Heteroleptic enantiopure Pd(ii)-complexes derived from halogen-substituted Schiff bases and 2-picolylamine: Synthesis, experimental and computational characterization and investigation of the influence of chirality and halogen atoms on the anticancer acti

Article abstract of DOI:10.1039/d1nj01491a

Seven enantiomeric pairs of palladium complexes, [Pd(pic)(R or S)-N-1-(phenyl)ethyl-2,4-X1,X2-salicylaldiminate)]NO3, [Pd(pic)(R or S)]NO3 (X1 = X2 = Cl, Br, I, H; X1/X2 = Br/Cl), were synthesized by the reaction of enantiopure halogen-substituted Schiff

Full text of DOI:10.1039/d1nj01491a

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Heteroleptic enantiopure Pd(II)-complexes derived
from halogen-substituted Schiff bases and
Cite this: DOI: 10.1039/d1nj01491a
2-picolylamine: synthesis, experimental and
computational characterization and investigation
of the influence of chirality and halogen atoms on
the anticancer activity†
a
a
b
Nazanin Kordestani, Hadi Amiri Rudbari, * Isabel Correia,
c
c
d
Andreia Valente, * Leonor Corte-Real, Mohammad Khairul Islam,
ˆ
e
f
f
g
Nicola Micale, Jason D. Braun,
Johan Wouters and Mohammed Enamullah
David E. Herbert,
Nikolay Tumanov,
g
d
*
Seven enantiomeric pairs of palladium complexes, [Pd(pic)(R or S)-N-1-(phenyl)ethyl-2,4-
,X -salicylaldiminate)]NO , [Pd(pic)(R or S)]NO (X = X = Cl, Br, I, H; X /X = Br/Cl), were synthesized
by the reaction of enantiopure halogen-substituted Schiff bases (R or S)-N-1-(phenyl)ethyl-2,4-
,X -salicylaldimine with [Pd(pic)Cl ] (pic = 2-picolylamine). The composition and structure of the
X
1
2
3
3
1
2
1
2
X
1
2
2
1
13
complexes were confirmed by means of FT-IR, H NMR and C NMR spectroscopy, elemental analysis
and X-ray crystallography. The electronic structure of the reported complexes was investigated using
UV–vis absorption and electronic circular dichroism (ECD) spectroscopy, complemented by DFT/TDDFT
modelling. To investigate the effect of chirality and different halogen substituents on the anticancer
activity of the complexes, the cytotoxic activity of all fourteen complexes was tested in the human
breast cancer cell lines MDA-MB-231 and MCF-7 at 24 h using the colorimetric MTT assay. Also, the cell
death mechanism was assessed using the annexin V/propidium iodide (AV/PI) cytometry-based assay.
Received 26th March 2021,
Accepted 16th April 2021
DOI: 10.1039/d1nj01491a
rsc.li/njc
1
essential insights into the design of new active drugs. Indeed,
many drugs currently on the market are chiral, and their
Introduction
Chirality is an important tool for modern drug discovery as the therapeutic activity depends on the stereospecific recognition
2
molecular recognition of chiral biological targets can provide of biomolecules. Besides, the biological activity of enantio-
mers can also differ drastically in terms of toxicity and
a
Department of Chemistry, University of Isfahan, Isfahan 81746-73441, Iran.
E-mail: h.a.rudbari@sci.ui.ac.ir, hamiri1358@gmail.com
Centro de Qu ´ı mica Estrutural, Departamento de Engenharia Qu ´ı mica,
Instituto Superior T ´e cnico, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal
Centro de Qu ´ı mica Estrutural and Departamento de Qu ´ı mica e Bioqu ´ı mica,
Faculdade de Ci ˆe ncias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa,
Portugal. E-mail: amvalente@fc.ul.pt
pharmacokinetics. Indeed, one of them may be beneficial,
whereas the other may be harmful or inactive. This is valid
not only for organic drugs but also for metallodrugs, in which
the enantiomeric resolution becomes necessary for their
b
c
3
medicinal applicability. An example of the latter is oxaliplatin,
one of the most commonly used and active chemotherapeutic
drugs utilized in oncology nowadays, that contains the chiral
ligand 1R,2R-cyclohexane-1,2-diamine (Fig. 1). This platinum-
based anticancer compound is more biologically active and
chemically reactive than its enantiomer (Fig. 1) containing the
d
e
Department of Chemistry, Jahangirnagar University, Dhaka1342, Bangladesh.
E-mail: enamullah@juniv.edu
Department of Chemical, Biological, Pharmaceutical and Environmental Sciences,
University of Messina, Viale Ferdinando Stagno D’Alcontres 31, I-98166 Messina,
Italy
f
4
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2,
Canada
ligand 1S,2S-cyclohexane-1,2-diamine. Other examples of potent
chiral antineoplastic agents are the platinum drugs heptaplatin
g
Department of Chemistry, Namur Institute of Structured Matter,
University of Namur, 5000 Namur, Belgium
(
administered as a single enantiomer) and lobaplatin (that is
5
used as a B1 : 1 diastereomeric mixture SSS, RRS).
†
Electronic supplementary information (ESI) available. CCDC 1989598 and
007895. For ESI and crystallographic data in CIF or other electronic format,
In addition, halogens (fluorine (F), chlorine (Cl), bromine
(Br), and iodine(I)) as the substituents of the pharmacologically
2
see DOI: 10.1039/d1nj01491a
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considerable potential for cancer therapy. It was recently
reported that Pd(II)-complexes have significant anticancer
activity and lower toxicity compared with some clinically used
1
5
chemotherapeutics. However, less attention has been focused
on the synthesis and characterization of enantiopure Pd(II)-
1
6
complexes endowed with biological activity. Indeed, according
to our search in the literature, there are no reports about
the synthesis of heteroleptic enantiopure Pd(II)-complexes for
biological investigations.
Fig. 1 Enantiomers of the Pt(II)-based drug oxaliplatin with chiral ligands.
(
A) 1R,2R-cyclohexane-1,2-diamine (oxaliplatin in clinical use); (B) 1S,2S-
cyclohexane-1,2-diamine (not in clinical use).
Schiff bases are useful chelating ligands for metal ions,
resulting in complexes with different physical and chemical
active ligands are widely used in drug design. Approximately properties. Thus, the metal complexes of Schiff bases have
6
17
5
0% of molecules in high-throughput screening are halogenated been extensively designed and investigated for biomedical
1
8
19
and around 40% of drugs currently on the market or in clinical applications, such as antitumor,
anti-inflammatory,
7
20
21
trials are halogenated. The large proportion of halogenated antibacterial and antifungal activities. Recently, our group
drugs in the clinic and clinical trials implies the existence of reported that Pd(II) and V(V) complexes containing chiral Schiff
7
,8
22
halogen bonds in drug–target complexes. A halogen bond (XB) base ligands exhibited unique biological characteristics.
Based on the above discussion, and in an attempt to find
in biomolecules can be referred to as a short C–Xꢀ ꢀ ꢀD–Z
interaction, where D is a halogen bond donor; C–X is a new potent anticancer agents, together with the consideration
carbon-bonded chlorine, bromine, or iodine; D–Z is a carbonyl, that pyridine moieties, Schiff base ligands, halogenation
hydroxyl, thiol, aromatic ring, charged carboxylate, phosphate and chirality are attractive in medicinal and pharmaceutical
group or amine; and the Xꢀ ꢀ ꢀD distance is less than or equal to chemistry, fourteen optically pure chiral heteroleptic Pd(II)-
9
the sum of the respective van der Waals radii. Halogen bonds complexes (i.e. seven enantiomeric pairs) were designed,
(
XBs) are attracting increasing attention in the design of drugs synthesized and characterized, with the aim of exploring their
due to their ability to improve the binding affinity towards the potential stereospecific anticancer activities.
intended target and tune the ADME/T (absorption, distribution,
The enantiopure Schiff bases, (R or S)-N-1-(phenyl)ethyl-2,4-
metabolism, excretion, and toxicity) profile of the resulting X ,X -salicylaldimine (X , X = Cl, Br, I), were also recently used
1
2
1
2
8
,10
compounds.
Halogen bonding can also be exploited for for the synthesis of pseudotetrahedral chiral copper(II)
2
3
designing metal complex ligands or biologically active organic complexes in our research group. For the synthesis of the
compounds to overcome the drug-resistance phenomenon.
8
,11
heteroleptic Pd(II)-complexes, we also used a bidentate amine
On the basis of the structural and thermodynamic analogy ligand having a heteroaromatic nitrogen base (2-picolylamine)
1
2
between platinum(II) and palladium(II) complexes, a variety of that possesses p-accepting properties, which is believed to be
studies on palladium(II) derivatives as potential anticancer drugs involved in the p–p stacking effects with purine and pyrimidine
1
3
have been performed. The aquation and ligand-exchange rates bases. In this respect, it is very interesting to note that Farrell
5
24
of the Pd(II)-complexes are about 10 times faster than those of et al. found a considerable increase in the cytotoxicity of
the Pt(II) analogues. Moreover, the Pd(II)-complexes have overall a trans-[Pt(py)
Cl
]
complexes in comparison to inactive
] by introducing aromatic nitrogen ligands.
2
2
2
1
4
better solubility compared to that of the Pt(II) complexes.
trans-[Pt(NH
3
)
2
Cl
Therefore, it seems that at least some Pd(II)-complexes may have Also, a systematic variation of the N-heterocyclic ligands in
1 2
Fig. 2 Synthesis of the enantiopure Schiff bases, (R or S)-N-1-(phenyl)ethyl-2,4-X ,X -salicylaldimine.
