Synthesis and Cytotoxic Activity of Hexahydro-1,3,5-triazine Derivatives through Ring Condensation

Article abstract of DOI:10.1002/bkcs.12266

As a part of a research program for pharmacologically interacting sym-triazine derivatives with hexahydrotriazine ring, novel hexahydrotriazine derivatives (1?13) were successfully synthesized. The synthesis of hexahydrotriazines was carried out from the assembly of three molecules of various amines and three molecules of formaldehyde by “1 + 1 + 1 + 1 + 1 + 1” cycloaddition. The synthesis mechanism is probably passes through imines, which trimerizes to give the hexahydrotriazine ring. All synthesized hexahydrotriazines (1–13) were screened for their cytotoxic activity by cancer cells. As a result, compound 1–9 reduced the viability of T98G cells in a concentration-dependent manner. At a concentration of 100 μM, compound 9 exhibited the greatest cytotoxicity at 94.6%. The reason is that compound 9 is C₅H₅N that has a resonance hybrid structure and exhibits aromaticity. It is considered to be active because it has a structure that is a raw material for pyridine derivatives as mainly medicines and pesticides.

Full text of DOI:10.1002/bkcs.12266

Article
DOI: 10.1002/bkcs.12266
BULLETIN OF THE
S. M. Bae et al.
KOREAN CHEMICAL SOCIETY
Synthesis and Cytotoxic Activity of Hexahydro-1,3,5-triazine
Derivatives through Ring Condensation
*
Song Mi Bae, Sung Young Kang, and Ju Hyun Song
Department of Chemistry, Dong-A University, Busan 604-714, South Korea. *E-mail: jhsong@dau.ac.kr
Received December 21, 2020, Accepted March 2, 2021
As a part of a research program for pharmacologically interacting sym-triazine derivatives with
hexahydrotriazine ring, novel hexahydrotriazine derivatives (1−13) were successfully synthesized. The
synthesis of hexahydrotriazines was carried out from the assembly of three molecules of various amines
and three molecules of formaldehyde by “1 + 1 + 1 + 1 + 1 + 1” cycloaddition. The synthesis mechanism
is probably passes through imines, which trimerizes to give the hexahydrotriazine ring. All synthesized
hexahydrotriazines (1–13) were screened for their cytotoxic activity by cancer cells. As a result, com-
pound 1–9 reduced the viability of T98G cells in a concentration-dependent manner. At a concentration
of 100 μM, compound 9 exhibited the greatest cytotoxicity at 94.6%. The reason is that compound 9 is
C5H5N that has a resonance hybrid structure and exhibits aromaticity. It is considered to be active because
it has a structure that is a raw material for pyridine derivatives as mainly medicines and pesticides.
Keywords: Hexahydrotriazine, Sym-triazine, Cytotoxic activity, Cyclocondensation, Pharmaceutical
activity
Introduction
1,5-dicarboxylic acid. Here we report the synthesis of
hexahydrotriazine derivatives from the assembly of three
molecules of various amines[aniline, 4-ethylaniline, 6-
methylpyridin-2-amine, 3-aminoquinoline, 2-furfurylamine,
2-(aminomethyl)-thiophene, thiazol-2-amine, benzylamine,
3-picolylamine, 1-(3-aminopropyl)pyrrolidin-2-one,13 3-
hydrazino-1,2-benzisothiazole 1,1-dioxide,14 L-cysteine,15
glucosamine16] and three molecules of formaldehyde by “1
+ 1 + 1 + 1 + 1 + 1” cyclocondensation originated from six
single different molecules. The synthesis mechanism is prob-
ably passes through imines, which trimerizes to give the
hexahydrotriazine ring. In recent decades, ultrasound has
been used more and more frequently in organic synthesis.
