Synthesis, screening as potential antitumor of new poly heterocyclic compounds based on pyrimidine-2-thiones

Background Continuing our interest in preparing of new heterocyclic compounds and examining their various biological activities, this work was designed to prepare new condensed and non-condensed heterocyclic compounds 9a-c, 10a-c, 11a-c, 13a-c and 14a-c were synthesized starting with pyrimidine-2-thiones 4a-c. Results Thiazolo[3,2-a]pyrimidines 9a-c were synthesized by S-alkylation of pyrimidine-2-thiones,4a-c, internal cyclization in alkaline medium with ammonia, condensation with benzaldehyde and finally reaction with hydroxylamine hydrochloride.[1,2,4]thiadiazolo[4,5-a]pyrimidines 11a-c were formed by heating of the 4a-c with benzoylcholride to afford 10a-c followed by reaction with sodium hypochlorite, ammonia and sodium hydroxide. Cyclocondensation of 4a-c with ethyl acetoacetate or formic acid yielded pyrazol-3-ones 13a-c or [1,2,4] triazolo[4,3-a]pyrimidines 14a-c, respectively Elements analysis, IR, 1H-NMR, 13C-NMR and mass spectra were used to validate the structures of newly synthesized heterocycles. Screening of the selected compounds 4a, 6a, 7a, 9a, 10a, 13a and 14a against colon carcinoma cell lines (HCT-116) and hepatocellular carcinoma cell lines (HepG-2). Conclusions Elements analysis, IR, 1H-NMR, 13C-NMR and mass spectra were used to validate the structures of newly synthesized heterocycles. Screening of the selected compounds 4a, 6a, 7a, 9a, 10a, 13a and 14a against colon carcinoma cell lines (HCT-116) and hepatocellular carcinoma cell lines (HepG-2) showed that compound 10a exhibited the most cytotoxic, while compounds 4a, 6a and 14a exhibited considerable cytotoxic activity. Supplementary Information The online version contains supplementary material available at 10.1186/s13065-022-00810-4.


Introduction
Continuing our interest in preparing of new heterocyclic compounds and examining their various biological activities [1][2][3][4][5][6], this work was designed to prepare new derivatives of condensed and non-condensed five-membered rings with pyrimidine. Pyrimidine derivatives have aroused the interest of researchers in recent years, as they have demonstrated a wide variety of biological activities such as antibacterial [6], antiallergic, antihypertensive [7] and antitumor activity [5,8], along with their cardiopulmonary and bronchodilating effect [9]. It has been observed that the substitution of the benzene ring in pyrimidine derivatives with heterocyclic moieties such as pyrrole and thiophene shows some biological activities such as anti-proliferative and anti-inflammatory activities [10][11][12]. In addition, the pyrazolpyrimidine and [1,2,4]triazolopyrimidine derivatives have antimicrobial, antioxidant, antimalarial, analgesic and antitumor activities [13][14][15][16][17]. Most classes of heterocyclic compound have been studied to show their role as strong and chelating ligands with most of transition metals as electron rich sites [1,18,19], this point is important in forming novel metal-complexes to be used in different industrial, Abdelrehim and El-Sayed BMC Chemistry (2022) 16:16 pharmaceutical and medicinal applications. This study aimed to synthesize and investigate a new heterocyclic class that has an important role in biological behavior based on its structure.

