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

VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY

MINISTRY OF EDUCATION AND TRAINING

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

thesis was completed

in Graduate University Science and

The 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

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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

1. 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".

1

2. 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. 3. 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

– 12 quaternary ammonium salts of anhydrovinblastine, vinblastine,

92a-92e selective reduction derivatives

2

performed at Department of Chemistry and Laboratory of Computational Chemistry and Modelling, Quy Nhon University. 4. 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: vincristine and 18 (S) -3 ', 5'-dimethoxyanilineecleavamine 81a - 84c. – 11 new derivatives of 3'-cyanoanhydrovinblastine include 5 vinca alkaloid 3'- via cyanoanhydrovinblastine 88. 6 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, 2D- NMR, 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 1 and vincristine 2. 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. 5. The main contents of the thesis The thesis consists of 138 pages: Introduction: 2 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 1. 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 Semisynthesis of vinca alkaloids 1.2.2.1. Total synthesis of vinca alkaloids 1.2.2.2. Biosynthesis and biotechnological approaches 1.2.2.3. 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 2. EXPERIMENTAL

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2.1. Chemicals and equipment 2.1.1. Chemicals and solvents 2.1.2. Research equipment

Infrared Spectroscopy IR Nuclear Magnetic Resonance Spectrum NMR

4

2.1.2.1. 2.1.2.2. 2.1.2.3. Mass spectrometry MS and HRMS Specific rotation [α]D 2.1.2.3. 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,β- unsaturated 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,β- unsaturated 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'S- cyano-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 3. RESULTS AND DISCUSSION

3.1. Synthesis of some new vinca alkaloid derivatives contain α,β-

unsaturated 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, β- unsaturated 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 5

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 in Scheme 3.1. The synthesis started by a Claisen–Schmidt outlined condensation of arylcarboxadehyde and acetone in methanol at room temperature followed by a selective monobromination of the transient α- methylketons 75a–75c using N-bromosuccinimide in the presence of p- toluensulfonic acid at room temperature for 2 h. These afforded 76a–c in 70– 73% yields for two steps.

Scheme 3.2. Synthesis of vinca alkaloids 12 và 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

and vindoline reaction between

Compounds 12, 77 are synthesized according to the method described previously by Vukovic with good yield (76-85%). Accordingly, we performed a coupling (or 3,5- catharanthine dimethoxyaniline) 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 6

18(S)-30,50-dimethoxyanilinecleavamine 12,

77, anhydrovinblastine vinblastine 1 and vincristine 2 (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

7

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,β- saturated ketone.

Figure 3.4. Structure of hybrids vinca alkaloid - ketone α,β-saturated 81a-c

The structure of the vinca alkaloid bisindole such as anhydrovinblastine has been demonstrated by Szantay, Kutney, Webb Andrews. The NMR data of compound 81a-c was compared with the original compound anhydrovinblastine 12. Proton resonance on the vindoline part change negligible. Some of the peaks are easily identified on the 1H NMR spectrum with their chemical shifts and interactions. These peaks are then used as a convenient starting point for assigning the next signal. The 1H, 13C NMR resonance signals on the vindoline part of compound 81b are listed in Table 3.1.

Figure 3.5. Structure and numbering according to IUPAC in the vindoline half

Table 3.1. NMR data on the vindoline part of compound 81b and anhydrovinblastine 12 in CDCl3

δC

Compound 81b δC

Anhydrovinblastine 12 δH, J (Hz)

Position 1 2 3 4 5 6

83,06 3,72 (s, 1H) 79,85 76,53 5,45 (s, 1H) 42,65 129,7 5,30 (d, J = 15,5, 1H)

83,2 79,7 76,4 42,7 130,0

δH, J (Hz) 3,81(s, 1H) 5,43 (s, 1H) 5,36 (d, J = 15,7, 1H)

8

7

124,6

125,20 5,86 (dd, J = 10,2/ 4,5, 1H)

8

50,3

50,02 2,82 (d, J = 16,0, 1H) 3,37 (m, 1H)

9 10

50,3

50,3

11

44,6

2,47 (m, 1H) 3,23 (m, 1H) 45,44 1,84 (m, 1H) 2,15 (m, 1H)

