key: cord-1009544-hwa7g29l
authors: Mohamed, S. F.; Abdel-Hafez, N. A.; Amr, A. E.; Awad, H. M.
title: Synthesis and antitumor activity against HepG-2, PC-3, and HCT-116 cells of some naphthyridine and pyranopyridinecarbonitrile derivatives
date: 2017-08-02
journal: Russ J Gen Chem
DOI: 10.1134/s1070363217060226
sha: 72a2c4c3bd55f9b37b2ba103b3cbf05e7fc1420f
doc_id: 1009544
cord_uid: hwa7g29l

A series of substituted and fused heterocyclic derivatives 2–17 were synthesized using 3,5-bis(4-methoxybenzylidene)-1-propylpiperidin-4-one (1) as starting material. Treatment of 1 with malononitrile or semicarbazide afforded compounds 2 and 3, respectively. Condensation of 1 with ethyl cyanoacetate afforded naphthyridine-3-carbonitrile derivative 4, which reacted with phosphorus pentachloride and phosphoryl chloride to give chloro derivative 5. Treatment of 5 with thiosemicarbazide afforded compound 6. The reaction of 1 with malononitrile gave cyano aminopyrane derivative 7 which was condensed with pyromellitic dianhydride, phthalic anhydride, succinic anhydride, or morpholine in glacial acetic acid to obtain imide derivatives 8–11. Additionally, the reaction of 7 with aromatic aldehydes gave derivatives 12a–12c. Acetylation of 7 with acetic anhydride in boiling acetic acid gave N-acetyl derivative 13 which was cyclized to pyridine derivative 14 by refluxing in dioxane in the presence of triethylamine. Treatment of 7 with hydrazine hydrate gave pyrazolo derivative 15. Finally, the reaction of 7 with triethyl orthoformate in the presence of acetic anhydride gave formimidate 16 which was treated with hydrazine hydrate to form N-amino derivative 17. Some of the synthesized compounds were examined in vitro for their antitumor activity against HepG-2, PC-3, and HCT-116 human carcinoma cell lines using MTT assay.

1 The text was submitted by the authors in English.

Pyridinones and their derivatives play an essential role in several biological processes and are of considerable chemical and pharmacological importance [1 -3] . Moreover, pyridinones represent a unique class of pharmacophores which are encountered in various therapeutic agents [4] , antibiotics [5] , and antibacterial [6] , antifungal [7] , and cardiotonic agents [8] . On the other hand, pyran and pyridine derivatives are widely used as anticoagulant, anticancer [9, 10] , antiviral [11] , antitumor [12] , and anti-inflammatory agents [13] . Additionally, pyran derivatives are well known for their antihistaminic [14] and antimicrobial activity [15] and inhibition of influenza virus sialidases [16] . Apart from biological importance, some 2-amino-4H-pyrans are widely used as photoactive materials [17] . We previously synthesized some new pyridine and pyran derivatives which were used as analgesic, anticonvulsant, anti-anxiety, and antiparkinsonian agents [18] [19] [20] . We have recently [21] [22] [23] [24] [25] [26] prepared some new heterocyclic compounds and evaluated them as antiinflammatory [21, 22] , anti-HSV-1 [23] , and antimelanoma [24] activities; they can also be used as activators of tumor suppressor protein p53 [25] and SARS-CoV 3C-like protease inhibitors [26] . In continuation of our previous work in heterocyclic chemistry, we synthesized some new heterocyclic compounds and tested them for anticancer activity.

A series of substituted and fused nitrogen heterocycles were synthesized using 3,5-bis(4-methoxybenzylidene)-1-propylpiperidin-4-one (1) as starting material, which was prepared from 1-propylpiperidin-4-one according to known procedure [27] . Treatment of 1 with malononitrile in the presence of alcoholic Thirteen compounds were examined in vitro for their antitumor activity against HepG-2, PC-3, and HCT-116 human carcinoma cell lines using MTT assay. The percentage of the intact cells was measured and compared to Doxorubicin ® used as control (see figure) . The results indicated dose-dependent anticancer activity of all compounds against the three cancer cells. Compound 3, 8, 11, and 15 at a concentration of 100 µg/mL showed good anticancer activities against HCT-116 carcinoma cells. Six compounds showed moderate activities (4-6, 12b, 14, 17) against HCT-116 cells, and the others showed weak activity. Good anticancer activity against PC-3 cancer cells was observed for three compounds (3, 12b, 15), compounds 6, 8, and 17 were moderately active, and the others showed weak or no antitumor activity. All the tested compounds were weakly active or inactive against HepG-2 liver cancer. The IC 50 values are given in table.

