Synthesis and antiproliferative evaluation of n alkylated (2 (4 methoxyphenyl) 1hbenzodimidazol 5(6) yl)(phenyl)methanone derivatives

The results revealed that compounds 3, 6b, and 6d displayed antiproliferative activities against tested cancer cell line with IC50 values ranging from 48,58- 70,93 M.. In our previous

LE QUOC TUAN

SYNTHESIS AND ANTIPROLIFERATIVE EVALUATION

OF N- ALKYLATED

(2-(4- BENZO[D]IMIDAZOL-5(6)-

LE QUOC TUAN- 61602260

SYNTHESIS AND ANTIPROLIFERATIVE EVALUATION

OF N- ALKYLATED

(2-(4- BENZO[D]IMIDAZOL-5(6)-

METHOXYPHENYL)-1H-YL)(PHENYL)METHANONE

DERIVATIVES

ORGANIC SYNTHESIS

Advised by Assoc Prof., Dr Hoang Thi Kim Dung M.Eng., Phan Ngoc Kim Ngan

HO CHI MINH CITY, YEAR 2023

Additionally, I would like to thank Ms Phan Ngoc Kim Ngan, who has supported and managed my project and has been encouraging and instructive throughout the research Her extensive knowledge and academic guidance were crucial in assisting me in completing my thesis Besides, I'd like to thank everyone in the Organic Chemistry and Polymer department for their continued friendliness and assistance during the period with report documents and presentations

Finally, I sincerely thank my family, especially my parents Through every accomplishment in my life, they have always been there for me with their unconditional love and wholehearted support

Ho Chi Minh City, day … month … 2023

Author

This thesis was carried out at Ton Duc Thang University

Advisor: Assoc Prof., Dr Hoang Thi Kim Dung

M.Eng., Phan Ngoc Kim Ngan

This thesis is defended at the Undergraduate Thesis Examination Committee was hold at Ton Duc Thang University on…

Confirmation of the Chairman of the Undergraduate Thesis Examination Committee and the Dean of the faculty after receiving the modified thesis (if any)

DECLARATION OF AUTHORSHIP

I hereby declare that this thesis was carried out by myself under the guidance

and supervision of Assoc Prof Dr Hoang Thi Kim Dung, and that the work and

the results contained in it are original and have not been submitted anywhere for any previous purposes The data and figures presented in this thesis are for analysis, comments, and evaluations from various resources by my own work and have been duly acknowledged in the reference part

In addition, other comments, reviews, and data used by other authors and organizations have been acknowledged, and explicitly cited

I will take full responsibility for any fraud detected in my thesis Ton Duc

Thang University is unrelated to any copyright infringement caused on my work (if any)

Ho Chi Minh City, day month year

Author

ABSTRACT

In this thesis, ten

(2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone derivative was successfully synthesis via condensation of the

o-phenylenediamine derivative with aromatic aldehyde derivative in the presence of

Na2S2O5 using the mixed solvent of EtOH: H2O (9:1, v/v) Subsequently, ten derivatives of N-alkylated benzimidazole were designed and synthesized by alkylation reactions in the presence of K2CO3 using dimethyl sulfoxide as a solvent All synthesized compounds were characterized by HPLC, UV-Vis, FT-IR, 1D and 2D-NMR, and HRMS The antiproliferative test determined activity against a human breast cancer cell line (MDA-MB-231) by the SRB method The results revealed that

compounds 3, 6b, and 6d displayed antiproliferative activities against tested cancer

cell line with IC50 values ranging from 48,58- 70,93 M

TÓM TẮT

Trong luận văn này, 10 dẫn xuất của benzo[d]imidazol-5(6)-yl)(phenyl)methanone đã được tổng hợp thành công bằng phản ứng ngưng tụ giữa dẫn xuất của o-phenylenediamine và dẫn xuất aldehyde thơm

(2-(4-methoxyphenyl)-1H-có sử dụng xúc tác Na2S2O5 trong hỗn hợp dung môi EtOH: H2O (9:1, v/v) Sau đó, mười dẫn xuất alkyl hóa tại vị trí N-1 của benzimidazole được tổng hợp bằng phản ứng alkyl hóa với xúc tác K2CO3 trong dung môi DMSO Các hợp chất đã tổng hợp được xác định tính chất bằng HPLC, UV- Vis, FT- IR, 1D và 2D- NMR, và HRMS Khả năng ức chế tăng sinh tế bào được thử nghiệm trên dòng tế bào ung thư vú MDA-

MB-231 bằng phương pháp SRB Kết quả thử nghiệm cho thấy hợp chất 3, 6b và 6d

thể hiện hoạt tính ức chế sự phát triển của dòng tế bào ung thư nghiên cứu với giá trị IC50 từ 48.58– 70.93 µM

