Manuscript Details

Manuscript number DYPI_2020_1055

Title Docking studies, antitumor and antioxidant evaluation of newly synthesized

porphyrin and metallo-porphyrin derivatives

Article type Research paper

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 metallo-porphrins, we employed our reported strategy in
which we utilized dimethyl formamide (DMF) as 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
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 relationship was 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.

Keywords Porphyrin; Capping mechanism; Metal Complex; Antioxidant; Antitumor; Docking

Studies

Corresponding Author Walaa Omar

Corresponding Author's The British University in Egypt

Institution

Order of Authors Asmaa Ahmed, Walaa Omar, Hala Elasmy, Laila Abou-Zeid, Ahmed Fadda

Suggested reviewers M. Wills, Juha Heiskanen, Hala Refat

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 Cover Letter

Dear Respected Editor,

I am pleased to submit an original research article entitled “Docking Studies,

antitumor and antioxidant evaluation of newly synthesized porphyrin and

metallo-porphyrin derivatives” to the journal “Dyes & Pigments”.

I/We confirm that this work is original and has not been published nor

currently under consideration for publication elsewhere.

Walaa A. E. Omar, PhD

Faculty of Energy & Environmental Engineering

The British University in Egypt

+201006109420
Graphic abstract
Docking studies, antitumor and antioxidant evaluation of newly synthesized porphyrin

and metallo-porphyrin derivatives

Asmaa Ahmeda,b, Walaa A. E. Omarc,d*, Hala A. El-Asmya, Laila Abou-Zeide, Ahmed A. Faddab

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.

NH

S
NH N
NH
NH
H
N
N
S

NH
1
2
3
Docking studies, antitumor and antioxidant evaluation of newly
4
5 synthesized porphyrin and metallo-porphyrin derivatives
6
7 Asmaa Ahmeda,b, Walaa A. E. Omarc,d*, Hala A. El-Asmyb, Laila Abou-Zeide,f,
8
9
Ahmed A. Faddab
10
11 aResearch
12
Unit of Sustainable Chemistry, Faculty of Technology, P.O.Box 8000
13
14 FI-90014 University of Oulu  Finland.
15
16 bChemistry Department, Faculty of Science, Mansoura University, 35516 Mansoura 
17
18 Egypt
19
20 cDepartment of Basic Sciences, Faculty of Energy & Environmental Engineering, The
21
22
23
British University in Egypt, El Sherouk City, P.O.Box 43, 11837 Cairo  Egypt.
24
25 Walaa.Omar@bue.edu.eg
26
dDepartment of Engineering Sciences and Mathematics, Faculty of Petroleum and
27
28
29 Mining Engineering, Suez University  Egypt.
30
31 ePharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Mansoura
32
33
University, 35516 Mansoura  Egypt
34
35 fPharmaceutical
36 Chemistry Department, Faculty of Pharmacy, Delta University, 35516
37
38 Gamassa, Egypt
39
40 Abstract
41
42 In this work we have synthesized a series of novel porphyrin derivatives, 1–5, in high
43
44 yields. The metal complexes of two of the newly synthesized porphyrin derivatives, 1a–d
45
46 and 2a–d, have also been synthesized in high yields and characterized. In the synthesis of
47
48 the new porphyrins and metallo-porphrins, we employed our reported strategy in which
49
50
we utilized dimethyl formamide (DMF) as capping agent in the reaction of pyrrole with
51
52
53
different hetero-aryl aldehydes. The new porphyrin derivatives are equipped with
54 1
55
56
57
58 different aromatic substituents and hetero-cycles at peripheral position. The structures of
59
60
the new compounds were confirmed by elemental and spectral analyses. The geometry
61
62
63 and magnetic properties of the new metalloporphyrins 1a–d and 2a–d have also been
64
65 studied. Antioxidant and cytotoxic activities of the new compounds were evaluated and
66
67 structure activity relationships were performed. Porphyrin derivatives 2a and 4 showed
68
69 exceptional antioxidant activity compared to ascorbic acid as a reference. While the
70
71 derivatives 2, 3 and 5 exhibited very strong cytotoxic activity against two human cell
72
73 lines, HePG-2 and MCF-7. Docking for the most promising antioxidant porphyrins, 2a
74
75 and 4, into the binding active site of antioxidant protein Human Peroxiredoxin (code:
76
77
1HD2) has been carried out to detect the degree of recognition antioxidant activity.
78
79
80
Molecular docking of the most cytotoxic active porphyrins, 3 and 5, into the biding site
81
82 of telomerase inhibitor enzyme has been carried out to assess the degree of recognition
83
84 cytotoxic activity.
85
86 Key Words: Porphyrin; Capping mechanism; Metal Complex; Antioxidant; Antitumor;
87
88 Docking Studies
89
90 1. Introduction
91
92 Porphyrins and related tetrapyrrolic macrocyclic pigments are important heterocyclic
93
94
compounds due to their wide chemical and biologically applications. They have been
95
96
97
employed successfully in the areas of catalysis, medicine, and material science [1–4].
98
99 Additionally, metallo-porphyrins that coordinated to transition metal ions such as iron,
100
101 cobalt and magnesium were able to perform a diversity of functions and applications [5–
102
103 8].
104
105 Traditional strategies described by Rothemund and Alder’s for porphyrins synthesis
106
107 allowed the synthesis of limited structures of porphyrins through simple condensation
108
109
110 2
111
112
113
114 between pyrrole and aldehydes and the yields were always very low (< 20%) [9–12]. The
115
116
difficulties of separation and the limited availability of suitable precursors are extra
117
118
119 limitations during porphyrin traditional synthetic protocols. Since then, many
120
121 modifications have been done to the Rothemund procedures in order to overcome the
122
123 drawback accompanied the synthesis [13–16]. Among the considerable attempts
124
125 dedicated to elaborate improved protocols for facilitating the synthesis of various useful
126
127 porphyrin systems in satisfactory yields, our reported procedure has shown a considerable
128
129 success. We succeeded to prepare a series of mesoporphyrins in high yields using DMF
130
131 as a capping reagent [17–19].
132
133
Although many synthetic routes for certain porphyrins are available so far [14–17], it is
134
135
136
still challenging to synthesize porphyrins bearing specific chemical groups in satisfactory
137
138 yield (> 20%) [20–22]. Meso-substituted porphyrins have been considered as a key
139
140 building block for porphyrin-based systems and molecular materials [3,13–15]. There is
141
142 an increased interest in the development of meso-substituted porphyrin synthesis as they
143
144 act as ligands for metal ions forming important metallo-porphyrins for therapeutic
145
146 purposes [3, 23–27].
147
148 Porphyrin derivatives have been tested as sensitizing drugs for application in tumor
149
150
diagnosis and treatments using photodynamic therapy (PDT) [24–29] and boron neutron
151
152
153
capture therapy (BNCT) [30, 31]. In PDT and BNCT therapies, light and low energy
154
155 neutrons are utilized for activation of a tumor-localized sensitizer, respectively.
156
157 In the above-mentioned therapeutical investigations, certain porphyrin derivatives
158
159 undergo selective localization in tumor tissues depending on their affinity for carrier
160
161 biomolecules and biological membranes [24–31]. Clearly, Positively charged porphyrins,
162
