Tóm tắt Luận văn Synthesis and evaluation of anticancer activity of tubulysin derivatives

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING 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 1 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. 2 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. 3 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 4 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 5 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 6 CHAPTER 3. RESULTS AND DISCUSSIONS 3.1. Orientable synthesis of derivatives and analogue tubulysins 3.2. Synthesis of tubuphenylalanine Tubuphenylalanine acid (Tup) 47a was synthesized from L- phenylalanine 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 Scheme 3.1. Orientable synthesis of derivatives and analogue tubulysins 7 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. 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 Pd- catalyzed 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. Scheme 3.2. Synthesis of Tup 47a 8 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. 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 Scheme 3.6. Synthesis of 2-bromethiazole 14. 9 silver benzoate catalyst and HCl*NH(OMe)Me in triethylamine, diazoketon 21 was converted to Weinred amide 22a in 84% yield. The key step was involed in the coupling of compound 73 with 2-bromthiazole 14. Employment of strong base nBuLi in THF at -78 oC gave the thiazolylketon 74 in 56% yield. Reduction of keton 74 to 15a was used NaBH4 in MeOH/THF (1/1) or LiAlH4 gave epimeric alcohols 15a and 15b in ratio of 1:1. Alternatively, using the Corey, Bakshi and Shibato oxazaborolidine ((S)-(-)-2-methyl-CBS- oxazaborolidine) catalyst in presence of BH3*Me2S, pure diastereomer 15a was obtained preferentially (15a/15b ≈ 85/15 in ratio). Acetylation of 15a and subsequent desilylation using tetra-n-butylammonium fluoride afforded hydroxy 76. According to reported previous protocols, Scheme 3.14. Synthesis of 79 10 two-step oxidation of alcohol to carboxylic acid was performed by treatment with NaOCl and subsequent with NaClO2/NaH2PO4. However, this synthetic approach did not give product in high yield, and the pure product is needed to purify over column chromatography. In our synthetic route, hydroxy 76 was exposed with IBX under EtOAc reflux, followed by treatment by oxone in DMF at room temperature. Acid 78 was obtained quantitative yield. Methylation of 78 was carried out by treatment with diazomethane in ether at 0 oC for 12h providing ester 79. The structures were established by interpretation of their spectral data, including HRMS, 1D-NMR (1H, 13C, DEPT 135), as well as by comparison with literature data. 3.4. The removal of Boc protective group of Tuv and Tup The products 79, 47a, 42 were processed with the TFA in the presence of triisopropylsilane (TIPS) and water (TFA/TIPS/H2O = 95/2.5/2.5 in volume ratio) at 0 oC for 2h received the trifluoroacetic salt 80, 81, 82 with high performance (scheme 3.15). 3.5. Synthesis of dipeptide The reaction of Boc-N deprotected 80 with Boc-Ile-OH using HATU as coupling reaction afforded dipeptide 84 in 54% yield. (Scheme 3.17). Various coupling reagents, including DEPBT, PyBop, Scheme 3.15. Deprotected of 42, 47a, 79 11 HBTU and BOP-Cl always resulted in incomplete conversion and lower yield in compared to using HATU. Hydrolysis of methyl ester 84 with 5% LiOH gave the intermediate acid 85 in 90% yield. Acetylation of 85 in presence DMAP as catalyst afforded 86 with high performance. 