One pot-like regiospecific access to 1-aryl-1 H -pyrazol-3(2 H )-one derivatives and evaluation of the anticancer activity

With pleasure, we warmly acknowledge Professor Girolamo Cirrincione for the significant contribution to the chemistry of heterocyclic compounds. We are honored to have been part of his research team


Introduction
Cancer is a dangerous disease characterized by abnormal and uncontrolled cell growth that can occur in any part of the human body.Despite the excellent results obtained in the diagnosis and treatment of this disorder, the fight against cancer always remains an open challenge. 1rom a medicinal chemistry point of view, much modern research pays particular attention to the discovery of new anticancer agents with optimal biological activities, and many efforts have been directed towards the synthesis of small molecules characterized by a heterocyclic scaffold.In this context, a large number of derivatives containing nitrogen atoms in the central core, (for example, pyridine and pyrimidine, [2][3][4] indole, 5 imidazole, 6 quinoline, 7,8 pyrazole and pyrrolizine 9,10 and pyrazolone derivatives, [11][12][13] and others [14][15][16] ) have been described in literature and some of them have already been approved as therapeutics, thus showing great potential in the treatment of cancer.
Specifically, the pyrazolone heterocyclic ring is, and remains, an interesting scaffold for the development of new small molecules endowed with anticancer properties.Indeed, a wide number of pyrazolone-based compounds have been described in the literature, both as non-selective and selective (VEGFR and c-Met inhibitors) antiproliferative agents, 11,17 including some compounds demonstrating catalytic inhibition of human telomerase. 18n this context, we reported a revisited synthesis and the biological evaluation of a new series of substituted 1-aryl-pyrazol-3-one derivatives of type 1 (Figure 1).The research interest for these molecules relies on the assumption that these compounds represent a significant fragment of the related antiproliferative tricyclic pyrazolo [1,2-a]benzo [1,2,3,4]tetrazin-3-one analogues (PBTs), endowed with anticancer potential with GI50 values in the sub-micromolar range. 19,20In an attempt to get insight into the minimal structural requirements, necessary to maintain the antiproliferative activity exhibited by the PBTs, we moved towards the synthesis of 1-aryl-pyrazol-3-one derivatives to evaluate how the increased conformational flexibility, jointly with the possible tautomeric equilibrium, could affect the activity in function of our selected functionalizations.Depending on the physical state, the chemical environment and the effect of substituents on the aryl moiety, including the possible formation of internal H-bonds, the derivatives 1 can exist in one of the two tautomeric forms (Figure 1).
After the synthetic campaign, the new series of 1-aryl-pyrazol-3-one molecules was submitted to the National Cancer Institute (NCI) for antiproliferative in vitro screening to evaluate the influence of the de-azo modification on the biological profile of the compounds.Moreover, compared with the previous synthesis described before, 19,20 herein, we report a revisited more advantageous preparation strategy to obtain the 1-aryl-pyrazol-3-one derivatives 1, bearing also substitutions on the two ortho-aryl positions.