163 such as tetra-(trimethylaminophenyl)- and meso-tetra(methylpyridyl)- porphyrins showed
164
165
166 3
167
168
169
170 a strong interaction with the negatively charged groups of the biological targets , such as
171
172
certain proteins [32], RNA [33] and DNA [34, 35] , in addition to their effectiveness as
173
174
175 photosensitizers for PDT [24–29, 32–34]. It is also observed that the number and
176
177 distribution of the positive charges about the porphyrin macrocycle plays a very
178
179 important role in its photodynamic efficacy [32, 34]. For example, amphiphilic porphyrin
180
181 derivatives containing water-solubilizing groups, such as –NMe3+, showed an increasing
182
183 photodynamic efficacy compared to hydrophilic macrocycles [33–35].
184
185 The free radical mediated peroxidation of membrane and oxidative damage of DNA were
186
187 considered responsible for a variety of chronic health problems, such as cancer,
188
189
antherosclerosis, neurodegenerative diseases, and aging [36]. Consequently, during the
190
191
192
last years, many studies investigating biological attributes of different precursors, which
193
194 include mainly antioxidant activity, have been reported [36, 37].
195
196 Apparently, studies related to the kinetics and mechanisms of natural antioxidants have
197
198 shown that simple structural customization can lead to a noticeable enhancement in the
199
200 antioxidative activity [38, 39]. An interesting example for such observation was
201
202 resveratrol, an antioxidant component in red wine, in which chemical structure
203
204 modification could efficiently improve its antioxidative activity and cytotoxicity against
205
206
cancer cell [38].
207
208
209
Based on the above promising findings, we were motivated to use porphyrin as a basic
210
211 nucleus to design more potential antioxidants and chemo preventive agents against
212
213 cancer. The properties of porphyrin can also be modulated by the insertion of different
214
215 metal ions such as copper, silver, nickel, and Zinc [40–43]. Therefore, we have also
216
217 synthesized a novel series of porphyrin metal complexes in high yields and performed an
218
219 in-vitro study of their protective effects. The new porphyrins have been prepared in high
220
221
222 4
223
224
225
226 yields by using the previously reported modified one pot mixed solvent method in which
227
228
DMF is used as capping reagent [17–19]. All the synthesized compounds have been
229
230
231 characterized and assessed for the antioxidant and antitumor activities. The geometry and
232
233 magnetic interaction in the metal complexes have been also investigated using the
234
235 electron spin resonance (ESR) technique.
236
237 2. Experimental Section
238
239
240 2.1. General remarks
241
242
1H and 13C-NMR spectra were measured on 300 MHz JOEL ECA-300 spectrometer, using
243
244
245 DMSO or CDCl3 as solvents and TMS as the internal standard, at the Micro analytical Center,
246
247 Faculty of Science, Mansoura University. The IR spectra were recorded (KBr disk) on a
248
249 Mattson 5000 FTIR Spectrometer at Faculty of Science, Mansoura University. Elemental
250
251 analyses (C, H and N) were carried out at the Faculty of Science, Cairo University, the results
252
253
were found to be in good agreement with the calculated values.
254
255
256 Ultraviolet spectra were recorded using Unicam UV2 UV/Vis spectrometer at the
257
258
Microanalytical Unit, Faculty of Science, Mansoura University. Butylated hydroxyanisole
259
260
(BHA) and L-ascorbic acid were purchased from Sigma-Aldrich Company. 2,2-Azo-bis-(2-
261
262
263 amidinopropane) dihydrochloride (AAPH) and 2, 2-azino-bis(3-ethyl benzthiazoline-6-sulfonic
264
265 acid) (ABTS) were purchased from Wako. All other chemicals were of analytical grade and
266
267 purchased from sigma-aldrich, Germany.
268
269
270 2.2 Synthesis
271
272
273 2.2.1 Synthesis of porphyrin derivatives 1–5
274
275
276
General procedures for the synthesis of porphyrins 1–5:
277
278 5
279
280
281
282 A mixture of the appropriate aromatic aldehyde (0.72 mmol) and pyrrole (0.72 mmol) in DMF
283
284
(15 ml) were placed into a 50 mL three-necked flask. The mixture was flushed with nitrogen
285
286
287 gas for a couple of minutes and then heated to 100 ˚C for 10 min. P-toluene sulphonic acid
288
289 (0.72 mmol, dissolved in DMF) was then added to the reaction mixture. The colorless mixture
290
291 turned red over the next couple of minutes then heated at 150 ˚C for 1 hour. The reaction
292
293 mixture was then cooled and poured over ice with stirring for 15 min. the residue was collected,
294
295 dried under vacuum and purified by column chromatography using chloroform/hexane (1.5/1)
296
297 as eluent).
298
299 Synthesis of 5,10,15,20- mesotetrakis[2,4- dichlorophenyl]-21,23H- porphyrin (1)
300
301
(H2mdcpp)
302
303
304 Dark brown color, yield 85%, m.p. 160 ˚C. IR (cm-1): ν(N-H), 3279; ν(C=N), 1664; ν(C=C),
305
306
307
1584; ν(C-Cl), 784. 1H-NMR (DMSO-d6) (ppm):  2.27 (s, 1H, NH), 5.30 (d, 1H, 2 pyrrolic
308
309 CH), 5.81 (d, 2H, 2 pyrrolic CH), 6.53 (d, 2H, 2 pyrrolic CH), 7.00 (d, 2H, 2 pyrrolic CH), 7.32
310
311 (d, 4H, Ar-H), 7.50 (d, 4H, Ar-H), 7.95 (s, 4H, Ar-H), 10.64 (s, 1H, NH). 13C-NMR (DMSO-
312
313 d6) (ppm):  103.0, 120.5, 123.3, 125.0, 128.6, 130.5, 132.2, 135.7, 136.5, 137.7, 155.8, 181.1.
314
315 UV-Vis. Spectrum: ʎmax = 413 nm. Elemental Anal.: Calcd. C, 59.36; H, 2.49; N, 6.29;
316
317
318 (C44Cl8H22N4); Found: C, 59.70; H, 2.53; N, 6.30%.
319
320
321 Synthesis of 5,10,15,20- mesotetrakis[4-N,N-dimethylaminophenyl]-21,23H- porphyrin
322
323 (2) (mdmapp)
324
325
326 Dark violet color, yield 82%, m.p > 300˚C. IR (cm-1): ν(NH), 3240; ν(CH3), 2925; ν(C=N),
327
328 1659; ν(C=C), 1603. 1H-NMR (DMSO-d6) (ppm):  3.03 (s, 1H, NH), 3.90 (s, 24H, 4 N-
329
330 (CH3)2), 5.31 (d, 2H, 2pyrrolic CH), 6.23 (d, 2H, 2pyrrolic CH), 6.46 (d, 2H, 2pyrrolic CH),
331
332 6.54 (d, 2H, 2pyrrolic CH), 6.95 (d, 8H, Ar-H), 7.25 (d, 8H, Ar-H), 10.23 (s, 1H, NH). 13C-
333
334 6
335
336
337
338
NMR (DMSO-d6) (ppm):  106.4, 106.6, 107.4, 117.3, 128.6, 128.8, 124.4, 129.5, 132.2,
339
340
341 132.6, 149.3, 163.0, 167.6. UV-Vis. Spectrum: ʎmax = 421 nm. Elemental Anal.: Calcd. C,
342
343 79.37; H, 4.42; N, 14.26; (C52H50N8); Found: C, 79.76; H, 4.42; N, 14.20%.
344
345
346 Synthesis of 5,10,15,20- mesotetrakis [4- carboxylic]-21,23H- porphyrin (3)
347
348
349 Deep green color, yield 75%, m.p > 300˚C. IR (KBr): ύ /cm-1 = 3450 (OH), 3350 (NH), 1630
350
351 (C=N), 1570 (C=C), 1H-NMR (DMSO-d6) (ppm):  2.30 (s, 1H, NH), 5.91 (d, 2H, 2 pyrrolic
352
353
CH), 6.22 (d, 2H, 2 pyrrolic CH), 6.30 (d, 2H, 2 pyrrolic CH), 6.73 (d, 2H, 2 pyrrolic CH), 7.21
354
355
(d, 8H, Ar-H), 7.81 (d, 8H, Ar-H), 10.52 (s, 1H, NH), 10.56 (s, 1H, OH). UV-Vis. Spectrum:
356
357
358 ʎmax = 426 nm. Elemental Anal.; Calcd. for C48H30N4O8 (790), C 72.90; H 3.82; N 7.09%.
359
360 Found: C 72.52; H 3.55; N 7.50%.
361
362 Synthesis of 5,10,15,20- mesotetrakis [2-chloroquinolin-6-hydroxy-3-yl]-21,23H- porphyrin
363
364
(4)
365
366
367 Porphyrin (4) was prepared according to the general procedure using the aldehydes (A, 2-
368
369
chloroquinoline-6-hydroxy-3-carboxaldehyde) to give 4 as deep brown color, yield 64%, m.p
370
371
372
>300˚C. IR (KBr): ύ /cm-1 = 3340 (NH), 1630 (C=N), 1570 (C=C), 675 (C-Cl). 1H-NMR

