Next Article in Journal
Herpes Simplex Virus ICP27 Protein Inhibits AIM 2-Dependent Inflammasome Influencing Pro-Inflammatory Cytokines Release in Human Pigment Epithelial Cells (hTert-RPE 1)
Next Article in Special Issue
Design and Synthesis of 1,3-Diarylpyrazoles and Investigation of Their Cytotoxicity and Antiparasitic Profile
Previous Article in Journal
Identification of Indicator Genes for Agar Accumulation in Gracilariopsis lemaneiformis (Rhodophyta)
Previous Article in Special Issue
Recent Advances in the Development of Pyrazole Derivatives as Anticancer Agents
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 9
10.3390/ijms25094607
ijms-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

Structure–Activity Relationship Studies on Highly Functionalized Pyrazole Hydrazones and Amides as Antiproliferative and Antioxidant Agents

by
Matteo Lusardi
1,
Maria Grazia Signorello
1,
Eleonora Russo
1,
Debora Caviglia
1,
Marco Ponassi
2,
Erika Iervasi
2,
Camillo Rosano
2,
Chiara Brullo
1 and
Andrea Spallarossa
1,*
1
Department of Pharmacy, University of Genova, Viale Benedetto XV, 3, 16132 Genova, Italy
2
Proteomics and Mass Spectrometry Unit, IRCCS Ospedale Policlinico San Martino, Largo R. Benzi 10, 16132 Genova, Italy
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(9), 4607; https://doi.org/10.3390/ijms25094607
Submission received: 29 March 2024 / Revised: 19 April 2024 / Accepted: 20 April 2024 / Published: 23 April 2024
(This article belongs to the Special Issue Pharmaceutically-Active Pyrazole Compounds)
Browse Figures
Versions Notes

Abstract

:
Aminopyrazoles represent interesting structures in medicinal chemistry, and several derivatives showed biological activity in different therapeutic areas. Previously reported 5-aminopyrazolyl acylhydrazones and amides showed relevant antioxidant and anti-inflammatory activities. To further extend the structure–activity relationships in this class of derivatives, a novel series of pyrazolyl acylhydrazones and amides was designed and prepared through a divergent approach. The novel compounds shared the phenylamino pyrazole nucleus that was differently decorated at positions 1, 3, and 4. The antiproliferative, antiaggregating, and antioxidant properties of the obtained derivatives 10–22 were evaluated in in vitro assays. Derivative 11a showed relevant antitumor properties against selected tumor cell lines (namely, HeLa, MCF7, SKOV3, and SKMEL28) with micromolar IC50 values. In the platelet assay, selected pyrazoles showed higher antioxidant and ROS formation inhibition activity than the reference drugs acetylsalicylic acid and N-acetylcysteine. Furthermore, in vitro radical scavenging screening confirmed the good antioxidant properties of acylhydrazone molecules. Overall, the collected data allowed us to extend the structure–activity relationships of the previously reported compounds and confirmed the pharmaceutical attractiveness of this class of aminopyrazole derivatives.

1. Introduction

Pyrazole scaffold is a pharmaceutically relevant moiety [1,2,3,4,5,6], and pyrazole-containing compounds show antiviral [7], antibacterial [8,9], antimalarial [10], anti-inflammatory [11], antidiabetic [12], antiglaucoma [13,14], and anticancer [15,16,17,18,19,20,21] properties. Furthermore, pyrazole scaffolds are shared by several protein kinase inhibitors, including FDA-approved drugs Avapritinib, Asciminib, Crizotinib, Encorafenib, Erdafitinib, Pralsetinib, Pirtobrutinib, and Ruxolitinib (Figure 1) [22,23]. Among pyrazole series, aminopyrazoles (APs) represent an attractive framework in medicinal chemistry [24,25,26]; indeed, the decoration of the pyrazole ring with amino substituents at different positions led to the isolation of pharmacologically active derivatives including analgesic (e.g., Aminophenazone and Metamizole; Figure 1) and antitumor (e.g., AT7519, AT9283, Prexasertib, Pirtobrutinib/Jaypirca™; Figure 1) agents [23,27,28,29,30,31,32,33]. Additionally, the AP scaffold has been widely studied for its relevant activity in oxidative stress and inflammation. In detail, 3-AP I (Figure 1) showed weak antiproliferative activity against four tumor cell lines (i.e., HepG2, WI38, VERO, and MCF-7), but exhibited high antioxidant activity, due to the free amino group on the pyrazole ring [34]. 4-APs IIa,b (Figure 1) and their hydrochloride salts displayed excellent antiradical activity in the ABTS scavenging assay, with Trolox equivalent antioxidant capacity (TEAC) values of 1.35 and 1.10 and IC50 of 14.1 µM and 17.6 µM, respectively. Additionally, the two compounds confirmed their promising antioxidant properties in the oxygen radical absorbance capacity assay (ORAC) and in the oxidative erythrocyte hemolysis assay [35]. Structure–activity relationships (SARs) extension on IIa,b led to the isolation of pyrazole hydrochloride III (Figure 1) endowed with improved pharmacokinetic and antioxidant properties. Further statistical analysis and cytotoxicity studies confirmed the promising profile of the compound, which was taken as the lead structure for the development of effective agents against oxidative stress-related diseases [36].
More recently, 5-AP acylhydrazones Iva–d (Figure 2) proved to inhibit platelet aggregation and reactive oxygen species (ROS) production with IC50 values in the low micromolar range [37]. In particular, derivative IVd showed antioxidant and anti-inflammatory dual activity, inhibiting ROS production in fMLP-activated neutrophils and blocking PDE4B and PDE4D phosphodiesterase enzymes (IC50 = 1.05 µM and 0.55 µM, respectively), two PDE4 isoforms involved in inflammatory processes [37]. Furthermore, pyrazoles IVa–c strongly reduced superoxide anion production, lipid peroxidation, and NAPDH oxidase activity in H2O2-stimulated EA.hy926 cells, thus highlighting the potential of compounds on oxidative status and aerobic metabolism [38]. In previous work, the SARs of hydrazones IV were further extended through the preparation of amide derivatives V (Figure 2), able to inhibit both aggregation and ROS formation in platelets and p38MAPK phosphorylation [39].
To further exploit the pharmacological potentials of derivatives IV and V (Figure 2), a novel series of pyrazoles 10–22 have been studied for their antiproliferative and antioxidant activities. In particular, acylhydrazones 10–13 bear an anilino substituent on the pyrazole scaffold (not present in the structures of the lead derivatives IV and V) with or without concomitant variation of the pyrazole 2-hydroxy-2-phenylethyl chain (derivative 13: no modification; derivatives 10: removal of the chain; derivatives 11 and 12: replacement of the chain with a methyl substituent). The substituents of ring A were selected according to the SARs developed for compounds IV (X = H, OMe, OBn, OPh). Additionally, to evaluate the effects on activity of the acylhydrazone moiety, pyrazolyl amides 14–22 were prepared. These compounds share with their acylhydrazone congeners the anilino substituent and bear, as G groups, cyclopropyl, or differently substituted phenyl rings (namely, 4-Cl, 4-OMe, 2,6-(OMe)2; 3,4-(OMe)2; 3,4,5-(OMe)3), characterizing the most effective derivatives IV or V.

2. Results

2.1. Chemistry

The desired compounds 10–22 were obtained through a divergent, stepwise approach, starting from the common AP intermediates 1–5 (Scheme 1). These key building blocks were prepared through the condensation of cyanoacetic ester, phenyl isothiocyanate, and (un)substituted hydrazine, as previously reported [40,41,42]. Thus, synthons 2–5 were condensed with hydrazine monohydrate, leading to the corresponding carbohydrazide intermediates 6–9 in good yields (Scheme 1); these derivatives were then reacted with 4-methoxy benzaldehydes a–d (commercially available or prepared by alkylation or arylation of isovanillin according to the published procedures) [43,44] in absolute ethanol to afford the desired compounds 10–13 in moderate to good yields (13–80%, Scheme 1). Interestingly, this reaction proved to be stereoselective, allowing the unique isolation of the E-isomer, as assessed by proton NMR analysis. In fact, acylhydrazones 10–13 showed the signal of the acylhydrazone proton at chemical shift values lower than 12 ppm (chemical shift range: 9.59–10.65 ppm), typical of the E-isomer as recently reported for similar hydrazones [37].
Amides 14–22 were prepared through the condensation of APs 1–3 with the proper acyl chloride (namely, cyclopropyl carbonyl chloride, 4-chlorobenzoyl chloride, and differently methoxy-substituted benzoyl chlorides; Scheme 1), selected according to the SAR developed for the acylhydrazone series. The different reactivity of the acyl chlorides towards the pyrazole amino group required the definition of different experimental protocols. Thus, for derivatives 14–16 and 20–22, the reaction was carried out at rt in dichloromethane (DCM) with the addition of triethylamine (TEA), while pyrazoles 17 and 18 were prepared in anhydrous N,N-dimethylformamide (DMF) at 120 °C in the presence of N,N,N’,N’-tetramethylethylenediamine (TMEDA). Interestingly, TMEDA plays a dual role in the acylation reaction, acting as a HCl scavenger and further activating the acyl chloride through the formation of a pseudo-cyclic complex, as previously described in the literature [45,46]. Finally, compound 19 was prepared at rt in acetonitrile (ACN) using TEA as a base. The acylation of pyrazole 4 (regioisomer of 3) was tried in different experimental conditions. However, the amino group proved to be unreactive, possibly due to the steric hindrance of the proximal methyl group.

