Introduction

Clinical trials show that most medical drugs which are used in cancer chemotherapy fail to produce satisfactory outcomes because of poor selectivity, efficiency, or various side effects [1,2,3]. Therefore, there is still a need to improve the drug development process [4, 5]. Pyrimidine-based small molecules [6] are a group of chemotherapeutic agents [7], which include 5-FU, capecitabine [8], decitabine [9], gemcitabine [10], raltitrexed [11], floxuridine [12], tegafur [13], and others [14]. These anticancer agents induce apoptosis while progressing via the cell cycle by inhibiting appropriate targets. In addition, pyrimidine-based small molecules are also included in system nitrogen, oxygen, and sulfur heteroatoms.

Organosulfur compounds are components or fragments in pyrimidine-based small molecules [15]. Herein, among the organosulfur derivatives (both natural and synthetic), thio-analogs of ketones are important scaffolds from chemical, medical [16], and industrial [17] points of view. Literature reports show that most organosulfur derivatives are found as potential inhibitors of various major enzymes, indicating the importance of these class compounds in medicinal chemistry [18,19,20].

Naturally occurring alkaloids deoxyvasicinone [21, 22] and mackinazolinone [23] are isolated from the plants Adhatoda vasica and Mackilaya subulata Philipson, respectively. These pyrimidine-based derivatives have demonstrated potential biological properties [24,25,26]. The tricyclic deoxyvasicinone system is part of other biologically important alkaloids including isaindigotone [27], tryptanthrin [28], and luotonin A [29], while mackinazolinone is part of rutaecarpine [30] and evodiamine [31], respectively (Fig. 1). Our research group performed large studies on the synthetic analogs of deoxyvasicinone and mackinazolinone, which containing carbonyl (C=O) group in the pyrimidine ring [32,33,34,35,36,37,38]. Results revealed that several modified analogs of these alkaloids exhibited satisfactory higher antiproliferative activity against a panel of human cancer cell lines [32, 39].

Fig. 1
figure 1

Main approach of present work and structurally diverse condensed pyrimidinones

Full size image

Spurred by this preliminary success, we proceeded to examine the combinatorial and parallel transformation of furo- and pyrrolopyrimidinones under thionation agents. An important field of research in medicinal and organic chemistry involves heterocyclic thiones forming the backbone of more complex organic compounds, especially those using other sites for their attachment to specific types of rapidly forming molecules [40, 41]. Therefore, herein, we studied the formation of thiones via C=O group conversion into the C=S functional group-based tricyclic pyrimidinone system using Lawesson’s reagent [42,43,44] and phosphorus pentasulfide [45] as thionation agents. In addition, all synthesized thio-compounds were evaluated for their antitumor activity on human cancer cell cervical HeLa, breast MCF-7, and colon HT-29 lines.

Results and discussion

Chemistry

The thionation of bicyclic pyrimidines or related compounds with Lawesson’s reagent has been presented earlier [46, 47]. Other pathways have also been reported in the literature for the C=O group conversion into C=S [48, 49]. However, we turned our attention toward a milder route in order to perform systematic combinatorial and parallel thionation of the diverse molecular systems, along with their evaluation of the antitumor activity.

The formation of furo[2,3-d]pyrimidinone 6a-6aa and pyrrolo[2,3-d]pyrimidinone 11a-11aa intermediates were described in our recently reported research (Scheme 1) [50]. Proceeding from this research, we have studied a parallel thionation process of the obtained pyrimidinones 6a-6aa and 11a-11aa. First, in an example of compounds 6 and 11 g, thionation conditions were investigated. Subjecting pyrimidinones 6 and 11 g to reflux in the presence of Lawesson’s reagent in toluene or dioxane results in conversion of the oxygen of the carbonyl group convers to sulfur; under these conditions, the products 12 and 13 g are obtained in 56 and 88% yields, respectively (Table 1). In our research, we also used other thionation agents in order to convert C=O to C=S (for example with P2S5). All reactions using this reagent were performed in dioxane or toluene. Unfortunately, all efforts to introduce “thion” functionality into the tricyclic annulated system were not similar in both series of pyrimidinones. Herein, Lawesson’s reagent was more suitable for the thionation of pyrrolo[2,3-d]pyrimidinone (for compound 11 g), while using the P2S5 afforded a final thion-product (12 g) of furo[2,3-d]pyrimidinone derivatives yielded in quit good yields (74%, Table 1).

Scheme 1
scheme 1

Synthesis of furo[2,3-d]pyrimidinone 6a-6aa and pyrrolo[2,3-d]pyrimidinone 11a-11aa intermediates

Full size image
Table 1 Optimization of the conditions for thionation of 6 g and 11 g
Full size table

The direct one-pot formation of target thiones (12 and 13 g) from five-membered 2-amino esters (4c and 10c) was also investigated (Scheme 2). When 2-amino esters are subjected to condensation in the presence of lactam (5a) and POCl3 (dioxane used as reaction solvent) leads to intermediates (6 and 11 g) after 3–4 h, the addition of Lawesson’s reagent into the reaction mixture followed by a reaction time of 4 h gives thiones 12 and 13 g.

Scheme 2
scheme 2

One-pot formation of pyrimidine-thiones 12 and 13 g

Full size image

As discussed above, pyrimidine-thiones were produced in two cases, where dioxane or toluene and only dioxane (in the one-pot reaction, Scheme 2) were used as a solvent. Although we performed direct one-pot synthesis (from mono-cyclic esters), increased yields were observed in the synthetic pathways from the furo[2,3-d]pyrimidinone 6 g and pyrrolo[2,3-d]pyrimidinone 11 g intermediates. The direct one-pot synthesis from ester’s desired thiones was obtained at a low yield, as well as workup procedures were a bit difficult, because of the formed side products. However, further development of this method may serve to obtain pyrimidine-thiones via a simple pathway.

Thus, the combinatorial and parallel thionation of pyrimidinones was achieved using Lawesson’s reagent toward the pyrrolo[2,3-d]pyrimidinthiones (Scheme 3) and P2S5 in the furo[2,3-d]pyrimidinthiones (Scheme 4), respectively.

Scheme 3
scheme 3

A library of the synthesized tricyclic furo[2,3-d]pyrimidinthiones (12a-12aa)

Full size image
Scheme 4
scheme 4

A library of the synthesized tricyclic pyrrolo[2,3-d]pyrimidinthiones (13a-13aa)

Full size image

It should be noted that using a P2S5 in the furo[2,3-d]pyrimidinthiones formation, we observed the ability of substituents in the yield of final products. The methyl group at C-2 or C-2/C-3 positions of the furan ring influence to form of thiones in lower yield (18–44%), followed to increase compounds yield (up to 74%) by introducing a phenyl substituent at position C-2. Introduction of the 4-substituted phenyl group at C-2 gave desired thiones up to 99%. Therefore, phosphorus pentasulfide was found as a convenient thionation agent on furo[2,3-d]pyrimidinthiones 12j-12aa. Furthermore, using a P2S5 in the furo[2,3-d]pyrimidinthiones, only products were formed during checking at TLC (thin-layer chromatography), while with Lawesson’s reagent was formed several bi-products. In the case of pyrrolo[2,3-d]pyrimidinthiones, final products were yielded up to 99% using Lawesson’s reagent as a thionation agent, and no bi-products were observed.

