INTRODUCTION

According to the World Health Organization (WHO), tuberculosis is an infectious disease that causes the largest number of deaths worldwide. Recent studies conducted by WHO identified 9.6 million new cases and 2 million deaths in 2021 [1]. In addition, the emergence and growth of multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis arouse concern of authorities around the world. These strains are characterized by low cure rates and high mortality rates due to dif­ficulties in treatment [2, 3]. Furthermore, more cases of totally drug-resistant tuberculosis have been reported in clinics [4, 5]. Over the past few years, progress has been made in the search for new antitubercular agents [6, 7]. Drug development is currently underway to treat drug-sensitive and/or drug-resistant tuber­culosis. As regards MDR-TB, there are effective foreign drugs such as bedaquiline (Sirturo®, Janssen Therapeutics, Titusville, New Jersey, USA) and delamanid (Deltyba®, Otsuka Pharmaceutical, Tokyo, Japan). However, despite recent advances, strains resistant to these new drugs have already been reported, which increases the urgent need to develop new drugs for the treatment of tuberculosis [8–10]. Indeed, research on new drugs against different types of tuberculosis plays a critical role in reducing morbidity and mortality, which is necessary to achieve global targets set by WHO [1].

Analysis of the literature data on currently known compounds active against Mycobacterium tuberculosis and Mycobacterium bovis showed that uracil deriva­tives and nucleosides based thereon are effective inhibitors of these mycobacteria [11, 12]. Our ongoing efforts to develop new effective treatments for myco­bacterial infections on the basis of 5-alkynyl-sub­stituted pyrimidine bases and nucleosides led to the identification of uridine derivatives 1 with good inhibitory activity against M. bovis (MIC50 = 1.5– 50 µg/mL) and M. tuberculosis (MIC50 = 1.1– 50 µg/mL) in cell-based assays [13]. In the present study, we report the synthesis and in vitro antitubercular activity of 5-alkynyl-2-thiouridine derivatives II with various 5-alkyl-, 5-cycloalkyl-, and 5-arylethynyl sub­stituents against M. bovis and M. tuberculosis (Fig. 1). It should be noted that 2-thiouridine derivatives are of great interest since they are important modified units of natural nucleic acids, which play a significant role in tuning the translation process through codon–anticodon interactions [14, 15].

Fig. 1.
figure 1

Structures of 2-alkynyl derivatives of 2-thiouridine as efficient antimycobacterial agents.

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RESULTS AND DISCUSSION

The target 5-alkynyl-2-thiouridines 6 were syn­thesized in good yields by the palladium-catalyzed Sonogashira cross-coupling of 5-iodo-2-thiouridine 5 with various terminal acetylenes. It is known that cross-coupling reactions make it possible to directly introduce an alkyne moiety into the pyrimidine ring under mild conditions without formation of large amounts of byproducts. Initial 5-iodo-2-thiouridine 5 was prepared in 80% yield by regioselective C5-iodination of 2-thiouridine 4 in anhydrous methanol using iodine monochloride as iodinating agent (Scheme 1). It should be noted that the use of other iodinating agents such as molecular iodine and N-iodosuccinimide did not provide formation of 5-iodo-6-methyluridine in a good yield. 2-Thiouridine 4 is not a commercially available product, so that it was synthesized from 2-thiouracil 1 as shown in Scheme 1. Specifically, 2-thiouracil 1 was converted to trimethylsilyl ether 2 which was treated with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribose according to a modified Hilbert–Johnson procedure [16] in the presence of a Lewis acid (SnCl4). The resulting benzoylated nucleoside 3 was deprotected by the action of sodium methoxide in methanol to obtain 2-thio­uridine 4.

Scheme
scheme 1

1.

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We used in our study the complex tetrakis(tri­phenylphosphine)palladium(0) to catalyze the Sonogashira cross-coupling, and it showed excellent catalytic activity. Optimization of the conditions for the reaction of 5-iodo-2-thiouridine with phenylacetylene revealed that the best solvent was N,N-dimethyl­formamide (DMF), and triethylamine (Et3N) was found to be the optimal base. It should be noted that the use of polar solvents, such as DMF, N,N-dimethylacetamide (DMA), and acetonitrile, was associated exclusively with the poor solubility of the initial compound due to intermolecular hydrogen bonding.

