3-(6-Phenylimidazo [2,1-b][1,3,4]thiadiazol-2-yl)-1H-Indole Derivatives as New Anticancer Agents in the Treatment of Pancreatic Ductal Adenocarcinoma

A new series of imidazo[2,1-b][1,3,4]thiadiazole derivatives was efficiently synthesized and screened for their in vitro antiproliferative activity on a panel of pancreatic ductal adenocarcinoma (PDAC) cells, including SUIT-2, Capan-1 and Panc-1. Compounds 9c and 9l, showed relevant in vitro antiproliferative activity on all three pre-clinical models with half maximal inhibitory concentration (IC50) ranging from 5.11 to 10.8 µM, while the compounds 9e and 9n were active in at least one cell line. In addition, compound 9c significantly inhibited the migration rate of SUIT-2 and Capan-1 cells in the scratch wound-healing assay. In conclusion, our results will support further studies to increase the library of imidazo [2,1-b][1,3,4] thiadiazole derivatives for deeper understanding of the relationship between biological activity of the compounds and their structures in the development of new antitumor compounds against pancreatic diseases.


Introduction
Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with an increased incidence also in young adults and a mortality/incidence ratio around 98% [1,2]; therefore new therapeutic strategies to counteract this malignancy are urgently needed. [1,3,4]Thiadiazole, both uncondensed or annealed to other heterocyclic moieties, particularly with an imidazole ring, has been recognized as a valuable scaffold for the development of pharmacologically active derivatives as it is present in many molecules with biological properties antitubercular [7], anticonvulsant [8], antibacterial and antibiofilm [9][10][11]. In particular, this ring system showed interesting antitumor activity with different mechanisms of action [12][13][14].
All compounds (9a-p) were initially screened at three different concentration (0.1, 1 and 16 µM) in all cell lines. The compounds 9c, 9e, 9l and 9n emerged for their ability to inhibit the growth rate in one or more cell lines. Therefore, these compounds were selected for further screening using eight different increasing concentrations (in the range between 0.125 and 16 µM) in order to evaluate the half maximal inhibitory concentration (IC50) values.  [1,3,4]thiadiazole compounds 9a-p was evaluated by Sulforhodamine-B assay (SRB) on a panel of PDAC cells, including SUIT-2, Capan-1 and Panc-1. These cells have been used in a number of pharmacological studies and are representative of the typical inherent resistance of pancreatic cancer cells to most chemotherapeutic agents [29,30].
All compounds (9a-p) were initially screened at three different concentration (0.1, 1 and 16 µM) in all cell lines. The compounds 9c, 9e, 9l and 9n emerged for their ability to inhibit the growth rate in one or more cell lines. Therefore, these compounds were selected for further screening using eight different increasing concentrations (in the range between 0.125 and 16 µM) in order to evaluate the half maximal inhibitory concentration (IC 50 ) values. Table 2 summarizes the IC 50s reported as means ± SEM of three independent experiments.
The compounds 9c and 9l showed relevant antiproliferative activities in all the three preclinical in vitro models, with IC 50s ranging from 5.11 to 10.8 µM. Between these two derivatives, the compound 9c induced the most relevant inhibition of cell growth, showing similar activities in SUIT-2, Capan-1 and Panc-1 cells, with IC 50s of 5.5, 5.11 and 5.18 µM, respectively ( Figure 2).
The compound 9e was more active in Panc-1 cells, with IC 50 value of 10.26 µM, whereas SUIT-2 and Capan-1 cells were resistant to the same treatment and showed IC 50s > 16 µM. Conversely, the IC 50 of the compound 9n was above 16 µM in Panc-1 cells, while SUIT-2 and Capan-1 cells were more sensitive, with IC 50s of 11.8 and 10.49 µM, respectively.  The compounds 9c and 9l showed relevant antiproliferative activities in all the three preclinical in vitro models, with IC50s ranging from 5.   In parallel experiments we evaluated the IC 50 s of the conventional anticancer drugs gemcitabine and 5-fluorouracil, which were below 1 µM. These results were in agreement with previous studies [29,31]. However, because of PDAC chemoresistant nature, these drugs have very limited clinical activity, prompting studies on novel compounds.
Results highlighted that the introduction of a methyl group on the nitrogen atom of the indole moiety was detrimental for the antiproliferative activity against the three cancer cell lines, whereas, substitutions on the phenyl ring as well as the presence of a fluorine atom or a methoxy group on the indole scaffold were not relevant for the activity.