All synthesized furo[2,3-d]pyrimidinthiones (12a-12aa) were elucidated by 1H-NMR, 13C-NMR, and HRMS as described in the experimental part, as well as X-ray analysis for derivative 12c (Fig. 2).

Fig. 2
figure 2

Crystal structure of compound 12c (CCDC: 2,169,324)

Full size image

Antitumor activity

All the synthesized condensed pyrimidine-thiones 12a-12aa and 13a-13aa were tested for their cytotoxic activity against three types of human cancer cell: cervical HeLa, breast MCF-7, and colon HT-29 lines using the MTT method (Table 2). Doxorubicin (DOX) is used as a reference drug. As shown in Table 2, furo[2,3-d]pyrimidinthiones were inactive and showed IC50 values ≥ 50 µM, except of derivatives 12e (40.45 ± 2.03) and 12 g (17.68 ± 0.83) on HT-29 cell lines. In general, among the furopyrimidinthione series, there was no real trend between the various side-rings and substituents on the pyrimidinone system concerning the activity. However, several pyrrolo[2,3-d]pyrimidinthiones demonstrated weak to high cytotoxic activity against selected human cancer cell lines. Among the twenty-seven synthesized pyrrolo[2,3-d]pyrimidinthione derivatives, fourteen samples were quite active with IC50 values ≤ 50. In addition, results revealed that among the human cancer cell lines evaluated, the HT-29 cancer cell line was more sensitive. Derivatives 13j, 13 l, 13o, 13r, 13t, 13z, and 13aa have exhibited higher activity with IC50 values ranging from 2.09 ± 0.22 to 9.84 ± 0.88 µM. All these compounds contain phenyl or 4-substituted phenyl group at position C-2. It was observed that the increase of methylene groups effect cytotoxic activity (Fig. 3). For example, a compound with azepine fragment 13aa (1-methyl-2-(4-(trifluoromethyl)phenyl)-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione) was most active derivative (IC50 = 2.09 ± 0.22 µM) against the HT-29 cell line (Fig. 3). Therefore, a further detailed study is needed on this way, where obtained leading compounds may serve a good candidate as anticancer agents.

Table 2 In vitro cytotoxic activity of the synthesized pyrimidinthiones (12a-12aa and 13a-13aa) against selected cell lines
Full size table
Fig. 3
figure 3

Structure–activity relationship for active derivative 13aa

Full size image

Conclusion

We have designed and synthesized novel 2-substituted furo[2,3-d]pyrimidinthione and pyrrolo[2,3-d]pyrimidinthione derivatives under combinatorial and parallel synthesis strategy. This heterocyclic system is furan/pyrrole analogs of the naturally occurring deoxyvasicinone mackinazolinone alkaloids. In general, two thionation agents were used to study systematic thionation. Results revealed that Lawesson’s reagent was more suitable for the thionation of pyrrolo[2,3-d]pyrimidinones, while using P2S5 afforded a final thion-products of furo[2,3-d]pyrimidinones in a quit good yield. All of the newly designed thiones were evaluated against the human cancer cell lines Hela, MCF-7, and HT-29. The furo[2,3-d]pyrimidinthione series were not demonstrated antitumor activity against selected cell lines. However, several pyrrolo[2,3-d]pyrimidinthiones showed weak to high cytotoxic activity. Among the twenty-seven synthesized pyrrolo[2,3-d]pyrimidinthione derivatives, fourteen samples were quite active with IC50 values ≤ 50. Azepine containing derivative 13aa (1-methyl-2-(4-(trifluoromethyl)phenyl)-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione) displayed a high (IC50 = 2.09 ± 0.22 µM) antitumor activity against the HT-29 cell line, which further modification could be serve as leading compound in anticancer drug design investigations.

Experimental section

Chemistry

Materials and methods

All reagents and solvents were purchased from Sigma and used without further purification. Thin-layer chromatography (TLC) was performed on glass plates coated with silica gel (Qingdao Haiyang Chemical Co., G60F-254) and visualized by UV light (254 nm). The products were purified by column chromatography over silica gel (Qingdao Haiyang Chemical Co., 200–300 mesh). Melting points were determined on a Buchi B-540 apparatus and were uncorrected. Nuclear magnetic resonance (NMR) spectra were recorded with a Varian 400 MHz NMR spectrometer in CDCl3, using TMS as an internal standard. High-resolution mass spectra (HRMS) were recorded on an AB SCIEX QSTAR Elite quadrupole time-of-flight mass spectrometry.

General procedure for synthesis of compounds 12a-12aa

A solution of 1-mmol furo[2,3-d]pyrimidinones (6a-6aa) in 10 mL of dried toluene or dioxane was stirred under reflux with 1.1 mmol (222 mg) of phosphorus pentasulfide (P2S5). After 4 h, the reaction mixture was cooled to 20–25 °C, the solvent was removed under reduced pressure and the residue column chromatographed using petroleum ether: EtOAc (1:1 − 3:1) as an eluent to give solid thiones 12a-12aa.

2-Methyl-7,8-dihydrofuro[2,3-d]pyrrolo[1,2-a]pyrimidine-4(6H)-thione (12a) Yield 25%, yellow solid, m.p.159–160 °C. 1H NMR (400 MHz, CDCl3) δ 6.62 (s, 1H), 4.52 (t, J = 8.0 Hz, 2H), 3.32 (t, J = 8.1 Hz, 2H), 2.42 (s, 3H), 2.41 – 2.32 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 176.57, 159.91, 159.64, 154.09, 120.54, 104.06, 52.50, 33.02, 19.12, 14.09; HRMS (ESI): calcd for C10H10N2OS [M + H]+: 207.0592, found 207.0592.

2-Methyl-6,7,8,9-tetrahydro-4H-furo[2,3-d]pyrido[1,2-a]pyrimidine-4-thione (12b) Yield 18%, yellow solid, m.p.139–140 °C. 1H NMR (400 MHz, CDCl3) δ 6.61 (s, 1H), 4.57 (t, J = 6.2 Hz, 2H), 3.07 (t, J = 6.8 Hz, 2H), 2.39 (s, 3H), 2.09–2.00 (m, 2H), 1.99–1.90 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 179.58, 157.43, 156.87, 153.88, 121.18, 104.70, 48.83, 32.63, 22.37, 18.72, 13.98; HRMS (ESI): calcd for C11H12N2OS [M + H]+: 221.0749, found 221.0749.

