Abstract
Aim: Design and synthesis of pyrazole-based chemotherapeutic agents. Materials & methods: A series of novel diphenyl pyrazole–chalcone derivatives were synthesized and assessed for their cytotoxic activities against 14 cancer cell lines and their antimicrobial activities against MRSA and Escherichia coli along with their safety using HSF normal cell line. Results & conclusion: Majority of the compounds showed moderate-to-significant anticancer activity with selective high percentage inhibition (>80%) against HNO-97 while being nontoxic toward normal cells. Compounds 6b and 6d were the most potent congeners with IC50 of 10 and 10.56 μM respectively. The synthesized compounds exhibited moderate to potent antimicrobial activities. Interestingly, compound 6d exhibited a minimum inhibitory concentration of 15.7 μg/ml against MRSA; and a minimum inhibitory concentration of 7.8 μg/ml versus E. coli.
Keywords: : anticancer, antimicrobial, E. coli, MRSA, nontoxic, pyrazole chalcone
Plain language summary
Summary points.
Aim
Design and synthesis of nontoxic pyrazole–chalcone hybrids as dual anticancer and antimicrobial agents.
Materials & methods
A series of novel diphenyl pyrazole–chalcone derivatives were synthesized and evaluated for their cytotoxic activities against 14 cancer cell lines. All the synthesized compounds were further assessed for their antimicrobial activities against Gram-positive MRSA and Gram-negative Escherichia coli ATCC 25922. Finally, the synthesized compounds were further tested for their cytotoxicity against normal cell line HSF.
Most of the compounds showed moderate to significant anticancer activity against the tested cancer cell lines with a selective high percentage inhibition (>80%) against HNO-97 at a concentration of 100 μg/ml. Compounds (6b and 6d) were the most potent congeners of the series with IC50 of 10.5 and 10 μM, respectively.
The synthesized compounds evinced moderate to potent antimicrobial activities against both the tested strains; MRSA and E coli; with superior activities against E. coli being two to eightfold more potent than the reference compound; Ciprofloxacin. Interestingly, compound 6d exhibited a moderate MIC of 15.7 μg/ml against MRSA; and a potent MIC of 7.8 μg/ml versus E. coli. All synthesized compounds demonstrated a good safety profile, as they were proven to be nontoxic toward normal cells.
1. Background
Cancer is a major health concern with annihilating repercussions toward mankind. According to the WHO, cancer accounted for around 10 million deaths worldwide [1]. Oral squamous cell carcinoma (OSCC), mounted for being the most common type of oral cancer, it represents a great health risk and is considered as one of the foremost causes of mortality in developing countries [2]. This urged the need to research novel anti-cancer agents with great potency to treat cancer.
Inexorable bacterial infections are becoming great menaces to humanity. This is due to the overconsumption and misuse of antimicrobials [3]. Eventually, this leads to inevitable resistance developing toward marketed antibiotics [4]. Thus, the discovery and development of novel, effective and safer antibacterial agents is crucial for providing treatment to infection caused by resistant bacterial strains and expanding the repertoire of treatment options.
When both bacterial infections and cancer disease intersect, this becomes a global apprehension. Unfortunately, this was emphasized through divulging a considerable number of cancer deaths because of bacterial infections [5]. This can be explained by the debility of the immune system because of cancer or anti-cancer drugs. On the other hand, 2.2 million cases of cancer were diagnosed to be originating from an infectious agent [6]. Moreover, the existing anti-cancer and antimicrobial treatment regimens suffer from major setbacks including multi-drug resistance [7], nonspecificity as well as high toxicity [8]. These findings highlight the urgency of getting novel, effective and nontoxic chemotherapeutic agents possessing dual anti-cancer and anti-microbial activities.
Pyrazole is one of the common nitrogen-based heterocycles that manifests diverse biological activities. This is presented in many US FDA-approved drugs that incorporate pyrazole as a major pharmacophore [9,10]. Pyrazole along with its synthetic analogs including 1,3-diphenylpyrazole display a diversity of biological activities against multiple diseases as diabetes, depression, convulsions, bacteria, cancer in addition to having analgesic and anti-inflammatory activities [11,12]. Similarly, chalcone is a leading scaffold that proved their potential therapeutic activities. They are implicated in many drugs with versatile biological activities as anticancer, anti-inflammatory and antimicrobial agents [13,14].
Indeed, hybridization of bioactive pharmacophores is a well-settled strategy that demonstrated great efficacy in evolving novel agents with outpacing biological activities over the parent individual analogs [15]. Pyrazole–chalcone hybrids have been reported to inhibit the growth of numerous cancer cell lines which includes lung, breast, colon, pancreatic and prostate cancer cells [16]. The focus of previous research has been on examining pyrazole–chalcone hybrids and their N-acetyl pyrazole derivatives primarily as cytotoxic agents revealing broad effectiveness against various cancer cell lines. As such, compound I showed remarkable in vitro activity against leukemia, renal cancer and non-small-cell lung cancer cell lines, where GI50 values extended from 0.4 to 11.4 μM. However, the safety profile for such agents is yet to be investigated (Figure 1) [17]. Besides, Pyrazole–chalcone hybrids have been previously investigated as potential anticancer agents; especially compound II (Figure 1) which manifested moderate to powerful inhibitory activity toward cancer cell lines HELA, HC-6, MCF-7 and RPMI-822 [12].
