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Article

Synthesis, Characterization, and Anticancer Activities Evaluation of Compounds Derived from 3,4-Dihydropyrimidin-2(1H)-one

by
Ye Liu
1,
Jiaqi Liu
1,
Renmei Zhang
1,
Yan Guo
1,
Hongbo Wang
1,
Qingguo Meng
1,
Yuan Sun
2 and
Zongliang Liu
1,*
1
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China
2
Department of Biochemistry and Molecular Medicine, School of Medicine, University of California Davis, Sacramento, CA 95817, USA
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(5), 891; https://doi.org/10.3390/molecules24050891
Submission received: 9 January 2019 / Revised: 25 February 2019 / Accepted: 26 February 2019 / Published: 3 March 2019
(This article belongs to the Section Medicinal Chemistry)
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Versions Notes

Abstract

:
3,4-dihydropyrimidin-2(1H)-one compounds (DHPMs) possess extensive biological activities and are mainly prepared via Biginelli reaction and N-alkylation. In the present study, selective alkylation of N1 was investigated by using tetrabutylammonium hydroxide. In vitro cytotoxicity study on all synthesized compounds demonstrated that introduction of the aryl chain in the R3 as well as the low electron-donating group in the R1 of DHPMs contributed to the anti-proliferative potency. A larger value of the partition coefficient (Log P) and suitable polar surface area (PSA) values were both found to be important in order to maintain the antitumor activity. The results from in vivo study indicated the great potential of compound 3d to serve as a lead compound for novel anti-tumor drugs to treat glioma. Pharmacophore study regarding the structure-activity relations of DHPMs were also conducted. Our results here could provide a guide for the design of novel bioactive 3,4-dihydropyrimidin-2(1H)-one compounds.

1. Introduction

Noncommunicable diseases (NCDs) are a major threat to global health, causing a significant amount of death every year. Second only to cardiovascular disease, cancer is becoming a global burden which lead to an estimation of 8.7 million deaths in 2015 [1]. Moreover, cancer is expected to rank as the leading cause of death and the single most significant barrier for the increase of life expectancy in every country worldwide [2]. Contrary to common misperception, cancer is a major health challenge not only in high-income countries but also in low- and middle-income countries (LMICs), where the number of cancer occurrence is rapidly growing [3]. Unfortunately, almost all anticancer drugs are associated with serious side effects, making the search for novel chemical agents that are cytotoxic to cancer cells with less side effects an urgent need.
The interests of using DHPMs in medicinal chemistry is dramatically growing (Figure 1) due to their therapeutic and pharmacological properties [4,5]. It has been reported that DHPMs can possess various biological activities including antiviral [6,7], antitumor [8], anti-inflammatory [9], antidiabetic [10], antibacterial [11], antifungal [12], anti-epileptic [13], antimalarial [14], and antileishmanial [15] and others upon suitable structural modification. The highly functionalized DHPM 10, termed MAL3-101, had been observed with effect of inducing breast cancer cell apoptosis [16]. More recently, DHPMs have emerged as the integral backbone of several calcium channel blockers [17,18], antioxidant molecules [19], and radical scavengers [20,21,22]. In addition, Barbosa et al. reported the synthesis and biological evaluation of a series of DHPMs functionalized with selenocyanides as potential multi-targeted therapeutics against Alzheimer’s disease (AD) [23].
In DHPMs, R1 is usually an aryl group, such as phenyl or pyridyl, while R2 is an ester or amide group and R3 is an alkyl group, such as methyl or ethyl. General method for the synthesis of DHPMs starts with firstly obtaining the basic scaffold via Biginelli reaction followed by N alkylation. Mohammadi and Behbahani reviewed the synthesis of DHPMs and improved procedures for the preparation of DHPMs under solvent-free conditions or with the presence of solvent [24]. Dallinger and Kappe introduced a selective N1-alkylation method of DHPMs using Mitsunobu reaction [25]. However, the yield of Mitsunobu reaction was low, and the reagents were relatively expensive, making it not suitable for practical synthesis. Singh et al. provided another N-alkylation method catalyzed by inorganic strong base [26]. In the present report, not only did we find highly selective N1-alkylation of DHPMs in the presence of tetrabutylammonium hydroxide, but also we investigated the biological importance of the newly synthesized molecules both in vitro and in vivo.

2. Results and Discussion

2.1. Chemistry

The various non-alkylated DHPM moieties used in this report were synthesized through one pot Biginelli condensation reaction according to the reported method [27]. The effects of different choices of bases on the reaction, including sodium hydride (NaH), lithium hydroxide (LiOH·H2O), potassium carbonate (K2CO3) and some pKa similar organic base, were carried out to perform N1 and N3 dialkylation of DHPMs. While using a strong base, such as LiOH·H2O, NaH and potassium tert-butoxide (KTB), the reactions were proceeding very fast. However the dialkyl product was formed and detected even from the beginning of the reaction. In some reactions the yields of dialkylation were even higher than that of N1-alkylation. N-alkylation cannot be achieved when a weak base is used, such as K2CO3, triethylamine (Et3N), 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) and tetramethylguanidine (TMG). Interestingly, when tetrabutylammonium hydroxide was selected as the base, the yield was similar to that of cesium carbonate (Cs2CO3), while no dialkyl products was found (Table S1 in Supplementary Materials). A possible explanation for this phenomenon is that N3-alkylation of DHPMs had a large steric effect, so the steric-hindered base like tetrabutylammonium hydroxide would favor the mono-alkylation reaction. The yields of the DHPMs were reported in Table 1.

2.2. Structure–Activity Relationship (SAR) Studies

2.2.1. Cytotoxicity Activities with SAR

Cytotoxic activity of the DHPMs are strongly dependent on their structure. Yadlapalli et al. screened 21 compounds in vitro anticancer screening against MCF-7 human breast cancer cells, and found the maximum GI50 was 33.2 μM. The results indicated that presence of thio-urea functional group in DHPMs enhanced the in vitro anticancer activity [28]. In vitro cytotoxicity of all synthesized compounds containing X = O were assayed on four cell lines, namely U87, U251 human malignant glioma cell lines, HeLa human cervical cancer cell line and A549 human lung cancer cell line. Cancer cell lines were exposed to drug solution at concentration of 10 µM for 72 h, and results were summarized in Table 2. On the whole, no certain trend in inhibitory activity was observed in Hela and A549 cell lines, indicating that these compounds were selective toward certain tumor types. Some of the tested compounds showed effective cytotoxicity in U87 and U251 cell lines.
Table 2 showed that compound 1a resulted in cell viability of 87.34 ± 1.24 and 67.14 ± 4.61 in U87 and U251 cell lines. For SAR studies, we maintained the R1 as Br, R2 as ethyl acetate and explored R3 first with a series of halohydrocarbons. Compounds 1b, 1f, 1g, and 1i, with an alkyl side chain replacing the methyl 4-bromobutanoate of 1a, were found to have similar activity as 1a. Compound 1h, with an 1-bromohexane in R3, displayed decent activity, indicating that the length of the alkyl side chain in the R3 would affect the potency. Compound 1d, with a 4-bromobenzyl group in the R3, also demonstrated strong cytotoxicity, suggesting that the R3 may tolerate variations to some degree. We then explored the R2 with the goal to compare the ester group and the amide group on cell viability. Compared with 1e, compound 7e and 8e differ in R2, had no significant change in activity. Compounds 7c, 7d, 7f, 8a, and 8d were also tested, and not satisfactory performances were observed, suggesting that the amide group may not be compatible in that position. For R1 of DHPMs, we proposed that the capability of electron-donating group may affect the cell viability and the SAR of the R1 group was explored. Compound 3a, with a low electron-donating 4-biphenyl group instead of 4-phenylmorpholine group or 4-methoxyphenyl, was found to have better potency than 4a and 2a. Compound 1a, with the 4-bromo phenyl group in the R1, had significant activity in U251 cell lines, suggesting that a low electron-withdrawing group in the R1 may contribute to augment the activity. Compound 5a and 6a, with a 4-nitrophenyl group and 4-pyridinylphenyl group in the R1, were also not active in the cell study, suggesting that high electron-withdrawing substituent in the R1 may not be tolerated. Studies on different electron-donating groups in the R1 had shown that a maximum cytotoxic activity may be achieved for low electron-donating ability or low electron-withdrawing ability. Based on the above studies, the cell viability of compounds 3c, 3d, 3e, 3g, and 3h were tested and compound 3d and 3g, with 4-biphenyl low electron-donating group in the R1 and alkyl chain or 4-bromobenzyl in the R3, were found to have good activity.

2.2.2. Half Maximal Inhibitory Concentration (IC50) Study of Compounds 1d, 1h, 3d and 3g

The values of 50% inhibitory concentration (concentration of drug yielding a 50% cell viability decrease, IC50) measured for the distinct compounds investigated were comprised in Table 3, which confirmed that the active compounds can inhibit tumor cell growth. In tumor cells, inhibition of heat shock protein 90 (HSP90) results in the degradation of oncoproteins which is crucial to malignant progression [29]. Preclinical data suggest that synthetic HSP90 inhibitors such as BIIB021 may be active against tumors with acquired multidrug resistance [30]. All of the tested compounds had IC50 in the micromolar range against U87 and U251 cell lines. These results evidenced that although compounds 1d (9.72 ± 0.29 µM in U87 cell line, 13.91 ± 0.86 µM in U251 cell line), 1h (9.3 ± 0.81 µM in U87 cell line, 14.01 ± 0.76 µM in U251 cell line), 3d (12.02 ± 0.5 µM in U87 cell line, 6.36 ± 0.73 µM in U251 cell line), and 3g (9.52 ± 0.81 µM in U87 cell line, 7.32 ± 0.86 µM in U251 cell line) did not display stronger cytotoxic activity on the U87 and U251 cell lines compared to positive control, they can still possess certain cytotoxic activity in micromolar range. In the present study, it was verified that the alkyl chain or aryl chain in the R3, and low electron-donating ability or low electron-withdrawing ability in the R1 displayed obvious effect. As expected, all of the four compounds especially 3d displaying high Log P (5.01) values and suitable PSA (58.64).

