An efficient process to directly convert 1-hydroxymethyl-3,5-dimethylpyrazole to Cd(ii) complexes via C-N bond creation: Cytotoxicity and factors controlling the structures

Article abstract of DOI:10.1039/c4ra07960d

We have demonstrated a simple process that involves one-pot, one-step reactions leading to efficient preparation of new cadmium complexes with N4-donating ligands [CdX₂L¹] (X = I^- (1), Br^- (2) L¹ = tris(1-(3,5-dimethylpyrazolyl)methyl)amine). The most prominent feature of the synthesis is the in situ formation of a new organic tripodal ligand (L¹) in a condensation reaction between a starting ligand (1-hydroxymethyl-3,5-dimethylpyrazole) and ammonia. A single-crystal X-ray analysis confirmed that the complexes obtained are monomers (1, 2) with octahedral geometry of the cadmium centres. Complex 3 has a cationic-anionic structure [Cu(LOH)₂CH₃OH][CdCl₄] and has been synthesised by the reaction of CdO and NH₄Cl in the presence of zerovalent copper (powder). IR, EPR, ¹H and ¹³C NMR, as well as simultaneous TG/DTG were carried out to characterise the products. Moreover, we try to compare the cytotoxic profile of CdO and cadmium salts with Cd(ii) complexes. Besides [CdX₂L¹] (1, 2) we take into consideration [Cd₂(L²)₂(SCN)₄(MeOH)₂]_n (4) and [Cd(SCN)₂L¹] (5) complexes. Biological studies demonstrated that Cd(ii) complexes with the N-scorpionate ligand (1, 2 and 5) have similar cytotoxicity, which points to a structure-cytotoxicity relationship. Thus, all the complexes (except 4) exhibited a lower cytotoxic activity compared to a cadmium ion in salts. This journal is

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An efficient process to directly convert 1-
hydroxymethyl-3,5-dimethylpyrazole to Cd(II)
complexes via C–N bond creation: cytotoxicity and
factors controlling the structures†
Cite this: RSC Adv., 2014, 4, 43962
a
a
b
Joanna Masternak, Barbara Barszcz,* Wanda Sawka-Dobrowolska,
c
b
c
Joanna Wietrzyk, Julia Jezierska and Magdalena Milczarek
We have demonstrated a simple process that involves one-pot, one-step reactions leading to efficient
1
ꢀ
ꢀ
1
preparation of new cadmium complexes with N4-donating ligands [CdX
2
L ] (X ¼ I (1), Br (2) L ¼ tris(1-
(
3,5-dimethylpyrazolyl)methyl)amine). The most prominent feature of the synthesis is the in situ
1
formation of a new organic tripodal ligand (L ) in a condensation reaction between a starting ligand (1-
hydroxymethyl-3,5-dimethylpyrazole) and ammonia. A single-crystal X-ray analysis confirmed that the
complexes obtained are monomers (1, 2) with octahedral geometry of the cadmium centres. Complex 3
has a cationic–anionic structure [Cu(LOH)
2 3 4
CH OH][CdCl ] and has been synthesised by the reaction of
1
13
CdO and NH Cl in the presence of zerovalent copper (powder). IR, EPR, H and C NMR, as well as
4
simultaneous TG/DTG were carried out to characterise the products. Moreover, we try to compare the
1
cytotoxic profile of CdO and cadmium salts with Cd(II) complexes. Besides [CdX
2
L ] (1, 2) we take into
Received 1st August 2014
Accepted 2nd September 2014
2
1
consideration [Cd
2
(L )
2
(SCN)
4
(MeOH) ]
2 n
2
(4) and [Cd(SCN) L ] (5) complexes. Biological studies
demonstrated that Cd(II) complexes with the N-scorpionate ligand (1, 2 and 5) have similar cytotoxicity,
which points to a structure–cytotoxicity relationship. Thus, all the complexes (except 4) exhibited a
lower cytotoxic activity compared to a cadmium ion in salts.
DOI: 10.1039/c4ra07960d
www.rsc.org/advances
1a–c
1d,e
of different solvents. For instance, Co(II)
and Cu(II)/(I)
Introduction
complexes with neutral, N4-tripodal ligands were synthesized
By screening of various synthesis methods, we selected a one- following a modied version of the procedure reported by
2a,b
1d
pot, one-step reaction as an efficient process for the construc- Driessen.
A typical synthesis of complexes begins with a
tion of metal complexes containing nitrogen multipodal preparation of the ligands (Scheme 1).
ligands. The interest in the synthesis of such complexes which
Next, the resulting ligand reacts with a suitable metal salts to
can serve as a model of an enzyme has focused mainly on their generate proper complex (Scheme 2).
unusual structural properties. For example, the synthetic
The success of such synthesis depends on keeping reaction
analogues that mimic the structure and the function of active in very strict conditions. For example preparation and handling
sites have been used to study cobalt-substituted carbonic of N4-tripodal ligands under argon by employing standard
1a–c
1d
1e
anhydrase (CoCA)
or ‘blue-copper’ proteins. In all such Schlenk tube techniques was performed by authors. The
cases, the complexes were obtained by conventional methods.
Such methods for the formulation of a C–N bond during a
synthesis of multi-nitrogen ligands (N-scorpionate ligands or
1,3,5,7-tetraazaadamantane (urotropine, hmta)) and its
complexes with metal ions require long reaction times and use
a
´
Institute of Chemistry, Jan Kochanowski University, 15 G Swi ˛e tokrzyska Str., 25-406
Kielce, Poland. E-mail: Barbara.Barszcz@ujk.edu.pl
b
Faculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie Str., 50-383 Wrocław,
Poland
c
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, 12 Weigla Str.,
Wrocław 53-114, Poland
†
CCDC 902408 (1) 997910 (2) and 902407 (3). For crystallographic data in CIF or
Scheme
amine.
1
The synthesis of tris(1-(3,5-dimethylpyrazolyl)methyl)-
1
e
other electronic format see DOI: 10.1039/c4ra07960d
43962 | RSC Adv., 2014, 4, 43962–43972
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solvent methanol, ethanol, 2-propanol, acetone, chloroform
and octane were spectroscopic grade. It has been show that, the
conventional synthesis of polymeric cadmium complexes con-
ꢀ
taining hexamethylenetetramine (hmta), and SCN multi-
ligands demand one-step process where hmta as ligand is used
as purchased and without further purication.
