Anticancer Activity of New Substituted Pyrimidines , Their Thioglycosides and Thiazolopyrimidine Derivatives

Article history: Received on: 10/07/2017 Accepted on: 21/09/2017 Available online: 30/11/2017 Novel functionalized pyrimidine, thioxopyrimidine, iminopyrimidine derivatives and their derived bicyclic thiazolopyrimidine compounds were synthesized. The substituted arylidine derivatives of the thiazolopyrimidine compounds were also prepared. Glycosylation of the thiopyrimidine derivative resulted in formation of the acetylated thioglycosides which were deacetylated to the free hydroxythioglycosides. The synthesized compounds were studied for their anticancer activity against hepatocellular carcinoma HepG-2, human prostate adenocarcinoma PC-3 and human colorectal carcinoma HCT-116 cell lines. Compounds 7c, 8a and 12a showed high activity against PC-3 cancer cells while compounds 11b and 12a revealed higher activity against HCT-116 cell line.


INTRODUCTION
The chemistry and biological research on pyrimidines and their derivatives have attracted great attention because such ring system represents the main skeleton in alkaloids and nucleic bases in addition to their interesting potent biological activities.The anticancer (Abdel Mohsen et al., 2010;El-Sayed et al. 2009), antiviral (El-Sayed et al., 2009;2008), antibacterial (El-Sayed and Abdel-Rahman, 2010;Ramez et al., 2010) antifungal (Gholap et al., 2008), anti-inflammatory (Da et al., 2006) and central nervous activities (Gillespie et al., 2009) properties of many pyrimidine derivatives are well reported.New 4,6diarylpyrimidine compounds have been found to exhibit antitubercular, antibacterial and antiviral activity (Siddiqui et al., 2007).Pyrimidinedione derivatives have been reported to possess antibacterial and anticancer activities (Haiba et al., 2013;Singh. and Paul, 2006).Thiazolopyrimidenes represent an important class of fused pyrimidine compounds due to their inhibition activity of 2-methylerythritol-2,4-cyclodiphosphate synthase (Geist et al., 2010).Such type of compounds have been shown to hav antiparkinsonian, analgesic (Amr et al., 2008), anticancer (Flefel et al., 2007;Said et al., 2004) and antimicrobial (Rashad et al., 2010) agents, in addition to activity as phosphate (Kolb et al., 2009) and acetylcholinesterase inhibitors (Zhi et al., 2008).Aromatic thiophenes have attracted much attention as target molecules due to the wide spectrum of its biological activities.These compounds exhibit potent antitumor (Brault et al., 2005) and antibacterial effects (Alagarsamy et al., 2006).On the other hand, extensive interest has been gained to the chemistry and biological properties of glycosyl heterocycles incorporating modified sugar and/or and heterocyclic constituents as biological inhibitors (El Ashry et al., 2007;El-Sayed et al., 2016;2017).Interestingly, the possible potential hydrogen bond donors and acceptors as a result of presence of polyhydroxyl chain in thioglycosides are expected to promote their affinity toward protein and nucleic acid (Kumar et al., 2015).
Many pyrimidine nucleosides and analogues have been synthesized and studied for anticancer behaviour, and two are in general clinical use.A number of nucleosides and 5-fluorouracil were applied orderly for remediation of breast cancer, gastrointestinal tract tumours in addition to other solid tumours (MacCoss and Robins, 1990).Nucleosides bearing pyranosyl rings have been evaluated for their potential antiviral (Ostrowski et al. 1998;Maurinsh et al., 1997), antioxidant (Spanou et al., 2007) and antibiotic (Haouz et al., 2003) properties and as building blocks in nucleic acid synthesis (Vastmans et al., 2001).
Furthermore, a number of pyrimidine thioglycosides have been shown to have antischistosomal activity (Srour et al., 2009).Compounds I-VI (Abdel Mohsen et al., 2010;Chou et al., 1999;Alagarsamy et al., 2013;Jordheim et al., 2013;Fahmy et al., 2003) (fig. 1) represents examples of reported disubstituted pyrimidine, thiazolopyrimidine and pyrimidinyl sugar derivatives with their anticancer activity.In the same direction and following our program aiming for the synthesis of glycosylthio five and six membered heterocycles, with anticancer, antiviral and antimicrobial activity properties (El-Sayed et al., 2008;2017;Flefel et al., 2017) we report the synthesis of novel disubstituted pyrimidines, their bicyclic derivatives and their derived thioglycosides with cytotoxic activity evaluation.

