Summary of chemistry doctoral thesis: Synthesis and bioactivity evaluation of new vinca alkaloid derivatives

The objectives of the thesis: Synthesis of new vinca alkaloid derivatives containing different substituents on C-3'' and N-6'' positions in ring D of the velbanamine subunit, concurrently evaluating the biological activity of the derivatives synthesized.

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY - VO NGOC BINH SYNTHESIS AND BIOACTIVITY EVALUATION OF NEW VINCA ALKALOID DERIVATIVES Major: Organic chemistry Code: 9.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Ha Noi - 2018 The thesis was completed in Graduate University Science and Technology, Vietnam Academy of Science and Technology Supervisor 1: Assoc.Prof Dr Ngo Quoc Anh Supervisor 2: Dr Doan Duy Tien 1st Reviewer: 2nd Reviewer: 3rd Reviewer: The thesis will be presented before the Council for Evaluation of Ph.D thesis at the Academy, meeting at Graduate University Science and Technology, Vietnam Academy of Science at …………… PUBLICATIONS Ngo Quoc Anh, Vo Ngoc Binh, Nguyen Le Anh, Nguyen Van Tuyen Synthesis and antitumor activity of new vinca-alkaloid mimicking sarcodictyin features Viet Nam Journal of Chemistry, 2014, 52(6A) 242-246 Q A Ngo, L A Nguyen, N B Vo, T H Nguyen, F Roussi and V T Nguyen Synthesis and antiproliferativeactivity of new vinca alkaloids containing an α, β-unsaturated aromatic side chain, Bioorganic & Medicinal Chemistry Letters, 2015, 25, 5597-5600 Vo Ngoc Binh, Nguyen Le Anh, Nguyen Thuy Hang, Tran Thi Yen, Ngo Quoc Anh Stereoselective synthesis of new dihydrocyanoanhydrovinblastine derivatives, Viet Nam Journal of Chemistry, 2016, 54(6e2), 180-183 Vo Ngoc Binh, Nguyen Le Anh, Nguyen Thuy Hang, Tran Thi Yen, Ngo Quoc Anh Synthesis of new vinca-alkaloids derivatives from 3'cyanoanhydrovinblastine, Viet Nam Journal of Chemistry, 2016, 54(6e2), 184-188 N B Vo, L A Nguyen, T L Pham, D T Doan, T B Nguyen and Q A Ngo Straightforward access to new vinca-alkaloids via selective reduction of a nitrile containing anhydrovinblastine derivative, Tetrahedron Letters, 2017, 58, 2503-2506 Vo Ngoc Binh, Nguyen Le Anh, Nguyen Thuy Hang, Tran Thi Yen, Ngo Quoc Anh Synthesis and antitumor activity of new vinca alkaloids from 3’-cyanoanhydrovinblastine, Viet Nam Journal of Chemistry, 2018 (Just Accepted Manuscript) INTRODUCTION The urgency of the thesis Cancers are a group of diseases characterized by uncontrolled growth and spread of abnormal cells The worldwide incidence of cancer is estimated at 14 million new cases every year Tremendous resources are being invested all around the world for developing preventive, diagnostic, and therapeutic strategies for cancer Several pharmaceutical companies and government/non-government organizations are involved in the discovery and development of anticancer agents Vinca alkaloids are isolated from Madagascar periwinkle, Catharantus roseus G Don, containing about 130 terpenoids of indole alkaloid Their clinical value was recognized in the early 1965s So the compound has been used as an anti-cancer agent for more than 40 years and is a leading compound for drug development Today, two natural compounds, vinblastine (VLB) and vincristine (VCR) and two semi-synthetic derivatives, vindesine (VDS) and vinorelbine (VRLB), have been approved for use in the United States Due to the importance of pharmaceuticals and the low extraction of VLBs, VCRs and other alkaloids, Catharanthus roseus has