Novel coumarin cyclotriphosphazene derivatives: Synthesis, characterization, DNA binding analysis with automated biosensor and cytotoxicity

Journal Pre-proof Novel coumarin cyclotriphosphazene derivatives: Synthesis, characterization, DNA binding analysis with automated biosensor and cytotoxicity Yakup Eker, Elif Şenkuytu, Zehra Ölçer, Tuba Yıldırım, Gönül Yenilmez Çiftçi PII:

S0022-2860(20)30296-9

DOI:

https://doi.org/10.1016/j.molstruc.2020.127971

Reference:

MOLSTR 127971

To appear in:

Journal of Molecular Structure

Received Date: 17 December 2019 Revised Date:

14 February 2020

Accepted Date: 24 February 2020

Please cite this article as: Y. Eker, E. Şenkuytu, Z. Ölçer, T. Yıldırım, Göü.Yenilmez. Çiftçi, Novel coumarin cyclotriphosphazene derivatives: Synthesis, characterization, DNA binding analysis with automated biosensor and cytotoxicity, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/ j.molstruc.2020.127971. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

CREDİT AUTHOR STATEMENT Ideas; Writing- Original Prof. Dr. Gönül YENİLMEZ ÇİFTÇİ: Conceptualization draft preparation, Writing- Reviewing and Editing, Investigation Prof.Dr. Tuba YILDIRIM: Investigation, Writing- Reviewing and Editing, Responsible for Biological aplication. Dr. Yakup EKER: Resources, Writing- Reviewing and Editing He is reponsible for synthesis and characterisation. Dr. Zehra ÖLÇER: Writing- Reviewing and Editing Investigation, Responsible for Biological aplication Dr. Elif ŞENKUYTU: Investigation, Methodology, Writing- Reviewing and Editing.

Novel

Coumarin

Cyclotriphosphazene

Derivatives:

Synthesis, Characterization, DNA Binding Analysis with Automated Biosensor and Cytotoxicity Yakup Eker a, Elif Şenkuytu a, Zehra Ölçer a, Tuba Yıldırımb, Gönül Yenilmez Çiftçi* a

a

Department of Chemistry, Gebze Technical University, 41400 Gebze-Kocaeli, Turkey

b

Department of Biology, Faculty of Arts and Sciences, Amasya University, 05100 Amasya,

Turkey

Corresponding author: *G.Yenilmez Ciftci. Tel: +90 2626053011 E-mail: [email protected]

1

Abstract The synthesis of many new anti-cancer compounds is currently in drug discovery area, unfortunately the side effects of these compounds are not eliminated. In the efforts to develop suitable anticancer drugs, medicinal chemists have focused on coumarin derivatives. To understand the effects of the coumarin derivatives on DNA, it is necessary to analyze the interaction with in vitro tests due to DNA is a target molecule for anticancer drug development and investigation on DNA interactions with synthesized potential compounds. Electrochemical biosensors provide good alternative for the quantitative analysis of DNA/compound interactions. In this study, we have prepared and characterized tetra novel coumarin substituted cyclotriphosphazene derivatives (3-6). By expanding our study, we performed biological activity studies of all compounds together with the compounds our synthesized before (7-12). All compounds were characterized by elemental analysis, MALDI-TOF mass spectrometry, FT-IR, 1H and 31P NMR techniques and screened to effect on DNA using an automated biosensor device. Compounds were also evaluated for their anti-cancer potential against cancer cell lines to assess whether these derivatives may serve as a lead for the augmentation of anti-cancer drug. All compounds were assessed for cytotoxicity by the MTT assay against two different cancer cell lines, MCF-7 and DLD-1 and most of these compounds have cytotoxic effects for MCF-7 and DLD-1 cells.

Keywords: Coumarin, Phosphazene, Cyclotriphosphazene, Biosensor, Anticancer, Cytotoxicity.

2

Introduction Cancer, the disease that causes the most deaths in the world, is also referred to as the disordered replication of DNA and cell division. Several methods are used for the treatment of this disease, including surgical intervention, chemotherapy, immunotherapy, gene therapy, radiotherapy and photodynamic therapy. Anti-cancer drugs are developed for utilizing these therapies by designing molecules that will kill rapidly dividing cells1. Use of anticancer drugs is one of the effective methods in the treatment of cancer, and for all types of cancer, it is important to develop effective drugs. A number of compounds shown to be effective against cancer have been synthesized. Unfortunately, the side effects of these compounds are not eliminated. To understand the effects of a carcinogenic compound on DNA, it is necessary to analyze the interaction (of what? Are you talking about the carcinogen or the drug) with in vitro tests. The interaction of DNA with target molecules is based on the binding to DNA. There are a series of interactions associated with DNA and ligand molecules, such as hydrogen bonding, -stacking, van der Waals and hydrophobic interaction which can give rise to covalent binding/cross linking, intercalation, non-covalent groove binding, and DNA cleaving2. The interaction of potential drugs with DNA has been studied using in vitro techniques such as spectroscopic and voltammetry methods, absorption and fluorescence emission spectral studies, circular dichroism spectroscopy3-6. These kinds of analysis are difficult, costly and time-consuming. This has led the use of DNA interaction analyzes using biosensor devices that are easier, faster, cheaper and more specific. In this context, it is important to note that while the preparation of new drug candidate molecules is still ongoing, the effects of these newly synthesized compounds must be determined quickly by prescreening tests. Research and exploration into cancer drugs is almost a race. Biological activity tests include cytogenetic assays using cells and later as gel electrophoresis, etc. They are useful tools for monitoring DNA binding processes however the other good alternatives are biosensor based assays due to their fast, cost effective and quantitative nature together with potential of automation for high throughput screening7. There is a growing interest in the design of biosensors especially, the automated biosensor based pre-screening of synthesized molecules as potential drug candidates that followed by the further analysis of the selected compounds may pave the way for faster and targeted drug discovery process. Electrochemical DNA biosensors are often used for quantitative DNA/compound interaction analysis due to rapid, simple and efficient detection 3