12 13 14 15 16 17 18 19 20

53,3 122,8 123,5 121,1 158,0 94,2 152,7 65,4 30,9

21

53,4 124,08 122,4 6,55 (s, 1H) 118,4 157,91 94,22 5,45 (s, 1H) 153,6 65,00 2,66 (s, 1H) 30,85 1,35 (m, 1H) 1,79 (m, 1H) 0,80 (t, J = 7,4, 3H)

8,54

8,4

3,82 (s, 3H) 55,8 38,04 2,72 (s, 3H) 171,1 52,21 3,80 (s, 3H) 171,6 21,20 2,10 (s, 3H)

C16-OCH3 N-CH3 C3-COOCH3 C3-COOCH3 C4-OCOCH3 C4-OCOCH3

5,89 (dd, J = 10,1/ 4,4, 1H) 2,08 (d, J = 7,5, 1H) 3,33 (m, 1H) 2,72 (m, 1H) 3,33 (m, 1H) 2,06 (m, 1H) 2,22 (m, 1H) 6,55 (s, 1H) 6,14 (s, 1H) 2,83 (s, 1H) 1,38 (m, 1H) 1,78 (m, 1H) 0,87 (t, J = 7,4, 3H) 3,87 (s, 3H) 2,76 (s, 3H) 3,81(s, 3H) 2,13(s, 3H)

55,9 38,3 170,9 52,2 171,6 21,1

9

In the vindoline half, based on comparisons with spectral data anhydrovinblastine 12, easily localized the proton signals of methyl N-CH3, C16-OCH3, H-21, C3-COOCH3 and C4-OCOCH3 at 2.76, 3.87 (s, 3H), 0.87 (t, J = 7.4 Hz, 3H), 3.81 (s, 3H) and 2.13 (s, 3H). The doublet and double doublet signals of protons H-6 and H-7 are at 5.36 (d, J = 15.7 Hz, 1H) and 5.89 (dd, J = 10.1 / 4.4 Hz, 1H ), the COSY spectra both H-6 and H-7 proton interact with the two protons H-8. Two singlet resonance signals at 6.55 (s, 1H) and 6.14 (s, 1H) are assigned to the aromatic protons H-14 and H-17. The COSY spectra, two resonance signals at 1.78 (m, 1H, H-20b) and 1.38 (m, 1H, H-20a)

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.

Anhydrovinblastine 12 δH, J (Hz)

δC

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’

30,68 121,8 132,87 64,35

δC 34,3 32,9 123,5 140,0 52,1

7’

53,40

54,3

8’

19,93

25,9

33,85 2,40 (m, 1H) 3,04 (m, 1H) 1,30 (m, 1H) 5,45 (s, 1H) 3,28 (m, 1H) 3,52 (d, J = 16,0, 1H) 3,4 (m, 1H) 3,4 (m, 1H) 3,05 (m, 1H) 3,41 (m, 1H)

Compound 81b δH, J (Hz) 2,63 (m, 1H) 3,12 (m, 1H) 2,02 (m, 1H) 5,60 (s br, 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)

9’ 10’ 11’ 12’ 13’ 14’

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)

117,3 129,4 118,3 122,2 118,3 110,5

10

15’ N-H 17’ 18’ 19’

134,75 132,8 54,6 63,09

135,0 131,0 55,5 45,9

20’

8,36 (s, 1H) 4,11 (m, 1H) 4,53 (m, 1H) 2,04 (m, 2H)

27,26

27,8

1,07 (t, J = 7,4, 3H) 3,68 (s, 1H) 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)

8,04 (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) 3,62 (s, 3H)

12,2 11,50 21’ 70,5 22’ 191,18 23’ 123,90 24’ 147,27 25’ 132,1 26’ 130,6 27’, 31’ 129,4 28’, 30’ 137,9 29’ 174,6 173,09 C18’-COOCH3 53,3 52,83 C18’-COOCH3 3,68 (s, 3H) 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

Follow-up is an example of structural analysis of simple vinca alkaloid

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 4 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 HR- EI-MS high resolution mass spectrometry, we have demonstrated the structure of the remaining compounds 81a, 81c-d, 83a- 84d. 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 + formula confirms a alkylbromide 76b attached corresponding to C39H43ClN3O5 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, 4 resonant signals at 7.62 (d, J = 8.5 Hz, 2H, H-27', H- 31') 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, H- 11'), 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 6 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 Compound 82b 5’

