Synthesis, chemical and biological investigations of new Ru(III) and Se(IV) complexes containing 1,3,5-triazine chelating derivatives

Article abstract of DOI:10.1016/j.molstruc.2018.11.082

A series of vital complexes of [Se (Lⁿ) (Cl)_x]·yCl (where n = L¹, L², L³ and L⁴; x = 1, 2, or 3 and y = 1, 2, or 3) and [Ru (Lⁿ) (Cl)_x (H₂O)_y]zCl

Full text of DOI:10.1016/j.molstruc.2018.11.082

Accepted Manuscript
Synthesis, chemical and biological investigations of new Ru(III) and Se(IV) complexes
containing 1,3,5-triazine chelating derivatives
Fatima A.I. Al-Khodir, Tarfah Al-Warhi, Hana M.A. Abumelha, S.A. Al-Issa
PII:
S0022-2860(18)31395-4
DOI:
https://doi.org/10.1016/j.molstruc.2018.11.082
MOLSTR 25911
Reference:
To appear in: Journal of Molecular Structure
Received Date: 5 October 2018
Revised Date: 22 November 2018
Accepted Date: 23 November 2018
Please cite this article as: F.A.I. Al-Khodir, T. Al-Warhi, H.M.A. Abumelha, S.A. Al-Issa, Synthesis,
chemical and biological investigations of new Ru(III) and Se(IV) complexes containing 1,3,5-
triazine chelating derivatives, Journal of Molecular Structure (2018), doi: https://doi.org/10.1016/
j.molstruc.2018.11.082.
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ACCEPTED MANUSCRIPT
Cl
Cl
N
Cl
N
N
N
N
N
NH
HN
NH
.3Cl
HN
NH
Se
Cl
N
NH
Cl
Cl
N
N
N
N
N
N
N
N
.2Cl
HN
N
NH
N
Cl
Cl
N
NH
Se
HN
NH
N
N
Cl
Cl
.2Cl
.Cl
Se
Cl
Cl
3
Se
N
N
N
N
N
S
1
Cl
2
Cl
4
Cl
Cl
N
N
N
N
N
NH
N
HN
NH
Cl
Cl
.Cl
HN
NH
Ru
N
NH
Cl
N
N
N
OH2
N
N
N
N
N
HN
N
NH
Cl
N
OH2
N
Cl
H O
N
NH
Ru
HN
NH
N
Cl
Cl
7
2
Cl
N
.Cl
Ru
Cl
OH2
Ru
Cl
Cl
N
N
Cl
N
N
S
5
6
8
Structures of new Ru(III) and Se(IV) triazine complexes

ACCEPTED MANUSCRIPT
Synthesis, chemical and biological investigations of new Ru(III) and Se(IV)
complexes containing 1,3,5-triazine chelating derivatives
*
Fatima A.I. Al-Khodir , Tarfah Al-Warhi, Hana M.A. Abumelha, and S.A. AL-
ISSA
Department of Chemistry, College of Science, Princess Nourah Bint Abdulrahman
University
fatimaalkhodir@yahoo.com
Abstract
n
1
2
3
4
A series of vital complexes of [Se(L )(Cl) ].yCl (where n= L , L , L and L ;
x
n
x= 1, 2, or 3 and y= 1, 2, or 3) and [Ru(L )(Cl) (H O) ]zCl (where x= 2 or 3; y= 0,1 or
x
2
y
2
and z= 0 or 1) have been synthesized from the chemical reactions between
1
2
ruthenium(III) or selenium(IV) salts and tri-substituted s-triazine derivatives (L = N -
4
6
2
2
(4-chlorophenyl)-N ,N -di(pyrimidin-2-yl)-1,3,5-triazine-2,4,6-triamine; L = N -(4-
4
6
chlorophenyl)-N - (pyrimidin-2-yl)- N - (thiazol-2-yl)-1,3,5-triazine-2,4,6-triamine;
3
2
4
L =
diamine and L = 6-chloro-N -(4-chlorophenyl)-N -(pyrimidin-2-yl)-1,3,5-triazine-
,4-diamine). The structural was investigated using of elemental analyses, molar
6-chloro-N -(pyrimidin-2-yl)-N -(1H-1,2,4-triazol-3-yl)-1,3,5-triazine-2,4-
4
2
4
2
1
13
conductance, IR, UV–Vis, magnetic susceptibility, H, C−NMR spectra and thermal
analyses. The surface morphology behaviors of selenium(IV) and ruthenium(III)
complexes were studied based on scanning and transmittance electron microscopes.
The crystalline nature of s-triazine complexes have been investigated using X-ray
powder diffraction spectra. Spectral results were concluded that the ligand acts as a
1
4
2
3
neutral bidentate for (L and L ) and tridentate for (L and L ), and coordinates
through the nitrogen atom of triazine ring and nitrogen atoms of (pyrimidine, thiazole,
n
and triazole) rings towards metal ion. The cytotoxic IC results of the free L ligands
5
0
and its selenium(IV) complexes in vitro against the human colon and lung cancer cell
lines introduced a promising efficiency.
Keywords: s-triazine, complexity, ruthenium, selenium, spectroscopy, biological.
1
-Introduction
The 1,3,5−triazine derivatives have been widely used as herbicides [1], drug
delivery [2] and in synthetic polymeric materials [3-7] which have excellent thermal
and electrical properties. Many organic compounds containing triazines moiety are
shown an interesting biological and antiviral properties [8]. Numerous of s-triazine
derivatives have been formed colored complexes with various metal ions which can
be acts as analytical reagents [9-14]. 1,3,5-Triazine derivatives are of great current
attention in coordination chemistry and supramolecular chemistry [15]. Among the
many reasons for this are the electron deficient s-triazine ring has shown the ability to
exhibit unsual anion-π−contacts [16] and intricate H−bonding networks [17], s-
triazine is a rigid three-directional C −symmetric and its derivatives may serve as
3
versatile ligands towards transition metals [18] and the meta-phenylene disposition of
the triazine-N atoms may give rise to ferromagnetic interactions via the spin-
polarization mechanism in cases where several metal ions are bound to the triazine
ring [15-18]. Respecting to the latter two aspects, however, the electron-deficient
1
,3,5-triazine has proven quite reluctant to bind to three metal ions. As a result there
are only few structurally characterized complexes known to date in which all triazine-
1

ACCEPTED MANUSCRIPT
N are involved in metal coordination and very few of them feature paramagnetic
metal centers. Moreover, s-triazine appears to be quite susceptible to hydrolytic ring
cleavage upon metal activation [19]. Hence the potential of the s-triazine ring as a
magnetic coupler in the field of molecular magnetism is hardly exploited yet. A
further interesting aspect is the redox properties of s-triazine, which makes 2,4,6-
trisubstituted triazines important building blocks in electronic and polymeric materials
[
20]. This may also lead to new facets in triazine coordination chemistry in cases
where the triazine behaves as a non-innocent ligand. In the last two decades it has
been increasingly realized that oxidations of transition metal complexes can be either
metal or ligand-centered [21]. In view of the biological relevance of tyrosl radicals,
much work in this field has focused on the redox properties of metal phenolate
complexes [22,23]. In this article, new s-triazine ligands (Fig. 1) with different
substituents in 1,3,5−positions were synthesized. Syntheses and spectroscopic
characterizations of some relevant metal complexes using those lignads are discussed
in detail in this study.
The rapid development of pathogen resistance to most known antibiotics has
become a serious health problem [24]. One possible long-term solution is to develop
agents that work on untapped bacterial targets. S-triazine derivatives have received
considerable attention due to their active biological activity such as anti-cancer [25],
estrogen receptor modulators [26] and antimalarials [27]. It has been reported that
triazine derivatives possess potent antimicrobial activity [28]. The compounds
containing thiadiazole moiety have also been found to possess antitubercular and anti-
inflammatory activities [29]. Hence, our aim is to synthesis a new derivative of
triazine to enhance the pharmacological activities and nonlinear properties. The
development of new chelates is a very important step in the development of metal
complexes. These complexes have unique properties and an active novel. The nature
of these groups, such as steric properties, electronic properties, and geometric
properties, affect the metal orbits and thus affect its properties. In catalytic systems, a
small change in the electron donation characteristic of ligature or size of replacements
on ligand plays an important role in the optical selectivity and efficiency of the
ligament or its compound. This paper describes the synthesis of new multidentate s-
triazine ligands and discusses their coordination chemistry. A new s-triazine ligands
are presented, as well as their selenium(IV) and ruthenium(III) complexes.
2
-Materials and methods
2
-1-Chemicals
The cyanuric chloride, 2-aminopyrimidine, p-chloroaniline, 2-aminothiazole,
H-3-amino-1,2,4-triazole and metal chloride salts of RuCl .xH O and SeCl are of
1
3
2
4
pure materials that received from Sigma−Aldrich Chemical Corporation, St. Louis,
Mo, USA. The supported chemicals and solvents were of analytical grade and used
without further purifications.
2
-2-General method for the synthesis of trisubstituted s-triazine derivatives.
2
4
6
The synthesis of N ,N ,N -triaryl-1,3,5-triazine-2,4,6-triamine was carried out
as described in literature [30,31], 2,4,6-trichloro-1,3,5-triazine (cyanuric chloride; 1)
has been known for a long time as an excellent starting material for the synthesis of
multitopic molecules [31]. The unique feature of cyanuric chloride the ability to
replace each chlorine atom by any nucleophilic reagent under control of the reaction
2

