Synthesis of 4,4-Disubstituted 3,4-Dihydropyrimidin-2(1H)-ones and -thiones, the Corresponding Products of Biginelli Reaction Using Ketone, and Their Antiproliferative Effect on HL-60 Cells

Abstract

An efficient synthetic method for novel 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones 5 and -thiones 6 was developed. The cyclocondensation reaction of O-methylisourea hemisulfate salt 11 with 8 gives a tautomeric mixture of dihydropyrimidines 12 and 13 following acidic hydrolysis of the cyclized products to produce 5 in high yields. Thionation reaction of 5 at the 2-position smoothly proceeds to give 2-thioxo derivatives 6. These compounds 5 and 6, corresponding to the products of a Biginelli-type reaction using urea or thiourea, a ketone and a 1,3-dicarbonyl compound, have long been inaccessible and hitherto unavailable for medicinal chemistry. These methods are invaluable for the synthesis of 5 and 6, which have been inaccessible by conventional methods. Therefore, the synthetic methods established in this study will expand the molecular diversity of their related derivatives. These compounds were also assessed for their antiproliferative effect on a human promyelocytic leukemia cell line, HL-60. Treatment of 10 µM 6b and 6d showed high inhibitory activity similarly to 1 µM all-trans retinoic acid (ATRA), indicating that the 2-thioxo group and length of two alkyl substituents at the 4-position are strongly related to activity.

Introduction

The Biginelli reaction, a three-component cyclocondensation reaction involving urea or thiourea 1, aldehydes 2, and 1,3-dicarbonyl compounds 3, gives 3,4-dihydropyrimidin-2(1H)-ones or thiones 4^1–5) (Chart 1). These versatile heterocycles show a wide range of biological activities for medicinal applications.^6–10) For example, they display calcium channel inhibition,^11,12) anticancer,^13,14) antibacterial,¹⁵⁾ antifungal,¹⁶⁾ anti-human immunodeficiency virus (HIV),¹⁷⁾ antimalarial,¹⁸⁾ anti-inflammatory,¹⁹⁾ and antioxidation²⁰⁾ activities. However, this type of reaction has serious synthetic limitations. Unlike aldehydes 2, ketones are not applicable to the reaction because of their inertness.²¹⁾ Therefore, 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones 5 and -thiones 6 (Chart 2), corresponding to the products of a Biginelli-type reaction using ketones, have long been inaccessible and hitherto unavailable for medicinal chemistry.

Chart 1. Typical Biginelli Reaction for Synthesis of 3,4-Dihydropyrimidin-2(1H)-ones or -thiones 4

Chart 2. Synthetic Approach for a Series of 4,4-Disubstituted Dihydropyrimidines

Previously, we reported a general approach to the synthesis of novel 2-methylthio-4,4,6-trisubstituted 1,4-dihydropyrimidine 9 and its tautomer, 2-methylthio-4,6,6-trisubstituted 1,6-dihydropyrimidine 10²¹⁾ (Chart 2). This approach was achieved by applying the [3 + 3] cyclocondensation strategy for the synthesis of dihydropyrimidines, originally reported by Traube and Schwartz,^7,22) to the reaction of an S-methylisothiourea hemisulfate salt 7 with 2-(gem-disubstituted)methylene-3-oxoesters 8. The key intermediate 8 was prepared in advance by Lehnert’s modified procedure^23,24) using TiCl₄ and pyridine in a Knoevenagel-type condensation. In this paper, we describe the first synthesis of hitherto unavailable 5 and 6 from O-methylisourea hemisulfate salt 11 and 8; the outline of which is shown in Chart 2. For the synthesis of 5, the cyclocondensation reaction of 11 with 8 gives a tautomeric mixture of dihydropyrimidines 12 and 13 following the acidic hydrolysis of the cyclized products to produce 5 in high yields²⁵⁾ (Chart 2). A selective thionation reaction of 5 at the 2-position smoothly proceeds to give 2-thioxo derivatives 6 (Chart 2). These approaches present an effective solution to the problem of a Biginelli-type reaction using ketones. In our continuing medicinal chemistry program using dihydropyrimidines,^26,27) antiproliferative effect of 5 and 6 on a human promyelocytic leukemia cell line, HL-60 was also evaluated. According to a literature survey, there were some reports in terms of antiproliferative effect of dihydropyrimidine derivatives on leukemia cell lines such as HL-60, K-562, MOLT-4, and Jurkat.^28–31)

Results and Discussion

As a preliminary experiment for the synthesis of 5, 3-oxo-2-(2-propylidene)-butanoate 8a was prepared from ethyl 3-oxobutanoate and acetone under Lehnert conditions using TiCl₄ in the presence of pyridine.²¹⁾ When 8a (1.0 equivalent (equiv.)) and 11 (1.2 equiv.) were reacted in the presence of NaHCO₃ (4.0 equiv.) in N,N-dimethylformamide (DMF) at 65 °C for 12 h, the cyclocondensation reaction proceeded smoothly to provide ethyl 2-methoxy-4,4,6-trimethyl-1,4-dihydropyrimidine-5-carboxylate 12a and its tautomer, ethyl 2-methoxy-4,6,6-trimethyl-1,6-dihydropyrimidine-5-carboxylate 13a, as an inseparable mixture (12a : 13a = 1.0 : 1.4 in dimethyl sulfoxide (DMSO)-d₆) in a combined yield of 93% (Chart 3). The combined yields of the mixture of 12a and 13a were calculated after chromatographic purification and confirmation of their structure based on spectral and physiological data. The structure of each tautomer was assigned by nuclear Overhauser effect (NOE) experiment. For the major component 13a, its 6,6-dimethyl protons exhibited a significant NOE when the 1-NH proton was irradiated; hence, its structure was determined to be 1,6-dihydropyrimidine 13a, as shown in Chart 3. Irradiation of the 1-NH proton in the minor component 12a caused the NOE on the 6-methyl protons, which showed that its structure is 1,4-dihydropyrimidine 12a (Chart 3).

