A Short Review of Research Progress on the Synthesis Approaches of Aza-Dibenzocyclooctyne Derivatives

Cyclooctyne molecules have found wide applications in the strain-promoted azide–alkyne cycloaddition (SPAAC) reactions, which avoid the biotoxicity caused by the use of Cu(I) catalysts. Among the various cyclooctyne systems, dibenzocyclooctyne (DBCO) series have displayed the highest reaction activity. However, the synthesis processes of such structures are time-consuming, which to some extent limit their large-scale development and application. This review has summarized current synthesis routes of two DBCO molecules, aza-dibenzocyclooctyne (DIBAC) and biarylazacyclooctynone (BARAC).


Introduction
Bioorthogonal chemistry has emerged as a rapidly expanding field over the past few decades, driven by the need to study and manipulate biomolecules in their natural environment. It refers to the chemical transformations that can occur within living systems without perturbing natural biological processes, enabling researchers to label, detect, and manipulate biomolecules with high specificity and efficiency [1][2][3]. This approach has revolutionized many areas of biology and medicine, from basic research to drug discovery and diagnostics [4][5][6][7][8].
One of the most widely used bioorthogonal reactions is the Cu(I)-catalyzed azidealkyne cycloaddition (CuAAC), which enables efficient and selective conjugation of azide and alkyne functionalized molecules [9]. This reaction has been used to label and track biomolecules, such as proteins and nucleic acids, in live cells and animals, and to image biological processes in real-time. Other bioorthogonal reactions include tetrazine ligation, [10,11] strain-promoted alkyne-nitrone cycloaddition (SPANC), [12] and phototriggered reactions, [13] among others.
Recent advancements in bioorthogonal chemistry have focused on the development of new bioorthogonal reactions and probes that can be used for specific applications [14]. One of the most promising new bioorthogonal reactions is the inverse-electron-demand Diels-Alder (IEDDA) reaction, [15] which can be used for site-specific labeling of proteins and nucleic acids in live cells. Another area of active research in bioorthogonal chemistry is the development of new bioorthogonal probes that can be used for imaging and detection of biological molecules and processes [16]. These probes can be designed to target specific biomolecules or pathways, and can be used for both in vitro and in vivo imaging. For example, activatable probes have been developed that are only activated upon binding to a target biomolecule, allowing for highly specific and sensitive detection. Additionally, probes that emit light in response to biological stimuli, such as pH or redox potential, have been developed for in vivo imaging [17,18]. The 1,3-dipolar cycloaddition reaction between azides and alkynes is perhaps one of the most commonly used bioorthogonal reactions [19,20]. It allows for the formation of 1,2,3-triazoles, which have a wide range of applications in chemical biology [21]. Traditionally, this type of reaction required high temperatures and pressures. However, in 2001, Sharpless, Meldal and colleagues reported the development of copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), which could be carried out under mild conditions at room temperature [9,22]. This breakthrough led to the widespread use of click chemistry, a term used to describe the reaction's simplicity and efficiency. Although CuAAC is widely used in bioorthogonal chemistry, the cytotoxicity of copper(I) limits its use in biological systems. As a result, researchers have focused on developing copper-free alternatives. As early as in 1961, Wittig and Krebs noticed that cyclooctyne and azidobenzene could react by strain promotion to form triazole [23]. In 2004, Bertozzi and colleagues revisited this reaction and developed the first cyclooctyne OCT for copper-free click chemistry [7]. This type of reaction is known as strain-promoted azide-alkyne cycloaddition (SPAAC) (Figure 1). probes that emit light in response to biological stimuli, such as pH or redox potential, have been developed for in vivo imaging [17,18]. The 1,3-dipolar cycloaddition reaction between azides and alkynes is perhaps one of the most commonly used bioorthogonal reactions [19,20]. It allows for the formation of 1,2,3-triazoles, which have a wide range of applications in chemical biology [21]. Traditionally, this type of reaction required high temperatures and pressures. However, in 2001, Sharpless, Meldal and colleagues reported the development of copper(I)-catalyzed azidealkyne cycloaddition (CuAAC), which could be carried out under mild conditions at room temperature [9,22]. This breakthrough led to the widespread use of click chemistry, a term used to describe the reaction's simplicity and efficiency. Although CuAAC is widely used in bioorthogonal chemistry, the cytotoxicity of copper(I) limits its use in biological systems. As a result, researchers have focused on developing copper-free alternatives. As early as in 1961, Wittig and Krebs noticed that cyclooctyne and azidobenzene could react by strain promotion to form triazole [23]. In 2004, Bertozzi and colleagues revisited this reaction and developed the first cyclooctyne OCT for copper-free click chemistry [7]. This type of reaction is known as strain-promoted azide-alkyne cycloaddition (SPAAC) (Figure 1). SPAAC has become an essential tool in chemical biology because it can be used in live cells and in vivo. One of the benefits of SPAAC is its compatibility with a wide range of functional groups. The reaction's rate can also be increased by modifying the cyclooctyne's structure, which allows for the development of highly selective and efficient reactions (Table 1). In recent years, researchers have continued to develop and refine SPAAC. For example, cyclooctynes with different strain-promoting groups have been developed to increase the reaction rate. The use of SPAAC has also expanded to include SPAAC has become an essential tool in chemical biology because it can be used in live cells and in vivo. One of the benefits of SPAAC is its compatibility with a wide range of functional groups. The reaction's rate can also be increased by modifying the cyclooctyne's structure, which allows for the development of highly selective and efficient reactions ( Table 1). In recent years, researchers have continued to develop and refine SPAAC. For example, cyclooctynes with different strain-promoting groups have been developed to increase the reaction rate. The use of SPAAC has also expanded to include applications in drug discovery and materials science. For example, SPAAC has been used to develop drug conjugates and to create polymer networks. Compared with terminal alkynes, the reaction rate of OCT increases only slightly, and its water solubility is not good enough. Bertozzi improved cyclooctyne by introducing a fluorine atom with an electron-withdrawing effect at the propargylic position, resulting in DIFO (difluorinated cyclooctyne), which significantly increased the reaction rate with azides [24,25]. Subsequently, Boons reported DIBO (dibenzocyclooctyne), in which the dibenzene system can increase strain energy, making it significantly active [26][27][28]. In 2010, van Delft introduced a nitrogen atom into cyclooctyne to obtain DIBAC (azadibenzocyclooctyne) [8,29]. In the same year, Bertozzi et al. optimized cyclooctyne again by introducing the amide structure into the ring to obtain BARAC (biarylazacyclooctynone) [30]. These compounds are among the most reactive in SPAAC (Table 1). However, the synthesis of these compounds is time-consuming and always require harsh conditions, which limits their application. Considering that there has not yet been a comprehensive review in this area, herein we will briefly summarize the current synthesis approaches for the DIBAC and BARAC series.