2-Methyl-7,8,9,10-tetrahydrofuro[2’,3’:4,5]pyrimido[1,2-a]azepine-4(6H)-thione (12c) Yield 40%, yellow solid, m.p.119–120 °C. 1H NMR (400 MHz, CDCl3) δ 6.61 (s, 1H), 5.09 (t, J = 5.0 Hz, 2H), 3.20 (t, J = 6.8 Hz, 2H), 2.41 (s, 3H), 1.93–1.81 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 180.11, 161.19, 157.26, 154.23, 121.06, 105.24, 49.83, 38.07, 29.14, 26.15, 24.95, 14.02. HRMS (ESI): calcd for C12H14N2OS [M + H]+: 235.0905, found 235.0904.

2,3-Dimethyl-7,8-dihydrofuro[2,3-d]pyrrolo[1,2-a]pyrimidine-4(6H)-thione (12d) Yield 26%, yellow solid, m.p.223–224 °C. 1H NMR (400 MHz, CDCl3) δ 4.50 (t, J = 7.5 Hz, 2H), 3.29 (t, J = 8.1 Hz, 2H), 2.42 (s, 3H), 2.38–2.29 (m, 5H); 13C NMR (101 MHz, CDCl3) δ 177.32, 159.64, 159.43, 148.96, 118.90, 113.60, 52.15, 32.99, 18.96, 11.48, 9.74; HRMS (ESI): calcd for C11H12N2OS [M + H]+: 221.0749, found 221.0749.

2,3-Dimethyl-6,7,8,9-tetrahydro-4H-furo[2,3-d]pyrido[1,2-a]pyrimidine-4-thione (12e) Yield 44%, yellow solid, m.p. 179–180 °C. 1H NMR (400 MHz, CDCl3) δ 4.58 (t, J = 6.2 Hz, 2H), 3.06 (t, J = 6.8 Hz, 2H), 2.45 (s, 3H), 2.32 (s, 3H), 2.10–2.01 (m, 2H), 1.99–1.90 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 180.49, 157.39, 156.71, 148.78, 119.64, 113.92, 48.02, 32.69, 22.48, 18.73, 11.55, 10.17; HRMS (ESI): calcd for C12H14N2OS [M + H]+: 235.0905, found 235.0906.

2,3-Dimethyl-7,8,9,10-tetrahydrofuro[2’,3’:4,5]pyrimido[1,2-a]azepine-4(6H)-thione (12f) Yield 38%, yellow solid, m.p.114–115 °C. 1H NMR (400 MHz, CDCl3) δ 5.08 (t, 2H), 3.17 (t, J = 6.8 Hz, 2H), 2.44 (s, 3H), 2.31 (t, J = 5.0 Hz, 3H), 1.92–1.79 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 180.90, 160.93, 157.20, 149.01, 119.40, 114.13, 48.66, 38.06, 29.06, 26.05, 24.93, 11.52, 10.06; HRMS (ESI): calcd for C13H16N2OS [M + H]+: 249.1062, found 249.1062.

2-Phenyl-7,8-dihydrofuro[2,3-d]pyrrolo[1,2-a]pyrimidine-4(6H)-thione (12 g) Yield 74%, yellow solid, m.p. 234.5–235.7 °C. 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 7.2 Hz, 2H), 7.44 (t, J = 7.4 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 7.25 (s, 1H), 4.55 (t, J = 7.4 Hz, 2H), 3.36 (t, J = 8.1 Hz, 2H), 2.39 (dt, J = 15.7, 7.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 177.09, 160.62, 159.95, 154.38, 129.14, 128.99, 128.97, 124.66, 121.17, 102.38, 52.57, 33.16, 19.10; HRMS (ESI): calcd for C15H13N2OS [M + H]+: 269.0749, found 269.0750.

2-Phenyl-6,7,8,9-tetrahydro-4H-furo[2,3-d]pyrido[1,2-a]pyrimidine-4-thione (12 h) Yield 73%, yellow solid, m.p. 199.4–200.2 °C. 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 7.4 Hz, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.3 Hz, 1H), 7.25 (s, 1H), 4.60 (t, J = 6.1 Hz, 2H), 3.13 (t, J = 6.8 Hz, 2H), 2.13–2.05 (m, 2H), 2.03–1.94 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 180.06, 157.66, 154.05, 129.06, 128.92, 128.89, 124.64, 121.67, 103.03, 48.99, 32.77, 22.37, 18.71, 18.39; HRMS (ESI): calcd for C16H15N2OS [M + H]+: 283.0905, found 283.0908.

2-Phenyl-7,8,9,10-tetrahydrofuro[2’,3’:4,5]pyrimido[1,2-a]azepine-4(6H)-thione (12i) Yield 23%, yellow solid, m.p. 190.6–191.8 °C. 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 7.3 Hz, 2H), 7.44 (t, J = 7.4 Hz, 2H), 7.37 (t, J = 7.3 Hz, 1H), 7.23 (s, 1H), 5.11 (t, J = 4.1 Hz, 2H), 3.24 (t, J = 6.4 Hz, 2H), 1.95–1.85 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 180.61, 161.93, 157.51, 154.44, 129.15, 128.96, 124.96, 124.71, 121.59, 103.62, 49.90, 38.21, 29.14, 26.13, 24.93; HRMS (ESI): calcd for C16H15N2OS [M + H]+: 297.1062, found 297.1064.

2-(4-Fluorophenyl)-7,8-dihydrofuro[2,3-d]pyrrolo[1,2-a]pyrimidine-4(6H)-thione (12j) Yield 99%, yellow solid, m.p255.5–257.4.°C. 1H NMR (400 MHz, CDCl3) δ 7.71 (dd, J = 8.6, 5.3 Hz, 2H), 7.11 (t, J = 8.6 Hz, 2H), 7.01 (s, 1H), 4.21 (t, J = 7.3 Hz, 2H), 3.21 (t, J = 8.0 Hz, 2H), 2.40 –2.27 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 165.22, 164.02, 161.54, 160.89, 157.74, 151.40, 126.18, 126.10, 116.09, 115.87, 107.30, 99.89, 46.84, 32.39, 19.62. HRMS (ESI): calcd for C15H12FN2OS [M + H]+: 287.0654, found 287.0647.