1e
Scheme 2 Preparation of copper(II) complexes.
3
a,b
Compared with those multi-step reactions, here we used the
one-pot synthesis in which a copper powder, cadmium oxide, an
alcohol derivative of pyrazole, and an ammonium iodide/
bromide/chloride react in one step giving metal complexes
(Scheme 3).
It should be noted that the type and structure of obtained
complexes are additionally conditioned by the redox properties
of the halide anions of NH X salts (Scheme 3). Complex 3 has
cationic–anionic structure [Cu(LOH) CH OH][CdCl ] and was
synthesised by the reaction of CdO and NH Cl in the presence of
zerovalent copper (powder). The addition of NH I or NH Br
4
2
3
4
4
4
4
salts instead of NH Cl results in the generation of the cadmium
4
complexes 1, 2 only. Accordingly, we report here how the
ꢀ
ꢀ
ꢀ
reductive capability of the I , Br and Cl anions inuenced the
type of the obtained compounds.
Scheme 3 General scheme for the one-pot, one-step synthesis of 1–
complexes.
3
This work has also an important biological goal. We try to nd
effective chelating agents for treating cadmium intoxication
4
because of its obvious accumulation in the human body. Thus, we
take into consideration multiple mechanisms of cadmium toxicity
such as modulation of gene expression and signal transduction,
interference with enzymes of the cellular antioxidant system and
generation of reactive oxygen species (ROS), inhibition of DNA
5
repair and DNA methylation. For instance, a possible mechanism
for accumulation of reactive oxygen species (ROS) in cells is
caused by the displacement of copper by cadmium ions from
various intracellular sites. Additionally, cadmium is considered as
a human carcinogen by the International Agency for Research on
5
Cancer (IARC). To gain proper material for comparing cytotoxic
2+
properties of Cd ions and obtained complexes in our system, we
incorporated CdO as a cadmium ion source.
Results and discussion
Synthetic procedure
Scheme 4 Proposed pathway for the synthesis of complexes 1 and 2.
We found that practical one-pot, one-step synthesis of Cd(II)
complexes with N4-tripodal ligand (N-scorpionate ligand)
Fig. 1 TG/DTG curves of compounds 1–3.
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Table 1 Crystal data and structure refinement for complexes 1–3
1
2
3
Empirical formula
Formula weight
Crystal size (mm)
Wavelength
C
18
H
27
I
2
N
7
Cd
C
18
H
27Br
2
N
7
Cd
C
13 4 4 3
H24Cl N O CuCd
707.67
613.69
602.10
0.33 ꢁ 0.11 ꢁ 0.07
0.12 ꢁ 0.11 ꢁ 0.08
0.46 ꢁ 0.29 ꢁ 0.21
0.71073
0.71073
0.71073
Temperature (K)
Crystal system
Space group
100
Monoclinic
100
Monoclinic
100
Triclinic
P1ꢀ
P2
1
/n
P2
1
/n
Unit cell dimension
˚
a (A)
13.5701(2)
11.6825(2)
14.9163(3)
90.00
96.604(2)
90.00
2349.03(7)
4
2.001
13.5407(3)
11.2858(2)
14.6968(3)
90.00
97.550(2)
90.00
2226.46(8)
4
1.831
8.202(1)
8.470(1)
17.275(2)
103.01 (2)
99.07(1)
95.85(1)
1142.8(3)
2
1.747
2.344
˚
b (A)
˚
c (A)
ꢂ
a ( )
ꢂ
b ( )
ꢂ
g ( )
3
˚
V (A )
Z
D
ꢀ3
calc (mg m
m (mm
F(000)
)
ꢀ1
)
3.576
1352
4.589
1208
598
Max. and min. transmission
q limits ( )
Index ranges
0.7905 and 0.6858
2.60–27.50
ꢀ17 < ¼ h < ¼ 17
0.759 and 0.649
2.80–27.50
ꢀ17 < ¼ h < ¼ 14
ꢀ14 < ¼ k < ¼ 13
ꢀ12 < ¼ l < ¼ 19
10 219
5110 (0.021)
4263/259
1.003
0.678 and 0.435
3.03–27.50
ꢀ10 < ¼ h < ¼ 10
ꢀ11 < ¼ k < ¼ 10
ꢀ22 < ¼ l < ¼ 22
15 433
5224 (0.021)
4871/243
1.080
ꢂ
ꢀ
13 < ¼ k < ¼ 15
19 < ¼ l < ¼ 17
ꢀ
Reections collected
Unique reections, Rint
Data/parameters
16 069
5306 (0.028)
4179/259
0.974
2
Goodness-of-t on F
Final R and wR for [F > 4s(F)]
R
1
¼ 0.0242
wR
¼ 0.0523
¼ 0.0358
wR
¼ 0.0554
0.958, ꢀ0.454
R
1
¼ 0.0237
wR
¼ 0.0509
¼ 0.0314
wR
¼ 0.0532
0.421, ꢀ0.401
R
1
¼ 0.0285
wR
¼ 0.0661
¼ 0.0307
wR
¼ 0.0671
0.667, ꢀ0.417
2
2
2
Final R and wR for all reections
R
1
R
1
R
1
2
2
2
Dr (e A˚ 3
ꢀ
)
1
ꢀ
1
without isolating intermediates allowed us to: (1) avoid stan-
dard Schlenk tube techniques; (2) eliminate many solvents,
such as ethanol, 2-propanol, acetone, chloroform. The
CdO + L + 2X / [CdX L ]
(3)
2
It should be pointed out that by using NH I or NH Br, Cd(II)
4 4
following steps of our proposed one-step, direct synthesis of complexes were obtained from the solution. This suggests that
ꢀ
2+
ꢀ
Cd(II) complexes are outlined in Scheme 4.
According to Scheme 4, the formation of complexes is started allow the oxidation of Cu to Cu under these reaction condi-
with the following reaction:
tions. On the other hand, by the reaction of CdO and zerovalent
copper (powder) in the presence of NH Cl, a novel (3) complex
that consists of two coordinating ions, i.e. the cation
the reductive capability of the I , Br anions is too strong to
0
4
cat:;D
2þ
CdO þ 2NH
4
X ƒƒƒƒ ƒƒ ! Cd þ H
2
O þ 2NH
3
(1)
2
+
2ꢀ
[Cu(LOH)
2
CH
3
OH] and the anion [CdCl
The redox process that accompanies the direct synthesis of
complex 3 can be summarised in the following reaction:
4
]
was synthesised.
