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. 2025 Mar 20;10(12):12671–12678. doi: 10.1021/acsomega.5c00320

Derivatives of Pyrazole-Based Compounds as Prospective Cancer Agents

Lesetja V Ramoba †, Wakopo J Nzondomyo †, Karabo Serala ‡, Lucy W Macharia ‡, Supratim Biswas ‡, Sharon Prince ‡, Frederick P Malan §, Orbett T Alexander ∥, Amanda-Lee E Manicum †,*
PMCID: PMC11966572  PMID: 40191378

Abstract

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Five pyrazole-based compounds, 3,5-dimethyl-1H-pyrazole, L1; 3,5-diphenyl-1H-pyrazole, L2; 3-(trifluoromethyl)-5-phenyl-1H-pyrazole, L3; 3-(trifluoromethyl)-5-methyl-1H-pyrazole, L4; and 3,5-ditert-butyl-1H-pyrazole, L5 were synthesized from a typical condensation reaction of β-diketone derivatives with hydrazine hydrate reagent and characterized using various spectroscopic techniques such as FT-IR, UV–vis, 1H and 13C NMR, and LC–MS spectroscopy. L1 was further analyzed by single-crystal X-ray diffraction, and the N1–N1′ bond distance was found to be 1.361(3) Å and correlated well with other pyrazole-based compounds. The short-term cytotoxicity of 10 μM pyrazole compounds (L1–L5) was evaluated against pancreatic (CFPAC-1 and PANC-1), breast (MDA-MB-231 and MCF-7), and cervical (CaSki and HeLa) cancer cell lines using the MTT cell viability assay. Cisplatin and gemcitabine were included as positive control drugs followed by the determination of the half-maximal effective concentrations of prospective compounds. L2 and L3, respectively, displayed moderate cytotoxicity against CFPAC-1 (61.7 ± 4.9 μM) and MCF-7 (81.48 ± 0.89 μM) cell lines.

1. Introduction

Pyrazoles are five-membered heterocyclic compounds derived from a simple acid-catalyzed condensation reaction of β-diketone and hydrazine derivatives and are usually synthesized using a method introduced by Knorr (1883).1 Since the inception of his procedure, alternative synthetic pathways to obtain pyrazole compounds have been developed through numerous modifications of the method by the aforementioned pioneer. One of the modified procedures was conducted by Girish and co-workers,2 where they used a nano-ZnO catalyst for the synthesis of 1,3,5-substituted pyrazole derivatives. The main advantages of this protocol are the high yield (95%) and reduced reaction time, which are almost similar to the method employed in this study. The other method that can also be considered is the one brought forward by Huang and Katzenellenbogen,3 where a series of 4-alkyl-1,3,5-triarylpyrazoles in a regioselective fashion through the oxidation of pyrazolines were synthesized. The method first involves the preparation of pyrazolines via a cyclocondensation reaction between phenyl and 4-methoxyphenylhydrazine and chalcones, followed by alkylation at the C-4 position of the pyrazoline ring. The setback about this method, in contrast to the one employed in this study, is that it involves multiple steps prior to the final product as compared to the one-pot synthesis. Their structures involve the –C=N– functional group incorporated in a ring moiety with adjacent electron pair-bearing nitrogen atoms, whose basicity can be influenced by substituents on the ring backbone.4−6 These compounds are useful precursors for the synthesis of many heterocyclic products such as 1-aryl-1H-pyrazole derivative or as chelator ligands for the preparation of coordination compounds.7−10 In particular, the presence of the –C=N– functional moiety with two lone pairs of electrons on the nitrogen atoms makes these types of compounds significant in coordination and medicinal chemistry.11−15 A wide range of pharmacological activities of pyrazole compounds has been reported in the literature, including antibacterial, antifungal, anti-inflammatory, and anticancer properties for the treatment of various biological health concerns.16−23 Similar to pyrazole derivatives synthesized in this study, 4,5-dihydro-2H-pyrazole-2-hydroxyphenyl derivatives, namely, (1-(5-(5-chloro-2-hydroxyphenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone) and (1-(3-(4-chlorophenyl)-5-(3,5-dibromo-2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone), synthesized from hydrazine hydrate and chalcones by Liu et al. (2012) showed remarkable antiproliferative effects against WM266.5 (human melanoma cell line) and MCF-7 (human breast cancer cell line). The reported IC50 values for (1-(5-(5-chloro-2-hydroxyphenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone) were, respectively, 1.31 and 0.45 μM against MCF-7 and WM266.5 and 0.97 and 0.72 μM against MCF-7 and WM266.5 for (1-(3-(4-chlorophenyl)-5-(3,5-dibromo-2-hydroxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone).24 Due to the ability of pyrazoles to form stable complexes with metal ions, numerous pyrazole-based complexes exhibit excellent catalytic activity in various reactions. Over the past few years, there have been many research studies on their applications in homogeneous and heterogeneous catalysis.25−27

