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Article

New Derivatives of 5-Substituted Uracils: Potential Agents with a Wide Spectrum of Biological Activity

by
Vasily A. Kezin
1,
Elena S. Matyugina
1,
Mikhail S. Novikov
2,
Alexander O. Chizhov
3,*,
Robert Snoeck
4,
Graciela Andrei
4,
Sergei N. Kochetkov
1 and
Anastasia L. Khandazhinskaya
1,*
1
Engelhardt Institute of Molecular Biology, Russian Academy of Science, 119991 Moscow, Russia
2
Department of Pharmaceutical & Toxicological Chemistry, Volgograd State Medical University, 400131 Volgograd, Russia
3
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Science, Leninski pr. 47, 119991 Moscow, Russia
4
Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
*
Authors to whom correspondence should be addressed.
Molecules 2022, 27(9), 2866; https://doi.org/10.3390/molecules27092866
Submission received: 25 March 2022 / Revised: 20 April 2022 / Accepted: 27 April 2022 / Published: 30 April 2022
(This article belongs to the Special Issue Bioactive Compounds: Design, Synthesis and Biological Evaluation)
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Abstract

:
Pyrimidine nucleoside analogues are widely used to treat infections caused by the human immunodeficiency virus (HIV) and DNA viruses from the herpes family. It has been shown that 5-substituted uracil derivatives can inhibit HIV-1, herpes family viruses, mycobacteria and other pathogens through various mechanisms. Among the 5-substituted pyrimidine nucleosides, there are not only the classical nucleoside inhibitors of the herpes family viruses, 2′-deoxy-5-iodocytidine and 5-bromovinyl-2′-deoxyuridine, but also derivatives of 1-(benzyl)-5-(phenylamino)uracil, which proved to be non-nucleoside inhibitors of HIV-1 and EBV. It made this modification of nucleoside analogues very promising in connection with the emergence of new viruses and the crisis of drug resistance when the task of creating effective antiviral agents of new types that act on other targets or exhibit activity by other mechanisms is very urgent. In this paper, we present the design, synthesis and primary screening of the biological activity of new nucleoside analogues, namely, 5′-norcarbocyclic derivatives of substituted 5-arylamino- and 5-aryloxyuracils, against RNA viruses.

1. Introduction

Analogues and derivatives of nucleic acid (NA) components (nucleic bases, nucleosides, and mono- and oligonucleotides) were first tested for medicinal purposes in the middle of the last century [1]. This approach is still popular today, and its possibilities are far from being exhausted. The components of NA, primarily nucleosides and nucleotides, being the main structural units of DNA and RNA, also take part in a huge number of key metabolic processes playing the role of cofactors or regulators of hundreds of reactions of various types. In this regard, even small modifications of the nucleic base or sugar fragment of the nucleoside have a significant impact on the recognition and inhibition of the respective enzymes, and thus on its activity as an anti-pathogen. Nucleic acid analogues and derivatives are currently important elements of anticancer, antiviral, and antifungal therapy [1,2,3,4,5,6].
Pyrimidine nucleoside analogues are widely used to treat infections caused by the human immunodeficiency virus (HIV) and DNA viruses from the herpes family. The mechanism of antiviral activity of nucleoside inhibitors (NIs) is intracellular phosphorylation to the corresponding analogues of nucleoside 5′-triphosphates, which then act as terminator substrates for viral polymerases. Natural substrates for polymerases of retroviruses (reverse transcriptase) and DNA viruses (DNA polymerases) are 2′-deoxyribo nucleoside-5′-triphosphates. And only certain modifications of the pyrimidine base and the 2′-deoxyribose fragment make it possible to preserve the possibility of intracellular phosphorylation and manifestation of activity by this mechanism. Other pyrimidine derivatives belong to the class of non-nucleoside inhibitors (NNIs) and are capable of inhibiting viral polymerases by a noncompetitive mechanism [7,8,9]. These compounds bind to polymerases outside the active site, are hydrophobic, and are structurally much less similar to the natural components of NA.
Among the 5-substituted pyrimidine nucleosides, there are not only the classical NIs of the herpes family viruses, 2′-deoxy-5-iodocytidine [10,11] and brivudine (BVDU, 5-bromovinyl-2′-deoxyuridine) [12,13], but also derivatives of 1-(benzyl)-5-(phenylamino)uracil, which proved to be non-nucleoside inhibitors of HIV-1 and EBV [14] (Figure 1). Rigid amphipathic fusion inhibitors (RAFIs) have also been described [15,16,17], which inhibit unrelated enveloped viruses such as influenza, HCV, VSV, HSV-1, HSV-2, mCMV, and TBEV without cytotoxic or cytostatic effects (SI > 3000). The mechanism of action of RAFIs is biophysical, as they interact with virion envelope lipids and prevent the fusion of the viral and cell membranes by inhibiting the increased negative curvature required for the initial stages of fusion.
The antibacterial activity of 5-substituted pyrimidine analogues of nucleosides was revealed later [6]. Analogues with extended 1-alkynyl substituents demonstrated inhibitory activity against M. tuberculosis, M. avium, and M. bovis in vitro [5,18]. The study of the effect of the modification of 5-(1-alkynyl)pyrimidine nucleoside analogues carbohydrate fragment on anti-mycobacterial properties showed that changes in the carbohydrate fragment, including its replacement with acyclic or carbocyclic residues, do not affect the ability to inhibit the growth of mycobacteria [19,20,21,22,23]. The process of growth inhibition of M. tuberculosis (H37Rv strain) was accompanied by the accumulation of lipid intracellular vacuole-like inclusions in cells, the appearance of deep protrusions and depressions on the surface, and partial and/or complete destruction of the three-layer cell membrane [24]. The ability to inhibit the growth of laboratory and MDR strains of M. tuberculosis has also been described for 5′-norcarbocyclic derivatives of substituted 5-arylaminouracils [25].
Thus, it has been shown that 5-substituted uracil derivatives can inhibit HIV-1 herpes family viruses, mycobacteria and other pathogens through various mechanisms. It made this modification of nucleoside analogues very promising in connection with the emergence of new viruses and the crisis of drug resistance, when the task of creating effective antiviral agents of new types that act on other targets or exhibit activity by other mechanisms is very urgent. In this paper, we present the synthesis and primary screening of the biological activity of new nucleoside analogues, namely, 5′-norcarbocyclic derivatives of substituted 5-arylamino- and 5-aryloxyuracils, against RNA viruses.

2. Results and Discussion

Carbocyclic nucleosides are analogues of natural nucleosides in which the oxygen atom of the furanose ring is replaced by a methylene group. These compounds are highly stable against the pseudoglycoside bond cleavage reaction induced by phosphorylases and hydrolases compared to ribonucleoside analogues and exhibit a wide spectrum of biological activity [26,27,28,29,30,31]. A feature of the structure of 5′-norcarbocyclic analogues is the absence of a 5′-methylene group. The replacement of the hydroxymethyl (-CH2OH) group containing the primary hydroxyl with a secondary hydroxyl deprives the 5′-norcarbocyclic nucleoside analogues of the ability to be converted to phosphorylated forms by cellular enzymes. This feature is useful in studying the mechanism of action of compounds, since it allows one to determine the need for intracellular phosphorylation to realize the biological effect of the drug and to exclude some targets traditional for conventional modified nucleosides.

