DNA interaction and anticancer properties of new peripheral phthalocyanines carrying tosylated 4-morpholinoaniline units

Article abstract of DOI:10.1016/j.poly.2019.114319

In this paper, tosylated 4-morpholinoaniline units fused peripherally tetra-substituted free-base (5), copper(II) (6), zinc(II) (7), cobalt(II) (8) and magnesium(II) (9) phthalocyanines (Scheme 1) were reported and these new phthalocyanine conjugates were characterized through Fourier Transform–Infrared (FT–IR) with ATR sampling accessory, Mass Spectra Analysis [Matrix Assisted Laser Desorption/Ionization–Time of Flight–Mass Spectral (MALDI–TOF–MS)] and Ultraviolet–visible (UV–vis) (for all new phthalocyanines), elemental analysis, as well as ¹H and ¹³C NMR spectroscopic techniques [for the compounds (2), (4), (5), (7) and (9)]. The potential utilization of the new peripheral phthalocyanine compounds (7–9) as the new pharmaceutical agents in PDT applications (in oncology and molecular biology) were determined in aspects of pBR322 plasmid DNA cleavage on agarose gel electrophoresis. The results showed that compound (7) cleaved pBR322 plasmid DNA with irradiation. Compound (7) displayed hypochromism without any shift on the addition of increasing concentrations of ct-DNA and K_b of compound (7) was calculated as 2.45 ± (0.20) × 10⁴ M^?1. In photochemical studies, the Φ_Δ value of compound (7) was determined as 0.11. The cytotoxic/phototoxic properties of compound (7) which had the best photocleavage effects among tested compounds were investigated using MTT assay toward human colorectal (HCT-116) and cervical (HeLa) cancer cells. The cell viabilities of compound (7) were found to be 73 ± 1.6% (HCT-116) and 65 ± 5.5% (HeLa) at 100 μM with irradiation.

Full text of DOI:10.1016/j.poly.2019.114319

Journal Pre-proofs
DNA interaction and anticancer properties of new peripheral phthalocyanines
carrying tosylated 4-morpholinoaniline units
Halise Yalazan, Burak Barut, Beytullah Ertem, Can Özgür Yalç ın, Yasemin
Ünver, Arzu Özel, İpek Ömeroğlu, Mahmut Durmuş, Halit Kantekin
PII:
S0277-5387(19)30764-8
DOI:
https://doi.org/10.1016/j.poly.2019.114319
POLY 114319
Reference:
Polyhedron
To appear in:
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Accepted Date:
23 November 2019
18 December 2019
Please cite this article as: H. Yalazan, B. Barut, B. Ertem, C.O. Yalç ın, Y. Ünver, A. Özel, I. Ömeroğlu, M.
Durmuş, H. Kantekin, DNA interaction and anticancer properties of new peripheral phthalocyanines carrying
tosylated 4-morpholinoaniline units, Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.2019.114319
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DNA interaction and anticancer properties of new peripheral
phthalocyanines carrying tosylated 4-morpholinoaniline units
Halise Yalazan1, Burak Barut2, Beytullah Ertem3,*, Can Özgür Yalçın4,5 Yasemin Ünver1, Arzu
Özel2,5, İpek Ömeroğlu6, Mahmut Durmuş6, Halit Kantekin1
1Department of Chemistry, Karadeniz Technical University, 61080, Trabzon, Turkey
2Faculty of Pharmacy, Department of Biochemistry, Karadeniz Technical University, 61080,
Trabzon, Turkey
3Vocational School of Health Services, Karadeniz Technical University, 61080, Trabzon, Turkey
4Faculty of Pharmacy, Department of Toxicology, Karadeniz Technical University, 61080,
Trabzon, Turkey
5Drug and Pharmaceutical Technology Application and Research Center, Karadeniz Technical
University, 61080, Trabzon, Turkey
6Department of Chemistry, Gebze Technical University, Gebze, 41400, Kocaeli, Turkey
*Author for correspondence: Beytullah Ertem
Karadeniz Technical University, Vocational School of Health Services, 61080, Trabzon, Turkey
Tel.: +90 462 377 57 03, Fax: +90 462 325 29 33, e-mail: bertem@ktu.edu.tr
1

ABSTRACT
In this paper, tosylated 4-morpholinoaniline units fused peripherally tetra-substituted free-
base (5), copper(II) (6), zinc(II) (7), cobalt(II) (8) and magnesium(II) (9) phthalocyanines
(Scheme) were reported and these new phthalocyanine conjugates were characterized through
Fourier Transform–Infrared (FT–IR) with ATR sampling accessory, Mass Spectra Analysis
[Matrix Assisted Laser Desorption/Ionization–Time of Flight–Mass Spectral (MALDI–TOF–
MS)] and Ultraviolet-visible (UV–vis) (for all new phthalocyanines), elemental analysis, as well
1
13
as H and C NMR spectroscopic techniques [for the compounds (2), (4), (5), (7) and (9)]. The
potential utilization of the new peripheral phthalocyanine compounds (7 to 9) as the new
pharmaceutical agents in PDT applications (in oncology and molecular biology) were determined
in aspects of pBR322 plasmid DNA cleavage on agarose gel electrophoresis. The results showed
that compound (7) cleaved pBR322 plasmid DNA with irradiation. Compound (7) displayed
hypochromism without any shift on the addition of increasing concentrations of ct-DNA and Kb
of compound (7) was calculated as 2.45 ± (0.20) × 104 M−1. In photochemical studies, the 
value of compound (7) was determined as 0.11. The cytotoxic/phototoxic properties of compound
(7) which had the best photocleavage effects among tested compounds were investigated using
MTT assay toward human colorectal (HCT-116) and cervical (HeLa) cancer cells. The cell
viabilities of compound (7) were found to be 73 ± 1.6% (HCT-116) and 65 ± 5.5% (HeLa) at 100
µM with irradiation.
Keywords: DNA interaction; Anticancer studies; Phototoxicity; Cytotoxicity; Zinc(II)
phthalocyanine; 4-Morpholinoaniline.
2

