Functionalized 10-Membered Aza- and Oxaenediynes through the Nicholas Reaction

The scope and limitations of the Nicholas-type cyclization for the synthesis of 10-membered benzothiophene-fused heterocyclic enediynes with different functionalities were investigated. Although the Nicholas cyclization through oxygen could be carried out in the presence of an ester group, the final oxaenediyne was unstable under storage. Among the N-type Nicholas reactions, cyclization via an arenesulfonamide functional group followed by mild Co-deprotection was found to be the most promising, yielding 10-membered azaendiynes in high overall yields. By contrast, the Nicholas cyclization through the acylated nitrogen atom did not give the desired 10-membered cycle. It resulted in the formation of a pyrroline ring, whereas cyclization via an alkylated amino group resulted in a poor yield of the target 10-membered enediyne. The acylated 4-aminobenzenesulfonamide nucleophilic group was found to be the most convenient for the synthesis of functionalized 10-membered enediynes bearing a clickable function, such as a terminal triple bond. All the synthesized cyclic enediynes exhibited moderate activity against lung carcinoma NCI-H460 cells and had a minimal effect on lung epithelial-like WI-26 VA4 cells and are therefore promising compounds in the search for novel antitumor agents that can be converted into conjugates with tumor-targeting ligands.


Synthesis of Oxaenediyne I
A four-step synthesis of the ester-functionalized O-enediyne I was started from 3iodobenzothiophene 1. Desilylation of the functionalized alkyne 2 and the Sonogashira coupling were carried out in one pot using KF/MeOH/DMF as the desilylation source (Scheme 1) [56]. Further complexation of the enediyne 3 with Co2(CO)8 proceeded regioselectively with the formation of the cobalt complex 4 at the C2-triple bond. The higher selectivity for the C2-triple bond compared with the nonfunctionalized enediyne can be explained by the higher steric hindrance of the triple bond at the C3 position [46]. The Nicholas reaction of the ester-functionalized complex 4 proceeded under optimized conditions (1.5 equiv. of FB3·Et2O) to afford the cyclic product 5 in good yield. We recently showed that using tetrabutylammonium fluoride (TBAF) hydrate in an aqueous acetone solution increases the yield of cobalt-free 10-membered enediynes in the decomplexation step [43]. Therefore, we applied these conditions to the ester-functionalized Cocomplex 5, as well as to the previously reported Co-complex of the nonfunctionalized Oenediyne 6, which allowed us to obtain the enediyne I and noticeably increase the yield of the enediyne 7 at the decomplexation step compared with previous results [46]. However, the target ester-containing enediyne I was significantly less stable than its unsubstituted analog 7 and gave traces of the Bergman cyclization product in experiments with NMR detection. Then, the Bergmann cyclization of the enediynes I and 7 was carried out in i-PrOH at 45-50 °C, and both cyclization products 8 and 9 were isolated in high yields. Synthesis of all the target structures is based on a combination of electrophile-promoted cyclization of the starting diacetylene and the subsequent Sonogashira coupling to construct unsymmetrically substituted acyclic enediynes with the required functionalities at both triple bonds followed by the regioselective formation of Co 2 (CO) 6 -complexes for the further Nicholas cyclization [42,46,55] (Figure 3). The key intermediate compound for all the structures was 3-iodo-2-(3-methoxyprop-1-yn-1-yl)benzo[b]thiophene (1), which is available synthetically at the multigram scale as has been previously reported [46].

