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Nucleosides, Nucleotides & Nucleic Acids

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Synthesis of new uracil derivatives and their sugarhydrazones with potent antimicrobial, antioxidantand anticancer activities

Ibrahim F. Nassar, Ahmed F. El Farargy, Fathy M. Abdelrazek & Zeinab Hamza

To cite this article: Ibrahim F. Nassar, Ahmed F. El Farargy, Fathy M. Abdelrazek & ZeinabHamza (2020) Synthesis of new uracil derivatives and their sugar hydrazones with potentantimicrobial, antioxidant and anticancer activities, Nucleosides, Nucleotides & Nucleic Acids, 39:7,991-1010, DOI: 10.1080/15257770.2020.1736300

To link to this article: https://doi.org/10.1080/15257770.2020.1736300

Published online: 04 Mar 2020.

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Synthesis of new uracil derivatives and their sugarhydrazones with potent antimicrobial, antioxidantand anticancer activities

Ibrahim F. Nassara, Ahmed F. El Farargyb, Fathy M. Abdelrazekc, andZeinab Hamzad

aFaculty of Specific Education, Ain Shams University, Abassia, Cairo, Egypt; bDepartment ofChemistry, Faculty of Science, Zagazig University, Zagazig, Egypt; cDepartment of Chemistry,Faculty of Science, Cairo University, Giza, Egypt; dFood Toxicology and ContaminantsDepartment, National Research Centre, Dokki, Giza, Egypt

ABSTRACT6-(4-Chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyr-imidine-5-carbonitrile (4) was prepared and was reactedwith ethyl chloroacetate, hydrazine hydrate, 4-chloroaniline,formaldehyde, acetic anhydride, formic acid, carbon disulfide,4-cyanobenzaldehyde, triethyl orthoformate, D-sugars, 4-aminoacetophenone, benzoyl choride and cyclohexanone toafford a series of new uracil derivatives (5–18). Examination ofsome of the prepared compounds for their antimicrobial, anti-oxidant and anticancer activities was conducted. Among thetested samples, compound 17 was the most active substanceagainst the gram-positive bacteria and was more potent thanthe reference drug Cefoperazone. Moreover, the antibacterialactivity of 17 was higher against gram-negative bacteria.Compounds 6 and 13 reached a higher scavenging abilitytoward DPPH radicals and are better candidates for antioxi-dant activity. Also, compounds 6 and 13 had no significantanticancer activity toward liver cancer (Hep G2) and breastcancer (MCF-7) cell lines.

GRAPHICAL ABSTRACT

ARTICLE HISTORYReceived 27 October 2019Accepted 25 February 2020

KEYWORDSUracil; derivatives;antimicrobial; antioxidant;anticancer; activity

CONTACT Ibrahim F. Nassar [email protected] Faculty of Specific Education, Ain ShamsUniversity, 365 Ramsis Street, Abassia, Cairo, Egypt.� 2020 Taylor & Francis Group, LLC

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS2020, VOL. 39, NO. 7, 991–1010https://doi.org/10.1080/15257770.2020.1736300

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1. Introduction

Pyrimidines chemistry is of interest because these ring systems representthe main skeleton in alkaloids and nucleic bases in addition to their higherbiological activities. Many pyrimidine derivatives possess anticancer,[1,2]

antiviral,[2,3] antibacterial[4,5] antifungal,[6] anti-inflammatory[7] and centralnervous activities.[8] The pyrimidine derivatives have been investigatedfor their medicinal interest, for example the antithyroid activity of the5-fluoro-2-thiouracil.[9] Pyrimidine derivatives ethirimoland methirimolwere some of the earliest fungicides. Furthermore pyrimidine derivativesalso show the different pharmacological activities like antitumor,[10]

analgesic,[11] antineoplastic,[12] cardiovascular,[13] antiallergic.[14]

In general, nucleoside analogs are structurally, metabolically, andpharmacodynamically related agents that nevertheless have diversebiological actions and therapeutic effects. This class of agents affects thestructural integrity of DNA, usually after incorporation during replicationor DNA excision repair synthesis, leading to stalled replication forks andchain termination. One of the remarkable features that is still unexplainedabout nucleic antagonists is how drugs with such similar structural features,which share metabolite pathways and elements of their mechanismsof action, show such diversity in their clinical activities.[15] One of themost frequently used approaches to new antitumor drugs is a designof antimetabolites based on the similarity of structure to the naturallyoccurring pyrimidine and purines involved in the biosynthesis of DNA.Novel compounds should interfere with more biological applications ofnaturally occurring analogs. Since Carbon discovered in 1965 the presenceof 2-thiouracil tRNA of Escherichia coli[16] the role of thiopyrimidine nucle-obase and their biological activity has been investigated, although they havenot been studied as frequently as their oxygen analogs. It was foundthat also 4-thiouracil[17] and 2-thiocytosine[18–20] are present in tRNA ofseveral sources. These thionucleobases and their derivatives show biologicalactivity. Whereas the thionucleobases possess antiviral properties,[21,22]

derivatives of their nucleosides are potential antitumor agents.[23]

Thiopyrimidines show pronounced in vitro bacteriostatic activity[24] andthey are basic constituents of some t-RNAs.[25] They exhibit antitumor andantithyroidal activities because they are readily incorporated intonucleic acids.[26] Thiopyrimidine derivatives also act as potential inhibi-tors of protein and nucleic acid syntheses[27] as antimetabolites.[28]

From the above findings and our interest in the design and synthesis ofnew heterocyclic compounds with high impact as agrochemicals[29–35]

we synthesized new derivatives of a uracil ring linked to sugars andheterocyclic moieties and evaluating them for antimicrobial, antioxidantand anticancer activities.