373
374 (DMSO-d6) (ppm): 6.20 (d, 2H, pyrrolic protons), 6.46 (d, 4H, pyrrolic protons), 7.22 (s, 4H,
375
376 quinoline C5-H), 7.33 (4H, quinoline C7-H), 7.70 (d, 4H, quinoline C8-H), 7.82 (d, 2H, pyrrolic
377
378 protons), 8.24 (s, 4H, quinoline C4-H), 8.80 (s, 1H, NH), 9.40 (s, 4H, 4OH), 9.92 (s, 1H, NH).
379
380 UV-Vis. Spectrum: ʎmax = 424nm. Anal. data for C56H30Cl4N8O4 (1018), Calcd. C, 65.90; H,
381
382
383 2.96; 13.89; N, 10.98%. Found C, 65.88; H, 2.91; N, 10.99%
384
385
386
Synthesis of 5,10,15,20 tetrakis (4a, 10a-dihydro-10H-phenothiazin-3-yl) porphyrin (5)
387
388
389
390 7
391
392
393
394 Porphyrin (5) was prepared according to the general procedures using aldehyde (B, 10, 10a-
395
396
dihydro-4aH-phenothiazine-3 carbaldehyde) to give 5 as deep blue color, yield 72%, m.p >
397
398
399 300˚C. IR (KBr): ύ /cm-1 = 3340 (NH), 1630 (C=N) , 1570 (C=C), 1H-NMR (DMSO-d6)
400
(ppm): 3.03 (s, 1H, NH), 6.66- 7.51 (m, 29H, Ar-H) 8.57 (s, 1H, NH) 13C-NMR (DMSO-d6)
401
402
403 (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,
404
405 129.2, 134.9, 137.2, 142.0. UV-Vis. Spectrum: ʎmax = 422 nm. Anal. Calcd. for C68H50N8S4
406
407
408
(1106). C, 73.75; H, 4.55; N, 10.12%. Found C, 73.70; H, 4.50; N, 10.10%.
409
410
Synthesis of aldehydes, 2-chloroquinoline-6-hydroxy-3-carboxaldehyde (A) and 10, 10a-
411
412
413
dihydro-4aH-phenothiazine-3 carbaldehyde (B):
414
415
Synthesis of 2-chloroquinoline-6-hydroxy-3-carboxaldehydes (A)
416
417
418 Dimethylformamide (9.13 g, 9.6 mL, 0.125 mol) was cooled to 0 ˚C in a flask equipped with
419
420
dry tube and phosphoryl chloride (53.7 g, 32.2 mL, 0.35 mol) was added drop wise with
421
422
423 stirring. To this solution was added the N-Phenyl-acetamide (0.05 mol). After 30 min the
424
425 solution was heated under reflux for 15 hrs in a water bath. The reaction mixture was poured
426
427 onto ice-water (300 ml) and stirred for 30 min at 010 ˚C. The separated solid material was
428
429 filtered off and washed with water. The combined filtrate was adjusted to pH 9 with aqueous
430
431 sodium hydroxide, chloroform (200ml) was added and the mixture was stirred for 30 min and
432
433 then separated. The aqueous phase was extracted further with chloroform, combined organic
434
435
layer was dried with magnesium sulphate and evaporated to give oil which was treated with
436
437
438 HCl and extracted further with chloroform, the organic layer to give the corresponding
439
440 aldehyde (A). Deep yellow, yield 93%, m.p =185 ˚C. IR (KBr): ύ /cm-1 = 3400 (OH), 1730 (C-
441
442 C=O), 1630 (C=N), 695(C-Cl). 1H-NMR (DMSO-d6) (ppm):  7.26 (s, H4), 7.47 (d.d, H7) (J7,8
443
444
445
446 8
447
448
449
450 = 9), 7.87 (d.d, H8) (J8,7 = 2) 8.78 (s, H5), 9.22 (s, CHO), 9.78 (s, 1H, OH). Anal. Calced for
451
452
C10H6NO2Cl (207), C, 57.85, H, 2.91, N, 6.75% found C, 57.80; H, 2.89; N, 6.68 %.
453
454
455
Synthesis of 10, 10a-dihydro-4aH-phenothiazine-3 carbaldehyde (B)
456
457
458 Aldehyde (B) was synthesized following the general procedures used for the synthesis of
459
460
aldehydes (A) starting from the base phenothiazine. Off-white powder, yield 80%, m.p
461
462
463
175185 ˚C. IR (KBr): ύ /cm-1 = 3340 (NH), 1730 (C=O), 1570 (C=C), 1H-NMR (DMSO-d6)
464
465 (ppm): 6.88- 7.51 (m, benzene ring), 8.59 (s, 1H, NH), 8.70 (s, 1H, CHO) Anal. Calcd. for
466
467 C13H11NOS (229). C, 68.09; H, 4.84; N, 6.11%. Found C, 68.00; H, 4.79; N, 6.01%.
468
469
470 2.2.2 Synthesis of the new metalloporphyrins 1a–d and 2a–d
471
472 General procedure:
473
474 The metal complexes from the free porphyrins 1 and 2 have been prepared using the following
475
476 routes:
477
478
479 Route 1: The complexes were prepared by heating a mixture of porphyrin 1 or 2 (0.25 mmol)
480
481 dissolved in 10 mL ethanol and the metal salt (0.25 mmol of CuCl2.2H2O, VOSO4.H2O,
482
483 AgNO3 or K2PdCl4) dissolved in 10 mL H2O under reflux for 6–8 hours. After cooling to room
484
485
temperatures, the precipitate was filtered off, washed with ethanol, diethyl ether and dried.
486
487
488 Route 2: The proper aldehyde (0.01 mol) was dissolved in DMF (50 mL) and p-toluen sulfonic
489
490
acid (0.01 mol) was added. Pure pyrrole (0.01 mol) was then added dropwise. The mixture was
491
492
493
stirred under argon for one hour followed by the addition of 2.5 equiv. of the proper metal salt.
494
495 The whole reaction mixture was then refluxed for 8 hours in air. The solvent was removed
496
497 under vacuum and the crude product was washed with water, dried and purified by flash
498
499 chromatography over silica gel using chloroform as eluent.
500
501
502 9
503
504
505
506 [Cu(mdcpp)].3H2O (1a):
507
508
509 Dark green ppt. Yield: (85%). IR (cm-1): ν(C=N), 1604; ν(C=C), 1576; ν(C-N), 1335; ν(Cu-N),
510
511
502. UV-visible (nm): 536, 640. μeff= 1.9 B. M. Elemental Anal.: Calcd. C, 52.4; H, 2.6; N, 5.6;
512
513
(C44Cl8CuH26N4O3); Found: C, 52.7; H, 2.9; N, 5.52%.
514
515
516
[VO(mdcpp)] (1b):
517
518
519 Dark red ppt. Yield: (83%). IR (cm-1): ν(C=N), 1621; ν(C=C), 1598; ν(C-N), 1349; ν(V-N),
520
521
510. UV-visible (nm): 302, 410, 648. μeff= 2.1 B. M. . Elemental Anal.: Calcd. C, 55.3; H, 2.1;
522
523
524
N, 5.9; (C44Cl8H20N4OV); Found: C, 55.2; H, 2.0; N, 5.7%.
525
526
[Ag2(mdcpp)(H2O)2] (1c):
527
528
529
Dark violet ppt. Yield: (87%). IR (cm-1): ν(C=N), 1634; ν(C=C), 1580; ν(C-N), 1353; ν(Ag-N),
530
531 530. 1H-NMR (DMSO-d6) (ppm):  5.56 (d, 1H, 2 pyrrolic CH), 5.90 (d, 2H, 2 pyrrolic CH),
532
533 6.63 (d, 2H, 2 pyrrolic CH), 7.97 (d, 2H, 2 pyrrolic CH), 7.32 (d, 4H, Ar-H), 7.56 (d, 4H, Ar-
534
535 H), 8.31 (s, 4H, Ar-H). UV-visible (nm): 306, 416. Elemental Anal.: Calcd. C, 46.3; H, 2.1; N,
536
537 4.9; (Ag2C44Cl8H24N4O2); Found: C, 46.0; H, 1.9; N, 4.6%.
538
539 [Pd(mdcpp)](1d):
540
541
Dark blue-black ppt. Yield: (84%). IR (cm-1): ν(C=N), 1619; ν(C=C), 1584; ν(C-N), 1378;
542
543
544 ν(Pd-N), 510. 1H-NMR (DMSO-d6) (ppm):  5.33 (d, 1H, 2 pyrrolic CH), 5.87 (d, 2H, 2
545
546 pyrrolic CH), 6.51 (d, 2H, 2 pyrrolic CH), 6.96 (d, 2H, 2 pyrrolic CH), 7.35 (d, 4H, Ar-H), 7.55
547
548 (d, 4H, Ar-H), 8.11 (s, 4H, Ar-H). UV-visible (nm): 347, 460, 994.4. Elemental Anal.: Calcd.
549
550 C, 53.1; H, 2.0; N, 5.6; (C44Cl8H20N4Pd); Found: C, 52.9; H, 1.8; N, 5.3%.
551
552 [Cu(mdmapp)].4H2O (2a):
553
554
555
556
557
558 10
559
560
561
562 Red bloody ppt. Yield: (82%). IR (cm-1): ν(CH3), 2934; ν(C=N), 1659; ν(C=C), 1590; ν(C-N),
563
564
1318; ν(Cu-N), 540. UV-visible (nm): 318, 414, 470. μeff= 1.6 B. M. Elemental Anal. Calcd. C,
565
566
567 67.7; H, 6.1; N, 12.2; (C52CuH56N8O4); Found: C, 67.7; H, 5.9; N, 11.9%.
568
569
[Ni(mdmapp)(H2O)2].3H2O (2b):
570
571
572
Dark green ppt. Yield: (87%). IR (cm-1): ν(CH3), 2934; ν(C=N), 1659; ν(C=C), 1590; ν(C-N),
573
574
1335; ν(Cu-N), 540. UV-visible (nm): 318, 383, 438, 538, 799. μeff= 3.23 B. M. Elemental
575
576
577 Anal.: Calcd. C, 66.8; H, 6.2; N, 12.0; (C52H58N8NiO5); Found: C, 66.5; H, 6.3; N, 11.8%.
578
579
580
[Ag2(mdmapp)(H2O)2] (2c):
581
582
Dark blue ppt. Yield: (87%). IR (cm-1): ν(CH3), 2936; ν(C=N), 1600; ν(C=C), 1573; ν(C-N),
583
584
585
1320; ν(Ag-N), 520. 1H-NMR (DMSO-d6) (ppm): 3.92 (s, 24H, 4 N-(CH3)2), 5.32 (d, 2H,
586
587 2pyrrolic CH), 6.21 (d, 2H, 2pyrrolic CH), 6.46 (d, 2H, 2pyrrolic CH), 6.59 (d, 2H, 2pyrrolic
588
589 CH), 6.83 (d, 8H, Ar-H), 7.29 (d, 8H, Ar-H). UV-visible (nm): 306, 401, 471. Elemental Anal.
590
591 Calcd. C, 62.3; H, 5.2; N, 11.2; (Ag2C52H56N8O2); Found: C, 62.0; H, 5.1; N, 11.0%.
592
593
594 [Pd(mdmapp)].2H2O (2d):
595
596
597 Dark brown ppt. Yield: (82%). IR (cm-1): ν(CH3), 2934; ν(C=N), 1659; ν(C=C), 1590; ν(C-N),
598
599 1335; ν(Cu-N), 540. 1H-NMR (DMSO-d6) (ppm):  3.72 (s, 24H, 4 N-(CH3)2), 5.29 (d, 2H,
600
601 2pyrrolic CH), 6.22 (d, 2H, 2pyrrolic CH), 6.44 (d, 2H, 2pyrrolic CH), 6.55 (d, 2H, 2pyrrolic
602
603 CH), 6.87 (d, 8H, Ar-H), 7.32 (d, 8H, Ar-H). UV-visible (nm): 311, 437, 502. 928.4
604
605 Elemental Anal.: Calcd. C, 67.2; H, 5.6; N, 12.1; (C52H52N8O2Pd); Found: C, 67.2; H, 5.5; N,
606
607
12.0%.
608
609
610 2.3 Biochemical assays (Antioxidant properties, Cytotoxicity and antitumor assay)
611
612
613
614 11
615
616
617
618 2.3.1 Antioxidant properties
619
620
Free radical scavenging activity of all the new compounds was evaluated by measuring their
621
622
623 ability to neutralize DPPH and OH radicals. The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay
624
625 and hydroxyl-radical scavenging assays were applied as previously reported [17, 18].
626
627 2.3.2 Cytotoxicity and antitumor assay
628
629 Cytotoxicity and antitumor assay have been carried out according to the previously reported
630
631 procedures [17, 18].
632
633
634
635 2.4 Docking studies
636
637
638 2.4.1 Docking of antioxidant active porphyrin derivatives 2a and 4
639
640
641 All computational modeling were conducted with Schrödinger Suite 2015
642
643 (Schrödinger, LLC) that were run on dual core PC [44].
644
645
The newly synthesized compounds were comparatively evaluated in terms of their
