Comparative in vitro Studies of the Topoisomerase I Inhibition and Anticancer Activities of Metallated N-Confused Porphyrins and Metallated Porphyrins

Article abstract of DOI:10.1002/cmdc.201900633

Using the original approach, a series of metallated N-confused porphyrins and metallated porphyrins have been synthesized and characterized. For all the synthesized porphyrins, in vitro studies of cytotoxic activity against K562, U937, HL-60, Jurkat, A549 and HeLa cancer cell lines, the ability to induce apoptosis and effects on the cell cycle as well as the kinetics of proliferative activity of porphyrins and their respective metallated complexes in real time have been developed. The inhibitory activity of metallated porphyrins against human topoisomerase I and the possible mechanism of inhibition have been carried out by modelling using molecular docking.

Full text of DOI:10.1002/cmdc.201900633

Accepted Article
Title: Comparative In vitro Studies of the Topoisomerase I inhibition
and Anticancer activities of Metallated N-Confused porphyrins
and Metallated porphyrins
Authors: Harapriya Rath, Nyancy Halder, Prof. Lilya U. Dzhemileva,
Ilfir R. Ramazanov, Prof. Vladimir A. D’yakonov, and Usein M.
Dzhemilev
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Comparative In vitro Studies of the Topoisomerase I inhibition
and Anticancer activities of Metallated N-Confused porphyrins
and Metallated porphyrins
[
a]
* [b]
[b]
*[b]
Nyancy Halder, Lilya U. Dzhemileva, Ilfir R. Ramazanov, Vladimir A. D’yakonov,
Usein M.
[b]
*[a]
Dzhemilev, and Harapriya Rath,
Dedicated to Prof. Hiroyuki Furuta
[
a]
b]
Nyancy Halder, Prof (Dr.) Harapriya Rath
School of Chemical Sciences
Indian Association for the Cultivation of Science, 2A/2B Raja SC Mullick Road, Jadavpur, Kolkata 700032, India
E-mail: ichr@iacs.res.in
[
Prof. Lilya U. Dzhemileva, Ilfir R. Ramazanov, Prof. Vladimir A. D’yakonov, Dr. Usein M. Dzhemilev,
Laboratory of Catalytic Synthesis, Institute of Petrochemistry and Catalysis of RAS (IPC RAS), Prospect Octyabrya, 141, Ufa-450075, Russian Federation
Email:DyakonovVA@gmail.com
Supporting information for this article is given via a link at the end of the document.((Please delete this text if not appropriate))
Abstract: Using the original approach, a series of metallated N-
confused porphyrins and metallated porphyrins have been
synthesized and characterized. For all the synthesized porphyrins, in
vitro studies of cytotoxic activity against K562, U937, HL-60, Jurkat,
A549 and HeLa cancer cell lines, the ability to induce apoptosis and
effects on the cell cycle as well as the kinetics of proliferative activity
of porphyrins and their respective metallated complexes in real time
have been developed. The inhibitory activity of metallated porphyrins
against human topoisomerase I and the possible mechanism of
inhibition have been carried out by modelling using molecular
docking.
begun with the aim of overcoming the two key limiting factors in
development of the parent drug: high toxicity and low solubility in
water.
Introduction
Figure 1. The mechanism of Topoisomerase I action.
Effective cancer therapy evolves from the identification of a
molecule or process that can be targeted by a therapeutic agent
in a relatively selective and exploitable manner. Such endeavors
have led to the discovery of topoisomerases as potential
therapeutic targets. Topoisomerases (Topo) are universal
nuclear enzymes that modulate DNA supercoiling during DNA
replication, transcription, and chromatin assembly by forming a
This has resulted in the discovery of a number of water-soluble
camptothecin analogues that have now reached the clinic: CPT-l
1
(irinotecan), topotecan, 9-aminocamptothecin (9-AC) and
GI147211 (GG-211). CPT and its numerous derivatives
developed over the years thus display various antitumor
activities in a wide range of cancers, such as ovarian and lung
(topotecan), colon (irinotecan), or brain (gimatecan and
[
1-5]
reversible covalent Topo-DNA complex.
Topo I and Topo II
are two types of DNA topoisomerases, which cleave single or
double DNA strands leading to transient DNA single-strand
[
10−12]
belotecan).
Despite the fact that camptothecin and its
[
6-8]
breaks or double-strand breaks, respectively.
The mechanism
analogs (Fig. 2A) as Topoisomerase I poisons can effectively
treat cancers, residual secondary effects are still observed, such
as, hematological toxicity, headache, fever, and fatigue,
associated with the CPT-based chemotherapy. The
development of cellular resistance of these drugs thus has
impelled the discovery of novel non camptothecin catalytic Topo
1 inhibitors. To solve these problems, multi target ligand design
has been a recent surge of research. One such multi target
ligands are synthetic or unnatural porphyrins (Fig. 2B) which
of Topo inhibitors is to stabilize the DNA-enzyme cleavable
complex through intercalation between DNA base pairs. New
findings highlight that type I topoisomerases are interesting
targets for drug discovery for the treatment of several human
diseases, including multi drug resistant infections and genetic
disorders. Thus, over the past two decades, there has been an
explosion of interest in topo
I and the development of
compounds that can selectively inhibit this enzyme. Since its first
[
9]
discovery in 1966,
the pentacyclic alkaloid 20(S)
–
have been
anticipated to act as remarkably promising
camptothecin (CPT) has emerged as an important lead
compound in the field of anticancer drug development. CPT acts
by stabilizing the DNA-topoisomerase I binary complex and
enhancing apoptosis through blocking the advancement of
chemotype for development of anticancer agents and
photodynamic therapy which includes FDA-approved and
[13], [14]
clinically used sensitizer Photofrin.
Porphyrin derivatives
have diverse pharmaceutical properties and wide range of
biological activities such as selective modes of DNA binding,
4
replication forks. A search for analogues of camptothecin was
[
15-18]
mimicking photosynthetic centers, vitamin B12 and P-450.
1
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Recently, we reported the design and synthesis of a new class
of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-
bis(4-dimethylaminophenyl)- porphyrin (Fig. 2B) as a potent
pure compounds. In the next step, all three macrocycles were
metallated with copper and zinc salts for the formation of
metallated complexes 2, 3, 5, 6, 8, 9 following literature method.
[
19]
[47-52]
catalytic inhibitor of human Top1.
In contrast to CPT
Macrocycle 13 was synthesized following literature known
[
53]
(
Camptothecin (CPT) selectively traps topoisomerase 1-DNA
method
and the respective copper and zinc complexes 14
[
36],
cleavable complexes (Top1cc) to promote anticancer activity),
compound 8 was found to reversibly bind with the free enzyme
and inhibits the formation of Top1cc and promotes reversal of
the preformed Top1cc with CPT.
and 15 were synthesized following literature known methods.