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+
3 2
compounds of the formula cis-[Pt(NH ) (Am)Cl] (where Am
denotes a N-heterocyclic ligand), by Lippard et al., resulted in
the monofunctional DNA-binding phenanthriplatin, which
showed greater efficacy than cisplatin and oxaliplatin in
established human cancer cells and revealed a distinct pattern
2
5
of activity.
Results and discussion
Synthesis, stereochemistry and spectroscopic studies
The condensation of halogen-substituted salicylaldehydes with
enantiopure (R)-(+)-a-methylbenzylamine or (S)-(ꢁ)-a-methyl-
benzylamine yields the enantiopure Schiff bases (R or S)-N-1-
(phenyl)ethyl-2,4-X
1 2
,X -salicylaldimine (HR1–7 and HS1–7)
(Fig. 2). The purity and identity of the Schiff bases were
established using elemental analysis, and multinuclear NMR
2
3
and FT-IR spectroscopies. The reaction of [Pd(pic)Cl
2
] (pic = Fig. 4 Geometrical isomers for the synthesized Pd(II)-complexes that
may form.
2-picolylamine) with these asymmetric N^O-chelating Schiff
base ligands affords the asymmetric optically active palladium
complexes (Fig. 3).
group of 2-picolylamine (Fig. 4). However, a mixture of the two
Four-coordinated metal compounds with two asymmetrical isomers was not obtained in the synthesis due to steric
bidentate ligands in a square-planar geometry afford a mixture hindrance between the pyridine and –CH(C )CH groups.
of two geometrical isomers, A and B, as shown in Fig. 4. Only conformation A can be prepared from the reaction of
In isomer A, the oxygen atom of the Schiff base ligand is in [Pd(pic)Cl ] with the chiral Schiff base ligands. Configuration A
front of the –NH group of 2-picolylamine, while in isomer B, was confirmed using X-ray crystallography in [Pd(pic)R3]NO
6
H
6
3
2
2
3
the oxygen atom of the Schiff base ligand is in front of the –Npy and also racemic [Pd(pic)rac2]NO3.
Fig. 3 Synthetic route to enantiopure Pd(pic)–enantiopure Schiff bases complexes.
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1
Fig. 5 H NMR spectrum of [Pd(pic)R3]NO
3
in CDCl
3
at room tempera-
ture (** peak for CDCl and * peak for water in CDCl
3
3
).
Due to the above discussion, for our synthesized Pd(II) chiral
complexes, the existence of the two isomers in equimolar or
different quantities generally should produce two sets of
1
1
3 3
Fig. 7 H– H-2D COSY NMR spectra of [Pd(pic)R3]NO in CDCl at room
1
signals for each proton in the related H NMR spectrum, which temperature.
allows us to distinguish the isomers from each other. However,
1
in this case, the H NMR spectrum of all newly synthesized
Pd(II)-complexes showed only one pattern of signals for each
proton, as can be seen in Fig. 5 for [Pd(pic)R3]NO . Then, the
H NMR spectrum confirms the presence of a single isomer, A,
close to H4 and a long-range coupling is observed as seen in the
1
1
3
H– H-2D COSY NMR spectra (Fig. 7). Such a long-range
coupling was previously observed in the Pd(II)-complexes of the
1
2
2a
for the complexes in solution.
R/S-(1-phenylethylimino)methylnaphtalen-2-ol) ligand.
1
13
Both the H and C NMR spectra of the complexes are
The protons of the amino group of 2-picolylamine show two
multiplets at about 6.0 and 4.5 ppm after the coordination of
consistent with the assigned structures (for example: the
1
3
H NMR of [Pd(pic)R3]NO in Fig. 5). The resonance signal
[
Pd(pic)Cl
signals for the two protons of the amino group is due to the lack
of any symmetry elements in the complexes. Then, the H and
protons are diastereotopic and show two sets of multiplets.
2
] with the Schiff base ligands. The presence of two
corresponding to the phenolic (OH) proton disappears in the
complex spectra, indicating the deprotonation and coordination
of oxygen to the Pd(II) ion. In the ligands, the HC = N proton
appears as a sharp singlet at 8.15–8.45 ppm, while after
complexation, it is converted into a doublet that shifted down-
a
H 0
a
This connection between the H
a
a
and H 0
protons also is
1
1
observed in the H– H-2D COSY NMR spectra (Fig. 5 and 7).
Similarly, the –CH2 protons of 2-picolylamine show two
separate triplet of doublet signals at about 3.8 and 4.3 ppm
that shifted upfield after the coordination of the Schiff base
ligand with [Pd(pic)] . The signal of benzylic hydrogen,
CH(CH )(C )), appears as a quartet at about 5.1–5.4 ppm,
2
3
field by 0.2–1.0 ppm. The conversion of the singlet into a
doublet following complexation can be attributed to coupling
between the H1 and H4 protons (Fig. 6 and 7). The Pd(II)-
complexes are rigid, and therefore, the imine hydrogen is located
2
+
(
3
6 6
H
shifted downfield by 0.5–0.8 ppm with respect to the free Schiff
base. The methyl group of the ligand in the complexes shows a
doublet at about 1.6 ppm that remains nearly unshifted with
1
1
respect to the free Schiff base ligands. In the H– H-2D COSY
NMR spectra (Fig. 7), the connection between the methyl group
protons and benzylic hydrogen is also due to the vicinity of
these two classes of protons.
The infrared spectra of the Pd(II)-complexes feature a very
ꢁ
1
strong band at 1612–1620 cm
that is consistent with
2
2a
n(CHQN). The shift of the imine band to lower wavenumbers
upon complexation with respect to the free ligands suggests
2
2a
coordination via the imine nitrogen atoms.
Then, the most
relevant feature of the IR spectra of the ligands and complexes is
the red shift of the n(CHQN) bands upon the coordination of
2
6
ligands to the metal ions. This feature can be explained by the
withdrawal of electrons from the nitrogen atom to the metal ion
Fig. 6 Long-range coupling (between H1 and H4) and diastereotopic
0 0
protons (H , H , H and H ).
a a b b
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due to the coordination process. The presence of several O(1)–Pd(1)–N(1) and N(2)–Pd(1)–N(3) are very small, y = 0.451 in
ꢁ1
medium intensity bands in the range of 2910–3100 cm is [Pd(pic)S2]NO
3 3 3
of [Pd(pic)rac2]NO and 2.151 in [Pd(pic)R3]NO ,
due to the existence of the C–H stretching vibrations of indicating that the geometry around the metal ion is slightly
2
9–31
aliphatic and aromatic protons. The strong and sharp band at distorted square planar.
ꢁ1
B1383 cm is the characteristic of ionic nitrate, consistent with
The bond distances and angles are very similar in
[Pd(pic)rac2]NO and [Pd(pic)R3]NO as reported in Table 1.
This similarity can also be confirmed by overlaying the cationic
2
7
an outer sphere counter ion.
3
3
X-ray and optimized structures
1
1
3
part of [Pd(pic)R3] and [Pd(pic)R2] of [Pd(pic)rac2]NO .
The chirality of the Pd(II)-complexes was confirmed by single As shown in Fig. S1 (ESI†), most of the atom sites overlap well.
crystal X-ray diffraction analysis for the enantiopure The most significant difference is observed at the orientation of
[
Pd(pic)R3]NO3 and racemic [Pd(pic)rac2]NO3 complexes. the –CH(CH )(C H ) group, which can result from the inter-
3 6 5
Single crystals suitable for X-ray diffraction studies were molecular interactions.
obtained by the slow diffusion of diethyl ether into a DMF
The Pd(1)–N(2) bond lengths in both the structures (2.047(2)
3 3
solution of the complexes. A view of the molecular structures Å for [Pd(pic)rac2]NO and 2.051(2) Å for [Pd(pic)R3]NO ) are
with the common atom numbering scheme is shown in Fig. 8, longer than the Pd(1)–N(3) (aromatic N) bond lengths (2.028(2)
while the selected bond lengths and angles are collected in Å for [Pd(pic)rac2]NO and 2.015(2) Å for [Pd(pic)R3]NO ) and
Table 1. As shown in Fig. 8, it is only possible to form the comparable to the analogous Pd(II)-complexes. The Pd(1)–
isomer A for the Pd(II)-complexes. The crystal packing of O(1) (1.9811(18) Å for [Pd(pic)rac2]NO and 1.971(2) Å for
Pd(pic)rac2]NO3 is centrosymmetric (P1%), and therefore, the [Pd(pic)R3]NO ) and Pd(1)–N(1) (2.016(2) Å for [Pd(pic)rac2]
3
3
3
2
3
[
3
crystallized compound is a racemic mixture of the two enantiomers NO3 and 2.022(2) Å for [Pd(pic)R3]NO ) bond lengths are
3
2
2a,31
[
Pd(pic)R2]NO
S-conformation on C(8), [Pd(pic)S2]NO
The complex [Pd(pic)R3]NO crystallizes in the space group and [Pd(pic)R3]NO
P2 non-centrosymmetric unit cell, with
parameter
enantiopure crystals consistent with the model shown in characters, with the sp hybridization of the imino nitrogen
3
and [Pd(pic)S2]NO
3
. The enantiomer with similar to those reported for the related complexes.
