Design, Synthesis, and Biological Evaluation of Coupled Bioactive

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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 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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Journal of Medicinal Chemistry

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*,†, Wai-Keung 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:

Eng-Hui Chew (E-mail: [email protected]; Phone: +65

65161955; Fax: +65 67791554) and Wai-Keung Chui (E-mail: [email protected]; Phone: +65 65162933; Fax: +65 67791554)

1 ACS Paragon Plus Environment


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ABSTRACT

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 di-ether 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 multi-target inhibition at the cellular level.


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INTRODUCTION 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 multi-targeted agent named as a “designed multiple ligand”, bears the advantages of circumventing the problems of complicated pharmacokinetic and pharmacodynamics (PK/PD) relationships and drug-drug interactions associated with combination therapy.2 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 one-carbon transfer reactions leading to the synthesis of several nucleic acids and amino acids.3 The well-known 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 4,6-diamino-1,2-dihydro-1,3,5triazines are lipophilic DHFR inhibitors which have previously demonstrated antiproliferative activity on cancer cells.6-8 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 4,6-diamino-1,2-dihydro-1,3,5triazine, 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. 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.13-16 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.17-19 The nucleophilic selenocysteine (Sec) residue located on the flexible C-terminal active site of mammalian TrxR makes it a target for many electrophilic compounds.20-21 Chalcones are naturally-occurring compounds which have been reported to exert anticancer activity through a number of mechanisms including inhibition of tubulin polymerization,22 blockade of the nuclear factor-kappa B (NF-κB) signalling pathway,23 as well as inhibition of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR)24 and vascular endothelial growth factor receptor-2 (VEGFR-2)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,β-unsaturated 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 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 DHFRinhibiting

4,6-diamino-1,2-dihydro-1,3,5-triazine

and

TrxR-inhibiting

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 dihydrotriazinechalcone compounds (Figure 1A) that were designed based on merging the bioactive scaffolds.

28

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 dual-target inhibitors of DHFR and TrxR containing the bioactive 4,6-diamino-1,2-dihydro-1,3,5-triazine and chalcone scaffolds linked by flexible di-ether 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 human-derived HCT116 colorectal and MCF-7 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) dihydrotriazine-chalcone compounds 6a, 6b, 11a and 11b (synthesis reported in Ref No. 28) and (B) linked dihydrotriazine-chalcone compounds 1-15 containing di-ether linkers of varying lengths.


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RESULTS AND DISCUSSION Design and Synthesis of Compounds Scheme 1.

(A) Synthesis of 4,6-diamino-1-hydroxy-1,2-dihydro-1,3,5-triazine 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 1-15. 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, 4,6diamiino-1-hydroxy-1,2-dihydro-1,3,5-triazine hydrochlorides

(21) were

obtained as

previously reported8,29 through the synthesis of 4,6-diamino-N-benzyloxy-1,2-dihydro-1,3,5triazine hydrochloride (20) from refluxing O-benzylhydroxylamine 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, hydroxy-substituted chalcones (26, 27) were prepared from the sodium hydroxide-catalyzed Claisen-Schmidt condensation reaction between acetophenone (25) or hydroxy-substituted acetophenone (22) and benzaldehyde (23) or hydroxy-substituted 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 hydroxysubstituted 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 hydroxy-substituted 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). Finally, the two fragments were coupled via Williamson etherification (Scheme 1C). Sodium hydroxide was used to deprotonate the N-hydroxy group of the 4,6-diamino-1-hydroxy-1,2dihydro-1,3,5-triazine 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 1-15 as hydrochloride salts. The solid products were purified by recrystallization to obtain clean compounds at 4-50% yields. The



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compounds 16, 6h and 18 (Figure 2) were also synthesized for biological evaluation alongside 1-15 (Supporting information, Scheme S1 to S3).

Evaluation of in vitro DHFR and TrxR inhibitory activities of compounds 1-15 Compounds 1-15 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 1-15 showed potent DHFR inhibitory activity with IC50 values in the nanomolar range of concentrations. These values were comparable to that of the 4,6-diamino-1,2dihydro-1,3,5-triazines 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 4-carbon linkers were either improved by around 2-fold (for 9 and 10) or similar (for 11 and 12) when compared to the corresponding compounds bearing 3-carbon 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 O

O NH2

NH2 O N

O n

N

A

R H2N

Cpd

N

N

N

.HCl H2N

R

1

R

O n

R1

R2

n

O

or

B

1

Attachment to linker

2

Ring

N

B

A .HCl

R2

DHFR IC50 (µM)

TrxR IC50 (30 min) (µM)

TrxR IC50 (60 min) (µM)