Based on the above literature information, we decided to synthesize a series of derivatized pyrazole compounds: 3,5-dimethyl-1H-pyrazole, L1; 3,5-diphenyl-1H-pyrazole, L2; 3-(trifluoromethyl)-5-phenyl-1H-pyrazole, L3; 3-(trifluoromethyl)-5-methyl-1H-pyrazole, L4; and 3,5–3,5-ditert-butyl-1H-pyrazole, L5. The compounds were characterized using various spectroscopic techniques such as FT-IR, UV–vis, 1H and 13C NMR, and LC–MS. Furthermore, the crystallographic analysis on L1 gives an insight on the pyrazole solid-state intermolecular interactions, which highlights the observed high stability of these compounds through the observed dense hydrogen bonding network in the asymmetric unit cell. Additionally, the anticancer activities of the synthesized pyrazole compounds were investigated on different cancer cell lines to assess their potential cytotoxicity. The synthesized pyrazole derivatives, due to their understood strong coordinating abilities, will at a later stage be used for chelation to the rhenium tricarbonyl metal ion to evaluate the sensitization of these compounds toward the inert fac-[Re(CO)3]+ synthon.

2. Experimental Section

2.1. Materials and Methods

The following chemicals pentane-2,4-dione; 2,2,6,6-tetramethylheptane-3,5-dione; 1,1,1-trifluoropentane-2,4-dione; 1,5-diphenylacetylacetone; 4,4,4-trifluoro-1-phenylbutane-1,3-dione; hydrazine hydrate; and organic solvent (ethanol) used to synthesize the pyrazole compounds were purchased from Sigma-Aldrich and were used without any further purification. 1H and 13C NMR spectra were determined in CDCl3 and DMSO-d6 with an internal TMS standard using a 300 MHz Bruker Avance spectrometer. The UV–vis and IR data were obtained using PerkinElmer UV–vis and FT-IR spectrophotometers, respectively.

2.2. Synthesis of the Pyrazole-Based Compounds

The reported pyrazole compounds were synthesized according to the published procedure detailed by Knorr et al. (1883).1 They were synthesized from a simple condensation reaction of various β-diketone derivatives and hydrazine hydrate in ethanol at refluxing temperatures for the successful substitution of the carbonyl groups by amine analogues. 5.00 mL (26.3 mmol) of hydrazine hydrate was added dropwise to an equivalent amount (1:1) of various β-diketone (pentane-2,4-dione; 2,2,6,6-tetramethylheptane-3,5-dione; 1,1,1-trifluoropentane-2,4-dione; 1,5-diphenylacetylacetone; and 4,4,4-trifluoro-1-phenylbutane-1,3-dione) derivatives in 20 mL of ethanol (Scheme 1). The reaction was refluxed for 7 h, and the precipitates that formed were filtered and washed with cold ethanol, followed by drying of the solids. White cuboid crystals suitable for single-crystal X-ray diffraction were collected and analyzed for L1, L2, L3, and L5; however, only the crystal structure of L1 is reported since the other structures are already reported in the literature.