2.1. Synthesis of Target Compounds

Scheme 1 shows the preparation of a series of new 5-amino derivatives of uracil and their 5′-norcarbocyclic analogues. This synthesis of modified pyrimidines is based on the well-known bromine atom substitution reaction in 5-bromouracil. Thus, the literature describes the condensation of 5-bromouracil with amines of high nucleophilicity: aliphatic (n-butylamine, morpholine), alicyclic (cyclohexylamine) and aromatic fatty (benzylamine, N-methylbenzylamine). The reaction was carried out in the medium of an aminating agent at reflux for 1–2 h. The yields of target 5-amino derivatives of uracil were in the range of 75–100% [32]. The reaction time was reduced by an order of magnitude by using microwave irradiation of the reaction medium. However, in this case, the reaction was also carried out in the medium of an amination agent [33]. To obtain 5-phenylamino derivatives of uracil, Gerns and Perrota treated 5-bromouracil with a three-fold molar excess of the corresponding aniline in refluxing ethylene glycol, which was used as a solvent [34]. The use of ethylene glycol as a solvent was also successfully used in the amination of 1-benzyl-5-bromo derivatives of uracil with a 3.6-fold molar excess of a fatty aromatic amine (benzylamine and phenethylamine). The yield of target 5-benzylamino- and 5-phenethylamino- derivatives of uracil was in the range of 70–75% [14].
In order to reduce the consumption of amine, a 1.5-fold molar excess of quinoline as an acceptor of hydrogen bromide released during the reaction was added into the reaction mixture containing 2-fold molar excess of p-substituted aniline 1d–i, 5-bromouracil and ethylene glycol as a solvent. As a result, 5-arylaminouracils 2d–i were obtained in 68–80% yields. The corresponding 5-(azepan-1-yl)uracil 2a, 5-(4-phenylpiperazin-1-yl)uracil 2b, as well as 5-(3,4-dihydroisoquinolin-2(1H)-yl)uracil 2c were prepared under the same conditions using azepane 1a, 1-phenylpiperazine 1b, and 1,2,3,4-tetrahydroisoquinoline 1c. For NMR spectra of compounds 2a–i see Figures S1–S18 in Supplementary Materials.
5-Aminouracils 2a–i were then subjected to condensation with 6-oxobicyclo[3.1.0]hex-2-ene (1.3 molar equivalent) in the presence of tetrakis(triphenylphosphine)palladium(0) as a catalyst according to the Trost procedure [35] to give mono- and disubstituted 5′-norcarbocyclic derivatives, (±)-3a–i (in yields of 14–35%) and (±)-4a–i (in yields of 21–41%), respectively. The products 3a–i and 4a–i (see Figures S25–S60 Supplementary Materials) were obtained as racemic mixtures.
It is important to note that the use of a 1.3 molar equivalent of 6-oxobicyclo[3.1.0]hex-2-ene turned out to be the optimal solution for obtaining both target substances at the same time. A further increase in the molar ratio of 6-oxobicyclo[3.1.0]hex-2-ene and the modified pyrimidine base, the main product of the reaction becomes 1,3-di-(4-hydroxycyclopent-2-en-1-yl)-5-substituted uracil, and the 1-(4-hydroxycyclopent-2-en-1-yl)-5-substituted product is observed in trace amounts.
Scheme 2 illustrates the synthesis of 5-aryloxyuracils 5a–d by the methods described previously [36,37,38]. The starting 4-substituted phenols 6a–d were treated with ethyl bromoacetate in a DMF solution in the presence of K2CO3 to give the corresponding phenoxyacetic acid ethyl esters 7a–d. The esters 7a–d were treated with ethyl formate in a solution of THF or diethyl ether in the presence of NaH. The resulting formyl derivatives 8a–8d without additional purification were subjected to condensation with a 1.5-fold molar excess of thiourea in an anhydrous isopropanol solution in the presence of NaH to give the corresponding 5-phenoxy-2-thiouracils 9a–d. To obtain 5-phenoxyuracils 5a–d, 2-thiouracils 9a–d were subjected to desulfurization in an aqueous medium in the presence of a four-fold molar excess of monochloroacetic acid (MCA) and hydrochloric acid. The yields of 5-phenoxyuracils 5a–d (see Figures S19–S24 in Supplementary Materials) were of 77–85%.
It should be noted that the use of potassium tert-butoxide (tBuOK) instead of NaH reduces the yield of compounds 5a–d to 17%. The stage of synthesis of mono-10a–d and disubstituted 11a–d 5′-norcarbocyclic derivatives of 5-phenoxyuracils 5a–d (Scheme 2) is completely identical to the procedure described above for the preparation of 5′-norcarbocyclic derivatives of 5-aminouracils 3a–i and 4a–i (Scheme 1). The yields of the racemic target products (±)-10a–d and (±)-11a–d were 18–53% and 23–34%, respectively (see Figures S61–S76 in Supplementary Materials).
High regio and stereoselectivity of the Trost reaction is achieved due to the unique structure of (π-allyl)palladium complex that the 6-oxobicyclo[3.1.0]hex-2-ene forms with tetrakis(triphenylphosphine)palladium(0) [39]. The polar hydroxy group provides the regiocontrolled nucleophilic addition to the π-allyl system. The structure of the resulting palladium complex is such that the nucleophile adds to the carbocyclic moiety from the side opposite to palladium, to give only cis-products, thus providing the stereospecificity of the reaction. The formation of a monosubstituted product at the N-1 position is also due to the peculiarities of the Trost reaction mechanism. Attack of the complex with N-1 nitrogen is preferable, since the nucleophilicity of N-3 uracil is negatively affected by the presence of two neighboring carbonyl groups with pronounced electron-withdrawing properties. This pattern is generally observed for all types of N-substitution involving uracil and its derivatives [40,41]. On the other hand, the formation of N-1,3 substitution products occurs, apparently, due to the attack of the resulting N-1 nucleoside on the second epoxide molecule.
The use of 6-oxobicyclo[3.1.0]hex-2-ene in the Trost condensation affords racemic products, which can be used for primary activity screening. Individual enantiomers of the most active compounds can be latter catalytically cleaved using various lipases [42,43].

2.2. Biological Activity

Broad-spectrum antiviral activity screening was performed. Antiviral activity and cytotoxicity were assessed for all new compounds in cell-based assays against a variety of RNA viruses. The data in Table 1 show the presence of an antiviral effect against the causative agent of SARS-HCoV OC43. Mono- and disubstituted 5′-norcarbocyclic derivatives of 5-arylaminouracils (±)-3f, (±)-3i and (±)-4d, with an extended alkoxy or alkyl group in the p-position of the aryl fragment, and disubstituted aryloxyuracil derivative (±)-11a have moderate biological activity against this pathogen. Nucleoside analogue (±)-11a was the only compound that inhibited the respiratory syncytial virus RSV (strain A Long). 1,3-Bis-5′-norcarbocyclic derivative of 5-aryloxyuracial (±)-11d demonstrated activity against the yellow fever virus YFV (strain 17D). Testing of the other representatives of the coronavirus family (HCoV-229E, NL63), a number of influenza A virus serotypes (Influenza H1N1, H3N2) and influenza B virus showed no activity against these pathogens. None of the 5-aminouracils 2a–i and 5-phenoxyuracils 5a–d demonstrated either any antiviral activity or cytotoxicity.
It should be noted that although most of the compounds that showed antiviral activity on a particular infected cell line had no toxicity against that cell line up to concentration of 100 μM, a marked cytotoxicity was found in at least one of the other tested cell lines. Compounds (±)-3d, (±)-3e, (±)-4e, (±)-10a and (±)-11c did not show any activity but had marked cytotoxicity in at least one of the three tested cell lines.
Some of the synthesized compounds were additionally tested in HEL cells infected with the DNA viruses: herpes viruses (HSV-1 KOC), varicella-zoster virus (strains TK- and TK+) and cytomegalovirus (strains AD169 and Davis). Among them, only compound (±)-4e showed inhibitory activity. It inhibited the formation of viral plaques in the TK-varicella-zoster virus strain (EC50 = 48.89 μM) and in both cytomegalovirus strains (EC50 = 76.47 and 40.90 μM, respectively). The minimum concentration of compound (±)-4e that leads to the detection of changes in cell morphology was greater than 100 μM.
Thus, it can be assumed that the 5′-norcarbocyclic fragment of 5-substituted uracil derivatives plays an important role and is required for the biological activity. It has been shown that 5′-norcarbocyclic analogues of 2′,3′-dideoxy-2′,3′-didehydrouridine with different substituents in the 5 position of the heterocyclic base have activity against various viruses. To conclude, 5′-norcarbocyclic derivatives of 5-substituted uracils have a wide spectrum of activity and are promising for a more detailed study of the structure activity relationship.

3. Materials and Methods

3.1. Chemistry

All reagents used were of high quality, are commercially available, and do not require further purification (unless otherwise noted). Ethylene glycol, isopropanol, and DMF were supplied by Sigma-Aldrich Co. (Saint Louis, MO, USA). Anhydrous 1,2-dichloroethane and ethyl acetate was obtained by distillation over P2O5. The tetrakis(triphenylphosphine)palladium(0) catalyst was purchased from Alfa Aesar (Ward Hill, MA, USA). 6-Oxobicyclo[3.1.0]hex-2-ene was synthesized based on a previously published procedure [44].
Column chromatography was performed on Silica Gel 60 0.040–0.063 mm (Merck, Germany). Thin layer chromatography (TLC) was carried out on TLC Silica gel 60 F254 plates (Merck, Germany). For preparative thin-layer chromatography (PLC), PLC Silica gel 60 F254, 1 mm plates (Merck, Germany) were used. The results of separation on the plate, as well as the determination of the retention factor (Rf), were analyzed using a VL-6.LC UV lamp (Vilber Lourmat Deutschland, Eberhardzell, Germany). The Rf parameter was measured in the solvent system CHCl3: MeOH (95:5). Eluent systems for the separation of substances are indicated in the text.
NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz for 1H NMR, 100 MHz for 13C NMR) in CDCl3, CD3OD, a mixture of CD3OD/CDCl3, or DMSO-d6 containing tetramethylsilane as an internal standard. Chemical shifts are given in ppm. The product yields refer to spectroscopically (1H NMR and 13C NMR) homogeneous substances.
Melting points were determined using a Mel-Temp 3.0 (Laboratory Devices Inc., Auburn, CA, USA).
High resolution mass spectra were measured on a Bruker Daltonics micrOTOF II or maXis (Bruker, Germany) instruments using electrospray ionization (ESI HRMS). The measurements were done in a positive ion mode with the corresponding parameters: interface capillary voltage 4500 V, mass range from m/z 50 to 3000; internal calibration was done with ESI Tuning Mix (Agilent, Merck, Germany). A syringe injection was used for solutions in acetonitrile (flow rate 3 mL min−1). Nitrogen was applied as a drying gas (4.0 L/min); the interface temperature was set at 180 °C.

