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AL-ANI AWATEF A IBRAHIM (IQ)
SHAHEED DHURGHAM QASIM (IQ)
AL ANI AWATEF A IBRAHIM (IQ)
SHAHEED DHURGHAM QASIM (IQ)
AL-OBAIDI ET AL.: "Synthesis Of Novel Ibuprofen-Tranexamic Acid Codrug: Estimation Of The Clinical Activity Against Hctll6 Colorectal Carcinoma Cell Line And The Determination Of Toxicity Profile Against Mdck Normal Kidney Cell Line", IJDDT, vol. 9, no. 2, 25 June 2019 (2019-06-25), XP055802333
| Claims [Claim 1] Protective Element 1 : Synthesis and evaluation of the biological activity of ibuprofen derivative (2-(4-isobutylphenyl) N-(4-methyl-3-(4-(pyridine-3-yl) pyrimidine-2-yl-amino)phenyl) propanamide) against colon and breast cancer cell-lines. [Claim 2] Referring to the first protective component, an ibuprofen derivative was prepared by 6-methyl-N1-(4-(pyridin-3-yl) pyrimidin-2-yl) benzene-1 ,3- diamine. [Claim 3] Referring to the first protective component, the half maximal inhibitory concentration (IC50) was measured on colon cancer cell lines and found to be IC50 = 476.39 pg / ml. [Claim 4] Referring to the first protective element. The inhibitory concentration of the half maximal inhibitory concentration (IC50) was measured on breast cancer cell lines and it was found to be IC50 = 11.63 pg / ml. [Claim 5] Referring to the first protective element, the half maximal inhibitory concentration (IC50) was measured on the normal cell line of the kidneys, and it was found to be ICso= 31.9 gm / ml. |
Title of invention : Synthesis, characterization, and biological evaluation of ibuprofen derivative against colon and breast cancer cell-lines
Technical Field
[0001] The field of the invention related to the molecule kinase inhibitor anticancer drug.
Background Art
[0002] A previous study showed that there is an effect of ibuprofen in preventing tumor progression by affecting one of the anti-resurfacing molecules Hsp70, as ibuprofen has been shown to be a strong inhibitor of Hsp70 by inhibiting its expression (reduce and deplete heat shock factor 1 (HSF1) in A549 cells derived from adenocarcinoma pneumonia. The decrease in Hsp70 level by ibuprofen resulted in intracellular stimulation of the drug cisplatin, with an enhanced signaling of apoptosis induced by cisplatin. Ibuprofen not only facilitated post-mitochondrial events, including cisplatin-induced caspase-9 activation induced by cisplatin, but also activation of Bax, causing the release of cytochrome c [7]
[0003] So ibuprofen accelerates cisplatin-mediated apoptosis in multiple steps of the mitochondrial apoptosis pathway via Hsp70 inhibition. This indicate that ibuprofen is a chemotherapy agent which enables cisplatin to be used in the lowest concentration, and is less toxic by designing a new combination therapy for lung cancer.
Summary of Invention
[0004] The synthesis of novel conjugate of ibuprofen with the 6-methyl-N1-(4- (pyridin-3-yl) pyrimidin-2-yl) benzene-1 ,3-diamine derivative (that mimic Imatinib which is a small molecule kinase inhibitor anticancer drug) showed a potential antiproliferative activity against both colon cancer and breast cancer cell lines.
[0005] The chemical structure was characterized and confirmed by spectral (FTIR,1 H-NMR and 13C NMR), and the Differential Scanning Colorimetry (DSC) Thermal analyzer. While the biological activity was estimated by Genetic Optimization of Ligand Docking (GOLD) software supplied by Cambridge Crystallographic Data Centre (CCDC) and conducted in vitro with the MTT and the calculation of the IC50.
[0006] The docking process was successfully performed and the chemical synthesis yield was reasonably high. The spectral interpretations show characteristic identification of the target chemical compound.
[0007] The results indicated a powerful antitumor drug against breast cancer (MCF7) and colon (HCT116) cell lines. On the other hand, to evaluate the toxicity profile, the Madin-Darby Canine Kidney (MDCK) normal (noncancerous) cell line, was utilized.
Solution to Problem
[0008] Many clinical, epidemiological and experimental studies have noted strong chemopreventive activity by non-steroidal anti-inflammatory drugs (NSAIDs).
