Biguanide-based synthesis of 1,3,5-triazine derivatives with anticancer activity and 1,3,5-triazine incorporated calcium citrate nanoparticles

Article abstract of DOI:10.3390/molecules26041028

Twelve derivatives of biguanide-derived 1,3,5-triazines, a promising class of anticancer agent, were synthesised and evaluated for their anticancer activity against two colorectal cancer cell lines—HCT116 and SW620. 2c and 3c which are the derivatives con

Full text of DOI:10.3390/molecules26041028

molecules
Article
Biguanide-Based Synthesis of 1,3,5-Triazine Derivatives with
Anticancer Activity and 1,3,5-Triazine Incorporated Calcium
Citrate Nanoparticles
Monnaya Chalermnon 1, Sarocha Cherdchom 2,3 , Amornpun Sereemaspun 4, Rojrit Rojanathanes 5
and Tanatorn Khotavivattana 6,
*
1
2
3
4
5
6
Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand;
monnayach@yahoo.com
Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Chulalongkorn University,
Phayathai Road, Wangmai, Patumwan, Bangkok 10330, Thailand; sarocha.cherdchom@gmail.com
NanoMedicine Research Unit, Department of Anatomy, Faculty of Medicine, Chulalongkorn University,
Rama 4 Road, Patumwan, Bangkok 10330, Thailand
Chula Medical Innovation Centre (CMIC), Nanomedicine Research Unit, Department of Anatomy,
Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand; amornpun.s@gmail.com
Centre of Excellence in Materials and Bio-Interfaces, Faculty of Science, Chulalongkorn University,
Bangkok 10330, Thailand; rojrit@hotmail.com
Centre of Excellence in Natural Products Chemistry, Department of Chemistry, Faculty of Science,
Chulalongkorn University, Bangkok 10330, Thailand
*
Correspondence: tanatorn.k@chula.ac.th; Tel.: +66-2-218-7621
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Twelve derivatives of biguanide-derived 1,3,5-triazines, a promising class of anticancer
agent, were synthesised and evaluated for their anticancer activity against two colorectal cancer
cell lines—HCT116 and SW620. 2c and 3c which are the derivatives containing o-hydroxyphenyl
Citation: Chalermnon, M.;
Cherdchom, S.; Sereemaspun, A.;
Rojanathanes, R.; Khotavivattana, T.
Biguanide-Based Synthesis of
1,3,5-Triazine Derivatives with
Anticancer Activity and 1,3,5-Triazine
Incorporated Calcium Citrate
Nanoparticles. Molecules 2021, 26,
1028. https://doi.org/10.3390/
molecules26041028
substituents exhibited the highest activity with IC50 against both cell lines in the range of 20–27 µM,
which is comparable to the IC50 of cisplatin reference. Moreover, the potential use of the calcium
citrate nanoparticles (CaCit NPs) as a platform for drug delivery system was studied on a selected
1,3,5-triazine derivative 2a. Condition optimisation revealed that the source of citrate ions and
reaction time significantly influence the morphology, size and %drug loading of the particles. With
the optimised conditions, “CaCit-2a NPs” were successfully synthesised with the size of 148 ± 23 nm
and %drug loading of up to 16.3%. Furthermore, it was found that the release of 2a from the
synthesised CaCit-2a NPs is pH-responsive, and 2a could be control released under the acidic cancer
environment. The knowledge from this study is perceptive for further development of the 1,3,5-
triazine-based anticancer drugs and provide the platform for the incorporation of other drugs in the
CaCit NPs in the future.
Academic Editor: Alessia Irerra
Received: 25 January 2021
Accepted: 12 February 2021
Published: 15 February 2021
Keywords: 1,3,5-triazine; anticancer; calcium citrate; nanoparticles
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Cancer is a devastating disease with an increasing number of diagnosis and death
every year. In 2019, there were more than 1 million new cases, especially prostate, breast,
lungs and colorectal cancer [
methods despite several limitation of the currently available drugs including poor drug
selectivity, potential drug resistance and possibilities for recurrences [ ]. In order to mitigate
these limitations, the development of a new anticancer agent with an efficient delivery
system is necessary [ ]. The 1,3,5-triazine scaffold has been reported to possess many
biological activities especially anticancer activity [ ]. A variety of synthetic routes exists
for the 1,3,5-triazine derivatives, for example, three-component methods [ ] or reactions
between biguanides with alcohol [ ], amide [ ] or carboxylic acid derivatives [10 11]. As
1]. Chemotherapy is one of the most frequently used treatment
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
2
3
4
5,6
7,
8
9
,
Molecules 2021, 26, 1028. https://doi.org/10.3390/molecules26041028
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1028
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seen in many literatures, a biguanide like metformin has been frequently employed as
the starting material [8,10,12,13]. Metformin-derived 1,3,5-triazine derivative HL010183
was shown to be 100 times more cytotoxic than metformin with IC50 of 0.28 mM for both
MDA-MB-231 and Hs578T cell lines [14]. 1,3,5-Triazine synthesised from various other
aryl-biguanides were also reported as Rad6 ubiquitin-conjugating enzymes inhibitors and
they exhibited anticancer activity in the low micromolar range [15]. It is therefore very
intriguing to explore new structural modifications of 1,3,5-triazine derivatives to further
enhance the anticancer activity of this scaffold. However, one limitation of 1,3,5-triazine is
its ability to reach the target site which would affect its anticancer activity [4]. In 2016, one
example of the drug delivery system (DDS) was reported by coupling the 1,3,5-triazine
derivatives with bovine serum albumin in order to solve this issue [4].
DDS is a safe and efficient way to deliver drug while enhancing the therapeutic
efficiency and reducing side effects. Nanoparticles acting as drug carriers could benefit
from the enhanced permeability and retention (EPR) effect because cancer cells have leaky
vasculatures and an impaired lymphatic system [16]. One influential aspect of the EPR
effect is particles sized 30–300 nm, which has been reported as a suitable range. Particle
size outside of this range are prone to rapid clearance by the mononuclear phagocyte
system [17,18]. Additionally, the leakiness of tumour vessel could dictate the selectivity
of nanoparticles towards cancer cells. Another prominent characteristic of cancer cells is
their relatively lower pH environment compared to normal cells, therefore pH-responsive
nanoparticles would further reinforce the usefulness of nanoparticles for drug delivery
system [19]. Recently, our group has shown a potential use of calcium citrate nanoparticles,
a biocompatible and biodegradable material [20,21], as a promising platform for DDS.
Vancomycin, an antibiotic drug for bone spacer application, was successfully encapsulated
in the calcium citrate particles (VAN-CC) with a size of around 500 nm and an encapsulation
efficiency of 28% [22]. In another work, FITC, a fluorescent agent, was encapsulated
and slowly released from calcium citrate nanoparticles as experimented with human
keratinocytes [23].
Herein, we report the synthesis and anticancer activities of biguanide-derived 1,3,5-
triazine derivatives bearing various substituents on the triazine scaffold. Furthermore, the
DDS based on the incorporation of a selected 1,3,5-triazine derivative into calcium citrate
nanoparticles was investigated (Figure 1). We hypothesised that the nanoparticles would
benefit from the EPR effect as well as be pH-responsive and control released drug under
the acidic cancer environment.
Figure 1. Synthesis and incorporation of biguanide-derived 1,3,5-triazine derivatives in calcium
citrate nanoparticles (this work).

