Next Article in Journal
Influence of the Preliminary Storage on Methane Yield of Anaerobic Digestion of the Organic Fraction of Municipal Solid Waste
Next Article in Special Issue
Optimization of No-Wait Flowshop Scheduling Problem in Bakery Production with Modified PSO, NEH and SA
Previous Article in Journal
Use of the Solid By-Product of Anaerobic Digestion of Biomass to Remove Anthropogenic Organic Pollutants with Endocrine Disruptive Activity
Previous Article in Special Issue
A Case Study of Turbulent Free Jet Flows Issuing from Rectangular Slots on Process Performances and Quality of Hot-Air-Dried Apple
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Processes
Volume 9
Issue 11
10.3390/pr9112019
processes-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Synthesis of New 1-Aryl-2-(3,5-dimethylpyrazol-1-yl)ethanone Oxime Ether Derivatives and Investigation of Their Cytotoxic Effects

by
Mehmet Abdullah Alagöz
1,
Arzu Karakurt
1,
Ceylan Hepokur
2,
Emine Şalva
3,
Tijen Önkol
4,
Mohammed M. Ghoneim
5,
Mohamed A. Abdelgawad
6,
Ahmed Khames
7,
Hoon Kim
8,* and
Bijo Mathew
9,*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Inonu University, Malatya 44280, Turkey
2
Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Sivas Cumhuriyet University, Sivas 58100, Turkey
3
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Inonu University, Malatya 44280, Turkey
4
Department of Pharmacy, Faculty of Pharmacy, Gazi University, Ankara 06100, Turkey
5
Department of Pharmacy Practice, Faculty of Pharmacy, AlMaarefa University, Ad Diriyah 13713, Saudi Arabia
6
Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka 72341, Saudi Arabia
7
Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
8
Department of Pharmacy, and Research Institute of Life Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Korea
9
Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Vishwa Vidyapeetham, AIMS Health Sciences Campus, Kochi 682 041, India
*
Authors to whom correspondence should be addressed.
Processes 2021, 9(11), 2019; https://doi.org/10.3390/pr9112019
Submission received: 13 October 2021 / Revised: 2 November 2021 / Accepted: 8 November 2021 / Published: 11 November 2021
(This article belongs to the Special Issue Redesign Processes in the Age of the Fourth Industrial Revolution)
Browse Figures
Versions Notes

Abstract

:
In this study, 12 new 1-aryl-2-(3,5-dimethylpyrazol-1-yl)ethanone oxime ether derivatives were designed and synthesized to investigate their cytotoxic effects. The in vitro cytotoxic activities of the compounds were evaluated against cervix, colon, breast, glioma, neuroblastoma, and lung cancer cell lines, as well as a healthy cell line using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) assays with 5-fluorouracil (5-FU) as the reference compound. Compound 5f (IC50 = 5.13 µM) was found to be more effective than 5-FU (IC50 = 8.34 µM) in the C6 cancer cell line, and it had no cytotoxic effect on the L929 healthy cell line. Flow cytometry was used to investigate the mechanism of action of compound 5f on the cell cycle of the C6 cell line. The analysis showed that cell death was significantly due to apoptosis. These results indicate that compound 5f induces cell cycle arrest, and may be effective in treating glioma.

1. Introduction

Cancer is one of the most significant health problems worldwide. In addition, it is the second-leading cause of death in the world after cardiovascular diseases. According to the World Health Organization (WHO), approximately 9.6 million deaths worldwide were due to cancer in 2018 [1]. Surgery and radiotherapy are generally the primary treatments for early-stage local and non-metastatic cancers. The use of anticancer drugs (e.g., chemotherapy and hormone or biological treatments) is especially preferred in metastatic cancers. Chemotherapy is generally based on preventing cell division as a result of targeting rapidly growing cancer cells [2,3].
However, cancer treatment is still a challenging area due to low selectivity, side effects, the development of resistance in various strains, and a lack of efficacy. Therefore, intensive studies are consistently carried out in this field to develop safe, more effective, and selective anticancer agents [4].
Oxime ethers are a functional group frequently used for chemical modifications in medicinal chemistry and are included in the structure of active molecules. Specifically, the ant-Alzheimer’s agents, anticancer, anticonvulsant, antimicrobial, and anti-inflammatory activities of oxime derivatives are well known [5,6,7]. Moreover, they are found in the structures of antidepressant, antifungal, and antibacterial drugs, such as fluvoxamine (1), oxiconazole (2), cefuroxime (3), and aztreonam (4) (Figure 1).
Pyrazoles are heterocyclic aromatic compounds characterized by a five-membered ring (1,2-diazole) structure consisting of two nitrogen atoms and three carbon atoms adjacent to each other. Many biologically active compounds that carry pyrazole or substituted pyrazole derivatives, which exhibit interesting biological activities, such as antitumor, analgesic, anti-inflammatory, anticonvulsant, antipsychotic, antidepressant, anti-Alzheimer, anti-Parkinson, antihyperglycemic, antiobesity, antihypertensive, antibacterial, fungicidal, antiviral, and antitubercular activities, have been reported in the literature [8,9,10,11,12,13,14,15,16].
Azole compounds have gained their importance in cancer treatment. In addition, letrozole (5), ruxolitinib (6), fadrozole (7), crizotinib (8), and pralsetinib (9) are among the anticancer drugs with an azole moiety. Moreover, crizotinib (Figure 2) is currently undergoing clinical trials against neuroblastoma and other advanced solid tumors in both adults and children [4,17,18,19]. In the recent literature, some compounds with a dimethylpyrazole ring were reported to be potent anticancer agents (10–13) (Figure 3) [20,21,22,23,24].
Previously, the cytotoxic activities of some 2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-arylethanone derivatives (14) (Figure 4) have been evaluated, and they were found to be active against human colon cancer and lung adenocarcinoma cell lines [11].
In our previous study, some oxime ester derivatives carrying a pyrazole ring were determined to exhibit cytotoxic activity against the SH-SY5Y (e.g., human neuroblast cells) cell line (15). These aryl oxime esters bearing the naphthalene and pyrazole ring did not show a better activity than the standard compounds. In addition, they did not affect healthy mouse fibroblast cells (Figure 4) [25].
In the present study, 12 new 1-aryl-2-(3,5-dimethylpyrazol-1-yl)ethanone derivatives were synthesized (Figure 5). This study is based on the ketone compounds prepared by Kumar et al., which were cytotoxic [11]. For this reason, the 3,5-dimethylpyrazole group was protected, which was thought to be effective in the activity. In addition, the structure-activity relationship (SAR) was established by synthesizing oxime derivatives with different aryl groups. Then, methyl and isobutyl oxime ethers were prepared to investigate the effect of lipophilicity on the activity. Since the naphthalene and aryl oxime esters with a pyrazole ring in our previous study did not have significant cytotoxic activity, we decided to prepare oxime ether compounds containing a limited number of alkyl halides.
The series include 2-naphthyl, phenyl, and substituted phenyl groups as the aryl groups. Moreover, this study investigated the effect of various substituents (e.g., -F, -Cl, -CH3, and -OCH3) bound to the phenyl group on the cytotoxic activity. Ketone and oxime derivatives were used as the starting material in the synthesis of the new oxime ether derivatives. Furthermore, in the present study, the in vitro cytotoxic activities of the compounds were evaluated against A549, HCT 116 (i.e., human colon cancer cells), HeLa (i.e., human cervical cancer cells), MCF7 (i.e., human breast adenocarcinoma cells), C6 (i.e., rattus norvegicus brain glioma), SH-SY5Y, and L929 (i.e., mouse fibroblast) cell lines.
These 12 new oxime ether compounds (4a-4f; 5a-5f) were synthesized from the appropriate oxime compounds (3a-3f). Since the -OH group of the oxime compound did not react with the alkyl halide, the -OH group was treated with Na0 and -ONa was obtained. This reaction is the step with the lowest yield. The synthesis of 3a-3f began from the appropriate ketone compounds (2a-2f). The compounds 3a-3f were synthesized as a result of an easy reaction of 2a-2f with hydroxylamine hydrochloride in ethanol at pH = 11. Compounds 2a-2f were obtained by the substitution of 1-(aryl)-2-bromoethanone (1a-1f) with 3,5-dimethylpyrazole.