In addition, the synthetic processes for compounds 1 and 2
is based on condensation reaction of 1-hydroxymethyl-3,5-
dimethylpyrazole (L) with ammonia resulting in new organic
0
Cu + 2NH
+
4
2+
1
+ 0.5O
2
/ Cu + H
2
O + 2NH
3
(4)
tripodal ligand (L
amine):
¼
tris(1-(3,5-dimethylpyrazolyl)methyl)
Moreover, the starting organic ligand 1-hydroxymethyl-3,5-
dimethylpyrazole acts as a N,O-chelator of Cu ions and does
not decompose or undergo deprotonation.
2
+
1
NH
3
+ 3L / L + 3H
2
O
(2)
1
The nal [CdX
2
L ] complexes are composed by the reaction
between Cd(II) ion and two kinds of ligands: organic compound
Characterisation of complexes
Thermal stability
1
(
L – intermediate product of second reaction) and inorganic
ꢀ
ꢀ
ꢀ
ion X (I , Br ).
The thermal stability of complexes (1–3) was studied using TG/
DTG in the air atmosphere as well as powder XRD
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Table 2 Selected bond lengths ( A˚ ) and angels ( ) for 1–3
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ꢂ
1
2
3
˚
Bond lengths (A)
Cd–I(1)
Cd–I(2)
Cd–N(1)
Cd–N(3)
2.9674(3)
2.7714(3)
2.351(3)
2.581(2)
2.557(3)
2.332(2)
Cd–Br(1)
Cd–Br(2)
Cd–N(1)
Cd–N(3)
Cd–N(21)
Cd–N(31)
2.7463(3)
2.5860(3)
2.329(2)
2.5961(19)
2.5666(19)
2.316(2)
Cu–N(1)
Cu–N(11)
Cu–O(1)
Cu–O(2)
Cu–O(3)
Cd–Cl(1)
Cd–Cl(3)
Cd–Cl(4)
Cd–Cl(2)
1.986(2)
1.970(2)
2.025(2)
2.048(2)
2.336(2)
2.4706(7)
2.4394(8)
2.4576(7)
2.4572(8)
Cd–N(21)
Cd–N(31)
ꢂ
Valence angels ( )
N(31)–Cd–N(1)
N(31)–Cd–N(21)
N(1)–Cd–N(21)
N(31)–Cd–N(3)
N(1)–Cd–N(3)
N(21)–Cd–N(3)
N(31)–Cd–I(2)
N(1)–Cd–I(2)
N(21)–Cd–I(2)
N(3)–Cd–I(2)
N(31)–Cd–I(1)
N(1)–Cd–I(1)
N(21)–Cd–I(1)
N(3)–Cd–I(1)
I(2)–Cd–I(1)
137.78(9)
82.90(9)
76.73(9)
68.70(8)
69.59(8)
69.45(8)
110.32(6)
110.20(6)
103.42(6)
172.82(5)
93.08(6)
90.83(6)
155.61(6)
86.64(5)
100.536(9)
N(2)–Cd–N(1)
N(31)–Cd–N(21)
N(1)–Cd–N(21)
N(31)–Cd–N(3)
N(1)–Cd–N(3)
N(21)–Cd–N(3)
N(31)–Cd–Br(2)
N(1)–Cd–Br(2)
N(4)–Cd–Br(2)
N(3)–Cd–Br(2)
N(31)–Cd–Br(1)
N(1)–Cd–Br(1)
N(21)–Cd–Br(1)
N(3)–Cd–Br(1)
Br(1)–Cd–Br(2)
138.00(7)
82.23(6)
76.34(7)
68.92(6)
69.67(6)
68.21(6)
109.56(5)
110.19(5)
102.81(4)
170.94(4)
93.85(5)
89.64(5)
153.10(4)
85.49(4)
103.558(9)
N(11)–Cu–N(1)
N(11)–Cu–O(1)
N(1)–Cu–O(1)
N(11)–Cu–O(2)
N(1)–Cu–O(2)
O(1)–Cu–O(2)
N(11)–Cu–O(3)
N(1)–Cu–O(3)
O(1)–Cu–O(3)
O(2)–Cu–O(3)
Cl(3)–Cd–Cl(2)
Cl(3)–Cd–Cl(4)
Cl(2)–Cd–Cl(4)
Cl(3)–Cd–Cl(1)
Cl(2)–Cd–Cl(1)
Cl(4)–Cd–Cl(1)
178.57(9)
95.98(9)
82.78(9)
82.34(9)
99.06(9)
161.86(9)
88.94(8)
90.55(8)
99.66(9)
98.37(9)
112.24(3)
106.86(3)
110.98(2)
113.42(2)
105.02(2)
108.32(3)
decomposed. Similarly, fragmentation peaks were accompanied
by endothermic effects. Samples obtained by prolonged pyrolysis
ꢂ
ꢀ
ꢀ
at 800 C also lead to disintegration of the I and Br ions from
the cadmium complexes.
ꢂ
The rst step, at 140–220 C for 3 was assigned to the thermal
decomposition of one methanol molecule (obs. 5.31%, calcd
5
.38%). Moreover, in complex 3, next two steps can be assigned
ꢀ
2ꢀ
to the release of Cl anions from the [CdCl
4
]
and successive
decomposition of LOH molecules (obs. 66.27%, calcd 65.47%).
The collected solid residues of heated samples 1–3 were
evidenced by powder XRD. The polycrystalline products con-
6
7
tained CdO (1, 2), and a mixture of CuO and CdO (3). We
should point out that the differences between the experimental
mass loss and the loss expected from calculation were as
follows: for 1, D ¼ 0.42%; 2, D ¼ 0.39%; 3, D ¼ 0.71%.