2-(4-Fluorophenyl)-6,7,8,9-tetrahydro-4H-furo[2,3-d]pyrido[1,2-a]pyrimidine-4-thione (12 k) Yield 99%, yellow solid, m.p. 220.9–222.0 °C. 1H NMR (400 MHz, CDCl3) δ 7.73 (dd, J = 8.1, 5.6 Hz, 2H), 7.15 (s, 1H), 7.11 (t, J = 8.5 Hz, 2H), 4.59 (t, J = 6.1 Hz, 2H), 3.12 (t, J = 6.7 Hz, 2H), 2.14–1.94 (m, 4H); 13C NMR (101 MHz, CDCl3) δ 180.07, 164.32, 161.83, 157.73, 153.11, 126.61, 126.61, 125.27, 121.65, 116.20, 115.98, 102.72, 49.04, 32.80, 22.38, 18.71. HRMS (ESI): calcd for C16H14FN2OS [M + H]+: 301.0811, found 301.0810.

2-(4-Fluorophenyl)-7,8,9,10-tetrahydrofuro[2’,3’:4,5]pyrimido[1,2-a]azepine-4(6H)-thione (12 l) Yield 99%, yellow solid, m.p. 201.6–202.5 °C. 1H NMR (400 MHz, CDCl3) δ 7.76 (dd, J = 8.7, 5.3 Hz, 2H), 7.16 (s, 1H), 7.13 (t, J = 8.8 Hz, 2H), 5.15–5.06 (m, 2H), 3.23 (t, J = 5.0 Hz, 2H), 1.96–1.83 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 180.57, 164.37, 161.99, 157.46, 153.48, 126.64, 125.30, 121.53, 116.25, 116.03, 103.29, 49.89, 38.17, 29.10, 26.08, 24.88; HRMS (ESI): calcd for C17H16FN2OS [M + H]+: 315.0967, found 315.0961.

2-(4-Chlorophenyl)-7,8-dihydrofuro[2,3-d]pyrrolo[1,2-a]pyrimidine-4(6H)-thione (12 m) Yield 44%, yellow solid, m.p. 243.3–244.4 °C. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 8.5 Hz, 2H), 7.38 (d, J = 8.5 Hz, 2H), 7.06 (s, 1H), 4.22 (t, J = 7.3 Hz, 2H), 3.21 (t, J = 8.0 Hz, 2H), 2.39 – 2.28 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 165.31, 161.14, 157.69, 151.16, 134.32, 129.10, 127.92, 125.47, 107.30, 100.70, 46.86, 32.42, 19.61. HRMS (ESI): calcd for C15H12ClN2OS [M + H]+: 303.0359, found 303.0351.

2-(4-Chlorophenyl)-6,7,8,9-tetrahydro-4H-furo[2,3-d]pyrido[1,2-a]pyrimidine-4-thione (12n) Yield 99%, yellow solid, m.p. 220.6–222.0 °C. 1H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 8.5 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 7.24 (s, 1H), 4.60 (t, J = 6.2 Hz, 2H), 3.14 (t, J = 6.8 Hz, 2H), 2.10 (p, J = 6.2 Hz, 2H), 1.99 (p, J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 180.24, 157.94, 157.75, 152.91, 134.95, 129.21, 127.44, 125.84, 121.62, 103.54, 49.05, 32.82, 22.38, 18.71. HRMS (ESI): calcd for C16H14ClN2OS [M + H]+: 317.0515, found 317.0508.

2-(4-Chlorophenyl)-7,8,9,10-tetrahydrofuro[2’,3’:4,5]pyrimido[1,2-a]azepine-4(6H)-thione (12o) Yield 99%, yellow solid, m.p. 207.3–208.3 °C. 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 8.6 Hz, 2H), 7.39 (d, J = 8.6 Hz, 2H), 7.06 (s, 1H), 4.46–4.36 (m, 2H), 3.14–3.05 (m, 2H), 1.94–1.73 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 163.12, 161.75, 158.81, 151.12, 134.35, 129.12, 127.93, 125.52, 106.70, 101.05, 42.87, 37.64, 29.54, 27.49, 24.95. HRMS (ESI): calcd for C17H16ClN2OS [M + H]+: 331.0672, found 331.0665.

2-(4-Bromophenyl)-7,8-dihydrofuro[2,3-d]pyrrolo[1,2-a]pyrimidine-4(6H)-thione (12p) Yield 99%, yellow solid, m.p. 270.8–272.3 °C. 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.08 (s, 1H), 4.22 (t, J = 7.4 Hz, 2H), 3.21 (t, J = 8.0 Hz, 2H), 2.40 – 2.27 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 165.33, 161.19, 157.68, 151.18, 132.04, 128.34, 125.70, 122.50, 107.31, 100.82, 46.87, 32.43, 19.60. HRMS (ESI): calcd for C15H12BrN2OS [M + H]+: 346.9854, found 346.9841.

2-(4-Bromophenyl)-6,7,8,9-tetrahydro-4H-furo[2,3-d]pyrido[1,2-a]pyrimidine-4-thione (12q) Yield 89%, yellow solid, m.p. 234.4–235.7 °C. 1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H), 7.08 (s, 1H), 4.08 (t, J = 6.0 Hz, 2H), 3.02 (t, J = 6.6 Hz, 2H), 2.07 – 1.90 (m, 4H); 13C NMR (101 MHz, CDCl3) δ 163.41, 159.00, 157.02, 150.80, 132.03, 128.38, 125.72, 122.46, 106.66, 100.91, 42.57, 31.92, 22.02, 19.12. HRMS (ESI): calcd for C16H14BrN2OS [M + H]+: 361.0010, found 361.0001.

2-(4-Bromophenyl)-7,8,9,10-tetrahydrofuro[2’,3’:4,5]pyrimido[1,2-a]azepine-4(6H)-thione (12r) Yield 99%, yellow solid, m.p. 213.5–214.9 °C. 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.23 (s, 1H), 5.15–5.06 (m, 2H), 3.28–3.20 (m, 2H), 1.96–1.83 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 180.71, 162.22, 157.56, 153.27, 132.18, 127.85, 126.06, 123.24, 121.48, 104.19, 49.89, 38.20, 29.10, 26.08, 24.87. HRMS (ESI): calcd for C17H16BrN2OS [M + H]+: 375.0167, found 375.0161.

2-(p-Tolyl)-7,8-dihydrofuro[2,3-d]pyrrolo[1,2-a]pyrimidine-4(6H)-thione (12 s) Yield 73%, yellow solid, m.p. 262.9–264.3 °C. 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 7.02 (s, 1H), 4.21 (t, J = 7.4 Hz, 2H), 3.20 (t, J = 8.0 Hz, 2H), 2.37 (s, 3H), 2.36–2.26 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 165.08, 160.55, 157.82, 152.59, 138.65, 129.53, 126.71, 124.24, 107.34, 99.42, 46.81, 32.36, 21.33, 19.63. HRMS (ESI): calcd for C16H15N2OS [M + H]+: 283.0905, found 283.0898.

