Design, Synthesis, and Biological Evaluation of Coupled Bioactive Scaffolds as Potential Anticancer Agents for Dual Targeting of Dihydrofolate Reductase and Thioredoxin Reductase

Article abstract of DOI:10.1021/acs.jmedchem.6b01253

The dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) enzymes are involved in the process of tumor cell growth and survival. The 4,6-diamino-1,2-dihydro-1,3,5-triazine scaffold is well-established as a useful scaffold for DHFR inhibition, while chalcones have been reported to be inhibitors of TrxR. In this study, 15 novel compounds designed by the structural combination of the 4,6-diamino-1,2-dihydro-1,3,5-triazine and chalcone scaffolds via a diether linker were successfully synthesized and characterized. All of the compounds demonstrated dual inhibition against DHFR and TrxR when they were assessed by in vitro enzyme assays. The compounds also exhibited antiproliferative activity against the MCF-7 and HCT116 cells. The more potent analogs 14 and 15 were found to inhibit cellular DHFR and TrxR activities in HCT116 cells. Therefore, this study provided compelling evidence that 14 and 15 could exert their anticancer property via multitarget inhibition at the cellular level.

Full text of DOI:10.1021/acs.jmedchem.6b01253

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Article
Design, Synthesis and Biological Evaluation of Coupled
Bioactive Scaffolds as Potential Anticancer Agents for Dual
Targeting of Dihydrofolate Reductase and Thioredoxin Reductase
Hui-Li Ng, Xiang Ma, Eng-Hui Chew, and Wai-Keung Chui
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01253 • Publication Date (Web): 08 Feb 2017
Downloaded from http://pubs.acs.org on February 9, 2017
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Design, Synthesis and Biological Evaluation of Coupled Bioactive Scaffolds as Potential
Anticancer Agents for Dual Targeting of Dihydrofolate Reductase and Thioredoxin
Reductase
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†,
‡
*,†
*,†
∥
HuiꢀLi Ng , Xiang Ma , EngꢀHui Chew , WaiꢀKeung Chui
† Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science
Drive 4, Singapore 117543
‡
School of Pharmacy, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, Hubei 430030, China
∥ Present address: School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic,
535 Clementi Road, Singapore 599489
*
Corresponding Authors: EngꢀHui Chew (Eꢀmail: phaceh@nus.edu.sg; Phone: +65
65161955; Fax: +65 67791554) and WaiꢀKeung Chui (Eꢀmail: phacwk@nus.edu.sg; Phone:
+65 65162933; Fax: +65 67791554)
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ABSTRACT
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The dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) enzymes are involved
in the process of tumor cell growth and survival. The 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazine
scaffold is wellꢀestablished as a useful scaffold for DHFR inhibition, while chalcones have
been reported to be inhibitors of TrxR. In this study, 15 novel compounds designed by the
structural combination of the 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazine and chalcone scaffolds
via a diꢀether linker were successfully synthesized and characterized. All of the compounds
demonstrated dual inhibition against DHFR and TrxR when they were assessed by in vitro
enzyme assays. The compounds also exhibited antiproliferative activity against the MCFꢀ7
and HCT116 cells. The more potent analogs 14 and 15 were found to inhibit cellular DHFR
and TrxR activities in HCT116 cells. Therefore, this study provided compelling evidence that
14 and 15 could exert their anticancer property via multiꢀtarget inhibition at the cellular level.
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INTRODUCTION
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Combination chemotherapeutic regimens aiming at multiple molecular targets are commonly
used in the treatment of cancer to improve efficacy, decrease toxicity, and prevent the
development of drug resistance.1 An alternative strategy which is gaining interest in drug
discovery is the development of a single chemical entity that contains a combination of
pharmacophores that are capable of modulating multiple targets simultaneously. This strategy
of using a single multiꢀtargeted agent named as a “designed multiple ligand”, bears the
advantages of circumventing the problems of complicated pharmacokinetic and
pharmacodynamics (PK/PD) relationships and drugꢀdrug interactions associated with
combination therapy.2
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Dihydrofolate reductase (DHFR) has long been recognized as a target for the treatment of
certain cancers. DHFR is an essential enzyme component of the folate pathway that catalyzes
the reduction of dihydrofolic acid (DHF) to tetrahydrofolic acid (THF). THF and its
derivatives are cofactors in a number of oneꢀcarbon transfer reactions leading to the synthesis
of several nucleic acids and amino acids.3 The wellꢀknown DHFR inhibitor, methotrexate
(MTX), has been used for the treatment of cancer for more than 60 years. MTX is transported
into cells by the reduced folate carrier (RFC).4,5 MTX is then intracellularly polyglutamylated
by folylpolyglutamate synthetase (FPGS), leading to its intracellular accumulation.4,5
However, emergence of acquired resistance to MTX through a reduction in its active
transport, a decrease in its polyglutamylation, overexpression of DHFR, or mutations in the
DHFR gene has limited MTX’s uses in the clinical setting.5 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀ
triazines are lipophilic DHFR inhibitors which have previously demonstrated antiproliferative
activity on cancer cells.6ꢀ8 These compounds cross the cell membrane via passive diffusion
and are not polyglutamylated like MTX. Hence, they are unaffected by drug resistance
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arising from an impaired active transport mediated by RFC or a decrease in
polyglutamylation due to low levels of FPGS.9 Previously, our laboratory has reported the
synthesis and antiproliferative activity of 17 (Figure 2), a 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀ
triazine, which is a spiro analogue of the DHFR inhibitor WR99210.10 17 was found to
possess strong in vitro DHFR inhibition with an IC50 value of 2.3 nM against bovine
DHFR.7,8 Therefore, 17 forms the lead for the design of the new compounds in this study.
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More recently, thioredoxin reductase (TrxR) has been recognized as an attractive target for
anticancer drug development. Together with its substrate thioredoxin (Trx), TrxR maintains
redox homeostasis in cells, preventing oxidative damage and mutation.11,12 Both Trx and
TrxR have been reported to be overexpressed in numerous cancer cells, and observed to be
associated with poor prognosis and resistance to chemotherapy.13ꢀ16 The validity of TrxR as a
target for anticancer treatment has been demonstrated by experiments showing that
knockdown of TrxR in cancer cells brings about reversal of tumor phenotype, as well as
inhibition of DNA replication and cancer cell growth.17ꢀ19 The nucleophilic selenocysteine
(Sec) residue located on the flexible Cꢀterminal active site of mammalian TrxR makes it a
target for many electrophilic compounds.20ꢀ21
Chalcones are naturallyꢀoccurring compounds which have been reported to exert anticancer
activity through a number of mechanisms including inhibition of tubulin polymerization,22
blockade of the nuclear factorꢀkappa B (NFꢀκB) signalling pathway,23 as well as inhibition of
receptor tyrosine kinases such as epidermal growth factor receptor (EGFR)24 and vascular
endothelial growth factor receptorꢀ2 (VEGFRꢀ2)25. Chalcones have recently been reported to
be selective, irreversible inhibitors of TrxR that demonstrated antiproliferative activity
against selected human cancer cell lines, with the α,βꢀunsaturated carbonyl moiety of
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chalcones postulated to react with the Sec residue of the TrxR enzyme via Michael addition
reaction to form covalent adducts.26,27
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Based on the prior knowledge that DHFR and TrxR are valid molecular targets for anticancer
therapies, our laboratory has been focusing on exploiting the combination of DHFRꢀ
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inhibiting
4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazine
and
TrxRꢀinhibiting
chalcone
pharmacophoric scaffolds to produce novel single molecules that would elicit
antiproliferative effects on cancer cells at least partly through simultaneous inhibition of
DHFR and TrxR. In a previous study, we reported the synthesis of four dihydrotriazineꢀ
chalcone compounds (Figure 1A) that were designed based on merging the bioactive
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scaffolds. These compounds were found to display growth inhibitory effects like their
parent dihydrotriazine component, but lack cytotoxicity that is characteristic of their parent
chalcone component. Moreover, the growth inhibitory activities failed to correlate with their
in vitro DHFR and TrxR inhibitory activities, which underscored the suboptimal structural
design of combining the two pharmacophores in a single chemical compound using the
merging approach. In this study, we report the design and synthesis of fifteen dualꢀtarget
inhibitors of DHFR and TrxR containing the bioactive 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazine
and chalcone scaffolds linked by flexible diꢀether linkers of varying lengths (Figure 1B). This
work has led to the identification of 14 and 15 as the compounds having the greatest
cytotoxic activities against humanꢀderived HCT116 colorectal and MCFꢀ7 breast carcinoma
cells. The cytotoxic effect is correlated to strong inhibitory activities against DHFR and
TrxR. Therefore the results provided evidence to support the design strategy of including a
flexible linker between the bioactive scaffolds, which has likely facilitated their optimal
interaction with the targets.
