{"id":53922,"date":"2023-12-31T11:40:15","date_gmt":"2023-12-31T11:40:15","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=53922"},"modified":"2024-01-05T06:09:41","modified_gmt":"2024-01-05T06:09:41","slug":"synthesis-characterization-and-mechanistic-anticancer-evaluation-of-novel-analogues-of-pyrazoles-derived-from-substituted-3-acetyl-coumarins","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no4\/synthesis-characterization-and-mechanistic-anticancer-evaluation-of-novel-analogues-of-pyrazoles-derived-from-substituted-3-acetyl-coumarins\/","title":{"rendered":"Synthesis, Characterization and Mechanistic Anticancer Evaluation of Novel Analogues of Pyrazoles Derived from Substituted 3-Acetyl Coumarins."},"content":{"rendered":"\n

Introduction <\/strong><\/p>\n\n\n\n

Various\nillnesses with the potential to infiltrate or spread to different body parts\ninclude cancer, which is a category of diseases characterized by abnormal cell\nproliferation. About 90-95% of cancers are due to genetic mutations, which transform\nnormal cell into malignant cells.1<\/sup>Cancer cells acquire a degree of\nautonomy from mutations of tumor suppressor gene, resulting in uncontrolled\ncell growth and its proliferation.<\/p>\n\n\n\n

According to the\nWHO study 2020, cancer cases found to be 18 million and 10 million mortalities\nworldwide in 2018 and by 2040, the global prevalence is projected to double, to\n29-37 million new cancer cases2<\/sup>. It is also a big health concern\nthat needs to be tackled. The development of new anticancer therapeutics is one\nof medicinal chemistry’s top priorities because cancer accounts for roughly 70%\nof all fatalities. Because of the high demand for anticancer drugs, medicinal\nchemistry researchers have focused their efforts on the chemistry and biology\nof new anticancer agents3<\/sup>.<\/p>\n\n\n\n

In recent years anticancer agents derived from natural products have gained the attention of researchers due to their wide range of therapeutic activity4<\/sup>. Coumarin, a versatile phenolic nucleus consisting of \u03b1 -pyrone ring fused to a benzene ring that occurs naturally. Vogel first isolated it from tonka beans in 1820. Since tonka beans are rich in coumarins, the name came from the French word Coumarou. Coumarin-based compounds (Figure 1), both natural and synthetic, have anti-inflammatory, antibacterial, antiviral, antioxidant, and anticancer effects5<\/sup>.<\/p>\n\n\n\n\n\n
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Figure 1: Basic ring structure of Coumarin<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Along with coumarin, pyrazole analogs are\nfound to possess anticancer antioxidant and anticancer activity6<\/sup>. In\nthe last decade pyrazole gained much interest of researchers since the its\nstructure is frequently found as active ingredient in commercial drugs. <\/p>\n\n\n\n

Because of its wide variety of biological activities, Pyrazole and its derivatives have attracted a lot of interest in recent decades7<\/sup>. Molecular modeling  is used by thousands of researchers to design novel pyrazole analogues that can target cancer-related receptors such as protein kinase, tyrosine kinase, and vascular endothelial growth factor (VEGF)8<\/sup>.<\/p>\n\n\n\n

Pyrazole, also called as 1,2 diazoles, contains three carbon atom and two nitrogen atoms present adjacent to each other which was denoted by the molecular formula C3<\/sub>H4<\/sub>N2<\/sub>. The pKb value for pyrazole is 11.5 which explains the weak basic nature of the ring and pKa of conjugated acid of pyrazole was found to be 2.49 at 25\u00b0C. Ludwig Knorr invented the word pyrazole in 1883. The Pyrazole ring itself possesses therapeutic anticancer, analgesic, anti-inflammatory, antioxidant, antimicrobial, anticonvulsant, antiviral etc. activities (Figure-2)9<\/sup>.<\/p>\n\n\n\n\n\n
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Figure 2: Therapeutic properties of pyrazole nucleus.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Ruxolitinib (blood cancer), Axitinib\n(renal cancer), Crizotinib (lung cancer), and other authorized anticancer\nmedications all include the pyrazole ring as their fundamental core structure10<\/sup>\n.<\/p>\n\n\n\n