Chemistry
The reaction of 2-acetyl-1-methylpyrrole 1 with a series of 5-substituted-thiophene-2-carbaldehyde 2a-c in alcoholic sodium hydroxide afforded a new series of chalcones 3a-c as shown in Scheme 1 [20]. Melting points, yield % and IR spectral data of compounds 3a-c are included in the Additional file 1. The known 3,4-dihydro-1H-pyrimidine-2-thiones 4a-c nuclei taken as the key synthons for this work were synthesized by cyclocondensation of chalcones 3a-c with thiourea in the presence of alcoholic potassium hydroxide, Scheme 1 [21]. The structure of compounds 4a-c was established by their elemental analysis data and their IR spectra which showed two characteristic bands at ύ (3364-3394) and (3215-3275) cm −1 for the two NH groups. The 1 H-NMR spectra of compounds 4a-c indicated the chemical shifts (δ) at (3.36-3.56) corresponding to the protons of NCH 3 , (4.76-4.94) for H-4 of pyrimidine, (6.08-6.13) for H-5 of pyrimidine, (6.87-7.56) for aromatic protons of pyrrole, (7.51-8.19) for aromatic protons of thiophene and two D 2 O exchangeable singlet peaks at (8.89-10.08) ppm for 2NH groups. The 13 C-NMR of compound 4a indicated a group of signals at 39.57 for NCH 3 , 65.37 for C-4 of pyrimidine, 108.30 for C-5 of pyrimidine and a characteristic signal at 176.13 ppm for C=S group, (for more details see the experimental section).
S-alkylation, instead of N-alkylation was performed by heating 3,4-dihydro-1H-pyrimidine-2-thione 4a-c with ethyl chloroacetate to produce ethyl 1,6-dihydro-pyrimidin-2-ylsulfanyl]acetate 6a-c, Scheme 2 [22]. The structure of the compounds 6a-c was mainly confirmed from the 13 C-NMR spectrum of compound 6a which showed two characteristic signals at 163.74 and 169.87 ppm for C=N and C=O with absence of C=S group signal. The IR spectra of the compounds 6a-c exhibited stretching bands at (3222-3271) and (1722-1739) cm −1 for NH and C=O groups. The 1 H-NMR of the compounds 6a-c contained a set of peaks for ethyl, NCH 3 , SCH 2 , pyrimidine, pyrrole, thiophene and NH protons, (for more details see the experimental section). The internal cyclization of dihydropyrimidine esters 6a-c took place in an alkaline medium using ammonia affording the corresponding thiazolo[3,2-a]pyrimidin-3-ones 7a-c. The structure of the compounds 7a-c was confirmed by the disappearance of the NH signals in both the IR and 1 H-NMR spectra of these compounds, along with the disappearance of ethyl protons in the 1 H-NMR spectra compared to those in the compounds 6a-c. In order to build up a fused heterocyclic to the compounds 7a-c, the compounds 7a-c were condensed with benzaldehyde in the presence of freshly prepared sodium acetate to give the corresponding 2-Benzylidenethiazolo[3,2-a]pyrimidin-3-ones 8a-c. Heating under reflux of the compounds 8a-c with hydroxylamine hydrochloride in the presence of freshly prepared sodium acetate yielded the corresponding isoxazolo[5′,4′:4,5]thiazolo[3,2-a]pyrimidinse 9a-c [23]. The mass spectrum of 8-(5-Chloro-thiophen-2-yl)-6-(1-methyl-1H-pyrrol-2-yl)-3-phenyl-2,3-dihydro-8H-isoxazolo[5′,4′:4,5]thiazolo[3,2-a]pyr-imidine 9c has molecular ion peaks at 452 and 454 with in a ratio of 3:1 which is consistent with the molecular formula and the existence of chlorine isotopes of compound 9c. Also the spectral data of the compounds 9a-c indicated the presence of NH group at (3207-3233) cm −1 and (9.98-10.73) ppm for the IR and 1 H-NMR spectra, respectively.
The second path way of this work was heating the key synthons 4a-c under reflux with benzoyl chloride and a few drops of triethylamine to provide compounds 10ac, Scheme 3. The structure of the compounds 10a-c was elucidated by their correct elemental analysis and spectral data, where the IR spectra showed two characteristic bands at (3224-3363) and (1682-1697) cm −1 for the NH and C=O groups, respectively. 1 H-NMR spectra of the compounds 10a-c showed a characteristic signal at (10.98-11.42) ppm for NH group, along with two characteristic signals at 169.55 and 181.16 ppm for C=O and C=S groups in the 13 C-NMR spectrum of the compound 10a. The reaction of the compounds 10a-c with sodium hypochlorite, ammonia and sodium hydroxide passed through the formation of non-isolable intermediates sulphenyl chloride and sulphenamide which underwent an intramolecular dehydration to produce the corresponding [1,2,4]thiadiazolo [4,5-a]pyrimidine 11a-c, as shown in Scheme 3 [24]. The elemental analysis of the compounds 11a-c is consistent with their molecular formula. IR spectra indicated the disappearance of the C=O groups, and the 13 C-NMR spectrum of the product 11a also showed the disappearance of the C=S group with two new signals appearing at 150.11 and 156.34 ppm for the two C=N groups which suggesting the formation of thiadiazole ring.
The third part of this work was designed to syntheise the non-condensed system namely: pyrimidopyrazol-3-ones 13a-c and fused heterocylics compounds triazolo[4,3-a]pyrimidines 14a-c. Hydrazinolysis of pyrimidine-2-thiones 4a-c with hydrazine hydrate under reflux gave the corresponding hydrazino compounds 12a-c, Scheme 4. The structures of 12a-c were established on the basis of their elemental analysis and spectral data, the IR spectra of the compounds 12a-c showed absorption bands at (3376-3173) cm −1 for NH 2 and NH groups. 1 H-NMR spectra indicated D 2 O exchangeable singlet signals at (4.46-4.65), (8.79-9.23) and (9.85-10.44) ppm corresponding to the NH 2 , NH of pyrimidine and NH of hydrazine, respectively. Cyclocondensation of the hydrazinopyrimidine compounds 12a-c with ethyl acetoacetate in acetic acid gave the corresponding [pyrimidin-2-yl]-2,4-dihydro-pyrazol-3-ones 13a-c. IR spectra of compounds 13a-c revealed absorption bands at (3178-3208) for NH group and at (1683-1691) cm −1 for C=O group. The low frequency of the C=O group in the IR spectra for the compounds 13a-c is due to internal H-bonding as shown in Fig. 1 leads to C=O lengthening and as a result the C=O frequency decreases. 1 H-NMR spectra of the compounds 13a-c indicated three singlet signals at (1.75-1.89), (2.73-3.09) and (3.36-3.51) ppm for pyrazolyl CH 3 , pyrazolyl CH 2 and NCH 3 , respectively along with D 2 O exchangeable singlet signals at (9.38-10.51) ppm corresponding to the NH of pyrimidine. Finally cyclocondensation of 12a-c with formic acid gave the corresponding [1,2,4]triazolo[4,3-a]pyrimidine 14a-c, the chemical structure of the compounds 14a-c was suggested by their elemental analysis and through the disappearance of the NH and NH 2 signals in both 1 H-NMR spectra of 12a-c.

