(Tiểu luận) 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

LITERATURE REVIEW

Benzimidazoles

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 subsequent years, Ladenburg and Philip discovered a synthetic pathway for benzimidazole by condensing o-phenylenediamine with carbonyl group compounds This method of preparing benzimidazole from ortho-amino aniline is known as the Ladenburg method or Phillips’s method.

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

1H-benzimidazole derivatives exist as isomers due to tautomerization, exemplified by 5-methylbenzimidazole and 6-methylbenzimidazole, which represent a pair of tautomers of the same substance However, when a substituent at the N-1 position is larger than hydrogen, tautomerization does not occur, resulting in the formation of a distinct isomer.

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, 66 o C)

[5] because benzimidazoles cannot form intermolecular hydrogen bonds because the hydrogen is at N- 1 was replaced

1H-benzimidazoles exhibit high solubility in polar solvents and limited solubility in nonpolar solvents The solubility of these compounds varies with the polarity of substituents attached to the benzimidazole ring For instance, 2-methylbenzimidazole demonstrates good solubility in ether, whereas 2-aminobenzimidazole is highly soluble in water.

Benzimidazole derivatives are utilized across various industries, including textile dyeing, semi-conduction, and anti-corrosion However, their significance in pharmaceuticals has surged due to notable biological activities Numerous commercial drugs featuring the benzimidazole moiety are employed in clinical settings, demonstrating antibacterial, antifungal, antiallergic, and analgesic properties, as well as the ability to block the proton pump (H+/K+-ATPase), as detailed in Table 1.1.

Table 1.1 Marketed medicines containing benzimidazole moiety

No Uses of drugs Example Structure

3 Blocking of the proton pump [15] Omeprazole

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

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

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]

The reaction between o-phenylenediamine and acid anhydride yields either benzimidazole or N, N'-diacylphenylenediamine, influenced by reaction conditions and duration Prolonging the reaction time significantly increases the yield of benzimidazole When conducted under reflux boiling conditions, the cyclization of o-phenylenediamine with acetic anhydride results in the complete conversion to 2-methylbenzimidazole, achievable using acetic anhydride, sodium acetate, or acetic acid.

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]

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 continuously heated at 225 °C for three hours, resulting in the formation of 5(6)-methylbenzimidazole hydrochloride, which is not further alkylated by the ethyl chloride produced.

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

The condensation reaction between o-phenylenediamine and aldehyde produces benzimidazole with a substituent at the second position Optimal reaction conditions usually involve the presence of air or oxidizing agents.

Binh Phung et al synthesized benzimidazole derivatives from o- arylenediamine and 4-hydroxy-5,8-dimethoxy-2-naphthaldehyde in DMSO at 100 °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 o- phenylenediamine and various aldehydes using NaHSO3 in DMF at 80 o C [25]

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]

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 colleagues (2011) identified six promising anticancer candidates, specifically 2-aryl-5(6)-nitro-1H-benzimidazole derivatives (1−6) Their antiproliferative activities were evaluated using the MTT assay against seven human neoplastic cell lines, including K562, HL60, MCF-7, MDA231, A549, HT29, and KB.

Scheme 1.9 General structures of compounds 1-6

Compound 6 (R3=Cl, R4=NO2) demonstrated the highest activity against all tested cancer cell lines, particularly MDA-MB-231, with an IC50 value of 4.0 µM, comparable to the positive control, carboplatin Notably, this compound exhibited a reduced antiproliferative effect on the non-neoplastic cell line HACAT.

Nayak et al [30] synthesized derivatives of the 2,6-disubstituted benzimidazole-oxindole conjugate and evaluated their effectiveness as chemotherapeutic agents against the MCF-7 human breast cancer cell line The study found that compounds 4a and 4b exhibited significant apoptosis rates of 43.7% and 43.6% at 1μM, and 64.8% and 62.7% at 2μM, respectively.

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

In 2020, we successfully synthesized twenty-nine 2,5(6)-disubstituted benzimidazole derivatives under mild conditions These compounds were evaluated for their 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

We are focused on discovering and developing biologically active agents, specifically through the synthesis of new benzimidazole derivatives inspired by compound 40 Our goal is to evaluate the impact of these new substitutions on anticancer activity, particularly against breast cancer.

Overview to N- alkylated benzimidazole derivatives

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, including alcohols (R-OH), alkyl halides (RX), alkyl sulfates, sulfonic acids, and esters, are commonly utilized in alkylation reactions, often in conjunction with catalyst agents like HF, H2SO4, H3PO4, and Lewis acids These reactions can proceed through various pathways depending on the specific conditions employed.

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

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]

Pham et al (2022) [40] designed and synthesized forty-two N-substituted 6- (chloro/ nitro)-1H-benzimidazole derivatives and evaluated them for their anticancer activities

Compound 4k demonstrated significant anticancer efficacy, with an IC50 of less than 10 µM across five cancer cell lines, including HepG2, MDA-MB-231, MCF7, C26, and RMS, outperforming the reference drug, PTX The research highlighted that the presence 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 contributes to improved antitumor activity.

Jagadeesha and colleagues developed a series of 1,2,5-trisubstituted benzimidazole derivatives (TJ01–TJ15) through a multistep synthesis process This involved reacting various aromatic amines at position 1, different substituted aromatic acids at position 2, and introducing ester, acid, and amide groups at position 5 of the benzimidazole structure The synthesized derivatives were evaluated for their efficacy against six types of cancer cells, including human leukemic 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

The compound TJ08 demonstrated significant anti-cancer activity, exhibiting an IC50 range of 1.88 to 3.82 μM, while doxorubicin displayed apoptotic effects on Jurkat cells at 4.89 μM Among the tested cell lines, Jurkat cells were the most sensitive to TJ08, in contrast to MIA PaCa-2 cells, which showed the least susceptibility.

