Summary Of Engineering Doctoral Thesis: Synthesis and cytotoxicity of bengamide analogues A and E

The objective of the thesis is to build the synthesis process of Analogs of Bengamide A and E, conduct the biological activity of the synthesized Analogs. To find out more about the details of the content, please consult the thesis summary.

MINISTRY OF EDUCATION

AND TRAINING

VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY OF SCIENCE AND

TECHNOLOGY -

PHI THI DAO

“ Synthesis and cytotoxicity of bengamide analogues A and E”

Major : Organic Chemistry Code : 62 44 01 14

SUMMARY OF CHEMISTRY DOCTORAL THESIS

HaNoi - 2018

Science and Technology - Vietnam Academy of Science and Technology

Technology, at hour date month 2018

Thesis can be found in:

- The library of the Graduated University of Science and Technology, Vietnam Academy of Science and

Technology

- National Library of Vietnam

1 Thi Dao Phi, Huong Doan Thi Mai, Van Hieu Tran, Bich

Ngan Truong, Tuan Anh Tran, Van Loi Vu, Van Minh Chau, Van

Cuong Pham Design, synthesis and cytotoxicity of bengamide analogues and their epimers Med Chem Commun, 2017,8, 445-451

2 Thi Dao Phi, Huong Doan Thi Mai, Van Hieu Tran, Van

Loi Vu, Bich Ngan Truong, Tuan Anh Tran, Van Minh Chau and

Van Cuong Pham Synthesis of bengamide E analogues and their cytotoxic activity Tetrahedron Letters 2017, 58, 1830-1833

3 Phi Thi Dao, Doan Thi Mai Huong, Le Thi Phuong, Chau Van

Minh, Pham Van Cuong Synthesis of methyl ethylidene)-6,7,8,9-tetradeoxy-D-gulo-6-nonenonic acid (6E)- - lactone Vietnam Journal of Chemistry, 2015,53 (2e), 154-157

8-methyl-2-O-methyl-3,5-O-(1-4 Phi Thi Dao, Doan Thi Mai Huong, Vu Van Loi, Chau Van

Minh, Pham Van Cuong Microwave-assisted synthesis of lactams from amino acids Vietnam Journal of Chemistry, 2015, 53 (2e), 198-201

5 Phi Thi Dao, Vu Van Loi, Nguyen Thi Bich, Doan Thi Mai

Huong, Nguyen Hien, Chau Van Minh, Pham Van Cuong Synthesis

of N-alkyl amino lactam derivatives Journal of Science and Technology, 2016, 54 (2C), 291-298

6 Phi Thi Dao, Doan Thi Mai Huong, Vu Van Loi, Chau Van

Minh, Pham Van Cuong. Synthesis and cytotoxicity of (2R,3R,4S,5R,6E)-3,4,5-trihydroxy-2-methoxy-8,8-dimetyl-N-((S)-2-

oxoazepan-3-yl)non-6-enamide Vietnam Journal of Chemistry, 2016,

54 (6e2),62-65

7 Phi Thi Dao, Doan Thi Mai Huong, Vu Van Loi, Nguyen

Thii Hue, Pham Van Cuong In vitro cytotoxic and antimicrobial

activities of some bengamide derivatives Vietnam Journal of

Chemistry 2017 55(3), 342-347

8 Patent for utinity solution Synthetic method of bengamide analogues Accepted application

I INTRODUCTION

1 Introduction

Natural compounds isolated from terrestrial plants have been studied for a long time and have been successful However, studies on marine compounds have only begun in the middle of the last century Currently, natural marine compounds are known to be a promising source of pharmaceuticals, and many highly biologically active compounds have been found in various marine organisms Difficulties in collecting large amounts of samples and requiring high funding are one

of the obstacles to research in the field of marine chemical compounds Therefore, organic synthesis is an effective alternative to generate larger amounts of active ingredients to serve biological studies, as well as to ensure their applicability Many marine-derived active compounds play

as lead compounds so that researchers can make new derivatives possess higher biological activity The bengamides isolated from marine sponges are known for their potent anti-cancer activity However, structural instability is one of the reasons limiting the applicability of this class In order to overcome this limitation of bengamides, we chose the research topic "Synthesis and cytotoxicity of bengamide analogues A and E" in this thesis

