pubs.acs.org/joc Article Conformationally Locked Carbocyclic Nucleosides Built on a 4′- Hydroxymethyl-3′-hydroxybicyclo[4.1.0]heptane Template. Stereoselective Synthesis and Antiviral Activity Sergio Jurado, Ona Illa, Angel Á lvarez-Larena, Christophe Pannecouque, Félix Busqué,* and Ramon Alibés* Cite This: J. Org. Chem. 2022, 87, 15166−15177 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: Two new families of enantiomerically pure carbocyclic nucleoside analogues based on a cyclohexane moiety with five chiral centers and a fused cyclopropyl ring have been synthesized. A highly regio- and stereoselective synthetic approach for the modular construction of the functionalized bicyclo[4.1.0]- heptyl azide intermediate 6 has been established. Key steps to achieve this asymmetric synthesis involved highly diastereoselec- tive allylic oxidation and hydroboration reactions. The first family of compounds, 1a,b and 2, presents different natural nucleobases, whereas the second one 3a−e bears functionalized 1,2,3-triazoles. These derivatives have been tested as antiviral agents, and compound 3d has shown to display moderate activity against coxsackie B4 virus. ■ INTRODUCTION Nucleoside analogues (NAs) are an important class of small molecule-based antivirals, which mainly act by interfering with the metabolism and function of natural nucleosides. These prodrugs constitute the backbone for the treatment of chronic infections provoked by HIV, herpes viruses, and hepatitis B or C viruses.1−3 However, only 10 human viral pathogens can be treated with antiviral drugs.4,5 Moreover, the SARS-CoV-2 pandemic has emphasized the need for compounds that can respond to future outbreaks provoked by other emerging viruses. Thus, the fight against COVID has renewed the researchers’ interest in NAs.6,7 In the search for novel antiviral NAs, natural nucleosides have been submitted to several chemical modifications. Among them, those resulting in changes on the sugar moiety and/or on the heterocyclic bases of endogenous nucleosides have led to the development of a series of compounds displaying a wide range of antiviral Figure 1. Selected biologically active nucleoside analogues: cyclo- activity.8 hexenyl-G (DCG) and ribavirin, and targeted carbocyclic analogues 1a,b, 2, and 3a−e. Carbocyclic NAs (CNAs) are structurally related to ribonucleosides in which a carbocycle is in the place of the sugar moiety.9,10 CNAs are recognized by the same enzymes as antiviral activity against some herpes viruses (HSV-1, HSV-2, natural nucleosides and are more stable toward hydrolysis by VZV, CMV) was found to be the same for both enantiomers of phosphorylases displaying enhanced biostability. This family of CNAs includes, among others, the cyclopentene derivatives abacavir and entecavir and the six-membered counterpart Received: July 14, 2022 cyclohexenyl G (DCG, Figure 1).11 In particular, cyclohexenyl Published: October 27, 2022 nucleosides are an interesting class of antiviral compounds, wherein the presence of a double bond instead of the oxygen atom of the furanose ring produces a comparable annular flexibility to that of the parent nucleoside.12 Interestingly, © 2022 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.joc.2c01661 15166 J. Org. Chem. 2022, 87, 15166−15177 The Journal of Organic Chemistry pubs.acs.org/joc Article DCG.13 The conformational restriction of a nucleoside has often been used to enhance selectivity and/or potency against ■ RESULTS AND DISCUSSION The synthesis started with alcohol 4 previously prepared in the target enzymes.14,15 Reducing the overall number of 80% yield in four steps from commercially available 1,4- flexible conformations could favor the adoption of a bioactive cyclohexanedione on a multigram scale by a methodology conformation and, therefore, molecular recognition by the devised in our group (Scheme 2).26 Benzyl etherification of the target enzyme. Some conformationally locked carbocyclic analogues by a fused cyclopropane have been described to Scheme 2. Synthesis of Key Azide 6 present similar conformations than those of the natural counterparts and have shown improved enzyme recogni- tion.16,17 On the other hand, modification of the natural nucleobases, including five-membered heterocyclic nucleobases such as a triazole ring, has resulted in the discovery of a series of compounds with a broad spectrum of activities against diverse viruses.2,5,8 A remarkable example of this group of compounds is ribavirin, which is a 1,2,4-triazole nucleoside drug with a wide range of antiviral activities.18 1,2,3-Triazole derivatives have also been reported as antimicrobial,19 anticancer,20 and potential antiviral21,22 agents even in the field of CNAs.23,24 Over the past years, our research group has worked on the development of new routes for the enantioselective preparation of cyclohexane NAs as prodrug candidates.25 We recently described the design and synthesis of a new class of CNAs based on a bicyclo[4.1.0]heptane scaffold.26 As part of our continuing research program to identify bioactive NAs, we were interested in developing novel cyclohexenyl-G derivatives bearing an appended cyclopropyl ring 1a,b and unnatural bases, such as a triazole heterocycle 3a−e (Figure 1). It was expected that the fusion to a cyclopropane would impart a significant rigidity to the resulting nucleosides similar to that of the cyclohexene moiety but should be less prone to hydrolytic processes and have more lipophilicity. Limiting flexible conformation with a fused cyclopropane is a useful strategy to increase the potency and selectivity of a nucleoside.16,17,27 Herein, we present a full account of the stereoselective synthesis and the antiviral testing of several enantiomerically pure six-membered CNAs with five chiral centers starting from 1,4-cyclohexanedione. The known enantiomerically pure bicyclo[4.1.0]heptane alcohol under standard conditions, followed by ketal removal alcohol 4 was envisaged as a suitable starting material (Scheme under acidic conditions (4:1 mixture of TFA/H2O), afforded 1).26 The synthetic plan contemplated the preparation of the ketone 8 in 77% for the two steps. Reaction of ketone 8 with freshly prepared ylide generated from methyltriphenylphos- Scheme 1. Synthetic Approach Toward CNAs 1a,b and 3a−e phonium iodide (Ph3PCH3I) and t-BuOK afforded the corresponding terminal alkene 5, which was submitted without further purification to allylic oxidation. Accordingly, compound 5 was treated with catalytic SeO2 and stoichiometric tert-butyl hydroperoxide28,29 to furnish the corresponding allylic alcohol 9 as a single diastereoisomer in 79% yield from ketone 8. The excellent regio- and stereoselectivity furnished by this oxidation reaction can be rationalized by the generally accepted mechanism that implies two consecutive pericyclic reactions, an ene reaction followed by a [2,3]-sigmatropic rearrangement.30 The latter process occurs through the less hindered face delivering the anti-adduct. The anti-relative configuration was determined by a 2D NOESY experiment, where cross peaks between H-3 and H-7endo of the pivotal azide 6 from which thymine and guanine nucleobases cyclopropane revealed the cis relative disposition between H- would be stepwise constructed and the substituted 1,2,3- 3 and cyclopropane. The next step of our synthetic plan was the stereoselective triazoles would be assembled via cycloaddition. A major hydroxymethyl installation by a hydroboration-oxidation challenge of the synthetic approach would be the regio- and procedure. In a first attempt, hydroboration of 9 using BH3· stereoselective allylic hydroxylation and olefin hydration on THF followed by alkaline hydrogen peroxide workup furnished compound 5. a 5:1 mixture of diols 10 and 11 in 90% yield. However, when 15167 https://doi.org/10.1021/acs.joc.2c01661 J. Org. Chem. 2022, 87, 15166−15177 The Journal of Organic Chemistry pubs.acs.org/joc Article Scheme 3. Synthesis of Bicyclic Nucleoside Analogues 1a, 2a, and 19 the reaction was carried out using 9-BBN, a 20:1 mixture of sponding primary amine, which was isolated as the ammonium diols 10 and 11 was obtained in good yield (92%). The origin chloride salt 17. Then, the primary amine was treated with the of the remarkable stereoselectivity observed might be freshly prepared acyl isocyanate 18 at low temperature in basic rationalized by an initial coordination of the borane reagent