The
3
, is shown in Fig. 8. imine bond lengths (1.292(4) Å and 1.288Å for [Pd(pic)rac2]NO
3
3
3
, respectively) and bond angles for C(7)–
1
2
1
2
1
,
a
a
3
Flack N(1)–C(8) (118.5(2)1 and 118.28(2)1 for [Pd(pic)rac2]NO and
2
8
of 0.034(8) that confirms the formation of [Pd(pic)R3]NO
3
,
respectively) confirm the double bond
2
2
2a,26
Fig. 8. The chiral center in the Schiff base ligands has the atom.
R-configuration, which is consistent with the chirality of the salicylaldimine fragment of the ligand is not planar. The Pd
ligand precursors used in the synthesis. atom is displaced from the plane constituted by the remaining
The crystallographic data reveal that Pd(II) is four-coordinated five atoms (0.405 Å for [Pd(pic)R3]NO
and ꢁ0.133 Å for
in both structures, [Pd(pic)rac2]NO and [Pd(pic)R3]NO , by the [Pd(pic)rac2]NO ). Thus, the chelate ring is folded along the
The six-membered chelate ring formed by the
3
3
3
3
phenolate oxygen and imine nitrogen atoms from the Schiff O(1) and N(1) line in the O(1)–C(1)–C(2)–C(7)–N(1)–Pd(1) ring
bases and two nitrogen atoms from the 2-picolylamine (pic). and has a half-chair like conformation. Due to the formation of
The dihedral angles between the two coordinating planes a five-membered chelate ring, the N(2)–Pd(1)–N(3) angle is
Fig. 8 Perspective view of the cationic D-[Pd(pic)S2]NO
drawn at the 50% probability level, while the hydrogen size is arbitrary. The counter ion (NO
refined with site occupancy factors 0.675 : 0.325. Only the major component of the disordered NO
3
(left) and K-[Pd(pic)R3]NO
3
(right) showing their asymmetric unit. Thermal ellipsoids are
ꢁ
3
) for K-[Pd(pic)R3]NO
3
is disordered over two sites and
ꢁ
3
group is shown.
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Table 1 Experimental and calculated bond lengths (Å) and angles (1) in the cationic Pd(II)-complexes
X-ray structures
DFT optimized structures
+
+
+a
+a
+b
Bond lengths (Å) and angles (1)
D-[Pd(pic)rac2]
L-[Pd(pic)R3]
L-[Pd(pic)R3]
D-[Pd(pic)R3]
L-[Pd(pic)R3]
Pd–N(1)
Pd–N(2)
Pd–N(3)
Pd–O(1)
2.016(2)
2.047(2)
2.028(2)
1.9811(18)
1.292(4)
87.95(8)
92.84(8)
179.18(8)
168.74(9)
80.80(9)
98.41(9)
118.5(2)
0.45
2.022(2)
2.051(2)
2.015(2)
1.971(2)
1.288(4)
87.66(8)
92.36(8)
178.01(12)
169.23(9)
81.61(10)
98.40(10)
118.28(2)
2.16
2.0445
2.1116
2.0596
1.9867
1.3182
87.90
2.05096
2.10812
2.06102
1.98841
1.32155
87.51234
92.81871
178.45179
166.99694
79.61692
100.09668
115.67320
2.35
2.1500
2.1234
2.0661
1.9991
1.3174
87.94
C(7)–N(1)
O(1)–Pd(1)–N(3)
O(1)–Pd(1)–N(1)
N(3)–Pd(1)–N(1)
O(1)–Pd(1)–N(2)
N(3)–Pd(1)–N(2)
N(1)–Pd(1)–N(2)
C(7)–N(1)–C(8)
93.11
93.03
178.96
167.94
80.08
98.91
119.36
0.98
179.02
167.78
79.88
99.14
119.76
1.08
c
y (1)
a
b
c
Calculated at B3LYP/SDD. B3LYP/LANL2DZ. Dihedral angle (y/1) between the two coordinating planes O(1)–Pd(1)–N(1) and N(2)–Pd(1)–N(3).
8
0.811 and 81.611 in [Pd(pic)rac2]NO and [Pd(pic)R3]NO3, are essentially equienergetic (Fig. S2, ESI†). Some selected bond
3
32,33
respectively.
The pyridine moiety and the planar coordination lengths and angles in the optimized structures are listed in Table 1
sphere are nearly co-planar, as indicated by the O(1)–Pd(1)–N(3)– and are comparable to the X-ray parameters obtained for
1
C(16) torsion angle of only 11.61 for both the structures.
3
K-[Pd(pic)R3]NO and D-[Pd(pic)rac2] . The dihedral angles for
The X-ray molecular structures for the four-coordinated non- the optimized structures are very small (0.98 and 2.351) and close to
planar metal(II)-complexes with asymmetric bidentate chiral the X-ray values (Table 1).
ligands exhibit induced chirality at the metal center and
provide two opposite configured D- and L-diastereomers.
As shown in Fig. S3 and S4 (ESI†), the crystal packing for
these two structures is different because of the existence of a
2
9–31
Hence, based on the dihedral angle y = 2.151 and the ligand racemic mixture in the crystal packing of [Pd(pic)rac2]NO and
3
folding angles f
Pd(1)–O(1) and the salicylal ring, upward plane with respect to [Pd(pic)R3]NO
the O(1)–N(1)–Pd(1)–N(2)–N(3) plane) and f = 5.801 (angle action is O
N–Oꢀ ꢀ ꢀH–NH in [Pd(pic)rac2]NO
between the planes of N(2)–Pd(1)–N(3) and the phenyl ring, Oꢀ ꢀ ꢀH–NH and O N–Oꢀ ꢀ ꢀH–C in [Pd(pic)R3]NO
downward plane with respect to the O(1)–N(1)–Pd(1)–N(2)–N(3) The cationic parts of the [Pd(pic)R3]NO
1
= 16.311 (angle between the planes of N(1)– an enantiopure compound in the crystal packing of
. The most important intermolecular inter-
, while O N–
3
2
2
3
2
2
3
(Table S1).
molecule,
3
+
plane), the structure for [Pd(pic)R3]NO could be best described [Pd(pic)R3] , interact with each other via N–Hꢀ ꢀ ꢀO N–O and
3
2
2
9b
as a L-diastereomer (Fig. 8).
for the diastereomeric pairs K-[Pd(pic)R3] and D-[Pd(pic)R3]
Fig. 9) show that the former one is relatively more stable by notation), where R
The DFT optimized structures C–Hꢀ ꢀ ꢀO N–O hydrogen bonds, while the crystal packing of
2
1
+
4
4
[Pd(pic)rac2]NO adopts a R (12) dimer ring (using a graph set
3
a
d
(
(r) is a ring of r atoms involving a acceptors
ꢁ1
34
58.36 kcal mol , in parallel to the X-ray structural results. In fact, and d donors due to the existence of the racemic mixture
the X-ray molecular structures along with the DFT calculations (Fig. S3 and S4, ESI†). Beyond these strong interactions, there
reveal, in general, the preferred formation of the L-diastereomer are several other weak interactions in the crystal packing of
in most enantiopure crystals of R-ligated complexes and the both complexes (see Table S1, ESI†). Therefore, the above
D-diastereomer in most enantiopure crystal structures of the discussion indicates that the most and the strongest inter-
S-ligated complexes, in the context of induced chirality at the metal molecular interactions in the crystal packing of both complexes
2
9–31
ꢁ
and diastereoselection phenomenona.
The optimized struc- occur between the cationic unit and the NO3 counter ion
1
1
tures for the enantiomeric pairs K-[Pd(pic)R3] and D-[Pd(pic)S3]
(Table S1 and Fig. S3, S4, ESI†), reflecting the importance of the
ꢁ
NO
3
units to the stabilization of the crystal structure.
Hirshfeld surface analyses
In order to quantify the various intermolecular interactions,
Hirshfeld surfaces (HSs) and their associated fingerprint plots
3
5
were calculated using Crystal Explorer.
The HSs of
[
Pd(pic)S2]NO and [Pd(pic)R3]NO with their fingerprint plots
3
3
are illustrated in Fig. S5 (ESI†), showing the surfaces that have
been mapped over a dnorm range of ꢁ0.5 to 1.2 Å. The percentages
of contributions to various types of contacts in the fingerprint
plots are summarized in Fig. S6 (ESI†). It should be noted
that due to the similarity in the crystal packing of the two
Fig. 9 DFT optimized structures (gas phase) for the diastereomeric pair
K-[Pd(pic)R3] and D-[Pd(pic)R3] , calculated at B3LYP/SDD.
+
+
3 3
enantiomeric complexes ([Pd(pic)R2]NO and [Pd(pic)S2]NO )
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3 3
in the structure of [Pd(pic)rac2]NO , only [Pd(pic)S2]NO was
investigated and reported in this section (see the ESI† for more
details and also Fig. S7).
Experimental and calculated UV–vis and ECD spectra
The UV–vis absorption spectra were recorded in dichloro-
methane solutions for all complexes and show consistent
similarities along the series (with R or S-ligands) with little
changes in the band maxima (Fig. 10). All spectra show strong
bands/shoulders below ca. 330 nm with two maxima (lmax) at
250–260 nm and 280–290 nm, assigned as the intra-ligand p -
p* and n - p* transitions (LL), respectively. The spectra show
less intense broad bands at 350–500 nm (lmax = 380–406 nm),
assigned as the metal-to-ligand charge-transfer (MLCT)
3
6,37
transitions.
