Synthesis and cellular effects of novel 1,3,5-triazine derivatives in DLD and Ht-29 human colon cancer cell lines

Article abstract of DOI:10.1007/s10637-019-00838-9

This study provides new information on the cellular effects of 1,3,5-triazine nitrogen mustards with different peptide groups in DLD and Ht-29 human colon cancer cell lines. A novel series of 2,4,6-trisubstituted 1,3,5-triazine derivatives bearing 2-chloroethyl and oligopeptide moieties was designed and synthesized. The most cytotoxic derivative was triazine with an Ala-Ala-OMe substituent on the ring (compound 7b). This compound induced time- and dose-dependent cytotoxicity in the DLD-1 and HT-29 colon cancer cell lines. The triazine derivative furthermore induced apoptosis through intracellular signaling pathway attenuation. Compound 7b may be a candidate for further evaluation as a chemotherapeutic agent against colorectal cancer.

Full text of DOI:10.1007/s10637-019-00838-9

Investigational New Drugs
https://doi.org/10.1007/s10637-019-00838-9
PRECLINICAL STUDIES
Synthesis and cellular effects of novel 1,3,5-triazine derivatives
in DLD and Ht-29 human colon cancer cell lines
Agnieszka Wróbel1 & Beata Kolesińska2 & Justyna Frączyk2 & Zbigniew J. Kamiński2 & Anna Tankiewicz-Kwedlo3
Justyna Hermanowicz4 & Robert Czarnomysy5 & Dawid Maliszewski1 & Danuta Drozdowska1
&
Received: 10 May 2019 /Accepted: 12 July 2019
#
The Author(s) 2019
Summary
This study provides new information on the cellular effects of 1,3,5-triazine nitrogen mustards with different peptide groups in
DLD and Ht-29 human colon cancer cell lines. A novel series of 2,4,6-trisubstituted 1,3,5-triazine derivatives bearing 2-
chloroethyl and oligopeptide moieties was designed and synthesized. The most cytotoxic derivative was triazine with an Ala-
Ala-OMe substituent on the ring (compound 7b). This compound induced time- and dose-dependent cytotoxicity in the DLD-1
and HT-29 colon cancer cell lines. The triazine derivative furthermore induced apoptosis through intracellular signaling pathway
attenuation. Compound 7b may be a candidate for further evaluation as a chemotherapeutic agent against colorectal cancer.
.
.
.
.
.
Keywords Colon cancer Hybrid anticancer drugs Triazine derivatives Nitrogen mustards Apoptosis Drug design
Introduction
process [5–8]. New analogues of nitrogen mustards with in-
creased selectivity and pharmacological effectiveness are con-
tinually sought [9].
Nitrogen mustards (NMs), such as chlorambucil and
mechlorethamine, are an extensively investigated and clinical-
ly used class of anticancer drugs. Although they are them-
selves highly carcinogenic, these bifunctional alkylating
agents can be used in the treatment of various cancers and
autoimmune diseases [1–4]. Their mechanism of activity is
connected to their ability to cross-link double strands of
DNA involving the distal guanine bases in the opposite strand
of 5’-GNC sequences, yielding bifunctional DNA lesions.
This eventually inhibits tumor growth and the proliferation
of cancer cells, by impeding the replication and transcription
Bifunctional hybrids of triazine nitrogen mustards are a
unique group of compounds with confirmed anti-
proliferative activity. Recent research suggests that derivatives
with one, two or three fragments bearing the 2-
chloroethylamine moiety characteristic for nitrogen mustards
introduced into the 1,3,5-triazine scaffold show anticancer
ability [10, 11]. Compounds containing a melamine fragment
in the structure inhibit a wide range of enzymes involved in
the transcription and translation process. The main common
feature of these compounds is the presence of a triazine ring
substituted by alkyl-(aryl)-amino residues. A number of stud-
ies have identified many new, highly potent agents which
include a 1,3,5-triazine core. It has been reported that these
compounds show inhibitory activity against important en-
zymes involved in cell proliferation cycles.
* Danuta Drozdowska
danuta.drozdowska@umb.edu.pl
1
Department of Organic Chemistry, Medical University of Bialystok,
Białystok, Poland
Structure-activity studies have revealed that one of the
molecular targets is phosphatidylinositol 3-kinases
( P I 3K ) . T h e m e l a m i n e d e r i v a t i v e Z S T K 47 4
[2-(2difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-
1,3,5-triazine] demonstrates strong antitumor activity
against human cancer and reduces the growth of tumor
cells by inhibiting PI3K [12–14]. It has been also reported
that the use of 2,4-diamino-6-(pyridine-4-yl)-1,3,5-tri-
azine (4PyDAT) impedes the process of angiogenesis
2
Institute of Organic Chemistry, Lodz University of Technology,
Lodz, Poland
3
Department of Monitored Pharmacotherapy, Medical University of
Bialystok, Białystok, Poland
4
Department of Clinical Pharmacy, Medical University of Bialystok,
Bialystok, Poland
5
Department of Synthesis and Technology of Drugs, Medical
University of Bialystok, Bialystok, Poland

Invest New Drugs
[15]. Furthermore, molecules based on a structurally mod-
ified triazine ring have been found to inhibit histone
deacetylase and topoisomerases, as well as blocking telo-
mere replication and stabilizing telomeric G-rich single-
stranded DNA (G-quadruplex), causing G2/M arrest and
apoptosis with the possible involvement of p53 [16–20].
Recently, many compounds containing a 1,3,5-triazine
ring have been obtained which have potential pharmaco-
logical effects. As only two of these derivatives have been
tested clinically so far, there is rich potential for finding
new therapeutic compounds in this group [21].
structure of the substituents on the 1,3,5- triazine ring [24].
It was also found that 1,3,5-triazine with three alkylating sub-
stituents induced time- and dose-dependent cytotoxicity in
LBC3, LN-18 and LN-229 glioma cell lines. Compound C
caused apoptosis, necrosis and strong cell shrinkage, reducing
the number of apoptotic bodies. It can therefore be considered
as a candidate for further evaluation as a chemotherapeutic
agent against glioma cells [25].
In our previous paper, we presented the synthesis of triazine
derivatives bearing one, two or three 2-chloroethylamine resi-
dues. The cytotoxicity of all the obtained compounds, as well as
the apoptotic index and cell viability, were studied for the stan-
dard cell line of mammalian tumor MCF-7. All of the obtained
triazines inhibited colony formation by 50% at concentrations
in the range of 13.88 to 146.79 μM. As a comparison, the IC50
of chlorambucil was 24.6 μM. In addition, it was found that the
triazine analogues had anti-proliferative activity against pros-
tate cancer LNCaP, lung cancer A549, breast cancer T47D,
colorectal cancer SW707 and Jurkat lymphoblastic leukemia.
A significant finding was that in the case of estrogen-dependent
breast cancer MCF-7, which is resistant to chemotherapy, ac-
tivity increased with the number of 2-chloroethyloamino moi-
eties [22]. It was found that the compounds with antitumor
cytotoxicity towards the standard cell line of mammalian tumor
MCF-7 were strongly alkylating agents, which reacted easily
with most of the nucleophilic functional groups typical for pro-
teins and nucleic acids [23].
Materials and methods
Chemistry
General information
Thin layer chromatography experiments (TLC) were carried
out on silica gel (Merck; 60 Å F254). Spots were located with
UV light (254 and 366 nm) and 1% ethanolic 4-(4-
nitrobenzyl) pyridine (NBP). Analytical RP-HPLC was per-
formed on a Waters 600S HPLC system (Waters 2489 UV/
VIS detector, Waters 616 pump, Waters 717 plus autosampler,
HPLC manager software from Chromax) using a Vydac C18
column (25 cm × 4.6 mm, 5 mm; Sigma). HPLC was per-
formed with a gradient of 0.1% TFA in H2O (A) and 0.08%
TFA in MeCN (B), at a flow rate of 1 mL/min with UV
detection at 220 nm, tR in min.
MS analysis was performed on an MS Bruker micrOTOF-
QIII.
In our further studies, dual hybrid molecules were synthe-
sized by the functionalization of a melamine scaffold with 2-
chloroethyloamino (one, two or three) fragments attached to
1,3,5- triazine via the piperazine ring (Fig. 1). The anti-
proliferative activity of the compounds was evaluated using
two tumor cell lines: MCF-7 and MDA-MB-231. The results
confirmed the inhibitory effect of the tested compounds on
MCF-7 cancer cells. This effect was dependent on the
Infrared spectra were recorded as KBr pellets or on film
using a Bruker ALPHA spectrometer or a PerkinElmer
Spectrum 100.
1H-NMR and 13C-NMR, spectra were recorded on a Bruker
Avance DPX 250 (250 MHz) spectrometer and Varian
(300 MHz). Chemical shifts (ppm) were relative to TMS, used
as an internal standard. Multiplicities are marked as s = singlet,
d = doublet, t = triplet, q = quartet, qu = quintet, m = multiplet.
Fig. 1 Structures of triazine
mustards