3.1.1. General Procedure for the Preparation of 5-Aminouracil Derivatives (2a–i)

A mixture of 5-bromouracil (2.0 g, 10.47 mmol), the corresponding amine 1a–i (20.95 mmol), quinoline (1.9 mL, 16.08 mmol) and of ethylene glycol (50 mL) was refluxed for 1 h, cooled to room temperature, and cold water (250 mL) was added to the solidified reaction mass. The precipitate was filtered off, washed on the filter with water (3 × 50 mL) to remove ethylene glycol residues and ethyl acetate (3 × 15 mL) to remove traces of quinoline, unreacted amine, and its oxidation products, and dried on a Petri dish at 70°C for several hours. The raw material obtained was recrystallized from aqueous DMF.
5-(Azepan-1-yl)uracil 2a (1.79 g, 8,48 mmol, 81%). Decomposition at 310 °C. Rf 0.33. 1H NMR (DMSO-d6) δ, ppm: 10.92 (1H, s, HN3), 10.27 (1H, s, HN1), 6.63 (1H, s, H-6), 2.99–2.95 (4H, m, 2′-CH2, 7′-CH2), 169–1.64 (4H, m, 3′-CH2, 6′-CH2), 1.56–1.52 (4H, m, 4′-CH2, 5′-CH2). 13C NMR (DMSO-d6) δ, ppm: 162.4, 150.6, 128.0, 124.4, 52.0 × 2, 29.1 × 2, 27.3 × 2.
5-(4-Phenylpiperazin-1-yl)uracil 2b (2.25 g, 8.27 mmol, 79%). M.p. 352–354.5 °C. Rf 0.28. 1H NMR (DMSO-d6) δ, ppm: 10.72 (1H, s, HN3), 10.17 (1H, s, HN1), 7.25–7.20 (2H, m, H-3″, H-5″), 6.98–6.94 (2H, m, H-6, H-4″), 6.82–6.77 (2H, m, H-2″, H-6″), 3.25–3.21 (4H, m, 2′-CH2, 6′-CH2), 3.04–2.99 (4H, m, 3′-CH2, 5′-CH2). 13C NMR (DMSO-d6) δ, ppm: 161.9, 151.6, 150.7, 129.4 × 2, 127.0, 126.6, 119.41, 116.1 × 2, 50.2 × 2, 49.0 × 2.
5-(3,4-Dihydroisoquinolin-2(1H)-yl)uracil 2c (1.91 g, 7.85 mmol, 75%), M.p. 311–312 °C. Rf 0.29. 1H NMR (DMSO-d6) δ, ppm: 10.64 (1H, s, HN3), 10.11 (1H, s, HN1), 7.16–7.06 (4H, m, H-5′, H-6′, H-7′, H-8′), 6.81 (1H, s, H-6), 4.06 (2H, s, 2H-1′), 3.24–3.20 (2H, s, 2H-3′), 2.89–2.85 (2H, s, 2H-4′). 13C NMR (DMSO-d6) δ, ppm: 162.1, 150.8, 134.8, 134.2, 129.1, 127.1, 126.9, 126.7, 126.6, 126.1, 52.6, 47.9, 28.9.
5-((4′-Hexylphenyl)amino)uracil 2d (2.11 g, 7.33 mmol, 70%). M.p. 332–333 °C, Rf 0.38. 1H NMR (DMSO-d6) δ, ppm: 10.85 (1H, s, HN3), 10.14 (1H, s, HN1), 7.14 (1H, s, H-6), 6.99–6.96 (2H, m, H-3′, H-5′), 6.77–6.74 (2H, m, H-2′, H-6′), 2.52–2.46, 1.60–1.50 (2H, m, α-CH2), (2H, m, β-CH2), 1.34–1.30 (6H, m, (CH2)3), 0.90–0.86 (3H, m, CH3). 13C NMR (DMSO-d6) δ, ppm: 162.6, 150.5, 143.8, 133.3, 129.0 × 2, 129.0, 117.7, 116.0 × 2, 34.8, 31.5, 31.3, 28.7, 22.3, 14.1.
5-((4′-tert-Butylphenyl)amino)uracil 2e (1.85 g, 7.12 mmol, 68%). M.p. 354.5–356.5 °C. Rf 0.27. 1H NMR (DMSO-d6) δ, ppm: 10.93 (1H, s, HN3), 10.22 (1H, s, HN1), 7.20–7.15 (3H, m, H-3′, H-5′, H-6), 6.79–6.74 (2H, m, H-2′, H-6′), 1.26 (9H, s, 3CH3). 13C NMR (DMSO-d6) δ, ppm: 162.7, 150.6, 143.6, 141.7, 129.5, 125.9 × 2, 117.5, 115.5 × 2, 34.1, 31.8 × 3.
5-((4′-Heptylphenyl)amino)uracil 2f (2.24 g, 7.43 mmol, 71%). M.p. 328–330 °C. Rf 0.28. 1H NMR (DMSO-d6) δ, ppm: 10.92 (1H, s, HN3), 10.21 (1H, s, HN1), 7.14 (1H, s, H-6), 6.99–6.95 (2H, m, H-3′, H-5′), 6.77–6.72 (2H, m, H-2′, H-6′), 6.42 (1H, s, HN5), 2.50–2.45 (2H, m, α-CH2), 1.59–1.49 (2H, m, β-CH2), 1.33–1.24 (8H, m, (CH2)4), 0.90–0.86 (3H, m, CH3). 13C NMR (DMSO-d6) δ, ppm: 162.6, 150.6, 143.8, 133.1, 129.3, 129.1 × 2, 117.6, 115.9 × 2, 34.8, 31.6, 31.5, 29.0, 28.9, 22.4, 14.2.
5-((4′-iso-Propylphenyl)amino)uracil 2g (1.95 g, 7.96 mmol, 76%). M.p. 334.5–345 °C. Rf 0.29. 1H NMR (DMSO-d6) δ, ppm: 10.93 (1H, s, HN3), 10.22 (1H, s, HN1), 7.16 (1H, s, H-6), 7.05–7.00 (2H, m, H-3′, H-5′), 6.78–6.73 (2H, m, H-2′, H-6′), 6.42 (1H, s, HN5), 2.84–2.75 (1H, m, tCH), 1.18 (6H, d, (CH3)2). 13C NMR (DMSO-d6) δ, ppm: 162.7, 151.0, 143.9, 139.3, 129.4, 127.0 × 2, 117.6, 115.9 × 2, 33.0, 24.4 × 2.
5-((4′-Hexyloxyphenyl)amino)uracil 2h (2.22 g, 7.33 mmol, 70%) M.p. 322–324 °C. Rf 0.34. 1H NMR (DMSO-d6) δ, ppm: 10.92 (1H, s, HN3), 10.13 (1H, s, HN1), 7.02 (1H, s, H-6), 6.83–6.75 (4H, m, H-3′, H-5′, H-2′, H-6′), 6.25 (1H, s, HN5), 3.92–3.87 (2H, m, α-CH2), 1.73–1.64 (2H, m, β-CH2), 1.48–1.40 (2H, m, γ-CH2), 1.38–1.30 (4H, m, (CH2)2), 0.92–0.87 (CH3). 13C NMR (DMSO-d6) δ, ppm: 162.5, 153.1, 150.4, 139.1, 126.5, 118.8, 118.2, 116.1 × 2, 68.8, 31.4, 29.3, 25.6, 22.4, 14.1.
5-((4′-Heptyloxyphenyl)amino)uracil 2i (2.66 g, 8.38 mmol, 80%). Rf 0.34. 1H NMR (DMSO-d6) δ, ppm: 10.92 (1H, s, HN3), 10.13 (1H, s, HN1), 7.02 (1H, s, H-6), 6.84–6.75 (4H, m, H-3′, H-5′, H-2′, H-6′), 6.26 (1H, s, HN5), 3.92–3.87 (2H, m, α-CH2), 1.73–1.64 (2H, m, β-CH2), 1.47–1.28 (8H, m, γ-CH2, (CH2)3), 0.91–0.87 (CH3). 13C NMR (DMSO-d6) δ, ppm: 162.5, 153.1, 150.4, 139.1, 126.5, 118.8, 118.2 × 2, 116.1 × 2, 68.8, 31.6, 29.4, 28.8, 25.9, 22.3, 14.1.

3.1.2. General Procedure for the Preparation of 5-(Phenoxy)-2-Thiouracils 9a–d

Potassium carbonate (13.8 g, 99.85 mmol) and ethyl bromoacetate (8.5 mL, 76.65 mmol) were added to a solution of phenol 6a–d (69.40 mmol) in DMF (50 mL). The resulting mixture was stirred at 80 °C for 24 h, evaporated to dryness in vacuum, and the residue was dissolved in chloroform (100 mL) and washed with a 5% NaOH solution (3 × 50 mL) and water (100 mL). The organic fraction was dried over anhydrous Na2SO4, filtered and evaporated under reduced pressure. The resulting ethers 7a–d (45.02 mmol) was dissolved in THF (50 mL) without additional purification, ethyl formate (111 mmol) and NaH (113 mmol, a 60% suspension in oil) were added. The resulting mixture was stirred at room temperature overnight, the solvent was evaporated under reduced pressure, the residue was dissolved in anhydrous iPrOH (80 mL) and then thiourea (67.00 mmol) and NaH (66.67 mmol, a 60% suspension in oil) were added. About 30 min latter, after the end of gas evolution, the reaction mixture was refluxed for 24 h. Then it was evaporated to dryness, dissolved in 250 mL of water, acidified with hydrochloric acid to pH 2, and the precipitate formed was filtered off, dried in air and recrystallized from a mixture of DMF and water (1:1).
5-(3,5-Dimethylphenoxy)-2-thiouracil 9a. Yield 83%. M.p. 242–244 °C, Rf 0.60 (dioxane-NH4OH, 4:1). 1H NMR (DMSO-d6) δ, ppm: 12.71 (1H, s, HN3), 12.33 (1H, s, HN1), 7.52 (1H, s, H-6), 6.69–6.52 (4H, s, H-3′, H-5′, H-2′, H-6′), 2.23 (6H, s, 2CH3). 13C NMR (DMSO-d6) δ, ppm: 174.6, 157.9, 157.7, 139.4 x2, 134.5, 133.6, 124.6, 113.6 × 2, 21.3 × 2.
5-(4-Butoxyphenoxy)-2-thiouracil 9b. Yield 88%. M.p. 233–236 °C, Rf 0.76 (dioxane-NH4OH, 4:1). 1H NMR (DMSO-d6) δ, ppm: 12.71 (1H, s, HN3), 12.28 (1H, s, HN1), 7.43 (1H, s, H-6), 6.96–6.84 (4H, m, H-3′, H-5′, H-2′, H-6′), 3.93–3.89 (2H, m, α-CH2), 1.72–1.62 (2H, m, β-CH2), 1.49–1.36 (2H, m, γ-CH2), 0.95–0.89 (3H, m, CH3). 13C NMR (DMSO-d6) δ, ppm: 174.3, 157.9, 154.9, 151.1, 135.7, 132.4, 117.6 × 2, 115.7 × 2, 68.1, 31.3, 19.2, 14.2.
5-(4-Chlorophenoxy)-2-thiouracil 9c. Yield 76%. M.p. 206–209 °C, Rf 0.62 (dioxane-NH4OH, 4:1). 1H NMR (DMSO-d6) δ, ppm: 13.02 (1H, s, HN3), 7.36–7.30 (2H, m, H-3′, H-5′), 6.97–6.92 (2H, m, H-2′, H-6′), 4.69 (1H, s, H-6). 13C NMR (DMSO-d6) δ, ppm: 170.4, 157.1, 129.7 × 2, 129.2, 116.8 × 2, 65.2.