The spectra also show a very weak and broad
band in the visible region (450–600 nm; Fig. 10, inset),
3
6
attributed to the metal-centered d–d transitions. Indeed, the
ligand-field parameters for the square planar complexes are
relatively high, which overlap with the nearby strong MLCT
band, and thus, the forbidden d–d transition bands are not
separately visible (Fig. 10, inset). The n - p* and MLCT bands
shift to lower energy from 278 and 380 nm (H, H) to 284 and
395 nm (Cl, Cl) upon substitution with increasing electronegative
halogen atoms on the salicylal ring (Fig. 11). Here, the
destabilization of metal–ligand interactions with halogen
substituents weakens both the n - p* and MLCT bands and
results in a hypsochromic shift of ca. 6 and 15 nm, respectively.
The results suggest that the later band is relatively more charge-
3
7
transfer in nature (Fig. 11).
The electronic circular dichroism (ECD) spectra for all
complexes in dichloromethane (Fig. 12) show consistent
similarities for each enantiomeric pair along the series. The
Fig. 11 Shifts of the n - p* (top) and MLCT (bottom) bands maxima with
different substituents on the salicylal-ring in Pd(pic)-complexes with
spectra show several bands with opposite Cotton effects, R-ligands (ca. 0.022–0.083 mM) in dichloromethane at 25 1C.
associated with the different electronic transitions as discussed
in the UV–vis spectra. The observed mirror–image relationships
of the ECD spectra indicate the enantiopurity or enantiomeric
excess of R and S-ligated complexes in solution. The spectral
3
and [Pd(pic)S3]NO , are characterized by the following series of
bands (sign and strength) at ca. 400 nm (ꢂ, weak), 278 nm
(
ꢂ, weak) and 255 nm (ꢂ, strong), respectively.
data for the enantiomeric pairs, for example, [Pd(pic)R3]NO
3
We calculated the UV–vis spectra for the diastereomeric pair
+
+
K-[Pd(pic)R3] /D-[Pd(pic)R3] , which are identical (Fig. S8,
ESI†) and correspond well to the experimental spectra with
little shifts in the bands’ maxima (Fig. 13). The calculated
ECD spectra for the diastereomeric pair K-[Pd(pic)R3] /
D-[Pd(pic)R3] and the enantiomeric pair K-[Pd(pic)R3] /
+
+
+
+
D-[Pd(pic)S3] show the expected mirror–image relationships
(Fig. S14 and S9 in ESI†). The calculated spectra exhibit
different bands with opposite Cotton effects and correspond
well to the experimental spectra with little shifted band maxima
2
9–31
(Fig. 14), as reported in the related complexes.
Some
selected and simplified assignments on the calculated UV–vis
spectra are made on the basis of the orbital and population
+
analyses for K-[Pd(pic)R3] and data are listed in Table 2. Again,
the data are similar to the experimental results (Table 2). The
band at 373 nm with a significant oscillator strength, 0.0513
(
f ), arises mainly from a combination of the metal–metal and
Fig. 10 UV–vis spectra for the Pd(pic)-complexes with R-ligands
ca. 0.022–0.083 mM) in dichloromethane at 25 1C.
(
metal–ligand electronic transitions with the highest MO
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Fig. 12 ECD spectra for all enantiomeric pairs of [Pd(pic)(R)]NO
3
and [Pd(pic)(S)]NO
3
(ca. 0.20–0.30 mM) complexes in dichloromethane at 25 1C (cell
path length: 2.0 mm).
3
Fig. 13 Experimental spectrum for [Pd(pic)R3]NO (ca. 0.022 mM) in
+
dichloromethane at 25 1C. The calculated spectrum for [K-Pd(pic)R3] ,
calculated at B3LYP/SDD with PCM in dichloromethane (Gaussian band
shape with an exponential half-width, s = 0.33 eV).
Fig. 14 Experimental ECD spectra for the enantiomeric pair
Pd(pic)R3]NO and [Pd(pic)S3]NO (ca. 0.20 mM and 2 mm optical path)
in dichloromethane at 25 1C. The calculated ECD spectra for [L-
[
3
3
+
+
contributions of 72% for HOMO to LUMO transitions. Indeed, Pd(pic)R3] and [D-Pd(pic)S3] , calculated at B3LYP/SDD with PCM in
there are some other intense bands on the simulated spectra,
which are also close to the experimental bands (Fig. 10, Table 2).
dichloromethane (the Gaussian band shape with an exponential half-
width, s = 0.33 eV).
The frontier molecular orbitals, HOMO and LUMO are shown in
Fig. 15. The HOMO is only localized on the metal–d
moiety, while the LUMO is localized on both the metal–dz
ligand–p moieties. The energy gap between the HOMO and
z
2
electron
and with ligand folding angles (with respect to the coordination
plane around the metal ion) explore induced chirality at-metal
and afford mixtures of the two diastereomers (D and L), which
2
and
ꢁ1
LUMO is considerably low (DE = 17.58 kcal mol ) and thus
g
diastereoselectively favours one as the major diastereomer
involves a significant contribution in the excitation protocol.
29b
(Fig. 9),
as discussed above. Though the induced chirality
and diastereoselection phenomena are quantitative in the
solid-state (evidenced by using the X-ray and DSC analyses), a
dynamic diastereomeric equilibrium between the two diaster-
Excited state properties and D vs. K-chirality at-metal in
solution
The four-coordinated and slightly distorted square-planar eomers prevails in solution (evidenced by using the variable
1
29–31
metal complexes with asymmetrical bidentate chiral ligands time and temperature H NMR, ECD and VCD spectra).
We
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+
Table 2 Some selected and simplified assignments on the simulated spectrum for K-[Pd(pic)R3] , calculated at B3LYP/SDD with PCM in
dichloromethane
a
b
c
l/nm
Oscillator strength (f)
MO contribution (%)
Assignments
4
3
2
2
2
05 (410sh)
73 (390)
85 (280sh)
49 (255)
39
0.0168
0.0513
0.2885
0.1092
0.5760
Hꢁ1 - L + 1 (67), H - L (25)
Hꢁ1 - L + 1 (22), H - L (72)
Hꢁ3 - L (26), Hꢁ2 - L (62)
Hꢁ4 - L + 2 (17), H - L + 7 (39)
Hꢁ6 - L (24), H - L + 7 (28)
MM, ML
MM, ML
MM, ML, LL
MM, ML, LL
MM, ML, LL
a
b
c
The experimental values are in parentheses. H/L for HOMO/LUMO. MM, ML and LL for metal-centered (d–d), metal–ligand/ligand–metal and
ligand-centered transitions, respectively.
3
experimental ECD spectrum for [Pd(pic)R3]NO with the calculated
+
+
spectra for the diastereomeric pair K-[Pd(pic)R3] /D-[Pd(pic)R3]
shows the experimental spectrum to correspond well to
+
K-[Pd(pic)R3] (Fig. 14). The results suggest that the spectrum
for [Pd(pic)R3]NO
3
corresponds to a diastereomeric excess of
K-[Pd(pic)R3] (i.e., the major diastereomer) in solution. Similarly,
the spectrum for [Pd(pic)S3]NO corresponds to a diastereomeric
excess of D-[Pd(pic)S3] (i.e., the major diastereomer) in solution
+
3
+
1
(
(
Fig. 14). The H NMR spectrum for [Pd(pic)R3]NO
Fig. 5) shows one set of peaks for each proton, in contrast to the
expected two sets of peaks for each proton corresponding to the two
3 3
in CDCl
3
0a,b
diastereomers,
suggesting a rapid dynamic diastereomeric
equilibrium between the two diastereomers in the context of the
1
H NMR time scale in solution.
Biological evaluation of the compounds
Stability. All compounds were evaluated over time by
monitoring their stability in DMSO, the co-solvent used for
the biological assays. These studies were performed during 24 h
1
at room temperature by using H NMR in DMSO-d
6
.
From the X-ray studies of the [Pd(pic)R3]NO
3
and
[
Pd(pic)rac2]NO complexes, we proved that only isomer A
3
can be formed for the Pd(II)-complexes (Fig. 8). Also, a certain
configuration at the ligand, R or S, causes a quantitative
induction of a certain configuration at the metal ions (diaster-
eoselection) as L for the [Pd(pic)R]NO
3 3
and D for [Pd(pic)S]NO
complexes. The appearance of two signals in DMSO-d
6
,
different from the solid state and deuterated chloroform
solution, clearly indicates the existence of an equilibrium
between D- and L-diastereomers for all the Pd(II)-complexes
Fig. 15 The frontier molecular orbitals, HOMO and LUMO for
K-[Pd(pic)R3] , calculated at B3LYP/SDD with PCM in dichloromethane.
+
except [Pd(pic)R5]NO (not detectable) (for example see the
3
1
H NMR spectra of [Pd(pic)S2]NO3 and [Pd(pic)R5]NO3 in
attempted to correlate the experimental ECD spectra with the Fig. S10, ESI†). As shown in Fig. 16 and Fig. S10 (ESI†), we
calculated spectra and hence to estimate the equilibrium shift can see only one peak for the methyl proton in [Pd(pic)R5]NO
i.e., the preferred formation of diastereomer) in solution. It is Also, in all complexes except [Pd(pic)S1]NO , the equilibrium is
well documented that the overall nature of the chiral metal- towards the stable diastereomer (L-diastereomer for the
complexes is mainly reflected by their ECD spectra, which, in [Pd(pic)R]NO complexes and D-diastereomer for the [Pd(pic)S]
particular, are used to determine the absolute configuration at NO complexes) (Table 3).
3
.