2.2. Antiproliferative Properties

Pyrazole acylhydrazones 10–13 and amides 14–22 were tested by MTT assay to evaluate their antiproliferative and cytotoxicity properties against a panel of eight tumor cell lines and normal fibroblasts. The compounds were screened at a fixed concentration of 10 µM, and cisplatin (10 µM) was used as a reference drug. As reported in Table 1, the majority of acylhydrazones did not show any cytotoxic activity against tumor and fibroblast cells, displaying mean growth percentage values higher than 50%. However, AP 11a significantly inhibits the proliferation of HeLa (25.00%, Table 1), MCF7 (33.56%, Table 1), SKOV3 (43.60%, Table 1), and SKMEL28 (49.44%, Table 1) cancer cells, resulting in more efficacy than cisplatin against HeLa and MCF7 cells. The antiproliferative activity of the compound marginally affected the growth of normal fibroblasts, resulting in less cytotoxicity than the reference cisplatin (69.81% vs. 39.52% mean growth percentages, respectively).
The pyrazolyl amides 14–22 showed poor antiproliferative activity against all tested tumor cell lines, with exceptions made for derivatives 14 (active against SKMEL28, SKOV3, A549, and HeLa cells) and 16 (active against Hep-G2). In particular, 14 was found to be more effective than cisplatin against HeLa, A549, and SKMEL28 cells. Differently from its amide analogues, N-unsubstituted 3,4,5-trimethoxybenzoyl pyrazole 14 was as cytotoxic as cisplatin against normal GM6114 fibroblasts (mean growth percentage = 39.31%).
The remarkable antiproliferative, non-cytotoxic activity of 11a was further investigated, and the IC50 values against HeLa, MCF7, SKOV3, and SKMEL28 cell lines were determined. The compound showed cell proliferation inhibition values in the micromolar concentration range (IC50(HeLa) = 4.63 ± 0.41 µM; IC50(MCF7) = 6.90 ± 0.34 µM; IC50(SKOV3) = 6.88 ± 0.23 µM; IC50(SKMEL28) = 9.45 ± 0.66 µM), confirming its promising antiproliferative profile.
In addition, 11a and 17 (representative compounds of the acylhydrazone and amide series) were selected by the National Cancer Institute (NCI, Germantown, MD, USA) and tested at a fixed concentration of 10 µM against a panel of fifty-nine different cancer cell lines, including highly metastatic tumors (Table 2). Pyrazolyl amide 17 did not show any antiproliferative activity (growth percentage range = 81.47–118.49%), whereas 11a confirmed its promising antitumor properties, showing growth percentage values lower than 20% against leukemia (HL-60(TB), K-562, SR cells), NSCLC (NCI-H460 and NCI-H522 cells), colon (HCT-116, HCT-15, HT29, and SW-620 cells), breast (MCF7, HS 578T, MDA-MB-468 cells), and melanoma (LOX IMVI, M14, and MDA-MB-435 cells).

2.3. Antioxidant Evaluation

The antioxidant properties of acylhydrazones 10–13 and pyrazolyl amides 14–22 were tested by evaluating their inhibition of platelet aggregation and ROS production (Figure 3, Table S1). In fact, human platelets could represent a fast and low-cost biological model to screen compounds as anticancer, anti-inflammatory, and antiaggregating agents [47,48,49]. Moreover, ROS production inhibition, related to human platelet aggregation, could provide a good indication of the anti-inflammatory and antioxidant properties of the newly synthesized compounds [47,50,51,52]. N-acetylcysteine (NAC) and acetyl salicylic acid (ASA) were used as reference drugs for antioxidant and antiaggregant activities, respectively.
All derivatives blocked ROS production more effectively than NAC, and most of the tested compounds (18 out of 23) showed improved antiaggregant properties in comparison with ASA.

2.4. DPPH Radical-Scavenging Activity

The antioxidant activity of the representative compounds of the two series (namely, acylhydrazones 10b, 11a, 11d, 12d, 13d, and amides 14, 22) was measured in vitro using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [53]. The results were calculated as Trolox equivalent and expressed as a percentage of antioxidant activity (AA) (Table 3). Amides 14 and 22 showed poor antioxidant properties, whereas hydrazone compounds displayed significant AA values (range 15.53–76.45%). Derivatives 10b and 12d were endowed with the highest antioxidant activity, thus highlighting the relevance of both pyrazole N1 and acylhydrazone substituents on compounds’ radical scavenging properties.

3. Discussion

To further extend the SARs of lead derivatives IV and V, acylhydrazones 10–13 and amides 14–22 were prepared through a divergent, regioselective synthetic protocol. The novel acylhydrazone derivatives showed limited antiproliferative activity in cell-based assays, with an exception made for derivative 11a that significantly inhibits the duplication of leukemia, non-small cell lung cancer (NSCLC), colon cancer, CNS cancer, melanoma, and breast cancer cells, showing the highest inhibitory activity against the cell line (Table 1 and Table 2). Noteworthy, derivative 11a showed a lethal effect against melanoma MDA-MB-435 and breast cancer MDA-MB-468 cell lines, thus confirming the attractiveness of this molecule as a lead structure for the development of novel anticancer agents. Among amides, the N-unsubstituted compound 14 was more effective than cisplatin against cervical HeLa and lung A549 cancer cells, also affecting the proliferation of ovarian SKOV3. Unfortunately, the observed antiproliferative effects were coupled with significant cytotoxicity against normal fibroblasts.
Derivatives 10–22 showed a reduced antioxidant activity in comparison with lead compounds IV and V [37,39], still resulting in more effective than reference NAC (IC50 = 872 µM) in inhibiting ROS production. Moreover, the majority of the compounds showed increased anti-platelet aggregation properties in comparison to ASA (IC50 = 438 µM). The ability of acyl hydrazone compounds 10–13 to inhibit ROS formation and platelet aggregation appears to be affected by the substitution of both the pyrazole nucleus and the phenyl carbohydrazide ring. Thus, unsubstituted pyrazoles 10 and N-methyl pyrazoles 11 and 12 proved to be more active than their sterically hindered congeners 13. Moreover, compounds bearing a 4-methoxyphenyl or a 3,4-dimethoxyphenyl substituent (i.e., derivatives 10a,b, 11a,b, and 12a,b) were endowed with the lower IC50 values for both platelet aggregation (94–265 µM, Table S1) and ROS production (104–123 µM, Table S1) inhibition. Within the pyrazolyl amide series, the aromatic nature of the amide substituent emerged to be critical for activity. Thus, benzamido analogues 14–16, 18–22 showed anti-ROS and antiaggregant effects in a narrow IC50 range (ROS production IC50 range = 262–387 µM; antiaggregant IC50 range = 249–365 µM), resulting in greater effectiveness than the reference drugs. Conversely, the cyclopropyl amino analogue 17 was found to be less effective than its congeners (IC50 (ROS) = 573 µM; IC50 (aggregation) = 460 µM), with reduced antiaggregant properties in comparison with ASA.
In the DPPH radical-scavenging assay, amides 14 and 22 were less effective than their hydrazone analogues (Table 3), thus highlighting the relevance of this moiety for activity (Table 3). Among tested derivatives, pyrazole hydrazones 10b, 12d, and 13d proved to be more effective than their analogues 11a,d, indicating that the insertion of a methyl substituent on the pyrazole N-atom adjacent to the phenylamino group was detrimental for activity. Noteworthy, these data suggest that the antiproliferative activity of the prepared series (and, in particular, that of derivatives 11a and 14) does not correlate with the compounds’ in vitro anti-scavenging properties.
Conversely, the high radical scavenging properties of 10b (AA% = 76.45%) well correlate with the compound’s antiaggregant and ROS inhibitory activities observed in platelets.
The developed SARs for the two series of compounds are summarized in Figure 4.

4. Materials and Methods

4.1. Chemistry

Reagents were purchased by Alfa-Aesar and Sigma-Aldrich. 3,4-dimethoxybenzaldehyde, 3-methoxy-4-phenoxybenzaldehyde, and 4-(benzyloxy)-3-methoxybenzaldehyde were prepared according to published procedures [43,44,54]. All the solvents were reagent grade and were dried on molecular sieves (5 Å 1/16” inch pellets). Unless otherwise stated, all commercial reagents were used without further purification. Organic solutions were dried over anhydrous sodium sulphate. Aluminium-backed silica gel thin layer chromatography (TLC) plates (Merck DC-Alufolien Kieselgel 60 F254) were used for reaction monitoring and purity analyses. A DCM/MeOH 9:1 mixture was used as a developing solvent, and spots were detected by UV light and/or by iodine vapors. Melting points were measured on a Fisher-Johns apparatus and are uncorrected. 1H NMR and 13C NMR spectra were collected on a JEOL JNM-ECZR (Tokyo, Japan) instrument (Figures S1–S44); chemical shifts were reported in δ (ppm) units, and the splitting patterns were described as follows: s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). The first-order values reported for coupling constants J were given in Hz.The elemental composition of synthesized compounds was collected by an EA1110. Pyrazoles 1–5 were synthesized as previously reported [40,41,42].

4.1.1. General Synthesis of Intermediates 6–9

A mixture of the proper pyrazole 2–5 (2 mmol) and hydrazine monohydrate (2 mL) was refluxed for 4–6 h. After cooling at rt, water (15 mL) was added, and the solution was acidified with HCl 2 M. The precipitate was collected by filtration and used without further purification. For compound 6, the excess of hydrazine was removed under reduced pressure, and the crude mixture was purified by column chromatography (silica gel, eluent: Et2O-Et2O/20% EtOH).
  • 3-Amino-5-(phenylamino)-1H-pyrazole-4-carbohydrazide 6
Colourless oil. Yield 55%. Calcd for C10H12N6O: C = 51.72; H = 5.21; N = 36.19. Found: C = 36.07; H = 5.28. N = 5.18.
  • 3-Amino-1-methyl-5-(phenylamino)-1H-pyrazole-4-carbohydrazide 7
Mp 226–228 °C (H2O); yield 83%. Calcd for C11H14N6O: C = 53.65; H = 5.73; N = 34.13. Found: C = 53.34; H = 5.74; N = 34.07.
  • 5-Amino-1-methyl-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 8
Mp 200–204 °C (H2O); yield: 71%. Calcd for C11H14N6O: C = 53.65; H = 5.73; N = 34.13. Found: C = 53.88; H = 5.69; N = 34.21.
  • 5-Amino-1-(2-hydroxy-2-phenylethyl)-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 9
Mp 177–180 °C (H2O); yield 53%. Calcd for C18H20N6O2: C = 61.35; H = 5.72; N = 23.85. Found: C = 61.40; H = 5.67; N = 24.03.

