
C
3
3
4
1
1
1
1
2.0 (11-C), 115.1 (d, JPC 8.3, 1-C), 115.3 (9-C), 122.7 (7-C),
3
24.1 (5a-C), 124.6 (d, JPC 7.5, 4a-C), 127.4, 127.5 (6-C, 8-C),
3
1
28.6 (d, J 6.9, 15-C), 128.8 (16-C), 129.9 (d, J 10.2, 3-C),
PC
PC
2
2
2
32.6 (d, JPC 19.9, 2-C), 133.6 (d, JPC 22.5, 4-C), 133.6 (d, JPC
1
General procedure for the synthesis of [PdCl
2
{(10-alkyl-
9.3, 14-C), 137.4 (d,
9a-C). EI-MS: m/z 411 [M] .
JPC 10.4, 13-C), 144.5 (10a-C), 145.8
+
phenothiazine)-κS}
]
2
(
A solution of [PdCl
2 2 2
(cod)] in CH Cl was added dropwise with
stirring at room temperature to a CH Cl solution of N-substi-
2
2
tuted phenothiazine. The reaction mixture turned dark purple
and was stirred for one additional hour. On standing at room
temperature crystals suitable for X-ray measurements were
obtained.
2 2
[PdCl (10-Me-Ptz) ] (5). 79 mg 10-H-10-methyl-phenothi-
azine (0.37 mmol), 53 mg [PdCl (cod)] (0.19 mmol). 5 (92 mg,
2
82%). mp = 257–258 °C. Found: C 51.9, H 3.8, N 4.55, S 10.8.
Synthesis of 4-(diphenylphosphino)-10-ethyl-10H-phenothi- Calc. for C26
2 2 2
H22Cl N PdS : C 51.7, H 3.7, N 4.6, S 10.6%. UV-
3
−1
−1
azine (4). To a mixture of 10-ethyl-phenothiazine (1 g, vis: λmax(CH Cl )/nm 254, 309 and 565 (ε/dm mol cm
2
2
4
.40
mmol)
and
N,N,N′,N′-tetramethylethylenediamine 29 970, 7957 and 3260). Fluorescence: λexc = 254 nm, λem
=
, c = 10− M (standard: naphtha-
10
(TMEDA) (1.68 ml, 11.01 mmol) in 30 ml of dry THF, t-butyl- 366 nm, Φ = 5.2% in CH
2
Cl
2
−1
lithium (5.91 ml, 11 mmol) was added dropwise at 0 °C. The lene solution in cyclohexane). IR (KBr, ˜ν max/cm ) 2881 (w),
reaction mixture was allowed to stir overnight at room temp- 2854 (w), 2806 (w), 1645 (m), 1599 (m), 1571 (s), 1484 (m), 1455 (s),
erature and then cooled to 0 °C. Then chlorodiphenylphos- 1339 (m), 1286 (m), 1256 (m), 1166 (w), 1136 (w), 1098 (w),
phine (2.07 ml, 11.01 mmol) in 5 ml THF was added dropwise. 1039 (w), 996 (w), 871 (m), 756 (s). δH (600.13 MHz, CDCl3)
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3
3
3
.38 (6 H, s, CH
3
), 6.82 (4 H, d, JHH 8, 1-H, 9-H), 6.93 (4 H, t, (1 H, d,
J
HH 8.4, 2-H). δ
P 3 C
(242.94 MHz, CDCl ) 42.6. δ
3
3
3
JHH 7, 3-H, 7-H), 7.14 (4 H, d, JHH 8, 4-H, 6-H), 7.17 (4 H, t, (150.90 MHz, CDCl ) 48.3 (11-C), 128.2 (d, JPC 12.5, 4a-C),
3
3
3
J
HH 8, 2-H, 8-H). δ
C 3 3
(150.90 MHz, CDCl ) 35.4 (CH ), 114.2 128.3 (9-C), 128.4 (d, JPC 8.6, 3-C), 128.5 (7-C), 128.8 (6-C, 8-C),
1-C, 9-C), 122.6 (3-C, 7-C), 123.5 (4a-C, 5a-C), 127.3 (2-C, 8-C), 128.9 (5a-C), 129.2 (d, JPC 12.4, 14-C), 129.4 (d, JPC 12.1, 14-
27.6 (4-C, 6-C), 146.0 (9a-C, 10a-C).