Molecules 2021, 26, 1028
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2. Results and Discussions
2.1. Chemistry
1,3,5-Triazine derivatives of metformin (2a
–
2f) and phenylbiguanide (3a
–
3f) were syn-
thesised by the condensation between a biguanide and an ester. Metformin hydrochloride
was used as purchased, but phenylbiguanide hydrochloride (1a) was prepared according
to the previously reported method using aniline and dicyandiamide in excellent yield [24].
The five esters included in this work were isopropyl palmitate (R3 = C15H31), methyl ben-
zoate (R3 = Ph), methyl salicylate (R3 = Ph-o-OH), methyl cinnamate (R3 = C=CH-Ph) and
diethyl oxalate (R3 = COOC2H5). Initially, a one-pot reaction containing a biguanide hy-
drochloride, an ester and sodium methoxide (NaOMe) in methanol was used [10
After reaction optimisation by adjusting the ratio of reagents and reaction time, 2a
,
15
,
,
24
,
,
25].
2d
2b
,
2f and 3a were obtained in moderate yields (Figure 2, Pathway I). However, the synthetic
yields of other derivatives were very low. Carboxylic by-products of the ester starting
materials were detected even though anhydrous solvent and inert atmosphere were imple-
mented. We hypothesised that this could be due to the presence of base and water in the
reaction mixture simultaneously. Therefore, the method was slightly adjusted by starting
with the neutralisation step to generate biguanide in the free base form [10
the biguanide was isolated and reacted with an ester without the use of the additional base
(Figure 2, Pathway II). As a result, 2c 2e and 3b 3f were successfully synthesised with
low to moderate yields. In total, 12 1,3,5-triazine derivatives were obtained, of which four
compounds were novel (2e 2f 3d 3f); three compounds have been reported, but not fully
characterised (2c 3a 3c); and five compounds were known (2a 2b 2d 3b 3e). The struc-
,15,24,25]. Then
,
–
,
,
,
,
,
,
,
,
,
tures of all 1,3,5-triazine derivatives were elucidated by 1H and 13C-NMR spectroscopy. In
addition, the novel and not fully characterised derivatives were further characterised for
IR spectra, mass spectra and melting points as the information has yet been reported (see
Supplementary Materials).
[Pathway I]
R3
N
R3
N
O
R4
NH NH
R1
R3
O
HO
N
N
N
N
and
NH2
N
NH NH2
H3C
R3 =
i
N
N
NH2
C
15H31
R2
H
.HCl
CH3
2c
: 47%
3c: 39%
2b
: 52%
3b
: 7%
2a
: 64%
3a
: 42%
Metformin HCl: R1, R2 = CH3
1a: R1 = Ph, R2 = H
3a-3f
2a-2f
O
O
OCH3
OH
NH NH
NH NH2
ii
iii
O
R1
2d
: quant.
3d
: 17%
2e
3e
2f
: 65%
: quant.
: 7%
: 40%
N
R2
3f
R4
R3
O
[Pathway II]
Figure 2. Schematic diagram of the reaction of biguanides with several esters. Reagents and conditions: (
i) Ester (1–3 equiv.),
NaOMe (1–5 equiv.), anh. MeOH, reflux; (ii) NaOMe (1 equiv.), anh. MeOH, rt.; (iii) ester (1-excess equiv.), anh. MeOH.
2.2. In Vitro Anticancer Activities
The in vitro anticancer activities of 2a–2f and 3a–3f were evaluated against two colon
cancer cell lines to assess the influence of synthesised 1,3,5-triazine derivatives on cellular
viability (Table 1). Anticancer studies of 1,3,5-triazine derivatives is extensive, but only a
few studies had performed their evaluations with HCT116 and SW620 cell lines [26,27].
Cisplatin was used as a positive control and the result was consistent with reported lit-
erature value [28]. Initially, a preliminary screening of the cytotoxicity was performed
on all twelve 1,3,5-triazine derivatives at a specific concentration of 100 µM. All of the
synthesised 1,3,5-triazine derivatives were active at varied abilities, and some exhibited
comparable or higher cytotoxicity than cisplatin for both cell lines. The comparison of the
cytotoxicity of these compounds at 100 µM showed that the hydrophobic substituents re-

Molecules 2021, 26, 1028
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sulted in higher cytotoxicity than hydrophilic groups as reflected in the two most cytotoxic
compounds: 2a with the long alkyl substituent (%cytotoxicity = 96.02
67.43 1.82 (HCT116)) and 3d with the aryl group (87.47 1.54 (SW620) and 92.27
(HCT116)). In contrast, the %cytotoxicity of 2b with phenyl substituent, 2e 3e with the
ester substituent and 2f 3f with the carboxylic substituents were very low in the range
of 5–20%. Overall, the 1,3,5-triazine that are derived from the phenylbiguanide (3a 3f
showed significantly higher cytotoxicity compared with ones derived from the metformin
2a 2f). These results established a relationship between the increased hydrophobicity with
±
0.06 (SW620) and
±
±
±
0.04
/
/
–
)
(
–
enhanced cytotoxicity, but the reason for this relation do need further investigation. From
the %cytotoxicity values, 2a was selected for the further study on the drug incorporation
using calcium citrate nanoparticles.
Table 1. Anticancer activities of synthesised 1,3,5-triazine derivatives.
%Cytotoxicity at 100 µM 1
IC50 (µM) 2
1,3,5-Triazine Derivatives
SW620
HCT116
SW620
HCT116
3
3
4
4
1a
n.a.
n.a.
n.d.
n.d.
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
96.02 ± 0.06
15.86 ± 1.91
53.85 ± 1.40
27.39 ± 4.16
11.41 ± 3.52
5.64 ± 1.51
54.96 ± 1.46
52.60 ± 4.06
56.85 ± 3.18
87.47 ± 1.54
12.14 ± 3.40
15.36 ± 1.15
62.14 ± 5.05
67.43 ± 1.82
19.23 ± 5.94
50.77 ± 1.83
26.52 ± 3.77
7.16 ± 2.11
11.59 ± 4.03
66.56 ± 2.58
54.42 ± 8.48
65.46 ± 1.07
92.27 ± 0.04
5.54 ± 3.65
14.22 ± 2.81
78.20 ± 0.96
59.13 ± 1.46
59.35 ± 1.66
n.d.
n.d.
25.25 ± 1.12
26.29 ± 1.07
4
4
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4
4
4
4
54.55 ± 1.09
38.37 ± 1.05
22.80 ± 1.06
54.10 ± 1.05
59.17 ± 1.48
26.92 ± 1.31
20.79 ± 1.07
55.02 ± 1.04
4
4
n.d.
n.d.
n.d.
n.d.
4
4
Cisplatin
31.67 ± 1.13
19.18 ± 1.06
1
Results expressed in percentage taken as a mean of triplicates
in mol/L ( M), taken as a mean value of triplicates
Not determined.
±
standard deviation (SD). 2 Results expressed
µ
µ
±
standard deviation (SD). 3 Not active at the specified.
4
Six compounds with high %cytotoxicity were selected for further evaluation by vary-
ing the concentration between 0–100 M to calculate the 50% cell-growth inhibition (IC50)
values. (see Supplementary Materials) The most potent anticancer agents were 2c and
3c, both having the o-hydroxyphenyl substituent with the IC50 of 25.25 1.12 M and
22.80 ± 1.06 µM for SW620 cell line, and at 26.29 1.07 M and 20.79 1.07 M for
HCT116 cell line, respectively. These IC50 values of 2c 3c were comparable and slightly
µ
±
µ
±
µ
±
µ
/
better than the reference drug, cisplatin. Even though the synthesised compounds are yet
novel, the activity of 2c or 3c compared to the previously reported 1,3,5-triazine deriva-
tives with alike substituents and size, for example, HL010183 or 4-amino-6-(arylamino)-N-
phenyl-1,3,5-triazine-2-carbohydrazides, could be considered as decent competitors [14,29].
2.3. 1,3,5-Triazine Incorporated Calcium Citrate Nanoparticles
Despite the successful drug encapsulation in calcium citrate nanoparticles [23], the
method was not applicable for the incorporation of 2a. This was due to the use of water
as the only solvent in the system, leading to poor solubility of triazine drugs. Therefore,
we attempted to optimise the protocol for the preparation of 1,3,5-triazines-incorporated
calcium citrate nanoparticles (CaCit-triazine NPs) using EtOH-water as a binary solvent
system, adjusted from the synthesis of calcium carbonate particles previously reported [30].
There are many methods to prepare calcium citrate particles, but the two most common
reagents are CaCl2 and sodium citrate [21,23,31]. In our first attempt, a solution of CaCl2
in EtOH was slowly added to a solution of 2a and sodium citrate in EtOH-water under
vigorous stirring at room temperature. Unfortunately, the obtained product was large