The use of ultrasound in organic transformations is
well known because it can enhance the reaction rate and can
alter selectivity performance of the reaction.17–19 Comparing
with traditional methods under conventional condition, this
method is more convenient and easily controlled.20,21 It can
also felicitates reactions at ambient conditions that otherwise
require higher temperature, pressure, or concentrations; to the
best of our knowledge, the use of ultrasound has been
extended to the synthesis of hexahydrotriazine derivatives,
but they did not get enhanced yield for the reaction.22
Hexahydrotriazines as symtriazines are important building
blocks in high-explosive compounds1–4 and are reported to
show a broad spectrum of biological activity in particular,
antitumor, antimicrobial, and anticancer activities.5 They
are also used as herbicides, carcenolytics, and growth stim-
ulating and antidote activity.6 An increasing interest with
hexahydrotriazine core is attracted by hexahydrotriazine
derivatives, as witnessed in a recent review on their phar-
maceutical activity properties. Until now, despite the less
literature on hexahydrotriazines, they represent a useful
class of organic compounds. The close similarity with
hexahydrotriazine core suggested them as potential capabil-
ity of modifying substitutes of this ubiquitous moiety. As a
part of a research program related to the synthetic study of
pharmacologically interesting compounds and good chelat-
ing agents for transition metal ions, we were in the middle
of an experiment about 1,5,3,7-diazadiphosphocine-
1,5-dicarboxylic acids obtained from the synthesis of an
unusual medium-sized ring heterocyclic ligand with
carboxylic-amino-phosphonic donating groups.7–11 This
heterocyclic ligand results from the assembly of two mole-
cules of amine, two molecules of H3PO2, and four mole-
cules of formaldehyde; its striking feature is that each atom
of this eight-membered ring is originated from eight single
different molecules, representing a formal “1 + 1 + 1 + 1
+ 1 + 1 + 1 + 1” cyclocondensation.12
Experimental
Chemical Experimental. All chemicals were purchased
from commercial suppliers and used without further purifica-
tion unless otherwise stated. All solvents used in the reaction
were freshly distilled from a suitable dehydrating agent
under nitrogen. All solvents used in the chromatography
But according to a graduate student’s not to laughable
reaction except H3PO2, we fortunately were able to synthe-
size hexahydrotriazine, not 1,5,3,7-diazadiphosphocine-
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were purchased and used without further purification. H
NMR spectra were recorded on Varian Mercury 400 MHz
Fourier Transform Nuclear Magnetic Resonance (Palo
alto, CA, USA) and 100 MHz for 13C, with the chemical
shift (δ) reported in parts per million (ppm) downfield
relative to tetra-methyl silane and the coupling constants
(J) quoted in Hz. Chloroform‑d and dimethyl sulfoxide‑d6
were used as a solvent and an internal standard.
Mass spectra were recorded with an HP 5890 (Hewlett
Packard, Palo Alto, CA, USA) GC/Mass (70 eV, EI).
Melting points were measured on a MEL-TEMP II appara-
tus (Triad Scientific, Manasquan, NJ, USA) and were
uncorrected. Elemental analyses were measured on a
Thermo Scientific Flash 2000 (Thermo Scientific, Wal-
tham, MA, USA), Thin-layer chromatography (TLC) was
performed on DC-Plastikfolien 60, F254 (layer thickness
0.2 mm; Merck, Darmstadt, Germany) plastic-backed sil-
ica gel plates and visualized by UV light (254 nm). Chro-
matographic purification was carried out using Kieselgel
60 (60–120 mesh, Merck).
sulfoxide‑d6, 400 MHz): δ 7.86 (dd, J = 7.2 Hz and
3.2 Hz, 3H), 6.86 (d, J = 7.4 Hz, 3H), 6.48 (d, J = 7.4 Hz,
3H), 5.56 (s, 6H), 2.32 (s, 9H); 13C NMR (dimethyl sul-
foxide‑d6, 100 MHz): δ 158.45, 155.23, 138.32, 118.32,
101.21, 82.11, 24.22; Anal. Calcd. for C21H24N6: C,
69.97; H, 6.71; N, 23.31; Found: C, 69.96; H,
6.73; N, 23.30.
1,3,5-Tri(quinolin-3-yl)-1,3,5-triazine 4. Yield: 66.4%;
mp: 196ꢀ197 ꢁC; Rf: 0.52 (TLC eluent; n-Hexane: E.
A = 3:5); Mass (70 eV), m/z (rel. int.%): 468.21 (100),
469.21 (32.4), 470.21 (2.7); 1H NMR (dimethyl sul-
foxide‑d6, 400 MHz): δ 8.50 (s, 3H), 8.32 (d, J = 8.0 Hz,
3H), 7.65 (d, J = 7.4 Hz, 3H), 7.32–7.43 (m, 6H), 7.11 (s,
3H), 4.76 (s, 6H); 13C NMR (dimethyl sulfoxide‑d6,
100 MHz): δ 149.12, 148.32, 145.42, 133.22, 128.98,
125.32, 88.42; Anal. Calcd. for C30H24N6: C, 76.90; H,
5.16; N, 17.94; Found: C, 76.88; H, 5.19; N, 17.93.
1,3,5-Tris(furan-2-ylmethyl)-1,3,5-triazine 5.