ACCEPTED MANUSCRIPT
temperature (Fig. 2) [32]. In this study, we initially using 4,6-dichloro2-[Aryl or
1
2
3
4
heteroaryl]-1,3, 5-triazine as the precursor of L , L , L and L using 2,4,6-trichloro-
2
4
6
1
,3,5-triazine (cyanuric chloride) (1) as starting material. The synthesis of N ,N ,N -
triaryl-1,3,5-triazine-2,4,6-triamine was carried out as described in literature [30,33].
n
1
2
3
The following scheme describes the synthesis of the four ligands (L = L , L , L and
4
L ) [30,33].
2
-2-1-General procedures for synthesis of compounds triazin-2-amine 2a,b.
To a stirred solution of cyanuric chloride1 (10 mmol) in acetone (20 mL) at 0-
°C was added a solution of aryl amine(10 mmol) in acetone (10 mL) in drop wise
5
fashion. After addition is complete, the reaction mixture was stirred for 2 hrs. Sodium
bicarbonate (10% aq) solution was added to neutralize HCl resulted during the
reaction Reaction was monitored by TLC. Finally, the reaction mixture was poured
into crushed ice. The solid separated out was filtered, washed with water dried and
recrystallized from ethanol.
Synthesis of 4,6-dichloro-N-(4-chlorophenyl)-1,3,5-triazin-2-amine 2a.
−
1
−1
Yield, (73%), m.p.: 197 °C. IR: 3350 cm (NH stretching); 1630 cm
−
1
1
(
aromatic C=N stretching); and 1588 cm (aromatic C=C stretching). H-NMR
(
DMSO-d ): δ = 7.30 (d, 2H, J= 4.5 Hz, p-ChloroanilineC H), 7.38 (d, 2H, J= 9.9
6
3
1
3
Hz, p-ChloroanilineC H), 8.90 (br.s, 1H, NH),). C-NMR (DMSO-d ): δ = 110.3,
2
6
1
18.4, 123.1, 139.2, 158.2, and 162.2 (Ar-C, C=N,C-Cl). Anal. Calcd. for C H N Cl
9 5 4
3
(275.5): C, 39.20; H, 1.81; Cl, 9.03; N, 20.32.; Found: C, 39.40; H, 1.91; N, 20.52.
Synthesis of 4,6-dichloro-N-(1H-1,2,4-triazol-3-yl))-1,3,5-triazin-2-amine 2b.
−
1
−1
Yield, (65%), m.p.: 210 °C. IR: 3330-3256 cm (2NH stretching); 1620 cm
−
1
1
(
(
aromatic C=N stretching); and 1590 cm (aromatic C=C stretching). H-NMR
DMSO-d ): 7.55 (s, 1H, triazole C H), 10.22 (s, 1H, NH), 10.99 (s, 1H, NH) . C-
1
3
6
5
NMR (DMSO-d ): δ= 111.9, 158.8, 164.1and 169.8 (Ar-C, C=N,C-Cl) Anal. Calcd.
6
for C H N Cl (220): C, 21.81; H, 1.36; N, 44.54; Found: C, 21.71; H, 1.29; N, 44.50
4
3
7
2
2
4
2
2
-2-2-Synthesis of 6-chloro-N -(4-chlorophenyl)-N -(pyrimidin-2-yl)-1,3,5-triazine-
,4-diamine (L ).
4
2-Aminopyrimidine (1.27 g, 10 mmol) (10 mL) was added slowly to 4,6-
dichloro-N-( 4-chlorophenyl)-1,3,5-triazin-2-amine 2a (3.6 g, 10 mmol) in dioxane
with constant stirring within 4 hrs at 50°C. Sodium bicarbonate (10%aq) solution was
added to neutralize HCl evolved during the reaction. Finally, the contents were poured
in to crushed ice. The solid separated out was filtered, washed with water, dried and
−
1
recrystallized from ethanol. Yield, (76%), m.p.: 277°C. IR: 3244-3150 cm
−
1
−1
(
3NHstretching); 1619 cm (aromatic C=N stretching); and 1574 cm (aromatic C=C
stretching). H-NMR (DMSO-d ): δ = 7.19 (t, 2H, J = 4.5 Hz, pyrimidine C H), 7.22
1
6
5
(
d, 2H, J = 4.5 Hz, p-ChloroanilineC H), 7.32 (d, 2H, J = 9.9 Hz, p-
3
ChloroanilineC H), 8.58 (d, 4H, J = 4.5 Hz, pyrimidine C H), 9.42 (s, 1H, NH),
2
4,6
1
3
1
1
1.17 (s, H, NH). C-NMR (DMSO-d ): δ = 112.6, 112.9, 123.4, 129.1, 144.1, 159.3,
6
65.7, 168.2, 168.1, and 170.71 (Ar-C, C=N, C-Cl) Anal. Calcd. For C H Cl N
13
9
2
7
(334.16): C, 46.68; H, 2.69; Cl, 21.22; N, 29.32; Found: C, 46.64; H, 2.65; Cl, 21.08;
N, 29.31.
3