Scheme 1. Illustration of the Synthesized Pyrazole Compounds, L1–L5.

Scheme 1

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2.2.1. Synthesis of 3,5-Dimethyl-1H-pyrazole, L1

IR νC=N (cm–1) = 1600, νN–N (cm–1) = 1148; 13C NMR (CDCl3): δC (ppm) (–C=N–) 144.2, (=C(methine)–) 103.9, (−C(methyl)) 12.1; 1H NMR (400 MHz, CDCl3) δH (N–H) 12.38 (s, 1H), (–C=CH−) 5.83 (s, 1H), (−CH3) 2.31 (s, 6H); UV–vis: λmax = 218 nm, ε [0.0018 M] = 435 M–1·cm–1, LC–MS: m/z = found: 97.0759 ([M + H]+), 98.0814 ([M + 2H]+); calcd: 97.0766 ([M + H]+), 98.0844 ([M + 2H]+).

2.2.2. Synthesis of 3,5-Diphenyl-1H-pyrazole, L2

IR νC=N (cm–1) = 1568, νN–N (cm–1) = 1185; 13C NMR (CDCl3): δC(ppm) 154.2 (–C=N–), (−C–N–) 148.7, (–C=C(aromatic)) 130.9, 128.8, 128.2, 127.3, 126.5, 125.7, (–C=C–methine) 100.1; 1H NMR (400 MHz, CDCl3): δH (N–H) 10.96 (s, 1H), (−CH(aromatic)) 7.78–7.23 (s, 5H), (–C=CH−) 6.76 (s, 1H); UV–vis: λmax = 254 nm, ε [0.0005 M] = 1046 M–1·cm–1, LC–MS: m/z = found: 221.1051 ([M + H]+), 222.1132 ([M + 2H]+); calcd: 221.1079 ([M + H]+), 222.1157 ([M + 2H]+).

2.2.3. Synthesis of 3-(Trifluoromethyl)-5-phenyl-1H-pyrazole, L3

IR νC=N (cm–1) = 1509, νN–N (cm–1) = 1117; 13C NMR (CDCl3): δC (ppm) (–C=N–) 145.1, (–C=C–(aromatic)) 129.4, 129.3, 127.9, 125.6, 122.4, 119.7, (=C–(methine)) 101.2; 1H NMR (400 MHz, CDCl3): δH (N–H) 9.32 (s, 1H), (C–H(aromatic)) 7.62–7.34 (m, 4H), (=CH(aromatic)–) 6.73 (s, 1H); UV–vis: λmax = 252 nm, ε [0.0008 M] = 1029 M–1·cm–1, LC–MS: m/z – found: 213.0665 ([M + H]+), 214.0687 ([M + 2H]+); calcd: 213.0640 ([M + H]+), 214.0718 ([M + 2H]+).

2.2.4. Synthesis of 3-(Trifluoromethyl)-5-methyl-1H-pyrazole, L4

IR νC=N (cm–1) = 1587, νN–N (cm–1) – 1121; 13C NMR (CDCl3): δC (ppm) (–C=N–) 143.2, 142.70, 141.24 (CF3) 122.7, (–C=C–(methine))103.0, (−CH3) 10.5; 1H NMR (400 MHz, CDCl3): δH (N–H) 10.98 (s, 1H), (–C=CH–(methine)) 6.31 (s, 1H), (−CH3) 2.34 (s, 3H); UV–vis: λmax = 218 nm, ε [0.0011 M] – 685 M–1·cm–1, LC–MS: m/z = found: 151.0456 ([M + H]+), 152.0512 ([M + 2H]+); calcd: 151.0483 ([M + H]+), 152.0561 ([M + 2H]+).