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

Jurkat K562 MOLT-4 HeLa HCT116 MIA- PaCa-2 HEK

Pham et al (2023) synthesized twenty-three N,2,6-trisubstituted 1H-benzimidazole derivatives and evaluated their anticancer activities against various cancer cell lines, including HepG2, MDA-MB-231, MCF7, RMS, and C26, using the reference drug PTX for comparison.

Compound 4c demonstrated the highest antiproliferative activity against HepG2, MDA-MB-231, MCF7, RMS, and C26, with IC50 values of 3.22, 2.39, 5.66, 4.83, and 3.90 μM, respectively, outperforming PTX The study concluded that the presence of electron-withdrawing substituents on the phenyl ring, along with N-phenyl and N-(4-chlorobenzyl) groups, likely contributes to its superior biological activity compared to other compounds.

Structure-activity relationship studies of the benzimidazole ring system indicate that substitutions at the N-1, C-2, C-5, and C-6 positions significantly influence antiproliferative activity Based on this insight, we designed and synthesized a series of N-alkylated (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone This new series builds upon compound 40 from our previous research by repositioning the alkyl group at the N-1 position to enhance antiproliferative effects and identify the optimal alkyl substitution pattern.

EXPERIMENTAL

Materials and instrumentations

All chemicals were purchased commercially from manufacturers as listed in

Table 2.1 and were used without further purification unless otherwise noted

No Substances Molecular formula Manufacturer

10 Dichloromethane, > 99.7% CH2Cl2 Xilong (China)

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

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

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

1 Analytical balance Practum224- 1S Sartorius (Germany)

2 Vacuum oven VOS-301SD Eyela (Japan)

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

5 Vacuum pump GLD-37CC Ulvac (Japan)

6 HPLC 1260 Infinity Agilent (United States)

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

8 Melting point meters M5000 Kruss (Germany)

2.2 Procedure for synthesis of (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)- yl)(phenyl)methanone (3)

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 60 o C to afford the pure product as an opalescent solid

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

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 with potassium carbonate at room temperature, and upon completion of the reaction, indicated by TLC, the mixture was extracted with n-hexane The organic layer was then concentrated under reduced pressure, yielding a mixture of positional isomers, specifically 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 isolate the pure isomer, the residue underwent further purification via column chromatography on silica gel using a n-hexane/ethyl acetate (3:2, v/v) solvent system.

Isolation method and structure determination

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

A series of spectroscopic experiments were performed in a specific sequence, influenced by the quantity of purified sample available When over 50 mg of the sample was isolated, the order of analysis became less critical due to reduced risk of compound loss Conversely, for samples weighing less than 10 mg, a defined testing sequence was necessary to ensure accurate results.

UV spectroscopy is a simple method that requires minimal sample sizes (1 mg in 100 mL) and allows for sample recovery This technique is based on the excitation and relaxation of electrons between bonding or lone-pair orbitals and unfilled non-bonding or anti-bonding orbitals The absorption wavelength indicates the energy level separation of these orbitals, which varies for different molecular structures By analyzing the resulting chromophore, one can identify the types of functional groups present in the sample.

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

NMR experiments were conducted using a Bruker Advance 600MHz NMR Spectrometer in (CD3)2SO, with chemical shifts (δ) reported in ppm and referenced to the residual peak of the solvent as an internal standard The samples were submitted to the Institute of Chemistry at the 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 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.

Antiproliferative test

Compounds (3; 5a−e; 6a−e) were evaluated for their antiproliferative effects on the MDA-MB-231 human breast cancer cell line using the Sulforhodamine B (SRB) assay, following the methodology outlined by Skehan et al DMSO at 1% served as a negative control, while Camptothecin was tested at concentrations of 10 μM, 2 μM, 0.4 μM, and 0.08 μM as a positive control The cancer cells were dissociated with trypsin and adjusted to the appropriate density before testing Each compound was diluted to four different concentrations: 100 μM and 20 μM.

In this study, various concentrations of compounds (4 μM, 0.8 μM) in DMSO were added to 96-well culture plates containing cells (190 μL) to establish a no-growth control on day 0 After a one-hour incubation, cells on the no-growth control plate were fixed using 20% trichloracetic acid (TCA) Following a 72-hour incubation, the plates with target compounds were also treated with 20% TCA The TCA-fixed cells were then stained with SRB dye at 37°C for 30 minutes, followed by washing with 1% acetic acid to eliminate unbound dye The plates were air-dried at room temperature, and a 10 mM unbuffered Tris base was added to solubilize the dye bound to proteins The optical density at 540 nm (OD540) was measured using an ELISA Plate Reader (Biotek), and the percentage of cell growth inhibition was calculated using a specific formula.

𝑂𝐷 𝐷𝑀𝑆𝑂 − 𝑂𝐷 𝑑𝑎𝑦 0 Each experiment was conducted in triplicate to define the IC50 values calculated on the software TableCure2Dv4.

RESULT AND DISCUSSION

Chemistry

The compound (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone (3) was synthesized through a condensation reaction between 3,4-diaminobenzophenone and 4-methoxybenzaldehyde, yielding 53 to 82% after purification The synthetic pathway for ten target compounds (5a-e, 6a-e) is illustrated in Scheme 3.1, where these compounds were derived from compound 3 and long-chain alkyl bromides in the presence of sodium carbonate and DMSO as the solvent The overall yields of the N-alkylated benzimidazoles (5a-e, 6a-e) varied from 50 to 98%.

The target compounds were synthesized through a three-step process Initially, 4-methoxybenzaldehyde was treated with sodium disulfite in a solvent mixture of ethanol and water to produce the aldehyde bisulfite In the second step, a condensation reaction between 3,4-diaminobenzophenone and the aldehyde bisulfite yielded (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5(6)-yl)(phenyl)methanone Finally, the positional isomer mixture of N-alkylated (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone and N-alkylated (2-(4-methoxyphenyl)-1H-benzo[d]imidazol-6-yl)(phenyl)methanone was obtained through an alkylation reaction.