7’

19’

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)

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)

22’ 2

6 7,8 NH2

6,00 (s, 1H) 3,76 (s, 6H) 5,29 (s, 2H)

- 5,88 (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

Similarly, the structures of compounds 82a, 82c are also demonstrated by

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. 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 2 (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 và 87

14

Langlois and Potier disclosed the first synthetic nitrile vinca alkaloids derivatives (85, 86 and 87) as a mixture in poor yields (<30%). Nitrile containing compounds can serve as key precursors for a wide-range of synthetic applications, e.g. reduction of the nitrile group to access the aminomethyl

5’-cyanocatharanthine couple 85 or

group. The resulting nucleophilic amino group can undergo a wide-range of reactions with electrophilic agents. Naturally occurring nitriles such as bis- indole alkaloids from Tabernaemontana elegans, lahadinines A and B from Kopsia pauciflora, saframycin A, and cyanocycline A, exhibit both antimicrobial and antitumor activities. Furthermore, a survey of nitrile- containing pharmaceuticals and clinical candidates indicates the remarkable role of the nitrile group which can act as a bioisostere of carbonyl, halogen, hydroxyl and carboxyl functional groups. Nitrile groups were also showed to improve ADME-toxicology profiles. Herein, we report the synthesis of new nitrile containing vinca alkaloids from 3’-cyanoanhydrovinblastine 88. Langlois and Potier first published the synthesis of 3’-cyanoanhydrovinblastine 88 via conjugated iminium intermediate 15 using anhydrovinblastine N-oxide. Intermediate 15, resulting from a modified Polonovski reaction, was subsequently treated with a saturated methanolic solution of KCN, but only afforded 3’-cyanoanhydrovinblastine 88 as a mixture in low yield (32%). In another attempt, the Polonovski reaction to 3’-cyano-4’,5’- directly dihydrocatharanthine 86, 87 with vindoline failed to afford the corresponding bis-indolic compounds. 3’-cyanoanhydrovinblastine 88 was prepared exclusively in good yield (74%) via a modified Vukovic coupling reaction, coupling between catharanthine and vindoline in the presence of iron ion III in the presence of water acidity, with the nitril agent is the KCN in NH4OH is obtained 3'-cyanoanhydrovinblastine 88 (Scheme 3.5).

15

Scheme 3.5. Synthesis of 3’-cyanoanhydrovinblastine 88. Reagents and conditions: FeCl3.6H2O, glycine – NaCl 0,1M, HCl 0,1N, sau đó KCN/NH4OH Compound 88 is confirmed by IR, NMR and MS spectra. The absolute configuration is determined by the NOESY spectra.

The HR-ESI-MS spectra of 88 for the pseudo molecular ion peak [M+H]+ with m/z = 818.4124 corresponding compound of formula C47H55N5O8 with the exact mass [M+H]+ (m/z) is theoretically 818.4051. In the IR spectrum of compound 88, the band characteristics of the nitrile group at 2230 cm-1 and a strong band characterize an enamin at 1650 cm-1. 1H NMR spectra, in the low- field resonance signal appearing at 5.92 ppm proton singlet characterizing vinylic proton at position 5', so this proton near nitrogen atoms should shift toward the low field, this to distinguish it with the H-3' proton when the nitrile group is in position 5'. Thus, from the above analysis, combined with comparing the chemical shifts and coupling constant of this compound was Potier reported in document [90], compound 88 was identified as 3 - cyananhydrovinblastine.

lead exclusively this case, in

Vukovic and co-workers previously developed a method for coupling catharanthine 6 and vindoline 7 in the presence of Fe3+ in an acidic aqueous medium. They justified the exclusive obtention of the 18’S configuration by a concerted reaction mechanism. Addition of alkali metal cyanides such as to 1,4-addition. The potassium cyanide, regioselectivity of addition to α,β-unsaturated iminium ion 15 is normally attributed to the hard/soft character, with soft nucleophiles such as CN- preferring 1,4-addition. Conducting the reaction at high temperature under basic conditions also favored 1,4-adducts.