3.1.3. General Procedure for the Preparation of 5-(Phenoxy)uracils 5a–d

2-Thiouracil 9a–d (10.07 mmol) was mixed with a solution of MCA (3 g, 31.75 mmol) in a mixture of water (30 mL) and concentrated hydrochloric acid (10 mL), the resulting mixture was refluxed for 36 h, filtered, the solid residue was repeatedly washed with water, dried in air and recrystallized from a mixture of DMF and water (2:1).
5-(3,5-Dimethylphenoxy)uracil 5a (13.5 g, 58.3 mmol, 84%). M.p. 293–295 °C, Rf 0.27. 1H NMR (DMSO-d6) δ, ppm: 11.34 (1H, d, HN3), 10.79 (1H, m, HN1), 7.55 (1H, s, H-6), 6.64 (1H, s, H-4), 6.56 (2H, s, H-2′, H-6′), 2.22 (6H, s, 2CH3). 13C NMR (DMSO-d6) δ, ppm: 160.6, 158.5, 151.2, 139.2 × 2, 133.9, 129.4, 124.1, 113.2 × 2, 21.4 × 2.
5-(4-Butoxyphenoxy)uracil 5b (15.3 g, 55.5 mmol, 80%), M.p. 271–274 °C. Rf 0.27. 1H NMR (DMSO-d6) δ, ppm: 11.34–11.33 (1H, d, HN3), 10.77–10.75 (1H, m, HN1), 7.56–7.51 (1H, d, H-6), 6.96–6.82 (4H, m, H-3′, H-5′, H-2′, H-6′), 3.94–3.88 (2H, m, α-CH2), 1.71–1.62 (2H, m, β-CH2), 1.49–1.36 (2H, m, γ-CH2), 0.95–0.90 (3H, m, CH3). 13C NMR (DMSO-d6) δ, ppm: 160.7, 154.4, 152.2, 151.1, 133.5, 130.3, 116.7 × 2, 115.6 × 2, 68.0, 31.3, 19.2, 14.2.
5-(4-Chlorophenoxy)uracil 5c (14.1 g, 59.0 mmol, 85%). M.p. 331–333 °C, Rf 0.24. 1H NMR (DMSO-d6) δ, ppm: 11.40 (1H, d, HN3), 10.88–10.85 (1H, m, HN1), 7.68–7.66 (1H, d, H-6), 7.36–7.30 (2H, m, H-3′, H-5′), 7.03–6.98 (2H, m, H-2′, H-6′). 13C NMR (DMSO-d6) δ, ppm: 160.4, 157.3, 151.1, 134.4, 129.7 × 2, 129.2, 126.3, 117.4 × 2.
5-(4-tert-Butylphenoxy)uracil 5d (12.6 g, 48.9 mmol, 70%), M.p. 273–275 °C, Rf 0.27. 1H NMR (DMSO-d6) δ, ppm: 11.37–11.36 (1H, d, HN3), 10.82–10.79 (1H, m, HN1), 7.59–7.57 (1H, d, H-6), 7.32–7.27 (2H, m, H-3′, H-5′), 6.89–6.83 (2H, m, H-2′, H-6′), 1.26 (9H, s, (CH3)3). 13C NMR (DMSO-d6) δ, ppm: 160.7, 156.3, 151.1, 144.8, 134.0, 129.5, 126.6 × 2, 115.0 × 2, 34.3, 31.8 × 3.

3.1.4. General Procedure for the Preparation of Mono- and Di-Substituted 5′-Norcarbocyclic Derivatives of 5-Aminouracils ((±)-3a–i, (±)-4a–i) and 5-Phenoxyuracils ((±)-10a–d, (±)-11a–d)