PdCl (10-Et-Ptz) ] (6). 84 mg 10-H-10-ethyl-phenothiazine
0.37 mmol), 53 mg [PdCl (cod)] (0.19 mmol). 6 (91 mg, 78%). 2-C), 133.8 (d, JPC 11.3, 13-C), 134.1 (d, JPC 11.5, 13-C), 143.1
mp = 245–247 °C. Found: C 53.1, H 4.2, N 4.6, S 10.3. Calc. for (9a-C), 151.4 (d, J 16.8, 10a-C).
3
3
(
1
1
C), 131.7 (15-C), 131.9 (d, J 2.6, 1-C), 132.0 (15-C), 132.6 (d,
JPC 2.3, 12-C), 132.7 (d, JPC 3.2, 12-C), 133.4 (d, JPC 9.7, 4-C,
PC
1
1
2
[
2
2
2
2
(
2
2
PC
C
28
H
26Cl
2
N
2
PdS
2
: C 53.2, H 4.15, N 4.4, S 10.15%. UV-vis: λmax
2 2 2
[PtCl (10-Me-1-PPh -Ptz)] (8) and (9). 49 mg [PtCl (cod)]
3
−1
−1
−1
(CH Cl )/nm 256, 310 and 571 (ε/dm mol cm 51 833, (0.13 mmol), 52 mg 1 (0.13 mmol). IR (KBr, ˜ν max/cm ) 3055
2 2
1
3 473 and 5607). Fluorescence: λ = 256 nm, λem = 363 nm, (w), 3004 (w), 2955 (w), 2870 (w), 1471 (m), 1431 (s), 1402 (s),
exc
−
10
Φ = 4.1% in CH
2
Cl
2
, c = 10
M (standard: naphthalene solu- 1333 (w), 1183 (w), 1134 (w), 1097 (s), 1029 (w), 1002 (w), 904
−
1
tion in cyclohexane). IR (KBr, ˜ν max/cm ) 3061 (w), 2987 (w), (w), 867 (w), 750 (s), 694 (s). δ
571 (s), 1486 (s), 1456 (s), 1369 (m), 1288 (m), 1243 (m), 1172 s, CH 9), 4.27 (3 H, s, CH 8), 6.28 (2 H, d, J 8, 9-H 9),
HH
H
(600.13 MHz, CDCl
3
) 3.32 (6 H,
3
1
3
3
3
(
(
w), 1140 (w), 1104 (w), 1041 (w), 932 (w), 893 (m), 749 (s). δ
600.13 MHz, CDCl ) 1.42 (6 H, t, JHH 7, CH ), 3.93 (4 H, br s, 6.95–6.99 (5 H, m, 6-H (or 8-H) 8; 6-H, 8-H 9), 7.06–7.10 (4 H,
H
6.81–6.83 (3 H, m, 9-H 8; 7-H 9), 6.92 (1 H, t, JHH 8, 7-H 8),
3
3
3
3
CH ), 6.88–6.92 (8 H, m, 1-H, 9-H, 3-H, 7-H), 7.13 (4 H, d, J
m, 3-H, 4-H 9), 7.13–7.17 (7 H in total, m; 8-H (or 6-H) 8, 3-H,
4-H 8 (3 H); Ph 9 (4 H)), 7.22–7.25 (4 H, m, 14-H 8), 7.35–7.50
2
HH
3
3
7
(
,
J
HH 8, 4-H, 6-H), 7.15 (4 H, t,
150.90 MHz, CDCl ) 13.0 (CH ), 41.7 (CH
22.2 (3-C, 7-C), 123.4 (4a-C, 5a-C), 127.2 (2-C, 8-C), 127.3 (4-C, 9), 7.77 (3 H, dd, J 13, J
JHH 8, 2-H, 8-H). δ
C
3
3
3
3
2
), 115.0 (1-C, 9-C), (15 H, m, Ph 9), 7.57 (2 H, t, JHH 7, 2-H 9), 7.67 (1 H, d, JHH 8,
3
3
3
1
6
7, Ph 8), 7.81 (3 H, dd, J 13,
PH
PH
HH
3
3
-C), 144.9 (9a-C, 10a-C).