Molecules 2021, 26, 1028
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precipitate particles of 2a as confirmed by SEM and FTIR, and the desired CaCit-triazine
NPs was not observed, since there was no IR signal characteristic to calcium citrate, i.e.,
3400 cm−1 O-H stretching or peaks in the carbonyl group stretching region (Figure 3c).
This was due to the basicity of 2a and sodium citrate [32]. In alkali condition, calcium ions
have more affinity towards hydroxide than citrate ions, thus allowing the possibility to
form calcium hydroxide instead of calcium citrate precipitate [33]. Alternatively, calcium
citrate could be prepared via acid-base reaction between citric acid and calcium hydroxide
or calcium carbonate [34]. We decided to generate citrate ions in situ from the reaction
between citric acid and NaOH preventing the formation of calcium hydroxide in elevated
pH. The pH of citric acid solution was adjusted to pH 6.0 using 1:2 mole ratio of citric acid
to NaOH resulting in citrate ions in the divalent form [35]. Both the selected pH value
and the divalent form of citrate ions have been reported to allow the formation of calcium
citrate [32]. The FTIR spectrum of product obtained from using citric acid with NaOH
exhibited characteristic peaks of both calcium citrate (3452, 1543, 1429 and 1081 cm−1
)
and 2a (2920, 2851, 1570, 817, 804 and 720 cm−1) as depicted in Figure 3d. Even though
citric acid with NaOH could perform as the source of citrate, the obtained particles were
aggregated and not nano-sized. Further reaction optimisation continued with the study of
the effect of reaction time.
Figure 3. FTIR spectra of (
a) calcium citrate nanoparticles, (b) 2a, (c) reaction using sodium citrate
and (d) reaction using citric acid with NaOH.
Reaction time significantly influence the morphology and size of particles. SEM im-
ages in Figure 4 show a general trend that particle grew larger with longer reaction time.
Iafisco et al. has reported a similar finding stating that longer reaction time caused a growth
on the c-axis of citrate-apatite particles resulting in long needle-like morphology [36]. Re-
gardless of the trend, nanoparticles were obtained at two reaction times, at the second
(Figure 4ii) and the eighth hour (Figure 4v). This complication on the nanostructure forma-
tion emerged from having both the crystallisation of calcium citrate and the absorption of
2a occurring as well as the presence of ethanol. The experiment was verified again with
the exact same reaction condition at 2, 5 and 8 h (Table 2). We suspect that the obtained
nanoparticles at 2 h was the kinetic product that undergo phase change and reassembled
into more thermodynamically favourable nanoparticles at 8 h. The phase change with
respect to time is observed in calcium carbonate, where the amorphous calcium carbonate
transforms into more stable vaterite then calcite over time [37]. The double nanoparticle
formations could also be attributed to the presence of ethanol in the reaction. Ethanol
was found to be able to stabilise vaterite phase of calcium carbonate preventing its rapid

Molecules 2021, 26, 1028
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transformation to calcite. We suppose that this could be the case where the prior nanoparti-
cles formation was maintained long enough to be observed by SEM [38]. The synthesised
nanoparticles obtained from condition A and condition C had size consistent with those
particles presented in Figure 4, but nanoparticles from condition A was slightly smaller
than condition C at around 130 nm and 150 nm, respectively (Table 2). For condition
B, the obtained particles were aggregated and plate-like with particle size over 1000 nm.
Dynamic light scattering results also showed that the particles were monodispersed for
both condition A and condition C, which the hydrodynamic diameter of nanoparticles
were found to be in parallel to the SEM size (see Supplementary Materials). However,
the values were larger due to the tendency of nanoparticles to agglomerate in aqueous
solvent [39]. As the size of nanoparticles synthesised were between 300–370 nm, they still
correspond to the desired size for nanoparticles to take advantages of the EPR effect.
Figure 4. SEM images of CaCit-triazine at various synthesis time between 1–24 h at 10,000
iii (4 h), iv (6 h), v (8 h), vi (12 h), vii (16 h), viii (20 h) and ix (24 h).
×
magnification; i (1 h), ii (2 h),
The extent of drug incorporation in calcium citrate nanoparticles from condition A
and condition C was calculated using CHNS elemental analysis. This method was chosen
because 2a was the only compound containing nitrogen, thus the percentage of nitrogen
directly indicated the amount of 2a. Condition B had the highest %drug loading at 17.8%,
followed by condition C and condition A at 16.3% and 9.6%, respectively. It is noteworthy
that despite the similarity of particle size for condition A and C, longer reaction time
resulted in a higher %drug loading. Thermal gravimetric analysis additionally validated
the success of drug incorporation. The TGA curves of calcium citrate nanoparticles, 2a and
particles obtained from condition A–C are illustrated in Figure 5. The decomposition of
calcium citrate typically occurs in three stages. The first weight loss between 50–170 ◦C
was caused by the release of exterior and interior water molecules [21,31]. The second stage
between 420–600 ◦C indicated the change of calcium citrate to calcium carbonate. Lastly,
the decomposition of calcium carbonate to calcium oxide occurred at temperature beyond

Molecules 2021, 26, 1028
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700 ◦C. For 2a, it was almost a one-step decomposition starting at 200 ◦C. TGA curves of
all obtained products (condition A–C) exhibited weight loss behaviours of both calcium
citrate and 2a. The weight loss between 50–150 ◦C attributed to release of moisture and
the decrease in weight after 200 ◦C indicated the decomposition of 2a. %weight loss for
condition A and C agreed with the %drug loading calculated by CHNS analysis method
at 13.5% and 15.6%, respectively. Overall, the optimised reaction condition to synthesise
CaCit-triazine NPs preferred citric acid with NaOH as the source of citrate ion with reaction
time of 8 h.
Table 2. Characterisation of 1,3,5-triazine incorporated calcium citrate nanoparticles.
Reaction Time
(h)
SEM Size
(nm)
Particle Size
(nm) [PDI]
Zeta Potential Drug Loading
Condition
(mV)
(%)
369.30 ± 13.44
9.6 1
17.8 1
16.3 1
13.5 2
A
B
2
5
8
134 ± 18.87
>1000
−8.36
n.d.
[0.464]
3
3
3
n.d.
n.d.
300.30 ± 12.42
15.6 2
C
148 ± 23.69
−7.71
[0.322]
1
2
%drug loading calculated by CHNS elemental analysis. %drug loading calculated by thermal gravimetric
3
analysis. Not determined.
Figure 5. TGA curves of calcium citrate (CaCit), 2a and Condition A–C.
2.4. In Vitro Drug Release
The in vitro drug release study revealed that CaCit-2a NPs (condition C) was pH-
responsive with controlled drug release behaviour over 48 h. The drug release performance
was studied using dialysis method with 30%(v/v) EtOH in PBS as the release medium to
accommodate the release of the hydrophobic 1,3,5-triazine derivative 2a [40]. Typically,
the physiological pH of blood and inside normal tissues is 7.4 and the pH of the tumour
environment is slightly lower at 6.5–6.8 due to the metabolic adaptation called ‘Warburg
effect’ where there is a higher rate of glycolytic metabolism producing an increasing
number of protons (H+) [19]. Moreover, the lysosome of cancer cells has pH around 4.5 [41].
Therefore, we carried out the drug release at three conditions (pH = 7.4, 5.0, and 3.0) to
study the effect of pH on the drug release. The general drug release trend illustrated that
a higher %cumulative drug release was found at lower pH (Figure 6). The difference in
drug release was attributed to the higher rate of calcium citrate decomposition in low
pH condition [42]. After 24 h, 48.8% and 30.0% of 2a was released for pH 5.0 and pH 7.4,
respectively. A two-fold higher drug release was observed for pH 3.0 at 81.6%. The release