media35,36 and the formed acryloyl urea was successively (9-BBN) to the secondary hydroxyl group. The resulting heated at reflux in ethanolic acid and treated with 7 M borinate ester shields the top face, favoring the hydroboration methanolic ammonia solution to furnish the pyrimidine NA 1a of the olefinic functionality from the bottom face by a second in 79% yield. This compound provided an X-ray crystal borane reagent. Thus, the mainly formed hydroxymethyl group structure that confirmed the stereostructure. To prepare was found at the top face of the ring.31 The hydrolysis of the epimeric analogue 2, the unprotected NA 1a was subjected borate intermediate in the oxidation step under basic to standard Mitsunobu conditions using benzoic acid followed conditions delivers mainly alcohol 10.32,33 Therefore, in this by subsequent alcohol deprotection with an ammonia solution reaction, more than two equivalents of the borane reagent were in methanol.17 These conditions provided the epimeric NA 2 employed. in 37% yield along with alkene 19 in 18% yield. The inversion A pure fraction of 10 was obtained after several purifications of the configuration at C-3′ was assessed by a NOESY by column chromatography from the mixture of diols. The experiment, which exhibited a strong cross peak between H-3′ configuration of the new stereogenic center was determined by and H-6 of the thymine base.37 the 2D NOESY experiment, which displays cross peaks The guanine base was also assembled from carbocyclic between H-2, H-7endo, and H-4ax disclosing the cis relation- amine 17 (Scheme 4). The purine ring was efficiently built ship between these three protons. The relative configuration (76%) through a two-step procedure by reacting 17 first with was further confirmed by the cleavage of the benzyl protecting the diformyl derivative 20 at the reflux temperature and then group to deliver the corresponding triol 12, which provided immediately cyclizing the transiently isolated intermediate in adequate crystals for X-ray diffraction analysis. the presence of diethoxymethyl acetate, 21, at 140 °C to give Next, reaction of the diastereomeric mixture of diols 10 and 11 with an excess of benzoyl chloride furnished a chromato- Scheme 4. Synthesis of Bicyclic Guanine Analogue 1b graphically separable mixture of 13 and 14 in 92 and 5% yields, respectively. Hydrogenation of the benzyl protecting group in 13 under palladium catalysis furnished alcohol 15 in quantitative yield. Finally, alcohol 15 was transformed into azide 6 in 64% yield following a Mitsunobu protocol34 using diphenylphosphoryl azide (DPPA) in the presence of di-tert- butyl azodicarboxylate (DBAD) and triphenylphosphine (PPh3). Along with the desired azide, the elimination product 16 was also obtained in low yield (5%). The configuration of C-5′ was determined by the presence of strong cross peaks between H-5′ and H-7′endo and H-4′ax and between H-5′ and H-2′ and H-3′ in the NOESY spectrum and was further confirmed by X-ray diffraction analysis. Therefore, the stereocontrolled preparation of the chiral cyclohexane scaffold bearing the five stereogenic centers has been successfully accomplished starting from 1,4-cyclohexanedione. With 6 in hand, we next examined the stepwise construction of the thymine and guanine nucleobases (Scheme 3). First, catalytic hydrogenation delivered quantitatively the corre- 15168 https://doi.org/10.1021/acs.joc.2c01661 J. Org. Chem. 2022, 87, 15166−15177 The Journal of Organic Chemistry pubs.acs.org/joc Article compound 22.38,39 Acidic hydrolysis under reflux followed by diversity, efforts were focused on the synthesis of a 5-amino- reaction with a 7 M solution of MeNH2 in EtOH afforded the 4-carboxamide-1,2,3-triazolo carbanucleoside 3b related to desired NA 1b in 57% overall yield. Unexpectedly, all attempts acadesine41 using a known cycloaddition reaction under to epimerize C-3′ of 1b using standard Mitsunobu conditions basic conditions.42−44 Accordingly, azide 6 was treated with were unsuccessful, resulting in degradation products only. 2-cyanoacetamide 25 in the presence of K2CO3 in DMSO at Our next effort focused on exploiting the common 50 °C to afford directly the 1,4,5-trisubstituted-triazolo intermediate 6 to build a substituted 1,2,3-triazolo moiety deprotected carbanucleoside 3b in moderate yield. (Schemes 5 and 6). First, the preparation of 1,2,3-triazole-4- Finally, to expand the collection of NAs bearing triazole rings, the syntheses of various 4-substituted triazole com- Scheme 5. Synthesis of Bicyclic 1,2,3-Triazolo pounds were undertaken starting from terminal alkynes 27c−e Carbanucleosides 3a and 3b via CuAAC (Scheme 6). First, to avoid the previously described solubility issues of 6, the benzoyl protecting groups were removed using an ammonia solution in methanol providing the deprotected azide 26 in 85% yield. Then, 26 was reacted with alkynes 27c−e using a Cu0/CuSO4 mixture in t-BuOH/H2O (1:1) media under microwave (MW) irradiation delivering the expected 4-substituted-1,2,3-triazole carbanu- cleosides 3c−e in good yields in short reaction times. The regioselectivity of the cycloaddition reactions (Schemes 5 and 6) was confirmed by HMBC experiments in compounds 3a−3e, which show cross peaks between H-1′ of the bicyclo[4.1.0]heptane moiety and C-5 of the 1,2,3-triazole ring. The antiviral activity against various viruses and the cytotoxicity of the synthesized compounds 1a,b, 2, 19, and 3a−e have been tested, as shown in the Supporting Information. While all the carbocyclic nucleosides did not display any significant antiviral activity, the 1,2,3-triazole analogue 3d exhibited moderate antiviral activity [EC50 9.4 Scheme 6. Synthesis of Bicyclic 1,2,3-Triazolo μg/mL, selectivity index (SI) = 8.4] against coxsackie B4 virus. Carbanucleosides 3c−3e The coxsackie virus causes a great variety of diseases such as myocarditis45 or pericarditis,46 or aseptic meningitis,47 and is one of the main enteroviruses causing the hand, foot, and mouth disease in children under the age of 10.48 Moreover, coxsackie B4 virus has been related to the infection of β cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.49 Precedents of active NAs against coxsackie B4, using ribavirin derivatives, showed similar antiviral activity to our compounds.50 Remarkably, a seminal work using neplanocin-related CNAs described higher activities against coxsackie B4.51 Interestingly, another precedent using CNAs pointed out that the use of cyclopentenyl derivatives showed higher activity against coxsackie B4 than the corresponding saturated counterparts.52 Recently, other non-CNAs have been described with important antiviral activity toward the closely related coxsackie B3 virus.53,54 In one of these works, the use of 1,2,3-triazole-based carboxamide ribavirin analogues was attempted via copper(I)- non-natural nucleobases proved to be effective to improve the catalyzed azide alkyne cycloaddition (CuAAC) using standard antiviral activity.54 However, in other cases, the use of non- conditions.40 Thus, azide 6 was reacted with methyl propiolate natural nucleobases in linear CNAs has not led to significant 23 in the presence of CuSO4·5H2O (10 mol %) and sodium antiviral activity against coxsackie B4 virus.55,56 Despite all ascorbate (20 mol %) in a 2:1 EtOH/H2O mixture overnight. these precedents, at present, there is no approved specific Unfortunately, only starting material was recovered, as the treatment for coxsackie virus infections. The preliminary in reaction did not work. The same result was obtained using vitro antiviral activity of the novel synthesized carbocyclic MeOH or t-BuOH. Alcohol deprotection to increase the nucleoside 3d opens the door to carry out additional studies of solubility of 6 was precluded due to the known conjugated structure−activity relationships as well as studies of the mode addition of alcohols on the methyl propiolate.40 After some of action. experimentation, it was found that using a 2:1 mixture of ACN/H2O, the cycloaddition proceeded readily to deliver the 4-metylcarboxylate triazole 24 in 79% yield. The latter was ■ CONCLUSIONS In summary, a highly stereoselective route to enantiomerically further easily transformed into the 1,2,3-triazole-4-carboxamide pure 4′-hydroxymethyl-3′-hydroxybicyclo[4.1.0]heptyl NAs carbanucleoside 3a by treatment with an ammonia solution in has been finely tuned, from a common azide derivative 