992 I. F. NASSAR ET AL.

2. Results and discussion

Reaction of 4-chloro-3-nitrobenzaldehyde 1 with thiourea 2 and ethylcyanoacetate 3 in ethanol under reflux afforded 6-(4-chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4). The IRspectrum of compound 4 showed strong absorption bands at 3171 and1670 cm�1 assignable to NH and C¼O groups respectively. The 1H NMRspectrum of the same compound revealed signals at d 12.0, 13.10 ppmfor two NH groups (D2O exchangeable), in addition to the peaks of thearomatic protons which appeared as two douplets and one singlet. Also,the 13C NMR of 4 gave another proof of the structure (cf. Section 3).Stirring a solution of 4 and ethyl chloroacetate in dry acetone and potas-sium carbonate yielded ethyl 2-((4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-yl)thio)acetate (5) which in turn was con-verted to the hydrazinyl derivative 6 when refluxed with hydrazine hydratein absolute ethanol. Compound 6 also was afforded via refluxing ofcompound 4 with hydrazine hydrate in absolute ethanol. The 1H NMRspectrum of 5 showed signals at d 1.2 and 4.2 ppm attributed to the CH3(t)and the CH2(q) of the ethyl acetate ester group beside another singlet

Scheme 1. Synthesis of compounds 4–8.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS 993

signal for the other CH2 group at 4.0 ppm. The13C NMR of 5 revealed two

signals at d 15 and 60 ppm for CH3 and CH2 groups, respectively.The 1H NMR spectrum of 6 revealed signals at d 5.9 and 8.3 ppm for

NH2 and NH groups, respectively. Reaction of 4 with 4-chloroanilinein the presence of formaldehyde under Mannich conditions yielded (((4-chlorophenyl)amino)methyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrilederivative 7. Acetylation of 4 by refluxing with acetic anhydride afforded1-acetyl derivative 8. The 1H NMR spectrum of 7 showed signals at d 4.7,8.6 and 12 ppm attributable for one CH2 and two NH groups, respectively.The 13C NMR of 7 revealed signal at d 45ppm for CH2 group. The1H NMR spectrum of compound 8 revealed signals at d 1.9 ppm attributedto the acetyl CH3 group. Also, the

13C NMR of 8 showed a signal at d25 ppm for the CH3 group (cf. Section 3 and Scheme 1).The hydrazinyl derivative 6 was used as a key starting material for preparing

some interesting heterocyclic compounds. Thus, when the hydrazinyl derivative 6was refluxed with formic acid the [1,2,4] triazolo[4,3-a]pyrimidine derivative 9was produced. The 1H NMR spectrum of 9 showed signals at d 2.0 and 7.92ppmattributable to an exchangeable NH and triazole CH groups respectively.Compound 6 was reacted with carbon disulfide in pyridine and ethanol to yield3-thioxo-1,2,3,5-tetrahydro-[1,2,4]triazolo[4,3-a]pyrimidine derivative 10. The13C NMR of 10 showed a signal at d 180ppm for the triazole C¼S group.



Acetylation of 6 with acetic anhydride afforded 1,6-dihydropyrimidin-2-yl)acetohydrazide 11. The 1H NMR spectrum of 11 revealed signals at d1.9, 7.6, 10.6 and 11.0 ppm for one CH3 and three NH groups (D2Oexchangeable), respectively.Refluxing compound 6 with 4-cyanobenzaldehyde in ethanol and glacial

acetic acid afforded 2-(2-(4-cyanobenzylidene)hydrazinyl)-6-oxo-1,6-dihy-dropyrimidine-5-carbonitrile derivative 12. The 1H NMR spectrum of 12showed signals at d 8.2, 8.7 and 11.10 ppm for N¼CH and NH groups(D2O exchangeable), respectively. The

13C NMR of 12 showed signals forN¼CH group at d 161 ppm. Reaction of 6 with triethyl orthoformate inacetic anhydride yielded 5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)formo-hydrazonate 13. The 1H NMR spectrum of 13 inferred signals at d 1.2, 3.5and 8.0 ppm for CH3, CH2 and N¼CH groups respectively. The 13C NMRspectrum of 13 showed signals for the same groups, respectively (cf.Section 3 and Scheme 2).Similarly, the hydrazinyl derivative 6 was allowed to react with D-glucose

and D-xylose under the same conditions to afford the sugar hydrazonederivatives 14 and 15, respectively. The 1H NMR spectra of compounds 14and 15 revealed the signals of the sugar moiety at their specific regionsindicating the presence of the sugar parts. Also, the 13C NMR spectra



proved the presence of the sugar carbons and the C-1 of the sugar. Similarly,reaction of 6 with 4-aminoacetophenone or cyclohexanone under thesame conditions yielded 2-(2-(1-(4-aminophenyl)ethylidene)hydrazinyl)-4-(4-chloro-3-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 16,and 4-(4-chloro-3-nitrophenyl)-2-(2-cyclohexylidenehydrazinyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 17, respectively.The 1H NMR spectrum of 16 revealed signals for CH3 and NH2 groups

at d 2.3 and 5.4 ppm, respectively. The 13C NMR spectrum of the samecompound proved the presence of CH3 group at 15.5 ppm. In addition, the1H NMR spectrum of compound 17 showed two multiplet signals at d 1.59and 2.3 ppm for ten protons of the cyclohexyl moiety. The 13C NMR spec-trum of the same compound showed signals at d 25–34 ppm for the samemoiety. Finally, refluxing of compound 6 with benzoyl chloride yieldedN0-(4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)benzohydrazide 18 (cf. Section 3 and Scheme 3).