Yield:
66.8%; mp: sticky oil; Rf: 0.52 (TLC eluent; n-Hexane: E.
A = 5:3); Mass (70 eV), m/z (rel. int.%): 327.16 (100),
328.16 (19.5), 329.17 (1.8); 1H NMR (dimethyl sul-
foxide‑d6, 400 MHz): δ 7.56 (d, J = 7.3 Hz, 3H), 6.35 (dd,
J = 7.2 Hz and 3.2 Hz, 3H), 6.17 (d, J = 7.4 Hz, 3H), 3.80
(s, 6H), 3.62 (s, 6H); 13C NMR (dimethyl sulfoxide‑d6,
100 MHz): δ 148.22, 140.02, 110.04, 772.22, 50.42; Anal.
Calcd. for C18H21N3O3: C, 66.04; H, 6.47; N, 12.84; O,
14.66; Found: C, 66.01; H, 6.50; N, 12.82; O, 14.68.
1,3,5-Tris(thiophen-2-ylmethyl)-1,3,5-triazine 6. Yield:
65.6%; mp: sticky oil; Rf: 0.61 (TLC eluent; n-Hexane: E.
A = 5:3); Mass (70 eV), m/z (rel. int.%): 375.09 (100),
376.09 (20.5), 377.07 (13.6); 1H NMR (dimethyl sul-
foxide‑d6, 400 MHz): δ 7.36 (d, J = 7.3 Hz, 3H),
6.94–6.89 (m, 6H), 4.03 (s, 6H), 3.85 (s, 6H); 13C NMR
(dimethyl sulfoxide‑d6, 100 MHz): δ 140.22, 127.22,
125.55, 77.22, 50.12; Anal. Calcd. for C18H21N3S3: C,
57.57; H, 5.64; N, 11.19; S, 25.61; Found: C, 57.58; H,
5.62; N, 11.20; S, 25.60.
General
procedure
for
the
synthesis
of
1,3,5-hexahydrotriazine derivatives. Amine (0.03 mol)
was dissolved in 25 mL of ethanol in a 100 mL one necked
round-bottomed flask. Formaldehyde (0.11 mL, 0.03 mol),
trimethylamine (0.041 mL, 0.03 mol) as a catalyst were
added in mixture. The temperature of condensation reaction
is the reflux temperature of ethanol, the solvent of the reac-
tion. After mixing for 7 h, the reaction mixture was cooling
in room temperature, observed by TLC to the point of com-
pletion. After recrystallization of the mixture with ethanol,
the residual solution is removed by a vacuum rotary evapo-
rator. The products were purified by column chromatogra-
phy to give the compound 1–13.
1,3,5-Triphenyl-1,3,5-triazine 1.
Yield: 56.5%; mp:
ꢁ
150ꢀ151 C; Rf: 0.64 (TLC eluent; n-Hexane: E.A = 5:3);
Mass (70 eV), m/z (rel. int.%): 315.17 (100), 316.18 (22.7),
1
317.18 (2.5); H NMR (Chloroform‑d, 400 MHz): δ 7.21
(dd, J = 7.4 Hz and 3.4 Hz, 6H), 7.02 (d, J = 7.4 Hz, 6H),
6.86 (t, J = 7.4 Hz, 3H), 4.89 (s,6H); 13C NMR
(Chloroform‑d, 100 MHz): δ 148.45, 129.08, 120.032,
116.980, 67.13; Anal. Calcd. for C21H21N3: C, 79.97; H,
6.71; N, 13.32; Found: C, 80.01; H, 6.72; N,13.27.
1,3,5-Tri(thiazol-2-yl)-1,3,5-triazine 7. Yield: 72.3%; mp:
ꢁ
161ꢀ162 C; Rf: 0.54 (TLC eluent; n-Hexane: E.A = 5:3);
Mass (70 eV), m/z (rel. int.%): 336.03 (100), 338.02 (13.6),
1
337.03 (13.0); H NMR (dimethyl sulfoxide-d6, 400 MHz):
δ 7.05 (d, J = 7.4 Hz, 3H), 6.63 (d, J = 7.2 Hz, 3H), 3.3 2
(s, 6H); 13C NMR (dimethyl sulfoxide‑d6, 100 MHz): δ
155.02, 137.01, 112.38, 83.13; Anal. Calcd. for
C12H12N6S3: C, 42.84; H, 3.60; N, 24.98; S, 28.58;
Found: C, 42.82; H, 3.62; N, 24.97; S, 28.59.
