Tóm tắt Luận án Synthesis and cytotoxicity of bengamide analogues A and E

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ----------------------------- PHI THI DAO “ Synthesis and cytotoxicity of bengamide analogues A and E” Major : Organic Chemistry Code : 62 44 01 14 SUMMARY OF CHEMISTRY DOCTORAL THESIS HaNoi - 2018 The thesis was completed at: Graduated University of Science and Technology - Vietnam Academy of Science and Technology Scientific supervisor: Advisiors 1: Assoc. Prof. Dr. Habil Van Cuong Pham Advisiors 2. Dr. Huong Doan Thi Mai 1st Reviewer: 2 nd Reviewer: 3rd Reviewer: The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at hour date month 2018 Thesis can be found in: - The library of the Graduated University of Science and Technology, Vietnam Academy of Science and Technology - National Library of Vietnam Publications related to the thesis 1. Thi Dao Phi, Huong Doan Thi Mai, Van Hieu Tran, Bich Ngan Truong, Tuan Anh Tran, Van Loi Vu, Van Minh Chau, Van Cuong Pham. Design, synthesis and cytotoxicity of bengamide analogues and their epimers. Med. Chem. Commun, 2017,8, 445-451. 2. Thi Dao Phi, Huong Doan Thi Mai, Van Hieu Tran, Van Loi Vu, Bich Ngan Truong, Tuan Anh Tran, Van Minh Chau and Van Cuong Pham. Synthesis of bengamide E analogues and their cytotoxic activity. Tetrahedron Letters. 2017, 58, 1830-1833 3. Phi Thi Dao, Doan Thi Mai Huong, Le Thi Phuong, Chau Van Minh, Pham Van Cuong. Synthesis of 8-methyl-2-O-methyl-3,5-O-(1- methyl ethylidene)-6,7,8,9-tetradeoxy-D-gulo-6-nonenonic acid (6E)- - lactone. Vietnam Journal of Chemistry, 2015,53 (2e), 154-157 4. Phi Thi Dao, Doan Thi Mai Huong, Vu Van Loi, Chau Van Minh, Pham Van Cuong. Microwave-assisted synthesis of lactams from amino acids. Vietnam Journal of Chemistry, 2015, 53 (2e), 198-201. 5. Phi Thi Dao, Vu Van Loi, Nguyen Thi Bich, Doan Thi Mai Huong, Nguyen Hien, Chau Van Minh, Pham Van Cuong. Synthesis of N-alkyl amino lactam derivatives. Journal of Science and Technology, 2016, 54 (2C), 291-298. 6. Phi Thi Dao, Doan Thi Mai Huong, Vu Van Loi, Chau Van Minh, Pham Van Cuong. Synthesis and cytotoxicity of (2R,3R,4S,5R,6E)-3,4,5-trihydroxy-2-methoxy-8,8-dimetyl-N-((S)-2- oxoazepan-3-yl)non-6-enamide. Vietnam Journal of Chemistry, 2016, 54 (6e2),62-65. 7. Phi Thi Dao, Doan Thi Mai Huong, Vu Van Loi, Nguyen Thii Hue, Pham Van Cuong. In vitro cytotoxic and antimicrobial activities of some bengamide derivatives. Vietnam Journal of Chemistry 2017 55(3), 342-347. 8. Patent for utinity solution. Synthetic method of bengamide analogues . Accepted application 1 I. INTRODUCTION 1. Introduction Natural compounds isolated from terrestrial plants have been studied for a long time and have been successful. However, studies on marine compounds have only begun in the middle of the last century. Currently, natural marine compounds are known to be a promising source of pharmaceuticals, and many highly biologically active compounds have been found in various marine organisms. Difficulties in collecting large amounts of samples and requiring high funding are one of the obstacles to research in the field of marine chemical compounds. Therefore, organic synthesis is an effective alternative to generate larger amounts of active ingredients to serve biological studies, as well as to ensure their applicability. Many marine-derived active compounds play as lead compounds so that researchers can make new derivatives possess higher biological activity. The bengamides isolated from marine sponges are known for their potent anti-cancer activity. However, structural instability is one of the reasons limiting the applicability of this class. In order to overcome this limitation of bengamides, we chose the research topic "Synthesis and cytotoxicity of bengamide analogues A and E" in this thesis. 