Position

1

2

CH3 CH3 A

4

0.0141 ± 0.0022

43.3 ± 10.9

20.4 ± 3.4

2

2

CH3 CH3 A

3

0.0356 ± 0.0026

10.8 ± 2.7

6.9 ± 2.1

3

2

CH3 CH3 B

4

0.0534 ± 0.0014

29.8 ± 5.0

21.1 ± 8.4

4

2

CH3 CH3 B

3

0.0592 ±0.0141

5.7 ± 0.2

4.0 ± 0.6

5

3

CH3 CH3 A

4

0.0092 ± 0.0010

49.0 ± 15.0

32.4 ± 7.7

6

3

CH3 CH3 A

3

0.0090 ± 0.0012

10.8 ± 2.2

8.2 ± 2.7

7

3

CH3 CH3 B

4

0.0064 ± 0.0003

35.9 ± 5.4

25.7 ± 4.2

8

3

CH3 CH3 B

3

0.0032 ± 0.0003

4.1 ± 1.5

2.8 ± 0.9

9

4

CH3 CH3 A

4

0.0034 ± 0.0004

44.5 ± 9.1

27.0 ± 4.4

10

4

CH3 CH3 A

3

0.0042 ± 0.0002

11.2 ± 1.3

6.7 ± 1.8

11

4

CH3 CH3 B

4

0.0040 ± 0.0005

31.6 ± 10.1

21.1 ± 7.7

12

4

CH3 CH3 B

3

0.0048 ± 0.0012

6.0 ± 1.2

3.8 ± 1.2

13

2

-(CH2)4-

A

3

0.0131 ± 0.0023

8.0 ± 1.6

4.6 ±1.1

14

3

-(CH2)4-

A

3

0.0061 ± 0.0021

7.5 ± 0.9

4.6 ± 0.3

15

4

-(CH2)4-

A

3

0.0026 ± 0.0008

9.2 ± 1.2

5.9 ± 1.3

16

0.0053 ± 0.0012

N.D.

N.D.

17

0.0199 ± 0.0061

N.D.

N.D.

6h

0.0039 ± 0.0008

N.D.

N.D.

18

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 1-15 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,β-unsaturated 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 4,6-diamino-1,2-dihydro-1,3,5-triazines 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. 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 3-position of ring B > 3-position of ring A > 4-position of ring B > 4-position 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 meta-position of the phenyl ring gave rise to an inductive electron-withdrawing effect, while attachment to the para-position produced a mesomeric electron donating effect. The inductive electron-withdrawing effect of the alkoxy linker at the meta-position intensified the δ+ charge on the β-carbon atom of the α,βunsaturated carbonyl moiety, thereby potentially increasing its propensity to react with nucleophiles such as the Sec residue in the C-terminal active site of the TrxR enzyme. Since ring B of the chalcone is closer to the β-carbon atom, this electron-withdrawing effect is stronger when the alkoxy linker is attached to the 3-position 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 di-ether linker and the alkyl substituent at R1 and R2 did not affect TrxR inhibitory activity. Evaluation of antiproliferative activity against human-derived cancer cell lines Since the results from the in vitro enzyme activity assays provided strong evidence that compounds 1-15 were inhibitors of both DHFR and TrxR; the compounds were then evaluated for antiproliferative activity against the human HCT116 colorectal and MCF-7 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 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assays, are shown in Table 2. 1-15 exhibited a biphasic dose-response curve (dose-response curves of 14 and 15 for MCF-7 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 di-ether 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 13 ACS Paragon Plus Environment


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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. Replacement of the dimethyl substituent at R1 and R2 with the cyclopentyl substituent resulted in no difference in growth inhibitory activity against MCF-7 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,β-unsaturated 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 1,3-diphenylpropan-2-one, a compound similar to chalcone 18 but lacking the α,βunsaturation at the carbonyl moiety, did not display cytotoxic activity and in vitro TrxR inhibitory activity (results not shown). The key structure-activity relationships for the antiproliferative activity of compounds 1-15 are summarized in Figure 4.


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Figure 3. Representative dose-response curves of 14, 15, 17 and 18 against (A) MCF-7 cells and (B) HCT116 cells.

Figure 4. Key structure-activity relationships of 1-15.



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Table 2. Antiproliferative activity of compounds against HCT116 and MCF-7 cells O

O NH2

NH2 O N

O n

N

A

R1 H2N

Cpd

N

N

2

R

B

R1 H2N

1

O n

N

.HCl

R2

n R

O

or

B

Attachment linker

N

A .HCl

2

R

Antiproliferative activity

to HCT116 cells

MCF-7 cells

Ring

Position

GI50 (µM)

LC50 (µM)

GI50 (µM)

LC50 (µM)