6, methanol (Scheme 5). Next, to explore more structural which in turn derives from 1,4-cyclohexandione. Key achieve- 15169 https://doi.org/10.1021/acs.joc.2c01661 J. Org. Chem. 2022, 87, 15166−15177 The Journal of Organic Chemistry pubs.acs.org/joc Article ments in the asymmetric synthesis of these CNAs have been infected cell cultures that had not been treated with the test the highly diastereoselective allylic oxidation and hydro- compounds. boration reactions. Four of them, 1a, its epimer 2, the related Cytotoxic Assays. The cytotoxicity of the compounds was alkene derivative 19, and 1b, contain thymine and guanine, evaluated in parallel with their antiviral activity in uninfected cell cultures and is expressed as the 50% cytotoxic concentration (CC50) respectively, which have been constructed stepwise from 6. as determined by measuring the cell viability of normal cell The other five CNAs, 3a−3e, incorporate 1,2,3-triazoles as morphology (HEL, Hep-2, MDCK, and Vero cells) with the nucleobase and have been prepared from 6 in excellent yields colorimetric formazan-based MTS assay. via cycloadditions. It is noteworthy that, although most of the (1R,4′R,5R,5′R,6S)-5-(Benzyloxy)-4′,5′-diphenylspiro- compounds do not display relevant antiviral activity, [bicyclo[4.1.0]-heptane-2,2′-[1,3]dioxolane] (7). To an ice- compound 3d has shown interesting antiviral activity against cooled solution of alcohol 426 (3.02 g, 9.37 mmol) in anhydrous coxsackie B4 virus. Because to date there are no approved THF (90 mL), NaH (1.27 g, 31.8 mmol) was added in one portion treatments against this virus, this preliminary result prompts us and the mixture was stirred for 1 h at 0 °C. Then, benzyl bromide (1.3 to further study this privileged scaffold. mL, 11.24 mmol) and sodium iodide (1.73 g, 11.5 mmol) were added at 0 °C and the mixture was stirred at the reflux temperature for 3 h. ■ EXPERIMENTAL SECTION General Methods. Unless otherwise indicated, all reagents and After that time, water (60 mL) was carefully added at RT and stirred for 15 min. Then, EtOAc (30 mL) was added, and the aqueous layer was extracted with more EtOAc (2 × 30 mL). The organic layers were solvents were purchased from commercial sources and used directly as dried (Na2SO4), concentrated under reduced pressure, and purified received. Solvents were dried by distillation over the appropriate by column chromatography (SiO2, hexanes:EtOAc, 6:1) to furnish 7 drying agents. In the reaction mixtures that require heating, the (3.29 g, 7.96 mmol, 85% yield) as a pale oil. indicated temperatures refer to those of the used oil bath. All the 4. 1H NMR (400 MHz, CDCl3) δ 7.37−7.28 (m, 8H, H-Ar), reactions were monitored by analytical thin-layer chromatography 7.23−7.19 (m, 2H, H-Ar), 4.83 (d, J5′,4′ = 9.0 Hz, 1H, H-5′), 4.70 (d, (TLC) using silica gel 60 F254 pre-coated aluminum plates (0.25 mm J4′,5′ = 9.0 Hz, 1H, H-4′), 4.35 (q, J5,4ax = J5,4eq = J5,6 = 5.5 Hz, 1H, H- thickness). TLC spots were detected under UV light and/or by 5), 1.94−1.81 (m, 3H, 2H-3, H-4 eq), 1.70−1.55 (m, 2H, H-1, H-6), charring with a KMnO4/KOH aqueous solution or Vanillin solution. 1.56−1.41 (m, 1H, H-4ax), 0.96 (q, J7endo,1 = J7endo,6 = Jgem = 5.7 Hz, Flash column chromatography was performed using silica gel (230− 1H, H-7endo), 0.83 (td, J7exo,1 = J7exo,6 = 9.4 Hz, Jgem = 5.7 Hz, 1H, H- 400 mesh). 1H NMR spectra were recorded using 400 MHz and were 7exo). referenced to the residual proton signals of CDCl3, 7.26 ppm, and 7. Rf = 0.68 (hexanes:EtOAc, 1:1); [α]D20 + 45.6 (c 1.03, CHCl3); 1 MeOH-d4, 3.31 ppm. 13C[1H] NMR spectra were recorded at 100 H NMR (400 MHz, CDCl3) δ 7.46 (m, 3H, H-Ar), 7.43−7.34 (m, MHz and were referenced to the residual 13C signal of CDCl3, 77.16 8H, H-Ar), 7.32−7.28 (m, 4H, H-Ar), 4.94 (d, J5′,4′ = 8.4 Hz, 1H, H- ppm, and MeOH-d4, 49.00 ppm. Structural assignments were made 5′), 4.86−4.75 (m, 2H, H-4′, CH2-Ph), 4.59 (d, Jgem = 11.8 Hz, 1H, with additional information from gCOSY, gHSQC, and gHMBC CH2-Ph), 4.15 (q, J5,6 = J5,4 = 5.0 Hz, 1H, H-5), 1.99−1.80 (m, 4H, experiments. Melting points were determined on a hot stage and are H-4, H-3), 1.72−1.58 (m, 2H, H-6, H-1), 1.12 (q, Jgem = J7endo,1 = uncorrected. Optical rotations were measured at 20 ± 2 °C at the J7endo,6 = 5.7 Hz, 1H, H-7endo), 1.03 (td, J7exo,6 = J7exo,1 = 9.1 Hz, Jgem sodium D line (589 nm) in a microcell (0.1 dm). Infrared spectra = 5.7 Hz, 1H, H-7exo); 13C{1H} NMR (100 MHz, CDCl3) δ 139.1 were recorded on a spectrophotometer equipped with a Golden Gate (CAr), 137.1/136.9 (CAr), 128.5/128.5/128.4/128.3/127.8/127.5/ Single Refraction Diamond ATR (Attenuated Total Reflectance) 126.9/126.7 (CAr), 109.8 (C2), 85.6/85.3 (C4′,C5′), 70.7 (C5), 69.6 accessory. High-resolution mass spectra were recorded using electro- (CH2Ph), 30.2 (C3), 26.6 (C4), 22.4 (C1), 16.5 (C6), 5.5 (C7); IR spray ionization (ESI). (ATR) 3063, 2850, 2524, 2108, 1729, 1495, 1453, 1060, 697 cm−1; MW reactions were conducted on a CEM Discover Microwave HRMS (ESI+) m/z: [M + H]+ calcd for C28H29O3 413.2117; found synthesizer. The machine includes a continuous focused MW-power 413.2119. delivery system with operator-selectable power output from 0 to 300 (1R,5R,6S)-5-(Benzyloxy)bicyclo[4.1.0]heptan-2-one (8). To W. The temperature inside the vessel was monitored by a calibrated a stirred solution of ketal 7 (5.03 g, 12.19 mmol) in CH2Cl2 (120 infrared temperature control attached under the reaction vessel. All mL), a 4:1 mixture of TFA (2.8 mL, 36.57 mmol)/H2O (700 μL) was experiments were carried out under stirring by means of a rotating added at RT. The reaction mixture was allowed to stir for 2 h. Then, magnetic plate located below the floor of the MW cavity and a Teflon- additional CH2Cl2 (30 mL) was added, and the resulting solution was coated magnetic stir bar in the vessel. All the experiments were carried washed with saturated aqueous NaHCO3 solution (100 mL). The out in a sealed reaction vessel. organic layers were dried (Na2SO4), concentrated under reduced Evaluation of Antiviral Activity. Antiviral Activity Assays. The pressure, and purified by column chromatography (CHCl3 100%) to antiviral screening of compounds 1a, 1b, 2, 19, and 3a−e was provide 8 (2.401 g, 11.10 mmol, 91% yield) as a yellowish oil. Rf = performed against herpes simplex virus-1 (KOS) [HSV-1 KOS], 0.22 (CHCl3); [α]D20 + 62.6 (c 1.45, CHCl3); 1H NMR (400 MHz, herpes simplex virus-2 (G) [HSV-2 G], Vaccinia virus [VV], CDCl3) δ 7.39−7.34 (m, 4H, H-Ar), 7.32−7.28 (m, 1H, H-Ar), 4.73 adenovirus 2, and human coronavirus (229E) in HEL cell cultures; (d, Jgem = 11.9 Hz, 1H, CH2-Ph), 4.64 (d, Jgem = 11.9 Hz, 1H, CH2- vesicular stomatitis virus [VSV], coxsackie virus B4, and respiratory Ph), 4.15 (dt, J5,4ax = 9.3 Hz, J5,4eq = J5,6 = 4.9 Hz, 1H, H-5), 2.42 (dt, syncytial virus in Hep-2 cell cultures; Reovirus-1, Sindbis virus, Jgem = 17.7 Hz, J3eq,4 = 5.5 Hz, 1H, H-3 eq), 2.12 (ddd, Jgem = 17.7 Hz, coxsackie virus B4, Punta Toro virus, yellow fever virus, and Zika virus J3ax,4x = 10.7 Hz, J3ax,4eq = 6.5 Hz, 1H, H-3ax), 2.05−1.93 (m, 2H, H- in Vero cell cultures; Influenza A virus A/Ned/378/05A (H1N1), 4), 1.88−1.71 (m, 2H, H-1, H-6), 1.50 (q, Jgem = J7endo,6 = J7endo,1 = 5.4 Influenza A virus A/HK/7/87 (H3N2), and Influenza B virus B/ Hz, 1H, H-7endo), 1.24 (td, J7exo,6 = J7exo,1 = 9.1 Hz, Jgem = 5.4 Hz, Ned/537/05 in MDCK cell cultures; and human immunodeficiency 1H, H-7exo); 13C{1H} NMR (100 MHz, CDCl3) δ 208.2 (C2), 138.5 virus type 1 (HIV-1) (IIIB) and HIV-2 (ROD) in MT-4 cell cultures. (CAr), 128.6/128.2/127.9/127.8 (CAr), 71.3 (C5), 70.6 (CH2Ph), The results were expressed as the 50% effective concentration (EC50) 34.4 (C3), 26.5 (C4), 25.5 (C1), 21.3 (C6), 9.5 (C7); IR (ATR) or drug concentration required to inhibit virus-induced cytopathicity 3028, 2857, 1692, 1342, 1075, 1028, 881, 631 cm−1; HRMS (ESI+) by 50%. Read-out was through microscopical inspection or the MTS m/z: [M + H]+ calcd for C14H17O2 217.1229; found 217.1231. viability staining method. (1S,2R,6R)-2-(Benzyloxy)-5-methylidenebicyclo[4.1.0]- Confluent cell cultures in microtiter 96-well plates were inoculated heptane (5) and (1R,3R,5R,6S)-5-(benzyloxy)-2- with 100 CCID50 of the virus (one CCID50 being the dose of the methylidenebicyclo[4.1.0]heptan-3-ol (9). To a stirring solution virus sufficient to infect 50% of the cell cultures) in the presence of of Ph3PCH3I (11.82 g, 29.2 mmol) in anhydrous