4.1.2. General Synthetic Procedure for the Preparation of Pyrazole Acylhydrazones 10–13

To a solution of the proper intermediate 6–9 (1 mmol) in absolute EtOH (5 mL), the suitable benzaldehyde a–d (1 mmol) was added. The reaction mixture was stirred at reflux for 16 h and then cooled at rt. The precipitate was collected by filtration and crystallized with ethanol.
  • (E)-3-amino-N′-(4-methoxybenzylidene)-5-(phenylamino)-1H-pyrazole-4-carbohydrazide 10a.
Mp 227–232 °C (EtOH); Yield 24%. 1H NMR (400 MHz, DMSO-d6): δ 3.79 (s, 3H, OCH3); 6.15 (bs, 2H, NH2, exchangeable); 6.73–6.79 (m, 1H, arom. H); 6.97–7.02 (m, 2H, arom. H); 7.16–7.22 (m, 2H, arom. H); 7.35–7.41 (m, 2H, arom. H); 7.58–7.61 (m, 2H, arom. H); 8.13 (s, 1H, CH=C); 8.91 (bs, 1H, NH phenyl, exchangeable); 10.48 (bs, 1H, NH hydraz., exchangeable); 11.18 (bs, 1H, NH pyraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 55.32; 85.33; 114.45; 116.01; 118.96; 126.89; 128.37; 128.79; 142.53; 144.64; 148.48; 151.09; 160.62. Calcd for C18H18N6O2: C = 61.70; H = 5.18; N = 23.99. Found: C = 61.65; H = 5.11; N = 23.92.
  • (E)-3-amino-N′-(3,4-dimethoxybenzylidene)-5-(phenylamino)-1H-pyrazole-4-carbohydrazide 10b.
Mp 263–266 °C (EtOH); Yield 13%. 1H NMR (400 MHz, DMSO-d6): δ 3.75 (s, 3H, OCH3); 3.79 (s, 3H, OCH3); 6.06 (bs, 2H, NH2, exchangeable); 6.73–6.80 (m, 1H, arom. H); 7.01–7.28 (m, 5H, arom. H); 7.44–7.49 (m, 2H, arom. H); 8.22 (s, 1H, CH=C); 9.24 (bs, 1H, NH phenyl, exchangeable); 10.65 (bs, 1H, NH hydraz., exchangeable); 11.37 (bs, 1H, NH pyraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 55.29; 55.58; 83.60; 115.44; 115.90; 116.02; 118.79; 118.98; 121.45; 128.79; 128.91; 141.91; 146.17; 151.26; 152.14; 171.47. Calcd for C19H20N6O3: C = 59.99; H = 5.30; N = 22.90. Found: C = 59.82; H = 5.33; N = 22.75.
  • (E)-3-amino-N′-(4-methoxybenzylidene)-1-methyl-5-(phenylamino)-1H-pyrazole-4-carbohydrazide 11a.
Mp 208–210 °C (EtOH); Yield 51%. 1H NMR (400 MHz, DMSO-d6): δ 3.38 (s, 3H, NCH3); 3.78 (s, 3H, OCH3); 5.47 (bs, 2H, NH2, exchangeable); 6.58–6.63 (m, 2H, arom. H); 6.75–6.82 (m, 1H, arom. H); 6.93–7.00 (m, 2H, arom. H); 7.15–7.24 (m, 2H, arom. H); 7.53–7.59 (m, 2H arom. H); 7.91 (s, 1H, CH=C); 8.09 (bs, 1H, NH phenyl, exchangeable); 10.14 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 34.60; 55.30; 93.60; 114.35; 119.80; 126.74; 128.45; 129.54; 138.04; 144.49; 145.17; 155.32; 160.66. Calcd for C19H20N6O2: C = 62.62; H = 5.53; N = 23.06. Found: C = 62.59; H = 5.51; N = 22.94.
  • (E)-3-amino-N′-(3,4-dimethoxybenzylidene)-1-methyl-5-(phenylamino)-1H-pyrazole-4-carbohydrazide 11b.
Mp 218–220 °C (EtOH); Yield 60%. 1H NMR (400 MHz, DMSO-d6): δ 3.38 (s, 3H, NCH3); 3.77 (s, 3H, OCH3); 3.78 (s, 3H, OCH3); 5.47 (bs, 2H, NH2, exchangeable); 6.60–6.64 (m, 2H, arom. H); 6.77–6.82 (m, 1H, arom. H); 6.96–7.00 (m, 1H, arom. H); 7.10–7.14 (m, 1H, arom. H); 7.18–7.25 (m, 3H arom. H); 7.91 (s, 1H, CH=C); 8.09 (bs, 1H, NH phenyl, exchangeable); 10.16 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 34.58; 55.38; 55.55; 93.61; 108.18; 109.03; 111.49; 114.32; 119.81; 121.37; 123.56; 126.89; 129.54; 144.49; 145.47; 149.01; 150.51; 151.62; 155.30; 160.85. Calcd for C20H22N6O3: C = 60.90; H = 5.62; N = 21.31. Found: C = 61.40; H = 5.54; N = 21.05.
  • (E)-3-amino-N′-(4-methoxy-3-phenoxybenzylidene)-1-methyl-5-(phenylamino)-1H-pyrazole-4-carbohydrazide 11c.
Mp 226–228 °C (EtOH); Yield 49%. 1H NMR (400 MHz, DMSO-d6): δ 3.36 (s, 3H, NCH3); 3.79 (s, 3H, OCH3); 5.44 (bs, 2H, NH2, exchangeable); 6.57–6.62 (m, 2H, arom. H); 6.76–6.81 (m, 1H, arom. H); 6.87–6.90 (m, 2H, arom. H); 7.04–7.08 (m, 1H, arom. H); 7.16–7.22 (m, 3H arom. H); 7.26–7.35 (m, 3H, arom. H); 7.40–7.44 (m, 1H, arom. H); 7.90 (s, 1H, CH=C); 8.05 (bs, 1H, NH phenyl, exchangeable); 10.19 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 34.59; 55.87; 93.55; 113.35; 114.33; 116.80; 118.03; 119.80; 122.75; 124.88; 127.44; 129.53; 129.93; 138.16; 144.41; 152.56; 155.31; 157.29; 159.90. Calcd for C25H24N6O3: C = 65.78; H = 5.30; N = 18.41. Found: C = 65.55; H = 5.28; N = 18.21.
  • (E)-3-amino-N′-(3-(benzyloxy)-4-methoxybenzylidene)-1-methyl-5-(phenylamino)-1H-pyrazole-4-carbohydrazide 11d.
Mp 183–186 °C (EtOH); Yield 80%. 1H NMR (400 MHz, DMSO-d6): δ 3.34 (s, 3H, NCH3); 3.76 (s, 3H, OCH3); 5.05 (s, 2H, CH2Ph); 5.44 (bs, 2H, NH2, exchangeable); 6.55–6.61 (m, 2H, arom. H); 6.73–6.78 (m, 1H, arom. H); 6.96–7.00 (m, 1H, arom. H); 7.12–7.19 (m, 2H, arom. H); 7.28–7.44 (m, 7H arom. H); 7.86 (s, 1H, CH=C); 8.04 (bs, 1H, NH phenyl, exchangeable); 10.11 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 34.60; 55.65; 69.83; 93.65; 110.09; 111.82; 114.32; 119.81; 121.54; 126.86; 127.92; 128.01; 128.46; 129.55; 136.88; 144.49; 148.05; 150.79; 155.29; 160.78. Calcd for C26H26N6O3: C = 66.37; H = 5.57; N = 17.86. Found: C = 66.45; H = 5.48; N = 17.72.
  • (E)-5-amino-N′-(4-methoxybenzylidene)-1-methyl-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 12a.
Mp 195–198 °C (EtOH); Yield 50%. 1H NMR (400 MHz, DMSO-d6): δ 3.52 (s, 3H, NCH3); 3.79 (s, 3H, OCH3); 6.39 (bs, 2H, NH2, exchangeable); 6.74–6.79 (m, 1H, arom. H); 6.98–7.02 (m, 2H, arom. H); 7.16–7.22 (m, 2H, arom. H); 7.35–7.39 (m, 2H, arom. H); 7.58–7.63 (m, 2H arom. H); 8.12 (s, 1H, CH=C); 8.90 (bs, 1H, NH phenyl, exchangeable); 10.52 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 34.12; 55.32; 85.63; 114.45; 116.06; 119.06; 126.83; 128.36; 128.78; 130.03; 142.42; 144.58; 147.40; 149.62; 160.63. Calcd for C19H20N6O2: C = 62.62; H = 5.53; N = 23.06. Found: C = 62.56; H = 5.63; N = 22.99.
  • (E)-5-amino-N′-(3,4-dimethoxybenzylidene)-1-methyl-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 12b.
Mp 104–106 °C (EtOH); Yield 35%. 1H NMR (400 MHz, CDCl3): δ 3.59 (s, 3H, NCH3); 3.83 (s, 3H, OCH3); 3.92 (s, 3H, OCH3); 5.45 (bs, 2H, NH2, exchangeable); 6.82–6.85 (m, 1H, arom. H); 6.90–6.94 (m, 1H, arom. H); 7.01–7.05 (m, 1H, arom. H); 7.23–7.30 (m, 4H, arom. H); 7.32–7.36 (m, 2H, arom. H + CH=C); 7.72 (bs, 1H, NH phenyl, exchangeable); 9.80 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, CDCl3): δ 33.92; 55.94; 89.16; 108.12; 110.66; 116.70; 120.73; 122.31; 126.54; 129.34; 143.03; 145.57; 148.90; 149.41; 151.10; 163.16. Calcd for C20H22N6O3: C = 60.90; H = 5.62; N = 21.31. Found: C = 60.65; H = 5.38; N = 21.45.
  • (E)-5-amino-N′-(4-methoxy-3-phenoxybenzylidene)-1-methyl-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 12c.
Mp 234–236 °C (EtOH); Yield 68%. 1H NMR (400 MHz, DMSO-d6): δ 3.51 (s, 3H, NCH3); 3.80 (s, 3H, OCH3); 6.31 (bs, 2H, NH2, exchangeable); 6.74–6.78 (m, 1H, arom. H); 6.87–6.91 (m, 2H, arom. H); 7.04–7.09 (m, 1H, arom. H); 7.15–7.25 (m, 3H, arom. H); 7.30–7.37 (m, 5H arom. H); 7.44–7.48 (m, 1H, arom. H); 8.10 (s, 1H, CH=C); 8.79 (bs, 1H, NH phenyl, exchangeable); 10.50 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 34.09; 55.89; 85.70; 113.45; 116.05; 116.70; 118.13; 119.08; 122.69; 124.82; 127.60; 128.77; 129.91; 142.45; 144.11; 144.40; 147.36; 149.47; 152.54; 157.33; 162.61. Calcd for C25H24N6O3: C = 65.78; H = 5.30. N = 18.41; Found: C = 65.70; H = 5.30; N = 18.35.
  • (E)-5-amino-N′-(3-(benzyloxy)-4-methoxybenzylidene)-1-methyl-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 12d.
Mp 131–133 °C (EtOH); Yield 91%. 1H NMR (400 MHz, DMSO-d6): δ 3.53 (s, 3H, NCH3); 3.81 (s, 3H, OCH3); 5.01 (s, 2H, CH2Ph); 6.41 (bs, 2H, NH2, exchangeable); 6.74–6.80 (m, 1H, arom. H); 7.01–7.06 (m, 1H, arom. H); 7.16–7.22 (m, 3H, arom. H); 7.35–7.44 (m, 8H, arom. H); 8.09 (s, 1H, CH=C); 8.90 (bs, 1H, NH phenyl, exchangeable); 10.59 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 34.61; 56.16; 70.20; 86.14; 110.17; 112.37; 116.64; 119.63; 122.09; 127.42; 128.45; 128.93; 129.29; 137.32; 142.84; 145.11; 148.21; 148.65; 149.89; 151.25; 161.27. Calcd for C26H26N6O3: C = 66.37; H = 5.57; N = 17.86. Found: C = 66.34; H = 5.60. N = 17.68.
  • (E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N′-(4-methoxybenzylidene)-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 13a.
Mp 120–122 °C (EtOH); Yield 52%. 1H NMR (400 MHz, CDCl3): δ 3.78 (s, 3H, OCH3); 3.85–4.04 (m, 3H, CH2N + CHOH); 5.12–5.17 (m, 1H, OH, exchangeable); 5.43 (bs, 2H, NH2, exchangeable); 6.76–6.81 (m, 2H, arom. H); 6.83–6.89 (m, 1H, arom. H); 7.14–7.23 (m, 4H, arom. H); 7.27–7.39 (m, 5H, arom. H); 7.45–7.50 (m, 2H, arom. H); 7.55 (s, 1H, CH=C); 8.61 (bs, 1H, NH phenyl, exchangeable); 9.59 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, CDCl3): δ 55.26; 55.47; 73.50; 89.27; 114.36; 116.86; 120.71; 126.04; 126.18; 128.16; 128.75; 129.13; 129.32; 130.36; 140.97; 142.71; 145.55; 149.12; 161.45. Calcd for C26H26N6O3: C = 66.37; H = 5.57; N = 17.86. Found: C = 66.40; H = 5.30. N = 17.63.
  • (E)-5-amino-N′-(3,4-dimethoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 13b.
Mp 158–159 °C (EtOH); Yield 65%. 1H NMR (400 MHz, DMSO-d6): δ 3.74 (s, 3H, OCH3); 3.80 (s, 3H, OCH3); 3.92–4.12 (m, 2H, CH2N); 5.00–5.05 (m, 1H, CHOH); 5.73–5.76 (m, 1H, OH, exchangeable); 6.28 (bs, 2H, NH2, exchangeable); 6.75–6.80 (m, 1H, arom. H); 6.98–7.02 (m, 1H, arom. H); 7.12–7.22 (m, 3H, arom. H); 7.27–7.29 (m, 2H, arom. H); 7.32–7.36 (m, 4H, arom. H); 7.42–7.46 (m, 2H, arom. H); 8.09 (s, 1H, CH=C); 8.86 (bs, 1H, NH phenyl, exchangeable); 10.56 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 53.76; 55.29; 55.59; 71.33; 86.03; 107.84; 111.55; 116.13; 119.10; 121.47; 126.35; 126.98; 127.40; 128.14; 128.79; 142.53; 142.77; 144.72; 148.26; 149.11; 149.38; 150.49; 163.07. Calcd for C27H28N6O4: C = 64.79; H = 5.64; N = 16.79. Found: C = 64.55; H = 5.76. N = 16.52.
  • (E)-5-amino-1-(2-hydroxy-2-phenylethyl)-N′-(4-methoxy-3-phenoxybenzylidene)-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 13c.
Mp 144–145 °C (EtOH); Yield 59%. 1H NMR (400 MHz, DMSO-d6): δ 3.80 (s, 3H, OCH3); 3.90–4.10 (m, 2H, CH2N); 4.97–5.03 (m, 1H, CHOH); 5.71–5.74 (m, 1H, OH, exchangeable); 6.18 (bs, 2H, NH2, exchangeable); 6.73–6.79 (m, 1H, arom. H); 6.87–6.91 (m, 2H, arom. H); 7.03–7.09 (m, 1H, arom. H); 7.14–7.25 (m, 3H, arom. H); 7.27–7.37 (m, 8H, arom. H); 7.39–7.47 (m, 3H, arom. H); 8.09 (s, 1H, CH=C); 8.72 (bs, 1H, NH phenyl, exchangeable); 10.46 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 53.69; 55.91; 71.32; 86.12; 113.47; 116.09; 116.69; 118.17; 119.11; 122.69; 124.86; 126.33; 127.39; 127.61; 128.12; 128.78; 129.91; 142.60; 142.71; 144.11; 144.38; 148.12; 149.27; 152.56; 157.35. Calcd for C32H30N6O4: C = 68.31; H = 5.37; N = 14.94. Found: C = 68.01; H = 5.15; N = 15.00.
  • (E)-5-amino-N′-(3-(benzyloxy)-4-methoxybenzylidene)-1-(2-hydroxy-2-phenylethyl)-3-(phenylamino)-1H-pyrazole-4-carbohydrazide 13d.
Mp 170–173 °C (EtOH); Yield 74%. 1H NMR (400 MHz, DMSO-d6): δ 3.81 (s, 3H, OCH3); 3.92–4.11 (m, 2H, CH2N); 5.02 (s, 2H, CH2Ph); 5.12–5.16 (m, 1H, CHOH); 5.73–5.76 (m, 1H, OH, exchangeable); 6.29 (bs, 2H, NH2, exchangeable); 6.74–6.81 (m, 1H, arom. H); 7.02–7.06 (m, 1H, arom. H); 7.16–7.22 (m, 3H, arom. H); 7.32–7.237 (m, 5H, arom. H); 7.38–7.45 (m, 8H, arom. H); 8.08 (s, 1H, CH=C); 8.87 (bs, 1H, NH phenyl, exchangeable); 10.58 (bs, 1H, NH hydraz., exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 53.78; 55.66; 69.73; 71.33; 85.99; 109.55; 111.86; 116.14; 119.09; 121.66; 123.73; 126.34; 126.92; 127.38; 128.01; 128.11; 128.43; 128.48; 128.79; 136.82; 142.49; 142.75; 148.17; 149.34; 150.75; 151.91; 160.77. Calcd for C33H32N6O4: C = 68.73; H = 5.59; N = 14.57. Found: C = 68.78; H = 5.52; N = 14.67.