1,3,5-Tribenzyl-1,3,5-triazine 8. Yield: 66.5%; mp: sticky
oil; Rf: 0.52 (TLC eluent; n-Hexane: E.A = 5:1); Mass
(70 eV), m/z (rel. int.%): 357.22 (100), 358.22 (26.0),
359.23 (2.7); 1H NMR (Chloroform‑d, 400 MHz): δ
7.30–7.20 (m, 15H), 3.65 (s, 6H), 2.16 (s, 6H); 13C NMR
(Chloroform‑d, 100 MHz): δ 140.32, 130.22, 125.21,
80.22, 58.21; Anal. Calcd. for C24H27N3: C, 80.63; H,
7.61; N, 11.75; Found: C, 80.61; H, 7.63; N, 11.75.
1,3,5-Tribenzyl-1,3,5-triazine 2.
Yield: 60.5%; mp:
ꢁ
159ꢀ160 C; Rf: 0.58 (TLC eluent; n-Hexane: E.A = 5:1);
Mass (70 eV), m/z (rel. int.%): 402.55(100), 403.26(26.0),
1
404.26(3.2); H NMR (Chloroform‑d, 400 MHz): δ 7.05
(d, J = 7.3 Hz, 6H), 6.95 (d, J = 8.8 Hz, 6H), 4.78 (s, 6H),
4.89 (s, 6H), 2.51–2.57 (m, 6H), 1.15–1.19 (m, 9H); 13C
NMR (Chloroform‑d, 100 MHz):
δ 150.43, 138.25,
130.42, 115.56, 82.12, 24.42, 15.52; Anal. Calcd. for
C27H33N3: C, 81.16; H, 8.32; N, 10.52; Found: C,
81.13; H, 8.34; N, 10.53.
1,3,5-Tris(6-methylpyꢁridin-2-yl)-1,3,5-triazine 3. Yield:
60.2%; mp: 121ꢀ122 C; Rf: 0.58 (TLC eluent; n-Hexane:
E.A = 5:4); Mass (70 eV), m/z (rel. int.%): 360.21 (100),
361.22 (22.5), 362.21 (3.4); 1H NMR (dimethyl
1,3,5-Tris(pyridin-3-ylmethyl)-1,3,5-triazine 9.
Yield:
72.6%; mp: sticky oil; Rf: 0.54 (TLC eluent; MeOH = 1);
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Cytotoxic Activity of Hexahydro-1,3,5-triazine Derivatives
KOREAN CHEMICAL SOCIETY
Mass (70 eV), m/z (rel. int.%): 360.21 (100), 361.21 (22.7),
362.21 (2.5); H NMR (Chloroform‑d, 400 MHz): δ 8.50
reaction is the reflux temperature of ethanol, the solvent
of the reaction. After completion of reaction
(as monitored by TLC), reaction solvent was removed
by vacuum rotary evaporator. The reaction mixture was
purified by column chromatography to give the com-
pound 1, 5–7. We tried different reactions other than the
four suggested reactions, but they did not proceed and
we no longer performed different reactions under differ-
ent reaction conditions. The result of these reactions
was not satisfied and shown in Table 1.
1
(s, 3H), 8.47 (d, J = 7.2 Hz, 3H), 7.62 (d, J = 7.4 Hz, 3H),
7.21 (dd, J = 7.2 Hz and 3.4 Hz, 3H), 3.64 (s, 6H), 2.16(s,
6H); 13C NMR (Chloroform‑d, 100 MHz): δ 152.12,
150.32, 148.45, 140.12, 124.03, 80.12, 58.21; Anal. Calcd.
for C21H24N6: C, 69.97; H, 6.71; N, 23.32; Found C,
69.96; H, 6.73; N, 23.31.
1,10,100-((1,3,5-Triazine-1,3,5-triyl)tris(propane-3,1-diyl))
tris(pyrrolidin-2-one) 10. Yield: 30.5%; mp: sticky oil;
Rf: 0.53 (TLC eluent; MeOH: E.A = 3:5); Mass (70 eV),
m/z (rel. int.%): 462.33 (100), 463.34 (26.0), 464.34 (2.7);
1H NMR (dimethyl sulfoxide‑d6, 400 MHz): δ 5.75 (s,
6H), 3.18–3.38 (m, 18H), 1.89–2.21 (m, 18H); 13C NMR
(dimethyl sulfoxide‑d6, 100 MHz): δ 158.45, 80.32, 55.45,
52.81, 50.22, 28.52, 25.52, 18.43; Anal. Calcd. for
C24H42N6O3: C, 62.31; H, 9.15; N, 18.17; O, 10.37;
Found: C, 62.28; H, 9.18; N, 18.15; O, 10.39.