The corresponding 5-substituted uracil derivative (100 mg) was dissolved in dry DMF (10 mL) and dried under vacuum to remove possible traces of water. This procedure was repeated twice. 6-Oxobicyclo[3.1.0]hex-2-ene (1.3 molar equivalents) was dissolved in freshly distilled THF (3 mL) and added to the resulting suspension in DMF, the reaction mixture was purged with argon and Pd(PPh3)4 (0.07 molar equivalents) was finally added. The reaction mixture was stirred at room temperature overnight, and then evaporated to dryness under vacuum. The target compounds were isolated by column chromatography on silica gel. The solid products were recrystallized from a mixture of CHCl3 and ethyl acetate.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-(azepan-1″-yl)uracil 3a and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-(azepan-1″-yl)uracil 4a were synthesized from starting 5-(azepan-1-yl)uracil 2a. Compound 3a was isolated by silica gel column chromatography in CHCl3:MeOH (98:2) eluent system. Remaining impurities were removed by PLC in CHCl3:MeOH (95:5) eluent system. The target 3a was obtained as yellow oil (47 mg, 0.16 mmol, 33.3%). Rf 0.30. 1H NMR (CDCl3:CD3OD, 3:1) δ, ppm: 6.91 (1H, m, H-6), 6.19–6.17 (1H, m, H-2′), 5.80–5.78 (1H, m, H-3′), 5.52–5.49 (1H, m, H-1′), 4.78–4.75 (1H, m, H-4′), 3.10 (4H, s, 2H-2″, 2H-7″), 2.86–2.76 (1H, m, αH-5′), 1.76 (4H, s, 2H-3″, 2H-6″), 1.55–1.52 (1H, m, βH-5′). 13C NMR (CDCl3) δ, ppm: 161.2, 149.9, 139.2, 132.0, 129.4, 122.9, 74.6, 59.7, 52.0 × 2, 39.6, 29.7×2, 28.8 × 2, HRMS, m/z: calculated for C15H21N3O3 [M + H]+ 292.1656; found 292.1655.
Compound 4a was isolated by column chromatography on silica gel in a CHCl3:MeOH (99:1) eluent system and then purified by PLC in a CHCl3:MeOH (95:5) system. The resulting compound was obtained as yellow oil (47.5 mg, 0.13 mmol, 27%). Rf 0.48. 1H NMR (CDCl3) δ, ppm: 6.22–6.20 (1H, m, H-2⁗), 6.12–6.09 (1H, m, H-2′), 5.98–5.94 (1H, m, H-3⁗), 5.82–5.80 (1H, m, H-3′), 5.77–5.74 (1H, m, H-1⁗), 5.60–5.57 (1H, m, H-1′), 4.87–4.83 (1H, m, H-4⁗), 4.73–4.67 (1H, m, H-4′), 3.07–3.04 (4H, m, 2H-2″, 2H-7″), 2.88–2.70 (2H, m, αH-5′, αH-5⁗), 1.98–1.91 (1H, m, βH-5⁗), 1.80–1.75 (4H, s, 2H-3″, 2H-6″), 1.64–1.56 (5H, m, 2H-4″, 2H-5′, βH-5′). 13C NMR (CDCl3) δ, ppm: 161.0, 149.9, 139.3, 136.7, 132.1, 131.2, 128.8, 122.8, 76.2, 74.6, 60.0, 56.3, 52.3 × 2, 39.9, 37.5, 29.7 × 2, 28.4×2. HRMS, m/z: calculated for C24H28N4O4 [M + H]+ 437.2183; found 437.2180.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-(4″-phenylpiperazin-1″-yl)uracil 3b and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-(4″-phenylpiperazin-1″-yl)uracil 4b were synthesized from the starting 5-(4-phenylpiperazin-1-yl)uracil 2b. Compound 3b was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (97:3), and then purified by PLC in CHCl3:7M NH3/MeOH (9:1). The target 3b was obtained as a gray powder (21.2 mg, 0.06 mmol, 16.2%). Decomposed at 235 °C. Rf 0.35. 1H NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.32–7.27 (2H, t, H-3‴, H-5‴), 7.09–7.03 (3H, s, H-6, H-2‴, H-6‴), 6.98–6.93 (1H, t, H-4‴), 6.23–6.20 (1H, m, H-2′), 5.83–5.80 (1H, m, H-3′), 5.58–5.54 (1H, m, H-1′), 4.80–4.76 (1H, m, H-4′), 3.35–3.32 (4H, m, 2H-3″, 2H-5″), 3.14–3.11 (4H, m, 2H-2″, 2H-6″), 2.86–2.76 (1H, m, αH-5′), 1.63–1.55 (1H, m, βH-5′). 13C NMR (CDCl3:CD3OD, 3:1) δ, ppm: 161.3, 150.2 × 2, 139.7, 131.5, 129.3, 128.0, 126.8 × 2, 121.8, 117.2 × 2, 73.9, 59.3, 50.2 × 2, 49.7 × 2, 39.5. HRMS, m/z: calculated for C19H22N4O3 [M + H]+ 355.1765 found 355.1764.
Compound 4b was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system and then purified by PLC in CHCl3:MeOH (95:5). The target 4b was obtained as yellowish transparent oil (66.7 mg, 0.15 mmol, 41%). Rf 0.4. 1H NMR (CDCl3) δ, ppm: 7.31–7.26 (2H, m, H-3‴, H-5‴), 7.04 (H, d, H-6), 6.98–6.88 (3H, m, H-2‴, H-6‴, H-4‴), 6.24–6.22 (1H, m, H-2′), 6.14–6.11 (1H, m, H-2⁗), 5.99–5.95 (1H, m H-3′), 5.86–5.82 (1H, m H-3⁗), 5.78–5.75 (1H, m, H-1′), 5.66–5.63 (1H, m, H-1⁗), 4.87–4.84 (1H, m, H-4′), 4.73 (1H, m H-4⁗), 3.33–3.29 (4H, m, 2H-3″, 2H-5″), 3.10–3.08 (4H, m, 2H-2″, 2H-6″), 2.87–2.73 (2H, m, αH-5′, αH-5⁗), 2.01–1.96 (1H, m, βH-5′), 1.67–1.60 (1H, m, βH-5⁗). 13C NMR (CDCl3) δ, ppm: 160.6, 150.8, 150.2, 139.5, 136.8, 132.1, 131.0, 129.3 × 2, 127.7, 125.2, 120.6, 116.6 × 2, 76.3, 74.4, 60.0, 56.4, 50.2 × 2, 49.5 × 2, 39.8, 37.4. HRMS, m/z: calculated for C20H27N3O4 [M + H]+ 374.2074; found 374.2072.
(±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-5-(3′,4′-dihydroisoquinolin-2′(1H)-yl)uracil 3c and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-(3″,4″-dihydroisoquinoline-2″(1H)-yl)uracil 4c were synthesized from the starting 5-(3,4-dihydroisoquinolin-2(1H)-yl)uracil 2c. Compound was 3c isolated by column chromatography on silica gel in the system of eluents CHCl3:MeOH (98:2). The target 3c was obtained as orange crystals (39.1 mg, 0.120 mmol, 29%). It was decomposed at 112 °C. Rf 0.29. 1H NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.13–7.01 (5H, m, H-5″, H-6″, H-7″, H-8″, H-6), 6.21–6.18 (1H, m, H-2′), 5.81–5.78 (1H, m, H-3′), 5.56–5.51 (1H, m, H-1′), 4.80–4.76 (1H, m, H-4′), 4.07 (2H, m, 1″-CH2), 3.27–3.23 (2H, m, 3″-CH2), 2.97–2.93 (2H, m, 4″-CH2), 2.87–2.77 (1H, m, αH-5′), 1.61–1.54 (1H, m, βH-5′). 13C NMR (CDCl3:CD3OD, 3:1): 161.4, 150.2, 139.7, 133.7, 133.5, 131.4, 128.7, 128.0, 126.8, 126.4, 126.3, 125.8, 73.9, 59.3, 52.5, 48.2, 39.6, 28.7. HRMS, m/z: calculated for C18H19N3O3 [M + H]+ 326.1499; found 326.1493.
Compound 4c was isolated by column chromatography on silica gel in CHCl3:MeOH (99:1) eluent system and then purified by PLC in CHCl3:MeOH (95:5) system. The target 4c was obtained as an orange oil (44.3 mg, 0.11 mmol, 26%). Rf 0.40. 1H NMR (CDCl3) δ, ppm: 7.17–7.10 (3H, m, H-8″, H-7″, H-6″), 7.07–7.02 (2H, H-5″, H-6), 6.23–6.19 (1H, m, H-2‴), 6.16–6.12 (1H, m, H-2′), 6.02–5.96 (1H, m, H-3‴), 5.85–5.81 (1H, m, H-3′), 5.79–5.76 (1H, m, H-1‴), 5.64–5.59 (1H, m, H-1′), 4.87–4.83 (1H, m, H-4‴), 4.76–4.71 (1H, m, H-4′), 4.11 (2H, s, 1″-CH2), 3.36–3.22 (2H, m, 3″-CH2), 2.98–2.95 (2H, m, 4″-CH2), 2.89–2.74 (2H, m, αH-5′, αH-5‴), 2.04–1.97 (1H, m, βH-5‴), 1.64–1.57 (1H, m, βH-5′). 13C NMR (CDCl3) δ, ppm: 160.3, 149.7, 138.8, 136.4, 133.4, 133.3, 131.6, 130.5, 128.3, 127.0, 125.9, 125.8, 125.3, 124.9, 75.8, 74.0, 59.6, 59.9, 52.3, 47.7, 39.3, 36.9, 28.2. HRMS, m/z: calculated for C23H25N3O4 [M + Na]+ 430.1737; found 430.1734.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-((4′-hexylphenyl)amino)uracil 3d and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-(-((4″-hexylphenyl)amino)uracil 4d were synthesized from the starting 5-((4′-hexylphenyl)amino)uracil 2d. Compound 3d was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (98:2) and then purified by PLC in CHCl3:MeOH (95:5) to give an orange powder (38.1 mg, 0.10 mmol, 29.5%). M.p. 165.2 °C. Rf 0.32. 1H NMR (CDCl3) δ, ppm: 9.25 (1H, s, N1H), 7.54 (1H, s, H-6), 7.13–7.10 (2H, m, H-2″, H-6″), 7.03–7.01 (2H, m, H-3″, H-5″), 6.26–6.24 (1H, m, H-2′), 5.87–5.85 (1H, m, H-3′), 5.56–5.53 (1H, m, H-1′), 4.88–4.86 (1H, m, H-4′), 2.93–2.85 (1H, m, αH-5′), 2.58–2.53 (2H, m, α-CH2), 1.77–1.72 (1H, m, βH-5′), 1.64–1.54 (2H, m β-CH2), 1.31 (6H, s, (CH2)3), 0.93–0.88 (3H, m CH3). 13C NMR (CDCl3) δ, ppm: 160.5, 148.9, 139.5, 138.7, 131.8, 129.4×2, 128.1, 121.7, 119.7, 118.3 × 2, 74.8, 60.3, 39.7, 35.2, 31.7, 31.5, 29.0, 22.6, 14.1. HRMS, m/z: calculated for C21H27N3O3 [M + H]+ 370.2125; found 370.2123.