2.2.5. Synthesis of 3,5-Ditert-butyl-1H-pyrazole, L5

IR νC=N (cm–1) – 1587, νN–N (cm–1) = 1129; 13C NMR (DMSO-d6): δC (ppm) (–C=N–) 160.5, (–C=C–(methine)) 96.8; C(CH3) 30.9; 1H NMR (400 MHz, CDCl3): δH (N–H) 12.00 (s, 1H), (–C=CH–(methine)) 5.82 (s, 1H), (−CH3) 1.22 (s, 18H); UV–vis: λmax = 282 nm, ε [0.0007 M] = 685 M–1·cm–1, LC–MS: m/z = found: 181.1694 ([M + H]+), 182.1763 ([M + 2H]+); calcd: 181.1705 ([M + H]+), 182.1783 ([M + 2H]+).

2.3. Single-Crystal X-ray Crystallography

A single crystal of the synthesized pyrazole compound, L1, was analyzed on a Rigaku XtaLab Synergy R diffractometer, with a rotating-anode X-ray source and a HyPix CCD detector. Data reduction and absorption were carried out using the CrysAlisPro (version 1.17.40.23a) software.28 X-ray diffraction measurement was performed at 150.00(2) K, using an Oxford Cryogenics Cryostat. The structure was solved by intrinsic phasing with SHELXTS-201329 and refined using the SHELXL-201329 algorithm. All H atoms were placed in geometrically idealized positions and were constrained to ride on their parent atoms. The X-ray crystallographic coordinates for L1 have been deposited at the Cambridge Crystallographic Data Centre (CCDC), with deposition number CSD: 2235800. The data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

2.4. Biological Studies

2.4.1. Cell Culture

The human PANC-1 pancreatic cancer (PC) cell line, MDA-MB-231 triple-negative breast cancer (BC) cells, and HeLa (HPV-18) cervical cancer (CC) cell line were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies, New York). The CFPAC-1 PC cells were grown in Iscove’s modified Dulbecco’s medium (IMDM), and the CaSki (HPV-16) CC and MCF-7 (estrogen receptor-positive) BC cell lines were grown in Roswell Park Memorial Institute medium (RPMI) (Gibco, Life Technologies, New York). The culture media were supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin and 100 μg/mL streptomycin. All cells were cultured at 37 °C in a 5% CO2-95% air-humidified incubator. Media were replaced every 2–3 days, and cells were routinely subjected to mycoplasma tests. Only mycoplasma-free cells were used in the experiments.

2.4.2. Cell Treatments

The compounds, L1–L5, were dissolved in dimethyl sulfoxide (DMSO) (Merck 48856212719) to achieve a stock concentration of 5 mM and subsequently stored at −20 °C for a maximum of 5 days before use. For cell culture treatments, the 5 mM stock solutions were diluted to final concentrations of 10 μM using respective supplemented cell culture media, and the percentage of DMSO in the 10 μM concentration was prepared and used as a vehicle control. Cisplatin (Pfizer Ltd., New York, USA) was included and used as the positive control for MCF-7, MDA-MB-231, CaSki, and HeLa cells, while gemcitabine (Sigma-Aldrich, USA) was used as a positive control for CFPAC-1 and PANC-1 cells.

2.4.3. Short-Term Cell Viability Assay

The effects of the experimental compounds L1–L5 on the viability of CFPAC-1 and PANC-1 PC cells, MDA-MB-231 and MCF-7 BC cells, and CaSki and HeLa CC cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltrazolium bromide (MTT) assay (M21281G, Sigma-Aldrich), following the manufacturer’s guidelines. Briefly, cells were seeded in 96-well plates, and 24 h later, they were treated with 10 μM of the experimental compounds or the vehicle (DMSO) for 48 h, followed by the addition of 10 μL of the 5 mg/mL MTT solution to each well and incubation for further 4 h at 37 °C. This was followed by the addition of 100 μL solubilization buffer (10% SDS in 0.01 M HCl) followed by overnight incubation at 37 °C. The spectrophotometric absorbance of the samples was determined at a wavelength of 595 nm using the Glomax plate reader (Promega, USA). The mean cell viability was calculated and expressed as a percentage of the vehicle control.

