Meso-piperidine linked bodipys: Synthesis, fluorescent properties and biological evaluation

Author’s Accepted Manuscript Meso-Piperidine Linked Bodipys: Synthesis, Fluorescent Properties and Biological Evaluation Esra Tanrıverdi Eçik, Hasan Huseyin Kazan, Ibrahim F. Sengul, Hakan Kandemir, Bünyemin Çoşut www.elsevier.com/locate/jlumin

PII: DOI: Reference:

S0022-2313(18)31097-4 https://doi.org/10.1016/j.jlumin.2018.09.024 LUMIN15907

To appear in: Journal of Luminescence Received date: 20 June 2018 Revised date: 25 July 2018 Accepted date: 9 September 2018 Cite this article as: Esra Tanrıverdi Eçik, Hasan Huseyin Kazan, Ibrahim F. Sengul, Hakan Kandemir and Bünyemin Çoşut, Meso-Piperidine Linked Bodipys: Synthesis, Fluorescent Properties and Biological Evaluation, Journal of Luminescence, https://doi.org/10.1016/j.jlumin.2018.09.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. 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.

Meso-Piperidine Linked Bodipys: Synthesis, Fluorescent Properties and Biological Evaluation

Esra Tanrıverdi Eçik*,a, Hasan Huseyin Kazanb, Ibrahim F. Sengula, Hakan Kandemirc, Bünyemin Çoşuta

a

Department of Chemistry, Faculty of Science, Gebze Technical University, Gebze, Kocaeli,

Turkey b

c

Department of Biological Sciences, Middle East Technical University, Ankara, Turkey

Department of Chemistry, Faculty of Art and Science, Namık Kemal University, Tekirdag,

Turkey

*

Author for correspondence: Dr. Esra Tanrıverdi Eçik

Department of Chemistry, Gebze

Technical University, P.O.Box: 141, Gebze 41400, Kocaeli, Turkey Tel: 6053083 Fax:

00 90 262 6053105 e-mail:

00

90

262

[email protected]

Abstract The synthesis and characterization of meso-piperidine linked Bodipy starting from 4-(2(piperidine-1-yl)ethoxy)benzaldehyde was described. Meso-piperidine linked Bodipy was subsequently used for the construction of distyryl-piperidine-Bodipy and distyryl-morpholineBodipy via Knoevenagel type reaction. The photophysical properties including molar extinction

coefficient, fluorescence lifetime and fluorescence quantum yield of Bodipys were investigated in ethanol solution. The targeted compounds were also assessed in live cell imaging and cytotoxicity studies by using breast cancer cell line, MCF-7 in vitro. Graphical abstract:

Keywords: Bodipy, Piperidine, Fluorescence, Live cell imaging

1.Introduction Over the past two decades, the design and development of new Bodipy moieties have been continuously growing research interest of chemists due to their unique photophysical properties, good thermal and photochemical stability, intense absorption, high fluorescent quantum yield and good solubility [1-3]. Bodipy-based molecules have been shown to possess a range of applications including organic solar cells (OSCs), dye-sensitized solar cells (DSSCs), organic thin film transistors (OTFT), light harvesting materials, near infrared absorption materials and chemosensors [4-9]. In addition to advantages given above, Bodipy family of compounds have a multitude of interesting biological activity, ranging from biochemical labelling to photosensitizers for photo dynamic therapy (PDT) due to their singlet oxygen generation potential [3, 10-12]. It is well established that Bodipy dyes often display absorption and fluorescence emission in the region of 480 and 540 nm which are not useful bands for biological properties [2, 13]. Significant effort has been performed to extend their characteristic bands to 600-700 nm or near infrared (NIR) region [1, 2, 10, 13-15]. Since methyl protons at C3 and C5 positions of the Bodipy core is relatively acidic, the presence of the acidic protons located at C3 and C5 positions of Bodipy dyes creates an opportunity for the extending of the conjugated skeleton to red-shifted excitation and emission wavelengths [13-16]. For this purpose, the most commonly used synthetic approach typically entails the condensation of Bodipy with an aryl aldehyde under Knoevenagel type reaction to form double bound [1, 2]. In addition to the extension of conjugation of the Bodipy

