168
Acta Chim. Slov. 2015, 62, 168-180
DOI: 10.17344/acsi.2014.867
Scientific paper
Synthesis and Antitumor Evaluation of Novel Dihydropyrimidine, Thiazolo[3,2-a ]pyrimidine and Pyrano[2,3-^]pyrimidine Derivatives
Nadia Y. Megally Abdo
Department of Chemistry, Faculty of Education, Alexandria University, 21526, Alexandria, Egypt. * Corresponding author: E-mail: nadiamegally@yahoo.com Received: 30-07-2014
Abstract
A simple and efficient method has been developed for the synthesis of 4,5-dihydro-2-mercapto-4-oxo-6-substituted arylpyrimidine derivatives 2a-e and their fused rings 3b, 4b, 5b, 6b and also 1,4-dihydro-2-mercaptopyrimidine derivatives 7a-e, 9a-e using triethylamine as a catalyst. The structure of the newly synthesized compounds was confirmed on the basis of their spectral data and elemental analyses. All the synthesized compounds were evaluated for their in vitro anticancer activity against six human cancer cell lines and normal fibroblasts. Nine of the tested compounds (i.e. 2a, 2c, 2d, 3b, 4b, 5b, 8, 9a and 9c) exhibited significant cytotoxicity against most cell lines. Among these derivatives compounds 2a, 3b and 9c are the most potent, they exhibited cytotoxic effect against the six cancer cell lines with IC50 values < 330 nM compared to the standard CHS 828. Normal fibroblast cells (WI38) were affected to a much lesser extent (IC50 >10,000 nM). Toxicity of the most potent compounds was measured against shrimp larvae; the results showed that compounds 2a and 3b are not toxic towards the tested organisms.
Keywords: 4,5-dihydropyrimidine; 1,4-dihydropyrimidine; thiazolo[3,2-a]pyrimidine; pyrano[2,3-d]pyrimidine; anti-cancer activity
1. Introduction
Cancer is one of the most dreadful disease in the world and despite immense advances in the field of basic and clinical research, which have resulted in higher cure rates for a number of malignancies, cancer remains the second leading cause of death in developing as well as developed countries.1-3 Although chemotherapy is the mainstay of cancer therapy, the use of available chemothera-peutics is often limited mainly due to the undesirable side effects and this clearly underscores the need of developing novel chemotherapeutic agents for more effective cancer treatments.4
Among the wide range of compounds tested as potential anticancer agents, pyrimidine and fused pyrimidine derivatives comprise an important class of therapeutic agents. They were reported as antitumor,5-10 antimicrobial,1112 antiinflammatory,13 anti HIV,14 antinociceptive,1516 and antioxidant agents.1718 Various drugs containing pyrimidine nucleus were synthesized and used as an-
ticancer agents like 5-fluorouracil (5-FU), tegafur and thioguanine (Figure 1).19
In recent years dihydropyrimidinones/thiones (DHPMs) and their derivatives have occupied an important position in natural and synthetic organic chemistry because of their wide range of biological activities, such as antioxidant, antiinflammatory, anthelminic, antimicro-bial,20-22 antituberculoses23 and anticancer.24
Although a number of papers have been concerned with the synthesis of pyrimidine derivatives,1718 just a few one pot syntheses have been published using aromatic aldehydes, ethyl cyanoacetate and thiourea.11'20-24 Also, it was reported that 3,4-dihydropyrimidino-ne/thione have been synthesized by a three-component condensation of aldehydes, ethyl acetoacetate and urea/thiourea using different catalysts and new methods to improve and modify this reaction.25-29 Moreover, the one step synthesis of 6-amino-5-cyano-4-phenyl-2-mer-captopyrimidine using aldehydes, malononitrile and urea/thiourea in the presence of a catalytic amount of
Figure 1. Pyrimidine derivatives as anticancer agents.
phosphorus pentoxide has been reported.30 Therefore, in the view of the facts mentioned above and to discover potentially active new agents, we have synthesized some new dihydropyrimidine derivatives by a three-component condensation (MCR) of aromatic aldehydes, thi-ourea and either ethyl cyanoacetate, ethyl acetoacetate or malononitrile using triethylamine under refluxing condition. The cytotoxicity of the newly synthesized products was investigated against six cancer and one normal human cell lines.
2. Results and Discussion
2. 1. Chemistry
In the present work, we have developed an efficient method to generate the dihydropyrimidine derivatives 2a-d and oxopyrimido[5,4-c]quinoline 2e by the cycliza-tion of three components, like aryl aldehydes 1a-e, ethyl cyanoacetate and thiourea in ethanol in the presence of triethylamine in a one-pot reaction (Scheme 1). The structure assigned to the synthesized compounds was
Scheme 1. Reaction of 1a-e, ethyl cyanocetate and thiourea in absolute EtOH, Et3N, heat 3 h.
substantiated by their analytical and other spectral data. The IR spectrum of the synthesized compound 2a showed characteristic signals at 1691, 1685 cm-1 for two C=O, 2228 cm-1 for CN and 3450 cm-1 for NH2. Similarly the 1H NMR spectrum, showed the presence of a triplet at 5 1.28 ppm and a quartet at 5 4.31 ppm indicating the presence of the ester CH3 and CH2 groups respectively, beside the appearance of a singlet at 5 8.41 ppm corresponding to the presence of a CH-pyrimidine moiety, proving its structure.
The formation of the oxopyrimido[5,4-c]quinolin31 2e was proved by the presence of two stretching C=O signals at 1727, 1720 cm-1 and a CN signal at 2227 cm-1, beside the absence of any signal corresponding to the OH group either in the IR or the 1H NMR spectra. Moreover, 1H NMR spectrum showed the presence of the D2O exchangeable signal at 5 3.83 ppm corresponding to the NH group, the triplet and quartet signals at 5 1.29 and 4.29 ppm corresponding to the CH3 and CH2 groups, respectively, the CH pyrimidine signal at 5 8.95 ppm beside the SH signal at 5 10.75 ppm. In addition its 13C NMR spectrum revealed the presence of signals at 5 14.09 (CH3),
32.04 (CH-CN), 61.05 (CH2), 76.40 (C-5 pyrimidine), 112.02 (CN), 164.98, 169.8 (2 C=O). The appearance of a signal at 5 32.04 ppm in the 13C NMR spectrum corresponding to the CH-CN proved the formation of the tautomeric form 2e'.
It was reported that the three-component reaction of aromatic aldehydes, ethyl cyanoacetate and thiourea in ethanol using potassium carbonate afforded 6-oxo-4-sub-stituted aryl-2- sulfanyl-1,6- dihydropyrimidine-5- car-bonitrile.11,20
Thiazolo[3,2-a]pyrimidine 3b and pyrano[2,3-d]pyrimidines32 4b, 5b (Scheme 2) were obtained from the reaction of 4,5-dihydropyrimidine derivative 2b with ra-bromo-4-chloroacetophenone, hydrazine hydrate and phenyl hydrazine, respectively. The structure of these compounds was elucidated by IR, 1H and 13C NMR spectra beside their analytical and mass spectral data. Thus, the IR spectrum of 3b showed the absence of any CN signal beside the expected signals. Meanwhile, 1H NMR spectrum showed a singlet at 5 6.09 ppm for CH of the thiazole ring with the disappearance of signals corresponding to SH and pyrimidine H-5. At the same time 13C
Scheme 2. Reactions of 4,5-dihydropyrimidine 2b and reaction of 4b with ethyl cyanoacetate. Reagents and conditions; (i) 4-ClC6H4COCH2Br, absolute EtOH, Et3N, heat 3 h; (ii) NH2NH2.H2O, absolute EtOH, heat 2 h; (iii) PhNHNH2, absolute EtOH, heat 2 h; (iv) ethyl cyanoacetate, DMF, heat 4 h.