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Figure 1. Structures of (A) dihydrotriazineꢀchalcone compounds 6a, 6b, 11a and 11b
(synthesis reported in Ref No. 28) and (B) linked dihydrotriazineꢀchalcone compounds 1ꢀ15
containing diꢀether linkers of varying lengths.
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RESULTS AND DISCUSSION
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Design and Synthesis of Compounds
Scheme 1.
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(A) Synthesis of 4,6ꢀdiaminoꢀ1ꢀhydroxyꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazine hydrochlorides. Reagents
and conditions: (a) EtOH, Conc. HCl; (b) H2 (1 atm), 50% aq EtOH, PtO2. (B) Synthesis of
bromoalkoxychalcones. Reagents and conditions: (a) NaOH, EtOH, H2O, r.t.; (b) K2CO3,
acetone, reflux. (C) Synthesis of 1ꢀ15. Reagents and conditions: (a) NaOH, MeOH, reflux;
(b) Bromoalkoxychalcone, DMF, r.t.; (c) Conc HCl, pH = 1.
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Figure 2. Structures of 16, 17, 6h, 18 and MTX.
Compounds 1-15 were prepared by independent synthesis of the dihydrotriazine ring and
chalcone, followed by a convergent fragment coupling step (Scheme 1). Firstly, 4,6ꢀ
diamiinoꢀ1ꢀhydroxyꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazine hydrochlorides (21) were obtained as
previously reported8,29 through the synthesis of 4,6ꢀdiaminoꢀNꢀbenzyloxyꢀ1,2ꢀdihydroꢀ1,3,5ꢀ
triazine hydrochloride (20) from refluxing Oꢀbenzylhydroxylamine hydrochloride (19),
cyanoguanidine and a ketone in the presence of concentrated hydrochloric acid. This was
followed by the reduction of the benzyloxy dihydrotriazine hydrochloride (20) using
hydrogen gas in the presence of the catalyst platinum (IV) oxide (Scheme 1A).
At the same time, hydroxyꢀsubstituted chalcones (26, 27) were prepared from the sodium
hydroxideꢀcatalyzed ClaisenꢀSchmidt condensation reaction between acetophenone (25) or
hydroxyꢀsubstituted acetophenone (22) and benzaldehyde (23) or hydroxyꢀsubstituted
benzaldehyde (24) (Scheme 1B). The reaction was quenched by the addition of water and
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neutralization of the reaction mixture using a 2N HCl solution. The obtained hydroxyꢀ
substituted chalcones (26, 27) were then alkylated with suitable dibromoalkanes in the
presence of potassium carbonate to yield bromoalkoxychalcones (28, 29) using a method
adapted from that reported by Sodani et al.30 Excess dibromoalkane was used in a molar ratio
of 3:1 (dibromoalkane to chalcone) to decrease the formation of the disubstituted byproduct
produced by the reaction of two chalcone molecules with one molecule of dibromoalkane. In
addition, a dropping funnel was used to add the hydroxyꢀsubstituted chalcones (26, 27)
slowly into the reaction mixture to ensure that the dibromoalkanes were constantly in excess
during the reaction. When the reaction was complete (as determined by TLC), acetone and
excess dibromoalkane were removed under vacuum, and the residue was washed with cold
water and stirred well. The solid residue was filtered and washed with dilute NaOH solution,
followed by water. The residue was then dissolved in boiling ethanol and filtered hot to
remove any disubstituted byproduct present, which would have been insoluble in ethanol.
The filtrate was collected and ethanol was removed under vacuum to yield the solid
bromoalkoxychalcones (28, 29).
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Finally, the two fragments were coupled via Williamson etherification (Scheme 1C). Sodium
hydroxide was used to deprotonate the
Nꢀhydroxy group of the 4,6ꢀdiaminoꢀ1ꢀhydroxyꢀ1,2ꢀ
dihydroꢀ1,3,5ꢀtriazine hydrochloride (21). After the deprotonation, methanol was removed
and DMF was used for the SN2 substitution reaction. Bromoalkoxychalcone (28, 29) was
added together with DMF and the mixture was stirred overnight at room temperature. For
spiropentyl compounds 13, 14 and 15, potassium carbonate was also added to the reaction
mixture to increase the yield of the target compounds. Finally, the pH of the reaction mixture
was adjusted to pH 1 to obtain the target compounds 1ꢀ15 as hydrochloride salts. The solid
products were purified by recrystallization to obtain clean compounds at 4ꢀ50% yields. The
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compounds 16, 6h and 18 (Figure 2) were also synthesized for biological evaluation
alongside 1ꢀ15 (Supporting information, Scheme S1 to S3).
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Evaluation of in vitro DHFR and TrxR inhibitory activities of compounds 1-15
Compounds 1ꢀ15 were evaluated for DHFR inhibitory activity using recombinant human
DHFR, and the obtained IC50 values are shown in Table 1. The DHFR IC50 values of 16, 17,
6h, 18, and MTX were also evaluated and presented for comparison. All of the synthesized
compounds 1ꢀ15 showed potent DHFR inhibitory activity with IC50 values in the nanomolar
range of concentrations. These values were comparable to that of the 4,6ꢀdiaminoꢀ1,2ꢀ
dihydroꢀ1,3,5ꢀtriazines 16 and 17. Most of the compounds also exhibited DHFR inhibitory
efficacy comparable to MTX, especially compounds bearing linkers containing 3 or 4 carbon
atoms (5-12, 14-15). As expected, the chalcone compound 18 did not possess DHFR
inhibitory effect. In general, DHFR inhibitory activity improved with increasing linker length
from 2 to 4 carbon atoms with inhibitory activity against the enzyme significantly augmented
by 1.5 to 18 folds as the linker length increased from 2 to 3 carbon atoms. When linker length
was further increased from 3 to 4 carbon atoms, DHFR inhibitory activity of the compounds
bearing 4ꢀcarbon linkers were either improved by around 2ꢀfold (for 9 and 10) or similar (for
11 and 12) when compared to the corresponding compounds bearing 3ꢀcarbon linkers (5-8).
13, 14 and 15 bearing the cyclopentyl substituent at R1 and R2 of the triazine moiety had
produced stronger DHFR inhibition than their corresponding compounds (2, 6, 10) bearing
the dimethyl substituent. No consistent trend in correlation was observed between DHFR
inhibitory activity and the position of attachment of the linker to the chalcone moiety.
