- Email: [email protected]
Synthesis, molecular structure and cytotoxic studies of fluorene compound with potential anti-cancer properties
Accepted Manuscript Synthesis, molecular structure and cytotoxic studies of fluorene compound with potential anti-cancer properties Syarmila Ishak, Guan-Yeow Yeap, Shanmugapriya, Sreenivasan Sasidharan, Masato M. Ito PII:
S0022-2860(18)30979-7
DOI:
10.1016/j.molstruc.2018.08.030
Reference:
MOLSTR 25551
To appear in:
Journal of Molecular Structure
Received Date: 23 June 2018 Revised Date:
4 August 2018
Accepted Date: 9 August 2018
Please cite this article as: S. Ishak, G.-Y. Yeap, Shanmugapriya, S. Sasidharan, M.M. Ito, Synthesis, molecular structure and cytotoxic studies of fluorene compound with potential anti-cancer properties, Journal of Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.08.030. 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 proof before it is published in its final 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.
ACCEPTED MANUSCRIPT
SYNTHESIS, MOLECULAR STRUCTURE AND CYTOTOXIC STUDIES OF FLUORENE COMPOUND WITH POTENTIAL ANTI-CANCER PROPERTIES
Masato M Ito3
Liquid Crystal Research Laboratory, School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.
3
SC
2
Faculty of Science & Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan
ABSTRACT
M AN U
1
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Syarmila Ishak1, Guan-Yeow Yeap1*, Shanmugapriya2, Sreenivasan Sasidharan2 and
A new fluorene derivative, 1,1’-(9,9-dihexyl-9H-fluorene-2,7-diyl)bis(N,N-bis(pyridine-2ylmethyl)methanamine
has been successfully synthesized through condensation reaction of and
2,7-bis(bromomethyl)-9,9-dihexyl-9H-fluorene
with
TE D
bis(2-pyridylmethyl)amine
exceptionally good yield (60%). The molecular structure of the synthesized compound was well characterized by nuclear magnetic resonance (NMR), infrared (FTIR), UV-vis absorption and
EP
fluorescence techniques. The in-vitro anticancer activity of the title compound against human cervical (HeLa) cancer cell line was validated wherein the target molecule exhibits IC50 value of
AC C
28.58 µg/mL (37.76 µM).
Keywords: fluorene compound, nuclear magnetic resonance, fluorescence, in-vitro anticancer activity, human cervical, IC50 value
*Author for correspondence:‑G.Y.Yeap. Email: [email protected]. Fax : +60-4-6574854
ACCEPTED MANUSCRIPT
1.
INTRODUCTION Fluorene is categorized as an isocyclic aromatic hydrocarbon containing two benzene
SC
of which the molecular structure can be shown as follows:
RI PT
rings which are connected via a direct carbon-carbon bond and an adjacent methylene bridge [1]
The presence of a bridging methylene group at position 9 forces two phenyl rings to adopt a
M AN U
planar conformation that increases overlapping of their orbitals and the degree of conjugation of the aromatic system. As such, fluorene was found to absorb at longer wavelengths than the closely related biphenyl group.
Although fluorene can be isolated from coal tar but attempts to synthesize this compound
TE D
at laboratory scale had also been documented. Intramolecular Friedel−Crafts alkylation promoted by Brønsted or Lewis acids is the classical method in synthesizing fluorene [2]. This method is among the most powerful C–C bond-forming processes for the synthesis of functionalized
EP
aromatic compounds [3]. Despite intramolecular Friedel−Crafts alkylation, there are many other methods to synthesize fluorene including transition-metal-mediated cyclizations [4-6].
AC C
Polycyclic aromatic hydrocarbons like fluorene seemed to attract vast attention from
many researchers. The alteration of the structure of polycyclic aromatic hydrocarbons can lower the deleterious effect that promotes their interaction with specific cell organelles to evoke specific cytotoxic reactions. In the past, various biological activities such as antimicrobial, antibacterial as well as anticancer of fluorene derivatives had been reported [7-9].
ACCEPTED MANUSCRIPT
It is well accepted that the development of anticancer drugs requires the input of toxicity information as it provides important preliminary data to support the compounds thus synthesized possess potential anticancer properties. In general, the mechanism of toxic action on the target
RI PT
tissue can be based on specialized cell cultures [10]. A considerable studies had been carried out on different cell types; e.g. fibroblasts, HeLa (cervical adenocarcinoma cells) and hepatoma cells [11-12]. Among these studies, the fluorene derivatives with various substituted positions that
SC
exhibited cytotoxic activity on specialized cell cultures had been reported [13]. The typical examples of fluorene derivatives can be shown as following (a) and (c):
M AN U
O
N
NH
S
O
N
(b)
TE D
In spite of a remarkable advancement in the identification of numerous active cancer chemotherapeutic agents, the synthesis and biological evaluation of 2,7-substituted fluorene has hitherto not been reported. Hence, in this research, we are prompted to synthesize and investigate
2.1
EXPERIMENTAL
AC C
2.
EP
the cytotoxicity of title compound that can be grouped as 2,7-substituted fluorene derivative.
Material and instrument
All solvents and reagents were purchased commercially and used without any further
purification. 2,7-Bis(bromomethyl)-9,9-dihexyl-9H-fluorene was purchased from Sigma Aldrich. Bis(2-pyridylmethyl)amine, potassium carbonate and potassium iodide were obtained from TCI and Merck.
ACCEPTED MANUSCRIPT
1D (1H NMR and
13
C NMR) and 2D (DEPT 45, DEPT 90, DEPT 135, COSY, HMBC,
HMQC) NMR spectra were recorded in CDCl3 and deuterated DMSO using a Bruker 500 MHz Ultrashield TMFT-NMR spectrometer. Tetramethylsilane (TMS) was used as an internal standard.