NMR spectrum exhibited signals at 5 25.70, 53.25 (ethyl group), 96.07 (CH thiazole), 158.45, 159.97 and 162.15 (3 C=O). The IR spectra of pyrano[2,3-d]pyrimidines 4b, 5b showed the presence of only one stretching C=O signal at 1736 and 1744 cm-1, respectively. Similarly, their 1H NMR spectra showed the absence of any signals corresponding to ethyl ester, SH, H-5 pyrimidine beside the appearance of signals corresponding to H-4 and H-5 pyrimi-dine within the aromatic range. Compound 4b exhibited the presence of two singlets for the two NH2 at 5 3.73 and 8.69 ppm and a singlet for NH at 5 13.12 ppm (D2O exchangeable). In addition, for 5b there is a singlet appearing at 5 3.73 ppm indicating the presence of the NH2 and the two singlets at 5 6.73, 10.40 (D2O exchangeable) for the two NH groups of the -NHNHPh moiety. Moreover, the formation of pyrano[2,3-d]pyrimidines 4b and 5b was confirmed by their 13C NMR spectra. 13C NMR spectrum of 4b showed signals at 5 65.29, 67.12 (pyrimidine C-4 and C-5), 113.43 (CN), 160.28 (C=O). At the same time
13C NMR spectrum of 5b showed signals at 5 65.29, 67.4 (pyrimidine C-4 and C-5), 113.43 (CN), 160.28 (C=O).
On the other hand, the hydrazino moiety present in 4b showed high activity towards cyanomethylene reagents. Thus, its reaction with ethyl cyanoacetate in di-methylformamide solution gave the pyrazole derivative33 6b. The IR spectrum of 6b showed beside the former data the presence of the two C=O at 1791, 1683 cm-1 in addition to the presence of three signals corresponding to two NH2 groups at 5 3.79, 13.16 ppm and one NH at 8.70 ppm in the 1H NMR spectrum. Moreover, the 13C NMR spectrum showed beside the expected signals, a signal at 5 96.67 ppm corresponding to the pyrazole CH group, the presence of two CO and two C=N groups at 5 161.95, 162.15, 164.04, 166.38 ppm, respectively.
The reaction of the arylaldehydes 1a-e, ethyl ace-toacetate and thiourea in 1,4 dioxane using triethylamine as the catalyst afforded 1,4-dihydromercaptopyrimidines 7a-d and chromeno[4,3-d]pyrimidine 7e (Scheme 3). The
Scheme 3. Reaction of 1a-e, ethyl acetoacetate and thiourea and reaction of 7c with 4-ClC6H4COCH2Br. Reagents and conditions; (i) 1,4-dioxane, Et3N, heat 6-8 h; (ii) absolute EtOH, Et3N, heat 1 h.
1H NMR spectrum of 7a showed beside the expected signals the presence of singlets at 5 5.17, 9.6 and 10.29 ppm corresponding to the presence of CH pyrimidine, NH and SH, respectively. In our work, the pyrimidine derivative exists in the thiol form and not in the thione form as it was reported.25-29 This was confirmed through the reaction of the pyrimidine derivative 7c with a-halocarbonyl compounds. Thus, reaction of 7c with ra-bromo-4-chloroace-tophenone afforded thiazolo[3,2-a]pyrimidine 8 (Scheme 3). The 1H NMR spectrum of 8 showed the absence of SH and NH signals with the presence of CH-thiazole singlet at 5 7.15 ppm.
The structure of chromeno[4,3-d]pyrimidine 7e was elucidated on the basis of its IR spectrum which showed the absence of OH signal beside the 1H NMR spectrum which revealed the absence of both OH and COOE-t signals. Moreover, the 13C NMR spectrum of 7e showed 5 at 26.06 (CH3), 64.56 (CH pyrimidine) and 160.28 (C=O).
Condensation of each of the arylaldehydes 1a-e with the malononitrile and thiourea in ethanol in the presence of a catalytic amount of triethylamine produced 1,4-dihydromercaptopyrimidines 9a-d and chromeno[4,3-d]pyrimidine 9e (Scheme 4).
The structure of these compounds was based on their IR and 1H NMR spectra. Thus, the 1H NMR spectrum of 9a showed the presence of singlets at 5 7.90 and 10.14 ppm corresponding to the presence of NH and SH, respectively, beside the presence of a multiplet at 5 7.15-7.64 ppm corresponding to the aromatic protons and one pyri-midine proton. In our work we obtained the 1,4-dihydrop-yrimidine derivatives instead of pyrimidine derivatives as reported.30 The structure of 1,4-dihydropyrimidine 9a-e was confirmed by the reaction of 9c with ю-bromoacetop-henone to afford thiazolo[3,2-a]pyrimidine 10. The 1H NMR spectrum of 10 showed beside the expected signals the absence of any SH and NH with the presence of a singlet at 5 5.21 ppm corresponding to the presence of CH-
9a, R=C6Hä; 9b, R=4-C1C6H4; 9c, R=4-OCH3C6H4; 9d, R=2-Furyl
Scheme 4. Reaction of 1a-e, malononitrile and thiourea and reaction of 9c with C6H5COCH2Br. Reagents and conditions; (i) absolute EtOH, Et3N, heat 2 h; (ii) absolute EtOH, Et3N, heat 2 h.
thiazole. The formation of chromeno[4,3-d]pyrimidine derivative 9e was proved by the absence of any CN and OH signals in the IR spectrum beside the presence of a singlet at 5 6.96 ppm corresponding to the presence of two NH2 in the 1H NMR spectrum. Moreover, 13C NMR spectrum of 9e showed signal at 5 61.05 ppm (CH pyrimidine).
2. 2. In vitro Cytotoxicity
2. 2. 1. Effect on the Growth of Human Cancer Cell Lines
The heterocyclic compounds prepared in this study were evaluated according to standard protocols for their in vitro cytotoxicity against six human cancer cell lines including cells derived from human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), nasopharyngeal carcinoma (HONE1), human breast cancer (MCF) and normal fibroblast cells (WI38). For comparison, CHS 828 was used as a standard antitumor drug. The IC50 values (the sample concentration that produces 50% reduction in cell growth) were in nano-molar (nM) range and are listed in Table 1. Nine of the tested compounds showed cytotoxic activity with IC50 values < 650 nM and the results are represented in Figures 2-4. Compounds 2a, 3b, 9c were found to be the most potent derivatives. All the synthesized compounds were tested for their cytotoxicity against normal fibroblast cells as many anticancer drugs are toxic not only against cancer cells but also normal ones. The results obtained showed that normal fibroblast cells (WI38) were affected to a much lesser extent (IC50 >10,000 nM).