MTT assay. Cytotoxicity is the effect of being toxic to
cells caused by toxic agent. Cytotoxicity activity was moni-
tored using 3-(4,5-dimethyl-2-thiazolil)-2, 5-diphenyl-2H-
tetrazolium bromide (MTT). Cancer cells (T98G) and Nuff
(human newborn foreskin fibroblast) were purchased from
the Korean Cell Line Bank on March 14, 2015, and cyto-
toxicity tests were conducted in the Department of Genet-
ics, Dong-A University School of Medicine. Cell
cytotoxicity of the compounds were adjusted by MTT.
First, compounds were dissolved in dimethyl sulfoxide.
Then, compounds were dissolved in the free-medium
immediately before treatment with the cells, followed by
70% sonication, and the material was filtered through a
0.45 μM filter (Millipore, Billerica, MA, USA). All cells
3,30,300-((1,3,5-Triazine-1,3,5-triyl)tris(azanediyl))tris
(benzo[d]isothiazole 1,1-dioxide) 11. Yield: 10.5%; mp:
sticky oil; Rf: 0.53 (TLC eluent; n-Hexane: E.A = 5:3);
Mass (70 eV), m/z (rel. int.%): 633.12 (100), 634.13 (26.3),
635.12 (12.7); 1H NMR (dimethyl sulfoxide‑d6, 400 MHz):
δ 7.32–7.35 (m, 7H), 7.22–7.26 (m, 11H), 5.05 (s, 6H);
13C NMR (dimethyl sulfoxide‑d6, 100 MHz): δ 182.77,
137.34, 128.45, 128.41, 127.14, 127.11, 127.05, 75.10;
Anal. Calcd. for C24H21N9O6S3: C, 45.93; H, 3.37; N,
20.07; O, 15.29; S, 15.31; Found: C, 45.93; H, 3.38; N,
20.06; O, 15.30; S, 15.30.
were seeded into 96-well plates at
a density of
1 × 104 cells/well prior to treatment. Next day, medium
was replaced with complement medium containing various
ꢁ
concentration compounds at 37 C in a humidified atmo-
sphere of CO2 incubator. After 24 h, media replaced the
100 μL of thiazolyl blue tetrazolium solution (Sigma
Chemicals, St. Louis, MO, USA) at 5 mg/mL in 1X phꢁos-
phate buffered saline and cells incubated for 4 h at 37 C.
Thiazolyl blue tetrazolium–formazan was dissolved in
100 μL dimethyl sulfoxide, and the absorbance at 550 nm
was adjusted using a microplate reader. The cell viability
(%) was calculated as (O.D.550 of treated cells/O.D.550 of
nontreated cells) × 100.
First, when the synthesized compound 1–9 was adminis-
tered to normal cells (Nuff) at 25 and 50 μM concentrations
and analyzed by MTT analysis, there was almost no toxic-
ity to normal cells. (Figure 1) So then, the synthesized com-
pound 1–9 were administered to Cancer cell (human
(2S,20S,200S)-2,20,200-(1,3,5-triazine-1,3,5-triyl)tris
(3-mercaptopropanoic acid) 12. Yield: 20.2%; mp: sticky
oil; Rf: 0.43 (TLC eluent; MeOH: E.A = 5:2); Mass (70 eV),
m/z (rel. int.%): 399.06 (100), 401.06 (13.3), 400.06 (12.9);
1H NMR (dimethyl sulfoxide‑d6, 400 MHz): δ 4.64–4.79
(m, 7H), 4.28 (d, J = 7.4 Hz, 6H), 3.29–3.40 m, 18H); 13C
NMR (dimethyl sulfoxide‑d6, 100 MHz): δ 169.78, 62.30,
48.45, 32.07; Anal. Calcd. for C12H21N3O6S3: C, 36.08; H,
5.30; N, 10.52; O, 24.02; S, 24.08; Found: C, 36.06; H,
5.32; N, 10.52; O, 24.03; S, 24.07.
1,3,5-Triglucosyl-1,3,5-triazine 13. Yield: 30.5%; mp:
sticky oil; Rf: 0.53 (TLC eluent; MeOH: E.A = 3:5); Mass
(70 eV), m/z (rel. int.%): 573.24 (100), 574.24 (22.7),
575.24 (3.1); H NMR (dimethyl sulfoxide‑d6, 400 MHz): δ
4.95 (d, J = 7.4 Hz, 5H), 4.82 (s, 6H), 3.32–3.62 (m, 15H),
3.06–3.17 (m, 6H); 13C NMR (dimethyl sulfoxide‑d6,
100 MHz): δ 92.67, 89.08, 76.12, 71.96, 71.53, 69.63,
69.53, 60.25, 56.67, 54.23, 46.63; Anal. Calcd. for
C21H39N3O15: C, 43.98; H, 6.85; N, 7.33; O, 41.84;
Found: C, 43.95; H, 6.88; N, 7.35; O, 41.82.
