Docking studies, antitumor and antioxidant evaluation of newly synthesized porphyrin and metalloporphyrin derivatives

Article abstract of DOI:10.1016/j.dyepig.2020.108728

In this work, we have synthesized a series of novel porphyrin derivatives, 1–5, in high yields. The metal complexes of two of the newly synthesized porphyrin derivatives, 1a–d and 2a–d, have also been synthesized in high yields and characterized. In the synthesis of the new porphyrins and metalloporphrins, we employed our reported strategy in which we utilized N,N-dimethylformamide (DMF) as a capping agent in the reaction of pyrrole with different hetero-aryl aldehydes. The new porphyrin derivatives are equipped with different aromatic substituents and hetero-cycles at the peripheral position. The structures of the new compounds were confirmed by elemental and spectral analyses. The geometry and magnetic properties of the new metalloporphyrins 1a–d and 2a–d have also been studied. Antioxidant and cytotoxic activities of the new compounds were evaluated and structure-activity relationships were performed. Porphyrin derivatives 2a and 4 showed exceptional antioxidant activity compared to ascorbic acid as a reference. While the derivatives 2, 3, and 5 exhibited very strong cytotoxic activity against two human cell lines, HePG-2 and MCF-7. Docking for the most promising antioxidant porphyrins, 2a and 4, into the binding active site of antioxidant protein Human Peroxiredoxin (code: 1HD2) has been carried out to detect the degree of recognition antioxidant activity. Molecular docking of the most cytotoxic active porphyrins, 3 and 5, into the biding site of telomerase inhibitor enzyme has been carried out to assess the degree of recognition cytotoxic activity.

Full text of DOI:10.1016/j.dyepig.2020.108728

Journal Pre-proof
Docking studies, antitumor and antioxidant evaluation of newly synthesized porphyrin
and metalloporphyrin derivatives
Asmaa Ahmed, Walaa A.E. Omar, Hala A. El-Asmy, Laila Abou-Zeid, Ahmed A.
Fadda
PII:
S0143-7208(20)31425-X
DOI:
https://doi.org/10.1016/j.dyepig.2020.108728
DYPI 108728
Reference:
To appear in: Dyes and Pigments
Received Date: 14 May 2020
Revised Date: 23 June 2020
Accepted Date: 21 July 2020
Please cite this article as: Ahmed A, Omar WAE, El-Asmy HA, Abou-Zeid L, Fadda AA, Docking studies,
antitumor and antioxidant evaluation of newly synthesized porphyrin and metalloporphyrin derivatives,
Dyes and Pigments, https://doi.org/10.1016/j.dyepig.2020.108728.
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Author statement
For the manuscript titled: “Docking studies, antitumor and antioxidant
evaluation of newly synthesized porphyrin and metalloporphyrin
derivatives”
We declare that,
All persons who meet authorship criteria are listed as authors, and all authors certify
that they have participated sufficiently in this work
We also confirm that the manuscript has been read and approved by all
named authors.
We understand that the Corresponding Author is the sole contact for the Editorial
process. He/she is responsible for communicating with the other authors about
progress, submissions of revisions and final approval of proofs
Authors:
a,b
c,d*
b
e,f
Asmaa Ahmed , Walaa A. E. Omar , Hala A. El-Asmy , Laila Abou-Zeid ,
b
Ahmed A. Fadda
a
Research Unit of Sustainable Chemistry, Faculty of Technology, P.O. Box 8000
FI-90014 University of Oulu
−
Finland.
b
Chemistry Department, Faculty of Science, Mansoura University, 35516 Mansoura
−
Egypt
c
Department of Basic Sciences, Faculty of Energy & Environmental Engineering, The
British University in Egypt, El Sherouk City, P.O.Box 43, 11837 Cairo
− Egypt.
Walaa.Omar@bue.edu.eg
d
Department of Engineering Sciences and Mathematics, Faculty of Petroleum and
Mining Engineering, Suez University
− Egypt.
e
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Mansoura
University, 35516 Mansoura
− Egypt
f
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Delta University,
5516 Gamassa – Egypt
3

Graphic abstract
Docking studies, antitumor and antioxidant evaluation of newly synthesized porphyrin
and metallo-porphyrin derivatives
a,b
c,d*
a
e
b
Asmaa Ahmed , Walaa A. E. Omar , Hala A. El-Asmy , Laila Abou-Zeid , Ahmed A. Fadda
Compound 5 with gigantic phenothiazine functions is one of the promising lead in the treatment of cancer as it
showed highest cytotoxic activity against MCF7 cell line and proper complementarity with G-quartet 2A5R
protein.

Docking studies, antitumor and antioxidant evaluation of newly
synthesized porphyrin and metalloporphyrin derivatives
a,b
c,d*
b
e,f
Asmaa Ahmed , Walaa A. E. Omar , Hala A. El-Asmy , Laila Abou-Zeid , Ahmed
b
A. Fadda
a
Research Unit of Sustainable Chemistry, Faculty of Technology, P.O. Box 8000
FI-90014 University of Oulu
− Finland.
b
Chemistry Department, Faculty of Science, Mansoura University, 35516 Mansoura
−
Egypt
c
Department of Basic Sciences, Faculty of Energy & Environmental Engineering, The
British University in Egypt, El Sherouk City, P.O.Box 43, 11837 Cairo
− Egypt.
Walaa.Omar@bue.edu.eg
d
Department of Engineering Sciences and Mathematics, Faculty of Petroleum and Mining
Engineering, Suez University
− Egypt.
e
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Mansoura
University, 35516 Mansoura
− Egypt
f
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Delta University, 35516
Gamassa – Egypt
Abstract
In this work, we have synthesized a series of novel porphyrin derivatives, 1–5, in high
yields. The metal complexes of two of the newly synthesized porphyrin derivatives, 1a–d
and 2a–d, have also been synthesized in high yields and characterized. In the synthesis of
the new porphyrins and metalloporphrins, we employed our reported strategy in which we
utilized N,N-dimethylformamide (DMF) as a capping agent in the reaction of pyrrole with
different hetero-aryl aldehydes. The new porphyrin derivatives are equipped with different
1

aromatic substituents and hetero-cycles at the peripheral position. The structures of the new
compounds were confirmed by elemental and spectral analyses. The geometry and magnetic
properties of the new metalloporphyrins 1a–d and 2a–d have also been studied. Antioxidant
and cytotoxic activities of the new compounds were evaluated and structure-activity
relationships were performed. Porphyrin derivatives 2a and 4 showed exceptional
antioxidant activity compared to ascorbic acid as a reference. While the derivatives 2, 3, and
5
exhibited very strong cytotoxic activity against two human cell lines, HePG-2 and MCF-
. Docking for the most promising antioxidant porphyrins, 2a and 4, into the binding active
7
site of antioxidant protein Human Peroxiredoxin (code: 1HD2) has been carried out to
detect the degree of recognition antioxidant activity. Molecular docking of the most
cytotoxic active porphyrins, 3 and 5, into the biding site of telomerase inhibitor enzyme has
been carried out to assess the degree of recognition cytotoxic activity.
Key Words. Porphyrin; Capping mechanism; Metal Complex; Antioxidant; Antitumor;
Docking Studies
1. Introduction
Porphyrins and related tetrapyrrolic macrocyclic pigments are important heterocyclic
compounds due to their wide chemical and biologically applications. They have been
employed successfully in the areas of catalysis, medicine, and material science [1–4].
Additionally, metalloporphyrins that coordinated to transition metal ions such as iron,
cobalt, and magnesium were able to perform a diversity of functions and applications [5–8].
Traditional strategies described by Rothemund and Alder’s for porphyrins synthesis allowed
the synthesis of limited structures of porphyrins through simple condensation between
pyrrole and aldehydes and the yields were always very low (< 20 %) [9–12]. The difficulties
of separation and the limited availability of suitable precursors are extra limitations during
porphyrin traditional synthetic protocols. Since then, many modifications have been done to
2