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Table 1. Inhibitory activities of compounds on recombinant human DHFR and rat liver TrxR
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O
O
NH2
NH2
B
O
O
n
A
O
O
n
A
B
or
N
N
N
N
R1
R1
.HCl
9
.HCl
H2N
N
H2N
N
R2
R2
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Attachment to
linker
TrxR IC50
(30 min)
(µM)
TrxR IC50
(60 min)
(µM)
DHFR IC50
(µM)
Cpd
n
R1
R2
Ring
Position
1
2
2
2
2
3
3
3
3
4
4
4
4
2
3
4
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
CH3 CH3
ꢀ(CH2)4ꢀ
A
A
B
B
A
A
B
B
A
A
B
B
A
A
A
4
3
4
3
4
3
4
3
4
3
4
3
3
3
3
0.0141 ± 0.0022 43.3 ± 10.9 20.4 ± 3.4
2
0.0356 ± 0.0026 10.8 ± 2.7
0.0534 ± 0.0014 29.8 ± 5.0
0.0592 ±0.0141 5.7 ± 0.2
6.9 ± 2.1
21.1 ± 8.4
4.0 ± 0.6
3
4
5
0.0092 ± 0.0010 49.0 ± 15.0 32.4 ± 7.7
6
0.0090 ± 0.0012 10.8 ± 2.2
0.0064 ± 0.0003 35.9 ± 5.4
0.0032 ± 0.0003 4.1 ± 1.5
0.0034 ± 0.0004 44.5 ± 9.1
0.0042 ± 0.0002 11.2 ± 1.3
8.2 ± 2.7
25.7 ± 4.2
2.8 ± 0.9
27.0 ± 4.4
6.7 ± 1.8
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8
9
10
11
12
13
14
15
16
17
6h
18
0.0040 ± 0.0005 31.6 ± 10.1 21.1 ± 7.7
0.0048 ± 0.0012 6.0 ± 1.2
0.0131 ± 0.0023 8.0 ± 1.6
0.0061 ± 0.0021 7.5 ± 0.9
0.0026 ± 0.0008 9.2 ± 1.2
0.0053 ± 0.0012 N.D.
0.0199 ± 0.0061 N.D.
0.0039 ± 0.0008 N.D.
3.8 ± 1.2
4.6 ±1.1
4.6 ± 0.3
5.9 ± 1.3
N.D.
ꢀ(CH2)4ꢀ
ꢀ(CH2)4ꢀ
N.D.
N.D.
N.D.
46.4 ± 9.9
26.7 ± 4.3
MTX
0.0079 ± 0.0002 50.8 ± 16.7 20.9 ± 3.6
Data reported are mean values taken from 3 independent experiments and standard deviation.
N.D.: Not detected.
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The compounds were also evaluated for in vitro inhibitory activity against the rat TrxR by
incubating with the enzyme for 30 and 60 min separately and the IC50 values obtained are
presented in Table 1 as well. Compounds 1ꢀ15 exhibited in vitro TrxR inhibition in the
micromolar range of concentrations for both time points. The compounds were either as
potent as or more potent than the unsubstituted chalcone 18. The experiment design also
confirmed that the presence of the α,βꢀunsaturated carbonyl moiety was crucial for TrxR
inhibitory activity, as demonstrated by the lack of activity in 6h which did not contain the
enone moiety (Table 1). As expected, the 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazines 16 and 17
did not inhibit TrxR. These observations are essential to demonstrate that the dual enzymatic
inhibitory action is primarily due to the presence of the two pharmacophores in the single
molecule.
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The position of attachment of the linker to the chalcone moiety was observed to be a crucial
factor influencing TrxR inhibitory activity. It was observed that the TrxR inhibitory efficacy
followed a decreasing trend with the following analogs: attachment of linker to 3ꢀposition of
ring B > 3ꢀposition of ring A > 4ꢀposition of ring B > 4ꢀposition of ring A. A possible
explanation for this observation could be the electronic effect of the alkoxy linker.
Attachment of the alkoxy linker to the metaꢀposition of the phenyl ring gave rise to an
inductive electronꢀwithdrawing effect, while attachment to the paraꢀposition produced a
mesomeric electron donating effect. The inductive electronꢀwithdrawing effect of the alkoxy
linker at the metaꢀposition intensified the δ+ charge on the βꢀcarbon atom of the α,βꢀ
unsaturated carbonyl moiety, thereby potentially increasing its propensity to react with
nucleophiles such as the Sec residue in the Cꢀterminal active site of the TrxR enzyme. Since
ring B of the chalcone is closer to the βꢀcarbon atom, this electronꢀwithdrawing effect is
stronger when the alkoxy linker is attached to the 3ꢀposition of ring B than to ring A, hence
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giving rise to analogs possessing stronger TrxR inhibitory activity. Notably, the length of the
diꢀether linker and the alkyl substituent at R1 and R2 did not affect TrxR inhibitory activity.
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Evaluation of antiproliferative activity against human-derived cancer cell lines
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Since the results from the in vitro enzyme activity assays provided strong evidence that
compounds 1ꢀ15 were inhibitors of both DHFR and TrxR; the compounds were then
evaluated for antiproliferative activity against the human HCT116 colorectal and MCFꢀ7
breast cancer cell lines. These two cell lines are known to be sensitive to MTX with IC50
values in the nanomolar range.31 In addition, chalcones have previously been shown to inhibit
TrxR in these two cell lines, resulting in cell death.26 The GI50 values (concentration of drug
at which 50% of growth inhibition was achieved) and LC50 values (concentration at which
50% of cells were killed) of the compounds, as determined from 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀ
yl)ꢀ2,5ꢀdiphenyl tetrazolium bromide (MTT) cell viability assays, are shown in Table 2.
1-15 exhibited a biphasic doseꢀresponse curve (doseꢀresponse curves of 14 and 15 for MCFꢀ7
and HCT116 cells are presented in Figure 3A and 3B respectively), causing growth inhibition
of cancer cells at low concentrations with GI50 values ranging from 0.011 to 2.18 µM and
causing cell kill at higher concentrations with LC50 values ranging from 9.7 to 81.9 µM. In
general, the growth inhibitory effects of the target compounds were affected by the length of
the diꢀether linker. Compounds with a linker length of 3 carbon atoms possessed the strongest
growth inhibitory activities (based on GI50 values shown in Table 2). Upon comparison with
their in vitro DHFR inhibitory activities, the increase in growth inhibitory effect when linker
length was increased from 2 to 3 carbon atoms was consistent with a gain in DHFR inhibitory
efficacies. However, when linker length was further increased from 3 to 4 carbon atoms, a
mild decrease in growth inhibitory effects was observed despite a marginal gain in in vitro
DHFR inhibitory activity. This marginal reduction in growth inhibitory activity could be due
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to a slight decrease in permeability of the compounds into the cells as the molecular weight
increased, approaching 500 g/mol.
7
Replacement of the dimethyl substituent at R1 and R2 with the cyclopentyl substituent
resulted in no difference in growth inhibitory activity against MCFꢀ7 cells, but weaker
growth inhibitory activity against HCT116 cells. The drop in growth inhibitory effects of the
compounds with cyclopentyl substituent (13, 14 and 15) could be due to decreased
permeability of the compounds into the cells as their molecular weight increased,
approaching 500 g/mol. Cell kill was observed to improve with increasing lipophilicity of the
compounds, as evident from the decrease in LC50 values of the test compounds as linker
length increased from 2 to 4 carbon atoms. Replacement of the dimethyl substituent at R1 and
R2 with the bulkier cyclopentyl substituent also resulted in decreased LC50 values. As
discussed earlier, the in vitro TrxR IC50 values of 1-15 were observed to be in the micromolar
range of concentrations, which coincided with their micromolar LC50 values obtained from
the MTT viability assay. Hence, it was deduced that TrxR inhibition contributed to cell kill.
