Efficient synthesis of meso-substituted porphyrins and molecular docking as potential new antioxidant and cytotoxicity agents

Article abstract of DOI:10.1002/ardp.201800221

An improved methodology is reported for the synthesis of new series of mesotetrakis[aryl]-21H,23H-porphyrin derivatives 2a–h and was considered as a model to study their antioxidant and cytotoxic activities. The structures of the novel compounds were dete

Full text of DOI:10.1002/ardp.201800221

Received: 26 July 2018
Revised: 22 October 2018
Accepted: 4 November 2018
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DOI: 10.1002/ardp.201800221
FULL PAPER
Efficient synthesis of meso-substituted porphyrins and
molecular docking as potential new antioxidant and
cytotoxicity agents
Sraa Abu-Melha
Faculty of Science of Girls, Department of
Abstract
Chemistry, King Khaled University, Abha,
Saudi Arabia
An improved methodology is reported for the synthesis of new series of mesotetrakis-
[aryl]-21H,23H-porphyrin derivatives 2a–h and was considered as a model to study
Correspondence
Sraa Abu-Melha, Faculty of Science of Girls,
their antioxidant and cytotoxic activities. The structures of the novel compounds were
determined in 1H and 13C NMR, UV-Vis, and elemental analyses. Among the
Department of Chemistry, King Khaled
University, Abha 35516, Saudi Arabia.
Email: sraa201313@yahoo.com
derivatives, compounds 2c, 2d, and 2h showed strongest radical-scavenging activity.
Moreover, according to our results, compounds 2c, 2d, 2g, and 2h have very strong
activity against the HepG2 hepatoma cell line, with IC50 values from 9 to 25 μg/mL.
Molecular docking was performed to investigate the binding between the most active
porphyrin derivatives 2c, 2d, 2g, 2h and the two molecular targets Bcl-2 and caspase-3.
Compounds 2c and 2d seem to have better affinities to both proteins than 2g and 2h.
K E Y W O R D S
aldehydes, antioxidant, cytotoxicity, molecular docking, porphyrins, pyrrole
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1
INTRODUCTION
biological and pharmacological activities. The desirable cancer
preventive or putative therapeutic properties of porphyrins have
also been considered to be associated with their antioxidant
properties, since free radical-mediated peroxidation of membrane
lipids and oxidative damage of DNA were believed to be associated
with a variety of chronic health problems, such as cancer, atheroscle-
rosis, neurodegenerative diseases, and aging.[4] Therefore, the past
few years have witnessed intense research devoted to the antioxidant
activity of porphyrin. For instance, studies pertaining to the kinetics
and mechanisms of natural antioxidants[5] have demonstrated that
simple structural modifications of resveratrol, which is an antioxidative
component in red wine, could significantly enhance its antioxidative
activity[6] and cytotoxicity against cancer cells.[7] This motivated us to
use porphyrin as a lead compound to design more active potential
antioxidants and chemopreventive agents against cancer.
The porphyrins have been recognized as one of the most important
prosthetic groups in biological systems. The diverse chemistry
performed by natural porphyrins has inspired works in various fields
of chemistry.[1] In the past decades, much interest has been focused on
the synthesis of well-defined porphyrins for potential application as
photosensitizer in photodynamic therapy (PDT) of cancer.[2] Though
there are many aspects to attain to this purpose, one important issue to
porphyrin derivatives is their availability. Up to now, the most
porphyrins investigated are obtained from naturally occurring
porphyrins like hematoporphyrin and protoporphyrin, involving
complicated modification reaction and tedious separation. Porphy-
rin-type compounds have been actively investigated as sensitizing
drugs for application in cancer diagnosis and treatment using
photodynamic therapy and also using boron neutron capture therapy
(BNCT).[3] Porphyrins have been reported to possess a variety of
Porphyrins substituted at the meso-position have been synthe-
sized and used as molecular materials.[8,9] The synthesized porphyrins
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Arch Pharm Chem Life Sci. 2018;e1800221.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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showed interesting physicochemical properties, electrochemical
characters, medical treatment,[10] photosensitizers,[2] photocata-
lysts[11] and wide range of different technology. The general methods
for the synthesis of porphyrins involved condensation of dipyrro-
methanes with different aldehydes in acid medium. Most of the
reported methods led to the formation of porphyrins in low yields due
to the side reactions and the formation of polymeric products. It has
been published that porphyrins have different biological and
pharmacological activities. This prompted us to synthesize new
porphyrin derivatives to be used as antioxidant and anticancer agents.
sole (BHA) as positive controls. The results showed that compounds
2c, 2d, and 2h have strongest antioxidant activity. This high