the Rothemund procedures to overcome the drawback accompanied the synthesis [13–16].
Among the considerable attempts dedicated to elaborate improved protocols for facilitating
the synthesis of various useful porphyrin systems in satisfactory yields, our reported
procedure has shown considerable success. We succeeded to prepare a series of meso-
porphyrins in high yields using DMF as a capping reagent [17–19].
Although many synthetic routes for certain porphyrins are available so far [14–17], it is still
challenging to synthesize porphyrins bearing specific chemical groups in satisfactory yield
(> 20 %) [20–22]. Meso-substituted porphyrins have been considered as a key building
block for porphyrin-based systems and molecular materials [3,13–15]. There is an increased
interest in the development of meso-substituted porphyrin synthesis as they act as ligands
for metal ions forming important metalloporphyrins for therapeutic purposes [3, 23–27].
Porphyrin derivatives have been tested as sensitizing drugs for application in tumor
diagnosis and treatments using photodynamic therapy (PDT) [24–29] and boron neutron
capture therapy (BNCT) [30, 31]. In PDT and BNCT therapies, light and low energy
neutrons are utilized for the activation of a tumor-localized sensitizer, respectively.
In the above-mentioned therapeutic investigations, certain porphyrin derivatives undergo
selective localization in tumor tissues depending on their affinity for carrier biomolecules
and biological membranes [24–31]. Clearly, positively charged porphyrins, such as tetra-
(trimethylaminophenyl)- and meso-tetra(methylpyridyl)- porphyrins showed a strong
interaction with the negatively charged groups of the biological targets , such as certain
proteins [32], RNA [33] and DNA [34, 35] , in addition to their effectiveness as
photosensitizers for PDT [24–29, 32–34]. It is also observed that the number and
distribution of the positive charges about the porphyrin macrocycle plays a very important
role in its photodynamic efficacy [32, 34]. For example, amphiphilic porphyrin derivatives
3

+
containing water-solubilizing groups, such as –NMe , showed an increasing photodynamic
3
efficacy compared to hydrophilic macrocycles [33–35].
The free radical-mediated peroxidation of the membrane and oxidative damage of DNA
were considered responsible for a variety of chronic health problems, such as cancer,
antherosclerosis, neurodegenerative diseases, and aging [36]. Consequently, during the last
years, many studies investigating biological attributes of different precursors, which include
mainly antioxidant activity, have been reported [36, 37].
Apparently, studies related to the kinetics and mechanisms of natural antioxidants have
shown that simple structural customization can lead to a noticeable enhancement in the
antioxidative activity [38, 39]. An interesting example of such observation was resveratrol,
an antioxidant component in red wine, in which chemical structure modification could
efficiently improve its antioxidative activity and cytotoxicity against cancer cells [38].
Based on the above promising findings, we were motivated to use porphyrin as a basic
nucleus to design more potent antioxidants and chemopreventive agents against cancer. The
properties of porphyrin can also be modulated by the insertion of different metal ions such
as copper, silver, nickel, and zinc [40–43]. Therefore, we have also synthesized a novel
series of porphyrin metal complexes in high yields and performed an in-vitro study of their
protective effects. The new porphyrins have been prepared in high yields by using the
previously reported modified one-pot mixed solvent method in which DMF is used as a
capping reagent [17–19]. All the synthesized compounds have been characterized and
assessed for the antioxidant and antitumor activities. The geometry and magnetic interaction
in the metal complexes have been also investigated using the electron spin resonance (ESR)
technique.
2
. Experimental Section
.1 General remarks
2
4

1
13
H and C-NMR spectra were measured on 300 MHz JOEL ECA-300 spectrometer, using
DMSO-d or CDCl as solvents and TMS as the internal standard, at the Microanalytical
6
3
Center, Faculty of Science, Mansoura University. The IR spectra were recorded (KBr disk)
on a Mattson 5000 FTIR Spectrometer at Faculty of Science, Mansoura University.
Elemental analyses (C, H, and N) were carried out at the Faculty of Science, Cairo
University, the results were found to be in good agreement with the calculated values.
Ultraviolet spectra were recorded using Unicam UV2 UV/Vis spectrometer at the
Microanalytical Unit, Faculty of Science, Mansoura University. Butylated hydroxyanisole
(BHA) and L-ascorbic acid were purchased from Sigma-Aldrich Company. 2,2-Diphenyl-1-
●
picrylhydrazyl radical (DPPH ) was purchased from Wako. All other chemicals were of
analytical grade and purchased from Sigma-Aldrich, Germany.
2
2
-
.2 Synthesis
.2.1 Synthesis of porphyrin derivatives 1–5
General procedures for the synthesis of porphyrins 1–5
A mixture of the appropriate aromatic aldehyde (0.72 mmol) and pyrrole (0.72 mmol) in
DMF (15 ml) was placed into a 50 mL three-necked flask. The mixture was flushed with
nitrogen gas for a couple of minutes and then heated to 100 ˚C for 10 min. P-toluene
sulphonic acid (0.72 mmol, dissolved in DMF) was then added to the reaction mixture. The
colorless mixture turned red over the next couple of minutes then heated at 150 ˚C for 1 h.
The reaction mixture was then cooled and poured over ice with stirring for 15 min. the
residue was collected, dried under vacuum and purified by column chromatography using
chloroform/hexane
Synthesis
dihydroporphyrin (1), (H mdcpp)
(1.5/1)
as
eluent).
-
of
(1,4',9'',15''')-5,10,15,20-tetrakis(2,4-dichlorophenyl)-21,23-
2
5

-1
Dark brown color, yield 85 %, m.p. 160 ˚C. IR (cm ): ν(N-H), 3279; ν(C=N), 1664;
1
ν(C=C), 1584; ν(C-Cl), 784. H-NMR (DMSO-d
6
) (ppm):
δ
2.27 (s, 1H, NH), 5.30 (d, J =
10.5 Hz, 2H, 2 pyrrolic CH), 5.81 (d, 2H, J = 11 Hz, 2 pyrrolic CH), 6.53 (d, 2H, J = 11
Hz, 2 pyrrolic CH), 7.00 (d, 2H, J = 7.5 Hz, 2 pyrrolic CH), 7.32 (d, 4H, J = 9 Hz, Ar-H),
1
3
7
.50 (d, 4H, J = 9 Hz, Ar-H), 7.95 (s, 4H, Ar-H), 10.64 (s, 1H, NH). C-NMR (DMSO-d )
6
(ppm):
δ 103.0, 120.5, 123.3, 125.0, 128.6, 130.5, 132.2, 135.7, 136.5, 137.7, 155.8, 181.1.
UV-Vis. Spectrum: λmax = 413 nm. Elemental Anal.: Calcd. C, 59.36; H, 2.49; N, 6.29;
(
C Cl H N ); Found: C, 59.70; H, 2.53; N, 6.30 %.
44 8 22 4
-
Synthesis of N,N-dimethyl-4-[(5,10',14'',19''')-10,15,20-tris(4-dimethylaminophenyl)-
21,23-dihydroporphyrin-5-yl]aniline (2), (mdmapp)
-1
Dark violet color, yield 82 %, m.p > 300 ˚C. IR (cm ): ν(NH), 3240; ν(CH ), 2925; ν(C=N),
3
1
1
659; ν(C=C), 1603. H-NMR (DMSO-d
6
) (ppm):
δ
3.03 (s, 1H, NH), 3.90 (s, 24H, 4 N-
(
CH ) ), 5.31 (d, 2H, J = 10 Hz, 2pyrrolic CH), 6.23 (d, J = 10 Hz, 2H, 2pyrrolic CH),
3 2
6.46 (d, J = 8 Hz, 2H, J = 7.5 Hz, 2pyrrolic CH), 6.54 (d, 2H, J = 7.5 Hz, 2pyrrolic CH),
1
3
6
.95 (d, 8H, J = 8 Hz, Ar-H), 7.25 (d, 8H, J = 8 Hz, Ar-H), 10.23 (s, 1H, NH). C-NMR
) (ppm): 106.4, 106.6, 107.4, 117.3, 128.6, 128.8, 124.4, 129.5, 132.2, 132.6,
49.3, 163.0, 167.6. UV-Vis. Spectrum: λmax = 421 nm. Elemental Anal.: Calcd. C, 79.37;
(DMSO-d
6
δ
1
H, 4.42; N, 14.26; (C H N ); Found: C, 79.76; H, 4.42; N, 14.20 %.
5
2
50
8
-
Synthesis
of
4-[(1,4',9'',15''')-10,15,20-tris(4-carboxyphenyl)-21,23-
dihydroporphyrin-5-yl]benzoic acid (3)
-1
Deep green color, yield 75 %, m.p > 300 ˚C. IR (KBr): ύ /cm = 3450 (OH), 3350 (NH),
1
1
630 (C=N), 1570 (C=C), H-NMR (DMSO-d ) (ppm):
δ
2.30 (s, 1H, NH), 5.91 (d, 2H, J =
6
11 Hz, 2 pyrrolic CH), 6.22 (d, 2H, J = 11 Hz, 2 pyrrolic CH), 6.30 (d, 2H, J = 8 Hz, 2
pyrrolic CH), 6.73 (d, 2H, J = 8 Hz, 2 pyrrolic CH), 7.21 (d, 8H, J = 7.5 Hz, Ar-H), 7.81 (d,
6