A proposed mechanism for synthesizing benzimidazoles involves a nucleophilic attack by the amine group of o-phenylenediamine on the carbon atom of the aldehyde metabisulfite adduct, resulting in the elimination of one mole of water This reaction produces an alkyl sulphonate, which subsequently reacts with another amine group of o-phenylenediamine to form a dihydroimidazole intermediate The final step of the process is aromatization, leading to the formation of the benzimidazole nucleus.

The benzimidazole derivative (3) was reacted with various alkyl halides (4a-e) in DMSO, leading to the extraction of the final product using hexane Subsequently, N-alkylated benzimidazole derivatives (5a-e, 6a-e) were synthesized through alkylation at position 1 of benzimidazole in the presence of K2CO3, utilizing DMSO as the solvent In this process, a negatively charged nucleophile is formed under basic conditions, allowing the benzimidazole to use its lone-pair electrons to attack the carbon of the alkyl halide, resulting in the formation of a C-N bond and the departure of the bromide ion.

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

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)

- 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

Figure 3.3 Sample and TLC of 5b

Figure 3.4 Sample and TLC of 6b

Figure 3.5 Sample and TLC of 5c

Figure 3.6 Sample and TLC of 6c

Figure 3.7 Sample and TLC of 5d

Figure 3.8 Sample and TLC of 6d

Figure 3.9 Sample and TLC of 5e

- System C: CH2Cl2: EtOH (95:5, v/v); Rf= 0.77

Figure 3.10 Sample and TLC of 6e

- System C: CH2Cl2: EtOH (95:5, v/v); Rf= 0.88

Structure determination

The formation of target compounds 5a−e and 6a−e was characterized by spectrometric and spectroscopic data: UV- Vis, FT- IR, NMR, and HRMS

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

UV-Vis (max, MeCN/nm) (app AB.1 ): 267; 324

IR (KBr, cm -1 ) (app AC.1): 3063 (νC-H, aromatic), 2962−2835 (νC-H, aliphatic), 1639 (νC=O, νC=C, aromatic), 1612 (νC=N), 1492 (νC=C, aromatic), 1369 (δCH3), 1319 (νC−N), 1245 (δC=O), 1176- 1026 (νC-O), 714 (δCH2, aliphatic)

HRMS (ESI+, acetone) m/z calcd for C25H24N2O2+ [M+H] + := 329,1284; found 329,1291 (app AE.1)

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

1 H-NMR (500 MHz, DMSO-d 6 , δ-ppm, J=Hz)

1 H-NMR (600 MHz, DMSO-d 6 , δ-ppm, J=Hz)

3.39 (s, 3 H, O–CH3) 3,85 (s, 3 H, O–CH3) 3,85 (s, 3 H, O–CH3) 6.12-8.31 (m, 12 H, CHAr) 7,16- 8,16 (m, 12 H, CHAr) 7,14- 8,16 (m, 12 H, CHAr)

Based on previous studies about 1 H-NMR chemical shift, we can conclude that compound 3 was synthesized successfully

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

UV-Vis (max, MeCN/nm) (app AB.2 ): 262; 310

IR (KBr, cm -1 ) (app AC.2): 3051 (νC-H, aromatic), 2962−2929 (νC-H, aliphatic), 1647 (νC=O, νC=C,aromatic), 1606 (νC=N), 1475 (νC=C, aromatic), 1381 (δCH3), 1284 (νC−N), 1245 (δC=O), 1178- 1028 (νC-O), 715 (δCH2, aliphatic)

1H-NMR (500 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.7): 0.74 (3 H; t; J= 7,2 Hz; 3’’-H); 1,70 (2 H; sextet; J= 7,2 Hz; 2’’-H); 3,85 (3 H; s; OCH3); 4,31 (2 H; t; J= 7,2 Hz; 1’’-H); 7,14 (2 H; d, J= 8,5 Hz; 3’-H; 5’-H); 7.58 (2 H; t; J= 7.5 Hz; 13-H; 15-H); 7,67 (1 H; tt; J= 1,5 Hz; J= 7,5 Hz; 14-H); 7,74 (5 H; m; 2’-H; 6’-H; 12-H; 16-H; 6-H); 7,81 (1 H; d; J= 8,5 Hz; 7-H)

13C-NMR (125 MHz, DMSO-d 6 , δ-ppm) (app AD.8): 16,80 (C3’’); 22,45 (C2’’); 45,84 (C1’’); 55,29 (OCH3); 110,97 (C7); 114,26 (C3’; C5’); 121,56 (C4); 122,04 (C5); 124,02 (C6); 128,41 (C13; C15); 129,41 (C12; C16); 130,61 (C2’; C6’); 131,02 (C1’); 132,13 (C14); 137,97 (C11); 138,89 (C8); 141,73 (C9); 155,21 (C2); 160,49 (C4’); 197,75 (C10)

HRMS (ESI+, acetone) m/z calcd for C24H22N2O2 + [M+H] + := 371,1754, found 371,1757 (app AE.3)

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

UV-Vis (max, MeCN/nm) (app AB.3): 260; 312

1H-NMR (500 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.2): 0,72 (3 H; t; J= 7,5 Hz; 3’’-H); 1,68 (2 H; sextet; J= 7,5 Hz; 2’’-H); 3,85 (3 H; s; OCH3); 4,32 (2 H; t; J= 7,5 Hz; 1’’-H); 7,15 (2 H; d, J= 9 Hz; 3’-H; 5’-H); 7,58 (2 H; m; 13-H; 15-H); 7,63 (1 H; dd; J= 1,5 Hz; J= 8,5 Hz; 5-H); 7,68 (1 H; tt; J= 1 Hz; J= 7,5 Hz; 14-H); 7,76 (5 H; m; 2’-H; 6’-H; 12-H; 16-H; 4-H); 8,03 (1 H; d; J= 1 Hz; 4-H)