In the presence of bases other than triethylamine, the substrate conversion was low or the yield of the target product was reduced due to formation of byproducts. The progress of the Sonogashira reaction was monitored by TLC and HPLC/MS (ESI-TOF). In all cases, the conversion of initial 5-iodo-2-thiouridine was complete, and the presence of a sulfur atom at C2 of the pyrimidine ring did not affect the yield of the target products. Noteworthy is that the high efficiency of the catalytic system made it possible to carry out the Sonogashira reaction at room temperature without protecting the hydroxy groups of 5-iodo-2-thiouridine.

After optimization of the conditions for the Sonogashira cross-coupling between 5-iodo-2-thio­uridine and phenylacetylene, a series of 5-alkynyl-2-thiouridine derivatives with various substituents at the triple bond were successfully synthesized (Scheme 2) with the goal of establishing structure–activity relation­ship. It was also found that the side formation of both bicyclic furano[2,3-d]pyrimidine derivative and oxidative dimerization product of the initial acetylene (as a result of Glaser coupling) could be avoided by using fairly small amounts of the catalyst and copper(I) iodide cocatalyst.

Scheme
scheme 2

2.

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The structure of the obtained compounds was un­am­biguously confirmed by NMR and high-resolution mass spectra, and their purity was checked by HPLC/MS and elemental analysis. It was found that the positions of signals in the aromatic region of the 13C NMR spectra of 5-alkynyluridines strongly differed from those typical of furano[2,3-d]pyrimidines. The m/z values in the high-resolution mass spectra of com­pounds 6–16 completely coincided with the calculated values, and the observed fragmentation patterns were consistent with the assigned structures.

5-Alkynyl-2-thiouridines 6–16 were evaluated for their in vitro antimycobacterial activity at concentra­tions of 0.1 to 100 µg/mL against Mycobacterium bovis (BCG) and Mycobacterium tuberculosis (H37Rv) using microplate Alamar Blue assay (MABA). Rifampicin, isoniazid, and D-cycloserine were used as reference drugs. The results (minimum concentrations causing 50% inhibition of the bacterial growth, MIC50) obtained in different media are collected in Table 1. As seen in Table 1, most of the synthesized 5-alkynyl-2-thiouridine derivatives showed high efficiency in the inhibition of bacterial strains tested. Compounds con­taining a bulky alkyl or aromatic substituent at the triple bond turned out to be especially potent. The obtained results correlate with our previous data for 5-alkynyl derivatives of uracil and uridine [13]. The most active were 5-phenylethynyl (6), 5-cyclopropyl­ethynyl (14), 5-cyclohexylethynyl (15), and 5-(3-hy­droxy-3-phenylprop-1-yn-1-yl) (16) derivatives with MIC50 values in the range of 0.2–1.5 μg/mL. Replacement of the 5-phenylethynyl substituent by 3-hydroxy-3-phenylprop-1-yn-1-yl fragment signif­icantly improved the activity against both mycobac­terial strains. The increased activity of compound 16 is likely to be related to enhancement of interaction (H-bonding) between the heteroaryl ring and myco­bacterial enzymes. It should be noted that the presence of a thioxo group at the 2-position of the pyrimidine ring significantly increases the antimycobacterial activity against M. bovis and M. tuberculosis (H37Rv) in comparison to our previous data and reference compounds. Therefore, we can conclude that further studies in this field are promising and quite important.

Table 1. In vitro antitubercular activity (MIC50, μM) of compounds 6–16 against BCG and H37Rv-TB in different media
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All regents and solvents were prepared and purified according to standard procedures. Commercial reagents were purchased from Sigma–Aldrich and were used without further purification unless otherwise stated. Methylene chloride and methanol were dried over calcium hydride under argon, N,N-Dimethylformamide (DMF) was dried over phosphoric anhydride under argon. All solvents were stored in an argon atmosphere.