[
54]
The UV-vis absorption spectra, NMR spectra and chemical
analysis of all the macrocycles (both in free base forms and
zinc-complexes) are well consistent with the proposed structures.
Figure 2. Camptothecin and its derivatives widely used in cancer
Chemotherapy (A); Constitutional isomers of porphyrin (B).
Scheme 1. Synthesis of porphyrins and Metallated porphyrins under Biological
Studies.
It is noteworthy to mention that among the versatile unnatural or
synthetic porphyrinoids, N-confused porphyrin derivatives have Biological Studies
been anticipated to exhibit potential PDT photosensitizers
[
20],[21]
because of capability to generate singlet oxygen.
Singly N-
[22-28]
[29-
confused porphyrins
and doubly N-confused porphyrins
have witnessed unusual metallation chemistry leading to the
formation of metal-carbon bond inside the porphyrin cavity,
Cytotoxicity and induction of apoptosis
3
2]
The cytotoxic activity of porphyrins was studied on six cell lines
Jurkat, K562, HL-60, HeLa, A549 and U937. To study of
cytotoxicity, standard MTT test with (3-(4, 5 dimethylthiazol-2-yl)-
[33-39]
stabilization of unusual oxidation states of metals.
It is a
well-known fact that based on the interface between molecular
biology and bioinorganic chemistry, the design of coordination
complexes for cancer treatment, design strategies and
2, 5-diphenyl-2H-tetrazolium bromide) was used. The IC50 value
was determined from plots of % viability against the dose of the
compound added. The percentage of cell growth inhibition was
calculated as 100 minus (mean cell culture optical density in the
test/mean cell culture optical density in the control) multiplied by
[40-42]
mechanisms of action has been a recent surge of research.
We were thus intrigued by the robust activity and the potential
utility of metallated N-confused porphyrins and hence through
this manuscript, we have attempted to explore the efficiency of
100. The value obtained for the first three wells without addition
N-confused porphyrins, their metallated complexes and
a
of the test compound (control triplet), which was measured in
parallel for each test compound, was taken to be 100%. The
mean value and the error of mean were calculated separately for
each concentration of the test compound. The results were used
to plot the cell viability (%) versus the test compound
concentration, and the dose inhibiting the cell viability by 50%
(IC ) and the standard error (SE) of IC were calculated using
comparative study with those of normal porphyrins as inhibitors
towards Topoisomerase I that has remained hitherto unexplored
so far in literature.
Results and Discussion
50
50
the GraphPad Prism 7.0 software (GraphPad Inc. USA). The
data obtained in the experiment are presented in Table 1.
The macrocycles under biological investigation described in the
present manuscript are shown in Scheme 1. We have taken into
consideration, the parent basic porphyrin i.e. tetraphenyl
porphyrin and variations in its periphery with other meso-
substituents. Synthesis of porphyrins 1, 4, and 7 were carried
The cytotoxic effect of macrocycles 13 and 14 determined by
flow cytometry, with respect to the Jurkat tumor line has a
pronounced dose-dependent character, individual for each
compound has been represented (Table 1). It can be seen from
the table that in general, the value of the inhibitory concentration
of IC50 for the porphyrin 14 obtained by exposing the test
compounds to Jurkat cells, followed by staining the cells with the
7AAD dye, is 0.018 μM. All tested compound are somewhat less
active with respect to the Jurkat cell line compared to 13 and 14,
but all compounds exhibit rather high cytotoxic activity. It should
be noted that the studied compounds are more active on cell
[
43]
out following the Adler method.
All the three macrocycles
[
44]
bearing four identical meso-substituents such as phenyl,
4-
were obtained by
[
45]
[46]
pyridyl
and 4-carboxy phenyl
condensation of freshly distilled pyrrole and respective aryl
aldehydes under reflux condition using propionic acid as an
acidic solvent and were then purified by silica gel column
chromatography followed by recrystallization to arrive at extra
2
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ChemMedChem
10.1002/cmdc.201900633
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lines of mesodermal origin, which include Jurkat, U937, K562,
and HL 60, while A549 and HeLa, having endoderm origin,
turned out to be more resistant. Thus, the compound 14 was
selected as the targeted lead compound for the subsequent
study of apoptosis-inducing activity and its effect on the cell
cycle.
Cell cycle analysis
Figure 4 shows the results of the analysis of the kinetics of the
cell cycle of Jurkat cell lines determined by the flow cytometric
DNA method after treatment with compound 14 at various
concentrations and incubation with the test compound for 48
hours. It can be seen from Fig. 4 that the compound 14 has a
predominant effect on the S and G2 phases of the cell cycle,
and this effect is dose-dependent. Thus, the largest changes in
the S and G2 phases occur when the test substance is exposed
IC50
K562
U937
HL-60
Jurkat
A549
HeLa
2
Not dissolved in DMSO, H
2
O, DMF, MeOH
0
0
.031 ±
.005
0.043 ±
0.003
0.019 ±
0.003
0.024 ±
0.004
0.071 ±
0.002
0.084 ±
0.004
3
5
6
at a concentration of 0.6 μM (Fig. 4). Also, when the
concentration of the compound 14 increases, a subG0 peak
appears, indicating that the tumor cells die by apoptosis. The
greatest effect of enriching the subG0 cell population causes a
concentration of 0.8 μM.
0.095 ±
0.099 ±
0.013
0.081 ±
0.011
0. 089 ±
0.014
0.181 ±
0.034
0.204 ±
0.025
0
.012
0.112 ±
0.127 ±
0.021
0.094 ±
0.016
0. 104 ±
0.027
0.217 ±
0.043
0.231 ±
0.014
0
.003
0
0
.026 ±
.002
0.029 ±
0.001
0.022 ±
0.004
0.031 ±
0.003
0.086 ±
0.012
0.091 ±
0.013
8
9
Not dissolved in DMSO, H
2
O, DMF, MeOH
0
0
.083 ±
.017
0.096 ±
0.027
0.074 ±
0.015
0. 086 ±
0.014
0.197 ±
0.021
0.189 ±
0.034
13
14
15
0.012 ±
0.015 ±
0.002
0.014 ±
0.002
0.018 ±
0.004
0.042 ±
0.002
0.051 ±
0.002
0
.002
0
0
.021±
.003
0.027±0.
003
0.017±0.
002
0.019±0.
002
0.061±0.
003
0.076±0.