However, the trend in LC50 values did not correspond well with the trend of in vitro TrxR
inhibitory activity. Furthermore, compound 6h that lacks the α,βꢀunsaturated carbonyl moiety
essential for TrxR inhibitory activity also displayed cytotoxicity that was comparable to that
of 6, suggesting that besides TrxR inhibition, the cytotoxic activities of the compounds could
be attributed to additional mechanisms. This finding was unexpected, since we had found that
1,3ꢀdiphenylpropanꢀ2ꢀone, a compound similar to chalcone 18 but lacking the α,βꢀ
unsaturation at the carbonyl moiety, did not display cytotoxic activity and in vitro TrxR
inhibitory activity (results not shown). The key structureꢀactivity relationships for the
antiproliferative activity of compounds 1ꢀ15 are summarized in Figure 4.
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Figure 3. Representative doseꢀresponse curves of 14, 15, 17 and 18 against (A) MCFꢀ7 cells
and (B) HCT116 cells.
Figure 4. Key structureꢀactivity relationships of 1ꢀ15.
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Table 2. Antiproliferative activity of compounds against HCT116 and MCFꢀ7 cells
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6
7
8
O
O
NH2
NH2
B
O
O
n
A
O
O
n
A
B
or
N
N
N
N
9
R1
.HCl
R1
.HCl
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12
13
14
15
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17
18
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H2N
N
H2N
N
R2
R2
Antiproliferative activity
MCFꢀ7 cells
Attachment to
linker
Cpd
n R1
R2
HCT116 cells
Ring Position GI50 (µM)
LC50 (µM) GI50 (µM)
LC50 (µM)
1
2 CH3
2 CH3
2 CH3
2 CH3
3 CH3
3 CH3
3 CH3
3 CH3
4 CH3
4 CH3
4 CH3
4 CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
A
A
B
B
A
A
B
B
A
A
B
B
A
A
A
4
3
4
3
4
3
4
3
4
3
4
3
3
3
3
0.572 ± 0.144
0.170 ± 0.036
0.706 ± 0.142
0.145 ± 0.036
0.016 ± 0.004
0.011 ± 0.004
0.035 ± 0.010
0.011 ± 0.001
0.039 ± 0.009
0.051 ± 0.008
0.026 ± 0.008
0.034 ± 0.011
0.260 ± 0.069
0.026 ±0.007
0.116 ± 0.014
0.036 ±0.002
0.021 ± 0.007
0.010 ± 0.001
15.7 ± 2.2
50.1 ± 2.5
49.9 ± 5.6
0.584 ± 0.141
0.719 ± 0.063
40.1 ± 1.8
40.7 ± 7.0
47.7 ± 8.4
38.8 ± 8.3
34.0 ± 1.8
20.2 ± 4.2
35.3 ± 4.8
29.8 ± 2.5
14.2 ± 1.9
11.3 ± 1.4
14.8 ± 0.1
15.5 ± 1.1
16.1 ± 0.8
11.2 ± 1.5
9.7 ± 0.5
>100
2
3
81.9 ± 10.1 0.512 ± 0.101
4
52.2 ± 2.2
55.2 ± 3.5
42.7 ± 3.2
39.0 ± 3.1
0.623 ± 0.138
0.085 ± 0.020
0.076 ± 0.029
0.062 ± 0.013
5
6
7
8
48.2 ± 18.4 0.013 ± 0.004
9
24.0 ± 4.9
30.3 ± 6.0
42.9 ± 3.7
34.7 ± 5.8
24.1 ± 2.1
14.1 ± 1.0
13.5 ± 0.9
>100
0.087 ± 0.013
0.122 ± 0.009
0.141 ± 0.033
0.086 ± 0.016
0.770 ± 0.156
0.080 ± 0.025
0.127 ± 0.010
0.107 ± 0.006
0.077 ± 0.025
0.036 ± 0.006
12.4 ± 2.0
10
11
12
13
14
15
16
17
6h
18
2 ꢀ(CH2)4ꢀ
3 ꢀ(CH2)4ꢀ
4 ꢀ(CH2)4ꢀ
>100
90.8 ± 18.7
25.9 ± 1.3
30.7 ± 5.1
>100
34.2 ± 3.5
22.5 ± 1.5
>100
MTX
0.015 ± 0.001
0.024 ± 0.007
Data reported are mean values taken from 3 independent experiments and standard deviation.
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Evaluation of selected compounds on DHFR activity in cells
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14 and 15 were selected as lead compounds for evaluation of their inhibitory effects on
cellular DHFR and TrxR activities based on the fact they possessed the greatest cytotoxic
effects and exhibited strong in vitro DHFR and TrxR inhibitory activities. Together, 6 and 10,
which are structurally similar to 14 and 15 respectively (bearing the dimethyl substituent at
R1 and R2 instead of the cyclopentyl substituent in 14 and 15), 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ
1,3,5ꢀtriazines 16 and 17, as well as the chalcone compound 18, were also selected for
biological evaluation for cellular DHFR and TrxR inhibitory activities.
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The cellꢀbased DHFR assay was modified from an inꢀhouse protocol developed by Sigmaꢀ
Aldrich Co. for assessing in vitro activity of DHFR enzyme.32 At growth inhibitory
concentrations of 1 nM to 1 µM, 6, 10, 14 and 15 caused a doseꢀdependent decrease in DHFR
activity in HCT116 cells (Figure 5A). 6 brought about the strongest decline in cellular DHFR
activity, followed by 14, 10 and lastly, 15. This trend of cellular DHFR inhibition in HCT116
cells corresponded well with the trend in GI50 values of the compounds against MCFꢀ7 and
HCT116 cells, where the recorded GI50 values were also in the order of 6 < 14 < 10 < 15.
Hence, it was concluded that the growth inhibitory activities of the compounds were
attributed to their DHFR inhibitory activities. As expected, the 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ
1,3,5ꢀtriazines 16 and 17 also effected a doseꢀdependent decrease in cellular DHFR activity
whereas the chalcone compound 18 did not inhibit cellular DHFR (Figure 5A).
At cytotoxic concentrations of 40ꢀ70 µM, the activity of cellular DHFR in HCT116 cells was
completely inhibited by 6, 10, 14 and 15, as well as the 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀ
triazines 16 and 17 (Figure 5B). Western blot analyses revealed that protein levels of DHFR
were higher in cells treated with 14 and 15 than those observed in DMSOꢀtreated cells
(Figure 5C), indicating that the drop in cellular DHFR activity was mediated by 14 and 15
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inhibiting DHFR and not due to a downregulation of DHFR protein levels. The upregulation
of DHFR levels might be a cellular prosurvival mechanism triggered in response to the cell
death inducing effects of high doses of 14 and 15.
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Figure 5. Effects of selected dihydrotriazineꢀchalcone compounds on DHFR activity in
HCT116 cells. DHFR activity in lysates of HCT116 cells treated with 6, 10, 14, 15, 16, 17
and 18 at (A) concentrations of 0.001 to 1 µM and (B) 40 to 70 µM for 16 h was determined
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and expressed as a percentage of DHFR activity in DMSOꢀtreated cells. Each point
represents the average value from three independent experiments. (C) The lysates mentioned
in (B) were analyzed by Western blotting for detection of levels of DHFR. Western blot
images are representative of three independent experiments.
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Evaluation of selected compounds on TrxR activity in cells
At growth inhibitory concentrations of 1 nM to 1 µM, all four of the linked dihydrotriazineꢀ
chalcone compounds failed to cause significant TrxR inhibition in HCT116 cells (Figure 6A).