properties8-10. MiSens has been used as an automated biosensor device for the investigation of DNA interaction properties of the newly synthesised compounds by measuring DNA hybridization efficiency on the biochip surface7,11,12. Phosphazenes have become one of the leading fields of research owing to their flexibility towards substitution and the corresponding properties depending on the nature of the substituted groups13-16. Among phosphazenes, cyclotriphosphazenes have received more attention due to their better biological activity responses and wider application palettes while

discovering

the

improved

next

generation

novel

compounds.

The

cyclotriphosphazene ring not only provides a platform to bring groups together such as coumarins, spermines, tyramines and/or others which are exhibiting biological activity but also promotes synergy and increased efficacy on the final product17,18. Coumarins have recently attracted a great deal of attention as a result of their broad spectrum of pharmacological properties. They have been reported to exhibit antiviral19 antioxidant20, anti-inflammatory21 and anti-cancer properties22,23. Moreover, the unique structure of coumarin allows its derivatives to readily interact with a wide range of diverse biomacromolecules through weak interactions24,25. Compared to the protein-based targeted drugs, the number of known DNA-based targeted drugs is still very limited. Coumarins are one of the most strategic molecules serving this purpose. Increase in the knowledge regarding molecular and cellular mechanisms involved in the development of metastasis and disease progression. Discovery of new therapeutic targets should offer a new opportunity to improve clinical practices or develop new strategies for cancer treatments. With the aim of preparing new drugs with anticancer activity, we prepared and characterized tetra novel coumarin derivatives of phosphazene (3-6) (Scheme 1). We also extended our study to six known similar compounds (7-12) (Table 2.) since no investigations about their biological activity have been reported. All compounds were characterized by elemental analysis, mass spectrometry, 1H and 31P NMR. The chemical characterizations of the newly synthesized compounds and their DNA effects as potential drug candidates have been investigated. Cost effective and rapid methods are needed to speed up drug screening. To answer this need, in the current study an automated biosensor device was used to investigate molecule/DNA interaction properties. We synthesized compounds and analysed cytotoxic properties to assess anti-cancer capabilities against MCF-7 and DLD-1 cells.

4

CH3 O

O H3C

O

O

CH3

O

O P

O

O

CH3

CH3

CH3

N O H3C

O

N

P

O

N

N

P

O

O

O H3C

O

H3C

P

O

O

O

N

P

O

N

P

O

O

O O

O O

O O

H3C (3)

O CH3

(4)

OH

OH

H3C O

H3C

H3C

Cs2CO3 / THF

O

H 3C

Cl Cl

P N

P

O Cs2CO3 / THF

N

P

Cl

O O

O

N

Cl (1)

Cs2CO3 / THF OH

O O

H3C

P N

O

H3C

O

N

P

O P N

Cs2CO3 / THF

O

OH H3C O O

Cl

Cl (2)

CH3 O

H3C

O

H3C

CH3 O

O

N

P

O

P

O

O

O

O

N

P N

N

P N

P

O

O

O

(6)

(5)

Scheme 1. Coumarin substituted cyclotriphosphazene derivatives

5

O

CH3

O N

P O O

O

H3C

O O O

Results and discussion Synthesis and Characterization of the Compounds 3-6.

In this study, new four coumarin substituted (3-6) cyclotriphosphazene compounds have been synthesized and characterizated. For this purpose, monospiro- (1), dispiro- (2) substituted cyclotriphosphazene compounds were reacted with 4-Hydroxy 6,7dimethylcoumarin

and

4-Hydroxy

6-methylcoumarin,

respectively.

Then,

four

compounds (3-6) were obtained (Scheme 1). In addition to this we have extended in this study we have resynthesized six compounds26,27 (7-12), for purpose of DNA binding works (Table 1). All the products were purified by column chromatography and/or preparative TLC techniques. The isolated all compounds (3-12) were characterized by elemental analysis, MALDI-TOF Mass Spectrometry, FT-IR,

1

H and

31

P NMR

techniques. The elemental analyses, mass spectrometric and 1H NMR results for each new compound are provided as part of the analytical data in the experimental section. The proton decoupled 34

31

P NMR spectra of compounds 3-4 were observed as AB2 spin

31

system whereas P NMR spectra of compounds 5-6 were observed as A2B spin system due to the different environments for the two different phosphorus nuclei on the cyclotriphosphazene ring (Figure 1a, 1b as an example for compound 3 and compound 5). 31

P NMR Spectrums for newly synthesized all the compounds (3-6) were given in Fig.S1-

S4. For compound 3 –PO2(naphthoxy) was observed at δ=8.18 ppm, and (coumarin) are observed at δ=6.97 ppm (JAB = 97.95 Hz). Furthermore,