ACCEPTED MANUSCRIPT
2
4
6
2
-2-3-Synthesis of N -(4-chlorophenyl)-N - (pyrimidin-2-yl)-N - (thiazol-2-yl)-1,3,5-
2
triazine-2,4,6-triamine (L ).
2
-Aminothiazole (1.27 g, 10 mmol) in dioxane (15 mL) was added slowly to 6-
2
4
chloro-N -(4-chlorophenyl)-N -(pyrimidin-2-yl)-1,3,5-triazine-2,4-diamine (4.24 g, 10
mmol), in dioxane (25 mL), with constant stirring within 4 hrs at refluxed
temperature. Sodium bicarbonate (10% aq) solution was added to neutralize HCl
evolved during the reaction. Finally, the reaction mixture was cooled and poured in to
crushed ice. The solid separated out was filtered, washed with water, dried and
−
1
recrystallized from acetic acid. Yield, (73%), m.p.: 344 °C. IR: 3249-3112 cm (3NH
−
1
−1
stretching); 1623 cm (aromatic C=N stretching); and 1559 cm (aromatic C=C
1
stretching). H-NMR (DMSO-d ): δ = 6.85 (d, 1H, J = 7.2 Hz, thiazole C H), 7.17 (t,
6
5
2
H, J = 4.5 Hz, pyrimidine C H), 7.21 (d, 2H, J = 4.5 Hz, p-Chloroaniline C H), 7.30
5
3
(
d, 2H, J = 9.9 Hz, p-Chloroaniline C H), 7.49 (d, 1H, J = 6.3 Hz, thiazole C H),
2
4
8
1
1
.70 (d, 4H, J = 4.5 Hz, pyrimidine C H ), 9.09 (s, 1H, NH), 10.88 (s, 1H, NH),
4, 6
1
3
1.27 (s, 1H, NH). C-NMR (DMSO-d ): δ = 112.3, 12.9, 114.9, 123.2, 129.8, 139.8,
6
44.2, 147.2, 159.3, 164.3, 165.9, 166.1, and 171.0 (Ar-C, C=N, C-S, C-Cl). Anal.
Calcd. for C H N ClS (397.84): C, 48.30; H, 3.04; Cl, 8.91; N, 31.69; S, 8.06;
1
6
12
9
Found: C, 48.20; H, 3.00; Cl, 8.79; N, 31.51; S, 7.82.
2
4
6
2
-2-4-Synthesis of N -(4-chlorophenyl)-N N -di(pyrimidin-2-yl1,3,5-triazine-2,4,6-
1
triamine (L ).
2
-Aminopyrimidine (1.27 g, 10 mmol) in dioxane (15 mL) was added slowly
2
4
to
6-chloro-N -(4-chlorophenyl)-N -di(pyrimidin-2-yl)-1,3,5-triazine-2,4-diamine4
(4.4 g, 10 mmol), in dioxane (25 mL), with constant stirring within 4 hrs at refluxed
temperature. Sodium bicarbonate (10% aq) solution was added to neutralize HCl
evolved during the reaction. Finally, the reaction mixture was cooled and poured in to
crushed ice. The solid separated out was filtered, washed with water, dried and
−
1
recrystallized from ethanol. Yield, (73%), m.p.: 269°C. IR: 3249-3112 cm
−
1
−1
(
3NHstretching); 1623 cm (aromatic C=N stretching); and 1559 cm (aromatic C=C
stretching). H-NMR (DMSO-d ): δ = 7.14 (t, 2H, J = 4.5 Hz, pyrimidine C H), 7.23
1
6
5
(
d, 2H, J = 4.5 Hz, p-ChloroanilineC H), 7.26 (d, 2H, J = 9.9 Hz, p-
3
ChloroanilineC H), 8.66 (d, 4H, J = 4.5 Hz, pyrimidine C H ), 9.53 (s, 1H, NH),
2
4,
6
1
3
1
1
1.08 (s, 2H, 2NH). C-NMR (DMSO-d ): δ = 116.3, 117.5, 122.3, 123.4, 127.1,
6
39.0, 141.2, 157.2, and 163.8 (Ar-C, C=N, C-Cl). Anal. Calcd. For C H N Cl
17
13 10
(392.10): C, 52.02; H, 3.31; Cl, 9.04; N, 35.70; Found: C, 51.84; H, 3.28; Cl, 8.93; N,
3
5.53.
2
4
2
-2-5-Synthesis of 6-chloro- N -(1H-1,2,4-triazol-3-yl)- N -(pyrimidin-2-yl)1,3,5-
3
triazine-2,4-diamine (L ).
2
-aminopyrimidine (0.84 g, 10 mmol) in acetone (10 mL) was added slowly to
2
4
,6-dichloro-N -(1H-1,2,4-triazol-3-yl)-1,3,5-triazin-2-amine 2b in dioxane with
constant stirring within 3 hrs at 50 °C. Sodium bicarbonate (10%aq) solution was
added to neutralize HCl evolved during the reaction. Finally, the contents were poured
in to crushed ice. The solid separated out was filtered, washed with water, dried and
−
1
recrystallized from methanol. Yield, (74%), m.p.: 348 239 °C. IR: 3251-3156 cm
−
1
−1
(
3NHstretching); 1621 cm (aromatic C=N stretching); and 1586 cm (aromatic C=C
stretching). H-NMR (DMSO-d ): δ =7.14 (t, 1H, J = 4.5 Hz, pyrimidine C H), 7.62
1
6
5
(
1
s, 1H, triazoleC H), 8.77 (d, 4H, J = 4.5 Hz, pyrimidine C H), 9.88 (s, 1H, NH),
5 4,6
1
3
1.52 (s, 1H, NH), 13.97 (s, 1H, NH). C-NMR (DMSO-d ): δ = 112.7, 147.9, 155.8,
6
4

ACCEPTED MANUSCRIPT
1
59.1, 165.1, 166.1, 169.1, and 172.8 (Ar-C, C=N,C-Cl) Anal. Calcd. for C H ClN
10
9
7
(290.67): C, 37.19; H, 2.43; Cl, 12.20; N, 48.19; Found: C, 37.05; H, 2.33; Cl, 12.15;
N, 48.10.
2
-3-Synthesis of selenium(IV) and ruthenium(III) complexes
A 1.0 mmol of metal chlorides (RuCl .xH O and SeCl ) and tri-substituted s-
3
2
4
n
1
2
3
4
triazine derivatives ligands (L = L , L , L and L ) (1.0 mmol) were dissolved in 40
mL of methanol and the reaction mixtures were refluxed for 6 hrs. The colored
solutions were cooled at room temperature. Slow evaporation of the solvent afforded
solid colored precipitates after left one day.
2
-4-Instrumental analyses
The elemental analyses of % carbon, % hydrogen and % nitrogen contents
were performed using a Perkin Elmer CHN 2400 (USA). The percentage of respected
metal ions were estimated gravimetrically. Melting point was determined using a
MPS10−120 Melting point apparatus. The molar conductivities of freshly prepared
-
3
3
1
4
.0×10 mol/cm dimethylsulfoxide (DMSO) solutions were measured using Jenway
010 conductivity meter. The infrared spectra were recorded on Bruker Alpha FTIR
-1
Spectrophotometer (4000–400 cm ). The UV-Vis absorption spectra were recorded in
DMSO solvent within 800-200 nm range using a UV2 Unicam UV/Vis
Spectrophotometer fitted with a quartz cell of 1.0 cm path length. Magnetic moments
were calculated using the Magnetic Susceptibility Balance, Sherwood Scientific,
Cambridge Science Park, Cambridge, England, at Temp 25 C. The H, C-NMR
spectra were recorded on Oxford YH-300 NMR spectrometer. H spectra were run at
o
1
13
1
3
00 MHz spectra in deuterated (DMSO-d ). Chemical shifts are quoted in δ and were
6
related to that of the solvents. Mass spectra were carried out at 70 eV using AEI MS
0 mass spectrometer. The thermal studies TG/DTG–50H was carried out on a
3
Mettler Toledo AG thermogravimetric analyzer, Analytical CH-8603, Schwer
o
Switzerlad under nitrogen till 800 C. Scanning electron microscopy (SEM) images
were taken in Quanta FEG 250 equipment. The X-ray diffraction patterns were
recorded on X 'Pert PRO PANanalytical X-ray powder diffraction, target copper with
secondary monochromate. The transmission electron microscopy images (TEM) were
performed using JEOL 100s microscopy.
2
-5-Antimicrobial study
Antimicrobial activity of the tested samples was determined using a modified
Kirby-Bauer disc diffusion method [34]. Briefly, 100 ꢀL of the tested bacteria
Staphylococcus aureus (G+), Escherichia coli (G−))and fungi (Aspergillus flavus,
(
and Candida albicans) were grown in 10 mL of fresh media until they reached a
count of approximately108 cells/mL for bacteria/fungi [35]. 100 ꢀL of microbial
suspension was spread onto agar plates corresponding to the broth in which they were
maintained. Isolated colonies of each organism that might be playing a pathogenic
role should be selected from primary agar plates and tested for susceptibility by disc
diffusion method [34].
2
-6-Anti-cancer study
All the tested compounds were screened against the human colon and lung
cancer cell lines using neutral red (NR) technique in order to evaluated the cytotoxic
5