Figure 1.
Concept of designation of pyrazole–chalcone hybrids (compounds 6a–j and 7a,b) with potential dual anticancer and antimicrobial activities based on the structures of anticancer (compounds I,II) and antimicrobial (compounds III–V) pyrazole–chalcone derivatives.
Noteworthy, both compounds I and II bear a nitro group in their structures, which is categorized as a toxicophore with serious toxicity issues associated with serious mutagenicity and genotoxicity despite used extensively in therapeutic field. Thus, additional research is needed to devise safer alternatives when designing drug-like compounds [18].
On another aspect, hybridization of pyrazoles and chalcones has resulted in compounds with enhanced antimicrobial activity. Studies have shown that pyrazole chalcone hybrids exhibit significant activity against a wide range of bacterial and fungal strains [19].
In 2012, Shelke et al. [20] synthesized a number chalcone derivatives with compounds III and IV included displaying considerable antibacterial activity; particularly for Mycobacterium tuberculosis H37Rv with the minimum inhibitory concentration (MIC) of 6.25 μg/ml [20].
Pyrazolyl chalcones' series, developed by Siddiqui et al. [21], also revealed potential antibacterial activity with the MIC of 12.5 μg/ml against MRSA (Compound V) [21].
Based on the aforementioned facts and inspired by the promising anticancer and antimicrobial activities of pyrazole–chalcone hybrids, design, synthesis and biological evaluation of pyrazole–chalcone hybrids based on compounds I to V (Figure 1) was attempted. This was accomplished through substituting the nitro group in compounds I and II with other safer functional groups. In addition, manipulating the chalcone moiety with either substituted benzyl phenoxy group or benzimidazole derivatives was conducted. Cytotoxic activities against a vast variety of cancer cell lines as well as antimicrobial activities against virulent microbial strains were thoroughly investigated for the synthesized compounds. Moreover, safety profiles for the synthesized compounds were also studied.
2. Experimental
2.1. Chemistry
All chemicals either starting materials or reagents were obtained from Alfa-Aesar Organics and Aldrich (MA, USA) then used directly without further purification. Thin layer chromatography (TLC), purchased from Merck (Merck, Darmstadt, Germany) and performed on 0.255 mm silica gel plates, was used to monitor all the reactions using analytical with visualization under U.V. light (254 nm). At the Center for Drug Discovery and Development Research, Ain Shams University 1H NMR spectra were achieved at Bruker 400 MHz spectrometer (MA, USA) where TMS was used as reference. The proton of NH was not noticed in the 1H NMR unless noted. Elemental analysis was carried out confirming >95% purity of the final compounds.
(E)-1-(4-unsubstituted/chlorophenyl)-2-(1-phenylethylidene)hydrazine (1a–b) were prepared as previously reported [22]. 1,3-diphenyl-1H-pyrazole-4-carbaldehyde (2a) and 1-(4-chlorophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde (2b) were prepared as previously reported [22].
2.1.1. General synthetic method for compounds (3a–e)
A reaction mixture of a suspension of 4-hydroxyacetophenone (1 g, 7.34 mmol) in isopropanol, the corresponding benzyl halides (14.69 mmol, 2 eq.) and K2CO3 (3.05 g, 3 eq) was refluxed overnight. The obtained solid was filtered, washed by water then recrystallized from ethanol and used for the following step.
1-(1H-benzo[d]imidazol-2-yl)ethanol (4) and 1-(1H-benzo[d]imidazol-2-yl)ethanone (5) were prepared as previously reported [23,24].
2.1.2. General synthetic method for compounds (6a–j) & (7a–b)
In ethanol, compounds 2a–b (3.12 mmol) were suspended and the corresponding intermediates 3a–e and 5 (3.75 mmol, 1.2 eq.) were added to the suspension. In a drop-wise manner and through stirring, 10% KOH (6.24 mmol, 2 eq.) was added. At room temperature, the reaction mixture was left stirred overnight. After filtering the afforded solid, it was washed with water and recrystallized using ethanol providing the corresponding compounds.