31

–PO2

P NMR

Spectrum of Compound 5 –PO2 (naphthoxy) was observed at δ=8.93 ppm, PO2(coumarin) was observed at δ=7.81 ppm (JAB = 74.03 Hz) (Fig 1). The 1H NMR data also confirmed the structure of the compound 3. The 1H NMR chemical shifts and coupling constants were shown in Table S1. The aromatic protons for compound 3 were observed between δ=6.32 ppm and δ=8.25 ppm, -CH2 protons δ=4.98 ppm. In addition – CH3 protons δ=2.36 ppm, δ=2.28 ppm. 1H NMR Spectrums of compounds (3-6) were given in Fig.S5-S8. 1H NMR Spectrums of compounds 3 and 4 were given in Fig 2. The most important difference was seen for –CH3 groups protons in the spectrum. For compound 3 every coumarin groups include two methyl groups whereas for compound 4 every coumarin groups include one methyl group. 1H NMR spectrum of compound 3 showed that every single methyl group’s chemical shifts were different (Fig 2). For all compounds (3-6) 1H NMR data were given in table S1-S2. MALDI-TOF Spectrum of compounds 3 and 5 were shown the protonated molecular-ion [M+H]+ peaks, whereas 6

compounds 4 and 6 were shown the molecular-ion [M+] peaks. MALDI-TOF spectra of all compounds (3-6) were given in Fig S9-S12.

Figure 1. The proton decoupled

31

P NMR spectra of the (a) compound 3 and (b)

compound 5 in CDCl3 solution.

7

Figure 2. (a) 1H NMR spectra of the compound 4 (b) 1H NMR spectra of the compound 3 in CDCl3 solution

Synthesis of Compounds 7-12. Compounds 7-12 were synthesized according to literature27.

Table 1. Re-synthesized compounds (7-12) for biological applications. Comp. No

Structure of Compound

Comp. No

(7)a

(8)a

(9)b

(10)b

8

Structure of Compound

(11)b

a

(12)b

[26], b [27]

Characterization of the Sensor Chip Surface

Biochip surface was coated by alkanethiol. To investigate its effects, cyclic voltammetry measurements for the electrodes were performed using potassium ferrocyanide (K4[Fe(CN)6]) solution. SAM (self-assembled monolayer) on the biochip surface must be completed in order to establish a strong NeutrAvidin coverage. The evidence of the formation of SAM can be seen in Fig. 3 voltammograms. The area for electrochemical oxidation and reduction of ferrocyanide was larger on bare gold sensor chip compared to SAM coated chip. SAM surface coverage θ, was calculated with Equation 1 and correlated with bare gold electrode. Q is the charge over the course of cyclic voltammetry reduction/oxidation cycle29,30.

(Eq. 1) Surface coverage of MUDA was found as 93.91% ± 3.62 by using Equation 1.

9

Figure 3. The cyclic voltammetry using bare and MUDA coated gold electrode arrays (1 mM K4[Fe(CN)6]/KCI at 100 mV/s scan rate). DNA Interaction Analysis of Compounds 3-12. The investigation of anti-carcinogen compounds using electrochemical biosensors compared with the other techniques such as UV-vis, quartz crystal microbalance, fluorescent and electrochemiluminescence, is an effective way to discover potential drug candidates9,10,31,32. In our previous works, we were used MiSens®, a new automated electrochemical biosensor with sensitivity and fast response that has a microfluidic, automatic injection system and an integrated biochip [7, 12, 33]. In this work, firstly the reaction of 1 and 2 with 4-hydroxy 6,7-dimethylcoumarin and 4-hydroxy 6methylcoumarin were performed and compound 3, 4 and 5, 6 were obtained, respectively. Later, new synthesized compounds (3-6) and previously synthesized compounds26,27 (712) have been tested with the biosensor device and their ability to interact with DNA has been investigated. DNA interaction assays with compounds (3-12) were performed using device which on microfluidic channel integrated biochip during flow. This assay is consist of several steps that have capture of surface probe DNA, hybridization and modified gold nanoparticles. Firstly, the biotinylated surface probe DNA was captured on NeutrAvidin immobilized biochip surface. Second step was achieved hybridization of two complementary target sequence and biotinylated detection probe DNA and then incubation of hybridized DNA with varying concentrations (0-200 µM) of compounds. This hybridized mixture was injected to the surface probe immobilized biochip surface to assess the hybridization of the target to its complementary surface probe. Last step,

10

enzyme and NeutrAvidin modified gold nanoparticles were injected to the biochip surface for binding and then an amperometric biosensor signal was measured via substrate injection33 (Fig. 4).

Figure 4. Schematics of the DNA-interaction analysis using biosensor. Compounds 1-6; both the individual reactive (1, 2) and the synthesized products (3–6) were tested by biosensor device and the hybridization activity results are shown Fig. 5. When the hybridization activity of the reactive 1 has been compared to its synthesized compounds 3 and 4, 52% and 87% further activity reduction respectively has been observed. The other reactive 2 has been compared to its synthesized compounds 5 and 6. In the 50 µM compound concentration, reactive 2 resulted in 27% activity reduction whereas compound 5 and 6, 78% and 80% activity reduction have been observed. In the low compound concentrations (12.5-50 µM), the synthesized products (3–6) were more reactive than individual reactives (1-2). It was observed the most reactive compound is 4 among these compounds. Previously synthesized compounds (8-12) were tested and compared with the individual reactive (1, 2 and 7). It was used 0, 50, 100 and, 200 µM concentration range. When all the synthesized compounds were compared (7–12), it was found that both of compounds 10 and 12 resulted in the 77% further activity reduction (Fig. 6). In this concentration range, the hybridization activity of the reactive compounds 1 and 2 resulted in 55% and 45% activity reduction. In the 100 µM concentration, 11

compound 10 was considerably more reactive than the other compounds, resulted in 70% activity reduction. The reason for the high concentration study with these compounds was that they did not show activity at low concentrations (0-50 µM) as much as new synthesized compounds (3–6), especially compound 4 showed good activity (87%) reduction.