8
4
C
-
H, J = 7.5 Hz, Ar-H), 10.52 (s, 1H, NH), 10.56 (s, 1H, OH). UV-Vis. Spectrum: λmax =
26 nm. Elemental Anal.; Calcd. for C H N O (790), C 72.90; H 3.82; N 7.09 %. Found:
4
8
30
4
8
72.52;
H
3.55;
N
7.50
%.
Synthesis of 3-chloro-4-[4-[(1,4',9'',15''')-10,15,20-tris[4-(3-chloro-6-hydroxy-4-
isoquinolyl)phenyl]-21,23-dihydroporphyrin-5-yl]phenyl]isoquinolin-6-ol (4)
Porphyrin (4) was prepared according to the general procedure using the aldehydes (A, 2-
chloroquinoline-6-hydroxy-3-carboxaldehyde) to give 4 as deep brown color, yield 64 %,
-1
1
m.p > 300 ˚C. IR (KBr): ύ /cm = 3340 (NH), 1630 (C=N), 1570 (C=C), 675 (C-Cl). H-
NMR (DMSO-d ) (ppm): 6.20 (d, 2H, J = 10.5 Hz, pyrrolic protons), 6.46 (d, 4H, J = 9 Hz,
6
pyrrolic protons), 7.22 (s, 4H, quinoline C
d, 4H, J = 7.5 Hz, quinoline C -H), 7.82 (d, 2H, J = 8 Hz, pyrrolic protons), 8.24 (s, 4H,
quinoline C -H), 8.80 (s, 1H, NH), 9.40 (s, 4H, 4OH), 9.92 (s, 1H, NH). UV-Vis. Spectrum:
λmax = 424nm. Anal. data for C H Cl N O (1018), Calcd. C, 65.90; H, 2.96; 13.89; N,
5 7
-H), 7.33 (d, 4H, J = 6 Hz, quinoline C -H), 7.70
(
8
4
56
30
4
8
4
10.98%. Found C, 65.88; H, 2.91; N, 10.99 %
-
Synthesis of 3-[4-[(1,4',9'',15''')-10,15,20-tris(4-phenothiazin-3-ylphenyl)-21,23-
dihydroporphyrin-5-yl]phenyl]phenothiazine (5)
Porphyrin (5) was prepared according to the general procedures using aldehyde (B, 10, 10a-
dihydro-4aH-phenothiazine-3 carbaldehyde) to give 5 as deep blue color, yield 72 %, m.p >
-
1
1
3
00 ˚C. IR (KBr): ύ /cm = 3340 (NH), 1630 (C=N) , 1570 (C=C), H-NMR (DMSO-d
6
)
)
1
3
(
ppm): 3.03 (s, 1H, NH), 6.66- 7.51 (m, 29H, Ar-H) 8.57 (s, 1H, NH) C-NMR (DMSO-d
6
(
ppm) d: 114.4, 115.3, 121.7, 122.5, 125.6, 126.2, 127.0, 127.1, 127.2, 127.5, 128.2, 128.7,
29.2, 134.9, 137.2, 142.0. UV-Vis. Spectrum: λmax = 422 nm. Anal. Calcd. for C H N S
8 4
1
68
50
(1106). C, 73.75; H, 4.55; N, 10.12%. Found C, 73.70; H, 4.50; N, 10.10%.
-
Synthesis of aldehydes, 2-chloroquinoline-6-hydroxy-3-carboxaldehyde (A) and 10,
10a-dihydro-4aH-phenothiazine-3 carbaldehyde (B)
7

-
Synthesis of 2-chloroquinoline-6-hydroxy-3-carboxaldehydes (A)
N,N-Dimethylformamide (9.13 g, 9.6 mL, 0.125 mol) was cooled to 0 ˚C in a flask
equipped with dry tube and phosphoryl chloride (53.7 g, 32.2 mL, 0.35 mol) was added
dropwise with stirring. To this solution was added the N-Phenyl-acetamide (0.05 mol).
After 30 min the solution was heated under reflux for 15 hrs in a water bath. The reaction
mixture was poured onto ice-water (300 ml) and stirred for 30 min at 0−10 ˚C. The
separated solid material was filtered off and washed with water. The combined filtrate was
adjusted to pH 9 with aqueous sodium hydroxide, chloroform (200ml) was added and the
mixture was stirred for 30 min and then separated. The aqueous phase was extracted further
with chloroform, combined organic layer was dried with magnesium sulfate and evaporated
to give oil which was treated with HCl and extracted further with chloroform, the organic
layer to give the corresponding aldehyde (A). Deep yellow, yield 93 %, m.p = 185 ˚C. IR
-
1
1
(
(
KBr): ύ /cm = 3400 (OH), 1730 (C-C=O), 1630 (C=N), 695(C-Cl). H-NMR (DMSO-d )
6
ppm):
4 7 8
δ 7.26 (s, 1H, H ), 7.47 (dd, 1H, J = 2, 9Hz, H ) 7.87 (dd, 1H, J =8, 2Hz, H ) 8.78 (s,
1
H, H ), 9.22 (s, 1H, CHO), 9.78 (s, 1H, OH). Anal. Calced for C H NO Cl (207), C,
5
10
6
2
57.85, H, 2.91, N, 6.75 % found C, 57.80; H, 2.89; N, 6.68 %.
-
Synthesis of 10, 10a-dihydro-4aH-phenothiazine-3 carbaldehyde (B)
Aldehyde (B) was synthesized following the general procedures used for the synthesis of
aldehydes (A) starting from the base phenothiazine. Off-white powder, yield 80 %, m.p
-1
1
1
75−185 ˚C. IR (KBr): ύ /cm = 3340 (NH), 1730 (C=O), 1570 (C=C), H-NMR (DMSO-
d6) (ppm): 6.88- 7.51 (m, 7H, Ar-H), 8.59 (s, 1H, NH), 8.70 (s, 1H, CHO) Anal. Calcd. for
C H NOS (229). C, 68.09; H, 4.84; N, 6.11%. Found C, 68.00; H, 4.79; N, 6.01 %.
13
11
2
.2.2 Synthesis of the new metalloporphyrins 1a–d and 2a–d
General procedure
-
8