2.1.2.1. (E)-3-(1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)-1-(4-((4-methylbenzyl)oxy)phenyl)prop-2-en-1-one (6a)
Yellowish white solid. Yield: 56% 1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.14–8.01 (m, 2H), 8.01–7.91 (m, 2H), 7.83 (d, J = 15.4 Hz, 1H), 7.69–7.61 (m, 5H), 7.60–7.48 (m, 3H), 7.36 (d, J = 7.6 Hz, 2H), 7.21 (d, J = 7.7 Hz, 2H), 7.19–7.12 (m, 2H), 5.17 (s, 2H), 2.31 (s, 3H). Anal. Calcd. For C32H25ClN2O2 C, 76.11; H, 4.99; N, 5.55. Found: C, 76.32; H, 5.02; N, 5.31
2.1.2.2. (E)-1-(4-(benzyloxy)phenyl)-3-(1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)prop-2-en-1-one (6b)
White solid. Yield: 75%. 1H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.94 (dd, J = 7.2, 5.0 Hz, 2H), 7.82 (d, J = 15.4 Hz, 1H), 7.69–7.61 (m, 5H), 7.58–7.33 (m, 8H), 7.17 (d, J = 8.4 Hz, 2H), 5.22 (s, 2H). Anal. Calcd. For C31H23ClN2O2 C, 75.83; H, 4.72; N, 5.71; Found: C, 75.97; H, 4.89; N, 5.53
2.1.2.3. (E)-3-(1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)-1-(4-((3,4-dichlorobenzyl)oxy)phenyl)prop-2-en-1-one (6c)
Yellow solid. Yield: 64%. 1H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H), 8.15–8.02 (m, 2H), 8.01–7.90 (m, 2H), 7.81 (d, J = 15.5 Hz, 1H), 7.74 (d, J = 2.0 Hz, 1H), 7.70–7.60 (m, 6H), 7.59–7.44 (m, 4H), 7.21–7.15 (m, 2H), 5.23 (s, 2H). Anal. Calcd. For C31H21Cl3N2O2: C, 66.50; H, 3.78; N, 5.00; Found: C, 66.41; H, 3.73; N, 4.98.
2.1.2.4. (E)-1-(4-((4-bromobenzyl)oxy)phenyl)-3-(1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)prop-2-en-1-one (6d)
White solid. Yield: 70%. 1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.07 (d, J = 8.5 Hz, 2H), 8.02–7.91 (m, 2H), 7.82 (d, J = 15.5 Hz, 1H), 7.69–7.49 (m, 10H), 7.44 (d, J = 8.1 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H), 5.21 (s, 2H). Anal. Calcd. For C31H22BrClN2O2: C, 65.34; H, 3.89; N, 4.92; Found: C, 65.21; H, 3.64; N, 4.83.
2.1.2.5. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-1-(4-((4-fluorobenzyl)oxy)phenyl)prop-2-en-1-one (6e)
Off-white solid. Yield: 45%. 1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.0 Hz, 2H), 7.86 (d, J = 15.4 Hz, 1H), 7.74–7.62 (m, 3H), 7.65–7.49 (m, 7H), 7.41 (t, J = 7.4 Hz, 1H), 7.30–7.13 (m, 4H), 5.21 (s, 2H). Anal. Calcd. For C31H23FN2O2: C, 78.46; H, 4.89; N, 5.90; Found: C, 79.02; H, 4.93; N, 5.87.
2.1.2.6. (E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-1-(4-((4-methylbenzyl)oxy)phenyl)prop-2-en-1-one (6f)
Light brown solid. Yield: 60%. 1H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 8.08 (d, J = 8.3 Hz, 2H), 7.95 (d, J = 7.9 Hz, 2H), 7.86 (d, J = 15.5 Hz, 1H), 7.73–7.63 (m, 3H), 7.55 (dd, J = 26.0, 8.0 Hz, 5H), 7.43 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.19 (dd, J = 18.0, 8.0 Hz, 4H), 5.19 (s, 2H), 2.31 (s, 3H). Anal. Calcd. For C32H26N2O2: C, 81.68; H, 5.57; N, 5.95; Found: C, 81.73; H, 5.64; N, 5.82.
2.1.2.7. (E)-1-(4-(benzyloxy)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)prop-2-en-1-one (6g)
Yellow solid. Yield: 69%. 1H NMR (400 MHz, DMSO-d6) δ 9.41 (s, 1H), 8.10 (d, J = 8.5 Hz, 2H), 7.95 (d, J = 8.1 Hz, 2H), 7.87 (d, J = 15.4 Hz, 1H), 7.72–7.65 (m, 3H), 7.58 (q, J = 7.9 Hz, 4H), 7.52–7.47 (m, 3H), 7.45–7.35 (m, 4H), 7.19 (d, J = 8.3 Hz, 2H), 5.24 (s, 2H). Anal. Calcd. for C31H24N2O2: C, 81.56; H, 5.30; N, 6.14; Found: : C, 81.42; H, 5.37; N, 6.12.
