Parallel synthesis of condensed pyrimidine-thiones and their antitumor activities

Herein, we studied the formation of thiones via C=O group conversion into the C=S functional group-based tricyclic pyrimidinone systems using Lawesson’s reagent and phosphorus pentasulfide as thionation agents. Naturally occurring alkaloids deoxyvasicinone and mackinazolinone were selected as templates for the modification of furo[2,3-d]pyrimidinone and pyrrolo[2,3-d]pyrimidinone scaffold. Research work was performed under the combinatorial and parallel synthesis of pyrimidine-based small molecules, along with a one-pot reaction strategy. All synthesized 54 novel pyrimidine-thiones were elucidated by 1H-NMR, 13C-NMR, and HRMS analysis. In addition, both series of thiones were evaluated for their antitumor activity against three types of the human cancer cell: cervical HeLa, breast MCF-7, and colon HT-29 lines. 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.

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].
Parallel synthesis of condensed pyrimidine-thiones and their… 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]. 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.

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 Combinatorial parallel thionation of five-membered analogues of Deoxyvasicinone/Mackinazolinone alkaloids containing diverse single heteroatoms Fig. 1 Main approach of present work and structurally diverse condensed pyrimidinones 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 P 2 S 5 ). All reactions using this reagent were performed in dioxane or toluene. Unfortunately, all efforts to introduce "thion" functionality into the tricyclic Parallel synthesis of condensed pyrimidine-thiones and their… 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 P 2 S 5 afforded a final thion-product (12 g) of furo [2,3-d] pyrimidinone derivatives yielded in quit good yields (74%, Table 1).
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 POCl 3 (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.
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.
It should be noted that using a P 2 S 5 in the furo[2,3-d]pyrimidinthiones formation, we observed the ability of substituents in the yield of final products. The methyl Phosphorus pentasulfide (P 2 S 5) 11 g reflux 6 13 g 49

Scheme 2
One-pot formation of pyrimidine-thiones 12 and 13 g 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 P 2 S 5 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 1 H-NMR, 13 C-NMR, and HRMS as described in the experimental part, as well as X-ray analysis for derivative 12c (Fig. 2).

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 P 2 S 5 afforded a final thion-products of furo [2,3-d] Parallel synthesis of condensed pyrimidine-thiones and their… Table 2 In vitro cytotoxic activity of the synthesized pyrimidinthiones (12a-12aa and

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 CDCl 3 , using TMS as an internal standard. High-resolution mass spectra (HRMS) were recorded on an AB SCIEX QSTAR Elite quadrupole timeof-flight mass spectrometry.