P
JHH 7, Ph 8), 9.51 (1 H, d, JHH 8, 2-H 8). δ (242.94 MHz,
CDCl ) 14.8, JPtP = 3808 Hz for 8 and 14.9, JPtP = 2714 Hz
3
1
1
General procedure for the synthesis of complexes 7–15
A solution of [MCl (cod)] (M = Pd, Pt) in CH Cl was added
dropwise to a solution of the ligand in CH Cl at room temp-
for 9.
[MCl
2
2
{(10-methyl-3-diphenylphosphino-phenothiazine)-κP} ]
2
2
2
(
alkyl = methyl, ethyl; M = Pd, Pt).
2
2
erature. The reaction mixture was allowed to stir overnight,
and then the volume of the obtained solution was reduced to
1
/4 of the initial volume by removing the solvent in vacuo. Suit-
able crystals for X-ray measurements were obtained by slow
crystallisation from CH Cl /Et O.
MCl {(10-methyl-1-diphenylphosphino-phenothiazine)-κ N,
P}] (M = Pd, Pt).
2
2
2
2
[
2
[
PdCl
0.13 mmol), 103 mg 2 (0.26 mmol). 10 (115 mg, 91%). mp >
36 °C (decomp.). Found: C 61.9, H 4.1, N 2.8, S 6.7. Calc. for
PdS : C 61.8, H 4.15, N 2.9, S 6.6%. UV-vis: λmax
CH Cl )/nm 227, 263, 335 and 400 (ε/dm mol cm 58 690,
2
(10-Me-3-PPh
2
-Ptz)
2
]
(10). 37
mg
2
[PdCl (cod)]
(
2
C
(
50
H
40Cl
2
N
2
P
2
2
3
−1
−1
2
2
7
3
0 600, 22 360 and 9530). Fluorescence: λexc = 263 nm, λem =
2 2
75 nm, Φ = 4.8% in CH Cl
, c = 10− M (standard: naphtha-
10
−1
lene solution in cyclohexane). IR (KBr, ˜ν max/cm ) 2960 (w),
[
PdCl
2
(10-Me-1-PPh
2
-Ptz)]
(7). 37
mg
[PdCl
2
(cod)] 2871 (w), 1566 (w), 1462 (s), 1430 (m), 1386 (m), 1331 (m), 1253
(0.13 mmol), 52 mg 1 (0.13 mmol). 7 (60 mg, 80%). mp > (m), 1155 (w), 1104 (m), 998 (w), 929 (w), 879 (w), 810 (w), 745
2
07 °C (decomp.). Found: C 47.5, H 3.3, N 2.3, S 5.0. Calc. for (s), 698 (m). δH (600.13 MHz, CDCl ) 3.38 (6 H, s, 11-H),
3
C
25
H
20Cl
2
NPPdS·CH
Cl )/nm 228, 281 and 356 (ε/dm mol
89 600, 138 520 and 18 800). Fluorescence: λ = 281 nm, λem 6-H, 8-H, 14-H), 7.48 (4 H, br s, 15-H), 7.67–7.72 (8 H, m,
2 2
Cl : C 47.3, H 3.4, N 2.1, S 4.9%. UV-vis: 6.80–6.83 (4 H, m, 1-H, 9-H), 6.94–6.97 (2 H, m, 7-H), 7.10
3
−1
−1
λ
max (CH
2
2
cm
