Synthesis of BODIPY-Appended 2-(2-pyridyl) Benzimidazole Ligands for Potent Photoinduced Cytotoxicity
BODIPYs are one of the potent photosensitizers which generates singlet oxygen (1o2) for PhotoDynamic therapy (PDT). In PDT the absorption of light by a photosensitizer generates cytotoxic species such as Reactive Singlet Oxygen (ROS), leading to irreversible destruction of treated tissue. Herein, a series of BODIPY appended organic compounds and their iodinated derivatives are synthesised and well characterised which absorbs in the Green and Red regions of visible light. Further complexation of these ligands with several metals like Pt, Fe, Zn can lead to effective anticancer drugs.
Keywords: BODIPY, Photosensitizer, Photodynamic therapy (PDT), Reactive oxygen species (ROS), singlet oxygen.
|HOMO||Highest occupied molecular orbital|
|IC50||Half maximal Inhibitory concentration|
|NMR||Nuclear magnetic Resonance|
|ROS||Reactive Oxygen species|
|TLC||Thin Layer Chromatography|
Contemporary society bears the burden of cancer and a growing number of individuals die out of cancer in every year. Normal cells, after a certain number of cell divisions, would be metabolically active and mitotically inactive. Mutations in genes lead to adverse biological changes and if such alterations cannot be repaired, it would pave the way to the formation of cancerous cells. Current methodologies for cancer treatment includes surgery, chemotherapy, radiation therapy, immunotherapy and photodynamic therapy. Among the multiple therapies against cancer, photodynamic therapy (PDT) is a promising one due to its advantages of low systemic damage, non invasion and controllable characters. PDT uses a light of particular wavelength and photosensitizer which activates oxygen forming reactive species (ROS) which are lethal to cells and results in anticancer effects. Photosensitizer is a molecule capable of absorbing light energy and transferring that energy to an adjacent molecule. A wide variety of dye sensitizers have been developed for PDT. Suitable dye sensitizers for PDT are mainly, porphyrinoid compounds including chlorins, bacteriochlorins etc. Beside these, there are several non-porphyrin derivatives which has tunable photophysical and biological properties such as Anthraquinones, Xanthenes, Curcuminoids etc. One among them is BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene). Bodipy derivatives are recognized as potential candidates for dual use as bioimaging and PDT therapeutic agents. However BODIPY derivatives suffer from a low quantum yield of 1o2 . But rational design of BODIPY configuaration and incorporation of heavy atoms like iodine into its structure can improve the 1o2 quantum yield of photosensitizer. Herein ,we synthesize BODIPY conjugates of 2-(2-pyridyl) benzimidazole absorbing in green and red region.
Cancer and its characteristics
Cancer can be defined as genetic instability in a cell which results in an uncontrolled and sustained cell division. For normal cells after a certain number of cell division, they would be metabolically active and mitotically inactive. They undergo programmed cell death called apoptosis. Mutations in genes led to adverse biological changes and if such alterations cannot be repaired, it would pay way to the formation of cancerous cells.
Methodologies for cancer treatment
a) surgery: removal of cancerous cells by surgery
b) chemotherapy: use of chemical compounds capable of exerting cytotoxic effects. These chemotherapeutic agents lack selectivity and result in unwanted side effects.
c) Radiation therapy: Ionising radiations are used to destroy the cancerous cells.
d) Immunotherapy: They generate ,strengthen or stimulate immune signals to fought against cancerous cells.
e) Stem cell transplant: patient receives healthy blood forming cells to replace their own that have been destroyed by disease.
f) Hyperthermia: Infected area is regionally exposed to high temperatures
g) Photodynamic therapy: Mode of cancer treatment which uses a light of particular wavelength and photosensitizer. Photosensitizers used would activate oxygen forming reactive species (ROS) which are lethal to cells and results in anticancer effects.
Objectives of the Research
✸ Synthesis of BODIPY-Appendid 2(2-pyridyl) Benzimidazole ligands
✸ To characterize the synthesised sample via NMR, mass spectrometry and UV.