Synthesis Approaches for DIBAC
DIBAC was initially developed by van Delft, [8] who used 2-iodobenzyl alcohol 1 and 2-ethynylaniline 2 as the starting materials ( Figure 2). A Sonogashira coupling reaction was performed to construct the internal alkyne 3, followed by the protection of the aniline and semi-hydrogenation catalyzed by Pd/BaSO 4 to deliver the cis-alkene 5. The terminal hydroxyl methyl was oxidized to aldehyde using the Dess-Martin oxidation reaction and then passed through a reductive amination reaction with the intramolecular amino group to obtain the key intermediate 7. The synthetic route contained multiple steps, however, each step exhibited a relatively high yield, and the total yield for preparing intermediate 7 can reach as high as 70%.  With the key intermediate 7 in hand, the most commonly used method for synthesizing DIBAC could be achieved in three steps ( Figure 3). A simple alkylation would introduce functional probes (azide, alkyne, biotin, etc.) onto the nitrogen under various conditions. Following this, bromination and base-prompted elimination would then give the corresponding product, DIBAC. With the key intermediate 7 in hand, the most commonly used method for synthesizing DIBAC could be achieved in three steps ( Figure 3). A simple alkylation would introduce functional probes (azide, alkyne, biotin, etc.) onto the nitrogen under various conditions. Following this, bromination and base-prompted elimination would then give the corresponding product, DIBAC.  (Figure 4) [31]. Dibenzosuberone 10, a commonly fused tricyclic framework, was used as the starting material. It was converted to oxime 11 and then treated by polyphosphoric acid, which functioned both as an acid catalyst and a dehydrating agent, at 125 °C for 1 h to afford the amide 12 in 97% yield. A selective reduction in the amide linkage with lithium aluminum hydride would then deliver the expected amine 7 in 91%  (Figure 4) [31]. Dibenzosuberone 10, a commonly fused tricyclic framework, was used as the starting material. It was converted to oxime 11 and then treated by polyphosphoric acid, which functioned both as an acid catalyst and a dehydrating agent, at 125 • C for 1 h to afford the amide 12 in 97% yield. A selective reduction in the amide linkage with lithium aluminum hydride would then deliver the expected amine 7 in 91% yield. Although there were fewer steps in Popik's pathway, the overall yield was not superior (40% for three steps from 10). However, the utilization of cheap reagents (hydroxylamine and polyphosphoric acid) and easy operation without low temperature experiments made it more appealing.
Molecules 2023, 28, x FOR PEER REVIEW 5 of 12 yield. Although there were fewer steps in Popik's pathway, the overall yield was not superior (40% for three steps from 10). However, the utilization of cheap reagents (hydroxylamine and polyphosphoric acid) and easy operation without low temperature experiments made it more appealing. The key step in the Popik's route is the Beckmann rearrangement, which was promoted by polyphosphoric acid under a high temperature. Adronov et al. believed that the poor solubility of the polyphosphoric acid may be the reason for the low yield of this step, and therefore used the Eaton reagent instead for this key rearrangement [32]. It turned out that the 10 wt% of phosphorus pentoxide solution in methanesulfonic acid [33] is an excellent alternative to polyphosphoric acid for the nitrilium ion formation, with which the intermediate 7 could be obtained in 90% yield, even in gram-scale processes ( Figure  4). Only one chromatography purification step was needed in this modified DIBAC synthesis process, which greatly simplified the whole process.
In 2013, Schubert et al. reported a different method for constructing the DIBAC skeleton using Pd-catalyzed cyclization ( Figure 5) [34]. This method relied on the regioselectivity of the intramolecular Heck reaction between 2-vinylbenzaldehyde 14 and 2-bromoaniline 13. The former intermediate was prepared via a vinylation of bromo-benzaldehyde by Suzuki coupling with vinylboroxine [35] or via the Hiyama reaction with vinylsiloxane [36]. A subsequent reductive imine condensation with the 2-chloroaniline 13 The key step in the Popik's route is the Beckmann rearrangement, which was promoted by polyphosphoric acid under a high temperature. Adronov et al. believed that the poor solubility of the polyphosphoric acid may be the reason for the low yield of this step, and therefore used the Eaton reagent instead for this key rearrangement [32]. It turned out that the 10 wt% of phosphorus pentoxide solution in methanesulfonic acid [33] is an excellent alternative to polyphosphoric acid for the nitrilium ion formation, with which the intermediate 7 could be obtained in 90% yield, even in gram-scale processes (Figure 4). Only one chromatography purification step was needed in this modified DIBAC synthesis process, which greatly simplified the whole process.