To determine the IC50 values of selected compounds (L2, L3, and L4), cells were seeded as above and treated with a range of concentrations (10–50 μM) of these compounds followed by MTT assays, as previously described. The IC50 values were determined using GraphPad Prism version 8.0 software (GraphPad Prism software, USA) from sigmoidal plots of data obtained from two independent experiments performed in quadruplicate.

2.4.4. Statistical Analysis

Data were obtained from three independent experimental repeats and analyzed by an unpaired parametric t-test using the GraphPad Prism version 8.0 software (GraphPad Prism software, USA). Error bars represent the standard deviation (SD), and significance was accepted at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

2.5. Results and Discussion

2.5.1. Synthesis

Five pyrazole compounds, L1–L5, are reported in this study. The compounds were synthesized using a typical condensation reaction of hydrazine with various β-diketone derivatives (pentane-2,4-dione; 2,2,6,6-tetramethylheptane-3,5-dione; 1,1,1-trifluoropentane-2,4-dione; 1,5-diphenylacetylacetone; and 4,4,4-trifluoro-1-phenylbutane-1,3-dione) to investigate the biological activity that is believed to have been rendered by the pyrazole function within the heterocyclic ring. Experimental data of these compounds will be discussed in IR, UV–vis, 1H and 13C NMR, LC–MS, SC-XRD, and biological analysis sections. Table 1 represents the FT-IR data of L1–L5.

Table 1. FT-IR Stretching Frequency Data of L1–L5.
functional group IR stretching frequencies (cm–1)
  L1 L2 L3 L4 L5
C–C 1422 1410 1426 1436 1415
C=N 1600 1568 1509 1587 1570
C–N 1307 1272 1253 1246 1256
N–N 1148 1185 1117 1121 1129
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2.5.2. IR Characterization

In the FT-IR spectra (see Figure SI4a–e), all the synthesized pyrazole compounds show typical C=N stretching frequencies (medium peak) at 1600 cm–1 for L1, 1568 cm–1 for L2, 1509 cm–1 for L3, 1587 cm–1 for L4, and lastly, 1570 cm–1 for L5. All these stretching frequencies, impacted by respective substitution on the backbone of the pyrazole moiety, are in good agreement with other related IR data reported in the literature.30−32 The presence of the medium peaks attributed to C–N stretching at a region ranging between 1307 and 1246 cm–1 further supports the formation of these compounds. The −C–C– stretching of L3 (1426 cm–1) and L4 (1436 cm–1) occurs more downfield as compared to the −C–C– of L1 (1422 cm–1), L2 (1410 cm–1), and L5 (1415 cm–1). This is due to the presence of electron-withdrawing atoms (fluorides) at the terminal carbons of the former pyrazole compounds (L3 and L4).

2.5.3. UV–Vis Characterization

The UV–vis spectra of the pyrazole-based compounds under study in the exploited wavelength region (200–400 nm) are shown in Figure SI3a–e. The wavelength of maximum absorbance for all the compounds ranges from 218 to 254 nm with their corresponding calculated molar absorptivities (ε) ranging between 435 and 1045 M–1·cm–1, respectively, to L1 and L2. A red-shifted broad absorption band is observed around 250 nm on L2 and L3 spectra as compared to the narrow absorption profiles (around 218 nm) of L1, L4, and L5. The observed bathochromic shift in the λmax from 218 to 250 nm is a sure indication of aromatic moieties on the compound backbone since L1, L4, and L5 are free from phenyl scaffolds. Moreover, a weak secondary peak is observed around 200 nm for the UV–vis spectra of L2 and L3. All these noticeable absorption profiles are characterized by π–π* (aromatic) transition according to the literature.30