Chart 3. Cyclocondensation for Synthesis of of 12a and 13a, and Hydrolysis of the Mixture of Tautomers for Synthesis of 5a

The tautomeric relationship of 12a and 13a was confirmed by ¹H-NMR analysis. Namely, the tautomeric ratio was changed in a solvent-dependent manner; when the ¹H-NMR spectra were measured in CDCl₃ instead of DMSO-d₆, the 12a : 13a ratio changed from 1.0 : 1.4 to 1.0 : 2.9 (Chart 3). In addition, a tautomeric mixture of 12a and 13a was hydrolyzed using 3 M HCl aqueous solution in tetrahydrofuran (THF)-MeOH at 50 °C for 24 h to give the desired 2-oxo product 5a as a sole product in 95% yield. These results indicated that 12a and 13a were interconvertible tautomers. The thermodynamic preference of 1,6-dihydropyrimidine 13a in CDCl₃ and DMSO-d₆ is attributable to the extended conjugation along three double bonds of 13a. Its preference for the 1,6-dihydro tautomer is supported by the findings of our previous experimental and theoretical studies on 2-substituted dihydropyrimidine tautomers.³²⁾

Now that two-step synthesis of 5a by cyclocondensation of 8a with 11 following hydrolysis proceeded successfully in high yield, we applied this approach to the synthesis of a range of 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones 5, which were otherwise inaccessible. With all substrates 8b–i, the cyclocondensation reaction with 11 (1.2 or 3.0 equiv.) in the presence of NaHCO₃ (4.0 equiv.) in DMF at 65 °C for 12 h proceeded uneventfully to give inseparable mixtures of 12 and 13 in a fair to good combined yield, as listed in Table 1. The combined yields of the tautomers 12 and 13 were calculated after chromatographic purification and confirmation of their purity by ¹H-NMR analysis. The substrate 8b prepared from heptan-2-one and ethyl 3-oxobutanoate was also used in the reaction (entry 1). The bulky alkylidene substituents of 8 were sensitive to the cyclocondensation. Sterically demanding 8c, 8d, and 8e with two CH₂R units as alkylidene substituents, showed low reactivities; therefore, 11 was used in an excess amount of 3.0 equiv. to afford the products 12c/13c, 12d/13d, and 12e/13e in acceptable yields (entries 2–4), respectively. Furthermore, a fluorenylidene group could also be accommodated at the 4-position, as shown by 12f/13f (entry 5). When starting from 8g prepared from ethyl benzoylacetate, a phenyl group could be incorporated instead of methyl group (entry 6). In addition to ethyl ester 8a, the methyl and benzyl esters 8h and 8i, respectively, were tolerated in the cyclocondensation to give the respective products 12h/13h and 12i/13i (entries 7 and 8). Successive hydrolysis of the mixture of tautomers 12/13 proceeded smoothly to give various desired 2-oxo products 5b–i.

Table 1. Synthesis of 4,4-Disubstituted 3,4-Dihydropyrimidin-2(1H)-ones 5^a,b)

a) General conditions of cyclocondensation: a mixture of 11 (1.2 equiv.), 8 (1.0 equiv.), NaHCO₃ (4.0 equiv.) in DMF was heated at 65 °C for 12 h under an atmosphere of argon unless otherwise specified. b) Reaction conditions of hydrolysis: a mixture of 12 and 13, 3 M HCl aqueous solution (15 equiv.) in THF-MeOH was heated at 50 °C for 24 h. c) 11 (3.0 equiv.) was used.

With a variety of novel 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones 5 now available, we carried out the thionation reaction of 5 to synthesize 2-thioxo congeners 6. The reaction of 5a with Lawesson reagent³³⁾ (1.2 equiv.) in toluene at reflux for 2 h proceeded smoothly to give a desired 6a in 88% yield (Chart 4). To confirm that the thionation occurred at the 2-position, 6a was prepared through another synthetic route.³⁴⁾ Namely, the cyclocondensation reaction of S-(4-methoxybenzyl)isothiourea hydrochloride salt 14 with 8a produced ethyl 2-[(4-methoxybenzyl)thio]-4,4,6-trimethyl-1,4-dihydropyrimidine-5-carboxylate 15 and ethyl 2-[(4-methoxybenzyl)thio]-4,6,6-trimethyl-1,6-dihydropyrimidine-5-carboxylate 16 as an inseparable mixture (1.7 : 1.0 in DMSO-d₆) in a combined yield of 88%; the structure of each tautomer was not assigned (Chart 4). The deprotection of the 4-methoxybenzyl (PMB) group of 15 and 16 using trifluoroacetic acid (TFA) and ethanethiol at 70 °C for 24 h gave 6a.³⁴⁾ The ¹H- and ¹³C-NMR spectra of the products 6a obtained through the two synthetic routes matched. Therefore, the thionation of 5a using the Lawesson reagent at the 2-position was confirmed. Similar thionation reactions of pyrimidin-2(1H)-ones having ester group at the 5-position were reported, in which the reactions selectively proceeded at the 2-position in any case.^35,36) The selectivity is probably derived from higher reactivity of cyclic urea like amide than ester.³³⁾ Although the synthetic route for 6 from 14 and 8 using a PMB group for protection was attractive, we chose the cyclocondensation–hydrolysis–thionation reactions starting from 11 and 8 as a preferable and practical route for the synthesis of 6 because of unreproducible results of 21–89% yields in the deprotection step. In our experiments, highly volatile nature (bp 35 °C) of ethanethiol under the reaction condition of 70 °C probably caused the problem of reproducibility. The thionation procedure using the Lawesson reagent also worked well for other substrates 5b–d, and the corresponding 2-thioxo products 6b–d were obtained in good yields (Chart 5).