Instruments and reagents
All melting points were measured by Electro-thermal IA 9100 apparatus (Shimadzu, Tokyo, Japan) and are uncorrected.Infra-red spectra were investigated (KBr) by means of a Perkin-Elmer 1650 spectrophotometer (Norwalk, CT, USA).Nuclear Magnetic Resonance ( 1 H and 13 C NMR) of prepared compounds was carried out on a Jeol-Ex-400 NMR spectrometer (Jeol, Tokyo, Japan) at 25 ºC and chemical shifts were expressed as part per million; ppm (δ values) with respect to TMS as internal standard.
Mass spectrometry was performed using VG 2AM-3F spectrometer (Thermo electron corporation, USA).Microanalyses were determined using Mario El Mentar apparatus.Following up the reactions and checking the purity of the compounds were performed by means of TLC which was implemented by aluminum plates pre-coated with silica gel 60 or 60 F254 (Merck) and visualized using UV light (254 nm).
All chemicals which have been used were of reagent grade and used as provided directly unless otherwise stated.Synthesis of compounds 1a-c and 2a was performed according to reported procedure (Ramesh and Rao, 2010;Greiner-Bechert and Otto, 1991;Qiang et al., 2013).

General procedure for preparation of compounds 2b,c
To a solution of enone derivatives 1b,c (10 mmole) in dry acetone (40 mL)/methanol (15 mL) mixture, H 2 O 2 (20%, 15 mL) and anhydrous NaOH (2 g) were added portion-wise.The resulting mixture was stirred in an ice bath until the yellow color is disappeared, then, the temperature was allowed to raise to 30-40 °C for 45 minutes until white clear solution was achieved, then stirring was continued at room temperature for 3 h.Crushed icewater mixture was added and the resulting solid substance was filtered and re-crystallized from ethanol affording the oxirane 2b,c.

General method for preparation of dihydropyrimidines 3a-c
Thiourea (10 mmole) was added to a stirred mixture of chalcone derivatives 1a-c (10 mmole) and anhydrous sodium hydroxide (1.5 g) in absolute ethanol (10 mL) then the reaction mixture was heated under reflux till completion of reaction (TLC; Pet ether/ethyl acetate: 3:1).The resulting mixture was poured onto hydrochloric acid solution (5%, 15 mL) in an ice bath resulting in a yellowish precipitate which was filtered and recrystallized to afford substituted dihydropyrimidine 3a-c, respectively.

General procedure for preparation of compounds 5b,c
A mixture of compound 3b,c (10 mmole), chloroacetic acid (15 mmole), anhydrous sodium acetate (2 g), acetic anhydride (8 mL) and glacial acetic acid (16 mL) was heated at reflux temperature for 2-3 hours.The mixture was poured onto water to afford a brownish solid which was filtered and re-crystallized from acetic acid/water mixture (1:1).

General method for preparation of compounds 8a-c
A mixture of the substituted epoxides 2a-c (10 mmole), thiourea (10 mmole) and dry KOH (0.5 g) in ethyl alcohol (10 mL) was refluxed for 45 minutes.The reaction mixture was added portion-wise in a conical flask containing cold hydrochloric acid (20 mL), filtered, dried and re-crystallized from ethanol/water mixture (1:1).

General method for preparation of compounds 9a-c
To the thiopyrimidine derivatives 8a-c (10 mmole) was added chloroacetic acid (10 mmole) and the mixture was heated under reflux (TLC control; ethyl acetate/pet.ether: 1/3) till perfecting of reaction.The mixture was poured into ice-water, filtered, and then re-crystallization from acetic acid/water afforded the thiazolopyrimidines 9a-c, respectively.