core at C3 and C5 positions, derivatization of the Bodipy backbone at meso position can also lead to extend the conjugation. Considering this, a few synthetic approaches have been recently reported for the construction of meso-substituted Bodipy dyes with outstanding photophysical properties [17-19]. Piperidine and its derivatives are an important class of nitrogen containing heterocyclic compounds, which show potent biological activity in many cases [20]. These include anti-HIV, histamine H3R ligands, antimalarial, antimicrobial and antifungal activities [21-24]. Piperidinecontaining Bodipy compound such as Bodilisant was reported as the novel fluorescent human histamine H3 receptor ligand [25]. As a bioactive substructure, the incorporation of piperidine groups into Bodipy platform produces new type of Bodipy dyes which potentially illustrate important phtophysical and biological activities. The central aim of the current study was to develop novel Bodipy units bearing piperidine and morpholine scaffolds, which can serve as useful platforms for photophysical and biological applications. As an integral part of achieving this aim, the piperidine based Bodipy compounds (3-5) were successfully designed and synthesized starting from 4-(2-(piperidine-1-yl)ethoxy)benzaldehyde under standard reaction conditions. The second part of the project was to investigate the photophysical activities of the newly synthesized Bodipy molecules. In addition, we were also interested in biological evaluation of the piperidine linked Bodipys. The targeted piperidine and morpholine linked Bodipy derivatives were tested for the live cell imaging abilities in breast cancer cell line, MCF7. Moreover, the cytotoxicity of the compounds against this cell line was also reported.

Fig. 1. Meso-piperidine linked Bodipy (3), distyryl-piperidine-Bodipy (4) and distyryl-morpholine-Bodipy (5) 2.Experimental section 2.1. General methods All reagents were purchased from Aldrich and used without further purification and all solvents were obtained from Merck. All reactions were monitored by thin layer chromatography using Merck TLC Silica gel 60 F254. Silica gel 60 (particle size: 0.040-0.063 mm, 230-400 mesh ASTM) for column chromatography was obtained from Merck. All reactions were carried out under an argon atmosphere. 1H and 13C NMR spectra were recorded for all compounds in CDCl3 solutions on a Varian INOVA 500 MHz spectrometer. Electronic absorption spectra in the UV– Vis. region were recorded with a Shimadzu 2101 UV-Vis spectrophotometer. Fluorescence excitation and emission spectra were recorded on a Varian Eclipse spectrofluorometer using 1 cm path length cuvettes at room temperature. Fluorescence lifetimes were measured using a time correlated single photon counting setup (TCSPC) (Horiba Fluorolog 3 equipment) HORIBA

Model: FLUOROLOG 3. Signal acquisition was performed using a TCSPC module (NanoLED 390 emitting 390 nm). 2.2. The parameters for fluorescence quantum yields The fluorescence quantum yields value of the compounds 3-5 were determined in ethanol by comparing with the fluorescence of Rhodamine 6G for 3; ZnPc for 4 and 5 as a standard. Fluorescence quantum yields (ΦF) were calculated by the comparative method (Eq. 1) [26].

ΦF  ΦF(Std)

F . AStd . n 2 2 FStd . A . n Std

(1)

Where ΦF(Std) is the fluorescence quantum yield of standard. Rhodamine 6G (ΦF = 0.95 in water) and ZnPc (ΦF = 0.18 in DMSO) were employed as the standard [27, 28]. F and FStd are the areas under the fluorescence emission curve of compounds (3-5) and the standard, respectively. A and AStd are the respective absorbance of the compounds and standard at the excitation 2 wavelengths. η 2 and ηStd are the refractive indices of solvents used for the sample and

compounds. The concentration of the solutions at the excitation wavelength fixed at 2x10-6 mol.dm-3. 2.3. Synthesis Compound 1 and 2 were synthesized according to literature [29]. 2.3.1. Synthesis of compound 3 CH2Cl2 (300 mL) was purged with Ar for 30 min. 2,4-dimethyl pyrrole (0.45 g, 4.7 mmol) and compound 1 (0.50 g, 2.2 mmol) were added. The color of the solution turned into red after the