16

It is noteworthy that compound 88 is relatively stable, which enabled its isolation in the crystalline form. Langlois and Potier did not establish the configuration of the C-3’ carbon bearing the nitrile group in compound 88, although the C-3’ stereochemistry in compound 86 was reported as R by single- crystal X-ray analysis. In our case, compounds 88 was selectively obtained as a simple diastereomer, and its C-3’ configuration determined by a NOESY experiment. According to the X-ray structure of vinblastine, the absolute configuration at C-2’ is R. A nuclear Overhauser effect (NOE) between H2’ and H3’ was observed, such correlations are only consistent with an absolute S configuration for the nitrile group. We have synthesized the new nitrile containing vinca alkaloids from 3'- cyanoanhydrovinblastine 88, using various reduction reactions (Scheme 3.8, Table 3.4) which allows synthetic new derivatives had interesting biological activities.

Solvent

Catalyst

Reductant

Pd/C Ni2B CoCl2 Ni2B Co2B NiCl2 -

THF MeOH EtOH EtOH EtOH EtOH THF

Time (h) 12 12 5 5 5 5 3

Yield (%) 98(92a) 72(92b) 65(92c) - - - 37(92d/92e)

Temp. (οC) 40 40 40 40 40 40 0 οC-RT

Product ratio (%) 92a, 92b, 92c, 92d, 92e 100:0:0:0:0 10:90:0:0:0 10:0:80:10:0 5:0:5:40:50 5:0:5:40:50 10:0:40:50:0 0:0:0:50:50

Scheme 3.8. New vinca-alkaloid derivatives 92a–e via selective reduction of 88 Table 3.4. Reduction reaction of 3’-cyanoanhydrovinblastine 88 Ent ry 1a HCOOH-NEt3 2b NaBH3CN 3c NaBH4 4c NaBH4 5c NaBH4 6c NaBH4 7d LiAlH4 a nitril (0.05 mmol), Pd/C (10 mol%), THF (0,2 mL) và HCOOH-NEt3 (0,2 mL, 18,5 : 1), 40 οC. b nitril (0.05 mmol), NaBH3CN (20 equiv), Ni2B (2 equiv) MeOH, 40 οC. c nitril (0.06 mmol), NaBH4 (20 equiv), xúc tác (2 equiv) EtOH, 40 οC. d nitril (0.06 mmol), LiAlH4 (3 equiv) THF 0 οC - RT. Initially, compound 88 was reduced by H2 with Pd/C catalyst, we did not observe any product formation. However, when using Pd/C (10%) with HCOOH-NEt3 as the source of hydrogen at the previously optimized conditions [154]. The catalytic transfer hydrogenation reaction using Pd/C is only selective for reducing aromatic nitriles to the corresponding primary amines and attempts to hydrogenate acrylonitrile derivatives often affords a mixture of products. In this case, although formation of the amine product was not detected, we obtained a product unique selective reduction at position C-4' 92a with excelllent yield (98%),

17

The structure of compound 92a was demonstrated by the IR, NMR and HR-ESI-MS high-resolution mass spectrometry. Absolute configuration at position C-4' is verified by the NOESY spectrum.

The IR spectrum of 92a showed a loss of absorption bands at 1650 cm-1 for the enamine, while the cyano band at 2229 cm-1 was still present. The 1H NMR spectrum of 92a also indicated the absence of proton H-5’ at 5.92 ppm. The HR- ESI-MS spectrum of compound 92a for pseudo molecular ion peak m/z= 820.42383 corresponding compounds of formula C47H58N5O8 with the exact mass [M + H]+ (m / z) in the theory is 820.42854. The combination of ESI-MS, 1H NMR, 13C NMR, and 2D-NMR spectroscopic data indicated hydrogenation of the double bond in the C-4’ position. Additionally, a NOESY correlation between H-4’ and H- 3’ confirmed the absolute S configuration of C-4’ in 92a.

Next, we examined catalytic reduction using different strong hydride donors, including NaBH4, NaBH3CN and LiAlH4 in the presence of cobalt(II) and nickel (II) halides or their corresponding borides, to selectively reduce nitrile, ester and olefin functional groups. These functional groups are known to be inert to such reducing agents alone. First, with the reducing agent LiAlH4, there was no trace of nitril reduction instead we obtained two products with ratio (50:50), using MS mass spectrometry and the disappearance of the CH3OCO- methoxycarbonyl group and CH3COO- acetate group at the C-3, C-4 position on the NMR we identified the 4-deacetyl 92d product and the deacetyl product at C-4 and reducing ester at C-3 92e. The similar reduced derivatives were afforded when vincristine was treated with NaBH4.

