Investigations of Antioxidant and Anti-Cancer Activities of 5-Aminopyrazole Derivatives

To further extend the structure-activity relationships (SARs) of 5-aminopyrazoles (5APs) and identify novel compounds able to interfere with inflammation, oxidative stress, and tumorigenesis, 5APs 1–4 have been designed and prepared. Some chemical modifications have been inserted on cathecol function or in aminopyrazole central core; in detail: (i) smaller, bigger, and more lipophilic substituents were introduced in meta and para positions of catechol portion (5APs 1); (ii) a methyl group was inserted on C3 of the pyrazole scaffold (5APs 2); (iii) a more flexible alkyl chain was inserted on N1 position (5APs 3); (iv) the acylhydrazonic linker was moved from position 4 to position 3 of the pyrazole scaffold (5APs 4). All new derivatives 1–4 have been tested for radical scavenging (DPPH assay), anti-aggregating/antioxidant (in human platelets) and cell growth inhibitory activity (MTT assay) properties. In addition, in silico pharmacokinetics, drug-likeness properties, and toxicity have been calculated. 5APs 1 emerged to be promising anti-proliferative agents, able to suppress the growth of specific cancer cell lines. Furthermore, derivatives 3 remarkably inhibited ROS production in platelets and 5APs 4 showed interesting in vitro radical scavenging properties. Overall, the collected results further confirm the pharmaceutical potentials of this class of compounds and support future studies for the development of novel anti-proliferative and antioxidant agents.

In the effort to synthetize new pyrazole-based compounds able to interfere with inflammation, oxidative stress, and tumorigenesis, we recently reported derivatives I (Figure 1) endowed with excellent anti-proliferative activity.Molecular docking and molecular dynamic simulations suggested the ability of the most active compound Ia (Figure 1) to interact with polymeric tubulin α/tubulin β/stathmin4 complex at the colchicine binding site [34].By applying a ring opening strategy, we obtained 5APs II (Figure 1), able to (Figure 1) to interact with polymeric tubulin α/tubulin β/stathmin4 complex at the colchicine binding site [34].By applying a ring opening strategy, we obtained 5APs II (Figure 1), able to strongly inhibit ROS and superoxide anion production, lipid peroxidation, and NADPH oxidase in thrombin-stimulated human platelets and ROS formation in EAhy926 cells [35].To further extend the structure-activity relationships (SARs) of this class of compounds and verify their biological profile, a new library (eighteen compounds) of 5APs 1-4 was designed and synthesized (Figure 2 and Table 1).In detail: (i) in 5APs 1, smaller, bigger, and more lipophilic substituents, similar to previous II, were introduced in meta and para positions of catechol portion; (ii) in 5APs 2, a methyl group on C3 of the pyrazole scaffold was inserted to increase steric hindrance; (iii) in 5APs 3, a more flexible alkyl chain was inserted on N1 position; (iv) finally, in 5APs 4, the acylhydrazonic substituent was shifted from position 4 to position 3 of the pyrazole scaffold.To further extend the structure-activity relationships (SARs) of this class of compounds and verify their biological profile, a new library (eighteen compounds) of 5APs 1-4 was designed and synthesized (Figure 2 and Table 1).In detail: (i) in 5APs 1, smaller, bigger, and more lipophilic substituents, similar to previous II, were introduced in meta and para positions of catechol portion; (ii) in 5APs 2, a methyl group on C3 of the pyrazole scaffold was inserted to increase steric hindrance; (iii) in 5APs 3, a more flexible alkyl chain was inserted on N1 position; (iv) finally, in 5APs 4, the acylhydrazonic substituent was shifted from position 4 to position 3 of the pyrazole scaffold.
(Figure 1) to interact with polymeric tubulin α/tubulin β/stathmin4 complex at the colchicine binding site [34].By applying a ring opening strategy, we obtained 5APs II (Figure 1), able to strongly inhibit ROS and superoxide anion production, lipid peroxidation, and NADPH oxidase in thrombin-stimulated human platelets and ROS formation in EAhy926 cells [35].To further extend the structure-activity relationships (SARs) of this class of compounds and verify their biological profile, a new library (eighteen compounds) of 5APs 1-4 was designed and synthesized (Figure 2 and Table 1).In detail: (i) in 5APs 1, smaller, bigger, and more lipophilic substituents, similar to previous II, were introduced in meta and para positions of catechol portion; (ii) in 5APs 2, a methyl group on C3 of the pyrazole scaffold was inserted to increase steric hindrance; (iii) in 5APs 3, a more flexible alkyl chain was inserted on N1 position; (iv) finally, in 5APs 4, the acylhydrazonic substituent was shifted from position 4 to position 3 of the pyrazole scaffold.With the aim to investigate the antioxidant and anti-cancer activity of this novel library, in analogy with previous derivatives I and II, all new derivatives 1-4 have been tested for: (i) in vitro radical-scavenging activity (DPPH test); (ii) anti-aggregating/antioxidant activity in human platelets; and (iii) cell growth inhibitory activity.In addition, in silico pharmacokinetics, drug-likeness properties, and toxicity have been calculated.
With the aim to investigate the antioxidant and anti-cancer activity of this novel library, in analogy with previous derivatives I and II, all new derivatives 1-4 have been tested for: (i) in vitro radical-scavenging activity (DPPH test); (ii) antiaggregating/antioxidant activity in human platelets; and (iii) cell growth inhibitory activity.In addition, in silico pharmacokinetics, drug-likeness properties, and toxicity have been calculated.
As previously reported, the final reaction proved to be stereoselective and only hydrazones E were isolated, as assessed by NMR spectral analyses [34].As previously reported, the final reaction proved to be stereoselective and only hydrazones E were isolated, as assessed by NMR spectral analyses [34].