1

2 CH3

CH3

A

4

0.572 ± 0.144

50.1 ± 2.5

0.584 ± 0.141

40.1 ± 1.8

2

2 CH3

CH3

A

3

0.170 ± 0.036

49.9 ± 5.6

0.719 ± 0.063

40.7 ± 7.0

3

2 CH3

CH3

B

4

0.706 ± 0.142

81.9 ± 10.1

0.512 ± 0.101

47.7 ± 8.4

4

2 CH3

CH3

B

3

0.145 ± 0.036

52.2 ± 2.2

0.623 ± 0.138

38.8 ± 8.3

5

3 CH3

CH3

A

4

0.016 ± 0.004

55.2 ± 3.5

0.085 ± 0.020

34.0 ± 1.8

6

3 CH3

CH3

A

3

0.011 ± 0.004

42.7 ± 3.2

0.076 ± 0.029

20.2 ± 4.2

7

3 CH3

CH3

B

4

0.035 ± 0.010

39.0 ± 3.1

0.062 ± 0.013

35.3 ± 4.8

8

3 CH3

CH3

B

3

0.011 ± 0.001

48.2 ± 18.4

0.013 ± 0.004

29.8 ± 2.5

9

4 CH3

CH3

A

4

0.039 ± 0.009

24.0 ± 4.9

0.087 ± 0.013

14.2 ± 1.9

10

4 CH3

CH3

A

3

0.051 ± 0.008

30.3 ± 6.0

0.122 ± 0.009

11.3 ± 1.4

11

4 CH3

CH3

B

4

0.026 ± 0.008

42.9 ± 3.7

0.141 ± 0.033

14.8 ± 0.1

12

4 CH3

CH3

B

3

0.034 ± 0.011

34.7 ± 5.8

0.086 ± 0.016

15.5 ± 1.1

13

2 -(CH2)4-

A

3

0.260 ± 0.069

24.1 ± 2.1

0.770 ± 0.156

16.1 ± 0.8

14

3 -(CH2)4-

A

3

0.026 ±0.007

14.1 ± 1.0

0.080 ± 0.025

11.2 ± 1.5

15

4 -(CH2)4-

A

3

0.116 ± 0.014

13.5 ± 0.9

0.127 ± 0.010

9.7 ± 0.5

16

0.036 ±0.002

>100

0.107 ± 0.006

>100

17

0.021 ± 0.007

>100

0.077 ± 0.025

90.8 ± 18.7

6h

0.010 ± 0.001

34.2 ± 3.5

0.036 ± 0.006

25.9 ± 1.3

18

15.7 ± 2.2

22.5 ± 1.5

12.4 ± 2.0

30.7 ± 5.1

MTX

0.015 ± 0.001

>100

0.024 ± 0.007

>100

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 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), 4,6-diamino-1,2-dihydro1,3,5-triazines 16 and 17, as well as the chalcone compound 18, were also selected for biological evaluation for cellular DHFR and TrxR inhibitory activities. The cell-based DHFR assay was modified from an in-house protocol developed by SigmaAldrich 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 dose-dependent 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 MCF-7 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 4,6-diamino-1,2-dihydro1,3,5-triazines 16 and 17 also effected a dose-dependent decrease in cellular DHFR activity whereas the chalcone compound 18 did not inhibit cellular DHFR (Figure 5A). At cytotoxic concentrations of 40-70 µM, the activity of cellular DHFR in HCT116 cells was completely inhibited by 6, 10, 14 and 15, as well as the 4,6-diamino-1,2-dihydro-1,3,5triazines 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 DMSO-treated cells (Figure 5C), indicating that the drop in cellular DHFR activity was mediated by 14 and 15 17 ACS Paragon Plus Environment


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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.

Figure 5. Effects of selected dihydrotriazine-chalcone 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 DMSO-treated 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.

Evaluation of selected compounds on TrxR activity in cells At growth inhibitory concentrations of 1 nM to 1 µM, all four of the linked dihydrotriazinechalcone 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 40-70 µM, 6, 10, 14 and 15 brought about a dose-dependent 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 MCF-7 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, chalcone-containing 10, 14 and 15 produced stronger cellular TrxR inhibition than the parent chalcone compound 18, while the 4,6-diamino-1,2-dihydro-1,3,5-triazines 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 dose-dependent decrease in cellular TrxR activity was due to inhibition of the enzyme and not due to downregulation of the enzyme’s protein levels. 19 ACS Paragon Plus Environment


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Figure 6. Effects of selected dihydrotriazine-chalcone 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 DMSO-treated 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. 20 ACS Paragon Plus Environment

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CONCLUSION The previous attempt to design dihydrotriazine-chalcone compounds by merging the DHFRinhibiting 4,6-diamino-1,2-dihydro-1,3,5-triazine and TrxR-inhibiting 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 di-ether 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 MCF-7 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 cell-free and cell-based assays, it is therefore proposed that the strategy of combiing the dihydrotriazine and chalcone chemical scaffolds via di-ether linker has generated novel single molecules that exert antiproliferative effects on cancer cells at least partly through targeting DHFR and TrxR.

EXPERIMENTAL SECTION Chemistry All reagents were purchased from Sigma-Aldrich, Tokyo Chemical Industry, Alfa Aesar, or Merck and were used directly without any purification. Thin layer chromatography (TLC) was performed using silica gel-coated aluminium plates with fluorescent indicator and



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visualized with ultraviolet light at 254 nm. Melting points were determined using a Gallenkamp melting point apparatus and were uncorrected. 1H NMR and

13

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 (ESI-MS) 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 RP-18 250 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.