become one of the most studied medicinal plants The research efforts of scientists to find more compounds with lower toxicity and higher therapeutic potential are continuing Based on research results and the urgency in practice, we have carried out the thesis "Synthesis and Bioactivity Evaluation of New Vinca-alkaloid Derivatives" The objectives of the thesis Synthesis of new vinca alkaloid derivatives containing different substituents on C-3' and N-6' positions in ring D of the velbanamine subunit, concurrently evaluating the biological activity of the derivatives synthesized The research methods All the compounds were synthesized following modern synthetic methods with some improvements to adapt with each specific situation The synthesized products were purified by column chromatography and their structures were determined by modern spectroscopic methods: IR, HR-MS, NMR Biological activity was evaluated by Monks method on two KB and HepG2 cell lines Tests on the leukemia cell line HL-60 with regard to cytotoxicity, proliferation, apoptosis and cell cycle were performed at the Institute of Pharmacology and Toxicology, University of Würzburg, Germany Molecular docking studys were performed at Department of Chemistry and Laboratory of Computational Chemistry and Modelling, Quy Nhon University The new contributions of this thesis  23 new vinca alkaloid compounds have been synthesized from natural vinca alkaloids such as catharanthine, vindoline, vinblastine and vincristine, including: – 12 quaternary ammonium salts of anhydrovinblastine, vinblastine, vincristine and 18 (S) -3 ', 5'-dimethoxyanilineecleavamine 81a - 84c – 11 new derivatives of 3'-cyanoanhydrovinblastine include vinca alkaloid derivatives 92a-92e via reduction selective 3'cyanoanhydrovinblastine 88 vinca alkaloid derivatives 93a-93f via reductive alkylation aminomethyl 92c  The first time, the chemical shifts of proton and carbon is fully assigned to the 3'-cyanoanadrovinblastine 88 compound and the absolute configuration at the C-3 'position of compound 88 A new synthetic method of compounding 88 gives higher yield than the old synthetic method (74% versus 32%)  The structure of the new compounds was determined by 1D-NMR, 2DNMR, IR and HRMS data In particular, For the first time using the 2D-NMR spectra: COSY, HSQC, HMBC, NOESY determine the stereochemistry of the five new compounds 92a - 92e  23 new derivatives were tested for cytotoxic activity on two KB and Hep-G2 cell lines As a result, the four compounds 83a, 83b, 84a, 84b exhibited selective and potent cytotoxicity on the KB cell line with the IC50 equivalent to vinblastine and vincristine The three new vinca alkaloid derivatives 81a-c from anhydrovinblastine 12 had better cytotoxic activity KB than 12 and even better than Ellipcitine in case 81b The simple vinca alkaloids compounds 82a-c by replacing vindoline with 3,5-dimethoxyaniline (DMA) did not lose their activity but significantly improved their activity compared to 18 (S) -3' 5'-dimethoxyanilineecleavamine 77  Eight potent cytotoxic compounds 81a–c, 82a-b, 92a-c were selected for docking on tubulin Results showed that 02 new vinca alkaloid derivatives 92b and 82a have the strongest cytotoxic activity also have the strongest affinity with tubulin, equivalent to standard vinblastine  The first time, 02 chlorochablastine 83b and chlorochacristine 84b were tested for biological mechanisms in apoptosis and cell cycle, proliferation