2.1.2.8. (E)-1-(4-((2-chlorobenzyl)oxy)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)prop-2-en-1-one (6h)
Light brown solid. Yield: 72%. 1H NMR (400 MHz, DMSO-d6) δ 9.39 (s, 1H), 8.17–8.05 (m, 2H), 7.99–7.92 (m, 2H), 7.86 (d, J = 15.4 Hz, 1H), 7.74–7.49 (m, 10H), 7.46–7.36 (m, 3H), 7.27–7.17 (m, 2H), 5.29 (s, 2H). Anal. Calcd. For C31H24N2O2: C, 81.56; H, 5.30; N, 6.14; Found: C, 81.46; H, 5.42; N, 6.12.
2.1.2.9. (E)-1-(4-((4-bromobenzyl)oxy)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)prop-2-en-1-one (6i)
White solid. Yield: 76%. 1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.13–8.04 (m, 2H), 7.94 (d, J = 8.1 Hz, 2H), 7.85 (d, J = 15.4 Hz, 1H), 7.66 (dd, J = 11.1, 4.1 Hz, 3H), 7.63–7.54 (m, 6H), 7.54–7.49 (m, 1H), 7.47–7.39 (m, 3H), 7.19–7.15 (m, 2H), 5.21 (s, 2H). Anal. Calcd. For C31H23BrN2O2: C, 69.54; H, 4.33; N, 5.23; Found: C, 69.32; H, 4.45; N, 5.33.
2.1.2.10. (E)-1-(4-((3,4-dichlorobenzyl)oxy)phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)prop-2-en-1-one (6j)
Light brown solid. Yield: 73%. 1H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.08 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 15.4 Hz, 1H), 7.77–7.36 (m, 12H), 7.18 (d, J = 8.5 Hz, 2H), 5.24 (s, 2H). Anal. Calcd. For C31H22Cl2N2O2: C, 70.86; H, 4.22; N, 5.33; Found: C, 71.02; H, 4.32; N, 5.26.
2.1.2.11. (E)-1-(1H-benzo[d]imidazol-2-yl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)prop-2-en-1-one (7a)
Yellow solid. Yield: 56%. 1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.13 (d, J = 15.9 Hz, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 15.9 Hz, 1H), 7.78–7.65 (m, 4H), 7.57 (dd, J = 13.1, 6.0 Hz, 5H), 7.40 (t, J = 7.4 Hz, 1H), 7.28 (d, J = 6.9 Hz, 2H). Anal. Calcd. for C25H18N4O: C, 76.91; H, 4.65; N, 14.35; Found: C, 76.79; H, 4.72; N, 14.21.
2.1.2.12. (E)-1-(1H-benzo[d]imidazol-2-yl)-3-(1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl)prop-2-en-1-one (7b)
Light brown solid. Yield: 58%. 1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.16 (d, J = 15.9 Hz, 1H), 8.07 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 15.8 Hz, 1H), 7.74–7.48 (m, 9H), 7.33–7.20 (m, 2H). Anal. Calcd. For C25H17ClN4O: C, 70.67; H, 4.03; N, 13.19; Found: C, 70.74; H, 4.21; N, 13.21.
2.2. Biological evaluation
2.2.1. In vitro cytotoxic activity against cancer cell lines
The tested human cancerous cell lines were appraised for their chemosensitivity to pyrazolo-derivatives and were attained from American Type Culture Collection (ATCC; DC, USA) while maintained frozen in liquid nitrogen (-180°C). They were conserved at National Cancer Institute as monolayer cultures in RPMI-1640 complemented with 1% penicillin-streptomycin and 10% FBS. Preparation of samples was accomplished through dissolving 1:1 Stock solution and storage was performed at -20°C in dimethylsulfoxide (DMSO). Different concentrations of the drug were used (Range of concentration used by μg/ml). The cytotoxicity was performed using Sulphorhodamine-B (SRB) assay as reported by Skehan, 1990 [25]. Cytotoxicity screening was accomplished using Sulforhodamine B colorimetric assay. Nat. Protoc. 2006:1, 1112–1116. SRB is a bright pink aminoxanthrene dye encompassing two sulphonic groups where it can bind to the intracellular proteins' amino groups under acidic conditions providing a sensitive index of cellular protein content. The detailed procedure, the reagents and buffers and the calculations are provided in the Supplementary material.
2.2.2. Evaluation of antimicrobial activity
Employing the agar well diffusion method, preliminary screening for antimicrobial activity was performed versus the Gram-negative E. coli ATCC 25922 and the Gram-positive Staphylococcus aureus ATCC 43300 (Methicillin resistant) [26]. Of each standard strain, a standardized count of 107 colony forming unit (CFU) was balanced spectrophotometrically and inoculated on Muller Hinton agar (MHA) plates and afterward the needed wells (12 mm diameter) were formed. All the compounds were dissolved in DMSO. 100 μl of solution were used to fill the wells. After allowing incubation at 37°C overnight, visual examination of the plates was attempted for the inhibition zones along with measuring the zone diameters. DMSO and Ciprofloxacin (1 mg/ml) were the negative and positive controls, respectively.