1. Introduction
Being a member of aromatic heterocyclic macrocyclic compounds, phthalocyanines (Pcs)
consist of four pyrrole ring where 18 -electron cloud is delocalized over carbon and nitrogen
atoms with a coplanar configuration [1,2]. Since their accidental discovery during the production
of phthalimide from phthalic anhydride, phthalocyanines have been of great significance because
of their unique intense color, high inertness, thermal stability, and low solubility [3–5]. As such,
phthalocyanines have been used extensively as a coloring agent in the industrial productions of
paint, printing, and textile. Depending on the developments in the chemistry of Pcs, they have
been discovered to own exciting and tunable electronic, photochemical, photophysical, optical,
self-assembling and catalytic features in various disciplines [4,5]. To put it another way, above
mentioned properties made phthalocyanines to broaden their utilization from industrial dyestuffs
and pigments to various medicinal, biomedical, chemical, and high-technological applications
serving as photosensitizing agents for photodynamic therapy [6,7], fluorescent probes for
bioimaging and bioanalysis [8–10], photovoltaics [11,12], sensors [12–14], molecular electronics
[15], electrocatalysis [16], electrochromic displays [17], electrophotography [18], dye-sensitized
solar cells [19], and as well as nonlinear optical (NLO) materials [20]. To that end, there has been
a growing interest and effort to investigate the synthesis, features, and applications of this
aromatic heterocycles over the last few decades.
The nitrogen-containing heterocycles such as morpholine and its varied derivatives have attracted
exceptional attention owing to their valuable antimicrobial [21,22], anti-inflammatory [23–25],
central nervous system [26–29], therapeutic [30], anticancer (i.e., mammalian target of
rapamycin, non-small cell lung cancer, human colorectal adenocarcinoma, prostate cancer,
metastatic human breast cancer and gastric cancer) [31,32], antioxidant, pesticide and bactericide
activities [33,34] during the past few decades. According to The World Drug Index, nowadays,
morpholine based compounds have been made use of over 100 drugs (i.e., Gefitinib, Linezolid,
and Aprepitant) [35–37] which contain morpholine ring system as a scaffold, a side-chain or a
component of the fused-ring systems [38]. Other than its medicinal and pharmaceutical
applications, morpholine compounds have been of considerable significance and used in a wide
range of industrial applications such as in textile industry, waxes and in the preservation of book
paper, air conditioners, a chemical intermediate in catalysts, an anticorrosive agent in water
3

boiling systems, and photographic developers [33,34]. Due to their high potential and
importance, morpholine based compounds have been of great interest and a large number of
studies have been carried out including morpholine bearing phthalocyanines such as water-
soluble silicon phthalocyanine derivatives with DNA/BSA binding properties [39],
unsymmetrical octasubstituted zinc and cobalt phthalocyanines with electrochemical and optical
properties [40], water-soluble quaternized zinc phthalocyanine with PDT and protein binding
properties [41] and octasubstituted quaternized magnesium phthalocyanine with antimicrobial
and anticancer activities of adenocarcinomic alveolar basal epithelial cells and human oral
squamous carcinoma cells [42]. To the best of our knowledge, there is no published study that
contains the investigation of anticancer properties of tetra-substituted peripheral
metallophthalocyanine carrying tosylated morpholine derivative in the literature. In line with this
goal, we preferred choosing a different type of morpholine derivative, 4-methyl-N-(4-
morpholinophenyl)benzenesulfonamide, fused to the phthalocyanine scaffold to investigate the
anticancer properties toward human colorectal (HCT-116) and cervical (HeLa) cancer cells.
In today's modern life, cancer is among the prominent diseases that are responsible for
mortality [43,44]. In combat against cancer, several applicable treatment methods exist in
practice such as surgery, chemotherapy and radiation therapy as well as phytotherapy (as the
complementary medicine) [45,46] have been applied currently in medicine. For the treatment of
cancer, in general, a combination of surgery with the aforementioned therapeutic methods is
applied. In addition to the foregoing treatments, an alternative therapeutic modality has been used
to treat many kinds of cancer types. This innovative and attractive modality is called
photodynamic therapy (PDT) depending upon the use of a composition of a photosensitizer (a
non-toxic drug or dye), a long wavelength of red visible light (620–690 nm) [47–49] and
molecular oxygen to bring about to selective destruction or damage to the localized cancer
tumors [50–52]. Nowadays, PDT has been clinically approved and utilized for the treatment of
some cancerous tumors (such as skin tumors, head and neck tumors, digestive system tumors,
urinary system tumors, non-small cell lung cancer, and brain tumors) in medicine [53].
In light of the above-mentioned knowledge, after synthesis and structural verification, our
research group has focused on the investigation of DNA cleavage and cytotoxic/phototoxic
properties of morpholine bearing metallated phthalocyanine compounds to determine their
suitability in the treatment of cancer diseases as the second generation photosensitizers in PDT
4

