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CHEMICAL PROBLEMS 2024 no. 3 (22) ISSN 2221-8688
369
UDC 547.56.563.364
SYNTHESIS OF ACETYLENIC AMINO DERIVATIVES OF 2-(2-((2,6-DICHLOROPHENYL)AMINO)PHENYL)ACETIC ACID
Nagham M. Zaki Dawood1*, Zainab Faiyq Saeed2, Banan Borhan Saeed1
department of Pharmaceutical Chemistry, College of Pharmacy, University of Mosul, Mosul, Iraq 2Department of Physiology, Biochemistry and Pharmacology, College of Veterinary Medicine, University of
Mosul, Mosul, Iraq *e-mail: n3_m3_zmz@uomosul. edu. iq
Received 02.02.2024 Accepted 08.04.2024
Abstract: After reviewing a number of researches that demonstrate the effectiveness of a number of compounds containing the acetylenic amine group as antimicrobials and antiproliferative activity, our goal is to prepare new acetylenic amine derivatives from the compound 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetic acid using the Mannich reaction by the react salt of a carboxylic acid with propargyl bromide to produce the compound (prop-2-yn-l-yl 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate) (1), which was refluxed with a number of secondary amines and formaldehyde in presence of the copper chloride as a catalyst to obtain 7 compounds (4-(dialkylamino)but-2-yn-1-yl 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate)(2-8). The physical and spectral properties of the prepared compounds were measured, and the bioassay conducted for some substances by investigating their antibiotic activity. The two compounds (1,2) were also selected to test their anticancer activity against one of its types, a breast cancer cell line (MCF-7), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using multiple concentrations compared to the normal cell line (WRL-68). The previous results showed the effect of the two compounds on the cancer cell line more than its effect on the normal cell line, the results showed significant differences (p<0.0001) by calculating the (IC50) when treating compounds (1,2) for MCF-7 cancer cells at (250,300) ug/ml and for normal cells at (983,1292) ug/ml respectively. It is believed that the reason for its effectiveness is because contains acetylenic amine in compound (1) in addition to morpholine ring in compound (2), which research has proven to be effective in multiple medicines. Some pharmacokinetic activities of the prepared compounds have been predicted to support the discovery of new drugs using the Swiss ADME website. Keywords: Propargyl bromide, Mannich base, MTT method, Cytotoxicity Assay, ADME. DOI: 10.32737/2221-8688-2024-3-369-381
1. Introduction
Many derivatives containing an acetylene moiety over the years were studied and many of them had drug effectiveness in the clinical test phase. Compounds derived from acetylene have shown promising results as a treatment for various diseases. These derivatives have been found to possess anti-malarial [1], antituberculosis, anti-cancer, and anti-microbial properties [2]. Acetylene compounds are also included in the composition of some plants, such as the mushrooms Hydnum repandum and Polyporus biformis, which have been used to
enhance and strengthen immunity as antimicrobials and anticancers [3].
Not only are acetylene compounds essential in the preparation of various substances, but they can also be found in a plethora of marine sponges, corals, and plant species, as well as in fungal and bacterial cultures [4, 5]. These aforementioned compounds play a fundamental role in medicinal chemistry due to the presence of electronic effects mimicking the influences of aromatic rings, of which present in sedative-
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CHEMICAL PROBLEMS 2024 no. 3 (22)
hypnotic drugs and synthetic retinoids; tazarotene, various contraceptives, and natural pesticides [4, 6]. These compounds have been found to have pharmacological activity, due to their advantages summarized in their activity and low toxicity [7], in addition to being rich in
electrons, which facilitates their association with receptor proteins within living tissues [8]. Some research has demonstrated their efficacy as anticancer agents [9, 10], hypertensive agents [11], and antiproliferative activity [7, 12, 13].
2. Materials and methods
Chemicals and reagents: All the reagents and solvents were purchased; [2-(2-((2,6-dichlorophenyl)amino)phenyl)acetic acid], Cu2Cl2, Dioxane, formaldehyde, secondary amines, glacial acetic acid and Propargyl bromide from Flulka Co. All solvents used were checked for purity before use by measuring their boiling points. Instrumentation: The products were characterized by FT-IR spectra recorded on Burker's Alpha-platinum ATR (Germany) FT-IR spectrophotometer, melting points were uncorrected and measured with a thermo-digital device IA 9300.
