Catalyst-Free One-Pot Synthesis of Densely Substituted Pyrazole-Pyrazines as Anti-Colorectal Cancer Agents

The first catalyst-free post-Ugi cascade methodology was developed for expeditious access to structurally diverse and complex pyrazole-pyrazines in one-pot. This novel cascade reaction features an intramolecular N2-arylation of pyrazoles with allenes at the C-β position of triple bond. Screening in the colorectal cancer cell lines HCT116 and SW620 validated the feasibility of the methodology for generating bioactive compounds. The lead compound 7h which is active against HCT116 and SW620 with IC50 of 1.3 and 1.8 µM, respectively, can be synthesized and purified in a gram process synthetic scale in 7 hours. The mechanical studies indicated that compound 7h can induce cell cycle arrest in the G2/M phase and inhibit proliferation and viability in human colon cancer cells. Overall, compound 7h is represented as a promising starting point for the development of new anti-colorectal cancer drugs.

Encouraged by good yields for both the Ugi and cyclization reactions, we investigated the cyclization reaction with the unpurified Ugi product 5a. In one pot, the cascade reaction produced the final product 7a in 64% yield for two steps. We then investigated the scope of this one-pot procedure by varying starting materials (in Scheme 2). First, either electron withdrawing or donating phenyl amines and aromatic or aliphatic substituted isocyanides were well tolerated. Strikingly, compounds 7d and 7h with small cyclopropanamine were also obtained with good yields. Phenyl pyrazoles (7j-m, 7o and 7s) or methyl indazoles (7n, 7p-r) didn't make a significant difference for the reaction yield. It is worth to emphasize that the Ugi adduct 5 did not require purification by column chromatography, with the crude product having no discernible impact on the overall yield. In all cases, the initial Ugi products 5a-s were used directly in the next step without further purification after the removal of solvent removal. A variety of different starting materials were successfully employed under the optimized conditions for the construction of structurally diverse pyrazole-pyrazines 7a-s with yields in the range of 55-68% shown in Scheme 2, indicating a good functional group tolerance. The structure of compound 7i was unequivocally confirmed by X-ray crystallography, as shown in Scheme 2 as well.
Mechanistically, under microwave irradiation the tautomer 8 could be formed. Microwave irradiation would promote an isomerization at the triple bond to generate the intermediate allene 8', which in turn would react with the pyrazole to afford the unstable compound 6. It was surprising to find that the intramolecular N2-arylation occurred without a catalyst, which is obviously different from the previous reports [18][19][20][21][22][23][24]30,31 . This two-step operation provided the facile access to a variety of complex fused pyrazole-pyrazines 7 in good yields, making it suitable for the construction of small compound libraries and scale-up synthesis.
To expand this catalyst-free cyclization for the synthesis of other scaffolds, we investigated if the triple bond would still react with a 2H-indazole when it was present in different Ugi inputs. We replaced aniline with 2-propynylamine 10 to provide a triple bond in the amino-Ugi input as depicted in Scheme 3. Similarly to the synthesis of compound 7, the U-4CR with dazole carboxylic acid 1, 2-propynylamine 10, aldehyde 9 and isocyanide 4 progressed smoothly (Scheme 3). The U-4CR was completed at room temperature overnight and evaporated to dryness to give the crude product 11, which was then subjected to the optimal conditions with the microwave irradiation at 110 °C for 20 min in DMF without a catalyst to give compound 14. With this condition, we proceeded to investigate the synthesis scope of this new series of pyrazine-pyrazines 14. In all cases, initial Ugi products 11 were subjected to the cyclization reaction following the solvent removal. A variety of different starting www.nature.com/scientificreports www.nature.com/scientificreports/ Biology and structure activity relationship (SAR). To evaluate the potential for developing a drug lead from compounds 7a-s and 14a-j, the MTT assay was used to measure cancer cell viability upon the drug treatment ( Table 2) 33 . Compounds 7a and 7c with an unsubstituted phenyl and compound 7i with 2-chloro-substituted phenyl at the R 1 position of 7A (Scheme 2) were completely inactive, while compounds 7b, 7f and 7g with either an electron donating (-OMe) or withdrawing group (-Br) at the 4-position of the phenyl group were potent with IC 50 less than 5 µM in both cancer cells, indicating the importance of substituents at the 4-position of the phenyl group. The critical role of a 4-substituted phenyl group at the R 1 position was further confirmed with derivatives 7j, 7k, 7l, 7m, 7o, and 7s, which possess a different pyrazolopyridone core structure. Compounds 7l and 7o with a 4-substituted phenyl group demonstrated a good activity; in contrast, other compounds including compound 7m with a 3-substituted phenyl group was not active at 20 µM. Surprisingly, four compounds (compounds 7n and 7p-7r) with a methyl substituted indazole at the R 3 position of 7A (Scheme 2) were all inactive against HCT116 and SW620 cell lines at 20 µM. Furthermore, replacement of the non-substituted phenyl (R 1 of 7A) group of compound 7a with a simple cyclopropyl group (7d) increased the activity significantly. Especially when the R 2 group (7A, Scheme 2) was switched from the phenylmethyl group (7a) to a longer phenylethyl group, compound (7h) is equally potent for both cell lines with IC 50 of 1.3 µM and 1.8 µM against HCT116 and SW620 cell lines, respectively. Finally, all fused pyrazole-pyrazines 14a-j (Scheme 3) were not active at 20 µM.
As shown in Fig. 2A, the compound 7h efficiently inhibited colon cancer cell viability in a time-and dose-dependent manner. Moreover, soft agar assay in vitro was performed to evaluate the effect of the compound 7h in colony formation, and the results demonstrated that smaller and lesser colonies were formed in treated groups (1, 2 and 4 μmol/L) compared with control groups in both glioblastoma cell lines (Fig. 2B,C). These results supported that the compound 7h dramatically inhibited cell viability and proliferation in human colon cells.
To explore the underlying mechanism responsible to compound 7h-induced cell proliferation, cell cycle distribution was analyzed using flow cytometry in colon cancer cells treated with or without the inhibitor. Control