Interestingly, in the presence of NaBH3CN/Ni2B, the reduction did not lead to the methylamino product but rather to hydrogenation products 92a and 92b (10:90). The IR spectrum of 92b showed a loss of absorption bands at 1650 cm-1 for the enamine, while the cyano band at 2228 cm-1 was still present. 1H-NMR spectra of compound 92b, appeared full of resonance signals of the protons on the structural framework of the molecule. In the low field, the singlet resonance signal at 5.92 ppm characterizing the vinylic proton signal at the 5' position of compound 88 that has been lost. The combination of ESI-MS, 1H NMR, 13C NMR, and 2D- NMR spectroscopic data indicated hydrogenation of the double bond in the C-4’ position. Unlike compound 92a, no NOESY correlation between H-4’ and H-3’ was observed for 92b, suggesting the R absolute configuration of C-4’. Notably, high chemoselectivity and stereoselectivity were achieved for olefin versus ester and nitriles functionalities in the case of 92a,b.

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Treating compound 88 with NaBH4 and CoCl2, NiCl2 or their corresponding borides in EtOH, afforded a novel amine product along with the

2-naphthaldehyde, (p-vanillin,

4-imidazolecarboxaldehyde, benzaldehyde,

19

products of olefin hydrogenation 92a, ester reduction 92e or deacetylation 92d in different ratios (Table 3.4). To our delight, amine 92c was formed as the main product in good yield using CoCl2/NaBH4. The IR spectrum of amine 92c did not display the absorption bands for an enamine (1650 cm-1) or cyano (2228 cm-1) group. The 1HNMR spectrum also indicated the absence of proton H-5’ at 5.92 ppm. The HR-ESI-MS spectrum of compound 92c appear pseudo molecular ion peak at m/z 824.4597 [M+H]+ (theoretical calculations for the formula C47H61N5O8 824.4520). These data suggested that both the nitrile and C-4’-C-5’ double bond of compound 88 were reduced under these conditions. As for compound 92a, a NOESY correlation between H-4’ and H-3’ was observed, confirming the S absolute configuration of the amine product 92c. In summary, the synthesis of 3'S-cyanoanhydrovinblastine 88 from two natural vinca-alkaloids (catharanthine and vindoline) in one step with good yield was carried out. The reduction of stereochemistry and chemoselective of compound 88 led to the formation of two new vinca alkaloids 92a and 92b by two different methods. Successfully reduction of compound 88 to methylamino derivative 92c provided the precursor for next reactions. In addition, the reduction of 88 by LiAH4 obtained simultaneous two product 4-deacetyl 92d and product deacetyl at C-4 and reducing ester at C-3 92e. 3.2.2. Synthesize of some new vinca alkaloid derivatives through the reductive alkylation of aminomethyl 92c In the previous section we have presented the reduction reaction compound nitrile 88 and has obtained an important result that the reduction of compound 88 in the presence of NaBH4 catalyst by CoCl2 obtained products methylamino 92c with good yield. The nucleophilic methylamino group can undergo a series of reactions with electrophilic agents to new derivatives with interesting activity. Accordingly, we condensed the amine 92c with several 4- 4-chlorobenzaldehyde, andehyde (trifluoromethyl) indole-3- carboxaldehyde) and reduction with NaBH4 obtain the new vinca alkaloid derivatives according to scheme 3.12.

Scheme 3.12. Synthesize of new vinca alkaloid derivatives through the reductive alkylation of aminomethyl 92c