The metal complexes from the free porphyrins 1 and 2 have been prepared using the
following routes:
Route 1: The complexes were prepared by heating a mixture of porphyrin 1 or 2 (0.25
mmol) dissolved in 10 mL ethanol and the metal salt (0.25 mmol of CuCl
2 2
.2H O,
VOSO .H O, AgNO or K PdCl ) dissolved in 10 mL H O under reflux for 6–8 h. After
4
2
3
2
4
2
cooling to room temperatures, the precipitate was filtered off, washed with ethanol, diethyl
ether, and dried.
Route 2: The proper aldehyde (0.01 mol) was dissolved in DMF ( 50 mL) and p-toluen
sulfonic acid (0.01 mol) was added. Pure pyrrole (0.01 mol) was then added dropwise. The
mixture was stirred under argon for 1 h followed by the addition of 2.5 equiv. of the proper
metal salt. The whole reaction mixture was then refluxed for 8 h in the air. The solvent was
removed under vacuum and the crude product was washed with water, dried and purified by
flash chromatography over silica gel using chloroform as eluent.
-
[Cu(mdcpp)].3H O (1a)
2
-1
Dark green ppt. Yield: (85 %). IR (cm ): ν(C=N), 1604; ν(C=C), 1576; ν(C-N), 1335;
ν(Cu-N), 502. UV-visible (nm): 536, 640. μ = 1.9 B. M. Elemental Anal.: Calcd. C, 52.4;
eff
H, 2.6; N, 5.6; (C Cl CuH N O ); Found: C, 52.7; H, 2.9; N, 5.52 %.
4
4
8
26
4
3
-
[VO(mdcpp)] (1b)
-1
Dark red ppt. Yield: (83 %). IR (cm ): ν(C=N), 1621; ν(C=C), 1598; ν(C-N), 1349; ν(V-N),
5
2
-
10. UV-visible (nm): 302, 410, 648. μeff= 2.1 B. M. . Elemental Anal.: Calcd. C, 55.3; H,
.1; N, 5.9; (C Cl H N OV); Found: C, 55.2; H, 2.0; N, 5.7 %.
4
4
8
20
4
[Ag (mdcpp)(H O) ] (1c)
2
2
2
-1
Dark violet ppt. Yield: (87 %). IR (cm ): ν(C=N), 1634; ν(C=C), 1580; ν(C-N), 1353;
1
ν(Ag-N), 530. H-NMR (DMSO-d
6
) (ppm):
δ
5.56 (d, J = 10 Hz, 2H, 2 pyrrolic CH), 5.90
(d, 2H, J = 10.5 Hz, 2 pyrrolic CH), 6.63 (d, 2H, J = 8 Hz, 2 pyrrolic CH), 7.97 (d, 2H, J =
9

8
Hz, 2 pyrrolic CH), 7.32 (d, 4H, J = 7.5 Hz, Ar-H), 7.56 (d, 4H, J = 7.5 Hz, Ar-H), 8.31
s, 4H, Ar-H). UV-visible (nm): 306, 416. Elemental Anal.: Calcd. C, 46.3; H, 2.1; N, 4.9;
Ag ); Found: C, 46.0; H, 1.9; N, 4.6 %.
[Pd(mdcpp)](1d)
(
(
-
2 8 24 4 2
C44Cl H N O
-1
Dark blue-black ppt. Yield: (84 %). IR (cm ): ν(C=N), 1619; ν(C=C), 1584; ν(C-N), 1378;
1
ν(Pd-N), 510. H-NMR (DMSO-d ) (ppm): δ 5.33 (d, 1H, J = 10.5 Hz, 2 pyrrolic CH), 5.87
6
(
d, 2H, J = 10.5 Hz, 2 pyrrolic CH), 6.51 (d, 2H, J = 8 Hz, 2 pyrrolic CH), 6.96 (d, 2H, J =
Hz, 2 pyrrolic CH), 7.35 (d, 4H, J = 7.5 Hz, Ar-H), 7.55 (d, 4H, J = 7.5 Hz, Ar-H), 8.11
s, 4H, Ar-H). UV-visible (nm): 347, 460, 994.4. Elemental Anal.: Calcd. C, 53.1; H, 2.0;
N, 5.6; (C Cl H N Pd); Found: C, 52.9; H, 1.8; N, 5.3 %.
8
(
4
4
8
20
4
-
[Cu(mdmapp)].4H O (2a)
2
-1
Red bloody ppt. Yield: (82 %). IR (cm ): ν(CH ), 2934; ν(C=N), 1659; ν(C=C), 1590; ν(C-
3
N), 1318; ν(Cu-N), 540. UV-visible (nm): 318, 414, 470. μ = 1.6 B. M. Elemental Anal.
eff
Calcd. C, 67.7; H, 6.1; N, 12.2; (C CuH N O ); Found: C, 67.7; H, 5.9; N, 11.9 %.
52
56
8
4
-
2 2 2
[Ni(mdmapp)(H O) ].3H O (2b)
-1
Dark green ppt. Yield: (87 %). IR (cm ): ν(CH
3
), 2934; ν(C=N), 1659; ν(C=C), 1590; ν(C-
N), 1335; ν(Cu-N), 540. UV-visible (nm): 318, 383, 438, 538, 799. μeff= 3.23 B. M.
Elemental Anal.: Calcd. C, 66.8; H, 6.2; N, 12.0; (C H N NiO ); Found: C, 66.5; H, 6.3;
5
2
58
8
5
N, 11.8 %.
[Ag (mdmapp)(H O) ] (2c)
-
2
2
2
-1
Dark blue ppt. Yield: (87 %). IR (cm ): ν(CH ), 2936; ν(C=N), 1600; ν(C=C), 1573; ν(C-
3
1
N), 1320; ν(Ag-N), 520. H-NMR (DMSO-d
6
) (ppm): 3.92 (s, 24H, 4 N-(CH
3
)
2
), 5.32 (d,
2H, J = 10 Hz, 2pyrrolic CH), 6.21 (d, 2H, J = 10 Hz, 2pyrrolic CH), 6.46 (d, 2H, J = 7.5
Hz, 2pyrrolic CH), 6.59 (d, 2H, J = 7.5 Hz, 2pyrrolic CH), 6.83 (d, 8H, J = 8 Hz, Ar-H),
10

7
6
-
.29 (d, 8H, J = 8 Hz, Ar-H). UV-visible (nm): 306, 401, 471. Elemental Anal. Calcd. C,
2.3; H, 5.2; N, 11.2; (Ag C H N O ); Found: C, 62.0; H, 5.1; N, 11.0 %.
2
52 56
8
2
2
[Pd(mdmapp)].2H O (2d)
-
1
Dark brown ppt. Yield: (82 %). IR (cm ): ν(CH
3
), 2934; ν(C=N), 1659; ν(C=C), 1590;
3.72 (s, 24H, 4 N-(CH ) ), 5.29
1
ν(C-N), 1335; ν(Cu-N), 540. H-NMR (DMSO-d ) (ppm):
δ
6
3 2
(d, 2H, J = 10 Hz, 2pyrrolic CH), 6.22 (d, 2H, J = 10 Hz, 2pyrrolic CH), 6.44 (d, 2H, J =
7.5 Hz, 2pyrrolic CH), 6.55 (d, 2H, J = 7.5 Hz, 2pyrrolic CH), 6.87 (d, 8H, J = 8 Hz, Ar-
H), 7.32 (d, 8H, J = 8 Hz, Ar-H). UV-visible (nm): 311, 437, 502. 928.4 Elemental Anal.:
Calcd. C, 67.2; H, 5.6; N, 12.1; (C52 Pd); Found: C, 67.2; H, 5.5; N, 12.0 %.
.3 Biochemical assays (Antioxidant properties, Cytotoxicity and antitumor assay)
.3.1 Antioxidant properties
52 8 2
H N O
2
2
The free radical scavenging activity of all the new compounds was evaluated by measuring
their ability to neutralize DPPH and OH radicals. The DPPH (2,2-diphenyl-1-
picrylhydrazyl) assay and hydroxyl radical scavenging assays were applied as previously
reported [17, 18].
2.3.2 Cytotoxicity and antitumor assay
Cytotoxicity and antitumor assay have been carried out according to the previously reported
procedures [17, 18].
2.4 Docking studies
2.4.1 Docking of antioxidant active porphyrin derivatives 2a and 4
All computational modeling was conducted with Schrödinger Suite 2015 (Schrödinger,
LLC) that was run on dual-core PC [44].
The newly synthesized compounds were comparatively evaluated in terms of their binding
mode to the Human Peroxiredoxin 5, antioxidant enzyme (1HD2) pocket. Compounds 2a
and 4 showed potential antioxidant activity therefore they were considered for further
11