Invest New Drugs
Preparation of dipeptides
6.88 Hz, J2 = 3.22 Hz, HO-CH2-); 3.72 (s, 3H, CH3O-); 4.35
(q, 1H, J = 7.1 Hz, CH3-CH-); 4.37 (t, 1H, J = 7.2 Hz, -CH-
CH2-); 4.55 (t, 1H, J = 6.88 Hz, -CH2-CH-) [ppm].
Anal. RP-HPLC (10–97%B in 30 min): tR = 7.25 min (pu-
rity 98.7%).
Boc-Aib-Ala-OMe (1); typical procedure Boc-Aib-OH
(0.406 g, 2 mmol) and NMM (0.110 mL, 1 mmol) were
added to a vigorously stirred solution of DMT/NMM/
TosO− (0.826 g, 2 mmol) in CH2Cl2 (10 mL) cooled to
0 °C. Stirring was continued until the condensing reagent
disappeared (TLC analysis, staining with 0.5% solution of
NBP) (1 h), after which HCl*Ala-OMe (0.278 g, 2 mmol)
and NMM (0.220 mL, 2 mmol) were added. The mixture
was stirred for an additional 2 h at 0 °C and left overnight
at room temperature. The mixture was diluted with
CH2Cl2 (10 mL), then the solution was washed succes-
sively with water, 0.5 M aqueous NaHSO4, water, 0.5 M
aqueous NaHCO3 and water again. The organic layer was
dried with Na2SO4, filtered and concentrated to dryness.
The residue was dried under a vacuum with P2O5 and
KOH to constant weight, to obtain the neutral peptide.
Finally, 0.559 g was obtained, corresponding to a 97%
yield.
Fmoc-Lys(Boc)-Ala-OMe (1d) Starting materials: Fmoc-
Lys(Boc)-OH (0.937 g, 2 mmol), HCl*Ala-OMe (0.278 g,
2 mmol), DMT/NMM/TosO− (0.826 g, 2 mmol), NMM
(0.330 mL, 3 mmol). Product: Fmoc-Lys(Boc)-Ala-OMe.
Activation time: 1 h, yield 1.041 g (94%).
IR (film): ν = 3401, 3087, 2994, 1753, 1748, 1710, 1674,
1546, 1452, 1373 [cm−1].
1H-NMR (250 MHz; CDCl3) δ = 1.41 (d, 3H, J = 7.2 Hz,
CH3-CH-); 1.43 (s, 9H, (CH3)3C-); 1.48–1.55 (m, 4H, -CH2-
CH2-); 1.88 (qu, 2H, J = 7.4 Hz, -CH-CH2-); 3.21 (t, 2H, J =
7.0 Hz, NH-CH2-); 3.71 (s, 3H, CH3O-); 4.36 (q, 1H, J =
7.2 Hz, CH3-CH-); 4.46 (t, 1H, J = 3.1 Hz, -CH-CH2-); 4.62
(d, 2H, J = 3.1 Hz, -CH2-O-); 7.36–8.11 (m, 8H, arom.) [ppm].
Anal. RP-HPLC (10–97%B in 30 min): tR = 10.25 min
(purity 98.1%).
IR (film): ν = 3678, 3608, 3430, 3017, 2965, 2398, 1734,
1672, 1512, 1451, 1420 [cm−1].
1H-NMR (250 MHz; CDCl3) δ = 1.39 (s,9H, (CH3)3-C-);
1.41 (d, 3H, J = 7.2 Hz, CH3 CH-); 1.57 (s, 6H, (CH3)2-C-);
3.72 (s, 3H, CH3O-); 4.36 (q, 1H, J = 7.2 Hz, CH3-CH-)
[ppm].
Fmoc-Asp(OtBu)-Ala-OMe (1e) Starting materials: Fmoc-
Asp(OtBu)-OH (0.823 g, 2 mmol), HCl*Ala-OMe (0.278 g,
2 mmol), DMT/NMM/TosO− (0.826 g, 2 mmol), NMM
(0.330 mL, 3 mmol). Product: Fmoc-Asp(OtBu)-Ala-OMe.
Activation time: 1 h, yield 0.934 g (94%).
Anal. RP-HPLC (10–97%B in 30 min): tR = 9.55 min (pu-
rity 98.8%).
IR (film): ν = 3410, 3090, 2990, 1750, 1740, 1710, 1675,
1540, 1450, 1375 [cm−1].
Boc-Ala-Ala-OMe (1b) Starting materials: Boc-Ala-OH
(0.378 g, 2 mmol), HCl*Ala-OMe (0.278 g, 2 mmol), DMT/
NMM/TosO− (0.826 g, 2 mmol), NMM (0.330 mL, 3 mmol).
Product: Boc-Ala-Ala-OMe. Activation time: 1 h, yield
0.527 g (96%).
1H-NMR (250 MHz; CDCl3) δ = 1.40 (d, 3H, J = 7.1 Hz,
CH3- CH-); 1.46 (s, 9H, -OC(CH3)3); 2.57 (dd, 1H, J1 =
17.2 Hz, J2 = 7.3 Hz, -OCO-CH2-CH-); 2.92 (dd, 1H, J1 =
17.2 Hz, J2 = 4.1 Hz, -OCO-CH2-CH-); 3.73 (s, 3H,
CH3O-); 4.26 (t, 1H, J = 7.1 Hz, -CH-CH2O-); 4.42 (d, 2H,
J = 7.1 Hz, -CH-CH2O-); 4.48–4.60 (m, 2H, CH3CH- + OCO-
CH2-CH-); 5.97 (d, 1H, J = 7 Hz, NH); 7.07 (d, 1H, J = 5 Hz,
NH); 7.31 (t, 2H, J = 7 Hz, arom.); 7.41 (t, 2H, J = 7 Hz,
arom.); 7.58 (d, 2H, J = 7 Hz, arom.); 7.77 (d, 2H, J = 7 Hz,
arom.) [ppm].
IR (film): ν = 3119, 3008, 1740, 1718, 1675, 1504, 1450,
1376, 1314 [cm−1].
1H-NMR (250 MHz; CDCl3) δ = 1.36 (d, 3H, J = 6.8 Hz,
CH3-CH-); 1.41 (d, 3H, J = 6.8 Hz, CH3 CH-); 1.45 (s, 9H,
(CH3)3-C-); 3.75 (s, 3H, CH3O-); 4.57 (q, 1H, J = 6.8 Hz, CH3
CH-); 4.61 (q, 1H, J = 6.8 Hz, CH3 CH-); 4.97 (1H, bs, -NH-),
6.49 (1H, bs, -NH-) [ppm].
Anal. RP-HPLC (10–97%B in 30 min): tR = 10.05 min
(purity 97.9%).
Anal. RP-HPLC (10–97%B in 30 min): tR = 9.03 min (pu-
rity 98.1%).
Fmoc-Trp(Boc)-Ala-OMe Starting materials: Fmoc-Trp(Boc)-
OH (1.053 g, 2 mmol), HCl*Ala-OMe (0.278 g, 2 mmol),
DMT/NMM/TosO− (0.826 g, 2 mmol), NMM (0.330 mL,
3 mmol). Product: Fmoc-Trp(Boc)-Ala-OMe. Activation
time: 1 h, yield 1.187 g (97%).
Boc-Ser-Ala-OMe (1c) Starting materials: Boc-Ser-OH
(0.410 g, 2 mmol), HCl*Ala-OMe (0.278 g, 2 mmol), DMT/
NMM/TosO− (0.826 g, 2 mmol), NMM (0.330 mL, 3 mmol).
Product: Boc-Ser-Ala-OMe. Activation time: 1.5 h, yield
0.540 g (93%).
IR (film): ν = 3412, 3088, 2987, 1754, 1743, 1711, 1679,
1538, 1452, 1375 [cm−1].
IR (film): ν = 3110, 3002, 1748, 1722, 1648, 1501, 1444,
1372, 1314 [cm−1].
1HNMR (250 MHz; CDCl3) δ = 1.41 (d, 3H, J = 7.2 Hz,
CH3-CH-); 1.43 (s, 9H, (CH3)3C-); 3.15 (d, 2H, J = 6.7 Hz, -
CH-CH2-); 3.71 (s, 3H, CH3O-); 4.91 (t, 1H, J = 6.7 Hz, CH-
CH2-); 4.36 (q, 1H, J = 7.2 Hz, CH3-CH-); 4.46 (t, 1H, J =
1H-NMR (250 MHz; CDCl3) δ =1.38 (d, 3H, J = 7.12 Hz,
CH3-CH-); 1.43 (s, 9H, (CH3)3-C-); 3.65 (dd, 2H, J1 =