2. 2. 2. Structure Activity Relationship
In this study when comparing the structures of the synthesized compounds with their anticancer activity it has been observed that nine of the tested compounds, namely: 2a, 2c, 2d, 3b, 4b, 5b, 8, 9a and 9c showed significant broad cytotoxic effect with IC50 values < 650 nM. Normal
fibroblast cells (WI38) were affected to a much lesser ex-
tent (IC50 >10,000 nM). Considering the 4,5-dihydromer-captopyrimidine derivatives 2a-e, the unsubstituted derivative 2a showed high potency against the six cancer cell lines. It exhibited potent activity against gastric cancer NUGC (IC50 89 nM), colon cancer DLDI (IC50 49 nM) and liver cancer HA22T (IC50 64 nM). Compounds 2c and 2d displayed cytotoxicity against three cancer cell lines. Compound 2c bearing the 4-methoxy moiety was toxic against colon DLD1 (IC50 188 nM), liver cancer HEPG2 (IC50102 nM) and breast cancer MCF (IC50 239 nM), while 2d, substituted by a furan group, showed cytotoxic activity against gastric cancer NUGC (IC50 228 nM), colon cancer DLD1 (IC50 126 nM) and nasopharyngeal carcinoma HONE1 (IC50 1 38 nM). Moreover, 2b and 2e demonstrated selective toxicity against gastric cancer NUGC with IC50 323 and 128 nM, respectively. The cytotoxicity of compounds 2a-e
might be attributed to the presence of cyanoaminopyrimi-dine moiety.34 Comparing the cytotoxicity of the mercap-topyrimidine derivative 2b and its cyclized product the thiazolo[3,2-a]pyrimidine derivative 3b, it is obvious that 3b possessed potential cytotoxic effect to all cancer cell lines. It demonstrated significant cytotoxicity against gastric cancer NUGC (IC50 3 8 nM) compared to the standard CHS 828 (IC50 25 nM).5
Pyrano[2,3-d]pyrimidines 4b, 5b obtained by the reaction of 2b with hydrazine hydrate and phenyl hydrazi-ne, respectively, showed remarkable increase of the toxicity against most cancer cell lines. Such increase in toxicity can be attributed to the presence of the hydrazino moiety.35
The 1,4-dihydropyrimidine derivatives 7a-d are almost devoid of activity. However, compound 7d substituted by a furan ring was the only active compound, it showed selective cytotoxic activity against nasopharyngeal carcinoma HONE1 with (IC50 44 nM) compared to the standard CHS 828 (IC5015 nM). Thiazolo[3,2-a]pyrimidi-ne 8 obtained by the reaction of 7c with ra-bromo-4-chlo-roacetophenone exhibited significant cytotoxic activity against gastric cancer NUGC (IC50 226 nM), colon cancer DLD1 (IC50 254 nM) and liver cancer HA22T (IC50 94 nM). On the other hand, among the1,4-dihydropyrimidine derivatives 9a-d, the 4-methoxy derivative 9c possessed potent cytotoxic activity against the six cancer cell lines. The latter compound showed cytotoxicity against gastric cancer NUGC (IC50 3 8 nM), colon cancer DLD1 (IC50 3 5 nM), liver cancer HA22T (IC50 98 nM) and breast cancer MCF (IC50 103 nM). Moreover, the unsubstituted derivative 9a was toxic against liver cancer HEPG2, nasopharyn-geal carcinoma cancer HONE1 and breast cancer MCF cell lines, while the furano derivative 9d was toxic only against liver cancer HEPG2 (IC50 128 nM) cell line.36
Thus, even though some of the compounds were not the most potent, their specific activity against particular cell lines makes them of interest for further development as anticancer drugs.
2. 2. 3. Toxicity
Bioactive compounds are often toxic to shrimp larvae. In order to establish in vivo lethality of these cytoto-xic compounds to shrimp larvae (Artemia salina), Brine-Shrimp Lethality Assay37 was used. Results were analysed with LC50 program to determine LC50 values and 95% confidence intervals.38 Results for the compounds which exhibited optimal cytotoxic effect against cancer cell lines (i.e. 2a, 3b, 5b, 7d and 9c) are given in Table 2. The shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity and it has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity testing of dental materials,39 natural and synthetic organic compounds.37 It has also been shown that A. salina toxi-city test results have a correlation with rodent and human
acute oral toxicity data. Generally, a good correlation was obtained between A. salina toxicity test and the rodent data. Likewise, the predictive screening potential of the aquatic invertebrate tests for acute oral toxicity in man,
including A. salina toxicity test, was slightly better than the rat test for the tested compounds.40
In order to prevent the toxicity results from possible false effects originating from solubility of compounds and
Table 1. Cytotoxicity of compounds 2a-e, 3b, 4b, 5b, 6b, 7a-e, 8, 9a-e and 10 against a variety of cancer cell lines a [IC50b (nM)]
Compound			Cytotoxicity (IC50 in nM)				
No.	NUGC	DLDI	HA22T	HEPG2	HONE1	MCF	WI38
2a	89	49	64	238	272	328	NA
2b	323	3129	1155	2424	1242	1252	NA
2c	2179	188	2120	102	2380	239	NA
2d	228	126	1248	2269	138	1170	NA
2e	128	2115	3180	2312	1284	3260	1277
3b	38	364	175	122	206	274	NA
4b	328	608	282	523	201	348	NA
5b	55	146	1413	263	536	285	222
6b	2208	2128	2153	1236	1138	2240	NA
7a	2166	4840	1320	3266	4385	1428	NA
7b	1214	1640	2183	2809	2266	2285	NA
7c	2470	2482	1226	1483	3576	2328	1068
7d	1220	1240	1203	1237	44	2119	NA
7e	1029	2294	2069	2244	3327	2436	NA
8	266	254	94	1679	2114	1277	3679
9a	1242	2140	3220	634	428	256	NA
9b	2213	2146	2120	2110	3290	1368	NA
9c	38	35	98	308	210	103	NA
9d	1122	3224	24241	128	11220	2136	NA
9e	2253	2690	2166	3309	2213	2318	NA
10	2244	1770	3148	2182	1880	1290	1089
CHS 828	25	2315	2067	1245	15	18	NA
a NUGC: gastric cancer; DLDI: colon cancer; HA22T: liver cancer; HEPG2: liver cancer; HONE1: nasopharyngeal carcinoma; MCF: breast cancer; WI38: normal fibroblast cells. b The sample concentration produces a 50% reduction in cell growth
Figure 2. Cytotoxicity of 2a-e and CHS 828 against NUGC (gastric cancer); DLDI (colon cancer); HA22T (liver cancer); HEPG2 (liver cancer); HONE1 (nasopharyngeal carcinoma); MCF (breast cancer).
Figure 3. Cytotoxicity of 3b, 4b, 5b and CHS 828 against NUGC (gastric cancer); DLDI (colon cancer); HA22T (liver cancer); HEPG2 (liver cancer); HONE1 (nasopharyngeal carcinoma); MCF (breast cancer).
Figure 4. Cytotoxicity of 7d, 8, 9a, 9c, 9d and CHS 828 against NUGC (gastric cancer); DLDI (colon cancer); HA22T (liver cancer); HEPG2 (liver cancer); HONE1 (nasopharyngeal carcinoma); MCF (breast cancer).
DMSO's possible toxicity effect, compounds were prepared by dissolving in DMSO in the suggested DMSO volume ranges. It is clear from Table 2 that compounds 2b and 3b showed no toxicity against the tested organisms. On the other hand, compound 5b is very toxic, in addition, compounds 7b, 9c are harmful.
3. Experimental
3. 1. Chemistry
All melting points were determined on a Stuart apparatus and the values given are uncorrected. IR spectra
(KBr, cm-1) were determined on a Shimadzu IR 435 spectrophotometer (Faculty of Science, Cairo University, Egypt). 1H NMR spectra were recorded on Varian Gemini 300 MHz (Microanalysis Center, Cairo University, Egypt) using TMS as the internal standard. Chemical shift values are recorded in ppm on 5 scale. Mass spectra were recorded on a Hewlett Packard 5988 spectrometer (Microa-nalysis Center, Cairo University, Egypt). Elemental analyses were carried out at the Microanalysis Center, Cairo University, Egypt; found values were within ±0.35% of the theoretical ones. Progress of the reactions was monitored using thin layer chromatography (TLC) sheets coated with UV fluorescent silica gel Merck 60F 254 and were visualized using UV lamp.