molecular modeling study to explore their recognition profile at the human 1HD2 binding
active site.
-
Preparation of enzyme
The starting coordinates of the X-ray crystal structure of Human Peroxiredoxin in complex
with the BEZ 201A (PDB code: 1HD2) that retrieved from the RCSB Protein Data Bank of
Brookhaven National Laboratory [45]
-
Preparation of tested porphyrin
Molecular docking for compounds 2a and 4 into the biding site of oxidase enzyme was
performed using the Schrödinger Suite 2015 (Schrödinger, LLC) [44]. The three-
dimensional structures of both compounds were constructed using a building module.
Gasteiger–Hückel charges of ligands were assigned. AMBER with 100 iterations was
utilized for energy minimization. The active site of the protein was defined to contain
residues within a 10.0-Å radius around any of the inhibitor atoms. All hydrogens were
added and the enzyme structure was exposed to a refinement protocol in which the
constraints on the enzyme were systematically eliminated and minimized until the RMS
gradient was 0.01kcal/mol Å. The conformer with the lowest energy, the “global-minima,”
was pre-positioned using the crystal structure ligand “BEZ 201A” as a template at the
enzyme-binding pocket.
2.4.2 Docking antitumor active porphyrin derivatives 3 and 5
Molecular docking of compounds (3 and 5) into the biding site of telomerase was explored
using Schrödinger Suite 2015 (Schrödinger, LLC). The three-dimensional structures of the
aforementioned compounds were constructed using Chem. Draw 3D Ultra 11.0 software.
Using a compute module to perform Gasteiger–Hückel, charges of ligands were assigned.
Porphyrin derivatives, 3 and 5, were energetically minimized by using AMBER with 100
12

iterations and minimum RMS gradient of 0.10. The template (PDB code: 2A5R) was
obtained from the RCSB Protein Data Bank.
-
Preparation of the enzyme
The starting coordinates of the X-ray crystal structure of the complex of tetra(4-n-
methylpyridyl) porphin with monomeric parallel-stranded DNA Tetraplex (PDB code
2
A5R: in complex with the POH 25A) that retrieved from the RCSB Protein Data Bank of
Brookhaven National Laboratory [46]
Preparation of tested porphyrin
-
Molecular docking of compounds (3 and 5) into the binding site of the telomerase inhibitor
enzyme was investigated using the Schrödinger Suite 2015 (Schrödinger, LLC). The three-
dimensional structures of the aforementioned compounds were constructed using a building
module. Gasteiger–Hückel charges of ligands were assigned. They were energetically
minimized by using AMBER with 100 iterations. The active site of the protein was defined
to contain residues within a 10.0-Å radius around any of the inhibitor atoms. All hydrogens
were added and the enzyme structure was exposed to a refinement procedure in which the
constraints on the enzyme were gradually eliminated and minimized until the RMS gradient
was 0.01kcal/mol Å. Conformer with the lowest energy, the “global-minima”, was pre-
adjusted using the crystal structure ligand “POH 25A” as a template at the enzyme-binding
pocket.
3.
Result and discussion
3.1 Synthesis and characterization
This work reports the synthesis of new porphyrin derivatives including the free base
compounds 1–5 (Scheme 1) and metal complexes 1a–d, and 2a–d (Scheme 2) which were
evaluated as antitumor and antioxidant agents.
13

<
Scheme 1>
Scheme2>
.1.1 Synthesis and characterization of the free porphyrins 1−5
<
3
The low yields (6–20 %) in porphyrin chemistry is a persistent obstacle so far, although the
continuous advancement in the porphyrin synthesis [20–22]. Recently, Fadda et al. [17, 18]
developed a synthetic method that gives 80–90 % yield with minimal chromatography. We
used the capping mechanism to prevent the pyrrole polymerization process. N,N-
Dimethylformamide (DMF) was used as a solvent and capping reagent (Scheme 3). The
DMF-pyrrole intermediate reacts with more pyrrole to keep constructing the corresponding
porphorengon, and the DMF molecule would act as a good leaving group in each time
pyrrole is added [17–19].
<
Scheme 3>
Our reported strategy facilitated the synthesis of various derivatives of porphyrin and
metalloporphyrin in high yields [17, 18]. In this work, we were able to obtain the free-base
porphyrins 1−5 in high yields (64–85 %). The newly synthesized porphyrins were well
1
characterized by spectral data. In the H-NMR spectra, porphyrin derivatives 1−5 showed
two sets of doublets between δ 5.00 and 8.00 ppm that correspond to the pyrrolic CH
protons which are different from the signals for porphyrins normally found above δ 8.00
ppm. This shift in peaks position may be related to the increased shielding of the pyrrolic
protons resulting from the interference of electron delocalization within the macrocycle.
1
The H-NMR of porphyrin 1 showed a singlet signal at δ 10.64 ppm that corresponds to the
N-H proton, two doublet signal at δ 7.32 and 7.22 ppm and a singlet signal at δ 7.48 due to
the deshielded aromatic protons, in addition to the doublets signals at δ 6.24, 6.38, 6.44, and
7.84 ppm characterizing the pyrrolic protons.
14

1
The H-NMR of porphyrin 2 showed a characteristic singlet signal at δ 10.23 ppm
corresponding to the N-H proton. Aromatic protons displayed a characteristic two doublets
due to (AB system) at δ 6.77 and 7.16 ppm, while pyrrolic protons appeared as doublets at δ
3 2
6.24, 6.38, 6.44, and 7.84 ppm. The singlet signal for (-N(CH ) ) protons appeared at δ 3.02
ppm.
1
The H-NMR of porphyrin 3 showed a characteristic singlet signal at δ 12.71 ppm due to
carboxylic proton and another singlet signal at δ 10.52 ppm that corresponds to the N-H
proton. The aromatic system represented as two doublets at δ 7.83 and 7.55 ppm due to “AB
system”. Pyrrolic protons appeared at its normal position at δ 6.00−7.50 ppm as
1
3
doublets. C-NMR displayed an obvious signal at δ 167.6 ppm characterizing the carbonyl
carbon.
1
The H-NMR of porphyrin 4 displayed a singlet signal at δ 9.92 ppm due to -NH proton, a
singlet at δ 9.40 corresponding to the O-H group, in addition to pyrrolic and quinolinic
protons at the range δ 6.20−8.40 ppm.
1
The HNMR of porphyrin 5 displayed a singlet signal at δ 8.57 ppm due to N-H protons, in
addition to a complex pattern of multiplet signals due to pyrrolic and aromatic protons at δ
5.60- 7.80 ppm.
3.1.2 Synthesis and characterization of metalloporphyrins 1a−d, and 2a−d
Recently, the biochemical and photo-electro properties of porphyrin have attracted growing
interest. Substituted metalloporphyrins are now playing crucial roles in the fields of
iatrology [47], medicinal chemistry, [48–50], analytical chemistry [49] and electronic
devices [13–15, 51, 52]. It has been observed that when a porphyrin complex is applied in
the natural enzyme peroxidase, the dioxygen gets activated under mild conditions therefore,
15

