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MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCES AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ----------------------------- LE VAN HAI SYNTHESIS AND EVALUATION OF ANTICANCER ACTIVITY OF TUBULYSIN DERIVATIVES Major: Organic Chemistry Code: 9.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS HA NOI, 2020 The thesis was completed at: Institute of Chemistry Vietnam Academy of Science and Technology Supervisor: 1. Asso. Prof. Dr. Tran Van Loc Institute of Chemistry-Vietnam Academy of Science and Technology 2. Dr. Tran Van Chien Institute of Chemistry-Vietnam Academy of Science and Technology Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be presented in front of the doctoral thesis council at: Graduate University of Science and Technology - Vietnam Academy of Science and Technology- 18 Hoang Quoc Viet Road, Cau Giay, Hanoi. At time ........, …………………… The thesis can be found at: - Library of Academy of Science and Technology - National Library of Vietnam OPENING 1. The essential of the thesis The natural compounds have considered infinite supplies of bioactive substances for research and medical application. The abundance and diversity of the substance-frames had highly bioactive. Especially, the mechanism of action of each layer of these frames attracted of researchers so far. For the purpose of research on drug development, compounds from microbial sources are often achieved by total synthesis or will be modified in chemical structure to create large quantities of products as well as new derivatives. Tubulysins are the tetrapeptide class (figure 1.8) isolated from two myxobacteria strains Angiococcus disciformis An d48 and Archangium gephyra Ar 315. Studies have shown that tubulysins are the best class of anticytokines known to nowaday. The cancer cell inhibitory of tubulysins are show a wide range of human cancer cell lines such as ovarian cancer, breast cancer, prostate cancer, colon, lung and blood cancer. In vitro and in vivo studies have shown that tubulysins inhibit the growth of cancer cells higher than anticancer drugs in use such as vinblastine, epothilone or taxol by approximately 20- to 1000 fold. Figure 1.8. Chemical formula of the tubulysins From the point of view of pharmaceutists, natural tubulysins are a leading class for research and development of new anticancer drugs. However, their content in bacteria are very low, which were not enough for intensive research. Thus, the total synthesis of tubulysins and their derivatives are very necessary and scientific significance. 1 2. Aim of the thesis Synthesis of several derivatives and tubulysin analogues with the replacement of the amino acid methylpipecolic (Mep) at the N-terminal, replacing N,O-acetyl group by methyl group, and researching the role of tubuphenylalanine (Tup) group at the C-terminal . Evaluation of cytotoxic activity on a number of cancer cell lines in order to further clarify the correlation between the activity and the structure of the tubulysins, and to find new compounds with remarkable activity. 3. The main research content of the thesis + Overview of the myxobacteria. Overview of tubulysin: Biological activity, active-structure correlation. + Overview of synthesis of ɤ-amino acid tubuphenylalanine (Tup) and tubuvaline (Tuv). + Synthesize Tuv + Synthesize Tup + Synthesize dipeptides. + Synthesize tripeptides. + Synthesize tetrapeptides (derivatives and analogues of tubulysin) + Evaluation of cytotoxic activity of tetrapeptides Layout of the Thesis The thesis includes 133 pages: Opening (2 pages), Chapter 1: Overview (28 pages), Chapter 2: Research Methods and Experimental (37 pages), Chapter 3: Results and discussions (55 pages), conclusions (1 page), The list of published related to thesis (1 page). The reference contains of thesis have 96 documents, which were updated until the year 2020. The appendix 81 pages have composed the spectrum of synthetic substances. 