Synthesis and biological evaluation of new 3(2H)-pyridazinone derivatives as non-toxic anti-proliferative compounds against human colon carcinoma HCT116 cells

Abstract Novel 3(2H)-pyridazinone derivatives were designed, synthesised in satisfactory yields and evaluated in different experimental assays to assess their preliminary toxicity in vivo and anti-proliferative effects against HCT116 cell lines in vitro. Artemia salina lethality test provided LC50 values >100 µg/mL for all compounds. Successive assays revealed that some compounds were endowed with a promising anti-proliferative effect against HCT116 cells, alone or stimulated by serotonin as a pro-inflammatory factor in order to mimick an inflamed model in vivo of cancer cell microenvironment. Moreover, the kinurenic acid level after treatment with these newly synthesised compounds was monitored as a marker of anti-proliferation in colon carcinoma models. The IC50 values obtained for the best-in-class compounds were comparable to that of daunorubicin as a reference drug. Conversely, these compounds were not able to counteract the spontaneous migration of human cancer HCT116 cell line in the wound healing paradigm.


Introduction
Cancer consists of an uncontrolled proliferation of cells in different tissues and organs; it is a disease whose clinical appearance, treatment and approach are different from each other. Cancer is a major global health problem and it is currently the second leading cause of death in the world being expected to surpass cardiovascular diseases in the next few years 1,2 . Many factors, from bacteria to viruses, from radiation to heredity, from environmental factors to nutritional habits and chemicals, are accused of cancer formation. In the data announced by the World Health Organisation (WHO), approximately 18 million people were diagnosed with cancer in 2018, and around 10 million people died from cancer. According to data of Global Cancer Obervatory (GLOBOCAN), the most common types are lung (2.1 million), breast (2.09 million), colorectal (1.8 million), prostate (1.3 million), stomach (1 million) cancer. According to cancer-related deaths, lung (1.8 million), colorectal (881 thousand), stomach (783 thousand), liver (782 thousand) and breast (627 thousand) are listed. Colorectal carcinomas (CRC) are one of the most common types of cancer in the world that cause death. CRC metastases account for 40-50% of recently diagnosed cases and are correlated with high morbidity 3,4 .
In medicinal chemistry pyridazinones have been the subject of intensive synthetic investigations, because they possess a wide spectrum of pharmacological activities and gained importance in recent years 5 . A number of compounds such as zardaverine/imidazole, bemoradan, indolindan, pimobendan are examples of pyridazinones that are biologically active. Literature survey revealed that substituted pyridazinones have reported to possess pharmacological activities, which can be rationalised in the SAR study reported in Figure 1 6-12 . There are also compounds which were shown to have anti-cancer or cytostatic activity in the literature against HEP3B (liver cancer cells), HCT116 (colon cancer cells), SH-SY5Y (neuroblastoma cells) and promising selectivity index with respect to human fibroblasts [13][14][15][16] . These results suggest that pyridazinone compounds may be useful in cancer chemotherapy, depending on the type of cancer, and that derivatives bearing different substituents may exhibit varying degrees of cytotoxic effect.
Pursuing our efforts to discover novel anti-cancer compounds [17][18][19] and with the aim of enlarging the SAR knowledge within this chemical scaffold, we designed fifteen new 3(2H)-pyridazinones investigating the anti-proliferative effects against the human HCT116 cell line, their toxicity in the Artemia salina lethality assay in vivo, the HCT116 viability after serotonin challenging and compound treatment, the release of kynurenic acid after compound treatment and, lastly, the capability to limit the spontaneous migration of HCT116 cells in the wound healing paradigm.

Chemical studies
Melting points of the compounds were determined on Electrothermal 9200 melting points apparatus (Southent, Great Britain) and the values given are uncorrected. The IR spectra of the compounds were recorded on a Bruker Vector 22 IR Spectrophotometer (Bruker Analytische Messtechnik, Karlsruhe, Germany). The 1 H-NMR and 13C-NMR spectra of the compounds were recorded on a Bruker 400 MHz-NMR Spectrometer (Rheinstetten, Karlsruhe, Germany) using tetramethylsilane as an internal standard. All the chemical shifts were recorded as d (ppm). The mass spectra (HRMS) of the compounds were recorded on Waters Acquity Ultra Performance Liquid Chromatograpy Micromass which combined LCT PremierTM XE UPLC/MS TOFF spectrophotometer (Waters Corp, Milford, USA) by ESI þ and ESI-techniques.

Synthesis of 4-(3-fluoro-4-methoxyphenyl)-4-oxobutanoic acid (I)
A mixture of 0.275 mol aluminium chloride, 20 mL carbon disulphide and 0.25 mol succinic anhydride was added portionwise in standard conditions to a mixture of 0.25 mol 2-fluoroanisole and 50 mL of carbon disulphide. Then, the mixture was refluxed for 4 h at 40-50 C. After cooling, the residue was poured onto ice water and the precipitate was collected, dried and recrystallized from water. M.P.: 164 C.

