2-Thiopyrimidine/chalcone hybrids: design, synthesis, ADMET prediction, and anticancer evaluation as STAT3/STAT5a inhibitors

Abstract

A novel 2-thiopyrimidine/chalcone hybrid was designed, synthesised, and evaluated for their cytotoxic activities against three different cell lines, K-562, MCF-7, and HT-29. The most active cytotoxic derivatives were 9d, 9f, 9n, and 9p (IC₅₀=0.77–1.74 µM, against K-562 cell line), 9a and 9r (IC₅₀=1.37–3.56 µM against MCF-7 cell line), and 9a, 9l, and 9n (IC₅₀=2.10 and 2.37 µM against HT-29 cell line). Compounds 9a, 9d, 9f, 9n, and 9r were further evaluated for their cytotoxicity against normal fibroblast cell line WI38. Moreover, STAT3 and STAT5a inhibitory activities were determined for the most active derivatives 9a, 9d, 9f, 9n, and 9r. Dual inhibitory activity was observed in compound 9n (IC₅₀=113.31 and 50.75 µM, against STAT3 and STAT5a, respectively). Prediction of physicochemical properties, drug likeness score, pharmacokinetic and toxic properties was detected.

Graphical Abstract

Keywords:

• 2-Thiopyrimidine
• chalcone
• STAT
• cytotoxicity
• computational analysis

Introduction

One of the main causes of mortality all over the world is cancer^1,2. The highest prevalence for cancer death is being for stomach, breast, prostatic, lung, and colon³.

The most common female cancer around the world is breast cancer. It represents for 16% of all female cancers and 18.2% of all cancer death causes including both males and females⁴.

On the other hand, about two million new cases are diagnosed every year for colorectal cancer. Thus, making it as one of the most common causes of cancer-related death^5,6.

Another common cause of cancer death is leukaemia, cancer in blood-forming cells of the bone marrow, which is chemoresistant^7–11. Although, treatment of cancer using chemotherapeutic agents is still used for several cancer types including breast, colon and leukaemia cancers, high toxicity level of chemotherapeutic drugs limit their use¹².

A critical signalling intermediate in cancer cells, specially leukaemia, breast and colon cancer cells is called signal transducer and activator of transcription (STAT) protein family^13–17.

They are cytoplasmic transcription factors. STAT family consists of seven members, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. STAT2, STAT4, and STAT6 are responsible for regulation of immune response. While, STAT1, STAT3, and STAT5 can control cell cycle (cyclin D1, D2, c-Myc), cell survival (Bcl-xl, Bcl2, Mcl-1), and angiogenesis (HIF1α, VEGFR) through regulation of gene expression^18,19.

STAT can be activated either by receptor tyrosin kinases like JAKs, PDGFR, EGFR, and FLT3, or through non-receptor tyrosin kinases, Src, Brk, and Bcr-Abl. Also, activation of STAT may be from activation of cytokines (IL-6), growth factors or negative feedback mechanisms^20–24.

Phosphorylation of STATs transforms them to active form causing their homo- or heterodimerisation then migration to the nucleus to control gene expression. Over activation of STAT level can lead to tumorigenesis²⁰.

Several studies have demonstrated that blocking STAT3 or STAT5 signalling pathway led to apoptosis in tumour cells. While, normal cells were able to survive even under a very low concentration of STAT3 or STAT5 and also capable of growing using other mechanisms^21,25.

Therefore, development of new anti-cancer agents with less toxicity and overcoming chemotherapeutic drug resistance can be achieved by: (1) using drugs that target two or three activators of STAT3²⁴ or (2) combined targeting of STAT3 and STAT5⁸.

It was found that a potent STAT3 inhibitor, S31-201 (I, Figure 1), could inhibit proliferation of hepatocellular and breast carcinoma in mice¹⁶.

Figure 1. The designed strategy for 2-TP/chalcone hybrids as new anticancer STAT inhibitors.

Moreover, compound S3I-201.1066 (II, Figure 1), containing sulphonamide group could inhibit STAT3 function in both breast and myeloma cancer cells (EC₅₀=10 and 16 µM, respectively)²⁶. Another compound, curcumine analogue, FLLL32 (III, Figure 1), showed potent inhibitory activity in many human cancer cell lines such as breast, colorectal, melanoma, and myeloma by preventing STAT3 dimerisation and downstream functioning^20,27.

Moreover, treatment with chalcone IV (Figure 1) caused significant decrease in STAT3 level in leukaemia HL-60 cell line²⁸.

Pyrazolo[1,5-a]pyrimidine/chalcone hybrid V (Figure 1) showed promising anti-proliferative activity by down regulation of STAT3 in MDA-MB-231 cells²⁹.

Additionally, NCI library identified two compounds (BP-1108 and BP-1075) as the most potent STAT5 in K562 leukaemia cell lines through down regulation of STATs-defendant genes³⁰.

In medicinal chemistry, a very well-known heterocycle is pyrimidine. It takes its importance from its presence in thymidine, cytosine and uracil bases, the building blocks of DNA and RNA nucleic acids^31,32.

2-Thiopyrimidines (2-TPs), also named as 2-mercaptopyrimidines, are one of the most important class of pyrimidines.

They attract the biochemists attention due to their wide range of applications in preparation of cardiotonic drugs, antitubercular and anti-inflammatory agents^33,34.

Moreover, 2-TPs were evaluated for their anticancer activity³³. They were reported to have potent antitumor activity against leukaemia, colon and breast cell lines such as compounds VI–IX (Figure 1)^35–38.

Synthesis of 2-TPs derivatives could be achieved from reaction of chalcone derivatives with thiourea³⁹. As chalcones constitute an important group of natural products, their biological activities were arisen from their chemical structure, α,β-unsaturated carbonyl group⁴⁰.

Many synthesised chalcones were reported to have potent in vitro anticancer activity against human colon carcinoma, non-small cell lung carcinoma, and breast cancer^41–44.

Thus, both chloro- and dimethylamino-derivatives of compound X (Figure 1), showed cytotoxic activity against human leukaemia cells with CC₅₀=2.17 and 2.06 μM, respectively⁴⁰. While, compound XI (Figure 1) was apoptosis inducer in A549 cells⁴⁵.

In light of the above facts, and as a part of our previously published anticancer research articles^46,47, our scope in this research was to design and synthesised a new series of 2-TP/chalcone hybrids (Figure 1), through molecular hybridisation, by merging:

(i) 2-Thiopyrimidine scaffold, such as in compounds (VI–IX), (ii) chalcone part from compounds (III–V, X, XI), (iii) choosing substituents on phenyl rings of pyrimidine C-4, pyrimidine C-6, and chalcone as in compounds (I–V), and (iv) amide linkage to mimic that in compounds (I, II, VI, X, XI). The cytotoxic activities of the synthesised derivatives were evaluated against leukaemia (K-562), breast (MCF-7), and colon (HT-29) cancer cell lines. Inhibitory activities of the most potent bioactive molecules against STAT3 and STAT5a were measured, aiming at finding more effective anticancer therapeutics.

Experimental

Chemistry

Melting points were measured on the Griffin apparatus and were uncorrected. Determination of IR spectra was achieved using Shimadzu IR-435 spectrophotometer with KBr discs and values were obtained in cm⁻¹. ¹H NMR and ¹³C NMR were recorded on Bruker instrument at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR spectrophotometer (Faculty of Pharmacy, Mansoura University, Mansoura, Egypt), in DMSO-d₆ (as a solvent), D₂O using TMS as an internal standard and chemical shifts (δ) were expressed in parts per million (ppm) compared to internal standard, TMS (δ = 0 ppm). Coupling constant (J) values were expressed in Hertz (Hz). Signal splitting patterns were designated as follows: s, singlet; d, doublet, t, triplet; q, quartette; m, multiplet. The electron impact (EI) mass spectra were carried out using Hewlett Packard 5988 spectrometer (Palo Alto, CA) at Faculty of Science, Cairo University, Giza, Egypt. Microanalysis was calculated for C, H, N on Perkin-Elmer 2400 at the Microanalytical centre, Faculty of Science, Cairo University, Egypt and was within ±0.4% of theoretical values. The progress of the reaction and purity of products were monitored by thin layer chromatography (TLC), pre-coated plastic sheets, 0.2 mm silica gel with UV indicator (Macherey-Nagel, Düren, Germany). All used reagents and solvents were purchased from the Aldrich Chemical Company (Milwaukee, WI).

General method for preparation of compounds 4a–f

A mixture of the appropriate chalcone derivative 3a–f (0.01 mol), thiourea (0.76 g, 0.01 mol), and KOH (0.11 g, 0.02 mol) in absolute ethanol (20 ml) was heated under reflux temperature for 12 h. The resulting solution was evaporated to dryness or (a precipitate in case of 3c and 3f). The obtained residue was solubilised in water, filtered and dried. The crude product was crystallised from ethanol/DMF (8:2) to get compounds 4a–f.

4-(4-Methoxyphenyl)-6-p-tolylpyrimidine-2-thiol (4a)

Yield 82%; yellow powder; (ethanol 95%); mp 149–151 °C; IR (cm⁻¹): 3413 (NH), 3192 (CH aromatic), 2958 (CH aliphatic); ¹H NMR (400 MHz, DMSO-d₆) δ 2.50 (s, 3H, CH₃), 4.43 (s, 3H, OCH₃), 7.24–7.59 (m, 4H, p-methoxyphenyl H-3, H-5, p-tolyl H-3, H-5), 7.79–7.91 (m, 4H, pyrimidine H-5, p-methoxyphenyl H-2, H-6, NH, D₂O exchangeable), 8.21 (d, J= 8.4 Hz, 2H, p-tolyl H-2, H-6); ¹³C NMR (100 MHz, DMSO-d₆) δ 21.2 (CH₃), 55.6 (OCH₃), 101.0 (pyrimidine C-5), 114.7 (p-methoxyphenyl C-3, C-5), 126.1 (p-tolyl C-2, C-6), 128.1 (p-tolyl C-3, C-5), 129.4 (p-methoxyphenyl C-2, C-6), 130.9 (p-methoxyphenyl C-1), 134.5 (p-tolyl C-1), 136.7 (p-tolyl C-4), 159.2 (p-methoxyphenyl C-4), 164.6 (pyrimidine C-4) 172.9 (pyrimidine C-6), 175.2 (pyrimidine C-2); EIMS (m/z): 309.00 (M + 1, 31.75%), 308.00 (M⁺, 74.33%), 307.00 (100%); Anal. Calcd. for C₁₈H₁₆N₂OS (308.40): C, 70.10; H, 5.23; N, 9.08. Found: C, 70.31; H, 5.07; N, 8.88.

4-(4-Methoxyphenyl)-6-(4-nitrophenyl)pyrimidine-2-thiol (4b)

Yield 75%; yellow crystals; mp 63–65 °C³⁹.

4-[4-(2-Chloroethoxy)phenyl]-6-(4-methoxyphenyl)pyrimidine-2-thiol (4c)

Yield 69%; yellow powder; (ethanol 95%); mp 282–284 °C; IR (cm⁻¹): 3436 (NH), 3192 (CH aromatic), 2927 (CH aliphatic); ¹H NMR (400 MHz, DMSO-d₆) δ 3.83–4.37 (m, 7H, OCH₃, OCH₂, CH₂Cl), 6.96–7.10 (m, 7H, p-chloroethoxyphenyl H-2, H-3, H-5, H-6, p-methoxyphenyl H-3, H-5, NH, D₂O exchangeable), 8.22–8.30 (m, 3H, p-methoxyphenyl H-2, H-6, pyrimidine H-5); ¹³C NMR (100 MHz, DMSO-d₆) δ 42.9 (CH₂Cl), 55.8 (OCH₃), 68.1 (OCH₂), 105.0 (pyrimidine C-5), 114.8 (p-methoxyphenyl C-3, C-5), 115.4 (p-chloroethoxyphenyl C-3, C-5), 121.8 (p-chlorophenyl C-2, C-6), 127.9 (p-methoxyphenyl C-1), 128.5 (p-methoxyphenyl C-2, C-6), 129.4 (p-chloroethoxyphenyl C-2, C-6), 130.0 (p-chloroethoxyphenyl C-1), 160.6 (p-methoxyphenyl C-4), 161.7 (p-chloroethoxyphenyl C-4), 162.5 (pyrimidine C-4) 176.1 (pyrimidine C-6), 179.3 (pyrimidine C-2); Anal. Calcd. for C₁₉H₁₇ClN₂O₂S (372.87): C, 61.20; H, 4.60; N, 7.51. Found: C, 61.41; H, 4.57; N, 7.75.

