At the outset of our studies, we were interested to investigate whether the PhICl₂/NH₄SCN reagent system could be applied to the intramolecular cyclization as well as oxythio/selenocyanation of o-alkynylbenzoates, in hope of achieving the biologically interesting C4-thio/selenocyanated isocoumarins. Our study on condition optimization was commenced with the simplest o-alkynylbenzoate 1a. To our delight, the desired C4-thiocyanated isocoumarin 2a could be obtained in 86% yield from the reaction of 1a with 2 equivalents of PhICl₂ and 2 equivalents of NH₄SCN in DCM for 12 h at room temperature (Table 1, entry 1). The solvents screening showed DCE to be superior to other commonly used solvents, including DCM, MeOH, EtOAc, toluene, and MeCN (Table 1, entries 2–6). Neither increasing nor decreasing the dosage of PhICl₂/NH₄SCN were beneficial for improving the yield of the product (Table 1, entries 7–8). The other oxidants including PIDA, PIFA, PhIO, I₂, and NBS were also applied to take the place of PhICl₂, however, lower yield, no reaction or none desired product were observed in these cases (Table 1, entries 9–13). The other SCN-containing inorganic salts including NaSCN, KSCN, AgSCN and CuSCN have also been investigated, but none of them provided better outcome than NH₄SCN (SI, Supplementary Table S1, entries 14–17). We have also found that the reaction temperature had an obvious influence on the outcome of the reaction. Performing the reaction of 1a (0.20 mmol), PhICl₂ (0.4 mmol) and NH₄SCN (0.4 mmol) in DCE at 50°C not only improved the reaction yield to 96%, but also shorten the reaction time to 2 h (Table 1, entry 14). However, when the reaction temperature was further elevated to 60°C, it was found that product 2a was obtained in relatively lower yield, with the starting substrate 1a recovered in a yield of 5% (SI, Supplementary Table S1, entry 19). Furthermore, the reaction does not need a N₂ atmosphere (Table 1, entry 14 vs 15). Based on the outcomes of the above screening experiments, the best yield of product 2a (96%) could be obtained by subjecting NH₄SCN (2 equiv), PhICl₂ (2 equiv) to DCE at 50°C for 2 h (Table 1, entry 14).

With the optimized conditions in hand, we came to investigate the substrate scope of this reaction by subjecting various o-alkynylbenzoates to the standard condition (Table 2). First, the electronic effect of R¹ substituent on the phenyl ring of o-alkynylbenzoates was explored. Substrates with either electron-donating or -withdrawing groups on the phenyl ring were found to well participate the cyclization/thiocyanation reaction and the desired C4 thiocyanated ioscoumarins 2b-f were obtained in good to excellent yields. Moreover, it is worth noting that substrates bearing electron-donating groups (1a-c) exhibited better performance than those bearing electron-withdrawing substituents (1d-f). For instances, when R¹ is a strong electron-withdrawing -NO₂ group, the reaction smoothly afforded the corresponding isocoumarin 2d in 56% yield. Replacement of the -NO₂ group with -Cl or -F groups provided better results, with corresponding products 2e-f obtained in 87 and 85%, respectively. The electronic effect of substituents on the alkyne motif was carefully tested next. To our satisfaction, this transformation was applicable to alkynoates bearing diverse substituents ranging from phenyl, naphthyl, thienyl to alkyl, with the corresponding C4 thiocyanated products 2g-t accomplished in good to excellent yields. Most strikingly, TMS functionality in the substrate was also well tolerated under the reaction conditions and the target product 2r was obtained in 80% yield. The method was also applicable to terminal alkyne, which could afford the corresponding C3-unsubstituted isocoumarin 2s in 84% yield.

Encouraged by the feasibility of oxythiocyanation of o-alkynylbenzoates, we further explored the synthesis of C4 selenocyanated isocoumarins, a closely related analogue of thiocyanated isocoumarins. Since the NH₄SeCN was not commercially available, we had to resort to other selenocyanate sources (SI, Supplementary Table S2). To our delight, when KSeCN was applied, the reaction worked equally well as the oxythiocyanation and the corresponding selenocyanated product 3a was obtained in 92% yield. Under the adjusted conditions, a series of o-alkynylbenzoates were converted to the corresponding selenocyanated analogs 3b-m in high to excellent yields, regardless of the electronic effect of substituents on the phenyl ring. Notably, functional groups such as -F, -Cl, -NO₂, and -CO₂Me remained intact during the transformation, thereby facilitating late-stage functionalization of the obtained products. Furthermore, heterocycle-fused substrate 1n also reacted smoothly under the newly optimized reaction conditions, affording the corresponding product 3n in good yield. Finally, good to excellent yields were observed for substrates with R² being substituents ranging from aliphatic, hetero/aromatic ring to TMS functionality (3o-t). The structure of 2h (CCDC: 2126012) and 3h (CCDC: 2126018) were unambiguously confirmed through X-ray crystallographic analysis, for details, see supporting information.