4.1.3. General Synthetic Procedure for the Synthesis of Pyrazole Amides 14–16 and 20–22

To a DCM solution (10 mL) of 1 or 2 (1 mmol), TEA (211 µL, 1.5 mmol) and the suitable acyl chloride (1.2 mmol) were sequentially added. After stirring at rt for 24h, the reaction mixture was washed with saturated NaHCO3 (2 × 10 mL), water (1 × 10 mL), and dried with anhydrous Na2SO4. Evaporating in vacuo gave crude product that was purified by crystallization from the suitable solvent or solvent mixture.
  • Methyl 5-(phenylamino)-3-(3,4,5-trimethoxybenzamido)-1H-pyrazole-4-carboxylate 14.
Mp 164–166 °C (DCM/MeOH); Yield 55%. 1H NMR (400 MHz, DMSO-d6): δ 3.85 (s, 12H, OCH3); 6.97–7.05 (m, 1H, arom. H); 7.21–7.34 (m, 4H, arom. H); 7.68–7.76 (m, 2H, arom. H); 10.96 (s, 1H, CONH, exchangeable); 11.24 (s, 1H, NH phenyl, exchangeable). Calcd for C21H22N4O6: C = 59.15; H = 5.20; N = 13.14. Found: C = 59.40; H = 5.38; N = 12.86.
  • Ethyl 3-(4-methoxybenzamido)-5-(phenylamino)-1H-pyrazole-4-carboxylate 15.
Mp 155–157 °C (Et2O); Yield 65%. 1H NMR (400 MHz, DMSO-d6): δ 1.35 (t, 3H, J = 7.1 Hz, CH3); 3.88 (s, 3H, OCH3); 4.34 (q, 2H, J = 7.1 Hz, CH2O); 6.88–6.94 (m, 1H, arom. H); 7.08–7.14 (m, 2H, arom. H); 7.23–7.30 (m, 2H, arom. H); 7.51–7.57 (m, 2H, arom. H); 7.67 (bs, 1H, CONH, exchangeable); 8.13–8.20 (m, 2H, arom. H); 8.25 (bs, 1H, NH phenyl, exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 14.45; 55.60; 59.83; 82.87; 113.22; 117.17; 120.91; 124.67; 128.94; 133.18; 140.17; 151.07; 153.56; 162.76; 163.78; 168.15. Calcd for C20H20N4O4: C = 63.15; H = 5.30; N = 14.73. Found: C = 63.51; H = 5.23; N = 15.11.
  • Ethyl 3-(3,4-dimethoxybenzamido)-5-(phenylamino)-1H-pyrazole-4-carboxylate 16.
Mp 143–145 °C (EtOH); Yield 71%. 1H NMR (400 MHz, DMSO-d6): δ 1.35 (t, 3H, J = 7.1 Hz, CH3); 3.79 (s, 3H, OCH3); 3.88 (s, 3H, OCH3); 4.34 (q, 2H, J = 7.1 Hz, CH2O); 6.87–6.96 (m, 1H, arom. H); 7.10–7.18 (m, 1H, arom. H); 7.21–7.30 (m, 2H, arom. H); 7.53–7.60 (m, 2H, arom. H); 7.69 (bs, 1H, CONH, exchangeable); 7.78–7.88 (m, 2H, arom. H); 8.30 (bs, 1H, NH phenyl, exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 14.45; 55.48; 55.76; 59.87; 82.86; 110.64; 114.08; 117.15; 121.00; 124.53; 125.33; 128.90; 140.18; 147.46; 151.18; 152.65; 153.67; 163.80; 168.08. Calcd for C21H22N4O5: C = 61.46; H = 5.40; N = 13.65. Found: C = 61.08; H = 5.07; N = 13.72.
  • Ethyl 3-(2,6-dimethoxybenzamido)-1-methyl-5-(phenylamino)-1H-pyrazole-4-carboxylate 20.
Mp 158–160 °C (DCM/MeOH); Yield 30%. 1H NMR (400 MHz, DMSO-d6): δ 0.92 (t, 3H, J = 7.1 Hz, CH3); 3.57 (s, 3H, CH3N); 3.78 (s, 6H, 2 x OCH3); 3.96 (q, 2H, J = 7.1 Hz, CH2); 6.66–6.75 (m, 3H, arom. H); 6.78–6.84 (m, 1H, arom. H); 7.15–7.22 (m, 2H, arom. H); 7.33–7.44 (m, 2H, arom. H); 8.18 (bs, 1H, NH phenyl, exchangeable); 9.56 (bs, 1H, NH amide, exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 14.11; 35.80; 56.34; 56.60; 59.95; 95.10; 104.71; 110.97; 115.44; 120.15; 129.58; 133.28; 142.77; 144.86; 157.38; 157.86; 160.77; 163.44. Calcd for C22H24N4O5: C = 62.25; H = 5.70; N = 13.20. Found: C = 62.18; H = 5.53; N = 13.60.
  • Ethyl 3-(3,4-dimethoxybenzamido)-1-methyl-5-(phenylamino)-1H-pyrazole-4-carboxylate 21.
Mp 186–187 °C (DCM/Et2O); Yield 39%. 1H NMR (400 MHz, DMSO-d6): δ 0.86 (t, 3H, J = 7.1 Hz, CH3); 3.59 (s, 3H, CH3N); 3.82 (s, 3H, OCH3); 3.83 (s, 3H, OCH3); 3.91 (q, 2H, J = 7.1 Hz, CH2); 6.63–6.70 (m, 3H, arom. H); 6.76–6.85 (m, 1H, arom. H); 7.04–7.11 (m, 1H, arom. H); 7.15–7.24 (m, 2H, arom. H); 7.51–7.62 (m, 2H, arom. H); 8.21 (bs, 1H, NH phenyl, exchangeable); 10.13 (bs, 1H, NH amide, exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 13.70; 35.34; 55.57; 55.66; 59.18; 98.82; 110.79; 110.99; 114.71; 119.56; 120.96; 126.17; 129.13; 142.95; 144.49; 145.20; 148.31; 151.70; 162.09; 164.74. Calcd for C22H24N4O5: C = 62.25; H = 5.70; N = 13.20. Found: C = 62.18; H = 5.37; N = 13.08.
  • Ethyl 1-methyl-5-(phenylamino)-3-(3,4,5-trimethoxybenzamido)-1H-pyrazole-4-carboxylate 22.
Mp 135–138 °C (Et2O); Yield 22%. 1H NMR (400 MHz, DMSO-d6): δ 0.87 (t, 3H, J = 7.1 Hz, CH3); 3.59 (s, 3H, CH3N); 3.73 (s, 3H, OCH3); 3.85 (s, 6H, 2 x OCH3); 3.91 (q, 2H, J = 7.1 Hz, CH2); 6.63–6.70 (m, 2H, arom. H); 6.76–6.85 (m, 1H, arom. H); 7.16–7.24 (m, 2H, arom. H); 7.28–7.31 (m, 2H, arom. H); 8.21 (bs, 1H, NH phenyl, exchangeable); 10.21 (bs, 1H, NH amide, exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 13.72; 35.35; 56.03; 59.16; 60.15; 99.35; 105.13; 114.67; 119.57; 129.13; 140.33; 143.03; 144.51; 144.87; 152.66; 161.88; 164.87. Calcd for C23H26N4O6: C = 60.78; H = 5.77; N = 12.33. Found: C = 60.97; H = 5.67; N = 12.65.