Figure 5. The amperometric responses were shown as percent relative hybridization responses in the figure. Compounds (0, 12.5, 25, 50 µM) were incubated with DNA probes prior to the injection on to the biochip.

12

80 60 40 20 0

50 1

2

7

8

9 Compounds

200 10

11

μM

Relative Response %

100

12

Figure 6. The amperometric responses were shown as percent relative hybridization responses in the figure. Compounds (0, 50, 100, 200 µM) were incubated with DNA probes prior to the injection on to the biochip. The Cytotoxic Effects of Compounds

We used the MTT assay to determine whether the compound is cytotoxic in the following cell lines. Cell viabilities were measured using the MTT assay as noted, and the IC50 (the dose of compound that inhibits cell proliferation by 50%) value for cells. The 10 compounds were evaluated against two tumor cell lines derived from different tissues. Compounds were tested for their cytotoxic effects in vitro against MCF-7 and DLD-1 cancer cell lines. For this purpose, the cells were incubated with compounds (1-9 and 11) at different concentrations (12, 25, 50, 100, 200 µM) for 24 h at 37oC. The optical density (OD) values of the purple solution that represented cell viability were measured at 570 nm. Obtaining results were evaluated by ANOVA analysis (Table 2). After three independent experiments, cell survival was calculated using the following formula: survival (%) =(mean experimental OD value/mean control OD value) x100%. The values were expressed as the 50% inhibitory concentration which was calculated by the regression method in the linear range. Some of the compounds synthesized in the study attract particular attention as anti-cancer agents. We obtained varying results in terms of different concentrations of other compounds in MCF-7 and DLD-1 cells (Table 2 and Fig.7). Remarkable differences were observed that exceeded our expectations, with the

13

compounds 3, 4 and 5 for MCF-7 cells and 3 and 4 for DLD-1 cells exhibiting much higher cytotoxicity than the other compounds which were almost non-toxic. Based on the IC50 values, the in vitro antiproliferative efficacies of the compounds are in the following order. 3> 4 >5 In addition, compounds 3, 4 and 5 exhibited a moderate cytotoxicity against the cancer cell lines. Compound 4 displayed an IC50 value that was lower than compound 3 against the cancer cell line. Compounds exhibited IC50 values approximately lower than the ones of control under the same conditions, indicating improved selectivity for cancer cells.

MCF-7 Cell Absorbance 570 nm

2.5 2 1.5 1 0.5 0 Cell Cont.

200

100

50

25

12.5

DMSO

Pos. Cont

12.5

DMSO

Pos. Cont

Concentration (µM) 3

4

5

DLD-1 Cell Absorbance 570 nm

2.5 2 1.5 1 0.5 0 Cell Cont.

200

100

50

25

Concentration (µM) 3

4

Figure 7. MTT Assay of Compounds 3-5.

14

Table 2. In vitro cytotoxic activity of the cell lines were examined by MTT assay after treating MCF-7 and DLD-1 cell lines with varying concentrations of compounds for 24 h. The obtained data were evaluated using SPSS 20.0 analysis and described as IC50 values. 1 indicates 200 mM; 2 indicates 100 mM; 3 indicates 50 mM; 4 indicates 25 mM; 5 indicates 12.5 mM Compounds