Compound 4d was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (99:1), and then purified by PLC in CHCl3:MeOH (95:5) to give a red-orange oil (53 mg, 0.12 mmol, 34%). Rf 0.46. 1H NMR (CDCl3) δ, ppm: 7.38–7.37 (1H, m, H-6), 7.10–7.07 (2H, m, H-2″, H-6″), 6.93–6.90 (2H, m, H-3″, H-5″), 6.24–6.20 (1H, m, H-2′), 6.17–6.14 (1H, m, H-2′′′), 6.04–5.98 (1H, m, H-3′), 5.85–5.82 (1H, m, H-3‴), 5.80–5.77 (1H, m, H-1′), 5.68–5.61 (1H, m, H-1′′′), 4.88–4.84 (1H, m, H-4′), 4.76–4.72 (1H, m, H-4‴), 2.92–2.75 (2H, m, αH-5′, αH-5‴), 2.56–2.51 (2H, m, α-CH2), 2.04–1.98 (1H, m, βH-5‴), 1.71–1.64 (1H, m, βH-5′), 1.61–1.53 (2H, m, β-CH2), 1.35–1.28 (6H, m, (CH2)3), 0.92–0.87 (3H, m, CH3). 13C NMR (CDCl3) δ, ppm: 160.7, 149.3, 139.5, 137.2, 136.2, 132.2, 132.0, 130.7, 129.3 × 2, 128.5, 119.9, 118.2, 117.6 × 2, 76.2, 74.6, 60.4, 56.8, 39.9, 37.4, 35.2, 31.7, 31.5, 29.0, 22.6, 14.1. HRMS, m/z: calculated for C26H33N3O4 [M + Na]+ 474.2363; found 474.2361.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-((4″-tert-butyl)phenylamino)uracil 3e and (±)-1-(1-(4′-hydroxycyclopent-2′-ene-1′-yl))-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-((4″-tert-butyl)phenylamino)uracil 4e were synthesized from the starting 5-((4′-tert-butylphenylamino)uracil 2e. Compound 3e was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system and then purified by PLC in CHCl3:MeOH (95:5) system. The target 3e was obtained as a light yellow powder (71.2 mg, 0.17 mmol, 22%). M.p. 165 °C. Rf 0.30. 1H NMR (CDCl3) δ, ppm: 9.22 (1H, s, HN1), 7.46 (1H, s, H-6), 7.33–7.30 (2H, m, H-2″, H-6″), 7.01–6.99 (2H, m, H-3″, H-5″), 6.27–6.24 (1H, m, H-2′), 5.88–5.86 (1H, m, H-3′), 5.57–5.54 (1H, m, H-1′), 4.90–4.87 (1H, m, H-4′), 2.94–2.84 (1H, m, αH-5′), 1.79–1.71 (1H, m, βH-5′), 1.31 (9H, s, 3CH3). 13C NMR (CDCl3) δ, ppm: 160.6, 148.8, 144.0, 139.4, 138.7, 131.9, 126.4 × 2, 121.10, 119.8, 117.5 × 2, 74.8, 60.3, 39.7, 34.2, 31.4 × 3. HRMS, m/z: calculated for C19H23N3O3 [M + H]+ 342.1812; found 342.1809.
Compound 4e was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (99:1), and then purified by PLC in CHCl3:MeOH (95:5) to give dark red oil (55.4 mg, 0.26 mmol, 24%). Rf 0.46. 1H NMR (CDCl3) δ, ppm: 7.40–7.39 (1H, d, H-6), 7.30–7.26 (2H, m, H-2″, H-6″), 6.95–6.91 (2H, m, H-3″, H-5″), 6.25–6.21 (1H, m, H-2‴), 6.16–6.13 (1H, m, H-2′), 6.03–5.98 (1H, m, H-3‴), 5.85–5.82 (1H, m, H-3′), 5.80–5.77 (1H, m, H-1‴), 5.67–5.62 (1H, m, H-1′), 4.88–4.84 (1H, m, H-4‴), 4.76–4.72 (1H, m, H-4′), 2.94–2.74 (2H, m, αH-5′, αH-5‴), 2.04–1.97 (1H, m, βH-5‴), 1.71–1.64 (1H, m, βH-5′), 1.30 (9H, s, 3CH3). 13C NMR (CDCl3) δ, ppm: 160.8, 149.3, 144.2, 139.5, 139.3, 137.1, 131.9, 130.8, 126.3 × 2, 119.7, 118.5, 116.9 × 2, 76.2, 74.6, 60.4, 56.8, 39.9, 37.4, 34.2, 31.4 × 3. HRMS, m/z: calculated for C24H29N3O4 [M + Na]+ 446.2050; found 446.2048.
(±)-4′-Hydroxycyclopent-2′-en-1′-yl)-5-((4′′-heptylphenyl)amino)uracil 3f and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-(-((4″-heptylphenyl)amino)uracil 4f were synthesized from the starting 5-((4′-heptylphenyl)amino)uracil 2f. Compound 3f was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system. The target 3f was obtained as a light brown powder (30.5 mg, 0.08 mmol, 24%) Decomposed at 192 °C. Rf 0.29. 1H NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.44 (1H, s, H-6), 7.06–7.03 (2H, m, H-2″, H-6″), 6.88–6.83 (2H, m, H-3″, H-5″), 6.20–6.17 (1H, m, H-2′), 5.81–5.78 (1H, m, H-3′), 5.58–5.54 (1H, m, H-1′), 4.79–4.75 (1H, m, H-4′), 2.90–2.80 (1H, m, αH-5′), 2.52–2.47 (2H, m, α-CH2), 1.62–1.52 (3H, m, βH-5′, β-CH2), 1.29–1.24 (8H, m, (CH2)4), 0.88–0.83 (3H, m, CH3). 13C NMR (CDCl3:CD3OD, 3:1) δ, ppm: 160.4, 151.5, 139.7, 135.5, 131.2, 129.2 × 2, 129.1, 121.0, 118.7, 116.9×2, 74.0, 59.3, 39.7, 35.0, 31.7, 31.5, 29.1, 22.5, 13.8. HRMS, m/z: calculated for C22H29N3O3 [M + H]+ 384.2282; found 384.2272.
Compound 4f was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (99:1), and then purified by PLC in CHCl3:MeOH (95:5) to give a brown oil (33.6 mg, 0.07, mmol, 21%). Rf 0.38. 1H NMR (CDCl3) δ, ppm: 7.41–7.40 (1H, d, H-6), 7.14–7.08 (2H, m, H-2″, H-6″), 6.99–6.93 (2H, m, H-3″, H-5″), 6.24–6.22 (1H, m, H-2‴), 6.18–6.15 (1H, m, H-2′), 6.04–5.98 (1H, m, H-3‴), 5.85–5.83 (1H, m, H-3′), 5.80–5.78 (1H, m, H-1‴), 5.66–5.64 (1H, m, H-1′), 4.88–4.85 (1H, m, H-4‴), 4.76–4.74 (1H, m, H-4′), 2.93–2.76 (2H, m, αH-5‴, αH-5′), 2.57–2.52 (2H, m, α-CH2), 2.04–1.98 (1H, d, βH-5‴), 1.72–1.65 (1H, m, βH-5′), 1.61–1.54 (2H, m, β-CH2), 1.32–1.28 (8H, m, (CH2)4), 0.92–0.88 (3H, m, CH3). 13C NMR (CDCl3) δ, ppm: 160.7, 149.3, 139.4, 139.3, 137.3, 132.1, 130.7, 129.4 × 2, 128.7, 119.8, 118.4, 117.8 × 2, 76.2, 74.7, 60.4, 56.8, 39.9, 37.5, 35.2, 31.8, 31.6, 29.7, 29.3, 22.7, 14.1. HRMS, m/z: calculated for C27H35N3O4 [M + H]+ 466.2700; found 466.2690.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-((4″-isopropylphenyl)amino)uracil 3g and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-((4″-isopropylphenyl)amine)uracil 4g were synthesized from the starting 5-((4′-iso-propylphenyl)amino)uracil 2g. Compound 3g was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system. The target 3g was obtained as a light orange powder (47 mg, 0.14 mmol, 35%). Decomposed at 201 °C. Rf 0.27. 1H NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.41 (1H, s, H-6), 7.12–7.08 (2H, m, H-2″, H-6″), 6.91–6.86 (2H, m, H-3″, H-5″), 6.21–6.17 (1H, m, H-2′), 5.81–5.78 (1H, m, H-3′), 5.58–5.53 (1H, m, H-1′), 4.79–4.75 (1H, m, H-4′), 2.90–2.77 (2H, m, αH-5′, -CH-), 1.62–1.54 (1H, m, βH-5′), 1.20–1.17 (6H, d, 2CH3). 13C NMR (CDCl3:CD3OD, 3:1) δ 161.5, 149.5, 141.6, 140.0, 139.7, 131.2, 127.2 × 2, 121.1, 120.3, 116.9 × 2, 74.0, 59.4, 39.8, 33.3, 23.8 × 2. HRMS, m/z: calculated for C18H21N3O3 [M + H]+ 328.1656; found 328.1652.
Compound 4g was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (99:1), and then purified by PLC in CHCl3:MeOH (95:5) to give a red-orange oil (44.5 mg, 0.11 mmol, 27%). Rf 0.4. 1H NMR (CDCl3) δ, ppm: 7.34–7.33 (1H, d, H-6), 7.17–7.12 (2H, m, H-2″, H-6″), 6.95–6.90 (2H, m, H-3″, H-5″), 6.24–6.21 (1H, m, H-2‴), 6.19–6.15 (1H, m, H-2′), 6.05–5.99 (2H, m, H-3‴, 5-NH), 5.87–5.84 (1H, m, H-3′), 5.81–5.78 (1H, m, H-1‴, 5.69–5.62 (1H, m, H-1′), 4.90–4.86 (1H, m, H-4‴), 4.76–4.74 (1H, d, H-4′), 2.96–2.79 (3H, m, αH-5‴, αH-5′, -CH-), 2.05–1.98 (1H, d, βH-5‴), 1.72–1.65 (1H, m, βH-5′), 1.25–1.22 (6H, m, 2CH3). 13C NMR (CDCl3) δ, ppm: 160.8, 149.2, 141.9, 139.6, 139.6, 137.3, 132.2, 130.8, 127.4 × 2, 120.0, 117.5, 117.4 × 2, 76.2, 74.7, 60.4, 56.8, 39.8, 37.5, 33.4, 24.1 × 2. HRMS, m/z: calculated for C23H27N3O4 [M + H]+ 410.2074; found 410.2066.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-((4″-(hexyloxy)phenyl)amino)uracil 3h and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-((4″-(hexyloxy) phenyl)amino)uracil 4h were synthesized from the starting 5-((4′-hexyloxyphenyl)amino)uracil 2h. Compound 3h was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system. The target 3h was obtained as dark brown crystals (25 mg, 0.06 mmol, 20%). M.p. 175 °C. Rf 0.34. 1H NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.21 (1H, s, H-6), 6.95–6.90 (2H, d, H-2″, H-6″), 6.84–6.78 (2H, m, H-3″, H-5″), 6.17–6.14 (1H, m, H-2′), 5.78–5.75 (1H, m, H-3′), 5.55–5.48 (1H, m, H-1′), 4.77–4.72 (1H, m, H-4′), 3.91–3.87 (2H, m, α-CH2), 2.87–2.77 (1H, m, αH-5′), 1.77–1.68 (2H, m, β-CH2), 1.60–1.52 (1H, m, βH-5′), 1.47–1.38 (2H, m, γ-CH2), 1.35–1.28 (4H, m, (CH2)2), 0.90–0.86 (3H, m, CH3). 13C NMR (CDCl3:CD3OD, 3:1) δ, ppm: 161.1, 154.7, 149.5, 139.8, 134.9, 131.1, 123.9, 120.7, 120.1×2, 115.5×2, 74.0, 68.6, 59.4, 39.7, 31.5, 29.2, 25.6, 22.5, 13.7. HRMS, m/z: calculated for C21H27N3O4 [M + H]+ 386.2074; found 386.2065.
Compound 4h was isolated by column chromatography on silica gel in a CHCl3:MeOH eluent system (99:1), and then purified by PLC in a CHCl3:MeOH (95:5) to give dark red oil (47 mg, 0.11 mmol, 30%). Rf 0.42. 1H NMR (CDCl3) δ, ppm: 7.19–7.18(1H, d, H-6), 6.97–6.94 (2H, m, H-2″, H-6″), 6.86–6.82 (2H, m, H-3″, H-5″), 6.20–6.14 (2H, m, H-2‴, H-2′), 6.04–5.98 (1H, m, H-3‴), 5.83–5.77 (2H, m, H-3′, H-1‴), 5.64–5.58 (1H, m, H-1′), 4.85–4.82 (1H, d, H-4‴), 4.76–4.72 (1H, m, H-4′), 3.93–3.89 (2H, m, α-CH2), 2.90–2.75 (2H, m, αH-5‴, αH-5′), 2.03–1.98(1H, d, βH-5‴), 1.81–1.72 (2H, m, β-CH2), 1.68–1.61 (1H, m, βH-5′), 1.50–1.43 (2H, m, γ-CH2), 1.37–1.32 (4H, m, (CH2)2), 0.94–0.89 (3H, m, CH3). 13C NMR (CDCl3) δ, ppm: 160.6, 154.6, 149.2, 139.3, 137.2, 134.6, 132.0, 130.8, 121.3, 120.5 × 2, 116.1, 115.5 × 2, 76.2, 74.6, 68.5, 60.4, 56.8, 39.8, 37.5, 31.6, 29.3, 25.7, 22.6, 14.0. HRMS, m/z: calculated for C26H33N3O5 [M + Na]+ 490.2312; found 490.2303.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-((4″-(heptyloxy)phenyl)amino)uracil 3i and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-((4″-(heptyloxy)phenyl)amino)uracil 4i were synthesized from the starting 5-((4′-heptyloxyphenyl)amino)uracil 2i. Compound 3i was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system. The target 3i was obtained as small dark-brown crystals (18 mg, 0.045 mmol, 14%). M.p. 162 °C. Rf 0.27. 1H-NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.20 (1H, s, H-6), 6.92–6.89 (2H, m, H-2″, H-6″), 6.83–6.79 (2H, m, H-3″, H-5″), 6.17–6.14 (1H, m, H-2′), 5.78–5.75 (1H, m, H-3′), 5.56–5.50 (1H, m, H-1′), 4.77–4.72 (1H, m, H-4′), 3.91–3.87 (2H, m, α-CH2), 2.87–2.77 (1H, m, αH-5′), 1.77–1.68 (2H, m, β-CH2), 1.59–1.51 (1H, m, βH-5′), 1.46–1.37 (2H, m, γ-CH2), 1.35–1.27 (6H, m, (CH2)3), 0.89–0.84 (3H, m, CH3). 13C NMR (CDCl3:CD3OD, 3:1) δ, ppm: 160.8, 153.6, 148.9, 139.1, 134.8, 130.7, 121.0, 119.1 × 2, 118.5, 115.0 × 2, 73.4, 68.1, 58.8, 39.2, 31.2, 28.7, 28.4, 25.4, 21.9, 13.2. HRMS, m/z: calculated for C22H29N3O4 [M + H]+ 400.2231; found 400.2221.
