Design and chemical synthesis of selen–gemcitabine against multidrug-resistant Staphylococcus aureus

Abstract

Multidrug-resistant Staphylococcus aureus infection is a significant challenge to global health systems. We designed selen–gemcitabine to provide a dual inhibition mechanism and to reduce metabolic degradation by linking the amino group of gemcitabine with a selenoline ring, aiming to enhance antibacterial action. The synthetic routes were designed and optimized to adapt green chemistry using non-polluting reagents, such as cetyltrimethylammonium bromide and sodium hydroxymethylsulfonate to synthesize selenoline. In addition, the tetrabutylammonium fluoride process was optimized to selectively deprotect the tertiary butyldimethylsilyl group from gemcitabine’s 3′,5′-hydroxyl to gain high yield and purity. The half inhibitory concentration values of the synthesized selen–gemcitabine against several multidrug-resistant S. aureus ranged from 0.04 to 5.8 μM.

Introduction

Staphylococcus aureus is a gram-positive bacterium that may cause life-threatening illnesses. ¹ The prevalence of multidrug-resistant (MDR) S. aureus is due to the heavy and often inappropriate use of antibiotics, ² and its resistance to β-lactams, tetracyclines, aminoglycosides, and fluoroquinolones has been found.^3,4 Therefore, there is an urgent need to develop new antimicrobial agents with novel mechanisms of action.^5,6 In our early drug screening, gemcitabine and Ebselenoline were discovered as potent inhibitors for an MDR methicillin-resistant S. aureus (MRSA).

Ebselen is a low-molecular-weight organic selenium compound with antioxidant, anti-inflammatory, cytoprotective, and antimicrobial activity.^{7 –9} Ebselen could bind to a wide range of protein/enzyme targets and the inhibition is usually produced by covalent modification of cysteine residues through opening the benzisoselenazolone ring and forming S–Se bonds. Inhibition of some essential microbial enzymes accounts for its antimicrobial activities, and structural modification of the benzene ring in ebselen could significantly enhance the antimicrobial activity. ¹⁰

Gemcitabine is a cytidine analog and inhibitor of DNA polymerase with broad-spectrum antitumor and antiviral activity.^{11 –15} However, when administered intravenously, gemcitabine may be quickly metabolized into 2′,2′-difluoro deoxyuridine by the deoxycytidine deaminase (CDA) enzymes present in the body, leading to rapid renal clearance. ¹⁶ Gemcitabine has not been employed for the treatment of bacteremia or systemic infections due to its high toxicity, rapid metabolic inactivation, limited oral bioavailability, and innate or acquired resistance. ¹⁷ To overcome these challenges, several gemcitabine prodrugs have been developed, most of which are modified at the N4 of the nucleobase and C5′-hydroxyl positions of gemcitabine.^{18 –21}

To generate a molecule with a novel mechanism and high potency, we designed to build ebselen and gemcitabine together via the amino group to reduce metabolic clearance (Figure 1), in which selenoline replaces the amino group of gemcitabine to act as a protective group while it also introduces a new functional motif to enhance the antibacterial activity. The synthetic route is shown in Scheme 1, and the bioassay results showed that this bimolecular structure exhibited relatively effective antibacterial activity against several MRSA in vitro.

Figure 1.

Design scheme of selen–gemcitabine.

Scheme 1.

Synthesis route of selen–gemcitabine.

Results and discussion

Synthesis and optimization of selen–gemcitabine

The synthesis of the target molecule 8 is shown in ^{Scheme 1.} First, the hydroxyl group of gemcitabine was protected with tertiary butyldimethylsilyl (TBDMS); then, the amine group of gemcitabine was employed to construct the selenoline ring; and finally, the TBDMS protection was removed to obtain the selen–gemcitabine compound.

The selenoline ring is sensitive to acid, base, and redox reagent treatment. ²² Therefore, to incorporate it into the multifunctional gemcitabine molecule, we attempted two approaches to generate ebselenoline derivatives.^{23 –26} The first method involves reacting selenochloride with secondary amines which results in a long synthesis process. The second method involves copper-catalyzed Se–N coupling which offers a broad range of substrates but requires precise reaction conditions.