The results were in agreement with the in vitro TrxR IC50 values being greater than 1 µM for
all compounds tested against rat liver TrxR. On the other hand, at cytotoxic concentrations of
40ꢀ70 µM, 6, 10, 14 and 15 brought about a doseꢀdependent decrease in cellular TrxR
activity. 15 possessed the greatest cellular TrxR inhibitory activity, followed by 14, 10 and
lastly, 6 (Figure 6B). This trend of cellular TrxR inhibition corresponded well with the trend
in LC50 values of the compounds against MCFꢀ7 and HCT116 cells, where the recorded LC50
values were also in the order of 15 < 14 < 10 < 6. The results had therefore indicated that
inhibition of cellular TrxR could have at least played a role in causing cell death. Of note,
chalconeꢀcontaining 10, 14 and 15 produced stronger cellular TrxR inhibition than the parent
chalcone compound 18, while the 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazines 16 and 17
expectedly did not inhibit cellular TrxR activity even at high concentrations.
When the aforementioned lysates of HCT116 cells treated with lead compounds 14 and 15
were analyzed by western blotting, protein levels of TrxR were observed to be comparable to
the levels expressed in DMSO control cells (Figure 6C). This demonstrated that the observed
doseꢀdependent decrease in cellular TrxR activity was due to inhibition of the enzyme and
not due to downregulation of the enzyme’s protein levels.
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Figure 6. Effects of selected dihydrotriazineꢀchalcone compounds on TrxR activity in
HCT116 cells. TrxR activity in lysates of HCT116 cells treated with 6, 10, 14, 15, 16, 17 and
18 at (A) concentrations of 0.001 to 1 µM and (B) 40 to 70 µM for 16 h was determined and
expressed as a percentage of TrxR activity in DMSOꢀtreated cells. Each point represents the
average value from three independent experiments. (C) The lysates mentioned in (B) were
analyzed by Western blotting for detection of levels of TrxR. Western blot images are
representative of three independent experiments.
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CONCLUSION
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The previous attempt to design dihydrotriazineꢀchalcone compounds by merging the DHFRꢀ
inhibiting 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazine and TrxRꢀinhibiting chalcone scaffolds had
yielded single molecules that failed to possess potent cytotoxicity,28 suggesting a suboptimal
structural design in hybridizing the two pharmacophores into a single chemical entity. In this
study, stemming from the hypothesis that a flexible linker would enable better interaction of
the scaffolds with the targets, fifteen compounds having a diꢀether linker between the two
scaffolds were synthesized and evaluated for their antiproliferative and enzyme inhibitory
activities. Among the compounds, 14 and 15 were identified to possess the greatest cytotoxic
activities against HCT116 and MCFꢀ7 cancer cell lines that were correlated to strong in vitro
inhibitory activities against recombinant human DHFR and rat TrxR enzymes. In addition,
cellular DHFR and TrxR activities in lysates of HCT116 cells treated with growth inhibitory
and cytotoxic doses of 14 and 15 respectively were significantly reduced. Considering the
consistent results obtained from the cellꢀfree and cellꢀbased assays, it is therefore proposed
that the strategy of combiing the dihydrotriazine and chalcone chemical scaffolds via diꢀether
linker has generated novel single molecules that exert antiproliferative effects on cancer cells
at least partly through targeting DHFR and TrxR.
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EXPERIMENTAL SECTION
Chemistry
All reagents were purchased from SigmaꢀAldrich, Tokyo Chemical Industry, Alfa Aesar, or
Merck and were used directly without any purification. Thin layer chromatography (TLC)
was performed using silica gelꢀcoated aluminium plates with fluorescent indicator and
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visualized with ultraviolet light at 254 nm. Melting points were determined using a
1
13
Gallenkamp melting point apparatus and were uncorrected. H NMR and C NMR spectra
were recorded on a Bruker UltrashieldTM 400 Plus NMR spectrometer. Chemical shift values
(δ) were expressed as parts per million (ppm) relative to tetramethylsilane as the internal
standard. Electrospray Ionisation Mass Spectrometry (ESIꢀMS) was recorded on an AB
SCIEX API 2000 Q Trap mass spectrometer. High performance liquid chromatography
(HPLC) was performed for purity checking using Agilent 1100 Series HPLC on a Lichrosorb
10 RPꢀ18 250 x 4.0mm 10 µm column with gradient elution (gradient of 50% acetonitrile and
0.1% TFA to 100% acetonitrile over 20 min, followed by maintenance of 100% acetonitrile
for 5 min). All the final compounds were found to be pure up to 95% or higher.
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General Procedure for the synthesis of 1 to 12
4,6ꢀDiaminoꢀ1ꢀhydroꢀ1,2ꢀdihydroꢀ1,3,5ꢀtriazine hydrochloride and NaOH (1 molar
equivalent) were dissolved in methanol (8 to 20 mL) and refluxed for 30 minutes. After the
mixture was cooled to room temperature, methanol was removed under vacuum. The
remaining white solid was dissolved in N,Nꢀdimethylformamide (2 to 5 mL) and
bromoalkoxyꢀsubstituted chalcone (1 molar equivalent) was added. The reaction mixture was
stirred overnight at room temperature. After completion of the reaction, the pH of the solution
was adjusted to pH 1 using concentrated HCl. Water (approximately 50 mL) was added to the
mixture. If the product precipitated out as a solid, the solid was collected by filtration and
washed with water and ethyl acetate or dichloromethane. If a sticky substance was formed,
the substance was washed with ethyl acetate to remove excess unreacted bromoalkoxyꢀ
substituted chalcone. If the product dissolved in water, brine was added to the solution and
the product was extracted with ethyl acetate. The ethyl acetate solution was refrigerated to
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allow precipitation of the desired product. The collected products were then purified by
recrystallization with ethanol/dilute HCl.