Results and Discussion
Chemistry.The general synthetic route for the preparation of the variously substituted 1-aryl-pyrazol-3-one derivatives started from the initial preparation of the furan-3-one intermediate 4, as firstly described by Venturello and D'Alosio 21 (Scheme 1).Scheme 1. Synthesis of the 2,5-dimethyl-furan-3-one.
In detail, the dimerization of the diacetyl 2 occurred in aqueous NaOH solution and afforded compound 3 in good yields.Subsequent heating, under aqueous acidic conditions, allowed the elimination of a molecule of acetic acid and the rearrangement of 3 into the dimethyl-furanone 4, that can exist in equilibrium with the tautomeric form 4'.
In the next steps, the commercially available 2-nitroanilines, converted into the corresponding diazonium salts 5a-i upon treatment with sodium nitrite in concentrated hydrochloric acid, were coupled with the furanone 4 to give derivatives 7a-i in appreciable yields after 24-48 h.The synthesis of the title derivatives accomplished here, with respect to the previous ones, was conducted in a one-pot manner, avoiding the isolation and purification of the aza-furanone intermediate 6a-i or other intermediates.Thus, a step work-up economy is gained (Scheme 2), without appreciable variation of the overall yields.Scheme 2. Synthesis of 1-aryl-substituted-pyrazol-3-one derivatives 7a-i and 8.
Moreover, in this revisited procedure, we selected particularly 5a and 5c reactants, two di-ortho substituted nitro-anilines, R 3 = Cl and R 3 = Me groups, respectively, in order to evaluate how their electronic or steric effect could interfere with the entire rearrangement process.The isolation of the corresponding pyrazolones (7a, 7c) demonstrated that these groups are well tolerated, involving only a slight decrease in yields.
The synthetic process above was carried out under strong acidic reaction and mild temperature conditions, necessary for all the ordered rearrangement process, and sometimes caused the appearance of a by-product.Specifically, in the reaction involving the aniline 5a, we isolated, beside the expected product, a new compound firstly detected by GC-MS spectroscopy, where the fragmentation pattern agreed with the typical isotope rate in agreement with a tri-Cl derivative of type 8 [presence of peaks M+ m/z 276 (100%), 278 (96.8%), 280 (31.5%)].Further confirmations were furnished by 1 H and 13 C NMR spectroscopic data.Thus, in the course of the above sequential reaction carried out in a one pot procedure, a competitive nucleophilic substitution of the nitro group occurred, leading to the 1-(2,4,6-trichlorophenyl)-pyrazolone derivative 8.
Although the observed nucleophilic substitution of the nitro group by chloride ion (Cl -), could take place at different levels of the reaction sequence, it is reasonable to suppose that the pyrazolone 7a, once formed, undergoes the nucleophilic attack by the chloride ion, in large excess in the reaction media, affording the 2,4,6-trichloro derivative 8 as a minor by-product (yield 10%).Based on our investigations until now performed, some clues support the following supposition: a) aryl-pyrazol-3-ones are particularly stable in strong acid media; b) pyrazolones possess basic nitrogen centers which can be protonated at low pH values and thus withdraw electron density on the aryl portion; and c) additional electronic effects exerted by the two chlorine atoms play a synergistic role in this sense.Therefore, we hypothesized that these combined electronwithdrawing effects, exerted by the combination of protonated pyrazolone portion and the di-chloro-aryl moiety, could favor the nitro group displacement.For this purpose, to validate our supposition, derivative 7a was submitted to ultrasound stress in concentrated HCl (37%) for 6 h at 60 °C (Scheme 3).Notwithstanding all the favorable conditions above, after work-up, we recovered only the starting compound 7a.Then, we considered the possibility that derivative 8 could originate during the rearrangement of the intermediate 6a, not isolated in our one pot procedure.In this regard, we repeated the reaction in the classical procedure already described, by stopping the reaction at the aza-furanone step (by TLC monitoring).Thus, we isolated derivative 6a and submitted it to rearrangement in HCl 37%.After 1.5 h of sonication at 60 °C, we were pleased to detect by GC-MS, beside the expected 7a as major compound, a by-product corresponding to the identical fragmentation pattern as compound 8. Scheme 3. Stability of 7a after sonication under HCL 37% at 60 °C.Rearrangement of 6a under HCL 37% at 60 °C promoted by sonication to furnish 7a, along with minor extent of NO2 replacement by chloride ion.
Due to the relevance of the amino group in biological interactions, we proceeded with the nitro conversion.Thus, to reach the 1-(o-aminoaryl)-pyrazol-3-one derivatives 9a-i, we performed the reduction of the nitro group with Fe/AcOH for the chlorine derivatives, and for the others by using classical catalytic conditions with Pd/C 10% in EtOH under H2 (4 atm) in a Paar-like reactor (Scheme 4).All compounds were fully characterized by 1 H, 13 C NMR, GC-MS, IR spectroscopy and microanalysis.All experimental data agreed with the proposed structures.