With this background, it was believed to\nsynthesis some novel pyrazole analogues from substituted 3-Acetyl coumarin and\ntest for antioxidant and antitumor activity.<\/p>\n\n\n\n

Material and methods<\/strong><\/p>\n\n\n\n

Chemicals and analytical instruments <\/strong><\/p>\n\n\n\n

All the chemicals and\nreagents used in this investigation were from TCI and Sigma Aldrich. Using\nCDCl3, DMSO as the solvent, tetramethyl silane as an internal standard, and a\nBruker Avance II spectrophotometer, the spectra for 1<\/sup>H NMR and 13<\/sup>C\nNMR were obtained. The units used to report chemical changes were parts per\nmillion (ppm). Waters LCMS equipment were used to obtain all mass spectra.\nMelting points were also measured using an Electro Thermal 9100 tool without\nany post-processing.<\/p>\n\n\n\n

 The infrared spectra were captured using a Shimadzu spectrometer, and the absorptions were measured using a wave number (cm-1) scale that spanned from 400 to 4000 cm-1.The synthesis plan for the target compounds is shown in Figure 3.<\/p>\n\n\n\n\n\n
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Figure 3: General scheme for Coumarinyl pyrazole carbaldehyde synthesis.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

General procedure<\/strong><\/p>\n\n\n\n

Step 1 (Preparation of 3- acetyl coumarin):<\/strong><\/p>\n\n\n\n

Drop by drop, with constant\nstirring for five minutes, several salicylaldehyde derivatives each containing\none equivalent (0.08 mole) of ethyl acetoacetate were added to the cool\nsolution. About 5-6 drops of piperidine, the catalytic quantity, were added dropwise\nto the reaction mixture. Stirring continued for 6 to 48 hours. TLC was used to\nmonitor the reaction’s completion while utilising a 3:2 n-hexane:ethyl acetate\nsolvent solution. The methanol-based solvent was extinguished when the reaction\nwas finished, and the reaction mixture was then poured over crushed ice.\nPurification was achieved by recrystallizing the obtained precipitates in\ntoluene after filtering them out to obtain the crude product.<\/p>\n\n\n\n

Step 2 (Preparation of Coumarinyl hydrazones).<\/strong><\/p>\n\n\n\n

One equivalent of the\n3-acetyl coumarin derivatives (Product I) was put to a round-bottomed flask and\ndissolved in 10 ml of glacial acetic acid. With constant stirring, one\nequivalent of methanol-dissolved phenyl hydrazine derivatives was added to the\nsolution. Using the solvent system n-hexane: ethyl acetate 3:2, stirring was\ncontinued for 1-2 hours until the reaction was complete. Orange-colored\nprecipitates were seen after the reaction mixture was placed onto crushed ice.\nTo achieve a pure product, the precipitates were filtered and washed with\nmethanol.<\/p>\n\n\n\n

Step 3 (Preparation of Coumarinyl pyrazole carbaldehyde\u2019s)<\/strong>11<\/sup><\/p>\n\n\n\n

POCl3 (5ml) was added dropwise while stirring to a 25ml cold solution of DMF to create the Vilsmeier reagent. 30 minutes later, 5 mmo1 of Coumarinyl pyrazoles were added, portion by portion, and stirred continuously for 24 hours. After the reaction was finished, the liquid was poured over crushed ice, where yellow solid precipitates were seen. The mixture was then neutralised with a strong solution of NaOH. Precipitates were obtained, filtered to yield crude product, and refined using column chromatography with a ratio of 7:3 n-hexane to ethyl acetate.<\/p>\n\n\n\n