13C-NMR (125 MHz, DMSO-d 6 , δ-ppm) (app AD.3): 10,84 (C3’’); 22,56 (C2’’); 45,73 (C1’’); 55,31 (OCH3); 113,14 (C7); 114,29 (C3’; C5’); 118,55 (C4); 122,06 (C6); 124,14 (C5); 128,44 (C13; C15); 129,56 (C12; C16); 130,68 (C2’; C6’); 130,97 (C1’); 132,26 (C14); 135,34 (C8); 137;91 (C11); 145,79 (C9); 156,13 (C2); 160,58 (C4’); 195,68 (C10)

HRMS (ESI+, acetone) m/z calcd for C25H24N2O2+ [M+H] + := 371,1754, found 371,1768 (app AE.2)

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

IR (KBr, cm -1 ) (app ): 3066 (νC-H, aromatic), 2935- 2874 (νC-H, aliphatic), 1639 (νC=O, νC=C, aromatic), 1612 (νC=N), 1471 (νC=C, aromatic), 1373 (δCH3), 1301 (νC−N), 1250 (δC=O), 1176- 1026 (νC-O), 723 (δCH2, aliphatic)

1H-NMR (600 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.17): 0,78 (3 H; t; J= 7,2 Hz; 4’’-H); 1,18 (2 H; sextet; J= 7,2 Hz; 3’’-H); 1,68 (2 H; quintet; J= 7,2 Hz; 2’’-H); 3,86 (3 H; s; OCH3); 4,35 (2 H; t; J= 7,2 Hz; 1’’-H); 7,14 (2 H; d, J= 8,4 Hz; 3’-H; 5’-H); 7,58 (2 H; m; J= 7.5 Hz; 13-H; 15-H); 7,68 (1 H; t; J= 7,2 Hz; 14-H); 7,75 (5 H; m; 2’-H; 6’-H; 12-H; 16-H; 6-H); 7,81 (1 H; d; J= 8,5 Hz; 7-H); 7,99 (1 H; d; J= 1,2 Hz; 4-H)

13C-NMR (150 MHz, DMSO-d 6 , δ-ppm) (app AD.18): 13,27 (C4’’); 19,17 (C3’’); 31,14 (C2’’), 44,07 (C1’’); 55,31 (OCH3); 110,91 (C7); 114,24 (C3’; C5’); 121,63 (C4); 122,14 (C5); 123,96 (C6); 128,38 (C13; C15); 129,42 (C12; C16); 130,62 (C2’; C6’); 131,02 (C1’); 132,07 (C14); 138,04 (C11); 138,87 (C8); 144,87 (C9); 155,16 (C2); 160,50 (C4’); 195,63 (C10)

HRMS (ESI+, acetone) m/z calcd for C25H24N2O2 + [M+H] + := 385,1911; found 385,1916 (app AE.5)

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

IR (KBr, cm -1 ) (app AC.5): 3062 (νC-H, aromatic), 2950−2868 (νC-H, aliphatic), 1651 (νC=O, νC=C, aromatic), 1606 (νC=N), 1460 (νC=C, aromatic), 1340 (δCH3), 1257 (δC=O), 1176-

1H-NMR (600 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.12): 0,75 (3 H; t; J= 7,2 Hz; 4’’-H); 1,15 (2 H; sextet; J= 7,2 Hz; 3’’-H); 1,66 (2 H; quintet; J= 7,2 Hz; 2’’-H); 3,86 (3 H; s; OCH3); 4,35 (2 H; t; J= 7,2 Hz; 1’’-H); 7,14 (2 H; d, J= 9 Hz;

3’-H; 5’-H); 7,57 (2 H; m; 13-H; 15-H); 7,64 (1 H; dd; J= 1,2 Hz; J= 8,4 Hz; 5-H); 7,68 (1 H; t; J= 7,2 Hz; 14-H); 7,77 (5 H; m; 2’-H; 6’-H; 12-H; 16-H; 4-H); 8,03 (1 H; d; J= 1,2 Hz; 4-H)

13C-NMR (150 MHz, DMSO-d 6 , δ-ppm) (app AD.13): 13,26 (C4’’); 19,17 (C3’’); 31,22 (C2’’); 43,92 (C1’’); 55,32 (OCH3); 113,18 (C7); 114,27 (C3’; C5’); 118,60 (C4); 122,13 (C6); 124,05 (C5); 128,38 (C13; C15); 129,55 (C12; C16); 130,68 (C2’; C6’); 130,92 (C1’); 132,18 (C14); 135,30 (C8); 138,00 (C11); 145,93 (C9); 156,08 (C2); 160,59 (C4’); 195,55 (C10)

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

IR (KBr, cm -1 ) (app AC.5): 3064 (νC-H, aromatic), 2935- 2860 (νC-H, aliphatic), 1639 (νC=O, νC=C, aromatic), 1612 (νC=N), 1469 (νC=C, aromatic), 1373 (δCH3), 1302 (νC−N), 1247 (δC=O), 1173- 1027 (νC-O), 717 (δCH2, aliphatic)

1H-NMR (600 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.28): 0,75 (3 H; t; J= 7,2 Hz; 5’’-H); 1,14 (4 H; m; J= 7,2 Hz; 4’’-H; 3’’-H); 1,68 (2 H; quintet; J= 7,2 Hz; 2’’-H); 3,86 (3 H; s; OCH3); 4,34 (2 H; t; J= 7,2 Hz; 1’’-H); 7,14 (2 H; d, J= 8,4 Hz; 3’-H; 5’-H); 7,58 (2 H; m; 13-H; 15-H); 7,68 (1 H; t; J= 7,2 Hz; 14-H); 7,75 (5 H; m; 2’-H; 6’-H; 12-H; 16-H; 6-H); 7,80 (1 H; d; J= 8,4 Hz; 7-H); 7,99 (1 H; d; J= 1,2 Hz; 4-H)