Molecules 2021, 26, 1028
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of 2a was sustained for 48 h with the release of over half of the nanoparticles’ loaded
content at 63.9% for pH 5.0 and to a lesser extent of 41.9% for pH 7.4. It is probable that 2a
could still be released from CaCit-2a NPs even after 48 h.
Figure 6. Release profile of 1,3,5-triazine incorporated calcium citrate nanoparticles (Condition C) at
pH 3.0, 5.0 and 7.4.
3. Materials and Methods
3.1. Materials
Metformin hydrochloride and methyl cinnamate were purchased from Fluorochem
(Hadfield, Derbyshire, UK); dicyandiamide, aniline, diethyl oxalate, methyl salicylate and
sodium methoxide were purchased from TCI Chemical (Tokyo, Japan); isopropyl palmitate
was purchased from Sigma Aldrich (St. Louis, MO, USA); methyl benzoate was gifted by
the Department of Chemistry, Chulalongkorn University. Calcium chloride dihydrate and
trisodium citrate dihydrate were purchased from Merck (Darmstadt, Germany). Citric acid
anhydrous was purchased from Loba Chemie PVT. LTD (Mumbai, India). All of solvents
used were distilled or analytical grade.
HCT116 (Human colorectal carcinoma) and SW620 (Human colorectal adenocarci-
noma) cells were obtained from Nanomedicine RU, Faculty of Medicine, Chulalongkorn
University. DMEM (Dulbecco’s Modified Eagle Medium), RPMI, fetal bovine serum (FBS),
antibiotics, trypsin-EDTA, PBS (Phosphate Buffered Saline) (1X, pH 7.4) and Prestoblue
™
cell viability reagent were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Cisplatin was purchased from Sigma Aldrich (St. Louis, MO, USA) and dialysis bag Spec-
tra/Por membranes (MWCO = 12–14 kDa) were purchased from Spectrum Laboratories,
Inc. (Rancho Dominguez, CA, USA).
3.2. Methods
3.2.1. Chemical Synthesis
The protocol for the synthesis of 2a
reaction reported in the literature with slight adjustment of the ratio of the reagents and
reaction time [10 15]. 2c 3b 3c 3d and 3e were synthesised using a modified two-step
, 2b, 2d, 2f and 3a was modified from the one-pot
,
,
,
,
reaction with slight adjustment of the ratio of the reagents and reaction time [24,25].
N2,N2-Dimethyl-6-pentadecyl-1,3,5-triazine-2,4-diamine (2a): Metformin hydrochloride
(497 mg, 3 mmol, 3 equiv.), NaOMe (1 mL, 5 mmol, 5 equiv.) and isopropyl palmitate
(350 µL, 1 mmol, 1 equiv.) in anh. MeOH (4 mL) were mixed and heated under reflux for
2 h. Purification via extraction with EtOAc/H2O and silica gel column chromatography
(eluent: 1:3 EtOAc:hexane) yielded 2a as a white solid (225 mg, 64% yield). 1H-NMR
(400 MHz, CDCl3)
1.78–1.64 (m, 2H), 1.32–1.19 (m, 24H), 0.87 (t, J = 6.7 Hz, 3H).; 13C-NMR (101 MHz, CDCl3)
178.1, 166.3, 165.6, 38.8, 36.3, 32.1, 29.8, 27.7 22.8, 14.2. Data are consistent with literature
values [43].
δ 5.17 (brs, 2H), 3.15 (brs, 3H), 3.11 (brs, 3H), 2.49 (t, J = 7.7 Hz, 3H),
δ

Molecules 2021, 26, 1028
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N2,N2-Dimethyl-6-phenyl-1,3,5-triazine-2,4-diamine(2b): Metformin hydrochloride (497 mg,
3 mmol, 3 equiv.), NaOMe (1 mL, 5 mmol, 5 equiv.) and methyl benzoate (126 µL, 1 mmol,
1 equiv.) in anh. MeOH (4 mL) were heated under reflux for 2 h. The crude mixture purified
by silica gel column chromatography (eluent: 1:2 EtOAc:hexane) yielded 2b as a white-yellow
solid. (112 mg, 52% yield). 1H-NMR (400 MHz, CDCl3)
δ
8.37 (d, J = 7.5 Hz, 2H), 7.53–7.38 (m,
3H), 5.26 (brs, 2H), 3.30 (brs, 3H), 3.17 (brs, 3H); 13C-NMR (101 MHz, CDCl3)
δ
171.0, 167.4,
166.0, 131.4, 130.1, 128.5, 128.3, 36.4. Data are consistent with literature values [7].
2-(4-Amino-6-(dimethylamino)-1,3,5-triazin-2-yl)phenol (2c): Metformin in the free base
form was prepared with the same protocol as 1b using metformin hydrochloride (3.313 g,
20 mmol, 1 equiv.) and NaOMe (4 mL, 20 mmol, 1 equiv.) in 15 mL of anh. MeOH. Next,
excess amount of methyl salicylate (5 mL) was added and heated at 115 ◦C for 2 h with
a notable formation of basic fume. Upon the completion of reaction, the crude mixture
was acidified with NH4Cl to neutral pH then purified by extraction with EtOAc/H2O and
silica gel column chromatography (eluent: 1:3, 1:2 EtOAc:hexane). The product was further
purified by recrystallisation using EtOAc and hexane, which yielded 2c as a greenish yellow
1
solid. (2.153 g, 47% yield). H-NMR (400 MHz, CDCl3)
δ 8.33 (d, J = 7.9 Hz, 1H), 7.37 (t,
J = 7.8 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.89 (t, J = 7.5 Hz, 1H), 5.21 (brs, 2H), 3.22 (s, 3H),
3.17 (s, 3H); 13C-NMR (101 MHz, CDCl3)
δ
171.3, 165.8, 164.4, 162.2, 134.1, 129.7, 119.1,
118.4, 118.1, 36.9, 36.7; IR (neat): 3390, 3324, 3200, 2926, 1652, 1566, 1499, 1031, 746; HRMS
(ESI+): m/z calcd for C11H13N5O [M+H] + 232.1198, found 232.1191; Mp:194–197 ◦C.
(E)-N2,N2-dimethyl-6-styryl-1,3,5-triazine-2,4-diamine (2d): Metformin hydrochloride
(331 mg, 2 mmol, 1 equiv.), NaOMe (800
µL, 4 mmol, 2 equiv.), and methyl cinnamate
(446 L, 3 mmol, 1.5 equiv.) in anh. MeOH (4 mL) were stirred under reflux for 3 h. The
µ
crude mixture purified by extraction using EtOAc/hexane yielded 2d as a yellow solid
1
(302 mg, quant.). H-NMR (400 MHz, CDCl3)
δ 7.95 (d, J = 15.9 Hz, 1H), 7.58 (d, J = 7.0 Hz,
2H), 7.43–7.30 (m, 3H), 6.83 (d, J = 15.9 Hz, 1H), 5.28 (brs, 2H), 3.22 (brs, 3H), 3.15 (brs, 3H);
13C-NMR (101 MHz, CDCl3)
δ
171.0, 167.2, 166.1, 139.7, 136.3, 129.6, 129.2, 128.3, 127.5, 36.7.
Data are consistent with literature values [8].
Methyl 4-amino-6-(dimethylamino)-1,3,5-triazine-2-carboxylate (2e): Metformin in the free
base form was prepared with the same protocol as 1b using metformin hydrochloride
(497 mg, 3 mmol, 1.5 equiv.) and NaOMe (400
MeOH. Metformin dissolved in anh. MeOH (7.5 mL) was added dropwise into diethyl
oxalate (1218 L, 9 mmol, 3 equiv.) also dissolved in anh. MeOH (7.5 mL) with constant
µL, 2mmol, 1 equiv.) in 6 mL of anh.
µ
stirring motion at room temperature. Subsequently, the mixture was heated under reflux
overnight. The mixture was purified by silica gel column chromatography (eluent: 3:5
1
EtOAc:hexane), which yielded 2e as a yellow solid (27 mg, 7% yield). H-NMR (400 MHz,
CDCl3)
δ
5.68 (brs, 2H), 3.95 (s, 3H), 3.23 (s, 3H), 3.13 (s, 3H); 13C-NMR (101 MHz, CDCl3)
δ
166.5, 165.4, 164.0, 163.1, 53.1, 36.4, 36.2; IR (neat): 3426, 3303, 3170, 2953, 1732, 1640, 1566,
1439, 1247, 1209, 1011, 793; HRMS (ESI+): m/z calcd for C7H11N5O2 [M+Na] + 220.0810,
found 220.0815; Mp:219–221 ◦C.
4-Amino-6-(dimethylamino)-1,3,5-triazine-2-carboxylic acid (2f): Metformin hydrochloride
(167 mg, 1 mmol, 1 equiv.), NaOMe (400 µL, 2 mmol, 2 equiv.) and diethyl oxalate (2975 µL,
22 mmol, 22 equiv.) in anh. MeOH (5 mL) were stirred under reflux for 4 h. The white
precipitate was filtered, washed with MeOH and dried under vacuum to yield 2f as a
1
white-yellow solid (118 mg, 65% yield). H-NMR (400 MHz, DMSO)
δ
11.08 (s, 1H), 9.01
171.2,
(brs, 1H), 8.02 (brs, 1H), 3.11 (s, 3H), 3.02 (s, 3H); 13C-NMR (101 MHz, DMSO)
δ
169.9, 163.8, 160.4, 37.9, 37.2; IR (neat): 3304, 3168, 1763, 1708, 1563, 1479, 1324, 1040, 819;
◦
HRMS (ESI+): m/z calcd for C6H9N5O2 [M+Na] + 206.0654, found 206.0645; Mp: >300 C.
6-pentadecyl-N2-phenyl-1,3,5-triazine-2,4-diamine (3a): 1a (6.410 g, 30 mmol, 3 equiv.),
NaOMe (10 mL, 50 mmol, 5 equiv.) and isopropyl palmitate (10.511 mL, 30 mmol, 3 equiv.)
in anh. MeOH (40 mL) were stirred under reflux for 2 h. Purification via extraction with
EtOAc/H2O and silica gel column chromatography (eluent: 1:3, 1:2 EtOAc:hexane) yielded