4.1.4. Synthesis of Pyrazole Amides 17 and 18

To a dry DMF solution (5 mL) of pyrazole 3 (266 mg, 1 mmol), TMEDA (169 µL, 1.1 mmol) and the proper acyl chloride (1.1 mmol) were sequentially added. After stirring at 120 °C for 2 h, the reaction mixture was cooled at rt, and water (40 mL) was added. The precipitated solid was collected by filtration and recrystallized from the proper solvent or solvent mixture.
  • Ethyl 3-(cyclopropanecarboxamido)-1-methyl-5-(phenylamino)-1H-pyrazole-4-carboxylate 17.
Mp 144–145 °C (EtOH); Yield 43%. 1H NMR (400 MHz, CDCl3): δ 0.82–0.93 (m, 2H, CH2-cycloprop); 1.08–1.18 (m, 2H, CH2-cycloprop); 1.33 (t, 3H, J = 7.1 Hz, CH3); 1.44–1.74 (m, 1H, CHCO); 3.46 (s, 3H, CH3N); 4.30 (q, 2H, J = 7.1 Hz, CH2O); 6.75–6.85 (m, 3H, arom. H + NH amide, exchangeable); 6.99–7.08 (m, 1H, arom. H); 7.23–7.35 (m, 2H, arom. H); 9.18 (bs, 1H, NH phenyl, exchangeable). Calcd for C17H20N4O3: C = 62.18; H = 6.14; N = 17.06. Found: C = 61.86; H = 5.96; N = 16.65.
  • Ethyl 3-(4-chlorobenzamido)-1-methyl-5-(phenylamino)-1H-pyrazole-4-carboxylate 18.
Mp 158–161 °C (Et2O/ligroin); Yield 36%. 1H NMR (400 MHz, DMSO-d6): δ 0.85 (t, 3H, J = 7.1 Hz, CH3); 3.59 (s, 3H, CH3N); 3.91 (q, 2H, J = 7.1 Hz, CH2); 6.62–6.70 (m, 2H, arom. H); 6.75–6.86 (m, 1H, arom. H); 7.12–7.24 (m, 2H, arom. H); 7.55–7.65 (m, 2H, arom. H); 7.94–7.99 (m, 2H, arom. H); 8.23 (bs, 1H, NH phenyl, exchangeable); 10.34 (bs, 1H, NH amide, exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 13.69; 35.40; 59.21; 98.96; 114.76; 119.62; 128.66; 129.14; 129.54; 132.74; 136.67; 143.12; 144.41; 144.77; 161.93; 164.45. Calcd for C20H19ClN4O3: C = 60.23; H = 4.80; N = 14.05. Found: C = 59.87; H = 4.81; N = 14.39.

4.1.5. Synthesis of Ethyl 3-(4-Methoxybenzamido)-1-methyl-5-(phenylamino)-1H-pyrazole-4-carboxylate 19

To a dry ACN solution (10 mL) of 3 (266 mg, 1 mmol), TEA (214 µL, 1.5 mmol), and p-methoxybenzoyl chloride (164 µL, 1.2 mmol) dissolved in dry ACN (2 mL) were sequentially added. After stirring at rt for 72 h, the reaction mixture was refluxed for 0.5 h. After cooling at rt, the solvent was evaporated in vacuo and saturated NaHCO3 (10 mL) was added. The mixture was extracted with DCM (2 × 10 mL), and the pooled organic phases were washed with water (1 × 10 mL), dried and filtered. Evaporating in vacuo gave a crude residue, which was purified by column chromatography (silica gel, eluent: Et2O-Et2O/5% EtOH).
Mp 143–145 °C (Et2O); Yield 30%. 1H NMR (400 MHz, DMSO-d6): δ 0.85 (t, 3H, J = 7.1 Hz, CH3); 3.58 (s, 3H, CH3N); 3.83 (s, 3H, OCH3); 3.91 (q, 2H, J = 7.1 Hz, CH2); 6.63–6.71 (m, 2H, arom. H); 6.76–6.85 (m, 1H, arom. H); 7.02–7.10 (m, 2H, arom. H); 7.15–7.24 (m, 2H, arom. H); 7.90–7.97 (m, 2H, arom. H); 8.21 (bs, 1H, NH phenyl, exchangeable); 10.11 (bs, 1H, NH amide, exchangeable). 13C NMR (101 MHz, DMSO-d6): δ 13.67; 35.34; 55.44; 59.19; 98.46; 113.75; 114.75; 119.58; 126.15; 129.12; 129.48; 142.93; 144.45; 145.30; 162.04; 162.19; 164.57. Calcd for C21H22N4O4: C = 63.95; H = 5.62; N = 14.20. Found: C = 63.56; H = 5.55; N = 14.39.

4.2. Biology

4.2.1. MTT Assays

All reagents were purchased from EuroClone, Milan, Italy). The following cell lines were used for MTT assays: SKOV-3 (ovarian adenocarcinoma, ATCC, Manassas, VA, USA); MCF-7 (breast adenocarcinoma, Biologic Bank and Cell Factory, IRCCS Policlinico San Martino, Genoa, Italy); Hep-G2 (hepatocellular carcinoma, ATCC, Manassas, VA, USA); SK-MEL28 (skin melanoma, Biologic Bank and Cell Factory, IRCCS Policlinico San Martino, Genoa, Italy), GM-6114 (embryonic human fibroblast, ATCC, Manassas, VA, USA); MDA-MB231 (breast adenocarcinoma, Biologic Bank and Cell Factory, IRCCS Policlinico San Martino, Genoa, Italy); HeLa (cervical adenocarcinoma, Biologic Bank and Cell Factory, IRCCS Policlinico San Martino, Genoa, Italy); SK-BR3 (breast andenocarcinoma, Biologic Bank and Cell Factory, IRCCS Policlinico San Martino, Genoa, Italy); A549 (lung carcinoma, Biologic Bank and Cell Factory, IRCCS Policlinico San Martino, Genoa, Italy); HUVEC (Human Umbilical Vein Endothelial Cells, ATCC, Manassas, VA, USA). All cell lines were grown in their medium with 10% FBS, 2 mM Glutamine, and 1% penstrep and incubated at 37 °C in 5% CO2 in a humidified environment. The cell lines were plated in 96-well plates at an adequate number to reach 80–90% confluence at the end of the assay. 16 h after cell plating, a 10 mM DMSO stock solution of the compounds was diluted in growth medium and added at a final working concentration of 10 μM. After 48h of incubation, a 2 mg/mL PBS solution of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) was added (30 μL/well). After 4h, the supernatant was removed, and the Formazan precipitates were dissolved in DMSO (100 μL/well). The 96-well plates were incubated for 20 min, and absorbance was measured at 570 nm using a plate reader. The results are expressed as a percentage ratio over control samples (100%) in which the cells were incubated with the same amount of DMSO but without compounds. Each value is the mean of three independent experiments run in six replicates.
The IC50 values were extrapolated from nonlinear regression analysis of concentration–response curves (used concentrations: 1, 5, 10 μM), using the MS Excel software (Microsoft 365 suite). Each IC50 value is the mean of three independent experiments run in duplicate.

4.2.2. Blood Collection

Freshly drawn venous blood from healthy volunteers from “Centro Trasfusionale” (IRCCS Policlinico San Martino, Genoa, Italy) was collected into a 130 mM aqueous trisodium citrate anticoagulant solution (9:1). The donors claimed to not have taken drugs known to interfere with platelet function during the two weeks prior to blood collection and gave their informed consent. Whole blood was centrifuged at 100× g for 20 min to afford platelet-rich plasma that was then spun at 1100× g for 15 min. The obtained pellet was washed once with a pH 5.2 ACD solution (75 mM trisodium citrate, 42 mM citric acid, and 136 mM glucose), centrifuged at 1100× g for 15 min, and then re-suspended in pH 7.4 Hepes buffer (145 mM NaCl, 5 mM KCl, 1 mM MgSO4, 10 mM glucose, and 10 mM HEPES).