Scheme 4. Reduction of nitro derivatives 7a-c.
Preliminary in vitro studies on pyrazol-3-ones.The set of pyrazol-3-one intermediates synthesized, 7a-i, 8, and 9a-i, were submitted to the National Cancer Institute for one dose screening (concentration of 10 μM) against the full NCI60 library, including sixty cell lines belonging to nine different tumor panels (leukemia, lung, renal, CNS, prostate, breast, melanoma, ovarian and colon).In general, the results for each tested compound are reported as the percent of growth (G%) of the treated cells when compared to the untreated control cells.This parameter strictly expresses the anticancer potential of the compound, indeed: for G%>100, the compound does not affect cancer cell proliferation; 0<G%<100, the compound inhibits the cell proliferation of a percentage given by 100-G%; if G%<0, the compound is cytotoxic and lethal for the cancer cells.Furthermore, to graphically appreciate the most sensitive panels/cell lines, a mean growth percent is provided.Among all, six pyrazol-3-one derivatives (9d-g 19 , 9h,i 20 ) were selected and assayed against the NCI cancer cell panel (chemical structures reported in Figure 2).
In Table 1 the overall results obtained by the six selected compounds in the single-dose assay are reported: none of the tested compounds exhibited appreciable antiproliferative activity (expressed as G%) against the full NCI60 panels; indeed, all reported a mean G% higher than 100, meaning no reduction of cancer cell growth.Two compounds, 9e 19 (NSC752656) and 9g 19 (NSC752658), with the lowest mean G% (both 105.7) exhibited some modest results against UO-31 renal cancer cell line, with G% of 81.2 and 73.3, respectively.*N.T.: not tested against the cell line.
In addition, although moderate, the derivative 9g exhibited a growth inhibition (GI) against CNS tumor (SNB-75) and melanoma (UACC-257) cell lines, with 18% and 15% GI, respectively.Lastly, derivative 9i deserves attention with 20% of inhibition growth, also against SNB-75 cells.These outcomes, even if modest, could be a basis for further developments in cell selectivity and decorations on new pyrazolone series.

Conclusions
Due to the increasing interest towards pyrazolone derivatives in the anticancer field, either as key intermediates or by incorporation in polycyclic heterocycles, 22 herein new insights into the synthesis and the antiproliferative properties of the 1-aryl-pyrazol-3-ones 7a-i, 8 and 9a-i are reported.In this light, we propose a more advantageous synthetic procedure, involving a step economy with respect to the previously reported methods in the literature, including the possibility to introduce functionalities in the hindered position of the 1-aryl moiety.In addition, this ordered regiospecific pathway can be alternatively performed under sonication stress, maintaining the N1-aryl isomer formation.Moreover, in some cases, depending on the effects of the substituents on the aryl portion, a nitro displacement by a chlorine could also be observed.Thus, six of the isolated compounds were selected for the NCI in vitro screening.Generally, none of them exhibited appreciable anticancer activity, with the exception of 9e and 9g, which reported a modest, but promising G% against the UO-31 renal cancer cell line.Deeper investigation will be conducted to investigate the reactivity of aryl-pyrazol-3-one derivatives, as well as analyze their potential biological activities, other than the anticancer one.

Experimental Section
General. 1 H and 13 C NMR spectra were recorded in CDCl3 or DMSO-d6 solution as specified below, using a Bruker AC-E series 200/300/400 MHz spectrometer.As internal reference, the residual peak of the solvent was calibrated, at d 7.26 and 2.50 ppm, respectively for CDCl3 and DMSO-d6 or using TMS as internal reference.The chemical shifts (δ) and coupling constants (J) are expressed in ppm and hertz respectively."a" means "apparent" for close coupling constant and the number of equivalent group are indicated after the sign "x".Carbon attribution, namely C, CH, CH2 and CH3 were determined by 13 C and DEPT 135.IR spectra were recorded with bromoform on a NaCl window with a Bruker Alpha FT/IR spectrophotometer.All melting points were measured on a Sanyo-Gallenkamp capillary apparatus.Mass spectroscopy was performed using a GC-MS Shimadzu QP5050 with EI (75ev), equipped with a ZB5 Phenomenex 20m column.Column chromatography was performed with Merck silica gel 230 or 400Mesh ASTM.Thin layer chromatography was performed on precoated (0.25 mm) silica gel GF254 plates (Merck) and compounds were detected with 254/366 nM UV lamp.All reactions were carried out under hood and air exposed with commercially reagents purchased from Aldrich or Alfa Aesar and directly used.Sonication was performed with Transonic T460/H, ELMA (HF-Frequ 35KHz, A 1.30, F 50/60 Hz).