In the lab at the University of Ega-Oxford, England, a few tools were utilized to conduct our experiment. First, TMS was used as an internal reference to ensure measurements were accurate. Secondly, d6-DMSO was utilized as a solvent to dissolve the sample. To capture the data needed, the proton NMR spectra need to be recorded on a Bruker (AS 400 MHz). (1H-NMR) spectra provide insight into the various atoms in a molecule and how they are arranged. To better understand the data, signals assigned using the following abbreviations: s for singlet, d for doublet, t for triplet, and m for multiplet. These signals helped us analyze the molecular structure of tested sample. 2.1. Chemicals
2.1.1. General procedure for preparation of sodium 2-(2-((2,6-
dichlorophenyl)amino)phenyl)acetate (I). The
carboxylic acid was converted to its salt by mixing (0.01 mol, 3 g) of the starting material [2-(2-((2,6-dichlorophenyl)amino)phenyl)acetic acid] with an aqueous solution of sodium bicarbonate (0.01 mol, 0.84 g) dissolved in 15 ml of distilled water and stirred for half an hour until a clear solution was obtained. Filter and evaporate the water from the filtrate completely under vacuum pressure to obtain the acid salt in the form of a pure white powder.
1. Synthesis of prop-2-yn-1-yl 2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (1) [14].
In a 100 mL round flask, a mixture of (0.02 mol, 6.4 g) of acid salt (I), (10 mL) of propargyl bromide and (10 ml) of DMSO was refluxed for 3 h in a water bath at 80°C, and the solution was poured onto a beaker containing crushed ice (40 ml) with stirring to obtain an oily product which was treated by adding more crushed ice with continuous rubbing and stirring until it turned into a precipitate, then filtered , dried and recrystallized with benzene to obtain a precipitate with yellow color 76 % yield, and a melting point (87-89 °C). IR (umax cm-1): 3318 (N-H), 3279 (=C-H), 2977-2898 (C-H), 2160 (C=C), 1716 (C=O ester), 1206,1138 (C-O-C asy, sym ) ; (1H-NMR) (DMSO-d6 ppm): 5= 9.23 (s, 1H, NH), 7.53-6.75 m, 7H, Ar-H), 4.65 (m, 2H, OCH2C=C), 3 .61 (t, 2H, CH2CO2), 3 .42 (s,1H, C=C-H).
2. A general method for the synthesis of acetylenic amine compounds (2-8) [14, 15]:
Mannich bases were prepared by mixing (0.002 mol, 0.67 g) of acetylene compound (1) with a secondary amine (0.003 mol) in a round bottom flask fitted with a reflux condenser and after cooling the mixture to (0 °C), adding (0.1 g) of Cu2Cl2 and paraformaldehyde (0.003 mol) with (15 mL) dioxane free peroxide at 0°C, then slowly added dropwise glacial acetic acid (0.5 mL) with stirring for five minutes, refluxing with continues stirring for 2 hours in dry condition. To obtain a pure product, the solution is acidified by adding dilute hydrochloric acid (1:1) to pH = 1 with continuous stirring, then the mixture is extracted three times by adding (10 ml) of diethyl ether and after collecting the aqueous layer, it is neutralized by adding sodium bicarbonate to form a precipitate that is filtered, dried and then recrystallized with a
mixture of benzene and petroleum ether (80-100 °C) as shown in Scheme 1 and Table 1. 4-morpholinobut-2-yn-1-yl-2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (2):
Pale yellow, yield 68 %, m.p (139-141°C), IR (cmax cm-1 ): 3312 (N-H), 2972-2909 (C-H), 2196 (C=C), 1722 (C=O ester), 1211,1129 (C-O-C asy, sym ) ; (1H-NMR) (DMSO-d6 ppm): 5= 9.36 (s, 1H, NH), 7.64-6.86 (m, 7H, Ar-H), 4.77 (m, 2H, OC^C^C), 3.67 (t, 2H, CH2CO2), 3.58 (t, 4H, CH2O morpholine ring), 3.36 (t, 2H, C=C-CH2N), 2.52 (t, 4H, CH2N morpholine ring).