2.5.4. NMR Characterization

The pyrazole compounds L1–L5 are readily identified by their respective 1H and 13C NMR chemical shifts. The analyses for all of the compounds were done in CDCl3 and DMSO solvents. All NMR data are consistent with the proposed structures and with other structures already reported in the literature.30 In 13C NMR, all these compounds, L1–L5, denote a downfield chemical shift (peak) at δC 144.2, 148.7, 145.1, 143.2, and 160.5 ppm, respectively, typically attributed to aromatic carbons within the pyrazole ring that are influenced by the adjacent nitrogen atoms, resulting in the carbon being deshielded and appearing downfield (C=N). In L5, the imine carbon signal (C=N) appears quite small; however, this could be due to the analysis time and also the amount of sample that was used for the 13C NMR analysis (see Figure SI2b’). In the 1H NMR spectra, a small prominent peak observed at δH 12.38, 10.96, 9.32, 10.98, and 11.20 ppm attributable to N–H for L1–L5, respectively, further advocates for the successful synthesis of these compounds. The methyl proton peaks for L1 (2.31 ppm) and L4 (2.34 ppm) are observed more downfield as compared to those of L5 (1.15 ppm) and this might be due to the fact that the protons of the former (L1 and L4) are much closer to the electron-withdrawing atoms (adjacent nitrogens) within the pyrazole moiety in contrast to the latter (L5).

2.5.5. Single-Crystal XRD Characterization

The molecular crystal structure of the pyrazole compound, L1, was validated by single-crystal X-ray diffraction. The solid-state molecular structures along with crystal packing with intermolecular hydrogen interactions of the pyrazole derivative mentioned above are shown in Figure 1. This compound crystallized in the trigonal crystal system within the R̅3c space group, corresponding to the previously determined structure of the same compound by Smith et al.31 in 1989 at 295 K. In the structure reported herein, determined at 150 K, 36 molecules are present in the unit cell (Z = 36), and a head–head arrangement is noted in the packing of the molecules. In the asymmetric unit of L1, half a molecule exists, whereby a complete molecule is generated by means of a range of symmetry elements, including inversion center, rotation, and screw axes. The implication is that atoms C3, H3 (which resides on C3), and H1 (which resides on N1) are observed with associated 50% site occupancies to allow for a full occupancy in the grown structure (using the 2/3 – x, 1/3 – x + y, and 5/6 – z symmetry operator).

Figure 1.

Figure 1

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(A) Molecular structure of L1. Some hydrogen atoms are omitted for clarity, (B) asymmetric unit of the molecule, and (C) unit cell showing crystal packing and intermolecular hydrogen interactions. Thermal ellipsoids are displayed at 50% probability.

The molecular crystal structure consists of a heterocyclic moiety composed of three carbons and two adjacent nitrogen atoms, with methyl groups attached to the backbone of the molecule. The N1–N1′ bond distance is found to be 1.361(3) Å, while C1–C2 and C2–C3 bond distances are found to be 1.493(2) and 1.391(2) Å, respectively. On the other hand, the C2–N1 and C2′=N1′ are both reported as 1.342(2) Å. This is due to the 50% probability of double bond tautomerism happening within the pyrazole ring. In comparison, the bond distances of the reported structure are in good agreement with other pyrazole compounds already reported in the literature.32−35Figure 1 and Table SI3 show that this compound is further stabilized by intermolecular hydrogen bonding (N–H···N), with a bond distance of 2.03(4) Å.