2. Objectives of the thesis - Constructing synthesis process of bengamide analgoues A and E - Evaluating the biological activity of the synthesized analogs. 3. Scientific significance and new contributions of the thesis 3.1. Scientific significance - Synthesis of new analogues of bengamide A and E. - Application of microwave irradiation in organic synthesis. 3.2. New contributions of the thesis - Stereochemical synthesis process of bengamide A and E analogues was described. - 30 bengamide A and E analogues were synthesized, including 16 bengamide E analogues, 4 bengamide A analogues and 10 fluorine containing compounds. Among them, there are 27 new analogues. 2 - Microwave irradiation method was effectively used in the reactions of intra-molecular cyclization of amine acids, protection of primary amines and coupling reactions of ketide side chains and amino lactams. Reaction times are shorten and reaction yields were improved remarkably. - Cytotoxic activities of these analogues were evaluated against several cancer cell lines (Lu, NCI-H1975, A549, MCF7, MDA-MB-231, HepG2, Hep3B, KB, HL60 and Hela). The analogues containing R configuration at C-2’ always exhibited higher activity than the analogues containing S configuration at C-2’. Many analogues have IC50 values less than 1µM for cytotoxicity tests. Beside that, anti-microbial activity of several analogues against 7 bacterial and yeast strains were also investigated. Among them, 7 analogues showed high activity against Gram positive bacteria and Candida albicans. 4. The main content of the thesis Thesis includes 153 pages, 25 tables, 65 figures and 57 references as following: Introduction: 2 pages Chapter 1: Overview 32 pages Chapter 2: Experimental and methodology 64 pages Chapter 3: Results and discussion 41 pages Conclusion: 2 pages 57 documents were referenced in the thesis, the documents were updated to 2017. The appendix includes 234 pages, including spectra of synthesized derivatives. II. CONTENT THESIS General introduction Refers to the scientific meaning, practicality, object and research task of the thesis. CHAPTER 1: OVERVIEW Overview included 32 pages, summarizing the literature of naturally isolated bengamides, previously synthesized methods of bengamide analogues and 3 their biological activities up to now. CHAPTER 2: EXPERIMENTAL AND METHODOLOGY The four-pages of research methods described organic synthesis methods, chemical structure determination methods and biological activity assay methods. The 64-page experiment details the synthetic process of benamide analogs. Physical properties and spectral data of the synthesized substances. We have developed the method of synthesis of the following substances: - Synthesis of polyketide chain - Synthesis of N-ankyl substituted 6 and 7-membered aminolactam rings. - Synthesis of bengamide analogues A and E - Synthesis of fluorine containing bengamide analogues. - Evaluated cytotoxic activity of 30 synthesized analogues against 10 cancer cell lines (Lu1, NCI-H1975, A549, MCF7, MDA-MB-231, HepG2, Hep3B, KB, HL60 và Hela). Evaluated antimicrobial activity against 07 strains, including: Gram (+), Gram (-) and yeast. CHAPTER 3: RESULTS AND DISCUSSION Previous studies on the synthesis of bengamide analogues and their evaluation of tumor inhibitory activity have shown that the structural modification of bengamide skeleton remarkably effected to their biological activity. Especially, previous studies have shown that the presence of the hydroxyl groups and the configuration of C-3, C-4 and C- 5 of ketide side chains play an important role to the cytotoxic activity of bengamide analogues. In order to study the relationship between the structure and cytotoxic activity of bengamide analogues, the conversion of bengamide skeleton is carried out by the following ways i) replacing the