Characterization <\/strong><\/p>\n\n\n\n

The\nsynthesized compounds were examined using physical and spectroscopic techniques. Spectroscopic characterization was performed using\nUV, IR, MASS and NMR spectroscopic methods12<\/sup>. UV spectroscopy helps\nin identifying conjugation in molecules as conjugation is directly linked to UV\nabsorption. The infrared spectroscopy helps in identifying the various\nfunctional groups present in molecules with specific bending or stretching\nvibrations. <\/p>\n\n\n\n

The mass spectrum identifies the molecular ion peak\nthat further helps in identifying the molecular weight of any sample molecules.\nNMR spectrum identifies the various type of magnetically equivalent protons\npresent in the given structure. Through the spectral assignment, one can deduce\nthe structure of a given organic molecule. <\/p>\n\n\n\n

Anticancer\nactivity using MTT assay method using A-549(Lung cancer cell lines) <\/strong><\/p>\n\n\n\n

 <\/sup>According to the established protocol described in\nthe literature, the MTT assay was carried out13<\/sup>. The cells were\nseeded on the 96 well plates along with media and test solution. Cell cultures’\nmedia should be discarded. Aspirate the media slowly to check for adhering\ncells. For suspension cells, spin the 96-well plate at 1,000 x g at 4 \u00b0C for 5\nminutes in a centrifuge that is compatible with microplates, then carefully\naspirate the media. Each well should include 50 mL of serum-free medium and 50\nmL of MTT solution. The plate should be incubated for three hours at 37 \u00b0C.\nAfter incubation, pour 150\u00b5L\nof MTT solvent into each well. The plate should be covered with foil and shook for\n15 minutes using an orbital shaker. To completely dissolve the MTT formazan,\nthe liquid might occasionally need to be pipetted. Read the absorbance at\nOD=590 nm.<\/p>\n\n\n\n

PI Annexin V-FITC labelling for A549 cell\napoptosis detection by flow cytometry.<\/strong><\/p>\n\n\n\n

According to the established protocol described in\nthe literature, the apoptosis detection was carried out14<\/sup>.<\/p>\n\n\n\n

Stepwise procedure<\/strong><\/p>\n\n\n\n

On the day before apoptosis was induced, 1 X106<\/sup> cells per well for a 6-well plate were seeded using medium containing 10% FBS and 1% Pen Strep, respectively. These cells were then incubated overnight at 37\u00b0C with 5% CO2.<\/p>\n\n\n\n

Test solutions in medium containing 10% FBS were substituted for the original media.<\/p>\n\n\n\n

Under standard culture conditions, the treated cells were incubated for 24 hours.<\/p>\n\n\n\n

After the cells were removed from the wells, the entire contents were transferred to the sterile FACS tubes.<\/p>\n\n\n\n

The supernatant from the centrifugation of the cell contents at 2000 rpm for 5 minutes was discarded.<\/p>\n\n\n\n

After centrifugation, rinsed the cells twice with cold PBS before resuspending them in 1 mL of 1X Binding Buffer at a concentration of approximately 1 x 106<\/sup> cells\/mL. <\/p>\n\n\n\n

Transfer 500 mL (5 x 105<\/sup> cells) of the cell suspension to a fresh FACS tube.<\/p>\n\n\n\n

The tubes were filled with 8. 5 l Annexin V and 10 LPI, and the cells were then gently mixed before being incubated for 20 minutes at RT in the dark.<\/p>\n\n\n\n

As quickly as feasible (within an hour), flow cytometry was used to analyze the cells.<\/p>\n\n\n\n

Cell\nCycle studies using A549 cells.<\/strong><\/p>\n\n\n\n

According to the established protocol described in\nthe literature, the cell cycle analysis was carried out15<\/sup>.<\/p>\n\n\n\n