Fig. 2 Molecular structure of 1 with coordination polyhedron.
measurements for solid residues. Solid state thermal analyses
have been performed in order to (i) understand the thermal
decomposition patterns of the complexes, (ii) to verify the
molecular composition, and (iii) to synthesise the thermal
stable end products. The TG/DTG analysis of the compounds
1
ꢀ
ꢀ
The molecular structure of [CdX
2
L ] (X ¼ I for 1, Br for 2)
Both complexes are isostructural and crystallise in the mono-
1
clinic space group P2
(tris(1-(3,5-dimethylpyrazolyl)methyl)amine) obtained in situ
Scheme 4). The crystallographic data and detailed information
1
/n with one molecule of tripodal ligand L
–
(
(Fig. 1) reveals different endothermic mass losses.
for the structure solution and renement for the complexes are
given in Table 1. The ligand L is tetradentate and bonds to the
metal through the lone pair on the nitrogen of the tertiary
amine group and through three nitrogen atoms of the pyrazolyl
rings, that act as true scorpionates. Selected bond distances and
bond angles are listed in Table 2. The crystal structure of 1 is
The rst step of thermal decomposition for 1, 2 occurs
1
ꢂ
between 150 and 430 C and was attributed to the decomposi-
1
tion of L (obs. 35.19%, calcd 34.38% (1); obs. 40.97%, calcd
39.60% (2)). In the second and third steps of degradation, the
rest of the organic moiety, except the coordinated atoms,
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Table 3 Selected short contacts in the structure of complexes ( A˚ and )
Paper
ꢂ
a
d(D–H) ( A˚ )
d(H/A) ( A˚ )
d(D/A) ( A˚ )
<(DH/A) ( )
ꢂ
D–H/A
I
1
2
3
C(41)–H(411)/I(1)
0.99
0.99
0.99
0.99
0.99
0.98
0.99
0.95
0.97
0.97
0.97
0.97
0.96
0.96
0.96
0.93
0.82
0.82
0.82
0.97
0.96
0.96
0.97
0.97
0.97
0.97
3.18
2.47
3.03
3.12
3.03
2.83
3.05
2.99
2.87
2.50
2.89
2.98
2.97
2.84
2.94
2.87
2.46
2.58
2.33
2.93
2.99
2.83
2.92
2.84
2.92
2.75
4.083(3)
2.892(4)
3.740(3)
4.061(3)
3.744(3)
3.641(4)
3.972(4)
3.909(3)
3.545(2)
2.909(3)
3.580(2)
3.703(3)
3.773(2)
3.606(3)
3.858(3)
3.777(3)
3.258(2)
3.279(2)
3.137(2)
3.513(3)
3.794(3)
3.750(3)
3.526(3)
3.525(3)
3.412(3)
3.495(3)
154
105
130
159
130
141
154
163
128
105
129
133
141
137
161
163
166
145
167
120
142
161
121
128
113
134
C(41)–(H412)/N(32)
C(42)–H(41)/I(1)I
C(42)–H(42)/I(1)
C(43)–H(431)/I(1)
C(32)–H(323)/N(21)
C(52)–H(523)/I(2)
II
III
C(4)–H(4)/N(31)
C(42)–H(41)/Br(1)
C(41)–H(412)/N(32)
C(43)–H(432)/Br(1)
I
C(43)–H(431)/N(1)I
C(13)–H(33)/Br(1)
C(32)–H(323)/N(21)
C(52)–H(523)/Br(2)
II
III
C(4)–H(4)/N(31)I
O(1)–H(1)/Cl(3)
O(2)–H(2)/Cl(1)
O(3)–H(3)/Cl(4)
II
III
C(1)–H(11)/Cl(2)I
C(6)–H(63)/Cl(2)
IV
C(71)–H(27)/Cl(3)
C(11)–H(211)/Cl(2)
C(11)–H(211)/Cl(3)
C(11)–H(222)/Cl(1)
C(11)–H(222)/Cl(4)
II
II
a
I
II
III
I
II
Symmetry codes: (1) ꢀx + 3/2, y + 1/2, ꢀz + 1/2; ꢀx + 1, ꢀy + 1, ꢀz + 1; x + 1/2, ꢀy + 1/2, z + 1/2; (2) ꢀx + 1/2, y + 1/2, ꢀz + 1/2; ꢀx + 1, ꢀy + 1, ꢀz;
I
II
III
IV
(
3) x, y ꢀ 1, z; x ꢀ 1, y, z; x ꢀ 1, y ꢀ 1, z; ꢀx + 1, ꢀy + 1, ꢀz + 1.
8
9
˚
˚
shown in Fig. 2, the coordination environment of the central 2.750 A, and 2.706(1) A, but it is similar to the Cd–I bond
˚
˚
11
Cd(II) ion adopts a highly distorted octahedral geometry, with lengths of 2.8162(9) A (ref. 10) and 3.030(1) A. However, the
the basal plane comprising three nitrogen atoms of the three Cd–Br distances are slightly longer to literature data
12a,b
and fall
˚
pyrazole rings (N(1), N(21), and N(31)) and one iodide/bromide in the range 2.55960(3)–2.7463(3) A. The Cd–N bond lengths
˚
˚
ion (I/Br(1)). The axial sites are occupied by the tertiary nitrogen (2.332(2)–2.581(2) A and 2.316(2)–2.5961(19) A for 1 and 2,
atom of tris(1-(3,5-dimethylpyrazolyl-methyl)amine) and the respectively) are greater than the average Cd–N bond length
ꢀ
ꢀ
13
˚
second I /Br anion (I/Br(2)). The degree of distortion of 1 and 2 (2.207(2)–2.268(2) A) because of steric factors that determine
1
from the ideal octahedral geometry is reected not only in bond the coordination of L in a tetradentate manner to Cd(II) ions.
length but also in the bond angles (see Table 2) and is derived Distortions of the Cd polyhedra in both complexes also conrm
1
ꢂ
ꢂ
from the peculiar chemical structure of the organic ligand L . the X–Cd–X angles (100.536(9) (1), 103.558(9) (2)).