General Procedure for the synthesis of 1 to 12 4,6-Diamino-1-hydro-1,2-dihydro-1,3,5-triazine

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,N-dimethylformamide (2 to 5 mL) and bromoalkoxy-substituted 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 bromoalkoxysubstituted 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. (2E)-1-{4-[2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-ethoxy]-phenyl}-3phenylprop-2-en-1-one hydrochloride (1). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.58 g, 3 mmol) and (2E)-1-[4-(2-bromoethoxy)phenyl]-3phenylprop-2-en-1-one (0.99 g, 3 mmol), 1 was obtained as a yellow solid, 0.32 g, yield 23.9%. mp: 188-190°C. 1H NMR (400MHz, DMSO-d6) δ 9.07 (s, 1H, NH), 8.70 (s, 1H, NH), 8.18 (d, 2H, J = 9.2 Hz), 8.092 (s, 1H, NH), 7.92-7.71 (m, 4H), 7.79 (br s, 1H, NH), 7.507.49 (m, 3H), 7.16 (d, 2H, J = 8.8 Hz), 7.12 (br s, 1H, NH), 4.42 (br s, 2H), 4.36-4.35 (m, 2H), 1.58 (br s, 3H), 1.35 (br s, 3H). 13C NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 408.2 (M+1)+ (2E)-1-{3-[2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-ethoxy]-phenyl}-3phenylprop-2-en-1-one hydrochloride (2). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.39 g, 2 mmol) and (2E)-1-[3-(2-bromoethoxy)phenyl]-3phenylprop-2-en-1-one (0.66 g, 2 mmol), 2 was obtained as a pale yellow solid, 0.13 g, yield 14.3%. mp: 132-134°C. 1H NMR (400MHz, DMSO-d6) δ 9.02 (s, 1H, NH), 8.71 (s, 1H, NH), 8.06 (s, 1H, NH), 7.90-7.86 (m, 3H), 7.82-7.74 (m, 2H), 7.63-7.62 (m, 1H), 7.56 (t, 1H, J = 7.8 Hz), 7.51-7.49 (m, 3H), 7.31-7.29 (m, 1H), 7.08 (br s, 1H, NH), 4.40 (br s, 2H), 4.35-4.34 (m, 2H), 1.58 (br s, 3H), 1.34 (br s, 3H).

13

C NMR (100MHz, DMSO-d6) 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. ESI-MS 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}-1phenylprop-2-en-1-one hydrochloride (3). From 4,6-diamino-1,2-dihydro-2,2-dimethyl23 ACS Paragon Plus Environment


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[1,3,5]triazin-1-ol hydrochloride (0.58 g, 3 mmol) and (2E)-3-[4-(2-bromoethoxy)phenyl]-1phenylprop-2-en-1-one (0.99 g, 3 mmol), 3 was obtained as a yellow solid, 0.47 g, yield 35.5%. mp: 141-143°C. 1H NMR (400MHz, DMSO-d6) δ 9.03 (s, 1H, NH), 8.70 (s, 1H, NH), 8.14-8.12 (m, 2H), 8.07 (s, 1H, NH), 7.87 (d, 2H, J = 8.8 Hz), 7.82-7.66 (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, DMSO-d6) 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. ESI-MS m/z 408.2 (M+1)+ (2E)-3-{3-[2-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-ethoxy]-phenyl}-1phenylprop-2-en-1-one hydrochloride (4). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.39g, 2 mmol) and (2E)-3-[3-(2-bromoethoxy)phenyl]-1phenylprop-2-en-1-one (0.66 g, 2 mmol), 4 was obtained as a yellow solid, 0.45 g, yield 50.4%. mp: 139-141°C. 1H NMR (400MHz, DMSO-d6) δ 9.20 (s, 1H, NH), 8.73 (s, 1H, NH), 8.15-8.13 (m, 2H), 8.06 (s, 1H, NH), 7.90 (d, 1H, J = 15.6 Hz), 7.78 (br s, 1H, NH), 7.757.70 (m, 2H), 7.64-7.60 (m, 2H), 7.50-7.42 (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).

13

C NMR

(100MHz, DMSO-d6) 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. ESIMS m/z 408.2 (M+1)+ (2E)-1-{4-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-propoxy]-phenyl}-3phenylprop-2-en-1-one hydrochloride (5). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.97 g, 5 mmol) and (2E)-1-[4-(3-bromopropoxy)phenyl]3-phenylprop-2-en-1-one (1.73 g, 5 mmol), 5 was obtained as a yellow solid, 0.68 g, yield 29.8%. mp: 196-198°C. 1H NMR (400MHz, DMSO-d6) 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), 7.87-7.85 (m, 2H), 7.78 (br s, 1H, NH), 7.72 (d, 1H, J = 15.6 Hz), 7.50-7.48 (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).

