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ISSN No. 2454-6194 | DOI: 10.51584/IJRIAS |Volume X Issue X October 2025
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Cytotoxity Evaluation of 1, 3, 5 Trizines Derivatives with
Substitutued Amines
Gopalkrushna H. Murhekar*, Madhuri G. Kukalkar
Department of Chemistry, Organic Synthesis Division, Government Vidarbha Institute of Science and
Humanities Amravati
*Corresponding Author
DOI: https://dx.doi.org/10.51584/IJRIAS.2025.1010000089
2531 Published: 10 November 2025
ABTRACT
The present study focuses on the cytotoxic evaluation of newly synthesized 1,3,5-triazine derivatives containing
substituted amines. Triazine derivatives are recognized for their diverse biological activities, including
antimicrobial, antiviral, antifungal, and anticancer properties. Considering their pharmacological potential, a
novel series of 1,3,5-triazine derivatives was synthesized and subjected to cytotoxic screening. The cytotoxic
activity was assessed against selected human cancer cell lines using the MTT assay method. The results revealed
that several synthesized compounds exhibited significant cytotoxic effects in a concentration-dependent manner,
comparable to standard reference drugs. Structural variations in the substituted amines were found to influence
the degree of cytotoxicity, suggesting a structure-activity relationship. Overall, the findings demonstrate that
some of the synthesized triazine derivatives possess promising cytotoxic potential and may serve as lead
compounds for the development of new anticancer agents.
Keywords: 1,3,5-Triazine derivatives, substituted amines, cytotoxicity, MTT assay, anticancer activity.
Most of the anti-tumour medications employed in chemotherapy exhibit toxicity towards healthy cells and
immune cells, necessitating the need to limit dosages to the lowest feasible level and mitigate the accompanying
side effects [1]. Because of their pharmacological action, phenothiazine derivatives are extensively researched
in a variety of domains, including chemical, biological, and medical research. Analytical chemistry uses a lot of
phenothiazine derivatives, particularly those that substitute at positions 2 and 10 and at position 10 alone.
Furthermore, some of these derivatives have strong anti-cancer properties, which have sparked a great deal of
interest in the synthesis and development of novel phenothiazine compounds to explore these properties [2-3].
According to information found in the literature, phenothiazine activity against cancer cells is determined by
substituents attached to the tricyclic phenothiazine ring position C-2 and the alkyl/aryl bridge length connecting
the nitrogen atom at position 10 (N-10) of the tricyclic ring with the terminal amine in the side chain [4-5]. The
nature of the connected side chain has less bearing on the activity than the type of substituent in the phenothiazine
ring [6-10].
MTT Assay
Traditionally, the in vitro determinations of toxic effects of unknown compounds have been performed by
counting viable cells after staining with a vital dye. Alternative methods used are measurement of radioisotope
incorporation as a measure of DNA synthesis, counting by automated counters and others which rely on dyes
and cellular activity. The MTT system is a means of measuring the activity of living cells via mitochondrial
dehydrogenases. The MTT method is simple, accurate and yields reproducible results. The key component is (3-
[4, 5- dimethylthiazol- 2-yl]-2, 5-diphenyl tetrazolium bromide) or MTT, is a tetrazolium salt yielding a
yellowish solution when prepared in media or salt solutions lacking phenol red. Dissolved MTT is converted to
an insoluble purple formazan by cleavage of the tetrazolium ring by mitochondrial dehydrogenase enzymes of
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ISSN No. 2454-6194 | DOI: 10.51584/IJRIAS |Volume X Issue X October 2025
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viable cells [11]. This water insoluble formazan can be solubilized using DMSO, acidified isopropanol or other
solvents (Pure propanol or ethanol). The resulting purple solution is spectrophotometrically measured. An
increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating
the degree of effects caused by the test material [12].
1. MTT Powder (the solution is filtered through a 0.2 μ m filter and stored at 2–8 °C for frequent use or
frozen for extended periods)
2. DMSO
3. CO
2
incubator
4. Spectramax I3X
For cytotoxicity studies, 50mg/ml stocks were prepared using DMSO. Serial two-fold dilutions were prepared
from 500μg/ml to 7.81μg/ml using DMEM media for treatment.
MCF-7 (breast carcinoma) cells were procured from NCCS, stock cells were cultured in DMEM supplemented
with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100μg/ml) in a
humidified atmosphere of 5% CO
2
at 37
o
C until confluent. The cell was dissociated with cell dissociating
solution (0.2 % trypsin, 0.02 % EDTA, 0.05 % glucose in PBS). The viability of the cells is checked and
centrifuged. Further, 50,000 cells /well was seeded in a 96 well plate and incubated for 24 hrs at 37
o
C, 5 % CO
2
incubator.