✸ Flourescence study of synthesised samples.
✸ To study the singlet oxygen generation of synthesised sample via DPBF titration.
Use of prepared samples as potent photoinduced ligands for platinum complexes. Thus it possess double targeting properties against cancerous cells by production of ROS due to the presence of ligands and intrastrand linking of DNA of cancerous cells with platinum will destruct it.
A photosensitizer is a molecule that produces a chemical change in another molecule in a photochemical process. Photosensitizers have large delocalized π system which lowers the energy of HOMO orbitals and it’s absorption of light might be able to ionize the molecule. The way it works in cancer treatment is described below.
Photosensitizing agent is injecting into the bloodstream accumulates in cancerous cells. Tumour is exposed to light ,photosensitizer get excited to higher singlet state. This in turn converted to triplet state. The triplet state can either interact directly with ground state triplet oxygen (type 2) which gives singlet oxygen species or the excited photosensitizer accept electron from adjacent molecule forming a radical anion (type 1) which on reaction with oxygen gives superoxide anion, hydroxyl radicals which ultimately causes the damage of cells.
Properties of an ideal photosensitizer in PDT
✸ Selectivity for tumours
✸ Short time interval between administration and maximal accumulation within tumour tissues.
✸ Low skin photosensitivity and pain after irradiation.
✸ Low dark toxicity
✸ High efficiency of crossing from singlet to triplet state
Potency of a photosensitizer is determined by IC50 values in light and dark conditions. It is given by phototoxic index
Phototoxic index =IC50 (light) /IC50 (dark)
High value of phototoxic index is favourable. Most essential feature of PDT agent is to have less or no dark toxicity.
Photosensitizers can widely classified into five classes.
1. porphyrins (eg: photophrin)
5. non-porphyrin derivatives
Majority of PDT agents are cyclic tetrapyrolles . To modulate photophysical and biological properties are correspondingly difficult for them. So this is the interest for non-porphyrin photosensitizers. One among them is BODIPY.
Dipyrromethene is complexed with a disubstituted boron center.
✸ Small stoke’s shift.
✸ Relatively chemically inert.
✸ High Environment Independent ,fluorescence Quantum yield.
Pyrolle which is synthesised from knorr-pyrolle synthesis is treated with carboxylic acid derivative (RCOX) to obtain dipyromethene . This dipyrromethene precursor react with boron triflouride etherate in the presence of tertiary amine gives BODIPY.
Significance of BODIPY in PDT
To use BODIPY as PDT agent firstly high quantum yield of fluorescence to be reduced and enhance singlet to triplet inter system crossing. Halogenated BODIPY would meet this requisite. Spin-orbit coupling of heavy atoms is the reason for enhancing the population of triplet BODIPY. High oxidation potential of halogen would help BODIPY from self oxidation.
Among the halogens used the iodine atom shows highest efficiency than the bromine atom does in most cases. It has 1o2 Quantum yield of 73% and lowest half minimal inhibitory concentration (IC50).Besides these BODIPY (without quenching fluorescence) can act as a good biosensor.
Techniques used for Experimental section
Thin layer chromatography
Chromatography is used to separate substances from a mixture. They all have a stationary and mobile phase. Components are separated on the basis of polarity differences. All kind of chromatographic technique has a stationary phase and mobile phase. In TLC uniform layer of silica gel or alumina on a piece of glass ,metal or plastic is coated serve as stationary phase. The mobile phase is suitable liquid solvent or mixtures of solvent. A pencil line is drawn near the bottom of the plate and a small drop of a solution of the dye mixture is placed on it. When the spot of mixture is dry, the plate is stood in a shallow layer of solvent in a covered beaker. It is important that the solvent level is below the line with the spot on it.