 Procedure\n<\/strong><\/p>\n\n\n\n

In a 6-well plate with 2 ml of medium, 1 x 106<\/sup>\ncells were planted and grown for 24 hours. Following that, cells were given the\nappropriate concentrations of the specified samples, prepared in medium, and\ncultured for an additional 24 hours. The cells were then collected, centrifuged\nfor 5 minutes at room temperature at 2000 rpm, and the supernatant carefully\ndiscarded while still holding the cell pellet. After resuspending the cell\npellet in 2mL of 1XPBS, it was cleaned. \nAnother time, the washing was done under the same circumstances. The\nparticulate was kept in the supernatant, which was discarded. After\nresuspending the cells in 300 l of Sheath fluid, 1 mL of cold 70% EtOH was\nadded drop by drop while being continuously gently shaken, and a final 1 mL of\nchilled 70% EtOH was added slowly all at once. The cells were then kept at 4 \u00b0C either overnight or\nfor 30 minutes. The cells were centrifuged at 2000 rpm for 5 minutes after\nfixation. 2 ml of cold 1XPBS was used to wash the cell pellet twice. After\nthat, the cell pellet was resuspended in 500 l of sheath fluid that included\n0.05 mg\/ml of PI and 0.05 mg\/ml of RNaseA, and it was left to work for 20\nminutes in the dark. Using FACS Calibre (BD Biosciences, San Jose, CA), it was\npossible to compare populations treated and untreated with drugs to assess the\nproportion of cells at different stages of the cell cycle.<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

Synthesis<\/strong><\/p>\n\n\n\n

The target compounds were synthesized in 3 steps via formation of variety of intermediates. In the step 1, 3-acetyl coumarin was synthesized from the reaction of various substituted salicyl aldehydes and ethyl acetoacetate via Knoevenagel condensation. In the step 2, Coumarinyl hydrazones were synthesized by the nucleophilic addition of substituted phenyl hydrazine to 3-acetyl coumarin. In the final cyclization step, Vilsmeier Haack reaction was utilized for the preparation of various substituted Coumarinyl pyrazole derivatives. The structures and important physical properties for the synthesized compounds were presented in the table 1.<\/p>\n\n\n\n\n\n
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Table 1: Physico-chemical properties of synthesized test compounds<\/strong><\/p>\n

<\/p>\n

Click here to view Table<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Characterization<\/strong><\/p>\n\n\n\n

The\nsynthesized test compounds were characterized by various spectroscopic methods\nincluding UV, IR, MASS, and NMR spectroscopic methods16<\/sup>.<\/p>\n\n\n\n

In\norganic chemistry, the presence of free electrons or double (pi) bonds within a\nmolecule can be determined using the UV\/Visible spectroscopy approach. The term\n“Lamda-max” refers to the wavelength that a molecule absorbs most of,\ntherefore it is possible to compare several compounds using this value17<\/sup>.<\/p>\n\n\n\n

The\nlambda max of all the 8 synthesized coumarin-pyrazole carbaldehydes were\npresented in Table 2.      <\/p>\n\n\n\n

The UV spectra of one of the representative compound P-01 was presented in the Figure 4a.<\/p>\n\n\n\n

Table 2: \u03bb-max of synthesized compounds <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Code <\/strong><\/p>\n<\/td>\n

\n

\u03bbmax(nm)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

P-01<\/p>\n<\/td>\n

\n

201.50<\/p>\n<\/td>\n<\/tr>\n

\n

P-02<\/p>\n<\/td>\n

\n

203.50<\/p>\n<\/td>\n<\/tr>\n

\n

P-03<\/p>\n<\/td>\n

\n

206.50<\/p>\n<\/td>\n<\/tr>\n

\n

P-04<\/p>\n<\/td>\n

\n

209.50<\/p>\n<\/td>\n<\/tr>\n

\n

P-06<\/p>\n<\/td>\n

\n

202<\/p>\n<\/td>\n<\/tr>\n

\n

P-07<\/p>\n<\/td>\n

\n

219<\/p>\n<\/td>\n<\/tr>\n

\n

P-08<\/p>\n<\/td>\n

\n

219<\/p>\n<\/td>\n<\/tr>\n

\n

P-09<\/p>\n<\/td>\n

\n

215<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
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Figure 4a: UV Spectra of  the compound P-01<\/strong><\/p>\n

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Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