Chart 4. Synthesis of 2-Thioxo Product 6a by Two Synthetic Routes

Chart 5. Synthesis of 4,4-Disubstituted 3,4-Dihydropyrimidin-2(1H)-thiones 6b–d

With novel 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones 5 and -thiones 6 now available, the antiproliferative effect of the thirteen compounds on HL-60 cells was assessed (Fig. 1). As shown in Fig. 1B, a significant decrease in cell viability was observed for 10 µM 6b-, 6d-, and 1 µM all-trans retinoic acid (ATRA)-treated HL-60 cells; the strong inhibitory effect of 6b was comparable to that of ATRA. These results suggest that 2-thioxo derivatives 6 showed higher antiproliferative activities than 2-oxo analogs 5 (Fig. 1A). Importantly, length of two alkyl substituents at 4-position of 6 had a crucial effect on activity; 4-methyl-4-pentyl 6b and 4,4-dipropyl 6d showed high inhibitory activity. Therefore, we further examined the IC₅₀ values of 6b (Fig. 2A) and 6d (Fig. 2B) with various concentrations in 0.5–10.0 µM. As shown in Fig. 2, a dose-dependent decrease in cell viability was observed in 6b- and 6d-treated HL-60 cells; the IC₅₀ values of 6b and 6d were calculated to 3.4 and 7.9 µM, respectively. The antiproliferative effect of 6b and 6d was comparable to the effect of reported dihydropyrimidines of monastrol analogs on HL-60 cells because their IC₅₀ values were 4.7–17.8 µM.³⁰⁾ Although the effects of 6b and 6d was not so high in comparison with those of 2-aminodihydropyrimidines²⁷⁾ and thiazolodihydropyrimidines²⁸⁾ on HL-60 cells in regard to the IC₅₀ values, further structure–activity relationship studies and detail analyses of the effects of 6b and 6d in terms of the type of cell death, cell cycle arrest, and cell differentiation are ongoing.

Fig. 1. Antiproliferative Effect of 5a–i and 6a–d on HL-60 Cells

Following 96 h treatment with 10 µM 5a–i (A), 10 µM 6a–d (B), and 1 µM ATRA, positive control, the viability of HL-60 cells was determined by XTT assay as described in “Cell Viability Assay” in Experimental. Relative cell viability was calculated as the ratio of the absorbance at 450 nm of each treatment group to that of the corresponding untreated control group. Data are shown as the mean and standard deviation (S.D.) from three independent experiments. ^§ p < 0.01 vs. control.

Fig. 2. A Dose-Dependent Decrease in Cell Viability of HL-60 Cells Treated with 6b (A) and 6d (B)

Following 96 h treatment with various concentrations of 6b and 6d, the viability of HL-60 cells was determined by XTT assay as described in “Cell Viability Assay” in Experimental. Relative cell viability was calculated as the ratio of the absorbance at 450 nm of each treatment group to that of the corresponding untreated control group. Data are shown as the mean and S.D. from nine independent experiments. ^† p < 0.01 vs. control.

Conclusion

As shown in Table 1, the first synthesis of 5 was achieved by cyclocondensation of 11 with 8 following hydrolysis of a tautomeric mixture of dihydropyrimidines 12 and 13. Also, as shown in Charts 4 and 5, thionation of 5 smoothly proceeds to give hitherto unavailable 2-thioxo analogs 6. Although these synthetic approaches described in this paper require multiple steps, they are invaluable methods for the synthesis of novel 4,4-disubstituted 3,4-dihydropyrimidin-2(1H)-ones 5 and -thiones 6, which have been inaccessible by Biginelli-type reactions and other conventional synthetic methods. Evaluation of the antiproliferative effect of 5 and 6 on HL-60 cells established that 6b showed high activity similarly to ATRA. Clearly, the 2-thioxo group and methyl and pentyl substituents at the 4-position have a strong effect on activity. Therefore, our synthetic method for 5, 6, and related 4,4-disubstituted derivatives should have a positive impact on dihydropyrimidine-based medicinal chemistry.

Experimental

General Information

All reaction reagents and solvents for synthesis were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) or Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) or Kanto Chemical Co., Inc. (Tokyo, Japan). All-trans retinoic acid (ATRA), Penicillin–Streptomycin solution (10000 U/mL penicillin and 10000 µg/mL streptomycin), phenazine methosulfate (PMS), and RPMI-1640 (Cat# 186-02155) were purchased from FUJIFILM Wako Pure Chemical Corporation. Dimethyl sulfoxide (DMSO, Cat# D4540), fetal bovine serum (FBS), and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) were purchased from Sigma-Aldrich (St. Louis, Mo, U.S.A.). TLC was performed using pre-coated silica gel 60 F₂₅₄ plates (Merck KGaA, Germany) using the indicated solvents. Column chromatography was performed on silica gel 60 [70–230 mesh, NACALAI TESQUE, Inc. (Kyoto, Japan)] or Silica gel 60 N (spherical, neutral, 40–50 µm, Kanto Chemical Co., Inc.) using the indicated solvents. All melting points were determined with an AS ONE melting point apparatus ATM-02 (AS ONE Corporation, Osaka, Japan) or Yanaco melting point apparatus MP-J3 without correction. ¹H-NMR spectra were recorded on a Bruker AVANCE™ III 600 (600 MHz) or JEOL JNM-ECZ500R (500 MHz) with tetramethylsilane (0 ppm) in CDCl₃ or dimethylsulfoxide (2.49 ppm) in DMSO-d₆ as internal standards. ¹³C-NMR spectra were recorded on a Bruker AVANCE™ III 600 (150 MHz) or JEOL JNM-ECZ500R (125 MHz) with chloroform (77.0 ppm) in CDCl₃ or dimethylsulfoxide (39.7 ppm) in DMSO-d₆ as internal standards. IR spectra were measured on a JASCO FT/IR-6100 or JASCO FT/IR-4100 Fourier Transform Infrared Spectrophotometer (JASCO Corporation, Tokyo, Japan). Mass spectra were recorded on a JEOL JMS-700 mass analyzer (JEOL Ltd., Tokyo, Japan). High-resolution spectroscopy (HRMS) was performed using a JEOL JMS-700 mass analyzer. Absorbance spectra were measured on an absorbance microplate reader, Wallac 1420 ARVOsx multilabel counter (PerkinElmer, Inc. Life Sciences, MA, U.S.A.).