After the sample has been applied on the plate ,the eluent which is mobile phase is drawn up the plate via capillary action. The solvent level must be below the base line. The components moves with different rates because of their different degrees of interaction with mobile and stationary phase. The solvent allowed to rise until it reaches the top. That will give maximum separation of components for this particular combination of solvent and stationary phase.
Measuring Rf values
To find out what all are the components present in the mixture we have to measure Rf values. When the solvent front gets close to the top of the plate, the plate is removed from the beaker and the position of the solvent is marked with another line before it has a chance to evaporate. Rf value is given by the formula as follows.
Rf = Distance travelled by the component (from base line)/Distance travelled by the solvent front (from base line)
Column chromatography works on a much larger scale by packing the same materials into a vertical glass column. The separation is based on the differential adsorption of the components to the adsorbent. compounds move through the column at different rates and separated into fractions. The base of column tube contains a filter or cotton to ensure that adsorbent does not wash away. Adsorbent used in the column is silica which is made to a slurry using any suitable solvent. The silica is allowed to settled well and the side of column is tapped with a rubber stopper or tubing which helps the silica gel to settle uniform . Ensure that the column is always wet. The mixture is added into the column and eluent (mixture of suitable solvents) is poured into the column to transfer it into silica gel .Once the sample mixture is rinsed into silica ,the eluent is added carefully to the column. The eluent which is collected just prior to elution of sample can be reused. The composition of the eluent can be changed can be changed as the column progresses. The addition of eluent starts from least polar to more molar. Individual components in the sample separate from each other and the eluent along with our component are collected as a series of fractions.
Each fraction is analysed by TLC whether the fraction contains more than one component and it’s purity level.
Some other techniques like rotary evaporation , solvent distillation and reflux condenser are also used.
Techniques used for characterisation
Nuclear magnetic Resonance spectroscopy
Nuclear magnetic Resonance (NMR) spectroscopy is an analytical technique used to determine the content and purity of a sample as well as its molecular structure. It is applicable to any kind sample containing nuclei possessing spin. The most common types of NMR are 1H (proton) and 13C NMR. This technique measures the local magnetic field around the nuclei. An external magnetic field applied will split the degenerate spin states of the nuclei in the sample and excitation of nuclei will takes place from lower to higher states with radio waves. Those nuclei with high shielding effect require low energy of radio waves for resonance and nuclei has low shielding effect require high energy radio waves for resonance. These radio waves are detected by sensitive radio receivers which is helpful to interpret the sample.
The sample that is taken for analysis of NMR spectroscopy must dissolve in the taken solvent. Most of the solvents contain hydrogen and the signals due to these protons interfere with the protons in the sample. Hence deuterated solvents like CDCl3 ,dimethyl sulphoxide (DMSO d6) are used.
UV Visible spectroscopy
Absorption of UV Visible light radiation is associated with excitation of electrons , in both atoms and molecules, from lower to higher energy levels. The larger the energy gap between the energy levels ,greater the energy required to promote the electron into higher energy state. The absorption of UV Visible region corresponds to electronic transitions from n (lone pair) →π *(antibonding) and π(bonding)→ π* orbitals. As the amount of delocalisation in the molecule increases the energy gap between π bonding orbitals and π antibonding orbitals decreases therefore light of lower energy or lower wavelength is absorbed.
UV visible spectrometers can be used to measure the absorbance of ultraviolet or visible light by a sample , either at single wavelength or perform a scan over a range in the spectrum.
Mass spectroscopy is an analytical technique which ionizes the chemical species and sorts the ions on the basis of charge to mass ratio. In simpler terms it measures the mass of a given sample. In this technique a sample which may be solid, liquid or gas is bombarded with electrons and get fragmented ions of parent sample (usually unipositive ions). These ions are accelerated and allowed to move through a magnetic field. Different ions are deflected by magnetic field by different amount. The amount of deflection depends on:
✸ The mass of the ion. Lighter ions are deflected most.
✸ Charge on the ion.