RI PT
The FT-IR spectrum of the compound was recorded using the Perkin Elmer 2000 FT-IR spectrophotometer, at the range of frequency 4000-400cm-1. Electronic spectra (600–200 nm) were recorded on a Shimadzu 2600 UV–Vis spectrophotometer. Fluorescence spectra were
SC
obtained from a Perkin-Elmer LS 55 fluorescence spectrophotometer with quartz cuvette (path
2.2
Cell line and culture condition
M AN U
length = 1 cm).
Cervical adenocarcinoma cells (HeLa cell line) were obtained from Tissue Culture Laboratory of Institute for Research in Molecular Medicine (INFORMM). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100
5% CO2 incubator at 37 ºC.
Preparation of 1,1’-(9,9-dihexyl-9H-fluorene-2,7-diyl)bis(N,N-bis(pyridine-2-ylmethyl)-
methanamine (1)
EP
2.3
TE D
µg/mL streptomycin. Cell line was maintained as adherent culture in a humidified atmosphere of
AC C
2,7-Bis(bromomethyl)-9,9-dihexyl-9H-fluorene (0.1g, 0.19 mmol) was first dissolved in acetone. Potassium carbonate, K2CO3 (0.212g, 1.52 mmol) and a catalytic amount of potassium iodide, KI were heated on reflux for 30 minutes and subsequently added with bis(2pyridylmethyl)amine (0.115g, 0.57 mmol) and the mixture was heated through reflux for 48 hours. The resulted solution was left at room temperature to allow complete evaporation of the solvent. The product was then washed with petroleum ether several times to give a brown sticky solid. Yield 60%. IR ʋ/cm-1: 2924 (C-H aromatic stretch), 2855 (C-H alkane stretch), 1587 (C=C
ACCEPTED MANUSCRIPT
aromatic stretch), 1433 (C-N aromatic amine stretch), 1364 (C-N aliphatic amine stretch). 1HNMR (CDCl3) δ/ppm: 0.65 (t, 6H, CH3), 0.7-1.01 (m, 16H, CH2), 2.17 (t, 4H, CH2), 3.75 (s, 4H, CH2), 3.83 (s, 8H, CH2), 7.13 (t, 4H, CH), 7.35 (d, 4H, CH), 7.58 (d, 6H, CH), 7.60 (t, 4H, CH),
RI PT
8.51 (d, 4H, CH). 13C NMR (125 MHz, CDCl3). δ, ppm: 13.91, 22.50, 23.80, 29.73, 31.44, 40.46 (aliphatic chain), 54.71 (cyclopentane), 59.03, 59.95 (aliphatic), 119.30, 121.92, 122.78, 123.28, 127.51, 136.35, 138.00 (aromatic), 140.06, 150.99 (cyclopentane), 148.95, 159.90 (aromatic). synthesis
of
1,1’-(9,9-dihexyl-9H-fluorene-2,7-diyl)bis(N,N-bis(pyridine-2-
SC
The
AC C
EP
TE D
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ylmethyl)methanamine (1) can be depicted by the following Scheme 1:
I
Scheme 1: Synthetic route toward formation of compound 1
2.4
Preparation of cell culture
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Initially, cells (HeLa) were allowed to grow under optimal incubator conditions. Subculturing of cells was performed after the cells achieved 70-80% confluence. Old medium was first aspirated out of the plate. The cells were then washed twice with sterile phosphate
RI PT
buffered saline (PBS) (pH=7.4) and then completely discarded. A solution of 2 mL trypsineEDTA was then added and distributed evenly onto the surface of the cells in 75 cm2 cell culture flask. The cells were then placed in incubator at 37ºC in 5% CO2 for 5 min. To aid cells
SC
segregation, the flask containing the cells was gently tapped. Trypsin activity was inhibited by adding 2 mL of fresh complete media (10% FBS). The cells were then transferred to a sterile
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centrifuge tube and upon centrifugation, the cell pellet was resuspended with media and counted with the aid of haemocytometer. Cells were then plated into fresh tissue culture flask containing fresh media and incubated at 37ºC with an internal atmosphere of 5% CO2. 2.5
MTT assay
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The cells (HeLa) were harvested and approximately 4000 cells in 100 µL media were seeded in each well of a sterile 96-well plate and incubated overnight using CO2 incubator. Different concentrations of fluorene compound were prepared from the stock by serial dilution in
EP
media ranging from 200 µg/mL to 0.781 µg/mL which were then used to perform cell treatment in triplicates for each concentration. The media was then carefully aspirated out and the
AC C
formazon crystals thus formed in each well were dissolved using 100 µL of DMSO. Finally, the absorbance was taken at 540 nm by using a standard ELISA microplate reader (Molecular Devices Inc., USA). Fluorouracil was used as positive control and the negative control was represented by the untreated medium containing vehicle DMSO.
3.
RESULT AND DISCUSSION
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3.1
Synthesis A new fluorene compound 1 was prepared following the synthetic route as shown in 1.
2,7-Bis(bromomethyl)-9,9-dihexyl-9H-fluorene
was
reacted
with
bis(2-
RI PT
Scheme
pyridylmethyl)amine in the presence of potassium carbonate and potassium iodide in acetone. The mixture was refluxed for 48 hours. The resulted product was left at room temperature to
SC
allow complete evaporation of the solvent. The sticky brown solid thus formed was then washed with petroleum ether to yield the desired compound. It was well characterized by IR, NMR, Uv-
3.2
M AN U
Vis and fluorescence spectroscopic techniques before its cytotoxic activity was investigated. Characterization
In the IR spectrum, the diagnostic infrared absorption bands can be assigned to the aromatic C-H (2924 cm-1), alkyl C-H (2855 cm-1), aromatic C=C (1587 cm-1), aromatic C-N (1433 cm-1) and aliphatic amine C-N (1364 cm-1). Most of the bands as observed in the IR
TE D
spectrum of compound 1 suggest the existence of all the functional groups present in the compound.