General Procedure for the Synthesis of 2a-e
The dihydropyrimidines 2a-e were synthesized by reflu-xing the aprropriate arylaldehyde 1a-e (0.01 mol), ethyl cyanoacetate (1.13 g, 0.01 mol) and thiourea (0.76 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (1.0 mL) for 3 h. The solution was left to cool to room temperature, poured onto ice/water and neutralized with hydrochloric acid. The precipitated solid was filtered off, washed with cold water and crystallized from ethanol.
Ethyl 3-Amino-2-cyano-3-(2-mercapto-4-oxo-6-phenyl-4,5-dihydropyrimidin-5-yl)acrylate (2a). Yield: 65%; m.p.: 296-298 °C; IR (KBr, cm-1): 3450-3320 (NH2, NH), 3082 (CH, aromatic), 2228 (CN), 1691-1685 (2 C=O); 1H NMR (DMSO-d6): 5 1.28 (t, 3H, J = 7.2 Hz, CH2-CH3), 3.89 (s, 2H, NH2,D2O exchangeable), 4.31 (q, 2H, J = 7.2 Hz, CH2-CH3), 7.542-8.07 (m, 5H, CH aromatic), 8.41 (s, 1H, pyrimidine H-5), 13.17 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 342 (M+, 42). Anal. Calcd. for C16H14N4O3S: C, 56.13; H, 4.12; N, 16.36; S, 9.37. Found: C, 564.43; H, 4.46; N, 16.72; S, 9.59.
Table 2. LC50 of compounds 2a, 3b, 5b, 7d and 9c against shrimp larvae
Compound No.	Cons. (^g/mL)	Mortality3	Toxicity	LC50	Upper 95% lim.	Lower 95% lim.
2a	10	0	Non toxic	996.27	-	-
	100	0				
	1000	5				
3b	10	0	Non toxic	880.42	-	-
	100	1				
	1000	4				
5b	10	1	Very toxic	18.38	-	-
	100	6				
	1000	10				
7d	10	0	Harmful	22.7	210.59	160.22
	100	6				
	1000	8				
9c	10	0	Harmful	420.28	112.23	90.55
	100	5				
	1000	10				
a Ten organisms (A. salina) tested for each concentration
Ethyl 3-Amino-3-(6-(4-chlorophenyl)-2-mercapto-4-oxo-4,5-dihydropyrimidin-5-yl)-2-cyanoacrylate (2b). Yield: 55%; m.p.: 155-157 °C; IR (KBr, cm-1): 3431-3321 (NH2,NH), 3075 (CH, aromatic), 2221 (CN), 1725-1718 (2 C=O); 1H NMR (DMSO-d6): 5 1.28 (t, 3H, J = 7.2 Hz, CH2-CH3), 3.86 (s, 2H, NH2, D2O exchangeable), 4.31 (q, 2H, J = 7.2 Hz, CH2-CH3), 7.67-8.08 (m, 4H, CH aromatic), 8.41 (s, 1H, pyrimidine H-5), 13.09 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 376 (M+, 34). Anal. Calcd. for C16H13ClN4O3S: C, 51.00; H, 3.48; N, 14.87; S, 8.51. Found: C, 50.67; H, 3.33; N, 14.62; S, 8.73.
Ethyl 3-Amino-2-cyano-3-(-2-mercapto-6-(4-methox-yphenyl)-4-oxo-4,5-dihydropyrimidin-5-yl)acrylate
(2c). Yield: 63%; m.p.: 116-118 °C; IR (KBr, cm-1): 3418-3324 (NH2,NH), 3094 (CH, aromatic), 2212 (CN), 1716-1710 (2 C=O); 1H NMR (DMSO-d6): 5 1.27 (t, 3H, J = 7.2 Hz, CH2-CH3), 3.83 (s, 3H, OCH3), 3.86 (s, 2H, NH2, D2O exchangeable), 4.25 (q, 2H, J = 7.2 Hz, CH2-CH3), 7.13-8.09 (m, 4H, CH aromatic), 8.30 (s, 1H, pyrimidine H-5), 13.10 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 372 (M+, 21). Anal. Calcd2 for C17H16N4O4S: C, 54.83; H, 4.33; N, 15.04; S, 8.61. Found: C, 54.69; H, 4.22; N, 15.37; S, 8.93
Ethyl 3-Amino-2-cyano-3-(6-(furan-2-yl)-2-mercapto-4-oxo-4,5-dihydropyrimidin-5-yl)acrylate (2d). Yield: 59%; m.p.: 96-98 °C; IR (KBr, cm-1): 3421-3329 (NH2, NH), 3040 (CH, aromatic), 2221 (CN), 1719-1712 (2 C=O); 1H NMR (DMSO-d6): 5 1.32 (t, 3H, J = 7.2 Hz, CH2-CH3), 3.89 (s, 2H, NH2,D2O exchangeable), 4.31 (q, 2H, J = 7.2 Hz, CH2-CH3), 6.92- 8.21 (m, 3H, CH furan), 8.27 (s, 1H, pyrimidine H-5), 13.14 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 332 (M+, 62). Anal. Calcd. for C14H12N4O4S: C, 50.60; H, 3.64; N, 16.86; S, 9.65. Found: C, 50.38; H, 3.32; N, 16.69; S, 9.95.
Ethyl 2-Cyano-2-(2-mercapto-4-oxo-4,4a-dihydropyri-mido[5,4-c]quinolin-5(6H)-ylidene)acetate (2e). Yield: 62%; m.p.: 185-187 °C; IR (KBr, cm-1): 3436 (NH), 3043 (CH, aromatic), 2227 (CN), 1727-1720 (2 C=O); 1H NMR (DMSO-d6): 5 1.29 (t, 3H, J = 7.2 Hz, CH2-CH3), 3.83 (s, 1H, NH, D2O exchangeable), 4.29 (q, 2H, J = 7.2 Hz, CH2-CH3), 7.41-7.93 (m, 4H, CH aromatic), 8.95 (s, 1H, pyrimidine H-5), 10.75 (s, 1H, SH, D2O exchangeable); 13C HMR (DMSO): 5 14.09, 32.04, 6L05, 76.40, 112.02, 126.70, 129.25, 130.43, 132.09, 134.80, 162.04, 164.98, 169.8, 172.75, 173.40; MS: m/z (%) 340 (M+, 24). Anal. Calcd. for C16H12N4O3S: C, 56.46; H, 3.55; N, 16.46; S, 9.42. Found: C, 56.(46; H, 3.29; N, 16.77; S, 9.29.
Synthesis of Ethyl 4-Amino-2-(3,5-bis(4-chlorophenyl) -7-oxo-7_ff-thiazolo[3,2-a]pyrimidin-6-yl)-5-(4-chloro-benzoyl)-1ff-pyrrole-3-carboxylate (3b)
A solution of 4,5-dihydropyrimidine 2b (2.63 g, 0.01 mol) and ra-bromo-4-chloroacetophenone (2.34 g, 0.01 mol) in
absolute ethanol (40 mL) containing triethylamine (1.0 mL) was heated under reflux for 3 h, left to cool to room temperature, poured onto ice/water and neutralized by hydrochloric acid. The precipitated solid was filtered, washed with water and crystalized from ethanol.