4-(9H-carbazol-9-yl)but-2-yn-1-yl-2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (3):
Pale orange, yield 59 %, m.p (209-211°C), IR (cmax cm-1 ): 3317 (N-H), 2976-2911 (C-H), 2183 (C=C), 1726 (C=O ester), 1208,1133 (C-O-C asy, sym ) ; (1H-NMR) (DMSO-d6 ppm): 5= 9.41 (s, 1H, NH), 8.22-6.63 (m, 15H, Ar-H), 4.69 (m, 2H, OCH2C=C), 4.51 (t, 2H, C=C-CH2N), 3.65 (t, 2H, CH2CO2). 4-(diphenylamino)but-2-yn-1-yl-2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (4): Yellow, yield 53 %, m.p (101-104°C), IR (cmax cm-1 ): 3310 (N-H), 2979-2915 (C-H), 2187 (C=C), 1728 (C=O ester), 1210,1143 (C-O-C asy, sym ). (1H-NMR) (DMSO-d6 ppm): 5= 9.39 (s, 1H, NH), 7.62-6.57 (m, 15H, Ar-H), 4.66 (m, 2H, OCH2C=C), 4.54 (t, 2H, C=C-CH2N), 3.72 (t, 2H, CH2CO2). 4-(2-ethylpiperidin-1-yl)but-2-yn-1-yl-2-(2-((2,6-dichlorophenyl)amino)phenyl) acetate (5): Pale yellow, yield 62 %, m.p (153-155°C), IR (cmax cm-1 ): 3289 (N-H), 2976-2857 (C-h), 2182 (C=C), 1730 (C=O ester), 1211,1132 (C-
O-C asy, sym ) ; ^H-NMR) (DMSO-d6 ppm): 5= 9.34 (s, 1H, NH), 7.52-6.58 (m, 7H, Ar-H),
4.63 (m, 2H, OCH2C=C), 3.67 (t, 2H, CH2CO2), 3.22 (t, 2H, C=C-CH2N), 2.46-1.28 (m, 10H, CH2), 0.91 (m, 3H, CH3 ) for secondary amine ring).
4-(dibutylamino)but-2-yn-1-yl-2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (6):
Pale brown, yield 64 %, m.p (123-125°C), IR (cmax cm-1 ): 3293 (N-H), 2975-2893 (C-H), 2189 (C=C), 1729 (C=O ester), 1205,1144 (C-O-C asy, sym ); (1H-NMR) (DMSO-d6 ppm): 5= 9.42 (s, 1H, NH), 7.46-6.43 (m, 7H, Ar-H), 4.57 (m, 2H, OCH2C=C), 3.63 (t, 2H, CH2CO2), 3.34 (t, 2H, C=C-CH2N), 2.52-1.36 (m, 12H, CH2) & 0.99 (t, 6H, CH3) for secondary amine. 4-(dipropylamino)but-2-yn-1-yl-2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (7): Pale yellow, yield 58 %, m.p (108-110 °C), IR (cmax cm-1 ): 3291 (N-H), 2973-2858 (C-H), 2185 (C=C), 1724 (C=O ester), 1201,1146 (C-O-C asy, sym ); (1H-NMR) (DMSO-d6 ppm): 5= 9.37 (s, 1H, NH), 7.58-6.47 (m, 7H, Ar-H),
4.64 (m, 2H, OCH2C=C), 3.59 (t, 2H, CH2CO2), 3.28 (t, 2H, C=C-CH2N), 2.42-1.48 (m, 8H, CH2) & 1.07 (t, 6H, CH3) for secondary amine. 4-(diethylamino)but-2-yn-1-yl-2-(2-((2,6-dichlorophenyl)amino)phenyl)acetate (8): Pale yellow, yield 67 %, m.p (98-100 °C), IR (cmax cm-1 ): 3302 (N-H), 2978-2867 (C-H), 2191 (C=C), 1729 (C=O ester), 1209,1145 (C-O-C asy, sym ) ; (1H-NMR) (DMSO-d6 ppm): 5= 9.41 (s, 1H, NH), 7.43-6.52 (m, 7H, Ar-H), 4.68 (m, 2H, OCH2C=C), 3.64 (t, 2H, CH2CO2), 3.36 (t, 2H, C=C-CH2N), 2.51 (t, 4H, CH2) & 1.1 (t, 6H, CH3) for secondary amine).
aq NaHC03
ArCH2C02H
AT =
0 © propargyl bromide
ArCH2C02Na -^ ArCH2CC)2CH2OCH
reflux (J)
(I)
reflux
secondary amine
parafprmaldehyde
Cu2Cl2
o o
ArCH2CC)2CH2OCCH2-NR2 (2-8)
Scheme 1. Route of the synthesis of acetylenic amine compounds (2-8)
Table 1. The derivatives of acetylenic amine compounds (2-8)
Compounds No. -NR2 Compounds No. -NR2
2 —N\ /° 6 —N(C4H9)2
3 C¿0 7 -N(C3H7)2
4 ___—ph ph 8 —N(C2H5)2
5
2.2. Antimicrobial activity:
To evaluate the antimicrobial activity of the compounds numbered (1, 2, 3, and 5); disk diffusion method (16) was performed (Table 2). Two G+ and two G- bacteria were submitted to the current study (Streptococcus pneumonia, Staphylococcus aureus, Klebsiella spp and E coli; respectively), added to one pathogenic fungus, Candida albicans. The microbial isolates were obtained and authenticated in the
Microbiology Laboratory, College of Science -University of Mosul. Each isolate was sub-cultured in a selective media, then the dilution method was applied to assume Minimum inhibitory concentration (MIC). After being inoculated, the Petridishes were placed in a warm incubator where they could grow and thrive. They were given plenty of fresh air and left to incubate for a full day at a toasty 37°C.