The proposed molecular structure of compound 1 along with its atomic-numbering is
EP
illustrated in Figure 1. Inspection on the 1H NMR spectrum of compound 1 (Figure 2) shows two
AC C
singlets at chemical shifts of δ 3.75 and 3.82 ppm indicating two identical units of (2pyridylmethyl)amine connected to 2,7-bis(bromomethyl)-9,9-dihexyl-9H-fluorene at the respective C2 and C7 atoms. A comparison between the 1H NMR spectra of compound 1 and 2,7-bis(bromomethyl)-9,9-dihexyl-9H-fluorene shows the appearance of additional signals at the chemical shifts ranging from 7.0-8.5 ppm which can be attributed to the presence of pyridyl moiety. The
13
C NMR spectrum (Figure 3) shows unambiguously the sharp peaks at chemical
ACCEPTED MANUSCRIPT
shifts of δ 59.03 and 59.95 ppm which can be ascribed to C16 and C17 (that is also identical with C18), respectively. The UV-Visible absorption spectrum for compound 1 (Figure 4) shows the absorption
RI PT
maximum at 260 nm. The shape of the absorption spectrum for compound 1 is found to be almost identical to that of fluorene [14]. However, the fluorescence spectrum (Figure 5) shows that compound 1 does not emit high fluorescence. MTT assay
SC
3.3
The cytotoxic effect of compound 1 on HeLa cell line was evaluated by MTT assay at a
M AN U
range of 0–200 µg/mL after 24 h of treatment period, which is based on the ability of live cells to convert tetrazolium salt into purple formazan. The cell viability can be qualitatively assessed by the color intensity of the purple formazan dye. The intensity of the purple formazan dye increased when the concentration of the drug decreased.
TE D
The cytotoxicity of compound 1 was expressed as the half maximal inhibitory of concentration (IC50). The absorbance values obtained from the microplate reader for respective concentration of the drug were used to plot the percentage cell viability graph (Figure 6). The
EP
IC50 value which was the concentration of the drug resulting in inhibition of 50% of cell viability was determined to be 28.58 µg/mL (37.76 µM), indicating that compound 1 exhibits moderate
AC C
cytotoxic effect [15].
It had earlier been reported by Miri et al. that thirty novel derivatives of aza-
cyclopenta[b]fluorene-1,9-dione thus synthesized were tested against HeLa, LS180, MCF-7, and Raji cancer cell lines by MTT assay [16]. One of the noticeable features is that the IC50 value obtained from our study is comparable with those IC50 values (from 3.1 to 100 µM) as reported earlier [16]. In the present study, fluorouracil, a commercial drug for the treatment of cancer was
ACCEPTED MANUSCRIPT
used as a positive control and it exhibited an inhibition of cell viability activity with IC50 value of 27.79 µg/mL (213.63 µM) in comparison to the treatment using compound 1 (Figure 6). However, the negative control was merely represented by the untreated medium containing
RI PT
vehicle DMSO of which the IC50 value was found to be more than 1000.00 µg/mL (12799.1 µM) by maintaining cell viability above 95% (Figure 6). This piece of information is a strong evidence that the vehicle negative control failed to exhibit any significant inhibition of cell
4.
SC
viability. Conclusion
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A new fluorene derivative was synthesized and the characterization by physical and spectroscopy techniques were reported. Its spectroscopic properties supported the proposed structure. The prepared compound was tested for its cytotoxicity against HeLa cell line, showing
Acknowledgement
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moderate activity with IC50 value of 28.58 µg/mL (37.76 µM).
The main author (G.-Y. Yeap) gratefully acknowledges Malaysian Ministry of Higher Education
EP
(MOHE) for funding this project through FRGS Grant No.203/PKIMIA/6711422 and USM for
AC C
partial support from RU Research Grant No.1001/PKIMIA/8011034.
References [1]
R. Abbel, A.P.H.J. Schenning, E.W. Meijer, Fluorene-Based Materials and Their Supramolecular Properties, J. Polym. Sci. A1. 47 (17) (2009) 4215-4233.
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[2]
S. Xu, X. Shangguan, H. Li, Y. Zhang, J. Wang, Pd(0)-Catalyzed Cross-Coupling of 1,1Diboronates with 2,2′-Dibromobiphenyls: Synthesis of 9H Fluorenes, J. Org. Chem. 80 (15) (2015) 7779-7784. S. Sarkar, S. Maiti, K. Bera, S. Jalal, U. Jana, Highly Efficient Synthesis of Polysubstituted
RI PT
[3]
Fluorene via Iron-catalyzed Intramolecular Friedel–Crafts Alkylation of Biaryl Alcohols, Tetrahedron Lett. 53 (41) (2012) 5544-5547.
D. Chen, G. Shi, H. Jiang, Y. Zhang, Y. Zhang, Sequential Difunctionalization of 2
SC
[4]
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Iodobiphenyls by Exploiting the Reactivities of a Palladacycle and an Acyclic Arylpalladium Species, Org. Lett. 18 (9) (2016) 2130-2133. [5]
T.P Liu, C. H. Xing, Q. S. Hu, Synthesis of Fluorene and Indenofluorene Compounds: Tandem Palladium-Catalyzed Suzuki Cross-Coupling and Cyclization**, Angew. ChemGer. Edit. 122 (16) (2010) 2971-2974.
