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

Dealing with evaluating synthesized derivatives for antiproliferative activity against breast cancer cell line MDA-MB-231.. Synthetic method The most common way to synthesize benzimidazo

HO CHI MINH CITY, YEAR 2023

Headforemost, I want to express my thankful gratitude to Assoc Prof., Dr.Hoang Thi Kim Dung, for all of her guidance and support throughout the writing of

my thesis and her kindness, patience, and knowledge In addition to writing andconducting academic research in organic chemistry, I have received many helpfuland constructive recommendations about my field

Additionally, I would like to thank Ms Phan Ngoc Kim Ngan, who hassupported and managed my project and has been encouraging and instructivethroughout the research Her extensive knowledge and academic guidance werecrucial in assisting me in completing my thesis Besides, I'd like to thank everyone

in the Organic Chemistry and Polymer department for their continued friendlinessand assistance during the period with report documents and presentations

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

Ho Chi Minh City, day … month … 2023

Author

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)

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

In addition, other comments, reviews, and data used by other authors andorganizations 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 mywork (if any)

year Author

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, tenderivatives of N-alkylated benzimidazole were designed and synthesized byalkylation reactions in the presence of K2CO3 using dimethyl sulfoxide as a solvent.All synthesized compounds were characterized by HPLC, UV-Vis, FT-IR, 1D and2D-NMR, and HRMS The antiproliferative test determined activity against ahuman 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

(2-(4-methoxyphenyl)-1H-thơm 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 hợpbằng 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 NMR, và HRMS Khả năng ức chế tăng sinh tế bào được thử nghiệm trên dòng tếbào ung thư vú MDA-MB-231 bằng phương pháp SRB Kết quả thử nghiệm cho

2D-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

doublet of doubletsDeion water

dimethyl sulfoxidedoublet of tripletsethanol

Fourier transform Infrared spectroscopyn- hexane

Heteronuclear Multiple Bond Coherence

High- performance Liquid Chromatography

High-Resolution Mass SpectroscopyHeteronuclear Single Quantum CoherenceCoupling constant

multipletacetonitrilemethanolNuclear Magnetic Resonance Spectroscopyretardation factor

singlettriplettrichloracetic acidThin Layer Chromatographytriplet of triplets

1 Introduction

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

Historically, nitrogen-containing heterocycles are an exciting research topicbecause they are bioactive compounds Due to their reputation, N-heterocycles areessential for studying biological activities Among them, benzimidazoles greatlyinterest many research groups investigating drug development Mounting evidenceindicates that benzimidazole and its derivatives, with substitutions at the 1, 2, 5,and/or 6-positions, have a vast medicinal profile in multiple categories oftherapeutic 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

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 andN-alkylated benzimidazole derivatives and characterizing them by physical and spectralanalysis

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 positional isomers

iv To establish the structure based on melting point, UV-Vis, FT-IR, NMR, and HRMS spectra

v To evaluate compounds for antiproliferative activity

CHAPTER 1 LITERATURE REVIEW

1.1 Benzimidazoles

1.1.1 Introduction

Benzimidazole is a compound consisting of a phenyl ring attached to animidazole 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 ofbenzimidazole 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 theligand during the degradation of vitamin B12 Consequently, the connection betweenbenzimidazole 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 oftautomers and describe the same substance However, no tautomerization occurswhen a substituent at the N-1 position is larger than the hydrogen, but acharacteristic 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 thehydrogen 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, methylbenzimidazole is well-soluble in the ether, while 2-aminobenzimidazole ishighly soluble in water [14]

2-1.1.3 Application

Benzimidazole derivatives have been applied in a variety of industries: textiledying [6], semi-conduction [7], and anti-corrosion [8], However, benzimidazolederivatives gain more attention in pharmaceuticals due to their biological activities.Several commercial drugs containing benzimidazole moiety are used in clinicalapplications, 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]

6 Anticancer

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 betweenbenzene derivatives containing substitution groups of nitrogen at 1,2 position andcarbonyl 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 morecomplex benzimidazole derivatives In 1983, J Gerald Wilson and Frederick C Huntsuccessfully synthesized iminodiacetic acid derivatives of benzimidazole [21], [22]

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

1.2.1.2.Reaction with anhydrideThe reaction products between o-phenylenediamine and acid anhydride arebenzimidazole or N, N'-diacylphenylenediamine, depending on the operatingconditions and reaction duration A high yield of benzimidazole is achieved whenthe reaction time is sufficiently prolonged Under refluxed boiling conditions, the o-phenylenediamine cyclization process with acetic anhydride is entirely converted to2-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 fromo-phenylenediamine and an ester Nevertheless, this approach is not commonlyused In a sealed vessel, 3,4-diamino-toluene dihydrochloride and ethyl formatewere heated continuously at 225oC for three hours to produce 5(6)-methylbenzimidazole hydrochloride This product is not further alkylated by theethyl 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 phenylenediamine and aldehyde yields benzimidazole with a substituent at thesecond position The best circumstances are typically reaction conditions in thepresence 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

o-°C using Na2S2O5 [24]

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 modernmethods to synthesize benzimidazole, specially synthesized with microwavesupport Navarrete‐Vázquez et al., by microwave-assisting, successfullysynthesized benzimidazole deliveries [27]

Scheme 1.8 Benzimidazole synthesis with microwave- assisting

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

1.2.2 Biological activities

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

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 againstseven human neoplastic cell lines (K562, HL60, MCF-7, MDA231, A549, HT29 vàKB.)