2-(p-Tolyl)-6,7,8,9-tetrahydro-4H-furo[2,3-d]pyrido[1,2-a]pyrimidine-4-thione (12t) Yield 99%, yellow solid, m.p. 202.0–203.6 °C. 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H), 7.20 (s, 1H), 4.61 (t, J = 6.1 Hz, 2H), 3.14 (t, J = 6.7 Hz, 2H), 2.39 (s, 3H), 2.10 (p, J = 6.2 Hz, 2H), 1.99 (p, J = 6.5 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 179.87, 157.56, 157.42, 154.42, 139.32, 129.62, 126.22, 124.64, 121.79, 102.23, 49.00, 32.77, 22.40, 21.40, 18.74. HRMS (ESI): calcd for C17H17N2OS [M + H]+: 297.1062, found 283.0898.

2-(p-tolyl)-7,8,9,10-tetrahydrofuro[2’,3’:4,5]pyrimido[1,2-a]azepine-4(6H)-thione (12u) Yield 99%, yellow solid, m.p. 185.6–186.6 °C. 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.2 Hz, 2H), 7.18 (s, 1H), 5.16–5.07 (m, 2H), 3.27–3.20 (m, 2H), 2.39 (s, 3H), 1.95–1.83 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 180.36, 161.68, 157.35, 154.77, 139.38, 129.64, 126.21, 124.66, 121.66, 102.77, 49.87, 38.16, 29.12, 26.11, 24.91, 21.40. HRMS (ESI): calcd for C18H19N2OS [M + H]+: 311.1218, found 311.1211.

2-(4-Methoxyphenyl)-7,8-dihydrofuro[2,3-d]pyrrolo[1,2-a]pyrimidine-4(6H)-thione (12v) Yield 99%, yellow solid, m.p. 219.2–221.0 °C. 1H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.7 Hz, 2H), 7.11 (s, 1H), 6.97 (d, J = 8.7 Hz, 2H), 4.55 (t, J = 7.5 Hz, 2H), 3.86 (s, 3H), 3.35 (t, J = 8.0 Hz, 2H), 2.39 (p, J = 7.8 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 176.63, 160.42, 160.11, 159.69, 154.60, 126.23, 121.77, 121.36, 114.45, 100.56, 55.38, 52.54, 33.08, 19.09. HRMS (ESI): calcd for C16H15N2O2S [M + H]+: 299.0854, found 299.0847.

2-(4-Methoxyphenyl)-6,7,8,9-tetrahydro-4H-furo[2,3-d]pyrido[1,2-a]pyrimidine-4-thione (12w) Yield 99%, yellow solid, m.p. 206.6–207.6 °C. 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 8.8 Hz, 2H), 7.13 (s, 1H), 6.97 (d, J = 8.8 Hz, 2H), 4.62 (t, J = 6.2 Hz, 2H), 3.86 (s, 3H), 3.14 (t, J = 6.8 Hz, 2H), 2.10 (p, J = 6.2 Hz, 2H), 1.99 (p, J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 179.69, 160.43, 157.49, 157.17, 154.39, 126.28, 121.97, 121.78, 114.44, 101.26, 55.38, 49.00, 32.76, 22.42, 18.76. HRMS (ESI): calcd for C17H17N2O2S [M + H]+: 313.1011, found 313.1002.

2-(4-Methoxyphenyl)-7,8,9,10-tetrahydrofuro[2’,3’:4,5]pyrimido[1,2-a]azepine-4(6H)-thione (12x) Yield 99%, yellow solid, m.p. 191.8–193.3 °C. 1H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.8 Hz, 2H), 7.08 (s, 1H), 6.96 (d, J = 8.8 Hz, 2H), 5.15–5.07 (m, 2H), 3.85 (s, 3H), 3.27–3.19 (m, 2H), 1.94–1.83 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 180.10, 161.44, 160.42, 157.24, 154.66, 126.26, 121.77, 121.72, 114.43, 101.75, 55.37, 49.87, 38.14, 29.12, 26.10, 24.91. HRMS (ESI): calcd for C18H19N2O2S [M + H]+: 327.1167, found 327.1158.

2-(4-(Trifluoromethyl)phenyl)-7,8-dihydrofuro[2,3-d]pyrrolo[1,2-a]pyrimidine-4(6H)-thione (12y) Yield 99%, yellow solid, m.p. 237.7–238.9 °C. 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.35 (s, 1H), 4.54 (t, J = 7.4 Hz, 2H), 3.38 (t, J = 8.1 Hz, 2H), 2.41 (p, J = 8.0 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 177.43, 161.38, 160.24, 152.44, 132.11, 130.42, 125.94, 124.64, 122.49, 120.86, 104.35, 52.58, 33.19, 18.99. HRMS (ESI): calcd for C16H12F3N2OS [M + H]+: 337.0622, found 337.0613.

2-(4-(Trifluoromethyl)phenyl)-6,7,8,9-tetrahydro-4H-furo[2,3-d]pyrido[1,2-a]pyrimidine-4-thione (12z) Yield 99%, yellow solid, m.p. 224.8–226.4 °C. 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.37 (s, 1H), 4.60 (t, J = 6.1 Hz, 2H), 3.15 (t, J = 6.7 Hz, 2H), 2.11 (p, J = 6.1 Hz, 2H), 2.00 (p, J = 6.7 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 180.59, 158.44, 158.02, 152.24, 132.15, 130.42, 125.94, 124.70, 122.50, 121.46, 105.14, 49.08, 32.87, 22.36, 18.68. HRMS (ESI): calcd for C17H14F3N2OS [M + H]+: 351.0779, found 351.0770.

2-(4-(Trifluoromethyl)phenyl)-7,8,9,10-tetrahydrofuro[2’,3’:4,5]pyrimido[1,2-a]azepine-4(6H)-thione (12aa) Yield 99%, yellow solid, m.p. 178.6–180.2 °C. 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.2 Hz, 2H), 7.19 (s, 1H), 4.48–4.37 (m, 2H), 3.15–3.06 (m, 2H), 1.94–1.74 (m, 6H); 13C NMR (101 MHz, CDCl3) δ 163.44, 162.30, 158.77, 150.52, 132.62, 129.96, 125.88, 124.35, 122.57, 106.67, 102.68, 42.91, 37.68, 29.53, 27.47, 24.92. HRMS (ESI): calcd for C18H16F3N2OS [M + H]+: 365.0935, found 365.0929.