1

2

3

4

5

6

Concentration

Cell line IC50 [µm] ± SE MCF-7

DLD-1

1

1.51 ± 0.17

1.18 ± 0.39

2

1.42 ± 0.27

1.27 ± 0.33

3

1.27 ± 0.12

1.16 ± 0.27

4

1.21 ± 0.21

1.11 ± 0.27

5

1.21 ± 0.22

1.52 ± 0.56

1

1.42 ± 0.34

1.46 ± 0.57

2

1.28 ± 0.22

1.46 ± 0.54

3

1.25 ± 0.15

1.35 ± 0.39

4

1.16 ± 0.24

1.24 ± 0.46

5

1.04 ± 0.15

1.45 ± 0.68

1

0.70 ± 0.15

0.46 ± 0.08

2

0.79 ± 0.21

0.44 ± 0.06

3

0.86 ± 0.22

0.46 ± 0.08

4

0.92 ± 0.29

0.44 ± 0.04

5

0.88 ± 0.22

0.43 ± 0.04

1

1.05 ± 0.13

0.56 ± 0.09

2

1.08 ± 0.08

0.54 ± 0.02

3

1.19 ± 0.22

0.78 ± 0.11

4

1.06 ± 0.25

0.93 ± 0.25

5

0.95 ± 0.29

0.76 ± 0.11

1

1.22 ± 0.33

1.46 ± 0.63

2

1.23 ± 0.21

1.35 ± 0.56

3

1.14 ± 0.11

1.39 ± 0.48

4

1.05 ± 0.17

1.40 ± 0.53

5

1.29 ± 0.40

1.41 ± 0.47

1

1.56 ± 0.20

1.28 ± 0.28

2

1.52 ± 0.32

1.48 ± 0.51

3

1.51 ± 0.34

1.51 ± 0.46

4

1.44 ± 0.44

1.36 ± 0.29

15

7

8

9

11

5

1.53 ± 0.41

1.27 ± 0.23

1

1.42 ± 0.35

1.31 ± 0.56

2

1.39 ± 0.28

1.33 ± 0.53

3

1.43 ± 0.25

1.37 ± 0.65

4

1.61 ± 0.31

1.0 ± 0.42

5

1.40 ± 0.31

1.37 ± 0.69

1

1.67 ± 0.52

1.51 ± 0.64

2

1.42 ± 0.24

1.50 ± 0.52

3

1.45 ± 0.27

1.52 ± 0.74

4

1.56 ± 0.42

1.59 ± 0.80

5

1.60 ± 0.26

1.32 ± 0.64

1

1.44 ± 0.29

1.36 ± 0.22

2

1.56 ± 0.25

1.43 ± 0.28

3

1.66 ± 0.46

1.33 ± 0.23

4

1.62 ± 0.35

1.37 ± 0.36

5

1.64 ± 0.25

1.48 ± 0.32

1

1.38 ± 0.28

1.65 ± 0.49

2

1.29 ± 0.22

1.36 ± 0.31

3

1.41 ± 0.21

1.19 ± 0.19

4

1.52 ± 0.35

1.38 ± 0.26

5

1.24 ± 0.09

1.13 ± 0.16

Experimental Materials and Methods Hexachlorocyclotriphosphazene (Otsuka Chemical Co., Ltd) was purified by fractional crystallization from n-Hexane. 4-Hydroxy 6-methylcoumarin (98%), 4-Hydroxy 6,7dimethylcoumarin

(98%)

and

Cs2CO3 were

obtained

from

Sigma

Aldrich.

Tetrahydrofuran (≥99.0%), n-Hexane (≥95.0%) were obtained from Merck. THF was distilled over sodium-potassium alloy under an atmosphere of dry argon. Silica gel 60 (230–400 mesh) for column chromatography was obtained from Merck. CDCl3 for NMR spectroscopy was obtained from Goss Scientific. Phosphate buffered saline tablets, mercaptoundecanoic acid, ethanolamine, horseradish peroxidase (HRP), 3,3′,5,5′Tetramethylbenzidine (TMB) ready to use reagent, hydrochloric acid (HCI), Tris–HCI, N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. 1-ethyl-3-(316

dimethylaminopropyl)-carbodiimide (EDC) and NeutrAvidin (NA) was purchased from Thermo Scientific. Au nanoparticles (15nm) were obtained from BBI International. The oligonucleotide sequences were obtained from TIB Molbiol.

Chemical analysis methods Elemental analytical data was obtained using a Thermo Finnigan Flash 1112 Instrument. Positive ion and linear mode MALDI-MS of complexes were obtained in 2,5dihydroxybenzoic acid as MALDI matrix using nitrogen laser accumulating 50 laser shots using Bruker Microflex LT MALDI-TOF mass spectrometer. All reactions were monitored using thin-layer chromatography (TLC) on Merck silica gel plates (Merck, Kieselgel 60, 0.25 mm thickness) with F254 indicator. Column chromatography was performed on silica gel (Merck, Kieselgel 60, 230-400 mesh; for 3g. crude mixture, 100g. Silica gel was filled in a column of 3 cm in diameter and 60 cm in length). 1H and

31

P

NMR spectra were recorded in CDCl3 solutions on a Varian INOVA 500 MHz spectrometer using TMS as an internal reference for 1H NMR and 85 % H3PO4 as an external reference for

31

P NMR. FT-IR spectra were recorded on a Perkin Elmer

Spectrum 100 FT-IR spectrometer. DNA interaction analysis tests were performed using an automated MiSens biosensor device and its sensor chips (BILGEM-TUBITAK, Kocaeli, Turkey).

Synthesis Synthesis of compounds 1 and 2. Compounds 1 and 2 were synthesized according to literature26.

4.3.2. Synthesis of compounds 3. Compound 1 (150 mg, 0.269 mmol) and Cs2CO3 (357.78 mg, 1.10 mmol) were dissolved in 20 mL of dry THF in a 100 mL three-necked round-bottomed flask. 4-Hydroxy 6,7dimethylcoumarin (229.63 mg, 1.10 mmol) in THF (20 ml) was added to the stirred solution. The reaction mixture was refluxed in an oil bath with stirring for 5 days and followed TLC on silica gel plates using THF/n-hexane (1:1) as mobile phase. Cesium chloride and other insoluble material such as oligomers or polymers were filtered off and the THF was removed under reduced pressure. The resulting colorless solid was washed with THF, DCM and then precipitated in n-hexane. Compound 3 (104 mg, 0.087 mmol, 34%). Elemental analyses: Calc. (%) for C65H50N3O14P3: C, 65.60; H, 4.23; N, 3.53; 17

found C, 65.40; H, 4.33; N, 3.60. MS (MALDI-TOF) m/z Calc. 1190; found 1191 [M+H]+ (Fig. S9).

31

P NMR (proton decoupled) (202 MHz, CDCl3) P(O-spiro) δ =8.18

ppm (1P, 2JP-P = 97.95 Hz,); P(OCoum)2 δ =6.97 ppm (2P, 2JP-P = 97.95 Hz) (Fig. 1a and S1). 1H NMR, CDCl3, 298 K: δ ppm, 8.25 d, 2H, Hb (3JHb-Ha = 8.35 Hz); 7.83 d, 2H, Hc (3JHc-Hd = 7.78 Hz); 7.68 d, 2H, Hf (3JHf-He = 8.70 Hz); 7.58 t, 2H, He (3JHe-Hf = 8.70 Hz, 3

JHe-Hd = 7.40 Hz); 7.50 t, 2H, Hd (3JHd-He = 7.40 Hz, 3JHd-Hc = 7.78 Hz); 7.36 s, 4H, H2;

7.22 d, 2H, Ha (3JHa-Hb = 8.35 Hz); 7.05 s, 4H, H3; 6.32 s, 4H, H1; 4.98 s, 2H, CH2; 2.36 s, 12H, CH3; 2.28 s, 12H, CH3 (Fig. 1b and S5). FT-IR: ʋ(CH2)=2920 cm-1, ʋ(C=O)= 1718 cm1

, ʋ(C-C)aro= 1627 cm-1, ʋ(P-N)= 1210, 1183 cm-1, ʋ(P-O)=1058 cm-1, 1013 cm-1.