2-(gem-Disubstituted)methylene-3-oxoester (8a–i)

The 2-(gem-disubstituted)methylene-3-oxoesters 8a–i, were all assembled under the conditions in our previous report.²¹⁾

Ethyl 2-Methoxy-4,4,6-trimethyl-1,4-dihydropyrimidine-5-carboxylate (12a) and Ethyl 2-Methoxy-4,6,6-trimethyl-1,6-dihydropyrimidine-5-carboxylate (13a)

Under an atmosphere of argon, a mixture of 11 (74 mg, 0.601 mmol), 8a (85 mg, 0.499 mmol), and NaHCO₃ (168 mg, 2.00 mmol) in dry DMF (1.0 mL) was heated at 65 °C for 12 h. To the reaction mixture was added EtOAc (20 mL) followed by water (10 mL), and the organic layer was separated. The aqueous layer was extracted with EtOAc (20 mL), and the combined organic layer and extracts were washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography [n-hexane–EtOAc (3 : 1)] to give 12a and 13a as an inseparable mixture of tautomers (105 mg, 0.464 mmol, 93%) in a ratio of 1.0 : 1.4 in DMSO-d₆ in favor of 13a. Colorless crystals; mp 71–72 °C (n-hexane); ¹H-NMR (600 MHz, DMSO-d₆) of the major component, 13a δ: 1.19 (3H, t, J = 7.2 Hz), 1.31 (6H, s), 1.96 (3H, s), 3.67 (3H, s), 4.06 (2H, q, J = 7.2 Hz), 7.58 (1H, s); ¹H-NMR (600 MHz, DMSO-d₆) of the minor component, 12a δ: 1.19 (3H, t, J = 7.2 Hz), 1.28 (6H, s), 1.92 (3H, s), 3.56 (3H, s), 4.05 (2H, q, J = 7.2 Hz), 8.84 (1H, s). Structural assignment was made by NOE experiment: With 13a, the significant NOE was observed between 1-NH proton (δ 7.58) and 6,6-dimethyl protons (δ 1.31) and as such, its structure was determined to be 13a (Fig. 3). With 12a, the significant NOE was observed between 1-NH proton (δ 8.84) and 6-methyl protons (δ 1.92) and as such, its structure was determined to be 12a (Fig. 3). ¹³C-NMR of mixture of 12a and 13a (150 MHz, DMSO-d₆) δ: 14.32, 14.33, 17.9, 23.6, 29.7, 31.7, 52.6, 53.4, 54.1, 55.3, 59.1, 59.2, 105.0, 108.7, 143.1, 148.2, 153.1, 157.5, 167.2, 167.4; IR (KBr) cm⁻¹: 3297, 2973, 1664, 1618, 1562, 1499, 1369, 1344, 1276, 1208, 1154, 1078, 1059; electron ionization-mass spectra (EI-MS) m/z: 226.1324 (Calcd for C₁₁H₁₈N₂O₃: 226.1317).

Fig. 3. NOE Observed with 12a and 13a

Ethyl 4,4,6-Trimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5a)

A mixture of 12a and 13a (46.0 mg, 0.203 mmol) of 3 M HCl aqueous solution (1.5 mL) in THF-MeOH (0.5 mL + 0.5 mL) was heated at 50 °C for 24 h. To the reaction mixture was added EtOAc (20 mL) followed by 2 M NaOH aqueous solution (2.2 mL), and the organic layer was separated. The aqueous layer was extracted with EtOAc (10 mL), and the combined organic layer and extracts were washed with water, brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by flash column chromatography [n-hexane–EtOAc (1 : 2)] to give 5a (41.0 mg, 0.193 mmol, 95%). Colorless crystals; mp 156–158 °C (n-hexane-EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 1.31 (3H, t, J = 7.2 Hz), 1.49 (6H, s), 2.13 (3H, s), 4.21 (2H, q, J = 7.2 Hz), 5.20 (1H, s), 7.74 (1H, s); ¹³C-NMR (150 MHz, CDCl₃) δ: 14.2, 18.8, 29.9, 54.9, 60.0, 106.9, 142.8, 153.9, 166.5; IR (KBr) cm⁻¹: 3357, 3222, 3110, 2974, 1697, 1629, 1417, 1374, 1287, 1214, 1096; EI-MS m/z: 212.1159 (Calcd for C₁₀H₁₆N₂O₃: 212.1161).

Alkyl 2-oxo-4,4,6-trisubstituted 1,2,3,4-Tetrahydropyrimidine-5-carboxylates (5b–i)

The 2-oxo products 5b–i were synthesized according to the same procedures as described for the synthesis of 5a; below listed are their isolated yield and their physicochemical and spectral data for 5b–i. Structural assignments of each tautomer of 12 and 13 were not made.

Ethyl 4,6-Dimethyl-2-oxo-4-pentyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5b)

The reaction of 8b (113 mg, 0.499 mmol) gave a tautomeric mixture of ethyl 2-methoxy-4,6-dimethyl-4-pentyl-1,4-dihydropyrimidine-5-carboxylate 12b and ethyl 2-methoxy-4,6-dimethyl-6-pentyl-1,6-dihydropyrimidine-5-carboxylate 13b (106 mg, 0.375 mmol, 75%) in a ratio of 1.0 : 1.2 in DMSO-d₆. Colorless oil; ¹H-NMR (600 MHz, DMSO-d₆) of the major component δ: 0.82 (3H, t, J = 7.2 Hz), 1.02–1.32 (13H, m), 1.86–1.95 (1H, m), 1.97 (3H, s), 3.66 (3H, s), 3.99–4.09 (2H, m), 7.44 (1H, s); ¹H-NMR (600 MHz, DMSO-d₆) of the minor component δ: 0.81 (3H, t, J = 7.2 Hz), 1.02–1.32 (13H, m), 1.86–1.95 (1H, m), 1.93 (3H, s), 3.56 (3H, s), 3.99–4.09 (2H, m), 8.76 (1H, s). Hydrolysis of the mixture of 12b and 13b (48.0 mg, 0.170 mmol) gave 5b (44.5 mg, 0.166 mmol, 98%). Colorless crystals; mp 150–152 °C (n-hexane-EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 0.86 (3H, t, J = 7.2 Hz), 1.14–1.44 (10H, m), 1.45 (3H, s), 2.11–2.18 (1H, m), 2.14 (3H, s), 4.20 (2H, q, J = 7.2 Hz), 4.94 (1H, s), 7.63 (1H, s); ¹³C-NMR (150 MHz, CDCl₃) δ: 14.0, 14.2, 18.9, 22.5, 23.9, 30.0, 31.8, 42.2, 58.4, 59.9, 104.7, 143.8, 154.0, 166.6; IR (KBr) cm⁻¹: 3350, 3227, 3110, 2960, 2925, 1703, 1681, 1629, 1423, 1370, 1290, 1259, 1211, 1096; FAB-MS m/z: [M + H]⁺ 269.1869 (Calcd for C₁₄H₂₅N₂O₃: 269.1865).