Table 1. Yields of 1,3,5-triazacyclohexanes by sonochemistry.
Yield
Yield
Yield
Compounds (%)a Compounds (%)a Compounds (%)a
1
2
3
4
5
46.5
-
-
-
6
7
8
9
10
55.8
52.6
-
-
-
11
12
13
-
-
-
b
General
procedure
for
the
synthesis
of
1,3,5-hexahydrotriazine derivatives by ultrasound
assisted method. A mixture of amine (0.03 mol) and form-
aldehyde (0.11 mL, 0.03 mol) was dissolved in 25 mL of
ethanol in a 100 mL one necked round-bottomed flask intro-
duced into sonicator. The reaction mixture was stirred with
gentle refluxing for 30 min. The temperature of condensation
46.2
a Isolated yield.
b No reaction; after reaction, TLC was unable to determine the
product’s spot.
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Cytotoxic Activity of Hexahydro-1,3,5-triazine Derivatives
KOREAN CHEMICAL SOCIETY
Table 2. Summary of cytotoxic activity of the compounds against
T98G cell line at various concentrations.
Cytotoxic activity (%)
Concentration (μM)
Compounds
12.5 μM
25 μM
50 μM
100 μM
1
2
3
4
5
6
7
8
9
60.3
30.8
58.1
74.5
85.0
88.8
53.3
64.9
79.7
61.4
36.0
67.7
83.0
94.4
89.0
57.0
80.2
87.7
64.5
45.1
68.5
94.6
94.3
89.2
60.0
84.1
90.4
75.7
55.9
79.8
94.1
94.1
90.1
63.5
68.2
94.6
Figure 1. Cytotoxic activity of the 1,3,5-triazacyclohexane deriva-
tive compounds against Normal cell line at various concentration.
glioblastoma, T98G) and analyzed by MTT assay Experi-
mental results shown in Figure 2 and Table 2. Human glio-
blastoma (T98G) showed the following activities. At a low
concentration of 12.5 μM, compound 5,6 exhibited greater
than 80% cytotoxicity, compound 2 showed a low cytotox-
icity of 30.8%, Also the same concentrates, the values for
T98G were 60.3% for compound 1, 58.1% for compound
3, 74.5% for compound 4, 53.3% for compound 7, 64.9%
for compound 8, 79.7% for compound 9. At a concentra-
tion of 25 μM, compound 4,5,6,8,9 exhibited greater than
80% cytotoxicity, 83.0% for compound 4, 94.4% for com-
pound 5, 89.0% for compound 6, 80.2% for compound 8,
87.7% for compound 9. Compound 2 showed a low cyto-
toxicity as well. Compound 2 showed of 36.0%. Also the
same concentrates, the values were 61.4% for compound 1,
67.7% for compound 3, 57.0% for compound 7. At a con-
centration of 50 μM cytotoxicity graphs look like 25 μM,
the values were 64.5% for compound 1, 45.1% for com-
pound 2, 68.5% for compound 3, 94.6% for compound 4,
94.3% for compound 5, 89.2% for compound 6, 60.0% for
compound 7, 84.1% for compound 8, and 90.4% for com-
pound 9. In particular, at a concentration of 100 μM, shows
that greatest cytotoxicity is the compound 9 (94.6%),
followed by compound 4,5 (94.1%). Also the same concen-
trates, the values were 75.7% for compound 1, 55.9% for
compound 2, 79.8% for compound 3, 90.1% for compound
6, 63.5% for compound 7, 68.2% for compound 8. For the
IC50 values, compound 1 was 6.30 μM, compound 2 was
67.61 μM, compound 3 was 7.08 μM, compound 4 was
5.12 μM, compound 7 was 7.59 μM, compound 9 was
2.82 μM, and compound 5, 6, 8’s IC50 values could not be
unpredictable (Table 3; Supporting Information).
Results and Discussion
The 1,3,5-hexahydrotriazines are the subject of several
structure studies considering their use in the industrial and
pharmacological chemistry. The symmetrically substituted
triazines product number 1–13, were synthesized from the
condensation reaction of various amines and formaldehyde.