Yield: 75%; m.p.: 255-257 °C; IR (KBr, cm-1): 3439-3307 (NH2, NH), 3046 (CH, aromatic), 1743-1665 (3 C=O); 1H NMR (DMSO-d6): 5 0.91 (t, 3H, J = 7.2 Hz, CH2-CH3), 3.49 (s, 2H, NH2, D2O exchangeable), 4.20 (q, 2H, J = 7.2 Hz, CH2-CH3), 6Ю9 (s, 1H, CH thiazole), 7.26-8.21 (m, 13H, 12H aromatic and 1H, NH, D2O exchangeable); 13C HMR (DMSO): 5 25.70, 53.25, 96.027,
114.04,	120.56, 123.09, 124.69, 129.20, 129.53, 131.95,
132.05,	133.14, 134.49, 138.3, 139.22, 140.33, 158.45, 159.97, 162.15, 164.50, 166.39; MS: m/z (%) 663 (M+, 51). Anal. Calcd. for C32H2lCl3N4O4S: C, 57.89; H, 3.19; N, 8.44; S, 4.83. Found: C 5X55; H, 3.09; N, 8.09; S, 4.64.
General Procedure for the Synthesis of Pyrano[2,3-d] pyrimidine Derivatives 4b, 5b. A mixture of 4,5-dihydropyrimidine 2b (2.63 g, 0.01 mol) with either of hydrazine hydrate (0.05 g, 0.01 mol) or phenyl hydrazine (1.08 g, 0.01 mol) in absolute ethanol (40 mL), was heated under reflux for 2 h. The solid was precipitated by cooling to room temperature, filtered off, and crystalized from ethanol.
5-Amino-4-(4-chlorophenyl)-2-hydrazinyl-7-oxo-4a,7-dihydro-4_ff-pyrano[2,3-d]pyrimidine-6-carbonitrile
(4b). Yield: 73%; m.p.: 265-267 °C; IR (KBr, cm-1): 3483-3200 (2 NH2, NH), 3094 (CH, aromatic), 2225 (CN), 1736 (C=O); 1H NMR (DMSO-d6): 5 3.73 (s, 2H, NH2, D2O exchangeable), 7.20-7.91 (m, 6H, 4H aromatic and 2H pyrimidine), 8.69 (s, 2H, NH2, D2O exchangeable), 13.12 (s, 1H, NH, D2O exchangeable); 13C HMR (DMSO): 5 65.29, 67.12, 113.43, 121.38, 127.73, 129.47, 130.59, 148.93, 150.74, 160.28, 163.19, 164.46; MS: m/z (%) 330 (M+, 11). Anal. Calcd. for C14HnClN6O2: C, 50.84; H, 3.35; N, 25.41. Found: C, 50.53; H, 3.09; NN, 25.09.
5-Amino-4-(4-chlorophenyl)-7-oxo-2-(2-Phenylhydra-zinyl)-4a,7-dihydro-4_ff-pyrano[2,3-d]pyrimidine-6-carbonitrile (5b). Yield: 76%; m.p.: 149-151°C; IR (KBr, cm-1): 3457-3307 (NH2, 2 NH), 3092 (CH, aromatic), 2260 (CN), 1744 (C=O); 1H NMR (DMSO-d6): 5 3.73 (s, 2H, NH2, D2O exchangeable), 6.73-7.67 (m, 11H, 9H aromatic and 2H pyrimidine), 7.84 (s, 1H, NH, D2O exchangeable), 10.40 (s, 1H, NH, D2O exchangeable); 13C HMR (DMSO): 5 65.29, 67.03, 113.43, 118.12, 121.01, 123.37, 124.22, 127.73, 129.47, 130.59, 140.02, 148.93, 150.74, 160.28, 163.23, 164.46; MS: m/z (%) 406 (M+, 13). Anal. Calcd. for C20H15ClN6O2: C, 59.05; H, 3.72; N, 20.66. Found: C, 59.36; H, 3.87; N, 20.82.
Synthesis of 5-Amino-2-(3-amino-5-oxo-2,5-dihydro-1_ff-pyrazol-1-yl)-4-(4-chlorophenyl)-7-oxo-4a,7-dihy-
dro-4.ff-pyrano[2,3-d]pyrimidine-6-carbonitrile (6b). A mixture of pyrano[2,3-d]pyrimidine 4b (3.3 g, 0.01 mol) and ethyl cyanoacetate (1.13 g, 0.01 mol) in dimethyl formamide (20 mL) was heated under reflux for 4 h. The solution was left to cool, poured onto ice/water, the solid product was collected by filtration and crystalized from EtOH/DMF.
Yield: 40%; m.p.: 256-258 °C; IR (KBr, cm-1): 3446-3192 (2 NH2, NH), 3090 (CH, aromatic), 2223 (CN), 1791-1683 (2 C=O); 1H NMR (DMSO-d6): 5 3.79 (s, 2H, NH2, D2O exchangeable), 7.39-7.91 (m6, 7H, 4H aromatic, 2H pyrimidine and 1H pyrazole H-4), 8.70 (s, 1H, NH, D2O exchangeable), 13.16 (s, 2H, NH2, D2O exchangeable); 13C HMR (DMSO): 5 66.01, 66.12, 96.07, 114.06, 116.07, 120.56, 124.69, 129.20, 133.14, 138.02, 140.33, 161.95, 162.15, 164.04, 166.38; MS: m/z (%) 397 (M+, 65). Anal. Calcd. for C17H12 ClN7O3: C, 51.33; H, 3.04; N, 24.65 Found: C, 51.09; H, 3.1; N, 24.79.
General Procedure for the Synthesis of 7a-e
A solution of the appropriate arylaldehyde 1a-e (0.01 mol), ethyl acetoacetate (1.3 g, 0.01 mol) and thiourea (0.76 g, 0.01 mol) in 1,4-dioxan (40 mL) containing triethylamine (1.0 mL) was heated under reflux for 6-8 h, then left to cool. The solid product formed upon pouring onto ice/water containing few drops of hydrochloric acid was collected by filtration and crystallized from ethanol.
Ethyl 2-Mercapto-6-methyl-4-phenyl-1,4-dihydropyri-midine-5-carboxylate (7a). Yield: 81%; m.p.: 275-277 °C; IR (KBr, cm-1): 3325 (NH), 3090 (CH, aromatic), 2982 (CH, aliphatic), 1670 (C=O); 1H NMR (DMSO-d6): 5 1.07 (t, 3H, J = 7.2 Hz, CH2-CH3), 2.28 (s, 3H, CH3), 3.97 (q, 2H, J = 7.2 Hz, CH2-CH3), 5.17 (s, 1H, pyrimidine H-4), 7.20-7.37 (m, 5H2 CH aromatic), 9.60 (s, 1H, NH, D2O exchangeable), 10.29 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 276 (M+, 27). Anal. Calcd. for C14H16N2O2S: C, , 60.85; H, 5.84; N, 10.14; S, 11.60. Found: C, 60.68; H, 5.99; N, 10.38; S, 11.89.
Ethyl 4-(4-Chlorophenyl)-2-mercapto-6-methyl-1,4-dihydropyrimidine-5-carboxylate (7b). Yield: 85%; m.p.: 210-212 °C; IR (KBr, cm-1): 3302 (NH), 3094 (CH, aromatic), 2982 (CH, aliphatic), 1731 (C=O); 1H NMR (DMSO-d6): 5 0.94 (t, 3H, J = 7.2 Hz, CH2-CH3), 2.30 (s, 3H, CH3), 3.91 (q, 2H, J = 7.2 Hz, CH2-CH3), 6.11 (s, 1H, pyrimidine H-4), 7.30-7.40 (m, 4H, CH aromatic), 8.38 (s, 1H, NH, D2O exchangeable), 8.74 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 310 (M+, 41). Anal. Calcd. for C14H15ClN2O2S: C, 54.10; H, 4.86; N, 9.01; S, 10.32. Found: C, 53.88; H, 4.99; N, 9.35; S, 10.03.