2-Thiouracil, 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose, hexamethyldisalazane (HMDS), chloro­(trimethyl)silane (TMSCl), tin(IV) chloride, sodium methoxide, Dowex 50X8, copper(I) iodide, tetrakis­(triphenylphosphine)palladium(0), triethylamine, phenylacetylene, trimethylsilylacetylene, cyclopropyl­acetylene, propyne, but-1-yne, pent-1-yne, hex-1-yne, oct-1-yne, cyclohexylacetylene, 1-phenylprop-2-yn-1-ol, and 4-methylpent-1-yne were commercial products (ABCR Chemical, Merck) and were used without further purification.

The 1H and 13C NMR spectra were recorded at 25°C on a Bruker Ascend 600 spectrometer (Germany) at 600 and 150 MHz, respectively, using DMSO-d6 and CDCl3 as solvents and tetramethylsilane as internal standard. Thin-layer chromatography was performed on Silica F254 plates (Merck) using methylene chloride as eluent; spots were visualized under UV light. The high-resolution mass spectra (electrospray ionization) were recorded on a Bruker LC-QTOF maXis II instru­ment. Elemental analysis was performed with a Euro Vector EA-3000 automated CHNS analyzer (Italy).

4-[(Trimethylsilyl)oxy]-2-[(trimethylsilyl)sul­fanyl]pyrimidine (2). A suspension of 2-thiouracil 1 (1.28 g, 10 mmol) in a mixture of 30 mL of 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and 1 mL of trimethylsilyl chloride was refluxed for 5 h until it became homogeneous. The solution was cooled, excess HMDS was removed under reduced pressure on a rotary evaporator, and the product was stored under argon until further use. A small sample of the oily syrup was dissolved in CDCl3 and checked for the complete­ness of silylation by NMR spectroscopy. Yield 2.70 g (99%), white powder. 1H NMR spectrum (CDCl3), δ, ppm: 0.33 s (9H), 0.44 s (9H), 6.3 d (1H, J = 5.6 Hz), 8.1 d (1H, J = 5.8 Hz). 13C NMR spectrum (CDCl3), δC, ppm: 0.10, 0.25, 106.0, 145.3, 157.64, 167.49. Mass spectrum (HRMS): m/z 273.0872 [M + H]+. C10H20N2OSSi2. Calculated: M + H 273.0868.

2-Sulfanylidene-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-2,3-dihydropyrimidin-4(1H)-one (3). Silylated thiouracil 2 (1.36 g, 5 mmol; kept under argon) was dissolved in 50 mL of 1,2-dichloroethane, and 1.43 g (0.65 mL, 5.5 mmol) of SnCl4 (10% excess) was added with vigorous stirring to the solution. A solution of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribo­furanose (4.4 g, 8.8 mmol) in 50 mL of 1,2-dichloro­ethane was then added dropwise to obtain a slightly yellowish solution, and the mixture was stirred at room temperature for 5 h; the progress of the reaction was monitored by TLC (CH2Cl2–MeOH, 9:1). The resulting solution was poured into a saturated aqueous solution of NaHCO3 with vigorous stirring, and the mixture was left overnight. The suspension was filtered through a layer of silica gel, and the sorbent was washed with ethyl acetate (2×50 mL) and chloroform (2×100 mL). The organic phase was separated, dried over anhydrous Na2SO4, and passed through a column charged with silica gel. The eluate was evaporated to dryness, and the residue was purified by recrystallization from ethanol. Yield 2.46 g (86%), white powder or crystals. 1H NMR spectrum (DMSO-d6), δ, ppm: 4.72 d (2H, J = 4.7 Hz), 4.85 q (1H, J = 5.1 Hz), 5.83 t (1H, J = 6.1 Hz), 5.8–5.9 m (1H), 5.97 d (1H, J = 8.2 Hz), 7.22 d (1H, J = 4.7 Hz), 7.4–8.0 m (16H), 12.7 s (1H). 13C NMR spectrum (DMSO-d6), δC, ppm: 63.63, 70.23, 73.88, 79.50, 90.19, 107.36, 128.54–134.05, 140.56, 159.43, 164.55, 164.65, 165.58, 176.16. Mass spectrum (HRMS): m/z 573.1281 [M + H]+. C30H24N2O8S. Cal­culated: M + H 573.1287.