Molecules 2021, 26, 1028
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1
3a as a white solid (5.0 g, 42% yield). H-NMR (400 MHz, DMSO)
δ
9.38 (s, 1H), 7.77 (d,
J = 7.9 Hz, 2H), 7.24 (t, J = 7.9 Hz, 2H), 6.94 (t, J = 7.2 Hz, 3H), 2.41 (t, J = 7.6 Hz, 2H),
1.73–1.60 (m, 2H), 1.29–1.20 (m, 23H), 0.84 (t, J = 6.7 Hz, 3H); 13C-NMR (101 MHz, DMSO)
δ
177.8, 166.6, 164.2, 140.0, 128.3, 121.7, 119.7, 37.9, 31.2, 29.0, 26.9, 22.0, 13.9; IR (neat): 3462,
3311, 3106, 2913, 2848, 1655, 1626, 1528, 1470, 1429, 1385, 1028, 815, 747, 720, 699; HRMS
(ESI+): m/z calcd for C24H39N5 [M+H] + 398.3284, found 398.3291; Mp: 118–121 ◦C.
N2,6-diphenyl-1,3,5-triazine-2,4-diamine (3b): 1b was used without further purification.
Methyl benzoate (792 µL, 6 mmol, 2 equiv.) was added into 1b (532 mg, 3 mmol, 1 equiv.)
dissolved in anh. MeOH (4 mL). The mixture was stirred under reflux for 24 h. Purified
by silica gel column chromatography (eluent: 1:3 EtOAc:hexane) yielded 3b as a yellow
1
solid (54 mg, 7% yield). H-NMR (400 MHz, DMSO)
δ 9.52 (s, 1H), 8.31 (d, J = 6.8 Hz, 2H),
7.84 (d, J = 8.0 Hz, 2H), 7.58–7.44 (m, 3H), 7.31 (t, J = 7.8 Hz, 2H), 7.12 (brs, 2H), 6.99 (t,
J = 7.3 Hz, 1H); 13C-NMR (101 MHz, DMSO)
δ
170.7, 167.6, 165.1, 140.3, 137.2, 131.8, 128.9,
128.7, 128.2, 122.5, 120.4. Data is consistent with literature values [44].
2-(4-amino-6-(phenylamino)-1,3,5-triazin-2-yl)phenol (3c): 1b was used without further
purification. A mixture of 1b (355 mg, 2 mmol, 1 eqiv.) and an excess amount of methyl
◦
salicylate (500
µ
L) was stirred and heated at 115 C for 7 h with a notable formation
of basic fume. Upon the completion of reaction, the crude mixture was acidified with
NH4Cl to neutral pH then purified by extraction with EtOAc/H2O and silica gel column
chromatography (eluent: 1:4–1:2 EtOAc:hexane). The product was further purified by
recrystallisation using EtOAc and hexane, which yielded 3c as a greenish yellow solid
1
(220 mg, 39% yield). H-NMR (400 MHz, DMSO)
δ 13.49 (s, 1H), 9.77 (brs, 1H), 8.26 (d,
J = 7.3 Hz, 1H), 7.76 (brs, 2H), 7.55 (brs, 1H), 7.44–7.35 (m, 2H), 7.33 (t, J = 7.2 Hz, 2H), 7.04
(t, J = 7.5 Hz, 1H), 6.90 (t, J = 8.0 Hz, 2H); 13C-NMR (101 MHz, DMSO)
δ
169.9, 164.7, 160.8,
138.8, 133.2, 128.3, 128.1, 122.3, 120.2, 117.9, 117.2, 117.1; IR (neat): 3475, 3302, 3181, 1667,
1595, 1533, 1443 1429, 1031, 811, 752, 691; HRMS (ESI+): m/z calcd for C15H13N5O [M+H] +
280.1198, found 280.1205; Mp: 222–224 ◦C.
(E)-N2-phenyl-6-styryl-1,3,5-triazine-2,4-diamine (3d): 1b was used without further pu-
rification. Methyl cinnamate (447 µL, 3 mmol, 1 equiv.) was added into 1b (709 mg, 4 mmol,
1.33 equiv.) dissolved in anh. MeOH (4 mL). The mixture was stirred under reflux for
24 h. Purification by silica gel column chromatography (eluent: 1:2, 1:1 EtOAc:hexane)
1
yielded 3d as a yellow solid (154 mg, 17% yield). H-NMR (500 MHz)
δ
9.47 (s, 1H), 7.87
(d, J = 15.9 Hz, 1H), 7.81 (dd, J = 8.7, 1.2 Hz, 2H), 7.66 (d, J = 7.5 Hz, 2H), 7.46–7.36 (m,
3H), 7.28 (t, J = 7.6 Hz, 2H), 7.03 (brs, 2H), 6.97 (tt, J = 7.3, 1.2 Hz, 1H), 6.82 (d, J = 16.0 Hz
1H); 13C-NMR (101 MHz, CDCl3)
171.0, 166.6, 164.3, 141.2, 138.3, 135.5, 129.9, 129.1,
,
δ
129.0, 128.2, 125.5, 124.0, 121.0; IR (neat): 3462, 3276, 3058, 1638, 1574, 1526, 1490, 1442, 970,
735, 691; HRMS (ESI+): m/z calcd for C17H15N5 [M+H] + 290.1406, found 290.1409; Mp:
186–189 ◦C.
Methyl 4-amino-6-(phenylamino)-1,3,5-triazine-2-carboxylate (3e): 1b was used without
further purification. 1b (4.430 g, 25 mmol, 1 equiv.) dissolved in anh. MeOH (62.5 mL) was
added dropwise into diethyl oxalate (10.15 mL, 75 mmol, 3 equiv.) also dissolved in anh.
MeOH (62.5 mL) with constant stirring motion at room temperature. Subsequently, the
mixture was stirred under reflux for 14 h. Purification by silica gel column chromatography
(eluent: 1:1 EtOAc/hexane to 5% EtOAc/MeOH) yielded 3e as a yellow solid (2.428 g,
1
40% yield). H-NMR (400 MHz, DMSO) δ 9.93 (brs, 1H), 7.78 (d, J = 7.9 Hz, 2H), 7.56 (brs,
1H), 7.43 (brs, 1H), 7.28 (t, J = 6.8 Hz, 2H), 7.01 (t, J = 7.9 Hz, 1H), 3.83 (s, 3H); 13C-NMR
(101 MHz, DMSO)
δ 167.3, 164.5, 164.2, 164.1, 139.5, 128.7, 122.7, 120.3, 52.6. Data are
consistent with literature values [29].
4-amino-6-(phenylamino)-1,3,5-triazine-2-carboxylic acid (3f) was synthesised using a
modified protocol from the literature [45]: NaOH (aq.) (50 mL, 1 M) was added to a
solution of 3e (2.023 g, 8.25 mmol) dissolved in EtOH (100 mL). The mixture was stirred
under reflux for 3 h, cooled to room temperature, followed by the addition of HCl (aq.)