IR\nspectroscopic analysis<\/strong><\/p>\n\n\n\n

IR\nspectra is useful to determine the functional groups present in the sample.\nFingerprint region16<\/sup> of IR spectra is unique for each compound since\ndifferent compounds have different natural frequencies of vibrations, no two\norganic compounds will produce a similar spectrum.<\/p>\n\n\n\n

The IR spectra for the representative compound P-01 is given in Figure 4b, the IR functional group values are provided in Table 3.  <\/p>\n\n\n\n\n\n
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Figure 4b: IR spectra for the synthesized test compounds<\/strong><\/p>\n

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Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Table 3: IR functional group frequency values for the synthesized compounds <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n
\n

Functional groups<\/strong><\/p>\n<\/td>\n

\n

IR values (cm<\/strong>-1<\/strong>)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

C=O<\/p>\n<\/td>\n

\n

1724.36<\/p>\n<\/td>\n<\/tr>\n

\n

Aldehydic C=O<\/p>\n<\/td>\n

\n

1685.79<\/p>\n<\/td>\n<\/tr>\n

\n

C=N<\/p>\n<\/td>\n

\n

1608.63 and 2357.01<\/p>\n<\/td>\n<\/tr>\n

\n

Aldehydic CH<\/p>\n<\/td>\n

\n

2781.35 and 2856.58<\/p>\n<\/td>\n<\/tr>\n

\n

C=C<\/p>\n<\/td>\n

\n

1527.62<\/p>\n<\/td>\n<\/tr>\n

\n

Aromatic CH<\/p>\n<\/td>\n

\n

3055.24<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

MASS Spectrometric\nanalysis <\/strong><\/p>\n\n\n\n

The LC-MS technique makes use of HPLC to\nisolate individual components from the mixture, followed by ionization and\naccording to mass\/charge ration ions are separated, directed into detector\nwhich recognizes and quantifies each ion18,19<\/sup>.<\/p>\n\n\n\n

The popular Atmospheric Pressure Chemical\nionization is the ion source used in LC-MS to generate ions from intact\nmolecules. Since the LC-MS technique is precise, specific, sensitive the\nanalysis is made at molecular level, it is easy to figure out structural\ndetails of the injected analyte.   <\/p>\n\n\n\n

The LCMS spectra for the representative\ncompound P-01 is represented in Figure 5.<\/p>\n\n\n\n

LC-MS (ITMS + cAPCI corona Full ms): Calculated for C19H12N2O3 [M+H]+ 317.32, found 317.11<\/p>\n\n\n\n\n\n
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Figure 5: Mass spectrum of the representative compound. <\/strong><\/p>\n

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Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

NMR spectroscopic analysis<\/strong><\/p>\n\n\n\n

NMR helps us by providing the information about different\nmagnetically distinct atoms of the provided be it hydrogen or carbon. Also, it\ngives brief idea about the nature of immediate environment of each proton. So,\nit helps in determining the structure of the compound.<\/p>\n\n\n\n

In the 1<\/sup>H NMR of the representative compound\nP-01 given below the characteristic peaks20<\/sup>. corresponding to\nprotons were as follows <\/p>\n\n\n\n

1<\/sup>H NMR (400MHz, CDCl3, \u03b4 in ppm): 10.061(1H, s, CHO),\n8.525(1H, s, CH-pyrazole), 8.223(1H, s, CH of pyrone ring), 7.26-7.77 (9H, m,\nAr-H)<\/p>\n\n\n\n

The 1<\/sup>H NMR spectrum for the representative compound P-01 is presented in Figure 6.<\/p>\n\n\n\n\n\n
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Figure 6: 1<\/sup>H NMR spectrum for the synthesized compound P-01<\/strong><\/p>\n

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Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Anticancer activity <\/strong><\/p>\n\n\n\n

The cytotoxicity studies of the synthesized test compounds were performed on A549 lung cancer cell lines. All the synthesized compounds possessed appreciable anticancer activity against lung cancer cell lines. Out of the 8 compounds tested, the compound P-03 showed the most prominent anticancer activity with an IC50 <\/sub>of 13.5 mmol in comparison to the standard doxorubicin that showed an IC50<\/sub> value of 3.63 mmol. Results of anticancer studies were presented in Table 4.<\/p>\n\n\n\n