Invest New Drugs
3.1 Hz, -CH-CH2-); 4.62 (d, 2H, J = 3.1 Hz, -CH2-O-); 7.03–
8.11 (m, 13H, arom.) [ppm].
Anal. RP-HPLC (10–97%B in 30 min): tR = 11.14 min
(purity 98.1%).
weight, giving 0.491 g of 2-chloro-4-methoxy-6-(NH-Aib-
Ala-OMe)-1,3,5-triazine (4a), yield = 98.7% as colorless oil.
Analysis: for C12H18ClN5O4 (331.76): calculated: C
43.4%, H 5.5%, Cl 10.7%, N 21.1%, found: C 43.2%, H
5.6%, Cl 10.7%, N 21.2%.
1H-NMR (250 MHz; CDCl3) δ = 1.36 (s, 6H, ((CH3)2-C-);
1.41 (s, 3H, d, J = 7.2 Hz, CH3-CH-); 3.72 (s, 3H, CH3O-);
4.02 (s, 3H, CH3O-); 4.36 (q, 1H, J = 7.2 Hz, CH3-CH-)
[ppm].
Removal of protected groups
Boc group deprotection A Boc-protected dipeptide solution
(1 mmol) dissolved in 1,4-dioxane (5 mL) was cooled in a
water-ice bath with vigorously stirring, and a 4 M solution of
HCl in 1,4-dioxane (5 mL) was added. Stirring was continued
for 4 h. The solution was then removed in a vacuum evapora-
tor and 1,4-dioxane (10 mL) was added to the residue. The
solvent was removed again using a vacuum evaporator. This
operation was repeated four times. The residue was dried in a
vacuum desiccator to constant weight. The procedure gave
HCl*Aib-Ala-OMe, HCl*Ala-Ala-OMe and HCl*Ser-Ala-
OMe in quantitative yield. The derivatives obtained were used
in the subsequent reaction steps without additional purifica-
tion procedures.
13C-NMR (75 MHz, CDCl3): δ = 17.7, 27.1, 48.1, 52.3,
54.6, 58.1, 159.7, 166.0, 172.9, 175.6 [ppm].
Synthesis of 2-chloro-4-methoxy-6-(NH-Ala-Ala-OMe)-1,3,5-
triazine (4b) Starting material: solid NaHCO3 (0.42 g,
5 mmol), DCMT (0.270 g, 1.5 mmol), HCl*H-Ala-Ala-
OMe (0.316 g, 1.5 mmol). Product: 0.465 g 2-chloro-4-
methoxy-6-(NH-Ala-Ala-OMe)-1,3,5-triazine (4b), yield =
97.6%, colorless oil.
Analysis: for C11H16ClN5O4 (317.73): calculated: C
41.6%, H 5.1%, Cl 11.2%, N 22%, found: C 41.5%, H
5.2%, Cl 11.2%, N 22.1%.
1H-NMR (250 MHz; CDCl3) δ = 1.39 (d, 3H, J = 7.0 Hz,
CH3-CH-); 1.41 (d, 3H, J = 7.2 Hz, CH3-CH-); 3.71 (s, 3H,
CH3O-); 4.02 (s, 3H, CH3O-); 4.35 (q, 1H, J = 7.2 Hz, CH3-
CH-); 4.38 (q, 1H, J = 7.0 Hz, CH3-CH-) [ppm].
Fmoc-group deprotection A solution of Fmoc-protected di-
peptide derivatives (1 mmol) dissolved in DCM (5 mL) was
cooled in a water-ice bath with intensive stirring and a 25%
piperidine solution in DCM (10 mL) was added. Stirring was
continued for 15 min, then MeOH (20 ml) was added to the
solution. Stirring was continued for 5 min. The solvent was
removed using a vacuum evaporator. To the residue was
added 10 mL of DCM and the solvent was removed again in
a vacuum evaporator. This operation was repeated four times.
The solid residue was dried under a vacuum to constant
weight. The procedure gave H-Lys(Boc)-Ala-OMe, H-
Asp(OtBu)-Ala-OMe and H-Trp(Boc)-Ala-OMe in quantita-
tive yield. The derivatives obtained were used immediately in
subsequent reaction steps, without additional purification
procedures.
13C-NMR (75 MHz, CDCl3): δ = 17.4, 17.6, 47.8, 49.2,
52.3, 54.6, 159.8, 166.0, 172.2, 172.8 [ppm].
Synthesis of 2-chloro-4-methoxy-6-(NH-Ser-Ala-OMe)-1,3,5-
triazine (4c) Starting material: solid NaHCO3 (0.42 g, 5 mmol),
DCMT (0.270 g, 1.5 mmol), HCl*H-Ser-Ala-OMe (0.340 g,
1.5 mmol). Product: 0.480 g 2-chloro-4-methoxy-6-(NH-Ser-
Ala-OMe)-1,3,5-triazine (4c), yield = 95.8%, colorless oil.
Analysis: for C11H16ClN5O5 (333.73): calculated: C
39.6%, H 4.8%, Cl 10.6%, N 21%, found: C 39.7%, H
4.7%, Cl 10.6%, N 21%.
1H-NMR (250 MHz; CDCl3) δ = 1.41 (d, 3H, J = 7.2 Hz,
CH3-CH-); 3.67 (dd, 2H, J1 = 6.8 Hz, J2 = 3.2 Hz, -CH-CH2-
OH); 3.78 (s, 3H, CH3O-); 4.05 (s, 3H, CH3O-); 4.35 (1, 1H,
J = 7.2 Hz, CH3-CH-); 4.48 (t, 1H, J = 6.8 Hz, -CH-CH2-)
[ppm].
Synthesis of 4a-f from DCMT and dipeptides
Synthesis of 2-chloro-4-methoxy-6-(NH-Aib-Ala-OMe)-1,3,5-
triazine (4a). General procedure To a vigorously stirred slurry
of solid NaHCO3 (0.42 g, 5 mmol) in dichloromethane
(DCM) (3 mL) cooled to 0 °C was added 2,4-dichloro-6-
methoxy-1,3,5-triazine (DCMT) (0.270 g, 1.5 mmol) in
DCM (3 mL), followed by the slow addition of a solution of
HCl*NH-Aib-Ala-OMe (0.337 g, 1.5 mmol). Stirring was
continued until complete consumption of the DCMT, as
shown by the disappearance of a DCMT spot stained with
0.5% solution of NBP in ethanol at room temperature, moni-
tored by TLC analysis. After filtration of the solid deposit, the
solvent was removed using vacuum evaporation and the res-
idue dried under a vacuum with P2O5 and KOH to a constant
13C-NMR (75 MHz, CDCl3): δ = 17.6, 38.3, 47.8, 52.3,
54.6, 61.4, 159.5, 166.0171.3, 172.9 [ppm].
Synthesis of 2-chloro-4-methoxy-6-(NH-Lys(Boc)-Ala-OMe)-
1,3,5-triazine (4d) Starting material: solid NaHCO3 (0.42 g,
5 mmol), DCMT (0.270 g, 1.5 mmol), H-Lys(Boc)-Ala-OMe
(0.497 g, 1.5 mmol). Product: 0.685 g 2-chloro-4-methoxy-
6-(NH-Lys(Boc)-Ala-OMe)-1,3,5-triazine (4d), yield =
96.2%, colorless oil.
Analysis: for C19H31ClN6O6 (474.94): calculated: C 48%,
H 6.6%, Cl 7.5%, N 17.7%, found: C 48.1%, H 6.7%, Cl
7.4%, N 17.6%.