Additionally, the equatorial Cd–I(1) bond length of 2.9674(3) A˚
There are no conventional hydrogen bonds in the crystal
is signicantly greater than the respective axial Cd–I(2) distance structure of both complexes, however, an analysis of the crystal
˚
(
2.7714(3) A) and greater than the average Cd–I bond length of structure shows that there are short contacts of the C–H/I/Br
Table 4 Selected C–H/p type and p/p interaction in the structures
D–H/Cg
H/Cg ( A˚ )
<D–H/Cg ( )
ꢂ
D/Cg ( A˚ )
2
3
C(43)–H(431)/Cg(N(21)–C(25))
C(7)–H(73)/Cg(N1–C5))(ꢀx,ꢀy,ꢀz)
C(8)–H(83)/Cg(N11–C51)
2.88
2.95
2.99
133
146
132
3.613(3)
3.785(3)
3.694(3)
Cg/Cg ( A˚ )
Offset ( A˚ )
1
2
Cg(N(21)–C(25))/Cg(ꢀx+1,ꢀy+1,ꢀz+1)
Cg(N(21)–C(25))/Cg(N(1)–C(5))
Cg(N(1)–C(5))/Cg(ꢀx+1,ꢀy+1,ꢀz)
3.942(3)
3.488(3)
3.722(3)
1.592
1.234
1.143
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i
ii
i
Fig. 3 The p–p interactions in complexes 1 (a) ( ꢀx + 1, ꢀy + 1, ꢀz; ꢀx + 1, ꢀy + 1, ꢀz + 1), 2 (b) ( ꢀx + 1, ꢀy + 1, ꢀz) and the C–H/p interaction
in complex 2 (b).
type, respectively (see Table 3). All of these intermolecular
contacts are long when compared with the sum of the respective
van der Waals radii, but these interactions may be important for
the molecular packing. Additionally, in crystal structure of 2,
the C–H/p interactions C(43)–H(431)/Cg (see Table 4), can
all be classied in the V group of Malone's classication for
14a,b
X–H/p interactions.
In both molecular structure are also
observed intermolecular p/p interactions between pyrazole
1
rings of L in the two neighbouring complex molecules, which
also contribute to the structure stabilization (Fig. 3a and b).
The molecular structure of [Cu(LOH)
2 3 4
CH OH][CdCl ] (3)
Fig. 4 shows a perspective view of complex 3, whose structure
2
+
2ꢀ
4
2 3
consists of the [Cu(LOH) CH OH] cation and the [CdCl ]
anion. The crystallographic data and detailed information for
the structure solution and renement for 3 are given in Table 1. Fig. 4 Combined polyhedra/ball-and-stick representation of 3.
Selected bond distances and bond angles are listed in Table 2.
In the cation, one molecule of methanol and two N,O-chelating
molecules of 1-hydroxymethyl-3,5-dimethylpyrazole (LOH) are that result in an N(1)–Cu–O(1) angle of 82.78(9) and an N(11)–
ꢂ
ꢂ
bound directly to the copper ion to give a {CuN O } chromo- Cu–O(2) angle of 82.34(9) . The two Cu–N bond lengths are
2
3
˚
phore. Thus, the metal atom is in a ve-coordinate environ- approximately equal 1.970(2) vs. 1.986(2) A, and both are normal
16
ment, and the coordination polyhedron can best be described Cu–N coordination bonds. The Cu–O (hydroxymethyl) bonds
as a distorted square-pyramid with an Addison parameter of s ¼ are slightly longer but approximately equal to 2.025(2) vs.
˚
0
.269 (where: (s ¼ (b ꢀ a)/60), b and a are the largest coordi- 2.048(2) A. The apical Cu–O (methanol) coordination bond
˚
nation angles; s ¼ 0 for the perfect square-pyramidal and s ¼ 1 measures 2.336(2) A and is the longest bond among the coor-
15
for the trigonal-bipyramidal geometry). The Cu(II) ion lies dination atomic bonds. The cadmium atom in the anionic
2
ꢀ
ꢀ
˚
0
.155(3) A above the basal least-squares plane dened by N(1), complex [CdCl
N(11), O(1) and O(2), in the direction of the apical oxygen atom anions, and the bonding angles Cl(3)–Cd–Cl(2) 112.34(5) ,
of the methanol molecule. The N,O-donor ligands and the Cl(2)–Cd–Cl(1) 105.02(2) , Cl(3)–Cd–Cl(1) 113.42(2) and
central Cu(II) ion form two ve-membered coordination rings, Cl(2)–Cd–Cl(4) 110.98(2) reect some distortion of the
4
]
is tetrahedrally coordinated by four Cl
ꢂ
ꢂ
ꢂ
ꢂ
i.e., N(1)–N(2)–C(1)–O(1)–Cu and N(11)–N(21)–C(11)–O(2)–Cu, coordination polyhedron.
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The analysis of the crystal structure shows that there are no
strong hydrogen bonds in the structure of 3. However, O–H/Cl
interactions, which are important for this molecular structure,
can be found between the cation and anion of 3 (Table 3). Addi-
tionally, it appears that a particular structure of the LOH organic
ligand facilitates the formation of the intermolecular or intra-
17
molecular C–H/Cl interactions of C(6) or C(71) (the methyl
groups) and C(1) or C(11) of hydroxymethyl groups (Fig. 5).
Spectroscopic investigations
The IR spectra conrm the coordination of Cd(II) ions with
1
tris(1-(3,5-dimethylpyrazolyl)methyl)amine (L ) obtained in the
condensation reaction of 1-hydroxymethyl-3,5-dimethylpyrazole
Fig. 6 The EPR spectra of compound 3 in frozen (77 K) methanol
solution together with the spectra simulated using Bruker program,
WIN EPR SimFonia version 1.25.
(
LOH) with ammonia. It is displayed by disappearing the broad
ꢀ
1
n(OH) stretching band at 3152 cm in the spectrum of the
starting organic ligand LOH. Additionally, the spectra of
complexes exhibit corresponding IR bands: 2956, 2916 (2952,
Biological results
2
921) (n(CH
2
)), 1552 (1557) (n(C]C)pyrazole), 1459 (1468)
III 18
(n(C]N)
), 1271 (1276) (n(C–N )), and 1191 (1193) Bearing in mind cytotoxic properties of Cd(II) ion we try to
pyrazole
III
ꢀ1
(
n(C–N )) cm for 1 and 2, respectively, which are character- compare cytotoxic prole of CdO and cadmium salts with Cd(II)
1e
1
1
istic for the new scorpionate ligand L . However, the spectrum complexes. Besides [CdX
2
L ] (1, 2) we take into consideration
2
1
of complex 3 conrms that the starting 1-hydroxymethyl-3,5- [Cd
2
(L )
2
(SCN)
4
(MeOH)
2
]
n
(4) and [Cd(SCN)
2
L ] (5) complexes
dimethylpyrazole takes part in the coordination process and which were structurally characterised by us in short commu-
19
acts as N,O-chelator of Cu(II) ion (n(C]N) frequencies of the nication. The mentioned above compounds (4, 5) were syn-
ꢀ1
pyrazole ring are shied by 29 cm to lower wavenumbers and thesised in one-pot synthesis according to Scheme 5.
