1 Department of Medicinal Chemistry, Isfahan University of Medical Sciences, Iran
2Isfahan Pharmaceutical Sciences Research Center, Isfahan, Iran
3Physiology Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
Submission: August 16, 2019; Published: September 11, 2019
*Corresponding author: Farshid Hassanzadeh, Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, Iran
How to cite this article: Shirin Banitalebi Dehkordi, Farshid Hassanzadeh, Marzieh Rahmani Khajouei, Hojat Sadeghi, Nasim Dana. Synthesis and
Cytotoxicity Evaluation of Some New 4-(4oxo-4H-Quinazoline-3yl)- Thiobenzoic Acid-S-(1-H-Benzimidazole-2-Yl) Ester Derivates. Organic & Medicinal
Chem IJ. 2019; 8(5): 555750. DOI: 10.19080/OMCIJ.2019.08.555750
Quinazolines are a group of heterocyclic compounds that have different biological activities such as cytotoxicity, anti-bacterial, anti-fungal. benzimidazoles on the other hand are also a group of heterocyclic compounds with anti-tumor, anti-virus, anti-fungal and anti-inflammatory effects. Due to significant cytotoxic effects of both quinazoline and benzimidazole derivatives , in this work a group of quinazolinone-benzimidazole hybrids were prepared. The structures of synthesized compounds were confirmed by IR and 1H-NMR. Cytotoxic activity of the compounds was evaluated at 1, 10, and 100 μM concentrations against MCF-7 and HT-29 cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The results show that compound 9d has highest cytotoxic activity against both MCF-7 and HT-29 cell lines.
Cancer is the leading cause of death in the developed world . Despite discovery of numerous drugs for treating cancer and lots of progresses, due to the toxicity and drug resistance associated with the common treatments of cancer continued efforts to discover new anticancer drugs is still a very important issue among medicinal chemists and the efforts to develop new leading compounds is of great importance .
In order to discover new target drugs, a new approach; combining two or more pharmacophores can be effective ,as there’s one single molecule with different mechanism of actions that can also eliminate resistance problems and may reduce the side-effects . Quinazolines are a group of heterocyclic compounds and have a broad spectrum of biological effects including sedative , anti-compulsive [4-6], anti-inflammatory [4,7], antitumor [4,8], antibacterial [4,9-11], antifungal[5,6], anti-Tuberclosis [6,8,11], anti-malaria , anti-virus [5,7], anti-HIV [4,9-11], and lowering of blood lipid [12-14]. iproqualone,Pazosin,Doxazosin,Febrifungine,Evodiamine,Lutonin A are some examples of synthetic and natural Quinazolines with good therapeutic effect .
Thymitaq and lapatinib are two examples of Quinazoline structured anticancer drugs. Lapatinib has tyrosin kinase(dual kinase) inhibition activity, it also inhibits EGFR(ErbB1) and HER2(ErbB2) by reversibly binding to tyrosine kinase [16-18]. Anticancer effect of quinazolinone derivatives are mainly attributed to their multi target activities including inhibition of topoisomerase I, EGFR tyrosine kinase and dihydrofolate reductase inhibition [19-21].
Benzimidazoles are an important class of heterocyclic compounds with a wide variety of effects such as anticancer,anti-inflammatory,antibiotics,antiviral,anti-fungal,anti-HIV,antihistamine, antioxidant and lowering of blood pressure
. Some well-known anti-fungal drugs such as clotrimazol,
miconazole and ketoconazole have imidazole structures .
According to the biological activities and cytotoxic effects of both
Quinazolines and Benzimidazoles, in this study we try to combine
these two pharmacophores in one single molecule in order to
reach new compounds with better cytotoxic effects and hopefully
less side-effects .
All initial materials, solvents and regents were prepared from
commercial suppliers such as Merck (Germany) and Aldrich (USA).
By using different solvents, the purity of purchased compounds
was proved via thin layer chromatography (TLC). 1HNMR spectra
of synthetized compounds were determined by (Bruker 400
MHz, Germany) spectrometer, and chemical shifts were shown
as δ (ppm) with tetramethyl silane (TMS) as internal standard.