These compounds are stable at room temperature and ade-
quate yield with a trans parent color. This heterocyclic
compound results from the assembly of three molecules of
amine and three molecules of formaldehyde; its striking
feature is that each atom of this six-membered ring is origi-
nated from six single different molecules, representing a
formal 1 + 1 + 1 + 1 + 1 + 1; cyclocondensation. The
obtained yield is satisfactory in spite of the number of
essential steps involved in the significant transformation
and of the ring size, usually unfavorable for entropic
T98G (giloblastoma)
100
Table 3. IC50 of the 1,3,5-triazacyclohexane derivative
compounds against T98G cell line at various concentration.
80
60
40
20
0
Compounds
Cell lines
T98G
IC50 values (μM)a
1
2
3
4
5
6
7
8
9
6.30
67.61
7.08
5.12
>100
>100
7.59
>100
2.82
12.5 (μM)
25 (μM)
50 (μM)
100 (μM)
Figure 2. Cytotoxic activity of the 1,3,5-triazacyclohexane deriva-
tive compounds against T98G cell line at various concentration.
a p < 0.0001.
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Cytotoxic Activity of Hexahydro-1,3,5-triazine Derivatives
KOREAN CHEMICAL SOCIETY
Table 4. Structures and yields of 1,3,5-triazacyclohexanes by thermal reaction.
Compounds
Amines
Structures
Yield (%)a
56.5
1
2
60.5
3
4
60.2
66.4
5
66.8
6
7
65.6
72.3
8
66.5 24
(continued overleaf )
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Cytotoxic Activity of Hexahydro-1,3,5-triazine Derivatives
KOREAN CHEMICAL SOCIETY
Table 4 (continued)
Compounds
Amines
Structures
Yield (%)a
72.6 25
9
10
30.5
11
10.5
12
13
20.2
30.5
a Isolated yield.
reasons. The reaction mechanism about formation of
hexahydrotriazines is that the first reaction step begins with
the lone-pair electron of amine attacking the carbon of
formaldehyde. Subsequently, through a series of reversible
reaction processes, intermediate imine is formed. It can then
be inferred from the reaction steps of amine, imine, and
formaldehyde. Finally, hexahydrotriazine as a final product
is produced. In fact, hexahydrotriazines synthesized by
amines with various functional groups are expected to have
high biological activity. Evidence that hexahydrotriazines
were synthesized is as follows (Table 4). In this study,
various amines were used to react with formaldehyde and
have physiological activity. In bioactivity, to investigate the
effect of compounds in T98G, the cells were treated with
designated concentrations of compounds for 24 h. The cyto-
toxicity of compound 1–9 was measured by MTT assays
(Figure 2, Table 2). As a result, compound 1–9 reduced the
viability of T98G cells in a concentration-dependent manner.
At a concentration of 100 μM, compound 9 exhibited the
greatest cytotoxicity at 94.6%. The reason is that compound
9 is C5H5N that has a resonance hybrid structure and
exhibits aromaticity. It is considered to be active because it
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Cytotoxic Activity of Hexahydro-1,3,5-triazine Derivatives
KOREAN CHEMICAL SOCIETY
Supporting Information. Additional supporting informa-
tion may be found online in the Supporting Information
section at the end of the article.
References
Scheme 1. Structures of 1,5,3,7-diazadiphosphosines23 and
hexahydrotriazines (1–13)
1. G. Kvesitadze, B. Meskhi, G. Khatisashvili, BAK 2018, 1, 53.
2. G. Adamia, M. Ghoghoberidze, D. Graves, G. Khatisashvili,
Ecotoxicol. Environ. Saf. 2006, 64, 136.
has a structure that is a raw material for pyridine derivatives
as mainly medicines and pesticides. Synthetic compound
3 has pyridine ring directly attached to the N atoms of the
triazine ring, but product compound 9 is a form where the
pyridine rings can be freely rotated by methylene group as
you can see. With this structural difference, there is a clear
difference in cytotoxicity activity. To solve this problem, we
will demonstrate this effect through a structure-activity rela-
tionship in the near future. We expressed the aforementioned
sentence based on this thought. Although it cannot be accu-
rately expressed, the high cytotoxicity activity (94.6%) of
product 9 can be described as the role of the N atom of pyri-
dine attached to a freely rotating methylene group. Synthesis
of hexahydrotriazine derivatives was performed by means of
the effect of ultrasound in forming acoustic cavitation in
reaction solution and resulting in the initiation or enhance-
ment of the chemical activity in the reaction solution. The
result of these reactions was not satisfied and shown in
Table 1. Ethanol is not a suitable solvent for this condensa-
tion reaction. However, the results of the first reaction of
sonochemistry were not good, so we stopped it, and we per-
formed cyclocondensation with ethanol, which melts the
starting material well, as a solvent (Scheme 1).