2.2.3. Determination of minimum inhibitory concentrations
The compounds with auspicious results in the preliminary anti-microbial screening were promoted for MIC exploration. MICs were calculated versus both Escherichia coli ATCC 25922, and MRSA ATCC 43300 using the broth microdilution method as reported [27]. Using Mueller Hinton Broth in microtiter plates, serial dilutions of the compounds to be tested were achieved in DMSO. The inocula of the microorganisms were added on to have an inoculum density of 106 CFU/ml per well. Negative controls were the unioculated wells and Ciprofloxacin was used as the positive control. Plates were examined thoroughly at the end of the incubation period where MICs were documented being the minimum concentration inhibiting microorganisms' growth. Experiments were executed in triplicates and the average of the data was determined.
3. Results & discussion
3.1. Chemistry
The synthesis of the 12 targeted compounds was accomplished as stated in the outlined synthetic schemes 1–4 (Figures 2 and 3). 1,3-Diphenyl-1H-pyrazole-4-carbaldehyde (2a) and 1-(4-chlorophenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde (2b) were synthesized through reacting the purchased 4-chloro/unsubstituted phenylhydrazine with acetophenone in glacial acetic acid to afford the hydrazone derivatives 1a–b in a considerable yield as reported [22]. Cyclized 1H-pyrazole-4-carbaldehyde derivatives (2a–b) were produced through Vilsmeier-Haack reaction in a good yield (Figure 2A). Benzylation of 4-hydroxyacetophenone utilizing various benzyl halide congeners, in the presence of anhydrous K2CO3, afforded 1-(4-(Benzyloxy)phenyl)ethenone derivatives (3a–e) (Figure 2B) [28]. Meanwhile, 1-(1H-benzo[d]imidazol-2-yl)ethanol (4) was synthesized through reacting lactic acid and o-phenylenediamine using conc. HCl as reported [23]. Oxidation of compound (4) was conducted using K2Cr2O7 in conc. H2SO4 giving 1-(1H-benzo[d]imidazol-2-yl)ethanone (5) (Figure 2C) [24]. The synthesized intermediates 3a–e and 5 were reacted with 2a–b to bring out the targeted compounds 6a–j and 7a–b as per Claisen-Schmidt condensation reactions (Figure 3) [29]. As well known, chalcones can exist as cis or trans isomer [30]. In our study, the trans isomer was confirmed by the appearance of the doublet signal in the 1H NMR spectra with j-coupling constant of 15.4 or 15.9 Hz. Moreover, the benzyloxy protons appeared as singlet signal at 5.17 to 5.24 Hz.
Figure 2.
Synthetic schemes 1–3. (A) Scheme 1, for preparation of compounds 2a,b. (B) Scheme 2, for preparation of compounds 3a–e. (C) Scheme 3, for preparation of compound 5.
rt: Room temperature.
Figure 3.
Synthetic scheme 4.
rt: Room temperature.
3.2. Biological evaluation
3.2.1. In vitro cytotoxic activity
The in vitro cytotoxic assay was performed using MTT assay against 14 cancer cell lines representing seven human cancers: breast, colo-rectal, cervical, lung, melanoma, oral and prostate cancer. The cancer cell lines grew up according to the NCI standard protocols. The screening began with the assessment of all compounds versus the 14 cell lines at a single-dose of 100 μg/ml [31]. The results are presented as ‘Percentage Growth Inhibition’ (GI%) representing growth relative to the negative control.
The cytotoxic activities of the synthesized compounds ranged from weak (GI% is less than 45%) to potent (GI% more than 80%) against the cell lines to be tested. Whereas the targeted compounds showed moderate quasi non-selective inhibition against cell lines HELA, HEP2, MDA-MB-231, PC3 with GI% ranging from 55 to 60% in HELA; 54–71% in HEP2, 51–70% in MDA-MB-231 and 54–65% in PC3. Interestingly, all the tested compounds exhibited potent selective activities against HNO-97 oral cancer cell line with GI% ranging from 69 to 92%. Compounds 6a, 6b, 6d, 6e and 7a were the most active compounds against a number of tested cell lines. Compound 6a exhibited a range of activities against the tested cells ranging from moderate activity (GI% 51.95 ± 1.36 and 51.32 ± 1.24) against both MDA-MB-231 and T47D, respectively to potent activity (GI% of 85.43 ± 0.98) against HNO-97 cell line.
The in vitro cytotoxic activity results unveiled the correlation between the substitution in the structure of pyrazole–chalcone hybrids and the cytotoxic activities against a considerable number of cancer cell lines. In series (6a–j), the results revealed that the cytotoxic activities against cancerous cell lines is well maintained when the R substituent is a chloro group as seen in compounds 6a, 6b and 6d. With the exception of compound 6e; when R is hydrogen (series 6f–6j); the cytotoxic activities are restricted mainly to HNO-97 cell line as shown in Table 1. On the other hand, R1 substituents are well tolerated when present in 4-position only when R is chloro group (4-methyl and 4-bromo in 6a and 6d, respectively or unsubstituted as shown in compound 6b). The lack of a wide range of antineoplastic activities against the tested cell lines is manifested when R1 is either in 2-position (as in compound 6h) or 3,4-di substitution (as in compounds 6c and 6j). The cytotoxic activities of series 6f–6j, when R is hydrogen; except for compound 6e when it's R1 is 4-Floro; are again restricted to HNO-97.