compared to commercial vinca alkaloids The results of the two selected compounds have the same effect as vinflunine, which is the latest commercially available semi-synthesis vinca alkaloid, which opens up the possibility of further research into these compounds for clinical use The main contents of the thesis The thesis consists of 138 pages: Introduction: pages Chapter 1: Overview (27 pages) Chapter 2: Experimental (38 page) Chapter 3: Results and discussion (52 pages) Conclusions: (1 pages) The reference section consists of 16 pages of documents cited, documents updated to 2018 CHAPTER OVERVIEW 1.1 Microtubule - An important target for the treatment of cancer drugs 1.1.1 Definitions 1.1.2 Dynamics of microtubule 1.1.3 Classification drugs interfered with microtubule 1.2 Vinca alkaloid 1.2.1 Introduction of vinca alkaloids 1.2.2 Synthesis of vinca alkaloids 1.2.2.1 Semisynthesis of vinca alkaloids 1.2.2.2 Total synthesis of vinca alkaloids 1.2.2.3 Biosynthesis and biotechnological approaches 1.2.3 The structure-activity relationship of vinca alkaloids 1.2.3.1 Modifcations of the vindoline moiety 1.2.3.2 Modifcations of the velbanamine moiety 1.2.4 Clinical applications of vinca alkaloids 1.3 Orientation and objectives of the thesis CHAPTER EXPERIMENTAL 2.1 Chemicals and equipment 2.1.1 Chemicals and solvents 2.1.2 Research equipment 2.1.2.1 Infrared Spectroscopy IR 2.1.2.2 Nuclear Magnetic Resonance Spectrum NMR 2.1.2.3 Mass spectrometry MS and HRMS 2.1.2.3 Specific rotation [α]D 2.2 Research methods 2.2.1 Organic synthesis methods 2.2.2 Biological Activity Methods 2.2.3 Methods of refining and determination structure 2.3 Synthesis of some new vinca alkaloid derivatives contain α,βunsaturated ketone 2.3.1 Synthesis of anhydrovinblastine 12 2.3.2 Synthesis of 18(S)-3’,5'-dimethoxyanilinecleavamine 77 2.3.3 Synthesis of some new vinca alkaloid derivatives contain α,βunsaturated ketone 2.4 Synthesize of some new vinca alkaloid derivatives from 3'cyanoanhydrovinblastine 88 2.4.1 Synthesis of 3’-cyanoanhydrovinblastine 88 2.4.2 Synthesis of new vinca alkaloid derivatives via selective reduction of 3'cyanoanhydrovinblastine derivative 88 2.4.2.2 Synthesis of 3'R-cyano-(4’S,5’-dihydro)-anhydrovinblastine 83a 2.4.2.2 Synthesis of 3'R-cyano-(4’R,5’-dihydro)-anhydrovinblastine 92b 2.4.2.3 Synthesis of (3'R-aminomethyl)-(4’S,5’-dihydro)-anhydrovinblastine 92c 2.4.2.4 Synthesis of 3'S-cyano-4-deacetyl-anhydrovinblastine 92d and 3'Scyano-4-deacetyl-3-hydroxymethyl-anhydrovinblastine 92e 2.4.3 Synthesize of some new vinca alkaloid derivatives through the reductive alkylation of aminomethyl 92c 2.5 Cytotoxic activity evaluation methods Biological activity was evaluated by Monks (1991) method on two KB and HepG2 cell lines Tests on the leukemia cell line HL-60 with regard to cytotoxicity, proliferation, apoptosis and cell cycle were performed at the Institute of Pharmacology and Toxicology, University of Würzburg, Germany Molecular docking studys were performed at Department of Chemistry and Laboratory of Computational Chemistry and Modelling, Quy Nhon University CHAPTER RESULTS AND DISCUSSION 3.1 Synthesis of some new vinca alkaloid derivatives contain α,βunsaturated ketone α,β -unsaturated ketones are compounds found in nature such as alkaloids, terpene, sesquiterpenes, triterpenoids, chalcones and flavones such as daphniapylmines in Daphliphyllum paxianum, myrtenal from Citrus reticulata, zerumbone from Zingiber