K. Morimoto, M. Itoh, K. Hirano, T. Satoh, Y. Shibata, K. Tanaka, M. Miura, Synthesis of
TE D
[6]
Fluorene Derivatives through Rhodium-Catalyzed Dehydrogenative Cyclization**, Angew. Chem. Int. Edit. 51 (22) (2012) 5359-5362. B. K. Banik, C. Mukhopadhyay, F. F. Becker, Synthesis and Biological Evaluation of
AC C
Novel
EP
[7]
Dibenzofluorene Derivatives as Anticancer Agents, Oncol. Lett. 1 (2) (2010) 309-311. [8]
N. S. Youssef, E. A. El Zahany, M. M. Anwar, S. A. Hassan, Synthesis, Characterization, and Antitumor Activity of Some Metal Complexes with Schiff Bases Derived from 9Fluorenone as a Polycyclic Aromatic Compound, Phosphorus Sulfur. 184 (1) (2008) 103125.
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[9]
P. E. Marinova, M. Marinov, M. Kazakova, Y. Feodorova, A. Slavchev, D. Blazheva, N. Stoyanov, Study on the Synthesis, Characterization and Bioactivities of 3-Methyl-9’fluorenespiro-5-hydantoin, Acta. Chim. Slov. 63 (1) (2016) 26-32.
RI PT
[10] Y. P. Kwan, (2014). In Vitro Cytotoxicity Activity and in Vitro Oral Toxicity of Euphorbia Hirta, (Doctoral dissertation, Universiti Sains Malaysia) (2014).
[11] B. Ekwall, V. Silano, A. Paganuzzi-Stammati, F. Zucco, Toxicity Tests with Mammalian
SC
Cell Cultures, Short-term Toxicity Tests for Non-genotoxic Effects, (1990) 75-99.
M AN U
[12] M. S. Islam, Y. Kusumoto, M. A. Al-Mamun, Cytotoxicity and Cancer (HeLa) Cell Killing Efficacy of Aqueous Garlic (Allium sativum) Extract, J. Sci. Res. 3 (2) (2011) 375-382. [13] H. Chabane, A. Pierre, S. Leonce, B. Pfeiffer, P. Renard, V. Thiery, Y. Besson, Synthesis and Cytotoxic Activity of Thiazolofluorenone Derivatives, J. Enzyme Inhib. Med. Ch. 19
TE D
(6) (2004) 567-575.
[14] C. W. Lee, H. W. Rhee, C. H. Kim, M. S. Gang, Chemiluminescent Properties of Fluoreneand Carbazole-containing Polymeric Fluorophores, B. Korean Chem. Soc. 21 (7) (2000)
EP
701-704.
[15] M. A. Salem, M. I. Marzouk, A. M. El-Kazak, Synthesis and Characterization of Some
AC C
New Coumarins with In Vitro Antitumor and Antioxidant Activity and High Protective Effects against DNA Damage, Molecules. 21(2) (2016) 249. [16] R.1. Miri, O. Firuzi, P. Peymani, M. Zamani, A.R. Mehdipour, Z. Heydari, M.M. Farahani, A. Shafiee, Synthesis, Cytotoxicity, and QSR Study of New Aza-cyclopenta[b]fluorene1,9-dione Derivatives, Chemical Biology & Drug Design, 79(1) (2012), 68-75.
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Table 1: Functional Group
Characteristic Absorption (cm-1)
Intensity
C-H aromatic
2924.3
Strong, Sharp
C-H alkane
2855.2
C=C aromatic
1587.4
Strong, Sharp
C-N aromatic amine
1433.7
Strong, Sharp
C-N aliphatic amine
1364.6
Characteri stic
IR
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absorption
Medium
frequencie
AC C
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TE D
M AN U
SC
s
of
compound
Medium 1
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ACCEPTED MANUSCRIPT
AC C
EP
TE D
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SC
Figure 1: Structure of compound 1 with atomic numbering scheme
Figure 2: Comparison of 1H NMR spectra for starting material and compound 1
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SC
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ACCEPTED MANUSCRIPT
Figure 3: 13C NMR spectrum of compound 1
-0.017 200.00
1
0.992
2
Abs.
AC C
3
EP
2.000
400.00 nm.
Figure 4: UV-Vis spectrum of compound 1
600.00
ACCEPTED MANUSCRIPT
250
RI PT
200 150 100 50 0 340
390
440
490
SC
Fluorescence Intensity (a.u)
300
540
M AN U
wavelength (nm)
590
AC C
EP
TE D
Figure 5: Fluorescence spectrum of compound 1
640
ACCEPTED MANUSCRIPT
120 y = -1.9449x + 95.901 R² = 0.9292
100
Positive Control (5FU) Negative Control (Vehicle) Compound 1
y = -36.437x + 102.63 R² = 0.9753
60
RI PT
Cell Viability (%)
80
y = -27.756x + 90.407 R² = 0.976
SC
40
0 -0.5
0
0.5
1
M AN U
20
1.5
2
2.5
Log [concentration] (µg/mL)
IC50 (µg/mL): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), above 100 (non-cytotoxic).
Figure 6: Cell viability of HeLa cells treated with compound 1, fluorouracil (positive control)
EP
TE D
and DMSO (negative control) by using MTT assay
AC C
a
ACCEPTED MANUSCRIPT
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SYNTHESIS, MOLECULAR STRUCTURE AND CYTOTOXIC STUDIES OF FLUORENE COMPOUND WITH POTENTIAL ANTI-CANCER PROPERTIES
Highlights •
A new fluorene derivative 1,1’-(9,9-dihexyl-9H-fluorene-2,7-diyl)bis(N,N-bis(pyridine2-ylmethyl)methanamine had successfully been isolated and characterized.