2.1. In vitro antimicrobial activity

The antibacterial activity of compounds 4, 6, 8, 10, 12, 13, 15, 16, 17 and18 was evaluated against gram positive bacteria S. aureus, B. cereus,L. monocytogenes and gram negative bacteria as, E. coli, S. typhimurium andY. enterocolitica in terms of the diameter of the inhibition zone diameterin mm relative to Cefoperazone, which was used as a reference drug.The results of the antimicrobial activity showed that the compounds 4, 6,

8, 10, 12, 13, 15, 16, 17 and 18 had antibacterial activities that variedamong the tested microbial strains (Table 1). Antibacterial and antifungalactivity of the synthesized derivatives was done in comparison withCefoperazone as standard to reveal the potency of the synthesizedcompounds. Compound 17 was the most active substance among the testedsamples against the following gram-positive bacteria L. monocytogenes

Table 1. In vitro antibacterial activity of compounds 4, 6, 8, 10, 12, 13, 15, 16, 17 and 18against gram positive and gram negative bacteria in terms of the diameter of the inhibitionzone diameter in mm.

Test microorganisms

Zone of inhibition (mm)a antibacterial activity (1mg/mL)

4 6 8 10 13 12 15 17 16 18Cefoperazone(100 mg/mL)

Gram1 ve bacteriaS. aureus – – – – 7.5 – – – – – 12B. cereus – – – – – – – 9 – – 12L. monocytogenes 12 14 13 12 14 13 11 11Gram2 ve bacteriaE. coli 11 13 8 12 9 8.5 11 13 12 13 15S. typhimurium – – – – – – – – – – 11Y. enterocolitica 16 – – 7.5 9 – – 11 – – 12aThe values (average of triplicate) are diameter of zone of inhibition at 1mg/mL


(14mm) and was more potent than the reference drug. Moreover, its anti-bacterial activity was significant against gram-negative bacteria E. coli, withan inhibition zone of 13mm comparing to the other compounds (Table 1).Compound 4 showed a high inhibition zone (16mm) against Y. enterocoli-tica as compared to the other compounds that showed considerable inhibi-tory zones 7.5–12mm (Figure 1a). The synthesized compounds showed noactivity against S. typhimurium, also all compounds are inactive against S.aureus and B. cereus except 13 and 17, respectively. An entirely differenttrend in antifungal activity was observed, with all compounds beinginactive against A. flavus and A. niger. The findings of the present studyshowed that there were differences between the antimicrobial activitiesamong the compounds, and the nature of the substituent on the benzenering and the heterocyclic skeleton attached to the pyrimidine molecules incompounds 4, 17 and 16 has a strong influence on the extent of antimicro-bial activity. The mechanism of bactericidal action is thought to be due todisruption of intermolecular interactions. This can cause dissociation of cel-lular membrane lipid bilayers, which compromises cellular permeabilitycontrols and induces leakage of cellular contents. The antimicrobial activityof the synthesized compounds was related to the inactivation of cellularenzymes, which depend on the penetration of the compounds into the cellor caused by membrane permeability changes. The antibacterial activityseemed to be dependent on the nature of substituents that had a prominenteffect on the activity profile of compounds rather the basic skeleton of themolecules. Our findings are in line with Fang et al.[36] who tested the

Figure 1. The effect of the synthesized compounds on the growth of pathogenic bacteria bydisc diffusion assay. (a) E. coli. (b) Y. enterocolitica, (c) L. monocytogenes.


antibacterial activity of a new 2,4-disubstituted-6-thiophenyl-pyrimidinederivative against Gram-positive bacteria through different biological assays.

2.2. In vitro antioxidant activity

The antioxidant activity of compounds (4, 6, 8, 10, 12, 13, 15, 16, 17 and18) was determined against DPPH, which is a free radical compound thathas been widely used to determine the free radical-scavenging ability forthe compounds.

2.2.1. The DPPH assay for evaluation of antioxidant activityFree radicals are involved in the normal physiology of living organisms.The excess of free radicals and reactive oxygen species have been proposedto induce cellular oxidative damage, which results in a variety of chronicdiseases such as cancer, arteriosclerosis, inflammatory disorders as hereffects on the aging process.[37] DPPH is a free radical compound that hasbeen widely used to determine the free radical-scavenging ability of varioussamples and decreases significantly depending on the exposure to protonradical scavengers.[38] The reduction capability of DPPH radicals are deter-mined by the decrease in the absorbance at 517 nm, which is induced byantioxidants.[39] In Figure 2, the lowest antioxidant activity was observedwith 15, 17 and 16, while 6 and 13 recorded the highest scavenging results.Both 6 and 13 reached the maximum scavenging ability against DPPH rad-icals of 99.6% and 99.5%, respectively at 75 mg/mL. There are noticeableeffects on the scavenging of free radical in a concentration-dependentmode with higher scavenging activity upon increasing the concentration ofthe other compounds. The results are higher activity than those of com-mercial phenolic antioxidants such as BHA and TBHQ. The increasing

0

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50

60

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25 ug/ml 50 ug/ml 75 ug/ml

)%(

ytiv itcat nadi xoitnA

4 6 8 10 13 12 15 17 16 18 BHA TBHQTreatment

Figure 2. DPPH radical Scavenging activity of compounds 4, 6, 8, 10, 12, 13, 15, 16, 17 and 18.Values are expressed as mean (n¼ 3) of the percent inhibition of the absorbance of DPPH radicals.


scavenging activity upon increasing concentration of the compounds 4–18from 25mg/mL to 75 mg/mL is due to the decrease in the concentrationof the DPPH radical, which indicates the reduction capability of DPPHradicals by the antioxidant activity.The DPPH antioxidant assay measures the hydrogen-donating capacity of

the molecules in the sample. On the other hand, the chemiluminescencemethod is based on the light emission produced by a chemical reaction. TheDPPH radical loses its chromophore upon receiving a proton from a hydro-gen donor. The scavenging potential of the synthesized compounds 6 and 13was appraised through investigating their DPPH reduction against positivecontrol BHA and TBHQ. The compounds that had a high antioxidant activityexert their effects via several basic mechanisms which include scavenging thespecies that initiate peroxidation, quenching singlet oxygen, breaking the freeradical chain reaction and reducing the concentration of O2.