The isocoumarin skeleton commonly exists in many naturally-occurring compounds and potent pharmaceutical agents displaying anti-tumor, antifungal, and anti-inflammatory properties (Pal and Pal, 2019). For instance, Xyridin A (Ruangrungsi et al., 1995) was isolated in 1995 from Xyris indica, and was found to possess antibacterial activity against various bacteria (Saeed, 2003). To further demonstrate the synthetic utility of our method, a gram-scale reaction was performed with substrate 1u under standard conditions and the corresponding C4 thio/selenocyanated Xyridin A derivatives 2u and 3u were obtained in 87 and 90%, respectively (Scheme 2). Ultimately, compounds 2u and 3u could be readily transformed into SCF₃- (Liang et al., 2015) and SeCF₃-containing Xyridin A derivatives 2v and 3v by treatment with TMSCF₃ and Cs₂CO₃ in acetonitrile. Moreover, [3 + 2] cycloaddition of compounds 2u/3u with sodium azide was also performed to afford Xyridin A bearing thiotetrazole moiety 2w/3w in 94 and 96% yields (Liang et al., 2015), respectively.

We have previously postulated that the reaction of PhICl₂ with NH₄SCN could generate PhI(SCN)₂ as a reactive hypervalent iodine (III) species via a ligand-exchange process (Scheme 3). In order to further corroborate whether PhI(SCN)₂ species was indeed formed, the reaction of TolICl₂ with NH₄SCN in CDCl₃ was carried out and ¹H NMR analysis was implemented. The outcome revealed that only the peak of 2.46 ppm (s), which can be attributed to the methyl group of TolICl₂, and the peak of 2.29 ppm(s), which can be attributed to the methyl group of p-iodotoluene, were observed throughout the whole reaction process (see SI for details). No signal from PhI(SCN)₂ was detected, though. In order to further understand the most appropriate pathway adopted by the reaction between PhICl₂ with NH₄SCN, a computational study was carried out and the result is shown in Scheme 3. The reaction pathway b involving formation of PhI(SCN)₂ obviously requires more energy than other pathways. Even though pathway a is also theoretically possible, the ¹H NMR experiment result did not support the formation of PhICl(SCN). It is not the reaction pathway with the lowest energy, either. Pathway c with the formation of ClSCN is not only consistent with the ¹³C NMR experiment (Tao et al., 2021), but also preferred thermodynamically (see SI for details).

Based on these newly gained experimental (control experiments see ESI, Supplementary Scheme S1, SupplementaryFigure S1, S2 and computational calculation) and computational results as well as previous literature reports (Kita et al., 1997; Woon et al., 2006; Ito et al., 2019; Xing et al., 2019a; An et al., 2020), a possible mechanistic pathway for the formation of the C4 thiocyanated isocumarins was proposed (Scheme 4). Differing from the previous mechanism (Kita et al., 1997), we tentatively proposed that intermediate A, an ionic form of PhICl₂, reacted with thiocyanate directly to give the reactive thiocyanogen chloride, which could be supported by the observation of peak of 109.1 ppm in its ¹³C NMR analysis (Tao et al., 2021). Then the reaction of thiocyanate with thiocyanogen chloride provides (SCN)₂, which further reacts with the oxidative PhICl₂ to give thiocyanogen chloride (Tao et al., 2021). Next, electrophilic addition between the reactive thiocyanogen chloride with substrate 1a gave rise to intermediate B (Xing et al., 2019a; An et al., 2020; Hellwig et al., 2020; Lynch and Scanlan, 2020; Sun et al., 2020; Jurinic et al., 2021; Slivka and Onysko, 2021). Due to the presence of the adjacent electron-withdrawing methoxycarbonyl group which makes the C (sp²) connecting with the Ph substituent more electron-deficient, a favored intramolecular 6-exo cyclization occurred in intermediate B, leading to formation of the cyclic intermediate C. Finally, removal of the methyl group by the nucleophilic attack of chloride ion gave the title product 2a (Scheme 4).

Finally, those synthesized 4-thio/selenocyanated isocoumarins were screened in vitro for antitumor activity test using CCK-8 assay against HCT 116 and MCF 7 cell lines (for details, see the ESI, Supplementary Figure S5). The results in Figure 2 indicated that these compounds showed moderate activity against HCT 116 after 48 h exposure. Compound 2l was found to be the most potent candidate of the series, which exerted 69% inhibition at 10 μM concentration against HCT 116. Moreover, the screening results revealed that those compounds were active against MCF 7 cell line. Especially, compound 2f, 3h, and 3r seem to be equally potent with 84, 82, and 81% antiproliferative inhibition at 10 μM concentration against MCF 7 cell line, respectively.

To an oven-dried 25 ml round-bottom flask were added NH₄SCN or KSeCN (0.4 mmol), PhICl₂ (0.4 mmol) and DCE (5 ml). The mixture was stirred at rt for 30 min. Then, substrate 1 (0.2 mmol) in DCE (5 ml) was added to the reaction mixture in one portion. The reaction was heated to 50°C in an aluminum heating block and stirred for another 2 h, poured into the saturated brine solution (20 ml). The product was extracted with DCM (20 ml), dried with Na₂SO₄ and concentrated. The crude product was purified using silica gel column chromatography.Table 2