The synthesized compound was well characterized with nuclear magnetic resonance
SC
•
The synthesized compound was tested for its cytotoxicity against HeLa cell line, showing
EP
TE D
moderate activity with IC50 value of 28.58 μg/mL(37.76 µM).
AC C
•
M AN U
(NMR), infrared (FTIR), UV-vis absorption and fluorescence techniques.
S0022-2860(18)30979-7
DOI:
10.1016/j.molstruc.2018.08.030
Reference:
MOLSTR 25551
To appear in:
Journal of Molecular Structure
Received Date: 23 June 2018 Revised Date:
4 August 2018
Accepted Date: 9 August 2018
Please cite this article as: S. Ishak, G.-Y. Yeap, Shanmugapriya, S. Sasidharan, M.M. Ito, Synthesis, molecular structure and cytotoxic studies of fluorene compound with potential anti-cancer properties, Journal of Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.08.030. 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 proof before it is published in its final 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.
ACCEPTED MANUSCRIPT
SYNTHESIS, MOLECULAR STRUCTURE AND CYTOTOXIC STUDIES OF FLUORENE COMPOUND WITH POTENTIAL ANTI-CANCER PROPERTIES
Masato M Ito3
Liquid Crystal Research Laboratory, School of Chemical Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia. Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.
3
SC
2
Faculty of Science & Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8577, Japan
ABSTRACT
M AN U
1
RI PT
Syarmila Ishak1, Guan-Yeow Yeap1*, Shanmugapriya2, Sreenivasan Sasidharan2 and
A new fluorene derivative, 1,1’-(9,9-dihexyl-9H-fluorene-2,7-diyl)bis(N,N-bis(pyridine-2ylmethyl)methanamine
has been successfully synthesized through condensation reaction of and
2,7-bis(bromomethyl)-9,9-dihexyl-9H-fluorene
with
TE D
bis(2-pyridylmethyl)amine
exceptionally good yield (60%). The molecular structure of the synthesized compound was well characterized by nuclear magnetic resonance (NMR), infrared (FTIR), UV-vis absorption and
EP
fluorescence techniques. The in-vitro anticancer activity of the title compound against human cervical (HeLa) cancer cell line was validated wherein the target molecule exhibits IC50 value of
AC C
28.58 µg/mL (37.76 µM).
Keywords: fluorene compound, nuclear magnetic resonance, fluorescence, in-vitro anticancer activity, human cervical, IC50 value
*Author for correspondence:‑G.Y.Yeap. Email: [email protected]. Fax : +60-4-6574854
ACCEPTED MANUSCRIPT
1.
INTRODUCTION Fluorene is categorized as an isocyclic aromatic hydrocarbon containing two benzene
SC
of which the molecular structure can be shown as follows:
RI PT
rings which are connected via a direct carbon-carbon bond and an adjacent methylene bridge [1]
The presence of a bridging methylene group at position 9 forces two phenyl rings to adopt a
M AN U
planar conformation that increases overlapping of their orbitals and the degree of conjugation of the aromatic system. As such, fluorene was found to absorb at longer wavelengths than the closely related biphenyl group.
Although fluorene can be isolated from coal tar but attempts to synthesize this compound
TE D
at laboratory scale had also been documented. Intramolecular Friedel−Crafts alkylation promoted by Brønsted or Lewis acids is the classical method in synthesizing fluorene [2]. This method is among the most powerful C–C bond-forming processes for the synthesis of functionalized
EP
aromatic compounds [3]. Despite intramolecular Friedel−Crafts alkylation, there are many other methods to synthesize fluorene including transition-metal-mediated cyclizations [4-6].
AC C
Polycyclic aromatic hydrocarbons like fluorene seemed to attract vast attention from
many researchers. The alteration of the structure of polycyclic aromatic hydrocarbons can lower the deleterious effect that promotes their interaction with specific cell organelles to evoke specific cytotoxic reactions. In the past, various biological activities such as antimicrobial, antibacterial as well as anticancer of fluorene derivatives had been reported [7-9].
ACCEPTED MANUSCRIPT
It is well accepted that the development of anticancer drugs requires the input of toxicity information as it provides important preliminary data to support the compounds thus synthesized possess potential anticancer properties. In general, the mechanism of toxic action on the target
RI PT
tissue can be based on specialized cell cultures [10]. A considerable studies had been carried out on different cell types; e.g. fibroblasts, HeLa (cervical adenocarcinoma cells) and hepatoma cells [11-12]. Among these studies, the fluorene derivatives with various substituted positions that
SC
exhibited cytotoxic activity on specialized cell cultures had been reported [13]. The typical examples of fluorene derivatives can be shown as following (a) and (c):
M AN U
O
N
NH
S
O
N
(b)
TE D
In spite of a remarkable advancement in the identification of numerous active cancer chemotherapeutic agents, the synthesis and biological evaluation of 2,7-substituted fluorene has hitherto not been reported. Hence, in this research, we are prompted to synthesize and investigate
2.1
EXPERIMENTAL
AC C
2.
EP
the cytotoxicity of title compound that can be grouped as 2,7-substituted fluorene derivative.
Material and instrument
All solvents and reagents were purchased commercially and used without any further
purification. 2,7-Bis(bromomethyl)-9,9-dihexyl-9H-fluorene was purchased from Sigma Aldrich. Bis(2-pyridylmethyl)amine, potassium carbonate and potassium iodide were obtained from TCI and Merck.