antioxidant activity of compounds 2c and 2d can be attributed to
the presence of nitrogen and sulfur atoms in their structures. This
result agrees with the previously reported work showing that the
sulfur atom in porphyrin derivatives acts as good radical scaven-
ger.[14] The deoxyribose assay was used to measure the hydroxyl
radical scavenging activity of the synthesized compounds (Table 2).
Protective effect of the new compounds was followed as they can
remove hydroxyl radicals from the tested solution and inhibit the
degradation. It was found that all tested compounds, especially 2c,
2d, and 2h have high prevention of degradation. IC50 values of
compounds 2c, 2d, and 2h showed that they are very strong
scavengers of OH radicals (generated in Fenton's reaction). The
prevention of lipid peroxidation (LP) was characterized by determin-
ing the formation of MDA, using liposomes as an oxidizable
substance.[15] Compounds 2c and 2d showed the highest inhibition
of some of Fe2+/ascorbate-induced LP in liposomes. From Table 2, it
was noticed that compounds 2c and 2d are very good inhibitors of
LP and the most active compounds in this assay as antioxidant
agents comparable to the positive controls. These results showed
great agreement with the previous reported work that sulfur
compounds are good antioxidants at low concentration.[14]
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
Many methods deal with the synthesis of porphyrin derivatives.[8] All
the reported methods have two long standing problems associated
with low yield reactions (6:20%) and purification.[12] In our lab, we
synthesized new porphyrin derivatives in very good yield 70:85% with
successful purification on silica gel using eluent (1.5:1 chloroform/
hexane). We successfully used the capping process which prevents the
formation of polymeric pyrrole. We used DMF as solvent and capping
agent.[13]
It involves the acid-catalyzed addition of the pyrrole to the
substituted benzaldehyde carbonyl group followed by acid-catalyzed
dehydration. Repeating this process adds the next benzaldehyde
moiety. Ring closure results in the formation of the reduced form of
porphyrin (porphyrinogen) followed by oxidation to furnish the
porphyrin building blocks. In the presence of DMF, a reversible cap
forms which protects this intermediate species, while allowing the
reaction with pyrrole.
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2.2.2 Cytotoxic activity of new porphyrin
derivatives against human cell lines
The porphyrin mode(s) of action, their cytotoxicity are due to caspase
3/7 activation and subsequent induction of apoptosis. Additionally,
different phenotypical changes were observed and these included
endoplasmic reticulum, actin cytoskeleton, and cellular morphology
alterations as well as cell cycle arrest and various biochemical changes
(e.g., ROS and GSH levels).[16–19]
Aldehyde derivatives 1a–h were condensed with pyrrole in
presence of p-toluenesulfonic acid in DMF as solvent and capping
agent afforded the porphyrin derivatives 2a–h. The rate and yield of
reaction depend on the concentration of p-toluenesulfonic acid,
solvent, temperature, and the presence of atmospheric oxygen and
initial concentration of reactants (Scheme 1). Schemes 2 and 3 show
the reaction capping mechanism.
The cytotoxicity of the newly synthesized compounds are tested
against HepG2, MCF-7, Vero and normal cells (WI-38) and their effect
on the expression levels of caspase-3 and Bcl-2 molecular biomarkers
is evaluated.
The metabolic activity of the cells was measured after 48 h of
incubation with different concentrations of the investigated com-
pounds by means of MTT assay. The IC50 (µM) was determined from
the dose–response curves as mean of two parallel experiments; 5-
fluorouracil (5-Fu) was used as positive control; growth inhibition
100 µM (inactive).
The relation between the observed absorption maximum shifts
and the polar characteristics of substituents is shown in Table 1. From
Table 1, it was noticed that there is no any increase or decrease in λmax
in the visible region and there is no any marked change for log ε due to
the presence of electron donating or electron withdrawing groups.
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2.2 Pharmacology
2.2.3 Structure–activity relationship
From Table 3, it was noticed that compounds 2c and 2d displayed the
highest cytotoxic activity against HepG2 cell line. Also, compounds 2g,
2h, and 2b showed cytotoxic activity against the same cell line. The rest
of other compounds exhibited low activity against HepG2. From these
results, we can conclude that sulfonyl group enhances the cytotoxic
activity against all cell lines. Moreover, it was found that compounds 2c
and 2d displayed the strongest cytotoxic activity against WI-38 cell
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2.2.1 Antioxidant activities
The newly synthesized compounds 2a–h were evaluated for their
antioxidant properties. We used 2,2-diphenyl-1-picrylhydrazyl (DPPH)
assay. The purple-colored radical DPPH• was reduced to yellow-
colored DPPH-H form (Table 2) by all tested compounds. We used 3,5-
di-tert-butyl-4-hydroxytoluene (BHT) and 3-tert-butyl-4-hydroxyani-

