A Facile Ugi/Ullmann Cascade Reaction to Access Fused Indazolo-Quinoxaline Derivatives with Potent Anticancer Activity

A facile methodology for the construction of a complex heterocycle indazolo-fused quinoxalinone has been developed via an Ugi four-component reaction (U-4CR) followed by an intramolecular Ullmann reaction. The expeditious process features an operationally simple approach, time efficiency, and a broad substrate scope. Biological activity was evaluated and demonstrated that compound 6e inhibits human colon cancer cell HCT116 proliferation with an IC50 of 2.1 μM, suggesting potential applications for developing a drug lead in medicinal chemistry.

As an evergreen in organic chemistry, multicomponent reactions (MCRs) never became old-fashioned or tedious [29][30][31][32][33], because they always inspire creative spirits by following the fundamental quest: more than two compounds are reacted in a one-pot fashion to form two or more bonds.The post-Ugi reactions [34][35][36][37], as typical MCRs, are well suited for the construction of important heterocycles, macrocycles, polymers, and other compounds in drug discovery and natural product synthesis [38][39][40][41][42][43].In the course of our continued study on the construction of novel N-fused heterocyclic chemical spaces and activity evaluation from Ugi adductive [28,36,[44][45][46], we hoped to establish a highly efficient diversity-oriented synthetic route to indazole-fused polycyclics utilizing a sequential one-pot Ugi/Ullmann reaction, as shown in Scheme 1d.We reasoned that the Ugi product involving 3-carboxyindazole and 2-bromoaniline would undergo an intramolecular Ullmann cyclization cascade process to yield the indazolo-quinoxaline derivatives.As excepted, indazolo-quinoxaline derivatives were synthesized by a facile and efficient process and are discussed below.As an evergreen in organic chemistry, multicomponent reactions (MCRs) never became old-fashioned or tedious [29][30][31][32][33], because they always inspire creative spirits by following the fundamental quest: more than two compounds are reacted in a one-pot fashion to form two or more bonds.The post-Ugi reactions [34][35][36][37], as typical MCRs, are well suited for the construction of important heterocycles, macrocycles, polymers, and other compounds in drug discovery and natural product synthesis [38][39][40][41][42][43].In the course of our continued study on the construction of novel N-fused heterocyclic chemical spaces and activity evaluation from Ugi adductive [28,36,[44][45][46], we hoped to establish a highly efficient diversity-oriented synthetic route to indazole-fused polycyclics utilizing a sequential one-pot Ugi/Ullmann reaction, as shown in Scheme 1d.We reasoned that the Ugi product involving 3-carboxyindazole and 2-bromoaniline would undergo an intramolecular Ullmann cyclization cascade process to yield the indazolo-quinoxaline derivatives.As excepted, indazolo-quinoxaline derivatives were synthesized by a facile and efficient process and are discussed below.

Results and Discussion
The starting materials, 3-carboxyindazole 1a, 4-chlorobenzaldehyde 2a, 2-bromoaniline 3, and tert-butyl isocyanide 4a, were mixed in methanol at room temperature and under air to afford the desired Ugi product 5a.With compound 5a in hand, we proceeded to optimize the conditions for the subsequent Ullmann reaction, and the results are shown in Table 1.Compound 5a was treated in N,N-dimethylformamide (DMF) with different bases under a variety of microwave irradiation conditions.When an inorganic base was used, the target compound 6a was observed in a relatively low yield (55%) (Table 1, entry 1-5).The results of these screening experiments revealed that when organic bases 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU), 4-dimethylaminopyridine (DMAP), and triethylamine) (TEA) were used, target compound 6a was observed in the LC/MS spectrum with very low yield (Table 1, entries 6-8).Addition of a copper catalyst and ligand resulted in a considerable increase in the yield (Table 1, entries 9-17).Different ligands were screened by performing the reaction with K 2 CO 3 and CuI (Table 1, entries 9-14), and the results showed that when tetramethylethylenediamine (TMEDA) was used as the ligand, compound 6 was obtained with a high yield (95%).When we carried out the reaction using another copper catalyst, such as CuBr, Cu 2 O, and Cu(OTf) 2 , in the presence of TMEDA, we only achieved yields of 49-65% (entries 15-17).The results proved that the optimal conditions for the Ullmann reaction were K 2 CO 3 as the base in the presence of CuI and TMEDA in DMF under microwave at 150 • C for 30 min.After the Ullmann reaction conditions were optimized, we continued to investigate the possibility of carrying out this reaction as a one-pot procedure.