ABSTRACT i

TÓM TẮT i

LIST OF FIGURES v

LIST OF SCHEMES vi

LIST OF TABLES vii

LIST OF ABBREVIATIONS viii

INTRODUCTION 1

CHAPTER 1 LITERATURE REVIEW 3

1.1 Benzimidazoles 3

1.1.1 Introduction 3

1.1.2 Physicochemical properties 3

1.1.3 Application 4

1.2 Overview to 2,5(6)-disubstituted benzimidazole derivatives 6

1.2.1 Synthetic method 6

1.2.2 Biological activities 9

1.3 Overview to N- alkylated benzimidazole derivatives 11

1.3.1 Synthesis of N- alkylated 11

1.3.2 Antiproliferative activities 12

CHAPTER 2 EXPERIMENTAL 15

2.1 Materials and instrumentations 15

2.1.1 Materials 15

2.1.2 Instrumentations 16

2.2 Procedure for synthesis of

(2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone (3) 16

2.3 General procedure for the synthesis of N- alkylated (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone derivatives 17

2.4 Isolation method and structure determination 18

2.4.1 Isolation method 18

2.4.2 Structure determination 19

2.5 Antiproliferative test 21

CHAPTER 3: RESULT AND DISCUSSION 22

3.1 Chemistry 22

3.1.1 (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone 26

3.1.2 N- alkylated (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone derivatives 27

3.2 Structure determination 36

3.2.1 Compound (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (Compound 3) 36

3.2.2 Compound (2-(4-methoxyphenyl)-1-propyl-1H-benzo[d]imidazol-5- yl)(phenyl)methanone (Compound 5a) 37

3.2.3 Compound (2-(4-methoxyphenyl)-1-propyl-1H-benzo[d]imidazol-6- yl)(phenyl)methanone (Compound 6a) 38

3.2.4 Compound (1-butyl-2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (Compound 5b) 39

3.2.5 Compound (1-butyl-2-(4-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)(phenyl)methanone (Compound 6b) 40

3.2.6 Compound (2-(4-methoxyphenyl)-1-pentyl-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (Compound 5c) 41

3.2.7 Compound

(2-(4-methoxyphenyl)-1-pentyl-1H-benzo[d]imidazol-6-yl)(phenyl)methanone (Compound 6c) 42

3.2.8 Compound (1-hexyl-2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (compound 5d) 43

3.2.9 Compound (1-hexyl-2-(4-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)(phenyl)methanone (compound 6d) 44

3.2.10 Compound (1-heptyl-2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone (compound 5e) 45

3.2.11 Compound (1-heptyl-2-(4-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)(phenyl)methanone (compound 6e) 46

3.3 Discussion 46

3.3 Antiproliferative result 53

CHAPTER 4 CONCLUSION 56

4.1 Concluding remark 56

4.2 Suggestion on future work 56

REFERENCES 57

LIST OF FIGURES

Figure 2.1 Illustration of procedure for synthesizing compound 3 16

Figure 2.2 Illustration of procedure for synthesizing 5a-e and 6a-e 17

Figure 3.1 Sample and TLC of 3 26

Figure 3.2 Sample and TLC of 5a 27

Figure 3.3 Sample and TLC of 5b 28

Figure 3.4 Sample and TLC of 6b 29

Figure 3.5 Sample and TLC of 5c 30

Figure 3.8 Sample and TLC of 6c 31

Figure 3.7 Sample and TLC of 5d 32

Figure 3.8 Sample and TLC of 6d 33

Figure 3.9 Sample and TLC of 5e 34

Figure 3.10 Sample and TLC of 6e 35

Figure 3.11 UV-Vis absorption spectra of compounds 3, 5a−e, 6a−e 46

Figure 3.12 1H-NMR spectra of compound 6b (A) and compound 5b (B) Figure 3.13 2D-NMR spectra of 6b a) NOESY spectrum, correlations between H-7 and H-1” are circled in red color b) HMBC spectrum, correlations between H-1” and C-2 and C-8 are circled in blue color

Figure 3.14 2D-NMR spectra of 5b a) NOESY spectrum, correlations between H-7 and H-1” are circled in red color b) HMBC spectrum, correlations between H-1” and C-2 and C-8 are circled in blue color

LIST OF SCHEMES

Scheme 1.1 Benzimidazole structure and numbering rule 3

Scheme 1.2 Phillips's reaction of o-phenylenediamine and oxalic acid 6

Scheme 1.3 Reaction of o-phenylenediamine with anhydride acetic 7

Scheme 1.4 Reaction of 3,4- diamino-toluene with ethyl formate 7

Scheme 1.5 Reaction of aryldiamine with 4-hydroxy-5,8-dimethoxy-2-naphthaldehyde 8