Inhibiting Effect on Human Platelet Aggregation and ROS Production
As previously reported for compounds I and II, platelets could be considered inflammatory cells and could represent a simple, economic, and suitable cellular model based on a causal relationship between inflammation and tumorigenesis [34].Inflammation and thrombosis are two critical, closely interconnected processes in the response to injury and infection.This correlation has long been recognized, particularly in atherosclerotic cardiovascular disease [44] as well in cancer [45].
Oxidative stress has been associated with several pathological conditions (e.g., cancer, diabetes, metabolic disorders, atherosclerosis, and cardiovascular diseases) [46] and plays a significant role in promoting inflammation and platelet activation [47].Thus, we tested the new 5AP library on human platelets to verify the inhibitory activity on aggregation and ROS production.As reported in Table 3, the newly synthetized compounds affected platelet aggregation and ROS production.Thus, the majority of the tested derivatives displayed IC 50 ≤ 200 µM on both tested parameters; 3b and 3c showed the higher inhibitory properties against both platelet aggregation and ROS production (IC 50 values between 113 and 139 µM).It should also be noted that three compounds have intermediate IC 50 values (around 400 µM), while five are virtually inactive.

Cell Growth Inhibitory Activity
APs 1-4 were tested for anti-proliferative activity on a panel of 60 different cancer cell lines by National Cancer Institute (Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Table 4) [48].
Compounds 2 (3-methyl substituted pyrazoles) and 4 (3-benzylidenecarbohydrazide pyrazoles) did not show a relevant anti-cancer effect, thus indicating that 3-unsubstitued pyrazole scaffold is as key determinant for anti-proliferative activity.Conversely, compounds 1, close analogues of previous II, and derivatives 3, characterized by a more flexible chain on N1, evidenced a good percentage of cancer cell growth inhibition in different cancer cell lines (Table 4).
Interestingly, among compounds 1, only derivatives with hindered substituents on catechol portion (i.e., OPh, OCH 2 Ar) and more strictly related to previous I showed growth percent inhibition values lower than 40% against selected cancer cell lines (1c and 1f on breast cancer cell lines; 1d on leukemia cell lines and breast cancer cell lines, 1e on renal cell lines; Table 4).Remarkably, compound 1e proved to selectively block the growth of renal cancer cell line CAKI-1.
Within series 3, compounds 3a and 3c showed a relevant anti-proliferative activity against different leukemic cell lines.
The significant cell growth inhibitory activity of 1c, 1d, 1f, and 1g against breast cancer cell lines prompted us to further evaluate (at fixed concentration of 10 µM, MTT assay) their effect against other breast adenocarcinoma cancer cell lines (namely, MCF7, MDA-MB231, and SK-BR3) using Cisplatin as reference compound (Table 5).5-APs 1c and 1d were inactive against all three cell lines, 1f selectively inhibited SK-BR3 cell lines (65% of cell growth), while 1g showed remarkable action against all breast cancer cell lines, particularly against SKBR3, evidencing a GI 50 value lower than reference compound Cisplatin (14.4 µM versus 26 µM).

Pharmacokinetic Properties, Druglikness and Toxicity Prediction
To evaluate the pharmaceutical relevance of this new library of 5APs, the pharmacokinetics and drug-likeness properties of all compounds were calculated by SwissADME (Tables S1 and S2, Supporting Information) [49].
Collectively the considered compounds are characterized by eight to twelve rotatable bonds, five to eight H-bond acceptors, three H-bond donors, and TPSA values of 123.99A 2 except 1a and 2a (114.76A 2 ), thus supporting a good capacity of all derivatives to permeate lipophilic barriers.In detail, none of the novel 5APs are able to pass brain-blood barrier (BBB), whereas gastrointestinal (GI) absorption is predicted high for derivatives 1a-g, 2a,3a, and 4a.Except for 1i, all 5APs are predicted to be moderately soluble with a further gain in water solubility for derivatives 3a,b (ESOL method) [50].A Lipinski violation (MW > 500 Da) was identified for compounds 1e-i, 2b, 2c, 3c, 4b, and 4c, but no compound showed any pan-assay interference compound (PAINS) alerts.Finally, the presence of a C=N functionality was identified as a limitation, according to the Brenk filter [51].