Molecules 2021, 26, 1028
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(50 mL, 1M). The resulting precipitate was filtered, washed with DI and dried under
1
vacuum to yield 3f as a pale-yellow solid (2.416 g, quant.). H-NMR (400 MHz, DMSO)
δ
9.89 (brs, 1H), 7.78 (d, J = 8.1 Hz, 2H), 7.47 (brs, 2H), 7.28 (t, J = 7.3 Hz, 2H), 7.00 (t, J = 7.5
,
6.7 Hz, 1H); 13C-NMR (101 MHz, DMSO)
δ
166.9, 165.4, 165.0, 164.3, 139.4, 128.5, 122.6,
120.2; IR (neat): 3319, 3129, 1675, 1622, 1594, 1568, 1489, 1451, 1005, 788, 761, 691; HRMS
(ESI+): m/z calcd for C10H9N5O2 [M+2Na-H] + 276.0473, found 276.0473; Mp: 248–249 ◦C.
3.2.2. Characterisation of 1,3,5-Triazine Derivatives
1H and 13C-NMR were recorded with Bruker Avance (III) 400WB spectrometer (400 mHz)
and JEOL JNM-ECZS NMR spectrometer (500 mHz); FTIR spectra were analysed by Thermo
Scientific™ Nicolet 6700 FT-IR Spectrometer with ATR mode (32 scans); high-resolution mass
spectra (HRMS) were recorded with micrOTOF-Q II mass spectrometer (Bruker Daltonics)
with electrospray ionization; and melting points (Mp) were measured using a Stuart SMP20
melting point apparatus.
3.2.3. In vitro Anticancer Activity of 1,3,5-Triazine Derivatives
HCT116 were cultured in a growth medium of RPMI. SW620 were cultured in a
growth medium of DMEM. Both cell culture mediums were supplemented with 10% FBS
and 1% antibiotic, and the cells were maintained in an incubator at 37 ◦C in a humidified
atmosphere of 5% CO2.
HCT116 and SW620 were seeded in 96-well plates at a density of 1
cell culture medium and incubated for 24 h to allow cell adherence. Cells were treated
with the synthesised 1,3,5-triazine derivatives at concentration between 0–100 M for 48 h.
Following incubation, 10 L PrestoBlue solution was added to each well, and then plates
×
104 per well in
µ
µ
™
were placed back into the incubator for a further 30 min incubation [46]. Fluorescence was
measured using a microplate reader at 560 nm excitation and 590 nm emission (Thermo,
Varioskan Flash, England). The results were analysed by one-way ANOVA and Tukey
post-test using GraphPad Prism 5.0 (Graph-Pad Software Inc., San Diego, CA, USA).
3.2.4. Preparation of 1,3,5-Triazine Incorporated Calcium Citrate Nanoparticles (Condition
A–C)
Solution A: citric acid was first dissolved in DI water, followed by the addition of 1M
NaOH until pH of solution reached pH 6.0 then the volume was adjusted to 5 mL with
EtOH.; Solution B: 2a was dissolved in 5 mL EtOH while heating at 50–60 ◦C.; Solution C:
calcium chloride was dissolved in 5 mL EtOH. Solution A was added into solution B which
turned cloudy straightaway. EtOH and DI water were added to the mixture to obtain
a clear solution. Solution C was added into the AB mixture with vigorous stirring then
moved to stir at room temperature for 2, 5 or 8 h. The white suspension was centrifuged,
washed with EtOH/DI water and freeze dried for 24 h.
3.2.5. Characterisation of 1,3,5-Triazine Incorporated Calcium Citrate Nanoparticles
Size and morphology of the synthesised products were recorded with JEOL JSM-
6480LV scanning electron microscope at 15kV; hydrodynamic size and zeta potential were
measured with the Zetasizer Nano ZS, Malvern Instrument (IR of 1.660); functional groups
were analysed by Thermo Scientific
Scientific, Waltham, MA, USA) with ATR mode; chemical changes or decomposition temper-
ature by Perkin Elmer Pyris 1 TGA; elemental analysis by Thermo Flash 2000 CHNS/O
™ Nicolet 6700 FT-IR Spectrometer (Thermo Fisher
™
analyser; and %cumulative drug release was measured with UV/Vis spectrometer (Hewlett
Packard 8453, Agilent Technology, Santa Clara, CA, USA).
3.2.6. Drug Release Study
The drug release profile of ◦the synthesised nanoparticles was determined by the
dialysis method carried out at 37 C and stirring speed of 100 rpm. Thirty percent (v/v) of
EtOH in PBS at pH 3.0, 5.0 and 7.4 were used as the release medium [40]. Nanoparticles

Molecules 2021, 26, 1028
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(weight equivalent to 1 mg of 2a) were dispersed in 10 mL 30% (v/v) EtOH/PBS at
pH 3.0, 5.0 and 7.4. The dialysis bag was immersed into 190 mL of release medium. At
predetermined time, the dialysis bag was moved into another container with 190 mL of
fresh release medium [22]. To extract 2a out of the release medium, the solution was
evaporated under vacuum to remove EtOH and extracted with dichloromethane [47]. For
the UV-Vis spectroscopy measurement, 10 mL of 60% EtOH/PBS acidified with HCl to
pH 3.0, 5.0 and 7.4 was added to each sample and measured at 200–320 nm. The calibration
curve was drawn using a standard which was 2a dissolved in 60% EtOH/PBS at various
concentrations.
4. Conclusions
In conclusion, we have reported a new study that integrated drug discovery with
drug delivery. Twelve 1,3,5-triazine derivatives of metformin (2a
–2f) and phenylbiguanide
(
3a–3f) were synthesised via two pathways using various esters. The anticancer activity
of the derivatives ranged widely depending on the hydrophobicity of the substituent.
It was found that some of the derivatives were more potent as anticancer agents with
IC50 comparable to the values of the reference drug, cisplatin. The optimisation for the
preparation of CaCit-2a NPs under EtOH-water binary solvent system revealed that the
use of 1:2 citric acid/NaOH as the citrate source with a reaction time of 8 h provided the
best result. The obtained nanoparticles had appropriate size satisfying the EPR effect,
moderate %drug loading, as well as showing a controlled drug release behaviour under
pH 5.0 environment. The knowledge from both the biological evaluations and the DDS
studies could be valuable for further development of triazine-based anticancer drug and
could serve as a platform for potential calcium citrate nanoparticles drug delivery system
in the future.
Supplementary Materials: The following are available online, 1H and 13C-NMR spectra for com-
pounds 1a, 1b, 2a–2f and 3a–3f; IR and HRMS spectra for compound 2c, 2e, 2f, 3a, 3c, 3d, and 3f;
biological evaluations of 2a–2f and 3a–3f; size distribution graphs for Condition A and C.
Author Contributions: Conceptualization, R.R. and T.K.; methodology, M.C. and S.C.; validation,
A.S., R.R. and T.K.; formal analysis, S.C., M.C. and T.K.; investigation, M.C.; resources, A.S., R.R. and
T.K.; data curation, M.C.; writing—original draft preparation, M.C.; writing—review and editing,
R.R. and T.K.; project administration, T.K.; funding acquisition, R.R. and T.K. All authors have read
and agreed to the published version of the manuscript.
Funding: TheresearchgrantfundshavebeenprovidedbyChulalongkornUniversity: CU_GR_63_159_23_25,
the 72nd Anniversary of His Majesty King Bhumibol Adulyadej Scholarship, and the 90th Anniversary
Chulalongkorn University Fund (Ratchadapiseksomphot Endowment Fund).
Data Availability Statement: Data is contained within the article or Supplementary Material.
Acknowledgments: Special thanks to members of the RR Lab and TK Lab at the Faculty of Science,
and Nanomedicine RU at the Faculty of Medicine, Chulalongkorn University for their assistances
throughout this project.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 2a, 2c, and 3c are available from the authors.
References
1.
2.
Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2019. Ca: Cancerj. Clin. 2019, 69, 7–34. [CrossRef] [PubMed]
Das, M.; Mohanty, C.; Sahoo, S.K. Ligand-based targeted therapy for cancer tissue. Expert Opin. Drug Deliv. 2009, 6, 285–304.
[CrossRef]
3.
4.
Banerjee, A.; Pathak, S.; Subramanium, V.D.; Dharanivasan, G.; Murugesan, R.; Verma, R.S. Strategies for targeted drug delivery
in treatment of colon cancer: Current trends and future perspectives. Drug Discov. Today 2017, 22, 1224–1232. [CrossRef] [PubMed]
Singla, P.; Luxami, V.; Paul, K. Synthesis and in vitro evaluation of novel triazine analogues as anticancer agents and their
interaction studies with bovine serum albumin. Eur. J. Med. Chem. 2016, 117, 59–69. [CrossRef] [PubMed]