Scheme 1 illustrates the process of synthesizing selenium chloride (4) from amino benzoate (1) using the diselenium compound 3. The process involves reacting 2 with newly prepared Na₂Se₂, which will emit a large amount of heat and hydrogen gas (which contains a small amount of selenium powder, and the highly toxic H₂Se). The formation of 4 is through the reaction of thionyl chloride with 3, and the reaction emits a large amount of strongly irritating and corrosive hydrogen chloride gas.

To avoid the formation of H₂Se or HCl during the process of generating the 3 generation process, we employ the two environmentally friendly optimization strategies described below. Initially, we added the quaternary ammonium surfactant cetyltrimethylammonium bromide (CTAB) to decrease the surface tension of the liquid, facilitate dispersion, and ensure more effective contact and collision between reactant molecules, thus accelerating the rate of chemical reaction and improving yield. Furthermore, the surfactants reduced the danger of explosion by reducing the intensity of the exothermic reaction. An alternative option is to use sodium hydroxymethyl sulfonate (PN) as the reducing agent in the reduction of selenium powder. This is because sodium borohydride has a tendency to react with water in the presence of air, resulting in the production of hydrogen gas and subsequent decomposition. When PN was employed, we observed minimal or no generation of hydrogen gas and heat, while still achieving a satisfactory yield (Table 1).

Table 1.

Screening of reaction conditions for Na₂Se₂.

Entry	Reaction condition	Reaction phenomenon	Yield a (%)
1	NaBH4	The reaction is violently exothermic, and emitting large amounts of hydrogen gas	85.2
2	NaBH4, CTAB	Slightly exothermic, no gas generation	78.6
3	PN	Slightly exothermic, no gas generation	79.1

a

Since sodium diselenide could not be detected by LC-MS, to calculate the yield, we continued the reaction of the prepared Na₂Se₂ solution with the diazonium salt (2) to obtain selenoic acid (3), and the yield was calculated from the total two-step yield of 1–3.

Selenium tetrachloride (4) is typically synthesized using sulfoxide chloride as both a reagent and a solvent. However, during the reaction, sulfoxide emits highly irritating and corrosive hydrogen chloride gas, and sulfur dioxide, hydrogen sulfide, and other hazardous substances that may overflow into the reaction system and endanger operational safety and the environment. Therefore, its use must be strictly controlled. We attempted to replace a portion of the sulfoxide chloride with toluene as an organic solvent since it had superior solubility and stability. The experimental findings revealed that when the amount of toluene grew, the amount of sulfoxide fell, the hydrogen chloride gas was gradually reduced, and the reaction yield improved as shown in Table 2.

Table 2.

The effect of the dosage of reactants on the formation of compound 4.

Entry	Amount of SOCl2 (eq)	Reaction solvent	Reaction phenomenon	Yield (%)
1	22	No other solvents	Generation of large quantities of hydrochloric acid gas	66.9
2	9	Toluene	Small amount of hydrochloric acid gas generation	71.3
3	5	Toluene	No generation of hydrochloric acid gas	72.5

The second route for selenochlorine 4 generation involved copper atom-catalyzed Se–N coupling. Unfortunately, the Se–N coupling reaction usually demands a high temperature and a long reaction time, which results in additional by-products and a low yield of less than 30%. Taken together, Route 1 was chosen for green chemical synthesis of 7 from 6, and the one-step process yielded 75%.

Hydroxyl group protection/deprotection is another issue for multifunctional gemcitabine molecules. Due to its reactive nature, the hydroxyl group is often necessary to be protected during the derivatization of glycoconjugates. ²⁷ In this investigation, we used silyl groups, namely TBDMS, to protect the hydroxyl group on gemcitabine. This choice of protection was advantageous due to its ease of effective linking, stability under various reaction circumstances, and mild deprotection properties.^{28 –30}

However, the effect of the bulky groups on the substrate conformation has been reported. Tamao et al. and Eliel et al.’s^31,32 work demonstrated that two consecutive silyloxy groups could induce a drastic conformational change in the cyclohexane ring. Marzabadi et al.’s ³³ work computationally calculated the preferred conformation of 1,2-bis(trialkylsilyloxy)cyclohexanes for a wide range of silyloxy substitutions. During our experiment, we sequentially introduced imidazole and TBDMSCl to gemcitabine in a dimethylformamide (DMF) solution. The mixture was then stirred at room temperature for 12 h. Following ethyl acetate extraction and column chromatography purification, a white solid of component 6 was obtained with a yield of 75.0%. Subsequently, the 6 was dissolved in dry tetrahydrofuran, treated with DIPEA, and then added dropwise to the tetrahydrofuran solution of selenochlorine 4 in a cold bath. Following the 4 addition, the reaction was gradually brought to room temperature before being quenched with methanol 4 h later. The substance was purified by column chromatography and gave a pale yellow solid 7 with a 75.0% yield.