Table 1.
Cytotoxicity against cancer cells.
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|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Cpd # | R | R1 | Cytotoxicity against cancer cells (% inhibition) at 100 μg/ml |
|||||||||||||
| HELA | HEP2 | HEPG2 | HuH7 | T47D | MCF7 | A549 | H460 | CaCo2 | HCT116 | MDA-MB-231 | HNO-97 | PC3 | A375 | |||
| 6a | Cl | 4-CH3 | 59.1 ± 0.6 | 70. ± 3.2 | NA | NA | 51.3 ± 1.2 | NA | NA | NA | NA | NA | 51.9 ± 1.3 | 85.4 ± 0.9 | 54.4 ± 1.3 | NA |
| 6b | Cl | H | 59.4 ± 0.6 | 65.3 ± 2.7 | NA | NA | NA | NA | NA | NA | NA | NA | 59.1 ± 1.7 | 92.1 ± 0.7 | NA | NA |
| 6c | Cl | 3,4-di-Cl | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | 76.4 ± 1.4 | NA | NA |
| 6d | Cl | 4-Br | 60.1 ± 2.2 | 71.6 ± 0.5 | NA | NA | NA | NA | NA | NA | NA | NA | 51.7 ± 1.58 | 86.22 ± 1.9 | 59.5 ± 3.7 | NA |
| 6e | H | 4-F | 60.6 ± 1.0 | 61.9 ± 2.8 | NA | 50.8 ± 2.4 | NT | NA | NA | NA | 53.9 ± 3.9 | NA | 70.1 ± 1.7 | 85.6 ± 1.8 | 64.6 ± 3.5 | 57.3 ± 1.9 |
| 6f | H | 4-CH3 | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | 69.5 ± 2.6 | NA | NA |
| 6g | H | H | NA | 61.7 ± 1.8 | NA | NA | NA | NA | NA | NA | 51. ± 0.95 | 50.5 ± 0.98 | 84.8 ± 1.8 | NA | NA | |
| 6h | H | 2-Cl | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA |
| 6i | H | 4-Br | NA | 56.1 ± 5.1 | NA | NA | NA | 52.3 ± 2.1 | NA | 56.2 ± 5.3 | NA | NA | 53.4 ± 2.5 | 87.9 ± 1.6 | 55.1 ± 5.7 | NA |
| 6j | H | 3,4-diCl | 55.6 ± 3.6 | 54.6 ± 2.9 | 49 ± 2.5 | NA | NA | NA | NA | 61.0 ± 1.4 | NA | NA | NA | 74.1 ± 1.2 | NA | NA |
| 7a | H | – | NA | 67.1 ± 3.8 | NA | NA | NA | NA | NA | NA | NA | NA | 64.7\ ± 1.6 | 86.3 ± 3.5 | 61.2 ± 4.7 | 67.3 ± 3.7 |
| 7b | Cl | – | 59.6 ± 0.78 | NA | NA | NA | NA | NA | 52.9 ± 2.26 | NA | NA | NA | 46.9 ± 3.6 | NA | NA | NA |
| Dox | – | – | ||||||||||||||
A375: Skin melanoma cell line; A549: Non-small-cell lung cancer; Caco2: Colorectal adenocarcinoma cell; H460: Non-small-cell lung cancer; HELA: Cervical cancer cell line; HEP-2: Human laryngeal carcinoma cell line; HEP2: Hyman Larynx; Carcinoma cell; HNO-97: Head and neck squamous cell carcinoma cell line; MCF-7: Human breast carcinoma cell line; MDA-MB-231: Metastatic mammary adenocarcinoma1; PC3: Human prostate cancer cell line; T47D: Human breast cancer cell line; human colorectal carcinoma cell line. Values are the means ± SD of three independent experiments performed in triplicates.
NA: Not active (% inhibition is less than 50%).
As for benzimidazole based derivatives (compounds 7a,b), only compound 7a; where R is H; exhibited potent to moderate GI% against a number of cancer cell lines (GI% against cell lines HEP2, MDA-MB-231, HNO-97, PC3 and A375 are 67.09 ± 3.8, 64.69 ± 1.66, 86.36 ± 3.57, 61.19 ± 4.71 and 67.29 ± 3.77, respectively. On the other hand, compound 7b displayed moderate cytotoxic activities against MDA-MB-231, A549 and HELA cells with GI% ranging from 46.9 ± 3.6.1 to 59.6 ± 0.78.