applications. To accomplish this goal, we synthesized the new free-base (5), copper(II) (6),
zinc(II) (7), cobalt(II) (8) and magnesium(II) (9) Pcs connected with tosylated 4-
morpholinoaniline functionalities as peripheral substituents (Scheme). An agarose gel
electrophoresis was employed to examine the DNA cleavage effects of the compounds (6–9). The
DNA binding effect and singlet oxygen quantum yield of compound (7) were investigated using
UV-vis spectroscopy. Besides, cytotoxic and phototoxic properties of compound (7) against
human colorectal (HCT-116) and cervical (HeLa) cancer cells were investigated using (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
2. Experimental
The equipment, materials, chemicals, DNA interaction, photochemical parameters,
cytotoxicity, and phototoxicity experiments were supplied as Supplementary information. The
tosylated 4-morpholinoaniline derivative (2) [54] and 4-Nitrophthalonitrile (3) [55] were
prepared according to the relating articles.
2.1. Synthesis
2.1.1. Phthalonitrile derivative (4)
The compound (4) was synthesized by the modification of the procedure described in the
literature [54]. Color: Light brown. Yield: 1.40 g (79.1%), m.p. 158–161 °C. Elemental analysis:
Calc. (%) for C25H22N4O3S: C: 65.48; H: 4.84; N: 12.22; S: 6.99, Found (%) C: 64.91; H: 4.66;
N: 12.00; S: 6.63. Fouirer Transform Infrared: υmax (cm-1) = 3078–3051 (Ar C–H), 2973–2920–
2887 (C–H), 2228 (C≡N), 1594 (C=C), 1509, 1492, 1451, 1361–1159 (SO2), 1261, 1236, 1123
1
(CH2–O–CH2), 986, 908, 834, 733, 670. H NMR (ppm, CDCl3): δ = 7.65–7.43 (m, 4H,
Tosylated, Ar–H), 7.34–7.23 (m, 3H, Ar–H), 7.03–6.78 (m, 4H, Ar–H), 3.88–3.86 (t, 4H, O–
13
CH2), 3.25–3.22 (t, 4H, N–CH2), 2.46–2.45 (s, 3H, CH3). C NMR (ppm, CDCl3): δ = 151.68,
151.66, 146.96, 145.08, 145.07, 135,87, 134.14, 134.13, 130.96, 130.95, 130.05, 130.04, 128.92,
127.77, 127.76, 126.63, 126.62, 126.13, 126.12, 116.60, 115.82–115.81–115.24–115.07 (C≡N),
110.24, 66.66–66.65–65.84 (O–CH2), 48.19–48.18 (N–CH2), 21.67–21.66 (Ar–CH3). Mass
Spectra Analysis m/z: 458.21 [M]+.
5

2.1.2. Free-base phthalocyanine (5)
The new free-base phthalocyanine (5) was synthesized by the common synthetic
procedure in which a phthalonitrile compound and a strong base (1,8-diazabicyclo[5.4.0]undec-7-
ene) heated in a high boiling solvent (pentan-1-ol) under inert atmosphere as described in the
literature [7]. The crude product was purified by washing it with hot ethanol to give the free-base
compound (5). Color: Light Green. Yield: 20.0 mg (20.0%), m.p. >300 ºC. Elemental analysis:
Calc. (%) for C100H90N16O12S4: C: 65.41; H: 4.94; N: 12.21; S: 6.99, Found (%) C: 64.89; H:
4.83; N: 11.79; S: 6.71. Fouirer Transform–Infrared: υmax (cm-1) = 3292 (N–H), 3047 (Ar C–H),
2956–2921 (C–H), 1600, 1509, 1449, 1348–1160 (SO2), 1119 (CH2–O–CH2), 1052 (N–H), 927,
1
814, 762, 662. H NMR (ppm, CDCl3): δ = 7.89–7.52 (m, 16H, Ar–H), 7.31–7.21 (m, 16H, Ar–
H), 7.08–6.84 (m, 12H, Ar–H), 3.88–3.82 (t, 16H, O–CH2), 3.25–3.16 (t, 16H, N–CH2), 2.45 (s,
12H, CH3). 13C NMR (ppm, CDCl3): δ = 150.84, 146.78, 144.07, 138.13, 136.60, 130.90, 130.55,
129.90, 129.85, 128.15, 127.74, 123.80, 121.27, 100.95, 66.82–66.73–65.83 (O–CH2), 48.67–
48.59 (N–CH2), 21.73–21.63–21.51 (Ar–CH3). Ultraviolet–Visible (CHCI3), λmax, nm (logε): 341
(4.91), 611 (4.41), 646 (4.57), 671 (4.96) and 706 (5.04). Mass Spectra Analysis m/z: 1836.05
[M]+.
2.1.3. Syntheses of metallated phthalocyanines (6 to 9)
The compound (4) (0.1 g, 0.244 mmol, 2 eq.) and anhydrous metal salts [CuCI2 (14.76
mg, 0.109 mmol, 1 eq., for 6); Zn(OAc)2 (22.39 mg, 0.122 mmol, 1 eq., for 7); CoCI2 (14.15
mg, 0.109 mmol, 1 eq., for 8) and MgCI2 (11.62 mg, 0.122 mmol, 1 eq., for 9)] were solvated in
dry pentan-1-ol (3.0 mL) inside a Schlenk tube and finally, 6 drops of 1,8-
diazabicyclo[5.4.0]undec-7-ene were added. Subsequently, the flask content was purged with
nitrogen gas and refluxed at 160 ºC for 12 h for all compounds (6 to 9). Followed by chilling to
room temperature, the obtained product was precipitated with the addition of 10 mL ethanol and
mixed at room temperature for 1 hour. After the filtration of the precipitate, it was then dried
under vacuum. Then, it was chromatographed on basic alumina column with chloroform:ethanol
solvent system (5.0 mL:5 drops for 6 and 8); (50.0:5.0 v/v for 7) and (50.0:1.5 v/v for 9). The
collected solution was condensed in an evaporator to obtain the metallophthalocyanines as blue
6