ꢀ1
n(OH) are shied by 72 cm to higher wavenumbers compared
with free ligand).
To assessed cytotoxicity of the cadmium compounds we
used: (1) three cancer cell lines with various origin (lung, colon
The effective magnetic moment of complex 3 at room cancer and leukaemia, signed as A549, HT-29, K562, respec-
temperature was 1.77 B.M., which is similar to the spin-only tively); (2) normal mice broblasts (BALB/3T3). According to
value (1.73 B.M.). Simultaneously, the EPR spectrum of reference salts which were used to evaluate their toxic ability,
compound 3 (Fig. 6) was simulated using gII ¼ 2.234, g
t
t
¼
the cadmium chloride demonstrated the most cytotoxic effect
ꢀ
4
ꢀ1
ꢀ4
ꢀ1
2
.055, AII ¼ 190 ꢁ 10 cm , and A
¼ 15 ꢁ 10 cm . The on the all tested cell lines.
1
4
superhyperne splitting caused by the two N nuclei (amplied
Each of the tested compounds possessed similar to CdO
spectrum) indicates that the complex with two N,O-chelating cytotoxicity against cancer cells. The cytotoxic properties of
ligands is stable in methanol. The EPR parameters conrmed a complexes (1) against normal mice broblasts BALB/3T3 was
strong chelating effect of ligands possessing an aromatic char- even 6 times lower.
acter and showed that the metal atom is in a ve-coordinate
The highest cytotoxicity against leukaemia and normal
environment. Thus the coordination polyhedron can best be broblasts (similar to CdO) possessed compound 4 (Scheme 5).
16
described as a distorted square-pyramid.
Fig. 5 Perspective view (along b axis) of 3 that emphasizes the O–H/ Scheme 5 Plausible mechanism for the synthesis of complexes 4
Cl (red-coloured) type or C–H/Cl (green) interaction. and 5.
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Table 5 Cytotoxic properties of tested compounds against A549, HT-29, K562 and BALB/3T3 cell lines
Mean IC50 ꢄ standard deviation [mg mlꢀ1
]
Compound/cell line
A549
HT-29
K562
BALB/3T3
1
2
4
5
30.19 ꢄ 1.16
30.44 ꢄ 1.84
19.62 ꢄ 7.85
27.49 ꢄ 4.70
3.19 ꢄ 0.01
13.67 ꢄ 10.34
26.93 ꢄ 4.00
3.27 ꢄ 1.36
26.38 ꢄ 5.86
30.41 ꢄ 2.47
30.86 ꢄ 1.73
30.70 ꢄ 2.00
28.46 ꢄ 3.38
31.69 ꢄ 0.45
32.14 ꢄ 0.91
2.99 ꢄ 0.73
25.89 ꢄ 1.18
21.92 ꢄ 4.93
3.85 ꢄ 1.19
25.31 ꢄ 8.93
1.98 ꢄ 0.97
3.08 ꢄ 0.21
11.41 ꢄ 7.17
0.95 ꢄ 0.45
8.55 ꢄ 6.00
5.54 ꢄ 2.83
2.74 ꢄ 0.46
6.88 ꢄ 3.39
2.71 ꢄ 0.46
2.95 ꢄ 0.39
1.37 ꢄ 0.97
0.81 ꢄ 0.43
CdCl
CdBr
CdO
2
$2.5H
2
O
2
$4H O
2
Cisplatin
that why only the cadmium complexes 1, 2 are generated under
these reaction conditions. However, in the presence of NH Cl,
4
2 3 4
cationic–anionic complex [Cu(LOH) CH OH][CdCl ] (3) was
synthesised.
As stated in the introduction, our interest in the synthesis of
cadmium complexes was motivated by searching for effective,
chelating agents to treat cadmium intoxication. The compar-
ison of cytotoxic prole of CdO and cadmium salts with the
obtained Cd(II) complexes showed that all the coordination
compounds exhibit lower cytotoxic activity comparing to
cadmium ion in salts. An exception is compound 4 possessing
high cytotoxicity against leukaemia and normal broblasts
(similar to CdO). This may suggest that the obtained in situ
ligands can be considered as good toxic metal chelators.
Fig. 7 Assessment of cadmium-induced inhibition of cells growth.
Experimental section
Physical measurements
The comparison of cytotoxicity of tested compounds with the
same properties of cisplatin – transition metal complex used
worldwide in nearly 50% of all cancer therapies – showed that
tested complexes was nearly 10 times less cytotoxic to cancer
cells than cisplatin.
Elemental analyses were performed on Model 240 Perkin-Elmer
1
13
CHN Analyzer. The H{ C} spectra were performed with a
Varian INOVA-500 NMR spectrometer. IR spectra were recorded
with Nicolet 380 FTIR spectrophotometer in the region 4000–
ꢀ
1
4
00 cm using KBr pellets. Fourier transform Raman scat-
The exception is the compound 4 with the IC50 value 3.8 and
tering measurements (FT-RS) were performed at room temper-
2.7 on K562 leukaemia and BALB/3T3 broblasts respectively
ature with a PerkinElmer RamanStation™ 400F spectrometer,
(see Table 5). In summary, it can be stated that the Cd(II)
ꢀ
1
resolution 4 cm . The incident radiation (l ¼ 785 nm) came
from a diode laser. The thermal decompositions of ꢃ5 mg of the
prepared complexes were measured under the static air atmo-
complexes with N-scorpionate ligand (1, 2, 5), which are iso-
structural, exhibited similar cytotoxicity (Fig. 7) which points to
structure–cytotoxicity relationship. All the complexes (except of
e
sphere with Mettler-Toledo Star TG/SDTA 851 thermal analyzer
at a heating rate of 5 C min . For all complexes in the
temperature range of 25–1000 C alumina open crucibles were
used. The thermoanalytical curves were obtained using STAR
4) exhibited lower cytotoxic activity compared to cadmium ion
ꢂ
ꢀ1
in salts (excluding HT-29 line, for which ability to inhibit the
growth of cancer cell are at the same level).