3.6. Synthesis of tripeptide 3.6.1. Synthesis of tripeptide 88 Dipeptide 88 was prepared from 84 by treatment 84 with trifluoroacetic for 2h at 0oC, gave the intermediate salt. The reaction of trifluoroacetic salt with N-methylpipecolic acid (Mep) using HATU as coupling agent afforded tripeptide 88 in 55% yield. (Scheme 3.18). Structure of 88 was confirmed by analysis of the 1H-, 13C-NMR spectra. Scheme 3.17: Synthesis of dipeptide 84, 85, 86 Scheme 3.18. Synthesis of tripeptide 88 12 3.6.2. Synthesis of tripeptide 89 The coupling reaction of acid 86 with Boc-N-deprotected 82 using HATU in DMF and DIPEA for 12h at room tempareture obtained tripeptide 89 in 60% yield. Analysis of the 1H-NMR spectroscopy of 89 show that singlet signal of proton thiazole ring at 8.05 ppm (1H, H-9), signals of protons phenyl ring at 7.29-7.21 ppm (5H). The doublet signal of the proton E- olefin (H-4) at 6.67 ppm (J = 9 Hz). Singlet signal of proton CH3-N at 3.01 ppm (3H). Signal of proton acetyl group (OAc) at 2.16 ppm (3H). Singlet signals at 1.41 ppm of protons Boc group (9H). 3.6.3. Synthesis of tripeptide 90 and 90b In a similar synthesis, dipeptides 85 and 86 were coupled with Boc-N deprotected Tup (81) using HATU providing tripeptide 90b and 90 in 60-65% yield (Sheme 3.20). Structure of 90, 90b were confirmed by analysis of the 1H, 13C-NMR spectra. Scheme 3.19: Synthesis of tripeptide 89 Scheme 3.20: Synthesis of tripeptide 90 and 90b 13 3.6.4. Synthesis of tripeptide 91 and 92 Similarly, from the dipeptide 85 and 86 coupling reaction with phenylalanine methyl ester hydrochloride in the DMF and DIPEA at room temperature for 14h (Sheme 3.21) afforded tripeptide 91 and 92 in 55-60% yield. Structures of 91 and 92 were confirmed by analysis of the 1H, 13C-NMR spectra. 3.7. Synthesis of tubulysin derivatives The synthesis of tetrepeptide tubulysin derivatives was prepared from tripeptide 90 and 90b (Scheme 3.22). The deprotective reaction of 90 and 90b by treatment with TEA/TIP/H2O afforded trifluoroacetic salts. Coupling of trifluoroacetic salts.with N-methylpipecolic acid (Mep) using HATU as coupling reagent and DIPEA in DMF formed tetrapeptide 93 and 93a. Safonification of menthyl ester group of 93 and 93a was performed upon treatment with 10% KOH in THF/H2O (2/1) at 45 oC for 48 h providing N-methyltubulysin V (94). Treatment of hydroxy group of 94 with acetic anhydride in TEA at room temperature overnight afforded N-methyltubulysin U (95) in 90% yield The structures were established by interpretation of their spectral data, including HRMS, 1D-NMR (1H, 13C, DEPT 135), as well as by comparison with literature data. Sheme 3.21. Synthesis of tripeptide 91 and 92 14 Mass spectrometer HRMS-ESI of 95 was appeared molecule ion peak m/z = 728.4051 [M+H]+, (calculated for C38H58N5O7S: 728.4057), this result is compliant with C38H57N5O7S formula. The analysis of the 1H-NMR spectrum of 95 show that the signal of proton thiazole ring at 8.10 ppm (H-9), the signals of the proton phenyl ring at 7.26-7.17 ppm (5H). The doubtlet-doublet signals at 5.73-5,70 ppm features of the proton H-11. The proton's singlet signal of CH3-N on Tuv at 3.10 ppm (H-17), and signal of CH3-N on pipecolinic (H-6') at 2.40 ppm. In addition, the singlet signals characteristic of group CH3-acetyl at 2.15 ppm. The 1H-NMR spectrum of 95 show that similar with the 1H-NMR spectrum of previously published in reference [11, 48, 49]. 