In 2013, Schubert et al. reported a different method for constructing the DIBAC skeleton using Pd-catalyzed cyclization ( Figure 5) [34]. This method relied on the regioselectivity of the intramolecular Heck reaction between 2-vinylbenzaldehyde 14 and 2-bromoaniline 13. The former intermediate was prepared via a vinylation of bromobenzaldehyde by Suzuki coupling with vinylboroxine [35] or via the Hiyama reaction with vinylsiloxane [36]. A subsequent reductive imine condensation with the 2-chloroaniline 13 afforded the N-benzylaniline 15 containing a vinyl and halide moiety, which then underwent an intramolecular Suzuki-Heck coupling to obtain the key intermediate 7. The reaction conditions (phosphine ligand, aryl halide, and temperature) were carefully optimized to suppress side reactions and the anticipated product 7 was isolated in 73% (two steps) when Pd(dba) 2 and S-PHOS was applied. Considering the modular nature of this method, Schubert further extended it to a one-pot synthesis ( Figure 5). They used commercially available 2-bromo-benzyl bromide 16 and 2-chloroaniline 13 as the starting materials. The mixture was subjected to the standard intramolecular amination and Suzuki-Heck coupling and the final product 7 was obtained in 33% yield. The yield was a little lower than that of the two step route due to unavoidable double alkylation of the amino group, and thus further research and optimization may be required.
Unlike the strategies mentioned above that involved dibromination and dehydrohalogenation to generate the triple bond within the dibenzosuberane, Pietzsch et al. utilized ultraviolet irradiation to promote the elimination of carbon monoxide from the 2-cyclopropen-1-one motif, resulting in the release of the C-C triple bond ( Figure 6) [37]. The key step was the ring closure with tetrachlorocyclopropene 21. Starting from 3,4,5-trimethoxyaniline 17 and 3-methoxybenzaldehyde 18, a reductive amination delivered the N-benzylaniline 19, followed by a dual Friedel-Crafts reaction with tetrachlorocyclopropene 21 to produce the key intermediate 22. The 2-cyclopropen-1-one motif was highly unstable under ultraviolet light irradiation, which then led to the decomposition of the cyclopropene with the release of carbon monoxide and the formation of the corresponding triple bond. This method was simple, rapid, started from inexpensive materials and delivered the final product in a three-step synthesis. Although the overall yield was low (23%), further improvements may be explored, such as the Friedel-Crafts acylation efficiency. Considering the modular nature of this method, Schubert further extended it to a onepot synthesis ( Figure 5). They used commercially available 2-bromo-benzyl bromide 16 and 2-chloroaniline 13 as the starting materials. The mixture was subjected to the standard intramolecular amination and Suzuki-Heck coupling and the final product 7 was obtained in 33% yield. The yield was a little lower than that of the two step route due to unavoidable double alkylation of the amino group, and thus further research and optimization may be required.
Unlike the strategies mentioned above that involved dibromination and dehydrohalogenation to generate the triple bond within the dibenzosuberane, Pietzsch et al. utilized ultraviolet irradiation to promote the elimination of carbon monoxide from the 2-cyclopropen-1-one motif, resulting in the release of the C-C triple bond ( Figure 6) [37]. The key step was the ring closure with tetrachlorocyclopropene 21. Starting from 3,4,5-trimethoxyaniline 17 and 3-methoxybenzaldehyde 18, a reductive amination delivered the N-benzylaniline 19, followed by a dual Friedel-Crafts reaction with tetrachlorocyclopropene 21 to produce the key intermediate 22. The 2-cyclopropen-1-one motif was highly unstable under ultraviolet light irradiation, which then led to the decomposition of the cyclopropene with the release of carbon monoxide and the formation of the corresponding triple bond. This method was simple, rapid, started from inexpensive materials and delivered the final product in a three-step synthesis. Although the overall yield was low (23%), further improvements may be explored, such as the Friedel-Crafts acylation efficiency.
While van Delft's method has been commonly used for the synthesis of DIBAC, recently Hosoya et al. designed new alkyne−cobalt complexes to prepare DIBAC and its derivatives (Figure 7) [38]. This strategy is based on the utilization of Pictet−Spengler reac-tion, in which N-methoxymethyl amide 24 was generated from o-iodoaniline 23 through amidation and N-methoxymethylation. A Sonogashira coupling reaction was performed to construct diarylacetylene 25 that was later treated with Co 2 (CO) 8 to yield an alkyne−cobalt complex 26. The Pictet−Spengler reaction was applied to afford the DIBAC−cobalt complex 27. Finally, the cobalt complex 27 was disassociated using the Me 3 NO/pyridine/air system to deliver the DIBAC derivative 28. The overall yield was 72% (six steps), which is higher than van Delft's synthesis method. While van Delft's method has been commonly used for the synthesis of DIBAC, recently Hosoya et al. designed new alkyne−cobalt complexes to prepare DIBAC and its derivatives (Figure 7) [38]. This strategy is based on the utilization of Pictet−Spengler reaction, in which N-methoxymethyl amide 24 was generated from o-iodoaniline 23 through amidation and N-methoxymethylation. A Sonogashira coupling reaction was performed to construct diarylacetylene 25 that was later treated with Co2(CO)8 to yield an alkyne−cobalt complex 26. The Pictet−Spengler reaction was applied to afford the DIBAC−cobalt complex 27. Finally, the cobalt complex 27 was disassociated using the Me3NO/pyridine/air system to deliver the DIBAC derivative 28. The overall yield was 72% (six steps), which is higher than van Delft's synthesis method.