addition of two drops of trifluoroacetic acid. The reaction mixture was stirred at room temperature for 14h. Then, p-chloranil (0.53 g, 2.2 mmol) was added and the reaction mixture was stirred at room temperature for 1h. Then triethyl amine (5.0 mL) and boron trifluoride diethyl etherate (5.0 mL) were added sequentially. After stirring at room temperature for 3h, it was extracted with water. Organic layer was dried with Na2SO4 and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography using CH2Cl2CH3OH (10:1) as mobile phase. Fraction containing compound 3 was collected then the solvent was removed under reduced pressure (0.35 mmol, 160.0 mg, 16 %). MALDI TOF (m/z) Calc. 451.29, Found: 451.03 [M+]. 1H NMR (500 MHz, CDCl3) δH 7.19 (d, J = 8.4 Hz, 2H), (Ar-CH); 7.03 (d, J = 8.4 Hz, 2H), (Ar-CH); 5.99 (s, 2H), (Ar-CH); 4.28 (broad, 2H), (OCH2); 3.01 (broad, 2H), (NCH2); 2.76, (broad, 4H), (CH2NCH2); 2.57 (s, 6H), (CH3), 1.74 (m, 4H), (CH2); 1.50 (m, 2H), (CH2); 1.44 (s, 6H), (CH3) ppm.13C NMR (126 MHz, CDCl3) δC 159.51, 152.36, 141.59, 138.22, 135.59, 133.43, 129.7, 117.42, 114.91, 65.86, 57.77, 55.01, 30.33, 25.74, 24.05, 15.95 ppm. 2.3.2. Synthesis of Compound 4 Compound 1 (67.1 mg, 0.28 mmol) and compound 3 (65.0 mg, 0.14 mmol) were dissolved in benzene (45 mL). Piperidine (350 µl) and acetic acid (350 µl) were added. The reaction mixture was reflux using Dean-Stark apparatus until all aldehyde was consumed. Then, crude product was extracted with CH2Cl2 and water. Organic layer was dried with Na2SO4 and evaporated in vacuo. The crude product was purified by silica gel column chromatography using CH2Cl2-CH3OH (2:1) as mobile phase. Fraction containing compound 4 was collected then the solvent was removed under reduced pressure (0.02 mmol, 21.0 mg, 6 %). MALDI TOF (m/z) Calc. 881.52, Found: 881.33 [M+]. 1H NMR (500 MHz, CDCl3) δH 7.62 (d, J = 16.3 Hz, 2H), (trans-CH); 7.58 (d, J =

8.4 Hz, 4H), (Ar-CH); 7.24 (d, J = 16.3 Hz, 2H), (trans-CH); 7.20 (d, J = 8.3 Hz, 2H), (Ar-CH); 7.03 (d, J = 8.3 Hz, 2H), (Ar-CH); 6.94 (d, J = 8.4 Hz, 4H), (Ar-CH); 6.62 (s, 2H), (Ar-CH); 4.20 (t, J = 4.6 Hz, 4H+2H), (OCH2); 2.86, (m, 4H+2H), (NCH2); 2.59, (m, 8H+4H), (NCH2); 1.67 (m, 8H+4H), (CH2); 1.50 (m, 6H), (CH2); 1.45 (s, 6H), (CH3) ppm.13C NMR (126 MHz, CDCl3) δC 159.41, 159.30, 152.55, 141.81, 138.12, 135.49, 133.66, 129.67, 129.15, 127.35, 125.51, 117.42, 117.31, 115.11, 114.91, 66.03, 65.89, 57.90, 57.77, 55.17, 55.01, 30.33, 29.60, 25.74, 24.05, 14.85 ppm. 2.3.3. Synthesis of Compound 5 Compound 2 (84.7 mg, 0.36 mmol) and compound 3 (65.0 mg, 0.14 mmol) were dissolved in benzene (45 mL). Piperidine (350 µl) and acetic acid (350 µl) were added. The reaction mixture was reflux using Dean-Stark apparatus until all aldehyde was consumed. Then, crude product was extracted with CH2Cl2 and water. Organic layer was dried with Na2SO4 and evaporated in vacuo. The crude product was purified by silica gel column chromatography using CH2Cl2-CH3OH (8:1) as mobile phase. Fraction containing compound 5 was collected then the solvent was removed under reduced pressure (0.04 mmol, 40.0 mg, 31 %). MALDI TOF (m/z) Calc. 885.48, Found: 885.30 [M+].1H NMR (500 MHz, CDCl3) δH 7.62 (d, J = 16.2 Hz, 2H), (trans-CH); 7.58 (d, J = 8.5 Hz, 4H), (Ar-CH); 7.22 (d, J = 16.2 Hz, 2H), (trans-CH); 7.19 (d, J = 8.3 Hz, 2H), (Ar-CH); 7.02 (d, J = 8.3 Hz, 2H), (Ar-CH); 6.94 (d, J = 8.5 Hz, 4H), (Ar-CH); 6.62 (s, 2H), (Ar-CH); 4.27 (broad, 2H), (OCH2); 4.17 (t, J = 5.1 Hz, 4H), (OCH2); 3.77 (m, 8H), (CH2OCH2); 2.95 (broad, 2H), (NCH2); 2.84, (m, 4H), (NCH2); 2.71, (broad, 4H), (NCH2); 2.61 (m, 8H), (CH2NCH2); 1.74 (m, 4H), (CH2); 1.54 (m, 2H), (CH2); 1.49 (s, 6H), (CH3) ppm.13C NMR (126 MHz, CDCl3) δC 159.46, 159.04, 152.49, 141.84, 138.16, 135.64, 135.68, 133.63, 129.63, 129.00, 127.22, 117.46, 117.30, 115.01, 114.83, 66.93, 65.83, 57.60, 54.98, 54.12, 29.73, 29.70, 25.20, 23.63, 14.87 ppm.