13

C NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 422.2 (M+1)+ (2E)-1-{3-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-propoxy]-phenyl}-3phenylprop-2-en-1-one hydrochloride (6). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.58 g, 3 mmol) and (2E)-1-[3-(3-bromopropoxy)phenyl]3-phenylprop-2-en-1-one (0.99 g, 3 mmol), 6 was obtained as a yellow solid, 0.31 g, yield 22.9%. mp: 164-166°C. 1H NMR (400MHz, DMSO-d6) δ 8.89 (s, 1H, NH), 8.62 (s, 1H, NH), 8.087 (s, 1H, NH), 7.89-7.85 (m, 3H), 7.79-7.73 (m, 2H), 7.68 (br s, 1H, NH), 7.60-7.50 (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, J = 5.8 Hz), 2.21 (br s, 2H), 1.43 (br s, 6H). 13C NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 422.2 (M+1)+ (2E)-3-{4-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-propoxy]-phenyl}-1phenylprop-2-en-1-one hydrochloride (7). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.77 g, 4 mmol) and (2E)-3-[4-(3-bromopropoxy)phenyl]1-phenylprop-2-en-1-one (1.38 g, 4 mmol), 7 was obtained as a yellow solid, 0.92 g, yield 50.0%. mp: 193-195°C. 1H NMR (400MHz, DMSO-d6) δ 9.11 (s, 1H, NH), 8.63 (s, 1H, NH), 8.12-8.10 (m, 2H), 8.09 (s, 1H, NH), 7.84 (d, 2H, J = 8.8 Hz), 7.79-7.68 (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).

13

C NMR (100MHz,



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DMSO-d6) 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. ESI-MS m/z 422.2 (M+1)+ (2E)-3-{3-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-propoxy]-phenyl}-1phenylprop-2-en-1-one hydrochloride (8). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.39 g, 2 mmol) and (2E)-3-[3-(3-bromopropoxy)phenyl]1-phenylprop-2-en-1-one (0.69 g, 2 mmol), 8 was obtained as yellow crystals, 0.095 g, yield 10.4%. mp: 132 °C (decomposed). 1H NMR (400MHz, DMSO-d6) δ 8.94 (s, 1H, NH), 8.63 (s, 1H, NH), 8.17-8.15 (m, 2H), 8.08 (s, 1H, NH), 7.95 (d, 1H, J = 15.6 Hz), 7.74-7.67 (m, 2H), 7.67 (br s, 1H, NH), 7.59 (t, 2H, J = 7.4 Hz), 7.50-7.46 (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).

13

C NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 422.3 (M+1)+ (2E)-1-{4-[4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-butoxy]-phenyl}-3phenylprop-2-en-1-one hydrochloride (9). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.77 g, 4 mmol) and (2E)-1-[4-(4-bromobutoxy)phenyl]-3phenylprop-2-en-1-one (1.44 g, 4 mmol), 9 was obtained as a pale yellow solid, 0.31 g, yield 16.5%. mp: 196-198 °C. 1H NMR (400MHz, DMSO-d6) 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), 7.89-7.87 (m, 2H), 7.71 (d, 1H, J = 15.6 Hz), 7.76 (br s, 1H, NH), 7.47-7.45 (m, 3H), 7.10 (br s, 1H, NH), 7.09 (d, 2H, J = 8.8 Hz), 4.15-4.00 (m, 4H), 1.85 (br s, 4H), 1.44 (br s, 6H). 13C NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 436.5 (M+1)+ (2E)-1-{3-[4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-butoxy]-phenyl}-3phenylprop-2-en-1-one hydrochloride (10). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.97 g, 5 mmol) and (2E)-1-[3-(4-bromobutoxy)phenyl]-3phenylprop-2-en-1-one (1.80 g, 5 mmol), 10 was obtained as a white solid, 0.21 g, yield 8.7%. mp: 192-194 °C. 1H NMR (400MHz, DMSO-d6) δ 9.00 (s, 1H, NH), 8.58 (s, 1H, NH), 8.02-7.46 (m, 10H), 7.67 (br s, 2H, NH), 7.25 (dd, 1H, J = 8.0, 2.0 Hz), 7.07 (br s, 1H, NH), 4.11-4.00 (m, 4H), 1.85 (br s, 4H), 1.44 (br s, 6H). 13C NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 436.4 (M+1)+ (2E)-3-{4-[4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1(2H)-yloxy)-butoxy]-phenyl}-1phenylprop-2-en-1-one hydrochloride (11). From 4,6-diamino-1,2-dihydro-2,2-dimethyl[1,3,5]triazin-1-ol hydrochloride (0.68 g, 3.5 mmol) and (2E)-3-[4-(4-bromobutoxy)phenyl]1-phenylprop-2-en-1-one (1.26 g, 3.5 mmol), 11 was obtained as a yellow solid, 0.067 g, yield 4.1%. mp: 194-196 °C. 1H NMR (400MHz, DMSO-d6) δ 9.07 (s, 1H, NH), 8.59 (s, 1H, NH), 8.15-8.13 (m, 2H), 8.03 (s, 1H, NH), 7.87-7.55 (m, 7H), 7.72 (br s, 1H, NH), 7.13 (br s, 1H, NH), 7.02 (d, 2H, J = 8.8 Hz), 4.08-3.99 (m, 4H), 1.84 (m, 4H), 1.44 (m, 6H). 13C NMR (100MHz, DMSO-d6) 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. ESI-MS 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}-1phenylprop-2-en-1-one hydrochloride (12). From 4,6-diamino-1,2-dihydro-2,2-dimethyl27 ACS Paragon Plus Environment