zerumbet, licorisoflavane A, quercetin, kaemferol in Morus alba L or isolaquirigenin from Glycyrrhiza glabra, Curcumin from Curcuma longa L In particular, sarcodictyin 71, 72 and eleutherobin 73 were isolated from some soft corals, Even the DNA of the living organism is made up of compounds containing α, β-saturated ketones such as thymine and uracil The α,β-saturated ketone group plays an important role both in terms of chemistry and biology Chemically, α,β-unsaturated ketones are the key intermediates for the synthesis of many important substances, such as flavonoids, pyrazoline, diazepines, pyrimidines, etc Biologically, compounds containing α,β-unsaturated ketones have been identified as having many biological activities including anti-inflammatory activity, anti-malarial activity, anti-parasitic activity, anti-parasitic activityhypotension or NF-κB elimination causes a variety of diseases, particularly cytotoxic activity, which is considered by the Michael acceptor for thiol groups of certain proteins or the ability to orient the cancer cells apoptosis Therefore, compounds containing ketone α, βunsaturated ketone are always attractive subjects of scientists, some drugs containing this group have also been used effectively in the treatment of diseases such as AZT, Edoxudine, Zalcitabine, Griseofulvin and many other naturally occurring substances are used in the treatment of cancer Figure 3.3 Hybridization of ketone α,β-unsaturated and vinca alkaloids Thus, we aimed to elaborate a new series of vinca-alkaloids that contains an α,β -unsaturated aromatic side chain linked to the tertiary amine of velbanamine via an ammonium salt in order to determine their anti-cancer activity The synthesis of additional simplified compounds was also envisaged, replacing the vindoline moiety by a simplified aromatic (3,5-dimethoxyaniline – DMA) Scheme 3.1 General procedure for the synthesis of compound 76a–c Reagents and conditions: (a) ArCHO, MeOH, room temp.; (b) NBS, p-TsOH, CH3CN, rt First of all, three various α,β-unsaturated aromatic compounds 76a–c were elaborated in a straightforward manner according to a generic procedure outlined in Scheme 3.1 The synthesis started by a Claisen–Schmidt condensation of arylcarboxadehyde and acetone in methanol at room temperature followed by a selective monobromination of the transient αmethylketons 75a–75c using N-bromosuccinimide in the presence of ptoluensulfonic acid at room temperature for h These afforded 76a–c in 70– 73% yields for two steps Scheme 3.2 Synthesis of vinca alkaloids 12 77 Reagents and conditions: (a) (i) Vindoline (V) or 3,5-dimethoxyaniline (DMA), FeCl3, glycine-NaCl 0,1M, HCl 0,1 N, (ii) NaBH4, NH4OH Compounds 12, 77 are synthesized according to the method described previously by Vukovic with good yield (76-85%) Accordingly, we performed a coupling reaction between catharanthine and vindoline (or 3,5dimethoxyaniline) in the presence of iron ion in acidic water, then reduced by NaBH4 to obtain compound 12 and 77 (Scheme 3.2) In organic Chemistry, the Menshutkin reaction is an easy and effective way to convert a tertiary amine to a quaternary ammonium salt through an alkylhalide By the Menshutkin reaction, twelve new ammonium salts 81a–84c were then obtained by stirring one equivalent of the alkylbromide 76a–c at room temperature in THF with various vinca compounds that is, either anhydrovinblastine 12, 18(S)-30,50-dimethoxyanilinecleavamine 77, vinblastine and