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9
(2
phenylprop-2-en-1-one hydrochloride (1). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.58 g, 3 mmol) and (2 )ꢀ1ꢀ[4ꢀ(2ꢀbromoethoxy)phenyl]ꢀ3ꢀ
E)-1-{4-[2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-ethoxy]-phenyl}-3-
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E
phenylpropꢀ2ꢀenꢀ1ꢀone (0.99 g, 3 mmol), 1 was obtained as a yellow solid, 0.32 g, yield
23.9%. mp: 188ꢀ190°C. 1H NMR (400MHz, DMSOꢀd6) δ 9.07 (s, 1H, NH), 8.70 (s, 1H, NH),
8.18 (d, 2H, J = 9.2 Hz), 8.092 (s, 1H, NH), 7.92ꢀ7.71 (m, 4H), 7.79 (br s, 1H, NH), 7.50ꢀ
7.49 (m, 3H), 7.16 (d, 2H, J = 8.8 Hz), 7.12 (br s, 1H, NH), 4.42 (br s, 2H), 4.36ꢀ4.35 (m,
13
2H), 1.58 (br s, 3H), 1.35 (br s, 3H). C NMR (100MHz, DMSOꢀd6) δ 187.3, 162.0, 161.3,
156.2, 143.2, 134.7, 130.9, 130.7, 130.4, 128.8, 128.7, 121.9, 114.5, 75.9, 72.3, 65.4. Purity:
97.9%, HPLC tR = 5.24 min. ESIꢀMS m/z 408.2 (M+1)+
(2
phenylprop-2-en-1-one hydrochloride (2). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.39 g, 2 mmol) and (2 )ꢀ1ꢀ[3ꢀ(2ꢀbromoethoxy)phenyl]ꢀ3ꢀ
E)-1-{3-[2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-ethoxy]-phenyl}-3-
E
phenylpropꢀ2ꢀenꢀ1ꢀone (0.66 g, 2 mmol), 2 was obtained as a pale yellow solid, 0.13 g, yield
14.3%. mp: 132ꢀ134°C. 1H NMR (400MHz, DMSOꢀd6) δ 9.02 (s, 1H, NH), 8.71 (s, 1H, NH),
8.06 (s, 1H, NH), 7.90ꢀ7.86 (m, 3H), 7.82ꢀ7.74 (m, 2H), 7.63ꢀ7.62 (m, 1H), 7.56 (t, 1H, J =
7.8 Hz), 7.51ꢀ7.49 (m, 3H), 7.31ꢀ7.29 (m, 1H), 7.08 (br s, 1H, NH), 4.40 (br s, 2H), 4.35ꢀ4.34
(m, 2H), 1.58 (br s, 3H), 1.34 (br s, 3H). 13C NMR (100MHz, DMSOꢀd6) δ 188.8, 161.3,
158.4, 156.2, 144.1, 139.0, 134.5, 130.6, 130.0, 128.9, 128.8, 122.0, 121.5, 119.8, 113.5,
76.1, 72.3, 65.4. Purity: 96.7%, HPLC tR = 5.61 min. ESIꢀMS m/z 408.2 (M+1)+
(2E)-3-{4-[2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-ethoxy]-phenyl}-1-
phenylprop-2-en-1-one hydrochloride (3). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
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[1,3,5]triazinꢀ1ꢀol hydrochloride (0.58 g, 3 mmol) and (2E)ꢀ3ꢀ[4ꢀ(2ꢀbromoethoxy)phenyl]ꢀ1ꢀ
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8
phenylpropꢀ2ꢀenꢀ1ꢀone (0.99 g, 3 mmol), 3 was obtained as a yellow solid, 0.47 g, yield
35.5%. mp: 141ꢀ143°C. 1H NMR (400MHz, DMSOꢀd6) δ 9.03 (s, 1H, NH), 8.70 (s, 1H, NH),
8.14ꢀ8.12 (m, 2H), 8.07 (s, 1H, NH), 7.87 (d, 2H, J = 8.8 Hz), 7.82ꢀ7.66 (m, 3H), 7.75 (br s,
1H, NH), 7.59 (t, 2H, J = 7.4 Hz), 7.10 (br s, 1H, NH), 7.07 (d, 2H, J = 8.8 Hz), 4.37 (br s,
2H), 4.32 (br s, 2H), 1.56 (br s, 3H), 1.35 (br s, 3H). 13C NMR (100MHz, DMSOꢀd6) δ 189.0,
161.3, 160.1, 156.2, 143.8, 137.7, 132.9, 130.8, 128.7, 128.3, 127.6, 119.8, 114.9, 75.9, 72.3,
65.3. Purity: 98.5%, HPLC tR = 5.21 min. ESIꢀMS m/z 408.2 (M+1)+
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(2
phenylprop-2-en-1-one hydrochloride (4). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.39g, 2 mmol) and (2 )ꢀ3ꢀ[3ꢀ(2ꢀbromoethoxy)phenyl]ꢀ1ꢀ
E)-3-{3-[2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-ethoxy]-phenyl}-1-
E
phenylpropꢀ2ꢀenꢀ1ꢀone (0.66 g, 2 mmol), 4 was obtained as a yellow solid, 0.45 g, yield
50.4%. mp: 139ꢀ141°C. 1H NMR (400MHz, DMSOꢀd6) δ 9.20 (s, 1H, NH), 8.73 (s, 1H, NH),
8.15ꢀ8.13 (m, 2H), 8.06 (s, 1H, NH), 7.90 (d, 1H, J = 15.6 Hz), 7.78 (br s, 1H, NH), 7.75ꢀ
7.70 (m, 2H), 7.64ꢀ7.60 (m, 2H), 7.50ꢀ7.42 (m, 3H), 7.18 (br s, 1H, NH), 7.10 (dd, 1H, J =
8.0, 1.6), 4.39 (br s, 2H), 4.314 (br s, 2H), 1.59 (br s, 3H), 1.35 (br s, 3H). 13C NMR
(100MHz, DMSOꢀd6) δ 189.2, 161.4, 158.4, 156.2, 143.8, 137.4, 136.1, 133.1, 130.0, 128.7,
128.5, 122.4, 122.1, 117.2, 113.9, 76.1, 72.3, 65.2. Purity: 98.0%, HPLC tR = 5.64 min. ESIꢀ
MS m/z 408.2 (M+1)+
(2
phenylprop-2-en-1-one hydrochloride (5). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.97 g, 5 mmol) and (2 )ꢀ1ꢀ[4ꢀ(3ꢀbromopropoxy)phenyl]ꢀ
E)-1-{4-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-propoxy]-phenyl}-3-
E
3ꢀphenylpropꢀ2ꢀenꢀ1ꢀone (1.73 g, 5 mmol), 5 was obtained as a yellow solid, 0.68 g, yield
29.8%. mp: 196ꢀ198°C. 1H NMR (400MHz, DMSOꢀd6) δ 8.95 (s, 1H, NH), 8.63 (s, 1H, NH),
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8.17 (d, 2H, J = 8.8 Hz), 8.10 (s. 1H, NH), 7.91 (d, 1H, J = 15.6 Hz), 7.87ꢀ7.85 (m, 2H), 7.78
(br s, 1H, NH), 7.72 (d, 1H, J = 15.6 Hz), 7.50ꢀ7.48 (m, 3H), 7.13 (d, 2H, J = 8.8 Hz), 7.06
(br s, 1H, NH), 4.23 (t, 2H, J = 6.0 Hz), 4.13 (t, 2H, J = 5.8 Hz), 2.21 (br s, 2H), 1.42 (br s,
6H). 13C NMR (100MHz, DMSOꢀd6) δ 187.3, 162.2, 161.1, 156.1, 143.1, 134.7, 130.9,
130.5, 130.3, 128.8, 128.7, 122.0, 114.4, 74.2, 72.2, 64.5, 26.9. Purity: 98.5%, HPLC tR =
5.71 min. ESIꢀMS m/z 422.2 (M+1)+
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(2
phenylprop-2-en-1-one hydrochloride (6). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.58 g, 3 mmol) and (2 )ꢀ1ꢀ[3ꢀ(3ꢀbromopropoxy)phenyl]ꢀ
E)-1-{3-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-propoxy]-phenyl}-3-
E
3ꢀphenylpropꢀ2ꢀenꢀ1ꢀone (0.99 g, 3 mmol), 6 was obtained as a yellow solid, 0.31 g, yield
22.9%. mp: 164ꢀ166°C. 1H NMR (400MHz, DMSOꢀd6) δ 8.89 (s, 1H, NH), 8.62 (s, 1H, NH),
8.087 (s, 1H, NH), 7.89ꢀ7.85 (m, 3H), 7.79ꢀ7.73 (m, 2H), 7.68 (br s, 1H, NH), 7.60ꢀ7.50 (m,
5H), 7.29 (dd, 1H, J = 8.2, 2.2 Hz), 7.04 (br s, 1H, NH), 4.21 (t, 2H, J = 5.8 Hz), 4.15 (t, 2H,
13
J = 5.8 Hz), 2.21 (br s, 2H), 1.43 (br s, 6H). C NMR (100MHz, DMSOꢀd6) δ 188.8, 161.2,
158.6, 156.0, 144.1, 139.0, 134.5, 130.6, 130.0, 128.9, 128.8, 122.0, 121.2, 119.6, 113.5,