To deprotect TBDMS ethers, tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) solvent is typically used. ³⁴ However, the strong alkalinity of TBAF causes various adverse effects, limiting its widespread usage. ³⁵ Numerous researchers have attempted to enhance the deprotection reaction, including the work by Barnych et al., ³⁶ Bothwell et al., ³⁷ Zubaidha et al., ³⁸ and Thopate et al., ³⁹ and formulated the acid-based media protocols; the work by Karimi et al. ⁴⁰ and Glória et al. ⁴¹ reported the halide source protocols (especially fluoride), and de Vries et al. ⁴² developed reduction protocols. Furthermore, moderate alkaline media were used for selective deprotection reactions of phenolic TBDMS ethers, although the process was slow and inefficient. Overall, selective cleavage of phenolic TBDMS ethers remains a challenge. ⁴³

In preliminary studies, we treated the TBDMS ether-protected selen–gemcitabine 7 with the TBAF in THF solution, obtaining a pale yellow solid 8 following column chromatography purification in 49% yield. The primary difficulty was the excessive amount of by-product.

To reduce the amount of the by-product, we tested applying some weak acids to neutralize the strong alkalinity of TBAF to keep the system in a mild environment. As indicated in Table 3, LC-MS analysis demonstrated that without acetic acid, the by-product can reach 30%, but the addition of acetic acid or a lower reaction temperature could significantly lower the by-product formation. At 0 °C–5 °C, with 3.0 equivalents of TBAF and acetic acid to pH 7–8, the reaction showed a significant increase in the reaction yield, low by-product formation, and low level of single TBDMS-protected product which could be recovered and deprotected subsequently.

Table 3.

The effects of temperature, the amount of TBAF, and AcOH on the formation of Compound 8.

Entry	Reaction temperature (°C)	TBAF (eq.)	pH adjusted by AcOH	Reaction time (h)	Yield (%)	Impurity content (%)	Single TBDMS yield (%)
1	30	3.0	/	1	49	30	/
2	30	3.0	7–8	6	85	12	/
3	30	3.0	6–7	6	80	8	10
4	0–5	3.0	7–8	5.5	74	4	16
5	0–5	3.0	6–7	14	75	2	14
6	0–5	2.0	6–7	9	48	1	50
7	0–5	2.5	6–7	15	55	6	34

Under optimal conditions (Entry 4 in Table 3), we also investigated the efficacy of various acids aiming to reduce the by-product production (Table 4). Acetic acid condition was the best and generated the least impurities. Furthermore, the acetic acid condition resulted in reliable deprotection at both the 50 mg and 10 g levels.

Table 4.

The effect of other acids on the reaction.

Entry	Acid types	Reaction time (h)	Yield (%)	Impurity content (%)	Single TBDMS
1	Acetic acid	5.5	74	4	16
2	Maleic acid	3	82	12	/
3	Citric acid	1.5	74	11	9
4	Methanesulfonic acid	2.5	80	11	/
5	Trifluoroacetic acid	4	83	10	/

Biological activity testing of selen–gemcitabine

In a prior study, we illustrated that 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazo-lium-3-ium-5-yl)benzenesulfonate sodium salt (EZMTT) reagent can enhance bacterial viability signals and improve the sensitivity of the turbidity assay. ⁴⁴ Depending on the time of EZMTT addition to the culture medium, the bioactivity of selen–gemcitabine was tested using tracking and endpoint methods.

The in vitro antimicrobial activity test results showed that Compound 8 exhibited stronger antimicrobial activity against MDR MRSA bacteria with IC₅₀ values of 0.04–5.8 μM compared with gemcitabine (Table 5). Furthermore, the tracking assay illustrated that selenium–gemcitabine exhibited good antibacterial activity against all five strains, although the endpoint assay showed that selen–gemcitabine has weaker activity than gemcitabine against ATCC 29213 and MRSA-XHX.

Table 5.

Comparison of IC₅₀ (μM) between gemcitabine and selen–gemcitabine by tracking method and endpoint method.