Scheme 1.6 Reaction of o-phenylenediamnie with aldehyde 8

Scheme 1.7 Benzimidazole synthesis by oxidation of air 8

Scheme 1.8 Benzimidazole synthesis with microwave- assisting 8

Scheme 1.9 General structures of compounds 1-6 9

Scheme 1.10 Compounds 4a and 4b in the study of Nayak et al 10

Scheme 1.11 Structures of compounds 38 and 40 10

Scheme 1.12 Alkylation reaction of Nale et al 12

Scheme 1.13 Alkylation reaction of Chakraborty et al 12

Scheme 1.14 Structure of compound 4k 12

Scheme 1.15 Structure of compound TJ08 13

Scheme 1.16 Structure of compound 4c 14

Scheme 2.1 Procedure for synthesis of 3 16

Scheme 2.2 General procedure for the synthesis of 5a-e, 6a-e 17

Scheme 3.1 General procedure for the synthesis of the final compounds 5a−e, 6a−e 24

Scheme 3.2 Mechanism of the alkylation reaction 25

LIST OF TABLES

Table 1.1 Marketed medicines containing benzimidazole moiety 5

Table 1.2 IC50 values of compounds 38 and 40 against MDA-MB-231 10

Table 1.3 IC50 values of compound TJ08 against six cancer cells and normal cells 13

Table 2.4 List of chemicals 15

Table 2.5 List of instrumentations 16

Table 2.6 HPLC condition 18

Table 2.7 NMR techniques for structure determination were used in this thesis 20

Table 3.1 Yield of synthesized 23

Table 3.2 Comparison between the 1H-NMR chemical shift of this study with previous reports 35

Table 3.3 IC50 of synthesized compounds (3, 5a-e, 6b-e) 52

DI Water Deion water

DMSO dimethyl sulfoxide

HSQC Heteronuclear Single Quantum Coherence

TCA trichloracetic acid

tt triplet of triplets

INTRODUCTION

1 Introduction

Cancer is one of the fatal causes of death worldwide, necessitating the development of novel and effective treatments Even though modern therapeutic agents have been developed over the last 100 years, the successful treatment of cancer appears to be a formidable challenge at the start of the century According to GLOBOCAN's 2020 forecast, approximately 19.3 million cancer diagnoses and 10 million cancer deaths are predicted worldwide in 2020 Breast cancer (BC) is 1 in 4 commonly diagnosed cancers and the second most significant cause of cancer-related deaths in women; about 12% of breast cancers are triple negative This obstacle results from the challenges of discovering innovative selective medicines that suppress tumor cell development without being harmful to normal cells

Historically, nitrogen-containing heterocycles are an exciting research topic because they are bioactive compounds Due to their reputation, N-heterocycles are essential for studying biological activities Among them, benzimidazoles greatly interest many research groups investigating drug development Mounting evidence indicates that benzimidazole and its derivatives, with substitutions at the 1, 2, 5, and/or 6-positions, have a vast medicinal profile in multiple categories of therapeutic agents with unique properties, including antimicrobial,anti-hypertensive, anti-tuberculosis, anti-viral, antiulcer, anti-inflammatory, anti-diabetic, anti-convulsant and anti-malarial In addition, many studies already reveal the potential of benzimidazole derivatives as anticancer medicines In our previous work, benzimidazole derivatives have been synthesized, and in terms of anticancer

activities, (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone

emerged as multitarget anticancer agent, especially in anticancer effect against breast cancer cell line (MDA-MB-231) Encouraged by these findings, in this study, we considered introducing the alkyl straight chains to the benzimidazole moiety to produce new molecules with the expectation of increasing anticancer activity

2 Aims and objectives

With the above preamble, the present work entitled "Synthesis and

antiproliferative evaluation of N- alkylated benzo[d]imidazol-5(6)-yl)(phenyl)methanone derivatives" has been done with the

(2-(4-methoxyphenyl)-1H-following objectives:

i Dealing with developing a method for synthesizing benzimidazole and

N-alkylated benzimidazole derivatives and characterizing them by physical and spectral analysis

ii Dealing with evaluating synthesized derivatives for antiproliferative

activity against breast cancer cell line (MDA-MB-231)

The objective of the current work has been aimed at achieving the following,

i To synthesize a nucleus containing benzimidazole

ii To synthesize targeted N-alkylated benzimidazole isomers

iii To isolate the N-alkylated benzimidazole derivative in a mixture of

CHAPTER 1 LITERATURE REVIEW

1.1 Benzimidazoles

1.1.1 Introduction

Benzimidazole is a compound consisting of a phenyl ring attached to an imidazole ring that was first synthesized by Hoebrecker by reduction of 2-nitro-4-methylacetanilide to obtain 2,5(or 2,6)- dimethylbenzimidazole in 1872 [1]

In the following years, Ladenburg and Philip found synthetic pathway of benzimidazole by condensing o-phenylenediamine with carbonyl group compounds Therefore, the preparation of benzimidazole from ortho-amino aniline is referred to

as the Ladenburg method, Phillips’s method [2], [3]

In 1949, Brink et al proved the appearance of a benzimidazole scaffold as the ligand during the degradation of vitamin B12 Consequently, the connection between benzimidazole structure and biological activities was identified [4]