2.4. Biological experiments 2.4.1. Cell lines and cell culture In this study, breast cancer cell line MCF-7 was used. Cells were grown in RPMI 1640 medium (Lonza, Switzerland) containing 10% FBS (Biochrom, Germany) and 0.1% gentamycin at 37°C with 5% CO2. 2.4.2. Live cell imaging 2x105 cells per well were seeded into 6-well plates and incubated for 24 h. Cells were then washed with PBS twice, and medium was renewed. Cells were treated with 25 μg/ml compounds for 2 h and, untreated and ethanol-treated groups were used as control. Then, cells were washed with PBS twice and medium was renewed, and images were taken by FLoid Cell Imaging Station (Thermo Fischer Scientific, USA) under white light, and by FITC filter (Ex: 482/18 nm and Em: 532/59 nm) for compound 3 and Texas Red filter (Ex: 586/15 nm and Em: 646/68 nm) for compound 4 and 5. 2.4.3. Cell viability assay Cell viabilities were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. 1x104 cells were seeded into each wells of 96-well microplates and incubated for 48 h. Next, cells were treated with ethanol alone or increasing concentrations (0.78-25 μg/ml) of compounds for 48 h. Cells were washed with PBS twice and medium was renewed. 10 μl of MTT solution (5 mg/ml) was added into each well and incubated for 4 h. Finally, cells were disrupted by 100 μl SDS-HCl solution (1.0 g SDS in 0.01 M HCI in 10.0 mL final volume) and incubated overnight at 37 C and the microplates were read by microplate spectrophotometer (Multiskan

GO; Thermo Fisher Scientific, USA) at wavelength 570 nm. Optical densities were converted to % viability by using untreated cells as reference (100%). 2.4.5. Statistical analyses All data were representative of three independent experiments with each set containing triplicates of the same sample (n=9). Results were analyzed by GraphPad Prism 7 (GraphPad Software, Inc., USA) with One-Way ANOVA and post-hoc Tukey’s test, and were significant when p<0.05.