004
Figure 4. Cell cycle phases in Jurkat cells treated with porphyrine 14. (A)
Control; (B) 14 (0.8 µM); (C) 14 (0.6 µM); (D) 14 (0.4 µM); (E) 14 (0.2 µM); (F)
Flow cytometry histogram represents the percentage of cells in each cell cycle
phase. The time of incubation of substance 14 with the cells is 48 hours. Flow
Table 1. Cytotoxic activity of porphyrins studied on cultures of tumor cells
(Jurkat, K562, U937, HL-60, A549, HeLa) (µМ).
The determination of apoptosis induced by the action of
cytometric histograms are representative of
3 separate experiments.
porphyrins 13 and 14 using the method of intravital double
staining of AnnexinV/7AAD with the detection of flow cytometry
was performed on tumor cells of the Jurkat line in the
concentration of test compounds from 0.15 μM to 0.6 μM. It was
established that both compounds cause the death of tumor cells
of the studied lines along the apoptotic pathway. The highest
apoptosis-inducing activity was registered for 14 (Fig. 3).
Proliferating index (PI) value = [S+(G2+M))/[(G0/G1)+S+(G2+M))×100%. Data
are presented with the means ±SD and mean values of three independent
experiments. **p<0.01, compared with the control group (n=4).
Monitoring Cell Adherence and Proliferation
In our work, we used the iCELLigence RTCA system as a
simple, reliable and universal method for quantitative detection
and visualization of changes in cell adhesion in response to
addition of test compounds 13 and 14 into the nutrient medium.
This technology is based on measuring the flow of electrons
transferred between gold microelectrodes fused to the bottom
surface of a micro titration plate in the presence of an electrically
conductive solution, such as a culture medium. Adherent cells
interfere with the interaction between the electrodes and
interfere with the flow of electrons. This impedance (resistance
to alternating current) is expressed in arbitrary units, called the
cell index, the value of which depends on the strength of the
attachment of the cells to the substrate covering the plate. The
advantage of using RTCA iCELLigence compared to traditional
[
55]
microscopy-based cell adhesion analysis
is that cell adhesion
Figure 3. Tumor cell line Jurkat, treated with various concentrations of
compound 14 and stained with annexine/7AAD and analyzed using flow
cytometry. (A) Control; (B) 14 (0.6 µМ); (C) 14 (0.3 µМ); (D) 14 (0.15 µМ); (E)
Flow cytometry histogram represents the percentage of cells in each apoptosis
phase. (Q7-3 quadrant - normal cells, Q7-4 quadrant - early apoptosis, Q7-2 -
late apoptosis, Q7-1-necrosis). The histogram shows the apoptosis rate (%);
is continuously read from the moment it attaches to the
substrate until the time of their death, i.e. detachment from the
substrate for various reasons. In addition, the measurements are
based on the entire cell population, and not on the limited
number of selected cells and therefore, the results are
statistically more valid.
*
*P<0.05 when compared with the control group (n = 4).
The adhesion and proliferation of A549 cell line was
monitored in real time using the system iCELLigence. DMEM
3
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medium containing 10% bovine serum was added to each well,
followed by inoculation of A549 cells (18,000 per well). The
impedance value of each well was automatically monitored by
the xCELLigence system for 48 hours and expressed as a CI
value (Cell Index). Data on cell adhesion were normalized after
inhibitors of one of the key enzymes of the cell cycle,
topoisomerase II, cause lethal damage to chromosomes, such
as breaks and translocations. When Jurkat cells were stimulated
with etoposide for
2 hours, a marked increase in the
phosphorylated fraction of histone H2A.X was observed (Fig. 6,
histogram B). When analyzing Jurkat cells treated with test
compounds, there was a pronounced accumulation of the cell
population with the phosphorylated fraction of the histone H2A.X
in the presence of compound 14 (96.01%) (Fig. 6, histogram D),
while compound 13 affects the process of H2A.X
phosphorylation slightly by increasing the population of cells with
phosphorylated protein by only 6.75% (Fig. 6, histogram C).
Thus, it can be argued that the main mechanism of action of
compound 14 in the cell is the initiation of DNA breaks, which is
confirmed by the presence of sufficiently high levels of the
phosphorylated fraction of histones H2A.X and this compound
acts in slightly lower concentrations (1.5 mM / ml) than
etoposide.
40 min. The normalized CI is calculated by dividing the CI in the
normalized time by the original CI. The growth rate of the cells
was determined by calculating the slope of the line between two
given time points.
Figure 5. Time-dependent/dose-dependent cell response profiles of samples
with different concentrations of compounds 13 and 14 acting on A549 cells
plotted versus time with a mean of three experiments (iCELLigence RTCA
system). Control (pink line); Compound 13 at a concentration of 1.5 mM/ml
(blue line); Etoposide at a concentration of 2 mM/ml (red line); Compound 14
at a concentration of 1.5 mM/ml (green line).
Figure 6. Detection of phosphorylated H2A.X accumulation in Jurkat cells
when exposed to compounds 13 and 14. Analyzed 10,000 cells in each
experiment. Control (A) (cells with non-phosphorylated H2A.X protein fraction);
cells treated with etoposide at a concentration of 2 mM/ml (B); cells treated
with compound 13 at a concentration of 1.5 mM/ml (C); cells treated with
compound 14 at a concentration of 1.5 mM/ml (D).
The growth curves of cells treated with compounds 13 and 14
acting on the lung cancer cell line A549 (Fig. 5), show the
dynamic of cell growth in real time. Compared with etoposide
(
control preparation), test compounds 13 and 14 showed
pronounced inhibition of the growth of A549 cell lines, which
indirectly reflects their cytotoxic effect and in the case of Human topoisomerase I inhibition studies:
compound 14, the cytotoxic effect is more pronounced than in
compound 13. As can be seen in Fig. 5, all cells died almost
immediately after adding the test compound in the experiment
with etoposide and after adding compound 14, while the wells
with compound 13 maintained cell adhesion until the end of the
second day. Thus, it can be argued that compound 14 has a
more pronounced cytotoxicity compared with 13 probably due to
the presence of a Cu metal.
Taking into account the literature data on the inhibitory activity
exhibited by metal-containing compounds against human
[19]
topoisomerase I and our previous results
on the high activity
of hTopo1 inhibition by synthesized porphyrins, as well as the
proven mechanism for initiating DNA breaks (based on the
detection of high levels of the phosphorylated fraction of H2A.X)
with these compounds, we have attempted to study the effect of
the presence of Cu in the porphyrin structure on the inhibition
activity of human topoisomerase I (hTop1). Thus, we studied the
ability of the synthesized porphyrins to inhibit the topoisomerase
I enzymes in vitro in the relaxation reaction of supercoiled
plasmid DNA under standard conditions (Fig. 7). An increase of
the compounds (13 and 14) concentration introduced into the
reaction from 1 to 2 μM caused a gradual decrease in the
number of formed topoisomers and lead to increase in the
proportion of both supercoiled DNA and the open circular form,
which indicated a slowdown in the relaxation process, that is, a
decrease in the topoisomerase activity I. In the absence of
substances, no similar effect was observed (Fig. 7A, Lane 3).