4-(4-Chlorophenyl)-6-p-tolylpyrimidine-2-thiol (4d)

Yield 91%; yellow crystals; mp 180–182 °C⁴⁷.

4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidine-2-thiol (4e)

Yield 65%; yellow powder; (ethanol 95%); mp 263–265 °C; IR (cm⁻¹): 3434 (NH), 3064 (CH aromatic); ¹H NMR (400 MHz, DMSO-d₆) δ 7.60 (d, J= 8.4 Hz, 2H, p-chlorophenyl H-3, H-5), 7.96 (s, 1H, NH, D₂O exchangeable), 8.33–8.34 (m, 4H, p-chlorophenyl H-2, H-6, p-nitrophenyl H-2, H-6), 8.54 (d, J= 8.4 Hz, 2H, p-nitrophenyl H-3, H-5), 8.66 (s, 1H, pyrimidine H-5); ¹³C NMR (100 MHz, DMSO-d₆) δ 101.3 (pyrimidine C-5), 123.6 (p-chlorophenyl C-2, C-6), 128.0 (p-nitrophenyl C-3, C-5), 128.9 (p-chlorophenyl C-1), 129.3 (p-chlorophenyl C-3, C-5), 129.5 (p-nitrophenyl C-2, C-6), 133.5 (p-chlorophenyl C-4), 139.2 (p-nitrophenyl C-1), 150.2 (p-nitrophenyl C-4), 164.6 (pyrimidine C-4) 176.3 (pyrimidine C-6), 180.4 (pyrimidine C-2); Anal. Calcd. for C₁₆H₁₀ClN₃O₂S (343.79): C, 55.90; H, 2.93; N, 12.22. Found: C, 60.12; H, 2.87; N, 12.46.

4-[4-(2-Chloroethoxy)phenyl]-6-(4-chlorophenyl)pyrimidine-2-thiol (4f)

Yield 62%; yellow powder; (ethanol 95%); mp 242–244 °C; IR (cm⁻¹): 3417 (NH), 3066 (CH aromatic), 2927 (CH aliphatic); ¹H NMR (400 MHz, DMSO-d₆) δ 3.98 (t, J= 8.4 Hz, 2H, CH₂Cl), 4.36 (t, J= 8.4 Hz, 2H, OCH₂), 7.07–7.12 (m, 4H, p-chloroethoxyphenyl H-3, H-5, p-chlorophenyl H-2, H-6), 7.31 (s, 1H, NH, D₂O exchangeable), 7.60 (d, J= 8.4 Hz, 2H, p-chlorophenyl H-3, H-5), 7.96 (s, 1H, pyrimidine H-5), 8.31 (d, J= 8.4 Hz, 2H, p-chloroethoxyphenyl H-2, H-6); ¹³C NMR (100 MHz, DMSO-d₆) δ 43.4 (CH₂Cl), 68.5 (OCH₂), 103.9 (pyrimidine C-5), 115.3 (p-chloroethoxyphenyl C-3, C-5), 120.2 (p-chlorophenyl C-2, C-6), 128.5 (p-chlorophenyl C-1), 129.4 (p-chloroethoxyphenyl C-2, C-6), 129.5 (p-chlorophenyl C-3, C-5), 130.7 (p-chloroethoxyphenyl C-1), 136.6 (p-chlorophenyl C-4), 161.7 (p-chloroethoxyphenyl C-4), 162.8 (pyrimidine C-4) 176.3 (pyrimidine C-6), 179.0 (pyrimidine C-2); EIMS (m/z): 376.95 (M + 1, 16.83%), 375.90 (M⁺, 19.31%), 55.10 (100%); Anal. Calcd. for C₁₈H₁₄ClN₂OS (377.29): C, 57.30; H, 3.74; N, 7.42. Found: C, 57.41; H, 3.57; N, 7.68.

General method for preparation of compounds 9a–r

A mixture of pyrimidine derivatives 4a–f (0.01 mol), acetyl chloride derivatives 8a–c (0.01 mol), and catalytic amount of TEA is stirred in acetonitrile (20 ml) for 24 h. The solution was evaporated to dryness. The obtained residue was solubilised in ice cold water and neutralised with conc. HCl. The obtained solid was filtered, dried and crystallised from ethanol/DMF (8:2).

(ZE)-2-[4-(4-Methoxyphenyl)-6-p-tolylpyrimidin-2-ylthio]-N-{4-[3-(4 methoxyphenyl)acryloyl]phenyl}acetamide (9a)

Yield 82%; yellow powder; mp 226–228 °C; IR (cm⁻¹): 3257 (NH), 3039 (CH aromatic), 2925 (CH aliphatic), 1795, 1663 (2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 1.86 (s, 3H, CH₃), 4.41 (s, 3H, OCH₃), 4.43 (s, 3H, OCH₃), 4.84 (s, 2H, CH₂), 6.67 (d, J= 8.4 Hz, 2H, p-methoxyphenylacroyl H-3, H-5), 7.10 (d, J= 7.6 Hz, 2H, p-methoxyphenyl H-3, H-5), 7.24 (d, J= 8.4 Hz, 2H, p-methoxyphenylacroyl H-2, H-6), 7.33 (d, J= 6.0 Hz, 2H, p-tolyl H-3, H-5), 7.44–7.74 (m, 7H, COCH=CH, pyrimidine H-5, p-methoxyphenyl H-2, H-6, p-tolyl H-2, H-6), 7.91–7.94 (m, 4H, phenyl H-2, H-3, H-5, H-6), 10.84 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 22.3 (CH₃), 36.2 (CH₂), 56.4 (2OCH₃), 113.7 (p-methoxyphenylacroyl C-3, C-5), 118.8 (p-methoxyphenyl C-3, C-5), 120.1 (COCH=CH), 121.7 (phenyl C-2, C-6), 127.5 (p-methoxyphenylacroyl C-1), 127.6 (p-tolyl C-2, C-6), 129.0 (p-methoxyphenyl C-2, C-6), 129.1 (p-methoxyphenyl C-1), 130.2 (p-tolyl C-3, C-5), 130.8 (p-methoxyphenylacroyl C-2, C-6), 131.4 (phenyl C-3, C-5), 132.0 (p-tolyl C-4), 133.5 (phenyl C-4), 136.9 (p-tolyl C-1), 144.3 (phenyl C-1), 154.4 (COCH=CH), 155.1 (p-methoxyphenylacroyl C-4), 155.4 (p-methoxyphenyl C-4), 164.1 (pyrimidine C-6), 167.8 (pyrimidine C-4), 172.3 (pyrimidine C-2), 173.0 (CONH), 190.0 (CO); Anal. Calcd. for C₃₆H₃₁N₃O₄S (601.20): C, 71.86; H, 5.19; N, 6.98. Found: C, 71.67; H, 5.07; N, 6.93.

(ZE)-N-{4-[3-(3,4-Dimethoxyphenyl)acryloyl]phenyl}-2-[4-(4-methoxyphenyl)-6-p-tolylpyrimidin-2-ylthio]acetamide (9b)

Yield 65%; yellow powder; mp 165–167 °C; IR (cm⁻¹): 3324 (NH), 3061 (CH aromatic), 2925 (CH aliphatic), 1663 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 2.36 (s, 3H, CH₃), 3.81 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.23 (s, 2H, CH₂), 6.97 (d, J= 8.4 Hz, 2H, p-methoxyphenyl H-3, H-5), 7.03 (d, J= 8.0 Hz, 1H, dimethoxyphenyl H-5), 7.28 (d, J= 8.0 Hz, 1H, dimethoxyphenyl H-6), 7.37 (d, J= 12.0 Hz, 1H, COCH=CH), 7.54 (s, 1H, dimethoxyphenyl H-2), 7.69 (d, J= 12.0 Hz, 1H, COCH=CH), 7.83–7.87 (m, 3H, p-tolyl H-3, H-5, pyridine H-5), 8.18–8.23 (m, 6H, phenyl H-2, H-6, p-methoxyphenyl H-2, H-6, p-tolyl H-2, H-6), 8.31 (d, J= 8.4 Hz, 2H, phenyl H-3, H-5), 10.84 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 21.4 (CH₃), 36.4 (CH₂), 55.8 (OCH₃), 56.0 (OCH₃), 56.1 (OCH₃), 107.4 (pyrimidine C-5), 111.0 (dimethoxyphenyl C-2), 111.9 (dimethoxyphenyl C-5), 114.6 (dimethoxyphenyl C-6), 118.8 (p-methoxyphenyl C-3, C-5), 119.8 (COCH=CH), 124.4 (phenyl C-2, C-6), 127.7 (p-tolyl C-2, C-6), 128.0 (p-methoxyphenyl C-2, C-6), 129.6 (p-methoxyphenyl C-1), 130.3 (p-tolyl C-3, C-5), 130.4 (p-tolyl C-1), 133.0 (dimethoxyphenyl C-1), 133.6 (phenyl C-3, C-5), 141.7 (p-tolyl C-4), 143.9 (phenyl C-4), 144.4 (phenyl C-1), 149.4 (dimethoxyphenyl C-3), 150.0 (dimethoxyphenyl C-4), 151.6 (COCH=CH), 162.3 (p-methoxyphenyl C-4), 164.2 (pyrimidine C-6), 164.3 (pyrimidine C-4), 167.7 (pyrimidine C-2), 170.8 (CONH), 187.8 (CO); Anal. Calcd. for C₃₇H₃₃N₃O₅S (631.21): C, 70.34; H, 5.27; N, 6.65. Found: C, 70.54; H, 5.07; N, 6.71.

(ZE)-2-[4-(4-Methoxyphenyl)-6-p-tolylpyrimidin-2-ylthio]-N-{4-[3-(3,4,5-trimethoxyphenyl)acryloyl]phenyl}acetamide (9c)

Yield 56%; yellow powder; mp 195–197 °C; IR (cm⁻¹): 3290 (NH), 2998 (CH aromatic), 2927 (CH aliphatic), 1665 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 2.36 (s, 3H, CH₃), 3.72 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 3.87 (s, 6H, 2OCH₃), 4.24 (s, 2H, CH₂), 6.95 (s, 2H, trimethoxyphenyl H-2, H-6), 6.98–7.28 (m, 4H, p-methoxyphenyl H-3, H-5, p-tolyl H-3, H-5), 7.69 (d, J= 12.0 Hz, 1H, COCH=CH), 7.87–8.19 (m, 3H, COCH=CH, p-tolyl H-2, H-6), 8.22–8.31 (m, 7H, p-methoxyphenyl H-2, H-6, pyrimidine H-5, phenyl H-2, H-3, H-5, H-6), 10.86 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 21.4 (CH₃), 36.4 (CH₂), 55.8 (OCH₃), 56.5 (2OCH₃), 60.6 (OCH₃), 106.8 (trimethoxyphenyl C-2, C-6), 107.4 (pyrimidine C-5), 114.5 (p-methoxyphenyl C-3, C-5), 118.8 (phenyl C-2, C-6), 121.5 (COCH=CH), 126.9 (p-tolyl C-2, C-6), 127.7 (trimethoxyphenyl C-1), 128.5 (p-methoxyphenyl C-1), 129.6 (p-methoxyphenyl C-2, C-6), 130.4 (p-tolyl C-3, C-5), 131.0 (p-tolyl C-4), 132.8 (phenyl C-3, C-5), 133.5 (p-tolyl C-1), 140.0 (phenyl C-4), 141.7 (trimethoxyphenyl C-4), 144.0 (phenyl C-1), 144.4 (COCH=CH), 153.5 (trimethoxyphenyl C-3, C-5), 162.3 (p-methoxyphenyl C-4), 164.2 (pyrimidine C-6), 164.3 (pyrimidine C-4), 167.7 (pyrimidine C-2), 170.7 (CONH), 187.9 (CO); EIMS (m/z): 662.05 (M + 1, 5.50%), 661.05 (M⁺, 12.94%), 322.05 (100%); Anal. Calcd. for C₃₈H₃₅N₃O₆S (661.22): C, 68.97; H, 5.33; N, 6.35. Found: C, 68.78; H, 5.17; N, 6.24.