ACCEPTED MANUSCRIPT
effects of gradient concentration (0.1 to 500 ꢀg/mL) from the tested compounds in
terms of cell viability and IC values. Avastin compound was used as standard drug
5
0
against colon anticancer and the Abitrexate as lung anticancer standard drug.
2
-6-1-Chemicals
All chemical materials were received from Gibco® life technologies, USA.
2
-6-2-NR reagent
A 0.33 g of NR was added in 100 mL H O filter sterilized and stored at room
2
2
temperature, protected from light. On the day of use, the reagent was diluted in media
to obtain a final concentration of 33 ꢀg/mL. The NR desorb: 1% % glacial acetic acid
and 50% ethanol solution made in water.
2
-6-3-Cell culture
Human transformed cell lines from colon or lung were obtained from
Vaccera (Giza, Egypt). Cells were maintained in RPMI-1640 supplemented with 100
g/mL streptomycin, 100 ꢀg/mL penicillin and 10% (w/v) heat-inactivated fetal
ꢀ
o
bovine serum in a humidified, 5% (v/v) CO atmosphere at 37 C.
2
2
-6-4-Cytotoxicity assay
Neutral Red (NR) assay was used to evaluate the cytotoxicity effects of
prepared compounds as previously described [36]. A 25.000 cells/well were plated
in 96 well plates in quadruplets for each treatment. The 24 hrs later, cells were
washed with pre warmed PBS and 125ꢀL of media with appropriate concentration of
each tested compounds. After the treatments, cells were washed with pre warmed PBS
and 125 ꢀL of the NR reagent was added in each well. The plates were incubated at
o
3
7 C with 5% CO , for further 5 hrs. After incubation, plates were taken out, gently
2
washed 3 times with PBS and 100 ꢀL of NR desorb added per well. Plates were
protected from light and shaken on a shaker at 60 rpm for 30 min followed by keeping
the plates still for 5 min. Plates were taken to 96-well plate reader and their
absorbance measured in each well at 540 nm. Each value is based on means of the
absorbance values acquired from quadruplet of treatments and normalized to the mean
value of untreated control and expressed as % cell death with the following formula.
The data is based on three independent experiments.
1
00 – [(Abs540 treated sample/Abs540 untreated sample) × 100]
2
-6-5-Calculation of IC50 values
The IC values (50 % cell viability maximum inhibition concentration) of the
5
0
tested and control compounds was calculated using XLfit5 software (IDBS) and
-1
expressed in µg mL at 95% confidence intervals.
3
-Results and discussion
3
-1- Micro−analytical and physical data
The micro−analytical data and physicochemical properties of s-triazine
n
1
2
3
4
derivatives ligands (L = L , L , L and L ) and their complexes of Ru(III) and
6

ACCEPTED MANUSCRIPT
Se(IV) are in good agreement with calculated values and are listed in Table 1. The
−3
molar conductance values were registered in 10 M freshly prepared in dimethyl
sulfoxide solutions (Table 1) display that all the synthesized complexes are 1:1
electrolytes and different number of ionized chloride ions are existed in the outer
2
4
sphere of coordination. Unlike all complexes, [Ru(L )(Cl) ] (6) and [Ru(L )(Cl)
3
3
(
H O)] complex (8) have no conductive properties. These data confirmed that the
2
s-triazine derivatives ligands presence as neutral charge after complexation toward
metal ions [37]. Thus, the complexes of Se(IV) and Ru(III) may be formulated as
1
2
3
4
[
[
Se(L )(Cl) ]2Cl (1), [Se(L )(Cl) ]Cl (2), [Se(L )(Cl)]3Cl (3), [Se(L )(Cl) ]2Cl (4),
2 3 2
1
3
Ru(L )(Cl) (H O) ]Cl (5), [Ru(L )(Cl) (H O)]Cl (7).
2
2
2
2
2
Table 1. Micro−analytical and physicochemical data of ligands and their complexes
Elemental analyses found(Calc.)
Yield, %
Compounds
Color
Beige
Λ(ꢀS)
92.3
%
C
%H
(3.34)
3.31
%N
(35.66)
35.61
%M
1
(51.98)
1.87
(48.30)
8.22
(37.19)
7.11
(53.59)
3.51
33.28)
3.21
31.06)
1.03
21.14)
1.09
28.14)
8.07
32.09)
2.00
31.75)
1.70
20.94)
0.90
27.90)
7.87
L
-
73
71
74
76
68
72
70
69
71
73
71
69
5
2
(3.04)
3.00
(31.69)
31.62
L
Light brown
Yellow
31.32
33.40
62.10
181
-
-
-
4
3
(2.43)
2.40
(48.19)
48.12
L
3
4
(3.86)
3.81
(31.25)
31.20
L
Orange
5
(
(
(
(
(
(
(
(
(2.14)
2.11
(22.83)
22.80
(12.87)
12.81
1
2
3
4
5
6
7
8
Violet
3
(1.96)
1.90
(20.38)
20.31
(12.76)
12.71
Red brown
63.30
3
(1.38)
1.31
(27.39)
27.32
(15.44)
15.40
Yellow green 146.4
2
(1.63)
1.60
(17.67)
17.66
(14.23)
14.21
Dark blue
Dark brown
Black
138.7
107.8
42
2
(2.69)
2.64
(22.01)
21.96
(15.88)
15.80
3
(2.00)
1.96
(20.83)
20.80
(16.70)
16.45
3
(1.76)
1.74
(27.14)
27.11
(19.58)
19.50
Dark brown
Dark green
70.60
57.30
2
(1.89)
1.78
(17.52)
17.45
(18.06)
18.05
2
1
2
3
4
Solubility test of the Ru(III) and Se(IV) complexes of L , L , L and L s-triazine
derivatives ligands shows that they are insoluble in most organic solvents
(chloroform, dichloromethane, alcohol, benzene, and ethers) but soluble in DMF and
DMSO solvents with gently heating. All complexes have a higher melting points
o
above >300 C, this leads to thermal stability behavior. Despite the use of various
crystallization techniques, we have not been able to obtain the appropriate crystals to
identify the structures with X-ray crystallography. So, the structures of the
synthesized complexes (Fig. 3) were confirmed by using different tools of analyses,
1
13
like FTIR, H, C−NMR, magnetic, electronic and thermal analyses measurements.
1
2
3
4
3
-2-Molecular modeling studies of free L , L , L and L s-triazine new ligands
The theoretical calculations of quantum chemical and molecular modeling
1
2
3
4
studies of the new L , L , L and L s-triazine derivatives ligands are useful to
7