Results and Discussion
The starting materials, 3-carboxyindazole 1a, 4-chlorobenzaldehyde 2a, 2-bromoaniline 3, and tert-butyl isocyanide 4a, were mixed in methanol at room temperature and under air to afford the desired Ugi product 5a.With compound 5a in hand, we proceeded to optimize the conditions for the subsequent Ullmann reaction, and the results are shown in Table 1.Compound 5a was treated in N,N-dimethylformamide (DMF) with different bases under a variety of microwave irradiation conditions.When an inorganic base was used, the target compound 6a was observed in a relatively low yield (55%) (Table 1, entry 1-5).The results of these screening experiments revealed that when organic bases 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 4-dimethylaminopyridine (DMAP), and triethylamine) (TEA) were used, target compound 6a was observed in the LC/MS spectrum with very low yield (Table 1, entries 6-8).Addition of a copper catalyst and ligand resulted in a considerable increase in the yield (Table 1, entries 9-17).Different ligands were screened by performing the reaction with K2CO3 and CuI (Table 1, entries 9-14), and the results showed that when tetramethylethylenediamine (TMEDA) was used as the ligand, compound 6 was obtained with a high yield (95%).When we carried out the reaction using another copper catalyst, such as CuBr, Cu2O, and Cu(OTf)2, in the presence of TMEDA, we only achieved yields of 49-65% (entries 15-17).The results proved that the optimal conditions for the Ullmann reaction were K2CO3 as the base in the presence of CuI and TMEDA in DMF under microwave at 150 °C for 30 min.After the Ullmann reaction conditions were optimized, we continued to investigate the possibility of carrying out this reaction as a one-pot procedure.With the optimized intramolecular Ullmann cyclization reaction conditions in hand, we investigated the cyclization reaction with the unpurified Ugi product 5a.In all cases, the initial Ugi products were used directly in the next step without further purification after the removal of the solvent.In one pot, the cascade reaction produced the final product 6a with a 73% yield for two steps, which showed no discernible impact on the overall yield.We then investigated the scope of this one-pot procedure by varying the starting materials (in Scheme 2).First, different aldehydes were tested.The results showed that different substituents on the aryl aldehydes exhibited good performance, affording compounds 6a-6e with 62-78% yield, and the electronic properties of the substituent did not impact the reactivity.Strikingly, when heterocyclic aldehydes and aliphatic aldehydes were used as input in the Ugi reaction, compounds 6f and 6g were also obtained, with 53% and 62% yields, respectively.Next, different isocyanides were used as Ugi inputs, and the results showed that the corresponding indazolo-quinoxalines 6i-6h were generated smoothly with a 56-76% yield.The substituents of the benzene of aniline were also investigated for obtaining compounds 6m-6q.Further investigations of the substituents on carboxyindazole revealed that different substituents didn't make a significant difference in the reaction yield.The Cl-or Me-substituted carboxyindazoles were involved in the protocol, and the products 6r-6u were obtained in 76-85% yields.It is worth noting that when formaldehyde was used, the product 6v was obtained with a 77% yield.All the results showed that a variety of different starting materials were successfully employed under the optimized conditions for the construction of structurally diverse indazolo-quinoxalines 6a-6v, with yields in the range of 53-78% (Scheme 2), indicating a good functional group tolerance.To extend the application of the synthesized compound in medical chemistry for developing a drug lead from compounds 6, the antiproliferative effect of compounds 6 was evaluated in several human cancer cell lines.To our delight, a significant cell growth inhibitory effect was observed, especially compound 6e, which exhibited better anticancer activities in the human colorectal cancer HCT116 cell lines than other cell lines (Figure 2).It is worth noting that compounds 6e, 6h, and 6i showed excellent anticancer activities, with IC50 of 2.1 μM, 2.7 μM, and 2.8 μM, respectively (Figure 3), which is comparable to 36.80 for etoposide, as reported in our previous work [44].Further efforts Scheme 2. Scope of the Ugi/Ullmann reaction route leading to indazolo-quinoxaline derivatives 6a-6v a .a Reaction conditions: 1 (1.0 mmol), 2 (1.0 mmol), 3 (1.0 mmol), and 4 (1.0 mmol) in MeOH (2.0 mL) under air.After the solvent removed, added K 2 CO 3 (2.0 mmol), CuI (0.1 mmol), and TMEDA (0.2 mmol) in DMF (5.0 mL) under microwave irradiation at 150 • C for 30 min.Overall yield of isolated product.
To extend the application of the synthesized compound in medical chemistry for developing a drug lead from compounds 6, the antiproliferative effect of compounds 6 was evaluated in several human cancer cell lines.To our delight, a significant cell growth inhibitory effect was observed, especially compound 6e, which exhibited better anticancer activities in the human colorectal cancer HCT116 cell lines than other cell lines (Figure 2).It is worth noting that compounds 6e, 6h, and 6i showed excellent anticancer activities, with IC 50 of 2.1 µM, 2.7 µM, and 2.8 µM, respectively (Figure 3), which is comparable to 36.80 for etoposide, as reported in our previous work [44].Further efforts are ongoing to explore the mechanism of action of drug leads and optimize their potency and drug properties.