vincristine (Scheme 3.4) All the final compounds 81a–84c were obtained in 63–72% yields Scheme 3.4 Synthesis of new vinca alkaloid containing α,β-unsaturated ketone Compounds are fully described using 1D, 2D NMR and high-resolution mass spectrometry HR-EI-MS In general, when compared to the spectrum of the original compound, significant changes in their NMR spectrum were observed around the N-6' position, especially for the 5', 7', 19' and 22', the proton and carbon resonances on the vidoline moiety change insignificantly Vinca alkaloids are complex molecules, so the assignment of the NMR spectrum of vinca alkaloids must be approached with caution Structural analyzes of the obtained compounds are approached in terms of structural part in the molecule, firstly the vindoline part and then velbanamine part contain the α,βsaturated ketone interacted and interacted with the H-21 proton HMBC spectra appear to have proton interactions at 3.81 (s, 1H, H-2) with carbon atoms at 38.1 (N-CH3), 45.5 (C-11), 53.5 C-12), 76.5 (C-4) and 79.9 (C-3) The singlet signal at 5.43 (s, 1H) is assigned to the H-4 proton due to this proton next to the –OCOCH3 group, which moves towards the lower field The HMBC spectrum, H-4 proton interacts with carbon atoms at 30.9 (C-20), 42.7 (C-5), 129.7 (C-6) and 171.1 (C3-COOCH3) The singlet resonance signal at 2.83 (s, 1H) is assigned to the H-19 proton, the HMBC spectrum, the H-19 interacts with 30.9 carbon atoms (C-20), 50,1 (C -10), 53.5 (C-12), 76.5 (C-4) and 83.1 (C-2) The COSY spectrum, H-10 protons interact with the H-11 Figure 3.6 Structure and numbering according to IUPAC in the velbanamine half Table 3.2 NMR data in the velbanamine half of compound 81b and anhydrovinblastine 12 in CDCl3 Position 1’ 2’ 3’ 4’ 5’ 7’ 8’ 9’ 10’ 11’ 12’ 13’ 14’ Compound 81b δH, J (Hz) 2,63 (m, 1H) 3,12 (m, 1H) 2,02 (m, 1H) 5,60 (s br, 1H) δC 33,85 Anhydrovinblastine 12 δH, J (Hz) 2,40 (m, 1H) 3,04 (m, 1H) 1,30 (m, 1H) 5,45 (s, 1H) 30,68 121,8 132,87 64,35 3,28 (m, 1H) 3,52 (d, J = 16,0, 1H) 53,40 3,4 (m, 1H) 3,4 (m, 1H) 19,93 3,05 (m, 1H) 3,41 (m, 1H) 107,66 129,1 117,44 7,51 (d, J = 7,7, 1H) 120,6 7,20 – 7,10 (m, 1H) 123,6 7,20 – 7,10 (m, 1H) 111,27 7,20 – 7,10 (m, 1H) 4,45 (m, 1H) 4,56 (m, 1H) 4,45 (m, 1H) 4,47 (m, 1H) 3,30 (m, 1H) 3,87 (m, 1H) 7,57 (d, J = 8,1, 1H) 7,16 – 7,26 (m, 1H) 7,16 – 7,26 (m, 1H) 7,16 – 7,26 (m, 1H) 10 δC 34,3 32,9 123,5 140,0 52,1 54,3 25,9 117,3 129,4 118,3 122,2 118,3 110,5 15’ N-H 17’ 18’ 19’ 20’ 21’ 22’ 23’ 24’ 25’ 26’ 27’, 31’ 28’, 30’ 29’ C18’-COOCH3 C18’-COOCH3 134,75 8,36 (s, 1H) 135,0 8,04 (s, 1H) 132,8 54,6 63,09 4,11 (m, 1H) 4,53 (m, 1H) 2,04 (m, 2H) 27,26 1,07 (t, J = 7,4, 3H) 3,68 (s, 1H) 2,55 (br d, J = 14,0, 1H) 3,31 (m, 1H) 1,92 (dd, J = 14,5/7,5, 1H) 0,98 (t, J = 7,5, 3H) 11,50 70,5 191,18 123,90 147,27 132,1 130,6 129,4 137,9 173,09 52,83 3,62 (s, 3H) 6,90 (d, J = 16,5, 1H) 8,25 (d, J = 16,5, 1H) 7,66 (d, J = 8,4, 2H) 7,40 (d, J = 8,5, 2H) 3,68 (s, 3H) 131,0 55,5 45,9 27,8 12,2 174,6 53,3 In the velbanamine half, the N-H and H-3 signals can be easily located at 8.36 (s, 1H) and 5.60 (s br, 1H) Using H-3' as the starting point, it is possible to identify protons H-1', H-2' and H-19' based on COSY spectra Three resonance signal methyl protons H-21', C18'-COOCH3 at 1.07 (t, J = 7.4 Hz, 3H) and 3.68 (m, 3H) were not significantly different than in anhydrovinblastine 12 The COSY spectrum, proton methylene H-20' resonance at 2.02 ppm interacts with the H-21' protons