13C-NMR (150 MHz, DMSO-d 6 , δ-ppm) (app AD.29): 13,66 (C5’’); 21,43 (C3’’); 28,02 (C4’’); 28,71 (C2’’); 44,26 (C1’’); 55,33 (OCH3); 110,90 (C7); 114,26 (C3’;C5’); 121,65 (C4); 122,16 (C5); 124,00 (C6); 128,40 (C13; C15); 129,43 (C12; C16); 130,61 (C2’; C6’); 131,03 (C1’); 132,09 (C14); 138,05 (C11); 138,87 (C8); 141,87 (C9); 155,18 (C2’); 160,53 (C4’); 195,66 (C10)

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

IR (KBr, cm -1 ) (app AC.6): 3064 (νC-H, aromatic), 2928−2860 (νC-H, aliphatic), 1639 (νC=O, νC=C, aromatic), 1612 (νC=N), 1469 (νC=C, aromatic), 1371 (δCH3), 1248 (δC=O), 1173-

1H-NMR (500 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.22): 0,74 (3 H; t; J= 6,5 Hz; 5’’-H); 1,10 (4 H; m; J= 7,2 Hz; 4’’-H; 3’’-H); 1,65 (2 H; quintet; J= 7,2 Hz; 2’’-H); 3,86 (); (3 H; s; OCH3); 4,35 (2 H; t; J= 7,5 Hz; 1’’-H); 7,15 (2 H; d, J= 9 Hz; 3’-H; 5’-H); 7,57 (2 H; m; 13-H; 15-H); 7,64 (1 H; dd; J= 1,5 Hz; J= 8,5 Hz; 5- H); 7,68 (1 H; tt; J= 1 Hz; J= 7 Hz; 14-H ); 7,76 (5 H; m; 2’-H; 6’-H; 12-H; 16-H; 4- H); 8,02 (1 H; d; J= 1 Hz; 7-H)

13C-NMR (150 MHz, DMSO-d 6 , δ-ppm) (app AD.23): 13,61 (C5’’); 21,40 (C3’’); 28,01 (C4’’); 28,74 (C2’’); 44,12 (C1’’); 55,35 (OCH3); 113,23 (C7); 114,29 (C3’;

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

UV-Vis (max, MeCN/nm) (app AB.8): 258; 306;

1H-NMR (600 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.39): 0,76 (3 H; t; J= 7,2 Hz; 6’’-H); 1,13 (6 H; m; 5’’-H; 4’’-H; 3’’-H); 1,66 (2 H; quintet; J= 6 Hz; 2’’- H); 3,85 (3 H; s; OCH3); 4,34 (2 H; t; J= 7,2 Hz; 1’’-H); 7,14 (2 H; d, J= 8,4 Hz; 3’- H; 5’-H); 7,57 (2 H; m; 13-H; 15-H); 7,67 (1 H; t; J= 7,2 Hz; 14-H); 7,74 (5 H; m; 2’-H; 6’-H; 12-H; 16-H; 6-H); 7,79 (1 H; d; J= 8,4 Hz; 7-H); 7,99 (1 H; s; 4-H)

13C-NMR (150 MHz, DMSO-d 6 , δ-ppm) (app AD.40): 13,68 (C6’’); 21,82 (C5’’); 25,47 (C3’’); 28,91 (C2’’); 30,42 (C4’’); 44,20 (C1’’); 55,31 (OCH3); 110,88 (C7); 114,22 (C3’; C5’); 121,64 (C4); 122,16 (C5); 123,97 (C6); 128,38 (C13; C15); 129,42 (C12; C16); 130,61 (C2’; C6’); 138,04 (C11); 138,84 (C8); 141,88 (C9); 155,18 (C2’); 160,51 (C4’); 195,63 (C10)

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

IR (KBr, cm -1 ) (app AC.6): 3063 (νC-H, aromatic), 2937- 2860 (νC-H, aliphatic), 1649 (νC=O, νC=C, aromatic), 1606 (νC=N), 1460 (νC=C, aromatic), 1325 (δCH3), 1258 (δC=O), 1173-

1H-NMR (600 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.33): 0,74 (3 H; t; J= 6,6 Hz; 6’’-H); 1,11 (6 H; m; 5’’-H; 4’’-H; 3’’-H); 1,65 (2 H; quintet; J= 6,6 Hz; 2’’- H); 3,86 (3 H; s; OCH3); 4,35 (2 H; t; J= 7,2 Hz; 1’’-H); 7,15 (2 H; d, J= 8,4 Hz; 3’- H; 5’-H); 7,57 (2 H; m; 13-H; 15-H); 7,64 (1 H; dd; J= 1,2 Hz; J= 8,4 Hz, 5-H);

13C-NMR (150 MHz, DMSO-d 6 , δ-ppm) (app AD.34): 13,68 (C6’’); 21,78 (C5’’); 25,45 (C3’’); 28,95 (C2’’); 30,39 (C4’’); 44,07 (C1’’); 55,34 (OCH3); 113,23 (C7); 114,27 (C3’; C5’); 118,64 (C4); ; 122,15 (C6); 124,01 (C5); 128,40 (C13; C15); 129,42 (C12; C16); 130,67 (C2’; C6’); 130,92 (C1’); 132,19 (C14); 135,25 (C8); 138,01 (C11); 145,93 (C5); 156,16 (C2); 160,61 (C4’); 195,56 (C10)

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

1H-NMR (600 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.49): 0,79 (3 H; t; J= 7,2 Hz; 7’’-H); 1,13 (6 H; m; 6’’-H; 5’’-H; 4’’-H; 3’’-H); 1,67 (2 H; m; 2’’-H); 3,86