In the presence of the studied compounds in the same
Detection DNA Damage Histone H2A.X:
Next, we investigated the possible mechanism of cytotoxic
action of compounds 13 and 14, evaluating their effect on DNA
damage in the cell using a flow cytofluorimetry kit. FlowCellect™
DNA Damage Histone H2A.X Dual Detection Kit.
Protein H2A.X is a member of the histone H2A family. The
process of phosphorylation of serine in the 139 position of the
[
56]
protein H2A.X is a reliable indicator of DNA damage.
As the
level of DNA damage increases, the level of phosphorylated
H2A.X (also known as γH2AX) increases simultaneously,
accumulating specifically at the sites of damage to the DNA
molecule. It is the accumulation of phosphorylated H2A.X that is
often used as a marker of the level of DNA damage within cells.
reaction,
a marked inhibition was observed already at a
concentration of 1 μM (Lanes 4-5) for compound 14, whereas
compound 13 at this concentration did not inhibit the enzyme.
Both compounds behaved almost identically at a concentration
of 2 µM, inhibiting of topoisomerase I at a given and higher
concentrations. The results of our experiments show that the
synthesized porphyrins even at micromolar concentrations are
[
57]
H2A.X also plays an important role in DNA repair processes.
It is known that actively proliferating cells are particularly
vulnerable to DNA damage due to the inability to pass the
control of verification points during mitosis. For example,
4
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able to suppress the catalytic activity of topoisomerase I. The
mechanism of interaction of porphyrins with topoisomerase I
remain as before not quite clear. Presumably, the action of
metallated porphyrins can include both the stabilization of the
covalent DNA topo I complex (specific inhibition) and the
competition between porphyrin and the enzyme for DNA binding
sites (non-specific inhibition), while the free base porphyrins
this compound locally changes the DNA conformation. This
assumption was supported by the data of molecular docking of
the protonated form of the compound 13 with DNA duplex and
topo I performed using XP Glide. Docking score for 13-DNA
complex is -3.8 kcal/mol while for 13-topo I complex is 1.1
kcal/mol. This result is quite consistent with the idea of the
preferential interaction of positively charged substrates with a
negatively charged sugar-phosphate backbone DNA. To clarify
the mechanism of action of the metallated complexes 14 and 15,
molecular modeling was performed using a three-dimensional
binding model of the tested compound with the topoisomerase I–
DNA covalent complex. The initial model for docking, 70-
kilodalton human DNA topoisomerase I in complex with the
poison topotecan and covalent complex with a 22 base pair DNA
duplex (PDB ID 1K4T), from which were excluded topotecan and
water molecules was used.
[
19]
have been shown earlier
inhibits topo1 in a non-specific way,
without stabilizing the DNA-topo1 complex.
Figure 7. (A) Electrophoregram of relaxation products of 250 ng plasmid DNA
(pHOT1) in vitro under the action of topoisomerase I (Topoisomerase I Drug
Screening Kit TG-1018-2, Topogen, USA) in the presence of compounds 13
and 14 (the substance was added before adding the enzyme topoisomerase I)
(A). 1. Supercoiled plasmid DNA (pHOT1). 2. Relaxed DNA 3. Plasmid DNA
relaxation reaction in the presence of camptothecin (5 μM). 4-5. The result of
the influence of various concentrations of substance 13 (1 and 2 μM) on the
plasmid DNA relaxation reaction (pHOT1). 6-7. The result of the influence of
various concentrations of substance 14 (1 and 2 μM) on the plasmid DNA
relaxation reaction (pHOT1). 8. Negative control with DMSO (at concentration
of 3%) (B) Modification of gel electrophoretic mobility of pHOT1 supercoiled
plasmid DNA when incubated (30 min, 37 oC) and (1 hour 30 min, 37 oC) with
various concentrations of compounds 13 and 14. Concentrations (in µM) are
as follows: (lane 1) untreated pHOT1 supercoiled plasmid DNA; (lanes 2-4,
incubated with 13): 10 and 25 μM (lanes 4-5, incubated with 14): 10 and 25
μM. The top and the bottom bands correspond to open circular form,
covalently closed circular form, and linear conformation form of plasmids,
respectively.
Figure 8. Docking of the macrocycle 14 in DNA minor groove.
The molecular docking of the compounds into the
topoisomerase I and DNA-binding sites was carried out using
AutoDock Vina. Topoisomerase–ligand–DNA complexes with
the best values of scoring functions calculated by the indicated
program were found. Predicted binding energy of the interaction
of the compounds 14 and 15 with hTopI+DNA covalent complex
is -11.4 and -8.6 kcal/mol, respectively. Given the obtained
experimental data, as well as the docking results, it can be
assumed that, unlike topotecan, metal-complexes 14 and 15
stabilize the topoisomerase–DNA covalent complex due to minor
groove binding (Fig. 8).
To check the viability of porphyrins interacting with DNA, we
investigated the electrophoretic mobility of DNA treated with
compounds 13 and 14. We needed to determine in what range
of concentrations the studied compounds are able to influence
the electrophoretic mobility of DNA themselves. For this purpose,
the mobility of supercoiled DNA was studied in the presence of
the compounds under study without the addition of the topo I
enzyme. The results presented in Fig. 7B indicate that the
electrophoretic mobility of DNA molecules does not change
under the influence of 13 and 14 (Fig. 7B) even at
concentrations that are tens of times higher than the minimal
inhibitory ones (10 and 25 μM). The results of our study has
been strongly supported by Molecular docking of compounds 13,
Conclusions
The porphyrins reported in the manuscript exhibit high cytotoxic
activity in vitro against K562, U937, HL-60, Jurkat, A549 and
HeLa cancer cell lines along the apoptotic pathway. Additionally,
the compounds have a predominant dose-dependent effect on
the S and G2 phases of the cell cycle. Simultaneously, it has
been shown that the main mechanism of action of metallated
porphyrins in the cell is the initiation of DNA breaks, which is
confirmed by the presence of sufficiently high levels of the
phosphorylated fraction of histones H2A.X. In addition, a high
inhibitory activity of the synthesized porphyrins with respect to
human topoisomerase I has been determined. Using molecular
docking it has been assumed that, unlike topotecan, metallated
N-confused porphyrins stabilize of topoisomerase–DNA covalent
complex due to minor groove binding. The study allowed not
14 and 15 that has been carried out in a refined computer model
for the region of binding of the analyte with the active center of
[
58]
topoisomerase I and DNA using crystallographic methods.