	ATCC 29523		ATCC 29213 a		CYZH		ZHF		XHX b
	T	E c	T	E	T	E	T	E	T	E
Gemcitabine	4.55	0.23	0.839	1.86	0.779	0.416	0.296	0.046	0.725	0.627
Selenium–gemcitabine	2.26	0.136	0.326	5.797	0.238	0.121	0.244	0.044	0.244	0.762

a

ATCC 29523 and ATCC 29213 are two different strains of S. aureus.

b

MRSA-CYZH, MRSA-XHX, and MRSA-ZHF are the ID number of the MRSA strain.

c

T: Tracking Method; E: Endpoint Method.

Conclusion

In conclusion, we have successfully explored a strategy for the development of a novel antimicrobial drug for MDR S. aureus. By protecting the amino group of gemcitabine through a selenoline ring, we obtained a novel compound that is effective against drug-resistant bacteria. In addition, we have developed an effective and green chemical synthetic route for selen–gemcitabine. Especially, we have achieved the selective removal of the TBDMS protecting group on the 3′,5′-hydroxyl group of gemcitabine by the improved TBAF method, and the selen–gemcitabine was obtained in 74% yield. Importantly, selen–gemcitabine showed submicromolar strong antimicrobial activity against clinically isolated MDR S. aureus. Our results provide a valid approach for a novel drug for MDR microbial.

Experimental

General materials and methods

Gemcitabine and other chemicals were from Sigma-Aldrich (China) and used without further purification unless otherwise noted. S. aureus and MRSA strains were obtained from the strain collection from Zhejiang Provincial People’s Hospital; EZMTT was from JNF Bioscience, Inc., (Hangzhou, China).

¹H and ¹³C NMR spectra were obtained from a solution in CDCl₃ or d₆-DMSO with TMS (tetramethylsilane) as an internal standard using a 400/101 MHz (¹H/¹³C) spectrometer. The chemical shifts were reported in δ (ppm) using the δ 7.26 signal of CDCl₃ and δ 2.50 signal of DMSO-d₆ (¹H NMR), the δ 77.16 signal of CDCl₃ and δ 39.52 signal of DMSO-d₆ (¹³C NMR) as internal standards. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. High-resolution mass spectra were acquired on a X500R Quadrupole time-of-flight mass spectrometer. Melting points were determined using a WRS-1C High-temperature type melting-point tester and are uncorrected. Automatic Enzyme Labeling Instrument was from Molecular Devices.

General procedure for the synthesis

The synthesis of compound 3

Preparation of Sodium Diselenide Solution: Sodium borohydride (1.0 g, 26.4 mmol) is dissolved in 40 mL of water and stirred until completely dissolved. Under ice bath cooling, black selenium powder (2.1 g, 26.7 mmol) is slowly added, and the reaction will release a significant amount of heat and produce bubbles. It is necessary to maintain the temperature below 15 °C. Once the selenium powder has been added completely, the reaction is continued at room temperature for 3 h. Afterward, 15 mL of a sodium hydroxide solution (6.9 g, 173 mmol) in water is added to the reaction mixture, resulting in a dark red solution that can be used without further treatment.

Preparation of Diazonium Salt Solution: To a reaction flask, add methyl anthranilate (4.0 g, 26.5 mmol) and 40 mL of water. Then, add 6 N hydrochloric acid (10.6 mL, 63.6 mmol) and cool the mixture under an ice bath, maintaining the temperature below 5 °C. Slowly add 10 mL of an aqueous solution of sodium nitrite (2.2 g, 31.9 mmol) dropwise to the methyl anthranilate solution. Continue stirring at a temperature below 5 °C for 20 min to obtain a pale yellow diazonium salt solution.

Synthesis of Diseleninic Acid 3: With the temperature controlled below 15 °C under an ice bath, the diazonium salt solution is slowly added dropwise to the sodium diselenide solution, resulting in the generation of a significant amount of bubbles. After the addition is complete, stir the mixture at room temperature for 1 h, then heat it to 65 °C and react for 3 h. Once the reaction is finished, cool the mixture to room temperature, add activated carbon, stir, and filter. Adjust the pH of the collected filtrate to 1–2 using hydrochloric acid, which will cause a large amount of solid to precipitate. Filter and dry the solid to obtain 4.5 g of a gray solid with a yield of 85.4%.