Compound 9c Inhibited the Migration Rate in SUIT-2, Capan-1 and Panc-1 Cells
It is well known that the poor prognosis of PDAC is caused by its early metastatic behaviour. Although many efforts have been made in the multi-omic fields to study the promoters of metastatic events and identify new drug targets, the key drivers of the metastatic properties of PDAC remain unclear [32,33]. However, new therapeutic agents are needed to overcome PDAC aggressiveness and combat PDAC metastasis. Therefore, considering the metastatic nature of SUIT-2, Capan-1 and Panc-1 cells [34,35], we assessed whether or not the most promising compound 9c was able to affect cell migration. To this goal, the inhibition of cell migration was examined through a high-throughput screening using the scratch wound healing assay, as described previously [36]. SUIT-2, Capan-1 and Panc-1 cells were treated with the compound 9c at a concentration of 3xIC 50 and the migration rate was monitored over time within 24 h. Images of the wound closure were taken immediately after scratch (T = 0) and at 4, 8, 20 and 24 h from the treatment. As shown in the Figure 3, the compound 9c led to a net reduction of the migration in SUIT-2 and Capan-1 cells, whose the percentages of migration compared to the control (set to 100%) were 59.09% and 27.71%, respectively.

Chemistry
All melting points were taken on a Büchi-Tottoly capillary apparatus and are uncorrected. IR spectra were determined in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. 1   Of note these results showed that this compound was able to inhibit migration more than gemcitabine, which in parallel experiments marginally (i.e., less than 15% inhibition) affected PDAC cell migration, as also reported in our previous studies in Panc-1 cells [37]. Conversely, the compound 9c accelerated migration up to 142.85% in Panc-1 cells. The latter, unexpected results, might be explained by the tendency of PANC-1 cells to clump, a feature which might affect the analysis of the scratch after 24 h. Moreover, though most previous studies, including ours, showed the ability of tyrosine kinase inhibitors (TKIs) to reduce migration in different PDAC cancer cell lines [38], a seminal study on sunitinib and other TKIs reported metastatic acceleration depending on treatment schedule and tumour models [39].
Statistical analyses showed that the above described inhibition of migration in treated cells was significant in all three pre-clinical models, compared to untreated cells. Importantly, to exclude that the wound areas were covered by cell proliferation, we determined the doubling time of each cell lines used, which were above 24 h and we calculated that the area of the wound track which was approximately 10 6 µm 2 , too large to be covered by only cell proliferation in 24 h, since the average tumor adherent cell surface is around 100-150 µm 2 . Finally, we used concentrations of the compound 9c that did not induce cell death during 24 h of exposure to the treatments, in fact, we did not observe detached cells after 24-h drug treatment.

Chemistry
All melting points were taken on a Büchi-Tottoly capillary apparatus and are uncorrected. IR spectra were determined in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. 1 H and 13 C NMR spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d 6 solution, using a Bruker Avance II series 200 MHz spectrometer. Column chromatography was performed with Merck silica gel 230-400 mesh ASTM or with Büchi Sepacor chromatography module (prepacked cartridge system). Elemental analyses (C, H, N) were within ±0.4% of theoretical values and were performed with a VARIO EL III elemental analyser. Purity of all the tested compounds was greater than 95%, determined by HPLC (Agilent 1100 Series).
Analytical and spectroscopic data for compounds 5a,b are in agreement with those reported in literature [40].

Synthesis of 1-methylindole-3-carbonitriles (6a,b)
To a solution of the suitable 3-cyanoindole 5a,b (7.03 mmol) in anhydrous DMF (10 mL) 3.61 mmol of K 2 CO 3 and dimethyl carbonate (1.8 mL, 21.4 mmol) were added and the mixture was heated at 130 • C for 3.5 h. After cooling (3 • C), water and ice (25 mL) was slowly added under stirring. The suspension obtained was extracted with diethyl ether (3 × 10 mL) and the organic phase was washed with water and brine, was dried over Na 2 SO 4 and the solvent evaporated at reduced pressure to obtain the 3-cyano-1-methylindole 6a,b in excellent yields.
Analytical and spectroscopic data are in accordance to those reported in literature [41].

Cell Growth Inhibition
To evaluate the inhibitory effects of the imidazo[2,1-b] [1,3,4]thiadiazole compounds 9a-p on cell growth, we performed the Sulforhodamine-B (SRB) assay, as previously described [42]. Cells were seeded into 96-well flat-bottom plates in triplicate at a density of 3 × 10 3 cells/well for SUIT-2 and Panc-1, while 5 × 10 3 cells/well were used for Capan-1. Cells were incubated at 37 • C for 24 h to create a confluent monolayer and then they were treated with 100 µL of increasing concentrations of the compounds dissolved in DMSO.
After 72 h of treatment, the cells were fixed with 25 µL of 50% cold trichloroacetic acid (TCA) and kept for at least 60 min at 4 • C. Then, the plates were washed gently with deionized water, dried at room temperature (RT) overnight and stained with 50 µL of 0.4% SRB solution in 1% acetic acid for 15 min at RT. The excess of SRB stain was removed on dried tissues and the plates were washed with 1% acetic acid and let dry at RT overnight. The SRB was dissolved in 150 µL of tris(hydroxymethyl)aminomethane solution pH = 8.8 (TRIS base) and the optical density (OD) was detected at a wavelength of 490 nm and 540 nm. Cell growth inhibition was calculated as the percentage versus vehicle-treated cells ("negative control") OD (corrected for OD before drug addiction). Finally, the half maximal inhibitory concentration (IC 50 ) was calculated by non-linear least squares curve fitting (GraphPad Prism 7, Intuitive Software for Science, San Diego, CA, USA).