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SCHEME 1 Synthesis of porphyrin derivatives 2a–h
line in which these results were attributed to the effect of sulfonyl
group present in their structure. Compound 2b displayed mild
cytotoxicity due to the presence of fluorine atom in its structure,
which acts as electron withdrawing group, the rest of other
compounds exhibited weak activity. Compounds 2h, 2c, and 2d
displayed good activity against Vero cell line, while the other
compounds exhibited weak cytotoxicity against the same cell line. In
addition, compounds 2c and 2d showed the highest activity against
MCF-7 cell line owing to the sulfonyl group and rich in nitrogen atom.
Of course, such initial studies are not directly transferable and
therefore require more investigations using a wider arsenal of normal
and tumor cells, and eventually organismic experiments.
compounds 2c, 2d, 2g, and 2h at the molecular level, the expression
levels of selected tumor proliferation, anti-apoptotic and apoptotic
protein markers were assessed in HepG2 cells. In view of the former,
pro-apoptotic caspase-3 and anti-apoptotic Bcl-2 proteins were
selected to monitor apoptosis induction.
As shown in Figure 1, compounds 2c, 2d, 2g, and 2h were able to
upregulate the expression of caspase-3 and down regulate the
expression of Bcl-2 compared with untreated cells. It is worthwhile
to mention that a distinct correlation was observed between the
chemical structures of test compounds and their corresponding
expression modulation activity showing that it is not a general
porphyrin cytotoxic effect. Not surprisingly, and in agreement with
SARs.
Interestingly, 2d exhibited a superior activity within its series
analogues and this worth further study. These results were in line with
our previous studies, in which porphyrin-based aromatic amine
derivatives induced a chain of biochemical alterations among which
is the cell caspases activation and apoptosis induction.[12,20,21] These
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2.2.4 Evaluation of caspase-3 and Bcl-2 molecular
biomarkers in HepG2 cells
In order to further study the possibly addressed signaling pathways
and obtain hints on the mode(s) of action of the most promising

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SCHEME 2 Proposed nucleophilic attack by pyrrole on protonated aldehyde following nucleophilic attack by the DMF nitrogen on the
carboncation to form a reactive species with DMF as a good leaving group of DMF as a capping agent during porphyrin formation
alterations seem to take place primarily in specific cells (e.g., HepG2)
with a disturbed intracellular redox balance.
in Figure 1 are for expression levels, they cannot be correlated to the
binding affinities. Nevertheless, at least for caspase-3, porphyrins have
been shown to have considerable binding using enzyme assays and
molecular docking.[22] The predicted affinities for the most populated
clusters per each docking calculation are shown in Table 4. Compounds
2d and 2c seem to have better affinities to both proteins than
compounds 2g and 2h. As a representative for the two molecules with
largest affinity, we further discuss the molecular interactions made by
2d and the active site of each enzyme.
While it is premature to explain why compound 2d was among the
most active agents in these assays, one may speculate that this
compound may hit more than one specific cellular target(s) and cause
widespread modification of proteins and enzymes for the benefit of
activation. Furthermore, it is likely that 2d might also be taken up by
cells and modified in vivo into active metabolic intermediates. As these
are unknown, it is too early to speculate over details on its exact
metabolism, pharmacokinetics in animals and enrichment in specific
tissues or degradation, although these issues are clearly important and
will form part of our future studies. Ultimately, as the structure of these
compounds provides considerable scope for modifications, and the
synthesis of derivatives is now straight forward, this will become a
promising starting point for future studies of structural variants.
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2.3.1 Docking of 2d in caspase-3
It has been demonstrated that porphyrins fit well in the active site of
caspase-3.[22] In accordance with the previous results, it seems that the
porphyrin ring itself is not involved in strong interactions with the
amino acids of the active site. Rather, it acts as a spacer to position H-
bond donors and acceptors to interact with the side chains of arginine,
serine, threonine and asparagine in the orthosteric active site of
caspase-3. An illustration of the best pose of 2d in the pocket of
caspase-3 is given in Figure 2. A clearer 2D projection of the H-bonds
network between 2d and the surrounding amino acids is depicted in
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2.3 Molecular docking
Docking was performed to investigate the binding between the most
active porphyrin derivatives (2c, 2d, 2g, and 2h) and the two molecular
targets: Bcl-2 and caspase-3. Since the experimental values presented
SCHEME 3 Proposed capping mechanism for the synthesis of porphyrin derivatives 2a–h showing how DMF acts as a good leaving group
as pyrrole is added

