Development of New Effective Methods for the Synthesis of Lembehynes A–C Exhibiting Cytotoxic and Neuritogenic Activity †

: This report presents data on our studies on the preparation of the precursor lembehyne A and the complete stereoselective synthesis of natural lembehynes B and C, which have cytotoxic and neuritogenic activity. All methods and approaches to the synthesis of the above-mentioned lembehynes presented in this report are based on the use of the catalytic cross-cyclomagnesiation of 1,2-dienes (Dzhemilev reaction) at the key stage of the synthesis.


Introduction
To date, a wide range of unique molecules have been isolated from marine organisms, and some of them are used as drug candidates and reagents for biomedical research [1][2][3][4]. Sponges have been recognized as the richest sources of biologically active compounds among marine invertebrates, and their metabolites are diverse in structure, many have antibacterial, antiviral, antifungal, antimalarial, anthelmintic, immunosuppressive, muscle relaxant and anti-inflammatory activity. Currently, there is a steady increase in the drug resistance of microorganisms, so the search and development of new pharmaceuticals is of the utmost importance. Some compounds derived from sea sponges are undergoing preclinical and clinical trials as agents against cancer, microbial infections, and inflammation; however, the development of drugs based on them is very difficult due to the low content of the corresponding compounds in sponge tissues [5][6][7][8].
Among the wide variety of compounds found in sea sponges, acetylenic alcohols lembehynes A, B, and C ( Figure 1) are of particular interest. Lembehyne A has been shown to exhibit neuritogenic activity against pheochromacetoma PC12 and neuroblastoma Neuro 2A cells. These data allow us to consider natural acetylenic alcohols as the basis for the development of modern drugs for the treatment of such neurodegenerative diseases as Alzheimer's disease, Parkinson's disease, and Huntington's chorea. Meanwhile, the inaccessibility and lack of effective methods for the synthesis of natural lembehynes are a significant constraint for a detailed study of their properties and the full disclosure of their biomedical potential [6][7][8][9][10][11][12]. An analysis of the structure of lembehynes and known approaches to their complete synthesis showed that the most complex and multistage stage is the stage of stereoselective formation of the 1Z,5Z diene fragment in lembehyne molecules. Thus, during the implementation of the complete 11-stage synthesis of lembehyne A, six stages were spent to obtain the key synthon, 4Z,8Z-pentacose-4,8-dien-1-ol.
Based on data from the literature, the most common and popular approaches to the formation of the 1Z,5Z-diene fragment are methods based on the use of the Wittig reaction, alkene metathesis, alkyne alkylation, and stereoselective reduction of 1,5-diynes.

Results and Discussion
Based on our previous results [13][14][15][16], we put forward the idea of the possibility of using the cross-cyclomagnesiation reaction of O-containing and aliphatic allenes in the synthesis of neuritogenic acetylenic alcohols and lembehynes A, B and C.
Based on data from the literature, the most common and popular approaches to the formation of the 1Z,5Z-diene fragment are methods based on the use of the Wittig reaction, alkene metathesis, alkyne alkylation, and stereoselective reduction of 1,5-diynes.
Based on data from the literature, the most common and popular approaches to the formation of the 1Z,5Z-diene fragment are methods based on the use of the Wittig reaction, alkene metathesis, alkyne alkylation, and stereoselective reduction of 1,5-diynes.

Results and Discussion
Based on our previous results [13][14][15][16], we put forward the idea of the possibility of using the cross-cyclomagnesiation reaction of O-containing and aliphatic allenes in the synthesis of neuritogenic acetylenic alcohols and lembehynes A, B and C.