Invest New Drugs
1H-NMR (250 MHz; CDCl3) δ = 1.26–1.57 (m, 4H, -CH2-
CH2-); 1.41 (d, 3H, d, J = 7.2 Hz, CH3-CH-); 1.43 (s, 9H,
(CH3)3C-); 1.93 (dt, 2H, J1 = 7.5 Hz, J2 = 5.1 Hz, -CH-
CH2-); 3.22 (t, 2H, J = 7.1 Hz, -CH2-NH-); 3.71 (s, 3H,
CH3O-); 4.05 (s, 3H, CH3O-); 4.28 (t, 1H, J = 7.5 Hz, -CH-
CH2-); 4.36 (q, 1H, J = 7.2 Hz, CH3-CH-) [ppm].
DCM (10 mL) cooled to 0 °C in an ice-water bath was added
1,4-diazabicyclo[2.2.2]octane (DABCO, 5) (0.112g, 1 mmol).
Stirring at 0 °C was continued until complete consumption of
4a, as shown by TLC analysis (Rf = 0.45, DCM; staining with
0.5% solution of NBP in ethanol). The cooling bath was re-
moved and stirring was continued at room temperature until
all 6a salt was consumed, as shown by TLC analysis (Rf =
0.00, pure DCM, colored with 0.5% solution of NBP in eth-
anol). The solvent was removed by evaporation and the resi-
due was dried under a vacuum with P2O5 and KOH to con-
stant weight, giving 0.442 g 2-[4-(2-chloroethyl) piperazin-1-
yl]-4-methoxy-6-(NH-Aib-Ala-OMe)-1,3,5-triazine (7a),
yield = 99.1%, colorless oil.
13C-NMR (75 MHz, CDCl3): δ = 17.7, 21.4, 28.2, 29.3,
29.6, 40.5, 47.8, 52.3, 53.6, 54.6, 80.5, 156.3, 159.5, 166.0,
172.5, 172.9 [ppm].
Synthesis of 2-chloro-4-methoxy-6-(NH-Asp(OtBu)-Ala-OMe)-
1,3,5-triazine (4e) Starting material: solid NaHCO3 (0.42 g,
5 mmol), DCMT (0.270 g, 1.5 mmol), H-Asp(OtBu-Ala-
OMe (0.411 g, 1.5 mmol). Product: 0.607 g 2-chloro-4-
methoxy-6-(NH-Asp(OtBu)-Ala-OMe)-1,3,5-triazine (4e),
yield = 96.9%, colorless oil.
Analysis: for C18H30ClN7O4 (443.93): calculated: C
48.7%, H 6.8%, Cl 8%, N 22.1%, found: C 48.6%, H 6.7%,
Cl 8%, N 22%.
Analysis: for C16H24ClN5O6 (417.84): calculated: C 46%,
H 5.8%, Cl 8.5%, N 16.8%, found: C 46%, H 5.9%, Cl 8.5%,
N 16.8%.
1H-NMR (250 MHz; CDCl3) δ = 1.36 (s, 6H, (CH3)2-C-);
1.41 (d, 3H, J = 7.2 Hz, CH3-CH-); 2.66 (t, 2x2H, J = 7.8 Hz, -
N-CH2-); 2.85 (t, 2H, J = 6.2 Hz, -N-CH2-); 3.60 (t, 2x2H, J =
7.8 Hz, -N-CH2-); 3.86 (2, 2H, J = 6.2 Hz, Cl-CH2-); 3.71 (s,
3H, CH3O-); 4.02 (s, 3H, CH3O-); 4.36 (q, 1H, J = 7.2 Hz,
CH3-CH-) [ppm].
1H-NMR (250 MHz; CDCl3) δ = 1.38 (s, 9H, (CH3)3C-);
1.41 (d, 3H, J = 7.2 Hz, CH3-CH-); 3.01 (d, 2H, J = 6.3 Hz, -
CH-CH2-); 3.72 (s, 3H, CH3O-); 4.02 (s, 3H, CH3O-); 4.36 (1,
1H, J = 7.2 Hz, CH3-CH-); 5.02 (t, 1H, J = 6.3 Hz, -CH-CH2-)
[ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 27.1, 41.2, 44.1,
48.1, 51.8, 52.3, 53.1, 54.6, 58.1, 167.8, 171.7, 172.9, 175.6
[ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 28.1, 37.9, 47.8,
52.3, 53.6, 54.6, 81.4, 159.6, 166.0, 172.3, 172.5, 172.9
[ppm].
LC/MS: 444.9373 ([M + H]+, C18H31ClN7O4+; calc.
443.93).
Synthesis of 2-chloro-4-methoxy-6-(NH-Trp(Boc)-Ala-OMe)-
1,3,5-triazine (4f) Starting material: solid NaHCO3 (0.42 g,
5 mmol), DCMT (0.270 g, 1.5 mmol), H-Trp(Boc)-Ala-
OMe (0.584 g, 1.5 mmol). Product: 0.776 g 2-chloro-4-
methoxy-6-(NH-Trp(Boc)-Ala-OMe)-1,3,5-triazine (4f),
yield = 97.1%, colorless oil.
Analysis: for C24H29ClN6O6 (532.98): calculated: C
54.1%, H 5.5%, Cl 6.7%, N 15.8%, found: C 54%, H 5.4%,
Cl 6.7%, N 15.7%.
Synthesis of 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-
(NH-Ala-Ala-OMe)-1,3,5-triazine (7b) Starting material: 2-
chloro-4-methoxy-6-(NH-Ala-Ala-OMe)-1,3,5-triazine (4b)
(0.318 g, 1 mmol), DABCO (5) (0.112 g, 1 mmol). Product:
0.419 g 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-
6-(NH-Ala-Ala-OMe)-1,3,5-triazine (7b), yield = 97.4%, col-
orless oil.
Analysis: for C17H28ClN7O4 (429.91): calculated: C
47.5%, H 6.6%,Cl 8.2%, N 22.8%, found: C 47.6%, H
6.7%,Cl 8.2%, N 22.7%.
1H-NMR (250 MHz; CDCl3) δ = 1.41 (d, 3H, J = 7.2 Hz,
CH3-CH); 1.44 (s, 9H, (CH3)3C-); 3.15 (d, 2H, J = 6.7 Hz, -
CH-CH2-); 3.71 (s, 3H, CH3O-); 4.02 (s, 3H, CH3O-); 4.37 (q,
1H, J = 7.2 Hz, CH3-CH-); 4.91 (t, 1H, J = 6.7 Hz, CH-CH2-);
7.03–7.98 (m, 5H, arom) [ppm].
1H-NMR (250 MHz; CDCl3) δ = 1.37 (d, 3H, J = 7.0 Hz,
CH3-CH-); 1.41 (d, 3H, J = 7.2 Hz, CH3-CH-); 2.66 (t, 2x2H,
J = 7.8 Hz, -N-CH2-); 2.85 (t, 2H, J = 6.2 Hz, -N-CH2-); 3.60
(t, 2x2H, J = 7.8 Hz, -N-CH2-); 3.86 (2, 2H, J = 6.2 Hz, Cl-
CH2-); 3.71 (s, 3H, CH3O-); 4.02 (s, 3H, CH3O-); 4.35 (q, 1H,
J = 7.2 Hz, CH3-CH-); 4.38 (q, 1H, J = 7.0 Hz, CH3-CH-)
[ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 27.7, 28.2, 47.9,
52.3, 53.6, 54.6, 80.6, 115.4, 117.1, 118.8, 120.1, 122.8,
123.7, 129.6, 135.4, 149.2, 166.0, 162.1, 172.3, 172.9 [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.4, 17.7, 41.2, 44.0,
47.8, 49.2, 51.8, 52.3, 53.0, 163.4, 171.1, 172.1, 172.9 [ppm].
MS: 430.9118 ([M + H]+, C17H29ClN7O4+; calc. 429.91).
Synthesis of 2-[4-(2-chloroethyl)piperazin-1-yl]
-4-methoxy-6-(NH-dipeptydyl)-1,3,5-triazines 7a-f
Synthesis of 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-
(NH-Aib-Ala-OMe)-1,3,5-triazine (7a). General procedure To a
vigorously stirred solution of 2-chloro-4-methoxy-6-(NH-
Aib-Ala-OMe)-1,3,5-triazine (4a) (0.332 g, 1 mmol) in
Synthesis of 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-
(NH-Ser-Ala-OMe)-1,3,5-triazine (7c) Starting material: 2-
chloro-4-methoxy-6-(NH-Ser-Ala-OMe)-1,3,5-triazine (4c)
(0.334 g, 1 mmol), DABCO (5) (0.112 g, 1 mmol). Product:

Invest New Drugs
0.438 g 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-
6-(NH-Ser-Ala-OMe)-1,3,5-triazine (7c), yield = 98.2%, col-
orless oil.
Analysis: for C17H28ClN7O5 (445.90): calculated: C
45.8%, H 6.3%, Cl 8%, N 22%, found: C 45.7%, H 6.4%,
Cl 8%, N 22%.
1H-NMR (250 MHz; CDCl3) δ = 1.38 (s, 9H, (CH3)3C-);
1.41 (d, 3H, J = 7.2 Hz, CH3-CH-); 3.01 (d, 2H, J = 6.3 Hz, -
CH-CH2-); 2.66 (t, 2x2H, J = 7.8 Hz, -N-CH2-); 2.85 (t, 2H,
J = 6.2 Hz, -N-CH2-); 3.60 (t, 2x2H, J = 7.8 Hz, -N-CH2-);
3.86 (2, 2H, J = 6.2 Hz, Cl-CH2-); 3.71 (s, 3H, CH3O-); 4.02
(s, 3H, CH3O-); 4.36 (1, 1H, J = 7.2 Hz, CH3-CH-); 5.02 (t,
1H, J = 6.3 Hz, -CH-CH2-) [ppm].
1H-NMR (250 MHz; CDCl3) δ = 1.40 (d, 3H, J = 7.2 Hz,
CH3-CH); 2.66 (t, 2x2H, J = 7.8 Hz, -N-CH2-); 2.85 (t, 2H,
J = 6.2 Hz, -N-CH2-); 3.60 (t, 2x2H, J = 7.8 Hz, -N-CH2-);
3.68 (d, 2H, J = 6.8 Hz, -CH2-OH); 3.86 (2, 2H, J = 6.2 Hz,
Cl-CH2-); 3.71 (s, 3H, CH3O-); 4.02 (s, 3H, CH3O-); 4.35 (q,
1H, J = 7.2 Hz, CH3-CH-); 4.47 (t, 1H, J = 6.8 Hz, -CH-CH2-
OH) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 37.9, 41.1, 44.0,
47.8, 51.8, 52.3, 53.1, 53.5, 54.6, 81.1, 162.2, 171.7, 172.1,
172.5, 172.9 [ppm].
+
MS: 531.1256 ([M + H]+, C22H37ClN7O6 ; calc. 530.02).
Synthesis of 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-
(NH-Trp(Boc)-Ala-OMe)-1,3,5-triazine (7f) Starting material: 2-
chloro-4-methoxy-6-(NH-Trp(Boc)-Ala-OMe)-1,3,5-triazine
(4f) (0.533 g, 1 mmol), DABCO (5) (0.112 g, 1 mmol).
Product: 0.638 g 2-[4-(2-chloroethyl)piperazin-1-yl]-4-
methoxy-6-(NH-Trp(Boc)-Ala-OMe)-1,3,5-triazine (7f),
yield = 98.9%, colorless oil.
Analysis: for C30H41ClN8O6 (645.15): calculated: C
55.9%, H 6.4%, Cl 5.5%, N 17.4%, found: C 55.9%, H
6.4%, Cl 5.5%, N 17.4%.
13C-NMR (75 MHz, CDCl3): δ = 17.6, 38.3, 41.2, 44.0,
47.8, 51.8, 52.3, 53.0, 54.6, 61.4, 163.5, 171.3, 171.7, 172.9
[ppm].
MS: 446.9342 ([M + H]+, C17H29ClN7O5 ; calc. 445.90).
+
Synthesis of 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-
(NH-Lys(Boc)-Ala-OMe)-1,3,5-triazine (7d) Starting material: 2-
chloro-4-methoxy-6-(NH-Lys(Boc)-Ala-OMe)-1,3,5-triazine
(4d) (0.475 g, 1 mmol), DABCO (5) (0.112 g, 1 mmol).
Product: 0.580 g 2-[4-(2-chloroethyl)piperazin-1-yl]-4-
methoxy-6-(NH-Lys(Boc)-Ala-OMe)-1,3,5-triazine (7d),
yield = 98.8%, colorless oil.
Analysis: for C25H43ClN8O6 (587.11): calculated: C
51.1%, H 7.4%, Cl 6%, N 19.1%, found C 51.1%, H 7.4%,
Cl 6%, N 19.1%.
1H-NMR (250 MHz; CDCl3) δ = 1.41 (d, 3H, J = 7.2 Hz,
CH3-CH-), 1.44 (9H, s, (CH3)3-C-), 2.66 (t, 2x2H, J = 7.8 Hz,
-N-CH2-); 2.85 (t, 2H, J = 6.2 Hz, -N-CH2-); 3.15 (d, 2H, J =
6.7 Hz, -CH2-CH-); 3.60 (t, 2x2H, J = 7.8 Hz, -N-CH2-); 3.86
(2, 2H, J = 6.2 Hz, Cl-CH2-); 3.72 (s, 3H, CH3O-); 4.02 (s, 3H,
CH3O-); 4.36 (q, 1H, J = 7.2 Hz, CH3-CH-); 4.91 (t, 1H, J =
6.7 Hz, -CH2-CH-); 7.03–7.98 (m, 5H, arom) [ppm].
13C-NMR (75 MHz, CDCl3): δ = 17.7, 27.7, 28.1, 41.2,
44.1, 47.9, 51.8, 52.3, 53.0, 53.6, 54.5, 81.1, 115,1, 117.1,
118.7, 120.6, 122.7, 123.7, 129.6, 135.4, 149.5, 163.3,
172.4, 172.9 [ppm].
1H-NMR (250 MHz; CDCl3) δ = 1.26–1.57 (m, 4H, -CH2-
CH2-); 1.41 (d, 3H, d, J = 7.2 Hz, CH3-CH-); 1.43 (s, 9H,
(CH3)3C-); 1.93 (dt, 2H, J1 = 7.5 Hz, J2 = 5.1 Hz, -CH-
CH2-); 2.66 (t, 2x2H, J = 7.8 Hz, -N-CH2-); 2.85 (t, 2H, J =
6.2 Hz, -N-CH2-); 3.22 (t, 2H, J = 7.1 Hz, -CH2-NH-); 3.60 (t,
2x2H, J = 7.8 Hz, -N-CH2-); 3.86 (2, 2H, J = 6.2 Hz, Cl-
CH2-); 3.71 (s, 3H, CH3O-); 4.02 (s, 3H, CH3O-); 4.28 (t,
1H, J = 7.5 Hz, -CH-CH2-); 4.36 (q, 1H, J = 7.2 Hz, CH3-
CH-) [ppm].
+
MS: 646.15864 ([M + H]+, C30H42ClN8O6 ; calc. 645.15).
Biology
13C-NMR (75 MHz, CDCl3): δ = 17.7, 21.4, 28.2, 29.3,
29.9, 40.5, 41.2, 44.0, 47.8, 51.8, 52.3, 53.1, 53.6, 54.6,
80.1, 156.2, 162.2, 171.7, 172.5, 172.9 [ppm].
Reagents
RPMI-1640 medium, McCoy’s 5a medium, fetal bovine se-
rum, penicillin and streptomycin were obtained from the
American Type Culture Collection (ATCC) (Manassas, VA).
5-Fluorouracil (5-Fluorouracil-Ebewe) was obtained from
Ebewe (Unterach, Austria).
MS: 588.2016 ([M + H]+, C25H44ClN8O6 ; calc. 587.11).
+
Synthesis of 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-
(NH-Asp(OtBu)-Ala-OMe)-1,3,5-triazine (7e) Starting materi-
al: 2-chloro-4-methoxy-6-(NH-Asp(OtBu)-Ala-OMe)-
1,3,5-triazine (4e) (0.418 g, 1 mmol), DABCO (5)
(0.112 g, 1 mmol). Product: 0.520 g 2-[4-(2-
chloroethyl)piperazin-1-yl]-4-methoxy-6-(NH-
Asp(OtBu)-Ala-OMe)-1,3,5-triazine (7e), yield = 98.2%,
colorless oil.
Cell culture
Cells were purchased from ATCC. Cell line DLD-1 was cul-
tured in RPMI 1640 medium (Sigma, USA), line HT-29 in
McCoy’s 5a medium (Sigma, USA) supplemented with 10%
fetal calf serum (Sigma, USA), penicillin (50 IU, Sigma,
USA) and streptomycin (50 μg/l, Sigma, USA), in an incuba-
tor with 5% CO2 (normoxia), at a relative humidity of 95%, at
Analysis: for C22H36ClN7O6 (530.02): calculated: C
49.9%, H 6.8%, Cl 6.7%, N 18.5%, found: C 49.8%, H
6.7%, Cl 6.7%, N 18.5%.