(6 and 8); dark green (7) or light green (9) solids. Eventually, the acquired metal complexes were
dried in vacuo.
2.1.3.1. Peripherally copper (II) phthalocyanine (6). Color: Blue. Yield: 31.0 mg (29.9%), m.p.
>300 ºC. Elemental analysis: Calc. (%) for C100H88N16O12S4Cu: C: 63.29; H: 4.67; N: 11.81; S:
6.76, Found (%) C: 62.41; H: 4.40; N: 11.52; S: 6.47. Fouirer Transform–Infrared: υmax (cm-1) =
3045 (Ar C–H), 2955–2920 (C–H), 1607, 1508, 1469, 1449, 1341–1160 (SO2), 1119 (CH2–O–
CH2), 926, 812, 764, 659. Ultraviolet–Visible (CHCI3), λmax, nm (logε): 342 (4.64), 618 nm
(4.31) and 686 (4.97). Mass Spectra Analysis m/z: 1897.76 [M]+.
2.1.3.2. Peripherally zinc (II) phthalocyanine (7). Color: Dark Green. Yield: 60.0 mg (57.9%),
m.p. >300 ºC. Elemental analysis: Calc. (%) for C100H88N16O12S4Zn: C: 63.23; H: 4.67; N: 11.80;
S: 6.75, Found (%) C: 62.44; H: 4.60; N: 11.63; S: 6.51. Fouirer Transform–Infrared: υmax (cm-1)
= 3044 (Ar C–H), 2956–2920 (C–H), 1606, 1509, 1488, 1449, 1332–1159 (SO2), 1118 (CH2–O–
CH2), 926, 813, 762, 660. 1H NMR (ppm, CDCl3): δ = 7.81–7.60 (m, 20H, Ar–H), 7.30–7.25 (m,
4H, Ar–H), 6.85–6.79 (m, 20H, Ar–H), 3.79–3.76 (t, 16H, O–CH2), 3.16–3.14 (t, 16H, N–CH2),
2.48 (s, 12H, CH3). 13C NMR (ppm, CDCl3): δ = 150.48, 147.58, 146.58, 143.78, 143.33, 140.56,
137.68, 136.30, 132.96, 130.29, 129.87, 129.74, 128.11, 124.63, 120.84, 112.16, 105.82, 102.30,
100.97, 66.85–66.77– 66.56–65.83 (O–CH2), 48.94–48.54–48.42 (N–CH2), 22.68–22.59–21.70–
21.52–20.81 (Ar–CH3). Ultraviolet–Visible (CHCI3), λmax, nm (logε): 350 (4.60), 617 (4.28) and
684 (4.99). Mass Spectra Analysis m/z: 1899.62 [M]+.
2.1.3.3. Peripherally cobalt (II) phthalocyanine (8). Color: Blue. Yield: 12.0 mg (11.6%), m.p.
>300 ºC. Elemental analysis: Calc. (%) for C100H88N16O12S4Co: C: 63.45; H: 4.69; N: 11.84; S:
6.78, Found (%) C: 62.68; H: 4.47; N: 11.63; S: 6.54. Fouirer Transform–Infrared: υmax (cm-1) =
3048 (Ar C–H), 2956–2915 (C–H), 1604, 1508, 1449, 1346–1160 (SO2), 1119 (CH2–O–CH2),
926, 813, 765, 660. Ultraviolet–Visible (CHCI3), λmax, nm (logε): 332 (4.90), 618 nm (4.52) and
680 (5.03). Mass Spectra Analysis m/z: 1893.91 [M]+.
2.1.3.4. Peripherally magnesium (II) phthalocyanine (9). Color: Light Green. Yield: 40.0 mg
(39.5%), m.p. >300 ºC. Elemental analysis: Calc. (%) for C100H88N16O12S4Mg: C: 64.63; H: 4.77;
7

N: 12.06; S: 6.90, Found (%) C: 64.26; H: 4.62; N: 11.74; S: 6.62. Fouirer Transform–Infrared:
υmax (cm-1) = 3044 (Ar C–H), 2956–2917–2894 (C–H), 1606, 1509, 1483, 1332–1159 (SO2),
1
1234, 1119 (CH2–O–CH2), 926, 813, 752, 661. H NMR (ppm, CDCl3): δ = 7.75–7.53 (m, 22H,
Ar–H), 7.31–7.25 (m, 4H, Ar–H), 6.91–6.78 (m, 18H, Ar–H), 3.89–3.82 (t, 16H, O–CH2), 3.18–
13
3.15 (t, 16H, N–CH2), 2.46 (s, 12H, CH3). C NMR (ppm, CDCl3): δ = 150.63, 148.82, 148.24,
144.22, 143.76, 142.93, 139.56, 139.44, 137.62, 132.91, 132.62, 130.41, 129.79, 129.71, 128.02,
126.19, 123.37, 104.86, 103.63, 66.74–65.83 (O–CH2), 48.67 (N–CH2), 21.68 (Ar–CH3).
Ultraviolet–Visible (CHCI3), λmax, nm (logε): 355 (4.57), 619 (4.27) and 686 (5.00). Mass Spectra
Analysis m/z: 1858.37 [M]+.
3. Results and discussion
3.1. Syntheses and characterization
In Scheme, the syntheses route associated with the tosylated 4-morpholinoaniline
functionalities fused at the peripheral positions of phthalonitrile compound (4) and their
corresponding free-base (5), copper(II) (6), zinc(II) (7), cobalt(II) (8) and magnesium(II) (9) Pcs
were demonstrated. In this work, the synthesis, structural verification, DNA cleavage effects of
the new phthalocyaninato copper(II) (6), zinc(II) (7), cobalt(II) (8) and magnesium(II) (9)
compounds were reported. Compound (7) which had the best photocleavage effects among tested
compounds was investigated DNA binding effect and singlet oxygen quantum yield using UV-vis
spectroscopy. Also, cytotoxic and phototoxic properties of compound (7) against human
colorectal (HCT-116) and cervical (HeLa) cancer cells were investigated using MTT assay. The
main goal of this research was to acquire new potent photosensitizing agents for PDT
applications for the treatment of cancerous tumors. Common spectroscopic techniques such as
Fourier Transform–Infrared (Attenuated Total Reflection sampling accessory), Matrix Assisted
Laser Desorption/Ionization–Time of Flight Mass spectral data (Dithranol, abbreviated as DIT
1
13
used as the matrix), elemental analysis, Ultraviolet-Visible (UV–vis), H NMR and C NMR
were utilized for the verification of the newly synthesized chemical structures and the results
were found in accordance with the proposed chemical structures in Scheme.
8