Results and Discussion
Based on our previous results [13][14][15][16], we put forward the idea of the possibility of using the cross-cyclomagnesiation reaction of O-containing and aliphatic allenes in the synthesis of neuritogenic acetylenic alcohols and lembehynes A, B and C.
We carried out the synthesis of the key monomer of lembehyne A, (4Z,8Z)-pentacose-4,8-dien-1-ol, by the reaction of cross-cyclomagnesiation of 1,2-nonadecadiene (1) with 4,5-hexadienol tetrahydropyran ester (2) with EtMgBr in the presence of metallic Mg (a halogen ion acceptor) and a Cp 2 TiCl 2 catalyst (10 mol %) to give the intermediate magnesacyclopentane As a follow-up to these studies, an original scheme for the complete stereoselective synthesis of natural lembehyne B was developed for the first-time catalyst Cp2TiCl2 (10 mol %) to give magnesacyclopentane (7a), hydrolysis of which gives (13Z,17Z)-tetraconta-13,17-dienol tetrahydropyran ester (8) in 88% yield. Successive reactions of deprotection of tetrahydropyranyl protection and oxidation of unsaturated alcohol (9) with Dess-Martin periodinane led to the key monomer (13Z,17Z)-tetraconta-13,17-dienal (10) in ~64% yield in two stages (Scheme 3). Thus, the yield of (13Z,17Z)-tetracont-13,17-dienal was 64% by the first route and 77% by the second. At the final stage in the complete synthesis of lembehyne B, aldehyde (10) was reacted with lithium trimethylsilylacetylenide, which was preliminarily prepared by the reaction of equimolar amounts of trimethylsilylacetylene with n-BuLi in THF. After keeping the reaction mixture for 3 days at room temperature, silane (11) was obtained with a yield of 90%. Removal of trimethylsilyl protection with tetrabutylammonium fluoride (TBAF) in THF in 4 hours allowed us to obtain racemic lembehyne B (14) in almost quantitative yield (Scheme 4).  Thus, the yield of (13Z,17Z)-tetracont-13,17-dienal was 64% by the first route and 77% by the second. At the final stage in the complete synthesis of lembehyne B, aldehyde (10) was reacted with lithium trimethylsilylacetylenide, which was preliminarily prepared by the reaction of equimolar amounts of trimethylsilylacetylene with n-BuLi in THF. After keeping the reaction mixture for 3 days at room temperature, silane (11) was obtained with a yield of 90%. Removal of trimethylsilyl protection with tetrabutylammonium fluoride (TBAF) in THF in 4 hours allowed us to obtain racemic lembehyne B (14) in almost quantitative yield (Scheme 4). Thus, the yield of (13Z,17Z)-tetracont-13,17-dienal was 64% by the first route and 77% by the second. At the final stage in the complete synthesis of lembehyne B, aldehyde (10) was reacted with lithium trimethylsilylacetylenide, which was preliminarily prepared by the reaction of equimolar amounts of trimethylsilylacetylene with n-BuLi in THF. After keeping the reaction mixture for 3 days at room temperature, silane (11) was obtained with a yield of 90%. Removal of trimethylsilyl protection with tetrabutylammonium fluoride (TBAF) in THF in 4 hours allowed us to obtain racemic lembehyne B (14) in almost quantitative yield (Scheme 4). Subsequently, starting from racemic lembehyne B, we synthesized its natural stereoisomer with the 3R configuration of the hydroxyl group at the C-3 carbon atom. Thus, oxidation of alkynol (12) with Dess-Martin periodinane in CH2Cl2 at room temperature for 1 hour yielded (15Z,19Z)-hexaconta-15,19-dien-1-yl-3-one (13) with a yield of 86% (Scheme 4). Stereoselective reduction of ketone (13) was carried out using the reagent B-3-pinanyl-9-borabicyclo[3.3.1]nonane (Alpine-borane), previously prepared from (+)-α-pinene (98%ee) and 9-borabicyclo[3.3.1]nonane. As a result, lembehyne B (14) was obtained with a yield of 84% and an enantiomeric purity (95%ee).
The reactions developed by us made it possible to synthesize natural lembehynes B, C and the key precursor of lembehyne A. For the synthesized natural lembehynes B, C, apoptosis-inducing activity against five tumor cell lines Jurkat, K562, U937, HeLa and HEK293 and neuritogenic activity against cell cultures were studied in detail. PC12, PC9 and Neuro2A.

Conclusions
Thus, for the first time, we synthesized natural lembechins B and C and a precursor of lembehyne A using the cross-cyclomagnesiation reaction of aliphatic and O-containing 1,2-dienes catalyzed by Cp2TiCl2 at the key stage of the synthesis, and studied their antitumor activity using modern methods of flow cytometry and multiplex analysis.