2 Objectives of the thesis

- Constructing synthesis process of bengamide analgoues A and E

- Evaluating the biological activity of the synthesized analogs

3 Scientific significance and new contributions of the thesis 3.1 Scientific significance

- Synthesis of new analogues of bengamide A and E

- Application of microwave irradiation in organic synthesis

3.2 New contributions of the thesis

- Stereochemical synthesis process of bengamide A and E analogues was described

- 30 bengamide A and E analogues were synthesized, including 16 bengamide E analogues, 4 bengamide A analogues and 10 fluorine containing compounds Among them, there are 27 new analogues

- Microwave irradiation method was effectively used in the reactions of intra-molecular cyclization of amine acids, protection of primary amines and coupling reactions of ketide side chains and amino lactams Reaction times are shorten and reaction yields were improved remarkably

- Cytotoxic activities of these analogues were evaluated against

several cancer cell lines (Lu, NCI-H1975, A549, MCF7, MDA-MB-231,

HepG2, Hep3B, KB, HL60 and Hela) The analogues containing R

configuration at C-2’ always exhibited higher activity than the analogues

containing S configuration at C-2’ Many analogues have IC50 values less than 1µM for cytotoxicity tests Beside that, anti-microbial activity of several analogues against 7 bacterial and yeast strains were also investigated Among them, 7 analogues showed high activity against

Gram positive bacteria and Candida albicans

4 The main content of the thesis

Thesis includes 153 pages, 25 tables, 65 figures and 57 references as following:

Introduction: 2 pages

Chapter 1: Overview 32 pages

Chapter 2: Experimental and methodology 64 pages

Chapter 3: Results and discussion 41 pages

their biological activities up to now

CHAPTER 2: EXPERIMENTAL AND METHODOLOGY

The four-pages of research methods described organic synthesis methods, chemical structure determination methods and biological activity assay methods

The 64-page experiment details the synthetic process of benamide analogs Physical properties and spectral data of the synthesized substances

We have developed the method of synthesis of the following

substances:

- Synthesis of polyketide chain

- Synthesis of N-ankyl substituted 6 and 7-membered aminolactam

rings

- Synthesis of bengamide analogues A and E

- Synthesis of fluorine containing bengamide analogues

- Evaluated cytotoxic activity of 30 synthesized analogues against

10 cancer cell lines (Lu1, NCI-H1975, A549, MCF7, MDA-MB-231, HepG2, Hep3B, KB, HL60 và Hela)

Evaluated antimicrobial activity against 07 strains, including: Gram (+), Gram (-) and yeast

CHAPTER 3: RESULTS AND DISCUSSION

Previous studies on the synthesis of bengamide analogues and their evaluation of tumor inhibitory activity have shown that the structural modification of bengamide skeleton remarkably effected to their biological activity Especially, previous studies have shown that the presence of the hydroxyl groups and the configuration of C-3, C-4 and C-

5 of ketide side chains play an important role to the cytotoxic activity of bengamide analogues In order to study the relationship between the structure and cytotoxic activity of bengamide analogues, the conversion

of bengamide skeleton is carried out by the following ways i) replacing

the isopropyl group by the tert-butyl group leading to the obtainment of

more stable structures by avoiding olefin isomerization ), ii) modification

of configuration at C-2’ carbon, thereby evaluating the effect of C-2'

configuration to biological activities, iii) N-alkylated amide groups of the

lactam ring, iv) synthesis of some analogs with the hydroxyl group at 5', v) changing the lactam ring size, vi) replacing the terminal olefinic chain by FCH2-CH(OH) to evaluate its role to biological activity (Figure 3.1)

C-Figure 3.1 Modification of the bengamide skeleton

The synthesis process of new bengamide analogs was carried out

from commercial chemicals, such as: α-D-glucoheptonic γ-lactone and

amine acids (L-ornithine monohydrochloride, D-ornithine monohydrochloride, L-lysine, D-lysine and D, L-5-hydroxylysine hydrochloride) through 3 main stages