[0009] Anticancer properties of NSAIDs related to inhibiting cyclooxygenase (COX), knowing that the doses are much higher than NSAIDs to obtain an anti-tumor effect from COX inhibition [1], indicating that they also work through Mechanisms are independent of Cox.
[0010] NSAIDs, such as aspirin, salicylate, and sulindac sulfide, showed inhibition in cell proliferation and stimulation of apoptosis in various cancer cell lines, which is an important component of their anti-tumor activity, and increase the sensitivity of cancer cells to anti-cancer drugs [2, 3,4,5] .There is currently a fact that NSAIDs are able to directly lower levels of NSAIDs molecules, such as 14-3-3, Bcl-2, family30 [6] that inhibit mitochondrial-dependent apoptosis in different cancer cells.
[0011 ] Working Methods
[0012] 1- Chemistry
[0013] Preparation of 2-(4-isobutylphenyl) N-(4-methyl-3-(4-(pyridine-3-yl) pyrimidine- 2-yl-amino) phenyl) propanamide [8-10]:
[0014] To a solution of 6-methyl-N1-(4-(pyridin-3-yl) pyrimidin-2-yl) benzene-1 , 3- diamine (compound 1) (500 mg, 1.8 mmol)) and K2CO3(1240 mg , 9 mmol) in 20 ml_ of tetrahydrofuran at freezing point, the ibuprofen acid chloride ((compound 2) (493 mg, 2.2 mmol)) was added with continued stirring at (0°C) for 0.75 hr. Add the reaction mixture drop by drop to a cooled solution of sodium bicarbonate (10%) (20 mL) while stirring at (0°C) for 0.5 hr. A yellow solid precipitated that was obtained by filtration. This yellow solid was washed with cooled water, and the hexane. After that the solid was dissolved in a methanol / dichloromethane mixture (1 :1, 10 mL) and forced to be concentrated employing vacuumed pressure.
[0015] The obtained residuum was scattered in cooled purified water (20 ml). Thereafter, triethylamine Et3N was added to this suspension and extracted with two portions of (20 ml) of ethyl acetate. The ethyl acetate extracts were joined and washed with cold water (10 ml), and concentrated under vacuum in a desiccator to obtain ((compound 3 (697 mg, 83%)) (Scheme 1). The compounds were characterized and confirmed by spectroscopic methods (FT-IR, 13 C-NMR, 1 H-NMR,) and DSC.
[0016] 2 General methods of chemical analyses
[0017] A DSC thermal analyzer was used in the characterization of the melting point of compound (3), which was observed as 125.99°C. Moreover, fourier transformed infrared (FT-IR) spectroscopy (FT-IR spectrophotometer / Shimadzu). The results of the infrared spectrum gave the following peaks (cm 1 ) and they were as follows: 1625 cm 1 (str. vib. C=0), amide and the disappearance of 3500 (str. vib. NH, NFI2).
[0018] The method for calculating protons with ( 1 FINMR- 400 MFIz spectrometer / Bruker, Switzerland) gave the following results: 0.8 d (6H, triplet) (CH3)2, 1.4 d (3H, triplet) CH 3 , 1.9 d (2H, septet) CH2, 2.1 d and 2.2 d (1 H, singlet) R2NH, 2.4 d (1 H, quartet) CFI -C=0, 6.8-7.5 d (multiplet) aromatics. 13 C-NMR; 175 d C=0 amide, 100- 160 d aromatic, 50 d CH-C=0, 40 d CH2, 30 d CH3-CH, 22 d CH (CH 3 ) 2 , 18 d (CH 3 ) .
[0019] Chemicals and Reagents:
[0020] 6-methyl-N1-(4-(pyridin-3-yl) pyrimidin-2-yl)benzene-1 ,3-diamine was purchased from BLDpharm (China), TFIF and potassium carbonate were obtained from SCR (China). The ibuprofen acid chloride was obtained from Dr. Zaid Mahdi Jaber Al-Obaidi's Medicinal Chemistry Laboratory, University of Alkafeel as a gift. While the methanol (HPLC grade) was purchased from Biosolve Chimie SARL (France). The sodium hydrogen carbonate was obtained from Himedia (India) while the triethylamine was purchased from Thermo (Canada). Finally, the ethyl acetate (HPLC-grade), n-Hexane, Dichloromethane (HPLC-grade) were purchased from GCC (UK).