2. Results and Discussion

2.1. Chemistry

The structures and general synthetic routes of the compounds are shown in Scheme 1.
First, we attempted the ring closure reaction presented in Kumar et al. to obtain the 1-aryl-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone derivatives [11]. Although the reaction time was extended, the reaction did not end completely, and the reaction yield was very low. Therefore, the synthesis of these compounds was carried out via a N-alkylation reaction of 1-aryl-2-bromoethanone with 3,5-dimethylpyrazole. The spectral data of the compounds (2a-2f) were compatible with the results reported in the literature [11]. The oxime derivatives (3a-3f) were prepared via the reaction of 1-aryl-2-(3,5-dimethylpyrazol-1-yl)ethanone and hydroxylamine hydrochloride [26]. The compounds were obtained in high yields ranging from 81 to 93%. We adjusted our synthesis method, which used NaOH rather than NaHCO3, to a pH of 11, and the reaction time was extended to 90 min. Therefore, we obtained the same compounds with a much higher yield than the reaction yields (32–80%) of Sharma et al. [27]. Two of these oximes (3a and 3f) are novel compounds, and their spectral data are provided in the experimental section. The spectral data of the other oxime derivatives were in accordance with the results reported in the literature [27]. The oxime compounds were obtained as E/Z isomer mixtures.
The synthesis of the 12 novel oxime ether derivatives (4a-4f–5a-5f) was carried out via O-alkylation of the respective oximes with the appropriate alkyl halides. They were purified through column chromatography, and their yields ranged between 45–80%. A double spot was observed in the TLC analysis of the reaction medium. However, a single isomer was obtained for each oxime ether derivative as a result of purification. This was probably due to the solvent effect, which converted one isomer into another.
In the IR spectra of the oxime and oxime ether derivatives, in accordance with the data reported in the literature, the peaks of C-O were observed at 1009–1072 cm−1, C=N at 1513–1612 cm−1, and N-O at 851–982 cm−1. The disappearance of the flat peak of the -OH group at 3000–3400 cm−1 and the formation of a new peak belonging to the C-O group at 1100–1000 cm−1 showed the transformation of the oxime structures into their respective oxime ether derivatives.
The C=N-OH protons in the oxime derivatives were observed as a singlet in the range of 10.6–11.9 ppm. The aromatic protons were observed as multiples between 6.8–8.1 ppm. The protons of the -CH2 attached to the pyrazole ring resonated as a singlet between 4.9–5.5 ppm. The =CH proton of the pyrazole was observed as a singlet between 5.67–5.72 ppm. Since the chemical environment of the protons of the -CH3 attached to the pyrazole was different, they were observed as two separate singlets between 1.98–2.21 ppm.
The oximes (3a-f) were obtained as mixtures of the E and Z isomers. Therefore, the peaks of both isomers were seen together in the 1H NMR spectra. The percentages of isomers were calculated using the ratios of the integral values of both isomer peaks for specific protons. According to the 1H NMR spectrum analysis of compound 3b (see Supplementary Data), the -OH proton was observed as two singlets at 10.68 and 11.11 ppm, with a relative integration of 23 and 77%, respectively. Likewise, the -CH proton of the pyrazole produced two singlets at 5.67 and 5.72 ppm.
In the 13C NMR spectra of the oxime derivatives, the -CH3 groups attached to the pyrazole were observed at 10–20 ppm, and the -CH at the 4th position of the pyrazole was within 100–110 ppm. The aromatic carbons of the benzene were seen at 120–160 ppm. The C=N of the pyrazole resonated at a higher chemical shift value than the =CH due to the nitrogen atom. The C=N-O carbon was observed between 140–165 ppm. The -O-C of the oxime ether derivatives were in the range of 60–65 ppm, and the aliphatic carbon peaks were in the range of 10–40 ppm. Since the two isomers were together in the oxime compounds, two isomer peaks with different intensities for each carbon could also be seen in the 13C NMR spectra. In the 13C NMR spectra of the compounds carrying fluorine atoms, the peaks of some carbon atoms in the phenyl ring were split in two due to coupling with the fluorine.
The HRMS spectra of the compounds were recorded using the positive ion (ESI+) electrospray ionization technique. In this method, only the [M + H]+ (molecular ion + H) peaks and isotope peaks can be seen. The calculated [M + H]+ values were compatible with the analysis results. In the HRMS spectra of 5a-5e, a [M + H]+ peak at 100% was observed, while [M + 2 + H]+ peaks at 33% confirmed the presence of one chlorine atom in the structure (see Supplementary Data).

2.2. Cytotoxic Activity Assay

In this study, the cytotoxic activity of the compounds was studied on six different cancer cell lines (MCF-7, A549, HCT116, HeLa, C6, SH-SY5Y) and one healthy cell line (L929). The cell lines were treated with five different concentrations of the compounds (5, 10, 25, 50, and 100 µM) for 24 h, and 5-fluorouracil (5-FU) was used as a positive control. Although 14 of the 30 compounds were original, they were all included in the cytotoxic activity screening tests.
The cytotoxic activity of the 12 compounds were screened, among which nine compounds were found to be more active against the C6 cell line and three compounds against the SH-SY5Y cell line, compared with the standard 5-FU. No activity was seen against the MCF-7, A549, HCT116 or HeLa cell lines, as presented in Table 1. According to the results, the compounds were significantly effective against the SH-SY5Y and C6 cell lines in comparison with the other cell lines and positive control 5-FU. Moreover, it was observed that the compounds did not exhibit cytotoxic effects on the L929 healthy cell line. The IC50 values of compounds 4a–5f on the C6 and SH-SY5Y cell lines are presented in Table 2.
There was no clear structure activity relationship established between the changes in the aromatic structure on the compound for the activity against the C6 cell line. However, a significant potential could be seen in the naphthyl and nonsubstituted phenyl derivatives against the SH-SY5Y cell line, which was reversed for the methyl and methoxyphenyl compounds.
The IC50 values of compounds 4a–5f ranged from 5.00–29.85 µM in the SH-SY5Y cell line and 5.13–24.31 µM in the C6 cell line. However, the IC50 values of 5-FU were calculated as 8.34 and 8.53 µM in the C6 and SH-SY5Y cell lines, respectively.
Further studies were performed on the C6 cells, as these compounds have a better cytotoxic activity against C6, compared to the other cells. Compound 5f is the best cytotoxic compound, with 5.13 µM on the C6 cell line, and 5a is the best cytotoxic compound, with 5.00 µM on the SH-SY5Y cell line.
In the L929 cell line, the % viability values ranged between 71.28 and 115.74 µM with a 10 µM-concentration (Table S1). The fact that all of the compounds exhibited activity against glioblastoma and neuroblastoma with close IC50 values while not being active against the other cell lines shows that the effects of the compounds are selective.

2.2.1. Flow Cytometry Analysis

The flow cytometry analysis was used to investigate the mechanical effect of compound 5f (at a concentration of IC50 value), which had the lowest IC50 value in the C6 cell line, on the cell cycle. With this method, it is possible to determine the number of cells in each division phase. In the analysis performed on the C6 cell line, it was found that compound 5f killed 79.02% of the cancer cells. Cell line deaths were mostly caused by apoptosis. In this case, 4.88% of these deaths were early apoptotic, 69.27% were late apoptotic, and 4.88% were necrosis (Figure 6). The apoptosis-necrosis ratio was calculated as 15:19. These results show that compound 5f had a significant cytotoxic effect on the C6 cell line.

2.2.2. Cell Cycle Analysis

The cell cycle is the process in which biochemical activities and morphological changes are observed in the cell. It begins with the division of one cell, continuing with the division of the other cells. The cell cycle consists of two parts: Interphase (G1, S, and G2) and mitosis (M). Until the cells receive a division signal, they remain in the resting phase of the cell cycle, the G0 (Gap) phase. When the division signal is received, the cell divides by passing through the G1, S, G2, and M phases, respectively.
In the cell cycle analysis performed on the C6 cell line, it was determined that compound 5f inhibited the cell cycle by 45.1% in the G0/G1 phase, 32.9% in the S phase, and 19.5% in the G2/M phase (Figure 7). These results indicate that compound 5f induces cell cycle arrest, and might be effective in the glioma treatment.

3. Materials and Methods

3.1. Chemistry

All of the chemicals used in this study were purchased from Merck and Sigma-Aldrich. Dimethylformamide was dried with a 4A molecular sieve. All of the reactions were carried out in atmospheric conditions. The reactions were monitored by thin layer chromatography (TLC) with Merck Kieselgel 60 F254 aluminum plates. Preparative TLC for the purification of the compounds was performed on Kieselgel 60 plates (Merck). The melting points were determined with a Stuart SMP30 capillary melting point apparatus and were uncorrected. The infrared (IR) spectra were recorded in KBr disks with a Perkin Elmer Spectrum One FTIR Spectrometer. 1H- and 13C-nuclear magnetic resonance (NMR) spectra were recorded on an Agilent WB UltrashieldTM NMR Spectrometer in DMSO-d6 or CDCl3 at 400 MHz. All of the chemical shifts were expressed in δ (ppm). The splitting patterns were designated as follows—s: Singlet; d: Doublet; dd: Doublet of doublets; t: Triplet; q: Quartet; and m: Multiplet. The high-resolution mass spectrometry (HRMS) spectra of the solutions of the compounds in methanol were recorded via positive ion (ESI +) electrospray ionization techniques using the Waters LCT Premier XE UPLC/MS TOFF system and MassLynx 4.1 software.