3.2.2. IC50 determination against HNO-97
Striking compounds 6a, 6b, 6d, 6e and 7a exhibited a wide range of cytotoxic activities as demonstrated by GI% against a number of cell lines; also, showing potent GI% (more than 80%) against HNO-97, were further evaluated for their IC50 against HNO-97 cell line.
As illustrated in Table 2, the IC50 values of the tested compounds ranged from 10 μM for compound 6b to 48.6 μM for compound 7a. Compounds 6b and 6d; where R is Chloro in both substituents; while R1 is H and 4-Bromo respectively; displayed the most potency with IC50 values of 10 and 10.56 μM, respectively. Nevertheless, results further divulged that the benzimidazole derivative 7a (IC50 of 48.6 μM) is less potent when compared with the phenyl benzyloxy derivatives (6a, 6b, 6d and 6e) which exhibited IC50 values ranging from 10 to 35 μM (Supplementary Figure S13).
Table 2.
IC50 values against HNO-97 cell line for compounds 6a, 6b, 6c, 6d, 7a and doxorubicin.
| Compound | Structure | IC50 against HNO-97 (μM) |
|---|---|---|
| 6a |
|
35 |
| 6b |
|
10 |
| 6d |
|
10.56 |
| 6e |
|
25.2 |
| 7a |
|
48.66 |
| Doxorubicin | – | 8.6 |
3.2.3. Anti-microbial activities evaluation versus MRSA & Escherichia coli
In vitro antibacterial activities of the targeted compounds (series 6a–j) and (series 7a,b) were determined by broth microdilution (BMD) method. The in vitro antimicrobial activity was assessed versus Gram-positive bacteria S. aureus ATCC 43300 (methicillin resistant-human isolates) (MRSA) and Gram-negative E. coli ATCC 25922. Ciprofloxacin was used as positive control (C+) for MRSA and E. coli. The data from Table 3 unveils that some of our pyrazole–chalcone hybrids proved their efficacy toward the tested bacterial species. Our hybrid compounds manifested a much prominent antibacterial activity against Gram-negative E. coli in comparison to gram-positive MRSA. All compounds evinced extreme activity against E. coli demonstrating MIC values superior to Ciprofloxacin (MICs ranging from 7.8 to 31.5 μg/ml compared with ciprofloxacin’ MIC which is 64 μg/ml). Regarding S. aureus, only compounds 6b and 6d exhibited moderate antimicrobial activity with MICs of 15.7 μg/ml compared with ciprofloxacin (MIC equals to 1 μg/ml).
Table 3.
Anti-microbial activity against Staphylococcus aureus ATCC 43300 (Methicillin resistant) and Escherichia coli ATCC 25922.
|
|
|
|---|---|---|
| Cpd. # | Staphylococcus aureus ATCC 43300 (methicillin resistant) (MIC in μg/ml) | Escherichia coli ATCC 25922 (MIC in μg/ml) |
| 6a | 250 | 7.8 |
| 6b | 15.7 | 31.5 |
| 6c | 31.5 | 31.5 |
| 6d | 15.7 | 7.8 |
| 6e | 500 | 31.5 |
| 6f | 62.5 | 7.8 |
| 6g | 62.5 | 7.8 |
| 6h | 31 | 31.5 |
| 6i | 31 | 15.7 |
| 6j | 31.5 | 15.7 |
| 7a | 62.5 | 15.7 |
| 7b | 31.5 | 31.5 |
| Ciprofloxacin | 1 | 64 |
MIC: Minimum inhibitory concentration.
Though antimicrobial activities were rather moderate against MRSA strains, few insights regarding the effect of substituents (R and R1) in series (6a–j) were observed.
While the antimicrobial activities were maintained when R is Chloro, only compound 6a exhibited lower MIC value (250 μg/ml) compared with compounds (6b, 6c and 6d) (MICs were 15.7, 31.5 and 15.7, respectively). The most potent MIC values were observed in compounds 6b and 6d, when R1 is Hydrogen or a bulky electron withdrawing 4-Bromo group, respectively. Interestingly, a small lipophilic electron donating substitution (like methyl group) resulted in a pronounced decline in the activity as observed in compound 6a.
Instead, when R is hydrogen; the antimicrobial activity declined significantly compared with the chloro derivatives (6a–d) as observed in the MIC values of compounds (6e–j). The benzimidazole derivatives 7a,b, were moderately active. Noticeably, the chloro derivative (7b) was two folds more active than the unsubstituted derivative 7a (MICs were 62.5 and 31.5 μg/ml, respectively).
As per mentioned, MIC values were superior against E. coli strains compared with Ciprofloxacin. Compounds (6a, 6d, 6f and 6g) displayed the most potent growth inhibition with MIC values of 7.8 μg/ml (compared with MIC of 64 μg/ml with Ciprofloxacin). The growth inhibition was decreased by two folds in compounds 6i, 6j and 7a with MICs of 15 μg/ml. It declined by three folds in compounds 6b, 6c, 6e, 6h and 7b with MIC values of 31.5 μg/ml. Notably, MIC values in E. coli were more influenced with R1 substituents compared with R. whereas, the most active compounds with MIC values of 7.8 μg/ml when R was either 4-methyl (as in 6a and 6f), 4-bromo (as in 6d) or hydrogen (as in 6g), regardless whether R was chloro (as in 6a and 6d) or hydrogen (as in 6f and 6g).