General procedure for synthesis of compounds 13a-13aa

The solution of 1-mmol pyrrolo[2,3-d]pyrimidinones (11a-11aa) in 10 mL of dried toluene or dioxane was stirred under reflux with 0.6 mmol (243 mg) of Lawesson’s reagent. After 4 h (in 8–12 h for dioxane), the reaction mixture was cooled to 20–25 °C, the solvent was removed under reduced pressure and the residue column chromatographed using petroleum ether: EtOAc (1:1 − 3:1) as an eluent to give solid thiones 13a-13aa.

1,2-Dimethyl-1,6,7,8-tetrahydro-4H-dipyrrolo[1,2-a:2’,3’-d]pyrimidine-4-thione (13a) Yield 77%, yellow solid, m.p. 272–273 °C. 1H NMR (400 MHz, CDCl3) δ 6.55 (s, 1H), 4.54 (t, J = 7.5 Hz, 2H), 3.62 (s, 3H), 3.26 (t, J = 8.0 Hz, 2H), 2.38 – 2.24 (m, 5H); 13C NMR (101 MHz, CDCl3) δ 174.56, 156.52, 143.87, 135.43, 119.37, 102.26, 52.03, 32.87, 28.30, 19.46, 12.75; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 220.0908, found 220.0903.

1,2-Dimethyl-6,7,8,9-tetrahydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-4(1H)-thione (13b) Yield 76%, yellow solid, m.p. 184–186 °C. 1H NMR (400 MHz, CDCl3) δ 6.57 (s, 1H), 4.70 (t, J = 6.3 Hz, 2H), 3.62 (s, 4H), 3.04 (t, J = 6.8 Hz, 2H), 2.35 (s, 3H), 2.02 (p, J = 6.6, 6.1 Hz, 2H), 1.94 (p, J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 177.32, 153.50, 141.87, 135.61, 120.14, 103.03, 47.84, 32.47, 28.04, 22.46, 18.92, 12.70; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 234.1065, found 234.1059.

1,2-Dimethyl-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione (13c) Yield 80%, yellow solid, m.p. 223–224 °C. 1H NMR (400 MHz, CDCl3) δ 6.54 (s, 1H), 5.11 (s, 2H), 3.61 (s, 3H), 3.21 – 3.13 (m, 2H), 2.34 (s, 3H), 1.84 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 177.79, 157.73, 141.67, 135.82, 119.80, 103.60, 49.38, 38.09, 29.19, 28.03, 26.63, 25.44, 12.65; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 248.1221, found 248.1217.

1,2,3-Trimethyl-1,6,7,8-tetrahydro-4H-dipyrrolo[1,2-a:2’,3’-d]pyrimidine-4-thione (13d) Yield 79%, yellow solid, m.p. 258–260 °C. 1H NMR (400 MHz, CDCl3) δ 4.52 (t, J = 7.2 Hz, 2H), 3.60 (s, 3H), 3.23 (t, J = 8.0 Hz, 2H), 2.57 (s, 3H), 2.34–2.21 (m, 5H); 13C NMR (101 MHz, CDCl3) δ 175.32, 156.12, 143.61, 131.00, 117.52, 111.84, 51.87, 32.89, 28.24, 19.32, 10.98, 9.84; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 234.1065, found 234.1060.

1,2,3-Trimethyl-6,7,8,9-tetrahydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-4(1H)-thione (13e) Yield 82%, yellow solid, m.p. 179–180 °C. 1H NMR (600 MHz, CDCl3) δ 4.04 (t, J = 4.2 Hz, 2H), 3.58 (s, 3H), 2.90 (t, J = 6.7 Hz, 2H), 2.36 (s, 3H), 2.21 (s, 3H), 1.94 (p, J = 6.1 Hz, 2H), 1.88 (p, J = 6.2 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 159.64, 152.30, 146.01, 127.29, 109.59, 104.28, 41.00, 31.71, 28.11, 22.38, 19.48, 9.74, 9.51; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 248.1221, found 248.1216.

1,2,3-Trimethyl-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione (13f) Yield 86%, yellow solid, m.p. 138–140 °C. 1H NMR (400 MHz, CDCl3) δ 5.10 (s, 2H), 3.59 (s, 3H), 3.14 (d, J = 7.2 Hz, 2H), 2.58 (s, 3H), 2.24 (s, 3H), 1.83 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 178.56, 157.39, 141.51, 131.30, 117.94, 112.38, 48.39, 38.16, 29.16, 28.01, 26.60, 25.50, 11.41, 9.91; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 262.1378, found 262.1372.

1-Methyl-2-phenyl-1,6,7,8-tetrahydro-4H-dipyrrolo[1,2-a:2’,3’-d]pyrimidine-4-thione (13 g) Yield 88%, yellow solid, m.p. 195–196 °C. 1H NMR (400 MHz, CDCl3) δ 7.52–7.37 (m, 5H), 6.90 (s, 1H), 4.58 (t, J = 7.5 Hz, 2H), 3.75 (s, 3H), 3.31 (t, J = 8.0 Hz, 2H), 2.34 (p, J = 7.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 175.75, 157.28, 144.85, 139.68, 131.41, 128.86, 128.72, 128.38, 119.77, 104.01, 52.09, 33.00, 30.28, 19.45; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 282.1065, found 282.1060.

1-Methyl-2-phenyl-6,7,8,9-tetrahydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-4(1H)-thione (13 h) Yield 87%, yellow solid, m.p. 157–158 °C. 1H NMR (400 MHz, CDCl3) δ 7.53 – 7.37 (m, 5H), 4.71 (t, J = 6.1 Hz, 2H), 3.74 (s, 3H), 3.09 (t, J = 6.8 Hz, 2H), 2.05 (p, J = 6.5 Hz, 2H), 1.96 (p, J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 178.52, 154.30, 142.79, 139.80, 131.45, 128.84, 128.71, 128.36, 120.49, 104.82, 47.94, 32.59, 29.98, 22.47, 18.92; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 296.1221, found 296.1216.

1-Methyl-2-phenyl-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione (13i) Yield 90%, yellow solid, m.p. 140–141 °C. 1H NMR (400 MHz, CDCl3) δ 7.52–7.38 (m, 5H), 6.88 (s, 1H), 5.19–5.10 (m, 2H), 3.74 (s, 3H), 3.26–3.18 (m, 2H), 1.87 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 179.00, 158.51, 142.60, 140.02, 131.42, 128.84, 128.71, 128.38, 120.17, 105.41, 49.44, 38.18, 30.00, 29.20, 26.64, 25.44; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 310.1378, found 310.1373.