Artemia salina lethality test in vivo
In Artemia salina lethality bioassay, brine shrimp larvae were incubated for 24 h with compounds VIa-o (0.01-10 mg/mL) dissolved in the incubation medium (artificial sea water). The detailed protocol was described in our previous article 20 .

2.2.2.
Human colon cancer HCT116 cell culture and experiments in vitro HCT116 cell line (ATCCV R CCL-247 TM ) was cultured in DMEM (Euroclone) supplemented with 10% (v/v) heat-inactivated foetal bovine serum and 1.2% (v/v) penicillin G/streptomycin in 75 cm 2 tissue culture flask (n¼5 individual culture flasks for each condition) as previously reported with or without serotonin treatment 20 .
In the same condition, the kynurenic acid (KA) extracellular level was determined through a validated high performance liquid chromatography (HPLC)-fluorimetric method 21 . To assess the cytotoxicity of synthesised compounds (VIa-o), a viability assay was performed on 96 microwell plates, using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) test. Cells were incubated with compounds (10 mg/mL) for 24 h. An aliquot of 10 mL of MTT (5 mg/mL) was added to each well and incubated for 3 h. The viability of HCT116 cell line was evaluated both in basal conditions and after challenging with serotonin (5-HT) at 1 ng/mL. The anti-proliferative effects were compared to that induced by daunorubicin (0.1-20 mg/mL), used as reference drug.
Finally, the effects of the most potent compounds were evaluated on the spontaneous migration of HCT116 cells, in the 48 h following the experimental lesion of cell monolayer (wound healing paradigm). The detailed protocol related to wound healing experimental model was described in our previous article 20 .

Statistical analysis
Results of in vitro studies were expressed as means ± standard error (SE) of three experiments performed in triplicate. Statistical analysis was determined through analysis of variance (ANOVA), followed by post hoc Newman-Keuls comparison multiple test. The level of significance was set at p < 0.05.