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TABLE 1 The UV absorption bands of porphyrin derivatives 2a–h
TABLE 3 Cytotoxicity (IC50) of tested compounds on different
cancer cell lines
Compound
λmax (nm)
425
log ε
4.18
4.30
4.17
4.23
4.17
4.22
4.23
4.30
a
IC50 (µg/mL)
2a
2b
2c
2d
2e
2f
Compound
HepG2
75
26
10
9
WI-38
80
51
23
21
61
61
43
24
0
Vero
41
44
19
17
71
69
56
12
7
MCF-7
75
35
12
9
427
2a
423
2b
426
2c
423
2d
425
2e
36
31
25
12
9
51
54
60
18
3
2g
2h
422
2f
427
2g
2h
5-Flub
Figure 3. The oxygen atoms in the sulfonamide groups act as H-bond
acceptors, while the nitrogen atom acts as a H-bond donor. This
rationalizes the low score obtained by compound 2g, as it lacks any
protruding H-bond donors or acceptors.
aIC50 (mg/mL): 1–10 (very strong), 11–25 (strong), 26–50 (moderate), 51–
100 (weak), 100–200 (very weak), 200 (non-cytotoxicity).
b5-Flu = 5-fluorouracil.
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2.3.2 Docking of 2d in Bcl-2
acids while the porphyrin structure acts to position the H-bond donors
and acceptors without being involved in major interactions (Figure 6).
In contrast to caspase-3, porphyrins have not been reported to directly
bind to the Bcl-2 protein. Rather, studies have been conducted on the
interactions between porphyrins and the quadruplex DNA in the
promoter region of Bcl2.[23] We herein, however, study the possibility
of having interactions between the active site of Bcl-2 and porphyrin
representatives. Similar to the docking in caspase-3, 2d has the largest
affinity to Bcl-2. The superposition of the best docking pose of 2d and
the co-crystallized ligand in the crystal structure demonstrates that the
porphyrin fits properly in the active site of Bcl-2, see Figure 4.
Nevertheless, the number of interactions made by 2d and the amino
acids in the active site are significantly less than those made by the
native ligand. A 2D depiction of the interaction map is given in Figure 5.
Similar to their role in the interaction with caspase-3, the sulfonamide
groups of 2d are involved in H-bonding with the neighboring amino
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3
CONCLUSION
Although the eight porphyrin derivatives 2a–h are new, they were
synthesized via a new method, the capping mechanism, which led to
high-yield porphyrins, and these derivatives were evaluated for
antioxidant and antitumor activity in which compounds 2c, 2d, and
2h showed strongest radical scavenging. On the other hand,
compounds 2c, 2d, 2g, and 2h have very strong activity against
HepG2 cell line with IC50 values from 9 to 25 μg/mL. Docking was
performed to investigate the binding between the most active
porphyrin derivatives (2c, 2d, 2g, and 2h) and the two molecular
targets: Bcl-2 and caspase-3.
TABLE 2 Antioxidant assay of the tested compounds relative to the standard commercial antioxidants BHA and BHT
Compound
DPPH (IC50, mM/1 h)
HO (IC50, mM)
3.011
LP (IC50, mM)
naa
2a
1.74
2b
1.33
2.38
0.190
0.030
0.054
naa
2c
0.061
0.051
2.60
0.230
2d
0.155
2e
1.520
2f
1.320
1.720
0.098
0.012
0.040
1.930
0.190
0.185
0.053
0.048
0.210
2g
3.111
2h
0.210
BHA
BHT
2.130
1.940
BHA, 3-tert-butyl-4-hydroxyanisole; BHT, 3,5-di-tert-butyl-4-hydroxy-toluene; DPPH, 2,2-diphenyl-1-picrylhydrazyl; HO, hydroxyl; LP, lipid peroxidation.
a50% inhibition not achieved.

