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

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
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".
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
1


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:
– 12 quaternary ammonium salts of anhydrovinblastine, vinblastine,
vincristine and 18 (S) -3 ', 5'-dimethoxyanilineecleavamine 81a - 84c.
– 11 new derivatives of 3'-cyanoanhydrovinblastine include 5 vinca
alkaloid
derivatives
92a-92e
via
reduction
selective
3'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, 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 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
2


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
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 2. EXPERIMENTAL
2.1. Chemicals and equipment
2.1.1. Chemicals and solvents
2.1.2. Research equipment
3


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.

4

CHAPTER 3. 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
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
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 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
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
6


anhydrovinblastine
12,

18(S)-30,50-dimethoxyanilinecleavamine
77,
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
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.
7


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
Position
Compound 81b
Anhydrovinblastine 12
δH, J (Hz)
δC
δH, J (Hz)
1
2
3,81(s, 1H)
83,06 3,72 (s, 1H)
3
79,85
4
5,43 (s, 1H)
76,53 5,45 (s, 1H)
5
42,65
6
5,36 (d, J = 15,7, 129,7 5,30 (d, J = 15,5, 1H)
1H)
8

δC
83,2
79,7
76,4
42,7
130,0

7
8

9
10
11
12
13
14
15
16
17
18
19
20
21
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)

125,20 5,86 (dd, J = 10,2/
4,5, 1H)
50,02 2,82 (d, J = 16,0, 1H)
3,37 (m, 1H)

50,3
45,44
53,4
124,08
122,4
118,4
157,91
94,22

153,6
65,00
30,85
8,54
55,8
38,04
171,1
52,21
171,6
21,20

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

6,55 (s, 1H)

5,45 (s, 1H)
2,66 (s, 1H)
1,35 (m, 1H)
1,79 (m, 1H)
0,80 (t, J = 7,4, 3H)
3,82 (s, 3H)
2,72 (s, 3H)
3,80 (s, 3H)
2,10 (s, 3H)

124,6
50,3

50,3
44,6
53,3
122,8
123,5
121,1
158,0
94,2
152,7
65,4
30,9
8,4
55,9
38,3
170,9
52,2
171,6
21,1

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)
9

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 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 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, 4 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 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
5’
7’
19’

22’
2

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)

6
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 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
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
14


group. The resulting nucleophilic amino group can undergo a wide-range of
reactions with electrophilic agents. Naturally occurring nitriles such as bisindole 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 nitrilecontaining 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

directly
couple
5’-cyanocatharanthine
85
or
3’-cyano-4’,5’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).

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.
15


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 lowfield 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.

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
potassium cyanide, in this case, lead exclusively to 1,4-addition. The
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.
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 singlecrystal 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.

16


Scheme 3.8. New vinca-alkaloid derivatives 92a–e via selective reduction of 88
Table 3.4. Reduction reaction of 3’-cyanoanhydrovinblastine 88
Ent
Reductant
Catalyst
Solvent

Temp.
Time
Product ratio (%)
Yield
ry
(οC)
(h)
92a, 92b, 92c, 92d, 92e
(%)
a
1
HCOOH-NEt3
Pd/C
THF
40
12
100:0:0:0:0
98(92a)
b
2
NaBH3CN
Ni2B
MeOH
40
12
10:90:0:0:0
72(92b)
3c
NaBH4
CoCl2

EtOH
40
5
10:0:80:10:0
65(92c)
c
4
NaBH4
Ni2B
EtOH
40
5
5:0:5:40:50
5c
NaBH4
Co2B
EtOH
40
5
5:0:5:40:50
c
6
NaBH4
NiCl2
EtOH
40
5
10:0:40:50:0
d
ο

7
LiAlH4
THF
0 C-RT
3
0:0:0:50:50
37(92d/92e)
a
ο
nitril (0.05 mmol), Pd/C (10 mol%), THF (0,2 mL) và HCOOH-NEt3 (0,2 mL, 18,5 : 1), 40 C.
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%),
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.
17

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 HRESI-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,
13
C 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 H3’ 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 2DNMR 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.
Treating compound 88 with NaBH4 and CoCl2, NiCl2 or their
corresponding borides in EtOH, afforded a novel amine product along with the
18


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
andehyde
(p-vanillin,
4-chlorobenzaldehyde,
2-naphthaldehyde,
4(trifluoromethyl)
benzaldehyde,
4-imidazolecarboxaldehyde,
indole-3carboxaldehyde) and reduction with NaBH4 obtain the new vinca alkaloid
derivatives according to scheme 3.12.

19


Scheme 3.12. Synthesize of new vinca alkaloid derivatives through the
reductive alkylation of aminomethyl 92c
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.
Similarly, the structure of compound 93b-f, also confirmed by 1D NMR,
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
Cytotoxic activity of new vinca alkaloid derivatives containing α,βunsaturated ketone
20


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,βunsaturated ketone
Entry
Compound
KB (IC50 µM) HepG2 (IC50 µM)
1
1 (VLB.H2SO4)

0,02
0,34
2
2(VCR.H2SO4)
0,02
0,69
3
2,06
4,53
12
4
11,5
20,4
77
5
0,64
4,31
81a
6
0,28
1,75
81b
7
0,64
51,5
81c
8
4,16
10,9
82a

9
6,12
20,2
82b
10
9,11
86,8
82c
11
0,03
18,05
83a
12
0,06
7,77
83b
13
1,69
7,23
83c
14
0,08
7,94
84a
15
0,03
14,24
84b
16
1,67

2,44
84c
17
Ellipticine
1,66
2,07
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.
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
21


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 improved activity compared 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
KB
HepG2
(IC50 M)
(IC50 M)
1
0,41
0,43
88
2
0,55
0,55
92a
3
0,41
0,48
92b
4
16,84
24,49

92c
5
0,37
0,29
92d
6
2,26
2,34
92e
7
13,87
11,93
93a
8
1,63
1,10
93b
9
8,79
6,90
93c
10
1,89
14,73
93d
11
12,69
72,49
93e
12

11,09
1,86
93f
13
Vinblastine
0,0099
0,011
sulfate
22