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[1,3,5]triazin-1-ol hydrochloride (0.77 g, 4 mmol) and (2E)-3-[3-(4-bromobutoxy)phenyl]-1phenylprop-2-en-1-one (1.44 g, 4 mmol), 12 was obtained as a pale yellow solid, 0.18 g, yield 9.3%. mp: 177-179 °C. 1H NMR (400MHz, DMSO-d6) δ 9.00 (s, 1H, NH), 8.59 (s, 1H, NH), 8.18-8.15 (m, 2H), 8.03 (s, 1H, NH), 7.95 (d, 1H, J = 15.6 Hz), 7.74-7.67 (m, 2H), 7,69 (br s, 1H, NH), 7.59 (t, 2H, J = 7.6 Hz), 7.49-7.44 (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), 4.10-4.00 (m, 4H), 1.84 (br s, 4H), 1.44 (br s, 6H). 13

C NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 436.2 (M+1)+ 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. (2E)-1-(3-{2-[(7,9-diamino-6,8,10-triazaspiro[4.5]deca-7,9-dien-6-yl)oxy]ethoxy}phenyl)3-phenylprop-2-en-1-one

hydrochloride

(13).

From

7,9-diamino-6,8,10-

triazaspiro[4.5]deca-7,9-dien-6-ol hydrochloride (0.44 g, 2 mmol) and (2E)-1-[3-(2bromoethoxy)phenyl]-3-phenylprop-2-en-1-one (0.66 g, 2 mmol), 13 was obtained as an offwhite solid, 0.36 g, yield 38.4%. mp: 192-194 °C. 1H NMR (400MHz, DMSO-d6) δ 9.34 (s, 1H, NH), 8.73 (s, 1H, NH), 8.14 (s, 1H, NH), 7.94-7.74 (m, 5H), 7.65 (br s, 1H, NH), 7.637.47 (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), 1.76-1.66 (m, 6H).

13

C NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 434.5 (M+1)+


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(2E)-1-(3-{3-[(7,9-diamino-6,8,10-triazaspiro[4.5]deca-7,9-dien-6yl)oxy]propoxy}phenyl)-3-phenylprop-2-en-1-one hydrochloride (14). From 7,9-diamino6,8,10-triazaspiro[4.5]deca-7,9-dien-6-ol hydrochloride (0.66 g, 3 mmol) and (2E)-1-[3-(3bromopropoxy)phenyl]-3-phenylprop-2-en-1-one (1.04 g, 3 mmol), 14 was obtained as an off-white solid, 0.35 g, yield 24.2%. mp: 191-192 °C. 1H NMR (400MHz, DMSO-d6) δ 9.14 (s, 1H, NH), 8.65 (s, 1H, NH), 8.14, (s, 1H, NH), 7.95-7.89 (m, 3H), 7.80-7.73 (m, 2H), 7.637.62 (m, 1H), 7.75 (br s, 1H, NH), 7.53-7.47 (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), 2.21-2.19 (m, 2H), 2.00 (br s, 2H), 1.74-1.70 (m, 2H), 1.57-1.51 (m, 4H).

13

C NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 448.5 (M+1)+ (2E)-1-(3-{4-[(7,9-diamino-6,8,10-triazaspiro[4.5]deca-7,9-dien-6yl)oxy]butyloxy}phenyl)-3-phenylprop-2-en-1-one hydrochloride (15). From 7,9-diamino6,8,10-triazaspiro[4.5]deca-7,9-dien-6-ol hydrochloride (1.10 g, 5 mmol) and (2E)-1-[3-(4bromobutoxy)phenyl]-3-phenylprop-2-en-1-one (1.80 g, 5 mmol), 15 was obtained as an offwhite solid, 0.78 g, yield 31.4%. mp: 186-187 °C. 1H NMR (400MHz, DMSO-d6) δ 9.25 (s, 1H, NH), 8.61 (s, 1H, NH), 8.06 (s, 1H, NH), 7.94-7.89 (m, 3H), 7.79-7.73 (m, 2H), 7.71 (br s, 1H, NH), 7.61-7.60 (m, 1H), 7.52-7.47 (m, 4H), 7.25 (dd, 1H, J = 8.0, 2.0 Hz), 6.98 (br s, 1H, NH), 4.13-4.10 (m, 2H), 4.02-3.99 (m, 2H), 2.01 (br s, 2H), 1.88-1.69 (m, 10H).