ACCEPTED MANUSCRIPT
support the suggestion structures [38,39]. Geometry optimizations have been
estimated based on semi-empirical PM3 level [38,39] as implemented in software of
hyperchem 7.5 program [40] (Fig. 4 and Table 2).The quantum chemical parameters;
the highest occupied molecular orbital energy (EHOMO), the lowest unoccupied
molecular orbital energy (ELUMO), the difference between HOMO and LUMO energy
levels (ꢁE), Mulliken electronegativity (χ), chemical potential (P ), global hardness
i
(η), global softness (S), global electrophilicity (ω), absolute softness (σ) and
electronic charge (ꢁNmax) have been estimated [41-45].
1
2
3
4
Table 2: The quantum chemical parameters of L , L , L and L s-triazine derivatives
ligands
1
2
3
4
Parameters
L
L
L
L
Total energy (a.u)
Binding energy (a.u)
Heat formation (a.u)
Electronic energy (a.u)
Dipole moment/ debye
EHOMO (eV)
171.57
310.57
317.05
-1298.46
785.5
-7.7697
1.2872
9.0569
3.24125
4.52845
0.220826
-3.24125
0.110413
1.159967
0.715753
178.07
313.92
320.06
-1215.66
745.1
-8.3860
2.8568
11.2428
2.7646
-72.37
33.93
38.22
-995.05
7.825
-6.2541
1.8392
8.0933
2.20745
4.04665
0.247118
-2.20745
0.123559
0.602083
0.545501
-76.71
32.65
37.77
-1038.42
9.739
-7.4731
0.3719
7.845
3.5506
3.9225
0.254939
-3.5506
0.12747
1.60698
0.905188
ELUMO (eV)
ꢁE (eV)
χ (eV)
η (eV)
σ (eV)
Pi (eV)
S (eV)
ω (eV)
5.6214
0.177892
-2.7646
0.088946
0.679814
0.491799
ꢁNmax (eV)
The high value of energy gap (ꢁE) led to hard molecules as well as low
reactivity, but, the low energy gap data led to soft molecules, which is more reactivity.
The lower values of ELUMO give the highly acceptability for electrons [38-45], as well
as the highly values of EHOMO which easily to electrons to liberated. The high values
1
2
3
4
of EHOMO were confirmed that the L , L , L and L s-triazine ligand have a powerful
donation behavior. The difference between energy gap for ligand reflect to the
presence of complexation status. The global electrophilicity (ω) of ligand is increasing
that attributed to highest capacity of accepted electrons. The calculation of both global
hardness (η) and absolute softness (σ) parameters are useful to recognize the
molecular stability and reactivity. The negative data of both ELUMO and EHOMO were
assigned to the stability of synthetic complexes.
3
-3-FTIR spectra
In order to investigate the coordination mode of the synthetic L , L , L and
1
2
3
4
L s-triazine derivatives ligands towards the ruthenium(III) and selenium(IV) metal
ions in situ complexity, FTIR spectra of the free ligands were compared with the
spectra of the associated metal(II) complexes as assigned in Table 3 and for example
display in Fig. 5. The FTIR spectra of the ligands shows a strong-to-medium strong
bands at 1488 cm (L ), 1485 cm (L ), 1510 cm (L ), and 1484 cm (L ) are
assigned to ν(C=N) group of triazine [46]. These bands are shifted to lower
−
1
1
−1
2
−1
3
−1
4
8

ACCEPTED MANUSCRIPT
−1
wavenumbers in the spectra of all the complexes (1386–1480 cm ) attributed to the
binding of triazine ring nitrogen to the metal ions [47]. The observed bands presence
−
1
1
−1
2
−1
3
−1
4
at 1623 cm (L ), 1619 cm (L ), 1621 cm (L ), and 1619 cm (L ) in the free
ligands are assigned to ν(C=N) of pyrimidine and triazole rings are shifted to lower
−1
wavenumbers (1600–1618 cm ) dependent on complexity, confirming the
coordination of nitrogen atom of pyrimidine and triazole rings towards the central
metal ion [48]. Furthermore, the ν(N−H) stretching bands of the pyrimidine/triazole
rings presence at 3260−3112 cm are shifted to lower wavenumbers in case of FTIR
spectra of synthesized complexes. This shift in wavenumbers may be assigned to the
reduction of lone pair lone pair repulsive forces in the adjacent nitrogen atoms upon
complexation [49]. The assignments of the suggested binding locations are supported
−
1
−1
by appearance of small-to-medium bands in case of metal complexes at 538–450 cm
which could be assigned to ν(M−N) [50].
1
2
3
4
Table 3. FTIR spectral band assignments of L , L , L and L ligands and their
complexes
−1
FTIR spectral assignments (cm )
ν(C=N)aromatic ν(C=C)aromatic ν(C=N)triazine ν(M−N)
Compounds
ν(N−H)
1
L
L
L
L
3249-3112 1623
3260-3142 1619
3251-3156 1621
3244-3150 1619
3245
3046
3047
3245
3070
3250
3100
3100
1559
1555
1586
1574
1560
1547
1585
1570
1552
1546
1580
1571
1488
1485
1510
1484
1478
1471
1466
1470
1472
1480
1386
1480
-
-
-
-
2
3
4
1
2
3
4
5
6
7
8
1618
1617
1616
1604
1604
1610
1600
1600
538
456
531
450
502
500
450
470
3
-4-Electronic and magnetic studies
The electronic absorption spectral data of the free s-triazine derivatives L , L ,
1
2
3
4
L and L ligands and its selenium(IV) and ruthenium(III) complexes were measured
within 200−800 nm range and their absorption bands have been assigned. The L , L ,
L and L ligands display an absorption bands at (281and 294 nm), (296 and 376 nm),
1
2
3
4
3
04 nm, and (296 and 370 nm)), respectively, that are due to intra−ligand charge
transfer transitions [51]. In DMSO solvent, UV−Vis spectra of ruthenium(III)
complexes (5−8) shows a different electronic transition bands at 668−565, 512−450,
2
4
2
4
2
2
and 390−320 nm assigned to T → T (ν1), T → T (ν2), and T → A (ν3),
2
g
1g
2g
2g
2g
2g
respectively. The inter−electronic repulsion parameters and ligand field parameters
were calculated based on three complementary equations [52,53] as follows:
ν1 = 10Dq − 5B − 4C; ν2 = 10Dq + 3B − 4C; ν3 =10Dq – 2B – C
−1
The Racah inter−electronic repulsion parameter (B = 570−565 cm ), 10Dq value
−1
(
29559−26940 cm ) high and nephelauxetic parameter (β = ~ 0.9) ≤ 1.0 [45,46]. The
9

ACCEPTED MANUSCRIPT
low value of β is due to reduction of effective positive charge of the metal ion and
increasing the ability of reduction to the lower oxidation state [53]. These outcome
data confirm the associated of strong covalent bond between s-triazine derivatives
1
2
3
4
3+
ligands (L , L , L and L ) and Ru ions [53]. The magnetic susceptibilities of the
3
+
synthesized Ru complexes are 1.84−1.16 B.M., so, the experimental magnetic
moment data confirm that ruthenium(III) complex has been found in (+3) oxidation
state with octahedral geometry [54]. The electronic absorption spectra of selenium
n
1
2
3
4
complexes [Se(L )(Cl) ].yCl (where n= L , L , L and L ; x= 1, 2, or 3 and y= 1, 2, or
x
3
) have different bands at (296, 399 and 565 nm), (311 and 563 nm), (299 and 499
1
2
3
4
nm), and (293, 393 and 581 nm) for L , L , L and L ligands, respectively, due to Se-
L
n
charge−transfer transitions. Selenium(IV) complexes have a diamagnetic
CT
properties.
1
13
3
-5- H
−
NMR and C
−NMR spectra
1
13
The chemical shifts (δ, ppm) assignments of H−NMR and C−NMR spectral
1
data for the selenium(IV) and ruthenium(III) complexes [Se(L )(Cl) ]2Cl (1),
2
2
3
4
1
[
(
Se(L )(Cl) ]Cl (2), [Se(L )(Cl)]3Cl (3), [Se(L )(Cl) ]2Cl (4), [Ru(L )(Cl) (H O) ]Cl
3 2 2 2 2
2
3
4
5), [Ru(L )(Cl) ] (6), [Ru(L )(Cl) (H O)]Cl (7) and [Ru(L )(Cl) (H O)] (8) were
3
2
2
3
2
collected in DMSO solvent and can be assigned as follows:
Complex 1
1
H-NMR (DMSO-d ): δ = 7.23 (t, H, J = 4.5 Hz, pyrimidine C H), 7.27 (t, H,
6
5
J = 4.5 Hz, pyrimidine C H), 7.32 (d, 2H, J = 4.5 Hz, p-Chloroaniline C H), 7.38 (d,
5
3
2
8
1
1
H, J = 9.9 Hz, p-Chloroaniline C H), 8.75 (d, 2H, J = 4.5 Hz, pyrimidine C H),
2 4,6
.88 (d, 2H, J = 4.5 Hz, pyrimidine C H), 9.81 (s, 1H, NH), 11.14 (s, 1H, NH),
4
,6
1
3
2.01 (s, 1H, NH). C-NMR (DMSO-d ): δ = 112.7, 114.0, 118.5, 123.2, 129.2,
6
39.2, 145.8, 149.1, 151.4, 158.2, 159.1, 162.1, 163.5, 165.1, and 170.4 (Ar-C, C=C,
C=N).
Complex 2
1
H-NMR (DMSO-d ): δ = 6.97 (d, 1H, J = 7.2 Hz, thiazole C H), 7.24 (t, 2H,
6
5
J = 4.5 Hz, pyrimidine C H), 7.25 (d, 2H, J = 4.5 Hz, p-Chloroaniline C H), 7.31 (d,
5
3
2
H, J = 9.9 Hz, p-Chloroaniline C H), 7.61 (d, 1H, J = 6.3 Hz, thiazole C H), 8.71
2 4
(
1
1
d, 4H, J = 4.5 Hz, pyrimidine C H), 9.62 (s, 1H, NH), 11.12 (s, 1H, NH), 12.02 (s,
4,6
1
3
H, NH). C-NMR (DMSO-d ): δ = 107.3, 112.2, 112.9, 117.2, 123.2, 129.7, 144.5,
6
49.1, 150.7, 151.4, 157.8, 158.6, 160.5 and 172.5 (Ar-C, C=C, C=N, C-S).
Complex 3
1
H-NMR (DMSO-d ): δ = 7.26 (t, 1H, J = 4.5 Hz, pyrimidine C H), 7.69 (s,
6
5
1
H, triazole C H), 8.86 (d, 4H, J = 4.5 Hz, pyrimidine C H), 9.99 (s, 1H, NH), 11.69
5 4,6
1
3
(s, 1H, NH), 14.21 (s, 1H, NH). C-NMR (DMSO-d ): δ = 117.5, 140.9, 146.2,
6
1
55.8, 157.9, 159.0, 162.3, 162.8, and 168.2 (Ar-C, C=C, C=N, C-Cl).
Complex 4
1
H-NMR (DMSO-d ): δ = 7.25 (t, 2H, J = 4.5 Hz, pyrimidine C H), 7.26 (d,
6
5
2
8
H, J = 4.5 Hz, p-Chloroaniline C H), 7.34 (d, 2H, J = 9.9 Hz, p-Chloroaniline C H),
3 2
1
3
.62 (d, 4H, J = 4.5 Hz, pyrimidine C H), 9.51 (s, 1H, NH), 11.30 (s, H, NH). C-
4
,6
1
0