Our results suggests that enhanced anticancer activity of some tested compounds against HCT-116 (3, 8, 11, 15) and PC-3 (3, 12b, 15) human carcinoma cell lines may be determined by the following factors: heteroaromaticity, large number of nitrogen atoms, naphthyridine moiety, meta substitution, and/or fused five-membered pyrazole or pyrrole ring. 

The melting points were determined in open glass capillaries with an Electro Thermal IA9100 digital melting point apparatus and are uncorrected. The elemental microanalyses for carbon, hydrogen, and nitrogen (Microanalytical Unit, NRC) were within the acceptable limits. The IR spectra (KBr) were recorded on a Nicolet Nexus 670 FTIR spectrometer. The 1 H and 13 C NMR spectra were run in DMSO-d 6 (unless otherwise stated) on a Jeol instrument (500 MHz). The mass spectra (electron impact, 70 eV) were obtained on a Finnigan MAT SSQ 7000 spectrometer. Analytical thin-layer chromatography (TLC) was performed on silica gel 60 F 254 aluminum sheets (E. Merck). (3E,5E)-3,5-bis(4-methoxybenzylidene)-1-propylpiperidin-4-one (1) was synthesized from 1-propylpiperidin-4-one according to the reported procedure [27] .

(8E)-2-Ethoxy-8-(4-methoxybenzylidene)-4-(4methoxyphenyl)-6-propyl-5,6,7,8-tetrahydro-1,6naphthyridine-3-carbonitrile (2). A mixture of 3.77 g (0.01 mol) of compound 1 and 0.66 g (0.01 mol) of malononitrile in a solution of sodium ethoxide prepared from 1 g of sodium metal and 30 mL of ethanol was refluxed for 4 h. The mixture was cooled, poured into ice water, and acidified with aqueous HCl to pH ~3.5. The precipitate was filtered off, washed with water, and recrystallized from methanol. Yield 86%, mp 236°C. IR spectrum: ν 2225 cm -1 (C≡N). 1 

Concentrated aqueous HCl (2 mL) was added to a mixture of compound 1 (3.77 g, 0.01 mol) and semicarbazide (0.75 g, 0.01 mol) in ethanol (10 mL). The mixture was refluxed for 6 h and cooled, and the precipitate was filtered off, dried, and recrystallized from methanol. Yield 78%, mp 172°C. IR spectrum, ν, cm -1 : 3326 (NH), 3250 (NH 2 ). 1 

A mixture of compound 1 (3.77 g, 0.01 mol), ethyl cyanoacetate (0.12 g, 0.01 mol), and anhydrous ammonium acetate (7.04 g, 0.08 mol) in ethanol (30 mL) was refluxed for 2 h. After cooling, the precipitate was filtered off, dried, and recrystallized from dioxane. Yield 87%, yellow crystals, mp 265°C. IR spectrum, ν, cm -1 : 3447 (NH), 2219 (CN), 1698 (C=O). 1 (20 mL) , and the mixture was refluxed for 2 h. After cooling, the mixture was poured into ice water with stirring, and the precipitate was filtered off, washed with water, dried, and recrystallized from methanol. Yield 71%, yellow-green powder, mp 186°C. IR spectrum: ν 2219 cm -1 (CN). 1 

A mixture of compound 5 (4.60 g, 0.01 mol) and thiosemicarbazide (0.91 g, 0.01 mol) in ethanol (30 mL) containing a few drops of triethylamine was refluxed for 5 h. The mixture was cooled and poured into ice water, and the precipitate was filtered off, washed with water, and recrystallized from methanol. Yield 82%, mp 278°C. IR spectrum, ν, cm -1 : 3447 (NH), 3348 (NH), 3265 (NH 2 ), 2219 (CN). 1 

A mixture of compound 1 (3.77 g, 0.01 mol), malononitrile (0.66 g, 0.01 mol), and piperidine (10 mL) in ethanol ( 