Finally, the overall antibacterial activities against both MRSA and E. coli strains of the benzimidazole derivatives (7a, 7b) are less potent compared with phenyl benzyloxy derivatives (6a–6j).
3.2.4. Cytotoxicity assay versus normal cell lines
Out of urgency to have nontoxic compounds, cytotoxicity assay against normal cells (HSF) was performed using concentration of 100 μg/ml for all the tested compounds. The results were indicated as % growth inhibition and revealed that all the synthesized compounds showed % inhibition ranging from 34 ± 4.5% in compound 6i to 2.05 ± 1.43% in compound 7b (Table 4). These results emphasized the safety profile of synthesized compounds against normal cells.
Table 4.
Cytotoxicity against normal cells (HSF)% growth inhibition (100 μg/ml).
| Cpd. # | HSF cytotoxicity % inhibition |
|---|---|
| 6a | 15.32 ± 2.97 |
| 6b | 10.5 ± 1.84 |
| 6c | 8.4 ± 3.02 |
| 6d | 10.18 ± 5.14 |
| 6e | 19.93 ± 1.92 |
| 6f | 5.04 ± 2.32 |
| 6g | 20.06 ± 3.2 |
| 6h | NT |
| 6i | 34 ± 4.5 |
| 6j | 23.8 ± 4.68 |
| 7a | 30.6 ± 2.5 |
| 7b | 2.05 ± 1.43 |
NT: Not tested.
3.2.5. Structure–activity relationship analysis
To gain further insight related to structure–activity relationship of the synthesized compounds, a few observations were noted as summarized in Figure 4:
-
▪
The 4-phenyl benzyloxy derivatives (6a–j) manifested more diverse cytotoxic activity against a panel of cancer cell lines as well as better antimicrobial profile compared with benzimidazole derivatives (7a,b).
-
▪
For R substituent: Chloro derivatives showed more potent anticancer activity as well as antimicrobial activities against MRSA except for compound 6a.
-
▪
For R1 substituent: Having R as Cl maintained the cytotoxic activity whether it's unsubstituted (as in compound 6b) or anchored in the terminal benzene ring with an electron withdrawing group in the para position as in compounds 6d and 6e. Activity declined upon di-substitution as shown in compound 6c or upon para substitution with lipophilic; electron donating group as in methyl group in compound 6a.
Figure 4.
Schematic representation of structure–activity relationship study of the synthesized pyrazole–chalcone hydrids.
Captivatingly, the antimicrobial activity against MRSA and E coli was seamlessly influenced by R and R1 substitution pattern in benzyloxy derivatives (6a–6j) as noticed in the cytotoxic activities of the same series.
Thus, compound 6d emerged as a novel pyrazole–chalcone hybrid with potent cytotoxic and anti-microbial activities.
4. Conclusion
Novel pyrazole–chalcone hybrids (compounds 6a–j and 7a,b) were synthesized and evaluated for their cytotoxic activities against 14 cancer cell lines. The synthesized compounds exhibited moderate to potent anticancer activities against HELA, HEP2, MDA-MB-231, PC3 and HNO-97 cell lines. Outstanding anticancer activities were observed with HNO-97 cell line with GI% ranging from (69–93%). IC50 of the most potent compounds (6a, 6b, 6d, 6e and 7a) against HNO-97 were calculated where compounds 6b and 6d stood out as the most potent cytotoxic agents in the series with IC50 of 10 and 10.56 μM, respectively. Meanwhile, to further explore the compounds dual activity as antimicrobial beside its anticancer activity; MICs were revealed for the targeted compounds against MRSA and E. coli strains. All the compounds unveiled better E. coli growth inhibition when compared with Ciprofloxacin. Compounds 6a, 6d, 6f and 6g expressed MIC of 7.8 μg/ml whereas compounds 6i, 6j and 7a showed 15.7 μg/ml as MIC. Noteworthy, compounds 6b and 6d manifested moderate antimicrobial activity against MRSA with MIC value of 15.7 μg/ml. Based on the importance of having safe agents on normal cells, all the targeted compounds were examined against HSF normal cells and interestingly they divulged GI% <30%, so can be considered as safe and nontoxic agents. Out of the aforementioned findings, the synthesized pyrazole–chalcone hybrids can be anticipated as powerful and safe chemotherapeutic agents with dual anticancer-antimicrobial activities paving the way to have more potent derivatives upon further optimization.
Supplementary Material
Supplemental material
Supplemental data for this article can be accessed at https://doi.org/10.1080/17568919.2024.2347090
Financial disclosure
The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Competing interests disclosure
The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Writing disclosure
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