ACCEPTED MANUSCRIPT
NMR (DMSO-d ): δ = 112.1, 118.2, 123.2, 129.8, 144.5, 149.7, 151.9, 158.2, 161.4,
6
1
67.8, and 172.9 (Ar-C, C=C, C=N, C-Cl).
Complex 5
1
H-NMR (DMSO-d ): δ = 7.22 (t, H, J = 4.5 Hz, pyrimidine C H), 7.25 (t, H,
6
5
J = 4.5 Hz, pyrimidine C H), 7.34 (d, 2H, J = 4.5 Hz, p-Chloroaniline C H), 7.34 (d,
5
3
2
8
1
1
H, J = 9.9 Hz, p-Chloroaniline C H), 8.72 (d, 2H, J = 4.5 Hz, pyrimidine C H),
2 4,6
.81 (d, 2H, J = 4.5 Hz, pyrimidine C H), 9.79 (s, 1H, NH), 11.10 (s, 1H, NH),
4
,6
1
3
2.05 (s, 1H, NH). C-NMR (DMSO-d ): δ = 112.7, 113.9, 116.0, 123.2, 129.8,
6
46.6, 146.9, 149.2, 151.6, 159.1, 160.1, 163.2, 165.1, and 171.3 (Ar-C, C=C, C=N).
Complex 6
1
H-NMR (DMSO-d ): δ = 7.07 (d, 1H, J = 7.2 Hz, thiazole C H), 7.25 (t, 2H,
6
5
J = 4.5 Hz, pyrimidine C H), 7.27 (d, 2H, J = 4.5 Hz, p-Chloroaniline C H), 7.33 (d,
5
3
2
H, J = 9.9 Hz, p-Chloroaniline C H), 7.66 (d, 1H, J = 6.3 Hz, thiazole C H), 8.79
2 4
(
1
1
d, 4H, J = 4.5 Hz, pyrimidine C H), 9.63 (s, 1H, NH), 11.19 (s, 1H, NH), 12.11 (s,
4,6
1
3
H, NH). C-NMR (DMSO-d ): δ = 108.2, 112.6, 114.5, 123.2, 129.8, 144.5, 149.0,
6
51.6, 151.7, 154.6, 158.6, 160.6, 161.7, and 172.5 (Ar-C, C=C, C=N, C-S).
Complex 7
1
H-NMR (DMSO-d ): δ = 7.28 (t, 1H, J = 4.5 Hz, pyrimidine C H), 7.72 (s,
6
5
1
1
1
H, triazole C H), 8.88 (d, 4H, J = 4.5 Hz, pyrimidine C H), 10.02 (s, 1H, NH),
5 4,6
1
3
1.72 (s, 1H, NH), 14.23 (s, 1H, NH). C-NMR (DMSO-d ): δ = 114.4, 126.1, 142.9,
6
51.7, 153.7, 157.5, 158.2, 160.4 and 169.6 (Ar-C, C=C, C=N, C-Cl).
Complex 8
1
H-NMR (DMSO-d ): δ = 7.26 (d, 2H, J = 4.5 Hz, p-Chloroaniline C H), 7.30
6
3
(
8
t, 2H, J = 4.5 Hz, pyrimidine C H), 7.35 (d, 2H, J = 9.9 Hz, p-Chloroaniline C H),
.66 (d, 4H, J = 4.5 Hz, pyrimidine C H), 9.55 (s, 1H, NH), 11.37 (s, H, NH). C-
4,6
5
2
1
3
NMR (DMSO-d ): δ =112.1, 115.8, 123.2, 129.8, 144.5, 145.9, 148.6, 157.5, 159.7,
6
1
68.7, and 174.3 (Ar-C, C=C, C=N, C-Cl).
1
1
2
3
The H−NMR spectral data of the synthesized s-triazine derivatives L , L , L
4
and L free ligands recorded in DMSO in comparison with eight new triazine
complexes of Ru(III) and Se(IV) metal ions affected by downfield chemical shifts due
to formation complexity through the nitrogen atom of triazine ring and nitrogen atoms
of (pyrimidine, thiazole, and triazole) rings towards metal ion.
3
-6- Thermogravimetric and kinetic thermodynamic studies
Thermogravimetric analyses were displayed under N atmosphere. TG curves
2
1
2
3
4
of the s-triazine derivatives L , L , L and L free ligands and synthesized Se(IV) and
Ru(III) complexes were performed (Fig. 1S and 2S). Table 4 gives the temperature
maximum differential thermogravimetric DTGmax, total mass loss, total residual
materials, and assignments of these lost species of free ligands and their complexes.
The resulted data refer to the decomposition modes of all compounds occur as
expected in two-to-three main degradation steps. TG analysis of the free ligand and its
complexes show that, mass loss started from above >80 ºC, these results confirmed
the absence of any crystalline water molecule. At 800 ºC, complexes 1−8 were
1
1