ACCEPTED MANUSCRIPT
1D (1H NMR and
13
C NMR) and 2D (DEPT 45, DEPT 90, DEPT 135, COSY, HMBC,
HMQC) NMR spectra were recorded in CDCl3 and deuterated DMSO using a Bruker 500 MHz Ultrashield TMFT-NMR spectrometer. Tetramethylsilane (TMS) was used as an internal standard.
RI PT
The FT-IR spectrum of the compound was recorded using the Perkin Elmer 2000 FT-IR spectrophotometer, at the range of frequency 4000-400cm-1. Electronic spectra (600–200 nm) were recorded on a Shimadzu 2600 UV–Vis spectrophotometer. Fluorescence spectra were
SC
obtained from a Perkin-Elmer LS 55 fluorescence spectrophotometer with quartz cuvette (path
2.2
Cell line and culture condition
M AN U
length = 1 cm).
Cervical adenocarcinoma cells (HeLa cell line) were obtained from Tissue Culture Laboratory of Institute for Research in Molecular Medicine (INFORMM). Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100
5% CO2 incubator at 37 ºC.
Preparation of 1,1’-(9,9-dihexyl-9H-fluorene-2,7-diyl)bis(N,N-bis(pyridine-2-ylmethyl)-
methanamine (1)
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µg/mL streptomycin. Cell line was maintained as adherent culture in a humidified atmosphere of
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2,7-Bis(bromomethyl)-9,9-dihexyl-9H-fluorene (0.1g, 0.19 mmol) was first dissolved in acetone. Potassium carbonate, K2CO3 (0.212g, 1.52 mmol) and a catalytic amount of potassium iodide, KI were heated on reflux for 30 minutes and subsequently added with bis(2pyridylmethyl)amine (0.115g, 0.57 mmol) and the mixture was heated through reflux for 48 hours. The resulted solution was left at room temperature to allow complete evaporation of the solvent. The product was then washed with petroleum ether several times to give a brown sticky solid. Yield 60%. IR ʋ/cm-1: 2924 (C-H aromatic stretch), 2855 (C-H alkane stretch), 1587 (C=C
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aromatic stretch), 1433 (C-N aromatic amine stretch), 1364 (C-N aliphatic amine stretch). 1HNMR (CDCl3) δ/ppm: 0.65 (t, 6H, CH3), 0.7-1.01 (m, 16H, CH2), 2.17 (t, 4H, CH2), 3.75 (s, 4H, CH2), 3.83 (s, 8H, CH2), 7.13 (t, 4H, CH), 7.35 (d, 4H, CH), 7.58 (d, 6H, CH), 7.60 (t, 4H, CH),
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8.51 (d, 4H, CH). 13C NMR (125 MHz, CDCl3). δ, ppm: 13.91, 22.50, 23.80, 29.73, 31.44, 40.46 (aliphatic chain), 54.71 (cyclopentane), 59.03, 59.95 (aliphatic), 119.30, 121.92, 122.78, 123.28, 127.51, 136.35, 138.00 (aromatic), 140.06, 150.99 (cyclopentane), 148.95, 159.90 (aromatic). synthesis
of
1,1’-(9,9-dihexyl-9H-fluorene-2,7-diyl)bis(N,N-bis(pyridine-2-
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The
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ylmethyl)methanamine (1) can be depicted by the following Scheme 1:
I
Scheme 1: Synthetic route toward formation of compound 1
2.4
Preparation of cell culture
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Initially, cells (HeLa) were allowed to grow under optimal incubator conditions. Subculturing of cells was performed after the cells achieved 70-80% confluence. Old medium was first aspirated out of the plate. The cells were then washed twice with sterile phosphate
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buffered saline (PBS) (pH=7.4) and then completely discarded. A solution of 2 mL trypsineEDTA was then added and distributed evenly onto the surface of the cells in 75 cm2 cell culture flask. The cells were then placed in incubator at 37ºC in 5% CO2 for 5 min. To aid cells
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segregation, the flask containing the cells was gently tapped. Trypsin activity was inhibited by adding 2 mL of fresh complete media (10% FBS). The cells were then transferred to a sterile
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centrifuge tube and upon centrifugation, the cell pellet was resuspended with media and counted with the aid of haemocytometer. Cells were then plated into fresh tissue culture flask containing fresh media and incubated at 37ºC with an internal atmosphere of 5% CO2. 2.5
MTT assay
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The cells (HeLa) were harvested and approximately 4000 cells in 100 µL media were seeded in each well of a sterile 96-well plate and incubated overnight using CO2 incubator. Different concentrations of fluorene compound were prepared from the stock by serial dilution in
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media ranging from 200 µg/mL to 0.781 µg/mL which were then used to perform cell treatment in triplicates for each concentration. The media was then carefully aspirated out and the
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formazon crystals thus formed in each well were dissolved using 100 µL of DMSO. Finally, the absorbance was taken at 540 nm by using a standard ELISA microplate reader (Molecular Devices Inc., USA). Fluorouracil was used as positive control and the negative control was represented by the untreated medium containing vehicle DMSO.
3.
RESULT AND DISCUSSION
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3.1
Synthesis A new fluorene compound 1 was prepared following the synthetic route as shown in 1.