3.8. Synthesis of tubulysin analogues 3.8.1. Synthesis of tubulysin analogues with isoleucine substitution by leucine Tetrapeptide 96 was prepared from tripeptide 87 through out five-step reactions (Scheme 3.24). Fist, tripeptide 87 was removed Boc protection by TFA, next, coupling reaction of trifluoroacetic salt with N-methylpipecolic acid using HATU in DMF to furnish intermediate Scheme 3.22. Synthesis of tubulysin derivatives 93a,93,94,95 15 tripeptide. Safonification of tripeptide and coupling reaction with trifluoroacetic salt 81 formed tetrapeptide 96a. Acetylation 96a gave tetrapeptide 96 in overal 45% yield. The structure of 96 was defined by analysis of their spectral data, including HRMS, 1D-NMR (1H, 13C, DEPT 135). 3.8.2. Synthesis of tubulysin analogues with with the replacements of amino acid at the top of N-terminal From tripeptide 90 through out four-step reactions (Scheme 3.25), afforded tetrapeptide 97, 98. Tripeptide 90 was removed Boc protected and coupling reaction with 3-methylpicolinic acid and isoquinoline acid using HATU in DMF gave intermediate tetrapeptides that were safonified with KOH in THF/H2O, followed by acetylated reaction with acetic anhydride to provide tetrapeptides 97 and 98 . Scheme 3.24: Synthesis of tetrapeptide 96 16 Structure of 97 and 98 were confirmed by analysis of HRMS, 1H-, 13C- NMR spectra. 1H-NMR spectra of 97 and 98 have similar signal spectrum of 90. Beside, in the 1H-NMR spectra of 97 and 98 had appeared signals of proton nitrogen heterocyclic of 3-methylpicolinic and isoquinoline. In similar strategy, tetrapeptide 99, 100, 101 were prepared from tripeptide 90 and 90b by treatment with TFA/TIPs/H2O and coupling reaction with 5-methylpyrazin cacrboxylic acid and N- allylpipecolic acid providing 99, 100 and 101 in 55-60% yield. (Sheme 3.26 and 3.27) Scheme 3.25. Synthesis of tetrapeptide 97, 98 Scheme 3.26. Synthesis of tetrapeptide 99 17 The structure of the 99, 100 and 101 were confirmed by analysis of the HRMS-ESI, 1H-, 13C-NMR spectra. 3.8.3. Synthesis of tubulysin analogues with the replacements of amino acid at the top of C-terminal. From tripeptide 89, coupling reaction with N-methylpipecolic acid afforded tetrapeptide 102 in 60% yield. Saponification of ethyl ester with 5% LiOH in THF/H2O at room temperature provided acid 103 in 92% yield (Scheme 3.28) In another strategy, owing to the complexity of the synthesis of Tup amino acid, a replacement of this amino acid by 4-(2- aminoethyl)benzen-sulfonamide was carried out. Saponification of 88 with LiOH in THF/H2O and coupling reaction with 4-(2- Scheme 3.27. Synthesis of tetrapeptide 100, 101 Scheme 3.28: Synthesis of tetrapeptide 102 and 103 18 aminoethyl)benzen-sulfonamide using HATU in DMF followed by acetylation with acetic anhydride in TEA afforded 104 in the best yield (Scheme 3.29). The structure of the 102, 103 and 104 were confirmed by analysis of the HRMS-ESI, 1H-, 13C-NMR spectra. 