Synthesis of compounds 4 Compound 1 (143 mg, 0.249 mmol) and Cs2CO3 (331.18 mg, 1.02 mmol) were dissolved in 20 mL of dry THF in a 100 mL three-necked round-bottomed flask. 4-Hydroxy 6methylcoumarin (179.60 mg, 1.02 mmol) in THF (20 ml) was added to the stirred solution. The reaction mixture was refluxed in an oil bath with stirring for 7 days and followed TLC on silica gel plates using THF/n-hexane (1:1) as mobile phase. Cesium chloride and other insoluble material such as oligomers or polymers were filtered off and the THF was removed under reduced pressure. The resulting colorless oil was subjected to column chromatography using THF/n-hexane (1:1) as mobile phase. Compound 4 (90 mg, 0.079 mmol, 32%), Elemental analyses: Calc. (%) for C62H44N3O14P3: C,64.87; H,3.86; N,3.66; found C,64.83; H,3.81; N,3.68. MS (MALDI-TOF) m/z Calc. 1135; found 1135 [M]+ (Fig. S10). 31P NMR (proton decoupled) (202 MHz, CDCl3) P(O-spiro) δ =8.03 ppm (1P, 2JP-P = 97.62 Hz,); P(OCoum)2 δ =6.93 ppm (2P, 2JP-P = 97.62 Hz) (Fig. S2). 1H NMR, CDCl3, 298 K: δ ppm, 8.25 d, 2H, Hb (3JHb-Ha = 8.54 Hz); 7.83 d, 2H, Hc (3JHc-Hd = 7.63 Hz); 7.66 d, 2H, Hf (3JHf-He = 8.21 Hz); 7.60 t, 2H, He (3JHe-Hf = 8.21 Hz, 3JHe-Hd = 7.05 Hz); 7.51 t, 2H, Hd (3JHd-He = 7.05 Hz, 3JHd-Hc = 7.63 Hz); 7.44 s, 4H, H4; 7.39 d, 4H, H2 (3JH2-H3 = 8.38 Hz); 7.20 d, 2H, Ha (3JHa-Hb = 8.54 Hz); 7.16 d, 4H, H3 (3JH3-H2 = 8.38 Hz); 6.41 s, 4H, H1; 4.98 s, 2H, CH2; 2.40 s, 12H, CH3 (Fig. S6). FT-IR: ʋ(CH2)=2925 cm-1, ʋ(C=O)= 1722 cm-1, ʋ(C-C)aro= 1631 cm-1, ʋ(P-N)= 1276, 1262 cm-1, ʋ(P-O)= 1057 cm-1.

Synthesis of compounds 5 Compound 2 (100 mg, 0.125 mmol) and Cs2CO3 (85.31 mg, 0.263 mmol) were dissolved in 20 mL of dry THF in a 100 mL three-necked round-bottomed flask. 4-Hydroxy 6,718

dimethylcoumarin (49.83 mg, 0.262 mmol) in THF (20 ml) was added to the stirred solution. The reaction mixture was refluxed in an oil bath with stirring for 4 days and followed TLC on silica gel plates using THF/n-hexane (1:1) as mobile phase. Cesium chloride and other insoluble material such as oligomers or polymers were filtered off and the THF was removed under reduced pressure. The resulting colourless oil was subjected to column chromatography using THF/n-hexane (1:1) as mobile phase. Compound 5 (45 mg, 0.040 mmol, 46%), Elemental analyses: Calc. (%) for C64H46N3O10P3: C,69.25; H,4.18; N,3.79; found C,69.29; H,4.11; N,3.66. MS (MALDI-TOF) m/z Calc. 1109; found 1110 [M+H]+ (Fig. S11).

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P NMR (proton decoupled) (202 MHz, CDCl3) P(O-

2

spiro) δ =8.93 ppm (2P, JP-P = 74.03 Hz,); P(OCoum)2 δ =7.81 ppm (1P, 2JP-P = 74.03 Hz) (Fig. S3). 1H NMR, CDCl3, 298 K: δ ppm, 8.22 d, 4H, Hb (3JHb-Ha = 8.47 Hz); 7.81 d, 4H, Hc (3JHc-Hd = 8.05 Hz); 7.63 d, 4H, Hf (3JHf-He = 8.79 Hz); 7.54 t, 4H, He (3JHe-Hf = 8.79 Hz, 3JHe-Hd = 7.56 Hz); 7.46 t, 4H, Hd (3JHd-He = 7.56 Hz, 3JHd-Hc = 8.05 Hz); 7.42 s, 2H, H2, 7.28 s, 2H, H3, 7.13 d, 4H, Ha (3JHa-Hb = 8.47 Hz); 6.55 s, 2H, H1; 3.77 s, 4H, CH2; 2.34 s, 6H, CH3; 2.20 s, 6H, CH3 (Fig. S7). FT-IR: ʋ(CH2)=2920, 2851 cm-1, ʋ(C=O)= 1713 cm-1, ʋ(C-C)aro= 1627, 1596 cm-1, ʋ(P-N)= 1184, 1138 cm-1, ʋ(P-O)=1052.