(2 H, br s, 4-H), 7.17–7.20 (2 H, m, 2-H), 7.36–7.42 (12 H, m,
2
=
exc
−
10
375 nm, Φ = 0.9% in CH
2
Cl
2
, c = 10
P 3 C 3
M (standard: naphtha- 13-H). δ (242.94 MHz, CDCl ) 22.3. δ (150.90 MHz, CDCl )
−1
lene solution in cyclohexane). IR (KBr, ˜ν max/cm ) 3053 (w), 35. 7 (11-C), 114.5 (2-C), 114.8 (1-C, 9-C), 123.1 (4a-C), 123.4
3
1
2
958 (w), 2921 (w), 2848 (w), 1515 (w), 1483 (s), 1459 (s), 1436 (7-C), 128.2 (t, J 5.1, 14-C), 128.6 (d, J 12.1, 12-C), 129.9
PC PC
1
(
(
4
s), 1408 (s), 1133 (w), 1101 (s), 1031 (w), 997 (w), 958 (w), 910 (5a-C), 130.6 (4-C), 131.1 (d, JPC 11.6, 3-C), 131.8 (15-C), 134.8
2
w), 787 (m), 751 (s), 721 (m), 692 (w). δ
H
(600.13 MHz, CDCl
3
)
(6-C, 8-C), 135.1 (t, JPC 6.2, 13-C), 147.9 (10a-C), 148.8 (9a-C).
[PtCl (10-Me-3-PPh -Ptz) ] (11). 49 mg PtCl (cod)
m, 14-H, 6-H, 7-H, 8-H), 7.49–7.52 (3 H, m, 14-H, 4-H), 7.60 (0.13 mmol), 103 mg 2 (0.26 mmol). 11 (115 mg, 83%). mp =
3
.05 (3 H, s, 11-H), 7.29 (1 H, d, J
HH
8, 9-H), 7.39–7.44 (5 H,
2
2
2
2
3
3
(
1 H, t, JHH 8, 3-H), 7.72 (2 H, t, JHH 8, 15-H), 7.77 (2 H, dd, 255–256 °C. Found: C 56.5, H 3.9, N 2.8, S 5.9. Calc. for
JHH 8, J 13, 13-H), 7.82 (2 H, dd, J 8, J 11, 13-H), 9.42 C H Cl N P PtS : C 56.6, H 3.8, N 2.6, S 6.0%. UV-vis: λ
3
3
3
3
PH
HH
PH
50 40
2
2
2
2
max
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3
(
5
CH
2
Cl
2
)/nm 234, 268 and 318 (ε/dm mol−1 cm−1 48 100, (m), 1155 (w), 1105 (m), 928 (w), 878 (w), 808 (m), 745 (m), 699
3
7 750 and 10 440). Fluorescence: λ = 268 nm, λem = 371 nm, (m). δH (600.13 MHz, CDCl ) 1.36 (6 H, t, J
7, 12-H), 3.84
HH
exc
3
−
10
3
3
Φ = 3.5% in CH
2
Cl
2
, c = 10
M (standard: naphthalene solu- (4 H, q, JHH 9, 11-H), 6.48 (2 H, d, JHH 8, 1-H), 6.84 (2 H, d,
−
1
3
3
3
tion in cyclohexane). IR (KBr, ˜ν max/cm ) 3051 (w), 2964 (w),
J
HH 8, 9-H), 6.91 (2 H, t, JHH 8, 7-H), 6.94 (2 H, dd, JHH 11,
4
3
3
1
1
6
d, J
7
7
594 (m), 1464 (s), 1434 (m), 1389 (m), 1360 (m), 1258 (m),
162 (w), 1105 (s), 1021 (m), 998 (w), 891 (w), 804 (m), 748 (m), m, 6-H, 8-H), 7.22 (8 H, t, JHH 7, 15-H), 7.34 (4 H, t, JHH 7,