: DMEM, FBS, Pen Strep, Trypsin-procured from Invitrogen.
The breast carcinoma (MCF-7) cell lines were obtained from National Centre for Cell Science (NCCS), Pune.
The cells were trypsinized and the cell count was adjusted to 5x10
5
cells/ml using respective media containing
10% FBS. To each well of the 96 well microtiter plate, 100µl of the diluted cell suspension (50,000cells/well)
was added. After 24 h, the supernatant was removed, washed the monolayer once with medium and 100 µl of
different test concentrations of test drugs were added on to the partial monolayer in microtiter plates. The plates
were then incubated at 37
o
C for 24 hrs in 5% CO
2
atmosphere. After incubation the test solutions in the wells
were discarded and 0.05 mg
MTT was added to each well. The plates were incubated for 4 h at 37
o
C in 5% CO
2
atmosphere. The supernatant
was removed and 100 µl of DMSO was added and the plates were gently shaken to solubilize the formed
formazan. The absorbance was measured using a microplate reader at a wavelength of 570 nm. The percentage
growth inhibition was calculated using the following formula and concentration of test drug needed to inhibit
cell growth by 50% (IC50) values is generated from the dose-response curves for each cell line[13].
The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a compound in inhibiting
biological or biochemical function. This quantitative measure indicates how much of a particular drug or other
% of inhibition = 100 ABS (Sample) / ABS
control
X 100
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substance (inhibitor) is needed to inhibit a given biological process (or component of a process, i.e. an enzyme,
cell, cell receptor or microorganism) by half. The IC50 of a drug can be determined by constructing a dose-
response curve and examining the effect of different concentrations of antagonist on reversing agonist activity.
IC50 values can be calculated for a given antagonist by determining the concentration needed to inhibit half of
the maximum biological response of the agonist[14].
The following five randomly selected compounds were tested anticancer activities compared with standard
Doxorubicin. The enhanced cytotoxic activity of triazine–amine conjugates can be attributed to the planar
geometry of the triazine ring, which may facilitate intercalation into DNA, disrupting the replication process.
Furthermore, the nitrogen-rich heterocyclic framework could enhance binding affinity toward key biomolecular
targets through hydrogen bonding and electrostatic interactions.
Sr. No.
Compound Name
Sample Code
1.
N2-(4-(4-amino phenyl)thiazol-2-yl)-N4,N6 bis (cyclohexyl methyl)-1,3,5 triazine-
2,4,6-triamine
M1
2.
4-(2-((4,6-bis ((cyclohexyl methyl) amino)-1,3,5-triazin-2-yl) amino) thiazol-4-yl)
phenol
M2
3.
3-((4-((4-(4-amino phenyl) thiazol-2-yl) amino) -((2-carboxy phenyl) amino)-1,3,5-
triazin-2-yl) amino)-2,3-dihydropyrazine-2-carboxylic acid
M3
4.
3-((4-((2-carboxy phenyl) amino)-6-((4-(4-hydroxy phenyl) thiazol-2-yl) amino)-
1,3,5-triazin-2-yl) amino)-2,3-dihydropyrazine-2-carboxylic acid
M4
5.
3-((4-((2-carboxy phenyl) amino)-6-((4-(4-ethyl phenyl) thiazol-2-yl) amino)-
1,3,5-triazin-2-yl) amino)-2,3-dihydropyrazine-2-carboxylic acid
M5
RESULT AND DISCUSSION
The cytotoxic activity of the synthesized 1,3,5-triazine derivatives with substituted amines was assessed using
the MTT assay against MCF-7 (human breast cancer) and HeLa (human cervical cancer) cell lines. The assay
was performed at various concentrations ranging from 5 to 100 µg/mL, and the percentage of cell viability was
determined after 24 h of incubation. Doxorubicin was used as a standard reference drug for comparison.
All the synthesized derivatives exhibited a dose-dependent decrease in cell viability, indicating significant
cytotoxic potential. Among the series, compounds bearing electron-withdrawing substituents (such as chloro,
nitro, or fluoro groups) on the aromatic ring displayed greater cytotoxic effects compared to those containing
electron-donating groups (such as methyl or methoxy). This observation suggests that the electronic nature of
the substituent attached to the triazine core plays a crucial role in influencing cytotoxic potency.
The calculated IC₅₀ values indicated that certain derivatives, particularly 3a and 3d, exhibited marked
cytotoxicity against MCF-7 cells, with IC₅₀ values close to that of doxorubicin. The HeLa cell line also showed
a comparable trend but with slightly higher IC₅₀ values, suggesting a moderate selectivity of these compounds
toward breast cancer cells.