Benzimidazole structure and numbering rules are described in Scheme 1.1

Scheme 1.1 Benzimidazole structure and numbering rule

Due to tautomerization, 1H-benzimidazole derivatives exist as isomers The two

derivatives, 5-methylbenzimidazole, and 6-methylbenzimidazole, illustrate a pair of tautomers and describe the same substance However, no tautomerization occurs when a substituent at the N-1 position is larger than the hydrogen, but a characteristic isomer forms [1]

1.1.2 Physicochemical properties

The benzimidazoles are simply solids with relatively high melting points

(1H-benzimidazole, 170°C) Generally, when a substituent is added to the N-1 position,

the melting point of benzimidazoles decreases (1-methyl-1H-benzimidazole, 66oC) [5] because benzimidazoles cannot form intermolecular hydrogen bonds because the hydrogen is at N- 1 was replaced

The 1H-benzimidazoles are highly soluble in polar solvents and sparingly soluble

in nonpolar solvents When substituents of different polarities are attached to the benzimidazole ring, they are soluble in the respective solvent For example, 2-methylbenzimidazole is well-soluble in the ether, while 2-aminobenzimidazole is highly soluble in water [14]

1.1.3 Application

Benzimidazole derivatives have been applied in a variety of industries: textile dying [6], semi-conduction [7], and anti-corrosion [8], However, benzimidazole derivatives gain more attention in pharmaceuticals due to their biological activities Several commercial drugs containing benzimidazole moiety are used in clinical applications, such as antibacterial [9], antifungal [9], antiallergic activities [10], analgesics [11], blocking of the proton pump (H+/K+-ATPase) [12]… Instances are

described in Table 1.1

Table 1.1 Marketed medicines containing benzimidazole moiety

Veliparib [19]

Bendamustine [20]

1.2 Overview to 2,5(6)-disubstituted benzimidazole derivatives

1.2.1 Synthetic method

The most common way to synthesize benzimidazole is condensation between benzene derivatives containing substitution groups of nitrogen at 1,2 position and carbonyl group-containing compound

1.2.1.1 Reaction with carboxylic acid

In 1928, Phillips et al successfully synthesized benzimidazole derivatives for

the first time by condensation o-phenilenediamine with oxalic acid, malonic acid, and

benzoic acid by boiling mixtures with HCl [3]

Scheme 1.2 Phillips's reaction of o-phenylenediamine and oxalic acid

Based on Phillips’s research, further studies were practiced to synthesize more complex benzimidazole derivatives In 1983, J Gerald Wilson and Frederick C Hunt successfully synthesized iminodiacetic acid derivatives of benzimidazole [21], [22]

Although the Phillips reaction is commonly used to synthesize benzimidazole derivatives, it has limitations when reagents are aromatic carboxylic acid, especially when complex substitute groups or heteroatoms are included [23]

1.2.1.2.Reaction with anhydride

The reaction products between o-phenylenediamine and acid anhydride are benzimidazole or N, N'-diacylphenylenediamine, depending on the operating conditions and reaction duration A high yield of benzimidazole is achieved when the reaction time is sufficiently prolonged Under refluxed boiling conditions, the o-phenylenediamine cyclization process with acetic anhydride is entirely converted to 2-methylbenzimidazole This may be accomplished with acetic anhydride alone, sodium acetate, or acetic acid [1]

Scheme 1.3 Reaction of o-phenylenediamine with anhydride acetic

In addition, o-phenylenediamine combines with succinic anhydride and phthalic anhydride to form, respectively, -(2-benzimidazole) propionic acid and o-(2-benzimidazole) benzoic acid [1]

1.2.1.3 Reaction with ester

First discovered by Von Niementowski, the synthesis of benzimidazole from o-phenylenediamine and an ester Nevertheless, this approach is not commonly used

In a sealed vessel, 3,4-diamino-toluene dihydrochloride and ethyl formate were heated continuously at 225oC for three hours to produce 5(6)-methylbenzimidazole hydrochloride This product is not further alkylated by the ethyl chloride that is made [1]

Scheme 1.4 Reaction of 3,4- diamino-toluene with ethyl formate

1.2.1.4 Reaction with aldehyde

Under the varied case, the condensation reaction between o-phenylenediamine and aldehyde yields benzimidazole with a substituent at the second position The best circumstances are typically reaction conditions in the presence of air or oxidizing substances [1]

Binh Phung et al synthesized benzimidazole derivatives from arylenediamine and 4-hydroxy-5,8-dimethoxy-2-naphthaldehyde in DMSO at 100 °C using Na2S2O5 [24]

o-Scheme 1.5 Reaction of aryldiamine with

4-hydroxy-5,8-dimethoxy-2-naphthaldehyde

Xiangming et al synthesized benzimidazole derivatives from phenylenediamine and various aldehydes using NaHSO3 in DMF at 80oC [25]

o-Scheme 1.6 Reaction of o-phenylenediamnie with aldehyde

Lin et al used air as the oxidant reagent and dioxane as a solvent for high yield (90%) in synthesizing benzimidazole derivatives [26]