13

C

NMR (100MHz, DMSO-d6) 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. ESI-MS m/z 462.5 (M+1)+



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Chemicals, Reagents and Cell culture Stock solutions of the compounds were prepared by dissolving the compounds in DMSO (final concentration 50 mM), and stored at -20ºC. DMSO, dihydrofolate, NADPH, recombinant human DHFR, thioredoxin reductase from rat liver, 5,5'-dithiobis-(2nitrobenzoic acid) (DTNB), guanidine HCl and insulin from bovine pancreas were purchased from Sigma Aldrich (St. Louis, MO). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Duchefa Biochemie (Haarlem, The Netherlands). MTX and ribonuclease A were purchased from MP Biomedicals (Santa Ana, CA). Protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Mannheim, Germany). Recombinant human Trx was obtained from IMCO Corporation (Stockholm, Sweden). DHFR antibody (AB54612) was purchased from Abcam (Cambridge, UK), while TrxR (sc18220) and β-actin (sc-47778) antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Thermo Scientific (Rockford, IL) or Santa Cruz Biotechnology Inc. Human-derived HCT116 colorectal and MCF-7 breast carcinoma cells (ATCC, Rockville, MD) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2.

In vitro DHFR inhibition assay The assay was performed at room temperature using a Hitachi U-1900 UV/visible spectrophotometer. In a 1 ml quartz cuvette containing a solution of 50 µM dihydrofolate (DHF), 1.5 x 10-3 units of recombinant human DHFR and various concentrations of the test


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compounds in 0.15 M phosphate buffer (pH 7), NADPH (final concentration 60 µM) was added to initiate the enzymatic reaction. The rate of NADPH consumption during the conversion of DHF to THF was monitored by taking absorbance readings at 340 nm every 30 sec over 6 min. A graph of absorbance readings versus time was plotted and the slope of the graph represents the rate of reaction. The percentage inhibition at each concentration of inhibitor was calculated using the following formulae:

Activity % =

Slopecompound − Slopeblank × 100 Slopecontrol − Slopeblank

Inhibition % = 100 − Activity where Slopecompound is the slope of the graph for solutions containing DHFR, test compound, NADPH and DHF; Slopecontrol is the slope of the graph for solutions containing DHFR, NADPH and DHF; while Slopeblank is the slope of the graph for solutions containing NADPH and DHF only. Graphs of percentage inhibition against logarithmic concentration were plotted for each compound and IC50 values were calculated using GraphPad Prism Version 5.01.

In vitro TrxR inhibition assay (DTNB reduction assay) In 96-well plates, a range of concentrations of the test compounds in DMSO stock solutions were incubated with 2.1 units/ml of TrxR from rat liver and 200 µM of NADPH in a volume of 100 µl of 50 mM Tris-HCl and 1 mM EDTA, pH 7.5 (TE buffer) at room temperature for 30 or 60 min. A volume of 100 µl of TE buffer containing 5 mM of DTNB and 200 µM NADPH was added to initiate the reaction, where the increase in TNB absorbance at 412 nm was measured over the initial 2 min with a Molecular Devices VersaMax Microplate Reader.



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Maximal velocity (Vmax) values were obtained from the linear portion of the absorbance over time curve. TrxR activity was calculated as a percentage of enzyme activity compared to that of vehicle-treated sample using the formula:

Activity % =

Vmax compound × 100 Vmax control

Graphs of percentage activity against logarithmic concentration were plotted for each compound and IC50 values were calculated using GraphPad Prism Version 5.01.

Cell viability assay Cells were seeded at a density of 1.0 x 104/well into 96-well plates and allowed to adhere overnight before introduction of test compounds. Serial dilutions of the compounds were prepared in medium from DMSO stock solutions immediately before each assay. At the time of compound addition (T0) and after 72 h of incubation with the compounds, cell viability was determined by MTT assay. Briefly, MTT was added into each well at a final concentration of 400 µg/ml and the plates were incubated at 37°C for 4 h. The MTTcontaining medium was aspirated and the insoluble formazan product was solubilized by addition of 150 µl of DMSO:glycine buffer, pH 10.5 (ratio 4:1). Absorbance at 550 nm was measured using a Molecular Devices VersaMax Microplate Reader. To obtain GI50 and LC50 values, the absorbance readings were plotted against logarithmic concentration on GraphPad Prism Version 5.01. GI50 and LC50 values were interpolated from the graphs as concentrations corresponding to the following absorbance values:

Absorbance at GI$% =

Absorbance at T0 ' Absorbance of control 2


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Absorbance at LC50 =

Absorbance at T0 2

Preparation of cell lysates and Western blot analysis HCT116 cells were plated onto 100 mm culture plates at 1.5 x 106 cells per plate and incubated for 48 h to allow exponential growth before treatment with selected test compounds for 16 h. Following drug treatment, adherent and floating cells were pooled, pelleted, washed once in ice-cold PBS and lysed in Triton X-100 lysis buffer (25 mM TrisHCl pH 7.5, 100 mM NaCl, 2.5 mM EGTA, 2.5 mM EDTA, 20 mM NaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 20 mM sodium β-glycerophosphate, 0.5% Triton X-100) containing freshly added protease inhibitor cocktail. Prior to use for determination of DHFR or TrxR activity or Western blot analysis, the lysates were precleared by centrifugation and protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA) as described in the manufacturer’s manual. For Western blot analysis, equal amounts of protein in lysate samples were separated by SDS-PAGE and resolved proteins were electroblotted onto nitrocellulose membranes. The blots were probed with a primary antibody followed by a secondary HRP-conjugated antibody, and then developed using enhanced chemiluminescence system [Western Lightning, PerkinElmer (Boston, MA) or SuperSignal West Femto, Thermo Scientific (Rockford, IL)]. All Western blots shown are representative of three independent experiments.