Results and discussion
In order to enlarge our SAR on this chemical scaffold, we investigated the presence of different substituents on the hydrazone moiety to explore the chemical space in terms of electronic and steric effects. Moreover, we deleted the piperazine linker attached to the pyridazinone core nucleus aiming at limiting the conformational freedom of our compounds. The title compounds (VIa-o) were synthesised according to the literature methods as outlined in Scheme 1.
Synthesis of the compounds was initiated by obtaining benzoyl propanoic acid derivative (I) in the presence of succinic anhydride and 2-fluoroanisole by anhydrous aluminium chloride catalysis. Subsequently, the reaction of this compound with hydrazine hydrate led to the formation of 4,5-dihydro-3(2H)-pyridazinone (II). 6-Substituted-3(2H)-pyridazinone derivative (III) was obtained by oxidation of II with bromine in glacial acetic acid. Ethyl 6-substituted-3(2H)-pyridazinone-2-ylacetate derivative (IV) was obtained by the reaction of III with ethyl bromoacetate in the presence of K 2 CO 3 in acetone. Then, 6-substituted-3(2H)-pyridazinone-2-ylacetohydrazide derivative (V) was synthesised by the condensation reaction of IV with hydrazine hydrate. Ultimately, the title compounds bearing benzylidenhydrazide structure were obtained by the reaction of V with substituted/nonsubstituted benzaldehydes. All of the title compounds were reported for the first time in this study. The reaction yields ranged approximately from 38% to 96%. Compound VIe was synthesised with the highest yield (96%), while compound VIi with the lowest yield (38%). The physical and spectral properties of the starting compounds were in accordance with the literature. Molecular structures of title compounds were confirmed by IR, 1 H-NMR, 13 C-NMR, and mass spectral data. Their molecular structures, yields, and melting points are given in Table 1.
Firstly, the biocompatibility limit of the compounds was determined through the A. salina brine shrimp lethality test in vivo. The nauplii were stimulated with compounds VIa-o, in the range 0.01-10 mg/mL. The lethality test showed LC 50 values >100 mg/mL for all the compounds. Based on our previous investigations 20,21 , a 10-fold lower concentration (10 mg/mL) was selected for the subsequent in vitro cell-based tests. In this regard, the human colon cancer HCT116 cell line was selected and treated with the aforementioned molecules. The HCT116 viability was stimulated through 5-HT challenging. 5-HT has long been described as a proinflammatory factor, particularly in the gut 22 , with in vitro studies substantiating a mitogen role, mediated by different receptor types towards multiple cell lines 23 . According to these findings, a preliminary study was carried out in order to optimise the experimental conditions that could demonstrate a cell viability-stimulating effect of 5-HT, in a wide range of concentrations (10 pg/mL -1 mg/mL). We observed that HCT116 cell viability increased in a concentration-dependent manner, in the range 0.1-1 mg/mL, although it remained constant, at upper tested concentrations (Figure 2) given. Considering our previous ex vivo and in vitro studies focussed on inflamed colon specimens and hypothalamic cells, respectively, reporting 5-HT concentrations in the order of 1 ng/mL 20,21 , we have chosen the 5-HT concentration of 1 ng/mL as a reliable proliferative stimulus for the following tests. Specifically, compounds VIc-e and VIh-m were able to inhibit 5-HT-stimulated viability of HCT116 cells (Figure 3), thus substantiating the potential anti-proliferative effect of the compounds in the real in vivo colon cancer cell microenvironment, characterised by the up-regulated production of multiple pro-inflammatory and anti-apoptotic/mitogen factors, including 5-HT 24,25 . Furthermore, compounds were assayed for evaluating modulatory effects on the extracellular level of KA, one of the two main kynurenine metabolites. Kynurenic acid was reported to be    produced in multiple tissues, including brain and peripheral organs 26 , although pharmacokinetic studies excluded any possibility of the peripheral pool to cross blood-brain barrier 27 . In the brain, the kynurenine-derived kynurenic acid was described as a reliable marker of neuroprotection 28,29 , whereas it seems to be involved in inflammatory response at the peripheral level 30 . Kynurenic acid was also described as an anti-proliferative factor towards colon, renal, and glioma cells 31 . Specifically, this marker was considered as a potential chemopreventive agent against colon cancer 32,33 . The assessment of KA levels showed that the sole compound VIc was able to blunt 5-HT-induced KA depletion ( Figure 4) after treatment. Additionally, the KA level after compound VIc stimulation was even higher compared to basal condition (CTR group). Conversely, the other tested compounds (VIa, VIb and VId-m) failed to prevent the inhibition of KA level following the stimulation with 5-HT. They were able in potentiating 5-HT-induced KA depletion, as well. The results of this pharmacological screening suggest a minor role exerted by the compounds VIa, VIb and VId-m as anti-proliferative agents.
Considering the effects induced by all compounds on HCT116 viability and KA production, compounds VIc, VIe and VIk were further assayed in order to deepen our knowledge about their anti-proliferative effects, in basal conditions. Specifically, HCT116 cells were stimulated with the aforementioned compounds, in a wider concentration range (0.1-20 mg/mL), with respect to the initial test. Additionally, the anti-proliferative effects induced by these compounds were compared with that of daunorubicin, used as anti-tumoral reference drug in the same concentration range. The IC 50 values were calculated and, as evidenced in Figure  5(A-D), compounds VIe and VIk showed interesting potencies ( Figure 5(C,D)) with IC 50 values of 3.09 and 2.73 mg/mL respectively, that were very close to that shown by daunorubicin (1.39 mg/mL). Conversely, compound VIc showed an IC 50 value (15.03 mg/mL) that was at least 10-fold higher compared to daunorubicin ( Figure 5(A,B)). On the other hand, although the MTT test seems to exclude any application of VIc compound as anti-proliferative agent, the increased KA level (Figure 4), shown at a concentration even lower than the IC 50 , indicated a potential use as chemopreventive agent that deserves a further investigation.
Finally, considering their potencies in reducing HCT116 cell viability, compounds VIe and VIk were further studied in order to evaluate their effects on the spontaneous migration of HCT116 cells, through the wound healing paradigm. In this experiment, HCT116 cells were treated with compounds VIe and VIk at their respective IC 50 values. The spontaneous migration of HCT116 cells was monitored in the 48 h following the experimental lesion of cell monolayer. The null effects on sponstaneous migration ( Figure 6) rule out any involvement of the tested compounds in altering the invasion capacity of human colon cancer cells.

Conclusion
Fifteen new 3(2H)-pyridazinone derivatives were synthesised and studied for their ability to limit the proliferation of HCT116 cell line (colon carcinoma), alone or after stimulation with serotonin, a well-recognized pro-inflamamtory factor in the gut. In particular, compound VIc induced a strong release of kynurenic acid after treatment, thus representing a strong chemopreventive agent in this model. Moreover, all compounds resulted non-toxic up to 100 mg/mL in the A. salina lethality assay, whereas three of them (VIc, VIe and VIk) displayed a promising inhibitory action comparable to that of daunorubicin as a standard drug at basal conditions.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by intramural grants by Ministero Italiano dell'Universit a e della Ricerca (MIUR) FAR 2019 (ex 60%), held by Dr. Simone Carradori. Figure 6. Effects of compounds VIe (3.09 mg/mL) and VIk (2.73 mg/mL) on the spontaneous migration of human colon cancer HCT116 cell line (wound healing paradigm). The spontaneous migration was monitored in the 48 h following treatment. Data are expressed as percentage scratch area relative to the untreated CTR group.