The synthesis of compound 4

Diseleninic acid 3 (4.0 g, 10.0 mmol) is dried and then dispersed in 20 mL of thionyl chloride. A drop of DMF is added to the mixture. The temperature is slowly raised to reflux, causing the generation of a significant amount of gas. After reacting for 3 h, the solution becomes clear. Once cooled to room temperature, the solvent is evaporated to obtain an oily product. To this, 40 mL of n-heptane is added, and the mixture is heated to reflux for 1 h. The solution is then filtered while hot, and the filtrate is evaporated to yield 3.4 g of a yellow solid with a yield of 66.9%.

The synthesis of compound 6

Gemcitabine 5 (10 g, 38 mmol) was dissolved in 80 mL of DMF, and imidazole (7.8 g, 11.5 mmol) and TBDMSCl (15 g, 9.6 mmol) were added sequentially. The mixture was stirred at room temperature overnight. After that, the reaction solution was poured into 100 mL of ice water and extracted with 100 mL of ethyl acetate thrice. The layers were separated, and the organic phase was washed with 100 mL of water five times. The organic phase was then dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. The crude product was purified by column chromatography to yield 14 g of white solid with a yield of 75.0%.

The synthesis of compound 7 in route 1

Protected gemcitabine 6 (200 mg, 0.4 mmol) was dissolved in 3 mL of dry THF. DIPEA (155 mg, 1.2 mmol) was added, and under stirring in an ice bath, a 3 mL solution of selenium chloride 4 (152 mg, 0.6 mmol) in THF was slowly added dropwise. The reaction mixture was slowly warmed to room temperature and allowed to react for 4 h. After that, 1 mL of methanol was added to quench the reaction. The solvent was evaporated to dryness, and the crude product was purified by column chromatography to yield 202 mg of a pale yellow solid with a yield of 75.0%.

The synthesis of compound 6-1 in Route 2

In a reaction flask, 2-bromobenzoic acid (123 mg, 0.61 mmol) protected gemcitabine 6 (300 mg, 0.61 mmol), 5 mL of dichloromethane, DCC (188 mg, 0.92 mmol), and DMAP (110 mg, 0.92 mmol) were added sequentially. The reaction was monitored by TLC until completion. After that, the solid was filtered and washed with a small amount of dichloromethane. The filtrate was evaporated to dryness, and the crude product was purified by column chromatography to yield 368 mg of a white solid with a yield of 89.5%.

The synthesis of compound 7 in Route 2

In a dry round-bottom flask, 5 mL of anhydrous DMF, cuprous iodide (15 mg, 0.08 mmol), and 1,10-phenanthroline (14 mg, 0.08 mmol) were added sequentially. The mixture was stirred at room temperature for 15 min. Then, compound 6-1 (100 mg, 0.16 mmol), selenium powder (16 mg, 0.20 mmol), and anhydrous potassium carbonate (26 mg, 0.19 mmol) were added in order. The mixture was slowly heated to 110 °C under nitrogen and stirred for 5 h. After cooling to room temperature, it was poured into 20 mL of water, stirred for 1 h, filtered, and the filter cake was dried. Column chromatography was performed to yield 32 mg of a pale yellow solid with a yield of 29.7%.

The synthesis of compound 8

A solution of TBAF (70 mg, 0.27 mmol) in THF was slowly added dropwise to a solution of the protected selenium chloride–gemcitabine 7 (120 mg, 0.18 mmol) in THF. After reacting for 1 h, the solvent was evaporated to dryness, and the crude product was purified by column chromatography to yield 39 mg of a pale yellow solid with a yield of 49.0%.

General procedure for the biological activity testing

Freshly cultured bacteria colonies were selected from Mueller–Hinton agar plates and diluted to 0.5 McFarland concentration with sterile saline. In the tracking method, 1 mL of bacterial solution was added to 18.9 mL of MH medium and diluted 20-fold, then 100 μL of EZMTT (200-fold) was added to the medium, and then 100 μL of bacterial solution was added to each well of a drug plate. After 24-h incubation in the incubator, the absorbance values at 450 and 600 nm were measured by a plate reader. In the endpoint method, 1 mL of bacterial solution was added to 19 mL of MH medium for 20-fold dilution, and 100 μL of bacterial solution was added to each well after mixing. After incubation in the incubator for 20 h, 10 μL of 10x EZMTT was added to each well, and the absorbance values at 450 and 600 nm were determined by a plate reader after adding the detection reagent for 4 h. For comparison, incubation without compound or bacteria under the same conditions was performed as a control, and the half inhibitory concentration (IC₅₀) values were calculated.