Similarly, the structure of compound 93b-f, also confirmed by 1D NMR,

The structure of the compounds was demonstrated by the 1H-NMR spectra method, 13C-NMR and HR-ESI-MS. Spectral data of the obtained substances were compared with the original compound 92c. The 1H-NMR spectra of compound 93a appear full resonance of the proton signals are present in the molecule. In the low field, three proton signals at 6.76 (s, 1H, H-26'), 6.82 (d, J = 8.0 Hz, 1H, H-29') and 6.62 , J = 7.9 Hz, 1H, H-30'), which characterizes aromatic rings that have been substituted for meta and para. The COSY spectrum, two proton signals at 2.06 (m, 1H, H-22'a) and 2.60 (m, 1H, H-22'b) interacted and interacted with the H-3’ proton. Two singlet signals at 3.45 ppm and 3.43 ppm are assigned to the methylene proton H-24'b and H-24'a. The HSQC spectrum, resonance signal of a proton singlet at 1.92 ppm do not interact with the carbon assigned to proton secondary amine N-H. The HR-ESI-MS spectrum [M-H]- for pseudo molecular ion peak m/z = 858.4949 corresponding to the compound of formula C55H69N5O10 with the exact mass [MH]- (m/z) the theory is 858.5044. The combination data of HR-ESI-MS, 1D và 2D NMR spectroscopic allow the determination of the structure of 93a. 2D NMR and HR-ESI-MS spectra. Thus, from the compound amine 92c we have successfully synthesized six derivatives of 3'-cyanoanhydrovinblastine as compounds 93a-f. The structure of the products is demonstrated by modern spectrum analyzes. 3.3. Evaluation of biological activity of research substances 3.3.1. Evaluation of cytotoxic activity in vitro 3.3.1.1. Evaluation of the cytotoxic activity of KB and HepG2

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Cytotoxic activity of new vinca alkaloid derivatives containing α,β- unsaturated ketone

µM)

New vinca alkaloid derivatives containing α,β-unsaturated ketones were evaluated for cytotoxic activity on KB, Hep-G2 cell line. The results are given in Table 3.5. Table 3.5. Cytotoxic activity of new vinca alkaloid derivatives containing α,β- unsaturated ketone

µM) HepG2 (IC50 0,34 0,69 4,53 20,4 4,31 1,75 51,5 10,9 20,2 86,8 18,05 7,77 7,23 7,94 14,24 2,44 2,07

Entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Compound 1 (VLB.H2SO4) 2(VCR.H2SO4) 12 77 81a 81b 81c 82a 82b 82c 83a 83b 83c 84a 84b 84c Ellipticine

KB (IC50 0,02 0,02 2,06 11,5 0,64 0,28 0,64 4,16 6,12 9,11 0,03 0,06 1,69 0,08 0,03 1,67 1,66 Of these two cancer cell lines, compounds 83a, 83b, 84a, 84b exhibited selective and potent cytotoxicity for the KB cell line with the IC50 equivalent to vinblastine 1 and vincristine 2. While 83c and 84c exhibit a much weaker activity than the activity of 1 and 2 but still equate to ellipcitine. It should also be noted that three new vinca alkaloids are derivatives of 81a-c derived from anhydrovinblastine 12 that have better cytotoxic KB activity than 12 and are even better than those of Ellipcitine in case 81b.

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The results of the evaluation cytotoxic activity of the new vinca alkaloid derivatives of α,β-unsaturated ketone are also evaluated and compared with the series of vinca alkaloid – phomopsin previously elaboration by Ngo Quoc Anh and co-worker[117]. We found that both vinca alkaloid α,β-unsaturated ketone and vinca alkaloid – phomopsin with the same original compound (AVLB) gave strong cytotoxic activity (IC50 <1 μM). on the KB cell line. The IC50

improved activity compared

values of vinca alkaloid - α,β-unsaturated ketone in the range of 0.28-0.64 μM, the IC50 values of the vinca alkaloid – phomopsin ranges from 0.08 to 0.7 μM. Comparing the IC50 values of the vinca alkaloid – phomopsin series with all the vinca alkaloid - α,β-unsaturated ketone (Table 3.5), it was found that some of the compounds in series the vinca alkaloid - α,β-unsaturated ketone had better cytotoxic activity as compound 83a (0.03 μM), 83b (0.06 μM) and 84b (0.03 μM). It is worth noting that the simplification of vindoline on vinca alkaloid – phomopsin by replacing vindoline with 3,5-dimethoxyaniline (DMA) resulted in the loss of active compounds [118]. In contrast, the simplification of vindoline on the vinca alkaloid - α,β-unsaturated ketone by replacing vindoline with 3,5-dimethoxyaniline (DMA) without losing activity of the compounds obtained but significantly to 18(S)-3',5'- dimethoxyanilinecleavamine 77.