(3 H; s; OCH3); 4,35 (2 H; t; J= 7,2 Hz; 1’’-H); 7,14 (2 H; d, J= 8,4 Hz; 3’-H; 5’-H); 7,58 (2 H; m; 13-H; 15-H); 7,68 (1 H; t; J= 7,2 Hz; 14-H); 7,75 (5 H; m; 2’-H; 6’-H; 12-H; 16-H; 6-H); 7,80 (1 H; d; J= 8,4 Hz; 7-H); 7,99 (1 H; s; 4-H)

13C-NMR (150 MHz, DMSO-d 6 , δ-ppm) (app AD.50): 13,79 (C7’’); 21,88 (C6’’); 25,72 (C3’’); 27,87 (C4’’); 28,89 (C2’’); 30,95 (C5’’); 44,16 (C1’’); 55,31 (OCH3); 110,90 (C7); 114,22 (C3’; C5’); 121,64 (C4); 122,17 (C5); 123,97 (C6); 128,39 (C13;

HRMS (ESI+, acetone) m/z calcd for C28H30N2O2 + [M+H] + := 427,2380, found 427,2383 (app AE.11)

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

IR (KBr, cm -1 ) (app AC.10): 3063 (νC-H, aromatic), 2929- 2856 (νC-H, aliphatic),

1650 (νC=O, νC=C, aromatic), 1608 (νC=N), 1461 (νC=C, aromatic), 1340 (δCH3), 1255 (δC=O), 1174- 1028 (νC-O), 712 (δCH2, aliphatic)

1H-NMR (600 MHz, DMSO-d 6 , δ-ppm, J=Hz) (app AD.54): 0,77 (3 H; t; J= 6,6 Hz; 7’’-H); 1,11 (8 H; m; 6’’-H; 5’’-H; 4’’-H; 3’’-H); 1,66 (2 H; m; 2’’-H); 3,86

(3 H; s; OCH3); 4,35 (2 H; t; J= 7,2 Hz; 1’’-H); 7,15 (2 H; d, J= 8,4 Hz; 3’-H; 5’-H); 7,57 (2 H; m; 13-H; 15-H); 7,64 (1 H; dd; J= 1,2 Hz; J= 8,4 Hz, 5-H); 7,68 (1 H; t;

13C-NMR (150 MHz, DMSO-d 6 , δ-ppm) (app AD.55): 13,83 (C7’’); 21,91 (C6’’); 25,74 (C3’’); 27,87 (C4’’); 28,96 (C2’’); 30,95 (C5’’); 44,05 (C1’’); 55,36 (OCH3); 136,26 (C7); 114,22 (C3’; C5’); 118,67 (C4); 122,16 (C6); 124,06 (C7); 128,44 (C13;

Discussion

To investigate the spectroscopic properties of the synthesized compounds, their UV–VIS absorption spectra were recorded in MeCN, as illustrated in Fig 3.00 The UV–VIS spectra revealed two absorption bands within the low UV range of 252–324 nm The bands detected above 250 nm are attributed to n−π* transitions, which are characteristic of charge transfer between the C=N groups of the compounds.

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

The IR spectra of the studied compounds exhibited expected vibration signals corresponding to various functional groups, including C–H, C=O, C=C, C=N, and C–N Notably, compounds (5c−e, 6c−e) displayed a long-chain band around 720 cm\(^{-1}\) due to the rocking motion associated with four or more CH2 groups in a straight chain Additionally, the stretching vibrations of C=O were observed at frequencies 25-45 cm\(^{-1}\) lower than the base value of 1715 cm\(^{-1}\), attributed to the delocalization of π electrons in the adjacent C=C bond, which reduces the force constants and absorption frequencies of the ketone and double bonds The absence of the characteristic N−H vibration at 3273 cm\(^{-1}\) further confirmed the formation of compounds 5a−e and 6a−e, as indicated in the spectrum of precursor 3.

The appropriate deuterated solvent for NMR spectroscopy, DMSO-d6, was chosen based on the peak separation and the solubility of all analyzed compounds

The assignment of 1 H and 13 C chemical shifts was achieved through several key stages: (i) the splitting pattern and coupling constants of the trisubstituted benzimidazole ring differentiated the 5- from 6-CO-Ph derivatives; (ii) NOESY experiments provided further distinction between these derivatives; (iii) HSQC experiments clarified the hydrogen-to-carbon connections; (iv) HMBC experiments were conducted; and (v) comparisons among different isomers facilitated the identification of the remaining carbons Notably, HMBC spectroscopy revealed correlations for compounds only through three covalent bonds.

The 1 H-NMR spectra of N-alkylated (2-(4-methoxyphenyl)-1H-benzimidazol-5(6)-yl)(phenyl)methanone displayed proton signals in two distinct regions In the upfield region (δ 0.74−4.35 ppm), signals attributed to aliphatic substituents were observed, with protons H-2” to H-7” appearing between 0.74−1.68 ppm, while proton H-1" was noted at approximately 4.35 ppm due to deshielding effects from the electronegative nitrogen atom Additionally, a singlet signal for the protons of 4’-OCH3 on the 2-phenyl ring was recorded at 3.85-3.86 ppm The downfield region (δ 7.14−8.03 ppm) was characterized by signals from aromatic ring protons Notably, the NH signal peak at 13.12 ppm disappeared in the 1 H-NMR spectra of N-alkylated benzimidazole derivatives, indicating successful synthesis The 13 C-NMR spectra revealed signals at 10−46 ppm for methyl and methylene carbons in the N-1 position, while singly-oxygenated saturated carbon signals were found at 110−130 ppm Electron-rich CH carbons (C-4, C-5, C-6, C-7, etc.) shifted upfield (110-120 ppm), whereas electron-poor CH carbons (C-12, C-13, C-15, C-16, etc.) shifted downfield (120−130 ppm) Quaternary sp² carbons, including C-2, C-8, C-9, and C-4', were shifted downfield to around 160 ppm due to the inductive effect, with carbonyl carbons (C-10) appearing at approximately 195 ppm.