1-(β-D-Ribofuranosyl)-2-sulfanylidene-2,3-dihy­dropyrimidin-4(1H)-one (4). Benzoylated nucleoside 3 (69 mg, 1.2 mmol) was dissolved in a mixture of 20 mL of anhydrous methanol and 3.9 mL of 5% methanolic sodium methoxide, and the solution was stirred at room temperature for 3 h. After com­pletion of the reaction (TLC, CH2Cl2–MeOH, 3:1), the solution was neutralized by adding DOWEX-50 WX-8 (H form) preliminarily washed with methanol. The exchanger was filtered off, the solvent was evaporated, and 20 mL of water was added to the residue. Methyl benzoate (or methyl acetate) was removed by extrac­tion with diethyl ether (2×30 mL), and the aqueous phase was lyophilized to obtain the unprotected nucleoside. Yield 29 mg (93%), white powder. 1H NMR spectrum (DMSO-d6), δ, ppm: 3.60 m (2H), 3.88 m (1H), 3.95 q (1H, J = 4.6 Hz), 4.04 q (1H, J = 4.4 Hz), 5.07 d (1H, J = 5.0 Hz), 5.2 t (1H, J = 4.5 Hz), 5.37 d (1H, J = 5.3 Hz), 5.95 d (1H, J = 8.1 Hz), 6.53 d (1H, J = 3.7 Hz), 8.14 d (1H, J = 8.1 Hz), 12.50 s (1H). 13C NMR spectrum (DMSO-d6), δC, ppm: 60.03, 69.12, 74.71, 84.80, 92.74, 106.56, 141.13, 159.73, 176.53. Mass spectrum (HRMS): m/z 261.0514 [M + H]+. C9H12N2O5S. Calculated: M + H 261.0500.

5-Iodo-2-thiouridine (5). Iodine monochloride (0.6 g, 3.90 mmol) was added with stirring to a sus­pension of 2-thiouridine 4 (1 g, 2.60 mmol) in anhydrous methanol (50 mL) heated to 50°C, and the mixture was stirred at that temperature for 2 h. After completion of the reaction (TLC), the mixture was treated with a saturated aqueous solution of sodium thiosulfate and extracted with methylene chloride (2×100 mL). The combined extracts were dried over Na2SO4, filtered, and evaporated on a rotary evapo­rator. The crude product was added to a 7 N solution of ammonia in methanol cooled to 0°C. The mixture was allowed to slowly warm up to room temperature and was stirred for 5 h to obtain a yellow solution. The solvent was removed on a rotary evaporator, and the residue was purified by silica gel column chromatog­raphy (CH2Cl2–MeOH, 9:1). Yield 75 mg (75%), white powder. 1H NMR spectrum (DMSO-d6), δ, ppm: 3.68–3.65 m (1H, 4′-H), 3.69–3.55 m (2H, 5′-H), 4.08 q (1H, 3′-H, J = 6.0 Hz), 4.46 d (1H, 5′-OH, J = 5.5 Hz), 4.55 t (1H, 2′-H, J = 5.8 Hz), 4.82 d (1H, 3′-OH, J = 6.3 Hz), 4.99 d (1H, 2′-OH, J = 4.8 Hz), 6.03 d (1H, 1′-H, J = 3.7 Hz), 11.10 s (1H, NH). 13C NMR spec­trum (DMSO-d6), δC, ppm: 53.1, 62.5, 70.3, 71.0, 84.4, 87.3, 151.1, 152.4, 162.9. Mass spectrum (HRMS): m/z 386.9472 [M + H]+. Found, %: C 28.12; H 2.98; N 7.30. C9H11IN2O5S. Calculated, %: C 27.99; H 2.87; N 7.25. M + H 386.9467.