(
3
3
3
the metal center (i.e., L vs. D) following the MLCT and/or d–d
For a quantitative evaluation, we chose the methyl proton
transition bands in solution. In fact, combined studies on the represented by a doublet above 1.5 ppm for each diastereomer
experimental and calculated ECD spectra have successfully (Fig. 16). In accordance with the X-ray structures in the solid-
3 3
been used to determine the absolute configuration at-metal, state of [Pd(pic)R3]NO and [Pd(pic)rac2]NO , we assume that
resulting from induced chirality and diastereoselection in S or the methyl proton peak with a higher integration value
2
9–31
R-ligated complexes.
Thus, the comparison of the relatively downfield corresponds to the major diastereomer
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1
Fig. 16 Display of part of the VT H NMR spectrum of [Pd(pic)S2]NO
3 3 3 3 6
, [Pd(pic)R5]NO , [Pd(pic)S1]NO and [Pd(pic)R1]NO in DMSO-d solution.
The NMR signals around 1.75 ppm and 1.50 ppm account for the methyl proton of the D- and L-diastereomers.
Table 3 Change of diastereomers in L- or D-Pd-R (D- or L-Pd-S) at The variation in diastereomeric ratios suggests a unique equili-
1
variable times recorded by H NMR in DMSO-d
6
. The diastereomeric ratio
brium between the L- and D-diastereomers for each complex in
solution, which is obviously different for different substituents
on the salicylaldehyde ring. This suggests that the substitution
at the salicylaldehyde ring has a strong impact on the thermo-
dynamic equilibrium.
(
%) is from the integration of the methyl proton signals
%
Compound
L/D (in initial time)
L/D (after 24 h)
[
[
[
[
[
[
[
[
[
[
[
[
[
[
Pd(pic)R1]NO
Pd(pic)R2]NO
Pd(pic)R3]NO
Pd(pic)R4]NO
Pd(pic)R5]NO
Pd(pic)R6]NO
Pd(pic)R7]NO
3
3
3
3
3
3
3
78/22
72/28
77.5/22.5
86/14
100/0
78/22
78/22
74/26
40/60
19/81
10/90
7.5/92.5
23/77
34/66
78/22
71/29
77.5/22.5
84/16
100/0
78/22
78/22
74/26
41/59
19/81
10/90
5.5/94.5
22/78
34/66
Also, the ratio of the diastereomers remains unaltered
(or with small changes) during the whole experimental time
(Table 3). This result indicates that the kinetics for the L- to
D- interconversions do not depend on the substituents on the
salicylaldehyde ring.
Pd(pic)S1]NO
Pd(pic)S2]NO
Pd(pic)S3]NO
3
All compounds are stable in this solvent (DMSO-d ). The
6
3
3
only exception was observed for the compound [Pd(pic)S1]NO ,
3
Pd(pic)S
4
]NO
3
in which the precipitation of a blackish product was observed,
while the compound remaining in solution maintained its
original structure.
Pd(pic)S5]NO
Pd(pic)S6]NO
Pd(pic)S7]NO
3
3
3
1
According to the VT H NMR data, the equilibrium for both
the [Pd(pic)S1]NO
the L-diastereomer (Fig. 16 and Table 3). These results are in
L for ([Pd(pic)R]NO and D for ([Pd(pic)S]NO ) and the down- agreement with the other results for [Pd(pic)R1]NO3 and in
3 3
and [Pd(pic)R1]NO complexes is towards
(
3
3
field one to the minor diastereomer (D for ([Pd(pic)R]NO
3
and L contradiction with the other results for [Pd(pic)S1]NO
3
. It
for ([Pd(pic)S]NO ) in solution (Fig. 16). We estimated the ratios should be noted that the [Pd(pic)R1]NO
3
3
and [Pd(pic)S1]NO
3
of diastereomers (L:D in %) based on the integration values of complexes are the only complexes without halogen-substituted
the methyl proton peaks at ca. d 1.75 and 1.50 ppm (Table 3). on the salicylaldehyde ring.
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Cytotoxicity assessment of the Pd(II)-complexes in MDA-MB-231
and MCF-7 breast cancer cell lines
The cytotoxic activity of all fourteen complexes was tested in the
human breast cancer cell lines MDA-MB-231 and MCF-7 at 24 h,
using the colorimetric MTT assay. These cell lines were chosen
considering their different degrees of aggressiveness. While
MDA-MB-231 is hormone independent and invasive, being
highly aggressive, MCF-7 is hormone dependent and non-
invasive. The cells were treated with the compounds within
the concentration range of 0.1 mM to 100 mM. All compounds
are cytotoxic in the conditions tested with the IC50 values
ranging from 5.8 to 45.5 mM in MDA-MB-231 cells and 5.5 to
21.7 mM in MCF-7 cells (Table 4), thus being more active in the
hormone dependent cell lines. Importantly, the introduction of
the halogen(s) at the Schiff base led to an increase of cytotoxicity
up to B6 and 8-fold for R and S isomers, respectively, in the
MDA-MB-231 cell line, and up to B3 and 4-fold for R and S
isomers, respectively, in the MCF-7 cell line. Also, the double
functionalization afforded more cytotoxic compounds, evidencing
the role of halogen in the overall activity (Fig. 17). Compounds
Fig. 17 IC50 values (mM) obtained at 24 h incubation for the enantiomeric
pairs in the MDA-MB-231 and MCF-7 breast cancer cells.
significantly reduce the ability of the cells to form colonies at
the IC50 values (Fig. S11, ESI†). When the concentration was
decreased to 25% of the IC50 values, the differences between
the compounds could be noticed (Fig. 18). In this case, it seems
that the majority of the R enantiomers are more efficient in
inhibiting the colony formation ability of the MDA-MB-231 cells
than their S pairs. This could possibly indicate differences in
the mechanism of action of the different enantiomers. It is also
important to mention that the ability of the compounds to
inhibit the formation of colonies does not correlate with their
3 3
with two iodine substituents, [Pd(pic)R6]NO and [Pd(pic)S6]NO ,
exhibited the highest cytotoxicity. Compared with the data from
the literature, one can observe that some of the Pd(II)-complexes
presented here are among the best reported in the MDA-MB-231
38
cell line (taking also into consideration the incubation time) and
also much better than cisplatin in the same experimental
39
conditions (122 ꢂ 25 mM). Kumar et al. described a series
of palladium complexes of the 1,2,4-triazole-derived chiral
N-heterocyclic carbene ligands aiming at studying the influence
cytotoxicity. For example, the compound [Pd(pic)S7]NO is the
3
most cytotoxic compound; yet it is the compound where at 1/4
of the IC50 values, more colonies are detected. In contrast,
16a
of chirality on the compound’s anticancer activity. The authors
did not see any chirality-related influence for any of the enantio-
meric pairs, an outcome that is in agreement with what we
observed with our Pd(II)-complexes endowed with chiral
Schiff bases.
[
3
Pd(pic)R1]NO was the compound with the highest IC50, but it
showed remarkable ability to inhibit the formation of colonies.
Evaluation of the cell death mechanism induced by selected
compounds
The effects of the Pd(II)-complexes in the colony formation
potential of the MDA-MB-231 cells
The cell death mechanism was assessed using the annexin
V/propidium iodide (AV/PI) cytometry-based assay. Annexin V is
To evaluate the colony formation potential of the Pd(II)- a marker of early apoptosis, while PI is a marker of necrosis.
complexes, the MDA-MB-231 breast cancer derived cell line For this assay, three pairs of compounds were selected,
was exposed to 1/4 of the IC50 values and the IC50 values of the [Pd(pic)R6]NO
different compounds for 24 hours, after which the cellular NO given the good cytotoxicity presented by these compounds,
medium was removed and the cells were maintained in the and [Pd(pic)R2]NO /[Pd(pic)S2]NO , since these pairs of iso-
3 3 3
/[Pd(pic)S6]NO and [Pd(pic)R7]NO /[Pd(pic)S7]
3
3
3
culture for 8 days, mimicking what would happen in a chemo- mers were very efficient in inhibiting the formation of the
therapy cycle. Our results showed that all tested compounds MDA-MB-231 cell colonies. Thus, the cancer cells were
Table 4 IC50 values (mM) for the Pd(II)-complexes at 24 h incubation, in MDA-MB-231 and MCF-7 breast cancer cells
IC50
IC50
Compound
MDA-MB-231
MCF-7
Compound
MDA-MB-231
MCF-7
[
[
[
[
[
[
[
Pd(pic)R1]NO
Pd(pic)R2]NO
Pd(pic)R3]NO
Pd(pic)R4]NO
Pd(pic)R5]NO
Pd(pic)R6]NO
Pd(pic)R7]NO
3
3
3
3
3
3
3
45.5 ꢂ 5.3
42.2 ꢂ 2.7
31.0 ꢂ 2.2
9.6 ꢂ 1.2
13.6 ꢂ 0.9
7.8 ꢂ 0.5
8.5 ꢂ 0.7
21.5 ꢂ 1.1
16.8 ꢂ 1.2
15.5 ꢂ 0.9
6.7 ꢂ 0.8
12.2 ꢂ 0.6
6.9 ꢂ 0.3
7.3 ꢂ 0.4
[Pd(pic)S1]NO
[Pd(pic)S2]NO
[Pd(pic)S3]NO
[Pd(pic)S4]NO
[Pd(pic)S5]NO
[Pd(pic)S6]NO
[Pd(pic)S7]NO
3
3
3
3
3
3
3
43.7 ꢂ 10.0
29.1 ꢂ 2.0
27.6 ꢂ 2.3
12.6 ꢂ 0.8
9.7 ꢂ 0.7
5.8 ꢂ 0.4
8.8 ꢂ 0.8
20.2 ꢂ 1.3
20.2 ꢂ 1.1
20.4 ꢂ 1.5
7.3 ꢂ 0.4
8.1 ꢂ 0.9
5.5 ꢂ 0.4
6.7 ꢂ 0.4
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Fig. 18 Colony formation assay of the MDA-MB-231 cell line after exposure to the Pd(II)-complexes. (A) The analysis of the clonogenic ability, after 24 h
of incubation with 1/4 of the IC50 values. The values represent mean ꢂ S.D. of at least two independent experiments. Statistical analysis was performed by
one-way ANOVA with Dunnett’s multiple comparisons test. *P r 0.0001 compared with control. (B) The representative images of the colony formation
assay in the MDA-MB-231 cell line.