THF (30 mL) at 0 varying concentrations of the test compounds. Viral cytopathicity was °C, t-BuOK (3.31 g, 29.5 mmol) was added, under a nitrogen recorded as soon as it reached completion in the control virus- atmosphere, and the resulting yellow mixture was allowed to react for 15170 https://doi.org/10.1021/acs.joc.2c01661 J. Org. Chem. 2022, 87, 15166−15177 The Journal of Organic Chemistry pubs.acs.org/joc Article 1 h. At this time, a solution of ketone 8 (1.27 g, 5.8 mmol) in H-4 eq), 1.76 (tt, J2,1′ = 6.2 Hz, J2,1 = J2,3 = 2.8 Hz, 1H, H-2), 1.39 (tt, anhydrous THF (10 mL) was added and the mixture was stirred for 3 J6,7exo = J6,1 = 8.8 Hz, J6,7endo = J6,5 = 5.9 Hz, 1H, H-6), 1.18 (ddd, Jgem h. Then, diethyl ether (30 mL) was added, and the crude filtered = 13.8 Hz, J4ax,5 = 9.5 Hz, J4x,3 = 1.8 Hz, 1H, H-4ax), 0.93 (tdd, J1,7exo through a pad of silica and Celite, using more diethyl ether (100 mL) = J1,6 = 8.8 Hz, J1,7endo = 5.5 Hz, J1,2 = 2.8 Hz, 1H, H-1), 0.81 (td, as eluent. The volatiles were removed under vacuum to obtain 5 as an J7exo,6 = J7exo,1 = 8.8 Hz, Jgem = 4.9 Hz, 1H, H-7exo), 0.39 (q, Jgem = orange oil, which is unstable was directly used for the next step J7endo,6 = J7endo,1 = 5.5 Hz, 1H, H-7endo); 13C{1H} NMR (100 MHz, without further purification. Accordingly, crude alkene 5 was rapidly CDCl3) δ 139.0 (Cipso), 128.5/127.9/127.6 (CAr), 70.7 (C5), 69.9 dissolved in CH2Cl2 (75 mL) and SeO2 (128 mg, 1.16 mmol) and t- (CH2Ph), 69.0 (C3), 65.2 (C1′), 41.2 (C2), 33.0 (C4), 13.8 (C6), BuOOH (440 μL, 6.38 mmol, 70% in water) were sequentially added 13.0 (C1), 8.6 (C7); IR (ATR) 3350, 2872, 2365, 1497, 1454, 1068, at RT. After stirring for 20 min, water (50 mL) was added, and the 739 cm−1; HRMS (ESI+) m/z: [M + H]+ calcd for C15H21O3 aqueous phase was extracted with more CH2Cl2 (2 × 30 mL). Finally, 249.1491; found 249.1466. the organic layers were dried with anhydrous Na2SO4, concentrated (1S,2R,4R,5R,6R)-5-(Hydroxymethyl)bicyclo[4.1.0]heptane- under reduced pressure, and purified by column chromatography 2,4-diol (12). A stirred solution of a 20:1 mixture of diols 10 and 11 (hexanes:EtOAc, 5:1) to provide allylic alcohol 9 (1.05 g, 4.55 mmol, (101 mg, 0.40 mmol) in EtOH (4 mL) at RT was hydrogenated in 79% overall yield from 8) as brown oil. the presence of 10% Pd/C (10 mg, 10 wt %) at 2 atm for 24 h. Then, An aliquot of compound 5 was purified by column chromatography the mixture was filtered through a short pad of Celite and rinsed with (hexanes:EtOAc, 5:1); however, due to its instability, the obtained more EtOH. The solvent was evaporated under reduced pressure to HRMS analysis was not within the 10 ppm acceptable error: Rf = 0.75 give alcohol 12 (63 mg, 0.39 mmol, 95% yield) as a white solid, which (hexanes:EtOAc, 2:1); [α]D20 = +47.6 (c 0.98, CHCl3); 1H NMR was re-crystallized by vapor diffusion using MeOH/Et2O at RT Mp (400 MHz, CDCl3) δ 7.46−7.25 (m, 5H, H-Ar), 4.90 (br s, 1H, H- 166−167 °C (from MeOH−Et2O); [α]D20−52 (c 1.3, CHCl3); 1H 1′), 4.79 (br s, 1H, H-1′), 4.74 (d, Jgem = 11.9 Hz, 1H, CH2-Ph), 4.56 NMR (400 MHz, CDCl3) δ 4.47 (dt, J2,3ax = 10.8 Hz, J2,1 = J2,3eq = 5.8 (d, Jgem = 11.9 Hz, 1H, CH2-Ph), 4.05 (q, J2,3ax = J2,3eq = J2,1 = 5.9 Hz, Hz, 1H, H-2), 3.93 (br t, J4,3eq = J4,3eq = 4.3 Hz, 1H, H-4), 3.72 (dd, 1H, H-2), 2.21 (dddt, Jgem = 14.9 Hz, J4ax,3ax = 8.5 Hz, J4ax,3eq = 4.4 Hz, Jgem = 10.6 Hz, J1′a,5 = 7.0 Hz, 1H, H-1′a), 3.65 (dd, Jgem = 10.6 Hz, J4ax,1′ = 1.4 Hz, 1H, H-4ax), 2.03 (dddt, Jgem = 14.9 Hz, J4eq,3ax = 7.2 J1′b,5 = 7.0 Hz, 1H, H-1′b), 1.95 (dt, Jgem = 13.3 Hz, J3eq,2 = J3eq,4 = 5.8 Hz, J4eq,3eq = 4.3 Hz, J4eq,1′ = 1.4 Hz, 1H, H-4 eq), 1.88−1.75 (m, 1H, Hz, 1H, H-3 eq), 1.58 (tt, J5,1′a = J5,1′b = 7.0 Hz, J5,6 = J5,4 = 3.0 Hz, H-6), 1.71−1.50 (m, 3H, H-1, H-3), 0.94−0.84 (m, 2H, H-7); 1H, H-5), 1.28 (tt, J1,7exo = J1,6 = 8.8 Hz, J1,7endo = J1,4 = 5.8 Hz, 1H, H- 13 C{1H} NMR (100 MHz, CDCl3) δ 146.3 (C5), 139.1 (Cipso), 1), 1.00 (ddd, Jgem = 13.3 Hz, J3ax,2 = 10.8 Hz, J3ax,4 = 1.6 Hz, 1H, H- 128.4/127.7/127.5 (CAr), 108.1 (C1′), 72.0 (C2), 69.7 (CH2Ph), 3ax), 0.83 (tdd, J6,7exo = J6,1 = 8.8 Hz, J6,7endo = 5.3 Hz, J6,5 = 2.4 Hz, 28.8 (C4), 27.7 (C3), 19.9 (C1), 17.0 (C6), 8.7 (C7); IR (ATR) 1H, H-6), 0.73 (td, J7exo,1 = J7exo,6 = 8.8 Hz, Jgem = 4.9 Hz, 1H, H- 3070, 2930, 1471, 1427, 1106, 806, 741 cm−1; HRMS (ESI+) m/z: 7exo), 0.27 (q, Jgem = J7endo,6 = J7endo,1 = 5.3 Hz, 1H, H-7endo); [M + H]+ calcd for C15H19O 215.1436; found 215.1465. 13 C{1H} NMR (100 MHz, CDCl3) δ 68.4 (C4), 65.0 (C1′), 64.7 9. Rf = 0.35 (hexanes:EtOAc, 2:1); [α]D20 + 43 (c 1.1, CHCl3); 1H (C2), 43.9 (C5), 36.0 (C3), 17.9 (C1), 14.0 (C6), 9.6 (C7); IR NMR (400 MHz, CDCl3) δ 7.39−7.32 (m, 4H, H-Ar), 7.30−7.27 (m, (ATR) 3324, 3239, 3019, 2925, 1405, 1250, 1040, 906 cm−1; HRMS 1H, H-Ar), 5.06 (d, Jgem = 10.0 Hz, 2H, H-1′), 4.71 (d, Jgem = 11.8 Hz, (ESI-) m/z: [M + HCOO]− calcd for C9H15O5 203.0919; found 1H, CH2-Ph), 4.52 (d, Jgem = 11.8 Hz, 1H, CH2-Ph), 4.29 (q, J5,6 = 203.0919. J5,4eq = J5,4ax = 6.0 Hz, 1H, H-5), 4.22 (dd, J3,4ax = 8.3 Hz, J3,4eq = 3.2 [(1′R,2′R,3′R,5′R,6′S)-3′-(Benzoyloxy)-5′-(benzyloxy)- Hz, 1H, H-3), 1.87 (ddd, J1,7exo = 9.3 Hz, J1,6 = 7.8 Hz, J1,7endo = 5.0 bicyclo[4.1.0]hept-2′-yl]methyl benzoate (13) and Its (2′S)- Hz, 1H, H-1), 1.77 (ddd, Jgem = 13.5 Hz, J4ax,3 = 8.3 Hz, J4ax,5 = 6.0 Hz, diastereoisomer (14). To a 20:1 mixture of diols 10 and 11 (1.079 1H, H-4ax), 1.70 (ddd, Jgem = 13.5 Hz, J4eq,5 = 6.0 Hz, J4eq,3 = 3.2 Hz, g, 4.35 mmol) in dry pyridine (70 mL), benzoyl chloride (1.76 mL, 1H, H-4 eq), 1.57 (m, 1H, H-6), 0.90 (td, J7exo,6 = J7exo,1 = 9.3 Hz, Jgem 15.15 mmol) was added dropwise at RT. The solution was heated to = 5.0 Hz, 1H, H-7exo), 0.76 (q, Jgem = J7endo,6 = J7endo,1 = 5.0 Hz, 1H, reflux in an oil bath for 2 h. Then, the solution was cooled to 70 °C H-7endo); 13C{1H} NMR (100 MHz, CDCl3) δ 148.6 (C2), 138.8 and quenched with MeOH (6 mL). After 30 min, the solvent was (Cipso), 128.4 (CAr), 127.7 (CAr), 127.5 (CAr), 109.2 (C1′), 70.8 removed under reduced pressure and the residue was partitioned (C5), 69.8 (CH2Ph), 67.6 (C3), 37.0 (C4), 19.9 (C1), 16.4 (C6), 9.0 between EtOAc (100 mL) and water (100 mL). The organic layer (C7); IR (ATR) 3399, 3065, 2857, 1640, 1454, 1200, 1066, 824, 698 was washed with 1 M HCl (2 × 70 mL) and aqueous saturated cm−1; HRMS (ESI+) m/z: [M + H]+ calcd for C15H19O2 231.1385; NaHCO3 solution (2 × 70 mL) and dried with Na2SO4. The filtrate found 231.1375. was concentrated under reduced pressure, and the residue was (1R,2R,3R,5R,6S)-5-(Benzyloxy)-2-(hydroxymethyl)bicyclo- [4.1.0]heptan-3-ol (10) and Its (2S)-Diastereoisomer (11). To a purified by column chromatography (hexanes:EtOAc, 10:1 → 8:1 → stirred solution of allylic alcohol 9 (1.750 g, 7.60 mmol) in anhydrous 5:1) to afford alcohols 13 (1.826 g, 4.00 mmol, 92% yield) as a clear THF (80 mL), 9-borabicyclo[3.3.1]nonane solution (9-BBN, 38 mL, oil and 14 (98 mg, 0.218 mmol, 5% yield) also as a clear oil. 37.99 mmol, 0.5 M in THF) was added at −10 °C. The mixture was 13. Rf = 0.25 (hexanes:EtOAc, 5:1); [α]D20 + 16.9 (c 2.13, allowed to slowly warm to RT and stirred overnight. Then, water (18 CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.06−7.98 (m, 4H, H-Ar), mL), NaOH (35 mL, 3 M in water), and H2O2 (35 mL, 30% in 7.63−7.53 (m, 2H, H-Ar), 7.45 (m, 4H, H-Ar), 7.40−7.24 (m, 5H, H- water) were added at 0 °C. After stirring for 15 min at RT, the Ar), 5.50−5.44 (m, 1H, H-3′), 4.74 (d, Jgem = 11.5 Hz, 1H, CH2-Ph), mixture was diluted with brine (150 mL) and CH2Cl2 (150 mL) and 4.63−4.52 (m, 2H, CH2-Ph, H-1), 4.37 (dd, Jgem = 10.8 Hz, J1,2′ = 8.3 the aqueous phase was extracted with CH2Cl2 (2 × 100 mL). The Hz, 1H, H-1), 4.29 (dt, J5′,4′ax = 10.0 Hz, J5′,4′eq = J5′,6′ = 6.0 Hz, 1H, organic layers were dried (Na2SO4), concentrated under reduced H-5′), 2.46 (dt, Jgem = 14.1 Hz, J4′eq,5′ = J4′eq,3′ = 6.0 Hz, 1H, H-4′eq), pressure, and purified by column chromatography (CH2Cl2 100% to 2.34 (ddd, J2′,1 = 8.3 Hz, J2′,1 = 6.3 Hz, J2′,3′ = 3.3 Hz, 1H, H-2′), 1.56 CH2Cl2:MeOH 20:1 to 10:1) to provide a mixture of alcohols 10 and (tt, J6′,1′ = J6′,7′exo = 8.7 Hz, J6′,7′endo = J6′,5′ = 5.8 Hz, 1H, H-6′), 1.35 11 (1.724 g, 6.94 mmol, 92% overall yield, 2 steps) in a ca. 20:1 (ddd, Jgem = 14.1 Hz, J4′ax,5′ = 10.1 Hz, J4′ax,3′ = 1.7 Hz, 1H, H-4′ax), diastereomeric ratio as a white solid. After repeated purification by 1.11 (tdd, J1′,7′exo = J1′,6′ = 8.7 Hz, J1′,7′endo = 5.5 Hz, J1′,2′ = 2.4 Hz, 1H, column chromatography, it was possible to obtain a pure fraction of H-1′), 0.97 (td, J7′exo,1′ = J7′exo,6′ = 8.7 Hz, Jgem = 5.1 Hz, 1H, H-7′exo), 10: Rf = 0.1 (hexanes:EtOAc, 1:1); Mp 73−75 °C (from Et2O); 0.59 (q, Jgem = J7′endo,1′ = J7′endo,6′ = 5.5 Hz, 1H, H-7′endo); 13C{1H} [α]D20 + 104 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.39− NMR (100 MHz, CDCl3) δ 166.6 (CO), 165.8 (CO), 138.8/133.1/ 7.30 (m, 4H, H-Ar), 7.29−7.24 (m, 1H, H-Ar), 4.73 (d, Jgem = 11.8 130.3/130.1/129.74/129.67/128.6/128.50/128.48/127.9/127.6 Hz, 1H, CH2-Ph), 4.55 (d, Jgem = 11.8 Hz, 1H, CH2-Ph), 4.30 (dt, (CAr), 70.7 (C5′), 70.22 (C3′), 70.17 (CH2Ph), 65.2 (C1), 38.7 J5,4ax = 9.5 Hz, J5,4eq = J5,6 = 6.1 Hz, 1H, H-5), 4.08 (br m, 1H, H-3), (C2′), 30.0 (C4′), 14.3 (C6′), 13.5 (C1′), 9.1 (C7′); IR (ATR) 3066, 3.91−3.80 (m, 2H, H-1′), 2.60 (t, J = 5.2 Hz, 1H, OH), 2.47 (d, J = 3017, 1714, 1602, 1585, 1451, 1268, 1215, 747 cm−1; HRMS (ESI+) 3.8 Hz, 1H, OH), 2.06 (dt, Jgem = 13.8 Hz, J4eq,5 = J4eq,3 = 6.1 Hz, 1H, m/z: [M + H]+ calcd for C29H29O5 457.2015; found 457.2015. 