General Methods for the Preparation of the Compounds

Synthesis of 1-(Aryl)-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone (2a-2f)
A solution of 3,5-dimethylpyrazole (0.03 mol, 3.0 equiv., 288.39 mg) in dry dimethylformamide (2.5 mL) was cooled down to 0–5 °C in an ice bath. A solution of 1-(aryl)-2-bromoethanone (0.01 mol, 1.0 equiv., 233.10 mg 1a, 181.97 mg 1b, 201.03 mg 1c, 217.49 mg 1d, 195.98 mg 1e, 211.93 mg 1f) in dry dimethylformamide (2.5 mL) was then slowly added and stirred. After 2 h, the reaction was stirred at room temperature for 24 h. Thereafter, the reaction medium was poured into ice water in a dropwise manner. The formed precipitates were filtered, washed with distillated water, and dried [28].
1-(Naphthalen-2-yl)-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone (2a)
Yield: 81%; mp: 100–2 °C, white powder. 1H NMR (400 MHz, CDCl3-d): δ 2.25 (s, 3H, -CH3), 2.41 (s, 3H, -CH3), 6.15 (s, 2H, -CH2), 6.29, (s, H, pyrazole), 7.42–8.05 (m, 6H, naphthalene), 8.67 (s, H, naphthalene). 13C NMR (100 MHz, CDCl3-d) δ 189.44, 146.42, 145.16, 136.05, 132.25, 131.15, 130.56, 129.94, 128.91, 127.71, 123.17, 107.39, 54.33, 11.34, 10.97. HRMS (ESI): m/z calcd for C17H16N2O [M+H]+ 265.1296; found: 265.2000.
1-Phenyl-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone (2b)
Yield: 90%; mp: 95–8 °C, white powder. 1H NMR (400 MHz, CDCl3-d): δ 2.17 (s, 3H, -CH3), 2.29 (s, 3H, -CH3), 5.46 (s, 2H, -CH2), 5.92, (s, H, pyrazole), 7.39–7.70 (m, 3H, phenyl) 7.89–8.04 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d) δ 192.74, 148.31, 140.51, 134.61, 133.97, 128.92, 128.09, 105.84, 55.31, 13.56, 11.03. HRMS (ESI): m/z calcd for C13H14N2O [M+H]+ 215.1140; found: 215.0000.
1-(4-Fluorophenyl)-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone (2c)
Yield: 85%; mp: 110–11 °C, white powder. 1H NMR (400 MHz, CDCl3-d): δ 2.16 (s, 3H, -CH3), 2.24 (s, 3H, -CH3), 5.42 (s, 2H, -CH2), 5.91 (s, H, pyrazole), 7.09–7.28 (m, 2H, phenyl), 7.95–8.07 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d) δ 191.31, 167.86, 164.47, 148.41, 140.51, 131.03, 116.28, 115.98, 105.91, 55.22, 13.52, 11.01. HRMS (ESI): m/z calcd for C13H14FN2O [M+H]+ 233.1045; found: 233.1000.
1-(4-Chlorophenyl)-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone (2d)
Yield: 96%; mp: 108–109 °C, white powder. 1H NMR (400 MHz, CDCl3-d): δ 2.17 (s, 3H, -CH3), 2.25 (s, 3H, -CH3), 5.42 (s, 2H, -CH2), 5.92 (s, H, pyrazole), 7.43–7.53 (m, 2H, phenyl), 7.98–7.86 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d) δ 191.77, 148.49, 140.52, 140.50, 132.90, 129.57, 129.28 105.96, 55.30, 13.53, 11.03. HRMS (ESI): m/z calcd for C13H13ClN2O [M+H]+ 249.0750; found: 249.0000.
1-(4-Methylphenyl)-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone (2e)
Yield: 89%; mp: 83–85 °C, white powder. 1H NMR (400 MHz, CDCl3-d): δ 2.29 (s, 3H, -CH3), 2.44 (s, 3H, -CH3), 2.48 (s, CH3-C6H4), 6.10 (s, 2H, -CH2), 6.21, (s, H, pyrazole), 7.24–7.39 (m, 2H, phenyl), 7.98–7.87 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d) δ 188.80, 146.21. HRMS (ESI): m/z calcd for C14H16N2O [M+H]+ 229.1296; found: 228.0000.
1-(4-Methoxyphenyl)-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone (2f)
Yield: 87%; mp: 174–6 °C, white powder. 1H NMR (400 MHz, CDCl3-d): δ 2.27 (s, 3H, -CH3), 2.45 (s, 3H, -CH3), 3.85 (s, 3H, O-CH3), 6.10 (s, 2H, -CH2), 6.20 (s, H, pyrazole), 6.89–7.04 (m, 2H, phenyl), 8.06–7.93 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d) δ 187.47, 164.82, 146.62, 145.04, 131.01, 126.27, 114.40, 107.41, 55.67, 53.67, 11.28, 10.98. HRMS (ESI): m/z calcd for C14H16N2O2 [M+H]+ 245.1245; found: 245.1000.
Synthesis of 1-(Aryl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)ethanone oxime (3a-f)
1-(Aryl)-2-(3,5-dimethyl-1H-pyrazol-1-yl))ethanone (0.015 mol, 1.0 equiv., 265.12 mg 2a, 215.14 mg 2b, 233.10 mg 2c, 249.07 mg 2d, 229.12 mg 2e, 245.12 mg 2f) was dissolved in absolute ethanol (75 mL). The reaction was heated under reflux and hydroxylamine hydrochloride (0.03 mol, 3.0 equiv., 208.47 mg) was added. The medium was alkalized to pH = 11 with 3 mL 15 N sodium hydroxide solution and was stirred under reflux for 1–2 h. The ethanol was evaporated to dryness. The resulting solid was treated with ice water and filtered [26].
1-(2-Naphthyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone oxime (3a)
Yield: 84%; mp: 174–6 °C, white powder; IR (KBr) cm−1; 3135, 3051, 2939, 2845, 1600, 1552, 107, 897. 1H NMR (400 MHz, DMSO-d6): δ 1.99 (s, 3H, -CH3; 83%), 2.00 (s, 3H, -CH3, 17%), 2.08 (s, 3H, -CH3, 83%), 2.16 (s, 3H, -CH3, 17%), 5.10 (s, 2H, -CH2, 17%), 5.36 (s, 2H, -CH2, 83%), 5.67 (s, 1H, =CH, 83%), 5.68 (s, 1H, =CH, 17%), 7.43–7.91 (m, 6H, naphthyl), 8.08 (s, H, naphthyl), 11.88 (s, 1H, =N-OH). 13C NMR (100 MHz, DMSO-d6) δ 152.78, 151.94, 146.41, 146.32, 139.74, 133.29, 133.05, 132.73, 132.60, 139.70, 128.69, 128.59, 127.94, 127.90, 127.81, 127.52, 127.18, 127.02, 126.84, 126.74, 126.71, 126.37, 124.44, 105.37, 105.17, 52.37, 42.86, 13.73, 11.11, 11.01. HRMS (ESI): m/z calcd for C17H17N3O [M+H]+ 280.1450; found: 280.1444.
1-Phenyl-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone oxime (3b) [27]
Yield: 81%; mp: 132–5 °C, white powder; IR (KBr) cm−1; 3150, 3054, 2939, 1553, 1071, 897. 1H-NMR (CDCl3-d, 400 MHz): δ ppm 2.05 (3H, s, -CH3, 23%), 2.08 (3H, s, -CH3, 77%), 2.18 (3H, s, -CH3, 77%), 2.21 (3H, s, -CH3, 23%), 5.05 (2H, s, -CH2-, 23%), 5.47 (2H, s, -CH2-, 77%), 5.67 (H, s, =CH-, 77%, pyrazole), 5.72 (H, s, =CH-, 23%, pyrazole), 7.16–7.50 (5H, m, 23%, phenyl), 7.43–7.49 (5H, m, 77%, phenyl), 10.68 (H, s, =N-OH, 23%), 11.11 (H, s, =N-OH, 23%). 13C-NMR (CDCl3-d, 100 MHz), 154.24, 152.52, 148.09, 147.60, 140.13, 139.97, 133.91, 131.70, 129.00, 128.98, 128.23, 128.13, 127.80, 127.15, 105.49, 52.29, 43.90, 13.29, 10.94, 10.87. HRMS (ESI): m/z calcd for C13H15N3O [M+H]+ 230.1293; found: 230.1286.