The structures of the positional isomers 5 and 6 were validated through 1H, HMBC, and NOESY spectral analysis Notably, the proton signals within the benzimidazole ring were identified as an ABX spin system, with compound 6b exhibiting peaks at positions 4, 5, and 7 characterized by overlapping doublets, a doublet with a coupling constant of J=1.2 Hz, and a doublet of doublets with coupling constants of J=1.2 Hz and J=8.4 Hz In contrast, compound 5b displayed peaks at position 4.

6, and 7 with patterns of doublet (J=1.2 Hz), doublet (overlap) and doublet of doublets (J=8.4 Hz), respectively (as shown in Figure 3.12)

NOESY experiments revealed a correlation between the methylene protons (H-1") and the doublets of proton H-7 in compound 6b, confirming it as 6-COPh substituted Similarly, compound 5b exhibited a correlation between the methylene protons (H-1") and the doublet of proton H-7, validating it as 5-COPh substituted Additionally, HMBC experiments further confirmed the correlations between H-1" and C-2, C-.

8 ( Figure 3.13b and 3.14b) The NOESY and HMBC experiments clarified the 5-COPh and 6-COPh substitutions for compounds 5b and 6b

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

The HRMS analysis of the target compounds (3, 5a−e, 6a−e) confirmed the presence of a pseudo molecular ion peak [M+H]⁺, aligning with the proposed molecular formula weights and indicating the successful formulation of these compounds Detailed HRMS spectra for all compounds can be found in Appendix E.

Antiproliferative result

The synthesized compounds (3, 5a−e, 6b−e) were evaluated for their antiproliferative activity against the MDA-MB-231 human breast cancer cell line using the SRB method, with Camptothecin serving as a reference drug The findings are detailed in Table.

3.3 The compounds were synthesized based on the differences in the alkyl group on the N-1 position to clarify their influences over antiproliferative activity on the MDA- MB-231 cell line

Table 3.3 IC 50 of synthesized compounds (3, 5a-e, 6b-e)

No Compound Molecular structure IC 50 (M)

From the results of Table 3, compounds 3, 6b, and 6d showed an inhibitory effect on cancer cell proliferation with IC50 values ranging from 48.58 M to 70.93

M In contrast, other compounds 5a−e, 6c e showed poor antiproliferative activity against MBA-MB-231 (IC50>100 M) cell line compared to their precursor 3 with

The study found that the IC50 value for a series of N,2,5-trisubstituted-1H-benzimidazole derivatives was 69.44 µM, indicating inactivity against tested cancer cell lines This suggests that adding alkyl groups to these derivatives may hinder their biological activity In contrast, N,2,6-trisubstituted-1H-benzimidazole derivatives, such as compound 6b (N-butyl) and 6d (N-hexyl), demonstrated improved antiproliferative activity with IC50 values of 70.93 µM and 48.58 µM, respectively, surpassing the activity of precursor 3 The presence of alkyl substitutions and longer carbon chains in these derivatives appears to enhance their biological effects, aligning with previous findings by Pham and colleagues The disparity in activity between the two series may stem from steric hindrance affecting binding site accessibility and the more lipophilic nature of the N,2,5-trisubstituted compounds, which could impede their ability to penetrate cell membranes and exhibit biological activity.

CONCLUSION

Concluding remark

In summary, a 1H-benzimidazole derivative and ten positional isomers of N-alkylated 1H-benzimidazole derivatives were successfully synthesized, achieving yields of 88.8% and 41.6-53.8%, respectively The physicochemical properties and structures of the synthesized compounds were characterized using techniques such as melting point determination, TLC, HPLC, UV-Vis, FT-IR, 1D and 2D-NMR, and HRMS spectra Furthermore, all compounds were assessed for their antiproliferative activities against the MDA-MB-231 breast cancer cell line, with compounds 3, 6b, and 6d demonstrating significant antiproliferative effects, exhibiting IC50 values between 48.58 and 70.93.

Suggestion on future work

Future research can broaden the substrate scope to include various substituent types, such as long straight chains, branched chains, and heterocyclic or aromatic substituents Enhanced biological screening is essential to gather more data on antioxidant, antibacterial, antifungal, and antiproliferative effects across different cancer cell lines Moreover, employing modern synthetic pathways, including microwave-assisted or ultrasonic techniques, along with catalytic-free reactions and environmentally friendly solvents or reusable catalysts, is recommended for improved outcomes.

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Bài báo của P V Bình và các cộng sự trình bày về việc tổng hợp và đánh giá độc tính đối với tế bào của một số dẫn xuất naphthalenyl-benzimidazole Nghiên cứu được công bố trên Tạp chí Khoa học Trường Đại học Cần Thơ, số 53, trang 108.

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APPENDIX A: HPLC- DAD CHROMATOGRAM OF THE