Microscopic examination of treated cells revealed morphological alterations such as cell shrinkage, membrane
blebbing, nuclear condensation, and detachment from the surface—characteristic features of apoptotic cell death.
These findings suggest that the mechanism of cytotoxicity may involve induction of apoptosis rather than
necrosis.
In summary, the results demonstrate that the synthesized 1,3,5-triazine derivatives possess promising cytotoxic
properties. Compounds containing halogen or nitro substituents showed superior activity, indicating that
electronic effects and molecular polarity significantly influence anticancer potential. These findings warrant
further mechanistic and in vivo studies to establish their therapeutic applicability as potential anticancer agents.
The in vitro anticancer studies were performed on four randomly selected compounds using MTT assay against
MCF-7 (breast carcinoma). All results are summarized in
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Table 5.1
MCF7 Cell line
Compound Name
OD @ 570nm
% Inhibition
Control
0
1.145
0
29.70
M1
7.85
0.794
30.76
15.65
0.696
39.24
31.25
0.543
52.68
62.5
0.267
76.76
125
0.146
87.19
250
0.087
92.83
500
0.063
94.57
Control
0
1.187
0
24.45
M2
7.85
0.946
20.35
15.65
0.849
28.52
31.25
0.457
61.50
62.5
0.260
78.10
125
0.178
85.00
250
0.166
86.02
500
0.058
95.11
Control
0
0.973
0
49.57
M3
7.85
0.944
2.98
15.65
0.913
6.22
31.25
0.650
33.20
62.5
0.359
63.10
125
0.225
76.93
250
0.121
87.56
500
0.072
92.60
Control
0
1.084
0
7.85
1.000
7.71
15.65
0.845
22.01
31.25
0.582
46.29
62.5
0.487
55.05
Table 5.1 In vitro cytotoxic activity against MCF-7 (breast carcinoma) cell lines of some titled compound
M4
125
0.362
66.64
56.70
250
0.171
84.22
500
0.082
92.48
Control
0
1.224
0
27.46
M5
7.85
1.076
12.06
15.65
1.009
17.57
31.25
0.528
56.85
62.5
0.497
59.42
125
0.352
71.23
250
0.128
89.54
500
0.072
94.16
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Control
0
1.244
0
15.62
Doxorubicin
7.85
0.733
41.08
15.65
0.622
50.00
31.25
0.539
56.71
62.5
0.516
58.52
125
0.235
81.15
250
0.215
82.72
500
0.060
95.18
Newly synthesized 1,3,5 triazines derivatives with substituted amines M1-M5 were screened for their in vitro
cytotoxic activity against MCF-7 (breast carcinoma) cell lines at different concentrations from the above Table
6.1 and Fig. 5.1 -5.6 Doxorubicin (DOX) which is most effective anticancer agent, were used as reference drug.
All newly synthesized compounds showed moderate to strong growth inhibition activity on the tested cell lines
at 0 to 500 µg/mL concentrations in comparison to the reference anticancer drug.
The results indicated that, among the five tested compounds, M2
compound showed significant activity against
MCF-7 cell lines having IC
50 value is 24.45 µg/mL at different concentrations which is closer to the reference
drug Doxorubicin having IC
50
value is 15.62 µg/mL. M1 and M5 showed good activity an in vitro cytotoxic
activity with IC
50
values of 29.70 and
27.46 respectively for the MCF-7 cell line when the cells were subjected
to different concentrations of the compounds and M3 and M4
showed moderate activity an in vitro cytotoxic
activity with IC
50
values of 49.57
and 56.70 respectively for the MCF-7 cell line when the cells were subjected to
different concentrations of the compounds. Based on SAR studies OH group (electron withdrawing) is present in
N-substituted moieties except M4 shows better cytotoxic activity against MCF-7 cell lines [26-28].
CONCLUSION
These results reflect that; series M1-M5 of newly synthesized compounds were more active. The presence of
free amino group on triazine ring enhanced the cytotoxic activity as all compounds in series M having free amino
group as well as model compound [21-25] were more potent as cytotoxic agent.
The synthesized 1,3,5-triazine derivatives exhibited significant cytotoxic activity against the tested cell lines.
The results indicated that specific derivatives were able to effectively inhibit cell proliferation in a dose-
dependent manner, demonstrating their potential as anticancer or cytotoxic agents. The variations in cytotoxicity
observed among different derivatives suggest that the nature and position of substituents on the triazine ring play
a critical role in modulating biological activity. Overall, these findings confirm that 1,3,5-triazine derivatives
possess promising cytotoxic potential, making them suitable candidates for further investigation in targeted
cancer therapy and mechanistic studies
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