Molecular docking studies
According to the obtained experimental data, inhibition of
topoisomerase I using metal-complexes 14 and 15 proceeds
with the formation of a covalent topoisomerase – DNA complex.
At the same time, inhibition with metal free 13 prevents the DNA
helix from breaking. Probably, the inhibitory action of 13 is
determined by its high DNA binding affinity, which apparently
hampers the topo I interaction with DNA specific sequences or
only to find active topoisomerase
I inhibitors among the
synthesized porphyrin complexes, but also to identify the
relationship between their structure and inhibitory activity, which
5
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ChemMedChem
10.1002/cmdc.201900633
FULL PAPER
determines the most promising directions of chemical
modification of these compounds in order to improve their
chemotherapeutic properties.
Cell cycle was analyzed using the method of propidium iodide
staining. Briefly, cells were plated in 24-well round bottom plates
5
at density 10  10 cells per well, centrifuged at 450×g for 5 min,
◦
and fixed with ice-cold 70% ethanol for 24 h at 0 C. Cells were
then washed with PBS and incubated with 250 µL of Guava Cell
Cycle Reagent (Millipore, Burlington, MA, USA) for 30 min at
room temperature in the dark. Samples were analyzed on
NovoCyteTM 2000 FlowCytometry System (ACEA, San Diego,
CA, USA).
Experimental Section
Cell culture
Cells (Jurkat, K562, U937, HL-60, HeLa, and A549) were
purchased from the HPA Culture Collections (Salisbury, UK) and
cultured according to standard mammalian tissue culture
protocols and sterile technique. All cell lines used in the study
were tested and shown to be free of mycoplasma and viral
contamination. HeLa and A549 cell lines were cultured as
monolayers and maintained in Dulbecco’s modified Eagle’s
medium (DMEM, Gibco BRL, Waltham, MA, USA) supplemented
Monitoring Cell Adherence and Proliferation
Cell adherence and proliferation was monitored in real-time
using the iCELLigence system E-Plate L8. 18 000 A549 cells
were seeded in each well. Media taken from, A549 cultures
(
prepared as described above) or fresh DMEM was added to the
well. The impedance value of each well was automatically
monitored by the iCELLigence system for duration of 48 h and
expressed as a CI (cell index) value. Data for cell adherence
was normalized at 40 min. Normalized CI is calculated by
dividing CI at the normalized time into the original CI. The rate of
cell growth was determined by calculating the slope of the line
between two given time points.
with 10% foetal bovine serum and 1% penicillin-streptomycin
◦
solution at 37 C in a humidified incubator under a 5% CO
2
atmosphere. Cells were maintained in RPMI 1640 (Jurkat, K562,
U937, HL-60) (Gibco, Thermo Fisher Scientific, Waltham, MA,
USA) supplemented with 4 mM glutamine, 10% FBS (Sigma,
Burlington, MA, USA) and 100 units/mL penicillin-streptomycin
Detection DNA Damage Histone H2A.X
Millipore’s FlowCellect™ Histone H2A.X DNA Damage Dual
Detection kit includes two directly conjugated antibodies, a
phospho-specific Anti-phospho-Histone H2A.X (Ser139)-PerCP
and an Anti-Histone H2A.X-FITC conjugated antibody to
measure total levels of Histone H2A.X. This two color kit is
designed to detect the extent of Histone H2A.X pathway
activation by measuring H2A.X phosphorylation relative to the
total H2A.X expression in any given cell population. Ionizing
radiation (IR) and many chemotherapeutic agents like etoposide
kill cancer cells by induction of DNA DSBs. Several reports show
that the level of γ-H2A.X as detected by flow cytometry
correlates with the number of DNA strand breaks, to the level of
cell death and radiosensitivity. H2A.X phosphorylates in
response to a DNA damaging reagent (e.g. Etoposide) or UV
light, and its activation clearly indicates that DSBs have occurred.
Understanding when DSBs take place can help researchers
understand the mechanisms involved in DNA repair and the
DNA damage response. The phospho-specific Anti-phospho-
Histone H2A.X (Ser139)-PerCP and an Anti-Histone H2A.X-
FITC conjugated antibody provided in the kit have been carefully
titrated to ensure the ability to measure.
(
Sigma). All types of cells were grown in an atmosphere of 5%
◦
2
CO at 37 C. The cells were subcultures at 2–3 days intervals.
Adherent cells (A549, HeLa) were suspended using
trypsin/EDTA and counted after they have reached 80%
confluency. Cells were then seeded in 24 well plates at 510
cells per well and incubated overnight. Jurkat, K562, U937 cells
were subcultured at 2 day intervals with a seeding density of
1
Cytotoxicity assay
Viability (Live/dead) assessment was performed by staining cells
with 7-AAD (7-Aminoactinomycin D) (Biolegend, SanDiego, CA,
USA). Cells were treated of test compounds with different
concentrations. After treatment, cells were harvested, washed
4
5
10 cells per 24 well plates in RPMI with 10% FBS.
1
–2 times with phosphate-buffered saline (PBS) and centrifuged
at 400×g for 5 min. Cell pellets were resuspended in 200 µL of
2
+
2+
flow cytometry staining buffer (PBS without Ca and Mg , 2.5%
FBS) and stained with 5µL of 7-AAD staining solution for 15 min
at room temperature in the dark. Samples were acquired on
NovoCyte TM2000 Flow Cytometry System (ACEA, SanDiego,
CA, USA) equipped with 488 nm argon laser. Detection of 7-
AAD emission was collected through a 675/30 nm filter in FL4
channel.
Synthesis
Induction of Apoptosis
Apoptosis was determined by flow cytometric analysis of
Annexin V and 7-aminoactinomycin D staining. Briefly, 200 µL of
Guava Nexin reagent (Millipore, Bedford, MA, USA) was added
Macrocycle 1: 1 mL (10 mmol) of benzaldehyde and 50 mL of
propionic acid were mixed and the reaction mixture was
magnetically stirred. Freshly distilled pyrrole (0.7 mL; 10 mmol)
was then added to the mixture, the temperature then brought to
reflux and allowed to stir for 2 hrs at reflux. After allowing the
reaction mixture to cool to room temperature, the reaction flask
was placed in the freezer overnight to aid precipitation of the
porphyrin. The reaction mixture was then vacuum filtered using
a sintered funnel and a dark purple solid was collected, washed
with 5x50 mL of DCM, washed with methanol and dried
overnight and purified by silica gel chromatography to give 4.9 g,
of 5, 10, 15, 20-tetraphenylporphyrin (79% yields).
5
to 510 cells in 200 µL, and the cells were incubated with the
reagent for 20 min at room temperature in the dark. The plates
were treated with compounds at various concentrations for 24 h.