3.1 Synthesis of ketide chains

Synthesis of BG5 ketide is carried out using the commercially

available α-D-glucoheptonic γ-lactone (Figure 3.2) The acetonide

reaction using acetone and sulfuric acid as catalyst produces BG1 in

67.8%

Figure 3.2 Synthesis of BG5 compound

Selective hydrolysis of the isopropylidene group of BG2 with acetic acid yielded compound BG3 in 85% Study on the oxidation of

diol with NaIO4 in a mixture of different solvents showed that using 1.2

eq NaIO4 in a solution of MeCN and H2O (4/1, v/v) obtained the highest

yield of aldehyde BG4 in 91%

The final reaction is the olefination reaction of aldehyde BG4 The

reaction was investigated using (P(t-Bu)3HBF4) (1.5-3eq) in the presence

of TEA, potassium t-butoxide (CH3)3COK) or NaH (1.5 eq) in THF at room temperature for 3 - 24 h However, these reactions do not form the

desire product BG5 Then, the reaction was successfully carried out

using Takai olefination reaction in the presence of dimethylpropane and CrCl2 In fact, the reaction using 1,1-diiodo-2,2-dimethylpropane bought from Aldrich company gave the ketide

1,1-diiodo-2,2-compound BG5 in the highest yield of 45% Meanwhile, this 1,1-diiodo-2,2-compound

was prepared much effectively (73%), when the reaction was carried out with fresly prepared 1,1-diiodo-2,2-dimethylpropane The mechanism of

the oxidation of BG3 and the formation of BG5 from BG4 using Takai

reaction is shown in the following figure

Figure 3.3: Mechanism of oxidation of BG3 and Takai olefination of

BG4

Accordingly, in the oxidation reaction with NaIO4, the oxidation state of iodine is shifted from +7 (NaIO4) to +5 (NaIO3) For Takai olefination reaction, this is a combination of aldehyde and geminal dihaloalkane to form olefins In this reaction, Cr (II) is oxidized to Cr (III) when both halogen atoms are replaced The formed geminal carbodianion reacts with aldehyde to form the desire alkene

Structures of synthesized compounds was determined using MS,

1H-NMR, 13C-NMR spectra and compared with the published data

3.2 Synthesis of 2-amino-lactam compounds

3.2.1 Synthesis of BG6a-b and BG14a-b

The ring structure of α-amino lactams exist in many compounds exhibiting highly biological activity Thus, the synthesis of α-amino

lactams has attracted the attention of many research groups and typically,

one of them is the synthesis of α-aminocaprolactam from L-lysine

According to the publication of G Pifferi and co-workers., the

cyclization of L-lysine under heat conditions provided BG14a in 95% for

48 h In another study, Blade-Front reported that synthesis of BG14a

achieved in 70% using Al2O3 in toluene / pyridine mixture for a shorter

time of 20 h In addition, another article reported that synthesis of BG14a

from L-lysine was carried out under high temperature and pressure conditions with a yield of 88%

In order to overcome the disadvantages of using expensive, toxic agents, long reaction time or harsh conditions as high temperature and pressure, we have studied the intramolecular cyclization of amine acids

with the aid of microwave irradiation Accordingly, the

α-aminocaprolactam compound (BG14a) obtained in 79% from L-lysine

using mild conditiions in ethylene glycone under microwave irradiation

at 284 W for 1 h Meanwhile, the yield of BG14a was lower (48%) if the

reaction was carried out in butanol Using the same reaction conditions,

the BG14b compound was synthesized with 82% of yield from D-lysine Under microwave irradiation at 284 W, compounds BG6a and BG6b

were obtained in 55.6% for 1h using ethylene glycol and pyridine Replacement of pyridine by a solution of 10% NaHCO3 under

microwave irraditation gave BG6a in 78% Similarly, compound BG6b

was obtained in 72% from D-ornithine hydrochloride

3.2.2 Synthesis of rac-BG22a and rac- BG22b

(i): NaHCO 3 10%, ethylen glycon, pyridine, MW, 284W, 1h

(ii): (Boc) 2 O (1eq), TEA, THF, H 2 O, rt, 3h

Due to commercially unavailable chiral isomers of 5-hydroxylysine, the 6-hydroxycaprolactam isomers were prepared from a racemic mixture

of the 5-hydroxylysine compound Accordingly, the intramolecular reaction of D, L-5-hydroxylysine hydrochloride mixture was carried out with the aid of microwave irradiation at 284 W for 60 minutes gave a

racemic mixture of the two diastereomers rac-BG22a and rac-BG22b

The mixture then was purified on a silica gel column with acetone/ H2O/

NHOH (ratio of 9/ 1/ 0.1) obtained a racemic mixture of rac-BG22a

(53%, Rf = 0.49) and rac-BG22b (39%, Rf = 0.34) Due to impossible

determination of the relative configuration at C-3 and C-6 of rac-BG22a

and rac-BG22b, these compounds are converted into N-Boc derivatives

to compare with published NMR data The reaction of each rac-BG22a and rac-BG22b with Boc2O is carried out in THF/H2O mixture in the

presence of TEA to produce corresponding compounds rac-BG23a and rac-BG23b Compared to the published NMR data, rac-BG23a has a 3,