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FIGURE 1 Expression levels of caspace-3 and Bcl-2 in HepG2 cells after 48 h incubation with 2c, 2d, 2g, and 2h at their respective
IC50s compared to unreacted cells
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4
EXPERIMENTAL
4.1.2 Synthesis of porphyrin derivatives 2a–h
Porphyrin derivatives 2a–h were synthesized according to the
previously reported work.[13]
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4.1 Chemistry
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4.1.1 General
5,10,15,20-Mesotetrakis(3-aminophenyl)-21H,23H-porphyrin
Gallenkamp electric melting point apparatus was used to determine
melting points in degree centigrade, all melting points were
uncorrected. Mattson 5000 FTIR spectrometer was used to record
the IR spectra (KBr disk) at Mansoura University, Faculty of Science.
Bruker WP 300, 200 MHz in DMSO as solvent was used to measure
1H NMR spectra at Cairo University, Faculty of Science, Microanalyti-
cal Center. Unicam UV/Vis-spectrometer was used to record
ultraviolet spectra at Mansoura University, Faculty of Science.
Aldehydes: commercially available aldehydes were used as received.
m-Aminobenzaldehyde was obtained from Aldlab Chemicals. 2,2,2-
Trifluoro-N-(4-formylphenyl)acetamide was prepared according to the
previously reported work (Richard P. Bonar-Law, J. Org. Chem., 1996,
61, 3623–3634).[30] N-(4-Formylphenyl)methanesulfonamide from
Alfa Chemistry, N-(3-formylphenyl)methanesulfonamide from Sigma–
Aldrich, N-formylpiperidine and N-formylmorpholine from Alfa Aesar.
Julolidine-9-carbaldahyde was obtained from Tokyo Chemical Indus-
try. N-Formylphenothiazine was prepared by traditional formylation
method of Dűff (POCl3/DMF at 100°C for 4 h).
(2a)
Yield 81%; m.p. 193°C; IR (KBr): ν/cm−1 = 3350 (NH2), 3250 (NH),
1
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1661 (C N), 1575 (C C); H NMR (DMSO-d6) δ (ppm) = 5.22 (s, 8H,
4NH2), 6.24 (d, 2H, pyrrolic CH), 6.38 (d, 2H, pyrrolic CH), 6.44 (d, 2H,
pyrrolic CH), 6.78 (d, 1H, pyrrolic CH), 6.82 (d, 1H, pyrrolic CH), 6.62–
7.82 (m, 12H, Ar-H), 8.89 (s, 1H, NH), 9.60 (s, 1H, NH); 13C NMR: δ
(ppm): 103.1, 118.3, 119.5, 120.98, 121.35, 123.1, 130.1, 131.1,
132.4, 136.5, 136.6, 140.9, 145.8, 153.7, 160.1; UV-vis. spectrum:
(λmax) 350, 425, 520, 550, 590, 650 nm. Anal. calcd. for C44H34N8
(674.81): C, 78.32; H, 5.08; N, 16.61%. Found: C, 78.10; H, 4.99; N,
16.50%.
5,10,15,20-Mesotetrakis(4-trifluoro-acetamidophenyl)-
21H,23H-porphyrin (2b)
Yield 76%; m.p. 171°C; IR (KBr): ν/cm−1 = 3310 (NH), 1682 (CO), 1665
(C N), 1580 (C C); 1H NMR (DMSO-d6) δ (ppm) = 6.24 (d, 2H,
pyrrolic CH), 6.26 (d, 2H, pyrrolic CH), 6.41 (d, 2H, pyrrolic CH), 6.52 (d,
2H, pyrrolic CH), 7.45–7.83 (m, 16H, Ar-H), 8.83 (s, 1H, NH), 9.62 (s,
1H, NH), 10.00 (s, 1H, NH-CO); 13C NMR: δ (ppm): 103.1, 115.8, 119.5,
120.5, 122.0, 127.2, 129.8, 132.1, 136.7, 138.0, 141.1, 143.1, 155.0,
157.4, 161.1; UV-vis. spectrum: (λmax) 360, 430, 520, 550, 595,
650 nm. Anal. calcd. for C52H30N8F12O4 (1058.84): C, 58.99; H, 2.86;
N, 10.58%. Found: C, 58.81; H, 2.77; N, 10.50%.
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The original spectra of the investigated compounds are provided
as Supporting Information. The InChI codes of the compounds
together with some biological activity data are also provided as
Supporting Information.
5,10,15,20-Mesotetrakis(4-methylsulfonamidophenyl)-
21H,23H-porphyrin (2c)
TABLE 4 The docking scores of selected porphyrins in the active
Yield 71%; m.p. 210°C; IR (KBr): ν/cm−1 = 3350 (NH), 1660 (CN), 1570
(CC), 1300 (SO2); 1H NMR (DMSO-d6) δ (ppm) = 3.00 (s, 12H, 4CH3),
6.22 (d, 2H, pyrrolic CH), 6.24 (d, 2H, pyrrolic CH), 6.40 (d, 2H, pyrrolic
CH), 6.51 (d, 1H, pyrrolic CH), 6.91 (d, 1H, pyrrolic CH), 6.93–7.85 (m,
16H, Ar-H), 8.81 (s, 1H, NH), 9.63 (s, 1H, NH), 10.50 (s, 1H, NHSO2);
13C NMR: δ (ppm): 43,1, 103.1, 116.2, 119.7, 120.8, 128.1, 130.2,
132.3, 133.4, 136.0, 138.0, 141.1, 143.1, 155.7, 161.1; UV-vis.
spectrum: (λmax) 353, 425, 515, 550, 595, 650 nm. Anal. calcd. for
sites of caspase-3 and Bcl-2
Docking scores for
caspase-3 (kcal/mol)
Docking scores for
Bcl-2 (kcal/mol)
Porphyrin
2h
2d
2c
2g
−7.6
−5.6
−10.6
−10.1
−4.0
−10.7
−10.9
−9.8