Ethyl 4-Methyl-2-oxo-1,3-diazaspiro[5,5]undeca-2,5-diene-5-carboxylate (5c)

The reaction of 8c (105 mg, 0.499 mmol) with 3.0 equiv. of 11 (185 mg, 1.50 mmol) gave a tautomeric mixture of ethyl 2-methoxy-4-methyl-1,3-diazaspiro[5,5]undeca-1,4-diene-5-carboxylate 12c and ethyl 2-methoxy-4-methyl-1,3-diazaspiro[5,5]undeca-2,4-diene-5-carboxylate 13c (98.0 mg, 0.368 mmol, 74%) in a ratio of 3.0 : 1.0 in DMSO-d₆. Colorless crystals; ¹H-NMR (600 MHz, DMSO-d₆) of the major component δ: 0.98–1.94 (10H, m), 1.19 (3H, t, J = 7.2 Hz), 1.84 (3H, s), 3.61 (3H, s), 4.05 (2H, q, J = 7.2 Hz), 8.78 (1H, s); ¹H-NMR (600 MHz, DMSO-d₆) of the minor component δ: 0.98–1.94 (10H, m), 1.20 (3H, t, J = 7.2 Hz), 1.84 (3H, s), 3.68 (3H, s), 4.07 (2H, q, J = 7.2 Hz), 7.47 (1H, s). Hydrolysis of the mixture of 12c and 13c (562 mg, 2.11 mmol) gave 5c (480 mg, 1.90 mmol, 90%). Colorless crystals; mp 155–157 °C (n-hexane-EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 1.22 (1H, ddq, J = 3.6, 3.6, 13.2 Hz), 1.32 (3H, t, J = 7.2 Hz), 1.42 (2H, ddq, J = 3.0, 3.0, 13.8 Hz), 1.55–1.71 (3H, m), 1.73 (2H, d, J = 12.6 Hz), 2.06 (3H, s), 2.10 (2H, dt, J = 4.2, 13.2 Hz), 4.22 (2H, q, J = 7.2 Hz), 5.45 (1H, s), 7.16 (1H, s); ¹³C-NMR (150 MHz, CDCl₃) δ: 14.2, 18.5, 20.6, 24.6, 34.8, 56.5, 60.1, 107.8, 141.6, 153.8, 166.9; IR (KBr) cm⁻¹: 3299, 3220, 3101, 2931, 1703, 1676, 1431, 1313, 1173, 1100; EI-MS m/z: 252.1473 (Calcd for C₁₃H₂₀N₂O₃: 252.1474).

Ethyl 4-Methyl-2-oxo-4,4-dipropyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5d)

The reaction of 8d (105 mg, 0.499 mmol) with 3.0 equiv. of 11 (185 mg, 1.50 mmol) gave a tautomeric mixture of ethyl 2-methoxy-6-methyl-4,4-dipropyl-1,4-dihydropyrimidine-5-carboxylate 12d and ethyl 2-methoxy-4-methyl-6,6-dipropyl-1,6-dihydropyrimidine-5-carboxylate 13d (57.0 mg, 0.202 mmol, 51%) in a ratio of 1.0 : 1.1 in DMSO-d₆. Colorless oil; ¹H-NMR (600 MHz, DMSO-d₆) of the major component δ: 0.80 (3H, t, J = 7.2 Hz), 1.05–1.38 (6H, m), 1.17 (3H, t, J = 7.2 Hz), 1.77–1.87 (2H, m), 1.99 (3H, s), 3.65 (3H, s), 4.04 (2H, q, J = 7.2 Hz), 7.30 (1H, s); ¹H-NMR (600 MHz, DMSO-d₆) of the minor component δ: 0.81 (3H, t, J = 7.2 Hz), 1.05–1.38 (6H, m), 1.17 (3H, t, J = 7.2 Hz), 1.77–1.87 (2H, m), 1.93 (3H, s), 3.56 (3H, s), 4.03 (2H, q, J = 7.2 Hz), 8.67 (1H, s). Hydrolysis of the mixture of 12d and 13d (105 mg, 0.372 mmol) gave 5d (89.0 mg, 0.332 mmol, 89%). Colorless crystals; mp 151–153 °C (n-hexane-EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 0.89 (6H, t, J = 7.2 Hz), 1.18–1.33 (4H, m), 1.29 (3H, t, J = 7.2 Hz), 1.37–1.49 (2H, m), 2.03–2.12 (2H, m), 2.15 (3H, s), 4.19 (2H, q, J = 7.2 Hz), 4.77 (1H, s), 7.63 (1H, s); ¹³C-NMR (150 MHz, CDCl₃) δ: 14.15, 14.20, 17.3, 19.0, 44.8, 59.8, 62.2, 102.3, 144.9, 154.3, 166.6; IR (KBr) cm⁻¹: 3301, 3092, 2956, 1708, 1676, 1618, 1433, 1281, 1241, 1188, 1097; FAB-MS m/z: [M + H]⁺ 269.1869 (Calcd for C₁₄H₂₅N₂O₃: 269.1865).