At the end of incubation, the cells were analyzed on
NovoCyteTM 2000 FlowCytometry System (ACEA). Different
states of cell death were defined as follows: normal cells are
localized in the lower-left quadrant (Annexin V−/PI−); early
apoptotic cells are in the lower-right quadrant (Annexin V+/PI−);
late apoptotic cells and necrotic cells are in the upper-right
quadrant (Annexin V+/PI+); and necrotic cells are in the upper-
left quadrant (Annexin V−/PI+).
1
H NMR (500 MHz, CDCl 298 K, δ [ppm]):8.88 (s, 8H, py), 8.25
3
(m, 8H, Ph-CH), 7.79 (m, 12H, Ph-CH), -2.76 (s, 2H,-NH). UV-
-1
-1
5
2 2
vis (CH Cl , λ [nm]), 298 K, (ϵ [M cm x10 ]): 417 (4.13), 515
(
0.18), 548 (0.07), 590 (0.05), 645 (0.03). M.p.  350 C.
Cell Cycle Analysis
Elemental Analysis: C, 85.97; H, 4.92; N, 9.11; Found C, 86.01;
H, 4.94; N, 9.05. HR-ESI TOF MS (m/z):615.2546 [M+H] (Calc.
+
6
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ChemMedChem
10.1002/cmdc.201900633
FULL PAPER
-
1
for C44
H
30
N
4
, exact mass: 615.2543). FT-IR (CHCl
3
, cm ): 3318
filtered, the precipitate was washed with water and acetone and
dried under vacuum to obtain a red-purple solid. (0.09 g, yield
(
N-H), 1596, 1473 (C=C); 800 (C-H Py).
Macrocycle 2: A saturated methanolic solution of anhydrous
copper acetate (3.94 g, 0.217 mmol) was added to a 4 mL DCM
solution of 1 ( 0.1g, 0.162 mmol), and the reaction mixture was
stirred for 1 hour. After the complete metalation was confirmed
by TLC, indicated by the absence of free base spot on the TLC,
the mixture was diluted with water and extracted with DCM. The
organic layer was washed with water and brine to remove the
-1
-
8
2%). UV-vis (CH
2
Cl
2 3
:CH OH (95:5 (v/v)), λ [nm]), (ϵ [M cm
1
5
x10 ]), 298 K): 412 (4.12), 539 (0.23). HR-ESI TOF MS (m/z):
+
680.1492[M+H] (Calc. for C40
24 8
H N Cu, exact mass: 680.1493).
M.p.  350 C. Elemental Analysis: C, 70.63; H, 3.56; N, 16.47;
-1
Found: C, 70.68; H, 3.60; N, 16.43. FT-IR (CHCl
592(C=C); 793 (C-H Py).
3
, cm ):
1
unreacted salt and dried over anhydrous Na
was removed in vacuum and the residue was recrystallized in
CH Cl /MeOH to get purple powder. (0.092 g, yield 85%). UV-vis
CH Cl
79 (0.02). M.p.  350 C. Elemental Analysis: C, 78.15; H, 4.17;
N, 8.28; Found C, 78.25; H, 4.20; N, 8.24. HR-ESI TOF MS
2 4
SO . The solvent
Macrocycle 6: Anhydrous zinc acetate (0.179 g, 0.968 mmol)
was added to a suspension of 4 (0.1 g, 0.162 mmol) into 4 mL of
N, N-dimethylformamide and the reaction mixture was refluxed
for 48 hours. The suspension was filtered, the precipitate was
washed with water and acetone and dried under vacuum to
2
2
-
1
-1
5
(
5
2
2
, λ [nm]), (ϵ [M cm x10 ]), 298 K): 414 (1.42), 539 (0.3),
1
obtain as purple solid. (0.097 g, yield 87 %). H NMR (500 MHz,
+
(
6
m/z): 677.1764[M+H] (Calc. for C44
H
28
N
4
Cu, exact mass:
CDCl
3 3
/CD OD, 298 K, δ [ppm]): 9.07 (d, J= 5 Hz, 8H, Pyridyl-H),
-1
77.1761). FT-IR (CHCl , cm ): 1594, 1439 (C=C); 797 (C-H
3
8
.89 (br, 8H, Pyrrole -H), 8.19 (d, J= 5 Hz, 8H, Pyridyl- CH).
Py).
-1
-1
5
UV-vis (CH
98 K): 425 (5.18), 557 (0.23), 598 (0.10). M.p.  350 C.
Elemental Analysis: C, 70.44; H, 3.55; N, 16.43; Found: C,
2 2 3
Cl :CH OH (95:5 (v/v)), λ [nm]), (ϵ [M cm x10 ]),
2
Macrocycle 3: A saturated methanol solution of zinc acetate
(
(
3.98 g, 0.217 mmol) was added to a 4 mL DCM solution of 1
0.1 g, 0.162 mmol) and the reaction mixture was stirred for 1
7
0.46; H, 3.56; N, 16.42.
HR-ESI TOF MS (m/z):
Zn, exact mass: 681.1488).
3
, cm ): 1654, 1411 (C=C); 805 (C-H Py).
+
24 8
681.1487[M+H] (Calc. for C40H N
hour. After the complete mettalation was confirmed by TLC,
indicated by the absence of free base spot on the TLC, the
mixture was diluted with water and extracted with DCM. The
organic layer was washed with water and brine to remove the
-1
FT-IR (CHCl
Macrocycle 7: 1.5 g (10 mmol) of 4-formylbenzoic acid and 50
mL of propionic acid were added and the reaction mixture was
stirred at ambient temperature. To increase the solubility of 4-
unreacted salt and dried over anhydrous Na
2 4
SO . The solvent
was removed in vacuum and the residue was recrystallized in
o
formylbenzoic acid, the reaction mixture was heated to 80 C at
1
CH
NMR (500 MHz, CDCl
.27 (m, 8H, Ph-CH), 7.79 (m, 12H, Ph-CH). UV-vis (CH
[
(
2
Cl
2
/MeOH to get purple powder. (0.087 g, yield 80%).
298 K, δ [ppm]): 8.89 (s, 8H, Py-β-CH),
Cl , λ
nm]), (ϵ [M cm x10 ]), 298 K): 418 (5.64), 551 (0.29), 588
0.04). M.p.  350 C. Elemental Analysis: C, 71.99; H, 3.84; N,
5.26; Found C, 72.01; H, 3.86; N, 15.25. HR-ESI TOF MS
H
which point the aldehyde fully dissolved. Freshly distilled pyrrole
3
(
0.7 mL; 10 mmol) was then added to the mixture, and reaction
8
2
2
mixture was allowed to stir for 2 hrs under reflux. The reaction
mixture was cooled to room temperature, placed in the freezer
overnight to aid precipitation of the porphyrin. The reaction
mixture was then vacuum filtered using a sintered funnel and a
dark purple solid was collected, washed with 5x50 mL of DCM,
washed with methanol and dried overnight to give 1.1 g, of. 5,
-1
-1
5
1
+
(
m/z): 676.1608[M] (Calc. for
C
44
H
28
N
8
Zn, exact mass:
76.1605). FT-IR (CHCl , cm ):1593, 1439 (C=C); 798 (C-H Py).