Molecules 2021, 26, 1028
13 of 14
5.
Junaid, A.; Lim, F.P.L.; Tiekink, E.R.T.; Dolzhenko, A.V. Design, synthesis, and biological evaluation of new 6,N2-diaryl-1,3,5-
triazine-2,4-diamines as anticancer agents selectively targeting triple negative breast cancer cells. Rsc Adv. 2020, 10, 25517–25528.
[CrossRef]
6.
7.
8.
9.
Junaid, A.; Lim, F.P.L.; Tiekink, E.R.T.; Dolzhenko, A.V. New One-Pot Synthesis of 1,3,5-Triazines: Three-Component Condensa-
tion, Dimroth Rearrangement, and Dehydrogenative Aromatization. Acs Comb. Sci. 2019, 21, 548–555. [CrossRef]
Zeng, M.; Wang, T.; Cui, D.-M.; Zhang, C. Ruthenium-catalyzed synthesis of tri-substituted 1,3,5-triazines from alcohols and
biguanides. Newj. Chem. 2016, 40, 8225–8228. [CrossRef]
Zeng, M.; Xie, Z.P.; Cui, D.-M.; Zhang, C. Ruthenium-catalyzed synthesis of arylethyl 1,3,5-triazines from arylallyl alcohols and
biguanides. Org. Biomol. Chem. 2018, 16, 6140–6145. [CrossRef] [PubMed]
Xu, Y.; Shen, B.; Liu, L.; Qiao, C. Metal free [4+1] and [5+1] annulation reactions to prepare heterocycles using DMF and its
derivatives as one-carbon source. Tetrahedron Lett. 2020, 61, 151844. [CrossRef]
10. Cao, H.; Liao, S.; Zhong, W.; Xiao, X.; Zhu, J.; Li, W.; Wu, X.; Feng, Y. Synthesis, Characterization, and Biological Evaluations of
1,3,5-Triazine Derivatives of Metformin Cyclization with Berberine and Magnolol in the Presence of Sodium Methylate. Molecules
2017, 22, 1752. [CrossRef] [PubMed]
11. Makowska, A.; Sa˛czewski, F.; Bednarski, P.J.; Sa˛czewski, J.; Balewski, Ł. Hybrid Molecules Composed of 2,4-Diamino-1,3,5-
triazines and 2-Imino-Coumarins and Coumarins. Synthesis and Cytotoxic Properties. Molecules 2018, 23, 1616. [CrossRef]
12. Chaurasia, S.R.; Dange, R.; Bhanage, B.M. Graphene oxide as a carbo-catalyst for the synthesis of tri-substituted 1,3,5-triazines
using biguanides and alcohols. Catal. Commun. 2020, 137, 105933. [CrossRef]
13. Yao, W.; Duan, Z.-C.; Zhang, Y.; Sang, X.; Xia, X.-F.; Wang, D. Iridium Supported on Phosphorus-Doped Porous Organic Polymers:
Active and Recyclable Catalyst for Acceptorless Dehydrogenation and Borrowing Hydrogen Reaction. Adv. Synth. Catal. 2019
361, 5695–5703. [CrossRef]
,
14. Koh, M.; Lee, J.-C.; Min, C.; Moon, A. A novel metformin derivative, HL010183, inhibits proliferation and invasion of triple-
negative breast cancer cells. Bioorg. Med. Chem. 2013, 21, 2305–2313. [CrossRef] [PubMed]
15. Kothayer, H.; Spencer, S.M.; Tripathi, K.; Westwell, A.D.; Palle, K. Synthesis and in vitro anticancer evaluation of some 4,6-
diamino-1,3,5-triazine-2-carbohydrazides as Rad6 ubiquitin conjugating enzyme inhibitors. Bioorg. Med. Chem. Lett. 2016, 26,
2030–2034. [CrossRef] [PubMed]
16. Torchilin, V. Tumor delivery of macromolecular drugs based on the EPR effect. Adv. Drug Deliv. Rev. 2011, 63, 131–135. [CrossRef]
[PubMed]
17. Hickey, J.W.; Santos, J.L.; Williford, J.-M.; Mao, H.-Q. Control of polymeric nanoparticle size to improve therapeutic delivery.
J. Control. Release 2015, 219, 536–547. [CrossRef] [PubMed]
18. Bae, Y.H.; Park, K. Targeted drug delivery to tumors: Myths, reality and possibility. J. Control. Release 2011, 153, 198–205.
[CrossRef]
19. Estrella, V.; Chen, T.; Lloyd, M.; Wojtkowiak, J.; Cornnell, H.H.; Ibrahim-Hashim, A.; Bailey, K.; Balagurunathan, Y.; Rothberg, J.M.;
Sloane, B.F.; et al. Acidity Generated by the Tumor Microenvironment Drives Local Invasion. Cancer Res. 2013, 73, 1524. [CrossRef]
20. Zhang, W.; Wang, W.; Chen, Q.-Y.; Lin, Z.-Q.; Cheng, S.-W.; Kou, D.-Q.; Ying, X.-Z.; Shen, Y.; Cheng, X.-J.; Nie, P.-F.; et al. Effect of
calcium citrate on bone integration in a rabbit femur defect model. Asian Pac. J. Trop. Med. 2012, 5, 310–314. [CrossRef]
21. Li, J.; Liu, Y.; Gao, Y.; Zhong, L.; Zou, Q.; Lai, X. Preparation and properties of calcium citrate nanosheets for bone graft substitute.
Bioengineered 2016, 7, 376–381. [CrossRef] [PubMed]
22. Oungeun, P.; Rojanathanes, R.; Pinsornsak, P.; Wanichwecharungruang, S. Sustaining Antibiotic Release from a Poly(methyl
methacrylate) Bone-Spacer. Acs Omega 2019, 4, 14860–14867. [CrossRef]
23. Rimsueb, N.; Cherdchom, S.; Aksornkitti, V.; Khotavivattana, T.; Sereemaspun, A.; Rojanathanes, R. Feeding Cells with a Novel
“Trojan” Carrier: Citrate Nanoparticles. Acs Omega 2020, 5, 7418–7423. [CrossRef] [PubMed]
24. Kothayer, H.; Morelli, M.; Brahemi, G.; Elshanawani, A.A.; Abu Kull, M.E.; El-Sabbagh, O.I.; Shekhar, M.P.V.; Westwell, A.D.
Optimised synthesis of diamino-triazinylmethyl benzoates as inhibitors of Rad6B ubiquitin conjugating enzyme. Tetrahedron Lett.
2014, 55, 7015–7018. [CrossRef]
25. Shapiro, S.L.; Parrino, V.A.; Freedman, L. Guanamines. VIII. 6-(Substituted Phenyl)guanamines. J. Org. Chem. 1961, 26, 3331–3334.
[CrossRef]
26. Mallon, R.; Feldberg, L.R.; Lucas, J.; Chaudhary, I.; Dehnhardt, C.; Santos, E.D.; Chen, Z.; dos Santos, O.; Ayral-Kaloustian, S.;
Venkatesan, A.; et al. Antitumor Efficacy of PKI-587, a Highly Potent Dual PI3K/mTOR Kinase Inhibitor. Clin. Cancer Res. 2011
17, 3193. [CrossRef]
,
27. Moreno, L.M.; Quiroga, J.; Abonia, R.; Ramírez-Prada, J.; Insuasty, B. Synthesis of New 1,3,5-Triazine-Based 2-Pyrazolines as
Potential Anticancer Agents. Molecules 2018, 23, 1956. [CrossRef] [PubMed]
28. Zhou, J.; Li, P.; Xue, X.; He, S.; Kuang, Y.; Zhao, H.; Chen, S.; Zhi, Q.; Guo, X. Salinomycin induces apoptosis in cisplatin-resistant
colorectal cancer cells by accumulation of reactive oxygen species. Toxicol. Lett. 2013, 222, 139–145. [CrossRef] [PubMed]
29. Kothayer, H.; Elshanawani, A.A.; Abu Kull, M.E.; El-Sabbagh, O.I.; Shekhar, M.P.V.; Brancale, A.; Jones, A.T.; Westwell, A.D. De-
sign, synthesis and in vitro anticancer evaluation of 4,6-diamino-1,3,5-triazine-2-carbohydrazides and -carboxamides. Bioorg. Med.
Chem. Lett. 2013, 23, 6886–6889. [CrossRef]
30. Peng, H.; Li, K.; Wang, T.; Wang, J.; Wang, J.; Zhu, R.; Sun, D.; Wang, S. Preparation of hierarchical mesoporous CaCO3 by a facile
binary solvent approach as anticancer drug carrier for etoposide. Nanoscale Res. Lett. 2013, 8, 321. [CrossRef] [PubMed]