[40]

2.3. Anti-tumor activity

The anti-tumor activity of compounds 6 and 13 was determined againstLiver Hep G2 and Breast MCF-7 cancer cell lines using the MTT assay(n¼ 3). We found that there is no activity for the tested compounds againsteither Hep G2 or MCF-7 cell lines and so we cannot calculated the IC50values for the two tested compounds.

3. Experimental

3.1. Chemistry

All melting points were measured using a Reichert Thermovar apparatus and areuncorrected. Yields listed are of isolated compounds. The IR spectra wererecorded on a Perkin-Elmer model 1720 FTIR spectrometer for KBr disc.Routine NMR measurements were made on a Bruker AC-300 or DPX-300 spec-trometer. Chemical shifts were reported in d scale (ppm) relative to TMS as a ref-erence standard and the coupling constants J values are given in Hz. Theprogress of the reactions was monitored by TLC using aluminum silica gel plates60 F245. Spectral measurements and Elemental analyses were performed at theMicro-analytical center at the Faculty of science, Cairo University, Cairo, Egypt.

3.1.1. 6-(4-Chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4)

An equimolar mixture of 4-chloro-3-nitrobenzaldehyde 1, thiourea 2, ethylcyano-acetate 3 and anhydrous K2CO3 in absolute ethanol (30mL) wasrefluxed for 5 h. The mixture was cooled, filtered off; the precipitate formedwas dissolved in water and neutralized with dill HCl. The formed


precipitate was filtered off, washed with water, dried and crystallized fromethanol to give 4 as a Brown powder; Yield (75%), mp �C; IR (KBr cm�1)mmax: 3171 (NH), 1670 (C¼O); 1H NMR (DMSO-d6, 300MHz): d 7.74,7.96 (2d, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 12.0, 13.10 (2s, 2H, 2NH, D2Oexchangeable), 13C NMR (75MHz): d 74 (C-5), 115–145 (CN, Ar-C), 166(C¼O), 170 (C-NH), 175 (C¼S); Analysis Calcd. for C11H5ClN4O3S(308.70) C, 42.80; H, 1.63; N, 18.15; Found C, 42.75, H, 1.80, N, 18.0.

3.1.2. Ethyl 2-((4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-yl)thio)acetate (5)

A mixture of 4 (0.01mol) and anhydrous K2CO3 (0.01mol) in dry acetone(30mL) was stirred at room temperature for 1 h, ethyl chloroacetate (2mL)was added and the stirring was continued for 25 h at the same temperature.The solvent was removed under reduced pressure and water was added tothe residue. The formed solid was filtered off and crystallized from benzeneto form 5. Yield (65%), mp �C; IR (KBr cm�1) mmax: 3171 (NH), 1735(C¼O); 1H NMR (DMSO-d6, 300MHz): d 1.2 (t, 3H, J¼ 5.2, CH3), 4.0(s, 2H, CH2), 4.2 (q, 2H, J¼ 5.2, CH2), 7.74, 7.96 (2d, 2H, Ar-H), 8.05(s, 1H, Ar-H), 12.0 (s, 1H, NH, D2O exchangeable);

13C NMR (75MHz): d15 (CH3), 30, 60 (2CH2), 95–145 (pyriminine-C, CN, Ar-C), 160, 170(2CN), 165, 169 (2CO), 180 (CS); Analysis Calcd. for C15H11ClN4O5S(394.79) C, 45.64; H, 2.81; N, 14.19; Found C, 45.70, H, 2.80, N, 14.20.

3.1.3. 4-(4-Chloro-3-nitrophenyl)-2-hydrazinyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (6)

A solution of compound 5 (0.01mol) and hydrazine hydrate (0.150mol) inabsolute ethanol (30mL) was refluxed for 4 h. The reaction mixture wasallowed to cool at room temperature and the solvent was evaporated underreduced pressure, the solid that formed was collected and recrystallizedfrom benzene to afford 6. Yield (85%), mp �C; IR (KBr cm�1) mmax: 3420,3171 (NH2, NH), 1670 (C¼O); 1H NMR (DMSO-d6, 300MHz): d 5.9 (bs,2H, NH2, D2O exchangeable), 7.74, 7.96 (2d, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 8.3 (s, 1H, NH, D2O exchangeable), 11.5 (s, 1H, NH, D2O exchange-able); 13C NMR (75MHz): d 95–145 (pyriminine-C, CN, Ar-C), 160, 170(2CN), 165 (CO); Analysis Calcd. for C11H7ClN6O3 (306.67) C, 43.08; H,2.30; N, 27.41; Found C, 43.0, H, 2.35, N, 27.45.