These two factors are combined into mass/charge ratio. Ions with same mass to charge ratio will undergo same amount of deflection. These ions are detected by a mechanism capable of detecting the charged particles such as electron amplifier. Results were displayed as spectra of relative abundance of detected ions as a function of mass to charge ratio. Mass of the parent sample is correlated with the mass of fragmented ions.
Fluorescence spectroscopy is a type of electromagnetic spectroscopy which is used to determine the concentration of an analyte in solution based on its fluorescent properties. In fluorescence spectroscopy, a beam with a wavelength varying between 180 and ∼800 nm is allowed to pass through a solution in a cuvette. The light that is emitted by the sample is measured from an angle. In fluorescence spectrometry both an excitation spectrum (the light that is absorbed by the sample) and/or an emission spectrum (the light emitted by the sample) can be measured. The concentration of the analyte is directly proportional with the intensity of the emission.
Procedure: In fluorescence, the species is first excited by absorbing a photon from its ground state to any of its excited states. When it de-excites back to its ground state, collisions with other molecules cause the excited molecules to lose vibrational energy until it reaches the lowest energy ground state. The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. As molecules may drop down into any of several vibrational levels in the ground state, the emitted photons will have different energies, and thus frequencies. Therefore, by analyzing the different frequencies of light emitted in fluorescent spectroscopy, along with their relative intensities, the structure of the different vibrational levels can be determined.
In PDT, efficiency of photosensitizer is measured on the basis of singlet oxygen quantum yield. It measures the ability of photosensitizer to convert ground state oxygen to singlet oxygen. The ΦΔ is typically described as the number of molecules of singlet oxygen generated per number of photons absorbed by the sensitizer. A number of different techniques for the determination of measurement of this efficiency have been developed. The two most prevalent indirect methods includes chemical trapping and o2 consumption methods. A number of 1o2 chemical traps have been investigated for use in singlet oxygen studies.
One of the most well known compounds is 1,3-diphenylisobenzofuran (DPBF). The disappearence of DPBF and the formation of the product o-dibenzoylbenzene due to its reaction with 1o2 can be monitored by absortion or fluorescence spectroscopically. It is a good acceptor because it reacts with 1o2 ,it does not react with ground state molecular oxygen nor with superoxide anion, and its only reaction with 1o2 is a chemical one.
To determine the quantum yields for singlet-oxygen generation solutions were made in DMF. The photo-oxidation of DPBF sensitized by the compounds was monitored. The absorption spectra of DPBF were recorded after each photo-irradiation for 10 secs. Low concentrations were taken to minimize the possibility of singlet oxygen quenching by the dye, for the measurement of singlet oxygen quantum yield. The quantum yields of singlet oxygen generation (Φ[1O2]) were calculated by using a relative method with optically matched solutions and by comparing the quantum yield of the photo oxidation of DPBF that was sensitized by the compound of interest to the quantum yield of Rose Bengal (RB) (Φ[1O2]=0.76 in DMF) as a reference compound according to equation (1), where subscripts “c” and “RB” denotes the complex and Rose Bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein),which is a fluorescent stain, respectively, ΦΔ is the quantum yield of singlet oxygen, “m” is the slope of a plot with a difference in the change in the absorbance of DPBF (at 415 nm) with the irradiation time, and “F” is the absorption correction factor, which is given by F=1–10-OD, where OD is the optical density at the irradiation wavelength.
ΦΔc = ΦΔRB x (mc/mRB) x (FRB/Fc) ---- equation (1)
For the measurement of the slope and the OD, the absorption data is plotted in Origin.
Synthesis of PBI-GB
|Substrate||Molecular weight (g/mol)||No. of moles||No. of equivalents||Weight taken/volume|
|2,4-dimethyl pyrrole||95.14||19.42×10-3||2.10||2 ml|
|4-chloromethyl benzoyl chloride||189.04||9.25×10-3||1.00||1748 mg|
|Triethyl amine||101.19||54.39×10-3||5.88||5.6 ml|
|Boron trifluoride etherate (BF3Et2O)||141.93||58.28×10-3||6.30||6 ml|
1748 mg of 4-chloromethyl benzoyl chloride along with 150 ml of dry Dichloromethane (P2O5 distilled) were taken in a two necked RB. Added 2 ml of pyrrole through one neck of the RB which is covered with septum. Reflux the mixture at 45οC for 3 hours in N2 atmosphere. Added 5.6 ml of Triethyl amine and 6 ml of BF3.Et2O to the mixture and refluxed it for 1 hr.