Discussion and Conclusions
The biological results reported here pointed at 5AP scaffold as an interesting chemotype to obtain new potential antioxidant/anti-inflammatory/anti-proliferative agents.In detail, the following SARs have been defined (Figure 3): (i) The presence of the acylhydrazone moiety at position 3 of the pyrazole core increases the radical scavenging activity (compare 4 with 1-3); (ii) The best ROS inhibitors in human platelets are 5APs 3 (particularly 3b and 3c, with IC 50 values of 113-115 µM), characterized in N1 by a more flexible alkyl chain (hydroxyhexyl).Furthermore, the substituents on the cathecol portions of these compounds (difluoromethoxy in para position and phenoxy or benzyloxy in meta position) are the same of II, previously identified as potent ROS inhibitors.In addition, 3c showed significative anti-cancer profile against different leukemic cell lines.These data confirmed the key role of a flexible hydroxyalkyl chain on N1 position, not only for ROS production inhibition, but also for anti-cancer activity; (iii) 5APs 1 showed promising anti-proliferative activity, probably related to the chemical similarity of these compounds with previous derivatives I.Of note is the fact that 1g (active against breast cancer cell lines) bears the same substituents on the catechol fragment of its precursor Ia. (iv) Finally, compounds 2, bearing an additional methyl group on C3 position of pyrazole nucleus, did not show a relevant biological activity, confirming that the increase of steric hindrance in this position is detrimental not only for antioxidant activity and ROS production inhibition in platelets but also for anti-proliferative activity.Collectively, the results here reported extend the SARs on 5AP scaffold, confirming its role as important chemo-type to obtain compounds able to counteract cancer and inflammation.

General Information
Chiminord (Milan, Italy) and Aldrich Chemical (Milan, Italy) purchased all chemicals.Solvents were reagent grade.Unless otherwise stated, all commercial reagents were used without further purification.Organic solutions were dried over anhydrous sodium sulphate.A thin layer chromatography (TLC) system was used for routine monitoring of the course of reactions and confirming the purity of analytical samples.Detection of spots was performed using UV light.Merck silica gel, 230-400 mesh, was used for chromatography.Flash chromatography was performed using Isolera one instrument (Biotage, Uppsala, Sweden) using Silicagel column.Melting points are not "corrected" and were measured with a Buchi M-560 instrument (Buchi instruments, Flawil, Switzerland).NMR spectra were recorded on JEOL JNM-ECZR (400 MHz, Tokyo, Japan) instruments (Figures S1-S42, Supporting Information) using CDCl3 or DMSO-d6 as solvent; chemical shifts are reported as δ (ppm) and signals were characterized as s (singlet), d (doublet), t (triplet), n t (near triplet), q (quartet), m (multiplet), br s (broad signal); J are reported in Hz.
Elemental analysis was determined with an elemental analyzer EA 1110 (Fison-Instruments, Milan, Italy) and the purity of all synthesized compounds was > 95%; products are considered pure when the difference between calculated and found values is ± than 0.4.
Synthesis of Carbohydrazide 8a, 8c, and 8d Collectively, the results here reported extend the SARs on 5AP scaffold, confirming its role as important chemo-type to obtain compounds able to counteract cancer and inflammation.Solvents were reagent grade.Unless otherwise stated, all commercial reagents were used without further purification.Organic solutions were dried over anhydrous sodium sulphate.A thin layer chromatography (TLC) system was used for routine monitoring of the course of reactions and confirming the purity of analytical samples.Detection of spots was performed using UV light.Merck silica gel, 230-400 mesh, was used for chromatography.Flash chromatography was performed using Isolera one instrument (Biotage, Uppsala, Sweden) using Silicagel column.Melting points are not "corrected" and were measured with a Buchi M-560 instrument (Buchi instruments, Flawil, Switzerland).NMR spectra were recorded on JEOL JNM-ECZR (400 MHz, Tokyo, Japan) instruments (Figures S1-S42, Supporting Information) using CDCl 3 or DMSO-d 6 as solvent; chemical shifts are reported as δ (ppm) and signals were characterized as s (singlet), d (doublet), t (triplet), n t (near triplet), q (quartet), m (multiplet), br s (broad signal); J are reported in Hz.