6-trans configuration (3S*, 6S*) and thus, the 3,6-cis (3S*, 6R*)

configuration was assigned for rac-BG23b This allows to determine

3,6-trans (3S*, 6S*) and 3,6-cis (3S*, 6R*) configuration for compounds

rac-BG22a and rac-BG22b, respectively

3.2.3 Synthesis of N-ankyl aminolactam compounds

3.2.3.1 The reaction protects the amine group of 2-aminolactam

In order to protect the primary amine groups, 3-aminolactam compounds are reacted with phthalic anhydride The reaction between

BG6a and phthalic anhydride is carried out in acetic acid at 100°C for 6 hours to obtain BG7a in a low yield of 30% of Then with the aid of microwave irradiation, compound BG7a obtained with higher yield

(53%) for a shorter time (1 hour) Using the same reaction conditions

under microwave irradiation, amine groups of BG6b, BG14a and BG14b compounds were successfully protected, yielding BG7b, BG15a and BG15b compounds in the range of 52 - 57%

3.2.3.2 N-alkylation reactions

After the protection of the primary amines, the alkylation reactions were carried out with halide derivaties, such as: alkyl halides,

(bromomethyl)cyclohexane, benzyl bromide and cinnamyl bromide

Compound BG7 was selected for optimization study at the beginning The reaction of BG7a with (bromomethyl) cyclohexane or benzyl bromide or

cinnamyl bromide did not lead to the desired N-alkyl products when

using alkaline agents such as K2CO3, KOH or NaH in DMF or THF However, when the reaction was performed in DMSO, formation of the

desired N-alkylated products BG8a-BG10a was recorded The results

showed that the use of DMSO and KOH (2 eq), K2CO3 (2 eq) and KI (1 eq) at 50-60oC provided the best yield Using the same optimized

conditions, other N-alkyl compounds were synthesized with the yield in

the range of 45-55%

(a) (bromomethyl)cyclohexane or(bromomethyl)benzene or cinnamyl bromide,

Finally, the deprotection of phthalimide group was carried out by

treating the compounds BG8a - BG10a, BG8b - BG10b, BG16a - BG18a, BG16b - BG18b with hydrazine solution in acetonitrile at room

temperature, leading to the formation of the corresponding N -

alkylaminolactam BG11a-13a, BG11b-13b, BG19a-21a and 21b

BG19b-3.3 Synthesis of bengamide E analogue

Synthesis of bengamides was carried out by coupling between ketide

and aminolactams In fact, the lactone ring-opening reaction of BG5 has

been studied and published previously Accordingly, David D Xu and

co-workers studied the reaction between BG5 and LAF-A The authors

found that using sodium 2- ethyl hexanoate as a base in THF at room

temperature, yielded LAF-B products in 85-92% after 20 hours

Figure 3.9 Coupling reaction of BG5 and LAF-A by David and

co-workers

The method of David D Xu and co-workers has the advantage of using cheap agents, mild conditions, but prolonged reaction time (15 - 20

hours) The reaction between BG5 and BG6a is used to optimize the

reaction conditions Accordingly, with the use of sodium 2- ethyl hexanoate in THF or 1,4-dioxane, at temperatures between 50 and

100°C, for 10 to 24 hours, the yield of BG24a products is between 50

and 58% Then, the lactone ring opening was examined under microwave irradition at 100W, the reaction time was significantly shortened for 1

hour and the yield of the BG24a reached to 87% As can be observed, the reaction between BG5 and BG6a achieved the highest yield when using

1.2 - 1.5 eq of sodium 2- ethyl hexanoate, under microwave irradiation at

100 W for 1 hour

Table 3.7 Synthesis of BG24a

Solvent

sodium 2- ethyl hexanoate

MW Temperature Time

(hour)

Yield (%)