Compound characterization

2,2'-diselanediyldibenzoic acid (3): gray solid; 85.4%; m.p. 290.1 °C–293.9 °C; UV (nm⁻¹): 314, 256, 222; ¹ H NMR (400 MHz, DMSO-d₆) δ 8.0 (dd, J = 7.8, 1.5 Hz, 1H), 7.7 (d, J = 8.0 Hz, 1H), 7.5 (td, J = 7.6, 1.6 Hz, 1H), 7.4 (t, J = 7.4 Hz, 1H).¹³ C NMR (101 MHz, DMSO) δ 168.6, 133.6, 133.5, 131.6, 129.5, 128.8, 126.6; HRMS (ESI): m z⁻¹ calcd for C₁₄H₁₁O₄Se₂ [M+H]⁺ 402.8910, found 402.8870.

2-(chlorocarbonyl)phenyl hypochloroselenoite (4): yellow solid; 66.9%; m.p. 57.8 °C–59.7 °C; UV (nm⁻¹): 236; ¹ H NMR (400 MHz, Chloroform-d) δ 8.3 (dd, J = 8.0, 1.4 Hz, 1H), 8.1 (dd, J = 8.2, 1.0 Hz, 1H), 7.8 (ddd, J = 8.4, 7.2, 1.4 Hz, 1H), 7.5–7.5 (m, 1H).¹³ C NMR (101 MHz, DMSO) δ 173.3, 144.7, 134.0, 129.9, 126.9, 126.8, 126.6.

4-amino-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (5): white solid; m.p. 219.9 °C–221.1 °C; UV (nm⁻¹): 270, 240; ¹ H NMR (400 MHz, DMSO-d₆) δ 7.7 (d, J = 7.5 Hz, 1H), 7.4 (d, J = 12.5 Hz, 2H), 6.2 (d, J = 6.5 Hz, 1H), 6.1 (t, J = 8.2 Hz, 1H), 5.8 (d, J = 7.5 Hz, 1H), 5.2 (t, J = 5.5 Hz, 1H), 4.1 (tt, J = 12.8, 7.3 Hz, 1H), 3.8–3.7 (m, 2H), 3.6 (ddd, J = 12.5, 6.0, 3.8 Hz, 1H).¹³ C NMR (101 MHz, DMSO-d₆) δ 165.7, 154.7, 140.8, 130.2–116.7 (m), 94.6, 83.5, 80.4, 68.7 (t, J = 22.5 Hz), 59.0；HRMS (ESI): m z⁻¹ calcd for C₉H₁₂F₂N₃O₄ [M+H]⁺ 264.0718, found 264.0790.

4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (6): white solid; 75.0 %; m.p. 219.9 °C–221.1 °C; UV (nm⁻¹): 268, 244; ¹ H NMR (400 MHz, DMSO-d6) δ 7.53 (d, J = 7.5 Hz, 1H), 7.42 (d, J = 7.6 Hz, 2H), 6.16 (t, J = 8.0 Hz, 1H), 5.76 (d, J = 7.5 Hz, 1H), 4.35–4.25 (m, 1H), 3.96–3.85 (m, 2H), 3.76 (dd, J = 12.1, 3.0 Hz, 1H), 0.88 (d, J = 6.6 Hz, 19H), 0.11–0.06 (m, 12H). ¹³ C NMR (101 MHz, CDCl3) δ 165.7, 155.5, 140.6, 95.3, 84.2 (dd, J = 40.3, 23.3 Hz), 81.0 (d, J = 8.9 Hz), 69.8 (dd, J = 27.6, 18.1 Hz), 60.1, 25.8, 25.5, 18.3, 18.0, −4.7, −5.3, −5.5 (d, J = 4.3 Hz); HRMS (ESI): m z⁻¹ calcd for C₂₁H₄₀F₂N₃O₄Si₂ [M+H]⁺ 492.2447, found 492.2520.

