The objective of the thesis is to build the synthesis process of Analogs of Bengamide A and E, conduct the biological activity of the synthesized Analogs. To find out more about the details of the content, please consult the thesis summary.
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 Dr Huong Doan Thi Mai 1st Reviewer: 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 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 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 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-(1methyl ethylidene)-6,7,8,9-tetradeoxy-D-gulo-6-nonenonic acid (6E)- lactone Vietnam Journal of Chemistry, 2015,53 (2e), 154-157 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 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 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)-2oxoazepan-3-yl)non-6-enamide Vietnam Journal of Chemistry, 2016, 54 (6e2),62-65 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 Patent for utinity solution Synthetic method of bengamide analogues Accepted application I INTRODUCTION 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 Objectives of the thesis - Constructing synthesis process of bengamide analgoues A and E - Evaluating the biological activity of the synthesized analogs 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, bengamide A analogues and 10 fluorine containing compounds Among them, there are 27 new analogues - 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 bacterial and yeast strains were also investigated Among them, analogues showed high activity against Gram positive bacteria and Candida albicans The main content of the thesis Thesis includes 153 pages, 25 tables, 65 figures and 57 references as following: Introduction: pages Chapter 1: Overview 32 pages Chapter 2: Experimental and methodology 64 pages Chapter 3: Results and discussion 41 pages Conclusion: 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 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 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 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 C5 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' configuration to biological activities, iii) N-alkylated amide groups of the lactam ring, iv) synthesis of some analogs with the hydroxyl group at C5', 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 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 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 - 24 h However, these reactions not form the desire product BG5 Then, the reaction was successfully carried out using Takai olefination reaction in the presence of 1,1-diiodo-2,2dimethylpropane and CrCl2 In fact, the reaction using 1,1-diiodo-2,2dimethylpropane 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 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, H-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 with the aid of microwave irradiation Accordingly, the αaminocaprolactam compound (BG14a) obtained in 79% from L-lysine using mild conditiions in ethylene glycone under microwave irradiation at 284 W for 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 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 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 hour Table 3.7 Synthesis of BG24a Solvent 1,4dioxane 1,4- sodium 2ethyl hexanoate MW Temperature Time (hour) Yield (%) 2.0 eq - 100 oC 10 50 1.5 eq - 100 oC 10 50 11 Solvent dioxane THF THF THF THF THF sodium 2ethyl hexanoate MW Temperature Time (hour) Yield (%) 1.5 eq 1.5 eq 1.5 eq 1.2 eq 1.0 eq 284 W 100 W 100 W 100 W 50-60 oC >100 oC 50-60 oC 50-60 oC 50-60 oC 24 0.5 1h 1h 1h 58 55 87 87 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), methoxy group at C 59.8 (C12), methine groups, methylene groups, sp3 methine groups, methyl groups and quaternary carbons 12 Figure 3.12 13C-NMR spectrum of compound BG24a The 1H-NMR spectrum of BG24a appeared the signals of methyl groups at H 1.03 (9H, s, x H-9,10,11), 1.47 (3H, s, H-15), 1.49 (3H, s, H-16), 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 aminolactam via C-1/N-13 linkage was confirmed by the interaction of H-2' at H 4.33 with carbonyl group at C 171.1 on the HMBC spectrum Figure 3.14 Major interactions on COSY and HMBC spectra of BG24a The final step in the synthesis process