ACCEPTED MANUSCRIPT
thermally decomposed with different residual materials were formed as
ruthenium(IV) oxide (RuO ) and selenium metal polluted with carbon atoms.
2
*
*
Kinetic thermodynamic parameters (activation energy (E ), enthalpy (∆H ),
*
entropy (∆S ) and Gibbs free energy change) of free ligands and their Ru(III) and
Se(IV) complexes are calculated using Coats-Redfern [55] method and listed in Table
5
. The ruthenium(III) complexes have the higher value of activation energy, Ea,
suggesting a stronger complexity character. The entropy of activation, ꢁS*, values
have a negative values that indicates a more ordered activated state through the
chemisorption of oxygen and other decomposition products [56].
1
2
3
4
Table 4. Thermogravimetric data of L , L , L and L free ligands and synthesized
Se(IV) and Ru(III) complexes
Total mass loss
Total residual
Assignments
Few Carbons
Few Carbons
--
Compounds
DTGmax
Mass loss, %
Assignments
Residue, %
13.32
16.53
0.00
0.00
13.45
1.42
1
1
2
3
4
1
L
180, 380, 650
180, 380, 760
100, 390, 740
170, 360, 600
120, 350, 500
110, 360, 500
180, 400, 600
150, 330, 500
100, 360, 600
100, 370, 560
120, 380, 580
100, 370, 630
86.68
83.47
100
L
L
L
L
2
3
4
L
L
L
1
2
3
4
5
6
7
8
100
--
86.55
98.58
90.16
88.67
77.58
70.59
77.25
65.00
L +2Cl
Se + Few carbons
Few Carbons
Se + Few carbons
Se + Few carbons
2
2
2
2
2
L +2Cl
3
L +2Cl
9.84
4
L +2Cl
11.33
22.42
29.41
22.75
35.00
1
L +3Cl+2H
O
RuO
RuO
RuO
RuO
+ Few carbons
+ Few carbons
+ Few carbons
+ Few carbons
2
2
2
2
2
2
L +3Cl
3
L +3Cl+H
O
O
2
2
4
L +3Cl+H
1
2
3
4
Table 5. Kinetic thermodynamic parameters of L , L , L and L free ligands and
synthesized Se(IV) and Ru(III) complexes
Parameter
o
Compounds
DTGmax/ C
r
E
−1
ꢁS
ꢁH
ꢁG
(J mol
−1
A (s )
−1
−1
−1
−1)
(
J mol )
(J mol K )
-1.10E+02
-7.90E+01
2.80E+02
2.30E+02
1.86E+01
4.80E+01
-2.28E+02
-2.10E+02
3.16E+02
3.21E+02
5.14E+01
6.00E+01
(J mol )
1.30E+05
1.50E+05
3.95E+05
4.00E+05
2.11E+05
1
2
3
4
L
L
L
L
1
380
380
390
360
350
360
400
330
360
370
380
370
1.35E+05
1.52E+05
4.01E+05
4.05E+05
2.20E+05
2.41E+05
4.33E+04
4.96E+04
4.21E+05
4.23E+05
2.40E+05
2.45E+05
3.70E+08
1.13E+08
7.72E+28
4.63E+27
1.31E+15
5.00E+14
2.20E+02
1.54E+03
3.80E+28
6.62E+21
6.85E+14
2.06E+15
2.05E+05
2.01E+05
1.97E+05
1.90E+05
2.02E+05
0.9933
0.9871
0.9862
0.9834
0.9911
0.9928
0.9937
0.9890
0.9491
0.9461
0.9800
0.9675
2
2.32E+05 1.91E+05
3
3.84E+04
4.49E+04
4.18E+05
4.21E+05
2.41E+05
2.40E+05
1.69E+05
1.62E+05
1.97E+05
1.90E+05
2.01E+05
1.97E+05
4
5
6
7
8
1
2

ACCEPTED MANUSCRIPT
3
-7- X−ray diffraction spectra
1
X−ray powder diffraction patterns of Se(IV) and Ru(III) complexes 1−8 for L ,
L , L and L ligands have a semi-crystalline nature (Fig. 3Sa and 3Sb). Powder X-
2
3
4
ray diffraction patterns of the synthesized complexes showed the diffraction pattern at
2
1
θ values of (20.487, 22.615, 26.632, 28.052, 30.337, 31.749, 45.53, 56.651°), (10.20,
1.691, 13.968, 19.871, 22.631, 26.716, 28.229, 29.91, 30.543, 46.431, 55.876°),
(22.93, 27.185, 30.337, 44.875, 56.212°), (22.457, 23.639, 26.554, 27.894, 30.258,
4
2
4
5.653, 56.876°), (16.638, 25.233, 31.699, 42.333, 45.523, 51.689°), (19.857, 23.087,
4.112, 29.549, 42.546, 45.737, 51.476°), (16.72, 25.316, 27.844, 42.333, 45.736,
9.996°) and (16.722, 27.056, 29.58, 30.372, 43.305, 45.736, 51.689°) for the
selenium and ruthenium complexes 1−8, respectively, which indicated the
transformation into new crystalline phase. The particle size has been calculated using
Scherrer’s equation [57]. The diffractions at 31.749, 45.53 and 56.651° can be
indexed to the (101), (111) and (112) planes of the face-centered cubic (fcc) Se-NPs,
respectively [58]. The XRD of the Ru(III) complex has three significant peaks at
4
2.333, 45.523, and 51.689° due to the presence of (002), (100), and (101) planes of
ruthenium metal [59]. The crystallite sizes of synthesized complexes have been
calculated and are found to be 85−86 and nm, respectively for Se(IV) and Ru(III)
nanoparticle complexes according to highest distinguish peaks.
3
-8- Scanning and transmission electron microscopes
1
2
3
4
SEM images of L , L , L and L ligands and their (selenium(IV) and
ruthenium(III)) complexes 1−8 are scanned (Fig. 6). The SEM images have a
homogeneous morphology with different morphology in comparison with free ligands
due to the presence of metal ions which make rearrangement of grain size and
changing of morphological shapes. The existence of the metal ions works to fragment
the aggregation of granules and turn them from large to small particles.
Figure 7 is refers to the TEM images of the Se(IV) and Ru(III) complexes.
The average particle diameters for these complexes were of 12−60 nm within
nano−scale range.
3
3
-9-Biological studies
-9-1- Antimicrobial assessments
1
2
3
4
The s-triazine derivatives L , L , L and L free ligands and their
selenium(IV) (1−4) and ruthenium(III) (5−8) complexes were evaluated for bacterial
activity against one Gram positive bacteria Staphylococcus aureus, one Gram
negative bacteria Escherichia coli and two fungi (Aspergillus flavus and Candida
albicans) by using
a modified Kirby-Bauer disc diffusion method [34] and the results
were summarized in Table 6 and display in Fig. 8. Ampicillin and amphotericin B
were used as a standards regarding antibacterial agent and antifungal agent.
Antimicrobial studies were experimentally done in triplicate and the average was
taken as the final result. In vitro antifungal activity data of the ligands and their metal
complexes against fungal organisms display not significant activity except for
complexes 1−5. It is clearly show all selenium(IV) complexes have a higher activity
against Aspergillus flavus and moderate activity against Candida albicans rather than
1
the Amphotericin B standard agent. The [Ru(L )(Cl) (H O) ]Cl complex 5 exhibits
2
2
2
moderate activity against Aspergillus flavus. In vitro antibacterial activity data reveals
1
3