15171 https://doi.org/10.1021/acs.joc.2c01661 J. Org. Chem. 2022, 87, 15166−15177 The Journal of Organic Chemistry pubs.acs.org/joc Article 14. Rf = 0.31 (hexanes:EtOAc 5:1); [α]D20 + 5.42 (c 1.55, CHCl3); 4′ax), 1.30 (td, J6′,7′exo = J6′,1′ = 9.0 Hz, J6′,7′endo = 5.3 Hz, 1H, H-6′), 1 H NMR (400 MHz, CDCl3) δ 8.10−8.00 (m, 4H, H-Ar), 7.61−7.52 1.12 (td, J1′,7′exo = J1′,6′ = 9.0 Hz, J1′,7′endo = 5.3 Hz, 1H, H-1′), 1.04 (td, (m, 2H, H-Ar), 7.47−7.36 (m, 8H, H-Ar), 7.35−7.29 (m, 1H, H-Ar), J7′endo,6 = J7′endo,1 = 9.0 Hz, Jgem = 5.1 Hz, 1H, H-7′exo), 0.23 (q, Jgem = 5.24 (ddd, J3′,2′ = 10.2 Hz, J3′,4′eq = 7.7 Hz, J3′,4′ax = 3.1 Hz, 1H, H-3′), J7′endo,6′ = J7′endo,1′ = 5.3 Hz, 1H, H-7′endo); 13C{1H} NMR (100 4.76 (d, Jgem = 11.9 Hz, 1H, CH2-Ph), 4.58 (d, Jgem = 11.9 Hz, 1H, MHz, CDCl3) δ 166.6 (CO), 166.2 (CO), 133.2 (2C, Cpara), 130.3 CH2-Ph), 4.50 (dd, Jgem = 11.1 Hz, J1,2′ = 6.3 Hz, 1H, H-1), 4.45 (dd, (Cipso), 130.0 (Cipso), 129.8 (Corto), 129.7 (Corto), 128.52 Jgem = 11.1 Hz, J1,2′ = 6.5 Hz, 1H, H-1), 4.33 (dt, J5′,4′ax = 7.3 Hz, (Cmeta), 128.50 (Cmeta), 66.6 (C3′), 65.2 (C1), 55.2 (C5′), 38.3 J5′,4′eq = J5′,6′ = 5.3 Hz, 1H, H-5′), 2.73−2.66 (m, 1H, H-2′), 1.98 (C2′), 29.2 (C4′), 14.1 (C6′), 11.3 (C1′), 11.0 (C7′); IR (ATR) (ddd, Jgem = 13.6 Hz, J4′ax,5′ = 7.3 Hz, J4′ax,3′ = 3.1 Hz, 1H, H-4′ax), 2922, 2109, 1709, 1599, 1449, 1267, 1248 cm−1; HRMS (ESI+) m/z: 1.88 (ddd, Jgem = 13.6 Hz, J4′eq,3′ = 7.7 Hz, J4′eq,5′ = 5.3 Hz, 1H, H- [M + H]+ calcd for C22H22N3O4 392.1610; found 392.1607. 4′eq), 1.53−1.46 (m, 2H, H-1′, H-6′), 0.79 (q, Jgem = J7′endo,1′ = 16. Rf = 0.44 (hexanes:EtOAc, 3:1); [α]D20−35.6 (c 1.01, CHCl3); 1 J7′endo,6′ = 5.6 Hz, 1H, H-7′endo), 0.72 (td, J7′exo,6′ = J7′exo,1′ = 9.0 Hz, H NMR (400 MHz, CDCl3) δ 8.06−8.01 (m, 2H, H-orto), 7.94− Jgem = 5.4 Hz, 1H, H-7′exo); 13C{1H} NMR (100 MHz, CDCl3) δ 7.87 (m, 2H, H-orto), 7.53−7.44 (m, 2H, H-para), 7.40−7.29 (m, 4H, 166.6 (CO), 165.8 (CO), 138.7/133.0/132.9/130.4/130.2/129.7/ H-meta), 6.27 (ddd, J5′,4′ = 10.1 Hz, J5′,6′ = 4.7 Hz, J5′,3′ = 2.4 Hz, 1H, 128.4/128.4/127.7/127.5 (CAr), 70.7 (C5′), 69.7 (C3′), 69.6 H-5′), 5.51 (dt, J3′,2′ = 6.8 Hz, J3′,4′ = J3′,5′ = 2.4 Hz, 1H, H-3′), 5.47 (CH2Ph), 66.1 (C1), 37.7 (C2′), 33.4 (C4′), 15.5 (C6′), 13.4 (dd, J4′,5′ = 10.2 Hz, J4′,3′ = 2.0 Hz, 1H, H-4′), 4.57 (dd, Jgem = 11.0 (C1′), 3.5 (C7′); IR (ATR) 3065, 3017, 1716, 1605, 1585, 1453, Hz, J1a,2′ = 5.7 Hz, 1H, H-1a), 4.31 (dd, Jgem = 11.0 Hz, J1b,2′ = 7.0 Hz, 1272, 1211, 747 cm−1. HRMS (ESI+) m/z: [M + Na]+ calcd for 1H, H-1b), 3.10 (qd, J2′,3′ = J2′1a = J2′,1b = 6.7 Hz, J2′,1′ = 2.5 Hz, 1H, C29H28O5Na 479.1834; found 479.1832. H-2′), 1.49 (tdd, J1′,7′exo = J1′,6′ = 8.5 Hz, J1′,7′endo = 5.7 Hz, J1′,2′ = 2.5 [(1′R,2′R,3′R,5′R,6′S)-3′-(Benzoyloxy)-5′-hydroxybicyclo- Hz, 1H, H-1′), 1.42 (tt, J6′,7′exo = J6′,1′ = 8.5 Hz, J6′,7′endo = J6′,5′ = 4.7 [4.1.0]hept-2′-yl]methyl Benzoate (15). A stirred solution of 13 Hz, 1H, H-6′), 1.03 (td, J7′exo,6′ = J7′exo,1′ = 8.5 Hz, Jgem = 4.7 Hz, 1H, (1.872 g, 4.10 mmol) in EtOH (41 mL) at RT was hydrogenated in H-7′exo), 0.68 (dt, J7′endo,1′ = 5.7 Hz, Jgem = J7′endo,6′ = 4.7 Hz, 1H, H- the presence of 10% Pd/C (190 mg, 10 wt %) at 2 atm for 24 h. 7′endo); 13C{1H} NMR (100 MHz, CDCl3) δ 166.8 (CO), 166.0 Then, the mixture was filtered through a short pad of Celite and (CO), 133.2 (Cpara), 132.9 (Cpara), 132.3 (C5′), 130.2 (Cipso), rinsed with EtOH. The solvent was evaporated under reduced 130.2 (Cipso), 129.8 (Corto), 129.6 (Corto), 128.5 (Cmeta), 128.3 pressure to give alcohol 15 (1.501 g, 4.08 mmol, 100% yield) as a (Cmeta), 122.1 (C4′), 68.3 (C3′), 65.7 (C1), 33.0 (C2′), 13.7 (C1′), colorless syrup. Rf = 0.28 (hexanes:EtOAc, 1:1); [α]D20−25.0 (c 1.45, 13.2 (C7′), 9.7 (C6′); IR (ATR) 3063, 2922, 1712, 1601, 1265, 1109, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.03−7.98 (m, 4H, H-orto), 1025, 707 cm−1; HRMS (ESI+) m/z: [M + Na]+ calcd for 7.60−7.49 (m, 2H, H-para), 7.47−7.37 (m, 4H, H-meta), 5.43 (br t, C22H20O4Na 371.1254; found 371.1247. J3′,4′ax = J3′,4′eq = J3′,2′ = 4.3 Hz, 1H, H-3′), 4.57 (dd, Jgem = 10.8 Hz, [(1′R,2′R,3′R,5′S,6′S)-5′-Amino-3′-(benzoyloxy)bicyclo- J1,2′ = 6.7 Hz, 1H, H-1), 4.50 (dt, J5′,4′ax = 11.2 Hz, J5′,4′eq = J5′,6′ = 5.8 [4.1.0]hept-2′-yl]methyl Benzoate Hydrochloride (17). A Hz, 1H, H-5′), 4.33 (dd, Jgem = 10.8 Hz, J1,2′ = 8.8 Hz, 1H, H-1), 2.39 stirred solution of azide 6 (615 mg, 1.57 mmol) in EtOAc (16 mL) (dt, Jgem = 12.4 Hz, J4′eq,5′ = J4′eq,3′ = 5.8 Hz, 1H, H-4′eq), 2.26 (ddt, was hydrogenated in the presence of Pd/C (61.5 mg, 10 wt %) at 2 J2′,1 = 8.9 Hz, J2′,1 = 6.7 Hz, J2′,3′ = J2′1′ = 4.5 Hz, 1H, H-2′), 1.63 (s, atm for 24 h at RT. Then, the mixture was filtered through a short pad 1H, OH), 1.58−1.52 (m, 1H, H-6′), 1.28−1.14 (m, 1H, H-4′ax), 1.08 of Celite and rinsed with more EtOAc. The solvent was evaporated (tt, J1′,7′exo = J1′,6′ = 8.5 Hz, J1′,7′endo = J1′,2′ = 4.5 Hz, 1H, H-1′), 0.89 under reduced pressure, and the crude was treated with 2 M HCl· (td, J7′exo,1′ = J7′exo,6′ = 8.5 Hz, Jgem = 5.4 Hz, 1H, H-7′exo), 0.45 (q, Et2O (1 mL, 2 mmol) at 0 °C and stirred for 15 min. The suspension Jgem = J7′endo,6′ = J7′endo,1′ = 5.4 Hz, 1H, H-7′endo); 13C{1H} NMR was filtered to furnish a white solid identified as the ammonium salt (100 MHz, CDCl3) δ 166.5 (CO), 165.8 (CO), 133.2 (Cpara), 133.1 17 (618 mg, 1.54 mmol, 98% yield). Mp >210−215 °C (Cpara), 130.2 (Cipso), 130.0 (Cipso), 129.7 (Corto), 129.6 (Corto), (decomposes) (from Et2O); [α]D20-33.5 (c 0.82, CHCl3); 1H NMR 128.6 (Cmeta), 128.5 (Cmeta), 70.4 (C3′), 65.2 (C1), 63.9 (C5′), (400 MHz, MeOH-d4) δ 8.05 (d, Jorto,meta = 7.4 Hz, 2H, H-orto), 7.92 38.5 (C2′), 32.4 (C4′), 17.4 (C6′), 13.5 (C1′), 8.6 (C7′); IR (ATR) (d, Jorto,meta = 7.8 Hz, 2H, H-orto), 7.65−7.52 (m, 2H, H-para), 7.50− 3489, 3005, 2953, 2338, 1713, 1601, 1450, 1265, 1108, 708 cm−1; 7.36 (m, 4H, H-meta), 5.43 (dt, J3′,4′ax = 6.3 Hz, J3′,4′eq = J3′,2′ = 4.3 Hz, HRMS (ESI+) m/z: [M + Na]+ calcd for C22H22O5Na 389.1365; 1H, H-3′), 4.63−4.56 (m, 2H, H-1), 3.61 (br t, J5′,4′eq = J5′4′ax = 6.4 found 389.1363. Hz, 1H, H-5′), 2.49 (tt, J2′,1 = 7.7 Hz, J2′,3′ = J2′,1′ = 4.0 Hz, 1H, H-2′), [(1′R,2′R,3′R,5′S,6′S)-5′-Azido-3′-(benzoyloxy)bicyclo- 2.19 (dt, Jgem = 14.7 Hz, J4′eq,3′ = J4′eq,5 = 4.9 Hz, 1H, H-4′eq), 1.99 [4.1.0]hept-2-yl]methyl Benzoate (6) and [(1′R,2′R,3′R,6′R)-3′- (dt, Jgem = 14.7 Hz, J4′ax,3′ = J4′ax,5′ = 6.3 Hz, 1H, H-4′ax), 1.33−1.19 (Benzoyloxy)bicyclo[4.1.0]hept-4′-en-2′-yl]methyl Benzoate (m, 2H, H-1′,H-6′), 1.11 (td, J7′endo,6 = J7′endo,1 = 8.9 Hz, Jgem = 5.4 Hz, (16). To a stirred solution of Ph3P (1.25 g, 4.76 mmol) in dry 1H, H-7′exo), 0.52 (q, Jgem = J7′endo,6′ = J7′endo,1′ = 5.4 Hz, 1H, H- toluene (35 mL), DBAD (1.10 g, 4.76 mmol) was slowly added under 7′endo); 13C{1H} NMR (100 MHz, MeOH-d4) δ 167.7 (CO), 167.2 an argon atmosphere and the mixture was stirred for 45 min at 0 °C (CO), 134.6 (Cpara), 134.3 (Cpara), 131.1 (Cipso), 131.0 (Cipso), (after 15 min, a white suspension appeared). Then, DPPA (720 μL, 130.7 (Corto), 130.5 (Corto), 129.7 (Cmeta), 129.5 (Cmeta), 69.0 3.33 mmol) and a solution of 15 (1.161 g, 3.17 mmol) in dry toluene (C3′), 65.9 (C1), 47.6 (C5′), 38.3 (C2′), 30.6 (C4′), 13.9 (C6′), (15 mL) were sequentially added at −10 °C. The mixture was allowed 12.3 (C1′), 11.3 (C7′); IR (ATR) 3048, 2922, 1714, 1615, 1514, to slowly warm to RT and stirred overnight. Then, the