[0021] Biological evaluation [0022] 1 - Cell-lines
[0023] The cell-lines utilized in this invention are listed below:
[0024] 1 - HCT 116 colorectal cancerous cells [0025] 2- MCF-7 breast cancerous cells [0026] 3- MDCK normal kidney cells
[0027] The cell-lines were supplied and gained from the International Cell Line Group of Dr. Hamid Najeh Obaid, University of Babylon. Cell lines were grown in culture medium (Gibco-BRL, RPMI-1640), with 10% thermally denaturized fetal bovine serum (FBS) (Gibco). The cell-lines were incubated into Celltreat ® 96-well cell culture plates and customized to grow at 37°C. The cell culture time scale was improved from 72 hours to just one third (i.e. 24 hours, besides; it was found that at the time of culturing 48 hours and 72 hours, all cells were killed, so they could not give reliable statistical results and are suitable for scientific research). The steps involved in the cell line portion of this work are listed below [11 ]:
[0028] 2 Preparation of MTT stock solution [12-14]:
[0029] A 25-mg of MTT powder was accurately weighed and transferred to a suitable container. Thereafter, 5 mL of DMSO was added and the MTT was entirely dissolved. This resulted in a 5-mL solution of 5 mg / mL and the solution was filtered and placed in a 12-mL centrifuge-tube using sterile filters (0.22 pm). The later tube was coated by aluminum foil to reduce the photosensitivity of the MTT solution toward the light and was kept in the refrigerator to prepare the working solution.
[0030] 3 Preparation of MTT Work Solution:
[0031] Sticking to the protocols, the concentration of MTT working solution is 500 pg / mL. This represents a 1/10 (10% v / v) of the stock solution. Consequently, to obtain a total volume of 24 ml of the cell culture medium containing 10% MTT, the next dilution was performed. An exact volume of 21.60 ml of cell medium was precisely allocated into a suitable flask. Thereafter, 2.40 mL of MTT solution were added to the medium and homogenized appropriately. The freshly prepared culture medium with 10% MTT was ready to be used in cell-lines with an incubation period of 3 h.
[0032] 4 - Preparation of the working solution of compound 3 for cell-line testing:
[0033] An appropriate amount of compound 3 was dissolved in DMSO to obtain a stock solution of 5 mg / ml_ for compound 3 and the imatinib standard. After many cell-line experiments, the highest concentration was improved to be 50 pg / ml_ by which serial dilutions were prepared for both compounds. An exact volume of 990 pi of cell culture medium was precisely measured and 10 pi of 5 mg / ml_ were added and homogenized to get the required concentration. A final concentration of 50 pg / ml and a final concentration of 1% for DMSO were obtained. Lastly, serial dilutions were performed for each compound to obtain the next specified concentrations (50, 25, 12.5, 6.25, 3.125, and 1.5625) pg / mL.
[0034] 5 - The application of the chemicals to the cell lines:
[0035] The above serially-diluted solutions were added in portions of 0.20 mL per well in triplicates and were incubated for 24 hours. After this 24hr incubation time, the 96-well plates were photographed using an inverted microscope, which was utilized to get clear screen shots for each single well. After that, the medium was replaced by 10% MTT and incubated for 3 hours [15]. This was followed by removing of the media and rinsing the wells with phosphate buffer saline (PBS). Lastly, 200 mI (dimethyl sulfoxide) was added to each well and left for a half an hour and was read at 630 nm employing a plate reader.
[0036] Study of molecular receptors:
[0037] Compound 3 was studied using a molecular receptor method on Tyrosine- protein kinase ABL1 (PDB ID 3k5v). To find out the specific receptors responsible for their effectiveness we use: the genetic optimization of ligand docking (GOLD, version: 2020.2.0) supplied by the Cambridge crystallographic data centre (CCDC) and the chemical composition of the binding proteins from the Protein Data Bank PDB, www.rcsb.org (DOI: 10.2210/pdb3K5V/pdb). Where the docking of compound (3) shows its association with the target protein in figure (1). The coalescence study showed good receptor fitness that may enhance its effect in vitro [16].
Advantageous Effects of Invention
[0038] 1- This compound has a strong binding to the receptor protein.
[0039] 2- Tyrosine-protein kinase ABL1 (PDB ID 3k5v).