3. Results and Discussion 3.1. Synthesis and structural characterization

Scheme 1. Synthetic pathway of compounds 3-5 A variety of synthetic strategies for the preparation and modification of meso-substituted Bodipy backbones have so far been developed and reported (Scheme 1). Although the condensation of an aryl aldehyde and pyrrole is a classic route, it is still a good way for the preparation of Bodipy

derivatives in a facile one pot procedure. Accordingly, the treatment of compound 1 with 2,4dimethylpyrrole in the presence of trifluoroacetic acid in dichloromethane afforded the dipyrromethane which was subsequently oxidized into corresponding dipyrromethene, prior to its complexation in boron trifluoride, producing the targeted meso-piperidine linked Bodipy (3), with 16 % yield. 1H NMR spectrum of compound 3 showed the presence of two singlets at 1.4 and 2.5 ppm corresponding to two sets of methyl protons on the pyrrole rings. The pyrrole ring CH protons appeared as sharp singlet 5.9 ppm and meso-aromatic protons appeared as doublets ~7.0 ppm (Fig. S1). The disappearance of the -CHO protons in the spectrum of the starting material indicated that reaction had taken place. In the

13

C NMR, the aromatic carbons for the

compound 3 was observed between at 159.5-114.9 ppm along with aliphatic carbons between at 65.8-15.9 ppm (Fig. S2). Additionally, the molecular weight of compound 3 was determined by MALDI-TOF MS using 1,8,9-anthracenetriol as the MALDI matrix, which revealed the anticipated molecular ions at 451.03 Da and molecular ion rupture fluor at 432.19 Da (Fig. S3). With meso-substituted Bodipy (3) in hand, preparation of the distyryl bodipy compounds under Knoevenagel reaction conditions was under taken. The acidic nature of methyl protons at C3 and C5 positions on pyrrole rings allows to be condensed with aromatic aldehyde to form double bounds. Treatment of compound 1 with meso-piperidine linked Bodipy (3), at reflux in benzene in the presence of both acetic and piperidine yielded the corresponding distyryl Bodipy as a green solid. The successful preparation of new distyryl-piperidine-Bodipy (4) encouraged us to extend this work to build new morpholine conjugated distyryl Bodipy (5).

According to standard

procedure, the reaction of compound 1 with morpholine based aldehyde (2) gave the corresponding distyryl- morpholine-Bodipy (5) as a green solid in 31% yield.

The structure of compounds 4 and 5 were supported by 1H NMR,

13

C NMR and mass

spectrometry. The aromatic and aliphatic protons for distyryl Bodipy derivatives, 4 and 5 were observed between at 7.6–6.6 and at 4.2–1.4 ppm in the 1H NMR spectra (Fig. S4 and S7). Wellresolved 1H NMR spectra of Bodipy derivatives 4 and 5 exhibited sets of signals for mesoaromatic protons and distyryl benzene protons at around 7.7-7.0 ppm region. The observed 16.3 Hz proton-proton coupling constants at ~7.6 and ~7.2 ppm for distyryl bodipys (4 and 5) proved those trans positions of the double bonds as expected. The pyrrole rings -CH protons and methyl protons appeared as sharp singlets at ~6 ppm and ~1.4 ppm, respectively. Distyryl 4 and 5 aliphatic protons were observed ~4.3-1.5 ppm region. The aromatic carbons for the compounds 4 and 5 were observed between 159.5-114.9 ppm and aliphatic carbons 66.9-14.8 ppm in the

13

C

NMR spectra (Fig. S5 and S8). Further structural verification was obtained via mass spectrometry, with MALDI-TOF spectra revealing [M-F]+ and [M]+ ion peaks at m/z 881.33 and 862.22; 885.30 and 866.34, consistent for compounds 4 and 5, respectively (Fig. S6 and S9). 3.2. Photophysical properties The electronic absorption properties of the newly synthesized Bodipy derivatives (3-5) were characterized at room temperature in ethanol solution. The absorption spectra revealed the major bands centered at 500, 639 and 638 nm which are assigned to the S0-S1 transition for 3-5, respectively [1] (Fig.2a). The characteristic absorption bands of distyryl-piperidine and morpholine groups were observed at 368 nm. The ground state absorption spectra of the compounds (3-5) were recorded at different concentrations for determination of the molar extinction coefficient () (Fig.3). While the  value of meso-piperidine substituted derivative (3) was observed as 5.43x104 M−1 cm−1, the values of distyryl- Bodipys (4 and 5) increased to 9.35x104 and 9.94 x104 M−1 cm−1 (Table 1). This study revealed that the molar extinction

coefficient of targeted compounds (4 and 5) are found to be higher compared with many reported bodipy derivatives [1, 3]. 700