solvent was 1269, 1099, 708 cm−1; HRMS (ESI+) m/z: [M]+ calcd for removed, and the crude was purified by column chromatography C22H24NO4 366.1705; found 366.1703. (hexanes:EtOAc, 30:1 → 20:1 → 15:1) to provide azide 6 (794 mg, 1-((1′S,2′S,4′R,5′R,6′R)-4′-Hydroxy-5′-(hydroxymethyl) 2.03 mmol, 64% yield) as a white solid, which was re-crystallized by bicyclo[4.1.0]heptan-2′-yl)-5-methylpyrimidine-2,4(1H,3H)- vapor diffusion using MeOH/Et2O at RT, and the elimination dione (1a). To a suspension of silver cyanate (235 mg, 1.57 mmol), product 16 (98 mg, 0.28 mmol, 9% yield) as a colorless syrup. previously dried over phosphorus pentoxide at 80 °C in a laboratory 6. Rf = 0.31 (hexanes:EtOAc, 3:1); Mp 109−108 °C (from oven for 3 h, in dry toluene (3 mL), a solution of (E)-3-ethoxy-2- MeOH−Et2O); [α]D20−73.9 (c 0.98, CHCl3); 1H NMR (400 MHz, methylacryloyl chloride (196 mg, 1.32 mmol) was added dropwise in CDCl3) δ 8.08 (d, Jorto,meta = 7.5 Hz, 2H, H-orto), 8.00 (d, Jorto,meta = dry toluene (0.8 mL). The heterogeneous mixture was refluxed in an 7.5 Hz, 2H, H-orto), 7.58−7.52 (m, 2H, H-para), 7.42 (m, 4H, H- oil bath under an argon atmosphere for 1.5 h before allowing it to cool meta), 5.25 (ddd, J3′,4′eq = 6.5 Hz, J3′,2′ = 4.1 Hz, J3′,4ax = 2.8 Hz, 1H, to RT. The precipitate was allowed to settle, and the supernatant was H-3′), 4.62 (dd, Jgem = 10.9 Hz, J1,2′ = 7.2 Hz, 1H, H-1), 4.48 (dd, Jgem transferred via a cannula to a dry Schlenk flask. The precipitate was = 10.9 Hz, J1,2′ = 7.2 Hz, 1H, H-1), 4.02 (td, J5′,4ax = J5′,4eq = 5.4 Hz, further washed with a small quantity of dry CH2Cl2 (1 mL) and J5′,6′ = 1.5 Hz, 1H, H-5′), 2.33 (tt, J2′,1 = 7.2 Hz, J2′,3′ = J2′1′ = 3.4 Hz, transferred to the same flask to deliver a solution of the isocyanate 18. 1H, H-2′), 2.23 (dt, Jgem = 15.0, J4′eq,5′ = J4′eq,3′ = 5.4 Hz, 1H, H-4′eq), This solution was cooled to −78 °C, and a solution of the ammonium 1.71 (ddd, Jgem = 15.0 Hz, J4′ax,5′ = 5.4 Hz, J4′ax,3′ = 2.8 Hz, 1H, H- salt 17 (175 mg, 0.44 mmol) with Et3N (65 μL, 0.47 mmol) in dry 15172 https://doi.org/10.1021/acs.joc.2c01661 J. Org. Chem. 2022, 87, 15166−15177 The Journal of Organic Chemistry pubs.acs.org/joc Article CH2Cl2 (1.5 mL) was added dropwise over 3 min. The solution was 1H, H-1″a), 4.16−4.07 (m, 1H, H-1″b), 2.28 (ddd, Jgem = 18.2 Hz, allowed to warm slowly to RT and stirred overnight (16 h). EtOH (3 J3′ax,2′ = 6.9 Hz, J3′ax,4′ = 2.4 Hz, 1H, H-3′ax), 2.18 (ddd, Jgem = 18.2 mL) was added, and the reaction mixture was concentrated in vacuo. Hz, J3′eq,4′ = 5.5 Hz, J3′eq,2′ = 2.1 Hz, 1H, H-3′eq), 1.84 (d, JCH3,6 = 1.2 To the crude residue, EtOH (4 mL) and 2 M HCl (1.25 mL, 2.5 Hz, 3H, CH3), 1.72 (td, J6′,7exo = J6′,1′ = 8.7 Hz, J6′,7′endo = 4.8 Hz, 1H, mmol) were added. The reaction mixture was refluxed overnight in an H-6′), 1.50 (tdd, J1′,7exo = J1′,6′ = 8.7 Hz, J1′,7′endo = 4.8 Hz, J1′,2′ = 2.1 oil bath (20 h) then cooled to RT, and the solution was concentrated Hz, 1H, H-1′), 1.17 (td, J7′exo,6′ = J7′exo,1′ = 8.7 Hz, Jgem = 4.8 Hz, 1H, in vacuo. The residue was dissolved in a 33% solution of methylamine H-7′exo), 0.86 (q, Jgem = J7′endo,6′ = J7′endo,1′ = 4.8 Hz, 1H, H-7′endo); 13 in EtOH (50 mL) in a sealed flask and stirred for 48 h at RT. Then, C{1H} NMR (100 MHz, MeOH-d4) δ 166.4 (C4), 153.0 (C2), the mixture was concentrated under reduced pressure and purified by 143.0 (C5′), 140.6 (C6), 114.2 (C4′), 110.4 (C5), 66.5 (C1″), 47.6 column chromatography (CH2Cl2:MeOH, 20:1 → 15:1) to provide (C2′), 27.3 (C3′), 18.5 (C1′), 12.9 (C6′), 12.6 (CH3), 11.3 (C7′); the NA 1a (92 mg, 0.36 mmol, 79% overall yield) as a white solid, HRMS (ESI+) m/z: [M + Na]+ calcd for C13H16N2O3Na 271.1059; which was crystallized by vapor diffusion using the MeOH−Et2O found 271.1035. mixture at low temperature. Rf = 0.26 (CH2Cl2:MeOH 1:1); Mp ((1′R,2′R,3′R,5′S,6′S)-3′-(Benzoyloxy)-5′-[6″-chloro-2″-(for- 189−191 °C (from MeOH/Et2O); [α]D20 + 63.8 (c 0.47, MeOH); mylamino)-9″H-purin-9″-yl]bicyclo[4.1.0]hept-2′-yl)methyl 1 H NMR (400 MHz, MeOH-d4) δ 7.82 (q, J6,CH3 = 1.1 Hz, 1H, H-6), benzoate (22). A solution of the ammonium salt 17 (100 mg, 0.25 4.77 (td, J2′,3′ax = J2′,3′eq = 6.3, J2′,1′ = 1.5 Hz, 1H, H-2′), 3.89 (dd, Jgem mmol), 4,6-dichloro-2,5-diformamidopyrimidine, 20 (64 mg, 0.25 = 10.7 Hz, J1″a,5′ = 6.4 Hz, 1H, H-1″), 3.86−3.80 (m, 2H, H-4′, H- mmol), and N,N-diisopropylethylamine (DIPEA, 175 μL, 1.0 mmol) 1″), 1.89 (d, JCH3,6 = 1.1 Hz, 3H, CH3), 1.87−1.82 (m, 2H, H-3′eq, in dry 1,4-dioxane (5 mL) was stirred under argon at RT for 12 h and H-5′), 1.63 (ddd, Jgem = 14.2 Hz, J3′ax,2′ = 6.3 Hz, J3′ax,4′ = 2.9 Hz, 1H, then refluxed in an oil bath for 30 min. The solvent was evaporated to H-3′ax), 1.17 (tdd, J6′,7′exo = J6′,1′ = 8.5 Hz, J6′,7′endo = 5.5 Hz, J6′5′ = 2.6 dryness under reduced pressure, and the residue was dissolved in Hz, 1H, H-6′), 1.06 (tdd, J1′,7′exo = J1′6′ = 8.5 Hz, J1′7′endo = 6.2 Hz, J1′2′ EtOAc (7 mL), washed with water (5 mL) and brine (5 mL), dried = 1.5 Hz, 1H, H-1′), 0.91 (td, J7′exo,6′ = J7′exo,1′ = 8.5 Hz, Jgem = 5.3 Hz, (Na2SO4), filtered, and evaporated in vacuo. The crude was then 1H, H-7′exo), 0.23 (q, Jgem = J7′endo,1′ = J7′endo,6′ = 5.3 Hz, 1H, H- dissolved in diethoxymethyl acetate, 21 (5 mL), and the solution was 7′endo); 13C{1H} NMR (100 MHz, MeOH-d4) δ 166.7 (C2), 153.0 stirred at 140 °C in an oil bath under argon for 24 h. The mixture was (C4), 141.9 (C6), 110.2 (C5), 65.7 (C4′), 64.4 (C1″), 52.1 (C2′), cooled to RT and treated with MeOH (4 mL) and concentrated 42.4 (C5′), 33.6 (C3′), 14.5 (C1′), 13.6 (C6′), 12.5 (CH3), 11.1 (C- aqueous ammonia (0.5 mL) while stirring was continued. The solvent 7′); IR (ATR) 3483, 3270, 3019, 2885, 2360, 1700, 1660, 1269, 1002, was evaporated to dryness, and the residue was dissolved in EtOAc 847 cm−1; HRMS (ESI+) m/z: [M + H]+: calcd for C13H19N2O4 (30 mL) and extracted with water (3 × 15 mL). The organic layer was 267.1345; found 267.1344. dried (Na2SO4), filtered, and evaporated to dryness. The resultant 1-[(1′S,2′S,4′S,5′R,6′R)-4′-Hydroxy-5′-(hydroxymethyl)- yellow solid was purified by column chromatography (hexanes:E- bicyclo[4.1.0]hept-2′-yl]-5-methylpyrimidine-2,4(1H,3H)- tOAc, 5:1 → 1:1) to give compound 22 (103 mg, 0.19 mmol, 76% dione (2) and 1-[(1′S,2′S,6′R)-5′-(hydroxymethyl)-bicyclo- overall yield) as a yellowish solid. Rf = 0.11 (hexanes:EtOAc, 1:1); [4.1.0]hept-4′-en-2′-yl]-5-methylpyrimidine-2,4(1H,3H)-dione [α]D20 + 12.7 (c 0.79, CHCl3); 1H NMR (400 MHz, MeOH-d4) δ (19). A solution of compound 1a (30 mg, 0.11 mmol), 9.55 (d, JCHO,NH = 10.3 Hz, 1H, CHO), 8.94 (d, JNH,CHO = 10.3 Hz, triphenylphosphine (118 mg, 0.45 mmol), and benzoic acid (55 1H, NH), 8.12 (s, 1H, H-8″), 8.10−8.05 (m, 2H, H-orto), 7.90−7.84 mg, 0.45 mmol) in a mixture of dry benzene/acetonitrile (5:1, 2.5 (m, 2H, H-orto), 7.60−7.52 (m, 2H, H-para), 7.48−7.34 (m, 4H, H- mL) was stirred at 0 °C under an argon atmosphere. Diethyl meta), 5.35−5.25 (m, 2H, H-1a, H-3′), 4.94 (ddd, J5′,4′ax = 8.2 Hz, azodicarboxylate (DEAD, 71 μL, 0.45 mmol) was added dropwise J5′,4′eq = 6.7 Hz, J5′,6′ = 1.6 Hz, 1H, H-5′), 4.62 (dd, Jgem = 10.7 Hz, during 1 min, the ice bath was removed, and the mixture was stirred at J1b,2′ = 5.5 Hz, 1H, H-1b), 2.80 (dtd, J2′,3′ = 8.3 Hz, J2′,1b = J2′,1a = 5.5 RT for 12 h. The solvent was evaporated to dryness, and the residue Hz, J2′,1′ = 2.4 Hz, 1H, H-2′), 2.66 (dt, Jgem = 13.9 Hz, J4′ax,5′ = J4′ax,3′ = was purified by column chromatography (CH2Cl2:MeOH, 20:1) to 8.2 Hz, 1H, H-4′ax), 2.11 (ddd, Jgem = 13.9 Hz, J4′eq,5′ = 6.7 Hz, J4′eq,3′ provide a white solid still contaminated with Ph3P. The solid was = 3.5 Hz, 1H, H-4′eq), 1.53 (tdd, J1′,7′exo = J1′,6′ = 8.5 Hz, J1′,7′endo = dissolved in a solution of NH3 in MeOH (7 M, 2 mL) in a sealed flask 5.7 Hz, J1′,2′ = 2.4 Hz, 1H, H-1′), 1.33 (tdd, J6′,7′exo = J6′,1′ = 8.5 Hz, and stirred for 48 h at RT. Then, the mixture was concentrated under J6′,7′endo = 5.7 Hz, J6′,5′ = 1.6 Hz, 1H, H-6′), 1.10 (td, J7′exo,6′ = J7′exo,1′ = reduced pressure and purified by column chromatography (slow 8.5 Hz, Jgem = 5.8 Hz, 1H, H-7′exo), 0.59 (q, Jgem = J7′endo,6′ = J7′endo,1′ gradient of CH2Cl2:MeOH, 40:1 → 30:1 → 20:1 → 15:1 → 10:1) to = 5.7 Hz, 1H, H-7′endo); 13C{1H} NMR (100 MHz, MeOH-d4) δ provide the substitution product 2 (11 mg, 4.1 μmol, 37% overall 167.2 (CO), 165.7 (CO), 163.0 (CHO), 152.2/152.0/151.8 (C-4″, yield) as a white solid and the elimination product 19 (5 mg, 2.0 