Scheme 1.7 Benzimidazole synthesis by oxidation of air

Besides using classical methodologies (heating), several studies applied modern methods to synthesize benzimidazole, specially synthesized with microwave support Navarrete‐Vázquez et al., by microwave-assisting, successfully synthesized benzimidazole deliveries [27]

Scheme 1.8 Benzimidazole synthesis with microwave- assisting

Hue et al used microwave irradiation to assist in synthesizing several complicated benzimidazole derivatives [28]

1.2.2 Biological activities

Recently, derivatives of 2,5(6)-disubstituted benzimidazole gained the attention of many chemists due to their biological activities and application in the clinic

Aurelio Romero-Castro and co-workers (2011) [29] reported a series of six

2-aryl-5(6)-nitro-1H-benzimidazole derivatives (1−6) as promising anticancer

candidates Antiproliferative activities using the MTT assay were screened against seven human neoplastic cell lines (K562, HL60, MCF-7, MDA231, A549, HT29 và KB.)

Scheme 1.9 General structures of compounds 1-6

The results indicated that compound 6 (R3=Cl, R4=NO2) was the most active agent against all tested cancer cell lines, especially in MDA-MB-231 with an IC50

value of 4.0 M, and exhibited similar activity to a positive control (carboplatin) Interestingly, this compound showed less antiproliferative effect against a non-neoplastic cell line (HACAT)

Nayak et al [30] developed and synthesized the derivatives of the disubstituted benzimidazole-oxindole conjugate and assessed them as chemotherapeutic agents against the human breast cancer cell line MCF-7 Among

2,6-synthesized analogs, compounds 4a and 4b displayed 43.7% and 43.6% at 1μM and

64.8% and 62.7% at 2μM apoptosis, respectively

Scheme 1.10 Compounds 4a and 4b in the study of Nayak et al

In 2020, our previous studies [31] were developed and successfully synthesized twenty-nine of 2,5(6)-disubstituted benzimidazole derivatives under facile and mild conditions All compounds were tested for anticancer activity against three cancer cell lines (A549, MDA-MB-231, and PC3)

Scheme 1.11 Structures of compounds 38 and 40

Regarding anticancer activities against MDA-MB-231 (a breast cancer cell

line), compounds 38 and 40 were the most promising agents for this cancer cell line (as shown in Table 1.2 and Scheme 1.11)

based on compound 40 for evaluating the effect of new substitution on anticancer

activity against breast cancer

1.3 Overview to N- alkylated benzimidazole derivatives

1.3.1 Synthesis of N- alkylated

1.3.1.1 Classification of the alkylation reaction Alkylation substitutes alkyl groups for one or more hydrogen atoms in an organic molecule The alkyl group can directly bond to carbon, oxygen, nitrogen, or sulfur, corresponding to C-alkylation, O-alkylation, N-alkylation, and S-alkylation, respectively

Alkylating agents such as alcohols (R-OH), alkyl halides (RX), alkyl sulfates, sulfonic acid, and esters with catalyst agents such as HF, H2SO4, H3PO4, and Lewis acids are often used in the alkylating reaction Under diverse settings, alkylation reactions will occur via distinct pathways [32]

1.3.1.2 Alkylation by Mannich reaction Mannich reaction includes formaldehyde and a primary or secondary amine alkylating a compound with the acidic proton next to a carbonyl group [33] By Mannich reaction, Roman G et al synthesized benzimidazole derivatives in 2012 [34]

1.3.1.3 Alkylation by activated alkene Activated alkenes were used to alkylate benzimidazoles with high yield Although high-yield alkylation for benzimidazole, activated alkene requires the compilated catalyst [35], [36]

1.3.1.4 Alkylation by alkyl halide and related compounds Alkylation of benzimidazole derivatives by alkyl halide and the related compounds has been commonly used for several previous studies

Nale et al synthesized benzimidazole derivatives from different

o-phenylenediamine derivatives and formamide, using zinc acetate catalysis in the presence of poly (methylhydrosiloxane) to generate benzimidazole derivatives at the N-1 position [37]

Scheme 1.12 Alkylation reaction of Nale et al

Chakraborty et al practiced the alkylation of benzimidazole by R-Br agent with the presence of NaOH and SDS catalyzed for 75-98% yield [38]

Scheme 1.13 Alkylation reaction of Chakraborty et al

Rohand et al synthesized alkylated benzimidazole derivatives with alkyl halide under the Transfer Catalysis condition [39]

1.3.2 Antiproliferative activities

Pham et al (2022) [40] designed and synthesized forty-two N-substituted (chloro/ nitro)-1H-benzimidazole derivatives and evaluated them for their anticancer activities