Materials and Methods
Elemental analysis was determined with an elemental analyzer EA 1110 (Fison-Instruments, Milan, Italy) and the purity of all synthesized compounds was >95%; products are considered pure when the difference between calculated and found values is ± than 0.4.

Synthesis of Final Compounds 1-4
To a solution of suitable carbohydrazide 8a-d (1 mmol) in absolute ethanol (10 mL), the suitable aldehyde 9a-l (1 mmol), solved in absolute ethanol (2 mL), is added dropwise, then the reaction mixture is heated at reflux for 1-18 h.After cooling to room temperature, the solvent is removed under reduced pressure to obtain yellow/white solids, which are filtered and recrystallized from abs. ethanol (compounds 1) or diethyl ether (compounds 2-4).

In Vitro Antioxidant Activity (DPPH Assay)
The antioxidant activity was measured using the DPPH antioxidant assay.The assay is based on the bleaching rate of the stable radical DPPH [56].Briefly, ca 3 mg of single compound was dissolved with methanol, then 0.1 mL 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.Linear calibration curve was obtained using Trolox standards (range between 20 to 200 mg/L, R2 = 0.9988).The result was calculated as Trolox equivalents in

MTT Assay
To perform MTT assay, SK-BR3 (breast adenocarcinoma, Biologic Bank and Cell Factory, IRCCS Policlinico San Martino, Genoa, Italy), MCF-7 (breast adenocarcinoma, Biologic Bank and Cell Factory, IRCCS Policlinico San Martino, Genoa, Italy), and MDA-MB231 (breast adenocarcinoma, Biologic Bank and Cell Factory, IRCCS Policlinico San Martino, Genoa, Italy) cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) added to 10% Fetal bovine serum (FBS), 2 mM Glutamine, and 1% penstrep.Reagents were acquired from EuroClone (Milan, Italy) and incubated in a humidified environment at 37 • C with 5% CO 2 .All chemical compounds (1c, 1d, 1f, and 1g and Cisplatin, used as reference compound) were dissolved in DMSO to give a 10 mM stock solution.Then, after an intermediate dilution in growth medium, they were added to the cultured cells at a final working concentration of 10 µM and incubated for 48 h.At the end of the incubation, 30 µL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) at a concentration of 2 mg/mL in PBS, were added in each well and incubated 4 h.Finally, the supernatants were removed and 100 µL/well of DMSO were added to each well to dissolve the Formazan precipitates.After 20 min, the results were read at λ = 570 nm.Results are expressed as a percentage of the control samples, where cells have been treated with the same amount of DMSO but without any chemical compound.The assay was repeated three times, and a single compound was tested six times.Means and standard deviations were calculated.
The GI 50 values were calculated based on single concentration-response curves.Each experiment was repeated three times.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29102298/s1,Table S1.Predicted pharmacokinetics and druglike properties of compounds 1a-i.Table S2.Predicted pharmacokinetics and drug-like properties of compounds 2a-c, 3a-c, 4a-c.Table S3.Predicted toxicity of compounds 1a-i, 2a-c, 3a-c, 4a-c.Figures S1-S42 Institutional Review Board Statement: Since blood for the experiments is collected during the voluntary blood donation in the transfusion center of the Hospital, under the Italian legislation it's not necessary to obtain the Ethics Commission Authorization.Donors are healthy subjects who have not undergone any treatment.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Figure 1 .
Figure 1.Chemical structure of previous compounds I and derived 5APs II.

Figure 1 .
Figure 1.Chemical structure of previous compounds I and derived 5APs II.

Figure 1 .
Figure 1.Chemical structure of previous compounds I and derived 5APs II.

Figure 3 .
Figure 3. Schematic representation of SARs of novel 5APs here reported.

Figure 3 .
Figure 3. Schematic representation of SARs of novel 5APs here reported.

Table 3 .
Inhibitory effect of new APs 1-4 and reference compounds ASA (acetylsalicylic acid) and NAC (N-acetylcysteine) on platelet aggregation and ROS production on human platelets expressed as IC 50 (µM) values.
a Reported data are the mean +/− SD obtained in six different experiments each performed in duplicate.n.d.: not determined.

Table 4 .
Cell growth percent values of most active 5APS 1-4 on different cancer cell lines at 10 µM concentration.For each compound, only cell lines with a growth percent values ≤ 50% are indicated.

Table 5 .
Cell growth percent values of 1c, 1d, 1f, and 1g and Cisplatin, used as reference compound, on three breast cancer cell lines at 10 µM concentration.Data are mean values for three separate experiments.Variation among triplicate samples is less than 10%.For 1g, GI 50 values (µM) were also reported.