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FIGURE 2 (a) The highest affinity docking pose of 2d in the active site of caspase 3. (b) A zoom-in view showing the major amino acids
interacting with 2d
C48H42N8O8S4 (987.15): C, 58.40; H, 4.29; N, 11.35%. Found: C,
58.33; H, 4.11; N, 11.21%.
12H, 4CH3), 6.22 (d, 2H, pyrrolic CH), 6.24 (d, 2H, pyrrolic
CH), 6.40 (d, 2H, pyrrolic CH), 6.49 (d, 1H, pyrrolic CH), 6.63
(d, 1H, pyrrolic CH), 6.65–7.82 (m, 16H, Ar-H), 8.89 (s, 1H, NH),
5,10,15,20-Mesotetrakis(3-methylsulfonamidophenyl)-
9.60 (s, 1H, NH), 10.54 (s, 1H, NHSO2). UV-vis. spectrum: (λmax)
21H,23H-porphyrin (2d)
Yield 78%; m.p. 187°C; IR (KBr): ν/cm−1 = 3345 (NH), 1662 (C N),
360, 425, 515, 550, 595, 650 nm. Anal. calcd. for C48H42N8O8S4
(987.15): C, 58.40; H, 4.29; N, 11.35%. Found: C, 58.32; H, 4.25; N,
11.31%.
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1571 (C C), 1310 (SO2); 1H NMR (DMSO-d6) δ (ppm) = 3.05 (s,
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FIGURE 3 A 2D depiction of the interactions between 2d and the active site of caspase, as illustrated in Figure 2

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FIGURE 4 A 2D depiction of the interactions between 2d and the active site of Bcl-2. The green arrow refers to an interaction with the
backbone carbonyl of the alanine amino acid
5,10,15,20-Mesotetrakis(piperidin-1-yl)porphyrin (2e)
CH), 3.85 (t, 16H, morpholinic CH), 6.24 (d, 2H, pyrrolic CH), 6.26 (d,
2H, pyrrolic CH), 6.40 (d, 2H, pyrrolic CH), 7.78 (d, 2H, pyrrolic CH),
8.84 (s, 1H, NH), 9.62 (s, 1H, NH); 13C NMR: δ (ppm): 52.3, 53.3, 67.0,
113.8, 114.7, 117.3, 119.9, 123.2, 125.1, 127.0, 139.6, 158.1. UV-vis.
spectrum: (λmax) 355, 425 nm. Anal. calcd. for C36H42N8O4 (650.78): C,
66.44; H, 6.51; N, 17.22%. Found: C, 66.40; H, 6.47; N, 17.23%.
Yield 61%; m.p. 196°C; IR (KBr): ν/cm−1 = 3315 (NH), 1620 (C N),
1580 (CC); 1H NMR (DMSO-d6) δ (ppm) = 1.12–1.41 (m, 24H,
piperidine CH), 3.46 (t, 8H, piperidine CH), 3.65 (t, 8H, piperidine
CH), 6.26 (d, 2H, pyrrolic CH), 6.40 (d, 2H, pyrrolic CH), 6.50 (d, 2H,
pyrrolic CH), 7.80 (d, 2H, pyrrolic CH), 8.65 (s, 1H, NH), 9.60 (s, 1H,
NH), 10.54 (s, 1H, NHSO2); 13C NMR: δ (ppm): 24.5, 26.1, 53.7, 58.5,
113.8, 114.7, 117.3, 119.9, 123.2, 125.1, 127.0, 139.9, 158.1. UV-vis.
spectrum: (λmax) 355, 450 nm. Anal. calcd. for C40H50N8 (642.90): C,
74.73; H, 7.84; N, 17.43%. Found: C, 74.78; H, 7.73; N, 17.39%.

5,10,15,20-Tetrakis(2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]
quinolin-9-yl)porphyrin (2g)
Yield65%;m.p. 263°C;IR(KBr):ν/cm−1 = 3333(NH), 1621(C N), 1577

1

(C C); H NMR(DMSO-d6) δ (ppm) = 1.96 (m, 16H, quinolinic CH), 2.79
5,10,15,20-Mesotetrakis(morpholin-1-yl)porphyrin (2f)
Yield 70%; m.p. 221°C; IR (KBr): ν/cm−1 = 3310 (NH), 1625 (C N),
(t, 16H, quinolinic CH), 3.37 (t, 16H, quinolinic CH), 6.24 (d, 2H, pyrrolic
CH), 6.38 (d, 2H, pyrrolic CH), 6.44 (d, 2H, pyrrolic CH), 6.92 (s, 8H, Ar-
H), 7.84 (d, 2H, pyrrolic CH), 8.87 (s, 1H, NH), 9.64 (s, 1H, NH). UV-vis.
spectrum: (λmax) 355, 427 nm. Anal. calcd. for C68H66N8 (995.33): C,
82.06; H, 6.68; N, 11.26%. Found: C, 81.91; H, 6.58; N, 11.19%.