Melting points were recorded by utilizing electro thermal melting
point analyzer apparatus (IA 9000, UK) and are uncorrected. TheIR spectra were obtained on a Shimadzu 470 spectrophotometer
(potassium bromide disks). All cell lines were bought from Pasteur
Institute of Iran
4-(4-Oxo-4H-quinazolin-3-yl)-thiobenzoic acid S-(1Hbenzoimidazol-
2-yl)ester derivates(9a-e) were prepared through
two separate reactions one to produce the Benz imidazol 4 and
the other Benz oxazinone derivates 8a-e,Respectively. In the
first part,4-Nitro-thiobenzoic acid S-(1H-benzoimidazol-2-yl)
ester( 3) (Scheme 1) was prepared through addition of 4-Nitro
benzoyl chloride 2 to Benz imidazol 2-thiol 1 to give the Nitro
derivate 3 which was reduced to S-1H-benzoimidazol-2-yl
4-aminobenzothioate 4 using palladium-Charcoal powder [25-
27]. In the second part a group of Benzoxazinone derivatives with
different substituents at position 2 were synthesized from the
reaction of 5 with different acyl chlorides . Finally, the primary
amine 4 was reacted with the benz oxazinones 8a-8e to produce
the target compounds 9a-9e as presented in Scheme1.
MCF7 (human breast adenocarcinoma cell line) cells were
maintained at 37°C in a humidified atmosphere (90%) containing
5% CO2.MCF-7 cell line was cultured in Roswell Park Memorial
Institute medium (RPMI) with 5% v/v fetal bovine serum, 100
U/ml penicillin, and 100 mg/mL streptomycin. The medium was
changed every two to three days and sub- cultured when the cell
population density reached to 70–80% confluence. Cells were
seeded at an appropriate density according to each experimental
design (), and HT-29 (human colon adenocarcinoma cell line)
cells were maintained at 37°C in a humidified atmosphere (90%)
containing 5% CO2. HT-29 cell line was cultured in Dulbecco’s
modified Eagle’s medium (DMEM-F12) with 10% v/v fetal bovine
serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The
medium was changed every two to three days and sub-cultured
when the cell population density reached to 70-80% confluence,
Cells were seeded at an appropriate density according to each
HT29 and MCF-7 cells were seeded on 96-well tissue culture
plates (15 × 103 cells/well) and incubated overnight. Cells were
treated with different concentrations of the derivatives for 48h.
Then the medium was removed and the MTT substrate was
prepared in a physiologically balanced solution (PBS), added to
cells in culture, concentration of 0.5 mg/ml, and incubated for 1 to
4 h. The formazan crystals were solubilized in dimethyl sulfoxide
(DMSO) and the quantity of formazan (presumably directly
proportional to the number of viable cells) was measured by
recording changes in absorbance at 570 nm using a plate reading
spectrophotometer. Cell viability was calculated using following
formula: IC50 values were calculated by plotting the cell viability
against compound concentrations All statistical analyzes were
performed with the SPSS Statistics 24.
The mixture of 4-nitro benzoylChloride (9.6g,50mmol) in
Dimethylformamide(DMF)(5ml) was added to a mixture of
2-mercaptobenzimidazol(6g,40mmol) in DMF(5ml)and the rest
of DMF was added dropwise, the mixture was stirred for 24 hours
at room temperature. Water(200 ml) was added to the solution.
The product was filtered and the separated cake was washed with
water and dried to give the final product 3 as a yellow powder,
Yield: 85% ,mp:207- 210˚C lit. mp 206-208˚C ref (14)1H NMR (400
MHZ: d6-DMSO: 8.298-8.371(2H, m, H-C3’,H- C4’),8.012(2H ,dd,
J=6.8 Hz, j=2 Hz,H-C2’,H-C5’), 7.7402(1H,d,J=7.8 Hz,H-C6),7.310-
7.391(2H,m,H-C5H-C6),7.257(1H, dd, J=8.8 Hz, j=1.6 Hz,H-C7)(
A suspension of S-1H-benzoimidazol-2-yl-4-nitrobenzothioate
(1g, 3mmol) and palladium-charcoal powder(120mg,1.1mmol) in
Anhydrous methanol(20ml,99.8% v/v) was shake for 30 minutes,
the air was vacuumed and H2 gas was slowly added to the mixture.