porphyrin complexes with various metal ions have received much attention as antioxidants
53].
[
Metalloporphyrins were synthesized by the classical Alder two-step strategy, which
involves the reaction of the free base porphyrin with the proper metal salt in refluxing DMF
[12]. The yields of metalloporphyrins obtained by this method were unsatisfactory (< 20 %)
which is apparently a result of the long reaction time and pyrrole polymerization. On the
other hand, in the present work, the one-pot reaction method with DMF as a capping agent
was proved to be very successful. The latter strategy has the advantages of the short reaction
time, simple workup, and higher yield of the porphyrin complexes [40, 41]. In the one-pot
reaction method, pyrrole, a proper aldehyde and the metal salt were mixed and refluxed for
a reasonable short time to produce a variety of metalloporphyrins in high yields. In this
work, the addition of DMF as a capping reagent during the one-pot synthesis for the
metalloporphyrins 1a–d and 2a–d increased the yield dramatically (82–87 %) compared to
the two-steps method in which the free porphyrin has to be prepared before the following
step in which the metal ion is added.
2
+
Cu(II), Pd(II), Ag(I), and VO porphyrin metal complexes showed exceptional properties
for medicinal and biological applications [47–50]. While Nickel porphyrin complexes have
been employed successfully in catalytic reactions [54]. Therefore, in the synthesis of the
2
+
new complexes 1a–d, and 2a–d, metal II salts of Cu(II), Ag(I), Ni(II), VO , and Pd(II)
have been used along with the pyrrole and the proper aldehydes (Scheme 2). The purity of
1
the new complexes is confirmed by H-NMR and elemental analysis.
The structures conformation of the metal complexes 1a–d and 2a–d were examined by IR
spectroscopy and compared to that of the free porphyrins, (1,4',9'',15''')-5,10,15,20-
tetrakis(2,4-dichlorophenyl)-21,23-dihydroporphyrin
1
and
N,N-dimethyl-4-
[(5,10',14'',19''')-10,15,20-tris(4-dimethylaminophenyl)-21,23-dihydroporphyrin-5-
16

yl]aniline 2. The IR absorption frequencies for the complexed porphyrins 1a–d, and 2a–d
showed two main differences than the free porphyrins 1 and 2. Firstly, The N-H bond
-
1
-1
stretching and bending frequencies of free porphyrins located at ~3300 cm and ~960 cm .
Insertion of the metal ion into the porphyrin resulted in the disappearance of the N-H group
and alternatively, the band characteristic for the functional groups of M-N bond appeared at
-1
~
1000 cm . This confirmed the formation of metalloporphyrin derivative. Secondly, As the
coordination between the nitrogen and the metal ion reduces the electron density in the
-1
azomethine link, the band at ~1664, 1659 cm corresponding to the ν(C=N) group in the
free porphyrin is shifted in the complexes towards lower wavenumber values ∼ 1600–1630
-1
-1
cm . The bands observed in the region around 1310–1370 cm are attributed to υ (C-N)
stretching vibrations in the complexes. This value is less than the value of υ (C-N)
-1
stretching in ligands, which appears at 1383 and 1355 cm , respectively. This decrease by
-1
5
5–20 cm in the metalloporphyrin is probably due to the increase in electron density of
azomethine (C-N) bond due to π–electron delocalization from the metal to the nitrogen
--
atom and resonance interaction with porphyrin ring. Therefore, it can be inferred that 1 and
-
-
2
acted as bi-negative tetradentate ligands, coordinating the metal ions through the
azomethine nitrogen and the deprotonated imine nitrogen centers forming four six-
-1
membered rings. Moreover, the several bands at 550–500 cm due to ν (M-N) stretches
were observed in the IR spectra of the complexes [55].
1
The H-NMR spectroscopic data for the complexes 1c, 1d, 2c, and 2d in DMSO-d are
6
1
reported in the experimental section. The H-NMR spectra for metalloporphrins 1c, 1d, 2c,
and 2d confirmed the complexation of the free porphyrins 1 and 2 with the proper metal
ions. The spectra of free porphyrin 1 and 2 showed sharp singlet signals characterizing the
NH proton at δ 10.64 and δ 10.23 ppm, respectively. In the spectra of complexes 1c, 1d, 2c,
and 2d, The N-H signals disappeared due to the replacement of the imine proton of the free
17

porphyrins by the metal ions, which confirms the formation of complexes 1c, 1d, 2c and 2d.
On the other hand, the signals for pyrrolic -CH are slightly shifted to downfield due to the
-
-
--
complexation of 1 and 2 through the deprotonated imine nitrogen and the metal centers
[55]. In metalloporphyrins 1c, 2c, and 2b, we attributed the presence of two water molecules in
the coordination sphere to the proposed geometry for each complex. The coordinated water
molecules were confirmed by elemental analysis and thermal study [56], which gave an excellent
agreement with the proposed structure. Thermograms for porphyrins 1c, 2c, and 2b are in the
supplementary file.
3
.2 Electronic and geometrical studies of metalloporphyrins 1a–d and 2a–d
.2.1 Magnetic and electronic spectra for complexes 1a–d and 2a–d
3
The electronic spectra of the complexes 1a–d, and 2a–d in DMSO contain intense bands.
The bands between 430–900 nm are resulting from ligand-to-metal charge-transfer (LMCT)
transitions while weaker bands are assigned to d–d transitions. The intra-ligand charge
transfer (n → π* and π → π*) appeared as transitions below 430 nm. The electronic spectra
of the diamagnetic Pd(II) complexes 1d, and 2d exhibit bands near 465 and 340 nm due to
1
1
1
1
A → B and A → E transitions, respectively, which relevant to a square-planar
1
g
g
1g
g
configuration [57]. The magnetic moment of [Ni(mdmapp)(H O) ], 2b, is 3.24 BM
2
2
3
assigned for octahedral structure with A
electronic spectrum shows a broadband at 799 nm is assigned to the A
transition [58]. The electronic spectra of [Cu(mdcpp)] 1a and [Cu(mdmapp)] 2a exhibit
2
g ground term [57, 58]. Interestingly, Its
3
3
g→ T
g (F) (ν )
1 2
2
2
2
bands near 640 nm assigned to T g→ Eg. The band positions with magnetic moments of
2
1
.9 and 1.6 BM are designated to a square-planar geometry [57, 58]. The porphyrin in plane
geometry is a crucial factor of Cu(II) square planar geometry.
.2.2 Electron spin resonance (ESR) spectra of the Cu(II) and VO(IV) complexes
3
18