isopropyl group by the tert-butyl group leading to the obtainment of more stable structures by avoiding olefin isomerization ), ii) modification of configuration at C-2’ carbon, thereby evaluating the effect of C-2' 4 configuration to biological activities, iii) N-alkylated amide groups of the lactam ring, iv) synthesis of some analogs with the hydroxyl group at C- 5', v) changing the lactam ring size, vi) replacing the terminal olefinic chain by FCH2-CH(OH) to evaluate its role to biological activity (Figure 3.1). Figure 3.1. Modification of the bengamide skeleton The synthesis process of new bengamide analogs was carried out from commercial chemicals, such as: α-D-glucoheptonic γ-lactone and amine acids (L-ornithine monohydrochloride, D-ornithine monohydrochloride, L-lysine, D-lysine and D, L-5-hydroxylysine hydrochloride) through 3 main stages. 3.1. Synthesis of ketide chains Synthesis of BG5 ketide is carried out using the commercially available α-D-glucoheptonic γ-lactone (Figure 3.2). The acetonide reaction using acetone and sulfuric acid as catalyst produces BG1 in 67.8%. Figure 3.2. Synthesis of BG5 compound 5 Selective hydrolysis of the isopropylidene group of BG2 with acetic acid yielded compound BG3 in 85%. Study on the oxidation of diol with NaIO4 in a mixture of different solvents showed that using 1.2 eq NaIO4 in a solution of MeCN and H2O (4/1, v/v) obtained the highest yield of aldehyde BG4 in 91%. The final reaction is the olefination reaction of aldehyde BG4. The reaction was investigated using (P(t-Bu)3HBF4) (1.5-3eq) in the presence of TEA, potassium t-butoxide (CH3)3COK) or NaH (1.5 eq) in THF at room temperature for 3 - 24 h. However, these reactions do not form the desire product BG5. Then, the reaction was successfully carried out using Takai olefination reaction in the presence of 1,1-diiodo-2,2- dimethylpropane and CrCl2. In fact, the reaction using 1,1-diiodo-2,2- dimethylpropane bought from Aldrich company gave the ketide compound BG5 in the highest yield of 45%. Meanwhile, this compound was prepared much effectively (73%), when the reaction was carried out with fresly prepared 1,1-diiodo-2,2-dimethylpropane. The mechanism of the oxidation of BG3 and the formation of BG5 from BG4 using Takai reaction is shown in the following figure. Figure 3.3: Mechanism of oxidation of BG3 and Takai olefination of BG4 6 Accordingly, in the oxidation reaction with NaIO4, the oxidation state of iodine is shifted from +7 (NaIO4) to +5 (NaIO3). For Takai olefination reaction, this is a combination of aldehyde and geminal dihaloalkane to form olefins. In this reaction, Cr (II) is oxidized to Cr (III) when both halogen atoms are replaced. The formed geminal carbodianion reacts with aldehyde to form the desire alkene. Structures of synthesized compounds was determined using MS, 1H-NMR, 13C-NMR spectra and compared with the published data. 3.2. Synthesis of 2-amino-lactam compounds 3.2.1. Synthesis of BG6a-b and BG14a-b The ring structure of α-amino lactams exist in many compounds exhibiting highly biological activity. Thus, the synthesis of α-amino lactams has attracted the attention of many research groups and typically, one of them is the synthesis of α-aminocaprolactam from L-lysine. According to the publication of G. Pifferi and co-workers., the cyclization of L-lysine under heat conditions provided BG14a in 95% for 48 h. In another study, Blade-Front reported that synthesis of BG14a achieved in 70% using Al2O3 in toluene / pyridine mixture for a shorter time of 20 h. In addition, another article reported that synthesis of BG14a from L-lysine was carried out under high temperature and pressure conditions with a yield of 88%. In order to overcome the disadvantages of using expensive, toxic agents, long reaction time or harsh conditions as high temperature and