3 3 3
Table 5 Percentage of MDA-MB-231 cells in each state after treatment with complexes [Pd(pic)R2]NO , [Pd(pic)S2]NO , [Pd(pic)R6]NO ,
[
Pd(pic)S6]NO
3
, [Pd(pic)R7]NO
3
and [Pd(pic)S7]NO
3
at IC50 concentrations for 24 h incubation
%
Vital
% Apoptotic
cells AV /PI
% Late apoptotic
cells AV /PI
% Necrosis
AV /PI
ꢁ
ꢁ
+
ꢁ
+
+
ꢁ
+
cells AV /PI
Control
99.6
41.5
70.0
31.9
59.4
66.7
64.5
0.1
19.4
19.7
29.9
21.7
18.6
19.5
0.2
36.4
7.7
32.8
15.4
11.2
12.2
0.1
2.7
2.6
5.4
3.5
3.5
3.8
[
[
[
[
[
[
Pd(pic)R2]NO
Pd(pic)S2]NO
Pd(pic)R6]NO
Pd(pic)S6]NO
Pd(pic)R7]NO
3
3
3
3
3
Pd(pic)S7]NO
3
incubated with the compounds for 24 h at their IC50 values. The complexes were synthesized from the reaction between
results show that all compounds led to an increase in the [Pd(pic)Cl (pic 2-picolylamine) and the enantiopure
percentage of the AV /PI and AV /PI stained cells (Table 5) Schiff bases (R or S)-N-1-(phenyl)ethyl-2,4-X ,X -salicylaldimine.
in comparison to the negative control indicating that they The single crystal X-ray structural analyses revealed that the com-
2
]
=
+
ꢁ
+
+
1
2
induce cell death by apoptosis.
pounds have pseudo square-planar geometry around the metal
center. The observed mirror–image relationships of the ECD
spectra suggest enantiomeric excess of the [Pd(pic)R]NO and
3
[
Pd(pic)S]NO
3
in solution. The combined studies on the X-ray
Conclusions
structures, ECD spectra and DFT/TDDFT calculations explore the
A series of heteroleptic enantiopure [Pd(pic)(R or S)-N-1-(phe- induced chirality at the metal center, which diastereoselectively
+
nyl)ethyl-2,4-X
1
,X
2
-salicylaldiminate)]NO
3
, [Pd(pic)(R or S)]NO
3
prefers L-[Pd(pic)R3] as the major diastereomer in the solid-state,
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ꢁ
1
3
gas phase or solution. The anticancer activity of all compounds (CH ). The IR data (KBr, cm ): 3433m (br, nOH), 3062, 2924w
was assessed against MDA-MB-231 and MCF-7 cancer cells and (nC–H) and 1628vs (nCQN).
showed that all Pd(II)-complexes are cytotoxic in the conditions HR2. Yield: 93%. Analysis calculated for C H ClNO
1
5
14
they were tested. The introduction of halogens to the Schiff base (259.73): C, 69.37; H, 5.43; and N, 5.39. Found: C, 69.28; H,
1
ligands led to a clear increase of cytotoxicity. Moreover, all 5.50; and N, 5.67%. The H-NMR data [400 MHz, CDCl ] d
3
compounds inhibited the formation of MDA-MB-231 colonies (ppm): 13.54 [s, 1H (OH)]; 8.36 [s, 1H (imine)]; 7.56–6.93 [m, 8H
to different extents and induced cell death by apoptosis, (Ar)]; 4.61 [q, 1H (benzyl)]; and 1.66 [d, 3H (methyl)]. The
1
3
indicating in some cases the involvement of chirality-related
3
C-NMR data [100 MHz, CDCl , in ppm]: 162.2 (CQN), 160.1,
mechanisms. Further studies to elucidate the active species are 143.3, 136.9, 132.5, 128.7, 126.5, 119.4, 68.4 (chiral C), and 24.8
ꢁ1
presently underway.
3
(CH ). The IR data (KBr, cm ): 3434m (br, nOH), 2928w (nC–H)
and 1630vs (nCQN).
HR3. Yield: 97%. Analysis calculated for C H BrNO
1
5
14
(
303.05): C, 59.23; H, 4.64; and N, 4.60. Found: C, 59.89; H,
Experimental section
Chemicals and instrumentation
1
4
.58; and N, 6.39%. The H-NMR data [400 MHz, CDCl
(ppm): 13.60 [s, 1H (OH)]; 8.34 [s, 1H (imine)]; 7.41–6.87 [m, 8H
Ar)]; 4.60 [q, 1H (benzyl)]; and 1.66 [d, 3H (methyl)]. The
3
] d
(
PdCl
2 3
, AgNO , 5-bromosalicylaldehyde, 5-chlorosalicyladehyde,
1
3
C-NMR data [100 MHz, CDCl
3
, in ppm]: 162.2 (CQN), 160.0,
3,5-dibromosalicylaldehyde, 3,5-diiodosalicylaldehyde, 3,5-
1
43.3, 134.9, 133.9, 127.8, 127.4, 126.4, 120.2, 119.0, 68.4 (chiral
dichlorosalicylaldehyde, 3-bromo-5-chlorosalicylaldehyde, sali-
cylaldehyde, 2-picolylamine, (R)-(+)-a-methylbenzylamine and
ꢁ
1
C), and 24.7 (CH ). The IR data (KBr, cm ): 3429m (br, nOH),
2
3
919w (nC–H) and 1625vs (nCQN).
(
S)-(ꢁ)-a-methylbenzylamine were purchased from Sigma-
The other ligands were fully characterized in a previous
Aldrich and used without further purification. Commercial
solvents were distilled and then used for the preparation of
the ligands and complexes. The FT-IR spectra were recorded on
2
3
article.
Synthesis of the complexes
[Pd(pic)Cl was synthesized and purified as previously
described. The synthesis of all complexes followed a generic
synthetic route. Briefly, to a stirring aqueous solution of 1 mmol
of [Pd(pic)Cl ], 2 mmol of AgNO was added at 20 1C in the
ꢁ1
a JASCO, FT/IR-6300 spectrometer (4000–400 cm ) in KBr
1
13
2
]
pellets. The H and C NMR spectra were recorded on a Bruker
Avance III 400 spectrometer at room temperature. The elemental
analyses were performed on Leco, CHNS-932 and PerkinElmer
3
3
2
3
7300 DV elemental analyzers. The electronic absorption spectra
dark. After the white AgCl precipitate was filtered off, 1 mmol of
the appropriate ligand dissolved in methanol was added to the
yellow filtrate and the solution was refluxed for 2 h. After
reducing the volume of the solvent, the yellow precipitate was
formed. The precipitate was collected by filtration, washed with
cold methanol and diethyl ether and dried in air.
(
UV–vis) were recorded with a PerkinElmer Lambda 35 spectro-
photometer. The electronic circular dichroism (ECD) spectra
were recorded at ca. 25 1C on a Jasco J-720 spectropolarimeter
with a UV–vis (180–800 nm) photomultiplier (EXEL-308). The
stock solutions of the complexes were prepared in CH Cl at ca.
2
2
2
.0 mM. Dilutions were made to measure the spectra at different
R and S-enantiomers have identical IR- and NMR-spectra.
concentrations, typically 20–85 mM and 200 mM. The ECD
measurements were done using quartz Suprasil cells of 0.2 or
1
s
[
Pd(pic)S1]NO
Yield: 81% and 87%, respectively. Analysis calculated for
Pd: C, 50.36; H, 4.43; and N, 11.19. Found
for [Pd(pic)S-1]NO : C, 50.39; H, 4.41; and N, 11.23%. Found
for [Pd(pic)R-1]NO : C, 50.40; H, 4.45; and N, 11.21%. The
H-NMR data [400 MHz, CDCl ] d (ppm): 8.65 [d, 1H (imine)];
.78–6.62 [m, 13H (Ar)]; 5.94, 4.08 [m, 2H (NH )]; 5.36 [q, 1H
3 3
and [Pd(pic)R1]NO
.0 cm optical path. The DA values measured correspond to DA =
De ꢃ b ꢃ C, where b is the optical path and C is the concentration
21 22 4 4
C H N O
of the complex. The measuring conditions were as follows: data
ꢁ1
3
pitch of 0.5 nm, speed of 200 nm min , response of 2 s,
bandwidth of 0.5 nm, and 3 accumulations.
3
1
3
7
2
Synthesis of the chiral Schiff bases
(
benzyl)]; and 4.35, 4.14 [t of d, 2H (CH )], 1.77 [d, 3H (CH )].