4.2.3. ROS Assay

2′,7′-Dichlorofluorescein diacetate (DCFH-DA) and thrombin were purchased from Sigma-Aldrich/Merck Millipore. DMSO solutions of 10–22 were diluted in saline immediately before each experiment. ROS production was quantified by DCFH-DA, a ROS-sensitive probe that yields, upon oxidation, the fluorescent adduct DCF that is trapped inside the cells [55]. Briefly, washed platelets (1.0 × 108/mL), pre-incubated with saline solutions of 10–22 for 15 min at 37 °C, were stimulated by 0.1 U/mL thrombin. Incubation was stopped by cooling samples in an ice bath, and then samples were immediately analyzed in a Merck Millipore Bioscience Guava easyCyte flow cytometer (Merk Millipore, Burlington, MA, USA). The reported IC50 values represent the molar concentration of the compounds able to inhibit 50% of the maximal aggregation induced by the agonist and are calculated as the percentage inhibition of the maximal aggregation measured in the presence of the agent compared with the measure in a control sample containing saline, carried out under the same conditions. The IC50 values were extrapolated from nonlinear regression analysis of concentration–response curves (three points) using MS Excel software (Microsoft 365 suite). Each IC50 value is the mean of six independent experiments.

4.2.4. Platelet Aggregation

Thrombin was purchased from Sigma-Aldrich/Merck Millipore. A DMSO solution of compounds 10–22 was diluted in saline immediately before each experiment and added to the washed platelets (3.0 × 108/mL) at 37 °C. After 3 min, 0.1 U/mL thrombin was added, and platelet aggregation was quantified according to Born’s method [56] using a Bio-Data Aggregometer (Bio-Data Corporation, Horsham, PA, USA). The IC50 values were calculated as detailed above.

4.3. DPPH Radical-Scavenging Activity

Compounds 10b, 11a, 11d, 12d, 13d, 14, and 22 (ca. 3 mg) were dissolved in DMSO (1 mL), and then 100 µL of this solution was mixed with 3.9 mL of DPPH methanol solution (65 µM). Absorbance was measured at 517 nm after reacting for 30 min in the dark. The linear calibration curve was obtained using Trolox standards (ranging between 20 and 200 mg/L, R2 = 0.9955). The result was calculated as Trolox equivalent in mg/L, and the percentage of antioxidant activity (AA%) was calculated from the ratio of decreasing absorbance of sample solution (A0 − As) to absorbance of blank DPPH solution (A0), as expressed in Equation (1) [57,58].
A A % = A 0 − A s A 0 × 100
All analyses were carried out in duplicate (n = 2), and values are given as means ± standard deviation (SD).

5. Conclusions

To further extend the SARs of antioxidant derivatives IV and V, pyrazolyl acylhydrazones 10–13 and amides 14–22 were prepared from APs 1–5 through a divergent approach. The novel compounds were evaluated for (i) antiproliferative activity in cell-based assays; (ii) antioxidant and antiaggregating properties in platelets; and (iii) anti-scavenging efficacy. Compound 11a displayed micromolar IC50 values against selected tumor cell lines (namely, HeLa, MCF7, SKOV3, and SKMEL28 cells), and NCI screening on a large panel of tumor cell lines confirmed the promising cytotoxic activity of this derivative. Different from all its analogues, pyrazolyl amide 14 showed relevant and unexpected antiproliferative activity against melanoma (SKMEL28), lung (A549), and cervical (HeLa) tumors. Unfortunately, the compound was as cytotoxic as cisplatin against GM6114 normal fibroblasts. Despite resulting in less activity compared to lead compounds IV and V, selected pyrazole acylhydrazones and amides significantly inhibited aggregation and ROS production in platelets and proved to be more effective than ASA and NAC. Moreover, the antiproliferative activity does not seem to correlate with the antioxidant/antiaggregant values. Finally, DPPH experiments indicate relevant radical scavenging properties of acylhydrazones, which can, therefore, represent a privilege scaffold for the development of novel antiproliferative and antioxidant agents.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25094607/s1.

Author Contributions

Conceptualization, A.S.; chemical synthesis, M.L., C.B. and A.S.; platelet assays, M.G.S.; cell-based assays, M.P., E.I. and C.R.; radical-scavenging assays, E.R. and D.C.; writing—original draft preparation, A.S.; writing—review and editing, M.L., C.B., M.G.S., M.P., E.I., C.R., E.R. and D.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Università degli Studi di Genova, grant Fondi di ricerca di ateneo (FRA). The work of E.I., C.R., and M.P. has been partially supported by a grant from the Italian Ministry of Health (Ricerca Corrente).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Samples of the compounds are available from the authors.