) 3.28 (6 H, s, 11-H), 6.44 (2 H, 16-H), 7.56 (8 H, t,
8, 9-H), 6.91 (4 H, m, 4-H, CDCl ) 13.0, JPtP 3672. δC (150.90 MHz, CDCl ) 12.9 (12-C),
-H), 7.00 (2 H, d, JHH 8, 2-H), 7.16 (4 H, t, JHH 8, 6-H, 8-H), 42.1 (11-C), 100.3 (1-C), 113.9 (2-C), 115.4 (9-C), 123.2 (7-C),
JPH 2, 4-H), 6.98 (2 H, dd, J 8, J 2, 2-H), 7.13–7.17 (4 H,
HH PH
3
3
3
3
94 (m). δ
H
(600.13 MHz, CDCl
3
J
PH 2,
JHH 8, 14-H). δ
P
(242.94 MHz,
3
3
1
8, 1-H), 6.78 (2 H, d, J
HH
HH
3
3
3
3
3
3
3
.22 (8 H, t, JHH 7, 14-H), 7.35 (4 H, t, JHH 7, 15-H), 7.56 (8 H, 123.5 (4a-C), 123.7 (3-C), 127.6 (6-C, 8-C), 128.0 (t,
t, J 2, J 8, 13-H). δ (242.94 MHz, CDCl ) 13.2, J 3699. 15-C), 130.1 (5a-C), 130.9 (16-C), 132.9 (4-C), 134.7 (13-C), 134.9
JPC 5.7,
3
3
1
PH
HH
P
3
PtP
2
δ
C
(150.90 MHz, CDCl
3
) 35.5 (11-C), 100.3 (1-C), 113.2 (2-C), (t, JPC 5.1, 14-C), 143.7 (10a-C), 147.1 (9a-C).
[MCl {(10-ethyl-4-diphenylphosphin-phenothiazinyl)-κ S,P}]
6-C, 8-C), 128.0 (t, JPC 5.5, 14-C), 130.0 (5a-C), 131 (15-C), (M = Pd, Pt).
2
1
(
1
14.6 (9-C), 122.7 (4a-C), 123.1 (3-C), 123.3 (7-C), 127.3, 127.8
2
3
2
32.5 (4-C), 134.6 (12-C), 134.9 (t, JPC 5.1, 13-C), 144.7 (10a-C),
1
47.9 (9a-C).
[
MCl {(10-ethyl-3-diphenylphosphino-phenothiazine)-κP} ]
2
2
(alkyl = methyl, ethyl; M = Pd, Pt).
[
PdCl
0.13 mmol), 54 mg 4 (0.13 mmol). 14 (54 mg, 71%). mp >
10 °C (decomp.). Found C 52.85, H 3.8, N 2.5, S 5.5. Calc. for
26 2
C H22Cl NPSPd: C 53.0, H 3.8, N 2.4, S 5.45%. UV-vis: λmax
2
{(10-Et-4-PPh
2
-Ptz)-κS,P}] (14). 37 mg [PdCl
2
(cod)]
(
3
[
PdCl
2
(10-Et-3-PPh
2
-Ptz)
2
]
(12). 17
mg
[PdCl
2
(cod)]
3
−1
−1
(
0.06 mmol), 49 mg 3 (0.12 mmol). 12 (54 mg, 90%). mp > (CH Cl )/nm 228, 287 and 417 (ε/dm mol cm 26 450, 9565
2 2
2
C
60 °C (decomp.). Found: C 62.3, H 4.3, N 2.7, S 6.55. Calc. for and 1490). Fluorescence: λexc = 287 nm, λem = 370 nm, Φ =
H
44Cl
2
N
2
P
2
PdS
2
: C 62.4, H 4.4, N 2.8, S 6.4%. UV-vis: λmax 0.3% in CH
2
Cl
2
, c = 10− M (standard: naphthalene solution in