3.8.4. Synthesis of tubulysin analogues with the replacement of amino acids at the top of N-and C-terminal 3.8.4.1. Synthesis of tubulysin analogues from tripeptide 89 Coupling reaction of trifluoroacetic salt of 89 with 5- methylpyrazin-1-carboxylic and isoquinoline -1-carboxylic acid using HATU in DMF afforded tetrapeptide 105 and 106 in 55-65% yield (Scheme 3.30). Next, saponification of ethyl ester of 106 with 5% LiOH in THF/H2O at room temperature for 12h provided acid 107 in quantitative yield (93%) (Scheme 3.31) Scheme 3.29. Synthesis of tetrapeptide 104 Scheme 3.30. Synthesis of tetrapeptide 105, 106 19 The structure of the 105, 106 and 107 were confirmed by analysis of the HRMS-ESI, 1H-, 13C-NMR spectra. 3.8.4.2. Synthesis of tubulysin analogues from tripeptide 91 The tripeptide 91 was treated with TFA/TIPS/H2O at 0 oC for 2h gave trifluoroacetic salt 91a in high yield. The coupling reaction of 91a with 5-methylpyrazin cacboxylic acid using HATU in DMF at room temperature afforded tetrapeptide 108 in 60% yield (Scheme 3.32). Hydrolysis of methyl ester with 5% LiOH in THF/H2O provided acid 109 in 90% yield. Scheme 3.31. Synthesis of acid 107 Scheme 3.32. Synthesis of tetrapeptide 108 and 109 20 In a similar synthesis, trifluoroacetic salt of 92 was coupled with 5-methylpyrazin carboxylic acid providing ester 101 in 65% yield (Scheme 3.34) 3.9. Cytotoxic activity of derivatives and tubulysin analogues Tetrapeptides were evaluated for their cytotoxic activity in human breast carcinoma MCF7, human lung adenocarcinoma A549, human colorectal adenocarcinoma HT29, human acute leukemia HL60, human colon carcinoma SW480. The ellipticine was used as a positive control in assays. Table 3.1. Cytotoxic activity of derivatives and tubulysin analogues No Compounds IC50 (μM) HT29 A549 MCF-7 SW480 HL-60 1 93a 1.45 1.46 3.26 0.91 0.43 2 94 4.38 4.61 4.83 1.99 1.47 3 95 0.56 0.42 0.68 0.25 0.14 4 96 4.76 6.82 9.88 n.d n.d 5 97 32.37 44.75 34.53 33.28 26.54 6 98 15.12 17.91 13.34 13.13 10.57 7 99 > 50 > 50 > 50 n.d n.d 8 100 > 50 > 50 > 50 n.d n.d 9 101 24.38 25.87 29.84 n.d n.d 10 102 0.27 0.30 0.23 0.11 0.08 11 103 2.60 3.33 4.49 2.32 1.21 12 104 > 50 > 50 > 50 > 50 > 50 Scheme 3.34. Synthesis of ester 110 21 * n.d: Not determined The results in table 3.1 show that modification of Mep to nitrogen heterocyclic acid provide inactivity or weak cytotoxic on the tested cancer cell lines, indicating that the tertiary amine functionality at the N-terminal peptides is requisite for high cytotoxic activity of all tubulysin analogues. N-methyltubulysin U (95) is about 7-fold more active than its unacetyleted compound 94. Surprisingly, tetrapeptide 93a that links to menthyl group is tronger than 94 by three magnitude, and weaker 95 by two order of magnitude on the tested cancer cell lines. This is may be its increased lipophilicity and potential to across cell membrane and indicating that the important of the OAc group on the tubuvaline fragment for activity of tubulysin analogues. The replacement of α,β-unsaturated tubuphenylalanine to produce 102 and 103 was not significant decreased cytotoxicity. Unacetylated peptide 103 is about 5-fold less active than N- methyltubulysin U (95). Interestingly, analogues 102 is significant increased cytotoxicity by 2 to 3 order of magnitude compared to N- methyltubulysin U (95). This results indicating the modifications of tubulysin with Tup fragment are tolerated. In addition, synthesis of tubulysin analogues with the replacement of Tup fragment and Mep by phenylalanine and 5-methyl- 2-pyrazinecarboxylic acid have moderate or weak cytotoxicity. 