C-2″, C-6″), 143.7 (C-8), 133.7 (Cpara), 133.5 (Cpara), 129.9 μmol, 18% overall yield) as a white solid. (Corto), 129.7 (Cipso), 129.5 (Corto), 129.4 (Cipso), 129.3 (C5″), 2. Rf = 0.15 (CH2Cl2:MeOH, 15:1); [α]D20 + 39.8 (c 0.32, 128.6 (Cmeta), 128.6 (Cmeta), 67.4 (C3′), 65.0 (C1), 51.6 (C5′), MeOH); 1H NMR (400 MHz, MeOH-d4) δ 7.98 (q, J6,CH3 = 1.2 Hz, 36.3 (C2′), 29.5 (C4′), 14.3 (C1′), 14.2 (C6′), 10.3 (C7′); IR (ATR) 1H, H-6), 5.00 (td, J2′,3′ax = J2′3′eq = 3.8 Hz, J2′1′ = 1.2 Hz, 1H, H-2′), 3276, 2922, 2360, 1700, 1601, 1574, 1266, 1110, 749 cm−1; HRMS 3.88 (dd, Jgem = 10.6 Hz, J1″a,5′ = 3.5 Hz, 1H, H-1″a), 3.75 (dd, Jgem = (ESI+) m/z: [M + H]+ calcd for C28H25ClN5O5 546.1544; found 10.6 Hz, J1″b,5′ = 3.5 Hz, 1H, H-1″b), 3.61 (ddd, J4′,3′ax = 12.2 Hz, J4′,5′ 546.1546. = 9.1 Hz, J4′,3eq = 3.8 Hz, 1H, H-4′), 1.89 (d, JCH3,6 = 1.2 Hz, 3H, 2-Amino-9-[(1′S,2′S,4′R,5′S,6′S)-4′-hydroxy-5′-(hydroxy- CH3), 1.85 (dt, Jgem = 14.2 Hz, J3′eq,2′ = J3′eq,4′ = 3.8 Hz, 1H, H-3′eq), methyl) Bicyclo[4.1.0]hept-2′-yl]-1,9-dihydro-6H-purin-6-one 1.64 (dtd, J5′,4′ = 9.1 Hz, J5′,1″a = J5′,1″b = 3.5 Hz, J5′,6′ = 1.3 Hz, 1H, H- (1b). A solution of 22 (100 mg, 0.18 mmol) in 80% HCO2H (4.5 5′), 1.49 (ddd, Jgem = 14.2 Hz, J3′ax,4′ = 12.2 Hz, J3′ax,2′ = 3.8 Hz, 1H, mL) was stirred at reflux in an oil bath for 2 h. The solution was H-3′ax), 1.27 (dddd, J6′,7′exo = 9.3 Hz, J6′,1′ = 7.7 Hz, J6′,7′endo = 5.3 Hz, cooled at RT, and the solvent was evaporated under reduced pressure. J6′,5′ = 1.3 Hz, 1H, H-6′), 1.08 (dddd, J1′,7′exo = 9.3 Hz, J1′,6′ = 7.7 Hz, The residue was dissolved in a NH3 solution in MeOH (7 M, 20 mL) J1′,7′endo = 5.3 Hz, J1′,2′ = 1.2 Hz, 1H, H-1′), 0.91 (td, J7′exo,6′ = J7′exo,1′ = and stirred at RT for 3 days. Then, the mixture was concentrated 9.3 Hz, Jgem = 5.3 Hz, 1H, H-7′exo), 0.39 (q, Jgem = J7′endo,1′ = J7′endo,6′ under reduced pressure and purified by column chromatography = 5.3 Hz, 1H, H-7′endo); 13C{1H} NMR (100 MHz, MeOH-d4) δ (CH2Cl2:MeOH, 10:1 → 5:1 → 1:1) to provide guanine compound 166.6 (C2), 153.0 (C4), 141.2 (C6), 110.1 (C5), 64.1 (C4′), 63.0 1b (30 mg, 0.10 mmol, 57% yield) as a pale white solid. Rf = 0.09 (C1″), 53.7 (C2′), 46.1 (C5′), 33.3 (C3′), 15.3 (C1′), 14.9 (C6′), (CH2Cl2:MeOH, 15:1); [α]D20 + 26.5 (c 0.48, MeOH); 1H NMR 12.4 (CH3), 10.4 (C7′); HRMS (ESI+) m/z: [M + Na]+ calcd for (400 MHz, MeOH-d4) δ 7.99 (s, 1H, H-8), 4.73 (td, J2′,3′ax = J2′,3′eq = C13H18N2O4Na 289.1164; found 289.1164. 6.5 Hz, J2′,1′ = 1.6 Hz, 1H, H-2′), 3.94 (dd, Jgem = 10.7 Hz, J1″a,5′ = 6.5 19. Rf = 0.36 (CH2Cl2:MeOH, 15:1); 1H NMR (400 MHz, Hz, 1H, H-1″a), 3.91−3.83 (m, 2H, H-1″b, H-4′), 2.06 (dt, Jgem = MeOH-d4) δ 7.54 (q, J6,CH3 = 1.1 Hz, 1H, H-6), 5.37 (m, 1H, H-4′), 14.1 Hz, J3′ax,2′ = J3′ax,4′ = 6.5 Hz, 1H, H-3′ax), 1.87 (tdd, J5′,1″a = J5′,1″b 5.12 (dt, J2′,3′ax = 6.6 Hz, J2′,3′eq = J2′,1′ = 2.1 Hz, 1H, H-2′), 4.17 (m, = 6.7 Hz, J5′,3′ax = 6.5 Hz, J5′,3′eq = 3.0 Hz, 1H, H-5′), 1.76 (ddd, Jgem = 15173 https://doi.org/10.1021/acs.joc.2c01661 J. Org. Chem. 2022, 87, 15166−15177 The Journal of Organic Chemistry pubs.acs.org/joc Article 14.1 Hz, J3′eq,2′ = 6.5 Hz, J3′eq,4′ = 3.0 Hz, 1H, H-3′eq), 1.31−1.13 (m, carboxamide (3b). To a solution of 2-cyanoacetamide, 25 (35 mg, 2H, H-1′, H-6′), 0.96 (td, J7′exo,1′ = J7′exo,6′ = 9.2 Hz, Jgem = 5.0 Hz, 1H, 0.42 mmol), in dry DMSO (500 μL) at RT, K2CO3 (54 mg, 0.39 H-7′exo), 0.28 (q, Jgem = J7′endo,1′ = J7′endo,6′ = 5.4 Hz, 1H, H-7′endo); mmol) under an argon atmosphere was added. The mixture was 13 C{1H} NMR (100 MHz, MeOH-d4) δ 158.0 (C6), 153.6 (C2), stirred at the same temperature for 1 h. After this time, a DMSO 151.3 (C4), 138.1 (C8), 115.9 (C5), 64.4 (C4′), 63.0 (C1″), 49.2 solution (500 μL) of 6 (50 mg, 0.13 mmol) was added and the (C2′), 41.4 (C5′), 33.3 (C3′), 14.0 (C1′), 12.2 (C6′), 10.1 (C7′); stirring was continued for 2 d at 50 °C in an oil bath. After HRMS (ESI+) m/z: [M + H]+ calcd for C13H18N5O3 292.1410; evaporation of the solvent, the solid residue was purified by column found 292.1410. chromatography (CH2Cl2:MeOH, 15:1 → 10:1) to give 3b (19 mg, Methyl 1-{(1′S,2′S,4′R,5′R,6′R)-4′-(Benzoyloxy)-5′- 0.07 mmol, 55% yield) as whitish solid. Rf = 0.1 (CH2Cl2:MeOH, [(benzoyloxy)methyl]-bicyclo[4.1.0]hept-2′-yl}-1H-1,2,3-tria- 10:1); Mp 210−213 °C (from MeOH-CH2Cl2); [α]D20−35.6 (c 0.64, zole-4-carboxylate (24). The benzyl protected azido alcohol 6 (200 MeOH); 1H NMR (400 MHz, MeOH-d4) δ 4.72 (td, J2′,3′ax = J2′,3′eq = mg, 0.511 mmol) was suspended in a 2:1 mixture of acetonitrile and 7.5 Hz, J2′,1′ = 1.9 Hz, 1H, H-2′), 3.95 (dd, Jgem = 10.6 Hz, J1″a,5′ = 6.4 water (8 mL), and methyl propiolate, 23 (70 μL, 0.767 mmol), was Hz, 1H, H-1″a), 3.91−3.80 (m, 2H, H-4′, H-1″b), 2.12 (dt, Jgem = added. Then, a 1 M solution of sodium ascorbate in water (205 μL, 13.7 Hz, J3′ax,2′ = J3′ax,4′ = 8.2 Hz, 1H, H-3′ax), 1.97−1.87 (m, 2H, H- 0.205 mmol, 40 mol %) and a 1 M of CuSO4·5H2O in water (102 μL, 3′eq, H-5′), 1.29−1.17 (m, 2H, H-1′, H-6′), 0.95 (td, J7′exo,1′ = J7′exo,6′ 0.10 mmol, 20 mol %) were sequentially added. The round-bottom = 9.2, Jgem = 5.4 Hz, 1H, H-7′exo), 0.32 (q, Jgem = J7′endo,1′ = J7′endo,6′ = flask was wrapped with aluminum foil, and the reaction mixture was 5.4 Hz, 1H, H-7′endo); 13C{1H} NMR (100 MHz, MeOH-d4) δ stirred 16 h at RT. Then, the mixture was filtered through a short pad 167.1 (CO), 146.0 (C5), 123.3 (C4), 65.7 (C4′), 63.8 (C1″), 54.2 of Celite and washed with EtOAc. The solvent was evaporated under (C2′), 42.3 (C5′), 34.4 (C3′), 14.4/14.2 (C1′/C6′), 11.2 (C7′); IR reduced pressure and purified by column chromatography (hex- (ATR) 3308, 3174, 1662, 1633, 1561, 1517, 1226, 1096, 1021, 877, anes:EtOAc, 4:1 → 1:1) to afford triazole compound 24 (192 mg, 784 cm−1; HRMS (ESI+) m/z: [M + Na]+ calcd for C11H17N5O3Na 0.40 mmol, 79% yield) as a white solid. Rf = 0.27 (hexanes:EtOAc, 290.1229; found 290.1229. 1:1); [α]D20−115 (c 0.40, CHCl3); 1H NMR (400 MHz, CDCl3) δ (1R,2R,3R,5S,6S)-5-Azido-2-(hydroxymethyl)bicyclo[4.1.0]- 8.28 (s, 1H, H-5), 7.98 (dd, Jorto,meta = 8.4 Hz, Jorto,para = 1.3 Hz, 2H, H- heptan-3-ol (26). Compound 6 (320 mg, 0.82 mmol) was dissolved orto), 7.78 (dd, Jorto,meta = 8.4 Hz, Jorto,para = 1.3 Hz, 2H, H-orto), 7.58− in a solution of NH3 solution in MeOH (7 M, 50 mL) in a sealed 7.48 (m, 2H, H-para), 7.46−7.33 (m, 4H, H-meta), 5.30 (ddd, J4′,3′ax flask, and the solution was stirred for 3 days at RT. Then, the mixture = 5.8 Hz, J4′,5′ = 4.4 Hz, J4′,3′eq = 2.9 Hz, 1H, H-4′), 5.14 (ddd, J2′,3′eq = was concentrated under reduced pressure and purified by column 6.2 Hz, J2′,3′ax = 4.8, J2′,1′ = 1.6 Hz, 1H, H-2′), 4.72 (dd, Jgem = 10.9 Hz, chromatography (CH2Cl2:MeOH, 30:1 → 20:1 → 10:1) to provide J1″a,5′ = 7.3 Hz, 1H, H-1″a), 4.54 (dd, Jgem = 10.9 Hz, J1″b,5′ = 8.1 Hz, the deprotected azide compound 26 (128 mg, 0.70 mmol, 85% yield) 1H, H-1″b), 3.77 (s, 3H, OCH3), 2.78 (ddd, Jgem = 15.3 Hz, J3′ax,4′ = as a white solid. Rf = 0.32 (CH2Cl2:MeOH, 20:1); [α]D20−46.3 (c 1.0, 5.8 Hz, J3′ax,2′ = 4.8 Hz, 1H, H-3′ax), 2.49 (tdd, J5′,1″a = J5′,1″b = 7.3 Hz, MeOH); 1H NMR (400 MHz, MeOH-d4) δ 3.90−3.76 (m, 3H, H-3, J5′,4′ = 4.4 Hz, J5′,6′ = 2.8 Hz, 1H, H-5′), 2.08 (ddd, Jgem = 15.3 Hz, H-5, H-1′a), 3.68 (dd, Jgem = 10.6 Hz, J1′b,2 = 7.1 Hz, 1H, H-1′b), J3′eq,2′ = 6.2 Hz, J3′eq,4′ = 2.9 Hz, 1H, H-3′eq), 1.67−1.60 (m, 1H, H- 1.82−1.67 (m, 2H, H-4ax, H-2), 1.62 (ddd, Jgem = 14.3 Hz, J4eq,5 = 5.8 1′), 1.35 (tdd, J6′,7′exo = J6′,1′ = 9.3 Hz, J6′,7′endo = 5.5 Hz, J6′,5′ = 2.8 Hz, Hz, J4eq,3 = 3.0 Hz, 1H, H-4 eq), 1.13−1.03 (m, 1H, H-6), 0.95 (tdd, 1H, H-6′), 1.23 (td, J7′exo,6′ = J7′exo,1′ = 9.3 Hz, Jgem = 5.5 Hz, 1H, H- J1,6 = J1,7exo = 9.1 Hz, J1,7endo = 5.2 Hz, J1,2 = 2.9 Hz, 1H, H-1), 0.87 7′exo), 0.50 (q, Jgem = J7′endo,6′ = J7′endo,1′ = 5.5 Hz, 1H, H-7′endo); (td, J7exo,1 = J7exo,6 = 9.1 Hz, Jgem = 4.7 Hz, 1H, H-7exo), 0.11 (q, Jgem = 13 C{1H} NMR (100 MHz, CDCl3) δ 166.5 (CO), 165.5 (CO), 160.8 J7endo,1 = J7endo,6 = 5.2 Hz, 1H, H-7endo); 13C{1H} (101 MHz, (C(O)OCH3), 139.3 (C4), 133.3 (Cpara), 133.1 (Cpara), 129.8 MeOH-d4) δ 65.5 (C3), 64.5 (C1′), 57.5 (C5), 43.2 (C2), 33.4 (C4), (Cipso), 129.7 (Corto), 129.5 (Corto), 129.4 (Cipso), 128.6 (Cmeta), 14.9 (C6), 12.7 (C1), 11.0 (C7); HRMS (ESI+) m/z: [M + Na]+ 128.5 (Cmeta), 126.8 (C5), 66.5 (C4′), 64.8 (C1″), 54.8 (C2′), 52.0 calcd for C8H13N3O2Na 206.0905; found 206.0904. (OCH3), 38.0 (C5′), 30.7 (C3′), 14.1 (C1′), 11.4 (C7′), 11.2 (C6′); General Procedure for the CuAAC under MW Irradiation. To