Determination of DHFR Activity in HCT116 cell lysates



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In 96-well plates, each cell lysate sample containing 400 µg of protein was mixed in a final volume of 200 µl PBS containing 50 µM DHF and 200 µM NADPH. Controls containing all the reagents with additional 1 µM methotrexate were set up alongside. The decrease in absorbance at 340 nm was measured at room temperature over 15 min with a Molecular Devices VersaMax Microplate Reader. DHFR activity was calculated as percentage activity of untreated cells using the following formula:

Activity %=

Vmax treated − Vmax treated'MTX ×100 Vmax untreated − Vmax untreated'MTX

Determination of TrxR Activity in HCT116 cell lysates TrxR activity in cell lysates was measured using a published end point insulin assay method.33 In 96-well plates, each cell lysate sample containing 25 µg of protein was incubated in a reaction volume of 50 µl containing 85 mM HEPES (pH 7.6), 0.3 mM insulin, 660 µM NADPH, 3 mM EDTA and 10 µM human Trx for 40 min at room temperature. Controls containing cell lysate and all the reagents except human Trx were set up alongside. The reaction was quenched by addition of 200 µl of 1 mM DTNB and 240 µM NADPH in 6 M guanidine hydrochloride and 200 mM Tris-HCl solution (pH 8.0). The amount of free thiols generated from insulin reduction was determined by DTNB reduction measured at 412 nm with a Molecular Devices VersaMax Microplate Reader. TrxR activity was represented as the absorbance at 412 nm minus that of the corresponding control.

ASSOCIATED CONTENT Supporting Information. 34 ACS Paragon Plus Environment

Page 35 of 48

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Synthesis and characterization of 18, 16 and 6h. This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Authors *

E.-H. Chew (E-mail: [email protected]; Phone: +65 65161955; Fax: +65 67791554) and

W.-K. Chui (E-mail: [email protected]; Phone: +65 65162933; Fax: +65 67791554) Present Address ∥

H.-L. Ng: School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 535

Clementi Road, Singapore 599489 Author Contributions All authors have given approval to the final version of the manuscript and declare that they have no conflict of interest.

ACKNOWLEDGMENTS This work was supported by the following research grants: National University of Singapore Academic Research Fund Tier 1 R-148-000-153-112 to W.-K. Chui, National Medical Research Council Grant NMRC/NIG/0050/2009 and National University of Singapore Academic Research Fund Tier 1 R-148-000-184-112 to E.-H. Chew.

ABBREVIATIONS



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Page 36 of 48

DHF, dihydrofolate; DHFR, dihydrofolate reductase; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid; EtOH, ethanol; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTX, methotrexate; SN2, nucleophilic substitution bimolecular; NMR, nuclear magnetic resonance; Trx, thioredoxin; TrxR, thioredoxin reductase

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Sodani, R. S.; Choudhary, P. C.; Sharma, H. O.; Verma, B. L. Syntheses and

Reactions of 4 '-[(omega-Bromoalkyl)oxy]- and 4 ',4 '''-(Polymethylenedoxy)-bis Substituted Chalcones. E-J. Chem. 2010, 7, 763-769. 31.

Lu, X.; Errington, J.; Curtin, N. J.; Lunec, J.; Newell, D. R. The impact of p53 status

on cellular sensitivity to antifolate drugs. Clin Cancer Res 2001, 7, 2114-2123.



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32.

Dihydrofolate

reductase

assay

kit

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-

technical

bulletin.

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/cs0340bul.pdf (6 July 2016). 33.

Arnér, E. S.; Holmgren, A. Measurement of thioredoxin and thioredoxin reductase.

Curr. Protoc. Toxicol. 2001, Chapter 7, Unit 7.4.


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Graphical Abstract 47x26mm (600 x 600 DPI)

ACS Paragon Plus Environment


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Figure 2. Structures of 16, 17, 6h, 18 and MTX. 96x73mm (600 x 600 DPI)


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Figure 3. Representative dose-response curves of 14, 15, 17 and 18 against (A) MCF-7 cells and (B) HCT116 cells. 123x140mm (600 x 600 DPI)



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Figure 4. Key structure-activity relationships of 1-15. 59x27mm (600 x 600 DPI)


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Design, Synthesis, and Biological Evaluation of Coupled Bioactive

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