Synthesis of Oxaenediyne I
A four-step synthesis of the ester-functionalized O-enediyne I was started from 3iodobenzothiophene 1. Desilylation of the functionalized alkyne 2 and the Sonogashira coupling were carried out in one pot using KF/MeOH/DMF as the desilylation source (Scheme 1) [56]. Further complexation of the enediyne 3 with Co 2 (CO) 8 proceeded regioselectively with the formation of the cobalt complex 4 at the C2-triple bond. The higher selectivity for the C2-triple bond compared with the nonfunctionalized enediyne can be explained by the higher steric hindrance of the triple bond at the C3 position [46]. The Nicholas reaction of the ester-functionalized complex 4 proceeded under optimized conditions (1.5 equiv. of FB 3 ·Et 2 O) to afford the cyclic product 5 in good yield. We recently showed that using tetrabutylammonium fluoride (TBAF) hydrate in an aqueous acetone solution increases the yield of cobalt-free 10-membered enediynes in the decomplexation step [43]. Therefore, we applied these conditions to the ester-functionalized Co-complex 5, as well as to the previously reported Co-complex of the nonfunctionalized O-enediyne 6, which allowed us to obtain the enediyne I and noticeably increase the yield of the enediyne 7 at the decomplexation step compared with previous results [46]. However, the target ester-containing enediyne I was significantly less stable than its unsubstituted analog 7 and gave traces of the Bergman cyclization product in experiments with NMR detection. Then, the Bergmann cyclization of the enediynes I and 7 was carried out in i-PrOH at 45-50 • C, and both cyclization products 8 and 9 were isolated in high yields.

Synthesis of Azaenediynes II-V
We recently showed that the amino functional group protected by the tosyl group (NH-Ts) is very efficient for the synthesis of azaenediyne systems through the Nicholas cyclization, and there was an optimal balance between the stability and DNA-damaging activity of the resulting N-Ts-enediyne [42]. Therefore, we decided to use an arenesulfonamide fragment to introduce functional groups into the N-enediyne molecule. Two types of functionalized arenesulfonamide moieties were used: 2-nosyl as an easily removable protecting group and a sulfanilamide moiety with an NH2 group acylated with hex-5ynoic acid.
The N-Ns (2-nosyl, 2-nitrobenzenesulfonyl) enediyne III was synthesized similarly to the N-Ts enediyne [42] (Scheme 2). Thus, the starting Co-complex 12a for the Nicholas reaction was obtained without any difficulties and in high yield. However, the Nicholas reaction of the NH-Ns group formed the product 13a in a considerably lower yield (46%) compared with the NH-Ts function (76%) and required a higher quantity of a Lewis acid (8 equiv. instead of 1.5 equiv.) [42], which can be explained both by the steric hindrance and lower nucleophilicity of NH-Ns.

Synthesis of Azaenediynes II-V
We recently showed that the amino functional group protected by the tosyl group (NH-Ts) is very efficient for the synthesis of azaenediyne systems through the Nicholas cyclization, and there was an optimal balance between the stability and DNA-damaging activity of the resulting N-Ts-enediyne [42]. Therefore, we decided to use an arenesulfonamide fragment to introduce functional groups into the N-enediyne molecule. Two types of functionalized arenesulfonamide moieties were used: 2-nosyl as an easily removable protecting group and a sulfanilamide moiety with an NH 2 group acylated with hex-5-ynoic acid.