Invest New Drugs
37 °C (Heraeus, USA). The culture media was changed every
two days. The cells were generally kept in 75 cm3 flasks
(Sarstedt, USA). However, for the experiments the cells were
plated in 100-mm dishes (Sarstedt, USA) with 10 ml of me-
dium. The control was medium with PBS only.
with 10 μg/ml JC-1. The cells were then incubated for 15
min at room temperature in darkness, washed again and resus-
pended in PBS for immediate BD FACSCanto II flow cytom-
etry analysis (Bioscences Systems, CA). The percentage of
cells with disrupted MMP was calculated using FACSDiva
software (Bioscences Systems, CA).
Drug treatment
Western blot analysis for Bax and Bcl-2
Prior to each treatment, DLD-1 and Ht-29 cells were grown
until they reached 50–60% confluence. The cells were then
treated with 5-FU and 7b at different concentrations (1, 5, 10,
50 μM) for 24 or 48 h. Untreated cells were used as the
control.
Cells were lysed in NP-40 buffer (50 mM Tris-HCl (pH 8.0),
150 mM NaCl, 1% Triton X-100 and protease inhibitor cock-
tail (Roche)). The lysate was centrifuged at 10000×g for
20 min at 4 °C. An aliquot (10 μl) of the supernatant was
subjected to electrophoresis in a 10% SDS-polyacrylamide
gel, then transferred to a nitrocellulose membrane (Bio-Rad),
pore size 0.2-μm, according to the method described in the
manual accompanying the unit. The blots were blocked for 1 h
at room temperature with 5% nonfat milk (Bio-Rad) in Tris-
buffered saline, pH 8.0 (Sigma-Aldrich, St. Louis, MO). The
membrane was incubated with mouse anti-Bax (1:400),
mouse anti-Bcl-2 (1:400) or mouse anti-β-actin (1:3000) an-
tibodies from Sigma-Aldrich (St. Louis, MO) in TBS-T
(20 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl
and 0.05% Tween 20) overnight. Alkaline phosphatase-
conjugated goat anti-mouse secondary antibodies (Sigma-
Aldrich, St. Louis, MO) were added at a 1:10000 dilution in
TBS-T and incubated for 1 h with slow shaking. The nitrocel-
lulose was then washed with TBS-T (2 × 10 min) and exposed
to the Sigma-Fast BCIP/NBT reagent. The intensity of the
bands was quantified by densitometric analysis using Image
J 1.45 software (National Institute of Health, USA).
Proliferation assay
A proliferation assay was performed according to the method
described by Mosmann using 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) [26]. Confluent cells
cultured in 6-well plates for 24 or 48 h with various concen-
trations of the studied compounds 7a-7 f. The plates were
washed three times with PBS and then incubated for 4 h in
1 ml of MTT solution (0.5 mg/ml of PBS) at 37 °C in a 5%
CO2 incubator. The medium was removed and 1 ml of 0.1 M
HCl in absolute isopropanol was added to the attached cells.
The absorbance of the converted dye in the living cells was
measured at a wavelength of 570 nm. The viability of DLD-1
and HT-29 cells cultured in the presence of the studied com-
pounds was calculated as a percentage of the control cells. The
experiments were performed in triplicate. The ratio of living to
dead cells with and without the drugs (control) was calculated
for each drug concentration.
Statistical analysis
Quantitative assessment of apoptotic cells
The normally distributed data were presented as mean SD.
Normality of distribution was tested using the Shapiro–Wilk
test. Multiple group comparisons were performed by one-way
analysis of variance and significant differences between
groups were assessed by the Tukey–Kramer test.
Computations were performed using GraphPad 7 Prism soft-
ware (GraphPad Software, Inc., La Jolla, CA, USA).
Colon and breast cells were stained with annexin-V-Fluos and
propidium iodide (FITC Annexin VApoptosis Detection Kit I
BD, Pharmingen). After 10 min of incubation in ice and dark-
ness, the samples were analyzed with a FACSCalibur flow
cytometer (BD). The results were verified by microscopy
(Olympus CH30) (Annexin V-FITC Fluorescence
Microscopy Kit BD, Pharmingen), counting 100 consecutive
cells at 200× magnification. The percentage of apoptotic cells
was calculated based on the average taken from both study
methods.
Results
Preparation of new analogues of nitrogen mustard
derivatives containing dipeptide substituents
on 1,3,5-triazine ring 7a-f
Analysis of mitochondrial membrane potential
Dysfunction of the mitochondrial membrane potential (MMP)
was assessed using the lipophilic cationic probe 5,5′,6,6′-
tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine io-
dide (JC-1 MitoScreen kit; BD Biosciences). Briefly, unfixed
cells were washed and resuspended in PBS supplemented
The synthesis of new analogues of nitrogen mustards 7a-f
containing the 1,3,5-triazine ring substituted with dipeptide
residue is a multi-step process (Scheme 1). The first stage is
the synthesis of dipeptides 1. This was made in a solution

Invest New Drugs
Scheme 1 Multi-stage synthesis of nitrogen mustard derivatives 7a-f containing dipeptide substituents on the 1,3,5-triazine ring
using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium toluene-4-sulfonate (DMT/NMM/
TosO−) [27] as a coupling reagent. After removing the protec-
tive groups from the amine function, the dipeptide derivatives
2 were reacted with 2,4-dichloro-6-methoxy-1,3,5-triazine
(DCMT, 3) in the presence of solid NaHCO3 as a hydrogen
chloride acceptor. Derivatives 4 were obtained with yields
ranging from 95.8% to 98.7%. Due to the presence of halogen
on the ring of 1,3,5-triazine, derivatives 4 undergo
quaternization with tertiary amines, leading to the formation
of triazinylammonium chloride 6. However, due to their im-
permanence, they are easily dealkylated [28, 29]. Use of
DABCO as a tertiary amine enables derivatives containing
the 2-chloroethylamine substituent characteristic of nitrogen