General procedure for the synthesis of 5-alkynyl-substituted 2-thiouridine derivatives 6–16 via palladium-and-copper-catalyzed Sonogashira cross-coupling. Tetrakis(triphenylphosphine)palladium(0) (5 mol %), copper(I) iodide (10 mol %), triethylamine (3.0 equiv), and the corresponding terminal acetylene (1 equiv) were added to a solution of 5-iodo-2-thiouridine (1 equiv) in anhydrous dimethylformamide (20 mL). The orange mixture was stirred at room temperature for 12 h in a nitrogen atmosphere [the progress of the reaction was monitored by TLC using methanol–chloroform (1:4 v/v) as eluent]. After stirring for 12 h, 15 drops of 5% EDTA disodium salt in water was added, and the mixture was concentrated on a rotary evaporator. The residue was subjected to silica gel column chromatography using chloroform–methanol (95:5 v/v) as eluent. Compounds 6–16 were isolated as white solids.

5-(Phenylethynyl)-2-thiouridine (6) was synthe­sized from 50 mg of 5 and 13.2 mg of phenylacetylene. Yield 34.5 mg (74%), white powder, mp 192°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 3.32–3.28 m (1H, 4′-H), 3.54–3.47 m (2H, 5′-H), 4.10 q (1H, 3′-H, J = 6.0 Hz), 4.21 d (1H, 5′-OH, J = 5.5 Hz), 4.38 t (1H, 2′-H, J = 5.8 Hz), 4.47 d (1H, 3′-OH, J = 6.5 Hz), 4.62 d (1H, 2′-OH, J = 4.9 Hz), 5.98 d (1H, 1′-H, J = 3.8 Hz), 7.56–7.40 m (5H, Harom), 11.05 s (1H, NH). 13C NMR spectrum (DMSO-d6), δC, ppm: 61.5, 69.4, 70.1, 83.5, 86.4, 87.1, 89.2, 91.5, 122.4, 128.2, 128.6, 132.5, 150.7, 151.9, 161.7. Mass spectrum (HRMS): m/z 361.0817 [M + H]+. Found, %: C 56.83; H 4.60; N 7.81. C17H16N2O5S. Calculated, %: C 56.66; H 4.47; N 7.77. M + H 361.0813.

5-[(Trimethylsilyl)ethynyl]-2-tiouridin (7) was synthesized from 50 mg of 5 and 12.7 mg of trimethylsilylacetylene. Yield 36 mg (78%), white powder, mp 181°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 0.18 s (9H, CH3), 3.31–3.27 m (1H, 4′-H), 3.56–3.50 m (2H, 5′-H), 4.12 q (1H, 3′-H, J = 6.0 Hz), 4.20 d (1H, 5′-OH, J = 5.5 Hz), 4.41 t (1H, 2′-H, J = 5.8 Hz), 4.48 d (1H, 3′-OH, J = 6.5 Hz), 4.60 d (1H, 2′-OH, J = 4.9 Hz), 5.95 d (1H, 1′-H, J = 3.8 Hz), 11.08 s (1H, NH). 13C NMR spectrum (DMSO-d6), δC, ppm: 1.9, 60.9, 69.5, 70.0, 83.6, 86.5, 87.2, 89.3, 91.3, 150.4, 151.8, 161.5. Mass spectrum (HRMS): m/z 357.0893 [M + H]+. Found, %: C 47.32; H 5.79; N 7.90. C14H20N2O5SSi. Calculated, %: C 47.17; H 5.66; N 7.86. M + H 357.0896.

5-(Prop-1-yn-1-yl)-2-thiouridine (8) was synthe­sized from 50 mg of 5 and 5.2 mg of propyne. Yield 29.7 mg (77%), white powder, mp 186°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 1.82 s (3H, CH3), 3.30–3.28 m (1H, 4′-H), 3.57–3.51 m (2H, 5′-H), 4.11 q (1H, 3′-H, J = 6.0 Hz), 4.21 d (1H, 5′-OH, J = 5.5 Hz), 4.39 t (1H, 2′-H, J = 5.8 Hz), 4.47 d (1H, 3′-OH, J = 6.5 Hz), 4.65 d (1H, 2′-OH, J = 4.9 Hz), 5.90 d (1H, 1′-H, J = 3.8 Hz), 11.07 s (1H, NH). 13C NMR spectrum (DMSO-d6), δC, ppm: 3.6, 60.7, 69.7, 70.2, 83.8, 86.6, 87.0, 89.4, 91.5, 150.3, 151.7, 161.4. Mass spectrum (HRMS): m/z 299.0634 [M + H]+. Found, %: C 48.55; H 4.89; N 9.44. C12H14N2O5S. Calculated, %: C 48.31; H 4.73; N 9.39. M + H 299.0657.