3. B. Van Aken, J. Yoon, L. Just, L. Schnoor, Environ. Sci.
Technol. 2004, 38, 4572.
4. G. Kvesitadze, G. Khatisashvili, T. Sadunishvili, Naturforsch.
C. J. Biosci. 2005, 60, 340.
5. H. Khamees, Arch. Pharm. Res. 1990, 13, 19.
6. S. Mikhaylichenko, O. Kvak, S. Dalili, Chem. Eur. J. 2010,
1, 302.
7. L. Maier, M. Smith, J. Phosphorous Sulfur. 1980, 8, 67.
8. L. Xu, S. Retttig, C. Orvig, Inorg. Chem. 2001, 40, 3734.
9. T. Varga, Synth. Commun. 1997, 27, 2899.
10. (a) S. Aime, M. Botta, G. Crich, B. Giovenzana, R. Pagliarin,
M. Piccinini, J. Biol. Inorg. Chem. 1997, 2, 470. (b) S. Aime,
M. Botta, L. Frullano, S. Geninatti, G. Giovenzana, R.
Pagliarin, M. Sisti, J. Med. Chem. 2000, 43, 4017.
11. B. Song, T. Storr, S. Liu, C. Orvig, Inorg. Chem. 2002,
41, 685.
12. S. Aime, C. Cavallotti, E. Gianolio, B. Giovenzana, G.
Palmisano, M. Sisti, Tetrahedron Lett. 2002, 43, 8387.
13. N. Seiler, B. Knödgen, Int. J. Biochem. Mol. Biol. 1983,
15, 907.
14. W. Whitehead, J. Traverso, F. Bell, W. Willard, J. Med.
Chem. 1967, 10, 844.
15. (a) B. Sameem, F. Khan, K. Niaz, Nonvitamin and
Nonmineral Nutritional Supplements Book, Tehran, Iran
2019. Chapter 2.6, p. 53. (b) Y. Samuni, S. Goldstein, M.
Dean, M. Berk, Biochim. Biophys. Acta 2013, 1830, 1.
16. K. Khwaldia, Nonvitamin and Nonmineral Nutritional Sup-
plements Book, Tunisia 2019, Chapter 2.3, p. 27.
17. T. Mason, Advances in Sonochemistry, JAI Press,
London, 1990.
18. K. Shibata, I. Katsuyama, M. Matsui, H. Muramatsu, Bull.
Chem. Soc. Jpn. 1990, 63, 3710.
19. N. Nandurkar, Synthesis Organic Sonochemistry, Plenum
Press, New York, 1998.
20. S. Suslick, Sonochemistry 1990, 247, 1439.
21. S. Suslick, J. Flannigan, Annu. Rev. Phys. Chem. 2008,
59, 659.
22. M. Salman, K. Ansari, J. Haque, V. Srivastava, M. Quraishi,
M. Mazumder, J. Heterocycl. Chem. 2020, 57, 2157.
23. S. M. Bae, Y. Chen, I. S. Jeong, J. H. Song, D. I. Jung, Bull.
Kor. Chem. Soc. 2020, 42, 60.
24. M. Reis, V. Campos, A. Resende, F. Silva, V. Ferreira,
Beilstein J. Org. Chem. 2015, 11, 1235.
25. T. Naoyuki, T. Kazuyuki, S. Shinzo. Method for Selectively
Producing Primary Amine Compound. WO2007069685A1,
June 21, 2007.
Conclusion
We think that there is an immense need for new compounds
with new mode of drug action, for treatment of human dis-
ease. In conclusion, hexahydrotriazine derivatives (1–13)
were obtained in 10–72% yields by using a one-pot reaction
of various amines and formaldehyde. The preparation is very
easy, the molecule being assembled in one single reaction
from readily available and cheap starting materials. The most
outstanding result is that compound 9 exhibited the greatest
cytotoxic activity (94.6%) at a concentration of 100 μM.
Generally highly active pesticides and pharmaceuticals have
pyridine ring, compound 9 with pyridine ring has a resonance
hybrid structure and exhibits aromaticity. The need for new
pharmaceutical continues to be a still standing challenge.
Acknowledgment.
This work was supported by the
National Research Foundation of Korea(NRF) grant funded
by the Korea government (MSIT) (No. 2017RICIB5074137).
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