2-bromo-N-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (6-1): white solid; 89.5%; m.p. 149.5 °C–151.2 °C; UV (nm⁻¹): 371, 286, 243; ¹ H NMR (400 MHz, DMSO-d₆) δ 11.61 (s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.70 (dd, J = 7.8, 1.3 Hz, 1H), 7.57 (dd, J = 7.5, 1.9 Hz, 1H), 7.48 (td, J = 7.4, 1.3 Hz, 1H), 7.43 (td, J = 7.6, 1.9 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H), 6.25 (t, J = 7.2 Hz, 1H), 4.39 (td, J = 12.2, 8.6 Hz, 1H), 4.01 (ddd, J = 13.1, 7.5, 2.4 Hz, 2H), 3.83 (dd, J = 12.4, 3.3 Hz, 1H), 0.91 (d, J = 13.3 Hz, 18H), 0.12 (dd, J = 3.7, 1.6 Hz, 12H).¹³ C NMR (101 MHz, CDCl₃) δ 162.4, 144.8, 136.1, 134.1, 132.8, 129.9, 128.0, 119.6, 84.9 (dd, J = 40.5, 24.1 Hz), 81.7, 81.6, 69.6 (dd, J = 27.4, 18.2 Hz), 60.5, 60.1, 33.9, 26.0, 25.6, 21.2, 18.5, 18.1, 14.3, −4.6, −5.2, −5.3, −5.3; HRMS (ESI): m z⁻¹ calcd for C₂₈H₄₃BrF₂N₃O₅Si₂ [M+H]⁺ 674.1814, found 674.1887.

2-(1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzo[d][1,2]selenazol-3(2H)-one (7): pale yellow solid; 75.0%; m.p. 242.1 °C–247.2 °C; UV(nm⁻¹): 303, 256; ¹ H NMR (400 MHz, Chloroform-d) δ 8.12 (dd, J = 7.8, 3.7 Hz, 1H), 8.00 (td, J = 5.7, 2.8 Hz, 1H), 7.67–7.61 (m, 1H), 7.42–7.35 (m, 1H), 6.40 (dd, J = 10.4, 3.6 Hz, 1H), 4.39 (td, J = 11.6, 7.9 Hz, 1H), 4.09–3.99 (m, 1H), 3.84 (dd, J = 11.7, 1.8 Hz, 1H), 0.95 (d, J = 21.7 Hz, 10H), 0.19–0.11 (m, 6H).¹³ C NMR (101 MHz, CDCl₃) δ 1678.0, 163.0, 155.6, 142.9, 141.6, 134.0, 129.5, 128.6, 126.4, 126.0, 98.1, 85.2 (dd, J = 41.1, 23.7 Hz), 81.8, 81.7, 69.7 (dd, J = 27.4, 17.6 Hz), 60.1, 26.0, 25.7, 18.5, 18.1, −4.2, −5.1, −5.3, −5.3; HRMS (ESI): m z⁻¹ calcd for C₂₈H₄₂F₂N₃O₅SeSi₂ [M+H]⁺ 674.1718, found 674.1791.

2-(1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzo[d] [1,2]selenazol-3(2H)-one (8): pale yellow solid; 49%; m.p. 238.0 °C–243.7 °C; UV (nm⁻¹): 372, 288, 244; ¹ H NMR (500 MHz, DMSO-d₆) δ 8.35 (d, J = 7.5 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.79–7.71 (m, 2H), 7.47 (t, J = 7.5 Hz, 1H), 6.35 (d, J = 6.5 Hz, 1H), 6.20 (t, J = 7.3 Hz, 1H), 5.35 (t, J = 5.4 Hz, 1H), 4.29–4.14 (m, 1H), 3.92 (dt, J = 8.5, 2.8 Hz, 1H), 3.83 (d, J = 9.2 Hz, 2H). ¹³ C NMR (101 MHz, DMSO) δ 166.9, 162.6, 154.0, 145.3, 140.4, 134.6, 129.7, 128.7, 126.9, 126.5, 123.4 (t, J = 257.6 Hz), 96.5, 84.7 (t, J = 30.3 Hz), 81.6, 68.7 (t, J = 22.2 Hz), 59.2 HRMS (ESI): m z⁻¹ calcd for C₁₆H₁₄F₂N₃O₅Se [M+H]⁺ 445.9988, found 446.0061.

Supplemental Material

sj-docx-1-chl-10.1177_17475198251320459 – Supplemental material for Design and chemical synthesis of selen–gemcitabine against multidrug-resistant Staphylococcus aureus

Supplemental material, sj-docx-1-chl-10.1177_17475198251320459 for Design and chemical synthesis of selen–gemcitabine against multidrug-resistant Staphylococcus aureus by Guozhen Li, Yue Wan, Debao Chen and Benfang Helen Ruan in Journal of Chemical Research