646
647
binding mode to the Human Peroxiredoxin 5, antioxidant enzyme (1HD2) pocket.
648
649
650 Compounds 2a and 4 showed potential antioxidant activity therefore they were
651
652 considered for further molecular modeling study in order to explore their recognition
653
654 profile at the human 1HD2 binding active site.
655
656 - Preparation of enzyme:
657
658 The starting coordinates of the X-ray crystal structure of Human Peroxiredoxin in
659
660 complex with the BEZ 201A (PDB code: 1HD2) that retrieved from the RCSB Protein
661
662 Data Bank of Brookhaven National Laboratory [45]
663
664
- Preparation of tested porphyrin
665
666
667 Molecular docking for compounds 2a and 4 into the biding site of oxidase enzyme was
668
669 performed using the Schrödinger Suite 2015 (Schrödinger, LLC) [44]. The three-
670 12
671
672
673
674 dimensional structures of both compounds were constructed using building module.
675
676
Gasteiger–Hückel charges of ligands were assigned. AMBER with 100 iterations was
677
678
679 utilized for energy minimization. The active site of the protein was defined to contain
680
681 residues within a 10.0-Å radius around any of the inhibitor atoms. All hydrogens were
682
683 added and the enzyme structure was exposed to a refinement protocol in which the
684
685 constraints on the enzyme were systematically eliminated and minimized until the RMS
686
687 gradient was 0.01kcal/mol Å. The conformer with the lowest energy, the “global-
688
689 minima,” was pre-positioned using the crystal structure ligand “BEZ 201A” as a template
690
691 at the enzyme-binding pocket.
692
693
2.4.2 Docking antitumor active porphyrin derivatives 3 and 5
694
695
696
Molecular docking of compounds (3 and 5) into the biding site of telomerase was
697
698 explored using Schrödinger Suite 2015 (Schrödinger, LLC). The three-dimensional
699
700 structures of the aforementioned compounds were constructed using Chem. Draw 3D
701
702 Ultra 11.0 software. Using compute module to perform Gasteiger–Hückel, charges of
703
704 ligands were assigned. Porphyrin derivatives, 3 and 5, were energetically minimized by
705
706 using AMBER with 100 iterations and minimum RMS gradient of 0.10. The template
707
708 (PDB code: 2A5R) was obtained from the RCSB Protein Data Bank.
709
710
- Preparation of the enzyme
711
712
713
The starting coordinates of the X-ray crystal structure of complex of tetra(4-n-
714
715 methylpyridyl) porphin with monomeric parallel-stranded DNA Tetraplex (PDB code
716
717 2A5R: in complex with the POH 25A) that retrieved from the RCSB Protein Data Bank
718
719 of Brookhaven National Laboratory [46]
720
721 - Preparation of tested porphyrin
722
723
724
725
726 13
727
728
729
730 Molecular docking of compounds (3 and 5) into the binding site of telomerase inhibitor
731
732
enzyme was investigated using the Schrödinger Suite 2015 (Schrödinger, LLC). The
733
734
735 three-dimensional structures of the aforementioned compounds were constructed using
736
737 building module. Gasteiger–Hückel charges of ligands were assigned. They were
738
739 energetically minimized by using AMBER with 100 iterations. The active site of the
740
741 protein was defined to contain residues within a 10.0-Å radius around any of the inhibitor
742
743 atoms. All hydrogens were added and the enzyme structure was exposed to a refinement
744
745 procedure in which the constraints on the enzyme were gradually eliminated and
746
747 minimized until the RMS gradient was 0.01kcal/mol Å. Conformer with the lowest
748
749
energy, the “global-minima”, was pre-adjusted using the crystal structure ligand “POH
750
751
752
25A” as a template at the enzyme-binding pocket.
753
754 3. Result and Discussion
755
756 3.1 Synthesis and characterization
757
758 This work reports the synthesis of new porphyrin derivatives including the free base
759
760 compounds 1–5 (scheme 1) and metal complexes 1a–d, 2a–d (Scheme 2) which were
761
762 evaluated as antitumor and antioxidant agents.
763
764
765
766
<scheme 1>
767
768
769
<scheme2>
770
771 3.1.1 Synthesis and characterization of the free porphyrins 15:
772
773 The low yields (6–20%) in porphyrin chemistry is a persistent obstacle so far, although
774
775 the continuous advancement in the porphyrin synthesis [20–22]. Recently, Fadda et al
776
777 [17, 18] developed a synthetic method that gives 80–90% yield with minimal
778
779 chromatography. We used the capping mechanism to prevent the pyrrole polymerization
780
781
782 14
783
784
785
786 process. Dimethylformamide (DMF) was used as a solvent and capping reagent (Scheme
787
788
3). The DMF-pyrrole intermediate reacts with more pyrrole to keep constructing the
789
790
791 corresponding porphorengon, and the DMF molecule would act as good leaving group in
792
793 each time pyrrole is added [17–19].
794
795 <Scheme 3>
796
797
798
799 Our reported strategy facilitated the synthesis of various derivatives of porphyrin and
800
801 metalloporphyrin in high yields [17, 18]. In this work, we were able to obtain the free-
802
803
base prophyrins 15 in high yields (64–85%). The newly synthesized porphyrins were
804
805
806 well characterized by spectral data. In the 1H-NMR spectra, porphyrin derivatives 15
807
808 showed two sets of doublets between δ 5.00 and 8.00 ppm that corresponds to the
809
810 pyrrolic CH protons which is different from the signals for porphyrins normally found
811
812 above δ 8.00 ppm. This shift in peaks position may be related to the increased shielding
813
814 of the pyrrolic protons resulting from the interference of electron delocalization within
815
816 the macrocycle.
817
818 1H-NMR of porphyrin 1 showed a singlet signal at δ 10.64 ppm that corresponds to the
819
820
821 N-H proton, two doublet signal at δ 7.32 and 7.22 ppm and a singlet signal at δ 7.48 due
822
823 to the deshielded aromatic protons, in addition to the doublets signals at δ 6.24, 6.38, 6.44
824
825 and 7.84 ppm characterizing the pyrrolic protons.
826
827 1H-NMR of porphyrin 2 showed a characteristic singlet signal at δ 10.23 ppm
828
829 corresponding to the N-H proton. Aromatic protons displayed a characteristic two
830
831 doublets due to (AB system) at δ 6.77 and 7.16 ppm, while pyrrolic protons appeared as
832
833 doublets at δ 6.24, 6.38, 6.44, and 7.84 ppm. The singlet signal for (-N(CH3)2) protons
834
835
appeared at δ 3.02 ppm.
836
837
838 15
839
840
841
842 1H-NMR of porphyrin 3 showed a characteristic singlet signal at δ 12.71 ppm due to
843
844
carboxylic proton and another singlet signal at δ 10.52 ppm that corresponds to the N-H
845
846
847 proton. The aromatic system represented as two doublets at δ 7.83 and 7.55 ppm due to
848
849 “AB system”. Pyrrolic protons appeared at its normal position at δ 6.007.50 ppm as
850
851 doublets.13C-NMR displayed an obvious signal at δ 167.6 ppm characterizing the
852
853 carbonyl carbon.
854
855 1H-NMR of porphyrin (4) displayed a singlet signal at δ 9.92 ppm due to -NH proton, a
856
857 singlet at δ 9.40 corresponding to the O-H group, in addition to pyrrolic and quinolinic
858
859
860
protons at the range δ 6.208.40 ppm.
861
862 1HNMR of porphyrin (5) displayed a singlet signal at δ 8.57 ppm due to N-H protons, in
863
864
865 addition to a complex pattern of multiplet signals due to pyrrolic and aromatic protons at
866
867 δ 5.60- 7.80 ppm.
868
869 3.1.2 Synthesis and characterization of metalloporphyrins 1ad and 2ad
870
871
872 Recently, the biochemical and photo-electro properties of porphyrin have attracted a
873
874 growing interest. Substituted metalloporphyrins are now playing crucial roles in the fields
875
876 of iatrology [47], medicinal chemistry, [48–50], analytical chemistry [49] and electronic
877
878 devices [13–15, 51, 52]. It has been observed that when a porphyrin complex is applied in
879
880
the natural enzyme peroxidase, the dioxygen gets activated under mild conditions
881
882
883
therefore, porphyrin complexes with various metal ions have received much attention as
884
885 antioxidants [53].
886
887
888
Metalloporphyrins were synthesized by the classical Alder two-step strategy, which
889
890 involves the reaction of the free base porphyrin with the proper metal salt in refluxing
891
892 DMF [12]. The yields of metalloporyphrins obtained by this method were unsatisfactory
893
894 16
895
896
897
898 (< 20%) which is apparently a result of the long reaction time and pyrrole
899
900
polymerization. On the other hand, in the present work, one-pot reaction method with
901
902
903 DMF as a capping agent was proved to be very successful. The later strategy has the
904
905 advantages of the short reaction time, simple work up and higher yield of the porphyrin
906
907 complexes [40, 41]. In the one-pot reaction method, pyrrole, a proper aldehyde and the
908
909 metal salt were mixed together and refluxed for a reasonable short time to produce a