5-(But-1-yn-1-yl)-2-thiouridine (9) was synthe­sized from 50 mg of 5 and 7 mg of but-1-yne. Yield 31.9 mg (79%), white powder, mp 184°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 1.22 t (3H, CH3, J = 7.5 Hz), 2.38 q (2H, CH2, J = 7.5 Hz), 3.29–3.26 m (1H, 4′-H), 3.55–3.50 m (2H, 5′-H), 4.10 q (1H, 3′-H, J = 6.0 Hz), 4.23 d (1H, 5′-OH, J = 5.5 Hz), 4.38 t (1H, 2′-H, J = 5.8 Hz), 4.46 d (1H, 3′-OH, J = 6.5 Hz), 4.63 d (1H, 2′-OH, J = 4.9 Hz), 5.92 d (1H, 1′-H, J = 3.8 Hz), 11.10 s (1H, NH). 13C NMR spec­trum (DMSO-d6), δC, ppm: 15.6, 28.9, 60.8, 69.9, 70.3, 83.7, 86.8, 87.1, 89.6, 91.3, 150.5, 151.8, 161.5. Mass spectrum (HRMS): m/z 313.0821 [M + H]+. Found, %: C 50.14; H 5.30; N 9.02. C13H16N2O5S. Calculated, %: C 49.99; H 5.16; N 8.97. M + H 313.0813.

5-(Pent-1-yn-1-yl)-2-thiouridine (10) was synthe­sized from 50 mg of 5 and 8.8 mg of pent-1-yne. Yield 33.8 mg (80%), white powder, mp 187°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 1.08–0.98 m (3H, CH3), 1.59–1.42 m (2H, CH2), 2.26–2.07 m (2H, CH2), 3.30–3.26 m (1H, 4′-H), 3.56–3.50 m (2H, 5′-H), 4.12 q (1H, 3′-H, J = 6.0 Hz), 4.25 d (1H, 5′-OH, J = 5.5 Hz), 4.36 t (1H, 2′-H, J = 5.8 Hz), 4.45 d (1H, 3′-OH, J = 6.5 Hz), 4.62 d (1H, 2′-OH, J = 4.9 Hz), 5.91 d (1H, 1′-H, J = 3.8 Hz), 11.11 s (1H, NH). 13C NMR spectrum (DMSO-d6), δC, ppm: 13.3, 20.4, 21.9, 60.7, 70.0, 70.2, 83.6, 86.7, 87.1, 89.7, 91.2, 150.4, 151.9, 161.3. Mass spectrum (HRMS): m/z 327.0979 [M + H]+. Found, %: C 51.63; H 5.71; N 8.69. C14H18N2O5S. Calculated, %: C 51.52; H 5.56; N 8.58. M + H 327.0970.

5-(Hex-1-yn-1-yl)-2-thiouridine (11) was synthe­sized from 50 mg of 5 and 10.6 mg of hex-1-yne. Yield 32.2 mg (73%), white powder, mp 180°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 0.84 t (3H, CH3, J = 7.0 Hz), 1.40–1.25 m (4H, CH2), 2.31 t (2H, CH2, J = 7.0 Hz), 3.31–3.25 m (1H, 4′-H), 3.52–3.48 m (2H, 5′-H), 4.09 q (1H, 3′-H, J = 6.0 Hz), 4.22 d (1H, 5′-OH, J = 5.5 Hz), 4.33 t (1H, 2′-H, J = 5.8 Hz), 4.41 d (1H, 3′-OH, J = 6.5 Hz), 4.59 d (1H, 2′-OH, J = 4.9 Hz), 5.89 d (1H, 1′-H, J = 3.8 Hz), 11.12 s (1H, NH). 13C NMR spectrum (DMSO-d6), δC, ppm: 14.3, 20.2, 23.2, 29.4, 60.6, 69.8, 70.1, 83.4, 86.5, 86.8, 89.5, 91.0, 150.4, 151.8, 161.1. Mass spectrum (HRMS): m/z 341.1135 [M + H]+. Found, %: C 53.12; H 6.10; N 8.34. C15H20N2O5S. Calculated, %: C 52.93; H 5.92; N 8.23. M + H 341.1126.