(ZE)-2-[4-(4-Methoxyphenyl)-6-(4-nitrophenyl)pyrimidin-2-ylthio]-N-{4-[3-(4-methoxyphenyl)acryloyl]phenyl}acetamide (9d)

Yield 57%; yellow powder; mp 183–185 °C; IR (cm⁻¹): 3407 (NH), 3063 (CH aromatic), 2930 (CH aliphatic), 1664 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 3H, OCH₃), 3.84 (s, 3H, OCH₃), 4.24 (s, 2H, CH₂), 6.98 (d, J= 8.8 Hz, 2H, p-methoxyphenylacryloyl H-3, H-5), 7.03 (d, J= 8.8 Hz, 2H, p-methoxyphenyl H-3, H-5), 7.69 (d, J= 15.6 Hz, 1H, COCH=CH), 7.80–7.86 (m, 5H, COCH=CH, p-methoxyphenylacryloyl H-2, H-6, p-methoxyphenyl H-2, H-6), 8.18 (d, J= 8.8 Hz, 2H, phenyl H-2, H-6), 8.27 (d, J= 8.8 Hz, 2H, phenyl H-3, H-5), 8.33 (d, J= 8.8 Hz, 2H, p-nitrophenyl H-2, H-6), 8.40 (s, 1H, pyrimidine H-5), 8.56 (d, J= 8.8 Hz, 2H, p-nitrophenyl H-3, H-5), 10.86 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 36.4 (CH₂), 55.8 (OCH₃), 55.9 (OCH₃), 109.1 (pyrimidine C-5), 114.7 (p-methoxyphenylacryloyl C-3, C-5), 114.8 (p-methoxyphenyl C-3, C-5), 118.8 (phenyl C-2, C-6), 119.8 (p-nitrophenyl C-3, C-5), 124.2 (COCH=CH), 127.8 (p-nitrophenyl C-2, C-6), 128.2 (p-methoxyphenyl C-2, C-6), 129.1 (p-methoxyphenylacryloyl C-1), 129.8 (p-methoxyphenyl C-1), 130.0 (p-methoxyphenylacryloyl C-2, C-6), 130.3 (phenyl C-3, C-5), 131.1 (p-nitrophenyl C-1), 133.1 (phenyl C-4), 142.4 (phenyl C-1), 143.8 (COCH=CH), 149.3 (p-nitrophenyl C-4), 161.7 (p-methoxyphenylacryloyl C-4), 162.2 (p-methoxyphenyl C-4), 162.6 (pyrimidine C-6), 164.9 (pyrimidine C-4), 167.6 (pyrimidine C-2), 171.3 (CONH), 187.8 (CO); Anal. Calcd. for C₃₅H₂₈N₄O₆S (632.17): C, 66.44; H, 4.46; N, 8.86. Found: C, 66.42; H, 4.39; N, 8.74.

(ZE)-N-{4-[3-(3,4-Dimethoxyphenyl)acryloyl]phenyl}-2-[4-(4-methoxyphenyl)-6-(4-nitrophenyl)pyrimidin-2-ylthio]-acetamide (9e)

Yield 54%; yellow powder; mp 225–227 °C; IR (cm⁻¹): 3431 (NH), 3039 (CH aromatic), 2924 (CH aliphatic), 1656 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 3H, OCH₃), 3.87 (s, 6H, 2OCH₃), 4.27 (s, 2H, CH₂), 6.99 (d, J= 8.4 Hz, 2H, p-methoxyphenyl H-3, H-5), 7.03 (d, J= 8.8 Hz, 1H, dimethoxyphenyl H-5), 7.39 (d, J= 8.8 Hz, 1H, dimethoxyphenyl H-6), 7.55 (s, 1H, dimethoxyphenyl H-2), 7.68 (d, J= 15.2 Hz, 1H, COCH=CH), 7.82–7.88 (m, 3H, COCH=CH, p-methoxyphenyl H-2, H-6), 8.20 (d, J= 8.8 Hz, 2H, phenyl H-2, H-6), 8.28 (d, J= 8.8 Hz, 2H, phenyl H-3, H-5), 8.34 (d, J= 8.8 Hz, 2H, p-nitrophenyl H-2, H-6), 8.41 (s, 1H, pyrimidine H-5), 8.57 (d, J= 8.8 Hz, 2H, p-nitrophenyl H-3, H-5), 10.87 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 36.4 (CH₂), 55.9 (OCH₃), 56.0 (OCH₃), 56.2 (OCH₃), 109.1 (pyrimidine C-5), 111.0 (dimethoxyphenyl C-2), 112.0 (dimethoxyphenyl C-5), 114.7 (p-methoxyphenyl C-3, C-5), 118.8 (dimethoxyphenyl C-6), 119.8 (phenyl C-2, C-6), 124.2 (p-nitrophenyl C-3, C-5), 124.4 (COCH=CH), 128.0 (p-nitrophenyl C-2, C-6), 128.2 (p-methoxyphenyl C-2, C-6), 129.1 (dimethoxyphenyl C-1), 129.8 (p-methoxyphenyl C-1), 130.3 (phenyl C-3, C-5), 132.0 (phenyl C-4), 142.4 (phenyl C-1), 143.8 (p-nitrophenyl C-1), 144.4 (COCH=CH), 149.4 (dimethoxyphenyl C-4), 149.4 (dimethoxyphenyl C-3), 153.5 (p-nitrophenyl C-4), 162.2 (p-methoxyphenyl C-4), 162.6 (pyrimidine C-6), 165.0 (pyrimidine C-4), 167.6 (pyrimidine C-2), 171.3 (CONH), 187.8 (CO); Anal. Calcd. for C₃₆H₃₀N₄O₇S (662.18): C, 65.24; H, 4.56; N, 8.45. Found: C, 65.35; H, 4.71; N, 8.24.

(ZE)-2-[4-(4-Methoxyphenyl)-6-(4-nitrophenyl)pyrimidin-2-ylthio]-N-{4-[3-(3,4,5-trimethoxyphenyl)acryloyl]phenyl}acetamide (9f)

Yield 69%; yellow powder; mp 254–256 °C; IR (cm⁻¹): 3265 (NH), 3103 (CH aromatic), 2933 (CH aliphatic), 1663 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.71 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 3.87 (s, 6H, 2OCH₃), 4.27 (s, 2H, CH₂), 6.98 (d, J= 8.0 Hz, 2H, p-methoxyphenyl H-3, H-5), 7.23 (s, 2H, trimethoxyphenyl H-2, H-6), 7.69 (d, J= 12.0 Hz, 1H, COCH=CH), 7.86–7.92 (m, 3H, COCH=CH, p-methoxyphenyl H-2, H-6), 8.21 (d, J= 8.0 Hz, 2H, phenyl H-2, H-6), 8.27 (d, J= 8.0 Hz, 2H, phenyl H-3, H-5), 8.33 (d, J= 8.0 Hz, 2H, p-nitrophenyl H-2, H-6), 8.40 (s, 1H, pyrimidin H-5), 8.56 (d, J= 8.0 Hz, 2H, p-nitrophenyl H-3, H-5), 10.89 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 36.4 (CH₂), 55.8 (OCH₃), 56.5 (2OCH₃), 60.6 (OCH₃), 106.8 (trimethoxyphenyl C-2, C-6), 109.0 (pyrimidine C-5), 114.6 (p-methoxyphenyl C-3, C-5), 118.8 (phenyl C-2, C-6), 121.4 (COCH=CH), 124.2 (p-nitrophenyl C-3, C-5), 128.1 (trimethoxyphenyl C-1), 129.1 (p-nitrophenyl C-2, C-6), 129.8 (p-methoxyphenyl C-2, C-6), 130.4 (phenyl C-3, C-5), 130.7 (p-methoxyphenyl C-1), 132.9 (phenyl C-4), 140.0 (trimethoxyphenyl C-4), 142.3 (p-nitrophenyl C-1), 144.3 (phenyl C-1), 144.4 (COCH=CH), 149.3 (p-nitrophenyl C-4), 153.5 (trimethoxyphenyl C-3, C-5), 162.2 (p-methoxyphenyl C-4), 162.6 (pyrimidine C-6), 164.9 (pyrimidine C-4), 167.6 (pyrimidine C-2), 171.3 (CONH), 187.9 (CO); EIMS (m/z): 693.00 (M + 1, 0.93%), 692.00 (M⁺, 1.33%), 55.10 (100%); Anal. Calcd. for C₃₇H₃₂N₄O₈S (692.19): C, 64.15; H, 4.66; N, 8.09. Found: C, 63.98; H, 4.57; N, 7.89.

(ZE)-2-{4-[4-(2-Chloroethoxy)phenyl]-6-(4-methoxyphenyl)pyrimidin-2-ylthio}-N-{4-[3-(4-methoxyphenyl)acryloyl]phenyl}acetamide (9g)

Yield 52%; yellow powder; mp 280–282 °C; IR (cm⁻¹): 3431 (NH), 3039 (CH aromatic), 2935 (CH aliphatic), 1598 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.70 (s, 3H, OCH₃), 3.83–3.85 (m, 5H, OCH₃ and CH₂Cl), 4.21–4.23 (m, 4H, OCH₂ and CH₂), 7.02–7.16 (m, 6H, p-methoxyphenyl H-3, H-5, p-methoxyphenylacryloyl H-3, H-5 and p-chloroethoxyphenyl H-3, H-5), 7.71–7.96 (m, 9H, p-methoxyphenyl H-2, H-6, p-methoxyphenylacryloyl H-2, H-6, p-chloroethoxyphenyl H-2, H-6, phenyl H-2, H-6 and COCH=CH), 8.16 (d, J= 8.4 Hz, 2H, phenyl H-3, H-5), 8.18–8.20 (m, 2H, pyrimidin H-5 and COCH=CH), 10.89 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 36.2 (CH₂), 40.6 (CH₂Cl), 55.4 (OCH₃), 55.8 (OCH₃), 68.9 (OCH₂), 107.8 (pyrimidine C-5), 114.1 (p-methoxyphenylacroyl C-3, C-5), 114.7 (p-methoxyphenyl C-3, C-5), 114.8 (p-chloroethoxyphenyl C-3, C-5), 121.3 (COCH=CH), 122.1 (phenyl C-2, C-6), 127.4 (p-chloroethoxyphenyl C-1), 127.5 (p-methoxyphenylacroyl C-1), 128.3 (p-chloroethoxyphenyl C-2, C-6), 129.5 (p-methoxyphenyl C-2, C-6), 130.3 (p-methoxyphenylacroyl C-2, C-6), 131.1 (phenyl C-3, C-5), 133.5 (phenyl C-4), 144.0 (phenyl C-1), 144.3 (p-methoxyphenyl C-1), 145.4 (COCH=CH), 159.3 (p-chloroethoxyphenyl C-4), 159.8 (p-methoxyphenylacroyl C-4), 160.6 (p-methoxyphenyl C-4), 162.8 (pyrimidine C-6), 164.9 (pyrimidine C-4), 168.6 (pyrimidine C-2), 172.3 (CONH), 189.9 (CO); Anal. Calcd. for C₃₇H₃₂ClN₃O₅S (665.18): C, 66.71; H, 4.84; N, 6.31. Found: C, 66.98; H, 4.57; N, 6.28.