910
911 variety of metalloporphyrins in high yields. In this work, the addition of DMF as a
912
913 capping reagent during the one pot synthesis for the metalloporphyrins 1a–d and 2a–d
914
915 increased the yield dramatically (82–87%) compared to the two-steps method in which
916
917
the free porphyrin has to be prepared before the following step in which the metal ion is
918
919
920
added.
921
922
Cu(II), Pd(II), Ag(I) and VO2+ porphyrin metal complexes showed exceptional properties
923
924
925 for medicinal and biological applications [47–50]. While Nickel porphyrin complexes
926
927 have been employed successfully in catalytic reactions [54]. Therefore, in the synthesis of
928
929 the new complexes 1a–d and 2a–d, metal II salts of Cu(II), Ag(I), Ni(II), VO2+, and
930
931 Pd(II) have been used along with the pyrrole and the proper aldehydes (Scheme 2).
932
933
934 The structures conformation of the matal complexes 1a–d and 2a–d were examined by
935
936 IR spectroscopy and compared to that of the free porphyrins, 5,10,15,20- mesotetrakis
937
938 [2,4- dichlorophenyl]-21,23H- porphyrin (1) and 5,10,15,20- mesotetrakis[4-N,N-
939
940 dimethylaminophenyl]-21,23H- porphyrin (2). The IR absorption frequencies for the
941
942 complexed porphyrins 1a–d and 2a–d showed two main differences than the free
943
944
porphyrins 1 and 2. Firstly, The N-H bond stretching and bending frequencies of free
945
946
947 porphyrins located at ~3,300 cm-1 and ~960 cm-1. Insertion of the metal ion into the
948
949 porphyrin resulted in the disappearance of the N-H group and alternatively, the band
950 17
951
952
953
954 characteristic for the functional groups of M-N bond appeared at ~1,000 cm-1. This
955
956
obviously confirmed the formation of metalloporphyrin derivative. Secondly, As the
957
958
959 coordination between the nitrogen and the metal ion reduces the electron density in the
960
961 azomethine link, the band at ~1664, 1659 cm-1 corresponding to the ν(C=N) group in the
962
963 free porphyrin is shifted in the complexes towards lower wave number values  1600–
964
965 1630 cm-1. The bands observed in the region around 1310–1370 cm-1 are attributed to υ
966
967 (C-N) stretching vibrations in the complexes. This value is less than the value of υ (C-N)
968
969 stretching in ligands, which appears at 1383 and 1355 cm-1, respectively. This decrease
970
971
by 55–20 cm-1 in the metalloporphyrin is probably due to the increase in electron density
972
973
974 of azomethine (C-N) bond due to π–electron delocalization from the metal to the nitrogen
975
976 atom and resonance interaction with porphyrin ring. Therefore, it can be inferred that 1--
977
978 and 2-- acted as bi-negative tetradentate ligands, coordinating the metal ions through the
979
980 azomethine nitrogen and the deprotonated imine nitrogen centers forming four six-
981
982 membered rings. Moreover, the several bands at 550–500 cm-1 due to ν (M-N) stretches
983
984 were observed in the IR spectra of the complexes [55].
985
986
987 The 1H-NMR spectroscopic data for the complexes 1c, 1d, 2c and 2d in DMSO-d6 are
988
989 reported in the experimental section. The 1H-NMR spectra for metalloporphrins 1c, 1d,
990
991 2c and 2d confirmed the complexation of the free porphyrins 1 and 2 with the proper
992
993
metal ions. The spectra of free porphyrin 1 and 2 showed sharp singlet signals
994
995
996 characterizing the NH proton at δ 10.64 and δ 10.23 ppm, respectively. In the spectra of
997
998 complexes 1c, 1d, 2c and 2d, The NH signals disappeared due to the replacement of the
999
1000 imine proton of the free porphrins by the metal ions, which confirms the formation of
1001
1002 complexes 1c, 1d, 2c and 2d. On the other hand, the signals for pyrrolic -CH are slightly
1003
1004
1005
1006 18
1007
1008
1009
1010 shifted to downfield due to the complexation of 1-- and 2-- through the deprotonated imine
1011
1012
nitrogen and the metal centers [55].
1013
1014
1015 3.2 Electronic and geometrical studies of metalloporphyrins 1a–d and 2a–d
1016
1017 3.2.1 Magnetic and electronic spectra for complexes 1a–d and 2a–d
1018
1019 The electronic spectra of the complexes 1a–d and 2a–d in DMSO contain intense bands.
1020
1021 The bands between 430–900 nm are resulting from ligand-to-metal charge-transfer
1022
1023 (LMCT) transitions while weaker bands are assigned to d–d transitions. The intra-ligand
1024
1025 charge transfer (n → π* and π → π*) appeared as transitions below 430 nm. The
1026
1027 electronic spectra of the diamagnetic Pd (II) complexes (1d, 2d) exhibit bands near 465
1028
1029
and 340 nm due to 1A1g→1Bg and 1A1g→1Eg transitions, respectively, which relevant to a
1030
1031
1032
square-planar configuration [56]. The magnetic moment of [Ni(mdmapp)(H2O)2] 2b is
1033
1034 3.24 BM assigned for octahedral structure with 3A2g ground term [56, 57]. Interestingly,
1035
1036 Its electronic spectrum shows a broad band at 799 nm is assigned to the 3A2g→3T1g (F)
1037
1038 (ν2) transition [57]. The electronic spectra of [Cu(mdcpp)] 1a and [Cu(mdmapp)] 2a
1039
1040 exhibit bands near 640 nm assigned to 2T2g→2Eg. The band positions with magnetic
1041
1042 moments of 1.9 and 1.6 BM are designated to a square-planar geometry [56, 57]. The
1043
1044 porphyrins in plane geometry is crucial factor of Cu(II) square planar geometry.
1045
1046
3.2.2 Electron spin resonance (ESR) spectra of the Cu(II) and VO(IV) complexes
1047
1048
1049
ESR spectrum of the complexes 1a, 1b and 2a were recorded in the solid state in order to
1050
1051 identify the stereochemistry and the site of the metal-ligand bonding as well as the
1052
1053 magnetic interaction in the metal complexes. Generally, the mononuclear oxo-vanadium
1054
1055 (VO+2), (S = 1/2, I = 7/2) has a characteristic octet ESR spectrum showing the hyperfine
1056
1057 coupling owing to 51V nuclear magnetic moment [58]. In comparison with other reported
1058
1059 ESR spectrum of VO2+ [59], Compound 1b shows a broad single line with poorly
1060
1061
1062 19
1063
1064
1065
1066 resolved eight-line pattern. Specifically, in the powder sample, the spectrum shows
1067
1068
parallel and perpendicular features corresponding to axially symmetric anisotropy with
1069
1070
1071 poorly resolved sixteen-lines hyperfine splitting which confirms the interaction between
1072
1073 the electron and the vanadium nuclear spin (I = 7/2) [60]. The spin Hamiltonian
1074
1075 parameters for the complex are calculated as g|| = 1.93, g⊥ =1.96 and the hyperfine
1076
1077 coupling A|| = 195 × 10-4 (cm-1), A⊥ = 55. The ESR parameters indicate that the unpaired
1078
1079 electron (d1) of 1b complex is present in the dxy orbital with square-pyramidal geometry
1080
1081 [65, 66]. The values obtained for this complex is in complete agreement with those
1082
1083
reported for VO2+ square pyramidal geometry [61]. The spectra of the Cu (II) complexes
1084
1085
1086
1a, 2a at 300 K show a strong isotropic absorption band in the high field area due to the
1087
1088 tumbling motion of the molecules. These complexes at 77K show four well-resolved
1089
1090 peaks of low intensities in the low field area and one single intense peak in the high field
1091
1092 area. In square planar complexes, the unpaired electrons are in the dx2–y2 orbital and 2B1g
1093
1094 is the ground state with the g|| > g⊥. Based on the detected values, it is clear that g|| > g⊥
1095
1096 (2.37 > 2.08), which confirms square planar geometry of the complexes. Also, No band
1097
1098 for Cu–Cu interaction was observed which indicates the mononuclear Cu (II) [62].
1099
1100
3.3 Pharmacology of porphyrins
1101
1102
1103
3.3.1 Antioxidant activity:
1104
1105 The newly synthesized compounds 1–5, 1a–d, 2a, 2c and 2d were evaluated as
1106
1107 antioxidant agents using DPPH assay at different concentrations of 5 mg/mL of each
1108
1109 tested sample [63]. Ascorbic acid was used as a reference drug. The radical scavenging
1110
1111 activity was determined for the synthesized compounds using 1,1-dipheny l-2-
1112
1113 picrylhydrazyl radical (DPPH●) as a stable radical organic compound and its oxidative
1114
1115 method is widely used in the determination of the capacity of free radical scavengers or
1116
1117
1118 20
1119
1120
1121
1122 the capacity of hydrogen donors following in vitro assay. The type of antioxidant tests is
1123
1124
suggested for the compounds containing reactive nitrogen, oxygen and carbon atom
1125
1126
1127 species. The mechanism of action of antioxidant tests as reported previously [64] initiated
1128
1129 by the transfer of hydrogen atom, single electron and followed by proton transfer.
1130
1131 From the results of inhibitive concentrations (IC50) in Table 1, compounds 2a and 4 are
1132
1133 the most potent antioxidant compounds compared to the results of ascorbic acid. On the
1134
1135 other hand, compounds 5, 2c, 2d, 1a and 1b exhibited good antioxidant activity, while,