Synthesis of compounds 6 Compound 2 (100 mg, 0.125 mmol) and Cs2CO3 (85.31 mg, 0.263 mmol) were dissolved in 20 mL of dry THF in a 100 mL three-necked round-bottomed flask. 4-Hydroxy 6methylcoumarin (46 mg, 0.262 mmol) in THF (20 ml) was added to the stirred solution. The reaction mixture was refluxed in an oil bath with stirring for 4 days and followed TLC on silica gel plates using THF/n-hexane (4:5) as mobile phase. Cesium chloride and other insoluble material such as oligomers or polymers were filtered off and the THF was removed under reduced pressure. The resulting colourless oil was subjected to column chromatography using THF/n-hexane (4:5) as mobile phase. Compound 6 (58 mg, 0.054 mmol, 43%), Elemental analyses: Calc. (%) for C62H42N3O10P3: C,68.83; H,3.91; N,3.88; found C,68.80; H,3.86; N,3.87. MS (MALDI-TOF) m/z Calc. 1081; found 1081 [M+] (Fig. S12). 31P NMR (proton decoupled) (202 MHz, CDCl3) P(O-spiro) δ =8.91 ppm (2P, 2

JP-P = 74.71 Hz,); P(OCoum)2 δ =7.89 ppm (1P, 2JP-P = 74.71 Hz) (Fig. S4). 1H NMR,

CDCl3, 298 K: δ ppm, 8.35 m, 4H, Hb; 7.93 m, 4H, Hc; 7.63 m, 13H, Hd /He/Hf/H4; 7.40 m, 4H, H2/H3; 7.22 m, 4H, Ha; 7.14 s, 2H, H1; 5.04 s, 4H, CH2; 2.41 s, 6H, CH3 (Fig. S8). FT-IR: ʋ(CH2)=2917 cm-1, ʋ(C=O)= 1724 cm-1, ʋ(C-C)aro= 1675, 1629 cm-1, ʋ(P-N)= 1274, 1253 cm-1, ʋ(P-O)= 1052 cm-1. 19

Biological assay Biosensor device and biochip surface modification For the amperometric measurements, MiSens biosensor device and its biochips have been used and DNA interaction analyses performed. The biosensor device include biochip docking station, a pump, microfluidic tubing connected sample pick up needle and sample/reagent carousel MiCont™ software developed by the Bioelectronic Devices and System Development Group of BILGEM Research Center (BILGEM-TUBITAK, Kocaeli, Turkey). A biochip that has Au electrode arrays, consist of shared reference/counter electrodes and 8 working electrodes has been fabricated and used for the assays28. The biochip surface has been coated with a self-assembled monolayer (SAM) prior to the immobilization of neutravidin (NA) that has been used to capture the biotinylated surface probe. For this, plasma cleaned biochip was activated with 2 mM mercaptoundecanoic acid in ethanolic solution for overnight. Later it was rinsed with ethanol and water, dryed with nitrogen stream and was vacuum packed. The functionalised biochip is stored at +4°C until used.

DNA interaction tests using MiSens biosensor

The biochip was integrated to a microfluidics system and all the steps of the assay were been performed during fluid flow. The biochip surface activated with EDC/NHS and then neutravidin (NA) was immobilized to capture biotinylated surface probe. Newly synthesised chemicals with varying concentrations (0-200 µM) were diluted in dimethyl sulfoxide and this mixture was added to the of hybridised target (1 x 10_9 M) and detection probe (2 x 10_9 M) solution for 15 min incubation. Later, for the hybridization process the mixture was injected to biochip surface that the capture probe immobilized on. The subsequent injection of gold nanoparticles on which NA and enzyme (HRP) modified. This was followed with the injection of the enzyme substrate (3,3’,5,5’Tetramethylbenzidine) in the presence of H2O2 and against -0.1 V potential amperometric measurements, resulted in amperometric reading during the flow.

Cytotoxicity of the compounds Cell Lines: The following in vitro human cancer cell lines were used: MCF-7 (ATCC® HTB22™) (human breast adenocarcinoma) and DLD-1 (ATCC® CCL-221™) (Dukes'

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Type C, Colorectal Adenocarcinoma). The cell lines were purchased from the Anadolu University, Cell Culture Laboratory, Eskişehir, Turkey. Cell Culture: MCF-7 and DLD-1 cells were cultured in RPMI 1640 (Gibco) containing 2 mM Na2CO3 supplemented with 10% (v/v) FBS (foetal bovine serum) (Sigma, Steinheim, Germany). The cell culture media was supplemented with penicillin/streptomycin at 100 Units/mL (Sigma, Steinheim, Germany) as adherent monolayers. Cell lines were incubated at 37oC under 5% CO2 and 95% air in a humidified atmosphere. Stock solutions were prepared in dimethyl sulfoxide (DMSO) (Sigma, Steinheim, Germany) and dilutions were made with fresh culture medium. The concentration of DMSO in the final culture medium was 1%. After reaching 90% confluence, cells were treated with various concentrations of compounds, where the final concentration of DMSO was ≤0.1%. Controls were treated with RPMI 1640 medium with a final concentration of 0.1% of DMSO but without compounds. MTT assay: The cytotoxic response to the cell lines and the growth-inhibitory effects of compounds were determined by MTT (1 mg/mL in serum free medium) assay using a colorimetric substrate tetrazolium salt and 3-[4,5- dimethyl-thiazollyl-2-yl]-2,5diphenyltetrazolium bromide (Sigma, Steinheim, Germany). MCF-7 (5x103 cells/mL) and DLD-1 (1x104 cells/mL) cells were incubated with the compounds (12-200 µM) in 96-well plates (Costar, USA) for 24 h. After incubation, culture medium was removed and 100 µL of MTT reagent was added to each well. The plates were incubated for 2 h at 37oC. MTT containing medium was removed and each well was washed gently with 100 µL of PBS (Phosphate Buffered Saline). The blue formazan product was dissolved by addition of 100 µL of 100% DMSO per well. The plates were swirled gently for 10 min to dissolve the precipitate, and quantified by measuring the OD (optical density) of the plates at 570 nm on a microplate reader (Thermo Scientific, USA). The anti-cancer drug curcumin (200 µM) and mytomicine (200 µM) was used as a positive control. Each concentration was repeated in three wells and control cell viability was considered as 100%. The same experimental conditions were provided for all compounds and MTT analysis was also repeated three times for each cell line.