13 105 28.57 39.14 27.13 19.37 15.98 14 106 > 50 > 50 > 50 n.d n.d 15 107 > 50 > 50 > 50 n.d n.d 16 108 > 50 > 50 > 50 n.d n.d 17 109 7.57 9.38 12.46 n.d n.d 18 110 42.91 29.20 29.45 n.d n.d 19 Ellipticine 0.32 0.36 0.35 0.31 0.33 22 CONCLUSION 1. The thesis has used modern organic synthetic methods for synthesis of ɤ-amino acid tubuvaline and tubuphenylalanine, such as: Horner-Wadsworth-Emmons reaction, Arndt-Eistertn, Sandmeyer, Dondoni, Hantzsch, Steglich and synthesize Weinreb amide. 2. Designed and synthesized 4 dipeptides and 6 tripeptides were new intermediate compounds of tubulysin (include 84 - 92 compounds), and 19 tetrapeptides (have 17 new tetrapeptides) are derivatives and tubulysin analogues, include: + 04 tubulysin derivatives (93, 93a, 94 and 95 compounds), two new compounds 93 and 93a. + 01 new tubulysin analogue with the replacement of isoleucine by leucine (96 compound). + 05 new tubulysin analogues with the replacement of the N- terminal, include 97, 98, 99, 100, 101 compounds. + 03 new tubulysin analogues with the replacement of the C- terminal, include 102, 103, 104 compounds. + 06 new tubulysin analogues with the replacement of the N- and C- terminal, include 105, 106, 107, 108, 109, 110 compounds. 3. The structures of all products have been confirmed by the analysis of their spectral data, include HRMS, 1D-NMR (1H- ,13C, DEPT 135) spectra data. 4. The synthesized tetrapeptide (18 compounds) have tested their biological activity against cancer cell lines HT29, A549, MCF-7, HL60 and SW480. The results show that modification of Mep to nitrogen heterocyclic acid provide inactivity or weak cytotoxicity. Compounds that link the Mep at the N-terminal and have Tup or replacement of the Tup fragment by α,β-unsaturated 23 tubuphenylalanine ( in 93a, 94, 95, 102, 103 compounds) providing good cytotoxic activity on all tested cancer cells lines. THE NEW CONTRIBUTIONS OF THESIS 1. The thesis has chosen simple conditions and providing higher performance in the synthesis of two ɤ-amino acids tubuvalin and tubuphenylalanine as well as tubulysin derivatives. 2. Designed and synthesized ten new intermediate compounds of tubulysin (inculude 04 dipeptides and 06 tripeptides) and 17 new tetrapeptides are derivatives and tubulysin analogues. 3. Determined cytotoxic activity of 16 new tetrapeptides on human cancer cell lines (include HT29, A549, MCF-7, HL60 and SW480 lines). Detected 3 new tetrapeptides including 102, 103 and 93a compounds performed remarkable cytotoxic activity. In which, cytotoxic activity of 102 compound (IC50 : 0.27- 0.08 µM) is more potential than N-Methyltubulysin V (94) and N-Methyltubulysin U (95). 24 THE PUBLISHABLE LISTS OF THESIS 1. Hai Le Van, Loc Tran Van, Anh Tran Tuan, Thao Tran Thi Phuong, Sung Tran Van, Chien Tran Van. Biological Activity of Tubulysin Analogues. Tetrahedron, 2020. (Manuscript submitted) 2. Hai Le Van, Loc Tran Van, Anh Tran Tuan, Thao Tran Thi Phuong, Sung Tran Van, Chien Tran Van. Total synthesis and cytotoxicity evaluation of tubulysin analogues containing nitrogen heterocyclic acids. Natural Product Research, 2020. (Manuscript submitted) 3. Le Van Hai, Tran Tuan Anh, Tran Van Loc, Tran Van Chien. Synthesis of tubuphenylalanine acid (tup) derivatives. Journal of Chemistry, 2019, 57 (4E3, 4), 31-34. 4. Le Van Hai, Tran Tuan Anh, Tran Van Loc, Tran Van Chieng. Stereoselective synthesis of the tubuphenylalanine acid (Tup) of tubulysin. Journal of Chemistry, 2017, 55 (3), 384-387.