1

1570 (C C); H NMR (DMSO-d6) δ (ppm) = 3.55 (t, 16H, morpholinic
5,10,15,20-Tetra(10H-phenothiazin-10-yl)porphyrin (2h)
Yield71%;m.p. 237°C;IR(KBr):ν/cm−1 = 3315(NH), 1620(C N), 1585

(C C); 13C NMR: δ (ppm): 111.4, 114.8, 115.8, 118.3, 119.5, 120.5,

122.0, 123.2, 125.2, 127.2, 128.1, 132.2, 139.0, 140.6, 150.5. UV-vis.
spectrum: (λmax) 355, 427 nm. Anal. calcd. for C68H42N8S4 (1099.38): C,
74.29; H, 3.85; N, 10.19%. Found: C, 74.10; H, 3.78; N, 10.00%.
|
4.2 Biochemical assays
|
4.2.1 Antioxidant properties
The antioxidant properties were evaluated according to the reported
method.[24]
FIGURE 5 The superposition of the docked pose of 2d (red) and
the co-crystallized ligand (yellow) of the Bcl-2 crystal structure (PDB
ID: 4MAN) with respect to the amino acids of the active site
|
4.2.2 Cytotoxicity and antitumor assay
It was carried out according to the previously published work.[13]

ABU-MELHA
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FIGURE 6 A 2D depiction of the interactions between 2d and the active site of Bcl-2. The green arrow refers to an interaction with the
backbone carbonyl of the alanine amino acid
|
4.2.3 Detection of caspase-3 activity
CONFLICT OF INTEREST
Each IC50 of each compound was treated with HepG2 cells and
incubated for 48 h. The cells were detached by trypsin and lysed by
freezing at liquid nitrogen and then thawing with gentle mixing. Lysates
cell was incubated with horseradish peroxidase (HRP) conjugated anti-
CASP8 antibody for 30 min at 37°C. The end reaction product was
recorded at 450 nm using enzyme-linked immunosorbent assay
(Platinum ELISA; Biospes).
The author declares that there is no conflict of interest.
ORCID
Sraa Abu-Melha
http://orcid.org/0000-0001-5539-0163
REFERENCES
|
4.2.4 Detection of Bcl-2 protein levels
[1] Porphyrins and Metalloporphyrins (Ed: K. M. Smith), Elsevier, The
Netherlands 1976, pp. 29–58.
[2] K. M. Kanish, K. M. Smith, R. Guilard, The Porphyrin Handbook, Vol. 6,
Academic Press, San Diego 2000, pp. 45–118.
[3] R. F. Barth, A. H. Soloway, J. H. Goodman, R. A. Gahbauer,
Neurosurgery 1999, 44, 433.
Bcl-2 levels were evaluated in HepG2 cells treated with the
corresponding IC50 of each compound and incubated for 48 h and
compared with their levels in control untreated HepG2 cell line. The
cells were harvested by applying trypsin and lysed by freezing with
liquid nitrogen and then thawing with gentle mixing. According to the
instructions of the manufacturer of (Platinum ELISA; eBioscience©),
cell lysates were incubated with biotin-conjugate for 2 h at 25°C and
then with streptavidin-HRP for 1 h at 25°C. The reaction product was
detected at 450 nm.
[4] K. J. Barnham, C. I. Masters, A. I. Bush, Nat. Rev. Drug Discov. 2004, 3,
205.
[5] a) M. S. Cooke, M. D. Evans, M. Dizdaroglu, J. Lunez, FASEB J. 2003,
17, 1195; b) T. Finkel, N. G. Holbrook, Nature 2000, 408, 239; c) S. P.
Hussain, L. J. Hofseth, C. C. Harris, Nat. Rev. Cancer 2003, 3, 276.
[6] a) Y.-J. Cai, L. P. Ma, L. F. Hou, B. Zhou, L. Yang, Z.-L. Liu, Chem. Phys.
Lipids 2002, 120, 109; b) B. Zhou, Q. Miao, L. Yang, Z.-L. Liu, Chem.
Eur. J. 2005, 11, 680; c) B. Zhou, L.-M. Wu, L. Yang, Z.-L. Liu, Free
Radical Biol. Med. 2005, 38, 78.
|
4.3 Molecular docking
[7] Y. J. Cai, J. G. Fang, L. P. Ma, L. Yang, Z.-L. Liu, Biochim. Biophys. Acta
2003, 1637, 31.
The porphyrin structures were optimized using GAMESS software[25]
at the HF/6-31 level. The optimized structures were used in various
dockings. All dockings were done using AutoDock4[26] keeping side
chains at the active site rigid. The bonds in the porphyrin ring were also
rendered rigid during docking. We used the crystal structure 4MAN[27]
and 6BFJ[28] for Bcl-2 and caspase-3, respectively. Docking was
performed using 100 steps of genetic algorithm while keeping all the
default settings provided by AutoDock Tools. Visualization was done
using VMD.[29]
[8] D. Holten, D. F. Bocian, J. S. Lindsey, Acc. Chem. Res. 2002,
35(1), 57.
[9] O. Mongin, A. Schuwey, M. A. Vallot, A. Gossauer, Tetrahedron Lett.
1999, 40, 8347.
[10] A. Dudkowiak, E. Teslak, J. Habdas, J. Mol. Struct. 2006, 792–793, 93.
[11] G. Mele, R. Del Sole, G. Vasapollo, E. Garcia-Lopez, J. Catal. 2003,
217(2), 334.
[12] C. S. Chan, K. S. Chan, J. Org. Chem. 1994, 59, 6084.
[13] A. A. Fadda, R. E. El-Mekawy, A. I. El-Shafei, H. Freeman, Arch. Pharm.
Chem Life Sci. 2013, 346, 53.