2 CHAPTER 1. OVERVIEW 1.1. Myxobacteria, substances and biological activity 1.2. Microtube in drug research 1.3. Tubulysin: Isolation, determination of structure, biosynthesis and biological activity. Figure 1.10. Chemical formulas of natural tubulysins 3 1.4. Synthesis of ɤ-amino acids of tubulysin 1.5. The structure and biological activity correlation of the tubulysins CHAPTER 2. RESEARCH METHODOLOGY AND EXPERIMENTAL 2.1. Research methodology 2.1.1. Organic synthetic methods 2.1.2. Determination of organic compounds structure 2.1.3. Evaluation of cytotoxic activity 2.2. Experimental 2.2.1. Chemicals and solvents 4 2.2.2. The general scheme of synthesis of tetrapeptides 2.2.3. Synthesis of tubuphenylalanine 2.2.4. Synthesis of tubuvaline 2.2.5. Remove of Boc protected group 2.2.6. Synthesis of dipeptides 2.2.7. Synthesis of tripeptides 2.2.8. Synthesis of tubulysin derivatives 2.2.9. Synthesis of tubulysin analogues 2.2.10. Evaluate cytotoxic activity of tetrapeptide 5 CHAPTER 3. RESULTS AND DISCUSSIONS 3.1. Orientable synthesis of derivatives and analogue tubulysins Scheme 3.1. Orientable synthesis of derivatives and analogue tubulysins 3.2. Synthesis of tubuphenylalanine Tubuphenylalanine acid (Tup) 47a was synthesized from Lphenylalanine methyl ester hydrochloride (68), through 7-step of reactions process as described in scheme 3.2. First, the amino group of 68 was protected with tert-butoxycarbonyl group via the reaction with Boc2O in the THF and NaHCO3 at room temperature for 16 h to obtain the product 45 (94%). The conversion of ester 45 to aldehyde 52 were 6 effectuated through two steps reaction, by treated with NaBH4 in MeOH at room temperature during 20h, the N-Boc-phenylalaninol (45a) was obtained in 96% without purification. Next, conversion of hydroxy 45a to aldehyde 52 was alternatively performed by refluxing 45a with oxidative reagent IBX in EtOAc for 5h. Aldehyde 52 was obtained in quantitative yield. Scheme 3.2. Synthesis of Tup 47a The Horner-Wadsworth-Emmons reaction of aldehyde 52 with triethyl-2-phosphonopropionate and NaH in THF at 0oC to 25oC for 14h provided α,β-unsaturated ester 42, with E-isomer as a mạjor product in 75% yield. Configuration of E-isomer of ester 42 was determined based on data analysis of the 1H-NMR and NOESY spectrum, and comparison with the spectrum of the substance has been published. Transesterification of 42 to acid 69 by saponification, and menthyl esterification furnished α,β-unsaturated menthyl ester what Pdcatalyzed hydrogenation afforded diastereomers 47a and 47b in ratio 4:1, which were easily separated by flash chromatography to provide pure desired major diastereomer of 47a. 7 Analysis of 1H-, 13C-NMR spectra, and combined with published of spectral comparison for confirmed structure of 47a. 3.3. Synthesis of ɤ-amino acid tubuvaline 3.3.1. Synthesis of 2-Bromo-4-((tert-butyldimethylsilyloxy) methyl)thiazole (14) 2-bromethiazole 14 was prepared by reaction of thioure with ethyl bromopyruvate under EtOAc refluxing for 4h (Scheme 3.6). Conversion of 70 to 71 was proceeded by treatment with NaNO2, CuSO4 and KBr in the presence of H2SO4 30% at 0 oC to room temperature for 14h (Sandmeyer reaction), Ester 71 wass obtained with 65 % yield. Scheme 3.6. Synthesis of 2-bromethiazole 14. Ester 71 was reduced to hydroxy 72 that was protected with TBS by treatment with TBSCl in DCM and presence of DMAP as a catalyst in 12h at room tempareture afforded 14 with 97% yield. 3.3.2. Synthesis of N-methyltubuvaline-OMe (79) The synthesis of tubuvaline (Tuv) fragment was started from Boc-Val-OH (Scheme 3.14). The homologation reaction of valine amino acid was carried out by first treatment with ethyl chlorofomate in TEA at 0oC for 2 h, followed by exposure to diazomethane in ether to provide diazoketon 21. Under Wolff rearrangement conditions using 8