ESR spectrum of the complexes 1a, 1b, and 2a was recorded in the solid-state in order to
identify the stereochemistry and the site of the metal-ligand bonding as well as the magnetic
2
+
interaction in the metal complexes. Generally, the mononuclear oxo-vanadium (VO ), (S =
/2, I = 7/2) has a characteristic octet ESR spectrum showing the hyperfine coupling owing
1
5
1
to V nuclear magnetic moment [59]. In comparison with other reported ESR spectrum of
2
+
VO [60], Compound 1b shows a broad single line with a poorly resolved eight-line
pattern. Specifically, in the powder sample, the spectrum shows parallel and perpendicular
features corresponding to axially symmetric anisotropy with poorly resolved sixteen-lines
hyperfine splitting which confirms the interaction between the electron and the vanadium
nuclear spin (I = 7/2) [61]. The spin Hamiltonian parameters for the complex are calculated
-4
-1
as g|| = 1.93, g
⊥
=1.96 and the hyperfine coupling A|| = 195 × 10 (cm ), A⊥ = 55. The ESR
1
parameters indicate that the unpaired electron (d ) of 1b complex is present in the d orbital
xy
with square-pyramidal geometry. The values obtained for this complex is in complete
2
+
agreement with those reported for VO square pyramidal geometry [62]. The spectra of the
Cu(II) complexes 1a and 2a at 300 K show a strong isotropic absorption band in the high
field area due to the tumbling motion of the molecules. These complexes at 77K show four
well-resolved peaks of low intensities in the low field area and one single intense peak in
the high field area. In square planar complexes, the unpaired electrons are in the dx2–y2
2
orbital, and B
1
g is the ground state with the g|| > g
(2.37 > 2.08), which confirms the square planar geometry of the complexes.
Also, No band for Cu–Cu interaction was observed which indicates the mononuclear Cu(II)
63].
⊥
. Based on the detected values, it is clear
that g| > g
⊥
|
[
3
.3 Pharmacology of porphyrins
.3.1 Antioxidant activity
3
19

The newly synthesized compounds 1–5, 1a–d, 2a, 2c and 2d were evaluated as antioxidant
agents using DPPH assay at different concentrations of 5 mg/mL of each tested sample [64].
Ascorbic acid was used as a reference drug. The radical scavenging activity was determined
●
for the synthesized compounds using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH ) as a
stable radical organic compound and its oxidative method is widely used in the
determination of the capacity of free radical scavengers or the capacity of hydrogen donors
following in vitro assay. This type of antioxidant test is suggested for the compounds
containing reactive nitrogen, oxygen and carbon atom species. The mechanism of action of
antioxidant tests as reported previously [65] initiated by the transfer of hydrogen atom,
single electron and followed by proton transfer.
From the results of inhibitive concentrations (IC50) in Table 1, compounds 2a and 4 are the
most potent antioxidant compounds compared to the results of ascorbic acid. On the other
hand, compounds 5, 2c, 2d, 1a, and 1b exhibited good antioxidant activity, while,
compounds 2, 3, 1c, and 1d have moderate activity. Compound 1 has the highest value of
IC = 1.760 mg/ mL and considered as the less potent antioxidant agent.
5
0
The changes in the free radical scavenging capacity of the test compounds and their metal
complexes depending on the inhibition percent are shown in Table 1.
3.3.2 Structure-activity relationship (SAR) for the antioxidant activity
The order of free radical scavenging capacity of the tested compounds and their complexes
as followed: 2a > 4 > Vit. C > 5 > 2c > 2d > 1b > 1a > 2 > 1d > 1c > 3 > 1.
The metal complexes 1a–d showed an enhanced antioxidant activity with lower values of
IC compared to porphyrin 1. Similarly, complexes 2a, 2c, and 2d showed an improved
5
0
oxidative capacity compared to the free porphyrin 2. The scavenging activity of the formed
complexes, in general, is significantly higher than that of the corresponding free ligands,
indicating that the formed complexes are stronger than free radical scavengers [66]. Similar
20

high antioxidant activity of metalloporphyrins compared to their free bases is reported in
literature [67]. The increasing antioxidant activity of the coordinated porphyrins compared
to their corresponding free bases can be attributed to the presence of an additional
scavenging metal center, which increases the affinity of the molecule to stabilize the radical
[67].
It is also observed that, the presence of electron-donating substituents on the aromatic ring
increases the antioxidant activity while electron-withdrawing substituents decrease it [68].
This explains the higher potency of compound 2 as antioxidant agent compared to
compound 1 and 3 in this work. The chlorine atoms with their strong electron-withdrawing
character in porphyrin 1 decrease it antioxidant activity. Similarly, compound 3, which has
four carboxylic groups (strong electron-attracting group), possesses a lower antioxidant
activity compared to 2 (Table 3). Consequently, compound 2 enhanced antioxidant capacity
is attributed to the presence of the four electron-donating -N(CH ) groups.
3
2
The obtained results showed that compounds 2a and 4 can trap the free radicals and
displayed antioxidant capacity higher than ascorbic acid (Vitamin C). Compound 4 possess
two types of active hydrogen donors, the pyrrolic N-H and the phenolic O-H. Both groups
can undergo hydrogen transfer to the DPPH radical to form very stable radicals by
resonance on quinolone and porphyrin [69–72]. A plausible mechanism for the hydrogen
transfer from compound 4 to the DPPH radical is illustrated in Scheme 4. In the proposed
mechanism (Scheme 4), the hydrogen atom transfers from the phenolic O-H to the stable
radical 2,2-diphenyl-1-picrylhydrazyl (DPPH radical), followed by radical stabilization by
the porphyrin macrocycle [73].
<
Scheme 4>
21

The copper (II) complex 2a showed higher antioxidant activity than its corresponding
ligand 2. This result is in a great agreement with previously reported work [74]. The higher
antioxidant activity is attributed to the presence of additional scavenging metal center. In
complex 2a, the DPPH free radical is scavenged by reduction mechanism of Cu(II) to Cu(I),
2
z
in which the DPPH radical is scavenged by the d
orbital of Cu(II) ion [72], Scheme 5.
<
Scheme 5>
The oxidative potentials of the synthesized compounds are related to the presence of
compounds capable of exerting impact by breaking the chain of free radicals through
hydrogen atoms donation [63]. Consequently, the obtained antioxidant results from this
study prolong a link with the use of these compounds in the treatment of the pathological
disease, which arises from oxidative stress.
<Table1> and <Figure 1>
3.3.3 Cytotoxicity (anticancer screening)
Cytotoxicity studies of the newly synthesized compounds were performed against two
mammalian cancer cell lines, HepG2 and MCF-7 cells. The evaluation process was carried
out according to the previously reported work [17].
The newly synthesized compounds 1–5, 1a–d and 2a–d were screened for cytotoxic activity
(Table 2) against HepG2 (hepatoma cells or human liver hepatocarcinoma cell line) and
MCF-7 cells (human breast adenocarcinoma cell line). The results were expressed as
growth inhibitory concentration (IC50) values, which represent the concentrations of the
compound required to produce a 50 % inhibition of cell growth after 72 h of incubation,
compared with untreated controls (Table 2, Fig. 2). Generally, All the tested compounds
showed a cytotoxic activity that ranged from very strong to weak activity. Compound 5
showed an IC value (5.56 ± 0.4 µg/mL) that is very similar to DOX as a standard for the
5
0
two used cell lines. The most promising values were observed from compounds 2 and 3
22

(
very strong activity) with an IC value of 5.52 ± 1.1 and 6.34 ± 0.7 µg/mL for HePG-2 cell
50
line, respectively. Compounds 5, 2, and 3 exhibited very strong activity with an IC value
5
0
of 4.28 ± 0.3, 8.83 ± 1.2 and 7.51 ± 0.5 µg/mL for MCF-7 cell line, respectively.
Compounds 1a, 1b, 2b, and 4 exhibited strong activities for both cell lines. Compounds 1,
1
d, 2a, 2c, and 2d have moderate cytotoxic activities against the same cell lines.
Figure 2 >, < Table 2>
.3.4 Structure-activity relationship (SAR) from cytotoxicity studies
<
3
The cytotoxic activity of the new porphyrins towards different cell lines depends on the
following: Firstly, the intermolecular hydrogen bonds formed between the porphyrin
derivatives and DNA bases. Secondly, the positive charges on the tested porphyrins that are
attracted to the negative charges on the cell wall. By comparing the experimental cytotoxic
data of the new compounds to their structures, the following SAR was inferred: Compounds
5, 3, and 2 have exceptional strong activities, this can be attributed to the substituents, -NH,
-
N(CH ) and -COOH which may undergo addition to any unsaturated moiety in DNA or
3
2
form hydrogen bonds with any of the nucleo-bases of the DNA and causes its damage .
Compounds 1a and 1b showed strong activity towards the tested cell lines due to the
presence of strong electron attracting chlorine atoms which caused the molecules to be
positively charged resulting in electrostatic attraction with the DNA nucleobases. For
compound 4, the strong activity is probably caused by chlorine atom as an electron-
withdrawing substituent and one hydroxyl group which may also be added to any
unsaturated moiety in DNA or forming a hydrogen bond with either one of the nucleobases
of the DNA and causes its damage.
<Table2>, <Figure 2>
3
.4 Molecular docking studies
.4.1 Docking studies for the new porphyrins 2a and 4
3
23