(ZE)-2-{4-[4-(2-Chloroethoxy)phenyl]-6-(4-methoxyphenyl)pyrimidin-2-ylthio}-N-{4-[3-(3,4-dimethoxyphenyl)acryloyl]phenyl}acetamide (9h)

Yield 52%; yellow powder; mp 135–137 °C; IR (cm⁻¹): 3426 (NH), 3067 (CH aromatic), 2928 (CH aliphatic), 1657 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.70 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 3.96 (t, J= 7.2 Hz, 2H, CH₂Cl), 4.23 (s, 2H, CH₂), 4.34 (t, J= 7.2 Hz, 2H, OCH₂), 6.82–7.12 (m, 7H, p-methoxyphenyl H-3, H-5, p-chloroethoxyphenyl H-3, H-5, dimethoxyphenyl H-2, H-5, H-6), 7.38 (d, J= 15.2 Hz, 1H, COCH=CH), 7.67–7.86 (m, 5H, COCH=CH, p-methoxyphenyl H-2, H-6, p-chloroethoxyphenyl H-2, H-6), 8.18–8.29 (m, 5H, phenyl H-2, H-6, phenyl H-3, H-5, pyrimidin H-5), 10.89 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 42.2 (CH₂), 43.4 (CH₂Cl), 55.8 (OCH₃), 56.1 (OCH₃), 56.2 (OCH₃), 75.1 (OCH₂), 109.0 (pyrimidine C-5), 111.5 (dimethoxyphenyl C-2), 111.9 (dimethoxyphenyl C-5), 114.5 (p-methoxyphenyl C-3, C-5), 115.1 (p-chloroethoxyphenyl C-3, C-5), 118.8 (phenyl C-2, C-6), 119.8 (COCH=CH), 124.4 (dimethoxyphenyl C-6), 127.3 (dimethoxyphenyl C-1), 127.4 (p-chloroethoxyphenyl C-1), 128.0 (p-chloroethoxyphenyl C-2, C-6), 128.7 (p-methoxyphenyl C-1), 129.6 (p-methoxyphenyl C-2, C-6), 130.3 (phenyl C-3, C-5), 131.3 (phenyl C-4), 143.9 (phenyl C-1), 144.4 (COCH=CH), 149.4 (dimethoxyphenyl C-4), 151.6 (dimethoxyphenyl C-3), 157.2 (p-chloroethoxyphenyl C-4), 161.2 (p-methoxyphenyl C-4), 162.2 (pyrimidine C-6), 164.3 (pyrimidine C-4), 167.6 (pyrimidine C-2), 170.7 (CONH), 187.6 (CO); Anal. Calcd. for C₃₈H₃₄ClN₃O₆S (695.19): C, 65.56; H, 4.92; N, 6.04. Found: C, 65.74; H, 5.07; N, 5.99.

(ZE)-2-{4-[4-(2-Chloroethoxy)phenyl]-6-(4-methoxyphenyl)pyrimidin-2-ylthio}-N-{4-[3-(3,4,5-trimethoxyphenyl)acryloyl]phenyl}acetamide (9i)

Yield 54%; yellow powder; mp 116–118 °C; IR (cm⁻¹): 3417 (NH), 3039 (CH aromatic), 2934 (CH aliphatic), 1658 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.71 (s, 3H, OCH₃), 3.72 (s, 3H, OCH₃), 3.82 (t, J= 3.6 Hz, 2H, CH₂Cl), 3.88 (s, 6H, 2OCH₃), 4.23 (s, 2H, CH₂), 4.34 (t, J= 3.6 Hz, 2H, OCH₂,), 7.25 (s, 2H, trimethoxyphenyl H-2, H-6), 7.68–7.72 (m, 3H, p-chloroethoxyphenyl H-3, H-5 and COCH=CH), 7.81 (d, J= 8.8 Hz, 2H, p-methoxyphenyl H-3, H-5), 7.90 (d, J= 8.8 Hz, 2H, phenyl H-3, H-5), 7.95 (d, J= 8.2 Hz, p-chloroethoxyphenyl H-2, H-6) , 8.20–8.22 (m, 4H, phenyl H-2, H-6 and p-methoxyphenyl H-2, H-6), 8.29–8.31 (m, 2H, COCH=CH and pyrimidin H-5), 10.71 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 43.4 (CH₂), 55.4 (CH₂Cl), 55.8 (OCH₃), 56.5 (2OCH₃), 60.6 (OCH₃), 68.6 (OCH₂), 106.5 (trimethoxyphenyl C-2, C-6), 106.9 (pyrimidine C-5), 114.1 (p-methoxyphenyl C-3, C-5), 114.9 (p-chloroethoxyphenyl C-3, C-5), 118.9 (phenyl C-2, C-6), 119.8 (COCH=CH), 121.5 (trimethoxyphenyl C-1), 129.6 (p-chloroethoxyphenyl C-1), 129.7 (p-chloroethoxyphenyl C-2, C-6), 130.4 (phenyl C-3, C-5), 130.7 (p-methoxyphenyl C-1), 131.0 (p-methoxyphenyl C-2, C-6), 131.6 (phenyl C-4), 140.0 (trimethoxyphenyl C-4), 143.7 (phenyl C-1), 144.4 (COCH=CH), 153.5 (trimethoxyphenyl C-3, C-5), 160.6 (p-methoxyphenyl C-4), 161.7 (p-chloroethoxyphenyl C-4), 164.1 (pyrimidine C-6), 164.0 (pyrimidine C-4), 168.6 (pyrimidine C-2), 172.3 (CONH), 187.9 (CO); Anal. Calcd. for C₃₉H₃₆ClN₃O₇S (725.20): C, 64.50; H, 5.00; N, 5.79. Found: C, 64.38; H, 4.98; N, 5.53.

(ZE)-2-[4-(4-Chlorophenyl)-6-p-tolylpyrimidin-2-ylthio]-N-{4-[3-(4-methoxyphenyl)acryloyl]phenyl}acetamide (9j)

Yield 59%; yellow powder; mp 242–244 °C; IR (cm⁻¹): 3256 (NH), 3038 (CH aromatic), 2917 (CH aliphatic), 1663 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 2.36 (s, 3H, CH₃), 3.82 (s, 3H, OCH₃), 4.24 (s, 2H, CH₂), 7.02 (d, J= 8.8 Hz, 2H, methoxyphenyl H-3, H-5), 7.27 (d, J= 8.0 Hz, 2H, p-tolyl H-3, H-5), 7.52 (d, J= 8.8 Hz, 2H, p-chlorophenyl H-3, H-5), 7.69 (d, J= 11.6 Hz, 1H, COCH=CH), 7.82 (d, J= 11.6 Hz, 1H, COCH=CH), 7.83–7.85 (m, 4H, p-tolyl H-2, H-6, p-methoxyphenyl H-2, H-6), 8.16 (d, J= 8.8 Hz, 2H, phenyl H-2, H-6), 8.22 (d, J= 8.8 Hz, 2H, phenyl H-3, H-5), 8.30 (s, 1H, pyrimidine H-5), 8.34 (d, J= 8.8 Hz, 2H, p-chlorophenyl H-2, H-6), 10.84 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 21.4 (CH₃), 36.4 (CH₂), 55.8 (OCH₃), 108.3 (pyrimidine C-5), 114.8 (p-methoxyphenyl C-3, C-5), 118.8 (phenyl C-2, C-6), 119.8 (COCH=CH), 127.8 (p-tolyl C-2, C-6), 129.3 (p-chlorophenyl C-2, C-6), 129.6 (p-chlorophenyl C-3, C-5), 129.9 (p-tolyl C-3, C-5), 130.3 (p-methoxyphenyl C-2, C-6), 131.1 (phenyl C-3, C-5), 133.0 (p-methoxyphenyl C-1), 133.3 (p-tolyl C-4), 135.1 (p-tolyl C-1), 136.6 (phenyl C-4), 142.0 (p-chlorophenyl C-1), 143.8 (phenyl C-1), 143.8 (p-chlorophenyl C-4), 148.0 (COCH=CH), 161.7 (p-methoxyphenyl C-4), 163.4 (pyrimidine C-6), 164.9 (pyrimidine C-4), 167.6 (pyrimidine C-2), 171.1 (CONH), 187.8 (CO); EIMS (m/z): 607.20 (M + 2, 1.54%), 606.15 (M + 1, 1.28%), 605.15 (M⁺, 2.65%), 57.10 (100%); Anal. Calcd. for C₃₅H₂₈ClN₃O₃S (605.15): C, 69.35; H, 4.66; N, 6.93. Found: C, 69.41; H, 4.87; N, 7.13.

(ZE)-2-[4-(4-Chlorophenyl)-6-p-tolylpyrimidin-2-ylthio]-N-{4-[3-(3,4-dimethoxyphenyl)acryloyl]phenyl}acetamide (9k)

Yield 57%; yellow powder; mp 144–146 °C; IR (cm⁻¹): 3273 (NH), 3039 (CH aromatic), 2924 (CH aliphatic), 1665 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 2.35 (s, 3H, CH₃), 3.85 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 4.24 (s, 2H, CH₂), 7.01 (d, J= 8.4 Hz, 1H, dimethoxyphenyl H-5), 7.27 (d, J= 8.0 Hz, 2H, p-tolyl H-3, H-5), 7.37 (d, J= 8.4 Hz, 1H, dimethoxyphenyl H-6), 7.50 (d, J= 8.8 Hz, 2H, p-chlorophenyl H-3, H-5), 7.53 (s, 1H, dimethoxyphenyl H-2), 7.69 (d, J= 11.6 Hz, 1H, COCH=CH), 7.81 (d, J= 11.6 Hz, 1H, COCH=CH), 7.85 (d, J= 8.0 Hz, 2H, p-tolyl H-2, H-6), 8.18 (d, J= 8.0 Hz, 2H, phenyl H-2, H-6), 8.22 (d, J= 8.0 Hz, 2H, phenyl H-3, H-5), 8.28 (s, 1H, pyrimidine H-5), 8.34 (d, J= 8.8 Hz, 2H, p-chlorophenyl H-2, H-6), 10.91 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 21.4 (CH₃), 36.2 (CH₂), 56.1 (OCH₃), 56.8 (OCH₃), 108.3 (pyrimidine C-5), 111.0 (dimethoxyphenyl C-2), 111.9 (dimethoxyphenyl C-5), 118.8 (phenyl C-2, C-6), 119.6 (dimethoxyphenyl C-6), 119.8 (p-tolyl C-2, C-6), 124.3 (COCH=CH), 127.8 (p-chlorophenyl C-2, C-6), 128.0 (dimethoxyphenyl C-1), 129.3 (p-chlorophenyl C-3, C-5), 129.6 (p-tolyl C-3, C-5), 129.9 (phenyl C-3, C-5), 130.3 (p-tolyl C-4), 133.0 (p-tolyl C-1), 133.3 (p-chlorophenyl C-1), 134.0 (p-chlorophenyl C-4), 135.1 (phenyl C-4), 136.6 (phenyl C-1), 144.7 (COCH=CH), 149.4 (dimethoxyphenyl C-4), 151.6 (dimethoxyphenyl C-3), 163.4 (pyrimidine C-6), 164.9 (pyrimidine C-4), 167.6 (pyrimidine C-2), 171.1 (CONH), 187.9 (CO); Anal. Calcd. for C₃₆H₃₀ClN₃O₄S (635.16): C, 67.97; H, 4.75; N, 6.61. Found: C, 67.85; H, 4.58; N, 6.42.