Molecules 2021, 26, 1028
14 of 14
31. Li, J.F.; Gao, Y.; Zhong, L.Z.; Liu, Y.Q.; Liu, H.Q.; Zou, Q.; Lai, X.F. Facile Self-Assembly Synthesis of Hierarchical 3D Flowerlike
Calcium Citrate Microspheres. J. Nano Res. 2017, 45, 185–192. [CrossRef]
32. Garcia, A.C.; Vavrusova, M.; Skibsted, L.H. Supersaturation of calcium citrate as a mechanism behind enhanced availability of
calcium phosphates by presence of citrate. Food Res. Int. 2018, 107, 195–205. [CrossRef]
33. Um, N.; Hirato, T. Precipitation behavior of Ca(OH)2, Mg(OH)2, and Mn(OH)2 from CaCl2, MgCl2, and MnCl2 in NaOH-H2O
solutions and study of lithium recovery from seawater via two-stage precipitation process. Hydrometallurgy 2014, 146, 142–148.
[CrossRef]
34. Al-Khaldi, M.H.; Nasr-El-Din, H.A.; Mehta, S.; Al-Aamri, A.D. Reaction of citric acid with calcite. Chem. Eng. Sci. 2007, 62,
5880–5896. [CrossRef]
35. Ma, C.; Gerhard, E.; Lu, D.; Yang, J. Citrate chemistry and biology for biomaterials design. Biomaterials 2018, 178, 383–400.
[CrossRef] [PubMed]
36. Iafisco, M.; Ramírez-Rodríguez, G.B.; Sakhno, Y.; Tampieri, A.; Martra, G.; Gómez-Morales, J.; Delgado-López, J.M. The growth
mechanism of apatite nanocrystals assisted by citrate: Relevance to bone biomineralization. CrystEngComm 2015, 17, 507–511.
[CrossRef]
37. Xiao, H.; Hu, C.; Chen, C.; Tao, C.; Wu, Y.; Jiang, J. The advantage of alcohol–calcium method on the formation and the stability
of vaterite against ethanol–water binary solvent method. J. Mater. Res. 2020, 35, 289–298. [CrossRef]
38. Sand, K.K.; Rodriguez-Blanco, J.D.; Makovicky, E.; Benning, L.G.; Stipp, S.L.S. Crystallization of CaCO3 in Water–Alcohol
Mixtures: Spherulitic Growth, Polymorph Stabilization, and Morphology Change. Cryst. Growth Des. 2012, 12, 842–853.
[CrossRef]
39. Hebeish, A.; El-Rafie, M.H.; El-Sheikh, M.A.; El-Naggar, M.E. Ultra-Fine Characteristics of Starch Nanoparticles Prepared Using
Native Starch With and Without Surfactant. J. Inorg Organomet Polym Mater. 2014, 24, 515–524. [CrossRef]
40. Hardiansyah, A.; Yang, M.-C.; Liu, T.-Y.; Kuo, C.-Y.; Huang, L.-Y.; Chan, T.-Y. Hydrophobic Drug-Loaded PEGylated Magnetic
Liposomes for Drug-Controlled Release. Nanoscale Res. Lett. 2017, 12, 355. [CrossRef] [PubMed]
41. Zhou, C.; Chen, T.; Wu, C.; Zhu, G.; Qiu, L.; Cui, C.; Hou, W.; Tan, W. Aptamer CaCO3 Nanostructures: A Facile, pH-Responsive,
Specific Platform for Targeted Anticancer Theranostics. Chem. Asianj. 2015, 10, 166–171. [CrossRef] [PubMed]
42. Guo, Y.; Li, H.; Shi, W.; Zhang, J.; Feng, J.; Yang, X.; Wang, K.; Zhang, H.; Yang, L. Targeted delivery and pH-responsive release of
doxorubicin to cancer cells using calcium carbonate/hyaluronate/glutamate mesoporous hollow spheres. J. Colloid Interface Sci.
2017, 502, 59–66. [CrossRef] [PubMed]
43. Liu, C. Synthesis of the derivatives of 2-Amino-4-dimethylamino-1,3,5-triazine. J. Guangdong Coll. Pharm. 2005, 21, 2.
44. Liu, C.; Lin, J.; Leftheris, K. A novel one-pot synthesis of N,6-disubstituted 1,3,5-triazine-4,6-diamines from isothiocyanates and
amidines. Tetrahedron Lett. 2007, 48, 435–437. [CrossRef]
45. Lebel, O.; Perron, M.-È.; Maris, T.; Zalzal, S.F.; Nanci, A.; Wuest, J.D. A New Class of Selective Low-Molecular-Weight Gelators
Based on Salts of Diaminotriazinecarboxylic Acids. Chem. Mater. 2006, 18, 3616–3626. [CrossRef]
46. Xu, M.; McCanna, D.J.; Sivak, J.G. Use of the viability reagent PrestoBlue in comparison with alamarBlue and MTT to assess the
viability of human corneal epithelial cells. J. Pharmacol. Toxicol. Methods 2015, 71, 1–7. [CrossRef]
47. Wu, J.-L.; Wang, C.-Q.; Zhuo, R.-X.; Cheng, S.-X. Multi-drug delivery system based on alginate/calcium carbonate hybrid
nanoparticles for combination chemotherapy. Colloids Surf. B 2014, 123, 498–505. [CrossRef]