Synthesis Approaches for BARAC
BARAC is another useful cyclooctyne probe, which displays a constant reaction rate higher than that of DIBAC. However, its synthesis methods are quite limited. Bertozzi

Synthesis Approaches for BARAC
BARAC is another useful cyclooctyne probe, which displays a constant reaction rate higher than that of DIBAC. However, its synthesis methods are quite limited. Bertozzi et al. subjected the Fischer indole intermediate from 1-indanone 29 and phenylhydrazine to an acid-promoted rearrangement to obtain the eight-membered skeleton 30 (Figure 8).
The key intermediate was then alkylated with the corresponding linkage and installed in the TMS group as a placeholder. The decorated 31 was subjected to oxidative cleavage with m-CPBA and then converted to the triple bond precursor TMS substituted vinyl trifluoromethanesulfonate 33, which was then cleaved using fluoride ion to generate the BARAC. In 2019, Okano et al. improved Bertozzi's synthesis method ( Figure 9) [39]. Instead of introducing a TMS group onto 34, the eight-membered skeleton was firstly subjected to an oxidative cleavage of an amide-ketone 35. Using the KHMDS and PhNTf2 as the dehydrogenation combination, the target BARAC product was obtained in good yields (74% when R = Me). However, when the final step was conducted at 0 °C or higher, only trace amount of the product was observed.  Figure 9) [39]. Instead of introducing a TMS group onto 34, the eight-membered skeleton was firstly subjected to an oxidative cleavage of an amide-ketone 35. Using the KHMDS and PhNTf 2 as the dehydrogenation combination, the target BARAC product was obtained in good yields (74% when R = Me). However, when the final step was conducted at 0 • C or higher, only trace amount of the product was observed. In 2019, Okano et al. improved Bertozzi's synthesis method ( Figure 9) [39]. Instead of introducing a TMS group onto 34, the eight-membered skeleton was firstly subjected to an oxidative cleavage of an amide-ketone 35. Using the KHMDS and PhNTf2 as the dehydrogenation combination, the target BARAC product was obtained in good yields (74% when R = Me). However, when the final step was conducted at 0 °C or higher, only trace amount of the product was observed. As mentioned before, an amide intermediate 12 was generated during the synthesis of DIBAC via the Beckmann rearrangement ( Figure 4). Therefore, Adronov et al. considered using this process to prepare BARAC derivatives ( Figure 10) [32]. Similar to the synthesis method of DIBAC, they first prepared the alkylated amide and then subjected the As mentioned before, an amide intermediate 12 was generated during the synthesis of DIBAC via the Beckmann rearrangement ( Figure 4). Therefore, Adronov et al. considered using this process to prepare BARAC derivatives ( Figure 10) [32]. Similar to the synthesis method of DIBAC, they first prepared the alkylated amide and then subjected the 36 to dibromination to obtain 37, followed by base-facilitated elimination. However, the corresponding BARAC was generated in less than 10% (NMR and TLC) and could only be trapped by click reaction with benzyl azide. Therefore, further optimization was still required.