3.1.4. 4-(4-Chloro-3-nitrophenyl)-2-((((4-chlorophenyl)amino)methyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (7)

A mixture of 4 (0.01mol), primary amine such as 4-chloroaniline(0.01mol) and formaldehyde (2mL) was stirred in acetonitrile (20mL) at


room temperature for 2–3h. The formed precipitate was filtered off, dried andcrystallized from methanol to form 7. Dark brown solid; Yield (55%), mp144–146 �C; IR (KBr cm�1) mmax: 3150 (NH), 1670 (CO);

1H NMR (DMSO-d6,300MHz): d 4.7 (s, 2H, CH2), 6.54, 6.57 (2d, 4H, Ar-H), 7.74, 7.96 (2d, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 8.6 (s, 1H, NH, D2O exchangeable), 12.0 (s, 1H, NH, D2Oexchangeable); 13C NMR (75MHz): d 45 (CH2), 95–145 (pyriminine-C, CN, Ar-C), 160 (C-S), 165 (CO), 170 (C¼N), Analysis Calcd. for C18H11Cl2N5O3S(448.28) C, 48.23; H, 2.47; N, 15.62; Found C, 48.10, H, 2.45, N, 15.60.

3.1.5. 1-Acetyl-6-(4-chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (8)

A mixture of 4 (0.01mol), Acetic anhydride (20mL) and few drops of pyridinewas refluxed for 5–8 h. The reaction mixture was poured in dilute HCl, theformed precipitate was filtered off, dried and crystallized from methanol toafford 8. Dark brown solid; Yield (60%), mp above 300 �C; IR (KBr cm�1)mmax: 3150 (NH), 1670 (CO);

1H NMR (DMSO-d6, 300MHz): d 1.9 (s, 3H,CH3), 7.74, 7.96 (2d, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 10.9 (s, 1H, NH exchange-able); 13C NMR (75MHz): d 25 (CH3), 74 (C-5), 115–145 (CN, Ar-C), 160 (C-4), 166, 167 (2C¼O), 175 (C¼S); Analysis Calcd. for C13H7ClN4O4S (350.73)C, 44.52; H, 2.01; N, 15.97; Found C, 44.50, H, 2.0, N, 15.99.

3.1.6. 7-(4-Chloro-3-nitrophenyl)-5-oxo-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile (9)

A mixture of 6 (0.01mol) and formic acid (20mL) was refluxed for 8–10 h,the formed precipitate was filtered off, dried and crystallized from ethanolto form 9. Yield (70%), mp 100–102 �C; 1H NMR (DMSO-d6, 300MHz): d2.0 (s, 1H, NH exchangeable); 7.74, 7.91 (2d, 2H, Ar-H), 7.92 (s, 1H, CH),8.05 (s, 1H, Ar-H), 13C NMR (75MHz): d 94 (C-5), 120–145 (CN, Ar-C,tetrazole-C3,5), 166 (C¼O), 170 (C-N), Analysis Calcd. for C12H5ClN6O3(316.66) C, 45.52; H, 1.59; N, 26.54; Found C, 45.50, H, 1.55, N, 26.55.

3.1.7. 7-(4-Chloro-3-nitrophenyl)-5-oxo-3-thioxo-3,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile (10)

A solution of compound 6 (0.01mol) in pyridine (15mL), carbon disulfide(5mL) was added with stirring for 10min., the reaction mixture wasrefluxed for 18 h on water bath, then cooled to room temperature andacidified with ice cold HCl. The formed precipitate was filtered off, driedand crystallized from methanol to afford 10 as dark brown solid. Yield(50%), mp 170–172 �C; 1H NMR (DMSO-d6, 300MHz): d 7.74, 7.96 (2d,2H, Ar-H), 8.05 (s, 1H, Ar-H), 13C NMR (75MHz): d 94 (C-5), 120–145(CN, Ar-C, tetrazole-C5), 165 (C¼O), 180 (C¼S), Analysis Calcd. for


C12H3ClN6O3S (346.71) C, 41.57; H, 0.87; N, 24.24; Found C, 41.55, H,0.80, N, 24.25.

3.1.8. N0-(4-(4-Chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)acetohydrazide (11)

A mixture of 6 (0.01mol) and acetic anhydride (20mL) was refluxed for 8–10hthe formed solid was filtered, dried and crystallized from ethanol to yield 11.Brown solid, Yield (65%), mp 120–122 �C; 1H NMR (DMSO-d6, 300MHz): d 1.9(s, 3H, CH3), 7.60 (s, 1H, NH, D2O exchangeable), 7.74, 7.96 (2d, 2H, Ar-H),8.05 (s, 1H, Ar-H), 10.60 (s, 1H, NH, D2O exchangeable), 11.10 (s, 1H, NH, D2Oexchangeable); 13C NMR (75MHz): d 20 (CH3), 94 (C-5), 120–145 (CN, Ar-C,),153, 170 (C-2,4), 165, 168 (2C¼O); Analysis Calcd. for C13H9ClN6O4 (348.70) C,44.78; H, 2.60; N, 24.10; Found C, 44.75, H, 2.60, N, 24.15.

3.1.9. 4-(4-Chloro-3-nitrophenyl)-2-(2-(4-cyanobenzylidene)hydrazinyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (12)

A mixture of compound 6 (0.01mol) and 4-cyanobezaldehyde (0.01mol) inethanol (20mL) and few drops of glacial acetic acid was refluxed for 6 hand cooled to room temperature. The solvent was evaporated underreduced pressure; the formed solid was crystallized from methanol to yieldcompound 12. Brown solid; Yield (70%), mp 285–287 �C; 1H NMR(DMSO-d6, 300MHz): d 7.50, 7.64 (2d, 2H, Ar-H), 7.7, 7.9 (2d, 4H, Ar-H),8.05 (s, 1H, Ar-H), 8.2 (s, 1H, N¼CH), 8.7 (s, 1H, NH, D2O exchangeable),11.10 (s, 1H, NH, D2O exchangeable);

13C NMR (75MHz): d 94 (C-5),115–145 (2CN, Ar-C,), 153, 170 (C-2,4), 161 (N¼CH), 166 (C¼O);Analysis Calcd. for C19H10ClN7O3 (419.79) C, 54.36; H, 2.40; N, 23.36;Found C, 54.33, H, 2.40, N, 23.22.