Invest New Drugs
mustard to be obtained as a result of the bicyclic ring opening
process [24, 25]. Finally, six new analogues of nitrogen mus-
tards 7 with different dipeptide substituents on the 1,3,5-tri-
azine ring were prepared (Fig. 2).
IC50 values for 7b were at least 2-fold lower than those for 5-
FU in both tested lines after 48 h of incubation (Table 1).
Triazine derivative induces apoptosis
through intracellular signaling pathway attenuation
Triazine derivatives decrease colon cancer viability
To quantitate the extent of apoptosis, the cells were stained with
annexin V, a phospholipid-binding protein that has high affinity
for phosphatidylserine (PS) and enables identification of cells in
the early stages of apoptosis. Propidium iodide (PI) was used to
identify late apoptotic and dead cells. Dual-labeling annexin V/
propidiumiodide (PI) allowed us to assess the apoptosis status of
DLD-1 and HT-29 cells after 48 h of 7b and 5-FU treatment. We
observed that the tested compound significantly induced pro-
grammed cell death in DLD-1 and HT-29 cells in comparison
to the control (untreated cells). We also found that the pro-
apoptotic potential of 7b was similar to that of 5-FU. For 5-
In the first stage of our in vitro study, we investigated the
effective cytotoxic dose of triazine derivatives. The MTT test
indicated that all of the compounds exhibited cytotoxic ef-
fects, but there were differences in the sensitivity of colon
cancer cells (Fig. 3). Prolonged incubation for 48 h increased
the cytotoxic effects of all the compounds.
Of all the synthesized compounds, 7b showed the highest
activity against cells from both lines. Therefore, we used this
derivate in further research. The DLD-1 cell lines were found
to be more sensitive to compound 7b than the HT-29 line. The
Fig. 2 Structures of the new
triazine-peptide-mustards 7a-f

Invest New Drugs
a
DLD-1 cells - 24 h
b
HT-29 cells - 24 h
^^^
^^^
100
50
0
^
**
100
50
0
^
**
*
*
***
***
^^^
**
***
***
***
^^^
***
***
***
^^
***
^^
***
***
***
^^^
***
^^^
***
***
***
***
***
^^
***
***
***
^^
***
***
***
***
***
^^^
***
^^^
***
^^^
***
^^^
***
***
c
DLD-1 cells - 48 h
d
HT-29 cells - 48 h
^^^
^^^
***
100
50
0
^^^
^^
*
100
50
0
^^^
*
^^^
**
**
***
***
^^^
***
***
***
^^^
***
^^
***
***
***
***
***
***
^
***
***
***
***
***
***
^^^
***
^^^
***
^^^
***
^^
***
^^
***
^
***
^^^
***
***
^^^
***
***
***
***
***
***
***
***
***
Fig. 3 Effects of 5-fluorouracil (5-FU) and triazine derivatives 7a-b on the viability of DLD-1 (a, c) and HT-29 (b, d) cells after 24 h (a, b) or 48 h (c, d)
of incubation. Results are presented as means SD, n = 6, *P < 0.05 (vs. Con), ^P < 0.05 (vs. 5-FU)
FU and 7b in DLD-1, we observed 61.5% and 66.5% viable
cells and 32.6% and 30.0% apoptotic cells, respectively. For 5-
FU and 7b in HT-29, we observed 64.4% and 67.6% viable cells
and 29.6% and 30.1% apoptotic cells, respectively (Fig. 4a).
In order to investigate the cellular mechanism underlying
7b-induced intrinsic apoptosis in DLD-1 and HT-29 using
flow cytometry analysis and lipophilic fluorochrome JC-1,
we estimated the alterations of mitochondrial transmembrane
potential (MMP). As shown in Fig. 4b, 7b increased the num-
ber of cells with decreased levels of MMP in both DLD-1 (25
0.6%) and HT-29 (26,1 0.3%) cells after 48 h of incuba-
tion. For untreated cells, the decrease in mitochondrial poten-
tial was 7.1% in DLD-1 and 7.7% in HT-29. We observed that
the decreases in MMP caused by 7b and 5-FU were similar.
These results are in line with those obtained in the Annexin-V
and PI assay.
Treatment for 48 h with 7b contributed to a marked in-
crease in the expression of BAX compared with control cells
in both the DLD-1 and HT-29 lines. We observed a decrease in
the expression of BCL-2 after incubation with 7b compared
with the control cells. Incubation with 7b led to an increase of
BCL-2 expression both in DLD-1 and in HT-29 cells. This
effect was more pronounced than after incubation of cells with
Table 1 Antiproliferative activity of 5-fluorouracil (5-FU) and com-
pounds 7a-f
IC50 [μM]
DLD-1
HT-29
5-FU
7a
27.22
30.27
13.71
23.80
24.54
26.33
26.85
21.72
36.46
17.78
28.06
29.54
32.85
34.02
7b
7c
7d
7e
7f

Invest New Drugs
a
a
percent of cells with early and late
DLD-1
HT-29
late apoptoꢀc cells
necroꢀc cells
early apoptosis
viable cell
Bax
23 kDa
CON
5-FU
7b
propidium
iodide
48h
26 kDa
42 kDa
Bcl-2
Β-acꢀn
b
2.0
Annexin V-FITC
1.5
1.0
0.5
0.0
b
normal MMP
decrease MMP
percent of cells with
Con
5-FLU
7b
JC-1
red
b
b
7
48h
7
FU
-
FU
-
5
on
on
C
5
C
HT-29
DLD-1
Fig. 5 Immunoblotting analysis for BAX and Bcl-2 in DLD-1 and HT-29
cells treated with 7b (10 μM) for 48 h (a). The samples used for electro-
phoresis consisted of 20 μg of protein from six pooled cell extracts (n =
6). Bax/Bcl-2 protein expression was performed using a scanning
densitometer (b)
JC-1 green
Fig. 4 Representative flow cytometry dot-plots for Annexin V-FITC as-
say of DLD-1 and HT-29 cells incubated with 7b (10 μM) for 48 h (mean
SD; n = 3). Live cells appear in the lower left corner of the plots. Early
apoptotic cells appear in the lower right corner. Necrotic cells appear in
the upper-left corner. Dead cells appear in the upper-right corner (a).
Representative dot-plots presenting the loss of mitochondrial membrane
potential (MMP) of DLD-1 and HT-29 cells incubated with 7b (10 μM)
for 48 h (mean SD; n = 3). Cells with normal MMT are shown on the
right-hand side of the plots, cells with decreased MMT on the left-hand
side of the plots (b)
numerous reports, including our research. In this paper,
we reported the synthesis a new series of trisubstituted
triazine derivatives. The investigated compounds 7a-f
displayed an interesting spectrum of activity. All of
them exhibited cytotoxic effects against DLD-1 and
HT-29 colon cancer cells. Compound 7b, in which the
1,3,5-triazine ring was substituted with an Ala-Ala-OMe
group and one 2-chloroethylamino moiety, was the most
active, with IC50 values of 13.71 μM. This was at least
2-fold lower than the values for 5-FU, for which the
IC50 was equal to 27.22 μM. Because of its activity,
the mechanism of 7b action was examined in detail. It
was determined that this triazine derivative induces ap-
optosis through intracellular signaling pathway
attenuation.
5-FU. An increase in the BAX/BCL-2 ratio was linked with
increased apoptosis. The ratio of BAX to BCL-2 shifted in
favor of BAX compared with the control, which confirms
the intensification of this process (Fig. 5).
Our research confirms the value of triazine deriva-
tives as potential anticancer agents. Further investiga-
tions are needed to ascertain whether the studied com-
pounds should be considered for possible therapeutic
applications.
Conclusion
Hybrid compounds based on nitrogen mustard 1,3,5-tri-
azine have great therapeutic potential, as confirmed by