(ZE)-2-[4-(4-Chlorophenyl)-6-p-tolylpyrimidin-2-ylthio]-N-{4-[3-(3,4,5-trimethoxyphenyl)acryloyl]phenyl}acetamide (9l)

Yield 62%; yellow powder; mp 234–236 °C; IR (cm⁻¹): 3280 (NH), 3088 (CH aromatic), 2927 (CH aliphatic), 1663 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 2.36 (s, 3H, CH₃), 3.71 (s, 3H, OCH₃), 3.86 (s, 6H, 2OCH₃), 4.25 (s, 2H, CH₂), 7.23 (s, 2H, trimethoxyphenyl H-2, H-6), 7.28 (d, J= 8.0 Hz, 2H, p-tolyl H-3, H-5), 7.52 (d, J= 8.4 Hz, 2H, p-chlorophenyl H-3, H-5), 7.69 (d, J= 12.8 Hz, 1H, COCH=CH), 7.87 (d, J= 8.0 Hz, 2H, p-tolyl H-2, H-6), 7.93 (d, J= 12.8 Hz, 1H, COCH=CH), 8.19–8.24 (m, 4H, phenyl H-2, H-3, H-5, H-6), 8.31 (s, 1H, pyrimidine H-5), 8.35 (d, J= 8.4 Hz, 2H, p-chlorophenyl H-2, H-6), 10.86 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 21.4 (CH₃), 36.4 (CH₂), 56.0 (2OCH₃), 60.6 (OCH₃), 106.8 (trimethoxyphenyl C-2, C-6), 108.3 (pyrimidine C-5), 118.8 (phenyl C-2, C-6), 121.5 (COCH=CH), 127.9 (p-tolyl C-2, C-6), 129.3 (trimethoxyphenyl C-1), 129.9 (p-chlorophenyl C-2, C-6), 130.4 (p-chlorophenyl C-3, C-5), 130.4 (p-tolyl C-3, C-5), 130.7 (phenyl C-3, C-5), 132.8 (p-tolyl C-4), 133.3 (p-tolyl C-1), 135.1 (phenyl C-4), 136.6 (p-chlorophenyl C-1), 140.0 (p-chlorophenyl C-4), 142.0 (trimethoxyphenyl C-4), 144.0 (phenyl C-1), 144.4 (COCH=CH), 153.5 (trimethoxyphenyl C-3, C-5), 163.4 (pyrimidine C-6), 164.9 (pyrimidine C-4), 167.6 (pyrimidine C-2), 171.1 (CONH), 187.8 (CO); Anal. Calcd. for C₃₇H₃₂ClN₃O₅S (665.18): C, 66.71; H, 4.84; N, 6.31. Found: C, 66.57; H, 4.76; N, 6.37.

(ZE)-2-[4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-ylthio]-N-{4-[3-(4-methoxyphenyl)acryloyl]phenyl}acetamide (9m)

Yield 55%; yellow powder; mp 190–192 °C; IR (cm⁻¹): 3403 (NH), 3066 (CH aromatic), 2927 (CH aliphatic), 1658 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 3H, OCH₃), 4.29 (s, 2H, CH₂), 7.03 (d, J= 8.8 Hz, 2H, p-methoxyphenyl H-3, H-5), 7.55 (d, J= 8.8 Hz, 2H, p-chlorophenyl H-3, H-5), 7.72 (d, J= 11.6 Hz, 1H, COCH=CH), 7.77–7.93 (m, 5H, COCH=CH, p-methoxyphenyl H-2, H-6, phenyl H-2, H-6), 8.17 (d, J= 8.8 Hz, 2H, phenyl H-3, H-5), 8.28 (d, J= 8.8 Hz, 2H, p-nitrophenyl H-2, H-6), 8.40 (d, J= 8.8 Hz, 2H, p-chlorophenyl H-2, H-6), 8.50 (s, 1H, pyrimidine H-5), 8.58 (d, J= 8.8 Hz, 2H, p-nitrophenyl H-3, H-5), 10.89 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 38.8 (CH₂), 55.8 (OCH₃), 109.7 (pyrimidine C-5), 114.8 (p-methoxyphenyl C-3, C-5), 118.8 (phenyl C-2, C-6), 120.1 (COCH=CH), 123.2 (p-nitrophenyl C-3, C-5), 124.3 (p-nitrophenyl C-2, C-6), 125.8 (p-chlorophenyl C-2, C-6), 127.9 (p-methoxyphenyl C-1), 129.4 (p-chlorophenyl C-3, C-5), 129.4 (p-methoxyphenyl C-2, C-6), 133.5 (phenyl C-3, C-5), 133.8 (p-chlorophenyl C-1), 134.4 (p-chlorophenyl C-4), 134.5 (phenyl C-4), 141.9 (p-nitrophenyl C-1), 144.3 (phenyl C-1), 145.4 (COCH=CH), 149.2 (p-nitrophenyl C-4), 161.8 (p-methoxyphenyl C-4), 162.4 (pyrimidine C-6), 164.6 (pyrimidine C-4), 167.8 (pyrimidine C-2), 171.5 (CONH), 187.5 (CO); Anal. Calcd. for C₃₄H₂₅ClN₄O₅S (636.12): C, 64.10; H, 3.96; N, 8.79. Found: C, 64.24; H, 4.06; N, 8.47.

(ZE)-2-[4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-ylthio]-N-{4-[3-(3,4-dimethoxyphenyl)acryloyl]phenyl}acetamide (9n)

Yield 53%; yellow powder; mp 147–149 °C; IR (cm⁻¹): 3256 (NH), 3079 (CH aromatic), 2922 (CH aliphatic), 1660 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.82 (s, 3H, OCH₃), 3.87 (s, 3H, OCH₃), 4.29 (s, 2H, CH₂), 7.03 (d, J= 8.4 Hz, 1H, dimethoxyphenyl H-5), 7.39 (d, J= 8.4 Hz, 1H, dimethoxyphenyl H-6), 7.53–7.55 (m, 3H, p-chlorophenyl H-3, H-5, dimethoxyphenyl H-2), 7.70 (d, J= 15.6 Hz, 1H, COCH=CH), 7.82–7.87 (m, 3H, p-chlorophenyl H-2, H-6, COCH=CH), 8.19 (d, J= 8.8 Hz, 2H phenyl H-2, H-6), 8.27 (d, J= 8.8 Hz, 2H phenyl H-3, H-5), 8.38 (d, J= 8.4 Hz, 2H p-nitrophenyl H-2, H-6), 8.49 (s, 1H, pyrimidine H-5), 8.57 (d, J= 8.4 Hz, 2H, p-nitrophenyl H-3, H-5), 10.88 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 36.5 (CH₂), 56.0 (OCH₃), 56.1 (OCH₃), 109.9 (pyrimidine C-5), 111.0 (dimethoxyphenyl C-2), 111.9 (dimethoxyphenyl C-5), 118.8 (phenyl C-2, C-6), 119.8 (dimethoxyphenyl C-6), 124.2 (p-nitrophenyl C-3, C-5), 124.3 (COCH=CH), 128.0 (p-nitrophenyl C-2, C-6), 129.2 (dimethoxyphenyl C-1), 129.4 (p-chlorophenyl C-2, C-6), 129.8 (p-chlorophenyl C-3, C-5), 130.3 (phenyl C-3, C-5), 133.1 (p-chlorophenyl C-1), 134.7 (p-chlorophenyl C-4), 137.0 (phenyl C-4), 142.1 (p-nitrophenyl C-1), 143.8 (phenyl C-1), 144.4 (COCH=CH), 149.4 (p-nitrophenyl C-4), 151.6 (dimethoxyphenyl C-4), 152.3 (dimethoxyphenyl C-3), 162.7 (pyrimidine C-6), 164.1 (pyrimidine C-4), 167.5 (pyrimidine C-2), 171.6 (CONH), 187.8 (CO); Anal. Calcd. for C₃₅H₂₇ClN₄O₆S (666.13): C, 63.01; H, 4.08; N, 8.40. Found: C, 62.89; H, 4.15; N, 8.37.

(ZE)-2-[4-(4-Chlorophenyl)-6-(4-nitrophenyl)pyrimidin-2-ylthio]-N-{4-[3-(3,4,5-trimethoxyphenyl)acryloyl]phenyl}acetamide (9o)

Yield 47%; yellow powder; mp 268–270 °C; IR (cm⁻¹): 3371 (NH), 3059 (CH aromatic), 2935 (CH aliphatic), 1656 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.72 (s, 3H, OCH₃), 3.87 (s, 6H, 2OCH₃), 4.29 (s, 2H, CH₂), 7.24 (s, 2H, trimethoxyphenyl H-2, H-6), 7.55 (d, J= 8.8 Hz, 2H, p-chlorophenyl H-3, H-5), 7.70 (d, J= 15.6 Hz, 1H, COCH=CH), 7.87 (d, J= 8.8 Hz, 2H, p-chlorophenyl H-2, H-6), 7.92 (d, J= 15.6 Hz, 1H, COCH=CH), 8.21 (d, J= 8.8 Hz, 2H, phenyl H-2, H-6), 8.28 (d, J= 8.8 Hz, 2H, phenyl H-3, H-5), 8.39 (d, J= 8.8 Hz, 2H, p-nitrophenyl H-2, H-6), 8.51 (s, 1H, pyrimidine H-5), 8.58 (d, J= 8.8 Hz, 2H, p-nitrophenyl H-3, H-5), 10.89 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 36.5 (CH₂), 56.6 (2OCH₃), 60.6 (OCH₃), 106.9 (trimethoxyphenyl C-2, C-6), 110.0 (pyrimidine C-5), 118.8 (phenyl C-2, C-6), 121.5 (COCH=CH), 124.3 (p-nitrophenyl C-3, C-5), 129.3 (p-nitrophenyl C-2, C-6), 129.4 (p-chlorophenyl C-2, C-6), 129.8 (p-chlorophenyl C-3, C-5), 130.5 (trimethoxyphenyl C-1), 130.8 (phenyl C-3, C-5), 132.9 (p-chlorophenyl C-1), 134.8 (p-chlorophenyl C-4), 137.0 (phenyl C-4), 140.0 (trimethoxyphenyl C-4), 142.1 (phenyl C-1), 143.9 (p-nitrophenyl C-1), 144.4 (COCH=CH), 149.5 (p-nitrophenyl C-4), 153.5 (trimethoxyphenyl C-3, C-5), 162.8 (pyrimidine C-6), 164.2 (pyrimidine C-4), 167.5 (pyrimidine C-2), 171.6 (CONH), 187.8 (CO); EIMS (m/z): 696.20 (M⁺, 0.11%), 86.15 (100%); Anal. Calcd. for C₃₆H₂₉ClN₄O₇S (696.14): C, 62.02; H, 4.19; N, 8.04. Found: C, 61.98; H, 4.35; N, 7.98.