(4-Fluorophenyl)-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone oxime (3c) [27]
Yield: 91%; mp: 118–20 °C, white powder; IR (KBr) cm−1; 3142, 3021, 2870, 1509, 1060, 917. 1H-NMR (CDCl3-d, 400 MHz): δ ppm 2.05 (3H, s, -CH3, 17%), 2.07 (3H, s, -CH3, 83%), 2.18 (3H, s, -CH3, 83%), 2.20 (3H, s, -CH3, 17%), 5.02 (2H, s, -CH2-, 17%), 5.44 (2H, s, -CH2-, 83%), 5.69 (H, s, =CH-, 83%, pyrazole), 5.72 (H, s, =CH-, 17%, pyrazole), 6.85–7.04 (2H, m, phenyl), 7.25–7.46 (2H, m, phenyl), 11.21 (H, s, =N-OH). 13C-NMR (CDCl3-d, 100 MHz), 164.42, 161.95, 153.38, 151.37, 148.20, 147.76, 140.18, 140.04, 130.05, 130.00, 129.96, 129.12, 129.04, 127.39, 115.41, 115.25, 115.20, 115.04, 105.64, 105.60, 52.22, 43.93, 13.24, 10.96, 10.83. HRMS (ESI): m/z calcd for C13H14FN3O [M+H]+: 248.1199; found: 248.1177.
1-(4-Chlorophenyl)-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone oxime (3d) [27]
Yield: 92%; mp: 140–2 °C, white powder; IR (KBr) cm−1; 1H-NMR (DMSO-d6, 400 MHz) δ ppm: 1.98 (3H, s, -CH3, 77%), 1.99 (3H, s, -CH3, 23%), 2.06 (3H, s, -CH3, 23%), 2.12 (3H, s, -CH3, 77%), 4.99 (2H, s, -CH2-, 23%), 5.21 (2H, s, -CH2-, 77%), 5.69 (H, s, =CH-, 77%, pyrazole), 5.70 (H, s, =CH-, 23%, pyrazole), 7.30–7.62 (4H, m, phenyl), 11.28 (H, s, =N-OH, 23%), 11.88 (H, s, =N-OH, 77%). 13C-NMR (DMSO-d6, 100 MHz), 152.09, 150.97, 146.50, 146.42, 139.76, 139.70, 134.05, 133.88, 133.68, 131.06, 130.51, 128.82, 128.58, 128.39, 105.44, 105.21, 52.05, 42.80, 13.72, 13.70, 11.06, 10.93. HRMS (ESI): m/z calcd for C13H14ClN3O [M+H]+: 264.0904; found: 264.0786.
1-(4-Methylphenyl)-2-(1H-3,5-dimethylpyrazol-1-yl)ethanone oxime (3e) [27]
Yield: 87%; mp: 138–40 °C, white powder; IR (KBr) cm−1; 3128, 2991, 1512, 1030, 890. 1H-NMR (DMSO-d6, 400 MHz) δ ppm: 1.99 (3H, s, -CH3, 77%), 2.00 (3H, s, -CH3, 23%), 2.06 (3H, s, -CH3, 23%), 2.10 (3H, s, -CH3, 77%), 2.25 (3H, s, CH3-C6H4, 77%), 2.26 (3H, s, CH3-C6H4, 23%), 4.96 (2H, s, -CH2-, 23%), 5.20 (2H, s, -CH2-, 77%), 5.67 (H, s, =CH-, 77%, pyrazole), 5.69 (H, s, =CH-, 23%, pyrazole), 7.07–7.47 (4H, m, phenyl), 11.06 (H, s, =N-OH, 23%), 11.64 (H, s, =N-OH, 77%). 13C-NMR (DMSO-d6, 100 MHz), 152.74, 151.60, 146.25, 146.17, 139.64, 139.56, 138.58, 138.51, 132.36, 129.40, 129.12, 128.84, 128.53, 126.94, 105.31, 105.13, 52.30, 42.97, 21.33, 21.21, 13.76, 11.11, 10.96. HRMS (ESI): m/z calcd for C14H17N3O [M+H]+: 244.1450; found: 244.1442.
1-(4-Metoxyphenyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone oxime (3f)
Yield: 88%; mp: 150–2 °C, white powder; IR (KBr) cm−1, 3128, 3028, 2992, 1556, 1029, 890. 1H NMR (400 MHz, DMSO-d6): δ 2.00 (s, 3H, -CH3), 2.09 (s, 3H, -CH3, 20%), 2.11 (s, 3H, -CH3, 80%), 3.72 (s, 3H, -C6H4-OCH3, 80%), 3.73 (s, 3H, -C6H4-OCH3, 20%), 4.98 (s, 2H,-CH2, 20%), 5.19 (s, 2H, -CH2, 80%), 5.68 (s, 1H, =CH), 6.82–6.90 (m, 2H, phenyl), 7.40–7.55 (m, 2H, phenyl), 11.07 (s, 1H, =N-OH, 20%), 11.55 (s, 1H, =N-OH, 80%). 13C NMR (100 MHz, DMSO-d6) δ 160.08, 159.68, 152.35, 150.93, 146.19, 139.64, 139.59, 130.36, 128.39, 127.57, 124.28, 113.94, 113.62, 105.34, 105.13, 55.51, 55.49, 52.30, 42.96, 13.77, 13.75, 11.16, 10.96. HRMS (ESI): m/z calcd for C14H17N3O2 [M+H]+ 260.1399; found: 260.1390.
Synthesis of 1-(aryl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)ethanone oxime ethers (4a-4f; 5a-5f)
Specifically, 0.011 mol NaOH was dissolved in absolute ethanol (10 mL), and 1-(aryl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)ethanone oxime (0.01 mol, 1.0 equiv., 280.14 mg 3a, 230.12 mg 3b, 248.11 mg 3c, 264.09 mg 3d, 244.14 mg 3e, 260.13 mg 3f) was added. The mixture was stirred under reflux for 1 h. The ethanol in the flask was evaporated to dryness. The remaining solid was dissolved via the addition of dry dimethylformamide (10 mL). The appropriate alkyl halide (0.02 mol, 2.0 equiv., 283.88 mg Iodomethane, 271.96 mg 1-bromo-2-metylpropane) was added to the solution and stirred at room temperature for 3–4 h [28,29]. The reaction medium was poured into ice water, and the precipitate was filtered off and dried. The compounds were purified via column chromatography using ethyl acetate/n-hexane (1:3).
1-(2-Naphthyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone O-methyl oxime (4a)
Yield: 74%; mp: 69–72 °C, white powder; IR (KBr) cm−1: 3058, 2967, 1553, 1039, 907. 1H NMR (400 MHz, DMSO-d6): δ 2.05 (s, 3H, -CH3), 2.25 (s, 3H, -CH3), 4.05 (s, 3H, -OCH3), 5.35 (s, 2H, -CH2), 5.70 (s, 1H, =CH), 7.60–7.90 (m, 6H, naphthyl), 8.15 (s, 1H, naphthyl). 13C NMR (100 MHz, CDCl3-d): δ 143.75, 141.42, 139.30, 136.65, 134.50, 134.01, 129.50, 128.66, 126.89, 126.42, 123.38, 121.19, 60.86, 47.02, 13.67, 12.03. HRMS (ESI): m/z calcd for C18H19N3O [M+H]+: 294.1562; found: 294.1604.
1-Phenyl-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone O-methyl oxime (4b)
Yield: 53%, white viscous liquid, IR (KBr) cm−1: 3010, 2930, 1513, 1026, 891. 1H NMR (400 MHz, CDCl3-d): δ 2.05 (s, 3H, -CH3), 2.16 (s, 3H, -CH3), 4.04 (s, 3H, -OCH3), 5.31 (s, 2H, -CH2), 5.66 (s, 1H, =CH), 7.23–7.31 (m, 3H, phenyl), 7.46–7.51 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 154.01, 147.48, 139.61, 133.40, 129.21, 128.16, 127.18, 105.48, 62.42, 44.63, 13.44, 10.80. HRMS (ESI): m/z calcd for C14H17N3O [M+H]+: 244.1450; found: 244.1447.
1-(4-Fluorophenyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone O-methyl oxime (4c)
Yield: 69%, mp: 55–57 °C, white powder, IR (KBr) cm−1: 3011, 2983, 1612, 1026, 891. 1H NMR (400 MHz, CDCl3-d): δ 2.05 (s, 3H, -CH3), 2.16 (s, 3H, -CH3), 4.03 (s, 3H, -OCH3), 5.29 (s, 2H, -CH2), 5.68 (s, 1H, =CH), 6.89–6.98 (m, 2H, phenyl), 7.44–7.52 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 163.36 (d, 1JF-Ar = 249.17 Hz), 153.08, 147.61, 139.61, 129.50, 129.47, 129.07 (d, 2JF-Ar = 8.36 Hz), 115.30, 115.08, 105.59, 62.46, 44.59, 13.42, 10.77. HRMS (ESI): m/z calcd for C14H16FN3O [M+H]+: 262.1356; found: 262.1350.
1-(4-Chlorophenyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone O-methyl oxime (4d)