In the first step, we prepared the tosylated 4-morpholinoaniline compound (2 via the
reaction of 4-morpholinoaniline (1) and Ts–CI in dry pyridine at –5 ºC with 68.8% yield through
the modification of the synthetic method cited in the literature [56]. Then, the dicyano compound
(4) which was the starting reagent for the corresponding free-base (5) and its
metallophthalocyanines (6 to 9) were synthesized by the reaction of 4-nitrophthalonitrile (3) with
compound (2) and anhydrous K2CO3 in dry DMF with 79.1% yield. Following that, free-base (5)
was synthesized from the dicyano compound (4) in the presence of the catalytic amount of DBU
in dry pentan-1-ol at 160 °C under nitrogen stream inside a Schlenk tube. And lastly, the CuII (6),
ZnII (7), CoII (8) and MgII (9) Pcs were synthesized as blue (6 and 8); dark green (7) or light green
(9) solids via cyclotetramerization reaction of (4) in the existence of anhydrous salts [CuI2 for (6),
Zn(CH3COO)2 for (7), CoI2 for (8) and MgCI2 for (9)] with 6 drops of DBU as the strong base in
dry pentan-1-ol at 160 °C inside a Schlenk tube connected with N2(g) gas. The newly prepared
metallophthalocyanines (6 to 9) were chromatographed on the basic alumina column with
chloroform:ethanol solvent systems given in the experimental section for final purification
whereas the free-base Pc (5) was purified by washing the crude product with hot ethanol. The
unsubstituted phthalocyanines (free-bases and metallophthalocyanines) generally do not dissolve
in most of the solvents. All the new phthalocyanine metal complexes (5 to 9) synthesized for this
study were soluble in common organic solvents including tetrahydrofuran, chloroform,
dichloromethane, ethyl acetate, dimethylsulfoxide and N,N-dimethylformamide owing to the
substitution of four tosylated 4-morpholinoaniline functionalities at peripheral sites of
phthalocyanine scaffold.
The N–H vibration peaks at 3154 cm-1 and 1051 cm-1 in the FT–IR spectra of the
compound (2) disappeared after the synthesis of the peripheral dicyano derivative (4) and a new
vibration peak arose at 2228 cm-1 which was ascribed to the formation of C≡N functionality. The
aromatic C–H stretchings were monitored at 3078–3051 cm-1; aliphatic C–H stretches at 2973–
2920–2887 cm-1; the tosyl group (S=O asymmetric and symmetric vibrations) at 1361–1159 cm-1
for the compound (4). Other characteristic FT–IR vibration peaks concerning the compound (4)
1
were similar to starting compound (2) with small changes. In the H NMR spectra of dicyano
derivative (4), a singlet N–H proton signal, integrating 1 proton, of the tosylated compound (2) at
δ = 6.79 ppm vanished. As expected, aromatic protons were resonated at δ = 7.65–7.43, 7.34–
7.23 and 7.03–6.78 ppm. The signals of CH2 and methyl protons were resonated at δ = 3.88–3.86
9

1
(O–CH2), 3.25–3.22 (N–CH2) and 2.46–2.45 ppm (CH3) in the H NMR spectra of compound
(4). The observation of the signals in 13C NMR spectra belonging to the C≡N functionalities at δ
= 115.82–115.81–115.24–115.07 ppm was proved the proposed compound (4). The aromatic
carbon signals of phthalonitrile derivative (4) were observed between at δ = 151.68 and 110.24
ppm. Other 13C NMR signals of the compound (4) were observed at δ = 66.66–66.65–65.84 ppm
(O–CH2), 48.19–48.18 ppm (N–CH2) and 21.67–21.66 ppm (Ar–CH3) (Figure 1). The Mass
Spectra Analysis data of the compound (4) was measured via MALDI–TOF–MS technique and at
m/z: value was detected as 458.21 [M]+. All spectral data were in good accord with the newly
synthesized precursor compound (4).
To confirm the structural characterization of the new free-base phthalocyanine (5), we
firstly evaluated the disappearance of the sharp C≡N vibrations at 2228 cm-1 in the Fourier
transform infrared spectrum of dicyano compound (4). Besides, a new stretching vibration was
monitored in the FT–IR spectrum of (5) at 3292 and 1052 cm-1 belonging to the secondary amine
(N–H) functionalities inside the inner core of phthalocyanine ring. The rest of the FT–IR
spectrum of (5) had a similarity to that of the starting phthalonitrile compound (4) with slight
vibrational variations. The proton signals of aromatic rings in the 1H NMR spectrum of (5) were
detected at 7.89–7.52, 7.31–7.21 and 7.08–6.84 ppm. The CH2 and methyl protons were
monitored at 3.88–3.82 (O–CH2), 3.25–3.16 (N–CH2), 2.45 ppm (CH3), respectively. With small
1
changes in chemical shifts, the H NMR signals of (5) were similar to that of the precursor
phthalonitrile compound (4). During the NMR measurements, a common feature of the 1H NMR
spectra of the free-base Pc molecules is the broad absorption that is occurred most likely through
the strong aggregation of free-base Pc, which is most likely taken place at the concentration used
1
for H NMR samples [57–59]. The two inner core protons of free-base phthalocyanine (5) could
not be observed because of this broad absorption that is presumably happened through the strong
aggregation between phthalocyanine molecules in the deuterated chloroform during the
preparation and measurement of NMR samples [60,61]. In the case of 13C NMR spectrum of free-
base phthalocyanine (5), the C≡N functional groups for the dicyano compound (4) at δ = 115.82–
115.81–115.24–115.07 ppm disappeared. 13C NMR signals of the phthalocyanine (5) given in the
experimental section exhibited a resemblance to that of the dicyano compound (4). The molecular
ion peak was observed at m/z = 1836.05 [M]+ in the Mass Spectra Analysis data of (5) (Figure 2).
10