ꢂ
e
System METTLER-TOLEDO soware. The powder X-ray
diffraction (XRD) patterns of the products of decomposition
Conclusions
˚
were recorded by DRON-2 using CuK
a
radiation (l ¼ 1.54178 A)
ꢂ
We have demonstrated a simple one-step and one-pot protocol over 2q angle range 8–65 . Magnetic moment for 3 was
for the synthesis of N4-donating ligand (tris(1-(3,5-dimethyl- measured using a MSB-MKI instrument (Sherwood Scientic
4
pyrazolyl)methyl)amine) and cadmium(II) complexes (1, 2). It is Ltd) at ambient temperature with [Hg(Co(SCN) )] as standard.
really worth emphasizing that the use of in situ formed ligands, Electron paramagnetic resonance (EPR) spectra for complex 3
without isolating intermediates, allows shortening the pathway was performed at 77 K on a Bruker ESP 300E spectrometer at the
to a target compound.
X-band frequency (about 9.45 GHz) and equipped with the
It should be highlighted that the reductive capability of the Bruker NMR gaussmetter ER 035 M and the Hewlett-Packard
I , Br anions is too strong to allow the oxidation of Cu to Cu , microwave frequency counter HP 5350B. The spectra was
ꢀ
ꢀ
0
2+
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ꢀ1
analysed by a computer simulation program based on reso- 4.07; N, 9.50. IR (KBr, cm ): 3412 m, 3392 m, 3352 m, 3216 s,
nance elds calculated according to the method of Sakaguchi 3135 s, 2937 m, 2839 m, 2745 w, 1649 w, 1616 w, 1599 m, 1569
2
0
et al.
m, 1551 s, 1521 w, 1496 m, 1474 s, 1447 m, 1423 s, 1390 s, 1329
m, 1271 s, 1216 m, 1229 s, 1089 s, 1063 m, 1046 m, 1025 s, 1007
m, 968 w, 822 s, 808 s, 698 s, 680 m, 649 m, 615 w, 586 m, 527 m,
General remarks
4
CdO, NH I, NH Br, NH Cl were obtained from POCH S.A., and
454 m, 376 m, 318 m.
4
4
copper powder, 1-hydroxymethyl-3,5-dimethylpyrazole from
Sigma-Aldrich were used as received. Reagent and solvent
X-ray crystallography
(
CH OH) were purchased from commercial sources and were of The crystallographic measurements were performed on a Kuma
3
analytical grade.
KM4 CCD automated four-circle diffractometer with the
˚
graphite-monochromatized MoK
a
radiation (l ¼ 0.71073 A).
The data for the crystals were collected at 100 K using the
Oxford Cryosystem cooler. The summary of the conditions for
the data collection and the structure renement parameters are
given in Table 1. The structures of crystals were solved by direct
methods using the SHELXL-97 (ref. 21) program, and rened by
a full-matrix least-squared technique using SHELXS-97 (ref. 22)
with anisotropic thermal parameters for non-hydrogen atoms.
Synthesis of metal complexes
1
[
CdI L ] (1). Methanolic solution of copper powder (1 mmol),
2
cadmium oxide (1 mmol), ammonium iodide (6 mmol) and 1-
hydroxymethyl-3,5-dimethylpyrazole (4 mmol), were taken in a
100 ml round bottomed ask. The mixture was vigorous stirred
ꢂ
and heated at 60 C for 7 h. The crystals were collected by
vacuum ltration and washed with mother liquid. Yield: 63%.
Anal. calcd for C18
2 3
The H atoms of the –CH –, –CH groups were placed geomet-
ꢀ
1
H
27
I
2
N
7
Cd (M
r
¼ 707.67 g mol ): C, 30.55; H,
rically and rened as rigid groups. The hydrogen atoms were
introduced at calculated positions with the isotropic tempera-
ture factors xed at 1.2 or 1.5 times Ueq of their parent atoms.
The data were corrected for absorption with the use of CRYSA-
ꢀ1
3
3
1
6
.85; N, 13.85. Found: C, 30.35; H, 3.83; N, 13.76. IR (KBr, cm ):
127 w, 2956 m, 2916 m, 1552 s, 1519 w, 1459 s, 1419 m, 1393 m,
308 m, 1271 s, 1191 w, 1176 w, 1109 m, 1040 m, 1014 m, 980 w,
79 m, 804 s, 791 s, 627 m, 594 w, 567 w, 456 w, 405 w. Raman
23
LIS RED program. The gures were made using DIAMOND
ꢀ1
(cm ): 2963 m, 2952 m, 2922 s, 1558 m, 1467 s, 1435 w, 1388 m,
24
soware.
1
8
2
317 w, 1297 w, 1266 w, 1154 w, 1143 w, 1046 m, 1020 m, 989 w,
83 m, 848 m, 703 m, 631 w, 607 m, 595 s, 488 m, 407 m, 372 m,
83 w, 240 m, 210 m, 160 s; d (500 Hz; DMSO-d ; Me Si) 1.93
H 6 4
Cytotoxicity assay in vitro
), 5.86 (3H, s, Established in vitro, human cell line: K562 (leukaemia), A549
H(pz)). d (500 Hz; DMSO-d ; Me Si) 10.1 (Me), 13.50 (Me), 62.4 (lung cancer), HT-29 (colon cancer) and normal mice broblast
(9H, s, Me), 2.15 (9H, s, Me), 4.91 (6H, s, CH
2
4
C
6
4
(
CH ), 106.1 (4-c(pz)), 140.3 (3-C(pz)), 147.5 (5-C(pz)).
(BALB/3T3) was used. All lines were obtained from American
2
1
[
CdBr L ] (2). The coordination compound was obtained Type Culture Collection (Rockville, Maryland, USA) and are
2
following of the procedure described for complex 1. A colorless being maintained at the Institute of Immunology and Experi-
product was obtained aer 7 days upon evaporation of an mental Therapy, Wrocław, Poland.