A mixture of compound 7 (4.43 g, 0.01 mol) and pyromellitic dianhydride, phthalic anhydride, succinic anhydride, or morpholine (0.01 mol) in glacial acetic acid (30 mL) was refluxed for 6-10 h. The mixture was cooled and poured into ice water, and the solid was filtered off, washed with water, dried, and crystallized from methanol. (13) . A solution of compound 7 (4.43 g, 0.01 mol) in a 2 : 1 mixture of acetic acid and acetic anhydride (45 mL) was refluxed for 6 h. After cooling, the solid was filtered off and recrystallized from acetic acid. Yield 76%, mp 218°C. IR spectrum, ν, cm -1 : 3257 (NH), 2242 (CN), 1702 (C=O). 1 (9E)-4-Amino-9-(4-methoxybenzylidene)-5-(4methoxyphenyl)-7-propyl-1,5,6,7,8,9-hexahydro-2Hpyrano[2,3-b:5,6-c′]dipyridin-2-one (14) . A solution of compound 13 (4.86 g, 0.01 mol) in dioxane (50 mL) containing 5 mL of triethylamine was refluxed for 8 h. After cooling, the solid was filtered off and recrystallized from dioxane. Yield 64%, mp 247°C. IR spectrum, ν, cm -1 : 3348 (NH), 3297 (NH 2 ), 1682 (C=O). 1 H NMR spectrum, δ, ppm: 12.39 s (1H, NH, D 2 O exchangeable), 6.97-7.68 m (9H, H arom , 9-CH), 6.84 s (1H, 5-H), 6.25 s (1H, 3-H), 4.38 s (2H, NH 2 , D 2 O exchangeable), 3.88 s and 3.86 s (3H each, OCH 3 ), 3.14-3.42 m and 2.51-3.04 m (4H, 6-H, 8-H), 2.27 m (2H, 7-CH 2 ), 1.37 m (2H, 7-CH 2 CH 2 ), 0.87 t (3H, CH 3 CH 2 ). 13 

16 mol) in ethanol (30 mL) was refluxed for 10 h. After cooling, the mixture was poured dropwise into ice water, and the solid was filtered off and recrystallized from methanol. Yield 68%, mp 196°C. IR spectrum: ν 3384 cm -1 (NH). 1 H NMR spectrum, δ, ppm: 12

OCH 3 ), 3.28-3.48 m and 2.63-3.07 m (4H, 5-H, 7-H), 2.37 m (2H, 6-CH 2 ), 1.24 m (2H, 6-CH 2 CH 2 ), 0.85 t (3H, CH 3 CH 2 ). 13 C NMR spectrum, δ C

A mixture of compound 7 (4.43 g, 0.01 mol) and triethyl orthoformate (5.27 g, 0.04 mol) in acetic anhydride (20 mL) was refluxed for 12 h. After cooling, the solid was filtered off and recrystallized from ethanol. Yield 76%, mp 134°C. IR spectrum: ν 2233 cm -1 (C≡N). 1 H NMR spectrum

CH 3 CH 2 O), 1.25 m (2H, 6-CH 2 CH 2 ), 0.80 t (3H, CH 3 CH 2 CH 2 ). 13 C NMR spectrum, δ C

A mixture of compound 16 (5.00 g, 0.01 mol) and hydrazine hydrate (5.12 g, 0.16 mol) in ethanol (40 mL) was refluxed for 6 h. After cooling, the solid was filtered off and recrystallized from ethanol. Yield 72%, mp 224°C. IR spectrum, ν, cm -1 : 3395 (NH), 3232 (NH 2 ). 1 H NMR spectrum, δ

7-CH 2 CH 2 ), 0.80 t (3H, CH 3 CH 2 ). 13 C NMR spectrum, δ C

The test cultures, HepG-2 (human liver carcinoma), PC-3 (human prostate adenocarcinoma), and HCT116 (human colorectal carcinoma) cell lines, were purchased from the American Type Culture Collection (Rockville, MD) and were maintained in RPMI-1640 medium which was supplemented with 10% heat-inactivated FBS (fetal bovine serum), 100 U/mL penicillin, and 100 U/mL streptomycin. The cells were grown at 37°C in a humidified atmosphere of 5% CO 2 . The antitumor activity against HepG-2, PC-3, and HCT-116 human cancer cell lines was estimated using the 3-(4,5dimethyl-1,3-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, which is based on cleavage of the tetrazolium salt by mitochondrial dehydrogenases in viable cells [28] [29] [30] [31] [32] . The cells were dispensed in a 96-well sterile microplate (5 × 10 4 cells per well) and incubated at 37°C with series of each tested compound or Doxorubicin ® (positive control) in DMSO at different concentrations for 48 h in a serum-free medium prior to the MTT assay. After incubation, the media were carefully removed, 40 µL of MTT (2.5 mg/mL) was added to each well, and the plate was incubated for an additional 4 h. The purple formazan dye crystals were solubilized by addition of 200 µL of DMSO, and the absorbance was measured at λ 590 nm using a SpectraMax ® Paradigm ® Multi-Mode microplate reader. The relative cell viability was expressed as the mean percentage of viable cells compared to the untreated control cells. All experiments were conducted in triplicate and repeated in three different days. The IC 50 values were determined by probit analysis using SPSS software (SPSS Inc., Chicago, IL).