As we compared the infrared spectrum of dicyano derivative (4) with CuII (6), ZnII (7),
CoII (8) and MgII (9) phthalocyanines, the disappearance of C≡N vibration of compound (4)
indicated that the cyclotetramerization reactions from phthalonitrile derivatives to corresponding
metallophthalocyanines (6–9) were succeeded. Other vibrational bands of phthalocyanine metal
complexes (6 to 9) were alike to the FT–IR spectrum of corresponding phthalonitrile derivative
1
(4) with small changes. In the H NMR spectra of peripheral zinc(II) (7) and magnesium(II) (9)
phthalocyanines, aromatic protons were resonated at δ = 7.81–7.60, 7.30–7.25 and 6.85–6.79
ppm for (7); 7.75–7.53, 7.31–7.25 and 6.91–6.78 ppm for (9). Other proton signals were
monitored at δ = 3.79–3.76 (O–CH2), 3.16–3.14 (N–CH2) and 2.48 (CH3) ppm for (7); 3.89–3.82
13
(O–CH2), 3.18–3.15 (N–CH2) and 2.46 (CH3) ppm for (9). When we compare the C NMR
spectra of compounds (7 and 9) with corresponding dicyano compound (4), the C≡N signals of
(4) at δ = 115.82–115.81–115.24–115.07 ppm were disappeared, indicating that the targeted
metal complexes were successfully synthesized from their dicyano derivative (4). Aromatic
carbon signals were resonated between at δ = 150.48 and 100.97 ppm for (7); 150.63 and 103.63
13
ppm for (9), respectively. Other C NMR signals were as flows: 66.85–66.77–66.56–65.83 (O–
CH2), 48.94–48.54–48.42 (N–CH2) and 22.68–22.59–21.70–21.52–20.81 ppm (Ar–CH3) for (7);
66.74–65.83 (O–CH2), 48.67 (N–CH2) and 21.68 ppm (Ar–CH3) for (9). According to the Mass
Spectra Analysis data of the metallated phthalocyanine derivatives, the m/z: values were observed
at 1897.11 [M]+ for (6); 1893.23 [M]+ for (8); 1899.62 [M]+ for (7) and 1858.37 [M]+ for (9)
(Figure 3). All spectral data supported the newly synthesized phthalocyaninato copper(II) (6),
zinc(II) (7), cobalt(II) (8) and magnesium(II) (9) Pcs.
3.2. Ultraviolet-visible spectra
As it is known, one of the best verification methods for the formation of phthalocyanines
is obtained from UV–vis spectral measurements. To do so, we benefited from ground state
electronic spectra of newly synthesized free-base (5), CuII (6), ZnII (7), CoII (8) and MgII (9) Pcs
(C = 1.0×10−5 M) in chloroform in addition to aforementioned spectral techniques. As expected,
free-base Pc (5) showed two split Q Bands (Qx and Qx) with high intensity which was typical with
Ultraviolet-visible (UV–vis) spectra of free-base phthalocyanines with D2h symmetry. The Q
band and B band absorptions in the electronic absorption spectrum of compound (5) are as
11

follows: 706 nm (logε = 5.04) (Qx), 671 nm (logε = 4.96) (Qy), 646 nm (logε = 4.57), 611 nm
(logε = 4.41) and 341 nm (logε = 4.91) (Figure 4). The ground state electronic spectra of
compounds (6 to 9) recorded as the typical of metallated phthalocyanines with a single band of
high intensity at 686 nm (logε = 4.97) and 618 nm (logε = 4.31) for (6); 684 nm (logε = 4.99) and
617 nm (logε = 4.28) for (7); 680 nm (logε = 5.03) and 618 nm (logε = 4.52) for (8); 686 nm
(logε = 5.00) and 619 nm (logε = 4.27) for (9), respectively. Along with Q band absorption, the
studied metallophthalocyanines (6 to 9) displayed B band absorptions in the Soret region at 342
nm (logε = 4.64) for (6); 350 nm (logε = 4.60) for (7); 332 nm (logε = 4.90) for (8) and 355 nm
(logε = 4.57) for (9) as well. The UV–vis spectral measurements were consistent with the
proposed phthalocyanine compounds (5 to 9) (Figure 4).
3.3. DNA cleavage studies
The DNA cleavage assays were carried out as described previously with minor
modifications [62]. The pBR322 plasmid DNA (Thermo Fischer Scientific, SD0041) cleavage
activities of the compounds (6–9) were investigated on agarose gel electrophoresis and analyzed
using Image Lab Version 4.0.1 Software. The results of cleavage activities of the compounds (6–
9) without and with irradiation were given in Figures 5 and 6. The compounds (6–9) did not show
cleavage activities in the dark, as shown in Figure 5. The results suggested that the compounds
had a low cytotoxic effect in the dark, which is very important for acting as the photosensitizer.
On the other hand, compound (7) cleaved pBR322 plasmid DNA with light irradiation (white,
17.5 mW/cm2, 30 min) showing that DNA cleavage from supercoiled form to nicked form as
shown in Figure 6, Lane 5–7. The untreated pBR322 plasmid DNA was determined as 97.40%
(supercoiled form) and 2.60% (nicked form) with irradiation under our experimental conditions.
Upon increasing concentration of the compound (7) (25, 50 and 100 µM) with plasmid DNA, the
nicked form increased from 2.60% to 41.10%. The other tested compounds (6, 8 and 9) did not
show any photocleavage effects under our experimental conditions. It is well-known that zinc(II)
compounds have high singlet oxygen quantum yield due to d10 configuration [63]. Therefore,
compound (7) might show photocleavage activities via a singlet oxygen pathway against plasmid
DNA.
12