pressure, we have studied the intramolecular cyclization of amine acids 7 with the aid of microwave irradiation. Accordingly, the α- aminocaprolactam compound (BG14a) obtained in 79% from L-lysine using mild conditiions in ethylene glycone under microwave irradiation at 284 W for 1 h. Meanwhile, the yield of BG14a was lower (48%) if the reaction was carried out in butanol. Using the same reaction conditions, the BG14b compound was synthesized with 82% of yield from D-lysine. Under microwave irradiation at 284 W, compounds BG6a and BG6b were obtained in 55.6% for 1h using ethylene glycol and pyridine. Replacement of pyridine by a solution of 10% NaHCO3 under microwave irraditation gave BG6a in 78%. Similarly, compound BG6b was obtained in 72% from D-ornithine hydrochloride. 3.2.2. Synthesis of rac-BG22a and rac- BG22b (i): NaHCO3 10%, ethylen glycon, pyridine, MW, 284W, 1h (ii): (Boc)2O (1eq), TEA, THF, H2O, rt, 3h Due to commercially unavailable chiral isomers of 5-hydroxylysine, the 6-hydroxycaprolactam isomers were prepared from a racemic mixture of the 5-hydroxylysine compound. Accordingly, the intramolecular reaction of D, L-5-hydroxylysine hydrochloride mixture was carried out with the aid of microwave irradiation at 284 W for 60 minutes gave a racemic mixture of the two diastereomers rac-BG22a and rac-BG22b. The mixture then was purified on a silica gel column with acetone/ H2O/ NH4OH (ratio of 9/ 1/ 0.1) obtained a racemic mixture of rac-BG22a 8 (53%, Rf = 0.49) and rac-BG22b (39%, Rf = 0.34). Due to impossible determination of the relative configuration at C-3 and C-6 of rac-BG22a and rac-BG22b, these compounds are converted into N-Boc derivatives to compare with published NMR data. The reaction of each rac-BG22a and rac-BG22b with Boc2O is carried out in THF/H2O mixture in the presence of TEA to produce corresponding compounds rac-BG23a and rac-BG23b. Compared to the published NMR data, rac-BG23a has a 3, 6-trans configuration (3S*, 6S*) and thus, the 3,6-cis (3S*, 6R*) configuration was assigned for rac-BG23b. This allows to determine 3,6-trans (3S*, 6S*) and 3,6-cis (3S*, 6R*) configuration for compounds rac-BG22a and rac-BG22b, respectively. 3.2.3. Synthesis of N-ankyl aminolactam compounds 3.2.3.1. The reaction protects the amine group of 2-aminolactam In order to protect the primary amine groups, 3-aminolactam compounds are reacted with phthalic anhydride. The reaction between BG6a and phthalic anhydride is carried out in acetic acid at 100°C for 6 hours to obtain BG7a in a low yield of 30% of. Then with the aid of microwave irradiation, compound BG7a obtained with higher yield (53%) for a shorter time (1 hour). Using the same reaction conditions under microwave irradiation, amine groups of BG6b, BG14a and BG14b compounds were successfully protected, yielding BG7b, BG15a and BG15b compounds in the range of 52 - 57%. a) anhydride phthalic, CH3COOH, molecular sieve 4Ǻ, MW, 284 W, 1 h 3.2.3.2. N-alkylation reactions After the protection of the primary amines, the alkylation reactions were carried out with halide derivaties, such as: alkyl halides, 9 (bromomethyl)cyclohexane, benzyl bromide and cinnamyl bromide. Compound BG7 was selected for optimization study at the beginning. The reaction of BG7a with (bromomethyl) cyclohexane or benzyl bromide or cinnamyl bromide did not lead to the desired N-alkyl products when using alkaline agents such as K2CO3, KOH or NaH in DMF or THF. However, when the reaction was performed in DMSO, formation of the desired N-alkylated products BG8a-BG10a was recorded. The results showed that the use of DMSO and KOH (2 eq), K2CO3 (2 eq) and KI (1 eq) at 50-60oC provided the best yield. Using the same optimized conditions, other N-alkyl compounds were synthesized with the yield in the range of 45-55%. (a) (bromomethyl)cyclohexane