2.5.6. In Vitro Studies of Pyrazole Compounds

The treatment of most diseases remains an imperative and strenuous matter due to many issues associated with therapy. This includes the resistance of these diseases to currently known drugs and the rapid increase in virus and bacterial infections in immunoarbitrated patients (e.g., AIDS and cancer) after organ transplantation or during chemotherapy.36−38 Despite the existence of many groups of compounds with various biological activities, pyrazole compounds are comprehensively explored because they are rich in pharmacological activity to most diseases.39,40 Therefore, the cytotoxic effect of L1–L5 pyrazole compounds was investigated in pancreatic (CFPAC-1 and PANC-1), breast (MDA-MB-231 and MCF-7), cervical (CaSki), and HeLa cancer cell lines. To this end, the cells were treated for 48 h with vehicle (DMSO) or 10 μM of the L1–L5 compounds followed by MTT assays. Cisplatin and gemcitabine were included in these assays as reference drugs because they are used as a chemotherapeutic for these cancers.

In pancreatic cancer cell lines, compared to gemcitabine which showed a cell viability of 56.7% and 49.10% in CFPAC-1 and PANC-1 cells, respectively, none of the experimental compounds inhibited cell viability by more than 50%. However, L2 was the most promising compound, which showed a significant inhibition of cell viability in both cell lines, i.e., 72.8% for CFPAC-1 and 91.31% for PANC-1 cells (Figure 2A). As shown in Figure 2B, while compound L2 significantly inhibited the viability of both MDA-MB-231 (80.02%) and MCF-7 (90.36%) cells, L3 and L4 showed a statistically significant inhibition of cell viability (81.14% and 70.3%, respectively) in the estrogen receptor-positive MCF-7 cell line. However, none of the compounds inhibited cell viability by more than 50% or was more effective than cisplatin in the breast cancer cell lines tested. Similarly, none of the compounds were more effective than cisplatin in cervical cancer cell lines, but compounds L2 and L4 showed promising results in HeLa (88.09%) and CaSki (87.91%) cells, respectively (Figure 2C). Overall, the results summarized in Table 2 reveals that although L1–L5 were not as cytotoxic as the gold standard drugs gemcitabine and cisplatin, L2 showed promise across all of the cancer cell lines tested. These results are consistent with numerous literature reports on the potential biological activity of pyrazole compounds on various cancer cell lines.35−39

Figure 2.

Figure 2

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Single-dose treatments of pancreatic, breast, and cervical cancer cell lines with L1–L5 compounds. (A) CFPAC-1 and PANC-1 pancreatic cancer, (B) MDA-MB-23 and MCF-7 breast cancer, and (C) CaSki and HeLa cervical cancer cell lines were treated with vehicle (DMSO) or 10 μM of L1-L5 for 48 h followed by MTT cell viability assay. Graphs represent the mean cell viability ± SD of three independent experiments performed in quadruplicate. Data were analyzed using the parametric unpaired t-test by GraphPad Prism 8.0 software where *p < 0.05, **p < 0.01, ***0.001, and ****0.0001.

Table 2. Summary of Percentage Viability 48 h Post-treatment with 10 μM of L1–L5 in Various Cancer Cell Lines.
compounds CFPAC-1 PANC-1 MDA-MB-231 MCF-7 CaSki HeLa
vehicle 100 ± 3.98 100 ± 3.18 100 ± 2.14 100 ± 3.92 100 ± 6.05 100 ± 6.00
L1 103.39 ± 6.87 97.81 ± 4.62 98.00 ± 3.54 96.02 ± 4.01 95.93 ± 6.35 97.87 ± 2.84
L2 72.84 ± 4.31 91.31 ± 3.65 80.02 ± 2.29 90.36 ± 3.23 93.17 ± 4.40 88.09 ± 3.63
L3 104.10 ± 6.20 96.74 ± 3.94 98.42 ± 4.46 81.14 ± 4.14 92.52 ± 5.34 98.94 ± 3.68
L4 102.70 ± 4.05 95.64 ± 4.65 99.89 ± 4.30 70.30 ± 3.82 87.91 ± 4.40 97.08 ± 5.96
L5 89.74 ± 6.05 98.54 ± 5.75 90.47 ± 3.38 95.07 ± 4.25 102.09 ± 5.78 87.83 ± 3.45
gemcitabine 56.73 ± 7.79 49.10 ± 7.84        
cisplatin     48.63 ± 1.99 56.71 ± 4.45 44.41 ± 3.18 55.16 ± 4.22
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We therefore determined the half maximal inhibitory (IC50) concentrations of L2, L3, and L4 in the cancer cell lines where they showed most activity, and their results are listed in Table 3. Our results showed that L2 exhibited IC50 values of 61.7 and 104.7 μM in the CFPAC-1 and PANC-1 pancreatic cancer cell lines, respectively, and 189.3 μM in the MDA-MB-231 triple negative breast cancer cells. In the estrogen receptor positive MCF-7 breast cancer cells, IC50 values of 81.5 and 185.5 μM were obtained for L3 and L4, respectively. The IC50 values obtained for our compounds are much greater than those of cisplatin and gemcitabine, which are our reference standards, with IC50 values of 13.98 and 5.54 μM against