conclusions
In summary, two series of densely substituted and distinct fused pyrazole-pyrazines were synthesized by using an Ugi reaction following an intramolecular N2-arylation of pyrazoles with allenes at the C-β position in absence of a catalyst in one-pot. Under the mild reaction condition, simple operation procedure, commercial availability of starting materials, and good overall yield, the novel methodology will offer a quick access to diverse indazole and pyrazole analogues for biological testing in a cost-effective and environmental-friendly manner. The gram scale synthesis of compound 7 can be accomplished within in one day (see SI for details). To our best knowledge, this is the first report for post-Ugi cascade reaction to prepare pyrazole-pyrazine scaffolds without a catalyst in one-pot. This catalyst-free cascade methodology would be highly applicable to other MCRs such as Passerini 34 , Petasis 35 , Betti 36 , Kabachnik-Fields 37 , and GBBR 38 .
The preliminary screening results of compounds 7 in the colon cancer cell lines HCT116 and SW620 are promising. Considering that compounds 7b and 7h are only 10 times less active than the most popular anticancer drug paclitaxel, 7b and 7h provide starting points for the further optimization in the drug discovery value chain for the treatment of colorectal cancer. Further efforts are ongoing for applicable design strategy of this cascade cyclization to synthesize new scaffolds and optimize 7h toward a clinical candidate.

Experimental Section
General information. 1 H and 13 C NMR were recorded on a Bruker 400 spectrometer. 1 H NMR data are reported as follows: chemical shift in ppm (δ), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant (Hz), relative intensity. 13 C NMR data are reported as follows: chemical shift in ppm (δ). HPLC-MS analyses were performed on a Shimadzu-2020 LC-MS instrument using the following conditions: Paclitaxel 0.14 ± 0.05 - Table 2. Anticancer activities of compounds 7 and 14 a . a All MTT assays were repeated three times by using six samples per assay.
Shim-pack VPODS C18 column (reverse phase, 150 × 2.0 mm); 80% acetonitrile and 20% water over 6.0 min; flow rate of 0.4 mL/min; UV photodiode array detection from 200 to 300 nm. The reaction was irradiated at the required ceiling temperature (the reaction temperature was monitored by an external surface sensor using the Biotage Initiator reactor) using maximum power for the stipulated time. Then it was cooled to 50 °C with gas jet cooling. The products were purified by Biotage Isolera ™ Spektra Systems and Hexane/EtOAc solvent systems. All reagents and solvents were obtained from commercial sources and used without further purification.