Acknowledgments

The authors acknowledge the NCI DTP’s contributions to the project.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Becerra, D.; Abonia, R.; Castillo, J.C. Recent Applications of the Multicomponent Synthesis for Bioactive Pyrazole Derivatives. Molecules 2022, 27, 4723. [Google Scholar] [CrossRef]
  2. Ebenezer, O.; Shapi, M.; Tuszynski, J.A. A Review of the Recent Development in the Synthesis and Biological Evaluations of Pyrazole Derivatives. Biomedicines 2022, 10, 1124. [Google Scholar] [CrossRef] [PubMed]
  3. Li, M.M.; Huang, H.; Pu, Y.; Tian, W.; Deng, Y.; Lu, J. A close look into the biological and synthetic aspects of fused pyrazole derivatives. Eur. J. Med. Chem. 2022, 243, 114739. [Google Scholar] [CrossRef]
  4. Alam, M.A. Pyrazole: An emerging privileged scaffold in drug discovery. Future Med. Chem. 2023, 15, 2011–2023. [Google Scholar] [CrossRef]
  5. Kumar, R.; Sharma, R.; Sharma, D.K. Pyrazole; A Privileged Scaffold of Medicinal Chemistry: A Comprehensive Review. Curr. Top. Med. Chem. 2023, 23, 2097–2115. [Google Scholar] [CrossRef] [PubMed]
  6. Costa, R.F.; Turones, L.C.; Cavalcante, K.V.N.; Rosa Júnior, I.A.; Xavier, C.H.; Rosseto, L.P.; Napolitano, H.B.; Castro, P.F.D.S.; Neto, M.L.F.; Galvão, G.M.; et al. Heterocyclic Compounds: Pharmacology of Pyrazole Analogs From Rational Structural Considerations. Front. Pharmacol. 2021, 12, 666725. [Google Scholar] [CrossRef] [PubMed]
  7. Kumar, S.; Gupta, S.; Rani, V.; Sharma, P. Pyrazole Containing Anti-HIV Agents: An Update. Med. Chem. 2022, 18, 831–846. [Google Scholar] [CrossRef]
  8. Alam, M.A. Antibacterial pyrazoles: Tackling resistant bacteria. Future Med. Chem. 2022, 14, 343–362. [Google Scholar] [CrossRef]
  9. Verma, R.; Verma, S.K.; Rakesh, K.P.; Girish, Y.R.; Ashrafizadeh, M.; Sharath Kumar, K.S.; Rangappa, K.S. Pyrazole-based analogs as potential antibacterial agents against methicillin-resistance staphylococcus aureus (MRSA) and its SAR elucidation. Eur. J. Med. Chem. 2021, 212, 113134. [Google Scholar] [CrossRef]
  10. Ravindar, L.; Hasbullah, S.A.; Rakesh, K.P.; Hassan, N.I. Pyrazole and pyrazoline derivatives as antimalarial agents: A key review. Eur. J. Pharm. Sci. 2023, 183, 106365. [Google Scholar] [CrossRef]
  11. Turones, L.C.; Martins, A.N.; Moreira, L.K.D.S.; Fajemiroye, J.O.; Costa, E.A. Development of pyrazole derivatives in the management of inflammation. Fundam. Clin. Pharmacol. 2021, 35, 217–234. [Google Scholar] [CrossRef] [PubMed]
  12. Firdaus, J.U.; Siddiqui, N.; Alam, O.; Manaithiya, A.; Chandra, K. Pyrazole scaffold-based derivatives: A glimpse of α-glucosidase inhibitory activity, SAR, and route of synthesis. Arch. Pharm. 2023, 356, e2200421. [Google Scholar] [CrossRef]
  13. Asproni, B.; Murineddu, G.; Corona, P.; Pinna, G.A. Tricyclic Pyrazole-Based Compounds as Useful Scaffolds for Cannabinoid CB1/CB2 Receptor Interaction. Molecules 2021, 26, 2126. [Google Scholar] [CrossRef]
  14. Dorbabu, A. Pyrazole/pyrazoline as an excellent pharmacophore in the design of carbonic anhydrase inhibitors (2018–2022). Arch. Pharm. 2023, 356, e2200562. [Google Scholar] [CrossRef] [PubMed]
  15. Zhang, Y.; Wu, C.; Zhang, N.; Fan, R.; Ye, Y.; Xu, J. Recent Advances in the Development of Pyrazole Derivatives as Anticancer Agents. Int. J. Mol. Sci. 2023, 24, 12724. [Google Scholar] [CrossRef] [PubMed]
  16. Dube, Z.F.; Soremekun, O.S.; Ntombela, T.; Alahmdi, M.I.; Abo-Dya, N.E.; Sidhom, P.A.; Shawky, A.M.; Shibl, M.F.; Ibrahim, M.A.; Soliman, M.E. Inherent efficacies of pyrazole-based derivatives for cancer therapy: The interface between experiment and in silico. Future Med. Chem. 2023, 15, 1719–1738. [Google Scholar] [CrossRef] [PubMed]
  17. Ardevines, S.; Marqués-López, E.; Herrera, R.P. Heterocycles in Breast Cancer Treatment: The Use of Pyrazole Derivatives. Curr. Med. Chem. 2023, 30, 1145–1174. [Google Scholar] [CrossRef] [PubMed]
  18. Bennani, F.E.; Doudach, L.; Cherrah, Y.; Ramli, Y.; Karrouchi, K.; Ansar, M.; Faouzi, M.E.A. Overview of recent developments of pyrazole derivatives as an anticancer agent in different cell line. Bioorg. Chem. 2020, 97, 103470. [Google Scholar] [CrossRef]
  19. Yan, X.Q.; Wang, Z.C.; Qi, P.F.; Li, G.; Zhu, H.L. Design, synthesis and biological evaluation of 2-H pyrazole derivatives containing morpholine moieties as highly potent small molecule inhibitors of APC-Asef interaction. Eur. J. Med. Chem. 2019, 177, 425–447. [Google Scholar] [CrossRef]
  20. Xu, Z.; Zhuang, Y.; Chen, Q. Current scenario of pyrazole hybrids with in vivo therapeutic potential against cancers. Eur. J. Med. Chem. 2023, 257, 115495. [Google Scholar] [CrossRef]
  21. Mor, S.; Khatri, M.; Punia, R.; Sindhu, S. Recent Progress in Anticancer Agents Incorporating Pyrazole Scaffold. Mini Rev. Med. Chem. 2022, 22, 115–163. [Google Scholar] [CrossRef] [PubMed]
  22. El-Gamal, M.I.; Zaraei, S.O.; Madkour, M.M.; Anbar, H.S. Evaluation of Substituted Pyrazole-Based Kinase Inhibitors in One Decade (2011–2020): Current Status and Future Prospects. Molecules 2022, 27, 330. [Google Scholar] [CrossRef] [PubMed]
  23. Nitulescu, G.M.; Stancov, G.; Seremet, O.C.; Nitulescu, G.; Mihai, D.P.; Duta-Bratu, C.G.; Barbuceanu, S.F.; Olaru, O.T. The Importance of the Pyrazole Scaffold in the Design of Protein Kinases Inhibitors as Targeted Anticancer Therapies. Molecules 2023, 28, 5359. [Google Scholar] [CrossRef] [PubMed]
  24. Lusardi, M.; Spallarossa, A.; Brullo, C. Amino-Pyrazoles in Medicinal Chemistry: A Review. Int. J. Mol. Sci. 2023, 24, 7834. [Google Scholar] [CrossRef] [PubMed]
  25. Alam, M.J.; Alam, O.; Naim, M.J.; Nawaz, F.; Manaithiya, A.; Imran, M.; Thabet, H.K.; Alshehri, S.; Ghoneim, M.M.; Alam, P.; et al. Recent advancement in drug design and discovery of pyrazole biomolecules as cancer and inflammation therapeutics. Molecules 2022, 27, 8708. [Google Scholar] [CrossRef] [PubMed]
  26. Marinozzi, M.; Marcelli, G.; Carotti, A. N-Aryl-5-aminopyrazole: A versatile architecture in medicinal chemistry. Mini-Rev. Med. Chem. 2015, 15, 272–299. [Google Scholar] [CrossRef] [PubMed]
  27. Pérez-Fernández, R.; Goya, P.; Elguero, J. A review of recent progress (2002–2012) on the biological activities of pyrazoles. ARKIVOC 2013, 2014, 233–293. [Google Scholar] [CrossRef]
  28. Ansari, A.; Ali, A.; Asif, M.; Shamsuzzaman, S. Review: Biologically Active Pyrazole Derivatives. New J. Chem. 2017, 41, 16–41. [Google Scholar] [CrossRef]
  29. U.S National Library of Medicine. ClinicalTrials.gov. Available online: https://clinicaltrials.gov/ct2/results?cond=AT7519&term=&cntry=&state=&city=&dist= (accessed on 8 June 2023).
  30. U.S National Library of Medicine. ClinicalTrials.gov. Available online: https://clinicaltrials.gov/ct2/results?cond=&term=AT9283&cntry=&state=&city=&dist= (accessed on 8 June 2023).
  31. Gomez, E.B.; Ebata, K.; Randeria, H.S.; Rosendahl, M.S.; Cedervall, E.P.; Morales, T.H.; Hanson, L.M.; Brown, N.E.; Gong, X.; Stephens, J.R.; et al. Pirtobrutinib preclinical characterization: A highly selective, non-covalent (reversible) BTK inhibitor. Blood 2023, 142, 62–72. [Google Scholar] [CrossRef]
  32. Michot, J.-M.; Ribrag, V. Pirtobrutinib Shows Evidence to Inaugurate a Third Generation of BTK Inhibitors. Lancet 2021, 397, 855–857. [Google Scholar] [CrossRef]
  33. U.S National Library of Medicine. ClinicalTrials.gov. Available online: https://clinicaltrials.gov/ct2/results?term=pirtobrutinib (accessed on 8 June 2023).
  34. Metwally, M.A.; Gouda, M.A.; Harmal, A.N.; Khalil, A.M. Synthesis, Antitumor, Cytotoxic and Antioxidant Evaluation of some new pyrazolotriazines attached to antipyrine moiety. Eur. J. Med. Chem. 2012, 56, 254–262. [Google Scholar] [CrossRef]
  35. Burgart, Y.V.; Agafonova, N.A.; Shchegolkov, E.V.; Krasnykh, O.P.; Kushch, S.O.; Evstigneeva, N.P.; Gerasimova, N.A.; Maslova, V.V.; Triandafilova, G.A.; Solodnikov, S.Y.; et al. Multiple biological active 4-aminopyrazoles containing trifluoromethyl and their 4-nitroso-precursors: Synthesis and evaluation. Eur. J. Med. Chem. 2020, 208, 112768. [Google Scholar] [CrossRef] [PubMed]
  36. Burgart, Y.V.; Makhaeva, G.F.; Krasnykh, O.P.; Borisevich, S.S.; Agafonova, N.A.; Kovaleva, N.V.; Boltneva, N.P.; Rudakova, E.V.; Shchegolkov, E.V.; Triandafilova, G.A.; et al. Synthesis of 4-aminopyrazol-5-ols as edaravone analogs and their antioxidant activity. Molecules 2022, 27, 7722. [Google Scholar] [CrossRef] [PubMed]
  37. Brullo, C.; Massa, M.; Rapetti, F.; Alfei, S.; Bertolotto, M.B.; Montecucco, F.; Signorello, M.G.; Bruno, O. new hybrid pyrazole and imidazopyrazole antinflammatory agents able to reduce ROS production in different biological targets. Molecules 2020, 25, 899. [Google Scholar] [CrossRef] [PubMed]
  38. Brullo, C.; Russo, E.; Garibaldi, S.; Altieri, P.; Ameri, P.; Ravera, S.; Signorello, M.G. Inside the mechanism of action of three pyrazole derivatives in human platelets and endothelial cells. Antioxidants 2023, 12, 216. [Google Scholar] [CrossRef] [PubMed]
  39. Signorello, M.G.; Rapetti, F.; Meta, E.; Sidibè, A.; Bruno, O.; Brullo, C. New series of pyrazoles and imidazo-pyrazoles targeting different cancer and inflammation pathways. Molecules 2021, 26, 5735. [Google Scholar] [CrossRef] [PubMed]
  40. Lusardi, M.; Rotolo, C.; Ponassi, M.; Iervasi, E.; Rosano, C.; Spallarossa, A. One-pot synthesis and antiproliferative activity of highly functionalized pyrazole derivatives. ChemMedChem 2022, 17, e202100670. [Google Scholar] [CrossRef] [PubMed]
  41. Lusardi, M.; Profumo, A.; Rotolo, C.; Iervasi, E.; Rosano, C.; Spallarossa, A.; Ponassi, M. Regioselective synthesis, structural characterization, and antiproliferative activity of novel tetra-substituted phenylaminopyrazole derivatives. Molecules 2022, 27, 5814. [Google Scholar] [CrossRef] [PubMed]
  42. Lusardi, M.; Wehrle-Haller, B.; Sidibe, A.; Ponassi, M.; Iervasi, E.; Rosano, C.; Brullo, C.; Spallarossa, A. Novel 5-aminopyrazoles endowed with anti-angiogenetic properties: Design, synthesis and biological evaluation. Eur. J. Med. Chem. 2023, 260, 115727. [Google Scholar] [CrossRef]