Microorganisms used Species
Bacteria species. Gram Streptococcus pneumonia
Staphylococcus aureus
Gram Klebsiella spp.
E. coli
Yeast fungus Candida albicans
Table 2. Microorganisms used to test the antibiotic action of each substance (1, 2, 3, and 5).
Prepare ingredient concentrations: A stock solution was prepared from each of the synthesized compounds (1, 2, 3, and 5) in a concentration of (100 pg/mL), that used then to prepare ingredient concentrations (75, 50, 25 pg/mL) of each compound [16, 17]. Disc diffusion test:A suspension of 0.5 MacFarland was prepared from bacteria mixed with Muller-Hinton broth, and cultured on the Muller-Hinton agar plates exploiting a sterile cotton swab [16, 17].
Estimating antibacterial activity: The tested microbes were cultured on Muller-Hinton to perform a disc diffusion sensitivity test. In each Petridis, five wells were perforated in agar plates at 4 mm depth, and then 0.1 ml of each dilution of the studied compounds was put inside the well. Cultured (temperature 37 °C,
duration 24 hours), then the inhibition zone (mm) was estimated to estimate the antibiotic actions of each substance at various dilutions [17].
2.3. Cytotoxicity assay [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay] [18, 19]:
At the Center for Natural Product Research and Drug Discovery, the team selected two compounds (1 and 2) that showed promise for fighting breast cancer. But they didn't stop there. To truly test their potential, the compounds underwent rigorous cytotoxicity testing against both cancerous and normal cell lines (MCF-7 and WRL-68, respectively). The team used the MTT colorimetric method to evaluate the efficacy of these compounds.
Department of Pharmacology at the Faculty of Medicine, University of Malaya in Kuala Lumpur kindly provided the cell lines, which were carefully preserved in liquid nitrogen. The cancer cell lines were propagated, purified, and put to the test at the Biotechnology Research Center at Al-Nahrain University. The results of this testing could have far-reaching implications for the future of breast cancer treatment. 2.4. Swiss ADME calculations:
The synthesized substances were tested for their ability to predict appropriate pharmacokinetic properties. Our combined analogs' ADME was taken into account by efficient and precise techniques that predicted the physical and pharmacological features of the produced drugs. Several parameters and their recommended values have been determined, and are listed in Table 3.
Table 3. Some Swiss ADME parameters of compounds (1-8)
Mole cule No. Formula MW TPSA ESOL Class GI absorption BBB permeant Lipinski violations Bio availability Score Synthetic Accessi bility
1 C17H13C12NO2 334.2 38.33 MS High Yes 1 0.55 2.74
2 C22H22Cl2N2O3 433.33 50.8 MS High Yes 0 0.55 3.62
3 C30H22Cl2N2O2 513.41 43.26 PS Low No 2 0.17 3.75
4 C30H24C12N2O2 515.43 41.57 PS Low No 2 0.17 3.96
5 C25H28C12N2O2 459.41 41.57 PS High Yes 1 0.55 4.25
6 C26H32Cl2N2O2 475.45 41.57 PS Low No 1 0.55 4.02
7 C24H28 Cl2N2O2 447.4 41.57 PS High No 1 0.55 3.85
8 C22H24C12N2O2 419.34 41.57 MS High Yes 1 0.55 3.66
PS= Poorly soluble, MS= Moderately soluble
3. Results and Discussion
3.1. The chemistry: The validity of the methods used in this research has been proven by measuring some of the physical and spectral properties of the prepared compounds. It was found that there is a difference in the melting points between compound (1) and compounds (2-8) by more than 10 degrees. It is noted that some of the products were in small percentages, and this may be due to the reaction requiring a longer time to complete and not because of the side products, because the Mannich method used was taken from reliable sources and is a well-known method, especially using the catalyst (Cu2Cl2) in the reaction. The products were also processed by purifying them as described above in the synthesis of compounds (2-8). The infrared spectroscopy of the compounds (1-8), where they gave stretching bands of the N-H bond with limits (3318-3289) in the form of medium-intensity bands and at the range (2196-2160) they gave weakly-stretched of the (C=C) bond, while the ester bond stretching (C=O) appeared in the range
(1730-1716) of strong intensity accompanied by two asymmetrical and symmetrical bands of strong (C-O-C) bonding at (1211-1201) and (1146-1129) respectively, noting the disappearance of the stretching absorption (C=O) of the carboxylic acid of the starting material and also the disappearance of the stretching band which belongs to hydroxyl group. What confirmed the validity of the method is the appearance of the (=C-H) at (3279 cm-1) in compound (1) and its disappearance in the compounds (2-8) with the appearance of additional bands belonging to the secondary amine represented by the C-H stretch bands of the groups of CH2, CH3 in the compounds (2, 58). As for the (1H-NMR), several bands appeared, including: the N-H amine proton in all compounds at the region (9.42-9.23) in the form of a single band with the disappearance of the O-H band of carboxylic acid. Multiple bands also appeared at (8.22-6.43) belonging to the protons of the aromatic rings and multiple bands at (4.77-4.57) belonging to the CH2 protons