Ethyl 2-Mercapto-4-(4-methoxyphenyl)-6-methyl-1,4-dihydropyrimidine-5-carboxylate (7c). Yield: 82%; m.p. > 300 °C; IR (KBr, cm-1): 3380 (NH), 3087 (CH, aromatic), 2983 (CH, aliphatic), 1726 (C=O); 1H NMR
(DMSO-d6): 5 0.90 (t, 3H, J = 7.2 Hz, CH2-CH3), 2.12 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 3.85 (q, 2H, J = 7.2 Hz, CH2-CH3), 5.73 (s, 1H, pyrimidine H-4), 6.86-7.25 (m, 4H,2CH aromatic), 9.92 (s, 1H, NH, D2O exchangeable), 12.33 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 306 (M+, 16). Anal. Calcd. for C15H18N2O3S: C, 58.80; H, 5.92; N, 9.14; S, 10.47. Found: C, 58.93; H, 5.61; N, 9.29; S, 10.09.
Ethyl 4-(Furan-2-yl)-2-mercapto-6-methyl-1,4-dihy-dropyrimidine-5-carboxylate (7d). Yield: 86%; m.p.: 200-202 °C; IR (KBr, cm-1): 3336 (NH), 3089 (CH, aromatic), 2979 (CH, aliphatic), 1723 (C=O); 1H NMR (DMSO-d6): 5 1.03 (t, 3H, J = 7.2 Hz, CH2-CH3), 2.23 (s, 3H, CH3), 3.95 (q, 2H, J = 7.2 Hz, CH2-CH3), 630 (s, 1H, pyrimidine H-4), 6.33-7.71 (m, 3H, CH furan), 10.25 (s,
IH,	NH, D2O exchangeable), 10.58 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 266 (M+, 54). Anal. Calcd. for C12H14N2O3S: C, 54.12; H, 5.30; N, 10.52; S, 12.04. Found: C, 532.92; H, 5.09; N, 10.35; S, 11.98.
2-Mercapto-4-methyl-3.ff-chromeno[4,3-d]pyrimidin-5(10b#)-one (7e). Yield 84%; m.p.: 290-292 °C; IR (KBr, cm-1): 3381 (NH), 3088 (CH, aromatic), 2981 (CH, aliphatic), 1722 (C=O); 1H NMR (DMSO-d6): 5 2.49 (s, 3H, CH3), 6.98 (s, 1H, pyrimidine H-4), 7.41-8.40 (m, 4H, aromatic), 8.58 (s, 1H, NH, D2O exchangeable),
II.19	(s, 1H, SH, D2O exchangeable); 13C HMR (DMSO): 5 26.04, 64.56, 1132.43, 121.38, 123.37, 127.73, 129.47, 130.59, 148.93, 150.74, 160.28, 164.46; MS: m/z (%) 246 (M+, 20). Anal. Calcd. for C12H10N2O2S: C, 58.52; H, 4.09; N, 11.37; S, 13.02. Found: C, 58.28; H, 4.43; N, 11.09; S, 12.89.
Synthesis of Ethyl 3-(4-Chlorophenyl)-7-(4-methoxyp-henyl)-5-methyl-7.ff-thiazolo[3,2-a]pyrimidine-6-car-boxylate (8).
A mixture of 1,4-dihydropyrimidine 7c (3.06 g, 0.01 mol) with ra-bromo-4-chloroacetophenone (2.34 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (1.0 mL) was heated under reflux for 1 h, left to cool to room temperature, poured onto ice/water, and neutralized by hydrochloric acid. The precipitated solid was filtered, washed with water and crystalized from ethanol.
Yield: 65%; m.p. > 300 °C; IR (KBr, cm-1): 3022 (CH, aromatic), 2932 (CH, aliphatic), 1702 (C=O); 1H NMR (DMSO-d6): 5 1.15 (t, 3H, J = 7.2 Hz, CH2-CH3), 2.06 (s, 3H, CH3), 3.66 (s, 3H, OCH3), 3.75 (q, 2H, J = 7".2 Hz, CH2-CH3), 5.67 (s, 1H, CH pyrimidine), 7.15 (s, 1H, CH thiazole), 7.43-8.58 (m, 8H, CH aromatic); MS: m/z (%): 440 (M+, 100). Anal. Calcd. for C23H21ClN2O3S: C, 62.65; H, 4.80; N, 6.35; S, 7.27. Found: C, 62.33; H, 4.54; N, 6.12; S, 6.99.
General Procedure for the Synthesis of 9a-e
A mixture of the appropriate arylaldehyde 1a-e (0.1 mol),
malononitrile (0.66 g, 0.1 mol) and thiourea (0.76 g, 0.01 mol) were heated under reflux in ethanol (40 mL) containing triethylamine (1.0 mL) for 3 h. The reaction mixture was left to cool, poured onto ice water and neutralized by hydrochloric acid. The solid product was precipitated, filtered, washed with water, and crystalized from ethanol.
6-Amino-2-mercapto-4-phenyl-1,4-dihydropyrimidine-
5-carbonitrile	(9a). Yield: 87%; m.p.: 150-152 °C; IR (KBr, cm-1): 3444-3353 (NH2, NH), 3063 (CH, aromatic), 2191 (CN), 1633 (C=N); 1H NMR (DMSO-d6): 5 6.82 (s, 2H, NH2), 7.15-7.64 (m, 6H, 5H aromatic and pyrimidine H-4), 7.90 (s, 1H, NH, D2O exchangeable), 10.14 (s, 1H, SH, D2O exchangeable); IMS: m/z (%) 230 (M+, 35). Anal. Calcd. for C11H10N4S: C, 57.37; H, 4.38; N, 24.33; S, 13.92. Found: C, 57.09; H, 4.41; N, 24.68; S, 13.79.
6-Amino-4-(4-chlorophenyl)-2-mercapto-1,4-dihy-dropyrimidine-5-carbonitrile (9b). Yield: 82%; m.p.: 207-209 °C; IR (KBr, cm-1): 3380-3258 (NH2, NH), 3071 (CH, aromatic), 2190 (CN), 1628 (C=N); 1H NMR (DMSO-d6): 5 6.80 (s, 2H, NH2), 7.12-7.64 (m, 5H, 4H aromatic and pyrimidine H-4), 7.84 (s, 1H, NH, D2O exchangeable), 10.17 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 264 (M+, 29). Anal. Calcd. for C11H9ClN4S: C, 49.91; H, 3.43; N, 21.16; S, 12.11. Found: C, 49.93; H, 3.08; N, 21.42; S, 12.34.
6-Amino-2-mercapto-4-(4-methoxyphenyl)-1,4-dihy-dropyrimidine-5-carbonitrile (9c). Yield: 88%; m.p.: 158-160 °C; IR (KBr, cm-1): 3439-3367 (NH2, NH), 3073 (CH, aromatic), 2219 (CN), 1605 (C=N); 1H NMR (DMSO-d6): 5 3.94 (s, 3H, OCH3), 7.23 (s, 2H, NH2), 7.24-8.05 (m, 5H, 4H aromatic and pyrimidine H-4), 8.45 (s, 1H, NH, D2O exchangeable), 10.12 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 260 (M+, 11). Anal. Calcd. for C12H12N4OS: C, 55.37; H, 4.65; N, 21.52; S, 12.32. Found2 C, 554.59; H, 4.39; N, 21.55; S, 12.34.