Anticancer activity discussion
In this study, selected compounds 4a, 6a, 7a, 9a, 10a, 13a and 14a were tested for potential cytotoxity using the Mossman [25], Gangadevi and Muthumary [26] methods for anticancer activity against colon carcinoma cells lines (HCT-116) and hepatocellular carcinoma cells lines (HepG-2) using Vinblastine drug as standard. Data on antitumor activity were represented by the cytotoxic effect of the selected compounds. The inhibitory activities of the tested compounds against colon carcinoma cells (HCT-116) and hepatocellular carcinoma cells lines (HepG-2) were calculated by dissolving the selected compounds in DMSO and diluting with saline to appropriate volume using different concentrations of the samples (50, 25, 12.5, 6.25, 3.125 and 1.56 µg mL −1 ), and the cell viability (percent) of the studied compounds was determined using a colorimetric technique, Tables 1, 2. From Tables 1, 2, inhibitory concentration fifty (IC 50 ) which corresponds to the concentration necessary for 50% inhabitation of cell viability was calculated, Table 3. Screening of the selected compounds against human colon carcinoma cancer cell lines and hepatocellular carcinoma cells lines revealed that the compound 2-thioxo-3,6-dihydro-2H-pyrimidin-1-yl]-phenyl-methanone 10a was the most active among the group of selected compounds with IC 50 (10.72 and 18.95) µM in both human colon carcinoma cancer cell lines and hepatocellular carcinoma cells lines, respectively, Table 3 and 10a exhibit the highest cytotoxic activity and this activity increase with the inclusion of a polar group such as the carbonyl group (C=O). The IC 50 values also show that increased toxicity necessitates larger doses in the case of hepatocellular carcinoma cell lines compared to human colon carcinoma cancer cell lines. As a result, we recommended that the synthesized compound, particularly 2-thioxo-3,6-dihydro-2H-pyrimidin-1-yl]phenyl-methanone 10a, be used in the formulation of antibiotics as drugs to increase the sensitivity of antibiotics that stimulate cancer treatment and cause apoptosis in human colon carcinoma.