General methods for preparation of compounds 10b,c
A mixture of the thiopyrimidine 8b,c (10 mmole), 2chloroacetic acid (10 mmole) and acetic anhydride (10 mL) in acetic acid (20 mL) was stirred for 15 minutes, then the corresponding aromatic aldehyde (10 mmole) and dry CH 3 COONa (2 g) were provided separately.The reaction components were refluxed for 4 h at which TLC showed completion of the reaction.The flask content was then added portion-wise to crushed ice and the precipitated solid was collected washed with water then recrystallized from acetic acid/water, to afford compound 10b,c, respectively.

General procedure for the preparation of compounds 11a-c
To a suspension of compounds 2a-c (10 mmole) and thiosemicarbazide (10 mmole) in ethanol (20 mL) was added portion wise potassium hydroxide (1.5 g) then the mixture was heated under efflux till completion of the reaction (TLC) (ethyl acetate/pet.ether: 1/3).Cold diluted HCl (15 mL) was then added drop wise in an ice bath.The appeared precipitated solid m was filtered and re-crystallized from proper solvent.

General procedure for the preparation of compounds 12a,c
A mixture of the epoxide derivatives 2a,c (10 mmole), urea (10 mmole) and anhydrous KOH pellets (0.5 g) in absolute ethyl alcohol (10 mL) was heated at reflux temperature for 45 minutes.The reaction mixture poured on cold hydrochloric acid (15 mL), filtered, dried and crystallized from ethanol/water (1:1).

General method for preparation of compounds 14b,c
A mixture of compound 2b,c (10 mmole), carbon disulfide (10 mmole), potassium hydroxide (1 g) in ethanol (20 mL) was refluxed for 3 hours.The mixture was put onto crushed ice-water, filtered and crystallized.

General method for preparation of compounds 15a,b
A solution of the acetylated bromoglucose or acetylated bromoxylose (5 mmole) in acetone (15 mL) was inserted progressively to a stirred mixture of compound 8a (5 mmole) and KOH (5 mmole) in water (2 mL).Stirring was continued at r.t. for a time at which reaction was judged complete by TLC (chloroform/methanol; 99.7/0.3v/v).Evaporation of the solvent resulted in a residue that was washed with water (10 mL).The organic layer was extracted with chloroform, dried and evaporated under reduced pressure.Petroleum ether (b.p. 40-60 °C) (45 mL) was then provided to the remnant with stirring.The resulted solid was filtered and re-crystallized.

General method for preparation of compounds 16a,b
The acetylated thioglycosides 15a,b (5 mmol) was dissolved in dry saturated methanolic ammonia solution (20 mL) and stirred at 0 °C for 1 h, then stirring was persisted at r.t. for 5 h.Removal of the solvent under vacuum at 40 °C gave a solid residue, which was recrystallized from ethanol to give the corresponding free glycoside 16a,b.

Material
The cell lines namely; Human liver carcinoma (HepG-2), PC-3 (human prostate adenocarcinoma) and HCT116 (human colorectal carcinoma) cell culture were brought from the American Type Culture Collection (Rockville, MD, USA).

Cell culture
The cells were preserved in the medium of Roswell Park Memorial Institute (RPMI-1640) that was replenished 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 5% CO 2 and 95% moisture.
The cells were distributed in a 96-well sterile microplate (5 × 10 4 cells/well), and incubated with each of synthesized compound or Doxorubicin ® (positive control), prepared in a set of various concentrations in diemthyl sulfoxide, at 37 °C for 48 hours in a serum-free medium prior to the assay.The media were cautiously isolated after incubation, and then MTT (2.5 mg/mL, 40 µL) was provided to each well and then incubated for further four hours.The crystals of purple formazan dye were solubilized by providing dimethyl sulfoxide (200 µL).At 590 nm (SpectraMax ® Paradigm ® Multi-Mode microplate reader), the absorbance was determined.The average percentage of viable cells with respect to to the untreated control cells expresses the relative cell viability.