Yield: 80%; mp: 64–66 °C, white powder; IR (KBr) cm−1: 3005, 2995, 1552, 1026, 895. 1H NMR (400 MHz, CDCl3-d): δ 2.05 (s, 3H, -CH3), 2.16 (s, 3H, -CH3), 4.03 (s, 3H, -OCH3), 5.29 (s, 2H, -CH2), 5.68 (s, 1H, =CH), 7.21–7.25 (m, 2H, phenyl), 7.43–7.47 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 152.91, 147.65, 139.61, 135.30, 131.84, 129.42, 128.49, 128.40, 105.66, 62.56, 44.40, 13.44, 10.79. HRMS (ESI): m/z calcd for C14H16ClN3O [M+H]+: 278.1060; found: 278.1052.
1-(4-Methylphenyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone O-methyl oxime (4e)
Yield: 58%; yellow viscous liquid; IR (KBr) cm−1: 3008, 2962, 1555, 1035, 890. 1H NMR (400 MHz, CDCl3-d): δ 2.05 (s, 3H, -CH3), 2.17 (s, 3H, -CH3), 2.29 (s, 3H, C6H4-CH3), 4.02 (s, 3H, -OCH3), 5.30 (s, 2H, -CH2), 5.67 (s, 1H, =CH), 7.04–7.08 (m, 2H, phenyl), 7.36–7.41 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 153.84, 147.39, 139.61, 139.22, 130.52, 128.91, 127.00, 105.46, 62.33, 44.57, 21.28, 13.46, 10.81. HRMS (ESI): m/z calcd for C15H19N3O [M+H]+: 258.1606; found: 258.1603.
1-(4-Methoxyphenyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone O-methyl oxime (4f)
Yield: 49%; mp: 70–72 °C, white powder; IR (KBr) cm−1: 3133, 2995, 1555, 1028, 982. 1H NMR (400 MHz, CDCl3-d): δ 2.05 (s, 3H, -CH3), 2.17 (s, 3H, -CH3), 3.77 (s, 3H, C6H4-OCH3), 4.01 (s, 3H, -OCH3), 5.29 (s, 2H, -CH2), 5.67 (s, 1H, =CH), 6.75–6.80 (m, 2H, phenyl), 7.42–7.46 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 160.39, 153.48, 147.40, 139.66, 128.55, 125.87, 113.59, 105.50, 62.28, 55.19, 44.59, 13.46, 10.80. HRMS (ESI): m/z calcd for C15H19N3O2 [M+H]+: 274.1556; found: 274.1552.
1-(2-Naphthyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone O-2-methylpropyl oxime (5a)
Yield: 65%; mp: 67–69 °C, white powder; IR (KBr) cm−1: 3039, 2939, 1554, 1072, 897. 1H NMR (400 MHz, CDCl3-d): δ 0.99 (d, J = 6.72 Hz, 6H, -CH(CH3)2, 77%), 0.88 (d, J = 6.72 Hz, 6H, -CH(CH3)2, 23%), 2.00 (m, 1H, -CH-), 2.07 (s, 3H, -CH3, 77%), 2.14 (s, 3H, -CH3, 23%), 2.15 (s, 3H, -CH3, 23%), 2.19 (s, 3H, -CH3, 77%), 4.07 (d, 2H, -CH-CH2-, 77%), 3.90 (d, 2H, -CH-CH2-, 23%), 5.47 (s, 2H, -N-CH2-, 77%), 5.12 (s, 2H, -N-CH2-, 23%), 5.65 (s, 1H, =CH, 77%), 5.72 (s, 1H, =CH, 23%), 7.40–7.50 (m, 4H, naphthyl), 7.7–7.8 (m, 2H, naphthyl), 7.97 (s, 1H, naphthyl). 13C NMR (100 MHz, CDCl3-d): δ 153.14, 147.44, 139.68, 133.49, 132.99, 130.95, 128.64, 128.50, 127.73, 127.58, 127.49, 127.46, 127.21, 126.64, 126.56, 126.08, 125.60, 124.24, 105.74, 105.60, 81.15, 81.19, 53.01, 44.80, 28.13, 27.98, 19.23, 19.16, 13.45, 11.19. HRMS (ESI): m/z calcd for C21H25N3O [M+H]+: 336.2076; found: 336.2065.
1-Phenyl-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone O-2-methylpropyl oxime (5b)
Yield: 63%; white viscous liquid; IR (KBr) cm−1: 3013, 2959, 1557, 1026, 926. 1H NMR (400 MHz, CDCl3-d): δ 0.95 (d, J = 6.72 Hz, 6H, -CH(CH3)2), 2.04–2.10 (m, 4H, -CH, CH3), 2.16 (s, 3H, -CH3), 4.02 (d, J = 6.72, 2H, -CH-CH2-), 5.34 (s, 2H, -CH2-N), 5.66 (s, 1H, =CH), 7.22–7.30 (m, 3H, phenyl), 7.47–7.51 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 153.19, 147.32, 133.68, 133.45, 129.08, 128.14, 127.15, 105.45, 81.40, 44.85, 28.08, 19.17, 13.43, 10.83. HRMS (ESI): m/z calcd for C17H23N3O [M+H]+: 286.1919; found: 286.1910.
1-(4-Fluorophenyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanoneO-2-methylpropyl oxime (5c)
Yield: 56%; white viscous liquid; IR (KBr) cm−1: 3014, 2960, 1604, 1050, 926. 1H NMR (400 MHz, CDCl3-d): δ 0.97 (d, J = 6.72 Hz, 6H, -CH(CH3)2), 2.06 (s, 3H, -CH3-), 2.01-2.11 (m, 4H, -CH, CH3), 4.01 (d, J = 6.78, 2H, -CH-CH2-), 5.32 (s, 2H,-N-CH2), 5.68 (s, 1H, =CH), 6.90–6.97 (m, 2H, phenyl), 7.45–7.52 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 163.30 (d, 1JF-Ar = 248.83 Hz), 152.55, 147.55, 139.58, 129.74, 129.71, 129.17, 129.08, 115.27, 115.06, 105.56, 81.45, 44.81, 28.07, 19.16, 13.42, 10.80. HRMS (ESI): m/z calcd for C17H22FN3O [M+H]+: 304.1825; found: 304.18.
1-(4-Chlorophenyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanoneO-2-methylpropyl oxime (5d)
Yield: 63%; mp: 55–58 °C, white powder; IR (KBr) cm−1: 3010, 2960, 1551, 1009, 851. 1H NMR (400 MHz, CDCl3-d): δ 0.96 (d, J = 6.72 Hz, 6H, -CH(CH3)2), 2.01-2.11 (m, 4H, -CH, CH3), 4.02 (d, J = 6.76, 2H, -CH-CH2), 5.31 (s, 2H, N-CH2), 5.68 (s, 1H, =CH), 7.20–7.24 (m, 2H, phenyl), 7.44–7.48 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 157.37, 147.58, 139.56, 135.16, 132.10, 128.46, 128.37, 105.62, 81.55, 44.60, 28.07, 19.15, 13.43, 10.81. HRMS (ESI): m/z calcd for C17H22ClN3O [M+H]+: 320.1530; found: 320.1521.
1-(4-Methylphenyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanoneO-2-methylpropyl oxime (5e)
Yield: 52%; yellow viscous liquid; IR (KBr) cm−1: 3005, 2922, 1555, 1030, 874. 1H NMR (400 MHz, CDCl3-d): δ 0.97 (d, J = 6.74 Hz, 6H, -CH(CH3)2), 2.03–2.10 (m, 4H, -CH, CH3), 2.17 (s, 3H, -CH3), 2.29 (s, 3H, C6H4-CH3), 4.01 (d, J = 6.78, 2H, -CH-CH2), 5.33 (s, 2H, -N-CH2), 5.67 (s, 1H, =CH), 7.03–7.08 (m, 2H, phenyl), 7.37–7.42 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 153.28, 147.30, 139.56, 139.07, 130.78, 128.88, 126.98, 105.43, 81.30, 44.80, 28.07, 21.27, 19.19, 13.45, 10.83. HRMS (ESI): m/z calcd for C18H25N3O [M+H]+: 300.2076; found: 300.2078.
1-(4-Methoxyphenyl)-2-(1H-3,5-dimetylpyrazol-1-yl)ethanone O-2-methylpropyl oxime (5f)
Yield: 45%; yellow viscous liquid; IR (KBr) cm−1: 3004, 2957, 1608, 1040, 874. 1H NMR (400 MHz, CDCl3-d): δ 0.96 (d, J = 6.72 Hz, 6H, -CH(CH3)2), 2.02–2.09 (m, 4H, -CH, CH3), 2.17 (s, 3H, -CH3), 3.76 (s, 3H, C6H4-OCH3), 3.98 (d, J = 6.76, 2H, -CH-CH2), 5.32 (s, 2H, -N-CH2), 5.67 (s, 1H, =CH), 6.74–6.80 (m, 2H, phenyl), 7.42–7.47 (m, 2H, phenyl). 13C NMR (100 MHz, CDCl3-d): δ 160.30, 152.93, 147.32, 139.60, 128.52, 126.14, 113.57, 105.46, 81.24, 55.18, 44.82, 28.06, 19.20, 13.46, 10.82. HRMS (ESI): m/z calcd for C18H25N3O2 [M+H]+: 216.2025; found: 216.2020.