5-(4-Methylpent-1-yn-1-yl)-2-thiouridine (12) was synthesized from 50 mg of 5 and 10.6 mg of 4-methylpent-1-yne. Yield 31.3 mg (71%), white powder, mp 181°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 0.92 d (6H, CH3, J = 6.4 Hz), 1.75 sept (1H, CH, J = 6.4 Hz), 3.32–3.25 m (1H, 4′-H), 3.39 d (2H, CH2, J = 6.5 Hz), 3.51–3.48 m (2H, 5′-H), 4.07 q (1H, 3′-H, J = 6.0 Hz), 4.21 d (1H, 5′-OH, J = 5.5 Hz), 4.34 t (1H, 2′-H, J = 5.8 Hz), 4.39 d (1H, 3′-OH, J = 6.5 Hz), 4.60 d (1H, 2′-OH, J = 4.9 Hz), 5.90 d (1H, 1′-H, J = 3.8 Hz), 11.11 s (1H, NH). 13C NMR spectrum (DMSO-d6), δC, ppm: 18.8, 30.8, 60.5, 69.8, 69.9, 70.2, 83.4, 86.4, 86.9, 89.6, 91.0, 150.3, 151.6, 160.8. Mass spectrum (HRMS): m/z 341.1129 [M + H]+. Found, %: C 53.20; H 6.17; N 8.29. C15H20N2O5S. Calculated, %: C 52.93; H 5.92; N 8.23. M + H 341.1126.

5-(Oct-1-yn-1-yl)-2-thiouridine (13) was synthe­sized from 50 mg of 5 and 14.3 mg of oct-1-yne. Yield 37.2 mg (78%), white powder, mp 178°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 0.90 t (3H, CH3, J = 7.0 Hz), 1.38–1.27 m (6H, CH2), 1.61–1.54 m (2H, CH2), 2.38 t (2H, CH2, J = 7.0 Hz), 3.33–3.27 m (1H, 4′-H), 3.49–3.46 m (2H, 5′-H), 4.09 q (1H, 3′-H, J = 6.0 Hz), 4.22 d (1H, 5′-OH, J = 5.5 Hz), 4.33 t (1H, 2′-H, J = 5.8 Hz), 4.40 d (1H, 3′-OH, J = 6.5 Hz), 4.58 d (1H, 2′-OH, J = 4.9 Hz), 5.91 (1H, 1′-H, J = 3.8 Hz), 11.12 s (1H, NH). 13C NMR spectrum (DMSO-d6), δC, ppm: 18.9, 20.2, 23.3, 29.5, 29.7, 32.3, 60.5, 70.2, 70.4, 83.5, 86.5, 87.1, 89.8, 91.1, 150.4, 151.8, 160.6. Mass spectrum (HRMS): m/z 369.1428 [M + H]+. Found, %: C 55.68; H 6.81; N 7.71. C17H24N2O5S. Calculated, %: C 55.42; H 6.57; N 7.60. M + H 369.1439.

