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

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.


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 In DHPMs, R 1 is usually an aryl group, such as phenyl or pyridyl, while R 2 is an ester or amide roup and R 3 is an alkyl group, such as methyl or ethyl. General method for the synthesis of DHPMs tarts with firstly obtaining the basic scaffold via Biginelli reaction followed by N alkylation ohammadi and Behbahani reviewed the synthesis of DHPMs and improved procedures for the reparation of DHPMs under solvent-free conditions or with the presence of solvent [24]. Dallinger nd Kappe introduced a selective N 1 -alkylation method of DHPMs using Mitsunobu reaction [25] owever, the yield of Mitsunobu reaction was low, and the reagents were relatively expensive aking it not suitable for practical synthesis. Singh et al. provided another N-alkylation method atalyzed by inorganic strong base [26]. In the present report, not only did we find highly selective 1 -alkylation of DHPMs in the presence of tetrabutylammonium hydroxide, but also we nvestigated the biological importance of the newly synthesized molecules both in vitro and in vivo .

Results and Discussion
. 1

. Chemistry
The various non-alkylated DHPM moieties used in this report were synthesized through one ot Biginelli condensation reaction according to the reported method [27]. The effects of differen hoices of bases on the reaction, including sodium hydride (NaH), lithium hydroxide (LiOH·H2O) otassium carbonate (K2CO3) and some pKa similar organic base, were carried out to perform N nd N 3 dialkylation of DHPMs. While using a strong base, such as LiOH·H2O, NaH and potassium ert-butoxide (KTB), the reactions were proceeding very fast. However the dialkyl product was ormed and detected even from the beginning of the reaction. In some reactions the yields o ialkylation were even higher than that of N 1 -alkylation. N-alkylation cannot be achieved when a eak base is used, such as K2CO3, triethylamine (Et3N), 1,8-diazabicyclo [5,4,0]undec-7-ene (DBU nd tetramethylguanidine (TMG). Interestingly, when tetrabutylammonium hydroxide was selected s the base, the yield was similar to that of cesium carbonate (Cs2CO3), while no dialkyl products was The structure of a 3,4-dihydropyrimidin-2(1H)-one compound (DHPM) and its thione derivatives.
In DHPMs, R 1 is usually an aryl group, such as phenyl or pyridyl, while R 2 is an ester or amide group and R 3 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 N 1 -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 N 1 -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.

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·H 2 O), potassium carbonate (K 2 CO 3 ) and some pKa similar organic base, were carried out to perform N 1 and N 3 dialkylation of DHPMs. While using a strong base, such as LiOH·H 2 O, 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 N 1 -alkylation. N-alkylation cannot be achieved when a weak base is used, such as K 2 CO 3 , triethylamine (Et 3 N), 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 (Cs 2 CO 3 ), while no dialkyl products was found (Table S1 in Supplementary Materials). A possible explanation for this phenomenon is that N 3 -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.

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 GI 50 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.   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 R 3 , displayed decent activity, indicating that the length of the alkyl side chain in the R 3 would affect the potency. Compound 1d, with a 4-bromobenzyl group in the R 3 , also demonstrated strong cytotoxicity, suggesting that the R 3 may tolerate variations to some degree. We then explored the R 2 with the goal to compare the ester group and the amide group on cell viability. Compared with 1e, compound 7e and 8e differ in R 2 , 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 R 1 of DHPMs, we proposed that the capability of electron-donating group may affect the cell viability and the SAR of the R 1 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 R 1 , had significant activity in U251 cell lines, suggesting that a low electron-withdrawing group in the R 1 may contribute to augment the activity. Compound 5a and 6a, with a 4-nitrophenyl group and 4-pyridinylphenyl group in the R 1 , were also not active in the cell study, suggesting that high electron-withdrawing substituent in the R 1 may not be tolerated. Studies on different electron-donating groups in the R 1 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 R 1 and alkyl chain or 4-bromobenzyl in the R 3 , were found to have good activity.

Half Maximal Inhibitory Concentration (IC 50 ) Study of Compounds 1d, 1h, 3d and 3g
The values of 50% inhibitory concentration (concentration of drug yielding a 50% cell viability decrease, IC 50 ) 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 IC 50 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 R 3 , and low electron-donating ability or low electron-withdrawing ability in the R 1 displayed obvious effect. As expected, all of the four compounds especially 3d displaying high Log P (5.01) values and suitable PSA (58.64).

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 IC 50 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 R 3 , and 4-biphenyl low electron-donating group in the R 1 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.

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 R 3 , and 4-biphenyl low electron-donating group in the R 1 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.

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 R 2 position and X position. Hydrophobic center is the DHPMs parent ring, R 1 position and R 3 position.

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 R 2 position and X position. Hydrophobic center is the DHPMs parent ring, R 1 position and R 3 position.

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). 1 H NMR and 13 C 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).

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 GF 254 plates. Melting points were measured on an XT3A micro-melting point apparatus and are uncorrected (Beijing Keyi Company, Beijing, China). 1 H NMR and 13 C 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).

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.

MTT Assay
The in vitro antiproliferative effects were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium 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 × 10 4 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.

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 × 10 6 ) 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.

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

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.

Conclusions
A wide range of organic bases had been selected to study the N 1 and N3 dialkylation of DHPMs. Selective alkylation of N 1 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 R 3 , and low electron-donating group in the R 1 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.