6-Amino-4-(furan-2-yl)-2-mercapto-1,4-dihydropyrimi-dine-5-carbonitrile (9d). Yield 81%; m.p.: 280-282 °C; IR (KBr, cm-1): 3324-3247 (NH2, NH), 3076 (CH, aromatic), 2217 (CN), 1606 (C=N); 1H NMR (DMSO-d6): 5 6.46 (s, 2H, NH2), 6.50-8.08 (m, 4H, 3H furan and pyrimidine H-4), 8.81 (s, 1H, NH, D2O exchangeable), 10.01 (s, 1H, SH, D2O exchangeable); MS: m/z (%) 220 (M+, 43). Anal. Calcd. for C9H8N4OS: C, 49.08; H, 3.66; N, 25.44; S, 14.56. Found: C, 49.42; H, 3.75; N, 25.68; S, 14.42
4,5-Diamino-10bff-chromeno[4,3-d]pyrimidine-2-thi-
ol (9e). Yield 79%, m.p. > 300°C; IR (KBr, cm-1): 3438-3345 (2 NH2), 3080 (CH, aromatic), 1611 (C=N); 1H NMR (DMSO-d6): 5 6.96 (br s, 4H, 2 NH2, D2O exchangeable), 7.24-8.33 (m, 5H, 4H aromatic and pyri-midine H-4), 8.98 (s, 1H, SH, D2O exchangeable); 13C HMR (DMSO): 5 61.05, 76.12, 112.02, 129.25, 130.43,
134.80, 164.98, 169,47, 172.23, 173.40; MS: m/z (%) 246 (M+, 19). Anal. Calcd. for CnH10N4OS: C, 53.64; H, 4.09; N, 22.75; S, 13.02. Found: C, 53.82; H, 4.39; N, 22.47; S, 12.74.
Synthesis of 5-Amino-7-(4-methoxyphenyl)-3-phenyl-7_ff-thiazolo[3,2-a]pyrimidine-6-carbonitrile (10). A mixture of 1,4-dihydropyrimidine 9c (2.6 g, 0.01 mol) and ю-bromoacetophenone (2 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (1.0 mL) was heated under reflux for 2 h, left to cool to room temperature, poured onto ice/water, and neutralized by hydrochloric acid. The precipitated solid was filtered, washed with water and crystalized from ethanol.
Yield: 58%; m.p.: 148-150 °C; IR (KBr, cm-1): 3439 (NH2), 3073 (CH, aromatic), 2223 (CN), 1612 (C=N); 1H NMR (DMSO-d6): 5 3.87 (s, 3H, OCH3), 5.21 (s, 1H, CH thiazole), 7.12 (s, 2H, NH2), 7.15-8.15 (m, 10H, 9H aromatic and 1H pyrimidine); MS: m/z (%) 360 (M+, 30). Anal. Calcd. for C20H16N4OS: C, 66.65; H, 4.47; N, 15.54; S, 8.90. Found: C, 6(5.32; H, 4.22; N, 15.67; S, 8.78.
3. 2. In vitro Cytotoxic Assay
Chemicals: Fetal bovine serum (FBS) and L-gluta-mine were purchased from Gibco Invitrogen Co. (Scotland, UK). RPMI-1640 medium was purchased from Cambrex (New Jersey, USA). Dimethylsulfoxide (DM-SO), CHS 828, penicillin, streptomycin and sulforhoda-mine B (SRB) were purchased from Sigma Chemical Co. (Saint Louis, USA).
Cell cultures: were obtained from the European Collection of cell Cultures (ECACC, Salisbury, UK) and human gastric cancer (NUGC), human colon cancer (DLD1), human liver cancer (HA22T and HEPG2), human breast cancer (MCF), nasopharyngeal carcinoma (HONE1) and normal fibroblast cells (WI38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer and were routinely maintained in RPMI-1640 medium supplemented with 5% heat-inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL), at 37 °C in a humidified atmosphere containing 5% CO2. Exponentially growing cells were obtained by plating 1.5 x 105 cells/mL for the six human cancer cell lines followed by 24 h of incubation. The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.
4. Conclusion
In this study we synthesized a series of 4,5-dihydro-2-mercapto-4-oxo-6-substituted arylpyrimidine derivati-
ves 2a-e and their fused analogues 3b, 4b, 5b, 6b, as well as 1,4-dihydro-2-mercaptopyrimidine derivatives 7a-e and 9a-e using triethylamine as a catalyst. All the synthesized compounds were evaluated for their in vitro anticancer activity against six human cancer cell lines and normal fibroblast cells. Compounds 2a, 3b and 9c were found to be the most potent derivatives. Toxicity of the most potent compounds was measured against shrimp larvae; the results showed that compounds 2a and 3b are non-toxic towards the tested organisms. Most of the compounds that exhibited anticancer activity were nontoxic to normal fibroblast cells suggesting that they may serve as lead compounds for further development of new drugs.
5. Acknowledgement
The author would like to acknowledge the help, guidance and encouragement provided by Professor Dr. Rafat M. Mohareb and for offering the needed facilities.
6. References
1.	S. Eckhardt, Curr. Med. Chem. 2002, 2, 419-439.
2.	C. W. Lee, D. H. Hong, S. B. Han, S. H. Jong, H. C. Kim, R. L. Fine, S. H. Lee, H. M. Kim, Biochem. Pharmacol. 2002, 64, 473-480.
http://dx.doi.org/10.1016/S0006-2952(02)01105-X
3.	Y. S. Lee, S. M. Park, H. M. Kim, S. K. Park, K. Lee, C. W. Lee, B. H. Kim, Bioorg Med Chem Lett. 2009, 19, 46884691.
http://dx.doi.org/10.1016/j.bmcl.2009.06.072
4.	F. Rojo, J. Albanell, A. Rovira, J. M. Corominas, F. Manzar-beitia, Semin. Diagn. Pathol. 2008, 25, 245-261. http://dx.doi.org/10.1053/j.semdp.2008.08.001
5.	F. A. M. Al-Omary, G. S. Hassan, S. M. El-Messery, H. I. El-Subbagh, Eur. J. Med. Chem. 2012, 47, 65-72. http://dx.doi.org/10.1016/j.ejmech.2011.10.023
6.	H. T. Abdel-Mohsen, F. A. F. Ragab, M. M. Ramla, H. I. El Diwani, Eur. J. Med. Chem. 2010, 45, 2336-2344. http://dx.doi.org/10.1016/j.ejmech.2010.02.011
7.	A. Kamal, D. Dastagiri, M. J. Ramaiah, J. S. Reddy, E. V. Bharathi, M. K. Reddy, M. V. P. Sagar, T. L. Reddy, S. N. C. V. L. Pushpavalli, M. Pal-Bhadra, Eur. J. Med. Chem. 2011, 46, 5817-5824.
http://dx.doi.org/10.1016/j.ejmech.2011.09.039
8.	M. K. Abd El Hamid, M. D. Mihovilovic, H. B. El-Nassan, Eur. J. Med. Chem. 2012, 57, 323-328. http://dx.doi.org/10.1016/j.ejmech.2012.09.031
9.	A. E. Kassab, E. M. Gedawy, Eur. J. Med. Chem. 2013, 63, 224-230.
http://dx.doi.org/10.1016/j.ejmech.2013.02.011 10. S. E. Abbas, N. M. Abdel Gawad, R. F. George, Y. A. Akar, Eur. J. Med. Chem. 2013, 65, 195-204. http://dx.doi.org/10.1016/j.ejmech.2013.04.055
11.	A. M. Fargualy, N. S. Habib, K. A. Ismail, A. M. M. Hassan, M. T. M. Sarg, Eur. J. Med. Chem. 2013, 66, 276-295. http://dx.doi.org/10.1016/j.ejmech.2013.05.028
12.	L. Ballell, R. A. Field, G. A. Chung, R. J. Young, Bioorg Med Chem Lett. 2007, 17, 1736-1740. http://dx.doi.org/10.1016/j.bmcl.2006.12.066