Ethyl 4-Ethyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5e)

The reaction of 8e (123 mg, 0.499 mmol) with 3.0 equiv. of 11 (185 mg, 1.50 mmol) gave a tautomeric mixture of ethyl 4-ethyl-6-methyl-2-methoxy-4-phenyl-1,4-dihydropyrimidine-5-carboxylate 12e and ethyl 6-ethyl-4-methyl-2-methoxy-6-phenyl-1,6-dihydropyrimidine-5-carboxylate 13e (62.0 mg, 0.205 mmol, 41%) in a ratio of 1.5 : 1.0 in DMSO-d₆. Colorless crystals; ¹H-NMR (600 MHz, DMSO-d₆) of the major component δ: 0.82 (3H, t, J = 7.2 Hz), 0.91 (3H, t, J = 7.2 Hz), 1.75–1.86 (2H, m), 2.08 (3H, s), 3.55 (3H, s), 3.76–3.90 (2H, m), 7.10 (1H, t, J = 7.2 Hz), 7.22 (2H, t, J = 7.2 Hz), 7.31 (2H, d, J = 7.2 Hz), 9.04 (1H, s); ¹H-NMR (600 MHz, DMSO-d₆) of the minor component δ: 0.89 (3H, t, J = 7.2 Hz), 0.94 (3H, t, J = 7.2 Hz), 2.13 (3H, s), 2.40–2.50 (2H, m), 3.569 (3H, s), 3.76–3.90 (2H, m), 7.18 (1H, t, J = 7.2 Hz), 7.28 (2H, t, J = 7.2 Hz), 7.37 (2H, d, J = 7.2 Hz), 7.91 (1H, s). Hydrolysis of the mixture of 12e and 13e (78.0 mg, 0.258 mmol) gave 5e (74.0 mg, 0.257 mmol, 99%). Colorless crystals; mp 204–206 °C (n-hexane-EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 0.96 (3H, t, J = 7.2 Hz), 1.06 (3H, t, J = 7.2 Hz), 1.93 (1H, dq, J = 14.4, 7.2 Hz), 2.27 (3H, s), 2.70 (1H, dq, J = 14.4, 7.2 Hz), 3.91 (2H, q, J = 7.2 Hz), 4.88 (1H, s), 7.23 (1H, t, J = 7.8 Hz), 7.32 (2H, t, J = 7.2 Hz), 7.37 (1H, s), 7.47 (2H, d, J = 7.8 Hz); ¹³C-NMR (150 MHz, CDCl₃) δ: 8.5, 13.7, 19.0, 32.4, 59.7, 63.8, 103.3, 126.4, 127.1, 128.0, 145.4, 147.1, 153.0, 166.0; IR (KBr) cm⁻¹: 3339, 3220, 3103, 2970, 1691, 1629, 1425, 1370, 1294, 1259, 1098; EI-MS m/z: 288.1467 (Calcd for C₁₆H₂₀N₂O₃: 288.1474).

Ethyl 6′-Methyl-2′-oxo-2′,3′-dihydro-1′H-spiro[fluorene-9,4′-pyrimidine]-5′-carboxylate (5f)

The reaction of 8f (146 mg, 0.499 mmol) gave a tautomeric mixture of ethyl 2′-methoxy-6′-methyl-1′H-spiro[fluorene-9,4′-pyrimidine]-5′-carboxylate 12f and ethyl 2′-methoxy-6′-methyl-3′H-spiro[fluorene-9,4′-pyrimidine]-5′-carboxylate 13f (72.0 mg, 0.207 mmol, 41%) in a ratio of 1.0 : 1.6 in CDCl₃ while structural assignment of each tautomer was not made. Pale yellow amorphous; ¹H-NMR (600 MHz, CDCl₃) of the major component δ: 0.47 (3H, t, J = 7.2 Hz), 2.44 (3H, s), 3.50 (2H, q, J = 7.2 Hz), 3.89 (3H, s), 4.85 (1H, s), 7.22–7.39 (4H, m), 7.54 (2H, d, J = 7.2 Hz), 7.61 (2H, d, J = 7.2 Hz); ¹H-NMR (600 MHz, CDCl₃) of the minor component δ: 0.45 (3H, t, J = 7.2 Hz), 2.34 (3H, s), 3.45 (2H, q, J = 7.2 Hz), 3.59 (3H, s), 6.00 (1H, s), 7.22–7.39 (6H, m), 7.63 (2H, d, J = 7.2 Hz). Hydrolysis of the mixture of 12f and 13f (119 mg, 0.342 mmol) gave 5f (108 mg, 0.323 mmol, 95%). Colorless crystals; mp 243–245 °C (CHCl₃-EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 0.48 (3H, t, J = 7.2 Hz), 2.40 (3H, s), 3.51 (2H, q, J = 7.2 Hz), 4.94 (1H, s), 7.29 (2H, t, J = 7.8 Hz), 7.37 (2H, t, J = 7.8 Hz), 7.49 (2H, d, J = 7.8 Hz), 7.62 (2H, d, J = 7.8 Hz), 8.10 (1H, s); ¹³C-NMR (150 MHz, CDCl₃) δ: 12.9, 18.9, 59.3, 67.2, 101.6, 119.7, 123.9, 128.2, 128.9, 139.4, 147.8, 150.4, 153.1, 165.1; IR (KBr) cm⁻¹: 3264, 3080, 2972, 1703, 1631, 1449, 1406, 1382, 1367, 1284, 1104, 733; EI-MS m/z: 334.1320 (Calcd for C₂₀H₁₈N₂O₃: 334.1318).