adjacent to the high electronegative oxygen atom (OCH2C=C), protons of CH2CO2 appeared at (3.72-3.59) in the form of triple beams with the appearance of triples also at (4.54-3.22) belonging to the CH2 protons adjacent to the nitrogen atom C=CCH2N, which were entered by the Mannich reaction. The triple-terminal bond proton (=C-H) appeared only in compound (1) in the form of a single band at (3.42) and disappeared in compounds (2-8). 3.2. Biological Stady: To make the work more feasible, the biological activity of some of the synthesized compounds (1, 2, 3, 5) as antimicrobial compounds was tested. Two species of Gram+ bacteria (Streptococcus
The activity of compound (2) may be attributed to the presence of a heterocyclic morpholine ring, which has been proven to have multiple bioactivities. M. Marinescu and et.al. Was referred to a number of Mannich bases those prepared from benzimidazole and had antibacterial activity against some G- bacteria, like: (S. aureus, B. flexus, C sporogenes and S. mutans) [20]. Christina Zalaro and et.al were prepared a new series of hybrid Mannich bases of pyrazolo benzimidazole, they recorded antibacterial effects of the synthesized compounds against G+ bacteria (Staphylococcus aureus and enterococcus faecalis) and G-bacteria (Pseudomonas aeruginosa and Escherichia coli). They concluded that the synthesized compounds illustrated a higher inhibitory effect against all bacterial isolates than the antibiotic of choices (Metronidazole, Nitrofurantoin) [21]. As for the researcher (S.
pneumonia and Staphylococcus aureus) and two species of Gram- bacteria (Klebsiella spp and E.coli) , added to one species of fungus (Candida albicans) were chosen as targeted opportunistic microorganisms. According to disk diffusion test, it was found that the compounds 1, 3, and 5 had no considerable inhibitory effect against the four microbes submitted to the current study. On the other hands, compound (2) award inhibitory effect against Streptococcus pneumoniae and Staphylococcus aureos at concentrations (100, 75, 50, 25^g/ml) with inhibitory zones of (9.68, 8.53, 7.78, 7.32mm) and (14.92, 13.94, 8.77, 7.47 mm); respectively, as listed in Table 4.
Manap), he prepared number of derivatives that contain the morpholine ring; it was found that these compounds manifested a significant bioactivity against some pathogenic bacteria species [22].
3.3. Cytotoxicity Assay [23, 24]: The
cytotoxicity assay (MTT assay) was performed to determine the toxic effect of the two compounds (1,2) at different concentrations on (MCF-7) breast cancer cells compared to the healthy cell line (WRL-68). Compound (2) has been carefully selected to contain the powerful morpholine ring. This ring has proven to be a highly effective pharmaceutical compound, thanks to its unique physio-chemical properties that boost both hydrophilic and lipophilic reactions. These properties work together to enhance the solubility of blood and improve the permeability of the brain's overall structure. What's truly remarkable about the morpholine
Table 4. The antimicrobial effect of compounds (1,2,3, and 5) according disk diffusion test.
Micro organis ms 1 2 3 5
100 Mg/ml 75 Mg/ml 50 Mg/ml 25 Mg/ml 100 Mg/ml 75 Mg/ml 50 Mg/ml 25 Mg/ml 100 Mg/ml 75 Mg/ml 50 Mg/ml 25 Mg/ml 100 Mg/ml 75 Mg/ml 50 Mg/ml 25 Mg/ml
Strepto coccus pneumo nia R R R R 9.68 8.53 7.78 7.32 R R R R R R R R
Staphylo coccus aureus R R R R 14.92 13.94 8.77 7.47 R R R R R R R R
Klebsiell a spp R R R R R R R R R R R R R R R R
E-coli R R R R R R R R R R R R R R R R
Candida albicans R R R R R R R R R R R R R R R R
R = Resistance < 7 value
ring is its similarity to neurotransmitters found in the active compounds of the central nervous system. For example, aryl-morpholines have been shown to play a key role in crossing the blood-brain barrier and improving brain function [23]. It's no wonder that this ring has been the subject of many studies and reviews. So, while the biological activity of morpholine derivatives has been covered in other studies, the potential benefits of this powerful compound cannot be overstated. With compound (2), researchers have unlocked a new level of potential in the world of pharmaceuticals [23-27].