0,2

a)

b) 600 500

3

Intensity (a.u)

Absorbance

0,15

4 0,1

5

3

400

4

300

5 200

0,05

100 0

0 300

400

500

600

480

700

530

580

630

680

730

780

Wavelenght (nm)

Wavelenght (nm)

Fig. 2. a) Electronic absorption spectra of bodipys 3-5 b) Fluorescence emission spectra of compound 3 (λex= 470 nm), 4 and 5 (λex= 620 nm) (2.0 µM) in ethanol solution.

b) 0,6

Absorbance

0,5

0,4

1,2

0,4 1

0,2

y = 0,0543x - 0,0079 R² = 0,9997

0

0 0,3

5

10

15

Concentration (μM)

0,2

y = 0,0935x - 0,0049 R² = 0,9987

1,2 0,8

0,4 0 0

0,8

10μM 8μM 6μM 4μM 2μM

Absorbance

Max. abs

0,6

Max. abs.

a)

2 4 6 8 10 Concentration (µM )

12

12µM 10µM

0,6

8µM 6µM

0,4

4µM 2µM

0,1

0,2

0

0 300

350

400

450

500

550

600

300

350

400

450

500

550

600

650

700

Wavelength (nm)

Wavelenght (nm)

c) 1,6 max. abs.

1,2

Absorbance

1

y = 0,0994x - 0,001 R² = 0,9997

1,2 0,8 0,4

12µM

0

0,8

0

2

4

6

8

10µM

10 12 14

8µM

Concentration (µM) 0,6

6µM

4µM 0,4

2µM

0,2

0 300

350

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500

550

600

650

700

Wavelength (nm)

Fig. 3. Absorbance spectra of bodipys a) 3; b) 4; c) 5 in ethanol at different concentration (inset: plot of absorbance versus concentration).

Table 1 Photophysical properties of Bodipy derivatives 3-5a Compound

λab, nm

λem, nm

єb, 104 M-1 cm-1

∆Stokes, (nm)

3

500

510

5.43

10

4

368; 639

652

9.35

13

5

369; 638

655

9.94

17

a

τF (ns)c 3.38 CHISQ = 1.053881 3.78 CHISQ = 1.022854 3.72 CHISQ =0.99187

Φ Fd 0.58 0.35 0.36

Ethanol. b Molar extinction coefficients. c Lifetime, d Fluorescence quantum yield.

The fluorescence responses of compounds 3, 4 and 5 were examined in ethanol by employing steady state and time resolved fluorescence techniques. Upon excitation at 470, 620 and 620 nm, 3, 4 and 5 showed fluorescent emissions centered at 510, 652 and 655 nm, respectively (Fig.2b). It is worth noting here that compound 3 is more emissive (ΦF = 0.53) than 4 (ΦF = 0.35) and 5 (ΦF = 0.36), which might be attributed to the improved photoinduced electron transfer (PET) effect due to the increase in the piperidine and morpholine group in the 3,5-positions of 3 compared to compounds 4 and 5 [30]. It was observed from this study that the targeted Bodipy structures (3-5) were showed moderate fluorescence quantum yield compared with other bodipy compounds [1,2]. The lifetimes (τF) were also obtained using the time correlated single photon counting (TCSPC) technique in ethanol. Our values of the lifetimes were found to be 3.38, 3.78 and 3.72 ns for compounds 3, 4 and 5, respectively (Fig. 4).

10000

(a)

Prompt Fit 3

6000 4000

(b)

Prompt Fit 4

8000

Counts

Counts

8000

10000

2000

6000 4000 2000

0 80

85

90

95 100 Time (ns) 10000

105

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(c)

95 100 Time (ns)

105

110

Prompt Fit 5

8000

Counts

90

6000 4000 2000 0 80

85

90

95 100 Time (ns)

105

110

Fig. 4. The fluorescence decay profiles of bodipy derivatives a) 3 obtained using a laser excitation source of 390 nm; b) 4 and c) 5 obtained using a laser excitation source of 674 nm.