Figure AA.2 HPLC- DAD chromatogram of 5a and 6a at signal 260nm

Figure AA.1 HPLC- DAD chromatogram of 5a and 6a at signal 310nm

Figure AA.3 HPLC- DAD chromatogram of 5b and 6b at signal 260nm

Figure AA.4 HPLC- DAD chromatogram of 5b and 6b at signal 310nm

Figure AA.5 HPLC- DAD chromatogram of 5c and 6c at signal 260nm

Figure AA.6 HPLC- DAD chromatogram of 5c and 6c at signal 310nm

Figure AA.7 HPLC- DAD chromatogram of 5d and 6d at signal 260nm

Figure AA.8 HPLC- DAD chromatogram of 5d and 6d at signal 310nm

Figure AA.9 HPLC- DAD chromatogram of 5e and 6e at signal 260nm

Figure AA.10 HPLC- DAD chromatogram of 5e and 6e at signal 310nm

APPENDIX B: UV- VIS ABSORPTION SPECTRA OF

Figure AB.1 UV-Vis absorption spectrum of compound 3

Figure AB.2 UV-Vis absorption spectrum of 5a

Figure AB.3 UV-Vis absorption spectrum of 6a

Figure AB.4 UV-Vis absorption spectrum of compound 5b

Figure AB.5 UV-Vis absorption spectrum of compound 6b

Figure AB.6 UV-Vis absorption spectrum of compound 5c

Figure AB.7 UV-Vis absorption spectrum of compound 6c

Figure AB.8 UV-Vis absorption spectrum of compound 5d

Figure AB.9 UV-Vis absorption spectrum of compound 6d

Figure AB.10 UV-Vis absorption spectrum of compound 5e

Figure AB.11 UV-Vis absorption spectrum of compound 6e

APPENDIX C: FT- IR SPECTRUM OF COMPOUNDS

Figure AC.1 FT-IR spectra of compound 3

Figure AC.2 FT-IR spectra of compound 5a

Figure AC.3 FT-IR spectra of compound 5b

Figure AC.4 FT-IR spectra of compound 6b

Figure AC.5 FT-IR spectra of compound 5c

Figure AC.6 FT-IR spectra of compound 6c

Figure AC.7 FT-IR spectra of compound 5d

Figure AC.8 FT-IR spectra of compound 6d

Figure AC.10 FT-IR spectra of compound 6e Figure AC.9 FT-IR spectra of compound 5e

Figure AD.1 1 H-NMR spectra of compound 3

Figure AD.2 1 H-NMR spectra of compound 6a

Figure AD.3 13 C-NMR spectra of compound 6a

Figure AD.4 HSQC spectra of compound 6a

Figure AD.5 HMBC spectra of compound 6a

Figure AD.6 NOESY spectra of compound 6a

Figure AD.7 1 H-NMR spectra of compound 5a

Figure AD.8 13 C-NMR spectra of compound 5a

Figure AD.9 HSQC spectra of compound 5a

Figure AD.10 HMBC spectra of compound 5a

Figure AD.11 NOESY spectra of compound 5a

Figure AD.12 1 H-NMR spectra of compound 6b

Figure AD.13 13 C-NMR spectra of compound 6b

Figure AD.14 HSQC spectra of compound 6b

Figure AD.15 HMBC spectra of compound 6b

Figure AD.16 NOESY spectra of compound 6b

Figure AD.17 1 H-NMR spectra of compound 5b

Figure AD.18 13 C-NMR spectra of compound 5b

Figure AD.19HSQC spectra of compound 5b

Figure AD.20HMBC spectra of compound 5b

Figure AD.21 NOESY spectra of compound 5b

Figure AD.22 1 H-NMR spectra of compound 6c

Figure AD.23 13 C-NMR spectra of compound 6c

Figure AD.25 HSQC spectra of compound 6c

Figure AD.26HMBC spectra of compound 6c

Figure AD.27 NOESY spectra of compound 6c

Figure AD.28 1 H-NMR spectra of compound 5c

Figure AD.29 13 C-NMR spectra of compound 5c

Figure AD.30 HSQC spectra of compound 5c

Figure AD.31 HMBC spectra of compound 5c

Figure AD.32 NOESY spectra of compound 5c

Figure AD.33 1 H-NMR spectra of compound 6d

Figure AD.34 13 C-NMR spectra of compound 6d

Figure AD.35 HSQC spectra of compound 6d

Figure AD.37 HMBC spectra of compound 6d

Figure AD.38 NOESY spectra of compound 6d

Figure AD.39 1 H-NMR spectra of compound 5d

Figure AD.40 13 C-NMR spectra of compound 5d

Figure AD.41 HSQC spectra of compound 5d

Figure AD.42 HMBC spectra of compound 5d

Figure AD.43 NOESY spectra of compound 5d

Figure AD.49 1 H-NMR spectra of compound 5e

Figure AD.50 13 C-NMR spectra of compound 5e

Figure AD.51 HSQC spectra of compound 5e

Figure AD.52 HMBC spectra of compound 5e

Figure AD.53 NOESY spectra of compound 5e

Figure AD.54 1 H-NMR spectra of compound 6e

Figure AD.55 13 C-NMR spectra of compound 6e

Figure AD.56 HSQC spectra of compound 6e

Figure AD.57 HMBC spectra of compound 6e

Figure AD.58 NOSEY spectra of compound 6e

APPENDIX E HRMS SPECTRUM OF COMPOUNDS

Figure AE.1 HRMS spectra of compound 3

Figure AE.2 HRMS spectra of compound 6a

Figure AE.3 HRMS spectra of compound 5a

Figure AE.4 HRMS spectra of compound 6b

Figure AE.5 HRMS spectra of compound 5b

Figure AE.6 HRMS spectra of compound 6c

Figure AE.7 HRMS spectra of compound 5c

Figure AE.8 HRMS spectra of compound 6d

Figure AE.9 HRMS spectra of compound 5d

Figure AE.10 HRMS spectra of compound 6e

Figure AE.11 HRMS spectra of compound 5e

Tiêu đề	Synthesis and Antiproliferative Evaluation of n-Alkylated (2-(4-Methoxyphenyl)-1H-Benzodimidazol-5(6)Yl)(Phenyl)Methanone Derivatives
Tác giả	Le Quoc Tuan
Người hướng dẫn	Assoc. Prof., Dr. Hoang Thi Kim Dung
Trường học	Ton Duc Thang University
Chuyên ngành	Chemical Engineering
Thể loại	Undergraduate thesis
Năm xuất bản	2023
Thành phố	Ho Chi Minh City

Định dạng
Số trang	134
Dung lượng	12,68 MB