2 3
1
3
All chiral Schiff base ligands were synthesized using identical The C-NMR data [100 MHz, CDCl , in ppm]: 163.7 (CQN),
3
reaction conditions and were achieved by the Schiff condensation 163.0, 160.9, 146.1, 141.7, 140.0, 136.2, 135.0, 129.5, 128.3,
of (R)-(+)-a-methylbenzylamine and (S)-(ꢁ)-a-methylbenzylamine 126.8, 123.3, 121.3, 120.2, 119.6, 116.4, 68.3 (chiral C), 51.5,
ꢁ1
3
with the appropriate halogen substituted salicylaldehyde in and 23.1 (CH ). The IR data (KBr, cm ): 1612vs (nCQN),
2
3
aqueous solution as previously published.
HR1. Yield: 90%. Analysis calculated for C15
C, 79.97; H, 6.71; and N, 6.22. Found: C, 80.06; H, 6.52; and N, respectively. Analysis calculated for C H ClN O Pd: C, 47.12;
1383vs (nNO
3
), and 824vs (nNO
3
).
H
15NO (225.29):
[Pd(pic)S2]NO
3
and [Pd(pic)R2]NO . Yield: 92% and 86%,
3
2
1
21
4 4
1
6
.25%. The H-NMR data [400 MHz, CDCl ] d (ppm): 13.60 H, 3.95; and N, 10.47. Found for [Pd(pic)S-2]NO : C, 47.07; H,
3
3
[
[
[
s, 1H (OH)]; 8.44 [s, 1H (imine)]; 7.39–6.88 [m, 9H (Ar)]; 4.58 3.99; and N, 10.51%. Found for [Pd(pic)R-2]NO : C, 47.09; H,
3
1
3
1
q, 1H (benzyl)]; and 1.66 [d, 3H (methyl)]. The C-NMR data 3.94; and N, 10.43%. The H-NMR data [400 MHz, CDCl
100 MHz, CDCl , in ppm]: 162.9 (CQN), 160.5, 143.3, 131.8, (ppm): 8.56 [d, 1H (imine)]; 7.76–6.89 [m, 12H (Ar)]; 5.92, 4.37
30.9, 128.2, 128.8, 125.9, 118.3, 118.1, 68.0 (chiral C), and 24.5 [m, 2H (NH )]; 5.21 [q, 1H (benzyl)]; and 4.18, 3.98 [t of d, 2H
3
] d
3
1
2
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NJC
1
3
(
CH
in ppm]: 162.2 (CQN), 160.2, 143.3, 137.0, 132.5, 132.0, 130.5, 41.05; H, 3.32; and N, 9.10%. The H-NMR data [400 MHz,
28.8, 127.4, 126.4, 119.4, 68.5 (chiral C), and 24.8 (CH ). The IR CDCl ] d (ppm): 9.07 [d, 1H (imine)]; 7.90–7.24 [m, 11H (Ar)];
2
)], 1.67 [d, 3H (CH
3
)]. The C-NMR data [100 MHz, CDCl
3
,
3
41.05; H, 3.24; and N, 9.11%. Found for [Pd(pic)R-7]NO : C,
1
1
3
3
ꢁ
1
data (KBr, cm ): 1615vs (nCQN), 1384vs (nNO ), and 825vs 6.22, 4.42 [m, 2H (NH )]; 5.36 [q, 1H (benzyl)]; and 4.32, 4.09
3
2
1
3
(
nNO3).
Pd(pic)S3]NO
respectively. Analysis calculated for C21
H, 3.65; and N, 9.66. Found for [Pd(pic)S-3]NO
[t of d, 2H (CH )], 1.78 [d, 3H (CH )]. The C-NMR data
2 3
[
3
and [Pd(pic)R3]NO
3
3
. Yield: 85% and 90%, [100 MHz, CDCl , in ppm]: 163.1 (CQN), 160.5, 147.1, 140.2,
H21BrN
4
O
4
Pd: C, 43.51; 138.1, 132.9, 129.6, 128.6, 127.0, 123.5, 121.3, 117.2, 68.7 (chiral
ꢁ1
3
: C, 43.51; H, C), and 23.0 (CH
3
). The IR data (KBr, cm ): 1614vs (nCQN),
).
. This complex was prepared exclusively for
3
3
.66; and N, 9.64%. Found for [Pd(pic)R-3]NO
.61; and N, 8.69%. The H-NMR data [400 MHz, CDCl ] d [Pd(pic)rac2]NO
3
: C, 43.55; H, 1383vs (nNO
3 3
), 834vs (nNO
1
3
3
(
ppm): 8.52 [d, 1H (imine)]; 7.75–6.79 [m, 12H (Ar)]; 6.01, 4.55 the purpose of growing X-ray-quality crystals and theoretical
1
[m, 2H (NH )]; 5.17 [q, 1H (benzyl)]; and 4.18, 3.99 [t of d, 2H calculations. Its H NMR spectra were identical with those of
2
1
3
(
CH )], 1.64 [d, 3H (CH )]. The C-NMR data [100 MHz, CDCl3, [Pd(pic)R2]NO3 and [Pd(pic)S2]NO . The yield is more than
2
3
3
in ppm]: 162.2 (CQN), 162.2, 160.1, 143.4, 139.7, 135.6, 134.9, 85%. Other analytical data were not determined. The route
33.5, 128.8, 127.4, 126.4, 120.2, 119.0, 68.4 (chiral C), and 24.7 for the synthesis of this compound is similar to other
1
ꢁ
1
(
CH
and 822vs (nNO
Pd(pic)S4]NO
respectively. Analysis calculated for C H Cl N O Pd: C, 44.27; (phenyl)ethyl-2,4-Cl-salicylaldimine.
3
). The IR data (KBr, cm ): 1614vs (nCQN), 1384vs (nNO
3
), complexes. The only difference is the use of the racemic Schiff
3
).
base ligand, (R/S)-N-1-(phenyl)ethyl-2,4-Cl-salicylaldimine,
[
3 3
and [Pd(pic)R4]NO . Yield: 78% and 85%, instead of the enantiopure Schiff base ligand, (R or S)-N-1-
2
1
20
2 4 4
H, 3.54; and N, 9.83. Found for [Pd(pic)S-4]NO : C, 44.24; H,
3
3
3
(
[
(
.57; and N, 9.86%. Found for [Pd(pic)R-4]NO : C, 44.24; H, Single crystal X-ray details
3
1
.53; and N, 9.81%. The H-NMR data [400 MHz, CDCl
ppm): 8.71 [d, 1H (imine)]; 7.66–6.96 [m, 11H (Ar)]; 5.79, 4.28
3
] d
The X-ray crystal data for [Pd(pic)R3]NO were collected from
3
multi-faceted crystals of suitable size and quality selected from
a representative sample of crystals of the same habit using an
optical microscope. In each case, the crystals were mounted on
MiTiGen loops, and the data collection was carried out in a cold
stream of nitrogen (150(2) K; Bruker D8 QUEST ECO; Mo K
radiation). All diffractometer manipulations were carried out
m, 2H (NH
2
)]; 5.10 [q, 1H (benzyl)]; and 4.08, 3.84 [t of d, 2H
1
3
CH
2
3 3
)], 1.53 [d, 3H (CH )]. The C-NMR data [100 MHz, CDCl ,
in ppm]: 162.2 (CQN), 160.1, 143.4, 139.7, 135.6, 134.9, 133.5,
28.8, 127.4, 126.4, 123.6, 119.8, 68.8 (chiral C), and 23.1 (CH ).
1
3
a
ꢁ1
The IR data (KBr, cm ): 1620vs (nCQN), 1383vs (nNO ), and
3
8
20vs (nNO3).
Pd(pic)S5]NO
respectively. Analysis calculated for C21
H, 3.06; and N, 8.51. Found for [Pd(pic)S-5]NO
40
using the Bruker APEX3 software. The structure solution and
refinement were carried out using the XS, XT and XL software,
embedded within the Bruker SHELXTL suite. For each
structure, the absence of additional symmetry was confirmed
[
3
and [Pd(pic)R5]NO
3
. Yield: 86% and 91%,
Pd: C, 38.30;
: C, 38.32; H,
: C, 38.34; H,
.10; and N, 8.55%. The H-NMR data [400 MHz, CDCl ] d
H
20Br
2
N
4
O
4
41
3
3
3
(
[
(
.11; and N, 8.54%. Found for [Pd(pic)R-5]NO
3
42
using ADDSYM incorporated in the PLATON program.
1
3
The single-crystal X-ray diffraction data for [Pd(pic)rac2]NO
were collected using an Oxford Diffraction Gemini R Ultra
diffractometer (Cu K , multilayer mirror, Ruby CCD area detector)
3
ppm): 9.05 [d, 1H (imine)]; 7.92–7.34 [m, 11H (Ar)]; 6.34, 4.65
m, 2H (NH )]; 5.36 [q, 1H (benzyl)]; and 4.35, 4.13 [t of d, 2H
2
a
1
3
CH )], 1.77 [d, 3H (CH )]. The C-NMR data [100 MHz, CDCl3,
2
3
at 295(2) K. Data collection, unit cell determination and data
reduction were carried out using the CrysAlis PRO software
in ppm]: 163.8 (CQN), 160.7, 147.1, 142.0, 136.0, 134.9, 129.6,
28.6, 127.0, 123.5, 121.8, 121.2, 115.2, 68.7 (chiral C), and 23.0
1
43
44
package using the Olex2 interface. The structure was solved
ꢁ
1
(
CH
and 860vs (nNO
Pd(pic)S6]NO
respectively. Analysis calculated for C H I N O Pd: C, 33.51;
3
). The IR data (KBr, cm ): 1612vs (nCQN), 1384vs (nNO
3
),
45
with the SHELXT 2015 structure solution program by intrinsic
).