Synthesis of Azaenediynes II-V
We recently showed that the amino functional group protected by the tosyl group (NH-Ts) is very efficient for the synthesis of azaenediyne systems through the Nicholas cyclization, and there was an optimal balance between the stability and DNA-damaging activity of the resulting N-Ts-enediyne [42]. Therefore, we decided to use an arenesulfonamide fragment to introduce functional groups into the N-enediyne molecule. Two types of functionalized arenesulfonamide moieties were used: 2-nosyl as an easily removable protecting group and a sulfanilamide moiety with an NH2 group acylated with hex-5ynoic acid.
The N-Ns (2-nosyl, 2-nitrobenzenesulfonyl) enediyne III was synthesized similarly to the N-Ts enediyne [42] (Scheme 2). Thus, the starting Co-complex 12a for the Nicholas reaction was obtained without any difficulties and in high yield. However, the Nicholas reaction of the NH-Ns group formed the product 13a in a considerably lower yield (46%) compared with the NH-Ts function (76%) and required a higher quantity of a Lewis acid (8 equiv. instead of 1.5 equiv.) [42], which can be explained both by the steric hindrance and lower nucleophilicity of NH-Ns. Thus, the starting Co-complex 12a for the Nicholas reaction was obtained without any difficulties and in high yield. However, the Nicholas reaction of the NH-Ns group formed the product 13a in a considerably lower yield (46%) compared with the NH-Ts function (76%) and required a higher quantity of a Lewis acid (8 equiv. instead of 1.5 equiv.) [42], which can be explained both by the steric hindrance and lower nucleophilicity of NH-Ns.
To synthesize the N-hex-5-ynoyl enediyne III, the obtained p-NO 2 Ph-substituted enediyne 11b was reduced by Zn in the AcOH/DCM system to the NH 2 -derivative 11c, which was then converted to the Co-complex 12b. Then, the Co-complex 12b was acylated with hex-5-ynoyl chloride to produce the nontrivial triacetylenic compound 12c, in which only one triple bond out of three was converted to the Co complex. The Nicholas cyclization of the N-protected/functionalized Co-complex 12c proceeded smoothly to give the desired cyclic Co-complex 13b in good yield (64%). It should be emphasized that the Nicholas cyclization of the Co-complex with the free NH 2 group 12b did not proceed at all. Therefore, protection, along with functionalization at the stage of an acyclic Co-complex, is a necessary synthetic step for producing a 10-membered enediyne with a terminal triple bond. Finally, we investigated the last decomplexation step, which proceeded in good to high yields to give the N-enediynes II and III, which were stable under isolation and storage. We also tested decomplexation in aqueous acetone for the Co-complex of the N-Ts-enediyne 13c, which has been previously reported [42]. In this case, we succeeded in increasing the yield of 14 at the decomplexation step from 45% (in anhydrous acetone) [42] to 88% (in aqueous acetone).
It is known that the Nicholas-type cyclization can proceed using amide functional groups [57] and even through secondary amino groups in the presence of DIPEA [58]. Therefore, we decided to test these functional groups, which could also be useful for the functionalization of enediynes. Therefore, cyclization using NHBn and NHBz groups was also studied (Scheme 3). To synthesize the N-hex-5-ynoyl enediyne III, the obtained p-NO2Ph-substituted enediyne 11b was reduced by Zn in the AcOH/DCM system to the NH2-derivative 11c, which was then converted to the Co-complex 12b. Then, the Co-complex 12b was acylated with hex-5-ynoyl chloride to produce the nontrivial triacetylenic compound 12c, in which only one triple bond out of three was converted to the Co complex. The Nicholas cyclization of the N-protected/functionalized Co-complex 12c proceeded smoothly to give the desired cyclic Co-complex 13b in good yield (64%). It should be emphasized that the Nicholas cyclization of the Co-complex with the free NH2 group 12b did not proceed at all. Therefore, protection, along with functionalization at the stage of an acyclic Co-complex, is a necessary synthetic step for producing a 10-membered enediyne with a terminal triple bond. Finally, we investigated the last decomplexation step, which proceeded in good to high yields to give the N-enediynes II and III, which were stable under isolation and storage. We also tested decomplexation in aqueous acetone for the Co-complex of the N-Tsenediyne 13c, which has been previously reported [42]. In this case, we succeeded in increasing the yield of 14 at the decomplexation step from 45% (in anhydrous acetone) [42] to 88% (in aqueous acetone).
It is known that the Nicholas-type cyclization can proceed using amide functional groups [57] and even through secondary amino groups in the presence of DIPEA [58]. Therefore, we decided to test these functional groups, which could also be useful for the functionalization of enediynes. Therefore, cyclization using NHBn and NHBz groups was also studied (Scheme 3). The corresponding starting materials, the Co-complexes 17a and 17b, were synthesized without any difficulties starting from iodobenzothiophene 1 and the corresponding functionalized terminal alkynes 15a and 15b in two steps (Scheme 3); however, cyclization failed in both cases. Thus, the conditions tested for the Nicholas cyclization of the NHBn Co-complex 17a (BF3·Et2O; HBF4·Et2O and HBF4·Et2O/DIPEA) gave the desired cyclic compound 18 in low yields due to the complexation of the NHBn group with the acid. Even The corresponding starting materials, the Co-complexes 17a and 17b, were synthesized without any difficulties starting from iodobenzothiophene 1 and the corresponding functionalized terminal alkynes 15a and 15b in two steps (Scheme 3); however, cyclization failed in both cases. Thus, the conditions tested for the Nicholas cyclization of the NHBn Co-complex 17a (BF 3 ·Et 2 O; HBF 4 ·Et 2 O and HBF 4 ·Et 2 O/DIPEA) gave the desired cyclic compound 18 in low yields due to the complexation of the NHBn group with the acid. Even generation of the carbocation with HBF 4 ·OEt 2 followed by deprotonation of the [NH 2 Bn] + group with DIPEA only gave a 12% yield of the cyclic enediyne 18. Therefore, the basicity of the secondary amino function should be considered a strong limitation of the Nicholas reaction in the case of enediyne systems.
Cyclization of the complex 17b using an NH-benzoyl moiety as a nucleophilic group did not give the desired 10-membered enediyne at all (Scheme 3). The main product of the reaction was the pyrroline derivative 19 due to the electrophile-promoted cyclization of the NHBz functional group at the free triple bond. This result can be explained by the higher steric hindrance of the planar NHBz group compared with that of the tetrahedral arenesulfonamide functional group. Thus, we have proven that the sulfonamide moiety remains the functional group of choice when using the aza-Nicholas reaction to synthesize 10-membered N-enediynes.