of benamide analogues is to remove the acetonide protecting group This step was carried out in TFA at 0°C for 1.5 h for a reaction yield in the range of 35-69% Table 3.9 Deprotection of acetonide group The structure of bengamide analogues 32a-b - 39a-b has been confirmed by spectral methods Mass spectra obtained on the HRESI-MS spectra of these compounds are fully compatible with the expected molecular formula In addition, on the 1D -NMR spectrum of the 14 analogues 32a-b - 39a-b, there is no longer any signal of the acetal group related to the corresponding acetonide compounds At the same time, the chemical structure of 32a-b - 39a-b analogues was also confirmed by the 2D- NMR spectra Figure 3.18 13C-NMR spectrum of BG32a Hinh 3.19 Phổ 1H-NMR chât BG32a Figure 3.19 1H-NMR spectrum of BG32a 3.4 Synthesis of bengamide A analogues 15 (a):MW,100W, 120 mn, 1,4-dioxane, sodium-2 ethylhexanoate Applying the lactone ring-opening reaction of ketide BG5 with the aminolactam under microwave irradiation conditions as described above, the acetonide BG40a-d was also synthesized Compound BG5 when reacted with rac-BG22a (3S*, 6S*) racemic mixture forms two codiastereomer BG40a and BG40b The two diastereomers BG40a and BG40b were separated by preparative thin layer chromatography The chemical structures of the BG40a-d compounds were confirmed by spectral analysis, especially NMR spectra The formation of an amide bond between the ketide chain and the aminolactam portion was demonstrated by the interaction of C-1 with H-2' proton, and the interaction of amide proton (NH-13) with C-1 and C-1' on the HMBC spectrum Comparison of NMR data and optical rotation with bengamide Y allows to determine 2'S, 5'S configuration of compound BG40a Thus, 2'R, 5'R configuration is assigned to the remaining BG40b Similarly, the reaction between BG5 and the racemic mixture rac-BG22b (3S*, 6S*) yielded two diastereomers BG40c and BG40d by preparative thin layer chromatography 2'S, 5'R configuration was determined for BG40c and 16 2'R, 5'S configuration for BG40d was based on the comparison of NMR data and optical rotation with previous publication Figure 3.39 Major interactions of BG40a on COSY and HMBC spectrum Similar to the bengamide E analogues, the analogues of bengamide A were synthesized by the removal of the acetonide group of compounds BG40a-d in the presence of TFA and THF at 0-5°C for h Comparison of NMR data of BG41a-d with acetonides BG40a-d revealed that the disappearance of two methyl groups and one quaternary carbon confirmed the removal of the acetonide group 3.5 Synthesis of fluorine containing bengamide analogs In order to understand the role of terminal olefinic part to the biological activities of bengamides, we have synthesised and investigated 17 the biological activity of the analogue in which the terminal olefinic part is replaced by CH(OH) -CH2F group The process of synthesizing these analogs was carried out as described in Figure 3.45 Figure 3.45: Synthesis of fluorine containing bengamide analogs The synthetic process of fluorine containing bengamide analogs was first performed by the fluorinated reaction of compound BG3 with diethylaminosulfur trifluoride (DAST) at room temperature to obtain the BG3F in 42% This fluorination occurs selectively at the primary hydroxyl group The structure of was confirmed by analysis of spectral data The HRESI-MS of BG3F appeared at at m/z 263.0928 [M-H]- being suitable to the expected formula C11H16FO6 of BG3F In addition, the presence of fluorine in the structure of BG3F also expressed by the double signals induced by interaction of C-7 [δC 83.7 (d, JC-F = 166.25 Hz)], C-6 [δC 68.6 (d, 2J C-F = 18.75 Hz)] and C-5 [67.0 (d, 3J C-F = 6.25 Hz)] with the fluorine atom The fluorine BG3F derivative was then reacted with the abovedescribed aminolactams, in the presence of sodium 2-ethylhexanoate under microwave irradiation conditions at 100 W for hour obtained the 18 corresponding lactone ring-opening product Its chemical structure has also been confirmed by spectral methods, particularly by the HMBC spectrum In particular, the connection of the aminolactam ring and the polyketide chain was confirmed by the interaction of C-1, C-2 and C2’ with NH proton of the formed amide group Figure 3.50 Major interactions on COSY and HMBC spectrum of BG4Fa