The cytotoxicity of the two selected compounds (1, 2) was tested by measuring their invitro anti-proliferative activity against one of
the cancer cells lines (MCF-7). The assay is based on the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) yellow by the action of mitochondrial succinate dehydrogenase to insoluble formazan violet. The test results of compound (1) showed efficacy against MCF-7 breast cancer cells with inhibition rates (51.47, 36.58, 31.98, 9.95, 3.78, 3.36, 3.63) % treated with concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.75) pg/ml, respectively, while it gave less cytotoxicity on healthy cells (WR-68), where the percentage of inhibition (21.87, 11.73, 6.41, 5.05, 4.94, 4.13, 4.06) % ranged at concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.75) pg/ml as shown in Table 5.
Table 5. Effect of compound (1) on breast cancer cell line MCF-7 and normal cell line WRL-68 _using the MTT assay for a period of 24 hours at a temperature of 37°C._
Cell line Viability% (SD± mean)
The concentrations used (pg/ml)
1000 500 250 125 62.5 31.25 15.75
MCF-7 48.53±2.03 63.42±2.75 68.02±3.87 90.05±2.42 96.22±0.67 96.64±1.36 96.37±0.81
WRL68 78.13±1.44 88.27±1.45 93.59±2.10 94.95±0.92 95.06±0.63 95.87±1.12 95.94±0.20
In vitro cytotoxic activity results were determined by calculating the (IC50) value, which represents the concentration of a compound required to halt the growth of 50% of tumor cells in comparison to untreated cells. We used Cisplatin as a positive control for our test. This allowed us to compare our results with
known cytotoxic agents and determine the effectiveness of our compound. The results showed significant differences (p<0.0001) by calculating the (IC50) when treating compound (1) for MCF-7 cancer cells at (250) pg/ml and for normal cells at (983) pg/ml as shown in Fig.1.
Fig. 1. Effect of compound (1) on MCF-7 and WRL-68 cell line using MTT assay
When investigating the effect of the new the results showed effectiveness, the inhibition compound (2) on the MCF-7 cancer cell line, rates reached (45.10, 36.30, 22.38, 9.41, 5.21,
4.59, 3.86) % tagged with concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.75) |ig/ml, respectively. But when testing the toxicity of compound (2) on the normal cell line WRL-68,
it showed inhibition rates that ranged between (30.44, 13.97, 7.87, 3.82, 3.05, 5.09, 3.90) % at concentrations (1000, 500, 250, 125, 62.5, 31.25, 15.75) |ig/ml, as shown in Table 6.
Table 6. Effect of compound (2) on the breast cancer cell line MCF-7 and the normal cell line _WRL-68 using MTT assay for a period of 24 hours at a temperature of 37°C._
Cell line Viability% (SD± mean)
The concentrations used (^g/ml)
1000 500 250 125 62.5 31.25 15.75
MCF-7 54.90±1.00 63.70±2.12 77.62±2.41 90.59±1.80 94.79±1.33 95.41±1.41 96.14±1.05
WRL68 69.56±3.20 86.03±0.85 92.13±1.56 96.18±1.25 96.95±1.14 94.91±2.20 96.10±0.48
The results showed significant differences (p<0.0001) when calculating the (IC50) upon compound (2) treatment for (MCF-7) cancer cells (300) p,g/ml and for (WRL-68) normal
cells (1292) pg/ml. The previous results showed the effect of the two compounds on the cancer cell line more than its effect on the normal cell line, as shown in Fig. 2.