or(bromomethyl)benzene or cinnamyl bromide, DMSO, KOH, K2CO3, KI, 23h; (b) hydrazine, MeCN, 1h. Finally, the deprotection of phthalimide group was carried out by treating the compounds BG8a - BG10a, BG8b - BG10b, BG16a - BG18a, BG16b - BG18b with hydrazine solution in acetonitrile at room temperature, leading to the formation of the corresponding N - alkylaminolactam BG11a-13a, BG11b-13b, BG19a-21a and BG19b- 21b. 3.3. Synthesis of bengamide E analogue Synthesis of bengamides was carried out by coupling between ketide and aminolactams. In fact, the lactone ring-opening reaction of BG5 has been studied and published previously. Accordingly, David D. Xu and 10 co-workers studied the reaction between BG5 and LAF-A. The authors found that using sodium 2- ethyl hexanoate as a base in THF at room temperature, yielded LAF-B products in 85-92% after 20 hours. Figure 3.9. Coupling reaction of BG5 and LAF-A by David and co-workers. The method of David D. Xu and co-workers has the advantage of using cheap agents, mild conditions, but prolonged reaction time (15 - 20 hours). The reaction between BG5 and BG6a is used to optimize the reaction conditions. Accordingly, with the use of sodium 2- ethyl hexanoate in THF or 1,4-dioxane, at temperatures between 50 and 100°C, for 10 to 24 hours, the yield of BG24a products is between 50 and 58%. Then, the lactone ring opening was examined under microwave irradition at 100W, the reaction time was significantly shortened for 1 hour and the yield of the BG24a reached to 87%. As can be observed, the reaction between BG5 and BG6a achieved the highest yield when using 1.2 - 1.5 eq of sodium 2- ethyl hexanoate, under microwave irradiation at 100 W for 1 hour. Table 3.7. Synthesis of BG24a Solvent sodium 2- ethyl hexanoate MW Temperature Time (hour) Yield (%) 1,4- dioxane 2.0 eq - 100 oC 10 50 1,4- 1.5 eq - 100 oC 10 50 11 Solvent sodium 2- ethyl hexanoate MW Temperature Time (hour) Yield (%) dioxane THF 1.5 eq - 50-60 oC 24 58 THF 1.5 eq 284 W >100 oC 0.5 55 THF 1.5 eq 100 W 50-60 oC 1h 87 THF 1.2 eq 100 W 50-60 oC 1h 87 THF 1.0 eq 100 W 50-60 oC 1h 80 Using the same reaction conditions as synthesis of BG24a, the compounds BG24b, BG25a - BG31a and BG25b - BG31b are also successfully synthesized with the yields in the range of 64-95%. The structures of BG24a - BG31a, BG24b - BG31b were confirmed by MS and NMR spectral data analysis. For BG24a, the pseudomolecular ion mass at m/z 421 [M + Na]+ was observed on the ESI-MS spectrum. On the other hand, the 13C-NMR and DEPT spectra of BG24a appeared the signal of 20 carbon atoms including two carbonyl groups at C 171.1 (C-1’), 170.5 (C-1), 1 methoxy group at C 59.8 (C- 12), 2 methine groups, 3 methylene groups, 5 sp3 methine groups, 5 methyl groups and 2 quaternary carbons. 12 Figure 3.12. 13C-NMR spectrum of compound BG24a The 1H-NMR spectrum of BG24a appeared the signals of 5 methyl groups at H 1.03 (9H, s, 3 x H-9,10,11), 1.47 (3H, s, H-15), 1.49 (3H, s, H-16), 2 olefinic protons at H 5.77 (1H, dd, J = 0.5; 16.0 Hz, H-7), 5.55 (1H, dd, J = 7.0; 16.0 Hz, H-6) and a methoxy group at H 3.46 (3H, s). Furthermore, the signal of an NH group was recorded at H 7.32 (1H, d, J = 5.5 Hz, NH-13), along with the signals of a methin group at H 4.33 (1H, m, H -2'), a methylene group connected with a nitrogen atom at H 3.36 (3H, m, CH2-5' and OH) and four oxymethine groups in the range of H 3.91-4.25 were also observed on the 1H-NMR spectrum. In addition, the 1H-NMR spectrum also showed that four protons in the range of H 1.57-2.57 were identified for the two methylene groups by HSQC spectrum analysis 13 Figure 3.13. 1H-NMR spectrum of compound BG24a As observed, the signals obtained from the 1D-NMR spectra are fully consistent with the chemical structure of BG24a. Furthermore, the connection of ketide to aminol