2,7-Bis(bromomethyl)-9,9-dihexyl-9H-fluorene
was
reacted
with
bis(2-
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Scheme
pyridylmethyl)amine in the presence of potassium carbonate and potassium iodide in acetone. The mixture was refluxed for 48 hours. The resulted product was left at room temperature to
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allow complete evaporation of the solvent. The sticky brown solid thus formed was then washed with petroleum ether to yield the desired compound. It was well characterized by IR, NMR, Uv-
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Vis and fluorescence spectroscopic techniques before its cytotoxic activity was investigated. Characterization
In the IR spectrum, the diagnostic infrared absorption bands can be assigned to the aromatic C-H (2924 cm-1), alkyl C-H (2855 cm-1), aromatic C=C (1587 cm-1), aromatic C-N (1433 cm-1) and aliphatic amine C-N (1364 cm-1). Most of the bands as observed in the IR
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spectrum of compound 1 suggest the existence of all the functional groups present in the compound.
The proposed molecular structure of compound 1 along with its atomic-numbering is
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illustrated in Figure 1. Inspection on the 1H NMR spectrum of compound 1 (Figure 2) shows two
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singlets at chemical shifts of δ 3.75 and 3.82 ppm indicating two identical units of (2pyridylmethyl)amine connected to 2,7-bis(bromomethyl)-9,9-dihexyl-9H-fluorene at the respective C2 and C7 atoms. A comparison between the 1H NMR spectra of compound 1 and 2,7-bis(bromomethyl)-9,9-dihexyl-9H-fluorene shows the appearance of additional signals at the chemical shifts ranging from 7.0-8.5 ppm which can be attributed to the presence of pyridyl moiety. The
13
C NMR spectrum (Figure 3) shows unambiguously the sharp peaks at chemical
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shifts of δ 59.03 and 59.95 ppm which can be ascribed to C16 and C17 (that is also identical with C18), respectively. The UV-Visible absorption spectrum for compound 1 (Figure 4) shows the absorption
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maximum at 260 nm. The shape of the absorption spectrum for compound 1 is found to be almost identical to that of fluorene [14]. However, the fluorescence spectrum (Figure 5) shows that compound 1 does not emit high fluorescence. MTT assay
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3.3
The cytotoxic effect of compound 1 on HeLa cell line was evaluated by MTT assay at a
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range of 0–200 µg/mL after 24 h of treatment period, which is based on the ability of live cells to convert tetrazolium salt into purple formazan. The cell viability can be qualitatively assessed by the color intensity of the purple formazan dye. The intensity of the purple formazan dye increased when the concentration of the drug decreased.
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The cytotoxicity of compound 1 was expressed as the half maximal inhibitory of concentration (IC50). The absorbance values obtained from the microplate reader for respective concentration of the drug were used to plot the percentage cell viability graph (Figure 6). The
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IC50 value which was the concentration of the drug resulting in inhibition of 50% of cell viability was determined to be 28.58 µg/mL (37.76 µM), indicating that compound 1 exhibits moderate
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cytotoxic effect [15].
It had earlier been reported by Miri et al. that thirty novel derivatives of aza-
cyclopenta[b]fluorene-1,9-dione thus synthesized were tested against HeLa, LS180, MCF-7, and Raji cancer cell lines by MTT assay [16]. One of the noticeable features is that the IC50 value obtained from our study is comparable with those IC50 values (from 3.1 to 100 µM) as reported earlier [16]. In the present study, fluorouracil, a commercial drug for the treatment of cancer was
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used as a positive control and it exhibited an inhibition of cell viability activity with IC50 value of 27.79 µg/mL (213.63 µM) in comparison to the treatment using compound 1 (Figure 6). However, the negative control was merely represented by the untreated medium containing
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vehicle DMSO of which the IC50 value was found to be more than 1000.00 µg/mL (12799.1 µM) by maintaining cell viability above 95% (Figure 6). This piece of information is a strong evidence that the vehicle negative control failed to exhibit any significant inhibition of cell
4.
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viability. Conclusion
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A new fluorene derivative was synthesized and the characterization by physical and spectroscopy techniques were reported. Its spectroscopic properties supported the proposed structure. The prepared compound was tested for its cytotoxicity against HeLa cell line, showing
Acknowledgement
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moderate activity with IC50 value of 28.58 µg/mL (37.76 µM).
The main author (G.-Y. Yeap) gratefully acknowledges Malaysian Ministry of Higher Education
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(MOHE) for funding this project through FRGS Grant No.203/PKIMIA/6711422 and USM for
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partial support from RU Research Grant No.1001/PKIMIA/8011034.
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R. Abbel, A.P.H.J. Schenning, E.W. Meijer, Fluorene-Based Materials and Their Supramolecular Properties, J. Polym. Sci. A1. 47 (17) (2009) 4215-4233.
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[2]
S. Xu, X. Shangguan, H. Li, Y. Zhang, J. Wang, Pd(0)-Catalyzed Cross-Coupling of 1,1Diboronates with 2,2′-Dibromobiphenyls: Synthesis of 9H Fluorenes, J. Org. Chem. 80 (15) (2015) 7779-7784. S. Sarkar, S. Maiti, K. Bera, S. Jalal, U. Jana, Highly Efficient Synthesis of Polysubstituted
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Fluorene via Iron-catalyzed Intramolecular Friedel–Crafts Alkylation of Biaryl Alcohols, Tetrahedron Lett. 53 (41) (2012) 5544-5547.
D. Chen, G. Shi, H. Jiang, Y. Zhang, Y. Zhang, Sequential Difunctionalization of 2
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Iodobiphenyls by Exploiting the Reactivities of a Palladacycle and an Acyclic Arylpalladium Species, Org. Lett. 18 (9) (2016) 2130-2133. [5]
T.P Liu, C. H. Xing, Q. S. Hu, Synthesis of Fluorene and Indenofluorene Compounds: Tandem Palladium-Catalyzed Suzuki Cross-Coupling and Cyclization**, Angew. ChemGer. Edit. 122 (16) (2010) 2971-2974.