3.3. Biological evaluation In the biological part of this current study, since piperidine and morpholine-containing Bodipys have frequently been designed for cellular imaging [25, 31-33], live cell imaging ability and cytotoxic potential of the final compounds (3-5) were assessed using MCF-7 breast cancer cell line. For this purpose, 25 μg/mL of compounds 3, 4 and 5 were treated with MCF-7 cells for 2 h. We imaged the cells by FLoid Cell Imaging Station (Thermo Fischer Scientific, USA) using a green filter (Ex: 482/18 nm and Em: 532/59 nm) for compound 3 and a red filter (Ex: 586/15 nm

and Em: 646/68 nm) for compound 4 and 5 in order to evaluate the live cell imaging abilities of the products. The results revealed that the piperidine and morpholine-containing Bodipys successfully stained the cells (Fig. 5). Although the imaging platform was not enough to determine the subcellular localization of the compounds, similar to other Bodipy-based conjugates reported in the literature [34], the compounds could be concluded to stain the all cytoplasmic units and outer membranes of the cells. Moreover, the intensities of compound 3 and 4 was higher than that of compound 5 in cell culture conditions, which may point morpholinebased structural orientation and/or photophysical influence changing the fluorescent properties of the Bodipy molecule, which was previously underlined in the literature [35]. These results showed that the novel piperidine-linked Bodipy molecules would be used in live cell imaging and targeted imaging studies as further perspective.

Fig. 5. a) Live cell imaging by compound 3 and b) 4 and 5 in MCF-7 cell line. Ethanol-treated cells were used as negative control. After treatment with related compounds, cells were excited and the emissions were collected by the wavelengths given on the figure. Scale: 100 μm.

The cytotoxic potentials of the compounds 3-5 were also studied. Cells were treated with increasing concentrations of compounds (0.78-25 μg/mL) in the presence of untreated and ethanol-treated cell controls. It was revealed from the study that although both compounds 3 and 5 were not toxic to cells, compound 4 significantly decreased the viabilities of the cells with an inhibitory concentration 50 (IC50) value of approximately 6 μg/mL (Figure 6). Importantly, distyryl-piperidine-Bodipy (4) displayed significant cytotoxicity while distyryl-morpholineBodipy (5) was not toxic, pointing the toxicity was a result of piperidine platform.

Fig. 6. Cell viability assay. The viabilities of untreated cells were regarded as 100% and those of the treated cells were correlated to untreated group.

4. Conclusion

In summary, the condensation of 4-(2-(piperidine-1-yl)ethoxy)benzaldehyde with 2,4-dimethyl pyrrole led to formation of related meso-piperidine linked Bodipy (3) which was successfully converted into distyryl-piperidine-Bodipy (4) and distyryl-morpholine-Bodipy (5) employing Knoevenagel reaction. The new Bodipys exhibited favorable photophysical properties, including strong absorption bands in the visible spectrum with good molar extinction coefficients and high fluorescence quantum yields. In the biological studies, all compounds were able to stain all cellular compartments, especially cytoplasm and cell membrane. Contrary to compound 4, compounds 3 and 5 were not toxic against this cell line, making them candidates for cell imaging studies. Distyryl-piperidine-Bodipy (4) would be proposed as a well imaging agent for short-term cellular exposure. References [1] G. Ulrich, R. Ziessel, The chemistry of fluorescent bodipy dyes: versatility unsurpassed, Angew. Chem. Int. Ed. 47 (2008) 1184-1201. [2] A. Loudet, K. Burgess, BODIPY dyes and their derivatives: syntheses and spectroscopic properties, Chem. Rev. 107 (2007) 4891-4932. [3] A. Kamkaew, S.H Lim, H.B. Lee, L.V. Kiew, L.Y. Chungc, K. Burgess, BODIPY dyes in photodynamic therapy, Chem. Soc. Rev. 42 (2013) 77-88. [4] B. Kim, B. Ma, V.R. Donuru, H. Liu, J.M.J. Frechet, Bodipy-backboned polymers as electron donor in bulk heterojunction solar cells, Chem. Commun. 46 (2010) 4148-4150. [5] S. Kolemen, O.A. Bozdemir, Y. Cakmak, G. Barin, S. Erten-Ela, M. Marszalek, J.H.Yum, S.M. Zakeeruddin, M.K. Nazeeruddin, M. Gratzelc, E.U. Akkaya, Optimization of distyryl-

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