Wound-Healing Assays
The in vitro scratch wound-healing assay was performed as previously described [43]. SUIT-2, Capan-1 and Panc-1 cells were seeded into a 96-well plates at a density of 5 × 10 4 cells/well and incubated for 24 h at 37 • C, 5% CO 2 and 100% humidity. Then, cell monolayer was scratched through a specific needle to create a scratch of constant width. After removal of the detached cells by washing with phosphate buffered saline (PBS), we added only medium in the control wells and medium with the compounds of interest in the experimental wells. The wound closure was monitored by phase-contrast microscopy using a Universal Grab 6.3 software (digital Cell Imaging Labs, Keerbergen, Belgium) integrated to the Leica DMI300B migration station (Leica Microsystems, Eindhoven, Netherlands) and the pictures were captured immediately after scratch (T = 0) and at 4, 8, 20 and 24 h from the treatment. The results were analysed with the Scratch Assay 6.2 software (digital Cell Imaging Labs).

Statistical Analysis
All SRB assays were carried out in triplicate and repeated at least three times, whereas the percentages of cell migration were calculated taking into account at least six scratch areas. The data were evaluated using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Data were expressed as mean values ± SEM and analysed by the Student t test.

Conclusions
PDAC is one of the deadliest cancer types and despite enormous efforts in pancreatic cancer research, in 2019, the American Cancer Society estimated 1,762,450 new cancer cases and 606,880 cancer deaths in the United States (US) [44]. Due to the lack of clinical signs and symptoms, most patients are diagnosed at an advanced/unresectable stage of the disease and regimens with combinations of conventional chemotherapy drugs are the best option for the treatment of PDAC patients. [45]. However, PDACs are characterized by common inherent or acquired resistance to conventional treatment modalities and new therapeutic strategies are warranted [33,46]. A new series of imidazo [2,1-b] [1,3,4]thiadiazole derivatives 9a-p were efficiently synthesized and tested for their in vitro antiproliferative properties on a panel of PDAC cell lines, including SUIT-2, Capan-1 and Panc-1. Four out of sixteen compounds (9c, 9e, 9l and 9n), showed interesting in vitro antiproliferative activity. In particular, the compounds 9c and 9l were active in all three preclinical models with IC 50s ranging from 5.1 to 10.8 µM. Notably, the IC 50s of the compound 9c in SUIT-2, Capan-1 and Panc-1 cells were the lowest (around 5 µM). The compound 9e was active only in Panc-1 cells. Conversely, the compound 9n inhibited cell proliferation in SUIT-2 and Capan-1 cells, with IC 50s of 11.8 ± 0.54 and 10.49 ± 0.16 µM, respectively. Finally, using the scratch wound-healing assay, we demonstrated a relevant anti-migratory activity of the compound 9c in SUIT-2 and Capan-1 cells. Overall, the results of cytotoxicity and cell migration obtained with compound 9c could suggest its role as an interesting hit compound to create a library of new derivatives and study in deep the structure-activity relationship (SAR). This could indeed be extremely useful to guide the synthesis of future analogues. In particular, we focused our research on derivatives bearing at position 5 of indole scaffold a group (-OCH3) or atom (-F) with electron-withdrawing properties. Furthermore, we investigated how the N-methyl indole, as well as the effect of different substitutions on the phenyl ring, could influence activity. As reported in the Table 1, some of the compounds 1H-indole showed antiproliferative activity in one or more cell lines, unlike the 1-methyl-1H-indole analogues. Probably, this is due to the ability to create hydrogen bonds with the target. Concerning the substitution on phenyl ring, we did not observe differences between the presence or absence of electron-withdrawing groups, with the exception of nitro group (-NO 2 ) that, thanks to the delocalization of negative and positive charges, increased the cytotoxicity activity in all the cell lines.
Author Contributions: S.C., G.L.P., B.P., D.C., U.P. performed chemical research and analyzed the data. G.L.P., B.E.H., V.A., N.F., performed biological research and analyzed the data. G.J.P., G.C., A.P., P.D. and E.G. participated in the design of the research and the writing of the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This project was supported by a 2014-2020 PON Ricerca e Innovazione grant from the Italian Ministry of Education, University and Research, entitled "PROGEMA-Processi Green per l'Estrazione di Principi Attivi e la Depurazione di Matrici di Scarto e Non" (ARS01_00432) to P.D. and by the AIRC Start-up grant to E.G.

Conflicts of Interest:
The authors declare no conflict of interest.