and allylic interaction with the H-3' protons at 5.60 ppm The chemical shift of proton methylene H-20' is consistent with its allylic character Two resonant signals at 4.45 and 4.56 ppm are assigned to the H-5' protons because these two protons are weakly interacting with the two H-20 protons The remaining protons in the velbanamine half are H-7', H-8' and aromatic protons of the indole ring The COSY spectrum, the two H-7' protons are weakly interacting with the H-5' protons and interacting with the two H-8 protons Chemical shift of the two protons H-8' at 3.30 ppm and 3.87 ppm is not much different than in anhydrovinblasstine 12, whereas the chemical shift of the two H-7' protons in compound 81b at 4.45 and 4.56 ppm differed significantly from the two H-7' protons in anhydrovinblastine 12 at 3.61 and 3.44 ppm, which may be due to substitution at the N-6' position The aromatic protons on the indole ring from H-11' to H-14' can be easily identified based on 11 COSY, HSQC and HMBC spectra, starting from H-11' at 7.57 (d, J = 8.1 Hz, 1H, H-11') and at 7.16 - 7.26 (m, 3H, H-12', H-13', H-14') Thus, basically we have completed the assignment of proton spectra on two parts of the vindoline and velbanamine framework Compared to the original compound, the proton and carbon-13 resonance signals on the vindoline half were negligible, the significant change in the NMR spectrum of 81b was observed around the N-6 ' , especially for positions 5', 7', 19 ' and 22' (see Table 3.2) The 1H-NMR spectrum of compound 81b, the low-field area appears resonant signals at 7.66 (d, J = 8.4 Hz, 2H-H-27', H-31') and 7.40 (d, J = 8.5 Hz, 2H-H-28', H-30') characterizes the aromatic ring substituted at the para position The doublet signals at 8.25 ppm and 6.9 ppm have the same J = 16.5 Hz coupling constant characteristic for olefin H-25' and H-24' next to carbonyl groups High resolution mass spectrometry HR-EI-MS of compound 81b for molecular ion peak M+ with m/z 971.4366 (calculated for formula C56H64ClN4O9 M+, 971.4356) confirmed a alkylbromide substituent 76b was attached into anhydrovinblastine 12 Thus, the alkylbromide 76b substituent was attached to the velbanamine half of anhydrovinblastine 12 The configuration at the N6′ quaternary center is the determining factor for the orientation of the α,β-unsaturated ketone, which itself is fundamental for the interaction of these compounds with tubulin According to the X-ray structure of vinblastine, the nitrogen lone pair is oriented such that the absolute configuration of the amino group is S Thus, the absolute configuration at N-6' for compound 81b is configuration S Using the same approach, based on 1D, 2D NMR spectral data and HREI-MS high resolution mass spectrometry, we have demonstrated the structure of the remaining compounds 81a, 81c-d, 83a- 84d Follow-up is an example of structural analysis of simple vinca alkaloid compounds by replacing vindoline with 3,5-dimethoxyaniline (DMA) Figure 3.10 Structure of compound 82b and numbering according to IUPAC 12 Mass Spectrometer HR-EI-MS for molecular Peak M+ with 668.2866 m/z corresponding to C39H43ClN3O5+ formula confirms a alkylbromide 76b attached to 18 (S)-3', 5'-dimethoxyanilinecleavamine 77 The 1H-NMR spectrum of compound 82b, appear full resonance signals of the proton are present on the molecule The low field, resonant signals at 7.62 (d, J = 8.5 Hz, 2H, H-27', H31') and 7.38 (d, J = 8.5 Hz, 2H, H-28', H-30') characterizes the