Invest New Drugs
Funding The work was supported by Polish Ministry of Science and
Higher Education (grant N N405 355537).
12. Ben Abid F, Gazzah A, Ousbane A, Gutierrez M, Brain E (2007)
Les alkylants. Oncologie 9:751–757
13. Di Francesco AM, Hargreaves RH, Wallace TW, Mayalarp SP,
Hazrati A, Hartley JA, Butler J (2000) The abnormal cytotoxicities
of 2,5-diaziridinyl-1,4-benzoquinone-3-phenyl esters. Anticancer
Drug Des 15:347–359
14. Vedejs E, Naidu BN, Klapars A, Warner DL, Li VS, Na Y, Kohn H
(2003) Synthetic enantiopure aziridinomitosenes: preparation, reac-
tivity, and DNA alkylation studies. J Am Chem Soc 125:15796–
15806 and references cited therein
15. Maeda M, Ligo M, Tsuda H, Fujita H, Yonemura Y, Nakagawa K,
Endo Y, Sasaki T (2000) Antimetastatic and antitumor effects of 2,
4-diamino-6-(pyridine-4-yl)-1,3,5-triazine (4PyDAT) on the high
lung metastatic colon 26 tumor in mice. Anticancer Drug Des 15:
217–223
Compliance with ethical standards
Conflict of interest Agnieszka Wróbel declares that she has no conflict
of interest. Beata Kolesińska declares that she has no conflict of interest.
Justyna Frączyk declares that she has no conflict of interest. Zbigniew J.
Kamiński declares that he has no conflict of interest. Anna Tankiewicz-
Kwedlo declares that she has no conflict of interest. Justyna
Hermanowicz declares that she has no conflict of interest. Robert
Czarnomysy declares that he has no conflict of interest. Dawid
Maliszewski declares that he has no conflict of interest. Danuta
Drozdowska declares that she has no conflict of interest.
16. Paquin I, Raeppel S, Leit S, Gaudette F, Zhou N, Moradei O,
Saavedra O, Bernstein N, Raeppel F, Bouchain G, Fréchette S,
Woo SH, Vaisburg A, Fournel M, Kalita A, Robert MF, Lu A,
Trachy-Bourget MC, Yan PT, Liu J, Rahil J, MacLeod AR,
Besterman JM, Li Z, Delorme D (2008) Design and synthesis of
4-[(s-triazin-2-ylamino)methyl]-N-(2-aminophenyl)-benzamides
and their analogues as a novel class of histone deacetylase inhibi-
tors. Bioorg Med Chem Lett 18:1067–1071
17. Chandra M, Sahay AN, Pandey DS, Tripathi RP, Saxena JK, Reddy
VJM, Puerta MC, Valerga P (2004) Potential inhibitors of DNA
topoisomerase II. Ruthenium (II) poly-pyridyl and pyridyl-azine
complexes; potential DNA cleaving agents. J Organomet Chem
689:2256–2267
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed consent For this type of study, formal consent is not required.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
18. Riou JF, Guittat L, Mailliet P, Laoui A, Renou E, Petitgenet O,
Mégnin-Chanet F, Hélène C, Mergny JL (2002) Cell senescence
and telomere shortening induced by a new series of specific G-
quadruplex DNA ligands. Proc Natl Acad Sci U S A 99:2672–2677
19. Gomez D, Aouali N, Londono-Vallejo A, Lacroix L, Megnin-
Chanet F, Lemarteleur T, Douarre C, Shin-ya K, Mailliet P,
Trentesaux C, Morjani H, Mergny JL, Riou JF (2003) Resistance
to the short term anti-proliferative activity of the g-quadruplex li-
gand 12459 is associated with telomerase overexpression and telo-
mere capping alteration. J Biol Chem 278:50554–50562
20. Mandal S, Bérubé G, Asselin E, Mohammad I, Richardson VJ,
Gupta A, Pramanik SK, Williams AL, Mandal SK (2007) A novel
series of potent cytotoxic agents targeting G2/M phase of the cell
cycle and demonstrating cell killing by apoptosis in human breast
cancer cells. Bioorg Med Chem Lett 17:4955–4960
References
1. Sanderson BJ, Shield AJ (1996) Mutagenic damage to mammalian
cells by therapeutic alkylating agents. Mutat Res 355:41–57
2. Chabner BA, Allegra CJ, Curt GA, Calabresi P (1996)
Antineoplastic agents. In: Hardman JG, Gilman AG, Limbird LE
(eds) Goodman & Gilman’s the pharmacological basis of therapeu-
tics, 9th edn. McGraw-Hill Press, New York, pp 1233–1288
3. Colvin OM (1999) An overview of cyclophosphamide develop-
ment and clinical applications. Curr Pharm Des 5:555–560
4. Povirk LF, Shuker DE (1994) DNA damage and mutagenesis in-
duced by nitrogen mustards. Mutat Res 318:205–226
5. Rajski SR, Williams RM (1998) DNA cross-linking agents as anti-
tumor drugs. Chem Rev 98:2723–2796
21. Liu B, Sun T, Zhou Z, Du L (2015) A systematic review on antitu-
mor agents with 1,3,5-triazines. Med Chem 5:131–148
22. Kolesińska B, Barszcz K, Kamiński ZJ, Drozdowska D, Wietrzyk
J, Świtalska M (2012) Synthesis and cytotoxicity studies of bifunc-
tional hybrids of nitrogen mustards with potential enzymes inhibi-
tors based on melamine framework. J Enzyme Inhib Med Chem 27:
619–627
23. Kolesińska B, Drozdowska D, Kamiński ZJ (2008) The new ana-
logues of nitrogen mustard with one, two or three 2-
chloroethylamino fragments. Reactions with nucleophiles. Acta
Pol Pharm 65:709–714
6. Chabner BA, Longo DL (1996) Cancer chemotheraphy and
biotheraphy: Principles and practice. Lippincot-Raven,
Philadelphia
7. Balcome S, Park S, Quirk DR, Hafner L, Phillips L, Tretyakova N
(2004) Adenine-containing DNA−DNA cross-links of antitumor
nitrogen mustards. Chem Res Toxicol 17:950–962
8. Lawley PD, Brookes P (1965) Molecular mechanism of the cyto-
toxic action of difunctional alkylating agents and of resistance to
this action. Nature 206:480–483
24. Frączyk J, Kolesińska B, Świontek M, Lipiński W, Drozdowska D,
Kamiński ZJ (2016) Synthesis of arylamino-1,3,5-triazines func-
tionalized with alkylatin 2-chloroethylamine fragments and studies
of their cytotoxicity on the breast cancer MCF-7 cell line. Anti
Cancer Agents Med Chem 16:1435–1444
9. Chen Y, Jia Y, Song W, Zhang L (2018) Therapeutic potential of
nitrogen mustard based hybrid molecules. Front Pharmacol 9.
https://doi.org/10.3389/fphar.2018.01453
10. Nieto Y (2005) DNA-binding agents. Cancer Chemother Biol
Response Modif 22:163–203
11. Kaldor JM, Day NE, Hemminki K (1988) Quantifying the carcino-
genicity of antineoplastic drugs. Eur J Cancer Clin Oncol 24:703–
711
25. Krętowski R, Drozdowska D, Kolesińska B, Kamiński ZJ, Frączyk
J, Cechowska-Pasko M (2019) The cellular effects of novel triazine
nitrogen mustards in glioblastoma LBC3, LN-18 and LN-229 cell

Invest New Drugs
lines. Investig New Drugs. https://doi.org/10.1007/s10637-018-
synthesis of N,N-disubstituted melamines (triazine triskelions) un-
der mild reaction condition. Tetrahedron 65:3573–3576
0712-8
29. Kolesińska B, Kamiński ZJ (2008) Transformation of tertiary
amines into alkylating reagents by treatment with 2-chloro-4,6-
dimethoxy-1,3,5-triazine. A synthetic application of side-reaction
accompanying coupling by means of 4-(4,6-dimethoxy-[1,3,
5]triazin-2-yl)-4-methyl-morpholin-4-ium chloride (DMTMM).
Pol J Chem 82:2115–2123
26. Mosmann T (1983) Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63
27. Kolesińska B, Różniakowski KK, Fraczyk J, Relich I, Papini AM,
Kaminski ZJ (2015) The effect of counterion and tertiary amine on
the efficiency of N-triazinylammonium sulfonates in solution and
solid-phase peptide synthesis. Eur J Org Chem 2015(2):401–408
28. Kolesińska B, Kamiński ZJ (2009) The umpolung of substituent
effect in nucleophilic aromatic substitution. A new approach to the
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.