3.1.10. Ethyl N-(4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)formo hydrazonate (13)

A mixture of 6 (0.01mol), triethylorthoformate (10mL) and acetic anhyd-ride (10mL) was refluxed for 7 hr .The reaction mixture was poured tocold water, the formed precipitate was washed with water, filtered off andcrystallized from ethyl alcohol to yield compound 13. Brown solid; Yield(66%), mp 83–85 �C; 1H NMR (DMSO-d6, 300MHz): d 1.2 (t, J¼ 5.3, 3H,CH3), 3.5 (q, J¼ 5.3 2H, CH2), 7.7, 7.9 (2d, 4H, Ar-H), 7.96 (s, 1H, Ar-H),8.0 (s, 1H, N¼CH), 8.7 (s, 1H, NH, D2O exchangeable), 11.10 (s, 1H, NH,D2O exchangeable);

13C NMR (75MHz): d 15 (CH3), 63 (CH2), 94 (C-5),120–150 (CN, Ar-C, N¼CH), 153, 170 (C-2,4), 166 (C¼O), 161 (N¼CH);Analysis Calcd. for C14H11ClN6O4 (362.73) C, 46.36; H, 3.06; N, 23.17Found C, 46.33, H, 3.0, N, 23.15.


3.2. General procedure to prepare compounds 14 and 15

To a well stirred solution of the respective monosaccharide (5mmol) inwater (1mL), and glacial acetic acid (1mL) was added compound 6(5mmol) in ethanol (15mL). The mixture was heated under reflux for 3 hand the resulting solution was concentrated and left to cool. The formedprecipitate was filtered off, washed with water and ethanol, then dried andcrystallized from ethanol.

3.2.1. 4-(4-Chloro-3-nitrophenyl)-6-oxo-2-(2-D-Galactopentitolylidenehydrazinyl)-1,6-dihydropyrimidine-5-carbonitrile (14)

Dark brown solid; Yield (55%), mp 102–104 �C; 1H NMR (DMSO-d6): d3.30–3.39 (m, 2H, H-60,600), 3.73 (m, 1H, H-50), 4.12 (m, 1H, H-40), 4.25 (t,J¼ 7.4Hz, 1H, H-30), 4.36 (dd, J¼ 7.4Hz, J¼ 7.8Hz, 1H, H-20), 4.45 (m, 1H,OH), 4.48 (d, J¼ 6.4Hz, 1H, OH), 5.19 (m, 1H, OH), 5.63 (t, J¼ 4.6Hz, 1H,OH), 5.79 (t, J¼ 4.6Hz, 1H, OH), 7.7, 7.9 (2d, 4H, Ar-H), 7.94 (d, 1H, J10,20 ¼7.8Hz, H-10), 7.96 (s, 1H, Ar-H), 8.7 (s, 1H, NH, D2O exchangeable), 11.10(s, 1H, NH, D2O exchangeable);

13C NMR (75MHz): d 62.10 (C-60), 63.05(C-50), 69.21 (C-40), 74.44 (C-20), 75.71 (C-30), 94 (C-5), 120–135 (CN, Ar-C),152 (C-10), 153, 170 (C-2,4), 166 (C¼O); Analysis Calcd. for C17H17ClN6O8(468.81) C, 43.55; H, 3.66; N, 17.93; Found C, 43.53, H, 3.60, N, 14.95.

3.2.2. 4-(4-Chloro-3-nitrophenyl)-6-oxo-2-(2-D-Xylotetritolylidenehydrazinyl)-1,6-dihydropyrimidine-5-carbonitrile (15)

Dark brown solid; Yield (65%), mp 100–102 �C; 1H NMR (DMSO-d6): d3.49–3.53 (m, 2H, H-50,500), 3.73 (m, 1H, H-40), 4.12 (m, 1H, H-30), 4.36 (dd,J¼ 7.4Hz, J¼ 7.8Hz, 1H, H-20), 4.45 (m, 1H, OH), 4.88 (d, J¼ 6.4Hz, 1H,OH), 5.60 (t, J¼ 4.6Hz, 1H, OH), 5.72 (t, J¼ 4.6Hz, 1H, OH), 7.28 (m, 3H,Ar-H), 7.36 (m, 3H, Ar-H), 7.42 (m, 2H, Ar-H), 7.52 (d, 1H, J10,20 ¼ 7.8Hz,H-10), 7.7, 7.9 (2d, 4H, Ar-H), 7.96 (s, 1H, Ar-H), 8.7 (s, 1H, NH, D2Oexchangeable), 11.10 (s, 1H, NH, D2O exchangeable);

13C NMR (DMSO-d6):d 62.22 (C-50), 63.15 (C-40), 69.32 (C-30), 74.56 (C-20), 122–140 (CN, Ar-C),152 (C-10), 153, 170 (C-2,4), 166 (C¼O); Analysis calcd. for: C16H15 ClN6O7(438.78): C, 43.80; H, 3.45; N, 19.15 Found: C, 43.75; H, 3.40; N, 19.10%.

3.3. Synthesis of compounds 16 and 17

A mixture of compound 6 (0.01mol) and different ketones namely, 4-ami-noacetophenone and cyclohexanone in a mixture of ethanol and glacialacetic acid was refluxed for 4–5 h. The formed precipitate was washed withwater, filtered and crystallized to give compounds 16 and 17.