ACCEPTED MANUSCRIPT
that all the newly synthesized complexes display moderate to significant activity in
comparison to ampicillin standard drug. It is found that selenium(IV) complexes
(1−4) show significant activity against Staphylococcus aureus and Escherichia coli
when compared to other complexes and there are higher activity than ampicillin
standard antibacterial agent. Ruthenium(III) complexes (5−8) are moderate activity
2
3
against Staphylococcus aureus and Escherichia coli bacteria. Ligands L and L are
inactive towards bacteria and fungi species. Complexes (1−4) exhibits higher activity
against Staphylococcus aureus and Escherichia coli compared to standard reference.
Such increased activity of the synthesized complexes over the ligand can be explained
on the basis of Overton’s concept [60] and chelation theory [61]. The variation in the
activity of different metal complexes against different microorganisms depends on
either the impermeability of the cells of the microbes or the differences in ribosomes
in microbial cells.
1
2
3
4
Table 6: Inhibition zone diameter (mm/mg sample) of L , L , L and L ligands and
their complexes 1−8 against some kind of bacteria and fungi
Inhibition zone diameter (mm/mg Sample)
Sample
Bacteria
Fungi
Escherichia coli
Staphylococcus aureus
Aspergillus flavus Candida albicans
-
+
(G )
(G )
(Fungus)
(Fungus)
Ampicillin: Antibacterial
agent
3
0
24
--
--
Amphotericin B:
Antifungal agent
--
--
16
21
Control: DMSO
0.0
11
0.0
0.0
15
38
32
41
37
14
12
13
16
0.0
10
0.0
0.0
10
33
31
40
35
15
11
11
18
0.0
10
0.0
0.0
0.0
25
21
28
30
11
0.0
0.0
0.0
0.0
0.0
17
13
20
20
0.0
0.0
0.0
0.0
1
L
L
L
L
2
3
4
1
2
3
4
5
6
7
8
0.0
0.0
0.0
3
-9-2- Anticancer assessments
1
2
3
4
The neutral red cytotoxicity test for the free L , L , L and L ligands and their
selenium(IV) complexes (1−4) was examined on the human colon cancer cell lines
and human lung cancer A549. In Table 7 and as shown in Fig. 9, IC values were
5
0
obtained for the free ligands and their Se(IV) complexes, which are shown for the
sake of comparison using neutral red uptake assay for the estimation of cell
1
viability/cytotoxicity [36]. The complex [Se(L )(Cl) ]2Cl (1) inhibits the growth of
2
human colon cancer cells and, its activity is 142-fold higher than of the corresponding
1
free L ligand. However, in comparison to cisplatin, the effect of the complex is
slightly lower.
In general, all selenium(IV) complexes have a significant activity in
comparison with respected ligands except for complexes 3 and 4. In Fig. 9, the
percentage of growth of tumor cells and the method used for the estimation depends
on the red dye, which has a large absorption within the living cells only and therefore
1
4

ACCEPTED MANUSCRIPT
the images attached to the results show that the compounds high-impact against
cancer cells produce a light red color low value.
1
2
3
4
Table 7. Inhibitory activity IC of the free ligands (L , L , L and L ) and their
5
0
selenium(IV) complexes (1−4) against human colon and human lung cancer A549cell
lines.
Against human colon cancer cell lines
Concentration
ꢀg/mL)
Viability (%)
1
2
3
4
(
L
1
0
0
1.1
39.3
1.2
L
2
0
0
L
3
0.3
1.3
0
37
4
L
4
0
0
0
26.5
-
1
1
50
00
49.8
65
70
72
170
59.6
61
64.4
69.3
277
51.7
55.3
60
31
35.5
41.4
66.8
50.7
5
1
0
0
0
54.6
13.4
65
162.8
IC50
Against human lung cancer A549 cell lines
Viability (%)
Concentration
ꢀg/mL)
50
00
0
1
2
3
4
(
L
1
7.9
10.5
25
L
96
98.7
100
100
2
60.8
64
L
100
100
100
100
-
3
14.6
33.5
50
84.4
68
L
100
100
100
100
-
4
13.4
16
25.9
76
1
1
5
1
82.4
83.4
86
98.7
431.4
69
0
IC50
76
88.6
33
1305.7 189.8
35
Acknowledgement
This work was funded by the Deanship of Scientific Research at “Princess Nourah
bint Abdulrahman University. The authors would like to thank Prof. Dr. Ibrahim A.
Alsarra for his scientific guidance, also we would like to thank the assistant researcher
Mrs. Lina Almaliky for her participation in the preparation of experimental work in
lab.
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(
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Figure captions
Fig. 1. Structures of synthesized s-triazine ligands with different substituent's
Fig. 2. Synthesis of trisubstituted s-triazine derivatives from cyanuric chloride.
Scheme 1. Synthesis pathways of trisubstituted s-triazine derivatives ligands.
1
2
3
4
Fig. 3. Speculated structures of Ru(III) and Se(IV) complexes with L , L , L and L
s-triazine derivatives ligands
Fig. 4: HOMO and LUMO structure of L , L , L and L s-triazine derivatives
1
2
3
4
ligands
2
2
Fig. 5. FTIR spectrum of a: L and b: [Ru(L )(Cl) ] (6) complex
Fig. 6. SEM morphology of L , L , L and L ligands and their complexes 1−8.
3
1
2
3
4
1
2
3
Fig. 7. TEM images of selenium(IV) complexes 1−4 complexes with L , L , L and
4
L ligands.
Fig. 8: Inhibition zone diameter (mm/mg sample) of selenium(IV) complexes 1−4 for
1
2
3
4
L , L , L and L ligands against some kind of bacteria and fungi
Fig. 9a. Images of the neutral red cytotoxicity test of the free L , L , L and L
ligands and their selenium(IV) complexes (1−4) against human colon cancer cell lines
1
2
3
4
1
2
3
4
Fig. 9b. Images of the neutral red cytotoxicity test of the free L , L , L and L
ligands and their selenium(IV) complexes (1−4) against human lung cancer A549 cell
lines
1
8

ACCEPTED MANUSCRIPT
3
3
4
4
Cl
Cl
2
2
HN 1
1
2
N
HN 1
2
N
6
4
3
1
N
1
3
N
5
4
3
N
N
5
2
4
S
N
N 4 NH
6
N 2 N 6 N
NH
1
H
5
2
H
3
5
2
N 3
1
N
1N
N3
4
6
4
6
5
5
1
2
L
L
3
4
Cl
Cl
6
2
5
5
N
4
1
HN
N
N1
HN 1
6
N1
5
N
N 3 N 4 N 2 NH
2
H
3
2
1
N
N3
4
Cl 4 N 2 NH
3
2
6
1
N
N3
4
5
6
5
3
4
L
L
Fig. 1.
Fig. 2.
1

ACCEPTED MANUSCRIPT
Cl
Cl
H N
2
N
N
Cl
Acetone/ 0-5 oC
Cl
N
NH
N
N
str.
Cl
N
Cl
N
HN
Cl
1
N
N
NH2
2
a
Cl
N
N
N
HN
N
N
N
Cl
N
NH2
H
Cl
2b
Cl
HN
N
N
HN
S
NH2
N
N
N
N
N
NH2
N
N
S
N
H
N
NH
Reflux/Dioxane
Cl
N
NH
N
Cl
N
N
L2
N
N
N
L4
HN
N
N
N
NH
H
N
N
L3
N
N
NH2
Cl
HN
N
N
N
N
N
N
NH
H
N
N
L1
Scheme 1.
2

ACCEPTED MANUSCRIPT
Cl
N
N
N
NH
HN
N
N
N
NH
Cl
Cl
HN
N
Se
N
HN
Cl
NH
Cl
.Cl
Se
N
N
.2Cl
N
N
N
S
Cl
Cl
1
2
Cl
Cl
N
N
N
NH
HN
NH
.3Cl
N
N
.
2Cl
Se
Cl
Cl
Cl
N
NH
N
N
N
N
NH
Se
Cl
N
N
3
4
Cl
N
N
N
NH
HN
N
N
N
NH
Cl
HN
N
N
HN
NH
Cl
N
OH2
Cl
Cl
Ru
Ru
Cl
N
.Cl
N
N
N
S
OH2
Cl
5
6
3

ACCEPTED MANUSCRIPT
Cl
Cl
N
N
N
HN
NH
.Cl
NH
Cl
Cl
Ru
N
N
N
N
N
N
OH2
Cl
H O
N
NH
NH
2
Cl
N
Ru
Cl
N
Cl
7
8
Fig. 3.
4

ACCEPTED MANUSCRIPT
Ligand
HOMO
LUMO
1
L
2
L
3
L
4
L
Fig. 4:
5

ACCEPTED MANUSCRIPT
Fig.5a
6

ACCEPTED MANUSCRIPT
Fig.5b
Fig. 5.
7

ACCEPTED MANUSCRIPT
Fig. 6.
Fig. 7.
8

ACCEPTED MANUSCRIPT
Fig. 8:
Fig. 9a.
9

ACCEPTED MANUSCRIPT
Fig. 9b.
1
0