and BG4Fb Similar to the removal of the acetonide group in the synthesis of the bengamide A analogues described above, the deprotection of acetonide group of BG4Fa was carried out in the presence of TFA in THF, at 0oC to room temperature However, under these reaction conditions, the starting material was decomposed and the desired product was not observed Change of TFA into a solution of 1N HCl provided the desired product Accordingly, BG4Fa was obtained with a yield of 33-50% in THF for 24 hours Meanwhile, the use of MeOH instead of THF lead to BG4Fa in 19 higher yield (58 - 66%) and shorter time (5 hours) It can be observed that the use of HCl 1N in MeOH is more suitable for the removal of the acetonide group of the fluorine containing analogue bengamide 3.6 Biological activities of synthesized analogues 3.6.1 Cytotoxic activity All synthetic analogues were evaluated for their cytotoxicity against a panel of cancer cell lines (KB, HepG-2, LU-1, MCF-7, HL-60 and Hela) Cytotoxicity comparision of the tested compounds revealed that the 2’R series were generally more cytotoxic than the corresponding 2’Sepimer which possessed similar C-2’ configuration as the natural bengamides (Table 3.13) The analogues with 7-membered aminolactam rings are almost more active than the 6-membered ones These synthetic analogues were found to be less active against HL-60 and Hela cell lines in comparison with the other tested cell lines (KB, HepG-2, Lu-1 and MCF-7) Nine compounds, BG34b, BG35a, BG35b, BG36b, BG37b, BG38a, BG38b, BG39a and BG39b exhibited cytotoxic activity on all six cancer cell lines In particular, compound BG36b exhibited strong cytotoxic activity against six cancer cell lines KB, HepG-2, LU-1, MCF7, HL-60 and Hela with IC50 value of 1.1, 1.1, 0.5, 0.2, 5.4 and 2.3 μM, respectively On the other hand, compounds BG36a and BG36b exhibited selective cytotoxic activity against MCF-7 breast cancer cell 20 lines with IC50 values of 1.3 and 0.2 μM, while compounds BG37b and BG38b selectively inhibited against three cancer cell lines (KB, HepG-2 and LU-1) with an IC50 value in the range of 0.3-1.1 μM Beside that, compounds BG39a and BG39b exhibited selectively against KB and HepG-2 cancer cell lines with IC50 value of 0.4 and 1.9 μM Table 3.13 Result of cytotoxic activity of synthetic bengamide analogues IC50 (µM) Analog KB HepG-2 Lu-1 MCF-7 HL-60 Hela BG32a 22.1 >50 13.8 >50 >50 >50 BG32b 11.1 22.0 2.6 >50 >50 >50 BG33a 8.2 11.8 12.7 20.1 >50 >50 BG33b 2.4 6.6 2.6 8.8 >50 >50 BG34a 2.9 11.3 7.5 17.8 >50 39.7 BG34b 4.4 12.0 2.7 20.4 29.0 19.7 BG35a 2.3 6.6 5.8 4.3 36.8 36.8 BG35b 1.7 2.4 2.3 15.1 21.5 16.1 BG36a 21.0 21.1 4.3 1.3 >50 >50 BG36b 1.1 1.1 0.5 0.2 5.3 2.1 BG37a 11.8 >50 17.1 >50 >50 >50 BG37b 0.4 1.0 0.3 10.4 19.5 39.1 BG38a 5.7 4.8 7.7 32.5 45.6 41.5 BG38b 1.0 1.9 1.1 8.6 10.6 16.2 21 IC50 (µM) Analog KB HepG-2 Lu-1 MCF-7 HL-60 Hela BG39a 1.9 1.5 23.6 4.9 13.4 26.9 BG39b 1.1 0.4 25.1 2.7 8.9 9.5 BG41a 19.8 >50 10.0 >50 >50 >50 BG41b 5.1 21.3 17.2 >50 12.1 15.4 BG41c >50 >50 23.4 23.4 >50 >50 BG41d 23.7 >50 36.3 >50 >50 >50 BGF5a >50 >50 >50 >50 >50 >50 BGF5a >50 >50 >50 >50 >50 >50 Elipticine 1.2 1.6 1.2 2.4 2.0 1.6 Positive control: Elipticine Compounds with hydroxyl group at 5'-position exhibited lower activity than analogs without OH groups at the lactam ring Among these compounds, the two compounds BG41a and BG41b exhibited significant activity against Lu-1 and KB cell lines The remaining two compounds showed no significant activity against all six cancer cell lines On the other hand, the two fluorine compounds BG5Fa-b did not show cytotoxic activity against the six cancer cell lines In addition, the remaining fluorine compounds (BG7Fa-b, BG9Fa-b, BG11Fa-b, BG13Fa-b) were also tested against six other cancer cell lines, NCIH1299, A549, MCF-7, MDA-MB-231, Hep3B and Hela However, all of these compounds are not active This suggests that the olefinic terminal structure plays an important role in cytotoxic activity In addition, in order to understand the flexibility effects of the ketide side chain and free hydroxy groups at C-3, C-4 and C-5 to cytotoxic activity, some acetonide compounds BG24a, BG28a-b and BG31a-b have also been tested for 22 their activity against KB, HepG-2, LU-1, MCF-7, HL-60 and Hela cell lines However, all of them were much less active than