-1
6
3
1
0, 15, 20-tetrakis (4-carboxyphenyl) porphyrin (55% yields).
Macrocycle 4: 1.07 g (10 mmol) of 4-Pyridinecarboxaldehyde
and 50 mL of propionic acid were added and the reaction
mixture was magnetically stirred. Freshly distilled pyrrole (0.7
mL; 10 mmol) was then added to the mixture, the temperature
then brought to reflux and allowed to stir for 2 hrs at reflux. After
allowing the reaction mixture to cool to room temperature, the
reaction flask was placed in the freezer overnight to aid
precipitation of the porphyrin. The reaction mixture was then
vacuum filtered using a sintered funnel and a dark purple solid
was collected, washed with 5x50 mL of DCM, washed with
methanol and dried overnight to give 5,10,15, 20-tetra(4-
1
H NMR (500 MHz, DMSO-d
COOH), 8.86 (8H, s, Py β-H), 8.38 (16H, dd, phenyl H), -2.93
6
298 K, δ [ppm]): 13.14 (4H, br, -
-1
-1
5
(
2H, s, NH). UV-vis (CH
3
OH, λ [nm], (ϵ [M cm x 10 ]), 298 K):
4
3
7
13 (3.91), 513 (0.25), 545 (0.18), 589 (0.08), 646 (0.04). M.p. 
50 C. Elemental Analysis: C, 72.91; H, 3.82; N, 7.09; Found: C,
2.92; H, 3.83; N, 7.08. HR-ESI TOF MS (m/z): 791.2145
+
[
M+H] (Calc. for C48
H
30
N
4
O
8
, exact mass: 791.2142). IR (KBr,
-1
cm ): 3427 (NH), 1691 (C=O), 1411 (C=C), 790 (C-H Py).
Macrocycle 8: Anhydrous copper chloride (0.934 g, 6.949 mmol)
was added to a solution of 7 (0.1 g, 0.126 mmol) in 25 mL N, N-
dimethylformamide (DMF), and the reaction mixture was
refluxed for 5 hours. The reaction was monitored by checking
the absorption spectrum in DMF. The DMF was then evaporated
and the residue was washed many times with water remove the
unreacted salt. The residue was then recrystallized from DMF-
acetone solution to obtain compound 8 as brown solid. (0.092 g,
1
pyridyl)porphyrin. Yield 2.472 g (20%). H NMR (500 MHz,
CDCl
3
298 K, δ [ppm]): 8.94 (d, J= 5 Hz, 8H, Pyridyl-H), 8.79(b,
8
H, Pyrrole -H), 8.16 (d, J= 5 Hz, 8H, Pyridyl- H), -2.99 (b, 2H,
-1
-1
5
2 2
pyrrole NH). UV-vis (CH Cl , λ [nm]), (ϵ [M cm x10 )), 298 K):
4
3
15 (3.59), 511 (0.36), 545 (0.14), 587 (0.11), 642 (0.06). M.p. 
50 C. Elemental Analysis: C, 77.65; H, 4.24; N, 18.11; Found
C, 77.66; H, 4.25; N, 18.09. HR-ESI TOF MS (m/z):619.2351
-1
-1
5
yield 86%). UV-vis (DMF, λ [nm]), (ϵ [M cm x10 ]), 298 K): 417
4.62), 540 (0.33), 580 (0.10). M.p.  350 C. Elemental
Analysis: C, 67.64; H, 3.31N, 6.57; Found: C, 67.84; H, 3.61N,
+
-
[
M+H] (Calc. for C40
)
H
26
N
8
, exact mass: 619.2353). IR (KBr, cm
(
1
: 3311, 972 (NH), 1593 (C=C), 800 (C-H Py).
+
6.53. HR-ESI TOF MS (m/z): 853.1279[M+H] (Calc. for
Macrocycle 5: Anhydrous copper acetate (0.176 g, 0.968 mmol)
was added to a suspension of 4 (0.1 g, 0.162 mmol) into a 4 mL
-1
C
48
H
28
N
4
O
8
Cu, exact mass: 853.1276).FT-IR (CHCl
3
, cm ):
1738 (C=O), 1599, 1456 (C=C); 769 (C-H Py).
1:1 N, N-dimethylformamide: acetic acid solvent mixture and the
reaction mixture was refluxed for 48 hours. The suspension was
7
This article is protected by copyright. All rights reserved.

ChemMedChem
10.1002/cmdc.201900633
FULL PAPER
Macrocycle 9: Anhydrous zinc acetate (1.26 g, 6.949 mmol) was
added to a solution of 7 (0.1 g, 0.126 mmol) in 25 mL N,N-
dimethylformamide (DMF), and the reaction mixture was
refluxed for 5 hours. The reaction was monitored by checking
the absorption spectrum in DMF. The DMF was then evaporated
and the residue was washed many times with water remove the
unreacted salt. The residue was then recrystallized from DMF-
acetone solution to obtain compound 9 as brown solid. (0.092 g,
complete metalation was confirmed by TLC, indicated by the
absence of free base spot on the TLC, the solvent was removed
in vacuum. The residue was then dissolved in DCM and washed
with saturated brine. The organic layer was then dried over
anhydrous Na
2
SO
4
and evaporated in vacuum to obtain
compound 15 as green solid. (0.092 g, yield 73%). UV-vis
-1
-1
4
(CH
540 (0.04), 582 (0.05), 625 (0.07), 704 (1.02), 773 (1.96).