Biological Activity of Cyclic Enediynes
All the synthesized 10-membered enediynes (I-III, 7, and 14) were tested for their effect on the growth of NCI-H460 lung carcinoma and WI-26 VA4 lung epithelial-like cell lines using the MTT colorimetric test [59,60] with the cytotoxic drug etoposide as a positive control. All the enediynes at a concentration of 75 µM displayed moderate cytotoxicity toward cancer cells and had less effect on normal fibroblasts ( Figure 4). generation of the carbocation with HBF4·OEt2 followed by deprotonation of the [NH2Bn] + group with DIPEA only gave a 12% yield of the cyclic enediyne 18. Therefore, the basicity of the secondary amino function should be considered a strong limitation of the Nicholas reaction in the case of enediyne systems. Cyclization of the complex 17b using an NH-benzoyl moiety as a nucleophilic group did not give the desired 10-membered enediyne at all (Scheme 3). The main product of the reaction was the pyrroline derivative 19 due to the electrophile-promoted cyclization of the NHBz functional group at the free triple bond. This result can be explained by the higher steric hindrance of the planar NHBz group compared with that of the tetrahedral arenesulfonamide functional group. Thus, we have proven that the sulfonamide moiety remains the functional group of choice when using the aza-Nicholas reaction to synthesize 10-membered N-enediynes.

Biological Activity of Cyclic Enediynes
All the synthesized 10-membered enediynes (I-III, 7, and 14) were tested for their effect on the growth of NCI-H460 lung carcinoma and WI-26 VA4 lung epithelial-like cell lines using the MTT colorimetric test [59,60] with the cytotoxic drug etoposide as a positive control. All the enediynes at a concentration of 75 μM displayed moderate cytotoxicity toward cancer cells and had less effect on normal fibroblasts ( Figure 4). These data correspond with the previously estimated ability of benzothiophenefused enediynes to cleave plasmid DNA [41,42,46]. Therefore, the observed cytotoxic activity of enediynes is assumed to be associated with their DNA damaging effect. However, it is clear that for DNA to be affected and destroyed in cells, a molecule must have a sufficient hydrophilic-lipophilic balance and the ability to penetrate into cells and avoid various drug resistance mechanisms. Therefore, to improve the cytotoxic effect of enediynes, the functional design of benzothiophene-fused enediyne molecules should be elaborated. From this point of view, considering the absence of significant differences in cytotoxicity, N-enediynes are the most promising compounds for further development of antitumor agents. Thus, functionalized derivatives of N-enediynes are stable and synthetically accessible and can be used for further conjugation with ligands with an affinity for cancer cells.

Discussion
The scope and limitation of the Nicholas-type cyclization for the synthesis of various 10-membered azaenediynes, as well as oxa-analog were studied. We used two types of heteroatoms -xygen and nitrogen, to choose which type of heteroenediynes and which These data correspond with the previously estimated ability of benzothiophene-fused enediynes to cleave plasmid DNA [41,42,46]. Therefore, the observed cytotoxic activity of enediynes is assumed to be associated with their DNA damaging effect. However, it is clear that for DNA to be affected and destroyed in cells, a molecule must have a sufficient hydrophilic-lipophilic balance and the ability to penetrate into cells and avoid various drug resistance mechanisms. Therefore, to improve the cytotoxic effect of enediynes, the functional design of benzothiophene-fused enediyne molecules should be elaborated. From this point of view, considering the absence of significant differences in cytotoxicity, Nenediynes are the most promising compounds for further development of antitumor agents. Thus, functionalized derivatives of N-enediynes are stable and synthetically accessible and can be used for further conjugation with ligands with an affinity for cancer cells.