8
52
3
−1
−1
−1
(CH Cl )/nm 229, 263, 336 and 404 (ε/dm mol cm 51 850, cyclohexane). IR (KBr, ˜ν max/cm ) 3058 (m), 2981 (w), 2933 (w),
2 2
7
3
2 530, 23 890 and 9630). Fluorescence: λexc = 263 nm, λem
=
1593 (m), 1564 (m), 1478 (s), 1449 (m), 1413 (m), 1364 (m),
−
10
2 2
68 nm, Φ = 2.8% in CH Cl , c = 10
M (standard: naphtha- 1253 (s), 1165 (m), 1101 (w), 997 (w), 853 (s), 813 (m), 753 (m),
−1
3
lene solution in cyclohexane). IR (KBr, ˜ν max/cm ) 2872 (w), 695 (m). δH (600.13 MHz, CDCl ) 1.46 (3 H, t, J
7, 12-H),
3
HH
3
2
816 (w), 1644 (w), 1566 (m), 1463 (s), 1429 (m), 1387 (m), 1371 4.12 (2 H, t, JHH 7, 11-H), 7.12–7.15 (2 H, m, 1-H, 9-H), 7.23
3
3
(m), 1331 (m), 1253 (m), 1156 (w), 1104 (m), 878 (w), 808 (w), (1 H, t, JHH 8, 7-H), 7.27 (1 H, d, JHH 8, 6-H), 7.37–7.41 (3 H,
7
46 (m), 698 (m). δH (600.13 MHz, CDCl ) 1.42 (6 H, br s, 12- m, 8-H, 16-H), 7.48–7.51 (4 H, m, 15-H), 7.58 (1 H, br s, 2-H),
3
3
H), 3.93 (4 H, br s, 11-H), 6.87–6.93 (6 H, m, 1-H, 9-H, 7-H), 7.70 (2 H, br s, 14-H), 7.85 (2 H, br s, 14-H), 8.76 (1 H, d, JHH
.07 (2 H, br s, 4-H), 7.15–7.19 (4 H, m, 2-H, 6-H), 7.37–7.48 7, 3-H). δ (242.94 MHz, CDCl ) 59.5. δ (150.90 MHz, CDCl
10 H, m, 8-H, 15-H), 7.48 (2 H, br s, 16-H), 7.58 (2 H, br s, 16- 13.4 (12-C), 42.2 (11-C), 116.4 (1-C), 118.3 (2-C), 119.1 (9-C),
7
(
P
3
C
3
)
3
H), 7.68–7.71 (8 H, m, 14-H). δ
P
(242.94 MHz, CDCl
3
) 22.1. δ
C
125.3 (7-C), 127.6 (16-C), 128.3 (4-C), 129.2 (d, JPC 11.1, 15-C),
2
(150.90 MHz, CDCl
3
) 13.0 (12-C), 42.3 (11-C), 114.5 (2-C), 115.4 130.2 (8-C), 130.7 (6-C), 131.9 (d, JPC 8.4, 3-C), 132.6 (13-C),
3
2
(1-C), 115.6 (9-C), 122.9 (7-C), 123.3 (4a-C), 128.2 (t, J 5.4, 134.2 (d, J 11.3, 14-C), 141.8 (10a-C). Because of low solubi-
PC
PC
1
1
5-C), 128.6 (d, JPC 11.9, 13-C), 130.0 (5a-C), 130.6 (4-C), 131.0 lity, quaternary carbon atoms 4a-C, 5a-C and 9a-C could not be
1
(
d, JPC 11.9, 3-C), 131.8 (16-C), 134.7, 134.8 (6-C, 8-C), 135.0 (t, assigned.
2
JPC 6.3, 14-C), 147.2 (10a-C), 148.2 (9a-C).