methanolic solution containing Cu–CdO–NH
4
Br–L in molar
K562 cells were cultured in the RPMI 1640 (IIET, Poland)
ratio 2 : 1 : 3 : 3. Colourless solution was ltered and le in air. supplemented with 2 mM L-glutamine, 1.0 mM sodium pyruvate
Aer evaporation of the solvent solid colourless compound and 10% fetal bovine serum (all from Sigma-Aldrich, Germany),
was formed (2), which was collected on a lter and air dried. HT-29 and A549 cells were cultured in the RPMI 1640 + Opti-
The crystals were collected in 65% yield. Anal. calcd for MEM (50 : 50) medium (IIET, Poland) supplemented with 2 mM
ꢀ
1
C
18
H
27
N
7
Br
2
Cd (M
r
¼ 613.69 g mol ): C, 35.23; H, 4.43; L-glutamine and 5% fetal bovine serum (all from Sigma-Aldrich
ꢀ1
N, 15.98. Found: C, 34.90; H, 4.14; N, 15.85. IR (KBr, cm ): Germany), BALB/3T3 cells were cultured in Dulbecco medium
3
1
1
1
6
127 w, 2964 m, 2952 m, 2922 m, 1557 s, 1468 w, 1424 s, 1399 m, (IIET, Poland) supplemented with 2 mM L-glutamine and 1.0
375 m, 1317 m, 1299 s, 1276 w, 1265 w, 1243 w, 1230 w, 1193 m, mM sodium pyruvate, 10% fetal bovine serum (all from Sigma-
182 w, 1155 w, 1041 m, 1129 m, 1011 m, 1046 m, 1036 m, Aldrich, Germany). All culture medium was supplemented with
018 m, 980 w, 884 m, 840 m, 679 s, 810 s, 799 s, 691 m, 674 m, 100 units per ml penicillin (Sigma-Aldrich, Germany), and 100
ꢀ
1
61 m, 634 w, 599 w, 564 w, 456 w, 419 w.
mg ml streptomycin (Polfa Tarchomin S.A., Poland). All cell
ꢂ
[
Cu(LOH) MeOH][CdCl ] (3). Copper powder (1 mmol), CdO lines were grown at 37 C with 5% CO humidied atmosphere.
2
4
2
ꢀ1
(0.5 mmol), NH
4
Cl (1.5 mmol), 1-hydroxymethyl-3,5-dime-
Test solutions of the compounds tested (1 mg ml ) were
thylpyrazole (1.5 mmol) and methanol (60 ml) were placed in a prepared by dissolving the substances in 100 ml of DMSO
ꢂ

ask, heated to 60 C and magnetically stirred for 8 h with completed with 900 ml of tissue culture medium. Aerwards, the
accessibility to air. The solution was ltered to removed any tested compounds were diluted in culture medium to reach the
ꢀ
1
undissolved material. The resulting dark green solution was le nal concentrations of 100, 1, 0.1 and 0.01 mg ml . Twenty four
to crystallize at room temperature. Aer few days dark blue hours prior to the addition of the tested compounds, the cells
crystal suitable for X-ray were obtained. The precipitate was were plated in 96-well plates (Sarstedt, Germany) at a density of
4
separate by ltration and dried under vacuum. The product was 1 ꢁ 10 cells per well. The assay was performed aer 72 h of
collected in 43% yield. Anal. calcd for C13
H
24Cl
4
N
4
O
3
CuCd (M
r
exposure to varying concentrations of the tested agents. The in
ꢀ
1
¼
602.10 g mol ): C, 25.94; H, 3.99; N, 9,30. Found: C, 26.15; H, vitro cytotoxic effect of all agents was examined using the MTT
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K562) or SRB (A549, HT-29 and BALB/3T3) assay. The results
were calculated as an IC50 (inhibitory concentration 50) – the
concentration of tested agent which is cytotoxic for 50% of the
cancer cells population. IC values were calculated for each
experiment separately and mean values ꢄ SD are presented in
the Table 5. Each compound in each concentration was tested
in triplicate in a single experiment, which was repeated 3–5
times.
RSC Advances
25
(
1982, 21, 1014–1019; (e) K. Fujisawa, S. Chiba, Y. Miyashita
and K.-I. Okamoto, Eur. J. Inorg. Chem., 2009, 3921–3934.
2 (a) W. L. Driessen, Recl. Trav. Chim. Pays-Bas, 1982, 101, 441–
443; (b) W. L. Driessen, W. G. R. Wiesmeijer, M. Schipper-
Zablotskaja, R. A. G. de Graaff and J. Reedijk, Inorg. Chim.
Acta, 1989, 162, 233–238.
3 (a) Y. Bai, W.-L. Shang, B.-B. Dang, J.-D. Sun and H. Gao,
Spectrochim. Acta, Part A, 2009, 72, 407–411; (b) A. Banerjee,
P. Maiti, T. Chattopadhyay, K. S. Banu, M. Ghosh,
E. Suresh, E. Zangrando and D. Das, Polyhedron, 2010, 29,
51–958.
(a) P. O'Brien and H. J. Salacinski, Arch. Toxicol., 1998, 72,
90; (b) D. Beyersmann and S. Hechtenberg, Toxicol. Appl.
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Biochem., 2000, 79, 241–244; (d) P. Josph, Toxicol. Appl.
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Appl. Pharmacol., 2009, 238, 294–300; (f) D. Beyersmann,
Toxicol. Lett., 2002, 127, 63–68.
MTT assay
9
This technique was applied for the cytotoxicity screening
against leukaemia cells growing in suspension culture. An assay
was performed aer 72 hours exposure to varying concentra-
4
6
ꢀ
1
tions (from 0.1 to 100 mg ml ) of the tested agents. For the last
–4 hours of incubation 20 ml of MTT solution were added to
each well (MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
3
ꢀ1
zolium bromide; stock solution: 5 mg ml , Sigma-Aldrich,
Germany). The mitochondria of viable cells reduce the pale
yellow MTT to a navy blue formazan: the more viable cells are
present in well, the more MTT will be reduced to formazan.
When incubation time was completed, 80 ml of the lysing
mixture were added to each well (lysing mixture: 225 ml dimethyl-
formamide, POCh, Gliwice, Poland, 67.5 g sodium dodecyl
sulphate, Sigma-Aldrich, Germany, and 275 ml of distilled
water). Aer 24 h, when formazan crystals had been dissolved,
the optical densities of the samples were read on Synergy H4
photometer (BioTek Instruments, USA) at 570 nm wavelength.
5
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1
26
was performed aer 72 hour exposure of the cultured cells to
1
ꢀ
1
varying concentrations (from 0.1 to 100 mg ml ) of the tested
agents. The cells attached to the plastic were xed by gently
layering cold 50% TCA (trichloroacetic acid, Aldrich-Chemie,
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ꢂ
plates were incubated at 4 C for 1 h and then washed ve times
1
1
with tap water. The background optical density was measured
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