13.	E. A. Amr, M. S. Nermien, M. M. Abdulla, Monatsh Chem. 2007, 138, 699-707.
http://dx.doi.org/10.1007/s00706-007-0651-0
14.	N. Fujiwara, T. Nakajima, Y. Ueda, H. Ka. Fujita, H. Wakami, Bioorg Med Chem. 2008, 16, 9804-9816. http://dx.doi.org/10.1016/j.bmc.2008.09.059
15.	J. V. dos Anjos, R. M. Srivastava, J. H. Costa-Silva, L.Scotti, M. T. Scotti, A. G. Wanderley, E. S. Leite, S. J. Melo, F. J. B. Mendonga Jr., Molecules. 2012, 16, 809-819. http://dx.doi.org/10.3390/molecules17010809
16.	A. L. Xavier, A. M. Simas, E. P. da S. Falcäo, J. V. dos Anjos, Tetrahedron Lett. 2013, 54, 3462-3465. http://dx.doi.org/10.1016/j.tetlet.2013.04.099
17.	G. Vanessa, M. Sidnei, F. C. F. Alex, C. F. Darlene, C. Pio, P. Ernani, J. Braz. Chem. Soc. 2010, 21, 1477-1483. http://dx.doi.org/10.1590/S0103-50532010000800010
18.	M. Prasenjit, J. Soma, K. K. Lakshmi, T. Ph. Res. 2010, 3, 17-26.
19.	K. S. Jain, T. S, Chitre, P. B. Miniyar, M. K. Kathiravan, V. S. Bendre, V. S. Veer, S. R. Shahane, C. J. Shishoo, Curr. Sci. 2006, 90, 793-803.
20.	B. Ramesh, C. M. Bhalgat, Eur. J. Med. Chem. 2011, 46, 1882-1891.
http://dx.doi.org/10.1016/j.ejmech.2011.02.052
21.	B. Ramesh, D. R. Bharathi, H. S. Basavaraj, K. V. Jayadeva-iah. Asian J. Chem. 2008, 20, 2591-2596.
22.	M. S. Mohamed, S. M. Awad, N. M. Ahmed, Acta Pharm. 2011, 61, 171-185.
http://dx.doi.org/10.2478/v10007-011-0019-1
23.	S. B. Mohan, B. V. Ravi Kumar, S. C. Dinda, D. Naik, S. Prabu Seenivasan, V. Kumar, D. N. Rana, P. S. Brahmksha-triya, Bioorg. Med. Chem. Lett. 2012, 22, 7539-7542. http://dx.doi.org/10.1016/j.bmcl.2012.10.032
24.	O. A. Fathalla, I. F. Zeid, M. E. Haiba, A. M. Soliman, S. I. Abd-Elmoez, W. S. El-Serwy. World J. Chem. 2009, 4, 127-132.
25.	Y. Ma, C. Qian, L. Wang, M. Yang, J. Org. Chem. 2000, 65, 3864-3866.
http://dx.doi.org/10.1021/jo9919052
26.	H. Salehi, S. Kakaei, S. J. Ahmadi, M. A. Firooz Zareh, S. M. Sadat Kiai, H. R. Pakoyan, H. Tajik Ahmadi, J. Applied Chem. Res. 2010, 4, 5-10.
27.	A. Ghorbani-Choghamarani, P. Zamani, Chinese Chem. Lett. 2013, 24, 804-808.
http://dx.doi.org/10.1016/j.cclet.2013.05.033
28.	S. Asghari, M. Tajbakhsh, B. J. Kenari, S. Khaksar, Chinese Chem. Lett. 2011, 22, 127-130. http://dx.doi.org/10.1016/j.cclet.2010.09.030
29.	H. Khabazzadeh, E. T. Kermani, T. Jazinizadeh, Arab. J. Chem. 2012, 5, 485-488.
http://dx.doi.org/10.1016/j.arabjc.2010.09.015
30.	D. R. Patil, S. M. Salunkhe, M. B. Deshmukh, P. V. Anbhule, Open Catal. J. 2010, 3, 83-86. http://dx.doi.org/10.2174/1876214X01003010083
31.	L. G. Voskressensky, A. A. Festa, A. V. Varlamov, Tetrahedron. 2014, 70, 551-572. http://dx.doi.org/10.1016/j.tet.2013.11.011
32.	Y. Ünver, K. Sancak, F. £elik, E. Birinci, M. Küfük, S. Soylu, N. A. Burnaz, Eur. J. Med. Chem. 2014, 84, 639-650. http://dx.doi.org/10.1016/j.ejmech.2014.01.014
33.	A. Dandia, A. K. Laxkar, R. Singh, Tetrahedron Lett. 2012, 53, 3012-3017.
http://dx.doi.org/10.1016/j.tetlet.2012.03.136
34.	N. Zhang, S. Ayral-Kaloustian, T. Nguyen, R. Hernandez, C. Beyer, Bioorg. Med. Chem. Lett. 2007, 17, 3003-3005. http://dx.doi.org/10.1016/j.bmcl.2007.03.070
35.	M. T. Cocco, C. Congiu, V. Lilliu, V. Onnis, Bioorg. Med. Chem. 2006, 14, 366-372. http://dx.doi.org/10.1016/j.bmc.2005.08.012
36.	M. Kuramoto, Y. Sakata, K. Terai, I. Kawasaki, J. Kunitomo, T. Ohishi, T. Yokomizo, S. Takeda, S. Tanaka, Y. Ohishi, Org. Biomol. Chem. 2008, 6, 2772-2781. http://dx.doi.org/10.1039/b803313g
37.	M. I. Choudhary, W. J. Thomsen, Bioassay Techniques For Drug Development. 2001, pp 9-10.
38.	B. Brayn, M. Timothy, S. Tore, General and Applied Toxicology, 2nd Ed. vol. I, p 52.
39.	J. L. Carballo, Z. L. H. Inda, P. Pérez, M. D. Garcia-Grava-los, BMC Biotechnol. 2002, 2, 17. http://dx.doi.org/10.1186/1472-6750-2-17
40.	M. C. Calleja, G. Persoone, Atla. 1992, 20, 396-405.
Povzetek
Razvili smo enostavno in učinkovito metodo za sintezo 4,5-dihidro-2-merkapto-4-okso-6-substituiranih arilpirimi-dinskih derivatov 2a-e ter njihovih pripojenih analogov 3b, 4b, 5b, 6b kot tudi 1,4-dihidro-2-merkaptopirimidinskih derivatov 7a-e, 9a-e, ki temelji na uporabi trietilamina kot katalizatorja. Strukture novih produktov smo potrdili na osnovi njihovih spektroskopskih podatkov in elementnih analiz. Za vse pripravljene spojine smo določili njihovo in vitro delovanju proti šestim človeških rakastim celičnim linijam in normalnim fibroblastom. Devet izmed preiskovanih spojin (to so 2a, 2c, 2d, 3b, 4b, 5b, 8, 9a in 9c) je izkazovalo občutno citotoksičnost proti večini celičnih linij. Izmed teh derivatov so se spojine 2a, 3b in 9c pokazale kot najbolj učinkovite, saj so se citotoksični efekti proti šestim celičnim linijam pojavili že pri IC50 vrednostih < 330 nM, kar je zelo učinkovito v primerjavi s standardom CHS 828. Normalne fi-broblastne celice (WI38) pa so bile na preiskovane spojine bistveno manj občutljive (IC50 > 10,000 nM). Izmerili smo tudi morebitno toksičnost najbolj učinkovitih spojin pri ličinkah rakcev Artemia salina; rezultati so pokazali, da sta spojini 2a in 3b za preiskovani organizem nestrupeni.