The solution was filtered after cooling to remove the catalyst. The
solvent was then evaporated under reduced pressure to give the
final product S-1H-benzoimidazol-2-yl 4-aminobenzothioate 4 as
an orange-yellow powder, yield: 60%, mp: 290-295, lit. mp 292-
293 ref [14,15].
To a solution of anthranilic acid (1.37g, 10 mmol) in
dimethylformamide (DMF) (5 ml) different acyl chlorides 6a-6e
(15 mmole) were added drop wise and the resulting solutions
were stirred for 3 h. The end of the reactions was determined
by TLC. The mixtures were then poured into distilled water and
stirred for additional 1 h. Finally, the precipitated products were
collected by filtration and washed with water to furnish 7a-7e
(16). Each compound of the previous step (7a-7e) (2.5 mmol) was
added to acetic anhydride (2 mL) and refluxed at 140 °C until the
starting materials 7a-7e were disappeared from TLC. At the end of
the reaction the excess of acetic anhydride was removed from the
reaction medium under reduced pressure. The resulting products
were cooled to give solid mass. Finally, the products were washed
with hexane to give Benz oxazinones (8a-8e).
A mixture of compound 4 (0.6, 2mmol) and compounds 8a-8e
(1mmol) were refluxed for a period of 4-6 hours in glacial aceticacid (10 ml). The reaction progression was investigated using
TLC and at the end of the reaction, acetic acid was removed using
rotary evaporator to give target compounds 9a-9e.
In this study, we synthesized some new quinazoline
derivatives with substitute containing benzimidazole at position
3. Amine bearing benzimidazole moiety  was synthesized
via 2 steps. In the first step,4-Nitrobenzoyl chloride was added
to 2-Mercaptobenzimidazol, the water was used in order to
neutralize the acid. In the second step, reduction of the nitro group
to amine was carried out using palladium on activated charcoal in
Palladium on activated charcoal also referred as Pd/c is used
as catalyst. The metal is supported on activated carbon in order
to maximize its surface area and activity and is known to be used
for Reductive amination as in our case  Benz oxazinones are
highly reactive and should be used immediately after preparation.
In this study, benz oxazinones were prepared in two steps. In the
first part, anthranilic acid was treated with the acyl chlorides
to prepare N-acyl anthranilic acids. These compounds could
be prepared easily at room temperature via a nucleophilic
This could be because of the high electrophilicity of the
carbonyl group next to a powerful electron withdrawing group
(Cl) in acyl chloride. In addition, DMF could also facilitate this
reaction by removal of hydrogen from the amino group .
Subsequent reflux of N-acyl anthranilic acids in acetic anhydride
resulted in production of the corresponding benz oxazinones via
a dehydrative cyclization mechanism. The excess amount of acetic
anhydride was removed immediately to avoid its side reactions
with primary amines utilizing in the next step to prevent the
production of corresponding amide by product.
Finally, compounds 8a-8e were added to 4 to produce final
hybrids via a nucleophilic substitution mechanism. Cytotoxiceffects of the synthesized hybrids were investigated on HT-
29 and MCF-7 by MTT assay. According to the results shown in
Table 1, compound 9e bearing aromatic substituents on C2 of
the quinazolinone ring showed lowest cytotoxic activity on both
cell lines, i.e. IC50s 100 μM , while compounds 9a-9d containing
aliphatic substituents on C2 were more active on both cell lines
with IC50 values between 25-80 μM. It seems that the presence
of electron donating substituents on C2 could be in favor of the
activity for these compounds while electron withdrawing groups
have opposite effects, perhaps because of un favored electronic
effects on the site of action.
In summary, the novel derivatives of quinazolinone with
substituted benzimidazole at position 3 were synthesized in
several steps and their in vitro cytotoxic activities were evaluated
against MCF-7 and HT-29 cell lines. The cytotoxic evaluation
of synthesized derivatives on both MCF-7 and HT-29 cell lines
represented that compound with phenyl substitute 9e had the
lowest cytotoxic activity against both cell lines and compound 9d
with isopropyl substitute had the highest potency