Statistical Analysis
All experimental investigations were performed in triplicate with repeating at three different days.All the OBTAINED values were expressed as mean ± SD.IC 50 s have been determined by PROBIT analysis by means of SPSS software (version 20, SPSS Inc., Chicago, IL, USA).

Chemistry
α,β-Unsaturated ketones 1a-c were prepared by reaction of aromatic aldehyde (namely benzaldehyde, 3,4dimethoxybenzaldehyde, and 3,4,5-trimethoxybenzaldehyde) with 2-acetylthiophene.The corresponding epoxide derivatives 2a-c were prepared by reaction of α,β-unsaturated ketones 1a-c with hydrogen peroxide in basic medium.Compounds 1a-c and 2a-c were used as starting key compounds for the preparation of a variety of substituted pyrimidine derivatives.Reaction of compounds 1a-c with thiourea in sodium hydroxide afforded the corresponding thiopyrimidine derivatives 3a-c.The signals assigned to the aryl, pyrimidine ring and NH protons in their 1 H NMR spectra confirmed the assigned structure.Thiopyrimidine derivatives 3b,c were allowed to react with chloroacetic acid and resulted in the formation of thiazolo[3,2-a]pyrimidine derivatives 5b,c.The products revealed absorption band for carbonyl group in the infra-red spectrum and the corresponding 1 H NMR data revealed singlet peaks for -SCH 2 .Compounds 5b,c were reacted with 3,4-dimethoxybenzaldehyde and 3,4,5trimethoxybenzaldehyde (6a,b) and afforded the thiazolo[3,2a]pyrimidin-3(2H)-one 7b, and 7c, respectively.The 1 H NMR spectrum of compounds 7b,c revealed disappearance of singlet peak of the -SCH 2 and other assignments greed with their structures.Reaction of compounds 1a-c with guanidine hydrochloride in potassium hydroxide afforded the dihydropyrimidin-2(1H)-imine derivative 4c (scheme 1).The resulting iminopyrimidine derivative 4c showed the assigned absorption bands for NH functions in the IR spectrum in addition to the doublet signals attributed to the hydrogens (H-4 and H-5) in its 1 H NMR spectrum.
Scheme 1: synthesis of pyrimidine and thiazolopyrimidine derivatives.
Epoxide derivatives 2a-c were reacted with thiourea to furnish the thioxopyrimidinone derivatives 8a-c, respectively.The IR spectra for compounds 8a-c showed absorption bands for the NH and the 1 H NMR spectra showed signals for pyrimidine H-4 and H-6.The presence of such signals each as doublet in 1 H NMR data revealed that the pyrimidine moiety in such derivatives is alicyclic, with a twist boat conformation (Flefel et al., 2007).Pyrimidine derivative 8a-c were reacted with chloroacetic acid and formed compounds 9a-c (Scheme 2).The IR spectra of the latter compounds showed absorption band for carbonyl groups.The signals corresponding singlet signal of the CH 2 appeared in the 1 H NMR spectra in addition to aryl and pyrimidine protons.The thioxopyrimidine derivatives 8b,c were allowed to react with chloroacetic acid and 3,4-dimethoxybenzaldehyde or 3,4,5trimethoxybenzaldehyde and furnished the arylidine substituted thiazolo[3,2-a]pyrimidine derivatives 10b,c respectively.The oxirane derivatives 2a-c were reacted with thiosemicarbazide to produce compounds 11a-c.Also, compounds 2a,c were reacted with urea in presence of potassium hydroxide to form compounds 12a,c.Epoxide derivative 2a was reacted with guanidine hydrochloride in potassium hydroxide to afford the tetrahydropyrimidin-2(1H)-imine compound 13a.Reaction of the unsaturated ketones 2b,c with carbon disulfide in potassium hydroxide to afforded 1,3-oxathiolane derivatives 14b,c.The 1 H NMR of 14b,c showed the doublet signals of the oxathiolane ring in addition to aromatic signals.
Scheme 2: Synthesis of amino-and iminopyrimidine and thiazolopyrimidine derivatives.
When compound 8a was reacted with the acetylated glycopyranosyl bromide for glucose and xylose moieties, in basic medium, the corresponding acetylaed thioglycosides 15a,b were afforded in good yields.The infra-red spectra of the glycosides 15a,b indicated the bands in the range 1742-1750 cm -1 for the acetyl carbonyl groups.The 1 H NMR spectra showed peaks assigned for the protons of the sugar moiety and carbonyl methyl protons.The sugar moiety attachment at the sulfur center rather than the nitrogen atom was also confirmed by the disappearance of a signal corresponding to the C=S in the 13 C NMR spectrum of 15a.Deacetylation of the acetylated thioglycosides 15a,b in methanol saturated with ammonia produced the free hydroxy thioglycosides 16a,b (Scheme 3).The IR spectra of the deacetylated products 16a,b showed absorption bands for the hydroxyl groups and also revealed the absence of the acetyl carbonyl bands. 1 H NMR spectra of compounds 16a,b showed signals corresponding to the hydroxyl protons.