74.3, 72.3, 64.4, 27.0. Purity: 99.7%, HPLC tR = 6.13 min. ESIꢀMS m/z 422.2 (M+1)+
(2
phenylprop-2-en-1-one hydrochloride (7). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.77 g, 4 mmol) and (2 )ꢀ3ꢀ[4ꢀ(3ꢀbromopropoxy)phenyl]ꢀ
E)-3-{4-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-propoxy]-phenyl}-1-
E
1ꢀphenylpropꢀ2ꢀenꢀ1ꢀone (1.38 g, 4 mmol), 7 was obtained as a yellow solid, 0.92 g, yield
50.0%. mp: 193ꢀ195°C. 1H NMR (400MHz, DMSOꢀd6) δ 9.11 (s, 1H, NH), 8.63 (s, 1H, NH),
8.12ꢀ8.10 (m, 2H), 8.09 (s, 1H, NH), 7.84 (d, 2H, J = 8.8 Hz), 7.79ꢀ7.68 (m, 3H), 7.70 (br s,
1H, NH), 7.60 (t, 2H, 7.4 Hz), 7.14 (br s, 1H, NH), 7.06 (d, 2H, J = 8.8 Hz), 4.18 (t, 2H, J =
6.0 Hz), 4.13 (t, 2H, J = 5.8 Hz), 2.19 (br s, 2H), 1.43 (br s, 6H). 13C NMR (100MHz,
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DMSOꢀd6) δ 189.0, 161.1, 160.4, 156.2, 143.9, 137.7, 132.8, 130.8, 128.6, 128.3, 127.4,
119.6, 114.8, 74.2, 72.1, 64.3, 26.9. Purity: 99.6%, HPLC tR = 5.58 min. ESIꢀMS m/z 422.2
(M+1)+
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(2
phenylprop-2-en-1-one hydrochloride (8). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.39 g, 2 mmol) and (2 )ꢀ3ꢀ[3ꢀ(3ꢀbromopropoxy)phenyl]ꢀ
E)-3-{3-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-propoxy]-phenyl}-1-
E
1ꢀphenylpropꢀ2ꢀenꢀ1ꢀone (0.69 g, 2 mmol), 8 was obtained as yellow crystals, 0.095 g, yield
1
10.4%. mp: 132 °C (decomposed). H NMR (400MHz, DMSOꢀd6) δ 8.94 (s, 1H, NH), 8.63
(s, 1H, NH), 8.17ꢀ8.15 (m, 2H), 8.08 (s, 1H, NH), 7.95 (d, 1H, J = 15.6 Hz), 7.74ꢀ7.67 (m,
2H), 7.67 (br s, 1H, NH), 7.59 (t, 2H, J = 7.4 Hz), 7.50ꢀ7.46 (m, 2H), 7.39 (t, 1H, J = 7.8 Hz),
7.05 (dd, 1H, J = 8.0, 1.6 Hz), 7.04 (br s, 1H, NH), 4.18 (t, 2H, J = 6.2 Hz), 4.11 (t, 2H, J =
6.0 Hz), 2.19 (br s, 2H), 1.42 (br s, 6H). 13C NMR (100MHz, DMSOꢀd6) δ 189.2, 161.2,
158.6, 156.1, 143.9, 137.4, 136.0, 133.1, 129.9, 128.7, 128.5, 122.3, 121.7, 117.1, 114.0,
74.3, 72.2, 64.2, 27.0. Purity: 99.9%, HPLC tR = 5.77 min. ESIꢀMS m/z 422.3 (M+1)+
(2
phenylprop-2-en-1-one hydrochloride (9). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.77 g, 4 mmol) and (2 )ꢀ1ꢀ[4ꢀ(4ꢀbromobutoxy)phenyl]ꢀ3ꢀ
E)-1-{4-[4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-butoxy]-phenyl}-3-
E
phenylpropꢀ2ꢀenꢀ1ꢀone (1.44 g, 4 mmol), 9 was obtained as a pale yellow solid, 0.31 g, yield
1
16.5%. mp: 196ꢀ198 °C. H NMR (400MHz, DMSOꢀd6) δ 9.00 (s, 1H, NH), 8.58 (s, 1H,
NH), 8.17 (d, 2H, J = 9.2 Hz), 8.02 (s, 1H, NH), 7.95 (d, 1H, J = 15.6 Hz), 7.89ꢀ7.87 (m, 2H),
7.71 (d, 1H, J = 15.6 Hz), 7.76 (br s, 1H, NH), 7.47ꢀ7.45 (m, 3H), 7.10 (br s, 1H, NH), 7.09
(d, 2H, J = 8.8 Hz), 4.15ꢀ4.00 (m, 4H), 1.85 (br s, 4H), 1.44 (br s, 6H). 13C NMR (100MHz,
DMSOꢀd6) δ 187.3, 162.5, 161.1, 156.3, 143.0, 134.7, 130.9, 130.3, 130.3, 128.8, 128.7,
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122.0, 114.4, 77.2, 72.1, 67.7, 24.9, 24.0. Purity: 99.7%, HPLC tR = 6.25 min. ESIꢀMS m/z
436.5 (M+1)+
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(2E)-1-{3-[4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-butoxy]-phenyl}-3-
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phenylprop-2-en-1-one hydrochloride (10). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.97 g, 5 mmol) and (2E)ꢀ1ꢀ[3ꢀ(4ꢀbromobutoxy)phenyl]ꢀ3ꢀ
phenylpropꢀ2ꢀenꢀ1ꢀone (1.80 g, 5 mmol), 10 was obtained as a white solid, 0.21 g, yield
8.7%. mp: 192ꢀ194 °C. 1H NMR (400MHz, DMSOꢀd6) δ 9.00 (s, 1H, NH), 8.58 (s, 1H, NH),
8.02ꢀ7.46 (m, 10H), 7.67 (br s, 2H, NH), 7.25 (dd, 1H, J = 8.0, 2.0 Hz), 7.07 (br s, 1H, NH),
13
4.11ꢀ4.00 (m, 4H), 1.85 (br s, 4H), 1.44 (br s, 6H). C NMR (100MHz, DMSOꢀd6) δ 188.9,
161.1, 158.8, 156.2, 144.0, 138.9, 134.5, 130.6, 129.9, 128.9, 128.8, 122.0, 121.0, 119.6,
113.5, 77.2, 72.1, 67.5, 25.0, 24.0. Purity: 99.1%, HPLC tR = 6.62 min. ESIꢀMS m/z 436.4
(M+1)+
(2
phenylprop-2-en-1-one hydrochloride (11). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
[1,3,5]triazinꢀ1ꢀol hydrochloride (0.68 g, 3.5 mmol) and (2 )ꢀ3ꢀ[4ꢀ(4ꢀbromobutoxy)phenyl]ꢀ
E)-3-{4-[4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-butoxy]-phenyl}-1-
E
1ꢀphenylpropꢀ2ꢀenꢀ1ꢀone (1.26 g, 3.5 mmol), 11 was obtained as a yellow solid, 0.067 g,
yield 4.1%. mp: 194ꢀ196 °C. 1H NMR (400MHz, DMSOꢀd6) δ 9.07 (s, 1H, NH), 8.59 (s, 1H,
NH), 8.15ꢀ8.13 (m, 2H), 8.03 (s, 1H, NH), 7.87ꢀ7.55 (m, 7H), 7.72 (br s, 1H, NH), 7.13 (br s,
13
1H, NH), 7.02 (d, 2H, J = 8.8 Hz), 4.08ꢀ3.99 (m, 4H), 1.84 (m, 4H), 1.44 (m, 6H). C NMR
(100MHz, DMSOꢀd6) δ 188.9, 161.2, 160.6, 156.1, 143.9, 137.7, 132.8, 130.7, 128.6, 128.3,
127.2, 119.4, 114.8, 77.2, 72.1, 67.5, 24.9, 24.0. Purity: 99.4%, HPLC tR = 6.04 min. ESIꢀMS
m/z 436.3 (M+1)+
(2E)-3-{3-[4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-butoxy]-phenyl}-1-
phenylprop-2-en-1-one hydrochloride (12). From 4,6ꢀdiaminoꢀ1,2ꢀdihydroꢀ2,2ꢀdimethylꢀ
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[1,3,5]triazinꢀ1ꢀol hydrochloride (0.77 g, 4 mmol) and (2E)ꢀ3ꢀ[3ꢀ(4ꢀbromobutoxy)phenyl]ꢀ1ꢀ
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7
8
phenylpropꢀ2ꢀenꢀ1ꢀone (1.44 g, 4 mmol), 12 was obtained as a pale yellow solid, 0.18 g,
yield 9.3%. mp: 177ꢀ179 °C. 1H NMR (400MHz, DMSOꢀd6) δ 9.00 (s, 1H, NH), 8.59 (s, 1H,
NH), 8.18ꢀ8.15 (m, 2H), 8.03 (s, 1H, NH), 7.95 (d, 1H, J = 15.6 Hz), 7.74ꢀ7.67 (m, 2H), 7,69
(br s, 1H, NH), 7.59 (t, 2H, J = 7.6 Hz), 7.49ꢀ7.44 (m, 2H), 7.37 (t, 1H, J = 8.0 Hz), 7.06 (br
s, 1H, NH), 7.04 (dd, 1H, J = 8.2, 1.8 Hz), 4.10ꢀ4.00 (m, 4H), 1.84 (br s, 4H), 1.44 (br s, 6H).