[0040] 3- The compound has strong anti-cancer efficacy.
[0041] 4- It needs a small dose to be used as drug.
[0042] 5- It has no antiproliferative effect on normal kidney cells.
Brief Description of Drawings
[0043] Fig 1 : Demonstrates the interaction between Compound 3 and the protein tyrosine kinase PDB ID 3k5v
[0044] Fig 2: Percentage of viable cells versus compound 3 concentration after 24 hour incubation of colon cancer cell line - IC 50 = 476.39 pg / ml
[0045] Fig 3: Percentage of viable cells versus compound 3 concentration after 24 hour incubation of a breast cancer cell line - IC 50 = 11.63 pg/ml
[0046] Fig 4: Percentage of viable cells versus concentration of compound 3 after 24hour incubation for a normal renal cell line - IC50 = 31.9 g/ml
[0047] Fig 5: HCT 116 colorectal cancer cells - Concentrations in descending order:
50, 25, 12.5, 6.25, 3.125, 1.5625 pg / ml
[0048] Fig 6: MCF-7 breast cancer cells - Concentrations in descending order: 50, 25, 12.5, 6.25, 3.125, 1.5625 pg / ml
[0049] Fig 7: MDCK normal kidney cells - Concentrations in descending order: 50, 25, 12.5, 6.25, 3.125, 1.5625 pg / ml
Description of Embodiments
[0050] Fig 5:
[0051] 1- shows colon cancer cell line after exposure to 50 pg / ml of compound and was observed by inverted microscope after 24 hour incubation [0052] 2- shows colon cancer cell line after exposure to 25 pg / ml of compound and was observed by inverted microscope after 24 hour incubation
[0053] 3- shows the colon cancer cell line after exposure to12.5 pg/ ml of compound and was observed by the inverted microscope after 24 hour incubation
[0054] 4- shows a colon cancer cell line after exposure to 6.25 pg / ml of compound and was observed by inverted microscope after 24 hour incubation
[0055] 5- shows the colon cancer cell line after exposure to 3,125pg /ml of compound and was observed by an inverted microscope after 24 hour incubation
[0056] 6- shows a colon cancer cell line after exposure to 1 ,5625pg /ml of compound and was observed by an inverted microscope after 24 hour incubation
[0057] Fig 6:
[0058] 7- Shows the breast cancer cell line after exposure to 50 pg / ml of compound and was observed by inverted microscope after 24 hour incubation
[0059] 8- Shows the breast cancer cell line after exposure to 25pg / ml of compound and was observed by inverted microscope after 24 hour incubation
[0060] 9- shows the breast cancer cell line after exposure to 12.5 pg / ml of compound and was observed by an inverted microscope after 24 hour incubation
[0061 ] 10- shows a colon cancer cell line after exposure to 6.25pg / ml of compound and was observed by inverted microscope after 24 hour incubation
[0062] 11 - shows the breast cancer cell line after exposure to 3,125 pg / ml of compound and was observed by an inverted microscope after 24 hour incubation
[0063] 12- shows the breast cancer cell line after exposure to 1 ,5625pg / ml of compound and was observed by an inverted microscope after 24 hour incubation
[0064] Fig 7:
[0065] 13- shows the line of normal cells in the kidney after exposure to 50 pg / ml of the compound and was observed by the inverted microscope after 24 hour incubation
[0066] 14- shows the normal cells line in the kidney after exposure to 25 pg / ml of compound and was observed by inverted microscope after 24 hour incubation [0067] 15- shows the line of normal cells in the kidney after exposure to 12.5 pg / ml of the compound and was observed by the inverted microscope after 24 hour incubation
[0068] 16- shows the normal cells line in the kidney after exposure to 6.25 pg / ml of the compound and was observed by the inverted microscope after 24 hour incubation
[0069] 17- shows the normal cell line in the kidney after exposure to 3,125 pg / ml of compound and was observed by inverted microscope after 24 hours incubation
[0070] 18- shows the normal cell line in the kidney after exposure to 1 ,5625pg/ ml of compound and was observed by the inverted microscope after 24 hour incubation
Industrial Applicability
[0071] 1- This compound can be prepared as a pharmaceutical preparation.
[0072] 2- Human medical use as an anti-cancer with high quality effectiveness.
[0073] 3- Preparing the compound at a low cost compared to the currently available drugs.
Reference Signs List
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