10 of 10
ABU-MELHA
|
[14] S. Shaaban, A. Negm, M. A. Sobh, L. A. Wessjohann, Anti-Cancer
Agents Med. Chem. 2016, 16, 621.
[26] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. k. Belew, D. S.
Goodsell, A. J. Olson, J. Comput. Chem. 2009, 30, 2785.
[27] A. J. Souers, J. D. Leverson, S. W. Elmore, Nat. Med. 2013, 19,
202.
[15] N. Mimica-Dukin, B. Bozin, M. Sokolovic, N. Simin, J. Agric. Food Chem.
2004, 52, 2485.
[28] M. E. Thomas, R. Grinshpon, P. Swartz, A. C. Clark, J. Biol. Chem. 2018,
293, 5447.
[16] N. A. Abbas, S. Shaaban, H. E. Gaffer, E. Abdel-Latif, Res. J. Pharm. Biol.
Chem. Sci. 2015, 6, 1655.
[29] W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graph 1996, 14, 33.
[30] R. P. Bonar-Law, J. Org. Chem. 1996, 61, 3623.
[17] S. Shaaban, F. Sasse, T. Burkholz, C. Jacob, Bioorg. Med. Chem. 2014,
22, 3610.
[18] B. F. Abdel-Wahab, S. Shabaan, Synthesis 2014, 46, 1709.
[19] S. Shaaban, R. Diestel, B. Hinkelmann, Y. Muthukumar, R. P. Verma,
F. Sasse, C. Jacob, Eur. J. Med. Chem. 2013, 58, 192.
[20] A. A. Fadda, R. E. El-Mekawy, A. I. El-Shafei, J. Porphyr. Phthalocya-
nines 2015, 19, 735.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
[21] A. A. Fadda, R. E. El-Mekawy, A. I. El-Shafei, J. Chem. 2013, 2013, 1.
[22] S. B. Blumenthal, A. K. Kiemer, G. Tiegs, S. Seyfried, M. Holtje, B.
Brandt, H.-D. Holtje, S. Zahler, A. M. Vollmar, FASEB J. 2005, 19,
1272.
How to cite this article: Abu-Melha S. Efficient synthesis of
meso-substituted porphyrins and molecular docking as
potential new antioxidant and cytotoxicity agents. Arch
Pharm Chem Life Sci. 2018;1–10.
[23] M. del Toro, P. Bucek, A. Aviñó, J. Jaumot, C. González, R. Eritja, R.
Gargallo, Biochimie 2009, 91, 894.
[24] A. A. Fadda, R. E. El-Mekawy, N. N. Soliman, A. M. Allam, M. T.
Abdelaal, Dyes Pigments 2018, 155, 300.
[25] M. W. Schmidt, K. K. Baldridge, J. A. Boatz, S. T. Elbert, M. S. Gordon,
J. J. Jensen, S. Koseki, N. Matsunaga, K. A. Nguyen, S. Su, T. L. Windus,
M. Dupuis, J. A. Montgomery, J. Comput. Chem. 1993, 14, 1347.
https://doi.org/10.1002/ardp.201800221