3.2. Activity

3.2.1. MTT Assay

All of the cell lines were purchased from the American Type Cell Culture (ATCC). With this assay, it is possible to determine the ability of a cell line to grow and live in vitro. It is principally based on the staining of living cells with MTT. Additionally, it is taken up by living cells and metabolized to formazan crystals. These formed crystals are dissolved with acidic 2-propanol, which helps photometrically determine the number of viable cells after the treatment. This method was used to study the A549, HCT 116, HeLa, MCF7, C6, and SH-SY5Y cancer and L929 healthy cell lines [30]. Towards that end, 10 µL of sterile MTT solution (prepared in 5 mg/mL PBS) was pipetted into a 96-well plate (well) and remained in an incubator for 4 h. Then, the media in all of the wells were removed (adherent cells), and 110 µL (prepared with 0.04 N HCl and isopropanol) was pipetted into all of the wells. The formazan crystals were dissolved by removing the solution in the wells. It was measured at 540 nm (or 570 nm) (against the 690 nm reference) in an ELISA Plate Reader. Cells in the untreated wells were used as the reference [31].

3.2.2. Flow Cytometry

The flow cytometric analysis was performed using Annexin-V/PI commercial kits. Compound 5f (at a concentration of IC50 value) was treated on the C6 cell lines for 24 h. At the end of this period, the content of the plate was removed, and the cells were washed with phosphate-buffered saline (PBS), treated with trypsin-EDTA, and centrifuged at 800 rpm for 8 min. The obtained cells were counted with trypan blue, and then 5 μL of Annexin V and 5 μL of propidium iodide (PI) were added. They were kept in the dark at room temperature for 15 min. After incubation, 400 μL of the binding buffer was added over ice, and flow cytometry measurements were performed (emission: 530 nm, excitation: 488 nm) [32,33,34].

3.2.3. Cell Cycle Analysis

The C6 cells were seeded in a 6-well plate at a concentration of 106 cells per well and incubated for 24 h. Then, the IC50 values of the samples were applied and the cells were left to incubate again for 24 h. After incubation, the medium and sample were removed, the cells were washed three times with 5 mL of PBS, and 1 mL of trypsin-EDTA solution was added to the plates. Thereafter, the cells were collected in falcon tubes with 4 mL of Dulbecco’s Modified Eagle Medium (DMEM) in each tube, and the cell suspensions were centrifuged at 300 g for 5 min. Following this step, each 10 μL of the cell suspensions were mixed with 10 μL of trypan blue, and the cell counts were made in Neubauerlam. The number of cells in each tube was set to be 106. The cells were maintained at −20 °C for 3 h and all of the supernatants were carefully removed. After the supernatant was dissolved in 1 mL of PBS, it was placed in a 200 µL muse cell cycle kit, incubated for 30 min at room temperature, and read using the flow cytometry. The results were given as % cells [35,36].

4. Conclusions

In this study, 12 new 1-aryl-2-(3,5-dimethylpyrazol-yl)ethanone oxime ether derivatives were synthesized. Their cytotoxic effects were evaluated against the A549, HCT 116, HeLa, MCF7, C6, and SH-SY5Y cell lines. The methyl and isobutyl ether derivatives of the oxime groups were synthesized to obtain more lipophilic compounds. Moreover, it was determined that, in general, the isobutyl oxime ether derivatives had higher cytotoxic activities in the C6 cell line. This finding is interesting in terms of the development of new anticancer compounds with better properties stemming from improving the properties of the active molecules. In the L929 healthy cell line, it was observed that the compounds did not show cytotoxic effects. These results show that 1-aryl-2-(3,5-dimethylpyrazol-1-yl)ethanone derivatives may have anticancer effects. In particular, compound 5f induces cell cycle arrest and may be effective in the treatment of glioma.
Based on the literature, we thought that ethanone derivatives containing 3,5-dimethylpyrazole could have an anticancer activity. In this study, we first evaluated the effect of aryl groups on the activity. In future studies, the effect of methyl substitutions on the pyrazole ring on the activity will be evaluated by adding another substituted pyrazole ring rather than 3,5-dimethylpyrazole. Furthermore, the study is aimed at developing more effective compounds by synthesizing oxime ether derivatives containing various aryl and alkyl groups.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/pr9112019/s1. Table S1: Percent viability findings of synthesized compounds in the L929 cell line. Supplementary Figures (Data 1~72): 1H-NMR, 13C-NMR, and Mass spectra of the compounds tested.

Author Contributions

Conceptualization, M.A.A. (Mehmet Abdullah Alagöz), A.K. (Arzu Karakurt), and T.Ö.; synthesis, M.A.A. (Mehmet Abdullah Alagöz), A.K. (Arzu Karakurt), T.Ö., and B.M.; biological activity, M.A.A. (Mehmet Abdullah Alagöz), E.Ş., C.H., M.M.G., and M.A.A. (Mohamed A. Abdelgawad); original draft writing, M.A.A. (Mehmet Abdullah Alagöz), A.K. (Ahamed Khames), and B.M.; review and editing, H.K. and B.M.; supervision, H.K.; funding acquisition, H.K. and M.M.G. All authors have read and agreed to the published version of the manuscript.

Funding

Taif University Researchers Supporting Project number (TURSP-2020/68), Taif University, Taif, Saudi Arabia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study have been included in this article.