Compound 4i was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (99:1), and then purified by PLC in CHCl3:MeOH (95:5) to give a dark red oil (48 mg, 0.10 mmol, 31%). Rf 0.43. 1H-NMR (CDCl3) δ, ppm: 7.17–7.16 (1H, d, H-6), 6.98–6.93 (2H, m, H-2″, H-6″), 6.87–6.81 (2H, m, H-3″, H-5″), 6.20–6.15 (2H, m, H-3‴, H-3‴), 6.04–5.99 (1H, m, H-2‴), 5.84–5.77 (2H, m, H-2′, H-1‴), 5.64–5.58 (1H, m, H-1′), 4.87–4.83 (1H, m, H-4‴), 4.76–4.72 (1H, m, H-4′), 3.94–3.89 (2H, m, α-CH2), 2.91–2.76 (2H, m, αH-5‴, αH-5′), 2.04–1.98 (1H, d, βH-5‴), 1.81–1.72 (2H, m, β-CH2), 1.69–1.62 (1H, m, βH-5′), 1.51–1.39 (2H, m, γ-CH2), 1.38–1.30 (6H, m, (CH2)3), 0.94–0.89 (3H, m, CH3). 13C NMR (CDCl3) δ, ppm: 160.1, 154.1, 148.7, 138.6, 136.8, 134.0, 131.7, 130.2, 120.8, 120.1 × 2, 115.3, 115.0 × 2, 75.7, 74.2, 67.9, 59.9, 56.3, 39.3, 37.0, 31.3, 28.8, 28.5, 25.5, 22.1, 13.5. HRMS, m/z: calculated for C27H35N3O5 [M + Na]+ 504.2469; found 504.2461
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-(3″,5″-dimethylphenoxy)uracil 10a and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-(3″,5″-dimethylphenoxy)uracil 11a were synthesized from the starting 5-(3,5-dimethylphenoxy)uracil 5a. Compound 10a was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system. The target 10a was obtained as a light yellow powder (40 mg, 0.13 mmol, 30%). 188.9 °C. Rf 0.37. 1H-NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.49 (1H, s, H-6), 6.65 (1H, s, H-4″), 6.52 (2H, s, H-2″, H-6″), 6.19–6.16 (1H, m, H-2′), 5.80–5.77 (1H, m, H-3′), 5.55–5.50 (1H, m, H-1′), 4.77–4.72 (1H, m, H-4′), 2.94–2.84 (1H, m, αH-5′), 2.24 (6H, s, 2CH3), 1.57–1.49 (1H, m, βH-5′). 13C NMR (CDCl3:CD3OD, 3:1) δ, ppm: 159.9, 157.5, 150.3, 140.2 × 2, 139.4, 132.4, 131.5, 130.8, 124.7, 113.2 × 2, 74.0, 59.6, 40.1, 21.2 × 2. HRMS, m/z: calculated for C17H18N2O4 [M + H]+ 315.1339; found 315.1337.
Compound 11a was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (99:1) to give a yellowish oil (39 mg, 0.1 mmol, 23%). Rf 0.42. 1H-NMR (CDCl3) δ, ppm: 7.46 (1H, s, H-6), 6.70 (1H, s, H-4′′), 6.52 (2H, s, H-2″, H-6″), 6.23–6.19 (1H, m, H-2‴), 6.15–6.10 (1H, m, H-2′), 5.95–5.92 (1H, m, H-3‴), 5.85–5.81 (1H, m, H-3′), 5.79–5.75 (1H, m, H-1‴), 5.65–5.59 (1H, m, H-1′), 4.85–4.80 (1H, m, H-4‴), 4.73–4.70 (1H, d, H-4′), 2.94–2.74 (2H, m, αH-5‴, αH-5′), 2.28 (6H, s, 2CH3), 2.03–1.96 (1H, m, βH-5‴), 1.67–1.58 (1H, m, βH-5′). 13C NMR (CDCl3) δ, ppm: 159.5, 157.4, 150.4, 140.1, 139.5 × 2, 137.3, 131.4, 130.7, 130.6, 130.5, 124.9, 113.4 × 2, 76.2, 74.5, 60.3, 56.7, 40.2, 37.4, 21.4 × 2. HRMS, m/z: calculated for C22H24N2O5 [M + Na]+ 419.1577; found 419.1569.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-(4″-butoxyphenoxy)uracil 10b and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-(4″-butoxyphenoxy) uracil 11b were synthesized from the starting 5-(4-chlorophenoxy)uracil 5b. Compound 10b was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system. The target 10b was obtained as a light yellow powder (23 mg, 0.06 mmol, 18%). M.p. 174.8 °C. Rf 0.31. 1H-NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.46 (1H, s, H-6), 6.90–6.84 (2H, m, H-2″, H-6″), 6.82–6.78 (2H, m, H-3″, H-5″), 6.19–6.15 (1H, m, H-2′), 5.78–5.75 (1H, m, H-3′), 5.54–5.49 (1H, m, H-1′), 4.76–4.72 (1H, m, H-4′), 3.91–3.87 (2H, m, α-CH2), 2.92–2.81 (1H, m, αH-5′), 1.76–1.67 (2H, m, β-CH2), 1.55–1.52 (1H, m, βH-5′), 1.50–1.39 (2H, m, γ-CH2), 0.97–0.92 (3H, m, CH3). 13C NMR (CDCl3:CD3OD, 3:1) δ, ppm: 160.3, 155.0, 151.2, 150.4, 140.2, 132.6, 131.6, 130.8, 117.0 × 2, 115.4 × 2, 73.9, 68.3, 59.4, 40.0, 31.2, 19.1, 13.6. HRMS, m/z: calculated for C19H22N2O5 [M + H]+ 359.1601; found 359.1599.
Compound 11b was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (99:1) to give a yellowish oil (48.5 mg, 0.11 mmol, 31%). Rf 0.47. 1H-NMR (CDCl3) δ, ppm: 7.41 (1H, s, H-6), 6.89–6.78 (4H, m, H-2″, H-6″, H-3″, H-5″), 6.20–6.16 (1H, m, H-2‴), 6.13–6.09 (1H, m, H-2′), 5.95–5.89 (1H, m, H-3‴), 5.81–5.74 (2H, m, H-3′, H-1‴), 5.62–5.56 (1H, m, H-1′), 4.82–4.76 (1H, m, H-4‴), 4.71–4.69 (1H, d, H-4′), 3.94–3.89 (2H, m, α-CH2), 2.90–2.72 (2H, m, αH-5‴, αH-5′), 2.00–1.95 (1H, m, βH-5‴), 1.79–1.70 (2H, m, β-CH2), 1.64–1.56 (1H, m, βH-5′), 1.52–1.42 (2H, m, γ-CH2), 1.00–0.95 (3H, m, CH3). 13C NMR (CDCl3) δ, ppm: 159.5, 155.2, 150.8, 150.3, 140.1, 137.2, 132.1, 131.3, 130.7, 129.7, 117.3 × 2, 115.6 × 2, 76.2, 74.4, 68.2, 60.3, 56.7, 40.1, 37.3, 31.4, 19.2, 13.8. HRMS, m/z: calculated for C24H28N2O6 [M + H]+ 441.2020; found 441.2016.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-(4″-chlorophenoxy)uracil 10c and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-(4″-chlorophenoxy)uracil 11c were synthesized from the starting 5-(4-butoxyphenoxy)uracil 5c. Compound 10c was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system. The target 10c was obtained as a pale yellow powder (58 mg, 0.18 mmol, 43%). M.p. 190 °C. Rf 0.29. 1H NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.57 (1H, s, H-6), 7.24–7.18 (2H, m, H-2″, H-6″), 6.89–6.84 (2H, m, H-3″, H-5″), 6.20–6.17 (1H, m, H-2′), 5.79–5.77 (1H, m, H-3′), 5.56–5.51 (1H, m, H-1′), 4.77–4.72 (1H, m, H-4′), 2.92–2.82 (1H, m, αH-5′), 1.56–1.49 (1H, m, βH-5′). 13C NMR (CDCl3:CD3OD, 3:1) δ, ppm: 160.0, 156.2, 150.4, 140.3, 133.1, 131.1, 130.7, 129.4 × 2, 127.8, 116.9 × 2, 73.9, 59.5, 40.0 HRMS, m/z: calculated for C15H13ClN2O4 [M + H]+ 321.0637; found 321.0643.
Compound 11c was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (99:1) to give a yellowish oil (58 mg, 0.14 mmol, 34%). Rf 0.38. 1H NMR (CDCl3) δ, ppm: 7.54 (1H, s, H-6), 7.27–7.24 (2H, m, H-2″, H-6″), 6.88–6.84 (2H, m, H-3″, H-5″), 6.26–6.22 (1H, m, H-2‴), 6.16–6.12 (1H, m, H-2′), 5.95–5.90 (1H, m, H-3‴), 5.85–5.83 (1H, m, H-3′), 5.78–5.74 (1H, m, H-1‴), 5.65–5.63 (1H, m, H-1′), 4.85–4.83 (1H, m, H-4‴), 4.73–4.71 (1H, d, H-4‴), 2.92–2.74 (2H, m, αH-5‴, αH-5′), 2.00–1.95 (1H, d, βH-5‴), 1.67–1.58 (1H, m, βH-5′). 13C NMR (CDCl3) δ, ppm: 158.7, 155.5, 149.8, 139.7, 136.8, 130.8, 130.6, 130.1, 129.9, 129.1 × 2, 127.6, 116.5 × 2, 75.6, 73.8, 59.8, 56.3, 39.6, 36.8. HRMS, m/z: calculated for C20H19ClN2O5 [M + Na]+ 425.0875; found 425.0867.
(±)-1-(4′-Hydroxycyclopent-2′-en-1′-yl)-5-(4″-(tert-butyl)phenoxy)uracil 10d and (±)-1-(4′-hydroxycyclopent-2′-en-1′-yl)-3-(4‴-hydroxycyclopent-2‴-en-1‴-yl)-5-(4″-(tert-butyl) phenoxy)uracil 11d were synthesized from the starting 5-(4-tert-butylphenoxy)uracil 5d. Compound 10d was isolated by column chromatography on silica gel in CHCl3:MeOH (98:2) eluent system. The target 10d was obtained as a pale-yellow powder (68.6 mg, 0.2 mmol, 53%). M.p. 200 °C. Rf 0.27. 1H NMR (CDCl3:CD3OD, 3:1) δ, ppm: 7.52 (1H, s, H-6), 7.29–7.24 (2H, m, H-2″, H-6″), 6.86–6.81 (2H, m, H-3″, H-5″), 6.19–6.16 (1H, m, H-2′), 5.79–5.76 (1H, m, H-3′), 5.56–5.50 (1H, m, H-1′), 4.77–4.72 (1H, m, H-4′), 2.93–2.83 (1H, m, αH-5′), 1.56–1.48 (1H, m, βH-5′), 1.25 (9H, s, 3CH3). 13C NMR (CDCl3:CD3OD, 3:1) δ, ppm: 160.2, 155.3, 150.4, 145.7, 140.2, 132.67, 131.5, 130.7, 126.3 × 2, 114.9 × 2, 73.9, 59.4, 40.0, 34.1, 31.2 × 3. HRMS, m/z: calculated for C19H22N2O4 [M + H]+ 343.1652; found 343.1652.
Compound 11d was isolated by column chromatography on silica gel in CHCl3:MeOH eluent system (99:1) to give a yellow-green oil (69 mg, 0.13 mmol, 34%). Rf 0.38. 1H NMR (CDCl3) δ, ppm: 7.47 (1H, s, H-6), 7.34–7.29 (2H, m, H-2″, H-6″), 6.85–6.80 (2H, m, H-3″, H-5″), 6.22–6.17 (1H, m, H-2‴), 6.15–6.10 (1H, m, H-2′), 5.97–5.91 (1H, m, H-3‴), 5.83–5.75 (2H, m, H-3′, H-1‴), 5.65–5.59 (1H, m, H-1′), 4.83–4.78 (1H, m, H-4‴), 4.73–4.69 (1H, m, H-4′), 2.91–2.74 (2H, m, αH-5‴, αH-5′), 2.03–1.95 (1H, m, βH-5‴), 1.67–1.57 (1H, m, βH-5′), 1.31 (9H, s, 3CH3). 13C NMR (CDCl3) δ, ppm: 159.6, 155.2, 150.4, 145.8, 140.1, 137.3, 131.4, 130.9, 130.8, 130.6, 126.5 × 2, 115.0 × 2, 76.2, 74.45, 60.3, 56.8, 40.1, 37.4, 34.2, 31.5 × 3. HRMS, m/z: calculated for C24H28N2O5 [M + Na]+ 447.1890; found 447.1879.