NMR (500 MHz, CDCl , δ [ppm)): 8.34 (d,J = 5 Hz, 1H), 8.33 (d,
2 2
Cl , λ [nm), (ϵ [M cm x10 ]), 298 K): 415 sh, 461 (9.52),
1
H
-1
-1
5
yield 86%). UV-vis (DMF, λ [nm]), (ϵ [M cm x10 ]), 298 K): 422
3
1
(
5.11), 557 (0.32), 598 (0.11). H NMR (500 MHz, DMSO-d
6
298
J = 4.5 Hz, 1H), 8.28 (d, J = 5 Hz, 2H), 8.27 (d, J = 5 Hz, 2H),
7.85-7.77 (m, 6H), 7.75-7.58 (m, 8H), 7.58 (br, 6H), 6.24 (s, 1H),
K, δ [ppm]): 13.15 (4H, br, -COOH), 8.86 (s, 8H), 8.39 (br, 8H),
13
8.01 (br, 8H). M.p.  350 C. Elemental Analysis: C, 67.50; H,
3
3.22 (s, 3H), -0.59 (s, 1H). C NMR (125 MHz, CDCl ,
3
.30, N, 6.56; Found: C, 67.53; H, 3.31; N, 6.55. HR-ESI TOF
δ[ppm]):162.07,161.45, 157.79, 156.28, 153.27, 150.63, 141.73,
141.62, 140.51, 139.86, 138.08, 136.92, 135.02, 134.26, 134.04,
133.66, 133.54, 133.39, 133.26, 131.70, 129.84, 129.16, 128.89,
+
MS (m/z): 853.1273[M+H] (Calc. for C48
H
28
N
4
O
8
Zn, exact mass:
-1
3
853.1271). FT-IR (CHCl , cm ): 1735 (C=O), 772 (C-H Py).
128.75, 128.05, 127.64, 127.51, 127.45, 127.30, 127.23, 124.67,
Macrocycle 13: Tripyrrane 12 (0.391g, 1 mmol) was taken in a
round bottom flask, to it 740 mL of dry DCM was added and
stirred for 15 minutes under nitrogen atmosphere to get a clear
solution. p-Toluenesulfonic acid (0.035 g, 0.185 mmol) was
added to the reaction mixture and stirred for 90 minutes under
dark condition. After that p-chloranil (0.735 g, 3 mmol) was
added and the resulting mixture was refluxed for 90 minutes in
open air. After complete removal of solvent from crude mixture
by rotary evaporator, the compound was purified using basic
alumina followed by repeated silica gel column chromatography
120.76, 118.65, 116.51, 38.05. M.p.  350 C. Elemental
Analysis: C, 78.09; H, 4.37; N, 8.09; Found: C, 78.10; H, 4.38; N,
+
8.07. HR-ESI TOF MS (m/z): 691.1835[M+H] (Calc. for
-1
45 30 4 3
C H N Zn, exact mass: 691.1833). FT-IR (CHCl , cm ): 1508,
1471(C=C); 799 (C-H Py).
Acknowledgements
NH thanks IACS for senior research fellowship. HR thanks DST-
SERB (EMR/2016/004705), New Delhi, India for research grant.
This work was financially partly supported by the Russian
Foundation for Basic Research (Project number 18-29-09068),
Grants Council of the President of Russian Federation (Grant
NSh-5240.2018.3). The biological studies of porphyrins were
done in the Laboratory of Molecular Design and Drug
Bioscreening at the Institute of Petrochemistry and Catalysis of
RAS.
to obtain compound 13 as green solid. Yield. ~ 50 mg (~8%).
1
UV-vis (CH
NMR (500 MHz, CDCl
CH, Phenyl-CH),7.84 (br, 5H, Pyrrole-βCH, Phenyl-H), 7.74
br,2H, Pyrrole-βH), 7.62 (br, 6H, Phenyl-H), 7.56 (br, 6H,
2
Cl
2
, λ [nm]), 298K): 445, 361, 566, 603, 654, 711. H
3
, 298 K, δ [ppm)): 7.95 (br, 5H, Pyrrole-β-
(
Phenyl-H), 7.51 (d, J = 4.5 Hz, 1H, Pyrrole-βH), 7.48 (d, J = 4.5
Hz, 1H, Pyrrole-β-CH), 7.19 (s, 1H, N-Me Pyrrole- α CH), 3.70
(
br, 1H, pyrrole-NH), 3.39 (s, 3H, N-Methyl-CH), 0.90 (s, 1H, N-
1
3
Me-Pyrrole-β-CH). C NMR (125 MHz, CDCl
3
, δ[ppm)):163.62,
1
1
1
1
6
63.08,154.70, 153.85, 143,69, 143.26, 141.88, 141.45, 140.87,
36.46, 135.11, 135.00, 134.85, 133.72, 133.58, 131.86, 131.01,
30.61, 129.45, 129.20, 128.47, 128.04, 127.90, 127.34, 126.85,
Keywords: N-confused porphyrins • Cu-N confused porphyrin •
Topoisomerase I inhibitor • Anticancer Activities • in vitro studies
24.20, 115.95, 115.48, 106.42, 38.97. HR-ESI-TOF (m/z)
+
29.2704[M] (629.2700 calc. for C45
H
32
N
4
). M.p.  350 C. References:
Elemental Analysis: C, 85.96; H, 5.13; N, 8.91; Found: C, 85.97;
-1
H, 5.14; N, 8.89. FT-IR (CHCl
498 (C=C); 795 (C-H Py).
3
, cm ): 3346 (NH), 1557, 1544,
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2192.
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[
[
[
3]
4]
5]
6]
Y. Pommier, ACS Chem. Bio. 2013, 8, 82-95.
Macrocycle 14: Compound 14 was obtained by refluxing 13 (50
mg, 0.079 mmol) with an excess of Cu(OAc) (72 mg) in 15 mL
Y. Pommier, Chem. Rev. 2009, 109, 2894-2902.
2
Y. Pommier, Nat. Rev. Cancer 2006, 6, 789-802.
THF for 1 hour. Subsequently, solvent was removed, and then
hexane was used to extract a green residue that precipitated
after solvent volume reduction. This was washed several times
to remove unreacted salt. The residue was then recrystallized
from DCM- hexane solution. (0.097 g, yield 76%). UV-vis
P. Majumdar, C. Bathula, S. M. Basu, S. K. Das, R. Agarwal, S. Hati, A.
Singh, S. Sen, B. B. Das, Eur. J. Med. Chem. 2015,102, 540-551.
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[7]
[8]
-
1
-1
4
(
5
(
6
CH
43 (3.70), 587 (3.49), 682 (3.68), 740 (3.77). HR-ESI-TOF
m/z) : 690.1841[M+H]+ (Calc. for C45H30CuN4, exact mass:
2 2
Cl , λ [nm), (ϵ [M cm x10 ]), 298 K): 389 (4.40), 461 (9.31),
3688-3698.
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Entry for the Table of Contents
Comparative in vitro studies of the topoisomerase I inhibition and anticancer activities of metallated N-confused porphyrins and
metallated porphyrins have been developed. The synthetic porphyrins exhibit inhibitory activity against human topoisomerase I and
high cytotoxic activity in vitro against K562, U937, HL-60, Jurkat, A549 and HeLa cancer cell lines. Induction of the programmed cell
death in Jurkat cells by the metallated N-confused porphyrin corresponds to the apoptotic pathway and have a predominant dose-
dependent effect on the S and G2 phases of the cell cycle.
1
0
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