(ZE)-2-{4-[4-(2-Chloroethoxyphenyl)phenyl]-6-(4-chlorophenyl)pyrimidin-2-ylthio}-N-{4-[3-(4-methoxyphenyl)acryloyl]phenyl}acetamide (9p)

Yield 52%; yellow powder; mp 136–138 °C; IR (cm⁻¹): 3181 (NH), 3043 (CH aromatic), 2927 (CH aliphatic), 1663 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.83 (s, 3H, OCH₃), 3.97 (t, J= 6.9 Hz, 2H, CH₂Cl), 4.25 (s, 2H, CH₂), 4.32 (t, J= 6.9 Hz, 2H, OCH₂), 6.90–7.14 (m, 4H, methoxyphenyl H-3, H-5, p-chloroethoxyphenyl H-3, H-5), 7.43–7.69 (m, 10H, COCH=CH, COCH=CH, phenyl H-2, H-6, p-methoxyphenyl H-2, H-6, p-chloroethoxyphenyl H-2, H-6, p-chlorophenyl H-3, H-5), 8.12–8.35 (m, 5H, phenyl H-3, H-5, pyrimidine H-5, p-chlorophenyl H-2, H-6), 10.88 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 36.2 (CH₂), 43.4 (CH₂Cl), 54.6 (OCH₃), 55.8 (OCH₂), 107.8 (pyrimidine C-5), 114.8 (p-methoxyphenyl C-3, C-5), 115.3 (p-chloroethoxyphenyl C-3, C-5), 118.8 (phenyl C-2, C-6), 121.8 (COCH=CH), 127.4 (p-chloroethoxyphenyl C-1), 128.5 (p-methoxyphenyl C-1), 128.6 (p-chlorophenyl C-2, C-6), 129.4 (p-chlorophenyl C-3, C-5), 129.6 (p-methoxyphenyl C-2, C-6), 129.6 (p-chloroethoxyphenyl C-2, C-6), 130.7 (p-chlorophenyl C-1), 132.7 (phenyl C-3, C-5), 133.9 (p-chlorophenyl C-4), 140.1 (phenyl C-4), 143.0 (phenyl C-1), 144.4 (COCH=CH), 159.4 (p-chloroethoxyphenyl C-4), 159.9 (p-methoxyphenyl C-4), 162.8 (pyrimidine C-6), 164.3 (pyrimidine C-4), 167.3 (pyrimidine C-2), 171.4 (CONH), 187.8 (CO); Anal. Calcd. for C₃₆H₂₉Cl₂N₃O₄S (669.13): C, 64.48; H, 4.36; N, 6.27. Found: C, 64.36; H, 4.19; N, 6.35.

(ZE)-2-{4-[4-(2-Chloroethoxyphenyl)phenyl]-6-(4-chlorophenyl)pyrimidin-2-ylthio}-N-{4-[3-(3,4-dimethoxyphenyl)acryloyl]phenyl}acetamide (9q)

Yield 49%; yellow powder; mp 210–212 °C; IR (cm⁻¹): 3429 (NH), 3061 (CH aromatic), 2927 (CH aliphatic), 1663 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.72–3.82 (m, 5H, OCH₃ and CH₂Cl), 3.83–3.88 (m, 5H, OCH₃ and OCH₂), 4.34 (s, 2H, CH₂), 6.98–7.15 (m, 4H, dimethoxyphenyl H-5, H-6, p-chloroethoxyphenyl H-3, H-5), 7.31–7.40 (m, 2H, dimethoxy H-2 and COCH=CH), 7.52–7.70 (m, 4H, p-chlorophenyl H-3, H-5 and p-chloroethoxyphenyl H-2, H-6), 7.75–7.88 (m, 4H, phenyl H-2, H-6 and p-chlorophenyl H-2, H-6), 7.95 (d, J= 8.8 Hz, 2H, phenyl H-3, H-5), 8.28 (d, J= 12.0 Hz, 1H, COCH=CH), 8.46 (s, 1H, pyrimidine H-5), 10.76 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 38.4 (CH₂), 42.9 (CH₂Cl), 56.1 (OCH₃), 56.6 (OCH₃), 68.6 (OCH₂), 107.3 (pyrimidine C-5), 111.5 (dimethoxyphenyl C-2), 112.7 (dimethoxyphenyl C-5), 114.9 (p-chloroethoxyphenyl C-3, C-5), 121.1 (phenyl C-2, C-6), 121.3 (COCH=CH), 122.5 (dimethoxyphenyl C-6), 127.3 (dimethoxyphenyl C-1), 127.4 (p-chloroethoxyphenyl C-1), 128.1 (p-chloroethoxyphenyl C-2, C-6), 128.9 (p-chlorophenyl C-2, C-6), 129.3 (p-chlorophenyl C-3, C-5), 131.4 (phenyl C-3, C-5), 133.9 (p-chlorophenyl C-1), 134.3 (p-chlorophenyl C-4), 135.5 (phenyl C-4), 144.3 (phenyl C-1), 145.1 (COCH=CH), 149.3 (dimethoxyphenyl C-4), 149.7 (dimethoxyphenyl C-3), 151.1 (p-chloroethoxyphenyl C-4), 162.5 (pyrimidine C-6), 164.4 (pyrimidine C-4), 168.7 (pyrimidine C-2), 172.7 (CONH), 189.7 (CO); Anal. Calcd. for C₃₇H₃₁Cl₂N₃O₅S (699.14): C, 63.43; H, 4.46; N, 6.00. Found: C, 63.33; H, 4.27; N, 6.11.

(ZE)-2-{4-[4-(2-Chloroethoxyphenyl)phenyl]-6-(4-chlorophenyl)pyrimidin-2-ylthio}-N-{4-[3-(3,4,5-trimethoxyphenyl)acryloyl]phenyl}acetamide (9r)

Yield 53%; yellow powder; mp 221–223 °C; IR (cm⁻¹): 3421 (NH), 3061 (CH aromatic), 2931 (CH aliphatic), 1665 (broad, 2C=O); ¹H NMR (400 MHz, DMSO-d₆) δ 3.70 (s, 3H, OCH₃), 3.85 (s, 6H, 2OCH₃), 3.96 (t, J= 7.2 Hz, 2H, CH₂Cl), 4.24 (s, 2H, CH₂), 4.45 (t, J= 7.2 Hz, 2H, OCH₂), 7.03–7.20 (m, 4H, trimethoxyphenyl H-2, H-6, chloroethoxyphenyl H-3, H-5), 7.29 (d, J= 12.0 Hz, 1H, COCH=CH), 7.51–7.60 (m, 4H, p-chlorophenyl H-3, H-5, chloroethoxyphenyl H-2, H-6), 7.87–7.96 (m, 3H, phenyl H-2, H-6, COCH=CH), 8.18–8.32 (m, 5H, phenyl H-3, H-5, p-chlorophenyl H-2, H-6, pyrimidine H-5), 10.91 (s, 1H, NH, D₂O exchangeable); ¹³C NMR (100 MHz, DMSO-d₆) δ 36.8 (CH₂), 43.9 (CH₂Cl), 55.5 (2OCH₃), 57.8 (OCH₂), 60.8 (OCH₃), 106.7 (trimethoxyphenyl C-2, C-6), 109.4 (pyrimidine C-5), 114.9 (p-chloroethoxyphenyl C-3, C-5), 118.8 (phenyl C-2, C-6), 121.5 (COCH=CH), 126.4 (trimethoxyphenyl C-1), 127.4 (p-chloroethoxyphenyl C-1), 128.1 (p-chloroethoxyphenyl C-2, C-6), 129.5 (p-chlorophenyl C-2, C-6), 129.8 (p-chlorophenyl C-3, C-5), 132.7 (phenyl C-3, C-5), 135.4 (p-chlorophenyl C-1), 136.8 (p-chlorophenyl C-4), 140.3 (phenyl C-4), 141.7 (trimethoxyphenyl C-4), 143.0 (phenyl C-1), 144.4 (COCH=CH), 153.5 (trimethoxyphenyl C-3, C-5), 159.4 (p-chloroethoxyphenyl C-4), 162.2 (pyrimidine C-6), 164.1 (pyrimidine C-4), 167.7 (pyrimidine C-2), 171.7 (CONH), 187.7 (CO); EIMS (m/z): 728.10 (M-1, 0.28%), 58.10 (100%); Anal. Calcd. for C₃₈H₃₃Cl₂N₃O₆S (729.15): C, 62.47; H, 4.55; N, 5.75. Found: C, 62.53; H, 4.76; N, 5.58.

Biological evaluations

Cytotoxic assay

To investigate cytotoxic activity of the final target compounds 9a–r, MTT assay was performed. Three different cell lines were used, leukaemia (K-562), breast (MCF-7) and colon (HT-29) cell lines. Cisplatin and erlotinib were the reference drugs used in this study. Half maximal concentration at which 50% of cells were viable was calculated as IC₅₀ in μM, according to cytotoxic assay reported protocol⁴⁷.

STAT3/STAT5a assays

Both K-562 and MCF-7 cell lines were seeded overnight in plates, then 10 µM of test compounds (9a and 9r for MCF-7 cells; 9d, 9f, and 9n for K-562 cells) or reference drug pacritinib was added for 24 h. A nuclear extract kit was used to extract nuclear fractions from treated cells using the manufacture’s procedure. STAT3 and STAT5a activations were analysed using the collected nuclear extracts (20 µg) through TransAM STAT3 and STAT5a activation assay guided by the manufacture’s protocol. The obtained results were expressed in the form of mean ± SD. Each experiment was done in triplicate.

Biological properties

The target compounds 9a–r were drawn using ChemDraw Ultra 10.0. Biological properties and drug likeness were predicted using online computational tool Molinspiration⁴⁸.

Predicted pharmacokinetic and toxicity properties

Pharmacokinetic properties (absorption, distribution, metabolism, and excretion) through determination of human intestinal absorption (HIA), in vitro caco-2 cell permeability, in vitro Madin-Darby Canine Kidney (MDCK) cell permeability, plasma protein binding (PPB), blood–brain binding (BBB), skin permeability, p-glycoprotein (Pgp), and cytochrome p450 isoforms inhibition data, in addition to toxicity (Ames test, rodent carcinogenicity assay and hERG-inhibition) were evaluated through preADMET online server⁴⁹.

Statistical analysis

Data obtained were expressed as means ± standard deviations (SDs). The results were considered significant when *p ˂ 0.05 or **p ˂ 0.005 using Student’s t-test was compared to reference drugs. The obtained values were representative of triplicate independent experiments.

Results and discussion

Chemistry

The target 2-TP/chalcone hybrids 9a–r were prepared from two synthesised starting materials 4a–f and 8a–c, as depicted in Schemes 1–3.

Heating under reflux condition chalcone derivatives 3a–f (synthesised from condensation of p-methoxy/chlorobenzaldehyde 1a&b with p-methyl/nitro/or ethoxychloroacetophenone 2a–c) and thiourea in presence of KOH afforded 2-TP derivatives 4a–f. The method was reported for compounds 4b and 4d^39,50 (Scheme 1).

Scheme 1. Synthesis of 2-thiopyrimidine derivatives 4a–f.

The other starting materials, chloroacetyl chalcone derivatives 8a–c were obtained by stirring p-aminochalcone derivatives 7a–c with chloroacetyl chloride, K₂CO₃ in chloroform at room temperature²⁴ (Scheme 2).

Scheme 2. Synthesis of chloroacetyl aminochalcone derivatives 8a–c.

S-Alkylation of 2-TPs 4a–f with acetylated chalcones 8a–c was achieved in acetonitrile using TEA as a base catalysis to obtain the target compounds 9a–r in 47–82% yield.

¹H NMR and ¹³C NMR spectroscopic tools were used to confirm formation of the target derivatives 9a–r. Thus, ¹H NMR spectra of compounds 9a–r displayed a singlet signal at δ 3.82–4.84 ppm attributed to (SCH₂CO) protons. Additionally, protons of chalcone fragment appeared as two doublet signals at δ 7.29–7.72 ppm and 7.69–8.31 ppm with coupling constant J= 11.6–15.6 Hz. Furthermore, amide NH proton appeared as a singlet signal at δ 10.71–10.91 ppm.

¹³C NMR spectra of compounds 9a–r showed appearance of a peak at δ 36.21–43.44 ppm characterised to SCH₂ carbon. Moreover, two carbonyl carbons at δ 170.71–173.07 ppm and 187.58–190.01 ppm related to (NHCO) and (C=O), respectively, were also appeared (Scheme 3).