  43. Ashton, M.J.; Cook, D.C.; Fenton, G.; Karlsson, J.-A.; Palfreyman, M.N.; Raeburn, D.; Ratcliffe, A.J.; Souness, J.E.; Thurairatnam, S.; Vicker, N. Selective type IV phosphodiesterase inhibitors as antiasthmatic agents. the syntheses and biological activities of 3-(cyclopentyloxy)-4-methoxybenzamides and analogs. J. Med. Chem. 1994, 37, 1696–1703. [Google Scholar] [CrossRef]
  44. Crowder, J.R.; Glover, E.E.; Grundon, M.F.; Kaempfen, H.X. Bisbenzylisoquinolines. Part IV. the preparation of diaryl ethers from diaryliodonium salts. J. Chem. Soc. Resumed 1963, 1, 4578–4585. [Google Scholar] [CrossRef]
  45. Sano, T.; Ohashi, K.; Oriyama, T. Remarkably fast acylation of alcohols with benzoyl chloride promoted by TMEDA. Synthesis 1999, 7, 1141–1144. [Google Scholar] [CrossRef]
  46. Sano, T.; Ohashi, K.; Oriyama, T. ChemInform abstract: Remarkably fast acylation of alcohols with benzoyl chloride promoted by TMEDA. ChemInform 1999, 30, 1141–1144. [Google Scholar] [CrossRef]
  47. Broos, K.; Feys, H.B.; De Meyer, S.F.; Vanhoorelbeke, K.; Deckmyn, H. Platelets at work in primary hemostasis. Blood Rev. 2011, 25, 155–167. [Google Scholar] [CrossRef] [PubMed]
  48. Leslie, M. Beyond clotting: The power of platelets. Science 2010, 328, 562–564. [Google Scholar] [CrossRef] [PubMed]
  49. Cassano, V.; Armentaro, G.; Iembo, D.; Miceli, S.; Fiorentino, T.S.; Succurro, E.; Perticone, M.; Arturi, F.; Hribal, M.L.; Montalcini, T.; et al. Mean platelet volume (MPV) as new marker of diabetic macrovascular complications in patients with different glucose homeostasis. Cardiov. Diabetol. 2024, 23, 89. [Google Scholar] [CrossRef] [PubMed]
  50. Mittal, M.; Siddiqui, M.R.; Tran, K.; Reddy, S.P.; Malik, A.B. Reactive oxygen species in inflammation and tissue injury. Antioxid. Redox Signal. 2014, 20, 1126–1167. [Google Scholar] [CrossRef] [PubMed]
  51. Chen, L.; Deng, H.; Cui, H.; Fang, J.; Zuo, Z.; Deng, J.; Li, Y.; Wang, X.; Zhao, L. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget 2018, 9, 7204–7218. [Google Scholar] [CrossRef] [PubMed]
  52. Franco, A.T.; Corken, A.; Ware, J. Platelets at the interface of thrombosis, inflammation, and cancer. Blood 2015, 126, 582–588. [Google Scholar] [CrossRef]
  53. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 1995, 28, 25–30. [Google Scholar] [CrossRef]
  54. Gavrin, L.K.; Lee, A.; Provencher, B.A.; Massefski, W.W.; Huhn, S.D.; Ciszewski, G.M.; Cole, D.C.; McKew, J.C. Synthesis of pyrazolo[1,5-α]pyrimidinone regioisomers. J. Org. Chem. 2007, 72, 1043–1046. [Google Scholar] [CrossRef] [PubMed]
  55. Leoncini, G.; Maresca, C.C. Oxidative metabolism of human platelets. Biochem. Int. 1991, 25, 647–655. [Google Scholar] [PubMed]
  56. Born, G.V.R. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962, 194, 927–929. [Google Scholar] [CrossRef] [PubMed]
  57. Singleton, V.L.; Orthofer, R.; Lamuela-Raventós, R.M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. In Methods in Enzymology; Academic Press Ltd.: Cambridge, MA, USA; Elsevier Science Ltd: London, UK, 1999; Volume 299, pp. 152–178. [Google Scholar]
  58. Russo, E.; Spallarossa, A.; Comite, A.; Pagliero, M.; Guida, P.; Belotti, V.; Caviglia, D.; Schito, A.M. Valorization and potential antimicrobial use of olive mill wastewater (OMW) from italian olive oil production. Antioxidants 2022, 11, 903. [Google Scholar] [CrossRef] [PubMed]
Ijms 25 04607 g001
Figure 1. Selected examples of pyrazole compounds with relevant pharamacological activity. The pyrazole and aminopyrazole substructures are colored blue and red, respectively.
Figure 1. Selected examples of pyrazole compounds with relevant pharamacological activity. The pyrazole and aminopyrazole substructures are colored blue and red, respectively.
Ijms 25 04607 g001
Ijms 25 04607 g002
Figure 2. Developed SARs around pyrazolyl hydrazones IV.
Figure 2. Developed SARs around pyrazolyl hydrazones IV.
Ijms 25 04607 g002
Ijms 25 04607 sch001
Scheme 1. Synthesis of pyrazole acylhydrazones 10–13 and pyrazolyl amides 14–22. Reaction conditions: (a) hydrazine monohydrate, EtOHabs, reflux, 4–6 h; (b) aldehyde a–d, EtOHabs, reflux, 16 h; (c) acyl chloride, TEA or TMEDA, DMF or ACN or DCM, various temperatures and times.
Scheme 1. Synthesis of pyrazole acylhydrazones 10–13 and pyrazolyl amides 14–22. Reaction conditions: (a) hydrazine monohydrate, EtOHabs, reflux, 4–6 h; (b) aldehyde a–d, EtOHabs, reflux, 16 h; (c) acyl chloride, TEA or TMEDA, DMF or ACN or DCM, various temperatures and times.
Ijms 25 04607 sch001
Ijms 25 04607 g003
Figure 3. Bidimensional plot of ROS formation inhibition and antiaggregant activity of derivatives 10–22. Pyrazolyl amides are colored red, and acylhydrazones are reported as green dots. The dashed red line indicates the antiaggregant IC50 value of the reference drug ASA (IC50 = 438 µM). All compounds were found to be more effective ROS formation inhibitors than NAC (IC50 = 872 µM).
Figure 3. Bidimensional plot of ROS formation inhibition and antiaggregant activity of derivatives 10–22. Pyrazolyl amides are colored red, and acylhydrazones are reported as green dots. The dashed red line indicates the antiaggregant IC50 value of the reference drug ASA (IC50 = 438 µM). All compounds were found to be more effective ROS formation inhibitors than NAC (IC50 = 872 µM).
Ijms 25 04607 g003
Ijms 25 04607 g004
Figure 4. SARs developed for acylhydrazones 10–13 and pyrazolyl amides 14–22.
Figure 4. SARs developed for acylhydrazones 10–13 and pyrazolyl amides 14–22.
Ijms 25 04607 g004
Table 1. Antiproliferative activity of pyrazole acylhydrazones 10–22.
Table 1. Antiproliferative activity of pyrazole acylhydrazones 10–22.
Mean Growth Percentage a
CpdMCF7MDA-MB231SK-BR3SKMEL28SKOV3Hep-G2A549HeLaGM-6114
10a95.0699.1195.5186.73103.95115.01103.4798.41110.40
10b95.34100.3591.6194.45114.49115.0991.07105.04107.36
11a33.5671.9556.9649.4443.6075.0460.7725.0069.81
11b94.7785.7386.0093.00100.15116.1894.8499.47104.95
11c84.5393.3883.0787.64109.68113.1791.74101.22105.27
11d82.9098.5990.2189.00102.55111.19100.9595.1599.22
12a88.5497.8894.9876.17101.78110.7497.1494.80100.38
12b100.6391.7294.2473.3198.5992.7295.3092.8199.78
12c88.28100.4782.1394.56104.19111.3498.4685.20104.08
12d85.1794.1490.5980.78106.17105.32102.3385.68102.79
13a80.9996.3991.8281.8493.8085.9494.4883.6699.43
13b101.4992.1192.2495.69113.65106.33104.0798.84106.86
13c99.75103.8787.45103.71128.71124.7694.3897.8899.14
13d107.5793.7597.8793.89111.82119.5590.4096.6595.39
1466.1256.9853.7541.4938.5167.2435.109.4539.31
1565.94129.8499.7671.6588.3785.4867.91104.7068.40
1662.3995.9782.5454.0266.8749.1552.5857.6553.68
1792.75127.34106.0985.8986.92102.7475.80108.5265.92
1863.52140.9680.8768.4396.8878.4769.6491.4672.99
1995.25136.09103.3085.1897.34105.1292.92123.7766.69
2092.09123.59106.4583.6284.42108.0770.83110.9264.71
2190.28149.42107.3984.5990.2497.90112.33108.0785.88
22114.93140.34103.4384.1294.16102.14105.54108.7357.94
CisPt72.7486.0770.5944.4036.8338.0759.0929.3339.52
a Data mean values for three separate experiments; variation among triplicate samples was less than 10%.
Table 2. NCI screening of compounds 11a and 17. Negative values indicate lethality.
Table 2. NCI screening of compounds 11a and 17. Negative values indicate lethality.
Growth Percentage (%) Growth Percentage (%)
Panel/Cell Line11a17 Panel/Cell Line11a17
Leukemia Ovarian Cancer
CCRF-CEM38.4597.75 IGROV139.64100.75
HL-60(TB)15.4696.44 OVCAR-487.0399.35
K-56219.2596.41 OVCAR-576.8899.49
MOLT-449.41101.92 OVCAR-870.80101.92
RPMI-822661.6599.62 NCI/ADR-RES32.98103.29
SR18.9094.23 SK-OV-349.6499.73
Non-Small Cell Lung Cancer Renal Cancer
A549/ATCC57.09101.59 786-057.97101.26
EKVX69.7196.82 A49833.0892.87
HOP-6238.79110.36 ACHN49.99104.77
HOP-9246.7981.47 CAKI-139.8486.39
NCI-H22651.6198.86 RXF 39337.26101.07
NCI-H2366.8698.44 SN12C57.59104.27
NCI-H322M89.9694.41 TK-1089.16107.78
NCI-H46014.63101.02 UO-3151.1887.45
NCI-H52219.3497.23 Prostate Cancer
Colon Cancer PC-355.9095.84
COLO 20535.82106.91 DU-14576.34104.67
HCC-299859.18114.15 Breast Cancer
HCT-11614.99104.12 MCF717.5692.37
HCT-1519.1096.95 MDA-MB-231/ATCC65.06100.37
HT2918.5899.47 HS 578T3.8196.86
KM1227.82105.43 BT-54914.04118.46
SW-62016.0194.82 T-47D42.3893.07
CNS Cancer MDA-MB-468−12.56102.11
SF-26858.65101.17 Melanoma
SF-29598.52102.51 LOX IMVI18.93102.01
SF-53940.2097.42 MALME-3M44.7598.67
SNB-1936.7898.39 M1413.00102.31
SNB-7535.1494.70 MDA-MB-435-31.00101.76
U25133.33100.80 SK-MEL-270.70100.69
SK-MEL-2857.48107.16
SK-MEL-557.2199.45
UACC-25756.3599.58
UACC-6223.7293.55
Table 3. DPPH antioxidant activity of selected acylhydrazone and amide derivatives.
Table 3. DPPH antioxidant activity of selected acylhydrazone and amide derivatives.
CpdA (λ = 517 nm) aDPPH (%) bAA (%) b
10b0.211523.55 ± 0.3976.45 ± 0.39
11a0.668574.44 ± 2.4425.56 ± 2.44
11d0.758584.47 ± 0.2415.53 ± 0.24
12d0.277530.90 ± 0.3969.10 ± 0.39
13d0.392543.71 ± 1.1856.29 ± 1.18
140.865596.38 ± 0.083.62 ± 0.08
220.876597.61 ± 0.242.39 ± 0.24
a Absorbance. b Mean value ± standard deviation (SD) of two independent experiments (n = 2).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Lusardi, M.; Signorello, M.G.; Russo, E.; Caviglia, D.; Ponassi, M.; Iervasi, E.; Rosano, C.; Brullo, C.; Spallarossa, A. Structure–Activity Relationship Studies on Highly Functionalized Pyrazole Hydrazones and Amides as Antiproliferative and Antioxidant Agents. Int. J. Mol. Sci. 2024, 25, 4607. https://doi.org/10.3390/ijms25094607

AMA Style

Lusardi M, Signorello MG, Russo E, Caviglia D, Ponassi M, Iervasi E, Rosano C, Brullo C, Spallarossa A. Structure–Activity Relationship Studies on Highly Functionalized Pyrazole Hydrazones and Amides as Antiproliferative and Antioxidant Agents. International Journal of Molecular Sciences. 2024; 25(9):4607. https://doi.org/10.3390/ijms25094607

Chicago/Turabian Style

Lusardi, Matteo, Maria Grazia Signorello, Eleonora Russo, Debora Caviglia, Marco Ponassi, Erika Iervasi, Camillo Rosano, Chiara Brullo, and Andrea Spallarossa. 2024. "Structure–Activity Relationship Studies on Highly Functionalized Pyrazole Hydrazones and Amides as Antiproliferative and Antioxidant Agents" International Journal of Molecular Sciences 25, no. 9: 4607. https://doi.org/10.3390/ijms25094607

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