Table 4: Cytotoxicity of the synthesized test compounds against A549 lung cancer cell lines.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Compound<\/strong><\/p>\n<\/td>\n

\n

IC50<\/sub> mmol<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

P-03<\/p>\n<\/td>\n

\n

  13.5<\/p>\n<\/td>\n<\/tr>\n

\n

P-04<\/p>\n<\/td>\n

\n

   25<\/p>\n<\/td>\n<\/tr>\n

\n

P-01<\/p>\n<\/td>\n

\n

   18<\/p>\n<\/td>\n<\/tr>\n

\n

P-09<\/p>\n<\/td>\n

\n

   31.3<\/p>\n<\/td>\n<\/tr>\n

\n

P-07<\/p>\n<\/td>\n

\n

   37.7<\/p>\n<\/td>\n<\/tr>\n

\n

P-02<\/p>\n<\/td>\n

\n

   29.23<\/p>\n<\/td>\n<\/tr>\n

\n

P-06<\/p>\n<\/td>\n

\n

   32<\/p>\n<\/td>\n<\/tr>\n

\n

P-08<\/p>\n<\/td>\n

\n

   21.67<\/p>\n<\/td>\n<\/tr>\n

\n

Doxorubicin<\/p>\n<\/td>\n

\n

   3.63<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

To\nfurther substantiate their potential as anticancer agents, mechanistic studies\nincluding cell cycle analysis and apoptosis studies were performed. <\/p>\n\n\n\n

Cell\ncycle analysis<\/strong><\/p>\n\n\n\n

The treatment of A549 cells at the concentrations of 12.5\u00b5M and 25\u00b5M with sample P03<\/em> has shown S phase and G2<\/sub>M phase arrest of 13.48%, 24.64% and 12.74%, 29.42% respectively. <\/p>\n\n\n\n

Standard Colchicine <\/em>at 25\u00b5M showed a G2<\/sub>M arrest of 36.55% in A549 cells as shown in the Figure 7.<\/p>\n\n\n\n\n\n
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Figure 7: Cell cycle analysis of Compound P-03 with control and standard doxorubicin.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The cell cycle analysis suggested the potential of the synthesized compound to inhibit the cell cycle at G2\/M phase. The cell cycle analysis data was presented in Table 5 and Figure 8.<\/p>\n\n\n\n

Table 5: Flow cytometry analysis of cell cycle arrest in A549 cell <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

FACS analysis of Cell cycle arrest in A549 cells<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Samples<\/strong><\/p>\n<\/td>\n

\n

SUBG0<\/strong><\/p>\n<\/td>\n

\n

G0\/G1<\/strong><\/p>\n<\/td>\n

\n

S<\/strong><\/p>\n<\/td>\n

\n

G2M<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/p>\n<\/td>\n

\n

1.30<\/p>\n<\/td>\n

\n

86.08<\/p>\n<\/td>\n

\n

8.10<\/p>\n<\/td>\n

\n

4.88<\/p>\n<\/td>\n<\/tr>\n

\n

P03_12.5\u00b5M<\/p>\n<\/td>\n

\n

1.32<\/p>\n<\/td>\n

\n

73.14<\/p>\n<\/td>\n

\n

13.48<\/p>\n<\/td>\n

\n

12.74<\/p>\n<\/td>\n<\/tr>\n

\n

P03_25\u00b5M<\/p>\n<\/td>\n

\n

0.44<\/p>\n<\/td>\n

\n

46.38<\/p>\n<\/td>\n

\n

24.64<\/p>\n<\/td>\n

\n

29.42<\/p>\n<\/td>\n<\/tr>\n

\n

Colchicine_25\u00b5M<\/p>\n<\/td>\n

\n

0.12<\/p>\n<\/td>\n

\n

55.96<\/p>\n<\/td>\n

\n

5.85<\/p>\n<\/td>\n

\n

36.55<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 8: Flow cytometry analysis of cell cycle arrest in A549 cells<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Apoptosis detection <\/strong><\/p>\n\n\n\n