K. Morimoto, M. Itoh, K. Hirano, T. Satoh, Y. Shibata, K. Tanaka, M. Miura, Synthesis of
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Fluorene Derivatives through Rhodium-Catalyzed Dehydrogenative Cyclization**, Angew. Chem. Int. Edit. 51 (22) (2012) 5359-5362. B. K. Banik, C. Mukhopadhyay, F. F. Becker, Synthesis and Biological Evaluation of
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Novel
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Dibenzofluorene Derivatives as Anticancer Agents, Oncol. Lett. 1 (2) (2010) 309-311. [8]
N. S. Youssef, E. A. El Zahany, M. M. Anwar, S. A. Hassan, Synthesis, Characterization, and Antitumor Activity of Some Metal Complexes with Schiff Bases Derived from 9Fluorenone as a Polycyclic Aromatic Compound, Phosphorus Sulfur. 184 (1) (2008) 103125.
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P. E. Marinova, M. Marinov, M. Kazakova, Y. Feodorova, A. Slavchev, D. Blazheva, N. Stoyanov, Study on the Synthesis, Characterization and Bioactivities of 3-Methyl-9’fluorenespiro-5-hydantoin, Acta. Chim. Slov. 63 (1) (2016) 26-32.
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[10] Y. P. Kwan, (2014). In Vitro Cytotoxicity Activity and in Vitro Oral Toxicity of Euphorbia Hirta, (Doctoral dissertation, Universiti Sains Malaysia) (2014).
[11] B. Ekwall, V. Silano, A. Paganuzzi-Stammati, F. Zucco, Toxicity Tests with Mammalian
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Cell Cultures, Short-term Toxicity Tests for Non-genotoxic Effects, (1990) 75-99.
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[12] M. S. Islam, Y. Kusumoto, M. A. Al-Mamun, Cytotoxicity and Cancer (HeLa) Cell Killing Efficacy of Aqueous Garlic (Allium sativum) Extract, J. Sci. Res. 3 (2) (2011) 375-382. [13] H. Chabane, A. Pierre, S. Leonce, B. Pfeiffer, P. Renard, V. Thiery, Y. Besson, Synthesis and Cytotoxic Activity of Thiazolofluorenone Derivatives, J. Enzyme Inhib. Med. Ch. 19
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(6) (2004) 567-575.
[14] C. W. Lee, H. W. Rhee, C. H. Kim, M. S. Gang, Chemiluminescent Properties of Fluoreneand Carbazole-containing Polymeric Fluorophores, B. Korean Chem. Soc. 21 (7) (2000)
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701-704.
[15] M. A. Salem, M. I. Marzouk, A. M. El-Kazak, Synthesis and Characterization of Some
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New Coumarins with In Vitro Antitumor and Antioxidant Activity and High Protective Effects against DNA Damage, Molecules. 21(2) (2016) 249. [16] R.1. Miri, O. Firuzi, P. Peymani, M. Zamani, A.R. Mehdipour, Z. Heydari, M.M. Farahani, A. Shafiee, Synthesis, Cytotoxicity, and QSR Study of New Aza-cyclopenta[b]fluorene1,9-dione Derivatives, Chemical Biology & Drug Design, 79(1) (2012), 68-75.
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Table 1: Functional Group
Characteristic Absorption (cm-1)
Intensity
C-H aromatic
2924.3
Strong, Sharp
C-H alkane
2855.2
C=C aromatic
1587.4
Strong, Sharp
C-N aromatic amine
1433.7
Strong, Sharp
C-N aliphatic amine
1364.6
Characteri stic
IR
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absorption
Medium
frequencie
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s
of
compound
Medium 1
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Figure 1: Structure of compound 1 with atomic numbering scheme
Figure 2: Comparison of 1H NMR spectra for starting material and compound 1
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Figure 3: 13C NMR spectrum of compound 1
-0.017 200.00
1
0.992
2
Abs.
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3
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2.000
400.00 nm.
Figure 4: UV-Vis spectrum of compound 1
600.00
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250
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200 150 100 50 0 340
390
440
490
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Fluorescence Intensity (a.u)
300
540
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wavelength (nm)
590
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Figure 5: Fluorescence spectrum of compound 1
640
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120 y = -1.9449x + 95.901 R² = 0.9292
100
Positive Control (5FU) Negative Control (Vehicle) Compound 1
y = -36.437x + 102.63 R² = 0.9753
60
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Cell Viability (%)
80
y = -27.756x + 90.407 R² = 0.976
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40
0 -0.5
0
0.5
1
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20
1.5
2
2.5
Log [concentration] (µg/mL)
IC50 (µg/mL): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), above 100 (non-cytotoxic).
Figure 6: Cell viability of HeLa cells treated with compound 1, fluorouracil (positive control)
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and DMSO (negative control) by using MTT assay
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a
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SYNTHESIS, MOLECULAR STRUCTURE AND CYTOTOXIC STUDIES OF FLUORENE COMPOUND WITH POTENTIAL ANTI-CANCER PROPERTIES
Highlights •
A new fluorene derivative 1,1’-(9,9-dihexyl-9H-fluorene-2,7-diyl)bis(N,N-bis(pyridine2-ylmethyl)methanamine had successfully been isolated and characterized.
The synthesized compound was well characterized with nuclear magnetic resonance
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•
The synthesized compound was tested for its cytotoxicity against HeLa cell line, showing
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moderate activity with IC50 value of 28.58 μg/mL(37.76 µM).
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•
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(NMR), infrared (FTIR), UV-vis absorption and fluorescence techniques.