3.4. DNA binding studies of Zn(II) phthalocyanine derivative (7)
UV-vis spectroscopy is an important technique to estimate the binding mode of compounds
with calf-thymus DNA (ct-DNA). It is well-known that compounds interact with DNA via
covalent or non-covalent (intercalation, major/minor groove binding and electrostatic) bonds. The
spectral changes with the interaction of compounds with ct-DNA show the binding propensity in
the UV-vis spectrum [62]. Compound (7) showed a hypochromic effect (25.98%) without any
shift upon the addition of increasing concentrations of ct-DNA. Also, the intrinsic binding
constant (Kb) of compound (7) was determined as 2.45 ± (0.20) × 104 M−1. In our previous
studies, Kb values of tetra 4-(3-methyl-4-(3-morpholinopropyl)-5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl) substituted Zn(II) and morpholine substituted Zn(II) phthalocyanines were calculated
as 6.53 ± (0.04) × 104 and 4.76 (± 0.06) × 104 M−1, respectively [62,63]. Compound (7) had a
lower binding affinity than our previous studies due to the bulky structure. UV-vis spectroscopy
studies showed that compound (7) bound to ct-DNA via non-covalent interaction.
3.5. Photochemical parameters of Zn(II) phthalocyanine derivative (7)
One of the most important parameters for PDT applications of a photosensitizer is the
singlet oxygen quantum yield () [64]. In the present study, singlet oxygen quantum yield ()
of compound (7) in DMF was calculated with 1,3-diphenylisobenzofuran (DPBF) used as a
singlet oxygen quencher. The absorption decay of DPBF was monitored at 417 nm using a UV-
vis spectrophotometer during irradiation. The results were shown in Table 1 and Figure 7. Figure
7 showed that absorbance of the DPBF decreased at 417 nm due to singlet oxygen, but Q band
shapes of compound (7) did not change. This behavior showed that compound (7) was stable
against light irradiation. The  value of compound (7) was determined as 0.11, while
unsubstituted ZnPc was 0.56 in DMF.
3.6. Cytotoxicity and phototoxicity of Zn(II) phthalocyanine derivative (7)
In this study, cytotoxicity and phototoxicity of the compound (7) which showed the best
photocleavage effects among tested compounds were examined using MTT assay against HCT-
13

116 and HeLa cancer cells with/without irradiation. In this test, the water-soluble yellowish MTT
dye is metabolized by succinate dehydrogenase enzyme in the viable active cells; and produces
water-insoluble purple formazan crystals. This color change is used as an indicator of cell
viability [65]. The results were tabulated in Tables 2, 3 and Figures 8, 9. Table 2 showed that cell
viabilities of HCT-116 cells (%) at 50 and 100 µM of compound (7) were determined as 90 ±
1.30% and 73 ± 1.60%, respectively with irradiation (white, 17.5 mW/cm2, 1 h). In the dark, they
were found to be 93 ± 2.50% and 85 ± 4.40%, respectively. On the other hand, compound (7) on
HeLa cells had higher phototoxic activity than HCT-116 cells. Upon irradiation, cell viabilities of
HeLa cells (%) were 83 ± 3.80%, 71 ± 1.70% and 65 ± 2.50%, respectively at 10, 50 and 100
µM. As shown in Table 3, cell viabilities were determined as 90 ± 4.80%, 74 ± 2.00% and 69 ±
1.60% at same concentrations in the dark At 1 and 5 µM, compound (7) did not any cytotoxic and
phototoxic effects against HCT-116 and HeLa cells with/without irradiation.
In the previous studies demonstrated that quaternized 2, 9(10), 16(17), 23(24)-tetra-(4-(N-
propanesulfonic-acid) phenoxy) phthalocyaninto-zinc(II), non-ionic (2, 9(10), 16(17), 23(24)-
tetra-(4-(N-(2-methyls)) phenoxy) phthalocyaninato-zinc(II), and cationic (2, 9(10), 16(17),
23(24)-tetra-(4-(N-(2-methyls)) phenoxy) phthalocyaninato-zinc(II) showed phototoxic effects on
HeLa cells upon irradiation (665 nm LED) in a time-dependent manner [66]. Besides, Göksel
reported that the determined IC50 values for zinc(II) phthalocyanine containing [2-(tert-
butoxycarbonyl)amino]ethoxy and iodine groups on HeLa cells were 4.5 and 2.17 µM after
irradiation with 2 J.cm-2 due to high singlet oxygen quantum yields [67]. Moreover, tetra-
triethyleneoxysulfonyl substituted zinc phthalocyanine had phototoxic activities against HCT-116
cells with a broadband white light source at 10 J.cm-2 [68]. Comparing previous studies and our
studies, compound (7) had lower cytotoxic and phototoxic activities on HCT-116 and HeLa cells
despite effective DNA cleavage properties of it. This may trigger by the low cellular uptake and
intracellular reactive oxygen species due to its large structure. Several studies demonstrated that
the drug carrier system was helpful for phthalocyanines to uptake cells and improve their activity.
4. Conclusion
The synthetic procedures and chemical characterization of the new peripherally tosylated
4-morpholinoaniline functionalities attached to phthalocyanines were announced in this report.
14

The newly synthesized compounds were verified via elemental analysis, Fourier Transform–
1
Infrared, Matrix Assisted Laser Desorption/Ionization–Time of Flight Mass spectral data, H
13
NMR and C NMR, and as well as UV–vis. Due to the tosylated substituents at peripheral and
sites of phthalocyanine skeleton, these new tetra-substituted phthalocyanines (5 to 9) showed
good solubility in common organic solvents such as dimethylformamide, chloroform,
tetrahydrofuran, ethanol, dichloromethane, ethyl acetate, and dimethyl sulfoxide. The UV–vis
spectra of studied metallophthalocyanines (6–9) were recorded at room temperature with 1.00 x
10–5 M concentration in chloroform. Depending on the electronic absorption spectra, we obtained
monomeric species with D4h symmetry, as evidenced by a single narrow Q band with a short
wavelength shoulder in terms of working conditions.
The potential utilization of the new peripheral phthalocyanine compounds (6 to 9) as the
new pharmaceutical agents in PDT applications were determined in DMSO in aspects of pBR322
plasmid DNA cleavage on agarose gel electrophoresis. The results showed that compound (7)
cleaved significantly pBR322 plasmid DNA upon irradiation. Compound (7) displayed a
hypochromic effect without any shift on the addition of increasing concentrations of ct-DNA and
Kb of compound (7) was calculated as 2.45 ± (0.20) × 104 M−1. In photochemical studies, the 
value of compound (7) was determined as 0.11. The cytotoxicity and phototoxicity effects of
compound (7) were investigated using MTT assay on HCT-116 and HeLa cancer cells. The
compound (7) had a low cytotoxic and phototoxic effect against used cancer cell lines because
this may trigger by the low cellular uptake and intracellular reactive oxygen species due to its
large structure. Further studies are required to determine the potential therapeutic effect of
compound (7) using a drug carrier system such as nanoparticles liposomes and micelles etc.
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19