3.2. Biology

Cells and viruses. The compounds were evaluated against the following viruses: respiratory syncytial virus (RSV) strain A Long, Influenza virus A (subtypes H1N1, H3N2), influenza virus B, yellow fever virus (YFV) strain 17 D, coronaviruses (HcoV-OC43, HcoV-229E, NL63), herpes simplex virus type 1 (HSV-1) strain KOS, varicella-zoster virus (VZV) strains TK+ and TK−, human cytomegalovirus (HCMV) strains AD-169 and Davis. Cell lines used: human embryonic lung fibroblasts (HEL 299), human liver carcinoma (Huh 7) and Madin-Darby canine kidney (MDCK) were obtained from American Type Culture Collection (ATCC).
Cell toxicity evaluation in HEL299, Huh 7 and MDCK cells. Cells were seeded at a rate of 5 × 103 cells/well into 96-well plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds starting at 100 µM was added. After three days of incubation at 37 °C, the cell number was determined with a Beckman Coulter counter. The cytostatic concentrations or CC50 (compound concentration required reducing cell proliferation by 50%) were estimated from graphic plots of the number of cells (percentage of control) as a function of the concentration of the test compounds.
With regard to the cytopathicity or plaque reduction test, confluent cell cultures in 96-well plates were inoculated with 100 CCID50 of virus (CCID50 is a virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU). Following a 2 h adsorption period, viral inoculum was removed and the cell cultures were incubated in the presence of varying concentrations of the test compounds starting at 100 µM (solution in DMSO). Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC50 (compound concentration required reducing virus-induced cytopathicity or viral plaque formation by 50%).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27092866/s1, 1H and 13C NMR spectra of the target products: Figures S1–S78.

Author Contributions

Conceptualization, M.S.N. and A.L.K.; Methodology and Investigation, V.A.K., E.S.M., M.S.N. and A.O.C.; Formal Analysis, R.S. and G.A.; Data Curation, M.S.N. and A.L.K.; Writing—Original Draft Preparation, V.A.K.; Writing—Review & Editing, M.S.N., A.L.K. and A.O.C.; Supervision, S.N.K.; Project Administration, A.L.K. and S.N.K.; Funding Acquisition, A.L.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Russian Foundation for Basic Research, grant number 18-29-08010 (design and synthesis) and Russian Science Foundation, grant number 19-74-10048 (characterization of target products).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

This research was funded by Russian Foundation for Basic Research, grant number 18-29-08010 (design and synthesis) and Russian Science Foundation, grant number 19-74-10048 (characterization of target products).

Acknowledgments

High-resolution mass spectra were acquired in the Department of Structural Studies of N.D. Zelinsky Institute of Organic Chemistry. The biological part of the work was supported by KU Leuven.

Conflicts of Interest

The authors declare that they have no conflict of interest.

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Molecules 27 02866 g001 550
Figure 1. 5-Substituted pyrimidine analogues with different types of antiviral activity.
Figure 1. 5-Substituted pyrimidine analogues with different types of antiviral activity.
Molecules 27 02866 g001
Molecules 27 02866 sch001 550
Scheme 1. Synthesis of 5-amino derivatives of uracil and their 5′-norcarbocyclic analogues. Reaction conditions: (a) (CH2OH)2, quinoline, reflux for 1 h; (b) Pd(PPh3)4, THF, DMF.
Scheme 1. Synthesis of 5-amino derivatives of uracil and their 5′-norcarbocyclic analogues. Reaction conditions: (a) (CH2OH)2, quinoline, reflux for 1 h; (b) Pd(PPh3)4, THF, DMF.
Molecules 27 02866 sch001
Molecules 27 02866 sch002 550
Scheme 2. Synthesis of 5-phenoxyuracils and their 5′-norcarbocyclic derivatives. Reaction conditions: (a) BrCH2C(O)OC2H5, K2CO3, DMF; (b) ethyl formate, NaH, THF; (c) thiourea, NaH, iPrOH; (d) MCA, HCl, H2O; (e) 6-oxobicyclo[3.1.0]hex-2-ene, Pd(PPh3)4, THF, DMF.
Scheme 2. Synthesis of 5-phenoxyuracils and their 5′-norcarbocyclic derivatives. Reaction conditions: (a) BrCH2C(O)OC2H5, K2CO3, DMF; (b) ethyl formate, NaH, THF; (c) thiourea, NaH, iPrOH; (d) MCA, HCl, H2O; (e) 6-oxobicyclo[3.1.0]hex-2-ene, Pd(PPh3)4, THF, DMF.
Molecules 27 02866 sch002
Table
Table 1. Antiviral activity and cytotoxicity of compounds in cell cultures.
Table 1. Antiviral activity and cytotoxicity of compounds in cell cultures.
Comp.EC50 (μM) HCoV-OC43, HEL 299CC50 (μM)
HEL 299		EC50 (μM)
RSV A Long,
HEL 299		CC50 (μM)
Huh7		EC50 (μM) YFV 17D,
Huh7		CC50 (μM)
MDCK
2a>100>100>100>100>100>100
2b>100>100>100>100>100>100
2d>100>100>100>100>100>100
2e>100>100>100>100>100>100
(±)-3a>100>100>100>100>100>100
(±)-3b>100>100>100>100>100>100
(±)-3d>10069.5>10046.8>10030
(±)-3e>100>100>100>100>10063
(±)-3f22.4>100>100>100>10070.4
(±)-3g>100>100>100>100>100>100
(±)-3i36.8>100>1009.8>10034.0
(±)-4a>100>100>100>100>100>100
(±)-4b>100>100>100>100>100>100
(±)-4d28.7>100>100>100>10051.2
(±)-4e>10050.6>10011.7>10019.5
(±)-4f>100>100>100>100>100>100
5a>100>100>100>100>100>100
5b>100>100>100>100>100>100
5c>100>100>100>100>100>100
5d>100>100>100>100>100>100
(±)-10a>100>100>100>100>10033.8
(±)-10b>100>100>100>100>100>100
(±)-10c>100>100>100>100>100>100
(±)-10d>100>100>100>100>100>100
(±)-11a29.8>10028.72.5>10039.3
(±)-11c>100>100>100>100>10047.4
(±)-11d>10056.4>100>10047.940.3
Remde-
sivir		0.03>100.01>10n.dn.d.
Bolded numbers are active compounds.
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Kezin, V.A.; Matyugina, E.S.; Novikov, M.S.; Chizhov, A.O.; Snoeck, R.; Andrei, G.; Kochetkov, S.N.; Khandazhinskaya, A.L. New Derivatives of 5-Substituted Uracils: Potential Agents with a Wide Spectrum of Biological Activity. Molecules 2022, 27, 2866. https://doi.org/10.3390/molecules27092866

AMA Style

Kezin VA, Matyugina ES, Novikov MS, Chizhov AO, Snoeck R, Andrei G, Kochetkov SN, Khandazhinskaya AL. New Derivatives of 5-Substituted Uracils: Potential Agents with a Wide Spectrum of Biological Activity. Molecules. 2022; 27(9):2866. https://doi.org/10.3390/molecules27092866

Chicago/Turabian Style

Kezin, Vasily A., Elena S. Matyugina, Mikhail S. Novikov, Alexander O. Chizhov, Robert Snoeck, Graciela Andrei, Sergei N. Kochetkov, and Anastasia L. Khandazhinskaya. 2022. "New Derivatives of 5-Substituted Uracils: Potential Agents with a Wide Spectrum of Biological Activity" Molecules 27, no. 9: 2866. https://doi.org/10.3390/molecules27092866

APA Style

Kezin, V. A., Matyugina, E. S., Novikov, M. S., Chizhov, A. O., Snoeck, R., Andrei, G., Kochetkov, S. N., & Khandazhinskaya, A. L. (2022). New Derivatives of 5-Substituted Uracils: Potential Agents with a Wide Spectrum of Biological Activity. Molecules, 27(9), 2866. https://doi.org/10.3390/molecules27092866

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