Concentration
Fig. 2. Effect of compound (2) on MCF-7 and WRL-68 cell line using MTT assay
Upon conducting a thorough examination of the published research, it was discovered that Boryczka and their group had successfully synthesized a plethora of novel acetylene derivatives from betulin. These newly formulated compounds were then subjected to rigorous testing to determine their cytotoxic potential against a variety of human cancer cell lines including SW707 (colorectal adenocarcinoma), CCRF/CEM (leukemia), T47D (breast cancer), and P388 murine leukemia. Furthermore, the anti-proliferative activity of these compounds was evaluated using the MTT method in vitro, alongside normal Balb3T3 fibroblast cell lines. The results showed that there is a noticeable effect of the prepared compounds against the above-
mentioned cancer cell lines in varying proportions [28]. Research has also proven that natural and synthetic naphthoquinones possess many anti-cancer efficacies for a number of derivatives prepared from Lawsone (2-hydroxy-1, 4-naphthoquinone) obtained from dried henna leaves (Lawsonia inermis). New Lawone-derived aminonaphthoquinones were synthesized by Mannich reaction and after testing their anticancer activity using the MTT method, they gave toxic effect against human hepatocellular carcinoma cell line HepG2 [29]. There are other researchers who tested the effectiveness of some morpholine derivatives and gave their results which showed that both N-acetyl and N-benzoyl derivatives give high effectiveness against some cancer lines [30].
3.4. Swiss ADME prediction: According to the recorded values in Table (3), it is clear that the compounds (1,2,3,6, and 9) penetrate the blood brain barrier and are active in the CNS, while compound (1,3) does not violate any of
Lipinski's five principles. In terms of projected human gastrointestinal absorption, the bulk of the compounds had high GIT absorption, low solubility, and good bioavailability.
4. Conclusions
This study focuses on the preparation of acetylenic amine derivatives derived from 2-(2-((2,6-dichlorophenyl)ami no)phenyl)aceti c acid through the Mannich reaction using (Cu2Cl2) as catalyst, a series of compounds (1-8) were successfully prepared and characterized. The antimicrobial and antiproliferative activities of some compounds were evaluated, revealing promising results.
Testing the biological activity of some of the prepared compounds (1, 2, 3, 5) against five types of microbes at concentrations (25, 50, 75, 100 pg/ml), where the highest inhibitory activity was for compound 2 against Gram+ bacteria (Streptococcus pneumonia, Staphylococcus aureus) at a concentration of (100 pg/ml). A cytotoxicity test (MTT assay) was performed to determine the toxic effect of the two compounds (1,2) at different concentrations on breast cancer cells (MCF-7) compared to a healthy cell line (WRL-68). The compounds showed significant
effectiveness in inhibiting the growth of breast cancer cells compared to normal cells, indicating their potential as anti-cancer agents. Compound (2) was carefully selected because it contains a morpholine ring. This ring has proven to be a highly effective pharmaceutical compound, thanks to its unique physicochemical properties that promote hydrophilic and lipophilic interactions. These properties work together to enhance the solubility of the blood and improve the permeability of the overall structure of the brain.
The efficacy achieved might be linked to acetylinic amine group and morphine ring in compound 1 and 2, respectively. With the help of ADME website, these produced compounds might be a template for new drug discovery opening the horizons for new pharmaceutical product, research, and design.
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2-(2-((2,6-DiXLORFENiL)AMiN)FENiL)SiRKO TURÇUSUNUN ASETiLEN-AMiN
TÔROMOLORÎNÎN SiNTEZi
Nagham M. Zaki Dawood1*, Zainab Faiyq Saeed2, Banan Borhan Saeed1
1dczaçiliq Kolleci, dczaçiliq Kimyasi Departamenti, Mosul Universiteti, Mosul, iraq 2Fiziologiya, Biokimya vs Farmakologiya Departamenti, Baytarliq Kolleci, Mosul Universiteti, Mosul, iraq
*e-poçt: n3_m3_zmz@,uomosul. edu. iq