Table Captions
Table 1. Maximum electronic absorption and singlet oxygen quantum yield for zinc(II)
phthalocyanine (7) in DMF.
Table 2. Cytotoxic and phototoxic effects of compound (7) on HCT-116 cells expressed as cell
viability (%).
Table 3. Cytotoxic and phototoxic effects of compound (7) on HeLa cells expressed as cell
viability (%).
20

Table 1. Maximum electronic absorption and singlet oxygen quantum yield for zinc(II)
phthalocyanine (7) in DMF.
Compound
Solvent
Q band λmax, (nm)
(log ε)

ZnPc (7)
DMF
683
5.28
0.11
ZnPca
DMF
670
5.37
0.56
a Tetrahedron, 2010, 66, 3248–3258.
21

Table 2. Cytotoxic and phototoxic effects of compound (7) on HCT-116 cells expressed as cell
viability (%).
1 µM
5 µM
10 µM
50 µM
100 µM
(7) without irradiation
(7) with irradiation
100 ± 3.10
100 ±1.50
100 ± 1.50
93 ± 2.50
85 ± 4.40
100 ± 2.90 ns 100 ± 0.90 ns 100 ± 0.70 ns 90 ± 1.30ns 73 ± 1.60***
*** p < 0.001, ns: not significant compared to without irradiation at the same concentration.
22

Table 3. Cytotoxic and phototoxic effects of compound (7) on HeLa cells expressed as cell
viability (%).
1 µM
5 µM
10 µM
50 µM
100 µM
(7) without irradiation
(7) with irradiation
100 ± 4.60
100 ± 2.50
90 ± 4.80
74 ± 2.00
69 ± 1.60
100 ± 1.30 ns 100 ± 2.30 ns 83 ± 3.80 ns 71 ± 1.70ns 65 ± 5.50ns
ns: not significant compared to without irradiation at the same concentration.
23

Scheme and Figure Captions
Scheme. The synthesis of the peripheral tetra-substituted free-base (5), copper(II) (6), zinc(II)
(7), cobalt(II) (8) and magnesium(II) (9) phthalocyanine derivatives.
Figure 1. 1H and 13C NMR spectra of the precursor dicyano compound (4) in CDCI3.
Figure 2. Mass spectral data of the compounds (2) and (5).
Figure 3. Mass spectral data of the compounds (7) and (9).
Figure 4. The ground state electronic absorption spectra of new free-base (5) copper(II) (6),
zinc(II) (7), cobalt(II) (8) and magnesium(II) (9) phthalocyanines (C = 1.0×10−5 M) in
chloroform.
Figure 5. Agarose gel electrophoresis of pBR322 plasmid DNA in the absence and presence of
compounds without irradiation. Lane 1, DNA control; lane 2–4, (6) (25, 50 and 100 μM); lane 5–
7, (7) (25, 50 and 100 μM); lane 8–10, (8) (25, 50 and 100 μM); lane 11–13, (9) (25, 50 and 100
μM).
Figure 6. Agarose gel electrophoresis of pBR322 plasmid DNA in the absence and presence of
compounds with irradiation (white, 17.5 mW/cm2, 30 min). Lane 1, DNA control; lane 2–4, (6)
(25, 50 and 100 μM); lane 5–7, (7) (25, 50 and 100 μM); lane 8–10, (8) (25, 50 and 100 μM);
lane 11–13, (9) (25, 50 and 100 μM).
Figure 7. Absorption changes during the determination of singlet oxygen quantum yield for
zinc(II) phthalocyanine (7) in DMF using DPBF as a singlet oxygen quencher.
Figure 8. Cytotoxic and phototoxic effects of zinc(II) phthalocyanine (7) against HCT-116.
Figure 9. Cytotoxic and phototoxic effects of zinc(II) phthalocyanine (7) against HeLa.
24

CH3
O
S
H3C
Cl
CN
CN
O
S
O
O
O
N
NH2
O
N
N
+
O2N
H
dry Pyridine, N2(g), 68.8%
-5 ºC
1
2
3
N2(g) anhyrous K2CO3
ºC
55
79.1%
dry DMF
CH3
anhydrous CoI2, DBU, N2(g)
dry Pentan-1-ol, 11.6%, 160 ºC
dry n-Pentanol, DBU, N2 (g)
6
CuPc
CoPc 8
ZnPc 7
anhydrous CuCI , 29.9%, 160
ºC
2
O
S
O
O
N
N
CN
CN
dry Pentan-1-ol, DBU, 160
N2(g), 20.0%, 160 ºC
ºC
anhydrous Zn(OAc)2, DBU, N2(g)
dry Pentan-1-ol, 57.9%, 160 ºC
5 Free-base
4
anhydrous MgCI2
dry Pentan-1-ol
160 ºC
DBU
N2(g)
39.5%
O
N
O
S
N
CH3
O
CH3
N
N
N
N
N
N
N
O
S
O
O
N
N
Mg
N
N
S
N
O
O
O
O
CH3
H3C
S
N
O
N
O
9 MgPc
Scheme. The synthesis of the peripheral tetra-substituted free-base (5), copper(II) (6), zinc(II)
(7), cobalt(II) (8) and magnesium(II) (9) phthalocyanine derivatives.
25

Figure 1. 1H and 13C NMR spectra of the precursor dicyano compound (4) in CDCI3.
26

Figure 2. Mass spectral data of the compounds (2) and (5).
27

Figure 3. Mass spectral data of the compounds (7) and (9).
28

Figure 4. The ground state electronic absorption spectra of new free-base (5) copper(II) (6),
zinc(II) (7), cobalt(II) (8) and magnesium(II) (9) phthalocyanines (C = 1.0×10−5 M) in
chloroform.
29