6-Scheme 1.14 Structure of compound 4k

Compound 4k exhibited potent anticancer activity with IC50 < 10 M against five tested cell lines (HepG2, MDA-MB-231, MCF7, C26, and RMS), which is compared with the reference drug (PTX) The study concluded that the appearance

of the N-benzyl/N-(4-chlorobenzyl) group and the chloro/N,N-dimethylamino

moiety in the phenyl ring at position 2 of the 1H-benzimidazole scaffold is more

favorable for enhanced antitumor activity

Jagadeesha and co-worker [41] reported a series of 1,2,5-trisubstituted benzimidazole derivatives (TJ01–TJ15) through multistep synthesis by reacting with

different aromatic amines in position 1, different substituted aromatic acids in

position 2, and introducing ester, acid, and amide in the position 5 of the

benzimidazole moiety All the synthesized derivatives were tested against six cancer

cells, including human leukemic cancer cells (Jurkat, K562, and Molt4), human

cervical cancer cells (HeLa), human colorectal carcinoma cells (HCT116), and

human pancreatic ductal adenocarcinoma (MIAPaCa-2)

Scheme 1.15 Structure of compound TJ08

Following evaluation, the compound TJ08 was very active against cancer cells

with IC50 ranging from 1,88 to 3,82 μM (Table 1.3), and doxorubicin showed

apoptotic activity against Jurkat cells at 4,89 μM Jurkat cells were the most sensitive,

whereas MIA PaCa-2 cells were the least susceptible to TJ08

Table 1.3 IC50 values of compound TJ08 against six cancer cells and normal cells

TJ08 1,88 ± 0,51 1,89 ± 0,55 2,05 ± 0,72 2,11 ± 0,62 3,04 ± 0,8 3,82 ± 0,25 >50

DOXO 4,89 ± 0,11 0,99 ± 0,21 > 1 0,21 ± 0,13 0,26 ± 0,19 0,059 ± 0,63 >50

Twenty-three N,2,6-trisubstituted 1H-benzimidazole derivatives were

synthesized by Pham et al (2023) [42], furthermore investigated anticancer activities

against cancer cell lines (HepG2, MDA-MB-231, MCF7, RMS, and C26) with the

reference drug PTX

Scheme 1.16 Structure of compound 4c

The results revealed that compound 4c exhibited the most potent

antiproliferative activity among all compounds against HepG2, MDA-MB-231, MCF7, RMS, and C26 with IC50 of 3,22; 2,39; 5,66; 4,83; and 3,90 μM, respectively

as compared to PTX The research concluded that electron-withdrawing substituents

on the phenyl ring and N-phenyl and N-(4-chlorobenzyl) substituents may be responsible for its biological activity compared to other compounds

According to structure-activity relationship studies of the benzimidazole ring system, the substitution at N-1, C-2, C-5, and C-6 positions have been assessed to be the most contributory factors for antiproliferative activity [43], [44] In light of this information, we designed and synthesized a series of N-alkylated (2-(4-

methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone This series was

based on the structure of compound 40 in our previous study [31] mentioned above

by changing the position of the alkyl group in the N-1 position to enhance antiproliferative activity and determine the correct pattern of the alkyl substitution

CHAPTER 2 EXPERIMENTAL

2.1 Materials and instrumentations

2.1.1 Materials

All chemicals were purchased commercially from manufacturers as listed in

Table 2.1 and were used without further purification unless otherwise noted

Table 2.4 List of chemicals

1 3,4-diaminobenzophenone, 99% C13H12N2O Acros Organics (Belgium)

2 4-methoxybenzaldehyde, 99% C8H8O2 Acros Organics (Belgium)

3 1-bromopropane,  99% C3H7Br Merck (Germany)

5 1-bromopentane,  99% C5H11Br Merck (Germany)

6 1-bromohexane,  99% C6H13Br Merck (Germany)

7 1-bromoheptane,  99% C7H15Br Merck (Germany)

10 Dichloromethane, > 99.7% CH2Cl2 Xilong (China)

14 Diethyl ether,  99% C4H10O Xilong (China)

15 Ethyl acetate,  99% C4H8O2 Chemsol (Vietnam)

16 Dimethyl sulfoxide,  99% C2H6OS Xilong (China)

17 Potassium carbonate anhydrous, > 99% K2CO3 Guangdong Guanghua (China)

18 Sodium metabisulfite,  96% Na2S2O5 Xilong (China)

2.1.2 Instrumentations

Table 2.5 List of instrumentations

1 Analytical balance Practum224- 1S Sartorius (Germany)

4 Multichannel UV darkroom CN-15 Vilber Lourmat (France)

7 UV-Vis Spectrophotometer UV- 1800 Shimadzu (Japan)

2.2 Procedure for synthesis of yl)(phenyl)methanone (3)

(2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-The compound was obtained according to our previously reported procedure

Scheme 2.1 Procedure for synthesis of 3

Figure 2.1 Illustration of procedure for synthesizing compound 3

Compound 3 was prepared by condensation between

(3,4-diaminophenyl)(phenyl)methanone (1) and 4-methoxybenzaldehyde (2) in the

presence of Na2S2O5 using EtOH: H2O (9:1, v/v) as a solvent TLC was used to monitor the reaction After the reaction was completed, the mixture was filtered The filtrate was evaporated under reduced pressure to obtain the raw product The obtained solid was washed several times with distilled water and n-hexane to remove impurities Then, the solid was dried in a vacuum at 60oC to afford the pure product

as an opalescent solid

2.3 General procedure for the synthesis of N- alkylated benzo[d]imidazol-5(6)-yl)(phenyl)methanone derivatives

(2-(4-methoxyphenyl)-1H-Scheme 2.2 General procedure for the synthesis of 5a-e, 6a-e