2.2.3. Effects on Xenograft Model of Compounds 3d and 3g

We further investigated the efficacy of compounds 3d and 3g in xenograft tumor model on the basis of their good membrane permeability and IC50 value. In brief, GL261 mouse malignant glioma cells were inoculated subcutaneously in right frank regions. Mice were treated with either: control, positive control (30 mg/kg), or compound 3d (100 mg/kg), or compound 3g (100 mg/kg). The results of representative studies were summarized in Table 4, and examples were shown in Figure 2. These data indicated that in xenograft tumor model, compound 3d or 3g were able to significantly inhibit tumor growth, with inhibition ratios (IR) of 54.9% and 34.3%, respectively. The compound 3d produced a similar antitumor activity compared with BIIB021 (IR 59.7%). This study had shown that the aryl chain in the R3, and 4-biphenyl low electron-donating group in the R1 had a moderate growth inhibitory effected on xenograft tumor model. The compound 3d also had suitable Log P and PSA values. Although the compounds were less active when compared to the positive control, compound 3d had the potential to serve as lead compound and be further optimized to improve activity.

2.3. Pharmacophore Requirements

According to previous studies, thirteen substituted DHPMs with good anticancer activities were selected to generate pharmacophores and guide the design of novel DHPMs derivatives (Table S3 in Supplementary Materials). Galahad module of Sybyl-X 2.0 (Certara, Princeton, NJ, USA) was used to generate pharmacophore using population size of 20 and maximum generations as 10. Finally, 13 models were generated (Figure 3). The best pharmacophore model was chosen with low energy and high value of steric and hydrogen bonding. Eight pharmacophoric features, namely three acceptor atoms (AA-4, 5, 6), two donor atoms (DA-3, 8) and three hydrophobic center (HY-1, 2, 7) were identified. The two acceptor atoms were at the R2 position and X position. Hydrophobic center is the DHPMs parent ring, R1 position and R3 position.

3. Materials and Methods

3.1. General Information

All reagents were obtained from commercial suppliers and were used without any further purification unless otherwise stated. Column chromatography was performed using an SRL silica gel (200–300 mesh). Thin layer chromatography was performed using Merck silica gel GF254 plates. Melting points were measured on an XT3A micro-melting point apparatus and are uncorrected (Beijing Keyi Company, Beijing, China). 1H NMR and 13C NMR spectra were recorded with a Bruker AV-400 instrument or a Bruker AV-300 (Bruker, Ettlingen, Germany). Chemical shifts were reported as δ values (ppm) from internal reference tetramethylsilane (TMS). All coupling constants were reported in hertz (Hz), and proton multiplicities were labeled as br (broad), s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), and m (multiplet). HR-MS were performed on a Waters Vion IMS Q-tof (Waters, MA, USA).

3.2. Synthesis and the General Procedure for N1-alkylation

Tetrabutylammonium hydrogen sulfate (TBAS 0.1 eq) was added to a solution of ethyl 4-(4-bromophenyl)-6-methyl-2-oxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylate (200.0 mg, 0.59 mmol) and tetrabutylammonium hydroxide (0.26 mL, 1.0 mmol) in anhydrous DMF (4.0 mL). The mixture was stirred at 45 °C for 1.5 h under anhydrous condition. Then 2-chlorobenzyl chloride (0.13 mL, 1.06 mmol), potassium iodide (0.1 eq) were added to the reaction mixture slowly and stirred at 45 °C for 16 h. Saturated NaCl solution (25 mL) was added and then the reaction mixture was extracted with ethyl acetate (3 × 25 mL). The combined organic layer was washed with water (2 × 50 mL), followed by brine solution (1 × 50 mL). The organic layer was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. Column chromatographic purification using a methanol in dichloromethane gradient (dichloromethane: methanol = 60:1–5:1) yielded compounds 1a–8e.