Xülasa: Tarkibinda asetilen-amin qrupu olan bazi birla§malarin antimikrob va antiproliferativ tasir effektivliyini tasdiq edan bir sira tadqiqatlari tahlil edarak, bu i§da maqsadimiz 2-(2-((2,6-diklorofenil)amin)fenil)sirka tur§usu asasinda yeni asetilen-amin töramalarinin alinmasi olmu§dur. Karbon tur§usu duzunun propargil bromidla Mannix reaksiyasi naticasinda (prop-2-yn-1-yl 2-(2-((2,6-diklorofenil)amino)fenil)asetat) birla§masi (1) alinmi§dir. Alinmi§ birla§ma mis xlorid katalizatorunun i§tiraki ila bir sira ikinci daracali aminlar va formaldehidla qaynadilmi§ va naticada 7 (4-(dialkilamino)but-2-yn-1-il-2-(2-((2,6-diklorofenil)amino)fenil)asetat) birla§malari (2-8) alda edilmi§dir. Alinan birla§malarin fiziki va spektral xassalari öl9ülmü§, bazi maddalarin antibiotik aktivliyinin öyranilmasi ü9ün bioanalizi aparilmi§dir. 3-(4,5-dimetiltiazol-2-il)-2 birla§masindan istifada edarak onun növlarindan birina, dö§ xar9angi hüceyra xattina (MCF-7) qar§i antionkoloji faaliyyatini yoxlamaq ü9ün iki birla§ma (1 va 2) da se9ilmi§dir. 5-difeniltetrazolium bromidin (MTT) analizi normal hüceyra xatti (WRL-68) ila müqayisada bir ne9a qatiliqdan istifada etmakla aparilmi§dir. övvalki naticalar bu iki birla§manin xar9ang hüceyra xattina tasirinin normal hüceyra xattina nisbatan daha 9ox oldugunu göstarmi§dir. Naticalar göstarir ki, (250-300) pg/ml-da MCF-7 xar9ang hüceyralari va 983 va 1292 pg/ml-da normal hüceyralar ü9ün birla§malar (1 va 2) ila müalica edildikda (IC50) hesablandiqda ahamiyyatli farqlar (p<0.0001) alinir: müvafiq olaraq 983 va 1292 pg/ml. Hesab olunur ki, effektivliyin sababi, (1) va (2) birla§malarinin tarkibinda morfolin halqasinin va asetilen-aminin olmasidir ki, bunlarin da bir 9ox darmanlarda tasirli oldugu göstarilmi§dir. Alinmi§ birla§malarin bazi farmakokinetik xassalari isve9ra ADME veb saytindan istifada edilmakla darmanlarin kafina töhfa veracayi proqnozla§dirilmi§dir. A?ar söztari: propargil bromid, Mannix asaslari, MTT metodu, sitotoksiklik analiz, ADME.
СИНТЕЗ АЦЕТИЛЕНОВЫХ АМИНОПРОИЗВОДНЫХ 2-(2-((2,6-ДИХЛОРФЕНИЛ)
АМИНО)ФЕНИЛ)УКСУСНОЙ КИСЛОТЫ
Нагам М. Заки Давуд1 , Зайнаб Фаик Саид2, Банан Борхан Саид1
1 Кафедра фармацевтической химии, Фармацевтический колледж, Мосульский университет, Мосул,
Ирак
2Кафедра физиологии, биохимии и фармакологии, Колледж ветеринарной медицины, Университет
Мосула, Мосул, Ирак *e-mail: n3_m3_zmz@,uomosul. edu. iq
Резюме: Проанализировав ряд исследований, демонстрирующих эффективность некоторых соединений, содержащих ацетиленовую аминогруппу, в качестве антимикробного и антипролиферативного действия, нашей целью являлось получение новых производных ацетиленового амина на основе соединения 2-(2-((2,6-дихлорфенил))амино)фенил)уксусной кислоты реакцией Манниха взаимодействием соли карбоновой кислоты с пропаргилбромидом и получением соединения (проп-2-ин-1-ил 2-(2-((2,6-дихлорфенил)амино))фенил)ацетат) (1). Полученное соединение кипятили с рядом вторичных аминов и формальдегидом в присутствии хлорида меди в качестве катализатора с получением 7 соединений (4-(диалкиламино)бут-2-ин-1-ил-2-(2-((2,6-
дихлорфенил)амино)фенил)ацетат) (2-8). Измерены физические и спектральные свойства полученных соединений, а также проведен биоанализ некоторых веществ путем исследования их антибиотической активности. Два соединения (1 и 2) были выбраны также для проверки их противораковой активности против одного из его типов, линии клеток рака молочной железы (MCF-7), с использованием 3-(4,5-диметилтиазол-2-ил)-2. Анализ 5-дифенилтетразолия бромида (МТТ) проводили с использованием нескольких концентраций
по сравнению с нормальной клеточной линией (WRL-68). Предыдущие результаты показали, что влияние этих двух соединений на линию раковых клеток больше, чем на нормальную клеточную линию. Результаты показали значительные различия (р<0,0001) при расчете (1С50) при обработке соединениями (1,2) для раковых клеток MCF-7 при (250 300) мкг/мл и нормальных клеток при 983 и 1292 мкг/мл, соответственно. Считается, что причина его эффективности заключается в том, что в соединениях (1) и (2) содержится ацетиленовый амин в дополнение к морфолиновому кольцу, эффективность которого, как показали исследования, применяется во многих лекарствах. Было предсказано, что некоторая фармакокинетическая активность полученных соединений будет способствовать открытию новых лекарств с использованием швейцарского веб-сайта АОМЕ.
Ключевые слова: пропаргил бромид, основание Манниха, метод МТТ, анализ цитотоксичности, АОМЕ.