Cytotoxic activity
The cytotoxic activity of prepared compounds was investigated for their activity with respect to HepG-2, PC-3 and HCT-116 cell lines and such activity behavior has been estimated using MTT assay (Awad et al., 2014;Soliman et al., 2014).
The percentage of the intact cells was determined and compared to the control (table 1 and Fig. 2).The cytotoxicity of compounds under test towards the previously mentioned carcinoma cells were compared with that of Doxorubicin ® .
The afforded results indicated that the tested compounds exhibited dose-dependent behavior against the three cancer cell lines.From Table 1 we can deduce that, at 100 µg/mL, two compounds (11b and 12a) showed good cytotoxic activities against HCT-116 carcinoma cells.Compounds 3a, 5c, 7c, 8a and 11a showed moderate activities and the remaining compounds displayed weak activities with respect to HCT-116 cells.In addition, in case of PC-3 cancer cells, the synthesized compounds; 7c and 12a showed high cytotoxic activity when compared with the reference drug.Furthermore, compound 8a showed also good cytotoxic activity against such cell line.Three compounds showed moderate activities (3a, 11a and 11b) and the rest of the compounds showed weak or no cytotoxic activities against this type of cell lines.Furthermore, all the compounds showed weak or no cytotoxic activities against HepG-2 liver cancer.By correlation of the afforded cytotoxic activity results with structural features of tested compounds it may be deduced that substitution of the thiopyrimidine nucleus at ring nitrogen (N 3 ) with amino group resulted in an enhanced effects in case of incorporation of phenyl moiety as an aryl function at C-6 in the pyrimidine ring.Furthermore, substituted pyrimidine derivatives, with dimethoxyphenyl at C-6, revealed more activity against HCT-116 and PC-3 cell lines than their analogues incorporating trimethoxyphenyl moiety.The free 1,3-unsubstituted pyrimidinedione derivatives showed relatively higher activity against HCT-116 and PC-3 cell lines than the corresponding 2- Obviously, the substituted diarylthiazolopyrimidine nucleus free of oxo-substitution at C-6 was clearly found to be more active than the corresponding 6-oxo analogue as the activity was lost in compound 10 in which C-6 was functionalized with oxo-group.Furthermore, substitution at the methylene carbon in the thiazolopyrimidine system with arylidine function, in absence of oxo-group at C-6, led to more active derivatives than their precursors with free CH 2 group, especially in presence of trimethoxyphenyl moiety.

New
substituted oxo-and thioxopyrimidine, thiazolopyrimidine and pyrimidine thioglycoside derivatives were synthesized and structurally characterized.The prepared compounds showed cytotoxic activity against HCT-116 and PC-3 cell lines revealing moderate to good activities.A number of compounds revealed good effectiveness especially on PC-3 cell line and could be believed as valuable templates for further investigations to get more potent agents.
Financial support and sponsorship: Nil.

Fig. 2 :
Fig. 2: Cytotoxic activity of compounds against cancer cell lines at 100 ppm.

Table 1 :
Cytotoxic activity of compounds against cancer cell lines at 100 ppm.

Table 2 :
IC50 values for synthesized compounds against cancer cell lines.