Table 3. IC50 Values (μM) Determined for the Most Active Compounds, L2–L4, in Pancreatic and Breast Cancer Cell Linesa.
cell lines
 compound IC50 ± SD (μM)
    L2 L3 L4 cisplatin41 gemcitabine42
pancreatic cancer CFPAC-1 61.7 ± 4.9 – – – 1.5 ± 0.84
  PANC-1 104.7 ± 7.3 – – – 5.7 ± 0.19
breast cancer MDA-MB-231 189.3 ± 0.87 – – 13.98 –
  MCF-7 – 81.48 ± 0.89 185.5 ± 0.95 5.54 –
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a

NB: dashes (−) show that the compounds did not show significant cell viability on those specific cancer cell lines to be further screened in IC50 concentrations.

MDA-MB-231 and MCF-7 breast cancers for cisplatin and 1.5 ± 0.84 and 5.7 ± 0.19 μM against CFPAC-1 and PANC-1 pancreatic cancers for gemcitabine. Graphical representation of the results obtained is illustrated in the Supporting Information (Figure SI6).

2.6. Conclusions

In this study, five pyrazole compounds (L1–L5) were synthesized and analyzed by using FT-IR, 1H and 13C NMR, UV–vis, LC–MS, and single-crystal X-ray crystallography techniques. The short-term cytotoxicity of the synthesized pyrazole compounds was explored on CFPAC-1, PANC-1, MDA-MB-231, MCF-7, CaSki, and HeLa cancer cell lines via the MTT viability assay. Based on the outcomes obtained from anticancer screening, it is evident that none of the compounds exhibited more activity than the standard drugs in the cancer cell lines tested, which is the main limitation to this study. However, L2 showed the most promising outcome against pancreatic (CFPAC-1) cancer with IC50 value of 61.7 ± 4.9 μM. Therefore, further investigation such as coordinating these compounds to d-group metals (Re, Tc, and Mn) to evaluate their biological behavior remains to be investigated for drug discovery and development in the future. These pyrazole moieties can also be explored biologically on other diseases such malaria, TB, and Chagas disease.

Acknowledgments

This work is based on the research supported by the National Research Foundations of South Africa under the Thuthuka programme (Grant specific unique reference numbers 113629). We would like to express our gratitude toward the Tshwane University of Technology together with the Department of Chemistry for institutional support and the University of Pretoria for crystal data collection and refinements. The authors acknowledge that the opinions, findings, conclusions, or recommendations expressed in this publication do not necessarily reflect the views of the SA National Research Foundation.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c00320.

  • Crystallographic data; selected bond lengths and angles; hydrogen interactions; 1H and 13C NMR spectra of L1–L5; UV–vis spectra; IR spectra; MS spectra; and half-maximal inhibitory (IC50) concentrations of L2, L3, and L4 (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao5c00320_si_001.pdf (714.8KB, pdf)

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Supplementary Materials

ao5c00320_si_001.pdf (714.8KB, pdf)

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