3.2.1. N1-Substituted Ethyl4-(4-bromophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates 1a–1j

Ethyl 4-(4-bromophenyl)-1-(4-methoxy-4-oxobutyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1a). White solid, m.p.: 92.4–95.0 °C, yield: 8.0%, Rf value: 0.5 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.14 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 5.53 (s, 1H, NH), 5.35 (d, J = 2.9 Hz, 1H, Ar-CH), 4.12 (q, J = 7.1 Hz, 2H, OCH2), 3.98–3.88 (m, 1H, NCH2), 3.71 (d, J = 5.3 Hz, 3H, OCH3), 3.66 (dd, J = 9.5, 5.1 Hz, 1H, NCH2), 2.57 (s, 3H, =CCH3), 2.35–2.24 (m, 2H, COCH2), 2.00–1.77 (m, 2H, CH2), 1.20 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 173.35 (C=O), 165.98 (C=O), 153.11 (C=O), 150.50 (Ar-C), 143.74 (C-N), 131.86 (2 × Ar-C), 128.82 (2 × Ar-C), 120.95 (Br-Ar-C), 103.30 (C=C), 60.24 (CH2), 52.28 (CH), 51.88 (CH3), 41.43 (CH2), 30.67 (CH2), 24.98 (CH2), 16.08 (CH3), 14.56 (CH3). HRMS (ESI): m/z calcd for C19H23BrN2O5 [M + H]+ 439.0869, found 439.0868.
Ethyl 4-(4-bromophenyl)-1-(3-ethoxy-3-oxopropyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1b). White solid, m.p.: 126.8–127.0 °C, yield: 52.2%, Rf value: 0.6 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.48–7.42 (m, 2H, 2 × Ar-H), 7.25 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 5.37 (s, 1H, Ar-CH), 4.19–4.05 (m, 4H, 2 × OCH2), 3.83–3.73 (m, 1H, NCH2), 3.29–3.20 (m, 1H, NCH2), 2.70 (d, J = 16.4 Hz, 1H, CH2), 2.49–2.38 (m, 1H, CH2), 2.33 (s, 3H, =CCH3), 1.25 (q, J = 7.3 Hz, 6H, 2 × CH3). 13C NMR (101 MHz, DMSO-d6) δ 171.71 (C=O), 165.37 (C=O), 152.05 (C=O), 148.21 (Ar-C), 142.66 (C-N), 132.08 (2 × Ar-C), 129.58 (2 × Ar-C), 121.40 (Br-Ar-C), 100.21 (C=C), 60.57 (2 × CH2), 59.97 (CH2, CH), 33.09 (CH2), 18.10 (CH3), 14.54 (2 × CH3). HRMS (ESI): m/z calcd for C19H23BrN2O5 [M + H]+ 439.0869, found 439.0870.
Ethyl 4-(4-bromophenyl)-1-(2-chlorobenzyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1c). White solid, m.p.: 125.6–127.8 °C, yield: 35.1%, Rf value: 0.5 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 7.36 (d, J = 7.7 Hz, 1H, Ar-H), 7.26 (s, 1H, Ar-H), 7.16 (d, J = 8.2 Hz, 3H, 3 × Ar-H), 6.90 (d, J = 7.4 Hz, 1H, Ar-H), 6.48 (s, 1H, NH), 5.44 (s, 1H, Ar-CH), 5.06 (d, J = 7.8 Hz, 2H, NCH2), 4.11 (q, J = 7.1 Hz, 2H, OCH2), 2.38 (s, 3H, =CCH3), 1.18 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 165.33 (C=O), 152.34 (C=O), 148.46 (2 × Ar-C), 141.90 (C-N), 134.59 (Ar-C-Cl), 132.88 (2 × Ar-C), 132.15 (2 × Ar-C), 130.04 (Ar-C), 129.59 (Ar-C), 129.34 (Ar-C), 128.00 (Ar-C), 121.56 (Br-Ar-C), 100.15 (C=C), 60.01 (CH2), 59.16 (CH2, CH), 18.21 (CH3), 14.53 (CH3). HRMS (ESI): m/z calcd for C21H20BrClN2O3 [M + H]+ 463.0424, found 463.0439.
Ethyl 1-(4-bromobenzyl)-4-(4-bromophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1d). White solid, m.p.: 190.4–191.9 °C, yield: 32.3%, Rf value: 0.7 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.44 (d, J = 8.3 Hz, 4H, 4 × Ar-H), 7.13 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.01 (d, J = 8.3 Hz, 2H, 2 × Ar-H), 5.72 (s, 1H, NH), 5.42 (d, J = 2.6 Hz, 1H, Ar-CH), 5.10 (d, J = 16.7 Hz, 1H, NCH2), 4.87 (d, J = 16.3 Hz, 1H, NCH2), 4.12 (q, J = 7.1 Hz, 2H, OCH2), 2.44 (s, 3H, =CCH3), 1.19 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 165.90 (C=O), 153.29 (C=O), 150.16 (Ar-C), 143.76 (C-N), 138.63 (Ar-C), 131.90 (4 × Ar-C), 128.98 (4 × Ar-C), 121.06 (Br-Ar-C), 120.43 (Br-Ar-C), 103.75 (C=C), 60.33 (OCH2), 52.53 (CH-NH), 45.05 (N-CH2), 16.54 (CH3), 14.53 (CH3). HRMS (ESI): m/z calcd for C21H20Br2N2O3 [M + H]+ 508.9898, found 508.9901.
Ethyl 4-(4-bromophenyl)-1-(4-methoxybenzyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1e). White solid, m.p.: 136.8–139.7 °C, yield: 43.6%, Rf value: 0.5 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.43–7.37 (m, 2H, 2×Ar-H), 7.11 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.05 (d, J = 8.6 Hz, 2H, 2 × Ar-H), 6.86–6.80 (m, 2H, 2 × Ar-H), 5.75 (s, 1H, NH), 5.39 (d, J = 2.9 Hz, 1H, Ar-CH), 5.16 (d, J = 15.9 Hz, 1H, NCH2), 4.78 (d, J = 15.8 Hz, 1H, NCH2), 4.09 (q, J = 7.1 Hz, 2H, OCH2), 3.81 (s, 3H, OCH3), 2.48 (s, 3H, =CCH3), 1.18 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 165.95 (C=O), 158.70 (C=O), 153.42 (Ar-C), 150.43 (Ar-C), 143.86 (C-N), 131.83 (2 × Ar-C), 130.91 (2 × Ar-C), 128.99 (2 × Ar-C), 128.15 (Ar-C), 120.98 (Br-Ar-C), 114.41 (2×Ar-C), 103.66 (C=C), 60.27 (CH2), 55.68 (CH3), 55.44 (CH), 52.49 (CH2), 16.55 (CH3), 14.53 (CH3). HRMS (ESI): m/z calcd for C22H23BrN2O4 [M + H]+ 459.0919, found 459.0917.
Ethyl 4-(4-bromophenyl)-6-methyl-2-oxo-1-propyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1f). White solid, m.p.: 114.7–115.3 °C, yield: 20.0%, Rf value: 0.3 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.45 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.15 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 5.59 (s, 1H, NH), 5.36 (d, J = 2.8 Hz, 1H, Ar-CH), 4.12 (q, J = 7.1 Hz, 2H, OCH2), 3.88 (td, J = 9.9, 9.4, 5.0 Hz, 1H, NCH2), 3.56 (ddd, J = 14.7, 9.9, 5.5 Hz, 1H, NCH2), 2.54 (s, 2H, =CCH3), 1.69–1.54 (m, 2H, CH2), 1.27 (t, J = 7.1 Hz, 1H, OCH2), 1.21 (t, J = 7.1 Hz, 2H, OCH2), 0.92 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 167.36 (C=O), 154.39 (C=O), 151.87 (Ar-C), 145.26 (C-N), 133.17 (2 × Ar-C), 130.18 (2 × Ar-C), 122.22 (Br-Ar-C), 104.32 (C=C), 61.50 (OCH2), 53.73 (CH-NH), 45.12 (N-CH2), 24.38 (CH2), 17.51 (CH3), 15.90 (CH3), 12.72 (CH3). HRMS (ESI): m/z calcd for C17H21BrN2O3 [M + H]+ 381.0814, found 381.0807.
Ethyl 4-(4-bromophenyl)-1-butyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1g). White solid, m.p.: 117.5–119.3 °C, yield: 69.7%, Rf value: 0.3 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.42 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.13 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 5.39 (s, 1H, NH), 5.33 (s, 1H, Ar-CH), 4.10 (q, J = 7.1 Hz, 2H, OCH2), 3.90 (ddd, J = 15.1, 9.9, 5.9 Hz, 1H, NCH2), 3.58 (ddd, J = 14.7, 9.6, 5.4 Hz, 1H, NCH2), 2.52 (s, 3H, =CCH3), 1.64 (s, 2H, CH2), 1.30 (dd, J = 14.9, 7.4 Hz, 2H, CH2), 1.18 (t, J = 7.1 Hz, 3H, CH3), 0.92 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 166.02 (C=O), 153.12 (C=O), 150.58 (Ar-C), 143.92 (C-N), 131.81 (2 × Ar-C), 128.94 (Ar-C), 128.72 (Ar-C), 120.89 (Br-Ar-C), 103.17 (C=C), 60.18 (OCH2), 52.30 (CH-NH), 31.90 (N-CH2), 19.89 (CH2), 16.16 (CH3), 14.57 (CH3), 14.10 (CH3). HRMS (ESI): m/z calcd for C18H23BrN2O3 [M + H]+ 395.0970, found 395.0977.
Ethyl 4-(4-bromophenyl)-1-hexyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1h). White solid, m.p.: 161.3–161.6 °C, yield: 55.8%, Rf value: 0.5 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.42 (d, J = 8.1 Hz, 2H, 2 × Ar-H), 7.13 (d, J = 8.2 Hz, 2H, 2 × Ar-H), 5.50 (s, 1H, NH), 5.33 (s, 1H, Ar-CH), 4.10 (q, J = 7.1 Hz, 2H, OCH2), 3.90 (ddd, J = 14.8, 9.6, 5.9 Hz, 1H, NCH2), 3.57 (ddd, J = 14.7, 9.6, 5.4 Hz, 1H, NCH2), 2.52 (s, 3H, =CCH3), 1.59 (s, 1H, CH2), 1.49 (s, 1H, CH2), 1.27 (s, 6H, 3 × CH2), 1.19 (t, J = 7.1 Hz, 3H, CH3), 0.88 (t, J = 6.6 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 166.04 (C=O), 153.20 (C=O), 150.68 (Ar-C), 143.84 (C-N), 131.79 (2 × Ar-C), 128.78 (2 × Ar-C), 120.91 (Br-Ar-C), 103.25 (C=C), 60.19 (OCH2), 52.12 (CH-NH), 42.07 (N-CH2), 31.51 (CH2), 29.84 (CH2), 26.35 (CH2), 22.60 (CH2), 16.17 (CH3),14.51 (CH3). HRMS (ESI): m/z calcd for C20H27BrN2O3 [M + H]+ 423.1283, found 423.1270.
Ethyl 4-(4-bromophenyl)-1-(3-cyanopropyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1i). White solid, m.p.: 128.8–129.7 °C, yield: 27.2%, Rf value: 0.4 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.42 (dd, J = 8.4, 1.9 Hz, 2H, 2 × Ar-H), 7.09 (dd, J = 8.3, 1.4 Hz, 2H, 2 × Ar-H), 5.33 (s, 1H, Ar-CH), 4.14 – 4.03 (m, 2H, OCH2), 3.95–3.84 (m, 1H, NCH2), 3.74 (dd, J = 9.0, 5.4 Hz, 1H, NCH2), 2.52 (s, 3H, =CCH3), 2.38 – 2.18 (m, 2H, CH2), 1.96 (s, 1H, CH2), 1.84 (s, 1H, CH2), 1.16 (dd, J = 7.5, 6.8 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 166.04 (C=O), 153.20 (C=O), 150.69 (Ar-C), 143.84 (C-N), 131.78 (2 × Ar-C), 128.79 (2 × Ar-C), 120.91 (Br-Ar-C, C≡N), 103.24 (C=C), 60.19 (OCH2), 52.11 (CH-NH), 42.07 (N-CH2), 22.60 (CH3), 16.17 (CH2), 14.59 (CH2), 14.43 (CH3). HRMS (ESI): m/z calcd for C18H20BrN3O3 [M + Na]+ 428.0586, found 428.0577.
Ethyl 4-(4-bromophenyl)-6-methyl-1-(4-nitrobenzyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (1j). White solid, m.p.: 183.3–184.6 °C, yield: 9.4%, Rf value: 0.6 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.47–7.44 (m, 2H, 2 × Ar-H), 7.29 (s, 2H, 2 × Ar-H), 7.17–7.10 (m, 2H, 2 × Ar-H), 5.69 (d, J = 3.0 Hz, 1H, NH), 5.45 (d, J = 3.1 Hz, 1H, Ar-CH), 5.10 (q, J = 17.1 Hz, 2H, CH2), 4.11 (q, J = 7.1 Hz, 2H, OCH2), 2.40 (s, 3H, =CCH3), 1.18 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 165.88 (C=O), 153.21 (C=O), 149.95 (NO2-Ar-C), 147.20 (Ar-C), 147.00 (C-N), 143.70 (Ar-C), 131.96 (2 × Ar-C), 129.02 (2 × Ar-C), 127.77 (2 × Ar-C), 124.25 (2 × Ar-C), 121.12 (Br-Ar-C), 103.87 (C=C), 60.39 (OCH2), 52.59 (CH-NH), 45.51 (N-CH2), 16.56 (CH3), 14.52 (CH3). HRMS (ESI): m/z calcd for C21H20BrN3O5 [M + H]+ 474.0665, found 474.0658.

3.2.2. N1-Substituted Ethyl 4-(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate 2a

Ethyl 1-(4-methoxy-4-oxobutyl)-4-(4-methoxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2a). White solid, m.p.: 87.8–90.4 °C, yield: 21.7%, Rf value: 0.4 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.15 (d, J = 8.6 Hz, 2H, 2 × Ar-H), 6.80 (d, J = 8.6 Hz, 2H, 2 × Ar-H), 5.77 (s, 1H, NH), 5.30 (d, J = 1.9 Hz, 1H, Ar-CH), 4.14–4.03 (m, 2H, OCH2), 3.96–3.84 (m, 1H, NCH2), 3.77 (d, J = 5.7 Hz, 3H, OCH3), 3.67 (s, 3H, OCH3), 3.63 (dd, J = 9.0, 4.6 Hz, 1H, NCH2), 2.54 (s, 3H, =CCH3), 2.29 (td, J = 7.0, 2.8 Hz, 2H, COCH2), 1.98–1.87 (m, 1H, CH2), 1.86–1.76 (m, 1H, CH2),1.20–1.13 (m, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 173.38 (C=O), 166.18 (C=O), 159.01 (Ar-C), 153.29 (C-N), 149.61 (Ar-C), 136.54 (Ar-C), 127.75 (Ar-C), 127.71 (Ar-C), 114.24 (Ar-C), 104.21 (C=C), 60.11 (OCH2), 55.57 (OCH3), 52.27 (OCH3), 51.82 (CH-NH), 41.37 (N-CH2), 30.72 (CH2), 25.05 (CH2), 16.06 (CH3), 14.58 (CH3). HRMS (ESI): m/z calcd for C20H26BrN2O6 [M + H]+ 391.1869, found 391.1869.

3.2.3. N1-Substituted Ethyl 4-([1,1′-biphenyl]-4-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates 3a, 3c, 3d, 3e, 3g, 3h

Ethyl 4-([1,1′-biphenyl]-4-yl)-1-(4-methoxy-4-oxobutyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (3a). Yellow solid, m.p.: 141.5–142.7 °C, yield: 33.5%, Rf value: 0.5 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.55 (dd, J = 10.1, 8.0 Hz, 4H, 4 × Ar-H), 7.44 (t, J = 7.5 Hz, 2H, 2 × Ar-H), 7.35 (dd, J = 11.1, 7.8 Hz, 3H, 3 × Ar-H), 5.87 (d, J = 2.8 Hz, 1H, NH), 5.44 (d, J = 2.9 Hz, 1H, Ar-CH), 4.21–4.09 (m, 2H, OCH2), 4.04–3.90 (m, 1H, NCH2), 3.76–3.68 (m, 1H, NCH2), 3.68–3.65 (m, 3H, OCH3), 2.63–2.54 (m, 3H, =CCH3), 2.38–2.27 (m, 2H, OCH2), 2.04–1.78 (m, 2H, CH2), 1.23 (dd, J = 13.0, 5.9 Hz, 3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 173.29 (C=O), 166.08 (C=O), 153.25 (Ar-C), 150.06 (Ar-C), 143.48 (Ar-C), 140.20 (Ar-C), 139.68 (Ar-C), 129.37 (2 × Ar-C), 127.89 (2 × Ar-C), 127.21 (2 × Ar-C), 127.06 (2 × Ar-C), 103.72 (C=C), 60.16 (OCH2), 52.46 (OCH3), 51.74 (CH-NH), 41.41 (N-CH2), 30.67 (CH2), 24.99 (CH2), 16.07 (CH3), 14.56 (CH3). HRMS (ESI): m/z calcd for C25H28N2O5 [M + Na]+ 459.1896, found 459.1903.
Ethyl 4-([1,1′-biphenyl]-4-yl)-1-(2-chlorobenzyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (3c). White solid, m.p.: 189.8–190.9 °C, yield: 25.8%, Rf value: 0.5 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.57 (dd, J = 12.2, 4.8 Hz, 4H, 4 × Ar-H), 7.45 (t, J = 7.5 Hz, 2H, 2 × Ar-H), 7.41–7.33 (m, 4H, 4 × Ar-H), 7.15 (dtd, J = 13.6, 7.4, 6.0 Hz, 2H, 2 × Ar-H), 6.97 (d, J = 7.4 Hz, 1H, Ar-H), 5.77 (d, J = 3.0 Hz, 1H, NH), 5.55 (d, J = 3.0 Hz, 1H, Ar-CH), 5.11 (d, J = 2.5 Hz, 2H, NCH2), 4.14 (q, J = 7.1 Hz, 2H, OCH2), 2.40 (s, 3H, =CCH3), 1.21 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 166.03 (C=O), 153.25 (C=O), 149.75 (Ar-C), 143.39 (Ar-C), 140.25 (C-N), 139.88 (Ar-C), 135.98 (Ar-C), 131.35 (Ar-C), 129.83 (2 × Ar-C), 129.42 (2 × Ar-C), 129.03 (2 × Ar-C), 127.93 (2 × Ar-C), 127.78 (2 × Ar-C), 127.30 (2 × Ar-C), 127.13 (Ar-C), 104.07 (C=C), 60.33 (OCH2), 52.60 (CH-NH), 43.80 (N-CH2), 16.12 (CH3), 14.53 (CH3). HRMS (ESI): m/z calcd for C27H25ClN2O3 [M + H]+ 461.1632, found 461.1631.
Ethyl 4-([1,1′-biphenyl]-4-yl)-1-(4-bromobenzyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (3d). White solid, m.p.: 175.3–177.8 °C, yield: 13.3%, Rf value: 0.5 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.59 (dd, J = 5.2, 3.3 Hz, 2H, 2 × Ar-H), 7.56–7.52 (m, 2H, 2 × Ar-H), 7.47 (dd, J = 10.2, 4.7 Hz, 2H, 2 × Ar-H), 7.41 (dd, J = 7.7, 4.9 Hz, 2H, 2 × Ar-H), 7.38 (s, 1H, Ar-H), 7.33 (d, J = 8.2 Hz, 2H, 2 × Ar-H), 7.04 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 5.79 (d, J = 3.0 Hz, 1H, NH), 5.52 (d, J = 3.1 Hz, 1H, Ar-CH), 5.14 (d, J = 15.5 Hz, 1H, NCH2), 4.88 (d, J = 16.1 Hz, 1H, NCH2), 4.14 (q, J = 7.1 Hz, 2H, OCH2), 2.46 (s, 3H, =CCH3), 1.22 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (151MHz, DMSO-d6) δ 166.00 (C=O), 153.46 (C=O), 149.81 (Ar-C), 143.42 (C-N), 140.24 (Ar-C), 139.83 (Ar-C), 138.64 (Ar-C), 131.92 (2 × Ar-C), 131.82 (2 × Ar-C), 129.40 (2 × Ar-C),128.94 (2 × Ar-C), 127.92 (2 × Ar-C), 127.24 (Ar-C), 127.12 (2 × Ar-C), 120.34 (Br-Ar-C), 104.13 (C=C), 60.27 (OCH2), 52.59 (CH-NH), 44.97 (N-CH2), 16.50 (CH3), 14.52 (CH3). HRMS (ESI): m/z calcd for C27H25BrN2O3 [M + Na]+ 527.0946, found 527.0945.
Ethyl 4-([1,1′-biphenyl]-4-yl)-1-(4-methoxybenzyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (3e). White solid, m.p.: 163.5–165.8 °C, yield: 24.8%, Rf value: 0.5 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 7.2 Hz, 2H, 2 × Ar-H), 7.50 (d, J = 8.1 Hz, 2H, 2 × Ar-H), 7.44 (t, J = 7.5 Hz, 2H, 2 × Ar-H), 7.36 (d, J = 7.3 Hz, 1H, Ar-H), 7.32 (t, J = 6.4 Hz, 2H, 2 × Ar-H), 7.08 (d, J = 8.5 Hz, 2H, 2 × Ar-H), 6.81 (d, J = 8.6 Hz, 2H, 2 × Ar-H), 5.71 (s, 1H, NH), 5.48 (s, 1H, Ar-CH), 5.17 (d, J = 15.8 Hz, 1H, NCH2), 4.82 (d, J = 16.2 Hz, 1H, NCH2), 4.11 (q, J = 7.1 Hz, 2H, OCH2), 3.75 (d, J = 3.9 Hz, 3H, CH3), 2.46 (d, J = 14.7 Hz, 3H, OCH3), 1.19 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 166.06 (C=O), 158.59 (C=O), 153.60 (Ar-C), 150.08 (Ar-C), 143.52 (C-N), 140.26 (Ar-C), 139.76 (Ar-C), 130.92 (2 × Ar-C), 129.40 (2 × Ar-C), 128.10 (2 × Ar-C), 127.91 (2 × Ar-C), 127.35 (2 × Ar-C), 126.96 (2 × Ar-C), 114.32 (2 × Ar-C), 104.05 (C=C), 60.21 (OCH2), 55.40 (OCH3), 52.59 (CH-NH), 44.75 (N-CH2), 16.51 (CH3), 14.52 (CH3). HRMS (ESI): m/z calcd for C28H28N2O4 [M + Na]+ 457.2127, found 457.2127.
Ethyl 4-([1,1′-biphenyl]-4-yl)-1-butyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (3g). White solid, m.p.: 161.2–165.8 °C, yield: 19.9%, Rf value: 0.5 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.58–7.50 (m, 4H, 4 × Ar-H), 7.43 (t, J = 7.6 Hz, 2H, 2 × Ar-H), 7.37–7.30 (m, 3H, 3 × Ar-H), 5.42 (s, 2H, NH, Ar-CH), 4.13 (q, J = 7.1 Hz, 2H, OCH2), 3.98–3.87 (m, 1H, NCH2), 3.62 (ddd, J = 14.8, 14.3, 9.5 Hz, 1H, NCH2), 2.55 (s, 3H, =CCH3), 1.64–1.50 (m, 2H, CH2), 1.31 (dt, J = 15.1, 7.5 Hz, 2H, CH2), 1.21 (t, J = 7.1 Hz, 3H, CH3), 0.92 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 166.12 (C=O), 153.25 (C=O), 150.18 (Ar-C), 143.64 (C-N), 140.24 (Ar-C), 139.67 (Ar-C), 129.38 (2 × Ar-C), 127.87 (2 × Ar-C), 127.18 (2 × Ar-C), 127.09 (2 × Ar-C), 127.08 (Ar-C), 103.52 (C=C), 60.12 (OCH2), 52.49 (CH-NH), 41.87 (N-CH2), 31.91 (CH2), 19.88 (CH2), 16.15 (CH3), 14.56 (CH3), 14.10 (CH3). HRMS (ESI): m/z calcd for C24H28N2O3 [M + H]+ 393.2168, found 393.2178.
Ethyl 4-([1,1′-biphenyl]-4-yl)-1-hexyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (3h). White solid, m.p.: 114.4–117.4 °C, yield: 50.5%, Rf value: 0.4 (CH2Cl2: CH3OH=20:1). 1H NMR (400 MHz, CDCl3) δ 7.57–7.48 (m, 4H 4 × Ar-H), 7.42 (t, J = 7.5 Hz, 2H 2 × Ar-H), 7.33 (t, J = 8.0 Hz, 3H, 3 × Ar-H), 5.99 (d, J = 2.9 Hz, 1H, NH), 5.42 (d, J = 2.9 Hz, 1H, Ar-CH), 4.13 (q, J = 7.1 Hz, 2H, OCH2), 3.99–3.87 (m, 1H, NCH2), 3.57 (ddd, J = 14.6, 9.5, 5.4 Hz, 1H, NCH2), 2.54 (s, 3H, =CCH3), 1.68–1.44 (m, 2H, CH2), 1.25 (s, 6H, 3 × CH2), 1.21 (t, J = 7.1 Hz, 3H, CH3), 0.85 (t, J = 6.5 Hz, 3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 166.13 (C=O), 153.31 (C=O), 150.26 (Ar-C), 143.58 (C-N), 140.22 (Ar-C), 139.64 (Ar-C), 129.35 (2 × Ar-C), 127.86 (2 × Ar-C), 127.13 (3 × Ar-C), 127.04 (2 × Ar-C), 103.56 (C=C), 60.12 (OCH2), 52.34 (CH-NH), 42.04 (N-CH2), 31.48 (CH2), 29.83 (CH2), 26.3 (CH2), 22.54 (CH2), 16.15 (CH3), 14.57 (CH3), 14.33 (CH3). HRMS (ESI): m/z calcd for C26H32N2O3 [M + H]+ 421.2491, found 421.2488.

3.2.4. N1-Substituted Ethyl 6-methyl-4-(4-morpholinophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate 4a

1-(4-methoxy-4-oxobutyl)-6-methyl-4-(4-morpholinophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4a). Orange solid, m.p.: 123.1–125.4 °C, yield: 20.3%, Rf value: 0.7 (CH2Cl2: CH3OH=10:1). 1H NMR (500 MHz, CDCl3) δ 7.14 (d, J = 8.5 Hz, 2H, 2 × Ar-H), 6.83 (d, J = 8.0 Hz, 2H, 2 × Ar-H), 5.49 (s, 1H, NH), 5.29 (s, 1H, Ar-CH), 4.14–4.05 (m, 2H, OCH2), 3.95–3.86 (m, 1H, NCH2), 3.87–3.82 (m, 4H, 2 × OCH2), 3.68 (s, 3H, OCH3), 3.65 (dd, J = 8.9, 5.9 Hz, 1H, NCH2), 3.16–3.09 (m, 4H, 2 × NCH2), 2.55 (d, J = 16.9 Hz, 3H, =CCH3), 2.38–2.25 (m, 2H, CH2), 1.99–1.89 (m, 1H, CH2), 1.84 (qd, J = 13.3, 6.9 Hz, 1H, CH2), 1.22 – 1.14 (m, 3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 173.32 (C=O), 166.16 (C=O), 153.27 (Ar-C), 150.77 (C-N), 149.33 (Ar-C), 135.01 (Ar-C), 127.15 (2 × Ar-C), 115.33 (Ar-C), 104.18 (C=C), 66.52 (2 × OCH2), 60.02 (OCH3), 52.21 (N-CH2), 51.79 (N-CH2), 48.87 (N-CH2), 41.32 (CH-NH), 30.69 (CH2), 25.01 (CH2), 16.00 (CH3), 14.55 (CH3). HRMS (ESI): m/z calcd for C23H31N3O6 [M + Na]+ 468.2111, found 468.2105.

3.2.5. N1-Substituted Ethyl 6-methyl-4-(4-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate 5a

Ethyl 1-(4-methoxy-4-oxobutyl)-6-methyl-4-(4-nitrophenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5a). Yellow solid, m.p.: 108.4–111.2 °C, yield: 17.8%, Rf value: 0.7 (CH2Cl2: CH3OH=10:1). 1H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 7.42 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 5.66 (s, 1H, NH), 5.47 (d, J = 2.4 Hz, 1H, Ar-CH), 4.11 (q, J = 7.1 Hz, 2H, OCH2), 3.96–3.83 (m, 1H, NCH2), 3.73–3.60 (m, 4H, NCH2, OCH3), 2.56 (s, 3H, =CCH3), 2.32–2.23 (m, 2H, OCH2), 1.98–1.74 (m, 2H, CH2), 1.18 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (151 MHz, DMSO-d6) δ 173.24 (C=O), 165.77 (C=O), 152.93 (Ar-C), 151.64 (C-N), 151.16 (Ar-C), 147.21 (Ar-C), 127.89 (2 × Ar-C), 124.28 (Ar-C), 102.758 (C=C), 60.30 (OCH2), 52.37 (OCH3), 51.75 (N-CH2), 41.47 (CH-NH), 30.62 (CH2), 16.08 (CH3), 14.50 (CH3) HRMS (ESI): m/z calcd for C19H23N3O7 [M + H]+ 406.1614, found 406. 1611.

3.2.6. N1-Substituted Ethyl 6-methyl-2-oxo-4-(pyridin-4-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate 6a

Ethyl 1-(4-methoxy-4-oxobutyl)-6-methyl-2-oxo-4-(pyridin-4-yl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6a). Yellow solid, m.p.: 97.8–100.3 °C, yield: 21.7%, Rf value: 0.6 (CH2Cl2: CH3OH=10:1). 1H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 5.8 Hz, 2H, 2 × Pyridine-H), 7.20 (d, J = 6.0 Hz, 2H, 2 × Pyridine-H), 6.00 (d, J = 3.4 Hz, 1H, NH), 5.39 (d, J = 3.5 Hz, 1H, Ar-CH), 4.15 (q, J = 7.1 Hz, 2H, OCH2), 3.90 (ddd, J = 15.1, 9.7, 5.8 Hz, 1H, NCH2), 3.67 (s, 3H, OCH3), 3.66–3.60 (m, 1H, NCH2), 2.56 (s, 3H, =CCH3), 2.29 (td, J = 7.0, 3.9 Hz, 2H, CH2), 1.79 (ddt, J = 13.3, 9.7, 6.6 Hz, 2H, CH2), 1.21 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 173.14 (C=O), 165.73 (C=O), 153.04 (2 × Pyridine-C), 152.474 (C=O), 151.02 (2 × Pyridine-C), 150.27 (C-N), 121.45 (2 × Pyridine-C), 102.45 (C=C), 60.19 (CH2), 51.75 (Ar-CH), 41.40 (CH2), 30.57 (CH2), 24.83 (CH2 2), 15.97 (CH3), 14.41 (CH3). HRMS (ESI): m/z calcd for C18H23N3O5 [M + H]+ 362.1716, found 362.1704.

3.2.7. N1-Substituted 4-(4-bromophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide 7c, 7d, 7e, 7f

4-(4-bromophenyl)-1-(2-chlorobenzyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (7c). White solid, m.p.: 186.4–188.5 °C, yield: 14.6%, Rf value: 0.5 (CH2Cl2: CH3OH=10:1). 1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.38 (dd, J = 5.4, 3.8 Hz, 1H, Ar-H), 7.24 (dd, J = 8.9, 6.7 Hz, 4H, 4 × Ar-H), 7.08 – 7.04 (m, 1H, Ar-H), 5.83 (s, 1H, NH), 5.36 (s, 1H, Ar-CH), 5.05 (s, 2H, NCH2), 2.19 (s, 3H, =CCH3). 13C NMR (101 MHz, DMSO-d6) δ 169.09 (C=O), 153.52 (C=O), 143.36 (Ar-C), 138.64 (Ar-C), 136.65 (C-N), 131.83 (Ar-C-Cl), 131.39 (2 × Ar-C), 129.83 (2 × Ar-C), 129.28 (Ar-C), 129.00 (Ar-C), 127.82 (Ar-C), 127.30 (Ar-C), 121.10 (Br-Ar-C), 109.76 (C=C), 53.96 (CH), 43.34 (CH2), 16.36 (CH3). HRMS (ESI): m/z calcd for C19H17BrClN3O2 [M − H]− 432.0114, found 432.0110.
1-(4-bromobenzyl)-4-(4-bromophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (7d). White solid, m.p.: 280.3–280.7 °C, yield: 58.2%, Rf value: 0.7 (CH2Cl2: CH3OH=10:1). 1H NMR (400 MHz, DMSO) δ 7.56–7.43 (m, 4H, 4 × Ar-H), 7.15 (s, 2H, 2 × Ar-H), 7.03 (d, J = 7.1 Hz, 2H, 2 × Ar-H), 5.17 (s, 1H, Ar-CH), 4.92 (d, J = 17.6 Hz, 1H, NCH2), 4.67 (t, J = 14.5 Hz, 1H, NCH2), 2.02 (s, 3H, =CCH3). 13C NMR (101 MHz, DMSO-d6) δ 169.06 (C=O), 153.73 (C=O), 143.42 (Ar-C), 139.33 (C-N), 138.70 (Ar-C), 131.80 (4 × Ar-C), 129.19 (2 × Ar-C), 129.07 (2 × Ar-C), 121.03 (Br-Ar-C), 120.29 (Br-Ar-C), 109.88 (C=C), 53.91 (CH), 44.61 (CH2), 16.75 (CH3). HRMS (ESI): m/z calcd for C19H17Br2N3O2 [M + H]+ 477.9766, found 477.9769.
4-(4-bromophenyl)-1-(4-methoxybenzyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (7e). White solid, m.p.: 256.4–257.9 °C, yield: 73.8%, Rf value: 0.6 (CH2Cl2: CH3OH=10:1). 1H NMR (400 MHz, DMSO-d6) δ 7.80 (d, J = 3.0 Hz, 1H, NH2), 7.48 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.34 (s, 1H, NH2), 7.14 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 6.99 (d, J = 8.6 Hz, 3H, 2 × Ar-H, NH), 6.81 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 5.16 (d, J = 2.4 Hz, 1H, Ar-CH), 4.92 (d, J = 16.5 Hz, 1H, NCH2), 4.58 (d, J = 16.5 Hz, 1H, NCH2), 3.69 (s, 3H, OCH3), 2.04 (s, 3H, =CCH3). 13C NMR (101 MHz, DMSO-d6) δ 168.97 (C=O), 158.48 (Ar-C-O), 153.71 (C=O), 143.42 (Ar-C), 138.98 (C-N), 131.59 (2 × Ar-C), 131.52 (2 × Ar-C), 129.08 (2 × Ar-C), 128.01 (Ar-C), 120.81 (Br-Ar-C), 114.19 (2 × Ar-C), 109.58 (C=C), 55.44 (CH3), 53.73 (CH), 44.29 (CH2), 16.59 (CH3). HRMS (ESI): m/z calcd for C20H20BrN3O3 [M + Na]+ 452.0586, found 452.0571.
4-(4-bromophenyl)-6-methyl-2-oxo-1-propyl-1,2,3,4-tetrahydropyrimidine-5-carboxamide (7f). White solid, m.p.: 238.4–239.5 °C, yield: 21.7%, Rf value: 0.5 (CH2Cl2: CH3OH=10:1). 1H NMR (400 MHz, DMSO) δ 7.60 (d, J = 3.0 Hz, 1H, Ar-H), 7.49 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.15 (t, J = 7.8 Hz, 2H, 2 × Ar-H), 5.09 (d, J = 2.6 Hz, 1H, Ar-CH), 3.73–3.61 (m, 1H, NCH2), 2.47 (d, J = 1.6 Hz, 3H, =CCH3), 2.13 (s, 3H, NCH2, CH2), 0.72 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 169.36 (C=O), 153.59 (C=O), 143.56 (Ar-C), 138.96 (C-N), 131.74 (2 × Ar-C), 129.06 (2 × Ar-C), 120.88 (Br-Ar-C), 109.67 (C=C), 53.71 (CH), 43.37 (CH2), 23.24 (CH2), 16.60 (CH3), 11.46 (CH3). HRMS (ESI): m/z calcd for C15H18BrN3O2 [M + Cl]− 386.0271, found 386.0269.

3.2.8. N1-Substituted 4-(4-bromophenyl)-6-methyl-2-oxo-N-phenyl-1,2,3,4-hydropyrimidine-5-carboxamides 8a, 8d, 8e

Methyl 4-(4-(4-bromophenyl)-6-methyl-2-oxo-5-(phenylcarbamoyl)-3,4-dihydropyrimidin-1(2H)-yl)butanoate (8a). White solid, m.p.: 182.4–185.0 °C, yield: 25.2%, Rf value: 0.5 (CH2Cl2: CH3OH=10:1). 1H NMR (400 MHz, DMSO) δ 9.89 (s, 1H, NH2), 7.77 (d, J = 3.0 Hz, 1H, NH2), 7.54 (dd, J = 15.5, 8.1 Hz, 4H, 4 × Ar-H), 7.27 (t, J = 7.9 Hz, 2H, 2 × Ar-H), 7.22 (d, J = 8.4 Hz, 2H, 2 × Ar-H), 7.03 (t, J = 7.4 Hz, 1H, Ar-H), 5.22 (s, 1H, Ar-CH), 3.86–3.76 (m, 1H, NCH2), 3.59 (d, J = 4.8 Hz, 3H, CH3), 3.43 (ddd, J = 14.4, 8.9, 5.6 Hz, 1H, NCH2), 2.24 (d, J = 7.4 Hz, 2H, CH2), 2.15 (s, 3H, =CCH3), 1.80 (dd, J = 13.1, 5.2 Hz, 1H, COCH2), 1.70–1.60 (m, 1H, COCH2). 13C NMR (101 MHz, DMSO-d6) δ 173.33 (C=O), 166.18 (C=O), 153.36 (C=O), 143.19 (Ar-C), 139.38 (C-N), 138.66 (Ar-C-NH), 131.75 (2 × Ar-C), 129.02 (2 × Ar-C), 128.82 (2 × Ar-C), 123.80 (Ar-C), 120.91 (2 × Ar-C), 120.04 (Br-Ar-C), 110.23 (C=C), 53.86 (CH), 51.74 (CH3), 40.95 (CH2), 30.71 (CH2), 25.16 (CH2), 16.66 (CH3). HRMS (ESI): m/z calcd for C23H24BrN3O4 [M + H]+ 486.1028, found 486.1016.
1-(4-bromobenzyl)-4-(4-bromophenyl)-6-methyl-2-oxo-N-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxamide (8d). White solid, m.p.: 171.8–175.2 °C, yield: 16.7%, Rf value: 0.4 (CH2Cl2: CH3OH=10:1). 1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H, NH2), 7.94 (s, 1H, NH2), 7.54–7.45 (m, 6H, 6 × Ar-H), 7.26–7.15 (m, 4H, 4 × Ar-H), 7.10 (d, J = 8.2 Hz, 2H, 2 × Ar-H), 6.98 (t, J = 7.3 Hz, 1H, Ar-H), 5.28 (s, 1H, Ar-CH), 4.93 (d, J = 17.0 Hz, 1H, NCH2), 4.72 (d, J = 11.6 Hz, 1H, NCH2), 2.00 (s, 3H, =CCH3). 13C NMR (100 MHz, DMSO-d6) δ 165.58 (C=O), 152.83 (C=O), 144.02 (Ar-C), 139.51 (C-N), 139.06 (Ar-C-NH), 131.75 (Ar-C), 128.92 (4 × Ar-C), 123.55 (6 × Ar-C), 120.79 (Ar-C), 120.04 (4 × Ar-C), 105.40 (C=C), 55.00 (CH), 48.99 (CH2), 17.44 (CH3). HRMS (ESI): m/z calcd for C25H21Br2N3O2 [M + H]+ 556.0058, found 556.0043.
4-(4-bromophenyl)-1-(4-methoxybenzyl)-6-methyl-2-oxo-N-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxamide (8e). White solid, m.p.: 191.7–193.5 °C, yield: 28.7%, Rf value: 0.5 (CH2Cl2: CH3OH=10:1). 1H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H, NH2), 7.92 (d, J = 3.0 Hz, 1H, NH2), 7.53 (d, J = 8.4 Hz, 4H, 4 × Ar-H), 7.29–7.20 (m, 4H, 4 × Ar-H), 7.09 (d, J = 8.6 Hz, 2H, 4 × Ar-H), 7.03 (d, J = 7.4 Hz, 1H, Ar-H), 6.88 (d, J = 8.7 Hz, 2H, 2 × Ar-H), 5.31 (s, 1H, Ar-CH), 4.97 (d, J = 16.0 Hz, 1H, NCH2), 4.69 (d, J = 16.2 Hz, 1H, NCH2), 3.74 (s, 3H, OCH3), 2.06 (s, 3H, =CCH3). 13C NMR (101 MHz, DMSO-d6) δ 166.19 (C=O), 158.68 (Ar-C-O), 153.74 (C=O), 143.43 (Ar-C), 139.48 (C-N), 139.05 (Ar-C-NH), 131.86 (2 × Ar-C), 131.61 (2 × Ar-C), 129.15 (2 × Ar-C), 129.10 (2 × Ar-C), 128.21 (Ar-C), 123.95 (Ar-C), 121.09 (2 × Ar-C), 120.28 (Br-Ar-C), 114.39 (2 × Ar-C), 109.96 (C=C), 55.60 (CH3), 54.35 (CH), 44.57 (CH2), 16.94 (CH3). HRMS (ESI): m/z calcd for C26H24BrN3O3 [M + H]+ 506.1079, found 506.1069.

3.3. N1 and N3-Substituted Ethyl 4-(4-bromophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates

Ethyl 4-(4-bromophenyl)-1,3-bis(2-chlorobenzyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate. White solid, m.p.: 145.1-146.6 °C, yield: NA. 1H NMR (400 MHz, CDCl3) δ 7.48-7.41 (m, 2H, 2 × Ar-H), 7.41 -7.33 (m, 3H, 2 × Ar-H), 7.31-7.24 (m, 3H, 2 × Ar-H), 7.24-7.12 (m, 4H, 4 × Ar-H), 5.38 (s, 1H, Ar-CH), 5.29 (d, J = 15.7 Hz, 1H, NCH2), 5.12 (s, 2H, NCH2), 4.11 (q, J = 7.1 Hz, 2H, OCH2), 4.05 (d, J = 15.7 Hz, 1H, NCH2), 2.39 (s, 3H, =CCH3), 1.19 (t, J = 7.1 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) δ 165.47 (C=O), 153.08 (C=O), 149.52 (Ar-C), 140.96 (C-N), 135.74 (Ar-C), 134.47 (Ar-C), 133.00(Ar-C), 132.20(Ar-C), 131.56 (2 × Ar-C), 130.12 (2 × Ar-C), 129.95 (Ar-C), 129.87 (Ar-C), 129.78 (Ar-C), 129.41 (Ar-C), 129.22 (Ar-C), 127.95 (Ar-C), 127.83 (Ar-C), 127.43 (Ar-C), 121.70 (Br-Ar-C), 104.15 (C=C), 60.54 (CH), 57.84 (CH2), 47.89 (CH2), 45.28 (CH2), 16.29 (CH3), 14.48 (CH3). HRMS (ESI): m/z calcd for C28H25BrCl2N2O3 [M+H]+ 587.0504, found 587.0520.

3.4. The Information on Reaction Condition Optimization

In order to investigate the effects of reaction conditions on N1 selectivity, we designed orthogonal tests table of different aspects to optimize the reaction, including temperature, time, amount of solvent, dosing interval, the presence of a phase transfer catalyst, different mole equivalent of base and halohydrocarbons. Each condition set include two variables of low dose and high dose (Table S2 in Supplementary Materials). Orthogonal test is widely used in everyday studies and research, because it could handle a complex issue with much lower cost and less time. Orthogonal tests L8(27) were applied to analyze the influence of the seven factors above on the selectivity and yield of N1-alkylation of DHPMs.
Results of orthogonal tests for N1-alkylation of DHPMs in tetrabutylammonium hydroxide system were shown in Table S2 and statistics analysis wascarried out with the extremely analysis method. The change of all conditions had no effect on N1-alkylation selectivity. According to the importance of influence, they were sorted into temperature (R 36.9), dosing interval (R 22.3), reaction time (R 19.5), phase transfer catalyst (R 16.5) and mole ratio of halohydrocarbons (R 11.9). Two other factors, the mole equivalent of base and the amount of solvent, had little effect on yield (R<10). Therefore, the yield could be increased by raising the reaction temperature and prolonging the reaction time. Meanwhile the selectivity was maintained.
In accordance with the requirements of saving raw materials and lowering costs, 1.7 equivalents of tetrabutylammonium hydroxide, and other conditions selected better factors for orthogonal tests were selected. The N1-alkylation of DHPMs was synthesized from compound 1, compound 2. The optimal conditions were the mole ratio 1:1.8:1.7 (compound 1, compound 2 and tetrabutylammonium hydroxide), DMF 20 mL/g, TBA was added as a catalyst. After the reaction of compound 1 and TBA in DMF for 1.5 h, compound 2 and potassium iodide(KI) were added to the mixture and stirred for 16 h at 45 °C.

3.5. In Vitro Studies

3.5.1. Cell Culture

U87, U251, Hela and A549 cell lines were obtained from Stem Cell Bank, Chinese Academy of Sciences. They were maintained in 75 cm2 culture flasks at 37 °C in a humidified air incubator with 5% CO2. The high-glucose Dulbecco´s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gemini, New York, NY, USA), and penicillin-streptomycin (Solarbio; Beijing, China) was used to culture cells. For all cell lines, the medium was renewed every 2 days until cells reach approximately 90–95% confluence. Then, they were detached by trypsin (Beyotime, China) and before the experiments, cells were counted using a hemocytometer and suitably diluted in the adequate complete culture medium.

3.5.2. Preparation of Compounds Solutions

BIIB021 was purchased from Aladdin Industrial Co. (Shanghai, China). The remaining compounds were synthesized in the authors laboratory. All compounds were dissolved individually in DMSO in a concentration of 10 mM and stored at −20 °C. From this stock solution, the various working solutions of the compounds in different concentrations were prepared by adequate dilutions in the complete culture medium before each experiment.

3.5.3. MTT Assay

The in vitro antiproliferative effects were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St Louis, MO, USA) assay [31,32]. After reaching confluence, cells were trypsinized and counted using a hemocytometer. Then, cell suspension/well with density of 4 × 104 cells/mL was seeded in 96-well culture plates and left to adhere for 24 h. After adherence, the medium was replaced by the several solutions of the compounds in study (10 µM for preliminary studies and 1.25, 2.5, 5, 10, 20, and 40 µM for concentration-response studies) in the appropriate culture medium for approximately 72 h. Untreated cells were used as the negative control. Each experiment was performed in triplicate and independently repeated. Then, the medium was removed, 20 µL of the MTT solution (5 mg/mL), prepared in the appropriate serum-free medium, was added to each well, followed by incubation for approximately 3 h at 37 °C. Then, the MTT containing medium was removed and the formazan crystals were dissolved in DMSO. The absorbance was measured at 570 nm using a microplate reader Bio-rad Xmark spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). After background subtraction, cell proliferation values were expressed as percentage relatively to the absorbance determined in negative control cells.

3.6. In Vivo Studies on Xenograft Model

C57 mice (6−8 weeks old, male) were used to establish the xenograft tumors following our published Protocol [33,34]. In brief, GL261 cells (5 × 106) were inoculated subcutaneously in right frank regions. The mice were divided into four groups randomly as control, BIIB021 (30 mg/kg), 303 (100 mg/kg), and 305(100 mg/kg) with eight mice per group. The mice were intra-gastric administration once a day for compound 3d and 3g starting from the second day, and body weights was measured every 3 days. At the end of treatment, animals were euthanized and the tumors were stripped and weighed after two weeks. All data were expressed as mean ± SD (n = 5). * p < 0.05, compared with control group. The use of animals was approved by the Animal Experimentation Ethics Committee of Yantai University (protocol number 20180601) in accordance with the guidelines for ethical conduct in the care and use of animals.

3.7. Log P Properties

The logarithm of the partition coefficient (Log P) properties of compounds were calculated by ACD/labs 6.00.

3.8. Pharmacophore Requirements

The GALAHAD module of Sybyl-X 2.0 (Certara, Princeton, NJ, USA) was used to generate pharmacophore. Thirteen DHPMs derivatives were selected with good activity against anticancer. All the structures are attached in Table S3 [20,35,36]. The final pharmacophore models were achieved with follow operations, including a population size value of 20, a maximum generation value of 100 and the value of molecular required hitting was 8.

4. Conclusions

A wide range of organic bases had been selected to study the N1 and N3 dialkylation of DHPMs. Selective alkylation of N1 was achieved with the use of tetrabutylammonium hydroxide. All the synthesized derivatives were screened for their anti-proliferative activity in U87, U251, Hela and A549 cell lines using the MTT assay. The study demonstrated that these compounds were more selective toward glioma tumor types. Introduction of the aryl or alkyl chain in the R3, and low electron-donating group in the R1 of DHPMs exhibited potent anti-proliferative activity. The in vivo efficacy study showed that compound 3d may have the potential to serve as lead compound for novel anti-tumor drugs to treat glioma. The study may provide a foundation for the future development of DHPMs as a new anti-tumor drug.

Supplementary Materials

The following are available online. Copies of the 1H-NMR and 13C-NMR spectra of the compounds and Tables S1, S2 and S3 are available online.

Author Contributions

Conceptualization, Z.L., H.W. and Q.M.; Methodology, Y.L., R.Z., Y.G., and J.L.; Validation, Y.L., R.Z., Y.G., and J.L.; Formal Analysis, Y.L. and J.L.; Data Curation, Y.L. and J.L.; Writing—Original Draft Preparation, Y.L.; Writing—Review and Editing, Y.S. and Z.L.; Supervision, Z.L.; Project Administration, Z.L.

Funding

This research was funded by National Natural Science Foundation of China (Grant No. 81502983).

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.
Molecules 24 00891 g001 550
Figure 1. The structure of a 3,4-dihydropyrimidin-2(1H)-one compound (DHPM) and its thione derivatives.
Figure 1. The structure of a 3,4-dihydropyrimidin-2(1H)-one compound (DHPM) and its thione derivatives.
Molecules 24 00891 g001
Molecules 24 00891 g002 550
Figure 2. Effects of compounds 3d and 3g on the antitumor activity of GL261 xenograft tumors in C57 mice. (A): The tumors were stripped and photographed the experimental results after two weeks. (B): The body weight of the four groups of mice was changed for two weeks. (C): The tumor of the four groups of mice was weighed after two weeks. ** p < 0.01, compared with control group.
Figure 2. Effects of compounds 3d and 3g on the antitumor activity of GL261 xenograft tumors in C57 mice. (A): The tumors were stripped and photographed the experimental results after two weeks. (B): The body weight of the four groups of mice was changed for two weeks. (C): The tumor of the four groups of mice was weighed after two weeks. ** p < 0.01, compared with control group.
Molecules 24 00891 g002
Molecules 24 00891 g003 550
Figure 3. Pharmacophore Requirements.
Figure 3. Pharmacophore Requirements.
Molecules 24 00891 g003
Table
Table 1. Preparation of N1-alkylated DHPMs with different halohydrocarbons.
Table 1. Preparation of N1-alkylated DHPMs with different halohydrocarbons.
Molecules 24 00891 i001
DHPMs (1a–8e)R1R2R3N1-Alkylation Yield (%) aLog P (Partition Coefficient)
1aBrOEt Molecules 24 00891 i0028.03.33
1bBrOEt Molecules 24 00891 i00352.23.65
1cBrOEt Molecules 24 00891 i00435.15.27
1dBrOEt Molecules 24 00891 i00532.35.45
1eBrOEt Molecules 24 00891 i00643.64.59
1fBrOEt Molecules 24 00891 i00720.03.91
1gBrOEt Molecules 24 00891 i00869.74.44
1hBrOEt Molecules 24 00891 i00955.85.50
1iBrOEt Molecules 24 00891 i01027.22.71
1jBrOEt Molecules 24 00891 i0119.44.41
2aOCH3OEt Molecules 24 00891 i01221.72.47
3aPhOEt Molecules 24 00891 i01333.54.32
3cPhOEt Molecules 24 00891 i01425.86.26
3dPhOEt Molecules 24 00891 i01513.36.43
3ePhOEt Molecules 24 00891 i01624.85.58
3gPhOEt Molecules 24 00891 i01719.95.43
3hPhOEt Molecules 24 00891 i01850.56.49
4a Molecules 24 00891 i019OEt Molecules 24 00891 i02020.31.70
5aNO2OEt Molecules 24 00891 i02117.82.29
6a Molecules 24 00891 i022OEt Molecules 24 00891 i02321.71.07
7cBrNH2 Molecules 24 00891 i02414.63.92
7dBrNH2 Molecules 24 00891 i02558.24.09
7eBrNH2 Molecules 24 00891 i02673.83.24
7fBrNH2 Molecules 24 00891 i02721.72.55
8aBr Molecules 24 00891 i028 Molecules 24 00891 i02925.24.36
8dBr Molecules 24 00891 i030 Molecules 24 00891 i03116.76.47
8eBr Molecules 24 00891 i032 Molecules 24 00891 i03328.75.62
a The yields relate to the use of tetrabutylammonium hydroxide as a base.
Table
Table 2. Survival rate of all compounds against U87, U251, Hela, A549 cell lines at 72 h.
Table 2. Survival rate of all compounds against U87, U251, Hela, A549 cell lines at 72 h.
DHPMs (1a–8e)Survival Rate of Four Different Cells (%)
U87 aU251 aHela aA549 a
1a87.34 ± 1.2467.14 ± 4.6169.81 ± 2.0476.80 ± 1.76
1b84.93 ± 0.7278.25 ± 7.8871.44 ± 0.6759.48 ± 2.63
1c97.83 ± 4.3285.20 ± 1.1671.96 ± 0.9662.86 ± 0.97
1d50.83 ± 0.2551.07 ± 2.5653.71 ± 1.0873.26 ± 2.69
1e89.41 ± 1.4771.65 ± 4.6451.05 ± 1.5154.10 ± 1.44
1f95.09 ± 12.7674.75 ± 0.7962.48 ± 1.4161.86 ± 1.24
1g84.04 ± 3.0872.74 ± 9.0851.05 ± 1.4250.68 ± 1.22
1h60.05 ± 1.5556.40 ± 4.2151.05 ± 0.6359.28 ± 2.97
1i94.46 ± 5.3380.10 ± 7.9862.48 ± 1.5569.93 ± 0.34
1j60.69 ± 1.8963.27 ± 3.4065.72 ± 0.3966.60 ± 1.35
2a85.54 ± 3.1572.22 ± 9.3767.90 ± 1.6278.92 ± 1.62
3a65.62 ± 3.7748.23 ± 3.9759.56 ± 3.8758.92 ± 2.41
3c100.69 ± 2.7585.60 ± 4.0473.81 ± 5.7351.92 ± 3.35
3d51.98 ± 1.6449.49 ± 4.7363.57 ± 2.7464.94 ± 4.16
3e70.94 ± 5.1671.28 ± 3.7657.38 ± 0.4058.35 ± 4.25
3g54.27 ± 0.8851.07 ± 4.3244.85 ± 1.0843.70 ± 2.38
3h60.37 ± 2.7151.21 ± 0.5846.02 ± 0.8555.16 ± 2.43
4a71.26 ± 2.0877.28 ± 5.3862.32 ± 2.2681.36 ± 1.63
5a75.53 ± 4.8976.22 ± 5.4847.96 ± 5.0052.22 ± 0.19
6a81.20 ± 4.4482.03 ± 6.3868.80 ± 5.3968.04 ± 3.02
7c81.02 ± 2.9897.29 ± 3.4774.26 ± 7.2991.51 ± 1.21
7d78.91 ± 4.8184.79 ± 5.1465.73 ± 3.4474.22 ± 1.96
7e76.22 ± 3.2183.07 ± 2.2266.91 ± 2.7868.17 ± 7.73
7f77.11 ± 6.2281.09 ± 4.0178.32 ± 2.4771.78 ± 4.25
8a81.22 ± 4.5689.27 ± 3.6466.91 ± 4.9272.03 ± 5.56
8d68.21 ± 2.1671.53 ± 5.8578.32 ± 2.2871.29 ± 3.84
8e81.86 ± 0.7173.90 ± 2.2279.80 ± 3.7985.65 ± 2.39
a U87, U251, HeLa, A549 cell lines were exposed at concentrations 10 µM at 72 h.
Table
Table 3. Selected compounds studied for half maximal inhibitory concentration (IC50) in U87 and U251 cell lines.
Table 3. Selected compounds studied for half maximal inhibitory concentration (IC50) in U87 and U251 cell lines.
CompoundIC50 (µM)
U87U251
1d9.72 ± 0.2913.91 ± 0.86
1h9.30 ± 0.8114.01 ± 0.76
3d12.02 ± 0.56.36 ± 0.73
3g9.52 ± 0.817.32 ± 0.86
BIIB021 a2.07 ± 0.130.3 ± 0.043
aBIIB021 as a positive control.
Table
Table 4. Inhibitory effects of compounds 3d and 3g on the xenograft tumor growth of GL261 in C57 mice.
Table 4. Inhibitory effects of compounds 3d and 3g on the xenograft tumor growth of GL261 in C57 mice.
GroupsDosage (mg/kg)Number Initial/EndBody Weight (g)Tumor Weight (g)IR (%)
InitialEnd
Control08/820.2 ± 0.422.0 ± 1.20.83 ± 0.24
3d1008/819.6 ± 0.621.5 ± 1.50.37 ± 0.1954.9
3g1008/819.8 ± 0.521.8 ± 0.80.54 ± 0.2534.3
BIIB021308/820.1 ± 0.721.3 ± 1.00.33 ± 0.1759.7

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Liu, Y.; Liu, J.; Zhang, R.; Guo, Y.; Wang, H.; Meng, Q.; Sun, Y.; Liu, Z. Synthesis, Characterization, and Anticancer Activities Evaluation of Compounds Derived from 3,4-Dihydropyrimidin-2(1H)-one. Molecules 2019, 24, 891. https://doi.org/10.3390/molecules24050891

AMA Style

Liu Y, Liu J, Zhang R, Guo Y, Wang H, Meng Q, Sun Y, Liu Z. Synthesis, Characterization, and Anticancer Activities Evaluation of Compounds Derived from 3,4-Dihydropyrimidin-2(1H)-one. Molecules. 2019; 24(5):891. https://doi.org/10.3390/molecules24050891

Chicago/Turabian Style

Liu, Ye, Jiaqi Liu, Renmei Zhang, Yan Guo, Hongbo Wang, Qingguo Meng, Yuan Sun, and Zongliang Liu. 2019. "Synthesis, Characterization, and Anticancer Activities Evaluation of Compounds Derived from 3,4-Dihydropyrimidin-2(1H)-one" Molecules 24, no. 5: 891. https://doi.org/10.3390/molecules24050891

APA Style

Liu, Y., Liu, J., Zhang, R., Guo, Y., Wang, H., Meng, Q., Sun, Y., & Liu, Z. (2019). Synthesis, Characterization, and Anticancer Activities Evaluation of Compounds Derived from 3,4-Dihydropyrimidin-2(1H)-one. Molecules, 24(5), 891. https://doi.org/10.3390/molecules24050891

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