Ethyl 4,4-Dimethyl-2-oxo-6-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5g)

The reaction of 8g (116 mg, 0.499 mmol) gave a tautomeric mixture of ethyl 2-methoxy-4,4-dimethyl-6-phenyl-1,4-dihydropyrimidine-5-carboxylate 12g and ethyl 2-methoxy-6,6-dimethyl-4-phenyl-1,6-dihydropyrimidine-5-carboxylate 13g (103 mg, 0.357 mmol, 72%) in a ratio of 1.0 : 7.2 in DMSO-d₆. Colorless crystals; ¹H-NMR (600 MHz, DMSO-d₆) of the major component δ: 0.74 (3H, t, J = 7.2 Hz), 1.36 (6H, s), 3.72 (3H, s), 3.76 (2H, q, J = 7.2 Hz), 7.20–7.40 (5H, m), 7.76 (1H, s); ¹H-NMR (600 MHz, DMSO-d₆) of the minor component δ: 0.66 (3H, t, J = 7.2 Hz), 1.33 (6H, s), 3.60 (3H, s), 3.69 (2H, q, J = 7.2 Hz), 7.20–7.40 (5H, m), 9.12 (1H, s). Hydrolysis of the mixture of 12g and 13g (100 mg, 0.347 mmol) gave 5g (92.0 mg, 0.335 mmol, 97%). Colorless crystals; mp 185–186 °C (CHCl₃-EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 0.77 (3H, t, J = 7.2 Hz), 1.58 (6H, s), 2.18 (3H, s), 3.84 (2H, q, J = 7.2 Hz), 5.07 (1H, s), 6.52 (1H, s), 7.32 (2H, dd, J = 1.2, 7.8 Hz), 7.36–7.44 (3H, m); ¹³C-NMR (150 MHz, CDCl₃) δ: 13.3, 29.4, 55.0, 60.0, 108.5, 127.6, 128.3, 129.4, 135.2, 142.4, 153.4, 166.8; IR (KBr) cm⁻¹: 3323, 1702, 1678, 1632, 1396, 1366, 1320, 1142, 1087; EI-MS m/z: 274.1321 (Calcd for C₁₅H₁₈N₂O₃: 274.1317).

Methyl 4,4,6-Trimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5h)

The reaction of 8h (78 mg, 0.499 mmol) gave a tautomeric mixture of methyl 2-methoxy-4,4,6-trimethyl-1,4-dihydropyrimidine-5-carboxylate 12h and methyl 2-methoxy-4,6,6-trimethyl-1,6-dihydropyrimidine-5-carboxylate 13h (97.0 mg, 0.457 mmol, 92%) in a ratio of 1.0 : 1.4 in DMSO-d₆. Colorless crystals; ¹H-NMR (600 MHz, DMSO-d₆) of the major component δ: 1.30 (6H, s), 1.95 (3H, s), 3.59 (3H, s), 3.68 (3H, s), 7.63 (1H, s); ¹H-NMR (600 MHz, DMSO-d₆) of the minor component δ: 1.27 (6H, s), 1.92 (3H, s), 3.56 (3H, s), 3.58 (3H, s), 8.89 (1H, s). Hydrolysis of the mixture of 12h and 13h (92.0 mg, 0.433 mmol) gave 5h (82.6 mg, 0.417 mmol, 96%). Colorless crystals; mp 147–149 °C (n-hexane-EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 1.48 (6H, s), 2.12 (3H, s), 3.74 (3H, s), 5.26 (1H, s), 7.91 (1H, s); ¹³C-NMR (150 MHz, CDCl₃) δ: 18.9, 29.9, 50.9, 54.8, 106.6, 143.3, 153.9, 167.0; IR (KBr) cm⁻¹: 3335, 3222, 3102, 2966, 1702, 1619, 1421, 1293, 1214, 1098; EI-MS m/z: 198.1011 (Calcd for C₉H₁₄N₂O₃: 198.1005).

Benzyl 4,4,6-Trimethyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5i)

The reaction of 8i (116 mg, 0.499 mmol) gave a tautomeric mixture of benzyl 2-methoxy-4,4,6-trimethyl-1,4-dihydropyrimidine-5-carboxylate 12i and benzyl 2-methoxy-4,6,6-trimethyl-1,6-dihydropyrimidine-5-carboxylate 13i (125 mg, 0.434 mmol, 87%) in a ratio of 1.0 : 1.4 in DMSO-d₆. Colorless oil; ¹H-NMR (600 MHz, DMSO-d₆) of the major component δ: 1.30 (6H, s), 1.96 (3H, s), 3.67 (3H, s), 5.10 (2H, s), 7.28–7.40 (5H, s), 7.65 (1H, s); ¹H-NMR (600 MHz, DMSO-d₆) of the minor component δ: 1.27 (6H, s), 1.93 (3H, s), 3.56 (3H, s), 5.09 (2H, s), 7.28–7.40 (5H, m), 8.92 (1H, s). Hydrolysis of the mixture of 12i and 13i (90.0 mg, 0.312 mmol) gave 5i (84.5 mg, 0.312 mmol, 99%). Colorless crystals; mp 150–152 °C (n-hexane-EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 1.47 (6H, s), 2.11 (3H, s), 5.10 (1H, s), 5.19 (2H, s), 7.30–7.43 (5H, m), 7.58 (1H, s); ¹³C-NMR (150 MHz, CDCl₃) δ: 19.1, 30.0, 55.0, 66.0, 106.6, 128.2, 128.3, 128.6, 135.9, 143.5, 153.5, 166.3; IR (KBr) cm⁻¹: 3357, 3216, 3092, 2972, 1693, 1620, 1423, 1372, 1279, 1209, 1092; EI-MS m/z: 274.1321 (Calcd for C₁₅H₁₈N₂O₃: 274.1317).

Ethyl 4,4,6-Trimethyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6a)

A mixture of 5a (213 mg, 1.00 mmol) and Lawesson reagent (485 mg, 1.20 mmol) in toluene (13 mL) was heated at reflux for 2 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography twice [CH₂Cl₂–EtOAc (40 : 1 to 15 : 1) and n-hexane–CH₂Cl₂–EtOAc (14 : 6 : 1 to1 10 : 10 : 3)] to give 6a (202 mg, 0.883 mmol, 88%). Colorless crystals; mp 157–159 °C (n-hexane–EtOAc); ¹H-NMR (600 MHz, CDCl₃) δ: 1.32 (3H, t, J = 7.2 Hz), 1.52 (6H, s), 2.13 (3H, s), 4.22 (2H, q, J = 7.2 Hz), 6.49 (1H, s), 7.45 (1H, s); ¹³C-NMR (150 MHz, CDCl₃) δ: 14.1, 18.4, 29.6, 55.9, 60.4, 108.7, 139.6, 165.9, 173.5; IR (KBr) cm⁻¹: 3201, 2984, 1703, 1638, 1599, 1574, 1445, 1363, 1312, 1159, 1093; EI-MS m/z: 228.0928 (Calcd for C₁₀H₁₆N₂O₂S: 228.0933).