aromatic ring being substituted at the para position Two doublet signals at 8.03 ppm and 6.88 ppm have the same J = 16.2 Hz coupling constant characteristic for the H-25' and H-24' olefin proton Signal proton of indole resonance at 7.44 (m, 1H, H11'), 7.25 (m, 2H, H-13', H-14 '), 7.07 (t, J = 7,3 Hz, H-12') and 8.40 (s, NH) The aniline ring, the resonant singlet signal of the proton equivalents of the two methoxyl groups –OCH3 at 3.76 ppm and the two proton H-2 and H-6 proton resonance at 5.96 ppm and 6.00 ppm The 13C NMR and DEPT spectra of compound 82b, the carbon signal of the carbonyl group was clearly shown at 191.03 (C-23') and 172.76 (C18'-COOCH3) The 1H NMR spectrum and the HSQC confirm that the proton signal does not interact with carbon at 5.29 (s, 2H) which is the proton signal -NH2 on the aniline ring Table 3.3 1H NMR spectrum (CDCl3) of compounds 82b and 18(S)-3', 5'dimethoxyanilineecleavamine 77 Position 5’ 7’ 19’ 22’ Compound 82b 4,45 (m, 1H) 6,11 (m, 1H) 4,26 (m, 1H) 4,40 (m, 1H) 4,18 (d, J = 15,9 Hz, 1H) 4,65 (d, J = 15,9 Hz, 1H) 3,64 (s, 2H) 5,96 (s, 1H) 18(S)-3’,5'-dimethoxyanilinecleavamine 77 3,30 (m, 1H) 3,40 (m, 1H) 3,00 (dd, J = 13,6Hz/ 4,6Hz, 1H) 3,15 (m, 1H) 2,50 (d, J = 12,8Hz, 1H) 3,54 (m, 1H) 5,88 (s, 2H) 7,8 NH2 6,00 (s, 1H) 3,76 (s, 6H) 5,29 (s, 2H) 3,72 (s, 6H) - The spectrum data of compound 82b was compared with the original compound 18(S)-3', 5'-dimethoxyanilineecleavamine 77 (see Table 3.3) The proton and carbon-13 resonance signals on the velbanamine framework around 13 the N-6' position changed significantly compared to the original compound, especially for positions 5', 7', 19' and 22' From the above data, α,β-unsaturated ketone groups were attached to 18 (S)-3', 5'-dimethoxyanilineecleavamine 77 at the N-6' position on the velbanamine framework The configuration at the N6′ quaternary center is the determining factor for the orientation of the α,β-unsaturated ketone, which itself is fundamental for the interaction of these compounds with tubulin According to the X-ray structure of vinblastine, the nitrogen lone pair is oriented such that the absolute configuration of the amino group is S Thus, the absolute configuration at N-6' for compound 82b is configuration S Similarly, the structures of compounds 82a, 82c are also demonstrated by 1D, 2D NMR and HR-EI-MS high-resolution spectroscopy Thus, we have synthesized 12 new quaternary ammonium salts from anhydrovinblastine 12, 18 (S)-3', 5'-dimethoxyanilineecleavamine 77, vinblastine 1, vincristine (Figure 3.4) The products 81a-84c are obtained with 62-72% yield The advantage of this method is that the reaction occurs easily, good yield The products is more stable than the original compound, due to the reaction center is the nitrogen atom on the tertiary amine was alkylating In particular, the reaction occurs very selectively at the N-6' position on the velbanamine framework, which is explained by the T-structure shape of the vinca alkaloid structure that shields the N-9 position in vindoline half and its flexibility of the electron pair on the tertiary amine versus the primary amine on the aniline 3.2 Synthesize of new vinca alkaloids from 3'-cyanoanhydrovinblastine 88 3.2.1 Synthesis of new vinca alkaloids via selective reduction of 3'cyanoanhydrovinblastine 88 Figure 3.14 Compound 85, 86 87 Langlois and Potier disclosed the first synthetic nitrile vinca alkaloids derivatives (85, 86 and 87) as a mixture in poor yields (