In vitro studies
Human colon cancer (HCT-116) cells and hepatocellular carcinoma (HepG-2) cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The cells were cultured in RPMI-1640 media Scheme 4 Scheme of preparation of compounds 12a-c, 13a-c and 14a-c supplemented with 10% inactivated fetal calf serum and 50 g/mL gentamycin. The cells were kept in a humid environment with 5% CO 2 , 37 °C, and sub-cultured two to three times. Cytotoxic tests on the selected compounds 4a, 6a, 7a, 9a, 10a, 13a and 14a: Monolayers of 10,000 cells adhered to the bottom of wells in a 96-well microtiter plate cultured for 24 h at 37 °C in a humidified incubator with 5% CO 2 . The monolayers were then rinsed with sterile phosphate buffered saline (0.01 M pH 7.2), and the cells were incubated at 37 °C with 100 μL of various dilutions of the tested compounds or Vinblastine drug as a control. Six wells were utilized for each concentration of the tested compound, whereas control cells were produced in the absence of the tested compounds. Every 24 h, the observation was performed under an inverted microscope, and the number of (viable) surviving cells was counted by coloring cells with crystal violet, followed by cell lysis with glacial acetic acid (33%), and recording the absorbance at 495 nm, taking into account that the absorption of the untreated cell is 100%.
The following Eq. (1) was used to compute the percentage of cell viability.
where ODt denotes the mean optical density of test compound-treated wells and ODc denotes the mean optical density of untreated (control) cells. The 50% inhibitory concentration (IC 50 ), which is the concentration required   to cause toxic effect in 50% of inactivated cells, was estimated from graphic plots.

Materials and methods
The prepared compounds' melting points are uncorrected and were determined with MEL TEMP II equipment. A Perkin-Elmer FTIR spectrophotometer was used to record the IR spectra (KBr

-( 1 -M e t h y l -1 H -p y r r o l -2 -y l ) -5 -t h i op h e n -2 -y l -5 H -t hi a z o l o [ 3 , 2 -a ] p y r imi din -3 -o n e 7a
According to the previous general method, pale yellow crystals were obtained. Yield

Conclusion
New condensed and non-condensed heterocyclic compounds based on pyrimidine-2-thiones 4a-c were synthesized. The first synthetic path way took place through S-alkylation of pyrimidine-2-thiones 4a-c followed by reaction with ammonia to produce the corresponding thiazolo[3,2-a]pyrimidin-3-ones 7a-c which underwent condensation with benzaldehyde followed by heating under reflux with hydroxylamine afforded the corresponding isoxazolo [5′,4′:4,5]thiazolo[3,2-a]pyrimidinse 9a-c. The second path way of this work was the heatng of the key synthons 4a-c with benzoylcholride followed by reaction with sodium hypochlorite, ammonia and sodium hydroxide to produce [1,2,4]thiadiazolo[4,5-a]pyrimidine 11a-c. A final route of this work was the hydrazinolysis of 4a-c followed by the cyclocondensation with ethyl acetoacetate or formic acid to produce pyrazol-3-ones 13a-c or [1,2,4]triazolo[4,3-a]pyrimidine 14a-c, respectively. All newly synthesized heterocyclic structures were confirmed using various tools including, elemental analysis, IR, 1 H-NMR, 13 C-NMR and mass spectra. Screening of the selected compounds 4a, 6a, 7a, 9a, 10a, 13a and 14a against colon carcinoma cells lines (HCT-116) and hepatocellular carcinoma cells lines (HepG-2) showed that the compound 2-thioxo-3,6-dihydro-2H-pyrimidin-1-yl]-phenyl-methanone 10a was the most active among the group of selected compounds, meanwhile, compounds 4a, 6a and 14a exhibited considerable cytotoxic action, furthermore, compounds 7a, 9a and 13a were showed weak cytotoxic action. These results encourage us to suggest that the compound 10a be used in the formulation of antibiotics as a medication to improve the sensitivity of antibiotics that stimulate cancer therapy and cause apoptosis in both human colon carcinoma cancer and hepatocellular carcinoma.