Discussion
The scope and limitation of the Nicholas-type cyclization for the synthesis of various 10-membered azaenediynes, as well as oxa-analog were studied. We used two types of heteroatoms-xygen and nitrogen, to choose which type of heteroenediynes and which type of nucleophilic functional groups are most suitable for the synthesis of heteroenediynes that have additional functionality for further modification with tumor-targeting ligands.
We showed that functionalized O-enediyne with an ester group attached to the enediyne core is synthetically accessible through the O-Nicholas reaction. However, further functionalization is limited because of the low stability of O-enediynes.
N-Nicholas cyclization through three types of N-containing nucleophilic groupsamino, amido, and arenesulfonamido, was studied. We proved that an arenesulfonamide fragment is the optimal functional group to realize a high-yielded synthesis of 10-membered azaenediynes. Moreover, this group can serve as a site for the introduction of additional functional groups for further modification of cyclic enediynes using click chemistry. For this purpose, the 4-aminobenezenesulfonamide moiety should be acylated with acid derivatives containing a functional group tolerant to the Nicholas cyclization conditions. We demonstrated that this strategy could be applied to the synthesis of azaenediyne with a free terminal triple bond in the arenesulfonamide linker part.
While amino and amido nucleophilic groups also offer great potential as linkers for attaching clickable groups to an enediyne core, neither the secondary amino group nor the amido functional group is suitable for the closure of the 10-membered azaendiyne, which is a limitation of the aza-Nicholas cyclization.
All the synthesized cyclic enediynes were tested as potential anticancer compounds and showed moderate activity against NCI-H460 lung carcinoma and had a minimal effect on WI-26 VA4 lung epithelial-like cells. Thus, the modification of azaenediynes through the 4-aminobenezenesulfonamide moiety can be used in the future to synthesize enediyne conjugates with higher antitumor efficacy.
Solvents were dried under standard conditions. Purification and drying of DCM were carried out in accordance with the literature procedure using CaH 2 [65]. The Sonogashira coupling, the synthesis of Co-complexes, the Nicholas cyclization, and the Bergman cyclization were carried out under argon in oven-dried glassware. Other reactions were carried out under air unless stated otherwise. Evaporation of solvents and concentration of reaction mixtures were performed under vacuum at 20 • C (for the enediynes I-III, 7, 14) and 35 • C (for other compounds) on a rotary evaporator. TLC was carried out on silica gel plates (Silica gel 60, UV 254) with detection by UV or staining with a basic aqueous solution of KMnO 4 . A normal-phase silica gel (Silica gel 60, 230−400 mesh) was used for preparative column chromatography. 1 H and 13 C{ 1 H} and DEPT NMR spectra were recorded at 400 (or 500) and 101 (or 125) MHz, respectively, at 25 • C in CDCl 3 , acetone-d 6, or CD 3 CN without an internal standard. The 1 H NMR data are reported as chemical shifts (δ), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), coupling constants (J, given in Hz), and number of protons. The 13 C{ 1 H} NMR data are reported as chemical shifts (δ). Chemical shifts for 1 H and 13 C are reported as δ values (ppm) and referenced to residual solvents (δ = 7.26 ppm for 1 H; δ = 77.16 ppm for 13 C for spectra recorded in CDCl 3 and δ = 2.05 ppm for 1 H; δ = 29.84 ppm for 13 C for spectra recorded in acetone-d 6 and δ = 1.94 ppm for 1 H; δ = 1.32 ppm for 13 C for spectra in CD 3 CN). For copies of NMR spectra of all new compounds see the Supporting Information. High-resolution mass spectra were determined for solutions of all compounds in MeOH using ESI in the mode of positive ion registration with a TOF mass analyzer. For copies of ESI HRMS spectra of key products I-III, 18, 19 see the Supporting Information. To an argon-flushed, cooled (0 • C) stirred solution of Co 2 (CO) 6 -complex of an acyclic enediyne (1.00 equiv.) in anhydrous DCM (c = 0.001 M) was added boron trifluoride diethyl etherate (1.50-8.00 equiv.). The resulting mixture was allowed to warm to room temperature and was stirred at room temperature until the reaction was complete (TLC). Then the reaction mixture was quenched with a saturated aqueous solution of NaHCO 3 . The organic layer was separated, washed with brine, dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure to yield a crude product, which was purified by column chromatography.