Conclusions
In conclusion, we have summarized current synthetic methods of dibenzocyclooctyne (DBCO), i.e., the DIBAC and BARAC derivatives. Both of these probes are widely used in SPAAC reactions, but current synthetic routes still display some drawbacks, such as long synthetic routes, expensive starting materials and catalysts, harsh reaction conditions, and low yields, which makes it difficult to produce them on a large scale or generate a diverse library of derivatives. Therefore, there is still a lot of room for improvement in these synthetic aspects. Additionally, of course, with the advancements of bioorthogonal chemistry in the expansion of toolkits for studying complex biological systems, new structures and efficient approaches may also emerge in the future. We anticipate that in the near future, new structures based on DBCO will continue to be discovered, and their synthetic methods will also become more efficient. We hope that this mini review can bring some inspiration to those who are devoted to this area.

Conflicts of interest:
There are no conflicts to declare.

Conclusions
In conclusion, we have summarized current synthetic methods of dibenzocyclooctyne (DBCO), i.e., the DIBAC and BARAC derivatives. Both of these probes are widely used in SPAAC reactions, but current synthetic routes still display some drawbacks, such as long synthetic routes, expensive starting materials and catalysts, harsh reaction conditions, and low yields, which makes it difficult to produce them on a large scale or generate a diverse library of derivatives. Therefore, there is still a lot of room for improvement in these synthetic aspects. Additionally, of course, with the advancements of bioorthogonal chemistry in the expansion of toolkits for studying complex biological systems, new structures and efficient approaches may also emerge in the future. We anticipate that in the near future, new structures based on DBCO will continue to be discovered, and their synthetic methods will also become more efficient. We hope that this mini review can bring some inspiration to those who are devoted to this area.

Conflicts of Interest:
There are no conflict to declare.