1136
1137 compounds 2, 3, 1c and 1d have moderate activity. Compound 1 have the highest value
1138
1139 of IC50 = 1.760 mg/ mL and considered as the less potent antioxidant agent.
1140
1141
In addition, the changes in the free radical scavenging capacity of the test compounds and
1142
1143
1144
their metal complexes depending on the inhibition percent are shown in Table 1.
1145
1146 3.3.2 Structure activity relationship (SAR) for the antioxidant activity
1147
1148 The metal complexes 1a–d showed an enhanced antioxidant activity with lower values of
1149
1150 IC50 compared to porphyrin 1. Similarly, complexes 2a, 2c and 2d showed an improved
1151
1152 oxidative capacity compared to the free porphyrin 2. The scavenging activity of the
1153
1154 formed complexes, in general, is significantly higher than that of the corresponding free
1155
1156 ligands, indicating that the formed complexes are stronger than free radical scavengers
1157
1158
[65].
1159
1160
1161
It was noticed that compound 2 is more potent antioxidant agent compared to compound
1162
1163 1. Moreover the presence of electron donating substituents on aromatic ring increases the
1164
1165 antioxidant activity [66] and the electron withdrawing substituents decreases it. Chlorine
1166
1167 atoms have strong electron withdrawing character and are highly electronegative atoms.
1168
1169 So, compound 1 has eight chlorine atoms, which actually decrease the antioxidant
1170
1171 activity. In addition compound 3, which has four carboxylic groups (strong electron
1172
1173
1174 21
1175
1176
1177
1178 attracting group) also decrease the antioxidant activity (Table 3). On the other side,
1179
1180
compound 2 which contain four -N(CH3)2 groups that have electron donating character
1181
1182
1183 enhance antioxidant capacity of compound 2 compared to compound 1.
1184
1185 The obtained results showed that compounds 2a and 4 have the ability to trap the free
1186
1187 radicals and displayed antioxidant capacity higher than ascorbic acid (Vitamin C). The
1188
1189 copper (II) complex 2a showed high antioxidant activity than its corresponding ligand,
1190
1191 this result is in a great agreement with previously reported work [67]. Moreover, the
1192
1193 presence of four hydroxy groups in compound 4 as phenolic substituents increase the
1194
1195 antioxidant capacity.
1196
1197
The order of free radical scavenging capacity of the tested compounds and their
1198
1199
1200
complexes as followed: 2a > 4 > Vit. C > 5 > 2c > 2d > 1b > 1a > 2 > 1d > 1c > 3 > 1.
1201
1202 The oxidative potentials of the synthesized compounds are related to the presence of
1203
1204 compounds capable of exerting impact by breaking the chain of free radicals through
1205
1206 hydrogen atoms donation [62]. Consequently, the obtained antioxidant results from this
1207
1208 study prolong a link with the use of these compounds in the pathological diseases
1209
1210 treatment, which arise from oxidative stress.
1211
1212
1213
1214
<Table1> and <Figure 1>
1215
1216
1217
3.3.3 Cytotoxicity (anticancer screening):
1218
1219 Cytotoxicity studies of the newly synthesized compounds were performed against
1220
1221 two mammalian cancer cell lines, HepG2 and MCF-7 cells. The evaluation process was
1222
1223 carried out according to the previously reported work [17]
1224
1225 The newly synthesized compounds 1–5, 1a–d and 2a–d were screened for cytotoxic
1226
1227 activity (Table 2) against HepG2 (hepatoma cells or human liver hepato carcinoma cell
1228
1229
1230 22
1231
1232
1233
1234 line) and MCF-7 cells (human breast adenocarcinoma cell line). The results were
1235
1236
expressed as growth inhibitory concentration (IC50) values, which represent the
1237
1238
1239 compounds concentrations required to produce a 50% inhibition of cell growth after 72
1240
1241 hours of incubation, compared with untreated controls (Table 2, Fig. 2). Generally, All
1242
1243 the tested compounds showed a cytotoxic activity that ranged from very strong to weak
1244
1245 activity. Compound 5 showed an IC50 value (5.56 ± 0.4 µg/mL) that is very similar to
1246
1247 DOX as a standard for the two used cell lines. The most promising values were observed
1248
1249 from compounds 2 and 3 (very strong activity) with an IC50 value of 5.52 ± 1.1 and 6.34
1250
1251 ± 0.7 µg/mL for HePG-2 cell line, respectively. Compounds 5, 2 and 3 exhibited very
1252
1253
strong activity with an IC50 value of 4.28 ± 0.3, 8.83 ± 1.2 and 7.51 ± 0.5 µg/mL for
1254
1255
1256
MCF-7 cell line, respectively. Compounds 1a, 1b, 2b and 4 exhibited strong activities
1257
1258 for both cell lines. Compounds 1, 1d, 2a, 2c, 2d have moderate cytotoxic activities
1259
1260 against the same cell lines.
1261
1262 <Figure 2 >, < Table 2>
1263
1264
1265
1266 3.3.4 Structure activity relationship (SAR) from cytotoxicity studies:
1267
1268 The cytotoxic activity of the new porphorins towards different cell lines depends the
1269
1270
following: Firstly, the intermolecular hydrogen bonds formed between the porphorin
1271
1272
1273
derivatives and DNA bases. Secondly, the positive charges on the tested porphyrins that
1274
1275 are attracted to the negative charges on the cell wall. By comparing the experimental
1276
1277 cytotoxic data of the new compounds to their structures, the following SAR was inferred:
1278
1279 Compounds 5, 3 and 2 have exceptional strong activities, this can be attributed to the
1280
1281 substituents, -NH, -N(CH3)2 and -COOH which may undergo addition to any unsaturated
1282
1283
1284
1285
1286 23
1287
1288
1289
1290 moiety in DNA or form hydrogen bonds with any of the nucleo-bases of the DNA and
1291
1292
causes its damage .
1293
1294
1295
Compounds 1a and 1b showed strong activity towards the tested cell lines due to the
1296
1297
presence of strong electron attracting chlorine atoms which caused the molecules to be
1298
1299
1300 positively charged resulting in electrostatic attraction with the DNA nucleobases. For
1301
1302 compound 4, the strong activity is probably caused by chlorine atom as electron
1303
1304 withdrawing substituent and one hydroxyl group which may also be added to any
1305
1306 unsaturated moiety in DNA or forming hydrogen bond with either one of the nucleobases
1307
1308 of the DNA and causes it damage.
1309
1310
1311 <Table2>, <Figure 2>
1312
1313 3.4 Molecular Docking Studies:
1314
1315 3.4.1 Docking studies for the new porphyrins 2a and 4
1316
1317
1318 Free radical-scavenging activity properties using the 3-D crystallographic peroxiredoxin
1319
1320 5 (PRDX5) were carried out to explore the new porphyrins recognition in the active site
1321
1322
as potential antioxidant.
1323
1324
As porphyrins 2a and 4 showed the most powerful antioxidant activities, docking of the
1325
1326
1327 of 2a and 4 into the binding active site of antioxidant protein Human Peroxiredoxin
1328
1329 (code: 1HD2) has been conducted to check the degree of recognition antioxidant activity.
1330
1331 The tested compounds, 2a and 4 were prepared by partial charges and optimal
1332
1333 minimizations using the compute module of MOE.
1334
1335 Compound 2a and 4 showed proper and promising antioxidant activity by proper
1336
1337 recognition at the binding active site of Human Peroxiredoxin protein. By studying the
1338
1339
binding behavior of the new compounds relative to the antioxidant; BEZ 201A reference
1340
1341
1342 24
1343
1344
1345
1346 ligand that docked at the 1 HDC complex, it was found that hydrogen bonds, and
1347
1348
hydrophobic interactions formed with the surrounding amino acids are used to predict
1349
1350
1351 their binding modes. This active pocket consisted of conserved amino acid residues
1352
1353 including Thr44, Gly46, Cys47 and Arg127 that play fundamental roles in recognition of
1354
1355 the docked compounds by hydrogen bonding interaction and hydrophobic interactions.
1356
1357
1358 Metallo complex compound 2a is hocked by bifurcated two hydrogen bonds between the
1359
1360 conserved Arg127 residue and the two N-pyrrols that are facing Arg127. A third
1361
1362 heterocyclic pyrrole ring showed π-π stacking interaction with Arg127.
1363
1364
1365 Docking studies of compound 4 revealed that the crucial amino acid Arg127 played an
1366
1367 important role in recognition of the ligand by forming strong hydrogen bonds between N-
1368
1369 pyrrole and N-arginine. There are also hydrogen bonds that formed between hydroxy
1370
1371
aliphatic function group and the conserved amino acid, Val75. Moreover, the amino
1372
1373
1374
group of the key amino acid, Arg127, which is one of conserved residues at the binding
1375
1376 pocket, showed proper complementarity with the N-pyrrol atom in addition to the π-π
1377
1378 stacking interaction between Arg127 and the heterocyclic pyrrole ring (Fig.3).
1379
1380
1381 <Figure 3>
1382
1383
1384
3.4.2 Porphyrin derivatives and their interaction with Telomerase (Telomerase