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Statistics The significance of differences between data sets was analysed statistically by ANOVA with SPSS 20.0 program for DLD-1 and MCF-7. The results were indicated as ID50 ± SE (standard error of mean) for cell lines.

Conclusion In this study, four new coumarin substituted (3-6) cyclotriphosphazene and six compounds which were obtained in our laboratory synthesized and characterized by elemental analysis, mass spectrometry, FT-IR, 1H,

31

P NMR spectroscopies.

All

synthesized compounds (3-12) were screened to effect on DNA using an automated biosensor device. Compound 10 was considerably more reactive than the other compounds, resulted in 70% activity reduction (100 µM). Compound 4 showed the most activity (87%) reduction than all compounds at low concentrations (0-50 µM). Cytotoxic drugs have been used for cancer treatment in susceptible cells. Although, there are many studies about this area, only few drugs may be used for cancer treatment. Obtaining findings are expected to increase knowledge their potentials in this area. Further studies are required to provide detail information proliferation, cell cycle and apoptosis in cancer in terms of cell signalling pathways. Therefore, studies in this area are very important. This is the first study to analyse newly synthesised these chemical compounds as an anticancer drug (especially 3, 4 and 5 for MCF-7 cells and 3 and 4 for DLD-1 cells). In this presented study, cytotoxicity effects of compounds were investigated in human carcinoma cell lines. In particular, compound 4 is highly effective in both DNA binding activity and cytotoxic activity. This compound may be a potential candidate as anticancer agents. The data obtained from the biosensor assays and cytotoxicity studies can help researchers to select the most probable drug candidates for further investigations

Conflicts of interest There are no conflicts to declare Acknowledgements The authors would like to thanks the Gebze Technical University (GTU) for the provided financial support Grant no: BAP 2017-A102-16. We gratefully acknowledge to BILGEM-TUBITAK for their contribution to the fabrication of the biochip and the sensing platform 22

AUTHOR INFORMATION

Corresponding authors: * G.Yenilmez Çiftçi. Tel: +90 262 6053011. E-mail: [email protected]

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Highlights ► Coumarin substituted cyclotriphosphazene derivatives were synthesized. ► All compounds were assessed for cytotoxicity by MTT assay against two different cancer cell lines, (MCF-7 and DLD-1). ► All synthesized compounds (3-12) were screened to effect on DNA using an automated biosensor device.

Declaration of Interest Statement: There is no conflict of internet between authors. 1. Conflict of Interest Potential conflict of interest exists: We wish to draw the attention of the Editor to the following facts, which may be considered as potential conflicts of interest, and to significant financial contributions to this work: The nature of potential conflict of interest is described below: No conflict of interest exists. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

2. Funding Funding was received for this work. All of the sources of funding for the work described in this publication are acknowledged below: [List funding sources and their role in study design, data analysis, and result interpretation] No funding was received for this work. 3. Intellectual Property We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we 1

confirm that we have followed the regulations of our institutions concerning intellectual property.

Autors CV Gönül Yenilmez Çiftçi is a professor of chemistry at Gebze Technical University, Gebze, Turkey. She obtained Ph.D. degree in inorganic chemistry at Gebze Institute of Technology in 2002. Her research work has been mainly focused on chemical and biological active compounds synthesis, characterization and their properties of fluorescence and biological activities, development of electrochemical biosensors.

Elif Şenkuytu received her M.Sc. (2010) and Ph.D. (2014) degree in inorganic chemistry at Gebze Technical University, Gebze, Turkey. She is a specialist of chemistry at the same university. Her research interests are synthesis, characterization and structural investigation of advanced materials such as phosphazenes, spermine, coumarins, parabens and for different applications including biological activities, DNA interaction analysis, biosensors, chemosensors etc.

Tuba Yıldırım received her M.Sc 1996 and Ph.D. 2004 in biochemistry at Gebze Technical University, Gebze, Turkey. Her research interests are cell culture, monoclonal antibody (hybridoma technology), immunodiagnostic techniques (ELISA, SDS-PAGE, Western Blot), protein purification (gel filtration, ion exchange, affinity chromatography), recombinant DNA technology (PCR, RT-PCR) molecular typing methods (RAPD, RFLP, PFGE), molecular microbiology. She is working on Amasya University, Faculty of Arts and Sciences,, Department of Biology as a Associate Professor.

Zehra Ölçer received her M.Sc. (2004) and Ph.D. (2011) degree in biochemistry at Gebze Technical University, Gebze, Turkey. She worked for postdoctoral studies with Bioelectronic Devices and System Group at BILGEM-TUBITAK. Currently she is research assistant at the same university. Her research interests include immobilisation of enzymes, design of surface modification and development of electrochemical biosensors.

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Yakup Eker received his Ph.D. (2018) degree in inorganic chemistry at Gebze Technical University, Gebze, Turkey. He worked for doctoral studies with Bioelectronic Devices in GTU. Currently he is chemistry teacher at the college. His research interests include biological activities, DNA interaction analysis, biosensors, chemosensors etc..

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