Scheme 3. Synthesis of the target compounds 9a–r.

Biological activity

Cytotoxic activity

All target compounds 9a–r were screened against three different cancer cell lines, leukaemia (K-562), breast (MCF-7), and colon (HT-29). MTT assay was used. Both cisplatin and erlotinib were used as the reference drugs. Cytotoxicity results are recorded in Table 1.

Table 1. Cytotoxicity results of pyrimidine/chalcone hybrids 9a–r against three different cancer cell lines.

Compound	R	R^1	R^2	R^3	(IC50 μM)±SD
					K-562	MCF-7	HT-29
9a	OCH3	CH3	H	H	18.40 ± 0.76	3.56 ± 0.14	2.20 ± 0.04
9b	OCH3	CH3	CH3	H	3.62 ± 0.08	22.45 ± 1.57	16.36 ± 0.76
9c	OCH3	CH3	OCH3	OCH3	11.42 ± 0.04	4.25 ± 0.13	8.70 ± 0.28
9d	OCH3	NO2	H	H	0.77 ± 0.03	14.16 ± 0.74	25.92 ± 1.67
9e	OCH3	NO2	OCH3	H	7.05 ± 0.28	18.74 ± 0.92	7.31 ± 0.36
9f	OCH3	NO2	OCH3	OCH3	1.37 ± 0.03	5.77 ± 0.22	9.41 ± 0.43
9g	OCH3	OCH2CH2Cl	H	H	3.17 ± 0.11	10.40 ± 0.64	10.55 ± 0.47
9h	OCH3	OCH2CH2Cl	OCH3	H	7.07 ± 0.31	7.70 ± 0.32	18.77 ± 0.88
9i	OCH3	OCH2CH2Cl	OCH3	OCH3	9.71 ± 0.41	11.47 ± 0.81	11.47 ± 0.23
9j	Cl	CH3	H	H	4.26 ± 0.06	6.26 ± 0.24	6.26 ± 0.34
9k	Cl	CH3	OCH3	H	9.95 ± 0.29	28.65 ± 1.39	5.61 ± 0.18
9l	Cl	CH3	OCH3	OCH3	42.60 ± 1.99	13.46 ± 0.62	2.37 ± 0.07
9m	Cl	NO2	H	H	3.86 ± 0.14	3.90 ± 0.09	7.90 ± 0.22
9n	Cl	NO2	OCH3	H	1.05 ± 0.02	17.36 ± 0.75	2.10 ± 0.06
9o	Cl	NO2	OCH3	OCH3	12.35 ± 0.72	3.62 ± 0.084	3.62 ± 0.08
9p	Cl	OCH2CH2Cl	H	H	1.74 ± 0.04	11.64 ± 0.49	11.64 ± 0.63
9q	Cl	OCH2CH2Cl	OCH3	H	5.77 ± 0.16	6.81 ± 0.25	6.06 ± 0.29
9r	Cl	OCH2CH2Cl	OCH3	OCH3	10.67 ± 0.77	1.37 ± 0.07	4.74 ± 0.27
Cisplatin					2.31 ± 0.09	6.62 ± 0.29	1.12 ± 0.06
Erlotinib					9.85 ± 0.51	10.64 ± 0.58	9.20 ± 0.41

Regarding cytotoxic activity of the test compounds against leukaemia (K-562) cell line, compounds 9d, 9f, 9n, and 9p were the most potent compounds with IC₅₀ ranged from 0.77 to 1.74 μM if compared to cisplatin, the reference drug (IC₅₀=2.31 μM). Their common feature was presence of one or more para substituted phenyl ring(s) with electron withdrawing group (NO₂, Cl) at pyrimidine core.

Compounds 9b, 9e, 9g–j, 9m, and 9q exhibited potent inhibitory activity with IC₅₀ values ranged from 3.17 to 9.71 μM, if compared to the second reference erlotinib (IC₅₀: 9.85 μM). P-Methoxyacyloyl derivative 9k, with IC₅₀ value = 9.95 μM, was nearly equal in potency to erlotinib. Compounds 9a, 9c, 9o, and 9r showed moderate inhibitory activity (IC₅₀=10.67–18.40 μM). The lowest inhibitory activity was observed in compound 9l (IC₅₀=42.60 μM), bearing p-chlorophenyl and p-tolyl rings at pyrimidine scaffold, beside, trimethoxyphenyl chalcone hybrid.

Concerning MCF-7 cell line, the most active derivative was 9r (IC₅₀=1.37 μM) compared to the reference drug cisplatin (IC₅₀=6.62 μM). It is characterised by presence of p-chlorophenyl ring and p-chloroethoxyphenyl ring at pyrimidine core together with trimethoxy chalcone part.

Other compounds exerted excellent activity were 9a, 9c, 9f, 9j, 9m, and 9o (IC₅₀=3.56–6.26 μM). Additionally, compound 9q was nearly equipotent to the reference drug cisplatin (IC₅₀=6.81 μM). Derivatives 9g and 9h with IC₅₀=10.40 and 7.70 μM, respectively, were more potent than erlotinib (IC₅₀=10.64 μM).

Moderate activity was observed in compounds 9d, 9e, 9i, 9l, 9n, and 9p (IC₅₀=11.47–18.74 μM).

Compounds 9b and 9k showed weak inhibitory activity with IC₅₀ values equal to 22.45 and 28.65 μM, respectively. Both of them have dimethoxyphenyl ring on chalcone part, and p-tolyl ring at pyrimidine core.

By inspecting cytotoxicity results of HT-29 cell line, compounds 9a, 9l, and 9n showed IC₅₀ (2.10–2.37 μM) near in potency to the reference drug cisplatin (IC₅₀=1.12 μM). Compounds 9c, 9e, 9j, 9k, 9m, 9o, 9q, and 9r showed significant activity with IC₅₀ values between 3.62 and 8.70 μM, compared to erlotinib (IC₅₀=9.20 μM). While, rest of the compounds exhibited weak inhibitory activity (IC₅₀=9.41–25.92 μM). Results showed that no effect was observed regarding substituents on the two hybrid structures pyrimidine and chalcone.

Finally, dual cytotoxic activity was observed for compound 9f (against K-562 and MCF-7 cell lines), and for compound 9a (on MCF-7 and HT-29 cell lines) and 9n (against K-562 and HT-29 cell lines).

Cytotoxicity against normal cell line (WI38)

To know cytotoxicity of the most active compounds, they were tested against normal human fibroblast cell line (WI38) and IC₅₀ values are represented as in Figure 2. Cisplatin was used as a reference drug. All the test compounds showed higher IC₅₀ values (29.19–40.13 µM) than the reference drug (18.86 µM) except compound 9a which exerted cytotoxic activity (IC₅₀=17.09 µM) slightly less than cisplatin.

Figure 2. Cytotoxicity (IC₅₀) of the most active derivatives and cisplatin against WI38 cell line.

STAT3 and STAT5a inhibitory activity determination

The most active compounds in cytotoxic assay against leukaemia cell line K-562 and human breast adenocarcinoma cells MCF-7 were further tested as inhibitors for STAT3 and STAT5a enzymes. Pacritinib, an inhibitor for both STAT3 and STAT5a⁴⁹ was used in this study as a reference drug.

The results are listed in Table 2. They indicated that the test compounds showed inhibitory activity against both STAT3 and STAT5a. Compounds 9d, 9n, and 9r were the most active against STAT3. Additionally, compound 9n was the most effective as STAT5a inhibitor. Compounds 9a, 9f, and 9r had also strong inhibitory activity against STAT5a. Dual inhibitory activity against STAT3 and STAT5a was observed mainly in compound 9n. For this compound, both phenyl rings on pyrimidine core were para-substituted with electron withdrawing groups (NO₂ and Cl), beside presence of disubstituted methoxyphenyl ring at chalcone hybrid.

Table 2. STAT3 and STAT5a inhibitory activity of compounds 9a, 9d, 9f, 9n, 9r and reference drug pacritinib.

Compound/no.	Inhibition IC50 (µM)
	STAT3	STAT5a
9a/MCF7	242.53 ± 9.24	83.78 ± 3.28
9d/K562	160.01 ± 4.59	116.31 ± 4.13
9f/K562	244.74 ± 11.07	77.65 ± 2.91
9n/K562	113.31 ± 3.22	50.75 ± 1.26
9r/MCF7	148.69 ± 3.81	63.24 ± 1.57
Pacritinib/MCF7	79.47 ± 2.17	54.35 ± 1.09
Pacritinib/K562	65.49 ± 2.55	69.81 ± 1.82

Biological properties

Molinspiration was used to predict bioactivity scores for all the target compounds 9a–r. The obtained results are recorded in Table 3. It was found that most of the prepared compounds had bioactivity values in the range −0.5 to 0.00. This revealed that the designed pyrimidine/chalcone derivatives might be involved in moderate interactions with G-protein-coupled receptors (GPCRs) and protease inhibitors. However, the bioactivity prediction was not in the standard range against other receptors such as ion channel modulator, kinases and nuclear receptor ligand.

Table 3. Biological properties, prediction, and drug likeness of the target compounds.

Compound	GPCR ligand	Ion channel modulator	Kinase inhibitor	Nuclear receptor ligand	Protease inhibitor	Drug likeness score
9a	−0.43	−1.03	−0.58	−0.69	−0.43	0.49
9b	−0.56	−1.27	−0.78	−0.90	−0.51	0.54
9c	−0.73	−1.55	−1.01	−1.17	−0.62	0.96
9d	−0.61	−1.25	−0.83	−0.92	−0.53	0.07
9e	−0.78	−1.53	−1.07	−1.17	−0.65	0.11
9f	−0.99	−1.86	−1.35	−1.50	−0.80	0.49
9g	−0.69	−1.44	−0.81	−1.01	−0.56	0.83
9h	−0.88	−1.75	−1.08	−1.29	−0.70	0.79
9i	−1.11	−2.09	−1.39	−1.64	−0.87	1.18
9j	−0.39	−0.94	−0.52	−0.63	−0.44	0.82
9k	−0.50	−1.16	−0.68	−0.81	−0.50	0.80
9l	−0.64	−1.41	−0.89	−1.06	−0.58	1.07
9m	−0.55	−1.13	−0.75	−0.83	−0.52	0.43
9n	−0.69	−1.39	−0.95	−1.06	−0.62	0.40
9o	−0.88	−1.69	−1.21	−1.36	−0.74	0.63
9p	−0.61	−1.31	−0.71	−0.91	−0.54	1.14
9q	−0.79	−1.60	−0.96	−1.17	−0.66	1.05
9r	−1.00	−1.92	−1.24	−1.49	−0.81	1.40

Drug likeness is a complex balance between various molecular properties like, molecule size, hydrogen bonding characters, electronic distribution and hydrophobicity⁵¹. The results in Table 3 showed that all the final target compounds had positive predictable score values which stranded for good drug likeness behaviour, especially compound 9r as represented in Figure 3.

Figure 3. Drug likeness score value (1.40) for compound 9r.

Predicted pharmacokinetic and toxicity properties

Prediction of the major pharmacokinetic parameters such as absorption, distribution, metabolism, and excretion, in addition to toxicological properties, such as mutagenicity, carcinogenicity, and cardiac toxicity was estimated using Pharmacokinetics/PreADMET Toxicity Predictor⁵² (Table 4).

Table 4. Pharmacokinetic properties assessment of the target synthesised compounds 9a–r.