Ethyl 2-Thioxo-4,4,6-trisubstituted 1,2,3,4-Tetrahydropyrimidine-5-carboxylates (6b–d)

The 2-thioxo products 6b–d were synthesized according to the same procedures as described for the synthesis of 6a; below listed are their isolated yield and their physicochemical and spectral data for 6b–d.

Ethyl 4,6-Dimethyl-2-thioxo-4-pentyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6b)

Yield: 87%; Colorless oil; ¹H-NMR (600 MHz, CDCl₃) δ: 0.87 (3H, t, J = 7.2 Hz), 1.14–1.43 (7H, m), 1.30 (3H, t, J = 7.2 Hz), 1.49 (3H, s), 2.10–2.20 (1H, m), 2.14 (3H, s), 4.21 (2H, q, J = 7.2 Hz), 6.54 (1H, s), 7.61 (1H, s); ¹³C-NMR (150 MHz, CDCl₃) δ: 14.0, 14.1, 18.5, 22.4, 24.2, 29.7, 31.7, 41.8, 59.6, 60.3, 106.6, 140.4, 165.9, 173.7; IR (neat) cm⁻¹: 3187, 2959, 2927, 1702, 1637, 1577, 1459, 1438, 1322, 1304, 1150, 1091; EI-MS m/z: 284.1558 (Calcd for C₁₄H₂₄N₂O₂S: 284.1558).

Ethyl 4-Methyl-2-thioxo-1,3-diazaspiro[5,5]undeca-2,5-diene-5-carboxylate (6c)

Yield: 77%; Pale yellow crystals; mp 165–167 °C (n-hexane–EtOAc); ¹H-NMR (500 MHz, CDCl₃) δ: 1.18–1.30 (1H, m), 1.32 (3H, t, J = 7.0 Hz), 1.42–1.54 (2H, m), 1.61–1.75 (3H, m), 1.80 (2H, d, J = 15.0 Hz), 2.02–2.12 (1H, m), 2.05 (3H, s), 4.23 (2H, q, J = 7.2 Hz), 6.87 (1H, s), 7.43 (1H, s); ¹³C-NMR (125 MHz, CDCl₃) δ: 14.1, 18.2, 20.4, 24.4, 34.4, 57.3, 60.5, 109.7, 138.4, 166.1, 174.5; IR (KBr) cm⁻¹: 3184, 2926, 1699, 1650, 1565, 1454, 1437, 1314, 1156, 1134, 1096, 1057, 1032. FAB-MS m/z: [M + H]⁺ 269.1321 (Calcd for C₁₃H₂₁N₂O₂S: 269.1324).

Ethyl 4-Methyl-2-thioxo-4,4-dipropyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6d)

Yield: 93%; Colorless crystals; mp 160–162 °C (n-hexane–EtOAc); ¹H-NMR (500 MHz, CDCl₃) δ: 0.90 (6H, t, J = 7.2 Hz), 1.21–1.49 (6H, m), 1.30 (3H, t, J = 7.2 Hz), 2.00–2.12 (2H, m), 2.15 (3H, s), 4.21 (2H, q, J = 7.2 Hz), 6.47 (1H, s), 7.60 (1H, s); ¹³C-NMR (125 MHz, CDCl₃) δ: 13.96, 14.04, 17.6, 18.4, 44.1, 60.2, 63.5, 104.3, 141.4, 166.0, 173.7; IR (KBr) cm⁻¹: 3195, 2961, 1712, 1681, 1585, 1472, 1435, 1298, 1284, 1214, 1159, 1083, 758; FAB-MS m/z: [M + H]⁺ 285.1638 (Calcd for C₁₄H₂₅N₂O₂S: 285.1637).

Cell Culture and Treatment

HL-60 cells, a human cell line derived from promyelocytic leukemia cells, were obtained from the RIKEN BRC through the National Bio-Resource Project of the MEXT (Resource No. RBRC-RCB3683, Tsukuba, Japan). Cells were cultured in medium (RPMI-1640 supplemented with 10% heat-inactivated FBS and 100 U/mL penicillin and 100 µg/mL streptomycin) at 37 °C in a humidified atmosphere (5% CO₂ in air). All crystalline compounds 5 and 6 were purified by recrystallization prior to dissolution. ATRA, 5, and 6 were dissolved in DMSO to 10 mM and stored frozen (−20 °C) until use. HL-60 cells (1 × 10⁵ cells/mL) incubated for 96 h following treatment with DMSO (control, final concentration: 0.5%), ATRA (final concentration: 1 µM), and each sample (final concentration: 10 µM).

Cell Viability Assay

Cell viability was examined by the XTT assay as described previously.^26,27) Briefly, XTT/PMS mixed solution (1.5 mM XTT and 0.025 mM PMS in PBS) of a quarter of the medium in well were added following by incubation at 37 °C for 4 h. Next, the plate was mixed on a mechanical plate shaker, and absorbance at 450 nm was measured with a microplate reader. Cell viability was calculated using the following equation:

  

where A₄₅₀ sample denotes ATRA or each sample.

The IC₅₀ values were calculated using a software, GraphPad Prism Ver 8.4.3 (GraphPad Software, Inc., San Diego, CA, U.S.A.).

Statistical Analysis

Statistical analysis was performed with GraphPad Prism using a one-way ANOVA followed by Bonferroni’s multiple comparison test. p < 0.05 was considered to indicate a statistically significant difference. Data are presented as the mean ± S.D. of three or nine separate experiments.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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