5-(Cyclopropylethynyl)-2-thiouridine (14) was synthesized from 50 mg of 5 and 8,6 mg of cyclo­propylacetylene. Yield 31.5 mg (75%), white powder, mp 188°C (decomp.). NMR spectrum 1H (600 MHz, DMSO-d6), δ, ppm: 0.71–0.65 m (2H, CH2), 0.96– 0.92 m (2H, CH2), 1.89–1.84 m (1H, CH), 3.36–3.29 m (1H, 4′-H), 3.52–3.47 m (2H, 5′-H), 4.12 q (1H, 3′-H, J = 6.0 Hz), 4.25 d (1H, 5′-OH, J = 5.5 Hz), 4.36 t (1H, 2′-H, J = 5.8 Hz), 4.44 d (1H, 3′-OH, J = 6.5 Hz), 4.61 d (1H, 2′-OH, J = 4.9 Hz), 5.96 d (1H, 1′-H, J = 3.8 Hz), 11.15 s (1H, NH). 13C NMR spectrum (DMSO-d6), δC, ppm: 2.7, 13.4, 60.7, 70.3, 70.6, 83.6, 86.7, 87.4, 89.9, 91.3, 150.6, 152.0, 160.9. Mass spec­trum (HRMS): m/z 325.0817 [M + H]+. Found, %: C 51.99; H 5.11; N 8.73. C14H16N2O5S. Calculated, %: C 51.84; H 4.97; N 8.64. M + H 325.0813.

5-(Cyclohexylethynyl)-2-thiouridine (15) was synthesized from 50 mg of 5 and 14 mg of cyclo­hexylacetylene. Yield 36 mg (76%), white powder, mp 191°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 1.10–1.03 m (2H, CH2), 1.16–1.11 m (1H, CH), 1.27–1.21 m (2H, CH2), 1.69–1.62 m (1H, CH), 1.77–1.71 m (4H, CH2), 1.95–1.90 m (1H, CH), 3.37–3.29 m (1H, 4′-H), 3.53–3.47 m (2H, 5′-H), 4.14 q (1H, 3′-H, J = 6.0 Hz), 4.26 d (1H, 5′-OH, J = 5.5 Hz), 4.37 t (1H, 2′-H, J = 5.8 Hz), 4.45 d (1H, 3′-OH, J = 6.5 Hz), 4.60 d (1H, 2′-OH, J = 4.9 Hz), 5.95 d (1H, 1′-H, J = 3.8 Hz), 11.14 s (1H, NH). 13C NMR spec­trum (DMSO-d6), δC, ppm: 2.7, 13.4, 26.2, 26.5, 33.1, 42.1, 60.9, 70.4, 70.7, 83.7, 86.7, 87.3, 90.0, 91.4, 150.8, 152.1, 160.8. Mass spectrum (HRMS): m/z 367.1288 [M + H]+. Found, %: C 55.92; H 6.23; N 7.80. C17H22N2O5S. Calculated, %: C 55.72; H 6.05; N 7.64. M + H 367.1283.

5-(3-Hydroxy-3-phenylprop-1-yn-1-yl)-2-thio­uridine (16) was synthesized from 50 mg of 5 and 17.1 mg of 1-phenylprop-2-yn-1-ol. Yield 36.4 mg (72%), white powder, mp 185°C (decomp.). 1H NMR spectrum (DMSO-d6), δ, ppm: 3.24 s (1H, OH), 3.34–3.28 m (1H, 4′-H), 3.52–3.47 m (2H, 5′-H), 4.12 q (1H, 3′-H, J = 6.0 Hz), 4.26 d (1H, 5′-OH, J = 5.5 Hz), 4.35 t (1H, 2′-H, J = 5.8 Hz), 4.46 d (1H, 3′-OH, J = 6.5 Hz), 4.62 d (1H, 2′-OH, J = 4.9 Hz), 5.40 t (1H, CH, J = 2.2 Hz), 5.93 d (1H, 1′-H, J = 3.8 Hz), 7.56–7.23 m (5H, Harom), 11.15 s (1H, NH). 13C NMR spec­trum (DMSO-d6), δC, ppm: 14.5, 60.7, 61.9, 70.1, 70.6, 83.9, 86.8, 87.5, 90.3, 91.6, 126.8, 127.2, 129.1, 139.8, 150.5, 152.0, 160.7. Mass spectrum (HRMS): m/z 391.0920 [M + H]+. Found, %: C 55.47; H 4.72; N 7.27. C18H18N2O5S. Calculated, %: C 55.38; H 4.65; N 7.18. M + H 391.0919.