Compound	Absorption				Distribution		Metabolism (CYP) and excretion
	HIA (%)	SP LogP (cm/h)	Caco2 (nm/sc)	MDCK (nm/sc)	BBB (c.brain/c.blood)	PPB %	2C19	2C9	2D6	3A4	Pgp Inh.
9a	97.50	−1.72	54.34	0.05	0.03	98.31	No	Yes	No	Yes	Inh.
9b	97.36	−1.73	54.31	0.06	0.03	99.63	No	Yes	No	Yes	Inh.
9c	97.25	−1.74	54.26	0.06	0.03	97.83	No	Yes	No	Yes	Inh.
9d	98.78	−2.26	28.65	0.04	0.49	92.53	No	Yes	No	Yes	Inh.
9e	99.22	−2.25	29.60	0.04	0.45	96.23	No	Yes	No	Yes	Inh.
9f	99.39	−2.22	30.34	0.04	0.38	96.95	No	Yes	No	Yes	Inh.
9g	97.69	−2.09	37.98	0.05	0.02	92.10	No	Yes	No	Yes	Inh.
9h	97.54	−2.03	38.68	0.05	0.02	95.16	No	Yes	No	Yes	Inh.
9i	97.42	−1.96	39.36	0.05	0.02	95.17	No	Yes	No	Yes	Inh.
9j	97.95	−1.74	51.82	0.06	0.08	90.94	No	Yes	No	Yes	Inh.
9k	97.83	−1.74	52.19	0.07	0.05	94.17	No	Yes	No	Yes	Inh.
9l	97.69	−1.73	52.52	0.07	0.04	94.05	No	Yes	No	Yes	Inh.
9m	97.49	−2.28	33.88	0.04	0.37	93.03	No	Yes	No	Yes	Inh.
9n	97.96	−2.28	35.90	0.04	0.50	91.43	No	Yes	No	Yes	Inh.
9o	98.56	−2.27	37.81	0.04	0.57	92.17	No	Yes	No	Yes	Inh.
9p	98.11	−2.14	42.99	0.05	0.05	92.90	No	Yes	No	Yes	Inh.
9q	97.99	−2.10	43.47	0.05	0.03	91.65	No	Yes	No	Yes	Inh.
9r	97.86	−2.05	43.93	0.05	0.03	91.71	No	Yes	No	Yes	Inh.

Absorption refers to the process by which the drug can go to the systemic circulation through the organs of the body. Several routes for absorption such as oral absorption (human intestinal absorption, HIA), skin permeability (SP, logKp), and permeability through certain cells such as Caco2 (derived from human colon adenocarcinoma cells) and MDCK cells were measured.

By inspecting results recorded in Table 4, it was found that compounds 9a–r showed good intestinal absorption all above 97.25% (permissible limit: 70–100%abs). Skin permeability was found to be slightly less than acceptable range (−2.5 logKp). Moreover, moderate permeability through in vitro Caco2 cells ranged from 54.34 to 28.65 nm/sc were observed. While, low values were detected for in vitro MDCK cells.

The second property is the distribution, through which the transformation of the molecules from one tissue or organ to another can be predicted. Blood–brain barrier (BBB) and PPB were two distribution parameters used in this study. BBB permits the diffusion of hydrophobic and small molecules to the brain. It is an important predictor for central nervous system (CNS) drug discovery. Moreover, the measured of percentage of a molecule bound to plasma protein (%PPB) was also helpful in prediction of distribution for the novel target compounds.

Results showed that all the test compounds displayed strong PPB value (90.94–99.63%) indicating prolonged half-lives and limited brain penetration. Consequently, BBB (unbound brain-to-plasma ratio) was low in most compounds except in nitrophenyl containing derivatives 9d–9f and 9m–9o (0.37–0.57) which was medium and around the acceptable range to be CNS active compounds (>0.4).

Metabolism, the biotransformation or chemical modification of exogenous compounds to increase their water solubility by increasing their hydrophobicity facilitating their excretion can be predicted either in phase I or phase II. Cytochrome P450 isoforms, calculate the ability of the test compounds to be inhibitor to drug metabolising enzymes such as CYP2C19, CYP2C9, CYP2D6, CYP3A4, and CYP1A2. Moreover, glycoprotein (P-gp) inhibition measured to predict excretion property of the target compounds.

The test compounds showed good inhibitory behaviour for CYP2C9 and CYP3A4 and did not show inhibition behaviour for CYP2C19 and CYP3A4. All compounds had inhibitory effect on P-gp.

Prediction of toxicological behaviour of the test compounds was obtained by measuring AMES test (to predict mutagenicity of the compounds), carcino-Mouse/Rate (to test carcinogenicity of the compounds), and hERG-inhibition (to check cardiac toxicity of the target synthesised molecules) (Table 5). Half of test compounds showed non-mutagenic behaviour in AMES test. All compounds had negative carcinogenic effect in mouse and rats, in addition to medium risk as cardiotoxic agents. From the predicted ADMET properties of the novel synthesised compounds, it was justified that they may have good characters as lead compounds.

Table 5. Toxicity assessment of the target synthesised compounds 9a–r.

Compound	AMES	Carcino-Mouse	Carcino-Rat	hERG-inhibition
9a	Mutagen	Negative	Negative	Medium
9b	Non-mutagen	Negative	Negative	Medium
9c	Non-mutagen	Negative	Negative	Medium
9d	Mutagen	Negative	Negative	Medium
9e	Mutagen	Negative	Negative	Medium
9f	Mutagen	Negative	Negative	Medium
9g	Non-mutagen	Negative	Negative	Medium
9h	Non-mutagen	Negative	Negative	Medium
9i	Non-mutagen	Negative	Negative	Medium
9j	Mutagen	Negative	Negative	Medium
9k	Mutagen	Negative	Negative	Medium
9l	Non-mutagen	Negative	Negative	Medium
9m	Mutagen	Negative	Negative	Medium
9n	Mutagen	Negative	Negative	Medium
9o	Mutagen	Negative	Negative	Medium
9p	Non-mutagen	Negative	Negative	Medium
9q	Non-mutagen	Negative	Negative	Medium
9r	Non-mutagen	Negative	Negative	Medium

Structure–activity relationship of target compounds

Structure–activity relationship (SAR) study for the target compounds 9a–r focussed on two important scaffolds, pyrimidine and chalcone. There was a relationship between presence of small electron donating group such as –CH₃, (D_s), large electron donating group such as –OCH₂CH₂Cl, (D_l) or electron withdrawing group, –NO₂, (W) in para position of phenyl ring at pyrimidine C-4 and electron donating group, –OCH₃, (D) or electron withdrawing group, –Cl, (W) in para position of phenyl ring at pyrimidine C-6 with that of (mono-, di-, or tri-)methoxyphenyl ring of chalcone part, and between cytotoxic activities on the three different tested cell lines K-562, MCF-7, and HT-29, as represented in Figure 4.

Figure 4. SAR study of the target compounds 9a–r.

In compounds 9a–c, pyrimidine core carried p-tolyl group and p-methoxyphenyl group at C-4 and C-6, respectively. Cytotoxic activity against K-562 cell line was maximised in compound 9b bearing dimethoxyphenyl chalcone moiety then decreased in 9c and 9a (trimethoxyphenyl and methoxyphenyl chalcones, respectively).

The order of reactivity was altered when evaluated against MCF-7 or HT-29 cell lines, where 9a>9c>9b.

For compounds 9d–f, replacement of p-tolyl group at pyrimidine C-4 with p-nitrophenyl group and keeping p-methoxyphenyl group at pyrimidine C-6 constant, led to variation in cytotoxic activity. Thus, compound 9d (with p-methoxyphenyl chalcone part) was the most potent against K-562 cell line, than 9f (trimethoxyphenyl chalcone analogue) and finally 9e (dimethoxyphenyl chalcone analogue). While against MCF-7, the order was 9f>9d>9e. For HT-29, 9e was the most potent than 9f and at last 9d.

Regarding compounds 9g–i, they characterised by bearing electron donating groups at para position of two phenyl rings at pyrimidine C-4 and C-6; however, presence of large sized electron donating group such as –OCH₂CH₂Cl at phenyl ring of pyrimidine C-4 led to increase its lipophilic character.

The order of cytotoxic activity against K-562 was found to be 9g (methoxyphenyl chalcone)>9h (dimethoxyphenyl chalcone)>9i (trimethoxyphenyl chalcone), and for MCF-7 cell line was, 9h>9g>9i. While converted to be 9i>9g>9h in case of HT-29 cell line.

In 9j–l derivatives, electron withdrawing group (–Cl) was introduced to para position of phenyl ring at pyrimidine C-6, while pyrimidine C-4 carried small sized electron donating group (–CH₃) at para position of its phenyl ring.

The most active compound was 9j (methoxyphenyl chalcone) in both K-562 and MCF-7 cell lines, while 9l (trimethoxyphenyl chalcone) was the most potent against HT-29 cell line.

Compounds 9m–o, p-tolyl ring were replaced with p-nitrophenyl ring at pyrimidine C-4, while p-chlorophenyl ring at pyrimidine C-6 was kept constant.

The most potent derivatives against K-562 were dimethoxyphenyl chalcone 9n, than methoxyphenyl chalcone derivative 9m. While, trimethoxyphenyl chalcone derivative 9o and methoxyphenyl chalcone analogue 9m showed nearly equal potency against MCF-7. For HT-29, 9n was the most potent than 9o and finally 9m.

In compounds 9p–r, incorporation of p-chloroethoxyphenyl group at pyrimidine C-4, while keeping p-chlorophenyl group at pyrimidine C-6 constant, resulted in changing order of cytotoxic activity against K-562 to be 9p (methoxyphenyl chalcone)>9q (dimethoxyphenyl chalcone), and still the least potent compound was 9r (trimethoxyphenyl chalcone). However, for MCF-7 and HT-29, it was observed that 9r was the most potent than 9q and finally 9p.

Conclusions

A novel series of 2-TP/chalcone hybrids 9a–r was designed to be as anticancer agents. They were synthesised and identified using different spectroscopic techniques. Their cytotoxic activities against three different cancerous cell lines, K-562, MCF-7, and HT-29 were evaluated. The synthesised compounds showed strong to moderate cytotoxic activities especially against K-562 and MCF-7 cell lines. The highest cytotoxic activity against K-562 cell line was observed in compounds 9d, 9f, 9n, and 9p with IC₅₀ values in the range of 0.77–1.74 µM, compared to the reference drug, cisplatin (IC₅₀=2.31 µM). For cytotoxic activity against MCF-7 cell line, compounds 9a, 9c, 9f, 9j, 9m, 9o, and 9r exhibited the highest activities with IC₅₀ values of 1.37–6.26 µM (cisplatin IC₅₀=6.62 µM). While, moderate cytotoxic activity was noticed for test compounds against colon HT-29 cell line. The most potent derivatives between them were 9a, 9l, and 9n (IC₅₀=2.10–2.37 µM), if compared with cisplatin (IC₅₀=1.12 µM).

The most active derivatives 9a, 9d, 9f, 9n, and 9r (either against K-562 and/or MCF-7 cell lines) were selected for further evaluation against human normal fibroblast cells (WI38). All of them had IC₅₀ values (29.19–40.13 µM) higher than that of the reference cisplatin (IC₅₀=18.86 µM), except 9a analogue (IC₅₀=17.09 µM) which was slightly less than cisplatin.

Moreover, STAT3 and STAT5a inhibitory activities were determined for the five later compounds. Compounds 9d and 9n showed remarkable inhibitory activity against STAT3, while, compounds 9a, 9f, 9n, and 9r were the most effective at inhibiting STAT5a. Dual inhibitory activity at STAT3 and STAT5a was observed in compound 9n which beared p-nitrophenyl and p-chlorophenyl rings at pyrimidine core in addition to dimethoxyphenyl at chalcone part. On the other hand, physicochemical properties, drug likeness scores, pharmacokinetics and toxicity properties were predicted for all the synthesised compounds 9a–r.

Acknowledgements

The authors thank Dr. Essam Rashwan, head of the confirmatory diagnostic unit VACSERA-Egypt for evaluation of anticancer activity and enzyme assays.

Disclosure statement

The authors declare that there is no any conflict of interest.