13C NMR (100MHz, DMSOꢀd6) δ 189.1, 161.2, 158.9, 156.2, 143.9, 137.4, 136.0, 133.1,
129.9, 128.7, 128.5, 122.3, 121.6, 117.0, 114.0, 77.2, 72.1, 67.4, 25.0, 24.0. Purity: 97.4%,
HPLC tR = 6.41 min. ESIꢀMS m/z 436.2 (M+1)+
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General Procedure for the synthesis of 13 to 15
13 to 15 were synthesized using the same procedure as described in the general procedure for
synthesis of 1 to 12, but with potassium carbonate (1 molar equivalent) added to the reaction
mixture (before stirring overnight) to increase the yield of product.
(2
3-phenylprop-2-en-1-one
triazaspiro[4.5]decaꢀ7,9ꢀdienꢀ6ꢀol hydrochloride (0.44 g, 2 mmol) and (2
E
)-1-(3-{2-[(7,9-diamino-6,8,10-triazaspiro[4.5]deca-7,9-dien-6-yl)oxy]ethoxy}phenyl)-
hydrochloride (13). From 7,9ꢀdiaminoꢀ6,8,10ꢀ
)ꢀ1ꢀ[3ꢀ(2ꢀ
E
bromoethoxy)phenyl]ꢀ3ꢀphenylpropꢀ2ꢀenꢀ1ꢀone (0.66 g, 2 mmol), 13 was obtained as an offꢀ
1
white solid, 0.36 g, yield 38.4%. mp: 192ꢀ194 °C. H NMR (400MHz, DMSOꢀd6) δ 9.34 (s,
1H, NH), 8.73 (s, 1H, NH), 8.14 (s, 1H, NH), 7.94ꢀ7.74 (m, 5H), 7.65 (br s, 1H, NH), 7.63ꢀ
7.47 (m, 5H), 7.26 (dd, 1H, J = 8.0, 2.0 Hz), 7.03 (br s, 1H, NH), 4.42 (br s, 2H), 4.32 (br s,
2H), 2.06 (br s, 2H), 1.76ꢀ1.66 (m, 6H). 13C NMR (100MHz, DMSOꢀd6) δ 188.8, 162.1,
158.3, 156.6, 144.1, 139.0, 134.5, 130.6, 130.0, 128.9, 122.0, 121.4, 119.8, 113.4, 81.3, 76.2,
65.2, 33.3, 22.2. Purity: 96.4%, HPLC tR = 6.45 min. ESIꢀMS m/z 434.5 (M+1)+
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)-1-(3-{3-[(7,9-diamino-6,8,10-triazaspiro[4.5]deca-7,9-dien-6-
5
6
7
8
yl)oxy]propoxy}phenyl)-3-phenylprop-2-en-1-one hydrochloride (14). From 7,9ꢀdiaminoꢀ
6,8,10ꢀtriazaspiro[4.5]decaꢀ7,9ꢀdienꢀ6ꢀol hydrochloride (0.66 g, 3 mmol) and (2 )ꢀ1ꢀ[3ꢀ(3ꢀ
E
9
bromopropoxy)phenyl]ꢀ3ꢀphenylpropꢀ2ꢀenꢀ1ꢀone (1.04 g, 3 mmol), 14 was obtained as an
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offꢀwhite solid, 0.35 g, yield 24.2%. mp: 191ꢀ192 °C. H NMR (400MHz, DMSOꢀd6) δ 9.14
(s, 1H, NH), 8.65 (s, 1H, NH), 8.14, (s, 1H, NH), 7.95ꢀ7.89 (m, 3H), 7.80ꢀ7.73 (m, 2H), 7.63ꢀ
7.62 (m, 1H), 7.75 (br s, 1H, NH), 7.53ꢀ7.47 (m, 4H), 7.27 (dd, 1H, J = 8.0, 2.2 Hz), 6.92 (br
s, 1H, NH), 4.19 (t, 2H, J = 6.0 Hz), 4.12 (t, 2H, J = 5.8 Hz), 2.21ꢀ2.19 (m, 2H), 2.00 (br s,
13
2H), 1.74ꢀ1.70 (m, 2H), 1.57ꢀ1.51 (m, 4H). C NMR (100MHz, DMSOꢀd6) δ 188.8, 161.9,
158.6, 156.5, 144.1, 138.9, 134.5, 130.6, 129.9, 128.9, 128.8, 122.0, 121.2, 119.5, 113.5,
81.1, 74.2, 64.2, 33.2, 26.9, 22.0. Purity: 99.9%, HPLC tR = 6.74 min. ESIꢀMS m/z 448.5
(M+1)+
(2
yl)oxy]butyloxy}phenyl)-3-phenylprop-2-en-1-one hydrochloride (15). From 7,9ꢀdiaminoꢀ
6,8,10ꢀtriazaspiro[4.5]decaꢀ7,9ꢀdienꢀ6ꢀol hydrochloride (1.10 g, 5 mmol) and (2 )ꢀ1ꢀ[3ꢀ(4ꢀ
E)-1-(3-{4-[(7,9-diamino-6,8,10-triazaspiro[4.5]deca-7,9-dien-6-
E
bromobutoxy)phenyl]ꢀ3ꢀphenylpropꢀ2ꢀenꢀ1ꢀone (1.80 g, 5 mmol), 15 was obtained as an offꢀ
1
white solid, 0.78 g, yield 31.4%. mp: 186ꢀ187 °C. H NMR (400MHz, DMSOꢀd6) δ 9.25 (s,
1H, NH), 8.61 (s, 1H, NH), 8.06 (s, 1H, NH), 7.94ꢀ7.89 (m, 3H), 7.79ꢀ7.73 (m, 2H), 7.71 (br
s, 1H, NH), 7.61ꢀ7.60 (m, 1H), 7.52ꢀ7.47 (m, 4H), 7.25 (dd, 1H, J = 8.0, 2.0 Hz), 6.98 (br s,
1H, NH), 4.13ꢀ4.10 (m, 2H), 4.02ꢀ3.99 (m, 2H), 2.01 (br s, 2H), 1.88ꢀ1.69 (m, 10H). 13C
NMR (100MHz, DMSOꢀd6) δ 188.9, 161.9, 158.8, 156.6, 144.0, 138.9, 134.5, 130.6, 129.9,
128.9, 128.8, 122.0, 121.0, 119.6, 113.5, 81.1, 77.3, 67.5, 33.4, 24.9, 24.0, 22.2. Purity:
95.3%, HPLC tR = 7.59 min. ESIꢀMS m/z 462.5 (M+1)+
29
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