their corresponding deprotected compounds No inhibiyion was observed for the restricted compounds, even at the concentration of 150 µM This could be hypothesized due to the presence of the acetonide group limited the free rotation bonds for the C-3/C-4/C-5, thereby limiting the flexibility of the polyketide chain, and two hydroxyl groups at C-3 and C-5 are no in the free state This observation is in agreement with the previously published study by Xu W and co-workers about the role of hydroxyl groups at C-3, C-4 and C-5 of the polyketide chain which are involved in the activity of bengamides by forming complex with MetAps, enzyms playing important roles in cell proliferation and angiogenesis 3.6.2 Antimicrobial activity Some bengamide analogues have been tested for strains of Gram (+) (Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus), strains of Gram (-) (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica) and Candida albicans Table 3.12 Antimicrobial activity of some bengamide analogues Analog BG32a BG34b BG35b BG37a BG37b BG38a BG38b BG39a Gram (+) MIC (µg/ml) E.faec S.aure B.cere alis us us 64 128 32 128 256 32 32 64 64 32 128 64 64 32 - Gram (-) MIC (µg/ml) E.Co P.aerugino S li sa enterica - Yeast C.albica ns 128 256 64 128 256 128 - 23 Analog Gram (+) MIC (µg/ml) Gram (-) MIC (µg/ml) Yeast E.faec S.aure B.cere E.Co P.aerugino S C.albica alis us us li sa enterica ns BG39b 32 16 64 S 256 256 128 32 256 128 T 16 64 256 256 K 128 128 64 16 C 32 Positive control: S: Steptomycin; T: Tetramycin; K: Kanamycin; C: Cyclohexamide; (-): no activity CONCLUSIONS Using various organic synthesis methods provided 30 bengamide analogues, including: 16 analogues of bengamide E, analogues of bengamide A and 10 analogues containing fluorine During the synthesis of these compounds, many reaction steps were optimized and provided better yields than previously published methods Specifically, the microwave irradiation application was first implemented to shorten the reaction time and improve efficiency in the following reactions: + Synthesis of amino lactams from the corresponding amino acids + Protection of primary amines with phthalic anhydride + Coupling of aminolactams with polyketide chains through lactone ring opening The synthesized compounds have been evaluated for cytotoxic activity against 10 cancer cell lines and antimicrobial activity The results showed that 20 analogues exhibited cytotoxicity against several cancer cell lines Some of these compounds are strongly active with IC50 less than 1μM Results of antimicrobial activity tests on bacterial strains and Candida albicans showed some good activity for 24 Gram (+) strains such as BG32a, BG34b, BG35b, BG38b and BG39b In addition, the BG35b and BG39b compounds exhibited good activity against Candida albicans This is the first study of the antimicrobial activity of bengamide compounds The evaluation of structure - activity relationships showed that the C-2’R series showed better activity than the corresponding 2'S epimer which represented similar configuration at C-2’ as natural bengamides This is the first time the benamide analogues with the C-2'R configuration have been synthesized and investigated biologically Biological comparison between diastereomer isomers is very interesting and these results could contribute to the basis for further research orientations to find more bioactive compounds of this class Cytotoxic activity demonstrates that fluorine containing analogues are not active against all of tested cancer cell lines Futhermore, the acetonide compounds are much less active than their corresponding acetonide deprotection analogues This observation pointed out that the olefinic structure, the tert-butyl group, the presence of three free hydroxyl groups at C-3, C-4 and C-5, and their flexibility play an important role to the cytotoxic activity of bengamides ... E analogues was described - 30 bengamide A and E analogues were synthesized, including 16 bengamide E analogues, bengamide A analogues and 10 fluorine containing compounds Among them, there are... 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... 16 analogues of bengamide E, analogues of bengamide A and 10 analogues containing fluorine During the synthesis of these compounds, many reaction steps were optimized and provided better yields