Work up: Washed the reaction mixture with water followed by brine solution in a separating funnel. Dried it over sodium sulphate and filtered it. Purified the compound using column chromatography with solvent Dichloromethane in hexane and collect appropriate band of BOD Cl.
|Substrate||Molecular weight (g/mol)||No. of moles||No. of equivalents||weight taken/volume|
|Potassium carbonate||138.205||1.07×10-3||2||148.43 mg|
|2-(2-pyridyl) benzimidazole||195.220||5.37×10-4||1||104.80 mg|
|Potassium Iodide||166.003||1.07×10-3||2||178.28 mg|
Potassium iodide and potassium carbonate in minimum amount of water were dissolved in 100 ml of RB. 1 equivalent of BOD-Cl and 1 equivalent of 2-(2-pyridyl) benzimidazole was added to RB. 40 ml of acetonitrile was added.The mixture was refluxed for 24 hrs at 85Ο c.
Work up: solvent was evaporated using rota -vac. Dark green solid was dissolved in Dichloromethane and washed with water in separatory funnel. Dichloromethane layer was collected and again washed with brine solution. Lower layer of DCM was collected in a beaker containing sodium sulphate .The solution was filtered and concentrated using rota -vac.
The solid was dissolved in minimum amount of DCM and silica gel was added to it and kept for drying. The desired product PBI-GB is purified by column packed in Hexane. The product is eluted by 60-70 % DCM in Hexane. Dark green product is collected.
Characterisation data of PBI-GB
i) NMR Spectra
ii) Mass Spectra
iii) UV-Visible Spectra
iv) Fluorescence Spectra
Synthesis of PBI-GBI
|Substrate||Molecular weight (g/mol)||No .of moles||No. of equivalents||Weight taken/volume|
|N-Iodosuccinimide (NIS)||224.985||6.78×10-3||6||1530 mg|
1 Equivalent PBI-GB was taken in 100 ml dry Round bottom flask and dissolved in 50 ml of deoxygenated Dichloromethane and 6 equivalent N-Iodo succinimide (NIS) was stirred at room temperature until full consumption of PBI-GB was found by TLC.
Work up: The reaction mixture was washed with water, dried over anhydrous sodium sulphate ,filtered and the solvent was removed under reduced pressure
Characterisation data of PBI-GBI
i) NMR Spectra
ii) Mass Spectra
iii) UV- Visible Spectra
iv) Fluorescence Spectra
Synthesis of PBI-RB
|S.No.||Substrate||Molecular weight (g/ml)||No. of moles||No.of equivalents||Weight/volume taken|
|2||N,N-Dimethylamino Benzaldehyde||149.1930||5.69×10-4||1||85 mg|
1 equivalent of PBI-GB and 1 equivalent of N,N-Dimethyl aminobenzaldehyde were taken in 250 ml round bottom flask.Dean strack with sodium sulphate was attached to condenser and round bottom flask.Added 50 ml of dry toluene (Na distilled) to the mixture. Added 1 ml of glacial acetic acid and 1ml of piperidine. Refluxed the reaction mixture for 4 hrs.
work up: Evaporated toluene and dissolved the crude product in Dichloromethane. Washed it with distilled water and Brine solution. Dried it over sodium sulphate. Evaporated Dichloromethane using Rotavac.