1385
1386 inhibition activity):
1387
1388 The availability of a high-resolution crystallographic structure of the human telomerase
1389
1390 enzyme facilitated the research considering the small molecules with potential telomerase
1391
1392 inhibition activity that brings to light selective and safe cure of cancer. Telomerase
1393
1394 becomes one of the competitive targets in the field of cancer treatment. Additionally, it
1395
1396 plays a unique anti-aging role.
1397
1398 25
1399
1400
1401
1402 In this study, we aimed to find efficient G-quartets ligands that would be able at the same
1403
1404
time to discriminate between different forms of nucleic acids in order to be selective as
1405
1406
1407 telomeric DNA. The binding of porphyrins allows them to match perfectly large planar
1408
1409 surfaces of G-quartets.
1410
1411 <Figure 4>
1412
1413 G-quartet consists of four guanine bases, so its square is twice as large as the square of
1414
1415 the base pair. Large porphyrin ligands perfectly overlap with G-quartets and
1416
1417 professionally expressed proper quadruplex selectivity.
1418
1419 The standard porphyrin ligand, POH 25A, that aligned in the G-quartet 2A5R protein
1420
1421
showed appropriate recognition with the ceiling DG-4 and DG-8 bases by forming π-π
1422
1423
1424
stacking interactions with the two pyridine heterocyclic rings back protruded from the
1425
1426 minor groove. In the meantime, DG-13 and DG-17 bases in the major groove flooring
1427
1428 showed amplifying recognition with both pyridine heterocyclic rings exposed from the
1429
1430 major groove showed in figure 5
1431
1432 <Figure 5>
1433
1434 - Docking studies of Compound 3:
1435
1436 Telomerase and compound 3 have strong complementarities as it sandwiched between
1437
1438
the roof and floor of the major groove of telomeric DNA as the reference ligand POH
1439
1440
1441
25A. In comparative docking studies where POH 25A and porphyrin 3 were docked
1442
1443 (Figure 6), it was clear that the presence of four enriched electrophilic benzoic functions
1444
1445 properly approved the complementarity of compound 3.
1446
1447 <Figure 6>
1448
1449
1450 Two benzoic acid groups out of four in porphyrin 3, expressed professional alignment
1451
1452 extended out of the G-Q major groove. The other two benzoic acid substituents got
1453
1454 26
1455
1456
1457
1458 extended back through the minor groove forming two distinctive hydrophilic and
1459
1460
lipophilic interactions, respectively. A strong hydrogen bond was formed between DG13
1461
1462
1463 and the benzoic carboxyl group and augmented the hydrophilic recognition of compound
1464
1465 3. DG17 stabilized the phenyl group of the same benzoic function by expressing π-π
1466
1467 stacking interactions. Apparently, the ceiling DG-2 and DG-4 bases were recognized with
1468
1469 π-π stacking with the heterocyclic pyrrole backbones (Fig. 7).
1470
1471
1472 <Figure 7>
1473
1474
1475 - Docking of compound 5
1476
1477
1478 Docking of compound 5 showed certain selectivity including the following features:
1479
1480 Firstly, its global minima conformer identically aligned the POH-25A, the reference
1481
1482 crystallographic ligand, and also the docked compound 3 (Figure 8-A, B). Secondly:
1483
1484 Porphyrin 5 substituted with gigantic phenothiazine functions showed proper
1485
1486 complementarity with G-quartet 2A5R protein. [Figure 8-C]. Compound 5 binds not
1487
1488 only to the surrounding DG bases but also it performed four cation–π interactions with
1489
1490
the phosphorous backbones namely DG5, DG14 and bifurcated two bonds out of DG13
1491
1492
1493
(Figure 8-D). Thirdly, even there is no strong hydrogen bond between the phenothiazine
1494
1495 ring and the G-Q bases, an overall π - π stacking hydrophobic interaction between DG8
1496
1497 and the heterocyclic phenothiazine ring is clearly exist
1498
1499
1500 <Figure 8>
1501
1502
1503
1504 4 Conclusion:
1505
1506 A series of new porphyrin derivatives (1–5) have been synthesized in high yields (64-
1507
1508 85%). Porphyrins 1 and 2 have been complexed successively with different metal salts
1509
1510 27
1511
1512
1513
1514 such as Cu(II), VO2+, Ag(I), Ni(II) and Pd(II), by one-pot mixed solvent method. The
1515
1516
corresponding metalloporphyrins were obtained after 6–8 hours in high yield (> 80%).
1517
1518
1519 The relatively high yields obtained during the synthesis of the new porphyrins were
1520
1521 attributed to the use of DMF as a capping agent for the pyrrole during the reaction. The
1522
1523 newly synthesized porphyrin derivatives are equipped with different aryl and heterocyclic
1524
1525 substituents. The formation of the new compounds was confirmed by spectral and
1526
1527 elemental analysis. The new compounds have been tested for their pharmacological
1528
1529 activities. It can be concluded that the electronic properties of the peripheral substituents
1530
1531 on the porphyrin moiety had a major effect on the antioxidant and cytotoxic activities.
1532
1533
Compounds 4 and 2a were the most powerful antioxidants due to the presence of
1534
1535
1536
hydroxyl groups in compound 4 and the dimethylamino donating group along with Cu(II)
1537
1538 metal in complex 2a. Porphyrin 1 showed the weakest antioxidant activity among all the
1539
1540 newly synthesized porphyrins due to the presence of the electron withdrawing substituent
1541
1542 (Cl). In general, porphyrins with electron donating groups showed stronger antioxidant
1543
1544 activity than porphyrins equipped with electron withdrawing groups. In cytotoxicity tests,
1545
1546 compounds 2, 3 and 5 showed very strong activity towards both HePG2 and MCF-7 cell
1547
1548 lines, while 1a, 1b, 2b and 4 were only strong active antioxidant compounds for both cell
1549
1550
lines due to the electronic effect of the substituent on the porphyrin moiety. Finally the
1551
1552
1553
molecular docking studies of the most powerful antioxidant derivatives (2a and 4) and
1554
1555 the most active antitumor (3 and 5) were carried out to explore their binding modes in the
1556
1557 active sites of the studied proteins and results were reported. Compound 5 showed proper
1558
1559 complementarity with G-quartet 2A5R protein, and therefore considered a promising,
1560
1561 lead in the treatment of cancer
1562
1563
1564
1565
1566 28
1567
1568
1569
1570
1571
1572
1573
1574
1575 5 References
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1882 56. Z. Biyiklioglu, H. Kantekin, M. Ozil, Organomet. Chem. 2007, 692, 2436–2440.
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1884 https://doi.org/10.1016/j.jorganchem.2007.02.013
1885
1886 57. Z. Valicsek, O. Horvath, Microchemical Journal, 2013, 107,47–62. DOI:
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1888 10.1016/j.microc.2012.07.002
1889
1890 58. M. Nakamura, K. Kawai, Y. Fujiwara, J. Catalysis, 1974, 34(3), 345–355.
1891
1892 https://doi.org/10.1016/0021-9517(74)90047-5
1893
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59. B. T. Thaker, J. Lekhadia, A. Patel, P. Thaker, Transition Metal Chemistry, 1994,
1895
1896
19, 623–631. https://doi.org/10.1007/BF00980417
1897
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1900
1901
1902 34
1903
1904
1905
1906 60. S. Khasa, V. P. Seth, P. S. Gahlot. A. Agarwal, R. M. Krishna, S. K. Gupta, 2003,
1907
1908
Physica B, 334, 347–358. doi:10.1016/S0921-4526(03)00097-8
1909
1910
1911
61. N. Raman, A. Kulandaisamy, C. Thangaraja, K. Jeyasubramanian, 2003, Transition
1912
1913
Metal Chemistry volume 28, pages29–36. doi.org/10.1023/A:1022544126607
1914
1915
1916
62. U. Al-Ayaan, I. M. Gaber, Spectrochim. Acta Part A: Mol. Biomol. Spectroscopy,
1917
1918
2007, 67(1), 263–272. https://doi.org/10.1016/j.saa.2006.07.012
1919
1920
1921 63. A. Braca, C. Sortino, I, Morelli, J. Mendez, Journal of Ethnopharmacology, 2002,
1922
1923 79(3), 379–381. DOI: 10.1016/s0378-8741(01)00413-5
1924
1925 64. M. Szelag, D. Milkulski, M. Molski, J Mol Model., 2012, 18(7), 2907–2916.
1926
1927 doi: 10.1007/s00894-011-1306-y
1928
1929
1930 65. I. P. Ejidike, P. A. Ajibade, Bioinorg. Chem. Appl., 2015, 2015, 1–9.
1931
1932 https://doi.org/10.1155/2015/890734
1933
1934
1935 66. C. Y. Lee, C. N. Nanah, R. A. Held, A. R. Clark, U. G. Huynh, M. C. Maraskine et
1936
1937 al., Biochimie, 2015, 111:125–134. doi: 10.1016/j.biochi.2015.02.001
1938
1939
1940 67. N. M. Aburas, N. R. Stevanovic, M. K. Milcic, A. D. Lolic, M. M. Natic, Z. Lj. Tesic,
1941
1942 R. M. Baosic, J. Braz. Chem. Soc., 2013, 24, 1322–1328. doi.org/10.5935/0103-
1943
1944 5053.20130167
1945
1946
1947
1948
1949
1950
1951
1952
1953
1954
1955
1956
1957
1958 35
1959
1960
1961
1962
1963
1964
1965
1966 O
H
O
+
H
1967 H+ + Ar
·· +
1968 Ar H Ar H N N
OH
1969 Aldehyde
H H
1970
1971 H+
··
1972 N
H
1973 ·· ··
N H Ar -H2O
H
N
Ar
1974 -H + + +
H H N N
Ar
1975 H O
+
H H
1976 H
O H