PtCl (10-Et-3-PPh -Ptz) (13). 22
0.06 mmol), 49 mg 3 (0.12 mmol). 13 (56 mg, 86%). mp > 339 °C (decomp.). Found: C 46.2, H 3.4, N 2.3, S 4.9. Calc. for
[PtCl {(10-Et-4-PPh -Ptz)-κS,P}] (15). 50 mg [PtCl (cod)]
[PtCl (cod)] (0.14 mmol), 56 mg 4 (0.14 mmol). 15 (80 mg, 87%). mp >
2
2
2
2
[
2
2
2
]
mg
(
2
C
84 °C (decomp.). Found: C 57.5, H 4.0, N 2.7, S 5.8. Calc. for C H Cl NPSPt: C 46.1, H 3.3, N 2.1, S 4.7%. UV-vis: λ
26 22 2 max
3
−1
−1
52
H
44Cl
2
N
2
P
2
PtS
2
C 57.35, H 4.1, N 2.6, S 5.9%. UV-vis: λmax (CH
2
Cl
2
)/nm 229 and 327 (ε/dm mol
cm
33 610 and
3
−1
−1
−1
2 2
(CH Cl )/nm 227, 267 and 318 (ε/dm mol cm 53 390, 3375). IR (KBr, ˜ν max/cm ) 3056 (m), 2981 (w), 1593 (m), 1563
8
2 570 and 16 490). Fluorescence: λ = 267 nm, λem = 366 nm, (m), 1479 (s), 1449 (m), 1413 (m), 1364 (m), 1253 (s), 1164 (m),
exc
−
10
Φ = 2.9% in CH
2
Cl
2
, c = 10
H
M (standard: naphthalene solu- 1098 (m), 996 (w), 810 (m), 754 (s), 694 (m). δ (600.13 MHz,
−
1
3
3
3
tion in cyclohexane). IR (KBr, ˜ν max/cm ) 2963 (w), 2872 (w), CDCl ) 1.48 (3 H, t, JHH 7, 12-H), 4.12 (2 H, dd, JHH 7, 11-H),
3
2
817 (w), 1596 (w), 1565 (m), 1462 (s), 1383 (w), 1332 (m), 1253 7.13 (1 H, d, JHH 8, 1-H), 7.22–7.25 (3 H, m, 9-H, 6-H, 7-H),
Dalton Trans.
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7
.34–7.40 (3 H, m, 8-H, 16-H), 7.46–7.49 (4 H, m, 15-H), 7.55 Cytotoxic activity
3
3
3
(1 H, t, J
8, 2-H), 7.69 (2 H, dd, J
8, J 13, 14-H), 7.83
HH
HH
PH
Cell lines, culture. Three tumour cell lines were used to
evaluate the cytotoxic effect of the compounds: MCF7 (human
breast adenocarcinoma) and HepG2 (hepatocyte carcinoma)
cultured in Eagle’s MEM (minimum essential medium), and
DLD1 (colorectal adenocarcinoma) grown in RPMI 1640; all
media were supplemented with 10% foetal bovine serum, 1%
glutamine and antibiotics. All media were purchased from
Sigma. Cells seeded on 96-well plates were treated with
different concentrations of the compound for 24 h (each con-
centration in at least three wells). The concentrations were
3
3
2
(2 H, dd,
J
HH 8,
(242.94 MHz, CDCl
J
PH 13, 14-H), 8.63 (1 H, d,
JPH 8, 3-H).
1
δ
P
3 C
) 36.6, JPtP 3490. δ (150.90 MHz,
CDCl ) 13.4 (12-C), 42.0 (11-C), 116.4 (1-C), 117.7 (2-C), 119.2
3
(
(
9-C), 125.1 (7-C), 127.2 (3-C), 127.7 (4a-C), 128.2 (5a-C), 128.9
15-C), 129.0 (15-C), 129.1 (15-C), 129.9 (4-C), 130.1 (16-C),
1
1
30.2 (16-C), 131.9 (d, J 8.9, 13-C), 132.3, 132.4 (6-C, 8-C),
PC
3
1
34.1 (t, JPC 11.8, 14-C), 141.9 (10a-C), 145.6 (9a-C).
−
1
as follows: 162.5; 81.25; 40.62; 16.25; 6.5; 3.25; 1.63 µg ml
At least three wells were left untreated (control).