Free radical-scavenging activity properties using the 3-D crystallographic peroxiredoxin 5
PRDX5) were carried out to explore the new porphyrins recognition in the active site as a
(
potential antioxidant.
As porphyrins 2a and 4 showed the most powerful antioxidant activities, docking of the of
2a and 4 into the binding active site of antioxidant protein Human Peroxiredoxin (code:
1HD2) has been conducted to check the degree of recognition antioxidant activity. The
tested compounds, 2a and 4 were prepared by partial charges and optimal minimizations
using the compute module of MOE.
Compound 2a and 4 showed proper and promising antioxidant activity by proper
recognition at the binding active site of Human Peroxiredoxin protein. By studying the
binding behavior of the new compounds relative to the antioxidant; BEZ 201A reference
ligand that docked at the 1 HDC complex, it was found that hydrogen bonds and
hydrophobic interactions formed with the surrounding amino acids are used to predict their
binding modes. This active pocket consisted of conserved amino acid residues including
Thr44, Gly46, Cys47 and Arg127 that play fundamental roles in recognition of the docked
compounds by hydrogen bonding interaction and hydrophobic interactions.
Metallo complex compound 2a is hocked by bifurcated two hydrogen bonds between the
conserved Arg127 residue and the two N-pyrrols that are facing Arg127. A third
heterocyclic pyrrole ring showed π-π stacking interaction with Arg127.
Docking studies of compound 4 revealed that the crucial amino acid Arg127 played an
important role in the recognition of the ligand by forming strong hydrogen bonds between
N-pyrrole and N-arginine. There are also hydrogen bonds formed between the hydroxy
aliphatic function group and the conserved amino acid, Val75. Moreover, the amino group
of the key amino acid, Arg127, which is one of the conserved residues at the binding
24

pocket, showed proper complementarity with the N-pyrrole atom in addition to the π-π
stacking interaction between Arg127 and the heterocyclic pyrrole ring (Figure 3).
<Figure 3>
3.4.2 Porphyrin derivatives and their interaction with Telomerase (Telomerase
inhibition activity)
The availability of a high-resolution crystallographic structure of the human telomerase
enzyme facilitated the research considering the small molecules with potential telomerase
inhibition activity that brings to light the selective and safe cure of cancer. Telomerase
becomes one of the competitive targets in the field of cancer treatment. Additionally, it
plays a unique anti-aging role.
In this study, we aimed to find efficient G-quartets ligands that would be able at the same
time to discriminate between different forms of nucleic acids in order to be selective as
telomeric DNA. The binding of porphyrins allows them to match perfectly large planar
surfaces of G-quartets.
<Figure 4>
G-quartet consists of four guanine bases, so its square is twice as large as the square of the
base pair. Large porphyrin ligands perfectly overlap with G-quartets and professionally
expressed proper quadruplex selectivity.
The standard porphyrin ligand, POH 25A, that aligned in the G-quartet 2A5R protein
showed appropriate recognition with the ceiling DG-4 and DG-8 bases by forming π-π
stacking interactions with the two pyridine heterocyclic rings back protruded from the
minor groove. In the meantime, DG-13 and DG-17 bases in the major groove flooring
showed amplifying recognition with both pyridine heterocyclic rings exposed from the
major groove showed in Figure 5
<Figure 5>
25

-
Docking studies of compound 3
Telomerase and compound 3 have strong complementarities as it sandwiched between the
roof and floor of the major groove of telomeric DNA as the reference ligand POH 25A. In
comparative docking studies where POH 25A and porphyrin 3 were docked (Figure 6), it
was clear that the presence of four enriched electrophilic benzoic functions properly
approved the complementarity of compound 3.
<Figure 6>
Two benzoic acid groups out of four in porphyrin 3, expressed professional alignment
extended out of the G-Q major groove. The other two benzoic acid substituents got
extended back through the minor groove forming two distinctive hydrophilic and lipophilic
interactions, respectively. A strong hydrogen bond was formed between DG13 and the
benzoic carboxyl group and augmented the hydrophilic recognition of compound 3. DG17
stabilized the phenyl group of the same benzoic function by expressing π-π stacking
interactions. Apparently, the ceiling DG-2 and DG-4 bases were recognized with π-π
stacking with the heterocyclic pyrrole backbones (Figure 7).
<Figure 7>
-
Docking of compound 5
Docking of compound 5 showed certain selectivity including the following features: Firstly,
its global minima conformer identically aligned the POH-25A, the reference
crystallographic ligand, and also the docked compound 3 (Figure 8-A, B). Secondly:
Porphyrin 5 substituted with gigantic phenothiazine functions showed proper
complementarity with G-quartet 2A5R protein, (Figure 8-C). Compound 5 binds not only
to the surrounding DG bases but also it performed four cation–π interactions with the
phosphorous backbones namely DG5, DG14 and bifurcated two bonds out of DG13
(Figure 8-D). Thirdly, even there is no strong hydrogen bond between the phenothiazine
26

ring and the G-Q bases, and overall π - π stacking hydrophobic interaction between DG8
and the heterocyclic phenothiazine ring is clearly existing.
<Figure 8>
4
. Conclusion
A series of new porphyrin derivatives (1–5) have been synthesized in high yields (64–85
). Porphyrins 1 and 2 have been complexed successively with different metal salts such as
%
2
+
Cu(II), VO , Ag(I), Ni(II) and Pd(II), by a one-pot mixed solvent method. The
corresponding metalloporphyrins were obtained after 6–8 h in high yield (> 80 %). The
relatively high yields obtained during the synthesis of the new porphyrins were attributed to
the use of DMF as a capping agent for the pyrrole during the reaction. The newly
synthesized porphyrin derivatives are equipped with different aryl and heterocyclic
substituents. The formation of the new compounds was confirmed by spectral and elemental
analysis. The new compounds have been tested for their pharmacological activities. It can
be concluded that the electronic properties of the peripheral substituents on the porphyrin
moiety had a major effect on the antioxidant and cytotoxic activities. Compounds 4 and 2a
were the most powerful antioxidants due to the presence of hydroxyl groups in compound 4
and the dimethylamino donating group along with Cu(II) metal in complex 2a. Porphyrin 1
showed the weakest antioxidant activity among all the newly synthesized porphyrins due to
the presence of the electron-withdrawing substituent (Cl). In general, porphyrins with
electron-donating groups showed stronger antioxidant activity than porphyrins equipped
with electron-withdrawing groups. In cytotoxicity tests, compounds 2, 3, and 5 showed very
strong activity towards both HePG2 and MCF-7 cell lines, while 1a, 1b, 2b, and 4 were
only strong active antioxidants compounds for both cell lines due to the electronic effect of
the substituent on the porphyrin moiety. Finally, the molecular docking studies of the most
powerful antioxidant derivatives (2a and 4) and the most active antitumor (3 and 5) were
27