3.3.1. 2-(2-(1-(4-Aminophenyl)ethylidene)hydrazinyl)-4-(4-chloro-3-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (16)

Brown solid; Yield (65%), mp 200–202 �C; 1H NMR (DMSO-d6, 300MHz):d 2.3 (s, 3H, CH3), 5.4 (s, 2H, NH2), 6.8 (d, 2H, Ar-H), 7.6 (d, 2H, Ar-H),7.74, 7.96 (2d, 2H, Ar-H), 8.05 (s, 1H, Ar-H), 8.7 (s, 1H, NH, D2Oexchangeable), 11.10 (s, 1H, NH, D2O exchangeable);

13C NMR (75MHz):d 15.5 (CH3), 94 (C-5), 114–147 (CN, Ar-C, N¼C), 153, 170 (C-2,4), 166(C¼O); Analysis Calcd. for C19H14ClN7O3 (423.82) C, 53.85; H, 3.33; N,23.13; Found C, 53.83, H, 3.33, N, 23.12.

3.3.2. 4-(4-Chloro-3-nitrophenyl)-2-(2-cyclohexylidenehydrazinyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (17)

Yellow solid; Yield (67%), mp 290–292 �C; 1H NMR (DMSO-d6, 300MHz):d 1.59 (m, 6H, 3CH2), 2.3 (m, 4H, 2CH2), 7.74, 7.96 (2d, 2H, Ar-H), 8.05(s, 1H, Ar-H), 10.7 (s, 1H, NH exchangeable), 11.10 (s, 1H, NH exchange-able); 13C NMR (75MHz): d 25–34 (5CH2), 94 (C-5), 120–147 (CN, Ar-C),153,160,170 (C-2,4, N¼C), 166 (C¼O); Analysis Calcd. for C17H15ClN6O3(386.80) C, 52.79; H, 3.91; N, 21.73; Found C, 52.80, H, 3.93, N, 21.72.

3.3.3. N0-(4-(4-Chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)benzohydrazide (18)

A solution of compound 6 (0.01mol) and benzoylchloride (10mL) wasrefluxed for 3 hr, the mixture was allowed to cool and poured onto coldwater. The solid that formed was filtered off, dried and crystallized fromethanol to form compound 18. Brown solid; Yield (77%), mp 100–102 �C;1H NMR (DMSO-d6, 300MHz): d 7.49–7.63 (m, 4H, Ar-H), 7.93–7.96 (m,4H, Ar-H), 8.2 (s, 1H, NH, D2O exchangeable 12.0 (s, 1H, NH, D2Oexchangeable), 13.10 (s, 1H, NH, D2O exchangeable);

13C NMR (75MHz):d 94 (C-5), 120–147 (CN, Ar-C), 153,170 (C-2,4), 164, 166 (2C¼O);Analysis Calcd. for C19H14ClN7O3 (423.82) C, 53.85; H, 3.33; N, 23.13;Found C, 53.90, H, 3.33, N, 23.02.

4. Biology

4.1. Material and methods

4.1.1. In vitro antimicrobial assay4.1.1.1. Pathogenic strains. Bacillus cereus B-3711 and Asperagillus flavus3357 were provided by the Northern Regional Research Laboratory Illinois,USA (NRRL). Listeria monocytogenes 598 was provided by the Departmentof Food Science, University of Massachusetts, USA. Escherichia coli0157:H7, Salmonella typhmirum and Staphylococcus aureus were isolated


from serologically identified by Dairy microbiological Lab., NationalResearch Center. Yersinia enterocolitica was obtained from HungarianNational Collection of Medical Bacteria, OKI, Gyaliut 2-6, H-1966Budapest, Hungary.

4.1.1.2. Experimental. The stock solutions of the compounds under study(4–18) were prepared at conc. (1mg/mL) for antibacterial and antifungalassay. Sterile discs were impregnated with 10mL of each for anti-bacterialand antifungal assay 10lg/disk) a loading control was also prepared contain-ing 10mL of Dimethyl sulfoxide (DMSO) for each inoculated spread plate.The discs of (4–18) were placed on the surface of the agar plat using sterileforceps, gently press down each disc to ensure complete contact with theagar surface. Antimicrobial activity of the selected compounds (4–18) wasconducted against a wide range of human pathogenic microorganisms Grampositive bacteria (S. aureus, B. cereus and L. monocytogenes), Gram negativebacteria (Y. enterocolitica, S. typhmirum, E. coli) and fungal strain (A. flavusand A. niger).The inverted plates were incubated at (37 �C, 18 h) for bacteriaand (25 �C, 48 h) for fungi within 15min. after the discs are applied. Thediameter of zones of complete inhibition was measured to the nearest wholemillimeter, including the diameter of the disc, using sliding calipers, which isheld on the back of the inverted Petri plate. Plates were examined for growthinhibition and the diameter of the inhibition zone measured. The strength ofthe activity was classified as high activity for the inhibition zone havingdiameters of (10–15mm) and low activity for the diameter ranging from(7–10mm) and no activity for one with diameter less than 7mm.

4.1.2. The DPPH assay for evaluation of antioxidant activityIn vitro the antioxidant activity was evaluated by 2.2-diphenyl-1-picrylhydra-zyl free radical scavenging assay according to the method of Brand-Williamset al.[41] Briefly, 100, 200 and 300lL of each compound (1mg/mL) weretaken in different test tubes and 3.9 of 0.1mM ethanol solution of DPPH wasadded and shaken vigorously. The tubes were then incubated at 37Co for30min. Changes in the absorbance were measured at 517nm against a blank,i.e., without DPPH using UV-Vis Shimadzu (UV-1601, PC) spectrophotom-eter. Ethanol was used to zero spectrophotometer. Measurement was per-formed in triplicate and an average was used. The radical scavenging activitywas expressed as percentage inhibition of DPPH using the following formula:

%Inhibition ¼ ðAcontrol– Atreatment=AcontrolÞ½ � � 100where Acontrol is the absorbance of the control; Atreatment is the absorbanceof the treatments. Butylated hydroxyl anisol (BHA) and tert-Butylatedhydroxyl qunione (TBHQ) were used as positive controls.