Cytotoxic activity of the new vinca alkaloid derivatives from 3'- cyanoanhydrovinblastine New vinca alkaloid derivatives from 3'-cyano- dydrovinblastine were evaluated for cytotoxic activity on KB, HepG2 cell lines. The results are shown in Table 3.6. Table 3.6. Cytotoxic activity of 3'-cyanoanhydrovinblastine derivatives

Entry Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 HepG2 (IC50 M) 0,43 0,55 0,48 24,49 0,29 2,34 11,93 1,10 6,90 14,73 72,49 1,86 0,011 KB (IC50 M) 0,41 0,55 0,41 16,84 0,37 2,26 13,87 1,63 8,79 1,89 12,69 11,09 0,0099

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88 92a 92b 92c 92d 92e 93a 93b 93c 93d 93e 93f Vinblastine sulfate

Evaluation of biological activity of leukemia HL-60

Evaluation results cytotoxic activity of the compounds in this series shows. Compounds 92a, 92b and showed strong activity on KB and HepG2 cell lines with an IC50 equivalent 88 and weaker than the activity of vinblastine sulfate. Meanwhile, compound 92d exhibited better activity than that of the original compound 88. The derivatives 92c, 92e, 93a-f exhibited weaker compared with 88 and weaker compared with vinblastine sulfate. 3.3.1.2. Evaluation of HL-60 human lymphoma biological activity such as cytotoxic, proliferation, apoptosis, cell cycle analysis. We selected two cytotoxic activity assays for the best KB cell line, 4-chlorochablastine 83b and 4-chlorochacristine 84b. The results were compared with classic vinca alkaloid such as vinblastine, vincristine, vinorelbine and vinflunine.

Docking results

Results: Based on the score, 82a showed the best interaction with tubulin.

Results: Based on the score, 92b showed the best interaction with tubulin.

23

All in all, the novel vinca alkaloids chlorochablastine 83b and chlorochacristine 84b show effects that are similar to those caused by the classical vinca alkaloids. In general, the potency is lower than for VBL, VCR, and VRB, but similar to VFL which is also used in clinical practice. This can be taken as a first indication in vitro that these substances might prove efficient in cancer therapy. Further research needs to address whether there are cell-type specific differences, especially in neuronal cells, and whether these results can also be observed in vivo. 3.3.2. Docking results by software autodock in the first series shows. Compound 82a has the best binding capacity for tubulin due to the lowest estimated free energy of binding is -11.5 kcal/mol, the lowest inhibition constant is 3.69 (nM) and the root mean square deviation RMSD = 50.667 Å. This result is consistent with the lowest IC50 value of compound 82a on the KB cell line. Docking results by software autodock in the second series shows. Compound 92b has the best binding capacity for tubulin due to the lowest estimated free energy of binding is -12.8 kcal/mol, the lowest inhibition constant is 0.414 (nM) and the root mean square deviation RMSD = 51.227 Å. This result is consistent with the lowest IC50 value of compound 92b on the KB cell line. 3.3.2.2. Molecular docking results using Patchdock software This result is consistent with the lowest IC50 value of 82a on the KB cell line. This result is consistent with the lowest IC50 values of compounds 1 and 92b.

CONCLUSION

– 12 quaternary ammonium salts of anhydrovinblastine, vinblastine,

92a-92e selective reduction derivatives

24

 23 new vinca alkaloid compounds have been synthesized from natural vinca alkaloids such as catharanthine, vindoline, vinblastine and vincristine, including: vincristine and 18 (S) -3 ', 5'-dimethoxyanilineecleavamine 81a - 84c. – 11 new derivatives of 3'-cyanoanhydrovinblastine include 5 vinca alkaloid 3'- via cyanoanhydrovinblastine 88. 6 vinca alkaloid derivatives 93a-93f via reductive alkylation aminomethyl 92c.  The structure of the new compounds was determined by 1D-NMR, 2D- NMR, 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 and the initial compound 88.  Evaluation of cytotoxic activity on two KB and Hep-G2 cancer cell lines of 23 new compounds. Most compounds have good activity levels of 1-10 μM, some compounds have the activity same threshold as vinblastine and better than ellipticine.  Eight potent cytotoxic compounds 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.  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.