Acknowledgments

The authors acknowledge Taif University Researchers Supporting Project number (TURSP-2020/68), Taif University, Taif, Saudi Arabia. The authors also thank AlMaarefa University, Ad Diriyah 13713, Saudi Arabia, for its support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. World Health Organization. Cancer. 2021. Available online: https://www.who.int/news-room/fact-sheets/detail/cancer (accessed on 18 May 2021).
  2. Devan, A.R.; Kumar, A.R.; Nair, B.; Anto, N.P.; Muraleedharan, A.; Mathew, B.; Kim, H.; Nath, L.R. Insights into an Immunotherapeutic Approach to Combat Multidrug Resistance in Hepatocellular Carcinoma. Pharmaceuticals 2021, 14, 656. [Google Scholar] [CrossRef]
  3. Pérez-Herrero, E.; Fernández-Medarde, A. Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy. Eur. J. Pharm. Biopharm. 2015, 93, 52–79. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Alagöz, M.A.; Özdemir, Z.; Uysal, M.; Carradori, S.; Gallorini, M.; Ricci, A.; Zara, S.; Mathew, B. Synthesis, Cytotoxicity and Anti-Proliferative Activity against AGS Cells of New 3(2H)-Pyridazinone Derivatives Endowed with a Piperazinyl Linker. Pharmaceuticals 2021, 14, 183. [Google Scholar] [CrossRef] [PubMed]
  5. Oh, J.M.; Rangarajan, T.M.; Chaudhary, R.; Singh, R.P.; Singh, M.; Singh, R.P.; Tondo, A.R.; Gambacorta, N.; Nicolotti, O.; Mathew, B.; et al. Novel Class of Chalcone Oxime Ethers as Potent Monoamine Oxidase-B and Acetylcholinesterase Inhibitors. Molecules 2020, 25, 2356. [Google Scholar] [CrossRef]
  6. Abele, E.; Abele, R.; Golomba, L.; Višņevska, J.; Beresneva, T.; Rubina, K.; Lukevics, E. Oximes of six-membered heterocyclic compounds with two and three heteroatoms. ii. Reactions and biological activity (review). Chem. Het. Comp. 2010, 46, 769–792. [Google Scholar] [CrossRef]
  7. Abele, E.; Abele, R.; Lukevics, E. Oximes of five-membered heterocyclic compounds with three and four heteroatoms 2 synthesis of derivatives, reactions, and biological activity (review). Chem. Het. Comp. 2008, 44, 905–930. [Google Scholar] [CrossRef]
  8. Shamsuzzaman, S.; Siddiqui, T.; Alam, M.G.; Dar, A.M. Synthesis, characterization and anticancer studies of new steroidal oxadiazole, pyrrole and pyrazole derivatives. J. Saudi Chem. Soc. 2015, 19, 387–391. [Google Scholar] [CrossRef] [Green Version]
  9. Shen, S.L.; Zhu, J.; Li, M.; Zhao, B.Z.; Miao, J.Y. Synthesis of ferrocenyl pyrazole-containing chiral aminoethanol derivatives and their inhibition against A549 and H322 lung cancer cells. Eur. J. Med. Chem. 2012, 54, 287–294. [Google Scholar] [CrossRef] [PubMed]
  10. Ding, X.L.; Zhang, H.Y.; Qi, L.; Zhao, B.X.; Lian, S.; Lv, H.S.; Miao, J.Y. Synthesis of novel pyrazole carboxamide derivatives and discovery of modulators for apoptosis or autophagy in A549 lung cancer cells. Bioorg. Med. Chem. Lett. 2010, 20, 4766–4770. [Google Scholar] [CrossRef]
  11. Kumar, V.; Kaur, K.; Karelia, D.N.; Beniwal, V.; Gupta, G.; Sharma, A.K.; Gupta, A. Synthesis and biological evaluation of some 2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-arylethanones: Antibacterial, DNA photocleavage, and anticancer activities. Eur. J. Med. Chem. 2014, 81, 267–276. [Google Scholar] [CrossRef]
  12. Karrouchi, K.; Radi, S.; Ramli, Y.; Taoufik, J.; Mabkhot, Y.N.; Al-aizari, F.A.; Ansar, M. Synthesis and pharmacological activities of pyrazole derivatives: A review. Molecules 2018, 23, 134. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Khan, M.F.; Alam, M.M.; Verma, G.; Akhtar, W.; Akhter, M.; Shaquiquzzaman, M. The therapeutic voyage of pyrazole and its analogs: A review. Eur. J. Med. Chem. 2016, 120, 170–201. [Google Scholar] [CrossRef] [PubMed]
  14. Chauhan, S.; Paliwal, S.; Chauhan, R. Anticancer activity of pyrazole via different biological mechanisms. Synth. Comm. 2014, 44, 1333–1374. [Google Scholar] [CrossRef]
  15. Naim, M.J.; Alam, O.; Nawaz, F.; Alam, J.; Alam, P. Current status of pyrazole and its biological activities. J. Pharm. Bioallied. Sci. 2016, 8, 2–17. [Google Scholar] [CrossRef]
  16. Faria, J.V.; Vegi, P.F.; Miguita, A.C.G.; dos Santos, M.S.; Boechat, N.; Bernardino, A.M.R. Recently reported biological activities of pyrazole compounds. Bioorg. Med. Chem. 2017, 25, 5891–5903. [Google Scholar] [CrossRef]
  17. Yipel, M.; Ghica, M.V.; Kaya, M.G.A.; Spoiala, A.; Radulescu, M.; Ficai, D.; Ficai, A.; Bleotu, C.; Cornelia, N. Multifunctional materials for cancer therapy: From antitumoral agents to ınnovative administration. Curr. Org. Chem. 2016, 20, 2934–2948. [Google Scholar] [CrossRef]
  18. Elli, E.M.; Baratè, C.; Mendicino, F.; Palandri, F.; Palumbo, G.A. Mechanisms underlying the anti-inflammatory and immunosuppressive activity of ruxolitinib. Front. Oncol. 2019, 9, 1–10. [Google Scholar] [CrossRef] [Green Version]
  19. Lang, D.K.; Kaur, R.; Arora, R.; Saini, B.; Arora, S. Nitrogen-containing heterocycles as anticancer agents: An overview. Anticancer. Agents Med. Chem. 2020, 20, 2150–2168. [Google Scholar] [CrossRef] [PubMed]
  20. Aydin, S.; Kaushik-Basu, N.; Özbas-Turan, S.; Akbuğa, J.; Tiber, P.M.; Orun, O.; Gurukumar, K.R.; Basub, A.; Küçükgüzel, G. Synthesis of 1-aroyl-3,5-dimethyl-1H-pyrazoles as anti-HCV and anticancer agents. Lett. Drug Des. Discov. 2014, 11, 121–131. [Google Scholar] [CrossRef]
  21. Dai, H.; Ge, S.; Li, G.; Chen, J.; Shi, Y.; Ye, L.; Ling, Y. Synthesis and bioactivities of novel pyrazole oxime derivatives containing a 1,2,3-thiadiazole moiety. Bioorg. Med. Chem. Lett. 2016, 26, 4504–4507. [Google Scholar] [CrossRef]
  22. Kumar, K.S.; Priya, N.P.; Aravindakshan, K.K. Synthesis, crystal structure, and in vitro cytotoxic, antitumor and antimicrobial evaluation of a new pyrazole derivative N-3,5-trimethyl-N-phenyl-1-H-pyrazole-1-carbothioamide. Pharm. Chem. J. 2021, 54, 1079–1086. [Google Scholar] [CrossRef]
  23. Singh, P.; Paul, K.; Holzer, W. Synthesis of pyrazole-based hybrid molecules: Search for potent multidrug resistance modulators. Bioorg. Med. Chem. 2006, 14, 5061–5071. [Google Scholar] [CrossRef] [PubMed]
  24. Ali, A.R.; El-Bendary, E.R.; Ghaly, M.A.; Shehata, I.A. Synthesis, in vitro anticancer evaluation and in silico studies of novel imidazo[2,1-b]thiazole derivatives bearing pyrazole moieties. Eur. J. Med. Chem. 2014, 75, 492–500. [Google Scholar] [CrossRef] [PubMed]
  25. Karakurt, A.; Bozbey, İ.; Uslu, H.; Sari, S.; Özdemir, Z.; Şalva, E. Synthesis and cytotoxicity studies on new pyrazole-containing oxime ester derivatives. Trop. J. Pharm. Res. 2019, 18, 1315–1322. [Google Scholar] [CrossRef]
  26. Karakurt, A.; Dalkara, S.; Özalp, M.; Kendi, E.; Stables, J.P. Synthesis of some 1-(2-naphyl)-2-(imidazole-1yl)etanon oxime and oxime ether derivatives and their anticonvulsant and antimicrobial activities. Eur. J. Med. Chem. 2001, 36, 421–433. [Google Scholar] [CrossRef]
  27. Sharma, T.; Kumar, R.; Sahoo, S.C.; Sindhu, J.; Singh, J.; Singh, B.; Kumar-Mehta, S.; Umar, A.; Singh-Saini, T.; Kumar, V.; et al. Synthesis, structural and pharmacological exploration of 2-(3, 5-dimethyl-1H-pyrazol-1-yl)-acetophenone oximes and their silver complexes. Polyhedron 2021, 195, 114972. [Google Scholar] [CrossRef]