1977
Ar H
1978
1979 Cl
1980
Cl
1981 1, Ar =
Ar
1982
1983 2, Ar = N

1984
NH N
1985 3, Ar = COOH
1986 Ar Ar
H
1987 N
N Cl N
4, Ar =
1988
OH
1989 H
1990 Ar 5, Ar =
N
1991 Porphyrin derivatives 1-5
1992 S

1993
1994
1995
1996
1997 Scheme 1. Synthetic route for the preparation of porphyrin derivatives 1–5.
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014 36
2015
2016
2017
2018 Ar
2019 Metal salt, reflux 6-8 h
2020 Ethanolic solution of
porphyrin 1 and 2
2021
2022 N N
2023 M Ar
Ar
2024
1) DMF N
2025 2) p-Toluene sulphonic acid N
2026 3) Pyrrole, stepwise 1a-d
O 4) Stirr for 1 hr 2a-d
2027
2028 Ar
Ar H 5) Metal salt, reflux for 8 hrs
2029
2030 Aldehyde
2031
2032
2033 Cl N
2034
2035
2036 Cl
2037
2038 Cl
N N N N
2039
Cl M Cl N M N
2040
N N N N
2041
Cl
2042
2043 Cl
1a, M = Cu2+
2044 1b, M = VO2+ 2a, M = Cu2+
2045 1c, M = Ag1+2 2b, M = Ni2+
1d, M = Pd2+ 2c, M = Ag1+2
2046 N 2d, M = Pd2+
Cl
2047
2048
2049
Scheme 2. The synthetic routes for metal porphyrin complexes 1a–d and 2a–d
2050
2051
2052
2053
2054
2055
2056
2057
2058
2059
2060
2061
2062
2063
2064
2065
2066
2067
2068
2069
2070 37
2071
2072
2073
2074
Ar H
2075 H -H+ H
+
2076 O Ar Ar
·· + ··
2077 N N N
H
2078 H OH OH
H H
2079 H
2080
2081
2082 H
H H+
··
2083 + N
N
2084 O O
2085 H -H2O H
H
·· ·· Ar
2086 N
N
+ ··
2087 Ar N
Ar +
2088 H O H
H Ar
2089 H H
H
2090
··
2091 + N H
-DMF
2092 NH HN
2093
2094
Scheme 3: Proposed reaction mechanism shows capping mechanism for the synthesis of
2095
2096 porphyrin derivatives 1–5, 1a–d and 2a–d showing how DMF acts as a good leaving group as
2097 pyrrole added [17]
2098
2099
2100
2101
2102
2103
2104
2105
2106
2107
2108
2109
2110
2111
2112
2113
2114
2115
2116
2117
2118
2119
2120
2121
2122
2123
2124
2125
2126 38
2127
2128
2129
2130 Tables
2131
2132
2133 Table 1: Inhibitive concentration (IC50) and inhibition % values of the antioxidant activity of the
2134 investigated compounds.
2135
% Inhibition
2136 IC50 % Inhibition at % Inhibition at
2137 Compound no. at 0.01 mg/
(mg/ mL) 0.002 mg/ mL 0.005 mg/ mL
2138 mL
2139
2140 1 1.760 54.43 56.11 58.40
2141
2142 2 0.313 65.42 70.69 --
2143
2144 3 0.718 60.84 67.10 --
2145
2146 4 0.015 -- -- --
2147
2148 5 0.125 75.28 -- --
2149
2150 1a 0.239 70.69 71.80 80.0
2151
2152 1b 0.203 69.99 72.95 --
2153
1c 0.501 59.08 63.59 73.82
2154
2155
1d 0.346 64.05 69.27 75.73
2156
2157 2a 0.004 -- -- --
2158
2159
2c 0.155 75.24 76.05 83.40
2160
2161
2d 0.162 74.96 75.95 82.44
2162
2163 Vit. C 0.022 -- -- --
2164
2165
2166
2167
2168
2169
2170
2171
2172
2173
2174
2175
2176
2177
2178
2179
2180
2181 Table 2. Cytotoxicity (IC50) of tested compounds on different cancer cell lines
2182 39
2183
2184
2185
2186 IC50 (mg/ ml): 1-10 (very strong), 11-25 (strong), 26-50 (moderate), 51-100 (weak), 100-200
2187
(very weak), 200 (noncytotoxicity)
2188
2189
Cytotoxicity IC50 (µg/mL)
2190
Compounds
2191 HePG-2 MCF-7
2192
Doxorubicin 4.5±0.3 4.17±0.2
2193
2194 1 27.08±2.2 20.89±1.7
2195
1a 12.90±2.6 19.37±1.6
2196
2197 1b 18.31±3.1 21.02±2.8
2198
2199 1c 51.32±3.8 60.54±3.9
2200
1d 48.49±4.3 55.33±4.5
2201
2202 2 5.52±1.1 5.83±1.2
2203
2a 28.26±3.2 31.12±3.7
2204
2205 2b 18.71±1.7 16.43±2.0
2206
2207 2c 31.65±2.9 29.50±2.6
2208
2d 51.86±3.6 44.61±3.2
2209
2210 3 6.34±0.7 7.51±0.5
2211
4 14.81±1.5 15.38±1.3
2212
2213 5 5.56±0.4 4.28±0.3
2214
2215
2216
2217
2218
2219
2220
2221
2222
2223
2224
2225
2226
2227
2228
2229
2230
2231
2232
2233
2234
2235
2236
2237
2238 40
2239
2240
2241
2242
2243
2244
2245
2246
2247
2248
2249
2250
2251
2252 Figure 1. Comparison of the IC50 values of the tested compounds.
2253
2254
2255
2256
2257
2258
2259
2260
2261
2262
2263
2264
2265 2a 1d 1c 2c 2b 2d 1 2 1b 5 1a 3 4
2266
2267
2268 Figure 2: IC50 values in μg/mL of the compounds from MTT viability assays of HepG-2 and MCF-7
2269
cell lines.
2270
2271
2272
2273
2274
2275
2276
2277
2278
2279
2280
2281
2282
2283
2284
2285
2286
Figure 3: Docking of Compounds 2a & 4
2287
2288
2289
2290
2291
2292
2293
2294 41
2295
2296
2297
2298
2299
2300
2301
2302
2303
2304
2305
2306
2307
2308
2309
A B
2310
2311 Figure 4: A: 2-D chemical structure of ligand POH 25A that was in complex of DNA Tetraplex
2312 (PDB code 2A5R).
2313 B: the 2-D docking of POH 25A at the major groove of G-quartet DNA.
2314
2315
2316
2317
2318
2319
2320
2321
2322
2323
2324
2325
2326
2327
2328
2329
2330
2331
2332
2333
2334
2335 Figure 5: 3-D crystallographic interaction of ligand POH 25A that was in complex of G-
2336 quartet DNA (PDB code 2A5R).
2337
2338
2339
2340
2341
2342
2343
2344
2345
2346
2347
2348
2349
2350 42
2351
2352
2353
2354
2355
2356
2357
2358
2359
2360
2361
2362
2363
2364
2365
2366
2367
2368 Figure 6: Comparative docking studies of POH 25A with porphyrin-3
2369
2370
2371
2372
2373
2374
2375
2376
2377
2378
2379
2380
2381
2382
2383
2384
2385
2386
2387
2388
2389 A B
2390
2391
2392 Figure 7: A: The 2-D docking of POH 25A at the major groove of G-quartet DNA.
2393 B: 3-D crystallographic interaction of ligand POH 25A that was in complex of G-quartet DNA
2394 (PDB code 2A5R).
2395
2396
2397
2398
2399
2400
2401
2402
2403
2404
2405
2406 43
2407
2408
2409
2410
2411
2412
2413
2414
2415
2416
2417
2418
2419
2420
2421
2422
2423 A B
2424
2425
2426
2427
2428
2429
2430
2431
2432
2433
2434
2435
2436
C D
2437
2438 Figure 8: A) 2-D molecular structure of alignment of 2A5R docked ligand (cyano), compound 3 (green)
2439 and 5 (purple).
2440 B) 3-D molecular surface structure of aligned ligands namely POH25A, (cyano), compound 3 (green) and
2441 5 (purple).
2442 C) Porphyrin 5 substituted with gigantic phenothiazine functions showed proper complementarity with G-
2443 quartet 2A5R protein.
2444
D) Cation–π interactions with the phosphorous backbones namely DG5, DG14 and bifurcated two bonds
2445
out of DG13
2446
2447
2448
2449
2450
2451
2452
2453
2454
2455
2456
2457
2458
2459
2460
2461
2462 44
2463
2464
 Declaration of interests

The authors declare that they have no known competing financial interests or personal

relationships that could have appeared to influence the work reported in this paper.
 Supplementary Materials

Docking studies, antitumor and antioxidant evaluation of newly synthesized porphyrin and metallo-

porphyrin derivatives

Asmaa Ahmeda,b, Walaa A. E. Omarc,d*, Hala A. El-Asmya, Laila Abou-Zeide, Ahmed A. Faddab
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

1
1H-NMR for Compound 1(DMSO-d6)

2
13C-NMR for Compound 1

3
1H-NMR for compound 2

4
1H-NMR for compund 3

5
13C-NMR for compund 3

6
1H-NMR for aldehyde (A)

7
1H-NMR for compound (4)

8
1H-NMR for aldehyde (B)

9
1H-NMR for compound 5

10
Amplified spectum for compound 5

11
13C-NMR for compound 5

12
1H-NMR for compound 1c

13
1H-NMR for compound 1d

14
1H-NMR for compound 2c

15
1H-NMR for compound 2d

16
ESR spectrum for compound 1b

17
ESR for compound 1a

18
ESR spectrum for compound 2a

19
20