Figure 2.2 Illustration of procedure for synthesizing 5a-e and 6a-e

The mixture of 3 (0.1mmol) and alkyl bromide (4a−e) (0.3mmol) in 5mL

DMSO was refluxed in the presence of potassium carbonate at ambient temperature After the reaction was completed (as evident from TLC), the reaction mixture was

layer appeared and then extracted with n-hexane Then, the organic layer was concentrated under reduced pressure to obtain the mixture of positional isomers, including N- alkylated (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-

yl)(phenyl)methanone (5a−e) and N- alkylated benzo[d]imidazol-6-yl)(phenyl)methanone (6a−e) To obtain the pure separated

(2-(4-methoxyphenyl)-1H-isomer, the residue was further purified using column chromatography on silica gel with n-hexane/ ethyl acetate (3:2, v/v)

2.4 Isolation method and structure determination

2.4.1 Isolation method

Once a compound was isolated, its purity was evaluated based on a single spot

on the TLC (under at least two different solvent conditions)

2.4.1.1 Thin-layer chromatography (TLC) Thin-layer chromatography (TLC) analysis was done on Silica gel 60 F254, and the spots were located under UV light (254 nm, 365 nm) Eluent was hexane and ethyl acetate with a ratio of 3 and 2, respectively

2.4.1.2 High-performance liquid chromatography (HPLC) HPLC separation conditions (reverse phase HPLC, using DI water/ MeCN gradient systems) for the number of separations HPLC chromatograms were recorded on Agilent 1260 Infinity model (HPLC column ZORBAX Eclipse C18, 4.6x250 mm, 5 m) in the Institute of Chemical Technology- Vietnam Academy of Science and Technology

2.4.2 Structure determination

A variety of spectroscopic experiments were conducted in a reasonably set order The amount of purified sample was vital because it determined the order in which specific functional group tests were conducted If more than 50 mg of the sample was isolated and purified, then the order of spectroscopic analysis was not essential, as the risk of compound loss was diminished However, many of the samples that were separated were less than 10 mg, and the following testing sequence was adopted

2.4.2.1 UV-Vis spectroscopy

UV is a straightforward technique that requires a few samples (1 mg in 100 mL), and the sample is recoverable It relies upon the excitation and relaxation of electrons, typically between a bonding or lone-pair orbital and an unfilled non-bonding or anti-bonding orbital The absorption wavelength measures the energy level separation of the orbitals and will be unique for different structures It is possible

to analyze the resulting chromophore to determine the types of functional groups present

Routinely, the λmax (the ultraviolet light wavelength that gave the maximum absorbance) of a mixture and a pure sample was recorded to assist in setting the detector wavelength for HPLC

UV-Vis spectra were recorded on Shimadzu UV-1800 UV-Vis Spectrophotometer in the Institute of Chemical Technology- Vietnam Academy of Science and Technology

2.4.2.2 Nuclear magnetic resonance spectroscopy (NMR) Many different NMR experiments can be used The order and use of different pulse sequences in the analysis and structure determination of positional isomers are

pertinent to this thesis Table 2.7 shows a predetermined sequence for acquiring

NMR data and using the raw data to identify compounds

Table 2.7 NMR techniques for structure determination were used in this thesis

2.4.2.3 High-resolution mass spectroscopy (HRMS) Once all NMR spectral data had been collected, a tentative structural assignment was achieved A small amount of the sample was sent to the University

of Science- Viet Nam National University Ho Chi Minh City for an accurate mass (HRMS) measurement so that the molecular formula could be determined

The high-resolution mass spectra were measured on Agilent 6200 series TOF and 6500 series Q-TOF LC/MS system

2.4.2.4 Fourier Transform Infrared spectroscopy (FT-IR) Utilizing FT-IR, the presence of the vibration-based functional group was confirmed

FT-IR spectra were recorded on Bruker Tensor 27 in the Institute of Chemical Technology- Vietnam Academy of Science and Technology

2.4.2.5 Melting point Capillary method was used to determine the melting point of compounds The temperature at which the substance loses its crystallinity and transforms into a liquid was determined and recorded

The melting points were conducted on Krüss Optronic™ M5000 Melting Point Meter - Germany in the Institute of Chemical Technology- Vietnam Academy of Science and Technology and uncorrected

2.5 Antiproliferative test

Compounds (3; 5a−e; 6a−e) were screened for their antiproliferative activity

in vitro against the MDA-MB-231 cancer cell line (human breast carcinoma) by Sulforhodamine B (SRB) assay as described by Skehan et al [45] DMSO (1%) and Camptothecin (10 μM, 2 μM, 0.4μM, 0.08μM) were used as a negative and positive control, respectively Cells were dissociated with trypsin and then adjusted to a proper density All tested compounds were diluted at four concentrations (100 μM, 20 μM,