  28. Özdemir, Z.; Karakurt, A.; Çalış, Ü.; Günal, G.; Işık, Ş.; Şahin, Z.S.; Dalkara, S. Synthesis, anticonvulsant and antimicrobial activities of some new [1-(2naphthyl)-2-(pyrazol-1-yl)ethanone]oxime ethers. Med. Chem. 2015, 9, 41–49. [Google Scholar] [CrossRef]
  29. Karakurt, A.; Özalp, M.; Işık, Ş.; Stables, J.P.; Dalkara, S. Synthesis, anticonvulsant and antimicrobial activities of some new 2-acetylnaphthalene derivatives. Bioorg. Med. Chem. 2010, 18, 2902–2911. [Google Scholar] [CrossRef]
  30. Zhang, W.; Torabinejad, M.; Li, T. Evaluation of cytotoxicity of MTAD using the MTT-tetrazolium method. J. Endod. 2003, 29, 654–657. [Google Scholar] [CrossRef]
  31. van Meerloo, J.; Kaspers, G.J.; Cloos, J. Cell sensitivity assays: The MTT assay. Methods Mol. Biol. 2011, 731, 237–245. [Google Scholar] [CrossRef]
  32. Akalin-Çiftçi, G.; İşcan, A.; Kutlu, M. Escin reduces cell proliferation and induces apoptosis on glioma and lung adenocarcinoma cell lines. Cytotechnology 2015, 67, 893–904. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Han, Y.; Xu, J.; Li, Z.; Ren, G.; Yang, Z. In vitro toxicity of multi-walled carbon nanotubes in C6 rat glioma cells. Neurotoxicology 2012, 33, 1128–1134. [Google Scholar] [CrossRef]
  34. McKinnon, K.M. Flow Cytometry: An Overview. Curr. Prot. Immun. 2018, 120, 5-1. [Google Scholar] [CrossRef] [PubMed]
  35. Malumbres, M.; Barbacid, M. Cell cycle, CDKs and cancer: A changing paradigm. Nat. Rev. Cancer 2009, 9, 153–166. [Google Scholar] [CrossRef] [PubMed]
  36. Diaz-Moralli, S.; Tarrado-Castellarnau, M.; Miranda, A.; Cascante, M. Targeting cell cycle regulation in cancer therapy. Pharmacol. Ther. 2013, 138, 255–271. [Google Scholar] [CrossRef]
Processes 09 02019 g001 550
Figure 1. Structure of drugs with an oxime ether structure: fluvoxamine (1), oxiconazole (2), cefuroxime (3), and aztreonam (4).
Figure 1. Structure of drugs with an oxime ether structure: fluvoxamine (1), oxiconazole (2), cefuroxime (3), and aztreonam (4).
Processes 09 02019 g001
Processes 09 02019 g002 550
Figure 2. Chemical structures of anticancer drugs with an azole moiety: letrozole (5), ruxolitinib (6), fadrozole (7), crizotinib (8), and pralsetinib (9).
Figure 2. Chemical structures of anticancer drugs with an azole moiety: letrozole (5), ruxolitinib (6), fadrozole (7), crizotinib (8), and pralsetinib (9).
Processes 09 02019 g002
Processes 09 02019 g003 550
Figure 3. Chemical structures of cytotoxic compounds with a dimethylpyrazole ring. (2’,4’-difluoro-4-hydroxy-[1,1’-biphenyl]-3-yl)(3,5-dimethyl-1H-pyrazol-1-yl)methanone (10), (E)-1,3-dimethyl-5-phenyl-1H-pyrazole-4-carbaldehyde O-((1,2,3-thiadiazol-5-yl)methyl) oxime derivatives (11), N,3,5-trimethyl-N-phenyl-1H-pyrazole-1-carbothioamide (12), 3,5-dimethyl-4-nitroso-1-phenyl-1H-pyrazole (13).
Figure 3. Chemical structures of cytotoxic compounds with a dimethylpyrazole ring. (2’,4’-difluoro-4-hydroxy-[1,1’-biphenyl]-3-yl)(3,5-dimethyl-1H-pyrazol-1-yl)methanone (10), (E)-1,3-dimethyl-5-phenyl-1H-pyrazole-4-carbaldehyde O-((1,2,3-thiadiazol-5-yl)methyl) oxime derivatives (11), N,3,5-trimethyl-N-phenyl-1H-pyrazole-1-carbothioamide (12), 3,5-dimethyl-4-nitroso-1-phenyl-1H-pyrazole (13).
Processes 09 02019 g003
Processes 09 02019 g004 550
Figure 4. The 2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-arylethanone derivatives (14) and oxime ester derivatives carrying a pyrazole ring with cytotoxic activity (15).
Figure 4. The 2-(3,5-dimethyl-1H-pyrazol-1-yl)-1-arylethanone derivatives (14) and oxime ester derivatives carrying a pyrazole ring with cytotoxic activity (15).
Processes 09 02019 g004
Processes 09 02019 g005 550
Figure 5. Chemical structure of the designed compounds (4a-4f; 5a-5f).
Figure 5. Chemical structure of the designed compounds (4a-4f; 5a-5f).
Processes 09 02019 g005
Processes 09 02019 sch001 550
Scheme 1. Synthetic route.
Scheme 1. Synthetic route.
Processes 09 02019 sch001
Processes 09 02019 g006 550
Figure 6. Treatment of the C6 cell line with compound 5f for 24 h via quantification of apoptotic cells via the flow cytometric analysis.
Figure 6. Treatment of the C6 cell line with compound 5f for 24 h via quantification of apoptotic cells via the flow cytometric analysis.
Processes 09 02019 g006
Processes 09 02019 g007 550
Figure 7. Cell cycle analysis of C6 cells treated with compound 5f.
Figure 7. Cell cycle analysis of C6 cells treated with compound 5f.
Processes 09 02019 g007
Table
Table 1. Inhibitions of 4a–5f against MCF-7, A549, HCT116, and HeLa cancer cell lines after 24 h.
Table 1. Inhibitions of 4a–5f against MCF-7, A549, HCT116, and HeLa cancer cell lines after 24 h.
CompoundsIC50 (µM)
MCF-7A549HCT116Hela
4a218.80 ± 4.87117.20 ± 7.98185.70 ± 10.65126.30 ± 1.08
4b212.20 ± 3.89122.30 ± 6.54-190.10 ± 0.76
4c545.40 ± 6.98392.70 ± 7.08-211.00 ± 10.56
4d-302.20 ± 7.09-90.33 ± 9.06
4e438.10 ± 1.76369.90 ± 9.06404.30 ± 8.7990.14 ± 7.98
4f----
5a149.10 ± 10.08157.90 ± 9.0884.25 ± 7.4872.20 ± 12.67
5b--75.64 ± 4.9897.70 ± 15.78
5c456.70 ± 16.97185.30 ± 9.7558.99 ± 3.98137.50 ± 17.76
5d173.30 ± 2.98151.80 ± 6.98112.10 ± 10.8977.29 ± 9.65
5e250.00 ± 3.96145.50 ± 3.2099.10 ± 9.07113.10 ± 2.35
5f305.50 ± 3.9766.08 ± 2.89328.30 ± 13.31-
5-FU11.75 ± 0.348.34 ± 1.097.86 ± 0.5435.75 ± 2.98
“-“: Not determined.
Table
Table 2. Inhibitions of 4a–5f against C6 and SH-SY5Y cancer cell lines after 24 h.
Table 2. Inhibitions of 4a–5f against C6 and SH-SY5Y cancer cell lines after 24 h.
CompoundsIC50 (µM)CompoundsIC50 (µM)
C6SH-SY5YC6SH-SY5Y
4a7.93 ± 0.855.52 ± 0.235a19.01 ± 0.485.00 ± 0.37
4b24.31 ± 0.949.69 ± 0.875b6.02 ± 0.8510.08 ± 0.34
4c7.16 ± 0.848.71 ± 0.955c6.56 ± 0.928.55 ± 0.45
4d7.42 ± 0.399.52 ± 0.345d5.45 ± 0.199.02 ± 0.78
4e6.91 ± 0.0929.85 ± 1.235e14.15 ± 0.1320.28 ± 1.09
4f6.21 ± 0.9410.03 ± 0.545f5.13 ± 0.348.56 ± 0.78
5-FU8.34 ± 0.378.53 ± 0.43
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Alagöz, M.A.; Karakurt, A.; Hepokur, C.; Şalva, E.; Önkol, T.; Ghoneim, M.M.; Abdelgawad, M.A.; Khames, A.; Kim, H.; Mathew, B. Synthesis of New 1-Aryl-2-(3,5-dimethylpyrazol-1-yl)ethanone Oxime Ether Derivatives and Investigation of Their Cytotoxic Effects. Processes 2021, 9, 2019. https://doi.org/10.3390/pr9112019

AMA Style

Alagöz MA, Karakurt A, Hepokur C, Şalva E, Önkol T, Ghoneim MM, Abdelgawad MA, Khames A, Kim H, Mathew B. Synthesis of New 1-Aryl-2-(3,5-dimethylpyrazol-1-yl)ethanone Oxime Ether Derivatives and Investigation of Their Cytotoxic Effects. Processes. 2021; 9(11):2019. https://doi.org/10.3390/pr9112019

Chicago/Turabian Style

Alagöz, Mehmet Abdullah, Arzu Karakurt, Ceylan Hepokur, Emine Şalva, Tijen Önkol, Mohammed M. Ghoneim, Mohamed A. Abdelgawad, Ahmed Khames, Hoon Kim, and Bijo Mathew. 2021. "Synthesis of New 1-Aryl-2-(3,5-dimethylpyrazol-1-yl)ethanone Oxime Ether Derivatives and Investigation of Their Cytotoxic Effects" Processes 9, no. 11: 2019. https://doi.org/10.3390/pr9112019

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop