Next Article in Journal
Comparative Study on the Characteristics of Weissella cibaria CMU and Probiotic Strains for Oral Care
Next Article in Special Issue
Design, Synthesis and Evaluation of Naphthalimide Derivatives as Potential Anticancer Agents for Hepatocellular Carcinoma
Previous Article in Journal
Characterization of Fractional Polysaccharides from Gleditsia sinensis and Gleditsia microphylla Gums
Previous Article in Special Issue
The HK2 Dependent “Warburg Effect” and Mitochondrial Oxidative Phosphorylation in Cancer: Targets for Effective Therapy with 3-Bromopyruvate
Article Menu
Issue 12 (December) cover image

Export Article

Molecules 2016, 21(12), 1746; doi:10.3390/molecules21121746

Article
Targeting Cancer Stem Cells with Novel 4-(4-Substituted phenyl)-5-(3,4,5-trimethoxy/3,4-dimethoxy)-benzoyl-3,4-dihydropyrimidine-2(1H)-one/thiones
1
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
2
Stem Cell & Tissue Re-Engineering Program, Research Center, King Faisal Specialized Hospital & Research Center, MBC-03, P.O. Box 3354, Riyadh 11211, Saudi Arabia
3
Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
*
Author to whom correspondence should be addressed.
Academic Editor: Jean Jacques Vanden Eynde
Received: 25 September 2016 / Accepted: 13 December 2016 / Published: 19 December 2016

Abstract

:
Novel 4-(4-substituted phenyl)-5-(3,4,5-trimethoxy/3,4-dimethoxy)-benzoyl-3,4-dihydropyrimidine-2(1H)-one/thione derivatives (DHP 19) were designed, synthesized, characterized and evaluated for antitumor activity against cancer stem cells. The compounds were synthesized in one pot. Enaminones E1 and E2 were reacted with substituted benzaldehydes and urea/thiourea in the presence of glacial acetic acid. The synthesized compounds were characterized by spectral analysis. The compounds were screened in vitro against colon cancer cell line (LOVO) colon cancer stem cells. Most of the compounds were found to be active against side population cancer stem cells with an inhibition of >50% at a 10 μM concentration. Compounds DHP-1, DHP-7 and DHP-9 were found to be inactive. Compound DHP-5 exhibited an in vitro anti-proliferative effect and arrested cancer cells at the Gap 2 phase (G2) checkpoint and demonstrated an inhibitory effect on tumor growth for a LOVO xenograft in a nude mouse experiment.
Keywords:
cancer stem cells; antitumor activity; dihydropyrimidine; enaminones

1. Introduction

Compounds, which have potential anticancer activity, are often screened out in drug discovery programs for cancer research [1] due to the presence of cells which have the capability to regrow in vivo, called cancer stem cells (CSCs). Thus, the antitumor activity of the compounds in vivo is not adequate for the treatment of cancer in preclinical models. Tumors are maintained by a self-growing CSC population [2]. Research has confirmed the presence of cancer stem cells in leukemia [3], as well as in tumors of the breast [4], brain [5], lung [6] and colon [7]. In cancer relapse, CSCs must have resisted the primary drug action [8]. Literature has reported that aldehyde dehydrogenase 1 (ALDH-1) is a more potent marker of breast CSCs [9,10,11] and ALDH-1–positive cells are resistant to Epirubicin and Paclitaxel [12]. Adult stem cells can be predicted by a side population (SP) phenotype. A SP is confined to the tumorigenic part of the breast cancer cell line MCF-7 [13,14]. Normal chemotherapy could lead to augmentation of CSCs in treated patients [15,16]. Thus, there remains an urgent need to discover new drugs to effectively eliminate both proliferating cells and CSCs in order to treat cancer [17].
Multicomponent reactions (MCR) are important in the discovery of new lead compounds. The acid-catalyzed cyclocondensation reaction of a diketone with benzaldehyde and urea was reported in 1893 by Pietro Biginelli. The product obtained was identified as a dihydropyridimidine-2-one. Dihydropyrimidines presented a varied range of biological activities, e.g., calcium channel blockers, α-adrenoceptor selective antagonists and anti-mitotics [18]. Furthermore, (S)-Monastrol (1) has been identified as a novel molecule for the development of potentially new anticancer drugs [19]. Monastrol causes specific and reversible inhibition of kinesin Eg5. Oxo-Monastrol and its thio-analogues have been investigated for their anti-proliferative activity. The 4-methoxy derivative 2 and 3-methoxy-4-hydoxy derivative 3 of Monastrol have been synthesized as anticancer agents [20]. The 3,4-methylenedioxy derivative of Monastrol, Piperastrol (4), was found to be three times more potent than Monastrol [21]. Pyrimidinone peptoid hybrids have been reported as active against SKBr-3 breast cancer cells [22]. Improved efficiency was reported in cell-based assays by optimization of the Monastrol-based dihydropyrimidine (DHPM) analogue R-Monastrol-97 (5) [23]. The 3,4-difluoro derivative R-fluorestrol (6) was also reported to be a potent anticancer compound. Compound 7, derived from Monastrol-97, has been reported to be active in anticancer screens. Deaths of over 80% of cancer cells were observed after 72 h of treatment with the Biginelli adducts Enastron (8) and dimethyl Enastron (9) [24]. These compounds showed minute toxic effects against healthy fibroblast cells. Amide-derived Biginelli adducts exhibited moderate anti-proliferative activity against HepG2 epithelial carcinoma. Compounds 10 and 11 showed IC50 values of (190 μg/mL) against HeLa hepatocellular carcinoma cells [25]. Additionally, cinnamoyl derivatives of dihydropyrimidine have been reported as potent anticancer agents [26]. Examples of dihydropyrimidines demonstrating anticancer activities are presented in Figure 1.
There is a need for structural optimization of dihydropyrimidine derivatives with the aim of modifying the profile of current lead molecules. In an effort to discover novel dihydropyrimidine derivatives with potent anticancer activity against cancer stem cells, modulation of the Monastrol-97 structure was carried out as illustrated in Figure 2.
These dihydropyrimidine derivatives were then evaluated for antitumor activity.

2. Results and Discussion

Enaminones E1 and E2 were reacted with substituted benzaldehydes and urea/thiourea in the presence of acetic acid to yield dihydropyrimidinone/thione derivatives (DHP 19). The purity of the compounds was confirmed by elemental analysis and thin-layer chromatography. The compounds were characterized using spectroscopic methods. In the 1H-NMR spectra, the signals of the individual protons of the compounds were verified on the basis of multiplicity, chemical shifts and the coupling constant. All the compounds showed the D2O exchangeable broad singlet at 8.8–9.8 ppm and 9.5–10.5 ppm corresponding to the two NH protons. Analytical and spectral data for the compounds were in good agreement with the expected structures of the synthesized derivatives. The physicochemical properties of all compounds are given in Table 1.
The newly synthesized compounds (DHP 19) were evaluated for side population percent inhibition on colon cancer cell line (LOVO) at a 10 μM concentration (Figure 3, Table 2).
The structure-activity relationships of the compounds were studied. From the compounds (DHP 19), four compounds were found to be very effective, namely DHP-4, DHP-6, DHP-2 and DHP-3, when the side population inhibition percentage was measured at a 10 μM concentration. Compounds DHP-5 and DHP-8 were moderately active as indicated by a low value of the side population inhibition percentage. Most of the dihydropyrimidine compounds (DHP 19) presented significant activity against side population inhibition percentage. It was noted that most of the compounds having a methoxy group at R1 were active. Compounds with an oxygen atom at R2 were also active. Compound DHP-6, with a hydrogen at R1 and a sulfur atom at R2, displayed significant activity. Compound DHP-4 was found to be the most active compound of the series.
A side population analysis of tumor-derived cells of LOVO xenografts that were untreated, treated with the side population inhibitor reference drug Verapamil 200 μM, and with compound DHP-5 (50 μM) confirmed that DHP-5 had a more potent inhibitory effect on the side population cancer stem cells than the reference drug Verapamil (Figure 5).
The tumor growth of LOVO (colon cancer xenografts) was recorded in untreated mice groups and in DHP-5–treated (50 mg/kg) mice groups. A potent anti-tumor effect was demonstrated by a shrinking of tumors in the animals which were treated by compound DHP-5. A remarkable anti-tumor effect of compound DHP-5 was demonstrated on tumors of colon cancer xenografts (Figure 6).

3. Material and Methods

3.1. Experimental

All solvents were obtained from Merck (Kenilworth, NJ, USA). The homogeneity of the compounds was checked by TLC performed on silica gel G; An iodine chamber was used for visualization of TLC spots. The FT-IR spectra were recorded in KBr pellets on a Spectrum BX Perkin Elmer FT-IR spectrophotometer (Perkin Elmer, Hopkinton, MA, USA). Melting points were determined on a Gallenkamp melting point apparatus (Gallenkamp, Loughborough, UK), and are uncorrected. NMR spectra were scanned in DMSO-d6 on a Bruker NMR spectrophotometer (Bruker, Billerica, MA, USA) operating at 500 MHz for 1H and 125.76 MHz for 13C at the Research Center, College of Pharmacy, King Saud University, Saudi Arabia. Chemical shifts δ are expressed in parts per million (ppm) relative to TMS as an internal standard and D2O was added to confirm the exchangeable protons. Coupling constants (J) are in Hertz. The molecular masses of compounds were determined by UPLC/TQMS and all tested compounds yielded data consistent with a purity of ≥95%, as measured by HPLC (Agilent 1260 affinity). The elemental analyses (C, H, N (±0.4%); and S (±0.3%)) were performed on a CHN Elementar (Analysensysteme GmbH, Langenselbold, Germany).
The synthesis of dihydropyrimidine derivatives was carried out in single step as shown in Scheme 1.

3.2. General Synthesis of 4-(Substituted phenyl)-5-(3,4,5-trimethoxybenzoyl/3,4-dimethoxybenzoyl)-3,4-dihydropyrimidin-2(1H)-ones DHP 19

A solution of enaminone E1/E2 (0.01 mol), substituted benzaldehyde (0.01 mol), urea/thiourea (0.01 mol) and glacial acetic acid (10 mL) was heated under reflux for 3 h. The precipitates (DHP 19) thus formed were collected by filtration, washed with water and recrystallized from acetic acid.
4-Phenyl-5-(3,4,5-trimethoxybenzoyl)-3,4-dihydropyrimidin-2(1H)-one (DPH-1): Yield: 70%; m.p.: 153–155 °C; IR (KBr): 3412 (N-H), 2938 (ArC-H), 1700 (C=O), 1636 (C=O), 1618 (C=C), 1126 (C-O); 1H-NMR (500 MHz, DMSO-d6); δ = 3.80 (9H, s, 3× -OCH3), 5.40 (1H, d, J = 2.5 Hz, H-4), 6.73–7.36 (7H, m, Ar-H), 7.88 (1H, d, J = 2.5 Hz, =CH), 9.50 (1H, bs, NH, D2O exchg.), 10.00 (1H, bs, NH, D2O exchg.); 13C-NMR (125.76 MHz, DMSO-d6): δ = 54.0, 56.4, 56.5, 56.7, 60.5, 106.2, 112.4, 126.9, 127.8, 128.9, 134.4, 140.3, 142.3, 144.6, 151.8, 152.9, 153.0, 191.0; MS: m/z = 368.46 [M]+; Analysis: C20H20N2O5 for, calcd. C 65.21, H 5.47, N 7.60%; found C 65.45, H 5.48, N 7.62%.
4-(4-Chlorophenyl)-5-(3,4,5-trimethoxybenzoyl)-3,4-dihydropyrimidin-2(1H)-one (DPH-2): Yield: 75%; m.p.: 138–140 °C; IR (KBr): 3412 (N-H), 2938 (ArC-H), 1686 (C=O), 1654 (C=O), 1618 (C=C), 1123 (C-O); 1H-NMR (500 MHz, DMSO-d6); δ = 3.79 (9H, s, 3× -OCH3), 5.39 (1H, d, J = 3.0 Hz, H-4), 6.74–7.43 (6H, m, Ar-H), 7.91 (1H, d, J = 2.5 Hz, =CH), 9.50 (1H, bs, NH, D2O exchg.), 10.00 (1H, bs, NH, D2O exchg.); 13C-NMR (125.76 MHz, DMSO-d6): δ = 53.6, 56.4, 56.5, 56.7, 60.5, 60.7, 106.2, 108.2, 112.0, 128.4, 128.9, 128.9, 129.8, 131.6, 152.3, 134.2, 139.8, 140.2, 140.3, 142.5, 143.5, 151.6, 152.9, 153.0, 153.2, 191.0, 192.5, 193.0; MS: m/z = 402.8 [M]+, 403.8 [M + 1]+; Analysis: C20H19N2O5Cl for, calcd. C 59.63, H 4.75, N 6.95%; found C 59.45, H 4.73, N 6.97%.
4-(4-Nitrophenyl)-5-(3,4,5-trimethoxybenzoyl)-3,4-dihydropyrimidin-2(1H)-one (DPH-3): Yield: 65%; m.p.: 158–160 °C; IR (KBr): 3421 (N-H), 2936 (ArC-H), 1685 (C=O), 1654 (C=O), 1618 (C=C), 1125 (C-O); 1H-NMR (500 MHz, DMSO-d6); δ = 3.77 (9H, s, 3× -OCH3), 5.53 (1H, d, J = 2.5 Hz, H-4), 6.74–7.40 (6H, m, Ar-H), 8.20 (1H, d, J = 2.5 Hz, =CH), 9.47 (1H, bs, NH, D2O exchg.), 10.20 (1H, bs, NH, D2O exchg.); 13C-NMR (125.76 MHz, DMSO-d6): δ = 53.8, 56.5, 56.5, 56.7, 60.2, 60.5, 60.7, 65.3, 106.2, 124.7, 128.4, 131.0, 134.1, 138.2, 140.4, 143.0, 147.2, 151.4, 151.7, 153.0, 153.0, 153.2, 190.9; MS: m/z = 413.47 [M]+; Analysis: C20H19N3O7 for, calcd. C 58.11, H 4.63, N 10.16%; found C 58.32, H 4.62, N 10.19%.
4-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxybenzoyl)-3,4-dihydropyrimidin-2(1H)-one (DPH-4): Yield: 72%; m.p.: 165–167 °C; IR (KBr): 3367 (N-H), 2937 (ArC-H), 1700 (C=O), 1624 (C=O), 1578 (C=C), 1123 (C-O); 1H-NMR (500 MHz, DMSO-d6); δ = 3.81 (15H, s, 5× -OCH3), 5.36 (1H, d, J = 2.5 Hz, H-4), 6.75–7.28 (5H, m, Ar-H), 7.81 (1H, d, J = 2.5 Hz, =CH), 9.24 (1H, bs, NH, D2O exchg.), 9.84 (1H, bs, NH, D2O exchg.); 13C-NMR (125.76 MHz, DMSO-d6): δ = 53.5, 55.9, 56.0, 56.3, 56.4, 56.5, 56.7, 60.5, 60.7, 65.3, 106.1, 106.2, 108.2, 109.9, 111.0, 112.2, 116.4, 118.8, 120.3, 126.5, 130.1, 131.8, 134.4, 135.1, 136.9, 139.3, 140.1, 142.1, 147.6, 148.6, 149.6, 151.7, 152.9, 153.0, 154.6, 193.3; MS: m/z = 428.26 [M]+; Analysis: C22H24N2O7 for, calcd. C 61.67, H 5.65, N 6.54%; found C 61.45, H 5.66, N 6.56%.
4-(4-Ethoxyphenyl)-5-(3,4,5-trimethoxybenzoyl)-3,4-dihydropyrimidin-2(1H)-one (DPH-5): Yield: 60%; m.p.: 168–170 °C; IR (KBr): 3411 (N-H), 2938 (ArC-H), 1696 (C=O), 1648 (C=O), 1618 (C=C), 1126 (C-O); 1H-NMR (500 MHz, DMSO-d6); δ = 1.31 (3H, t, J = 7.0 Hz, -CH3), 3.80 (9H, s, 3× -OCH3), 4.20 (2H, q, J = 2.0 Hz, -OCH2), 5.32 (1H, d, J = 2.5 Hz, H-4), 6.75–7.25 (6H, m, Ar-H), 7.79 (1H, d, J = 2.5 Hz, =CH), 8.81 (1H, bs, NH, D2O exchg.), 9.50 (1H, bs, NH, D2O exchg.); 13C-NMR (125.76 MHz, DMSO-d6): δ = 15.1, 53.3, 56.5, 60.5, 63.4, 106.1, 112.7, 114.7, 128.1, 140.2, 153.0, 192.0; MS: m/z = 412.28 [M]+; Analysis: C22H24N2O6 for, calcd. C 64.07, H 5.87, N 6.79%; found C 64.25, H 5.88, N 6.76%.
(3,4-Dimethoxyphenyl)(4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methanone (DHP-6): Yield: 65%; m.p.: 248–250 °C; IR (KBr): 3413 (N-H), 2955 (ArC-H), 1653 (C=O), 1636 (C=O), 1595 (C=C), 1199 (C-O); 1H-NMR (500 MHz, DMSO-d6); δ = 3.81 (6H, s, 2× -OCH3), 5.45 (1H, d, J = 3.0 Hz, H-4), 6.97–7.28 (7H, m, Ar-H), 7.34 (1H, d, J = 3.0 Hz, =CH), 9.70 (1H, bs, NH, D2O exchg.), 10.40 (1H, bs, NH, D2O exchg.); 13C-NMR (125.76 MHz, DMSO-d6): δ = 54.2, 56.6, 56.1, 56.3, 111.1, 111.2, 111.7, 112.2, 113.6, 122.7, 127.1, 128.2, 129.1, 130.8, 136.7, 143.4, 149.1, 149.3, 152.2, 153.8, 162.7, 174.3, 191.0, 193.5; MS: m/z = 355.0 [M + 1]+; Analysis: C19H18N2O3S for, calcd. C 64.39, H 5.12, N 7.90, S 9.05%; found C 64.54, H 5.11, N 7.92, S 9.04%.
(3,4-Dimethoxyphenyl)(4-chlorophenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methanone (DHP-7): Yield: 65%; m.p.: 243–245 °C; IR (KBr): 3413 (N-H), 2933 (ArC-H), 1670 (C=O), 1647 (C=O), 1616 (C=C), 1195 (C-O); 1H-NMR (500 MHz, DMSO-d6); δ = 3.78 (6H, s, 2× -OCH3), 5.45 (1H, d, J = 3.0 Hz, H-4), 6.98–7.45 (7H, m, Ar-H), 7.96 (1H, d, J = 3.0 Hz, =CH), 9.76 (1H, bs, NH, D2O exchg.), 10.49 (1H, bs, NH, D2O exchg.); 13C-NMR (125.76 MHz, DMSO-d6): δ = 53.7, 56.0, 56.1, 111.1, 111.7, 113.2, 122.7, 129.0, 129.1, 130.7, 132.8, 137.0, 142.3, 149.1, 152.2, 162.7, 174.3, 190.9; MS: m/z = 387.99 [M]+; Analysis: C19H17N2O3ClS for, calcd. C 58.68, H 4.41, N 7.20, S 8.25%; found C 58.85, H 4.43, N 7.23, S 8.24%.
(3,4-Dimethoxyphenyl)(4-nitrophenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methanone (DHP-8): Yield: 68%; m.p.: 258–260 °C; IR (KBr): 3412 (N-H), 2933 (ArC-H), 1676 (C=O), 1654 (C=O), 1615 (C=C), 1141 (C-O); 1H-NMR (500 MHz, DMSO-d6); δ = 3.82 (6H, s, 2× -OCH3), 5.58 (1H, d, J = 3.0 Hz, H-4), 7.0–7.95 (7H, m, Ar-H), 8.26 (1H, d, J = 2.5 Hz, =CH), 9.85 (1H, bs, NH, D2O exchg.), 10.59 (1H, bs, NH, D2O exchg.); 13C-NMR (125.76 MHz, DMSO-d6): δ = 53.9, 56.0, 56.1, 111.1, 111.7, 112.6, 122.8, 124.4, 128.5, 130.6, 137.5, 147.4, 149.1, 150.3, 152.3, 162.7, 174.5, 190.8; MS: m/z = 402.23 [M + 3]+; Analysis: C19H17N3O5S for, calcd. C 57.13, H 4.29, N 10.52, S 8.03%; found C 57.23, H 4.28, N 10.55, S 8.01%.
(3,4-Dimethoxyphenyl)(3,4-dimethoxyphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methanone (DHP-9): Yield: 70%; m.p.: 228–230 °C; IR (KBr): 3410 (N-H), 2932 (ArC-H), 1684 (C=O), 1654 (C=O), 1611 (C=C), 1134 (C-O); 1H-NMR (500 MHz, DMSO-d6); δ = 3.81 (12H, s, 4× -OCH3), 5.40 (1H, d, J = 3.0 Hz, H-4), 6.82–7.21 (6H, m, Ar-H), 7.96 (1H, d, J = 2.5 Hz, =CH), 9.68 (1H, bs, NH, D2O exchg.), 10.38 (1H, bs, NH, D2O exchg.); 13C-NMR (125.76 MHz, DMSO-d6): δ = 43.7, 55.9, 56.0, 56.0, 56.1, 111.1, 111.7, 112.3, 113.5, 119.0, 122.7, 130.8, 135.7, 136.6, 148.9, 149.1, 149.1, 152.2, 162.7, 174.1, 191.0; MS: m/z = 413.6 [M − 1]+; Analysis: C21H22N2O5S for, calcd. C 60.85, H 5.35, N 6.76, S 7.74%; found C 61.05, H 5.36, N 6.78, S 7.73%.

3.3. Cell line and Tissue Culture

LOVO colon cancer cells were purchased from the American Type Culture Collection. LOVO cells were cultured in RPMI. The medium was supplemented with 10% FBS (Cambrex Bio Science, Franklin Lakes, NJ, USA), 100 IU/mL of Penicillin and 100 mg/mL of Streptomycin. Cell viability was assessed by trypan blue exclusion analysis. Cell numbers were determined by using a hemacytometer.

3.4. Flow Cytometric Analysis of Cellular DNA Content

Cells (2 × 106) were fixed in 1 mL of ethanol (70%) for 60 min at room temperature. Harvested cells were resuspended in 1 mL of sodium citrate (50 mM) containing 250 μg RNase A and incubated at 50 °C for 60 min Next, cells were resuspended in the same buffer containing 4 μg of propidium iodide (PI) and incubated for 30 min before being analyzed by flow cytometry (Becton Dickinson, San Jose, CA, USA). The percentage of cells in various cell cycle phases was determined by using Cell Quest Pro software (version 5.1, Becton Dickinson, East Rutherford, NJ, USA).

3.5. Side Population Staining by DYECYCLE Violet Stain

For DCV staining, cells were pelleted and suspended in DMEM cell culture medium at a concentration of 1 × 106 cells/mL. DCV (Invitrogen Molecular Probes®, Eugene, OR, USA) was added at a final staining concentration of 10 μM, as this concentration gave optimal separation between SP and non SP cells. PI staining was used to exclude dead cells. Functionally, to gate only side population cells, Verapamil 200 μM or Emtricitabine (FTC, 10 μg/mL) was used. All analyses were performed on a FACS LSRII (BD Biosciences, San Jose, CA, USA). Debris and cell clusters were excluded during side-scatter and forward-scatter analyses.

3.6. Antitumor Activity in Mice

Nude mice (Jackson Laboratories, Bar Harbor, ME, USA), six to seven weeks old, weighing 20 g, were obtained from the Animal Care and Use Committee of the King Faisal Specialist Hospital and Research Centre, Riyadh, KSA. All of the animals were acclimatized to laboratory conditions for one week before experiments. The animals were maintained under standard conditions, housed in a pathogen-free environment, and fed adequately. Each treatment and vehicle group consisted of six animals. The breeding, care and sacrifice of the animals were performed in accordance with the protocols approved by the Animal Care and Use Committee of the King Faisal Specialist Hospital and Research Centre. The mice were injected with 4 × 106 cells of LOVO subcutaneously in the right flank, and tumor size was measured weekly using a caliper. When the tumor reached approximately 400 mm3 diameter, the mice were divided into control, treated groups, the treatment including administration of DHP-5 (50 mg/kg) via intraperitoneal injection daily for 14 days. The general toxicity of the treatment was determined by measuring the total body weight of the treated and control mice.

4. Conclusions

In conclusion, we focused on the synthesis of dihydropyrimidine derivatives (DHP 19). The synthesized compounds were screened in vitro against LOVO colon cancer cells. DHP-4 was found to be the most active compound of the series in its side population inhibition percentage at 10 μM. The anti-tumor effect of compound DHP-5 was demonstrated on tumors of colon cancer xenografts. Compound DHP-5 was found to be a more potent inhibitor of side population cells than the reference drug Verapamil. Compound DHP-5 exhibited an in vitro anti-proliferative effect and arrested cancer cells at the G2 checkpoint. Furthermore, treatment with compound DHP-5 enabled blocking of the self-renewal ability of breast cancer cells in a dose-dependent manner. Compound DHP-5 induced apoptosis and blocked cell proliferation in vitro and presented superior efficacy compared to the reference drug Doxorubicin in advanced animal models of colon cancer without any sign of general toxicity.

Acknowledgments

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research group no. (RG 1435-006).

Author Contributions

Design, synthesis and analysis of the compounds was performed by M.A.B.; A.A.-D. performed the anti-proliferative activity, M.A.A.-O. helped in the preparation of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Winquist, R.J.; Furey, B.F.; Boucher, D.M. Cancer stem cells as the relevant biomass for drug discovery. Curr. Opin. Pharmacol. 2010, 10, 385–390. [Google Scholar] [CrossRef] [PubMed]
  2. McDermott, S.P.; Wicha, M.S. Targeting breast cancer stem cells. Mol. Oncol. 2010, 4, 404–419. [Google Scholar] [CrossRef] [PubMed]
  3. Bonnet, D.; Dick, J.E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3, 730–737. [Google Scholar] [CrossRef] [PubMed]
  4. Al-Hajj, M.; Wicha, M.S.; Benito-Hernandez, A.; Morrison, S.J.; Clarke, M.F. Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA 2003, 100, 3983–3988. [Google Scholar] [CrossRef] [PubMed]
  5. Singh, S.K.; Clarke, I.D.; Terasaki, M.; Bonn, V.E.; Hawkins, C.; Squire, J.; Dirks, P.B. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003, 63, 5821–5828. [Google Scholar] [PubMed]
  6. Ho, M.M.; Ng, A.V.; Lam, S.; Hung, J.Y. Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells. Cancer Res. 2007, 67, 4827–4833. [Google Scholar] [CrossRef] [PubMed]
  7. Ricci-Vitiani, L.; Lombardi, D.G.; Pilozzi, E.; Biffoni, M.; Todaro, M.; Peschle, C.; de Maria, R. Identification and expansion of human colon-cancer-initiating cells. Nature 2007, 445, 111–115. [Google Scholar] [CrossRef] [PubMed]
  8. Bomken, S.; Fiser, K.; Heidenreich, O.; Vormoor, J. Understanding the cancer stem cell. Br. J. Cancer 2010, 103, 439–445. [Google Scholar] [CrossRef] [PubMed]
  9. Ginestier, C.; Hur, M.H.; Charafe-Jauffret, E.; Monville, F.; Dutcher, J.; Brown, M.; Jacquemier, J.; Viens, P.; Kleer, C.G.; Liu, S.; et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 2007, 1, 555–567. [Google Scholar] [CrossRef] [PubMed]
  10. Morimoto, K.; Kim, S.J.; Tanei, T.; Shimazu, K.; Tanji, Y.; Taguchi, T.; Tamaki, Y.; Terada, N.; Noguchi, S. Stem cell marker aldehyde dehydrogenase 1-positive breast cancers are characterized by negative estrogen receptor, positive human epidermal growth factor receptor type 2, and high Ki67 expression. Cancer Sci. 2009, 100, 1062–1068. [Google Scholar] [CrossRef] [PubMed]
  11. Charafe-Jauffret, E.; Ginestier, C.; Iovino, F.; Tarpin, C.; Diebel, M.; Esterni, B.; Houvenaeghel, G.; Extra, J.M.; Bertucci, F.; Jacquemier, J.; et al. Aldehyde dehydrogenase 1-positive cancer stem cells mediate metastasis and poor clinical outcome in inflammatory breast cancer. Clin. Cancer Res. 2010, 16, 45–55. [Google Scholar] [CrossRef] [PubMed]
  12. Tanei, T.; Morimoto, K.; Shimazu, K.; Kim, S.J.; Tanji, Y.; Taguchi, T.; Tamaki, Y.; Noguchi, S. Association of breast cancer stem cells identified by aldehyde dehydrogenase 1 expression with resistance to sequential Paclitaxel and epirubicin-based chemotherapy for breast cancers. Clin. Cancer Res. 2009, 15, 4234–4241. [Google Scholar] [CrossRef] [PubMed]
  13. Kondo, T.; Setoguchi, T.; Taga, T. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc. Natl. Acad. Sci. USA 2004, 101, 781–786. [Google Scholar] [CrossRef] [PubMed]
  14. Patrawala, L.; Calhoun, T.; Schneider-Broussard, R.; Zhou, J.; Claypool, K.; Tang, D.G. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2 cancer cells are similarly tumorigenic. Cancer Res. 2005, 65, 6207–6219. [Google Scholar] [CrossRef] [PubMed]
  15. Li, X.; Lewis, M.T.; Huang, J.; Gutierrez, C.; Osborne, C.K.; Wu, M.F.; Hilsenbeck, S.G.; Pavlick, A.; Zhang, X.; Chamness, G.C.; et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J. Natl. Cancer Inst. 2008, 100, 672–679. [Google Scholar] [CrossRef] [PubMed]
  16. Yu, F.; Yao, H.; Zhu, P.; Zhang, X.; Pan, Q.; Gong, C.; Huang, Y.; Hu, X.; Su, F.; Lieberman, J.; et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 2007, 131, 1109–1123. [Google Scholar] [CrossRef] [PubMed]
  17. Al-Hajj, M.; Becker, M.W.; Wicha, M.; Weissman, I.; Clarke, M.F. Therapeutic implications of cancer stem cells. Curr. Opin. Genet. Dev. 2004, 14, 43–47. [Google Scholar] [CrossRef] [PubMed]
  18. Bokaeva, S.S. The effects of some pyrimidine derivates on the growth of transplanted tumors in animals. Tr. Kaz. Nauchno-Issled. Inst. Onkol. Radiol. 1967, 3, 305–309. [Google Scholar]
  19. Mayer, T.U.; Kapoor, T.M.; Haggarty, S.J.; King, R.W.; Schreiber, S.L.; Mitchison, T.J. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 1999, 286, 971–974. [Google Scholar] [CrossRef] [PubMed]
  20. Russowsky, D.; Canto, R.F.; Sanches, S.A.; D’Oca, M.G.; de Fátima, A.; Pilli, R.A.; Kohn, L.K.; Antônio, M.A.; de Carvalho, J.E. Synthesis and differential antiproliferative activity of Biginelli compounds against cancer cell lines: Monastrol, oxo-monastrol and oxygenated analogues. Bioorg. Chem. 2006, 34, 173–182. [Google Scholar] [CrossRef] [PubMed]
  21. Prokopcova, H.; Dallinger, D.; Uray, G.; Kaan, H.Y.; Ulaganathan, V.; Kozielski, F.; Laggner, C.; Kappe, C.O. Structure-activity relationships and molecular docking of novel dihydropyrimidine-based mitotic Eg5 inhibitors. ChemMedChem 2010, 5, 1760–1769. [Google Scholar] [CrossRef] [PubMed]
  22. Wright, C.M.; Chovatiya, R.J.; Jameson, N.E.; Turner, D.M.; Zhu, G.; Werner, S.; Huryn, D.M.; Pipas, J.M.; Day, B.W.; Wipf, P.; et al. Pyrimidinone-peptoid hybrid molecules with distinct effects on molecular chaperone function and cell proliferation. Bioorg. Med. Chem. 2008, 16, 3291–3301. [Google Scholar] [CrossRef] [PubMed]
  23. Garcia-Saez, I.; DeBonis, S.; Lopez, R.; Trucco, F.; Rousseau, B.; Thuéry, P.; Kozielski, F. Structure of human Eg5 in complex with a new monastrol-based inhibitor bound in the R configuration. J. Biol. Chem. 2007, 282, 9740–9747. [Google Scholar] [CrossRef] [PubMed]
  24. Ramos, L.M.; Guido, B.C.; Nobrega, C.C.; Corrêa, J.R.; Silva, R.G.; de Oliveira, H.C.; Gomes, A.F.; Gozzo, F.C.; Neto, B.A. The biginelli reaction with an imidazolium-tagged recyclable iron catalyst: Kinetics, mechanism, and antitumoral activity. Chem. Eur. J. 2013, 19, 4156–4168. [Google Scholar] [CrossRef] [PubMed]
  25. Soumyanarayanan, U.; Bhat, V.G.; Kar, S.S.; Mathew, J.A. Monastrol mimic Biginelli dihydropyrimidinone derivatives: Synthesis, cytotoxicity screening against HepG2 and HeLa cell lines and molecular modeling study. Org. Med. Chem. Lett. 2012, 2. [Google Scholar] [CrossRef] [PubMed]
  26. Kumar, B.R.P.; Sankar, G.; Nasir Baig, R.B.; Chandrashekaran, S. Novel Biginelli dihydropyrimidines with potential anticancer activity: A parallel synthesis and CoMSIA study. Eur. J. Med. Chem. 2009, 44, 4192–4198. [Google Scholar] [CrossRef] [PubMed]
  • Sample Availability: Samples of the compounds (DHP 19) in pure form are available from the authors.
Figure 1. Dihydropyrimidine derivatives demonstrating anticancer activity.
Figure 1. Dihydropyrimidine derivatives demonstrating anticancer activity.
Molecules 21 01746 g001
Figure 2. Lead compound Monstrol-97 and newly synthesized compounds (DHP 19).
Figure 2. Lead compound Monstrol-97 and newly synthesized compounds (DHP 19).
Molecules 21 01746 g002
Figure 3. Scatter plot showing results of side population analyses of tumor-derived cells of LOVO untreated, treated with DHP-1, DHP-4, DHP-5 and DHP-6. Furthermore, compound DHP-5 exhibit an in vitro anti-proliferative effect and arrested cancer cells at the G2 checkpoint (Figure 4). Blue color represents the percentage of cancer stem cells and red color represents the percentage of remaining cells other than cancer stem cells.
Figure 3. Scatter plot showing results of side population analyses of tumor-derived cells of LOVO untreated, treated with DHP-1, DHP-4, DHP-5 and DHP-6. Furthermore, compound DHP-5 exhibit an in vitro anti-proliferative effect and arrested cancer cells at the G2 checkpoint (Figure 4). Blue color represents the percentage of cancer stem cells and red color represents the percentage of remaining cells other than cancer stem cells.
Molecules 21 01746 g003
Figure 4. Compound DHP-5 arrested cancer cells at G2 checkpoint.
Figure 4. Compound DHP-5 arrested cancer cells at G2 checkpoint.
Molecules 21 01746 g004
Figure 5. Scatter plot showing results of side population analyses of tumor-derived cells of the LOVO xenograft that were untreated, treated with side population inhibitor reference drug Verapamil (200 μM) and with DHP-5 (50 μM).
Figure 5. Scatter plot showing results of side population analyses of tumor-derived cells of the LOVO xenograft that were untreated, treated with side population inhibitor reference drug Verapamil (200 μM) and with DHP-5 (50 μM).
Molecules 21 01746 g005
Figure 6. Graph showing tumor growth record of LOVO (colon cancer xenograft) in untreated mice group (red line) and DHP-5–treated (50 mg/kg) mice group (blue line).
Figure 6. Graph showing tumor growth record of LOVO (colon cancer xenograft) in untreated mice group (red line) and DHP-5–treated (50 mg/kg) mice group (blue line).
Molecules 21 01746 g006
Scheme 1. Synthetic route of compounds (DHP 19).
Scheme 1. Synthetic route of compounds (DHP 19).
Molecules 21 01746 sch001
Table 1. Physical data of the synthesized dihydropyrimidinone/thione compounds (DHP 19).
Table 1. Physical data of the synthesized dihydropyrimidinone/thione compounds (DHP 19).
Molecules 21 01746 i001
CompoundsRR1R2(Yield %)m.p. (°C)
DHP-1PhenylOCH3O70153–155
DHP–24-ChlorophenylOCH3O75138–140
DHP-34-NitrophenylOCH3O65158–160
DHP-43,4-DimethoxyphenylOCH3O72165–167
DHP-54-EthoxyphenylOCH3O60168–170
DHP-6PhenylHS65248–250
DHP-74-ChlorophenylHS65243–245
DHP-84-NitrophenylHS68258–260
DHP-93,4-DimethoxyphenylHS70228–230
Table 2. Side population inhibition on LOVO colon cancer cells (%) at 10 μM concentration.
Table 2. Side population inhibition on LOVO colon cancer cells (%) at 10 μM concentration.
Compounds* Side Population (%) at 10 μM# Side Population Inhibition (%) at 10 μM
DHP-14.90 ± 0.20
DHP-21.72 ± 0.164.7
DHP-31.76 ± 0.364
DHP-41.44 ± 0.570.5
DHP-52.01 ± 0.758.82
DHP-61.47 ± 0.670
DHP-74.90 ± 0.30
DHP-82.4 ± 0.150
DHP-94.90 ± 0.10
* Side population% as mean ± SD of three independent experiments; # Inhibition% = 100 − (SP% of treated cells/SP% of untreated cells) × 100.
Molecules EISSN 1420-3049 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top