2-(4-Fluorophenyl)-1-methyl-1,6,7,8-tetrahydro-4H-dipyrrolo[1,2-a:2’,3’-d]pyrimidine-4-thione (13j) Yield 94%, yellow solid, m.p. 230–232 °C. 1H NMR (400 MHz, CDCl3) δ 7.46 (dd, J = 8.6, 5.3 Hz, 2H), 7.16 (t, J = 8.6 Hz, 2H), 6.86 (s, 1H), 4.57 (t, J = 7.5 Hz, 2H), 3.71 (s, 3H), 3.31 (t, J = 8.0 Hz, 2H), 2.34 (p, J = 7.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 175.83, 164.01, 161.54, 157.41, 144.76, 138.54, 130.72, 127.51, 119.69, 115.96, 115.75, 104.06, 52.09, 32.99, 30.16, 19.43; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 300.0971, found 300.0967.

2-(4-Fluorophenyl)-1-methyl-6,7,8,9-tetrahydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-4(1H)-thione (13 k) Yield 94%, yellow solid, m.p. 179–180 °C. 1H NMR (400 MHz, CDCl3) δ 7.45 (dd, J = 8.4, 5.4 Hz, 2H), 7.14 (t, J = 8.5 Hz, 2H), 6.85 (s, 1H), 4.68 (t, J = 6.1 Hz, 2H), 3.69 (s, 3H), 3.07 (t, J = 6.8 Hz, 2H), 2.03 (p, J = 6.4 Hz, 2H), 1.95 (p, J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 178.54, 163.97, 161.50, 154.43, 142.72, 138.64, 130.60, 127.54, 120.37, 115.94, 115.72, 104.80, 47.95, 32.59, 29.88, 22.44, 18.89; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 314.1127, found 314.1120.

2-(4-Fluorophenyl)-1-methyl-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione (13 l) Yield 99%, yellow solid, m.p. 123–124 °C. 1H NMR (400 MHz, CDCl3) δ 7.46 (dd, J = 8.4, 5.4 Hz, 2H), 7.16 (t, J = 8.5 Hz, 2H), 6.85 (s, 1H), 5.15 (s, 2H), 3.71 (s, 3H), 3.22 (s, 2H), 1.88 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 179.09, 164.02, 161.55, 158.63, 142.56, 138.89, 130.71, 130.62, 127.53, 120.11, 115.96, 115.75, 105.46, 49.45, 38.20, 29.84, 29.20, 26.63, 25.43; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 328.1284, found 328.1279.

2-(4-Chlorophenyl)-1-methyl-1,6,7,8-tetrahydro-4H-dipyrrolo[1,2-a:2’,3’-d]pyrimidine-4-thione (13 m) Yield 92%, yellow solid, m.p. 199–200 °C. 1H NMR (400 MHz, CDCl3) δ 7.45 – 7.39 (m, 4H), 6.88 (s, 1H), 4.56 (t, J = 7.2 Hz, 2H), 3.72 (s, 3H), 3.31 (t, J = 8.0 Hz, 2H), 2.34 (p, J = 7.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 175.92, 157.55, 144.79, 138.30, 134.50, 130.01, 129.82, 129.01, 119.71, 104.34, 52.12, 32.99, 30.31, 19.40; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 316.0675, found 316.0672.

2-(4-Chlorophenyl)-1-methyl-6,7,8,9-tetrahydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-4(1H)-thione (13n) Yield 98%, yellow solid, m.p. 151–153 °C. 1H NMR (400 MHz, CDCl3) δ 7.42 (s, 4H), 6.89 (s, 1H), 4.69 (t, J = 6.1 Hz, 2H), 3.72 (s, 3H), 3.08 (t, J = 6.7 Hz, 2H), 2.05 (p, J = 6.6 Hz, 2H), 1.96 (p, J = 6.5 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 178.72, 154.57, 142.80, 138.42, 134.47, 130.00, 129.88, 129.00, 120.43, 105.16, 47.99, 32.57, 30.05, 22.45, 18.88; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 330.0832, found 330.0826.

2-(4-Chlorophenyl)-1-methyl-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione (13o) Yield 98%, yellow solid, m.p. 164–166 °C. 1H NMR (400 MHz, CDCl3) δ 7.42 (s, 4H), 6.86 (s, 1H), 5.13 (s, 2H), 3.71 (s, 3H), 3.21 (d, J = 6.0 Hz, 2H), 1.86 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 179.18, 158.75, 142.69, 138.62, 134.48, 129.99, 129.86, 129.00, 120.10, 105.73, 49.43, 38.17, 29.99, 29.16, 26.61, 25.40; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 344.0988, found 344.0984.

2-(4-Bromophenyl)-1-methyl-1,6,7,8-tetrahydro-4H-dipyrrolo[1,2-a:2’,3’-d]pyrimidine-4-thione (13p) Yield 99%, yellow solid, m.p. 210–212 °C. 1H NMR (400 MHz, CDCl3) δ 7.59 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 6.89 (s, 1H), 4.57 (t, J = 7.5 Hz, 2H), 3.73 (s, 3H), 3.31 (t, J = 8.0 Hz, 2H), 2.35 (p, J = 7.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 175.97, 157.55, 144.91, 138.31, 131.96, 130.30, 130.27, 122.70, 119.73, 104.37, 52.10, 33.00, 30.20, 19.41; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 360.0170, found 360.0170.

2-(4-Bromophenyl)-1-methyl-6,7,8,9-tetrahydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-4(1H)-thione (13q) Yield 99%, yellow solid, m.p. 196–197 °C. 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H), 6.90 (s, 1H), 4.69 (t, J = 6.3 Hz, 2H), 3.71 (s, 3H), 3.08 (t, J = 6.8 Hz, 2H), 2.05 (p, J = 7.0 Hz, 2H), 1.96 (p, J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 178.74, 154.56, 142.94, 138.41, 131.93, 130.36, 130.25, 122.66, 120.42, 105.15, 47.97, 32.61, 30.01, 22.45, 18.89; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 374.0327, found 374.0300.

2-(4-Bromophenyl)-1-methyl-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione (13r) Yield 99%, yellow solid, m.p. 188–190 °C. 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 6.87 (s, 1H), 5.13 (s, 2H), 3.71 (s, 3H), 3.21 (d, J = 6.9 Hz, 2H), 1.87 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 179.21, 158.77, 142.75, 138.63, 131.95, 130.33, 130.25, 122.68, 120.12, 105.75, 49.43, 38.19, 29.98, 29.17, 26.62, 25.41; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 388.0483, found 388.0478.

1-Methyl-2-(p-tolyl)-1,6,7,8-tetrahydro-4H-dipyrrolo[1,2-a:2’,3’-d]pyrimidine-4-thione (13 s) Yield 99%, yellow solid, m.p. 200–202 °C. 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 7.9 Hz, 2H), 6.86 (s, 1H), 4.57 (t, J = 7.5 Hz, 2H), 3.73 (s, 3H), 3.31 (t, J = 8.0 Hz, 2H), 2.41 (s, 3H), 2.34 (p, J = 7.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 175.51, 157.15, 144.74, 139.81, 138.39, 129.43, 128.74, 128.48, 119.73, 103.57, 52.09, 32.99, 30.24, 21.32, 19.44; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 296.1221, found 296.1215.