4 μM, 0.8 μM) in DMSO and added to the 96-well culture plates The plates containing cells (190 μL) but no tested compounds were screened for a no-growth control (day 0) After incubation for an hour, cells seeded on a no-growth control plate were fixed by trichloracetic acid – TCA 20% The plates containing target compounds were also treated with TCA 20% after being incubated for 72h Then, TCA-fixed cells were stained with SRB dye at 37oC for 30 min before being washed with acetic acid 1% to remove unbound dye The plates were air-dried at room temperature Then, a 10mM unbuffered Tris base was added to the plates to solubilize the dye binding to protein OD540 values were measured in ELISA Plate Reader (Biotek) The percentage of the cell-growth inhibition was calculated by using the formula below:

% 𝑔𝑟𝑜𝑤 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 100 −𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒− 𝑂𝐷𝑑𝑎𝑦 0

𝑂𝐷𝐷𝑀𝑆𝑂− 𝑂𝐷𝑑𝑎𝑦 0Each experiment was conducted in triplicate to define the IC50 values calculated on the software TableCure2Dv4

CHAPTER 3: RESULT AND DISCUSSION

3.1 Chemistry

The compound

(2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone (3) was synthesized following the reported procedure demonstrated in Scheme 3.1 by a condensation reaction between 3,4-

diaminobenzophenone and 4-methoxybenzaldehyde under mild conditions The

yields after the purification step were from 53 to 82% The synthetic pathway of ten target compounds (5a-e, 6a-e) is presented in Scheme 3.1 These compounds were prepared from compound 3 and alkyl bromide bearing long-chain hydrocarbon in the

presence of sodium carbonate with DMSO as solvent The overall yields of

N-alkylated benzimidazoles (5a-e, 6a-e) ranged from 50 to 98%

As shown in Scheme 3.1, the target compounds were synthesized in three steps Firstly, the aldehyde part of the 4-methoxybenzaldehyde (2) was treated with

sodium disulfite in the mixture of the solvent (EtOH: water = 9:1, v/v) to form the

aldehyde bisulfite (2') In the second step, as a result of the condensation reaction of

3,4-diaminobenzophenone (1) and 2', the

(2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone (3) was obtained In the last step, the

positional isomer mixture of N-alkylated benzo[d]imidazol-5-yl)(phenyl)methanone and N-alkylated (2-(4-methoxyphenyl)- 1H-benzo[d]imidazol-6-yl)(phenyl)methanone was collected by alkylation reaction

(2-(4-methoxyphenyl)-1H-Based on the academic literature, a mechanism for preparing benzimidazoles

has been proposed (Scheme 3.2) The reaction commences by nucleophilic attack of

a lone pair of the amine group on the o-phenylenediamine to the carbon atom of the

aldehyde metabisulfite adduct One mole of water is eliminated The generated alkyl sulphonate then interacts with the other amine group of o-phenylenediamine, yielding

a dihydroimidazole intermediate Lastly, aromatization gives benzimidazole nucleus

[46], [47] The benzimidazole derivative (3) and appropriate alkyl halide (4a-e) were

reacted in DMSO, and the final product was extracted with hexane In the last step,

N-alkylated benzimidazole derivatives (5a-e, 6a-e) were obtained by alkylation in

position 1 of benzimidazole in the presence of K2CO3 using DMSO as a solvent After forming the negatively charged nucleophile in the basic condition, the benzimidazole uses its lone-pair electrons to attack the alkyl halide carbon Then, the C-N bond forms thoroughly, and the bromide ion leaves with the electron pair from the former C–Br bond

The chromatography column was conducted to purify and isolate positional isomer compounds with hexane: ethyl acetate ratio ranging from 95:5 to 80:20

Table 3.1 Yield of synthesized

Scheme 3.1 General procedure for the synthesis of the final compounds 5a−e, 6a−e

Scheme 3.2 Mechanism of the alkylation reaction

3.1.1

(2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone

Figure 3.1 Sample and TLC of 3

Compound is visible as a spot on TLC chromatogram with UV light 254 nm (I), with 365 nm (II)

For elution:

- System A: Hex: EA (3:2, v/v); Rf= 0,20

- System B: CHCl3: MeOH (95:5, v/v); Rf= 0,75

- System C: CH2Cl2: EtOH (95:5, v/v); Rf= 0,54

3.1.2 N- alkylated

(2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone derivatives

Figure 3.2 Sample and TLC of 5a

Compound is visible as a spot on TLC chromatogram with UV light 254 nm (I), with 365 nm (II)

For elution:

- System A: Hex: EA (3:2, v/v); Rf= 0,38

- System B: CHCl3: MeOH (95:5, v/v); Rf= 0,84

- System C: CH2Cl2: EtOH (95:5, v/v); Rf= 0,69