General Information
1 H and 13 C NMR data were recorded on a Bruker (Billerica, MA, USA) 400 spectrometer. 1H 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. 13C NMR data are reported as follows: chemical shift in ppm (δ).HPLC-MS analyses were

General Information
1 H and 13 C NMR data were recorded on a Bruker (Billerica, MA, USA) 400 spectrometer. 1H 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. 13C NMR data are reported as follows: chemical shift in ppm (δ).HPLC-MS analyses were performed on a Shimadzu-2020 LC-MS instrument (Kyoto City, Japan) using the following conditions: 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 products were purified using Biotage (Uppsala, Sweden) Isolera™ Spektra Systems and hexane/EtOAc solvent systems.Unless otherwise specified, all chemicals were purchased from commercial sources and used without further treatment.All microwave irradiation experiments were carried out according to our previous report [44,45].

Cell Viability Assay
The cell viability and the IC 50 value of compounds 6a-v in the human tumor cells were evaluated using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, Beyotime, ST316, Shanghai, China) assay.Briefly, the tumor cells were harvested, counted, and seeded into the 96-well plate, with a density of 2 × 10 3 cells per well.After incubation for 24 h, 100 µL of medium with 20 µM compounds were added and incubated for 2 days for the initial screening.To assess anticancer activity, 20 µL of MTT solution (5 mg/mL in PBS) were added to each well and incubated for another 4 h.Then, the medium was removed, and 200 µL of DMSO was added to each well to the formazan cystals.The absorbance was measured at 570 nm using a microplate reader (Bio-Tek, Winooski, VT, USA).The experiments were repeated three times, and growth inhibition curves and IC 50 values were generated using GraphPad Prism 8.0.

General Procedure for Synthesis of Compounds 6a-v
Aldehyde (1.0 mmol, 1.0 eq.), MeOH (2 mL), and amine (1.0 mmol, 1.0 eq.) were sequentially added to a 10 mL microwave vial and stirred for 10 min.under air.Then, carboxylic acid (1.0 mmol, 1.0 eq.) and isocyanide (1.0 mmol, 1.0 eq.) were added separately.The mixture was stirred for 6-12 h at room temperature and monitored with TLC.When no isocyanide was present, the solvent was removed using rotary evaporation.The residue was dissolved in DMF (5 mL) in a 10 mL microwave vial, then, K 2 CO 3 (2.0 eq.), CuI (20 mol%), and TMEDA (20 mol%) were added.The vial was sealed and heated in a microwave at 150 • C for 30 min.The solvent was removed using rotary evaporation after the reaction was completed.The residue was dissolved in EtOAc (15 mL) and washed with brine.The EtOAc phase was dried over anhydrous Na 2 SO 4 and concentrated.The residue was purified using silica gel column chromatography using a gradient of EtOAc/hexane (0-100%) to afford the relative product 6.

Conclusions
In summary, we developed a facile and efficient methodology for the construction of an indazole-fused polyheterocycle using a unique Ugi/Ullmann cascade reaction strategy.With this protocol, complex functionalized indazolo-quinoxaline derivatives were obtained in good to excellent yields in one pot, with excellent functional group tolerance.Anticancer activity evaluation revealed that the synthesized new compounds show potential

Molecules 2024 ,
29, x FOR PEER REVIEW 6 of 13are ongoing to explore the mechanism of action of drug leads and optimize their potency and drug properties.

Figure 2 .
Figure 2. Anticancer activities of compounds 6a-v against different cancer cell lines.All MTT assays were repeated three times using six samples per assay.Proliferation inhibition activity of compounds 6a-v was assessed by MTT in breast cancer cells (MDA-MB-453), liver cancer cells (Hep3B), and colon cells (HCT116).These cells were treated with compounds 6 at 10 μM for 48 h, respectively, then cell viability was measured.

Figure 2 .
Figure 2. Anticancer activities of compounds 6a-v against different cancer cell lines.All MTT assays were repeated three times using six samples per assay.Proliferation inhibition activity of compounds 6a-v was assessed by MTT in breast cancer cells (MDA-MB-453), liver cancer cells (Hep3B), and colon cells (HCT116).These cells were treated with compounds 6 at 10 µM for 48 h, respectively, then cell viability was measured.

Figure 2 .
Figure 2. Anticancer activities of compounds 6a-v against different cancer cell lines.All MTT assays were repeated three times using six samples per assay.Proliferation inhibition activity of compounds 6a-v was assessed by MTT in breast cancer cells (MDA-MB-453), liver cancer cells (Hep3B), and colon cells (HCT116).These cells were treated with compounds 6 at 10 μM for 48 h, respectively, then cell viability was measured.

Table 1 .
Optimization of the Ullmann reaction conditions from 5a a .

Table 1 .
Optimization of the Ullmann reaction conditions from 5a a .