1-Methyl-2-(p-tolyl)-6,7,8,9-tetrahydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-4(1H)-thione (13t) Yield 95%, yellow solid, m.p. 159–161 °C. 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 7.9 Hz, 2H), 6.87 (s, 1H), 4.71 (t, J = 6.1 Hz, 2H), 3.72 (s, 3H), 3.08 (t, J = 6.7 Hz, 2H), 2.41 (s, 3H), 2.05 (p, J = 6.4 Hz, 2H), 1.96 (p, J = 6.5 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 178.24, 154.18, 142.71, 139.92, 138.36, 129.42, 128.71, 128.51, 120.44, 104.36, 47.92, 32.57, 30.00, 22.47, 21.33, 18.91; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 310.0378, found 310.1371.

1-Methyl-2-(p-tolyl)-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione (13u) Yield 99%, yellow solid, m.p. 149–151 °C. 1H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 7.9 Hz, 2H), 6.85 (s, 1H), 5.14 (s, 2H), 3.72 (s, 3H), 3.21 (s, 2H), 2.41 (s, 3H), 1.87 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 178.76, 158.36, 142.50, 140.14, 138.38, 128.72, 128.49, 120.15, 104.97, 49.41, 38.15, 29.96, 29.18, 26.64, 25.44, 21.32; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 324.1534, found 324.1528.

2-(4-Methoxyphenyl)-1-methyl-1,6,7,8-tetrahydro-4H-dipyrrolo[1,2-a:2’,3’-d]pyrimidine-4-thione (13v) Yield 99%, yellow solid, m.p. 224–225 °C. 1H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 6.82 (s, 1H), 4.56 (t, J = 7.5 Hz, 2H), 3.85 (s, 3H), 3.71 (s, 3H), 3.30 (t, J = 8.0 Hz, 2H), 2.33 (p, J = 7.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 175.39, 159.76, 157.05, 144.53, 139.64, 130.20, 123.77, 119.77, 114.17, 103.27, 55.33, 52.11, 32.97, 30.12, 19.44; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 312.1171, found 312.1166.

2-(4-Methoxyphenyl)-1-methyl-6,7,8,9-tetrahydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-4(1H)-thione (13w) Yield 95%, yellow solid, m.p. 178–179 °C. 1H NMR (400 MHz, CDCl3) δ 7.42 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H), 6.84 (s, 1H), 4.71 (t, J = 6.1 Hz, 2H), 3.86 (s, 3H), 3.71 (s, 3H), 3.08 (t, J = 6.8 Hz, 2H), 2.05 (p, J = 6.4 Hz, 2H), 1.96 (p, J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 178.15, 159.74, 154.06, 142.61, 139.77, 130.19, 123.84, 120.51, 114.16, 104.06, 55.39, 47.93, 32.56, 29.91, 22.47, 18.93; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 326.1327, found 326.1321.

2-(4-Methoxyphenyl)-1-methyl-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione (13x) Yield 91%, yellow solid, m.p. 220–221 °C. 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 6.80 (s, 1H), 5.13 (s, 2H), 3.84 (s, 3H), 3.69 (s, 3H), 3.20 (d, J = 5.6 Hz, 2H), 1.86 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 178.60, 159.75, 158.27, 142.42, 139.96, 130.16, 123.79, 120.15, 114.16, 104.64, 55.33, 49.41, 38.16, 29.89, 29.19, 26.65, 25.44; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 340.1484, found 340.1480.

1-Methyl-2-(4-(trifluoromethyl)phenyl)-1,6,7,8-tetrahydro-4H-dipyrrolo[1,2-a:2’,3’-d]pyrimidine-4-thione (13y) Yield 97%, yellow solid, m.p. 168–169 °C. 1H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 8.2 Hz, 2H), 6.94 (s, 1H), 4.54 (t, J = 7.6, 7.2 Hz, 2H), 3.74 (s, 3H), 3.30 (t, J = 8.0 Hz, 2H), 2.34 (p, J = 7.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 176.21, 157.89, 145.22, 137.78, 134.92, 129.97, 128.89, 125.75, 122.60, 119.66, 105.13, 52.11, 33.01, 30.38, 19.37; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 350.0939, found 350.0933.

1-Methyl-2-(4-(trifluoromethyl)phenyl)-6,7,8,9-tetrahydropyrido[1,2-a]pyrrolo[2,3-d]pyrimidine-4(1H)-thione (13z) Yield 88%, yellow solid, m.p. 178–179 °C. 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 8.1 Hz, 2H), 6.95 (s, 1H), 4.68 (t, J = 6.2 Hz, 2H), 3.74 (s, 3H), 3.08 (t, J = 6.8 Hz, 2H), 2.04 (p, J = 6.4 Hz, 2H), 1.95 (p, J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 179.04, 154.89, 143.17, 137.90, 135.00, 129.94, 128.87, 125.73, 122.62, 120.38, 105.98, 48.00, 32.63, 30.14, 22.43, 18.86; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 364.1095, found 364.1089.

1-Methyl-2-(4-(trifluoromethyl)phenyl)-1,6,7,8,9,10-hexahydro-4H-pyrrolo[2’,3’:4,5]pyrimido[1,2-a]azepine-4-thione (13aa) Yield 99%, yellow solid, m.p. 175–177 °C. 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 8.2 Hz, 2H), 7.60 (d, J = 8.2 Hz, 2H), 6.92 (s, 1H), 5.16–5.06 (m, 2H), 3.73 (s, 3H), 3.25–3.15 (m, 2H), 1.85 (s, 6H); 13C NMR (101 MHz, CDCl3) δ 179.45, 159.08, 142.95, 138.10, 134.94, 129.93, 128.88, 125.69, 122.61, 120.05, 106.54, 49.42, 38.17, 30.09, 29.12, 26.58, 25.37; HRMS (ESI): calcd for C19H19F3N3O [M + H]+: 378.1252, found 378.1248.

Biology

Materials

Doxorubicin was purchased from BBI Inc. (Shanghai, China). The human cancer HeLa, MCF-7, and HT-29 cell lines were obtained from Chinese Type Culture Collection, CAS (Shanghai, China).

Cell cultures

Human HeLa, MCF-7, and HT-29 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose and 0.37% sodium bicarbonate (Gibco, Rockville, MD, USA). All cell culture media contained with 10% FBS and antibiotic mix (1 × 100 μM penicillin A and 100 μM of streptomycin) and were grown at 37 °C in a humidified incubator (Binder, Germany) containing 95% air/5% CO2 and have been fed every 3–4 days [51, 52].