General Procedure (D) for the Synthesis of 10-Mebered Enediynes I-III, 7, 14 by the Deprotection of Acyclic Co 2 (CO) 6 -complexes from Cobalt
To a stirred solution of cyclic Co 2 (CO) 6 complex (1.00 equiv., c = 0.006 M) in a mixture of acetone/water (15:1, v/v), tetrabutylammonium fluoride (TBAF) hydrate (calculated for TBAF × H 2 O) or trihydrate (calculated for TBAF × 3H 2 O), was added in several portions until the starting Co-complex was consumed, as indicated by TLC. The total amount of TBAF hydrate or TBAF·trihydrate varied from 23.5 to 65.8 equiv. After completion of the reaction, the reaction mixture was filtered through a pad of Celite using fritted filter funnel, the sorbent was washed with acetone, and the resulting solution was concentrated under reduced pressure at 20 • C to~1/5 of the original volume; the resulting mixture was mixed with ethyl acetate and brine. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed three times with brine, dried over anhydrous Na 2 SO 4 , and the solvent was evaporated under reduced pressure at 20 • C. The crude product was purified by column chromatography.

Antiproliferative Assay
The effects of the synthesized compounds on cell viability were determined using the MTT colorimetric test. All examined cells were diluted with the growth medium to 3.5 × 104 cells per mL, and the aliquots (7 × 103 cells per 200 µL) were placed in individual wells in 96-multiplates (Eppendorf, Germany) and incubated for 24 h. The next day the cells were then treated with synthesized compounds separately at the final concentration of 75 µM and incubated for 72 h at 37 • C in a 5% CO 2 atmosphere. After incubation, the cells were then treated with 40 µL MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg mL −1 in PBS) and incubated for 4 h. After an additional 4 h incubation, the medium with MTT was removed, and DMSO (150 µL) was added to dissolve the crystals formazan. The plates were shaken for 10 min. The optical density of each well was determined at 560 nm using a microplate reader GloMax Multi+ (Promega, Madison, WI, USA). Each of the tested compounds was evaluated for cytotoxicity in three separate experiments.

Conclusions
The scope and limitation of the Nicholas-type cyclization for the synthesis of various 10-membered heteroenediynes fused to a benzothiophene ring were studied. We proved that an arenesulfonamide fragment is the optimal functional group to realize a high-yield synthesis of 10-membered enediynes. Moreover, this group can serve as a site for the introduction of additional functional groups for further modification of cyclic enediynes via click chemistry. For this purpose, the 4-(N-acylamino)benzenesulfonamide functional group can be used as a nucleophile for cyclization and functionalization. The crucial point is that neither the secondary amino group nor the amido functional group is suitable for the closure of the 10-membered azaendiyne, which is a limitation of the aza-Nicholas cyclization. Functionalized O-enediynes are also synthetically accessible through the O-Nicholas reaction. Still, the use of such functionalization is limited because of the low stability of O-enediynes compared with that of N-enediynes. All the synthesized cyclic enediynes were tested as potential anticancer compounds and showed moderate activity against NCI-H460 lung carcinoma and had a minimal effect on WI-26 VA4 lung epitheliallike cells, demonstrating that the synthesized enediynes can be further used to synthesize active molecules with antitumor activity based on enediyne conjugates. Azaenediynes modified through acylated 4-aminobenzenesulfonamide nucleophilic group should be considered the most suitable structures for ongoing studies. Data Availability Statement: Data supporting reported results (copies of 1 H, 13 C{ 1 H}, DEPT, 2D NMR spectra in PDF) can be found in a Supporting Information File or can be sent as original files upon request.