IR (ATR) 3007, 2164, 2111, 1712, 1601, 1265, 1175, 752, 710 cm-1; a solution of azide 26 in a 1:1 mixture of water and t-BuOH (10 mL/ HRMS (ESI+) m/z: [M + H]+ calcd for C26H26N3O6 476.1822; mmol) in a glass vial equipped with a magnetic stirring bar, copper found 476.1821. powder (80 mol %), a 1 M solution of copper sulfate in water (20 mol 1-[(1′S,2′S,4′R,5′R,6′R)-4′-Hydroxy-5′-(hydroxymethyl)- %), and finally the alkyne (1.05 eq) were added. The vial was sealed bicyclo[4.1.0]hept-2′-yl]-1H-1,2,3-triazole-4-carboxamide with a Teflon crimp top, and the reaction mixture was irradiated (3a). The ester-triazole 24 (130 mg, 0.27 mmol) was dissolved in a under MW at 125 °C. Upon completion of the reaction, the vial was solution of NH3 in MeOH (7 M, 6 mL) in a sealed flask and was cooled to 50 °C by air jet cooling before opening. The mixture was stirred for 2 days at RT. Then, the mixture was concentrated under filtered over a plug of Celite (rinsed with MeOH), and the filtrate was reduced pressure and purified by column chromatography evaporated under reduced pressure. (CH2Cl2:MeOH, 20:1 → 15:1 → 10:1) to provide carboxamide 3a (1R,2R,3R,5S,6S)-2-(Hydroxymethyl)-5-(4″-phenyl-1H- (61 mg, 0.24 mmol, 92% yield) as a white solid. Rf = 0.15 1″,2″,3″-triazol-1-yl)bicyclo[4.1.0]heptan-3-ol (3c). By following (hexanes:EtOAc, 1:1); [α]D20−78.6 (c 0.79, CHCl3); 1H NMR (400 the general procedure, the title compound was prepared from azide MHz, MeOH-d4) δ 8.63 (s, 1H, H-5), 5.07 (ddd, J2′,3′eq = 6.1 Hz, 26 (19 mg, 0.11 mmol) and phenylacetylene, 27c (12 μL, 0.110 J2′,3′ax = 5.5 Hz, J2′,1′ = 1.6 Hz, 1H, H-2′), 3.96−3.86 (m, 2H, H-4′, H- mmol), after 2 min of MW irradiation. Purification by column 1″a), 3.81 (dd, Jgem = 10.7 Hz, J1″b,5′ = 6.7 Hz, 1H, H-1″b), 2.12 (ddd, chromatography (EtOAc) delivered compound 3c (29 mg, 0.102 Jgem = 14.5 Hz, J3′ax,4′ = 6.3 Hz, J3′ax,2′ = 5.5 Hz, 1H, H-3′ax), 1.93 mmol, 97% yield) as a white solid. Rf = 0.21 (EtOAc); [α]D20−52 (c (ddd, Jgem = 14.5 Hz, J3′eq,2′ = 6.1 Hz, J3′eq,4′ = 3.0 Hz, 1H, H-3′eq), 0.8, CHCl3); 1H NMR (400 MHz, MeOH-d4) δ 8.52 (s, 1H, H-5″), 1.83 (tt, J5′,1″a = J5′,1″b = 6.7 Hz, J5′,4′ = J5′,6′ = 3.5 Hz, 1H, H-5′), 1.36− 7.86−7.79 (m, 2H, H-orto), 7.46−7.41 (m, 2H, H-meta), 7.37−7.31 1.26 (m, 1H, H-1′), 1.14 (tdd, J6′,7′exo = J6′,1′ = 8.9 Hz, J6′,7′endo = 5.4 (m, 1H, H-para), 5.02 (td, J5,4ax = J5,4eq = 6.6 Hz, J5,6 = 1.8 Hz, 1H, H- Hz, J6′,5′ = 3.5 Hz, 1H, H-6′), 1.01 (td, J7′exo,6′ = J7′exo,1′ = 8.9 Hz, Jgem 5), 4.02−3.91 (m, 2H, H-3, H-1′a), 3.84 (dd, Jgem = 10.7 Hz, J1′b,2 = = 5.4 Hz, 1H, H-7′exo), 0.33 (q, Jgem = J7′endo,6′ = J7′endo,1′ = 5.4 Hz, 6.8 Hz, 1H, H-1′b), 2.16 (dt, Jgem = 13.9 Hz, J4ax,3 = J4ax,5 = 7.0 Hz, 1H, H-7′endo); 13C{1H} NMR (100 MHz, MeOH-d4) δ 165.0 1H, H-4ax), 2.00 (ddd, Jgem = 14.2 Hz, J4eq,5 = 6.7 Hz, J4eq,3 = 3.3 Hz, (C(O)NH2), 143.1 (C4), 127.4 (C5), 65.1 (C4′), 64.4 (C1″), 57.2 1H, H-4 eq), 1.90 (tt, J2,1′ = 6.8 Hz, J2,3 = J2,1 = 3.6 Hz, 1H, H-2), (C2′), 43.1 (C5′), 35.1 (C3′), 15.4 (C1′), 12.8 (C6′), 11.5 (C7′); IR 1.39−1.31 (m, 1H, H-6), 1.20 (tdd, J1,6 = J1,7exo = 8.7 Hz, J1,7endo = 5.4 (ATR) 3339, 3195, 2886, 1661, 1603, 1289, 1046, 855, 721 cm−1; Hz, J1,2 = 3.2 Hz, 1H, H-1), 1.01 (td, J7exo,6 = J7exo,1 = 9.2 Hz, Jgem = 5.4 HRMS (ESI+) m/z: [M + Na]+ calcd for C11H16N4O3Na 275.1120; Hz, 1H, H-7exo), 0.37 (q, Jgem = J7endo,6 = J7endo,1 = 5.4 Hz, 1H, H- found 275.1121. 7endo), 13C{1H} NMR (100 MHz, MeOH-d4) δ 148.5 (C4″), 131.9 5-Amino-1-[(1′S,2′S,4′R,5′R,6′R)-4′-hydroxy-5′- (Cipso), 130.0 (Cmeta), 129.2 (Cpara), 126.6 (Corto), 121.7 (C5″), (hydroxymethyl)bicyclo-[4.1.0]hept-2′-yl]-1H-1,2,3-triazole-4- 65.5 (C3), 64.3 (C1′), 57.6 (C5), 43.0 (C2), 35.6 (C4), 15.6 (C6), 15174 https://doi.org/10.1021/acs.joc.2c01661 J. Org. Chem. 2022, 87, 15166−15177 The Journal of Organic Chemistry pubs.acs.org/joc Article 13.5 (C1), 11.4 (C7); HRMS (ESI+) m/z: [M + H]+ calcd for C16H20N3O2 286.1556, found 286,1555. (1R,2R,3R,5S,6S)-2-(Hydroxymethyl)-5-[4″-(4‴-propylphen- ■ AUTHOR INFORMATION Corresponding Authors yl)-1″H-1″,2″,3″-triazol-1″-yl]bicyclo[4.1.0]heptan-3-ol (3d). Félix Busqué − Departament de Química, Universitat By following the general procedure, the title compound was prepared Autònoma de Barcelona, Barcelona 08193, Spain; from azide 26 (20 mg, 0.11 mmol) and 1-ethynyl-4-propylbenzene, Email: felix.busque@uab.cat 27d (18 μL, 0.115 mmol), after 10 min of MW irradiation. Ramon Alibés − Departament de Química, Universitat Purification by column chromatography (EtOAc → EtOAc:MeOH, Autònoma de Barcelona, Barcelona 08193, Spain; 95:5) afforded compound 3d (33 mg, 0.10 mmol, 96% yield) as a orcid.org/0000-0002-7997-2691; Email: ramon.alibes@ white solid. Rf = 0.20 (EtAOc); [α]D20−25 (c 0.45, CHCl3); 1H NMR uab.cat (400 MHz, MeOH-d4) δ 8.47 (s, 1H, H-5″), 7.72 (d, J2‴/6‴,3‴/5‴ = 8.5 Hz, 2H, H-2‴/6‴), 7.25 (d, J3‴/5‴,2‴/6‴ = 8.5 Hz, 2H, H-3‴/5‴), 5.00 Authors (td, J5,4ax = J5,4eq = 6.6 Hz, J5,6 = 1.8 Hz, 1H, H-5), 4.00−3.90 (m, 2H, Sergio Jurado − Departament de Química, Universitat H-3, H-1′a), 3.84 (dd, Jgem = 10.7 Hz, J1′b,2 = 6.8 Hz, 1H, H-1′b), 2.62 Autònoma de Barcelona, Barcelona 08193, Spain (t, J1⁗,2⁗= 7.4 Hz, 1H, H-1⁗), 2.21 − 2.10 (m, 1H, H-4ax), 1.99 (ddd, Jgem = 14.1 Hz, J4eq,5 = 6.7 Hz, J4eq,3 = 3.3 Hz, 1H, H-4eq), 1.94 Ona Illa − Departament de Química, Universitat Autònoma de − 1.86 (m, 1H, H-2), 1.67 (h, J2⁗,1⁗= J2⁗,3⁗= 7.4 Hz, 1H, H-2⁗), 1.32 Barcelona, Barcelona 08193, Spain; orcid.org/0000- (tdd, J6,7exo = J6,1 = 9.2 Hz, J6,7endo = 5.6 Hz, J6,5 = 1.9 Hz, 1H, H-6), 0001-7390-4893 1.20 (tdd, J1,7exo = J1,6 = 8.7 Hz, J1,7endo = 5.4 Hz, J1,2 = 3.1 Hz, 1H, H- Angel Á lvarez-Larena − Servei de Difracció de Raigs X, 1), 1.01 (td, J7exo,1 = J7exo,6 = 9.2 Hz, Jgem = 5.4 Hz, 1H, H-7exo), 0.96 Universitat Autònoma de Barcelona, Barcelona 08193, Spain (t, J3⁗,2⁗= 7.4 Hz, 1H, H-3⁗), 0.36 (q, Jgem= J7endo,6= J7endo,1= 5.4 Hz, Christophe Pannecouque − Department of Microbiology and 1H, H-7endo); 13C{1H} NMR (100 MHz, MeOH-d4) δ 148.6 (C4′), Immunology, Laboratory of Virology and Chemotherapy, 144.1 (C4″), 130.0 (2C, C2″/C6″), 129.4 (C1″), 126.6 (2C, C3″/ Rega Institute for Medical Research, KU Leuven, Leuven B- C5″), 121.4 (C5′), 65.5 (C3), 64.3 (C1⁗), 57.6 (C5), 42.9 (C2), 3000, Belgium 38.8 (C1‴), 35.6 (C4), 25.7 (C2‴), 15.6 (C6), 14.1 (C3‴), 13.5 (C1), 11.4 (C7); HRMS (ESI+) m/z: [M + H]+ calcd for Complete contact information is available at: C19H26N3O2 328.2025; found 328.2027. https://pubs.acs.org/10.1021/acs.joc.2c01661 (1R,2R,3R,5S,6S)-2-(Hydroxymethyl)-5-[4″-(hydroxymethyl)- 1H-1″,2″,3″-triazol-1‴-yl]bicyclo[4.1.0]heptan-3-ol (3e). By Notes following the general procedure, the title compound was prepared The authors declare no competing financial interest. from azide 26 (49 mg, 0.27 mmol) and propargyl alcohol, 27e (16 μL, 0.278 mmol), after 1 min of MW irradiation. Purification by column chromatography (EtOAc:MeOH 95:5 → 90:10) furnished compound 3e (51 mg, 0.21 mmol, 80% yield) as a white solid. Rf = 0.1 ■ ACKNOWLEDGMENTS This work was partially supported by funds from the Spanish (CH2Cl2:MeOH, 10:1); [α]D20 62.9 (c 0.98, MeOH);1H NMR (400 Ministry of Economy, Industry, and Competitiveness (MINE- MHz, MeOH-d4) δ 8.14 (s, 1H, H-5″), 4.96 (td, J5,4ax = J5,4eq = 6.8 Hz, CO) (projects: CTQ2016-75363-R and PID2019-106403RB- J5,6 = 1.9 Hz, 1H, H-5), 4.68 (br s, 2H, H-1‴), 3.97−3.89 (m, 2H, H- I00) cofinanced with the European Fund for Regional 3, H-1′a), 3.81 (dd, Jgem = 10.7 Hz, J1′b,2 = 6.8 Hz, 1H, H-1′b), 2.09 Development (FEDER). We acknowledge additional support (dt, Jgem = 14.1 Hz, J4ax,3 = J4ax,5 = 6.8 Hz, 1H, H-4ax), 1.96 (ddd, Jgem = 14.1 Hz, J4eq,5 = 6.8 Hz, J4eq,3 = 3.4 Hz, 1H, H-4 eq), 1.88 (tt, J2,1′ = from Generalitat de Catalunya (2017-SGR-191). S.J. acknowl- 6.8 Hz, J2,3 = J2,1 = 3.7 Hz, 1H, H-2), 1.28−1.21 (m, 1H, H-6), 1.17 edges the financial support from the Universitat Autònoma de (tdd, J1,7exo = J1,6 = 8.7 Hz, J1,7endo = 5.4, J1,2 = 3.7 Hz, 1H, H-1), 0.97 Barcelona. (td, J7exo,1 = J7exo,6 = 9.2 Hz, Jgem = 5.4 Hz, 1H, H-7exo), 0.34 (q, Jgem = J7endo,1 = J7endo,6 = 5.4 Hz, 1H, H-7endo); 13C{1H} NMR (100 MHz, MeOH-d4) δ 148.9 (C4″), 123.6 (C5″), 65.5 (C3), 64.3 (C1′), 57.5 ■ REFERENCES (1) Ma, Y.; Frutos-Beltran, E.; Kang, D.; Pannecouque, C.; De (C5), 56.6 (C1‴), 42.9 (C4), 35.7 (C2), 15.6 (C6), 13.5 (C1), 11.3 Clercq, E.; Menéndez-Arias, L.; Liu, X.; Zhan, P. 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