.
Interactions with DNA
For the DNA electrophoresis tests, different volumes of stock
solutions of compounds 1 (c = 10 M), 3 (c = 10 M) and 4
Determination of cell survival. The cytotoxic effect of the
treatments was assessed using the MTT assay. This method is
based on the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-
−
3
−3
−
4
(
c = 5 × 10 M) and of the transition metal complexes 14 (c =
−
4
−4
2
× 10 M) and 15 (c = 4 × 10 M) prepared in DMSO were
2
,5-diphenyl-2H-tetrazolium bromide] to a coloured compound
formazan) only by living cells with active mitochondrial
hydrogenase, which represents a part of the respiratory chain.
4 h after treatments, 100 µl of MTT (Sigma) was added and
mixed with 200 ng of plasmid DNA (pTZ57R, 2836 bp) at
different ratios. The plasmid DNA was purified from an over-
night culture of Escherichia coli cells using the Gene JET
Plasmid Miniprep Kit (Thermo Scientific). The mixtures were
incubated for 30 min at room temperature and loaded into an
agarose gel 1% in TAE (tris-acetate ethylenediaminetetraacetic
acid) buffer (40 mM tris-acetate, 1 mM EDTA (ethylenediami-
netetraacetic acid), pH 8.0) without ethidium bromide. After
migration, gels were stained for 30 min in a TAE buffer con-
(
2
cells were incubated for 1 h. After that interval, formazan crys-
tals were dissolved by adding 150 µl of DMSO. The absorbance
was measured with a Biotek Synergy 2 microplate reader at
570 nm. Statistical analysis was done and graphs were plotted
using Graph Pad Prism 5 software.
−
1
taining ethidium bromide (0.5 μg ml ), according to standard
procedures.
5
0
Electronic absorption titration. The absorption titration of Acknowledgements
complexes 14 and 15 in DMSO was performed at a fixed con-
The authors thank Prof. R. Silaghi-Dumitrescu for useful dis-
centration (14: 0.025 mM; 15: 0.040 mM) with increments of
−
1
cussions. Financial support from the DAAD (Stability Pact for
South-Eastern Europe, MatCatNet project ID 54759562) and
the Romanian Ministry of Education, Research Youth and
Sports (grant ID PCCE 140/2008) is gratefully acknowledged.
1
0 μL CT-DNA (stock solution 1.054 mg ml ). The experiment
was followed by UV-Vis with a Perkin Elmer Lambda 35
spectrophotometer.
Ethidium bromide fluorescence quenching. Competitive
binding studies were carried out with a Perkin Elmer LS 55
spectrophotometer. The emission spectra of a DMSO solution
of free ethidium bromide (0.25 × 10–5 M) and ethidium
bromide bound-DNA (Tris buffer stock solution 1.054 mg
Notes and references
−
1
ml ) were recorded. Constant increments (100 μL) of complex
1 S. Hillebrand, J. Bruckmann, C. Krüger and M. W. Haenel,
Tetrahedron Lett., 1995, 36, 75–78.
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5 were added to a mixture of 400 μL ethidium bromide and
1
00 μL DNA. The samples were irradiated at 535 nm and the
2 M. Kranenburg, Y. E. M. van der Burgt, P. C. J. Kamer and
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emission maxima (650 nm) corresponding to ethidium
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Thermal denaturation. Thermal denaturation studies were
carried out with a CECIL CE7450 spectrophotometer equipped
3 L. A. van der Veen, M. D. K. Boele, F. R. Bregman,
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a Peltier temperature-controlling programmer. The
thermal denaturation of CT-DNA was performed with com-
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complexes at 1.5 : 1 ratio (DNA–complex). The samples were
−
1
heated from 40 to 90 °C at a rate of 1 °C min measuring the
absorbance at 260 nm. The melting temperatures (T ) for the
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− A ) versus temperature, where A is the initial, A is the
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o
(
A
f
o
o
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f
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