4.1.3. Anti-tumor activity4.1.3.1. Cell culture. Human Hepatocellular carcinoma cells (Hep G2) andHuman Breast adenocarcinoma (MCF-7) was purchased from ATCC, USA,were used to evaluate the cytotoxic effect of the tested samples. Cells wereroutinely cultured in Dulbecco’s Minimum Essential Media (DMEM),the media were supplemented with 10% fetal bovine serum (FBS), 2mML-glutamine, containing 100 units/mL penicillin G sodium, 100 units/mLstreptomycin sulfate, and 250 ng/mL amphotericin B. Cells were maintainedat sub-confluency at 37 �C in humidified air containing 5% CO2. Forsub-culturing, monolayer cells were harvested after trypsin/EDTA treatmentat 37 �C. Cells were used when confluence had reached 75%. Testedsamples were dissolved in dimethyl sulphoxide (DMSO), and then dilutedin the assay to reach the intended concentration. All cell culture materialwas obtained from Cambrex BioScience (Copenhagen, Denmark).All chemicals were from Sigma/Aldrich, USA, except mentioned. Allexperiments were repeated three times, unless mentioned.

4.1.3.2. Anti-tumor activity assay. Cytotoxicity of tested samples was meas-ured against Hep G2 and MCF-7 cells using the MTT Cell Viability Assay.MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) assayis based on the ability of active mitochondrial dehydrogenase enzymeof living cells to cleave the tetrazolium rings of the yellow MTT and forma dark blue insoluble formazan crystals which is largely impermeable tocell membranes, resulting in its accumulation within healthy cells.Solubilization of the cells results in the liberation of crystals, which arethen solubilized. The number of viable cells is directly proportional to thelevel of soluble formazan dark blue color. The extent of the reduction ofMTT was quantified by measuring the absorbance at 570 nm.[42]

4.1.3.3. Reagents preparationMTT solution: 5mg/mL of MTT in 0.9%NaCl and dimethylsulfox-ide (DMSO).

4.1.3.4. Procedure. Cells (0.5� 105 cells/well), in serum-free media, wereplated in a flat bottom 96-well microplate, and treated with 20 mL of differ-ent concentrations (100, 50, 25 and 12.5 mg/mL) of the tested samples for24 h at 37� C, in a humidified 5% CO2 atmosphere. After incubation, mediawere removed and 40 mL MTT solution/well were added and Incubatedfor an additional 4 h. MTT crystals were solubilized by adding 180 mLof dimethyl sulphoxide/well and plate was shacked at room temperature,followed by photometric determination of the absorbance at 570 nm usingmicroplate ELISA reader. Triplicate repeats were performed for each


concentration and the average was calculated. Data were expressed as thepercentage of relative viability compared with the untreated cells comparedwith the vehicle control, with cytotoxicity indicated by

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https://doi.org/10.1111/j.1365-2621.2005.tb07127.xhttps://doi.org/10.1111/j.1365-2621.2005.tb07127.xhttps://doi.org/10.1016/S0023-6438(95)80008-5https://doi.org/10.1016/S0023-6438(95)80008-5https://doi.org/10.1016/0022-1759(89)90397-9

AbstractIntroductionResults and discussionIn vitro antimicrobial activityIn vitro antioxidant activityThe DPPH assay for evaluation of antioxidant activity

Anti-tumor activity

ExperimentalChemistry6-(4-Chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4)Ethyl 2-((4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydro-pyrimidin-2-yl)thio)acetate (5)4-(4-Chloro-3-nitrophenyl)-2-hydrazinyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (6)4-(4-Chloro-3-nitrophenyl)-2-((((4-chlorophenyl)amino)methyl)thio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (7)1-Acetyl-6-(4-chloro-3-nitrophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (8)7-(4-Chloro-3-nitrophenyl)-5-oxo-1,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile (9)7-(4-Chloro-3-nitrophenyl)-5-oxo-3-thioxo-3,5-dihydro-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonitrile (10)N′-(4-(4-Chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)acetohydrazide (11)4-(4-Chloro-3-nitrophenyl)-2-(2-(4-cyanobenzylidene)hydrazinyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (12)Ethyl N-(4-(4-chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)formo hydrazonate (13)

General procedure to prepare compounds 14 and 154-(4-Chloro-3-nitrophenyl)-6-oxo-2-(2-D-Galactopentitolylidenehydrazinyl)-1,6-dihydropyrimidine-5-carbonitrile (14)4-(4-Chloro-3-nitrophenyl)-6-oxo-2-(2-D-Xylotetritolylidenehydrazinyl)-1,6-dihydropyrimidine-5-carbonitrile (15)

Synthesis of compounds 16 and 172-(2-(1-(4-Aminophenyl)ethylidene)hydrazinyl)-4-(4-chloro-3-nitrophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (16)4-(4-Chloro-3-nitrophenyl)-2-(2-cyclohexylidenehydrazinyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (17)N′-(4-(4-Chloro-3-nitrophenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-yl)benzohydrazide (18)

BiologyMaterial and methodsIn vitro antimicrobial assayPathogenic strainsExperimental

The DPPH assay for evaluation of antioxidant activityAnti-tumor activityCell cultureAnti-tumor activity assay

Reagents preparationProcedure

AcknowledgmentsReferences

Synthesis of new uracil derivatives and their sugar ... · Synthesis of new uracil derivatives and...

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