Characterisation data of PBI-RB
i) NMR Spectra
ii) Mass Spectra
iii) UV-Visible Spectra
iv) Fluorescence Spectra
Synthesis of PBI-RBI
|Substrate||Molecular weight (g/ml)||No. of moles||No.of equivalents||Weight/volume taken|
|N,N-Dimethylamino Benzaldehyde||149.193||8.299×10-4||1||123.81 mg|
1 equivalent of PBI-GBI and 1 equivalent of N,N-Dimethyl aminobenzaldehyde were taken in 250 ml round bottom flask.Dean strack with sodium sulphate was attached to condenser and round bottom flask.Added 50 ml of dry toluene to the mixture. Added 1 ml of glacial acetic acid and 1ml of piperidine. Refluxed the reaction mixture for 4 hrs.
work up: Evaporated toluene and dissolved the crude product in Dichloromethane. Washed it with distilled water and Brine solution. Dried it over sodium sulphate .Evaporated Dichloromethane using Rotavac.
Characterisation data of PBI-RBI
i) NMR Spectra
ii) Mass Spectra
iii) UV-Visible spectra
vi) Fluorescence Spectra
In this project, Four Bodipy- appended -Benzimidazole ligands were synthesized and characterised using 1H NMR, 13C NMR and mass spectroscopy. The photophysical properties of these ligands were studied using UV-Visible and emission spectroscopy. Various laboratory techniques like column chromatography, thin layer chromatography, solvent distillation and software applications like Origin, Topspin, and Chemdraw were learnt and used. The complexation of these ligands with metals like Pt, Fe, Zn could lead to the generation of new metal based anti tumour drugs for PDT agents. The characterisation of metal complexes and in vitro studies need to be done in order to evaluate their effectiveness as anticancer agents.
1) Jianhua Zou, Zhihui Yin, Kaikai Ding, Qianyun Tang, Jiewei Li, Weili Si, Jinjun Shao, Qi Zhang, Wei Huang, Xiaochen Dong, 2017, BODIPY Derivatives for Photodynamic Therapy: Influence of Configuration versus Heavy Atom Effect, ACS Applied Materials & Interfaces, vol. 9, no. 38, pp. 32475-32481
2) Dennis E.J.G.J. Dolmans, Dai Fukumura, Rakesh K. Jain, 2003, Photodynamic therapy for cancer, Nature Reviews Cancer, vol. 3, no. 5, pp. 380-387
3) Anyanee Kamkaew, Siang Hui Lim, Hong Boon Lee, Lik Voon Kiew, Lip Yong Chung, Kevin Burgess, 2013, ChemInform Abstract: BODIPY Dyes in Photodynamic Therapy, ChemInform, vol. 42, no. 18, pp. 77-88
4) Vanitha Ramu, Srishti Gautam, Aditya Garai, Paturu Kondaiah, Akhil R. Chakravarty, 2018, Glucose-Appended Platinum(II)-BODIPY Conjugates for Targeted Photodynamic Therapy in Red Light, Inorganic Chemistry, vol. 57, no. 4, pp. 1717-1726
5) Koushambi Mitra, Srishti Gautam, Paturu Kondaiah, Akhil R. Chakravarty, 2016, BODIPY-Appended 2-(2-Pyridyl)benzimidazole Platinum(II) Catecholates for Mitochondria-Targeted Photocytotoxicity, ChemMedChem, vol. 11, no. 17, pp. 1956-1967
I sincerely acknowledge the Indian Academy of Sciences, Bangalore for offering me the summer research fellowship programme-2019 in the Indian institute of science, Bangalore.
I extend my gratitude to almighty who gave blessings to complete this project successfully. With immense pleasure I acknowledge the Inorganic and physical chemistry department, Indian institute of science, Bangalore for providing me a platform for my scientific work. I sincerely thank my guide, Prof. Akhil R. Chakravarty for giving me an opportunity to work in my area of interest.
I extend my special gratitude to my co-guide Ms. Aarti Upadhyay for her guidance and support to complete my project.
I express my thanks to my labmates and all of my summer research friends for their encouragement. Finally I acknowledge my family for their prayers and wishes.