Scheme 1.9 General structures of compounds 1-6

The results indicated that compound 6 (R3=Cl, R4=NO2) was the most activeagent against all tested cancer cell lines, especially in MDA-MB-231 with an IC50value 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 aschemotherapeutic 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).

Table 1.2 IC 50 values of compounds 38 and 40 against MDA-MB-231

Continuing our work toward discovering and developing biologically active

agents, we envisaged the synthesis of new benzimidazole derivatives with a

structure 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 reactionAlkylation substitutes alkyl groups for one or more hydrogen atoms in anorganic molecule The alkyl group can directly bond to carbon, oxygen, nitrogen, orsulfur, corresponding to C-alkylation, O-alkylation, N-alkylation, and S-alkylation,respectively

Alkylating agents such as alcohols (R-OH), alkyl halides (RX), alkylsulfates, 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 reactionMannich reaction includes formaldehyde and a primary or secondary aminealkylating a compound with the acidic proton next to a carbonyl group [33] ByMannich reaction, Roman G et al synthesized benzimidazole derivatives in 2012[34]

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

1.3.1.4 Alkylation by alkyl halide and related compoundsAlkylation of benzimidazole derivatives by alkyl halide and the relatedcompounds 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 thepresence of poly (methylhydrosiloxane) to generate benzimidazole derivatives at theN-1 position [37]

Scheme 1.12 Alkylation reaction of Nale et al .

Chakraborty et al practiced the alkylation of benzimidazole by R-Br agent withthe 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 halideunder 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 theiranticancer activities

6-Scheme 1.14 Structure of compound 4k

Compound 4k exhibited potent anticancer activity with IC50 < 10 M againstfive tested cell lines (HepG2, MDA-MB-231, MCF7, C26, and RMS), which iscompared 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-trisubstitutedbenzimidazole 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

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

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

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 ascompared to PTX The research concluded that electron-withdrawing substituents onthe phenyl ring and N-phenyl and N-(4-chlorobenzyl) substituents may be responsiblefor its biological activity compared to other compounds

According to structure-activity relationship studies of the benzimidazole ringsystem, 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 ofthis 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 enhanceantiproliferative 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

formula

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

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

10 Dichloromethane, > 99.7% CH2Cl2 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 tomonitor the reaction After the reaction was completed, the mixture was filtered.The filtrate was evaporated under reduced pressure to obtain the raw product Theobtained solid was washed several times with distilled water and n-hexane toremove impurities Then, the solid was dried in a vacuum at 60oC to afford the pureproduct as an opalescent solid

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

(2-(4-methoxyphenyl)-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 waspoured into distilled water The resulting solution was stirred for 15 min until the oil

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

(2-(4-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)(phenyl)methanone (6a−e) To obtain the pure separated

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 setorder The amount of purified sample was vital because it determined the order inwhich specific functional group tests were conducted If more than 50 mg of thesample was isolated and purified, then the order of spectroscopic analysis was notessential, as the risk of compound loss was diminished However, many of thesamples that were separated were less than 10 mg, and the following testingsequence was adopted

2.4.2.1 UV-Vis spectroscopy

UV is a straightforward technique that requires a few samples (1 mg in 100mL), and the sample is recoverable It relies upon the excitation and relaxation ofelectrons, typically between a bonding or lone-pair orbital and an unfilled non-bonding or anti-bonding orbital The absorption wavelength measures the energylevel separation of the orbitals and will be unique for different structures It ispossible to analyze the resulting chromophore to determine the types of functionalgroups present

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

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

2.4.2.2 Nuclear magnetic resonance spectroscopy (NMR)Many different NMR experiments can be used The order and use of differentpulse 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

NMR experiments were operated in Bruker Advance 600MHz NMR

Spectrometer in (CD3)2SO The chemical shifts ( ) were expressed in ppm and

referred to the residual peak of solvent as an internal standard Samples were sent to

the Institute of Chemistry- Vietnam Academy of Science and Technology

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 pointCapillary method was used to determine the melting point of compounds.The temperature at which the substance loses its crystallinity and transforms into aliquid was determined and recorded.

The melting points were conducted on Krüss Optronic™ M5000 MeltingPoint 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) bySulforhodamine B (SRB) assay as described by Skehan et al [45] DMSO (1%) andCamptothecin (10 μM, 2 μM, 0.4μM, 0.08μM) were used as a negative and positivecontrol, respectively Cells were dissociated with trypsin and then adjusted to aproper 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 platescontaining cells (190 μL) but no tested compounds were screened for a no-growthcontrol (day 0) After incubation for an hour, cells seeded on a no-growth controlplate were fixed by trichloracetic acid – TCA 20% The plates containing targetcompounds 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 beingwashed with acetic acid 1% to remove unbound dye The plates were air-dried atroom temperature Then, a 10mM unbuffered Tris base was added to the plates tosolubilize the dye binding to protein OD540 values were measured in ELISA PlateReader (Biotek) The percentage of the cell-growth inhibition was calculated byusing the formula below:

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 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 alkylsulphonate then interacts with the other amine group of o-phenylenediamine, yielding adihydroimidazole 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