The sample P03 <\/em>treated at 12.5\u00b5M and 25\u00b5M has induced 5.89%, 29.82% early apoptosis and 13.42%, 11.22% late apoptosis in A549 respectively. Standard Doxorubicin<\/em> at 25\u00b5M has shown total apoptosis of 46.93% in A549 cellsas shown in the Figure 9.<\/p>\n\n\n\n\n\n
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Figure 9: Apoptosis detection with compound P-03 against A549 lung cancer cell lines with control and standard doxorubicin.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The apoptosis studies suggested the potential of the synthesized compound in inducing late apoptosis. The FACS apoptosis data was presented in Table 6 and Figure 10.<\/p>\n\n\n\n

Table 6: Flow cytometry analysis of Apoptosis detection in A549 cell lines <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

FACS analysis of Apoptosis detection in A549 cells<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Sample<\/strong><\/p>\n<\/td>\n

\n

Viable cells<\/strong><\/p>\n<\/td>\n

\n

Early Apoptotic<\/strong><\/p>\n<\/td>\n

\n

Late Apoptotic<\/strong><\/p>\n<\/td>\n

\n

Necrotic cells<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/p>\n<\/td>\n

\n

98.58<\/p>\n<\/td>\n

\n

0.01<\/p>\n<\/td>\n

\n

0.17<\/p>\n<\/td>\n

\n

1.24<\/p>\n<\/td>\n<\/tr>\n

\n

P03_12.5\u00b5M<\/p>\n<\/td>\n

\n

77.14<\/p>\n<\/td>\n

\n

5.89<\/p>\n<\/td>\n

\n

13.42<\/p>\n<\/td>\n

\n

3.55<\/p>\n<\/td>\n<\/tr>\n

\n

P03_25\u00b5M<\/p>\n<\/td>\n

\n

55.42<\/p>\n<\/td>\n

\n

29.82<\/p>\n<\/td>\n

\n

11.22<\/p>\n<\/td>\n

\n

3.54<\/p>\n<\/td>\n<\/tr>\n

\n

Doxorubicin_25\u00b5M<\/p>\n<\/td>\n

\n

51.98<\/p>\n<\/td>\n

\n

31.97<\/p>\n<\/td>\n

\n

14.96<\/p>\n<\/td>\n

\n

1.10<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 10: Flow cytometry analysis of Apoptosis detection in A549 cell lines<\/strong><\/p>\n

<\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

A series of novel\nsubstituted coumarin pyrazole hybrids were synthesized utilizing the concept of\nhybridization as a lead optimization approach. The synthesized compounds were\ncharacterized by physical as well as spectroscopic techniques. Anti cancer activity\nof the compunds was performed on A-549 lung cancer cell lines against standard\ndoxorubicin. Out of the 8 compounds synthesized, the compound P-03 emerged as a\npotent antiproliferative agent with an IC50 value of 13.5 mmol in comparison to\nstandard doxorubicin that possessed an IC50 value of 3.63 mmol. The presence of\nbromine attached to the coumarin moeity significantly enhanced its anticancer\npotential. To substantiate the results, mechanistic anticancer studies\nincluding cell cycle analysis and apoptosis detection studies were performed.\nCell cycle analysis further confirmed its ability to inhibit cell cycle at G2\/M\nphase. Apoptosis studies confirmed its role as a late apoptotic agent. <\/p>\n\n\n\n

In future studies, an\nattempt will be made to study their anticancer potential in animal models to\nreconfirm its ability against lung cancer models. <\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

The authors acknowledge the\nManipal Academy of Higher Education for the support provided to carry out the\npresent research.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors declare that there is\nno conflict of interest.<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

The current research is not\nfunded by any government of private funding agencies. <\/p>\n\n\n\n

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