2
3
phasing methods and refined by full-matrix least squares on |F|
[
3
and [Pd(pic)R6]NO
3
. Yield: 81% and 90%,
45
using SHELXL-2018/3. The non-hydrogen atoms were refined
2
1
20 2 4 4
anisotropically.
H, 2.68; and N, 7.44. Found for [Pd(pic)S-6]NO : C, 33.48; H,
3
More details concerning both crystal structures and their
refinements can be found in Table 6 or in the corresponding
CIF files (ESI).
2
2
.69; and N, 7.40%.; Found for [Pd(pic)R-6]NO : C, 33.50; H,
3
.67; and N, 7.44%. The H-NMR data [400 MHz, CDCl ] d
3
1
(
ppm): 9.14 [d, 1H (imine)]; 8.04–7.27 [m, 11H (Ar)]; 6.10, 4.51
[
(
m, 2H (NH
2
)]; 5.22 [q, 1H (benzyl)]; and 4.20, 4.00 [t of d, 2H
1
3
CH
2
3 3
)], 1.66 [d, 3H (CH )]. The C-NMR data [100 MHz, CDCl ,
Computational method
in ppm]: 163.7 (CQN), 160.4, 151.5, 147.9, 143.7, 140.2, 138.0,
1
29.5, 128.6, 127.1, 123.3, 121.3, 115.2, 68.5 (chiral C), and 22.8 To rationalize the experimental results, a computational pro-
ꢁ
1
46
(CH ). The IR data (KBr, cm ): 1612vs (nCQN), 1384vs (nNO3), cedure was performed with Gaussian 09. For optimization in
3
8
25vs (nNO3).
Pd(pic)S7]NO
respectively. Analysis calculated for C21
the gas phase, the initial geometry of the complex was
. Yield: 79% and 87%, generated from the X-ray structure of [Pd(pic)R3]NO . The
20BrClN Pd: C, four-coordinated and slightly distorted square-planar metal(II)-
1.07; H, 3.28; and N, 9.12. Found for [Pd(pic)S-7]NO : C, complexes with asymmetric bidentate chiral-Schiff base ligands
[
3
and [Pd(pic)R7]NO
3
3
H
4 4
O
4
3
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Paper
3 3
Table 6 Crystallographic data for [Pd(pic)R3]NO and [Pd(pic)rac2]NO
[
Pd(pic)R3]NO
21BrN
3
[Pd(pic)rac2]NO
3
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
a (1)
b (1)
C
21
H
4
O
4
Pd
C
21
H21ClN
4
O
4
Pd
579.73
150(2)
0.71073
535.27
295(2)
1.54184
Triclinic
P1%
9.0681(3)
10.7643(3)
11.5628(4)
90.106(3)
109.775(3)
93.914(3)
1059.25(6)
2/1.678
Orthorhombic
P2
1 1 1
2 2
7.0780(3)
14.5585(5)
20.1360(8)
g (1)
Volume (Å )
3
2074.92(14)
4/1.856
2.858
1152
ꢁ
ꢁ
3
Z/calculated density (g cm
Absorption coefficient (mm
F(000)
)
1
)
8.544
540
y range (1)
h; k; l ranges
Reflections collected/unique
Refinement method
Data/restraints/parameters
2.460 to 33.193
ꢂ10; ꢂ22; ꢁ31, 30
47 690/7903 R(int) = 0.0457]
Full-matrix least-squares on F
7903/0/312
4.118 to 67.003
ꢂ10; ꢂ12; ꢁ12, 13
8646/3732 [R(int) = 0.0285]
Full-matrix least-squares on F
3732/0/289
2
2
2
Goodness-of-fit on F
1.043
1.048
Final R indices [I 4 2s(I)]
R indices (all data)
Absolute structure parameter
Largest difference peak and hole (e Å
CCDC number
R
R
1
= 0.0279/wR
= 0.0377/wR
2
= 0.05
= 0.0533
R
R
1
= 0.0271/wR
= 0.0284/wR
2
= 0.0691
= 0.0704
1
2
1
2
0.034(8)
ꢁ3
)
0.763 and ꢁ0.675
0.569 and ꢁ0.441
1989598
2007895
and with ligand folding angles (i.e., angles between the Biological evaluation
co-ordination plane around metal ion and the phenyl- and
salicylal-rings) exhibit induced chirality at-metal and provide
Stability studies. All compounds were studied for their stability
in 100% DMSO-d6 by using NMR spectroscopy. Their H-NMR
spectra were recorded at time set intervals, over a period of 24 hours.
1
two diastereomers with opposite configurations (i.e., D-Pd and
2
9b
L-Pd) along the C
2
-symmetry of the molecule. Thus, based on
the X-ray structure (dihedral angles and ligand folding angles),
Cell lines and culture conditions
+
[
Pd(pic)R3] can be best described as a L-diastereomer (i.e.,
+
The MDA-MB-231 and MCF-7 breast cancer human tumour
cells were purchased from ATCC. The MDA-MB-231 and MCF-7
cells were grown at 37 1C in 5% CO in Dulbecco’s modified
2
K-[Pd(pic)R3] ). Therefore, the geometry of the opposite config-
ured diastereomer D-[Pd(pic)R3] was built by mirror inversion
of K-[Pd(pic)R3] , followed by manual inversion of the chiral-
carbon center.
meric pair K-[Pd(pic)R3] /D-[Pd(pic)R3] and the enantiomeric
pair K-[Pd(pic)R3] /D-[Pd(pic)S3] were carried out using
+
+
2
9–31
Eagles’s medium (DMEM high glucose) (Capricorn Scientific)
or Dulbecco’s modified Eagles’s medium – DMEM (1X) +
GlutaMAX (Gibco), respectively, supplemented with 10% fetal
bovine serum (Capricorn Scientific). All cells were adherent
in monolayers and, upon confluence, were washed with
phosphate buffer saline (PBS) 1ꢃ and harvested by digestion
with trypsin 0.05% (v/v). Trypsin was inactivated by adding fresh
complete culture media to the culture flask. The cells were then
suspended and transferred into new, sterile, culture flasks, or
seeded in sterile test plates for different assays. All cells were
manipulated under aseptic conditions in a flow chamber.
The DFT optimizations for the diastereo-
+
+
+
+
the functional B3LYP and the basis sets SDD and LANL2DZ,
3
6
respectively (Fig. 9 and Fig. S2, ESI†). Both methods provide
almost similar geometry parameters that are comparable to the
X-ray results (Table 1). However, the optimized structure with
ꢁ1
B3LYP/SDD is more stable by 883.06 kcal mol than B3LYP/
LANL2DZ and hence shows relatively better agreement with the
experimental results (Table 1), as reported for the related Pd(II)-
3
6b,36c
complexes.
The excited state properties (i.e., UV–vis. and
ECD spectra) by TDDFT were calculated for the diastereomeric
Compound dilution and storage
+
+
pair K-[Pd(pic)R3] /D-[Pd(pic)R3]
pair K-[Pd(pic)R3] /D-[Pd(pic)S3] at B3LYP/SDD with the
and the enantiomeric
+
+
All compounds were dissolved in 100% DMSO, divided in
aliquots of 100 mL each and stored at ꢁ20 1C until use.
polarization continuum model (PCM) using dichloromethane
3
6
as the solvent. For calculations, 72 excited states (roots) were
Compound cytotoxicity evaluated using the MTT assay
considered (Table S2, ESI†). The assignments on the UV–vis
spectra and molecular orbitals (MOs) calculations were carried The cells were adherent in monolayers and, upon confluency,
out at the same level of theory. The spectra were generated using were harvested by digestion with trypsin. The cytotoxicity of the
4
7
the program SpecDis by applying the Gaussian band shape complexes against the tumor cells was assessed using the
with an exponential half-width of s = 0.33 eV. colorimetric assay MTT (3-(4,5-2-yl)-2,5-ditetrazolium bromide),
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which measures the conversion of the yellow tetrazolium into work. Centro de Qu ´ı mica Estrutural acknowledges Fundaç ˜a o
purple formazan by the mitochondrial redox activity in living para a Ci ˆe ncia e Tecnologia (FCT) for the Project UIDB/00100/
3
3
cells. For this purpose, cells (10 ꢃ 10 or 20 ꢃ 10 in 200 mL of 2020. This work was also funded in the scope of the project
medium, for MDA-MB-231 or MCF-7, respectively) were seeded PTDC/QUI-QIN/28662/2017 (FCT). A. Valente acknowledges the
into 96-well plates and incubated in a 5% CO2 incubator at CEECIND 2017 Initiative (CEECIND/01974/2017). I. Correia
37 1C. The cells were settled for 24 h followed by the addition of a acknowledges the program FCT Investigator (IF/00841/2012).
dilution series of the complexes in medium (200 mL). The We are grateful to Professor ABP Lever, Department of
complexes were first solubilized in 100% DMSO, given a 10 mM Chemistry, York University, Toronto and the Computecanada.ca
stock solution, and then in medium within the concentration (https://ccdb.computecanada.ca/) Ontario, Canada for using
range of 0.1–100 mM. DMSO did not exceed 1% even for the higher computational resources.
concentration used and was without cytotoxic effects. After 24 h of
incubation, the treatment solutions were removed by aspiration,
ꢁ
1
and MTT solution (200 mL, 0.5 mg mL in PBS) was added to
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